CN108578583A - 一种中华被毛孢菌组合物及其制备方法与应用 - Google Patents
一种中华被毛孢菌组合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种中华被毛孢菌组合物及其制备方法与应用,属于生物医药技术领域。该组合物由中华被毛孢菌、铁皮石斛提取物、西洋参提取物组成。铁皮石斛加水煎煮,过滤,滤液浓缩后干燥即得铁皮石斛提取物;西洋参乙醇回流提取液浓缩后干燥得西洋参醇提取物,药渣加水煎煮,过滤,滤液浓缩后干燥得西洋参水提取物,将醇提取物和水提取物混合均匀,即得西洋参提取物;中华被毛孢菌发酵培养后菌液超声波破壁,浓缩后干燥即得破壁中华被毛孢菌。将破壁中华被毛孢菌、铁皮石斛提取物、西洋参提取物按相应的重量份数混合均匀,即得中华被毛孢菌组合物。中华被毛孢菌组合物在制备增强免疫力功能的特殊食品或药物中的应用。
Description
技术领域
本发明涉及生物医药技术领域,具体而言,涉及一种中华被毛孢菌组合物及其制备方法与应用。
背景技术
据世界卫生组织的一项调查结果表明,全世界真正健康的人仅占5%,经医生检查、诊断有病的人也只占20%,75%的人处于亚健康状态。可见,亚健康问题已经严重困扰着人们的工作和生活。据专家分析,85%以上的疾病和亚健康状态都与人体免疫力下降有关,同时认为有目的、适当的服用具有调节免疫机能的保健食品,对消除或避免亚健康状态是十分有益的。
铁皮石斛为兰科珍稀植物——石斛类中的极品,因表皮呈铁绿色而得名,具有独特的保健与药用价值,民间有“救命仙草”之称,被道家养生经典著作《道臧》誉为“中华九大仙草之首”,是医药界的“大熊猫”。作为养生极品,铁皮石斛自古以来为民间所推崇并深受皇宫贵族的青睐。铁皮石斛性味:甘,微寒,入胃、肾经。性属清润,清中有补,补中有清。铁皮石斛的功效作用,历代名医各有论述:《神农本草经》:主伤中、除痹、下气、补五脏虚劳羸瘦、强阴、久服厚肠胃。《本草纲目》:强阴益精。久服,厚肠胃,补内绝不足,平胃气,长肌肉,逐皮肤邪热痱气,脚膝疼冷痹弱,定智除惊,轻身延年。《纲目拾遗》:清胃除虚热,生津巳劳损,以之代茶,开胃健脾。定惊疗风,能镇涎痰,甘芳降气。《药品化义》:治肺气久虚,咳嗽不止。《别录》:益精补内绝不足,平胃气,长肌肉,逐皮肤邪热气,脚膝疼软弱,健阳,补肾积精。《日华子本草》:治虚损劣弱壮筋骨,暖水脏,益智,平胃气,逐虚邪。《本草再新》:理胃气,清胃火。除心中烦渴,疗肾经虚热,安神定惊,解盗汗,能散暑。
西洋参中的主要成分为人参皂苷,因此西洋参具有滋阴补气,宁神益智及清热生津,降火消暑的双重功效,西洋参还能够提高机体免疫功能,而且具有抗肿瘤的生物活性,可通过提高机体的免疫功能而抑制肿瘤的生长。
但是目前利用铁皮石斛、西洋参制备的保健品普遍存在,但是存在着铁皮石斛、西洋参制备有效成分难以被人体的吸收利用、且保健品中有效成分含量难以保证、效果不显著等问题。
针对上述问题,若能充分利用铁皮石斛、西洋参的药用功效,开发出一种以铁皮石斛和西洋参为主料,服用方便、使用效果更为显著的保健产品,无疑存在巨大的市场价值。
鉴于此,本发明提供一种中华被毛孢菌组合物及其制备方法与应用。
发明内容
本发明的目的在于提供一种中华被毛孢菌组合物及其制备方法与应用。
