具激性腺素效用中草藥之研究

Abstract

永續的繁殖管理在人類及其他動物中均是被受關注的課題，人類希望未來人口數降低而禽畜與野生動物則增加。快速的都市化消耗了天然的緩衝系統，人類不健康的生活型態，造成繁殖上的問題。而動物世界中，密集的飼養或棲息地的喪失，也傷害了其繁殖潛能。雖然人類嘗試用現代科技來解決，但由於人工合成內泌素或藥物的誤用，使此議題仍持續受重視。在此同時也逐漸引入永續醫學(Sustainable Medicine)方法例如草藥與針灸，也被認為是一種互補與可擇醫學(Complementary and Alternative Medicine)。 我們初步研究發現，一些具有植物性動情素與類固醇生成能力之草藥，我們利用體外細胞培養系統來篩選這些具有類固醇生成作用的草藥，主要使用牛黃體、豬腎上腺及老鼠睪丸的間質細胞來進行，顯示若干草藥萃取物對孕酮、皮質醇及睪固酮具有刺激、抑制或無作用等不同的效用。繼之，利用不同的化學同功劑或拮抗劑來瞭解其作用機制，其中我們選擇以刺蒺藜(Tribulus terrestris L)來做深入之探討，同時也以餵飼大鼠來觀察其對性行為與睪固酮的影響。我們發現刺蒺藜萃取物能增加睪固酮的生產，同時來自喜馬拉雅及保加利亞者有較強的效果，而臺灣出產者無任何效用。再者，刺蒺藜萃取物能維持由過氧化氫與加熱所造成間質細胞緊迫的睪固酮含量。 利用來自保加利亞的刺蒺藜進行活體試驗，以觀察對年輕(10週齡)及中年(48週齡)雄Long-Evans大鼠性行為的影響。並記錄其mount latency (ML)、intromission latency (IL)、intromission frequency (IF)、ejaculation latency (EL)、ejaculation frequency (EF) 及 post-ejaculatory interval (PEI)等。結果發現年輕大鼠之性能力較佳且血清與糞便睪固酮較高。在5 mg刺蒺藜處理組，除IL項目之外，其餘之表現均比10 mg者為佳。在中年鼠處理組，其EF與IF表現比對照組為佳。另外，試驗期間在不同組間之體重、器官重亦無顯著差異。糞便與血清中睪固酮之趨勢相似，老年者刺蒺藜處理組之睪固酮含量教對照組高。但年輕鼠血清中睪固酮無論刺蒺藜處理與否均無差異；而糞便睪固酮在年輕與老年大鼠中則無差異。在年輕與老年大鼠，來自不同動物的睪丸培養液其基礎量無差異；而在oLH刺激的細胞，顯示刺蒺藜處理顯著增加睪固酮含量。本研究提出了刺蒺藜增加性能力的證據，也暗示糞便類固醇的分析，可作為動物行為研究的一種非侵入方法。 經由本研究顯示具有類固醇生成與增加性能力的草藥，或許可作為改善人類與動物之繁殖的一種永續方法。然而仍須進一步深入研究，以瞭解其在藥理學及毒物學方面的作用。

