CN104068449B - A kind of isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer - Google Patents
A kind of isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer Download PDFInfo
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- 241001313734 Dictyophora Species 0.000 title claims abstract description 30
- 230000003385 bacteriostatic effect Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000000178 monomer Substances 0.000 title claims abstract description 17
- 238000002955 isolation Methods 0.000 title claims abstract description 7
- 238000000746 purification Methods 0.000 title claims abstract description 7
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 21
- 239000000284 extract Substances 0.000 claims abstract description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 42
- 239000012634 fragment Substances 0.000 claims description 29
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 238000005406 washing Methods 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 18
- 239000003208 petroleum Substances 0.000 claims description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000000741 silica gel Substances 0.000 claims description 16
- 229910002027 silica gel Inorganic materials 0.000 claims description 16
- 229960001866 silicon dioxide Drugs 0.000 claims description 16
- 238000011068 loading method Methods 0.000 claims description 12
- 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
- 238000000605 extraction Methods 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 7
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 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
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims 1
- 238000004062 sedimentation Methods 0.000 claims 1
- 239000002002 slurry Substances 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 5
- 230000003260 anti-sepsis Effects 0.000 abstract description 4
- 239000004599 antimicrobial Substances 0.000 abstract description 4
- 235000013372 meat Nutrition 0.000 abstract description 4
- 230000001603 reducing effect Effects 0.000 abstract description 4
- 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
- 150000001413 amino acids Chemical class 0.000 abstract description 2
- 230000000259 anti-tumor effect Effects 0.000 abstract description 2
- 230000010100 anticoagulation Effects 0.000 abstract description 2
- 239000008280 blood Substances 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract description 2
- 230000036772 blood pressure Effects 0.000 abstract description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 2
- 239000011707 mineral Substances 0.000 abstract description 2
- 235000008935 nutritious Nutrition 0.000 abstract description 2
- 230000004223 radioprotective effect 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
- 150000003722 vitamin derivatives Chemical class 0.000 abstract description 2
- 230000003014 reinforcing effect Effects 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241000194107 Bacillus megaterium Species 0.000 description 2
- 241001313688 Phallus rugulosus Species 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000004537 pulping Methods 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241001313710 Dictyophora indusiata Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000192128 Gammaproteobacteria Species 0.000 description 1
- 241001527087 Panax vietnamensis Species 0.000 description 1
- 235000017726 Panax vietnamensis Nutrition 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
- CDMXXXZRZWQJQE-UHFFFAOYSA-N acetic acid;2-methylprop-2-enoic acid Chemical compound CC(O)=O.CC(=C)C(O)=O CDMXXXZRZWQJQE-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 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
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006200 vaporizer Substances 0.000 description 1
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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)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
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Abstract
The present invention relates to the isolation and purification method of a kind of Cultivation of Dictyophora bacteriostatic activity monomer, the isolation and purification method of the Cultivation of Dictyophora bacteriostatic activity monomer of the present invention, including Cultivation of Dictyophora sporophore pretreatment, extract, extract, silica gel column chromatography, reversed-phase silica gel column chromatography.Cultivation of Dictyophora is the most nutritious, containing several amino acids, vitamin and mineral, also there is invigorating spleen and reinforcing stomach, blood fat reducing, blood pressure lowering, antitumor, anticoagulation, reducing swelling and alleviating pain, anti-AIDS, immune stimulatory, radioprotective and stronger antibacterial antisepsis, Caulis Bambusae In Taeniam is boiled altogether with meat, meat is unlikely to deteriorate, and is a kind of natural food preservative.Isolating the monomer with bacteriostatic activity from Cultivation of Dictyophora, it can be developed into a kind of natural antiseptic agent and be applied to food, has broad application prospects.
Description
Technical field
The present invention relates to the separation method of a kind of active substance, be specifically related to bacteriostatic activity monomer in a kind of Cultivation of Dictyophora
Isolation and purification method.
