CN104068449A - Separation and purification method for dictyophora indusia fisscher antibacterial active monomer - Google Patents
Separation and purification method for dictyophora indusia fisscher antibacterial active monomer Download PDFInfo
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- CN104068449A CN104068449A CN201410267889.XA CN201410267889A CN104068449A CN 104068449 A CN104068449 A CN 104068449A CN 201410267889 A CN201410267889 A CN 201410267889A CN 104068449 A CN104068449 A CN 104068449A
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- 241001313734 Dictyophora Species 0.000 title claims abstract description 37
- 239000000178 monomer Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000000746 purification Methods 0.000 title claims abstract description 12
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 10
- 238000000926 separation method Methods 0.000 title abstract description 8
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 63
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- 230000003385 bacteriostatic effect Effects 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- 239000000741 silica gel Substances 0.000 claims description 18
- 229910002027 silica gel Inorganic materials 0.000 claims description 18
- 229960001866 silicon dioxide Drugs 0.000 claims description 18
- 238000011068 loading method Methods 0.000 claims description 12
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 10
- 239000000287 crude extract Substances 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 241001313708 Dictyophora phalloidea Species 0.000 claims description 7
- 238000002955 isolation Methods 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 229920000742 Cotton Polymers 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000004537 pulping Methods 0.000 claims description 3
- 239000002002 slurry Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
- 235000013372 meat Nutrition 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 239000005452 food preservative Substances 0.000 abstract description 3
- 235000019249 food preservative Nutrition 0.000 abstract description 3
- 208000030507 AIDS Diseases 0.000 abstract description 2
- 235000017166 Bambusa arundinacea Nutrition 0.000 abstract description 2
- 235000017491 Bambusa tulda Nutrition 0.000 abstract description 2
- 235000015334 Phyllostachys viridis Nutrition 0.000 abstract description 2
- 150000001413 amino acids Chemical class 0.000 abstract description 2
- 239000011425 bamboo Substances 0.000 abstract description 2
- 239000008280 blood Substances 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 2
- 239000011707 mineral Substances 0.000 abstract description 2
- 230000036407 pain Effects 0.000 abstract description 2
- 210000000952 spleen Anatomy 0.000 abstract description 2
- 230000004936 stimulating effect Effects 0.000 abstract description 2
- 210000002784 stomach Anatomy 0.000 abstract description 2
- 230000008961 swelling Effects 0.000 abstract description 2
- 239000011782 vitamin Substances 0.000 abstract description 2
- 229940088594 vitamin Drugs 0.000 abstract description 2
- 229930003231 vitamin Natural products 0.000 abstract description 2
- 235000013343 vitamin Nutrition 0.000 abstract description 2
- 230000002335 preservative effect Effects 0.000 abstract 2
- 241001330002 Bambuseae Species 0.000 abstract 1
- 241000233866 Fungi Species 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 abstract 1
- 230000036772 blood pressure Effects 0.000 abstract 1
- 238000005345 coagulation Methods 0.000 abstract 1
- 230000015271 coagulation Effects 0.000 abstract 1
- 230000036039 immunity Effects 0.000 abstract 1
- 230000005764 inhibitory process Effects 0.000 abstract 1
- 235000016709 nutrition Nutrition 0.000 abstract 1
- 230000035764 nutrition Effects 0.000 abstract 1
- 239000003755 preservative agent Substances 0.000 abstract 1
- 230000005855 radiation Effects 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 230000003260 anti-sepsis Effects 0.000 description 3
- 239000004599 antimicrobial Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000194107 Bacillus megaterium Species 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 241001313710 Dictyophora indusiata Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 241001527087 Panax vietnamensis Species 0.000 description 1
- 235000017726 Panax vietnamensis Nutrition 0.000 description 1
- 241000288049 Perdix perdix Species 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 241001314279 Zoopagales Species 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a separation and purification method for a dictyophora indusia fisscher antibacterial active monomer. The separation and purification method for the dictyophora indusia fisscher antibacterial active monomer comprises the following steps: pre-treating a dictyophora indusia fisscher sporocarp, digesting, extracting, carrying out silica gel column chromatography and carrying out reverse phase silica gel column chromatography. The dictyophora indusia fisscher not only has abundant nutrition and contains various amino acids, vitamins and minerals, but also has the effects of tonifying spleen and stomach, lowering blood fat, lowering blood pressure, resisting tumors, resisting coagulation, relieving swelling and pain, resisting AIDS, stimulating the immunity and resisting radiation, and has strong antibacterial and preservative effects; bamboo fungi and meat are boiled together and the meat cannot go bad easily so that the dictyophora indusia fisscher is a natural food preservative. The monomer which has the bacterium inhibition activity is separated from the dictyophora indusia fisscher and can be developed into the natural preservative to be widely applied to foods; the application prospect is wide.
