Herbs
vs AIDS
Inhibition of HIV-1
replication by an aqueous extract of Spirulina platensis
Calcium-Spirulan
Reverse HIV Naturally with
Spirulina Filipina
ABAMAV vs HIV
Anti-HIV Activity of Medicinal
Herbs: Usage and Potential Development
Preparation of a novel molecularly
imprinted polymer for the highly selective extraction of
baicalin
Different extraction
pretreatments significantly change the flavonoid contents of
Scutellaria baicalensis
Patents : Extraction of
Baicalin
Black Seed Extract 'Cures' HIV
Patient Naturally
Nigella Sativa Concoction
Induced Sustained Seroreversion in HIV Patient
Olive Leaf Extract (20% oleuropein),
Oregano Oil, Turmeric Curcumin
Traditional Chinese Herbal
Medicines for Treating HIV Infections and AIDS
Randomized double-blinded and
controlled clinical trial on treatment of HIV/AIDS by
Zhongyan-4
Randomized, placebo-controlled trial
of Chinese herb therapy for HIV-1- infected individuals
Effects of Jingyuankang capsules
on leukocyte level in AIDS patients
YouTube : HIV/AIDS Herbal Remedy /
HIV/AIDS Herbal Remedy - Real testimonies (Part 2) / Dr.
Mitch interviews Dr. Paul Chepkwony
Dr. Paul Chepkwony : US7556830 --
Medicinal herbal composition for treating infection
US5837257 : Use of plant extracts
for treatment of HIV, HCV and HBV infections
US6696094 : Herbal pharmaceutical
composition for treatment of HIV/AIDS patients
US5178865 : CHINESE HERBAL EXTRACTS
IN THE TREATMENT OF HIV RELATED DISEASE IN VITRO
US6455078 : Medicinal herbal
composition for treating liver diseases and HIV
See also :
FARHADI, Mohammed,
et al : Urtica vs AIDS
~ Extract of Nettle treated with pulsed high
frequency EMF ( 45 W, 150 Tesla, 750 KHz )
being tested by Iran.
Spirulina
http://journals.lww.com/jaids/Abstract/1998/05010/Inhibition_of_HIV_1_Replication_by_an_Aqueous.2.aspx
http://www.ncbi.nlm.nih.gov/pubmed/9593452
J Acquir Immune Defic Syndr Hum Retrovirol. 1998 May
1;18(1):7-12.
Inhibition
of HIV-1 replication by an aqueous extract of Spirulina
platensis (Arthrospira platensis).
Ayehunie
S, Belay A, Baba TW, Ruprecht RM.
Abstract
An aqueous extract of the blue-green filamentous algae
Arthrospira platensis (previously called Spirulina platensis)
inhibited HIV-1 replication in human T-cell lines, peripheral
blood mononuclear cells (PBMC), and Langerhans cells (LC).
Extract concentrations ranging between 0.3 and 1.2 microg/ml
reduced viral production by approximately 50% (50% effective
concentration [EC50]) in PBMCs. The 50% inhibitory concentration
(IC50) of extract for PBMC growth ranged between 0.8 and 3.1
mg/ml. Depending on the cell type used, therapeutic indices
ranged between 200 and 6000. The extract inactivated HIV-1
infectivity directly when preincubated with virus before
addition to human T-cell lines. Fractionation of the extract
revealed antiviral activity in the polysaccharide fraction and
also in a fraction depleted of polysaccharides and tannins. We
conclude that aqueous A platensis extracts contain
antiretroviral activity that may be of potential clinical
interest.
http://www.lightparty.com/Health/Spirulina.html
...Another group of medical scientists has published new studies
regarding a purified water extract unique to Spirulina named
Calcium-Spirulan. It inhibits replication of HIV-1, herpes
simples, human cytomegalovirus, influenza A virus, mumps virus
and measles virus in-vitro, yet is very safe for human cells. It
protects human and monkey cells from viral infection in cell
culture. According to peer reviewed scientific journal reports
this extract, "...holds great promise for treatment of ...HIV-1,
HSV-1, and HCM infections, which is particularly advantageous
for AIDS patients who are prone to these life-threatening
infections."
Calcium-Spirulan is a polymerized sugar molecule unique to
Spirulina, containing both sulfur and calcium. Hamsters treated
with this water soluble extract had better recovery rates when
infected with an otherwise lethal herpes virus. How does it
work? When attacking a cell, a virus first attaches itself to
the cell membrane. However, because of spirulina extract, the
virus cannot penetrate the cell membrane to infect the cell. The
virus is stuck, unable to replicate. It is eventually eliminated
by the body's natural defenses. Spirulina extracts may become
useful therapeutics that could help AIDS patients lead longer,
more normal lives.
https://www.youtube.com/watch?v=ywwap1HNyxw
Reverse
HIV Naturally with Spirulina Filipina
THE SAN FRANCISCO MEDICAL RESEARCH FOUNDATION
The Study of Spirulina
Effects on the AIDS Virus, Cancer and the Immune System
Spirulina is gaining more attention from medical scientists as a
nutraceutical and source of potential pharmaceuticals. There are
several new peer reviewed scientific studies about Spirulina's
ability to inhibit viral replication, strengthen both the
cellular and humoral arms of the immune system and cause
regression and inhibition of cancers. While these studies are
preliminary and more research is needed, the results so far are
exciting.
In April 1996, scientists from the Laboratory of Viral
Pathogenesis, Dana-Farber Cancer Institute and Harvard Medical
School and Earthrise Farms, Calipatria, California, announced
on-going research, saying "Water extract of Spirulina platensis
inhibits HIV-1 replication in human derived T-cell lines and in
human peripheral blood mononuclear cells. A concentration of
5-10 mg/ml was found to reduce viral production." HIV-1 is the
AIDS virus. Small amounts of Spirulina extract reduced viral
replication while higher concentrations totally stopped its
reproduction. Importantly, with the therapeutic index of 100,
Spirulina extract was non-toxic to human cells at concentrations
stopping viral replication.
http://www.naturalpharmainternational.com/1/abamav_vs_hiv_1671420.html
Natural Pharma Gmbh
ABAMAV
VS HIV
How to cure HIV through ABAMAV® Application
Confidential Information
You can request information by sending email to:
info@naturalpharmainternational.com
Our ABAMAV is now, in the WORLD, the MOST POWERFUL
ANTIMICROBIAL existing. Origin is totally natural and non-toxic:
extracted compounds from 15 algae and from other medicinal
plants!
Attention information on ABAMAV can be ship ONLY to medical
doctors do not ship to private users. It is our interest to
cooperate with doctors and hospitals anywhere in the world!
http://www.worldscientific.com/doi/abs/10.1142/S0192415X01000083
Am. J. Chin. Med. 29, 69 (2001).
DOI: 10.1142/S0192415X01000083
Anti-HIV
Activity of Medicinal Herbs: Usage and Potential
Development
Ji An Wu
et al,
The acquired immunodeficiency syndrome (AIDS) is a result of
human immunodeficiency virus (HIV) infection which subsequently
leads to significant suppression of immune functions. AIDS is a
significant threat to the health of mankind, and the search for
effective therapies to treat AIDS is of paramount importance.
Several chemical anti-HIV agents have been developed. However,
besides the high cost, there are adverse effects and limitations
associated with using chemotherapy for the treatment of HIV
infection Thus, herbal medicines have frequently been used as an
alternative medical therapy by HIV positive individuals and AIDS
patients. The aim of this review is to summarize research
findings for herbal medicines, which are endowed with the
ability to inhibit HIV. In this article, we will emphasize a
Chinese herbal medicine. Scutellaria baicalensis Georgi
and its identified components (i.e. baicalein and baicalin)
which have been shown to inhibit infectivity and replication of
HIV. Potential development of anti-AIDS compounds using
molecular modeling methods will also be discussed.
http://onlinelibrary.wiley.com/doi/10.1002/jssc.201500865/abstract
DOI: 10.1002/jssc.201500865View
Preparation
of a novel molecularly imprinted polymer for the highly
selective extraction of baicalin
Xiao Liu,
et al
Abstract
The selective extraction of baicalin is important to its quality
control especially when the matrices are complicated. In this
work, a novel molecularly imprinted polymer was prepared for the
selective extraction of baicalin in herbs. The molecularly
imprinted polymer was synthesized by the copolymerization of
4-vinyl pyridine and ethylene glycol dimethacrylate in the
presence of baicalin by a precipitation polymerization method.
After the optimization of parameters for molecularly imprinted
polymer preparation, including the functional monomer, porogen,
sampling solvent, and washing solvent, good selectivity was
obtained, with an imprinting factor of about 4, which is much
better than that achieved by the bulk-polymerization method. The
performances of the prepared molecularly imprinted polymers were
systematically investigated, including adsorption kinetics,
isotherm experiment, and Scatchard analysis. On the basis of the
good adsorptive capability of the prepared molecularly imprinted
polymer, it was also applied for the pretreatment of baicalin in
Scutellaria baicalensis Georgi. The result showed that most of
the matrices were removed and baicalin was selectively enriched.
http://www.tandfonline.com/doi/abs/10.3109/13880209.2013.784922?journalCode=iphb20#.VpMTe6TR1FU
Pharmaceutical Biology Volume 51, Issue 10, 2013
DOI: 10.3109/13880209.2013.784922
Different
extraction pretreatments significantly change the
flavonoid contents of Scutellaria baicalensis
Chunhao
Yuabc, et al.
Abstract
Context: Scutellaria baicalensis Georgi (Labiatae) is one of the
most commonly used medicinal herbs, especially in traditional
Chinese medicine. However, compared to many pharmacological
studies of this botanical, much less attention has been paid to
the quality control of the herb’s pretreatment prior to extract
preparation, an issue that may affect therapeutic outcomes.
Objective: The current study was designed to evaluate whether
different pretreatment conditions change the contents of the
four major flavonoids in the herb, i.e., two glycosides
(baicalin and wogonoside) and two aglycones (baicalein and
wogonin).
Materials and methods: A high-performance liquid chromatography
assay was used to quantify the contents of these four
flavonoids. The composition changes of four flavonoids by
different pretreatment conditions, including solvent, treatment
time, temperature, pH value and herb/solvent ratio were
evaluated.
Results: After selection of the first order time-curve kinetics,
our data showed that at 50 °C, 1:5 herb/water (in w/v) ratio and
pH 6.67 yielded an optimal conversion rate from flavonoid
glycosides to their aglycones. In this optimized condition, the
contents of baicalin and wogonoside were decreased to 1/70 and
1/13, while baicalein and wogonin were increased 3.5- and
3.1-fold, respectively, compared to untreated herb.
Discussion and conclusion: The markedly variable conversion
rates by different pretreatment conditions complicated the
quality control of this herb, mainly due to the high amount of
endogenous enzymes of S. baicalensis. Optimal pretreatment
conditions observed in this study could be used obtain the
highest level of desired constituents to achieve better
pharmacological effects.
http://worldwide.espacenet.com/advancedSearch?locale=en_EP
Patents
: Extraction of Baicalin
CN104910225
Method for extracting baicalin from radix scutellariae
Inventor: CHEN XUESONG / DAI BAIAN
KR20150092954
COMPOSITION COMPRISING BAICALIN EXTRACTED FROM
SCUTELLARIA BAICALENSIS FOR INHIBITING DENDRITIC CELL
MATURATION
Inventor: LEE JUN SIK , et al.
CN104873573
Method for being suitable for industrial production and
extracting crude baicalin from scutellaria baicalensis
Inventor: SUN GUOQIANG, et al.
CN104829666
Method for preparing high purity baicalin from radix
scutellariae
Inventor: ZHOU YUZHI, et al.
CN104784254
Extraction method for producing baicalin with biological
enzyme method
Inventor: WEI YOULIANG, et al.CN104784254
CN104650165
Preparation method of high-purity baicalin
Inventor: GAO QIANSHAN, et al.
CN104610401
Method for simultaneously extracting baicalin, baicalein
and wogonin from scutellaria baicalensis
Inventor: YU BEIBEI, et al
CN104513285
Baicalin extracting technology process
Inventor: DU SHUQING
CN104356185
Method for quickly and efficiently extracting baicalin
Inventor: ZHANG LIWEI, et al.
Nigella
sativa
http://www.greenmedinfo.com/blog/black-seed-completely-cures-hiv-case-study
December 7th 2013
Black
Seed Extract 'Cures' HIV Patient Naturally
By
Sayer Ji
There are words you don't use in medicine today, such as "cure."
But a remarkable case study in an HIV positive patient treated
with black seed extract resulted in a sustained remission,
indicating a safe, accessible and affordable alternative to
highly toxic antiretroviral HIV drugs may already exist.
Nigella Sativa, also known as 'black seed,' has been studied for
a wide rage of health benefits, but not until recently was it
discovered to hold promise as a curative agent against
potentially lethal viral infections, including Hepatitis C[i]
and now HIV.
A remarkable case study published in August of this year in the
African Journal of Traditional, Complementary, and Alternative
Medicine described an HIV patient who after undergoing treatment
with a black seed extract experienced a complete recovery, with
no detectable HIV virus or antibodies against HIV in their blood
serum, both during and long after the therapy ended.[ii]
This was a remarkable and unexpected observation, described by
the researchers as follows:
"Nigella sativa had been documented to possess many therapeutic
functions in medicine but the least expected is sero-reversion
in HIV infection which is very rare despite extensive therapy
with highly active anti-retroviral therapy (HAART). "…
At the outset of the study, the patient presented with classical
symptoms of symptomatic HIV infection, "with [a] history of
chronic fever, diarrhoea, weight loss and multiple papular
pruritic lesions of 3 months duration." Examination identified
moderate weight loss, with laboratory confirmed tests showing
'sero-positivity' to HIV infection with a "pre-treatment viral
(HIV-RNA) load and CD4 count of 27,000 copies/ml and CD4 count
of 250 cells/ mm(3) respectively."...
The patient was administered a black seed concoction of 10 mls
twice daily for 6 months, resulting in a rapid improvement in
symptoms, and significant reductions in viral load:
"Fever, diarrhoea and multiple pruritic lesions disappeared on
5th, 7th and 20th day respectively on Nigella sativa therapy.
The CD4 count decreased to 160 cells/ mm3 despite significant
reduction in viral load (≤1000 copies/ml) on 30th day on N.
sativa."
By the 187th day on black seed therapy, testing indicating the
blood was entirely cleared of signs of infection, a so-called
'sero-negative status'. The post-therapy CD4 counts increased
from baseline to a normalized 650cells/ mm(3) with an
undetectable viral (HIV-RNA) load.
"This case report reflects the fact that there are possible
therapeutic agents in Nigella sativa that may effectively
control HIV infection."...
http://www.greenmedinfo.com/article/nigella-sativa-concoction-induced-sustained-seroreversion-hiv-patient
Afr J Tradit Complement Altern Med. 2013 Aug
12;10(5):332-5.
Nigella Sativa Concoction Induced Sustained
Seroreversion in HIV Patient
Abdulfatah
Adekunle Onifade, Andrew Paul Jewell, Waheed Adeola Adedeji.
Abstract:
Nigella sativa had been documented to possess many therapeutic
functions in medicine but the least expected is sero-reversion
in HIV infection which is very rare despite extensive therapy
with highly active anti-retroviral therapy (HAART). This case
presentation is to highlight the complete recovery and
sero-reversion of adult HIV patient after treatment with Nigella
sativa concoction for the period of six months. The patient
presented to the herbal therapist with history of chronic fever,
diarrhoea, weight loss and multiple papular pruritic lesions of
3 months duration. Examination revealed moderate weight loss,
and the laboratory tests of ELISA (Genscreen) and western blot
(new blot 1&2) confirmed sero-positivity to HIV infection
with pre-treatment viral (HIV-RNA) load and CD4 count of 27,000
copies/ml and CD4 count of 250 cells/ mm(3) respectively. The
patient was commenced on Nigella sativa concoction 10mls twice
daily for 6 months.. He was contacted daily to monitor
side-effects and drug efficacy. Fever, diarrhoea and multiple
pruritic lesions disappeared on 5th, 7th and 20th day
respectively on Nigella sativa therapy. The CD4 count decreased
to 160 cells/ mm3 despite significant reduction in viral load
(≤1000 copies/ml) on 30th day on N. sativa. Repeated EIA and
Western blot tests on 187th day on Nigella sativa therapy was
sero-negative. The post therapy CD4 count was 650cells/ mm(3)
with undetectable viral (HIV-RNA) load. Several repeats of the
HIV tests remained sero-negative, aviraemia and normal CD4 count
since 24 months without herbal therapy. This case report
reflects the fact that there are possible therapeutic agents in
Nigella sativa that may effectively control HIV infection.
http://www.destroydiseases.com/HIV.html
Olive
Leaf Extract (20% oleuropein), Oregano Oil, Turmeric
Curcumin
http://dx.doi.org/10.1155/2012/950757
Evidence-Based
Complementary and Alternative Medicine, Volume 2012 (2012),
Article ID 950757
Traditional
Chinese Herbal Medicines for Treating HIV Infections and
AIDS
Wen Zou, Ying Liu, Jian Wang, Hongjuan Li, and Xing Liao
To assess the effects of TCHM on patients with HIV infection and
AIDS, we reviewed eleven randomized placebo-controlled trials
involving 998 patients. Due to the limited number of RCTs for
included trials and the small sample size of each study, we are
not able to draw firm conclusions concerning TCHM therapy in
treating patients with HIV infection and AIDS. However, some
high-quality clinical studies do exist. Studies of diarrhea and
oral candidiasis, which are challenging symptoms of AIDS, were
demonstrated to have positive effects. Study of peripheral
leukocytes, which are a side effect of antiretroviral drugs,
suggested that an integrated treatment approach may be of
benefit. The overall methodological quality of the trials was
adequate; however, randomization methods should be clearly
described and fully reported in these trials according to the
Consolidated Standards of Reporting Trials (CONSORT)…
3.3.1.
IGM-1
A randomized trial tested a Chinese herbal formulation (IGM-1)
composed of 31 Chinese herbs (Table 1) in 30 HIV-infected adults
with symptoms and decreased CD4+ cell count (200–499/mm3) for
treatment of HIV-related symptoms for duration of 12 weeks [13].
The study found a significant better effect in improvement of
health-related QoL in terms of life satisfaction and symptoms
than placebo. The number of symptoms was reduced in patients
receiving herbs, but not in those receiving placebo. There were
no statistically significant differences in overall health
perception, symptom severity, CD4 counts, anxiety, or depression
between groups. No adverse events were reported among
participants. However, the above results need to be accounted
for with care due to the small sample in the trial.
3.3.2.
“35-Herb”
Interestingly, three years after the above trial was published,
the same investigator who prescribed IGM-1 prescribed another
Chinese herbal formulation that was tested in a trial in
Switzerland [17]. The formulation was composed of 35 Chinese
herbs containing most of the herbs listed in IGM-1 (Table 1). A
trial tested the Chinese herbal formulation in 68 HIV-infected
adults with decreased CD4+ cell count (less than 500/mm3) for a
treatment period of six months [17]. The participants were
randomized to receive “35-herb” ( ) or placebo ( ). Over 70% of
the patients had received previous antiretroviral therapy, the
two groups were comparable regarding sociodemographic
characteristics, previous antiretroviral use, viral load, CD4+
cell counts, and other clinical laboratory tests at entry. A
total of 53 (78%) patients completed treatment for 6 months,
including 24 in the herb group and 29 in the placebo group.
Analyses were based on complete data and on intention-to-treat
principle in the trial report. After six months, there was no
significant difference in CD4+ cell counts, viral load, new
AIDS-defining events, number of reported symptoms, psychosocial
measurements or QoL between two groups.
The total number of reported adverse events was 46 in the herb
group and 20 in the placebo group, and included diarrhea,
increased number of daily bowel movements, abdominal pain,
constipation, flatulence, and nausea. Hematological or serum
chemistry laboratory values showed no evidence of toxicity from
the study herbs. Two patients in the herb group died during the
study period and causes of death were believed to be due to
severe immunodeficiency and pre-enrolment history of severe
opportunistic complications, but not related to the study drugs.
3.3.3. Compound SH
Compound SH containing five herbs (Table 1) were combined with
zidovudine and zalcitabine in the treatment of 60 HIV-infected
Thai patients in a randomized trial [14]. The herbal formula was
made from more than 1000 chinese herbs from 120 plant families
by Kunming Institute of Botany of the Chinese Academy of
Science. The trial found that adding SH herbs to the two
nucleoside reverse transcriptase inhibitors has a greater
antiviral activity than antiretrovirals only. However, the data
analyses were based on participants, who had completed the
trial, 22 subjects who lost followup or withdrawal due to
adverse events were excluded, and the above benefits need to be
accounted for with care.
3.3.4.
Qiankunning
Qiankunning (Table 1) is a Chinese herb preparation extracted
from 14 herbs. A randomized, double blind placebo controlled
trial was conducted in 2003 in China [15], 36 adults with HIV
infection or AIDS were randomized to receive Qiankunning ( ) or
placebo ( ). Patients were comparable regarding age, body
weight, average duration of drug abuse, and pre trial HIV RNA
levels. No intention to treat analyses were applied, the data
analyses were based on participants who had completed the trial.
Significant decrease in HIV RNA levels was found in herb group
than placebo after the end of treatment for 7 months. In this
trial, the use of herbs was related to stomach discomfort and
diarrhea. No adverse effects were reported from the placebo
group. There were no serious adverse events observed.
3.3.5.
Zhongyan-4
Chinese herbal medicine zhongyan-4 (ZY-4) (Table 1) is prepared
by the Chinese Academy of Chinese Medical Sciences in Beijing,
China. A randomized, double blind placebo controlled trial
enrolled 72 patients with HIV infection or AIDS (36 with herbs
and 36 with placebo) [16]. CD4+ cell counts in the ZY-4 group
were increased by cells/mm3, while in the placebo group the CD4+
cell counts were decreased by cells/mm3 after treatment for 6
months ( ). A total of 15 out of 30 patients (6 dropped out) in
the ZY-4 group had their CD4 count increased compared with 8 out
of 33 patients (3 dropped out) in the placebo group ( ). The
study concludes that ZY-4 is effective in enhancing immunity
function based on CD4+ cell counts. However, this study showed
no significant difference in body weight or viral load after
treatment between ZY-4 and placebo.
3.3.6.
Aining Granule
The Chinese herbal medicine Aining Granule (AG) (Table 1) was
tested in 100 patients compared with placebo in a double blind
trial. Participants were randomized into two groups [18], AG
group ( ) received AG+HAART (d4T+ddI+NVP) and Placebo group ( )
received placebo + HAART (d4T+ddI+NVP). CD4+ cell counts in the
AG group were decreased by cells/mm3, while in the placebo
group the CD4+ cell counts were decreased by cells/mm3 after
treatment for 11 months ( ). Significant improvement of symptoms
such as fatigue, anorexia, nausea, diarrhea, skin rash was found
in AG group. The results showed that patients receiving Chinese
herb AG had a lower risk for the decrease of CD4+ cell counts.
However, this study showed no significant difference between two
groups in viral load after treatment.
3.3.7.
Xiaomi Granule
A randomized two arms positive-drug controlled open label trial
was conducted in 2009 in china, in which 80 AIDS participants
with oral candidiasis were included in the Xiaomi Granule (Table
1) plus Nystatin group ( ) and Nystatin group ( ) [19]. After
treatment for 2 weeks, significant improvement of symptoms of
oral candidiasis was found in herb group. No adverse event was
found. Xiaomi Granule is a Chinese herb preparation developed
from a prescription in classic Chinese medicine ancient book
“jin kui yao lve”. There is no description of CD4+ cell counts
and viral load in the paper available.
3.3.8.
Jingyuankang Capsule
In a double-blind, double-analogue trial, 116 participants with
HIV infection and peripheral leucopenia were randomized to
receive Jingyuankang Capsule (JC) (Table 1) plus AZT, ddI, NVP,
and analogue Leucogen Tablets ( ) or Leucogen Tablets plus AZT,
ddI, NVP and analogue JC ( ) for 6 months [20]. The application
of JC showed significant increase of peripheral leukocytes in
herb group. CD4+ cell count outcome was not reported. There were
no significant differences between the groups regarding adverse
effect in the trial report.
3.3.9.
Xielikang Capsule
A randomized, double-blind, double dummy and controlled clinical
trial was conducted between 2009 and 2011 in china, in which 158
AIDS-related chronic diarrhea patients were randomized into
Xielikang Capsule (XC) (Table 1) plus loperamide analogue group
( ) and loperamide capsule plus XC analogue group ( ) [21]. The
primary efficacy parameters were stool weight, abnormal stool
frequency and score of diarrhea questionnaire. All the patients
have no recognized enteritis or intestinal canal identified from
enteroscope or diarrhea resulted by protease inhibitors (PI)
drugs. According to an analysis of the treatment effect over 7
and 14 days based on daily measurements, Patients who were
treated with XC experienced a statistically significant
reduction in stool weight ( in 7 days and in 14 days) and in
diarrhea questionnaire score ( in 14 days). There were no
significant differences between groups with respect to stool
frequency. No serious adverse events were reported. There was no
major difference between XC and placebo in the occurrence of
adverse events or in laboratory abnormalities.
3.3.10.
Aikang Capsule
A randomized placebo controlled trial enrolled 102 patients
infected with HIV and AIDS with CD4+ cell counts between 250 and
600 cells/mm3 who were treated with Aikang Capsule (Table 1) or
placebo for 6 months [22]. There was no significant difference
in CD4+ cell counts between two groups.
3.3.11.
Tangcao Tablets
In a China phase III clinical muti-center trial conducted
between 2002 and 2003, 176 patients with CD4+ cell counts
200 cells/mm3 were randomized to receive a 6- month course of
treatment with Chinese herbal medicine Tangcao Tablets (Table 1)
( ) or placebo ( ) [23]. Patients receiving antiretroviral drugs
were excluded. Both intention to treat analysis and per-protocol
analysis showed significant increase in CD4 counts, CD4/CD8
ratio and weight in herb group, significant increase of viral
load in placebo group, improvement of symptoms in herb group.
The total number of reported adverse events was 21 in the herb
group and 27 in the placebo group, and included diarrhea, cold,
abdominal pain, flatulence, and nausea. Hematological or serum
chemistry laboratory values showed no evidence of toxicity from
the study herbs. Two patients in the placebo group died during
the study period and causes of death were believed to be due to
severe immunodeficiency and pre-enrolment history of severe
opportunistic complications, but not related to the placebo…
http://www.scopus.com/record/display.uri?eid=2-s2.0-33645549859&origin=inward&txGid=0
Chinese Journal of Integrative Medicine, vol. 12, no. 1,
pp. 6–11, 2006.
Randomized
double-blinded and controlled clinical trial on treatment
of HIV/AIDS by Zhongyan-4
J. Wang, F. Z. Yang, M. Zhao et al.
Objective: To assess the efficacy and safety of Zhongyan-4
((Chinese characters), ZY-4, a Chinese herbal preparation worked
out according to the therapeutic principle of supplementing qi,
nourishing Yin, clearing heat and detoxication) in treating
HIV/AIDS patients in the early or middle stage. Methods: Adopted
was randomized double-blinded and placebo-parallel-controlled
method, with 72 HIV/AIDS patients randomly divided into the ZY-4
group (36 patients) treated with ZY-4 and the control group (36
patients) treated with placebo. The treatment course was six
months. The index of CD 4 +, CD 8 + counts, body weight,
clinical symptom scoring were estimated at 4 time points (0, 1,
3 and 6 month in the course), and also the viral load before and
after treatment. The whole course of observation was completed
in 63 patients, 30 in the ZY-4 group and 33 in the control
group. Results: CD 4 + count in the ZY-4 group got elevated by
7.70 ± 150.96/mm 3 on average, while that in the control group
lowered by 27.33 ± 85.28/mm 3. Fifteen out of the 30 patients in
the ZY-4 group had their CD 4 + count increased, which was
evidently much higher than that in the control group (8/33,
P<0.05), suggesting that the efficacy of ZY-4 is superior to
that of placebo in elevating CD 4 + count. Moreover, ZY-4 showed
actions in elevating CD 45RA + and CD 8 + count, reducing HIV
virus load, improving clinical symptom/sign and increasing body
weight of patients. No obvious adverse reaction was found in the
clinical trial. Conclusion: ZY-4 has an immunity-protective
and/or rebuilding function in HIV/AIDS patients in the early and
middle stage, and also shows effects in lowering viral load,
increasing body weight and improving symptoms and signs to a
certain degree.
http://www.scopus.com/record/display.uri?eid=2-s2.0-0344549402&origin=inward&txGid=0
Journal of Acquired Immune Deficiency Syndromes and Human
Retrovirology, vol. 22, no. 1, pp. 56–64, 1999.
Randomized,
placebo-controlled trial of Chinese herb therapy for
HIV-1- infected individuals
R. Weber, L. Christen, M. Loy et al.,
Abstract
Context: Alternative medicine or complementary remedies that
have not been scientifically tested are nonetheless widely used
to treat chronic illnesses, particularly if curative options are
limited. Objectives: To assess the effectiveness of Chinese
medicinal herbs in reducing symptoms and improving the quality
of life of HIV-infected persons. Design: Prospective,
placebo-controlled double-blind study. Setting: University-based
HIV outpatient clinic. Patients: 68 HIV-infected adults with CD4
cell counts <0.5 x 109/L. Intervention: Participants were
randomized to receive four daily doses of seven pills containing
a standardized preparation of 35 Chinese herbs or placebo for 6
months. Main Outcome Measures: Symptoms, HIV disease
progression, HIV-1 RNA plasma vital loads, CD4 and CD8 cell
counts, and scores on standard questionnaires for quality of
life, depression, anxiety, and coping. Results: Intervention and
placebo groups were equivalent at baseline regarding,
respectively, previous antiretroviral therapy (74% versus 79%),
median CD4 cell counts (0.20 x 109/L versus 0.25 x 109/L), and
median HIV-1 plasma viral loads (35,612 copies/ml versus 52,027
copies/ml). At enrollment, none of the study subjects was
seriously ill or depressed, and average coping and quality of
life scores were in the normal range. In all, 53 (78%)
participants completed the study. Patients taking Chinese herbs
reported significantly more gastrointestinal disturbances (79%
versus 38%; p = .003) than those receiving placebo. No
therapy-related toxicities were observed. At completion of the
study, no significant differences between the intervention and
placebo groups were found regarding plasma viral loads, CD4 cell
counts, symptoms, and psychometric parameters. HIV-1 RNA level
was unchanged at study end. Among participants who were not on
concomitant antiretroviral therapy, median CD4 cell counts
declined by 0.05 x 109/L in both the intervention and placebo
groups. Conclusions: This standardized formulation of Chinese
herbs for HIV-infected individuals did not improve quality of
life, clinical manifestations, plasma virus loads, or CD4 cell
counts. The data suggest that this formulation of Chinese herbs
is not effective when administered in a Western medicine
setting.
http://www.scopus.com/record/display.uri?eid=2-s2.0-79956112856&origin=inward&txGid=0
Journal of Traditional Chinese Medicine, vol. 31, no. 1,
pp. 32–35, 2011.
Effects
of Jingyuankang capsules on leukocyte level in AIDS
patients
S. Q. Jiang, H. X. Sun, Y. M. Xu, Y. L. Jiang, J. W. Pei,
and H. L. Wang
Abstract
Objective: To observe the therapeutic effects of Jingyuankang
capsules for leukopenia in AIDS patients. Methods: In this
randomized double-blind trial, 58 patients orally took
Jingyuankang capsule, analog Leucogen tablet and the HAART
(highly active anti-retroviral therapy) drugs, and the other 58
patients took Leucogen tablet, analog Jingyuankang capsule and
the HAART drugs all for 6 months, during which the peripheral
hemogram was periodically examined to observe the therapeutic
effects of Jingyuankang capsule for leukopenia of the AIDS
patients. Results: With good therapeutic effect for leukopenia
of the AIDS patients, Jingyuankang capsule can enhance leukocyte
level as effective as Leucogen tablet in treating grade I and
grade II leukopenia, and more effectively than Leucogen tablet
in treating grade III leukopenia. No toxic side-effects and
adverse reactions were found during the treatment and in the
follow-up visit. Conclusion: Jingyuankang capsule can
effectively treat leukopenia of the AIDS patients.
Dr. Paul Chepkwony
https://www.youtube.com/watch?v=BHz3wZvhuug
HIV/AIDS
Herbal Remedy
https://www.youtube.com/watch?v=HzjZHlrkmXo
HIV/AIDS
Herbal Remedy - Real testimonies (Part 2)
https://www.youtube.com/watch?v=iiZhuFlcH-A
Dr.
Mitch interviews Dr. Paul Chepkwony
http://worldwide.espacenet.com/advancedSearch?locale=en_EP
US7556830
Medicinal herbal composition for treating infection
Inventor(s):
CHEPKWONY PAUL K, et al.
Abstract
Herbal compositions derived from Kenyan plants are provided for
the treatment of HIV and other infectious diseases. The herbal
compositions can include the extracts of up to 14 plants,
including the root of Dovyalis abyssinica and Clutia robusta.
Also provided are methods for extracting alkaloids and other
compounds from the plants. Also provided are methods of treating
a subject having an infectious disease, particularly HIV.
FIELD OF
THE INVENTION
The present invention relates to combinations of extracts from
plants that can be used in the treatment of infection.
BACKGROUND
OF THE INVENTION
This application claims the benefit of provisional application
Ser. No. 60/710,237, filed Aug. 22, 2005, incorporated herein by
reference in its entirety. The following discussion of the
background of the invention is merely provided to aid the reader
in understanding the invention and is not admitted to describe
or constitute prior art to the present invention.
Tens of millions of people world-wide are living with acquired
immunodeficiency syndrome (AIDS), or are infected with the
causative agent, human immunodeficiency virus (HIV). In some
countries in sub-Saharan Africa, up to one in four adults has
contracted the disease. Despite the costs and efforts spent
attempting to identify new methods of treatment, a cure for the
disease has remained elusive.
Ancient societies have traditionally turned to plants for their
health needs. Documented use of herbs to treat illnesses dates
back to as early as 2,000 B.C. Recently, individuals have
resorted to nature as remedies and medicines for the treatment
of modern illnesses have been derived from plants, such as for
example, treatment of HIV and other infectious diseases.
For example, U.S. Pat. No. 5,178,865 discloses an experimental
treatment with 56 herbs, and reports that 10 of the 56 herbs
exhibit anti-HIV activity in in vitro experiments. The 10 herbs
include: Coptis chineusis, Ligusticum wallichii, Ilicium
eanclolatum, Isatis tinctoria, Salvia miltiorrhiza, Erycibe
obtusifolia, Acanthopanax graciliatylus, Bostaurus domesticus,
Inula helenium and Lonicera japonica. Both Bostaurus
domesticus and Lonicera japonica are further described to
be able to combine with Scutellaria baicaleusis to exhibit
anti-HIV activity.
U.S. Pat. No. 5,837,257 discloses Chinese herbal medicines that
exhibit in vitro antiviral activity against murine leukemia
virus and HIV and for treatment of animals and humans infected
with HIV. In one of the preferred embodiments, the Chinese
herbal medicines contain hedyotis, Scutellarial barbatae
herba, Lonicera flos, Prunellae spica and Solani harba.
U.S. Pat. No. 5,989,556 discloses various herbal compositions
for treating viral infections which have shown in vitro
antiviral activities against HIV. A first herbal composition
contains Aeginetiae herba, Blechni rhizoma, Lespedezae
herba, Polygoni cuspidati rhizoma, Forsythiae fructus, and
Ligustri fructus. A second herbal composition contains Cirsii
rhizoma and radix, Breeae radix, Baphicacanthis rhizoma and
radix, Phellodendri cortex, and Bletillae tuber. A third
herbal composition disclosed in the patent includes Aeginetiae
Herba, Lonicerae, Flos, Prunellae spica and Lespedezal herba.
U.S. Pat. No. 6,696,094 discloses an herbal pharmaceutical
composition for treating HIV/AIDS. The pharmaceutical
composition contains 14 ingredients, including: diffuse
hedyotis, bistort rhizome, giant knotweed rhizome, Asiatic
moonseed rhizome, baical skullcap root, Bovine biliary powder,
milkvetch root, barbary wolfberry fruit, sanqi, figwort root,
Chinese magnoliavine fruit, turmeric root-tuber, hawthorn
fruit and Chinese angelica. Procedures are provided for
the preparation of an “HIVCIDE condensate”, which can be
formulated as an injectible solution or as capsules. Results
indicate that subjects injected with HIVCIDE solution showed no
symptoms of acute or chronic toxicity. Further, the HIVCIDE
injection solution was effective in inhibiting pathological
changes in cells caused by HIV-1 in vitro. In a third
experiment, the HIVCIDE injection solution was effective in
reducing symptoms of HIV-infected subjects in a treatment regime
together with administration of HIVCIDE capsules. HIV-positive
subjects did not show adverse reactions to HIVCIDE injection
solution. It was further reported three out of four subjects
showed improvement in fatigue after treatment with HIVCIDE, and
that HIV viral load studies indicated that all subjects
demonstrated reduced HIV viral loads.
U.S. Pat. No. 6,455,078 discloses a medical herbal composition
for treating liver diseases and HIV. The composition contains 15
ingredients, which includes diffuse hedyotis, bistort
rhizome giant knotweed rhizome, Asiatic moonseed rhizome,
baical skullcap root, bovine biliary powder, milkvetch root,
barbary wolfberry fruit, sanqi, red gingseng, figwort root,
Chinese magnoliavine fruit, turmeric root-tuber, hawthorn
fruit and Chinese angelica. Among the 15 ingredients, diffuse
hedyotis, bistort rhizome, giant knotweed rhizome, and Chinese
magnoliavine fruit are cited as being necessary to
contribute to the efficacy of the pharmaceutical composition.
In U.S. Pat. No. 5,366,725, an extract from the seeds of
Aeginetia indica was prepared which exhibited excellent
carcinostatic effects and possesses interleukin-2 and
interferon-gamma-inducing properties. The extract is believed to
be a macromolecular polysaccharide, which may or may not contain
Lipid A binding with protein depending on whether the extraction
is conducted using butanol or phenol. The extracted substance is
soluble in water, insoluble in n-butanol, and has a molecular
weight ranging from 100,000 to 200,000 Daltons.
U.S. Pat. No. 5,411,733 to Hozumi, et al., discloses a variety
of plant extracts for use as anti-herpes viral, anti-polioviral,
anti-varicella-zoster virus, anti-measles virus,
anti-cytomegalovirus (CMV), and anti-DNA and anti-RNA virus
agents.
U.S. Pat. No. 5,178,865 discloses the anti-HIV activity in vitro
of a variety of herbs known in China to exhibit anti-viral
activity. Water extractions of the mixtures, treatment with
ethanol for precipitation and charcoal adsorption are disclosed
for the preparation for the anti-HIV-active composition.
Two lignans, phyllamycin B and retrojusticiden B, have
been reported to have an inhibitory effect on HIV-1 reverse
transcriptase activity. The lignans are isolated from Phyllanthus
myrtifolius Moon, a plant widely grown in Southern China.
See, for example, Chang, et al., Antiviral Research, 27 (4),
367-374 (1995).
A mixture of aqueous extracts of Lonicera japonica flower
buds, Forsythia suspensa fruits, and Scutellaria baicalensis
rootbark have been shown to have antibacterial and
antiviral properties. Subjects with severe respiratory disease
treated with the mixture responded as well as a control group on
standard antibiotic therapy. See Houghton, et al., Phytother.
Res., 7(5), 384-386 (1993).
A water extract of Prunella vulgaris was reported to have
anti-HIVB activity when administered in combination with
zidovudine (AZT) and didanosine (dd1). Only a slight additive
effect was observed for the administration of an extract of Prunella
vulgaris and zalcitabine (ddC). See John, et al., Abstr.
Gen. Meet. Am. Sc. Microbiol., 94, 481 (1994).
Yamasaki et al. have reported the in vitro evaluation of 204
crude drugs commonly used in Japan for anti-HIV-1 activity and
studies indicate that hot water extracts of Lithospermum
erythrorhizon (root) and Prunella vulgaris (spike) showed
strong in vitro anti HIV-1 activity with an IC100 of 16 μg/mL.
See Yamasaki, et al., Yakugaku Zasshi, 113(11), 818-824 (1993).
Yao et al. have reported that water extracts of dried Prunella
vulgaris (whole plant) were active in vitro for
inhibiting HIV-1 replication, and showed relatively low
cytotoxicity to MT-4 cells. The extract also demonstrated
activity in the inhibition of reverse transcriptase. The active
factor was purified and identified as anionic with a molecular
weight of approximately 10,000 Daltons. This active component
may be the same as the prunellin, as described by Tabba, et al.,
(1989). The purified extract inhibited HIV-1 replication in the
lymphoid cell line MT-4, in the monocytoid cell line U937, and
in peripheral blood mononuclear cells (PBMC) at effective
concentrations of 6.3 and 12.5 μg/mL, respectively. Pretreatment
of uninfected cells with the extract prior to viral exposure did
not prevent HIV-1 infection upon subsequent exposure to the
virus. Preincubation with the purified extract decreased HIV-1
infectiousness. The purified extract also blocked cell-to-cell
transmission of HIV-1, prevented syncytium formation, and
interfered with the ability of both HIV-1 and purified gp 120 to
bind to CD4. PCR (polymerase chain reaction) analysis confirmed
the absence of HIV-1 proviral DNA in cells exposed to virus in
the presence of the extract, suggesting that the purified
extract antagonized HIV-1 infection of susceptible cells by
preventing viral attachment to the CD4 receptor. See Yao, et
al., Virology, 187(1), 56-62 (1992).
Tabba, et al. isolated and partially characterized prunellin, a
compound exhibiting anti-HIV properties, from aqueous
extracts of Prunella vulgaris, a Chinese herb. Prunellin
was identified as a carbohydrate (a partially sulfated
polysaccharide) with an minimum inhibition concentration of 2.2
μg/mL against HIV-1 in vitro. It was identified as having a
molecular weight of about 10,000 Dalton. See Tabba, et al.,
Antiviral Research, 11, 263-273 (1989).
