rexresearch.com



Nam DANG

Papaya vs Cancer








http://news.ufl.edu/2010/03/09/papaya-2/
March 9th, 2010

UF researchers find cancer-fighting properties in papaya tea

GAINESVILLE, FL. — The humble papaya is gaining credibility in Western medicine for anticancer powers that folk cultures have recognized for generations.

University of Florida researcher Dr. Nam Dang, and colleagues in Japan have documented papaya’s dramatic anticancer effect against a broad range of lab-grown tumors, including cancers of the cervix, breast, liver, lung and pancreas. The researchers used an extract made from dried papaya leaves, and the anticancer effects were stronger when cells received larger doses of the tea.

In a paper published in the Feb. 17 issue of the Journal of Ethnopharmacology, Dang and his colleagues also documented for the first time that papaya leaf extract boosts the production of key signaling molecules called Th1-type cytokines. This regulation of the immune system, in addition to papaya’s direct antitumor effect on various cancers, suggests possible therapeutic strategies that use the immune system to fight cancers.

The papaya extract did not have any toxic effects on normal cells, avoiding a common and devastating consequence of many cancer therapy regimens. The success of the papaya extract in acting on cancer without toxicity is consistent with reports from indigenous populations in Australia and his native Vietnam, said Dang, a professor of medicine and medical director of the UF Shands Cancer Center Clinical Trials Office.

“Based on what I have seen and heard in a clinical setting, nobody who takes this extract experiences demonstrable toxicity; it seems like you could take it for a long time — as long as it is effective,” he said.

Researchers exposed 10 different types of cancer cell cultures to four strengths of papaya leaf extract and measured the effect after 24 hours. Papaya slowed the growth of tumors in all the cultures.

To identify the mechanism by which papaya checked the growth of the cultures, the team focused on a cell line for T lymphoma. Their results suggested that at least one of the mechanisms employed by the papaya extract is inducing cell death.

In a similar analysis, the team also looked at the effect of papaya extract on the production of antitumor molecules known as cytokines. Papaya was shown to promote the production of Th1-type cytokines, important in the regulation of the immune system. For that reason, the study findings raise the possibility of future use of papaya extract components in immune-related conditions such as inflammation, autoimmune disease and some cancers.

Bharat B. Aggarwal, a researcher at the University of Texas M.D. Anderson Cancer Center in Houston, already is so convinced of papaya’s restorative powers that he has a serving of the fruit every day.

“We have always known that papaya has a lot of interesting things in there,” said Aggarwal, a professor in the center’s department of experimental therapeutics who was not involved in the UF research. Foremost among papaya’s health-promoting agents is papain, papaya’s signature enzyme, which is found in both the fruit and the leaves.

“This paper has not gone too much into identifying the components responsible for the activity, which is just fine. I think that is a good beginning,” Aggarwal said.

Aggarwal also noted that papaya extract’s success in reducing cancer in laboratory cell cultures must next be replicated in animal and human studies.

“I hope Dr. Dang takes it further, because I think we need enthusiastic people like him to move it forward,” Aggarwal said.

Dang and a colleague have applied to patent the process to distill the papaya extract through the University of Tokyo; the next step in the research is to identify the specific compounds in the papaya extract active against the cancer cell lines. For this stage, Dang has partnered with Hendrik Luesch, a fellow UF Shands Cancer Center member and a professor of medicinal chemistry. Luesch is an expert in the identification and synthesis of natural products for medicinal purposes, and recently discovered a coral reef compound that inhibits cancer cell growth in cell lines.


Nam Dang



http://www.youtube.com/watch?v=P7HLpzcjD3U

UF researchers find cancer-fighting properties in papaya tea

http://www.youtube.com/watch?v=YGe1UvF4bBU

Papaya tea may help fight cancer



https://papayaleaves.wordpress.com/tag/dr-nam-dang/
June 6, 2013    

Harvard Graduate Has Patent Pending on Papaya Leaves for Cancer

Dr Nam Dang is a medical oncologist with expertise in the study and treatment of lymphoma and chronic lymphocytic leukemia (CLL). He is Professor and Deputy Chief of the Division of Hematology & Oncology and also is Director of the University of Florida Shands Cancer Center Clinical Trials Office. Dr. Dang received his Phd in Immunology from Harvard.

WE are excited about Dr Dang’s work on papaya leaves and greatly appreciate what he has shown in research on their effect against cancer(s). The Julia Ruffin Project focuses on promoting, growing, and researching papaya leaves. We offer papaya leaves grown from non-genetically modified seeds… just the natural product to markets. The Julia Ruffin Project is non-profit and is not creating additional products from the leaves (such as selling extracted leaf juice) so the patent-pending is not applicable to the young papaya leaves that we grow for market.



www.papayaleavesforcancer.com

About

I am in the business of papaya leaf tea research, promotion, and production because it is, in my opinion, the most powerful and affordable anti-cancer natural product on earth.

My journey to this conclusion began with frustration that this natural product could have possibly kept my mother with me longer in this life.

As I learned more and more about enzyme therapy over years I could not understand why this was never a subject in school, or why it is not discussed more with doctors.

I want to start conversations with this blog that will change people’s lives. But I am a realist and get that it is hard to change our eating habits. This is another reason that I am so attached to the tea. This herb covers and makes up for a good amount of our bad eating habits.

I want the people who take the time to read this blog to become as confident as I am in the science of this leaf and why our bodies need it so much to fight disease… Not just cancer, but arthritis, diabetes, bronchitis, malaria, and many, many more.

This blog is dedicated to Jill Nicole Gallo who continues to inspire me to pursue this dream to help people.



http://www.ncbi.nlm.nih.gov/pubmed/19961915
J Ethnopharmacol. 2010 Feb 17;127(3):760-7. doi: 10.1016/j.jep.2009.11.024. Epub 2009 Dec 2.

Aqueous extract of Carica papaya leaves exhibits anti-tumor activity and immunomodulatory effects.

Otsuki N1, Dang NH, Kumagai E, Kondo A, Iwata S, Morimoto C.

Abstract

AIM OF THE STUDY:

Various parts of Carica papaya Linn. (CP) have been traditionally used as ethnomedicine for a number of disorders, including cancer. There have been anecdotes of patients with advanced cancers achieving remission following consumption of tea extract made from CP leaves. However, the precise cellular mechanism of action of CP tea extracts remains unclear. The aim of the present study is to examine the effect of aqueous-extracted CP leaf fraction on the growth of various tumor cell lines and on the anti-tumor effect of human lymphocytes. In addition, we attempted to identify the functional molecular weight fraction in the CP leaf extract.

MATERIALS AND METHODS:

The effect of CP extract on the proliferative responses of tumor cell lines and human peripheral blood mononuclear cells (PBMC), and cytotoxic activities of PBMC were assessed by [(3)H]-thymidine incorporation. Flow cytometric analysis and measurement of caspase-3/7 activities were performed to confirm the induction of apoptosis on tumor cells. Cytokine productions by PBMC were measured by ELISA. Gene profiling of the effect of CP extract treatment was performed by microarray analysis and real-time RT-PCR.

