CN111019838B - 一株内生真菌及其提取物与用途 - Google Patents
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Abstract
本发明公开了一株内生真菌及其提取物与用途。所述的真菌为马来西亚沉香内生真菌，命名为peniophora sp.A871，该菌株A871的保藏编号为GDMCC NO：60911。本发明通过实验发现，从peniophora sp.A871发酵培养物中分离得到的提取物具有明显的清除DPPH自由基的活性，该peniophora sp.A871提取物对DPPH自由基的清除能力与维生素C相当，能用于制备DPPH自由基清除剂，因此，peniophora sp.A871提取物在制备抗氧化药品、保健品及护肤品中具有广阔的应用开发前景。
Description
技术领域
本发明属于生物医药技术领域，具体涉及马来西亚沉香内生真菌peniophorasp.A871及从该菌株发酵液中制备的提取物和该提取物在制备DPPH自由基清除剂中的应用。
背景技术
自由基和人类多种疾病有着密切的关系，如果体内自由基过多，则可引起蛋白质、核酸(DNA)变性，导致细胞和组织器官损伤，诱发各种疾病，加速机体衰老。同时，自由基过量产生也是导致皮肤自然衰老和光致老化的主要原因，减少自由基的产生和清除老化代谢产物已成为目前延缓人体和皮肤衰老的有效方法。由于在药品、保健食品和护肤品中使用的合成抗氧化剂通常具有毒副作用，因此，寻找天然、高效、低毒的抗氧化剂成为了一种必然趋势。
植物内生真菌(endophytic fungi)是指生活史的一定阶段或全部阶段生活在健康植物组织内，但不对植物组织引起明显病害症状的真菌(Tan RX and Zou WX,2001)。内生真菌具有丰富的物种多样性和遗传多样性，迄今已从植物内生真菌中发现了不少新的或稀有的真菌物种，已成为国内外的研究热点。植物内生真菌生长于植物内部的特殊环境中，其产生活性化合物远远超出其植物代谢产物的范围，能够产生各种结构类型独特、具有潜在应用前景的活性物质，已成为发现新的天然活性物质的重要资源。
发明内容
本发明的第一个目的是提供一株内生真菌peniophora sp.A871，该菌株于2019年12月5日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC NO：60911。
本发明的第二个目的是提供一种具有清除DPPH自由基作用的内生真菌peniophora sp.A871提取物的制备方法，该制备方法是以上述的内生真菌peniophorasp.A871作为发酵菌株，液体发酵获得发酵培养物，分离菌丝体和发酵液，取发酵液用乙酸乙酯萃取，萃取液经浓缩后即得内生真菌peniophora sp.A871提取物。
优选，所述的液体发酵是将内生真菌peniophora sp.A871接种到发酵培养基中，于28℃、120r/min液体发酵培养，得到发酵培养物，所述的发酵培养基，每升是通过以下方法制备：用蒸馏水煮200g马铃薯20min，过滤得汁，再加入葡萄糖20g、KH2PO4 3g、MgSO40.75g、维生素B1 10mg，用水补足至1L，pH自然，得到发酵培养基。
本发明的第三个目的是提供一种由上述制备方法制得的内生真菌peniophorasp.A871提取物。
本发明的第四个目的是提供所述的内生真菌peniophora sp.A871提取物在制备DPPH自由基清除剂中的应用。
一种DPPH自由基清除剂，是以上述的内生真菌peniophora sp.A871提取物作为活性成分。
一种抗氧化药品、保健品或护肤品，含有上述的DPPH自由基清除剂。
本发明通过实验发现，该内生真菌peniophora sp.A871提取物在500μg/mL浓度下对DPPH自由基的清除率为96.31％，同样浓度下，阳性对照药物维生素C对DPPH自由基的清除率为99.67％。选择200mg/mL、100mg/mL、50mg/mL、25mg/mL、12.5mg/mL、6.25mg/mL、3.15mg/mL 7个作用浓度下对DPPH自由基进行最低抑制浓度测定，确定该内生真菌peniophora sp.A871提取物的DPPH自由基清除的IC50值为25.19，阳性对照药物维生素C对DPPH自由基清除的IC50值为12.06，清除能力与维生素C相当。实验结果表明：内生真菌peniophora sp.A871提取物具有显著清除DPPH自由基作用，能用于制备DPPH自由清除剂。因此，内生真菌peniophora sp.A871提取物在制备抗氧化药品、保健品及护肤品中具有广阔的应用前景。
