CN108030897A - 一种治疗前列腺癌的中药酵素及其制备方法 - Google Patents
一种治疗前列腺癌的中药酵素及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;所述中药原料包括:白花蛇舌草、半枝莲、水蛭、九香虫;黄芪、薏苡仁、莪术、熟地黄、苦参、土茯苓、槐角、虻虫、白头翁、藤梨根、元胡、甘草;所述复合菌包括酵母菌和醋酸菌。本发明还公开了治疗前列腺癌的中药酵素的制备方法。本发明通过引入多菌共生技术,对多种中药发酵后制成中药酵素,改进治疗直肠的中药的用药方式,得到小分子中药酵素,降低中药对肠胃的刺激作用、提高中药中有益成分的吸收率。
Description
技术领域
本发明属于生物酶制剂技术领域,具体涉及一种治疗前列腺癌的中药酵素及其制备方法。
背景技术
前列腺癌是指发生在前列腺的上皮性恶性肿瘤。2004年WHO《泌尿系统及男性生殖器官肿瘤病理学和遗传学》中前列腺癌病理类型上包括腺癌(腺泡腺癌)、导管腺癌、尿路上皮癌、鳞状细胞癌、腺鳞癌。其中前列腺腺癌占95%以上,因此,通常我们所说的前列腺癌就是指前列腺腺癌。
前列腺癌在我国的发病率和死亡率呈逐年增长态势。据统计,中国每年新发病例万人,是中国发病率排名前五的恶性肿瘤性疾病。前列腺癌在全球范围是发病率和死亡率第3位的恶性肿瘤,尤其值得关注的是,近年来西方国家凭借肿瘤早防早治的日趋完善,前列腺癌的发病率和死亡率已逐年下降。近年来,随着我国人口老龄化、工业化的进程和饮食结构的变化,前列腺癌的发病率却在逐年攀升。晚期前列腺癌的治疗大多釆用氟尿啼唆类单药化疗后,平均总生存期,在11~12个月左右,引入伊立替康和奥沙利销后,延长到16~18个月,如果再联合祀向治疗药物,目前的可达20~24个月,但其治疗费用昂贵,全程治疗至少需要万元左右,同时毒副作用大,对患者的生理、心理健康造成巨大伤害,给家庭和社会带来巨大负担。
随着现代中医药学的发展,中药有效成分的治疗参与到整个结前列腺癌治疗的过程当中,其在诱导结前列腺癌细胞凋亡,抑制癌细胞增殖,阻滞癌细胞的细胞周期,抑制新生血管形成,影响癌细胞的侵袭力及逆转耐药性等方面发挥了积极的作用,显示了中医药在治疗结直肠肿瘤方面的独特优势,中医药的善治未病、低毒价廉的优势取得疗效上的突破。而传统的中药大多是采用汤药或颗粒制剂等形式,传统的药剂方式导致中药利用率低、中药苦口、气味难闻、药物毒性、农药残留等弊端。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种治疗前列腺癌的中药酵素,通过引入多菌共生技术,对多种中药发酵后制成中药酵素,改进治疗直肠的中药的用药方式,得到小分子中药酵素,降低中药对肠胃的刺激作用、提高中药中有益成分的吸收率。
为解决上述问题,本发明所采用的技术方案如下:
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述中药原料包括:白花蛇舌草15~30重量份、半枝莲15~30重量份、水蛭15~30重量份、九香虫10~25重量份;黄芪10~25重量份、薏苡仁15~30重量份、莪术10~20重量份、熟地黄10~20重量份、苦参5~15重量份、土茯苓10~25重量份、槐角10~20重量份、虻虫15~30重量份、白头翁15~25重量份、藤梨根15~30重量份、元胡10~20重量份、甘草10~20重量份;
所述复合菌包括浓度为1.2×108~2.2×108CFU/mL的酵母菌和浓度为2.0×108~2.5×108CFU/m L的醋酸菌。
作为进一步的方案,本发明所述的中药原料还包括冬凌草12~25重量份、枸杞子10-20重量份。
作为进一步的方案,本发明所述的中药原料包括:白花蛇舌草18~25重量份、半枝莲18~25重量份、水蛭18~25重量份、九香虫15-20重量份;黄芪15~20重量份、薏苡仁18~25重量份、莪术12~18重量份、熟地黄12~18重量份、苦参8~12重量份、土茯苓15~20重量份、槐角12~18重量份、虻虫18~25重量份、、白头翁15~20重量份、藤梨根18~25重量份、元胡12~18重量份、甘草12~18重量份、冬凌草15~20重量份、枸杞子15-20重量份。
