CN108165499A - 一种猴头菌的培养基、山药生物转化菌丝体、山药生物转化菌丝体的提取物及其用途 - Google Patents
一种猴头菌的培养基、山药生物转化菌丝体、山药生物转化菌丝体的提取物及其用途 Download PDFInfo
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- CN108165499A CN108165499A CN201810066537.6A CN201810066537A CN108165499A CN 108165499 A CN108165499 A CN 108165499A CN 201810066537 A CN201810066537 A CN 201810066537A CN 108165499 A CN108165499 A CN 108165499A
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- chinese yam
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- hericium erinaceus
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Abstract
本发明公开了一种猴头菌的培养基、山药生物转化菌丝体、山药生物转化菌丝体的提取物及其用途。该猴头菌的培养基包括如下重量份的各组分:山药490~350份,麦麸140~100份,谷糠70~50份,水400~600份。该山药生物转化菌丝体是将猴头菌接入上述培养基经菌丝转化培养所得的产物。该山药生物转化菌丝体的提取物是将猴头菌丝体经水提后再减压浓缩,最后真空冷冻干燥而制备出来的。该山药生物转化菌丝体的提取物具有降血糖作用,可用于制备降血糖保健品或药物。
Description
技术领域
本发明涉及生物制药技术领域,特别涉及一种猴头菌的培养基、山药生物转化菌丝体、山药生物转化菌丝体的提取物及其用途。
背景技术
猴头菌是一种猴头菌科蘑菇(真菌),在中药用于治疗胃肠道疾病已经超过2000年的历史了,一系列小分子化合物如erinacines,被认为具有神经再生,并且可以透过血脑屏障修复受损神经组织。猴头菌还有抗溃疡,抗炎,抗微生物,免疫调节,提高肝功能,抗衰老,降低血糖和血脂,提高抗缺氧能力,增加心脏输出、和改善备注循环等作用,包含猴头菌的医用制剂在中国广泛使用。
猴头菌丝体中的活性物质最重要的是多糖及糖蛋白,目前国内外对猴头多糖的研究表明,猴头菌多糖具有多种生物活性和药理作用,能增强巨噬细胞的吞噬功能,促进溶血素的形成,抗白细胞下降,降血糖、抗凝血、抗血栓、抗突变和抗衰老等。因此,猴头菌多糖备受人们关注,成为近年来分子生物学、医药、食品科学等领域研究和开发应用的热点。《中国药典》记载了猴头菌丝体(参见2015年版一部的第1614-1615页),本发明所述的猴头菌丝体就是该标准记载的猴头菌丝体。
当前,从高等真菌及其培养物中进行筛选和发现活性成分,应用于降血糖、抗病毒和治疗糖尿病等,不仅成为国家重点研究新药的发展方向,也是当今世界各国开发生物医药的热点。但是,目前并未见到中药作为猴头菌的培养基经过生物转化后的新型药用菌丝体应用于降血糖的报道。
山药作为我国第一批药食同源的药物,为薯蓣科植物薯蓣(Dioscorea oppositaThunb.)的干燥根茎。山药味甘,性平,归肺、脾、肾经,具有补脾、养肺、固肾、益精之功效。薯蓣最早见于我国古代的《山海经》,在汉代《神农本草经》以及宋代的《图经本草》、《求薯蓣苗》、《种山药》,明代的《本草纲目》,清代的《植物名实图考》、现代《中华本草》等都有记载。现代研究发现,山药中的主要成分为薯蓣皂苷元、黏液质、糖蛋白、甘露聚糖、植酸、尿囊素、山药素、胆碱、多巴胺、粗纤维、果胶、淀粉酶、多种微量元素等活性成分,山药中起到药疗作用的主要成分是山药多糖,其具有很强的药理活性。
