CN101991580B - 以羊毛甾烷及茯苓萃取物治疗糖尿病的用途 - Google Patents
以羊毛甾烷及茯苓萃取物治疗糖尿病的用途 Download PDFInfo
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- CN101991580B CN101991580B CN2009101681199A CN200910168119A CN101991580B CN 101991580 B CN101991580 B CN 101991580B CN 2009101681199 A CN2009101681199 A CN 2009101681199A CN 200910168119 A CN200910168119 A CN 200910168119A CN 101991580 B CN101991580 B CN 101991580B
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Abstract
本发明提供一种用于治疗糖尿病的药物组合物,该组合物包含羊毛甾烷化合物作为有效成分。此羊毛甾烷化合物的合适来源为茯苓萃取物,其包含1-60重量%的羊毛甾烷化合物且实质上不含开环羊毛甾烷化合物。该萃取物萃取自多孔菌科植物茯苓菌(Poria cocos(Schw)Wolf)的代谢产物、菌丝或发酵产物。
Description
技术领域
本发明涉及一种以茯苓萃取物治疗糖尿病的用途,尤其涉及一种以萃取自茯苓的化合物治疗因血中胰岛素不足所引起的糖尿病的用途发明。
背景技术
糖尿病为富裕国家成人(尤指老年人)常见的慢性病。糖尿病人主要可分为两种:第一型糖尿病,或称为胰岛素依赖型糖尿病,其病因是免疫反应造成胰脏的β-细胞破坏而无法生产胰岛素,血中无胰岛素因此此种型病人必需借助于注射胰岛素补充才能治疗糖尿病;第二型糖尿病,或称为非胰岛素依赖型糖尿病,其病因不明,但是遗传基因问题为重要因素之一,另外生活方式影响(如肥胖)也是重要因素之一。
大约公元500年,唐朝医学书籍《备急千金要方》记载了用于治疗的糖尿病的多种复方,其中某些复方含有茯苓。2002年日本研究人员M.Sato发表茯苓皮部分的氢化去氢松苓酸(Dehydrotrametenolic acid)能够应用在第二型糖尿病(Biol.Pharm.Bull.2002,25(1),81-86)的治疗中,其作用原理是通过增加胰岛素敏感度(即减少人体对胰岛素抗药性)而达到治疗目的。但是从其体外脂肪细胞试验知道氢化去氢松苓酸(Dehydrotrametenolic acid)的有效浓度为10-5M,及从体内小老鼠动物实验的结果得知氢化去氢松苓酸(Dehydrotrametenolic acid)的有效剂量为110mg/kg,显然应用在人体其有效剂量至少高达大于700mg以上。700mg的有效剂量相当于目前临床使用用药剂量的10倍以上,因此发展成临床用药会造成困难。
发明内容
本发明的一个目的在于,本发明提供一种使用具有下列化学式(I)的羊毛甾烷或其药学上可接受的盐作为有效成分在制备用于治疗哺乳类动物因血中胰岛素不足所引起的糖尿病药物中的用途,
式中R1为H或CH3;R2为OCOCH3、=O或OH;R3为H或OH;R4为-C(=CH2)-C(CH3)2Ra,其中,Ra为H、OH或-CH=C(CH3)-Rb,其中,Rb为CH3或CH2OH;R5为H或OH;以及R6为CH3或CH2OH。
优选地,该羊毛甾烷(I)具有下列化学式:
优选地,该羊毛甾烷(I)具有下列化学式:
优选地,该药物为注射剂型。
优选地,该药物为口服剂型。
优选地,该糖尿病为第一型糖尿病。
优选地,该糖尿病为第二型糖尿病。
优选地,该哺乳类动物为人类。