作为进一步的方案,本发明所述的中药原料包括:白花蛇舌草20重量份、半枝莲20重量份、水蛭20重量份、九香虫18重量份;黄芪18重量份、薏苡仁20重量份、莪术15重量份、熟地黄15重量份、苦参10重量份、土茯苓18重量份、槐角15重量份、虻虫20重量份、、白头翁18重量份、藤梨根20重量份、元胡20重量份、甘草15重量份,冬凌草18重量份、枸杞子18重量份。
作为进一步的方案,本发明所述的复合菌还包括浓度为1.5×108~2.0×108CFU/mL乳酸菌。
一种治疗前列腺癌的中药酵素的制备方法,其特征在于,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌和1.0-1.5%醋酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
作为进一步的方案,本发明所述的超声萃取步骤中,采用20kHz的低频超声萃取8-12分钟,所述中药粉末与水的料液比为1:(100-300)。
作为进一步的方案,本发明所述的酶解步骤中,采用的复合酶的加酶量为100-150μg/g,所述复合酶是质量比为1:1:3的果胶酶、纤维素酶和蛋白质酶;酶解的时间为8-10小时,酶解的温度为42-50℃。
作为进一步的方案,本发明所述的接种、发酵步骤中,酵母菌的发酵时间为6-8天,醋酸菌的发酵时间为8-10天。
一种治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;乳酸菌接种于MRS培养基在温度37℃下活化l-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌、1.0-1.5%醋酸菌、1.0-1.5%乳酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,最后在35-40℃温度下继续发酵得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
相比现有技术,本发明的有益效果在于:
1.本发明所述的中药酵素,改进治疗前列腺癌的中药的用药方式,得到小分子中药酵素,改善了传统中药苦涩的口感,降低中药对肠胃的刺激作用、提高了中药中有益成分的吸收率;
2.本发明所述的中药酵素,用于治疗或防止前列腺癌,可减少晚期肠癌病死率,延长生存期、中位生存期,提高生存质量,可以减少中后期前列腺癌的复发和转移,同时相对于西药具有成本低的优点;
3.本发明所述的中药酵素也可以联合治疗前列腺癌的西药用药,可以是用于早期的治疗,也可以用于前列腺癌化疗后的修复。
下面结合具体实施方式对本发明作进一步详细说明。
具体实施方式
本发明所述的治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述中药原料包括:白花蛇舌草15~30重量份、半枝莲15~30重量份、水蛭15~30重量份、九香虫10~25重量份;黄芪10~25重量份、薏苡仁15~30重量份、莪术10~20重量份、熟地黄10~20重量份、苦参5~15重量份、土茯苓10~25重量份、槐角10~20重量份、虻虫15~30重量份、白头翁15~25重量份、藤梨根15~30重量份、元胡10~20重量份、甘草10~20重量份;
所述复合菌包括浓度为1.2×108~2.2×108CFU/mL的酵母菌和浓度为2.0×108~2.5×108CFU/m L的醋酸菌。
作为进一步的方案,本发明所述的中药原料还包括冬凌草12~25重量份、枸杞子10-20重量份。
在本发明中,所采用的白花蛇舌草成药味苦、淡,性,具有清热解毒、消痛散结、利尿除湿等功效,现代医学研究发现,白花蛇舌草也可通过调节免疫功能发挥抗肿瘤作用;在体内对白血病细胞如急性粒细胞白血病细胞、急性淋巴性白血病细胞有抑制作用;白花蛇舌草素对小白鼠腹水肝癌细胞有抑制、杀灭作用。半枝莲有清热解毒,活血化瘀,消肿止痛,抗癌之功效;可以治阑尾炎、肝炎、胃痛、早期肝癌、肺癌、子宫颈癌、乳腺炎等;外用治痛疖疗,跌打肿痛等症;经研究发现半枝莲能够抑制肿瘤细胞增长,通过凋亡通路诱导细胞死亡。