CN1283779C - Mucor for producing picropodophyllin and its glucoside and kaempferol flavone kind substance, its method and application - Google Patents

Mucor for producing picropodophyllin and its glucoside and kaempferol flavone kind substance, its method and application Download PDF

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CN1283779C
CN1283779C CN 200510042630 CN200510042630A CN1283779C CN 1283779 C CN1283779 C CN 1283779C CN 200510042630 CN200510042630 CN 200510042630 CN 200510042630 A CN200510042630 A CN 200510042630A CN 1283779 C CN1283779 C CN 1283779C
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mucor
chloroform
kaempferol
podophyllus
podophyllotoxin
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CN1718723A (en
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黄建新
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Northwest University
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Northwest University
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Abstract

The present invention relates to new mucor podophyllus, and a method and an application of producing substances of podophyllotoxins and glucosides thereof, and kaempferol flavones by using the new mucor podophyllus. A strain which can be used for producing substances of podophyllotoxins and glucosides thereof and kaempferol flavones is mucor podophyllus J. X. Huang sp. nov GJ-TW8 which is preserved in Chinese typical culture preserving center on April 8th, 2005, and the preservation code is CCTCC No. M205032; the new mucor podophyllus is a pure strain which is separated, sieved and purified from himalayan mayapple roots in Taibai Mountain in Shanxi province, has the physiological characteristic of production of various biologically active substances which have medicinal values and are identical with himalayan mayapple roots, and can be applied to the production of substances of podophyllotoxins and kaempferol flavones, which provides a new way for the development of podophyllum raw material resources.