本发明的另一个目的在于,本发明提供一种使用茯苓萃取物作为有效成分在制备用于治疗哺乳类动物因血中胰岛素不足所引起的糖尿病药物中的用途,其中,该茯苓萃取物包含1-60重量%的化学式(I)的羊毛甾烷(I)且实质上不含开环羊毛甾烷。
优选地,该茯苓萃取物包含5-35重量%的羊毛甾烷(I)。
优选地,该羊毛甾烷(I)具有下列化学式:
优选地,该药物为口服剂型。
优选地,该糖尿病为第一型糖尿病。
优选地,该糖尿病为第二型糖尿病。
优选地,该哺乳类动物为人类。
在本发明的一个实施方案中,所述茯苓萃取物为由茯苓分离纯化所得的羊毛甾烷型三萜类化合物,该化合物的结构如下:
R1 | 代号 | |
茯苓酸 | COCH3 | PA |
块苓酸 | H | TA |
本发明证明,从茯苓分离出来的有效成分能够发挥同胰岛素一样功能:(1)增加葡萄糖转运蛋白(GLUT4)的基因表达(mRNA);(2)葡萄糖转运蛋白(GLUT4)制造;(3)能够将转运蛋白(GLUT4)从细胞内转位(translocatin)到脂肪(或肌肉)细胞膜上;(4)葡萄糖转运蛋白能够将细胞外葡萄糖运送至细胞内;以及(5)同胰岛素功能一样能够制造三酸甘油脂储存在细胞内。故茯苓有效成分成功地扮演了胰岛素的角色,可将血糖运送至细胞内而降低血糖,可运用在第一型糖尿病及因血中胰岛素不足所引起的第二型糖尿病的治疗中。另外,最重要的是从脂肪体外试验说明在0.01μM浓度下就能产生葡萄糖吸收作用,相较于上述日学者Sato研究需要10-5M才能产生作用,二者之间剂量差1000倍。
附图说明
以下,结合附图来详细说明本发明的实施例,其中:
图1示出由茯苓分离纯化所得之羊毛甾烷型三萜类化合物的结构。
图2A显示茯苓萃取物促进3T3-L1脂肪细胞糖吸收。图2B显示胰岛素(0.1μM)作用30分钟及纯化的三萜类化合物于0.01μM剂量二小时反加糖测试下促进糖吸收。数据以平均值±标准误差(n=6)表示。*p<0.05, **p<0.01与对照组(不给药)比较。
图3A显示茯苓酸(PA)于0.01μM剂量下给予二小时吸收速率最高。图3B显示给予不同剂量的茯苓酸(pachymic acid,PA)随剂量增加,糖吸收有增加趋势。PA作用2小时后,再加入糖测验,数据以平均值±标准误差(n=6)表达,*p<0.05,**p<0.01与对照组(不给药)比较
图4显示PA促进糖吸收仅作用于分化成熟的脂肪细胞。如将未成熟与成熟脂肪细胞加入PA处理2小时外,另外加入或不加入葡萄糖转运蛋白抑制剂(PT)则显著抑制二种细胞的糖吸收作用也抑制PA的糖吸收促进作用。数据以平均值±标准误差(n=6)表示。*p<0.05,**p<0.01与对照组比较。
图5A显示PA不具有促进葡萄糖转运体1(GLUT1)转运蛋白表达的能力。而图5B显示PA具有促进葡萄糖转运体4(GLUT4)转运蛋白表达 的能力。分化成熟细胞以不同浓度PA处理24小时,然后分析GLUT1及GLUT4转运蛋白,数据以平均值±标准误差(n=3)表示。*p<0.05,**p<0.01与对照组(不给药)作比较。
图6显示PA具有促进GLUT4mRNA表达的能力,其中分化成熟细胞分别以胰岛素(0.1μM)及0.01μM PA处理24小时,然后分析GLUT1及GLUT4mRNA的表达,数据以平均值±标准误差(n=3)表示。*p<0.05, **p<0.01与对照组作比较。
图7显示对抑制GLUT4mRNA的表达的脂肪细胞PA不具促进糖吸收的能力。成熟脂肪细胞与抑制GLUT4mRNA表达的脂肪细胞(shG4-30)二者以不同浓度PA及胰岛素处理,然后分析GLUT4转运蛋白(图7A)及其糖吸收能力(图7B),数据以平均值±标准误差(n=6)表示。*p<0.05, **p<0.01与对照组作比较。
