CN103074234B - Marine fungus aspergillus sydowii and application thereof to preparation of anti-tumor medicines - Google Patents

Abstract

The invention provides a marine fungus with anti-tumor activity—Aspergillus sydowii (Aspergillus sydowii) MNP12010103 and its application. The strain is preserved in the China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, zip code 430072, preservation number: CCTCC No: M 2012391, preservation date October 8, 2012. The beneficial effects of the present invention are mainly reflected in: (1) the marine fungus Aspergillus salbui MNP12010103 of the present invention has simple nutritional requirements and is easy to cultivate; (2) the metabolites of the strain have anti-tumor activity; (3) the secondary metabolism of the strain The anti-tumor activity of the product is high, and the total extract of the fermented liquid obtained by culturing it has strong anti-tumor activity against HepG2 cells, PC12 cells and U937 cells.

Description

A marine fungus Aspergillus salvei and its application in the preparation of antitumor drugs

(1) Technical field

The invention relates to a marine fungus with anti-tumor activity—Aspergillus sydowii (Aspergillus sydowii) MNP12010103, and its application in the preparation of anti-tumor drugs. the

(2) Background technology

Cancer has always been a difficult problem for human beings to overcome. Finding substances with strong physiological activity has become one of the main tasks for human beings to overcome diseases. The study of marine microbial metabolites is an emerging research topic in the world in recent years. the

During the long evolution process of the marine ecosystem, marine microorganisms have formed a unique mechanism to adapt to the harsh living environment, have evolved a variety of genotypes, metabolic pathways, and physiological and ecological functions, and contain a large number of novel secondary metabolites. Some lead compounds from marine sources have been successfully developed into drugs, including anti-infective drug cephalosporins (cephalosporins) derived from marine fungi, anti-tumor drug cytarabine (AraC) derived from sponges, antiviral drugs Vidarabine (AraA), analgesic conotoxin (ziconotide, Prialt) derived from cone snails, etc. At present, there are more than 40 new marine drugs approved for clinical research in the world. the

In the past few decades, more than 20,000 new compounds have been isolated from marine microorganisms all over the world. A large number of studies have shown that the structural types of metabolites of marine fungi mainly include: cyclic peptides, sterols, anthraquinones, pyrones, ketals, carbonyl-containing hydroxyls (aldehydoketone compounds, esters and free carboxyl compounds, containing Hydroxyl compounds), nitrogen-containing compounds (sulfur-containing alkaloid compounds, piperazine and quinazole compounds, pyrroles, other aza-condensed ring and pyridine compounds, amides, amines and other nitrogen-containing compounds). The bioactive substances produced by marine fungi mainly include: antibacterial active substances and antitumor active substances. At present, the secondary metabolites of marine fungi, such as anti-tumor, anti-acetylcholinesterase, anti-bacteria and anti-oxidation, have become rich sources of natural compounds with biomedical significance, and all have broad medicinal prospects. Therefore, the discovery of highly active substances from the secondary metabolites of marine fungi is very important for the discovery of lead compounds, and it is also of great significance for the treatment of diseases. the

(3) Contents of the invention

The object of the present invention is to provide a marine fungus with anti-tumor activity—Aspergillus sydowii (Aspergillus sydowii) MNP12010103, and its application in the preparation of anti-tumor drugs. the

The technical scheme adopted in the present invention is:

A marine fungus with anti-tumor activity - Aspergillus sydowii (Aspergillus sydowii) MNP12010103, preserved in the China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, postcode 430072, preservation number: CCTCC No: M 2012391, The date of deposit was October 8, 2012. the

The characteristics of the colony of Aspergillus salbui MNP12010103 are as follows: it grows rapidly on seawater PDA by coating or streak inoculation, and cultured on seawater PDA at 28°C for 5 days. There are a large number of spore structures, the surface is grayish blue to dark blue, the edge is white, the color is dark after aging, there is no exudate, the opposite side of the colony is light yellowish brown, and the soluble pigment is lacking; In liquid culture medium, after 48 hours of culture at 28°C, it grows in granular suspension; the conidia heads of Aspergillus sarei MNP12010103 are spherical to radial, and the microconidia are penicillium-like in shape, scattered or loose columnar. The main features of this species are dense cylindrical conidia heads and smooth microconidia, and the mycelium is white. the

