CN1919257B - 老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用 - Google Patents
老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用 Download PDFInfo
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- CN1919257B CN1919257B CN 200610041486 CN200610041486A CN1919257B CN 1919257 B CN1919257 B CN 1919257B CN 200610041486 CN200610041486 CN 200610041486 CN 200610041486 A CN200610041486 A CN 200610041486A CN 1919257 B CN1919257 B CN 1919257B
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- actinodaphnes cupularis
- radix actinodaphnes
- ethanol
- ethanol extraction
- liver
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Abstract
本发明涉及老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用，即老鹰茶乙醇提取物的新用途。老鹰茶乙醇提取物中主要含有黄酮醇类化合物，黄酮醇类化合物分别为槲皮素-3β-D-半乳糖苷(I，2～4％)、山奈酚-3β-D-葡萄糖苷(II，12～14％)、异槲皮苷(III，5～8％)、山奈酚半乳糖苷(IV，3～5％)；黄烷醇类分别为儿茶素(28～30％)和表儿茶素(2～3％)。本发明对老鹰茶乙醇提取物发掘了新的医疗用途，开拓了一个新应用领域。老鹰茶乙醇提取物既能治标，又能治本，安全无毒，药理作用强，预示着很好的药用前景；其原料来源丰富、价廉、制备工艺成熟，可制成各种口服剂型，使用方便。
Description
技术领域
本发明涉及老鹰茶乙醇提取物的用途，尤其涉及老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用。
背景技术
老鹰茶是我国江南农村的一种传统饮料，千百年来深受人们的喜爱和欢迎，原因在于原料丰富，加工简单，便于保管，饮用方便，而且是一种无毒、无污染的纯天然饮料。近年来，由于环境受到一定程度的污染，人们回归自然的意识不断增加，这种纯天然饮料越来越受到人们的青睐。
老鹰茶的植物学分类：属于樟科、木姜子属、毛豹皮樟，俗称“白茶”，也叫老鹰茶。学名Litsea coreanaLeve.Var.，是一种常绿小乔木或乔木。常生长在海拔1000米左右的低山常绿阔叶林中，分布在四川、贵州、安徽、云南、重庆、湖南和江西等地。
当年生枝紫褐色或绿色，有卷曲柔毛，皮孔黄褐色或绿色，第二年枝秃净。芽卵行，鳞片外绿色或黄褐色绢毛。
叶革质、互生、椭圆形、狭椭圆形或倒卵状椭圆形，先端渐尖或突尖，基部锲形，长5-15cm，宽1.5-5.cm，背面灰白色，有卷曲柔毛，有时秃净，侧脉6-10条，叶柄长0.5-1.5cm，有毛。在自然环境下，年萌发新芽2轮，4-5月为春芽，8-9月为秋芽，以春芽产量为高。花，腊黄色、花序无梗，数个簇生在叶腋，总苞外有短毛，每序有花4-8朵，花朵直径4-5mm。果，近球状(有长椭圆形和倒椭圆形)，直径约6mm，果梗粗壮，长0.5-0.8mm，有毛，果托扁平膨大，有宿存的花被裂片。花期8-9月，果期次年5-8月。种子，由一层较薄的果皮包裹，种子坚硬，有蜡质。种子直径约6mm。
老鹰茶化学成分：
老鹰茶中含有丰富的氨基酸，总含量为0.82％，8种人体必需氨基酸齐全，含量以亮氨酸最高，赖氨酸次之，蛋氨酸最低，这和绿茶相似，其中蛋氨酸含量还略高于绿茶。在非必需氨基酸中，以谷氨酸含量最高，其次是天冬氨酸和精氨酸，与绿茶相比无明显差异。从氨基酸总量上看，绿茶的氨基酸高于老鹰茶，其主要原因是绿茶中特有茶氨酸的含量高，茶氨酸在一定程度上反映绿茶与老鹰茶的口感差异，另一方面也说明绿茶原植物与老鹰茶植物有不同的氨基酸代谢类型。
老鹰茶富含多种矿物质元素，其中钾(K)、磷(P)、钙(Ca)、镁(Mg)、钠(Na)、锌(Zn)、铁(Fe)、硒(Se)都是人体必需的营养元素，尤其是锌(Zn)、铁(Fe)、硒(Se)元素含量高于绿茶，对造血、健脑有较好作用。更值得一提的是有害元素铅的含量明显低于绿茶。
老鹰茶含有的水溶性维生素主要是Vc、VB1和VB2，其含量比绿茶稍低，可能与加工过程与代谢途径等因素有关。
据报道，老鹰茶中总黄酮(TF，total flavonoid)含量远高于绿茶，绿茶只有1.22％左右，而老鹰茶可高达3.45％.已经确定的有山柰酚(kaempferol)，山柰酚-3-O-β-D-葡萄糖苷(kaempferol-3-O-β-D-glucopyranoside)，槲皮素-3-O-β-D-葡萄糖苷(quercetin-3-O-β-D-glucopyranoside)、槲皮素-3β-D-半乳糖苷、山奈酚半乳糖苷、儿茶素和表儿茶素.
