CN111704658B - 具有消脂护肝功能的毛木耳糖肽及其应用 - Google Patents
具有消脂护肝功能的毛木耳糖肽及其应用 Download PDFInfo
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- CN111704658B CN111704658B CN202010516920.4A CN202010516920A CN111704658B CN 111704658 B CN111704658 B CN 111704658B CN 202010516920 A CN202010516920 A CN 202010516920A CN 111704658 B CN111704658 B CN 111704658B
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- auricularia polytricha
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Abstract
本发明公开了具有消脂护肝功能的毛木耳糖肽及其应用。该毛木耳糖肽是从毛木耳中提取出的糖肽APPII。APPII的N末端序列如序列表中序列25所示，具有序列表中序列25‑63所示的39个肽段，其单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖，摩尔比为1:72:99:10:5，其分子量为6.340×105道尔顿。APPII具有减少脂肪在肝内堆积，消脂护肝的功能。
Description
本申请是申请号为201810596739.1、申请日为2018年06月11日、发明创造名称为“具有消脂护肝功能的毛木耳提取物及其应用”的分案申请。
技术领域
本发明涉及生物领域中具有消脂护肝功能的毛木耳糖肽及其应用。
背景技术
肝脏是身体内以代谢功能为主的一个器官，脂肪肝在临床诊断中表现为肝脏细胞内含有大量的甘油三酯聚集，随着生活水平的提高，与膳食结构、肥胖、代谢综合症的增高，我国脂肪肝的发病率已占平均人口的10％，在肥胖和糖尿病高危人群中可高达50-60％。随着脂肪肝病程的加剧，在二次打击的作用下，形成了氧化应激和肝细胞释放异常细胞因子，导致脂肪变性的肝脏发生炎症反应、坏死和纤维化，进而演变为脂肪性肝炎、肝纤维化和肝硬化。目前对于造成脂肪堆积和脂毒性的具体分子机制仍未有明确的阐述，临床上多采取降脂功效的药物来治疗脂肪肝，针对病症的表征采用治疗方法，由于化学类药物的代谢仍然需要肝脏来完成，在降脂的同时增加了肝脏的负担，缺乏保护肝脏的效果。目前对于治疗脂肪肝药物的开发应向着降脂保肝低毒的方向发展，寻找天然产物做为化学药物的替代物。
发明内容
本发明所要解决的技术问题是如何消脂护肝。
为了解决以上技术问题，本发明提供了毛木耳提取物。
本发明所提供的毛木耳提取物是从毛木耳中提取出的名称为APPI的糖肽或名称为APPII的糖肽；所述APPI的N末端序列如序列表中序列1所示，所述APPI具有序列表中序列1-24所示的24个肽段，所述APPI的单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖，阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖的摩尔比为1:38:46:15:4(如1:38.3:46.2:15.4:4.4)，所述APPI的重均分子量为9.213×105道尔顿；所述APPII的N末端序列如序列表中序列25所示，所述APPII具有序列表中序列25-63所示的39个肽段，所述APPII单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖，阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖的摩尔比为1:72:99:10:5(如1:71.5:99.2:10:5.1)，所述APPII的重均分子量为6.340×105道尔顿。
上述毛木耳提取物中，所述毛木耳提取物是从毛木耳子实体中提取出的名称为APPI的糖肽或名称为APPII的糖肽。
上述毛木耳提取物中，所述毛木耳提取物是从毛木耳子实体水提液中提取出的名称为APPI的糖肽或名称为APPII的糖肽，所述毛木耳子实体水提液是用水从毛木耳子实体中提取出来的水溶性物质。
上述毛木耳提取物中，所述毛木耳提取物可按照下述毛木耳提取物的制备方法制备：所述毛木耳提取物是名称为APPI的糖肽或名称为APPII的糖肽；
所述APPI的制备方法可包括：I-1)制备名称为去除蛋白的毛木耳粗多糖，所述去除蛋白的毛木耳粗多糖的制备方法可包括用乙醇沉淀毛木耳子实体水提液，收集沉淀，去除所述沉淀中的蛋白后得到所述去除蛋白的毛木耳粗多糖；所述毛木耳子实体水提液是用水从毛木耳子实体中提取出来的水溶性物质；I-2)从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽，得到名称为APPI的糖肽；
所述APPII的制备方法可包括：II-1)制备名称为去除蛋白的毛木耳粗多糖，所述去除蛋白的毛木耳粗多糖的制备方法可包括用乙醇沉淀毛木耳子实体水提液，收集沉淀，去除所述沉淀中的蛋白后得到所述去除蛋白的毛木耳粗多糖；所述毛木耳子实体水提液是用水从毛木耳子实体中提取出来的水溶性物质；II-2)从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽，得到名称为APPII的糖肽。
上述毛木耳提取物的制备方法中，所述I-2)中，从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽可包括：I-2-1)对所述去除蛋白的毛木耳粗多糖进行阴离子交换柱层析，得到名称为D1组分的分离物；所述阴离子交换柱层析中所采用的阴离子交换基团是DEAE，所采用的洗脱程序为一步洗脱，所述一步洗脱用pH为6.8-7.5(如7.0)的磷酸缓冲溶液A进行洗脱，所述磷酸缓冲溶液A的溶质为3.5-4.5mM(如3.9mM)NaH2PO4和5.5-6.5mM(如6.1mM)Na2HPO4，溶剂为水；I-2-2)对所述D1组分进行分子筛层析(如层析介质为Superdex-200的分子筛层析)，得到重均分子量为9.213×105道尔顿的糖肽，该糖肽即为APPI；
所述II-2)中，从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽可包括：II-2-1)对所述去除蛋白的毛木耳粗多糖进行阴离子交换柱层析，得到名称为D2组分的分离物；所述阴离子交换柱层析中所采用的阴离子交换基团是DEAE，所采用的洗脱程序为两步洗脱，第一步洗脱用pH为6.8-7.5(如7.0)的磷酸缓冲溶液A进行洗脱，所述磷酸缓冲溶液A的溶质为3.5-4.5mM(如3.9mM)NaH2PO4和5.5-6.5mM(如6.1mM)Na2HPO4，溶剂为水；第二步洗脱用pH6.8-7.5(如7.0)的如下溶液洗脱：溶质是0.2M NaCl，溶剂是所述磷酸缓冲溶液A；II-2-2)对所述D2组分进行分子筛层析(如层析介质为Superdex-200的分子筛层析)，得到重均分子量为6.340×105道尔顿的糖肽，该糖肽即为APPII。
上文中，所述毛木耳子实体水提液可按照包括如下步骤的方法制备：用水浸泡粉碎后的毛木耳子实体8-12小时(如8小时)，加热至90-100℃(如90℃)保持3-4小时(如4小时)，收集水溶性物质，该水溶性物质即为毛木耳子实体水提液。
上述毛木耳子实体水提液中，所述水可为去离子水。
上述毛木耳子实体水提液中，毛木耳子实体可为新鲜的子实体也可为干燥的子实体。
