CN114107072A - 一种灵芝菌株及其用途 - Google Patents
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Abstract
本发明提供了一种灵芝菌株及其用途，涉及中药领域。该灵芝菌株是保藏于中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.23844的灵芝。该灵芝菌株具有遗传性状稳定、产量高、品质优的特点。同时，该灵芝菌株具有显著的抗炎作用，可用于制备抗炎的药物或化妆品，本发明灵芝菌株还可以用于制备食品或保健食品，具有良好的应用前景。
Description
技术领域
本发明涉及中药领域，具体涉及一种灵芝菌株及其用途。
背景技术
灵芝(Ganoderma sp.)隶属于担子菌纲(Basidiomycetes)多孔菌目(Polyporales)多孔菌科(Polyporaceae)灵芝属(Ganoderma)，是一种重要的药用真菌，在传统中医中被广泛应用，其药理作用多有论述。《神农本草经》中记载灵芝具有扶正固本、滋补强壮、延年益寿等功效，现代药理学与临床试验进一步证明灵芝具有抗衰老、抗病毒、抗氧化、抗肿瘤、增强免疫力、保肝等作用。其中赤芝是仅有的两个被《中华人民共和国药典》收录的灵芝属之一。
《中华人民共和国药典》(2020年版一部)规定灵芝水分不得过17.0％，总灰分不得过3.2％，浸出物不得少于3.0％，灵芝多糖不得少于0.9％，含三萜及甾醇以齐墩果酸计不得少于0.5％。然而，而灵芝的产地、品种使得我国灵芝品质良莠不齐，功效差异很大。
寻找到品质优良的灵芝菌株，对于灵芝产业的发展具有重要作用。
发明内容
本发明的目的在于提供一种灵芝菌株及其用途。
本发明提供了一种灵芝菌株，它是保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)的保藏编号为CGMCC NO.23844的灵芝。保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2021年11月8日，分类命名为灵芝(Ganoderma lucidum)。
本发明还提供了一种灵芝水提物，它是由前述的灵芝菌株经过水提取后得到的水提物。
本发明还提供了前述的灵芝水提物的制备方法，它包括如下步骤：灵芝粉碎后，加水煎煮提取，取滤液，即得。
进一步地，所述煎煮提取方法为加水浸泡0.5～1h后，加入8～10倍体积的水，煮沸1～3h，提取次数为1～3次。
进一步地，所述滤液浓缩至1～10g/mL。
本发明还提供了前述的灵芝菌株或前述的灵芝水提物在制备抗炎药物和/或具有抗炎功效的化妆品中的用途。
进一步地，所述抗炎药物为抑制NO、TNF-α生成的药物。
本发明还提供了前述的灵芝菌株或前述的灵芝水提物在制备保健食品和/或食品中的用途。
本发明还提供了一种保健食品、化妆品或药物制剂，它是以前述的灵芝菌株或前述的灵芝水提物为活性成分，加上保健食品、化妆品或药学上可接受的辅料或辅助性成分制备而成的保健食品、化妆品和药物制剂。
本发明还提供了一种食品，它是前述的灵芝菌株或前述的灵芝水提物加上食品上可接受的辅料或辅助性成分制备而成的食品。
本发明提供了一种新的灵芝菌株，该灵芝菌株具有遗传性状稳定、产量高、品质优的特点。同时，该灵芝菌株具有显著的抗炎作用，可用于制备抗炎的药物或化妆品，本发明灵芝菌株还可以用于制备食品或保健食品，具有良好的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
具体实施方式
除另有说明外，本发明所用原料与设备均为已知产品，通过购买市售产品所得。
数据统计与分析：
数据采用
表示。采用SPSS 26.0软件进行统计分析，采用单因素方差分析比较组间差异，两组数据差异比较采用LSD法。
实施例1、本发明的灵芝菌株