RESULTS:


We observed significant growth inhibitory activity of the CP extract on tumor cell lines. In PBMC, the production of IL-2 and IL-4 was reduced following the addition of CP extract, whereas that of IL-12p40, IL-12p70, IFN-gamma and TNF-alpha was enhanced without growth inhibition. In addition, cytotoxicity of activated PBMC against K562 was enhanced by the addition of CP extract. Moreover, microarray analyses showed that the expression of 23 immunomodulatory genes, classified by gene ontology analysis, was enhanced by the addition of CP extract. In this regard, CCL2, CCL7, CCL8 and SERPINB2 were representative of these upregulated genes, and thus may serve as index markers of the immunomodulatory effects of CP extract. Finally, we identified the active components of CP extract, which inhibits tumor cell growth and stimulates anti-tumor effects, to be the fraction with M.W. less than 1000.
CONCLUSION:

Since Carica papaya leaf extract can mediate a Th1 type shift in human immune system, our results suggest that the CP leaf extract may potentially provide the means for the treatment and prevention of selected human diseases such as cancer, various allergic disorders, and may also serve as immunoadjuvant for vaccine therapy.



http://www.bmj.com/rapid-response/2011/11/03/repapaya-leaves-speedy-rise-platelet-count-dengue#alternate#dengue

Dengue: an escalating prolem

http://cgpsl.org/downloads/pdf_cgpsl/7.pdf

Salutary effects of carica papaya leaf extract in dengue fever patients -a pilot study



pravda.ru
23.03.2010

Papaya: Natural Cure Against Cancer

by
Timothy Bancroft-Hinchey

Fresh research has revealed that the papaya plant and its extracts can provide protection against cancer by slowing the growth of cancerous cells without causing harmful side effects. The discovery has been made by a University of Florida researcher in a joint project with the University of Tokyo.

The research was published in Journal of Ethnopharmacology*. University of Florida researcher, the Vietnamese Dr. Nam Dang, conducted a research project with colleagues from the University of Tokyo and their findings indicate that extracts from the papaya plant affect the regulation of the immune system. In this way, the attack on the cancerous cells does not cause harmful side effects because it acts through boosting the immune system and therefore does not have any effect upon normal cells.

Dr. Dang’s research studied the application of the papaya plant for cures in patients suffering from cancer among indigenous communities in Vietnam and Australia. He then developed four strengths of extract from the papaya leaf, which when applied clinically, slowed the growth of cancerous cells in ten different types of cancer, namely those affecting the cervix, the breast, the liver, the lungs and the pancreas. The effects were visible within 24 hours.

While the research project concentrates on the application of the papaya leaf on cancerous cases in Asia, indigenous populations in Brazil have been using the papaya leaf for thousands of years in infusions to treat ailments of the liver, including hepatitis A. The research conducted by Dr. Dang was made using dried papaya leaves.

He proved that papaya leaf extract increases the level of signalling molecules which help to regulate the immune system, attacking the cancerous cells. By boosting the Th1-type cytokines, the immune system responds to invaders while not producing toxic effects on healthy cells.

This type of treatment, according to Dr. Dang, produces results which go hand in hand with reports from indigenous communities in Australia and Vietnam.



US2008069907
Compositions for Cancer Prevention, Treatment, or Amelioration Comprising Papaya Extract  


Also published as:     WO2006004226 (A1)   JP2008505887 (A)   EP1778262

The components of a brew/extract of leaves or other parts of the papaya plant (Carica papaya) or fractionated components thereof are effective in the prevention or treatment of stomach cancer, lung cancer, pancreatic cancer, colon cancer or other solid cancers, or lymphoma, leukemia or other blood cancers, and are very safe with few side effects, making their medical significance extremely high. In addition, food compositions comprising a papaya plant (Carica papaya) extract component as an effective ingredient for the prevention or amelioration of cancer are highly expected to become functional foods contributing to the preservation and promotion of human health.

TECHNICAL FIELD

[0001] The present invention relates to compositions or food compositions for cancer prevention, treatment, or improvement. More specifically, it relates to compositions or food compositions that comprise, as an active ingredient, components extracted by brewing leaves or other parts of the papaya plant (Carica papaya), and that are effective in the prevention, treatment, or improvement of stomach cancer, lung cancer, pancreatic cancer, colon cancer, uterine cancer, ovarian cancer or other solid cancers, or lymphoma, leukemia or other blood cancers.

BACKGROUND ART

[0002] Conventional cancer treatment has been widely done using chemotherapy employing chemotherapeutic agents including cyclophosphamide, busulfan and other alkylating agents; methotrexate, 5-fluorouracil and other agents that inhibit nucleic acid synthesis; and actinomycin D, daunomycin and other antibiotics. However, many of these chemotherapeutic agents are toxic not only to cancer cells but also to normal cells, and their side effects are viewed as problematic. Compared to these chemotherapeutic agents, immunotherapy using immune activators that treat cancer by activating the macrophages and T cells of the immune system has attracted attention as a treatment method having fewer side effects. However, therapeutic agents that are effective in the treatment of cancer and yet have few side effects and a high level of safety have not yet been developed.

DISCLOSURE OF THE INVENTION

[0003] Accordingly, the objective of the present invention is to provide compositions or food compositions for cancer prevention, treatment, or improvement that are highly effective in the treatment and prevention of cancer, and yet have few side effects and a high level of safety.

[0004] As a result of exhaustive research regarding the aforementioned objective, the present inventors discovered that components of papaya, preferably one or more extracted papaya components obtained by brewing papaya, have superior effects in the treatment of cancer, and that accordingly, they can become compositions or food compositions for cancer prevention, treatment, or improvement with few side effects and a high level of safety, thus completing the present invention. Accordingly, the present invention relates to compositions or food compositions for cancer prevention, treatment, or improvement that comprise as active ingredients one or more components of the papaya plant (Carica papaya), and preferably one or more components extracted by brewing a part of a papaya plant.

[0005] A more detailed explanation of the present invention is provided below.

[0006] The present invention uses components of the papaya plant (Carica papaya), preferably components extracted by brewing a part of a papaya plant tissue, as active ingredients. The tissue of the papaya plant may be any of its leaves, roots, stems or fruit, but the leaves are particularly preferable. Preferably, this papaya tissue is dried, and the dried material thus obtained is added to cold water or boiling water, brewed for a long time, and the brew thus obtained is used as an active ingredient. Alternatively, without drying the papaya leaves (or some other tissue), the leaves may be added to cold water or boiling water and brewed. More specifically, papaya leaves, for example, may be left in the sun, normally for one or two days, and dried to obtain the dried material. One to several of these dried leaves are added to cold water or boiling water (normally 400 ml to 3000 ml), and preferably 500 ml to 1000 ml, and brewed for normally two hours to 15 hours, and preferably three hours to 12 hours. The vessel used for brewing is preferably not a metal vessel, but rather a glass, wooden, plastic or other vessel.

