仙茅論文：仙茅 酚苷 提取 純化 分離 去卵巢 骨質(zhì)疏松 抗雌激素樣作用

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1、仙茅論文：仙茅 酚苷 提取 純化 別離 去卵巢 骨質(zhì)疏松 抗雌激素樣作用 【中文摘要】中藥仙茅來源于石蒜科植物仙茅Curculigo orchioides Gaertn的枯燥根莖,味辛、性熱、有毒,具有補(bǔ)腎陽、益精血、強(qiáng)筋骨、祛寒濕等成效,具有調(diào)節(jié)免疫、抗骨質(zhì)疏松、保肝、保護(hù)心血管系統(tǒng)等多種藥理活性。仙茅的主要化學(xué)成分為酚類成分及其衍生物、環(huán)菠蘿蜜烷型三萜皂苷、木脂素,黃酮等。本課題組研究發(fā)現(xiàn)仙茅乙醇提取物能夠減少去卵巢骨質(zhì)疏松大鼠骨質(zhì)的喪失,具有抗骨質(zhì)疏松作用?；钚猿煞趾Y選結(jié)果說明仙茅抗骨質(zhì)疏松的活性成分為酚苷類成分?；谏鲜鲅芯扛?本課題著重進(jìn)行了以下幾方面研究：建立了仙茅中苔黑酚

2、葡萄糖苷,仙茅苷,仙茅素A的HPLC含量測(cè)定方法。以仙茅中三種代表性酚苷類成分苔黑酚葡萄糖苷,仙茅苷,仙茅素A的含量為考察指標(biāo),以HPLC進(jìn)行含量測(cè)定,考察乙醇濃度、提取次數(shù)、提取時(shí)間、溶劑倍量對(duì)酚苷類成分提取效率的影響,通過四因素三水平L9(34)正交試驗(yàn)確定仙茅酚苷類成分的最正確提取方法。優(yōu)選出的最正確提取工藝為10倍量70%乙醇回流提取3次,每次120分鐘。以優(yōu)化的提取工藝提取仙茅藥材,調(diào)整提取液濃度為每毫升含1g原藥材,以1：1的上樣量(藥液體積：樹脂床體積),上樣于D101大孔樹脂,待吸附后,分別以水、10%、20%、30%、40%、50%、60%的乙醇洗脫,HPLC檢測(cè)洗脫液,確定

3、10%乙醇洗脫部位富含苔黑酚類酚苷,30%乙醇部位富含苯甲酸酯類酚苷仙茅苷和仙茅素A。將10%乙醇洗脫部位和30%乙醇洗脫部位用水溶解后分別再上D101大孔樹脂,分別收集10%乙醇洗脫部位和30%乙醇洗脫部位,濃縮枯燥后,10%乙醇洗脫部位苔黑酚葡萄糖苷含量達(dá)39%：30%乙醇洗脫部位仙茅苷和仙茅素A含量分別為46%和4%。通過D101大孔樹脂柱、凝膠柱、ODS反相柱、MCI柱,制備液相對(duì)10%乙醇洗脫部位和30%乙醇洗脫部位進(jìn)行化學(xué)成分的別離鑒定,共別離得到10個(gè)化合物。其中8個(gè)為仙茅酚苷類成分,2個(gè)為呋喃類化合物。以三月齡去卵巢雌性大鼠為模型考察仙茅提取物及仙茅酚苷類成分的抗骨質(zhì)疏松作用。

4、研究發(fā)現(xiàn)雌性大鼠去卵巢后,子宮萎縮,已造成絕經(jīng)狀態(tài)；血清堿性磷酸酶活性升高,U-Ca/Cr值升高,股骨骨密度降低,血清抗氧化酶活力水平下降,脂質(zhì)過氧化物含量升高。仙茅提取物及不同劑量的苯甲酸酯類酚苷成分均能提高去卵巢大鼠的骨密度,且中、高劑量組大鼠骨密度與去卵巢模型組大鼠比擬具有顯著性差異(P<0.01)。仙茅苔黑酚類酚苷成分對(duì)去卵巢大鼠骨密度的作用不顯著。這說明仙茅的抗骨質(zhì)疏松活性成分為其所含的苯甲酸酯類酚苷,如仙茅苷等,這些成分通過抑制骨吸收減少骨質(zhì)的喪失。代謝組學(xué)和血清抗氧化酶活性分析說明仙茅酚苷類成分通過提高機(jī)體的抗氧化能力改善骨代謝,減少骨質(zhì)的喪失。選用17天齡性未成熟的雌性SD大鼠