上述干燥的子实体是将新鲜的毛木耳子实体在常温(如20-25℃)下干燥或冷冻干燥得到的。
上述毛木耳提取物中，所述毛木耳提取物具有以下至少一种功能：A1)降低脂肪肝动物血清中血清总胆固醇(TC)的含量；A2)降低脂肪肝动物血清中血清甘油三酯(TG)的含量；A3)降低脂肪肝动物血清中血清低密度脂蛋白胆固醇(LDL-C)的含量；A4)降低脂肪肝动物血清中谷丙转氨酶(ALT)的含量；A5)降低脂肪肝动物血清中谷草转氨酶(AST)的含量；A6)使脂肪肝动物肝细胞内脂滴减少；A7)使脂肪肝动物肝细胞炎症降低；B1)提高受损肝细胞的存活率；B2)提高受损肝细胞的SOD酶活性；B3)降低受损肝细胞内的甘油三酯含量；B4)降低受损肝细胞的谷丙转氨酶的释放量；B5)降低受损肝细胞的谷草转氨酶的释放量。
含有所述毛木耳提取物的消脂护肝的产品(如药物、保健品或/食品)或保护受损肝细胞的产品(如药物、保健品或/食品)也属于本发明的保护范围。
上述产品中，所述消脂护肝的产品具有下述A1)-A7)中的7种、6种、5种、4种、3种、2种或1种功能：A1)降低脂肪肝动物血清中血清总胆固醇(TC)的含量；A2)降低脂肪肝动物血清中血清甘油三酯(TG)的含量；A3)降低脂肪肝动物血清中血清低密度脂蛋白胆固醇(LDL-C)的含量；A4)降低脂肪肝动物血清中谷丙转氨酶(ALT)的含量；A5)降低脂肪肝动物血清中谷草转氨酶(AST)的含量；A6)使脂肪肝动物肝细胞内脂滴减少；A7)使脂肪肝动物肝细胞炎症降低；
所述保护受损肝细胞的产品具有下述B1)-B5)中的5种、4种、3种、2种或1种功能：
B1)提高受损肝细胞的存活率；B2)提高受损肝细胞的SOD酶活性；B3)降低受损肝细胞内的甘油三酯含量；B4)降低受损肝细胞的谷丙转氨酶的释放量；B5)降低受损肝细胞的谷草转氨酶的释放量。
上述消脂护肝的产品和所述保护受损肝细胞的产品的活性成分可只为所述毛木耳提取物，也可含有其它成分，其它活性成分本领域技术人员可根据产品的消脂护肝效果确定。
上述消脂护肝的产品和所述保护受损肝细胞的产品可含有药学上可接受的载体。药学上可接受的载体可为稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；药学上可接受的载体可为湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；药学上可接受的载体可为崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
上述毛木耳提取物在制备上述消脂护肝的产品和所述保护受损肝细胞的产品中的应用也属于本发明的保护范围。
实验证明，上述毛木耳提取物——糖肽APPI和糖肽APPII既存在糖结构，又存在肽段结构，是一个糖肽纯品；通过体内和体外研究表明糖肽APPI和APPII都具有减少脂肪在肝内堆积，消脂护肝的功能。
附图说明
图1为10mM pH9.4 NH4HCO3-NH3H2O缓冲体系洗脱曲线。
图2为去离子水体系洗脱曲线。
图3为10mM pH7.0的磷酸缓冲体系洗脱曲线。
图4为D1组分Superdex-200纯化曲线。
图5为D2组分Superdex-200纯化曲线。
图6为名称为APPI的糖肽全水解还原乙酰化GC-MS质谱图。
图7为名称为APPII的糖肽全水解还原乙酰化GC-MS质谱图。
图8为糖肽APPI的红外光谱分析图。
图9为糖肽APPII的红外光谱分析图。
图10为糖肽APPI和糖肽APPII对受损肝细胞存活率的影响。图中，“*”表示与模型组相比具有显著差异(P＜0.05)。
图11为动物肝脏组织HE染色病理结果(400X)。图中，A为正常组，B为模型组，C为糖肽APPI(50mg/kg/d)组，D为糖肽APPI(100mg/kg/d)组，E为糖肽APPII(50mg/kg/d)组，F为糖肽APPII(100mg/kg/d)组，G为毛木耳粗多糖(613mg/kg/d)组，H为毛木耳粗多糖(202mg/kg/d)组，I为辛伐他汀组。图中的bar为50μm。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的毛木耳为毛木耳(Auricularia polytricha(Mont.)Sacc.)3号(赵爽等.毛木耳菌丝体产多糖发酵条件的优化.食品科技.2012年第37卷第4期)公众可从北京市农林科学院获得该菌种，以重复本申请实验。下述实施例中的L-O2细胞株(人肝细胞)为中国医学科学院肿瘤细胞库的产品。下述实施例中的RPMI1640培养液为Invitrogen公司产品，产品目录号为11875-093。下述实施例中的BCA蛋白定量试剂盒为北京博迈德生物技术有限公司产品，产品目录号为PP0103。下述实施例中的组织甘油三酯测定试剂盒为北京普利莱基因技术有限公司产品，产品目录号为E1013。下述实施例中的组织内活性氧测定试剂盒为北京普利莱基因技术有限公司产品，产品目录号为C1300。下述实施例中的血清内谷丙转氨酶测定试剂盒为南京建成科技有限公司产品，产品目录号为C009-2。下述实施例中的血清内谷草转氨酶测定试剂盒为南京建成科技有限公司产品，产品目录号为C010-2。
实施例1、毛木耳提取物(糖肽APPI和糖肽APPII)的制备
1、水提醇沉法提取毛木耳粗多糖
将新鲜的毛木耳子实体在20-25℃晾晒得到干燥的毛木耳子实体。将干燥的毛木耳子实体放入高速万能破碎机。反复破碎4次，每次20秒，制成100目的均一毛木耳子实体粉。向毛木耳子实体粉中加入是毛木耳子实体粉干重的25倍质量的去离子水，得到毛木耳子实体混合液。将该毛木耳子实体混合液在4℃的冰箱放置8小时后进行水浴。水浴前摇匀，封口，用水浴摇床对毛木耳子实体混合液进行90℃、4h的高温水浴。水浴后，对毛木耳子实体混合液进行6000g离心30min收集上清液(水溶性物质)，该上清液即为毛木耳子实体水提液。测定该上清液体积，向该上清液中加入是该上清液4倍体积的无水乙醇使多糖析出，搅拌均匀后盖上锡箔纸，静置12小时待固液分离，6000g离心20分钟收集沉淀，将沉淀放至60℃的烘箱中烘干直至质量恒定，研磨成粉末状,该粉末即为毛木耳粗多糖。
2、去除毛木耳粗多糖中的蛋白质
用Seveage法去除步骤1中的毛木耳粗多糖中的蛋白质得到去除蛋白的毛木耳粗多糖，具体方法如下：将步骤1中的毛木耳粗多糖溶于去离子水中，得到毛木耳粗多糖溶液。将是毛木耳粗多糖溶液1/3体积的Sevag试剂(由三氯甲烷和正丁醇按照4:1的体积比混合而成)加入毛木耳粗多糖溶液中，涡旋震荡5min，以4500g离心15min，吸取上清液，去除游离蛋白产生的凝胶状沉淀。向上清液中加入是上清液体积1/3的Sevag试剂涡旋震荡5min，以4500g离心15min，吸取上清液，如此重复多次直至获得无蛋白质层出现的上清液，该上清液即为去除蛋白的毛木耳粗多糖溶液。
3、去除蛋白的毛木耳粗多糖溶液的阴离子交换柱层析分离纯化
采用如下三种阴离子交换柱层析方法进行分离纯化。
3.1用10mM pH9.4 NH4HCO3-NH3·H2O缓冲溶液(溶质为10mM NH4HCO3和2.08mMNH3·H2O，溶剂为水)平衡DEAE-Cellulose层析柱(5×20cm，该层析柱的柱体积为100mL)，上样，样品为步骤2的去除蛋白的毛木耳粗多糖溶液，进行四步洗脱，流速均为1.5mL/min，部分收集器收集洗脱液，用硫酸苯酚法测定每管收集的洗脱液的多糖浓度。第一步洗脱用10mM pH9.4NH4HCO3-NH3·H2O缓冲溶液洗脱，收集第一步洗脱得到的洗脱峰(第一步洗脱出的所有液体)，将该洗脱峰定义为Da(即洗脱体积为0-224mL的洗脱液)；第二步洗脱用pH9.4的如下溶液洗脱：溶质是0.4M NaCl、溶剂是10mM pH9.4 NH4HCO3-NH3·H2O缓冲溶液，收集第二步洗脱得到的洗脱峰(第二步洗脱出的所有液体)，将该洗脱峰定义为Db(即洗脱体积为225mL-600mL的洗脱液)；第三步洗脱用pH9.4的如下溶液洗脱：溶质是0.8M NaCl、溶剂是10mM pH9.4NH4HCO3-NH3·H2O缓冲溶液，收集第三步洗脱得到的洗脱峰(第三步洗脱出的所有液体)，将该洗脱峰定义为Dc(即洗脱体积为601mL-900mL的洗脱液)；第四步洗脱用pH9.4的如下溶液洗脱：溶质是1.0M NaCl、溶剂是10mM pH9.4 NH4HCO3-NH3·H2O缓冲溶液，收集第四步洗脱得到的洗脱峰，第四步洗脱没有得到明显的多糖洗脱峰(图1)。
