Mohammed
FARHADI, et al.
Stinging Nettle vs HIV/AIDS
Stinging Nettle vs HIV/AIDS
http://www.silentcures.com/The-AIDS-Cure.html
Iran
unveils herbal remedy against AIDS
Tehran announces country’s scientists produced herbal medicine that boosts human's immunity against HIV.
TEHRAN - Health Minister Kamran Baqeri Lankarani on Saturday announced that Iran's scientists have produced a herbal medicine that boosts the human's body immunity system against the HIV/AIDS virus.
"The herbal-based medication, called IMOD, serves to control the AIDS virus and increases the body's immunity," Baqeri Lankarani was quoted as saying by the official news agency IRNA.
"It is not a medication to kill the virus, it rather can be used besides other anti-retroviral drugs," Baqeri Lankarani said on state radio.
The drug, made after five years of research, has been tested on 200 patients, IRNA said, adding that it is considered the fifth generation of medications helping control the HIV/AIDS virus.
"This is a substance good for both AIDS patients and those who carry the virus without showing the symptoms," the director of the project, Mohammad Farhadi, told state television.
Farhadi said the medication will now be tested on some 3,000 to 5,000 Iranian patients in the next year to monitor its efficacy.
Health Minister Baqeri Lankarani said that the number of HIV/AIDS cases in Iran stands at around 14,000 while 1,700 people have died of the disease.
Last June, Iranian officials warned about the rapid spread of HIV/AIDS infections in the country due to a surge in intravenous drug usage.
"If no action is taken against the spread of this disease as quickly as possible, the number of those infected will reach 100,000 by the end of the next Iranian year (March 2008)," said Iran's deputy health minister, Moayed Alavian.
Iran is believed to have at least two million regular drug users -- and possibly as many as 3.5 million. Alavian said addiction is growing by around eight percent a year.
Intravenous drug use is believed to be the main cause of HIV/AIDS infection at 62.3 percent, followed by "unknown causes" at 27.9 percent and sexual contact at 7.4 percent.
https://www.youtube.com/watch?v=sdqx1m1oS1k
Iranian
Herbal Drug IMOD Against HIV AIDS
http://www.ncbi.nlm.nih.gov/pubmed/22353002
Recent Pat Antiinfect Drug Discov. 2012 Apr;7(1):66-72.
Safety
and efficacy of Setarud (IMOD TM ) among people living
with HIV/AIDS: a review.
Paydary K, Emamzadeh-Fard S, Khorram Khorshid HR, Kamali K, SeyedAlinaghi S, Mohraz M.
Paydary K, Emamzadeh-Fard S, Khorram Khorshid HR, Kamali K, SeyedAlinaghi S, Mohraz M.
Abstract
The broad use of highly active anti-retroviral therapy (HAART), especially in developing world, has been associated with several problems such as lactic acidosis, lipodistrophy, pancreatitis, hyperlipidemia, insulin resistance and hepatotoxicity. Extensive use of HAART has also resulted in emergence of resistant HIV variants. Thereby, a pressing need for development of novel and cost-effective agents arises from these limitations. Setarud (IMOD™) is a safe, naturally- derived immunomodulator that was introduced for treatment of HIV patients in Iran. It is prepared as a mixture of herbal extracts including Tanacetum vulgare (tansy), Rosa canina and Urtica dioica (nettle) in addition to selenium, flavonoids and carotenes. Tanacetum vulgare may relieve anti-inflammatory symptoms and Rosa canina defers blood glucose and cholesterol elevation. Extracts from Urtica dioica may prevent maturation of myeloid dendritic cells and reduce T cell responses. A significant rise of CD4 count was observed in HIV patients treated by IMOD™ in clinical trial phases, which could be explained by its immunomodulatory effects. Anti-oxidative activity of compounds in IMOD™ might play a role in the clinical outcomes of patients treated with this drug. Moreover, IMOD™ may show improving activity upon lipid profile and liver metabolism. According to studies on IMOD™, it seems that IMOD™ has minor side effects. IMOD™ with international publication number WO 2007/087825 A1 is an herbal extract which includes Rosa canina, Urtica dioica, Tanacetum vulgare, and selenium comprising a treatment by pulsed electromagnetic field of high frequency and is useful in treatment of HIV infection and AIDS.
UA94082
HERBAL EXTRACT OF IMMUNOMODULATORY ACTION FROM ROSA SP., URTICA DIOICA AND/OR TANACETUM VULGARE
HERBAL EXTRACT OF IMMUNOMODULATORY ACTION FROM ROSA SP., URTICA DIOICA AND/OR TANACETUM VULGARE
The present invention refers to a method for preparing a herbal extract from sp., Urtica dioica and/or preferably comprising a treatment by pulsed electromagnetic field of high frequency. The herbal extract, optionally comprising selenium and/or urea, is useful in the treatment of conditions associated with impaired immune system, e.g. in HIV infection and AIDS.
EP2921175
Herbal extracts having anti-recurrent respiratory papillomatosis properties
Herbal extracts having anti-recurrent respiratory papillomatosis properties
The present invention relates to a herbal extract obtainable by a method, comprising the following steps: (a) providing a plant material derived from Rosa sp., Urtica dioica and/or Tanacetum vulagare; (b) drying a plant material; (c) adding an organic solvent; (d) incubating the mixture of plant material and organic solvent; (e) obtaining the herbal extract; (f1) adding selenium and/or an organic or inorganic salt thereof; and/or (f2) adding urea; and (g) exposing the herbal extract to a pulsed electromagnetic field, for use in the treatment of Recurrent Respiratory Papillomatosis.
[0001] The present invention refers to a herbal extract for use in the treatment of Recurrent Respiratory Papillomatosis.
Background of the invention
[0002] Recurrent Respiratory Papillomatosis (RRP), formerly known as juvenile laryngeal papillomatosis or glottal papillomatosis, is the most common benign tumor of the respiratory tract in children and adolescent. The cause of the disease is Human papilloma virus (HPV) types 6 and 11 for which there is, unfortunately, no definitive cure. The frequency of RRP occurrence in children and adult is 1-4/100,000 and 1.8-3.9/100,000 persons, respectively. RRP is most often located in the larynx. The most typical symptoms are hoarseness and sever stridor and dyspnea in progressive cases. Diagnosis of the disease involves physical examination, bronchoscopy, histopathologic detection and PCR.
[0003] The mainstay of treatment for RRP is surgical removing of the lesions by different methods, including physical debridement, CO2laser vaporization, and a newer method employing the laryngeal microdebrider. The clinical course is not predictable but usually infected patients typically require repeat surgical excisions due to hoarseness or respiratory obstruction signs every 3-4 years.
[0004] Patient's immune system seems to have main role in infection pathogenesis and spontaneous remission. Thus, a number of adjuvant therapies have been tried to control aggressive papillomatosis. These therapies include topical chemotherapy, steroids, cidofovir, cis retinoic acid, podophyllin, autogenous vaccine, immune stimulators, acyclovir, accutane, and interferon.
[0005] Although interferon therapy has shown significant problems, it is considered to be the most successful treatment (30-50% eradication & 70-80% partial response).
[0006] Thus, it is an object of the present invention to provide a pharmaceutically active composition for use in the treatment of Recurrent Respiratory Papillomatosis which overcomes drawbacks of the prior art, particularly a composition being safe and effective in the treatment of RRP. It is also an object of the present invention to provide a pharmaceutically active composition for use in the treatment of RRP which can advantageously be used in combination with classic surgical operation.
Summary of the invention
[0007] This object has been solved by a herbal extract obtainable by a method, comprising the following steps: (a) providing a plant material derived fromRosa sp., Urtica dioica and/or Tanacetum vulgare;( b) drying a plant material; (c) adding an organic solvent; (d) incubating the mixture of plant material and organic solvent; (e) obtaining the herbal extract; (f1) adding selenium and/or an organic or inorganic salt thereof; and/or (f2) adding urea; and (g) exposing the herbal extract to a pulsed electromagnetic field, for use in the treatment of Recurrent Respiratory Papillomatosis.
[0008] The plant material to be extracted may be provided separately or in any mixed combination. Preferably, the plant material ofUrtica dioicaandTanacetum vulagareis extracted jointly, with the plant material ofRosa sp.is extracted separately. After respective extracts are obtained, these may be combined.
[0009] It is also preferred that the extract ofRosa sp.is exposed to a pulsed electromagnetic field before being combined with the other extract(s).
[0010] In a preferred embodiment, the electromagnetic field pulse has a sinusoidal, rectangular and/or stochastic shape.
[0011] More preferred, the pulsed electromagnetic field has a frequency in the range of about 5 to 750 kHz, preferably of about 50 to 350 kHz, most preferably of about 250 kHz.
[0012] In a further embodiment, the pulsed electromagnetic field has a power in the range of about 10 to 200 Watt, preferably of about 20 to 100 Watt, most preferably of about 45 Watt. In another embodiment, the pulsed electromagnetic field has a magnetic field strength in the range of 100 to 150 µTesla.
[0013] Preferably, the exposing in step (g) is carried out for a time period of about 2 to 5 minutes.
[0014] More preferably, the exposing in step (g) is repeated, and preferably is carried out for three times.
[0015] In a further embodiment, the treatment is treatment in a subject.
[0016] Most preferred, the subject is a vertebrate, preferably a mammal, most preferably a human.
[0017] Preferably, the subject is not pregnant.
[0018] In one embodiment, the plant material derived from Rosa sp, is from Rosa canina.
[0019] In one embodiment, the plant material derived from Rosa sp.is a fruit.
[0020] In one embodiment, the plant material derived from Urtica dioica and/or Tanacetum vulgare is a leave and/or a small stem.
[0021] In one embodiment, the drying in step (b) is carried out at a temperature in the range of about 20 to 50°C, preferably of about 37 to 45°C, most preferably of about 42°C.
[0022] In one embodiment, the drying in step (b) is carried out for a time period of about 3 to 4 days.
[0023] In one embodiment, the organic solvent is ethanol, preferably of about 60 to 96 % (v/v), more preferably of about 80 to 96 % (by volume), most preferably of about 96 % (by volume).
[0024] In one embodiment, the incubating in step (d) is carried out for a time period in the range of about 20 to 40 days, preferably of about 22 to 38 days, most preferably of about 25 to 35 days. In one embodiment, the incubating in step (d) is carried out at a temperature in the range of about 20 to 50°C, preferably of about 37 to 45°C, most preferably of about 42°C.
[0025] Preferably, the treatment is post-surgical treatment of Recurrent Respiratory Papillomatosis.
[0026] In one embodiment, selenium is added to a concentration of free selenium in the range of about 1-100 mg/l, preferably of about 5-50 mg/l, most preferably of about 10-20 mg/l.
[0027] The object of the present invention is further solved by a pharmaceutical composition, comprising the herbal extract for use in the treatment of Recurrent Respiratory Papillomatosis according to the present invention.
[0028] In one embodiment, the pharmaceutical composition additionally comprises a pharmaceutically acceptable carrier.
[0029] In one embodiment, the pharmaceutical composition is formulated for oral administration.
[0030] The object of the present invention is further solved by a kit comprising the pharmaceutical composition according to the present invention.
[0031] The term "stochastic shape" comprises the meaning that the electromagnetic field pulse is in the form of a noise. Preferably, the electromagnetic field pulse is of rectangular shape and is combined with a sinusoidal wave inside. The "power" (Watt) of the pulsed electromagnetic field means e.g. effective power. The value of the "magnetic field strength" (Tesla) of the pulsed electromagnetic field indicates e.g. from peak to peak.
[0032] The term "pharmaceutical composition", as used herein, is intended to comprise the herbal extract of the present invention. Also considered is a pharmaceutical composition comprising at least one pharmaceutically active component of the herbal extract of the present invention and/or at least one derivative or analog of said active component and corresponding salts thereof.
[0033] The pharmaceutical composition can be, for example, in a liquid form, e.g. a solution, syrup, elixier, emulsion and suspension, or in a solid form, e.g. a capsule, caplet, tablet, pill, powder, and suppository. Granules or semi-solid forms and gel caps are also considered. In case that the pharmaceutical composition is a liquid or a powder, the dosage unit optionally is to be measured, e.g. in the dosage unit of a teaspoonful. In addition to the herbal extract or the pharmaceutically active component, the pharmaceutical composition can comprise, for example, flavouring agents, sweeteners, dyes, preservatives, stabilizers, colouring agents, diluents, suspending agents, granulating agents, lubricants, binders and disintegrating agents. A tablet, for example, can be coated. A liquid to be injected should be sterile. Also considered are transdermal delivery systems and liposomal systems. All of the formulations mentioned can be intended for immediate release, timed release and sustained release.
[0034] The term "pharmaceutically acceptable", as used herein, means at least non-toxic. The "pharmaceutically acceptable carrier", as meant in the present disclosure, may take a wide variety of forms depending upon the desired route of administration. The term comprises conventional pharmaceutical diluents such as water or ethanol and conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums.
[0035] Administration of the pharmaceutical composition of the present invention can use different routes, such as oral, sublingual, parenteral, intravenous, intraperitoneal, nasal, vaginal, rectal, subcutaneous, intradermal, intramuscular and topic. A dosage unit can be administered once or several times a day, week or month. The delivery can also be continuously by infusion or through a transdermal sustained release system, for example.
[0036] Thus, the present invention provides a combinatory herbal extract from Rosa sp., Urtica dioica and/or Tanacetum vulgare treated by electromagnetic field radiation. Clinical data showed a beneficial effect of the extract in the treatment of RRP. Studies conducted bothin vitroand in experimental animal models revealed that the extract does not exert toxicity, mutagenicity, or oncogenicity. Pregnancy, however, is a contraindication.
Detailed description of the invention
[0037] The invention will now be described in more detail by the following examples with the intention to exemplify the invention. The examples, however, are not intended to have any limiting effect on the subject-matter of the claims or on the scope of protection.
EXAMPLE 1: Preparation of raw herbal extracts
[0038] Leaves and small stems of nettle (Urtica dioica) and tansy (Tanacetum vulgare) are collected from wild fields. After separation of useful parts and initial cleaning, the material is dried on a wooden network in a dark place for 3-4 days, preferably at 42°C. In dried condition, the plant material should be green without any change in colour, and leaves and stems should be brittle.
