CN104673676A - Harmful mycogone perniciosa and ergot sterane type triterpenoid prepared by fermentation as well as method - Google Patents
Harmful mycogone perniciosa and ergot sterane type triterpenoid prepared by fermentation as well as method Download PDFInfo
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- CN104673676A CN104673676A CN201310648426.3A CN201310648426A CN104673676A CN 104673676 A CN104673676 A CN 104673676A CN 201310648426 A CN201310648426 A CN 201310648426A CN 104673676 A CN104673676 A CN 104673676A
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- mycogone perniciosa
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Abstract
The invention discloses harmful mycogone perniciosa 88362 with the preservation number of CGMCC NO.8352. Ergot sterane type triterpenoid prepared by fermenting the harmful mycogone perniciosa has a structural formula shown in the specification. The invention provides a new strain, two new components and a method for preparing the new compounds by utilizing the new strain, and a material base is provided for developing and utilizing natural active substances from microorganisms.
Description
Technical field
The present invention relates to a kind of Mycogone perniciosa bacterium and the ergostane type triterpene utilizing this bacterium to prepare and preparation method.
Background technology
Triterpene compound is the very important natural product of a class, lot of documents report their chemical structure, biological activity, biosynthesizing and chemosynthesis.Its structure is changeable, and part has antibacterial, antitumor, AntiHIV1 RT activity isoreactivity significantly and improves the effect of immunity, shows potential drug development and is worth, receive the concern of organic chemist and biochemist.
Mycogone perniciosa bacterium is the pathogenic bacteria of the saprophytic edible mushrooms brown heart of the excrement such as mushroom, straw mushroom, Pleurotus sajor-caju grass, and this disease makes further progress the trend spread in recent years, becomes one of adverse factor of Edible Fungi development.Its pathogeny may be that certain meta-bolites secreted due to it acts on mushroom body and causes the generation of brown heart.
At present, the report of ergostane type triterpene is not still prepared with Mycogone perniciosa bacterium.
Summary of the invention
The object of this invention is to provide the Mycogone perniciosa bacterium preparing ergostane type triterpene.
Second object of the present invention is to provide the standby ergostane type triterpene of Mycogone perniciosa bacterium fermentation.
3rd object of the present invention is to provide the method for Mycogone perniciosa bacterium fermentation for ergostane type triterpene.
Technical scheme of the present invention is summarized as follows:
Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 preserving numbers are: CGMCC NO.8352.
The ergostane type triterpene that above-mentioned Mycogone perniciosa bacterium fermentation is standby, the structural formula of described ergostane type triterpene is:
Mycogone perniciosa bacterium fermentation, for the method for ergostane type triterpene, comprises the steps:
(1) by Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 preserving numbers be: CGMCC NO.8352 ferments in solid medium, obtains fermenting culture;
(2) in described fermenting culture, add ethyl acetate to extract, obtain extracting solution, by described extracting solution underpressure distillation, obtain crude extract;
(3) crude extract is through silica gel column chromatography, be that eluent carries out gradient elution with the methylene chloride-methanol that volume ratio is 100:0 ~ 98:2, collect the fraction A that methylene chloride-methanol volume ratio is 100:0 wash-out, collected volume is than the fraction B of the methylene chloride-methanol wash-out for 99:1 ~ 98:2;
(4) after described fraction A decompression spin concentration through gel Sephadex LH-20 column chromatography, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (I) compound; Through purification on normal-phase silica gel column chromatography after fraction B decompression spin concentration, take volume ratio as the petroleum ether-ethyl acetate of 10:1 ~ 5:1 be eluent gradient wash-out, collected volume is than the cut of the petroleum ether-ethyl acetate wash-out for 8:1 ~ 5:1; Again through gel Sephadex LH-20 column chromatography after concentrating under reduced pressure, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (II) compound.
Mycogone perniciosa bacterium fermentation is for the method for ergostane type triterpene, step (1) is preferably: accessed by Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 in seed culture medium, cultivate 3 ~ 5 days in 20-25 DEG C, obtain seed culture fluid, by seed culture fluid by volume 1:20 ~ 1:30 ratio access solid medium in, in 20-25 DEG C of static gas wave refrigerator 50-60 days, obtain fermenting culture; Described seed culture medium often rises and contains: glucose 4 grams, malt extract 10 grams, yeast extract paste 4 grams, and surplus is water, pH value 6.0 ~ 7.0; Described solid medium is made up of 160 grams of rice and 240mL distilled water.
