CN104447915A - 一种从番石榴叶中制备番石榴苷和广寄生苷的方法及用途 - Google Patents
一种从番石榴叶中制备番石榴苷和广寄生苷的方法及用途 Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明提供一种从番石榴叶中同时制备番石榴苷和广寄生苷的方法及用途,是将番石榴叶原料粉碎,用5-10倍体积30-90%低碳醇提取2-3次,每次2-3h,合并提取液,真空浓缩回收溶剂,得提取物浸膏;将提取物浸膏加水溶解,用乙酸乙酯充分萃取,合并乙酸乙酯萃取液,真空浓缩得乙酸乙酯萃取物;乙酸乙酯萃取物进行硅胶柱层析分离,洗脱剂选用石油醚与乙酸乙酯进行梯度洗脱,真空浓缩石油醚和乙酸乙酯体积比为60:40、40:60的洗脱液;将上述浓缩液中加入低碳醇真空浓缩结晶,过滤干燥即得番石榴苷、广寄生苷。本发明方法简便,且本发明产品能够显著降低链脲佐菌素诱导的高血糖,可用于制备降血糖药物或保健品。
Description
技术领域
本发明涉及一种降糖保健品领域,特别是一种从番石榴叶中同时制备番石榴苷和广寄生苷的方法及用途。
背景技术
糖尿病(Diabetes Mellitus, DM)是由单一或多种致病因子共同作用于机体所导致的生物体胰岛功能衰退、胰岛素分泌不足和胰岛素抵抗等继而引发的以高血糖水平为主要特征、并伴随“三多一少”症状的代谢紊乱综合病征。随着糖尿病的发病率不断提高,在全球范围内已经成为继心脑血管疾病、肿瘤之后严重危害人类健康的第三大慢性病。
番石榴(Psidium guajava Linn.)是桃金娘科(Myrtaceae)番石榴属植物,原产于美洲,广泛分布于美洲、亚洲等的热带和亚热带地区,在我国南方各省区如广东、广西等地区有较大的种植面积。番石榴叶作为民间用药用来预防和治疗糖尿病已有悠久的历史,有大量的关于番石榴叶降血糖的文献报道,且大量的临床试验证明其疗效确切。中国专利申请公开的“降血糖药物制剂及其制备方法”(201110147738.7),提供了一种具有降血糖作用的药物制剂,但其并未说明其中的番石榴提取物降血糖的具体活性成分及其提取工艺。本实验室前期研究通过体外实验证实了番石榴叶中降血糖活性成分物质基础,但番石榴叶中降糖活性成分的提取工艺公开较少,且没有工业化工艺,多为实验室分离提取。
发明内容
本发明的目的在于提供一种从番石榴叶中同时制备番石榴苷和广寄生苷的方法,且两种化合物均具有降血糖的用途,可有效用于降糖药物或功能食品的大量生产制备,为高血糖患者的健康提供保障。
本发明提供的从番石榴叶中制备的化合物为番石榴苷、广寄生苷,结构式如下式所示:
番石榴苷
广寄生苷
两种化合物的制备方法包括以下步骤:
(1)提取:将番石榴叶原料粉碎,用5-10倍体积30-90%低碳醇提取2-3次,每次2-3h,合并提取液,真空浓缩回收溶剂,得提取物浸膏;
(2)萃取:将提取物浸膏加水溶解,用乙酸乙酯充分萃取,合并乙酸乙酯萃取液,真空浓缩得乙酸乙酯萃取物;
(3)硅胶柱层析富集:乙酸乙酯萃取物进行硅胶柱层析分离,选用石油醚与乙酸乙酯为洗脱剂进行梯度洗脱,分别真空浓缩石油醚和乙酸乙酯体积比为60:40、40:60的洗脱液;
(4)结晶:将上述浓缩液中加入低碳醇真空浓缩结晶,过滤干燥即得产品番石榴苷、广寄生苷。
步骤(1)、(4)中,所述低碳醇为甲醇或乙醇。
步骤(3)中,硅胶柱层析分离,硅胶选100-300目硅胶,洗脱的流速为1/2-1/5BV/h,用石油醚与乙酸乙酯进行梯度洗脱时,石油醚和乙酸乙酯的体积配比依次为100:0、80:20、60:40、50:50、40:60、20:80、0:100,真空浓缩石油醚和乙酸乙酯体积比为60:40、40:60的洗脱液,即为番石榴苷、广寄生苷粗品。
步骤(4)中,所述低碳醇浓度30-50%。
经高效液相色谱分析测定番石榴苷、广寄生苷,纯度均大于95%。
本发明方法简便,低碳醇既减少黄酮苷元溶出又减少了多糖和蛋白的溶出,有利于后续的分离纯化;硅胶柱层析吸附量大,上柱简单易操作,可以减少萃取溶剂的使用量,降低生产成本;低碳醇结晶,产品纯度高。经动物实验表明,本发明产品番石榴苷、广寄生苷能够显著降低糖尿病小鼠的血糖,可用于制备降血糖药物或保健品。
具体实施方式
以下结合实际情况,对本发明的具体实施方式进行详细说明。
实施例1:
称取番石榴叶原料10kg,80L 50%低碳醇提取3次,每次2小时,提取液真空浓缩得提取物浸膏;提取物浸膏分散于水中,用乙酸乙酯萃取3次,合并浓缩萃取液得乙酸乙酯萃取物400g,萃取物过100目硅胶柱,石油醚和乙酸乙酯的体积配比依次为100:0、80:20、60:40、50:50、40:60、20:80、0:100,每个比例5BV,洗脱的流速为1/2BV/h,真空浓缩60:40、40:60洗脱液,50%乙醇结晶得番石榴苷20.6g、广寄生苷21.3g,液相检测含量番石榴苷92.5%,广寄生苷95.7%。
