CN114717269B - 香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用 - Google Patents
香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用 Download PDFInfo
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Abstract
本发明公开了一种香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用,所述代谢产物是由橘绿木霉(Trichoderma citrinoviride)Tg.Z4‑01发酵液经超声波细胞破碎、硅胶柱层析、ODS柱层析、制备型薄层色谱获得的。香榧内生真菌代谢产物Fr.6‑8‑8的IC50=3.91µg/mL,阳性对照Vc的IC50=1.52µg/mL,Fr.6‑8‑8对DPPH的清除能力与抗坏血酸效果相似,而本发明通过微生物发酵的方法可制备得到,在一定程度上避免了植被的破坏,更加安全,环保。
Description
技术领域
本发明涉及一种对DPPH自由基具有较强清除能力的抗氧化剂,具体涉及一种香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用。
背景技术
香榧(Torreya grandis),别名榧树、榧子等,为裸子植物门,红豆杉科榧树属的常绿针叶乔木,为我国特有树种、国家二级保护植物,主产于江浙等地。榧树花期4月,种子翌年10月成熟。研究发现,榧树中化学成分的主要结构类型有黄酮类、木脂素类、二萜类、脂肪酸类、挥发油类和氨基酸类等。现代药理学研究发现其具有抗菌、驱虫、镇咳、抗病毒和抗肿瘤作用,此外香榧叶提取物被报道具有抗疲劳和抗氧化作用。
植物内生菌(Endophyte)是特指一类在其整个生活史或者生活史的部分阶段寄生于健康植物的部分细胞间隙、器官或者组织中,而不使宿主植物出现任何反常症状或者病症的微生物。从植物中能分离出具有多种生物活性的内生菌,其中部分内生菌通过次生代谢途径可以产生与植物次生代谢产物相似的产物。利用植物内生菌筛选生物活性成分或先导化合物已成为寻找天然药物的又一重要途径,也是当前植物内生菌研究的热点。癌症、衰老或其它疾病大都与过量自由基的产生有关联。研究抗氧化可以有效克服其所带来的危害,所以抗氧化被保健品、化妆品企业列为主要的研发方向之一,也是市场最重要的功能性诉求之一。
发明内容
本发明目的是提供一种香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用。
本发明采用的技术方案是:
本发明提供一种香榧内生真菌代谢产物,所述代谢产物按如下方法制备:(1)将橘绿木霉(Trichoderma citrinoviride)Tg.Z4-01发酵液经超声波细胞破碎后抽滤,滤液及滤渣用乙酸乙酯萃取,取有机相浓缩至恒重,获得代谢粗产物;所述橘绿木霉(Trichodermacitrinoviride)Tg.Z4-01保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M2022156,保藏日期为2022年2月25日,保藏地址:湖北省武汉市武昌区珞珈山武汉大学生命科学学院,邮编:430072;(2)将步骤(1)代谢粗产物用乙酸乙酯溶解后进行硅胶柱层析,以体积比100:0、100:1、100:2、100:5、100:10、100:20、50:50、0:100的二氯甲烷-甲醇梯度洗脱,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;(3)将步骤(2)组分Fr.6用乙酸乙酯溶解后再次进行硅胶柱层析,以体积比10:1-5:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;(4)组分Fr.6-8以体积比70:30的甲醇-水为洗脱剂进行ODS柱分离,收集Rf值为0.6的流出液,浓缩至干,记为组分Fr.6-8-8;(5)步骤(4)组分Fr.6-8-8用少量乙酸乙酯溶解,上样于20cm×20cm制备型薄层色谱板中,以体积比20:1的二氯甲烷:甲醇为展开剂进行薄层层析,将Rf为0.6的黄色条带用刀片刮下,收集刮下的硅胶用二氯甲烷为溶剂进行洗脱,收集洗脱液,浓缩至干,获得内生真菌代谢产物。
