CN108471749A - 氨基聚糖酯及其应用 - Google Patents
氨基聚糖酯及其应用 Download PDFInfo
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- CN108471749A CN108471749A CN201780005546.8A CN201780005546A CN108471749A CN 108471749 A CN108471749 A CN 108471749A CN 201780005546 A CN201780005546 A CN 201780005546A CN 108471749 A CN108471749 A CN 108471749A
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- aminoglycan
- animal
- ester
- phosphate
- virus
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Classifications
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Abstract
一种生物新材料,其是将氨基聚糖与磷酸或磷酸酯或盐或衍生物在催化剂作用下于pH2.1到pH4.9的液体反应介质中制成的,得到的材料具有增强人体和动物免疫抗性,治疗病毒或细菌引起的人体和动物疾病,增强动物抗应激能力,增加动物食欲,促进动物生长,抑制肿瘤生长和降低血脂水平以及防治植物病毒病的作用。
Description
本发明属于生物技术领域,涉及用于人体健康使用和动物饲喂或饮用的免疫抗性增强剂,即一种新的氨基聚糖酯及其应用。
随着社会文明的不断进步,人们更加意识到环境中的毒素对人体健康的影响,从空气,饮用水和食品都含有破坏细胞的物质,人类现代生活节奏快,工作压力大,时间长,睡眠不足等问题又容易引起免疫能力下降,从而极易被这些物质伤害,受到病菌侵染而罹病。而抗生素和许多药物的大量使用导致病原菌抗性发展严重,甚至出现变异而难以被药物杀灭,例如禽流感病毒等。研究发现,如果能够及时补充保护细胞的健康营养成分,将有助于细胞抵御有害物质和病菌的侵蚀,进而保护人体健康。
同样的,在养殖业中激素或抗生素等的大量使用,动物生长速度快,动物抗应激的能力显著减弱,免疫抗性低,也非常容易造成疾病的流行,导致动物大量死亡。在应激环境下,动物机体摄食中枢的兴奋性降低,神经内分泌失调,甲状腺素、肾上腺素等激素分泌紊乱,机体免疫机能下降等。例如,猪养殖方面的主要表现在以下几方面:1)采食量降低,生长猪增重缓慢。2)种猪繁殖障碍。母猪卵巢机能减退、流产、受胎率下降、死胎数增加、泌乳量下降、仔猪成活率降低等;种公猪性欲低下、精液品质下降,精子数量减少、活力降低、畸形率升高等。3)皮炎肾病综合症、仔猪下痢、无名高热病、慢性回肠炎等疾病增多。4)猪肉品质降低。5)病毒病流行。在水产品方面则出现虾蟹等生长缓慢,脱壳困难,疾病流行,大量死亡。
传统方法主要通过添加抗应激添加剂如脂肪、合成氨基酸、电解质、矿物质、维生素和有机酸类等增强动物的抗应激能力,或通过使用抗生素和疫苗来增强动物的抗病能力,可以起到一定作用。但由于这些产品的大量使用,不仅带来了愈加受到关注的兽药残留等食品安全问题,也导致了病原微生物日益严重的抗性产生,对养殖动物和人体健康均构成了严重挑战。
