CN110639011A - 治疗乳腺增生症的含重组水蛭素的中药组合物及制备方法 - Google Patents
治疗乳腺增生症的含重组水蛭素的中药组合物及制备方法 Download PDFInfo
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Abstract
本发明涉及乳腺增生治疗的相关技术领域,更具体地,本发明提供一种治疗乳腺增生症的含重组水蛭素的中药组合物及其制备方法。本发明的第一方面提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括3~10%的水相组分、0.5~3%的油相组分、5~15%重组水蛭素发酵液、0.5~2%中药组分、0.2~1%的助剂以及余量水。本发明提供的治疗乳腺增生症的含重组水蛭素的中药组合物利用重组水蛭素发酵液与中药组分相结合的技术方案,可以在较短时间内缓解或治愈乳房肿胀感或胀痛感,且本发明提供的组合物具有稳定的理化性能,储存稳定性较好,可以较好的延长相关产品的保存时间。
Description
技术领域
本发明涉及乳腺增生治疗的相关技术领域,更具体地,本发明提供一种治疗乳腺增生症的含重组水蛭素的中药组合物及其制备方法。
背景技术
乳腺增生病是临床常见病、多发病,其发病率占乳腺疾病之首,是正常乳腺小叶生理性增生与复旧不全,乳腺正常结构出现紊乱,属于病理性增生,是既非炎症又非肿瘤的良性疾病。发病年龄集中于20-50岁,其发病率占育龄妇女的40%左右,占全部乳房疾病的75%,其发生乳腺癌的危险性较健康妇女高2-3倍。由于本病病程较长,且易复发,以乳房疼痛和乳房肿块为主要临床表现,疼痛这一主观症状显著,部分患者对于增生转化癌变忧虑重重,严重危害了女性身心健康。
西医认为乳腺增生是由于卵巢内分泌紊乱导致孕酮水平降低而雌激素水平过高,过高的雌激素作用于乳腺的靶点,引起乳腺腺叶组织不得增生而出现乳房疼痛及乳房内肿块的产生。因此西医治疗乳腺增生以药物为主,必要时进行手术治疗,服用的药物以激素类药物为主,如他莫昔芬,甲基睾丸素,丹那唑以及孕激素类药物,另外还有维生素类和非甾体类药物,这些药物有一定的疗效,但也带来很多的副作用,例如他莫昔芬可能造成烦躁、血栓、子宫内膜癌等严重问题,丹那唑可能带来体重增加、头痛、水肿等问题,甲状腺素可能引起心悸、汗多、、失眠、呕吐等不良反应。
与西医不同,中医治疗和预防疾病都是从整体入手,中医认为乳房虽然属于局部器官,但通过与十二经脉及奇经八脉的纵横联系,和内在的脏腑形成了一个有机整体,因此对乳腺增生病要从整体与局部关系出发去认识病情,从脏腑辨证角度分析病理变化,在本病发病过程中,主要与肝、脾、肾三脏功能失调关系最为密切。中医学认为乳头属肝,乳房属胃。肝主疏泄,若郁怒伤肝,肝气郁滞,肝旺侮土,脾虚失于健运,或思虑伤脾,肝脾不和,可导致气滞、血瘀、痰凝结聚与乳房。肾藏精,肾中精气的盛衰决定着乳房生长、发育及分泌功能。若肾阳不足,推动无力,则肝失疏泄,脾失健运,致使气滞血瘀痰凝结聚于乳房;肾阴不足,肝失所养,致使气血失和,痰凝血瘀,乳络不通而发病。因此中医治疗常采用疏肝理气,活血化瘀,补肾健脾,化痰散结等中药方剂,国内有大量的研究和临床实践结果表明中药治疗乳腺增生具有疗效确切,副作用小等特点。而中药治疗乳腺增生多采用内治法,即口服中药或者中成药,虽然疗效确切但中药煎煮不便且口服给药可能发生肝脏首过效应再加上胃肠道消化,药效会打折扣,需要较长的治疗期限,另外也会增加胃肠道等脏器的负担。
发明专利公布了一项利用中药复方结合水蛭素生物发酵液治疗女性乳腺增生症的组合物,制剂及制备方法和应用,该组合物可用于药物外敷,药物穴位贴敷,药物乳罩等形式。
发明内容
本发明的第一方面提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括3~10%的水相组分、0.5~3%的油相组分、5~15%重组水蛭素发酵液、0.5~2%中药组分、0.2~1%的助剂以及余量水。
作为本发明的一种优选技术方案,其中,中药组的制备原料分包括第一组分与第二组分;其中,第一组分选自泽兰、漏芦、黄柏、大黄、冰片、柴胡、川穹、白芍、川楝子、莪术、三棱中的任一种或多种的组合;第二组分选自香附、蒲公英、甘草、当归、延胡素、瓜蒌、乳香、没药中的任一种或多种的组合。
作为本发明的一种优选技术方案,其中,水相组分包括小分子醇与络合剂。
作为本发明的一种优选技术方案,其中,油相组分为保湿剂,保湿剂选自木糖醇类物质、羟基乙酸、黄原胶、甘油辛酸酯、聚乙二醇、海藻糖、赤藓醇、吡咯烷酮羧酸钠、乙氧基葡萄糖衍生物、透明质酸钠中的任一种或多种的组合。
作为本发明的一种优选技术方案,其中,重组水蛭素发酵液的制备过程为:将种子液置于初始培养基中,当DO值大于55%时,滴加补料培养基;其中,初始培养基的制备原料包括糖溶液、微量金属溶液以及维生素溶液。
作为本发明的一种优选技术方案,其中,糖溶液包括无氨基酵母氮源、碳源以及氨基酸溶液。
作为本发明的一种优选技术方案,其中,微量金属溶液包括锌盐、锰盐、钴盐、铁盐、钙盐、钠盐、铜盐中的至少一种。
作为本发明的一种优选技术方案,其中,补料培养基包括微量金属溶液、维生素溶液以及碳源。
本发明的第二方面提供一种含有所述治疗乳腺增生症的含重组水蛭素的中药组合物的制剂。
本发明的第三方面提供一种所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法,包括以下步骤:
(1)将水相组分与水混合均匀,加热;
(2)将油相组分混合均匀,加热;
(3)将油相组分加入到水相组分中,高速剪切;
(4)降温,再加入重组水蛭素发酵液与中药组分;
(5)再降温,降入助剂,搅拌均匀,即得。
与现有技术相比,本发明提供的治疗乳腺增生症的含重组水蛭素的中药组合物利用重组水蛭素发酵液与中药组分相结合的技术方案,可以在较短时间内缓解或治愈乳房肿胀感或胀痛感,且本发明提供的组合物具有稳定的理化性能,储存稳定性较好,可以较好的延长相关产品的保存时间。
附图说明
图1:志愿者治疗前对应的诊断结果;
图2:志愿者治疗后对应的诊断结果。
具体实施方式
除非另有说明、从上下文暗示或属于现有技术的惯例,否则本申请中所有的份数和百分比都基于重量,且所用的测试和表征方法都是与本申请的提交日期同步的。如果现有技术中披露的具体术语的定义与本申请中提供的任何定义不一致,则以本申请中提供的术语定义为准。
下面结合具体实施方式对本发明提供技术方案中的技术特征作进一步清楚、完整的描述,并非对其保护范围的限制。
本发明中的词语“优选的”、“优选地”、“更优选的”等是指,在某些情况下可提供某些有益效果的本发明实施方案。然而,在相同的情况下或其他情况下,其他实施方案也可能是优选的。此外,对一个或多个优选实施方案的表述并不暗示其他实施方案不可用,也并非旨在将其他实施方案排除在本发明的范围之外。本发明中未提及的组分的来源均为市售。
