CN103074234B - Marine fungus aspergillus sydowii and application thereof to preparation of anti-tumor medicines - Google Patents
Marine fungus aspergillus sydowii and application thereof to preparation of anti-tumor medicines Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a marine fungus-aspergillus sydowii MNP12010103 with anti-tumor activity and application thereof. The marine fungus was collected into China Center for Type CultureCollection (CCTCC) on October 8th, 2012, wherein the collection number is CCTCC No: M2012391. The invention has the following benefits: (1) the marine fungus-aspergillus sydowii MNP12010103 is simple in nutritional requirement and easy to cultivate; (2) the metabolite of the marine fungus has anti-tumor activity; and (3) the secondary metabolite of the marine fungus has high anti-tumor activity, and the fermented liquid total extractum cultivated by the secondary metabolite has high anti-tumor activity on HepG2, PC12 and U937 cells.
Description
(1) technical field
The present invention relates to the thalassiomycetes that a strain has anti-tumor activity---aspergillus sydowi (Aspergillus sydowii) MNP12010103, and preparing the application in antitumor drug.
(2) background technology
Cancer always is the difficult problem that the mankind are difficult to capture, and the material finding physiologically active strong becomes one of mankind's groundwork of capturing disease.Marine microbial technology research is research topic emerging in the world in recent years.
In the evolutionary process that Marine ecosystems are very long, marine microorganism defines the unique mechanism adapting to harsh living environment, the diversity of genotype of evolving out, pathways metabolism and physiological ecological function, contains a large amount of novel secondary metabolite.The lead compound of some marine sources is successfully developed to medicine, comprise the anti-infectives cephamycin (cephalo sporins) deriving from thalassiomycetes, derive from the antitumor drug cytosine arabinoside (cytarabine of sponge, AraC), Antivirus Agent Adenine Arabinoside (vidarabine, AraA), the anodyne conotoxin (ziconotide, Prialt) etc. of cone shell is derived from.At present, the whole world also have more than 40 plant marine drug approval enter clinical study.
In the past few decades, in worldwide from marine microorganism be separated obtain novel cpd kind more than 20000.Large quantity research shows, the meta-bolites structure type of thalassiomycetes mainly contains: cyclic peptide, sterols, Anthraquinones, pyranone, ketal class, containing carbonyl hydroxy kind (Carbonyl compounds, ester and free carboxy chemical combination, hydroxy-containing compounds), nitrogenous class (sulfur-bearing alkaloid compound, piperazines and quinoline azole compounds, pyroles, other azepine condensed ring classes and pyridine compounds and their, acid amides, amine and other nitrogenous compounds).The biologically active substance that thalassiomycetes produces mainly contains: antibacterial substance, anti-tumor active substance.More antitumor, the acetylcholine esterase inhibition of current research, antibacterium and the thalassiomycetes secondary metabolite such as anti-oxidant have become the abundant source of the natural compounds with biomedical meaning, all have wide prospect in medicine.Therefore from the secondary metabolite of thalassiomycetes, find that highly active material is for finding lead compound very important, also has an important significance to capturing of disease.
(3) summary of the invention
The object of the invention is to provide the thalassiomycetes that a strain has anti-tumor activity---aspergillus sydowi (Aspergillus sydowii) MNP12010103, and preparing the application in antitumor drug.
The technical solution used in the present invention is:
One strain has the thalassiomycetes of anti-tumor activity---aspergillus sydowi (Aspergillus sydowii) MNP12010103, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M 2012391, preservation date on October 8th, 2012.
The colony characteristics of described aspergillus sydowi MNP12010103 is as follows: coating or streak inoculation grow rapidly on seawater PDA, and seawater PDA cultivates 5d, colony diameter 31mm for 28 DEG C, smooth, central protrusion, quality is velvet-like to cotton-shaped, and conidium structure is a large amount of, surface dusty blue is to faint blue, edge white, color depth after old, without transudate, bacterium colony reverse side is khaki, and soluble pigment lacks; The thalli growth feature of described aspergillus sydowi is as follows: in liquid medium within, in particulate state suspension growth after 28 DEG C of cultivation 48 h; Described aspergillus sydowi MNP12010103 conidial head is spherical to Radiation, and microconidium head is mould shape seemingly, at random or loose cylindricality.The main feature of this kind is fine and close cylindricality conidial head and smooth microconidium, and mycelium is in white.