[0039] For extraction, airtight glass vessels are used. The dried plant material is broken into small pieces (2-5 cm) and placed into the glass vessels such that there is no space left. After packing (compressing), EtOH (96 %; herein, % of an ethanolic solution refers to % by volume)) is added until the vessel is filled completely. The vessels are placed into an incubator (37-45°C, preferably 42°C) for 20-40 days until a dark green solution appears.
[0040] For the extraction of wild rose (Rosa canina), dried fruits are used. In further embodiments, other species of Rosa sp.can be used alternatively or in addition. The fruits are filled into airtight vessels up to a half and EtOH is added. The vessels are kept in an incubator (37-45°C, preferably 42°C) for 20-40 days until an orange-red coloured extract appears.
[0041] After the incubation period, when the plant material is colourless, the extracts are collected by separating them from plant material using a cloth filter.
EXAMPLE 2: Electromagnetic treatment
[0042] The extract ofRosa caninais exposed to an electromagnetic field for 3 min. Then, 50-70 ml of the radiated Rosa canine extract is transferred to 2 1 of Urtica dioica and Tanacetum vulgare extracts, respectively. To each litre of the combined Urtica/Rosa and Tanacetum/Rosa extracts, respectively, 16 mg of selenium and 150 mg urea is added. In alternative embodiments, either selenium or urea is added. Then, the vessels are sealed again and kept in the incubator for 24 h at 42°C. After incubation, the vessels are exposed 4 times to an electromagnetic field, 3 min each, and are pooled together. The resulting extract is passed sequentially through a 5, 0.45 and 0.22 µm filter, respectively, and partitioned to sterile vials. After labelling and packaging, the herbals extract is ready for use.
[0043] The electromagnetic field, to which the raw extracts are exposed, is pulsed, powerful and monopolar in that the direction of the electric current generated in a Magnetic Impulse Generator (MIG) apparatus doesn't change. The pulsed magnetic field has a very high frequency ranging from 5 kHz-750 kHz. In this example, the pulse of a rectangular shape is used. Nevertheless, in other embodiments, a sinusoidal or stochastic shape is considered as well.
[0044] Preferably, the pulse is of rectangular shape and is combined with a sinusoidal wave inside. Although it is not intended to be bound to any theory, it is hypothesised that the special kind of the produced pulse causes some changes in the physical configuration of atoms in the molecules and/or arrangements of molecules thus leading to altered chemical properties.
[0045] In the preparation of the herbal extracts, 3-4 times radiation of electromagnetic pulses of high frequency is used for 2-5 min each. The electrical power (e.g. effective power) of the pulses is about 20 to 100 Watt, and the best effect is obtained at 45 Watt.
EXAMPLE 3: Pre-clinical studies
[0046] Pre-clinical studies to investigate the drug's acute toxicity, chronic toxicity, mutagenicity, embryotoxicity and teratogenicity, effects on the reproductive function, and immuno-related effects, were done as described in details in prior art WO 2007/087825 Al .
[0047] In conclusion, the drug at the doses of 0.07 ml/kg and 0.18 ml/kg, did not show any serious side effects, and based on all of the experiments conducted and the obtained results, the herbal extract was recommended for clinical trials with the only contraindication of pregnancy.
EXAMPLE 4: Pharmacologic effects related to RRP of the herbal extract
[0048] Nine newly infected and/or previously treated RRP patients with surgical intervention 3 times through one year, received the inventive herbal extract, also called IMOD. Patients were 2-56 years old. The medical protocol was categorized in two phases; 1. Intravenous administration of IMOD, 1.5 mg/kg for 10 days and then increased to 6 mg/kg for a six months period; 2. IMOD oral solution administration.
[0049] A complete blood chemistry profile, including blood cell count, was obtained for all patients before the start of study as well as 3 months post-treatment. These patients were followed for 12 months to evaluate the side effects and improvement in clinical signs. The results of treatment were estimated by decreasing the recurrence of symptoms and comparing pre- and post-treatment severity score.
[0050] Nine patients (4 males and 5 females, with the average age of 13.05 years) received IMOD along with surgical excision within two years. One of these patients was excluded from the study because of unwillingness to continue treatment.
Table 1
4 M 4.87±4.17
5 F 19.60±20.94
Total 9
[0051] Six patients had been previously treated by surgical excision from 1 to 5 times. In these patients improvement in clinical signs were detected after IMOD administration.
[0052] In 3 patients (supra-glott and glott lesions were diagnosed in 2/3 cases) the clinical score decreased to 2-3.
[0053] Neither in one patient suffering from lesions in lower airway tract nor in the 56-year-old, interventions was effective.
[0054] In 6/8 patients improvement in clinical signs were evaluated. IMOD administration was effective for 2 patients with glott and supra-glott lesions which clinical scoring decreased to 2-3. This finding is related to anatomical location of lesions, better post-surgical prognosis and lower disease severity.
[0055] Two patients did not show decreasing in severity scores which may be due to senile factors in one and lower respiratory tract involvement in another.
[0056] Based on existing observations, IMOD administration is an effective immunomudulatory drug for treatment of recurrent respiratory papillomatosis in human.
EXAMPLE 5: Determination of the maximum tolerable dose of the herbal extract
[0057] This study was conducted to determine the maximum tolerable dose (MTD) of the herbal extract in HIV infected patients and its possible side-effects and toxicity that can cause dose limitation (dose limiting toxicities, DLTs).
[0058] The study protocol was based on the dose escalation method. The effects of the herbal extract on viral load and CD4 count of patients were evaluated as by-products. Four cohorts of patients (3 patients each) were selected and treated for 28 days (4 weeks) with escalated doses of the extract. A base dose of the extract has been determined according to LD10(10 % of the lethal dose) in former animal experiments. Patients were observed carefully for signs and symptoms of side-effects and toxicity by physical examination and laboratory workups according to the protocol.
[0059] All patients were male in the age of 28-60 years (mean: 41.6 years.). In the first cohort, the daily dose of 2 ml of extract in 100 ml warm normal saline was infused over 0.5-1 hr intravenously for 28 days. No toxicity or major side-effects were observed except for an increase in sweating and weight loss in 2 patients. In the second cohort, three other patients received a daily dose of 4 ml. There were no major side-effects and toxicity in this group. In the third cohort of originally 4 patients, one patient was excluded due to non-compliance and inability for regular daily attendance and the daily dose of 6.7 ml administered. In this group there were not only no major dose limiting toxicity and side-effects but also no minor ones. In the fourth cohort, three other patients received the daily dose of 10 ml, and there were no major side-effects and toxicity in this group too.
[0060] For the MTD study, a total of 12 patients were included in the study, who were treated for 4 weeks with escalated doses of the herbal extract. There was not toxicity or side-effects in all cohorts.
[0061] The features disclosed in the foregoing description and in the claims may both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.
US2009208598
USE OF A COMBINATION OF ETHANOLIC ROSA SP . , URTICA DIOICA AND TANACETUM VULGARE EXTRACTS, FURTHER COMPRISING SELENIUM AND UREA AND HAVING BEEN EXPOSED TO A PULSED ELECTROMAGNETIC FIELD, FOR THE PREPARATION OF A MEDICAMENT FOR IMMUNOSTIMULATION AND/
USE OF A COMBINATION OF ETHANOLIC ROSA SP . , URTICA DIOICA AND TANACETUM VULGARE EXTRACTS, FURTHER COMPRISING SELENIUM AND UREA AND HAVING BEEN EXPOSED TO A PULSED ELECTROMAGNETIC FIELD, FOR THE PREPARATION OF A MEDICAMENT FOR IMMUNOSTIMULATION AND/
[0001] The present invention refers to a method for preparing a herbal extract from Rosa sp., Urtica dioica and/or Tanacetum vulgare, preferably comprising a treatment by pulsed electromagnetic field. The present invention further refers to a herbal extract prepared by said method, optionally comprising selenium and/or urea, and to its use in the treatment of a disease associated with impaired immune system, preferably HIV infection and AIDS. The present invention also refers to a pharmaceutical composition and to a kit.
BACKGROUND OF THE INVENTION
[0002] The acquired immunodeficiency syndrome (AIDS) is a chronic life threatening disease caused by the human immunodeficiency virus (HIV). This retrovirus can be further specified, wherein HIV-1 is the cause of AIDS in the Western hemisphere and in Europe, while HIV-2 is the major cause of AIDS in Africa and Southeast Asia.
[0003] In more detail, AIDS is characterised by a series of symptoms becoming evident at later stages of the HIV infection. Without any treatment, the incubation period, i.e. the time period between HIV infection and clinical manifestation of AIDS, is about 10 years. An HIV infection causes progressive impairment of the immune system, finally resulting in immunodeficiency. Accordingly, the most important clinical symptoms of AIDS are opportunistic infections and, furthermore, characteristic malignancies such as Kaposi's sarcoma, HIV encephalopathy and HIV-associated wasting syndrome. The immunodeficiency is based on the loss of the CD4<+> T-cells that are essential for both cell-mediated and antibody-mediated immunity. The quantitative analysis of CD4<+> T-lymphocytes in the circulating blood has been the crucial method for detecting and evaluating HIV infection and AIDS since the beginning of the epidemic. For determination of the severity of the disease, prognosis and therapeutic observation, the percentage of CD4<+> T-lymphocytes and their changes over time (slope) serve as valuable parameters.
[0004] First cases of AIDS were reported in the early eighties of the last century. Meanwhile, AIDS has spread worldwide and, moreover, is epidemic in some regions, particularly in some of the developing countries. Today, about 40 million persons are infected by HIV with approximately 2.2 million children among them. In 2004, about five million new infections were noted, and about 3.1 million persons died from AIDS, 510 000 of them were children (according to the World Health Organization WHO).
[0005] Since 1995, the number of medicaments for the treatment of AIDS has trebled. Nowadays, combination therapies are of common use providing increased efficiency and deceased side-effects. The development of new active agents or drugs and therapeutic concepts were successful in slowing the progression of the disease, reversing the symptoms of the late stages of the disease and preventing the infection of babies born to infected mothers. The so-called “highly active anti-retroviral therapy” (HAART), a combined therapy comprising three or more drugs, has become a standard therapy. It was shown that HAART delays the progression of AIDS and reduce mortality. Usually, two of the drugs involved in HAART target the reverse transcriptase and one drug targets the viral protease. Well established reverse transcriptase inhibitors are nucleoside analogues such as zidovudine (AZT, Retrovir®), lamivudine (Epivir®), and didanosine (Videx®). Such transcriptase inhibitors are incorporated into the growing DNA strand which the consequence that further DNA synthesis is prevented. Other reverse transcriptase inhibitors are known, e.g., Viramune®, that inhibit the enzyme by other mechanisms. Protease inhibitors block the viral protease so that the proteins needed for assembly of new viruses cannot be cleaved from the large protein precursor. Examples of these kind of drugs are indinavir (Crixivan®), saquinavir (Invirase®), ritonavir (Norvir®), and nelfinavir mesylate (Viracept®). Further drugs useful for therapeutic intervention are fusion inhibitors, e.g. enfuvirtide (Fuzeon®) and integrase inhibitors.
[0006] Despite the great advances in the therapy of HIV infection and AIDS, there are still several disadvantages and drawbacks. The drugs currently used are very expensive and thus, they do not only drain resources in affluent countries, but are simply unavailable in the many poor countries where the epidemic rages. They have many unpleasant side-effects (e.g. nausea, diarrhea), however, they also may exert severe side-effects (e.g. liver and pancreas damage, sometimes with fatal outcome). They demand a very complicated dosing regimen, e.g. over a dozen pills a day (not counting those needed to cope with the accompanying opportunistic infections). Finally, they often lose effectiveness as they select for the emergence of drug-resistant virions in the patient.
[0007] Consequently, there is a need in alternative, improved or superior pharmaceutical means providing an amelioration of the AIDS symptoms, a retardation of AIDS manifestation and/or an intervention in the HIV infection. Furthermore, there is a need in more cost efficient pharmaceutical means.
[0008] Thus, it is an object of the present invention to provide a pharmaceutically active composition useful in the treatment of viral infections and associated symptoms and conditions, preferably HIV infection and AIDS.
SUMMARY OF THE INVENTION
[0009] The object of the present invention is solved by a method for preparing a herbal extract, comprising the following steps:
(a) providing a plant material derived from Rosa sp. and/or Urtica dioica and/or Tanacetum vulgare;
(b) drying the plant material;
(c) adding an organic solvent;
(d) incubating the mixture of plant material and organic solvent;
(e) obtaining the herbal extract.
[0015] In one embodiment, the plant material is derived from Rosa sp., Urtica dioica and Tanacetum vulgare.
[0016] In one embodiment, the plant material derived from Rosa sp. is from Rosa canina.
[0017] In one embodiment, the plant material derived from Rosa sp. is a fruit.
[0018] In one embodiment, the plant material derived from Urtica dioica and/or Tanacetum vulgare is a leave and/or a small stem.
[0019] In one embodiment, the drying in step (b) is carried out at a temperature in the range of about 20 to 50° C., preferably of about 37 to 45° C., most preferably of about 42° C.
[0020] In one embodiment, the drying in step (b) is carried out for a time period of about 3 to 4 days.
[0021] In one embodiment, the organic solvent is ethanol, preferably of about 60 to 96% (v/v), more preferably of about 80 to 96% (by volume), most preferably of about 96% (by volume).
[0022] In one embodiment, the incubating in step (d) is carried out for a time period in the range of about 20 to 40 days, preferably of about 22 to 38 days, most preferably of about 25 to 35 days.
[0023] In one embodiment, the incubating in step (d) is carried out at a temperature in the range of about 20 to 50° C., preferably of about 37 to 45° C., most preferably of about 42° C.
[0024] In one embodiment, the method additionally comprises the following step:
(f) adding selenium and/or an organic or inorganic salt thereof.