[0007] A component thus obtained, particularly a component extracted by brewing papaya, comprises the effect of suppressing the proliferation of cancer cells, and can be used as it is as an active ingredient of the composition or food composition according to the present invention. Furthermore, the composition can also be provided as a botanical drug. According to the present invention, this brew may be further subjected to a refining process. The components that comprise the effect of suppressing the proliferation of cancer cells can be fractionated, and the fractionated components thus obtained may be used as an active ingredient. Such fractionated components are, for example, preferably obtained from an extract of papaya leaves that, when subjected to gel filtration chromatography using a gel filtration column filled with cross-linked polyvinyl alcohol gel, the gel filtration column having an exclusion limit molecular weight of 40,000 when pullulan is used as a sample, is eluted in a portion of the eluate equivalent to 50-70 vol. % of the volume of the column. As an example of such gel filtration chromatography, the column used for gel filtration chromatography may be a column that is a stainless-steel tube with an inside diameter of 7.6 mm*length of 500 mm, filled with cross-linked polyvinyl alcohol gel with a grain diameter of 9 [mu]m, and having an exclusion limit molecular weight of 40,000 when pullulan is used as a sample (Shodex Asahipak GS-310 7G) (Showa Denko K. K.). A neutral buffer solution, for example a phosphate buffer solution, may be used as the eluate. A specific example is obtained by dissolving 9.6 g of powdered Dulbecco's PBS (-) "Nissui" (Nihon Pharmaceutical Co., Ltd.) in 1 L of deionized water and autoclaving for 15 minutes at 121[deg.] C. to obtain a sterilized buffer solution (pH 7.3 to 7.65).

[0008] Under these conditions, when the extract is subjected to gel filtration chromatography, it is possible to obtain those components comprising the effect of suppressing the proliferation of cancer cells by collecting fractionated components obtained by eluting a portion of the eluate equivalent to 50-70 vol. % of the volume of the column. As recited in the subsequent Examples, these fractionated components were put through a gel filtration column similar to that described above, the chromatogram was measured with a differential refractometer (RI) and ultraviolet spectrophotometer (SPD-10AVp), and the approximate calculation of the molecular weight and evaluation of the physical properties of the included components was performed based on a comparison of the molecular weights relative to pullulan and oligosaccharide markers with known molecular weights. Components with molecular weights of 1700, 1000, 700, 600, 400, and 300 were detected by an RI detector. Peaks were detected for molecules (groups) at molecular weights of 1700 and 1000 under UV (260 nm), and were also found at low molecular weights (300 to 700), also under UV (260 nm). Accordingly, the fractionated components comprising anti-tumor effects further refined from the extract of papaya leaves are contained within molecules (groups) with molecular weights of 1700, 1000 and 700 to 300 that absorb UV (260 nm), and the molecule (groups) with molecular weights of 1700, 1000, 700, 600, 400, 300 and 200 that are detectable by RI, each of which are thought to act independently or in combination to achieve the anti-tumor effect.

[0009] The brews/extracts thus obtained or further fractionated components may be ingested as they arein order to prevent, treat, or ameliorate cancer. The brews/extracts or fractionated components may also be mixed with appropriate ingredients ordinarily used in drinks and taken as a drink. Alternatively, the brews/extracts or fractionated components may be dried if necessary, and then taken in a powder or tablet or other form after the addition of excipients or other additives ordinarily used in powders, tablets or the like.

[0010] In addition, when being stored, the papaya components of brews/extracts, or fractionated components thereof, are preferably stored not in a metal vessel, but rather in a glass, wooden, plastic or other vessel under low-temperature conditions, for example around 4[deg.] C.

[0011] The present invention's brew/extract components, prepared from, for example, a papaya leaf, or fractionated components thereof, are effective in the prevention or treatment of stomach cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer, uterine cancer, ovarian cancer, neuroblastoma or other solid cancers, or lymphoma, leukemia or other blood cancers. In a preferred embodiment, treatment is administered to those suffering from a disease. In another embodiment, treatment is administered as prophylaxis. The dose of the brew/extract components or fractionated components thereof to be administered will depend on the dosage form, symptoms of the subject, type of cancer or the like. However, for example, when the brew/extract is taken, it is normally preferable to take an amount of 100 ml to 750 ml per day, every day for between one month and three months. When the fractionated components are taken, it is preferable to take an amount of 10 ml to 200 ml per day, every day for between one month and three months.

[0012] Also, the present invention provides food compositions that comprise a papaya (Carica papaya) brew/extract as an effective ingredient for preventing or treating cancer. In addition to general foods, a food composition of the present invention may include, for example, a health food, a functional food, a specified health food, a nutrient supplement, an enteral nutrient, and the like, but is not limited to these foods so long as it is effective in preventing or ameliorating cancer. Methods for manufacturing the food compositions are usual techniques known to those skilled in the art. That is, a papaya (Carica papaya) brew/extract component according to the present invention can be combined with an additive acceptable in view of food sanitation, and processed to make a general food, a health food, a functional food, a specified health food, a nutrient supplement, an enteral nutrient, etc. For example, an additive such as a stabilizer, preservative, colorant, perfume, vitamin can be appropriately added to a papaya (Carica papaya) brew/extract component, mixed, and processed by standard methods into a form suitable for a food composition, such as a tablet, pill, granule, powder, capsule, liquid, cream, drink, etc. Furthermore, the food compositions of the present invention include those sold with a description or indication written on the food composition's packaging container and/or in a promotional pamphlet, to the effect that the food composition, and/or an ingredient in the food composition, comprises the effect of preventing, or ameliorating cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts graphs showing the anti-tumor effect of a papaya leaf extract according to the present invention on AGS (a stomach cancer cell line: 1000 cells/well and 2000 cells/well, cultured for three days).

[0014] FIG. 2 depicts graphs showing the anti-tumor effect of the papaya leaf extract according to the present invention on Capan-1 (a pancreatic cancer cell line: 1000 cells/well and 2000 cells/well, cultured for five days; 40000 cells/well, cultured for four days).

[0015] FIG. 3 is a graph showing the anti-tumor effect of the papaya leaf extract according to the present invention on DLD-1 (a colon cancer cell line: 20000 cells/well, cultured for four days).

[0016] FIG. 4 is a graph showing the anti-tumor effect of the papaya leaf extract according to the present invention on DOV-13 (ovarian cancer cell line: 3000 cells/well, cultured for two days).

[0017] FIG. 5 is a graph showing the anti-tumor effect of a 50-fold-diluted papaya leaf extract according to the present invention on Karpas (a lymphoma cell line).

[0018] FIG. 6 depicts graphs showing the anti-tumor effect of the papaya leaf extract according to the present invention on MCF-7 (breast cancer cell line: 2500 cells/well and 7500 cells/well, cultured for six days).

[0019] FIG. 7 depicts graphs showing the anti-tumor effect of the papaya leaf extract according to the present invention on T98G (a neuroblastoma cell line: 2000 cells/well and 4000 cells/well, cultured for three days).

[0020] FIG. 8 is a graph showing the proliferation suppression effect of the papaya leaf extract according to the present invention on Hela (a uterine cancer cell line).

[0021] FIG. 9 depicts graphs showing the proliferation suppression effect of the papaya leaf extract according to the present invention on Karpas (a lymphoma cell line).

[0022] FIG. 10 depicts graphs showing the proliferation suppression effect of the papaya leaf extract according to the present invention on CD26 negative Jurkat (T cell leukemia cell line).