近年,国内外一些专家学者开始关注多糖类物质作为抗应激免疫增强剂在提高人体和动物机体的免疫抵抗力方面的作用。国内外研究发现,多糖如壳聚糖可以增加动物的抗应激免疫力,应用在动物的饲料添加剂中,可以增强动物的抗应激能力和免疫抗性。但是,由于单一成分的应用在实际应用中作用不稳定,多糖的效果不突出,动物已经出现应激反应或生病后使用根本没有效果。
发明内容
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
为了克服现有技术的不足,本发明将氨基聚糖与磷酸或磷酸酯或它们的盐或衍生物在催化剂(特别是金属盐或微生物)作用下生成的氨基聚糖酯制剂,发现其相比于单一多糖能显著地激活人体和动物细胞免疫功能,从而提高人体和动物机体免疫力,具有治疗病毒和细菌引起的人体和动物疾病,并具有抑制肿瘤和防治植物病害的作用。
本发明提供的氨基聚糖酯具有如下所示结构单元中的至少一种:
其中,X各自独立地为-OH、-O-CH2-CH2-N-(CH3)3、-O-CH2-CH2-NH2、金属元素或非金属元素;Y各自独立地为-OH、(例如,R1和R2可以各自独立地为-CH3)、金属元素或非金属元素。根据本发明的一种优选实施方式,所述氨基聚糖酯为基团修饰的几丁聚糖(即,具有几丁聚糖骨架结构)。
本发明提供的氨基聚糖酯可以由氨基聚糖与磷酸或磷酸酯或它们的盐或衍生物在pH2.1到pH4.9的液体反应介质中在催化剂的作用下制得。为了获得纯度更高的氨基聚糖酯,还可以进行乙醇沉淀。将氨基聚糖与磷酸或磷酸酯或它们的盐或衍生物在催化剂作用下制成的新材料的功能包括但不局限于用于激活人体和动物细胞免疫功能,从而提高人体和动物机体免
疫力,治疗病毒和细菌引起的人体和动物疾病,并具有抑制肿瘤的作用,还具有防治植物病害(特别是花叶病毒)的作用。
选用的氨基聚糖可以是由含有氨基的2-7000个单糖聚合的。构成这些聚糖的基础分子式为含有氨基的6个碳原子的单糖,可以来自植物、动物或微生物,包括但不局限于:氨基多糖,氨基寡糖,几丁聚糖,糖胺聚糖,壳聚糖,黄芪多糖,人参多糖,三七多糖,竹荪多糖,灵芝多糖,香菇多糖,酵母聚糖等。最优选地,所述氨基聚糖为几丁聚糖。
优选地,所选用的磷酸或磷酸酯或它们的盐或衍生物,包括但不局限于磷酸钠,磷酸氢钠,磷酸钾,磷酸氢钾,磷脂酰乙醇胺,磷脂酰胆碱,磷脂酰丝氨酸,磷脂酰肌醇,磷脂酰甘油,二磷脂酰甘油,磷脂酸,卵磷脂等,最优选为卵磷脂。
本发明中,所述催化剂可以包括但不局限于:金属盐和/或微生物。优选地,所述催化剂包括但不局限于氯化钙、氯化钠、氯化钾、氧化锌、氧化钙、过磷酸钙、三磷酸钠、碳酸氢钠、磷酸一氢钠,多磷酸钠、磷酸二氢钾、磷酸氢二钾、硫酸钠、硫酸锌、硫酸铜、醋酸锌、硫酸钾、碳酸钙、氨基酸钙、氨基酸锌、葡聚糖钙、葡聚糖锌、碳酸钠、碳酸氢钙、碳酸氢钾、硫酸钙、硝酸钙、葡萄糖钙、碳酸钾和生物酶中的至少一种。更优选地,所述催化剂为磷酸氢盐(如磷酸一氢钠和/或磷酸二氢钾)和/或木霉菌(如哈茨木霉菌)的发酵产物。所述发酵产物是指将微生物(如木霉菌,例如哈茨木霉菌)接种至发酵培养基中发酵培养后获得的产物。
上述所用的原料成分的材料均可以从市场上购买。
本发明提供的氨基聚糖酯制剂由氨基聚糖与磷酸酯或它们的盐或衍生物在木霉菌的发酵产物和磷酸氢盐的存在下且pH为2.1-4.9的条件下温育后固液分离而获得。根据本发明优选的实施方式,所述氨基聚糖为几丁聚糖,所述磷酸酯或它们的盐或衍生物为卵磷脂,所述木霉菌为哈茨木霉菌(trichodermaharzianum),所述磷酸氢盐为磷酸一氢钠和/或磷酸二氢钾,
所述氨基聚糖、磷酸酯或它们的盐或衍生物和磷酸氢盐的重量比为1.5-3∶0.8-1.5∶0.8-1(优选2∶1∶1)。所述温育的条件可以包括:温度为25-30℃,时间为70-80h。固液分离后还可以经无水乙醇沉淀(取固形物),以获得纯度更高的氨基聚糖酯。