5、幼鼠,以子宮濕重與100g體重之比作為評(píng)價(jià)仙茅雌激素樣活性的指標(biāo),考察了仙茅乙醇提取物及各D101大孔樹脂的純化部位的雌激素樣作用,研究結(jié)果說明仙茅總提物及其各純化部位均無明顯的雌激素樣作用。選用雌激素受體陽性細(xì)胞模型(MCF-7)考察了仙茅中的酚類化合物：仙茅苷、仙茅素A、2,6-二甲氧基苯甲酸、苔黑酚葡萄糖苷、苔黑酚龍膽二糖苷對(duì)MCF-7細(xì)胞的增殖作用的影響,結(jié)果發(fā)現(xiàn)陽性藥雌二醇(E2)那么具有顯著促細(xì)胞增殖作用,各受試藥物對(duì)細(xì)胞增殖無明顯作用,推斷仙茅各受試藥物即無雌激素樣作用。去卵巢大鼠給予苔黑酚類酚苷和苯甲酸酯類酚苷12周后,其雌激素受體陽性器官乳腺、陰道和子宮的組織構(gòu)造與去卵巢對(duì)照

6、大鼠相比擬沒有顯著變化,但其孕激素受體、雌激素受體P和β的表達(dá)有一定程度的降低,這些結(jié)果說明仙茅苔黑酚類酚苷和苯甲酸酯類酚苷具有抗雌激素樣作用。綜上所述,本課題獲得了仙茅酚苷類成分的制備方法,別離鑒定了其中主要的酚苷類化合物,說明了其抗骨質(zhì)疏松和抗雌激素樣作用的物質(zhì)根底。通過抗骨質(zhì)疏松和雌激素樣活性分析,初步明確了仙茅酚苷類成分通過抗氧化和抗雌激素樣作用抑制骨喪失的作用機(jī)制。缺乏之處在于仙茅酚苷類成分抗雌激素樣作用的研究尚不夠全面深入,有待在以后的研究中補(bǔ)充、完善,以揭示仙茅酚苷類成分抗雌激素樣作用的特點(diǎn),明確其可能對(duì)生物或人體產(chǎn)生的有益或不利作用。 【英文摘要】Curculigo orch

7、ioides is the dry rhizome of Curculigo orchioides Gaertn., one of the traditional Chinese medicine. Curculigo orchioides are considered to be pungent flavor, hot-natured and poisonous in traditional Chinese medicine. They have the effects of kidney-tonifying and yang-boosting, maintaining healthy en

8、ergy, strengthening the muscles and bone, expelling wind and rheumatism, etc. People usually use Curculigo orchioides for immunoregulation, anti-osteoporotic, protecting the liver and cardiovascular system. Curculigo orchioides are used in many of traditional Chinese prescriptions for anti-osteoporo

9、tic, so cause great interest and argument. The main constituents are phenol and phenolic glycoside, cycloartane-type triterpene saponin, lignan, flavone, etc. In previous we used OVX rat model to investigate the antiosteoporotic activity of the ethanol extract of Curculigo orchioides, and used the o

10、steoblast and osteoclast to investigate the antiosteoporotic activity of phenolic glycosides in vitro. The main contents of our study are as follows:The method of determination orcinol glucoside, curculigoside and curculigine A in Curculigo orchioides by HPLC was established. The contents of orcinol

11、 glucoside, curculigoside and curculigine A were used as parameters, and they were determined by HPLC. Study of the concentration of ethanol, extraction time, times of extraction and solvent volum on the impact of extraction efficiency. The extraction conditions of phenolic glycosides in Curculigo o

12、rchioides were optimized by orthogonal design. The optimal extraction process was refluxed three times with 70% ethanol (10 times) for 2h each time.Curculigo orchioides were refluxed with optimal extraction process. Then the solvent was evaporated under vacuum and dissolved into distilled water to g

13、ive 1g/ml (crude medicine/volume) sample solution. The solution was instilled into the prepared D101 macroporous resin, till the sample was absolutely adhered to the resin. After that the resin was washed with distilled water and ethanol solutions of different concentrations (water, 10%,20%,30%,40%,

14、50%,60%) successively. Resin elution was detected by HPLC to determine the time of replacing the eluent. Orcinol glucoside was found in the 10% ethanol solution and Curculigoside and curculigine A in the 30% ethanol solution.The desorption solution was collected and concentrated to dryness under vac

15、uum. Then the dryness was dissolved into distilled water and purified taking the same approach. Finally, purity of Orcinol glucoside was 39%, Curculigoside A and curculigine A were 46%and 4%, respectively.10 compounds were isolated and identified from 10% and 30% ethanol solution, by D101 macroporou

16、s resin column, gel column, ODS reversed phase column, MCI column, preparative liquid.8 of them are phenolic compounds, others are furan compounds. We selected the 3 months old ovariectomized rat model to investigate the anti-osteoporosis effect of Curculigo orchioides and its phenolic glycosides. I

