CN103536652A - 冬凌草叶水提物的纳米结构及应用 - Google Patents
冬凌草叶水提物的纳米结构及应用 Download PDFInfo
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Abstract
本发明涉及冬凌草叶用蒸馏水80℃提取制备水提物、涉及该水提物浓缩得到的固体在水溶液中的纳米结构、进一步涉及纳米结构对抗骨质疏松活性的影响。
Description
发明领域
本发明涉及冬凌草叶用蒸馏水80 oC提取制备水提物、涉及该水提物浓缩得到的固体在水溶液中的纳米结构、进一步涉及纳米结构对抗骨质疏松活性的影响。本发明属于生物医药领域。
背景技术
冬凌草(DLC)，是唇形科香茶菜属植物碎米桠 Rabdosia rubescens (Hemsl.) Hara 的全草。冬凌草为多年生草本植物，广泛分布于我国黄河长江流域，主产于河南济源太行山、王屋山区一带。因具有清热解毒、清咽利喉和消肿止痛功能， 民间常年将冬凌草叶当作茶叶煮水饮用。在过去的30多年里，冬凌草的化学成分及药理作用研究主要集中在叶的脂溶性部分，代表性成分为冬凌草甲素和冬凌草乙素。通过长期研究积累，发明人披露了冬凌草叶水提物的21种酚酸。它们是单咖啡酰酒石酸, 丹参素, 丹参素咖啡酰酒石酸, 2-绿原酸-3-对羟基桂皮酸酒石酸酯, 单阿魏酰酒石酸, 绿原酸, 咖啡酰苹果酸, 二咖啡酰酒石酸, 迷迭香酰丹参素酒石酸酯, yuannaneic acid E, 紫草酸, 咖啡酸, 咖啡酰对羟基桂皮酰酒石酸, 氢化紫草酸。2-丹参素-3-乙酰莽草酸酒石酸酯, 丹参素咖啡酰酒石酸, 2-(丙二酰奎尼酸单酯)-3-丹参素酒石酸奎尼酸单酯, 水化丹酚酸B, 丹参素酰丹参素酰迷迭香酸酯, 丹酚酸B和迷迭香酸。这些酚酸在冬凌草叶水提物中的相对含量接近83%。应当指出的是，到目前为止还没有任何与冬凌草叶水提物纳米结构的报道。
通过长期研究积累，发明人认识到这些酚酸在水溶液中可以通过π-π相互作用，氢键相互作用和疏水性相互作用发生共组装，形成纳米结构。恰恰是这些酚酸在水溶液中共组装形成的纳米结构，使得冬凌草叶水提物显示重要的生物活性。根据这些认识，发明人提出了本发明。
发明内容
本发明的第一个内容是提供冬凌草叶水提物。
本发明的第二个内容是测定冬凌草叶水提物的纳米结构。
本发明的第三个内容是阐明冬凌草叶水提物的纳米结构与抗骨质疏松活性的关系。
附图说明
图1在浓度为 78.75 mg/ml的超纯水溶液中，冬凌草叶的水提物以直径为102.3 nm的纳米球的聚集体存在。
图2在浓度为 15.75 mg/ml的超纯水溶液中冬凌草茎的水提物以直径为46.4-110.7 nm的纳米球存在。
图3在浓度为 3.15 mg/ml的超纯水溶液中冬凌草茎的水提物以直径为58.8-147.1 nm的纳米球存在。
图4在浓度为 0.63 mg/ml的超纯水溶液中冬凌草叶的水提物以直径为81.8-218.2 nm的纳米球存在。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1 冬凌草叶水提物的制备
21 g干冬凌草叶自来水洗净之后加500 ml双蒸水于60 - 100 oC, 优选80 oC搅拌提取10 - 60分钟, 优选30分钟。过滤得到的清液于40 oC减压浓缩, 得到6.3 g 冬凌草叶水提物, 为淡黄色固体, 研成粉末, 供后面的研究使用。
实施例2 冬凌草叶水提物的纳米结构
为了描述冬凌草叶水提物的纳米结构，本发明将冬凌草叶水提物的淡黄色固体配成浓度为 78.75 mg/ml, 15.75 mg/ml, 3.15 mg/ml 和 0.63 mg/ml的超纯水溶液，然后将溶液滴在铜网上，35 °C挥发 24 h至干溶后按标准操作(20 eV 能量窗口，6000 - 400,000倍) 在透射电镜 (JSM- 6360 LV, JEOL, Tokyo, Japan)下观察纳米形态。测定的透射电镜照片显示在图1 – 4中。
图1说明在浓度为 78.75 mg/ml的超纯水溶液中，冬凌草叶的水提物以直径为102.3 nm的纳米球的聚集体存在。
图2说明在浓度为 15.75 mg/ml的超纯水溶液中，冬凌草叶的水提物以直径为46.4-110.7 nm的纳米球存在。
图3说明在浓度为3.15 mg/ml的超纯水溶液中，冬凌草叶的水提物以直径为58.8-147.1 nm的纳米球存在。
图4说明在浓度为0.63 mg/ml的超纯水溶液中，冬凌草叶的水提物以直径为81.8-218.2 nm的纳米球存在。
图2-4说明当浓度从15.75 mg/ml降至3.15 mg/ml再降至0.63 mg/ml时，冬凌草叶的水提物形成的纳米球的直径逐步增大。这种现象揭示，我们可以通过控制冬凌草叶的水提物的浓度来控制冬凌草叶的水提物形成的纳米球的直径。图1-2说明当浓度从 78.75 mg/ml降至15.75 mg/ml时，不仅冬凌草叶的水提物形成的纳米球的直径减小，而且纳米球的聚集体也不再存在。这种现象同样揭示，我们可以通过控制冬凌草叶的水提物的浓度来控制冬凌草叶的水提物形成的纳米球的直径和聚集状态。
