CN115466334A - 一种饲用速溶黄芪多糖粉剂的制备方法及应用 - Google Patents
一种饲用速溶黄芪多糖粉剂的制备方法及应用 Download PDFInfo
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- CN115466334A CN115466334A CN202210990031.0A CN202210990031A CN115466334A CN 115466334 A CN115466334 A CN 115466334A CN 202210990031 A CN202210990031 A CN 202210990031A CN 115466334 A CN115466334 A CN 115466334A
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- astragalus
- polysaccharide
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- astragalus polysaccharide
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Abstract
本发明一种饲用速溶黄芪多糖粉剂制备方法及应用，包括以下工艺步骤：取黄芪水提醇沉药渣加入65℃去离子水，使酒精体积分数控制在20%，充分搅拌使黄芪水提醇沉药渣溶解，冷却至室温后，经3500转/分钟离心除去不溶的热敏性大分子蛋白质、多糖及固体残渣，得到澄清上清液。取离心后得到的上清液通过中空纤维膜超滤处理，收集10‑100 kDa的多糖组分。并通过夹层冷风冷却喷雾干燥工艺，得到低分子量速溶高免疫活性黄芪多糖粉剂，活性多糖含量在87%以上。本发明得到的速溶高免疫活性黄芪多糖粉生产成本低，生物活性高，可作为饲料添加剂应用于畜禽养殖，替代或减少抗生素的使用，具有较高的经济价值。
Description
技术领域
本发明属于畜禽养殖功能性饲料添加剂领域，具体涉及一种利用中药工厂黄芪水提醇沉药渣提取黄芪高活性多糖组分并制备速溶黄芪多糖免疫增强粉剂的方法及应用。
背景技术
我国是畜禽、水产养殖大国，也是兽用抗菌药物生产和使用大国，兽用抗菌药物在防治动物疾病、提高养殖效益、保障畜禽水产品有效供给中，发挥了重要的作用。寻找合适的饲用抗生素替代品一直是研究的热点，具有深远的意义和非常广阔的应用前景。中药及其植物提取物属于天然的活性物质，中药兼具营养和药物的双重作用，具有无抗药性、功能多样性、经济实用等特点，是目前临床上应用最广的抗生素替代产品，也是重要的抗生素替代品研究热点之一。
黄芪是豆科紫云英膜荚黄芪及内蒙黄芪的根，是我国广泛使用的一种中草药，临床上最常用的补气药，具有2000多年的历史。现代药学研究表明，黄芪含有多种活性成分，包括黄芪皂苷、黄芪黄酮、黄芪多糖等主要功效成分。在免疫调节、抗肿瘤、抗病毒、糖尿病、延缓衰老等方面具有重要地位。其中黄芪多糖是黄芪中重要活性成分之一，具有安全、高效、无残留等特点。提高动物生长性能、改善免疫性能、提高抗氧化能力等多种药理作用。以往对黄芪多糖的应用或药理活性研究主要集中在总多糖方面.工业产品如黄芪多糖口服液、黄芪多糖饲料添加剂等均使用总多糖。而总多糖的制备方法较为粗放，即便采用同一提取方法也会因具体的生产原料或批次间的参数差异而导致所得的总多糖成分有所不同，必然导致粗多糖药理活性的稳定性，因此将黄芪多糖按照分子量的大小进行分段分离获得相对均一的组分，对提高黄芪多糖的药理活性和稳定性有重要的意义。黄芪多糖主要分为大分子成分和小分子成分。其中平均分子量为10-100kDa 的多糖组分是黄芪多糖中最主要的活性成分。但由于热敏性高，普通干燥方法难以制备。同时我国黄芪的资源有限，直接采用中药黄芪来提取黄芪多糖工艺成本高，难以广泛地应用于家禽、猪、反刍动物以及水产动物的生产中。