进一步,步骤(1)所述发酵液按如下方法制备:将橘绿木霉Tg.Z4-01接种于发酵培养基中,28℃,180r/min培养5d,获得发酵液;所述发酵培养基组成:蛋白胨10g/L,蔗糖40g/L,溶剂为蒸馏水,pH为5.6。
进一步,步骤(1)所述代谢粗产物按如下方法制备:在40%功率条件下,每超声3s,间歇4s,进行30min对发酵液超声波细胞破碎处理后抽滤,滤液用1倍体积乙酸乙酯萃取3次,滤渣用乙酸乙酯浸泡12h,合并有机相,浓缩至恒重,获得代谢粗产物。
进一步,所述步骤(2)具体操作方法为:将步骤(1)代谢粗产物用乙酸乙酯溶解,加入硅胶,混合均匀后,真空干燥,即为吸附样品的硅胶;将吸附样品的硅胶上样于硅胶色谱柱中,采用体积比100-0:0-100的二氯甲烷-甲醇梯度洗脱,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;所述硅胶与发酵粗产物质量比为1:1。
进一步,所述步骤(3)具体操作方法为:将步骤(2)组分Fr.6用乙酸乙酯溶解,加入硅胶,混合均匀后,真空干燥,即为吸附组分Fr.6的硅胶;将吸附组分Fr.6的硅胶上样于硅胶色谱柱中,以体积比10:1-5:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;所述硅胶与组分Fr.6质量比为1:1。
进一步,所述步骤(4)洗脱分离后收集Rf值为0.6的流出液,浓缩至干,记为组分Fr.6-8-8。
进一步,步骤(5)中制备型薄层色谱板为20cm×20cm,二氯甲烷:甲醇的体积比20:1。
更进一步,本发明所述香榧内生真菌代谢产物按如下方法制备:(1)将橘绿木霉Tg.Z4-01接种于发酵培养基中,28℃,180r/min培养5d,获得发酵液;将发酵液超声波细胞破碎后抽滤,滤液用1倍体积乙酸乙酯萃取3次(优选萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相),滤渣用乙酸乙酯浸泡12h,合并有机相,浓缩至恒重,获得代谢粗产物;(2)将步骤(1)代谢粗产物用微量乙酸乙酯溶解,加入硅胶(200~300目),混合均匀后,置于减压真空干燥器中干燥,即为吸附样品的硅胶,所述硅胶与发酵粗产物质量比为1:1;将吸附样品的硅胶上样于硅胶色谱柱(优选8cm×60cm)中,装柱量3/4,采用体积比100-0:0-100的二氯甲烷-甲醇梯度洗脱,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;(3)将步骤(2)组分Fr.6用乙酸乙酯溶解后再次进行硅胶柱层析,以体积比10:1-5:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;(4)组分Fr.6-8以体积比70:30的甲醇-水为洗脱剂进行ODS柱分离,收集Rf值为0.6的流出液,浓缩至干,记为组分Fr.6-8-8;(5)步骤(4)组分Fr.6-8-8用少量乙酸乙酯溶解,上样20cm×20cm制备型薄层色谱板中,以体积比20:1的二氯甲烷:甲醇为展开剂进行薄层层析,将Rf为0.6的黄色条带用刀片刮下,收集刮下的硅胶用二氯甲烷为溶剂进行洗脱,收集洗脱液,浓缩至干,获得香榧内生真菌代谢产物记为代谢产物Fr.6-8-8。
本发明所述橘绿木霉Tg.Z4-01,菌落呈白色疏松绒毛状,干燥,产大量肉眼可见黄色色素,菌落老化后成黄绿色。
本发明还提供一种香榧内生真菌代谢产物作为抗氧化剂的应用。
与现有技术相比,本发明有益效果主要体现在:香榧内生真菌代谢产物Fr.6-8的IC50=3.91μg/mL,阳性对照Vc的IC50=1.52μg/mL,Fr.6-8-8对DPPH的清除能力与抗坏血酸效果相似,而本发明通过微生物发酵的方法可制备得到,在一定程度上避免了植被的破坏,更加安全,环保。
附图说明
图1为Tg.Z4-01系统发育树。
图2为香榧内生真菌代谢产物对DPPH的清除能力。
图3为产物Fr.6-8-8主要结构。
图4为香榧内生真菌代谢产物的1H-NMR图谱。
图5为香榧内生真菌代谢产物的13C-NMR图谱。
图6为香榧内生真菌代谢产物的MS图谱。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明所述香榧,别名榧树、榧子等,为裸子植物门,红豆杉科榧树属的常绿针叶乔木,为我国特有树种、国家二级保护植物,主产于江浙等地。