本发明的第一方面提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括3~10%的水相组分、0.5~3%的油相组分、5~15%重组水蛭素发酵液、0.5~2%中药组分、0.2~1%的助剂以及余量水;优选地,包括5~8%的水相组分、0.1~2%的油相组分、8~12%重组水蛭素发酵液、0.1~1.5%中药组分、0.4~0.8%的助剂以及余量水;更优选地,包括7%的水相组分1.5%的油相组分、10%重组水蛭素发酵液、1%中药组分、0.6%的助剂以及余量水。
在一种实施方式中,水相组分包括小分子醇与络合剂;优选地,小分子醇与络合剂的重量比为1:(0.01~0.003);更优选地,小分子醇与络合剂的重量比为1:0.006。
在一种实施方式中,小分子醇选自丙二醇、1,3-丁二醇、1,2-已二醇、甘油中的任一种或多种的组合;优选地,小分子醇为1,3-丁二醇与1,2-已二醇;进一步优选地,1,3-丁二醇与1,2-已二醇的重量比为1:(0.05~0.2);更优选地,1,3-丁二醇与1,2-已二醇的重量比为1:0.1。
在一种实施方式中,络合剂为EDTA二钠和\或EDTA四钠;优选地,络合剂为EDTA二钠。
在一种实施方式中,水相组分还包括精氨酸;优选地,精氨酸与络合剂的重量比为1:(1.2~1.6);更优选地,精氨酸与络合剂的重量比为1:1.5。
在一种实施方式中,油相组分为保湿剂,保湿剂选自木糖醇类物质、羟基乙酸、黄原胶、甘油辛酸酯、聚乙二醇、海藻糖、赤藓醇、吡咯烷酮羧酸钠、乙氧基葡萄糖衍生物、透明质酸钠中的任一种或多种的组合;优选地,保湿剂为木糖醇类物质、黄原胶以及透明质酸钠;进一步优选地,木糖醇类物质、黄原胶以及透明质酸钠的重量比为1:(0.2~0.5):(0.05~0.15);更优选地,木糖醇类物质、黄原胶以及透明质酸钠的重量比为1:0.3:0.1。
在一种实施方式中,木糖醇类物质包括木糖醇、无水木糖醇以及木糖醇基葡糖苷,本发明对木糖醇类物质的购买厂家不做限制,一种实施方式中,木糖醇类物质购自法国SEPPIC公司,AQUAXYL木糖醇。
本发明对透明质酸钠的购买厂家不做特别限制,一种实施方式中,透明质酸钠纳诺HA,购自华熙福瑞达。
在一种实施方式中,中药组分的制备原料包括第一组分与第二组分;其中,第一组分选自泽兰、漏芦、黄柏、大黄、冰片、柴胡、川穹、白芍、川楝子、莪术、三棱中的任一种或多种的组合;第二组分选自香附、蒲公英、甘草、当归、延胡素、瓜蒌、乳香、没药中的任一种或多种的组合;优选地,第一组分与第二组分的重量比为1:(0.5~1.5);更优选地,第一组分与第二组分的重量比为1:1。
在一种实施方式中,第一组分包括泽兰、漏芦、黄柏以及大黄;第二组分包括香附与蒲公英;优选地,泽兰、漏芦、黄柏以及大黄的重量比为1:(0.5~1.5):(0.5~1.5):(0.5~1.5);香附与蒲公英的重量比为1:(0.5~1.5);更优选地,泽兰、漏芦、黄柏以及大黄的重量比为1:1:1:1;香附与蒲公英的重量比为1:1。
在一种实施方式中,第一组分包括柴胡、川穹、白芍以及川楝子;第二组分包括香附与延胡素;优选地,柴胡、川穹、白芍以及川楝子的重量比为1:(0.5~1.5):(0.5~1.5):(0.5~1.5);香附与延胡素的重量比为1:(0.5~1.5);更优选地,柴胡、川穹、白芍以及川楝子的重量比为1:1:1:1;香附与延胡素的重量比为1:1。
在一种实施方式中,中药组分的制备方法如下:
(1)将中药组分的制备原料用冷水浸泡,干燥后备用;
(2)将步骤(1)所得产物放入蒸馏水中蒸煮,过滤得滤液、滤渣,将滤液浓缩,得A组分提取液;
(3)将步骤(2)中的滤渣放入乙醇\蒸馏水的混合液中,回流提取,浓缩得B组分提取液,即得,所述乙醇与蒸馏水的体积比为1:(3~5)。
泽兰
别名地瓜儿苗、地笋、甘露子、方梗泽兰;本品为唇形科植物毛叶地瓜儿苗Lycopuslucidus Turcz.var.hirtus Regel的干燥地上部分。夏、秋季茎叶茂盛时采割,晒干。中药材泽兰是唇形科植物地瓜儿苗或毛叶地瓜儿苗的干燥地上部分,采收于夏、秋两季,割取地上部分,切段晒干即可;其性温,味苦、辛,有活血通经、利水消肿的功效,可以治疗月经不调、经闭、通经、水肿、产后瘀血腹痛等病症;主要成分为挥发油和鞣质,经气质联用鉴定46种成分;还含三萜化合物、熊果酸、葡萄糖、泽兰糖、虫漆蜡、白桦脂酸等成分。
漏芦
别名狼头花;来源本品为菊科植物祁州漏芦Rhaponticum uniflorum(L.)DC.的干燥根。春、秋二季采挖,除去须根及泥沙,晒干。漏芦含β-蜕皮甾酮等脱皮甾酮类化合物、挥发油及一些脂溶性成分。另含卡多帕亭、异卡多帕亭、漏芦甾酮、甘草苷等成分。《本草纲目》中记载:漏芦,下乳汁、消热毒、排脓、止血、生肌、杀虫,故东垣以为手、足阳明药,而古方治痈疽发背,以漏芦汤为首称也。庞安常《伤寒论》治痈疽及预解时行痘疹热,用漏芦叶,云无则以山栀子代之,亦取其寒能解热,盖不知其能入阳明之故也。漏芦可抗动脉粥样硬化;降血糖;抑制过氧化脂质生成;抗氧化;抑制大鼠B型单胺氧化酶活性;保肝等作用。
香附
别名莎草、香附子、雷公头、三棱草、香头草、回头青、雀头香;本品为莎草科植物莎草Cyperus rotundus L.的干燥根茎。秋季采挖,燎去毛须,置沸水中略煮或蒸透后晒干,或燎后直接晒干。香附中含有葡萄糖、果糖、淀粉40~41.1以及挥发油,用于肝气郁结之胸胁及胃腹胀痛。配柴胡、青皮治胸胁痛。配高良姜(名良附丸)治胃寒痛;也可用于肝气郁结之月经不调、小腹胀痛。配艾叶治寒凝气滞之行经腹痛;此外还有解热镇痛与降温作用。
黄柏
别名黄檗、元柏、檗木;本品为芸香科植物黄皮树Phellodendron chinenseSchneid.或黄檗Phellodendron amurense Rupr.的干燥树皮。前者习称“川黄柏”,后者习称“关黄柏”。剥取树皮后,除去粗皮,晒干。黄柏中含小檗碱,并含木兰碱、黄柏碱等多种生物碱以及内酯、甾醇、黏液质、黄酮类、生物碱、挥发油、固醇、糖类等化学成分。其中有盐酸小檗碱、黄柏苷、豆固醇、黄柏酮等成分;黄柏具有抗病原微生物及病原虫的作用;具有一定的解热和抗炎作用;有降低血压、兴奋心脏的作用,具有明显抗心律失常作用;影响消化系统功能及抑制中枢神经系统,还有抗血小板凝聚作用,有降低血糖、镇痛、祛痰等作用。
黄连
根状茎为著名中药“黄连”,含小檗碱、黄连碱、甲基黄连碱、掌叶防己碱等生物碱;用于湿热痞满,呕吐吞酸,泻痢,黄疸,高热神昏,心火亢盛,心烦不寐,血热吐衄,目赤,牙痛,消渴,痈肿疔疮;外治湿疹,湿疮,耳道流脓。酒黄连善清上焦火热。用于目赤,口疮。姜黄连清胃和胃止呕。用于寒热互结,湿热中阻,痞满呕吐。萸黄连舒肝和胃止呕。用于肝胃不和,呕吐吞酸;抗菌、抗炎、抗腹泻、解热、降血糖、抗血脂、抗氧化以及抗溃疡等作用。
甘草
