CN115557960B - 一种异戊烯基化吲哚生物碱类化合物及其制备方法与应用 - Google Patents
一种异戊烯基化吲哚生物碱类化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于植物化学技术领域,具体涉及一种异戊烯基化吲哚类生物碱类化合物及其制备方法与应用。所述化合物分子式为:C18H21NO3。本发明还公开了上述化合物的制备方法和用途。通过核磁共振、质谱、紫外等波谱数据鉴定为异戊烯基化吲哚类生物碱类化合物。且该异戊烯基化吲哚生物碱类化合物具有很好的抗烟草花叶病毒活性:经对抗烟草花叶病毒的实验,发现该异戊烯基化吲哚生物碱类化合物的相对抑制率达到58.4%,其活性高于对照品宁南霉素的相对抑制率(33.2%)。本发明的化合物在制备抗烟草花叶病毒药物中有良好的应用前景。
Description
技术领域
本发明属于天然产物化学技术领域,具体涉及一种烟草内生花斑曲霉真菌发酵产物中异戊烯基化吲哚生物碱类化合物及其制备方法与应用。
背景技术
烟草是茄科烟草属植物。根据文献报道,目前从烟草中鉴定出的化合物高达 4000多种,主要成分包括二萜类化合物、倍半萜类化合物、黄酮类化合物、生物碱、香豆素等。同时有研究证明,烟草内源真菌中分离鉴定的化合物具有不同的药理学作用,如抗菌、抗氧化、抗肿瘤、抗烟草花叶病毒等。因此,加强烟草内生真菌代谢产物研究,对发现活性显著的新骨架类型代谢产物具有重要的科学意义。
发明内容
本发明从烟草中分离鉴定的花斑曲霉菌株培养液分离得到了一种具有抗烟草花叶病毒活性的新异戊烯基化生物碱类化合物,该化合物至今尚未见到相关报道。
本发明第一方面提供一种异戊烯基化吲哚生物碱类化合物,所述化合物分子式为:C18H21NO3,其具有下述结构:
曲霉属真菌广泛存在于自然界中。其中,米曲霉是一种能产生复合酶的菌株,该菌株除可产生蛋白酶外,还可产淀粉酶、糖化酶、纤维素酶、植酸酶等多种功能的酶,因而广泛应用于食品、饲料、酿酒等发酵工业。同时,花斑曲霉次生代谢产物也被认为是一个亟待开发的重要资源。从不同来源的花斑曲霉发酵产物中也分离得到了一系列具有生物活性的天然产物,包括了生物碱、多肽、萜类化合物以及多酚类化合物。本发明化合物为从烟草内源花斑曲霉 (Aspergillus versicolor)YATS1111真菌发酵产物中分离得到一种新的异戊烯基化吲哚生物碱化合物。值得一提的是,该异戊烯基化吲哚生物碱类化合物具有显著的抗烟草花叶病毒活性,其对烟草花叶病毒的相对抑制率达到58.4%,显著高于对照品宁南霉素的相对抑制率(33.2%)。
该化合物为棕色胶状物,命名为:异花斑曲霉生物碱-A,英文名为:isoaspergilline A。
本发明第二方面提供所述的异戊烯基化吲哚生物碱类化合物的制备方法,具体包括以下步骤:
1)、浸膏提取
对烟草中分离鉴定的花斑曲霉菌株YATS1111进行固体发酵,其发酵产物用乙醇超声提取并过滤,滤液浓缩,然后加入乙酸乙酯与酒石酸混合液充分搅拌均匀,静置分层后分出水相,水相再用Na2CO3调节pH至8.0~10.0,并用乙酸乙酯再次萃取,乙酸乙酯相减压浓缩成浸膏;
2)、硅胶柱层析
将步骤1)得到的浸膏用200~300目硅胶干法装柱,进行硅胶柱层析;以氯仿-甲醇溶液进行梯度洗脱,合并相同极性的部分,收集各部分洗脱液并浓缩,其中,收集第一浓度梯度的洗脱液,称为第一洗脱液;
将第一洗脱液用硅胶层析柱继续分离,用氯仿-丙酮溶液进行梯度洗脱,收集第三浓度梯度的洗脱液,称为第二洗脱液;
3)、高效液相色谱分离
将步骤2)最终得到的第二洗脱液通入高效液相色谱进行分离纯化,收集每次进样后色谱峰对应的洗脱液,得第三洗脱液;将第三洗脱液脱除溶剂后即得所述的异戊烯基化吲哚生物碱类化合物粗品。
