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Tuesday, February 10, 2009

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Monday, August 11, 2008

History of Chill Peper

History of Chill Peper

Chilli peppers have been a part of the human diet in the Americas since at least 7500 BC and perhaps earlier. There is archaeological evidence at sites located in southwestern Ecuador that chilli peppers were already well domesticated more than 6000 years ago, and is one of the first cultivated crops in the Americas that is self-pollinating.

Chilli peppers are thought to have been domesticated at least five times by prehistoric peoples in different parts of South and North America, from Peru in the south to Mexico in the north and parts of Colorado and New Mexico (Ancient Pueblo Peoples).

Christopher Columbus was one of the first Europeans to encounter them (in the Caribbean), and called them "peppers" because of their similarity in taste (though not in appearance) with the Old World peppers of the Piper genus.

Chillis were cultivated around the globe after Columbus' time. Diego Álvarez Chanca, a physician on Columbus' second voyage to the West Indies in 1493, brought the first chili peppers to Spain, and first wrote about their medicinal effects in 1494.

From Mexico, at the time the Spanish colony that controlled commerce with Asia, chili peppers spread rapidly into the Philippines and then to India, China, Korea and Japan with the aid of European sailors. The new spice was quickly incorporated into the local cuisines.

An alternate sequence for chili peppers' spread has the Portuguese picking up the pepper from Spain, and thence to India, as described by Lizzie Collingham in her book Curry. The evidence provided is that the chili pepper figures heavily in the cuisine of the Goan region of India, which was the site of a Portuguese colony (e.g. Vindaloo, an Indian interpretation of a Portuguese dish). Collingham also describes the journey of chili peppers from India, through Central Asia and Turkey, to Hungary, where it became the national spice in the form of paprika.

There are speculations about pre-Columbian chili peppers in Europe. In an archaeological dig in the block of St. Botulf in Lund, archaeologists claimed to have found a Capsicum frutescens in a layer dating to the 13th century. Hjelmqvist also claims that Capsicum was described by the Greek Therophrasteus (370-286 BC). He also mentions other antique sources. The Roman poet Martialis (around the 1st century) described "Piper crudum" (raw pepper) to be long and containing seeds. The description of the plants does not fit pepper (Piper nigrum), which does not grow well in European climates.

The Black Habanero or as it is sometimes known, the Chocolate Habanero or Habanero Negra, is thought to be the closest to the original peppers that grew in the South American coastal plains.It is known to gourmets but rarely available, due to its long maturity and general rarity. Seeds are more readily available today but care is needed when purchasing as many sub species are sold under the same name.

Chilli Pepper

The chili pepper, chilli pepper, or chili, is the fruit of the plants from the genus Capsicum, which are members of the nightshade family, Solanaceae. Even though chilis may be thought of as a vegetable, their culinary usage is, generally, a spice, the part of the plant that is usually harvested is the fruit, and botany considers the plant a berry shrub.

The name, which is spelled differently in many regions (chili, chile, or chilli), comes from Nahuatl chīlli via the Spanish word chile. The term chili in most of the world refers exclusively to the smaller, hot types of capsicum. The mild larger types are called bell pepper in the United States, Canada (and sometimes the United Kingdom), sweet pepper in Britain and Ireland, capsicum in India and Australasia, and paprika in many European countries. Bell peppers are often named simply by their colour (e.g. green or red pepper).

Chili peppers and their various cultivars originate in the Americas; they are now grown around the world because they are widely used as spices or vegetables in cuisine, and as medicine.

Saturday, August 2, 2008

Requirement of Metabolic Activation for Estrogenic Activity of Pueraria mirifica

Title Requirement of Metabolic Activation for Estrogenic Activity of Pueraria mirifica

Author Y.S. Lee

Subject Requirement of Metabolic Activation for Estrogenic Activity of Pueraria mirifica

Institute of Research

Date Issue

Keyword pueraria mirifica (PM), endocrine disrupter, metabolic activation

AbstractTitle Requirement of Metabolic Activation for Estrogenic Activity of Pueraria mirifica

Author Y.S. Lee

Subject Requirement of Metabolic Activation for Estrogenic Activity of Pueraria mirifica

Institute of Research

Date Issue

Keyword pueraria mirifica (PM), endocrine disrupter, metabolic activation

Abstract

Abstract: A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activities. The present study was performed to examine the estrogenic effect of Kwao Keur, Pueraria mirifica (PM), that has been vised as a rejuvenating folk medicine in Thailand, using recombinant yeast, MCF-7 cell proliferation and HepGSJ cell transient transfection assay. In recombinant yeast assay, 0.025, 0.25, 23, 25, 2.5 xlO2, 2.5 xlO3, 2.5x10* ngfail concentrations of PM did not shov/ any estrogenic activities, while 10\"8 of 17 /?-estradiol (positive contrcl) showed high estrogenic activity. Estrogenic activities were induced at 2.5ng/ml to 25^ml concentrations of FM in a dose-dependent manner on MCF-7 cells and the estrogenic effect of PM was blocked by tamoxifen treatment, a well-known anti-estrogen. FM also showed estrogenic effect on human hepatoma cell line, FIepG2 cells, containing estrogen receptor and luciferase reporter gene. Taken together, PM in itself may have no estrogenidty in yeast system, but it has estrogenicity in MCF-7 & HepG2 cells that have human metabolic enzymes. The results indicated that PM may require metabolic activation for estrogenic activity

Keywords : pueraria mirifica (PM), endocrine disrupter, metabolic activation

Introduction

The steroid hormones influence the growth, differentiation, and functioning of many target tissues. Estrogens also play an important role in bone maintenance, in central nervous

system and in cardiovascular system where estrogens have certain cardioprotective effects [5, 7, 20], Estrogen receptors (ERs) belong to the nuclear receptor superfamily, and are ligand-inducible transcription factors that mediate the biological effects of estrogens and anti-estrogens. Two ER subtypes, encoded by different genes have been isolated in mammals, ERa and ER/? [6, 9, 19]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that ER/? is highly expressed in prostate and ovary [9, 16], but moderate expression was detected in other tissues including testis and uterus, some of which also seem to express ERa [10]. In the presence of estrogen or estrogen-like ligands, a ronformational change in the ER is induced, an event that promotes ER homodimerization and high-affinity binding of ER to specific sites on DNA. Once bound to DNA, the estrogen-responsive genes, results in tissue-specific estrogenic responses.

Human diet contains several plant-derived, nonsteroidal weakly estrogenic compounds [8]. Chemically, the phy-toestrogens may be divided into three main classes; flavonoids (genistein, naringenin, and kaempferol); coumestans (cou-mestrol); and lignans (enterodiol and enterolactone) [11]. Fhytoestrogens act as weak mitogens for breast tumor cells in vitro, compete with 17 /? -estradiol for binding ER protein, and induce activity of estrogen- responsive reporter gene constructs in the presence of ER protein [12, 13, 15]. It may also act as chemopreventive agents by the fact that intake of phytoestrogens is significantly higher in countries where the incidence of breast and prostate cancers is low [14].

Pueraria mirifica (PM) is an indigenous herb of Thailand, known as "Kwao Kreu" or "Kwao Kreu Kao" (White Kwao Kreu). Similar to soybean, it belongs to the same subfamily and possesses several compounds that act as phytoestrogens like phenol miroestrol and deoxymiroestrol [3]. For over a century, the tuberous root of PM has been used by local Thai people for rejuvenating and enhancing endurance and vigor. Chansakaow et aL [4] reported that nine isoflavonoids including a new pterocarpene, puemiricarpe were isolated from the tuberous root of PM and showed estrogenic activity in - MCF-7 human breast cancer cells.

In the present study, estrogerric activity of PM was evaluated in recombinant yeast assay expressing human estrogen receptor (hER) and corresponding β-galactoeidase reporter-gene, in MCF-7 human breast cancer cells proliferation assay, and in transient transfection assay using HepG2 human hepatoma cells. Estrogenic response is created by cotransfectian with reconirinant rat ER a cDNA in the presence of an estrogen-dependent luciferase reporter plasmid (C3-luc).

Materials and Methods

1. Chemicals

17 B -Estradiol (E2) and 4-hydroxytamoxifen (OHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pueraria mirifica (PM) as test material was obtained from Cheil Jedang (In-chon, Korea). All test materials were dissolved with appropriate solvents for each experiment

2. Recombinant yeast assay

2-1. Recombinant yeast cell

Saccharomyces cerevisiae ER+ LYS 8127 (YER) was obtained from Dr. Donald P. McDonnell (Duke University Medical Center, USA). The yeast cells were stored in 20% giycerol at -SOT. The YER cells were grown in a shaking incubator at 30*0 with 300rpm in a selective growth medium containing yeast nitrogen base (without amino acid, 67mg/ml), 1% dextrose, L-lysine (36/igtol), and L-histidine (24/igfail,). The yeast cells were then allowed to grow until the OD values at 600nm reached between 1.0 and 2.0.