本品为豆科植物甘草Glycyrrhiza uralensis Fisch.、胀果甘草Glycyrrhizainflata Bat.或光果甘草Glycyrrhiza glabra L.的干燥根。春、秋二季采挖,除去须根,晒干。甘草含有多种化学成分,主要成分有甘草酸、甘草甙等。甘草的化学组成极为复杂,目前为止从甘草中分离出的化合物有甘草甜素、甘草次酸、甘草甙、异甘草甙、新甘草甙、新异甘草甙、甘草素、异甘草素以及甘草西定、甘草醇、异甘草醇、7-甲基香豆精、伞形花内酯等数十种化合物;甘草可用于治疗心气虚,心悸怔忡,脉结代,以及脾胃气虚,倦怠乏力;痈疽疮疡、咽喉肿痛;也可用于抗炎,抗过敏,能保护发炎的咽喉和气管粘膜;甘草还可抑制皮质醇的转化,从而导致血压上升和低血钾。
蒲公英
别名黄花地丁、婆婆丁;本品为菊科植物蒲公英Taraxacum mongolicum Hand.-Mazz.、碱地蒲公英Taraxacum sinicum Kitag.或同属数种植物的干燥全草。春至秋季花初开时采挖,除去杂质,洗净,晒干。蒲公英,又叫黄花地丁、婆婆丁,为菊科蒲公英属多年生草本植物蒲公英的带根全草。全国大部分地区有分布。为常用中药材,其营养成分极为丰富。蒲公英性寒、味甘苦,有清热解毒、消肿散结、利湿退黄之功效。
从蒲公英干料中提取的黄酮类物质,具有治疗痈肿疔毒、治疗肺痈咳痰、治疗湿热黄疸、可治疗小便淋沥涩痛、治疗乳痈等作用,可作保健品及食品原料。
蒲公英又称尿床草,对于利尿可是有非常好的效果,它具有丰富的维生素A和C及矿物质,对消化不良、便秘都有改善的作用,另外叶子还有改善湿疹、舒缓皮肤炎、关节不适的净血功效,根则具有消炎作用,可以治疗胆结石、风湿。
中医认为,本品味甘苦性寒,入肝、胃二经。具有清热解毒、消痈散结之功效。现代药理研究证明,此药花含叶黄呋喃素、维生素B2,根含多种三帖醇,本草含肌醇,天冬酰胺、苦味质、皂甙、树脂、菊糖,此外,尚含果胶、胆碱等。药理实验发现蒲公英煎剂对金黄色葡萄球菌有较强的抑制作用,对致病性皮肤真菌也有抑制作用。水浸剂有相当强的利胆作用。另外,还有抗肺癌之作用。
大黄
本品为蓼科植物掌叶大黄Rheum palmatum L。、唐古特大黄Rheum tanguticumMaxim。ex Balf。或药用大黄Rheum officinale Baill。的干燥根及根茎。秋末茎叶枯萎或次春发芽前采挖,除去细根,刮去外皮,切瓣或段,绳穿成串干燥或直接干燥。大黄苦,寒。归脾、胃、大肠、肝、心包经;泻下攻积,清热泻火,凉血解毒,逐瘀通经,利湿退黄。用于实热积滞便秘,血热吐衄,目赤咽肿,痈肿疔疮,肠痈腹痛,瘀血经闭,产后瘀阻,跌打损伤,湿热痢疾,黄疸尿赤,淋证,水肿;外治烧烫伤。酒大黄善清上焦血分热毒。用于目赤咽肿,齿龈肿痛。熟大黄泻下力缓,泻火解毒。用于火毒疮疡。大黄炭凉血化瘀止血。用于血热有瘀出血症。
冰片
别名合成龙脑、梅片、艾粉、结片;分机制冰片与艾片两类。机制冰片以松节油、樟脑等为原料经化学方法合成的龙脑;艾片为菊科艾纳香属植物大风艾的鲜叶经蒸气蒸馏、冷却所得的结晶,又称“艾粉”或“结片”。冰片味辛、苦,微寒;归心、肝、肺经;清香宣散,具有开窍醒神,清热散毒,明目退翳的功效,主治热病高热神昏,中风痰厥惊痫,暑湿蒙蔽清窍,喉痹耳聋,口疮齿肿,疮痈疳痔,目赤肿痛,翳膜遮睛;凡痰火郁闭,喉痹音哑,或火热壅滞,口疮齿肿者,冰片可与朱砂、硼砂、玄明粉配伍吹搽患处,以散火解毒;热闭神昏者,冰片可与麝香、牛黄、黄连、郁金等配伍,以清热开窍;暴发火眼,或翳膜胬肉者,冰片可与炉甘石、玄明粉、硼砂配伍,为末点眼。
柴胡
本品为伞形科植物柴胡Bupleurum chinense DC.或狭叶柴胡Bupleurumscorzonerifolium Willd.的干燥根。按性状不同,分别习称“北柴胡”及“南柴胡”。春、秋二季采挖,除去茎叶及泥沙,干燥。柴胡含有戊酸、亚麻酸、棕榈酸、硬脂酸、山柰酚、山柰苷、槲皮素、芸香苷、香橙烯等成分,其性微寒,味苦。归肝经、胆经、肺经;柴胡具有抗炎、解热、镇静、镇痛、镇咳及抗惊厥作用;可减轻肝损伤和促进胆汁分泌;具有降血压、降低血清胆固醇以及溶血作用;具有抗溃疡、抗菌、抗病毒、抗肿瘤、升高血糖、降低血中脂肪含量、抗辐射损伤等作用。
当归
本品为伞形科植物当归Angelica sinensis(Oliv.)Diels的干燥根。秋末采挖,除去须根及泥沙,待水分稍蒸发后,捆成小把,上棚,用烟火慢慢熏干。伞形科植物当归Angelica sinensis(Oliv、)Diels的干燥根。秋末采挖,除去须根及泥沙,待水分稍蒸发后,捆成小把,上棚,用烟火慢慢熏干。全当归根略呈圆柱形,根上端称“归头”;主根称“归身”或“寸身”。支根称“归尾”或“归腿”,全体称“全归”。全当归既能补血,又可活血,统称和血;当归身补血,当归尾破血。当归补血;活血;调经止痛;润燥滑肠。主血虚诸证;月经不调;经闭;痛经;症瘕结聚;崩漏;虚寒腹痛;痿痹;肌肤麻木;肠燥便难;赤痢后重;痈疽疮疡;跌扑损伤。
川芎
别名为芎藭、小叶川芎;本品为伞形科植物川芎Ligusticum chuanxiong Hort.(或Ligusticum wallichii Franch.)的干燥根茎。夏季当茎上的节盘显着突出,并略带紫色时采挖,除去泥沙,晒后炕干,再去须根。用于感冒头痛,属于风寒者,可与荆芥、防风、白芷同用;属于风热者,可与菊花、薄荷同用。川芎在生活中对于人体的功效是非常的多的。川芎有明显的镇静作用。川芎挥发油少量时对动物大脑的活动具有抑制作用,而对延脑呼吸中枢、血管运动中枢及脊髓反射中枢具有兴奋作用。用于眼部产品主要也是行气活血,促进眼部的血液循环。
白芍
别名芍药;本品为毛茛科植物芍药Paeonia lactiflora Pall.的干燥根。夏、秋二季采挖,洗净,除去头尾及细根,置沸水中煮后除去外皮或去皮后再煮,晒干。白芍性微寒,味苦、酸。归肝经、脾经。含芍药苷、羟基芍药苷、苯甲酰芍药苷、芍药内酯苷、氧化芍药苷、白芍苷、常春藤皂苷元、芍药苷元酮、没食子酰芍药苷、山柰酚-3,7-二-O-β-D-葡萄糖苷、胡萝卜苷、β-谷固醇、芍药乳糖酮等,挥发油中主要含苯甲酸、牡丹酚等。养血敛阴,柔肝止痛,平肝阳。平肝止痛,养血调经,敛阴止汗。用于头痛眩晕、胁痛、腹痛、四肢挛痛、血虚萎黄、月经不调、自汗、盗汗。
延胡索
别名玄胡素、元胡;本品为罂粟科植物延胡索Corydalis yanhusuo W.T.Wang的干燥块茎。夏初茎叶枯萎时采挖,除去须根,洗净,置沸水中煮至恰无白心时,取出,晒干。延胡索性温,味辛、苦。归肝经、脾经;含20多种生物碱,如紫堇碱、异紫堇球碱等,镇痛、催眠、镇静、安定;抗惊厥;扩张冠状动脉;抑制心脏;抗心律失常;抗溃疡;促进垂体分泌促肾上腺皮质激素。含有的总碱镇痛效价是吗啡的40%,总碱中以延胡索甲素、延胡素乙素、延胡素丑素的镇痛作用最为明显,其中延胡素乙素最强,延胡素丑素次之,延胡素甲素再次,较大用量对兔、犬、猴等均有镇静催眠作用,延胡索乙素有对抗中枢兴奋和增强中枢抑制作用,静注延胡素乙素可缩小脑梗塞范围、脑水肿及钙离子抑制。
瓜蒌