Advantage of the present invention:
The invention provides a strain new strains, two new compounds and utilize this new strains to prepare the method for new compound.Basic substance is provided for developing microbe-derived natural active matter.
Accompanying drawing explanation
Fig. 1 is the NOESY spectrum of formula (I) compound.
Fig. 2 is the NOESY spectrum of formula (II) compound.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.The test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The separation andpreconcentration of embodiment 1, bacterial strain Mycogone perniciosa bacterium
One, the separation of bacterial strain
To be in Augusts, 2008 be separated speckle wire cover umbrella (the Inocybe maculata Boud) basidioma that collects obtain from the Jilin Province Changbai Mountain woods bacterial strain 88362.Basidioma is put into aseptic plastics bag, laboratory is taken back from forest land, be placed on testing table and dry in the air under room temperature 2 hours, make its surface drying, Bechtop tears cap epidermis off with the tweezers through sterilization under spirit lamp, cuts a little bacterial context with sterile scalpel and insert on PDA inclined-plane and cultivate, treat that it grows bacterium colony, cut fritter mycelia with inoculating needle from colony edge, be transferred to another PDA inclined-plane, purifying, preservation.
Two, the qualification of bacterial strain
1, identification of morphology
Bacterial strain 88362 grows comparatively fast on PDA substratum, and under 25 DEG C of dark conditions, grow 9 days, colony diameter is 75 ~ 80mm; Bacterium colony is just white, fades to burgundy red to clay powder, and it is raw that mycelia pastes substratum, and aerial hyphae is undeveloped, and the bacterium colony back side is shallow burgundy.The hyphae colorless of this bacterial strain, tool is separated, and branch is wide 2.3 ~ 3.1 μm.This bacterium produces chlamydospore and conidium on PDA substratum.Chlamydospore end is raw, mostly is two cell, color depth, and upper cell is comparatively large, wall thickness, there is wart on surface, subsphaeroidal, 22.3 ~ 35.9(31.5) × 18.2 ~ 33.8(29.0) μm, lower cell, wall is thin, smooth surface, semisphere, 11.2 ~ 22.1(18.0) × 5.9 ~ 14.9(11.1) μm.Minority chlamydospore is by 3 cellularities, and 2 of upper end comparatively large, and there is wart on surface, and 1 of lower end less, smooth surface.Conidiophore ampuliform, takes turns dendritic arrangement.Conidium is colourless, oval, long oval to club shape, unicellular or two cell.Single celled, 5.3 ~ 17.3(9.3) × 3.8 ~ 13.3(7.6) μm; Bicellular, slightly hang and contract or contracting of not hanging in separated place, 11.3 ~ 29.9(17.7) × 2.5 ~ 14.4(7.8) μm.Minority three cell.
2, Molecular Identification
The ITS gene order of bacterial strain 88362 is shown in SEQ ID NO.1.
The result of comprehensive morphological qualification and Molecular Identification, bacterial strain 88362 belongs to Mycogone perniciosa bacterium (Mycogone perniciosa Magnus)
Three, the preservation of bacterial strain
Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number: CGMCC NO.8352.