2-2. Estrogenicity assay in yeast

The yeast cells were diluted to an ODeoonm value of 0.03 in selective medium plus 50 ^M CuSQt to induce receptor production. The diluted yeasts were aliquoted into 50-ml conical tube (5 ml/tube) and 5 /^ of 0.025, 0.25, 2.5, 25, 250, 2.5 x 103 and 2.5 x 104 ngAnl concetrations of FM and E2 (positive control) in absolute ethanol (0.1%) were added. The cultures were incubated for 18 h in a shaking incubator at 30 °C with 300 rpm. After incubation the yeast culture samples were diluted with appropriate selective medium to an ODeoonm value of 0.25 and 100 \il was added to each well of a 96-well microtiter plate. Each sample was assayed in quadruplicate. /?-Galactosidase activity was induced by the addition of 100 <d of a Z buffer (60 mM NasHPOi, 40 mM NaHaPCu, 10 mM KC1 and 1 mM MgSQ,, pH 7.0) containing 2 mgfail O-nitrophenyl-^-D-galactopyranoside (ONPG), 0.1% sodium dodecyl sulfate (SDS), 50 mM -mercaptoethanol, and 200 UAnl oxalyticase (Enzogenetics, Cornavillis, OR, USA). The 0D42>nm and ODsgonm values of each well were measured using Titertek Multiscan MCC/344 plate reader after allowing the tube to stand for 20 min. The OD420nm value of each-well--was corrected by subtracting the ODssonm value.

3. MCF-7 cell proliferation assay

MCF-7 cells were grown in phenol red-free D-media

(EMEM containing 50% increase of all essential ammn acids except glutamine, 50% increase of all vitamins, and 100% increase of all non-essential amino acids) supplemented with 5% fetal bovine serum (FBS) and 3ml/L of PSN antibiotic mixture (Gibco, NY, USA). The cells were placed in an incubator maintained at 5% COs, 95% air and 10C% humidity at 37 "C. PM was then diluted with the phenol red-free D-media supplemented with 5% dextran-coated charcoal-stripped FBS (DCC-FBS; Hyclone, UT, USA) and 3mVL PSN antibiotic mixture (test media). The concentrating DMSO in the vehicle control media was 0.1% E2 was used as positive conu-ol and OKT was co-treated with E2.

The cells (5 x lCf/ml) were plated in 6-well culture plate (2mlAvell) in triplicate, and allowed to attach for 24 h. The phenol red-free D-media was replaced with phenol red-free D-media supplemented with 5% DCC-FBS, followed by incubation for 24h, then the medium was removed and replaced by test medium (prepared as above) containing various concentrations of PM. The cells were incubated for 3 days at 37T, and the test media were changed once. The cells were then washed three times with phosphate-buffered" saline (PBS) and lysed with lml of 0.1N NaOH. The lysates were transferred into a 1.5-ml microcentrifuge tube and centrifuged for 2 minutes. Tne OD260nm value of the dear lysate was measured with a spectrophotometer (Du 650, Beckman, Pullerton, USA).

4. Transient transfection assay in HepG2 cell

4-1. Plating and transfection

HepG2 human hepatoma cells (Korean Cell line Bank, Korea) were plated in triplicate in 24-well plate at a density of 5 x 104 cells/well in complete medium consisting of phenol red-free Eagle's minimal essential medium (GIBCC/BRL, Grand Island, NY, USA) supplemented with 10 % DCC-FBS, 2% L-glutamine, and 0.1 % sodium pyruvate. Cells were incubated for 24 h at 37 "C in a humidified atmosphere of 5 % CO2 air and then transfected Mowing the Superfect procedure (Qiagen, Valencia, CA, USA) with two plasmids: (1) 0.4 gtel receptor plasmid encoding rat ERa, (2) 0.8 g/well C3-luc, reporter plasmid. Transfected cells were then rinsed with PBS and treated with various concentrations of PM or with absolute alcohol (vehicle control) in complete medium. After 24 h incubation, treated cells were rinsed with PBS and lysed with 65 fit of passive lysis buffer (Promega, Madison, WI, USA). Lysate was plated into 96-well plates for luciferase determination.

4-2. Dual Luciferase reporter assay A 100 fit volume of Lucifease assay reagent II (Promega) was added into each well containing 20 pi of lysate and then firefly luciferase activity was determined immediately using microplate luminometer LB96P (Berthold technologies, Germany). After determination of firefly luciferase activity, 100 ^ of Stop & Glo reagent (Promega) was added and Renilla luciferase activity was determined. Using the DLR™Assay System (Promega), the luminescence from the firefly ludferase reaction is 'experimental' reporter and the luminescent reaction of Renilla ludferase is 'control' reporter.

Results

1. Recombinant yeast assay '

A two-plasmid system consisting of human Estrogen receptor (hER) expression plasmid and a reporter plaamid containing estrogen response element (EHE) was employed to study estrogenic property of PM (Fig. 1). The reporter gene /? -galactosidase gave a measure for ligand-dependent transactivation. In recombinant yeast assay, 0.025, 0.25, 2.5, 25, 2.5 xlO2, 2.5 xlO3, 2.5 xu)4 ng/ml concentrations of PM did not induce any estrogenic activities while 10' of E2 as positive control had strong estrogenic activity compared with control (p>0.05).

Fig. 1. Effect of Pueraria mirifica on the yeast expressing human estrogen receptor. C, untreated; V, vehicle (1% EtOH); E2, 109 M 17,3-estradiol; PM1, 0.025 ng/ml; PM2, 0.25 ng/ml; PM3, 2.5 ng/ml; PM4, 25 ng/ml; PM5, 2.5 xlO2 ng/ml, PM6, 2.5xltf ng/ml; PM7, 2.5xlO4 ng/ml. •, significantly different from control (p>0.05)

2. MCF-7 cell proliferation assay

Estrogenic activity of PM was estimated in terms of its proliferation-promoting effects in MCF-7 human breast cancer cells (Fig. 2). Estrogenic activity was observed significantly (p>0.05) compared with control from 2.5 ng/ml concentration of PM in a dose-dependent manner. Tne PM concentration of maximal estrogen activity was 2.5 x 103 ng/ml and exhibited strong proliferation similar to E2 at the concentration of 2.5 x 10 M. DNA contents also decreased to as low as the level of vehicle control when OHT, estrogen receptor antagonist, was co-treated with PM in a dose of 2.5 xKT ng/ml, maximal effective concentration of PM or 2.5 X10 M of E2, respectively.

3. HepG2 cell transient transfection assay

Estrogenic activity of PM was characterized in HepG2 human hepatama cells transfected with rER a plus an estrogen-responsive ludferase reporter gene (Fig. 3). The estrogenic activity of PM in HepG2 cell was similar to that of PM in MCF-7 cells. PM was complete agonists at the ERa and 2.5 xlO3 ng/ml of PM, maximal effective concentration and showed stronger estrogenic activity than E2 at the concentration of 10 M.

Fig. 2. Effect of Pueraria mirifica on the proliferation of MCF-7 human breast cancer cells. C, untreated; V, vehicle (l%EtOH); E2, 2.5 x Iff10 M 170-estradiol; PM3, 2.5 ng/ml; PM4, 25 ng/ml; PM5, 2.5xlO2 ng/ml, PM6, 2.5103 ng/ml; PM7, 2.5x10* ng/ml; OHT, 10* M 4-hydroxytamoxuea *, significantly different from control (p>0.05)

Fig. 3, Effect of Pueraria mirifica on the HepG2 human hepatoma cells. C, E2, 1.0 xlO"8 M 17 0 -estradiol; PM1, 0.025 ng/ml; PM2, 0.25 ng/ml; PM3, 2.5 ng/ml; FM4, 25 ng/ml; PM5, 2.5xlO2 ng/ml, PM6, 2.5xlO3 ng/ml; PM7, 2.5 x 104 ng/fcol. *, significantly different from control (p>0.05)

Discussion

A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activity and nowadays there are increasing tendencies to get hormonal recipes using some natural products phytoestrogens. Pueraria mirifica (PM) is commonly known in Thai as White Kwao Krua, that has been used as a rejuvenating folk medicine. The enlarged underground tuber accumulates phytoestrogens comprising isoflavones such as daidzin, daidzein, genistin, genistein and puerarin. Recent studies have evaluated estrogenic activity of the isolated phytoestrogens from Pueraria mirifica (PM) such as kwakhurin, miroestrol, and deoxymiroestrol in MCF-7 human beast cancer cells [4],

In this study, three in vitro assay systems were used to evaluate the estrogenic activity of PM, and present results showed that PM did not induce estrogenic effects in recombinant yeast assay system, which PM in itself did not bind estrogen receptor. However, PM promoted MCF-7 cell proliferation in a dose-dependent manner and co-treatment PM with 4-hydroxytamoxifen inhibited PM-induced cell proliferation. Chansakaow et aL [4] supported our result on the estrogenic activity of PM in MCF-7 human breast cancer cell. This indicates that PM may be metabolized to a form capable of binding to the estrogen receptor in MCF-7 cells, whereas the yeast system may not have the capability to metabolically activate PM by the lack of mammalian metabolic enzymes using HepG2 human hepatoma cell would be able to know whether PM is metabolized before induction of estrogenic activity. The evaluation of PM in HepG2 cell lines confirmed that PM may be metabolized to induced estrogenic activity.

