CN109620884A - 一种靶向治疗禽流感的活性组合物的制备方法 - Google Patents
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Abstract
本发明涉及一种靶向治疗禽流感的活性组合物的制备方法,具体为:首先将包括金银花、红景天、返魂草、绞股蓝、石香薷、木犀草、藤黄、黄芩、冬虫夏草、白英、半枝莲在内的各中药成分进行粉碎处理、煎煮、过滤、调整碳氮比,得到调整后滤液;再将调整后滤液配制为培养基,并以除藤黄和冬虫夏草外其他各中药植物的茎和叶作为待培养物质,将待培养物质在上述培养基中进行培养,再依次进行扩大繁殖、药效筛选,即得本发明所述活性组合物。本发明所述靶向治疗禽流感的活性组合物是通过生物炮制发酵的手段获得,避免了因使用化学方法而产生的污染;本活性组合物对禽流感病毒能够靶向治疗,不会产生耐药性,药效可靠,无毒副作用。
Description
技术领域
本发明属于中药应用领域,具体涉及一种靶向治疗禽流感的活性组合物的制备方法。
背景技术
禽流感,全称为鸟禽类流行性感冒,是由禽流感病毒引起的一种急性传染病,一般只在鸟类之间传播,少见情况会感染猪,在罕有情况下会跨越物种障碍感染人。禽类感染禽流感病毒主要会出现全身性或者呼吸系统性疾病,鸡、火鸡、鸭和鹌鹑等家禽及野鸟、水禽、海鸟等野禽均可感染,发病情况从急性败血性死亡到无症状带毒等极其多样,主要取决于带病体的抵抗力及其感染病毒的类型及毒力。禽流感病毒在禽类之间主要通过空气、粪便、饲料、饮水等传播,病毒可以随病禽的呼吸道、眼鼻分泌物、粪便等排出,其他禽类通过消化道和呼吸道感染发病。被病禽粪便、分泌物污染的人和物体,如饲料、禽舍、笼具、饲养管理用具、饮水、空气、运输车辆、人、昆虫等都可能传播病毒。禽类感染禽流感病毒后,一般3-5天发病,发病率和死亡率都很高,最高可达100%,给养禽业造成巨大的经济损失。
现阶段,针对禽类感染禽流感病毒的药物种类很多,但部分有效药物已经出现了耐药性,这对于治疗禽流感来说非常不利,或者部分药物副作用较大,治疗效果不佳。因此,亟需我们研制一种安全可靠且有效的治疗禽类感染禽流感病毒且不产生耐药性的药物,以应对市场的需求。
发明内容
为了解决现有技术存在的上述问题,本发明提供了一种靶向治疗禽流感的活性组合物的制备方法。
本发明所采用的技术方案为:
一种靶向治疗禽流感的活性组合物的制备方法,包括如下步骤:
(1)取金银花、红景天、返魂草、绞股蓝、石香薷、木犀草、藤黄、黄芩、冬虫夏草、白英、半枝莲依次进行清洗、烘干、粉碎,混合后得到粉碎原料;
(2)向所述粉碎原料中加入水,进行煎煮、过滤,得到中药滤液;
(3)调整所述中药滤液的碳氮比,得到调整后滤液;
(4)将所述调整后滤液制备为培养基;
(5)取步骤(1)中除藤黄和冬虫夏草外其他各中药植物的茎和叶依次进行消毒、清洗、裁剪,得到待培养物质,将所述待培养物质放入所述培养基中进行培养得到培养产物,将所述培养产物经摇床扩繁、药效筛选后,得到所述靶向治疗禽流感的活性组合物。
进一步地,步骤(1)中,所述各原料的配比为金银花10%、红景天9%、返魂草10%、绞股蓝8%、石香薷10%、木犀草8%、藤黄9%、黄芩9%、冬虫夏草7%、白英10%、半枝莲10%。
进一步地,步骤(1)中,所述各原料粉碎至150目-250目。
进一步地,步骤(2)中,粉碎原料与水的质量比为1:(8-12)。
进一步地,步骤(2)中,所述煎煮为文火煎煮,所述煎煮时间为1.5-2.5h。
进一步地,步骤(3)中,使用硫酸铵和葡萄糖将所述中药滤液的碳氮比调整至(25-29):1。
进一步地,步骤(4)中,所述培养基的组分组成为:调整后滤液96.248%-96.948%、葡萄糖1.2%、磷酸氢二钾0.3%、硫酸镁0.15%、酵母膏0.1%、维生素B10.01‰、维生素B60.01‰、琼脂1.3%-2%。
进一步地,步骤(4)中,或者所述培养基的组分组成为:调整后滤液94.3%-94.7%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸氢二钾0.1%、丹宁0.1%、蛋白胨0.1%、琼脂1.6%-2%。
