CN102911877B - Marine fungi cladosporium sphaerospermum and application thereof - Google Patents
Marine fungi cladosporium sphaerospermum and application thereof Download PDFInfo
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Abstract
The invention provides a marine fungi-cladosporium sphaerospermum MNP100102A with antineoplastic activity and application thereof. The bacterial strain is preserved in a China Center for Type Culture Collection, the address is Wuhan university of Wuhan in China, the postcode is 430072, the preservation serial number is CCTCC No: M 2012290, and the preservation data is July 18, 2012. The marine fungi-cladosporium sphaerospermum has the advantages that: (1) the bacterial strain is simple in nutritional requirements and easy to cultivate; (2) metabolic products of the bacterial strain has the antineoplastic activity; and (3) a secondary supersession product of the bacterial strain is high in antineoplastic activity, fermentation broth total extract prepared by cultivation has certain antineoplastic activity on hepatoma carcinoma cells (HepG2), HP-20 macroporous resin column treatment is carried out on the total extract, the restrain rate of inhibitory activity of a small polarity portion obtained by collection on HepG2 and PC12 cells can achieve more than 95% when concentration is 200 mu g/ml, and the marine fungi-cladosporium sphaerospermum has strong antineoplastic activity.
Description
(1) technical field
The present invention relates to the thalassiomycetes that a strain has anti-tumor activity---Cladosporium fungal ball spore cladosporium (Cladosporium sphaerospermum) 100102A and application thereof.
(2) background technology
Cancer always is the difficult problem that the mankind are difficult to capture, and the strong material of searching physiologically active becomes the mankind and captures one of groundwork of disease.Marine microorganism meta-bolites research is emerging research topic in the world in recent years.
Because the medicine Feed Discovery to terrestrial organism is more and more rare, ocean has become the virgin land that the mankind open up wasteland new drug.Ocean accounts for the earth total area 71%, is containing extremely abundant Biological resources, is the largest production person of organic substance.In the source of various natural bioactive molecules, marine microorganism is the most ergastic.Unlike terrestrial plant, marine microorganism can produce the halo secondary metabolite.In the treatment of finding first to make it to infect on bacterium of nineteen twenty-eight penicillin, a very big breakthrough is arranged, fungi just becomes one of important sources of the medicine for the treatment of disease.Separated in 1971 the ciclosporin obtained thereupon and promoted it further developing aspect immunopharmacology.The meta-bolites of thalassiomycetes is not only essential for the exploitation of medicine, and it is also extremely important aspect plant protection simultaneously, makes the Research Prospects of thalassiomycetes more extensive.More antitumor, the acetylcholine esterase inhibition of at present 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 find that from the secondary metabolite of thalassiomycetes highly active material, for finding very important of lead compound, has an important significance to capturing also of disease.
(3) summary of the invention
The present invention seeks to the thalassiomycetes that a strain has anti-tumor activity---Cladosporium fungal ball spore cladosporium (Cladosporium sphaerospermum) 100102A, and the application in preparing antitumor drug.
The technical solution used in the present invention is:
One strain has the thalassiomycetes of anti-tumor activity---ball spore cladosporium (Cladosporium sphaerospermum) 100102A, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M 2012290, preservation date: on July 18th, 2012.
The colony characteristics of described ball spore cladosporium (Cladosporium sphaerospermum) 100102A is as follows: coating or streak inoculation grows rapidly on plate culture medium, and 28 ℃ of cultivations grow the bacterium colony of circle, blackish green, diameter 0.3 ~ 0.5 cm after 48 h; This bacterial strain thalli growth feature is as follows: in liquid medium within, after 28 ℃ of cultivation 48 h, be the particulate state adherent growth; These bacterial strain physio-biochemical characteristics are as follows: Gram-positive.
Ball spore cladosporium of the present invention (Cladosporium sphaerospermum) the 100102A bacterial strain that Isolation and screening obtains in the seawater of Zhejiang Province's Area of The East China Sea collection.
The screening purification process of bacterial strain is: use method of dilution butteron on plate to be applied on the potato plate culture medium in the seawater of collection, 28 ℃ are cultured to colony counts and no longer increase, and picking list bacterium colony is to slant medium.Cultivate 2d for 28 ℃, selected with the difference of strain morphology feature, obtain this bacterial strain, and this bacterial strain is carried out to strain identification.
The composition of described plate culture medium and slant medium is identical, consists of: potato 150 ~ 350g/L, and glucose 10 ~ 30g/L, agar 15 ~ 35g/L, solvent is water, pH7.2 ~ 8.0.Ball spore cladosporium (Cladosporium sphaerospermum) 100102A that the present invention obtains through screening, coating or streak inoculation grows rapidly on plate culture medium, and 28 ℃ of cultivations grow the bacterium colony of circle, blackish green, diameter 0.1 ~ 0.3 cm after 48 h.
