CN109336987A - 一种羊肚菌和牛肝菌复方粗多糖及制备方法和医用用途 - Google Patents
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Abstract
本发明提供了一种羊肚菌和牛肝菌复方粗多糖及制备方法,同时还公开了该多糖在保护酒精性肝病中的医用用途,相比于羊肚菌和牛肝菌的单一粗多糖,羊肚菌和牛肝菌复方作用后,会发生多种反应,如通过氢键、范德华力和化学键的重新键合出现新的单糖链,具体反应为检测峰出现数量增多和合并现象,从而为后续酒精性肝病活性的考察能够产生相比于羊肚菌和牛肝菌的单一粗多糖更好的效果奠定基础。制成的羊肚菌和牛肝菌复方粗多糖冻干粉安全、无副作用,具有显著保健功效,可长期服用,具有很好的市场开发前景。
Description
技术领域
本发明提供了一种羊肚菌和牛肝菌复方粗多糖及制备方法,同时还公开了该多糖在保护酒精性肝病中的医用用途,属于中药和保健食品技术领域。
背景技术
酒精中毒死亡率和癌症及冠心病的死亡率相仿,是中青年死亡的主要原因之一;酒精性肝病,通常发生于多年的过度饮酒者。饮酒时间越长,饮酒量越大,酒精性肝病发生的可能性就越大。短时间内大量的饮酒也能导致酒精性肝炎,在极端情况下甚至会危及生命。酒精性肝病初期并无症状,发展到酒精性肝炎时会出现体重减轻、食欲不振、恶心呕吐、全身乏力、腹痛腹泻、黄疸、肝肿大和压痛等症状。我国近年来临床酒精性肝病的患病率也逐年升高,成为仅次于病毒性肝炎的第二大肝病,因此重视酒精性肝病的防治具有重要意义。目前对于酒精性肝病最重要的治疗手段是禁酒,药物治疗方面目前还没有理想的药物。抗炎物质能够防止促炎症细胞因子的释放,被认为是一类有希望成为有效的治疗酒精性肝病的药物。
羊肚菌(Morchella)属隶属于子囊菌亚门(Ascomycotina),盘菌纲(Discomycetes),盘菌目(Pezizales),羊肚菌科(Morchellaceae),是一种稀珍的食(药)用真菌。据目前报道,我国羊肚菌共有 20 种,在西北、西南、华中、华南和华东等地均有分布。羊肚菌不但滋味鲜香,而且营养物质非常丰富。有研究发现,羊肚菌中氨基酸含量高达19种;还含有大量矿物质元素和糖类以及各种维生素。研究发现羊肚菌有降血脂、抗炎、调节免疫功能、抗疲劳、抗辐射、抗肿瘤作用,并能减轻癌症患者放化疗引起的毒副作用。牛肝菌(Boletus)属伞菌目牛肝菌科真菌,为野生菌,尚未实现人工栽培,其中除少数品种有毒或味苦而不能食用外,大部分品种均可食用。牛肝菌因肉质肥厚,极似牛肝而得名,是名贵稀有的野生食用菌,为“四大菌王”之一。其菌体较大,柄粗壮,可入药,出口欧美、日本等国,是一种世界性著名食用菌。牛肝菌富含多糖、黄酮类、多酚类物质和麦角甾醇等功能成分,具有抗氧化、抗炎、抗肿瘤、预防、治疗伛偻病和机体各种黏膜炎及皮肤炎等功效。
经检索未见二者在酒精性肝病具有医疗作用的文献报道。
发明内容
本发明提供了一种羊肚菌和牛肝菌复方粗多糖及制备方法,用于对酒精性肝病具有治疗保健功能。
本发明所述的一种羊肚菌和牛肝菌复方粗多糖冻干粉,是由如下方法制备得到的:
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15~30倍的去离子水,在80℃~100℃下提取两次,合并提取液,得羊肚菌水提液;取牛肝菌子实体100g,按照同样上述同样方法制备得到牛肝菌水提液;取上述羊肚菌水提液和牛肝菌水提液按照体积比1:1~3:1混合,4℃以50~75 r/min混合24~48h,让其充分相互作用。随后,减压浓缩至50 mL~100 mL,按体积比:1:3~1:5加入95%的乙醇,室温静置过夜,随后以3000~4000 r/min离心10 min,收集沉淀,并以-30~-50℃冻干60~80小时,得到羊肚菌和牛肝菌复方粗多糖冻干粉。