Antiviral agents have been isolated from Syzygium aromatica,
Sapium sebiferum (Chinese tallow tree leaves), Scutellaria
baicalensis, and Scutellaria rivularis. Eugeniin, (a tannin
isolated from Syzygium aromatica), and methyl gallate,
(isolated from Sapium sebiferum), exhibited anti-herpes
simplex virus (HSV-2) activity in vitro. Plant flavonoids, such
as 5,7,4-truhydroxyflavone, extracted from the whole herb
Scutellaria rivularis, were reported to have anti-influenza
virus activity. See Hozumi, et al., U.S. Pat. No. 5,411,733;
Takechi, et al., Planta Medica, 42, 69-74 (1981); Kane, et al.,
Bioscience Report, 8, 85-94 (1988); and Nagai, et al., Chem.
Pharm Bull. 38(5), 1329-1332 (1990).
Ethiopian medicinal plants known for treatment of a variety of
ailments were screened for activity against HIV-1 and HIV-2, as
reported by Asres, et al. Extracts from Bersama abyssinica
root bark, Combretum paniculatum leaves, Dodonaea angustfolia
leaves, and Ximenia Americana stem bark each displayed
anti-viral activity at concentrations that were non-toxic to
MT-4 cells. Anti-viral activity of the extracts is noted to be
more effective against HIV-1 than HIV-2. See Asres, et al.,
Phytother. Res., 15, 62-69 (2001).
Selected plants used in traditional Rwandan medicine for
treatment of infections and/or rheumatoid diseases were
investigated for antiviral activity in vitro against HIV-1. See
Cos, et al., Phytomedicine 9, 62-68 (2002). Of 38 plant extracts
tested, extracts from the leaves of Aspilia pluriseta and
Rumex bequaertii had the highest antiviral activities.
SUMMARY OF
THE INVENTION
The present invention is based upon the discovery of the unique
antiviral properties of a herbal remedy composition prepared
from a variety of plants native to Kenya. The herbal composition
of the present invention can include plant material from between
two and 14 different plants preferably including roots of
abyssinica (representative seed of said line having been
deposited under ATCC Accession No. PTA-6769) and Clutia robusta
(representative seed of said line having been deposited under
ATCC Accession No. PTA-6970). For treatment of infectious
disease, the herbal composition of plant material may be
extracted to produce a liquid herbal composition or further
purified to obtain alkaloid compounds from the plant material.
The liquid herbal composition prepared from aqueous extracts
from the plants has demonstrated effectiveness in treating
HIV-positive subjects, as subjects treated with the liquid
herbal composition have experienced improvements in CD4+ cell
counts, and in some cases, complete reversal of HIV positive
status.
In one aspect, the invention provides a herbal composition for
treating infectious diseases, such as for example, HIV. The
composition containing plant material includes the roots of
abyssinica and the roots of Clutia robusta. In other embodiments
of the invention, the herbal pharmaceutical composition may also
include plant material, as indicated, from one or more of the
following: stem bark of Prunus africanastem bark of Croton
macrostachyus, stem bark of Acacia nilotica (representative
seed of said line having been deposited under ATCC Accession
No. PTA-7378), roots of Rhamnus prinoides, roots of Adenia
gummifera, roots of Asparagus africanus, stem bark of
Anthocleista grandiflora, whole plant of Plantago palmata
(representative seed of said line having been deposited under
ATCC Accession No. PTA-7377), roots of Clematis hirsuta, stem
bark of Ekebergia capensis, stem bark of Bersama abyssinica,
and roots of Periploca linearifolia.
In another aspect, the invention provides a method for preparing
a liquid extract of the solid herbal composition of the
invention. The extraction of plant material can be done with hot
water. In one embodiment, hot aqueous extraction is done under
basic conditions, followed by hot aqueous extraction under
acidic conditions. In further embodiments, desired alkaloid
compounds purified from the liquid extracts are provided or
produced from direct chemical synthesis.
The invention further provides aqueous extracts of the herbal
compositions of the invention. Also provided are alkaloid
compounds purified from aqueous extracts and the chemical
synthesis of the herbal compositions of the invention.
In another aspect of the present invention a method for treating
HIV-positive subjects is provided. Subjects are administered an
effective amount of a herbal composition of the invention
prepared from the aqueous extracts of Dovyalis abyssinica
and Clutia robusta, alone or in combination with one or more
of the following: Prunus africana, Croton macrostachyus,
Acacia nilotica, Rhamnus prinoides, Adenia gummifera,
Asparagus africanus, Anthocleista grandiflora, Plantago
palmata, Clematis hirsuta, Ekebergia capensis, Bersama
abyssinica, and Periploca linearifolia, in doses based on
subjects' body weights. In other embodiments the herbal
composition of the invention is prepared from purified alkaloid
compounds obtained from the aqueous extracts. The herbal
compositions are administered at least once a day. In other
embodiments, the herbal composition is administered twice or
three times daily, based upon the health of the subject. In
other embodiments, the composition may be administered as a
beverage, capsule, tablet, powder, candy, gel, nutritional
product or pharmaceutical product.
In another aspect of the present invention provides an herbal
composition for treating subjects having infection, such as for
example, HIV or AIDS. The herbal composition consists
essentially of extracts of abyssinica and Clutia robusta, and
optionally one or more of the following: Prunus africana,
Croton macrostachyus, Acacia nilotica, Rhamnus prinoides,
Adenia gummifera, Asparagus africanus, Anthocleista
grandiflora, Plantago palmata, Clematis hirsuta, Ekebergia
capensis, Bersama abyssinica, and Periploca linearifolia.
In one embodiment, the herbal composition of the invention is
prepared from purified alkaloid compounds obtained from aqueous
extracts.
In another aspect of the present invention a method is provided
for treating subjects having infection, such as for example, HIV
or AIDS. Subjects are administered an effective amount of a
herbal composition consisting essentially of extracts of abyssinica
and Clutia robusta, and optionally the extract of one or more
of the following: Prunus africana, Croton macrostachyus,
Acacia nilotica, Rhamnus prinoides, Adenia gummifera,
Asparagus africanus, Anthocleista grandiflora, Plantago
palmata, Clematis hirsuta, Ekebergia capensis, Bersama
abyssinica, and Periploca linearifolia, in doses based on
subjects' body weights. In other embodiments the herbal
composition of the invention is prepared from purified alkaloid
compounds obtained from the aqueous extracts. The herbal
compositions can be administered at least once a day. In other
embodiments, the herbal composition can be administered twice or
three times daily, based upon the health of the subject. In
other embodiments, the composition may be administered as a
beverage, capsule, tablet, powder, candy, gel, nutritional
product or pharmaceutical product...
BRIEF
DESCRIPTION OF THE DRAWING
FIG. 1 shows relationships between observed clinical
symptomatology and CD4+ count results.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the discovery that combinations
of extracts from plants native to Kenya can be used in the
treatment of infection, such as for example, HIV and AIDS.
Herbal compositions prepared from combinations of the extracts
of the following: the roots of abyssinica and Clutia
robusta, Rhamnus prinoides, Adenia gummifera, Asparagus
africanus, Clematis hirsuta, and Periploca linearifolia, the
stem bark of Ekebergia capensis, Bersama abyssinica, Prunus
africana, Croton macrostachyus, Acacia nilotica, and
Anthocleista grandiflora, and the whole plant of Plantago
palmata have been shown to be particularly effective in
improving the health of infected subjects. Specifically, herbal
compositions of the present invention are particularly well
suited for the treatment of infectious diseases including HIV.
Compositions of the invention can be prepared from plant
material collected from the Mau Forest Complex in Western Kenya.
Herbal compositions prepared from aqueous extractions and
purified extracts of plants from this region of Kenya exhibit
increased potency in the treatment of infectious diseases. The
Mau Forest Complex is located at 0° 30′ South, 35° 20′ East and
in the Rift Valley Province, and spans four Kenyan
administrative districts: Narok, Nakuru, Bomet and Kericho. Mean
annual rainfall varies from 1000 to 1500 mm with peaks in April
and August. The rainfall pattern at the western flanks is
governed by the moist monsoon winds from the Indian Ocean and
dry winds from the Great Rift Valley. The western flanks of the
Mau Forest Complex are influenced by the Lake Victoria
macroclimatic region and are generally wetter with annual
rainfall greater than 2000 mm and more evenly distributed. Mean
annual temperatures for the Mau Forest Complex range from 12-16°
C. The soil of the Mau Forest Complex is rich volcanic loam
having a pH between 3.8-5.8.
The vegetational pattern follows an altitudinal gradient with
local topographical ecolines. The closed canopy moist mountain
forest at lower altitudes becomes increasingly intermixed with
bamboo from 2200 m onwards. Between 2300 and 2500 m, pure bamboo
(Arundinaria alpina) swards are found. Above 2500 m this gives
way to mixed bamboo/tree stands, both associated with grass
clearings that usually represent a sub-climax resulting from
burning and cutting of bamboo. A marginal type of mountain
sclerophyll forest, wherein the plants generally have hard
leaves to prevent wilting during dry conditions, occupies the
highest altitudes of the Mau complex.
Plants in the Western flank of the Mau Forest Complex have shown
the highest potency for the herbal compositions. Plants growing
in the Western flank, (which is generally a high rainfall, high
altitude region), have fewer environmental stresses. It is
therefore possible that plants of the Western flank have more
biosynthetic pathways, which may in turn lead to the production
of a greater number of diverse compounds, which may in turn
explain the greater potency of plants from the Western flank (as
compared to other regions of the Mau Forest Complex).
Alternatively, the greater potency plant extracts from the
Western flank plants may be a result of a greater variety and
number of alkaloids and other compounds in the plant extracts,
such that the combined effect is greater than the sum of their
individual effects.
The East Mau Forest Complex has a drier vegetation of Cedar and
Podo. Wherever these species have been extracted, colonizing
species such as Neuboutonia marcrocalyx and Macaranga
capensis can be found.
The compositions of the invention may be prepared using plants
collected from three altitude ranges of the Mau Forest Complex:
2000 m (annual rainfall of 1000 mm), 2300 m (1500 mm), 2500 m
(western Mau flank, annual rainfall greater than 2000 mm) above
sea level. The Western flanks of the Mau Forest contain plants
that are particularly preferred for preparing the herbal
compositions of the invention. The plants grown in the drier.
Eastern flank of the Mau Forest Complex also may be used.
Plant material for preparing compositions of the invention may
also be obtained from plants grown in a greenhouse environment.
The germination of the seeds of particular plants may be
altitude or soil dependent. Seeds for greenhouse planting may
require collection from the natural dispersal agents as they
exist in the wild. Additionally, simulation of rainfall,
sunlight (an average of 12 hours per day in the Mau Forest
Complex), and soil conditions of the Mau Forest Complex (i.e.,
rich volcanic loam having a pH between 3.8-5.8) may be required
to obtain plants of similar potency.
The seeds of abyssinica (representative seed of said line having
been deposited under ATCC Accession No. PTA-6969) are contained
in a fleshy fruit. There are about 4 seeds enclosed by the
flesh. A ripe fleshy fruit can be soaked in water for about 4
days, to make it possible to squeeze with minimum force to
release the small seeds, each being approximately the size of a
tomato seed or slightly larger. The seeds are then washed, dried
and stored, awaiting germination under Mau Forest-like
environmental conditions. In the wild, the fruit flesh is soaked
by rain water, which results in the release of the seeds. The
seeds grow naturally under the environmental conditions of the
Mau Forest Complex as described above.
The Clutia robusta (representative seed of said line
having been deposited under ATCC Accession No. PTA-6970) seeds
are much smaller and encased in berries having a nut-like outer
covering which encases approximately 3 to 4 seeds the size of a
grain of sand. When mature seeds are exposed direct sunlight,
they disperse rapidly in a process called explosive dispersal.
This is not a problem in the wild, but if one is interested in
collecting the seeds, care and intelligence are required, or
else all the seeds will fly away under the scattering effect of
the hot sun.
To recover the clutia robusta seeds, the berries should
be placed in a metallic container, and covered with a material
that allows sunlight to enter, such as a transparent
polyethylene film surrounding a container of appropriate wire
mesh. Exposure to light will cause the shells to break open,
releasing the seeds which can then be separated from the chaff.
The optimal time for planting the clutia and dovyallis seeds in
their natural environment is during the long rains, typically
around the month of April. However, in the wild, the plants will
generally grow throughout the year, except during the dry
season, as the plants require a considerable amount of water and
light to grow.
Croton
macrostachyus produces pale pea-sized capsules, on
drooping spikes to 30 cm long, splitting open on the tree to
release 3 shiny grey seeds, covered at one end by a soft, creamy
aril, or envelope.
Prunus
africana produces spherical fruit, about 10 mm in diameter
and is pinkish brown in color.
The Acacia nilotica (representative seed of said line
having been deposited under ATCC Accession No. PTA-7378) plant
produces straight or curved pods measuring approximately 17×2
cm. When young, the pods are green and fleshy but get darker
with age, and are usually velvety. Pods have a fruity odor and
open on the ground to release seeds.
Ekebergia
capensis produces rounded, thin skinned berries, up to 2.5
cm in diameter, on long stalks in heavy bunches, which are
yellow to red in color when mature.
The berry-like fruits of Rhamnus prinoides are
approximately the size of a pea (about 5 mm in diameter),
roundish and clearly divided into three compartments. They are
fleshy and green, turning red and then purple as they ripen.
The fruit of the Asparagus africanus is a round berry,
approximately 0.5 cm in diameter, green aging to orange, found
most of the year. It is spread mainly by birds carrying the
seeds.
The Anthocleista grandiflora produces fruits that are
oval in shape, measuring approximately 3 cm×2 cm, glossy, smooth
and brown when mature. Multi-seeded, large fruits are found
throughout the year.
The Bersama abyssinica produces a smooth, spherical
capsule, measuring approximately 2.5 cm in diameter, golden
velvety at first, losing most of the hair and becoming brown by
maturity; splitting into four valves to reveal attractive bright
red seeds, about 10 mm long, enveloped for about their half
length by a yellow, cup-shaped aril.
Adenia
gummifera produces a fruit which is a stalked 3-valved
capsule, leathery or fleshy, often red; seeds compressed with
bony testa in a fleshy aril.
Plantago
palmata (representative seed of said line having been
deposited under ATCC Accession No. PTA-7377) produces a
capsule-like fruit with two seeds per capsule.
Periploca
linearifolia (representative seed of said line having
been deposited under ATCC Accession No. PTA-7375) produces black
seeds measuring approximately 10 mm long and 2 mm wide with
white wool measuring around 3 cm attached to the tips of the
seeds. The seeds are enclosed in pods measuring about 12 cm
long. Upon maturity, the pods break open upon exposure to
sunlight. This releases the seeds, which are borne aloft by the
wool as they are dispersed by wind. Alternatively, these plants
may be cultivated from stem cuttings, which when laid on or
planted in the ground, grow roots and propagate new plants.
Clematis
hirsuta (representative seed of said line having been
deposited under ATCC Accession No. PTA-7383) produces yellowish
seeds measuring approximately 3mm in length and 1 mm in breadth.
The seeds are surrounded by yellowish-white wool which measures
about 5 mm long. The wool carries the seeds upon the wind, which
is the dispersal agent.
HIV Testing
As noted previously, for purposes of this application, a person
is considered HIV-negative if the subject tested negative on a
two-part HIV screening tests, consisting of an initial screening
test and a confirmatory test.
An infected individual usually goes for testing for one or more
of the following reasons: 1) the individual feels ill, 2) the
individual's sexual partner is ill and has tested positive, 3)
the individual's sexual partner died of AIDS; or 4) the
individual suspects his/her sexual partner is sexually
promiscuous.
The initial screening test is ELISA (Enzyme-Linked Immunosorbent
Assay), an enzyme immunoassay (EIA) to determine the presence of
HIV antibodies. The ELISA test uses artificial HIV proteins that
capture antibodies to the virus and is more than 99 percent
accurate. If antibodies to HIV are present (positive result),
the test is typically repeated. However, other antibodies can
cause a false-positive result.
Generally, HIV-1 antibodies are detectable approximately 25 days
after acute infection, with nearly all infected subjects testing
HIV positive 12 weeks after infection. The process of developing
antibodies to a virus is termed seroconversion, and individuals
who become antibody-positive are often called seroconverters.
Two types of HIV have been identified: HIV-1 and HIV-2, of
which, HIV-1 is more common. HIV-1 and HIV-2 are similar in the
modes of transmission (sexual contact, sharing needles, etc.)
and infected individuals are generally subject to the same
opportunistic infections. However, HIV-2 appears to weaken the
immune system more slowly than HIV-1.
In Kenya, individuals are generally tested for antibodies to
both HIV-1 and HIV-2. HIV-1 is generally more common in the
Western world and HIV-2 is more common in Africa. In Kenya
however, most HIV-positive individuals have the HIV-1 infection.
It is believed that 90% of the HIV-positive cases in Kenya are
HIV-1, with the remaining 10% of HIV-positive cases being the
HIV-2. While rare, subjects occasionally are HIV
antibody-positive to both types of HIV (i.e. HIV-1 and HIV-2).
The second part of the HIV screening test is called the
confirmatory test. In the U.S., the most often used confirmatory
test is the Western blot, wherein an electrical field is used to
separate the various components by their molecular weight prior
to evaluating antibody binding. This allows identification of
antibodies to specific viral antigens, which show up as
identifiable “bands” on a strip of test paper. The Western blot
test is more difficult to perform and accurately interpret than
the ELISA test, but it is less likely to give a false-positive
result because it can distinguish HIV antibodies from other
antibodies that may react to the ELISA. Other confirmatory tests
may be used, including the indirect fluorescent antibody assay
(IFA) and the radioimmunoprecipitation assay (RIPA).
One major drawback of antibody tests is the “window” period
(i.e. the time it takes the body to produce antibodies after
infection has begun). The screening tests do not correlate to
the presence or absence of symptoms. The standard HIV tests do
not detect the virus itself, but instead detect the antibodies
that the body produces in response to the virus. During the
period before the antibodies are produced, a person may be
infected with HIV and can infect others, and still test negative
on the HIV antibody test. It is therefore important to tell
subjects who test negative to avoid engaging in high-risk
behavior and to return for retesting at a later date.
The p24 antigen test can be used in diagnosing HIV early in the
course of infection. It is primarily used to screen the blood
supply but in some places it is used for testing for HIV. The
p24 antigen is a protein that is part of the HIV. Early in the
infection, it is produced in excess and can be detected in the
blood serum by a commercial test. The p24 test can detect HIV
infection before the HIV antibody test can and it is recommended
2-3 weeks after a risk exposure.
Individuals that test positive for HIV are regularly
administered two tests to monitor HIV levels in the blood and to
determine how the virus is affecting the immune system. These
tests are: (1) a viral load measurement, and (2) CD4+ cell
counts.
Viral load measurement (also called the HIV plasma RNA test)
determines how many HIV viral particles are present in a given
amount of a person's blood. Test results help determine the best
treatment for the HIV infection as the viral load test shows how
fast the virus is multiplying in the body. Because HIV
reproduces by making copies of itself, the results are given as
copies per milliliter (mL). Viral load testing can also reveal
the presence HIV infection before antibodies can be detected and
can also accurately determine whether a baby born to an infected
mother has HIV.
CD4+ cell counts (T-lymphocyte measurements) provide an estimate
of the immunologic status of an individual and help determine
the immediate risk of opportunistic infection. The CD4+ count
measures the number of a certain type of white blood cell that
is most affected by HIV, and are measured every 3 to 4 months in
individuals infected with HIV. On average, an individual
infected with HIV loses approximately, 30-60 CD4+ cells per
year, although in some subjects, CD4+ T-lymphocyte counts may
remain stable for years followed by rapid decline.
CD4(T4) or CD4+ cells are a type of T cell involved in
protecting against infections, such as for example, viral,
fungal, and protozoal infections. Destruction of these cells is
the major cause of immunodeficiency observed in AIDS, and
decreasing CD4+ lymphocyte counts appear to be the best
indicator for the potential development of opportunistic
infections. In judging the severity of HIV/AIDS cases, the CD4+
lymphocyte count is more indicative of the severity of the
disease than gross symptomalogy, although it is also true that
certain symptoms may be associated with particular CD4+
lymphocyte levels. See, for example, FIG. 1. Average normal
adult CD4+ cell counts typically ranges from 500 to 1,500/2,000
cells per cubic milliliter of blood.
As CD4+ cell counts decrease below the normal adult levels
during primary HIV infection, CD8+ or cytotoxic T-lymphocytes
also increase. However, most studies indicate that an increase
in CD8 count is not a prognostic indicator of disease
progression. Some clinicians in the U.S. use the CD4/CD8 ratio
as an indicator of disease progression, however, this ratio
varies not only with the severity of the disease, but with the
ethnicity of the subject.
There are several systems for classifying and staging HIV
infection. The most commonly-used system is the CDC (Centers for
Disease Control) Scheme. The CDC scheme has three
classifications based upon CD4 counts. The definitions of the
three CD4+ T-lymphocyte categories I as follow: Category 1:
>500 cells/mm<3 >(or CD4%>28%); Category 2: 200-499
cells/mm<3 >(or CD4% 14% -28%); and Category 3: <200
cells/mm<3 >(or CD4%<14%).
In addition to the CDC classification scheme, there are also 3
possible categories of clinical conditions, which are designated
by the letters A, B and C. Therefore, a given individual can
have the following CDC classification and clinical
categorization designation: 1-A, or 1-B, or I-C, 2-A, 2-B, 2-C,
3-A, 3-B or 3-C.
An individual in category A is identified as an adolescent or
adult (>13 years) with documented HIV infection having one or
more of the following conditions (and lacking any of the
conditions associated with categories B and C): asymptomatic HIV
infection; persistent generalized lymphadenopathy; and acute
(primary) HIV infection with accompanying illness or history of
acute HIV infection.
An individual in category B is identified as an adolescent or
adult (>13 years) with documented HIV infection having one or
more of the following conditions (and lacking any of the
conditions associated with category C) and that meet at least
one of the following criteria: (a) the conditions are attributed
to HIV infection or are indicative of a defect in cell-mediated
immunity; or (b) the conditions are considered by physicians to
have a clinical course or to require management that is
complicated by HIV infection. Examples of conditions in clinical
category B include but are not limited to: bacillary
angiomatosis; candidiasis (oropharyngeal, i.e. thrush);
candidiasis (vulvovaginal, persistent, frequent, or poorly
responsive to therapy); cervical dysplasia (moderate or
severe/cervical carcinoma in situ); constitutional symptoms,
such as fever (body temperature of 38.5° C. or greater) or
diarrhea lasting longer than 1 month; hairy leukoplakia (oral);
herpes zoster (shingles), involving at least two distinct
episodes or more than one dermatome; idiopathic thrombocytopenic
purpura; listeriosis; pelvic inflammatory disease (particularly
if complicated by tubo-ovarian abscess); and (11) peripheral
neuropathy. For classification purposes, Category B conditions
take precedence over Category A conditions. For example, an
individual previously treated for oral or persistent vaginal
candidiasis (but not exhibiting a Category C disease or
condition) who is now asymptomatic, should be classified in
Category B.
An individual in category C is identified as an adolescent or
adult (>13 years) with documented HIV infection having one or
more of the following conditions Category C conditions include
the following: candidiasis of bronchi, trachea, or lungs;
candidiasis (esophageal); invasive cervical cancer;
coccidioidomycosis (disseminated or extrapulmonary);
cryptococcosis (extrapulmonary); cryptosporidiosis (chronic
intestinal, greater than 1 month's duration); cytomegalovirus
disease (other than liver, spleen, or nodes); cytomegalovirus
retinitis (with loss of vision); encephalopathy (HIV-related);
herpes simplex: chronic ulcer(s) (greater than 1 month's
duration), or bronchitis, pneumonitis, or esophagitis;
histoplasmosis (disseminated or extrapulmonary); isosporiasis
(chronic intestinal, greater than 1 month's duration); Kaposi's
sarcoma; lymphoma (Burkitt's, or equivalent term), lymphoma,
(immunoblastic, or equivalent term); Lymphoma (primary, of
brain); mycobacterium avium complex or M. kansasii, disseminated
or extrapulmonary; mycobacterium tuberculosis, (any site,
pulmonary or extrapulmonary); mycobacterium, (other species or
unidentified species, disseminated or extrapulmonary);
pneumocystis carinii pneumonia; pneumonia (recurrent);
progressive multifocal leukoencephalopathy; Salmonella
septicemia (recurrent); toxoplasmosis of brain; and wasting
syndrome due to HIV. For classification purposes, once a
Category C condition has occurred, the individual will remain in
Category C.
One method of treatment for HIV-positive individuals is the
highly active antiretroviral therapy (HAART) regimen. HAART is a
therapeutic treatment regime consisting of the combination of
anti-HIV drugs, that is prescribed to HIV-positive individuals
even before they develop symptoms of AIDS. The therapy usually
includes one nucleoside analog, one protease inhibitor and
either a second nucleoside analog or a non-nucleoside reverse
transcription inhibitor (NNRTI). Frequently, the HAART regime is
toxic to the individual, resulting in adverse side effects. For
example, HAART can be toxic to blood because it almost always
includes one or two nucleoside analogs, like AZT that are
notorious for their toxicity to red and white blood cells and
blood cell production. Various forms of anemia are very common
and sometimes are irreversible. However, it is extremely rare
for a subject on the HAART regimen reverse his/her HIV status in
Kenya.
Examples of drugs administered for the HAART treatment regime
include: azidovudine (AZT), didanosine (dideoxyinosine, ddI),
zalcitabine (dideoxycytosine, ddC), lamivudine (epivir, 3TC),
nevirapine (Viramune), abacavir (Ziagen), stavudine (Zerit,
d4T), tenofovir (Viread), efavirenz (Sustiva), amprenavir
(Agenerase), lopinavir (Kaletra), nefinavir (Viracept),
saquinavir (Invirase), ritonavir (Norvir), indinavir (Crixivan),
and delavirdine (Rescriptor).
Method for
Extracting Alkaloid Compounds and Preparing Herbal Composition
The compositions of the invention are prepared using roots of
abyssinica and Clutia robusta, and optionally one or more of the
following: the stem bark of Prunus africana, stem bark of
Croton macrostachyus, stem bark of Acacia nilotica, roots of
Rhamnus prinoides, roots of Adenia gummifera, roots of
Asparagus africanus, stem bark of Anthocleista grandiflora,
whole plant of Plantago palmata, roots of Clematis hirsuta,
stem bark of Ekebergia capensis, stem bark of Bersama
abyssinica, and roots of Periploca linearifolia.
Preferably, the ingredients collected are fresh, although dried
samples may also be used. The ingredients are combined and
chopped into small pieces and dried. Preferably, the dried
ingredients are ground into a fine powder after drying.
Alternatively, each ingredient may be processed individually and
combined at a later stage. Preferably, if combined for the
extraction process, the ingredients are combined in equal weight
ratios. Optionally, Dovyalis abyssinica, Clutia robusta, Prunus
africana, Croton macrostachyus, Acacia nilotica, Rhamnus
prinoides, Adenia gummifera, Asparagus africanus, Anthocleista
grandiflora, Plantago palmata, Clematis hirsuta, Ekebergia
capensis, Bersama abyssinica and Periploca linearifolia can be
present in a weight ratio of 2:2:2:2:2:2:1:2:2:1:2:2:2:2.
The herbal plant material mixture may be extracted with a
non-polar solvent to remove fats from the chopped herbal
ingredients. Preferably, approximately 20% by volume non-polar
solvent is added to the herbal ingredient mixture. Non-polar
solvents are generally organic solvents having a dielectric
constant less than 20. Non-polar solvents that may be used
include, but are not limited to: alkanes, 1,4-dioxane, carbon
tetrachloride, chloroform, methylene chloride, benzene, ethers,
ethyl acetate, tetrahydrofuran, acetic acid, butanol,
chlorobenzene, cycloalkanes, xylene, and the like. Preferred
non-polar solvents are xylene and ether.
The non-polar solvent is decanted and discarded. The defatted
herbal solids, are then allowed to dry. Sufficient base is added
to the defatted herbal material to achieve a pH of approximately
8. The concentration of the base added can be adjusted to
provide sufficient liquid volume to cover the defatted herbal
solid mixture. Any suitable base may be used, with preferred
bases including NaOH, KOH, Ca(OH)2, Mg(OH)2, NH4OH, and the
like. The base extract is then heated for 2-4 hours. Preferably,
the ingredients are slowly simmered under reflux conditions,
although the same effect can be achieved by simmering the
mixture in a covered pot.
Acid is added to the base extract to achieve a pH of
approximately 3. Preferably the acid is HCl, although other
acids, including but not limited to, HBr, HNO3, H2SO4, H3PO4, or
any other acid suitable for achieving a pH of approximately 3
may be used as well. The concentration of the acid can be
adjusted as necessary to provide sufficient volume to the
mixture. The acidified solution is then boiled for approximately
2-4 hours under the same conditions employed for the heating of
the basic solution. After heating, the mixture is cooled, and
the aqueous layer is separated from the mixture, such as for
example, by decanting the liquid from the remaining solids. Acid
is then added to the remaining residue sufficient to achieve a
pH of approximately 3, and the mixture is then reheated for
approximately 2-4 hours under the same conditions previously
employed. The aqueous layer is separated from the ingredients
and the two acidified layers are combined. If necessary,
additional acid extractions may be performed.
The acidic filtrate is extracted several times with a non-polar
solvent until little or no emulsion forms. Preferable non-polar
solvents are ether and xylene. Base is added to the aqueous
layer to precipitate the alkaloid compounds. Preferably, base is
added to achieve a pH of approximately 9. The precipitate is
separated from the aqueous solution, neutralized and dried.
The precipitate is preferably collected in either crystalline or
powder form, and may administered to an subject as a beverage,
capsule, tablet, powder, candy, gel, nutritional product or
pharmaceutical product.
The precipitate can be further purified as desired to isolate
individual alkaloid compounds by any known chromatographic
means.
It is understood that at any point during the process of
extracting the alkaloid compounds from the herbal ingredients
that the aqueous solution can be concentrated and stored for
later use without the need for precipitation of the compounds
from solution.
Alternatively, the alkaloid compounds for use in the present
invention can be synthesized by known methods once the chemical
structure has been determined. Isolated compounds can be
analyzed by chemical analysis, mass spectroscopy, infrared
spectroscopy, X-ray diffraction, NMR (including <1>H NMR,
<13>C NMR, COSY, NOSEY, and the like), and other known
analytical techniques to obtain the chemical structures. For
example, chemical structures for four extracts obtained from
abyssinica have been previously determined. (See, for example,
http://www.dfuni.dk/uploads/media/Naturstofgruppen_BonnieRasmussen.pdf).
The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Determination of Bioactivity of Plant Extracts
The efficacy of the individual plant extracts were tested
against Escherichia coli and Staphylococcus aureus. Plant
extracts were obtained as described above. Solutions containing
100 ppm (parts per million) of each plant extract were prepared
for use in the anti-bacterial assay.
Preparation of bacterial culture of Escherichia coli and
Staphylococcus aureus.
Standard cultures of E. coli (representing gram-negative strains
of bacteria) and Staphylococcus aureus (representing
gram-positive bacteria) were obtained from Moi University
Teaching and Referral Hospital. Assays were conducted at the Moi
University Department of Botany.
Bioassay procedure (Diffusion Method)
Nutrient agar was used as growth medium for both bacteria
samples. The agar was sterilized in an autoclave at 120° C.,
cooled and poured into sterile Petri dishes and allowed to set.
Sterile conditions were achieved and maintained by exposing the
area to a UV lamp during sample preparation and the assay the
procedure.
The cooled agar medium was streaked on the surface with each
bacteria culture. Wells were dug in the middle of the medium,
using a cork borer, where the prepared plant extract was
deposited. A control experiment was also performed, using plain
sterile water in place of the plant extracts.
Cultures were incubated for 12 hours, after which zones of
inhibition of bacterial growth were determined and measured.
Bacteria-growth inhibition was expressed in diameters (mm), and
was determined by measuring the distance from edge of the well
to area where the bacteria begin to show growth. Generally, the
larger inhibition diameter indicates greater potency of the
particular extract against the bacteria.
Of the 23 plants were screened in this assay, 14 of the plants
had bacteria growth inhibition diameters greater than 8 mm,
which was previously determined to be the minimum activity
required for adoption of the extract for the herbal remedy. The
anti-bacterial activities of the plants were compared with
standard antibiotics. Of the 14 plants having inhibition
diameters greater than 8 mm, abyssinica and Clutia robusta
demonstrated the greatest anti-bacterial activity. Results for
plant extracts exhibiting inhibition diameters greater than 8 mm
are provided in the Table 1.
TABLE 1
Zones of Inhibition Expressed as Inhibition Diameter (mm)
Plant Name E. coli S. aureus
1. Dovyalis abyssinica 17.2 16.6
2. Clutia robusta 16.7 15.8
3. Prunus Africana 14.7 14.6
4. Croton macrostachyus 14.7 14.4
5. Acacia nilotica 13.6 13.2
6. Ekebergia capensis 12.8 13.0
7. Clematis hirsuta 11.9 12.8
8. Adenia gummifera 11.7 12.8
9. Asparagus africanus 11.3 11.2
10. Plantago palmata 11.0 11.0
11. Rhamnus prinoides 10.9 10.8
12. Periploca linearifolia 10.9 10.6
13. Bersama abyssinica 10.5 10.3
14. Anthocleista grandiflora 10.0 9.7
Administration of the Herbal Composition
The plant extract precipitates are preferably purified and
collected in either crystalline or powder form. The precipitates
can administered to a subject as a beverage, capsule, tablet,
powder, candy, gel, nutritional product or pharmaceutical
product. Preferably, between 0.1 and 25 grams of alkaloids are
administered per day to an infected subject. The herbal
composition is preferably administered as a beverage wherein
approximately 1 tbsp of powdered extract is dissolved in
approximately 250 mL of hot water, and drunk. Dosing is either
twice daily at 12 hour intervals, or three times daily at eight
hour intervals (depending on the level of infection of the test
subject), and is preferably administered with a meal.
Subjects in the current trials were screened at the Walter Reed
Hospital of the U.S. Army in Kericho, Kenya, the Moi University
Hospital in Eldoret, and at various Voluntary Counseling and
Testing (VCT) Centers scattered throughout the country.
Subjects' CD4 and CD8 counts were measured using a FACSCount™
system following procedures provided in the FACSCount White
Paper (July 1994). HIV-1 and HIV-2 antibodies were detected
using a bioMérieux Vironostika® HIV Uni-Form II Ag/Ab ELISA
system.
All subjects administered the herbal composition were
HIV-positive adults. Prior to administration of the herbal
composition, an initial CD4 count for each subject was
determined, followed by an assessment of the level of
opportunistic infections. Those with fewer opportunistic
infections were administered the herbal composition twice daily
after meals, at twelve hour intervals. Those with more
opportunistic infections were administered the herbal
composition three times daily, at 8 hours intervals. Each
subject was given one week's dosage during each visit to the
clinic. This was done to make it possible to monitor compliance,
and to avoid the possibility of subjects sharing the drug with
others.
Example 1
Initial studies for the treatment of HIV positive subjects with
herbal remedy were conducted by treating four HIV positive
subjects with two different herbal remedies. Two subjects were
administered a herbal composition which included the extract of
abyssinica, while the other two subjects were administered a
herbal remedy which included the extract of Clutia robusta. The
subjects were each treated for a period of three months. The CD4
counts of both sets of subjects (i.e., those administered either
abyssinica or Clutia robusta) increased by approximately 10 per
month of treatment.
Example 2
In another study, three subjects were administered a herbal
composition prepared with a 1:1 ratio by weight mixture of
Doyalis abyssinica and Clutia robusta for a period of
approximately three months. The CD4 counts of the subjects
treated with the mixture increased by approximately 30 per
month.
Example 3
In yet another experiment, 20 subjects were administered a
herbal composition containing extracts of abyssinica, Clutia
robusta, Prunus africana, Croton macrostachyus, Acacia nilotica,
Ekebergia capensis, Clematis hirsuta and Adenia gummifera. The 8
plant extracts were selected from 23 total plant extracts which
had been previously assayed against E. coli and S. aureus. As
shown in Table 2, CD4 counts increased of subjects by up to 100
per month, but none of the subjects tested HIV negative within
the three-month period.
TABLE 2
CD4/ul per month
Subject ID Month 1 Month 2 Month 3 Month
4 Month 5
1b 118 150 399 420 —
2b 100 250 420 460 —
3b 04 93 190 320 —
4b 667 550 815 830 —
5b 160 120 480 620 —
6b 210 190 520 510 —
7b 420 500 780 780 —
8b 128 108 310 304 —
9b 110 150 380 348 —
10b 380 460 716 716 —
11b 300 410 390 560 —
12b 100 120 310 318 —
13b 250 180 340 420 —
14b 80 70 260 380 —
15b 140 110 300 420 —
16b 250 180 290 360 —
17b 300 380 460 580 —
18b 280 290 290 410 —
19b 118 190 170 320 —
20b 160 160 220 299 360
Example 4
In another experiment, 26 HIV-positive subjects were treated
with a herbal composition consisting of the 14 herbal
ingredients identified in Table 1. Subjects were administered a
composition prepared by dissolving approximately 1 tbsp. (or 15
ml) of the powdered ingredients (a mixture prepared the 14
plants listed in Table 1) in approximately 8 ozs. (250 ml) of
hot water. The supernatant liquid was then ingested by the
subject.
The subjects were divided into two groups: the first group
having 10 subjects (subject ID Nos. 1-10) and the second group
having 16 subjects (Subject ID Nos 11-26). In the first group,
each the 14 plants was present in the composition in equal
weight ratios. In the second group, the concentrations of
abyssinica and Clutia robusta were approximately half of the
other 12 ingredients as disclosed.
As shown in Table 3, CD4 counts for each subject were measured
on a monthly basis. The CD4 counts of the test subjects treated
with the 14 ingredient herbal composition increased by up to 100
per month. Six subjects tested HIV-negative after four months of
treatment. Two subjects tested HIV-negative after two months of
treatment.
TABLE 3
Subject CD4/uL per month
ID Month 1 Month 2 Month 3 Month 4
1 420 450 570 HIV negative
2 320 390 480 520
3 100 115 250 —
4 80 150 310 —
5 340 370 480 560
6 120 180 299 —
7 118 350 360 HIV negative
8 125 105 225 —
9 300 200 400 HIV negative
10 280 399 410 HIV negative
11 400 500 520 HIV negative
12 250 250 310 —
13 250 460 600 —
14 400 520 780 —
15 250 330 480 HIV negative
16 667 550 815 830
17 150 250 380 —
18 620 640 660 —
19 310 400 480 —
20 243 245 280 —
21 180 216 434 —
22 280 390 — —
23 360 420 — —
24 190 280 — —
25 630 720; — —
HIV negative
26 N/A; N/A;
HIV positive HIV negative
By comparison with the results achieved with the present
invention, in a study conducted on subjects on HAART in Moi
University Teaching and Academic Model for Prevention and
Treatment of HIV (AMPATH), the CD4 count increases were gradual,
generally taking several years to reach above 500. The subjects
were treated with conventional antiretroviral (ARV) therapy,
consisting of twice daily dosing of Stavudine, Lamivudine and
Nevirapine (d4T-3TC-NVP). Other ARV regimes include treatment
with combinations consisting of ZDV-3TC-NVP, d4T-3TC-EFV and
ZDV-3TC-EFV (wherein ZDV is Zidovudine and EFV is Efavirenz).
Treatment guidelines are provided in the publication “Integrated
Management of Adolescent and Adult Illness,” published in
January 2004 by the World Health Organization. ARV therapy
subjects rarely reverse their seroconversion status, and among
those listed in Table 4, none did so.
TABLE 4
Comparative Results of CD4 Count Increases in Subjects Under
Conventional ARV Therapy.
6 Months 1 Year 11⁄2 Years 2 Years 21⁄2
Years 3 Years
1. 247 207 264 197 138 367
2. 315 327 150 260 — —
3. 268 199 195 360 — —
4. 99 163 — — — —
5. 265 40 36 247 332 397
6. 138 311 584 578 — —
7. 37 298 — — — —
8. 201 261 — — — —
9. 21 52 74 309 — —
10. 2 156 — — — —
11. 43 200 — — — —
12. 169 295 — — — —
13. 75 144 179 — — —
US5837257
/ US5989556
Use of plant extracts for treatment of HIV, HCV and HBV
infections
Inventor(s): TSAI HSIU-HSIEN [TW]; HWANG
SHIE-MING [US]; KUNG PAI-CHU [TW] +
Abstract
This invention relates to compositions derived from Chinese
herbal medicines, medicinal plants and extracts thereof, and to
their use for the treatment of animals infected with viruses,
especially with hepatitis B virus (HBV), hepatitis C virus
(HCV), and human immunodeficiency virus (HIV). More
specifically, the compositions of the present invention are
derived from various Chinese herbal medicines or medicinal
plants which have a long history of human consumption. The
compositions of the invention are obtained through specific
techniques and have demonstrated outstanding efficacy for
treating human HBV carriers and hepatitic C patients.
Compositions according to the invention have also exhibited in
vitro antiviral activities against murine leukemia virus (MuLV)
and HIV. HIV is the virus known to cause acquired
immunodeficiency syndrome (AIDS) in humans and AIDS presents
special problems to the medical community which the present
invention addresses.
RELATED
APPLICATIONS
This application claims priority to a provisional application
filed Jul 9, 1996, Ser. No. 60/016,100 entitled: ANTI-VIRAL
AGENTS; and to a provisional application filed Jul. 10, 1996,
Ser. No. 60/021,467 entitled: ANTI-VIRAL AGENTS FROM CHINESE
MEDICINAL HERBS.
TECHNICAL
FIELD
This invention relates to compositions derived from Chinese
herbal medicines, medicinal plants and extracts thereof, and to
their use for the treatment of animals infected with viruses,
especially with hepatitis B virus (HBV), hepatitis C virus
(HCV), and human immunodeficiency virus (HIV). More
specifically, the compositions of the present invention are
derived from various Chinese herbal medicines or medicinal
plants which have a long history of human consumption. The
compositions of the invention are obtained through specific
techniques and have demonstrated outstanding efficacy for
treating human HBV carriers and hepatitic C patients.
Compositions according to the invention have also exhibited in
vitro antiviral activities against murine leukemia virus (MuLV)
and HIV. HIV is the virus known to cause acquired
immunodeficiency syndrome (AIDS) in humans and AIDS presents
special problems to the medical community which the present
invention addresses.