九香虫,即半翅目,异翅亚目,蝽科的瓜黑蝽;九香虫中的九香虫油,油中含硬脂酸、棕榈酸、油酸,九香虫的臭味来源于醛或酮,九香虫中还含蛋白质、甲壳质等物质;中医上认为九香虫具有理气止痛,温中助阳的功效;现代医学研究证明:九香虫对金黄色葡萄球菌、伤寒杆菌、副伤寒杆菌、福氏痢疾杆菌有较强的抗菌作用;并有促进机体新陈代谢作用。黄芪中主要含有黄芪多糖、黄芪酸、黄芪素、黄芪酚等物质,还含有脂肪酸、草酸钙、葡萄糖、果糖和淀粉;其中黄芪多糖自身可直接抑制癌细胞的增殖,并与抗癌药物联合应用具有协同抗肿瘤作用。薏苡仁富含淀粉、蛋白质、多种维生素及人体所需的多种氨基酸,从薏苡仁热提取物分得的中性多糖葡聚糖混合物及酸性多糖均具有示抗补体活性,研究表明其具有抗肿瘤的作用。冬凌草含有冬凌草甲素、冬凌草乙素和迷迭香酸3种主要活性成分,可有效地抑制甲型、乙型溶血性链球菌、金黄色葡萄球菌等,从而提高肌体抵抗力,迅速降低因炎症引起的白血球增高;研究表明冬凌草甲素能抑制细胞的生长并诱导细胞发生调亡在本发明中冬凌草与与其他成分配合治疗癌症有明显地增效作用。莪术具有行气止痛、破血祛癖的功能,集抑瘤、活血、调节免疫三大功能于一身,其主要有效部位为挥发油和姜黄素;姜黄素在国内外均有深入研究,除了传统的诱导肿瘤细胞凋亡和自噬效应,还已证实有抗新血管生成作用、挥发油中的莪术二酮、吉马酮、姜黄素、莪术醇、β-榄香稀均具有抗肿瘤活性。苦参是常用的清热解毒类药物,苦参中的苦参素可以通过下调瘤细胞中对NK细胞起负调节的关键因子TGF-βi和PGE2的分泌量,增强NK细胞杀伤肿瘤细胞的作用,研究发现苦参不仅能抗癌而且能够改善胃癌患者的免疫功能。槐角含黄酮类和异黄酮类化合物;此外,槐角还含有杀菌物质,对葡萄球菌和大肠杆菌有抑制作用。虻虫又称牛蜢,牛虻,牛蚊子,中华虻、白斑虻、灰虻。属虻科昆虫;研究发现虻虫提取液具弱的抗凝血酶作用和抗癌作用;水蛭主要含蛋白质,亦含铁、锰、锌等多种微量元素,新鲜水蛭唾液中含有一种抗凝血物质名水蛭素(Hirudin),属于多肽,由于其分子组成中没有色氨酸和精氨酸,故有抗凝血作用,水蛭素对肿瘤细胞有抑制作用,对小鼠肝癌生长有一定的抑制作用;由于水蛭有高抗凝作用,因而有利于抗癌药及免疫活性细胞浸入癌组织杀伤癌细胞。虻虫攻血之力峻猛,急而短暂;水蛭破血之力迟缓,稳而持久;二药合用具有很强的破血逐瘀功效,现代药理证实,虻虫、水蛭、虫在抗凝血、纤溶、扩张血管、保护组织缺血等方面的作用相似。藤梨根具有清热解毒、清热利湿、防肿瘤抗癌、祛风除湿、利尿止血、解毒消肿等功效,可以用治疗消化不良、呕吐、风湿痹痛、风湿骨痛、消化道癌肿、消化道肿瘤、痈疡疮疖、风湿骨痛及黄疸等症。败酱草具有清热解毒、祛瘀排脓的功效,可用于阑尾炎、痢疾、肠炎、肝炎、眼结膜炎、产后瘀血腹痛、痈肿疔疮等症。元胡中所含的紫堇碱、四氢巴马亭有镇痛作用,四氢巴马亭镇痛指数较高,四氢巴马亭对大脑皮层及皮层下的电活动都能抑制,尤以皮层运动区较为敏感。甘草主要化学成分为甘草酸、黄酮、香豆素等;甘草酸可通过调节Th2细胞、抑制二乙基亚硝胺等机制,广泛应用于黑色素瘤、肝癌等恶性肿瘤;同时甘草酸具有非特异性免疫调节作用,可增强M细胞的吞噬功能,还可选择性增强辅助性T淋巴细胞的增殖能力和活性。
在本发明中,通过上述多种中药配伍,其功能之间存在相互协同增效的作用。
作为进一步的方案,本发明所述的中药原料包括:白花蛇舌草18~25重量份、半枝莲18~25重量份、水蛭18~25重量份、九香虫15-20重量份;黄芪15~20重量份、薏苡仁18~25重量份、莪术12~18重量份、熟地黄12~18重量份、苦参8~12重量份、土茯苓15~20重量份、槐角12~18重量份、虻虫18~25重量份、、白头翁15~20重量份、藤梨根18~25重量份、元胡12~18重量份、甘草12~18重量份、冬凌草15~20重量份、枸杞子15-20重量份。
作为进一步的方案,本发明所述的中药原料包括:白花蛇舌草20重量份、半枝莲20重量份、水蛭20重量份、九香虫18重量份;黄芪18重量份、薏苡仁20重量份、莪术15重量份、熟地黄15重量份、苦参10重量份、土茯苓18重量份、槐角15重量份、虻虫20重量份、、白头翁18重量份、藤梨根20重量份、元胡20重量份、甘草15重量份,冬凌草18重量份、枸杞子18重量份。
作为进一步的方案,本发明所述的复合菌还包括浓度为1.5×108~2.0×108CFU/mL乳酸菌。
