CN103768594A - 中药桑杜糖免疫增强剂及其应用 - Google Patents

中药桑杜糖免疫增强剂及其应用 Download PDF

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CN103768594A
CN103768594A CN201410010826.6A CN201410010826A CN103768594A CN 103768594 A CN103768594 A CN 103768594A CN 201410010826 A CN201410010826 A CN 201410010826A CN 103768594 A CN103768594 A CN 103768594A
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folium mori
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蒋春茂
陈晓兰
王德云
杨海峰
武彩虹
郑义
戴建华
陈未
何祥来
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Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.
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Abstract

本发明涉及一种由中药桑叶多糖和杜仲多糖制成的免疫增强剂,由桑叶多糖和杜仲多糖制成,本发明的桑杜糖増免剂在体外能显著刺激鸡淋巴细胞增殖;配合新城疫疫苗免疫雏鸡,能显著提高血清抗体效价,促进淋巴细胞增殖,增强雏鸡细胞免疫和体液免疫;配合猪蓝耳疫苗免疫仔猪,能显著提高血清抗体效价;配合猪蓝耳疫苗免疫蛋鸡,能显著提高蓝耳卵黄抗体效价,提高疫苗的免疫应答。

Description

中药桑杜糖免疫增强剂及其应用
技术领域
本发明涉及一种中药桑杜糖免疫增强剂,由桑叶多糖和杜仲多糖组成,简称桑杜糖増免剂,属于畜禽免疫佐剂领域。
背景技术
动物的病毒性传染病,如高致病性禽流感、新城疫、传染性法氏囊病等,严重危害畜牧业的发展,造成严重的经济损失,一直受到世界的广泛关注。免疫接种是防控这些疫病暴发的最好措施。然而单独接种疫苗通常产生相对较弱的免疫力。免疫佐剂能增强疫苗的免疫应答,使保护时间延长。许多免疫佐剂如弗氏佐剂和脂多糖因易引起全身性反应(如恶心,发热,过敏反应,嗜酸性粒细胞增多,中毒,麻痹以及自身免疫性疾病)和局部炎症反应(炎症,疼痛,肿胀,坏死,溃疡和脓肿)而限制了其广泛应用。因此,研制高效安全的新型免疫佐剂成为防控动物传染病的热点。许多中药证明具有免疫增强作用,且高效安全。但大多数组方复杂,给药不便。随着集约化养殖业的发展,动物群体饮水给药具有更明显的优势。本发明,进行了组分药筛选、方剂筛选和剂量筛选,结合便于应用等方面综合比较,研制成功由桑叶多糖和杜仲多糖制成的桑杜糖増免剂。
发明内容
本发明针对当前动物疫病防控现有技术中急需高效、安全的免疫增强剂或疫苗佐剂的问题,提供一种由桑叶多糖和杜仲多糖制成的新型中药免疫增强剂。
本发明一种新型中药免疫增强剂,其特征在于,每1000ml药液由桑叶150g和杜仲皮145g制成。药液中的桑叶多糖含量不低于0.2%,杜仲多糖的含量不低于0.1%。
生产每1000mL药液的工艺流程为:桑叶水煎2次,合并滤液,醇沉后干燥,640mL水溶解;杜仲乙醇脱脂后,水煎2次,合并滤液,醇沉后干燥,360mL水溶解。两种溶液混匀,分装,即得。
桑叶多糖的提取工艺为桑叶加30倍质量水煎煮2次,第一次1.5小时,第二次1小时。杜仲多糖的提取工艺为杜仲皮加4倍质量无水乙醇回流脱脂2次,后水煎2次,第一次1.5小时,第二次1小时。
有益效果:本发明的基础工作,首先选择有免疫增强作用的中药多糖,比较它们的增强免疫作用,筛选出作用较好的桑叶多糖、杜仲多糖,按不同比例组成多个复方,通过系列试验比较,证明复方的效果强于单味药,筛选出效果最好的组方配比(表1)。
本领域的技术人员可以通过正交试验确定了桑叶多糖和杜仲多糖的提取条件,优化多糖溶解性条件,确定了桑杜糖増免剂的生产工艺。
与现有技术相比,本发明优点如下:
1.与化学佐剂相比,桑杜糖増免剂效果可靠,且来源于天然中药,无毒副作用,无药残隐患,保障动物源食品安全;生产对环境无污染,符合绿色环保的要求。
2.与传统中药方剂相比,桑杜糖増免剂处方精炼,成分明确,工艺简化,质量可控,给药方便,成本较低。
3.与同类中药成分方剂相比,桑杜糖増免剂功效提高,配合新城疫疫苗免疫雏鸡,能显著增强细胞免疫和体液免疫,提高疫苗的免疫应答。
具体实施方式
1.制备工艺
(1)桑叶多糖的制备以提取物中的桑叶多糖得率为指标,对加水量、提取时间、提取次数、醇沉用乙醇浓度进行正交试验,确定桑叶多糖的最佳提取工艺为加30倍水,煎煮2次,第一次1.5h,第二次1h。取桑叶150g,按以上工艺水煎,合并滤液,加入95%乙醇使醇含量达75%,静置12h,沉淀65℃干燥,640mL水溶解,得桑叶多糖水溶液。
(2)杜仲多糖的制备以提取物中的桑叶多糖得率为指标,对脱脂乙醇量、加水量、提取时间、提取次数进行正交试验,确定杜仲多糖的最佳提取工艺为加4倍质量95%乙醇回流脱脂2次,加20倍水,煎煮2次,第一次1.5h,第二次1h。取杜仲皮145g,按以上工艺脱脂,水煎,合并滤液,加入95%乙醇使醇含量达75%,静置12h,沉淀65℃干燥,360mL水溶解,得桑叶多糖水溶液。
(3)药液制备将桑叶多糖溶液和杜仲多糖溶液混合,分装。用苯酚-硫酸法测定多糖含量,HPLC法测定芦丁和松脂醇二葡萄糖苷的含量。药液中多糖含量不低于0.3%、芦丁含量不低于0.025%,松脂醇二葡萄糖苷不低于0.01%。
2.增强免疫效果比较
