CN116077549B - 木豆提取物及其活性成分银松素单甲醚在制备黄曲霉抑制产品中的应用 - Google Patents
木豆提取物及其活性成分银松素单甲醚在制备黄曲霉抑制产品中的应用 Download PDFInfo
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Abstract
本发明公开了木豆提取物及其活性成分银松素单甲醚在制备黄曲霉抑制产品中的应用。研究发现木豆提取物具有明显的抗黄曲霉活性。同时，木豆叶中活性成分银松素单甲醚能够抑制黄曲霉生长、孢子萌发以及黄曲霉毒素累积，抑菌IC50为0.26mg/mL。在浓度为0.5mg/mL时能够完全抑制黄曲霉生长，且溶血活性可忽略。进一步机理研究发现，PME能够靶向细胞膜，导致内容物外渗。鉴于木豆提取物及银松素单甲醚强抗黄曲霉活性，因此在制备抗黄曲霉产品中具有良好的开发应用前景。
Description
技术领域：
本发明属于天然产物领域，具体涉及木豆提取物及其活性成分银松素单甲醚(PME)在制备抗黄曲霉产品中的应用。
背景技术：
黄曲霉(Aspergillus flavus)是一种常见腐生真菌，它对人类最大的危害是能够产生强毒性的次级代谢物黄曲霉毒素。黄曲霉毒素可破坏人类及动物肝脏组织，严重时可引发肝癌甚至死亡。据统计，黄曲霉毒素引发的肝癌比例高达世界总肝癌的28.2％。因其强毒性，黄曲酶毒素被国际癌症研究机构(IARC)列为Ⅰ类致癌物，同时世界上已有100多个国家和地区对食品和饲料中黄曲霉毒素的最大允许含量作了立法限制。我国国家标准GB2761-2017《食品安全国家标准食品中真菌毒素限量》也对食品中黄曲霉毒素允许含量进行了严格规定。因此，黄曲霉防治是受法律制约的。
寻找新型、高效、绿色抗菌化合物依然是目前人类防治黄曲霉的首选措施。经过长期进化过程，天然产物被赋予了多样的化学结构和丰富的药理活性，成为寻找新型药物不可替代的宝库。木豆(Cajanus cajan)是一种药食两用的豆类植物。在民间，木豆常被用作主粮和菜肴(嫩叶)，木豆是一种高蛋白食品，木豆叶是一种优质饲料。在药用领域方面，木豆具有清热解毒、补中益气、利水消食、排痈肿、止血止痢、治心虚、水肿、痔血、痢疾及脚气等功效。木豆最初是印度地区的一种经济作物，现已在我国得到了广泛种植，这为木豆进一步开发和利用奠定物质基础。研究发现，木豆中含有丰富的芪类化合物，包括银松素单甲醚(Pinosylvin monomethyl ether，PME)。虽已有关于这个化合物的文献报道，但都未涉及其对黄曲霉的抑菌活性。
发明内容：
本发明的目的是提供木豆提取物及活性成分银松素单甲醚在制备抗黄曲霉产品中的应用。
本发明通过实验发现，木豆叶提取物对黄曲霉具有明显的抑制活性，其中乙酸乙酯提取物活性最佳。木豆叶中分离得到的活性成分银松素单甲醚能够抑制黄曲霉菌丝生长(IC50为0.26mg/mL)、孢子萌发以及黄曲霉毒素累积。进一步作用机理研究发现，银松素单甲醚能够靶向细胞膜，导致细胞膜渗透性增强和流动性降低，甚至引起细胞膜结构完全破坏，导致孢子结构坍塌。抑菌浓度梯度显示，在含有0.5mg/mL银松素单甲醚的PDA培养基上，黄曲霉生长被完全抑制，而溶血活性实验显示，在该浓度下银松素单甲醚溶血活性可忽略。鉴于木豆提取物和其活性成分银松素单甲醚强抗黄曲霉活性，因此可以被用于制备抗黄曲霉的产品。