进一步地,步骤(5)中,所述茎和叶的重量为各中药植物重量的0.5%-1.5%。
进一步地,步骤(5)中,所述消毒使用的是浓度为8%-12%的次氯酸钠。
进一步地,步骤(5)中,所述消毒的时间为8-12min。
进一步地,步骤(5)中,将所述茎或叶裁剪至1cm×1cm。
进一步地,步骤(5)中,所述培养温度为30-37℃,所述培养湿度为40%-50%,所述培养时间为5-7天。
进一步地,步骤(6)中,所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液95.42%、培养产物1.5%、葡萄糖2%、磷酸二氢钾0.5%、硫酸镁0.25%、蛋白胨0.2%、苯丙氨酸0.07%、酪氨酸0.06%。
进一步地,步骤(6)中,或者所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液94.3%、培养产物2%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸二氢钾0.1%、丹宁0.1%、蛋白胨0.1%。
进一步地,步骤(6)中,所述摇床扩繁的温度为25-37℃,所述转速为130-170rpm,所述扩繁天数为5-7天。
进一步地,所述靶向治疗禽流感的活性组合物在制备治疗禽流感药物中的应用。
下面对本发明中原料所产生的作用进行简单叙述。
金银花,性寒,味甘,归肺、心、胃经。功能主治:具有清热解毒、抗炎、补虚疗风的功效,主治胀满下疾、温病发热,热毒痈疡和肿瘤等症。
红景天,性寒,味甘、涩,归肺经。功能主治:有补气清肺、益智养心、收涩止血、散瘀消肿的功效。主治气虚体弱、病后畏寒、气短乏力、肺热咳嗽、咯血、白带腹泻、跌打损伤等。
返魂草,性温,味苦、辛,归心、肺二经。功能主治:清热解毒,散血消肿,下气通径,瘀血胀痛,跌打损伤,高热,肝炎等症。
绞股蓝,性寒,味苦,归肺、脾、肾经。功能主治:消炎解毒,止咳祛痰。现多用作滋补强壮药。
石香薷,性温,味辛、苦,归肺、肝、脾、胃四经。功能主治:祛暑,活血,理气,化湿,主治夏月感冒,中暑呕恶,腹痛泄泻,跌打瘀痛,湿疹,疖肿。
木犀草,含有木犀草素,具有多种药理活性,如消炎、抗过敏、降尿酸、抗肿瘤、抗菌、抗病毒等,临床主要用于止咳、祛痰、消炎、降尿酸、治疗心血管疾病、治疗“肌萎缩性脊髓侧索硬化症”,SARS,肝炎等。
藤黄,性凉,味酸、涩。功能主治:消肿,攻毒,祛腐敛疮,止血,杀虫,用于痈疽肿毒,溃疡,湿疮,肿瘤,顽癣,跌打肿痛,疮伤出血及烫伤。
黄芩,性寒,味苦,归肺、胆、脾、大肠、小肠经。功能主治:清热燥湿,泻火解毒,止血,安胎,用于湿温、暑湿,胸闷呕恶,湿热痞满,泻痢,黄疸,肺热咳嗽,高热烦渴,血热吐衄,痈肿疮毒,胎动不安。
冬虫夏草,性平,味甘,归肺、肾经。功能主治:补肾益肺,止血化痰,主要治疗阳痿遗精、腰膝酸痛、久咳虚喘、劳嗽痰血。
白英,性微寒,味苦,有小毒,归肝、胃经。功能主治:清热解毒,利湿消肿,抗癌,主要用于感冒发热,乳痈、恶疮,湿热黄疸、腹水,白带,肾炎水肿,外用治痈疖肿毒。
半枝莲,性寒,味辛、苦,归肺、肝、肾经。功能主治:清热解毒,化瘀利尿,用于疔疮肿毒,咽喉肿痛,跌扑伤痛,水肿,黄疸,蛇虫咬伤。
本发明的有益效果为:
本发明所述靶向治疗禽流感的活性组合物的制备方法,具体为:首先将包括金银花、红景天、返魂草、绞股蓝、石香薷、木犀草、藤黄、黄芩、冬虫夏草、白英、半枝莲在内的各中药成分进行粉碎处理、煎煮、过滤、调整碳氮比,得到调整后滤液;再将调整后滤液配制为培养基,并以除藤黄和冬虫夏草外其他各中药植物的茎和叶作为待培养物质,将待培养物质在上述培养基中进行培养,再依次进行扩大繁殖、药效筛选,即得本发明所述的靶向治疗禽流感的活性组合物。本发明所述靶向治疗禽流感的活性组合物是通过生物炮制发酵的手段获得,避免了因使用化学方法而产生的污染;本活性组合物通过各组分之间的协同作用对禽流感病毒能够靶向治疗,且不产生耐药性,药效可靠,无任何毒副作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1是实施例3所得活性组合物对H7N9病毒血抑试验结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1