目前，对黄芪多糖的相关产品主要集中在黄芪总多糖上，导致产品药理活性及稳定性差，10-100kDa低分子黄芪多糖组分是黄芪多糖中生物活性最高的部分。但其热敏性极高，在高温条件下极易发粘、发泡、褐变。因此少量的黄芪多糖样品通常采用冷冻干燥法，该种方法生产成本较高，处理量较小。采用喷雾干燥法是大量处理黄芪多糖的一种较好的方法，但由于黄芪多糖热敏性，粘壁较高，难以得到干粉，通常加入30％以上麦芽糊精进行混合处理，但很难得到黄芪多糖产品纯品，并有部分厂家利用目前多糖的检测方法上的缺陷，以大量的麦芽糊精混充黄芪多糖。
黄芪水提醇沉药渣是中药企业利用水提醇沉法从黄芪中提取黄芪甲苷等水溶性小分子成分后残留的废弃物，因该药渣为半固体膏状物，粘度极高，不利于运输及堆放，属于环保公司最难处理的一类废弃物，同时也是药渣处理过程中费用最高的部分。但黄芪水提醇沉半固体废渣中则含有大量的多糖等生物活性大分子，经分析，其中多糖含量占干物质的70％以上。利用黄芪水提醇沉残渣中提取的高活性黄芪多糖组分可减少中药企业环保压力，同时可降低黄芪多糖生产成本，有利于在畜禽养殖业中的广泛应用。本发明制备的速溶黄芪多糖免疫增强粉剂不仅成本低、产品溶解速度快，并经动物试验证明了其有较高的食用安全性，并对机体有良好的免疫增强作用。
发明内容
针对现有技术中存在的问题，本发明设计的目的在于提供一种饲用速溶黄芪多糖粉剂的制备方法及应用。以解决现有技术中对黄芪多糖的相关产品主要集中在黄芪总多糖上，导致产品药理活性及稳定性差的问题。
本发明具体通过以下技术方案加以实现：
一种饲用速溶黄芪多糖粉剂制备方法，包括以下工艺步骤：
1)取黄芪水提醇沉药渣加入65℃去离子水，使酒精体积分数控制在20％；
2)充分搅拌使黄芪水提醇沉药渣溶解，冷却至室温后，经3500 转/分钟离心除去不溶的热敏性大分子蛋白质、多糖及固体残渣，得到澄清上清液；
3)取离心后得到的上清液加入2倍质量的去离子水，充分搅拌后通过孔径为100KDa中空纤维膜进行超滤处理，收集100kDa以下组分。再通过10kDa中空纤维膜超滤处理，收集10-100kDa的多糖组分。
优选地，步骤3)中两次超滤处理后取分子量为10-100kDa的多糖组分，真空低温浓缩至该组分的固形物含量为40％，喷雾干燥，得到高活性低分子黄芪多糖粉。
优选地，喷雾干燥条件为：雾化器频率70Hz，进风温度控制在 140℃，出风温度控制在60℃以下，干燥塔夹层通入冷风冷却，每隔5分钟振动锤干燥塔底部振动一次，防止干燥塔壁及底部积粉并液化发泡。
上述任一制备方法制备的饲用速溶黄芪多糖粉剂在饲料添加剂中的应用。
本发明中的黄芪水提醇沉药渣为中药黄芪饮片经热水浸提后除去固体残渣部分，再加入3-4倍体积的无水乙醇后得到的醇沉半固体残渣。
本发明制备方法不仅能有效的利用废弃黄芪水提醇沉药渣，减少环境污染，而且制备出的速溶高免疫活性黄芪多糖粉，喷雾干燥出粉率可达90％，活性多糖含量在87％以上，干燥失重6％以下。本发明得到的速溶高免疫活性黄芪多糖粉生产成本低，生物活性高，可作为饲料添加剂应用于畜禽养殖，替代或减少抗生素的使用，具有较高的经济价值。
附图说明
图1为黄芪水提醇沉药渣；
图2为黄芪水提醇沉渣中乙醇醇沉分级比例；
图3为黄芪水提醇沉渣20％醇沉物中分子量分布；
图4为速溶高活性低分子量黄芪多糖粉；
图5为小鼠30天饲喂体重量变化图；
图6为30天饲喂小鼠脏器系数图；
