CN103083368A - Preparation method of ginkgo leaf lipoid components having antibacterial activities - Google Patents
Preparation method of ginkgo leaf lipoid components having antibacterial activities Download PDFInfo
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Abstract
The invention discloses a preparation method of ginkgo leaf lipoid components having antibacterial activities. Ginkgo leaf which is a raw material is extracted with a non-polar solvent, and undergoes molecular distillation separation, column chromatography, re-crystallization and purification to obtain eight compounds (sequentially comprising isophytol, nerolidol, linalool, beta-sitosterol acetate, beta-sitosterol, stigmasterol, ergosterol and daucosterol), polypentenol, ginkgo leaf lipoid total unsaponifiable matters, ginkgo leaf lipoid crystals, a ginkgo leaf lipoid light fraction and a ginkgo leaf lipoid heavy friction. The eight compounds, polypentenol, the ginkgo leaf lipoid total unsaponifiable matters, the ginkgo leaf lipoid crystals, the ginkgo leaf lipoid light fraction and the ginkgo leaf lipoid heavy friction have inhibition effects on salmonellas, Staphylococcus aureus and Aspergillus niger.
Description
Technical field
The present invention relates to have the preparation method of the Folium Ginkgo lipoid composition of bacteriostatic activity, belong to biomedicine field.
Technical background
The leaf that Folium Ginkgo is Ginkgoaceae Ginkgo plant Ginkgo biloba (Ginkgo biloba L.), the recent domestic scholar has carried out broad research to the chemical composition of flavonoid, terpene lactones, polysaccharide, polyprenol, volatile oil and sterol in Folium Ginkgo.Folium Ginkgo lipoid composition mainly contains the compounds such as hydro carbons, polypenthylene alcohols, terpene alcohols, sterols, their polar phase seemingly, more difficult separation; Folium Ginkgo lipoid composition is mainly to exist with the grease form, and the adularescent crystal is separated out usually at low temperatures, according to report, is mainly the sterols composition.The report of Folium Ginkgo sterol is studied the chemical constitution separately of its mixture by gas chromatography mass spectrometry (GC-MS) technology.We utilize molecular distillation technique, by the light fraction obtained after Folium Ginkgo lipoid component separating, by cracking-gas chromatography mass spectrometry (Py-GC-MS), analyze, wherein monoterpene, sesquiterpenoids GC content are about 23%, long-chain alcohol (ketone, ester) and Diterpenes relative amount are about 47%, and alkyl phenol, steroid GC content are about 30%; In heavy distillat, main component is polyprenol, and content is more than 80%.The compound that number molecular weight is large, boiling point is high and be subject to chromatographic separation condition and the restriction such as mass spectral analysis, can not entirely accurate by GC-MS, HPLC-MS determine its chemical constitution separately, therefore to the separation of Folium Ginkgo lipoid composition, and utilize the technology such as nuclear magnetic resonance, NMR (NMR) to its Structural Identification, the chemical composition of understanding this chemical fraction extremely is necessary.
The Folium Ginkgo bacteriostatic active ingredients of having reported is mainly the compositions such as flavone, phenolic acids, is mainly the chemical fraction of water solublity and pure dissolubility, and particularly lipoid composition bacteriostatic activity report is very few for liposoluble constituent.
Summary of the invention
For achieving the above object, invented the preparation method of the Folium Ginkgo lipoid composition with bacteriostatic activity, formed by following steps:
The first step, the preparation of Folium Ginkgo class fat ointment: get drying and crushing ginkgo leaf powder (diameter is below 0.25～5 millimeter), any solvent that adds acetone or petroleum ether (60～90 ℃) or ethyl acetate or dehydrated alcohol, ginkgo leaf powder is 1: 5～50 with the solvent quality ratio; 30～75 ℃ of heating and refluxing extraction, time 2～8h, repeat 3 times or above extraction; Merge extractive liquid,, reclaim solvent, obtains Folium Ginkgo liposoluble constituent ointment; The methanol or the alcoholic solution that add 1～15%NaOH or KOH, (kg: L), adopt 30～75 ℃ of hot refluxs to stir, mixing speed is 20～200r/min to ointment and ratio of solvent 1: 2～15, and time 0.5～5h, obtain saponification liquor; Any solvent that adds acetone or petroleum ether (60～90 ℃) or ethyl acetate, saponification liquor and ratio of solvent 1: 2～10 (V/V), merge extractive liquid,, reclaim solvent, thereby obtain the total non-saponifiable matter of Folium Ginkgo lipoid.