[0026] In one embodiment, selenium is added to a concentration of free selenium in the range of about 1 to 100 mg/l, preferably of about 5 to 50 mg/l, most preferably of about 10 to 20 mg/l.
[0027] In one embodiment, the method additionally comprises the following step:
(g) adding urea.
[0029] In one embodiment, the method additionally comprises the following step:
(h) exposing the herbal extract to a pulsed electromagnetic field.
[0031] In one embodiment, the electromagnetic field pulse has a sinusoidal, rectangular and/or stochastic shape.
[0032] In one embodiment, the pulsed electromagnetic field has a frequency in the range of about 5 to 750 kHz, preferably of about 50 to 350 MHz, most preferably of about 250 MHz.
[0033] In one embodiment, the pulsed electromagnetic field has a power in the range of about 10 to 200 Watt, preferably of about 20 to 100 Watt, most preferably of about 45 Watt.
[0034] In one embodiment, the pulsed electromagnetic field has a magnetic field strength in the range of 100 to 150 μTesla.
[0035] In one embodiment, the exposing in step (h) is carried out for a time period of about 2 to 5 minutes.
[0036] In one embodiment, the exposing in step (h) is repeated, and is preferably carried out for three times.
[0037] The object of the present invention is further solved by a herbal extract prepared by the method according to the present invention.
[0038] The object of the present invention is further solved by a use of the herbal extract according to the present invention, prepared by the method according to the present invention for stimulating the immune system of a subject.
[0039] The object of the present invention is further solved by a use of the herbal extract according to the present invention, prepared by the method according to the present invention, for the treatment of a disease associated with impaired immune system in a subject.
[0040] The object of the present invention is further solved by a use of the herbal extract according to the present invention, prepared by the method according to the present invention, for the manufacture of a pharmaceutical composition for the treatment of a disease associated with impaired immune system in a subject.
[0041] In one embodiment, the disease is an HIV infection and/or AIDS.
[0042] In one embodiment of the use, the subject is a vertebrate, preferably a mammal, most preferably a human.
[0043] In one embodiment, the subject is not pregnant.
[0044] The object of the present invention is solved by a use of the herbal extract according to the present invention, prepared by the method according to the present invention, for the treatment of a viral infection in a subject.
[0045] The object of the present invention is solved by a use of the herbal extract according to the present invention, prepared by the method according to the present invention, for the manufacture of a pharmaceutical composition for the treatment of a viral infection in a subject.
[0046] In one embodiment, the disease is an RNA viral infection, preferably a retroviral infection, most preferably an HIV infection.
[0047] In one embodiment of the use, the subject is a vertebrate, preferably a mammal, most preferably a human.
[0048] In one embodiment of the use, the subject is not pregnant.
[0049] The object of the present invention is further solved by a pharmaceutical composition, comprising the herbal extract according to the present invention, prepared by the method according to the present invention.
[0050] In one embodiment, the pharmaceutical composition additionally comprises a pharmaceutically acceptable carrier.
[0051] In one embodiment, the pharmaceutical composition is formulated for oral administration.
[0052] The object of the present invention is further solved by a kit comprising the pharmaceutical composition according to the present invention.
[0053] The term “stochastic shape” comprises the meaning that the electromagnetic field pulse is in the form of a noise. Preferably, the electromagnetic field pulse is of rectangular shape and is combined with a sinusoidal wave inside. The “power” (Watt) of the pulsed electromagnetic field means e.g. effective power. The value of the “magnetic field strength” (Tesla) of the pulsed electromagnetic field indicates e.g. from peak to peak.
[0054] The effect of “stimulating the immune system” and the conditions of an “impaired immune system” can be determined using methods and parameters known in the art. Targets of such a determination can be any component of the cell-mediated and antibody-mediated immune system such as T-lymphocytes (CD4 and/or CD8 T-lymphocytes), B-lymphocytes, antibodies and components of the complement system. An example of a method for determination is the FACS (fluorescence activated cell sorting) analysis. Preferably, CD4 T-lymphocytes are determined as counts or percentage, most preferably in a time dependent manner.
[0055] The term “AIDS”, as used herein, refers to a clinical condition having characteristic symptoms associated with later stages of HIV infection.
[0056] The term “RNA viral infection”, as used herein, refers to an infection by an RNA virus, preferably a retrovirus. Examples for RNA viruses considered by the present invention are be Polio, Coxsackie, Calici, Hepatitis A, Hepatitis C, Hepatitis D, Hepatitis E, Entero, Rhino, Rubella, CEE (central European encephalitis), Influenza, RS (respiratory syncitial), Parainfluenza, Measle, Mumps, Corona, Arena, Lassa, Bunya, Hanta, Rhabdo, Filo, Borna, HTLV (human T-cell leukaemia), and Rota virus.
[0057] The term “pharmaceutical composition”, as used herein, is intended to comprise the herbal extract of the present invention. Also considered is a pharmaceutical composition comprising at least one pharmaceutically active component of the herbal extract of the present invention and/or at least one derivative or analogon of said active component and corresponding salts thereof.
[0058] The pharmaceutical composition can be, for example, in a liquid form, e.g. a solution, syrup, elixir, emulsion and suspension, or in a solid form, e.g. a capsule, caplet, tablet, pill, powder, and suppository. Granules or semi-solid forms and gelcaps are also considered. In case that the pharmaceutical composition is a liquid or a powder, the dosage unit optionally is to be measured, e.g. in the dosage unit of a teaspoonful. In addition to the herbal extract or the pharmaceutically active component, the pharmaceutical composition can comprise, for example, flavouring agents, sweeteners, dyes, preservatives, stabilizers, colouring agents, diluents, suspending agents, granulating agents, lubricants, binders and disintegrating agents. A tablet, for example, can be coated. A liquid to be injected should be sterile. Also considered are transdermal delivery systems and liposomal systems. All of the formulations mentioned can be intended for immediate release, timed release and sustained release.
[0059] The term “pharmaceutically acceptable”, as used herein, means at least non-toxic. The “pharmaceutically acceptable carrier”, as meant in the present disclosure, may take a wide variety of forms depending upon the desired route of administration. The term comprises conventional pharmaceutical diluents such as water or ethanol and conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums.
[0060] Administration of the pharmaceutical composition of the present invention can use different routes, such as oral, sublingual, parenteral, intravenous, intraperitoneal, nasal, vaginal, rectal, subcutaneous, intradermal, intramuscular and topic. A dosage unit can be administered once or several times a day, week or month. The delivery can also be continuously by infusion or through a transdermal sustained release system, for example.
[0061] Thus, the present invention provides a combinatory herbal extract from Rosa sp., Urtica dioica and/or Tanacetum vulgare treated by electromagnetic field radiation. Clinical data showed a beneficial effect of the extract in the treatment of AIDS. Studies conducted both in vitro and in experimental animal models revealed that the extract does not exert toxicity, mutagenicity, or oncogenicity. Pregnancy, however, is a contraindication.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The invention will now be described in more detail by the following examples with the intention to exemplify the invention. The examples, however, are not intended to have any limiting effect on the subject-matter of the claims or on the scope of protection.
Example 1
Preparation of Raw Herbal Extracts
[0063] Leaves and small stems of nettle (Urtica dioica) and tansy (Tanacetum vulgare) are collected from wild fields. After separation of useful parts and initial cleaning, the material is dried on a wooden network in a dark place for 3-4 days, preferably at 42° C. In dried condition, the plant material should be green without any change in colour, and leaves and stems should be brittle. For extraction, airtight glass vessels are used. The dried plant material is broken into small pieces (2-5 cm) and placed into the glass vessels such that there is no space left. After packing (compressing), EtOH (96%; herein, % of an ethanolic solution refers to % by volume) is added until the vessel is filled completely. The vessels are placed into an incubator (37-45° C., preferably 42° C.) for 20-40 days until a dark green solution appears.
[0064] For the extraction of wild rose (Rosa canina), dried fruits are used. In further embodiments, other species of Rosa sp. can be used alternatively or in addition. The fruits are filled into airtight vessels up to a half and EtOH is added. The vessels are kept in an incubator (37-45° C., preferably 42° C.) for 20-40 days until an orange-red coloured extract appears.
[0065] After the incubation period, when the plant material is colourless, the extracts are collected by separating them from plant material using a cloth filter.
Example 2
Electromagnetic Treatment
[0066] The extract of Rosa canina is exposed to an electromagnetic field for 3 min. Then, 50-70 ml of the radiated Rosa canina extract is transferred to 2 1 of Urtica dioica and Tanacetum vulgare extracts, respectively. To each litre of the combined Urtica/Rosa and Tanacetum/Rosa extracts, respectively, 16 mg of selenium and 150 mg urea is added. In alternative embodiments, either selenium or urea is added. Then, the vessels are sealed again and kept in the incubator for 24 h at 42° C. After incubation, the vessels are exposed 4 times to an electromagnetic field, 3 min each, and are pooled together. The resulting extract is passed sequentially through a 5, 0.45 and 0.22 μm filter, respectively, and partitioned to sterile vials. After labelling and packaging, the herbals extract is ready for use.
[0067] The electromagnetic field, to which the raw extracts are exposed, is pulsed, powerful and monopolar in that the direction of the electric current generated in a Magnetic Impulse Generator (MIG) apparatus doesn't change. The pulsed magnetic field has a very high frequency ranging from 5 kHz-750 kHz. In this example, the pulse of a rectangular shape used. Nevertheless, in other embodiments, a sinusoidal or stochastic shape is considered as well. Preferably, the pulse is of rectangular shape and is combined with a sinusoidal wave inside. Although it is not intended to be bound to any theory, it is hypothesised that the special kind of the produced pulse causes some changes in the physical configuration of atoms in the molecules and/or arrangements of molecules thus leading to altered chemical properties.
[0068] In the preparation of the herbal extracts, 3-4 times radiation of electromagnetic pulses of high frequency is used for 2-5 min each. The electrical power (e.g. effective power) of the pulses is about 20 to 100 Watt, and the best effect is obtained at 45 Watt.
Example 3
Pre-Clinical Studies
[0069] In pre-clinical studies, the herbal extract of the present invention was studied for acute toxicity and chronic toxicity. For studying the drug acute toxicity, BALB/c mice and Wistar rats were injected with a single intramuscular (i.m.) injection. The drug chronic toxicity was studied during 3 months in Wistar rats and during 1 month in dogs. The potential mutagenic, embryotoxic, teratogenic, allergenic and immunotoxic properties of the herbal extract as well as its effect on the reproductive function were investigated.
[0070] As a result of the conducted experiments, it was evident that the herbal extract of the present invention is a low-toxic drug following a single i.m. injection to BALB/c mice and Wistar rats. Under acute toxicity experimental conditions, the LD50 of the extract administered intraperitoneally (i.p.) as an 1:5 dilution in normal saline was 51-54 ml/kg in rats and 56-59 ml/kg in mice.
[0071] When BALB/c mice or Wistar rats were injected i.m. or i.p. with the herbal extract, no specific or sexual differences were observed in the sensitivity of the test animals to the toxic effects of the extract. The pattern of intoxication of BALB/c mice and Wistar rats in doses equal to LD50 was similar to the pattern of ethyl alcohol poisoning; ethanol was used as a solvent at high concentrations in the herbal extract preparation. Studying the chronic toxicity of the herbal extract following its application i.m. to rats in doses of 0.07 and 0.21 ml/kg once a day for 3 months (10 and 30 times higher than the daily dose for humans) and to dogs in a dose of 0.07 ml/kg (10 times higher than the daily dose for humans) once a day for 1 month, no damaging effect was observed in the basic organs and systems of the animal body.
[0072] The herbal extract of the present invention obviously does not possess mutagenicity and it doesn't affect the reproductive function of animals. When the herbal extract was applied i.m. once a day to rats during pregnancy (from the first day of pregnancy up to birth) in doses of 0.21 ml/kg, the drug exhibited embryotoxic and teratogenic properties. Therefore it is contraindicated in pregnancy.
[0073] Furthermore, using doses of 0.07 and 0.14 ml/kg administered to guinea pigs every other day within 10 days, the herbal extract did not exhibit allergenic or immunotoxic properties.
[0074] In conclusion, the results of the toxicological experiments indicate that the herbal extract of the present invention can be used for systematic clinical trials with considering its contraindication in pregnancy.
3.1. Acute Toxicity
[0075] The studies were performed with 128 BALB/c mice (males and females, body weight 18-20 g) and 47 Wistar rats (males and females, body weight 180-220 g) using i.m. injection of the herbal extract in mice and i.p. injection in mice and rats. The herbal extract was diluted 1:10 and 1:5 in sterile normal saline, and then different volumes of these dilutions were applied to the test animals. The experimental animals were then observed for 14 days to determine possible toxic effects of the preparation.
[0076] The toxicity of the herbal extract following a single injection to the test animals were determined using a double-stage method: first, an approximate LD50 was established by the Deihman and Leblanc technique followed by the determination of the precise indices of LD16, LD50±SD and LD84 by probit-analysis according to Litchfield and Wilcoxon.
[0077] The conducted experiments revealed that a single i.m. injection of the herbal extract diluted 1:10 in normal saline in doses of 25-50 ml/kg to BALB/c mice doesn't cause intoxication and death of animals. When the doses increased to 75-100 ml/kg, a decrease in motor activity was observed, but no animal death. Injection of the herbal extract diluted 1:5 in normal saline in doses of 25-50 ml/kg was followed by pain, profound depression and finally animal death after few hours. The intoxication profile of the herbal extract in mice was similar to that of their poisoning by ethyl alcohol (37.3 ml/kg) which is present at high concentrations in the herbal extract as a vehicle. The results in Wistar rats were similar to those observed in BALB/c mice.
[0000]
TABLE 1
Parameters of acute toxicity of the herbal extract and ethanol (96%) diluted 1:5 in normal saline at different concentrations following i.m. or i.p. administration to experimental animals.