老鹰茶经过常压水蒸气蒸馏提取老鹰茶的挥发性成分，得到浅黄色油状液体，香味浓郁，出油率0.1％左右。经色谱和质谱分析，鉴定出32种成分(其中萜类化合物及其衍生物20种)，已鉴定的含量占挥发油组成的95％，主要成分是正癸醛(decanal)含量为71.53％；其次是10-烯-十一醛(10-undecenal)，含量为5.02％；壬醛(n-nonaldehyde)，含量3.58％；正十二酸乙烯酯(dodecanoic acid，ethenyl ester)，含量2.63％。香味的主要成分为萜类，其中主要是分子量为204的萜类。
老鹰茶中还含有多糖、皂甙、生物碱、多酚和天然色素等。
另外，尤其值得一提的是，与绿茶相比，老鹰茶的化学成分不含咖啡碱物质，无兴奋作用，不影响睡眠，这对需要控制咖啡碱摄入的人更是一种良好的选择。
老鹰茶(Litsea coreana Leve.Var.)在我国江南广大农村饮用由来已久，深受广大劳动人民的喜爱，但仅仅作为一种饮料。近年来，四川、贵州等地进行了一些开发和利用，但也仅限于粗加工，还没有充分认识到它的药理功能，它所潜在的药理作用机制更无前人探讨。根据文献报道，长期饮用有防馊、防腐、消暑解渴、消食化积、解毒、消肿利尿，明目、健胃、益思的作用。
本发明的目的在于提供老鹰茶乙醇提取物的新用途，即在制药中的新应用。
实际上，本发明涉及老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用。
酒精性肝病及其药物治疗现状
长期的过度饮酒，通过乙醇本身和它的衍生物乙醛可使肝细胞反复发生脂肪变性，坏死和再生，而导致酒精性肝病(Alcoholic liver disease，ALD)，包括酒精性脂肪肝(Alcoholicfatty liver)、酒精性肝炎(Alcoholic hepatitis)、肝纤维化(Alcoholic fibrosis)和肝硬化(Alcoholic cirrhosis)等多种表现。其组织学诊断分为酒精性脂肪肝、酒精性肝炎、酒精性肝纤维化和酒精性肝硬化4型。在欧美国家，酒精性肝病是中青年死亡的主要原因之一。据估计1993年美国约有1530万人酗酒，酒精性肝病有200多万人，每年有2万6千人死于肝硬化，至少40％或许达90％的患者有酗酒史。在我国，随着生活条件的改善，酗酒有增多趋势，尽管酒精性肝病的发生率尚无精确的统计，但并不少见。由于国内肝病主要由肝炎病毒引起，肝炎病毒携带者的数量更多，可能掩盖了实际上是酒精作为病因的肝病。因此正确认识酒精引起的肝损害，及时诊断和防治具有重要的意义。
酒精性肝病的治疗主要针对：(1)减轻酒精性肝病的严重度；(2)阻止或逆转肝纤维化；(3)改善已存在的继发性营养不良；(4)酒精性肝硬化的治疗。近年来，治疗酒精性肝病的药物有：
1.糖皮质激素酒精性肝病时肝内有炎症反应，肝细胞肿胀坏死以及胶原生成和沉积；有证据表明，酒精性肝病的起始和发展有免疫因素参与。糖皮质激素能抑制花生四烯酸代谢的脂氧合酶和环氧合酶的途径，从而抑制白三烯类及前列腺素的促炎症作用，尚可促进白蛋白合成和阻止I型胶原生成。因此有人提出可用来治疗酒精性肝病，但目前许多研究结果不一致。许多因素包括性别，肝病严重度，肾功能，营养状态甚至地域可造成不同的结局。一般认为激素对轻中型病例无明显效果，而仅仅严重病例才能从激素受益。至于激素是否对病人的远期生存率有影响，或长程激素治疗是否能阻止发展为肝硬化，尚缺乏研究。
2.胰岛素与胰高血糖素胰岛素及胰高血糖素静滴对酒精性肝病有一定疗效，但在治疗过程中应检测血糖，防止发生致命性低血糖。
3.丙基硫氧嘧啶丙基硫氧嘧啶治疗的疗效主要见于严重病例，而且是酒精量相对较少者。
4.抗氧化剂还原型谷胱甘肽，牛磺酸，胡萝卜素，维生素E，r-月见草，硒有机化合物等，有可能减少氧应激损害及脂质过氧化诱致的肝纤维化，可解除外源性有毒物质的毒性。
5.多不饱和卵磷脂为肝窦内皮和肝细胞膜稳定剂，可降低脂质过氧化，但因富含不饱和脂肪酸，对不能戒酒者应慎用。
6.降脂药有异议，需待解释。许多降血脂药可能趋使血脂更集中于肝脏进行代谢，反而促使脂质贮积并损害肝功能。