Second step, the molecular distillation separation method of Folium Ginkgo lipoid composition: get the total non-saponifiable matter of Folium Ginkgo lipoid, be put in 0～-40 ℃ of lower freezing and crystallizing 1～5h, after taking out, rapid sucking filtration, obtain crystal; The grease filtered separates by the wiped film molecular distillation device, knifing rotating speed 100～300r/min, and 120～250 ℃ of vapo(u)rizing temperatures, under high vacuum condition, (vacuum is more than or equal to 1.0 * 10
5), light component flies to straight interconderser and condenses into liquid with gaseous state, enters the light fraction catcher, obtains light fraction; Heavy constituent enters the heavy distillat catcher along cylinder inboard wall, obtains heavy distillat.
The 3rd step, the chromatography separating method of Folium Ginkgo lipoid composition: the crystal of gained in second step and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.04～0.1mm), with eluant chloroform/methanol (100%～85%: 0～15%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, 100% chloroform fraction, volatilize after solvent with isopropyl alcohol or dehydrated alcohol or acetonitrile under 5～-20 ℃, and recrystallization obtains compound 4 (cupreol acetas) repeatedly, chloroform/methanol (99%: 1%, V/V) fraction, go up Sephadex LH-20 post after volatilizing solvent, with eluant chloroform/methanol (30～50%: 70～50%, VN) system eluting, the fraction of collecting is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, get chloroform/methanol (30～35%: 70～65%, V/V) fraction, volatilize after solvent with Ketohexamethylene or acetone under 0～-20 ℃, recrystallization obtains compound 5 (cupreol) repeatedly, residue volatilizes after solvent with n-amyl alcohol or n-butyl alcohol under 5～-10 ℃, recrystallization obtains compound 6 (stigmasterol) repeatedly, residue uses ether under 0～-20 ℃ after volatilizing solvent, recrystallization obtains compound 7 (ergosterol) repeatedly, chloroform/methanol (90%: 10%, V/V) fraction, volatilize after solvent with chloroform or dehydrated alcohol under 0～-20 ℃, and recrystallization obtains compound 8 (daucosterol) repeatedly, in second step, the light fraction of gained and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.04～0.1mm), by eluant petrol ether/ethyl acetate (100%～90%: 0～10%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, petrol ether/ethyl acetate (95%: 5%, V/V) fraction, prepare the plate preparation with fluorescence (GF254) silica gel thin-layer after volatilizing solvent, developing solvent is petrol ether/ethyl acetate (100%～85%: 0～15%, V/V), under the 365nm ultraviolet light, develop the color, get Rf value at 0.50～0.65 place's silica gel, with ethyl acetate or dehydrated alcohol extraction, low temperature volatilizes solvent and obtains compound 1 (different vegetable alcohol), petrol ether/ethyl acetate (92%: 8%, V/V) fraction, go up Sephadex LH-20 post after volatilizing solvent, with eluant chloroform/methanol (40～50%: 60～50%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, gets chloroform/methanol (40～45%: 60～55%, V/V) fraction, low temperature volatilizes solvent and obtains compound 2 (nerolidol), petrol ether/ethyl acetate (90%: 10%, V/V) fraction, prepare the plate preparation with fluorescence (GF254) silica gel thin-layer after volatilizing solvent, developing solvent is petrol ether/ethyl acetate (95%～75%: 5～25%, V/V), under the 365nm ultraviolet light, develop the color, get Rf value at 0.40～0.55 place's silica gel, with ethyl acetate or dehydrated alcohol extraction, low temperature volatilizes solvent and obtains compound 3 (linalool), the heavy distillat of gained in second step and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.04～0.1mm), by eluant petrol ether/ethyl acetate (100%～95%: 0～5%, V/V) system eluting, the fraction of collecting is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, get petrol ether/ethyl acetate (99%～97%: 1～3%, V/V) fraction, volatilize after solvent that to add mass ratio be 1: 5～15 acetone or ethyl acetate, stirring at room is dissolved, then the adsorbent it mixed with kieselguhr 1: 1 in mass ratio～5 with active carbon, by the solid-liquid mass ratio, be 1: 10～100 mixing, at-40 ℃～30 ℃, stir 4～20 hours, 1000～10000 rev/mins of centrifugal rear filtrations, supernatant reclaims after solvent the polyprenol lipoid be after purification.