本发明的内生真菌peniophora sp.A871，于2019年12月5日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，保藏编号为：GDMCCNO：60911。
附图说明
图1为本发明的内生真菌peniophora sp.A871菌株与相关菌株的系统发育树，其中A871代表peniophora sp.A871。
具体实施方式
根据下述实施例，可以使本领域的技术人员更好地理解本发明。实施例所描述的仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：菌株A871(peniophora sp.)的分离、纯化及鉴定
(1)菌株的分离、纯化：
将采自马来西亚沉香树的叶用自来水冲洗干净，置于滤纸上自然风干。放入0.1％(m/v g/ml)的升汞溶液中浸泡7min后用无菌水漂洗4次，再放入体积分数75％的乙醇水溶液中浸泡3～5min后用无菌水漂洗3次。用剪刀剪成约1cm2大小。置于分离培养基(含20μg/mL硫酸卡那霉素和20μg/mL氨苄青霉素)中，28℃恒温箱培养7d后挑取边缘菌丝转接到新鲜的分离培养基上，继续纯化直至获得纯菌株，即得本发明的菌株A871。
所述的分离培养基是通过以下方法获得：用蒸馏水煮200g马铃薯20min，过滤得汁，再加入葡萄糖20g、KH2PO4 3g、MgSO4 0.75g、维生素B1 10mg，琼脂20g，用水补足至1L，pH自然，灭菌备用。
(2)菌株的鉴定：
将所述的菌株A871采用真菌基因组DNA提取试剂盒提取菌株的基因组DNA，作为PCR扩增的模板。PCR扩增采用真菌rDNA内转录间隔区(rDNAITS)通用引物ITS1(5'-TCCGTAGGTGAACCTGCGG-3'，正向)和ITS4(5'-TCCTCCGCTTATTGATATGC-3'，反向)扩增分离菌株的rDNA ITS区。PCR采用20μL反应体系，通过Ex Taq(TaKa-Ra)进行。反应条件：93℃预变性3min；93℃变性45s，55℃复性45s，72℃延伸1.5min，共30个循环；最后72℃延伸10min。PCR产物由上海美吉生物医药科技有限公司广州分公司进行测序，其序列如SEQ ID NO.1所示。获得的序列通过BLAST程序在GenBank上进行相似性序列检索，下载相关序列，运用MEGA5软件通过邻接法(Neighbor-Joining)，Bootstrap设置为1000次重复，构建系统发育树(图1)。菌株A871 ITS区的碱基序列如SEQ ID NO.1所示。将测序获得的序列信息进行BLAST，BLAST结果显示，菌株A871与peniophora sp.(KM246215)的相似度为99.02％。从构建的系统发育树看，菌株A871与peniophora sp.(KM246215)序列聚为高度自展支持的一支，彼此间遗传距离极小(图1)。因此，通过分子系统学分析确定该菌株为peniophora sp.，命名为peniophora sp.A871。
该菌株peniophora sp.A871，于2019年12月5日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC NO：60911。
实施例2：菌株A871(peniophora sp.)的活化、发酵培养及提取物制备
(1)将实施例1得到的纯菌株A871接种于斜面培养基，于28℃培养5d，得到活化后的菌种。所述斜面培养基通过以下方法获得：用蒸馏水煮200g马铃薯20min，过滤得汁，再加入葡萄糖20g、KH2PO4 3g、MgSO4 0.75g、维生素B1 10mg，琼脂20g，用水补足至1L，pH自然，灭菌备用。
(2)将步骤(1)活化培养后的菌株A871接种至种子培养基中，于28℃、120r/min振荡条件下培养7d，得到种子液，所述种子培养基通过以下方法获得：用蒸馏水煮200g马铃薯20min，过滤得汁，再加入葡萄糖20g、KH2PO4 3g、MgSO4 0.75g、维生素B1 10mg，用水补足至1L，pH自然，灭菌备用。
(3)将步骤(2)所得种子液以10％(v/v)的接种量接种到发酵培养基中，于28℃、120r/min培养7d，得到发酵培养物。所述的发酵培养基与种子培养基相同。
(4)提取物制备：将步骤(3)所得的发酵培养物除菌丝体后得到的滤液用乙酸乙酯萃取3次，萃取液经减压浓缩后即为本发明的内生真菌peniophora sp.A871提取物。
实施例3：内生真菌peniophora sp.A871提取物清除DPPH自由基能力的测定