研究发现,山药多糖对糖尿病小鼠的血糖有明显降低作用,说明山药多糖具有明显的降低血糖作用。通过研究山药水煎剂对实验性小鼠的降血糖作用。结果发现,山药水煎剂对四氧嘧啶引起的小鼠糖尿病有预防及治疗作用,并可对抗由肾上腺素或葡萄糖引起的小鼠血糖升高,其降糖作用随给药剂量的增加而增加。其次,山药多糖能明显升高2型糖尿病小鼠的HK、SDH、MDH活性,说明山药多糖对2型糖尿病的治疗机制可能为其直接或间接地提高了糖代谢或关键酶的酶活性。山药多糖不仅对糖尿病小鼠的血糖有明显降低作用,同时能升高C肽含量,说明山药多糖对糖尿病的治疗作用可能与增加胰岛素分泌有关。
糖尿病属于慢性内分泌代谢疾病,除了遗传因素外,它是人的胰岛素β-细胞合成和分泌胰岛素的功能均下降或障碍,同时可因饮食不当、体力活动过少、能量消耗不足,造成体重增加,腹部脂肪堆积,导致胰岛素抵抗。到目前还没有根治方法,需要长期坚持服药,所以糖尿病患者需要有信心、有恒心才能将血糖控制到理想程度。尽管新口服降糖药不断涌现,胰岛素广泛用于临床,使得大部分糖尿病患者的血糖得到了控制,但且并发症多发、脏器损害还一直困扰着人们。因而饮食疗法既是基础中的基础,又是重中之重,减少食物总热卡量及减少脂肪,增加纤维素和多维类食物。
生物转化“菌丝体”是指利用菌丝体(包括真菌)的代谢功能,使有机物分解的生物化学反应过程。以适宜的培养基为营养,通过菌丝体的生长代谢和生命活动产生丰富的次生代谢产物。借鉴中医药组方思想,将单味中药、具有类似或协同作用的中药作为部分培养基进行菌丝体转化,不同菌株诱变“次生”代谢不同的生物活性成分,再实行“定向培养、双向转化”生物转化工艺,可获得不同生物活性物质原料,目的是增强功效或者降低药物毒副作用。
发明内容
本发明旨在克服现有技术的不足,跨学科创新,结合微生物学、中药学等多学科,提供一种新原料(山药)作为猴头菌的培养基成分。即本发明的目的在于提供一种新材料(新原料),即一种猴头菌的培养基、山药生物转化菌丝体、山药生物转化菌丝体的提取物及其用途,所述山药生物转化菌丝体的提取物的制备方法简单,成本较低,提取物用于制备降血糖药物或保健品。
基于上述目的,本发明的内容包括一种猴头菌的培养基,包括如下重量份的组分:
山药50~70份,麦麸40~20份,谷糠10份,水40~50份。
优选地,所述基础培养基包括如下重量份的各组分:山药60份,麦麸30份,谷糠10份,水45份。
本发明还提供一种山药生物转化菌丝体,制备方法为将猴头菌类菌株接种至所述猴头菌的培养基中,在20~27℃下,猴头菌培育30~40天,烘干,得到山药生物转化菌丝体。
所述猴头菌为猴头菌科真菌猴头菌Hericium erinaceum(Bull.ex Fr.)Pers.。
所述山药生物转化菌丝体是猴头菌科真菌猴头菌(Hericium erinaceum(Bull.exFr.)Pers.)的菌丝体与其附生含有山药成份的固体培养基的干燥混合物。
本发明还提供一种山药生物转化菌丝体的提取物,制备方法为:
(1)将所述山药生物转化菌丝体进行水提,40-60℃下用水浸泡2-3次,每次浸泡2-5小时,水的重量为山药生物转化菌丝体重量的6~10倍,得到提取液;
(2)将步骤(1)中的提取液50℃减压浓缩,将减压浓缩所得的浓缩液冷冻干燥,即得山药生物转化菌丝体的提取物。
优选地,步骤(1)所述用水浸泡的温度为50℃。
步骤(2)所述冷冻干燥的工艺为:-40℃冷冻4-6小时,-10℃一次升华2-5小时,0℃二次升华5-10小时,30℃三次升华24-36小时。
优选地,所述冷冻干燥的工艺为:-40℃冷冻5小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时。