一种治疗前列腺癌的中药酵素的制备方法,其特征在于,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌和1.0-1.5%醋酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
作为进一步的方案,本发明所述的超声萃取步骤中,采用20kHz的低频超声萃取8-12分钟,所述中药粉末与水的料液比为1:(100-300)。
作为进一步的方案,本发明所述的酶解步骤中,采用的复合酶的加酶量为100-150μg/g,所述复合酶是质量比为1:1:3的果胶酶、纤维素酶和蛋白质酶;酶解的时间为8-10小时,酶解的温度为42-50℃。
作为进一步的方案,本发明所述的接种、发酵步骤中,酵母菌的发酵时间为6-8天,醋酸菌的发酵时间为8-10天。
一种治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;乳酸菌接种于MRS培养基在温度37℃下活化l-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌、1.0-1.5%醋酸菌、1.0-1.5%乳酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,最后在35-40℃温度下继续发酵得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
本发明中,采用复合的乳酸菌进行发酵,由于酵母菌和醋酸菌共生发酵时,先于20℃条件下酵母菌成为优势菌,使得酵液中的酶活性和总酸含量上升,但酵母菌活菌数达到饱和程度时,酵母菌因营养物质消耗受到限制不能继续扩增;当温度转到25℃时,醋酸菌大量繁殖,醋酸菌利用糖源或酵母菌代谢产物继续发酵产生大量有机酸,间接的使发酵液的总酸含量较单菌发酵要高,这样利于酵液的发酵。作为进一步的方案,本发明所述的复合菌还包括浓度为1.2×108~2.5×108CFU/mL乳酸菌。乳酸菌是在37℃是开始大量繁殖,可以有效持续发酵液的发酵过程。
以下是本发明具体的实施方式,在下述实施例中所采用的原料、试剂以及设备均可以通过商业方式获得。
实施例1
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述中药原料包括:白花蛇舌草30重量份、半枝莲30重量份、水蛭30重量份、九香虫25重量份;黄芪25重量份、薏苡仁30重量份、莪术20重量份、熟地黄20重量份、苦参15重量份、土茯苓25重量份、槐角20重量份、虻虫30重量份、白头翁25重量份、藤梨根30重量份、元胡20重量份、甘草20重量份;
所述复合菌包括浓度为1.2×108CFU/mL的酵母菌和浓度为2.0×108CFU/m L的醋酸菌;
治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将上述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,药粉末与水的料液比为1:200,然后在每种中药粉末单独采用20kHz的低频超声萃取8分钟,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25%,在浓缩后的酶解液接种2.0%的酵母菌和1.5%醋酸菌后先在22℃温度下摇床发酵,然后在28℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
实施例2
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述中药原料包括:白花蛇舌草15重量份、半枝莲15重量份、水蛭15重量份、九香虫10重量份;黄芪10重量份、薏苡仁15重量份、莪术10重量份、熟地黄10重量份、苦参5重量份、土茯苓10重量份、槐角10重量份、虻虫15重量份、白头翁15重量份、藤梨根15重量份、元胡10重量份、甘草10重量份;
所述复合菌包括浓度为2.2×108CFU/mL的酵母菌和浓度为2.0×108CFU/m L的醋酸菌;
