WO2016082186A1 - 一种具有细胞毒活性的组合物及其制备方法和应用 - Google Patents

一种具有细胞毒活性的组合物及其制备方法和应用 Download PDF

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WO2016082186A1
WO2016082186A1 PCT/CN2014/092485 CN2014092485W WO2016082186A1 WO 2016082186 A1 WO2016082186 A1 WO 2016082186A1 CN 2014092485 W CN2014092485 W CN 2014092485W WO 2016082186 A1 WO2016082186 A1 WO 2016082186A1
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ethanol
fermentation
concentrated
composition
endophytic
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郭文娟
王娜娜
董利鹏
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天津工业大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

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  • the invention belongs to the field of biotechnology, and particularly relates to a composition having cytotoxic activity, a preparation method and application thereof.
  • Tumors are the most serious diseases that endanger human life and health. Their prevention and treatment has become a problem related to the future and destiny of civilization, and has attracted the attention of the medical community all over the world. In China, the number of malignant tumor deaths among urban residents has ranked first among various causes.
  • the development of new anti-tumor drugs has become an urgent issue.
  • the development of natural products is an effective way to develop new anti-tumor drugs.
  • 61% are derived from natural products.
  • the large-scale development and demand of natural products have caused serious damage to natural wild drug resources, such as yew, which is an evergreen coniferous plant of Taxus, a rare and endangered species left over from the Quaternary glaciers.
  • Mangrove plants are also one of the world's key conservation targets. Mangrove plants are unique plant communities in the intertidal zone of tropical and subtropical seas. They are land-to-ocean transitional ecosystems. They are in the intertidal zone of high salt, frequent tidal, strong winds, high temperatures, strong ultraviolet radiation and anoxic sludge. surroundings.
  • Marine mangrove plants have many biological activities such as anti-AIDS, anti-tumor, anti-bacterial and anti-oxidation.
  • two cardiac glycoside compounds isolated from sea mango have anti-value-adding and anti-estrogen activity, and can significantly inhibit human colon cancer.
  • Growth of cell lines; a diterpenoid isolated from sea lacquer has antitumor effects and the like.
  • the mangroves due to its unique living environment, the mangroves have ecological functions such as wind protection, wave elimination, bank protection and environmental purification. The large-scale harvesting of mangroves has caused great damage to the marine wetlands and their surrounding ecological environment, and led to the coast. Protection and loss of ecological regulation.
  • Endophytic fungi are widely found in plants and refer to a certain stage or all of their life history. The stage lives in the intercellular spaces of cells or organs of organs of healthy plants. The medicinal value of endophytic fungi is derived from the long-term co-evolution and gene exchange with host plants and the results of their own intentional gene expression.
  • Stierle et al. first isolated an endophytic fungus, Taxus cepacia, from the phloem of Taxus chinensis, which produces the antitumor compound paclitaxel. This provides a possibility for plant endophytic fungi to replace plants for the development of natural medicines. Subsequently, endophytic fungi capable of producing anti-tumor components such as vinblastine, podophyllotoxin and maytansine were isolated from plant tissues such as periwinkle, peach seven and Meidengmu [9]. At the same time, endophytic fungi as new species usually have new functional genes, and the expression of their own specific genes can produce new natural products.
  • endophytic fungi are rich in ecological diversity and various biological activities, such as the new compound Botryorhodines A-D with weak cytotoxic activity isolated from the endophytic fungus Botryosphaeria rhodina of the medicinal plant Bidens pilosa. Seven new compounds were isolated from the endophytic fungus Alternaria sp, a medicinal plant of Polygonum senegalense, of which five had cytotoxic activity against the L5178Y cell line, and so on.
  • endophytic fungi also have the advantages of easy cultivation, easy control, rapid growth and reproduction, no restrictions on seasons, climate and geography, and can be improved by means of mutation breeding, cell fusion and genetic engineering. Performance, increase the yield of active ingredients, and become a biological source with great development potential.