图8A通过分析由离心方法分离的不同细胞膜层的GLUT4转运蛋白,显示PA具有促进葡萄糖转运体4转运蛋白从细胞内胞器转运到细胞膜的能力。图8B以荧光分析完整细胞的细胞膜层的GLUT4转运蛋白,显示PA具有促进GLUT4从细胞内胞器转运到细胞膜的能力。数据以平均值±标准误差(n=3)(图8A),(n=6)(图8B)表示。*p<0.05,**p<0.01与对照组(不给药)作比较。
图9显示PA具有促进脂肪细胞三酸甘油酯合成及抑制脂肪分解(产物甘油)产生的能力。成熟的脂肪细胞以胰岛素(0.1μM)及不同浓度PA处理24小时,数据以平均值±标准误差(n=6)表示。*p<0.05,**p<0.01与对照组作比较。
具体实施方式
本发明所揭示的从茯苓制备具有治疗因血中胰岛素不足所引起的糖尿病的有效成分的一种合适方法例如为EP 1535619A1所揭示的方法,包括利用传统萃取法萃取茯苓得到一粗萃取物,再经由层析法,分成极性小的羊毛甾烷(lanostane)类部位(以二氯甲烷∶甲醇(96∶4)为洗脱液)和极性大的开环羊毛甾烷(secolanostane)类部位(以二氯甲烷∶甲醇(90∶10或0∶100)为洗脱液),其中,利用硅胶薄层层析法,显示出羊毛甾烷(lanostane)类部位的所在位置,即展开溶媒为二氯甲烷-甲醇(96∶4)时,层析值(Rf)为≥0.1;至于开环羊毛甾烷(secolanostane)类成分, 则层析值小于0.1。用硅胶管柱层析法可进一步分离该羊毛甾烷类部位,其中洗脱液使用二氯甲烷∶甲醇(97∶3至95∶5),分离出数种羊毛甾烷(lanostane)类化合物。
下面结合实施例对本发明做进一步详细的描述,但不以此限制本发明。
实施例1
云南产茯苓26公斤,以260升含75%的酒精,加热萃取三次,合并酒精萃取液,经减压浓缩后可得225,2克萃取物。萃取物经定量分析知道每克萃取物含有76.27毫克羊毛甾烷,其中,K1(茯苓酸(Pachymic acid))33.4亳克,K1-1(去氢茯苓酸(dehydropachymic acid))9.59亳克,K2-1(块苓酸(tumulosic acid))19.01毫克,K2-2(去氢块苓酸(dehydrotumulosic acid))6.75亳克,K3(多孔覃酸C(poylporenic acidC))5.06mg,K4(3-表氢块苓酸(3-epidehydrotumulosic acid))2.46毫克。
实施例2
实施例1的酒精萃取物125克以1.3升二氯甲烷萃取6次,合并二氯甲烷萃取液,经浓缩后可得22.26克萃取物。以95%的热酒精溶解二氯甲烷萃取物,放冷后过滤不溶物,滤液加入少量水直到酒精含量为45%为止,此时会有沉淀产生,用离心方式获取沉淀物,可得17.4克沉淀物。经定量分析可知每克沉淀物含有264.78毫克羊毛甾烷,其中,K1-1159.7毫克,K1-256.96毫克,K2-124.43毫克,K2-28.8毫克,K39.84毫克,K45.05毫克。该沉淀物经硅胶薄层层析法检测证明其不含有开环羊毛甾烷。
实施例3
取茯苓药材100公斤,以800公斤水煮沸3小时后,静置冷却至50℃,以5N NaOH调节溶液至pH=11,再搅拌溶液3小时。接着以离心机分离液体和固体,固体再加入800公斤水,同上述方法,以NaOH调至pH=11、搅拌和离心机分离,去掉固体。合并两次液体,在50℃将液体真空浓缩至100公斤溶液,再加入3N HCl至pH=6.5,产生沉淀物。分离出该沉淀物,再以40L H2O清洗,接着以离心机分离出沉淀物,加入8L水喷雾干燥(spray dry),得到约380g粉末。再以4L的酒精萃取该粉末三 次,合并萃取液并浓缩可得238.9克酒精萃取物。该萃取物经硅胶薄层层析法(TLC)检测证明其不含有开环羊毛甾烷。该萃取物再经过HPLC分离,每克该萃取物可得主成分为K2214mg、K323mg、K424mg及少量成分K14.52mg,即萃取物每克约含265mg羊毛甾烷。