治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将上述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,药粉末与水的料液比为1:100,然后在每种中药粉末单独采用20kHz的低频超声萃取12分钟,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为30%,在浓缩后的酶解液接种1.5%的酵母菌和1.0%醋酸菌后先在25℃温度下摇床发酵,然后在30℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
实施例3
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述的中药原料包括:白花蛇舌草18重量份、半枝莲18重量份、水蛭18重量份、九香虫15重量份;黄芪15重量份、薏苡仁18重量份、莪术12重量份、熟地黄12重量份、苦参8重量份、土茯苓15重量份、槐角12重量份、虻虫18重量份、白头翁15重量份、藤梨根18重量份、元胡12重量份、甘草12重量份、冬凌草15重量份、枸杞子15重量份;
所述复合菌包括浓度为2.2×108CFU/mL的酵母菌、浓度为2.5×108CFU/m L的醋酸菌以及1.2×108CFU/mL乳酸菌;
治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将上述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,药粉末与水的料液比为1:300,然后在每种中药粉末单独采用20kHz的低频超声萃取10分钟,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25%,在浓缩后的酶解液接种1.6%的酵母菌和1.3%醋酸菌后先在20℃温度下摇床发酵,然后在28℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为22%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
实施例4
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述的中药原料包括:白花蛇舌草25重量份、半枝莲25重量份、水蛭25重量份、九香虫20重量份;黄芪20重量份、薏苡仁25重量份、莪术18重量份、熟地黄18重量份、苦参12重量份、土茯苓20重量份、槐角18重量份、虻虫25重量份、白头翁20重量份、藤梨根25重量份、元胡18重量份、甘草18重量份、冬凌草20重量份、枸杞子20重量份;
所述复合菌包括浓度为21.8×108CFU/mL的酵母菌、浓度为2.0×108CFU/m L的醋酸菌以及2.0×108CFU/mL乳酸菌;
治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将上述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,药粉末与水的料液比为1:280,然后在每种中药粉末单独采用20kHz的低频超声萃取10分钟,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为28%,在浓缩后的酶解液接种1.2%的酵母菌、1.2%醋酸菌、1.2%乳酸菌后先在22℃温度下摇床发酵,然后在30℃温度下静置发酵,最后在37℃温度下继续发酵得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
实施例5
一种治疗前列腺癌的中药酵素,是由中药原料经复合菌发酵制成的酵素;
所述的中药原料包括:白花蛇舌草20重量份、半枝莲20重量份、水蛭20重量份、九香虫18重量份;黄芪18重量份、薏苡仁20重量份、莪术15重量份、熟地黄15重量份、苦参10重量份、土茯苓18重量份、槐角15重量份、虻虫20重量份、、白头翁18重量份、藤梨根20重量份、元胡20重量份、甘草15重量份,冬凌草18重量份、枸杞子18重量份;