  • a first object of the present invention is to provide a composition having cytotoxic activity which is prepared by the following method:
  • composition is prepared by the following method:
  • the endophytic fungus Diaporthe sp. of marine mangrove plant is fermented to obtain mycelium and fermentation broth, and concentrated. And drying, the fermentation medium is glucose 0.5%, peptone 0.2%, yeast extract 0.5%, crude sea salt 0.5%, the fermentation method is specifically: the initial pH of the medium is 7.4, the fermentation temperature is 25 ° C, The inoculation amount was 3%, the culture rotation speed was 180 r/min, the culture was carried out for 5 days, and filtration;
  • the dried product obtained in the step 1) is refluxed 2-3 times with a concentration of 20-50 times by volume of 70-80% ethanol, extracted for 1-2 hours each time, filtered, and the combined extracts are used to recover ethanol. And concentrated to a non-alcoholic taste, the sample concentration is about 1g dry weight / 20mL;
  • the concentrated extract is passed through a macroporous resin column, the loading amount is 1-2 times of the column volume, the loading speed is 3-5 mL/min, the adsorption is 15-30 min, and the column volume is 3-5 times. Rinse and elute with 3-5 column volumes with 70-80% ethanol. The resulting eluate is concentrated and obtained.
  • the endophytic fungus Diaporthe sp. is prepared by collecting marine red sea sang (Sonneratia hainanensis), after surface disinfection, cutting into small pieces of 0.5 cm ⁇ 0.5 cm, and placing it in an endophytic culture solid medium. The hyphae were picked and purified, and the strains were identified, and 16 endophytic fungi were isolated, and the strains were stored in an ultra-low temperature refrigerator; the endophytic culture solid medium was glucose 0.5%, peptone 0.2%, Yeast extract 0.5%, crude sea salt 0.5%; agar 2%.
  • the endophytic fungus is inoculated into the plate by the test tube strain before the culture, and then inoculated into the shake flask for liquid culture, and the isolation, identification and activity detection of the endophytic bacteria of the fragrant aconite (Wang Nana, Qin Baofu, Liu Jiandang, etc.) Isolation, Identification and Activity Detection of Endophytes from Aconite[J].Chinese Agricultural Science Bulletin,2009,25(8):46-49)
  • the macroporous adsorption resin is any one of styrene, phenylpropionic acid, and acrylamide, and is preferably an AB8 type macroporous resin or a D101 type macroporous resin.
  • Another object of the present invention is to provide a process for the preparation of the above composition, which comprises the following steps:
  • Another object of the present invention is to provide the use of the above composition for the preparation of a medicament for treating tumors.
  • the raw material is derived from the endophytic fungus Diaporthe sp. of marine mangrove plant, and the microorganism can be artificially cultured, the fermentation cycle is short and is not controlled by seasons and climate, and the fungus Diaporthe sp. Fermentation is carried out and extraction is carried out to obtain a cytotoxic active composition.
  • compositions provided herein can treat tumors.
  • the endophytic fungus Diaporthe sp can be prepared by the method of the prior literature or by the following method: collecting marine red sea sang (Sonneratia hainanensis), after surface disinfection, cut into small pieces of 0.5 cm ⁇ 0.5 cm, placed inside The fungus is cultured in a solid medium, and the hyphae are picked and purified, until the strain becomes a single colony, and the strain is identified, and 16 endophytic fungi are isolated, and the strain is stored in an ultra-low temperature refrigerator.
  • the endophytic fungus needs to be pretreated, inoculated into the plate by the test tube strain, and then inoculated into a shake flask for liquid culture, which are prepared by a conventional method.
  • the endophytic fungus Diaporthe sp of marine mangrove plants was fermented.
  • the initial pH of the medium was 7.4, the inoculum was 3%, the fermentation temperature was 25 ° C, the culture speed was 180 r/min, the culture was carried out for 5 days, and the nylon cloth was filtered.
  • the cells and the fermentation broth are separated to obtain a mycelium, and the cells are dried and pulverized by a pulverizer, and passed through a 20 mesh sieve.