为了实现本发明的上述目的,特采用以下技术方案:
第一方面,本发明提供一种中华被毛孢菌组合物,中华被毛孢菌组合物包括中华被毛孢菌、铁皮石斛、西洋参。
进一步地,中华被毛孢菌为破壁中华被毛孢菌,发酵虫草菌粉生产用菌种为蝙蝠蛾拟青霉。
进一步地,铁皮石斛、西洋参分别为铁皮石斛、西洋参的提取物。
进一步地,按重量份数计,破壁中华被毛孢菌为1~3份、铁皮石斛提取物为2~5份、西洋参提取物为2~5份。
更进一步地,在本发明较佳的实施例中,按重量份数计,破壁中华被毛孢菌为2份、铁皮石斛提取物为3份、西洋参提取物为3份。
破壁中华被毛孢菌、铁皮石斛提取物、西洋参提取物,可以从市场上购买,也可以自行制备。
铁皮石斛提取物可以通过以下步骤获得:取铁皮石斛,切成长为2~3cm的小段,加水煎煮1~2次,第一次加8~10倍量水,煎煮25~35min,第二次加6~8倍量水,煎煮20~30min,过滤,合并滤液,真空浓缩到比重1.05~1.07,喷雾干燥即得。
西洋参提取物可以通过以下步骤获得:西洋参药材饮片后加8~10倍量40~60%乙醇浸泡1~3h,加热回流3~5h,收集提取液;药渣加入6~8倍量40~60%乙醇继续加热回流提取1~3h,收集提取液,合并提取液,回收乙醇,真空浓缩至比重1.02~1.04,16000r/min离心,直至药液澄清,真空浓缩到比重1.05~1.07,喷雾干燥得西洋参醇提取物;醇提后的西洋参50~70℃烘干后,加水煎煮1~2次,第一次加8~10倍量水,煎煮25~35min,第二次加6~8倍量水,煎煮20~30min,过滤,合并滤液,真空浓缩到比重1.05~1.07,喷雾干燥得西洋参水提取物,将西洋参醇提取物和西洋参水提取物混合均匀,即得西洋参提取物。
破壁中华被毛孢菌可以通过以下步骤获得:发酵虫草菌粉生产用菌种经分离培养,接入固体试管斜面培养基上培养,培养温度为17~19℃,长满斜面后放入1~4℃冰箱待用;按常规工艺配制蔗糖、葡萄糖、豆粕粉、酵母膏、牛肉膏、蛋白胨粉培养基,经121℃灭菌20~30min,冷却后接入试管菌种,摇瓶培养,摇瓶培养后经显微镜等检测确定菌丝生长旺盛后移入不锈钢种子罐培养,生长旺盛后再移入不锈钢发酵罐培养,发酵完成后菌液,超声波破壁,真空浓缩到比重1.05~1.07,喷雾干燥即得。
第二方面,本发明提供上述中华被毛孢菌组合物的制备方法,将破壁中华被毛孢菌、铁皮石斛提取物、西洋参提取物按相应的重量份数混合均匀,即得中华被毛孢菌组合物。
第三方面,本发明还提供一种含有中华被毛孢菌、铁皮石斛、西洋参的组合物的应用,主要在制备增强免疫力功能的特殊食品或药物中的应用。
与现有技术相比,本发明的有益效果包括:
本发明中华被毛孢菌组合物在增强免疫力功能方面,与相同重量的中华被毛孢菌、铁皮石斛提取物或西洋参提取物单一组分即现有技术相比,均有显著提高。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步详细描述:
实施例1
将破壁中华被毛孢菌为100g、铁皮石斛提取物为500g、西洋参提取物为200g混合均匀,即得中华被毛孢菌组合物。
铁皮石斛提取物通过以下步骤获得:取铁皮石斛干条5kg,切成长为2cm的小段,加水煎煮2次,第一次加8倍量水,浸泡2h后煎煮25min,第二次加6倍量水,煎煮30min,过滤,合并滤液,真空浓缩到比重1.05,喷雾干燥得759g铁皮石斛提取物。
