CN103074233A - Marine fungus penicillium chrysogenum and application thereof to preparation of anti-tumor medicines - Google Patents
Marine fungus penicillium chrysogenum and application thereof to preparation of anti-tumor medicines Download PDFInfo
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- CN103074233A CN103074233A CN2012105740836A CN201210574083A CN103074233A CN 103074233 A CN103074233 A CN 103074233A CN 2012105740836 A CN2012105740836 A CN 2012105740836A CN 201210574083 A CN201210574083 A CN 201210574083A CN 103074233 A CN103074233 A CN 103074233A
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Abstract
The invention provides a marine fungus-penicillium chrysogenum MNP07010101 with anti-tumor activity and application thereof. The marine fungus was collected into China Center for Type CultureCollection (CCTCC) on August 28th, 2012, wherein the collection number is CCTCC No: M2012317. The invention has the following benefits: (1) the marine fungus 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, the fermented liquid total extractum cultivated by the secondary metabolite has certain anti-tumor activity on HepG2, PC12 and U937 cells, and after the total extractum is treated by an HP-20 macroporous resin column, the highest inhibition rate of the collected small polar section on the HepG2, PC12 and U937 cells can reach over 90 percent in the aspect of inhibition activity when the concentration is 200 ug/ml, and high anti-tumor activity is achieved.
Description
(1) technical field
The present invention relates to the thalassiomycetes that a strain has anti-tumor activity---Penicllium chrysogenum (Penicillium chrysogenum) MNP07010101 and application thereof.
(2) background technology
Thalassiomycetes is an important branch of marine microorganism, because living environment and the ecosystem of their uniquenesses, guarantee that not only it survives in extreme environment, and the throughput of the meta-bolites that does not have in the Lu Sheng microorganism is provided, thereby be the fabulous resource of varied compounds and medicine.The mycetogenetic meta-bolites of marine source is mainly the compound of the types such as alkaloid, peptide class, polyketone class, steroidal and terpene; wherein much have significantly antitumor, antibacterium, antimycotic, pest-resistant, suppress the biological activity such as micro algae growth; have important fundamental research meaning in medicine and the fields such as agricultural chemicals research and development, environment protection, also shown the brilliant prospect of from marine microorganism, seeking pharmacological active substance.
(3) summary of the invention
The object of the invention provides the thalassiomycetes bacterial strain that a strain has anti-tumor activity---Penicllium chrysogenum (Penicillium chrysogenum) MNP07010101, and the application in the preparation antitumor drug.
The technical solution used in the present invention is:
One strain has the thalassiomycetes of anti-tumor activity---Penicllium chrysogenum (Penicillium chrysogenum) MNP07010101, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M 2012317, preservation date on August 28th, 2012.
The colony characteristics of this bacterial strain is as follows: coating or streak inoculation are grown on the PDA plate culture medium rapidly, behind 28 ℃ of cultivation 48 h, grow to diameter 21-25mm, the edge white mycelium, and quality is velvet-like, blue-greenish colour, the bacterium colony reverse side becomes light tan; The thalli growth feature of this bacterial strain is as follows: in the liquid medium within, be the particulate state adherent growth behind 28 ℃ of cultivation 48 h.
Penicllium chrysogenum of the present invention (Penicillium chrysogenum) MNP07010101 is obtained by Isolation and screening 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 plate culture medium in the seawater that gathers, 28 ℃ are cultured to colony counts and no longer increase, and picking list bacterium colony is to slant medium.Cultivate 2d for 28 ℃, select with the difference of strain morphology feature, namely get this bacterial strain, and this bacterial strain is carried out 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 are water, pH7.2 ~ 8.0.Penicllium chrysogenum (Penicillium chrysogenum) MNP07010101 that the present invention obtains through screening, coating or streak inoculation are in the plate culture medium growth rapidly, behind 28 ℃ of cultivation 48 h, grow to diameter 21-25mm, the edge white mycelium, quality is velvet-like, blue-greenish colour, and the bacterium colony reverse side becomes light tan.The state-run bioinformation of 16sRNA partial nucleotide sequence and U.S. center (National Center for Biotechnology Information USA with this bacterial strain, NCBI) the microorganism 16sRNA sequence alignment in the gene pool confirms as Penicllium chrysogenum (Penicillium chrysogenum).The partial nucleotide sequence of the 16s rRNA of this bacterial strain is as follows:
CTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCT?GAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTCCGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGAGGAGGAA。
The invention still further relates to the application of described Penicllium chrysogenum MNP07010101 in the preparation antitumor drug.