  • the medium of the fermentation culture is: glucose 0.5%, peptone 0.2%, yeast paste 0.5%, Crude sea salt 0.5%;
  • Alcohol extraction 30 times of 70% ethanol added to the raw material is refluxed and extracted 3 times, each time for 1 hour, filtered, combined with extract, and the extract is subjected to vacuum recovery of ethanol, and concentrated to a macroporous resin loading concentration. (1 g dry weight / 20 mL);
  • Fermentation fermentation of endophytic fungus Diaporthe sp of marine mangrove plants (the initial pH of the medium is 7.4, the fermentation temperature is 25 ° C, the inoculum is 3%, the culture speed is 180 r/min, the culture is 5 days, nylon The cloth is filtered, the bacteria and the fermentation liquid are separated, and the fermentation liquid is obtained, and concentrated to (about 500 mL), wherein the medium formula is: glucose 0.5%, peptone 0.2%, yeast extract 0.5%, crude sea salt 0.5%;
  • Alcohol extraction ethanol precipitation, adjusting the final concentration of ethanol to 70%, standing overnight, filtering, taking the supernatant, recovering the ethanol under reduced pressure, and concentrating to a macroporous resin loading concentration (1 g dry weight / 20 mL).
  • the preparation process is, the pH is 5.5, the loading flow rate is 4mL / min, the maximum sample loading is 1.5 times the resin bed volume, the adsorption time is 20min, first use 3 times the resin bed The volume of water is eluted to remove impurities, and then 70% ethanol is used as an eluent. The elution rate is 3 mL/min, and the column volume is eluted by 3 times. The eluent is recovered and concentrated, and finally dried. .
  • Fermentation fermentation of the endophytic fungus Diaporthe sp of marine mangrove plants.
  • the initial pH of the medium is 7.4, the fermentation temperature is 25 ° C, the inoculum is 3%, the culture speed is 180 r/min, and the culture is 5 days.
  • Filtration separation of the cells and fermentation broth, after which the mycelium is obtained.
  • the cells are dried and pulverized by a pulverizer and passed through a 20 mesh sieve.
  • the medium formula is: glucose 0.5%, peptone 0.2%, yeast extract 0.5%, coarse Sea salt is 0.5%.