4)、凝胶柱层析分离
上述异戊烯基化吲哚生物碱化合物粗品再次用纯甲醇溶剂,以甲醇为流动性进行凝胶柱层析分离,即可得到纯品的异戊烯基化吲哚生物碱化合物。
作为上述技术方案的优选,步骤1)中,乙醇的浓度为90~99wt%;更优选的,乙醇的浓度为95wt%。
作为上述技术方案的优选,步骤2)中,所述浸膏在经硅胶柱层析粗分前,用甲醇溶解后用重量1.5~2.5倍的80~120目硅胶拌样。
作为上述技术方案的优选,步骤3)中,高效液相色谱法分离纯化后,所得化合物再次用纯甲醇溶解,再以纯甲醇为流动相,用凝胶柱层析分离,以进一步分离纯化。
作为上述技术方案的优选,步骤2)中,氯仿-甲醇溶液进行梯度洗脱时,浓度梯度设置为体积配比20:1、8:2、7:3、6:4、5:5;所述第一洗脱液为20:1氯仿- 甲醇溶液洗脱时的洗脱液。
作为上述技术方案的优选,步骤2)中,氯仿-丙酮溶液进行梯度洗脱时,浓度梯度设置为体积配比9:1、8:2、7:3、6:4、5:5;所述第三洗脱液为7:3的氯仿- 丙酮溶液洗脱时的洗脱液。
作为上述技术方案的优选,步骤3)中,所述高效液相色谱法分离纯化是采用21.2mm×250mm,5μm的ZorbaxPrepHT GF色谱柱,流速为20mL/min,流动相为52wt%的甲醇水溶液,紫外检测器检测波长为359nm,第二洗脱液每次进样200μL,收集每次进样后色谱峰在保留时间为23.6min时所对应的洗脱液,将该洗脱液脱除溶剂,得所述的异戊烯基化吲哚生物碱类化合物粗品。
本发明的再一个目的是提供所述的异戊烯基化吲哚生物碱类化合物在制备抗烟草花叶病毒药物中的应用。
综上所述,本发明具有如下有益效果:
1、本发化合物从烟草内生花斑曲霉真菌菌株发酵产物中分离得到,由于内生真菌容易实现批量化发酵生产,因此,本发明的化合物原料易得;化合物的提取方法简单,容易分离得到,产业化制备容易实现。
2、本发明化合物具有很好的抗烟草花叶病毒活性。经对抗烟草花叶病毒的实验,发现该异戊烯基吲哚生物碱类化合物的相对抑制率达到58.4%,其活性高于对照品宁南霉素的相对抑制率(33.2%)。以上结果揭示了本发明的化合物在制备抗烟草花叶病毒药物中有良好的应用前景。
3、本发明化合物分子结构也简单,容易实现人工合成,后续的产业化也可通过人工合成实现。
4、采用了常规柱层析和高效液相色谱结合的制备方法,化合物制备操作流程简单,所获得的本发明化合物纯度高,后续的工业化生产中化合物的品质、纯度有保障。
5、本发明化合物安全无毒,展现出良好的抗烟草花叶病毒活性,可为烟草花叶病的防治提供理想的新骨架类型药源分子。
附图说明
图1为所述异戊烯基化吲哚生物碱类化合物的核磁共振碳谱。
图2为所述异戊烯基化吲哚生物碱类化合物的核磁共振氢谱。
图3为所述异戊烯基化吲哚生物碱类化合物的主要HMBC和1H-1HCOSY相关。
具体实施方式
下面对本发明通过实施例作进一步说明,但不仅限于本实施例。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件所用的通用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明所用原料不受培养基类型影响,下面以来源于云南的烟草中分离鉴定的花斑曲霉菌株培养基对本发明做进一步说明。
菌株分离鉴定
内生真菌花斑曲霉(Aspergillus versicolor)YATS1111的分离
将75%乙醇消毒后的烟草根茎放入无菌的研钵中进行研磨,研磨后转入无菌的塑料管内,1000~3000rpm离心2~10min,吸取1~100微升上清液,涂布在 BL平板上,倒置于培养箱中,25~30℃黑暗培养2~10天,反复挑取单菌落培养并编号保存菌种,直到获得单一内生真菌菌落;确定为曲霉属真菌花斑曲霉 (Aspergillus versicolor)。