本实施例提供一种靶向治疗禽流感的活性组合物的制备方法,包括如下步骤:
(1)取金银花10kg、红景天9kg、返魂草10kg、绞股蓝8kg、石香薷10kg、木犀草8kg、藤黄9kg、黄芩9kg、冬虫夏草7kg、白英10kg、半枝莲10kg依次进行清洗、烘干,粉碎至150目,混合后得到粉碎原料;
(2)向所述粉碎原料中加入8倍质量的水,文火煎煮1.5h,过滤,得到中药滤液;
(3)使用硫酸铵和葡萄糖将所述中药滤液的碳氮比调整至25:1,得到调整后滤液;
(4)取适合重量的调整后滤液、葡萄糖、磷酸氢二钾、硫酸镁、酵母膏、维生素B1、维生素B6和琼脂混合均匀,得到培养基,所述培养基组成为:调整后滤液96.948%、葡萄糖1.2%、磷酸氢二钾0.3%、硫酸镁0.15%、酵母膏0.1%、维生素B10.01‰、维生素B60.01‰、琼脂1.3%;
(5)取步骤(1)中除藤黄和冬虫夏草外其他各中药植物重量0.5%的茎和叶,使用8%的次氯酸钠消毒8min,清洗后裁剪至1cm×1cm大小,随后放入培养基中进行培养,30℃培养5天,培养湿度为40%,得到培养产物;
(6)所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液95.42%、培养产物1.5%、葡萄糖2%、磷酸二氢钾0.5%、硫酸镁0.25%、蛋白胨0.2%、苯丙氨酸0.07%和酪氨酸0.06%,将上述各组分混合均匀后进行摇床扩繁,摇床转速为130rpm,25℃培养5天,经过药效筛选后得到所述靶向治疗禽流感的活性组合物。
实施例2
本实施例提供一种靶向治疗禽流感的活性组合物的制备方法,包括如下步骤:
(1)取金银花10kg、红景天9kg、返魂草10kg、绞股蓝8kg、石香薷10kg、木犀草8kg、藤黄9kg、黄芩9kg、冬虫夏草7kg、白英10kg、半枝莲10kg依次进行清洗、烘干,粉碎至250目,混合后得到粉碎原料;
(2)向所述粉碎原料中加入12倍质量的水,文火煎煮2.5h,过滤,得到中药滤液;
(3)使用硫酸铵和葡萄糖将所述中药滤液的碳氮比调整至29:1,得到调整后滤液;
(4)取适合重量的调整后滤液、蔗糖、硝酸钠、硫酸镁、氯化钾、磷酸二氢钾、丹宁、蛋白胨和琼脂混合均匀,得到培养基,所述培养基组成为:调整后滤液94.3%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸氢二钾0.1%、丹宁0.1%、蛋白胨0.1%和琼脂2%;
(5)取步骤(1)中除藤黄和冬虫夏草外其他各中药植物重量1.5%的茎和叶,使用12%的次氯酸钠消毒12min,清洗后裁剪至1cm×1cm大小,随后放入所述培养基中进行培养,37℃培养7天,培养湿度为50%,得到培养产物;
(6)所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液94.3%、培养产物2%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸二氢钾0.1%、丹宁0.1%和蛋白胨0.1%,将上述各组分混合均匀后进行摇床扩繁,摇床转速为170rpm,37℃培养7天,经过药效筛选后得到所述靶向治疗禽流感的活性组合物。
实施例3
本实施例提供一种靶向治疗禽流感的活性组合物的制备方法,包括如下步骤:
(1)取金银花10kg、红景天9kg、返魂草10kg、绞股蓝8kg、石香薷10kg、木犀草8kg、藤黄9kg、黄芩9kg、冬虫夏草7kg、白英10kg、半枝莲10kg依次进行清洗、烘干,粉碎至200目,混合后得到粉碎原料;
(2)向所述粉碎原料中加入10倍质量的水,文火煎煮2h,过滤,得到中药滤液;
(3)使用硫酸铵和葡萄糖将所述中药滤液的碳氮比调整至27:1,得到调整后滤液;
(4)取适合重量的调整后滤液、葡萄糖、磷酸氢二钾、硫酸镁、酵母膏、维生素B1、维生素B6和琼脂混合均匀,得到培养基,所述培养基组成为:调整后滤液96.548%、葡萄糖1.2%、磷酸氢二钾0.3%、硫酸镁0.15%、酵母膏0.1%、维生素B10.01‰、维生素B60.01‰、琼脂1.7%;