The bacterial strain of the present invention is obtained by separating and screening the sea water collected in the sea area of Sanmen Bay, Zhejiang Province. the

The screening and purification method of the bacterial strain is as follows: apply the collected seawater on the potato plate medium by the dilution coating method, cultivate at 28°C until the number of colonies no longer increases, and pick a single colony to the slant medium. Cultivate at 28°C for 2 days, select the strains based on their morphological characteristics, and then obtain the strains, and identify the strains. the

The composition of the plate medium and the slant medium is the same, and the composition is: 150-350 g/L of potatoes, 10-30 g/L of glucose, 15-35 g/L of agar, water as a solvent, and pH 7.2-8.0. the

The rDNA-ITS sequence of Aspergillus sydowii MNP12010103 was homologously compared in the GenBank of the National Center for Biotechnology Information USA (NCBI), and identified as Aspergillus sydowii. The sequencing sequence of its rDNA-ITS is as follows:

CTTCCGTAGGTGAACCTGCGGAAGGATCATTACTGAGTGCGGGCTGCCTCCGGGCGCCCAACCTCCCACCCGTGAATACCTAACACTGTTGCTTCGGCGGGGAACCCCCTCGGGGGCGAGCCGCCGGGGACTACTGAACTTCATGCCTGAGAGTGATGCAGTCTGAGTCTGAATATAAAATCAGTCAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCGACTAGGGCCGGCCGGGCGCCAGCCGACGTCTCCAACCATTTTTCTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA。 the

The present invention also relates to the application of said Aspergillus salbui MNP12010103 in the preparation of antitumor drugs. the

Specifically, the antitumor drug is a drug for treating liver cancer, nerve cancer or lymphoma. the

Specifically, the Aspergillus salvei MNP12010103 extract is used to prepare antitumor drugs. the

Preferably, the extract is an ethyl acetate extract, which is obtained by inoculating Aspergillus salbui MNP12010103 into a liquid fermentation medium and cultivating it at 25~35°C and shaking at 150~250 r/min for 14 ~30d, obtain the fermentation broth, remove the bacterial cells from the fermentation broth, extract it with ethyl acetate, concentrate and evaporate to dryness to obtain the extract, which is the ethyl acetate extract of the Aspergillus salbui MNP12010103; the fermentation medium consists of: Glucose 8~20 g/L, peptone 1.5~3 g/L, yeast extract 0.8~2g/L, solvent is a mixture of water:artificial seawater volume ratio 1:1~4, pH 7~8. the

The composition of the artificial seawater per 100 mL is: 2.448 g NaCl, 0.3917 g Na ₂ SO ₄ , 0.0664 g KCl, 0.0096 g KBr, 0.0024 g SrCl ₂ , 0.4981 g MgCl 6H ₂ O, 0.1102 g CaCl ₂ H ₂ O , NaHCO ₃ 0.0192 g, H ₃ BO ₃ 0.0026 g, NaF 0.0004 g, distilled water 100 mL.

Before the fermentation culture, the strains usually need to be activated by slant culture, and then cultured by seeds to obtain the seed strains and then inserted into the liquid fermentation medium for enzyme production culture. the

The slant culture medium is composed of: 150-350 g/L of potatoes, 10-30 g/L of glucose, 15-35 g/L of agar, water as a solvent, and pH 7.2-8.0. the

Described planar seed culture medium consists of: glucose 8 ~ 15g/L, peptone 1.5 ~ 4g/L, yeast extract 0.5 ~ 1.5g/L, agar 15 ~ 20g/L, solvent is water: artificial seawater volume ratio 1: The mixture of 1~4, pH7.2~8.0. the

The liquid fermentation medium is composed of: glucose 8-15g/L, peptone 1.5-3g/L, yeast extract 0.8-2g/L, solvent is a mixture of water:artificial seawater volume ratio 1:1-4, pH7~ 8.0. the

Specifically, the extract is obtained by the following method:

(1) The marine fungus Aspergillus salvei MNP12010103 strain was inoculated on the slant medium, and cultured at 25-35°C for 24-48 hours to obtain the activated strain; the composition of the slant medium was: potatoes 150-350g/L , glucose 10~30g/L, agar 15~35g/L, solvent is water, pH7.2~8.0; 

(2) Inoculate the marine fungus Aspergillus salvei MNP12010103 after the activation culture in step (1) into the plane seed medium, and culture it at 25-35°C for 16-48 hours to obtain the seed fungus; the plane seed medium consists of For: glucose 8~15 g/L, peptone 1.5~4 g/L, yeast extract 0.5~1.5 g/L, agar 15~20g/L, the solvent is a mixture of water:artificial seawater volume ratio 1:1~4, pH7.2~8.0;

(3) Transplant the seed strain of step (2) into the liquid fermentation medium with an inoculum volume of 1%~10% by volume, and culture it at 25~35°C and 150~250 r/min for 14~ After 30 days, the fermented liquid was obtained; the liquid fermented medium consisted of: 8-20 g/L of glucose, 1.5-3 g/L of peptone, 0.8-2 g/L of yeast extract, and the solvent was water:artificial seawater volume ratio 1: 1~4 mixture, pH 7~8;

(4) Break the cells of the fermentation liquid in step (3), centrifuge or filter at 4°C, separate and remove the bacteria, extract the obtained fermentation liquid with ethyl acetate, collect the upper layer extract, concentrate and evaporate to dryness to obtain an oily extract The total extract, that is, ethyl acetate extract. the

The beneficial effects of the present invention are mainly reflected in: (1) the marine fungus Aspergillus salbui MNP12010103 of the present invention has simple nutritional requirements and is easy to cultivate; (2) the metabolites of the strain have anti-tumor activity; (3) the secondary metabolism of the strain The anti-tumor activity of the product is high, and the total extract of the fermented liquid obtained by culturing it has strong anti-tumor activity against HepG2 cells, PC12 cells and U937 cells. the

(4) Description of drawings

Fig. 1 is the bacterium colony morphology of Aspergillus salvei MNP12010103 on the plate culture medium;

Figure 2 is the cell morphology of Aspergillus salvei MNP12010103 under the light microscope. the

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1: Screening, purification and identification of bacterial strains

(1) Apply the seawater collected from the Sanmen Bay of the East China Sea on the potato plate medium by dilution coating method, cultivate at 28°C until the number of colonies no longer increases, and pick a single colony and transfer it to a new potato plate medium. Cultivate at 28°C for 2 days, and select the strains based on their morphological characteristics to obtain the strain. The potato plate culture medium is prepared according to the following composition: 200 g of potatoes, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water. the

(2) Comparing the nucleotide sequence of the PCR product sequence of the rDNA-ITS of the strain MNP12010103 obtained through screening, combined with morphological observation, named it Aspergillus sydowii MNP12010103 (Aspergillus sydowii MNP12010103), and submitted it to the Chinese Type Culture Collection Center, deposit number: CCTCC No: M 2012391, deposit date: October 8, 2012. the

Embodiment 2: Activation and large-scale cultivation of bacterial strains

(1) Inoculate the strain obtained by screening in Example 1 on the slant medium, and culture it at 28°C for 24-48h to obtain the activated strain. The slant medium is prepared according to the following composition: 200g of potatoes, 20g of glucose, and 20g of agar , distilled water 1000mL. the

(2) Inoculate the strain after the activated culture in step (1) into a flat culture medium, and culture it for 48 hours at 28°C under shaking conditions of 150-250 r/min to obtain a seed liquid. The liquid seed culture medium is prepared according to the following composition: Glucose 10g, peptone 2g, yeast extract 1g, agar 15g, distilled water 400mL, artificial seawater 600mL, pH 7.5. the

(3) Transplant the seed strain of step (2) into a large-scale liquid fermentation medium with an inoculum volume of 10% by volume, and culture it at 28°C for 21 days under shaking conditions of 150-250 r/min to obtain a fermentation broth . The liquid fermentation medium is prepared as follows: glucose 10g, peptone 2g, yeast extract 1g, distilled water 400mL, artificial seawater 600mL, pH 7.5. the