本发明还提供一种所述的山药生物转化菌丝体的提取物在制备降血糖保健品或药物中的用途。
本发明的有益效果有:
(1)本发明的山药生物转化菌丝体的提取物采用简单的处理工艺制备得到,其降血糖效果就达到预料不到的技术效果,经生物转化的“菌丝体”的降血糖效果好于所有的天然植物来源的中药材,如果进一步分离纯化富集有效成分,其降血糖效果将更好,可用于制备抗降血糖药物。
(2)猴头菌是一种药用真菌,能很好地吸收培养基中营养,彻底转化培养基,产生新的活性物质及代谢物,为此,本发明将中药山药加入到猴头菌的基础培养基中,作为猴头菌的培养基成分之一,并对中药材山药掺入猴头菌的培养基的比率进行调配,最终选择中药材山药最佳掺入的比率为60%,山药占60%比率时,菌丝体生长情况较好,山药多糖含量较高。
(3)猴头菌对山药进行中药菌丝体转化,得到新的菌丝体,本发明对所得的山药生物转化菌丝体进行提取,得到的山药生物转化菌丝体的提取物进行了降血糖试验,结果表明:山药生物转化菌丝体的提取物对各种原因引起的高血糖均有较好的抑制作用,降血糖作用由强到弱为:山药生物转化菌丝体>山药药材>猴头菌丝体≈猴头菌丝体+山药,该山药生物转化菌丝体的提取物降血糖作用明显优于猴头菌丝体或山药的抗降血糖作用,这说明猴头菌中丰富的酶系通过结构修饰或降解作用,把山药中的活性成分转化成活性更强的成分,或增强了山药中的活性成分(如山药多糖)的产量,或把非活性成分转化成活性成分,活性成分相互促进达到增强降血糖功效的目的。因此,生物转化菌丝体的提取物可用于制备抗降血糖药物。
(4)本发明低温水浸泡山药生物转化菌丝体,在40-60℃下用水浸泡,无高温破坏,既能溶解有效成分且又不破坏有效成分;为了不影响有关活性物质,本发明优选采用真空冷冻干燥方法,不会破坏有效成分,特别是活性酶类物质。
(5)本发明的山药生物转化菌丝体的提取物对四氧嘧啶致高血糖以及其他原因导致的高血糖都有疗效。
具体实施方式
实施例1
一种山药生物转化菌丝体的提取物,其制备方法包括如下步骤:
(1)取山药500g、麦麸400g、谷糠100g,混匀,加入400g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得山药生物转化菌丝体;
(2)将山药生物转化菌丝体进行水提,40℃下用水浸泡2次,每次浸泡5小时,水的重量为山药生物转化菌丝体重量的6倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻4小时,-10℃一次升华2小时,0℃二次升华10小时,30℃三次升华24小时,得到干燥的山药生物转化菌丝体的提取物(代号:HS-5)。
实施例2
一种山药生物转化菌丝体提取物,其制备方法包括如下步骤:
(1)取农副产品山药600g、麦麸300g、谷糠100g,混匀,加入450g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得山药生物转化菌丝体;
(2)将山药生物转化菌丝体进行水提,50℃下用水浸泡2次,每次浸泡4小时,水的重量为山药生物转化菌丝体重量的8倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻5小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时,得到干燥的山药生物转化菌丝体的提取物(代号:HS-6)。
实施例3
一种山药生物转化菌丝体提取物,其制备方法包括如下步骤:
(1)取农副产品山药700g、麦麸200g、谷糠100g,混匀,加入500g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得山药生物转化菌丝体;