Toxicity indices [ml/kg]
Route of LD16 LD50 ± SD LD84 LD16 LD50 ± SD LD84
administration Males Females
BALB/c mice, herbal extract
i.m. 53 66 ± 4.6 82 49 62 ± 4.3 78
i.p. 46 59 ± 3.8 75 43 56 ± 3.7 72
BALB/c mice, ethyl alcohol
i.p. 30.4 37.3 ± 2.1 44.8 ND* ND ND
Wistar rats, herbal extract
i.p. 42 54 ± 4.3 71 38 51 ± 4.2 67
*ND—not determined.
[0078] The data presented in Table 1 above show e.g. that the toxicity of the herbal extract according to the parameters of toxicometry doesn't differ significantly between i.m. and i.p. injection.
3.2. Chronic Toxicity
3.2.1. Studies in Rats
[0079] The toxicity studies on the herbal extract of the present invention were performed during 3 months by i.m. injection of the preparation to Wistar rats. These experiments were performed with 90 Wistar rats (males and females, body weight 180-200 g), which were divided into 3 groups of 30 animals (15 males and 15 females). The first group served as a control (normal saline), the second group received 0.07 ml/kg of the herbal extract, and the third group received 0.21 ml/kg of the herbal extract. The studied doses of the herbal extract were 10 and 30 times higher than the daily therapeutic dose recommended for humans (0.5 ml of the herbal extract diluted in 5 ml of normal saline or 0.007 ml/kg of the preparation).
[0080] The results show that i.m. injection of the herbal extract in doses of 0.07 and 0.21 ml/kg has no effect on the general state and behaviour of rats. During the chronic toxicity experiments, statistically significant differences in the amount of erythrocytes, leukocytes, platelets, and hemoglobin levels were not observed in animals receiving 0.07 and 0.21 ml/kg, respectively, of the herbal extract compared to the control (Tables 2 and 3).
[0000]
TABLE 2
Hematologic parameters in male rats following 3 months of i.m. injection with the herbal extract.
Herbal extract
Observation period Control 0.07 ml/kg 0.21 ml/kg
Erythrocytes [×10<12>
/l]
Initial value 7.3 ± 0.6 7.4 ± 0.4 7.3 ± 0.2
After 1 month 7.3 ± 0.5 7.5 ± 0.4 7.7 ± 0.5
After 3 months 7.6 ± 0.4 7.8 ± 0.3 7.7 ± 0.6Leukocytes [×10<9>
/l]
Initial value 11.4 ± 0.7 11.0 ± 0.5 11.0 ± 0.5
After 1 month 11.7 ± 0.6 11.1 ± 0.3 11.2 ± 0.4
After 3 months 11.2 ± 0.7 11.2 ± 0.4 11.3 ± 0.6Platelets [×10<9>
/l]
Initial value 660 ± 30 660 ± 30 660 ± 30
After 1 month 672 ± 33 663 ± 23 669 ± 24
After 3 months 667 ± 29 669 ± 31 655 ± 28
Hemoglobin [g/l]
Initial value 111 ± 4 110 ± 3 111 ± 2
After 1 month 114 ± 3 115 ± 4 117 ± 5
After 3 months 116 ± 4 115 ± 3 118 ± 3
[0000]
TABLE 3
Hematologic parameters in female rats following 3 months i.m. injection with herbal extract.
Herbal extract
Observation period Control 0.07 ml/kg 0.21 ml/kg
Erythrocytes [×10<12>
/l]
Initial value 7.2 ± 0.4 7.2 ± 0.3 7.0 ± 0.3
After 1 month 7.1 ± 0.4 7.4 ± 0.5 7.6 ± 0.4
After 3 months 7.3 ± 0.4 7.7 ± 0.4 7.8 ± 0.3Leukocytes [×10<9>
/l]
Initial value 11.3 ± 0.4 11.6 ± 0.5 11.4 ± 0.3
After 1 month 11.5 ± 0.5 11.2 ± 0.3 11.5 ± 0.5
After 3 months 11.2 ± 0.6 11.1 ± 0.4 11.2 ± 0.3Platelets [×10<9>
/l]
Initial value 670 ± 27 660 ± 20 671 ± 25
After 1 month 678 ± 24 669 ± 27 673 ± 22
After 3 months 667 ± 25 668 ± 21 662 ± 22
Hemoglobin [g/l]
Initial value 112 ± 4 110 ± 3 111 ± 4
After 1 month 114 ± 3 118 ± 4 119 ± 4
After 3 months 113 ± 5 119 ± 6 119 ± 7
[0081] Under the conditions of chronic toxicity experiments in rats, i.e. i.m. injection of the preparation in doses of 0.07 ml/kg and 0.21 ml/kg, no significant changes in total protein blood serum level were observed. The absence of damaging effects by prolonged treatment with the herbal extract in doses of 0.07 and 0.21 ml/kg to rats is supported by the stable activity of hepatic enzymes, such as aspartate and alanine aminotransferases, lactate dehydrogenase, and alkaline phosphatase during 3 months of the chronic toxicity experiments. Prolonged administration of the herbal extract to rats didn't influence the levels of bilirubin, cholesterol, triglycerides, urea, creatinine, and glucose in the blood serum of the experimental animals.
[0082] Prolong injection of the preparation in doses of 0.07 and 0.21 ml/kg, diluted 1:10 in normal saline, into the thigh muscles of the animals didn't have locally irritating effects.
[0083] Macroscopic examinations did not show any toxic or toxico-allergic effects of the herbal extract on this group of animals.
[0084] No pathological changes of the internal organs (brain, pituitary gland, thymus, trachea, lungs, heart, esophagus, spleen, liver, pancreas, adrenal glands, kidney, stomach, and reproduction organs in female and male) were observed in this group of animals during 3 months after i.m. injection of the herbal extract in a dose of 0.21 ml/kg.
3.2.2. Studies in Dogs
[0085] The experiments were conducted in 8 dogs (males, initial body weight 12-14.5 kg) which were divided into 2 groups, 4 animals in each: first group—control, second group—herbal extract 0.07 ml/kg. The applied dose of the preparation was 10 times higher than the highest daily therapeutic dose for humans. The studied preparation in a dose of 0.07 ml/kg was diluted in sterile normal saline 1:10, and then it was injected into thigh muscles of dogs once a day for 1 month.
[0086] Electrocardiogram (ECG) examination performed before the beginning of the experiments in dogs and 1 month after the beginning of the treatment using a dose of 0.07 ml/kg did not reveal an increase in the heart rate or changes in the ECG parameters. All the ECG parameters didn't change before and after experiments in treated compared to control animals (Table 4).
[0000]
TABLE 4
ECG parameters of the dogs receiving the herbal extract by i.m. injection in a dose of 0.07 ml/kg during 1 month.
After 1 month
Initial value Herbal
ECG Herbal extract extract
Parameters Control 0.07 ml/kg Control 0.07 ml/kg
R-R, m sec 467 ± 38 458 ± 35 466 ± 32 462 ± 22
P-Q, m sec 86 ± 12 89 ± 16 85 ± 21 89 ± 16
Q-T, m sec 139 ± 19 146 ± 18 138 ± 15 139 ± 15
QRS, m sec 40 ± 6 39 ± 3 39 ± 4 39 ± 6
ST, m sec 0.4 ± 0.2 0.4 ± 0.2 0.4 ± 0.2 0.4 ± 0.2
TP, m sec 189 ± 19 1392 ± 28 186 ± 22 194 ± 23
P, m sec 0.30 ± 0.03 0.29 ± 0.05 0.26 ± 0.04 0.28 ± 0.04
R, m sec 1.38 ± 0.12 1.39 ± 0.24 1.42 ± 0.22 1.38 ± 0.23
T, m sec 0.30 ± 0.06 0.32 ± 0.06 0.29 ± 0.06 0.28 ± 0.05
Heart 139 ± 15 145 ± 15 138 ± 12 146 ± 12
rate/min
[0087] The results showed that administration of the herbal extract to dogs by i.m. injection every day during 1 month in a dose of 0.07 ml/kg (10 times higher than a daily dose for humans) doesn't have any effect on the general state and behaviour of animals, and this treatment also doesn't change the functional state of the main organs and systems of the animal body.
[0088] According to our data of histological examinations no toxic or local irritating effects of the herbal extract were revealed during 1 month of i.m. injection in a dose of 0.07 ml/kg in dogs.
3.3. Mutagenicity
3.3.1. Gene Mutations in Microorganisms (Ames Test)
[0089] Evaluation of the mutagenic activity of the herbal extract was carried out by means of the technique of the ability of a substance to induce gene mutations in indicator microorganisms in the system of metabolic activation in vitro and without such system. Plate method of identification of mutations was used. This method was provided by Ames et al., and we used three autotrophic stains on histidine, namely Salmonella typhimurium TA 98, TA 100 and TA 1537, as indicator microorganisms.
[0090] In order to conduct the Ames test, dilutions of the herbal extracts were obtained in the following way: the initial solution of the preparation was measured and weighed and diluted with distilled water to a concentration of 10 mg/ml. Further dilutions were prepared in distilled water and added to Petri dishes. The concentrations of the preparation from 0.1 to 1000 μg/dish were examined. Distilled water was used as negative control.
[0000]
TABLE 5
The effect of the herbal extract on the bacterial indicator strain TA 98 in the Ames test.
TA 98 strain
Studied Dose −S9 +S9
substance μg/dish Mi1, 2, 3 Mean MO/MK MA Mi1, 2, 3 Mean MO/MK MA
Negative 0 33 29 37 32.8 1 − 40 36 31 35.5 1 −
control
(H2O)
Positive 20 0 0 0 0 0 0 0 0 0 − 1211 1280 1140 1209 34.0 +
control
(2AA)
Positive 0.5 568 567 614 6 17.8 + 0 0 0 0 0 0 0 0 0 −
control
(ANQO)
Herbal 0.1 35 32 35 34.0 1.04 − 30 42 45 38.4 1.08 −
extract 1.0 29 32 37 32.5 0.99 − 40 35 29 34.4 0.97 −
10.0 28 34 38 33.1 1.01 − 36 35 32 34.3 0.97 −
100.0 27 30 26 27.6 0.84 − 19 27 31 25.1 0.71 −
1000.0 29 28 33 29.9 0.91 − 30 28 39 32 0.90 −
[0091] Conventional signs and abbreviations: Mi—the number of revertants per dish, M—average geometrical number, M0/Mk—the ratio of the number of revertants in the test and the number of revertants in negative control, MA mutagenic activity: “+”—the presence of activity, “-”—the absence of activity.
[0000]
TABLE 6
The effect of the herbal extract on the bacterial indicator strain TA 100 in the Ames test.
TA 100 strain
Studied Dose −S9 +S9
substance μg/dish Mi1, 2, 3 Mean MO/MK MA Mi1, 2, 3 Mean MO/MK MA
Negative 0 144 223 228 194.2 1 − 188 287 206 223.2 1 −
control
(H2O)
Positive 20 0 0 0 0 0 0 0 0 0 − 2712 2008 2200 2288.2 10.2 +
control
(2AA)
Positive 2.0 1240 1368 1288 1297.6 6.68 + 0 0 0 0 0 0 0 0 0 −
control
(Sodium azide)
Herbal 0.1 185 154 225 185.8 0.96 − 190 253 284 239.0 1.07 −
extract 1.0 154 169 213 177 0.91 − 176 185 222 193.3 0.87 −
10.0 153 201 242 195.2 1.00 − 175 185 193 184.2 0.82 −
100.0 165 221 213 198.0 1.02 − 146 202 201 181.0 0.81 −
1000.0 209 207 202 206 1.06 − 131 136 194 151.2 0.68 −
[0000]
TABLE 7
The effect of the herbal extract on the bacterial indicator strain TA 1537 in the Ames test.
TA 1537 strain
Studied Dose −S9 +S9
substance μg/dish Mi1, 2, 3 Mean MO/MK MA Mi1, 2, 3 Mean MO/MK MA
Negative 0 6 6 10 7.11 1 − 9 9 11 9.62 1 −
control
(H2O)
Positive 20 0 0 0 0 0 0 0 0 0 − 126 144 118 128.9 13.4 +
control
(2AA)
Positive 20 5448 5632 5752 5609.3 788.5 + 0 0 0 0 0 0 0 0 0 −
control
(9AA)
Herbal 0.1 6 6 4 5.24 0.74 − 7 6 8 6.95 0.72 −
extract 1.0 8 7 7 7.32 1.03 − 10 8 8 8.62 0.90 −
10.0 10 10 9 9.65 1.36 − 5 3 3 3.56 0.37 −
100.0 5 5 5 5.00 0.70 − 8 7 9 7.96 0.83 −
1000.0 10 8 9 8.96 1.26 − 7 6 5 5.94 0.62 −
[0092] From the data given above we can conclude that the herbal extract in concentrations of 0.1-1000 μg/dish does not cause an increase in the number of revertants in Salmonella typhimurium strains TA 98, TA 100, TA 1537. Thus, the herbal extract does not have a mutagenic effect according to the Ames test.
3.3.2. Dominant Lethal Mutations in Murine Germ Cells
[0093] The experiments were carried out in order to evaluate the potential mutagenic properties of the herbal extract in the experiments aimed at studying dominant lethal mutations in mice hybrids F1 (CBA×C57BI6).
[0094] The herbal extract diluted in normal saline was administered i.m. to male mice in a dose of 0.7 ml/kg. This dose was 100 times higher than the recommended daily dose for humans (0.007 ml/kg).
[0000]
TABLE 8
The results of the ability of the herbal extract to induce dominant lethal mutations in mice germ cells.
Total No. of Post
Stages of No. of pregnant Fertility implantationχ<2>
Spermatogenesis Dose females females (%) losses
Normal 0 (control) 41 33 80.5 0.112
spermatozoids Herbal extract 39 34 75.6 0.011
700 μl/kg
Late 0 (control) 42 30 71.4 0.082
spermatids Herbal extract 39 29 74.4 0.037
700 μl/kg
Early 0 (control) 42 38 90.5 0.055 9.87
spermatids Herbal extract 38 29 76.3 0.143
700 μl/kg
Repeated Herbal extract 32 24 75.0 0.061 0.018
experiments 700 μl/kg
Average of 2 Herbal extract 35 27 90.5 0.099 2.93
experiments 700 μl/kg
Sum of the Herbal extract 70 53 76.0 0.097
experiments 700 μl/kg
and repeated experiments
Results of the Herbal extract 33 33 100 0.0404th
week 700 μl/kg
[0095] As can be seen from Table 8, the level of post implantation losses in animals undergoing the effect of a single i.m. injection of the herbal extract in dose of 700 μl/kg doesn't exceed the level in control animals.