西洋参提取物通过以下步骤获得:西洋参药材饮片2kg,加8倍量60%乙醇浸泡1h,加热回流5h,收集提取液;药渣加入6倍量60%乙醇继续加热回流提取1h,收集提取液,合并提取液,回收乙醇,真空浓缩至比重1.04,16000r/min离心,直至药液澄清,真空浓缩到比重1.05,喷雾干燥得115g西洋参醇提取物;醇提后的西洋参50℃烘干后,加水煎煮2次,第一次加8倍量水,煎煮25min,第二次加8倍量水,煎煮30min,过滤,合并滤液,真空浓缩到比重1.05,喷雾干燥得251g西洋参水提取物,将西洋参醇提取物和西洋参水提取物混合均匀,即得366g西洋参提取物。
破壁中华被毛孢菌通过以下步骤获得:发酵虫草菌粉生产用菌种经分离培养,接入固体试管斜面培养基上培养,培养温度为17℃,长满斜面后放入4℃冰箱待用;按常规工艺取蔗糖、葡萄糖、豆粕粉、酵母膏、牛肉膏、蛋白胨粉共0.5kg配制培养基,经121℃灭菌20min,冷却后接入试管菌种,摇瓶培养,摇瓶培养后经显微镜等检测确定菌丝生长旺盛后移入不锈钢种子罐培养,生长旺盛后再移入不锈钢发酵罐培养,发酵完成后菌液16000r/min离心,沉淀用PBS洗涤2遍,然后按原菌液体积的1/5加入裂解液。裂解液成分:50mMTris-HCl,pH8.0,0.2mM EDTA,100mM NaCl,加溶菌酶100ug/ml,0.1%Triton X-100。超声波功率300w,破壁20分钟,16000r/min离心,沉淀用2000ml水洗涤2遍,分别16000r/min离心,然后按原菌液体积的1/5加入水,真空浓缩到比重1.07,喷雾干燥得131g破壁中华被毛孢菌。
实施例2
将破壁中华被毛孢菌为300g、铁皮石斛提取物为200g、西洋参提取物为500g混合均匀,即得中华被毛孢菌组合物。
铁皮石斛提取物通过以下步骤获得:取铁皮石斛干条2kg,切成长为3cm的小段,加10倍量水浸泡2h后煎煮1次,煎煮35min,过滤,滤液,真空浓缩到比重1.05,喷雾干燥得261g铁皮石斛提取物。
西洋参提取物通过以下步骤获得:西洋参药材饮片3kg,加10倍量40%乙醇浸泡3h,加热回流3h,收集提取液;药渣加入8倍量40%乙醇继续加热回流提取3h,收集提取液,合并提取液,回收乙醇,真空浓缩至比重1.02,16000r/min离心,直至药液澄清,真空浓缩到比重1.07,喷雾干燥得181g西洋参醇提取物;醇提后的西洋参70℃烘干后,加10倍量水煎煮1次,煎煮35min,过滤,滤液真空浓缩到比重1.07,喷雾干燥得358g西洋参水提取物,将西洋参醇提取物和西洋参水提取物混合均匀,即得539g西洋参提取物。
破壁中华被毛孢菌通过以下步骤获得:发酵虫草菌粉生产用菌种经分离培养,接入固体试管斜面培养基上培养,培养温度为19℃,长满斜面后放入1℃冰箱待用;按常规工艺取蔗糖、葡萄糖、豆粕粉、酵母膏、牛肉膏、蛋白胨粉共1kg配制培养基,经121℃灭菌30min,冷却后接入试管菌种,摇瓶培养,摇瓶培养后经显微镜等检测确定菌丝生长旺盛后移入不锈钢种子罐培养,生长旺盛后再移入不锈钢发酵罐培养,发酵完成后菌液用超声细胞破碎仪破壁,条件是功率800w,超声5s,间隔5s,超声30次。菌液真空浓缩到比重1.05,喷雾干燥得302g破壁中华被毛孢菌。