本发明所述超纯水是指电阻率达到18MΩ·cm(25℃)的水,水中除了水分子外,几乎没有什么杂质,更没有细菌、病毒、含氯二噁英等有机物,也没有人体所需的矿物质微量元素。
实施例1:橘绿木霉Tg.Z4-01的分离
1.植物样本采集:新鲜健康的香榧枝条采于浙江省金华市,香榧种子用自来水清洗10分钟,然后用体积浓度75%乙醇水溶液漂洗一次。用质量浓度2%次氯酸钠水溶液浸泡10分钟后,用无菌水反复洗涤种子,再用体积浓度75%乙醇水溶液浸泡15分钟,然后用无菌水漂洗三次,收集最后一次漂洗液,用干燥的无菌吸收纸干燥,获得表面消毒后的香榧种子。在生物安全柜中,分别放置无菌PDA培养基平板作为空白对照1,用于检查生物安全柜的洁净程度;将最后一次漂洗液分别接种于无菌PDA培养基平板作为空白对照2,用于漂洗液的检查;将表面消毒后的香榧种子,置于PDA培养基的无菌平板中滚一圈后取出,作为空白对照3,用于植物组织印迹法筛选无菌的组织块。
2.内生真菌的筛选以及分离纯化:于生物安全柜中,将步骤1中消毒后的香榧种子从中间切成薄片,取假种皮部位,作为无菌组织,然后接种在PDA培养基中,在30℃倒置培养,待出现内菌丝沿着组织切口向外生长时,与空白对照1、2以及3进行比较,采用尖端菌丝挑选的方法,将不同形态的菌落划线接种于PDA培养基中,待长出单菌落后,将单菌落再次划线接种于无菌PDA培养基中,反复多次接种,直到菌落形态一致且只有一种真菌生长时说明纯化完成,得到8株真菌菌株,1号菌株为白色疏松绒毛状,干燥,产大量肉眼可见黄色色素,菌落老化后成黄绿色,无肉眼可见孢子,菌落背面为深黄色;2号菌株为白色绒毛状,有大量灰绿色孢子,菌落背面成点状,代谢物无明显颜色;3号菌株为浅橙色絮状,有大量橙色孢子,菌落背面为橙色;4号菌株为白色疏松绒毛状,干燥,菌落背面为白色;5号菌株为棕色绒毛状,无肉眼可见孢子,菌落背面为棕黑色;6号菌株为白色点状,无肉眼可见孢子,菌落背面为深黄色,菌落老化后成棕黄色;7号菌株为橙红色点状,无肉眼可见孢子,菌落背面为红色;8号菌株为白色绒毛状,无肉眼可见孢子,菌落背面为粉色点状,代谢物无明显颜色;1号菌株记为菌株Tg.Z4-01,PDA培养基组成:马铃薯200g,葡萄糖20g,琼脂15~20g,蒸馏水1000mL,pH为5.6。
3.总DNA的提取:将菌株Tg.Z4-01接种于PDA培养基中,于28℃恒温培养箱中倒置培养5d,采用Ezup柱式真菌基因组DNA抽提试剂盒(购自生工生物工程(上海)股份有限公司,产品编号:B518259-0050,以及相关操作说明提取基因组DNA:①取50-100mg新鲜真菌或20mg干燥子实体或菌丝,液氮中充分研磨成粉末后放入到1.5mL离心管中,依次加入200μLBuffer Digestion,2μlβ-巯基乙醇和20μl Proteinase K溶液,震荡混匀,65℃水浴1h至细胞完全裂解。②加入100μl Buffer PF,充分颠倒混匀,-20℃冰箱放置5min。③室温10000rpm离心5min,将上清液转移到新的1.5ml离心管中。④加入200μl Buffer BD,充分颠倒混匀。⑤加入200μl无水乙醇,充分颠倒混匀。⑥将吸附柱放入收集管中,用移液枪将溶液和半透明纤维状悬浮物全部加入吸附柱中,静置2min,再10000rpm室温离心1min,倒掉收集管中的废液。⑦将吸附柱放回收集管,加入500μl PW Solution,10000rpm离心30s倒掉收集管中的废液。⑧将吸附柱放回收集管,加入500μl Wash Solution,10000rpm离心30s倒掉收集管中的废液。⑨将吸附柱重新放回收集管中,于12000rpm室温离心2min,离去残留的WashSolution。⑩取出吸附柱,放入一个新的1.5ml离心管中,加入50μl TE Buffer静置3min,12000rpm室温离心2min,收集DNA溶液。提取的DNA可立即进行下一步实验或-20℃保存。
4.菌株Tg.Z4-01的ITS序列扩增:采用真菌扩增通用引物ITS1和ITS4扩增内部转录区间序列
ITS1:5’-TCCGTAGGTGAACCTGCGG-3’
ITS4:5’-TCCTCCGCTTATTGATATGC-3’
表1PCR反应体系
试剂 | 体积(μL) |
DNA模板 | 1 |
上游引物 | 1 |
下游引物 | 1 |
2×Hieff PCR Master Mix | 12.5 |