将该洗脱峰Da、Db和Dc分别在蒸馏水中透析10-12小时后，加入4倍体积无水乙醇静置12小时后，离心收集沉淀，将该沉淀60℃烘干，研磨成粉末状，分别得到Da组分、Db组分和Dc组分。
3.2用去离子水平衡DEAE-Cellulose层析柱(5×20cm，该层析柱的柱体积为100mL)，上样，样品为步骤2的去除蛋白的毛木耳粗多糖溶液，进行四步洗脱，流速均为1.5ml/min，部分收集器收集洗脱液，用硫酸苯酚法测定每管收集的洗脱液的多糖浓度。第一步洗脱用去离子水洗脱，收集第一步洗脱得到的洗脱峰(第一步洗脱出的所有液体)，将该洗脱峰定义为Dd(即洗脱体积为0-250mL的洗脱液)；第二步洗脱用如下溶液洗脱：溶质是0.2M NaCl、溶剂是水，收集第二步洗脱得到的洗脱峰(第二步洗脱出的所有液体)，将该洗脱峰定义为De(即洗脱体积为251mL-550mL的洗脱液)；第三步洗脱用如下溶液洗脱：溶质是0.5M NaCl、溶剂是水，收集第三步洗脱得到的洗脱峰(第三步洗脱出的所有液体)，第三步洗脱没有得到明显的多糖洗脱峰；第四步洗脱用如下溶液洗脱：溶质是1.0M NaCl、溶剂是水，收集第四步洗脱得到的洗脱峰(第四步洗脱出的所有液体)，第四步洗脱没有得到明显的多糖洗脱峰(图2)。
将该洗脱峰Dd和De分别在蒸馏水中透析10-12小时后，加入4倍体积无水乙醇静置12小时后，离心收集沉淀，将该沉淀60℃烘干，研磨成粉末状，分别得到Dd组分和De组分。
3.3用10mM pH7.0的磷酸缓冲溶液(溶质为3.9mM NaH2PO4和6.1mM Na2HPO4，溶剂为水)平衡DEAE-Cellulose层析柱(1.5×20cm，该层析柱的柱体积为30mL)，上样，样品为步骤2的去除蛋白的毛木耳粗多糖溶液，进行四步洗脱，流速均为1.0ml/min，部分收集器收集洗脱液，用硫酸苯酚法测定每管收集的洗脱液的多糖浓度。第一步洗脱用上述10mM pH7.0的磷酸缓冲溶液洗脱，收集第一步洗脱得到的洗脱峰(第一步洗脱出的所有液体)，将该洗脱峰定义为D1(即洗脱体积为0-135mL的洗脱液)；第二步洗脱用pH7.0的如下溶液洗脱：溶质是0.2M NaCl、溶剂是上述10mM pH7.0的磷酸缓冲溶液，收集第二步洗脱得到的洗脱峰(第二步洗脱出的所有液体)，将该洗脱峰定义为D2(即洗脱体积为136mL-255mL的洗脱液)；第三步洗脱用pH7.0的如下溶液洗脱：溶质是0.4M NaCl、溶剂是上述10mM pH7.0的磷酸缓冲溶液，收集第三步洗脱得到的洗脱峰(第三步洗脱出的所有液体)，第三步洗脱没有得到明显的多糖洗脱峰；第四步洗脱用如下溶液洗脱：溶质是1.0M NaCl、溶剂是上述10mMpH7.0的磷酸缓冲溶液，收集第四步洗脱得到的洗脱峰(第四步洗脱出的所有液体)，第四步洗脱没有得到明显的多糖洗脱峰(图3)。
将该洗脱峰D1和D2分别在蒸馏水中透析10-12小时后，加入4倍体积无水乙醇静置12小时后，离心收集沉淀，将该沉淀60℃烘干，研磨成粉末状，分别得到D1组分和D2组分。
4、洗脱组分活性初筛
4.1人受损肝细胞模型的建立
4.1.1细胞培养
在RPMI1640培养液中加入青霉素、链霉素混合液和胎牛血清(FBS)，使得青霉素和链霉素混合液在培养液中的最终体积百分含量为1％，FBS在培养液中的最终体积百分比含量为10％，即配制成含10％FBS的RPMI1640培养液。将对数生长期的L-O2细胞采用0.25％的胰蛋白酶进行消化并用含10％FBS的RPMI1640培养液收集重悬细胞，接种于96孔培养板中(每孔100μL，细胞密度为8×103个/孔)，置于温度为37℃，相对湿度为95％，CO2浓度为5％的培养箱中培养24h。
4.1.2建模诱导因子配置
向含10％FBS的RPMI1640培养液中加入无水乙醇和脂肪酸(该脂肪酸由油酸和棕榈酸组成)，配置成乙醇体积百分含量为1.8％、油酸含量为0.33mmol/L和棕榈酸的含量为0.17mmol/L培养液，将其称为含诱导因子培养液。
4.1.3诱导因子孵育得到人受损肝细胞模型
实验组：细胞培养结束后，吸除培养孔中的细胞培养液，加入200μL的4.1.2中的含诱导因子培养液，置于温度为37℃，相对湿度为95％，CO2浓度为5％的培养箱中继续培养48h，诱导因子孵育结束，得到的细胞为人受损肝细胞模型。实验设置三个复孔。
对照组：细胞培养结束后，吸除培养孔中的细胞培养液，加入200μL含10％FBS的RPMI1640培养液(乙醇的体积百分含量为0，油酸含量为0.0mmol/L和棕榈酸的含量为0.0mmol/L)，置于温度为37℃，相对湿度为95％，CO2浓度为5％的培养箱中继续培养48h后，作为对照组。实验设置三个复孔。
4.1.4MTT检测
诱导因子孵育结束后，将培养液吸除，每孔加入200μL MTT稀释液(MTT浓度为0.5mg/mL)，置于37℃,相对湿度为95％，CO2浓度为5％的培养箱中孵育4h。孵育结束后，吸除每孔中的液体，每孔加入200μL DMSO溶液，混合均匀，在荧光全波长酶标仪测定每孔的OD562值。
细胞存活率公式：细胞存活率(％)＝实验组的OD562值/对照组的OD562值×100％。
将对照组的细胞存活率设定为100％。实验结果表明，实验组的细胞存活率为52.67％，显微镜下观察发现细胞大量浮起，说明实验组中的细胞为人受损肝细胞模型。
4.2各组分对人受损肝细胞模型的细胞存活率的影响
将Da组分、Db组分、Dc组分、Dd组分、De组分、D1组分和D2组分用去离子水配置成以多糖浓度计为10mg/ml的溶液，采用对人受损肝细胞模型的细胞存活率的影响评价护肝功效。
实验设置对照组(用4.1.1的含10％FBS的RPMI1640培养液培养L-O2细胞)、模型组(用4.1.1的含10％FBS的RPMI1640培养液培养4.1的人受损肝细胞模型)、Da组分组(用含Da组分的培养液培养4.1的人受损肝细胞模型)、Db组分组(用含Db组分的培养液培养4.1的人受损肝细胞模型)、Dc组分组(用含Dc组分的培养液培养4.1的人受损肝细胞模型)、Dd组分组(用含Dd组分的培养液培养4.1的人受损肝细胞模型)、De组分组(用含De组分的培养液培养4.1的人受损肝细胞模型)、D1组分组(用含D1组分的培养液培养4.1的人受损肝细胞模型)、D2组分组(用含D2组分的培养液培养4.1的人受损肝细胞模型)。培养细胞24h后，利用4.1的MTT法检测细胞的存活率。设定对照组的细胞存活率为100％。实验设三次重复，每次重复设置三个复孔。试验所有数据采用SPSS12.0(SPSS Inc.，USA)统计软件的独立样本t检验处理统计。