或以4L的50%酒***溶液萃取该粉末,去除50%的酒***溶液部分,收取不溶的粉末,重复三次可得245.7克的50%酒***溶液不溶物,经TLC法检测显示该不溶物不含开环羊毛甾烷,再经过HPLC纯化分离该不溶物,每克可得主成分为K2214mg、K323mg、K424mg及少量成分K14.52mg,即萃取物每克约含261mg羊毛甾烷。
实施例4
以云南产茯苓30公斤,磨成粉后,利用120L酒精(浓度95%)萃取24小时,并过滤分离。再重复前述萃取及固液分离三次。合并滤液,并将其浓缩后得干燥萃取物265.2克。再利用两相萃取剂(己烷∶95%甲醇=1∶1)对该干燥萃取物进行分配萃取。取出甲醇层并将其浓缩后得到干燥固体246.9克。利用硅胶管柱层析对该干燥固体进行分离,该硅胶管柱填充有该干燥固体重量10-40倍的硅胶,购自Merck公司,Silica gel 60,70-230mesh。以二氯甲烷/甲醇混合液作为洗脱剂(eluent),依次以96∶4、90∶10、0∶100比例的混合液进行洗脱,洗脱液(eluate)以硅胶薄层层析法(Thin Layer Chromatography)(紫外光灯及碘作检测,展开液为二氯甲烷∶甲醇=96∶4)检测成分,将相同成分合并。
以二氯甲烷-甲醇(96∶4)混合液进行硅胶管柱层析,可得到本发明的茯苓萃取物的PCM部分78克。PCM部分依上述硅胶薄层层析法可明显看到6个迹点。以二氯甲烷∶甲醇(90∶10)及(0∶100)洗脱液层析合并可得到PCW部分168克。
PCM部分进一步以二氯甲烷∶甲醇(96.5∶3.5)作为洗脱剂进行硅胶管柱层析(同上述硅胶管柱),进一步分离可得纯化的羊毛甾烷(lanostane)类成分K1(K1-1及K1-2)、K2(K2-1及K2-2)、K3、K4、K4a、K4b、K5、K6a及K6b。详细分离步骤及鉴定分析数据请参见EP 1535619A1。
上述K1至K6b化合物,其结构如下:
从PCM部分分离出来羊毛甾烷化合物K1至K6b的产量如下表所示。PCM部分含有约15重量%的羊毛甾烷化合物K1至K6b。
K1 | K2 | K3 | K4 | K4a | K4b | K5 | K6a | K6b |
3.0g | 6.2g | 1.93g | 0.55g | 66mg | 86.8mg | 47.6mg | 21.4mg | 90.7mg |
实施例5:胶囊制备
依下列组成制备含有实施例4所制得的茯苓萃取物PCM成份的胶囊:
成份 | 每胶囊 | 每30,000胶囊 |
以实施例4的方法制备的茯苓萃取物 PCM(含约15wt%的K1-K6化合物) | 11.2mg | 336.0g |
硅铝酸钠(Sodium silicoaluminate) | 5.0mg | 150.0g |
马铃薯淀粉(Starch Potato) | 378.8mg | 11,364.0g |
硬脂酸镁(Mangensium Sterate) | 5.0mg | 150.0g |
小计 | 400mg | 12,000.0g |
将茯苓萃取物PCM与硅铝酸钠分别以#80目(mesh)筛网过筛,马铃薯淀粉以#60目筛网过筛,硬脂酸镁以#40目筛网过筛后,置入混合机搅拌均匀,接着填充入1号空胶囊,每颗胶囊含有约1.68mg(0.42wt%)的有效成份K1-K6。
实施例6茯苓三萜类化合物预防及治疗第一型糖尿病试验
进行下列细胞实验的茯苓萃取物由实施例2制得者或示于图1的纯化合物。它们被溶解在酒精:DMSO(9∶1)的溶媒中,所得溶液加入到培养盘上,其中每一个孔(well)仅加入最后体积的千分之一。
1.脂肪细胞培养
