Research On The Phytoestrogenic Effects And Their Mechanisms Of Ten Kinds Of Chinese Medicine

As one of the most important sex hormone in female, estrogen plays important role notonly in the hypothalamic-pituitary-gonadal axis and the growth, development anddifferentiation of female reproductive system, but also in regulation of normal function ofskeleton, cardiovascular, central neural system. The incidence of osteoporosis, cardiovasculardisease and dementia rise up with the decline of ovarian function and the falling down of theinternal level of estrogen in postmenopausal women. Such menopausal symptoms can bealleviated by estrogen replacement therapy (HRT). But on the other hand, estrogen alsocontributes to the development of hypertension, oedema and some estrogen-dependentcancers such as breast cancer and endometrial cancer. A substitute of estrogen: phytoestrogensare a group of substances that possess most positive functions of estrogen with less negativeeffects.Phytoestrogens are a group of biologically active plant substances with a similar chemicalstructure to estradiol. This structural similarity accounts for the ability of these compounds tobind ERs and exert various estrogenic or antiestrogenic effects. The estrogenic orantiestrogenic effects of phytoestrogens depend on the original level of internal estrogen.Therefore phytoestrogens are called selective estrogen receptor modulators (SERMs).Many kinds of Chinese medicine have been widely applied in female health protectionand treatment of gynecological diseases. Phytoestrogens in these Chinese medicine accountfor their pharmacological effects. Among many kinds of Chinese medicine containingphytoestrogen, medicine promoting blood circulation to remove blood statis and tonifying thekidney were the two most important kinds. In our research, ten of these kinds of medicinewere taken into consideration. Their estrogenic effects and possible mechanisms were testedand discussed by means of animal test, external cell culture experiment and ERE-controledluciferase reportor gene technique. The result offers experimental foundation and academicdata for gynecological applications of the medicine discussed. The methods and results are asfollows:1. Screening of the estrogenic effects of ten kinds of chinese medicine including Flos carthami by uterus growth test in miceTo evaluate and explore the phytoestrogenic effects of ten kinds of Chinese medicineincluding Herba epimedii by uterus growth test in rats. E₂,FSH,LH level in serum and theexpression level of ER_αand ERβin uterus were analyzed by Elisa test and immuno-histochemistry respectively.Result: (1) Administration of herba epimedii significantly increased uterus rate and bodyweight of mice, while herba cistanches and fructus psoraleae significantly decreased uterusrate and body weight of mice (P＜0.01). (2) Flos carthami significantly decreased theaugment of uterus rate caused by diethylstilbestrol. Fructus psoraleae showed markedlycooperative effect with Diethylstilbestrol. Fructus lycii and radix clycyrrhizae obviously resistthe augment of body weight caused by diethylstilbestrol. (3) Diethylstilbestrol inducedpositive expression of ERαand ERβ. Flos carthami, radix cyathulae, radix salviaemiltiorrhizae, fructus ligustri lucidi, radix clycyrrhizae, herba cistanches, herba epimedii,fructus psoraleae and semen cuscutae also led to changement in expression of ERαand/orERβat different side and degree. (4) Administration of diethylstilbestrol resulted in augmentof estrodial level and decrease of LH and FSH level in serum. Administration of semencuscutae, flos carthami, radix salviae miltiorrhizae and herba cistanches resulted in augmentof estrodial and LH level while FSH level in serum decreased. Administration of herbaepimedii, fructus ligustri lucidi and radix cyathulae resulted in decrease of estrodia, LH andFSH level.2. Evaluation on the phytoestrogen effects of ten kinds of chinese medicine includingflos carthami by MCF7 proliferation testTo explore and evaluate the phytoestrogenic effects of ten kinds of Chinese medicineincluding flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus ligustri lucidi,fructus lycii, radix clycyrrhizae, herba cistanches, herba epimedii, fructus psoraleae andsemen cuscutae by MTT assay.Result: In group A (administered diethylstilbestrol or Chinese medicine respectively),proliferation rates of MCF7 cells treated with serum from eight Chinese medicine groupsincluding flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus lycii, herbacistanches, herba epimedii, fructus psoraleae and semen cuscutae were markedly increased,while serum from fructus ligustri lucidi group markedly decreased the proliferation rate ofMCF7 cells. In group B (administered both diethylstilbestrol and Chinese medicine), theincreased proliferation rate of MCF7 cells caused by diethylstilbestrol significantly reduced inseven Chinese medicine antagonistic groups including flos carthami, radix cyathulae, radixsalviae miltiorrhizae, radix clycyrrhizae, herba epimedii, fructus psoraleae and semencuscutae, while the increased proliferation rate could be markedly enhanced in herbacistanches group. 