  • Alcohol extraction 30 times of 70% ethanol added to the raw material is refluxed and extracted 3 times, each time for 1 hour, filtered, combined with extract, and the extract is subjected to vacuum recovery of ethanol, and concentrated to a macroporous resin loading concentration. (1 g dry weight / 20 mL).
  • the preparation process is, the pH is 5.5, the loading flow rate is 3mL / min, the maximum sample volume is 1 times the resin bed volume, the adsorption time is 20min, first use 3 times the resin bed The volume of water is eluted to remove impurities, and then 70% ethanol is used as an eluent. The elution rate is 2 mL/min, and the column volume is eluted by 4 times. The eluent is recovered and concentrated, and finally dried. .
  • the composition for cytotoxic activity disclosed in the present invention is obtained by extracting the fermentation product of the endophytic fungus Diaporthe sp. of marine mangrove plant, and the tumor can be treated alone or together with other components.
  • the raw material is derived from endophytic fungi of marine mangrove plants Diaporthe sp., which utilizes microorganisms for artificial culture, has a short fermentation cycle and is not subject to seasonal and climate control.

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Abstract

一种具有细胞毒活性的组合物，其由以下方法制备：1)海洋红树植物内生真菌 Diaporthe sp.发酵得到菌丝体和发酵液；2)将发酵得到的菌丝体和发酵液用浓度为60-90％的乙醇回流提取2-3次，每次提取0.5-3小时，滤过，合并提取液，回收乙醇并浓缩；3)将浓缩后的提取液过大孔树脂柱，用水冲洗，用乙醇洗脱，得到的洗脱液浓缩，即得。

Description

一种具有细胞毒活性的组合物及其制备方法和应用 技术领域
本发明属于生物技术领域，具体涉及一种具有细胞毒活性的组合物及其制备方法和应用。
背景技术
肿瘤是危害人类生命和健康最严重的一类疾病，其防治已成为关系人类前途和命运的问题，被全世界医学界所关注。在我国，城市居民恶性肿瘤死亡人数已居各类病因的首位。抗肿瘤新药的研发已经成为刻不容缓的问题。天然产物的开发是研发抗肿瘤新药的一条有效途径，近年来上市的抗癌新药中，61％来自于天然产物。然而，天然产物的大规模开发与需求导致天然野生药物资源遭到严重破坏，例如红豆杉，是红豆杉科红豆杉属常绿针叶植物，是第四纪冰川遗留下来的世界珍稀濒危物种，素有“植物黄金”之称。1995年，从红豆杉中开发出抗肿瘤天然产物紫杉醇上市后，使红豆杉的资源急剧减少，致使我国不得不立法保护红豆杉资源。
红树植物(Mangrove plants)也是世界的重点保护对象之一。红树植物是热带、亚热带海区潮间带特有的植物群落，是陆地到海洋过渡的生态系，处于高盐、频繁的潮汐、强风、高温、强紫外线辐射和缺氧污泥的海陆潮间带环境。
海洋红树植物具有抗艾滋病，抗肿瘤、抗菌和抗氧化等多种生物活性，如从海芒果中分离的两个强心苷类化合物具有抗增值和抗雌激素活性，能明显抑制人体结肠癌细胞系的生长；从海漆中分离的一个二萜类化合物具有抗肿瘤作用等。同时，因其独特的生存环境，使红树林具有防风、消浪、护岸、净化环境等生态功能，而红树林的大规模采伐对海洋湿地及其周围生态环境造成了巨大的破坏，并导致海岸的防护与生态调节功能丧失。
因此，海洋红树林已成为全球海洋生物多样性保护的重点对象，开发红树林的替代药用资源成为保护红树林的有效途径之一。
植物内生真菌广泛存在于植物体内，是指在其生活史的一定阶段或全部 阶段生活于健康植物的各种组织和器官的细胞间隙或细胞内的真菌。内生真菌的药用价值，来源于其与寄主植物长期共同进化与基因相互交换以及自身特意基因表达的结果。