实施例2:
称取番石榴叶原料50kg,300L 50%低碳醇提取3次,每次2小时,提取液真空浓缩得提取物浸膏;提取物浸膏分散于水中,用乙酸乙酯萃取3次,合并浓缩萃取液得乙酸乙酯萃取物12kg,萃取物过200目硅胶柱,石油醚和乙酸乙酯的体积配比依次为100:0、80:20、60:40、50:50、40:60、20:80、0:100,每个比例5BV,洗脱的流速为1/3BV/h,真空浓缩60:40、40:60洗脱液,40%乙醇结晶得番石榴苷101.6g、广寄生苷103.5g,液相检测含量番石榴苷93.1%,广寄生苷97.4%。
为阐明、验证本发明降低高血糖的效果,特作了以下降糖实验。
取雄性小鼠100只,体重20±2克左右,适应性喂养一周后随机分为两组,其中正常组 10 只,造模组 90只。正常组喂养正常饲料,造模组喂养高脂高糖饲料(猪油 10%,蔗糖 20%,蛋黄粉10%,胆酸盐0.5%,麦芽糊精5% ,基础饲料 54.5%)6周后,各组小鼠禁食不禁水 12 小时,正常组小鼠腹腔注射 0.1M,pH4.2 的柠檬酸-柠檬酸钠缓冲液;造模组小鼠腹腔注射链脲佐菌素溶液(40mg/kg),连续注射 4 天。2 周后,测量各组小鼠空腹血糖(FPG)值、其中,FPG>11.1 mmol/L 的小鼠为建模成功的 2 型糖尿病模型(T2DM)小鼠,将造模成功的糖尿病小鼠分为模型组、吡格列酮(10mg/kg)、广寄生苷低(25mg/kg)、广寄生苷中(50mg/kg)、广寄生苷高(100mg/kg)、番石榴苷低(25mg/kg)、番石榴苷中(50mg/kg)、番石榴苷高(100mg/kg)剂量组,每组10只。连续灌胃给药8周,禁食12h后,小鼠眼眶静脉取血,分离血清,葡萄糖氧化酶法测定血糖浓度,具体结果如下表1。
表1. 本发明番石榴降糖活性成分对糖尿病小鼠血糖的影响(X±SD,n=10)
组别 | 动物数量(只) | 剂量(mg/kg) | 血糖浓度(mmol/L) |
正常对照组 | 10 | 4.23±0.54 | |
模型组 | 10 | 19.51±1.24 | |
吡格列酮组 | 10 | 10 | 13.29±2.31* |
广寄生苷低剂量 | 10 | 25 | 15.61±2.19* |
广寄生苷中剂量 | 10 | 50 | 14.74±1.98* |
广寄生苷高剂量 | 10 | 100 | 13.41±1.69* |
番石榴苷低剂量 | 10 | 25 | 16.12±1.25* |
番石榴苷中剂量 | 10 | 50 | 15.63±2.45* |
番石榴苷高剂量 | 10 | 100 | 14.32±1.89* |
*与模型组比较P<0.01
由上表1可知,与正常对照组比较,模型组血糖显著升高(P<0.01),阳性降糖药物吡格列酮能够显著下降血糖,与模型组比较P<0.01。本发明所获得番石榴降糖活性成分对糖尿病小鼠血糖有显著下调作用,与模型组比较有显著意义,且体现量效关系,即所给药剂量越大,降糖效果越明显。
Claims (6)
1. 一种从番石榴叶中制备番石榴苷和广寄生苷的方法,其特征在于,包括以下步骤:
(1)将番石榴叶原料粉碎,用5-10倍体积30-90%低碳醇提取2-3次,每次2-3h,合并提取液,真空浓缩回收溶剂,得提取物浸膏;
(2)将提取物浸膏加水溶解,用乙酸乙酯充分萃取,合并乙酸乙酯萃取液,真空浓缩得乙酸乙酯萃取物;
(3)乙酸乙酯萃取物进行硅胶柱层析分离,洗脱剂选用石油醚与乙酸乙酯进行梯度洗脱,分别真空浓缩石油醚和乙酸乙酯体积比为60:40、40:60的洗脱液;
(4)将上述浓缩液中加入低碳醇真空浓缩结晶,过滤干燥即得番石榴苷、广寄生苷。
2.根据权利要求1所述的一种从番石榴叶中制备番石榴苷和广寄生苷的方法,其特征在于,步骤(1)(4)中,低碳醇为甲醇或乙醇。
3.如权利要求1所述的一种从番石榴叶中制备番石榴苷和广寄生苷的方法,其特征在于,步骤(3)中,硅胶柱层析分离,硅胶选100-300目硅胶,洗脱的流速为1/2-1/5BV/h,用石油醚与乙酸乙酯进行梯度洗脱时,石油醚和乙酸乙酯的体积配比依次为100:0、80:20、60:40、50:50、40:60、20:80、0:100。
4.根据权利要求1所述的一种从番石榴叶中制备番石榴苷和广寄生苷的方法,其特征在于,步骤(4)中,所述低碳醇浓度为30-50%。
5.一种从番石榴叶中制备番石榴苷和广寄生苷在制备降糖保健品方面的用途。
6.一种从番石榴叶中制备番石榴苷和广寄生苷在制备降糖药品方面的用途。
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