Background technology
Caulis Bambusae In Taeniam (Dictyophora indusiata), also known as bamboo ginseng, veil bacterium, Bambusicolae thoracicae egg, is under the jurisdiction of Eumycota, load
Gammaproteobacteria, Phallus rugulosus Fisch. Zoopagales, Phallus rugulosus Fisch. Cordycepps, Caulis Bambusae In Taeniam belong to.Indusium is the distinctive morphological characteristic of Caulis Bambusae In Taeniam, is made up of spongioplasm, more than 10cm
Title Cultivation of Dictyophora.Cultivation of Dictyophora is the most nutritious, containing several amino acids, vitamin and mineral, also has spleen invigorating benefit
Stomach, blood fat reducing, blood pressure lowering, antitumor, anticoagulation, reducing swelling and alleviating pain, anti-AIDS, immune stimulatory, radioprotective and stronger pressing down
Bacterium antisepsis, is a kind of natural food preservative.Caulis Bambusae In Taeniam is boiled altogether with meat, and meat is unlikely to deteriorate.Our experimental result also table
Bright: Cultivation of Dictyophora crude extract is to staphylococcus aureus, escherichia coli, bacillus subtilis, Salmonella enteritidis, Liszt
The antibacterial such as bacterium, vibrio parahaemolytious has fungistatic effect clearly, and relatively low concentration i.e. can play the most antibacterial and sterilization
Effect；And antibacterial substance at the weak acid of pH5.0-8.5 to stable under weak basic condition, the most stable to High Temperature High Pressure, this with very
Many preservative competence exertion antisepsises the most in acid condition are compared the biggest advantage.
Isolating the monomer with bacteriostatic activity from Cultivation of Dictyophora, it can be developed into a kind of natural antiseptic agent and be applied to
Food, has broad application prospects.
Summary of the invention
It is an object of the invention to provide and a kind of separate the method for bacteriostatic activity monomeric substance in Cultivation of Dictyophora, provide for food
A kind of safe natural antiseptic agent.
The isolation and purification method of the Cultivation of Dictyophora bacteriostatic activity monomer of the present invention, including Cultivation of Dictyophora sporophore pretreatment,
Extraction, extraction, silica gel column chromatography, reversed-phase silica gel column chromatography, its operating procedure is as follows:
(1). Cultivation of Dictyophora sporophore pretreatment: Caulis Bambusae In Taeniam sporophore dry product toasts 3h in 60 DEG C of baking ovens, then uses plant
Pulverizer is pulverized, and crosses 80 mesh sieves, obtains sporophore powder；
(2). extraction: weigh Cultivation of Dictyophora sporophore powder, add dehydrated alcohol by liquid-solid weight fraction than 26 ~ 24:1,
At 72 DEG C, after water-bath 1.5h, sucking filtration obtains filtrate, is spin-dried for by liquid by rotary evaporator 100rpm, at 60 DEG C, and gained solid content is for long
Skirt Caulis Bambusae In Taeniam crude extract, standby；
(3). extraction: Cultivation of Dictyophora crude extract is dissolved in water, extracts by petroleum ether, ethyl acetate successively, every layer of extraction
3-5 time；Take ethyl acetate layer extract and be placed in rotary evaporator solvent evaporated at 60 DEG C, obtain ethyl acetate layer solid
Thing, standby；
(4). silica gel column chromatography:
1) silica gel column chromatography for the first time: 1. fill post: accurately weigh appropriate 200-300 mesh silica gel in beaker, add oil
Ether stirring pulping, fills in a Cotton Gossypii in chromatographic column bottom, adds 5mL petroleum ether, be slowly poured in post by serosity, make silicon in post
Glue natural subsidence, taps chromatographic column gently to get rid of bubble simultaneously, is opened by chromatographic column lower switch, make flowing phase with Glass rod
Naturally flow down, until liquid level about 3cm above cylinder, balance each other with first flowing of 700mL；2. loading: sample is dissolved in few