其中，含Da组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入Da组分得到的Da组分含量以其所含的多糖含量计为2mg/ml的液体；含Db组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入Db组分得到的Db组分含量以其所含的多糖含量计为2mg/ml的液体；含Dc组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入Dc组分得到的Dc组分含量以其所含的多糖含量计为2mg/ml的液体；含Dd组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入Dd组分得到的Dd组分含量以其所含的多糖含量计为2mg/ml的液体；含De组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入De组分得到的De组分含量以其所含的多糖含量计为2mg/ml的液体；含D1组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入D1组分得到的D1组分含量以其所含的多糖含量计为2mg/ml的液体；含D2组分的培养液是向4.1.1的含10％FBS的RPMI1640培养液中加入D2组分得到的D2组分含量以其所含的多糖含量计为2mg/ml的液体。
结果如表1所示，在设定对照组细胞存活率为100％的条件下，与模型组相比较，Da组分组、Db组分组、Dc组分组、Dd组分组和De组分组的细胞存活率与模型组相比无显著差异，对照组、D1组分组和D2组分组的细胞存活率显著高于模型组。说明D1组分和D2组分含有活性多糖。
表1.各组的细胞存活率
| 组别 | 细胞存活率(％) | 组别 | 细胞存活率(％) |
| 对照组 | 100±2.36* | 模型组 | 52.67±6.32 |
| Da组分组 | 50.82±6.32 | Db组分组 | 60.12±5.82 |
| Dc组分组 | 63.49±4.03 | Dd组分组 | 61.38±2.04 |
| De组分组 | 52.76±4.69 | D1组分组 | 85.98±2.45* |
| D2组分组 | 81.74±1.36* |
注：“*”表示与模型组相比具有显著差异(P＜0.05)。
5、分子筛纯化D1组分和D2组分
D1组分经FPLC-Superdex 200凝胶过滤层析，洗脱液为pH 8.5 0.2M NH4HCO3溶液，层析柱规格30cm(柱长)×1cm(内径)，上样体积为0.2ml，流速为0.4mL/min。部分收集器收集洗脱液，收集多糖集中的洗脱峰(洗脱体积为14mL-23mL的洗脱液)，将该洗脱峰定义为APPI洗脱峰(图4)。
D2组分经FPLC-Superdex 200凝胶过滤层析，洗脱液为pH 8.5 0.2M NH4HCO3溶液，层析柱规格30cm(柱长)×1cm(内径)，上样体积为0.2ml，流速为0.4mL/min。部分收集器收集洗脱液，收集多糖集中的洗脱峰(洗脱体积为12mL-23mL的洗脱液)，将该洗脱峰定义为APPII洗脱峰(图5)。
6、超滤浓缩
将APPI洗脱峰在蒸馏水中透析10-12小时，在4℃进行截留分子量为5000道尔顿的超滤浓缩后，于-80℃条件下至完全冰冻，将冰冻样品冻干成粉备用，该干粉即为名称为APPI的糖肽(简称糖肽APPI)。
将APPII洗脱峰在蒸馏水中透析10-12小时，在4℃进行截留分子量为5000道尔顿的超滤浓缩后，于-80℃条件下至完全冰冻，将冰冻样品冻干成粉备用，该干粉即为名称为APPII的糖肽(简称糖肽APPII)。
糖肽APPI的提取率是16.31％(即100g步骤1的毛木耳粗多糖提取得到16.31g糖肽APPI)，糖肽APPII的提取率是49.46％(即100g步骤1的毛木耳粗多糖提取得到49.46g糖肽APPII)。
7、糖肽APPI和糖肽APPII的单糖组成和摩尔比测定
利用多糖糖醇醋酸盐衍生物的方法通过气相色谱-质谱联用技术对糖肽APPI和糖肽APPII的单糖组成和摩尔比进行测定。
以鼠李糖、葡萄糖、木糖、半乳糖、阿拉伯糖、甘露糖等单糖为标准样品进行标准曲线的制作，其后称取多糖样品5mg，以2mol/L的三氟乙酸(TFA)溶解，99℃水解5h，旋蒸除酸，加入4％的硼氢化钠溶液0.5mL，室温放置1.5h，滴加乙酸至无气泡产生，反复浓缩除酸。将浓缩至干的样品真空干燥，加吡啶及正丙胺各1mL，55℃水浴30min，真空干燥，加吡啶及乙酸酐各0.5mL，95℃水浴1h，氮气吹干，真空干燥后以氯仿溶解，进行GC-MS分析。GC-MS分析条件如下所列，色谱柱：DB-5(30m×0.25mm×0.25μm)；检测器：质谱检测器；进样口温度：250℃；检测器温度：280℃；氦气流速：0.6mL/min；分流比：20:1；进样量：5μL；升温程序：200℃保持2min，以3℃/min的速率升至245℃，再以10℃/min的速率升至270℃，保持2min。
结果表明糖肽APPI的单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖，阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖的摩尔比为1:38.3:46.2:15.4:4.4(图6)；糖肽APPII的单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成，阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖的摩尔比为1:71.5:99.2:10:5.1(图7)。
8、糖肽APPI和糖肽APPII的红外光谱(IR)分析
分别取1-2mg的糖肽APPI和糖肽APPII，利用KBr压片法进行制片，采用傅里叶变换红外光谱仪Nicolet iS5进行检测分析。
红外分析结果显示糖肽APPI和糖肽APPII具有-OH，羟基的伸缩振动吸收峰，C＝O，C-H吸收峰等明显的糖特征基团的结构，说明糖肽APPI和糖肽APPII具有多糖组分(图8和图9)。
9、糖肽APPI和糖肽APPII的N-端部分氨基酸序列测定
采用自动化EDMAN降解法对糖肽APPI和糖肽APPII的N-端部分氨基酸序列进行测定，用Hewlett Packard 1000A配有HPLC系统的蛋白序列测定仪测定N-端部分序列。结果显示APPI糖肽N-端氨基酸序列为DLYEVVEGEI(序列表中序列1)，糖肽APPII N-端氨基酸序列为VPSSMVVVVG(序列表中序列25)，说明糖肽APPI和糖肽APPII具有多肽结构。
10、糖肽APPI和糖肽APPII的内部氨基酸序列分析
将糖肽APPI和糖肽APPII的SDS-PAGE条带回收，送检到清华大学实验公共平台检测，结果显示糖肽APPI具有序列表中序列1-24所示的24个肽段；糖肽APPII具有序列表中序列25-63所示的39个肽段。分子特性分析说明糖肽APPI和糖肽APPII是既具有多糖结构又存在多肽结构的纯品。
11、糖肽APPI和糖肽APPII的分子量
凝胶渗透色谱(GPC)测定糖肽APPI和糖肽APPII的分子量，结果表明糖肽APPI的Mw(重均分子量)为9.213×105道尔顿，糖肽APPII的Mw(重均分子量)为6.340×105道尔顿。
实施例2、糖肽APPI和糖肽APPII保护受损肝细胞
1、对受损肝细胞存活率的影响