因此，本发明的第一个目的是提供木豆提取物在制备抗黄曲霉产品中的应用；
本发明的第二个目的是提供化合物银松素单甲醚在制备抗黄曲霉产品中的应用；
所述的化合物银松素单甲醚的结构式如下所示：
本发明的第三个目的是提供一种抗黄曲霉的产品，其含有木豆提取物或化合物银松素单甲醚为活性成分；
所述的化合物银松素单甲醚的结构式如下所示：
优选，所述的木豆提取物包括但不限于木豆叶、根、茎或全株的乙酸乙酯、正己烷、丙酮、乙醇、甲醇等有机溶剂提取物。
优选，所述的木豆提取物是以木豆叶为原料，将其粉碎，10倍体积乙醇浸泡、过滤、减压蒸馏，得到乙醇提取物，乙醇提取物再分别经丙酮、乙酸乙酯、正己烷、甲醇萃取得到丙酮、乙酸乙酯、正己烷、甲醇提取物。
优选，所述的产品包括但不限于抗黄曲霉药物、抑制黄曲霉生长的产品，防止黄曲霉引起食品腐败的产品，如食品防腐剂。
本发明研究发现木豆提取物具有明显的抗黄曲霉活性。同时，木豆叶中活性成分银松素单甲醚(Pinosylvin monomethyl ether，PME)能够抑制黄曲霉生长、孢子萌发以及黄曲霉毒素累积，抑菌IC50为0.26mg/mL。在浓度为0.5mg/mL时能够完全抑制黄曲霉生长，且溶血活性可忽略。进一步机理研究发现，PME能够靶向细胞膜，导致内容物外渗。鉴于木豆提取物及银松素单甲醚强抗黄曲霉活性，因此在制备抗黄曲霉产品中具有良好的开发应用前景。
附图说明
图1是银松素单甲醚抑制黄曲霉活性；
图2是黄曲霉毒素含量测定；
图3是黄曲霉菌丝伊文思蓝染色；
图4是蛋白和核酸外渗；
图5是银松素单甲醚溶血活性。
具体实施方式：
下面通过实施例进一步描述本发明，但本发明并不局限于这些实施方式，任何在本实施例基本精神层面的改进或代替，仍属于本发明权利要求所保护的范围。
实施例1：木豆提取物抑菌活性测定
1、菌株
黄曲霉A.flavus BNCC 142788，购买于北纳生物。
2、试剂配制
(1)PDA固体培养基：马铃薯200g切成小块，加入800mL去离子水煮沸20min，纱布过滤，称取葡萄糖20g和琼脂15～20g，加入到滤液中，去离子水定容至1L，高温高压灭菌。
(2)木豆叶乙酸乙酯提取物工作液配制：准确称取膏状木豆叶乙酸乙酯提取物100mg，加入2mL DMSO溶解，配制得到50mg/mL木豆叶乙酸乙酯提取物。
木豆提取物是以木豆叶为原料，将其粉碎，10倍体积乙醇浸泡、过滤、减压蒸馏，得到乙醇提取物，乙醇提取物再分别经丙酮、乙酸乙酯、正己烷、甲醇萃取得到丙酮、乙酸乙酯、正己烷、甲醇提取物。
(3)木豆叶丙酮、正己烷、甲醇、乙醇提取物工作液配制同木豆叶乙酸乙酯提取物工作液配制。
3、孢子悬浮液制备
黄曲霉接种于PDA平板，28℃培养7天，用无菌水清洗平板，调节孢子悬浮液浓度1×106孢子/mL。
4、实验方法
将直径6mm的灭菌滤纸片放置于PDA平板中间，随后在滤纸片上滴加7μL 50mg/mL的木豆叶提取物，阴性对照滴加等体积DMSO。滤纸片晾干后再滴加5μL待测真菌孢子悬浮液，28℃恒温培养24h后统计菌落直径，并计算抑制率。抑制率＝(阴性对照组菌落直径-提取物组菌落直径)/阴性对照组菌落直径。
5、实验结果
表1
如表1所示，木豆叶提取物对黄曲霉生长具有抑制活性，且乙酸乙酯提取物活性最佳。
实施例2：银松素单甲醚抑菌浓度梯度测定
1、试剂配制
2mg/mL银松素单甲醚溶液：称取木豆中提取的银松素单甲醚(纯度>95％)4mg，溶解于2mL DMSO。
