인삼의효능> 갱년기장애 갱년기 更年期, climacteric) and... · 2016. 4. 16. · 인삼의효능> 갱년기장애 갱년기(更年期, climacteric) 1. 의학적으로여성의생식기능이소실하는징후로나타나는월경폐지의시기

인삼의 효능 > 갱년기 장애인삼의 효능 > 갱년기 장애

갱년기 (更年期, climacteric)

1. 의학적으로 여성의 생식기능이 소실하는 징후로 나타나는 월경폐지의 시기

2 갱년기에 폐경이 되면 호르몬 변화에 의해 여러 가지 신체적인 변화가 일어난다2. 갱년기에 폐경이 되면 호르몬 변화에 의해 여러 가지 신체적인 변화가 일어난다.

3. 원 인 > 에스트로겐 분비감소

> 시상하부의 온도 조절기능 불안정

정신적인 스트레스와 사회적 불안감> 정신적인 스트레스와 사회적 불안감

갱년기 & 호르몬

호르몬(에스트로겐, 프로게스테론)의 감소가 폐경의 원인이다. 여성은 사춘기를 맞으면

난소에서 여성호르몬인 에스트로겐(estrogen)과 프로게스테(progesterone)이 분비되는

데 에스트로겐은 유방의 성장 자궁을 형성하는 등의 대표적 여성 호르몬이고데 에스트로겐은 유방의 성장,자궁을 형성하는 등의 대표적 여성 호르몬이고

프로게스테론은 수정된 난자를 자궁에 착상시키고 보호하는 호르몬이다.

갱년기가 되면 이 호르몬의 생성은 감소하고 월경이 중단된다.

1


폐경기호르몬변화

폐경- 에스트로겐 결핍의 시작

2


갱년기장애증상

기 관 증 상

부인과 폐경, 대하와 외음부의 가려움증 악취 등 성생활 장애 등

혈관, 신경계통안면홍조, 발한, 냉증, 심장두근거림, 숨이참, 두통, 어깨결림,

초조감, 무력감, 식욕감퇴

소화기관 스트레스성궤양, 과민성 대장증후군, 구토, 식용부진

운동기관 골다공증 요통 손발저림 어깨결림운동기관 골다공증, 요통, 손발저림, 어깨결림

비뇨기계 요실금, 빈뇨

정신신경계통 불안공포, 초조, 우울증, 집중력저하, 불면, 두통, 현기, 귀울림

피부 원형탈모증, 가려움

3


국내갱년기장애환자수

국내국내 5050세세 이상이상 여성여성 인구수의인구수의 변화변화

900

1000

1100 10,650,0002020년예상

고령화고령화 사회로사회로 접어접어 들면서들면서 5050대대 이상이상노령노령 인구의인구의 급격한급격한 증가증가((국내국내 5050세세 이상의이상의 여성여성 인구는인구는20022002년년 약약 66백만명백만명 20202020년이면년이면

600

700

800

900

구수

20022002년년 약약 66백만명백만명, 2020, 2020년이면년이면약약 천만명에천만명에 이를이를 것으로것으로 예상예상))

국내는국내는 폐경기폐경기 여성의여성의 7%7%인인 5050만명의만명의

300

400

500인구

5,980,0002002년현재

1 650 000

경경 성성 명명여성이여성이 폐경기질환으로폐경기질환으로 인해인해 의사의사처방을처방을 받고받고 있다있다..

매년매년 1010만명의만명의 신규신규 폐경기질환자가폐경기질환자가

1950 1960 1970 1980 1990 2000 2010 2020 2030100

2001,650,000 매년매년 1010만명의만명의 신규신규 폐경기질환자가폐경기질환자가

발생발생

4


기존의 치료법

한방 – 허열을 끄는 처방 위주

호르몬 대체 요법 – 가장 보편적 치료, 에스트로겐과 프로게스테론의 병합 사용

솔코스프렌 – 호르몬 생성 촉진제 사용

기존의치료(호르몬대체요법)의문제점

지난 2002년 7월 발표된 미국 국립보건원(NIH)의 연구 결과

호르몬 대체 요법을 받은 여성은 받지 않는 여성에 비해 유방암 26%, 관상동맥질환(심장병) 29%,

뇌졸중 41% 정맥혈전증 111% 높게 발생 반대로 직장 대장암은 37% 자궁내막암은 17%뇌졸중 41%, 정맥혈전증 111% 높게 발생, 반대로 직장-대장암은 37%, 자궁내막암은 17%,

대퇴부 골절은 34% 낮게 발병.

5


식물성 Estrogen을이용한치료요법

합성 Estrogen(기존 호르몬 대체요법)

식물성 Estrogen

체내에 머무르는 시간이 길다.

효과가 광범위하다.

적절치 못한 농도로 사용시

체내에 머무르는 시간이 짧다.

부작용이 거의 없다.