(1)体外增强免疫效果
以桑杜糖増免剂为试验材料,另制备对照方1(桑叶提取物+杜仲多糖)和对照方2(桑叶多糖+杜仲提取物2份)。首先测定3个方剂对鸡外周血淋巴细胞的安全浓度,然后将3个方剂用细胞培养液(RPMI1640)分别稀释成250、125、62.5、31.25和15.625μg/mL一共5种个工作浓度,分别加入到培养的鸡外周血淋巴细胞中,用MTT法测定淋巴细胞增殖(A570值),作为淋巴细胞增殖的指标。并按公式计算淋巴细胞刺激指数(SI):SI=(药物组值—细胞对照组
Figure BDA0000455220000000034
值)/细胞对照组
Figure BDA0000455220000000035
值(其中
Figure BDA0000455220000000036
为平均值),比较3个方剂的作用强弱。
结果:①淋巴细胞增殖的变化:桑杜糖増免剂在250、125、62.5和31.25μg·mL-1时A570值显著大于细胞对照组(P﹤0.05),对照方1和对照方2在250、125和62.5μg·mL-1时A570值显著大于细胞对照组(P﹤0.05)。
表1各组淋巴细胞增殖的变化(A570值)
Figure BDA0000455220000000031
注:同列数据标注不含相同字母者差异显著(P﹤0.05),以下表同。
②淋巴细胞刺激指数的比较:桑杜糖増免剂在250、125、62.5和31.25μg·mL-1时刺激指数显著大于两个对照方组(P﹤0.05),在15.625μg·mL-1稍大于2个对照方组(表2)。
表2各方剂刺激淋巴细胞增殖效果的比较(SI)
Figure BDA0000455220000000032
(2)体内增强免疫试验
方法:以桑杜糖増免剂为试验材料,另制备对照方1(桑叶提取物+杜仲多糖)和对照方2(桑叶多糖+杜仲提取物2份)。取14日龄非免疫健康罗曼蛋公鸡150羽,随机均分为5组,除空白对照组外均用新城疫IV苗滴鼻、点眼免疫,2羽份/羽,28日龄二免。在首次免疫和二次免疫的同时,3个中药组同时饮水给以相应药物,连用3天,疫苗对照组和空白对照组自由饮水,分别于免疫后第7(D7)、14(D14)、21(D21)和28(D28)天每组随机抽取6羽采血,分离血清,用β-微量法检测ND-HI抗体效价,用MTT法测定外周血T淋巴细胞增殖。
结果:
①桑杜糖增免剂组在免疫后各时间点的的抗体效价均最高,在首次免疫后第14天(D14)比对照方2高0.53个滴度,且显著高于除对照方2外其余各组(P<0.05);在首免后第21天(D21),显著高于其余各组,分别比对照方1高1.43个滴度,对照方2高1.09个滴度;在首免后第28天(D28),显著高于其余各组,分别比对照方1高1.43个滴度,对照方2高1.23个滴度(表3),表明桑杜糖増免剂能显著提高血清抗体效价,效果优于两个对照方。
表3各组抗体效价的动态变化
Figure BDA0000455220000000041
②淋巴细胞增殖变化:
桑杜糖増免剂组在首次免疫后4个时间点的A570值均为最高,在首次免疫后第7天(D7)显著高于免疫对照和空白对照组(P﹤0.05),略高于两个对照方组;在首免后第14、28天(D14、D28)显著高于对照方1(P﹤0.05),略高于对照方2;在首免后第21天(D21),显著高于其余各组(P﹤0.05)(表4)。
表4各组淋巴细胞增殖的动态变化(A570
Figure BDA0000455220000000042
以上结果表明,桑杜糖増免剂配合新城疫疫苗免疫雏鸡,能显著促进淋巴细胞增殖,增强雏鸡的细胞免疫,提高新城疫疫苗的免疫应答。
3.桑杜糖増免剂最佳剂量的筛选
方法:取14日龄免疫健康罗曼蛋公鸡210羽,随机均分为9组,除空白组外均用新城疫IV苗滴鼻、点眼免疫(2羽份/羽),28日龄二免。在首免和二免的同时,桑杜糖増免剂5个剂量组,按照8、6、4、2和1mg/羽饮水给药,连用3天,免疫对照组和空白对照组正常饮水,分别于首次免疫后第7(D7)、14(D14)、21(D21)和28(D28)天采血,用β-微量法检测ND-HI抗体效价,用MTT法测定6、4、2mg组的淋巴细胞增殖。
结果:①血清抗体效价的变化:桑杜糖増免剂各剂量组在各时间点多明显或显著高于免疫对照组和空白对照组。在首次免疫后第7天(D7),4mg组抗体效价最高,且显著高于2mg、1mg剂量组(P﹤0.05);在首免后第14天(D14),6mg组抗体效价最高,且4、6、8mg剂量组均显著高于其它给药组(P﹤0.05);在首免后第21、28天(D21、D28),4mg组显著高于其它给药组(P﹤0.05)(表5)。
表5各组血清ND-HI抗体效价的变化(log2
Figure BDA0000455220000000051
②淋巴细胞增值的变化:桑杜糖増免剂各剂量组在各时间点A570值多明显或显著高于免疫对照组和空白对照组。在首次免疫后第7天(D7),桑杜糖増免剂三个给药剂量组的A570值均显著高于免疫对照组(P﹤0.05);在D14,6、4mg组显著高于免疫对照组和空白对照组(P﹤0.05);在D21,6、4mg组显著高于其余3组(P﹤0.05);在D28,4mg剂量组显著高于其余各组(P﹤0.05)(表6)。
表6淋巴细胞增殖的变化
Figure BDA0000455220000000052
以上结果表明,桑杜糖増免剂在合适剂量时能显著提高新城疫疫苗免疫雏鸡的血清抗体效价,促进外周血淋巴细胞增殖,增强雏鸡的体液免疫和细胞免疫功能,能显著提高新城疫疫苗的免疫效果;各剂量相比,雏鸡饮水给药4mg为最佳剂量。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何不经过创造性劳动想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应该以权利要求书所限定的保护范围为准。