本发明还提供了上述氨基聚糖酯或氨基聚糖酯制剂在增强人体和动物免疫抗性、治疗病毒或细菌引起的人体和动物疾病、增强动物抗应激能力、增加动物食欲、促进动物生长、抑制肿瘤生长、降低血脂水平以及防治植物病毒病中的应用,特别是在制备用于增强人体和动物免疫抗性、治疗病毒或细菌引起的人体和动物疾病、增强动物抗应激能力、增加动物食欲、促进动物生长、抑制肿瘤生长、降低血脂水平以及防治植物病毒病的药剂中的应用。
本发明中,述及的人体或动物疾病包括但不局限于:为害人体健康的疾病如病毒引起的疾病,所述病毒包括但不限于流感病毒、甲肝病毒、乙肝病毒、丙肝病毒、艾滋病毒和疱疹病毒,以及细胞癌变引起的肿瘤疾病,以及造成动物的疾病,包括但不局限于为害禽畜和鱼虾蟹水产的病毒和细菌性疾病,如禽流感、猪流感、蟹环爪病、水霉病和链球菌病。
本发明解决的技术问题是氨基聚糖与磷酸盐或其衍生物在在pH2.1到pH4.9的液体反应介质中催化制成的生物新材料,这种新材料可以用于抑制肿瘤生长,降低血脂水平,增强人体和动物免疫抗性,治疗病毒或细菌引起的人体和动物疾病,增强动物抗应激能力,增加动物食欲,促进动物生长,防治植物病害。
本发明的创新点一是将含有氨基的2-7000的单糖聚合的多糖与一种或多种磷酸或磷酸酯或它们的盐或衍生物反应制成的氨基聚糖酯。创新点二是这种反应是在pH2.1到pH4.9的液体介质中反应完成的。创新点三是本发明的氨基聚糖酯具有显著激活人体和动物细胞免疫功能,提高人体和动物机体免疫力,从而治疗病毒和细菌引起的人体和动物疾病,并具有抑制
肿瘤的作用,以及防治植物病毒病的作用。
本发明根据下述实例做进一步的描述,本领域技术人员知道,下述实施例对本发明仅仅起到说明的作用。在不背离本发明精神的前提下,对本发明所做的任意改进和替代均在本发明保护的范围内。
实施例1
本发明的氨基聚糖酯制剂的制备
以可溶性淀粉25g,碳酸钙5g,磷酸二氢钾2g,硫酸铵5g配制组合,另加玉米油4.0mL,pH自然,加水1000mL制成液体发酵培养基,在121℃灭菌20min。用接种环将纯化的哈茨木霉菌(购自成都特普生物科技股份有限公司)孢子转移到含无菌水的试管中,搅拌使孢子从菌丝上分散下来,用无菌纱布过滤除去菌丝后,将孢子浓度调节至107cfu/mL,按照1%的接种量接种到液体发酵培养基中,在28℃、150r/min培养48小时后,pH达到2.1,加入已灭菌的几丁聚糖(购自Sigma-Aldrich公司)10g、卵磷脂(购自Sigma-Aldrich公司)5g和磷酸一氢钠5g,继续在28℃、150r/min培养72小时,过滤收集反应液,加入2-3倍无水乙醇沉淀,抽滤,固形物用水稀释,获得氨基聚糖酯微乳剂,调整氨基聚糖酯的含量为0.01mg/ml。
经检测,该实施例制得的氨基聚糖酯微乳剂中含有结构单元如
所示的氨基聚糖酯(X为-O-CH2-CH2-N-(CH3)3,Y为)。
实施例2
本发明的氨基聚糖酯制剂的增强免疫抗性
将15只食蟹猴(云南灵长类实验动物有限公司)按照国际标准的实验动物的饲养管理和实验操作《实验动物饲养管理和使用指南》进行管理,分为3组,A组以灭菌水作为对照,将实施例1获得的氨基聚糖酯加水稀释后按B和C两个剂量给药,B组为氨基聚糖酯(3mg/3ml/kg),C组为氨基聚糖酯(9mg/3ml/kg),每组5只。每天早上10:00(±30min)对动物给药。各组给予的药物和剂量如下表所示:
表一试验分组
分组 | 处理 | 剂量(药量/水/体重) |