또는 장기 사용시 부작용 동반

식물성 Estrogen (Phytoestrogen)

식물에 존재하는 호르몬 유사물질로 내분비장애를 일으키는 물질

식물성 에스트로겐인 피토에스트로겐을 에스트로겐 대체제로 하여, 치료 목적으로 활용되고 있음

인삼 석류 감초 등에 다량 존재

6

인삼, 석류, 감초 등에 다량 존재


Reference 1

그림은그림은 alkaline alkaline phosphastasephosphastase(AK)(AK)로로 유래된유래된 자궁내막의자궁내막의 분비선분비선 종양종양 세포인세포인 Ishikawa cellIshikawa cell에서에서estradiolestradiol과과 인삼인삼 ((에탄올에탄올 추출물추출물))과의과의 관계이다관계이다. . EstradiolEstradiol은은 인삼에인삼에 의해의해 활성화활성화 되어되어 갱년기갱년기 장애를장애를 일으키는일으키는 호르몬호르몬 부족부족 현상을현상을 완화완화 하여하여예방에예방에 효과를효과를 보인다보인다

7

예방에예방에 효과를효과를 보인다보인다. .


Reference 2

Hormone replacement therapyHormone replacement therapy는는 갱년기갱년기 장애를장애를 유발하는유발하는 estrogenestrogen 부족을부족을 식물성식물성 estrogneestrogneHormone replacement therapyHormone replacement therapy는는 갱년기갱년기 장애를장애를 유발하는유발하는 estrogen estrogen 부족을부족을 식물성식물성 estrogneestrogne인인 phytoestrogenphytoestrogen을을 이용하여이용하여 치료하는치료하는 치료법이다치료법이다. .

GinsenosideGinsenoside Rb1Rb1은은 estrogenestrogen에에 특이적으로특이적으로 반응하는반응하는 estrogneestrogne--responsive responsive luciferaseluciferasegenegene를를 활성하므로활성하므로 estrogenestrogen의의 효과를효과를 보이고보이고 있음을있음을 알알 수수 있다있다

8

genegene를를 활성하므로활성하므로 estrogenestrogen의의 효과를효과를 보이고보이고 있음을있음을 알알 수수 있다있다. .


ReferenceReferenceReferenceReferenceJournal of Woman’s Health, V 13(4), 2004, 427~430 Estrogen Bioassay of Ginseng Extract and Arginmax, a Nutritional Supplement for the Enhancement of Female Sexual FunctionMary Lake Polan, M.P.H., Richard B. Hochberg

The Journal of Clinical Endocrinology & Metabolism, V 89(7), 3510~ 3515Ginsenoside-Rb1 from Panax ginseng C.A. Meyer Activates Estrogen Receptor-α and –β, Independent of Ligand BindingJungyoon Cho, Wankyu Park, Seungki Lee, Woongshick Ahn

9

Ginsenoside-Rb1 from Panax ginseng C.A. MeyerActivates Estrogen Receptor-� and -�, Independent ofLigand Binding

JUNGYOON CHO, WANKYU PARK, SEUNGKI LEE, WOONGSHICK AHN, AND YOUNGJOO LEE

College of Life Science (JY.C., W.P., YJ.L.), Institute of Biotechnology, Department of Bioscience and Biotechnology, SejongUniversity, Seoul 143-747, Korea; College of Pharmacy (SK.L.), Seoul National University, Seoul 151-742, Korea; andDepartment of Obstetrics and Gynecology (W.A.), Catholic Research Institutes of Medical Science, College of Medicine, TheCatholic University of Korea, Seoul 137-701, Korea

We studied the estrogenic activity of a component of Panaxginseng, ginsenoside-Rb1. The activity of ginsenoside-Rb1was characterized in a transient transfection system, usingestrogen receptor isoforms and estrogen-responsive lucif-erase plasmids, in COS monkey kidney cells. Ginsenoside-Rb1activated both � and � estrogen receptors in a dose-dependentmanner with maximal activity observed at 100 �M, the highestconcentration examined. Activation was inhibited by the es-trogen receptor antagonist ICI 182,780, indicating that theeffects were mediated through the estrogen receptor. Treat-

ment with 17�-estradiol or ginsenoside-Rb1 increased expres-sion of the progesterone receptor, pS2, and estrogen receptorin MCF-7 cells and of AP-1-driven luciferase genes in COScells. Although these data suggest that it is functionally verysimilar to 17�-estradiol, ginsenoside-Rb1 failed to displacespecific binding of [3H]17�-estradiol from estrogen receptorsin MCF-7 whole-cell ligand binding assays. Our results indi-cate that the estrogen-like activity of ginsenoside-Rb1 is in-dependent of direct estrogen receptor association. (J Clin En-docrinol Metab 89: 3510–3515, 2004)

GINSENG HAS BEEN used for over 2000 yr in orientalcountries to enhance stamina and immune function,where it has been suggested to have pharmacological activ-ities in the cardiovascular, endocrine, immune, and centralnervous systems (1). Its use has expanded to Western coun-tries and continues to rise with the increasing popularity ofcomplementary and alternative medicine. It is one of thebest-selling herbs in the United States (2). Although manystudies have examined the pharmacological action of gin-seng extracts, a detailed mechanism has yet to be determined.The major pharmacologically active components of ginsengare ginsenosides, which are steroidal saponins comprising3–6% of ginseng (3). It has been shown that ginsenosidesdecrease the levels of total cholesterol and triglyceride viacAMP production and inhibit the accumulation of calciumions in liver cells (4). Ginsenosides potentiate analgesia andinhibit analgesic tolerance (5). The cardioprotective action ofginsenosides is because of effects on vasodilation via nitricoxide (NO) release (6, 7). Other activities, such as anticarci-nogenic and neurological effects, have also been reported forginsenosides (8, 9).