花斑曲霉(Aspergillus versicolor)YATS1111的微生物学特征
1)、在PDA培养基上培养5天,菌落直径约2cm。菌落小而紧密,凸起,边缘白色,中间微绿,有紫红色透明分泌物,干燥不透明,不易挑取,生长速度慢。
2)、菌丝发达,分枝少,菌丝光滑无隔。
3)、分生孢子穗扇形,分生孢子梗光滑,顶囊椭圆形,小梗辐射状,两层,生于顶囊3/4处,分生孢子球形。
花斑曲霉(Aspergillus versicolor)YATS1111的菌种培养
将分离得到的花斑曲霉菌种在室温下接种在马铃薯葡萄糖琼脂培养基上, 25~30℃培养7~10天,再接种在50~500毫升的三角瓶中,每个三角瓶含10~100毫升马铃薯葡萄糖培养基,置于25~30℃下震荡培养5~10天得到液体发酵种;菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期:2020年6月8日,保藏地点:中国北京,保藏编号为 CGMCC No.19910。
实施例1
将培养得到的液体发酵种进行规模化发酵,规模化发酵在500个500mL的冯巴赫瓶中进行,每个瓶含有180g大米和180mL蒸馏水,每个瓶中接种2.5mL 培养得到的液体发酵种,于25~30℃培养30天得到花斑曲霉发酵物。发酵产物用95%的乙醇超声提取3次,每次提取30min;提取液合并,加入到乙酸乙酯与3%的酒石酸混合液(乙酸乙酯:酒石酸=97:3,质量比)中充分搅拌,待混合溶液静置分层后分离出水相,水相再用Na2CO3溶液将水层pH值调节至9.0,并用乙酸乙酯再次萃取;分离出乙酸乙酯相减压浓缩成浸膏,得浸膏580g。该浸膏用1.0kg的80~120目硅胶拌样,用3.0kg200目硅胶装柱进行硅胶柱层析,用体积配比为20:1,8:2,7:3,6:4,5:5的氯仿-甲醇梯度洗脱,TLC监测合并相同的部分,得到5个部分,其中体积配比为7:3的氯仿-甲醇洗脱部分浓缩,再次以体积配比分别为9:1,8:2,7:3,6:4,1:1的氯仿-丙酮溶液进行洗脱后,再用安捷仑1100制备高效液相色谱分离,以59%的甲醇为流动相,ZorbaxPrepHT GF柱 (21.2 250mm,5m)制备柱为固定相,流速为20mL/min,紫外检测器检测波长为 359nm,每次进样200L,收集23.6min的色谱峰,多次累加后蒸干可得化合物粗品;所得产物粗品再次用纯甲醇溶解,再以纯甲醇为流动相,用Sephadex LH-20凝胶柱层析分离,即得该新化合物纯品。
实施例2
将培养得到的液体发酵种进行规模化发酵,规模化发酵在250个1.0L的冯巴赫瓶中进行,每个瓶含有360g大米和360mL蒸馏水,每个瓶中接种5.0mL培养得到的液体发酵种,于25~30℃培养30天得到花斑曲霉发酵物。发酵产物用 95%的乙醇超声提取3次,每次提取30min;提取液合并,加入到乙酸乙酯与3%的酒石酸混合液(乙酸乙酯:酒石酸=97:3,质量比)中充分搅拌,待混合溶液静置分层后分离出水相,水相再用Na2CO3溶液将水层pH值调节至9.0,并用乙酸乙酯再次萃取;分离出乙酸乙酯相减压浓缩成浸膏,得浸膏610g。该浸膏用1.0kg 的80~120目硅胶拌样,用3.2kg 200目硅胶装柱进行硅胶柱层析,用体积配比为20:1,8:2,7:3,6:4,5:5的氯仿-甲醇梯度洗脱,TLC监测合并相同的部分,得到5个部分,其中体积配比为8:2的氯仿-甲醇洗脱部分浓缩,再次以体积配比分别为9:1,8:2,7:3,6:4,1:1的氯仿-丙酮溶液进行洗脱后,再用安捷仑1100制备高效液相色谱分离,以59%的甲醇为流动相,ZorbaxPrepHT GF柱(21.2 250mm, 5m)制备柱为固定相,流速为20mL/min,紫外检测器检测波长为359nm,每次进样200L,收集23.6min的色谱峰,多次累加后蒸干可得化合物粗品;所得产物粗品再次用纯甲醇溶解,再以纯甲醇为流动相,用Sephadex LH-20凝胶柱层析分离,即得该新化合物纯品。