实验设置对照组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养L-O2细胞)、模型组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养实施例1中4.1的人受损肝细胞模型)、9μg/mL糖肽APPI组(9μg/mL组)(用含9μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、12μg/mL糖肽APPI组(12μg/mL组)(用含12μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、15μg/mL糖肽APPI组(15μg/mL组)(用含15μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、18μg/mL糖肽APPI组(18μg/mL组)(用含18μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、9μg/mL糖肽APPII组(9μg/mL组)(用含9μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)、12μg/mL糖肽APPII组(12μg/mL组)(用含12μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)、15μg/mL糖肽APPII组(15μg/mL组)(用含15μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)、18μg/mL糖肽APPII组(18μg/mL组)(用含18μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)。培养细胞24h后，利用实施例1中4.1的MTT法检测细胞的存活率。设定对照组的细胞存活率为100％。实验设三次重复，每次重复设置三个复孔。
其中，含9μg/mL糖肽APPI的培养液、含12μg/mL糖肽APPI的培养液、含15μg/mL糖肽APPI的培养液和含18μg/mL糖肽APPI的培养液分别是向实施例1中4.1.1的含10％FBS的RPMI1640培养液中加入糖肽APPI得到的糖肽APPI含量分别为9μg/mL、12μg/mL、15μg/mL和18μg/mL的液体；含9μg/mL糖肽APPII的培养液、含12μg/mL糖肽APPII的培养液、含15μg/mL糖肽APPII的培养液和含18μg/mL糖肽APPII的培养液分别是向实施例1中4.1.1的含10％FBS的RPMI1640培养液中加入糖肽APPII得到的糖肽APPII含量分别为9μg/mL、12μg/mL、15μg/mL和18μg/mL的液体。
结果如图10所示，在设定对照组细胞存活率为100％的条件下，与模型组相比较，糖肽APPI和糖肽APPII在9μg/mL和12μg/mL的浓度下、糖肽APPII在15μg/mL的浓度下、糖肽APPI在18μg/mL的浓度下可显著提高人受损肝细胞的存活率。糖肽APPI的最佳作用浓度为12μg/mL，糖肽APPII的最佳作用为15μg/mL。
2、糖肽APPI和糖肽APPII对肝细胞指标的影响
实验设置对照组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养L-O2细胞)、模型组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养实施例1中4.1的人受损肝细胞模型)、12μg/mL糖肽APPI组(用步骤1的含12μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、18μg/mL糖肽APPII组(用步骤1的含18μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)，培养细胞24h后，反复冻融法裂解细胞，按照组织细胞甘油三酯酶法测定试剂盒(北京普利莱基因技术有限公司)说明书检测胞内甘油三酯(TG)含量；按照总超氧化物歧化酶测试盒(南京建成生物工程研究所)说明书检测胞内SOD活性。同时采用BCA蛋白定量试剂盒(北京博迈德生物技术有限公司产品)测定样品中的蛋白含量，具体操作参见说明书。实验设三次重复，每次重复设置三个复孔。
实验设置对照组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养L-O2细胞)、模型组(用实施例1中4.1.1的含10％FBS的RPMI1640培养液培养实施例1中4.1的人受损肝细胞模型)、12μg/mL糖肽APPI组(用步骤1的含12μg/mL糖肽APPI的培养液培养施例1中4.1的人受损肝细胞模型)、18μg/mL糖肽APPII组(用步骤1的含18μg/mL糖肽APPII的培养液培养施例1中4.1的人受损肝细胞模型)，培养细胞24h后，收集细胞培养液，按照谷草转氨酶和谷丙转氨酶测试盒(南京建成生物工程研究所，产品目录号为C009-2和C010-2)说明书检测胞外转氨酶的含量。试验所有数据采用SPSS12.0(SPSS Inc.，USA)统计软件的独立样本t检验处理统计。
结果如表2所示，12μg/mL糖肽APPI组和18μg/mL糖肽APPII组与模型组相比，均显著降低受损肝细胞内的甘油三酯含量，显著提高受损肝细胞的SOD酶活性，显著降低受损肝细胞的谷丙转氨酶(ALT)的释放量，显著降低受损肝细胞的谷草转氨酶(AST)的释放量。说明糖肽APPI和糖肽APPII均能显著降低受损肝细胞内的甘油三酯含量，显著提高受损肝细胞的SOD酶活性，显著降低受损肝细胞的谷丙转氨酶(ALT)的释放量，显著降低受损肝细胞的谷草转氨酶(AST)的释放量。
表2.APPI和APPII对肝细胞的保护作用
注：“*”表示与模型组相比具有显著差异(P＜0.05)。
3、糖肽APPI和糖肽APPII体内护肝消脂功能研究
220g-280g雄性Wistar大鼠共90只，随机分为9组，每组10只，即正常组、模型组、APPI(50mg/kg/d)组、APPI(100mg/kg/d)组、APPII(50mg/kg/d)组、APPII(100mg/kg/d)组、毛木耳粗多糖(613mg/kg/d)组、毛木耳粗多糖(202mg/kg/d)组和辛伐他汀组。除正常组给予基础饲料外，其余各组均给予高脂饲料。其中，基础饲料购自北京华阜康生物科技股份有限公司。高脂饲料组成为：在该基础饲料中添加2％基础饲料质量的胆固醇、25％基础饲料质量的猪油、0.05％基础饲料质量的胆酸盐和10％基础饲料质量的蛋黄粉，得到高脂饲料。
采取大鼠自由取食的方式，饮水不限。60天后，根据各组的药物剂量进行灌胃给药，药物处理4周后，检测动物血脂中各项指标(血清总胆固醇(TC)，血清甘油三酯(TG)，血清高密度脂蛋白胆固醇(HDL-C)，血清低密度脂蛋白胆固醇(LDL-C))和转氨酶(谷丙转氨酶(ALT)和谷草转氨酶(AST))的变化，通过肝脏组织的病理切片分析糖肽的护肝消脂作用。试验所有数据采用SPSS12.0(SPSS Inc.，USA)统计软件的独立样本t检验处理统计。其中，各组的药物剂量如下：
正常组：喂以基础饲料，不给药，只给予等体积的生理盐水.
模型组：喂以高脂饲料，不给药，只给予等体积的生理盐水。
APPI(50mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的糖肽APPI的剂量为50mg/kg体重/天，糖肽APPI溶于生理盐水后给药。
APPI(100mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的糖肽APPI的剂量为100mg/kg体重/天，糖肽APPI溶于生理盐水后给药。
APPII(50mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的糖肽APPII的剂量为50mg/kg体重/天，糖肽APPII溶于生理盐水后给药。
APPII(100mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的糖肽APPII的剂量为100mg/kg体重/天，糖肽APPII溶于生理盐水后给药。
