CN114568437A - 从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用 - Google Patents
从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种从余甘子中提取的1‑O‑没食子酸酰基‑β‑D‑葡萄糖苷在植物病害防治中的应用,所述植物病害为马铃薯疮痂病。1‑O‑没食子酸酰基‑β‑D‑葡萄糖苷从余甘子果渣中提取、分离纯化得到,并首次发现具有抑制铃薯疮痂链霉菌活性作用,可用于防治马铃薯疮痂病。
Description
技术领域
本发明涉及活性药物技术领域,具体涉及一种从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用。
背景技术
余甘子(phyllanthus emblica L.)是大戟科(Euphorbiaceae)叶下珠属(Phyllanthus)的果实。主要分布在热带、亚热带;原产于印度,分布于巴基斯坦、马来西亚、中国等国家;国内以福建和云南两省产量最多。早在1998年就被***列入“既是食品又是药品”的名单,收载于多版《中华人民共和国药典》,并被***卫生组织指定其为世界范围内推广种植的3种保健植物之一。余甘子的特殊成分构成和药理活性使其具有很好的开发和应用价值,近年来相关产业也发展迅速。
目前对余甘子的开发主要是以提取和鲜果榨汁为主,然后加工成饮料、精粉及含片等产品。其生产过程中会产生果核、果渣或药渣等废弃物,在提取物制备过程中也会产生大量沉淀物、过滤固形物及其他类型的副产品。有数据表明,仅余甘子果渣重量就能占原果重的65%左右,药企每年会产生近百吨的余甘子药物固体残渣,若仅丢弃或掩埋处理,不仅处理成本高、环境压力大,还造成了大量资源的浪费,故研究余甘子果渣的开发,不仅有利于防止环境污染,还能够生产出高附加值产品,有助于从根本上解决果渣的利用问题,形成余甘子果渣资源化利用的可持续发展之路。
马铃薯是仅次于小麦、水稻和玉米的全球第四大重要粮食作物。马铃薯既可以作为粮食作物供人们生活所需,也可以作为经济作物应用到多种行业中,而且其营养价值高,对环境的适应能力强,单产增产潜力大,发展前景十分广阔。已报道的马铃薯疮痂病菌有20余种,最常见的是疮痂链霉菌(Streptomyces scabies)、酸性疮痂链霉菌(S.acidiscabies)、肿痂链霉菌(S.turgidiscabies),其中S.scabies是马铃薯疮痂病的主要致病菌,该病的症状包括块茎表面结痂状、***或凹陷性病变,严重影响马铃薯的质量,导致其市场价值降低,给种植者造成了巨大的经济损失。疮痂链霉菌还能侵染萝卜、甜菜、胡萝卜和防风草等主根作物。由致病性链霉菌引发的土传细菌性植物病害,仍然是制约中国乃至全世界马铃薯产业积极向好发展的重要因素。目前,使用化学药剂防治该疾病虽见效快,但也会产生一些副作用,比如会造成马铃薯块茎减小、产量降低以及对环境和人类健康造成威胁。因此,从天然植物来源中寻找高效低毒的植物源农药是一种较好的替代方法,具有周期短、低毒、无污染和便于生产等优点,是农产品绿色防控的有效途径。
发明内容
为了解决上述问题,本发明提出一种从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用,1-O-没食子酸酰基-β-D-葡萄糖苷从余甘子果渣中提取、分离纯化得到,并首次发现具有抑制铃薯疮痂链霉菌活性作用,可用于防治马铃薯疮痂病。
为了实现上述目的,本发明的实施例提出了一种从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用,所述植物病害为马铃薯疮痂病。
根据本发明实施例的应用,1-O-没食子酸酰基-β-D-葡萄糖苷从余甘子果渣中提取、分离纯化得到,并首次发现具有抑制铃薯疮痂链霉菌活性作用,可用于防治马铃薯疮痂病。
可选地,所述1-O-没食子酸酰基-β-D-葡萄糖苷的制备包括:
将余甘子果渣用70%乙醇浸泡并用超声加速浸提,重复浸提3次,合并浸提液,旋转蒸发仪抽干溶剂,得到乙醇层粗提物EA;
将乙醇层粗提物EA用50%甲醇水溶解,用中低压反相C18柱分离纯化,流速设置30mL/min,25%甲醇水溶液洗脱,收集第15~31min组分,为组分EA-1;