Concrete, described antitumor drug is the medicine of Hepatoma therapy, neural cancer or lymphatic cancer.
Concrete, described Penicllium chrysogenum MNP07010101 extract is for the preparation of antitumor drug.
Preferably, described extract is ethyl acetate extract, make by the following method: Penicllium chrysogenum MNP07010101 is seeded to liquid fermentation medium, in 25 ~ 35 ℃, cultivated 14 ~ 30 days under 150 ~ 250 r/min oscillating conditions, obtain fermented liquid, fermented liquid is through cytoclasis, separate and remove thalline, use ethyl acetate extraction, the concentrated medicinal extract that volatilizes to get of extraction liquid, HP-20 macroporous resin chromatography is crossed post on the medicinal extract, and take methyl alcohol: water volume ratio carries out gradient elution as 2:8 ~ 10:0 as elutriant, collects the elutriant of methyl alcohol volumetric concentration more than 60%, revolve the inspissation contracting and volatilize, namely get described ethyl acetate extract; Described liquid fermentation medium consists of: Fructus Hordei Germinatus soaks powder 3.0 ~ 7.0g/L, maltose 1.5 ~ 2.0g/L, and glucose 3.0 ~ 7.0 g/L, yeast soak powder 0.8 ~ 1.5 g/L, and solvent is water: the mixed solution of artificial seawater volume ratio 1:0.25 ~ 4, pH7.2 ~ 8.0.
Per 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 activates through slant culture first, then accesses liquid fermentation medium through seed culture, acquisition seed liquor again and produces the enzyme cultivation.
Described slant medium consists of: potato 150 ~ 350g/L, and glucose 10 ~ 30g/L, agar 15 ~ 35g/L, solvent are water, pH7.2 ~ 8.0.
Described liquid seed culture medium consists of: Fructus Hordei Germinatus soaks powder 3.0 ~ 7.0g/L, maltose 1.5 ~ 2.0g/L, and glucose 3.0 ~ 7.0 g/L, yeast soak powder 0.8 ~ 1.5 g/L, and solvent is water: the mixed solution of artificial seawater volume ratio 1:0.25 ~ 4.0, pH7.2 ~ 8.0;
Described liquid fermentation medium consists of: Fructus Hordei Germinatus soaks powder 3.0 ~ 7.0g/L, maltose 1.5 ~ 2.0g/L, and glucose 3.0 ~ 7.0 g/L, yeast soak powder 0.8 ~ 1.5 g/L, and solvent is water: the mixed solution of artificial seawater volume ratio 1:0.25 ~ 4.0, pH7.2 ~ 8.0;
Concrete, described extract obtains by the following method:
(1) with thalassiomycetes Penicillium chrysogenum MNP07010101 inoculation in slant medium, cultivate 24 ~ 48 h, the bacterial classification after obtaining activating in 25 ~ 35 ℃; Described slant medium consists of: potato 150 ~ 350g/L, and glucose 10 ~ 30g/L, agar 15 ~ 35g/L, solvent are water, pH7.2 ~ 8.0;
(2) thalassiomycetes Penicillium chrysogenum MNP07010101 thalline after step (1) activation culture is seeded in the seed culture medium of plane, under 25 ~ 35 ℃, 150 ~ 210r/min concussion condition, cultivates 24 ~ 48h, obtain seed liquor; Described liquid seed culture medium consists of:
(3) with the inoculum size of step (2) seed bacterial strain with 1% ~ 10% volume ratio, culture transferring was cultivated 14 ~ 30 days under 25 ~ 35 ℃, 150 ~ 250 r/min oscillating conditions in liquid nutrient medium, obtained fermented liquid; Described liquid nutrient medium consists of: Fructus Hordei Germinatus soaks powder 3.0 ~ 7.0g/L, maltose 1.5 ~ 2.0g/L, and glucose 3.0 ~ 7.0 g/L, yeast soak powder 0.8 ~ 1.5 g/L, and solvent is water: the mixed solution of artificial seawater volume ratio 1:0.25 ~ 4.0, pH 7 ~ 8;
(4) with fermented liquid cytoclasis under 4 ℃ of conditions of step (3), centrifugal or filter, separate and remove thalline, the gained fermented liquid is collected upper layer of extraction liquid, the concentrated total medicinal extract of the oil-like extracts that makes that volatilizes after with ethyl acetate extraction;
(5) the total medicinal extract of oil-like extracts with step (4) carries out the processing of HP-20 macroporous resin column, take ethanol: water volume ratio as 2:8 ~ 10:0 as moving phase, gradient elution is collected the elutriant of ethanol volumetric concentration more than 60%, revolve the inspissation contracting and volatilize, namely get described ethyl acetate extract.