经动物实验证明(详见试验例),本发明羊肚菌和牛肝菌复方粗多糖冻干粉具有保护酒精性肝病的功能,因而可以在作为保健食品方面得到应用。其主要的功效学作用如下:经高效液相色谱检测结果表明,相比于羊肚菌和牛肝菌的单一粗多糖,羊肚菌和牛肝菌复方作用后,会发生多种反应,如通过氢键、范德华力和化学键的重新键合出现新的单糖链,具体反应为检测峰出现数量增多和合并现象,从而为后续酒精性肝病活性的考察能够产生相比于羊肚菌和牛肝菌的单一粗多糖更好的效果奠定基础。经白酒建模后的C57BL/6J小鼠经过灌胃给予复方粗多糖冻干粉15天后,小鼠体重下降有明显减轻,血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、白细胞介素-7(IL-7)、趋化因子3(CXCL3)、视黄醇结合蛋白4(RBP4)因子的水平发生显著变化,且病理学观察结果表明相较于单独给予羊肚菌子粗多糖冻干粉和单独给予羊肚菌粗多糖冻干粉,复方粗多糖冻干粉更能改善肝脏中脂肪细胞堆积及炎症因子浸润。
本发明的积极效果在于:提供利了羊肚菌和牛肝菌复方粗多糖冻干粉新的保健医用用途;相比于羊肚菌和牛肝菌的单一粗多糖,羊肚菌和牛肝菌复方作用后,会发生多种反应,如通过氢键、范德华力和化学键的重新键合出现新的单糖链,具体反应为检测峰出现数量增多和合并现象,从而为后续酒精性肝病活性的考察能够产生相比于羊肚菌和牛肝菌的单一粗多糖更好的效果奠定基础。制成的羊肚菌和牛肝菌复方粗多糖冻干粉安全、无副作用,具有显著保健功效,可长期服用,具有很好的市场开发前景。
附图说明
图1为羊肚菌粗多糖冻干粉高效液相色谱图;
图2为牛肝菌粗多糖冻干粉高效液相色谱图;
图3为羊肚菌和牛肝菌复方粗多糖冻干粉高效液相色谱图;
图4为不同粗多糖冻干粉对酒精性肝病小鼠肝脏组织病理学的影响。
具体实施方式
下面结合实施例对本发明作进一步描述,但本发明不受具体实施例限制。
实施例1
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15倍的去离子水,在80℃下提取两次,合并提取液,得羊肚菌水提液。取牛肝菌子实体100g,按照同样上述同样方法制备得到牛肝菌水提液。取上述羊肚菌水提液和牛肝菌水提液按照体积比1:1混合,4℃以50 r/min混合24小时,让其充分相互作用。随后,减压浓缩至60 mL,按体积比:1:3加入95%的乙醇,室温静置过夜,随后以3500 r/min离心10 min,收集沉淀,并以-50℃冻干72小时,得到羊肚菌和牛肝菌复方粗多糖冻干粉。
备注:下文试验例中使用到的羊肚菌粗多糖冻干粉和牛肝菌粗多糖冻干粉制备方法如下:
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15倍的去离子水,在80℃下提取两次,合并提取液,得羊肚菌水提液。减压浓缩至50mL,按体积比:1:3加入95%的乙醇,室温静置过夜,随后以3000 r/min离心10 min,收集沉淀,并以-50℃冻干80小时,得到羊肚菌粗多糖冻干粉。同样称取牛肝菌子实体100g,按照上述羊肚菌粗多糖冻干粉制备方法,得到牛肝菌粗多糖冻干粉。
实施例2
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加20倍的去离子水,在90℃下提取两次,合并提取液,得羊肚菌水提液。取牛肝菌子实体100g,按照同样上述同样方法制备得到牛肝菌水提液。取上述羊肚菌水提液和牛肝菌水提液按照体积比2:1混合,4℃以60 r/min混合36小时,让其充分相互作用。随后,减压浓缩至50 mL,按体积比:1:4加入95%的乙醇,室温静置过夜,随后以3000 r/min离心10 min,收集沉淀,并以-40℃冻干80小时,得到羊肚菌和牛肝菌复方粗多糖冻干粉。
备注:下文试验例中使用到的羊肚菌粗多糖冻干粉和牛肝菌粗多糖冻干粉制备方法如下:
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15倍的去离子水,在80℃下提取两次,合并提取液,得羊肚菌水提液。减压浓缩至50mL,按体积比:1:3加入95%的乙醇,室温静置过夜,随后以3000 r/min离心10 min,收集沉淀,并以-50℃冻干80小时,得到羊肚菌粗多糖冻干粉。同样称取牛肝菌子实体100g,按照上述羊肚菌粗多糖冻干粉制备方法,得到牛肝菌粗多糖冻干粉。
实施例3
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加30倍的去离子水,在100℃下提取两次,合并提取液,得羊肚菌水提液。取牛肝菌子实体100g,按照同样上述同样方法制备得到牛肝菌水提液。取上述羊肚菌水提液和牛肝菌水提液按照体积比3:1混合,4℃以70 r/min混合48h,让其充分相互作用。随后,减压浓缩至75mL,按体积比:1:5加入95%的乙醇,室温静置过夜,随后以4000 r/min离心10 min,收集沉淀,并以-30℃冻干65小时,得到羊肚菌和牛肝菌复方粗多糖冻干粉。
备注:下文试验例中使用到的羊肚菌粗多糖冻干粉和牛肝菌粗多糖冻干粉制备方法如下:
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15倍的去离子水,在80℃下提取两次,合并提取液,得羊肚菌水提液。减压浓缩至50mL,按体积比:1:3加入95%的乙醇,室温静置过夜,随后以3000 r/min离心10 min,收集沉淀,并以-50℃冻干80小时,得到羊肚菌粗多糖冻干粉。同样称取牛肝菌子实体100g,按照上述羊肚菌粗多糖冻干粉制备方法,得到牛肝菌粗多糖冻干粉。
通过以下试验例用于说明本发明的羊肚菌和牛肝菌复方粗多糖冻干粉相比于羊肚菌和牛肝菌单一粗多糖峰形发生明显变化,产生意想不到的协同效应,且羊肚菌和牛肝菌复方粗多糖冻干粉相比于羊肚菌和牛肝菌单一粗多糖对酒精性肝病的保护作用的影响。
1 实验方法
1.1 高效液相色谱测多糖
应用HPLC法测定菌丝体中羊肚菌粗多糖冻干粉、牛肝菌粗多糖冻干粉、羊肚菌和牛肝菌复方粗多糖冻干粉中多糖含量,色谱条件为:
色谱柱: C18柱
流动相: pH6.5的磷酸盐缓冲液:甲醇(85:15)
流速: 1mL/min
检测波长:620nm
柱温:35℃
进样量: 20μL
1.2动物饲养及分组给药。
本试验例用于说明本发明的羊肚菌和牛肝菌复方粗多糖冻干粉对酒精引起的肝病小鼠的保护作用。准备健康C57BL/6J雄性小鼠40只,体重18~22g,饲养温度23±1℃,相对湿度55±5%,随机分为5组,每组8只,分别作为空白对照组、模型组、实施例1、实施例2、实施例3羊肚菌粗多糖冻干粉组(ME组)、牛肝菌粗多糖冻干粉组(BA组)、羊肚菌和牛肝菌复方粗多糖冻干粉组(MB组)。
每天上午9时空白组灌胃给予10 ml/kg生理盐水,其余组均灌胃给予13 g/kg 56度红星二锅头,下午4时,空白组和模型组灌胃给予10 ml/kg生理盐水;实施例1、实施例2、实施例3羊肚菌粗多糖冻干粉组灌胃给予同样体积的羊肚菌粗多糖冻干粉溶液(50 mg/kg);牛肝菌粗多糖冻干粉组灌胃给予同样体积的粗多糖冻干粉溶液(50 mg/kg);羊肚菌和牛肝菌复方粗多糖冻干粉组灌胃给予同样体积的羊肚菌和牛肝菌复方粗多糖冻干粉溶液(50 mg/kg)。
1.3样本的制备方法
用药结束,各组小鼠禁食、不禁水6 h后摘眼球取血,血液静置30 min后3500 r/min离心10 min,取上清,置于-20℃冰箱保存待用。采血后,将实验动物处死、迅速分离小鼠肝脏,于-80℃冰箱保存,备用。取部分小鼠肝脏,加入定量生理盐水制备成10%肝组织匀浆液,待测。
1.4组织病理学观察
肝脏标本在10%中性福尔马林中固定,4℃保存。常规石蜡包埋、切片,采用HE染色,通过400倍光镜观察进行病理分析。
