CN104435058B - 一种免疫增强剂及其制备方法 - Google Patents
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Abstract
本发明公开了一种免疫增强剂及其制备方法，该免疫增强剂由活性成分和药学上可用的辅料制备而成，其中的活性成分是黄瓜叶的水部位提取物，制备方法为：将黄瓜叶原料用乙醇溶液回流提取，过滤，滤液减压浓缩至干，得粗提取物，用水溶解，加石油醚萃取，弃掉石油醚萃取液，所得水萃余液浓缩后得黄瓜叶的水部位提取物。本发明的免疫增强剂的原料来源稳定，价廉高效，制备方法简单。
Description
技术领域
本发明属于中药提取技术领域，具体涉及一种免疫增强剂及其制备方法。
背景技术
免疫是机体的一种生理功能，机体依靠这种功能识别“自己”和“非已”成分，从而破坏和排斥进入机体的抗原物质，或机体本身所产生的损伤细胞和肿瘤细胞等，以维持机体的健康，抵抗或防止微生物或寄生物的感染或其它所不希望的生物侵入的状态。
免疫系统是机体执行免疫应答及免疫功能的重要系统。由免疫器官、免疫组织、免疫细胞和免疫分子组成，是防卫病原体入侵最有效的武器，它能发现并清除异物、外来病原微生物等引起内环境波动的因素。胸腺和脾是机体主要的中枢和外周免疫器官，胸腺是T淋巴细胞发育成熟的主要场所，脾是B淋巴细胞发育成熟的主要场所。胸腺和脾指数可直接反映免疫器官的发育状态和机体的免疫水平。
免疫增强剂是通过不同方式，达到增强机体免疫力的一类免疫治疗药物。对于免疫力低下的人群或畜禽，服用免疫增强剂可以提高其免疫力，从而预防疾病的发生。因此，寻求一种增强机体抵抗病原体感染能力免疫增强剂，这显得尤为重要。
发明内容
本发明的目的在于通过对天然产物进行研究，从而提供一种提高机体免疫功能的免疫增强剂及其制备方法。
为了实现本发明的第一个目的，发明人通过大量试验研究并不懈探索，最终获得了如下技术方案：
一种免疫增强剂，由活性成分和药学上可用的辅料制备而成，所述的活性成分是黄瓜叶的水部位提取物。其中，黄瓜叶选用葫芦科黄瓜Cucumis sativus L.的叶。
本发明的第二个目的在于提供一种根据上述免疫增强剂的制备方法，其中该方法包括如下步骤：
(1)将黄瓜叶原料用体积浓度为58％-65％乙醇溶液回流提取，过滤，滤液减压浓缩至干，得粗提取物；
(2)将步骤(1)所得粗提取物用水溶解，加石油醚萃取1-4次，得到石油醚萃取液和水萃余液，将水萃余液浓缩成稠膏，干燥，得黄瓜叶的水部位提取物；
(3)将步骤(2)所得黄瓜叶的水部位提取物与药学上可用的辅料混匀制备成口服液或口服固体制剂。
优选地，如上所述免疫增强剂的制备方法，其中步骤(1)中所述的回流提取的次数为1-3次，每次1.5-3h，每次乙醇溶液的加入量为黄瓜叶原料的5-10倍。
优选地，如上所述免疫增强剂的制备方法，其中所述的口服固体制剂包括片剂、胶囊剂或颗粒剂。
胸腺和脾是机体主要的中枢和外周免疫器官，胸腺是T淋巴细胞发育成熟的主要场所，脾是B淋巴细胞发育成熟的主要场所。胸腺和脾指数可直接反映免疫器官的发育状态和机体的免疫水平。本发明人在动物试验中发现，每日给予小鼠高、低剂量的黄瓜叶提取物，小鼠的胸腺和牌指数均较空白对照组显著增高。通过MTT法测脾淋巴细胞的增殖活性，发现两个提取物干预组均高于空白对照组，说明本发明制备的黄瓜叶提取物能提高小鼠的免疫功能。因此，本发明的第三个目的在于提供一种工业新用途，即上述的黄瓜叶的水部位提取物在制备增强免疫功能或提高免疫力的药物、保健品和食品中的应用。由于本发明制备的黄瓜叶提取物既可以用于具有免疫缺陷的患者，也可以用于提高畜禽的免疫力，尤其是幼龄畜禽，因此在工业应用时所述的药物可制备成人用医药或兽药。
与现有技术相比，本发明的免疫增强剂原料来源稳定，价廉高效，制备方法简单，且天然无毒无残留，可用于制备成提高机体免疫力的人用医药或兽药，以及制备成各种保健品、营养强化食品等。
具体实施方式
以下是本发明所涉及黄瓜叶提取物的具体制备例和临床应用例，对本发明的技术方案做进一步作描述，但是本发明的保护范围并不限于该实施例。需要说明的是，各实施例中的黄瓜叶选用葫芦科黄瓜Cucumis sativus L.的叶。
实施例1黄瓜叶提取物的制备