Beneficial effect of the present invention is mainly reflected in: (1) thalassiomycetes bacterial strain of the present invention 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, its total medicinal extract of fermented liquid of cultivating gained has certain anti-tumor activity to HepG2, PC12 cell and U937 cell, its total medicinal extract was carried out the HP-20 macroporous resin column to be processed, the little polarity paragraph that collection obtains reaches as high as more than 90% HepG2, PC12, U937 cell inhibitory activity inhibiting rate when the concentration 200 μ g/mL, has very strong anti-tumor activity.
(4) description of drawings
Fig. 1 is the colonial morphology of Penicllium chrysogenum MNP07010101 on the plate culture medium;
Fig. 2 is the thalli morphology of Penicllium chrysogenum MNP07010101 under the opticmicroscope.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, 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 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 ℃, select with the difference of strain morphology feature, namely get this bacterial strain.Described potato plate culture medium is prepared by following composition: potato 200g, glucose 20g, agar 20g, distilled water 1000mL.
(2) the bacterial strain MNP07010101 that screening is obtained carries out the PCR product nucleotide sequence comparison of 16sRNA, with its called after Penicllium chrysogenum MNP07010101(Penicillium chrysogenum MNP07010101), submit Chinese Typical Representative culture collection center to, preserving number: CCTCC No:M 2012317, preservation date on August 28th, 2012.
Embodiment 2: the activation of bacterial strain and large scale culturing
(1) inoculation that screening among the embodiment 1 is obtained is in slant medium, and in 28 ℃ of cultivation 48h, the bacterial strain after obtaining activating, described slant medium are pressed following composition preparation: potato 200g, glucose 20g, agar 20g, distilled water 1000mL;
(2) with the inoculation after step (1) activation culture to liquid nutrient medium, under 28 ℃, 150 ~ 250 r/min oscillating conditions, cultivate 248h, obtain seed liquor, described liquid seed culture medium consists of: Fructus Hordei Germinatus soaks powder 5.0g/L, maltose 2.0g/L, glucose 5.0g/L, yeast soak powder 1.0g/L, solvent is water: the mixed solution of artificial seawater volume ratio 6:4, pH 7 ~ 8.
(3) with the inoculum size of step (2) seed liquor with 8% volume ratio, culture transferring is cultivated 21d under 28 ℃, 150 ~ 250 r/min oscillating conditions in the mass liquid fermention medium, obtain fermented liquid.Described liquid fermentation medium consists of: Fructus Hordei Germinatus soaks powder 5.0g/L, maltose 2.0g/L, and glucose 5.0 g/L, yeast soaks powder 1.2g/L, and solvent is water: the mixture of artificial seawater volume ratio 8:2, pH 7 ~ 8.
Embodiment 3: the antitumor activity of thalassiomycetes Penicllium chrysogenum MNP07010101
(1) will cultivate the fermented liquid of gained among the embodiment 2 prior to carrying out bacterial cell disruption 20min in the ultrasonic cell disruption instrument, then centrifugal (9000r/min under 4 ℃ of conditions, 15min) or filter, remove thalline, extract with ethyl acetate, extraction phase is revolved steaming, gained medicinal extract is the total medicinal extract of secondary metabolite of this bacterial strain.
(2) the total medicinal extract of fermented liquid with embodiment 2 gained carries out rough separation, get the total medicinal extract of 5.0g and carry out upper prop (the high 170cm of post, diameter 1cm, filler HP-20 macroporous adsorbent resin), use eluent methyl alcohol: the mixed solvent of water volume ratio 2 ︰ 8,4 ︰ 6,6 ︰ 4,8 ︰ 2,10 ︰ 0 washes successively, and each gradient flushing 500mL revolves steaming to flushing gained liquid and processes, final 5 paragraphs are denoted as A, B, C, D, E.