The compound 1～8 of gained in the present invention, be followed successively by different vegetable alcohol (inhibition zone scope 9.9-13.4 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 62.5-125 μ g/mL, 125 μ g/mL), nerolidol (inhibition zone scope 16.2-20.1 μ g/mL, MIC, MBC and MFC value are followed successively by 3.9-15.6 μ g/mL, 31.3-5-62.5 μ g/mL, 62.5 μ g/mL), linalool (inhibition zone scope 14.9-17.9 μ g/mL, MIC, MBC and MFC value are followed successively by 7.8-31.3 μ g/mL, 62.5 μ g/mL, 31.3 μ g/mL), cupreol acetas (inhibition zone scope 10.9-11.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 250 μ g/mL), cupreol (inhibition zone scope 12.1-14.1 μ g/mL, MIC and MBC value are followed successively by 31.3 μ g/mL, 125 μ g/mL), stigmasterol (inhibition zone scope 7.7-9.2 μ g/mL, MIC and MBC value are followed successively by 125 μ g/mL,>250 μ g/mL), ergosterol (inhibition zone scope 8.1-10.0 μ g/mL, MIC and MBC value are followed successively by 62.5-125 μ g/mL, >=250 μ g/mL) and daucosterol (inhibition zone scope 11.1-12.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125-250 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.Compound 1 and 5 and Polyprenols From Ginkgo Biloba L collaborative bacteriostasis is arranged.Compound 1 (different vegetable alcohol) shows as inhibition zone scope 14.1-17.1mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 7.8-15.6 μ g/mL, and the FIC index range is 0.25-0.5; Compound 5 (cupreol) shows as 10.4-16.3mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 15.6-31.3 μ g/mL, and the FIC index is 0.5.The total non-saponifiable matter of Folium Ginkgo lipoid (inhibition zone scope 14.4-16.9 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3 μ g/mL, 62.5 μ g/mL, 125 μ g/mL), lipoid crystal (inhibition zone scope 12.3-12.8 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125 μ g/mL), lipoid light fraction (inhibition zone scope 12.9-14.8 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 125 μ g/mL, 125 μ g/mL), lipoid heavy distillat (inhibition zone scope 15.0-16.8 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3 μ g/mL, 62.5 μ g/mL, 62.5 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.
The specific embodiment
Following examples are more of the present invention giving an example, and should not be seen as limitation of the invention.
The extraction separation method of embodiment 1 Folium Ginkgo lipoid composition
There is the preparation method of the Folium Ginkgo lipoid composition of bacteriostatic activity, formed by following steps:
The first step, the preparation of Folium Ginkgo class fat ointment: get drying and crushing ginkgo leaf powder (diameter is below 0.5 millimeter) 10kg, add petroleum ether (60～90 ℃) 30L, 60 ℃ of heating and refluxing extraction, time 4h, repeat 3 times and extract; Merge extractive liquid,, reclaim solvent, obtains Folium Ginkgo liposoluble constituent ointment 500g; Add 5%NaOH methanol solution 6L, adopt 65 ℃ of hot refluxs to stir, mixing speed is 60r/min, and time 2h, obtain saponification liquor; Add petroleum ether (60～90 ℃) 30L extraction 3 times, merge extractive liquid,, reclaim solvent, thereby obtain the total non-saponifiable matter 350g of Folium Ginkgo lipoid.