[0023] FIG. 11 depicts graphs showing the proliferation suppression effect of the papaya leaf extract according to the present invention on CD26 positive Jurkat (T cell leukemia cell line).

[0024] FIG. 12 shows the results of measurement of the suppression effect of Jurkat T cell proliferation by components of papaya leaf extract fractionated by gel filtration chromatography.




BEST MODE FOR CARRYING OUT THE INVENTION

[0025] Herein below, the present invention will be specifically described using Examples, however, it is not to be construed as being limited thereto.

WORKING EXAMPLE 1

Preparation of an Extract From Papaya Leaves

[0026] Papaya leaves were left in the air for approximately one to two days under the sun and thus dried, and approximately 27 g, that is, about one dried papaya leaf, was added to 500 ml of water in a glass vessel, brewed for twelve hours, until the total quantity was 50 ml. The brew thus obtained was filter-sterilized to obtain the papaya extract according to the present invention.

EXPERIMENTAL EXAMPLE 1

Test of the Anti-Cancer Effects of Papaya Leaf Extract by the MMT Assay Method

[0027] Various types of cancer cells were used to examine the anti-cancer effects of the papaya leaf extract, prepared according to Working Example 1, by means of the MMT assay method (Hansen et al., J. Immunol. Methods 119:203-210, 1989). (1) Method

[0028] Cancer cells from the AGS (stomach cancer cell line), Capan-1 (pancreatic cancer cell line), DLD-1 (colon cancer cell line), Dov-13 (ovarian cancer cell line), Karpas (lymphoma cell line), MCF-7 (breast cancer cell line) and T98G (neuroblastoma cell line) were dispensed into a 96-well microtiter plate and cultured in a RPMI-1640 solution medium containing 10% fetal calf serum, and cultured until confluent. Next, the papaya leaf extract prepared according to Working Example 1 was diluted with culture medium by 20 times (1:20), 150 times (1:150) and 400 times (1:400) respectively, and a volume of 100 [mu]l of the dilute solution was added to the cancer cell lines in each well, which were cultured at 37[deg.] C. for two to eight days. Thereafter, 25 [mu]l of 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was added to each well to make the final concentration 1 mg/ml, and they were cultured at 37[deg.] C. for an additional two hours, and then 100 [mu]l of isopropanol containing 0.04 N hydrochloric acid was added. After culturing overnight at 37[deg.] C., the absorbance at 570 nm was measured and compared against that of the control to examine just how many of the cells were killed by the papaya leaf extract. (2) Results

[0029] The results thus obtained are shown in FIG. 1 through FIG. 7. As is evident from these figures, the papaya leaf extract had a concentration-dependent anti-cancer effect on each of the cancer cell lines. In particular, as shown in FIG. 5, the maximum value of cell death of 40% appeared on the fourth day of culture in Karpas (lymphoma cell line) at a 50 times dilution of the concentration of the extract.

EXPERIMENTAL EXAMPLE 2

Test of the Anti-Cancer Effects of Papaya Leaf Extract by <3> H-Thymidine Incorporation (1) Method

[0030] Cancer cells from the Hela (uterine cancer cell line), Karpas (lymphoma cell line), CD26 negative Jurkat (T cell leukemia cell line) and CD26 positive Jurkat (CD26 transfectant T cell leukemia cell line) were dispensed at 5*10<3> /100 [mu]l into a 96-well microtiter plate. Next, the papaya leaf extract prepared according to Working Example 1 was diluted with culture medium (RPMI-1640 solution containing 10% fetal calf serum) to various dilution ratios, and 10 [mu]l of each was added to the cancer cell lines in each well, which were cultured at 37[deg.] C. in a 5% CO2 incubator. After 24 and 48 hours of culturing, <3> H-thymidine was added in the amount of 1 [mu]Ci (1 microcurie)/10 [mu]l. After four hours, the amount of <3> H-thymidine (cpm) incorporated into the cancer cells for the purpose of DNA synthesis was measured with a scintillation counter to examine the effect of suppressing the proliferation of cancer cells. (2) Results

[0031] The results thus obtained are shown in FIG. 8 through FIG. 11. As is evident from these figures, the papaya leaf extract suppressed the incorporation of <3> H-thymidine by various cancer cell lines, and thus suppressed DNA synthesis. This clearly exhibited a suppressive effect on proliferation.

EXPERIMENTAL EXAMPLE 3

Application to Patients

[0032] A 47-year-old female patient had stomach cancer that had metastasized to the pancreas. Her tumor marker value CEA was 49 and alpha fetoprotein was 369. One dried papaya leaf was placed in 3000 ml of water and boiled in a wooden vessel for approximately three hours until concentrated to 750 ml. The patient drank approximately 750 ml of the papaya leaf extract every day for 90 days, then took a break from drinking for 90 days, and then continued to drink for 90 days. As a result, the pancreatic metastasis of the stomach cancer disappeared, the CEA level dropped to 2.3, and the alpha fetoprotein also dropped to 2.0, with no relapse found thereafter.

[0033] In addition, long-term survival from drinking papaya leaf extract was seen in five lung cancer patients, three stomach cancer patients, three breast cancer patients, one pancreatic cancer patient, one liver cancer patient, and one blood cancer patient.

WORKING EXAMPLE 2

Fractionation of Papaya Leaf Extract, Measurement of the Anti-Tumor Effects of Fractionated Components, and Analysis of Fractionated Components (1) Fractionation by Gel Filtration Chromatography

[0034] Dried papaya leaf (approximately 27 g) was heated for one hour to 100[deg.] C. in 500 ml of distilled water using a glass beaker, and then further heated for ten hours at 55[deg.] C. to prepare an extract concentrated to a volume of 50 ml. Thereafter, fractionation by molecular weight was performed by gel filtration chromatography, and further analysis of the anti-tumor active substances was performed.

[0035] Gel filtration chromatography was performed at room temperature using 500 [mu]l of extract. Fluid transport was performed using an LC-10AVp (Shimadzu Corp.), where the moving-phase flow rate was 1 ml/min, and the fractionation method involved manually collecting the eluate from the detector outlet at one-minute intervals, thereby obtaining a total of 60 fractions. Note that the column used for gel filtration chromatography was a column that is a stainless-steel tube with an inside diameter of 7.6 mm*length of 500 mm, filled with cross-linked polyvinyl alcohol gel (grain diameter of 9 [mu]m), and having a molecular weight exclusion limit of 40,000 when pullulan is used as a sample (Shodex Asahipak GS-310 7G) (Showa Denko K. K.). Moreover, the eluate used was obtained by dissolving 9.6 g of powdered Dulbecco's PBS (-) "Nissui" (Nikon Pharmaceutical Co., Ltd.) in 1 L of deionized water, and autoclaving for 15 minutes at 121[deg.] C. to obtain a sterilized buffer solution (pH 7.3 to 7.65). (2) Measurement of the Anti-Tumor Effects of Fractionated Components