本发明灵芝菌株来源于四川峨眉山的一株野生灵芝ZL2，经系统选育而得。基原为多孔菌科真菌赤芝(Ganoderma lucidum(Leyss.ex Fr.)Karst.)，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2021年11月8日，保藏编号为CGMCC NO.23844，分类命名为灵芝(Ganodermalucidum)。
该灵芝菌株的主要特征特性：
1、外观
具典型的赤芝[Ganoderma lucidum(Leyss.ex Fr.)Karst.]形态特征。子实体外形呈伞状，菌盖肾形，直径10-16cm，厚1-2cm，皮壳坚硬，浅黄褐色，有光泽，具环状棱纹和辐射状皱纹，边缘薄而平截，常稍内卷，腹面黄白色。
2、生产周期：约100天。
3、产量：按常规袋料栽培技术，产量≥63.32g/kg.袋料。
4、品质：经检测，浸出物6.42％，多糖1.33％，三萜及甾醇0.69％，各项检验应符合《中华人民共和国药典》(2020年版，一部)标准。
实施例2、本发明灵芝水提物的制备
实施例1的灵芝预先打粉，加水浸泡0.5h，倒出浸泡的水后，再加入10倍体积的水，煮沸1h，过滤得提取液，滤渣重复提取2次，收集3次提取液，将提取液过滤，浓缩至1g/ml，得灵芝水提物。
以下通过具体试验例证明本发明的有益效果。
实验例1、灵芝菌株特性和品质的研究
1、生产周期：约100天。
2、外观形态：具典型的赤芝[Ganoderma lucidum(Leyss.ex Fr.)Karst.]形态特征。子实体外形呈伞状，菌盖肾形，直径10-16cm，厚1-2cm，皮壳坚硬，浅黄褐色，有光泽，具环状棱纹和辐射状皱纹，边缘薄而平截，常稍内卷，腹面黄白色。
3、产量：按常规袋料栽培技术，产量≥63g/kg.袋料。
4、品质：经检测，浸出物6.53％，多糖1.37％，三萜及甾醇0.65％，各项检验应符合《中华人民共和国药典》(2020年版，一部)标准。
实验结果表明：该灵芝菌株遗传性状稳定一致，符合《中华人民共和国药典》标准，具有高产、优质的特点。
实验例2、灵芝菌株抗炎活性研究
1、细胞实验
取实施例2制备的灵芝水提物进行细胞实验。
(1)Griess法检测LPS诱导RAW264.7细胞NO的生成
实验方法：
<1>细胞铺板
取对数生长期的RAW264.7细胞吸出原有1640FBS培养液，加入0.05％胰蛋白酶-0.53mM EDTA消化，2-5min待细胞能被培养液吹掉为止。加入新鲜培养液反复吹打，混匀转移至培养瓶中。取20μl细胞悬液至EP管中，加入180μl台盼蓝溶液，混匀。显微镜下镜检、计数。
取适量细胞悬液至50ml玻璃瓶中，以新鲜培养液补足体积至24ml，24孔细胞培养板中，每孔加入稀释后的细胞悬液1ml，使每孔细胞数为1×106个。细胞板置CO2培养箱中孵育过夜。
<2>加样
吸出原细胞板上加入的培养液，加900μl无酚红的新鲜培养液，取100μl得到的样品，室温解冻后，震荡溶解，混匀，得样品母液。根据预测实验结果，确定各样品起始浓度范围，并根据样品加样浓度设立溶剂对照。
每个样品设置5个样品浓度梯度，每个梯度重复3次。在24孔细胞培养板中，加入实施例2制备的灵芝水提物以及对照灵芝样品液，并以PBS补充至100μl，使样品终浓度分别为6.25ug/ml、12.5μg/ml、25μg/ml、50μg/ml、100μg/ml。空白对照孔加入100μl PBS，混匀。
对照灵芝样品液由保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC NO.23881的灵芝ZL214(保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2021年11月29日，分类命名为灵芝(Ganoderma lucidum))制备而得，制备方法同实施例2，得到对照灵芝水提物。
<3>诱导