7.抑制肝纤维化的中药在我国应用活血化瘀中药治疗慢性肝病已有悠久历史，如桃仁，丹参，当归，汉防己甲素，何首乌，山楂，姜黄，枸杞子，川芎，泽泻，黄岑，黄精，大黄等，分别有改善肝脏微循环，防止肝细胞变性坏死，减少胶原纤维的产生或增强胶原酶活性等作用，有助于酒精性肝炎肝纤维化的治疗。
老鹰茶乙醇提取物物理性状：为墨绿色粉末，易吸潮，水溶性较差，易溶于亲脂性有机溶剂。具有特殊香味。
老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清TG、TC、FFA、AST、ALT、ALP、γ-GT以及肝匀浆TG、TC、FFA水平(与模型组比较，p＜0.05)；老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清MDA、TNF-α、IL-1β、iNOS、NO以及肝匀浆MDA、TNF-α和IL-1β的含量，并升高SOD，GSH-Px水平(与模型组比较，p＜0.05)；病理组织学检查结果提示老鹰茶乙醇提取物(200，400mg·kg-1)可明显减轻肝脏脂肪变性程度，并减轻炎症反应。
为了更好地理解本发明的实质，下面将用老鹰茶乙醇提取物的药理试验及结果来说明其在制药领域中的新用途。
老鹰茶乙醇提取物对酒精性肝病的防治作用试验如下：
(一)材料
动物Spague-Dawlay(SD)大鼠60只，♂，体重200-220g，由安徽医科大学动物中心提供，普通级。
药物和试剂老鹰茶总黄酮(自制)，凯西莱(河南省新谊药业股份有限公司，批号：H41020799)，无水乙醇为国产分析纯试剂，胆固醇(购自国药集团化学有限公司)、脱氧胆酸钠(购自北京奥博星生物技术责任有限公司)，吐温80(蚌埠化学试剂厂，符合药用标准，批号20030602)，1-2丙二醇(淮南市化学试剂厂，AR级，批号20021020)，玉米油、白砂糖、全脂奶粉、动物用复合维生素(市售)；ALT、AST测定试剂盒(购自上海荣盛)，γ-GT、ALP测定试剂盒(购自长春汇力公司)，TC、TG测定试剂盒(购自北京福瑞生物工程公司)，SOD、MDA、考马斯亮兰蛋白测定试剂盒(南京建成生物工程研究所)。
仪器电子天平FA2004(上海精天天平厂)，79HW-1恒温磁力搅拌器(江苏金坛市金城国胜实验仪器厂)，722紫外分光光度计(上海分析仪器厂制造)。
(二)方法
模型制备及分组
以酒精联合脂肪乳剂(10ml·kg-1·d-1)灌胃，一天两次。酒精剂量计算采用下列公式：酒精剂量(g)＝无水乙醇体积(ml)*预配酒精浓度(％)*0.8(g/ml)。第1～3天以30％(v/v)酒精5g·kg-1·d-1适应性灌胃，自第4天起，每三天酒精的浓度增加5％，剂量增加0.6g·kg-1·d-1。至酒精浓度50％，剂量8g·kg-1·d-1止。脂肪乳剂组成如下：玉米油35g，胆固醇10g，蔗糖10g，胆盐1g，奶粉10g，复合维生素0.3g，矿物质0.2g，吐温803.1g，丙二醇3.64g，加蒸馏水至70ml。
正常喂养一周后的大鼠，按体重随机分为6组(每组10只)：正常对照组、模型组、老鹰茶低剂量组(100mg·kg-1)、老鹰茶中剂量组(200mg·kg-1)、老鹰茶高剂量组(400mg·kg-1)、凯西莱阳性药对照组。模型组按上述方法制备模型。用药组在制备酒精性肝病模型的同时，按相应药物给药剂量灌胃给药。模型组和用药组自由饮用15％的糖水。对照组则灌服同剂量生理盐水，自由饮水，给予普通饲料。实验持续6周。
标本采取于第六周末，所有大鼠禁食12h，第二天上午称重，麻醉，腹主动脉采血，常规制备血清。取肝脏相同部位，常规制备肝匀浆，并行病理组织学检查。
血清生化指标测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、γ-GT、甘油三酯(TG)、总胆固醇(TC)、超氧化物岐化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)测定按试剂盒规范操作。
