CN116478121B - 从金花茶内生菌代谢产物中提取的新化合物及其制备方法 - Google Patents
从金花茶内生菌代谢产物中提取的新化合物及其制备方法 Download PDFInfo
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Abstract
本发明属于天然药物化学技术领域，具体涉及一种从金花茶内生菌代谢产物中提取的新化合物及其制备方法，本发明中以金花茶为原材料，从金花茶内生菌代谢产物中提取获得2种新化合物，经过鉴定分别为CamelliaNigerⅠ(式I)、CamelliaNigerⅡ(式Ⅱ)，本发明的2种新化合物均为首次从金花茶内生菌Aspergillusniger发酵产物中得到；2种化合物均具有比较显著的体外抗肿瘤活性，为进一步开展深入的药理和临床研究，开发疗效强、副作用小的新型抗肿瘤药物提供了基础。
Description
技术领域
本发明属于天然药物化学技术领域，具体涉及一种从金花茶内生菌代谢产物中提取的新化合物及其制备方法。
背景技术
金花茶(Camellia nitidissima)属于山茶科山茶属植物，具有极高的药用价值，文献报告其花和叶部位具有抑菌、抗肿瘤、抗炎、降血糖、提高免疫力等功效，金花茶具有广阔的市场前景和开发价值。
植物内生菌是一定阶段或全部阶段生活于健康植物的组织和器官内部的真菌或细菌，普遍存在于高等植物中。现有技术对金花茶内生菌研究处于空白领域，本发明所涉及从金花茶内生菌Aspergillus niger的发酵产物中分离纯化得到两种化合物，并通过体外抗肿瘤活性筛选发现其具有显著抑制活性，为抗肿瘤药物的研发提供重要物质来源。同时通过发酵技术，解决肿瘤药物生产的成本问题，扩大了内生菌的使用范围。
发明内容
本发明旨在解决上述技术问题，提供一种从金花茶内生菌代谢产物中提取的新化合物及其制备方法。
本发明的技术方案为：
一种从金花茶内生菌代谢产物中提取的新化合物，结构如式I和/或式Ⅱ所示：
分别命名为：CamelliaNigerⅠ(式I)、CamelliaNigerⅡ(式Ⅱ)。
一种本发明的从金花茶内生菌代谢产物中提取的新化合物的制备方法，包括以下步骤：
(1)金花茶内生菌的分离纯化：对金花茶茎部位消毒，切成短茎，然后将短茎接种于含有青霉素-链霉素的琼脂培养基上，当短茎从内部向培养基周围长出菌落时，挑出单菌落进行纯化，得到金花茶内生菌Aspergillus niger；
(2)金花茶内生菌Aspergillus niger发酵产物的制备：将步骤(1)的金花茶内生菌Aspergillus niger接种至大米培养基表面进行发酵，得到金花茶内生菌Aspergillusniger发酵产物；
(3)将步骤(2)金花茶内生菌Aspergillus niger发酵产物捣碎，加入甲醇浸泡，过滤，收集滤液，减压浓缩，得到甲醇浸膏；
(4)将步骤(3)的甲醇浸膏用水搅拌分散成混悬液，加入乙酸乙酯萃取，过滤，收集滤液，减压浓缩，得到乙酸乙酯提取物；
(5)将步骤(4)所述乙酸乙酯提取物进行硅胶柱层析色谱分离，以体积比为100：1、50：1、20：1、10：1、5：1的三氯甲烷-甲醇梯度洗脱，收集合并相同馏分，得到6个馏分，分别记为其中馏分Fr.4为体积比为20：1的三氯甲烷-甲醇洗脱得到；
(6)将步骤(5)的馏分Fr.4浓缩，进行葡聚糖凝胶色谱分离，以体积比为5：5、7：3、9：1、1：0的甲醇-水梯度洗脱，收集合并相同馏分，得到4个馏分，分别记为 其中馏分Fr.4.2为体积比为7：3的甲醇-水洗脱得到，馏分Fr.4.3为体积比为9：1的甲醇-水洗脱得到；
(7)对步骤(6)的馏分Fr.4.2浓缩后，采用半制备高效液相色谱分离，得到式I的新化合物；
(8)对步骤(6)的馏分Fr.4.3浓缩后，采用半制备高效液相色谱分离，得到式Ⅱ的新化合物。
进一步地，步骤(2)中，所述大米培养基组成为：60g大米、3g蛋白胨、37.5g水。
进一步地，步骤(2)中，所述发酵条件为：温度：湿度：/>
进一步地，步骤(7)中，所述半制备高效液相色谱的条件为：色谱柱选择WelchUltimate XB-C18半制柱；流动相为乙腈和水的混合液，所述乙腈和水的体积比25：75；检测波长为210nm。