Description

Produce mucormycosis, the methods and applications of podophyllotoxin and glucoside and kaempferol Flavonoid substances
Technical field
The present invention relates to the methods and applications of the new mucormycosis of a strain and production podophyllotoxin and glucoside and kaempferol Flavonoid substances, belong to microbial technology field.
Background technology
Podophyllotoxin (Podophyllotoxin, PPT) and kaempferol (Kaempferol) flavonoid compound be to be present in Berberidaceae (Berheridaceae), promptly the class in the Podophyllum emodi var chinense class plant has the biologically active substance of pharmaceutical use.Podwyssotzkii was separated to the podophyllotoxin crystallization first from the Chinese podophyllum root resin of America in 1880.The forties in 19th century, people such as King have reported that podophyllotoxin has the effect of colchicine sample, can cause cytometaplasia.A series of afterwards medical science, biological study have confirmed that podophyllotoxin has the anti-tumor activity effect.But because its toxicity is bigger, be restricted in Clinical Application, furtherd investigate its active relation of imitating with structure the 1950's, synthesizing and filtering out with it is a large amount of derivative of parent, some derivative biologically active height, the characteristics that toxicity is low can be used for the multiple disease of clinical treatment.As: etoposide (etoposid.VP16, etoposide) and teniposide (tenipeside.VM26, Vumon) they are the broad-spectrum anti-cancer drugs with clinical application.Podophillotoxines also can be used for agricultural chemical insecticide in addition.
Podophyllotoxin mainly is to extract from wild Podophyllum emodi var chinense class plant rhizome at present, and this class plant only has 4 to belong to 15 kinds in the whole world, and China has 3 to belong to 12 kinds, mainly is grown in the Qinling Mountains, high altitude localitiess such as Tibet, and its poor growth, and the Podophyllum emodi var chinense substances content is low.Because Podophyllum emodi var chinense class material has broad application prospects aspect preventing and treating at medicine and agricultural, its demand increases day by day, to a large amount of collections of this class plant material, this class plant resources is seriously damaged; Cause species diversity and ecotope irreparable damage.Therefore it is significant to solve resource problem.
In recent years, utilize some microorganisms to have the characteristics of generation and the same or similar physiologically active substance of host, endophyte of plant has been carried out a large amount of research, the existing endophyte culture that is distributed in clump stalk spore genus and Penicillium that from Chinese podophyllum root, unmrellaleaf, is separated to, report (the Chinese medicine and clinical that has the Rf identical (mobility) value through chromatographic analysis with podophyllotoxin, 2003.3 (3), biotechnology, 2004.14 (2)).
Summary of the invention
One of purpose of the present invention provides the mucormycosis that a strain can be produced podophyllotoxin and glucoside and kaempferol Flavonoid substances.
Two of purpose of the present invention provides a kind of method of utilizing above-mentioned bacterial strains to produce podophyllotoxin and glucoside and kaempferol Flavonoid substances.
Three of purpose of the present invention provides the application that utilizes above-mentioned bacterial strains to produce podophyllotoxin and glucoside and kaempferol Flavonoid substances, for exploitation Podophyllum emodi var chinense class raw material resources provide new way.
The bacterial strain that can produce podophyllotoxin and glucoside thereof and kaempferol Flavonoid substances is Mucor (Mucorpodophyllus) J.X.Huang sp.nov GJ-TW8, be preserved in Chinese typical culture collection center (being called for short CCTCC) on April 8th, 2005, deposit number is CCTCC № M 205032.
Mucor (Mucor podophyllus) J.X.Huang sp.nov GJ-TW8 (hereinafter to be referred as the GJ-TW8 bacterium) is that separation in the Chinese podophyllum root roots of plants of Mount Taibai, Shaanxi, screening, purifying obtain pure bacterial strain, and its identification mark is as follows:
(1) colony characteristics
On potato dextrose agar (PDA) substratum and Cha Shi substratum: mycelia spreads growth, is loose cotton-shaped, just is white, and the PDA substratum was full of plate after last 5 day, and the Cha Shi substratum was full of plate after last 7 day, was canescence (seeing accompanying drawing 1).
(2) morphological features
Sporangiophore is born by mycelia, is dummy shaft branch or monopodial branching, upright or tool elastic bending, and surface smoothing, diameter 4.5～12.3 μ m, long 750～2160 μ m, long branch 450～1200 μ m, idol has a barrier film in bifurcation.The sporocyst sphere, the sporocyst size is uneven, big sporangiocyst diameter 80～120 μ m, microsporangium diameter 24～80 μ m are filbert after the maturation.The sporangiocyst wall is very easily cleared up (seeing accompanying drawing 2).
The columella sphere, diameter 12～30 μ m, colourless or filbert.