实施例3
将破壁中华被毛孢菌为200g、铁皮石斛提取物为300g、西洋参提取物为300g混合均匀,即得中华被毛孢菌组合物。
铁皮石斛提取物通过以下步骤获得:取铁皮石斛干条2kg,切成长为2.5cm的小段,加水煎煮2次,第一次加9倍量水,浸泡2h后煎煮30min,第二次加7倍量水,煎煮25min,过滤,合并滤液,真空浓缩到比重1.06,喷雾干燥得326g铁皮石斛提取物。
西洋参提取物通过以下步骤获得:西洋参药材饮片2kg,加9倍量50%乙醇浸泡2h,加热回流4h,收集提取液;药渣加入7倍量50%乙醇继续加热回流提取2h,收集提取液,合并提取液,回收乙醇,真空浓缩至比重1.03,16000r/min离心,直至药液澄清,真空浓缩到比重1.06,喷雾干燥得119g西洋参醇提取物;醇提后的西洋参60℃烘干后,加水煎煮2次,第一次加9倍量水,煎煮30min,第二次加7倍量水,煎煮25min,过滤,合并滤液,真空浓缩到比重1.06,喷雾干燥得252g西洋参水提取物,将西洋参醇提取物和西洋参水提取物混合均匀,即得371g西洋参提取物。
破壁中华被毛孢菌可以通过以下步骤获得:发酵虫草菌粉生产用菌种经分离培养,接入固体试管斜面培养基上培养,培养温度为18℃,长满斜面后放入2℃冰箱待用;按常规工艺取蔗糖、葡萄糖、豆粕粉、酵母膏、牛肉膏、蛋白胨粉共1kg配制培养基,经121℃灭菌25min,冷却后接入试管菌种,摇瓶培养,摇瓶培养后经显微镜等检测确定菌丝生长旺盛后移入不锈钢种子罐培养,生长旺盛后再移入不锈钢发酵罐培养,发酵完成后菌液,4000r/min离心10min,除去上清液,得菌体细胞悬浮液于冰浴中,利用40kHz的超声波,时间50min,使细胞破碎,按原菌液体积的1/5加入水,真空浓缩到比重1.06,喷雾干燥得319g破壁中华被毛孢菌。
对比例1~3组样品制备:
参照实施例3的工艺流程分别制得铁皮石斛提取物、西洋参提取物和破壁中华被毛孢菌样品,作为对比例1~3组样品。
实验例1
1、实验动物:SPF级昆明种雌性小鼠,体重18g~22g。
2、实验分组:ConA诱导的小鼠淋巴细胞转化实验、抗体生成细胞检测、NK细胞活性的测定均分别取70只小鼠,分为7组,每组10只。每日摄入2次,剂量0.15g/kg/日,共30天。实施例1~3组对应摄入实施例1~3制备得到的中华被毛孢菌组合物;对比例1~3组对应摄入对比例1~3组铁皮石斛提取物、西洋参提取物和破壁中华被毛孢菌样品;对照组摄入与中华被毛孢菌组合物等量的糊精。
3、ConA诱导的小鼠淋巴细胞转化实验(MTT法)
无菌取脾,置于盛有适量无菌Hank液的小平皿中,制成细胞悬液,经200目筛网过滤。用Hank液洗2次,每次离心10分钟(1000r/min)。然后将细胞悬浮于1mL完全培养液中,计数活细胞数,用RPMI1640培养液调整细胞浓度为3×106个/mL。再将细胞悬液分两孔加入24孔培养板中,每孔1mL,在其中一孔加ConA液75μL(相当于7.5μg/mL),另一孔作为对照,置5%二氧化碳,37℃培养72h。培养结束前4h,每孔轻轻吸去上清液0.7mL,加入0.7mL不含小牛血清的RPMI1640培养液,同时加入MTT(5mg/mL)50μL/孔,继续培养4h。培养结束后,每孔加入1mL酸性异丙醇,吹打混匀,使紫色结晶完全溶解。然后分装到96孔培养板中,每个孔作3个平行孔,用酶标仪,测570nm波长处的光密度值。淋巴细胞的增殖能力用加ConA孔的光密度值减去不加ConA孔的光密度值表示。样品对小鼠淋巴细胞转化能力的影响如表1所示。