(5)取步骤(1)中除藤黄和冬虫夏草外其他各中药植物重量1%的茎和叶,使用10%的次氯酸钠消毒10min,清洗后裁剪至1cm×1cm大小,随后放入所述培养基中进行培养,34℃培养6天,培养湿度为45%,得到培养产物;
(6)所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液95.42%、培养产物1.5%、葡萄糖2%、磷酸二氢钾0.5%、硫酸镁0.25%、蛋白胨0.2%、苯丙氨酸0.07%和酪氨酸0.06%,将上述各组分混合均匀后进行摇床扩繁,摇床转速为150rpm,31℃培养6天,经过药效筛选后得到所述靶向治疗禽流感的活性组合物。
实验例
对实施例3所得靶向治疗禽流感的活性组合物进行效果试验。
一、活性组合物的药效试验
1、研究目的
通过血凝抑制实验与鸡胚接种实验,考察实施例3所得活性组合物对H7N9病毒的杀毒效果,以评价该药物对H7N9病毒是否具有抵抗能力。
2、试验指南与规程
《中华人民共和国药典》2015年版三部(以下简称:《中国药典》三部)
3、研究地点与主要人员(见表1)
表1研究地点与主要人员
4、试验材料及方法
4.1研究用样品、试剂信息(见表2)
表2研究用样品试剂基本信息
4.2试验方法
4.2.1实施例3所得活性组合物对H7N9病毒血抑试验
通过血抑实验(HI实验),考察实施例3所得活性组合物对H7N9病毒是否具有破坏作用。将药物不进行霍乱滤液处理,药物离心后进行2倍梯度稀释,各稀释度加入25μl稀释药物,再加入四价抗原25μl,漩涡混合器震荡混匀后室温静置45分钟。每孔加入1%鸡红细胞25μl,漩涡混合器震荡混匀后室温静置30分钟,读取血凝结果。同时设置药物对照、病毒对照和空白对照。
4.2.2实施例3所得活性组合物鸡胚接种试验
将实施例3所得活性组合物除菌过滤后与梯度稀释的H7N9病毒混合后接种鸡胚,对比添加和不添加药物的鸡胚血凝滴度,考察药物对病毒是否具有破坏效果。将实施例3所得活性组合物采用生理盐水1:25稀释,除菌过滤后再1:10稀释;病毒采用生理盐水1:100稀释后除菌过滤再1:500,1:2500,1:12500 5倍梯度稀释三个稀释度。
将1:25,1:250稀释的药物与稀释后的病毒等体积混合,同时采用生理盐水与稀释病毒等体积混合作为病毒对照,药物与生理盐水等体积混合作为药物对照,室温放置30min后接种SPF鸡胚,0.2ml/胚。每组4枚鸡胚,药物对照接种3枚鸡胚。34℃培养60小时,照胚,冷胚,收获尿囊液检测血凝滴度。
5、结果及分析
5.1实施例3所得活性组合物对H7N9病毒血抑试验结果
血抑实验结果见图1。由结果可见,空白孔无血凝素HA,血球均凝集;病毒对照前2孔为4价抗原和2价抗原未凝集,1价抗原及更大稀释度抗原凝集,实验成立。
药物对照无血凝素HA,应出现血球沉淀。但1:8及之前未凝集,表明上述浓度对实验存在干扰。1:16及以后浓度血球凝集,说明此浓度及以后稀释度对血球凝集无干扰。
实施例3所得活性组合物加H7N9病毒时,1:16、1:32与1:64三个药物浓度时,由于药物破坏了血凝素HA,无血凝素HA时血球凝集,1:128以后药物浓度过低无法破坏血凝素HA,4价血凝素HA抗原存在血球无凝集。
初步判定,该实施例3所得活性组合物有一定的抗H7N9病毒的效果。
5.2实施例3所得活性组合物鸡胚接种试验结果
药物与病毒混合后接种鸡胚,培养60小时,照胚,检测尿囊液的血凝滴度。结果显示,鸡胚接种不同浓度的H7N9病毒感染程度与病毒浓度有量效关系,1:200,1:1000,1:5000接种鸡胚全部产生血凝,1:25000接种25%鸡胚出现阳转,实验成立。
药物1:50终浓度实验组,药物对照接种鸡胚,三胚死一胚,表明该浓度药物对鸡胚存在毒性。H7N9病毒1:200,1000,5000时,鸡胚除一枚外均为阴性,推测可能由于药物破坏了病毒导致病毒无法感染鸡胚所致。