The recoinbinant veast system can accurately predict the estrogenic activity of various phytoestrogens in the mammalian cell system, and it is useful for testing and detecting of novel estrogenic substances in the environment and natural specimens [1]. Also, there are many advantages of yeast system to study estrogen receptor function such as ease of manipulation, rapid attainment of stable transformants, and ability to process large sample numbers quickly and inexpensively. However, the yeast assay system cannot completely address metabolism of the compound. Some results relevant to the metabolic competence of recombinant yeast assay have been reported previously [17]. For example, Methoxychlor is metabolically converted to the active estrogenic product HPTE [2]. Shelby et aL[l8] showed that methoxychlor (proestrogen) was inactive in the yeast assay system, whereas HPTE was active. This suggests that yeast assay system lack the ability to demethylate methoxychlor and may miss certain proestrogens, leading to negative results. HepG2 cell based system is apparently less sensitive to the action of 17 # -estradiol compared to the yeast system but this system has the. known properties of hepatocytes to metabolize estrogens [1], Based on the results that PM did not induce estrogenic activity in recombinant yeast cells, but induced estrogenic activity in MCF-7 human breast cancer cells and HepG2 human hepatoma cells, therefore, PM in itself may neither bind estrogen receptor nor show estrogenic effect, but may require metabolic activation for estrogenic activity that may not be observed properly by yeast system.

Acknowledgement

This work was supported by G7 project from Ministry of Environment and partially supported by Research Institute for Veterinary Science (RIVS) of College of Vet Med. SNU.

References

1. Breithofer A, Grauroann K, Scicchitano MS., Kara-thanasis SJL, Butt TJL, Jungbauer A., Regulation

of Human Estrogen receptor by phytoestrogens in Yeast and Human Cells. J. Steroid. Biochem. Mol. Biol. 1998,

67(5-6), 421-429.

2. Bulger WJi, Muccitelli RM,, and Kupfer D., Studies on the in vivo and in vitro estrogenic activities

of methoxychlor and its metabolites; role of hepatic monooxygenase in methoxychlor activation. Biochem.

Pharmacol. 1978, 28, 2417-2423.

3. Chansakaow S., Ishikawa T., Sekine K, Okada M, Kguchi Y, Kudo M, Chaichantipvuth C, Isoflavonoids

from Pueraria mirifica and their estrogenic activity. Planta Med 2000, 66(6), 572-575.

4. Chansakaow S., Ishikawa T., Seki H., Sekine K, Okada M^ and Chaichantipyuth C, Identification of deoxymiroestrol as the actual rejuvenating principle of "Kwao Keur". Pueraria mirifica. The known miroestrol may be an artifact. J. Nat. Prod., 2000, 63(2), 173-175.

5. Farhai MY., Lavigne M.C., Rarawell P.W., The vascular protective effects of estrogen. FASEB J 1996,

10, 615-624.

6. Greene G.L, Gilna P., Waterfield M, B&kor A., Hort Y. and Shine Y., Sequence and expression of human estrogen receptor complementary DNA,. Science, 1986, 231, 1150-1154.

7. Iafrati MX)., Karas RJL, Aronovitz M, Kim S., Sullivan TH, Lubahn IXB., CDonnell TJF., Korach KS., Mendelsohn ME., Estrogen inhibits the vascular injury response in estrogen receptor deficient mice. Nature Med 1997, 3, 545-548.

8. Korach KS., Migliaccio S., Davis VX», Estrogen. In:Munson PL (ed) Principles of Pharmacology-Basic Concepts

and Clinical Applications. Chapman and Hall New York 1994, 809-825.

9 Kuiper G.G., Enmark E., Felto-huikko M, Nilsson S. and Gu8tafsson J-A, Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996, 93, 5925-5930.

10. Kuiper G.G., Cariesson B., Grandien K, Enmark E., Haggblad J., Nilsson S., and Gustafsson J-A,

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and j9. Endocrinology 1997, 183, 863-870.

11. Kuiper G.G., Lemmen J.G., Carlsson B.O., Corton J.C., Safe SJL, van der Saag P.T., van der Burg. B., and Gustafsson J-A, Interaction of estrogenic chemicals and phytoestrtigens with estrogen receptor Endocrinology 1998, 139, 4252-4263

12. Makela S., Davis VJL, Tally W.G, Korkman J.,Salo L, Vihko R, Santti R, Korach K.S., Dietary estrogens act through estrogen receptor-mediated processes and show no antiestrogenicity in cultured breast cancer

cells. Environ Health Perspect 1994, 102, 572-581. 18.

13. iMarMewicz L., Garey J., Adlercreutz H., Gurpide E., In vitro bioassays of non-steroidal phytoestrogens. J Steroid Biochem Mol Biol 1993, 45, 399-405.

14. Messina MJ., Persky V., Setchell KDR Barnes S., 19. Soy intake and cancer risk: a review of the in uitro and in vivo data. Nutr Cancer 1994, 21, 113-131.

15. Mksicek RJ., Commonly occurring plant Qavonoids have estrogenic activity. Mol Fharmacol 1993, 44, 37-43.

16. Mosselman S., Polraan J., and Dijkeraa R, 20. Identification and characterization of a novel human estrogen receptor. FEBS Lett 1996, 392, 49-53.

17. Odum J., Lefevre P.A., Tittensor S., Paton D., Routledge EJ., Beresfard N.A., Sumpter J.P. and Ashby J., The rodent uterotrophic assay: critical protocol features, studies with nonyl phenols, and comparison with a yenned estrogenicity assay. Regul. Toxicol. Pharmacol., 1997,25, 176-188.

18. Shelby M.D. Newbold R.R., Tully D.B., Chae K., Davis V.L., Assessing environmental chemicals for estrogenicity using a commutation of in vitro and in vivo assays, Environ Health prospect 1996, 104(12):1296-1300.

19. Tremblay G.B., Tremalay A., Copeland N.G., Gilbert D.J., Jenkina P.A., Labrie F. and Giguere V., Cloning, chromosomes localization, and functional analysis of the murine estrogen receptor β. Mol Endocrinol 1997, 11, 252-465.

20. Turner R.T., Riggs B.J., Spelsberg T.C., Skeletal effects of estrogens. Enarred Rev 1994, 15, 275-300

Abstract: A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activities. The present study was performed to examine the estrogenic effect of Kwao Keur, Pueraria mirifica (PM), that has been vised as a rejuvenating folk medicine in Thailand, using recombinant yeast, MCF-7 cell proliferation and HepGSJ cell transient transfection assay. In recombinant yeast assay, 0.025, 0.25, 23, 25, 2.5 xlO2, 2.5 xlO3, 2.5x10* ngfail concentrations of PM did not shov/ any estrogenic activities, while 10\"8 of 17 /?-estradiol (positive contrcl) showed high estrogenic activity. Estrogenic activities were induced at 2.5ng/ml to 25^ml concentrations of FM in a dose-dependent manner on MCF-7 cells and the estrogenic effect of PM was blocked by tamoxifen treatment, a well-known anti-estrogen. FM also showed estrogenic effect on human hepatoma cell line, FIepG2 cells, containing estrogen receptor and luciferase reporter gene. Taken together, PM in itself may have no estrogenidty in yeast system, but it has estrogenicity in MCF-7 & HepG2 cells that have human metabolic enzymes. The results indicated that PM may require metabolic activation for estrogenic activity

Keywords : pueraria mirifica (PM), endocrine disrupter, metabolic activation

Introduction

The steroid hormones influence the growth, differentiation, and functioning of many target tissues. Estrogens also play an important role in bone maintenance, in central nervous

system and in cardiovascular system where estrogens have certain cardioprotective effects [5, 7, 20], Estrogen receptors (ERs) belong to the nuclear receptor superfamily, and are ligand-inducible transcription factors that mediate the biological effects of estrogens and anti-estrogens. Two ER subtypes, encoded by different genes have been isolated in mammals, ERa and ER/? [6, 9, 19]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that ER/? is highly expressed in prostate and ovary [9, 16], but moderate expression was detected in other tissues including testis and uterus, some of which also seem to express ERa [10]. In the presence of estrogen or estrogen-like ligands, a ronformational change in the ER is induced, an event that promotes ER homodimerization and high-affinity binding of ER to specific sites on DNA. Once bound to DNA, the estrogen-responsive genes, results in tissue-specific estrogenic responses.

Human diet contains several plant-derived, nonsteroidal weakly estrogenic compounds [8]. Chemically, the phy-toestrogens may be divided into three main classes; flavonoids (genistein, naringenin, and kaempferol); coumestans (cou-mestrol); and lignans (enterodiol and enterolactone) [11]. Fhytoestrogens act as weak mitogens for breast tumor cells in vitro, compete with 17 /? -estradiol for binding ER protein, and induce activity of estrogen- responsive reporter gene constructs in the presence of ER protein [12, 13, 15]. It may also act as chemopreventive agents by the fact that intake of phytoestrogens is significantly higher in countries where the incidence of breast and prostate cancers is low [14].