Example 3: Research on the antitumor activity of the marine fungus Aspergillus salvei MNP12010103

(1) The fermented liquid obtained from the cultivation in Example 2 was crushed in an ultrasonic cell disruptor for 20 minutes, and then centrifuged (9000r/min, 15min) or filtered at 4°C to remove the bacteria, and then washed with ethyl acetate The ester is extracted, and the extract phase is subjected to rotary steaming, and the obtained extract is the total extract of the secondary metabolites of the strain. the

(2) Determining the antitumor activity of the total extract of the fermentation broth obtained in step (1). The specific steps are as follows: select three tumor cell lines, namely adrenal pheochromocytoma cells (PC12 cells), liver cancer cells (HepG2 cells) and human histiocytic lymphoma cells (U937 cells), and take logarithmic phase cell lines to make Cell suspension, inoculated in a 96-well plate, cultured for 48 hours, the sample to be tested (total extract) was dissolved by adding 0.1% DMSO 10 μL, diluted with serum-free 1640 cell culture medium, and added 100 μL to the test group to make the final fermentation broth The concentration of the total extract was 50 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL, 800 μg/mL, the negative control group was added with the same amount of serum-free medium without samples, and the blank control group was cell-free and The serum-free medium of the sample, etoposide (VP-16) was used as a positive control substance, and 5 replicate wells were set up for each concentration. The tumor cells were cultured in a CO2 incubator (37°C) for 48 hours, and 20 μL of 5 mg/mL MTT was added to each well. After continuing to culture for 4 hours, carefully remove the supernatant, add 150 μL of DMSO to each well, shake for 10 minutes, shake fully with a microplate reader to completely dissolve the purple MTT product, measure the A value at 490 nm, according to the formula: inhibition rate = (A negative control Group-A blank control group)-(A sample group-A blank control group)/(A negative control group-A blank control group) to obtain the inhibition rate. the

(3) See Tables 1-3 for the data obtained according to steps (1)-(3). the

Table 1: Inhibitory effect of strain fermentation liquid extract on HepG2 cells

Table 2: Inhibitory effect of strain fermentation broth extract on U937 cells

Table 3: Inhibitory effect of strain fermentation broth extract on PC12 cells

From the data in the table, it can be concluded that the secondary metabolites of the marine fungus Aspergillus salvei MNP12010103 have high anti-tumor activity, and the total extract of the fermentation broth obtained from its cultivation has a strong effect on liver cancer cells (HepG2 cells) and human histiocyte lymphoma cells (U937 cells). ) has strong anti-tumor activity, and also has certain anti-tumor activity against adrenal pheochromocytoma cells (PC12 cells). the

Claims (3)

1. A marine fungus with anti-tumor activity - Aspergillus sydowii MNP12010103, preserved in the China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, postcode 430072, preservation number: CCTCC No: M 2012391, date of deposit October 8, 2012. 2. The application of the Aspergillus salvei MNP12010103 described in claim 1 in the preparation of anti-tumor drugs, characterized in that the anti-tumor drugs are drugs for treating liver cancer, nerve cancer or lymphoma. 3. The application according to claim 2, characterized in that the Aspergillus salbui MNP12010103 extract is used to prepare antitumor drugs, and the Aspergillus salvei MNP12010103 extract is an ethyl acetate extract, which is prepared by the following method: Aspergillus sp. MNP12010103 was inoculated into the liquid fermentation medium, cultured at 25-35°C, 150-250r/min shaking conditions for 14-30 days to obtain a fermentation broth, the fermentation broth was removed from the bacterial cells, extracted with ethyl acetate, concentrated and evaporated to dryness The extract is obtained, which is the ethyl acetate extract of the Aspergillus salbui MNP12010103; the fermentation medium consists of: glucose 8-20g/L, peptone 1.5-3g/L, yeast extract 0.8-2g/L, solvent It is a mixture of water:artificial seawater volume ratio 1:1~4, pH 7~8.