本品为葫芦科植物栝楼Trichosanthes kirilowii Maxim.或双边栝楼Trichosan-thes rosthornii Harms的干燥成熟果实。秋季果实成熟时,连果梗剪下,置通风处阴干。瓜蒌性寒,味甘、微苦。归肺经、大肠经、胃经;果实含丝氨酸蛋白酶、多种氨基酸及含挥发油,种子含三萜皂苷等多种成分;有利于扩张动脉冠;抗心肌缺血;改善微循环;抑制血小板凝集;耐缺氧;抗心律失常;抗溃疡;抗菌;抗癌;抗衰老。
川楝子
别名金铃子、川楝实;本品为楝科植物川楝Melia toosendan Sieb.et Zucc.的干燥成熟果实。冬季果实成熟时采收,除去杂质,干燥。川楝子性寒,味苦,有小毒。归肝经、小 肠经、膀胱经;用治肝气郁滞或肝郁化火所致的胸胁胀痛、脘腹疼痛及疝痛,多与延胡索同用,如金铃子散;若用治寒疝少腹胀痛,可配伍小茴香、木香等同用;对于肝肾阴虚、肝气不舒之胸脘胁痛、吞酸口苦、舌赤少津等症,可与沙参、麦冬等配伍为用,如一贯煎;也可用于燥闷烦热、疝气上涌、热厥心痛、膀胱疝气、小肠疝痛、小便不利、下腹疼痛等。川楝子苦寒性降,能够导湿热下走渗道,行气解瘀,气血运行畅通了,自然可以止痛;川楝子可以导小肠、膀胱的燥热,辅之引心胞(即心包,是心脏外面的包膜,具有保护心脏的作用)相火下行,所以是治疗心痛、腹痛、疝气的要药。用于急性乳腺炎、头癖、肝火旺盛、淋病、茎痛、遗精等症;医学典籍《本经逢原》有记载称,川楝子主要用于温疾烦狂,取以引火毒下泄,从而心烦意乱、肝气郁结自然就消除了。古代有一个“金铃子散”就是运用川楝子清肝泻火的功效,用于治疗女性产后血瘀、心疼,配伍散结血的元胡,效果显著。
三棱
本品为黑三棱科植物黑三棱Sparganium stoloniferum Buch.-Ham.的干燥块茎。冬季至次年春采挖,洗净,削去外皮,晒干。三棱块茎含挥发油,主要成分为苯乙醇、对苯二酚、十六酸等,三棱水提物可使凝血酶对纤维蛋白的凝聚时间显著延长;有抗体外血栓形成的作用;水煎剂对离体兔子宫平滑肌呈兴奋作用;抗肿瘤;对心脏有降低心肌细胞耗氧量、减少冠脉阻力、增加冠脉流量、改善心肌缺氧耐受力等作用。
莪术
别名温莪术、蓬莪术、山姜黄、芋儿七、臭屎姜;本品为姜科植物蓬莪术Curcumaphaeocaulis Val.、广西莪术Curcuma Kwangsiensis S.G.Lee et C.F.Liang或温郁金Curcuma wenyujin Y.H.Chen et C.Ling的干燥根茎。后者习称“温莪术”。冬季茎叶枯萎后采挖,洗净,蒸或煮至透心,晒干或低温干燥后除去须根及杂质。莪术性和味属于经络,苦,暖。回到肝脾经络。功效指征,活血益气,祛积止痛。用于血瘀腹痛、肝脾肿大、心腹肿痛、积淀、女性血瘀闭经、跌伤为痛的饮食停滞,行气止痛。莪术活血化瘀,适用于闭经、胸闷、腹痛及气滞血瘀的症状性团块。它通常有三条边,消化用于胃和腹部因饮食停滞而引起的膨胀和疼痛,常伴有焦麦芽和槟榔。
没药
别名末药、明没药;橄榄科没药属植物没药树Commiphora myrrha Engl.[C.molmol Engl.]及同属他种植物的树干皮部渗出的油胶树脂。一般在11月至次年2月间采收,但亦有在6~7月间采收,采收后拣净树皮及其他杂质即得。没药具有抗菌、抗微生物、抗炎、收敛、具香胶特质、除臭、祛肠胃胀气、消毒、利尿、通经、化痰、杀霉菌、激励、利胃、催汗、补身、利子宫、治创伤,皮肤有助益;主治跌打损伤、筋骨疼痛。做茶喝,则体香,通血脉、舒筋络,固牙齿,长须发;防止组织退化很有效果,尤其是有伤口坏疽的情况。它清凉的功能可帮助皮肤溃疡与疮,改善流汤的伤口及龟裂的皮肤。有效对抗流汤的湿疹及香港脚;没药味苦平无毒。然平应作辛,气应微寒。没药苦能泄,辛能散,寒能除热。故能入血分,散瘀血,治血热诸疮及卒然下血证也。肝经血热,则目为亦痛、肤翳,散肝经之血热,则目病除矣。
乳香
别名滴乳香、熏陆香;橄榄科乳香树属植物乳香树Boswellia carterii Birdw.[B.sacra Fluek.]、药胶香树B.bhawdajiana Birdw.及野乳香树B.neglecta M.Moore等,以其树干皮部伤口渗出的油胶树脂入药。春夏均可采。乳香调气活血,定痛,追毒。治气血凝滞、心腹疼痛,痈疮肿毒,跌打损伤,痛经,产后瘀血刺痛;治产后瘀滞不清,攻刺心腹作痛:乳香、没药(俱瓦上焙出油)各三钱,五灵脂、延胡索、牡丹皮、桂枝各五钱(俱炒黄),黑豆一两(炒成烟炭)。共为末,每服三钱,生姜泡汤调下。
在一种实施方式中,助剂选自油醇聚醚、聚丙烯酸酯、硬脂醇、氢化蓖麻油、吐温、司盘中的任一种或多种的组合;优选地,助剂为油醇聚醚。
在一种实施方式中,油醇聚醚选自油醇聚醚-12、油醇聚醚-15、油醇聚醚-20中的任一种或多种的组合;优选地,油醇聚醚包括油醇聚醚-12、油醇聚醚-15、油醇聚醚-20;进一步优选地,油醇聚醚-12、油醇聚醚-15、油醇聚醚-20的重量比为1:(0.8~1.2):(0.8~1.2);更优选地,油醇聚醚-12、油醇聚醚-15、油醇聚醚-20的重量比为1:1:1。
申请人在实验过程中发现当选用特定的油醇聚醚时可以提高组合的稳定性,可能由于油醇聚醚-12、油醇聚醚-15、油醇聚醚-20,其亲水性基团与疏水性基团与体系中的中药组分的亲水、亲油达到平衡,一方面不同物质之间的界面力较小,一方面不同组分之间作用力均衡,从而使得组合物体系均匀、稳定。
重组水蛭素发酵液
水蛭素是迄今为止世界上所发现最强的凝血酶天然特异抑制剂。它在降血稠、血脂、血糖、血压、尿酸方面效果极佳,现已在治疗三高症、心脑血管疾病、痛风、中风、糖尿病等疾病上效果显著。但是天然的水蛭素只存在水蛭的唾液中,含量极低,通过野外捞取和人工培养水蛭,然后获取水蛭素,就在成本高,杂质多,活性低等种种问题。
我们采用基因工程技术,将水蛭素基因克隆到酿酒酵母菌株中,经过发酵培养产生了重组水蛭素发酵液,该发酵液中的水蛭素能促进皮肤内层的血液循环,加快代谢,提高肌肤清除自由基的能力和细胞活力,而发酵液还富含糖分,小分子肽和维生素,为皮肤提供营养成分。
在申请人完成技术方案的过程中发现,当利用重组水蛭素发酵液与中药组分共同作用时可以有效提高对乳腺增生症的治疗效果,可能由于重组水蛭素发酵液活性较高,有利于促进皮肤对中药组合物的吸收,促进血液循环,进而提高疗效。
在一种实施方式中,重组水蛭素发酵液的制备过程为:将种子液置于初始培养基中,当DO值大于55%时,滴加补料培养基;其中,初始培养基的制备原料包括糖溶液、微量金属溶液以及维生素溶液。
在一种优选地实施方式中,重组水蛭素的制备方法,其中,重组水蛭素的制备过程包括:将种子液置于初始培养基中,通无菌空气,并保持DO值大于40%;当DO值大于55%时,于初始培养基中通过流加的方式滴加补料培养基。
在一种实施方式中,无菌空气的通气量为3~7L/min;优选地,无菌空气的通气量为5L/min。
在一种实施方式中,初始培养基的温度为25~40℃;优选地,初始培养基的温度为27~35℃;更优选地,初始培养基的温度为30℃。