进一步地，步骤(8)中，所述半制备高效液相色谱的条件为：色谱柱选择WelchUltimate XB-C18半制柱；流动相为乙腈和水的混合液，所述乙腈和水的体积比37：63；检测波长为210nm。
本发明还提供一种从金花茶内生菌代谢产物中提取的新化合物在制备治疗肿瘤药物中的应用。
具体地，所述肿瘤为肺癌、结肠癌、乳腺癌或宫颈癌。
本发明的有益效果为：
1、本发明以金花茶为原材料，从金花茶内生菌代谢产物中提取获得2种新化合物，经过鉴定分别为CamelliaNigerⅠ(式I)、CamelliaNigerⅡ(式Ⅱ)，为金花茶药材的药理活性研究提供了化学依据和物质参考，并为金花茶制剂的开发和质量控制提供了基础。
2、本发明的2种新化合物均为首次从金花茶内生菌Aspergillus niger发酵产物中得到；2种化合物均具有比较显著的体外抗肿瘤活性，为进一步开展深入的药理和临床研究，开发疗效强、副作用小的新型抗肿瘤药物提供了基础。
附图说明
图1为本发明所述内生菌Aspergillus niger的单菌落图；
图2为本发明所述内生菌Aspergillus niger纯化后的菌落图；
图3为本发明所制备新化合物CamelliaNigerⅠ的正离子HR-ESI-MS谱图；
图4为本发明所制备新化合物CamelliaNigerⅠ的1H-NMR谱图；
图5为本发明所制备新化合物CamelliaNigerⅠ的13C-NMR谱图；
图6为本发明所制备新化合物CamelliaNigerⅠ的DEPT135谱图；
图7为本发明所制备新化合物CamelliaNigerⅠ的1H 1H-COSY谱图；
图8为本发明所制备新化合物CamelliaNigerⅠ的HSQC谱图；
图9为本发明所制备新化合物CamelliaNigerⅠ的HMBC谱图；
图10为本发明所制备新化合物CamelliaNigerⅡ的正离子HR-ESI-MS谱图
图11为本发明所制备新化合物CamelliaNigerⅡ的1H-NMR谱图；
图12为本发明所制备新化合物CamelliaNigerⅡ的13C-NMR谱图；
图13为本发明所制备新化合物CamelliaNigerⅡ的DEPT135谱图；
图14为本发明所制备新化合物CamelliaNigerⅡ的1H 1H-COSY谱图；
图15为本发明所制备新化合物CamelliaNigerⅡ的HSQC谱图；
图16为本发明所制备新化合物CamelliaNigerⅡ的HMBC谱图；
图17为本发明所制备新化合物CamelliaNigerⅠ的高效液相半制备色谱图；
图18为本发明所制备新化合物CamelliaNigerⅡ的高效液相半制备色谱图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本发明实施提供了从金花茶内生菌代谢产物中提取的两种新化合物CamelliaNigerⅠ和CamelliaNigerⅡ，其结构式分别如式I和式Ⅱ所示：
本发明实施例还提供了一种上述从金花茶内生菌代谢产物中提取的新化合物的制备方法，包括以下步骤：
利用金花茶培养得到Aspergillus niger操作如下：对金花茶茎部位消毒，切成长度1cm左右短茎，然后将短茎接种于含有青霉素-链霉素的φ10cm的琼脂培养基上，每个培养基放2根短茎，并放置于30℃培养箱中培养。每12小时观察，当金花茶短茎从内部向培养基周围长出菌落时，根据菌落形态、颜色挑出差异明显的单菌落进行纯化。
上述差异明显的单菌落包括但不限于具有以下特征：菌丝为白色，菌落中央为白色，稀疏，呈放射状生长，如图1所示。
纯化的条件如下：挑取菌落前言白色菌丝至另一块新的培养皿上Z形划线，新培养皿Z形划线长出菌丝后，挑取菌落前言白色菌丝至另一块新的培养皿上，重复三次，在显微镜下观察菌丝形态，如形态均一，则纯化完成。
纯化后的Aspergillus niger具有但不仅限于以下生理生化特征：菌落中央为白色，周围呈放射状，培养3天后分生孢子为球形，黑色或黑褐色颗粒，如图2所示。
接着将有Aspergillus niger菌落的培养基切成1×1cm琼脂块，接种至大米培养基表面，在温度：湿度：/>的条件下发酵28天，得到金花茶内生菌Aspergillusniger发酵产物。其中，大米培养基的制备方法为：取500mL广口锥形瓶，加入60g大米、3g蛋白胨、37.5g纯净水，浸泡4小时后，121℃高压灭菌20分钟即得。