实施例3
以实施例1制备的异戊烯基化吲哚生物碱类化合物的结构是通过以下方法鉴定出来的:
外观观测发现:本发明化合物为棕色胶状物。
如图1~3所示,紫外-可见吸收光谱显示其在215与358nm处有最大吸收,证明该化合物中存在芳香环结构;高分辨质谱(HRESIMS)给出准分子离子峰 322.1409[M+Na]+,可确定化合物的分子式为C18H21NO3,不饱和度为9。结合1H 和13C与HSQC NMR数据,显示该化合物包括一个3,7,8-四取代的吲哚环,两个异丙基(C-10、C-11、C-12、C-13,14,H-10,H-11,和H6-13,14;C-15、C-16、C-17、 C-18,19,H-16,H2-17,和H6-18,19),一个-NHCO-结构片段(C-2-和NH-1)。为了满足化合物的9个不饱和度,一个异戊烯基结构片段应和苯环形成1个环。该推断可进一步由H-10与C-7,C-8,C-11,和C-12的HMBC相关得到证实,这也确定了这个异戊烯基取代在C-8位。化合物的母体骨架确定后,剩余的取代基位置可通过进一步分析其HMBC相关确定。根据H-16与C-3,C-15,和C-18的HMBC相关,可确定另外一个异戊烯基取代在C-3位以及羟基取代在C-3位。至此,本发明化合物的结构得到确认。化合物命名为:异花斑曲霉生物碱-A,英文名为: isoaspergilline A。
化合物的波谱数据:UV(甲醇),λmax(logε)195(4.18)、221(3.89)、280(3.83) nm;1H and 13C NMR数据(CDCl3,600和150MHz),表-1;HRESIMS(正离子模式)m/z 322.1409[M+Na]+(计算值322.1414,C18H21NO3Na)。
表-1、化合物的1H NMR和13C NMR数据(CDCl3)
实施例4
取实施例2制备的化合物,为棕色胶状物。测定方法与实施例3相同,确认实施例2制备的化合物为所述的异戊烯基化吲哚生物碱类化合物–异花斑曲霉生物碱-A。
实施例5
取实施例1~2制备的任一异戊烯基化吲哚生物碱类化合物进行抗烟草花叶病毒活性试验,试验情况如下:
采用半叶法,在药剂的质量浓度均为20M时对本发明化合物进行抗烟草花叶病毒活性测定。
在5~6株烤烟的植株上,选取适用于测试的叶片(叶行正常,无病无虫),先将叶片均匀撒上细金刚砂,用毛笔将备用的烟草花叶病毒源(3.0×10-3)均匀抹在撒有金刚砂的叶片上,待所有中选的叶片接毒结束后,立即放在盛有药液的培养皿中处理20min,取出,洒去叶片上水珠和约液,将两个半叶复原排放在铺有卫生纸保湿的搪瓷衙内加盖玻璃,控温(23±2)℃,放在温室自然光照射,2~3d即可见枯斑。每个处理都设另一半叶为对照,另外设有1组为商品宁南霉素的处理作为对比,按下公式计算相对抑制率。
XI%=(CK-T)/CK×100%
X:相对抑制率(%),CK:浸泡于清水中半片接毒叶的枯斑数(个),T浸泡于药液中半片接毒叶的枯斑数(个)。
结果明本化合物的相对抑制率为58.4%,高于对照宁南霉素的相对抑制率33.2%,说明化合物有很好的抗烟草花叶病毒活性。
Claims (2)
1.一种异戊烯基化吲哚生物碱类化合物,其特征在于,所述化合物分子式为:C18H21NO3,其具有下述结构:
2.权利要求1所述的异戊烯基化吲哚生物碱类化合物在制备抗烟草花叶病毒药物中的应用。
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