The long avette or little lemon shape of sporangiospore, two ends are blunt, size 4.8～7.2 * 2.4～3.6, spore long-width ratio 1.5～2: 1, surface smoothing, colourless (seeing accompanying drawing 3).Rounded, the oval yeast cell type that expands during visible spore germination in the substratum, diameter 5.2～16 μ m or 6.4～14.5 * 4.2～12 μ m grow germ tube and form underdeveloped pseudohypha (seeing accompanying drawing 4).
The mycelia tubulose do not have every, cultivate 3 days mycelia to ask that cross occurrence connects and make mycelium be reticulated structure, connect spherical in shape or irregular spherical 14～24 μ m of joint or 24～27 * 14～24 μ m (seeing accompanying drawing 5), zygospore is difficult for seeing.Substrate mycelium tubulose branch, it is filbert that maturation is, and a large amount of globular adiponectins occur and drip.
(3) physiological characteristic
8 ℃～35 ℃ of growth temperature ranges, 25 ℃～29 ℃ of optimum growth temperatures.
Well-grown on organonitrogen, ammonium nitrogen synthetic medium, the growth of nitric nitrogen, nitrite nitrogen is slightly poor.
Comprehensive above-mentioned feature, according to " fungi identification handbook " the classification of fungi document of etc.ing, above-mentioned bacterial strains has the Mucor essential characteristic, but with this genuss is various obvious different characteristics, its affiliated mucorales of classifying, Mucoraceae, Mucor, mucor hiemalis group novel species is arranged relatively.
Produce the method for podophyllotoxin and glucoside and kaempferol Flavonoid substances, comprise the steps: that (A) is connected to seed culture medium with Mucor (Mucor podophyllus) J.X.Huang sp.nov GJ-TW8 CCTCC № M 205032 bacterial strains, cultivated 5-6 days 28-30 ℃ (scope), transfer in fermention medium fermentation culture 7-9 days; (B) above-mentioned fermenting culture is collected mycelium through suction filtration, and freezing, broken mycelium extracts broken mycelium successively with chloroform, propyl carbinol respectively, collects extract, concentrating under reduced pressure; (C) silica gel column chromatography separates on the chloroform extract, uses the chloroform-methanol mixed solvent wash-out of chloroform, 9.5: 0.5 and 9: 1 successively, collects 9.5: 0.5 chloroform-methanol elutriant, concentratedly can get podophyllotoxin; (D) collect 9: 1 chloroform-methanol wash-out parts, concentrate kaempferol and flavonoid crude product; (E) spissated n-butyl alcohol extract, last silica gel column chromatography separates, and uses chloroform, 9: 1 oxygen imitation-carbinol mixed solvent wash-out respectively successively, collects 9: 1 chloroform-methanol elutriant, concentratedly can get NSC 163024.
Beneficial effect of the present invention: a strain Mucor novel species that provides has produces the multiple physiological property that the biologically active substance of pharmaceutical use arranged identical with the Chinese podophyllum root plant, can be applicable to the production of podophillotoxines material and kaempferol Flavonoid substances; Bacterial classification nutritional requirement of the present invention is simple, easily cultivate, with short production cycle, application potential with commercial scale production; Provide new way to exploitation Podophyllum emodi var chinense class raw material resources, significant to the protective plant ecological diversity.
Description of drawings
Accompanying drawing 1 is the colonial morphology of GJ-TW8 bacterium;
Accompanying drawing 2 is ripe sporangiocyst of GJ-TW8 bacterium and branched sporangiophore structure (10 * 10 power microscope);
Accompanying drawing 3 is a GJ-TW8 bacterium sporangiospore form (10 * 40 power microscope);
Accompanying drawing 4 is yeast type cellular form (10 * 20 power microscope) for the spore germination of GJ-TW8 bacterium;
Accompanying drawing 5 is a GJ-TW8 bacterium mycelia connecting structure (10 * 20 power microscope);
The podophyllotoxin that accompanying drawing 6 produces for the GJ-TW8 bacterium 13The C nuclear magnetic resonance map;
The podophyllotoxin that accompanying drawing 7 produces for the GJ-TW8 bacterium 1The H nuclear magnetic resonance map;
Accompanying drawing 8 is a kaempferol HPLC standard diagram;
The kaempferol HPLC collection of illustrative plates that accompanying drawing 9 produces for the GJ-TW8 bacterium;
Embodiment:
Product characterizes instrument and the condition of using:
The INOVA-400 nuclear magnetic resonance spectrometer (Varian, USA)
Testing conditions: solvent: deuterochloroform; Temperature: 25 ℃; Interior mark: TMS;
Frequency: 1H 400MHZ, 13C 125MHZ; Add up: 64 times.
The HITACHI high performance liquid chromatograph
Detector: HITACHI L-7420
Chromatographic column: YWG-C 1810um 250mm * 4.6mm
Moving phase: methyl alcohol-0.4% phosphoric acid 60: 40
Flow velocity: 1ml/min
Detect wavelength: 360nm
Temperature: 25 ℃
Embodiment 1
Adopt back the Chinese podophyllum root plant from the Mount Taibai, use tissue isolation, separation, purifying, filter out the pure bacterial strain of GJ-TW8 and preserve standby.
The PDA substratum: 20% potato juice 100ml, sucrose 2g, pH 6.5.Seed culture medium adds 2g agar; Fermention medium is a liquid.