Bacterial strain of the present invention is that in the seawater gathered by Zhejiang Province's nutrients, Isolation and screening obtains.
The screening purification process of bacterial strain is: use method of dilution butteron on plate to be applied on potato plate culture medium in the seawater of collection, 28 DEG C are cultured to colony counts and no longer increase, and picking list bacterium colony is on slant medium.Cultivate 2d for 28 DEG C, select with the difference of strain morphology feature, obtain this bacterial strain, and strain identification is carried out to this bacterial strain.
Described plate culture medium is identical with the composition of slant medium, consists of: potato 150 ~ 350g/L, glucose 10 ~ 30g/L, agar 15 ~ 35g/L, and solvent is water, pH7.2 ~ 8.0.
By the rDNA-ITS sequence of described aspergillus sydowi Aspergillus sydowii MNP12010103 at US National Bioinformatics Institute (National Center for Biotechnology Information USA, NCBI) carry out homology comparison in GenBank, be accredited as aspergillus sydowi (Aspergillus sydowii).The sequencing sequence of its rDNA-ITS is as follows:
CTTCCGTAGGTGAACCTGCGGAAGGATCATTACTGAGTGCGGGCTGCCTCCGGGCGCCCAACCTCCCACCCGTGAATACCTAACACTGTTGCTTCGGCGGGGAACCCCCTCGGGGGCGAGCCGCCGGGGACTACTGAACTTCATGCCTGAGAGTGATGCAGTCTGAGTCTGAATATAAAATCAGTCAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCGACTAGGGCCGGCCGGGCGCCAGCCGACGTCTCCAACCATTTTTCTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA。
The invention still further relates to described aspergillus sydowi MNP12010103 and prepare the application in antitumor drug.
Concrete, described antitumor drug is the medicine of Hepatoma therapy, neural cancer or lymphatic cancer.
Concrete, described aspergillus sydowi MNP12010103 extract is for the preparation of antitumor drug.
Preferably, described extract is ethyl acetate extract, obtain by the following method: aspergillus sydowi MNP12010103 is seeded in liquid fermentation medium, in 25 ~ 35 DEG C, cultivate 14 ~ 30d under 150 ~ 250 r/min oscillating conditions, obtain fermented liquid, fermented liquid is extracted with ethyl acetate after removing somatic cells, concentrates and volatilizes to obtain medicinal extract, be the ethyl acetate extract of described aspergillus sydowi MNP12010103; Described fermention medium consists of: glucose 8 ~ 20 g/L, peptone 1.5 ~ 3 g/L, yeast extract paste 0.8 ~ 2g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH 7 ~ 8.
Every 100 mL of described artificial seawater consist of: NaCl 2.448 g, Na
2sO
40.3917 g, KCl 0.0664 g, KBr 0.0096 g, SrCl
20.0024 g, MgCl6H
2o 0.4981 g, CaCl
2h
2o 0.1102 g, NaHCO
30.0192 g, H
3bO
30.0026 g, NaF 0.0004 g, distilled water 100 mL.
Described bacterial strain is before fermentation culture, and usual needs first activates through slant culture, then accesses liquid fermentation medium again carry out the cultivation of product enzyme through seed culture, acquisition seed bacterial strain.
Described slant medium consists of: potato 150 ~ 350g/L, glucose 10 ~ 30g/L, agar 15 ~ 35g/L, and solvent is water, pH7.2 ~ 8.0.
Described plane seed culture medium consists of: glucose 8 ~ 15g/L, peptone 1.5 ~ 4g/L, yeast extract paste 0.5 ~ 1.5g/L, agar 15 ~ 20g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH7.2 ~ 8.0.
Described liquid fermentation medium consists of: glucose 8 ~ 15g/L, peptone 1.5 ~ 3g/L, yeast extract paste 0.8 ~ 2g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH7 ~ 8.0.