In the United States, ginseng is used to alleviate meno-pausal symptoms, as are black cohosh (Cimicifuga racemosa),chaste tree berry (Vitex agnus-castus), dong quai (Angelicasinensis), evening primrose oil (Oenethera biennis), mother-wort (Leonurus cardiaca), red clover (Trifolium pratense), and

licorice (Glycyrrhiza glabra) (10). One recent randomized con-trolled clinical trial showed that only black cohosh had abeneficial effect on postmenopausal hot flashes (10). Other invitro studies have measured estrogenic activity in red cloverand licorice, which was not demonstrated in black cohosh(11). Various studies have indicated that ginseng has estro-genic activity, although no clinical trials have demonstratedreal efficacy as an estrogen-replacement therapy (10). Ginsengextracts are able to stimulate the growth of estrogen receptor(ER)-positive cells (12). Ginsenoside-Rg1 and -Rh1 have beenshown to contain estrogen-like activity (13, 14), but more com-prehensive data are needed to adequately evaluate this activity.

Among 26 identified ginsenosides, ginsenoside-Rb1, -Ro,-Rg1, -Rc, and -Re are highly abundant. In particular,ginsenoside-Rb1 makes up 0.37–0.5% of ginseng extracts,depending on manufacturing and processing methods,and belongs to the protopanaxadiol class of ginseno-sides (http://www.netnam.vn/icasia/english/products/redkogin/redkogind/redkogind.htm). We have previouslyreported that ginsenoside-Rb1 has estrogenic activity (15).In the present study, we aimed to characterize the differen-tial activity of ginsenoside-Rb1 toward ER isoforms � and �.We examined its ability to induce endogenous ER-responsive genes and to act as an ER ligand. Our data showthat ginsenoside-Rb1 activates both ERs � and � in theabsence of receptor binding.

Materials and MethodsReagents

Ginsenoside-Rb1 was provided by the Korea Ginseng and TobaccoResearch Institute (Daejeon, Korea). 17�-Estradiol (E2) was purchasedfrom Sigma Chemical Co. (St. Louis, MO). ICI 182,780 (ICI) was obtainedfrom ZENECA Pharmaceuticals (Tocris, UK). Ginsenoside-Rb1 was dis-

Abbreviations: CD-FBS, Charcoal-dextran stripped fetal bovine se-rum; E2, 17�-estradiol; ER, estrogen receptor; ERE, estrogen-responsiveelement; PR, progesterone receptor.JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the en-docrine community.

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(7):3510–3515Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/jc.2003-031823

3510

by on September 24, 2009 jcem.endojournals.orgDownloaded from

http://jcem.endojournals.org

solved in 20% ethanol at a concentration of 15 mg/ml. E2 was dissolvedin 100% ethanol. All the compounds were added to the medium suchthat the total ethanol concentration was never higher than 0.15%. Anuntreated group served as a control.

Plasmids

ERE2-tk81-luc, constructed by inserting the fragment of the herpessimplex thymidine kinase promoter and two copies of the vitellogeninestrogen-responsive element (ERE) into pA3luc (16), was a kind gift ofDr. Larry Jameson. Expression vector for ERs � and � were from Dr.Pierre Chambon and Dr. Vincent Giguere, respectively. pAP-1-LUCplasmid was purchased from Stratagene (La Jolla, CA).

Cell cultures

ER-positive human breast adenocarcinoma, MCF-7, cells were pur-chased from the Korea Cell Line Bank. MCF-7 cells were maintained inphenol red-free DMEM containing 1� antibiotic/antimycotic mix (In-vitrogen, Gaithersburg, MD), 5 mm HEPES, and 0.37% sodium bicar-bonate, supplemented with 10% fetal bovine serum (FBS) (HyClone,Logan, UT). COS cells were maintained under identical conditions ex-cept that 10% calf serum was used instead of FBS. Cells were grown at37 C in a humidified atmosphere of 95% air/5% CO2 and fed every 3–4d. Before hormone induction, the cells were washed with PBS andcultured in DMEM/10% charcoal-dextran stripped FBS (CD-FBS) for 2 dto eliminate any estrogenic source before treatment. All treatments weredone with DMEM/10% CD-FBS, and 10 nm E2 was used to maximizethe response unless otherwise noted.

Transient transfection and luciferase assays

Cells were seeded in 24-well plates at a density of 7 � 104 cells perwell. After 24 h, plasmids were transiently transfected into the cell bya calcium phosphate-DNA coprecipitation method. A total of 0.5 �g ofDNA in 25 �l of CaCl2�H2O (250 mm CaCl2) was mixed with 25 �l of 2�HBS (280 mm NaCl, 10 mm KCl, 1.5 mm Na2HPO4�2H2O, 12 mm dex-trose, and 50 mm HEPES) with constant bubbling, and within 5–10 min

this solution was added to each well. The next day, transfected cells werewashed with PBS and treated with compounds. Luciferase activity wasdetermined 24 or 48 h after drug treatments with an AutoLumaat LB953luminometer using the luciferase assay system (Promega Corp., Mad-ison, WI) and expressed as relative light units. The mean and ses oftriplicate or quadruplicate samples are shown for representative exper-iments. All transfection experiments were repeated three or more timeswith similar results.