肝脏生化指标测定在相同部位取肝脏精确称取0.3g，在冰水中制成10％的肝匀浆，4℃3000r·min-1离心20min，提取上清液，用酶法测定TG、TC、SOD、MDA、GSH-PX含量。
肝脏组织病理学检查肝脏组织以10％的中性福尔马林固定，石蜡包埋切片进行常规HE染色，在光镜下观察肝脂肪变性和炎症活动情况。肝脏组织学诊断标准参考中华医学会肝脏病学分会脂肪肝和酒精性肝病学组所制订的酒精性肝病诊断标准。
(三)结果
1.老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆TG、TC、FFA水平的影响
由表1可见，老鹰茶乙醇提取物(100，200，400mg·kg-1)均能显著降低酒精性肝病大鼠血清以及肝匀浆TG、TC、FFA水平，说明其能够改善肝脏的脂肪变，减少肝脏对脂质的吸收，并能够减轻FFA对肝脏的细胞毒性作用。
表1老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆TG、TC、FFA水平的影响(x±s，n＝10)
△p＜0.05，△△p＜0.01 vs normal group；*p＜0.05，**p＜0.01vs model group
2.老鹰茶乙醇提取物对酒精性肝病大鼠血清AST、ALT、ALP、γ-GT含量的影响
由表2可见，老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清AST、ALT、ALP、γ-GT水平。表明其具有稳定肝细胞膜以及抑制、修复肝脏炎症的作用。
表2老鹰茶乙醇提取物对酒精性肝病大鼠血清AST、ALT、ALP、γ-GT含量的影响(x±s，n＝10)
△p＜0.05，△△p＜0.01 vs normal group；*p＜0.05，**p＜0.01 vs model group
3.老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆SOD，MDA，GSH-Px含量的影响
由表3可见，老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清以及肝匀浆MDA含量，并升高SOD，GSH-Px水平。表明本品具有一定的抑制过氧化物质形成以及促进抗氧化物质生成的作用。并可通过提高肝组织GSH-Px活力，阻遏酒精性脂肪肝的发展与转归。
表3老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆SOD，MDA，GSH-Px含量的影响(x±s，n＝10)
△p＜0.05，△△p＜0.01 vs normal group；*p＜0.05，**p＜0.01 vs model group
4.老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆TNF-α和IL-1β含量的影响
由表4可见，老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清以及肝匀浆TNF-α和IL-1β的含量。提示其对细胞因子的调节作用可能是其治疗酒精性肝病的重要机制。
表4老鹰茶乙醇提取物对酒精性肝病大鼠血清及肝匀浆TNF-α和IL-1β含量的影响(x±s，n＝10)
△p＜0.05，△△p＜0.01vs normal group；*p＜0.05，**p＜0.01 vs model group
5.老鹰茶乙醇提取物对酒精性肝病大鼠血清iNOS，NO含量的影响
由表5可见，老鹰茶乙醇提取物(100，200，400mg·kg-1)能显著降低酒精性肝病大鼠血清iNOS、NO的含量。提示抑制iNOS和NO的增加可能是其减轻肝脏脂质过氧化损伤的机制之一。
表5老鹰茶乙醇提取物对酒精性肝病大鼠血清iNOS、NO含量的影响(x±s，n＝10)
△p＜0.05，△△p＜0.01vs control group；*p＜0.05，**p＜0.01vs model group
6.老鹰茶乙醇提取物对酒精性肝病大鼠肝脏病理组织学变化的影响