BACKGROUND
OF THE INVENTION
Modem medical science is constantly searching for new and more
powerful agents to prevent, treat or retard bacterial and viral
infections and cure the diseases they cause. Bacterial and viral
infections of humans and domestic animals cost billions of
dollars annually. Vast sums of money are spent each year by
pharmaceutical companies to identify, characterize, and produce
new antibiotics and antivirals to combat the emerging drug
resistant strains which have become a serious problem. Reliable
prophylactic treatments for disease prevention are also of major
interest. Yet, despite the costs and efforts to identify
treatments for viral infections, such as hepatitis and AIDS,
effective therapies remain elusive.
Hepatitis is a disease of the human liver. It is manifested with
inflammation of the liver and is usually caused by viral
infections and sometimes from toxic agents. Hepatitis may
progress to liver cirrhosis, liver cancer, and eventually death.
Several viruses such as hepatitis A, B, C, D, E and G are known
to cause various types of viral hepatitis. Among them, HBV and
HCV are the most serious. HBV is a DNA virus with a virion size
of 42 nm. HCV is a RNA virus with a virion size of 30-60 nm. See
D. S. Chen, J. Formos. Med. Assoc., 95(1), 6-12 (1996).
Hepatitis B is a major health problem worldwide, especially in
Asia and Africa. Approximately 300 million people are
chronically infected with HBV worldwide. More than one million
carriers of HBV are found in the United States and HBV infection
is currently the main cause of liver cirrhosis and cancer. HBV
carriers not only become long-term reservoirs of the virus but
also may develop chronic liver disease and have a greatly
increased risk of developing liver cirrhosis and cancer. The
progression from chronic hepatitis B to cirrhosis is frequently
insidious and occurs without a noticeable change in symptoms.
Once the symptoms of cirrhosis or cancer are manifested,
therapies are of little value.
Current prevention of HBV infection is a hepatitis B vaccination
which is safe and effective. However, vaccination is not
effective in treating those already infected (i.e., carriers and
patients). Many drugs have been used in treating chronic
hepatitis B and none have been proven to be effective, except
interferon. Treatment with interferon has limited success and
has frequently associated adverse side effects such as fatigue,
fever, chills, headache, myalgias, arthralgias, mild alopecia,
psychiatric effects and associated disorders, autoimmune
phenomena and associated disorders and thyroid dysfunction.
Treatment with interferon for sixteen (16) weeks has been shown
to be effective with a sustained loss of viral replication in
approximately 40% of hepatitis B patients. The great majority of
responders had normal serum aminotransferase levels and relapse
rates appear to be low. See R. P. Perrillo, Digestive Diseases
and Sciences, 38(4), 577-593 (1993). However, a higher long-term
relapse rate (24%) was reported in Chinese patients with chronic
hepatitis B who underwent interferon therapy. See A. S. F. Lok,
H. T. Chung, V. W. S. Liu, & O. C. K. Ma, Gastroenterology,
105(6), 1833-1838 (1993).
Moreover, serum hepatitis B surface antigen (HBsAg) disappeared
in 10-15% of patients treated with interferon. The loss of HBsAg
coincided with the disappearance of HBV. Improvement in liver
histology was sustained years later in HBsAg-negative patients.
The lack of disease progression could thus conceivably result in
the prevention of liver cancer when treatment is provided in the
pre-cirrhotic stage of infection. See R. P. Perrillo, Digestive
Diseases and Sciences, 38(4), 577-593 (1993).
Hepatitis C has been previously described as a non-A non-B
hepatitis, which is caused by HCV. There are approximately 100
million HCV carriers worldwide. An estimated 3.5 million people
have chronic hepatitis C in the United States. HCV infection
will lead to liver cirrhosis and cancer with less clinical
manifestation. Most hepatitis C patients do not have particular
symptoms and can thus be easily overlooked until it is too late
for therapy. This poses a potentially more serious problem than
hepatitis B. HCV carriers also become long-term reservoirs of
the virus and eventually develop chronic liver disease and have
a greatly increased risk of developing liver cirrhosis and
cancer. See D. S. Chen, Science, 262, 369-370 (1993).
No effective immunization is currently available, and hepatitis
C can only be controlled by other preventive measures such as
improvement in hygiene and sanitary conditions and interrupting
the route of transmission. At present, the only acceptable
treatment for chronic hepatitis C is interferon which requires
at least six (6) months of treatment. Treatment with interferon
has limited long term efficacy with a response rate about 25%.
Initial treatment has a response rate of about 50% however, half
of those which respond relapse after cessation of interferon
treatment. Therefore, only about 25% of patients had a sustained
response. See D. S. Chen, J. Formos. Med. Assoc., 95(1), 6-12
(1996) and N. Terrault & T. Wright, New Engl. J. Med.,
332(22), 1509-1511 (1995).
Because the interferon therapy has limited efficacy and frequent
adverse effects, a more effective regimen is needed. New
antivirals and immune modulators are presently undergoing
clinical trials.
AIDS is a deadly disease of an acquired immunodeficiency
syndrome in humans caused by HIV. It has been plaguing the world
since the first description of the disease in 1981 and the
discovery of its causative agent, HIV, in 1983. About 13 million
people were infected with HIV worldwide in 1993 and the number
has increased to about 21 million in 1996. See B. Jasny,
Science, 260(5112), 1219 (1993) and P. Piot, Science, 272(5270),
1855 (1996).
Several drugs have been approved for treatment of this
devastating disease, including azidovudine (AZT), didanosine
(dideoxyinosine, ddI), d4T, zalcitabine (dideoxycytosine, ddC),
nevirapine, lamivudine (epivir, 3TC), saquinavir (Invirase),
ritonavir (Norvir), indinavir (Crixivan), and delavirdine
(Rescriptor). See M. I. Johnston & D. F. Hoth, Science,
260(5112), 1286-1293 (1993) and D. D. Richman, Science,
272(5270), 1886-1888 (1996).
All drugs currently approved for AIDS treatment utilize
inhibition of viral proliferation and are viral reverse
transcriptase inhibitors or viral protease inhibitors. More
protease inhibitors, such as nelfinavir and improved saquinavir,
are in development. An AIDS vaccine (Salk's vaccine) has been
tested and several proteins which are chemokines from CD8 have
been discovered to act as HIV suppressors.
In addition to the above synthetic nucleoside analogs, proteins,
and antibodies, several plants and substances derived from
plants have been found to have in vitro anti-HIV activity, such
as Lonicera japonica and Prunella vulgaris, and glycyrrhizin
from Glycyrrhiza radix. See R. S. Chang & H. W. Yeung,
Antiviral Research, 9, 163-175 (1988) and M. Ito, et al.,
Antiviral Research, 7, 127-137 (1987).
Despite all of the available pharmaceuticals for the treatment
of HIV, there is still no cure for the deadly disease. HIV
viruses continue to mutate and become resistant to existing
drugs such as the reverse transcriptase inhibitors and protease
inhibitors. Recently, a therapy of using two (2) or three (3)
anti-HIV drugs in combination has been found effective in
significantly lowering the HIV loads in AIDS patients. The
results have been promising, however the virus continues to
develop resistance to the drugs and the long-term outcome
(survival and cure rates) is still unknown. Thus, the medical
communities throughout the world continue to search for drugs
that can prevent HIV infections, treat HIV carriers to prevent
them from progressing to full-blown deadly AIDS, and treat the
AIDS patient.
The use of herbal drugs and folk medicines have been known for
thousands of years in China. These herbal approaches to the
treatment of numerous illnesses, from arthritis to viral
infections, have been viewed by western modern medicine as
ineffective and dangerous. Records of the use of herbs date from
ancient China, Egypt and Biblical times. Early physicians used
hundreds of herbs to treat a variety of ailments. The practice
is still widespread, especially in Asia and Europe. During the
19th century, many home remedies containing herbs were patented
and sold. Modern drugs have replaced those remedies, but many
modern drugs contain ingredients derived from herbs.
In 1776, the English botanist and physician William Withering
learned that an herbal tea made by an old farm woman was
effective in treating dropsy, or excess water in the tissues,
which is caused by the inability of the heart to pump strongly
enough. He found that one ingredient of the tea, which was made
with leaves of the foxglove plant, strengthened the heart's
pumping ability. The drug made from the foxglove plant is now
known as digitalis.
Folk medicine is a relatively modern term to the West and has
come to mean the care and treatment of the sick through a
variety of herbal medicines. In recent years, folk medicines
have become of increasing interest to many people in the western
scientific medical community.
PRIOR ART
A Chinese herbal medicine known as AEGINETIAE HERBA (a.k.a.
GOLDEN LOCK KEY or LOTUS HERBA); has traditionally been
used to treat illnesses such as swollen and sore throat, urinary
tract infection, osteomyelitis, boils, tonsillitis, goiter,
pharyngitis, thyroiditis, enteritis, liver disease, cancer,
rheumatism, hematemesis, neurasthenia, eye redness, piles,
menstruation irregularity, dropsy, jaundice, hernia, snake bite,
and child developmental retardation. AEGINETIAE HERBA is
prepared from the dried whole plant of Aeginetia indica which
belongs to the family Orobanchaceae Dichondra micrantha, Striga
lutea and Dichondra repens are also used to prepare this herbal
medicine. Treatment dosage using the dried plant is typically
from 4 to 150 g per day. It should be noted that the plant
tastes bitter and is toxic.
Okubo et al. disclose that a phosphate buffered saline (PBS)
extract (pH 7.2 at ambient to 4 DEG C.) from the seeds of
Aeginetia indica exhibits excellent carcinostatic effect and
possesses interleukin-2 and interferon-.gamma. inducing
properties. The PBS was a 0.1M phosphate buffered physiological
saline at pH 7.2, not containing calcium or magnesium ions. The
extracted substance is taught to be a macromolecular
polysaccharide which may or may not contain lipid A binding with
protein depending on whether the extraction is conducted using
butanol or phenol. The extracted substance was soluble in water
and insoluble in n-butanol. Its molecular weight was within the
range of 100,000 to 200,000 Dalton. See S. Okubo, M. Sato, &
K. Himeno, U.S. Pat. No. 5,366,725, issued on Nov. 22, 1994.
A Chinese herbal medicine known as BAPHICACANTHIS RHIZOMA ET
RADIX has traditionally been used to treat illnesses such
as fever, abscesses, erysipelas, swollen sore throat,
hematemesis, epistaxis, typhus, typhoid, mumps, puerperal fever,
flu, measles, beriberi, headache, jaundice, plague, leucorrhea,
and syphilis. BAPHICACANTHIS RHIZOMA ET RADIX is prepared from
the dried rhizoma and root of Baphicacanthes cusia,
Strobilanthes cusia, Isatis tinctoria, Isatis indigotica, or
Polygonum tinctorium. It has been reported that this herbal
medicine has exhibited inhibition of flu virus in vitro. Aqueous
extracts from boiling the root of Isatis tinctoria have also
exhibited antibacterial effect.
The dried leaf of Baphicacanthes cusia, Isatis tinctoria, Isatis
indigotica, or Polygonum tinctorium have been used to prepare
another herbal medicine known as BAPHICACANTHIS FOLIUM.
BAPHICACANTHIS FOLIUM has traditionally been used to treat
illnesses such as typhus, typhoid, measles, fever, erysipelas,
sore throat, tonsillitis, dysentery, acute laryngitis,
stomatitis, gum bleeding, and various infectious diseases with
fever. It has also exhibited antibacterial effects and
antipyretic effects. The leaf of Isatis tinctoria has been used
as an antipyretic in the past.
The leaf of Baphicacanthes cusia, Isatis tinctoria, Isatis
indigotica, or Polygonum tinctorium with additional processing
has also been used to prepare a third related herbal medicine
known as INDIGO PULVERATA LEVIS. INDIGO PULVERATA LEVIS
has traditionally been used to treat illnesses such as
epistaxis, rashes, sores, mumps, chronic skin boils, dermatitis,
anemia, fever, swollen sores, stomatitis, acute laryngitis,
tonsillitis, gingivitis, parasitic oral mucosa inflammation,
snake or dog bites, malignant sores, and erysipelas. Ethanol
extracts of INDIGO PULVERATA LEVIS have exhibited bacterial
inhibition properties.
Baphicacanthes cusia and Strobilanthes cusia belong to the
family of Acanthaceae. Isatis tinctoria and Isatis indigotica
belong to the family of Cruciferae. Polygonum tinctorium belongs
to the family of Polygonaceae. BAPHICACANTHIS RHIZOMA ET RADIX
tastes bitter while BAPHICACANTHIS FOLIUM tastes bitter and
salty, and is nontoxic. INDIGO PULVERATA LEVIS tastes salty and
is also nontoxic. Treatment doses are typically 10 to 19 g per
day for BAPHICACANTHIS RHIZOMA ET RADIX, 8 to 30 g per day for
BAPHICACANTHIS FOLIUM, and 0.4-1.1 g per day for INDIGO
PULVERATA LEVIS.
Ho et al. disclose the use of an extract from a mixture of herbs
for the in vitro inhibition of HIV infection in human T
lymphocyte cells and mononuclear phagocytic lineage cells. The
activity was based on the test results of a water extract from a
mixture of three herbs: Isatis tinctoria (or Isatis
indigotica), Lonicera japonica, and Polygonum bistorta.
See D. D. Ho & X. S. Li, U.S. Pat. No. 5,178,865, issued on
Jan. 12, 1993.
The compound known as tryptanthrin has been identified as the
principal antifungal agent in the leaf of Strobilanthes cusia
and as the main antidermatophytic substance in the leaf of Polygonum
tinctorium and Isatis tinctoria. See H. Y. Hsu, Y. P.
Chen, & M. Hong, The Chemical Constituents Of Oriental
Herbs, Vol. 2, Oriental Healing Arts Institute, Los Angeles,
Calif., U.S.A., 758-759 (1985).
A Chinese herbal medicine known as BLECHNI RHIZOMA or
DRYOPTERIS CRASSIRHIZOMAE RHIZOMA has traditionally been
used to treat conditions such as cuts, swelling, fever, measles,
hematemesis, menorrhagia, dysentery, stool with traces of blood,
abdominal pain caused by parasites, wound bleeding, uterus
bleeding, puerperal abdominal pain, and erysipelas. BLECHNI
RHIZOMA is prepared from the dried root and stem of Blechnum
orientate which belongs to the family of Polypodiaceae or
Blechnaceae. DRYOPTERIS CRASSIRHIZOMAE RHIZOMA is prepared from
the dried root and stem of Dryopteris crassirhizoma which
belongs to the family of Aspidiaceae. Osmunda
japonica(Osmundaceae family), Woodwardia orientalis and
Woodwardia unigemmata (Blechnaceae family), Athyrium
acrostichoides (Aspidiaceae or Athyriaceae family),
Sphaeropteris lepifera (Cyatheaceae family), Cyrtomium falcatum,
and Cyrtomium fortunei (Aspidiaceae family) have also been used
for preparation of the herbal medicines. These herbal medicines
taste bitter and astringent, and are slightly toxic. Treatment
dosage is typically 4-11 g per day.
The sprout of Blechnum orientate has been used to treat swelling
while the sprouts of Sphaeropteris lepifera (also known
as (hereinafter "a.k.a.", Alsophila pustulosa) have been used to
treat carbuncles. Blechnum orientate has also shown a strong
inhibition effect against the influenza virus. Filmarone,
filicin, aspidin, albaspidin, and filicic acid which are found
in Dryopteris crassirhizoma have been characterized as having an
anthelmintic effect. See H. Y. Hsu, Y. P. Chen, S. G. Hsu, J. S.
Hsu, C. J. Chen, & H. C. Chang, Concise Pharmacognosy, New
Medicine Publishing Co., Taipei, R.O.C., 577-578 (1985); and H.
Y. Hsu, Y. P. Chen, & M. Hong, The Chemical Constituents Of
Oriental Herbs, Oriental Healing Arts Institute, Los Angeles,
Calif., U.S.A., 249-250 (1982).
Hozumi et al. disclosed that the rhizome of Dryopteris
crassirhizoma was an antiherpesviral agent, antipolioviral
agent, and anti-varicella-zoster virus agent. The rhizome of
Cyrtomium fortunei and the rhizome of Woodwardia orientalis were
also disclosed as antiherpesviral, antipolioviral, anti-measles
virus, anti-varicella-zoster virus, anti-cytomegalovirus (CMV),
and an anti-DNA and anti-RNA virus agents. See T. Hozumi, T.
Matsumoto, H. Ooyama, T. Namba, K. Shiraki, M. Hattori, M.
Kurokawa, & S. Kadota, U.S. Pat. No. 5,411,733, issued May
2, 1995.
A Chinese herbal medicine known as BLETILLAE TUBER has
traditionally been used to treat illnesses such as hemoptysis,
epistaxis, hematemesis, abscesses, burns, dry and chapped skin,
tuberculosis, gastric ulcers, and sores. BLETILLAE TUBER has
astringent, antibacterial and antifungal properties. BLETILLAE
TUBER is prepared from the dried tuber of Bletilla striata which
belongs to the family of Orchidaceae. BLETILLAE TUBER tastes
bittersweet, astringent and is nontoxic. Treatment dose is
typically 2-11 g per day for an average human.
Bletilla-glucomannan is a mucilage in the tuber of Bletilla
striata which has astringent properties (can be used to stop
bleeding and decrease swelling). See H. Y. Hsu, Y. P. Chen, S.
G. Hsu, J. S. Hsu, C. J. Chen, & H. C. Chang, Concise
Pharmacognosy, New Medicine Publishing Co., Taipei, R.O.C., 381
(1985); and H. Y. Hsu, Y. P. Chen, & M. Hong, The Chemical
Constituents Of Oriental Herbs, Oriental Healing Arts Institute,
Los Angeles, Calif., U.S.A., 114-115 (1982).
Chinese herbal medicines known as CIRSII RHIZOMA ET RADIX
and BREEAE RADIX have traditionally been used to treat
illnesses such as hematemesis, urine with traces of blood, stool
with traces of blood, gonorrhea with traces of blood,
menorrhagia, leucorrhoea, boils, acute infectious hepatitis,
cuts, bleeding sores, and abscesses. CIRSII RHIZOMA ET RADIX is
prepared from the dried rhizoma or root or the whole plant of
plants such as Cirsium japonicum, Cirsium albescens, and Cirsium
japonicum var. australe which are from the Compositae family.
BREEAE RADIX is prepared from the dried root of Compositae
family plants such as Breea segetum (a.k.a., Cephalanoplos
segetum) and Breea setosum. Both herbal medicines taste sweet
and slightly bitter, and are nontoxic. Treatment dose is
typically 5 to 75 g per day for the average human.
A Chinese herbal medicine known as FORSYTHIAE FRUCTUS
has traditionally been used to treat illnesses such as sores,
abscesses, lymph node swelling, neck lymph node tuberculosis,
erysipelas, gonorrhea, measles, ecchymosis, urethritis, and
hypertension. It was also found to inhibit several bacteria and
influenza viruses. FORSYTHIAE FRUCTUS is prepared from the dried
mature fruit of Forsythia suspensa, Forsythia viridissima, or
Forsythia koreana which belong to the family Oleaceae. The
herbal medicine tastes bitter and is nontoxic. Treatment dosage
is typically 3 to 11 g per day.
Hozumi et al. disclose that the fruit of Forsythia suspensa
is an antipolioviral agent and an anti-measles virus agent
useful in treating these viral infections. See T. Hozumi, T.
Matsumoto, H. Ooyama, T. Namba, K. Shiraki, M. Hattori, M.
Kurokawa, & S. Kadota, U.S. Pat. No. 5,411,733,
issued May 2, 1995.
The compounds Forsythoside A (found in the leaf of Forsythia
suspensa), forsythoside B (found in the stem of Forsythia
koreana), forsythoside C and forsythoside D (found in the fruit
of Forsythia suspensa) have been reported to exhibit
antibacterial activity against Staphylococcus aureus at a
concentration less than 2 mM. Suspensaside (found in the fruit
of Forsythia suspensa, likely the same as forsythoside C) has
also been reported to exhibit antibacterial activity against
Staphylococcus aureus Terashima with a minimum inhibition
concentration (MIC) of 2.6 mg/mL. See H. Y. Hsu, Y. P. Chen,
& M. Hong, The Chemical Constituents Of Oriental Herbs, Vol.
2, Oriental Healing Arts Institute, Los Angeles, Calif., U.S.A.,
53-55, 142-143 (1985).
A Chinese herbal medicine known as HEDYOTIS (a.k.a.,
OLDENLANDIAE HERBA) has traditionally been used to treat
illnesses such as malignant swelling, urethra infection,
pharyngitis, laryngitis, tonsillitis, toxic snake bites,
subacute or chronic coccygodynia, prurigo, carbuncle,
appendicitis, intestinal cancer, contusion injuries and eye
diseases. It has also been found to have weak antibacterial
activity in vitro. HEDYOTIS is prepared from the dried whole
plant of Hedyotis diffusa (a.k.a., Oldenlandia diffusa) which
belongs to the family Rubiaceae. The herbal medicine tastes
sweet and is nontoxic. Treatment dosage is typically 19 to 300 g
per day.
The Chinese herbal medicines known as LESPEDEZAE HERBA and
SENECINIS HERBA have traditionally been used to treat
illnesses such as urine incontinence, gonorrhea, leucorrhoea,
asthma, stomach ache, general weakening and exhaustion, a
children's disease characterized by swelling of the belly and
limbs caused by malnutrition or parasitic worms, diarrhea,
contusion injuries, eye diseases, visual impairment, eye
redness, renal disease, breast abscess, acute inflammatory
disease, cataracts, dysentery, enteritis, jaundice, flu,
septicemia, abscesses, boils, ringworm, erysipelas, snake or dog
bites, rheumatic pains, sores, swelling and a disease of the
palm. LESPEDEZAE HERBA is prepared from the dried whole plant of
Lespedeza cuneata which belongs to the family Leguminosae.
SENECINIS HERBA is prepared from the dried whole plant of
Senecio scandens which belongs to the family Compositae. The
extracts of Lespedeza cuneata and Senecio scandens have been
shown to have antibacterial effects. Both herbs taste sour,
astringent and bitter. Treatment dose is typically 4 to 40 g per
day.
A Chinese herbal medicine known as LIGUSTRI FRUCTUS has
traditionally been used as a tonic and to treat illnesses such
as debility, knee limpness, tinnitus and dizziness, palpitation,
insomnia, constipation, early white hair, neck lymph node,
tuberculosis, lung tuberculosis, intermittent fever and dropsy.
LIGUSTRI FRUCTUS is prepared from the dried mature fruit of
Ligustrum lucidum or Ligustrum japonicum which belongs to the
family Oleaceae. The leaves of Ligustrum lucidum have been used
as an antipyretics, analgesics and anti-inflammatory agents. The
leaves of Ligustrum japonicum have also been used to treat
illnesses such as ophthalmalgia, ulcerative stomatitis,
mastitis, swelling, and burns. The fruit of Ligustrum lucidum
taste bitter and are nontoxic. Typical treatment dosage of the
dried fruit is typically 6 to 20 g per day. That of the dried
leaves is typically 40 to 75 g per day.
A Chinese herbal medicine known as LONICERAE FLOS has
traditionally been used to treat illnesses such as fever,
febrile diseases, acute infectious diseases, measles, carbuncle,
dysentery, malignant sores and swelling, abscesses, boils,
gonorrhea, syphilis, poisoning, enteritis, swelling, ringworm
and similar skin diseases. LONICERAE FLOS is prepared from the
dried flower bud of Lonicera japonica or Lonicera confusa. Both
plants belong to the family Caprifoliaceae. The flower of
Lonicera japonica has diuretic, antipyretic, anti-inflammatory,
anti-convulsive, antibacterial and antiviral properties. The
flower bud has also been used as a diuretic. The herbal medicine
tastes sweet and is nontoxic. Treatment dosage is typically 11
to 75 g per day for the typical human.
The dried vine, stem and leaf of Lonicera japonica is used for
preparation of another herbal medicine called LONICERAE
CAULIS ET FOLIUM, which has traditionally been used to
treat illnesses such as paralysis and pain caused by rheumatism,
rheumatism swelling, rheumatic pain, carbuncle swelling,
arthritis, gonorrhea, enteritis, and various symptoms with pus,
such as abscesses. Extracts have exhibited the ability to raise
blood sugar levels in rabbits. The root of Lonicera japonica has
also been used to treat illnesses such as venereal disease,
syphilis, gonorrhea, lymph node tuberculosis, contusion injury,
and skin disease. Treatment doses are typically 8 to 75 g per
day for the stem or leaf and 110 to 150 g per day for the root.
Ho et al. disclose the anti-HIV activity in vitro of a mixture Lonicera
japonica, Isatis tinctoria (or Isatis indigotica) and
Polygonum bistorta or a mixture of Lonicera japonica with
Scutellaria baicalensis. Water extractions of the
mixtures, treatment with ethanol for precipitation and charcoal
adsorption are disclosed for the preparation for the anti-HIV
active composition. See D. D. Ho & X. S. Li, U.S. Pat. No.
5,178,865, issued on Jan. 12, 1993. Several tannins such as
caffeoylquinates isolated from Lonicera japonica have been
reported to have an inhibitory effect on HIV-1 reverse
transcriptase activity. See C. W. Chang, M. T. Lin, S. S. Lee,
K. C. S. C. Liu, F. L. Hsu, & J. Y. Lin, Antiviral Research,
27(4), 367-374 (1995).
A mixture of aqueous extracts of Lonicera japonica flower
buds and Forsythia suspensa fruits with the crude
flavonoids from Scutellaria baicalensis have been shown to have
antibacterial and antiviral properties. A group of patients with
severe respiratory disease were treated with the mixture and
they responded as well as a control group on standard antibiotic
therapy. See P. J. Houghton, Z. Boxu, & Z. Xisheng,
Phytother. Res., 7(5), 384-386 (1993).
A Chinese herbal preparation which consisted of ten (10) herbs
such as Prunus armeniacae, Scutelaria baicalensis, Lonicera
japonica, etc. was shown to have strong inhibitory effects
in vitro against Streptococcus hemolyticus, Staphylococcus
aureus, Flexners Dysentery bacillus, Diplococcus pneumoniae and
Pseudomonas aeruginosa. The preparation was shown to be as
effective as penicillin and aminophylline in treating
bronchopneumonia and acute bronchitis patients. See Y. Q. Li, W.
Yuan, & S. L. Zhang, Chung Kuo Chung Hsi I Chieh Ho Tsa
Chih, 12(12), 708, 719-721, 737 (1992).
Another Chinese herbal preparation which consisted of Lonicera
japonica, Ophiopogon japonicus, and Astragalus membranaceus
was shown to be effective in treating viral myocarditis. The
authors reported that the preparation could directly inactivate
the virus of Coxsackie B3, protect heart cells in mice, prevent
attack by Coxsackie B3, promote the production of interferon and
increase the functionality of NK cells to regulate immunity in
experimental mice. See H. J. Yan, Chung Hsi I Chieh Ho Tsa Chih,
11(8), 452, 468-470 (1991).
A Chinese herbal medicine known as PHELLODENDRI CORTEX
has traditionally been used to treat illnesses such as
dysentery, diarrhea, jaundice, stools with blood, piles,
tinnitus, mouth and tongue boils, abscesses, sores, leucorrhea
with blood, abdominal pain, indigestion, bacteroid enteritis,
and tuberculoid diarrhea. The herbal medicine has also been used
as an eye wash, for strengthening stomach and intestine,
stimulate appetite, and as an astringent, anti-inflammatory,
etc. It has antibacterial, anti-inflammatory, and wound healing
properties. PHELLODENDRI CORTEX is prepared from the dried
cortex of plants from the Rutaceae family such as Phellodendron
amurense, Phellodendron chinense, Phellodendron amurense var.
sachalinense, and Phellodendron wilsonii. PHELLODENDRI CORTEX
tastes bitter and is nontoxic. Treatment dose is typically 1 to
11 g per day.
Hozumi et al. disclose the bark of Phellodendron amurense
as an antiherpesviral, antipolioviral, anti-measles virus,
anti-varicella-zoster virus, anti-CMV and anti-DNA virus and
anti-RNA virus agents. See T. Hozumi, T. Matsumoto, H. Ooyama,
T. Namba, K. Shiraki, M. Hattori, M. Kurokawa, & S. Kadota,
U.S. Pat. No. 5,411,733, issued on May 2, 1995.
A Chinese herbal medicine known as POLYGONI CUSPIDATI RHIZOMA
has traditionally been used to treat illnesses such as
dysentery, leucorrhea, fever, headache, menorrhagia,
dysmenorrhea, breast abscesses, sores, boils, contusion injury,
menstruation irregularity, puerperal ecchymotic abdominal
distension and pain, dysuria, infantile growth and appendicitis.
POLYGONI CUSPIDATI RHIZOMA is prepared from the dried rhizoma of
Polygonum cuspidatum, Polygonum runcinatum, or Polygonum
reynoutria (a.k.a. Reynoutria japonica) which belong to the
family Polygonaceae. The tender leaf has also been used to treat
contusion and cut injuries. Extracts of the herbal medicine have
exhibited antibacterial and antiviral effects in vitro.
Excessive use of the herbal medicine may cause a slight
diarrhea. The herbal medicine tastes bitter and the treatment
dose is typically 6 to 40 g per day.
Hozumi et al. disclose the root and rhizome of Polygonum
cuspidatum as an antiherpesviral, antipolioviral,
anti-varicella-zoster virus, and anti-CMV agent. See T. Hozumi,
T. Matsumoto, H. Ooyama, T. Namba, K. Shiraki, M. Hattori, M.
Kurokawa, & S. Kadota, U.S. Pat. No. 5,411,733,
issued on May 2, 1995.
Resveratrol has also been reported as an antifungal and
antibacterial component in the root of Polygonum cuspidatum. See
H. Y. Hsu, Y. P. Chen, & M. Hong, The Chemical Constituents
Of Oriental Herbs, Vol. 2, Oriental Healing Arts Institute, Los
Angeles, Calif., U.S.A., 51 (1985).
A Chinese herbal medicine known as PRUNELLAE SPICA has
traditionally been used to treat illnesses such as goiter,
scrofula, neck lymph node tuberculosis, lymph node swelling, eye
redness, pain, abscesses, sores, hemorrhoids, swollen eye,
ophthalmalgia, leucorrhoea with traces of blood, gonorrhea,
uterine disease, mastitis, breast abscesses, breast cancer, foot
swelling, paralysis, chronic arthritis, conjunctivitis, and
hypertension. PRUNELLAE SPICA is prepared from the dried spica
or whole plant of Prunella vulgaris or Prunella vulgaris subsp.
asiatica (a.k.a., Prunella vulgaris var. lilachina). Both plants
belong to the family Labiatae. The whole plant can be used as a
diuretic and also has antibacterial effects in vitro. The herbal
medicine tastes bitter and is nontoxic. Treatment dosage is
typically 4 to 110 g per day for the average human.
Hozumi et al. disclose that the spike of Prunella vulgaris as an
antiherpesviral agent for treating herpes virus infection. See
T. Hozumi, T. Matsumoto, H. Ooyama, T. Namba, K. Shiraki, M.
Hattori, M. Kurokawa, & S. Kadota, U.S. Pat. No.
5,411,733, issued May 2, 1995. The water extract of
Prunella vulgaris (boiling 3 g in 100 mL water for 45 minutes)
was also reported to have anti-HIV (strain H9/3B) activity. The
extract also exhibited synergistic anti-HIV activity with
zidovudine (AZT) and didanosine (ddl). Only a slight additive
effect was observed for Prunella vulgaris and zalcitabine (ddC).
See J. F. John, R. Kuk, & A. Rosenthal, Abstr. Gen. Meet.
Am. Soc. Microbiol., 94, 481 (1994).
Yamasaki et al. evaluate in vitro, two hundred and four (204)
crude drugs of common use in Japan for anti-HIV-1 activity and
reported that the hot water extract of Prunella vulgaris (spike)
showed a strong in vitro anti-HIV-1 activity with an IC100 of 16
.mu.g/mL. See K. Yamasaki, T. Otake, H. Mori, M. Morimoto, N.
Ueba, Y. Kurokawa, K. Shiota, & T. Yuge, Yakugaku Zasshi,
113(11), 818-824 (1993).
Yao et al. report that the water extract of the dried entire
plant of Prunella vulgaris was active in vitro in inhibiting
HIV-1 replication with relatively low cytotoxicity towards the
MT-4 cells. The extract was also active in reverse transcriptase
inhibition. The active factor was purified and identified as
anionic with a molecular weight of approximately 10,000 Dalton.
This active component may be the same as the prunellin, as
described below by Tabba et al. The purified extract inhibited
HIV-1 replication in the lymphoid cell line MT-4, in the
monocytoid cell line U937, and in peripheral blood mononuclear
cells (PBMC) at effective concentrations of 6, 30, and 12.5
.mu.g/mL, respectively. Pretreatment of uninfected cells with
the extract prior to viral exposure did not prevent HIV-1
infection. Preincubation of HIV-1 with the purified extract
dramatically decreased infectiousness. The purified extract was
also able to block cell-to-cell transmission of HIV-1, prevented
syncytium formation, and interfered with the ability of both
HIV-1 and purified gp120 to bind to CD4. PCR (polymerase chain
reaction) analysis confirmed the absence of HIV-1 proviral DNA
in cells exposed to virus in the presence of the extract. The
results suggested that the purified extract antagonized HIV-1
infection of susceptible cells by preventing viral attachment to
the CD4 receptor. See X. J. Yao, M. A. Wainberg, & M. A.
Pamiak, Virology, 187(1), 56-62 (1992).
Tabba et al. isolated and partially characterized an anti-HIV
component, prunellin, from aqueous extracts of Prunella
vulgaris. Prunellin is a carbohydrate with an MIC (minimum
inhibition concentration) of 2.2 .mu.g/mL against HIV-1 in
vitro. It was identified as a partially sulfated polysaccharide
with a molecular weight of about 10,000 Dalton. See H. D. Tabba,
R. S. Chang, & K. M. Smith, Antiviral Research, 11, 263-273
(1989).
Zheng evaluated four hundred seventy two (472) traditional
medicinal herbs for antiviral effect on type 1 herpes simplex
virus (HSV1). Prunella vulgaris was one of the ten herbs found
to be highly effective in vitro. Clinically, 78 cases of
herpetic keratitis due to HSV1 were treated with Prunella
vulgaris and Pyrrosia lingua eye drops. Among them, 38 cases
were effectively cured, 37 cases showed an improvement, and 3
cases showed no benefit. See M. Zheng, J. Tradit. Chin. Med.,
8(3), 203-206 (1988).
Triterpene 1 and Triterpene 2 which have been isolated from
Prunella vulgaris have shown antiviral activity against HSV1.
Triterpene 1 was identified as betulinic acid and triterpene 2
was identified as 2.alpha.,3.alpha.-dihydroxyurs-12-en-28-oic
acid. The EC50 was estimated to be 30 .mu.g/mL for triterpene 1
and 8 .mu.g/mL for triterpene 2 by plaque reduction assay. See
S. Y. Ryu, C-K. Lee, C. O. Lee, H. S. Kim, & O. P. Zee,
Arch. Pharmacal Res. (Seoul), 15(3), 242-245 (1992).
A Chinese herbal medicine known as SCUTELLARIAE BARBATAE
HERBA has traditionally been used to treat illnesses such
as hematemesis, gonorrhea with traces of blood, jaundice, sore
throats, lung abscesses, boils, carbuncles, abscesses, neck
lymph node swelling, sores, cancer, contusion or cut injuries,
snake bite injuries, dysentery with traces of blood,
convulsions, pneumonia, abdominal pains, congenital diseases,
enteritis, coccygodynia, appendicitis, asthma, malaria, and
rheumatism. It was also found to have antibacterial effect.
SCUTELLARIAE BARBATAE HERBA is prepared from the dried whole
plant of Scutellaria barbata, Scutellaria rivularis, or
Scutellaria dependens which belong to the family Labiatae. The
herbal medicine tastes bitter and should not be consumed by
those who have anemia. Pregnant women should avoid taking this
herb. Treatment dosage is typically 4 to 300 g per day.
Dried whole plants of Scutellaria rivularis have been
used in folk medicine for the treatment of tumors, hepatitis,
liver cirrhosis, and other diseases in China and Taiwan. See Y.
L. Lin, Y. H. Kuo, G. H. Lee, and S. M. Peng, J. Chem. Research
(S), 320-321.(1987).
Apigenin, isolated from the whole herb of Scutellaria rivularis,
was found to have anti-influenza virus activity. See T. Nagai,
et al., Chem. Pharm. Bull., 38(5), 1329-1332 (1990).
A Chinese herbal medicine known as SOLANI HERBA has
traditionally been used to treat illnesses such as boils,
abscesses, erysipelas, contusion or sprain injuries, chronic
bronchitis, acute nephritis, cancer, swelling, hernia, ulcers,
carbuncles with swelling and sores. SOLANI HERBA is prepared
from the dried whole plant of Solanum nigrum which belongs to
the family Solanaceae. Extracts of SOLANI HERBA have
demonstrated anti-inflammatory properties. The fruit has also
exhibited the effects of suppressing coughs and relieving
bronchial inflammation. The herbal medicine tastes bitter and
slightly sweet and is nontoxic. Treatment dosage is typically 11
to 60 g per day.
The root of Solanum nigrum was believed to have antipyretic
activity and has been used for treating high fevers by some
primitive tribes of western Ghats in India. A decoction prepared
from Solanum nigrum plants, Glycosmis Mauritania seeds
and/or Santalum album wood chips was believed to have
expectorant activity and has been used for coughs and to treat
hemoptysis. See P. Pushpangadan and C. K. Atal, J.
Ethnopharmacol., 11(1), 59-77 (1984).
The compound solasonine (found in the whole herb, fruit,
leaf, and fresh immature berries of Solanum nigrum) has an
anti-inflammatory effect similar to cortisone. Solasonine and
solanine (also found in Solanum nigrum) possesses the ability of
raising or lowering the blood sugar level in rats depending on
the situation of the animals. Solasonine was also reported to
have a stimulating effect on the heart, while solanine had a
suppressive effect. When administered at small doses, solasonine
enhances the stimulative process of the central nerve system in
animals (i.e., rat and rabbit). On the other hand, it enhances
the suppressive process when administered at large doses.
Solasonine can also lower the blood coagulability. See (1) H. Y.
Hsu, Y. P. Chen, S. G. Hsu, J. S. Hsu, C. J. Chen, & H. C.
Chang, Concise Pharmacognosy, New Medicine Publishing Co.,
Taipei, R.O.C., 176-177 (1985); (2) H. Y. Hsu, Y. P. Chen, &
M. Hong, The Chemical Constituents Of Oriental Herbs, Oriental
Healing Arts Institute, Los Angeles, Calif., U.S.A., 1400-1401,
1406 (1982); and (3) H. Y. Hsu, Y. P. Chen, & M. Hong, The
Chemical Constituents Of Oriental Herbs, Vol. 2, Oriental
Healing Arts Institute, Los Angeles, Calif., U.S.A., 742 (1985).
Additionally, Yamasaki et al. report that the hot water extract
of Lithospermum erythrorhizon (root) showed a strong in
vitro anti-HIV-1 activity with an IC100 of 16 .mu.g/mL. Yao et
al. reported that the water extracts of the dried root of
Arctium lappa and the dried aerial parts of Andrographis
paniculata were anti-HIV-1 active in vitro and cytotoxic towards
the MT-4 cells. Both extracts were also active in reverse
transcriptase inhibition. See K. Yamasaki, T. Otake, H. Mori, M.
Morimoto, N. Ueba, Y. Kurokawa, K. Shiota, & T. Yuge,
Yakugaku Zasshi, 113(11), 818-824 (1993); and X. J. Yao, M. A.
Wainberg, & M. A. Parniak, Virology, 187(1), 56-62 (1992).
Glycyrrhizin
is reported to have an inhibitory effect on the in vitro
infectivity and cytopathic activity of HIV. See M. Ito, et al,
Antiviral Research, 7, 127-137 (1987). Glycyrrhizin is a saponin
found in the herbal medicine GLYCYRRHIZAE RADIX.
GLYCYRRHIZAE RADIX is prepared from the dried root of
Glycyrrhiza uralensis, Glycyrrhiza glandulifera, Glycyrrhiza
echinata, or Glycyrrhiza glabra all of which belong to the
family Leguminosae.
Chang and Yeung screened the boiling water extracts of twenty
seven (27) medicinal herbs for anti-HIV activity. They found
eleven (11) of the extracts were active in inhibiting HIV in the
H9 cells. Lonicera japonica, Prunella vulgaris, Woodwardia
unigemmata, and Senecio scandens were among those active
ones with moderate activities. Forsythia suspensa, Isatis
tinctoria, and Polygonum cuspidatum were among those
tested which did not display activity in the anti-HIV assay. The
anti-HIV active extract of Viola yedoensis was further tested
and found to be fairly specific. The extract did not inactivate
HIV extracellularly and did not inhibit the growth of herpes
simplex, polio, or vesicular stomatitis viruses in human
fibroblast culture. See R. S. Chang & H. W. Yeung, Antiviral
Research, 9, 163-175 (1988).
Antiviral agents have been isolated from Syzygium
aromaticum, Sapium sebiferum, Scutellaria baicalensis, and
Scutellaria rivularis. Eugeniin (a tannin) isolated from
Syzygium aromaticum and methyl gallate isolated from Sapium
sebiferum exhibited anti-herpes simplex virus activity in vitro.
Plant flavonoids, such as 5,7,4'-trihydroxy-8-methoxyflavone
from the root of Scutellaria baicalensis and apigenin
(5,7,4'-trihydroxyflavone) from the whole herb Scutellaria
rivularis, were also reported to have anti-influenza virus
activity. See (1) T. Hozumi, et al., U.S. Pat. No. 5,411,733
(1995); (2) M. Takechi & Y. Tanaka, Planta Medica, 42, 69-74
(1981); (3) C. J. M. Kane, et al, Bioscience Reports, 8, 85-94
(1988); and (4) T. Nagai, et al., Chem. Pharm. Bull., 38(5),
1329-1332 (1990).
Hozumi et al. disclose ninety one (91) herbal medicines which
demonstrated antiviral activity. More specifically, fifty two
(52) of them had antiherpesviral activity, sixty four (64) had
antipolioviral activity, thirty seven (37) had anti-measles
virus activity, twenty seven (27) had anti-varicella-zoster
virus activity, twenty three (23) had anti-CMV activity, and
twenty eight (28) had anti-DNA virus and anti-RNA virus
activity. See T. Hozumi, T. Matsumoto, H. Ooyama, T. Namba, K.