所述复合菌包括浓度为1.5×108CFU/mL的酵母菌和浓度为2.0×108CFU/m L的醋酸菌;
治疗前列腺癌的中药酵素的制备方法,包括
超声萃取步骤:按照比例将上述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,药粉末与水的料液比为1:300,然后在每种中药粉末单独采用20kHz的低频超声萃取10分钟,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为425%,在浓缩后的酶解液接种1.5%的酵母菌和1.0%醋酸菌后先在22℃温度下摇床发酵8天,然后在30℃温度下静置发酵8天,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
效果评价
1.急毒性试验
1)小鼠口服急毒性试验:选取60只小鼠,雌雄各半,在人工照明12h、通风良好的条件下饲养观察3d后,随机分成5个试验组(对应实施例1-5)和1个对照组,每组10只,给药前8h和给药后4h内禁食,但不禁水。口服按0.5m L/20g剂量灌胃给药。连续观察10d,记录小鼠中毒症状,小鼠死亡或试验结束后进行剖解,观察各组织器官的变化,取心脏、肝脏、脾脏、肾脏、肺脏及肠组织做病理切片,并计算半数致死量(LD50)。
结果显示:用灌胃针经口腔灌入复方苦木注射液后,个别小鼠蜷缩在角落,活动减少,但第3d观察,发现有38只小鼠活动、饮水和采食恢复正常;连续观察10天后,50只小鼠均健康存活,且全部小鼠的精神、食欲、肤色、分泌物、呼吸、食欲、肌肉运动等方面均未观察到异常变化。试验结束后剖检,发现各试验组小鼠脏器变化与对照组无明显眼观差异。按照毒理学安全性评价标准得知,LD50>10000mg/kg,属无毒性物质。
2.抗肿瘤作用的试验
1)试验材料:昆明种小白鼠、Balb/c种小白鼠,雌雄各半,体重(20±2)g;瘤株:S37小鼠,体重(20±2)g,同一性别。
2)试验方法:
按常规方法制造S37荷瘤小鼠的移植瘤模型,即取接种7天生长良好的S37传代小鼠,无菌条件下抽取腹水,用灭菌生理盐水4:1稀释、混匀制成肿瘤细胞混悬液(1.6×107个/ml),癌细胞混悬液应为乳白色半透明状态,若有血性腹水不可使用,接种于昆明种小鼠、Balb/c种小鼠右侧腋窝皮下,(每只0.2ml)。接种后的小白鼠每天正常饲养。待7天后接种小鼠腋窝有肿块形成时开始随机分组:中药酵素按照实施例1-5分5个给药组(分别命名为给药组1-5,每组各10中),分别ip给药,每日1次,每次每只小鼠0.8ml;生理盐水组(10只小鼠):ip生理盐水注射液,每日1次,每次每只小鼠0.8m;给药21天左后,待对照生理盐水组小鼠瘤块生长情况已基本大于1g后,即停止给药,对荷瘤小鼠血象进行检测后处死小鼠,剥取瘤块、脾脏、称重、记录并求其均值、标准差,与对照组作比较。实验结果均以表示,采用SPP16.0统计软件进行数据处理,以p<0.05为差异有统计学意义。实验结果参见下表1。
表1:实施例1-5所述的中药酵素对Balb/c种荷瘤小鼠体内抑瘤作用
表1的实验结果显示:血象测定结结果显示本发明的中药酵素有升高外周血细胞数目的趋势;根据记录结果表明相对于生理盐水组,给中药酵素的组对小鼠瘤块的重量有所减轻,显示本发明的中药酵素有抑制肿瘤的作用。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (10)
1.一种治疗前列腺癌的中药酵素,其特征在于,是由中药原料经复合菌发酵制成的酵素;
所述中药原料包括:白花蛇舌草15~30重量份、半枝莲15~30重量份、水蛭15~30重量份、九香虫10~25重量份;黄芪10~25重量份、薏苡仁15~30重量份、莪术10~20重量份、熟地黄10~20重量份、苦参5~15重量份、土茯苓10~25重量份、槐角10~20重量份、虻虫15~30重量份、白头翁15~25重量份、藤梨根15~30重量份、元胡10~20重量份、甘草10~20重量份;
所述复合菌包括浓度为1.2×108~2.2×108CFU/mL的酵母菌和浓度为2.0×108~2.5×108CFU/mL的醋酸菌。
2.根据权利要求1所述的治疗前列腺癌的中药酵素,其特征在于,所述中药原料还包括冬凌草12~25重量份、枸杞子10-20重量份。