1993年，Stierle等首次从短叶红豆杉的韧皮部分离到一株产抗肿瘤化合物紫杉醇的内生真菌安德氏紫杉霉，这为植物内生真菌替代植物进行天然药物的开发提供了可能，随后，从长春花、桃儿七和美登木等植物组织内分离出分别能产长春碱、鬼臼毒素和美登素等抗肿瘤成分的内生真菌[9]。同时，内生真菌作为新的物种通常都会有新的功能基因，自身特意基因的表达可产生新的天然产物。研究表明内生真菌具有丰富的生态多样性和多种生物活性，如从药用植物Bidens pilosa的内生真菌Botryosphaeria rhodina分离到具有弱细胞毒活性的新化合物Botryorhodines A-D。从埃及一种药用植物Polygonum senegalense的内生真菌Alternaria sp分离到7个新化合物，其中5个对L5178Y细胞株具有细胞毒活性，等等。
除此之外，内生真菌还具有微生物易培养、易控制、生长繁殖迅速、不受季节、气候和地域的限制等优点，并可以通过诱变育种、细胞融合和基因工程等手段改良菌种性能，提高活性成分产量，成为一种开发潜力巨大的生物药源。
发明内容
本发明的目的是提供一种具有细胞毒活性的组合物。
本发明的第一目的是提供一种具有细胞毒活性的组合物，其由以下方法制备：
1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液；
2)将发酵得到的菌丝体和发酵液用浓度为60-90％的乙醇提取2-3次，每次提取0.5-3小时，滤过，合并提取液，回收乙醇并浓缩；
3)将浓缩后的提取液过大孔树脂柱，用水冲洗，用乙醇洗脱，得到的洗脱液浓缩，即得。
优选地，所述组合物由以下方法制备：
1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液，浓缩， 并干燥，所述发酵用的培养基为葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％，发酵的方法具体为：培养基的初始pH值为7.4，发酵温度为25℃，接种量为3％，培养转速为180r/min，培养5天，过滤；
2)将步骤1)得到的干燥物用其重量20-50倍体积的浓度为70-80％乙醇回流提取2-3次，每次提取1-2小时，滤过，合并提取液，回收乙醇并浓缩至无醇味，样品浓度约为1g干重/20mL；
3)将浓缩后的提取液过大孔树脂柱，上样量为柱体积的1-2倍，上样速度为3-5mL/min，吸附15-30min，用3-5倍柱体积的水冲洗，用浓度为70-80％的乙醇洗脱3-5个柱体积，得到的洗脱液浓缩，即得。
上述组合物中：
所述内生真菌Diaporthe sp.由以下方法制备：采集海洋红树海桑(Sonneratia hainanensis)，表面消毒后，切成0.5cm×0.5cm的小块，放置于内生真菌培养固体培养基中培养，挑取菌丝进行分离纯化，并对菌种进行鉴定，共分离16种内生真菌，菌种保存于超低温冰箱中；所述内生真菌培养固体培养基为葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％；琼脂2％。
所述内生真菌在培养前，由试管菌种接种到平板，再接种到摇瓶当中进行液体培养，参见香附子内生菌的分离鉴定及活性检测(王娜娜，秦宝福，刘建党，等.香附子内生菌的分离鉴定及活性检测[J].中国农学通报，2009,25(8):46-49)
所述大孔吸附树脂为苯乙烯类、苯丙酸类、丙烯酰胺类中的任何一种，优选为AB8型大孔树脂或D101型大孔树脂。
本发明的另一目的是提供上述组合物的制备方法，该方法包括以下步骤：
1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液；
2)将发酵得到的菌丝体和发酵液用浓度为60-90％的乙醇回流提取2-3次，每次提取0.5-3小时，滤过，合并提取液，回收乙醇并浓缩；
3)将浓缩后的提取液过大孔树脂柱，用水冲洗，用乙醇洗脱，得到的洗脱液浓缩，即得。
本发明的另一目的是提供上述组合物在制备治疗肿瘤的药物中的应用。
本发明提供的具有细胞毒活性的组合物具有以下优点：
1、本发明提供的组合物中，其原料来自于海洋红树植物的内生真菌Diaporthe sp.，利用微生物可以人工培养、发酵周期短且不受季节、气候控制的优点，对真菌Diaporthe sp.进行发酵，并进行提取获得细胞毒活性组合物。
2、本发明提供的组合物可以治疗肿瘤。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
内生真菌Diaporthe sp，可以用现有文献的方法制备，或由以下方法分离：采集海洋红树海桑(Sonneratia hainanensis)，表面消毒后，切成0.5cm×0.5cm的小块，放置于内生真菌培养固体培养基中培养，挑取菌丝进行分离纯化，直到菌种成为单菌落，并对菌种进行鉴定，共分离16种内生真菌，菌种保存于超低温冰箱中。
在进行下面的发酵前，需要对内生真菌进行前处理，由试管菌种接种到平板，再接种到摇瓶当中进行液体培养，均采用常规方法制备。
实验例1
1、海洋红树植物的内生真菌Diaporthe sp发酵，培养基的初始pH值为7.4，接种量为3％，发酵温度为25℃，培养转速为180r/min，培养5天，尼龙布过滤，分开菌体和发酵液，得到菌丝体，菌体烘干后用粉碎机粉碎，过20目筛，所述发酵培养的培养基配方为：葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％；
2、醇提：加入原料重量30倍的70％乙醇回流提取3次，每次提取1小时，滤过，合并提取液，对提取液进行减压回收乙醇，并浓缩至大孔树脂上样浓度(1g干重/20mL)；
3、选择AB8型大孔树脂，制备工艺为，上样pH为6.5，上样流速为4mL/min，最大上样量为1.5倍树脂床体积，吸附时间20min，先用3倍树脂床体积的水洗脱除去杂质，再用80％乙醇作为洗脱剂，洗脱速度为3mL/min，洗脱5个柱体积，对洗脱剂进行回收溶剂和浓缩处理，最后进行干燥，即得。