Pueraria mirifica (PM) is an indigenous herb of Thailand, known as "Kwao Kreu" or "Kwao Kreu Kao" (White Kwao Kreu). Similar to soybean, it belongs to the same subfamily and possesses several compounds that act as phytoestrogens like phenol miroestrol and deoxymiroestrol [3]. For over a century, the tuberous root of PM has been used by local Thai people for rejuvenating and enhancing endurance and vigor. Chansakaow et aL [4] reported that nine isoflavonoids including a new pterocarpene, puemiricarpe were isolated from the tuberous root of PM and showed estrogenic activity in - MCF-7 human breast cancer cells.

In the present study, estrogerric activity of PM was evaluated in recombinant yeast assay expressing human estrogen receptor (hER) and corresponding β-galactoeidase reporter-gene, in MCF-7 human breast cancer cells proliferation assay, and in transient transfection assay using HepG2 human hepatoma cells. Estrogenic response is created by cotransfectian with reconirinant rat ER a cDNA in the presence of an estrogen-dependent luciferase reporter plasmid (C3-luc).

Materials and Methods

1. Chemicals

17 B -Estradiol (E2) and 4-hydroxytamoxifen (OHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pueraria mirifica (PM) as test material was obtained from Cheil Jedang (In-chon, Korea). All test materials were dissolved with appropriate solvents for each experiment

2. Recombinant yeast assay

2-1. Recombinant yeast cell

Saccharomyces cerevisiae ER+ LYS 8127 (YER) was obtained from Dr. Donald P. McDonnell (Duke University Medical Center, USA). The yeast cells were stored in 20% giycerol at -SOT. The YER cells were grown in a shaking incubator at 30*0 with 300rpm in a selective growth medium containing yeast nitrogen base (without amino acid, 67mg/ml), 1% dextrose, L-lysine (36/igtol), and L-histidine (24/igfail,). The yeast cells were then allowed to grow until the OD values at 600nm reached between 1.0 and 2.0.

2-2. Estrogenicity assay in yeast

The yeast cells were diluted to an ODeoonm value of 0.03 in selective medium plus 50 ^M CuSQt to induce receptor production. The diluted yeasts were aliquoted into 50-ml conical tube (5 ml/tube) and 5 /^ of 0.025, 0.25, 2.5, 25, 250, 2.5 x 103 and 2.5 x 104 ngAnl concetrations of FM and E2 (positive control) in absolute ethanol (0.1%) were added. The cultures were incubated for 18 h in a shaking incubator at 30 °C with 300 rpm. After incubation the yeast culture samples were diluted with appropriate selective medium to an ODeoonm value of 0.25 and 100 \il was added to each well of a 96-well microtiter plate. Each sample was assayed in quadruplicate. /?-Galactosidase activity was induced by the addition of 100 <d of a Z buffer (60 mM NasHPOi, 40 mM NaHaPCu, 10 mM KC1 and 1 mM MgSQ,, pH 7.0) containing 2 mgfail O-nitrophenyl-^-D-galactopyranoside (ONPG), 0.1% sodium dodecyl sulfate (SDS), 50 mM -mercaptoethanol, and 200 UAnl oxalyticase (Enzogenetics, Cornavillis, OR, USA). The 0D42>nm and ODsgonm values of each well were measured using Titertek Multiscan MCC/344 plate reader after allowing the tube to stand for 20 min. The OD420nm value of each-well--was corrected by subtracting the ODssonm value.

3. MCF-7 cell proliferation assay

MCF-7 cells were grown in phenol red-free D-media

(EMEM containing 50% increase of all essential ammn acids except glutamine, 50% increase of all vitamins, and 100% increase of all non-essential amino acids) supplemented with 5% fetal bovine serum (FBS) and 3ml/L of PSN antibiotic mixture (Gibco, NY, USA). The cells were placed in an incubator maintained at 5% COs, 95% air and 10C% humidity at 37 "C. PM was then diluted with the phenol red-free D-media supplemented with 5% dextran-coated charcoal-stripped FBS (DCC-FBS; Hyclone, UT, USA) and 3mVL PSN antibiotic mixture (test media). The concentrating DMSO in the vehicle control media was 0.1% E2 was used as positive conu-ol and OKT was co-treated with E2.

The cells (5 x lCf/ml) were plated in 6-well culture plate (2mlAvell) in triplicate, and allowed to attach for 24 h. The phenol red-free D-media was replaced with phenol red-free D-media supplemented with 5% DCC-FBS, followed by incubation for 24h, then the medium was removed and replaced by test medium (prepared as above) containing various concentrations of PM. The cells were incubated for 3 days at 37T, and the test media were changed once. The cells were then washed three times with phosphate-buffered" saline (PBS) and lysed with lml of 0.1N NaOH. The lysates were transferred into a 1.5-ml microcentrifuge tube and centrifuged for 2 minutes. Tne OD260nm value of the dear lysate was measured with a spectrophotometer (Du 650, Beckman, Pullerton, USA).

4. Transient transfection assay in HepG2 cell

4-1. Plating and transfection

HepG2 human hepatoma cells (Korean Cell line Bank, Korea) were plated in triplicate in 24-well plate at a density of 5 x 104 cells/well in complete medium consisting of phenol red-free Eagle's minimal essential medium (GIBCC/BRL, Grand Island, NY, USA) supplemented with 10 % DCC-FBS, 2% L-glutamine, and 0.1 % sodium pyruvate. Cells were incubated for 24 h at 37 "C in a humidified atmosphere of 5 % CO2 air and then transfected Mowing the Superfect procedure (Qiagen, Valencia, CA, USA) with two plasmids: (1) 0.4 gtel receptor plasmid encoding rat ERa, (2) 0.8 g/well C3-luc, reporter plasmid. Transfected cells were then rinsed with PBS and treated with various concentrations of PM or with absolute alcohol (vehicle control) in complete medium. After 24 h incubation, treated cells were rinsed with PBS and lysed with 65 fit of passive lysis buffer (Promega, Madison, WI, USA). Lysate was plated into 96-well plates for luciferase determination.

4-2. Dual Luciferase reporter assay A 100 fit volume of Lucifease assay reagent II (Promega) was added into each well containing 20 pi of lysate and then firefly luciferase activity was determined immediately using microplate luminometer LB96P (Berthold technologies, Germany). After determination of firefly luciferase activity, 100 ^ of Stop & Glo reagent (Promega) was added and Renilla luciferase activity was determined. Using the DLR™Assay System (Promega), the luminescence from the firefly ludferase reaction is 'experimental' reporter and the luminescent reaction of Renilla ludferase is 'control' reporter.

Results

1. Recombinant yeast assay '

A two-plasmid system consisting of human Estrogen receptor (hER) expression plasmid and a reporter plaamid containing estrogen response element (EHE) was employed to study estrogenic property of PM (Fig. 1). The reporter gene /? -galactosidase gave a measure for ligand-dependent transactivation. In recombinant yeast assay, 0.025, 0.25, 2.5, 25, 2.5 xlO2, 2.5 xlO3, 2.5 xu)4 ng/ml concentrations of PM did not induce any estrogenic activities while 10' of E2 as positive control had strong estrogenic activity compared with control (p>0.05).

Clinical Trial of the Phytoestrogen-rich Herb; Pueraria mirifica as a Crude Drug in the Treatment of Symptoms in Menopausal Women

Title Clinical Trial of the Phytoestrogen-rich Herb; Pueraria mirifica as a Crude Drug in the Treatment of Symptoms in Menopausal Women

Author Verasing Muangman, M.D.* Wichai Cherdshewasart, D.Sc.**

Subject Clinical Trial of the Phytoestrogen-rich Herb; Pueraria mirifica as a Crude Drug in the Treatment of Symptoms in Menopausal Women

Institute of Research

Date Issue

Keyword

Abstract

Abstract : The clinical trial to evaluate the estrogenic effects of the crude drug derived form dry powder of a phytoestrogen-rich Thai herb Pueraria mirifica (White Kwao Krua) in five female volunteers with menopausal symptoms showed that the crude drug clarly improved the signs and symptoms resated to menopause such as, hot flushes, frustration, sleep disorder, skin dryness, high blood cholesterol, oligomenorrhoea and amenorrhoea; with no change in the blld cells, liver and kidney functions, as well as other physiological status after four months of treatment. In four volunteers, treatment were continued to complete a one-year test period with half the dose and was found to maintain their satisfied menopausal relief status. The crude drug dosage was administered at 200 mg daily for three weeks a month during the first four months to treatment and 200 mg every other day for 20 days per month for the remaining eight months. These doses were effective and safe as phytoestrogen treatment of menopausal symptoms.