3T3-L1为一种老鼠前脂肪细胞,型态为纺缍状,当加入诱导剂培养2-3天后,可以观察到细胞型态转为圆型,而且细胞内有油滴累积,随着分化天数增加,会分化的越完全。分化前的细胞,主要的葡萄糖转运蛋白为GLUT1,而分化后的细胞,则主要作用的葡萄糖转运蛋白为GLUT4, 如细胞膜上的GLUT4蛋白数目愈多,血液中葡萄糖穿越细胞膜被细胞吸收的速度与量也愈大,血糖下降速度越快。此3T3-L1脂肪细胞内具有完整的胰岛素活化葡萄糖吸收的***,可以进行糖类代谢与胰岛素信息路径的研究,另外也可以观察完整的脂质新生调节过程,因此分化的3T3-L1脂肪细胞为一具代表性且应用广泛的模式细胞株,由于人体组织内真正的脂肪细胞难以继代培养,故学术界一般使用此模式进行各项试验及评估。
1.1脂肪细胞培养
3T3-L 1细胞于DMEM(Dulbecco′s minimal essential medium)及37℃培养箱(5%二氧化碳,95%空气)中进行培养,向该DMEM添加10%成牛血清(calf serum)、100IU/mL青霉素、100μg/mL链霉素及1%非必需性胺基酸。待细胞完全长满后,加入含脂肪细胞分化促进剂[含0.5mM 3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthane,IBMX),1μM皮质类固醇激素(dexamethasone),10μg/mL胰岛素]的10%FBS/DMEM培养液培养2天,更换成含有10μg/mL胰岛素的10%FBS/DMEM培养液中2天后,再以每2天更换无胰岛素培养液依次进行培养4-6天。此时3T3-L1细胞将近90%形成脂肪细胞型态(adipocyte phenotype)后即可进行试验。试验进行前先以PBS溶液清洗3T3-L1细胞,再以无血清及无胰岛素的0.2%BSA/DMEM培养液培养过夜,以去除血清及胰岛素的干扰。
1.2抑制葡萄糖转运体4基因表达(mRNA)实验的脂肪细胞培养
利用携带抑制葡萄糖转运体4核醣核酸的病毒载体(TRCN0000043630shRNA,中央研究院基因体研究中心,台湾)感染3T3-L1前脂肪细胞(shG4-30),以建立长期性抑制葡萄糖转运体4基因表达,并以此株细胞分化后的脂肪细胞进行试验。
1.3检测葡萄糖转运体4转运蛋白质转位实验的脂肪细胞培养
将带有流行感冒病毒蛋白HA标志的葡萄糖转运体4(HA-GLUT4-GFP,Timothy E.McGraw赠送,美国纽约Well Gornell医学院)载体以Lipofectamine 2000(Invitrogen,CA,USA)转染到3T3-L1前脂肪细胞,并利用G418筛检出持续表达带有流行感冒病毒蛋白HA标志的葡萄糖转运体4转运蛋白的脂肪细胞株,分化成脂肪细胞后进行葡萄糖转运体4转运蛋白转位试验评估。
2.2-脱氧葡萄糖吸收评估
茯苓三萜类化合物促进3T3-L1脂肪细胞对于葡萄糖吸收的测试中, 将3T3-L1前脂肪细胞于6-孔培养盘上培养,待细胞长满以脂肪细胞分化促进剂进行分化刺激,待7-12天3T3-L1细胞成熟分化成脂肪细胞后,可用以进行葡萄糖吸收的测试。将前述脂肪细胞先置于无血清培养液(含0.2%BSA/DMEM)过夜后,以含不同浓度的茯苓三萜类化合物的无血清细胞培养液,培养2-6小时后,用PBS溶液洗涤一次,改换以KRP缓冲液(20mM HEPES,137mM NaCl,4.7mM KCl,1.2mM MgSO4,1.2mMKH2PO4,2.5mM CaCl2和2mM丙酮酸盐(pyruvate),pH 7.4及0.2%BSA)于37℃培养3小时后,再加入0.2μCi/mL的[14C]2-脱氧葡萄糖[2-deoxy-D-[14C]-glucose(2-DG,Amersham Biosciences,Little Chalfont,Bucks,U.K.)]和无放射性0.1mM 2DG的0.2m1葡萄糖缓冲液来取代KRP缓冲液以开始葡萄糖的吸收实验。5分钟后移出细胞并以PBS清洗来终止葡萄糖摄取。