毛木耳粗多糖(613mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的毛木耳粗多糖的剂量为613mg/kg体重/天，毛木耳粗多糖溶于生理盐水后给药。613mg毛木耳粗多糖含有100mg的糖肽APPI。
毛木耳粗多糖(202mg/kg/d)组：喂以高脂饲料，每天每只大鼠给予实施例1的毛木耳粗多糖的剂量为202mg/kg体重/天，毛木耳粗多糖溶于生理盐水后给药。202mg毛木耳粗多糖含有100mg的糖肽APPII。
辛伐他汀组：喂以高脂饲料，每天每只大鼠给予辛伐他汀的剂量为2mg/kg体重/天，辛伐他汀溶于生理盐水后给药。
结果如表3所示，表明模型组的血清总胆固醇(TC)含量、血清甘油三酯(TG)含量和血清低密度脂蛋白胆固醇(LDL-C)含量均显著高于正常组(p＜0.05)，模型组的血清高密度脂蛋白胆固醇(HDL-C)含量显著低于正常组(p＜0.05)；说明模型组为高血脂模型。与模型组相比，糖肽APPI和糖肽APPII可以显著降低脂肪肝动物血清中TG、TC、LDL-C、AST和ALT的水平，糖肽APPI显著提高了血清高密度脂蛋白胆固醇(HDL-C)含量，说明糖肽APPI和糖肽APPII都具有保护肝脏的作用，毛木耳粗多糖的作用效果相对较低，虽然在血脂指标中呈现下降趋势，但在脂肪肝动物血清中TG含量、HDL-C含量、AST含量和ALT含量方面没有显著差异，效果显著低于糖肽APPI和糖肽APPII。
表3.糖肽APPI和糖肽APPII对血清中肝脏指标的保护作用
注：“*”表示与模型组相比具有显著差异(P＜0.05)。
从HE病理切片可见，正常组肝细胞，肝小叶呈多边形，索状整齐排列，细胞核明显(图11中A)。而模型组细胞呈现严重水肿，胞质内充满大小不等却清亮的脂滴；部分细胞核固缩、溶解并消失，细胞坏死；肝窦扩张、淤血，局部枯否氏细胞增生；门管区纤维组织增生及炎细胞浸润，说明伴有轻度肝炎(图11中B)。经过糖肽APPI和糖肽APPII给药后，肝细胞内脂滴显著减少，肝窦缩小，炎症症状明显缓解(图11中C-F)。而毛木耳粗多糖的切片显示，细胞中脂滴虽然较模型组比较有所减少，但是依然存在较为清亮的油滴，而且仍然伴随炎症反应，作用效果显著低于糖肽APPI和糖肽APPII(图11中G和H)。
实施例1和实施例2的实验结果表明糖肽APPI和糖肽APPII既存在糖结构，又存在肽段结构，是一个糖肽纯品；通过体内和体外研究表明糖肽APPI和APPII都具有减少脂肪在肝内堆积，消脂护肝的功能。
SEQUENCE LISTING
<110> 北京市农林科学院
<120> 具有消脂护肝功能的毛木耳糖肽及其应用
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<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 18
Leu Ile Ser Trp Tyr Asp Asn Glu Phe Gly Tyr Ser Asn Arg
1 5 10
<210> 19
<211> 11
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 19
Ile Gly Gly Ile Gly Thr Val Pro Val Gly Arg
1 5 10
<210> 20
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 20
Gly Ile Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg
1 5 10
<210> 21
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 21
Val Ile Pro Gln Phe Met Ile Gln Gly Gly Asp Phe Thr Lys
1 5 10
<210> 22
<211> 6
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 22
Cys Ala Tyr Gly Asp Thr
1 5
<210> 23
<211> 24
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 23
Val Val Met Glu Thr Ala His Trp Ser Ser Ser Asp Glu His Met Thr
1 5 10 15
Trp Leu Ala Ser Glu Glu Tyr Lys
20
<210> 24
<211> 6
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 24
Cys Ala Tyr Gly Glu Ser
1 5
<210> 25
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 25
Val Pro Ser Ser Met Val Val Val Val Gly
1 5 10
<210> 26
<211> 17
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 26
His Gln Thr Ser Gly Asp Gln Val Thr Ser Ser Thr Gln His Ser Phe
1 5 10 15
Arg
<210> 27
<211> 15
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 27
Val Gln Asn Val Gly Asn Gly Val Leu Leu Gly Phe His Gly Arg
1 5 10 15
<210> 28
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 28
Asp Thr Pro Asn Thr Leu Phe Ser Leu Gly Ser Ile Phe Arg
1 5 10
<210> 29
<211> 18
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 29
Glu Leu Ala Thr Gly Gln Asn Gly Phe Gly Tyr Ala Gly Ser Ser Phe
1 5 10 15
His Arg
<210> 30
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 30
Leu Ala Val Asn Leu Ile Pro Phe Pro Arg
1 5 10
<210> 31
<211> 19
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 31
Gly His Tyr Thr Glu Gly Ala Glu Leu Ile Asp Ser Val Leu Asp Val
1 5 10 15
Val Arg Lys
<210> 32
<211> 12
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 32
Ile Asn Val Tyr Phe Asn Glu Ala Thr Gly Gly Arg
1 5 10
<210> 33
<211> 16
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 33
Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys
1 5 10 15
<210> 34
<211> 13