Description
technical field
The present invention relates to a kind of separation method of active material, be specifically related to the isolation and purification method of bacteriostatic activity monomer in a kind of Cultivation of Dictyophora.
background technology
Dictyophora phalloidea (
dictyophora indusiata) claim again bamboo ginseng, veil bacterium, bamboo partridge egg, be under the jurisdiction of Eumycota, Basidiomycetes, Phallus Zoopagales, Phallus Cordycepps, dictyophora phalloidea genus.Indusium is the distinctive morphological feature of dictyophora phalloidea, consists of title Cultivation of Dictyophora more than 10cm spongioplasm.Cultivation of Dictyophora is not only nutritious, contain several amino acids, vitamin and mineral matter, also there is invigorating spleen and reinforcing stomach, reducing blood lipid, hypotensive, antitumor, anticoagulation, swelling and pain relieving, anti-AIDS, immune stimulatory, radioresistance and stronger antibacterial antisepsis are a kind of natural food preservatives.Dictyophora phalloidea and meat boil altogether, and meat is not perishable.Our experimental result also shows: Cultivation of Dictyophora crude extract has fungistatic effect clearly to bacteriums such as staphylococcus aureus, Escherichia coli, bacillus subtilis, Bacterium enteritidis, Listeria, vibrio parahaemolytious, and lower concentration can play antibacterial and bactericidal effect preferably; And antibacterial substance at the weak acid of pH5.0-8.5 to stable under weak basic condition, also very stable to HTHP, this with very many anticorrisive agents only under acid condition competence exertion antisepsis compare and have very large advantage.
From Cultivation of Dictyophora, isolate the monomer with bacteriostatic activity, it can be developed to a kind of natural antiseptic agent and be applied to food, has broad application prospects.
summary of the invention
The method that the object of this invention is to provide bacteriostatic activity monomeric substance in a kind of separated Cultivation of Dictyophora, for food provides a kind of safe natural antiseptic agent.
The isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer of the present invention, comprises the pretreatment of Cultivation of Dictyophora fructification, lixiviate, extraction, silica gel column chromatography, reversed-phase silica gel column chromatography, and its operating procedure is as follows:
(1). Cultivation of Dictyophora fructification pretreatment: dictyophora phalloidea fructification dry product toasts 3h in 60 ℃ of baking ovens, then with plant pulverizer, pulverize, cross 80 mesh sieves, obtain fructification powder;
(2). lixiviate: take Cultivation of Dictyophora fructification powder, by liquid-solid weight fraction, than 26 ~ 24:1, add absolute ethyl alcohol, after water-bath 1.5h, suction filtration obtains filtrate at 72 ℃, with at rotary evaporator 100rpm, 60 ℃, liquid is spin-dried for, gained solid content is Cultivation of Dictyophora crude extract, standby;
(3). extraction: Cultivation of Dictyophora crude extract is water-soluble, with benzinum, ethyl acetate, extract successively, every layer extracts 3-5 time; Get ethyl acetate layer extract and be placed on rotary evaporator solvent evaporated at 60 ℃, obtain ethyl acetate layer solid content, standby;
(4). silica gel column chromatography:
1) silica gel column chromatography for the first time: 1. fill post: accurately take appropriate 200-300 order silica gel in beaker, add petroleum ether and stirring pulping, in a cotton of chromatographic column bottom plug, in post, add 5mL benzinum, by slurries slowly in impouring post, make silica gel natural subsidence, with glass bar, knock gently chromatographic column to get rid of bubble simultaneously, by chromatographic column lower end switch opens, mobile phase is flowed down naturally, until liquid level about 3cm above cylinder, by first mobile phase balance of 700mL; 2. loading: sample is dissolved in a small amount of methyl alcohol, is ground with 4 times of silica gel to sample, volatilize solvent, make it loose, slowly add chromatographic column top through funnel, then use a small amount of mobile phase drip washing post jamb, make silicagel column surfacing; 3. drip washing: distinguish successively 5 column volumes of drip washing with benzinum: ethyl acetate=6.5-7:2.8-3,0.8-1:0.8-1, two kinds of each plant demands of ratio leacheate are 1500 mL, with conical flask, collect eluent, collect 300mL for every bottle, obtain altogether 10 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