以0.2mL的0.2%SDS中溶解细胞并将10μL的细胞洗脱液转置至含有过滤底盘的UniFilter盘中(Perkim-Elmer,Wellesley,MA,USA)于37℃真空烘箱中干燥,并将在每孔中加入30μL的计数溶液,利用微盘液体闪烁计数器(TopCount,Packard NXT,Packard BioScience Company,Meriden,CT,USA)分析。计算出累积在细胞内的葡萄糖含量,并除以蛋白质浓度,所得摄取速率系以每分钟每毫克细胞蛋白质的纳摩尔葡萄糖(nmol/min/mg)来表示。蛋白质浓度可利用标准二辛可宁酸(Bicinchoninicacid;BCA)试剂(Pierce,Rockford,IL,USA)分析来测定。加入0.2μCi的L-[14C]-葡萄糖以测量非特定葡萄糖的摄取,并与每个测定值中相减,而得到特定葡萄糖摄取量。观察3T3-L1脂肪细胞在不同浓度的茯苓三萜类化合物的作用下对葡萄糖的吸收是否有所影响。
3.对葡萄糖转运体1及4(GLUT1 & 4)转运蛋白的评估
在茯苓三萜类化合物促进3T3-L1脂肪细胞葡萄糖转运体表达的测试中,同上述说明分化成熟的3T3-L1脂肪细胞以无血清细胞培养液培养过夜后,以含不同浓度茯苓三萜类化合物的无血清细胞培养液培养24小时,再以PBS溶液清洗,接着以0.2ml溶解细胞液[1%NP-40,150mM NaCl,0.1%SDS,50mM Tris-HCl pH 7.6,10mM EDTA,0.5%脱氧胆酸盐(deoxycholate),1mM PMSF,1mM Na3VO4,10mM NaF,10mM β-磷酸甘油酯(glycerophosphate),10μg/mL蛋白酵素抑制剂和磷酸酯酶(phosphatase)抑制剂]作用30分钟(4℃温度下)。每个样品经SDS-10%聚丙烯酰胺(polyacrylamide)电泳分离,转移至PVDF膜(Millipore,Bedford, MA,USA)。利用西方点墨法以专一性抗体,葡萄糖转运体1抗体(GLUT1,Abcam,Cambridge,MA),葡萄糖转运体4抗体(GLUT4,R&D systems,Minneapolis,MN),及β-肌动蛋白抗体(β-Actin,Chemicon,Temecula,CA,USA),来观察3T3-L1脂肪细胞在不同浓度的茯苓三萜类化合物的作用下对葡萄糖转运体蛋白质表达是否有影响。每个样品(蛋白)以化学冷光处理,再暴露在X光片,再以软件分析含量。
4.葡萄糖转运体1及4(GLUT1 & 4)转运蛋白的基因表达评估
利用实时定量聚合酵素链锁反应(Q-PCR)对3T3-L1脂肪细胞在不同浓度的茯苓三萜类化合物的作用下对葡萄糖转运体信息核醣核酸(mRNA)的表达进行评估。将分化完全的3T3-L1脂肪细胞,给予不同浓度的茯苓三萜类化合物24小时后,去除细胞培养液后,用Trizol试剂(Invitrogen,Irvine,CA,USA)提取总RNA,并取1μgRNA利用反转录试剂(High-Capacity cDNA Reverse Transcription Kits,Applied Biosystems,Darmstadt,Germany)将mRNA反转录成cDNA。分别针对葡萄糖转运体1、葡萄糖转运体4及β-肌动蛋白,设计出专一引子(primer),及利用SYBRGreen Q-PCR分析(Applied Biosystems,Foster City,CA,USA)扩增待测基因(GLUT1及GLUT4)及内参基因(β-actin),并以StepOne v2.0 software(Applied Biosystems)软件以ΔΔCT方法计算相对因表达值。
5.对葡萄糖转运体4转运蛋白转位的评估
5.1对葡萄糖转运体4转运蛋白转位分析(一)