GJ-TW8 bacterial strain provided by the invention is connected to seed culture medium, cultivates 5 days for 28 ℃, transfer in the 500ml triangular flask that the 200ml fermention medium is housed, 29 ℃, 180r/min, shake-flask culture 7 days, collect fermenting culture 40L altogether, through suction filtration, collect the about 4250g of wet mycelium, take out behind the freezing 4h, broken mycelium is used the mycelium of equal-volume chloroform and n-butanol extraction fragmentation successively respectively.Collect chloroform extract and n-butyl alcohol extract respectively, be evaporated to 10ml respectively at 60 ℃.
Spissated chloroform extract, adopt silica gel column chromatography separation (silica gel 200～300 orders on the wet method, column length 32cm, diameter 4cm), use chloroform, chloroform-methanol (9.5: 0.5,9: 1,8: 2), methyl alcohol wash-out successively respectively, with standard reagent podophyllotoxin (sigma company) and kaempferol (glucoside that tower natural organic-compound company) is contrast, thin layer color developing detection elutriant.Collect chloroform-methanol (9: 1) wash-out part, concentrating under reduced pressure is treated to obtain yellow powder after the solvent evaporates, and high performance liquid phase detects to kaempferol and Flavonoid substances (seeing accompanying drawing 8,9), obtains 369mg altogether.Chloroform-methanol (9.5: the 0.5) elutriant of collecting concentrates silicagel column (silica gel 200～300 orders on the secondary of back, column length 26cm, diameter 2cm) separation and purification, use chloroform, chloroform-methanol (9.5: 0.5,9: 1) wash-out successively, collect 9.5: 0.5 elutriants, concentrating under reduced pressure, obtain the podophyllotoxin crystal after the solvent evaporates, further use the ether recrystallization purifying to get 108mg, purity is the white, needle-shaped crystals more than 93%, nuclear-magnetism detects identical with the standard substance collection of illustrative plates (seeing accompanying drawing 6,7).The podophyllotoxin productive rate is the 0.48mg/g dry mycelium.
Spissated n-butyl alcohol extract, last silica gel column chromatography separates (silica gel 200～300 orders, column length 32cm, diameter 4cm) uses chloroform, chloroform-methanol (9: 1 respectively successively, 8: 2) wash-out, with standard NSC 163024 contrast thin layer color developing detection elutriant, collect 9: 1 elutriants and concentrate, again with preparation thin-layer chromatography purifying (thin layer silica gel GF 254, developing agent is chloroform-methanol (9: 1)), with the NSC 163024 standard control, the Rf value is identical, and with the spot shovel down, methyl alcohol embathes collection, and the methyl alcohol washing lotion concentrates, and product is a NSC 163024.
Embodiment 2
ISP fermention medium: Zulkovsky starch 10g, K 2HPO 41.0g, MgSO 41.0g, NaCl1.0g, (NH 4) 2SO 42.0g, liquid microelement 1ml, distilled water 1000ml, pH6.8 (liquid microelement: FeSO 47H 2O 0.1g, MnCl 24H 2O 0.1g, ZnSO 47H 2O 0.1g, distilled water 100ml)
GJ-TW8 inoculation provided by the invention in 28 ℃ of PDA seed culture mediums, cultivate 5 days after, transfer in the 500ml triangular flask that 200ml ISP fermention medium is housed, 29 ℃, 180r/min, shake-flask culture 7 days is collected wet mycelium 3160g, freezing 4h, broken mycelium is used the mycelium of equal-volume chloroform and n-butanol extraction fragmentation successively respectively.Collect chloroform extract and n-butyl alcohol extract respectively, be evaporated to 10ml respectively at 60 ℃.
Spissated chloroform extract, adopt silica gel column chromatography separation (silica gel 200～300 orders on the wet method, column length 32cm, diameter 4cm) uses chloroform, chloroform-methanol (9.5: 0.5 respectively successively, 9: 1,8: 2), methyl alcohol wash-out successively, be contrast with standard reagent podophyllotoxin and kaempferol, thin layer color developing detection elutriant.Collect chloroform-methanol (9: 1) wash-out part, concentrating under reduced pressure gets yellow powder after the solvent evaporates, is the common 263mg of kaempferol and flavonoid crude product material.Collect chloroform-methanol (9.5: 0.5) elutriant and concentrate silicagel column (silica gel 200～300 orders, column length 26cm, diameter 2cm) on the secondary, use chloroform, chloroform-methanol (9.5: 0.5,9: 1) wash-out successively, collect 9.5: 0.5 elutriants, concentrating under reduced pressure obtains the podophyllotoxin crystal after the solvent evaporates.Further to get 95mg purity be 95% podophyllotoxin white needle-like crystals to the ether recrystallization purifying, and productive rate is the 0.51mg/g dry mycelium.
Spissated n-butyl alcohol extract, last silica gel column chromatography separates (silica gel 200～300 orders, column length 32cm, diameter 4cm) uses chloroform, chloroform-methanol (9: 1 respectively successively, 8: 2) wash-out, with standard NSC 163024 contrast thin layer color developing detection elutriant, collect 9: 1 elutriants and concentrate, again with preparation thin-layer chromatography purifying (thin layer silica gel GF 254, developing agent is chloroform-methanol (9: 1)), with the NSC 163024 standard control, the Rf value is identical, and with the spot shovel down, methyl alcohol embathes collection, and the methyl alcohol washing lotion concentrates, and product is a NSC 163024.