11-D
1no. 10, merge D
12-D
1the component of 8 fragments, standby;
2) silica gel column chromatography for the second time: 1. fill post: with 1); 2. loading: with 1); 3. drip washing: distinguish successively 3 column volumes of drip washing with Shi You Mi ﹕ ethyl acetate=6.5-7:2.8-3,5.6-6:3.6-4, two kinds of each plant demands of ratio leacheate are that 750 mL collect eluent with conical flask, collect 250mL for every bottle, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
21-D
2no. 6, merge D
23-D
24 fragment components, standby;
3) silica gel column chromatography for the third time: 1. fill post: with 1); 2. loading: with 1); 3. drip washing: distinguish successively 3 column volumes of drip washing with Shi You Mi ﹕ ethyl acetate=7.5-8:1.8-2,7-7.5:3-3.5, two kinds of each plant demands of ratio leacheate are 750 mL, with conical flask, collect eluent, collect 250mL for every bottle, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
31-D
3no. 6, merge D
34-D
35 fragment components, standby;
(5). reversed-phase silica gel column chromatography: with 10g silica gel dress post, wash post with 300mL methyl alcohol, 300mL ultra-pure water balance, then by the D of above-mentioned merging
34-D
35 fragment components are dissolved in 2mL methyl alcohol, add chromatographic column top, and coutroi velocity is 10mL/min, with the drip washing of 300mL ultra-pure water, with conical flask, collect, and on rotary evaporator, 50 ℃ of evaporate to dryness liquid obtain white crystals body and are the monomer with bacteriostatic activity.
In step (3), wherein the ratio of weight and number of crude extract and water is 1:18 ~ 20, and benzinum is 1:1 ~ 2 with ethyl acetate weight fraction ratio.
The present invention is for the protection of bacteriostatic activity monomer in the Cultivation of Dictyophora that utilizes this method separation and purification to obtain.
The invention has the advantages that: with the monomer of the bacteriostatic activity of method separation and purification of the present invention, it is a kind of natural antibacterial substance, can be developed to a kind of natural antiseptic agent and be applied to food, overcome the side effect that artificial synthetic food preservative brings to consumer, therefore on food, have broad application prospects.Simultaneously this antibacterial monomer at weak acid to stable under weak basic condition, also very stable to HTHP, this with very many anticorrisive agents only under acid condition competence exertion antisepsis compare and have very large advantage.
the specific embodiment
In order fully to disclose the separation method of Cultivation of Dictyophora bacteriostatic activity monomer of the present invention, below in conjunction with embodiment, be illustrated.
embodiment 1
A separation method for bacteriostatic activity monomer in Cultivation of Dictyophora, comprises the following steps:
1. Cultivation of Dictyophora fructification pretreatment: dictyophora phalloidea fructification dry product toasts 3h in 60 ℃ of baking ovens, then pulverizes with plant pulverizer, crosses 80 mesh sieves, obtains fructification powder.
2. lixiviate: take Cultivation of Dictyophora fructification powder 1000g, by liquid-solid weight fraction, than 25:1, add absolute ethyl alcohol, after water-bath 1.5h, suction filtration obtains filtrate at 72 ℃, with at 60 ℃ of rotary evaporators, liquid is spin-dried for, gained solid content 190g is Cultivation of Dictyophora crude extract, standby.
3. extraction: get 100g Cultivation of Dictyophora crude extract and be dissolved in 2000 mL water, with benzinum, ethyl acetate, extract successively, general every layer of extraction 3-5 time, the consumption of each extractant is 4000mL.Get ethyl acetate layer extract and be placed on rotary evaporator solvent evaporated at 60 ℃, obtain ethyl acetate layer solid content 8.7g, standby.