3. Evaluation on the phytoestrogen effects of five kinds of chinese medicine includingfructus psoraleaeTo evaluate the phytoestrogenic effects of five kinds of Chinese medicine includingfructus psoraleae by uterus growth test in rats and analysis of MCF7 (ER+) proliferation ratechangement caused by their pharmacological serum.Result: (1) Administration of fructus psoraleae and radix cyathulae significantly increaseduterus rate of rats(P＜0.01), while fructus psoraleae, radix cyathulae and flos carthamisignificantly decreased the elevation of uterus rate of rats causing by diethylstilbestrol (P＜0.01). (2) Compared with the control group, proliferation rates of MCF7 cells treated withserum from fructus psoraleae, radix cyathulae, radix salviae miltiorrhizae groups weremarkedly increased respectively(P＜0.01). Their effects could be attenuated by ERβagonistDPN. Serum from three resisting groups including radix cyathulae, flos carthami, radixsalviae miltiorrhizae markedly decreased the proliferation of MCF7 cells compared with thediethylstilbestrol control group. Their effects could be enhanced by ERβagonist DPN. (3)Serum from fructus psoraleae, radix cyathulae, radix salviae miltiorrhizae and semen cuscutaegroup could significantly increase the proliferation rate of MCF7 cells. While Serum fromfive resisting groups showed no distinct effects on proliferation rate of MCF7 cells.4. Research on the phytoestrogenic effects of four kinds of chinese medicine and sixactive components in chinese medicineTo evaluate the estrogenic effects and their mechanisms of four kinds of ChineseMedicine and six active components in chinese medicine through the test of their influence onMCF7 proliferation.Result: The proliferation rates of the cells treated with radix cyathulae, flos carthami, radixsalviae miltiorrhizae, fructus psoraleae and four active components such as ecdysterone,saffcomin A, psoralen and isopsoralen were markedly increased. While the cell cycle ofMCF7 treated with ecdysterone, saffcomin A, psoralen and isopsoralen was impulsed from G₁to S phase. DNA synthesis was also inhanced and PI was increased. The above function onboosting proliferation could be inhibited by adding estrogen receptor antagonism ICI182,780.Tanshinoneâ… and tanshinoneâ…¡A showed inhibitory effects on proliferation of ER positiveMCF7 cell. However, they didn't have distinct effects on cell cycle. At the same time, theinhibitory effects of tanshinoneâ… and tanshinoneâ…¡A could be significantly decreased byadding the estrogen receptor antagonist ICI182,780.5. The influence of isopsoralen and eedysterone on proliferation and ER subtype, pS2,PR expression of breast cancer T47D cellsTo observe and evaluate the effects of isopsoralen and ecdysterone on proliferation andER subtype, pS2 and PR expression of T47D cells. Result: The proliferation rates of the cells treated with 10^-6mol·L^-1-10^-7mol·L^-1 isopsoralenand ecdysterone were increased significantly. While their proliferation indexes were alsoincreased. The RT-PCR result showed that isopsoralen and ecdysterone at 10^-6mol·L^-1 and10^-7mol·L^-1 can increase ER-αmRNA expression, while ER-βmRNA expression wasmarkedly increased when treated with isopsoralen, pS2 mRNA expression was also increasedwhen treated with isopsoralen and ecdysterone at 10^-6mol·L^-1. The Isopsoralen andecdysterone at 10^-6mol·L^-1 could also increase PR mRNA expression. The result throughFlow Cytometry showed that Levels of ER subtype and PR were all increased.6. Research on the phytoestrogenic effects and its mechanism of psoralenTo observe and evaluate the phytoestrogenic effects and its mechanism of psoralen inT47D cells by means of MTT assay, PR mRNA examination and test of ERα, ERβ, PRexpression level.Result: The proliferation rates of T47D cells treated with 10^-5mol·L^-1-10^-7mol·L^-1 psoralenwere increased significantly. The RT-PCR result showed that 10^-6mol·L^-1 and 10^-7 mol·L^-1psoralen could increase PR mRNA level. The above effects could be blocked by ICI182,780.Psoralen could also induce the augment of ER-α, ER-βand PR expression significantly.7. Research on the phytoestrogenic effects of several active components in chinesemedicine by reportor gene techniqueTo evaluate the estrogenic effects and their mechanisms of ecdysterone, psoralen andisopsoralen through the luciferase reportor gene assay.Result: All phytoestrogens tested showed distinct potencies to activate ERE induced lucexpression either through ERαor ERβ(P＜0.01). Compared to E₂ (EC50 value towards ERαand ERβ: 5.91×10^-11 M and 6.17×10^-11M), EC₅₀ value of ecdysterone, psoralen andisopsoralen were much higher. All the luciferase express induced by E₂ or the activecomponents in Chinese medicine could be blocked by ICI182,780.Conclusion:1. All the ten kinds of Chinese medicine tested show phytoestrogenic or anti-phytoestrogenic effects. But their target organs, intensity, and pathways are different.2. Six kinds of Chinese medicine such as flos carthami, radix cyathulae, radix salviaemiltiorrhizae, herba epimedii, fructus psoraleae and semen cuscutae show both estrogeniceffects (when administered indepently) and antiestrogenic effects (when administeredtogether with diethylstilbestrol) in test of their pharmacologic serum. Such bidirectionaleffects depend on the internal estrogen level.3. Radix cyathulae, flos carthami, radix salviae miltiorrhizae, fructus psoraleae and fouractive components such as ecdysterone, saffcomin A, psoralen and isopsoralen can improve MCF7 proliferation, while tanshinoneâ… and tanshinoneâ…¡A have inhibitoryeffects on MCF7 proliferation. All of their effects are realized by estrogen receptor.4. Isopsoralen and ecdysterone can accelerate ER(+) T47D cell proliferation.The expressionlevels of ER subtype, pS2 and PR in T47D change significantly with certainconcentrations of isopsoralen and ecdysterone. Isopsoralen and ecdysterone havephytoestrogenic effects.5. Psoralen can improve T47D proliferation and promote ER subtype and PR expression. Itseffects can be blocked by ICI182,780. Psoralen has phytoestrogenic effects.6. In recombined luciferase reportor gene assay, ecdysterone, psoralen and isopsoralen allshowed phytoestrogenic effects and their influences are much lower than estrogen withhigher affinity upon ERβthan ERα.