CN114854804B - 球毛壳菌素e的制备方法及应用 - Google Patents
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Abstract
本发明属于天然药物化学技术领域，公开了球毛壳菌素E的制备方法及应用。所述球毛壳菌素E可应用于制备抗肿瘤药物和抗炎药物，将球毛壳菌素E作为有效成分与药用辅料混合并制成药物，本发明球毛壳菌素E能够抑制U937细胞增殖，能够显著抑制NO的生成。
Description
技术领域
本发明属于天然药物化学技术领域，具体涉及球毛壳菌素E的制备方法及应用。
背景技术
真菌是生物多样性最为丰富的类群之一，目前近一半的具有生物活性的微生物次级代谢产物由丝状真菌产生，真菌已成为生物活性次级代谢产物的重要来源。毛壳属(Chaetomium)是子囊菌门中一个较大的属，超过400多个种。毛壳属真菌经常在植物、土壤、动物和粪便中发现。近年来发现毛壳属真菌能够产生具有多种生物学活性的次级代谢产物，其次级代谢产物化学结构多样、化学成分复杂，且具有多种生物活性，例如抗微生物，免疫调节和抗癌等。毛壳属真菌的主要代谢产物有细胞松弛素(Cytochalasans)类、嗜氮酮(Azaphilones)类、酚酸环醚类(Depsidones)、蒽醌类(Anthraquinones)和甾体类(Steroids)等，其中细胞松弛素类和嗜氮酮类化合物为毛壳属真菌的主要次级代谢产物，表现出细胞毒、抗微生物和抗氧化等多种生物活性。
发明内容
为了克服现有技术的不足，本发明提供一种球毛壳菌素E的制备方法及应用。
本发明的上述目的是通过以下技术方案实现的：球毛壳菌素E的制备方法，包括以下步骤：
S1.将PDA培养基中的菌种用接种环接种于真菌培养基中培养，将其放置于摇床上，在温度28℃、转速120r/min条件下震荡培养7天；
S2.将步骤S1所述真菌培养基发酵液及步骤S1中菌种的菌丝体以10％比例的接种量接种于固体培养基中，28℃下发酵40天；
S3.步骤S2所述固体培养基中的菌株发酵完成后，用甲醇对发酵产物超声提取3次，每次15min，过滤后合并滤液，减压浓缩得浸膏，随后将浸膏分散在水中，用等体积的乙酸乙酯萃取3次，减压浓缩得乙酸乙酯萃取物；
S4.取步骤S3所述的乙酸乙酯萃取物经正相硅胶柱色谱分离，分别以二氯甲烷-乙酸乙酯比为10:1、5:1、3:1、1:1、0:1，乙酸乙酯-甲醇比为9:1、8:2、7:3作为流动相梯度洗脱；将洗脱下来的流分经硅胶薄层板检识，合并相同流分，其中F20为二氯甲烷-乙酸乙酯比为0:1洗脱流分经检识后合并的部分；
S5.将步骤S4所得的F20经正相硅胶柱色谱分离，以三氯甲烷-丙酮比为10:1、5:1、3:1、1:1、0:1作为流动相梯度洗脱，将洗脱下来的流分经硅胶薄层板检识，合并相同流分。其中由三氯甲烷-丙酮比为10:1洗脱下的流分有白色沉淀析出，经洗涤过滤，得化合物球毛壳菌素E；
其中步骤S1所述的菌种为毛壳属真菌菌种Chaetomium globosum。
本发明的另一个目的是保护上述球毛壳菌素E的应用，包括球毛壳菌素E在制备抗肿瘤药物和抗炎药物上的应用。
进一步的，所述球毛壳菌素E在制备抗肿瘤药物和抗炎药物上的应用包括通过将球毛壳菌素E作为有效成分与药用辅料混合并制成药物的应用。
本发明与现有技术相比的有益效果是：本发明球毛壳菌素E能够抑制人白血病U937细胞增殖；球毛壳菌素E能够显著抑制LPS诱导的RAW264.7细胞NO的生成，降低炎症反应；本发明球毛壳菌素E的制备方法提高了球毛壳菌素E的收率，从50g乙酸乙酯萃取物中分离得到1.45g球毛壳菌素E。