在一种实施方式中,重组水蛭素的制备过程在本领域常规或熟知的装置和条件下进行。
在一种实施方式中,重组水蛭素的制备过程在摇瓶中进行,转速为300~1000r/min;优选地,转速为650r/min。
在一种实施方式中,重组水蛭素的制备过程在5L发酵罐中进行。
在一种实施方式中,通过滴加氨水控制初始培养基的pH为4.5~5.5;优选地,通过滴加氨水控制初始培养基的pH为5.0。
在一种实施方式中,氨水的浓度为20~30wt%;优选地,氨水的浓度为25wt%。
在一种实施方式中,种子液的接种量为10~15%;优选地,种子液的接种量为11~13%。
在一种实施方式中,种子液的制备方法为:接种工程菌甘油保藏菌至装有种子培养基的瓶中,培养,即得待接种的种子液。
优选地,种子液的制备方法为:种子液的制备方法为:①接种工程菌甘油保藏菌至装有5mL种子培养基的100mL三角瓶中,于30℃,220rpm摇床培养24小时;②转接至装有50mL种子培养基的250mL三角瓶中,于30℃,220rpm摇床培养24小时;③转接至装有250mL种子培养基的1000mL三角瓶中(共两瓶),于30℃,220rpm摇床培养12小时,即得待接种的种子液。
在一种实施方式中,种子培养基包括葡萄糖、YNB、氨基酸以及水。
在一种实施方式中,种子培养基中的葡萄糖含量为初始培养基的溶液的总体积的10~30g/L;优选地,种子培养基中的蔗糖含量为初始培养基的溶液的总体积的20g/L。
在一种实施方式中,葡萄糖、YNB、氨基酸的重量比为(10~30):(5~9):(1~2);优选地,葡萄糖、YNB、氨基酸的重量比为20:7:1.5。
在一种实施方式中,种子培养基中的水含量为400~550mL;优选地,种子培养基中的水含量为480mL。
在一种实施方式中,氨基酸选自L-精氨酸、L-天冬氨酸、L-谷氨酸、L-组氨酸、L-亮氨酸、L-赖氨酸、L-蛋氨酸、L-苯丙氨酸、L-丝氨酸、L-苏氨酸、L-色氨酸、L-酪氨酸、L-缬氨酸中的任一种或多种的组合;优选地,氨基酸为L-精氨酸、L-组氨酸以及L-色氨酸;更优选地,氨基酸为L-精氨酸、L-组氨酸以及L-色氨酸的重量比为1:(0.8~1.2):(0.8~1.2);更优选地,L-精氨酸、L-组氨酸以及L-色氨酸的重量比为1:1:1。
在一种实施方式中,接种工程菌为INVSC1酿酒酵母菌株。
在一种实施方式中,糖溶液包括无氨基酵母氮源(YNB)、碳源以及氨基酸溶液;优选地,糖溶液还包括铵盐、磷酸盐、镁盐、锌盐、锰盐、钴盐、铁盐、钙盐、钠盐、铜盐中的至少一种;更优选地,糖溶液还包括消泡剂。
在一种实施方式中,糖溶液包括无氨基酵母氮源(YNB)、碳源、氨基酸溶液、铵盐、磷酸盐、镁盐以及消泡剂。
在一种实施方式中,无氨基酵母氮源(YNB)含量为初始培养基的溶液的总体积的4~10g/L;优选地,无氨基酵母氮源(YNB)含量为初始培养基的溶液的总体积的6~8g/L;更优选地,无氨基酵母氮源(YNB)含量为初始培养基的溶液的总体积的7g/L。
在一种实施方式中,碳源含量为初始培养基的溶液的总体积的20~50g/L;优选地,碳源含量为初始培养基的溶液的总体积的30~40g/L;更优选地,碳源含量为初始培养基的溶液的总体积的35g/L。
在一种实施方式中,氨基酸溶液含量为初始培养基的溶液的总体积的10~40mL/L;优选地,氨基酸溶液含量为初始培养基的溶液的总体积的15~30mL/L;更优选地,氨基酸溶液含量为初始培养基的溶液的总体积的20mL/L。
在一种实施方式中,糖溶液中的铵盐、磷酸盐以及镁盐的重量比为(1~1.5):(1.5~3):1;优选地,糖溶液中的铵盐、磷酸盐以及镁盐的重量比为1.3:2.5:1。
在一种实施方式中,糖溶液中的铵盐含量为初始培养基的溶液的总体积的10~30g/L;优选地,糖溶液中的铵盐含量为初始培养基的溶液的总体积的15g/L。
在一种实施方式中,消泡剂含量为初始培养基的溶液的总体积的0.1~1mL/L;优选地,消泡剂含量为初始培养基的溶液的总体积的0.5mL/L。
在一种实施方式中,消泡剂为有机硅类消泡剂和/或聚醚类消泡剂;优选地,消泡剂为吐温80。
在一种实施方式中,氨基酸溶液包括L-精氨酸、L-天冬氨酸、L-谷氨酸、L-组氨酸、L-亮氨酸、L-赖氨酸、L-蛋氨酸、L-苯丙氨酸、L-丝氨酸、L-苏氨酸、L-色氨酸、L-酪氨酸、L-缬氨酸中的至少一种;优选地,氨基酸溶液包括L-组氨酸、L-亮氨酸、L-赖氨酸以及L-蛋氨酸;进一步优选地,L-组氨酸、L-亮氨酸、L-赖氨酸以及L-蛋氨酸的重量比为1:(2~4):(1~3):(0.5~1.5);更优选地,L-组氨酸、L-亮氨酸、L-赖氨酸以及L-蛋氨酸的重量比为1:3:1.5:1。
在一种实施方式中,L-组氨酸含量为100X氨基酸溶液的1~3g/L;优选地,L-组氨酸含量为氨基酸溶液的2g/L。
在一种实施方式中,初始培养基的制备原料中的微量金属溶液含量为初始培养基的溶液的总体积的5~20mL/L;优选地,初始培养基的制备原料中的微量金属溶液含量为初始培养基的溶液的总体积的10mL/L。
在一种实施方式中,初始培养基的制备原料中的维生素溶液含量为初始培养基的溶液的总体积的5~20mL/L;优选地,初始培养基的制备原料中的维生素溶液含量为初始培养基的溶液的总体积的10~15mL/L;初始培养基的制备原料中的维生素溶液含量为初始培养基的溶液的总体积的12mL/L。
在一种实施方式中,补料培养基包括微量金属溶液、维生素溶液以及碳源;优选地,补料培养基还包括磷酸盐、铵盐、磷酸盐、镁盐、锌盐、锰盐、钴盐、铁盐、钙盐、钠盐、铜盐、钾盐中的至少一种。
在一种实施方式中,补料培养基包括微量金属溶液、维生素溶液、碳源、磷酸盐、镁盐以及钾盐。
申请人意外发现,在特定的初始培养基以及补料培养基协同作用下,利用本发明提供的制备方法所得水蛭素的活性较高,可能由于有利于在菌体发酵过程的不同阶段营养成分充足,活性物产量较高,同时避免新生水蛭素的分解,最后有效表达的水蛭素产率较高。
在一种实施方式中,补料培养基中的碳源含量为初始培养基的溶液与补料培养基的溶液的总体积的300~500g/L;优选地,补料培养基中的碳源含量为初始培养基的溶液与补料培养基的溶液的总体积的350~450g/L;更优选地,补料培养基中的碳源含量为初始培养基的溶液与补料培养基的溶液的总体积的400g/L。
在一种实施方式中,补料培养基中的维生素溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的5~20mL/L;优选地,补料培养基中的维生素溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的10~15mL/L;更优选地,补料培养基中的维生素溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的12mL/L。