在因胰岛素促进脂肪细胞或肌肉细胞的葡萄糖吸收机制中,葡萄糖转运体4(GLUT4)转运蛋白从细胞内胞器转位(translocation)到质膜(plasmamembrane(PM))扮演重要角色,故进行以茯苓三萜类化合物促进3T3-L1脂肪细胞的葡萄糖转运体4转运蛋白转位到质膜的测试。分化成熟的3T3-L1脂肪细胞以无血清细胞培养液培养过夜后,再以含不同浓度的茯苓三萜类化合物的无血清细胞培养液培养2小时,接着以不同转速高速离心(16,000g~200,000g),将细胞质膜部分(plasma membrane(PM)fraction)及低密度微粒体(low density microsome(LDM))分离出(Liu,L.Z.等;Mol Biol.Cell 17,(5),2322-2330,2006)。利用西方点墨法以葡萄糖转运体4的专一性抗体来观察3T3-L1脂肪细胞在不同浓度的茯苓三萜类化合物的作用下对葡萄糖转运体4转运蛋白从细胞LDM转位到质膜(PM)是否有影响。
5.2对葡萄糖转运体4转运蛋白转位分析(二)
将稳定表达HA-GLUT4-GFP蛋白的3T3-L1前脂肪细胞置于96孔培养盘,待长满后以分化促进剂进行分化(Govers,R.等;Mol Cell Biol.24(14),6456-6466,2004)。将分化完全的3T3-L1脂肪细胞,给予不同浓度的茯苓三萜类化合物2小时,去除细胞培养液,用冰PBS清洗细胞。再以4%三聚甲醛(paraformaldehyde)于室温下固定细胞15分钟,接着以冰PBS清洗2-3次,再加入针对HA的一级抗体(12CA5)培育2小时。以冰PBS清洗2-3次,加入共轭架接荧光染料盐基桃红精的二级抗体(rhodamine-conjugated secondary antibody)(Leinco,Ballwin,MO)培育细胞1小时。以冰PBS清洗2-3次,再以荧光免疫分析仪(POLARstar Galaxy;BMG Labtechnologies,Offenburg,Germany)分别探测盐基桃红精(rhodamine)及绿色荧光蛋白(GFP)的激发波长强度(Em.480/Ex.425nm及Em.576nm/Ex.550nm),并以盐基桃红精对绿色荧光蛋白之比值评估HA-GLUT4-GFP转位质膜的相对量。因为仅有在HA-tagged GLUT4移位到质膜上时,才会被盐基桃红精标识,故此比值可用来评估葡萄糖转运体4转运蛋白转位到质膜的情形。
6.三酸甘油酯堆积及甘油释放的影响
在茯苓三萜类化合物对3T3-L1脂肪细胞内的三酸甘油酯堆积和甘油的释出的测试中,已分化成熟的3T3-L 1脂肪细胞以无血清细胞培养液培养过夜,再以含不同浓度的茯苓三萜类化合物的无血清细胞培养液培养24小时。收集培养液以甘油检测试剂(glycerol assay kit(Randox Laboratories,Antrim,UK))进行甘油释出检测,观察3T3-L1脂肪细胞在不同浓度的茯苓三萜类化合物的作用下对脂肪分解的甘油释出是否有所影响。脂肪细胞内三酸甘油酯检测利用油红染色法(Oil-Red O staining)进行(Ramirez-Zacarias,J.L.等;Histochemistry 97,(6),493-497,1992)。将细胞内脂质堆积形成的脂肪油滴染色,经60%异丙醇洗涤二次,再以100%异丙醇萃取后,检测490nm吸光值。以未给予茯苓三萜类化合物的脂肪细胞的吸光值进行比较,来评估不同浓度的茯苓三萜类化合物作用下对脂肪细胞的三酸甘油酯堆积的影响。
实验结果说明茯苓三萜类化合物如同胰岛素一样具备下列四种性质,因此具有治疗第一型糖尿病患的能力:
(1)茯苓成分或萃取物于脂肪细胞模式下,具有促进葡萄糖从细胞 外吸收进入细胞内的能力:
茯苓萃取物(实施例2)在成熟脂肪细胞的评估,如图2A所示,结果显示茯苓萃取物具有显著增加葡萄糖吸收的作用,其促进吸收效果随给予剂量增加而增加。如给予100nM胰岛素则一样看到葡萄糖吸收。进一步以实施例2的纯化合物作试验。如图2B所示,纯化合物给予脂肪细胞2小时后,其中三个化合物茯苓酸(PA)、块苓酸(TA)及多孔覃酸C(PPA)于0.01μM显著增加糖吸收,分别增加至165.89%、142.5%及147.9%。其中以PA增加程度最为显著,故后续试验评估均以PA进行试验。