金花茶内生菌Aspergillus niger发酵产物共462g，捣碎，加入5L甲醇，浸泡24h，过滤，重复3次，收集合并3次滤液，减压浓缩，得到甲醇浸膏。
将上述甲醇浸膏用40℃热水搅拌分散成混悬液，加入混悬液3倍体积的乙酸乙酯萃取，过滤，重复3次，收集合并3次滤液，减压浓缩，得到乙酸乙酯提取物；
将上述乙酸乙酯提取物进行柱层析硅胶色谱分离，以体积比为100：1、50：1、20：1、10：1、5：1的三氯甲烷-甲醇进行洗脱，通过TLC及碘熏显色反应结合合并相同馏分，得到其中馏分Fr.4为体积比为20：1的三氯甲烷-甲醇洗脱得到。
将馏分Fr.4进行葡聚糖凝胶色谱分离，以体积比5：5、7：3、9：1、1：0的甲醇-水进行洗脱，通过TLC及碘熏显色反应结合合并相同馏分，得到4个馏分，分别记为 其中馏分Fr.4.2为体积比为7：3的甲醇-水洗脱得到，馏分Fr.4.3为体积比为9：1的甲醇-水洗脱得到。
对步骤(6)的馏分Fr.4.2浓缩后，采用半制备高效液相色谱分离，得到式I的新化合物，命名为CamelliaNigerⅠ，其中，半制备高效液相色谱的条件为：色谱柱选择WelchUltimate XB-C18半制柱；流动相为乙腈和水的混合液，所述乙腈和水的体积比25：75；检测波长为210nm，色谱图如图17所示。
对步骤(6)的馏分Fr.4.3浓缩后，采用半制备高效液相色谱分离，得到式Ⅱ的新化合物，命名为CamelliaNigerⅡ，其中，半制备高效液相色谱的条件为：色谱柱选择WelchUltimate XB-C18半制柱；流动相为乙腈和水的混合液，所述乙腈和水的体积比37：63；检测波长为210nm，色谱图如图18所示。
实施例2结构鉴定
利用高分辨质谱(HR-ESI-MS)及核磁共振的一维(1H-NMR、13C-NMR、DEPT 135)和二维技术(1H-1H COSY、HSQC、HMBC、NOESY)鉴定化合物结构。
CamelliaNigerⅠ(式I)为黄色无定形粉末，HR-EISI-MS中，m/z 557.1442可见[M+H]+峰(M＝556.1369)，m/z 579.1263可见[M+Na]+峰(M＝556.1366)，计算可得分子式为C31H24O10(M＝556.1363)，根据核磁共振技术，确定化合物结构，核磁数据见表1，图谱如3-9所示。
表1化合物1核磁数据(500/126MHz，CDCl3)：
CamelliaNigerⅡ(式Ⅱ)为白色无定形粉末，HR-EISI-MS中，m/z 244.0970可见[M+H]+峰(M＝243.0891)，m/z 266.0787可见[M+Na]+峰(M＝243.0890)，计算可得分子式为C14H13NO3(M＝243.0895)，根据核磁共振技术，确定化合物结构，核磁数据见表2，，图谱如10-16所示。
表2化合物2核磁数据(500/126MHz，CDCl3)：
实施例3体外抗肿瘤活性测试
对上述制备得到的两种新化合物进行体外抗肿瘤活性测试，实验细胞为肺癌细胞株H460、LLC、A549，结肠癌细胞株CT-26，乳腺癌细胞株231，宫颈癌细胞株Hela，采用MTT法进行细胞毒体外活性实验。
具体实施方法如下：
将实施例1的新化合物(即为：CamelliaNigerⅠ(式I)、CamelliaNigerⅡ(式Ⅱ))用DMSO及1640胎牛双抗培养基(或DMEM胎牛双抗培养基)配制成浓度为0.1、0.5、1.0、2.0、4.0、8.0、16.0、32.0、64.0、128.0μg/mL待测液。
处于对数期状态良好的H460、LLC、A549、CT-26、231、Hela细胞以个细胞/孔(A549为1000个细胞/孔)密度接种于96孔板中，放置于37℃、5％二氧化碳培养箱中24小时。而后向96孔板中加入上述不同浓度待测液，每孔100μL，空白组和对照组给予不完全培养基，继续培养48小时，后向每孔中加入20μL四氮噻唑蓝-磷酸盐缓冲液，放置于37℃、5％二氧化碳培养箱中4小时，在酶标仪492nm处测定每孔吸光度OD值，重复3次，取平均值。使用Logit法计算化合物对每种肿瘤细胞抑制的IC50值，结果如表3所示。
表3本发明所制备两种新化合对受试肿瘤细胞的抑制作用