RNA extraction and RT-PCR

MCF-7 cells were grown in six-well plates in phenol red-free DMEMcontaining 10% CD-FBS. Near-confluent monolayers were treated withthe compounds for 24 h. The wells were rinsed in PBS, and total RNAwas isolated by lysing the cells in guanidinium isothiocyanate using theTRIzol reagent (Invitrogen) according to the manufacturer’s instruc-tions. After extraction, RNA was precipitated by recommended proce-dures and dissolved in diethylpyrocarbonate-treated water. To synthe-size first-strand cDNA, 5 �l total RNA was incubated in 0.5 �g ofoligo(dT)18 primer (Invitrogen) and 5 �l deionized water at 70 C for 5min. RT reactions were performed using 40 U of Moloney murine leu-kemia virus reverse transcriptase (Promega Corp.) in 5� reaction buffer[250 mm Tris-HCl (pH 8.3) at 25 C, 375 mm KCl, 15 mm MgCl2, and 50mm dithiothreitol] and 20 mm dNTP mixtures at 37 C for 60 min. Thereaction was terminated by heating at 70 C for 10 min, followed bycooling at 4 C. The resulting cDNA was added to the PCR mixturecontaining 10� PCR buffer [100 mm Tris-HCl (pH 8.3), 500 mm KCl, and15 mm MgCl2], 25 U of rTaq polymerase (TaKaRa, Japan), 4 �l of 2.5 mmdNTP mixtures, and 10 pmol of primers each. The final volume was 50�l. The sequences for the human progesterone receptor (PR) and pS2primers were 5�-CCA TGT GGC AGA TCC CAC AGG AGT T-3� and5�-TGG AAA TTC AAC ACT CAG TGC CCG G-3� for PR (17) and5�-CAT GGA GAA CAA GGT GAT CTG -3� and 5�-CAG AAG CGT GTCTGA GGT GTC-3� for pS2 (18), and those of human �-actin were 5�-CCTGAC CCT GAA GTA CCC CA-3� and 5�-CGT CAT GCA GCT CAT AGCTC-3� (19). The PCR product for PR is 271 bp and 365 bp for pS2 and 550

FIG. 1. Chemical structure of ginsen-oside-Rb1.

Cho et al. • Estrogenic Activity of Ginsenoside-Rb1 J Clin Endocrinol Metab, July 2004, 89(7):3510–3515 3511



bp for �-actin. The reactions were initiated by 3 min of denaturation at94 C followed by amplification at 94 C for 45 sec, 55 C for 45 sec, and72 C for 45 sec; 24 cycles for PR or pS2 and 20 cycles for �-actin. The PCRwas ended by elongation at 72 C for 5 min. The PCR products wereanalyzed by 2% agarose gel electrophoresis and visualized by ethidiumbromide staining, quantified using a bio-imaging analyzer (Bio-RadLaboratories, Inc., Hercules, CA), and band intensity was normalized tothe intensity of �-actin mRNA.

Western blotting

Protein was isolated by using radioimmune precipitation buffer [con-taining 150 mm NaCl, 50 mm Tris-HCl, 5 mm EDTA, 1% Nonidet P-40,0.5% deoxycholate, 1% SDS with protease inhibitor cocktail (Sigma)] onice for 1 h and then centrifuged for 20 min at 13,000 � g. Supernatantwas collected, and protein concentrations were measured using theBradford method (Bio-Rad). Fifty micrograms of protein were dissolvedin sample buffer and boiled for 5 min before loading onto an 8% acryl-amide gel. After SDS-PAGE, proteins were transferred to a polyvinyli-dene difluoride membrane, blocked with 5% nonfat dry milk in Tris-buffered saline/0.05% Tween, and incubated with rabbit anti-polyclonalantibody to ER (0.4 mg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz,CA) for 2 h at 1:500. After washing with Tris-buffered saline/0.05% Tween, blots were incubated with goat antirabbit horseradishperoxidase-conjugated secondary antibody and visualized with en-hanced chemiluminescence ECL kits (Amersham Bioscience, LittleChalfont, UK).

ER binding assay

MCF-7 cells were stripped of any estrogen by keeping them in phenolred-free DMEM supplemented with 10% CD-FBS for 2 d. Cells wereincubated with 1 nm [2,4,6,7-3H]E2 (89 Ci/mmol; Amersham Bioscience)and a 100-fold excess of nonlabeled E2 (100 nm) or 25–50 �m ginsenoside-Rb1 for 1 h at 37 C in a humidified atmosphere of 95% air/5% CO2.Aliquots of the medium were measured before and after the incubationwith the cells to determine the total uptake of E2 into the cells. Afterremoval of the medium, cells were washed with ice-cold PBS/0.1%methylcellulose twice, harvested by scraping and centrifugation, andlysed with 100% ethanol, 500 �l per 60 mm dish, for 10 min at roomtemperature (20). The radioactivity of extracts was measured by liquidscintillation counting.