药物1:500终浓度实验组,药物对照接种鸡胚无死亡,表明该浓度药物对鸡胚无毒性;病毒1:1000,5000,25000时,鸡胚的血凝较仅接种病毒的鸡胚出现更多阴性,且阳性的血凝滴度较仅接种病毒的低,分析可能由于药物破坏了病毒导致病毒无法感染或者感染能力下降所致;初步分析药物对于H7N9病毒存在一定的杀毒效果。对鸡胚感染的半数有效剂量1:500左右,具体实验结果见表3。
表3实施例3所得活性组合物接种鸡胚血凝与存活情况
6、结论
6.1血抑实验结果显示实施例3所得活性组合物于1:16、1:32与1:64浓度时对四价H7N9抗原有破坏作用,导致血球凝集,认为实施例3所得活性组合物有一定的抗H7N9病毒的效果。
6.2实施例3所得活性组合物鸡胚接种试验结果显示药物1:500终浓度时,可抑制或者降低1:1000,5000,25000的H7N9病毒对鸡胚的感染,初步分析药物对于H7N9病毒存在破坏能力。
二、第一养殖场药效试验
1、试验相关信息(见表4)
表4试验相关信息
2、试验目的
实施例3所得活性组合物对肉鸡疑似病毒感染治疗效果试验。
3、试验方法及内容:
戚相平鸡场于2018年11月28日进雏27200只,入舍鸡为三层笼养,种源为承德融融。本批鸡入舍正常,9天免疫后出现呼吸道症状,用药物治疗两个疗程后呼吸道症状没有彻底消失,2周后死淘率上升,至12月23日(26日龄)到场观察呼吸道症状有所减轻,但鸡群日死淘率达到0.19%,大群精神较差,眼形变长,鸡舍尾端咳音明显,有白色稀便,当日采食量为3000公斤。
解剖死淘鸡只可见,嗉囊内饲料稀少,肌胃内容物有的呈绿色,腺胃乳头肿胀出血,肝脏淡褐色,有腹气囊炎和胸气囊炎,瘦小鸡只有心包炎及腹水,气管环均出血,为禽流感。
根据以上症状初步判定本批鸡免疫力较差,新城疫及流感的抗体降低,同时伴有大肠杆菌及气囊炎病,拟定采取如下措施:
使用实施例3所得活性组合物4天:使用时间为12月25日至28日(28~31日龄),连续饮水4天,每天用量15瓶,每瓶兑水1000斤,每天集中4小时内将当天用药饮完。
另配合使用痢泻停4天,每天9.5袋控制大肠杆菌感染。
使用中注意上午用实施例3所得活性组合物,下午用痢泻停饮水,两种药物分开使用,并适当提高鸡舍管理温度。
使用药物前后鸡群死淘及采食情况如下,如表5所示:
表5使用药物前后鸡群死淘及采食情况表
日龄/天 | 26 | 27 | 28 | 29 | 30 | 31 |
当日死淘数:只 | 51 | 60 | 62 | 76 | 68 | 79 |
当日采食量:公斤 | 3000 | 3000 | 3000 | 3200 | 3350 | 3500 |
用药前两天(第26、27天),鸡群采食量为每日3000公斤,不见增料,死淘率增加,用药后第二天采食量均有所增加,死淘率没有降低,现场观察鸡群精神有好转,不再出现新的病重鸡,呼吸道症状没有变化。
4、试验结果分析与讨论
本次试验鸡群在投药两天后,采食量呈现每日增加趋势,但还没有达到标准采食量。鸡群死淘率没有降低,分析饲养员没有及时将病弱鸡只及时挑出所致。由此本次试验认为,实施例3所得活性组合物对本批肉鸡疑似病毒感染的治疗有一定效果。
三、第二养殖场药效试验
1、试验有关信息(见表6)
表6试验相关信息
2、试验目的
实施例3所得活性组合物对鸡群因受凉引起的流感的实际作用。
3、试验方法及内容:
基本情况:田连仲单栋规模为19000只,于10月22日进美咯雏鸡19000只,22日龄前鸡群长势良好,随着日龄的增大,鸡群产热增多,于23日龄开始调整风机的排风量,由时控风机转换为温控风机,由于操作失误加上外界温度的变化,造成鸡群受凉感冒,表现症状为出现眼角变形、呆立、羽毛松乱、闭眼、精神沉郁、采食量不增,两栋鸡舍一栋轻一栋重,日死亡率分别为0.26%和0.1%,剖检症状为肺脏有坏死,水肿,轻微气囊炎,胰脏边缘轻微出血。
用药情况:实施例3所得活性组合物10瓶/天于下午集中4个小时饮水。用药四天后,鸡群总体恢复正常,但仍有个别症状较重的鸡只,死淘没有降低,现场观察鸡群精神有好转,不再出现新的病重鸡。
4、试验结果分析与讨论
实施例3所得活性组合物对因鸡群受凉引起的流感有良好的治疗作用。