经线性回归计算IC50值，结果提示本发明所制备化合物CamelliaNigerⅠ、CamelliaNigerⅡ对H460细胞作用IC50值分别是0.11±0.02、85.11±6.42ug/mL(阳性对照组顺铂IC50值是10.09±1.14ug/mL)；CamelliaNigerⅠ、CamelliaNigerⅡ对LLC细胞作用IC50值分别是7.36±1.41、26.18±2.29ug/mL(阳性对照组环磷酰胺IC50值是17.71±0.40ug/mL)；CamelliaNigerⅠ、CamelliaNigerⅡ对CT-26细胞作用IC50值分别是0.87±0.04、46.15±5.45ug/mL(阳性对照组环磷酰胺IC50值是22.15±0.71ug/mL)；CamelliaNigerⅠ、CamelliaNigerⅡ对231细胞作用IC50值分别是8.10±1.24、＞100ug/mL(阳性对照组顺铂IC50值是74.05±9.92ug/mL)；CamelliaNigerⅠ、CamelliaNigerⅡ对Hela细胞作用IC50值分别是14.15±0.91、74.44±6.20(阳性对照组奥沙利铂IC50值是18.15±2.26ug/mL)；CamelliaNigerⅠ、CamelliaNigerⅡ对A549细胞作用IC50值分别是4.08±0.33、＞100ug/mL(阳性对照组奥沙利铂IC50值是71.15±13.38ug/mL)。
综上所述，本发明所制备得到的发酵新化合物CamelliaNigerⅠ(式I)、CamelliaNigerⅡ(式Ⅱ)具有体外抗肿瘤活性，能应用于制备预防和治疗肿瘤药物。
上述说明是针对发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
Claims (6)
1.一种从金花茶内生菌代谢产物中提取的新化合物，其特征在于，结构如式I所示：
2.一种权利要求1所述的从金花茶内生菌代谢产物中提取的新化合物的制备方法，其特征在于，包括以下步骤：
(1)金花茶内生菌的分离纯化：对金花茶茎部位消毒，切成短茎，然后将短茎接种于含有青霉素-链霉素的琼脂培养基上，当短茎从内部向培养基周围长出菌落时，挑出单菌落进行纯化，得到金花茶内生菌Aspergillusniger；
(2)金花茶内生菌Aspergillusniger发酵产物的制备：将步骤(1)的金花茶内生菌Aspergillusniger接种至大米培养基表面进行发酵，得到金花茶内生菌Aspergillusniger发酵产物；
(3)将步骤(2)金花茶内生菌Aspergillusniger发酵产物捣碎，加入甲醇浸泡，过滤，收集滤液，减压浓缩，得到甲醇浸膏；
(4)将步骤(3)的甲醇浸膏用水搅拌分散成混悬液，加入乙酸乙酯萃取，过滤，收集滤液，减压浓缩，得到乙酸乙酯提取物；
(5)将步骤(4)所述乙酸乙酯提取物进行硅胶柱层析色谱分离，以体积比为100：1、50：1、20：1、10：1、5：1的三氯甲烷-甲醇梯度洗脱，收集合并相同馏分，得到6个馏分，分别记为其中馏分Fr.4为体积比为20：1的三氯甲烷-甲醇洗脱得到；
(6)将步骤(5)的馏分Fr.4浓缩，进行葡聚糖凝胶色谱分离，以体积比为5：5、7：3、9：1、1：0的甲醇-水梯度洗脱，收集合并相同馏分，得到4个馏分，分别记为 其中馏分Fr.4.2为体积比为7：3的甲醇-水洗脱得到，馏分Fr.4.3为体积比为9：1的甲醇-水洗脱得到；
(7)对步骤(6)的馏分Fr.4.2浓缩后，采用半制备高效液相色谱分离，得到式I的新化合物。
3.根据权利要求2所述的从金花茶内生菌代谢产物中提取的新化合物的制备方法，其特征在于，步骤(2)中，所述大米培养基组成为：60g大米、3g蛋白胨、37.5g水。
4.根据权利要求3所述的从金花茶内生菌代谢产物中提取的新化合物的制备方法，其特征在于，步骤(2)中，所述发酵条件为：温度：湿度：/>
5.根据权利要求2所述的从金花茶内生菌代谢产物中提取的新化合物的制备方法，其特征在于，步骤(7)中，所述半制备高效液相色谱的条件为：色谱柱选择WelchUltimateXB-C18半制备柱；流动相为乙腈和水的混合液，所述乙腈和水的体积比25：75；检测波长为210nm。
6.权利要求1所述的从金花茶内生菌代谢产物中提取的新化合物在制备治疗肿瘤药物中的应用，其特征在于，所述肿瘤为肺癌、结肠癌、乳腺癌或宫颈癌。
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