Introduction

Menopause sometimes causes symptoms derived from decreased blood estrogen levels among middle-aged females. Estrogen Replacement Therapy (ERT) has been employed to protect and relieve symptoms but usually results tin substantial cost and long-term treatment has a certain risk of estrogen-related cancer1. Some recommendations such as lifestyle changes were proposed to minimize the menopausal symptoms2,3. Dietary and other natural therapies, especially soy products which contain significant levels of phytoestrogens, namely isoflavones, were shown to be not only effective in eliminating certain menopausal symptoms4-7, but also act as a potent anticancer therapy8-10. P. mirifica (Airy Shaw et Suvatabandhu) or White Kwao Krua is a Thai indigenous herb with a Long history of domestic consumption as a rejuve- Nating herb to promote youthfulness in both women and men11. The herb was first brought to public at-Tention in 1932 by a report that the tuberous root of

The kwao krua herb found in the Northern Thailand contained active constituent with such rejuvenating property12. That report elicited a study of the benefits of the herb and it was later proven in both animal experiment and clinical trial in a hospital by administering alcoholic crude extract of the herb13.

Such human benefit was also confirmed by another study14. The herb was initially recognized as Butea superba, and was finally identified and clearly established as Pueraria mirifica15.

Active ingredient were isolated and studied

From the herbal tuber and was found to contain miroestrol6,17 which exhibited the key estrogenic effect as reported from the studies in immature female mice18 and ovariectomized rats19. A very interesting result was obtained form a clinical trial in nine female volunteers at the Chelsea Hospital

Women in London, U.k., who were suffering from amenorrhea, as well as one volunteer with artificial menopause. The results clearly demonstrated that this active ingredient from P.mirifica could be administered as an estrogen supplement20. Other chemical constituents were also characterized including daidzin, daidzein, genistein, coumestrol, mirificain21, genistin22, puerarin23 and kwakhurin24. The phytochemical daidzin from soy source was shown to prevent bone loss25. Daidzein was reported to have immune enhancing activity26, inhibitory action on induced lung metastasis27 and on specific mutagenicity28. Genistein was shown to have a negative result in Ames test for mutagenesis and act as a specific inhibitor of tyrosine kinase30, an inhibitor of human breast cancer cell proliferation31-33, as well as reducing bone loss34. Coumestrol was shown to be an estrogen supplement35 and an anti-osteoporosis agent36. Stigmasterol was reported to have cholesterol lowering action.37 β-Sitosterol was shown to reduce benign prostate hyperplasia38,39 and inhibit human colon cancer growth40, as were mirificaoumestan and its derivatives41,42. Recently, deoxymiroestrol, with stronger estrogenic effects than that of miroestrol was also isolated43.

Thailand is the main natural habitat of P.mirifica with a long history of consumption record. While soy is currently used for prevention and treatment of menopausal symptoms, this preliminary clinical trial with P.mirifica in Thai women could help evaluate its potential use for long-term phytoestrogen supplementation and provide basic information to intitiate a full-scale clinical trial. Such a trial could help us to clearly understand how the crude drug works in menopausal women.

MATERIALS AND METHODS

1. Crude Drug Preparation

The fresh tuber of P. mirifica cultivar

“Wichai-III” was collected, cleaned, peeled, sliced into pieces, dried in a hot air oven until nearly completely dried, ground into fine powder of 100 mesh size particles and finally filled into capsules with the net filling filled into capsules with the net filling amount of 200 mg per capsule. Isoflavone contents were analyzed by high performance liquid Osaka, Japan, from the powder with the aid of puerarin, daidzin, daidzein, genistin and genistein as references and subsequently used as active ingredient markers.

2. The Volunteers

Menopausl women with a history of cyst formation at the breast, uterus and / or ovary were excluded. Five accepted volunteers were the outpatients of Ramathibodi Hospital, Faculty of Medicine, Mahidol University, Bangkok, Thailand, with an age range from 35 to 52 years. These women suffered from oligomenorrhoea or amenorrhoea, hot flushes, frustration, skin dryness, weakness and/or sleep disorder. The volunteers had complete blood cell count and blood chemistry analysis including haemoglobin, haematocrit, blood urea nitrogen, creatinine, SGOT, SGPT, cholesterol, triglycerides and urinalysis. The selected volunteers had to be in a normal health with normal blood chemical analysis results. They were verbally informed about the detail of the crude drug and the consumption of one 200 mg capsule a day for the first 21 days of the month for a 4 months period. If drug administration had to be extended, 1 capsule would be consumed every other day for the first 20 days of the month until the end of the 12th month. The volunteers were recruited for the first 4 months trial period, whereas 4 volunteers were asked to complete the one-year test period.

3. Evaluation Criteria

31. Physical Examination Record

The body weight, height, breast, waist and hip sizes of the volunteers were recorded just prior to the test, as well as monthly when they came on appointment to receive a new capsule batch, and again at the end of the test.

3.2 Medical Interview

A medical doctor interviewed the volunteers with standard questions just prior to the test, as well as monthly, and firnally at the end or the test.

33. Total Blood Count and Blood Chemis-try Analysis

Blood was collected for total blood count and blood chemistry analysis. The results were ana-lyzed just priod, and at the end of the 12th month test period.

RESULTS

The results from the four-month and one-year study from five selected volunteers are indivi-dually described as follows:-

Volunteer No. 1, 46 years old, weight 48 kg, height 1.47 m, had experienced amenorrhoea pe-riod for 4 months with clear menopausal symptoms exhibited as hot flushes, frustration, sleep disorder and skin dryness. Physical examination as well as total blood count and blood chemistry analysis, were normal.

After completion of the 1st month test pe-riod, it was found that the patient’s appetite increased, the body weight increased by 0.5 kg and recovered from hot flushes, frustration and skin dryness (especially facial skin).

After completion of the 2nd month test pe-riod, the patient had recovered from sleep disorder. After completion of the 3rd month test period, all recovering symptoms were maintained.

After completion of the 4th month test pe-riod, the patient had recovered from sleep disorder. After completion of the 3rd month test period, all recovering symptoms were maintained.

After completion of the 4th month test pe-riod, the recorde menopausal symptoms showed remarkable recovering from the hot flushes, frustra-tion, sleep disorder, and skin dryness. Total blood count and blood chemistry analysis revealed that the body responded to the crude drug normally, while the patient’s recorde weight increased by 0.5 kg and no recurrence of menstruation period was observed. The volunteer was satisfied with the test results and stopped consumption of the crude drug at that time.

Volunteer No. 2, 52 years old, weight 47

kg, height 1.47 m, had experienced amenorrhoea period for 5 years with obvious menopausal symptoms as exemplified as hot flushes, frustration, sleep disorder and wrinkled facial and body skin. Her physical examination was normal .The total blood count and blood chemistry analysis were normal except blood cholesterol which was recorded as 247 mg%.

After completion of the 1st month test period, the facial skin had become firm while the body weight increased by 1 kg.

After completion of the 2nd month test period, full recovery from the sleep disorder symptom had occurred and her frustration had been abolished. The body weight increased by 3 kg. Her breasts were firm and showed a 2.5 cm increase in size. Two and a half cm increase in size of the waist and the hip had occurred.

After completion of the 3rd month test period, the skin was firm and hot flushes no longer occurred. Her total blood count was normal. The blood chemistry analysis showed a sharp decrease in blood cholesterol level from 247 to 205 mg% (17.0% decrease). After completion of the 4th month test period, the patient showed full recovery from all recorded menopausal symptoms. The facial and body skin were firm and shiny. The net weight gain was 3 kg and recurrence of the menstruation was not observed.The volunteer kept taking the crude drug until the end of 12th month. It was found that the previously recorded recovery from menopausal symptoms as well as the facial and body skin firmness were all maintained while the menstruation did not recur. The volunteer was satisfied with the test results.

Volunteer No. 3, aged 35 years old, weighed 62 kg, 1.66 m height. This patent had had amenorrhoea for 7 months, was submitted to heart surgery (PDA ligation) 18 years ago, and also regularly suffered from constipation, sleep disorder, frustration, itching and a moderate amount of facial acne. Her total blood count and blood chemistry analysis were normal.

After completion of the 1st month test pe-riod, the breast size had increased by 2.5 cm and Were firm; her body skin became healthy and shiny; her facial acne disappeared resulting in clear face appearance. After completion of the 2nd month test pe-riod, the recovered symptoms were maintained as recorded in the 1st month. After completion of the 3rd month test pe-riod, the amenorrhoea symptoms were fully disap-peared as the volunteer exhibited menstruation pe-riod, itching was fully recovered, as well as sleep disorder andconstipation. The body weight incre-ased by 0.4 kg. After completion of the 4th month test per-iod, the menstruation period was recorded for2 days, the body weight had increased by 1.5 kg, the breast size remained the same as that recorded before the test, but became firmer. The hip and waist circum-ferences had increased by 2.5 cm. Total blood count and blood chemistry analysis were normal. The volunteer kept taking the crude drug until the end of 12th month. It was found that sleep disorder and frustrate symptoms were completely absent and the volunteer felt that she was in a good mood. Facial and body skin had recovered from dryness and retained firmness. The menstruation period was finally found to be irregular with increased volume. The final body weight increased by 2 kg. The volunteer was satisfied with the test results.