表1
由表1可见,实施例1~3组和对比例1~3组均能增加小鼠淋巴细胞转化能力,与对照组比较差异有显著性,实施例1~3组优势更明显。
4、抗体生成细胞检测(Jerne改良玻片法)
取羊血用生理盐水洗涤3次,每次离心(2000r/min)10min,将压积绵羊红细胞SRBC用生理盐水配成2%(v/v)的细胞悬液,每鼠腹腔注射0.2mL,免疫后5天,将小鼠处死,取脾,轻轻磨碎,用Hank液制成细胞悬液,200目筛网过滤,洗涤、离心2次,最后将细胞悬浮在8mLHank液中。计数细胞,并将细胞浓度调整为5×106个/mL。将表层培养基加热溶解后与等量pH值7.4、2倍浓度的Hank液混合,分装小试管,每管0.5mL,再向管内加入用SA液配制的10%SRBC 50μL(v/v)、20μL脾细胞悬液(5×106个/mL),迅速混匀后倾倒于已刷薄层琼脂糖的玻片上,待琼脂糖凝固后将玻片平扣放在玻片架上,放入二氧化碳培养箱中温育1.5h,将SA液稀释的补体(1:8)加入到玻片凹槽内继续温育1.5h后,计数溶血空斑数。样品对小鼠抗体生成细胞数的影响如表2所示。
表2
由表2可知,实施例1~3组和对比例1~3组均能增加小鼠抗体生成细胞数,均具有增强小鼠体液免疫的功能,与对照组比较差异有显著性,实施例1~3组优势更明显。
5、NK细胞活性的测定(乳酸脱氢酶测定法)
受试小鼠颈椎脱臼处死,无菌取脾,制成脾细胞悬液,用Hank液洗2次,每次离心10min(1000r/min),弃上清将细胞浆弹起,加入0.5mL灭菌水20秒裂解红细胞后,再加入0.5mL 2倍Hank液及8mL Hank液,1000rpm转速下,离心10min,用1mL含10%小牛血清的RPMI1640完全培养液重悬,用台酚兰染色计数(活细胞数应在95%以上),调整细胞浓度为2×107个/mL做为效应细胞,取传代后24h生长良好的YAC—1细胞,用RPMI1640完全培养液调整细胞浓度为4×105个/mL做为靶细胞;取靶细胞和效应细胞各100μL(效、靶比50:1),加入Μ型96孔培养板中;靶细胞自然释放孔加靶细胞和培养液各100μL,靶细胞最大释放孔加靶细胞和2.5%Triton各100μL;上述各项均设三个平行孔,于37℃,5%二氧化碳培养箱中培养4h,然后将96孔培养板以1500r/min的转速离心5min,每孔吸取上清液100μL置平底96孔培养板中,同时加入LDH基质液100μL/孔,根据室温反应3~10min,每孔加入30μL 1mol/L的HCl,在酶标仪490nm波长处测定光密度(OD)。
NK细胞活性=[(反应孔OD值-自然释放孔OD值)/(最大释放孔OD值-自然释放孔OD值)]×100%。
样品对小鼠NK细胞活性的影响如表3所示。
表3
由表3可见,实施例1~3组和对比例1~3组均能增加小鼠NK细胞活性有明显提高作用,与对照组比较差异有显著性,实施例1~3组优势更明显。
总之,就相同数量而言,本发明组合物较破壁中华被毛孢菌、铁皮石斛提取物、西洋参提取物,在增强免疫力等方面均有显著提高。