Concrete, described extract obtains by the following method:
(1) by thalassiomycetes aspergillus sydowi MNP12010103 inoculation in slant medium, in 25 ~ 35 DEG C cultivate 24 ~ 48 h, obtain activate after bacterial classification; Described slant medium consists of: potato 150 ~ 350g/L, glucose 10 ~ 30g/L, agar 15 ~ 35g/L, and solvent is water, pH7.2 ~ 8.0;
(2) thalassiomycetes aspergillus sydowi MNP12010103 thalline after step (1) activation culture is seeded in plane seed culture medium, under 25 ~ 35 DEG C of conditions, cultivates 16 ~ 48h, obtain kind of a daughter bacteria; Described plane seed culture medium consists of: glucose 8 ~ 15 g/L, peptone 1.5 ~ 4 g/L, yeast extract paste 0.5 ~ 1.5 g/L, agar 15 ~ 20g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH7.2 ~ 8.0;
(3) by step (2) seed bacterial strain with 1% ~ 10% the inoculum size of volume ratio, culture transferring in liquid fermentation medium, in 25 ~ 35 DEG C, cultivate 14 ~ 30 days under 150 ~ 250 r/min oscillating conditions, obtain fermented liquid; Described liquid fermentation medium consists of: glucose 8 ~ 20 g/L, peptone 1.5 ~ 3 g/L, yeast extract paste 0.8 ~ 2g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH 7 ~ 8;
(4) by fermented liquid cytoclasis under 4 DEG C of conditions of step (3), centrifugal or filtration, be separated removing thalline, gained fermented liquid is extracted with ethyl acetate rear collection upper layer of extraction liquid, concentrates and volatilizes the total medicinal extract of obtained oil-like extracts, i.e. ethyl acetate extract.
Beneficial effect of the present invention is mainly reflected in: (1) thalassiomycetes aspergillus sydowi of the present invention MNP12010103 nutritional requirement simply, is easily cultivated; (2) meta-bolites of this bacterial strain has anti-tumor activity; (3) anti-tumor activity of the secondary metabolite of this bacterial strain is high, and its total medicinal extract of fermented liquid cultivating gained has stronger anti-tumor activity to HepG2 cell, PC12 cell and U937 cell.
(4) accompanying drawing explanation
Fig. 1 is the colonial morphology of aspergillus sydowi MNP12010103 on plate culture medium;
Fig. 2 is the thalli morphology of aspergillus sydowi MNP12010103 under opticmicroscope.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of bacterial strain, Isolation and characterization
(1) use method of dilution butteron on plate to be applied on potato plate culture medium by from the seawater of East Sea nutrients collection, 28 DEG C are cultured to colony counts and no longer increase, and picking list bacterium colony is on new potato plate culture medium.Cultivate 2d for 28 DEG C, select with the difference of strain morphology feature, obtain this bacterial strain.Described potato plate culture medium is by forming preparation as follows: potato 200g, glucose 20g, agar 20g, distilled water 1000mL.
(2) nucleotide sequence comparison is carried out to the PCR primer sequence of the rDNA-ITS of the bacterial strain MNP12010103 that screening obtains, combining form is observed, by its called after aspergillus sydowi MNP12010103(Aspergillus sydowii MNP12010103), submit China typical culture collection center to, preserving number: CCTCC No:M 2012391, preservation date on October 8th, 2012.
Embodiment 2: the activation of bacterial strain and large scale culturing
(1) will screen the inoculation of acquisition in embodiment 1 in slant medium, cultivate 24-48h in 28 DEG C, obtain the bacterial strain after activating, described slant medium is by forming preparation as follows: potato 200g, glucose 20g, agar 20g, distilled water 1000mL.
(2) by the inoculation after step (1) activation culture in plating medium, in 28 DEG C, cultivate 48h under 150 ~ 250 r/min oscillating conditions, obtain seed liquor, institute's liquid seed culture medium is by forming preparation as follows: glucose 10g, peptone 2g, yeast extract paste 1g, agar 15g, distilled water 400mL, artificial seawater 600mL, pH 7.5.
(3) by step (2) seed bacterial strain with 10% the inoculum size of volume ratio, culture transferring in mass liquid fermention medium, in 28 DEG C, cultivate 21d under 150 ~ 250 r/min oscillating conditions, obtain fermented liquid.Described liquid fermentation medium is by forming preparation as follows: glucose 10g, peptone 2g, yeast extract paste 1g, distilled water 400mL, artificial seawater 600mL, pH 7.5.