[0036] In order to study the anti-tumor effects of the fractionated components, Jurkat T cells were suspended in RPMI-1640 medium (Sigma) (10% fetal bovine serum), and disseminated over a 96-well cell culture microplate (flat bottom) (Becton, Dickinson & Co.) at a concentration of 5*10<3 > cells/well (5*10<4 > cells/ml). Moreover, the fractionated components obtained by gel filtration chromatography were added to each well in the amount of 10 [mu]l per 100 [mu]l of medium, and culturing was performed. After 48 hours from the commencement of culturing, both the Jurkat T cells in the group with fractionated components added, and the group with no additions, were labeled with <3> H-thymidine (<3> H-TdR) (PerkinElmer Life Sciences) to 1 [mu]Ci/well and cultured for four hours. Thereafter, the amount of <3> H-TdR incorporated into the cells was measured, and the amount of DNA synthesized by each of the Jurkat T cells was evaluated, thereby examining the anti-tumor effect of each fractionated component. FIG. 12 illustrates the effects of the addition of fractionated components of papaya leaf infusion on the proliferation of Jurkat T cells. (3) Results

[0037] From the results illustrated in FIG. 12, the Jurkat T cells cultured in medium, and Jurkat T cells to which a quantity of PBS equal to the fractionated components was added, exhibited <3> H-TdR incorporation of 15192.33+-413.4 (cpm) and 16837.33+-288 (cpm) respectively. On the other hand, Jurkat T cells added with the fractionated components eluted between 12 minutes and 13 minutes of gel filtration chromatography elution time (fraction 13: the portion of eluate equivalent to 53 to 57 vol. % of the column volume), and the fractionated components eluted between 13 minutes and 14 minutes of gel filtration chromatography elution time (fraction 14: the portion of eluate equivalent to 57 to 62 vol. % of the column volume), exhibited <3> H-TdR incorporation of 2757+-153.7 (cpm) and 3080+-84.7 (cpm) respectively. This is comparable to the <3> H-TdR incorporation of 3316.7+-516.0 (cpm) by Jurkat T cells to which unfractionated (pre-gel filtration chromatography) extract, 100-fold diluted with culture medium, was added.

[0038] Accordingly, the present inventors found that the fractionated components of the papaya leaf extract that comprise the effect of suppressing the proliferation of Jurkat T cells (anti-tumor effect) are present in fractions 13 and 14. (4) Analysis of Fractionated Components

[0039] Furthermore, the fractionated components obtained by gel filtration chromatography that comprise anti-tumor effects (fraction 13 and fraction 14) were each placed in a similar column, and the chromatogram was measured with a differential refractometer (RI) (RID-6A) (Shimadzu Corp.) and ultraviolet spectrophotometer (SPD-10AVp) (Shimadzu Corp.). Approximate calculation of the molecular weight and evaluation of the physical properties of the included components was performed based on a comparison of the molecular weights relative to pullulan and oligosaccharide markers with known molecular weights.

[0040] As a result, in fraction 13, components with molecular weights of 1700, 1000, 700 and 300 were detected by an RI detector, and peaks were also detected for molecules (groups) at molecular weights of 1700 and 1000 under UV (260 nm). Thus this fraction is presumed to contain compounds with absorption in these regions. On the other hand, in fraction 14, components with molecular weights of 1700, 1000, 600, 400 and 200 were detected by the RI detector, and peaks were found for molecules (groups) at molecular weights of 1700 and 1000 and also at low molecular weights (300 to 700) under UV (260 nm).

[0041] Accordingly, the fractionated components comprising anti-tumor effects extracted from papaya leaves were extracted between 12 minutes and 14 minutes after the start of the gel filtration chromatography used in this experimental system. The components comprising anti-tumor effects are contained within the molecules (groups) with molecular weights of 1700, 1000 and 700 to 300 that absorb UV (260 nm), and the molecules (groups) with molecular weights of 1700, 1000, 700, 600, 400, 300 and 200 that are detectable by RI, each of which are considered to act independently or in combination to achieve anti-tumor effect.

INDUSTRIAL APPLICABILITY

[0042] As described above in detail, the components of an brew/extract of leaves or other parts of the papaya plant (Carica papaya) or fractionated components thereof, for example, are effective in the prevention or treatment of stomach cancer, lung cancer, pancreatic cancer, colon cancer or other solid cancers, or lymphoma, leukemia or other blood cancers, and are very safe with few side effects, making their medical significance extremely high. In addition, food compositions comprising a papaya plant (Carica papaya) extract component as an effective ingredient for the prevention or amelioration of cancer are highly expected to become functional foods contributing to the preservation and promotion of human health.



JP2008214299
COMPOSITION FOR PREVENTION, TREATMENT, OR AMELIORATION OF CANCER CONTAINING WATER-SOLUBLE EXTRACT CONSTITUENTS OF CARICA PAPAYA AND HEDYOTIS DIFFUSA  

[ Machine Translation ]

PROBLEM TO BE SOLVED: To provide a composition useful for the prevention, treatment, or amelioration of cancers which produces little side effects and is highly safe. ; SOLUTION: The composition comprises a combination of a water-soluble extract constituent from parts (e.g. leaves) of Carica papaya with a water-soluble extract constituent from parts (e.g. leaves) of Hedyotis Diffusa. This combination can effectively achieve prevention, treatment, or amelioration of solid cancers such as stomach cancer, lung cancer, pancreatic cancer, and colon cancer, produces little side effects, is highly safe, and is therefore of great significance

DESCRIPTION

[0001] Prevention of cancer, including water-soluble components extracted Shirohanahebi tongue grass and papaya, the present invention relates to compositions for the treatment or amelioration.
In particular, it contains a component extracted by decoction in hot water leaves Shirohanahebi tongue grass and papaya, while maintaining the immunostimulatory effects of the components of the two, to exert a synergistic effect in anti-tumor activity of cancer A composition for the amelioration, or treatment, to compositions and pharmaceutical can be used for food.

[0002] Of course the case of malignant tumors, until you get the assurance of remission after surgery in particular, chemotherapy is essential when surgery itself is difficult to carry out, or when resection of the tumor is impossible only surgery.

However, at present it to enhance the tumor specificity in chemotherapy is difficult, side effects on normal cells of the self is to always associated.

You can easily imagine that be faced with side effects in physical fitness in deep discharge is a major damage to the body and mind, but even would be an agent with the privation suffering how, people and the surrounding sufferers to face in every difficulty and pain, of connecting the desire to get better it is natural.

On the other hand, information on alternative therapies variety are flooding the society.

What you poor credibility, which imposes economic burden, are situations in which, such as those intake ongoing difficult there are also many, I can not help but want to believe even information unrealistic parable in it there also is a fact.

However, it is aims at the treatment of diseases of the mechanisms which can not be solved with the so-called modern medicine and alternative therapies original on heuristic scientific considerations is not always present, to recognize the significance difficult, it can not become the hope for recovery.

[0003] The extracts from plants transmitted from ancient times, it be effective is present in certain diseases is empirically known.

(Hedyotis Diffusa, Japanese name: Futabamugura) Shirohanahebishitakusa with a long history as a herbal medicine, is a plant that grows wild in our country also in an annual plant of the Rubiaceae, information as a folk medicine drugs is relatively large.