附图说明
下面结合附图与具体实施方式对本发明作进一步说明
图1是不同剂量的球毛壳菌素E对RAW 264.7炎症模型NO生成量的影响曲线图。
具体实施方式
下面通过具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。本实施例中涉及的毛壳属真菌菌种Chaetomiumglobosum为本领域已知菌种，下述实施例采用保藏编号为CGMCC:3.15616的毛壳属真菌菌种为例，详述本发明。
实施例1
球毛壳菌素E的制备方法：
(1)将PDA培养基中的菌种用接种环接种于真菌4号培养基(甘露醇20g，葡萄糖20g，酵母膏5g，蛋白胨10g，KH2PO40.5g，MgSO47H2O 0.3g，玉米浆1g，H2O 1000mL)中培养，将其放置于摇床上，在温度28℃、转速120r/min条件下震荡培养7天。
(2)将真菌4号培养基发酵液及菌丝体以10％比例的接种量接种于固体培养基中，28℃下发酵40天。
(3)菌株发酵完成后，用甲醇对发酵产物超声提取3次，每次15min，过滤后合并滤液，减压浓缩得浸膏，随后将浸膏分散在水中，用等体积的乙酸乙酯萃取3次，减压浓缩得乙酸乙酯萃取物。
(4)取乙酸乙酯萃取物经正相硅胶柱色谱分离，分别以二氯甲烷-乙酸乙酯(10:1、5:1、3:1、1:1、0:1)、乙酸乙酯-甲醇(9:1、8:2、7:3)为流动相梯度洗脱。将洗脱下来的流分经硅胶薄层板检识，合并相同流分，其中F20为二氯甲烷-乙酸乙酯(0:1)洗脱流分经检识后合并的部分。
(5)步骤(4)所得的F20，经正相硅胶柱色谱分离，以三氯甲烷-丙酮(10:1、5:1、3:1、1:1、0:1)为流动相梯度洗脱，将洗脱下来的流分经硅胶薄层板检识，合并相同流分。其中由三氯甲烷-丙酮(10:1)洗脱下的流分有白色沉淀析出，经洗涤过滤，得化合物球毛壳菌素E，产率比为2.9g/100g乙酸乙酯萃取物。
实施例2
球毛壳菌素E的结构鉴定
综合使用质谱分析技术(ESI-MS)、核磁共振分析技术(1H-NMR、13C-NMR)等技术对物质结构进行鉴定。
测定结果：
球毛壳菌素E：白色粉末，分子式为C32H38N2O5，溶于DMSO。ESI-MS：m/z 553.26[M+Na]+。其13C-NMR(125MHz，DMSO)显示在210.22处显示为羰基信号，而在174.04处同样也出现酰胺信号，在110.04～136.23之间有几个同样也显示了苯环上的碳信号。1H-NMR(500MHz，DMSO)在1.00，1.11，1.53和1.71处均显示了甲基信号，在6.98～7.40等处显示为苯环上的氢信号。将其与文献中的数据进行比对，鉴定该化合物为球毛壳菌素E(ChaetoglobosinE)，具有如下结构式，核磁数据归属见表1。
球毛壳菌素E的结构式
表1.化合物球毛壳菌素E的1H-NMR和13C-NMR数据
实施例3
球毛壳菌素E对U937细胞增殖的影响
1.U937细胞培养
人淋巴瘤白血病细胞株U937培养于含有10％热灭活小牛血清的RPMI 1640培养液中，37℃、5％CO2，饱和湿度条件下培养。
2.抑制U937细胞增殖体外实验
收集对数生长期的U937细胞，接种于96孔培养板，每孔细胞数4×103，设空白组、对照组、药物处理组，其中药物处理组每组加入不同浓度的球毛壳菌素E，其终浓度分别为0、12.5、25、50、100、200μM，对照组用DMSO代替药物，空白组用培养液代替细胞悬液和药物，各组设6个平行孔，置37℃，5％CO2条件下培养24h。培养结束前4h每孔加入20μL MTT溶液(5mg/mL)，继续培养4h，弃上清液，加入200μL DMSO充分溶解结晶物，置酶标仪上测490nm处吸光度值(OD值)。以对照组细胞存活率的均值为100％，按下式计算药物处理组细胞存活率：