Embodiment 3: the antitumor activity of thalassiomycetes aspergillus sydowi MNP12010103
(1) fermented liquid of gained will be cultivated in embodiment 2 prior to carrying out bacterial cell disruption 20min in ultrasonic cell disruption instrument, then centrifugal (9000r/min under 4 DEG C of conditions, 15min) or filter, removing thalline, extract by ethyl acetate, revolve steaming to extraction phase, gained medicinal extract is the total medicinal extract of secondary metabolite of this bacterial strain.
(2) the total medicinal extract of fermented liquid of step (1) gained is carried out antitumor cytolytic activity.Concrete steps are as follows: choose three strain tumour cells, be respectively Adrenal Pheochromocytoma cell (PC12 cell), liver cancer cell (HepG2 cell) and human tissue cell's lymphoma cell (U937 cell), the cell strain of phase of taking the logarithm makes cell suspension, be inoculated in 96 orifice plates, cultivate 48h, after testing sample (total medicinal extract) adds 0.1% DMSO10 μ L dissolving, with 1640 cell culture medium of serum-free, add 100 μ L in test group, the concentration of the total medicinal extract of final fermented liquid is made to be respectively 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/mL, 800 μ g/mL, negative control group adds equivalent not containing the serum free medium of sample, blank group is then acellular and serum free medium that is sample, Etoposide (VP-16) is as positive reference substance, each concentration establishes 5 multiple holes, tumour cell cultivates 48h at CO2 incubator (37 DEG C), every hole adds the MTT20 μ L of 5mg/mL, after continuing to cultivate 4h, carefully remove supernatant, every hole adds DMSO150 μ L, vibration 10min, fully vibrating by microplate reader makes MTT purple product dissolve completely, survey the A value of 490nm, according to formula: inhibiting rate=(A negative control group-A blank group)-(A sample sets-A blank group)/(A negative control group-A blank group) can try to achieve inhibiting rate.
(3) according to the data of step (1)-(3) gained in table 1 ~ 3.
Table 1: bacterial strain fermentation liquor medicinal extract is to the restraining effect of HepG2 cell
Table 2: bacterial strain fermentation liquor medicinal extract is to the restraining effect of U937 cell
Table 3: bacterial strain fermentation liquor medicinal extract is to the restraining effect of PC12 cell
Can be obtained by data in table: the anti-tumor activity of the secondary metabolite of thalassiomycetes aspergillus sydowi MNP12010103 is high, its total medicinal extract of fermented liquid cultivating gained has stronger anti-tumor activity to liver cancer cell (HepG2 cell) and human tissue cell's lymphoma cell (U937 cell), also has certain anti-tumor activity to Adrenal Pheochromocytoma cell (PC12 cell).
Claims (3)
1. a strain has the thalassiomycetes of anti-tumor activity---aspergillus sydowi (Aspergillus sydowii) MNP12010103, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M 2012391, preservation date on October 8th, 2012.
2. aspergillus sydowi MNP12010103 according to claim 1 is preparing the application in antitumor drug, it is characterized in that described antitumor drug is the medicine of Hepatoma therapy, neural cancer or lymphatic cancer.
3. apply as claimed in claim 2, it is characterized in that aspergillus sydowi MNP12010103 extract is for the preparation of antitumor drug, described aspergillus sydowi MNP12010103 extract is ethyl acetate extract, obtain by the following method: aspergillus sydowi MNP12010103 is seeded in liquid fermentation medium, in 25 ~ 35 DEG C, cultivate 14 ~ 30d under 150 ~ 250r/min oscillating condition, obtain fermented liquid, fermented liquid is extracted with ethyl acetate after removing somatic cells, concentrate and volatilize to obtain medicinal extract, be the ethyl acetate extract of described aspergillus sydowi MNP12010103; Described fermention medium consists of: glucose 8 ~ 20g/L, peptone 1.5 ~ 3g/L, yeast extract paste 0.8 ~ 2g/L, and solvent is water: the mixture of artificial seawater volume ratio 1:1 ~ 4, pH 7 ~ 8.
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