ddH2O | 9.5 |
体系 | 25 |
PCR扩增程序:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸1min,35个循环,72℃延伸10min,4℃低温保存。
5.PCR反应产物确认:取2.5μL PCR产物与0.5μL Loading Buffer混合后点样于1.2%琼脂糖凝胶,120V条件下电泳20分钟,于凝胶成像系统中观察条带,如出现600-750bp左右条带,则初步判断扩增成功。
6.PCR反应产物测序:将PCR产物送样北京擎科生物科技有限公司进行测序,双向测序结果使用DNAMAN进行序列拼接,菌株Tg.Z4-01的ITS序列见SEQ ID NO.1所示。
7.数据分析:将菌株Tg.Z4-01的序列在NCBI中与GenBank中的序列进行同源性比对,BLAST检索表明,菌株Tg.Z4-01的ITS序列与Trichoderma citrinoviride(橘绿木霉)(GenBank登录号NR077178.1)的序列相似度为96.73%,绘制系统发育树见图1所示,由图1可知支持率为91%,根据基因亲缘性对比结合形态学鉴定,确定菌株Tg.Z4-01为Trichoderma属,命名为橘绿木霉(Trichoderma citrinoviride),保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M 2022156,保藏日期为2022年2月25日,保藏地址:武汉大学,邮编:430072。
实施例2:香榧内生菌代谢产物的分离
1、菌株复苏活化:将保存在4℃冰箱中的橘绿木霉Tg.Z4-01接种于PDA培养基中,置于28℃恒温培养箱中培养5d;
2、香榧内生菌代谢产物的制备:在生物安全柜中,用无菌打孔器将步骤1的Tg.Z4-01菌株沿着菌落边缘打直径为5mm的菌饼,接种于含300mL发酵培养基的1500mL锥形瓶中,28℃,180r/min培养5d,以未接种菌饼的发酵培养基为空白对照。取发酵完成的发酵液,采用JY92-IIDN型超声波细胞破碎仪在40%功率条件下,每超声3s,间歇4s,进行30min对发酵液进超声破壁处理,然后抽滤得滤液,用1倍体积乙酸乙酯对浓缩液进行多次萃取,直至乙酸乙酯相肉眼观察无明显颜色变化,滤渣用乙酸乙酯浸泡12h,合并乙酸乙酯萃取相,利用旋转蒸发仪浓缩干燥至恒重,获得香榧内生菌代谢粗产物(简称:Tg-1Ea)62.82g,于-20℃保存。
所述发酵培养基组成:蛋白胨:10g/L,葡萄糖:40g/L,溶剂为蒸馏水,pH为5.6,利用高压蒸汽灭菌锅对其进行灭菌,条件为121℃,20min。
3、分离纯化代谢粗产物:(1)称取60g Tg-1Ea,用乙酸乙酯溶解,加入60g硅胶(200~300目),混合均匀后,置于减压真空干燥器中干燥,即为吸附样品的硅胶。将吸附样品的硅胶上样于硅胶色谱柱(8cm×60cm)中,装柱量3/4,采用二氯甲烷-甲醇(100:0、100:1、100:2、100:5、100:10、100:20、50:50、0:100,v/v)梯度洗脱,分别得到Fr.1~Fr.10,共10个组分,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;(2)将组分Fr.6用乙酸乙酯溶解,加入硅胶,研磨均匀后,真空干燥,即为吸附组分Fr.6的硅胶;将吸附组分Fr.6的硅胶上样于硅胶色谱柱中,以体积比7:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;(3)组分Fr.6-8用ODS柱以甲醇-水(70:30,v/v)的为洗脱剂进行柱分离流速为1drop/s,每10mL收集一份,相邻组分颜色相同进行合并浓缩,颜色不同者浓缩后点样于点样于薄层硅胶板进行层析(展开剂为二氯甲烷:甲醇=10:1,v/v),结果与相邻组分对比,具有相同Rf值的组分再次合并,收集得到8-20份均为黄色,层析后具有相似的Rf值,将Rf值为0.6的组分合并,浓缩至干,记为组分Fr.6-8-8;(4)步骤(3)组分Fr.6-8-8用少量乙酸乙酯溶解,上样于20cm×20cm制备型薄层色谱板中,以二氯甲烷:甲醇(20:1,v/v)的为展开剂进行薄层层析,将Rf为0.6的黄色条带用刀片刮下,收集刮下的硅胶用二氯甲烷为溶剂进行洗脱,收集洗脱液,浓缩至干,得到浅黄色粉末;(5)步骤(4)浅黄色粉末用甲醇溶解后,点样于薄层硅胶板(型号:GF254)上,以二氯甲烷:甲醇=10:1(v/v)为展开剂进行薄层层析,用硫酸乙醇显色观察均为一个点,Rf为0.6,获得21.38mg。香榧内生真菌代谢产物,记为代谢产物Fr.6-8-8。将样品溶于氘代氯仿中,进行核磁波谱以及质谱分析,波谱数据见于图4-6,主要成分为bisorbicillinolide。