Statistical analysis

Values shown represent mean � sd. Statistical analysis was per-formed by Student’s t test with a P value of less than 0.05 being con-sidered statistically significant.

ResultsGinsenoside-Rb1 activates estrogen-responsive luciferasegenes in the presence of either ER� or ER�

We have previously demonstrated that ginsenoside-Rb1(Fig. 1) activates ER in MCF-7 cells (15). It has been shownthat some phytoestrogens such as genistein and coumestrol

FIG. 2. Ginsenoside-Rb1-induced transactivation was assessed by cotransfection of the (ERE)-luciferase with either ER� or ER� expressionplasmids in COS cells. Cell extracts were prepared and analyzed by a luciferase assay. A, Ginsenoside-Rb1 activated both ER� and ER� in adose-dependent manner. Cells were exposed to E2 (10 nM), ginsenoside-Rb1 (100–0.5 �M) for 24 h. *, P � 0.05; **, P � 0.005. B, Ginsenoside-Rb1activation was inhibited by ICI. Cells were exposed to E2 (10 nM) or ginsenoside-Rb1 (100 �M) or in combination with ICI (1 �M) for 24 h. Thedata are representative of at least three independent experiments performed in triplicate with similar results, expressed as relative luciferaseunits and the SEM of triplicate samples. *, P � 0.05; **, P � 0.005.

3512 J Clin Endocrinol Metab, July 2004, 89(7):3510–3515 Cho et al. • Estrogenic Activity of Ginsenoside-Rb1



differ in their activity on ER� and ER� (21). In the presentstudy, we investigated whether there is differential activa-tion of these two receptor isoforms in response to gin-senoside-Rb1 by examining the transcription of an ERE-containing reporter plasmid in the presence of ER� or ER�in ER-negative COS cells. Ginsenoside-Rb1 activated bothER� and ER� in a dose-dependent manner (Fig. 2A). Lucif-erase gene activation was observed over two orders of gin-senoside-Rb1 concentration up to 100 �m, the highest con-centration tested. Higher concentrations would haveresulted in unacceptably high concentrations of ethanol inthe cell media. In the presence of the ER� isoform, ginsen-oside-Rb1 and E2 activated ERE-driven luciferase expressionto a similar extent (91% of E2 activity); approximately 60% of theE2 response was seen with the ER� isoform (Fig. 2A). Thesedata indicate that ginsenoside-Rb1 acts as a dose-dependentagonist, stimulating transcription through both receptors, butthat it has slightly higher affinity for ER� than for ER�.

To confirm that the activities of ginsenoside-Rb1 were ERmediated, we coincubated the cells with the pure antiestro-gen ICI, at a concentration sufficient to saturate almost all theERs in the cells (1 �m). Transcriptional activation of thereporter plasmid by ginsenoside-Rb1 was blocked by ICI(Fig. 2B) with either ER� or ER�, indicating that the geneactivation was estrogen-specific.

Ginsenoside-Rb1 activates AP-1 luciferase reporterplasmids, PR and pS2 mRNA, and ER protein in anER-dependent manner

To further characterize the effects of ginsenoside-Rb1, theeffects on ER-mediated AP-1 responses, endogenous estro-

gen-responsive PR and pS2 mRNAs, and ER protein wereexamined. Ligand-occupied ERs are known to mediate genetranscription from AP-1 enhancer elements (22). AP-1 is im-plicated in the inducible expression of a variety of genesinvolved in the regulation of proliferation and apoptosis, aswell as in cellular stress responses and inflammation (23).With transient ER� expression in COS cells, both ginsen-oside-Rb1 and E2 stimulated AP-1-driven luciferase plasmidtransactivation (Fig. 3A). Ginsenoside-Rb1 did not affect lu-ciferase production in the absence of ER (data not shown).After treatment of MCF-7 cells with the compounds for 24 h,steady-state mRNA levels were measured by RT-PCR of to-tal RNA, as indicated in Fig. 3B. Constitutively expressedhuman �-actin mRNA was used as an internal control.Ginsenoside-Rb1 increased steady-state mRNA levels of thePR and pS2 genes after 24 h of treatment, as did E2 (Fig. 3B).Coincubation with 1 �m ICI efficiently blocked the activationof PR and pS2 mRNA expression, indicating activationthrough ER. We also have examined the ER protein levels inMCF-7 cells by Western analysis. ER protein levels weredown-regulated after 24 h of either E2 or ginsenoside-Rb1treatments as compared with control (Fig. 3C). These dataindicate that ginsenoside-Rb1 is capable of activating an ER-mediated pathway.