3.3.3. Chromosomal Aberrations in Murine Bone Marrow Cells
[0096] The essence of this method consists in an evaluation of the effect of the examined substance introduced into the body of an animal on the genetic system of bone marrow cells sensitive to effects of chemical agents and physical factors. Chromosomal aberrations were analyzed after the administration of the herbal extract according to the scheme disclosed in “Instructions for experimental (pre-clinical) study of new pharmacologic substances”.
[0000]
TABLE 9
Structural disturbances of chromosomes in the bone marrow cells of mice under the effect of the herbal extract.
Cell number Aberrations With Fragments Ex-
Variants No. Counted aberrations Single Pair change Numbers Gaps
Herbal 1 100 0 0 0 0 0 0
extract, 2 100 1 0 0 0 0 1
700 μl/kg 3 100 2 2 0 0 0 0
(after 24 4 100 2 2 0 0 0 0
hours counted) 5 100 0 0 0 0 0 0
Total 500 5 4 0 0 0 1
Herbal extract, 1 100 0 0 0 0 0 0
700 μl/kg 2 100 1 0 0 0 0 1
4 days injection 3 100 2 2 0 0 0 0
(6 hours after 4 100 1 1 0 0 0 04th
day counted) 5 100 0 0 0 0 0 0
Total 500 4 3 0 0 0 1
Normal 1 100 2 2 0 0 0 0
saline 2 100 1 0 0 0 0 1
3 100 2 1 0 0 0 1
4 100 0 0 0 0 0 0
5 100 0 0 0 0 0 0
Total 500 5 3 0 0 0 2
[0097] As can be seen from the experimental data, statistically significant differences in the level of chromosomal aberrations were not observed in the bone marrow of mice undergoing the effect of the herbal extract in studied doses as compared to the control. Therefore, according to the test of the chromosomal aberrations in bone marrow cells, the herbal extract doesn't possess mutagenic activity.
[0098] 3.3.4. DNA Damaging Effect in the SOS-Chromotest
[0099] One of the tests of DNA damage is the test for determination of the induction of the SOS-response of a bacterial cell to the effect of the agent investigated, the so-called SOS-chromotest. The test is based on the knowledge of SOS-response to DNA damages. The basis of the test is the strain of E. coli PQ 37 constructed by means of the association of LacZ responsible for the synthesis of the enzyme beta-galactosidase with the gene sfiA controlled by the general repressor of the SOS-system. SfiA expression is induced after DNA damage as a part of the SOS-response. In this test, SOS expression is measured according to the qualitative determination of enzyme activity of beta-galactosidase, which can be measured according to colour reaction. The marker of cell growth in this strain is alkaline phosphatase, the activity of which can also be measured according to colour reaction. As a result of analysis, the curves of dependence of the beta-galactosidase synthesis on the concentration of the investigated substance and the curves characterising the changes of bacterial growth in such conditions are obtained. According to these indices, SOS-inducing potency is calculated. This potency reflects the ability of the substance to induce sfiA gene expression.
[0100] The results obtained showed that the herbal extract didn't induce the activation of DNA repair system in any of the investigated concentrations in E. coli PQ 37. Thus, the herbal extract doesn't possess a DNA damaging effect.
3.4. Embryotoxicity and Teratogenicity
[0101] The experiments were conducted in 36 pregnant Wistar rats divided into 2 groups, 18 rats in each: first group—control, second group—herbal extract (in a dose of 0.21 ml/kg that is 30 times higher than the maximal daily dose for humans). The preparation was diluted in normal saline and administered i.m. to rats once a day during all the period of pregnancy (from first day up to birth). The control animals received the appropriate amount of normal saline every day from the first day of pregnancy up to birth. At the 20th day of pregnancy, 70% of pregnant rats were sacrificed by means of dislocation of cervical vertebrate for the subsequent examination of the bony skeletons and internal organs of the fetuses and determination of the indices of pre-implantation and post-implantation death.
[0000]
TABLE 10
The changes of body weight of pregnant rats (% of initial).
Animal groups 1 week 2 weeks 3 week
Control 123 ± 1.9 136.7 ± 2.2 145.7 ± 2.8
Herbal extract, 0.21 ml/kg 112.5 ± 1.5* 120.5 ± 3.4* 131.6 ± 4.5*
Note: *indicates significant difference (P < 0.05)
[0102] As summarized in table 10, i.m. injection of the herbal extract to pregnant females in a dose of 0.21 ml/kg didn't influence the increase in the body weight of pregnant females as compared to the control group during all the period of pregnancy.
[0103] Such criteria of the evaluation of embryotoxicity of the herbal extract in the duration of pregnancy as the number of alive fetuses, implantation sites, yellow bodies, and embryo body weight were lower in the group of rats receiving the preparation. The index of pre-implantation death in the experimental group was significantly higher than that of the control, but post-implantation death level was lower. Cranio-caudal size of the fetuses of pregnant rats receiving the preparation in dose of 0.21 ml/kg was not statistically different from the appropriate size in the control group (Table 11).
[0000]
TABLE 11
The indices of embryotoxic effects of the herbal extract
in a dose of 0.21 ml/kg following i.m. administrationof the preparation from the first till the 20th day of pregnancy.
Herbal extract
Investigated indices Control 0.21 ml/kg
The duration of pregnancy (days) 24.5 ± 0.2 23.3 ± 0.2*
The number of embryos per rat 9.5 ± 1.3 5.5 ± 0.6*
The number of yellow bodies per rat 10.0 ± 1.2 6.0 ± 0.8*
Pre-implantation death (%) 13.5 40.8
Post-implantation death (%) 8 5.1
Cranio-caudal size of the fetus (cm) 3.3 ± 0.1 3.0 ± 0.1
The weight of the fetus (g) 3.0 ± 0.1 2.2 ± 0.2*
Note: *indicates significant difference (P < 0.05)
[0104] Macroscopic and microanatomic examinations (standard incisions according to Wilson-Diban) of fetuses undergoing the effect of the herbal extract in their pre-natal period in a dose of 0.21 ml/kg revealed underdevelopment of the fetus in 6.7% of cases. The frequency of such pathologies as hydronephrosis and hemopericardium in the group of rats receiving the herbal extract during pregnancy exceeded that of the control group.
[0105] When analyzing total fetus body preparations stained with alizarin with the purpose of studying the development of bony system in rat fetuses undergoing the effect of the herbal extract in a dose 0.21 ml/kg in the pre-natal period, no developmental defects of the skeleton were revealed. However, inhibition of ossification in the majority of the investigated foci of bone was observed.
[0106] In conclusion, it must be noted that i.m. administration of the herbal extract in a dose of 0.21 ml/kg (this dose is 30 times higher than the maximal therapeutic dose for humans) to pregnant rats from the first to the 20thday of pregnancy had a negative effect on the changes of the body weight of pregnant rats and on the duration of pregnancy. Effects of the preparation on indices of embryotoxicity such as the number of live fetuses, embryo weight, the number of implantation sites and yellow bodies, parameters of pre- and post-implantation death, and also the inhibition of ossification of the foci of bone were observed.
[0107] During the post-natal development of young rats, significant retardation of physical development was noted. In the experimental group, an underdevelopment of fetuses was revealed in 6.7%. This underdevelopment can be considered as developmental defect of embryos.
[0108] Thus, embryotoxic and teratogenic effects of the herbal extract in dose of 0.21 ml/kg were revealed. In connection with this, pregnancy must be considered to be a contraindication to the administration of the preparation.
3.5. Effect on the Reproductive Function
[0109] Investigations were carried out in Wistar rats (males and females; initial body weight 180-200 g). A group of females, consisting of 60 animals, was divided into two sub-groups: the control group (40 animals) and the experimental group (20 animals). Every day females of the experimental group were injected i.m. with 0.21 ml/kg of the herbal extract during the period of 2 weeks (3-4 of estrous cycle). Before injection, the preparation was diluted in normal saline.
[0110] Intramuscular injection of 0.21 ml/kg of the herbal extract to male and female rats did not change such indices of the reproductive function of rats as amounts of yellow bodies, implantations, live fetuses, and resorptions. The value of pre-implantation and post-implantation deaths was not very different from the control one; it was true both for the females who received the preparation and for the females who were impregnated by the males who had been injected the preparation.
[0111] The investigations helped to establish that i.m. injection of 0.21 ml/kg of the herbal extract (that is 30 times higher than the maximal daily dose recommended for humans) does not influence either sexual activity, reproductive indexes (amount of live fetuses, their body weight, amount of yellow bodies, cranio-caudal rate, places of implantation, resorptions), or neonatal development of the rats. Thus, influence of the preparation investigated on the reproductive function of sexually healthy mature rats was not revealed.
3.6. Immunorelated Effects
3.6.1. Allergenic and Anaphylactic Effects
[0112] This study was done in accordance with “Methodical instructions for evaluating allergenic properties of pharmacological substances” (Experimental study guide of new pharmacological substances. Moscow, 2000, pp. 25-32).
[0113] Investigations were carried out in 15 guinea-pigs (males, body weight is equal to 270-320 g), that were divided into three groups. Each group contained 5 animals: first group—control (normal saline), second group—0.07 ml/kg of the herbal extract, third group—0.14 ml/kg of the herbal extract. The preparation of doses used corresponded to 10 and 20 times more than daily doses recommended for a human. The investigations showed that 0.14 ml/kg of the herbal extract (20 times more than the daily therapeutic dose for a human) does not cause an anaphylactic shock upon i.m. injection on the 14thand on the 21<st >days of sensitization.
3.6.2. Delayed Type Hypersensitivity Reactions
[0114] Examinations were done on the skin of 15 guinea-pigs, that were of white-coloured skin (males, body weight is equal to 260-310 g). They were divided into 3 groups of 5 animals in each: first group—control, the second group—0.007 ml/kg of the herbal extract, third group—0.035 ml/kg of the herbal extract. The doses of the herbal extract mentioned above were diluted in sterile normal saline, then mixed with complete Freund's adjuvant in a ratio of 1:1 and then injected into the animals. According to the results of the previous investigations, reaction of hypersensitivity of a delayed-type in the herbal extract doses mentioned was negative for the guinea-pigs.
[0115] Forty-nine hybrid mice F1 (CBA*C57B16) (males, body weight is equal to 18-20 g) were divided into 7 groups, each group had 7 animals. The mice were immunized with a subcutaneous (s.c.) injection of RCR (dose: 2×10<8 >cells for a mouse) in an interscapular region. The difference in their mass characterised the degree of edema and intensity of the hypersensitivity reaction of a delayed type. The index of the reaction was calculated according to the formula:
[0000]
U=(P0−Pcontrol/Pcontrol)×100
[0000] where P0 is the mass of the experimental foot, and Pcontrol is the mass of the control foot.
[0116] Data analysis proved that the tested doses of the herbal extract, i.e. 0.18 and 0.07 ml/kg, did not influence the formation of hypersensitivity reaction of delayed type for mice or cellular immunity. Therefore, results of the experiments showed that the herbal extract did not have immunotoxic properties.
3.6.3. Effects on the Mass and Number of Cells of the Popliteal Lymph Nodes in Mice
[0117] To evaluate the allergenic properties of the herbal extract, the method of popliteal lymph nodes weight and mass change for rats was used as a response to an antigenic irritant, a so-called “popliteal lymph node assay”, PLNA. Ten hybrid mice F1 (CBA*C57B16) (males, body weight is equal to 18-20 g) were injected with 50 μl of sterile normal saline (control), and with 0.07 ml/kg of the herbal extract, respectively.
[0118] After 7 days weight and cellularity of right and left popliteal lymph nodes of the mice were determined. The relative index was calculated by means of division of left lymph node indices by similar indices of the right lymph node. Relative indices of weight and cellularity of lymph nodes both for the experimental and control group are equal to 0.95 and 0.98. Thus, evaluating the influence of the herbal extract on the weight and cellularity of lymph nodes, it was established that the preparation does not have allergenic properties.
3.6.4. Immunotoxic Effect
[0119] A study on different immunotoxic properties of the herbal extract was carried out in accordance with WHO recommendations. Examination of the influence of the herbal extract on the humoral immune response was determined by the number of antibody-forming cells in a spleen according to Erne. The influence of the herbal extract on cellular immunity was determined by hypersensitivity reaction of a delayed-type for mice. According to the recommendations of Labor Meeting in Arlington, the influence of the preparation on spleen cellularity against a background of anti-genic stimulus was defined.
3.6.5. Effect on the Amount of Antibody-Forming Murine Spleen Cells
[0120] To study the influence of the herbal extract on the amount of antibody-forming cells in spleens of mice, a direct method of local hemolysis was used. It helps to define the cells forming immunoglobulin M-antibody with a high hemolytic activity. Forty-nine hybrid mice F1 (CBA*C57B16) were selected (males with a body weight equal to 18-20 g). The mice were divided into 7 groups, each group consisted of 7 animals. The mice were immunized with an i.v. injection of sheep red blood cells (SRBC). On the forth day after immunization the number of antibody forming colonies (AFC) in a mice spleen was defined according to Jerne's method. From acquired results we understood that the herbal extract did not influence the amount of AFC in mice spleens, which were immunized by SRBC using the doses and scheme as mentioned, and correspondingly, did not influence the primary immune response.
3.6.6. Effect on the Number of Nucleus-Containing Murine Spleen Cells
[0121] Examinations were carried out on 49 mice of F1 (CBA*C57B16; males, the body weight of which was equal to 18-20 g). The animals were divided into 7 groups, every group consisted of 7 mice. They were immunized with an i.v. injection of SRBC in a dose of 5×10<8 >cells per mouse. The data proved that a single i.m. injection of the herbal extract to mice did not influence the cellularity of the spleen if doses of the preparation equal to 0.18, 0.07 ml/kg were given before the day of immunization, on the day and after the day as well.