图7为黄芪多糖粉对小鼠体重的影响,(与对照组比，*,P<0.05,**, P<0.01；与模型组比，#,P<0.05,##,P<0.01；同列数据无标记表示差异不显著(P＞0.05))；
图8为黄芪多糖粉对小鼠免疫器官脏器系数的影响，(与对照组比， *,P<0.05,**,P<0.01；与模型组比，#,P<0.05,##,P<0.01；同列数据无标记表示差异不显著(P＞0.05))；
图9为黄芪多糖粉对外周血γ-干扰素的影响，(与对照组比，*, P<0.05,**,P<0.01；与模型组比，#,P<0.05,##,P<0.01；同列数据无标记表示差异不显著(P＞0.05))；
图10为黄芪多糖粉对外周血IgG的影响，(与对照组比，*,P<0.05, **,P<0.01；与模型组比，#,P<0.05,##,P<0.01；同列数据无标记表示差异不显著(P＞0.05))；
图11为黄芪多糖粉对小鼠外周血T淋巴细胞亚群的影响，(与对照组比，*,P<0.05,**,P<0.01；与模型组比，#,P<0.05,##, P<0.01；同列数据无标记表示差异不显著(P＞0.05))。
具体实施方式
以下结合具体实施例对本发明做进一步详细描述，以便更好地理解本技术方案。
实施例1
取黄芪注射液生产企业的黄芪水提醇沉药渣50千克，加入已加热至65℃去离子水，用精密酒精计控制加水量，使最终的酒精体积分数控制在20％，搅拌20分钟至黄芪水提醇沉药渣溶解均匀。冷却至室温后，经3500转/分钟离心除去不溶性残渣，得到澄清上清液；将上清液加入2倍质量的去离子水，搅拌均匀后，以截留分子量为 100KDa的超滤膜进行超滤，收集100kDa以下组分液体。再通过10 kDa中空纤维膜超滤处理，收集10-100kDa的多糖组分。并通过真空低温蒸发浓缩，得到固形物含量为40％的浓缩截留液。采用喷雾干燥器进行干燥，雾化器频率为70Hz，进风温度控制在140℃，出风温度选择在60℃，进料速度为15千克/小时，干燥塔夹层通入20℃以下冷风冷却，每隔5min中用锤子锤干燥塔底部，防止干燥塔壁积粉及高温活性多糖液化发泡，在装有除湿设备的车间中收集干燥后的粉末得到高生物活性黄芪多糖粉。其外观呈浅黄色，易吸湿，极易溶于水，无不良气味。喷雾干燥出粉率约为90％，粗多糖含量为87％，干燥失重为6％，溶济残留未检出，重金属(砷+铅+镉+汞)含量≤10.0ppm，大肠菌群、沙门氏菌等致病菌未检出。高活性低分子黄芪多糖粉具有极高的吸湿性，收集后应密封保存。
试验例1：黄芪水提醇沉药渣成分分析及喷雾干燥工艺选择
黄芪水提醇沉药渣主要成分分析：黄芪水提醇沉药渣是黄芪采用水提醇沉法提取黄芪甲苷等水溶性小分子成分后残留的废弃物，因该药渣为半固体膏状物，粘度极大，见图1，其主要成分为水溶性活性多糖及蛋白质，见表1，黄芪多糖可作为免疫促进剂或调节剂，具有抗病毒、抗肿瘤、抗衰老、抗辐射、抗应激、抗氧化等作用。因此具有极高的保健价值和营养价值，可以开发成动物保健品或药品。
表1黄芪水提醇沉药渣成分分析
黄芪水提醇沉药渣中多糖醇沉分级：将黄芪水提醇沉渣加水溶解乙醇浓度在1％以下，离心收集上清液，上清液中加乙醇至浓度为10％，收集沉淀，再将上清液调节醇浓度为20％，收集沉淀，将上清液调节醇浓度为30％，依此类推至醇浓度为90％。将醇沉物冷冻干燥，分析各部分醇沉多糖比例。由图2可知，醇浓度30％的多糖所占比例最大，其次是20％和10％，而50％和40％醇沉物较少，表明黄芪多糖中不同醇沉部分的多糖分布是很不均匀的。不同分子量的多糖在不同浓度的乙醇中溶解度不一样，大分子多糖易在较低浓度的乙醇溶液中沉淀下来，而小分子多糖则易溶解于更高浓度的乙醇。