Shiraki, M. Hattori, M. Kurokawa, & S. Kadota, U.S. Pat. No.
5,411,733, issued on May 2, 1995.
The anti-DNA virus and anti-RNA virus activity of the twenty
eight (28) herbal medicines disclosed in the '733 patent solely
based upon their antiherpesviral, antipolioviral, anti-measles
virus, and/or anti-varicella-zoster virus and anti-CMV
activities. However, the extrapolation to cover both anti-DNA
virus and anti-RNA virus activities is unfounded from the work
conducted.
The data of the present invention presented below evidenced
little or no anti-HIV activity of the two herbal medicines at
2.5 and 0.5 mg/mL derived from the rhizome of Cyrtomium
fortunei and the bark of Phellodendron amurense. In
contrast, the three (3) herbal medicines using the spike of
Prunella vulgaris, the fruit of Forsythia suspensa, and the root
and rhizome of Polygonum cuspidatum, will be shown to have a
strong to moderate anti-HIV activity at 2.5 mg/mL.
Herbal medicines LONICERAE FLOS, BAPHICACANTHIS RHIZOMA ET
RADIX, and FORSYTHIAE FRUCTUS have been used separately
and/or in combination as antipyretic and detoxification agents
along with other herbal medicines for treating acute hepatitis.
The herbal medicines BLECHNI RHIZOMA and POLYGONI CUSPIDATI
RHIZOMA have been used along with other herbal medicines
in a formula for treating B hepatitis. The herbal medicines
SCUTELLARIAE BARBATAE HERBA and LIGUSTRI FRUCTUS have
occasionally been added to improve activity. Herbal medicine
LIGUSTRI FRUCTUS was occasionally used along with other herbal
medicines mainly as a tonic and HEDYOTIS was occasionally used
along with other herbal medicines as a detoxification agent. The
herbal medicine PRUNELLAE SPICA has also been used along with
other herbal medicines to relief liver stress.
It is noted that in the practice of Chinese traditional
medicine, herbal medicines were used to treat the symptoms of
the patients, not the disease entity itself, and were therefore
fairly nonspecific to a particular disease. Herbal medicines
were used depending on the symptoms of the individual patient.
The composition of herbal medicines would vary case by case and
may even change for each individual patient during the course of
the treatment according to each treatment result. It is
therefore very difficult to have a universal herbal composition
suitable for treating a specific disease within a population.
The present invention is directed to the discovery of antiviral
herb compositions, extracts thereof and the active chemical
constituents. The antiviral herb compositions of this invention
are derived from individual herbs, herb mixtures and
commercially available Chinese herbal medicines. These novel
herb compositions and their extracts and/or active principles
have demonstrated activities against viral diseases such as
hepatitis B, hepatitis C, HBV and HCV carriers, HIV infection
and AIDS.
SUMMARY OF
THE INVENTION
As used herein and in the claims, the following nomenclatures
will be used to identify the four (4) herb mixtures known as
HHT888-4, HHT888-5, HHT888-45 and HHT888-54. HHT888-4 is a
mixture of five single-herb Chinese herbal medicines at a
preferred ratio of No.4(1): No.4(2): No.4(3): No.4(4): No.4(5)
of about 3:3:3:3:4 (w/w). The weight ratio may vary up to 50%
per component. By "variance of the weight ratio by 50%" means
that each value of each component of the ratio may be increased
or decreased by 50%. Thus, as an example, 1:1 can range from
1.5:0.5 to 0.5:1.5 (or 3:1 to 1:3).
HHT888-5 is a mixture of eleven (11) single-herb Chinese herbal
medicines, No.5(1) to No.5(11) preferably at about equal
proportions by weight. The weight ratio may vary up to 50% per
component.
HHT888-45 is a mixture of four (4) to six (6) single-herb
Chinese herbal medicines at a ratio of No.4(3): No.4(4):
No.5(4): No.5(5): No.5(8): No.4(2) at a preferred ratio of about
1:1:1:1:0-1:0-1 (w/w). The weight ratio may vary up to 50% for
each component.
HHT888-54 is a mixture No.5(5) and at least one single herb
medicine selected from No. 4(2), No. 4(3), No. 4(4), No. 4(5),
No. 5(1), No. 5(2), No. 5(4), No. 5(7), No. 5(8) and No. 5(11)
wherein the weight ratio of No. 5(5) to each of the other single
herb medicines is 1:1. Thus, HHT888-54 consists of No. 5(5) plus
No. 4(3), No. 4(4) and No. 5(8); the most preferred weight ratio
is 1:1:1:1.
More generally, the weight ratio of No. 5(5) to the sum of the
other single herb medicines is from 1:10 to 10:1.
The single-herb components of HHT888-4 are:
No.4(1)=HEDYOTIS (a.k.a., OLDENLANDIAE HERBA) source: Hedyotis
diffusa (a.k.a., Oldenlandia diffusa)
No.4(2)=SCUTELLARIAE BARBATAE HERBA source: Scutellaria barbata,
Scutellaria rivularis, Scutellaria dependens
No.4(3)=LONICERAE FLOS source: Lonicera japonica, Lonicera
confusa
No.4(4)=PRUNELLAE SPICA source: Prunella vulgaris, Prunella
vulgaris subsp. asiatica (a.k.a., Prunella vulgaris var.
lilachina)
No.4(5)=SOLANI HERBA source: Solanum nigrum
The single-herb components of HHT888-5 are:
No.5(1)=HEDYOTIS (a.k.a., OLDENLANDIAE HERBA) source: Hedyotis
diffusa (a.k.a., Oldenlandia diffusa)
No.5(2)=BLECHNI RHIZOMA or DRYOPTERIS CRASSIRHIZOMAE RHIZOMA,
source: Blechnum orientale, Dryopteris crassirhizoma, Osmunda
japonica, Woodwardia orientalis, Woodwardia unigemmata, Athyrium
acrostichoides, Sphaeropteris lepifera, Cyrtomium falcatum,
Cyrtomium fortunei
No.5(3)=CIRSII RHIZOMA ET RADIX and BREEAE RADIX source: Cirsium
japonicum, Cirsium albescens, Cirsium japonicum var. australe,
Breea segetum (a.k.a., Cephalanoplos segetum), Breea setosum
No.5(4)=LESPEDEZAE HERBA or SENECINIS HERBA source: Lespedeza
cuneata, Senecio scandens
No.5(5)=AEGINETIAE HERBA(a.k.a. GOLDEN LOCK KEY or LOTUS HERBA).
source: Aeginetia indica, Dichondra micrantha, Striga lutea,
Dichondra repens
No.5(6)=BAPHICACANTHIS RHIZOMA ET RADIX source: Baphicacanthes
cusia, Strobilanthes cusia, Isatis tinctoria, Isatis indigotica,
Polygonum tinctorium
No.5(7)=POLYGONI CUSPIDATI RHIZOMA source: Polygonum cuspidatum,
Polygonum runcinatum, Polygonum reynoutria (a.k.a., Reynoutria
japonica)
No.5(8)=FORSYTHIAE FRUCTUS source: Forsythia suspensa, Forsythia
viridissima, Forsythia koreana
No.5(9)=PHELLODENDRI CORTEX source: Phellodendron amurense,
Phellodendron chinense, Phellodendron amurense var.
sachalinense, Phellodendron wilsonii
No. 5(10)=BLETILLAE TUBER source: Bletilla striata
No.5(11)=FLIGUSTRI FRUCTUS source: Ligustrum lucidum, Ligustrum
japonicum
The single-herb components of HHT888-45 are:
No.4(3)=LONICERAE FLOS source: Lonicera japonica, Lonicera
confusa
No.4(4)=PRUNELLAE SPICA source: Prunella vulgaris, Prunella
vulgaris subsp. asiatica (a.k.a., Prunella vulgaris var.
lilachina)
No.5(4)=LESPEDEZAE HERBA or SENECINIS HERBA source: Lespedeza
cuneata, Senecio scandens
No.5(5)=AEGINETIAE HERBA (a.k.a. GOLDEN LOCK KEY or LOTUS
HERBA). source: Aeginetia indica in addition to No.5(5) are at
least one selected from:
No.4(2)=SCUTELLARIAE BARBATAS HERBA (optional) source:
Scutellaria barbata, Scutellaria rivularis, Scutelaria dependens
No.5(8)=FORSYTHIAE FRUCTUS (occasionally used) source: Forsythia
suspensa, Forsythia viridissima, Forsythia koreana
The single herb components of HHT888-54 in addition to
No.5(5)are at least one selected from:
No.4(2)=SCUTELLARIAE BARBATAE HERBA source: Scutellaria barbata,
Scutellaria rivularis, Scutellaria dependens
No.4(3)=LONICERAE FLOS source: Lonicera japonica, Lonicera
confusa
No.4(4)=PRUNELLAE SPICA source: Prunella vulgaris, Prunella
vulgaris subsp. asiatica (a.k.a., Prunella vulgaris var.
lilachina)
No.4(5)=SOLANI HERBA source: Solanum nigrum
No.5(1)=HEDYOTIS (a.k.a., OLDENLANDIAE HERBA) source: Hedyotis
diffusa (a.k.a., Oldenlandia diffusa)
No.5(2)=BLECHNI RHIZOMA or DRYOPTERIS CRASSIRHIZOMAE RHIZOMA,
source: Blechnum orientale, Dryopteris crassirhizoma, Osmunda
japonica, Woodwardia orientalis, Woodwardia unigemmata, Athyrium
acrostichoides, Sphaeropteris lepifera, Cyrtomium falcatum,
Cyrtomiumfortunei
No.5(4)=LESPEDEZAE HERBA or SENECINIS HERBA source: Lespedeza
cuneata, Senecio scandens
No.5(7)=POLYGONI CUSPIDATI RHIZOMA source: Polygonum cuspidatum,
Polygonum runcinatum, Polygonum reynoutria (a.k.a., Reynoutria
japonica)
No.5(8)=FORSYTHIAE FRUCTUS source: Forsythia suspensa, Forsythia
viridissima, Forsythia koreana
No.5(11)=LIGUSTRI FRUCTUS source: Ligustrum lucidum, Ligustrum
japonicum
The names of the Chinese herbal medicines for the single-herb
components are shown in capital letters, followed by their plant
sources listed in italics.
As used herein and in the claims, the term HHT888-4, HHT888-5,
HHT888-45 and the like include the actual herbal blends, aqueous
extracts thereof and the individual active components or
principles of the extract. In similar fashion, the use of the
terms No.5(5), No. 5(8) and the like include the actual herb,
extracts thereof and the isolated active molecular agents.
As also used in the specification and in the claims, No.4(2),
No.4(3), No.4(4), No.4(5), No.5(1), No.5(2), No.5(3), No.5(4),
No.5(5), No.5(6), No.5(7), No.5(8), No.5(9), No.5(10), and
No.5(11) are the single-herb components described above,
including their respective source plants. It should be noted
that No.4(1) is the same as No.5(1) (HEDYOTIS).
Specific details and descriptions of the above recited Chinese
herbal medicines and medicinal herbs can be found in the
following references: (1) H. C. Chang, Medicinal Herbs I,
Holiday Publishing Co., Taipei, Taiwan, R.O.C., 15, 36, 100,
113, 127, 147 (1990); (2) H. C. Chang, Medicinal Herbs II,
Holiday Publishing Co., Taipei, Taiwan, R.O.C., 15, 131, 135,
155 (1991); (3) W. S. Kan, Pharmaceutical Botany, National
Research Institute Of Chinese Medicine, Taipei, Taiwan, R.O.C.,
113, 124-130, 200-201, 206-207, 289-290, 353-354, 442-444, 485,
487-488, 497, 505, 513-514, 522, 527-529, 558, 562-563, 648-649
(1971); (4) M. S. Lee, Frequently Used Chinese Crude Drugs And
Folk Medicines Handbook, 12th Ed., Sheng-Chang Medicinal Record
Magazine Publishing Co., Taipei, Taiwan, R.O.C., 4-6, 17, 21,
29, 36, 38, 40, 48, 64, 71, 79, 85 (1992); and (5) H. Y. Hsu, Y.
P. Chen, S. G. Hsu, J. S. Hsu, C. J. Chen, & H. C. Chang,
Concise Pharmacognosy, New Medicine Publishing Co., Taipei,
Taiwan, R.O.C., 90, 97, 105-106, 117-118, 126-127, 130-131, 133,
144-145, 152-153, 156-157, 161-162, 174, 176-177, 357-358,
381-382, 384-385, 456-457, 577-578 (1985).
The present invention in its broadest aspect relates to the use
of the described herbal medicines and various mixtures thereof
to prevent and treat viral infections. More specifically, the
viral infections are those caused by HBV, HCV and HIV. The
antiviral mixtures according to the invention have been
described above as HHT888-4, HHT888-5, HHT888-45 and HHT888-54.
In addition, the single herb agents designated No. 4(2), No.
4(5), No. 5(5), No. 5(7), No. 5(8) and No. 5(11) have been shown
to have antiviral activity. These single herb agents have not
been shown by the prior art to have antiviral activity.
A more specific aspect of the present invention resides in the
discovery that HHT888-5 is efficacious in reducing hepatitis B
viruses in HBV carriers. An additional aspect of the invention
resides in the discovery that HHT888-45 is efficacious in
treating hepatitis C patients and returning their liver function
to normal.
The herb mixtures HHT888-4 and HHT888-5 and their aqueous
extracts have both been shown by the inventors herein to also
have antiretroviral activities against MuLV and HIV in vitro. In
addition, eleven (11) of the fifteen (15) single-herb components
of HHT888-4 and HHT888-5, i.e., No.4(2), No.4(3), No.4(4),
No.4(5), No.5(1), No.5(2), No.5(4), No.5(5), No.5(7), No.5(8),
and No.5(11) have shown anti-HIV activities by effectively
suppressing viral proliferation in HIV infected human peripheral
blood lymphocytes (PBLs).
There is further disclosed as a composition of matters, the herb
mixtures HHT888-4, HHT888-5, HHT888-45 and HHT888-54. As
described above, HHT888-54 is No.5(5) or its extract or active
principle and at least one single-herb herbal medicine or its
extract or active principle selected from the group consisting
of No.4(2), No.4(3), No.4(4), No.4(5), No.5(1), No.5(2),
No.5(4), No.5(7), No.5(8), and No.5(11). These compositions of
matter have not been described before and are unobvious.
There is further disclosed a method of treating viral infections
in a mammal, said method comprising administering to said mammal
from 0.4 to 120 g per day of at least one composition selected
from the group consisting of HHT888-4, HHT888-5, HHT888-45,
HHT888-54, No. 4(2), No. 4(5), No. 5(1), No. 5(2), No. 5(4), No.
5(5), No. 5(7), No. 5(8), No. 5(11) and their respective
extracts or active principles.
More specifically, there is disclosed a method for reducing the
viral load of humans infected with hepatitis B virus, said
method comprising administering to said human a therapeutically
effective amount of a composition comprising HHT888-5.
There is also disclosed a method for reducing the viral load of
humans infected with hepatitis C virus, said method comprising
administering to said human a therapeutically effective amount
of a composition comprising HHT888-45.
There is also disclosed a method of reducing the viral load of a
human carrier of the hepatitis B virus and a method of
preventing hepatitis B in a human, said method comprising
administering to said human a therapeutically effective amount
of a composition comprising No.5(5) and at least one selected
from the group consisting of No.5(1), No.5(2), No.5(3), No.5(4),
No.5(6), No.5(7), No.5(8), No.5(9), No.5(10), and No.5(11).
There is further disclosed a method of treating a hepatitis C
virus carrier and a method of treating or preventing hepatitis C
in a human, said method comprising administering to said human a
therapeutically effective amount of a composition comprising the
mixture of the single-herb herbal medicine No.5(5), its extract
or active principle and at least one single-herb herbal
medicine, its extract or active principle selected from the
group consisting of No.4(2), No.4(3), No.4(4), No.5(4), No.5(8),
and No.5(11).
Also disclosed is a method of treating hepatitis B in a human,
said method comprising administering to said human a
therapeutically effective amount of at least one composition
selected from HHT888-45 and HHT888-5.
There is disclosed a method of treating hepatitis B in a human,
said method comprising administering to said human a
therapeutically effective amount of at least one composition
selected from: 1) a mixture of the single herb medicine No.
5(5), its extract or active principle and at least one
single-herb herbal medicine, its extract or active principle
selected from the group consisting of No.4(2), No.4(3), No.4(4),
No.5(4), No.5(8), and No.5(11); and 2) a mixture of the
single-herb herbal medicine No.5(5), its extract or active
principle and at least one single-herb herbal medicine, its
extract or active principle selected from the group consisting
of No.5(1), No.5(2), No.5(3), No.5(4), No.5(6), No.5(7),
No.5(8), No.5(9), No.5(10), and No.5(11).
There is further disclosed a method for treating humans infected
with HIV, said method comprising administering to said human a
therapeutically effective amount of a composition comprising
HHT888-4.
There is disclosed a method for treating humans infected with
HIV, said method comprising administering to said human a
therapeutically effective amount of a composition comprising
HHT888-5.
There is disclosed a method for treating humans infected with
HIV, said method comprising administering to said human a
therapeutically effective amount of a composition comprising
HHT888-45.
There is disclosed a method for treating humans infected with
HIV, HBV and HCV said method comprising administering to said
human a therapeutically effective amount of a composition
comprising HHT888-54.
There is also disclosed a method for treating humans infected
with HIV, said method comprising administering to said human a
therapeutically effective amount of a composition comprising at
least one single-herb herbal medicine, its extract or active
principle selected from the group consisting of No.4(2),
No.4(5), No.5(1), No.5(2), No.5(4), No.5(5), No.5(7), No.5(8),
and No.5(11).
There is also disclosed a method of treating humans infected
with HIV, said method comprising administering to said human a
therapeutically effective amount of a composition comprising the
mixture of the single-herb herbal medicine No.5(5), its extract
or active principle and at least one single-herb herbal
medicine, its extract or active principle selected from the
group consisting of No.4(2), No.4(3), No.4(4), No.4(5), No.5(l),
No.5(2), No.5(4), No.5(7), No.5(8), and No.5(11).
The dosage of the compositions of the invention can range from
0.4 to 120 g per day for the mammal in need of therapy. One
skilled in the art will appreciate that depending upon the
weight of the individual and the progression of the viral
infection, that higher doses of the compositions will be
required. As the compositions according to the invention have
demonstrated virtually no side effects, high doses may be
initiated with reduction of dosage upon manifestation (i.e.,
reduction of viral load) of therapeutic effect. One skilled in
the art can tailor each dosage rate for a given individual
without undue experimentation. More specifically, the dosages
for a given composition can range from 0.4 to 25 g per day.
Preferably, the compositions are administered at least three (3)
times per day however, bolus administration will be effective.
More specifically, oral dosages of 5.5 g three (3) times a day
(total 16.5 g per day) of the herb mixture HHT888-5 have been
found to be effective to reduce HBV load in carriers. Oral
dosages of 2.7-5.7 g three times a day (total 8-17 g per day) of
the herb mixture HHT888-45 have been found to be effective to
return normal liver function to hepatitis C patients. Dosages as
high as 121 g per day for HHT888-5 and 63 g per day for
HHT888-45 have not evidenced serious side effects. It will be
appreciated that the dosages recited herein are for the herbal
medicine (extract deposited on ground plant) in dry form.
Further, extracts of the inventive compositions will increase
the concentration of the actives and therefore reductions in the
dosage levels will be realized. Dosages as low as 10% of those
recited herein for the inventive compositions are contemplated.
The preferred dosage for No. 5(5) to treat HCV infection is from
0.4 to 17 g per day.
The compositions of the invention are preferably administered
enterally, however, intravenous (i.v.) and/or intramuscular
(i.m.) administration is also contemplated herein. Those skilled
in the art will understand how i.v. and i.m. formulations can be
prepared and how the effective dosages can be obtained.
In the method according to this invention a mammal may be a
human or animal. The human may be an adult, child or infant.
Thus, for infants, an infant formula containing the hereinafter
described plant extracts or active principles will be effective
in treating the infants infected with HBV, HCV, or HIV. For
children and adults, a medical food or nutritional product, such
as milks and yogurts, containing the plant extracts or active
principles described herein will also be effective in treating
humans infected with HBV, HCV, or HIV.
The herbs used as starting materials for this invention may be
obtained from commercial sources as single-herb herbal medicines
which may be mixed, or extracted and concentrated, and placed in
compositions for the administration to a human. The plant
extracts, once isolated from the plant material, may be
concentrated and then placed in compositions for the
administration to a human. The active principles, once isolated
from the plant material or herbal medicine, may be concentrated
and then placed in compositions for the administration to a
human. The compositions of this invention may take a variety of
forms such as capsules, tablets, powder, candies, gels,
beverages, teas, nutritional products, and the like.
Also disclosed is a medicinal product produced by the process
comprising the steps of: (a) contacting comminuted plant
material selected from the group consisting of No.5(1) to
No.5(11), No.4(2) to No.4(5), and mixtures thereof, with water
to form an aqueous dispersion; (b) heating the aqueous
dispersion to about 100 DEG C. and holding at that temperature
for about 0.5 to about 3 hours; (c) separating the insoluble
plant material from the aqueous phase; and (d) concentrating the
solute contained in the aqueous phase. The concentrated solute
may be obtained through freeze drying, spray drying, evaporation
and ultrafiltration.
As described in more detail in the following examples, the
herbal compositions of the invention contain components that are
active against viruses in vitro and in vivo.
Most impressively, the clinical effects of HHT888-5 on hepatitis
B virus carriers are shown in Table 1 while the clinical effects
of HHT888-45 on type C hepatitis patients are shown in Table 6.
In a preferred embodiment, the herb mixtures, individual
single-herb herbal medicines, their water extracts and/or active
principles are incorporated into oral dosage forms such as
capsules, tablets, teas, powders, candies, candy bars,
beverages, nutritional products, and the like.
This application sets forth the data available on the present
discoveries and fully describes the compositions of matter,
their preparation, and clinical applications. These and other
aspects of the invention will become apparent to those skilled
in the art as a result of the following examples which are
intended as illustrative of the invention and not limitative.
BEST MODE FOR CARRYING OUT THE INVENTION
To acquaint persons skilled in the art with the principles of
the invention, the following Examples are submitted.
EXAMPLE 1
Preparation of Herb Mixtures
In the preparation of the herbal compositions according to the
invention, Chinese herbal medicines in single herb format were
obtained from commercial sources in powder form. The individual
single-herb herbal medicines were mixed in the appropriate
proportions to prepare each herb mixture.
The herb mixture HHT888-4 was prepared by mixing five (5)
single-herb herbal medicines No.4(1), No.4(2), No.4(3), No.4(4),
and No.4(5) at a ratio of 3:3:3:3:4 by weight. The herb mixture
HHT888-5 was prepared by mixing equal weights of eleven (11)
single-herb herbal medicines No.5(1), No.5(2), No.5(3), No.5(4),
No.5(5), No.5(6), No.5(7), No.5(8), No.5(9), No.5(10), and
No.5(11).
The herb mixture HHT888-45 was prepared by mixing four (4) to
six (6) single-herb herbal medicines No.4(3), No.4(4), No.5(4),
No.5(5), No.5(8), and No.4(2) at a ratio of 1:1:1:1:0-1:0-1 by
weight. The single-herb herbal medicine No.5(8) or No.4(2), or
both, were not used in some cases in HHT888-45 for initial
administrations. One of the two single-herb herbal medicines or
both were added later when needed to enhance the therapy. The
weight ratio of the single-herb herbal medicine No.4(2) in the
herb mixture HHT888-45 also varied case-by-case between 0.5 and
1 when used.
It is noted that a mixture of decoctions prepared individually
from the source plants of the single-herb herbal medicines or a
decoction prepared from the pre-mixed source plants of the
single-herb components of each herb mixture is well within the
specification of the herb mixture.
EXAMPLE 2
Preparation of Single-herb Herbal Medicines
The single-herb herbal medicine used to prepare the herb
mixtures has been described in the Prior Art section of this
application. The plant source from which each single-herb herbal
medicine is obtained was also listed in the Prior Art section.
More than one species or genus of medicinal plant may be used to
prepare the same herbal medicine as indicated in the plant
source list of that herbal medicine. For example, the herbal
medicine No.5(8) or FORSYTHIAE FRUCTUS may be prepared from
either one of the three (3) species of Forsythia genus plants,
i.e., Forsythia suspensa, Forsythia viridissima, Forsythia
koreana or mixtures thereof. The herbal medicine No.5(6) or
BAPHICACANTHIS RHIZOMA ET RADIX may be prepared from one of the
five (5) plants of different genus and species, i.e.,
Baphicacanthes cusia, Strobilanthes cusia, Isatis tinctoria,
Isatis indigotica, Polygonum tinctorium or mixtures thereof The
herbal medicines were prepared from their respective plant
sources as follows.
A suitable part or parts or the whole herb of a medicinal plant
was obtained, washed with cold water, dried and comminuted. The
plant materials were then extracted with boiling water on a
basis of 1 part by weight of plant material to approximately 5
to 10 parts by weight of water. The amount of water used should
at least cover the plant material in the extraction vessel.
Samples were boiled for 0.5 to one hour, but not in excess of 3
hours, in order to allow effective extraction of the desired
components. Shorter or longer heating would not substantially
affect the extraction, except the yield and cost. The aqueous
solution was separated from the plant material by filtration.
The aqueous solution may be freeze dried or spray dried, or
reduced in volume by heating with or without an applied vacuum.
The concentrate may then be spray dried or freeze dried or
absorbed by powdered material of the same plant material or
starch and thus the single-herb herbal medicine is prepared in
powdered form.
It is noted that a decoction prepared from a source plant of the
single-herb herbal medicine is well within the specification. A
decoction is the aqueous solution of the plant material prepared
by boiling the plant material in water as described above for
about 0.5 to one hour. The decoction may be directly consumed
after it is prepared and cooled to warm or ambient temperatures
or preserved with proper sterilization for later consumption.
Sterilization may be accomplished by microfiltration or heat.
EXAMPLE 3
Treatment of Hepatitis B Virus Carriers
Twenty-nine (29) HBV carriers with normal levels of serum liver
enzymes, glutamine oxalacetate transferase (SGOT) and glutamine
pyruvate transferase (SGPT), were treated with HHT888-5. Several
HBV carriers who had elevated SGOT and SGPT levels were first
treated with other remedies which returned their serum liver
enzymes to normal levels (8-40 unit/mL for SGOT and 5-35 unit/mL
for SGPT) but failed to reduce the HBV load. Treatment with
HHT888-5 then began. HHT888-5 was prepared as described in
Example 1 by mixing eleven (11) single-herb herbal medicines
which were obtained from a commercial source and were
manufactured following good manufacture practice (GMP)
guidelines. Consent of the patients was obtained before their
treatment began.
Patients were instructed to take the HHT888-5 three (3) times a
day. Each dose was 5.5 g. Each 5.5 g packet of the herb mixture
was mixed with warm water and consumed orally. Serum hepatitis B
surface antigen (HBsAg) titers of each patient were determined
at intervals as shown in Table 1 to monitor the progress of the
treatment. Serum HBsAg titer was determined using a
reverse-passive hemagglutination test as described herein: (1)
Instruction of "Taifu" Serodia-HBs Test Reagent for HBsAg
Detection, Taifu Pharmaceutical Co., Ltd., Taoyuan, Taiwan,
R.O.C.; (2) D. S. Chen & J. L. Sung, J. Formosan Med.
Assoc., 77, 263-270 (1978); and (3) T. Juji & T. Yokochi,
Japan. J. Exp. Med., 39, 615-620 (1969).
Table 1 shows the treatment results of the twenty-nine (29) HBV
carriers. Individual patients showed progressive improvement in
their disease state over the course of treatment, as indicated
by their HBsAg titer reductions and well being. Fourteen (14)
carriers (48%) whose HBsAg titers ranged from 20 to 81,920 were
significantly lowered (four (4) to 256-fold reductions, or from
positive to negative) after 35 to 964 days of treatment. Four
(4) carriers (14%) reduced their HBsAg titers from 20, 40, and
2,560 to negative (i.e., below 20 ng/mL detection level) after
56-153 days of treatment. Fourteen (14) carriers (48%) had no
significant change (two-fold titer decrease or increase or no
change) in HBsAg titers. That means these carriers had static
HBsAg titers during the course of the treatment (63-284 days).
One carrier (3%) had a slightly four-fold titer increase.
The above HHT888-5 treatment results compare very favorably with
the current interferon therapy. The response rates for
interferon therapy and HHT888-5 treatment to lower the HBsAg
titers in patients infected with HBV were comparable,
approximately 40% vs. 48%. The serum HBsAg clearance rates were
also comparable for both, 10-15% for interferon therapy and
approximately 14% for HHT888-5 treatment. Furthermore, the
interferon therapy is administered intramuscularly or
intravenously and with frequent adverse effects. The HHT888-5
treatment is administered orally (like drinking a tea) and no
apparent side effects were observed in all patients treated.
Oral administration is a much more convenient and better way
than intramuscular or intravenous administration. HHT888-5 can
thus be safely and conveniently consumed even on a long-term
basis to reduce or control HBV proliferation in HBV carriers and
hepatitis B patients.
TABLE 1
Clinical Effects of HHT888-5 on Hepatitis B Virus Carriers
HBsAg Titer Duration
Patient Before After (days)
1 40 negative
56
2 2560 negative
72
3 20 negative
153
4 20 negative
88
5 2560 80 53
6 1280 320 101
7 2560 1280 32
1280 399
320 964
8 2560 1280 79
640 412
9 20480 5120 53
10 20480 5120 60
11 40960 10240 35
12 81920 40960 74
10240 461
13 81920 20480 6
14 5120 2560 170
2560 245
1280 556
1280 832
15 160 80 284
16 320 160 198
17 640 320 276
18 1280 640 120
19 2560 1280 69
20 5120 2560 263
21 20480 10240 77
22 40960 40960 120
20480 210
23 160 160 227
24 320 320 79
25 640 640 157
26 1280 1280 69
27 40960 40960 137
28 5120 10240 63
29 160 640 121
When the HBV viral load in an HBV carrier can be reduced or
maintained at a sufficiently low level, the carrier is less
likely to progress to hepatitis, liver cirrhosis, liver cancer,
and death. Thus, HHT888-5 may be used to prevent and treat
hepatitis B, or even prevent liver cirrhosis or liver cancer
caused by HBV infection.
Since HHT888-5 was administered in the above treatments by
mixing the powder in water first and then consumed orally, the
water extract of HHT888-5 or a decoction from the herbal mixture
comprising the single-herb components or plants of HHT888-5 is
expected to be also effective and safe. Isolation of the active
components of HHT888-5 and its administration to humans would
also be efficacious in the treatment of HBV.
It is noted that HHT888-5 may be administered "as is" or in
other solid dosage forms such as capsules, tablets, tea bags,
candies, etc. The powdered herb mixture is typically mixed with
warm or cold water and consumed orally. Its extracts may be
administered as capsules, tablets, teas, candies, beverages,
nutritional products, and the like.
Dosages range from 1 to 5 treatments per day at about 1 to 120 g
per dosage depending upon the form and concentration of the
herbal medicine. The effective minimum dose of a composition as
a dried water extract of HHT888-5 is 1 g per day. The effective
minimum dose of a composition comprising a more purified active
component or components would be lower. The water extract of the
tested HHT888-5 constituted 19% of the herb mixture by weight.
Dosages of the herb mixture HHT888-5 as high as 120 g per day
have been accomplished without serious side effects.
EXAMPLE 4
Antiretroviral Testing of Herb Mixtures and their Water
Extracts
Two herb mixtures, HHT888-4 and HHT888-5, were tested for their
antiretroviral activities and found to be active against EMuLV
and HIV in the in vitro assay. Two in-vitro assays,
anti-Ecotropic Murine Leukemia Virus (anti-EMuLV) and anti-HIV,
were used to test the antiretroviral activities of the inventive
compositions.
The anti-EMuLV assay uses a large, enveloped, RNA-containing
retrovirus, EMuLV, which belongs to the same virus family as HIV
and has many characteristics that are similar to HIV.
1. Anti-Ecotropic Murine Leukemia Virus Assay
The assay contained two parts, cytotoxicity test and virus
suppression test. See QBI Protocol 39014 Final Report and QBI
Protocol 39016 Final Report, Quality Biotech, Camden, N.J., USA,
1992. Each sample was initially tested for its cytotoxicity to
the SC-1 indicator cells which were used for titration of
infectious EMuLV in a XC plague assay. See QBI protocol C30015,
Quality Biotech, Camden, N.J., USA. Each sample was dispersed in
a virus resuspension buffer (50 mM Tris, pH 7.8, 10 mM KCL, 0.1
mM EDTA) without the virus. The solution was then subjected to
the XC plague assay under the same conditions as those for the
determination of EMuLV titer. A sample was considered cytotoxic
if the indicator cells for the assay were less than 50%
confluent. A noncytotoxic sample concentration was chosen for
the virus suppression test.
In the virus suppression test, each sample was incubated with
EMuLV (strain AKV623, titer 2.2-4.2.times.10@5 PFU/mL) in a
virus resuspension buffer at 23-25 mg/mL (e.g., 100 mg/4.0 mL)
for 12-32 minutes. The treated virus suspension was pH adjusted,
if necessary, to within 6.8-7.2 and then tested for its titer in
the XC plague assay.
An aliquot (1.5 mL) was diluted in the cell culture medium to
the endpoint (10@0, 10@-1, 10@-2, 10@-3, 10@-4, 10@-5, 10@-6,
10@-7, and 10@-8 dilutions, or as appropriate). Each dilution
was vortexed to resuspend any particulates if present and
assayed in duplicate for infectious viral particles by the XC
plaque assay. A positive control (virus suspension without
treatment) and a negative control (cell culture medium, no
virus) were also analyzed concurrently to validate the assay.
Anti-EMuLV activity of the sample was expressed in log10
reduction of the EMuLV titer when compared to the positive
control. A sample with log10 to titer reduction greater than 0.5
is considered to be active.
HHT888-4 and HHT888-5 were initially tested "as is" and
exhibited good antiviral activities (1.0 to 1.4 log10 reduction
in viral titer) at 25 mg/mL and 12 minutes of incubation with
the virus at room temperature. They were then tested again with
a longer incubation time (32 minutes) with the virus at the same
concentration. Each sample was also tested for its soluble and
insoluble fractions in the above virus resuspension buffer to
see if any active component was water soluble. The soluble
portion was separated from the insoluble one by centrifuge at
room temperature and 10,000x g for 10 minutes. The soluble
fraction was divided into two aliquots, one 0.45-.mu.m filtered
and one unfiltered, and tested to see if residual particulates
have any effect on the activity.
Table 2 summarizes the anti-EMuLV activity test results. The
results confirmed that both HHT888-4 and HHT888-5 and their
soluble and insoluble fractions have anti-EMuLV activities. The
samples caused 1.0 to 2.6 log10 reduction in viral titer when
they were incubated with the virus at 23-25 mg/mL for 32
minutes. Microfiltration did not significantly affect the
activity of either soluble fraction.
2. Anti-Human Immunodeficiency Virus Assay
This assay also contained two parts, a toxicity test and a HIV
suppression test. The sample was mixed in a cell culture medium,
e.g., 50 mg in 1.00 mL. The mixture was vortexed and centrifuged
to separate the soluble from the insoluble. The supernate was
filtered through a 0.45-.mu.m filter and then diluted with cell
culture medium to appropriate concentrations for the assay. The
cell culture medium used in the assay was RPMI 1640 (pH
7.3.±.0.3) supplemented with 10% fetal calf serum, 2 mM
glutamin, 50 U/mL penicillin and 50 .mu.g/mL streptomycin.
The sample was tested for its cytotoxicity and/or cytostatic
activity towards the target cells, human peripheral blood
lymphocytes (PBLs). A lymphocyte proliferation assay was used
for the toxicity test, where a 100 .mu.L sample was incubated
with 100 .mu.L of a cell suspension of uninfected PBLs
(3.times.10@5 cells) under the same conditions as the HIV
suppression test. Lymphocyte proliferation was measured by a
colorimetric assay
TABLE 2
Anti-Ecotropic Murine Leukemia Virus Activity
Cytotoxicity* Anti-EMuLV Activity
Sample
Treatment
25 2.5
0.25 mg/mL
Log10 Titer Reduction**
HHT888-4
"as is" Yes No No 1.02 (90%)***
"as is" Yes No No 1.04 (91%)****
Soluble -- -- -- 1.74 (98%)****
Soluble, filtered
-- -- -- 1.59 (97%)****
Insoluble
-- -- -- 2.64 (99.8%)****
HHT888-5
"as is" Yes No No 1.35 (96%)***
"as is" Yes No No 2.10 (99.2%)****
Soluble -- -- -- 2.05 (99.1%)****
Soluble, filtered
-- -- -- 1.71 (98.1%)****
Insoluble
-- -- -- 1.72 (98.1%)****
*Sample was considered cytotoxic if the SC1 indicator cells for
the assay were less than 50% confluent.
**As compared to a working virus suspension with a titer of
2.2-4.2.times. 10@5 PFU/mL, or Log10 (PFU/mL) = 5.34-5.62. The
values in parentheses indicate percent reductions in viral titer
from the workingvirus suspension.
***Incubation time 12 minutes, at 25 mg/mL test level. The
activity may be caused by the sample, by microbial contaminant,
or by a nonspecific
physical interaction between the particles of the sample and the
virus, since the samples were not sterile filtered before assay.
****Incubation time 32 minutes, at 25 mg/mL test level for the
"as is" unfractionated samples. For soluble, soluble &
sterile filtered, and insoluble fractions, the test level was
equivalent to 23 mg/mL of its unfractionated sample.
(MTT-Test). See T. Mosmann, J. Immunological Methods, 65, 55-63
(1983). A sample concentration which results in .gtoreq.70% of
the control in Iymphocyte proliferation is considered to be
acceptable for the HIV suppression test.
In the HIV suppression test, HIV-1 infected PBLs were cultivated
in the presence of the sample for four (4) days as in the
toxicity test. See H. Ruebsamen-Waigmann, et al., J. Med.
Virology, 19, 335-344 (1986). The secreted viral core protein
p24 and/or viral RNA were determined as indicators for virus
proliferation status on day 3 and day 4 by an HIV-1 p24 capture
ELISA technique and an HIV-RNA dot blot hybridization technique,
respectively. The concentration of p24 synthesized by the HIV
infected cells was determined by Sandwich ELISA. A standard
preparation of recombinant p24 (MicroGeneSys, USA) was used for
calibration of the ELISA. See Ch. Mueller, et al., Fresenius Z.
Anal. Chem., 330, 352-353 (1988).
HIV-RNA synthesized in the infected cells was determined by a
nucleic acid hybridization technique. Cellular RNA was prepared
from the infected cells and analyzed by a dot blot hybridization
technique. The hybridization solution contained the P@32
-labeled DNA probe which comprised a 5.5 kilobase DNA fragment
of the HIV isolate D31. See H. v. Briesen, et al., J. Med.
Virology, 23, 51-66 (1987). This fragment covering the gag/pol
region of the virus is labeled with P@32 alpha-d CTP by
oligonucleotide labeling. Plus-strand RNA transcripts derived
from the gag/pol region of the viral isolate D31 were used as
the external standard for the hybridization. These "run-off"
transcripts were generated by means of the T7 polymerase
reaction from negatively polarized HIV-DNA under T7-promotor
control. The concentration of RNA transcripts was determined
spectrophotometrically. The hybridized probe was detected by
autoradiography and the processed autoradiograms were evaluated
densitometrically.
A positive control, a negative control, and an AZT control were
conducted concurrently to assure the validity of the HIV
suppression test. All tests were performed in triplicates, and
96-well round bottom microtiter plates were used for all assays.
A positive control was HIV-1 infected lymphocytes cultivated in
the presence of the cell culture medium without the sample. A
negative control was lymphocytes infected with a
heat-inactivated virus inoculum incapable of replication. These
"mockinfected" lymphocytes were cultivated and assayed in the
same way as the infected cells. The amount of viral protein
being present in the cultures solely due to the remaining
inoculum was thus determined as the background level. The amount
of viral protein p24 in the test sample and in the positive
control due to viral replication was then determined by the
respective p24 levels less the background level.
The amount of viral protein being present in the cultures
containing the sample due to viral proliferation was compared
with that in the positive control, i.e., the culture without the
sample. The % suppression of HIV proliferation was determined by
the difference in p24 levels between the positive control and
the sample, divided by the p24 level of the positive control,
and timed 100%.
The AZT control was conducted via HIV-1 infected lymphocytes
that were cultivated in the presence of azidothymidine (AZT) at
concentrations of 100, 10, 1 and 0.1 ng/mL, respectively. This
provided an estimate of the sensitivity of the lymphocytes
towards AZT, a known inhibitor of HIV-1 replication. The
suppression of HIV-1 proliferation caused by AZT in a
concentration of 10 ng/mL should be greater than 50% as compared
to the untreated positive control.
TABLE 3
Anti-HIV Activities of HHT888-4 and HHT888-5
HIV Suppression
Test p24 RNA
Sample
Concentration
Cytotoxicity*
Day 3
Day 4
Day 3
Day 4
HHT888-4
2.5
mg/mL
>46% 100% 100% 100%
100%
50 .mu.g/mL
85% 1% 6% -- --
HHT888-5
5.0
mg/mL
75% 100% 97% 99%
100%
50 .mu.g/mL
86% 0% 12% -- --
AZT 100
ng/mL
-- 99-100%
100% -- --
10 ng/mL
-- 85-98%
77-96%
-- --
1 ng/mL
-- 20-39%
8-12%
-- --
0.1
ng/mL
-- 0% 0-3% -- --
*Percent proliferation of control. HHT8884 was 46% at 5.0 mg/mL.
Both HHT8884 and HHT8885 were cytotoxic (<50% of control) at
25 mg/mL level.
Table 3 summarizes the cytotoxicity and the HIV suppression test
results of HHT888-4 and HHT888-5, as well as the AZT controls.
Both herb mixtures were active in suppressing HIV proliferation
in infected human lymphocytes at 2.5-5.0 mg/mL, but not at 50
.mu.g/mL (50-100 times diluted). The AZT controls from all sets
of anti-HIV assays herein and thereinafter exhibited the
expected activities and thus assured the validity of the tests.