3.7. Conclusion
[0122] Summing up the result of the toxic study on the herbal extract that was considered to be an immunomodulating drug, we can note that the preparation was clean at a single i.m. injection of the herbal extract to laboratory animals and was well assimilated by Wistar rats and dogs during i.m. injection.
[0123] The investigation held showed that a single i.m. injection of the herbal extract diluted 1:10 in normal saline in doses of 0.5-1.0 ml per one mouse (BALB/c mice) did not cause intoxication and death of the animals. An increased dose of the preparation (diluted 1:10 in normal saline in doses of 1.5-2.0 ml per mouse, 75-100 ml/kg) led to a decrease of motor activity and depression in the animals, but their death was not observed.
[0124] Intramuscular and i.p. injection of the herbal extract dissolved with 1:5 physiological solution to BALB/c mice was accompanied by a great depression of animals, narcosis and sleep. Animal intoxication with the herbal extract on LD50 level was similar to their poisoning by ethyl alcohol, the latter being a part of the preparation.
[0125] According to LD50 indices, the herbal extract can be classified as belonging to the group of safe preparations if an i.m. injection of 51-66 ml/kg of the substance of the preparation after 1:5 dissolution with a physiological solution occurs. At the same time no significant specific and sexual differences in the sensitivity under conditions of a chronic experiment was observed in Wistar rats treated daily for 3 months by i.m. injection of 0.07 and 0.21 ml/kg of the preparation and in dogs treated daily for 1 month by i.m. injection of 0.07 ml/kg of the herbal extract. The marked doses of the preparation were diluted 1:10 in sterile normal saline before injection. The doses of the herbal extract tested on mice and dogs in chronic experiments exceeded the daily therapeutic dose for humans (0.5 ml/person or 0.007 ml/kg; 10 or 30 times).
[0126] Results of the studies showed that doses of 0.07 and 0.21 ml/kg of the herbal extract in a 3 month chronic experiment in mice and 0.07 ml/kg in a 1 month chronic experiment in dogs were well assimilated by animals and did not influence hematologic indices or functional conditions of main organs of the test animals (according to the data of the biochemical tests used and of the ECG). Absence of toxic damages in inner organs, general and local toxico-allergic reactions that are concerned with the effect of the herbal extract was confirmed by the results of pathomorphological investigations, held after the end of chronic experiments. Locally irritating effect of the preparation in chronic experiments in mice and dogs using doses of 0.07 and 0.21 ml/kg at a long-term i.m. injection of 1:10 dilutions in sterile normal saline was not observed. According to requirements of Pharmacological State Committee of Ministry of Public Health, an investigation of mutagenic properties of the herbal extract was held.
[0127] Besides, we studied the ability of the preparation to cause gene mutations at indicated cultures of Salmonella typhimurium in the Ames test, to stimulate chromosomal aberrations in the cells of hybrid F1(CBA*C57B16) mice bone marrow cells, to influence the amount of dominant lethal mutation in embryonic mice cells and to influence the system of DNA repair of E. coli PQ 37 in SOS-chromotest.
[0128] During the investigation held it was established that the herbal extract did not have mutagenic properties.
[0129] In a dose equal to 0.21 ml/kg (that is 30 times higher than the daily dose recommended for humans), the herbal extract reduced the increase of pregnant rat's body weight upon the i.m. injection for the first to the 20thdays of gestation, it also reduced the duration of pregnancy, amount of alive fetuses, places of implantation, yellow bodies and embryo's body weight. At the same time the index of pre-implant death was much lower for the mice that received 0.21 ml/kg of the herbal extract during pregnancy than for control mice, and the indices of post-implant death were lower for the first group.
[0130] During a macroscopic examination and microscopic investigation of standard sections of fetuses (according to Wilson-Diban) that underwent the influence of 0.21 ml/kg of the herbal extract during the prenatal period, in 6.7% of all the cases it was possible to suggest about underdevelopment of the fetuses. The effect can be evaluated as a defect of embryo's development.
[0131] An analysis of the preparations stained with alizarin that was necessary for studying the development of bone system in rat fetuses exposed to 0.21 ml/kg of the herbal extract during the prenatal period did not show defects of skeleton development. But at the same time a delay of ossification in the majority of points of calcification was observed.
[0132] Under the influence of i.m. injection of 0.21 ml/kg of the herbal extract from the first to the 20thdays of gestation, a decrease in new born rats and an increase in stillborn rats was noticed as compared with the control group. The body weight of rats exposed to the herbal extract during the prenatal period was lower than the indices of the control group. Results of the experiment on the development of the descendants did not deviate from the time constraints typical for a normal physiological development of this type of animals.
[0133] Therefore, the experiments held to establish that an i.m. injection of 0.21 ml/kg of the herbal extract (30-fold the highest daily dose for a human) from the first to the 20thdays of gestation has an embryotoxic and teratogenic effect in animals exposed to the herbal extract. Therefore, the pregnancy can be considered as a contraindication for prescription of the herbal extract.
[0134] At a daily i.m. injection of 0.21 ml/kg of the herbal extract to male rats during 10 weeks and female rats during 2 weeks, the influence of the preparation on the reproductive function of animals was not established.
[0135] Studying the allergenic properties of the herbal extract on guinea-pigs showed that at 5-fold i.m. injection of the herbal extract in sensitizing doses of 0.07 and 0.14 ml/kg and i.p. injection of a determinant dose of 0.14 ml/kg of the herbal extract at the 14thand 21<st >days after sensitization, the preparation did not cause anaphylactic shock.
[0136] The herbal extract in studied doses and schedules of sensitization did not have an allergenic effect of delayed type hypersensivity reaction in guinea-pigs and in the reaction of the popliteal lymphnode in mice.
[0137] In doses of 0.07 ml/kg and 0.18 ml/kg, the herbal extract did not influence the number of antibody-forming and nucleus-containing cells in the spleen, and it did not influences the reaction of hypersensitivity in mice. The data are evidence of the absence of a negative influence of the herbal extract on the humoral and cellular immunity and therefore of the absence of immunotoxicity of the preparation.
[0138] Finally, based on all of the experiments conducted and the obtained results, the herbal extract is recommended for clinical trials with the only contraindication of pregnancy.
Example 4
Pharmacologic Effects of the Herbal Extract
[0139] Studies were conducted in order to determine the herbal extract's potential in patients.
[0140] The first study was done in the year 2000. This study was designed for a clinical try on. The first aim of this project was the determination of toxicity or side-effects of the herbal extract in HIV patients, and the next aim was the determination of probable effectiveness of the herbal extract on the course of disease and humoral, cellular and non-specific immunities in the HIV-infected persons.
[0141] The 16 to 40 years old HIV positive patients who were at high risk to develop AIDS in general examination were selected for study. The patients used the herbal extract for 80 days. In this project, 0.4 ml of the herbal extract that had been diluted by normal saline up to 4 ml was injected i.m. and i.v. daily. During this period, the patients were examined daily and the effects of the treatment were registered. After a treatment period of 3 months, the patients were pursued and their preclinical factors were studied. The percentages of T-lymphocytes CD4 were determined at 21±1% at the first day of the study, 23±1.5% after 30 days of treatment with the herbal extract, 32±0.8% after 60 days of treatment, 32±0.7% after 80 days of treatment and 39±1.6% three months after the end of treatment period. These data show an increase in the amount of CD4 T-lymphocytes during treatment with the herbal extract. The percentages of CD8 lymphocytes in patients were 25±1.5% at the first day of the study, 24±1.5% after 30 days of treatment with the herbal extract, 22±0.8% after 60 days of treatment and after 80 days of treatment, it was 23±0.8%. In the follow-up of three months after the end of the treatment it was 20±2%.
[0142] The percentages of T-lymphocytes including CD95 were 40±9.2% at the first day of the study, 47±2% after 30 days of treatment with the herbal extract, 25±1.4% after 60 days of treatment, 30±1.3% after 80 days of treatment. In the follow-up of three months after the end of the treatment it was 25±1.5%. In the statistical comparison at the first day and 80 days after treatment with the herbal extract, there were an obvious differences between CD4, CD8 and CD95 in the patients (P<0.01).
[0143] The next study was done for considering the early and late side-effects of the herbal extract in the AIDS patients and HIV infected persons. In this project, six volunteers who were HIV positive were selected. The inclusion criteria were: HIV positive with severe fungi or other opportunistic infection, reduction of body weight more than 10%, secondary zoster, fever for more than one month. After selection of patients and entrance in the project, the preclinical parameters were measured initially and then the measurement was repeated weekly.
[0144] 0.4 ml of the herbal extract was taken in a 5 ml syringe, diluted with 3.5 ml of warm normal saline and was injected i.m. for two days and than i.v. for two more days. After completion of injections, the patients were examined for the drug side-effects and appearance of AIDS related clinical signs and symptoms. Data are shown in Table 12.
[0000]
TABLE 12
Clinical data obtained in the second clinical study on the herbal extract enrolling six volunteers.
CD4 CD8
CD4 % CD4 count CD8 % CD8 count3<rd>3<rd>3<rd>3<rd>
CD4 % count CD8 % count
No. Age Sex Weight before month before month before month before month
1 38 male 76 2 12 28 260 62 62 881 1345
2 42 male 63 21 21 177 318 58 56 490 894
3 48 male 56 22 23 352 636 15 47 688 799
4 23 male 67 22 23 350 360 23 38 318 574
5 23 female 54 24 41 352 651 41 43 632 683
6 28 male 56 19 51 221 1273 51 25 252 624
Mean 33.6 62 17.5 27.1 247 517 40 45 543 812
P* 0.04 0.02 0.1 0.02
*P-value by Wilcoxon signed ranks test
[0145] The next study was done for comparing the effectiveness and early side-effects of the herbal extract with routine HAART treatment in recovery of immunological factors in AIDS patients. In this study, 27 volunteers were studied. The patients were randomly divided into two treatment groups treated either with the herbal extract or HARRT (with selection of 4 blocks of randomization blocks). Two patients showed no tolerance in HAART, and thus, they were transferred to the herbal extract group. The patient's entrance and exit criteria were: HIV positive, severe affection by fungi or opportunistic infections now or before, reduction of body weight more than 10%, secondary zoster, fever for more than one month and minor opportunistic affections. These patients had not used any anti-AIDS drugs or enhancement drug for immune system. Antibiotics or other drugs, however, for elimination of AIDS effects was not prohibited. After selection of patients and their entrance into the project, the preclinical factors like blood cell count, triglyceride, cholesterol, uric acid, creatinine, blood urea nitrogen, alkaline phosphatase, aspartate transaminase, alanine transaminase, fasting blood sugar, RNA viral load, CD4 (Th1, Th2), CD4/CD8, CD8, urine analysis/urine culture were measured before treatment and 1, 2, and 3 months after treatment.
[0146] In the herbal extract group, 0.4 ml of the herbal extract was taken in a 5 ml syringe and was diluted with 3.5 ml of warm normal saline serum, and the mixture was injected i.m. or i.v. once a day for 90 days. The HAART treatment was done according to the standard regimen. The treatment was 9 tablets of Caplet Nelfinavir 250 mg for 90 days together with 2 capsules of Zidovudine 300 mg for 90 days together with 2 tablets of Lamivudine 150 mg for 90 days. All of the patients were examined for drug's side-effects and appearance of AIDS related clinical signs and symptoms. The data were registered in related questioners. 16 patients in the herbal extract group and 11 patients in the HAART group were examined. The comparisons of characteristics between 2 groups are shown in Table 13.
[0000]
TABLE 13
Comparison of the two study groups (WBC—white blood cells).
Variable Herbal extract HAART P-value
Age (Mean, SD) 34.9 (8.1) 38.6 0.4
Sex female/male 4/12 0/11 0.12
Weight 62.2 (8) 57.3 (8) 0.3
CD4 percent 14.0 (6.0) 18.3 (13) 0.5
(Mean, SD)
CD4 number 227 (90) 239 (136) 0.8
(Mean, SD)
CD8 percent 39.3 (28.1) 48.6 (30.1) 0.6
(Mean, SD)
CD8 number 686 (538) 624 (408) 0.8
(Mean, SD)
WBC (Mean, SD) 5500 (3238) 4662 (667) 0.8
Lymphocyte percent 35.5 (12.2) 31.0 (9.0) 0.5
(Mean, SD)
Viral load (Mean, SD) 2161721 (1278272) 270983 (228802) 0.4
[0147] The results in Table 14 indicate that the CD4 number has increased in both groups but there is no significant difference between the groups.
[0000]
TABLE 14
Comparison of CD4, CD8, white blood cells (WBC) and lymphocyte percentage in the two study groups.
Variable Herbal extract HAART P-value
CD4 percent (Mean, SD) 17.2 (6.6) 26.2 (8.6) 0.02
CD4 number (Mean, SD) 349 (232) 470 (191) 0.21
CD8 percent (Mean, SD) 39.5 (20.5) 30.4 (19.4) 0.3
CD8 number (Mean, SD) 871 (910) 507 (303) 0.4
WBC (Mean, SD) 5242 (1819) 4900 (2225) 0.2
Lymphocyte percentage 38.1 (7.7) 38.2 (5.3) 0.5
(Mean, SD)
[0148] Table 15 shows the different parameters in the group of patients who received the herbal extract. The increase in the percentage of CD4 is obvious and evident.
[0000]
TABLE 15
Comparison of CD4, CD8, white blood cells and lymphocyte percentage in the herbal extract treated group at the beginning and the end of study
Herbal extract Herbal extract
Variable (beginning) (end) P-value
CD4 percentage (Mean, SD) 14.0 (6) 17.2 (6.6) 0.01
CD4 Number (Mean, SD) 227 (90) 349 (232) 0.002
CD8 percentage (Mean, SD) 39.3 (28.1) 39.5 (20.5) 0.9
CD8 Number (Mean, SD) 686 (538) 871 (910) 0.3
WBC* (Mean, SD) 5500 (3238) 5242 (1819) 0.4
Lymphocyte percentage 35.5 (12.2) 38.1 (7.7) 0.06
(Mean, SD)
*WBC—white blood cells
Example 5
Determination of the Maximum Tolerable Dose of the Herbal Extract
[0149] This study was conducted to determine the maximum tolerable dose (MTD) of the herbal extract in HIV infected patients and its possible side-effects and toxicity that can cause dose limitation (dose limiting toxicities, DLTs).