By the state-run bioinformation of 16sRNA partial nucleotide sequence and the U.S. center of described ball spore cladosporium (Cladosporium sphaerospermum) 100102A (National Center for Biotechnology Information USA, NCBI) the microorganism 16sRNA sequence alignment in gene pool, by its name ball spore cladosporium (Cladosporium sphaerospermum) 100102A.The partial nucleotide sequence of the 16s rRNA of this bacterial strain is as follows:
TCCGTAGGGGTAACCTGCGGAGGGATCATTACAAGTTGACCCCGGCCCTCGGGCCGGGATGTTCACAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTCCGGGCGGGGGCCCCGGGTGGACATTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAATTTAATTAATAAATTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTTGGTATTGGGCGACGCGGTCCGCCGCGCGCCTCAAATCGACCGGCTGGGTCTTTCGTCCCCTCAGCGTTGTGGAAACTATTCGCTAAAGGGTGCCGCGGGAGGCCACGCCGTAAAACAACCCCATTTCTAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA
The invention still further relates to the application of described bacterium ball spore cladosporium 100102A in preparing antitumor drug.
The medicine that described antitumor drug is Hepatoma therapy or neural cancer.
Concrete, describedly be applied as the application of described ball spore cladosporium 100102A extract in preparing antitumor drug.
Preferably, described extract is n-butanol extract, obtain by the following method: ball spore cladosporium 100102A(CCTCC No:M 2012290) be seeded to liquid fermentation medium, in 25 ~ 35 ℃, under 150 ~ 250r/min oscillating condition, cultivate 14 ~ 30 days, obtain fermented liquid, fermented liquid is through cytoclasis, centrifuging and taking filtrate is used n-butanol extraction, extraction liquid concentrates to obtain medicinal extract, medicinal extract be take ethanol: water volume ratio as 2:8 ~ 10:0 be moving phase, carry out HP-20 macroporous resin chromatography and cross post, gradient elution, collect the elutriant of ethanol volumetric concentration more than 80%, concentrated by rotary evaporation volatilizes, obtain described n-butanol extract, described liquid nutrient medium consists of: glucose 8 ~ 20 g/L, and peptone 1.5 ~ 3 g/L, yeast extract paste 0.8 ~ 2g/L, solvent is water: the mixed solution 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 cultivation, and common the needs first activates through slant culture, then through seed culture, acquisition seed bacterial strain, accesses liquid fermentation medium again and is produced the enzyme cultivation.
Described slant medium consists of: potato 150 ~ 350g/L, and glucose 10 ~ 30g/L, agar 15 ~ 35g/L, solvent is water, pH7.2 ~ 8.0.
Described plane seed culture medium consists of: glucose 8 ~ 15g/L, and peptone 1.5 ~ 4g/L, yeast extract paste 0.5 ~ 1.5g/L, agar 15 ~ 20g/L, solvent is water: the mixed solution of artificial seawater volume ratio 1:1 ~ 4, pH7.2 ~ 8.0.
Described liquid fermentation medium consists of: glucose 8 ~ 15g/L, and peptone 1.5 ~ 3g/L, yeast extract paste 0.8 ~ 2g/L, solvent is water: the mixed solution of artificial seawater volume ratio 1:1 ~ 4, pH7 ~ 8.0.
Concrete, described extract can obtain by the following method:
(1) by thalassiomycetes ball spore cladosporium CCTCC No:M 2012290 inoculation in slant medium, cultivate 24 ~ 48 h, the bacterial classification after being activated in 25 ~ 35 ℃; Described slant medium consists of: potato 150 ~ 350g/L, and glucose 10 ~ 30g/L, agar 15 ~ 35g/L, solvent is water, PH7.2 ~ 8.0;
(2) thalassiomycetes spore cladosporium CCTCC No:M 2012290 thalline after step (1) activation culture are seeded in liquid seed culture medium, under 25 ~ 35 ℃ of conditions, cultivate 16 ~ 48h, obtain kind of a daughter bacteria; Described liquid seed culture medium consists of: glucose 8 ~ 15 g/L, and peptone 1.5 ~ 4 g/L, yeast extract paste 0.5 ~ 1.5 g/L, solvent is water: the mixed solution of artificial seawater volume ratio 1:1 ~ 4, pH7.2 ~ 8.0;
(3) inoculum size with 1% ~ 10% volume ratio by step (2) seed bacterial strain, culture transferring, in liquid fermentation medium, is cultivated 14 ~ 30 days under 25 ~ 35 ℃, 150 ~ 250 r/min oscillating conditions, obtains fermented liquid; Described liquid fermentation medium consists of: glucose 8 ~ 20 g/L, and peptone 1.5 ~ 3 g/L, yeast extract paste 0.8 ~ 2g/L, solvent is water: the mixed solution of artificial seawater volume ratio 1:1 ~ 4, pH 7 ~ 8;
(4) by the cytoclasis under 4 ℃ of conditions of the fermented liquid of step (3), centrifugal or filtration, separate and remove thalline, get filtrate with collecting upper layer of extraction liquid after n-butanol extraction, the concentrated total medicinal extract of the oil-like extracts made that volatilizes;
(5) the total medicinal extract of oil-like extracts of step (4) is carried out to the processing of HP-20 macroporous resin column, take ethanol: water volume ratio as 2:8 ~ 10:0 be moving phase, gradient elution, collect the elutriant of ethanol volumetric concentration more than 80%, concentrated by rotary evaporation volatilizes, and obtains described n-butanol extract.