The total medicinal extract of fermented liquid of step (1) gained and 5 paragraph A, B, C, D, the E of step (2) gained are carried out antitumor cytolytic activity.Concrete steps are as follows: choose three strain tumour cells, be respectively Adrenal Pheochromocytoma (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 is made cell suspension, be inoculated in 96 orifice plates, cultivate 48h, testing sample (total medicinal extract, 5 paragraph A, B, C, D, E) after the adding 0.1%DMSO10 μ L dissolving, 1640 cell culture mediums dilution with serum-free, add 100 μ L to test group, so that the concentration of the final total medicinal extract of fermented liquid is respectively 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/mL, 800 μ g/mL, 5 paragraph A, B, C, D, the concentration of E is 200 μ g/mL, negative control group adds the serum free medium that equivalent does not contain sample, the blank group then is acellular and serum free medium sample, Etoposide (VP-16) is as positive reference substance, each concentration is established 5 multiple holes, and tumour cell is at CO
2Incubator (37 ℃) is cultivated 48h, every hole adds the MTT20 μ L of 5mg/mL, after continuing to cultivate 4h, carefully remove supernatant, every hole adds DMSO150 μ L, and vibration 10min fully vibrates so that MTT purple product dissolves fully with 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.
(1) data according to step (1)-(3) gained see Table 1 ~ 2.
Table 1: the total medicinal extract of fermented liquid is to the restraining effect of 3 kinds of tumour cells
Record the total medicinal extract of its fermented liquid to PC12 tumour cell half inhibiting rate concentration IC with SPSS software
50Value is for 264.29ug/mL, to half inhibiting rate concentration IC of HepG-2 tumour cell
50Value is for 202.55ug/mL, to half inhibiting rate concentration IC of U937 tumour cell
50Value is 56.688 ug/mL.
Show 2:5 polarity paragraph to the restraining effect of 3 kinds of tumour cells
By the data obtained as can be known: the anti-tumor activity of the secondary metabolite of thalassiomycetes Penicllium chrysogenum MNP07010101 is high, and its total medicinal extract of fermented liquid of cultivating gained has certain anti-tumor activity to Adrenal Pheochromocytoma (PC12 cell), liver cancer cell (HepG2 cell) and human tissue cell's lymphoma cell (U937 cell).Its total medicinal extract is carried out the HP-20 macroporous resin column processed, and collected the little polarity paragraph that obtains the inhibition activity of HepG2, PC12 and U937 cell inhibiting rate when the concentration 200 μ g/mL is reached as high as more than 90%, had very strong anti-tumor activity.
Claims (6)
1. a strain has the thalassiomycetes of anti-tumor activity---Penicllium chrysogenum (Penicillium chrysogenum) MNP07010101, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number: CCTCC No:M 2012317, preservation date on August 28th, 2012.
2. Penicllium chrysogenum MNP07010101 as claimed in claim 1 is characterized in that the partial nucleotide sequence of its 16s rRNA is as follows:
CTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTCCGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGAGGAGGAA。
3. the application of Penicllium chrysogenum MNP07010101 claimed in claim 1 in the preparation antitumor drug.
4. application as claimed in claim 3 is characterized in that described antitumor drug is the medicine of Hepatoma therapy, neural cancer or lymphatic cancer.
5. such as claim 3 or 4 described application, it is characterized in that described Penicllium chrysogenum MNP07010101 extract is for the preparation of antitumor drug.
6. application as claimed in claim 5, it is characterized in that described extract is ethyl acetate extract, make by the following method: Penicllium chrysogenum MNP07010101 is seeded to liquid fermentation medium, in 25 ~ 35 ℃, cultivated 14 ~ 30 days under 150 ~ 250 r/min oscillating conditions, obtain fermented liquid, fermented liquid is through cytoclasis, separate and remove thalline, use ethyl acetate extraction, the concentrated medicinal extract that volatilizes to get of extraction liquid, HP-20 macroporous resin chromatography is crossed post on the medicinal extract, and take methyl alcohol: water volume ratio carries out gradient elution as 2:8 ~ 10:0 as elutriant, collects the elutriant of methyl alcohol volumetric concentration more than 60%, revolve the inspissation contracting and volatilize, namely get described ethyl acetate extract; Described liquid fermentation medium consists of: Fructus Hordei Germinatus soaks powder 3.0 ~ 7.0g/L, maltose 1.5 ~ 2.0g/L, and glucose 3.0 ~ 7.0 g/L, yeast soak powder 0.8 ~ 1.5 g/L, and solvent is water: the mixed solution of artificial seawater volume ratio 1:0.25 ~ 4.0, pH7.2 ~ 8.0.
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| CN104016982A (en) * | 2014-06-26 | 2014-09-03 | 华东理工大学 | Method for preparing fumigaclavine C by using macroporous resin |
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| CN115322169A (en) * | 2019-11-07 | 2022-11-11 | 自然资源部第三海洋研究所 | Polyketone compounds, preparation method and application thereof |
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