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 34
Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg
1 5 10
<210> 35
<211> 18
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 35
Asn Tyr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val Gly Ala Ser
1 5 10 15
Asp Arg
<210> 36
<211> 21
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 36
Asn Tyr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val Gly Ala Ser
1 5 10 15
Asp Arg Tyr Asp Arg
20
<210> 37
<211> 18
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 37
Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Ile Asn
1 5 10 15
Pro Lys
<210> 38
<211> 16
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 38
Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg
1 5 10 15
<210> 39
<211> 25
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 39
Gly Tyr Ala Asp Trp His Glu Ala Gly Val Phe Ser Asp Glu Gln Ile
1 5 10 15
Asn Gly Ile Gly Asn Ile Ala Gly Arg
20 25
<210> 40
<211> 13
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 40
Ser Thr Tyr Phe Ser Ser Ser Ser Pro Ser Phe Ile Lys
1 5 10
<210> 41
<211> 13
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 41
Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu Arg
1 5 10
<210> 42
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 42
Phe Glu Glu Leu Cys Ser Asp Leu Phe Arg
1 5 10
<210> 43
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 43
His Thr Lys Pro Gly Leu Leu Ser Met Ala Asn Ala Gly Lys
1 5 10
<210> 44
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 44
Val Ile Pro Gln Phe Met Leu Gln Gly Gly Asp Phe Thr Lys
1 5 10
<210> 45
<211> 18
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 45
Ala Ile Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val
1 5 10 15
Gly Lys
<210> 46
<211> 23
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 46
Asp Val Ser Gly Gly His Thr Leu Gln His Gly Glu Ser Tyr Ala Glu
1 5 10 15
Ala Phe Ala Val Asp Pro Lys
20
<210> 47
<211> 21
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 47
Phe Asp Gly Ala Leu Asn Val Asp Leu Thr Glu Phe Gln Thr Asn Leu
1 5 10 15
Val Pro Tyr Pro Arg
20
<210> 48
<211> 17
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 48
Glu Ala Asp Val Asp Gly Asp Gly Gln Ile Asn Tyr Glu Glu Phe Val
1 5 10 15
Lys
<210> 49
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 49
Val Pro Thr Val Asp Val Ser Val Val Asp Leu Thr Val Arg
1 5 10
<210> 50
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 50
Leu Ile Ala Gln Val Ile Ser Ser Ile Thr Ala Ser Leu Arg
1 5 10
<210> 51
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 51
Lys Glu Ser Thr Leu His Leu Val Leu Arg
1 5 10
<210> 52
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 52
Leu Ile Ser Trp Tyr Asp Asn Glu Phe Gly Tyr Ser Asn Arg
1 5 10
<210> 53
<211> 16
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 53
Phe Ile Gly Ser Asn Val Leu Thr Asn Glu Leu Gly Pro Asp Val Lys
1 5 10 15
<210> 54
<211> 9
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 54
Lys Ile Asn Asp Leu Asn Leu Lys Lys
1 5
<210> 55
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 55
Leu Gly Ile His Glu Asp Ser Gln Asn Arg
1 5 10
<210> 56
<211> 10
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 56
Phe Glu Glu Leu Asn Ala Asp Leu Phe Arg
1 5 10
<210> 57
<211> 12
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 57
