CI 植物萃取物抑制甲基安非他命引發老鼠的活動力增加與前脈衝抑制缺損之研究

Abstract

本研究源自於本研究團隊的醫師成員於國立台灣大學附設醫院小兒科門診所 發現之個案研究。一位十三歲的病人患有長期抽動症狀，對於多種抗抽動藥物的 反應不佳，直到她自己喝了苦藍盤（Clerodendrum inerme）所打成的汁液，發現 其症狀有明顯地緩解，而且追蹤兩年發現，其肝、腎和血液的功能均正常。紋狀 體的多巴胺 (Striatal dopaminergic)的過度活化被認為在抽動症狀的產生扮演 重要角色，妥瑞氏症也在其中，所以本研究利用甲基安非他命來引發老鼠的過動 症狀當作模型，在開放空間測試（open field test）了一系列由苦藍盤葉萃取出 來的萃取物。結果發現苦藍盤的乙醇萃取物 (10~300 mg/kg, i.p.)可以劑量依存 性地降低甲基安非他命所造成的活動性增加，但在滾輪 (rotarod)測試和抓力測 試 (grip tests)則是沒有影響。接下來又篩了乙醇萃取物下的分層，包含水 (water)、丁醇 (butanol)、二氯甲烷 (dichloromethane)和正己烷 (n-hexane) 分層，結果發現主要的作用成分存在於二氯甲烷 (dichloromethane)和正己烷 (n-hexane)層，再以相同的模型測試，在二氯甲烷 (dichloromethane)層發現了 粗毛豚草素 (hispidulin)是主要的作用成分，並且觀察到粗毛豚草素除了會降低 甲基安非他命所引發的活動性增加之外，還會產生鎮靜的效果。粗毛豚草素已被 研究過是一種γ- 胺基丁酸A 型接受器異位性致效劑 ( GABAA receptor allosteric agonist)，但跟一般的benzodiazepine 不同的地方是他會促進含α6 次單元γ-胺基丁酸A 型接受器 (α6-subunit-containing GABAA receptors)，而 這個接受器只有表現在小腦的顆粒細胞上，而且主要的作用是前突觸的抑制作用。 妥瑞氏症病人也表現出感覺運動門控（sensorimotor gating）不良。因此本 研究利用甲基安非他命來引發前脈衝抑制不良，測試粗毛豚草素 (hispidulin)能 否逆轉甲基安非他命所造成的效果。結果發現，粗毛豚草素 (hispidulin) (10~50 iv mg/kg i.p.) 可以逆轉甲基安非他命引發之前脈衝抑制不良，但diazepam (1 ~10 mg/kg i.p.)則無法逆轉。 然後，我們在老鼠小腦雙側微量注射粗毛豚草素 (hispidulin) 10 nmole/ side 發現可以逆轉甲基安非他命引發之前脈衝抑制不良。接著，將含α6 次單元 γ-胺基丁酸A 型接受器致效劑 (α6-subunit-containing GABAA receptors) 、 含 α1 次單元γ- 胺基丁酸A 型接受器反轉致效劑 (inversed α1-subunit-containing GABAA receptors) Ro154513 和全γ-胺基丁酸A 型接受 器致效劑 ( All GABAA receptors containg subunit agonist) (包含 α6-subunit) 的loreclezole，小腦微量注射10 nmole/side 發現皆可逆轉甲基安非他命所造成 的前脈衝抑制不良。Ro154513、loreclezole、粗毛豚草素 (hispidulin)以小腦 微量注射10 nmole/side 的效果可以被furosemide (50 nmole i.cb.)所抑制， furosemide 為α6次單元γ-胺基丁酸A 型接受器 (α6-subunit-containing GABAA receptors)的抑制劑，這樣的結果顯示粗毛豚草素 (hispidulin)可能是透過促進 小腦內含α6 次單元γ-胺基丁酸A 型接受器 (α6-subunit-containing GABAA receptors)而達到促進前脈衝抑制的效果。 由於一些神經精神疾病會發生前脈衝抑制不良，包括妥瑞氏症、精神分裂症、 注意力不集中過動症、強迫症，粗毛豚草素 (hispidulin)能否改善這些疾病的前 脈衝抑制功能，還需要進一步探討。

This study was inspired by a case report of one physician member of our research group in the pediatric neurology clinic of National Taiwan University Hospital. We found a pediatric patient with chronic motor tic disorder refractory to multiple anti-tic therapies showed dramatic improvement and remission after self administration of the grounded leaf juice of Clerodendrum inerme (CI) with normal hepatic, renal and hematological functions examined after 2 years＇ herb administration. Striatal dopaminergic hyperactivity is believed to play an important role in motor tic disorders, of which Tourette syndrom one of the spectrum. We, therefore, using the mouse model of methamphetamine-induced hyperlocomotor activity in the open-field test, examined the effects of various extraction fractions from the CI leaves. The ethanol extract of CI (10-300 mg/kg, i.p.) dose-dependently reduced methamphetamine-induced hyperactivity but had no significant effect in the rotarod, except at higher doses, and grip tests per se. We further screened the sub-fractionations of the ethanol extract, including water, butanol, dichloromethane and n-hexane extracts, and found the active component(s) were present in the fractions of dichloromethane vi and n-hexane. Through the bioassay guided isolation, we found that hispidulin was one of the active costituents. In addition to reducing methamphetamine-induced hyperactivity, hispidulin also had hypnotic /sedative effect. Hispidulin has been proved to be a GABAA receptor allosteric agonist, but unlike benzodiazepine, was effective in α6- subunit-containing GABAA receptors. These receptors are exclusively expressed in the cerebellar granule cells and mediate the presynaptic inhibitory tone on these neurons. Patients with Tourette syndrome also manifest with sensorimotor gating deficit. We, therefore, examined if hispidulin could rescue methamphetamine-induced impairment of prepulse inhibition (PPI) of the startle response in mice, an animal model of sensorimotor gating. Hispidulin (10~50 mg/kg), but not diazepam (1~10 mg/kg), when given by intraperitoneal injection rescued methamphetamine-induced PPI impairment. Bilateral microininjection of hispiduline, but not diazepam, at 10 nmole /side also rescued rescued methamphetamine-induced PPI impairment. Futhermore, microinjection of Ro154513, an agonist α6-subunit-containing GABAA receptors and an inversed α1-subunit-containing GABAA receptors, and vii loreclezole, all the GABAA receptors containg subunits, including α6-subunit, also rescued methamphetamine-induced PPI impairment. The effects of Ro154513, Loreclezole, hispidulin administered by intra-cerebellar injection (10 nmole/side) were reversed by intracerebellar microinjected furosemide (50 nmole), These results demonstrate that hispidulin is the major effective constituent in CI leave extract in rescuing methamphetamine induced hyperlocomotion and PPI impairment. The latter effect might be mediated by theα6-subunit-containing GABAA receptors in the cerebellum. Several neuropsychiatric disorders, including TS, schizophrenia, attention deficit hyerreactivity disorder and obsessive compulsive disorder, also have impaired PPI. It remains to be further elucidated if hispidulin might also be beneficial in improving the sensorimotor gating problems in these diseases.