2、PDA药物平板配制
200g土豆切块后放入锅中，煮沸20-30min。冷却后纱布过滤，滤液中加入葡萄糖20g和琼脂粉15-20g，定容至1L。高温高压灭菌，冷却至不烫手后加入终浓度为0、62.5、125、250、500μg/mL的银松素单甲醚，配制成药物平板，其中银松素单甲醚浓度为0μg/mL的平板中加入等体积DMSO。
3、实验方法
4μL孢子悬浮液接种于PDA药物平板中间部位，28℃恒温培养2天后统计菌落直径，计算抑制率并绘制抑菌曲线。抑制率＝(DMSO组菌落直径-药物组菌落直径)/DMSO组菌落直径。
4、实验结果
如图1所示，银松素单甲醚对黄曲霉生长具有显著的抑制作用，且存在浓度依赖性，抑制曲线为y＝0.1963x-0.8065，其中x代表PME浓度(单位：μg/mL)，y代表抑制率。抑制中浓度IC50为259μg/mL。
实施例3：黄曲霉毒素测定
1、实验方法
分别配制含有终浓度0.125mg/mL银松素单甲醚和等体积DMSO的YES培养基，培养基上铺一层灭菌玻璃纸。黄曲霉孢子接种于铺有玻璃纸的YES培养基，28℃培养三天，刮下菌丝，称重后溶解于10mL甲醇。28℃、180rpm摇2h，离心后取上清测定黄曲霉毒素含量。采用试剂盒Aflatoxin B1 ELISA Kits(Dogesce,DG90668Q-96)测定含量。
2、实验结果
如图2所示，银松素单甲醚(PME)处理后黄曲霉毒素累积明显减少。
实施例4：伊文思蓝染色
1、实验方法：
将灭菌后PDA培养基平铺在载玻片，使之形成1mm左右的PDA薄层，黄曲霉孢子悬浮液接种于PDA载玻片上，28℃培养2天后用0.25mg/mL的银松素单甲醚或者DMSO处理1h，PBS缓冲液(0.01M)清洗后用0.25％(w/v)伊文思蓝染色20min，PBS清洗后拍照。
2、实验结果
如图3所示，银松素单甲醚(PME)处理染色明显比DMSO组深，说明银松素单甲醚增加了菌丝渗透性。
实施例5：蛋白和核酸内容物外渗
1、实验方法
灭菌玻璃纸平铺于PDA平板，黄曲霉孢子悬浮液接种于玻璃纸之上，28℃培养，待菌丝长满平板四分之三区域时将其从玻璃之上取下并称重，之后放入浓度0.25mg/mL的银松素单甲醚的PDA液体培养基溶液中或者含有等体积DMSO的PBS溶液，28℃，180rpm，摇菌1h，离心后测定上清在260nm和280nm处的吸光度。
2、实验结果
如图4所示，银松素单甲醚处理组明显比DMSO组渗出更多蛋白和核酸，表明银松素单甲醚能够增强细胞膜渗透性。
实施例6：银松素单甲醚溶血活性测定
1、实验方法
无菌脱纤维羊血PBS清洗三次后加入二倍梯度稀释的银松素单甲醚，37℃孵育1h，2500rpm离心，吸取上清测定543nm吸光度。DMSO为阴性对照，0.2％ Triton-X-100为阳性对照。每个样品3个技术重复，实验进行3次生物学重复。
(2)实验结果：
如图5所示，在处理浓度为0.512μg/mL时，银松素单甲醚溶血活性可忽略。抑菌浓度梯度显示，在该浓度时银松素单甲醚可以完全抑制黄曲霉生长发育。
Claims (1)
1.化合物银松素单甲醚作为唯一活性成分在制备抑制黄曲霉药物中的应用；所述的化合物银松素单甲醚的结构式如下所示：
。
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