Ginsenoside-Rb1 fails to compete with estrogenbinding to ER

To determine whether ginsenoside activation occurs viadirect binding to ER, we examined the ability of ginsenoside-Rb1 to compete with 3H-labeled E2 for ER binding in MCF-7cells (Fig. 4). Specific binding was calculated as total binding

FIG. 3. Effects of ginsenoside-Rb1 on AP1-mediated activation, PR and pS2 mRNA, andER protein levels. A, Ginsenoside-Rb1-inducedexpression of AP1-driven reporter plasmids.COS cells were transiently transfected with anAP1-containing reporter and ER� expressionplasmids and treated with E2 (10 nM) or gin-senoside-Rb1 (50 �M) at the indicated concen-trations for 24 h. Cell extracts were preparedand analyzed by a luciferase assay. The data arerepresentative of at least three independent ex-periments performed in triplicate with similarresults, expressed as relative luciferase unitsand the SEM of triplicate samples. B, The semi-quantitative RT-PCR for PR and pS2 in theMCF-7 cells shown is a representative of threeindependent experiments. Cells were exposedto ginsenoside-Rb1 (50 �M) with and withoutICI (1 �M) for 24 h. PR amplification productwas detected at 271 bp (left) and 365 bp for pS2(right). C, The Western blot analysis shownis a representative of two independent experi-ments. Cells were treated with E2 (10 nM) orginsenoside-Rb1 (50 �M) alone for 24 h. ER pro-tein was detected at 62 kDa (right). Equal load-ing of protein in each lane was confirmed by�-actin protein (43 kDa). ER densitometry val-ues are expressed as a percentage of the control(left).




minus nonspecific binding (determined in the presence ofunlabeled E2 or ginsenoside-Rb1 at the concentration as in-dicated in the figure). At 50 �m, ginsenoside-Rb1 did notinhibit 3H-labeled E2 binding to ER (Fig. 4). We also failed toobserve any binding in a cell line that was stably transfectedwith ER (data not shown). In contrast, ginsenoside-Rh1, aweak phytoestrogen, displaced approximately 44% of 3H-labeled E2 binding to ER at a concentration of 50 �m inMCF-7 cells.

Discussion

Hormone replacement therapy is used to prevent or com-bat heart disease, stroke, osteoporosis, Alzheimer’s disease,and postmenopausal symptoms such as hot flashes and de-pression (24). However, such uses of estrogens are associatedwith side effects, including increased risks of breast andendometrial cancers (24). Owing to these problems, publicinterest in alternative medicines for hormone replacementtherapy has increased. The most commonly used alternativeherbal medicines for estrogen replacement are soy, blackcohosh, dong quai, chastetree berry, and ginseng (10). Phy-toestrogens that include isoflavones, lignans, and coumes-tans are found in some of these herbs (20, 25). This is likelyto be one reason for the lower prevalence of menopausalsymptoms in countries like Korea, Japan, and China, whereconsumption of soy is high (26). Although accumulatingstudies suggest important potential health benefits, both theclinical efficacies and mechanisms of action of these herbs arestill not fully known. We evaluated the estrogenic activity ofginseng, using purified ginseng components from Panaxginseng. Two ginsenosides with estrogenic activity, ginsen-oside-Rb1 and -Rh1, had been identified previously byscreening a panel of ginsenosides. In this study, we charac-terized the in vitro estrogenic activity of ginsenoside-Rb1,

providing a scientific foundation for potential clinical de-velopment. However, it should be noted that, as with otherphytoestrogens, ginsenoside-Rb1 is likely to contain biolog-ical activities that are independent of ER, such as antioxidant,antiproliferative, and antiangiogenic effects (27, 28).

In Asia, ginseng has been used for thousands of years asa tonic, to fight various aspects of stress, and to restorehomeostasis (1). In Western countries, it is being used as analternative herb for postmenopausal women. Accumulatingevidence suggests that ginseng contains either direct or in-direct estrogenic activity (29). Ginseng extracts activateestrogen-responsive genes and regulate the growth of hu-man breast cancer cells (12). Recent studies by Chan et al. (13)showed that picomolar ginsenoside-Rg1 from Panax noto-ginseng activated ER-mediated transcription without directreceptor interaction. Our group has reported estrogenic ac-tivity of ginsenoside-Rh1 in the micromolar range (14). Al-though ginsenosides share structural similarities with estro-gen and may activate ERs, more detailed studies are neededto fully characterize their activities (12, 29).

The results from other studies in different systems indi-rectly suggest the regulation of estrogen-responsive genes byginsenoside-Rb1. It was shown to decrease cardiac contrac-tion in adult rat ventricular myocytes, in part through anincrease in NO production (6). Although a correlation be-tween the increase in NO and ER activation was not evaluated,estrogen is known to enhance NO production (30). Ginsen-oside-Rb1 also regulates adrenal tyrosine hydroxylase (31),which is known to be under estrogen regulation (32).