A组 | 灭菌水 | 0mg/3ml/kg |
B组 | 氨基聚糖酯 | 3mg/3ml/kg |
C组 | 氨基聚糖酯 | 9mg/3ml/kg |
免疫学抗体检测证明,饲喂实施例1获得的氨基聚糖酯的试验B组和C组都促进了血中免疫球蛋白IgG和IgM抗体水平的增高,尤其对IgM抗体的增高作用较明显,在给药4周时IgM抗体水平达到最高,其中B组(3mg/kg)和C组(9mg/kg)的IgM抗体水平分别比A组(对照组)高出17%和28%。
免疫细胞因子检测证明本发明的新材料氨基聚糖酯显著增高了血中细胞因子IL10和TNF-a水平,而且随氨基聚糖酯浓度的提高而增强(如下表二所示)。
表二氨基聚糖酯增强免疫细胞因子的效果
实施例3
本发明的氨基聚糖酯制剂治疗猪病毒病的效果
选择养殖场出现有猪瘟病毒症状的猪只20头。分为氨基聚糖酯治疗组及对照组,两组患猪年龄、临床表现及病情轻重大体一致。治疗组和对照
组各10头,治疗组按3mg/kg体重加入氨基聚糖酯到饲料中混合均匀。对照组隔离饲养,常规管理。处理后每日观察猪只的精神状态、体温、采食情况。病猪用药5天后临床症状消失,体温、食欲、精神状态正常者判为治愈;病猪用药5天后食欲、精神状态好转,体温正常者判为好转;好转和治愈都判为有效;病猪用药10天后仍然精神不振、厌食或死亡判为无效。
表三氨基聚糖酯治疗猪病毒病的效果
通过饲料添加氨基聚糖酯,病猪有8头治愈,2头出现好转,治愈效果达到80%。对照组病猪有5头死亡,仅有1头好转,氨基聚糖酯有非常显著的治疗猪病毒病的效果。证明本发明的氨基聚糖酯比常规管理中采用注射抗生素类药物具有操作简便和效果优越的显著特点。
实施例4
本发明的氨基聚糖酯制剂防治植物病毒病的效果
取实施例1制备的氨基聚糖酯用于防治辣椒花叶病毒病。选取已经发生病毒病的辣椒作试验,氨基聚糖酯分为三个浓度稀释作为处理,每处理50株,重复4次。试验共设为5个处理:处理1:氨基聚糖酯稀释1000倍药液;处理2:氨基聚糖酯稀释500倍药液;处理3:氨基聚糖酯稀释300倍药液;处理4:6%寡糖·链蛋白(北京中保绿农科技集团有限公司)稀释1000倍药液;处理5:清水对照。间隔7天喷药一次,使用三次后调查发病率:观察各处理叶片发病情况,统计病情分级情况,并计算病情指数和
防治效果,结果如下表四所示。
调查分级标准:
0级:全株无病; 1级:病斑数2个以下;
3级:病斑数3-5个;5级:病斑数6-8个;
7级:病斑数9个以上;9级:病斑密布,叶片严重枯死。
病情指数=∑(各级病叶数×相对级数值)/(调查总叶总数×9)×100%
防治效果(%)=(对照处理病情指数-施药处理病情指数)/对照病情指数×100%。
表四氨基聚糖酯防治辣椒病毒病的效果
处理 | 病情指数 | 防治效果(%) |
氨基聚糖酯稀释1000倍药液 | 3.65 | 81.16 |
氨基聚糖酯稀释500倍药液 | 2.25 | 88.38 |
氨基聚糖酯稀释300倍药液 | 1.74 | 91.02 |
6%寡糖·链蛋白1000倍药液 | 5.21 | 73.10 |
清水对照 | 19.37 | —— |
从表四可以看出,清水对照植株发病严重,实施例1获得的氨基聚糖酯的防治效果较对照药剂更加显著,新生长出的辣椒叶片花叶病毒症状显著减少,而且随着氨基聚糖酯的浓度增加到500倍或300倍时,防治效果显著提高。
从以上表格示出的结果可以看出,本发明的氨基聚糖酯制剂能够有效增强动物免疫抗性、防治病毒或细菌引起的动物疾病和防治植物病害。
Claims (13)