(4 and Non-Patent Documents 1, 2, 3) which by oral administration to mice, anti-tumor effect, anti-viral effect, IgE production inhibition, and anti-parasitic effect has been reported.

In addition, as a herbal medicine that exerts an anti-tumor effect, Hanedahasu (Ren contrary, mint family, Scutellaria Barbatae) are known, the anti-tumor effect in the case of a combination of the Hanedahasu and Shirohanahebi Shitakusa also consider (6 and Non-Patent Document 5) are.

On the other hand, papaya (Carica Papaya), is distributed in tropical throughout now beginning Vitamin C with antioxidant, tannins, vitamin A, E, K, saponins, and iron contained in the leaves, digestion aid , anti-inflammatory effects, and antiparasitic effects have been reported (Non-Patent Document 7).

Further, the extract components leaf papaya, to have anti-tumor effects have been reported (Patent Document 1).

Yoshida Y, Wang MQ, Liu JN, Shan BE, Yamashita U. Immunomodulating activity of chinese medicinal herbs and oldenlandia diffusa in particular.
Int J Immunopharmacol 1997; 19 (7): 359-70Shan BE, Yoshida Y, Sugiura T, Yamashita U. Stimulating activity of chinese medicinal herbs on human lymphocytes in vitro.
. Int J Immunopharmacol 1999; 21 (3): 149-59Lin CC, Ng LT, Yang JJ, Hsu YF Anti-inflammatory and hepatoprotective activity of peh-hue-juwa-chi-cao in male rats.
Am J Chin Med 2002; 30 (2-3):. 225-34Lin CC, Ng LT, Yang JJ Antioxidant activity of extracts of peh-hue-juwa-chi-cao in a cell free system.
Am J Chin Med 2004; 32 (3): 339-49Wong BY, Lau BH, Wan CP, Oldenlandia diffusa and Scutellaria barbata augment macrophage oxidative burst and inhibit tumor growth, Cancer Biother Radiopharm 1996; 11 (1): 51-56Yoshida Y , Wang MQ, Liu JN, Sha Yamashita U., Imunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular, Int J Immunopharmacol 1997: 19 (7): 359-370Hsu S. Green tea and the skin.
No. WO2006/004226A1 pamphlet 1049-59 International Publication: 52 (6); 2005 J Am Acad Dermatol

[0004] The extract of the plant-derived handed down from ancient times, that the effective ones are present in certain diseases are known to experience, but the scientific evidence is poor, I unknown also the mechanism of the effect part in many cases.

Plant-derived extracts a long period of time, humans have been ingested, should you go beyond only wisdom empirical simply, it holds the promise significant potential towards drug development in the future.
Further, as long as the resulting extract Shirohanahebishitakusa, Hanedahasu, Great papaya exhibits an effective medicinal, because these plants are edible originally therapeutics highly safe less side effects This is what can be expected as.

Accordingly, an object of the present invention, as an active ingredient an extract from plants, prevention of cancer, is to provide a composition having high safety has fewer side effects or improvement effect on treatment and high.

[0005] When Shirohanahebi tongue grass with a long history as a herbal medicine and focused on water-soluble components extracted papaya tropical American origin, for these extracts were subjected to immunological analysis, the present inventors have found that two possibility that the cytotoxic activity, immunostimulating effect is a significant increase in water-soluble components of the papaya extract in a water-soluble component extraction White Flower snake tongue grass mainly when compared with between Sha has been shown.

Further, as a result of examination did on the details of this possibility, with the expectation that there is an effect synergistic or complementary by mixing both, it was analyzed, the mixing of the two, maintains immunostimulatory effect while, that the cytotoxic synergistic activity against tumor cell lines are derived was confirmed.

The present invention has been completed based on these findings.

Therefore, as an active ingredient a water-soluble component extraction Shirohanahebishitakusa and water-soluble extract components of papaya (Carica papaya) of (Hedyotis Diffusa), prevention of cancer, the present invention is a composition for the treatment or amelioration some.

[0006] Immunostimulatory activity is maintained, against the tumor cell line, composition comprising, as an active ingredient, both water-soluble components extracted Shirohanahebi tongue grass and water-soluble extract components of papaya exert cytotoxic activity synergistic.

Thus, the compositions of the present invention are useful pharmaceuticals highly safe with fewer side effects of cancer prevention, improvement or treatment is high and, as a health food and the like.

Further, the water-soluble extract obtained by boiling, in which easily is generally accepted as the "infusion" so to speak, relatively, it can be taken easily, regardless of the extent of the condition.

The usefulness high regard to the nature that must be taken continuously, it is excellent in convenience and economical efficiency, and safety.

[0007] The present invention will be described in detail below.

By way of active ingredient in the present invention, the use of both water-soluble components extracted Shirohanahebi tongue grass and water soluble extractives papaya.

Is used to obtain the extract, as the structure of the tongue Shirohanahebi grass and papaya, one leaf, root, stem, fruit may be any, but the leaves are particularly preferred.

In addition to hot water or a dried product dried tissue Shirohanahebi tongue grass papaya and these can be obtained, is used as an active ingredient, a liquid infusion which is boiled for a long time, are obtained.

It is also possible to obtain a liquid infusion without drying the tissue, such as leaves and grass Shirohanahebi tongue papaya, and boiled in addition to water or hot water as it is.

Specifically, for example, to obtain a dried product can be left to dry for 2 days from one day normal daylight, leaves Shirohanahebi tongue grass papaya and more.

1000ml usually 50ml, in addition to hot water or 500ml of 100ml, 5 hours 30 minutes usually, and preferably, to cook with boiling for 1 to 2 hours, preferably, an amount of 100g about 5g of the dried leaves  is preferred.

The container used when cooking, a glass container is not a container made of metal, wood, and plastic is preferred.

After boiling is preferably maintained at a temperature of about 70 ? from 60 ?, it is to concentrate to 250ml about 50ml.

Then, preferably by filtration, after removal of the leaves, further kept at a temperature of 70 ? about 60 ?, it's concentrated 125ml about 25ml.

Then, the water-soluble extract which was centrifuged at a high speed centrifuge, and the supernatants collected and passed through sequentially sterile filtration filters of different pore size, adjusted to 63ml about 13ml final concentration, and the desired It can be obtained preferably component.

[0008] It is possible to obtain a composition of the present invention by mixing thus obtained, both of the water-soluble components extracted Shirohanahebi tongue grass and water soluble extractives papaya.
4:1, preferably from 1:4 volume ratio, and further, 4:1, preferably from 2:1 mixing ratio of the water-soluble component extraction White Flower snake tongue grass and water-soluble extract components of papaya, 3 especially : 4:1, preferably from 1.

It should be noted that the volume ratio, based on the water-soluble extract components extracted under the same conditions using an equivalent amount of water from the leaves of equivalent each, a water-soluble extractives of Shirohanahebi tongue grass and water-soluble extract components of papaya is a volume obtained when.

[0009] It is possible by taking as a mixture of both water-soluble components extracted Shirohanahebi tongue grass and water soluble extractives papaya, prevention of cancer, use as a medicament for treating or improving.

Can also be taken as a health drink by mixing with a suitable component commonly used for beverage agent for the mixture.