黄芪水提醇沉渣20％醇沉物中分子量分布：将黄芪水提醇沉渣加去离子水溶解至酒精含量为20％。离心收集沉淀，沉淀再加去离子水至完全溶解，离心收集上清液，上清液并过截留分子量为100KDa 的中空纤维膜，收集截留液，并分别测定上清液与截留液中多糖含量，计算两者比例。由图3可知，黄芪水提醇沉渣20％醇沉物中，100KDa 以下的组分占不到5％，若黄芪水提醇沉渣加去离子水溶解至酒精含量为20％，并离心除去沉淀，对目标组分(10-100KDa分子量)的损失极少，但能有效的提高后续超滤的效率。
进风温度对黄芪多糖喷雾干燥的影响：将黄芪多糖10-100kDa 组分调整至固形物含量为40％左右，在此浓度下，即有较好的流动性，同时又有较高的固形物含量，有利于提高喷雾干燥的效率。出风温度控制60℃以下，雾化器频率70Hz，选择了140，160，180，200℃等四个温度梯度，结果见表2。
表2进风温度对黄芪多糖喷雾干燥的影响
由表2可知，当进风温度为140℃时，并配以夹层冷风。黄芪多糖略有粘壁，喷雾干燥效果好。所得到的黄芪多糖色泽较浅，见图4，呈浅黄色，无结块，易吸湿，极易溶于水。
试验例2：饲用速溶黄芪多糖免疫增强粉剂30天小鼠饲喂实验
试验方法：清洁级ICR小鼠80只，体重18-24g，随机分成4组，每组20只，雌雄各半。实验组分为高、中、低剂量三个剂量组，剂量分别为1.0、2.0、4.0mg·kg-1·d-1。再设对照组一个，每天灌胃同等剂量的蒸馏水。一般观察：实验期间每天观察大鼠一般状态、体征、摄食量、饮水及粪便状况。每7d测一次体重，按体重变化调整给药量，连续观察30d。血液学指标测定：给药期满30d，禁食禁水12h 后，摘眼球取血，检查血液学指标。血液学检查项目有：白细胞数量 (WBC)、嗜中性白细胞百分比(NE％)、淋巴细胞百分比(LY％)、单核细胞百分比(MO％)、嗜酸性粒细胞百分比(EO％)、嗜碱性粒细胞(BA％)、红细胞数量(RBC)、血红蛋白浓度(Hb)、红细胞压积(HCT％)、平均红细胞压积(MCV)、平均红细胞血红蛋白含量 (MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度 (RDW％)血小板数量(PLT)、平均血小板容量(MPV)。血液生化学指标测定：给药期满30d，禁食禁水12h后，摘眼球取血，检查血液生化学指标。血液生化学指标检查项目有：谷丙转氨酶(ALT/GPT)、谷草转氨酶(AST/GOT)、总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、肌酐(CRE)、总胆固醇(CHO)、甘油三脂(TG)、葡萄糖(GLU)。大体形态变化观察：取血后立即剖取动物肝、肾、脾、胃、睾丸或卵巢等脏器称重，根据体重计算器官重量系数。并进行肉眼观察形态变化。
试验结果：
小鼠一般毒性反应：高、中、低三个剂量给药组及对照组小鼠在 30d试验期内均活动正常，行为活泼，毛色光泽，进食饮水正常，大、小便无异常改变，无一死亡。
小鼠体重变化：30天饲喂体重量变化见图5所示，由图5可知，高、中、低三个剂量给药组体重增长正常，与对照组比较，体重量未见显著性差异(P>0.05)。
小鼠脏器系数：30天饲喂小鼠脏器系数见图6所示，由图6可知，黄芪多糖粉给药30d后，各剂量组与对照组结果比较，脏器系数未见显著性差异(P>0.05)。
血常规指标：黄芪多糖粉对小鼠血常规指标的影响结果见表3。
表3黄芪多糖粉对小鼠血常规的影响
注：给药组给药后体重与正常对照组比较无显著差异(P>0.05)