药物处理组细胞存活率＝药物处理组OD值/对照组OD值的均值×100％结果以细胞存活率的均值±标准差(n＝6)表示，结果表明球毛壳菌素E对U937细胞增殖具有抑制作用，其IC50为102.5±2.9μM。
实施例4
球毛壳菌素E的抗炎活性
1.RAW264.7细胞培养
巨噬细胞RAW 264.7培养于含有10％热灭活小牛血清的DMEM完全培养基中，37℃、5％CO2，饱和湿度条件下培养。
2.NO生成量的测定
收集对数生长期的RAW 264.7细胞，接种于96孔培养板，每孔细胞数2×104。分别设空白组、LPS模型组以及药物处理组。其中LPS模型组及药物处理组均加入同等浓度的LPS构建炎症模型，LPS浓度为100ng/mL。药物处理组球毛壳菌素E终浓度分别为1.56、3.12、6.25、12.50、25、50、100、200μM，处理24小时。培养结束后取培养液上清加入96孔板，每孔50μL，然后加入NO检测试剂盒中的Griess Reagent I，每孔50μL，再加入Griess Reagent II，每孔50μL，培养10min。最后使用酶标仪，在540nm处测定每孔的OD值，计算NO生成量，结果如图1。
结果表明球毛壳菌素E能够显著抑制NO生成，IC50值为7.10±2.11μM，提示球毛壳菌素E具有较强的抗炎活性。
以上所述实施方式仅为本发明的优选实施例，而并非本发明可行实施的全部实施例。对于本领域一般技术人员而言，在不背离本发明原理和精神的前提下对其所作出的任何显而易见的改动，都应当被认为包含在本发明的权利要求保护范围之内。
Claims (3)
1.球毛壳菌素E的制备方法，其特征在于，包括以下步骤：
S1.将PDA培养基中的菌种用接种环接种于真菌培养基中培养，将其放置于摇床上，在温度28℃、转速120r/min条件下震荡培养7天；
S2.将步骤S1所述真菌培养基发酵液及菌丝体以10％比例的接种量接种于固体培养基中，28℃下发酵40天；
S3.步骤S2所述固体培养基中的菌株发酵完成后，用甲醇对发酵产物超声提取3次，每次15min，过滤后合并滤液，减压浓缩得浸膏，随后将浸膏分散在水中，用等体积的乙酸乙酯萃取3次，减压浓缩得乙酸乙酯萃取物；
S4.取步骤S3所述的乙酸乙酯萃取物经正相硅胶柱色谱分离，分别以二氯甲烷-乙酸乙酯比为10:1、5:1、3:1、1:1、0:1，乙酸乙酯-甲醇比为9:1、8:2、7:3作为流动相梯度洗脱；将洗脱下来的流分经硅胶薄层板检识，合并相同流分，其中F20为二氯甲烷-乙酸乙酯比为0:1洗脱流分经检识后合并的部分；
S5.将步骤S4所得的F20经正相硅胶柱色谱分离，以三氯甲烷-丙酮比为10:1、5:1、3:1、1:1、0:1作为流动相梯度洗脱，将洗脱下来的流分经硅胶薄层板检识，合并相同流分；其中由三氯甲烷-丙酮比为10:1洗脱下的流分有白色沉淀析出，经洗涤过滤，得化合物球毛壳菌素E；
其中步骤S1所述的菌种为毛壳属真菌菌种Chaetomium globosum，保藏编号为CGMCC:3.15616。
2.基于权利要求1所述的球毛壳菌素E的应用，其特征在于，包括球毛壳菌素E在制备抗白血病药物和抗炎药物上的应用。
3.根据权利要求2所述的球毛壳菌素E的应用，其特征在于，包括通过将球毛壳菌素E作为有效成分与药用辅料混合并制成药物的应用。
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