Alternatively, it is possible, after drying if necessary, the mixture is taken in the form of powders, such as tablets by a conventional method, in addition to excipients such as commonly used powders, tablets, or the like.

[0010] A mixture of both water-soluble components extracted Shirohanahebi tongue grass and water-soluble extract components of papaya can also be used for health food, functional food, food for specified health use, dietary supplements, and enteral nutrition.

To a mixture of both water-soluble components extracted Shirohanahebi tongue grass and water soluble extractives papaya, formulations are typically used, for example, by the addition of stabilizers, preservatives, colorants, flavors, vitamins and the like, It is possible by conventional methods, can be processed to form a drink, powders, liquids, tablets, powders, and the like, to use health foods, functional foods, specified health food, nutritional supplements, as and enteral nutrition .

[0011] In particular, prevention of solid cancer, the composition of the present invention, including both water-soluble components extracted Shirohanahebi tongue grass and water-soluble extract components of the papaya is effective to treat or ameliorate, as solid cancer is , for example, large cell lung cancer, lung adenocarcinoma, lung cancer, breast cancer, cervical cancer, lung cancer mesothelioma, and pancreatic cancer.

Prevention of these cancers, the dose for the treatment or amelioration is, three months from one month the amount of 750ml from one day usually 100ml, a mixture of water-soluble extractives White Flower snake tongue grass and water-soluble extract components of papaya It is preferable to take a daily over.

In addition, even if you are edible health food, functional food, food for specified health use, dietary supplements, as such as enteral nutrition, as well, the water-soluble extractives White Flower snake tongue grass and water-soluble extract components of papaya It is preferred that the daily food over 3 months 1 month the amount of 750ml of normal daily 100ml, and the mixture.

[0012] Hereinafter, the present invention is described in more detail by Examples and Test Examples, but the present invention is not intended to be limited to the examples and test examples.

Preparation of water-soluble components extracted from the leaves of grass Shirohanahebi tongue and one papaya Example

It was carried out by the following method of preparing a water-soluble extract from the leaves of grass Shirohanahebi tongue and papaya.

Step 1: Put the water of 400ml each tea leaves 20g obtained by drying the leaves of each of Shirohanahebi tongue grass and papaya, it was boiled for one hour.

Step 2: I keep 60-70 ?, was concentrated over a period of about 3 hours to about 100ml before and after.

Was concentrated to about 50ml back and forth ,60-70 ? after it was filtered through a sterile gauze, to remove a large tea leaf: Step 3.

Step 4: To a high-speed centrifuge, followed by 20 minutes centrifugation 9000rpm, and the supernatant was recovered.
0 pore diameter: Step 5.
8,0.
The sequentially inserted through sterile filtration filter 45,0.22 · m, were prepared in the final volume of 20ml.
Step 6: 1ml dispensed into sterile 1.5ml tubes each extract, I was stored in 4 ?.
To prevent endotoxin contamination and throughout the entire process, using a disposable sterile instrument all.
In addition, as a standard concentration 20ml concentrate from tea leaves of 20g, were used in the following discussion.

[0013] Test Example 1

Measurement of cytokine production and isolation of human peripheral blood-derived mononuclear cells
1. How

Was separated by gravity separation method using LymphoprepTM a healthy person from fresh peripheral blood.
In 96well flat-bottom plate immobilized (OKT3, 1µg/ml) the anti-human CD3 antibody, I seeded the PBMC (2x105/well).
I was added to a final concentration of 5µg/ml CD28 antibody solubilized anti-human (4B10) further.
I was using the RPMI1640 (Sigma), including the FCS (Biowest) 10% culture solution.
The tea leaf extract, diluted with range of 800 times 200 times the final concentration, was added to the medium.
The supernatant was collected after 24 hours incubation, was measured cytokine production (OptEIATM, BD) by ELISA.
Further, the cell viability after culturing, after double stained and de 7-AAD FITC-Annexin V (BD) and with (Promega) cells were harvested and analyzed by flow cytometry.
[0014]

2. Result
Immunomodulatory effects of the extract on peripheral blood-derived lymphocytes
Presence or absence of induction of cell death for (1) healthy subject PBMC
To rule out a direct cytotoxicity of tea-derived components, and examined for viability of the cells after culture.
I shown in Figure 1 results.
In Figure 1, the H. D.
C., a water-soluble components extracted from the leaves of Shirohanahebi tongue grass P.
I refer to the water-soluble components extracted from the leaves of papaya.
Thereafter, the specification and the drawings may also be used the abbreviations.
Annexin V-positive cells at the point of 800 times added 65 additive-free 200-fold from the extract.
Think not that go down 65 percent for those 4%, was added, shall not be able to directly affect the survival rate as long as 24 hours at this concentration, it was determined this condition in subsequent analyzes.

[0015] (2) Analysis of cytokine production

Production is decreased in a concentration-dependent manner from the point of 800-fold, was inhibited 70% or more at 200-fold IL-2, IL-4 (Figure 2).
From the fact that decreased production of cell these growth factors was observed, the phenomenon of cell death has not got up yet, but against mediated activation (TCR) complex CD3 / T cell receptor in PBMC, and the very early stage may have been derived from the suppression stage was considered.
Enhancement of production is the most recognized at the point of 800 times with extracts of either, was reduced in a concentration-dependent manner and then IL-12p40 (Figure 3).
IL-12p70 is also similar, C. P.
H. than D.
More of addition, high inducibility slightly.
A similar trend was observed IFN-?, TNF-a, also IL-10 (FIG. 4).
Thus, is some material from the extract, the possibility that despite not contribute to the hyperproliferative, there is immunostimulatory effect of enhancing cell function was considered.
H. These results D.
And C. P.
It was observed alone both extracts in the state of mixing.
[0016]

Test Example 2

Measurement of proliferative capacity and culture of human tumor cell lines

As solid tumor line, Lu99 (large cell lung cancer), A549, PC14 (lung adenocarcinoma) HepG2, Huh-7 (liver cancer), MCF-7 (breast cancer), Hela (cervical cancer), H2452, JMN, MESO 4 (lung mesothelioma), I was using the (pancreatic cancer) CaPan1, Panc1.
Were cultured in (Sigma) DMEM or RPMI1640, including the FCS 10% cell lines, respectively.
Were plated in 96well flat bottom plates at 1X104/Well each tumor cell line was attached to the bottom surface sufficiently For adherent cells.
Then, add the extracts were diluted 400-fold 50-fold, were cultured for 24 hours.
18 hours until the end of the culture, measurement of proliferative capacity is [3H] - and then the radioactivity was measured by conduct labeling with thymidine.
[0017]

Result

It was considered that thymidine incorporation is low, significant results were not obtained, this analysis method is not suitable - [3H] For A549, Lu99.
On the other hand, both cell growth inhibition at high concentrations (50-fold) (cervical cancer, breast cancer, lung adenocarcinoma, lung mesothelioma, liver cancer, pancreatic cancer) with respect to the other cell lines with high uptake Shown, of course, H. up to the concentration of 400 times D.
Or C. P.
Growth inhibitory effect was observed in both (Figure 5).
Also in either cell line, and H. D.
C. than P.
There tends to be high inhibitory effect towards.
Showed growth suppression is added alone like a mixed addition.
When the point of the 400-fold high dilution most, we compared the additive mixture in the ratio of several single and added, by mixing, cervical cancer (FIG. 6A), breast (Fig. 6B), liver cancer (Figure 6C anti-tumor effect was observed synergistically lung adenocarcinoma (Figure 6D), lung mesothelioma (Fig. 6E), pancreatic cancer (Fig. 6F),).
A similar trend was observed for the other cell lines that Panc-1 with respect to pancreatic cancer, but low uptake itself of [3H]-Thymidine when compared with other cell lines to mention the presence or absence of a synergistic effect was determined (Figure 5), it is not appropriate.
Synergistic effect due to the mixing, H. D. : C. P.
It was found highest when it is used in a ratio of 1:3-1:4 ratio.