实施例3:香榧内生菌代谢产物DPPH清除能力的测定
1.称取10mg实施例2方法制备的香榧内生真菌代谢产物Fr.6-8-8,溶于1mL DMSO中,配置10mg/mL的样品储备液,取200μL置于96孔板中D2以及E2孔,用无水乙醇半倍稀释为不同浓度(5,2.5,1.25,0.625,0.3125,0.1562,0.0781,0.0391,0.0195mg/mL)分别置于D3-D11,E3-E11孔,D1、D12和E1、E12孔空白;
2.称取10mg抗坏血酸,1mL DMSO中,配置10mg/mL的阳性供试储备液,取200μL置于96孔板中F2以G2孔,用无水乙醇半倍稀释为不同浓度(5,2.5,1.25,0.625,0.3125,0.1562,0.0781,0.0391,0.0195mg/mL)分别置于F3-F11,G3-G11孔,F1、F12和G1、G12孔空白;
3.取200μL DMSO置于96孔板中B2以C2孔,用无水乙醇半倍稀释为不同浓度(5,2.5,1.25,0.625,0.3125,0.1562,0.0781,0.0391,0.0195mg/mL)分别置于B3-B11,C3-C11孔,B1、B12和C1、C12孔空白;
4.准确称取DPPH 8.0mg,用无水乙醇溶解转入200mL容量瓶中定容,配置成0.08mmol/L的DPPH溶液;
5.0.08mmol/L的DPPH溶液20μL加入B2-B11,D2-D11以及F2-F11孔中,C2-C11,E2-E11以及G2-G11中加入20μL无水乙醇为空白对照,轻微震荡后置避光反应30min;
6.30min后将96孔板置于酶标仪中测定517nm波长下的吸光度值,重复三次测量,根据公式计算清除率。
清除率%=(A0-As)/A0×100%(1)
式(1),A0是对照反应在517nm处的吸光度(包括除样品之外的所有试剂),而As是样品在517nm处的吸光度,测量重复三次,选择Vc作为阳性对照。
结果:由图2可得Fr.6-8-8的IC50=3.91μg/mL,阳性对照Vc的IC50=1.52μg/mL,Fr.6-8-8对DPPH的清除能力与抗坏血酸效果相似。
序列表
<110> 浙江工业大学
<120> 香榧内生真菌代谢产物的制备方法及其作为抗氧化剂的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 666
<212> DNA
<213> 橘绿木霉(Trichoderma citrinoviride)
<400> 1
acactcggcc aggtctccgt aggggtgacc tgcggaggga tcattacysa gwwwwyaact 60
cccaaaccca atgtgaacgt taccaatctg ttgcctcggc gggattctct gccccgggcg 120
cgtcgcagcc ccggatccca tggcgcccgc cggaggacca actcaaactc tttttttctc 180
tccgtcgcgg cctacgtcgc ggctctgttt tatttttgct ctgagccttt ctcggcgacc 240
ctagcgggcg tctcgaaaat gaatcaaaac tttcaacaac ggatctcttg gttctggcat 300
cgatgaagaa cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc 360
gaatctttga acgcacattg cgcccgccag tattctggcg ggcatgcctg tccgagcgtc 420
atttcaaccc tcgaacccct ccggggggtc ggcgttgggg atcggcccct caccgggccg 480
cccccgaaat acagtggcgg tctcgccgca gcctctcctg cgcagtagtt tgcacactcg 540