(2)将山药生物转化菌丝体进行水提,60℃下用水浸泡3次,每次浸泡2小时,水的重量为山药生物转化菌丝体重量的10倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻6小时,-10℃一次升华5小时,0℃二次升华5小时,30℃三次升华36小时,得到干燥的山药生物转化菌丝体的提取物(代号:HS-7)。
实施例4山药掺入比例对比试验
培养基山药的比率分别占50%、60%、70%,设计实验方案,总投料5000g,分别按上述实施例1、实施例2、实施例3配制培养基,加水混匀、灭菌、接种、培养,平行对照,培养条件完全一致,培养过程中观察菌丝体生长状况、生长周期、污染情况、生长速度,挖瓶后烘干称重计算收率,检测样品中的纤维素酶、麦角甾醇含量(考察转化是否彻底)、尿囊素、多糖、多肽的含量,干燥后的样品分别标注为HS-5、HS-6、HS-7。
参考申请号为201410393488.9的发明专利《一种猴头菌丝体及其培育方法》,同样灭菌、接种、培养,平行对照,培养条件完全一致,得到猴头菌丝体,将样品标注为HTJ。
试验结果统计如下表1。
表1
试验结果表示:山药占70%时污染率高,纤维素酶含量低,生物转化不彻底;而山药占50%时,菌类多糖、菌类多肽、尿囊素含量低;山药占60%比率时最为合适,菌丝体生长情况较好,兼顾各优势,为最佳比率。
此外,与HTJ组相比,山药占60%比率时的HS-6组的污染率低于HTJ组,且生长周期相对较短,生长速度快,纤维素酶、麦角甾醇、菌类多糖、菌类多肽以及尿囊素的含量也都高于HTJ组。由于菌类多糖、菌类多肽和尿囊素对降血糖有良好的作用,所以本发明在培养基中加入山药成分,使所得山药生物转化菌丝体的提取物可用于制备降血糖保健品或药物。
实施例5山药生物转化物降血糖试验
1.试验材料
1.1山药原药材
由湖南新汇制药股份有限公司提供。
1.2实验动物
18~22g,雄性小鼠,70只,昆明种小白鼠,由湖南斯莱克景达实验动物有限公司提供,实验动物生产许可证号:SCXK(湘)2011-0003;在湖南省药物安全评价研究中心屏障环境B区实验室饲养,实验动物质量合格证号:NO43004700004931,实验动物使用许可证号:SYXK(湘)2010-0008。实验期间环境温度20℃~26℃,湿度40%~70%。
1.3供试品
样品编号:HTJ表示猴头菌丝体,SY表示山药中药材,HTJ+SY表示40%的猴头菌丝体+60%山药中药材,HS-5、HS-6、HS-7分别表示猴头菌的培养基中山药掺入比例为50%、60%、70%的山药生物转化菌丝体。上述样品平行操作进行如下处理:山药生物转化菌丝体进行水提,50℃下用水浸泡2次,每次浸泡4小时,水的重量为山药生物转化菌丝体重量的8倍,得到提取液,50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻5小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时,即得6种提取物,分别编号为HTJ,SY,HTJ+SY,HS-5,HS-6和HS-7。
1.4主要试剂
四氧嘧啶,由西格玛奥德里奇贸易有限公司(Sigma-Aldrich)提供。
1.5主要仪器
自动生化分析仪(荷兰威图科学公司),TGL-16G台式高速离心机(强生有限公司),血糖试剂盒(上海荣盛生物技术有限公司),DL-50型超级恒温器,756MC型紫外可见分光光度计。
2.试验方法
2.1糖尿病模型的建立
昆明种雄性小鼠100只,体重20~25g,适应实验环境4d,禁食12h后,经腹腔一次性注射四氧嘧啶,剂量为150mg/(kg·bw),3d后禁食6h,测定空腹血糖。选择70只空腹血糖在10mmol/L以上者,作为糖尿病小鼠。
2.2实验分组及处理