Claims (7)

1.一种中药免疫增强剂,其特征在于由桑叶多糖和杜仲多糖制成,每1000ml免疫增强剂由桑叶100-200g和杜仲皮100-200g制成,优选由桑叶140-160g和杜仲皮135-155g制成;所述的免疫增强剂中包括或者不包括额外的中药提取物;进一步优选的,不含有其它化学活性成分。
2.根据权利要求1所述的中药免疫增强剂,其特征在于,药液中的桑叶多糖含量介于0.2-0.6%之间,杜仲多糖的重量百分含量介于0.1-0.5%之间。
3.权利要求1或2所述中药免疫增强剂,其特征在于,由以下方法制备而成:桑叶水煎2次,醇沉后,干燥醇沉物,水溶解,得桑叶多糖溶液;杜仲脱脂后,醇沉,干燥醇沉物,水溶解,得杜仲多糖溶液,两种溶液混匀,分装。
4.根据权利要求3所述中药免疫增强剂,其特征在于,桑叶多糖的提取工艺为桑叶加30倍质量水煎煮2次,第一次1.5小时,第二次1小时,多糖的提取工艺为杜仲皮加5倍质量无水乙醇回流脱脂2次,后20倍水煎2次,第一次1.5小时,第二次1小时。
5.权利要求1-4任意一项所述的中药免疫增强剂在增强疫苗免疫应当中或者在制备增强疫苗免疫应当的药物中的应用,优选的,所述的免疫疫苗为新城疫疫苗或猪蓝耳疫苗。
6.一种疫苗试剂盒,其特征在于所述的疫苗试剂盒包括权利要求1-4任意一项所述的中药免疫增强剂。
7.根据权利要求6所述的疫苗试剂盒,所述的疫苗试剂盒中包括新城疫疫苗或猪蓝耳疫苗,优选的,所述的中药免疫增强剂以每单位剂量疫苗配合1mL中药免疫增强剂的比例存在于试剂盒中。
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