在一种实施方式中,补料培养基中的微量金属溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的3~15mL/L;优选地,补料培养基中的微量金属溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的8~12mL/L;更优选地,补料培养基中的微量金属溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的10mL/L。
在一种实施方式中,补料培养基中的钾盐含量为初始培养基的溶液与补料培养基的溶液的总体积的1.5~6g/L;优选地,补料培养基中的钾盐含量为初始培养基的溶液与补料培养基的溶液的总体积的3~5g/L;更优选地,补料培养基中的钾盐含量为初始培养基的溶液与补料培养基的溶液的总体积的3.5g/L。
在一种实施方式中,补料培养基中的钾盐、镁盐以及磷酸盐的重量比为1:(1~3):(2~5);优选地,补料培养基中的钾盐、镁盐以及磷酸盐的重量比为1:1.5:3.5。
在一种实施方式中,碳源选自葡萄糖、蔗糖、半乳糖、淀粉、麦芽糖、乳糖中的任一种或多种的组合。
在一种实施方式中,微量金属溶液包括锌盐、锰盐、钴盐、铁盐、钙盐、钠盐、铜盐、钾盐中的至少一种;优选地,微量金属溶液包括钠盐、锌盐、钙盐以及锰盐;进一步优选地,钠盐、锌盐、钙盐以及锰盐的重量比为1:(50~90):(20~40):(3~10);更优选地,钠盐、锌盐、钙盐以及锰盐的重量比为1:80:30:5。
在一种实施方式中,微量金属溶液中的钠盐含量为微量金属溶液的0.05~0.2g/L;优选地,微量金属溶液中的钠盐含量为微量金属溶液的0.1g/L。
在一种实施方式中,微量金属溶液还包括0.5M EDTA;进一步优选地,EDTA含量为微量金属溶液的70~90mL/L;更优选地,EDTA含量为微量金属溶液的80mL/L。
在一种实施方式中,可列举的锌盐包括但不限于:硫酸锌、氯化锌;可列举的锰盐包括但不限于:硫酸锰、二氯化锰、甘氨酸锰;可列举的钴盐包括但不限于:二氯化钴;可列举的铁盐包括但不限于:硫酸亚铁、氯化亚铁;可列举的钙盐包括但不限于:氯化钙、甘氨酸钙;可列举的钠盐包括但不限于:氯化钠、钼酸钠;可列举的铜盐包括但不限于:无水硫酸铜、甘氨酸铜;可列举的钾盐包括但不限于:硫酸钾、氯化钾、钼酸钾;可列举的铵盐包括但不限于:硝酸铵、硫酸铵、氯化铵;可列举的磷酸盐包括但不限于:磷酸二氢钾、磷酸二氢钠、磷酸二氢钙。
在一种实施方式中,维生素溶液包括生物素、烟酸、盐酸硫胺、维生素B2中的至少一种;优选地,维生素溶液包括生物素、烟酸以及盐酸硫胺;进一步优选地,生物素、烟酸以及盐酸硫胺的重量比为1:(15~30):(10~30);更优选地,生物素、烟酸以及盐酸硫胺的重量比为1:22:20。
在一种实施方式中,生物素含量为维生素溶液的0.03~0.1g/L;优选地,生物素含量为维生素溶液的0.05g/L。
在一种实施方式中,初始培养基的制备过程包括以下步骤:
(1)将糖溶液进行灭菌处理,并添加于发酵罐中;
(2)将微量金属溶液过滤除菌,并添加于发酵罐中;
(3)将维生素溶液过滤除菌,并添加于发酵罐中,即得初始培养基。
在一种优选地实施方式中,初始培养基的制备过程包括以下步骤:
(1)将糖溶液进行灭菌处理,并通过流加的方式添加于发酵罐中;
(2)将微量金属溶液通过0.22μm的无菌过滤器过滤除菌,并添加于发酵罐中;
(3)将维生素溶液通过0.22μm的无菌过滤器过滤除菌,并添加于发酵罐中,即得初始培养基。
本发明的第二方面提供一种含有所述治疗乳腺增生症的含重组水蛭素的中药组合物的制剂。
本发明的第三方面提供一种所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法,包括以下步骤:
(1)将水相组分与水混合均匀,加热;
(2)将油相组分混合均匀,加热;
(3)将油相组分加入到水相组分中,高速剪切;
(4)降温,再加入重组水蛭素发酵液与中药组分;
(5)再降温,降入助剂,搅拌均匀,即得。
优选地,所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法,包括以下步骤:
(1)将水相组分与水混合均匀,加热至70~80℃;
(2)将油相组分混合均匀,加热至70~80℃;
(3)将油相组分加入到水相组分中,高速剪切,时间为4~10min;
(4)降温至50℃后,再加入重组水蛭素发酵液与中药组分;
(5)再降温至40℃后,降入助剂,搅拌均匀,即得。
实施例1
本发明的实施例1提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括7%的水相组分1.5%的油相组分、10%重组水蛭素发酵液、1%中药组分、0.6%的助剂以及余量水;
水相组分包括小分子醇与络合剂,小分子醇与络合剂的重量比为1:0.006;
小分子醇为1,3-丁二醇与1,2-已二醇,1,3-丁二醇与1,2-已二醇的重量比为1:0.1;络合剂为EDTA二钠;
水相组分还包括精氨酸;精氨酸与络合剂的重量比为1:1.5;
油相组分为保湿剂,保湿剂为木糖醇类物质、黄原胶以及透明质酸钠,木糖醇类物质、黄原胶以及透明质酸钠的重量比为1:0.3:0.1;木糖醇类物质购自法国SEPPIC公司,AQUAXYL木糖醇;
中药组分的制备原料包括第一组分与第二组分,第一组分与第二组分的重量比为1:1;第一组分包括泽兰、漏芦、黄柏以及大黄;第二组分包括香附与蒲公英;泽兰、漏芦、黄柏以及大黄的重量比为1:1:1:1;香附与蒲公英的重量比为1:1;
中药组分的制备方法如下:
(1)将中药组分的制备原料用冷水浸泡,干燥后备用;
(2)将步骤(1)所得产物放入蒸馏水中蒸煮,过滤得滤液、滤渣,将滤液浓缩,得A组分提取液;
(3)将步骤(2)中的滤渣放入乙醇\蒸馏水的混合液中,回流提取,浓缩得B组分提取液,即得,所述乙醇与蒸馏水的体积比为1:4;
助剂为油醇聚醚,油醇聚醚包括油醇聚醚-12、油醇聚醚-15、油醇聚醚-20;油醇聚醚-12、油醇聚醚-15、油醇聚醚-20的重量比为1:1:1;
重组水蛭素发酵液的制备过程为:将种子液置于初始培养基中,通无菌空气,并保持DO值大于40%;当DO值大于55%时,于初始培养基中通过流加的方式滴加补料培养基;其中,无菌空气的通气量为5L/min,初始培养基的温度为30℃,并通过滴加氨水控制初始培养基的pH为5.0,且氨水的浓度为25wt%;
重组水蛭素的制备过程在摇瓶中进行,转速为650r/min;
种子液的接种量为11%;
种子液的制备方法为:种子液的制备方法为:①接种工程菌甘油保藏菌至装有5mL种子培养基的100mL三角瓶中,于30℃,220rpm摇床培养24小时;②转接至装有50mL种子培养基的250mL三角瓶中,于30℃,220rpm摇床培养24小时;③转接至装有250mL种子培养基的1000mL三角瓶中(共两瓶),于30℃,220rpm摇床培养12小时,即得待接种的种子液;