At 2.5-5.0 mg/mL of HHT888-4 and HHT888-5, HIV proliferation in
infected human lymphocytes were essentially completely
suppressed: 97-100% suppression based on viral protein p24 and
99-100% suppression based on viral RNA determined on both day 3
and day 4 after treatment. The anti-HIV activity at 50 .mu.g/mL
was negligible, 0-12% suppression for both herb mixtures. The
activities could not be attributed to insoluble particulates
since they were filtered out by a 0.45-.mu.m filter before the
assay. The activities were not due to cytotoxicity. Repeat tests
on three lots of HHT888-4 showed 100% suppression at 2.5 mg/mL
on both day 3 and day 4 with acceptable cytotoxicity (71-100% of
control proliferation). Repeat tests on three lots of HHT888-5
at 2.5 mg/mL showed 93-98% suppression on day 3 and 89-99%
suppression on day 4 with acceptable cytotoxicity (85-91% of
control proliferation). Results of the repeat experiments are
shown in Table 4.
It is noted that Lot 3 of HHT888-4 or HHT888-5 was prepared by
mixing the respective single-herb components at equal proportion
by weight. Lot 3 of HHT888-5 was composed of nine (9)
single-herb components, excluding No.5(10) and No.5(11).
Water extracts of HHT888-4 and HHT888-5 from one to two lots
were further tested to see whether the active components were
extractable by water. Water extracts of HHT888-4 and 5 were
prepared by extracting 5 g of the powder with 25 mL of MilliQ
purified water twice. Each water suspension was vortexed for 1
minute, stood for 5 minutes, and vortexed again for 1 minute to
facilitate the extraction. The extract was separated from the
insoluble by centrifuge at 1,000-2,000 rpm for 20 minutes. The
supernate was transferred into a clean preweighed 50-mL
centrifuge tube, freeze dried, weighed, and tested for anti-HIV
activity.
The percent weight of material extracted was 17.3% for the first
25 mL extract and 10.8% for the second 25 mL extract of HHT888-4
(Lot 2). That was 14.2% for the first 25 mL extract and 4.6% for
the second 25 mL extract of HHT888-5 (Lot 2). The first (E1),
the second (E2) and the combined (E) extracts of HHT888-4 (Lot
2) were tested for anti-HIV activity. All the other extracts
were tested with the first and the second extracts combined. The
results are summarized also in Table 4.
TABLE 4
Anti-HIV Activities of HHT888-4 and HHT888-5 and their Water
Extracts
% Test HIV Suppression**
Sample Lot Weight
Concentration
Cytotoxicity*
Day 3
Day 4
HHT888-4
1 100% 2.5
mg/mL
>46% 100%
100%
2.5
mg/mL
98% 100%
100%
0.05
mg/mL
85% 1% 6%
2 100% 2.5
mg/mL
100% 100%
100%
3***
100% 2.5
mg/mL
71-79% 100%
100%
HHT888-4-E1
2 17% 1.0
mg/mL
98% 100%
96%
@ E2
2 11% 1.0
mg/mL
96% 100%
87%
@ E
2 28% 1.0
mg/mL
47% 100%
100%
0.5
mg/mL
78% 100%
100%
4 27 .±. 1% (2)
1.0
mg/mL
72% 100%
100%
1.0
mg/mL
100% 100%
93%
0.1
mg/mL
97% 34%
12%
0.02
mg/mL
82% 23%
2%
HHT888-5
1 100% 5.0
mg/mL
75% 100%
97%
2.5
mg/mL
89% 93%
91%
0.05
mg/mL
86% 0% 12%
2 100% 2.5
mg/mL
91% 94%
89%
3** 100% 2.5
mg/mL
44-85% 98%
99%
0.5
mg/mL
52-100%
0% 0%
HHT888-5-E
2 19% 1.0
mg/mL
91% 71%
26%
*Toxicity in percent of control proliferation.
**HIV suppression based on viral protein p24 levels.
***Composite of respective single herb components at equal
proportions.
No. 5(10) and No. 5(11) were not included in Lot 3 of HHT8885.
All three Lots of each of the herb mixtures were very active,
100% suppression at 2.5 mg/mL for HHT888-4 and 89-100%
suppression at 2.5-5.0 mg/mL for HHT888-5. The IC50 was between
0.05-2.5 mg/mL for HHT888-4 and between 0.5-2.5 mg/mL for
HHT888-5. IC50 is the concentration of the test substance at
which would cause 50% suppression of the viral proliferation.
The water extract of HHT888-4 showed very good activity: 93-100%
suppression at 0.5-1.0 mg/mL. The first (E1) and the second
water extract (E2) of Lot 2 exhibited comparable activities:
100% suppression on day 3 and 87-96% suppression on day 4 at 1.0
mg/mL. The IC50 of the water extract of HHT888-4 was between
0.1-0.5 mg/mL.
The water extract of HHT888-5 (lot 2) exhibited a substantially
lower activity: 71% suppression on day 3 which dropped to 26%
suppression on day 4 at 1.0 mg/mL. The main active component
apparently stayed behind in the insoluble fraction and was not
as easily extracted by water as that of HHT888-4 under the
aforementioned conditions. It is noted that the water extract of
HHT888-5 (Lot 2) constituted 19% by weight of the herb mixture.
The test concentration of the water extract of HHT888-5 (or
HHT888-5-E) at 1.0 mg/mL is equivalent to 5.3 mg/mL of HHT888-5
itself. HHT888-5 was tested very active at both 2.5 mg/mL
(93-98% suppression on day 3 and 89-99% on day 4) and 5.0 mg/mL
(100% suppression on day 3 and 97% on day 4).
The above results clearly demonstrated that both HHT888-4 and
HHT888-5 and their water extracts have in vitro antiretroviral
activities, more specifically anti-EMuLV and anti-HIV
activities. HHT888-5 has also been shown to be efficacious in
treating hepatitis B virus carriers, while HHT888-4 has not been
tested in vivo.
* EXAMPLE 5
Antiretroviral Testing of Individual Single-herb Herbal
Medicines
The individual single-herb components of HHT888-4 and HHT888-5
were tested for anti-HIV activity. Table 5 shows the test
results.
TABLE 5
Anti-HIV Activities of Single-herb
Components of HHT888-4 and HHT888-5
Test HIV Suppression**
Sample Lot Concentration
Cytotoxicity*
Day 3 Day 4
No. 4(1)**
1 2.5 mg/mL 98% 73% 50%
No. 4(2)
1 2.5 mg/mL 74-84% 92% 94%
No. 4(3)
1 2.5 mg/mL 75-78% 100% 100%
No. 4(4)
1 2.5 mg/mL 74-100% 100% 100%
No. 4(5)
1 2.5 mg/mL 41-79% 98% 92%
0.5 mg/mL 47-100% 0% 0%
No. 5(1)***
1 2.5 mg/mL 98% 73% 50%
No. 5(20
1 2.5 mg/mL 73-87% 18% 29%
No. 5(3)
1 2.5 mg/mL 89-100% 0% 0%
No. 5(4)
1 2.5 mg/mL 64% 100% 100%
1.0 mg/mL 69-91% 0% 0%
No. 5(5)
1 2.5 mg/mL 80-84% 93% 93%
No. 5(6)
1 2.5 mg/mL 94-100% 0% 0%
No. 5(7)
1 2.5 mg/mL 90-100% 50% 38%
No. 5(8)
1 2.5 mg/mL 32-59% 100% 100%
0.5 mg/mL 65-100% 0% 0%
No. 5(9)
1 0.5 mg/mL 24-78% 0% 0%
No. 5(10)
1 2.5 mg/mL 100% 65% 0%
No. 5(11)
1 2.5 mg/mL 100% 92% 74%
*Toxicity in percent of control proliferation.
**HIV suppression based on viral protein p24 levels.
No. 4(1) = No. 5(1)
All five (5) single-herb components of HHT888-4 exhibited
anti-HIV activities with various degrees: 73-100% suppression on
day 3 and 50-100% suppression on day 4 at 2.5 mg/mL. No. 4(3)
and No. 4(4) exhibited the best activity: 100% suppression at
2.5 mg/mL on both day 3 and day 4. No. 4(2) and No. 4(5) were
the next: 92-98% suppression on day 3 and 92-94% suppression on
day 4 at 2.5 mg/mL. No.4(1) exhibited a moderate activity: 73%
suppression on day 3 and 50% suppression on day 4 at 2.5 mg/mL.
No.4(5) exhibited a slight cytotoxicity (41-79% of control
proliferation) which was likely to contribute to the observed
activity with an ID50 between 0.5 and 2.5 mg/mL.
Three (3) of the eleven (11) single-herb components of HHT888-5:
No.5(4), No.5(5), and No.5(8) exhibited very good activities,
93-100% suppression of HIV proliferation on both day 3 and day 4
at 2.5 mg/mL. No.5(11) was the next: 92% suppression on day 3
and 74% suppression on day 4 at 2.5 mg/mL. Again, No.5(1), which
was the same as No.4(1), had a moderate activity: 73%
suppression on day 3 and 50% suppression on day 4 at 2.5 mg/mL.
No.5(2) and No.5(7) exhibited only marginal activities: 18-50%
suppression on day 3 and 29-38% suppression on day 4 at 2.5
mg/mL. No.5(10) exhibited a very slight activity: 65%
suppression on day 3 which dropped to 0% on day 4 at 2.5 mg/mL.
The remaining three (3) single-herb components, No.5(3),
No.5(6), and No.5(9) exhibited no activity at 0.5-2.5 mg/mL.
No.5(9) was not tested at 2.5 mg/mL level because of its
cytotoxicity: already 24-78% of control proliferation at 0.5
mg/mL.
Although No.5(4) and No.5(8) appeared to be slightly more active
than No.5(5) (100% vs. 93% suppression at 2.5 mg/mL), their
activities might be partially due to cytotoxicity (32-64% of
control proliferation at 2.5 mg/mL). This was supported by the
loss of activity (0% suppression) when tested at lower levels,
0.5-1.0 mg/mL, where the cytotoxicity was lower and more
acceptable to the assay.
EXAMPLE 6
Anti-HIV Testing of Medicinal Plant
The source plant of the single-herb herbal medicine No.5(5),
Aeginetia indica, was obtained from a local herbal store in
Taiwan and tested for its anti-HIV activity. This was to see
whether the activity can be reproduced in the herbal medicine
prepared directly from its source plant, instead of being
obtained from the commercial source.
The whole plant was washed with cold water, dried, comminuted,
and extracted with boiling water as described above in Example
2. The aqueous solution was separated from the plant material by
filtration. The aqueous solution was then reduced in volume by
heating. The concentrate was spray dried and absorbed onto
powdered material of the same plant material and thus was
prepared the herbal medicine in powder form, designated
hereinafter as raw No.5(5).
The powdered herbal medicine prepared from Aeginetia indica, or
raw No.5(5), was extracted with water at ambient temperature.
Two (2) 5.00 g samples were each extracted twice with about 40
mL of water each time in a separate 50-mL plastic centrifuge
tube by vortexing for one (1) minute, standing for ten (10)
minutes, and vortexing again for one (1) minute. The tubes were
centrifuged at 1500 rpm for twenty (20) minutes to separate the
extracts from the insoluble residues. The extracts were filtered
through a Whatman No.4 filter paper, freeze dried or nitrogen
dried, and weighed.
The above extraction of the raw No.5(5) with water
(pH.about.5.1) was repeated and the pH of the first extract was
measured to be 5.7. The first and the second extracts were
respectively separated from the residue, air dried, and weighed.
The percent weight of the extractable was determined to be
18.7.±.2.8% (n=2).
The first water extract of the raw No.5(5) was tested for
anti-HIV activity and found to be as active, 91% suppression on
day 3 and 97% suppression on day 4 at 1.0 mg/mL. Cytotoxicity
test showed that the extract was not cytotoxic at this level,
99% of control proliferation.
The above examples clearly demonstrate that both the herb
mixtures HHT888-4 and HHT888-5 are very active against HIV
proliferation. Complete (100%) or nearly complete (89-99%)
suppressions of HIV proliferation were achieved at 2.5 mg/mL.
The water extract of HHT888-4 is also very active. Complete
(100%) suppression of HIV proliferation was achieved at 0.5
mg/mL. The water extract of HHT888-5 is not as active as its
original mixture. It only suppressed 26-71% of HIV proliferation
at 1.0 mg/mL. Both HHT888-4 and HHT888-5 are not cytotoxic at
2.5 mg/mL. The water extracts of both HHT888-4 and HHT888-5 are
also not cytotoxic at 1.0 mg/mL.
HHT888-5 has been demonstrated to be effective and safe in
treating HBV infections in humans. That means, the active
principle or principles of HHT888-5 must be bioavailable in
humans through oral administration to cause the decrease of
hepatitis B virus in those patients treated, as indicated by the
decrease of their hepatitis B virus surface antigen (HBsAg)
exhibited in Example 3. In addition, Hozumi et al. provide
examples in U.S. Pat. No. 5,411,733 to support the belief that
substances exhibiting antiviral activity in vitro also possess
antiviral activity in vivo as described in the Prior Art
section. It is therefore logical to believe that HHT888-4 or
HHT888-5 and their water extracts or active principles should
also be effective for treating HIV infections in humans.
To test the belief, six (6) of the most anti-HIV active
single-herb components of HHT888-4 and HHT888-5 were selected to
treat hepatitis C patients caused by hepatitis C virus
infections. The logic is that both HCV and HIV are retroviruses.
Viral hepatitis C tends to become a chronic disease and is
therefore more suitable for the test of the treatment. If the
treatment works for patients infected with HCV, it will also
work for patients infected with HIV. Example 7 clearly
demonstrates the validity of this belief.
EXAMPLE 7
Treatment of Hepatitis C Patients
Six (6) of the most anti-HIV active single-herb components of
HHT888-4 and HHT888-5 were selected and mixed to treat hepatitis
C patients caused by hepatitis C virus infections. The six (6)
single-herb herbal medicines selected were No.4(2), No.4(3),
No.4(4), No.5(4), No.5(5), and No.5(8). No.4(5) was not included
although it exhibited a very good activity because it was
learned that the herb might have a certain unconfirmed toxicity.
The six (6) single-herb herbal medicines were obtained from a
commercial source and were manufactured following good
manufacture practice (GMP) guidelines. They were mixed according
to the desired ratio in various combinations and thus the herb
mixture HHT888-45 was prepared as further described in Example
1. Patients' consents were obtained before the initiation of
treatment.
Patients were instructed to take the herb mixture three (3)
times a day, 2.7-5.7 g each time. Unit dosages of the herb
mixture HHT888-45 were prepared in individual packets. Each unit
dose packet (2.7-5.7 g) of the herb mixture was mixed with warm
water and taken orally. All patients were treated with HHT888-45
containing No.4(3), No.4(4), No.5(4), and No.5(5). No.5(8) or
No.4(2) or both were added in HHT888-45 for the treatment of
some patients at the very beginning or during the course of the
treatment to enhance the effectiveness of the treatment. During
the course of the treatment, the daily dose of No.4(3), No.4(4),
No.5(4), and No.5(5) varied from two (2) to three (3) g each.
The daily dose of No.5(8) also varied from two (2) to three (3)
g when used. The daily dose of No.4(2) varied from 1.5 to two g
when used. The dose was varied according to the progress of the
disease.
Seven (7) viral hepatitis C patients were treated. Their serum
liver enzymes, SGOT and SGPT, were determined from time to time
by a local clinical laboratory during the course of the
treatment to monitor the progress of the disease. The SGOT and
SGPT were determined using an enzyme assay. See (1) Instruction
of Kyokuto TA-E Transaminase Assay Reagents, Permit No.
(62AM)0885, Kyokuto Pharmaceutical Industry Co., Ltd., Tokyo,
Japan, 1994; (2) Instruction of Yatrozyme TA-Lq
Transaminase-assay Reagent Solution (Enzyme Assay), Commodity
No. 817245 (RM163-K), Yatron Co., Ltd., Diayatron Co., Ltd.,
Tokyo, Japan; and (3) U. Lippi & G Guidi, Clin. Chim. Acta.,
28, 431-437 (1970).
The levels of serum GOT and GPT closely correlate with the
degree of cellular injury in the liver. These tests are widely
used in the diagnosis of liver diseases and as an indicator of
the liver function. The normal range for SGOT is 8-40 units/mL
and that for SGPT is 5-35 units/mL. Elevated SGOT and SGPT
levels usually indicate compromised liver functions.
The results of HHT888-45 treatment are shown in Table 6. All
seven (7) patients treated had their serum liver enzymes
returned from elevated levels (SGOT from 48 to 166 unit/mL and
SGPT from 41 to 291 unit/mL) to essentially normal range (SGOT
from 8 to 40 unit/mL and SGPT from 5 to 35 unit/mL) after 17 to
178 days of treatment. Thus, the liver functions of the patients
were returned to normal after consumption of the invention
composition.
The results clearly demonstrate that the herb mixture HHT888-45
is effective in treating hepatitis C patients. To accomplish
that, the causative hepatitis C virus needs to be eradicated or
reduced to a tolerable level. Since HHT888-45 components have
demonstrated very strong anti-HIV in vitro activity and several
of the components have demonstrated efficacy in reducing HBV in
carriers, the herb mixture will therefore be effective in
treating patients infected with HIV and HBV.
It is therefore an aspect of this invention that the antiviral
herbal medicines including the herb mixtures according to this
invention and their single-herb components at various
proportions and effective doses are effective in treating
hepatitis C, hepatitis B, and other retroviral diseases, such as
AIDS.
TABLE 6
Clinical Effect Of HHT888-45* On Type C Hepatitis Patients
SGOT**, unit/mL SGPT**, unit/mL
Duration
Patient
Before After Before After (days)
1 112 53 238 146 3
30 35 64
16 18 77
2 81 35 103 62 9
41 61 20
46 67 29
32 56 37
21 43 53
24 50 70
23 43 85
28 55 102
23 44 117
23 29 178
3 117 96 179 123 8
75 74 19
66 69 26
47 51 34
55 48 42
42 45 50
48 40 70
38 32 79
30 26 88
4 48 32 71 65 56
30 55 70
21 37 87
5 83 64 67 54 8
58 46 14
56 40 22
42 34 29
38 28 36
6 166 106 291 206 2
71 121 16
51 81 22
57 89 29
36 45 45
31 36 50
28 37 58
22 29 64
28 32 71
25 27 85
36 28 103
23 27 113
23 22 163
7 30 28 41 42 9
29 32 17
*Comprising mainly Nos. 4(3), 4(4), 5(4) and 5(5), and
occasionally 4(2) and 5(8).
**SGOT = serum glutamine oxalacetate transferase; normal range =
8-40 unit/mL.
SGPT = serum glutamine pyruvate transferase; normal range = 5-35
unit/mL.
Since the precise chemical composition and pharmacological
mechanism of the compositions of this invention have not yet
been eludicated, it is possible that the antiviral activity may
be due to a single herbal component, a combination of components
or the biological metabolite or derivative thereof.
Industrial
Applicability
The instant invention is directed in part, to the discovery that
specific medicinal plants or herbal medicines or their mixtures
possess surprising antiviral activities without causing damage
to the host cells. Further, the invention is directed to methods
of treating humans and mammals infected with viruses such as
HBV, HCV, or HIV. The data presented in this application clearly
demonstrate that the identified compositions possess antiviral
activity without toxicity to the host cells.
It can be concluded from the foregoing experiments that the herb
mixture designated HHT888-4 is effective in treating HBV
carriers and thus can be used to treat humans infected with HBV.
The reduction of viral load in HBV patients will thus result in
the prevention of HBV disease in the human and will also be
effective in the treatment of humans exhibiting HBV disease. The
clinical experiments have also shown that the herb mixture
HHT888-45 is effective in treating hepatitis C patients, and
thus is expected to be effective in treating hepatitis B
patients when administered alone or in combination with HHT888-5
or its antiviral single-herb components.
In addition, HHT888-5, HHT888-45, HHT888-54 and the individual
anti-HIV active single-herb components have demonstrated
efficacy in suppressing HIV proliferation in human cells.
Furthermore, HHT888-5, HHT888-45 and HHT888-54 have shown
efficacy in treating patients infected with HBV and HCV.
HHT888-5, HHT888-45, HHT888-4 and HHT888-54 are also effective
in treating humans infected with HIV, including HIV carriers and
AIDS patients.
The therapeutic effects described herein may be accomplished
through the administration of the herbal medicines "as is", or
as teas, decoctions, beverages, candies or other confections,
enteral liquid nutritional products such as infant formula and
adult nutritional products, medical foods, nutritional
supplements or neutraceuticals containing one or more of the
herbal medicines or their extracts or active principles. For
pharmaceutical preparations, one or more of the antiviral herbal
medicines or their extracts or active principles described above
may be administered in unit dosage forms such as capsules,
packets or tablets, with or without controlled-release
coating(s).
The medical community is constantly in search of methods and
products that will effectively treat viral infections,
especially methods and products for treating humans infected
with HBV, HCV, and HIV. The herb mixtures HHT888-4, HHT888-5,
HHT888-45, HHT888-54, the single-herb components, their
extracts, active principles, and products containing these
herbal compositions will be readily accepted by the medical
community as an additional tool in the prevention and treatment
of these devastating illnesses.
US6696094
Herbal pharmaceutical composition for treatment of
HIV/AIDS patients
Inventor(s): WU TZU-SHENG, et al.
The present invention provides a pharmaceutical composition for
treating patients with HIV infection. The pharmaceutical
composition is in the form of an intravenous injection solution
and optionally capsules. The pharmaceutical composition contains
fourteen (14) ingredients, i.e., diffuse hedyotis, bistort
rhizome, giant knotweed rhizome, Asiatic moonseed rhizome,
baical skullcap root, bovine biliary powder, milkvetch root,
barbary wolfberry fruit, sanqi, figwort root, Chinese
magnoliavine fruit, turmeric root-tuber, hawthorn fruit, and
Chinese angelica.
CROSS-REFERNCE
TO RELATED APPLICATION
The present application is a continuation-in-part (CIP) of U.S.
patent application Ser. No. 09/906,791 filed on Jul. 18, 2001,
now U.S. Pat. No. 6,455,078 which in turn claims the
priority of U.S. Provisional Application No. 60/240,963 filed on
Oct. 18, 2000. Both U.S. priority applications are herein
incorporated by reference.
FIELD OF
THE INVENTION
The present invention relates to a novel herbal pharmaceutical
composition and its use for treating patients with human
immunodeficiency virus (HIV) infection. The pharmaceutical
composition is in the form of an intravenous injection solution
or capsules and contains aqueous extracts of fourteen (14)
herbal ingredients, including diffuse hedyotis, bistort rhizome,
giant knotweed rhizome, Chinese magnoliavine fruit, Asiatic
moonseed rhizome, baical skullcap root, bovine biliary powder,
tumeric root-tuber, hawthorn fruit, sanqi, barbary wolfberry
fruit, figwort root, Chinese angelica, and milkvetch root. The
intravenous injection solution can be administered alone or
co-administered with the capsules. The present invention also
includes the methods for making the pharmaceutical composition
and for treating the patients with HIV infection.
DESCRIPTION
OF THE RELATED ART
The present invention is a continuation-in-part (CIP) of the
parent patent application, U.S. patent application Ser. No.
09/906,791, now U.S. Pat. No. 6,455,078, which is herein
incorporated by reference. In the parent application, novel
pharmaceutical compositions, which are particularly effective in
treating patients with human immunodeficeincy virus (HIV), were
described.
HIV infection causes acquired immunodeficiency syndromes (AIDS)
in humans which presents special problems to the medical
community. AIDS is a pandemic immunosuppressive disease which
results in life-threatening opportunistic infections and
malignancies. The retrovirus, HIV, has been isolated and
identified as the etiologic agent of this disease. HIV has been
detected in whole blood, plasma, lymphatic fluid, serum, semen,
vagina fluid, breast milk, tears, saliva, and central nervous
system tissue of infected patients. HIV can be transmitted
through sexual contact with an infected person, by sharing
needles or syringes (primarily for drug injection) with an
infected person, or, less commonly, through transfusions of
infected blood or blood clotting factors. Babies born to
HIV-infected women may become infected before or during birth or
through breast-feeding after birth. As of this time, complete
cure for HIV infection/AIDS has been diligently pursued by
scientists around the world, yet there has been no report of
absolute success.
Pharmaceutical compositions made from natural herbs have been
known and used for thousands of years to cure or ameliorate
various diseases and injuries. Some of these herbal compositions
have been disclosed for having medicinal properties for curing
or ameliorating symptoms associated with HIV infection. For
example, U.S. Pat. No. 5,178,865 discloses screening of
fifty-six (56) individual herbs and have found that ten (10) out
of 56 herbs exhibits anti-HIV activity in ex vivo experiments.
The ten (10) individual herbs include Coptis chineusis,
Ligusticum wallichii, Illicium lanceolatum, Isatis tinctoria,
Salvia miltiorrhiza, Erycibe obtusifolia, Acanthopanax
graciliatylus, Bostaurus domesticus, Inula helenium, and
Lonicera japonica. Both Bostaurus domesticus and Lonicera
japonica are further described to be able to combine with
Scutellaria baicaleusis to exhibit anti-HIV activity.
U.S. Pat. No. 5,837,257 discloses Chinese herbal medicines that
exhibit in vitro antiviral activities against murine leukemia
virus and HIV and for treatment of animals and humans infected
with HIV. In one of the preferred embodiments, the Chinese
herbal medicines contain hedyotis, scutellariae barbatae herba,
lonicerae flos, prunellae spica, and solani herba.
U.S. Pat. No. 5,989,556 discloses various herbal compositions
for treating viral infection. One of the herbal compositions
contains Aeginetiae Herba, Blechni Rhizoma, Lespedezae Herba,
Polygoni Cuspidati Rhizoma, Forsythiae Fructus, and Ligustri
Fructus. Another herbal composition contains Cirsii Rhizoma et
Radix, Breeae Radix, Baphicacanthis Rhizoma et Radix,
Phellodendri Cortex, and Bletillae Tuber. A third herbal
composition disclosed in the patent has Aeginetiae Herba,
Lonicerae Flos, Prunellae Spica, and Lespedezae Herba.
The present invention provides a novel pharmaceutical
composition for treatment of HIV, both in vitro and in vivo,
including treatment of AIDS patients, which are distinctively
different from prior art disclosures. This novel pharmaceutical
composition differs from the pharmaceutical compositions
described in the parent application for not containing red
ginseng (Radix ginseng rubra). The pharmaceutical composition of
the present invention is a natural Chinese medicine with little
or no side effects and has no toxicity.
SUMMARY OF
THE INVENTION
The present invention provides a pharmaceutical composition
which is particularly effective in treating HIV-positive or AIDS
patients. The pharmaceutical composition (in the name of
"HIVCIDE") is in the form of an injection solution (for
intravenous injection) or capsules. It contains aqueous extract
of the following herbal ingredients: (1) an entire plant of
Herba Hedyotidis diffusae (diffuse hedyotis); (2) a rhizome of
Rhizoma Bistortae (bistort rhizome); (3) a rhizome of Rhizoma
Polygoni Cuspidati (giant knotweed rhizome); (4) a ripe fruit of
Fructus Schisandrae (Chinese magnoliavine fruit); (5) a rhizome
of Rhizoma Menispermi (Asiatic moonseed rhizome); (6) a root of
Radix Scutellariae (baical skullcap root); (7) bovine biliary
powder; (8) a root tuber of Radix Curcumae (tumeric root-tuber);
(9) a ripe fruit of Fructus Crataegi (hawthorn fruit); (10) a
root of Radix Notoginseng (sanqi); (11) a ripe fruit of Fructus
Lycii (barbary wolfberry fruit); (12) a root of Radix
Scrophulariae (figwort root); (13) a root of Radix Angelicae
sinensis (Chinese angelica); and (14) a root of Radix Astragali
(milkvetch root). Examples of the aqueous solution for
extracting the pharmaceutical ingredients from the herbs
include, but are not limited to, water, ethanol, or a mixture
thereof. The preferred aqueous solution is water.
The herbal ingredients of HIVCIDE differ from that in the parent
application by not containing the root of Radix ginseng rubra
(red ginseng). Also, the weight ratio of the pharmaceutical
composition of the present invention is different from that of
the pharmaceutical composition of the parent application by
containing more quantity of diffuse hedyotis. The weight ratio
of the 14 ingredients listed above is about
4:3:1:2:1:1:0.1:1:2:1:3:2:1:2.
The aqueous extract of the herbal ingredients is further
filtered and condensed. The condensed aqueous extract is called
"herbal condensate" or "HIVCIDE condensate."The volume of the
herbal filtrate is about 1.4 fold of the herbal condensate.
The gram weight of the HIVCIDE for the purpose of determining
the dosage amounts for patient treatment as described
hereinafter is based on the weight of the "HIVCIDE condensate,"
not the weight of the original herbal ingredients.
The present invention also includes a method for preparing
HIVCIDE which includes the steps of: (1) grinding and mixing
diffuse hedyotis, bistort rhizome, giant knotweed rhizome,
Chinese magnoliavine fruit, Asiatic moonseed rhizome, baical
skullcap root, bovine biliary powder, tumeric root-tuber,
hawthorn fruit, sanqi, barbary wolfberry fruit, figwort root,
Chinese angelica, and milkvetch root to form a herbal mixture;
(2) boiling the herbal mixture in water to form a water extract;
(3) filtering the water extract to collect an herbal filtrate;
and (4) concentrating the herbal filtrate to form an herbal
condensate (also known as "HIVCIDE condensate"). The HIVCIDE
condensate is then either dissolved in a suitable solution to
form the intravenous injection solution or sprayed dried to form
herbal powders. The herbal powders are further mixed with a
binder, such as starch, and encapsulated.
The HIVCIDE injection solution contains about 0.1-1 g of the
HIVCIDE condensate per ml of the injection solution. When
administering to patients, the HIVCIDE is perferably diluted to
about 1:5 to 1:10 by volume of 5% glucose solution.
The present invention further contains a method for treating
patients with HIV infection which includes intravenous
administration of an effective amount of HIVCIDE to patients
with HIV infection. The preferred dosage is about 1 to 10 g of
HIVCIDE condensate, most preferably, about 2-6 g of HIVCIDE
condensate, per day.
Additionally, the present invention provides a method for
treating patients with HIV infection which comprises:
intravenous administering the HIVCIDE injection solution and
orally administering the HIVCIDE capsules to patients. The
preferred dosage of the HIVCIDE injection solution is about 1 to
10 g, most favorably, 2-6 g, of the HIVCIDE condensate per day,
preferrably in one injection.
The preferred dosage for HIVCIDE capsules is about 0.1 to 2 g of
HIVCIDE condensate per serving and for about 2-4 times a day.
BRIEF
DESCRIPTION OF THE DRAWING
FIG. 1 shows the HIV numbers in the five HIV positive patients
as described in EXAMPLE 5, infra. These five HIV-positive
patients were treated with daily intravenous injection of
HIVCIDE injection solution (Example 1, infra) for 3 months.
Patient #1 (, solid line with solid arrow) had received AZT and
DDI treatment for 3 months. Ten (10) days after the
discontinuance of the AZT and DDI treatment, patient #1 started
the HIVCIDE daily injection. Patient #5 (->, solid line with
open arrow) received the HIVCIDE treatment concurrently with
[alpha]-interferon. Results for Patents #2, #3 and #4 are
indicated as (dash line with solid arrow), -- (solid line with
solid circle) and - - - - (dash line with solid circle),
respectively.
DETAILED
DESCRIPTION OF THE INVENTION
Traditional Chinese medicine has been in existence for more than
two thousand years. It has a proven record of success for curing
many kinds of diseases. Traditional Chinese medicine utilizes a
variety of herbs and natural substances. Each herb/natural
substance has its unique characteristics. By combining and
balancing the unique characteristics of herbs, a doctor can
prescribe a formulation with enhanced medicinal activities and
with less or no toxicity by synergizing the medicinal effects
among various herbs, while in the meantime, cancelling out or
neutralizing the toxic effects of the herbs. This, in Chinese
herbal medicine, is regarded as to regulate between
negative/hypoactive characteristics ("yin") and
positive/hyperactive characteristics ("yang"),
Under the definitions set forth in the traditional Chinese
medicine, "yin" is defined as drugs which cure cold syndrome
(which itself has hot or warm property), and "yang" is defined
as drugs which cure heat syndrome (which itself has cold or cool
property).
The pharmaceutical combination of the present invention
comprises fourteen (14) ingredients, which are particularly
effective in treating patients with HIV. Out of the 14
ingredients, four (4) ingredients are the core ingredients which
contribute to the primary efficacy and healing effect of the
composition. They are: (1) diffuse hedyotis/spreading hedyotis
(Pharmaceutical name: Herba Hedyotidis diffusae; Botanical name:
Hedyotis diffusa Willd.); (2) bistort rhizome (Pharmaceutical
name: Rhizoma Bistortae; Botanical name: Polygonum bistorta L.);
(3) giant knotweed rhizome (Pharmaceutical name: Rhizoma
Polygoni Cuspidati; Botanical name: Polygonum cuspidatum Sieb.
et Zucc.), and (4) Chinese magnoliavine fruit (Pharmaceutical
name: Fructus Schisandrae Chinensis; Botanical name: Schisandra
chinensis (Turcz.) Baill., S. sphenanthera Rehd. et Wils.). The
core ingredients are functioned in clearing heat and toxic
substances while improving immune system and circulation, curing
symptoms of jaundice, and having beneficial effect on internal
organs.
There are six (6) additional ingredients that are used to
improve and balance the pharmaceutical effects activities
produced by the above named core ingredients. These six
ingredients also have toning effect and can improve blood
circulation in the liver. These six ingredients are: (1) Asiatic
moonseed rhizome (Pharmaceutical name: Rhizoma Menispermi;
Botanical name: Menisermum dauricum DC); (2) baical skullcap
root (Pharmaceutical name: Radix Scutellariae; Botanical name:
Scutellaria baicalensis Georgi); (3) bovine biliary powder
(Zoological name: Vesica Fellea Bovus); (4) tumeric root-tuber
(Pharmaceutical name: Radix Curcumae; Botanical name: Curcuma
wenyujin Y. H. Lee et Cl Ling); (5) Hawthorn Fruit
(Pharmaceutical name: Fructus Crataegi; Botanical name:
Crataegus pinnatifida Bge.); and (6) sanqui (Pharmaceutical
name: Radix Notoginseng; Botanical name: Panax notoginseng
(Burk.)).
Finally, there are additional five (4) ingredients which are
used to primarily provide nutrients and energy sources for
patients so as to expedite the recovery process. These
ingredients include: (1) barbary wolfberry fruit (Pharmaceutical
name: Fructus Lycii; Botanical name: Lycium barbarum L.); (2)
figwort root (Pharmaceutical name: Radix Scrophulariae;
Botanical name: Scrophularia ningpoensis); (3) Chinese angelica
(Pharmaceutical name: Radix Angelicae sinensis; Botanical name:
Angelica sinensis (Oliv.) Diels); and (4) milkvetch root
(Pharmaceutical name: Radix Astragali; Botanical name:
Astragalus membranaceus (Fisch.) Bge.). Among these ingredients,
milkvetch root (Radix Astragali) also has the capacity of
improving immunological functions of the body to fense off
diseases.
The pharmaceutical composition of the present invention differs
from the composition described in the parent application by not
containing red ginseng (Radix Ginseng Rubra). It was found that
the presence of red ginseng appeared to cause relapse of the
disease after termination of the use of the pharmaceutical
composition. Also, the pharmaceutical composition of the present
invention is particularly suitable for intravenous injection and
less effective for oral administration, which is also slightly
different from the pharmaceutical composition of the parent
application, where both capsules (in the form of HIVCIDE powder)
and intravenous injection are effective.
The pharmaceutical names, botanical or zoological names, family
names, common descriptions, and major ingredients of the herbs
used in the present invention are shown in Table 1.
TABLE 1
Herbs of the Present Pharmaceutical Composition
Botanical/
Pharmaceutical Zoological
Common Major
Name Name Family Description
Ingredients
Herba Hedyotidis Heydyotis Rubiaceae
heydyotis, hentriacontane,
Difffusae diffusa (Willd.)
oldenlandia stigmastatrienol,
Roxb.,
also ursolic acid,
known
as oleanolic acid,
[beta]-
Oldenlandia
sitosterol, [rho]-
diffusa coumaric,
[beta]-
sitosterol-D-
glucoside
Radix et Rhizoma Polygonum Polygonaceae
Giant Knotweed emodin, chryso-
Polygoni cuspidatum root
and phanol, rheic
Cuspidati Sieb. et Zucc.
Rhizome acid, emodin
monomethyl
ether,
polygonim, and
physcion-8-[beta]-D-
glucoside
Rhizoma Polygonum Polygonaceae Bistort
Rhizome n/a
Bistortae bistorta L.
Rhizoma Menispermum Menispermaceae
Asiatic n/a
Menispermi dauricum DC.
Moonseed
Rhizome
Radix Scutellaria Labiatae Baical
Skullcap baicalein,
Scutellariae baicalensis
Root baicalin,
Baicalensis
Georgi wogonin,
wogonoside,
neobaicalein,
oroxylin
aglucuronide,
camphesterol, [beta]-
sitosterol,
benzoic acid
Vesica Fellea
Bovine Biliary n/a
Bovus powder
Radix Astragali Astragalus Leguminosae
Milkvetch Root D-[beta]-asparagine,
membranaceus
2',4'-dihydroxy-
(Fisch.)
Bge. 5,6-
var.
dimethoxyisoflavane,
mongholicus.
calycosin,
(Bge.) Hsiao
or formononetin,
Astragalus
cycloastragenol,
membranaceus
astragalosides,
(Fisch.)
Bge. choline, betaine,
kumatakenin,
sucrose,
glucoronic acid,
[beta]-sitosterol
Fructus Lycii Lycium Solanaceae
Barbary betaine, carotene,
barbarum L.
Wolfberry Fruit physalien,
thiamine,
riboflavin,
vitamin C, [beta]-
sitosterol,
linoleic acid
Radix Panax noto- Araliaceae
San-chi, Arasaponin A,
Notoginseng ginseng (Burk.)
notoginseng, arasaponin B,
F.H. chen, P.
Tian qi, Shen san dencichine
pseudoginseng
qi
Wall, P. sanchi
Hoo.
Radix Scrophularia Scrophulariaceae
Figwort Root, 1-asparagine,
Scrophulariae ningpoensis
Scrophularia oleic acid,
Ningpoensis Hemsl.
orS. linoleic acid,
buergeriana stearic
acid,
Miq. carotene
Fructus Schisandra Magnoliaceae
Chinese sesquicarene, [beta]-
Schisandrae chinensis
Magnoliavine bisabolene, [beta]-
Chinensis (Turcz.) Baill.,
Fruit, schisandra chamigrene, [alpha]-
S. sphenanthera
fruit ylangene,
Rehd. et
Wils. schizandrin,
pseudo-[gamma]-
schizandrin,
deoxyschizandrin,
schizandrol,
citral,
stigmasterol,
vitamin C,
vitamin E
Tuber Curcumae Curcuma Zingiberaceae
Turmeric Root- d-camphene, d-
wenyujin Y. H.
tuber, curcuma camphor, 1-[alpha]-
Lee et C.
Ling., curcumene,
1-[beta]-
or
Curcuma curcumene,
Longa L.,
or curcumin,
Curcuma
demethoxycurcumin,
aromatica
bisdemethoxy-
Salisb.,
or curcumin,
Curcuma turmerone, ar-
zedoaria
Rosc., turmerone,
or
Curcuma carvone,
[rho]-
kwangsiensis
tolylmethyl-
S. G. Lee et
C. carbinoldiferuloyl-
F.
Liang methane
Fructus Crataegi Crataegus Rosaceae
Hawthorn Fruit crategolic acid,
pinnatifida
Bge.; citric acid,
C.
pinnatifida tartaric
acid,
Bge. var.
major flavone, sugars,
N.E. Br. or
C. glycosides,
suneata Sieb.
et vitamin C
Zucc.
Radix Angelicae Angelica Umbelliferae
Chinese butylidene
Sinensis sinensis (Oliv.) Angelica
root, phthalide, Diels tang-kuei ligustilide,
n-butylidene-phthalide, sequiterpenes, carvacrol,
dihydrophthalic anhydride, sucrose, vitamin B12, carotene,
[beta]- sitosterol
Diffuse hedyotis or spreading hedyotis (Herba Hedyotidis
Diffusae) belongs to the family of Rubiaceae. The entire plant
is used as an herbal medicinal component. The herb has no
toxicity. The herb is harvested in summer and autumn in mainland
China and in late spring or early winter in Taiwan. In "Materia
Medica" (Chinese Herbal medicine), compiled and translated by
Dan Bensky & Andrew Gamble, diffuse hedyotidis clears heat
and resolves dampness by promoting urination. It is particularly
useful for relieving hot painful urinary dysfunction and
damp-heat jaundice. Diffuse hedyotidis is the major ingredient
in the present herbal pharmaceutical composition which
contributes to the medicinal effect on liver diseases and HIV.
Bistort rhizome (Rhizoma Bistortae) is the dried rhizome of the
plant Polygonum bistorta L. It belongs to the family of
Polygonaceae. Bistort rhizome has moderate cool property
(meaning that bistor rhizome is an "yang" herb). It can be used
to remove toxic heat, to promote the subsidence of swelling and
to stop bleeding.
Giant knotweed rhizome (Radix et Rhizoma Polygoni Cuspidati) is
the dried rhizome and root of polygonum cuspidatum Sieb. et
Zucc. It belongs to the family of Polygonaceae. The plant is
grown throughout China, especially Jiangsu, Zhejiang, Anhui,
Guangdong, Guangxi, Sichuan, and Guizhou provinces. The plant is
harvested in spring and autumn. Giant knotweed rhizome is
normally used to dispel damp, to eliminate blood stasis and
alleviate pain, to relieve cough, and to resolve phlegm.
Chinese magnoliavine fruit (Fructus Schisandrae) is the dried
ripe fruit of Schisandra chinensis (Turcz.) Baill. or Schisandra
sphenanthera Rehd. et Wils. It belongs to the family of
Magnoliaceae. The former, the best of its kind, is produced in
northern parts of China and is habitually called "Northern
schisandra fruit"; the latter is commonly referred to as the
"Southern schisandra fruit" as it is produced in the southern
parts of China. Both kinds can be used for the pharmaceutical
preparation of the present invention. The fruit is collected in
autumn and dried under the sun after removing the fruit stalks.