Sustainable management of reproduction is an area of major concern in humans and animals. Ironic as it is for the moment in the human plan for future they envision reduction in human numbers but want the domestic and wild animals to increase. Rapid urbanization, depleting natural buffering system, unhealthy lifestyle in humans are all contributing to poor prospect of enjoying reproductive bliss to its most. While, for the part of our cousins in the animal world, intensification in raising or habitat loss is ruining their prolific potentiality. Equipped with technological and scientific knowledge humans are pursuing for solutions. Debatable but lets hope unintended misuse and misguided information about synthetic approaches like in hormones and other drugs do leaves room for concerns. Suitable and softer approaches as envisioned in the proposed Sustainable Medicine (SM) looks promising and worthy of gradual incorporation. Herbs and acupuncture do provide one such option and has been in use and promoted as Complementary and Alternative Medicine (CAM). Herbs with phytoestrogenic and steroidogenic capability are now subjects of detail investigation as preliminary findings reports encouraging results. To seek evidences in scientific platform, scores of medicinal herbs were screened for their potential effect on steroidogenesis in established in vitro cell culture system. Bovine luteal, porcine adrenal and small lab animals’ interstitial and Leydig cells obtained from the testis was used to conduct the experiment. Several medicinal herbs extracts showed various effects (stimulatory, inhibitory or no effect) on three main steroids investigated namely progesterone, cortisol and testosterone. In further detail study to understand the mechanism involved, signal transduction was trace at intracellular level employing several chemical agonists and antagonists. Among the list of several medicinal herbs, Tribulus terrestris L was chosen for in depth analysis in its testosterone synthesizing ability from isolated mice interstitial cells. In vivo experiment was carried out with protodioscin fortified Tribulus terrestris L feeding in young and middle-aged rats to observe their sexual behavior and their level of testosterone in serum and feces. HPLC-EC was use to measure the level of nordarenaline, adrenaline, dopamine from the medial preoptic area (MPOA), bed nucleus of stria terminalis (BSTMA) and mediam amygdal (MEA) pinced out section of the male rat brain parts from the different treatment in the young and middle-aged rats. We found that TT extract can increase testosterone production and by employing signal transduction tracing studies, we found the effect to be mild gonadotropin-like in action. On our investigation with different sources of TT, the material from Himalayas and commercial extract obtained from Bulgaria had stronger potentiality, while the material obtained from Taiwan did not show any significant effect. Furthermore, in our experiment TT extract countered the hydrogen peroxide (H2O2) and heating induced stress in the interstitial cells in maintaining the testosterone production ability. The study do establish that TT can in-fact contribute to the synthesis of testosterone by the Leydig cells in the interstitial tissue of the testes and one possible mechanism is by countering the reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2), which are detrimental to steroidogenic acute regulatory (StAR) protein. Furthermore, Bulgarian origin TT (TT-B) at higher doses cross-reacted with the enzyme immunoassay system, which demands further examination for possible presence of exogenous steroids or structural similarity of TT saponins to testosterone. TT-B was subjected to further in vivo experiment to understand if its administration had any impact on the sexual behavior of young (10 weeks) and middle-aged (48 weeks) male rats. Male Long–Evans rats used as experimental subjects and they were place in individual 10-gallon glass aquariums with a sexually receptive female for a test of sexual behavior. Ovariectomized females implanted with 5 mm long estradiol-filled sialistic capsules were brought into behavioral estrous by an injection of 500 μg progesterone 4 h prior to testing. The mount latency (ML), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), ejaculation frequency (EF) and post-ejaculatory interval (PEI) were observed and recorded in young and middle-aged rats pre and post treatment with five and ten mg/Kg body weight TT-B for four weeks. The finding showed that there was a clear difference in the sexual behavior of the young and middle-aged rats, with performances and T level in serum and feces being remarkably higher in the former. The sexual behavior observation of the old rats showed time and treatment effects for EF, IF PEI, IL in young and middle-aged rats at varying degree of significant differences. In the TT treatment groups, 5 mg showed better performance compared to the higher dose of 10 mg for all but IL parameters. In the middle-aged group of rats EF and IF was significantly improved in the TT treated group over the control at level of p<0.05 and even p<0.01. The finding also revels that there was no significant differences among the different groups in their live weight during the experiment period and wet organ weight in both young and middle-aged rats. Fecal and serum level of testosterone showed some resemblance in reflection of relative level, with treatment group in old rats showing significantly increased level over the control. However, in young rats, there was no difference in the serum testosterone level in either group, while fecal testosterone level was not significantly different in both young and old rats. The testis culture from different animals showed no difference in the basal level, but oLH stimulated cells showed significant (*p<0.05) increase in TT treated groups both in young and old rats. This study hence adds positive evidence to the aphrodisiac effect of TT; it also reports the possibility of employing fecal steroids assay in routine behavioral study with non-invasive approach. In the process of making EIA diagnostic procedure more integrated, I have assisted fellow students in establishing standard protocol for saliva steroids assay and developing biochips for faster and efficient assay. However, there are still some shortcomings to overcome, and hope EIA system’s integration with biotechnological advancement, linked with advancing techniques of biosensors will enable scientific pursuits like mine more viable. Resources are keys, financing for such projects that aims to understand the concept of scientific incorporation for evidence seeking in traditional knowledge and practices needs encouragements and initiations. Key is to achieve sustainable solutions to human progress in harmony with its surrounding by learning to respect it. Through this work, my intention is to highlight that herbs with steroidogenic and aphrodisiac activities can serve as potential candidates for improving reproduction in sustainable way both for use in humans and animals. However, more and deeper research and clear understanding on the pharmacological and toxicological interactions is necessary before coming to a final strong conclusion.