图3A显示PA随投予时间的增加而促使糖吸收随之增加,而以投予2小时的增加最为显著(增加165.89%),另外,PA随投予浓度的增加,糖吸收的促进程度亦随之增加,当PA为1μM时,增加至209.84%,如图3B所示。
如图4结果所示,PA促进糖吸收效果仅对已分化的脂肪细胞,对于前脂肪细胞PA无促进葡萄糖吸收效果。如对前脂肪细胞及成熟脂肪细胞二者给予葡萄糖转运体转运蛋白抑制剂(PT,phloretin)后观察到二种细胞的促进吸收能力被大幅抑制。根据文献前脂肪细胞仅有葡萄糖转运体-1(GLUT1)转运蛋白,而成熟脂肪细胞则为葡萄糖转运体-4(GLUT4)转运蛋白,故PA的作用应为增加GLUT4而增加糖吸收。
(2)茯苓三萜类化合物PA对成熟脂肪细胞有显著诱导葡萄糖转运体4转运蛋白质(GLUT4)及信息核醣核酸(mRNA)的表达
图5结果显示,对成熟脂肪细胞给予不同剂量的PA,作用24小时后,以西方点墨法分析葡萄糖转运体1及4转运蛋白(GLUT1,4),进行评估PA对GLUT1及GLUT4转运蛋白表达的影响,结果显示,PA具有促进GLUT4转运蛋白表达的效果(图5A),而其不具有促进GLUT1转运蛋白表达的效果(图5B)。
以定量聚合酵素链锁反应(Q-PCR)及以专一的探针来观察PA对成熟的3T3-L1脂肪细胞在不同浓度作用下对葡萄糖转运体信息核醣核酸(mRNA)的表达进行评估,图6结果显示,1μM PA促进GLUT4基因表达至228%。显示PA具有调节增加GLUT4基因及蛋白质表达的能力。另外,利用干扰信息核醣核酸技术建立持续性降低GLUT4转运蛋白的3T3-L1脂肪细胞(图7A),并以此脂肪细胞进行糖吸收评估,如图7B所示,所有PA的不同剂量均无法促进该脂肪细胞的糖吸收,进一步证实PA 促进糖吸收与GLUT4转运蛋白有直接关系。
(3)PA对成熟脂肪细胞具有促进GLUT4转运蛋白从细胞内转位移至质膜的功效
胰岛素促进糖吸收机制之一为促使大量GLUT4由细胞内胞器转位到质膜(PM)上进行糖吸收。故利用具备完整胰岛素活化葡萄糖吸收***的3T3-L1成熟脂肪细胞进行GLUT4转运蛋白转位效能评估。由图8A结果观察利用超高速离心方法所分离的质膜(PM)的西方点墨法,观察到0.01μM PA显著增加GLUT4于质膜量达到141%,随剂量增加到1μM则增加至328%。而利用持续表达HA-GLUT4-GFP蛋白的3T3-L1脂肪细胞方式及荧光检测,再次确认PA促进糖吸收为增加GLUT4转运蛋白转位移到质膜所导致。图8B结果观察到于PA剂量1.0μM时GLUT4转运蛋白转位到质膜显著增加达2.71倍。上述结果证实PA确实具有促进GLUT4转运蛋白转位到质膜的能力。
(4)PA具有促进脂肪细胞三酸甘油酯累积及减少脂肪细胞释放甘油至细胞培养液的能力
除观察PA促进糖吸收外,也观察脂肪细胞的影响,我们评估脂肪细胞三酸甘油酯合成(储存)及脂肪分解(甘油释出)情形。图9结果所示,给予不同剂量的PA 24小时后,以油红染色方法评估三酸甘油酯累积情形,观察到三酸甘油酯累积超过137%,PA给予也观察到甘油释出降至原本70%以下。显示PA具有促进脂肪新生及抑制脂肪分解的能力。
Claims (4)
1.一种使用具有如下化学式的羊毛甾烷或其药学上可接受的盐作为有效成分在制备用于治疗哺乳类动物因血中胰岛素不足所引起的糖尿病药物中的用途,
其中所述的糖尿病为第一型糖尿病。
2.权利要求1的用途,其中该药物为注射剂型。
3.权利要求1的用途,其中该药物为口服剂型。
4.权利要求1的用途,其中该哺乳类动物为人类。
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