实施例 3 冬凌草叶水提物的抗骨质疏松活性评价
5周龄ICR小鼠，体重25.7±2.1g, 48只随机分组，即卵巢切除组＋生理盐水 (称生理盐水组)、假手术组＋生理盐水 (称假手术组)、卵巢切除＋17β-*** (称***组)、卵巢切除＋冬凌草叶水提物 (称DLC组)。
用2%戊巴比妥钠按40 mg/kg剂量腹腔注射，麻醉小鼠，剪开小鼠腹腔，找到两侧的输卵管，在两侧输卵管的盲端找到卵巢，去除小鼠双侧卵巢，结扎输卵管(手术过程中如果小鼠苏醒过来，则用棉花蘸取适量无水***置于小鼠的鼻子处使之麻醉)。结扎好后，滴加几滴青霉素至小鼠腹腔，再将小鼠腹腔缝合好，于伤口外面涂上适量0.1%苯扎溴铵消毒液，以避免伤口感染。
术后5天开始口服给药。卵巢切除组和假手术组小鼠每天口服0.2 ml生理盐水。***组小鼠每天口服0.2 ml 17β-***的生理盐水溶液, 剂量为30 μg/kg。冬凌草叶水提物组小鼠每天口服0.2 ml冬凌草叶水提物的生理盐水溶液, 剂量为5 mg/kg, 25 mg/kg, 125 mg/kg和625 mg/kg。给药四周后用骨密度仪对小鼠股骨关节头下方0.2 - 0.4 mm 处进行定量断断层扫描，得到骨矿物质密度 (表4及图4)、称体重、处死、分离小鼠的肺、肝、脾、子宫，得它们的净重 (表5)。取小鼠左侧股骨、剔除干净肌肉组织、用氯仿:甲醇(2:1)浸泡两次（每次3小时）脱脂、置120 oC烘箱中烘6小时称干重、置800 o马福炉煅烧8小时称灰重、计算灰重与干重的比值(g/g)得骨矿含量 (表1)。
表1 口服冬凌草叶水提物对小鼠骨密度的影响
n=10; a) 与卵巢切除 + 生理盐水组，卵巢切除＋17β-***组, 5 mg/kg冬凌草叶水提物组，25 mg/kg冬凌草叶水提物组及625 mg/kg冬凌草叶水提物组比 p < 0.01，与假手术 + 生理盐水组比p > 0.05; b) 与卵巢切除 + 生理盐水组, 卵巢切除＋17β-***组及5 mg/kg冬凌草叶水提物组比 p < 0.05; c) 与卵巢切除 + 生理盐水组比 p < 0.05, 与卵巢切除＋17β-***组比p > 0.05; d) 与卵巢切除 + 生理盐水组, 卵巢切除＋17β-***组及5 mg/kg冬凌草叶水提物组比 p < 0.05, 与25 mg/kg冬凌草叶水提物组比p > 0.05.
实施例4 冬凌草茎水提物的纳米结构与抗骨质疏松活性的关系
实施例2揭示，当浓度从15.75 mg/ml降至3.15 mg/ml 再降至0.63 mg/ml时，冬凌草叶的水提物形成的纳米球的直径逐步增大。当浓度从78.75 mg/ml降至15.75 mg/ml时，冬凌草茎的水提物形成的纳米球的直径反而减小。浓度为78.75 mg/ml时冬凌草茎的水提物形成的纳米球发生聚集。按照每只小鼠的血量为2 ml估算，给药剂量为625 mg/kg, 125 mg/kg, 25 mg/kg和5 mg/kg时的血药浓度分别为78.75 mg/ml, 15.75 mg/ml, 3.15 mg/ml 和 0.63 mg/ml。这种估算说明，冬凌草叶的水提物形成的纳米球的直径在46.4-110.7 nm时，抗骨质疏松活性最好。冬凌草叶的水提物形成的纳米球的直径在大于200 nm时，抗骨质疏松活性变差。此外，当直径为100 nm左右的纳米球发生聚集时，抗骨质疏松活性也不理想。的因为冬凌草叶的水提物形成的纳米球的直径紧密依赖于浓度，所以我们可以通过浓度-粒径-活性三者的关系来谋取理想的疗效。这就是冬凌草叶水提物的纳米结构的应用价值。
Claims (3)
1. 冬凌草叶水提物的制备工艺，该工艺包括将干冬凌草叶自来水洗净之后按1:24的比例加水于60 - 100 oC, 优选80 oC搅拌提取10 - 60分钟, 优选30分钟，过滤得到的清液于40 oC减压浓缩, 得到冬凌草叶水提物干粉, 为淡黄色固体。
2. 冬凌草叶水提物在水溶液中的纳米结构。
3. 冬凌草茎水提物在水溶液中的纳米结构对抗骨质疏松活性的影响。
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| CN101167509A (zh) * | 2006-10-25 | 2008-04-30 | 张海 | 冬凌草茶的制造方法 |
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| CN112402407A (zh) * | 2020-11-06 | 2021-02-26 | 北京恒润泰生医药科技有限公司 | 冬凌草叶水提物粉末、其制备、治疗肺纤维化作用及应用 |
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