由表3可知，各剂量组小鼠白细胞数量(WBC)、嗜中性白细胞百分比(NE％)、淋巴细胞百分比(LY％)、单核细胞百分比(MO％)、嗜酸性粒细胞百分比(EO％)、嗜碱性粒细胞(BA％)、红细胞数量 (RBC)、血红蛋白浓度(Hb)、红细胞压积(HCT％)、平均红细胞压积(MCV)、平均红细胞血红蛋白含量(MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度(RDW％)血小板数量(PLT)、平均血小板容量(MPV)等指标与对照组比较差异均无显著性(P>0.05)。
血生化指标：由表4可知，各剂量组小鼠谷丙转氨酶(GPT)、谷草转氨酶(GOT)、总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、肌酐(CRE)、总胆固醇(CHO)、甘油三脂(TG)、葡萄糖(GLU) 等血生化指标和对照组比较差异均无显著性(P>0.05)。
表4黄芪多糖粉对小鼠血生化的影响
注：给药组给药后体重与正常对照组比较无显著差异(P>0.05)
30d喂养小鼠的病理组织学检查：全面细致观察各组心、肝、肾、脾、胃、睾丸及卵巢等器官大小，形态、色泽、质感等均未见异常。各主要脏器无明显变性、坏死，光泽正常。
30天小鼠饲喂实验表明饲用速溶黄芪多糖免疫增强粉剂具有较高的食用安全性。
试验例3：饲用速溶黄芪多糖免疫增强粉剂免疫保护试验
实验方法:动物适应一周后，分性别按体重将其随机分组。36只幼龄小鼠随机分为3组：阴性对照组，模型对照组，黄芪多糖给药组，每组12只，雌雄各半。以水为溶剂制成黄芪多糖粉悬浮液，每日一次，连续30d给小鼠灌胃。给药量200mg·kg-1·d-1，动物每周称一次体重，根据体重变化调整给药量。模型组小鼠腹腔注射环磷酰胺，剂量80mg/kg/d，连续注射3d后，给药实验，于实验后第9d，10d，11d， 19d，20d，21d后再分别强化一次，即腹腔注射环磷酰胺，剂量80 mg·kg-1·d-1。
试验结果
黄芪多糖粉对小鼠体重量的影响：小鼠体重量的测量结果见图7，由图7可知，经环磷酰胺造模以后，模型组及其它组小鼠进食饮水正常，大、小便无异常改变，在给的造模剂量内无非正常死亡动物。但模型组出现精神较差，毛色不一，体重增长明显下降，与对照组相比，差异显著(P<0.05)。给药组能改善小鼠的体重增长，与阴性对照无显著差异(P>0.05)。
黄芪多糖粉对小鼠免疫器官脏器系数的影响：小鼠免疫器官脏器系数测定结果见图8。由图8可知，与对照组比，模型组小鼠的胸腺及脾脏脏器指数发生了变化，其中脾指数升高极显著(P<0.01)，胸腺指数显著降低(P<0.05)，表明本实验所采用的造模方法能有效的改变小鼠的免疫器官指数。黄芪多糖粉能降低脾脏指数，升高胸腺指数，从而修复受损的免疫器官。
黄芪多糖粉对小鼠外周血γ-干扰素的影响：采用ELISA法，操作方法按试剂盒操作说明进行，其检测结果见图9。γ-干扰素是因T 细胞和NK(自然杀伤性)细胞受肿瘤、抗原和分裂因子的刺激激活后所分泌。具有抗病毒、免疫调节及抗肿瘤特性等生物学功能。此外，还可活化巨噬细胞，增强自然杀伤性细胞的细胞毒作用和刺激T细胞的细胞毒作用。由9可知，与对照组相比，模型组小鼠γ-干扰素含量降低极显著(P<0.01)，表明本实验所采用的免疫抑制剂环磷酰胺及造模方法能有效的降低小鼠的γ-干扰素水平，抑制小鼠的免疫功能。黄芪多糖样品组小鼠能极显著升高γ-干扰素水平，达到正常组范围 (P>0.05)，表明黄芪多糖粉样品有提高体内γ-干扰素的水平的作用，提高免疫功能。
黄芪多糖粉对小鼠外周血IgG的影响：小鼠外周血的血清IgG 测定：采用ELISA法，操作方法按试剂盒操作说明进行。其检测结果见图10。