称取黄瓜叶干品0.61kg，研磨至末，置于带有圆底烧瓶的提取装置中，用体积浓度为60％的乙醇溶液8L回流提取2h，过滤，得到醇提液，重复2次，合并醇提液，减压浓缩至干，得粗提取物，用0.5L蒸馏水溶解，用石油醚等体积萃取3次，弃掉石油醚萃取液，将水萃余液减压浓缩成稠膏，冷冻干燥，粉碎成细粉，过100目筛，得到黄瓜叶提取物。
实施例2黄瓜叶提取物增强小鼠免疫功能的试验研究
试验用36只6～8周龄清洁级雌性昆明小鼠随机分为如下三组：空白对照组，提取物高、低剂量组，每组12只。试验开始时称量小鼠体质量。将黄瓜叶提取物溶于水中，每天每只小鼠灌胃0.5mL左右。提取物各组灌胃给予本发明实施例1制备的黄瓜叶提取物进行干预，给药量为：提取物高剂量组小鼠1.8g/kg体质量；提取物低剂量组小鼠0.6g/kg体质量；空白对照组以等体积生理盐水灌胃。所用小鼠连续灌胃相应的受试物饲养14d，第15天颈椎脱臼处死，称小鼠体质量。解剖取出胸腺和脾，吸干表面血迹，用电子天平称取湿质量，计算胸腺和脾指数。
另外，按如下方法进行脾淋巴细胞增殖活性的测定：无菌取脾，在3mL预冷含1％双抗的PBS缓冲液中用注射器冲脾细胞，冲至脾发白。将冲洗液收集于5mL离心管中，1500r/min离心10min。弃上清，每管加2mL红细胞裂解液，重悬细胞。静置3min，1500r/min离心10min。用1％双抗-PBS洗2次。用含1％双抗的1640重悬细胞。调整细胞浓度至50万个/mL。取96孔板四周孔加200μL PBS(防止边缘效应)，其余各孔加入细胞悬液100μL，干预组加待测药物100μL(提取物高、低剂量组的药物浓度分别为72、14μg/μL)，空白对照组加培养液100μL，每组3个复孔。37℃孵育72h。培养结束前4h，于培养板各孔内加人5mg/mL MTT液，10μL/孔。37℃培养4h。1500r/min离心10min，弃上清，各孔内加入150μLDMSO，溶解10min，30min内用酶标仪测定490nm处的吸光度(OD)值。
根据表1的试验结果可见，试验前，各组小鼠体质量无明显差异，而试验后提取物高、低剂量组小鼠体质量均较空白对照组显著增加(P＜0.01)，这表明黄瓜叶提取物能促进小鼠的生长，且与使用剂量关系不大。
表1试验前后各组小鼠体质量比较
与空白对照组比较，★P＜0.05，★★P＜0.01。
根据表2的试验结果可见，提取物高、低剂量组的胸腺和脾指数都明显高于空白对照组，这表明本发明制备的黄瓜叶提取物能有效提高小鼠的免疫器官指数。
表2各组免疫器官指数比较
与空白对照组比较，★P＜0.05，★★P＜0.01。
通过表3的试验结果可以看出，脾淋巴细胞经黄瓜叶提取物诱导后，细胞活性增强，提取物组明显高于空白对照组(P＜0.05或P＜0.01)。
表3各组脾淋巴细胞增殖活性比较
与空白对照组比较，★P＜0.05，★★P＜0.01。
值得注意的是，试验过程中本发明人曾以实施例1制备的醇提物、石油醚部位分别作为受试物，结果发现醇提物组的免疫增强活性不明显，相比空白对照组不具有统计学意义(P＞0.05)：而石油醚部位则完全没有免疫增强活性，甚至免疫器官指数不如空白对照组。
Claims (3)
1.一种免疫增强剂的制备方法，其特征在于，该免疫增强剂由活性成分和药学上可用的辅料制备而成，所述的活性成分是黄瓜叶的水部位提取物；
该方法包括如下步骤：
(1)将黄瓜叶原料用体积浓度为58％-65％乙醇溶液回流提取，过滤，滤液减压浓缩至干，得粗提取物；
(2)将步骤(1)所得粗提取物用水溶解，加石油醚萃取1-4次，得到石油醚萃取液和水萃余液，将水萃余液浓缩成稠膏，干燥，得黄瓜叶的水部位提取物；
(3)将步骤(2)所得黄瓜叶的水部位提取物与药学上可用的辅料混匀制备成口服液或口服固体制剂。
2.根据权利要求1所述的免疫增强剂的制备方法，其特征在于，步骤(1)中所述的回流提取的次数为1-3次，每次1.5-3h，每次乙醇溶液的加入量为黄瓜叶原料的5-10倍。
3.根据权利要求1所述的免疫增强剂的制备方法，其特征在于，所述的口服固体制剂包括片剂、胶囊剂或颗粒剂。
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| CN103039622A (zh) * | 2011-10-14 | 2013-04-17 | 张延霖 | 一种黄瓜豆浆的制作方法 |
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