[0018] For immunostimulatory activity of the extract

Point of 400-fold diluted from 800-fold was observed with enhanced cell function and inhibition of growth factor-activated PBMC is believed to have a density slightly less than threshold cytotoxicity is expressed.
Will there is a possibility that it exceeds the concentration, resulting in damage directly to the cell, perhaps.
However, for oral ingestion is the basic, because it is present in the blood at high concentrations physiologically is impossible, there is a consistent result is obtained here, these extracts those very interesting is.
It is a cytokine produced from antigen presenting cells (APC) mainly, p40, a subunit of IL-12p70 is, (Brombacher F, Kastelein that the production is enhanced dramatically by activation of APC is known RA, Alber G. Novel il-12 family members shed light on the orchestration of th1 responses.
Trends Immunol 2003; 24 (4): 207-12).
On the other hand, be produced from a wide range of lymphocytes has been reported that p35 is (Watford WT, Moriguchi M, Morinobu A, O'Shea JJ The biology of il-12:. Coordinating innate and adaptive immune responses.
Cytokine Growth Factor Rev 2003; 14 (5): that stimulation through CD40-40L is essential in order to function as p70 361-8), subunits between these forms dimers CD40L-deficient mice (Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G. Ligation of cd40 on dendritic cells triggers production of high levels of interleukin-12 and enhances t cell stimulatory capacity has been reported by: Tt help via apc activation.
J Exp Med 1996; 184 (2): 747-52).
However, the extract-derived components in the state of "raw", so to speak, various substances of natural origin are included, could be recognized by the innate immune response as a mere "foreign matter" is difficult discarded.
Results were placed directly stimulate APC as a "foreign material", increasing the production of p40 can be considered thoroughly, and sufficient consideration to such dilution or purification method, in order to eliminate that possibility.
As a result, may result in the increased p70 production and p40 production correlated is obtained, are involved in modification of the adaptive immune response of the T-B cell-cell or T-APC, as well as activation of APC I was shown here.
In addition, inflammatory cytokines IFN-? or TNF-a is also an important factor to continue to modify the immune response that follows.
In particular, I hope to anti-tumor effect to have of these cytokines.
Further, from the viewpoint of TNF-a and IFN-? is induced in correlation with the IL-12p70, involvement of substance having Th1 induction activity was also unpredictable.

[0019] For anti-tumor effect of the extract

The growth inhibition was observed in solid tumor cell lines, there was no suitable analysis method, but the induction of cell death was determined to be contributed by microscopy.
The possibility when using these cell lines due to the synergistic mixture is observed, as shown in FIG 8, is enhanced by any factor inducing cytotoxicity is mixing was considered.
In the case of adherent cells, in order to avoid that the extract inhibits the direct adherence to culture plate bottom surface, can be added after it is adhered completely was essential.
Adhesion inhibitory effect by itself also extract, cells - contribution as contribute to the anti-tumor effect of suppressing the growth and metastasis of tumors to suppress the adhesion to the extracellular matrix between, expected in the in vivo - cell or cell-to-cell, likely to be thought about.

[0020] Summary

The H. by this test example D.

And C. the P has an immunostimulatory effect and cell proliferation inhibitory effect by itself, if it is the mixture was clearly without being canceled, and functions synergistically the effect.

Usefulness of mixing two liquids to cytotoxic activity against solid tumor cell lines was observed in the analysis of this test example.

[0021]

The PBMC were seeded at a concentration of 2x105cell / well in 96-well flat-bottom plates that had been immobilized (1µg/ml) anti-CD3 antibody was added (5µg/ml) solubilized anti-CD28 antibody.
The H. at the initiation of the culture D. , C. P.
H. alone or, D. + C. P.
I was added: (C.P. = 1:1,1:4,4:1 H.D.) mixture of.
The cells were harvested after 24 hours, were double stained with 7-AAD and FITC-annexin V, and analyzed by flow cytometry.
Dilution of the extract X-axis (and additive-free, 200 times 800 times), Y-axis illustrates the percentage of FITC-annexin V-positive cells.

Or I was examined by ELISA (A) IL-2, for (B) IL-4 cytokines in the supernatant with a mixture of their presence.
Dilution of the extract (and no addition, 200-fold diluted from 800-fold), X-axis illustrates the cytokine concentration Y-axis.
Data shows the results for 2 representative donor.

Or I was examined by ELISA (A) IL-12p40, for (B) IL-12p70 cytokines in the supernatant with a mixture of their presence.
In CD3 + CD28 stimulation presence, PBMC unstimulated, the lower data are subjected to extraction solution added data in the upper shown in A.
Dilution of the extract (and no addition, 200-fold diluted from 800-fold), X-axis illustrates the cytokine concentration Y-axis.
Data shows the results for 2 representative donor.

Or (A) IFN-?, (B) TNF-a I was examined by ELISA for (C) IL-10 cytokines in the supernatant with a mixture of their presence.
In CD3 + CD28 stimulation presence, PBMC unstimulated, the lower data are subjected to extraction solution added data in the upper shown in C and B.
Dilution of the extract (and no addition, 200-fold diluted from 800-fold), X-axis illustrates the cytokine concentration Y-axis.
Data shows the results for 2 representative donor.

I examined the incorporation of thymidine - 3 [H] after 24h of culture of tissue-derived tumor cell lines in the presence of extract.
Dilution of the extract (and additive-free, 50-fold diluted from 400-fold), X-axis illustrates the radioactivity Y axis.
From three experiments, the data shows the results of an example representative.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines of cervical cancer (Hela).
From three experiments, the data shows the results for a representative example.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines of breast cancer (MCF-7).
From three experiments, the data shows the results for a representative example.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines of liver cancer (HepG2, Huh-7).
From three experiments, the data shows the results for a representative example.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines of lung adenocarcinoma (PC-14).
From three experiments, the data shows the results for a representative example.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines (JMN, H2452) lung mesothelioma.
From three experiments, the data shows the results for a representative example.

It was compared to additive-free incorporation of thymidine, added alone, in addition mixing - 3 [H] after 24h of culture of tissue-derived tumor cell lines in extracts the presence of 400-fold dilutions.
Value of the graph shows the radioactivity.
I was using tumor cell lines of pancreatic cancer (CaPan-1).
From three experiments, the data shows the results for a representative example.




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