免疫球蛋白G(IgG)是在脾脏和淋巴结中合成，在血清中的含量最高(占Ig量的75％)，而且在血清中半衰期长，主要分布在血清和组织液中，是抗细菌、抗毒素和抗病毒抗体的主要组成部分，也是机体抗感染免疫的过程中的重要物质基础。由于IgG的含量最高，是免疫反应的最重要的物质基础。具有重要的免疫效应，是机体抗感染的主力军。由图9可知，与对照组相比，模型组小鼠IgG含量降低极其显著(P<0.01)，进一步证明本实验所采用的造模方法能有效抑制小鼠的免疫功能。与模型组相比，黄芪多糖能显提高IgG含量 (P<0.05)。
黄芪多糖粉对小鼠外周血T淋巴细胞亚群的影响：淋巴细胞亚群用流式细胞仪进行分析，利用BD CellQuest Pro数据分析软件对十字门内细胞的比例进行统计。黄芪多糖粉对小鼠外周血T淋巴细胞亚群的影响结果见图11。T淋巴细胞在机体细胞免疫和体液免疫中起着极为重要的作用，具有免疫防御、免疫调节、免疫监视等作用。研究资料表明，Th/Tc细胞的平衡在自身免疫性疾病的发生、发展过程中起重要作用。CD4是T辅助/诱导细胞(Th/Ti)的表面标志，CD4+T细胞具有辅助T细胞转变成效应细胞、B细胞转化成浆细胞以及活化巨噬细胞等功能，起辅助、诱导细胞及体液免疫的作用。CD8+是T抑制/杀伤细胞(Ts/Tc)的表面标志，CD8+T细胞具有细胞毒效应，它能抑制T细胞活化、抑制B细胞产生抗体，起抑制细胞及体液免疫的作用。Th细胞是机体免疫应答的中心细胞，CD4/CD8(Th/Tc) 比例变化反应机体的免疫功能状况，比例升高表明免疫功能亢进，比例降低表明免疫功能低下。由图10可知，模型组CD3+CD4+, CD3+CD8+及CD4/CD8降低极显著(P<0.01)，表明本实验所采用的免疫抑制剂环磷酰胺及造模方法能有效的抑制小鼠的细胞免疫功能。黄芪多糖粉可显著提高免疫抑制小鼠外周血中CD3+CD4+(Th)细胞的数量。此外，黄芪多糖粉还可显著提高免疫抑制小鼠外周血中 CD4/CD8的比值(P<0.01)，表明黄芪多糖粉具有显著的免疫增强作用。
Claims (6)
1.一种饲用速溶黄芪多糖粉剂的制备方法，其特征在于，该制备方法包括以下步骤：
1）取黄芪水提醇沉药渣加入65℃去离子水，搅拌至完全溶解，制得药渣水溶液；
2）待药渣水溶液冷却至室温后，离心除去不溶的热敏性大分子蛋白质、多糖及固体残渣，得到澄清上清液；
3）在澄清上清液加入2倍质量的去离子水，充分搅拌后依次通过孔径为100KDa和10kDa的中空纤维膜进行二次超滤处理，收集10-100 kDa的多糖组分；
4）将步骤3）收集的分子量在10-100 kDa之间的多糖组分，真空低温浓缩至该组分的固形物含量40%，喷雾干燥，得到速溶高活性黄芪多糖粉剂。
2.如权利要求1所述的一种饲用速溶黄芪多糖粉剂的制备方法，其特征在于步骤1）中药渣水溶液的酒精体积分数控制在20%。
3.如权利要求1所述的一种饲用速溶黄芪多糖粉剂的制备方法，其特征在于步骤1）中黄芪水提醇沉药渣为中药黄芪饮片经热水浸提后除去固体残渣部分，再加入3-4倍体积的无水乙醇后得到的醇沉半固体残渣。
4.如权利要求1所述的一种饲用速溶黄芪多糖粉剂的制备方法，其特征在于步骤2）中离心的转速为3500转/分钟。
5.如权利要求1所述的一种饲用速溶黄芪多糖粉剂的制备方法，其特征在于步骤4）中喷雾干燥条件为：雾化器频率70Hz，进风温度控制在140℃，出风温度控制在60℃以下，干燥塔夹层通入冷风冷却，每隔 5分钟振动锤干燥塔底部振动一次，防止干燥塔壁及底部积粉并液化发泡。
6.权利要求1-5任一所述的制备方法制备的饲用速溶黄芪多糖粉剂在饲料添加剂中的应用。
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