Beneficial effect of the present invention is mainly reflected in: (1) thalassiomycetes bacterial strain of the present invention---and ball spore cladosporium (Cladosporium sphaerospermum) 100102A nutritional requirement is simple, easily cultivation; (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 of cultivating gained has certain anti-tumor activity to liver cancer cell (HepG2).Its total medicinal extract was carried out to the HP-20 macroporous resin column and processed, and collected the little polar fraction that obtains and can reach more than 95% the inhibition activity of HepG2 and PC12 cell inhibiting rate when the concentration 200 μ g/ml, there is very strong anti-tumor activity.
(4) accompanying drawing explanation
The colonial morphology that Fig. 1 is ball spore cladosporium (Cladosporium sphaerospermum) 100102A on plate culture medium;
The thalli morphology that Fig. 2 is ball spore cladosporium (Cladosporium sphaerospermum) 100102A 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, purifying and evaluation
(1) will use method of dilution butteron on plate to be applied on the potato plate culture medium from the seawater of Area of The East China Sea collection, 28 ℃ be cultured to colony counts and no longer increase, and picking list bacterium colony is to slant medium.Cultivate 2d for 28 ℃, selected with the difference of strain morphology feature, obtain this bacterial strain.Described potato plate culture medium was prepared by following the composition: potato 200g, glucose 20g, agar 20g, distilled water 1000ml;
(2) bacterial strain 100102A screening obtained carries out the PCR product nucleotide sequence comparison of 16sRNA, by its called after ball spore cladosporium (Cladosporium sphaerospermum) 100102A, submit Chinese Typical Representative culture collection center to, preserving number: CCTCC No:M 2012290, preservation date on July 18th, 2012.
Embodiment 2: the activation of bacterial strain and large scale culturing
(1) inoculation screening in embodiment 1 obtained is in slant medium, cultivates 24 ~ 48h in 28 ℃, the bacterial strain after activate, and described slant medium was prepared by following the composition: potato 200g, glucose 20g, agar 20g, distilled water 1000ml.
(2) by the inoculation after step (1) activation culture to liquid seed culture medium, cultivate 24 ~ 48h under 28 ℃, 150 ~ 250 r/min oscillating conditions, obtain seed liquor, institute's liquid seed culture medium was prepared by following the composition: glucose 10g, peptone 2g, yeast extract paste 1g, distilled water 400ml, artificial seawater 600ml, pH7.5.
(3) inoculum size with 8% volume ratio by step (2) seed liquor, culture transferring, in the mass liquid fermention medium, is cultivated 21d under 28 ℃, 150 ~ 250 r/min oscillating conditions, obtains fermented liquid.Described liquid fermentation medium was prepared by following the composition: glucose 10g, peptone 2g, yeast extract paste 1g, distilled water 400ml, artificial seawater 600ml, pH7.5.
Embodiment 3: the application of thalassiomycetes ball spore cladosporium (Cladosporium sphaerospermum) 100102A in anti-tumor activity
(1) will cultivate the fermented liquid of gained in embodiment 2 prior to carrying out bacterial cell disruption 20min in ultrasonic cell disruption instrument, then centrifugal (9000r/min under 4 ℃ of conditions, 15min) (or filter also can), remove thalline, with the equal-volume propyl carbinol, extracted, extraction phase is revolved to steaming, and gained medicinal extract is the total medicinal extract of secondary metabolite of this bacterial strain.
(2) the total medicinal extract of fermented liquid of embodiment 2 gained is carried out to rough separation, get the total medicinal extract of 10g and carry out upper prop (the high 170cm of post, diameter 1cm, filler HP-20 macroporous adsorbent resin), use ethanol: water volume ratio is 2 ︰ 8,4 ︰ 6,6 ︰ 4,8 ︰ 2, the eluent of 10 ︰ 0 is rinsed respectively, and each gradient is rinsed 500ml, to rinsing gained liquid, is revolved the steaming processing, final 5 positions, be denoted as A, B, C, D, E.