Volunteer No. 4, aged 49 years old, had had right ovariectomy. Her weight was 68 kg, and the height was 1.56 m. She had an irregular menstrual-tion period, and thus, was classified as oligomenor-rhoea. She had hot flushes and rheumatism, but her total blood count and blood chemistry analysis were normal.

After completion of the 1st month test pe-riod, the volunteer felt breast pain starting from the 3rd day. The result of breast examination revealed no abnormality in term of cyst formation.

After completion of the 2nd month test pe-riod, the menstruation period was found to be nor-mal.The breast pain persisted but at a lower degree.

After completion of the 3rd month test pe-riod, the breast size had increased by 2.5 cm. She still had breast pain.

After completion of the 4th month test pe-riod, the skin became healthy and firm; backache remained; shorter menstruation period was found.

Urinary problems such as mild dysuria occurred for

some times. Laboratory results were normal.

The volunteer kept taking the crude drug until the end of one-year study period. Physical examination results were normal. The body weight was stable and the skin was healthy and firm. The regular menstruation period was maintained. The dysuria was investigated and white blood cells (6-10 cells/HPF) were found in urine. She was treated with ofloxacin for 7 days and the symptom was eliminated. The volunteer was satisfied with the test results.

Volunteer No.5, 39 years old, had a weight of 49.3 kg, a height of 1.58 m, and had been married for 12 years without having a baby. She had exhibited a fibroadenoma breast condition for 6 years. She was classified as oligomenorrhea due to the fact that the menstruation period had been reduced from 7 days to 3 days. She regularly had headaches and frustration. The total blood count and blood chemistry analyses were normal except blood cholesterol level which was normal except blood cholesterol level which was 237 mg%.

After completion of the 1st month test pe-riod, a fine acne had occurred on the face since the end of the 2nd week and thereafter developed into abundant acne for 1 week before completely disap-peared. The menstruation period became normal with fresh color.

After completion of the 2nd month test pe-riod, the frustration had abated; the headaches had disappeared; the skin became healthy and firm; clear secretion was observed within the vagina; the breasts had increased by 2.5 cm in size and were firm while the waist and hip circumferences and the body weight remained the same.

After completion of the 3nd month test pe-riod, the menstruation period remained normal, as well as other parameters.

After completion of the 4th month test pe-riod, the menstruation period remained normal; the skin was healthy and firm; the body weight had in-creased by 1 kg. Total blood count and blood che-mistry analysis were normal except blood choles-terol level which had decreased from 237 to 205 mg%(13.5% decrease).

The volunteer was asked to keep taking the crude drug until the end of the one-year study pe-riod. The normal menstruation period and the healthiness and firmness of the skin were fully maintained. The body weight had increased by 1 kg while the breasts were firm and had increased by 1 cm in size. The waist had also increased in size. The volunteer was satisfied with the test results.

The reported cases versus recovering cases in five menopausal women after 4 months consum-ption of P. mirifica crude drug are summarized in Tablel. The number of cases that exhibited adverse side effects is summarized in Table 2.

DISCUSSION

The crude drug prepared from the powder of P. mirifica cultivar Wichai-III was tested in five menopausal volunteers with oligomenorrhoea or amenorrhoea symptoms. The test was designed for 4 and 12 months test periods.

The results (Table 1) revealed that two volunteers with oligomenorrhoea symptom showed an estrogenic response to the crude drug that reflected in clear improvement of the menstruation period. The findings implied that phytoestrogens from P. mirifica cultivar Wichai-III exhibited trophic effects to the uterus similar to those from estrogen or phytoestrogens from other sources. The trophic effects were most likely fully functional if the uterus was still in a fresh status, as in the case of oligomenorhoea, but they might not fully work in the two cases with amenorrhoea at the age of 46, lasting for 4 months and another at the age of 52, lasting for 5 years. The uterus in such cases might be developing into an atrophic status while the younger woman at the age of 35 years, lasting for 7 months could recover for the menstruation peiod at the end of the 3rd month. This result was most likely to occur by the same reason as that of the oligomenorrhoea. Furthermore, in one case, the clear secretion released from the vagina was noted as a sign of adverse effect (Table 2). It was most likely to result from cervical and/or vaginal secretion due to the estrogenic effect that was normally found per se in normal menstrual cycle. This finding helps confirm that estrogen receptor (ER) β is present in the human cervix44 or vaginal lining and can respond to P. mirifica phytoestrogens as well.

Table 1.Number of cases suffering with reported symptoms and that recovered after four months consumption of P. mirifica crude drug.

Symptom	Reported Cases	Recovering Cases
Amenorrhoea	3	1
Oligomenorrhoea	2	2
Hot flushes	3	3
Frustration	4	4
Sleep disorder	3	3
Skin dryness / wrinkle	5	5
High blood cholesterol	2	2
Deformed breast	2	2
Acne	1	1
Headache	1	1
Rheumatism	1	1
Itching	1	1
Constipation	1	1

Table 2. Number of case exlibited the adverse effects after four months consumption of P. mirifica crude drug.

Adverse effects	Number of cases
Body weight increase	4
Hip and waist sizes increase	2
Breast pain	1
Breast size increase	3
Transient breast size increase	1
Transient development of acne	1
Vaginal secretion	1
Increased appetite	1

The breasts increased in size in three cases, and transient enlargement occurred in one case. The enhancement of breast firmness, a sign of breast restitution, resulted in an increase in breast elasticity in of breast tissues to P. Mirifica phytoestrogens by an increase in the elasticity of the breast skin as well as by grater accumulation of water and/or fat within the breast tissue. Breast pain which occurred rapidly in one case should result from increasing pressure in situ derived from water retention within the breast tissue, as always occurs by estrogen just prior to the menstrual period. In this case such pain was prolonged and finally partially habituated. It was mainly due to the body maintenance of high level phytoestrogens afterlong-term consumption of the crude drug.

The skin appeared to be healthy as shown to be shiny and firm in all volunteers after consu-mption of the crude drug. Its response to P.mirifica

Phytoestrogens was mediated by increasing water retention, and fat and/or collagen fiver accumula-treatment. Such accumulation would definitely re-sult in increasing firmness in that particular tissue and organ. Interestingly, all volunteers expressed this type of estrogenic response and thus should imply that ERβ is present abundantly in the skin and this type of response is very unique for P.mirifica phytoestrogen treatment.

The acne previously existed before treat-ment, or was initiated as a result of the crude drug consumption, would disappear soon. This pheno-menon might be mainly due to the novel balance of estrogen as phytoestrogens from P.mirifica could competitively bind to ERα. It could show an antag-onistic effect to estrogen throung an increase in the degradation of estrogen and/or a decrease in the ac-tivity of estrogen. The influence of P.mirifica upon acne formation and clearance as clearly demonstrated in this study could be explained by the presence of high amount of phytoestrogens in this plant.

The completed 4 months tested volunteers showed normal total blood count and blood chemi-stry analysis results especially the kidney and liver function tests. The results revaled that the designed dosage of 200 mg/day with maximum 21 times/month of the crude drug derived from P.mirifca cultivar Wichai-III was not toxic to the human body espec-ially female, whereas the 3 months subchronic tox-icity test in rats also revealed that the blood chemi-stry might change mildly at the dose of 1,000 mg /kg BW while normal findings were reported at the dosage of 10 and 100 mg/kg BW45. This dose emp-loyed should be most likely an effective dose as it could eliminate some meno-pausal symptoms wit-hin the 1st month of the test period and of nearly all menopausal symptoms within the 4th month of the test period without any serious adverse effects. One of the most interesting points was that this crude drug treatment fully abolished all previous menopausal symptoms, except the amoenorrhea which was resolved in only one out of 3 volunteers. It might imply that the crude drug at the employed dosage might not be strong enough to help reco-vering of such symptoms. In the contrary, it might be an advantage to administer this crude drug as it could help recovering the main nenopausal sym-ptoms without or mildly affecting the initiation or renewal of mens-truation period in amenorrhoea. Further-more the reduction of the crde drug consu-mption to approximately half the dose of the pre-vious one and keeping on of one year resulted in full recovery of menopausal symptoms. It could be implied from this study that half the dose was enough to maintain the recovering status whereas the full dose was necessary to boost the recovering within a definite period which was found to be not longer than 4 months. Some recovering symptoms could even be observed since the end of the 1st month test period.

The body weights of 4 volunteers were found to increase slightly at the end of the test. This phenomeno might be related to the increase in appetite which was clearly recorded in one volun-teer. It might also be related to the recovering of the facial and body skin dryness and/or wrinkles in all volunteers that was most likely to be due mainly to the increase of dermal fat and oil accumulation.

The decreases of blood cholesterol in volu-nteers that were 13.5 and 17.0% reduction, might result from suppression of cholesterol biosynthetic in specific tissue or increasing degradation of blo-od cholesterol or increasing uptake of blood cho-lesterol at peripheral tissues. The decreasing in blo-od and lesterol level was also reported in a toxi-cology In rats which was more marked in the male than fe-

male45. Our test results might open a new criterion to apply this phytoestrogen-rich herb to treat hy-percholesterolaemaia or even arthrosclerosis that is very common in aging population including meno-pausal women.