本发明中未详细说明的环节均为本领域普通技术人员可自行选择的公知常识。虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域普通技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种中华被毛孢菌组合物,其特征在于,所述中华被毛孢菌组合物包括中华被毛孢菌、铁皮石斛、西洋参。
2.根据权利要求1所述的中华被毛孢菌组合物,其特征在于,所述中华被毛孢菌为破壁中华被毛孢菌。
3.根据权利要求1所述的中华被毛孢菌组合物,其特征在于,所述铁皮石斛、西洋参分别为铁皮石斛、西洋参的提取物。
4.根据权利要求3所述的中华被毛孢菌组合物,其特征在于,按重量份数计,所述中华被毛孢菌为1~3份、所述铁皮石斛提取物为2~5份、所述西洋参提取物为2~5份。
5.根据权利要求4所述的中华被毛孢菌组合物,其特征在于,按重量份数计,所述中华被毛孢菌为2份、所述铁皮石斛提取物为3份、所述西洋参提取物为3份。
6.根据权利要求5所述的中华被毛孢菌组合物,其特征在于,所述铁皮石斛提取物通过以下步骤获得:取铁皮石斛,切成长为2~3cm的小段,加水煎煮1~2次,第一次加8~10倍量水,煎煮25~35min,第二次加6~8倍量水,煎煮20~30min,过滤,合并滤液,真空浓缩到比重1.05~1.07,喷雾干燥即得。
7.根据权利要求5所述的中华被毛孢菌组合物,其特征在于,所述西洋参提取物通过以下步骤获得:西洋参药材饮片后加8~10倍量40~60%乙醇浸泡1~3h,加热回流3~5h,收集提取液;药渣加入6~8倍量40~60%乙醇继续加热回流提取1~3h,收集提取液,合并提取液,回收乙醇,真空浓缩至比重1.02~1.04,16000r/min离心,直至药液澄清,真空浓缩到比重1.05~1.07,喷雾干燥得西洋参醇提取物;醇提后的西洋参50~70℃烘干后,加水煎煮1~2次,第一次加8~10倍量水,煎煮25~35min,第二次加6~8倍量水,煎煮20~30min,过滤,合并滤液,真空浓缩到比重1.05~1.07,喷雾干燥得西洋参水提取物,将西洋参醇提取物和西洋参水提取物混合均匀,即得西洋参提取物。
8.一种根据权利要求5所述的中华被毛孢菌组合物,其特征在于,所述破壁中华被毛孢菌通过以下步骤获得:发酵虫草菌粉生产用菌种经分离培养,接入固体试管斜面培养基上培养,培养温度为17~19℃,长满斜面后放入1~4℃冰箱待用;按常规工艺配制蔗糖、葡萄糖、豆粕粉、酵母膏、牛肉膏、蛋白胨粉培养基,经121℃灭菌20~30min,冷却后接入试管菌种,摇瓶培养,摇瓶培养后经显微镜等检测确定菌丝生长旺盛后移入不锈钢种子罐培养,生长旺盛后再移入不锈钢发酵罐培养,发酵完成后菌液,超声波破壁,真空浓缩到比重1.05~1.07,喷雾干燥即得。
9.一种根据权利要求1~8任一项所述的中华被毛孢菌组合物的制备方法,其特征在于,将所述中华被毛孢菌、所述铁皮石斛提取物、所述西洋参提取物按相应的重量份数混合均匀,即得中华被毛孢菌组合物。
10.一种如权利要求1~8所述的中华被毛孢菌组合物在制备增强免疫力功能的特殊食品或药物中的应用。
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