Chinese magnoliavine fruit is generally used to arrest
disharges, replenish qi, promote fluid secretion, tonify the
kidney, and induce sedation. Chinese magnoliavine fruit can also
decrease the level of GPT (glutamate-pyruvate transaminase) in
patients with hepatitis.
Asiatic moonseed rhizome (Rhizoma Menispermi) is the dried
rhizome of Menispermum dauricum DC. It belongs to the family of
Menispermaceae. Asiatic moonseed rhizome has cool property. It
can be used to remove toxic heat and relieve rheumatic pains.
Baical skullcap root (Radix Scutellariae) is the dried root of
Scutellaria baicalensis georgi. It belongs to the family of
Labiatae. The plant is produced in the provinces of Hebei,
Shanxi, Inner Mongolia, etc., and collected in spring or autumn.
Baical skullcap root is used to remove damp-heat, counteract
toxicity, arrest bleeding, and prevent abortion, in patients.
Bovine biliary powder is the gallbladder of the cow, Vesica
Fellea Bovus. It can clear heat and alleviate spasms.
Turmeric root-tuber (Radix Curcumae) is the dried root tuber of
Curcuma wenyujin Y. H. Lee et C. Ling., or Curcuma Longa L., or
Curcuma aromatica Salisb., or Curcuma zedoaria Rosc., or Curcuma
kwangsiensis S. G. Lee et C. F. Liang. The herb is mainly
produced in Sichuan, Zhejiang, Guangdong, and Guangxi provinces
in China, and harvested in winter or spring, washed clean after
the removal of the hairy rootlets, boiled thoroughly, and dried
in the sun. It belongs to the family of Zingiberaceae. Turmeric
root-tuber tastes bitter and had cool property. It can be used
to clear heat, alleviate spasms and chest pain, and resolve
phlegm.
Hawthorn fruit (Fructus Crataegi) is the dried ripe fruit of
Crataegus pinnatifida Bge. var major N. E. Br., or Crataegus
pinnatifida Bge., or Crataegus cuneata Sieb. It is produced
primarily in Henan, Jiangsu, and Shandong provinces of China. It
is harvested in autumn, sliced, and dried in sunlight. It
belongs to the family of Rosaceae. Hawthorn fruit is normally
used to stimulate digestion and promote the functional activity
of the stomach. It can also improve the normal blood flow and
dissipate blood stasis.
Sanqi, or San-chi, (Radix Notoginseng) belong to the family of
Araliaceae. Sanchi (Sanqi) is the dried root of Panax
notoginseng (Burk.) F. H. Chen. The plant is also known as P.
pseudoginseng Wall and P. sanchi Hoo. The plant grows in Yunnan,
Guangxi, Sichuan, Guizhou, and Jiangxi provinces of China, and
is harvested in the autumn or winter of the third or seventh
year, either before the flowers bloom (better) or after the
fruit is ripe. H. Gao et al., Pharmaceutical Research, (1996)
13(8): 1196-1200, disclose that polysaccharides from Panax
notoginseng (San-Chi) have immuno-stimulating activities in
vitro.
Barbary wolfberry fruit (Fructus Lycii) is the dried ripe fruit
of Lycium barbarum L. It belongs to the family of Solanaceae.
The plant is mainly produced in Ningxia, Gansu, and Qinghai
provinces of China. It is harvested in summer and autumn. It
nourishes and tonifies the liver and kidneys. It can also
replenish vital essence and improve eyesight.
Figwort Root (Radix Scrophulariae) is the dried root of
Scrophularia ningpoensis Hemsl. It belongs to the family of
Scrophulariaceae. The herb is chiefly produced in Zhejiang and
Sichuan provinces of China and harvested in winter when the part
of the plant above-ground has withered. The roots are piled and
dried in sunlight alternately until the inside becomes black and
then sliced for use. Figwort root can reduce heat from blood. It
als has nourishing capacity and can counteract toxicity.
Chinese angelica (Radix Angelicae Sinensis) is the dried root of
Angelica sinensis (Oliv.) Diels. It belongs to the family of
Umbelliferae. The herb is mainly produced in Gansu and Shanxi
provinces of China. It is harvested in late autumn, smoked dry
on slow fire after getting rid of the rootlets, sliced, or
stir-baked with wine. Chinese angelica can enrich blood, promote
blood circulation, regulate menstruation, relieve pain, and
relax bowels.
Milkvetch root (Radix Astragali) is the dried root of Astragalus
membranaceus (Fisch.) Bge. var. mongholicus. (Bge.) Hsiao or
Astragalus membranaceus (Fisch.) Bge. It belongs to the family
of Leguminosae. The herb is mainly produced in Shanxi, Gansu,
Heilongjiang, and Inner Mongolia of China. The plant of
four-year old or older is harvested in spring or autumn.
Milkvetch root can promote discharge of pus and the growth of
new tissue.
The pharmaceutical composition of the present invention is
suitable for preparation in a scale typical for pharmaceutical
industry as well as for smaller measure.
In the process for making the pharmaceutical composition of the
present invention, the individual herbal components are
pretreated according to the common procedures. The herbs are cut
and put in a container with water to boil and simmer twice. The
first time of simmering takes two hours, the solution is
collected, and water is added for the second time of simmering
for about 1.5 hour. The solutions from the simmering steps are
collected and then filtered by a sieve/filter. The filtrate is
then condensed from about 1.4 fold by volume to about 1.0 fold
by volume. The resulting condensate is pasty-like. Optionally,
the herbs can be simmered, filtered and condensed again. The
second condensate is then mixed with the first condensate to
form the "HIVCIDE condensate," which becomes the basic content
of the HIVCIDE injection solution. When used, the HIVCIDE
injection solution is further diluted 1:5 to 1:10 by volume with
a suitable solution. The gram weight of the HIVCIDE injection
solution described herein is referred to the weight of the
"HIVCIDE condensate."
The following example is illustrative, but not limiting the
scope of the present invention. Reasonable variations, such as
those occur to reasonable artisan, can be made herein without
departing from the scope of the present invention.
EXAMPLE 1
Pharmaceutical Preparation of HIVCIDE
The kinds and amounts of the herbal ingredients used in the
process of making the pharmaceutical composition of the present
invention are described in Table 1. The pharmaceutical
composition is called "HIVCIDE," which is named after
"HIV-killer" ("-cide" means "killer"). HIVCIDE was formulated as
injection solution and capsules.
Table 2. Ingredients Used in Making HIVCIDE Injection Solution
and Capsules
Component Amount (kg)
Diffuse hedyotis 3.32
Bistort Rhizome 2.49
Giant Knotweed root and 0.83
Rhizome
Asiatic Moonseed Rhizome 0.83
Baical Skullcap Root 0.83
Bovine Biliary powder 0.083
Milkvetch Root 1.66
Barbary Wolfberry Fruit 2.49
Sanchi 0.83
Figwort root 1.66
Chinese Magnoliavine Fruit 1.66
Turmeric Root-tuber 0.83
Hawthorn fruit 1.66
Chinese Angelica 0.83
(1) Quality Controls of Raw Materials
Quality control tests carried out for each individual raw
material were according to conventional methods used in the
herbal pharmaceutical field, which include, but are not limited
to, physical appearance, loss on drying, total ash, acid
insoluble ash, alcohol extracts, water extracts, TLC, HPLC,
heavy metals, microbial counts and residual pesticides. Bovine
biliary powder was tested for appearance, TLC and general
chemistry.
(2) Manufacturing Process
The individual herbal components were pretreated according to
common procedures. The herbs were weighed according to Table 2.
A flowchart of the manufacturing process for making HIVCIDE
injection solution is provided in Table 3:
TABLE 3
Manufacturing Process For Making HIVCIDE
Manufacturing Process Quality Control Procedure
Raw Herbs Delivered Quality Control of
Raw Herbs:
Physical Appearance
<img class="EMIRef" id="041702268-00010000" />
Preparation
(cutting, drying, etc.)
<img class="EMIRef" id="041702268-00020000" />
Quality Control of Raw Herbs: Physical Appearance Loss on
Drying Total Ash Acid Insoluble Ash Alcohol Extracts Water
Extracts TLC HPLC Heavy Metals Microbial Residues Pesticide
Note: Bovine biliary powder (also known as Bovis Bezoar) is only
tested for appearance, TLC and General Chemistry
1<st >Extraction
Parameter Set:
1. Dial Set Temperature at 95[deg.] C.
In-Process Quality Control:
with the Acceptable Range of
(take 10-15 g as test sample)
90[deg.] C.-100[deg.] C.
Concentration of Solid
2. Dial Set Steam Pressure at Content
2 kg/cm<2>. Concentration
of Water
3. Dial Read Out the Lid Pressure Content
at 0.2-0.4 kgf/cm<2 >Range.
4. Extract for 45 minutes.
<img class="EMIRef" id="041702268-00030000" />
1<st >Concentration
Parameter Set:
1. Dial Read Out Vacuum at -60≈76
cmHg.
2. Dial Read Out Temperature at
40[deg.] C. ± 5[deg.] C.
3. Process for 40 minutes.
<img class="EMIRef" id="041702268-00040000" />
Raw Herbs Add
Water 350 L ± 10%
2<nd >Extraction
Parameter Set:
1. Dial Set Temperature at 95[deg.] C. with
the Acceptable Range of
90[deg.] C.-100[deg.] C.
2. Dial Set Steam Pressure at
2 kg/cm<2>.
3. Dial Read Out the Lid Pressure at
0.2-0.4 kgf/cm<2 >Range.
4. Extract for 45 minutes
<img class="EMIRef" id="041702268-00050000" />
2<nd >Concentration
Parameter Set:
1. Dial Read Out Vacuum at -60-76
cmHg.
2. Dial Read Out Temperature at
40[deg.] C. ± 5[deg.] C.
3. Process for 40 minutes.
<img class="EMIRef" id="041702268-00060000" />
Combined Concentrated Extracts and
Pour into the Stainless Container
Final Yield: 6.1 kg ± 10%
Spraying Silo
Parameter Set:
1. Dial Set Temperature at 60[deg.] C.
2. Pre-heat for 15 minutes.
<img class="EMIRef" id="041702268-00070000" /
Fluid Bed Dryer
1. Add 3.9 kg of Starch (adj. Base on
water content of the starch).
2. Dial Set In-Temperature at 60[deg.] C.;
Out-Temperature at 50[deg.] C.
3. Pre-heat for 10 minutes.
4. Process for target of 2 hours ± 20
min. until LOD <5%.
<img class="EMIRef" id="041702268-00080000" />
Sievinig
Parameter Set:
1. Dial Read out Temperature
at 23 ± 4[deg.] C.
2. Dial Read out RH at 50 ± 10%.
3. Sieve with #100 followed by #80.
<img class="EMIRef" id="041702268-00090000" />
HIVCIDE Powder Quality Control of Drug
Substance:
Physical Appearance
Loss on Drying
Total Ash
Acid Insoluble Ash
Alcohol Extracts
Water Extracts
TLC
HPLC
Heavy Metals
Microbial
Residues Pesticide
Stability
The individual herb was cut into small pieces and thoroughly
mixed together. The mixed herbs were placed in bags with
sufficient space to spread out. The bags were placed in an
extractor with 350 L (±10%) of water and soaked for about 60±10
min. The herbs were first extracted at 95±5[deg.] C. under steam
pressure of 2 kg/cm<2 >and lid pressure of 0.2-0.4
kg/cm<2 >for 45 min. The water extract was collected into
a concentrator and concentrated at 40±5[deg.] C. under vacuum of
-60 to -76 cmHg for 40 min to form the first concentrate.
The herbs in the bags were recovered from the extractor, placed
into another 350 L (±10%) of water and extracted again at
95±5[deg.] C. under steam pressure of 2 kg/cm<2 >and lid
pressure of 0.2-0.4 kgf/cm<2 >for 45 min. The extract from
the second extraction was collected into a concentrator and
concentrated at 40±5[deg.] C. under vacuum of -60 to -76 cmHg
for 40 min to form the second concentrate.
The first and second concentrates were combined and poured into
a stainless container. The total weight of the combined
concentrates was about 6.1 kg±10%. The combined 1<st >and
2<nd >concentrates were called the "HIVCIDE condensate."
For the HIVCIDE injection solution, about 0.1 to 1 g, preferably
0.4 g of the "HIVCIDE condensate" was dissolved in about 1 ml of
the injection buffer. About 5 ml of the injection solution was
poured into an ampoule.
For the HIVCIDE powders (which were packaged into HIVCIDE
capsule), about 3.9 kg of starch (adjustable based on the water
content of the starch) were added to the HIVCIDE condensate and
spray dried in a fluidized bed setting at in-temperature of
60[deg.] C. and out-temperature of 50[deg.] C. for approximately
120±20 min until LOD (limit of detection)<5%.
The resultant powders were passed through a 100-mesh sieve and
then a 80-mesh sieve. The final yield of the HIVCIDE powders
were about 9.5≈10.5 kg. The HIVCIDE powders were further
packaged into capsules. Any conventional capsules, including,
but not limited to, natural gelatin, pectin, casein, collagen,
protein, modified starch, and polyvinyl pyrrolidone, were
suitable for encapsulation.
There were two dosage forms of HIVCIDE capsules: A 500 mg of the
HIVCIDE capsule, which contained about 305 mg of the "HIVCIDE
condensate" and about 195 mg of starch; and a 220 mg of the
HIVCIDE capsule, which contained about 134 mg of the "HIVCIDE
condensate" and about 86 mg of starch.
(3) In-Process Quality Controls
After the extracts were concentrated, a 10-15 g sample was
collected and the concentrations of the solid content and the
water content were determined by methods described in US
Pharmacopoeia, China Pharmacopoeia, and/or Japanese
Pharmacopoeia.
EXAMPLE 2
Acute Toxicity Study of HIVCIDE in Animals
Purpose:
The following experiment was conducted at the Toxicology
Laboratory of the Institute of Labor, Health, and Occupational
Disease of Heilungkiang Province in China to examine acute
toxicity of the HIVCIDE during intravenous injection in animals.
Methods:
Experimental animals were Japanese big-ear white rabbits
obtained from the Animal Center of Haerbin Medical University in
Haerbin, Heilungkiang Province, China. These rabbits were
characterized by the obvious blood vessels on ears which
facilitates the operation of injection during the experiments.
Ten (10) rabbits were obtained including six (6) males and four
(4) females, each weighing between 1900 g to 3000 g.
The rabbits were randomly divided into two (2) groups, five
rabbits in each group including two (2) females and three (3)
males. The HIVCIDE injection solution was intravenously injected
into the two groups of rabbits through the veins on their ears
at dosages of 10 g and 15 g of HIVCIDE condensate per kg of
rabbit body weight, respectively.
The HIVCIDE concentration was about 1 g/ml, so that the higher
dosage group was about 15 ml/kg, which was roughly equal to a
sixty (60) kg-weighted adult treated with 900 ml of the HIVCIDE
injection solution.
The rabbits were observed for behaviour continuously for a
period of two (2) weeks after intravenous injections.
Observation was conducted hourly at day 1; during the following
days, observation was conducted four to six (4-6) times per day.
At the end of the observation period, rabbits were sacrificed
and dissected to examine the eyes, liver, lung, and spleen to
determine adverse effects.
Results:
No abnormal behavior was found in the rabbits during the entire
observation period. All animals exhibited normal body weight
gains. No organ abnormality was observed. After sacrifice and
dissection of the animals, inspection of the eyes, liver, lung,
and spleen showed no extraordinary syndromes. The results
indicate that, when comparing with the general acute toxicity
index, the HIVCIDE injection solution showed no symptoms of
acute or chronic toxicity.
Conclusion:
The intravenous administration of HIVCIDE injection solution up
to 15 g/kg was well-tolerated, which was approximately equal to
injection of 900 ml of HIVCIDE into a 60-kg of human. The
HIVCIDE demonstrated no acute toxicity in rabbits.
EXAMPLE 3
Effects of the HIVCIDE on HIV in Cell Cultures
Purpose:
The following experiment was to determine the effectiveness of
the HIVCIDE injection solution gainst HIV in cell cultures.
Methods:
MT4 cells were cultured in HIV-1 suspension liquid of 100 TCID50
in a 96-orifice culture plate. MT4 is a human T-cell
lymphotropic virus type 1-transformed cell line. The culture
condition was set at a temperature of 37[deg.] C. and under 5%
CO2. The duration of the culture was seven (7) days.
The HIVCIDE injection solution of the present invention was
added to the cultural wells at various concentrations. The
morphology of the MT4 cells was observed under microscope by
conventional methods.
Results:
No pathological changes of MT4 cells were observed in the
cultural wells where the HIVCIDE injection solution was added to
in adequate concentrations. The inhibition of the pathological
changes of MT4 cells indicated that the HIVCIDE injection
solution had inhibitory effect on pathological changes of the
cultured cells caused by HIV.
The effective concentration of the HIVCIDE injection solution
for inhibition of the pathological changes of MT4 cells was more
than 12.5 mg/ml. To achieve a 50% of inhibition, the
concentration of the HIVCIDE herbal composition was 25 mg/ml.
Conclusion:
The HIVCIDE injection solution was effective in inhibiting
pathological changes in cells caused by HIV-1 in vitro.
EXAMPLE 4
A Case Study on an HIV-Patient Treated with HIVCIDE
Purpose:
The following clinical trial was conducted in the Infectious
Disease Hospital in Shanghai, China to test the effectiveness of
the herbal composition of the present invention in treating an
HIV-infected patient.
Methods:
A fifty-year Chinese male patient diagnosed with HIV infection
complicated by herpes zoster was treated with anti-virus
regimens by the combination of western medicine and the herbal
composition of the present invention during hospital stay.
Results:
The patient was confirmed of HIV-infection by Rapid Agglutinin
Assay. At the time of the initial diagnosis, the patient showed
no symptoms. Ten months after the initial diagnosis, the patient
quickly developed an herpetiform rash over the front of the left
side of the check extending over the nick, the shoulder, and the
upper left arm. The patient was then admitted into the Hospital
shortly thereafter.
At the hospital, the result of the physical examination was
normal except the skin rash. The pathology tests confirmed
normal renal function. The functional tests of the liver showed
a slightly increased level of serum [gamma] glutamyl
transpeptidase and acetyl glucuronidase. Hepatitis viral tests
showed negative for Hepatitis B virus and Hepatitis C virus
(HBV-DNA and HCV-RNA). However, Hepatitis G viral test showed
positive for HGV-RNA. The immunological studies showed that the
[beta]-2 microglobulin level was 2.4-2.5 mg/ml.
During the hospital stay, haemoglobin and erythrocytes levels of
the patient were slightly decreased, while the levels of the
leukocyte and platelet were normal. Peripheral blood lymphocytes
counts showed that T4 cells were decreased to 2.76*10/L (32.9%)
and the ratio of T4/T8 cell was 1.16. Thus, the diagnosis is
that the patient was with HIV infection complicated by herpes
zoster.
During hospital stay, the patient had diarrhea and dry cough for
a few days and was cured. The patient showed HIV antibody
positive by ELISA, and his T4 cells further decreased to 25.4%
and the ratio of T4/T8 cells was inverted to 0.94.
The patient's T4 cells and the ratio of T4/T8 gradually
increased after intravenously injected with the HIVCIDE
injection solution. In about three months of HIVCIDE treatment,
his T4 cells were returned to 40.7%, and the ratio of T4/T8 was
improved to 1.45. The skin rash gradually disappeared and
completely recovered by the end of November.
Conclusion:
The HIVCIDE injection solution was effective in reducing
symptoms of the HIV-infected patient in a treatment regime
together with western medicine.
EXAMPLE 5
Clinical Study of Five HIV-Positive Patients Treated with
HIVCIDE Injection Solution
Purpose:
The following clinical trial was conducted in De-Tang Hospital
(National AIDS Therapy Center) in Beijing, China to test the
effectiveness of HIVCIDE in HIV patients.
Methods:
Five (5) HIV-postive patients were intravenously injected with
HIVCIDE daily. The HIV infection was confirmed by western
blotting. The profile of the patients are shown in Table 4:
TABLE 4
Medical Profile of the HIV Patients Participated in the
Clinical Study
Patients Gender Age History
Diagnosis
1 Male 32 2 years AIDS
(Stage IV)
2 Male 25 0.5 year AIDS (Stage
II)
3 Female 32 1 year AIDS
(Stage IV)
4 Male 31 1 year AIDS
(Stage III)
5 Male 17 3 weeks HIV acute
infection
The patients were treated according to the following regimen:
The HIVCIDE injection solution was prepared according to EXAMPLE
1, supra. Prior to the injection, five (5) ml of HIVCIDE
injection solution was diluted in 250 ml of 5% glucose solution.
The diluted solution was injected intravenously once per day for
three (3) consecutive days. If no adverse reactions were
observed, the dosage was increased to 15 ml of HIVCIDE in 250 ml
of 5% glucose solution, and the patients were injected
intravenously once per day reactions for two (2)-three (3)
months.
Additionally, patient #1 was treated with AZT and DDI therapy
for 3 months. The AZT and DDI therapy was discontinued ten (10)
days prior to the HIVCIDE treatment. Patients #2, #3, and #4
were given HIVCIDE daily injection alone. Patient # 5 was given
combined treatment of [alpha]-interferon and HIVCIDE.
About 3 ml of blood sample were taken from the patients each
time before, during, and after the treatment and further tested
for HIV numbers. CD4 counts were determined in Patients #1, #2
and #4 before and after the respective treatment.
Results:
No adverse reactions to the HIVCIDE injection solution were
observed. Three out of the four AIDS patients had different
degrees of fatigue before the treatment of HIVCIDE. There was
marked improvement in the fatigue after the treatment with
HIVCIDE in the three AIDS patients.
The change in HIV viral load (copies/ml) in plasma before and
after HIVCIDE treatment is shown in Table 5 and FIG. 1.
TABLE 5
Change In HIV Viral Load In Plasma of HIV-Positive
Patients
before 1st month dur- 2nd
month dur- 3rd month, at the
Patients treatment ing treatment ing
treatment end of treatment
1 1.9 * 10<4>* 1.7 * 10<5>
6.3 * 10<3> 1.5 * 10<4>
2 1.5 * 10<4> 6.3 *
10<3> 3.8 * 10<2>
3 7.3 * 10<3> 3.2 *
10<3>
4 3.0 * 10<5> 3.7 *
10<3>*** 1.1 * 10<6>
5** 3.9 * 10<5> 2.6 * 10<3>
1.8 * 10<3>
*The lower HIV numbers observed in Patient #1 before
treatment might be contributed to the 3-month treatment of AZT
and DDI prior to the start of HIVCIDE treatment.
**Patient #5 received concurrent treatment of HIVCIDE and
[alpha]-interferon.
***The plasma HIV viral load of Patient #4 was detected 6
weeks after the HIVCIDE treatment.
As shown in Table 5 and FIG. 1, four of the five HIV-positive
patients (i.e., patients #1, #3, #4, and #5) showed decrease in
HIV viral load in two months. Two out of these four patients
(i.e., patients #1 and #4) showed a rebound in viral load after
the end of the third-month HIVCIDE treatment. The other two
patients, (i.e., patients #3 and #5), showed significant
clinical symptom relief, and did not provide plasma sample for
analysis at the end of the third-month treatment. Patient #2,
whose plasma sample was not taken at the end of the second month
treatment, showed a 40-time reduction in HIV viral load at the
end of the third-month HIVCIDE treatment. Patient #2 also
demonstrated significant clinical symptom relief at the end of
the third-month HIVCIDE treatment. However, because patient #5
received concurrent treatment with [alpha]-interferon, the
results of this patient were difficult to assess.
The results of the HIV viral load study indicate that all of the
patients demonstrated reduction in HIV viral load after HIVCIDE
treatment. However, great fluctuation in the replication of HIV
was observed in two of the five patients. For example, patient
#4's HIV viral load decreased significantly after 6 weeks of
treatment but rebounded after the end of the three-month HIVCIDE
treatment. Patient #1 also experienced a rebound (although to a
much lesser degree than that of patient #4) at the end of the
third-month treatment.
In addition to HIV viral load, CD4 count (count/mm<3>) was
measured in three patients (i.e., patients #1, #2, and #3) at
the end of the three-month HIVCIDE treatment. For example, the
CD4 count of patient #2 rose from 285 to 510/mm<3 >after 3
months of HIVCIDE treatment and continued to rise to 630/mm<3
>2 months after the discontinuance of the HIVCIDE treatment.
The CD4 count of Patient 4 was 190/mm<3 >prior to HIVCIDE
treatment, 40/mm<3 >after 3-month of HIVCIDE treatment,
and 360/mm<3 >2 months after the discontinuance of the
HIVCIDE treatment.
Conclusion:
The results indicated that HIVCIDE injection solution was
effective in treating HIV/AIDS patients.
EXAMPLE 6
Clinical Study of HIV-Positive Patients with HIVCIDE and HIVCIDE
Capsules Treatment in Russia
Purpose:
The following experiment was conducted in Hospital in Siberia,
Russia to examine the effectiveness of the HIVCIDE injection
solution and HIVCIDE capsules against HIV infection.
Methods:
Five (5) HIV-Positive patients were treated with the HIVCIDE
injection solution and HIVCIDE capsules obtained from Gongming
Pharmaceutical Co., Ltd, Heilongkiang Province, China. The
HIVCIDE injection solution and HIVCIDE capsules were prepared
according to EXAMPLE 1, supra.
The profile of the patients are shown in Table 6:
TABLE 6
Medical Profile of the HIV-Positive Patients
Participated in the Clinical Study in Russia
Patients Gender Age History
Diagnosis
1 Female 23 2 years AIDS (Grade
A3), adenitis, hepatitis
C, Syphilis, Citomegalo infection,
Gonorrhea
2 Female 28 2 year AIDS (Grade
A3), adenitis, hepatitis
B and C, Gerdeo and Citomegalo
infection, Gonorrhea, drug abuse
3 Male 35 1 year AIDS (Grade B2),
adenitis
4 Male 22 1 year AIDS (Grade B2),
adenitis, hepatitis
C, drug abuse
5 Male 34 several AIDS (Grade
A3), adenitis, hepatitis
months B and C, 10% weight loss, drug abuse
The patients were concurrently treated with HIVCIDE injection
solution and HIVCIDE capsules. For HIVCIDE injection solution,
about 5 ml of HIVCIDE injection solution (containing
approximately 0.4 g of HIVCIDE condensate per ml) were diluted
in 250 ml of 5% glucose. Each patient was given an intravenous
injection of the HIVCIDE injection solution once daily for 3
months. For HIVCIDE capsules, each patient was given 6 HIVCIDE
capsules (220 mg of HIVCIDE capsules) per time (before meal),
three times per day for 3 months.
Blood samples were taken from the patients before, during, and
after the HIVCIDE treatment for tests of CD4 cells count.
Results:
All patients experienced pain in the stomach and right rib area
during the first two weeks of the treatment period. Patient #5
also vomited every morning for a week in the third month of the
treatment period.
The CD4 cells count of the patients is shown in Table 7:
TABLE 7
Change in CD4 Cells Count in Patients With HIV Infection
2
months after the
2nd month
during completion of the 3-
Patients before treatment treatment
month treatment
1 477 641 849
2 740 1140 705
3 421 - 527
4 440 490 669
5 625 - 814
During the treatment period, all patients demonstrated positive
response to the HIVCIDE treatment except the minor adverse
reactions as mentioned above. The physical condition of all of
the patients had clearly improved after one month of the HIVCIDE
treatment. In particular, patients felt stronger, were happier
and showed improved appetite. Anaphylaxis, melancholy, stomach
pain and discomfort disappeared. For some of the patients who
had constipation, the symptom was greatly improved. For some of
the patients who had experienced insomnia, the sleepless problem
was also greatly improved. Also, patients #4 and #5 had gained
about 5 kg at the end of the three-month HIVCIDE treatment. And
patients # 2 and #4 stopped taking narcotic analgesics for pain
relief. The biochemical indicators, including HBV and HCV, in
some of the patients were also clearly improved. The M antibody
in Citomegalo virus, genorrhea antibody and HBC antibody also
disappeared.
Also, as shown in the last column of Table 7, 2 months after the
completion of the 3-month treatment period, all of the patients
CD4 cell counts were continued to increase, which indicated that
no relapse had occurred after the termination of the HIVCIDE.
Patients #1 and #2 had received a second 3-month course of
HIVCIDE treatment after the completion of the first 3-month
HIVCIDE program. No adverse effect was observed at the end of
the second course of HIVCIDE treatment.
Conclusion:
The combined use of HIVCIDE injection solution and HIVCIDE
capsules was effective in reducing symptoms in HIV-positive
patients.
US5178865
CHINESE HERBAL EXTRACTS IN THE TREATMENT OF HIV RELATED
DISEASE IN VITRO
Inventor(s): HO DAVID D [US]; LI XILING, et
al
The invention features herbal extracts from ten (10) Chinese
Herbal Medicines demonstrating significant in vitro and ex vivo
anti-HIV activity and their use for the diagnosis and treatment
of HIV and HIV-related disease.
TECHNICAL
FIELD
This invention is in the fields of medicine and pharmacology. In
particular, the invention features ten (10) commercially
available Chinese Herbal Extracts (CHEs) exhibiting in vitro
and/or ex vivo activity against the etiologic agent of Acquired
Immune Deficiency Syndrome (AIDS) and AIDS related complex
(ARC).
BACKGROUND
ART
Acquired Immune Deficiency Syndrome is a pandemic
immunosuppressive disease which results in life threatening
opportunistic infections and malignancies. A retrovirus,
designated human immunodeficiency virus (HIV-1(HTLV-III LAV)),
has been isolated and identified as the etiologic agent of this
disease. This virus has been shown to be harbored by T helper
lymphocytes and monocyte-macrophages, and it is detectable in
whole blood, plasma, lymphatic fluid, serum, semen, saliva and
central nervous system tissue. Ho et al., New England Journal of
Medicine, 321:1621-1625 (1989). Although cells of the
monocyte-macrophage lineage serve as important reservoirs of HIV
infection, most of the cell-associated virus in the blood is
contained within CD4+ T cells. Characteristically, then, AIDS is
associated with a progressive depletion of T cells, especially
the helper-inducer subset bearing the OKT4 surface marker.
Several agents have been reported to inhibit the growth of the
human immunodeficiency virus in vitro. Among the agents
exhibiting in vitro anti-HIV activity, some are now in clinical
use, including ribavirin, zidovudine (AZT), the 2',
3'-dideoxynucleosides (DDI and DDC); ganciclovir
alpha-interferon, interleukin-2, ampligen and isoprinosine.
Anand et al., Lancet i,97-98 (1986); Balzarini et al., Int. J.
Cancer 37:451-457 (1986); Ho et al., Lancet, i,602-604 (1985);
McCormick et al., Lancet ii,1367-1369 (1984); Mitchell et al.,
Lancet i,890-892 (1987); Mitsuya et al., Proc. Natl. Acad. Sci.
USA 83:1911-1915 and 82:7096-7100 (1985, 1986); Mitsuya et al.,
Science 226:172-174 (1984); Pert et al., Proc. Natl. Acad. Sci.
USA 83:9254-9258 (1986); Pizzi et al., Human Biol. 22:151-190
(1950); Rozenbaum et al., Lancet i,450-451 (1985); Sandstrom et
al., Lancet i,1480-1482 (1986); Veno and Kino, Lancet i,1379
(1987); Yamamoto et al., Interferon Res. 6:143-152 (1986), and
Antiviral Research 7:127-137 (1987). However, no therapy to date
is known to cure AIDS.
The majority of the compounds tested for use against HIV.
including those referenced above, appear to be either too toxic
for prolonged use or incapable of completely eliminating HIV
infection from the human host. Blanche et al., Lancet i,863
(1986); De Clercq et al., J. Med. Chem. 29:1561-1569 (1986);
Yarchoan et al., Lancet i,575-580 (1986); Wetterberg et al.,
Lancet i,159 (1987). In view of the severity of the AIDS
situation and the toxicity and limited clinical efficacy of the
compounds tested thus far, the scientists of the present
invention have begun investigating the anti-HIV activity of
extracts from Chinese medicinal herbs. Chang and Yeung,
Antiviral Research 9:163-176 (1988); Chang et al., Antiviral
Research 11:263-73 (1989). This interest in Chinese herbs was
prompted by Chinese folklore, wherein a number of these herbs
have been reputed to have anti-infective activity and to be well
tolerated by humans. A subset of these herbs now also appear to
exhibit anti-HIV activity, and are disclosed herein.
However, Chinese folk medicine is based largely on anecdotal
observations spanning the past several thousands of years.
Hence, the effectiveness of the medicinal herbs used by folk
medicine practitioners has, for the most part, not been
substantiated by scientific methods. Despite this lack of
scientific proof, it is quite possible that some herbal remedies
may have specific therapeutic action, as was proven to be the
case with the anti-malarial. qinghaosu, and perhaps even
anti-HIV activity. Klayman, Science, 228:1049-1055 (1985).
Consequently, with regard to the possible anti-HIV activity
among Chinese herbal extracts, an urgent need exists for: 1) the
identification of effective anti-HIV herbal extracts, 2) the
substantive documentation, by modern scientific methods, of the
effectiveness of these herbal extracts against HIV, and 3) the
identification of effective anti-HIV Chinese herbal extracts
that are less toxic than the currently available anti-HIV
agents. The present invention satisfies this need and provides
related advantages as well.
The papers cited throughout this application are incorporated
herein by reference.
DISCLOSURE
OF THE INVENTION
A total of fifty-six (56) herbal extracts. some of which are
known to have anti-infective properties and to be non-toxic in
clinical use in China, were screened for their anti-HIV activity
using in vitro techniques. Of these fifty-six (56) herbal
extracts, ten (10) were shown to have potent anti-HIV activity
in in vitro experiments, and two (2) of these ten (10) also
exhibited anti-HIV activity in ex vivo experiments.
These ten (10) include the extracts from: Sample #1--Coptis
chineusis, which can be located in Western, Southern and Central
China; Sample #8--Ligusticum wallichii, which can be found in
Northern and Southwestern China, and Salvia miltiorrhiza, which
can be located in most areas of China; Sample #21--Illicium
lanceolatum, which can be located in Eastern and Southern China;
Sample #30--Isatis tinctoria, which can be found in Central
China, Lonicera japonica, which can be located in most areas of
China, and Polygonum bistorta, which can be located in Northern,
Eastern and Southwestern China; Sample #32--Salvia miltiorrhiza,
which can be located in most areas of China; Sample #35--Erycibe
obtusifolia, which can be found in Southern China, Taiwan,
Japan, Indonesia and Northern Australia; Sample
#39--Acanthopanax graciliatylus--which can be located in Central
and Southwestern China and the Philippines; Sample
#41--Bostaurus domesticus, which can be found in most areas of
China and in Southern Africa, and Scutellaria baicaleusis, which
can be located in Northern, Western and Central China and
Southern Africa; Sample #44--Inula helenium, which can also be
located in most areas of Northern China, and Salvia
miltiorrhiza, which can be located in most areas of China; and
Sample #49--Lonicera japonica, which can be located in most
areas of China, and Scutellaria baicaleusis, which can be
located in Northern, Western and Central China, as well as in
Southern Africa. This information is reproduced in Table I
below, which also provides alternative means for identifying the
subject herbs.
TABLE I
SAMPLE
NAME OF HERB
CLASSIFICATION
MAJOR LOCATION
#1 Coptis chineusis
Ranunculaceae
Western, Southern and
Franch Central China
*#8 Ligusticum wallichii
Umbelliferae
Northern and
Franch and Southwestern China;
Salvia miltiorrhiza
Labiatae Most areas of China
Bunge
#21 Illicium lanceolatum
Illiciaceae
Eastern and Southern
A. C. Smith or China
Illicium henryi Diels
*#30 Isatis tinctoria L.
Cruciferae Central China
or Isatis indigotica
Fort.,
Lonicera japonica
Caprifoliaceae
Most areas of China
Thunb and
Polygonum bistorta L.
Polygonaceae
Northern, Eastern and
Southwestern China
#32 Salvia miltiorrhiza
Labiatae Most areas of China
Bunge
#35 Erycibe obtusifolia
Convolvulaceae
Southern China,
Benth Taiwan, Japan,
Indonesia and Northern
Australia
#39 Acanthopanax
Araliaceae Central and
graciliatylus Southwestern China,
W. W. Smith Philippines
*#41 Bostaurus domesticus
Bovine choleic
Most areas of China
Gmel. and
Scutellaria baicaleusis
Labiatae Northern, Western and
Georgi Central China, S.
Africa
*#44 Salvia miltiorrhiza
Labiatae Most areas of China
Bunge and
Inula helenium L.
Compositae Northern, Northeastern
and Northwestern China
*#49 Lonicera japonica
Caprifoliaceae
Most areas of China
Thunb and
Scutellaria baicaleusis
Labiatae Northern, Western and
Georgi Central China, S.
Africa
*A compound comprising more than one (1) herb.
In the context of the present specification, CHE is used to
refer to any species of any of the herbs delineated above which,
upon extraction, yields a fraction comprising a
pharmacologically active agent, whether a component, a
combination of components, a biological metabolite, a derivative
thereof or a combination of the above, that exhibits in vitro
and/or ex vivo anti-HIV activity. Since the precise chemical
composition and pharmacologic mechanism of the CHEs has not yet
been elucidated, it is possible that the anti-HIV activity may
be due to a single CHE component, a combination of CHE
components, or the biologic metabolite or derivative thereof.
By the terms "HIV," and "AIDS-related virus" is meant the
commonly designated HIV series (human immunodeficiency virus)
formerly called HTLV, LAV and ARV, and species thereof, as
described in the incorporated references.
Similarly, the terms "HIV-related disease" and "AIDS-related
disease" shall refer to any illness or syndrome, caused directly
or indirectly by HIV or AIDS-related virus, including but not
limited to infections whose source is fungal, viral and/or
bacterial.
It is therefore an object of the present invention to employ the
CHEs as therapeutic agents in hosts infected with HIV. In vitro
studies, ex vivo studies, including the therapeutic indices (TI)
calculated for each CHE, suggest that these CHEs will be useful
in pharmacological preparations as in vivo anti-HIV agents. The
pharmacological preparations may contain the pharmacological
active ingredient alone or in admixture with an appropriate
excipient or carrier, and administered to the HIV infected host
by enteral, such as oral or rectal, and parenteral, such as
intraperitoneal, intramuscular, intravenous or subcutaneous
route. The pharmacological agent may also be administered in
combination with a supplemental antiviral agent, an immune
modulator, any other chemotherapeutic agent, an antibody or a
combination thereof. In addition, the pharmacological
preparations according to the invention may be, for example, in
dosage unit form, such as tablets, capsules. suppositories or
ampoules.
It is another object of the invention to use a CHE component or
combination of CHE components, a biologic metabolite, a
derivative thereof or a combination of the above, in a
pharmacological preparation for the treatment of HIV-related
illness in infected hosts.
It is a further object of the invention to use the CHE, its
active component or combination of components, a biological
metabolite, a derivative thereof, or a combination of the above,
alone or conjugated to a label, in a diagnostic test for the
diagnosis of HIV related illness. Such a test could be an
immunofluorescent test, based upon a CHE's capacity to bind
either the HIV infected T cells or the anti-idiotypic antibody
derived from the CHE.
It is still a further object of the invention to use a CHE, a
CHE component, a combination of CHE components, a biological
metabolite, a derivative thereof, or a combination of the above
to produce a vaccine. Once the CHE's "active site" has been
determined, current immunologic techniques could be relied upon
to produce such a vaccine.
These and other objects will become readily apparent to those
skilled in the art from the following description and appended
claims.
BRIEF
DESCRIPTION OF THE DRAWINGS
The present invention will be described in connection with the
accompanying drawings in which:
FIG. 1 is a composite graph depicting the neutralization assay
results for each of the ten CHEs against the lab isolate IIIB.
FIG. 2 is a composite graph depicting the activity of the ten
(10) CHEs against the laboratory isolate, HIV-1 AC.
FIG. 3 is a composite graph depicting the neutralization assay
results for each of the ten CHEs against the lab isolate HIV-2.
FIG. 4 illustrates and compares the percent HIV neutralization
of CHE #1 for each of seven separate clinical isolates of the
virus.
FIG. 5 illustrates and compares the percent HIV neutralization
of CHE #8 for each of seven separate clinical isolates of the
virus.
FIG. 6 illustrates and compares the percent HIV neutralization
of CHE #21 for each of seven separate clinical isolates of the
virus.
FIG. 7 illustrates and compares the percent HIV neutralization
of CHE #30 for each of seven separate clinical isolates of the
virus.
FIG. 8 illustrates and compares the percent HIV neutralization
of CHE #32 for each of seven separate clinical isolates of the
virus.
FIG. 9 illustrates and compares the percent HIV neutralization
of CHE #35 for each of seven separate clinical isolates of the
virus.
FIG. 10 illustrates and compares the percent HIV neutralization
of CHE #39 for each of seven separate clinical isolates of the
virus.
FIG. 11 illustrates and compares the percent HIV neutralization
of CHE #41 for each of seven separate clinical isolates of the
virus.
FIG. 12 illustrates and compares the percent HIV neutralization
of CHE #44 for each of seven separate clinical isolates of the
virus.
FIG. 13 illustrates and compares the percent HIV neutralization
of CHE #49 for each of seven separate clinical isolates of the
virus.
FIG. 14 is a composite graph depicting the degree of HIV
replication inhibition exhibited by CHE #32.
FIG. 15 illustrates and compares the anti-HIV activity of CHEs
#21, #32 and #49 in chronically infected H9/IIIB cells.
FIG. 16 is a composite graph depicting each CHE's percent
neutralization of HIV IIIB reverse transcriptase activity.
FIG. 17 illustrates and compares the ex vivo anti-HIV activity
of CHE #32 in the peripheral blood mononuclear cells (PBMNs) and
plasma of three (3) patients.
FIG. 18 illustrates and compares the ex vivo anti-HIV activity
of CHE #49 in the PBMNs and plasma of three (3) patients.
DETAILED
DESCRIPTION
The following detailed description and procedures are provided
to illustrate the principles of the invention. They are not,
however, intended to limit the invention, which extends to the
full scope of the appended claims.
A. Preparation of Extracts
The fifty six (56) subject herbs were obtained from China in
extract form, packaged in ampoules for parenteral use. However,
the extracts of the present invention can be prepared from the
subject herbs by utilizing the procedures set forth below, or
any organic extraction procedure.