P.mirifica phytoestrogens also exhibited nervous response as the recorded frustration which happened in 4 cases, sleep disorder in 3 cases, and hot flushes in 3 cases, were fully recovered and re-placed by a better mood in those 4 cases, as well as better appetite in one case. These findings were hard to monitor. The elevation of mood in 4 cases contributed to the sign of better quality of life which was very important in menopausal women as stress from bad mood or frustration not only affected the subject itslf but also more or less affected nearby people. Although some minor adverse effects were found after consumption of the crude drug, such as backache in 1 case, some minor benefit effects were also found such as elimination of headache, rheumatism, itching, constipation and acne. These findings convinced that the crude drug could exhibit more benefit than adverse effects after consumption. For example, the vaginal and/or cervical secretion reported in one case was classified as adverse effect in one hand as it did not happen in the volunteer before the test, it might also be recognized as trophic effect in the other hand as it could help recovering from vaginitis and dyspareunia. The breast size increase was classified as adverse effect by the same criteria but this phenomenon might be recognized as a trophic effect as well, due to the fact that breast enlargement was the benefit criteria for cosmetic purpose.

P.mirifica cultivar Wichi-III contains sig-nificant amount of isoflavone as analyzed by high performance liquid chromatography. The 100g dried powder contains 169.1 mg total isoflavone whereas miroestrol, deoxymiroestrol and other minor chemi-cals are present in very small amount and can not be easily detected routinely by this method21-23,43. It was then deduced that isoflavones could at least be the main chemical marker in the crude drug, and part of the menopausal symptom recovering as observed in this study could result partially from the high isoflavone content in P.mirifica cultivar Wichai-III as it was demonstrated before in MCF-7 cell line that Isoflavone from P.mirifica could also exhibit estrogenic effects46.

Phytoestrogens can competitively bind to ERα in a premenopausal woman who still has sitnificant amount of estrogen. P.mirifica phytoestrogen suppl-ementation could result in reducing the risk of estrogen-related cancer in this group of woman. Sup-plementation of phytoestrogens including P. mirifica in menopausal women with trace amount of estrogen could be a natural choice for estrogen suppl-ementation in one hand. It may, in the other hand, increase the risk of estrogen-related cancer as found in estrogen supplementation, which is stronger on the other hand, due to the agonistic effects of phyto-estrogens which are nearly totally dominated in the absence of estrogen. Very low amount of phyto-estrogen supplementation especially genistein could promote supplementation especially genistein could promote the growth of human beast cancer cell in vitro47. In thes study, there was no volunteer who had breast cancer or any other cancer during the 4 or 12 months studied period. It may be reasonable to con-clude that P. mirifica cultivar Wichai-III crude drug at this dose may be in the high dosage level and thus ex-hibits mainly cytotoxic or neural but not trophic effect to the pre-existing breast cyst (if present). It also does not stimulate the occurrence of a new breast cyst. All animal treated with P.mirific or miroestrol, the key chemical of P.mirifica also exh-ibited no report of breast cancer occuring13,18-19,45, as well as the clinical trial with miroestrol20.

However, the number of the volunteers in this study is very small and thus may not be strong enough to conclude that P. mirifica can protect aga-inst breast cyst formation in menopausal women. It may be worth also testing the topical application of P. mirifica extract directly to the breast area whether it can protect against barest cancer or not if applied long term.

The number of menopausal women in cur-rently increasing in developed countries. ERT is at present the choice of effective treatment for such population. Phytoestrogen could be then a novel alternative as it could do both functions at the same time, exhibiting estrogenic effects as well as func-tioning as an anticancer agent. Previous studies have clearly indicated that the estrogenic effects of P. mirifica are far stronger than those of soy.whichi is a popular commercialized natural phytoestrogen

supplementation at present .We can therefore stress that this study has initiated an attractive invitation

for researchers to investigate deeply in to P.mirifica

phytoestrogens and manipulate them to serve the need for alternative medicine or Phytoestrogen Re-with a reduced cancer risk which has been pre-viously seen in those taking soy phytoestrogens48, and with a higher degree of success in relieving menopausal symptoms.

ACKNOWLEDGEMENTS

The authors wish to thank Japan Food Re¬search Laboratory, Osaka Branch, Japan, for HPLC isoflavone analysis with the sample of White Kwao Krua, P. mirifica cultivar Wichai-III, to Ramathibodi Hospital, Mahidol University and the Office of Aca¬demic Affairs, Chulalongkorn University for partial support.

References

1. Shoff SM, Newcomb PA, Mares-Periman JA, et al.Usual consumption of plant foods containing phytoestrogens and sex hormone levels in postmenopausl women in Wisconsin. Nutr Cancer 1998; 30:207-212

2. Ivarrson T, Spetz AC, Hammere M. Physical exercise and vasomotor symptoms in postmeno-pausal women. Maturitas 1998; 29: 139- 146.

3. Slaven L, Lee C. Mood and symptom reporting among middle-aged women: the relationship between meno¬pausal status, hormone replacement therapy and exer¬cise participation. Health Psychol 1997; 16:203 - 208.

4. Baird DD, Umbach DM, Landsedell L, et al. Dietary intervention study to access estrogenicity of dietary soy among postmenopausal women. J Clin Endocrinol Metab 1995; 80: 1685- 1690.

5. Cassidy A, Bingham S, Setchell KDR. Biological ef¬fects of a diet of soy protein rich in isoflavones on the menstruation cycle of premenopausal women. Am J Nutr 1994; 60: 333-340.

6. Albertazzi P, Pansini F, Bonaccorsi G, et al. The effect of dietary soy supplement on hot flushes. Obstet Gynecol 1998; 91: 6-11.

7. Brezinski A, Aldercreutz H, Shaoul R, et al. Short-term effects of phytoestrogen-rich diet on postme-nopausal women. Menopause 1997; 4: 89 - 94.

8. Messina MJ, Persky V, Setchell KDR, et al. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr Cancer 1994; 21: 113-131.

9. Kao YC, Zhou CB, Sherman M, et al. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-uireaed rnutagenesis study. Environ Health Persp 1998; 106:85-92.

10.Ingram D, Sanders K, KolybabaM, et al. Case-

control study of phyto-oestrogens and breast

cancer. Lancet 1997:350:990-994.

11. Suntara A. The remedy pamphlet of Kwao Krua tuber of Luang Anusarnsuntarakromkarnphiset, Chiangmai. Upatipongsa Press: Chiangmai, Thailand, 1931.

12. Kerr A. A reputed rejuvenator. J Siam Soc Nat Hist 1932;8(Suppl):336-338.

13. SukavattanaT. Oestrogenic principle of Butea-superba, preliminary report. J Med AssocThai 1940; 24: 183 -194.

14. Schoeller W, Dohrn M, Hohlweg W. An estro-genic substance from the tubers of the Siamese vine, Butea superba. Naturwissen-schaften 1934; 28: 532.

15. KasemsanlaMCL, Suvatabandhu K, Airy SHK. A new species of Pueraria (leguminosae) from Thailand, yield¬ing an oestrogenic principle. Kew Bull 1952:263 - 266.

16. Jones HEM, Pope GS. A method for the isolation of miroestrol from Pueraria mirifica. J Endocrinol 1961;22:303-312.

17. Tayler NE, Hodgkin DC, Rollett JS. The X - ray crys-tallographic determination of the structure of bromomi-roestrol. J Chem Soc I960; 33: 3685.

18. Jones HEM, Pope GS. A study of the action of miro-estrol and other oestrogens on the reproductive tract of the immature female mouse. J Endocrinol 1960; 20: 229 -235.

19.- Benson GK, Cowie AT, Hosking ZD. Mammog-enic activity of miroestrol. J Endocrinol 1961; 21: 401 -409.

20. Cain JC. Miroestrol: An oestrogen from the plant Pueraria mirifica. Nature I960; 188:774 -777.

21. Ingham JL. Tahara S, Dziedzic SZ. A chemical investtti¬gation of Pueraria mirifica roots. Z Natur-forsch Ser C 1986; 41: 403-408.

22. Ingham JL, Tahara S, Dziedzig SZ. Minor isofla-vonesfrom the root of Pueraria mirifica. Z Naturfo-rsch Ser C1989:44:724-726.

23. Ingham JL, Markham KR, Dziedzig SZ, et al. Pue-rarin6'-O-P - apiofuranoside, aC-glycosylisoflav-one- O-glycoside from Pueraria mirifica. Phytochemistry 36. 1986; 25: 1772- 1775.

24. Tahara S, Ingham JL, Dziedzig SZ. Structure elucidation of kwakhurin, a new phenylated isoflavone from Pueraria mirifica roots. Z Naturforsch Ser C 1987; 42:510-518.