将实施例2获得的peniophora sp.A871提取物和维生素C用无水乙醇分别配成500μg/mL溶液，DPPH用无水乙醇配成0.2mmoL/L溶液。吸取100μL peniophora sp.A871提取物溶液于96孔板中，加入100μLDPPH溶液作为样品组，以无水乙醇作为空白对照组，以无水乙醇代替DPPH溶液作为样品背景组，以无水乙醇代替peniophora sp.A871提取物溶液作为阴性对照组，以维生素C代替peniophora sp.A871提取物溶液作为阳性对照组，置于30℃培养箱中反应30min，用酶标仪测定每孔517nm处吸光值(A)，按以下公式计算DPPH自由基的清除率％：
清除率％＝[1-(A样品/阳性-A样品背景)/(A阴性对照-A空白对照)]×100％
每个样品3个重复。实验结果表明：在相同浓度条件下，peniophora sp.A871提取物对DPPH自由基清除率为96.31％，维生素C对DPPH自由基清除率为99.67％。
实施例4：内生真菌peniophora sp.A871提取物对DPPH自由基的半数清除浓度(IC50值)测定
将实施例3的peniophora sp.A871提取物溶液(500μg/mL)和维生素C溶液(500μg/mL)用无水乙醇分别稀释至200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL，3.15μg/mL，按实施例3的方法测定各浓度的peniophora sp.A871提取物溶液和维生素C溶液对DPPH自由基的清除率，采用SigmaPolot 14.0软件计算IC50值。每个样品3个重复。
实验结果表明：peniophora sp.A871提取物对DPPH自由基清除的IC50值为25.19，阳性对照药物维生素C对DPPH自由基清除的IC50值为12.06。从实验结果可以看出，peniophora sp.A871提取物具有明显的清除DPPH自由基的能力，其清除能力与维生素C相当，能用于制备DPPH自由基清除剂，因此，peniophora sp.A871提取物在制备抗氧化药品、保健品及护肤品中具有广阔的应用开发前景。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东省微生物研究所（广东省微生物分析检测中心）
<120> 一株内生真菌及其提取物与用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 320
<212> DNA
<213> 内生真菌A871(peniophora sp.A871)
<400> 1
gatggcgggg tcggatgcct gtccggtgct gagctgccca gcgatgggat gtgctcgtcc 60
gggccggtgt cccttcacta ttccacccca ctgtgaaccg agcgtgtgag cggaagagag 120
atcggaagct cgcatgcaca ctttaacata cccctaatga agtatcagaa tgtaccttgc 180
gttaactcgc acatatacaa ctttcaacaa cggatctctt ggctctcgca tcgatgaaga 240
acgcagcgaa atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg 300
aacgcacctt gcgccccttg 320
Claims (4)
1.一株内生真菌peniophora sp.A871，其保藏编号为：GDMCC NO：60911。
2.一种具有清除DPPH自由基作用的内生真菌peniophora sp.A871提取物的制备方法，其特征在于，以权利要求1所述的内生真菌peniophora sp.A871作为发酵菌株，液体发酵获得发酵培养物，分离菌丝体和发酵液，取发酵液用乙酸乙酯萃取，萃取液经浓缩后即得内生真菌peniophora sp.A871提取物。
3.根据权利要求2所述的制备方法，其特征在于，所述的液体发酵是将内生真菌peniophora sp.A871接种到发酵培养基中，于28℃、120r/min液体发酵培养，得到发酵培养物，所述的发酵培养基，每升是通过以下方法制备：用蒸馏水煮200g马铃薯20min，过滤得汁，再加入葡萄糖20g、KH2PO4 3g、MgSO4 0.75g、维生素B1 10mg，用水补足至1L，pH自然，得到发酵培养基。
4.权利要求2或3所述的制备方法制得的内生真菌peniophora sp.A871提取物在制备DPPH自由基清除剂中的应用。
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