[0150] The study protocol was based on the dose escalation method. The effects of the herbal extract on viral load and CD4 count of patients were evaluated as by-products. Four cohorts of patients (3 patients each) were selected and treated for 28 days (4 weeks) with escalated doses of the extract. A base dose of the extract has been determined according to LD10 (10% of the lethal dose) in former animal experiments. Patients were observed carefully for signs and symptoms of side-effects and toxicity by physical examination and laboratory workups according to the protocol.
[0151] All patients were male in the age of 28-60 years (mean: 41.6 years.). In the first cohort, the daily dose of 2 ml of extract in 100 ml warm normal saline was infused over 0.5-1 hr intravenously for 28 days. No toxicity or major side-effects were observed except for an increase in sweating and weight loss in 2 patients. In the second cohort, three other patients received a daily dose of 4 ml. There were no major side-effects and toxicity in this group. In the third cohort of originally 4 patients, one patient was excluded due to non-compliance and inability for regular daily attendance and the daily dose of 6.7 ml administered. In this group there were not only no major dose limiting toxicity and side-effects but also no minor ones. In the fourth cohort, three other patients received the daily dose of 10 ml, and there were no major side-effects and toxicity in this group too.
[0152] In summary, a total of 12 patients was included in the study who were treated for 4 weeks with escalated doses of the herbal extract. There was not toxicity or side-effects in all cohorts.
US2010233305
HERBAL EXTRACTS FOR TREATMENT OF CHRONIC WOUNDS
HERBAL EXTRACTS FOR TREATMENT OF CHRONIC WOUNDS
Inventor(s): FARZAMFAR BARDIA [IR]; MADANI HESSAMEDDIN [IR]; GHARIBDOUST FARHAD [IR]; FARHADI MOHAMMAD [IR]; NOVITSKY YURI ALEXEVICH [RU]; KHORRAMKHORSHID HAMID REZA [IR]; SADEGHI BEHNAM [IR]
The present invention refers to a method for preparing a herbal extract from Mellilotus sp. (Mellilotus officinalis), preferably comprising a treatment by pulsed electromagnetic field of high frequency. The herbal extract, optionally comprising selenium and/or urea and/or fructose and/or phosphoglycerol (or its sodium salt), is useful in the treatment of chronic wounds, in particular associated with states in which the normal wound repair ability is weakened, and preferably diabetic foot ulcers and/or bed sores.
https://en.wikipedia.org/wiki/Urtica_dioica
Urtica
dioica
Urtica dioica subsp. dioica
Scientific classification
Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Rosids
Order: Rosales
Family: Urticaceae
Genus: Urtica
Species: U. dioica
Binomial name -- Urtica dioica L.
Urtica dioica, often called common nettle or stinging nettle (although not all plants of this species sting), is a herbaceous perennial flowering plant, native to Europe, Asia, northern Africa, and western North America,[1] and is the best-known member of the nettle genus Urtica. The species is divided into six subspecies, five of which have many hollow stinging hairs called trichomes on the leaves and stems, which act like hypodermic needles, injecting histamine and other chemicals that produce a stinging sensation when contacted by humans and other animals.[2] The plant has a long history of use as a source of medicine, food, and fibre.
Description
Urtica dioica is a dioecious, herbaceous, perennial plant, 1 to 2 m (3 to 7 ft) tall in the summer and dying down to the ground in winter. It has widely spreading rhizomes and stolons, which are bright yellow, as are the roots. The soft, green leaves are 3 to 15 cm (1 to 6 in) long and are borne oppositely on an erect, wiry, green stem. The leaves have a strongly serrated margin, a cordate base, and an acuminate tip with a terminal leaf tooth longer than adjacent laterals. It bears small, greenish or brownish, numerous flowers in dense axillary inflorescences. The leaves and stems are very hairy with nonstinging hairs, and in most subspecies, also bear many stinging hairs (trichomes), whose tips come off when touched, transforming the hair into a needle that can inject several chemicals: acetylcholine, histamine, 5-HT (serotonin), moroidin,[3] leukotrienes,[3] and possibly formic acid.[4][5] This mixture of chemical compounds causes a painful sting or paresthesia from which the species derives one of its common names, stinging nettle, as well as the colloquial names burn nettle, burn weed, and burn hazel.[1]
Stinging nettle plants
Taxonomy
The taxonomy of Urtica species has been confused, and older sources are likely to use a variety of systematic names for these plants. Formerly, more species were recognised than are now accepted. However, at least six clear subspecies of U. dioica are described, some formerly classified as separate species:
U. dioica subsp. dioica (European stinging nettle), from Europe, Asia, and northern Africa, has stinging hairs.
U. dioica subsp. galeopsifolia (fen nettle or stingless nettle), from Europe, does not have stinging hairs.
U. dioica subsp. afghanica, from southwestern and central Asia, sometimes has stinging hairs or is sometimes hairless.[6]
U. dioica subsp. gansuensis, from eastern Asia (China), has stinging hairs.[6]
U. dioica subsp. gracilis (Ait.) Selander (American stinging nettle), from North America, has stinging hairs.
U. dioica subsp. holosericea (Nutt.) Thorne (hoary stinging nettle), from North America, has stinging hairs.[7]
Other species names formerly accepted as distinct by some authors but now regarded as synonyms of one or other subspecies include U. breweri, U. californica, U. cardiophylla, U. lyalli, U. major, U. procera, U. serra, U. strigosissima, U. trachycarpa, and U. viridis.
Distribution
U. dioica is abundant in northern Europe and much of Asia, usually found in the countryside. It is less widespread in southern Europe and north Africa, where it is restricted by its need for moist soil, but is still common. In North America, it is widely distributed in Canada and the United States, where it is found in every province and state except for Hawaii, and also can be found in northernmost Mexico. It grows in abundance in the Pacific Northwest, especially in places where annual rainfall is high. The European subspecies has been introduced into North America and South America.[8][9]
In Europe, nettles have a strong association with human habitation and buildings. The presence of nettles may indicate the site of a long-abandoned building. Human and animal waste may be responsible for elevated levels of phosphate[10] and nitrogen in the soil, providing an ideal environment for nettles.
Ecology
Nettles are the exclusive larval food plant for several species of butterflies, such as the peacock butterfly[11] or the small tortoiseshell, and are also eaten by the larvae of some moths including angle shades, buff ermine, dot moth, the flame, the gothic, grey chi, grey pug, lesser broad-bordered yellow underwing, mouse moth, setaceous Hebrew character, and small angle shades. The roots are sometimes eaten by the larva of the ghost moth Hepialus humuli.
Stinging nettle is particularly found as an understory plant in wetter environments, but it is also found in meadows. Although nutritious, it is not widely eaten by either wildlife or livestock, presumably because of the sting. It spreads by abundant seeds and also by rhizomes, and is often able to survive and re-establish quickly after fire.[12]
Nettle sting treatment
Anti-itch drugs, usually in the form of creams containing antihistaminics or hydrocortisone may provide relief from the symptoms of being stung by nettles.[13] Because of the combination of chemicals involved, though, other remedies may be required. Calamine lotion may be helpful, as well as urine, which has the advantage of usually being readily available. Many folk remedies exist for treating the itching, including dandelion, horsetail (Equisetopsida spp.), leaf of dock (Rumex spp.), greater plantain, jewelweed (Impatiens capensis and Impatiens pallida), the underside of a fern (the spores), mud, saliva, or baking soda, oil and onions, lemon juice, and topical use of milk of magnesia.[13]
Influence on language and culture
In Great Britain and Ireland, the stinging nettle (U. dioica subsp. dioica) is the only common stinging plant and has found a place in several figures of speech in the English language. Shakespeare's Hotspur urges that "out of this nettle, danger, we pluck this flower, safety" (Henry IV, part 1, Act II Scene 3). The figure of speech "to grasp the nettle" probably originated from Aesop's fable "The Boy and the Nettle".[14] In Sean O'Casey's Juno and the Paycock, one of the characters quotes Aesop "Gently touch a nettle and it'll sting you for your pains/Grasp it as a lad of mettle and soft as silk remains". The metaphor may refer to the fact that if a nettle plant is grasped firmly rather than brushed against, it does not sting so readily, because the hairs are crushed down flat and do not penetrate the skin so easily.[15] In the German language, the idiom sich in die Nesseln setzen, or to sit in nettles, means to get into trouble.[citation needed] In Hungarian, the idiom csalánba nem üt a mennykő (no lightning strikes the nettle) means bad things never happen to bad people.[citation needed] The same idiom exists in the Serbian language неће гром у коприве.[citation needed] In Dutch, a netelige situatie means a predicament.[citation needed]. The name urticaria for hives comes from the Latin name of nettle (urtica, from urure, to burn).
Uses
Food
The young leaves are edible and make a very good leaf vegetable, as with the purée shown in the above image.
U. dioica has a flavour similar to spinach mixed with cucumber when cooked, and is rich in vitamins A and C, iron, potassium, manganese, and calcium. Young plants were harvested by Native Americans and used as a cooked plant in spring when other food plants were scarce.[16] Soaking stinging nettles in water or cooking removes the stinging chemicals from the plant, which allows them to be handled and eaten without injury. After the stinging nettle enters its flowering and seed-setting stages, the leaves develop gritty particles called cystoliths, which can irritate the urinary tract.[16] In its peak season, nettle contains up to 25% protein, dry weight, which is high for a leafy green vegetable.[17] The leaves are also dried and may then be used to make a herbal tea, as can also be done with the nettle's flowers.
Nettles can be used in a variety of recipes, such as polenta, pesto, and purée.[18] Nettle soup is a common use of the plant, particularly in Northern and Eastern Europe. In Nepal (सिस्नो in Nepali) and the Kumaon and Gargwal region of northern India, stinging nettle is known as sisnu, kandeli, and bicchū-būṭī (Hindi: बिच्छू-बूटी), respectively. It is also found in abundance in Kashmir, where it is called soi.
Nettles are sometimes used in cheesemaking, for example in the production of Cornish Yarg[19] and as a flavouring in varieties of Gouda.[20]
Nettles are used in Albania as part of the dough filling for the börek. Its name is byrek me hithra. The top baby leaves are selected and simmered, then mixed with other ingredients such as herbs and rice, before being used as a filling between dough layers.[21][22]
Competitive eating
In the UK, an annual World Nettle Eating Championship draws thousands of people to Dorset, where competitors attempt to eat as much of the raw plant as possible. Competitors are given 60 cm (24 in) stalks of the plant, from which they strip the leaves and eat them. Whoever strips and eats the most stinging nettle leaves in a fixed time is the winner. The competition dates back to 1986, when two neighbouring farmers attempted to settle a dispute about which had the worst infestation of nettles.[23][24]
Drink
Nettle leaves are steeped in a concentrated sugar solution to extract the flavour. The leaves are then removed and a source of citric acid (usually lemon juice) is added to help preserve the cordial and add a tart flavour.
Commercially produced cordials are generally quite concentrated and are usually diluted by one part cordial to ten parts water – thus a 0.5 l (0.11 imp gal; 0.13 US gal) bottle of cordial would be enough for 5.5 litres (1.2 imp gal; 1.5 US gal) diluted. The high concentration of sugar in nettle cordial gives it a long shelf life.
Also, many recipes for alcoholic nettle beer are used, which is a countryside favourite in the British Isles.[25]
Medicinal
U. dioica herb has been used in the traditional Austrian medicine internally (as tea or fresh leaves) for attempted treatment of disorders of the kidneys and urinary tract, gastrointestinal tract, locomotor system, skin, cardiovascular system, hemorrhage, influenza, rheumatism, and gout.[26]
As Old English stiðe, nettle is one of the nine plants invoked in the pagan Anglo-Saxon Nine Herbs Charm, recorded in the 10th century. Nettle is believed to be a galactagogue, a substance that promotes lactation.[27]
Urtication, or flogging with nettles, is the process of deliberately applying stinging nettles to the skin in order to provoke inflammation. An agent thus used is known as a rubefacient (something that causes redness). This is done as a folk remedy in an attempt to treat rheumatism, providing temporary relief from pain.[28]
Textiles and fibre
Nettle stems contain a bast fibre that has been traditionally used for the same purposes as linen and is produced by a similar retting process. Unlike cotton, nettles grow easily without pesticides. The fibres are coarser, however.[29]
Historically, nettles have been used to make clothing for 2,000 years, and German Army uniforms were made from nettle during World War I due to a shortage of cotton. More recently, companies in Austria, Germany, and Italy have started to produce commercial nettle textiles.[30][31]
The fibre content in nettle shows a high variability and reaches from below 1% to 17%. Under middle-European conditions, stems yield typically between 45 and 55 dt / ha, which is comparable to flax stem yield. Due to the variable fibre content, the fibre yields vary between 0.2 and 7 dt / ha, but the yields are normally in the range between 2 and 4 dt / ha.[32] Fibre varieties are normally cloning varieties and therefore planted from vegetative propagated plantlets. Direct seeding is possible, but leads to great heterogeneity in maturity.[33]
Nettles may be used as a dye-stuff, producing yellow from the roots, or yello
Nutrient contents
Fresh leaves contain approximately 82.4% water, 17.6% dry matter, 5.5% protein, 0.7 to 3.3% fat, and 7.1% carbohydrates.[35] Despite the low fat content, especially mature leaves contain an interesting omega-3 (n-3): omega-6 (n-6) ratio, because about 40% of the fatty acids are α- Linolenic acid, a valuable omega-3 acid.[36] For exact fatty acid contents see Table 1. Seeds contain much more fatty acids than leaves.[36]
Minerals (Ca, K, Mg, P, Si, S, Cl) and trace elements (Mn, Cu, Fe) contents depend mostly on the soil and the season.[35]
Nettle contains different secondary metabolites. Carotenoids can be found primarily in the leaves, where different forms of lutein, xanthophyll and carotene are present (Table 2). Some carotenes are precursors of vitamin A (retinol), their retinol equivalents RE or retinol activity equivalents per g dry weight are 1.33 for mature leaves and 0.9 for yound leaves.[35] Nettle contains much less than carotenes and retinol than carrots, which contain 8.35 RE per g fresh weight.[37] Depending on the batch and the leave and stem content, nettle contains only traces of zeaxanthin or between 20 – 60 mg / kg of dry matter.[35][38] Nettle contains ascorbic acid (vitamin C), riboflavin (vitamin B2), pantothenic acid, vitamin K1[35] and tocopherols (vitamin E).[38] The highest vitamin contents can be found in the leaves.[35]
Poultry: Egg yolk colouring in laying hens
At first, whole shoot stinging nettle was fed experimentally to broiler chicken for its antioxidative properties. The broiler chicken fed 25 g stinging nettle per kg feed did not have improved antioxidative meat properties, but had a yellow skin colour.[39] Because of this findings, which indicated a good colouring property, nettle was used in laying hens instead. Another study that fed nettle powder (10 g / kg feed) or nettle essential oil to broilers did not find an improvement of the broilers' performance.[40]
In laying hens, nettle can effectively be used as an egg yolk colorant instead of artificial pigments or other natural pigments (derived from marigold for yellow). Nettle has high carotenoid contents, especially lutein, β-carotene and zeaxanthin, of which lutein and zeaxanthin act as yellow pigments.[38] Feeding as little as 6.25 g dry nettle per kg feed is as effective as the synthetic pigments to colour the egg yolk. Feeding nettle has no detrimental effect on the performance of the laying hens or the general egg quality.[38] The nettle dosage can be increased to 25 g per kg feed without impairing the performance.[38] Oxidation properties of the egg yolk lipids were not affected by the nettle addition, as the natural antioxidants (tocopherols and others) from nettle did not appear to be transferred to the egg. Because the carotenoid content varies, which is assumed to depend on climate, plant age and genotype, analyses of the carotenoid contents are recommended prior to large-scale application.[38]
Ruminants
Nettle sting not only humans, animals do not like to touch fresh stinging nettle. If the nettle are wilted or dry, voluntary intake can be high. Dry nettle shoots are said to be “as good as good alfalfa hay”. As with other leafy herbs with relatively hard stems, the harvest should be done carefully to avoid leave drop losses. Nettle can be conserved as hay or silage.