4. silica gel column chromatography:
(1) silica gel column chromatography for the first time: 1. fill post: accurately take appropriate 200-300 order silica gel in beaker, add petroleum ether and stirring pulping.In a cotton of chromatographic column bottom plug, in post, add 5mL benzinum, by slurries slowly in impouring post, make silica gel natural subsidence, with glass bar, knock gently chromatographic column to get rid of bubble, by chromatographic column lower end switch opens simultaneously, mobile phase is flowed down naturally, until liquid level about 3cm above cylinder.By first mobile phase balance of 700mL.2. loading: 8.7g extract is dissolved in 20mL methyl alcohol, is ground with the silica gel of 35g, volatilize solvent, make it loose, slowly add chromatographic column top through funnel, then use a small amount of mobile phase drip washing post jamb, make silicagel column surfacing.3. drip washing: get 130g silica gel wet method dress post, get ethyl acetate layer solid content 7.5g dry method loading, with benzinum: ethyl acetate be respectively 7:3 and 1:1 successively respectively 5 column volumes of drip washing with conical flask, collect eluent, obtain altogether 10 fragments, and on rotary evaporator 100rpm, the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
11-D
1no. 10.Merge wherein D
12-D
1the component of 8 fragments is 1.96g altogether, standby.
(2) silica gel column chromatography for the second time: 1. fill post: with (1).2. loading: with (1).3. drip washing: get 100g silica gel wet method
Dress post, the 1.96g component dry method loading merging in will (1), is respectively 7:3 and 6:4 distinguishes 3 column volumes of drip washing successively with the oily ether ﹕ of stone ethyl acetate, with conical flask, collect eluent, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
21-D
2no. 6.Merge wherein D
23-D
24 fragment components are 0.35g altogether, standby.
(3) silica gel column chromatography for the third time: 1. fill post: with (1).2. loading: with (1).3. drip washing: get 50g silica gel wet method dress post, by the 0.35g component dry method loading merging in (2), with Shi You Mi ﹕ ethyl acetate=8:2,7:3, distinguish successively 3 column volumes of drip washing, with conical flask, collect eluent, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
31-D
3no. 6.Merge D
34-D
35 fragment components are 100mg altogether, standby.
5. reversed-phase silica gel column chromatography: with 10g silica gel (model 2p-18) dress post, wash post with 300mL methyl alcohol, 300mL ultra-pure water balance, then by the D of merging
34-D
35 fragment component 100mg are dissolved in 2mL methyl alcohol, add chromatographic column top.Coutroi velocity is 10mL/min, use successively the drip washing of 300mL ultra-pure water, with conical flask, collect, on rotary evaporator, the liquid of 50 ℃ of each fragments of evaporate to dryness obtains white crystals body and is the monomer with bacteriostatic activity, this monomeric compound shows that through bacteriostatic experiment indicator bacteria Bacterium enteritidis and bacillus megaterium are had to very strong inhibitory action, to the 503nhibiting concentration of Bacterium enteritidis, is wherein 51.58 μ g/mL; To the 503nhibiting concentration of bacillus megaterium, be 83.06 μ g/mL.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. an isolation and purification method for Cultivation of Dictyophora bacteriostatic activity monomer, is characterized in that: comprise the pretreatment of Cultivation of Dictyophora fructification, lixiviate, extraction, silica gel column chromatography, reversed-phase silica gel column chromatography.
2. the isolation and purification method of Ju Cultivation of Dictyophora bacteriostatic activity claimed in claim 1 monomer, is characterized in that: concrete operation step is as follows:
(1) Cultivation of Dictyophora fructification pretreatment: dictyophora phalloidea fructification dry product toasts 3h in 60 ℃ of baking ovens, then pulverizes with plant pulverizer, crosses 80 mesh sieves, obtains fructification powder;
(2) lixiviate: take Cultivation of Dictyophora fructification powder, by liquid-solid weight fraction, than 26 ~ 24:1, add absolute ethyl alcohol, after water-bath 1.5h, suction filtration obtains filtrate at 72 ℃, with at rotary evaporator 100rpm, 60 ℃, liquid is spin-dried for, gained solid content is Cultivation of Dictyophora crude extract, standby;
(3) extraction: Cultivation of Dictyophora crude extract is water-soluble, extracts with benzinum, ethyl acetate successively, and every layer extracts 3-5 time; Get ethyl acetate layer extract and be placed on rotary evaporator solvent evaporated at 60 ℃, obtain ethyl acetate layer solid content, standby;
(4) silica gel column chromatography:
1) silica gel column chromatography for the first time: 1. fill post: accurately take appropriate 200-300 order silica gel in beaker, add petroleum ether and stirring pulping, in a cotton of chromatographic column bottom plug, in post, add 5mL benzinum, by slurries slowly in impouring post, make silica gel natural subsidence, with glass bar, knock gently chromatographic column to get rid of bubble simultaneously, by chromatographic column lower end switch opens, mobile phase is flowed down naturally, until liquid level about 3cm above cylinder, by first mobile phase balance of 700mL; 2. loading: sample is dissolved in a small amount of methyl alcohol, is ground with 4 times of silica gel to sample, volatilize solvent, make it loose, slowly add chromatographic column top through funnel, then use a small amount of mobile phase drip washing post jamb, make silicagel column surfacing; 3. drip washing: distinguish successively 5 column volumes of drip washing with benzinum: ethyl acetate=6.5-7:2.8-3,0.8-1:0.8-1, two kinds of each plant demands of ratio leacheate are 1500 mL, with conical flask, collect eluent, collect 300mL for every bottle, obtain altogether 10 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
11-D
1no. 10, merge D
12-D
1the component of 8 fragments, standby;
2) silica gel column chromatography for the second time: 1. fill post: with 1); 2. loading: with 1); 3. drip washing: distinguish successively 3 column volumes of drip washing with Shi You Mi ﹕ ethyl acetate=6.5-7:2.8-3,5.6-6:3.6-4, two kinds of each plant demands of ratio leacheate are that 750 mL collect eluent with conical flask, collect 250mL for every bottle, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
21-D
2no. 6, merge D
23-D
24 fragment components, standby;
3) silica gel column chromatography for the third time: 1. fill post: with 1); 2. loading: with 1); 3. drip washing: distinguish successively 3 column volumes of drip washing with Shi You Mi ﹕ ethyl acetate=7.5-8:1.8-2,7-7.5:3-3.5, two kinds of each plant demands of ratio leacheate are 750 mL, with conical flask, collect eluent, collect 250mL for every bottle, obtain altogether 6 fragments, and on rotary evaporator the liquid of 50 ℃ of each fragments of evaporate to dryness, number consecutively is D
31-D
3no. 6, merge D
34-D
35 fragment components, standby;
Reversed-phase silica gel column chromatography: with 10g silica gel dress post, wash post with 300mL methyl alcohol, 300mL ultra-pure water balance, then by the D of above-mentioned merging
34-D
35 fragment components are dissolved in 2mL methyl alcohol, add chromatographic column top, and coutroi velocity is 10mL/min, with the drip washing of 300mL ultra-pure water, with conical flask, collect, and on rotary evaporator, 50 ℃ of evaporate to dryness liquid obtain white crystals body and are the monomer with bacteriostatic activity.
3. the isolation and purification method of a kind of Cultivation of Dictyophora bacteriostatic activity monomer according to claim 2, it is characterized in that: in step (3), wherein the ratio of weight and number of crude extract and water is 1:18 ~ 20, and benzinum is 1:1 ~ 2 with ethyl acetate weight fraction ratio.
4. the monomer of the Cultivation of Dictyophora bacteriostatic activity that the isolation and purification method of an a kind of Cultivation of Dictyophora bacteriostatic activity monomer as claimed in claim 1 or 2 makes.
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| CN201410267889.XA CN104068449B (en) | 2014-06-17 | 2014-06-17 | A kind of isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer |
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| CN111494306A (en) * | 2020-05-29 | 2020-08-07 | 杭州百芮生物科技有限公司 | Female private gel microecological preparation composition and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102090620A (en) * | 2010-12-08 | 2011-06-15 | 江南大学 | Dictyophora indusiata fruiting body extract with antibacterial activity and uses thereof |
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| CN103435678A (en) * | 2013-08-27 | 2013-12-11 | 中国科学院华南植物园 | Preparation method of maslinic acid and application of maslinic acid in preparation of antibacterial agent |
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| CN101029087A (en) * | 2007-03-07 | 2007-09-05 | 浙江大学 | Method for separating polysaccharide against growth of cancer cells from dictyophord |
| CN102090620A (en) * | 2010-12-08 | 2011-06-15 | 江南大学 | Dictyophora indusiata fruiting body extract with antibacterial activity and uses thereof |
| CN102743322A (en) * | 2011-04-22 | 2012-10-24 | 上海家化联合股份有限公司 | Application of bamboo fungus extract in cosmetics |
| CN103435678A (en) * | 2013-08-27 | 2013-12-11 | 中国科学院华南植物园 | Preparation method of maslinic acid and application of maslinic acid in preparation of antibacterial agent |
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| 林陈强: "棘托竹荪菌托抑菌物质及多糖研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 1, 15 January 2012 (2012-01-15) * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111494306A (en) * | 2020-05-29 | 2020-08-07 | 杭州百芮生物科技有限公司 | Female private gel microecological preparation composition and preparation method thereof |
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