将组分EA-1用甲醇溶解,用Sephadex LH-20分离纯化,流速设置为0.25mL/min,甲醇做洗脱剂,收集第3540~4080min组分,为组分EA-1-3;
将组分EA-1-3用甲醇溶解,用高效液相色谱仪纯化,流动相:1%甲醇-水混合液,再加入0.075%的三氟乙酸;流速:10mL/min;进样量100μL;柱温25℃;检测波长:210nm和280nm;收集第42~44min组分,为组分A-1-3-6,经核磁鉴定,为1-O-没食子酸酰基-β-D-葡萄糖苷。由此,以余甘子果渣为原料进行分离,原料易得,能够对余甘子果渣进行充分回收利用,避免浪费;制备过程简单易操作;所得的1-O-没食子酸酰基-β-D-葡萄糖苷从余甘子果渣中提取、分离纯化得到,并首次发现具有抑制铃薯疮痂链霉菌活性作用,可制成马铃薯疮痂病植物源杀菌剂,为植物源农药开发提供了新的来源,对于药食同源的中药材天然产物有效成分的研究具有十分重要的意义。
本发明的实施例在另一方面还提出了从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在制备生物肥中的应用,所述1-O-没食子酸酰基-β-D-葡萄糖苷用于抑制马铃薯疮痂链霉菌的活性。由此,1-O-没食子酸酰基-β-D-葡萄糖苷从余甘子果渣中提取、分离纯化得到,并首次发现具有抑制铃薯疮痂链霉菌活性作用,可作为生物肥使用。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1为根据本发明实施例的乙醇层粗提物(EA)抑菌实验图;
图2为根据本发明实施例的提取物EA-1抑菌实验图;
图3为根据本发明实施例的提取物EA-1-3抑菌实验图;
图4为根据本发明实施例的提取物EA-1-3-6抑菌实验图;
图5为根据本发明实施例的阳性对照抑菌实验图;
图6为根据本发明实施例的溶剂对照抑菌实验图;
图7为根据本发明实施例的化合物1-O-没食子酸酰基-β-D-葡萄糖苷的HPLC分析图;
图8为根据本发明实施例的化合物1-O-没食子酸酰基-β-D-葡萄糖苷的1H-NMR图;
图9为根据本发明实施例的化合物1-O-没食子酸酰基-β-D-葡萄糖苷的13C-NMR图;
图10为根据本发明实施例的不同浓度1-O-没食子酸酰基-β-D-葡萄糖苷的最低抑菌浓度。
具体实施方式
以下通过特定的具体实例说明本发明的技术方案。应理解,本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以***其他方法步骤;还应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。而且,除非另有说明,各方法步骤的编号仅为鉴别各方法步骤的便利工具,而非为限制各方法步骤的排列次序或限定本发明可实施的范围,其相对关系的改变或调整,在无实质变更技术内容的情况下,当亦视为本发明可实施的范畴。
为了更好的理解上述技术方案,下面更详细地描述本发明的示例性实施例。虽然显示了本发明的示例性实施例,然而应当理解,可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。
本发明采用的试材皆为普通市售品,皆可于市场购得。
需要说明的是,以下实施例用到的牛津杯法为:在无菌操作台中,将配制好的高氏一号固体培养基置于酒精灯火焰旁,用无菌移液枪吸取200μL提前制备好的疮痂链霉菌孢子悬浮液,小心均匀地吹打在培养基表面,用无菌涂布棒均匀轻涂表面,直至表面没有明显水分,涂布棒有拉扯感即可。待涂布完成后,将灭菌的牛津杯放置在培养基表面,用无菌镊子轻压牛津杯,使之陷入培养基表面即可(不可过深触碰底部)。在每个牛津杯中加入200μL样品溶液,放入30℃的恒温培养箱中培养,48h后观察是否有抑菌圈。以氯霉素(1mg/mL)作为阳性对照组,甲醇为溶剂对照组。每次实验设置三个平行组。
下面参考具体实施例,对本发明进行描述,需要说明的是,这些实施例仅仅是描述性的,而不以任何方式限制本发明。
实施例1
一种从余甘子中制备1-O-没食子酸酰基-β-D-葡萄糖苷的方法,包括以下步骤:
步骤1、取740g的余甘子鲜果,去核榨汁后,得到234g果渣。将余甘子果渣置于5000mL烧杯中,以2000mL 70%乙醇浸泡并用超声加速浸提1h,重复浸提3次,合并浸提液,以旋转蒸发仪抽干溶剂,得到乙醇层粗提物(EA)19.74g。
步骤2、取步骤1得到的乙醇层粗提物(EA)19.74g,用50mL 50%甲醇水溶解,用中低压反相C18柱(填料为:YMC-GEL ODS-A-HG,s-50μm;柱长为4.5×35cm),流速设置为30mL/min,用25%甲醇水溶液洗脱,收集第15~31min组分(EA-1)。
步骤3、取步骤2得到的(EA-1)1.03g用15mL 100%甲醇溶解,用Sephadex LH-20(2.5×170cm),100%甲醇做洗脱剂,过柱流速为0.25mL/min,收集第3540~4080min组分(EA-1-3)。
步骤4、收集步骤3得到的(EA-1-3)336.5mg用5mL 100%甲醇溶解,用慧德易制备型高效液相色谱仪进行纯化;色谱柱:YMC-AQ,20×250mm,s-10μm,流动相:1%甲醇-水混合液,再加入0.075%的三氟乙酸;流速:10mL/min;进样量100μL;柱温25℃;检测波长:210nm和280nm;收集第42~44min组份(EA-1-3-6),浓缩得到化合物约82.0mg。
其过程采用牛津杯法,将粗提物及各个步骤得到的组分依次进行抑菌活性追踪,选取抑菌活性较高部分进行后续试验,其中,乙醇层粗提物(EA)的抑菌实验如图1所示,EA-1的抑菌实验如图2所示,EA-1-3的抑菌实验如图3所示,EA-1-3-6的抑菌实验如图4所示,阳性对照1mg/mL氯霉素)抑菌实验如图5所示,溶剂对照(甲醇)抑菌实验如图6所示,最终追踪分离纯化到化合物EA-1-3-6。
本实施例制得的化合物EA-1-3-6为白色粉末状,对其进行HPLC分析,HPLC参数如下:HPLC型号:Agilent Technologiees;检测器:1260Infinity II;色谱柱:Welch-LP-C18柱,4.6×250mm,s-5μm;液相条件:流动相:1%甲醇-水混合液,再加入0.1%的三氟乙酸,等度洗脱20min;流速:1mL/min,柱温:35℃,进样量:20.0μL;检测波长:280nm。
结果如图7所示,图7为化合物280nm检测波长下的高效液相色谱检测图,保留时间为10.370min。
对化合物EA-1-3-6进行质谱测定:
仪器型号:Q Exactive三重四极杆串联质谱仪,Bremen,德国Thermo;
质谱条件:离子源为ESI源,正离子模式,发射电流34.6μA;气帘气(CUR)20.0psi;离子源电压(IS)2500V;离子源温度(TEM)420℃;接口温度260℃;雾化气(GS1)40psi;辅助气(GS2)40psi;质量范围为100-1000m/z。
得到化合物EA-1-3-6分子量:ESI-MS(m/z):333.0801[M+H]+,355.0616[M+Na]+。
对化合物EA-1-3-6进行核磁测定:
NMR仪器:Bruker AvanceⅡ-500型核磁共振仪,测试温度:296K;溶剂:氘代甲醇;内标物:四甲基硅烷(TMS);1H-NMR:500MHz;13C-NMR:125MHz。。
结果如图8和图9。1H-NMR(500MHz,CD3OD)数据如下:δ:5.66(1H,d;J=5.2Hz,H-1),3.40(1H,m,H-2),3.42(1H,m,H-3),3.47(1H,m,H-4),3.48(1H,m,H-5),3.85(1H,d,J=12.0Hz,H-6a),3.70(1H,dd,J=12.0,4.2Hz,H-6b),7.13(2H,s,H-2,6);
13C-NMR(125MHz,CD3OD)数据如下:δ:96.1(C-1),74.3(C-2),79.0(C-3),71.2(C-4),78.3(C-5),62.5(C-6),120.9(C-G-1),110.7(C-G-2,6),146.7(C-G-3,5),140.4(C-G-4),167.2(C-G-7)。
综合化合物EA-1-3-6的核磁数据及质谱数据并与文献进行比对,推测化合物EA-1-3-6为1-O-没食子酸酰基-β-D-葡萄糖苷;英文名:1-O-galloyl-β-D-glucose;分子式:C13H16O10;相对分子量:332;结构如下:
实施例2
在超净工作台中,取斜面培养基保藏的菌种(马铃薯疮痂链霉菌AMCC400023),将灼烧冷却后的接种环在酒精灯火焰旁小心挑取单菌落,使用平板划线的方法,将病原菌菌体接种于高氏一号固体培养基中,封口膜封边,于30℃恒温生化培养箱中静置培养48h。其中,高氏一号固体培养基(g/L):硝酸钾:1.0;磷酸二氢钾:0.5;硫酸镁:0.5;硫酸亚铁:0.01;氯化钠:0.5;可溶性淀粉:20.0;琼脂:15.0。
用接种环挑取活化好的马铃薯疮痂链霉菌单菌落,较紧密的划在高氏一号培养基上,28℃恒温静置培养3天;吸取3mL无菌水于平板上,用刀片轻轻刮下表面孢子,少量多次冲洗培养基表面孢子,稀释至浓度为107CFU/mL,收集至50mL离心管中,得菌液,4℃保存待用。
取上述实施例1获得的1-O-没食子酸酰基-β-D-葡萄糖苷,用50%甲醇水溶液配置成浓度为10mg/mL的药液。取0.5mL药液加至装有0.5mL高氏一号液体培养基的2mL离心管中,为1号管。另取8支装有0.5mL高氏一号液体培养基的2mL离心管中分别加入0.5mL从前一管中吸取的药液,依次进行倍半稀释,最后一管弃0.5mL(也即,从1号管吸取0.5mL液体加到2号管,混匀;从2号管吸取0.5mL液体加到3号管,依次类推,一共稀释9管。为保每一管体积一致,故最后一管(9号管)吸取0.5mL丢弃),10号管以0.5mL甲醇水溶液为溶剂对照,11号管以0.5mL的1mg/mL氯霉素为阳性对照对照,这11支管最后分别加入0.5mL制备好的菌液,使各管药物的终浓度分别为5mg/mL、2.5mg/mL、1.25mg/mL、0.625mg/mL、0.3125mg/mL、0.15625mg/mL、0.078125mg/mL、0.0390625mg/mL、0.01953125mg/mL、0mg/mL和0mg/mL,置于摇床中30℃、150rpm震荡培养24h,观察各管菌体的生长情况,若前一管菌未生长而后一管有生长,则前者管中所对应的药物浓度即为1-O-没食子酸酰基-β-D-葡萄糖苷对该菌的最小抑菌浓度。结果如图10所示,测得1-O-没食子酸酰基-β-D-葡萄糖苷对马铃薯疮痂链霉菌的最小抑菌浓度为0.625mg/mL,并且1-O-没食子酸酰基-β-D-葡萄糖苷(15mg/mL)抑菌圈测得值为3.00cm(牛津杯直径为7mm)。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外,本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (3)
1.从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在植物病害防治中的应用,其特征在于,所述植物病害为马铃薯疮痂病。
2.如权利要求1所述的应用,其特征在于,所述1-O-没食子酸酰基-β-D-葡萄糖苷的制备包括:
将余甘子果渣用70%乙醇浸泡并用超声加速浸提,重复浸提3次,合并浸提液,旋转蒸发仪抽干溶剂,得到乙醇层粗提物EA;
将乙醇层粗提物EA用50%甲醇水溶解,用中低压反相C18柱分离纯化,流速设置30mL/min,25%甲醇水溶液洗脱,收集第15~31min组分,为组分EA-1;
将组分EA-1用甲醇溶解,用Sephadex LH-20分离纯化,流速设置为0.25mL/min,甲醇做洗脱剂,收集第3540~4080min组分,为组分EA-1-3;
将组分EA-1-3用甲醇溶解,用高效液相色谱仪纯化,流动相:1%甲醇-水混合液,再加入0.075%的三氟乙酸;流速:10mL/min;进样量100μL;柱温25℃;检测波长:210nm和280nm;收集第42~44min组分,为组分A-1-3-6,经核磁鉴定,为1-O-没食子酸酰基-β-D-葡萄糖苷。
3.从余甘子中提取的1-O-没食子酸酰基-β-D-葡萄糖苷在制备生物肥中的应用,其特征在于,所述1-O-没食子酸酰基-β-D-葡萄糖苷用于抑制马铃薯疮痂链霉菌的活性。
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