激黃體作用之中草藥研究
Studies on the Luteotropic Effect of Chinese Medicine Herbs
中文摘要
題目：激黃體作用之中草藥研究
第一部份 ：應用凍解牛黃體細胞篩選激黃體作用中草藥
中草藥廣泛的在生殖內分泌學的臨床上使用，然而其作用機轉方面則所知甚少。本研究應用凍 解牛黃體細胞探討中草藥對生殖內分泌學的影響。
以凍解牛黃體細胞培養系統和酵素免疫分析法分析孕酮，篩選51 種中草藥萃取物。於中草藥粗
萃液濃度1μg/ml 和 10μg/ml 下培養 4 小時，結果顯示下列中草藥具有激黃體效果：淫羊藿
（小薜科）、艾草、墨旱蓮（菊科）、絞股藍、栝樓根、栝樓（葫蘆科）、沙菀子、決明子（豆科）、
桑椹（桑椹科）、女貞子（木犀科）、石斛（蘭科）、蛇床子（繖形科）。而於濃度100μg/ml 時，
仙茅（石蒜科）、栝樓、銀杏（銀杏科）、桑椹、石斛、覆盆子（薔薇科）則具有抑制孕酮分泌的 作用。
於中草藥粗萃液濃度1μg/ml 和 10μg/ml 下培養 24 小時，結果顯示：黨參（桔梗科）、絞股藍 栝樓根、栝樓、銀杏、韭菜子（百合科）、女貞子、臺灣金線蓮（蘭科）、辣椒（茄科）、北沙參
（繖形科）能夠刺激孕酮之分泌；然而仙茅、淫羊藿、菟絲子（旋花科）、栝樓、鎖陽（鎖陽科）、
大蒜（百合科）、桑椹、石斛、覆盆子、枸杞子，於濃度100μg/ml 會抑制孕酮分泌。
藉由本研究，我們希望能發現一些具有激黃體作用的中草藥和提供臨床上使用的科學佐證。
第二部份 ： 絞股藍之激黃體成分的分離與鑑定
絞股藍Gynostemma pentaphyllum (Thunb.) Makino，屬於葫蘆科（Cucurbitaceae）
多年生攀緣性或匍匐狀藤本植物，廣佈於大陸、日本、韓國、及東南等地。絞股藍在醫學上的證實 是有包括抗氧化作用、增強免疫、抗癌、輻射保護、避免心血管疾病、降脂作用、抗潰瘍等作用。
由絞股藍分離純化之成分大約有90 種屬 dammarane 類的皂被確定，此類皂稱為 gypenosides (GP)，而其中的第 III、IV、VIII、XII、malonyl gypenoside III 和 VIII 皂和五 加科(Araliaceae)人參（P. ginseng）之人參皂Rb1、Rb3、Rd、F2、malonyl ginsenoside Rb1 和 Rd 完全相同；而 gypenoside-XVII 也和西洋參（P. quinquefolium），三七（P.
notoginseng）具相同之結構，因絞股藍和昂貴的人參有許多相似，因此絞股藍被認為是具有 潛力的新藥。
絞股藍以95％酒精萃取，以冷凍解凍牛黃體細胞培養系統作為激黃體效果成份的指引，絞股藍
酒精萃出物並經由水、己烷、正丁醇分配，其活性層為正丁醇層，將正丁醇層以管柱層析分析之。
結果顯示絞股藍 GpE 層、GpB 層及經由 Sephadex LH-20 層析得到三個活性層分別為 GpB1、GpB4、 GpB5。在 GpB1 部分對於激性腺作用強度為 GpB14> GpB13 >GpB15>
GpB12>GpB11；在 GpB4 部分其激性腺作用強度為 GpB46> GpB45> GpB43>
GpB42> GpB44 >GpB41；在 GpB5 部分其作用強度為 GpB52>GpB54> GpB53>
GpB51；由上顯示絞股藍有促性腺之因子，而其真正作用之成分仍然在純化中。
英文摘要
Studies on the luteotropic effect of Chinese medicine herbs
Part I： A screen test of Chinese medicine herbs for the luteotropic activity by using frozen -thawed bovine luteal cells
Chinese medicine herbs with a wide variety of clinical uses and relatively little knowledge on their actions mechanisms, has brought a great attention from the society and clinicians. Systematic study of Chinese medicine herbs on
reproduction endocrinology, this study was conducted to investigate this purpose.