17、t was found that uterines of female rats after ovariectomy were atrophy, indicated that menopausal status has been caused. Serum alkaline phosphatase activity, U-Ca/Cr value increased; femoral bone mineral density and the activity of serum antioxidant enzymes decreased, while MDA content was increas

18、ed. Curculigo orchioides and different doses of benzoic acid esters of phenolic glycosides could improve bone density in ovariectomized rats, and in the higher and highest dose groups, it was significantly different from the OVX rats (P <0.01). Orcinol glucosid had no significant effect on increasin

19、g bone density in ovariectomized rats. This indicates that the effective fraction of anti-osteoporosis is benzoic acid esters of phenolic glycosides, such as Curculigoside, etc. These compounds decrease bone loss by inhibiting bone resorption. Metabolomics and serum antioxidant enzyme activity analy

20、sis showed that the phenolic glycosides from Curculigo orchioides by increasing the antioxidant capacity to improve bone metabolism and reduce bone loss.We investigated the estrogenic effects of the ethanol extract of Curculigo orchioides, including each alcohol elution part through D101 macroporous

21、 resin by analysising the body weight and uterus growth of 17 days old rats. The results indicate that ethanol extract of Curculigo orchioides and each elution part have no significant estrogenic effect. Then we used of estrogen receptor-positive cell model (MCF-7) investigated the effect of phenoli

22、c compounds:Curculigoside, curculigine A,2,6 - dimethoxy benzoic acid, Orcinol glucoside, Orcinol gentiobioside. We found estradiol (E2) promoted cell proliferation significantly, while the tested drugs had no significant effect on cell proliferation, We concluded that the phenolic compounds had no

23、estrogen-like effects.Ovariectomized rats were administrated orcinol phenolic glycosides and benzoic acid esters of phenolic glycosides for 12 weeks, the tissue morphology of estrogen receptor-positive organs breast, vagina and uterus did not change significantly compared to OVX rats, but the expres

24、sion of progesterone receptor, estrogen receptorαandβdecreased. These results suggest that orcinol phenolic glycosides and benzoic acid esters of phenolic glycosides had anti-estrogenic effect.In summary, we obtained the method of preparation phenolic glycosides from Curculigo orchioides, isolated a

25、nd identificated the major phenolic glycosides, clarified the material basis of its anti-osteoporosis and anti-estrogenic effects. By anti-osteoporosis and estrogen-like activity analysis, the phenolic glycosides from Curculigo orchioides reduce bone loss by increasing the antioxidant capacity and a

26、nti-estrogenic effect. The disadvantage is that the sudy in anti-estrogenic effect of phenolic glycosides from Curculigo orchioides is still not comprehensive enough. In-depth study, we should reveal the characteristics of its anti-estrogenic, and define its beneficial or detrimental effect on human

27、 body. 【關(guān)鍵詞】仙茅 酚苷 提取 純化 別離 去卵巢 骨質(zhì)疏松 抗雌激素樣作用 【采買全文】 1.3.9.9.38.8.4.8 1.3.8.1.13.7.2.1 同時(shí)提供論文寫作一對(duì)一輔導(dǎo)和論文發(fā)表效勞.保過包發(fā). 【說明】本文僅為中國學(xué)術(shù)文獻(xiàn)總庫合作提供，無涉版權(quán)。作者如有異議請(qǐng)與總庫或?qū)W校聯(lián)系。 【英文關(guān)鍵詞】Curculigo orchioides phenolic glycosides extraction purification separat

28、ion ovariectomy osteoporosis anti-estrogenic effect 【目錄】仙茅酚苷類成分抗骨質(zhì)疏松及雌激素樣作用研究 摘要 5-7 Abstract 7-8 縮略詞表 9-10 前言 10-12 第一章 仙茅酚苷類成分的制備及別離 12-32 第一節(jié) 正交試驗(yàn)法優(yōu)選仙茅酚苷類成分的提取方法 12-21 第二節(jié) 仙茅酚苷類成分的制備 21-25 第三節(jié) 仙茅酚苷類成分的別離鑒定 25-32 第二章 仙茅酚苷類成分對(duì)去卵巢骨質(zhì)疏松大鼠的作用

29、 32-45 1 材料 32-33 2 方法 33-34 3 結(jié)果 34-42 4 討論 42-45 第三章 仙茅酚苷類成分雌激素樣作用的研究 45-64 第一節(jié) 仙茅酚苷類成分對(duì)乳鼠子宮重量的影響 45-50 第二節(jié) 仙茅酚苷類成分對(duì)MCF-7細(xì)胞增殖的影響 50-54 第三節(jié) 仙茅酚苷類成分對(duì)去卵巢大鼠雌激素受體陽性器官的影響 54-64 第四章 研究總結(jié) 64-66 參考文獻(xiàn) 66-69 致謝 69-70 文獻(xiàn)綜述 70-80 參考文獻(xiàn) 77-80 作者簡(jiǎn)歷 80