肉眼观察：正常组肝脏呈鲜红色，边缘锐利，表面光滑。模型组肝脏肿大，边缘变钝，颜色呈黄褐色，与周围组织有粘连，切面油腻感。老鹰茶乙醇提取物高、中、低剂量组及凯西莱组的大鼠肝脏颜色则略带黄，与周围组织无粘连。
病理组织学检查：正常组大鼠肝细胞形态正常，肝小叶结构清晰，肝细胞索排列整齐，肝细胞大小较一致，胞核居中，胞质淡红色，在中央静脉区偶可见少数散在脂滴，无明显变性、坏死(图1)。模型组大鼠出现典型脂肪肝病变：肝细胞索排列紊乱，肝细胞肿胀，细胞内充满大小不一的脂滴，小叶内出现碎片状坏死及散在的点状或灶型坏死，汇管区有炎细胞浸润，但未见肝纤维化(图2)。老鹰茶乙醇提取物(200mg·kg-1、400mg·kg-1)及凯西莱组明显减轻肝脏脂肪变性程度，并减轻炎症反应(图3，图4)。
从以上结果，可以得出本发明的有益效果在于：
A、本发明对老鹰茶乙醇提取物发掘了新的医疗用途，开拓了一个新应用领域。
B、本发明的老鹰茶乙醇提取物既能治标，又能治本，安全无毒，药理作用强，预示着很好的药用前景。
C、本发明的老鹰茶乙醇提取物原料来源丰富、价廉、制备工艺成熟，可制成各种口服剂型，使用方便。
D、本发明的老鹰茶乙醇提取物对酒精性肝病具有良好的防治作用，长期服药无危害。
附图说明
图1为正常组大鼠肝细胞形态病理组织学检查图，
图2为模型组大鼠出现典型肝脂肪肝病变病理组织学检查图，
图3为老鹰茶乙醇提取物明显减轻肝脏脂肪变性程度、并减轻炎症反应病理组织学检查图，
图4为凯西莱组明显减轻肝脏脂肪变性程度、并减轻炎症反应病理组织学检查图。
具体实施方式
下面结合实施例，对本发明作进一步地说明。
实施例：
老鹰茶(5kg)用80％乙醇，12倍体积，提取3次，每次提取2h，合并提取液，回收溶剂至无醇味(12L)，石油醚脱色，用正丁醇萃取，得正丁醇萃取物(1kg)，水层上AB-8大孔树脂(5kg)，用24L水洗脱，除去水溶性杂质，再用24L 90％乙醇洗脱，合并醇洗脱液，回收溶剂，该部分(500g)与正丁醇部分合并为老鹰茶乙醇提取物，主要成分为黄酮类化合物。
老鹰茶乙醇提取物中主要含有黄酮醇类化合物(25～30％，HPLC法；25～30％，比色法)、儿茶素(27～29％，HPLC法)、表儿茶素(2～4％，HPLC法)，皂苷(35～45％，比色法)。老鹰茶乙醇提取物中黄酮类化合物经进一步富集，分离，得到黄酮醇和黄烷醇类化合物。用HPLC法测定，黄酮醇类化合物分别为槲皮素-3β-D-半乳糖苷(I，2～4％)、山奈酚-3β-D-葡萄糖苷(II，12～14％)、异槲皮苷(III，5～8％)、山奈酚半乳糖苷(IV，3～5％)；黄烷醇类分别为儿茶素(28～30％)和表儿茶素(2～3％)。以上黄酮醇及黄烷醇类化合物为老鹰茶中主要的黄酮类化合物。
以上老鹰茶乙醇提取物临用前均以0.5％CMC-Na配成混悬液，所用剂量均以浸膏粉末量表示。
老鹰茶乙醇提取物推荐临床剂量：600-2400mg/日，口服，分三次服用。
Claims (1)
1.老鹰茶乙醇提取物在制备防治酒精性肝病的药物中的应用，其特征在于该老鹰茶乙醇提取物采用下述方法提取获得：
5kg老鹰茶用80％乙醇，12倍体积，提取3次，每次提取2h，合并提取液，回收溶剂至无醇味12L，石油醚脱色，用正丁醇萃取，得正丁醇萃取物1kg，水层上5kg的AB-8大孔树脂，用24L水洗脱，除去水溶性杂质，再用24L 90％乙醇洗脱，合并醇洗脱液，回收溶剂，该部分500g与正丁醇部分合并为老鹰茶乙醇提取物，主要成分为黄酮类化合物。
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| 张宇.茶多酚治疗慢性酒精性肝损伤的实验研究.中华肝脏病杂志13 2.2005,13(2),125-127. |
| 张宇.茶多酚治疗慢性酒精性肝损伤的实验研究.中华肝脏病杂志13 2.2005,13(2),125-127. * |
| 陶华君.茶叶与人体健康.中国食物与营养 2.2004,(2),45-46. |
| 陶华君.茶叶与人体健康.中国食物与营养 2.2004,(2),45-46. * |
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