In amount methanol, it is ground with the silica gel of 4 times of samples, volatilizes solvent so that it is be loose, be slowly added in chromatographic column through funnel
Side, then with a small amount of flowing phase drip washing post jamb, makes silicagel column surfacing；3. drip washing: use petroleum ether: ethyl acetate=6.5-7:
2.8-3,0.8-1:0.8-1 distinguish 5 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are 1500 mL, use taper
Eluent collected by bottle, collects 300mL, there are 10 fragments for every bottle, and on the rotary evaporator 50 DEG C be evaporated each fragment
Liquid, number consecutively is D11-D1No. 10, merge D12-D1The 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: use petroleum ether ethyl acetate=
6.5-7:2.8-3,5.6-6:3.6-4 distinguish 3 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are that 750 mL use
Conical flask collects eluent, collects 250mL, there are 6 fragments for every bottle, and on the rotary evaporator 50 DEG C be evaporated each sheet
The liquid of section, number consecutively is D21-D2No. 6, merge D23-D24 fragment components, standby；
3) silica gel column chromatography for the third time: 1. fill post: with 1)；2. loading: with 1)；3. drip washing: use petroleum ether ethyl acetate=
7.5-8:1.8-2,7-7.5:3-3.5 distinguish 3 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are 750 mL,
Collect eluent by conical flask, collect 250mL for every bottle, there are 6 fragments, and on the rotary evaporator 50 DEG C be evaporated each sheet
The liquid of section, number consecutively is D31-D3No. 6, merge D34-D35 fragment components, standby；
(5). reversed-phase silica gel column chromatography: fill post with 10g silica gel, wash post with 300mL methanol, 300mL ultra-pure water balances, so
After by the D of above-mentioned merging34-D35 fragment components are dissolved in 2mL methanol, add above chromatographic column, and coutroi velocity is 10mL/
Min, with 300mL ultra-pure water drip washing, collects by conical flask, and 50 DEG C are evaporated liquid and obtain white crystals body i.e. on the rotary evaporator
For having the monomer of bacteriostatic activity.
In step (3), wherein the ratio of weight and number of crude extract and water is 1:18 ~ 20, and petroleum ether divides with ethyl acetate weight
Number ratio is 1:1 ~ 2.
The present invention is bacteriostatic activity monomer in the isolated and purified Cultivation of Dictyophora obtained of Sustainable use this method.
The invention has the advantages that: with the monomer of the isolated and purified bacteriostatic activity of the method for the present invention, be a kind of natural pressing down
Fungus matter, can develop into a kind of natural antiseptic agent and be applied to food, overcomes the food preservative of synthetic to carry to consumer
The side effect come, therefore has broad application prospects on food.This antibacterial monomer is under weak acid to weak basic condition simultaneously
Stable, the most stable to High Temperature High Pressure, this is compared with the most many preservative competence exertion antisepsis the most in acid condition
There is the biggest advantage.
Detailed description of the invention
For the separation method of the Cultivation of Dictyophora bacteriostatic activity monomer of the fully open present invention, below in conjunction with embodiment in addition
Explanation.
Embodiment 1
The separation method of bacteriostatic activity monomer in a kind of Cultivation of Dictyophora, comprises the following steps:
1. Cultivation of Dictyophora sporophore pretreatment: Caulis Bambusae In Taeniam sporophore dry product toasts 3h in 60 DEG C of baking ovens, then uses Plant Powder
Broken machine is pulverized, and crosses 80 mesh sieves, obtains sporophore powder.
2. extraction: weigh Cultivation of Dictyophora sporophore powder 1000g, add dehydrated alcohol by liquid-solid weight fraction than 25:1,
At 72 DEG C, after water-bath 1.5h, sucking filtration obtains filtrate, is spin-dried for by liquid with at rotary evaporator 60 DEG C, and gained solid content 190g is longuette
Caulis Bambusae In Taeniam crude extract, standby.