3.根据权利要求2所述的治疗前列腺癌的中药酵素,其特征在于,所述中药原料包括:白花蛇舌草18~25重量份、半枝莲18~25重量份、水蛭18~25重量份、九香虫15-20重量份;黄芪15~20重量份、薏苡仁18~25重量份、莪术12~18重量份、熟地黄12~18重量份、苦参8~12重量份、土茯苓15~20重量份、槐角12~18重量份、虻虫18~25重量份、、白头翁15~20重量份、藤梨根18~25重量份、元胡12~18重量份、甘草12~18重量份、冬凌草15~20重量份、枸杞子15-20重量份。
4.根据权利要求2所述的治疗前列腺癌的中药酵素,其特征在于,所述中药原料包括:白花蛇舌草20重量份、半枝莲20重量份、水蛭20重量份、九香虫18重量份;黄芪18重量份、薏苡仁20重量份、莪术15重量份、熟地黄15重量份、苦参10重量份、土茯苓18重量份、槐角15重量份、虻虫20重量份、、白头翁18重量份、藤梨根20重量份、元胡20重量份、甘草15重量份,冬凌草18重量份、枸杞子18重量份。
5.根据权利要求1-4任一项所述的治疗前列腺癌的中药酵素,其特征在于,所述复合菌还包括浓度为1.5×108~2.0×108CFU/mL乳酸菌。
6.一种如权利要求1所述的治疗前列腺癌的中药酵素的制备方法,其特征在于,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
灭菌步骤:将上述酶解液在灭菌器中进行灭菌处理;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌和1.0-1.5%醋酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
7.根据权利要求6所述的制备方法,其特征在于,所述超声萃取步骤中,采用20kHz的低频超声萃取8-12分钟,所述中药粉末与水的料液比为1:(100-300)。
8.根据权利要求6所述的制备方法,其特征在于,所述酶解步骤中,采用的复合酶的加酶量为100-150μg/g,所述复合酶是质量比为1:1:3的果胶酶、纤维素酶和蛋白质酶;酶解的时间为8-10小时,酶解的温度为42-50℃。
9.根据权利要求6所述的制备方法,其特征在于,所述接种、发酵步骤中,酵母菌的发酵时间为6-8天,醋酸菌的发酵时间为8-10天。
10.一种如权利要求1所述的治疗前列腺癌的中药酵素的制备方法,其特征在于,包括
超声萃取步骤:按照比例将所述中药原料经过干燥后再分别粉碎得到中药粉末,中药粉末中加水搅拌均匀,然后在每种中药粉末单独用超声萃取,得到中药萃取物;
酶解步骤:将上述得到的每种中药萃取物混合后加入复合酶进行酶解,得到酶解液;
菌种活化和培养步骤:分别用液体培养基溶解酵母菌粉和醋酸菌,振荡使菌体溶解成悬浮液状态,然后将菌液在YDP培养基上进行涂布,酵母菌接种于PDA培养基在30℃恒温培养箱内培养2-3天,经过2次继代培养,菌液待用;醋酸菌接种于基础培养基活化1-2天,经过2次继代培养,菌液待用;乳酸菌接种于MRS培养基在温度37℃下活化l-2天,经过2次继代培养,菌液待用;
接种、发酵步骤:将上述酶解液浓缩至可溶性固形物含量为25-30%,在浓缩后的酶解液接种1.5-2.0%的酵母菌、1.0-1.5%醋酸菌、1.0-1.5%乳酸菌后先在20-25℃温度下摇床发酵,然后在28-30℃温度下静置发酵,最后在35-40℃温度下继续发酵得到发酵液;
真空冷冻干燥:上述发酵液浓缩至重量为20-25%后转入到真空干燥器中进行真空冷冻干燥;得到粉末状的中药酵素。
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Cited By (1)
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CN111281964A (zh) * | 2019-12-04 | 2020-06-16 | 红华御康生物科技(江苏)股份有限公司 | 一种中药发酵活性多肽口服液及其应用 |
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