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,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,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以所述权利要求的保护范围为准。
Claims (10)
1.一种靶向治疗禽流感的活性组合物的制备方法,其特征在于,包括如下步骤:
(1)取金银花、红景天、返魂草、绞股蓝、石香薷、木犀草、藤黄、黄芩、冬虫夏草、白英、半枝莲依次进行清洗、烘干、粉碎,混合后得到粉碎原料;
(2)向所述粉碎原料中加入水,进行煎煮、过滤,得到中药滤液;
(3)调整所述中药滤液的碳氮比,得到调整后滤液;
(4)将所述调整后滤液制备为培养基;
(5)取步骤(1)中除藤黄和冬虫夏草外其他各中药植物的茎和叶依次进行消毒、清洗、裁剪,得到待培养物质,将所述待培养物质放入所述培养基中进行培养得到培养产物,将所述培养产物经摇床扩繁、药效筛选后,得到所述靶向治疗禽流感的活性组合物。
2.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(1)中,所述各原料的配比为金银花10%、红景天9%、返魂草10%、绞股蓝8%、石香薷10%、木犀草8%、藤黄9%、黄芩9%、冬虫夏草7%、白英10%、半枝莲10%。
3.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(1)中,所述各原料粉碎至150目-250目。
4.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(2)中,粉碎原料与水的质量比为1:(8-12);
所述煎煮为文火煎煮,所述煎煮时间为1.5-2.5h。
5.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(3)中,使用硫酸铵和葡萄糖将所述中药滤液的碳氮比调整至(25-29):1。
6.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(4)中,所述培养基的组分组成为:调整后滤液96.248%-96.948%、葡萄糖1.2%、磷酸氢二钾0.3%、硫酸镁0.15%、酵母膏0.1%、维生素B10.01‰、维生素B60.01‰、琼脂1.3%-2%;
或者,所述培养基的组分组成为:调整后滤液94.3%-94.7%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸氢二钾0.1%、丹宁0.1%、蛋白胨0.1%、琼脂1.6%-2%。
7.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(5)中,所述茎和叶的重量为各中药植物重量的0.5%-1.5%;
所述消毒使用的是浓度为8%-12%的次氯酸钠,所述消毒的时间为8-12min。
8.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(5)中,将所述茎或叶裁剪至1cm×1cm;
所述培养温度为30-37℃,所述培养湿度为40%-50%,所述培养时间为5-7天。
9.根据权利要求1所述的靶向治疗禽流感的活性组合物的制备方法,其特征在于,步骤(5)中,所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液95.42%、培养产物1.5%、葡萄糖2%、磷酸二氢钾0.5%、硫酸镁0.25%、蛋白胨0.2%、苯丙氨酸0.07%、酪氨酸0.06%;
或者,所述培养产物进行摇床扩繁时的扩大培养体系组成为:调整后滤液94.3%、培养产物2%、蔗糖3%、硝酸钠0.3%、硫酸镁0.05%、氯化钾0.05%、磷酸二氢钾0.1%、丹宁0.1%、蛋白胨0.1%;
所述摇床扩繁的温度为25-37℃,所述转速为130-170rpm,所述扩繁天数为5-7天。
10.根据权利要求1所述靶向治疗禽流感的活性组合物在制备治疗禽流感药物中的应用。
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