By using frozen-thawed bovine luteal cells and EIA (Enzyme Immuno Assay) assay the progesterone. We have screened 51 Chinese medicine herb extracts. At the concentration of 1μg/ml and 10μg/ml with 4 hours incubation the following drugs had stimulation effects：Epimedium macroanthum (Berberidaceae), Astemisa argyi, Eclipta prostrata (Compositae), Gynostemma pentaphyllum, Trichosnthes kirilowii Maxin, Trichosnthes kirilowii Maximowicz (Cucurbitaceae), Astragalus complantatus, Cassia tora (Leguminosae), Morus alba (Moraceae), Ligustrum lucidum (Oleaceae), Dendrobium nobile (Orchidaceae), Cnidium monnieru
(Umbelliferae). At the concentration of 100μg/ml those inhibited the progesterone production：Cucrligo orchioides( Amaryllidacea), Trichosnthes kirilowii Maxin, Ginkgo biloba (Ginkgoaceae), Morus alba, Dendrobium nobile, Rubus chingii (Rosceae).
At the concentration of 1μg/ml and 10μg/ml with 24-hours incubation, Codonopsis pilpsila (Campanulaceae), Gynostemma pentaphyllum, Trichosnthes kirilowii Maximowicz, Ginkgo biloba, Allium tubeeosum (Liliaceae), Ligustrum lucidum (Oleaceae), Anoectochilus formosanus (Orchidaceae), Capsicum annuum (Solanaceae), Glehnia lottoralis (Umbelliferae) could stimulate progesterone production. However, Cucrligo orchioides, Epimedium macroanthum, Cuscuta officinalis (Convolvulaceae), Trichosnthes kirilowii Maxin, Cynomorium
songaricum(Cynomoriaveae), Astragalus complantatus, Cassia tora
(Leguminosae), Allium sativum (Liliaceae), Morus alba, Dendrobium nobile, Rubus chingii and Lycium chinese inhibit progesterone production at the concentration of 100μg/ml with 24-hours incubation
By doing so, we may provide scientific evidence of the luteotropic effects of some Chinese medicine herbs and may find some herbs for the new clinical use.
Part Ⅱ ： Isolation and characterization of luteotropic compounds of Gynostemma pentaphyllum (Thunb.) Makino
Gynosyemma pentaphyllum (Thunb.) Makino (Cucurbitaceae), a perennial creeping herb is widely distributed in China, Japan, Korea and Southeast Asia.
Gynostemma pentaphyllum (GP) are widely used in China for medicinal purposes.
Extracts from these plants have been reported to have a wide range of health benefits, including antioxidant effects, enhanced immunocompetence, anti- carraicogenic activity, radiation protective, prevention of cardiovascular disease, lipid-lowering, antiulcer and others.
Phytochemical studies of this plant have been identified about 90 dammarane -type glycosides (called gypenoside) closely to the ginseng saponins. Indeed, gypenosides III, IV, VIII XII and malonylgypenoside III and VIII are identical to ginsenosides Rb1, Rb3, Rd, F2, and malonylginsenoside Rb1 and Rd found in the roots of Panax ginseng C.A, Meyer (Araliaceae). Furthermore, gypenoside XVII was also isolated from P. quinopuefolium.and P. japoncius. Because of this similarity to the expensive ginseng root, G. pentaphyllum has been considering as a potential new drug.
By using a frozen-thawed bovine luteal cells system to direct luteotropic fractionation of G. pentaphyllum. The alcoholic extracts were partitioned with water, n-hexane and 1-butanol subsequently. The 1-butanol layer was most activating layer. Then for further investigation 1-butanol layer was
chromatographed with Sephadex LH-20 and MCI gel.
We found that the extracts of EtOH (GpE), 1-butanol layer (GpB) and Sephadex LH-20 chromatographed (GpB1, GpB4, and GpB5) had stronger gonadrotropic activities. The GpB11 fraction stimulation potency were GpB14> GpB13 >GpB15>
GpB12>GpB11. In the GpB4 fraction stimulation potency were GpB46> GpB45>
GpB43> GpB42> GpB44 >GpB41 and the GpB5 fraction were GpB52>GpB54>
GpB53> GpB51. The further purification is carrying out for isolating active compounds. These data suggest that G. pentaphyllum may have some luteotropic-stimulating factor.