实施例2：
1、发酵：海洋红树植物的内生真菌Diaporthe sp发酵，(培养基的初始pH值为7.4，发酵温度为25℃，接种量为3％，培养转速为180r/min，培养5天，尼龙布过滤，分开菌体和发酵液，后得到发酵液，浓缩至(约500mL)，其中培养基配方为：葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％；
2、醇提：乙醇沉淀，调节乙醇终浓度为70％，静置过夜，过滤，取上清液，减压回收乙醇，并浓缩至大孔树脂上样浓度(1g干重/20mL)。
3、过树脂柱：选择D101型大孔树脂，制备工艺为，pH为5.5，上样流速为4mL/min，最大上样量为1.5倍树脂床体积，吸附时间20min，先用3倍树脂床体积的水洗脱除去杂质，再用70％乙醇作为洗脱剂，洗脱速度为3mL/min，洗脱3倍柱体积，对洗脱剂进行回收溶剂和浓缩处理，最后进行干燥，即得。
实施例3：
1、发酵：海洋红树植物的内生真菌Diaporthe sp发酵，培养基的初始pH值为7.4，发酵温度为25℃，接种量为3％，培养转速为180r/min，培养5天，尼龙布过滤，分开菌体和发酵液，后得到菌丝体，菌体烘干后用粉碎机粉碎，过20目筛，其中培养基配方为：葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％。
2、醇提：加入原料重量30倍的70％乙醇回流提取3次，每次提取1小时，滤过，合并提取液，对提取液进行减压回收乙醇，并浓缩至大孔树脂上样浓度(1g干重/20mL)。
3、过树脂柱：选择D101型大孔树脂，制备工艺为，pH为5.5，上样流速为3mL/min，最大上样量为1倍树脂床体积，吸附时间20min，先用3倍树脂床体积的水洗脱除去杂质，再用70％乙醇作为洗脱剂，洗脱速度为2mL/min，洗脱4倍柱体积，对洗脱剂进行回收溶剂和浓缩处理，最后进行干燥，即得。
工业实用性
本发明公开的具有细胞毒活性的组合物，该组合物是从海洋红树植物的内生真菌Diaporthe sp.的发酵产物中提取得到，可以单独或与其他成分一起治疗肿瘤。本发明提供的组合物中，其原料来自于海洋红树植物的内生真菌 Diaporthe sp.，利用微生物可以人工培养、发酵周期短且不受季节、气候控制的优点。

Claims (7)

  1. 一种具有细胞毒活性的组合物，其由以下方法制备：
    1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液；
    2)将发酵得到的菌丝体和发酵液用浓度为60-90％的乙醇提取2-3次，每次提取0.5-3小时，滤过，合并提取液，回收乙醇并浓缩；
    3)将浓缩后的提取液过大孔树脂柱，用水冲洗，用乙醇洗脱，得到的洗脱液浓缩，即得。
  2. 根据权利要求1所述的组合物，其特征在于，所述组合物由以下方法制备：
    1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液，浓缩，并干燥，所述发酵用的培养基为葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％，发酵的方法具体为：培养基的初始pH值为7.4，发酵温度为25℃，接种量为3％，培养转速为180r/min，培养5天，过滤；
    2)将步骤1)得到的干燥物用其重量20-50倍体积的浓度为70-80％乙醇回流提取2-3次，每次提取1-2小时，滤过，合并提取液，回收乙醇并浓缩至无醇味，样品浓度约为1g干重/20mL；
    3)将浓缩后的提取液过大孔树脂柱，上样量为柱体积的1-2倍，上样速度为3-5mL/min，吸附15-30min，用3-5倍柱体积的水冲洗，用浓度为70-80％的乙醇洗脱3-5个柱体积，得到的洗脱液浓缩，即得。
  3. 根据权利要求1或2所述的组合物，其特征在于，所述大孔吸附树脂为苯乙烯类、苯丙酸类、丙烯酰胺类中的任何一种。
  4. 根据权利要求1或2所述的组合物，其特征在于，所述大孔吸附树脂为AB8型大孔树脂或D101型大孔树脂。
  5. 根据权利要求1或2所述的组合物，其特征在于，所述内生真菌Diaporthe sp.由以下方法制备：采集海洋红树海桑(Sonneratia hainanensis)，表面消毒后，切成0.5cm×0.5cm的小块，放置于内生真菌培养固体培养基中培养，挑取菌丝进行分离纯化，并对菌种进行鉴定，共分离16种内生真菌， 菌种保存于超低温冰箱中；所述内生真菌培养固体培养基为葡萄糖0.5％，蛋白胨0.2％，酵母膏0.5％，粗海盐0.5％；琼脂2％。
  6. 一种制备权利要求1-5任一项所述的组合物的方法，其特征在于，，该方法包括以下步骤：
    1)海洋红树植物内生真菌Diaporthe sp.发酵得到菌丝体和发酵液；
    2)将发酵得到的菌丝体和发酵液用浓度为60-90％的乙醇回流提取2-3次，每次提取0.5-3小时，滤过，合并提取液，回收乙醇并浓缩；
    3)将浓缩后的提取液过大孔树脂柱，用水冲洗，用乙醇洗脱，得到的洗脱液浓缩，即得。
  7. 权利要求1-5任一项所述的组合物在制备治疗肿瘤的药物中的应用。
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