The data presented here show that ginsenoside-Rb1 acti-vated both ER� and ER�, leading to the transactivation ofestrogen-responsive genes. However, this activation oc-curred in the absence of direct receptor binding, as examinedusing receptor competition assays. This indicates thatginsenoside-Rb1 activates ER via a mechanism or mecha-nisms other than that of classical, hormone-mediated ac-tivation. A variety of agents, including growth factors,neurotransmitters, and cAMP, activate ER in ligand-inde-pendent manners (33). Recent data on the pharmacoki-netics of ginsenoside-Rb1 show that its absolute oral bio-availability is as low as 4.35% in rats (34). Based on acalculation using the figures in the report by Xu et al. (34),approximately 180 mg/kg ginsenoside-Rb1 should betaken orally to obtain a serum concentration of 50 �m, theconcentration that activated estrogen receptors in our as-says. The ginsenoside persists for 3 d, but because of itslow absorption, initial administration or formulationchanges are necessary before clinical application (34). Ithas been shown recently that ginsenoside-Rb1 is terato-genic in the rat embryo at concentrations over 30 �m (35).Potential toxicity should be kept in mind during the clin-ical development of this compound. The exact cause of theestrogenic effects of ginseng may not be ginsenside-Rb1,because of its low serum concentration despite its in vitroestrogenic activity. However, these approaches are essen-tial to provide a scientific rationale for using ginseng forestrogen-related symptoms. In this report, we have ad-dressed the in vitro mechanism of one of the main com-ponents of ginseng. Studies investigating the upstreamtargets of ginsenoside-Rb1 that lead to ER activation and

FIG. 4. Competitive binding of ginsenside-Rb1 to ER in MCF-7 cells.Ginsenoside-Rb1 failed to compete E2 occupancy of ER. Cells wereincubated with 1 nM 3H-labeled E2 (89 Ci/mmol) plus vehicle, non-labeled E2 (100 nM), ginsenoside-Rb1 (25–50 �M), or ginsenoside-Rh1(50 �M) for 1 h. Radioactivity of ethanol extracts of cell lysates wasmeasured by scintillation counting. Specific binding was calculated astotal binding minus nonspecific binding. Data were expressed aspercentage of ligand binding, which is (total binding – nonspecificbinding)/total binding � 100. *, P � 0.05.

3514 J Clin Endocrinol Metab, July 2004, 89(7):3510–3515 Cho et al. • Estrogenic Activity of Ginsenoside-Rb1



in vivo studies will improve our understanding of theclinical application of ginseng.

Acknowledgments

Received October 20, 2003. Accepted March 18, 2004.Address all correspondence and requests for reprints to: YoungJoo

Lee, Ph.D., Department of Bioscience and Biotechnology, Sejong Uni-versity, Kwang-Jin-Gu, Seoul 143-747, Korea. E-mail: [email protected].

This work was supported in part by grants from the Korean Scienceand Engineering Foundation (2000-20700-005-3; SK.L.), Korea Tobaccoand Ginseng Corp. R&D Program 2000 (YJ.L.), Korean Ministry ofHealth and Welfare (HMP-00-O-21600-0009 to YJ.L.), and BK21 program(YJ.L.).

JY.C. and W.P. contributed equally to this work.

References

1. Attele AS, Wu JA, Yuan CS 1999 Ginseng pharmacology: multiple constitu-ents and multiple actions. Biochem Pharmacol 58:1685–1693

2. Yuan CS, Wu JA 2002 Ginsenoside variability in American ginseng samples.Am J Clin Nutr 75:600–601

3. Yoon M, Lee H, Jeong S, Kim JJ, Nicol CJ, Nam KW, Kim M, Cho BG, OhGT 2003 Peroxisome proliferator-activated receptor � is involved in the reg-ulation of lipid metabolism by ginseng. Br J Pharmacol 138:1295–1302

4. Park KH, Shin HJ, Song YB, Hyun HC, Cho HJ, Ham HS, Yoo YB, Ko YC,Jun WT, Park HJ 2002 Possible role of ginsenoside Rb1 on regulation of rat livertriglycerides. Biol Pharm Bull 25:457–460

5. Nemmani KV, Ramarao P 2003 Ginsenoside Rf potentiates U-50,488H-induced analgesia and inhibits tolerance to its analgesia in mice. Life Sci72:759–768

6. Scott GI, Colligan PB, Ren BH, Ren J 2001 Ginsenosides Rb1 and Re decreasecardiac contraction in adult rat ventricular myocytes: role of nitric oxide. Br JPharmacol 134:1159–1165

7. Chen X 1996 Cardiovascular protection by ginsenosides and their nitric oxidereleasing action. Clin Exp Pharmacol Physiol 23:728–732

8. Ernst E, Cassileth BR 1999 How useful are unconventional cancer treatments?Eur J Cancer 35:1608–1613

9. Choi S, Jung SY, Ko YS, Koh SR, Rhim H, Nah SY 2002 Functional expressionof a novel ginsenoside Rf binding protein from rat brain mRNA in Xenopuslaevis oocytes. Mol Pharmacol 61:928–935

10. Kronenberg F, Fugh-Berman A 2002 Complementary and alternative medi-cine for menopausal symptoms: a review of randomized, controlled trials. AnnIntern Med 137:805–813

11. Oerter Klein K, Janfaza M, Wong JA, Chang RJ 2003 Estrogen bioactivity inFo-Ti and other herbs used for their estrogen-like effects as determined by arecombinant cell bioassay. J Clin Endocrinol Metab 88:4077–4079

12. Duda RB, Zhong YZ, Navas V, Li MZC, Toy BR, Alavarez JG 1999 Americanginseng and breast cancer therapeutic agents synergistically inhibit MCF-7breast cancer cell growth. J Surg Oncol 72:230–239