1.5生化因子的测定
依照上海源叶试剂盒说明书,测定各组小鼠血清中ALT、AST、IL-7、CXCL3、RBP4等因子的水平。
2检测结果
2.1 高效液相色谱检测结果
如图1-3所示,当保留时间为22-23.5分钟时羊肚菌粗多糖冻干粉和牛肝菌粗多糖冻干粉均有一个峰出现,羊肚菌和牛肝菌复方粗多糖冻干粉出现明显变化,出现3个峰,但其中一个峰占据较大曲线下面积。从上述色谱图分析,相比于羊肚菌和牛肝菌的单一粗多糖,羊肚菌和牛肝菌复方作用后,产生协同效应,如通过氢键、范德华力和化学键的重新键合出现新的单糖链,具体反应为检测峰出现数量增多和合并现象,从而为后续酒精性肝病活性的考察能够产生相比于羊肚菌和牛肝菌的单一粗多糖更好的效果奠定基础。
2.2不同粗多糖冻干粉对酒精性肝病标志性酶类活力的影响
谷丙转氨酶(ALT)和谷草转氨酶(AST)是肝脏内的标志性酶类,当肝脏损伤后,二者会产生异常升高。本文考察各组对酒精性肝病小鼠ALT和AST含量的影响结果如表1所示。与空白组相比,模型组组小鼠的谷丙转氨酶和谷草转氨酶含量明显升高。给予羊肚菌和牛肝菌复方粗多糖冻干粉比单独给予羊肚菌和牛肝菌粗多糖冻干粉更能够降低ALT和AST水平。
表1 不同粗多糖冻干粉对酒精性肝病小鼠肝损伤标志性酶类的影响
2.3不同粗多糖冻干粉对酒精性肝病小鼠肝脏病理学的影响
短时间摄入过多酒精会引起小鼠肝脏组织病变,严重会引起肝细胞坏死和死亡,不同粗多糖冻干粉对小鼠肝脏病理组织的影响如下图所示:如图4所示,与空白组相比,酒精作用会引起肝脏组织中脂肪滴形成,出现炎症因子浸润和点状坏死。与模型组相比,三组给药组均能减少脂肪滴形成,减轻点状坏死和炎症因子浸润现象,但以羊肚菌和牛肝菌复方粗多糖冻干粉效果最好。
2.4不同粗多糖冻干粉对酒精性肝病小鼠血清中生化因子水平的影响
表2 不同粗多糖冻干粉对酒精性肝病小鼠血清中炎症因子水平的影响
白细胞介素-17(IL-17)是一种主要由活化的T细胞产生的致炎细胞因子;趋化因子配体3(CXCL3)能够吸引白细胞移行到感染部位,在炎症反应中具有重要作用。表2显示本品对酒精引起的小鼠血清中IL-17、CXCL3和RBP4蛋白含量的影响,与空白组相比,模型组中酒精作用能够显著升高IIL-17、CXCL3和RBP4水平(#:与空白组相比P<0.05)。与模型组相比,三类粗多糖冻干粉均能显著性降低IL-17、CXCL3和RBP4水平(*:与模型组相比P<0.05)。其中以羊肚菌和牛肝菌复方粗多糖冻干粉效果最为明显。
综合上述,连续建模及灌胃给予羊肚菌粗多糖冻干粉、牛肝菌粗多糖冻干粉、羊肚菌和牛肝菌复方粗多糖冻干粉15天后,考察对酒精引起的小鼠的肝组织损伤、炎症因子及组织病理学的变化的影响,以羊肚菌和牛肝菌复方粗多糖冻干粉效果最佳。
Claims (2)
1.一种羊肚菌和牛肝菌复方粗多糖冻干粉,是由如下方法制备得到的:
取羊肚菌子实体100g,利用高速粉碎机进行粉碎,过100目筛,制备的羊肚菌子实体粉,随后加15~30倍的去离子水,在80℃~100℃下提取两次,合并提取液,得羊肚菌水提液;
取牛肝菌子实体100g,按照同样上述同样方法制备得到牛肝菌水提液;
取上述羊肚菌水提液和牛肝菌水提液按照体积比1:1~3:1混合,4℃以50~75 r/min混合24~48h,让其充分相互作用,减压浓缩至50 mL~100 mL,按体积比:1:3~1:5加入95%的乙醇,室温静置过夜,随后以3000~4000 r/min离心10 min,收集沉淀,并以-30~-50℃冻干60~80小时,得到羊肚菌和牛肝菌复方粗多糖冻干粉。
2.如权利要求1所述的羊肚菌和牛肝菌复方粗多糖冻干粉在制备对酒精性肝病具有保护功能的保健食品和药品中的医用用途。
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