3. extraction: take 100g Cultivation of Dictyophora crude extract and be dissolved in 2000 mL water, extract by petroleum ether, ethyl acetate successively
Taking, general every layer extracts 3-5 time, and the consumption of each extractant is 4000mL.Take ethyl acetate layer extract and be placed in rotation
Solvent evaporated at 60 DEG C on vaporizer, obtains 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 weigh appropriate 200-300 mesh silica gel in beaker, add oil
Ether stirring pulping.Fill in a Cotton Gossypii in chromatographic column bottom, in post, add 5mL petroleum ether, serosity is slowly poured in post, makes silicon
Glue natural subsidence, taps chromatographic column gently to get rid of bubble simultaneously, is opened by chromatographic column lower switch, make flowing phase with Glass rod
Naturally flow down, until liquid level about 3cm above cylinder.Balance each other with first flowing of 700mL.2. loading: by 8.7g extract
It is dissolved in 20mL methanol, is ground with the silica gel of 35g, volatilizes solvent so that it is be loose, be slowly added in chromatographic column through funnel
Side, then with a small amount of flowing phase drip washing post jamb, makes silicagel column surfacing.3. drip washing: take 130g silica gel wet method dress post, take acetic acid
Methacrylate layer solid content 7.5g dry method loading, with petroleum ether: ethyl acetate is respectively 7:3 and 1:1 and distinguishes 5 column volumes of drip washing successively
Collecting eluent by conical flask, there are 10 fragments, and 100rpm on the rotary evaporator, 50 DEG C are evaporated each fragment
Liquid, number consecutively is D11-D1No. 10.Merge wherein D12-D1The component of 8 fragments 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: take 100g silica gel wet method
Dress post, the 1.96g component dry method loading that will merge in (1), it is respectively 7:3 and 6:4 by petroleum ether ethyl acetate and depends on
Secondary respectively 3 column volumes of drip washing, collect eluent by conical flask, there are 6 fragments, and on the rotary evaporator 50 DEG C be evaporated
The liquid of each fragment, number consecutively is D21-D2No. 6.Merge wherein D23-D24 fragment components 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: take 50g silica gel wet method dress
Post, the 0.35g component dry method loading that will merge in (2), 3 posts of drip washing are distinguished successively with petroleum ether ethyl acetate=8:2,7:3
Volume, collects eluent by conical flask, there are 6 fragments, and 50 DEG C of liquid being evaporated each fragment on the rotary evaporator,
Number consecutively is D31-D3No. 6.Merge D34-D35 fragment components 100mg altogether, standby.
5. reversed-phase silica gel column chromatography: with 10g silica gel (model 2p-18) dress post, washing post with 300mL methanol, 300mL is ultrapure
Water balance, the D that then will merge34-D35 fragment components 100mg are dissolved in 2mL methanol, add above chromatographic column.Coutroi velocity
For 10mL/min, successively with 300mL ultra-pure water drip washing, collecting by conical flask, 50 DEG C are evaporated each fragment on the rotary evaporator
Liquid obtain white crystals body and be the monomer with bacteriostatic activity, this monomeric compound shows indicator bacteria through bacteriostatic experiment
Salmonella enteritidis and bacillus megaterium have the strongest inhibitory action, to the 503nhibiting concentration of Salmonella enteritidis are wherein
51.58μg/mL；503nhibiting concentration to bacillus megaterium is 83.06 μ g/mL.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
Claims (1)
1. the isolation and purification method of a Cultivation of Dictyophora bacteriostatic activity monomer, it is characterised in that: include that Cultivation of Dictyophora sporophore is pre-
Process, extract, extract, silica gel column chromatography, reversed-phase silica gel column chromatography；
Concrete operation step is as follows:
(1) Cultivation of Dictyophora sporophore pretreatment: Caulis Bambusae In Taeniam sporophore dry product toasts 3h in 60 DEG C of baking ovens, then uses plant pulverizer
Pulverize, cross 80 mesh sieves, obtain sporophore powder；
(2) extraction: weigh Cultivation of Dictyophora sporophore powder, adds dehydrated alcohol by liquid-solid ratio of weight and number 26 ~ 24:1, at 72 DEG C
After water-bath 1.5h, sucking filtration obtains filtrate, is spin-dried for by liquid by rotary evaporator 100rpm, at 60 DEG C, and gained solid content is Cultivation of Dictyophora
Crude extract, standby；
(3) extraction: Cultivation of Dictyophora crude extract is dissolved in water, extracts by petroleum ether, ethyl acetate successively, every layer extracts 3-5 time
?；Take ethyl acetate layer extract and be placed in rotary evaporator solvent evaporated at 60 DEG C, obtain ethyl acetate layer solid content, standby
With；Wherein the ratio of weight and number of crude extract and water is 1:18 ~ 20, and petroleum ether and ethyl acetate ratio of weight and number are 1:1 ~ 2；
(4) silica gel column chromatography:
1) silica gel column chromatography for the first time: 1. fill post: accurately weigh appropriate 200-300 mesh silica gel in beaker, add petroleum ether and stir
Mix slurry, fill in a Cotton Gossypii in chromatographic column bottom, in post, add 5mL petroleum ether, serosity is slowly poured in post, make silica gel certainly
So sedimentation, taps chromatographic column gently to get rid of bubble simultaneously, is opened by chromatographic column lower switch, make flowing mutually naturally with Glass rod
Flow down, until liquid level 3cm above cylinder, balance each other with first flowing of 700mL；2. loading: sample is dissolved in a small amount of methanol
In, it is ground with the silica gel of 4 times of samples, volatilizes solvent so that it is be loose, be slowly added into above chromatographic column through funnel, then
With a small amount of flowing phase drip washing post jamb, make silicagel column surfacing；3. drip washing: use petroleum ether: ethyl acetate=6.5-7:2.8-3,
0.8-1:0.8-1 distinguishes 5 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are 1500 mL, collect by conical flask
Eluent, collects 300mL, there are 10 fragments, and 50 DEG C of liquid being evaporated each fragment on the rotary evaporator, depend on for every bottle
Secondary numbered D11-D1No. 10, merge D12-D1The 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: with petroleum ether ethyl acetate=6.5-
7:2.8-3,5.6-6:3.6-4 distinguish 3 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are 750 mL tapers
Eluent collected by bottle, collects 250mL, there are 6 fragments for every bottle, and on the rotary evaporator 50 DEG C be evaporated each fragment
Liquid, number consecutively is D21-D2No. 6, merge D23-D24 fragment components, standby；
3) silica gel column chromatography for the third time: 1. fill post: with 1)；2. loading: with 1)；3. drip washing: with petroleum ether ethyl acetate=7.5-
8:1.8-2,7-7.5:3-3.5 distinguish 3 column volumes of drip washing successively, and two kinds of each plant demands of ratio leacheate are 750 mL, with cone
Eluent collected by shape bottle, collects 250mL, there are 6 fragments for every bottle, and on the rotary evaporator 50 DEG C be evaporated each fragment
Liquid, number consecutively is D31-D3No. 6, merge D34-D35 fragment components, standby；
(5) reversed-phase silica gel column chromatography: fill post with 10g silica gel, washes post with 300mL methanol, and 300mL ultra-pure water balances, then by upper
State the D of merging34-D35 fragment components are dissolved in 2mL methanol, add above chromatographic column, and coutroi velocity is 10mL/min, use
300mL ultra-pure water drip washing, collects by conical flask, and 50 DEG C are evaporated liquid and obtain white crystals body and be and have on the rotary evaporator
The monomer of bacteriostatic activity.
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