- 一种氨基聚糖酯,其特征在于,该氨基聚糖酯具有如下所示结构单元中的至少一种:其中,X各自独立地为-OH、-O-CH2-CH2-N-(CH3)3、-O-CH2-CH2-NH2、金属元素或非金属元素;Y各自独立地为-OH、 金属元素或非金属元素。
- 一种氨基聚糖酯,其特征在于,该氨基聚糖酯由氨基聚糖与磷酸或磷酸酯或盐或衍生物混合后加入催化剂在pH2.1到pH4.9的液体反应介质中而制得。
- 根据权利要求2所述的氨基聚糖酯,其中,所述氨基聚糖是由含有氨基的由2-7000个单糖聚合的多糖,构成这些聚糖的基础分子式为6个碳 原子的单糖,可以来自植物、动物或微生物,包括但不局限于:氨基多糖、氨基寡糖、几丁聚糖、糖胺聚糖、壳聚糖、黄芪多糖、人参多糖、三七多糖、竹荪多糖、灵芝多糖、香菇多糖和酵母聚糖中的至少一种。
- 根据权利要求2所述的氨基聚糖酯,其中,所选用的磷酸或磷酸酯或盐或衍生物,包括但不局限于:磷酸钠、磷酸氢钠、磷酸钾、磷酸氢钾、磷脂酰乙醇胺、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰甘油、二磷脂酰甘油、磷脂酸和卵磷脂中的至少一种。
- 根据权利要求2所述的氨基聚糖酯,其中,所述催化剂包括但不局限于:金属盐和/或微生物。
- 根据权利要求5所述的氨基聚糖酯,其中,所述催化剂包括但不局限于氯化钙、氯化钠、氯化钾、氧化锌、氧化钙、过磷酸钙、三磷酸钠、碳酸氢钠、多磷酸钠、磷酸二氢钾、磷酸氢二钾、磷酸一氢钠、硫酸钠、硫酸锌、硫酸铜、醋酸锌、硫酸钾、碳酸钙、氨基酸钙、氨基酸锌、葡聚糖钙、葡聚糖锌、碳酸钠、碳酸氢钙、碳酸氢钾、硫酸钙、硝酸钙、葡萄糖钙、碳酸钾和生物酶中的至少一种。
- 根据权利要求5所述的氨基聚糖酯,其中,所述微生物包括但不局限于:根霉菌、曲霉菌、木霉菌、青霉菌、色杆菌、节杆菌、肠杆菌、乳酸杆菌、芽孢杆菌、酵母菌和假单胞菌中的至少一种。
- 一种氨基聚糖酯制剂,其特征在于,该制剂由氨基聚糖与磷酸或磷酸酯或盐或衍生物在木霉菌的发酵产物和磷酸氢盐的存在下且pH为2.1-4.9的条件下温育后固液分离而获得。
- 根据权利要求8所述的制剂,其中,所述氨基聚糖为几丁聚糖,所述磷酸或磷酸酯或盐或衍生物为卵磷脂,所述木霉菌为哈茨木霉菌(trichodermaharzianum),所述磷酸氢盐为磷酸一氢钠和/或磷酸二氢钾,所述氨基聚糖、磷酸或磷酸酯或盐或衍生物和磷酸氢盐的重量比为1.5-3∶0.8-1.5∶0.8-1。
- 根据权利要求8或9所述的制剂,其中,所述温育的条件包括:温度为25-30℃,时间为70-80h。
- 权利要求1-7中任意一项所述的氨基聚糖酯或权利要求8-10中任意一项所述的氨基聚糖酯制剂在增强人体和动物免疫抗性、治疗病毒或细菌引起的人体和动物疾病、增强动物抗应激能力、增加动物食欲、促进动物生长、抑制肿瘤生长、降低血脂水平以及防治植物病毒病中的应用。
- 权利要求1-7中任意一项所述的氨基聚糖酯或权利要求8-10中任意一项所述的氨基聚糖酯制剂在制备用于增强人体和动物免疫抗性、治疗病毒或细菌引起的人体和动物疾病、增强动物抗应激能力、增加动物食欲、促进动物生长、抑制肿瘤生长、降低血脂水平以及防治植物病毒病的药剂中的应用。
- 根据权利要求11或12所述的应用,其中,人体或动物疾病包括但不局限于:为害人体健康的疾病如病毒引起的疾病,所述病毒包括但不限于流感病毒、甲肝病毒、乙肝病毒、丙肝病毒、艾滋病毒和疱疹病毒,以及细胞癌变引起的肿瘤疾病,以及造成动物的疾病,包括但不局限于为害禽畜和鱼虾蟹水产的病毒和细菌性疾病,如禽流感、猪流感、蟹环爪病、水霉病和链球菌病。
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