Cut into small pieces, one kilogram of dried herb. Soak the cut
herb pieces in eight liters (8 L) of water at room temperature
for six to eight hours, and then boil under reflux for one (1)
hour. Decant the extract, filter it through a 0.45 .mu.m
membrane filter, and concentrate to one liter (1 L).
To the concentrated extract, add three liters (3 L) of 100%
ethanol, and maintain the mixture at room temperature for
forty-eight (48) hours. Decant, filter and concentrate the
extract to one liter (1 L) as above. Repeat this ethanol
precipitation two (2) more times.
Add 0.3% charcoal into the concentrated extract and boil the
charcoal-extract mixture for five (5) minutes. Filter the
extract again.
To the filtered extract, add 10% sodium hydroxide until pH7. The
final extract concentration obtained using these procedures
should be 1 g herb/ml.
B. Determination Of The Subtoxic Concentrations Of Herbal
Extracts
Before assessing the anti-HIV activity of the fifty-six (56)
CHEs, toxicity studies were performed to ensure that the
observed activity could not be attributed to the indiscriminate
destruction of the host lymphocytes by the CHE. For these
studies, the standard laboratory methods for T cell toxicity
testing were followed. Chang et al., Antiviral Research
9:163-176 (1988); Merchant et al., Handbook of Cell and Organ
Culture, Burgess Publishing Co., Minneapolis, Minn. (1960).
Briefly, the CHE extract to be tested was diluted two-fold
serially in medium. To 0.2 ml of the diluted extract, 0.8 ml of
a freshly prepared H9 cell suspension was added. (The H9 cells
had been obtained from the American Type Culture Collection
(A.T.C.C.).) This was done in duplicate; and a medium control
was included in every assay. This medium control consisted of
0.8 ml of the same H9 cell suspension added to 0.2 ml of medium;
and the control was done in quadruplicate. After 4 days of
incubation, the number of viable cells in each culture was
counted with a hemacytometer by dye exclusion. When the viable
count of extract-treated culture was 2 S.D. below the mean of
the medium control, the extract-treated culture was considered
to show evidence of cytotoxicity. The highest concentration of
an extract which showed no evidence of cytotoxicity was taken as
the subtoxic concentration, or maximum tolerated dose (MTD). The
MTD's for the ten (10) CHEs exhibiting anti-HIV activity are
disclosed in Table II which follows:
TABLE II
Maximum Tolerated Dose (MTD)
CHE MTD (.mu.l)
#1 5
#8 5
#21 40
#30 20
#32 20
#35 10
#39 80
#41 20
#44 40
#49 20
C. Neutralization Assay
Having determined their MTDs, the fifty-six (56) CHEs were then
screened for their inhibitory activity against HIV-IIIB in H9
cells. (The HIV-IIIB had been obtained from Drs. Popovic and
Gallo.) Employing a standard neutralization assay, which assay
is described in the literature, HIV expression was detected by
p24 production in the culture supernatant. Ho et al., Science
239:1021-1023 (1988); and Ho et al., J. Virol. 61:2024 (1987).
Specifically, the TCID50 (50% tissue culture infective doses)
for the HIV-IIIB isolate was placed in contact with 1.times.10@6
human T lymphocytes, one hour after the CHE under investigation
was added at varying doses. This culture was then followed for
seven days and observed for signs of viral expression, as
measured by the production of HIV core protein p24. A particular
CHE was not deemed to have anti-HIV activity unless 90% of viral
replication was blocked, as compared to control cultures.
An ID50 and ID90 (amount of CHE necessary to inhibit 50% and 90%
of viral replication, respectively) was also calculated for each
of the ten CHEs that exhibited anti-HIV activity. In addition,
by dividing the MTD by the ID50, a therapeutic index was
obtained. Generally, the therapeutic index (T.I.) is a measure
of both drug efficacy and safety, and a high therapeutic index
is desirable.
FIG. 1 demonstrates the anti-HIV activity of each of the ten
(10) CHEs against the lab isolate IIIB. In brief, the ID50 for
the ten (10) subject CHEs ranged from 0.15 .mu.l to 1.80 .mu.l,
while the ID90 ranged from 0.38 .mu.l to 2.70 .mu.l. The T.I.
for these same ten (10) ranged from 22 to 173. These values are
all presented in Table III below.
TABLE III
Ten CHEs With Positive Activity Against
HTLV-IIIB Infection Of H9 Cells
Anti-HTLV-IIIB Activity
CHE MTD (.mu.l)
ID50 (.mu.l)
ID90 (.mu.l)
T.I.
#1 05 0.15 0.84 33.33
#8 05 0.14 0.38 35.71
#21 40 1.80 2.30 22.22
#30 20 0.26 1.70 76.92
#32 20 0.45 0.66 44.44
#35 10 0.41 1.70 24.39
#39 80 1.00 5.60 80.00
#41 20 0.34 2.70 58.82
#44 40 0.23 0.65 173.91
#49 20 0.24 0.53 83.33
Using a similar method, the CHEs were then tested against seven
(7) clinical isolates (J, AP, L, B, P, C, F) and two (2)
additional HIV laboratory isolates, (AC and HIV2), in normal
stimulated PBMNs. (The clinical isolates, and the AC laboratory
isolate, had been obtained from AIDS patients treated at
Cedars-Sinai Medical Center, in Los Angeles, Calif. The HIV-2
(LAV-2ROD) had been obtained from Luc Montagnier, at the
Institute Pasteur, in France.) The ten (10) CHEs were found to
exhibit anti-HIV activity against most of the clinical isolates;
but with varying efficacy. Similarly, in FIG. 2, all ten (10)
CHEs exhibited activity against the AC laboratory isolates,
whereas only two (2) of the ten (10) CHEs (#41 and #49), in FIG.
3, showed appreciable inhibitory activity against the HIV2
isolate. The results of this method are discussed more
particularly as follows:
As illustrated in FIG. 4, CHE #1 exhibited greater than 90%
inhibition for six (6) of the seven (7) primary HIV-1 isolates,
with an ID90 ranging from 0.20 .mu.l to 3.5 .mu.l.
In FIG. 5, CHE #8 was found to have equal or greater than 90%
inhibition for the seven (7) primary isolates, with an ID90
ranging between 0.35 .mu.l to 5.00 .mu.l.
As illustrated in FIG. 6, CHE #21 exhibited greater than 90%
inhibition for all but one (1) of the seven (7) primary
isolates, with an ID90 ranging from 1.74 .mu.l to 7.6 .mu.l.
In FIG. 7, CHE #30 exhibited greater than 90% activity against
five (5) ofthe seven (7) primary isolates, with an ID90 of 0.52
.mu.l to 8.30 .mu.l.
FIG. 8 illustrates the neutralization activity for CHE #32. As
the graph illustrates, CHE #32 exhibited ID90 activity against
five (5) of the seven (7) primary isolates, with an ID90 ranging
from 0.52 .mu.l to 7.00 .mu.l.
In FIG. 9, CHE #35 inhibited six (6) of the seven (7) primary
isolates, with an ID90 ranging from 0.60 .mu.l to 8.2 .mu.l.
As illustrated in FIG. 10, only three (3) of the six (6) primary
isolates were inhibited more than 90% by CHE #39, with an ID90
ranging from 2.2 .mu.l to 10 .mu.l.
In FIG. 11, all but one (1) primary isolate were inhibited
greater than 90% by CHE #41, with an ID90 between 1.10 .mu.l to
5.00 .mu.l.
CHE #44 exhibited greater than 90% inhibition against three (3)
of the six (6) primary isolates in FIG. 12, with an ID90 ranging
from 1.00 .mu.l to 5.10 .mu.l.
In FIG. 13, CHE #49 inhibited all seven (7) primary isolates by
greater than 90%, with an ID90 of 0.62 .mu.l to 2.05 .mu.l.
D. Syncytial Inhibition
Formation of syncytia, with progression to cell death, is a
characteristic feature of in vitro cell cultures infected with
HIV. Syncytia formation depends upon the interaction of
HIV-expressing cells with neighboring cells bearing the CD4
differentiation antigen. Syncytial inhibition studies were
therefore performed to determine whether a particular CHE had
its primary effect upon the HIV envelope glycoproteins, or upon
the uninfected target cells. Following a standard method,
described in the literature, the two cell cultures Molt IIIB and
HPBALL were employed as the sources of infected and uninfected
cell specimens, respectively. See Lifson et al., Science
232:1123-7 (1986); Sodroski et al., Nature 322:470-4 (1986); and
Lifson et al., Nature 323:725-8 (1986).
Varying amounts (0.3 .mu.l, 1.0 .mu.l, 3.0 .mu.l) of each of the
ten (10) CHEs were preincubated separately with either the
infected Molt IIIB or the uninfected HPBALL cells for a standard
time period. The cells were then washed several times and the
two cell types were mixed in culture. The percent syncytial
inhibition of both methods was then evaluated for all CHEs by
light microscopy eighteen (18) hours after mixing. Table IV
lists the preliminary results of syncytial inhibition activity
exhibited by each CHE studied.
TABLE IV*
#1 #8 #21
#30
#32
#35
#39
#41
#44
#49
rsT4
A. CHE WAS PREINCUBATED WITH UNINFECTED CELLS (HPB-ALL)
3 .mu.l
96.2
98.1
3.7
70.9
93.6
98.1
67.9
77.4
50.9
86.1
32.1
(3 .mu.l)
1 .mu.l
62.3
26.4
1.9
62.3
74.7
83.0
15.1
58.5
35.8
62.3
35.8
(1 .mu.l)
0.3 .mu.l
28.3
15.1
0 28.3
37.7
58.5
0 16.9
15.5
50.9
0 (0.3 .mu.l)
B. CHE WAS PREINCUBATED WITH INFECTED CELLS (MoltIIIB)
3 .mu.l
0 20.8
7.6
47.2
73.6
26.4
39.6
50.6
92.5
73.6
92.5
(3 .mu.l)
1 .mu.l
0 26.4
13.2
39.6
37.2
20.8
16.9
50.9
88.7
47.2
62.3
(1 .mu.l)
0.3 .mu.l
0 9.4
0 16.9
33.9
0 18.3
43.4
77.4
32.1
35.8
(0.3 .mu.l)
*The results tabulated above were obtained from one (1) series
of experiments. These experiments have not yet been repeated to
verify the reproducability of the above results.
Briefly, as tabulated above in Section A of Table IV, four (4)
of the ten (10) CHEs (#1, #8, #32 and #35) exhibited greater
than 90% inhibition of syncytia formation when the CHE was
preincubated with the uninfected cells. However, only one (1)
(#44) exhibited greater than 90% inhibition of syncytia
formation when the CHE was preincubated with the infected cells.
An additional experiment was thereafter performed to determine
whether this anti-HIV activity produced by the CHEs occurred
inside the cells. In this experiment, the cells were infected
one (1) hour before adding the CHEs, using the reverse
transcriptase inhibitor, AZT, as a control. In FIG. 14, 10 .mu.l
of CHE #32 exhibited 100% inhibition of HIV replication in the
preinfected cells with both doses of HTLV-IIIB (50 TCID50 and
100 TCID50). This result appears to indicate that the observed
in vitro anti-HIV activity may actually occur within the cell,
although the precise mechanism for this activity is still being
investigated.
E. End-Point-Dilution Cultures
The end-point-dilution culture method, as described in the
literature, was used to determine whether the CHEs exhibited an
anti-HIV effect in chronically infected H9/IIIB cells. Ho et
al., NEJM 321:1621-1625 (1989). As indicated in FIG. 15, 1 .mu.l
of CHE #21 produced no viral titer change, although 10 .mu.l of
CHE #21 produced a 10 fold decrease in viral titer. Moreover, 1
.mu.l of CHE #32 produced a 2 fold decrease in viral titer,
while 10 .mu.l produced a 10 fold decrease; and 1 .mu.l of CHE
#49 decreased the viral titer 2 fold, while 10 .mu.l resulted in
a 100 fold decrease.
Hence, these results further strengthen the results obtained in
the pre-infection studies in FIG. 14, (the cells were infected
one hour before adding the CHEs), where it appeared that the
anti-HIV activity of the CHEs may actually occur within the cell
interior. However, as indicated above, the precise mechanism for
the CHE activities is still under investigation.
F. Reverse Transcriptase Assay
The HIV III-B RT enzyme was isolated and mixed with varying
amounts of each CHE, using a known reverse transcriptase (RT)
biochemical assay. Ho et al., Science 226:451-453 (1984);
Popovic et al., Proc. Natl. Acad. Sci. U.S.A., 77:7415 (1980).
Specifically, virus particles were precipitated from cell-free
supernatant as follows: 0.3 ml of 4M sodium chloride and 3.6 ml
of 30% (weight volume) polyethylene glycol (Carbowax 6000) were
added to 8 ml of harvested culture fluids and the suspension was
placed on ice overnight. The suspension was centrifuged at 2000
rev/min at 30 minutes. The precipitate was resuspended in 300
.mu.l of 50% (by volume) glycerol (25 mM tris-HCl, pH 7.5, 5 mM
dithiothreital, 150 mM potassium chloride and 0.025% Triton
X-100). Virus particles were disrupted by addition of 100 .mu.l
of 0.9% Triton X-100/1.5M potassium chloride solution.
The cell-free virus concentrate from a culture of H9/HIV III-B
was layered on a 20 to 60% (by weight) sucrose gradient in 10 mM
tris-HCl (pH 7.4) containing 0.1M sodium chloride and 1 mM EDTA
and centrifuged overnight at 35,000 rev/min. Fractions of 0.7 ml
were collected from the bottom of the gradient and 10 .mu.l
portions, in a final volume of 100 .mu.l containing 40 mM
tris-HCl (pH 7.8), 4 mM dithiothreital, 45 mM potassium chloride
and 50 .mu.g of template--primer poly (A).dT12-18 and poly
(C).dG12-18 per ml (with 10 mM Mg@2+) or 50 .mu.l of poly
(dA).dT12-18 per ml (with 0.25 mM Mn@2+) were assayed for RT at
37 DEG C. for 1 hour. The mixture also contained 15 .mu.M of the
appropriate labeled deoxyribonucleotide triphosphates, [@3
H]dTTP (16 Ci/mmole; 1 Ci-3.7.times.10@10 becquerels) or [@3
H]dGTP (12 Ci/mmole). The amount of each CHE necessary to
inhibit 50% and 90% of the HIV III-B RT activity is reported in
Table V below and FIG. 16. As illustrated, the results indicate
that seven (7) of the ten (10) CHEs exhibited greater than 90%
RT inhibition.
TABLE V
Anti-HTLV-IIIB RT Activity
CHE ID50 (.mu.l)
ID90 (.mu.l)
#1 0.53 >10.00
#8 2.40 9.00
#21 3.70 >10.00
#30 1.60 9.00
#32 3.00 9.00
#35 4.50 >10.00
#39 3.35 8.90
#41 1.20 9.20
#44 1.95 9.50
#49 1.12 6.80
G. Ex Vivo Experiments Utilizing CHEs #32 And #49, The Best Mode
CHEs
Having determined their in vitro anti-HIV activity, ex vivo
experiments were conducted in order to provide an experimental
model that resembles as closely as possible, in vivo conditions
for the CHEs. Ho et al., PNAS, 87:6574-6578 (1990). For these
experiments, CHEs #32 and #49 were utilized, as they were
considered the leading candidates among the ten (10) CHEs for
anti-HIV activity. These two (2) particular (CHEs) were selected
based upon the experimental data to date. However, this
selection of CHEs #32 and #49 as the best mode CHEs is not meant
to foreclose other possibilities, as future experiments may
identify other more effective anti-HIV agents among the ten (10)
CHEs.
Plasma and PBMNs were obtained from three (3) patients and
denoted as follows: A, for a patient with AIDS; R for a patient
with ARC; and H for a healthy patient. An end-point-dilution
culture method as described above, was used for serial
quantitation of HIV-1 in the PMBNs and plasma of the three (3)
patients, and serum p24 core antigen levels were measured as a
marker of viral burden. In FIG. 17, the HIV titers in PBMNs are
illustrated on the top graph; and the HIV titers in plasma are
illustrated on the bottom graph.
HIV-1 was recovered from the PBMNs of all three (3) patients,
with titers ranging from 10 to 1,000 TCID/10@6 cells, and a mean
titer of 370 TCID/10@6 cells. When 1 .mu.l of CHE #32 was added,
however, HIV-1 was detected in titers ranging from 10 to 100
TCID/10@6 cells, a 10 fold decrease in viral titers. When 10
.mu.l of CHE #32 was added, viral titers in all patients were
decreased to 1 TCID/10@6 cells. Meanwhile, the total HIV-1
titers in plasma before the addition of CHE ranged from less
than 1 to 10 TCID/ml, and a mean value of 3.3 TCID/ml. However,
the addition of 1 .mu.l or 10 .mu.l of CHE #32 decreased viral
titers to less than 1 to 1 TCID/ml.
As illustrated in FIG. 18, in PBMNs treated with 0.1 .mu.l of
CHE #49, HIV titers decreased from a mean titer of 370 TCID/10@6
to 10 TCID/10@6 cells. The addition of 1 .mu.l of CHE #49
further reduced the viral titer to 0.67 TCID/10@6 cells. In
plasma, however, 0.1 .mu.l of CHE #49 produced no change in HIV
titer, although 1 .mu.l reduced the HIV titer 10 fold.
These experiments and the resulting data demonstrate that CHEs
may be a rich source for potential in vivo anti-HIV therapy in
an infected host. As illustrated, ten (10) of the fifty-six (56)
CHEs tested were found to exhibit dose dependent anti-HIV
activity in vitro. Five (5) of these CHEs (#1, #8, #32, #35 and
#44) also demonstrated substantial syncytial inhibition
activity, while seven (7) CHEs (#8, #30, #32, #39, #41, #44 and
#49) exhibited inhibitory activity against reverse
transcriptase. Finally, CHEs #32 and #49 even exhibited ex vivo
dose dependent anti-HIV activity in patient plasma and PBMNs.
US6455078
Medicinal herbal composition for treating liver diseases
and HIV
Inventor(s): WU TZU-SHENG, et al
The present invention provides a herbal pharmaceutical
composition for treating patients with liver diseases and/or
HIV. The composition contains fifteen (15) ingredients, which
are diffuse hedyotis, bistort rhizome, giant knotweed rhizome,
Asiatic moonseed rhizome, baical skullcap root, bovine biliary
powder, milkvetch root, barbary wolfberry fruit, sanqi, red
ginseng, figwort root, Chinese magnoliavine fruit, turmeric
root-tuber, hawthorn fruit, and Chinese angelica. Among the
fifteen (15) ingredients, diffuse hedyotis, bistort rhizome,
giant knotweed rhizome, and Chinese magnoliavine fruit are the
required herbs which contribute to the efficacy of the
pharmaceutical composition.
CROSS-REFERNCE
TO RELATED APPLICATION
The present application claims the benefit of the filing date of
U.S. Provisional Application No. 60/240,963, filed on Oct. 18,
2000, which is herein incorporated by reference.
FIELD OF
THE INVENTION
The present invention relates to a novel herbal pharmaceutical
composition and its use for treating patients with liver
diseases (e.g., viral hepatitis [such as Hepatitis A, Hepatitis
B, Hepatitis C, Hepatitis D, and Hepatitis E], alcoholic or
fatty liver, liver cirrhosis, and liver cancer) and HIV. The
major ingredients in the herbal composition are diffuse
hedyotis, bistort rhizome, giant knotweed rhizome, and Chinese
magnoliavine fruit. The composition further contains Asiatic
moonseed rhizome, baical skullcap root, bovine biliary powder,
tumeric root-tuber, hawthorn fruit, sanqi, barbary wolfberry
fruit, red ginseng, figwort root, Chinese angelica, and
milkvetch root. The present invention also relates to a method
for making the medicinal herbal composition and methods for
treating patients with the medicinal herbal composition.
DESCRIPTION
OF THE RELATED ART
Liver diseases have great impact on human health. Hepatitis is a
kind of liver diseases, which is caused by liver inflammation
due to infection of a variety of pathogens, which include, but
are not limited to, viruses, bacteria, fungi, and protozoa.
Hepatitis can be categorized as acute, chronic, or fulminant.
Viral hepatitis is an enterically transmitted liver disease due
to viral infection. The major transmission means for viral
hepatitis is through ingestion. Viral hepatitis can also be
transmitted through blood transfusion or similar means of
hepatitis-virus-carrying blood or blood product such as blood
plasma. Viral hepatitis is widespread around the world. For
example, there are approximately thirty million (30,000,000)
viral hepatitis patients in China including an estimated number
of nine million (9,000,000) new patients each year, and about
one hundred million (100,000,000) hepatitis B virus (HBV)
carriers. It is estimated that 10% of the pregnant women in
China are HBV carriers. About one hundred thousand (100,000)
people in China die of liver cancer originated as liver diseases
each year.
Depending on the major etiologic agent, viral hepatitis is
categorized into Hepatitis A, Hepatitis B, Hepatitis C,
Hepatitis D, and Hepatitis E. Hepatitis A is caused by hepatitis
A virus (HAV); Hepatitis A can affect anyone and occur in
isolated cases as well as widespread epidemics. Hepatitis B is a
serious disease caused by hepatitis B virus (HBV). HBV attacks
the liver and can cause lifelong infection, cirrhosis (scarring)
of the liver, liver cancer, liver failure, and death. Hepatitis
C is caused by hepatitis C virus (HCV). Hepatitis D is caused by
the hepatitis D virus (HDV) which is a defective single-stranded
RNA virus that requires the helper function of HBV to replicate
and to synthesize envelope protein composed of HBsAg to
encapsulate HDV's genome. Hepatitis E is caused by hepatitis E
virus (HEV), which is an etiologic agent of enterically
transmitted non-A, non-B hepatitis. HEV is a spherical,
non-enveloped, single-stranded RNA virus of approximately 32 to
34 nm in diameter. HEV has been provisionally classified in the
Caliciviridae family; however, the organization of the HEV
genome is substantially different from that of other
Caliciviruses, and HEV may eventually be classified in a
separate family.
The most common types of viral hepatitis are Hepatitis A,
Hepatitis B, Hepatitis C, and Hepatitis E, which have similar
major symptoms including decreased appetite, nausea, unease
upper abdomen, lack of strength, etc. Acute jaundice is also one
of the common symptoms. Chronic hepatitis is very difficult to
cure. Severe hepatitis often comes on quickly and results in
high mortality.
Traditional Chinese herbal compositions have been developed and
shown success for preventing and treating various liver
diseases. The types of traditional Chinese herbal medicine for
treating hepatitis include medications having single or multiple
herbal components and medications made of active ingredients
extracted from the herbs.
For example, Qianglining injection solution is made of
glycyrrhizic acid extracted from licorice (Glycyrrhiza).
Glycyrrhizic acid reacts with ammonia to form a water-soluble
ammonium salt of glycyrrhizic acid, which then can compound with
amino acids. The injection solution is useful for treating
chronic viral hepatitis, liver cirrhosis, and hepatoma. The
total effective rate of qianglining injection solution is about
87.5%, in which 64.1% is significant, according to clinical
studies conducted on hepatitis patients provided by Shanghai
Huashan Hospital, Shanghai, China.
Yanhuanglian injection solution is derived from ground herb
Yanhuanglian grown in Guangxi Province in China. The solution is
useful for treating various types of hepatitis, liver cirrhosis,
and liver cancer, with a reported clinical efficacy rate of
81.47%. The solution has an effective rate of 93.88% in cases
involving acute jaundice patients, 87.50% in non-jaundice type
hepatitis patients, 87.09% in chronic active type hepatitis
patients, 69.23% in prolonged type hepatitis patients, and
80.95% in chronic cirrhosis patients. However, only 17.91% of
the patients show changes of HBV surface antigen from positive
to negative.
Shandougen (Radix Sophorae Tonkinensis) injection solution is
useful for both acute and chronic viral hepatitis, and
especially effective for chronic active hepatitis. As studied by
Guangxi Medical College in Guangxi province, China, the total
effective rate is 91.79% for chronic active hepatitis patients,
and the substantial effective rate is 54.23%. Also, 64.93% of
the patients' glutamate-pyruvate transaminase (GPT) level
returns to normal in two (2) months after the treatment.
However, some patients show recurring symptoms of hepatitis
after the treatment is discontinued.
Umbellate pore fungus (Polyporus umbellata) injection solution
has functions of improving immune function, inhibiting tumor,
lowering level of transaminase, and inhibiting replication of
hepatitis virus. After treating patients with chronic viral
hepatitis with umbellate pore fungus injection solution, 35.6%
of the patients return to normal serum GPT (SGPT) level, 76.61%
of the patients show some lowering effects on transaminase
level, 38.6% of the patients show HBV E antigen turning
negative, and 13.1% of the patients show surface antigen turning
negative.
Qidun fruit acid tablet has a total effective rate of 94.4% in
patients with acute jaundice-type hepatitis. The total recovery
rate is 64.8%. Qidun fruit acid tablet also shows an effective
rate of 69.8% in chronic active hepatitis, in which 43.7% of the
patients show a significant effect. The rate for HBsAg positive
turning negative is 16.8%.
Gandezhi (Liver-curing) capsule has Wuren alcohol, scutellarin,
mulberry fruit-spike (Fructus Mori Albae), salvia root (Radix
Salviae Miltiorrhizae), and licorice (Radix Glycyrrhizae
Uralensis) and is useful for lowering transaminase level. It has
an effective rate of 80.0% for treating prolonged hepatitis and
chronic hepatitis, according to studies reported by Guangzhou
Zhongshan Medical College Hospital in China. There has been no
report which shows that Gandzhi has effect on HBV Antigen
turning negative.
Danggui (Chinese angelica root) pill is made of Chinese angelica
root (Radix Angelicae Sinensis) and licorice (Radix Glycyrrhizae
Uralensis). In a study conducted by Beijing Medical College in
China, Danggui pill is effective for treating prolonged
hepatitis (with an effective rate of 84.4%), chronic hepatitis
(with an effective rate of 79.1%), and cirrhosis resulted from
hepatitis (with an effective rate of 73.6%).
Hugang (liver-protecting) tablet is made from schisandra fruit
(Fructus Schisandrae Chinensis) alcohol extractant,
liver-protecting extractant (including Junchen, Zihu, and woad
root (isatis root, Radix Isatidis seu Baphicacanthi)), and
biliary powder, etc. It has an effective rate of 95.08% for
treating chronic hepatitis (70% with significant effect), and
82.5% for treating cirrhosis (63% with significant effect).
Jigu ("chicken bone") grass pill is made of Jigu grass, billiary
powder, and bovine bezoar (Calculus Bovis). As studied by
Beijing Children's Hospital in China, Jigu grass pill has a
total effective rate of 100% in patients with acute viral
hepatitis, 73.3% in patients with chronic active hepatitis,
70.4% in patients with chronic prolonged type hepatitis.
However, Jigu grass pill does not appear to have any effect on
other types of prolonged hepatitis.
Wuzi ("five ester") capsule is made from schisandra fruit
(Fructus Schisandrae Chinensis) alcohol extractant. It shows
function of lowering GPT level and is useful for treating
chronic prolonged hepatitis. The total effective rate of wuzi
capsule is 95.33%, in which 74.21% is significant.
Ganfuneng (liver-healing) formula contains astragalus (Radix
astragali membranaceus), hawthorn fruit (Fructus crataegi),
pueraria (Radix puerariae), Cornu Bubali powder, San-qi, etc. It
has an effective rate of 88.7% for chronic hepatitis patients
and 79.1% for GPT recovery.
Biyansha Hepatitis B-curing formulation is made from diffuse
hedyotis (Hedyotis diffusa Willd.), rubia root (Radix Rubiae
Cordifoliae), Indigo Pulverata Levis, glabrous greenbrier
rhizome (Rhizoma Smilacis Glabrae), salvia root (Radix Salviae
Miltiorrhizae), finger citron fruit (Fructus Citri
Sarcodactylis), hawthorn fruit (Fructus Crataegi), Ganoderma
Lucidum, Ophiopogon tuber (Tuber Ophiopogonis Japonici), and
silkworm feces (Excrementum Bombycis Mori). The formulation has
been used for treating infectious HBV, acute and chronic
hepatitis, early-stage cirrhosis, swollen liver and spleen, etc.
It has a total effective rate of 84.75% and an HBsAg turning
negative rate of 41.35%, as shown in the study of 314 HBV
patients at Xian Medical University Second Affiliated Hospital
in China.
Ganpikang ("liver-spleen" health) capsule contains fourteen (14)
herbal components including bupleurum (Radix Bupleuri), San-qi,
and bear gallbladder (Vesica Fellea Ursi) powder. It has a
curing rate of 53.33% and an effective rate of 40.0 for chronic
active HBV, and a curing rate of 63.33% and an effective rate of
26.67 for chronic prolonged HBV.
Ruanjianhugan ("liver-protecting") tablet contains sophora root
(Radix Sophorae Tonkinensis), prunella (Spica Prunellae
Vulgaris), bushy knotweed root and rhizome (Radix et Rhizoma
Polygoni Cuspidati), scutellaria (Radix Scutellariae
Baicalensis), salvia root (Radix Salviae Miltiorrhizae),
astragalus (Radix Astragali Membranaceus), ligustrum (Fructus
Ligustri Lucidi), cardamon (Fructus Amomi), and hawthorn fruit
(Fructus Crataegi). It shows that 78% of the patients having
HBeAg turned negative, 28-57% of the patients having HBsAg
turned negative.
However, despite the effectiveness of the above herbal medicinal
compositions in treating hepatitis, none of these compositions
demonstrates significant effects on HBV antigen turning
negative.
The present invention provides a novel pharmaceutical
composition for treating liver diseases, particularly for
treating patients with viral hepatitis (e.g., HAV, HBV, HCV and
HEV), alcoholic or fatty liver, and liver cancer. The
compositions described in the present invention also
demonstrates significant clinical effects on patients with HIV.
This composition is a natural Chinese medicine with little or no
side effects and has no toxicity.
BRIEF
SUMMARY OF THE INVENTION
The novel medicinal composition of the present invention
comprises herb extracts from diffuse hedyotis, giant knotweed
rhizome, bistort rhizome, Asiatic moonseed rhizome, baical
skullcap root, bovine biliary powder, milkvetch root, barbary
wolfberry fruit, sanqi, red ginseng, figwort root, Chinese
magnoliavine fruit, turmeric root-tuber, hawthorn fruit, and
Chinese angelica. The composition is effective in treating
patients with liver diseases, including, but not limited to
viral hepatitis (e.g., HAV, HBV, and HCV, and HEV), alcoholic or
fatty liver, liver cirrhosis and liver cancer. It is also
effective for treating patients with HIV.
Among the herbs used in the composition, diffuse hedyotis,
bistort rhizome, giant knotweed rhizome, and Chinese
magnoliavine fruit are the necessary ingredients that provide
for the efficacy of the composition. Asiatic moonseed rhizome,
baical skullcap root, bovine biliary powder, tumeric root-tuber,
hawthorn fruit, and sanqi are used mainly to improve or enhance
the flavour, toning, and medicinal effects of, and to balance
the excessive effects cause by diffuse hedyotis, bistort
rhizome, giant knotweed rhizome, and Chinese magnoliavine fruit.
In addition, barbary wolfberry fruit, red ginseng, figwort root,
Chinese angelica and milkvetch root can be added to the
composition to provide further nutrition to the liver during the
recovery stage.
The weight ratio of diffuse hedyotis, bistort rhizome, giant
knotweed rhizome, and Chinese magnoliavine fruit is preferred to
be about 3:3:1:2. The weight ratio of diffuse hedyotis, bistort
rhizome, giant knotweed rhizome, Chinese magnoliavine fruit,
asiatic moonseed rhizome, baical skullcap root, bovine biliary
powder, tumeric root-tuber, hawthorn fruit, and sanqi is
preferred to be about 3:3:1:2:1:1:0.1:1:2:1. The weight ratio of
diffuse hedyotis, bistort rhizome, giant knotweed rhizome,
Chinese magnoliavine fruit, asiatic moonseed rhizome, baical
skullcap root, bovine biliary powder, tumeric root-tuber,
hawthorn fruit, sanqi, barbary wolfberry fruit, red ginseng,
figwort root, Chinese angelica, and milkvetch root is preferred
to be about 3:3:1:2:1:1: 0.1:1:2:1:3:1:2:1:3.
The present invention also provides a method for preparing the
herbal pharmaceutical composition, which comprises the steps of:
(1) grinding and mixing the entire plant of diffuse hedyotis,
the dried rhizome of bistort rhizome, the dried rhizome of giant
knotweed rhizome, and the dried ripe fruit of Chinese
magnoliavine fruit to form a herbal mixture; (2) boiling the
herbal mixture in water in two times (first by boiling the
mixture in water for 2 hours, then, after the mixture has cooled
down, boiling the mixture again for 1.5 hours); (3) filtering
the boiled herbal mixture to separate the herbs from the herbal
solution; (4) concentrating the herbal solution (preferrably
concentrating from about 1.4 fold by volume to about 1 fold by
volume); and (5) spray-drying and granulating the concentrated
herbal solution into granules, which can be further
encapsulated.
DETAILED
DESCRIPTION OF THE INVENTION
Traditional Chinese medicine has been in existence for more than
two thousand years. It has a proven record of success for curing
many kinds of diseases. Traditional Chinese medicine utilizes a
variety of herbs and natural substances. Each herb/natural
substance has its unique characteristics. By combining and
balancing the unique characteristics of herbs, a doctor can
prescribe a formulation with enhanced medicinal activities and
with less or no toxicity by synergizing the medicinal effects
among various herbs, while in the meantime, cancelling out or
neutralizing the toxic effects of the herbs. This, in Chinese
herbal medicine, is regarded as to regulate between
negative/hypoactive characteristics ("yin") and
positive/hyperactive characteristics ("yang"),
Under the definitions set forth in the traditional Chinese
medicine, "yin" is defined as drugs which cure cold syndrome
(which itself has hot or warm property), and "yang" is defined
as drugs which cure heat syndrome (which itself has cold or cool
property).
The pharmaceutical combination of the present invention
comprises fifteen (15) ingredients, in which four (4)
ingredients are the core ingredients which contribute to the
primary efficacy and healing effect of the composition. They
are: (1) diffuse hedyotis/spreading hedyotis (Pharmaceutical
name: Herba Hedyotidis diffusae; Botanical name: Hedyotis
diffusa Willd.); (2) bistort rhizome (Pharmaceutical name:
Rhizoma Bistortae; Botanical name: Polygonum bistorta L.); (3)
giant knotweed rhizome (Pharmaceutical name: Rhizoma Polygoni
Cuspidati; Botanical name: Polygonum cuspidatum Sieb. et Zucc.),
and (4) Chinese magnoliavine fruit (Pharmaceutical name: Fructus
Schisandrae Chinensis; Botanical name: Schisandra chinensis
(Turcz.) Baill., S. sphenanthera Rehd. et Wils.). The core
ingredients are functioned in clearing heat and toxic substances
while improving immune system and circulation, curing symptoms
of jaundice, and having beneficial effect on internal organs.
There are six (6) additional ingredients that are used to
improve and balance the pharmaceutical effects activities
produced by the above named core ingredients. These six
ingredients also have toning effect and can improve blood
circulation in the liver. These six ingredients are: (1) Asiatic
moonseed rhizome (Pharmaceutical name: Rhizoma Menispermi;
Botanical name: Menisermum dauricum DC); (2) baical skullcap
root (Pharmaceutical name: Radix Scutellariae; Botanical name:
Scutellaria baicalensis Georgi); (3) bovine biliary powder
(Zoological name: Vesica Fellea Bovus); (4) tumeric root-tuber
(Pharmaceutical name: Radix Curcumae; Botanical name: Curcuma
wenyujin Y. H. Lee et Cl Ling); (5) Hawthorn Fruit
(Pharmaceutical name: Fructus Crataegi; Botanical name:
Crataegus pinnatifida Bge.); and (6) sanqui (Pharmaceutical
name: Radix Notoginseng; Botanical name: Panax notoginseng
(Burk.)).
Finally, there are additional five (5) ingredients which are
used to primarily provide nutrients and energy sources for
patients so as to expedite the recovery process. These
ingredients include: (1) barbary wolfberry fruit (Pharmaceutical
name: Fructus Lycii; Botanical name: Lycium barbarum L.); (3)
figwort root (Pharmaceutical name: Radix Scrophulariae;
Botanical name: Scrophularia ningpoensis); (4) Chinese angelica
(Pharmaceutical name: Radix Angelicae sinensis; Botanical name:
Angelica sinensis (Oliv.) Diels); and (5) milkvetch root
(Pharmaceutical name: Radix Astragali; Botanical name:
Astragalus membranaceus (Fisch.) Bge.). Among these ingredients,
red ginseng (Radix Ginseng Rubra) and milkvetch root (Radix
Astragali) also have the capacity of improving immunological
functions of the body to fense off diseases.
The pharmaceutical names, botanical or zoological names, family
names, common descriptions, and major ingredients of the herbs
used in the present invention is shown in Table 1.
TABLE 1
Herbs of the Present Pharmaceutical Composition
Pharma- Botanical/
Common
ceutical Zoological
Descrip- Major
Name Name Family tion Ingredients
Herba Heydyotis Rubiaceae
heydyotis, hentriacontane,
Hedyotidis diffusa (Willd.)
olden- stigmastatrienol,
Diffusae Roxb., also
landia ursolic acid,
known
as oleanolic acid,
[beta]-
Oldenlandia
sitosterol, [rho]-
diffusa coumaric,
[beta]-
sitosterol-D-
glucoside
Radix et Polygonum Poly- Giant
emodin, chryso-
Rhizoma cuspidatum gonaceae
Knotweed phanol, rheic
Polygoni Sieb. et Zucc.
root and acid, emodin
Cuspidati
Rhizome monomethyl
ether,
polygonim, and
physcion-8-[beta]-D-
glucoside
Rhizoma Polygonum Poly- Bistort
n/a
Bistortae bistorta L. gonaceae Rhizome
Rhizoma Menispermum Meni- Asiatic
n/a
Meni- dauricum DC. sperm- Moonseed
spermi aceae Rhizome
Radix Scutellaria Labiatae Baical
baicalein,
Scutel- baicalensis
Skullcap baicalin,
lariae Georgi Root
wogonin,
Baica-
wogonoside,
lensis
neobaicalein,
oroxylin
aglucuronide,
camphesterol, [beta]-
sitosterol,
benzoic acid
Vesica Fellea
Bovine n/a
Bovus Biliary
powder
Radix Astragalus Leg- Milkvetch
D-[beta]-asparagine,
Astragali membranaceus uminosae
Root 2', 4'-dihydroxy-
(Fisch.)
Bge. 5,6-
var. dimethoxyiso-
mongholicus. flavane,
caly-
(Bge.) Hsiao
or cosin, formono-
Astragalus
netin,cyclo-
membranaceus
astragenol,astra-
(Fisch.)
Bge. galosides,
choline, betaine,
kumatakenin,
sucrose, glucoronic acid,
[beta]-sitosterol
Fructus Lycium Sol- Barbary
betaine, carotene,
Lycii barbarum L. anaceae
Wolfberry physalien,
Fruit thiamine,
riboflavin,
vitamin C, [beta]-
sitosterol,
linoleic acid
Radix Panax noto- Arali- San-chi,
Arasaponin A,
Noto- ginseng (Burk.) aceae noto-
arasaponin B,
ginseng F.H. chen, P.
ginseng, dencichine
pseudoginseng
Tian qi,
Wall, P. sanchi
Shen san
Hoo. qi
Radix Panax Ginseng Arali- Red
Panaxatriol,
Ginseng C. A. Mey aceae Ginseng
Panaxadiol,
Rubra
Other
Panoxisides,
Panoquilon,
Panaxin,
Ginsenin, [alpha]-
Panaxin,
Protopanaxadiol,
Protopanaxtriol,
Panacene,
Panaxynol,
Panaenic Acid,
Panose,
Dammarane,
Glucose,
Fructose,
Maltose,
Sucrose,
Nicrotinic Acid,
Riboflavin,
Thiamine
Radix Scroph- Scrophu- Figwort
1-asparagine,
Scrophu- ularia lariaceae Root,
oleic acid,
lariae ning- Scrophu-
linoleic acid,
Ning- poensis laria
stearic acid,
poensis Hemsl.
or carotene
S. buer-
geriana
Miq.
Fructus Schisandra Magno- Chinese
sesquicarene, [beta]-
Schis- chinensis liaceae
Magnolia- bisabolene, [beta]-
andrae (Turcz.) Baill.,
vine chamigrene, [alpha]-
Chinensis S. sphenanthera
Fruit, ylangene,
Rehd. et Wils.
schisandra schizandrin,
fruit pseudo-[gamma]-
schizandrin,
deoxyschizandrin,
schizandrol,
citral,
stigmasterol,
vitamin C,
vitamin E
Tuber Curcuma Zingi- Turmeric
d-camphene, d-
Curcumae wenyujin Y. H. beraceae
Root- camphor, 1-[alpha]-
Lee et C.
Ling., tuber, curcumene, 1-[beta]-
or Curcuma
curcuma curcumene,
Longa L.,
or curcumin,
Curcuma demethoxycurcu
aromatica min,
Salisb.,
or bisdemethoxycur
Curcuma cumin,
zedoaria
Rosc., turmerone,ar-
or
Curcuma turmerone,
kwangsiensis carvone,
[rho]-
S. G.Lee et
C. tolylmethylcarbi
F.
Liang noldiferuloyl-
methane
Fructus Crataegus Rosaceae
Hawthorn crategolic acid,
Crataegi pinnatifida Bge.;
Fruit citric acid,
C.
pinnatifida tartaric
acid,
Bge. var.
major flavone, sugars,
N.E. Br. or
C. glycosides,
suneata Sieb.
et vitamin C
Zucc.
Radix Angelica Umbel- Chinese
butylidene
Angelicae sinensis (Oliv.) liferae
Angelica phthalide,
Sinensis Diels root,
ligustilide, n-
tang-kuei butylidene-
phthalide,
sequiterpenes,
carvacrol,
dihydrophthalic
anhydride,
sucrose, vitamin
B12, carotene, [beta]-
sitosterol
Diffuse hedyotis or spreading hedyotis (Herba Hedyotidis
Diffusae) belongs to the family of Rubiaceae. The entire plant
is used as an herbal medicinal component. The herb has no
toxicity. The herb is harvested in summer and autumn in mainland
China and in late spring or early winter in Taiwan. In "Materia
Medica" (Chinese Herbal medicine), compiled and translated by
Dan Bensky & Andrew Gamble, diffuse hedyotidis clears heat
and resolves dampness by promoting urination. It is particularly
useful for relieving hot painful urinary dysfunction and
damp-heat jaundice. Diffuse hedyotidis is the major ingredient
in the present herbal pharmaceutical composition which
contributes to the medicinal effect on liver diseases and HIV.