Gardening
Nettles have a number of other uses in the vegetable garden, including the potential for encouraging beneficial insects. The growth of nettles is an indicator that an area has high fertility (especially phosphate and nitrate) and has been disturbed.[42][43][not in citation given]
Nettles contain a lot of nitrogenous compounds, so are used as a compost activator[44] or can be used to make a liquid fertilizer, which although somewhat low in phosphate, is useful in supplying magnesium, sulphur, and iron.[45][46] They are also one of the few plants that can tolerate, and flourish in, soils rich in poultry droppings.
Recent experiments have shown that nettles are a beneficial weed, having use as a companion plant.[47]
U. dioica can be a troubling weed, and mowing can increase plant density.[48] Regular and persistent tilling will greatly reduce its numbers, and the use of herbicides such as 2,4-D and glyphosate are effective control measures.[48]
Field cultivation
Sowing and planting[49]
Three cultivation techniques can be used for the stinging nettle: 1) direct sowing, 2) growing seedlings in nurseries with subsequent transplantation and 3) vegetative propagation via stolons or head cuttings.
Direct sowing: The seedbed should have a loose and fine structure, but should be reconsolidated using a packer roller imminently prior to sowing.[50] Sowing time can be either in autumn[51] or in spring.[52] Seed density should be 6 kg/ha with row spacing of 30 cm and 42–50 cm in autumn and spring, respectively.[50][51][53] The disadvantage of direct sowing is that it usually leads to incomplete plant coverage.[50][53] This drawback can be mitigated by covering the seedbed with a transparent perforated foil in order to improve seed germination.[50][51] Further, weed control can be problematic as the stinging nettle has a slow seedling development time.[50]
Growing seedlings: For this technique pre-germinated seeds are sown between mid-/end-February and beginning of April and grown in nurseries. Seedlings are grown in tuffs with 3-5 plants / tuff and a seed density of 1.2-1.6 kg / 1000 tuffs. A fastened germination is achieved by alternating high temperature during daytime (30 °C for 8 h) and lower temperature during nighttime (20 °C for 16 h).[50][53] Before transplanting, the seedlings should be fertilized and acclimated to cold temperatures.[50] Transplantation should start around Mid-April with row spacing of 42–50 cm and plant spacing within
rows of 25–30 cm.[52][53]
Vegetative propagation: Stolons (with several buds) of 10 cm should be planted from Mid-April in a depth of 5–7 cm.[52] Head cuttings are grown in nurseries starting between mid-May and mid-June. Growing tips with two leaf-pairs are cut from the mother plant and treated with root-growth inducing hormones. Transplantation can be delayed in comparison to the growing seedling technique.[50]
Greenhouse cultivation[54]
The stinging nettle can also been grown in controlled-environment agriculture (CEA) systems, such as soil-less medium cultivations or aeroponics.
According to a study,[54] CEA systems can achieve much higher yields, standardize the quality, as well as reduce harvesting costs and contamination
https://en.wikipedia.org/wiki/Tansy
Tansy
Not to be confused with tansy ragwort or tansy mustard.
Tanacetum vulgare - harilik soolikarohi Keilas2.jpg
Scientific classification
Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Asterids
Order: Asterales
Family: Asteraceae
Genus: Tanacetum
Species: T. vulgare
Binomial name Tanacetum vulgare L.
Tansy (Tanacetum vulgare) is a perennial, herbaceous flowering plant of the aster family, native to temperate Europe and Asia. It has been introduced to other parts of the world and in some areas has become invasive. It is also known as common tansy,[1] bitter buttons, cow bitter, or golden buttons.
Description
Blue tansy (Tanacetum annuum) essential oil in a clear glass vial, not to be confused with the oil from common Tansy (Tanacetum vulgare) which is not blue.
Tansy is a flowering herbaceous plant with finely divided compound leaves and yellow, button-like flowers. It has a stout, somewhat reddish, erect stem, usually smooth, 50–150 cm (20–59 in) tall, and branching near the top. The leaves are alternate, 10–15 cm (3.9–5.9 in) long and are pinnately lobed, divided almost to the center into about seven pairs of segments, or lobes, which are again divided into smaller lobes having saw-toothed edges, giving the leaf a somewhat fernlike appearance. The roundish, flat-topped, button-like, yellow flower heads are produced in terminal clusters from mid-to-late summer. The scent is similar to that of camphor with hints of rosemary. The leaves and flowers are toxic if consumed in large quantities; the volatile oil contains toxic compounds including thujone, which can cause convulsions and liver and brain damage. Some insects, notably the tansy beetle Chrysolina graminis, have resistance to the toxins and subsist almost exclusively on the plant.
History and distribution
Tansy is native to Eurasia; it is found in almost all parts of mainland Europe, as well as Britain and Ireland. It is absent from Siberia and some of the Mediterranean islands.[2] The ancient Greeks may have been the first to cultivate it as a medicinal herb.[3] In the sixteenth century it was considered to be "necessary for a garden" in Britain.[4]
History of uses
Tansy has a long history of use. It was first recorded as being cultivated by the ancient Greeks for medicinal purposes. In the 8th century AD it was grown in the herb gardens of Charlemagne and by Benedictine monks of the Swiss monastery of Saint Gall. Tansy was used to treat intestinal worms, rheumatism, digestive problems, fevers, sores, and to “bring out” measles.[3][5][6][7][8]
During the Middle Ages and later, high doses were used to induce abortions.[6][9][10] Contradictorily, tansy was also used to help women conceive and to prevent miscarriages.[5][6][11] In the 15th century, Christians began serving tansy with Lenten meals to commemorate the bitter herbs eaten by the Israelites.[8][11] Tansy was thought to have the added Lenten benefits of controlling flatulence brought on by days of eating fish and pulses[5][6] and of preventing the intestinal worms believed to be caused by eating fish during Lent.[12]
Tansy was used as a face wash and was reported to lighten and purify the skin.[5][6] In the 19th century, Irish folklore suggested that bathing in a solution of tansy and salts would cure joint pain.[13] Although most of its medicinal uses have been discredited, tansy is still a component of some medicines and is listed by the United States Pharmacopeia as a treatment for fevers, feverish colds, and jaundice.[3][6][11]
Insect repellent
Tansy has also been cultivated and used for its insect repellent and in the worm warding type of embalming.[3][6][7] It was packed into coffins, wrapped in funeral winding sheets, and tansy wreaths were sometimes placed on the dead.[3][6][7][11] Henry Dunster, the first president of Harvard University, was buried wearing a tansy wreath in a coffin packed with tansy; when “God’s Acre” was moved in 1846 the tansy had maintained its shape and fragrance, helping to identify the president’s remains.[3][6][11] By the 19th century, tansy was used so much at New England funerals that people began to disdain it for its morbid association with death.[6][8]
During the American colonial period, meat was frequently rubbed with or packed in tansy leaves to repel insects and delay spoilage.[3][6][11] Tansy was frequently worn at that time in shoes to prevent malaria and other fevers;[6][11] it has been shown, however, that some mosquito species including Culex pipiens take nectar from tansy flowers.[14]
Tansy can be used as in companion planting and for biological pest control. It is planted alongside potatoes to repel the Colorado potato beetle, with one study finding tansy reduced the beetle population by 60 to 100%.[6][15][16]
In England tansy is placed on window sills to repel flies; sprigs are placed in bed linen to drive away pests, and it has been used as an ant repellent.[17]
In the 1940s, distilled tansy oil mixed with fleabane, pennyroyal and diluted alcohol was a well known mosquito repellent; collectors were paid five cents a pound for tansy in full bloom.[6][18] Research has found that tansy extracts do indeed repel mosquitoes, but not as effectively as products containing DEET.[6][19] In 2008, researchers in Sweden investigated the use of tansy to repel ticks, showing a 64–72% repellency for each oil constituent.[20]
Toxicity
Many tansy species contain a volatile oil which can cause contact dermatitis in sensitive individuals. If taken internally, toxic metabolites are produced as the oil is broken down in the liver and digestive tract. It is highly toxic to internal parasites, and for centuries tansy tea has been prescribed by herbalists to expel worms. Tansy is an effective insecticide and is highly toxic to arthropods.[21] Because it contains thujone, the U.S. FDA limits the use of tansy to alcoholic beverages, and the final product must be thujone-free.[22] Tanacetum annuum is often confused with common tansy (Tanacetum vulgare) but the former produces an essential oil that is completely different chemically as it contains no thujone and high amounts of chamazulene making the oil dark blue in color, giving rise to it common name of Blue Tansy Oil.[23][24] Despite claims by some unethical resellers of essential oils who adulterate the very expensive Blue Tansy (Tanacetum annuum) oil with the much cheaper oil from Tanacetum vulgare, it should be noted that the oil from Tanacetum vulgare is never blue in color as it contains no chamazulene.[25][26] For this reason a high thujone oil from Tanacetum vulgare should never be referred to as "Blue Tansy" oil and any such blue oil containing significant thujone is an adulterated product.
The active components of the volatile oil include 1,8-cineole, trans-thujone, camphor and myrtenol, with the quantities and proportions of each varying seasonally and from plant to plant.[6][19][27][28][29]
1,8-Cineole is a toxin believed to defend the plant leaves against attacks by herbivores.[27][28]
Culinary uses
Tansy foil
Tansy was formerly used as a flavoring for puddings and omelets, but is now almost unknown. The herbalist John Gerard (c. 1545–1612) noted that tansy was well known as “pleasant in taste”, and he recommends tansy sweetmeats as “an especial thing against the gout, if every day for a certain space a reasonable quantitie thereof be eaten fasting.” In Yorkshire, tansy and caraway seeds were traditionally used in biscuits served at funerals.[12]
During the Restoration, a "tansy" was a sweet omelette flavoured with tansy juice. In the BBC documentary "The Supersizers go ... Restoration", Allegra McEvedy described the flavour as "fruity, sharpness to it and then there's a sort of explosion of cool heat a bit like peppermint." [30] However, the programme's presenter Sue Perkins experienced tansy toxicity.
According to liquor historian A. J. Baime, in the 19th century Tennessee whiskey magnate Jack Daniel enjoyed drinking his own whiskey with sugar and crushed tansy leaf.
Ethnomedical use
For many years, tansy has been used as a medicinal herb despite its toxicity. 19th-century Irish folklore suggests bathing in a solution of tansy and salt as a cure for joint pain.[13] A bitter tea made with tansy flowers has been used for centuries as an anthelmintic to treat parasitic worm infestations, and tansy cakes were traditionally eaten during Lent because it was believed that eating fish during Lent caused intestinal worms.[12] Various Tanacetum species are used ethnomedically to treat migraine, neuralgia and rheumatism and as anthelmintics. Traditionally, tansy was often used for its emmenagogue effects to bring on menstruation or end an unwanted pregnancy, and pregnant women are advised to not use this herb.[31] Research published in 2011 identified 3,5-dicaffeoylquinic acid (3,5-DCQA) and axillarin in tansy as antiviral compounds that are active against herpes simplex virus.[32][medical citation needed]
Among Native Americans
The Cherokee use an infusion of the plant for backache, use the plant as a tonic, and wear it around the waist and in shoes to prevent miscarriages.[33] The Cheyenne use an infusion of the pulverized leaves and blossoms for dizziness and weakness.[34]
Other uses
Some traditional dyers use tansy to produce a golden-yellow color.[35] The yellow flowers are dried for use in floral arrangements.
Tansy is also used as a companion plant, especially with cucurbits like cucumbers and squash, or with roses or various berries. It is thought to repel ants, cucumber beetles, Japanese beetles, squash bugs, and some kinds of flying insects, among others.
Dried tansy is used by some bee-keepers as fuel in a bee smoker.