种子培养基包括葡萄糖、YNB、氨基酸以及水,种子培养基中的蔗糖含量为初始培养基的溶液的总体积的20g/L;葡萄糖、YNB、氨基酸的重量比为20:7:1.5;种子培养基中的水含量为480mL;氨基酸为L-精氨酸、L-组氨酸以及L-色氨酸;L-精氨酸、L-组氨酸以及L-色氨酸的重量比为1:1:1;接种工程菌为INVSC1酿酒酵母菌株;
初始培养基的制备原料包括糖溶液、微量金属溶液以及维生素溶液;
糖溶液包括无氨基酵母氮源(YNB)、碳源、氨基酸溶液、铵盐、磷酸盐、镁盐以及消泡剂,无氨基酵母氮源(YNB)含量为初始培养基的溶液的总体积的7g/L,碳源含量为初始培养基的溶液的总体积的35g/L,氨基酸溶液含量为初始培养基的溶液的总体积的20mL/L,铵盐含量为初始培养基的溶液的总体积的15g/L;
糖溶液中的铵盐、磷酸盐以及镁盐的重量比为1.3:2.5:1;
消泡剂为吐温80;消泡剂含量为初始培养基的溶液的总体积的1mL/L;
氨基酸溶液包括L-组氨酸、L-亮氨酸、L-赖氨酸以及L-蛋氨酸,L-组氨酸、L-亮氨酸、L-赖氨酸以及L-蛋氨酸的重量比为1:3:1.5:1;其中,L-组氨酸含量为氨基酸溶液的2g/L;
初始培养基的制备原料中的微量金属溶液含量为初始培养基的溶液的总体积的10mL/L,初始培养基的制备原料中的维生素溶液含量为初始培养基的溶液的总体积的12mL/L;
补料培养基包括微量金属溶液、维生素溶液、碳源、磷酸盐、镁盐以及钾盐,补料培养基中的碳源含量为初始培养基的溶液与补料培养基的溶液的总体积的400g/L;补料培养基中的维生素溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的12mL/L;补料培养基中的微量金属溶液含量为初始培养基的溶液与补料培养基的溶液的总体积的10mL/L;补料培养基中的钾盐含量为初始培养基的溶液与补料培养基的溶液的总体积的3.5g/L;补料培养基中的钾盐、镁盐以及磷酸盐的重量比为1:1.5:3.5;
碳源为半乳糖;
微量金属溶液包括钠盐、锌盐、钙盐以及锰盐,钠盐、锌盐、钙盐以及锰盐的重量比为1:80:30:5;其中,微量金属溶液中的钠盐含量为微量金属溶液的0.1g/L;
微量金属溶液还包括0.5M EDTA,EDTA含量为微量金属溶液的80mL/L;
锌盐为硫酸锌,钠盐为钼酸钠,钙盐为氯化钙,锰盐为二氯化锰;钾盐为硫酸钾,镁盐为硫酸镁,磷酸盐为磷酸二氢钾;铵盐为硫酸铵;
维生素溶液包括生物素、烟酸以及盐酸硫胺,生物素、烟酸以及盐酸硫胺的重量比为1:22:20;其中,生物素含量为维生素溶液的0.05g/L;
初始培养基的制备过程包括以下步骤:
(1)将糖溶液进行灭菌处理,并通过流加的方式添加于发酵罐中;
(2)将微量金属溶液通过0.22μm的无菌过滤器过滤除菌,并添加于发酵罐中;
(3)将维生素溶液通过0.22μm的无菌过滤器过滤除菌,并添加于发酵罐中,即得初始培养基;
所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法,包括以下步骤:
(1)将水相组分与水混合均匀,加热至75℃;
(2)将油相组分混合均匀,加热至75℃;
(3)将油相组分加入到水相组分中,高速剪切,时间为7min;
(4)降温至50℃后,再加入重组水蛭素发酵液与中药组分;
(5)再降温至40℃后,降入助剂,搅拌均匀,即得。
实施例2
本发明的实施例2提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括10%的水相组分、3%的油相组分、15%重组水蛭素发酵液、2%中药组分、1%的助剂以及余量水;
水相组分包括小分子醇与络合剂,小分子醇与络合剂的重量比为1:0.01;
小分子醇为1,3-丁二醇与1,2-已二醇,1,3-丁二醇与1,2-已二醇的重量比为1:0.2;络合剂为EDTA二钠;
水相组分还包括精氨酸;精氨酸与络合剂的重量比为1:1.6;
油相组分为保湿剂,保湿剂为木糖醇类物质、黄原胶以及透明质酸钠,木糖醇类物质、黄原胶以及透明质酸钠的重量比为1:0.5:0.15;木糖醇类物质购自法国SEPPIC公司,AQUAXYL木糖醇;
中药组分包括第一组分与第二组分,第一组分与第二组分的重量比为1:1;第一组分包括泽兰、漏芦、黄柏以及大黄;第二组分包括香附与蒲公英;泽兰、漏芦、黄柏以及大黄的重量比为1:1:1:1;香附与蒲公英的重量比为1:1;中药组分的制备方法同实施例1;
助剂为油醇聚醚,油醇聚醚包括油醇聚醚-12、油醇聚醚-15、油醇聚醚-20;油醇聚醚-12、油醇聚醚-15、油醇聚醚-20的重量比为1:1.2:1.2;
重组水蛭素发酵液的制备方法同实施例1;
所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法同实施例1。
实施例3
本发明的实施例3提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,按照组合物的重量百分比计,包括3%的水相组分、0.5%的油相组分、5%重组水蛭素发酵液、0.5%中药组分、0.2%的助剂以及余量水;
水相组分包括小分子醇与络合剂,小分子醇与络合剂的重量比为1:0.003;
小分子醇为1,3-丁二醇与1,2-已二醇,1,3-丁二醇与1,2-已二醇的重量比为1:0.05;络合剂为EDTA二钠;
水相组分还包括精氨酸;精氨酸与络合剂的重量比为1:1.2;
油相组分为保湿剂,保湿剂为木糖醇类物质、黄原胶以及透明质酸钠,木糖醇类物质、黄原胶以及透明质酸钠的重量比为1:0.2:0.05;木糖醇类物质购自法国SEPPIC公司,AQUAXYL木糖醇;
中药组分包括第一组分与第二组分,第一组分与第二组分的重量比为1:1;第一组分包括泽兰、漏芦、黄柏以及大黄;第二组分包括香附与蒲公英;泽兰、漏芦、黄柏以及大黄的重量比为1:1:1:1;香附与蒲公英的重量比为1:1;中药组分的制备方法同实施例1;
助剂为油醇聚醚,油醇聚醚包括油醇聚醚-12、油醇聚醚-15、油醇聚醚-20;油醇聚醚-12、油醇聚醚-15、油醇聚醚-20的重量比为1:0.8:0.8;
重组水蛭素发酵液的制备方法同实施例1;
所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法同实施例1。
实施例4
本发明的实施例1提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,中药组分包括第一组分与第二组分,第一组分与第二组分的重量比为1:1;第一组分包括柴胡、川穹、白芍以及川楝子;第二组分包括香附与延胡素;柴胡、川穹、白芍以及川楝子的重量比为1:1:1:1;香附与延胡素的重量比为1:1。
实施例5
本发明的实施例5提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,重组水蛭素发酵液的含量为0。