Bistort rhizome (Rhizoma Bistortae) is the dried rhizome of the
plant Polygonum bistorta L. It belongs to the family of
Polygonaceae. Bistort rhizome has moderate cool property
(meaning that bistor rhizome is an "yang" herb). It can be used
to remove toxic heat, to promote the subsidence of swelling and
to stop bleeding.
Giant knotweed rhizome (Radix et Rhizoma Polygoni Cuspidati) is
the dried rhizome and root of polygonum cuspidatum Sieb. et
Zucc. It belongs to the family of Polygonaceae. The plant is
grown throughout China, especially Jiangsu, Zhejiang, Anhui,
Guangdong, Guangxi, Sichuan, and Guizhou provinces. The plant is
harvested in spring and autumn. Giant knotweed rhizome is
normally used to dispel damp, to eliminate blood stasis and
alleviate pain, to relieve cough, and to resolve phlegm.
Chinese magnoliavine fruit (Fructus Schisandrae) is the dried
ripe fruit of Schisandra chinensis (Turcz.) Baill. or Schisandra
sphenanthera Rehd. et Wils. It belongs to the family of
Magnoliaceae. The former, the best of its kind, is produced in
northern parts of China and is habitually called "Northern
schisandra fruit"; the latter is commonly referred to as the
"Southern schisandra fruit" as it is produced in the southern
parts of China. Both kinds can be used for the pharmaceutical
preparation of the present invention. The fruit is collected in
autumn and dried under the sun after removing the fruit stalks.
Chinese magnoliavine fruit is generally used to arrest
disharges, replenish qi, promote fluid secretion, tonify the
kidney, and induce sedation. Chinese magnoliavine fruit can also
decrease the level of GPT (glutamate-pyruvate transaminase) in
patients with hepatitis.
Asiatic moonseed rhizome (Rhizoma Menispermi) is the dried
rhizome of Menispermum dauricum DC. It belongs to the family of
Menispermaceae. Asiatic moonseed rhizome has cool property. It
can be used to remove toxic heat and relieve rheumatic pains.
Baical skullcap root (Radix Scutellariae) is the dried root of
Scutellaria baicalensis georgi. It belongs to the family of
Labiatae. The plant is produced in the provinces of Hebei,
Shanxi, Inner Mongolia, etc., and collected in spring or autumn.
Baical skullcap root is used to remove damp-heat, counteract
toxicity, arrest bleeding, and prevent abortion, in patients.
Bovine biliary powder is the gallbladder of the cow, Vesica
Fellea Bovus. It can clear heat and alleviate spasms.
Turmeric root-tuber (Radix Curcumae) is the dried root tuber of
Curcuma wenyujin Y. H. Lee et C. Ling., or Curcuma Longa L., or
Curcuma aromatica Salisb., or Curcuma zedoaria Rosc., or Curcuma
kwangsiensis S. G. Lee et C. F. Liang. The herb is mainly
produced in Sichuan, Zhejiang, Guangdong, and Guangxi provinces
in China, and harvested in winter or spring, washed clean after
the removal of the hairy rootlets, boiled thoroughly, and dried
in the sun. It belongs to the family of Zingiberaceae. Turmeric
root-tuber tastes bitter and had cool property. It can be used
to clear heat, alleviate spasms and chest pain, and resolve
phlegm.
Hawthorn fruit (Fructus Crataegi) is the dried ripe fruit of
Crataegus pinnatifida Bge. var major N. E. Br., or Crataegus
pinnatifida Bge., or Crataegus cuneata Sieb. It is produced
primarily in Henan, Jiangsu, and Shandong provinces of China. It
is harvested in autumn, sliced, and dried in sunlight. It
belongs to the family of Rosaceae. Hawthorn fruit is normally
used to stimulate digestion and promote the functional activity
of the stomach. It can also improve the normal blood flow and
dissipate blood stasis.
Sanqi, or San-chi, (Radix Notoginseng) belong to the family of
Araliaceae. Sanchi (Sanqi) is the dried root of Panax
notoginseng (Burk.) F. H. Chen. The plant is also known as P.
pseudoginseng Wall and P. sanchi Hoo. The plant grows in Yunnan,
Guangxi, Sichuan, Guizhou, and Jiangxi provinces of China, and
is harvested in the autumn or winter of the third or seventh
year, either before the flowers bloom (better) or after the
fruit is ripe. H. Gao et al., Pharmaceutical Research, (1996)
13(8): 1196-1200, disclose that polysaccharides from Panax
notoginseng (San-Chi) have immuno-stimulating activities in
vitro.
Barbary wolfberry fruit (Fructus Lycii) is the dried ripe fruit
of Lycium barbarum L. It belongs to the family of Solanaceae.
The plant is mainly produced in Ningxia, Gansu, and Qinghai
provinces of China. It is harvested in summer and autumn. It
nourishes and tonifies the liver and kidneys. It can also
replenish vital essence and improve eyesight.
Figwort Root (Radix Scrophulariae) is the dried root of
Scrophularia ningpoensis Hemsl. It belongs to the family of
Scrophulariaceae. The herb is chiefly produced in Zhejiang and
Sichuan provinces of China and harvested in winter when the part
of the plant above-ground has withered. The roots are piled and
dried in sunlight alternately until the inside becomes black and
then sliced for use. Figwort root can reduce heat from blood. It
als has nourishing capacity and can counteract toxicity.
Red ginseng (Radix Ginseng Rubra) is the steamed and dried root
of the cultivated form of Panax ginseng C. A. Mey (commonly
known as "Yuanshen"). The herb turns red after being steamed and
its properties become warmer in nature. It belongs to the family
of Araliaceae. The pharmaceutical effects of ginseng is in its
dried root. Ginseng has effects on central nervous system. It
enhances both stimulatory and inhibitory processes in the
central nervous system, thereby improving the adaptability of
nervous responses. Ginseng can also lower serum glucose and
cholesterol. It also shows therapeutic and preventive effect on
peptic ulcer.
Chinese angelica (Radix Angelicae Sinensis) is the dried root of
Angelica sinensis (Oliv.) Diels. It belongs to the family of
Umbelliferae. The herb is mainly produced in Gansu and Shanxi
provinces of China. It is harvested in late autumn, smoked dry
on slow fire after getting rid of the rootlets, sliced, or
stir-baked with wine. Chinese angelica can enrich blood, promote
blood circulation, regulate menstruation, relieve pain, and
relax bowels.
Milkvetch root (Radix Astragali) is the dried root of Astragalus
membranaceus (Fisch.) Bge. var. mongholicus. (Bge.)Hsiao or
Astragalus membranaceus (Fisch.) Bge. It belongs to the family
of Leguminosae. The herb is mainly produced in Shanxi, Gansu,
Heilongjiang, and Inner Mongolia of China. The plant of
four-year old or older is harvested in spring or autumn.
Milkvetch root can promote discharge of pus and the growth of
new tissue.
The herbal composition of the present invention was suitable for
preparation in a scale typical for pharmaceutical industry as
well as for smaller measure.
In the process for making the herbal composition of the present
invention, the individual herbal components are pretreated
according to the common procedures. The herbs are cut and put in
a container with water to boil and simmer twice. The first time
of simmering takes two hours, the solution is collected, and
water is added for the second time of simmering for 1.5 hour.
The solutions from the simmering steps are collected by passing
through a sieve/filter. The filtrate is then condensed from
about 1.4 fold by volume to 1.0 fold by volume. Subsequently,
the liquid condensate is spray-dried and granulated to form
particles. The particles are further packaged and preserved for
use or for further analysis by the conventional means of the
active ingredients to ensure their quality.
The composition of the present invention can further be
processed and formulated in a form suitable for oral
administration or intravenous injection.
The following example is illustrative, but not limiting the
scope of the present invention. Reasonable variations, such as
those occur to reasonable artisan, can be made herein without
departing from the scope of the present invention.
EXAMPLE 1
Pharmaceutical Preparation
The kinds and amounts of herbal ingredients used in the process
of making the pharmaceutical composition of the present
invention are described in Table 2.
TABLE 2
Ingredients Used In Example 1.
Amount Amount
Component (g) Component (g)
Diffuse heydyotis 90 Sanchi 30
Bistort Rhizome 90 Red Ginseng 30
Giant Knotweed root 30 Figwort root 60
and Rhizome
Asiatic Moonseed 30 Chinese
Magnoliavine 60
Rhizome Fruit
Baical Skullcap Root 30 Turmeric
Root-tuber 30
Bovine Biliary 3 Hawthorn fruit
60
powder
Milkvetch Root 60 Chinese Angelica 30
Barbary Wolfberry 90
Fruit
The individual herbal components are pretreated according to
common procedures. The herbs are weighed according to Table 2.
The herbs are cut into small pieces and put in a container with
water to boil and simmer twice, the first time for two hours,
and the second for 1.5 hour. After the first simmering, solution
is poured out and water is added to the container for the second
simmering. The solutions from the two simmering steps are
collected to pass through a sieve/filter, and then, condensed at
a ratio of 1:1.4. The liquid condensate is spray-dried and
granulated to form particles. The particles were further
packaged into about 1000 capsules. The capsules are called
"Yigan Kang capsules", abbreviated "YGK" capsules. The liquid
condensate can also be made for intravenous injection. The
injection solution is called "YGK" herbal injection solution.
The herbal composition of the present invention is called "YGK"
herbal composition.
EXAMPLE 2
Efficacy of the YGK Herbal Composition on Treatment of
Patients with Hepatitis B (HBV)
The clinical research was conducted in the Liberty Military
Hospital 211 in China. The course of hepatitis B is determined
by many factors, including immune response, host genetic
factors, and HBV mutations. The chronic hepatitis distinguishes
from the acute hepatitis. The acute hepatitis is the active and
symptomatic infection of the liver. A patient with the acute
hepatitis is contagious. Symptoms of acute HBV infection are
non-specific, but may include malaise, anorexia or jaundice. A
chronic hepatitis patient is asymptomatic. The HBV is present in
the liver and blood, although there are usually no obvious
physical symptoms. Specific blood tests will reveal the presence
of the virus, and the patient is also contagious via blood,
birth, sex, needles, etc. Cirrhosis is the pathological
dysfunctional state of the liver, the hardening of the liver as
the result of chronic hepatitis, chronic persistent hepatitis
(CPH) and chronic active hepatitis (CAH).
A total of 948 patients with acute HBV, chronic HBV, and liver
cirrhosis participated in a clinical comparative study. The
patients were divided into two (2) groups. The study group had
642 patients and the comparative group has 306 patients. The
data on patients who participated in this study are listed in
Table 3.
TABLE 3
Patients Data in the Clinical Study
Group Study Group Comparative Group
Total Number of 642 306
Patients
Sex Distribution of the Male: 482 Male: 229
Patients Female: 160 Female: 77
Age Distribution of 7 to 74 years old 8 to 70
years old
Patients (average age: 32.5) (average age:
30.5)
*Symptoms of Acute Hepatitis B: 282 Acute
Hepatitis B: 109
Patients' Chronic Hepatitis: 276 Chronic
Hepatitis B: 114
Liver Disease Cirrhosis: 84 Cirrhosis: 83
*According to the diagnosis criteria of Hepatitis revised
at the Shanghai Hepatitis Conference in 1980, Shanghai, China.)
The patients were treated according to the following regime:
(1) The patients in the study group were each orally
administered eight (8) YCK herbal composition containing the
herbal composition of the present invention per day.
(2) The patients in the comparative group were each orally
administered four (4) Hugang ("liver protecting") tablets per
day. A description of Hugang tablets has been provided in the
"Background" section, supra.
The treatment lasts for ninety (90) days.
Table 4 shows the results of this clinical comparative study.
TABLE 4
Effects of YGK Capsule Treatment
Group Number of Patients with Positive Effect* (%)
Study (642 patients) 456 (71.03%)
Comparative (306 patients) 104 (33.98%)
(p < 0.01)
*Positive effect means that the hepatitis B envelope
antigen (HBeAg) and HBV DNA of the patients turn negative after
taking the YGK herbal composition for 90 days.
As indicated in Table 4, approximately 71.03% of patients who
took the YGK herbal composition for 90 days show positive
responses to the herbal composition. This is contrary to the
comparative group where the patients were given a popular "liver
protecting" tablets which were available in the Chinese market.
Patients who had taken the "liver protecting" tablets only have
an effective rate of approximately 33.98% to show improvement in
their liver diseases.
The Hepatitis B virus (HBV) consists of a surface and a core.
The core contains a DNA polymerase and an e antigen. The DNA
structure is double stranded and circular. HBV has four (4)
genes encoding four (4) polypeptides: the S (surface), the C
(core), the P (polymerase), and the X (transcriptional
transactivating).
The S gene consists of three (3) regions, the pre-S 1 region,
the pre-S2 region, and the region that encodes the surface
protein (HBsAg). Very rarely a mutation occurs in the S gene
which aborts the production of HBsAg so that a person maybe
HBsAg negative but still has the virus present as determined by
HBV DNA. In addition, the HBsAg particles are antigenically
complex and the antigenic determinants have been identified as
one single common determinant designated a, and four (4) major
subdeterminants designated as d, y, w, and r. Thus, the four (4)
major determinants are adw, adr, ayw, and ayr.
The C gene consists of two (2) regions, the pre-core region and
the core region, which encodes for two different proteins, the
core antigen (HBcAg), and the e antigen (HBeAg). A mutation in
the pre-core region may stop the production of HBeAg, thus, a
person maybe HBeAg negative, but HBsAg positive and HBV DNA
positive. Another type of mutant in the core region is called
HBV2. The patients that have HBV2 mutant are HBsAg positive but
lack HBeAg and HBV DNA.
Because of the complexity and the antigenic differences among
the virus, there are a number of tests available for HBV
including:
(1) a test for HBsAg, which is an indicator of the presence of
the HBV;
(2) a test for HBeAg, which correlates with the viral
replication and infectivity, it indicates a high amount of the
virus in the blood, thus, is an indicator of the activity and
infectivity of the HBV; and
(3) a test for HBV DNA, which is an indication of the virus
presence and activity.
Tables 5-7 indicated the change of Hepatitis B envelope Antigen
("HBeAg"), Hepatitis B surface antigen ("HBsAg"), heptomegaly,
and splenomegaly in the patients after the treatment.
TABLE 5
Effect of Herbal Composition on HBeAg in Patients
Group Study Group Comparative Group
Acute Hepatitis Patients
Number of 260 78
Patients with
HbeAg(+)
Number of 48 59
Patients with
HbeAg(+) After
Treatment
Percentage of 81.5% 24.36%
Patients With
HbeAg Turning
Negative
Chronic Hepatitis Patients
Number of 206 82
HbeAg(+) Patients
Number of 74 64
HbeAg(+) Patients
After Treatment
Percentage of 64.0% 21.95%
Patients With
HbeAg Turning
Negative
Cirrhosis Patients
Number of 24 26
HbeAg(+) Patients
Number of 14 22
HbeAg(+) Patients
After Treatment
Percentage of 41.7% 15.38%
Patients With
HbeAg Turning
Negative
As indicated in Table 5, the percentages of patients with HBeAg
turning negative in all three (3) categories of patients
(including acute hepatitis, chronic hepatitis, and cirrhosis)
are 2.7-3.3 times higher than those of the comparative groups.
This demonstrates that the YGK herbal composition had
significant effect on HBeAg turning negative and inhibiting HBV
activity and infectivity.
TABLE 6
Effect of Herbal Composition on HBsAg in Patients
Group Study Group Control
Group
Acute Hepatitus
Patients
Number of 262 84
Patients with
HBsAg(+)
Number of 116 73
Patients with
HBsAg(+) After
Treatment
Percentage of 55.7% 13.09%
Patients With
HBsAg Turning
Negative
Chronic Hepatitis Patients
Number of 216 87
HBsAg(+) Patients
Number of 118 78
HBsAg(+) Patients
After Treatment
Percentage of 45.37% 10.30%
Patients With
HBsAg Turning
Negative
Cirrhosis Patients
Number of 64 43
HBsAg(+) Patients
Number of 50 40
HBsAg(+) Patients
After Treatrnent
Percentage of 21.88% 6.98%
Patients With
HBsAg Turning
Negative
As indicated in Table 6, the percentages of patients with HBsAg
turning negative in all three (3) categories of patients
including acute hepatitis, chronic hepatitis, and cirrhosis were
3.1-4.4 times of those of the comparative groups. This
demonstrates that the YGK herbal composition had significant
effect on HBsAg turning negative and inhibiting the HBV.
In addition to HBeAg and HBsAg turning negative, the YGK herbal
composition also show greater effects on increased appetite and
decreased various symptoms of liver diseases than the
comparative group using Hugang "liver protecting" tablets.
TABLE 7
Effect on Hepato-Splenmegaly
Group Reduced Hepatomegaly Reduced
Splenomegaly
Study Group 79.72% 58.54%
Comparative Group 30% 28.8%
Heptomegaly and splenomegaly are related to and possibly caused
by viral infection. The reduced hepatomegaly and splenomegaly in
patients was indicative to reduced symptoms of viral infection.
In summary, the YKG herbal composition demonstrates effect on
treating patients with HBV, which including acute hepatitis B,
chronic hepatitis B, and cirrhosis.
EXAMPLE 3
Effects of the YGK Herbal Composition on Treatement of Patients
with Chronic Hepatitis B (HBV)
The clinical research was conducted in the Liberty Military 302
Hospital, Ninth Section, China. The research was conducted on
treatment effects of the herbal composition of the present
invention on chronic hepatitis B patients.
Chronic Hepatitis is an ongoing injury to the cells of the liver
with inflammation which lasts for longer than six months. The
causes of chronic hepatitis include: viruses, metabolic or
immunologic abnormalities and medications. Symptoms resulted
from the injury of hepatocytes, the inflammation or from the
resulting scarring is called cirrhosis. Chronic hepatitis may
follow acute hepatitis B or C or may develop quietly without an
acute illness. Liver biopsy is helpful in that it confirms the
diagnosis, aids in establishing the cause (etiology) and can
demonstrate the presence of cirrhosis. It is less helpful in
judging the response to treatment. Approximately 25% patients
with chronic hepatitis B will develop cirrhosis, causing
permanent and serious liver damage. Chronic carriers of HBV are
far more likely to develop hepatocellular carcinoma than
non-carriers.
It is believed that chronic infections develop as the result of
a weak T helper (Th) cell response to the virus, in particular
to the HBsAg. The T cell response is responsible for clearing
the infected cells in the host's system. When the clearance is
inefficient and the infected cells persist in the body, a
chronic infection develops. As the HBsAg titer increases, the
patient moves into acute, symptomatic disease. When the titer of
anti-HBsAg rises, the symptoms of HBV begin to decline and
patient reaches the immune state.
Chronic hepatitis has been divided into two categories based on
histologic findings: chronic persistent hepatitis (CPH) and
chronic active hepatitis (CAH). Characteristically, specimens
from liver biopsy identified as CPH show inflammation confined
to the portal triad (does not penetrate the limiting plate).
Specimens identified as CAH show inflammation that penetrates
the limiting plate, extending to the surrounding individual
hepatocyte and yielding piecemeal necrosis. Under this schema,
CAH eventually reaches a point where lobular architecture is
destroyed, and bands of necrosis (bridging necrosis) are
replaced by scar tissue (bridging fibrosis), resulting in the
characteristic features of cirrhosis.
Sixty (60) patients with chronic hepatitis B are divided into
two (2) groups, one group for treatment with YKG herbal
composition and the other with Hugang ("Liver protecting")
tablets. The study was conducted and maintained for three (3)
months. The patients information in the two (2) groups are shown
in Table 8:
TABLE 8
Compositions of the Patients in the Clinical Study
Group Study Group Comparative Group
Total Number of Patients 30 30
Sex Distribution of the Male: 26 Male: 25
Patients Female: 4 Female: 5
Age Average Patients 32.8 35.1
Duration of Illness 2 months to 11 years 2
months to 9 years
*Symptoms of Patients' CPH: 13 CPH: 10
Liver Disease CAH: 17 CAH: 20
*According to the diagnosis criteria of hepatitis revised
at the Shanghai Hepatitis Conference in 1980.
Table 9 shows the changes in HBsAg, HBeAg, and HBV-DNA in
patients after treatment with the YGK herbal composition (the
study group) or Hugang tablets (the comparative group).
As indicated above, HBsAg can be detected in patients with acute
infection as well as patients who are chronic HBV carriers. In
the serological test, decreased titer of HBsAg indicates that
the symptoms of HBV are lessened and the patient is approaching
the immune state.
TABLE 9
The Changes of HBsAg, HBeAg, and HBV-DNA in Patients
HBsAg HbeAg HBV-
Sero- Decreased Sero-
Decreased DNA SGPT
Negative Titer
Negative Titer Sero- Recovery
Group (%) (%) (%) (%)
Negative Rate (%)
Study Group 1/30 6/30 12/26
6/26 9/15 73.33%
(3.33%) (20.00%)
(46.15%) (23.08%) (60.00%)
Comparative 0/30 2/30 5/27
2/27 4/18 71.43%
Group (0%) (6.67%) (18.52%)
(7.41%) (22.22%)
p<0.05.
As indicated in Table 9, the YGK herbal composition has
significant effects on chronic hepatitis patients. Patients
treated with the YGK herbal composition have Serum Glutamic
Pyruvic Transaminase (SGPT/ALT) recovery rate of 73.33%, HBeAg
turning negative rate of 46.15%, HBV-DNA turning negative rate
of 60.00%, suggesting that the YGK herbal composition has
significant effects on inhibition of HBV replication and
presence and depletion of aminotransferase. In addition, there
was no toxic adverse reaction on the patients treated with the
YGK herbal composition, accodring to clinical obervation.
EXAMPLE 4
Case Studies on Effects of the YGK Herbal Composition on
Patients with Hepatitis B
The clinical research was conducted in the Contagious Disease
Department of People's Liberation Army Hospital Branch 113 in
China. The research was conducted on treatment effects of the
YGK herbal composition on hepatitis B patients.
Each patient was tested for various markers. Serum alanine
aminotransferase (ALT) is an enzyme appears in liver cells, with
lesser amounts in the kidneys, heart, and skeletal muscles. When
such damage occurs, ALT is released from the liver cells into
the bloodstream, often before jaundice appears, resulting in
abnormally high serum level of ALT that last for days or weeks.
ALT is a relatively specific indicator of acute liver cell
damage. Serum bilirubin (BIL) is also tested as an indication of
liver diseases.
Case #1 was a twenty-four years old male patient with chronic
hepatitis B, with general weakness for more than one year. Table
10 shows the diagnoses of patient case #1 before and after
treatment with the YGK herbal composition:
TABLE 10
Diagnoses of the Patient #1 Before and After the
Treatment
TBIL
ALT
HBcAb PCR
(nmol/L) (U/L) HBSAg
HbsAb HBeAg HbeAb HbcAg (IgM)
HBV-DNA
Before 42 231 + - +
- + + ++
Treatment
(1:64)
After 18.6 66 - + -
+ + + --
Treatment
Table 10 indicates that the patient was in a state of immunity
towards HBV and with alleviated infection as shown by the
significant decrease of the viral DNA, and viral proteins,
HBsAg, HBeAg, HBcAg, with increased amount of the antibodies
against the viral protein in the serum.
Case #2 was a sixty-six years old male patient with recurrent
abdominal fullness and general weakness for about ten (10) years
with liver cirrhosis and splenomegaly. The following are the
diagnoses of the patient before and after treatment with the YGK
herbal composition (Table 11).
TABLE 11
Diagnoses of the Patient #2 Before and After
Treatment with the YGK herbal composition
TBIL ALT HBcAb PCR
(nmol/L) (U/L) HBsAg
HbsAb HBeAg HbeAb HbcAg (IgM)
HBV-DNA
Before 44.8 382 + - +
- + + +++
Treatment
(1:64)
After 25.3 43.8 + - -
+ + - +
Treatment
(1:32)
Table 11 shows that patient #2 was in a state of alleviated
infection symptoms towards HBV as shown by the significant
decrease of viral DNA, and viral proteins., The data also show
an increase in immunity as evidenced by reduced amount of HBsAg,
HBeAg, HBcAg, and an increased amount of the antibodies against
the viral proteins in the serum.
Case #3 was a thirty-one years old male patient with general
weakness for more than one (1) month, treated in local Chinese
Medicine clinic and subsequently hospitalized as acute biliary
hepatitis B patient. The following are the diagnoses of the
patient before and after treatment with the YGK herbal
composition (Table 12).
TABLE 12
Diagnoses of the Patient #3 Before and After
the Treatment With the YGK Herbal composition
TBIL ALT HBcAb PCR
(nmol/L) (U/L) HBsAg
HbsAb HBeAg HbeAb HBcAg (IgM)
HBV-DNA
Before 154 520 + - +
- + + +++
Treatment
(1:64)
After 22.1 29.1 + - -
+ + - +
Treatment
(1:32)
Table 12 shows that the patient was in a state of alleviated
infection symptoms towards HBV as shown by the significant
decrease of viral DNA, and viral proteins. The data also show an
increase in immunity as evidenced by reduced amount of HBsAg,
HBeAg, HBcAg, and an increased amount of the antibodies against
the viral proteins in the serum.
Case #4 was a forty-five years old male acute biliary hepatitis
B patient with recurrent abdominal fullness, abdominal pain and
general weakness for about one week. The following are the
diagnoses of the patient before and after the treatment with the
herbal composition of the present invention (Table 13).
TABLE 13
Diagnoses of the Patient #4 Before and After
the Treatment With the YGK Herbal composition
TBIL ALT HBcAb PCR
(nmol/L) (U/L) HBsAg
HbsAb HBeAg HbeAb HBcAg (IgM)
HBV-DNA
Before 143 966 + + +
- + + ++
Treatment
(1:64)
After 15.3 42.1 + - -
+ + - --
Treatment
(1:32)
Table 13 shows that the patient is in a state of alleviated
infection symptoms towards HBV as shown by the significant
decrease of viral DNA, and viral proteins. The data also show an
increase in immunity as evidenced by reduced amount of HBsAg,
HBeAg, HBcAg, and an increased amount of the antibodies against
the viral proteins in the serum.
Case #5 was a thirty-one years old male acute biliary hepatitis
B patient with abdominal fullness and general weakness for about
five (5) days and then admitted. The following are the diagnoses
of the patient before and after the treatment with the herbal
composition of the present invention (Table 14).
TABLE 14
Diagnoses of the Patient #5 Before and After
Treatment With the YGK Herbal composition
TBIL ALT HBcAb PCR
(nmol/L) (U/L) HBsAg
HbsAb HBeAg HbeAb HBcAg (IgM)
HBV-DNA
Before 47.7 694 + + +
- + + ++
Treatment
(1:64)
After 19.8 138 + + +
- + - -
Treatment
(1:32)
Table 14 shows that the patient is in a state of alleviated
infection symptoms towards HBV as shown by the significant
decrease of viral DNA, and viral proteins. The data also show an
increase in immunity as evidenced by reduced amount of HBsAg,
HBeAg, HBcAg, and an increased amount of the antibodies against
the viral proteins in the serum.
Table 15 shows the percentage of patients with therapeutic
effects in different markers.
TABLE 15
Therapeutic Effects on Patients After
Treatment with the YGK Herbal composition
Therapeutic Effects Percentage of
Patients*
Obvious therapeutic effects
80.9%
Improved therapeutic effects
19.10%
Hepatomegaly 75%
Splenomegaly 62.5%
Normalizationof liver function
ALT 93.7%
Bilirubin 91.1%
Seroconversion
HBsAg(+) to HbsAg(-) 33.3%
HBsAb(-) to HbsAb(+) 23.8%
HbeAg(+) to HbeAg(-) 68.6%
HbeAb(-) to HBeAb(+) 23.9%
HBcAb(+) to HbsAb(-) 43%
HBV-DNA(+) to HBV-DNA(-)
39.5%
*The study included a total number of
42 patients (male: 31; female: 11), who were aged between 16 and
63 (average age: 42). Before treatment, twenty six (26) of the
patients were diagnosed with acute hepatitis B, eight (8) with
chronic hepatitis B; and eight (8) with chronic active hepatitis
B. Thirty eight (38) patients had abnormal serum ALT. Thirty
four (34) patients had abnormal serum BIL. Forty two (42)
patients had HBV Marker (positive+).
#Thirty eight (38) patients had HBV-DNA
as tested by PCR (positive+). Thirty five (35) patients were
HBeAg positive. Thirty two (32) patients were anti-HAV,
anti-HCV, anti-HEV.
Results
The patients after being treated with the YGK herbal composition
showed improvement of subjective symptoms, especially pain on
liver area, fast normalization of liver function. Their ALT
levels started to fall in about sixteen (16) days generally.
Possible anti-viral activity was shown in the patients: the rate
of HBeAg turning negative was commonly found in the YGK herbal
composition treated patients (68.6%). No side-effects were noted
in the treated patients.
EXAMPLE 5
Effects of the YGK Herbal Composition On Animals With
Liver Diseases
The animal study was conducted at Korean Central Research
Center.
Experiment 5.1
Analysis of Effect on Alcoholic or Fatty Liver in White Rats
Purpose
The experiment was conducted to investigate effects of the
herbal composition on alcohol metabolism in white rats,
especially, the influence on the ability to transform alcohol to
triglyceride and cholesterol. The experimental dosage was 1
g/kg.
Method
The experimental animal used was male SD white rat with weight
of 200 g. Blood sampling of the experimental animal was taken
through orbital vein plexus. The animal was administered for the
herbal composition of the present invention three (3) times a
day for seven (7) days.
The experimental animals were divided into the control group and
the study group. The control group animals were administered
alcohol for one week. The study group animanls were administered
alcohol and concomitantly with 1 g/kg of the YGK herbal
composition for one week. The rats' livers were tested for
triglyceride and cholesterol level, lipid hyperoxidation, and
glutathione peptide.
Results
After one (1) week of alcohol administration, triglyceride and
cholesterol levels in the rats' liver were increased; lipid
hyperoxidation and diminished glutathione peptide occurred in
the control group. In contrast, in the study group, the fatty
metamorphosis of the liver was inhibited. Also, the processes of
lipid hyperoxidation and diminished glutathione peptide were
inhibited in the study group animals.
Conclusion
The YGH herbal composition prevents accumulation of triglyceride
and cholesterol levels in the liver which follows alcohol
consumption, thus providing beneficiary effects on the liver
functions.
Experiment
5.2
Analysis of Effect on Liver Cirrhosis in White Rats
Purpose
The experiment was conducted to investigate the effect of the
YGK herbal compositions on protein synthesis in white rats with
liver cirrhosis.
Method
The experimental animal used was male SD white rat with weight
of 200 g. Blood sampling of the experimental animal was taken
through orbital vein plexus. The animal was administered for the
herbal composition of the present invention three (3) times a
day for seven (7) days.
1. Induction of Liver Cirrhosis in the Rats
The rats were injected subcutaneously on the back with 1 ml/200
g 50% chloroform (CCl4) diluted in olive oil, for three (3)
times a week for four (4) weeks. Liver biopsy was conducted
through midline laparotomy. Most animals needed six (6) weeks of
injection to induce liver cirrhosis. The injection dosage was
adjusted each week in accordance to the weight of the rats.
Due to liver cirrhosis and partial liver resection, the serum
alanine aminotransferase (ALT) and serum aspartate
aminotransferase (AST) significantly increased in the rats.
2. Treating Rats with the YGK Herbal Composition
The rats in the study group were subdivided into three (3)
groups which were respectively administered the YGK herbal
composition of the present invention for 500 mg/kg, 1000 mg/kg,
or 2000 mg/kg.
Results
1. ALT and AST Levels: after the treatment with the YGK herbal
composition, the serum ALT and AST levels decreased in all three
(3) different dosage treatment groups. The liver cirrhosis
process was inhibited.
2. Hepatocyte Regeneration: after the administration of the
herbal composition in three (3) different doses, the rates of
liver regeneration in the rats were 19%, 30%, and 47%,
respectively, higher than the rats with liver cirrhosis and
partially resected livers which were not treated with the herbal
composition, and the rates of liver regeneration in the treated
rats were also 51%, 70%, and 92%, respectively, higher than the
partially liver resected rats with normal liver functions.
Conclusion
The YGK herbal composition was effective in liver regeneration
and had effectively inhibited the liver cirrhosis process.
EXAMPLE 6
Toxicity Study of the YGK Herbal Composition in Animals
Purpose
The following experiment was conducted at the Toxicology
Laboratory of the Institute of Labor, Health, and Occupational
Disease of Heilungkiang Province in China to examine acute
toxicity of the YGK herbal composition during intravenous
injection in animals.
Methods
Experimental animals were Japanese big-ear white rabbits
obtained from the Animal Center of Haerbin Medical University in
Haerbin, Heilungkiang Province, China. These rabbits were
characterized by the obvious blood vessels on ears which
facilitates the operation of injection during the experiments.
Ten (10) rabbits were obtained including six (6) males and four
(4) females, each weighing between 1900 g to 3000 g.
The rabbits were randomly divided into two (2) groups, five
rabbits in each group including two (2) females and three (3)
males. The YGK herbal composition was intravenously injected
into the rabbits through the veins on their ears at dosages of
10 g/kg and 15 g/kg, respectively, for two groups.
The concentration of injection fluid containing the herbal
composition was about 1 g/ml. So the higher dosage group at 15
g/kg has a concentration of about 15 ml/kg, which could be
calibrated as a sixty (60) kg-weighted adult who was treated by
900 ml of the herbal composition at a time.
The rabbits were observed for behaviour continuously for a
period of two (2) weeks after intravenous injections.
observation was conducted hourly at day 1; during the following
days, observation was conducted four-six (4-6) times per day.
At the end of the observation period, rabbits were sacrificed
and dissected to examine the eyes, liver, lung, and spleen for
adverse effects.
Results
No abnormal behavior was observed of the rabbits during the
observation period. The rabbits showed normal body weight
increase during the period. After the sacrifice and dissection,
inspection of the eyes, liver, lung, and spleen showed no
extraordinary syndromes. The results when compared to a general
acute toxicity index were normal and no acute toxicity.
EXAMPLE 7
Effects of the YGK Herbal Composition on HIV in Cell
Cultures
Purpose
The following experiment was conducted in the Military Medical
Research Institute in China to examine the effectiveness of the
YGK herbal composition of the present invention in the form of
intravenous product against HIV.
Methods
MT4 cells were cultured in HIV-1 suspension liquid of 100 TCID50
in a 96-hole culture plate. The culture condition was set at a
temperature of 37[deg.] C. and under 5% CO2. The culture time
was seven (7) days.
The YGK herbal composition of the present invention were added
into the wells at various concentrations. The morphology of the
MT4 cells were observed by conventional methods.
Results
No pathological changes of MT4 cells were observed in wells
where the YGK herbal composition was added to in adequate
concentrations. The inhibition of the pathological changes of
MT4 cells indicated that the YGK herbal composition had
inhibitory effect on pathological changes of the cultured cells
caused by HIV.
The effective concentration of the YGK herbal composition for
inhibition of the pathological changes of MT4 cells was more
than 12.5 mg/ml. To achieve a 50% of inhibition, the
concentration of the YGK herbal composition was 25 mg/ml.
Conclusion
The YGK herbal composition was effective in inhibiting
pathological changes in cells caused by HIV-1 in vitro.
EXAMPLE 8
A Case Study on an HIV-Patient Treated With the YGK
herbal Composition
Purpose
The following clinical trial was conducted in the Infectious
Disease Hospital in Shanghai, China to test the effectiveness of
the herbal composition of the present invention in treating an
HIV-infected patient.
Methods
A fifty-year Chinese male patient diagnosed with HIV infection
complicated by herpes zoster was treated with anti-virus
regimens by the combination of western medicine and the herbal
composition of the present invention during hospital stay.
Results
The patient was confirmed of HIV-infection by Rapid Agglutinin
Assay. At the time of the initial diagnosis in August 1996, the
patient showed no symptoms. Starting Jun. 1, 1997, the patient
quickly developed an herpetiform rash over the front of the left
side of the check extending over the nick, the shoulder, and the
upper left arm. The patient was then admitted into the Hospital
in Jun. 24, 1997.
At the hospital, the result of the physical examination was
normal except the skin rash. The pathology tests confirmed
normal renal function. The functional tests of the liver showed
a slightly increased levels of serum [gamma] glutamyl
transpeptidase and acetyl glucuronidase. Hepatitis viral tests
showed negative for Hepatitis B virus and Hepatitis C virus
(HBV-DNA and HCV-RNA). However, Hepatitis G viral test showed
positive for HGV-RNA. The immunological studies showed that the
[beta]-2 microglobulin level was 2.4-2.5 mg/ml.
During the hospital stay, haemoglobin and erythrocytes levels of
the patient were slightly decreased, while the levels of the
leukocyte and platelet were normal. Peripheral blood lymphocytes
counts showed that T4 cells were decreased to 2.76*10/L (32.9%)
and the ratio of T4/T8 cell was 1.16. Thus, the diagnosis is
that the patient was with HIV infection complicated by herpes
zoster.
During hospital stay, the patient had diarrhea and dry cough for
a few days and was cured. In September, 1997, the patient showed
HIV antibody positive by ELISA, and his T4 cells further
decreased to 25.4% and the ratio of T4/T8 cells was inverted to
0.94. Then, T4 cells and the ratio of T4/T8 gradually increased
after treatment with the YGK herbal composition and as tested in
November 1997, his T4 cells were 40.7%, and the ratio of T4/T8
1.45. The skin rash gradually disappeared and completely
recovered by the end of November.
Conclusion
The YGK herbal composition was effective in reducing symptoms of
the HIV-infected patient in a treatment regime together with
western medicine.
EXAMPLE 9
Clinical Trial on HIV-Infected Patients Treated with the
YGK Herbal Composition
Purpose
The following clinical trial was conducted in De-Tang Hospital
(National AIDS Therapy Center) in Beijing, China to test the
effectiveness of the herbal composition of the present invention
in treating HIV-infected patients.
Methods
Five (5) HIV-infected patients were treated with the YGK herbal
composition. The infection was confirmed by western blotting.
The profile of the patients were as follows:
Patients Sexuality Age History
Diagnosis
1 Male 32 2 years AIDS
(Stage IV)
2 Female 32 1 year AIDS
(Stage IV)
3 Male 31 1 year AIDS
(Stage III)
4 Male 25 0.5 year AIDS
(Stage II)
5 Male 17 3 weeks HIV Infection
The patients were treated according to the following regimen:
Five (5) ml injection fluid herbal composition of the present
invention was dissolved in 250 ml 5% glucose solution. The
solution was injected intravenously once per day for three (3)
days. Then, the dosage was increased to 15 ml injection fluid in
250 ml 5% glucose solution, and the patients were injected
intravenously once per day without uncomfortable reactions for
three (3) months.
Additionally, patient #1 was treated with AZT+DDI therapy for
ten (10) days before being treated with the YGK herbal
composition; patient #5 was treated with combination of
interferon and the herbal composition.
Three (3) ml blood sample was taken from the patients each time
before, during, and after the treatment and further tested for
HIV.
Results
The HIV counts of the patients are as
follows: 1st
month 2nd month 3rd month,
before during during
at the end
Patients treatment treatment
treatment of treatment
1 1.9 * 10<4> 1.7 * 10<5>
6.3 * 10<3> 1.5 * 10<4>
2 1.5 * 10<4> 6.3 *
10<3> 3.8 * 10<2>
3 7.3 * 10<3> 3.2 *
10<3>
4 3.0 * 10<5> 1.9 *
10<4> 1.9 * 10<4>
5 3.9 * 10<5> 2.6 * 10<3>
1.8 * 10<3> *
Note: the control level of HIV is 3,000.
Based on the above table, all patients showed decreased HIV
level and increased CD4 cells, except in patient #5 who was also
treated with interferon. Especially, patient #2 had significant
decrease of HIV; his CD4 counts also dropped from 285/mm<3
>to 510/mm<3>.
Conclusion
The herbal composition of the present invention is effective in
reducing HIV in serum in HIV-infected patients.
EXAMPLE 10
Clinical Trial on HIV-Infected Patients Treated with the
YGK Herbal Composition In Russia
Purpose
The following experiment was conducted in Hospital in Siberia,
Russia to amine the effectiveness of the YGK herbal composition
of the present invention against HIV.
Methods
Five (5) HIV-infected patients were treated with the YGK herbal
composition. The profile of the patients were as
follows:Patients Sexuality Age History
Diagnosis
1 Female 23 2 years AIDS (Phase
A3), adenitis,
hepatitis C, Syphilis,
Citomegalo infection, Gonorrhea
2 Female 28 2 year AIDS (Phase
A3), adenitis,
hepatitis B and C, Gerpec and
Citomegalo infection, Gonorrhea,
drug abuse
3 Male 35 1 year AIDS (Phase B2),
adenitis
4 Male 22 1 year AIDS (Phase B2),
adenitis,
hepatitis C, drug abuse
5 Male 34 several AIDS (phase
A3), adenitiis
months hepatitis B and C, 10% weight loss, drug abuse
2. The patients were treated with the herbal composition of the
present invention.
Samples were taken from the patients each time before, during,
and after the treatment and further tested for CD4 cells.
Results
The CD4 cells counts of the patients are as follows:
Patients before treatment / 2nd month
treatment / 5th month at the end of treatment
1 477 641 849
2 740 1140 705
3 421 . . . 527
4 440 490 669
5 625 . . . 814
Note: the normal level of CD4 cell count is about 500.
During the treatment process, all patients had positive response
except some minor side effects. The symptoms of the patients
were improved after one month of treatment including alleviation
of weakness, depression, and stegnosis. The abdominal region
pain and uncomfortable feeling also disappeared. Patients #4 and
#5 had 5 kg increase of body weight after three (3) months of
treatment. Patients #2 and #4 were disintoxicated. The
biological marker of the liver showed normal after all patients
after the treatment.
Based on the above table, all patients showed increased CD4 cell
counts except patient #2.
Conclusion
The herbal composition of the present invention is effective in
reducing symptoms in AIDS patients.