Embodiment 2
Mycogone perniciosa bacterium fermentation, for the method for ergostane type triterpene, comprises the steps:
(1) fermentation culture
1. bacterial strain activation: be inoculated on PDA flat board by Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 mycelium, cultivates 5 days for 25 DEG C.PDA is dull and stereotyped: by potato 200 grams, glucose 20 grams, and agar 15 grams of adding distil waters are to 1L, and 121 DEG C of high pressure steam sterilization 30min, make flat board;
2. seed culture:
Cover with after PDA flat board until bacterium, by 5 1cm
2the bacterium block of size is together with in culture medium inoculated to the 250mL triangular flask containing 50mL seed culture medium and on shaking table, cultivate 5 days (rotating speed: 200rpm) for 25 DEG C, obtain seed culture fluid, described seed culture medium often rises and contains: glucose 4 grams, malt extract 10 grams, yeast extract paste 4 grams, surplus is water, pH value 6.0; 50mL substratum is added, 121 DEG C of sterilizings 30 minutes in each 250mL triangular flask;
3. ferment:
By in the ratio access solid medium of seed culture fluid 1:20 by volume, in 25 DEG C of static gas wave refrigerator 50 days, obtain fermenting culture; Described solid medium is made up of 160 grams of rice and 240mL distilled water; Add in 1L triangular flask by 160 grams of rice and 240mL pure water, totally 12 bottles of about 2 kilograms of rice, soaked overnight, 121 DEG C of high pressure steam sterilization 30min, cool stand-by;
(2) smashed to pieces by described fermenting culture apparatus, add ethyl acetate and extract, static soak 1 day, obtains extracting solution, by described extracting solution underpressure distillation, obtains crude extract;
(3) crude extract is through silica gel column chromatography (200 ~ 300 order column chromatography silica gels, Ф 6 × 40cm), be that eluent carries out gradient elution with the methylene chloride-methanol that volume ratio is 100:0 ~ 98:2, collect the fraction A that methylene chloride-methanol volume ratio is 100:0 wash-out, collected volume is than the fraction B of the methylene chloride-methanol wash-out for 99:1 ~ 98:2;
(4) after described fraction A decompression spin concentration through gel Sephadex LH-20 column chromatography (Ф 1 × 100cm), take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (I) compound; Through purification on normal-phase silica gel column chromatography (200 ~ 300 order column chromatography silica gels after fraction B decompression spin concentration, Ф 1.5 × 30cm), take volume ratio as the petroleum ether-ethyl acetate of 10:1 ~ 5:1 be eluent gradient wash-out, collected volume is than the cut of the petroleum ether-ethyl acetate wash-out for 8:1 ~ 5:1; Again through gel Sephadex LH-20 column chromatography after concentrating under reduced pressure, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (II) compound.
The structural formula of described ergostane type triterpene is:
Embodiment 3
Mycogone perniciosa bacterium fermentation, for the method for ergostane type triterpene, comprises the steps:
(1) Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 is accessed in seed culture medium, in 20 DEG C cultivate 3 days, obtain seed culture fluid, by seed culture fluid by volume 1:30 ratio access solid medium in, in 20 DEG C of static gas wave refrigerator 60 days, obtain fermenting culture; Described seed culture medium often rises and contains: glucose 4 grams, malt extract 10 grams, yeast extract paste 4 grams, and surplus is water, pH value 6.0; Described solid medium is made up of 160 grams of rice and 240mL distilled water;
(2) in described fermenting culture, add ethyl acetate to extract, obtain extracting solution, by described extracting solution underpressure distillation, obtain crude extract;
(3) crude extract is through silica gel column chromatography (200 ~ 300 order column chromatography silica gels, Ф 3.5 × 40cm), be that eluent carries out gradient elution with the methylene chloride-methanol that volume ratio is 100:0 ~ 98:2, collect the fraction A that methylene chloride-methanol volume ratio is 100:0 wash-out, collected volume is than the fraction B of the methylene chloride-methanol wash-out for 99:1 ~ 98:2;
(4) after described fraction A decompression spin concentration through gel Sephadex LH-20 column chromatography (Ф 1 × 100cm), take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (I) compound; Through purification on normal-phase silica gel column chromatography (200 ~ 300 order column chromatography silica gels after fraction B decompression spin concentration, Ф 1.5 × 30cm), take volume ratio as the petroleum ether-ethyl acetate of 10:1 ~ 5:1 be eluent gradient wash-out, collected volume is than the cut of the petroleum ether-ethyl acetate wash-out for 8:1 ~ 5:1; Again through gel Sephadex LH-20 column chromatography after concentrating under reduced pressure, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (II) compound.
The sign of compound shown in formula (I)
The sign of dry formula (I) compound is as follows:
White powder; The molecular ion peak m/z461.3029 [M+Na] that high resolution mass spectrum provides
+(calcd.for C
29h
42o
3na, 461.3032); Hydrogen spectrum (
1h-NMR) and carbon spectrum (
13c-NMR) in table 1; NOESY spectrum is shown in Fig. 1.
Table 1: the NMR data (CDCl of compound shown in formula (I)
3)
δ: ppm, J:Hz, solvent C DCl
3,
1h:600MHz;
13C: 150MHz.
Comprehensively
1h,
13c, HMBC compose, and NOESY spectrum releases the structure of compound 1 as shown in the formula (I).