13. Chan RY, Chen WF, Dong A, Guo D, Wong MS 2002 Estrogen-like activityof ginsenoside Rg1 derived from Panax notoginseng. J Clin Endocrinol Metab87:3691–3695

14. Lee Y, Jin Y, Lim W, Ji S, Choi S, Jang S, Lee S 2003 A ginsenoside-Rh1, acomponent of ginseng saponin, activates estrogen receptor in human breastcarcinoma MCF-7 cells. J Steroid Biochem Mol Biol 84:463–468

15. Lee YJ, Jin YR, Lim WC, Park WK, Cho JY, Jang S, Lee SK 2003 Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells. ArchPharm Res 26:58–63

16. Gehm BD, McAndrews JM, Chien PY, Jameson JL 1997 Resveratrol, a poly-phenolic compound found in grapes and wine, is an agonist for the estrogenreceptor. Proc Natl Acad Sci USA 94:14138–14143

17. Mercier G, Turque N, Schumacher M 2001 Rapid effects of triiodothyronineon immediate-early gene expression in Schwann cells. Glia 2001 35:81–89

18. Liu J, Burdette JE, Xu H, Gu C, van Breemen RB, Bhat KP, Booth N, Con-stantinou AI, Pezzuto JM, Fong HH, Farnsworth NR, Bolton JL 2001 Eval-uation of estrogenic activity of plant extracts for the potential treatment ofmenopausal symptoms. J Agric Food Chem 49:2472–2479

19. Ren L, Marquardt MA, Lech JJ 1997 Estrogenic effect of nonylphenol on pS2,ER and MUC1 gene expression in human breast cancer cells-MCF-7. Chem BiolInteract 104:55–64

20. Lee YJ, Gorski J 1996 Estrogen-induced transcription of the progesteronereceptor gene does not parallel estrogen receptor occupancy. Proc Natl AcadSci USA 93:15180–15184

21. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, Saag PT, Burg BGustafsson JA 1998 Interaction of estrogenic chemicals and phytoestrogenswith estrogen receptor �. Endocrinology 139:4252–4263

22. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, ScanlanTS 1997 Differential ligand activation of estrogen receptors ER� and ER� atAP1 sites. Science 277:1508–1510

23. Maggi-Capeyron MF, Ceballos P, Cristol JP, Delbosc S, Le Doucen C, PonsM, Leger CL, Descomps B 2001 Wine phenolic antioxidants inhibit AP-1transcriptional activity. J Agric Food Chem 49:5646–5652

24. Woo J, Lau E, Ho SC, Cheng F, Chan C, Chan AS, Haines CJ, Chan TY, LiM, Sham A 2003 Comparison of Pueraria lobata with hormone replacementtherapy in treating the adverse health consequences of menopause. Meno-pause 10:352–361

25. Cos P, De Bruyne T, Apers S, Vanden Berghe D, Pieters L, Vlietinck AJ 2003Phytoestrogens: recent developments. Planta Med 69:589–599

26. Cassidy A 2003 Potential risks and benefits of phytoestrogen-rich diets. Int JVitam Nutr Res 73:120–126

27. Mishra SI, Dickerson V, Najm W 2003 Phytoestrogens and breast cancerprevention: what is the evidence? Am J Obstet Gynecol 188:66–70

28. Glazier MG, Bowman MA 2001 A review of the evidence for the use ofphytoestrogens as a replacement for traditional estrogen replacement therapy.Arch Intern Med 161:1161–1172

29. Amato P, Christophe S, Mellon PL 2002 Estrogenic activity of herbs com-monly used as remedies for menopausal symptoms. Menopause 9:145–150

30. Farhat MY, Lavigne MC, Ramwell PW 1996 The vascular protective effects ofestrogen. FASEB J 10:615–624

31. Kim HS, Zhang YH, Fang LH, Lee MK 1999 Effects of ginsenosides on bovineadrenal tyrosine hydroxylase. J Ethnopharmacol 66:107–111

32. Ivanova T, Beyer C 2003 Estrogen regulates tyrosine hydroxylase expressionin the neonate mouse midbrain. J Neurobiol 54:638–647

33. Pasapera Limon AM, Herrera-Munoz J, Gutierrez-Sagal R, Ulloa-Aguirre A2003 The phosphatidylinositol 3-kinase inhibitor LY294002 binds the estrogenreceptor and inhibits 17�-estradiol-induced transcriptional activity of an es-trogen sensitive reporter gene. Mol Cell Endocrinol 200:199–202

34. Xu QF, Fang Xl, Chen DF 2003 Pharmacokinetics and bioavailability of gin-senoside Rb1 and Rg1 from Panax notoginseng in rats. J Ethnopharmacol 84:187–192

35. Chan LY, Chiu PY, Lau TK 2003 An in-vitro study of ginsenoside rb1-inducedteratogenicity using a whole rat embryo culture model. Hum Reprod18:2166–2168

JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.




인삼과 갱년기 질환(김동현).pdfreference 1.pdfreference2.pdf

Documents

인삼의효능> 갱년기장애 갱년기 更年期, climacteric) and... · 2016. 4. 16. · 인삼의효능> 갱년기장애 갱년기(更年期, climacteric) 1. 의학적으로여성의생식기능이소실하는징후로나타나는월경폐지의시기