(3) 5 position A, B, C, D, the E of the total medicinal extract of fermented liquid of step (1) gained and step (2) gained are carried out to antitumor cytolytic activity.Concrete steps are as follows: choose two strain tumour cells, be respectively Adrenal Pheochromocytoma (PC12 cell) and liver cancer cell (HepG2 cell), the cell strain of phase of taking the logarithm is made cell suspension, be inoculated in 96 orifice plates, cultivate 48h, testing sample (total medicinal extract, 5 position A, B, C, D, E) after adding 0.1%DMSO10 μ L to dissolve, the 1640 cell culture medium dilutions with serum-free, add 100 μ L to test group, make the concentration of the total medicinal extract of final fermented liquid be respectively 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 400 μ g/ml, 800 μ g/ml, 5 position A, B, C, D, the concentration of E is 200 μ g/ml, negative control group adds equivalent and does not contain the serum free medium of sample, the blank group is acellular and serum free medium sample, Etoposide (VP-16) is as positive reference substance, each concentration is established 5 multiple holes, tumour cell is cultivated 48h at CO2 incubator (37 ℃), 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 vibrate and make MTT purple product dissolve fully by microplate reader, 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 be tried to achieve inhibiting rate.
(4) according to the data of step (1)-(3) gained in Table 1 ~ 3.
Table 1: the restraining effect of bacterial strain fermentation liquor medicinal extract to the HepG2 cell
| Drug level (μ g/ml) | Inhibiting rate (%) |
| 50 | 36.30 |
| 100 | 36.78 |
| 200 | 37.01 |
| 400 | 45.94 |
| 800 | 51.20 |
Table 2: the restraining effect of bacterial strain fermentation liquor medicinal extract to the PC12 cell
| Drug level (μ g/ml) | Inhibiting rate (%) |
| 50 | 3.85 |
| 100 | 13.03 |
| 200 | 11.94 |
| 400 | 17.28 |
| 800 | 22.38 |
Table 3: 5 polar fractions restraining effect to the HepG2 cell
| Sample | Concentration (μ g/ml) | Inhibiting rate (%) |
| A | 200 | 5.91 |
| B | 200 | 8.50 |
| C | 200 | 20.95 |
| D | 200 | 97.69 |
| E | 200 | 99.49 |
Show the restraining effect of 4:5 polar fraction to the PC12 cell
| Sample | Concentration (μ g/ml) | Inhibiting rate (%) |
| A | 200 | 4.54 |
| B | 200 | 7.10 |
| C | 200 | 23.01 |
| D | 200 | 95.78 |
| E | 200 | 96.07 |
In table, data can obtain: the anti-tumor activity of the secondary metabolite of thalassiomycetes ball spore cladosporium (Cladosporium sphaerospermum) 100102A is high, and its total medicinal extract of fermented liquid of cultivating gained has certain anti-tumor activity to liver cancer cell (HepG2).Its total medicinal extract was carried out to the HP-20 macroporous resin column and processed, and collected the little polar fraction that obtains and can reach more than 95% the inhibition activity of HepG2 and PC12 cell inhibiting rate when the concentration 200 μ g/ml, there is very strong anti-tumor activity.
Claims (3)
1. a strain has the thalassiomycetes of anti-tumor activity---ball spore cladosporium (Cladosporium sphaerospermum) 100102A, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M2012290, preservation date: on July 18th, 2012.
2. the application of ball spore cladosporium 100102A as claimed in claim 1 in preparing antitumor drug, the medicine that described antitumor drug is Hepatoma therapy or neural cancer.
3. application as claimed in claim 2, it is characterized in that the described application of extract in preparing antitumor drug that is applied as ball spore cladosporium 100102A, described extract is n-butanol extract, obtain by the following method: ball spore cladosporium 100102A is seeded to liquid fermentation medium, in 25～35 ℃, under 150～250r/min oscillating condition, cultivate 14～30 days, obtain fermented liquid, fermented liquid is through cytoclasis, centrifuging and taking filtrate is used n-butanol extraction, extraction liquid concentrates to obtain medicinal extract, medicinal extract be take ethanol: water volume ratio was moving phase as 2: 8～10: 0, carry out HP-20 macroporous resin chromatography and cross post, gradient elution, collect the elutriant of ethanol volumetric concentration more than 80%, concentrated by rotary evaporation volatilizes, obtain described n-butanol extract, described liquid fermentation medium consists of: glucose 8～20g/L, and peptone 1.5～3g/L, yeast extract paste 0.8～2g/L, solvent is water: the mixed solution of artificial seawater volume ratio 1: 1～4, pH7～8, the every 100ml of described artificial seawater consists of: NaCl2.448g, Na
2sO
40.3917g, KCl0.0664g, KBr0.0096g, SrCl
20.0024g, MgCl
26H
2o0.4981g, CaCl
2h
2o0.1102g, NaHCO
30.0192g, H
3bO
30.0026g, NaF0.0004g, distilled water 100ml.
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