将昆明种雄性小鼠分为7组,即正常对照组、山药多糖组(SY)、猴头菌丝体组(HTJ)、猴头菌丝体加山药组(HTJ+SY)、三种山药生物转化菌丝体(HS-5、HS-6、HS-7),每组10只小鼠。山药多糖组(SY)以20mg/(kg·bw)的山药多糖灌胃,正常对照组灌胃等量的蒸馏水,每日1次,连续给药30d。分别对小鼠不同时期的体重和空腹血糖进行测定。小鼠耐糖量的检测在实验结束时进行,将空腹6h后的小鼠用葡萄糖以25g/(kg·bw)的剂量灌胃处理后,分别测定小鼠口服葡萄糖后0min,60min,120min的血糖值。
2.3测定指标和方法
采集小鼠眼眶内眦静脉血,取血清用葡萄糖氧化酶-过氧化酶法进行血糖的测定,空腹血糖采集禁食6h后的小鼠静脉血。
2.4统计分析
实验数据用SPSS 11.5软件进行处理,结果用t检验,以x±s表示。
3山药多糖对正常小鼠和糖尿病小鼠血糖的影响结果
造型前各组小鼠空腹血糖值均无明显差异(P>0.05),给药前除空白对照组外,各组小鼠血糖明显升高(P<0.01)。给药后,除空白组外,各组给药后小鼠血糖值明显降低。
表2
与造型前比较**P<0.01,与给药前比较##P<0.01
从表2中可以看出,体外降血糖能力由强到弱为:山药生物转化菌丝体>山药药材>猴头菌丝体≈猴头菌丝体+山药,而且HS-6组最好。结合实施例4的结果说明猴头菌中丰富的酶系可能通过结构修饰或降解作用,把山药中的活性成分转化成活性更强的成分,或增强了山药中的活性成分(如多糖)的产量,或把非活性成分转化成活性成分,活性成分相互促进达到增强降血糖功效的目的。
Claims (10)
1.一种猴头菌的培养基,其特征在于,包括如下重量份的组分:
山药50~70份,麦麸40~20份,谷糠10份,水40~50份。
2.如权利要求1所述的猴头菌的培养基,其特征在于,所述培养基包括如下重量份的组分:
山药60份,麦麸30份,谷糠10份,水45份。
3.一种山药生物转化菌丝体,其特征在于,将猴头菌类菌株接种至如权利要求1或2所述的猴头菌的培养基中,在20~27℃下,猴头菌培育30~40天,烘干,即得山药生物转化菌丝体。
4.如权利要求3所述的山药生物转化菌丝体,其特征在于,所述猴头菌类菌株为猴头菌科真菌猴头菌Hericium erinaceum(Bull.ex Fr.)Pers。
5.如权利要求4所述的生物转化菌丝体,其特征在于,所述生物转化菌丝体是猴头菌科真菌猴头菌(Hericium erinaceum(Bull.ex Fr.)Pers.)的菌丝体与其附生含有山药成份的固体培养基的干燥混合物。
6.一种山药生物转化菌丝体的提取物,其特征在于,所述提取物通过以下步骤制备得到:
(1)将如权利要求3-5任意一项所述的山药生物转化菌丝体进行水提,40-60℃下用水浸泡2-3次,每次浸泡2-5小时,水的重量为山药生物转化菌丝体重量的6~10倍,得到提取液;
(2)将步骤(1)中的提取液50℃减压浓缩,将减压浓缩所得的浓缩液冷冻干燥,即得山药生物转化菌丝体的提取物。
7.如权利要求6所述的山药生物转化菌丝体的提取物,其特征在于,所述用水浸泡的温度为50℃。
8.如权利要求6或7所述的山药生物转化菌丝体的提取物,其特征在于,所述冷冻干燥的工艺为,-40℃冷冻4-6小时,-10℃一次升华2-5小时,0℃二次升华5-10小时,30℃三次升华24-36小时。
9.如权利要求8所述的山药生物转化菌丝体的提取物,所述冷冻干燥的工艺为:-40℃冷冻5小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时。
10.一种如权利要求6-9任意一项所述的山药生物转化菌丝体的提取物在制备降血糖保健品或药物中的用途。
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