caccgggagc gcggcgcggc cacagccgta aaacacccca aactctgaaa tgttgacctc 600
ggatcagktw gkwwtacccg ctgaacttaa gcatatcata aagcgggagg aattttattt 660
ttattt 666
Claims (8)
1. 一种香榧内生真菌代谢产物的制备方法,其特征在于所述代谢产物按如下方法制备:(1)将橘绿木霉(Trichoderma citrinoviride) Tg.Z4-01发酵液经超声波细胞破碎后抽滤,滤液及滤渣用乙酸乙酯萃取,收集有机相浓缩至恒重,获得代谢粗产物;所述橘绿木霉(Trichoderma citrinoviride) Tg.Z4-01保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M 2022156,保藏日期为2022年2月25日,保藏地址:湖北省武汉市武昌区珞珈山武汉大学生命科学学院,邮编:430072;(2)将步骤(1)代谢粗产物用乙酸乙酯溶解后进行硅胶色谱柱层析,以体积比依次为100:0、100:1、100:2、100:5、100:10、100:20、50:50、0:100的二氯甲烷-甲醇梯度洗脱,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;(3)将步骤(2)组分Fr.6用乙酸乙酯溶解后再次进行硅胶色谱柱层析,以体积比10:1-5:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;(4)组分Fr.6-8以体积比70:30的甲醇-水为洗脱剂进行ODS柱分离,收集Rf值为0.6的流出液,浓缩至干,记为组分Fr.6-8-8;(5)步骤(4)组分Fr.6-8-8用少量乙酸乙酯溶解,上样于制备型薄层色谱板中,以二氯甲烷和甲醇为展开剂进行薄层层析,将Rf为0 .6的黄色条带用刀片刮下,收集刮下的硅胶用二氯甲烷为溶剂进行洗脱,收集洗脱液,浓缩至干,获得内生真菌代谢产物。
2. 如权利要求1所述香榧内生真菌代谢产物的制备方法,其特征在于步骤(1)所述发酵液按如下方法制备:将橘绿木霉Tg.Z4-01接种于发酵培养基中,28 ℃,180 r/min培养5d,获得发酵液;所述发酵培养基组成:蛋白胨10 g/L,蔗糖40 g/L,溶剂为蒸馏水,pH为5.6。
3. 如权利要求1所述香榧内生真菌代谢产物的制备方法,其特征在于步骤(1)所述代谢粗产物按如下方法制备:在40%功率条件下,每超声3s,间歇4s,进行30 min对发酵液超声细胞破碎处理后抽滤,滤液用等体积乙酸乙酯萃取3次,滤渣用乙酸乙酯浸泡12 h,合并有机相,浓缩至恒重的制备方法,获得代谢粗产物。
4.如权利要求2所述香榧内生真菌代谢产物的制备方法,其特征在于步骤(2)的具体操作过程为:将步骤(1)代谢粗产物用乙酸乙酯溶解,加入硅胶,混合均匀后,真空干燥,即为吸附样品的硅胶;将吸附样品的硅胶上样于硅胶色谱柱中,采用体积比依次为100:0、100:1、100:2、100:5、100:10、100:20、50:50、0:100的二氯甲烷-甲醇梯度洗脱,收集体积比100:2二氯甲烷-甲醇的流出液,浓缩至干,记为组分Fr.6;所述硅胶与代谢粗产物质量比为1:1。
5.如权利要求1所述香榧内生真菌代谢产物的制备方法,其特征在于步骤(3)的具体操作过程为:将步骤(2)组分Fr.6用乙酸乙酯溶解,加入硅胶,混合均匀后,真空干燥,即为吸附组分Fr.6的硅胶;将吸附组分Fr.6的硅胶上样于硅胶色谱柱中,以体积比10:1-5:1石油醚-乙酸乙酯为流动相洗脱,收集第8个柱体积的流出液,浓缩至干,记为组分Fr.6-8;所述硅胶与组分Fr.6质量比为1:1。
6.如权利要求1所述香榧内生真菌代谢产物的制备方法,其特征在于步骤(5)中制备型薄层色谱板为20cm×20cm,二氯甲烷:甲醇的体积比20:1。
7.如权利要求1所述香榧内生真菌代谢产物的制备方法,其特征在于以体积比依次为100:0、100:1、100:2、100:5、100:10、100:20、50:50、0:100的二氯甲烷-甲醇梯度洗脱,每组梯度分别洗脱十个柱体积。
8.一种采用权利要求1所述的制备方法制备得到的香榧内生真菌代谢产物作为抗氧化剂的应用。
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