加样之后，细胞板放置CO2培养箱中孵育预先2h，再每孔细胞(空白组不加)加入终浓度为1mg/ml的脂多糖(LPS)，再将细胞板置CO2培养箱中孵育48h。
<4>NO含量检测
配制Griess A(奈乙基二胺盐酸盐0.1g，加水溶解定容至100ml)和Griess B(磺胺1g，加水溶解后再加入磷酸5ml，加水定容至100ml)试剂，保存备用。取细胞板上清液100μL至96孔板，再加入等体积混合的Griess试剂100ul，在室温下培养15min。在570nm波长的条件下检测吸光度，并利用已知浓度的亚硝酸钠作为标准，计算各组对应的实际浓度值。
(2)ELISA法检测LPS诱导RAW264.7细胞TNF-α的生成
实验方法参考“(1)”，TNF-α含量检测方法参考ELISA试剂盒。
2.实验结果
(1)Griess法检测LPS诱导RAW264.7细胞NO的生成
实验结果见表1所示：本发明灵芝水提物对LPS诱导的RAW264.7细胞NO的生成具有抑制作用，且有明显的量效关系；对照组灵芝水提物对LPS诱导的RAW264.7细胞NO的生成不具有抑制作用。在多种炎症性或免疫性的动物试验模型中，NO浓度显著增高，抑制NO合成可使典型的炎症症状得到改善。实验结果说明本发明灵芝对NO的生成具有显著的抑制作用，进而可以说明本发明灵芝有显著的抗炎作用。而对照组灵芝不具有抗炎作用。
表1.灵芝水提物对LPS诱导RAW264.7细胞NO生成的抑制作用的结果(％)
(2)ELISA法检测LPS诱导RAW264.7细胞TNF-α的生成
实验结果见表2所示：本发明灵芝水提物对LPS诱导的RAW264.7细胞TNF-α的生成具有抑制作用，且有明显的量效关系；对照组灵芝水提物对LPS诱导的RAW264.7细胞TNF-α的生成不具有抑制作用。TNF-α可抑制炎症信号通路中的重要蛋白，表现为促进炎症反应。实验结果说明本发明灵芝对TNF-α的生成具有显著的抑制作用，进而可以说明本发明灵芝有显著的抗炎作用。而对照组灵芝不具有抗炎作用。
表2.灵芝水提物对LPS诱导RAW264.7细胞TNF-α生成的抑制作用结果(％)
综上，本发明提供了一种新的灵芝菌株，该灵芝菌株具有遗传性状稳定、产量高、品质优的特点。同时，该灵芝菌株具有显著的抗炎作用，可用于制备抗炎的药物或化妆品，本发明灵芝菌株还可以用于制备食品或保健食品，具有良好的应用前景。
Claims (10)
1.一种灵芝菌株，其特征在于：它是保藏于中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.23844的灵芝。
2.一种灵芝水提物，其特征在于：它是由权利要求1所述的灵芝菌株经过水提取后得到的水提物。
3.权利要求2所述的灵芝水提物的制备方法，其特征在于：它包括如下步骤：灵芝粉碎后，加水煎煮提取，取滤液，即得。
4.根据权利要求3所述的制备方法，其特征在于：所述煎煮提取方法为加水浸泡0.5～1h后，加入8～10倍体积的水，煮沸1～3h，提取次数为1～3次。
5.根据权利要求4所述的制备方法，其特征在于：所述滤液浓缩至1～10g/mL。
6.权利要求1所述的灵芝菌株或权利要求2所述的灵芝水提物在制备抗炎药物和/或具有抗炎功效的化妆品中的用途。
7.根据权利要求6所述的用途，其特征在于：所述抗炎药物为抑制NO、TNF-α生成的药物。
8.权利要求1所述的灵芝菌株或权利要求2所述的灵芝水提物在制备保健食品和/或食品中的用途。
9.一种保健食品、化妆品或药物制剂，其特征在于：它是以权利要求1所述的灵芝菌株或权利要求2所述的灵芝水提物为活性成分，加上保健食品、化妆品或药学上可接受的辅料或辅助性成分制备而成的保健食品、化妆品和药物制剂。
10.一种食品，其特征在于：它是权利要求1所述的灵芝菌株或权利要求2所述的灵芝水提物加上食品上可接受的辅料或辅助性成分制备而成的食品。
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