Claims (3)

1, Mucor (Mucor podophyllus) J.X.Huang sp.nov GJ-TW8 CCTCC NO:M205032.
2, produce the method for podophyllotoxin and glucoside and kaempferol Flavonoid substances, comprise the steps:
(A) Mucor (Mucor podophyllus) J.X.Huang sp.nov GJ-TW8CCTCCNO:M 205032 bacterial strains are connected to seed culture medium, cultivated 5-6 days, transfer in fermention medium fermentation culture 7-9 days at 28-30 ℃;
Described seed culture medium is the PDA solid medium, and described fermention medium is PDA liquid nutrient medium or ISP fermention medium;
ISP fermentation culture based component is as follows: Zulkovsky starch 10g, K 2HPO 41.0g, MgSO 41.0g, NaCl 1.0g, (NH 4) 2SO 42.0g, liquid microelement 1ml, distilled water 1000ml, pH6.8, liquid microelement composition are FeSO 47H 2O 0.1g, MnCl 24H 2O 0.1g, ZnSO 47H 2O 0.1g, distilled water 100ml;
(B) above-mentioned fermenting culture is collected mycelium through suction filtration, and freezing, broken mycelium extracts broken mycelium successively with chloroform, propyl carbinol respectively, collects extract, concentrating under reduced pressure;
(C) silica gel column chromatography separates on the chloroform extract, uses the chloroform-methanol mixed solvent wash-out of chloroform, 9.5: 0.5 and 9: 1 successively, collects 9.5: 0.5 chloroform-methanol elutriant, concentratedly can get podophyllotoxin;
(D) collect 9: 1 chloroform-methanol wash-out parts, concentrate kaempferol and flavonoid crude product;
(E) spissated n-butyl alcohol extract, last silica gel column chromatography separates, and uses chloroform, 9: 1 chloroform-methanol mixed solvent wash-out respectively successively, collects 9: 1 elutriants and concentrates, and concentratedly can get NSC 163024.
3, the application of Mucor (Mucor podophyllus) J.X.Huang sp.nov GJ-TW8 CCTCC NO:M205032 in producing podophyllotoxin and glucoside and kaempferol Flavonoid substances.
CN 200510042630 2005-04-29 2005-04-29 Mucor for producing picropodophyllin and its glucoside and kaempferol flavone kind substance, its method and application Expired - Fee Related CN1283779C (en)

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CN102234669B (en) * 2010-04-29 2013-10-16 湖北工业大学 Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin
CN102133255B (en) * 2011-03-08 2013-03-13 暨南大学 Antineoplastic podophyllum traditional Chinese medicine extract and preparation method and application thereof
CN102559517B (en) * 2012-02-20 2013-04-10 北京师范大学 Endophytic fungi from podophyllum hexandrum plant and application of endophytic fungi
CN110172408B (en) * 2019-04-10 2020-09-15 甘肃省科学院生物研究所 Endophytic fungus of podophyllum hexandrum and application thereof

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