实施例6
本发明的实施例6提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,中药组分的含量为0。
实施例7
本发明的实施例7提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,中药组分与重组水蛭素发酵液的含量均为0。
实施例8
本发明的实施例8提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,油醇聚醚-12含量为0。
实施例9
本发明的实施例9提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,油醇聚醚-20含量为0。
实施例10
本发明的实施例10提供一种治疗乳腺增生症的含重组水蛭素的中药组合物,其具体实施方式同实施例1,不同之处在于,油醇聚醚-12与油醇聚醚-20含量均为0。
性能评估
1.理化性质评估:
A-目测观察实施例1~实施例4所得组合物均为淡黄色均匀乳液;
B-测定25℃下实施例1所得组合物的pH为6.52;
C-对实施例1~实施例4以及实施例8~实施例10的进行离心实验,即室温下3000r/min,离心30min,观察是否有分层现象,实验结果见表1;
2.观察实施例1~实施例7所得组合物对乳腺增生的患者的疗效观察,具体操作如下:
在两家美容院,挑选有乳腺增生的女性志愿者,年龄在25-40岁之间,每个实施例对应120人,分成两组,每组60人,使用治疗乳腺增生的产品,涂于胸部按摩,每天一次,七天一个疗程,做两个疗程。
方法:取实施例做的组合物,敷于患者全乳或乳房肿块上,根据乳房或肿块范围扩大2cm,厚度0.5~0.8cm,覆盖保鲜膜40~50min,然后进行乳房按摩。按摩方法:护理人员先把手掌搓热,将按摩油均匀涂于双侧乳房上,左手托住乳房,然后以右手的大鱼际、小鱼际自乳房基底部开始旋转至整个乳房螺旋式按摩,每次按摩5~10min,按摩完一侧按摩另一侧。同时用拇指、食指及中指自乳晕向乳头牵拉乳房及周围皮肤数次,使乳头向外突出,之后将两指放于乳头上下,对乳头进行上、下牵引。每天按摩一次,7天一个疗程,看志愿者的实际恢复情况,一般做两个疗程,测试结果见表2;
判断标准:由受测者主观判断乳房肿胀感或者胀痛感是否有所缓解或消失,是则判断有效,否则无效;也有部分患者在疗程前后去医院做了身体检查,已确定治疗前后的情况,结果见图1与图2。
表1
离心后的分层现象 | |
实施例1 | 无分层 |
实施例2 | 无分层 |
实施例3 | 无分层 |
实施例4 | 无分层 |
实施例8 | 分层 |
实施例9 | 分层 |
实施例10 | 分层 |
表2
临床实测效果:
史某,女,现年43岁,2019年9月19日去医院做彩色B超检查;
诊断结果:双侧乳腺增厚,回声不均,于中间回声区可见不规则条片状低回声,散在分布,左乳内见多个低回声结节,边界清,其中2点钟方向大小约0.8*0.3cm;右乳12点钟CDFI双乳低回声结节内未见明显血流信号。结果显示:双侧乳腺低回声结节,左侧多发,右侧单发,确诊为双侧乳腺增生,见图1。
该志愿者使用实施例1所得组合物一个疗程以后,自我感觉症状大为缓解,随后在2019年9月27日去医院进行彩色B超复查。
诊断结果:双侧乳腺增厚,回声不均,于中间回声区可见不规则条片状低回声,散在分布,左乳内2点钟方向大小约0.5*0.3cm,边界清,右乳未见局限性肿块回声,CDFI左乳低回声结节内未见明显血流信号,囊性结节内未见血流信号,见图2。临床B超的测试效果也证明了该按摩霜的确对乳腺增生有改善和治疗的效果。
前述的实例仅是说明性的,用于解释本发明所述方法的一些特征。所附的权利要求旨在要求可以设想的尽可能广的范围,且本文所呈现的实施例仅是根据所有可能的实施例的组合的选择的实施方式的说明。因此,申请人的用意是所附的权利要求不被说明本发明的特征的示例的选择限制。在权利要求中所用的一些数值范围也包括了在其之内的子范围,这些范围中的变化也应在可能的情况下解释为被所附的权利要求覆盖。
Claims (10)
1.一种治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,按照组合物的重量百分比计,包括3~10%的水相组分、0.5~3%的油相组分、5~15%重组水蛭素发酵液、0.5~2%中药组分、0.2~1%的助剂以及余量水。
2.根据权利要求1所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,中药组分的制备原料包括第一组分与第二组分;其中,第一组分选自泽兰、漏芦、黄柏、大黄、冰片、柴胡、川穹、白芍、川楝子、莪术、三棱中的任一种或多种的组合;第二组分选自香附、蒲公英、甘草、当归、延胡素、瓜蒌、乳香、没药中的任一种或多种的组合。
3.根据权利要求1所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,水相组分包括小分子醇与络合剂。
4.根据权利要求1所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,油相组分为保湿剂,保湿剂选自木糖醇类物质、羟基乙酸、黄原胶、甘油辛酸酯、聚乙二醇、海藻糖、赤藓醇、吡咯烷酮羧酸钠、乙氧基葡萄糖衍生物、透明质酸钠中的任一种或多种的组合。
5.根据权利要求1~4任一项所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,重组水蛭素发酵液的制备过程为:将种子液置于初始培养基中,当DO值大于55%时,滴加补料培养基;其中,初始培养基的制备原料包括糖溶液、微量金属溶液以及维生素溶液。
6.根据权利要求5所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,糖溶液包括无氨基酵母氮源、碳源以及氨基酸溶液。
7.根据权利要求5所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,微量金属溶液包括锌盐、锰盐、钴盐、铁盐、钙盐、钠盐、铜盐中的至少一种。
8.根据权利要求5所述治疗乳腺增生症的含重组水蛭素的中药组合物,其特征在于,补料培养基包括微量金属溶液、维生素溶液以及碳源。
9.一种含有如权利要求1~8任一项所述治疗乳腺增生症的含重组水蛭素的中药组合物的制剂。
10.一种根据权利要求1~8任一项所述治疗乳腺增生症的含重组水蛭素的中药组合物的制备方法,其特征在于,包括以下步骤:
(1)将水相组分与水混合均匀,加热;
(2)将油相组分混合均匀,加热;
(3)将油相组分加入到水相组分中,高速剪切;
(4)降温,再加入重组水蛭素发酵液与中药组分;
(5)再降温,降入助剂,搅拌均匀,即得。
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