Ala Ile Ala Ala Tyr Gly Pro Val Asp Trp Ser Arg
1 5 10
<210> 58
<211> 11
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 58
Ile Gly Leu Phe Gly Gly Ala Gly Val Gly Lys
1 5 10
<210> 59
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 59
Gly Thr Pro Ser Ser Tyr Ile Asp Asn Leu Thr Phe Pro Lys
1 5 10
<210> 60
<211> 11
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 60
Ile Val Val Ile Gly His Val Asp Ser Gly Lys
1 5 10
<210> 61
<211> 9
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 61
Gly Asp Phe Asn Gln His Tyr Val Arg
1 5
<210> 62
<211> 14
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 62
His Gly His Phe Arg His Arg Ser Ser Ser Ser Thr Pro Arg
1 5 10
<210> 63
<211> 36
<212> PRT
<213> 毛木耳（Auricularia polytricha (Mont.) Sacc.）
<400> 63
Gly Gln Ala Lys Arg Ile Glu Ser Ser Pro Val Leu Ala Ser Tyr Gln
1 5 10 15
Asn Pro Leu Tyr Ile Asp Ser His Thr Gly Tyr Ser Asp Glu Glu Ser
20 25 30
Glu Asp Glu Arg
35
Claims (5)
1.毛木耳提取物，其特征在于：所述毛木耳提取物是从毛木耳中提取出的名称为APPII的糖肽；
所述APPII的N末端序列如序列表中序列25所示，所述APPII具有序列表中序列25-63所示的39个肽段，所述APPII单糖组成为阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖，阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖的摩尔比为1:72:99:10:5，所述APPII的重均分子量为6.340×105道尔顿；
所述APPII按照包括如下步骤的方法制备：
II-1）制备名称为去除蛋白的毛木耳粗多糖，所述去除蛋白的毛木耳粗多糖的制备方法包括用乙醇沉淀毛木耳子实体水提液，收集沉淀，去除所述沉淀中的蛋白后得到所述去除蛋白的毛木耳粗多糖；所述毛木耳子实体水提液是用水从毛木耳子实体中提取出来的水溶性物质；
II-2）从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽，得到名称为APPII的糖肽；
所述II-2）中，从所述毛木耳粗多糖中分离纯化糖肽包括：
II-2-1）对所述去除蛋白的毛木耳粗多糖进行阴离子交换柱层析，得到名称为D2组分的分离物，所述D2组分为第二步洗脱得到的洗脱峰；所述阴离子交换柱层析中所采用的阴离子交换基团是DEAE，所采用的洗脱程序为两步洗脱，第一步洗脱用pH为6.8-7.5的磷酸缓冲溶液A进行洗脱，所述磷酸缓冲溶液A的溶质为3.5-4.5 mM NaH2PO4和5.5-6.5mMNa2HPO4，溶剂为水；第二步洗脱用pH6.8-7.5的如下溶液洗脱：溶质是0.2M NaCl，溶剂是所述磷酸缓冲溶液A；
II-2-2）对所述D2组分进行分子筛层析，得到重均分子量为6.340×105道尔顿的糖肽，该糖肽即为APPII。
2.根据权利要求1所述的毛木耳提取物，其特征在于：所述毛木耳提取物具有以下至少一种功能：
A1）降低脂肪肝动物血清中血清总胆固醇的含量；
A2）降低脂肪肝动物血清中血清甘油三酯的含量；
A3）降低脂肪肝动物血清中血清低密度脂蛋白胆固醇的含量；
A4）降低脂肪肝动物血清中谷丙转氨酶的含量；
A5）降低脂肪肝动物血清中谷草转氨酶的含量；
A6）使脂肪肝动物肝细胞内脂滴减少；
A7）使脂肪肝动物肝细胞炎症降低；
B1）提高受损肝细胞的存活率；
B2）提高受损肝细胞的SOD酶活性；
B3）降低受损肝细胞内的甘油三酯含量；
B4）降低受损肝细胞的谷丙转氨酶的释放量；
B5）降低受损肝细胞的谷草转氨酶的释放量。
3.毛木耳提取物的制备方法，其特征在于：所述毛木耳提取物是权利要求1或2所述的名称为APPII的糖肽；
所述APPII的制备方法包括：
II-1）制备名称为去除蛋白的毛木耳粗多糖，所述去除蛋白的毛木耳粗多糖的制备方法包括用乙醇沉淀毛木耳子实体水提液，收集沉淀，去除所述沉淀中的蛋白后得到所述去除蛋白的毛木耳粗多糖；所述毛木耳子实体水提液是用水从毛木耳子实体中提取出来的水溶性物质；
II-2）从所述去除蛋白的毛木耳粗多糖中分离纯化糖肽，得到名称为APPII的糖肽；
所述II-2）中，从所述毛木耳粗多糖中分离纯化糖肽包括：
II-2-1）对所述去除蛋白的毛木耳粗多糖进行阴离子交换柱层析，得到名称为D2组分的分离物，所述D2组分为第二步洗脱得到的洗脱峰；所述阴离子交换柱层析中所采用的阴离子交换基团是DEAE，所采用的洗脱程序为两步洗脱，第一步洗脱用pH为6.8-7.5的磷酸缓冲溶液A进行洗脱，所述磷酸缓冲溶液A的溶质为3.5-4.5 mM NaH2PO4和5.5-6.5mMNa2HPO4，溶剂为水；第二步洗脱用pH6.8-7.5的如下溶液洗脱：溶质是0.2M NaCl，溶剂是所述磷酸缓冲溶液A；
II-2-2）对所述D2组分进行分子筛层析，得到重均分子量为6.340×105道尔顿的糖肽，该糖肽即为APPII。
4.权利要求1或2所述的毛木耳提取物在制备含有权利要求1或2所述的毛木耳提取物的产品中的应用，所述产品为消脂护肝的产品或保护受损肝细胞的产品。
5.根据权利要求4所述的应用，其特征在于：所述消脂护肝的产品具有下述A1）-A7）中的7种、6种、5种、4种、3种、2种或1种功能：
A1）降低脂肪肝动物血清中血清总胆固醇(TC)的含量；
A2）降低脂肪肝动物血清中血清甘油三酯(TG)的含量；
A3）降低脂肪肝动物血清中血清低密度脂蛋白胆固醇(LDL-C)的含量；
A4）降低脂肪肝动物血清中谷丙转氨酶（ALT）的含量；
A5）降低脂肪肝动物血清中谷草转氨酶（AST）的含量；
A6）使脂肪肝动物肝细胞内脂滴减少；
A7）使脂肪肝动物肝细胞炎症降低；
所述保护受损肝细胞的产品具有下述B1）-B5）中的5种、4种、3种、2种或1种功能：
B1）提高受损肝细胞的存活率；
B2）提高受损肝细胞的SOD酶活性；
B3）降低受损肝细胞内的甘油三酯含量；
B4）降低受损肝细胞的谷丙转氨酶的释放量；
B5）降低受损肝细胞的谷草转氨酶的释放量。
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| CN108727474A (zh) | 2018-11-02 |
| CN108727474B (zh) | 2020-07-28 |
| CN111704658A (zh) | 2020-09-25 |
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