25. Ishida H, Uesugi T, Hirai K, et al. Prevention effects of the plant isoflavones, daidzin and genistin on bone loss in ovariectomized rats fed with a calcium-deficient diet. Biol Pharm Bull 1998; 21: 62 - 66.

26. Zhang R, Li Y, Wang W. Enhancement of immune func- tion in mice fed high doses of soya daidzein. Nutr Can¬cer 1988; 29: 24-48.

27. Masson LG, Kuttan R, Nair MG, et al. Effect of isoflavones genistein and daidzein in the inhib-itttion of lung metastasis in mice induced by B16 F - 10 mela¬noma cells. Natr Cancer 1998; 30: 74 - 79.

28. Weisberger JH, Dolan L, Pitman B. Inhibition of PhIP rhutagenicity by caffeine, lycopene, daid-zein and genistein. Mutat Res 1998; 416: 125- 128.

29. Batholomew RM, Ryan DS. Lack of mutagenicity

of some phytoestrogens in Salmonella mammal-

ian mi-crosome assay. Mutat Res 1980; 78:

317 - 321.

30. Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a specific inhibitor of tyrosine - specific protein kinase. J Biol Chem 1987; 262: 5592 - 5595.

31. Peterson G, Barnes S. Genistein inhibition of the growth of human breast cancer cells: Independence from estro¬gen receptors and multi-drug resistance gene. Biochem Biophys Res Commun 1991; 179: 661 - 667.

32. Verma SP, Salomone E, Goldin B. Curcumin and genistein plant natural products show synergistic in¬ hibitory effects on the growth of human breast cancer MCF-7 cells induced by estrogenic pesticides. Biochem Biophys Res Commun 1997; 233: 692 - 696.

33. Wang C, Kurzer MS. Phytoestrogen concen-tration de-termines effects on DNA synthesis in human breast cancer cells. Nutr Cancer 1997; 28: 236 - 247.

34. Fanti P, Monier - Faugere MC, Gerg Z, et al. The phytoestrogen genistein reduces bone loss in short -term ovariectomized rats. Osteoporos Int 1998; 8: 274-281.

35. Markaverich BM, Webb B, Densmore CL, et al. Effectsof coumestrol on estrogen receptor function and uterine growth in ovariectomized rats. Environ Health Perspect

1995:103:574-589.

36. Tsusumi N. Effect of coumestrol on bone

metabolism in organ culture. Bio Pharm Bull

1995; 18: 1012-1015.

37. Westrate JA, Meijer GW. Plant sterol - enriched marga¬rines and reduction of plasma total- and LDL-choles-terol concentration in mildly hypercholesterolaemic sub¬jects. Eur J Clin Nutr 1998; 52: 334 - 343.

38. Klippel KF, Hiltl DM, Schipp B. A multicenter, pla¬cebo-controlled, double-blind clinical trial of P-sitosterol (phytosterol) for the treatment of benign prostatic hy-perplasia. Brit J Urol 1997; 80: 427 - 432.

39. Von Holtz RL, Fink CS, Awad AB. P-sitosterol acti¬vates the sphingomyelin cycle and induces apoptosis in LNCaP Human Prostate Cancer Cells. Nutr and Cancer 1998; 32: 8- 12.

40. Awad AB, Von Holtz R, Conne JP, et al. P-sitosterol inhibits the growth of HT-29 human colon cancer cells by activating the sphingomyelin cycle. Anticancer Res 1998; 18:471-479.

41. Hoyodom M. Constituents of the tuberous roots of Pueraria mirifica. Thesis-M. Sc. Faculty of Science Chulalongkom University, Bangkok, Thailand, 1971.

42. Ingham JL, Tahara S, Dziedzic SZ. Coumestans from the roots of Pueraria mirifica. Z Naturforsch Ser C 1988; 43 (1/2): 5-10.

43. Chansakaow S, Ishikawa T, Seki H, et al. Identification of deoxymiroestrol as the actual rejuvenating principle of "Kwao Keur", Pueraria mirifica. The known miroestrol may be an artifact. J Nat Prod 2000; 63: 174-176.

44. Weihua Z, Saj S, Makinen S, et al. Estrogen receptor (ER) P, a modulation of ERa in the uterus. Proc Natl Acad Sci 2000; 97: 5936 - 5941. 45. Chivapat S, Chavalittumrong P, Rattanajarasroj S, et al. Toxicity study of Pueraria mirifica Airy "Shaw et Suvatabandhu. Bull Med Sci 2000; 42: 202 - 223.

46. Chansakaow S, Ishikawa T, Sekine K, et al. Isoflavo-noids from Pueraria mirifica and their estrogenic acti¬vity. Planta Med 2000; 66: 572 - 575.

47. Wang TTY, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen receptor mediated path¬ways. Carcinogenesis 1996; 17: 271 - 275.

48. Clarkson TB, Anthony MS, Williams JK, et al. The potential of soybean phytoestrogens for postmenopausal hormone replacement therapy. Proc Soc Exp Med Biol 1998; 217: 365-368.

Application of Pueraria Mirifica

Pueraria Mirifica has long been traditionally prepared in the form of pills for oral intakes. Currently, the dried roots are finely ground, sometimes mixed with other medicinal herbs, packed in capsules and marketed as a dietary supplement; however, for some distributors, the capsules are prepared from the root extract. The formulation that includes ingredient list and content of each ingredient varies from one supplier to another.

Pueraria Mirifica in the form of extract has been introduced just recently. With modern extraction technology, the extract from dried roots is standardized and prepared in the form of powder and solution, the extract solution is used in cosmetic industry such as preparation of breast cream, eye gel, and skin moisturizer. The extract powder is used as a dietary supplement similarly to the traditional method, but the product has more uniformity due to the advantage of standardization.

When Pueraria Mirifica is taken as a dietary supplement, its phyto-estrogen constituents will naturally alleviate symptoms occurring as a result of the aging process and a deficiency in estrogen levels, e.g., sagging breasts, wrinkled skin, bone loss, gray hair, etc. These aging signs and symptoms will, to a certain extent, be reversed.

When applied topically to the skin, Pueraria Mirifica will be beneficial to that respective part of the body.

Various forms and preparations of Pueraria Mirifica should provide an effective and inexpensive alternative to women who wish to take charge of their own health and to enhance their beauty or utilize Pueraria Mirifica for many cosmetic purposes.

With regard to the safety and efficacy of dermal preparations of Pueraria Mirifica extract, standard dermal toxicity tests were performed in various animal species as well as in humans, and no toxicity was observed, Moreover, the efficacy of breast cream was recently evaluated in a large group of human volunteers.

Source:Medicinal Plant Research Institute, Department of Medical Sciences, Ministry of Public Health, Thailand; July 2000

Effects of Pueraria Mirifica

High Isoflavones the phyto - Anti cancer

After many years of species selection, enrich with high isoflavones, the most interesting phyto-estrogens has been selected and submitted to organic farming in a huge area to guarantee the unique benefit of the phyto-estrogens.

Now this species is manufactured into our vast variety of Products and distributed in Japan, China, Thailand, Europe and U.S.A.

Our Selected Species

Phyto-estrogens especially isoflavones, enriched in Pueraria Mirifica only our selected species which this selected grade can make us differentiate from the other supplier with chemical compound approval If intake at a right dosage will support healthy female characteristics, bone structure, cardio - vascular system, breast and skin appearance in women and also health prostate function and keeps hair pattern in men.

According to our study, our researcher recommend women who are lack of endogenous estrogen including menopausal women and also mature men who are facing aging problems, should intake White Kwao Krua regularly to keep rejuvenate and the tonic felling.

What is Phyto-estrogen?

The term "natural" can refer to the source of the hormone or more importantly, it can mean that it is natural to a woman’s body; that its chemical structure is identical to the hormones produced by the human body.

The estrogens used in our formulation are the exact same molecules as the ones produced by a woman’s body. They are bio-identical. This means a woman’s body recognized it just as if she had produced it herself. It is not treated as a "foreign" molecule, her body can use it correctly, and side effects are minimal, or even eliminated!

Just as the hormone needs to be "bio-identical" to be natural, it should also be given in natural proportions. The term "estrogens" actually refers to several hormones, estriol, estrone, and estradiol being the ones natural to a woman’s body.

As a young woman, each is produced in certain amounts. When the body slows down making these estrogens, as in menopause, they should not only be replaced, but also in natural quantities. Our formulation provides all three estrogens, in the normal proportions, to basically imitate what a woman’s body produced before menopause.What is natural hormone?

What could be more natural?

One more hormone to consider “progesterone” that same young woman produced progesterone along with her three estrogens, and that same woman in menopause has quit making progesterone. Our formulation includes progesterone, also from natural sources and bio-identical, for several reasons. By replacing what is lost, a woman is getting back to what is most natural for her body. Progesterone actually helps the body use the estrogens better, so we can use the least amount of estrogens to be effective. Progesterone has been shown to actually build bone, act as a natural antidepressant, serve as a source of other hormones lost to aging; basically, making things more balanced.

Source: Medicinal Plant Research Institute, Department of Medical Sciences, Ministry of Public Health, Thailand; July 2000