The sign of dry formula (II) compound:
White powder; The molecular ion peak m/z493.2928 [M+Na] that high resolution mass spectrum provides
+(calcd for C
29h
42o
5na, 493.2930); Hydrogen spectrum (
1h-NMR) and carbon spectrum (
13c-NMR) in table 2; NOESY spectrum is shown in Fig. 2
Table 2: the NMR data (Pyridine-d of compound shown in formula (II)
5)
δ: ppm, J:Hz, solvent Pyridine-d
5,
1h:600MHz;
13C: 150MHz.
Comprehensively
1h,
13c, HMBC compose, and NOESY spectrum releases the structure of compound 2 as shown in the formula (II).
Claims (4)
1. Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 preserving numbers are: CGMCC NO.8352.
2. the ergostane type triterpene that the Mycogone perniciosa bacterium fermentation of claim 1 is standby, the structural formula of described ergostane type triterpene is:
3. Mycogone perniciosa bacterium fermentation is for the method for ergostane type triterpene, it is characterized in that comprising the steps:
(1) Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 is fermented in solid medium, obtain fermenting culture;
(2) in described fermenting culture, add ethyl acetate to extract, obtain extracting solution, by described extracting solution underpressure distillation, obtain crude extract;
(3) crude extract is through silica gel column chromatography, be that eluent carries out gradient elution with the methylene chloride-methanol that volume ratio is 100:0 ~ 98:2, collect the fraction A that methylene chloride-methanol volume ratio is 100:0 wash-out, collected volume is than the fraction B of the methylene chloride-methanol wash-out for 99:1 ~ 98:2;
(4) after described fraction A decompression spin concentration through gel Sephadex LH-20 column chromatography, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (I) compound; Through purification on normal-phase silica gel column chromatography after fraction B decompression spin concentration, take volume ratio as the petroleum ether-ethyl acetate of 10:1 ~ 5:1 be eluent gradient wash-out, collected volume is than the cut of the petroleum ether-ethyl acetate wash-out for 8:1 ~ 5:1; Again through gel Sephadex LH-20 column chromatography after concentrating under reduced pressure, take volume ratio as the methylene chloride-methanol wash-out of 1:1, obtain formula (II) compound.
4. Mycogone perniciosa bacterium fermentation according to claim 3 is for the method for ergostane type triterpene, it is characterized in that described step (1) is: accessed by Mycogone perniciosa bacterium (Mycogone perniciosa) 88362 in seed culture medium, cultivate 3 ~ 5 days in 20-25 DEG C, obtain seed culture fluid, by seed culture fluid by volume 1:20 ~ 1:30 ratio access solid medium in, in 20-25 DEG C of static gas wave refrigerator 50-60 days, obtain fermenting culture; Described seed culture medium often rises and contains: glucose 4 grams, malt extract 10 grams, yeast extract paste 4 grams, and surplus is water, pH value 6.0 ~ 7.0; Described solid medium is made up of 160 grams of rice and 240mL distilled water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109134573A (en) * | 2017-06-27 | 2019-01-04 | 中国科学院上海药物研究所 | A kind of lumistane type steroidal compounds, preparation method and the usage |
| CN114920792A (en) * | 2022-05-06 | 2022-08-19 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Steroid compound and preparation method and application thereof |
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| CN102614195A (en) * | 2011-01-26 | 2012-08-01 | 高雄医学大学 | Triterpenoid composition of antrodia cinnamomea fruiting body, preparation and analysis method |
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| DONG-ZE LIU: "A review of ergostane and cucurbitane triterpenoids of mushroom origin", 《NATURAL PRODUCT RESEARCH: FORMERLY NATURAL PRODUCT LETTERS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134573A (en) * | 2017-06-27 | 2019-01-04 | 中国科学院上海药物研究所 | A kind of lumistane type steroidal compounds, preparation method and the usage |
| CN109134573B (en) * | 2017-06-27 | 2021-03-23 | 中国科学院上海药物研究所 | Ergostane steroid compound, preparation method and application thereof |
| CN114920792A (en) * | 2022-05-06 | 2022-08-19 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Steroid compound and preparation method and application thereof |
| CN114920792B (en) * | 2022-05-06 | 2023-04-14 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Steroid compound and preparation method and application thereof |
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