CN112553088A - 内生真菌a871提取物及其制备方法和在制备治疗痛风药物中的应用 - Google Patents
内生真菌a871提取物及其制备方法和在制备治疗痛风药物中的应用 Download PDFInfo
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Abstract
本发明公开了内生真菌Peniophora sp.A871提取物在制备治疗痛风药物中的应用。以内生真菌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。本发明的Peniophora sp.A871提取物具有明显的抑制黄嘌呤氧化酶作用，表明该提取物具有很好的应用开发前景，因此本发明为研究与开发新的治疗痛风药物提供了物质基础，为开发利用植物内生真菌来源的天然活性物质提供了科学依据。
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提取物及其在制备治疗痛风药物中的应用。
背景技术
痛风是一种常见且复杂的关节炎类型，是由嘌呤合成代谢异常或尿酸排泄障碍导致的尿酸产生过多而引起体内血液尿酸水平升高的一种疾病，大脚趾为最常发病部位，手部的关节、膝盖、肘部等也是常见的发病部位。痛风患者关节部位出现严重的疼痛、水肿、红肿和炎症，经常会在夜晚出现突然性的关节疼，患者会在半夜熟睡中疼醒，成为威胁人类健康的严重代谢性疾病。
黄嘌呤氧化酶是一种重要的嘌呤代谢酶，可催化次黄嘌呤转化为黄嘌呤，并进一步催化后者氧化产生尿酸。黄嘌呤抑制剂可降低体内该酶的活性，减少尿酸生成。目前，临床中常用的黄嘌呤氧化酶抑制剂为别嘌醇，但别嘌醇存在如肝损伤、肾损伤等较为明显的副作用，因此，寻找天然、高效、低毒的黄嘌呤氧化酶抑制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
本发明的目的是提供内生真菌Peniophora sp.A871提取物及其制备方法和在制备治疗痛风药物中的应用。
本发明的内生真菌Peniophora sp.A871提取物是通过以下制备方法制备的：以内生真菌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。
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。
本发明的第二个目的是提供内生真菌Peniophora sp.A871提取物在制备治疗痛风的药物中的应用。
所述的治疗痛风的药物优选为对黄嘌呤氧化酶有抑制作用的药物。
本发明的第三个目的是提供一种抑制黄嘌呤氧化酶抑制剂，是以上述的内生真菌Peniophora sp.A871提取物作为活性成分。
一种治疗痛风药物，含有上述的黄嘌呤氧化酶抑制剂。
本发明通过实验发现，该内生真菌Peniophora sp.A871提取物在500μg/mL浓度下对黄嘌呤氧化酶抑制率为90.23％，同样浓度下，阳性对照药物别嘌醇对黄嘌呤氧化酶抑制率为95.62％，抑制效果与药物别嘌醇相当。因此，本发明的内生真菌Peniophora sp.A871提取物具有显著抑制黄嘌呤氧化酶作用，能用于制备治疗痛风药物，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。
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.)的活化、发酵培养及提取物制备
(1)将内生真菌Peniophora sp.A871接种于斜面培养基，于28℃培养5d，得到活化后的菌种。所述的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)所得的发酵培养物过滤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2：内生真菌Peniophora sp.A871提取物抑制黄嘌呤氧化酶活性的测定
(1)pH 7.5、浓度为0.2moL/L的磷酸盐缓冲溶液(PBS缓冲液)的配制：
称取10g K2HPO4、1g KH2 PO4用蒸馏水溶解，定容至250mL，得到pH 7.5、浓度为0.2moL/L的磷酸盐缓冲溶液(PBS缓冲液)。
(2)黄嘌呤氧化酶溶液的配制：
黄嘌呤氧化酶1支(5U/mg、1mg)溶于100mLPBS中，配制成50U/L浓度。
(3)黄嘌呤配制：称取2.5mg黄嘌呤用PBS溶解，定容至25mL，配成1.0mg/mL浓度。
将上述获得的Peniophora sp.A871提取物用PBS、别嘌醇用DMSO分别配成500μg/mL浓度。吸取已在25℃温浴10min的黄嘌呤100μL于96孔板中，加入Peniophora sp.A871提取物溶液5μL及在已25℃温浴10min的黄嘌呤氧化酶100μL作为样品组，以PBS代替样品及黄嘌呤氧化酶作为空白对照组，以PBS代替Peniophora sp.A871提取物溶液作为酶组，以别嘌醇代替Peniophora sp.A871提取物溶液作为阳性药物对照组，用酶标仪测定每孔290nm处吸光值，30S/次，共15次，分别计算酶斜率A、样品斜率B、空白对照斜率C，按以下公式计算对黄嘌呤氧化酶的抑制率％：
抑制率％＝(A-B)/(A-C)*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提取物对黄嘌呤氧化酶抑制率为90.23％％，别嘌醇对黄嘌呤氧化酶抑制率为95.62％。
实验结果表明：Peniophora sp.A871提取物对黄嘌呤氧化酶具有明显的抑制作用，其抑制率与别嘌醇相当，能用于制备黄嘌呤氧化酶抑制剂，因此，Peniophora sp.A871提取物在制备治疗痛风药物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。
Claims (7)
1.一种内生真菌Peniophora sp.A871提取物的制备方法，其特征在于，以内生真菌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。
2.根据权利要求1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 3 g、MgSO4 0.75 g、维生素B1 10 mg，用水补足至1L，pH自然，得到发酵培养基。
3.根据权利要求1或2所述的制备方法制备得到的内生真菌Peniophora sp.A871提取物。
4.权利要求3所述的内生真菌Peniophora sp.A871提取物在制备治疗痛风的药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述的治疗痛风的药物为对黄嘌呤氧化酶有抑制作用的药物。
6.一种抑制黄嘌呤氧化酶抑制剂，其特征在于，含有权利要求3所述的内生真菌Peniophora sp.A871提取物作为活性成分。
7.一种治疗痛风药物，其特征在于，含有权利要求6所述的黄嘌呤氧化酶抑制剂。
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| A HÄRTL ET AL.: "5-Hydroxy-3, 4, 7-triphenyl-2, 6-benzofurandione, a New Xanthine Oxidase Inhibitor from Peniophora sanguinea", 《THE JOURNAL OF ANTIBIOTICS》 * |
| 周静等: "一株枝网刺柳珊瑚共附生真菌Peniophora sp.次级代谢产物研究", 《中国海洋药物杂志》 * |
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