Second step, the molecular distillation separation method of Folium Ginkgo lipoid composition: get the total non-saponifiable matter 350g of Folium Ginkgo lipoid, be put in-20 ℃ of lower freezing and crystallizing 5h, after taking out, rapid sucking filtration, obtain crystal 35g; The grease filtered separates by the wiped film molecular distillation device, knifing rotating speed 200r/min, 180 ℃ of vapo(u)rizing temperatures, (vacuum 1.0 * 10 under high vacuum condition
5), light component flies to straight interconderser and condenses into liquid with gaseous state, enters the light fraction catcher, obtains light fraction 44g; Heavy constituent enters the heavy distillat catcher along cylinder inboard wall, obtains heavy distillat 168g.
The 3rd step, the chromatography separating method of Folium Ginkgo lipoid composition: the crystal of gained in second step and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.05～0.1mm), with eluant chloroform/methanol (100%～85%: 0～15%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, 100%CHCl
3fraction, volatilize after solvent with isopropyl alcohol under-10 ℃, and recrystallization obtains compound 4 (cupreol acetas) 290mg repeatedly, chloroform/methanol (99%: 1%, V/V) fraction, go up Sephadex LH-20 post after volatilizing solvent, with eluant chloroform/methanol (30～50%: 70～50%, V/V) system eluting, the fraction of collecting is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, get chloroform/methanol (35%: 65%, V/V) fraction, volatilize after solvent with under Ketohexamethylene-20 ℃, recrystallization obtains compound 5 (cupreol) 29g repeatedly, residue uses n-amyl alcohol under-10 ℃ after volatilizing solvent, recrystallization obtains compound 6 (stigmasterol) 116mg repeatedly, residue uses ether under-20 ℃ after volatilizing solvent, recrystallization obtains compound 7 (ergosterol) 105mg repeatedly, chloroform/methanol (90%: 10%, V/V) fraction, volatilize after solvent with under chloroform-20 ℃, and recrystallization obtains compound 8 (daucosterol) 203mg repeatedly, in second step, the light fraction 44g of gained and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.05～0.1mm), by eluant petrol ether/ethyl acetate (100%～90%: 0～10%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, petrol ether/ethyl acetate (95%: 5%, V/V) fraction, prepare the plate preparation with fluorescence (GF254) silica gel thin-layer after volatilizing solvent, developing solvent is petrol ether/ethyl acetate (95%: 5%, V/V), under the 365nm ultraviolet light, develop the color, get Rf value at 0.65 place's silica gel, be extracted with ethyl acetate, low temperature volatilizes solvent and obtains compound 1 (different vegetable alcohol) 178mg, petrol ether/ethyl acetate (92%: 8%, V/V) fraction, go up Sephadex LH-20 post after volatilizing solvent, with eluant chloroform/methanol (40～50%: 60～50%, V/V) system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, gets chloroform/methanol (40%: 60%, V/V) fraction, low temperature volatilizes solvent and obtains compound 2 (nerolidol) 51mg, (90%: 10%, V/V) fraction, volatilized after solvent with fluorescence (GF petrol ether/ethyl acetate
254) silica gel thin-layer prepares plate preparation, developing solvent be petrol ether/ethyl acetate (85%: 15%, V/V), under the 365nm ultraviolet light, develop the color, get Rf value at 0.40 place's silica gel, be extracted with ethyl acetate, low temperature volatilizes solvent and obtains compound 3 (linalool) 20mg, the heavy distillat 10g of gained in second step and silica gel G fully are mixed and finely ground in mass ratio at 1: 1, upper silicagel column (particle diameter 0.05～0.1mm), by eluant petrol ether/ethyl acetate (100%～95%: 0～5%, V/V) system eluting, the fraction of collecting is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, get petrol ether/ethyl acetate (98%: 2%, V/V) fraction, add 100ml acetone after volatilizing solvent, stirring at room is dissolved, then the adsorbent 20g it mixed with kieselguhr in mass ratio with active carbon at 1: 1, stir 5 hours at 0 ℃, 5000 rev/mins of centrifugal rear filtrations, and supernatant reclaims after solvent the polyprenol lipoid 7.2g be after purification.
The bacteriostatic activity investigation method of embodiment 2 Folium Ginkgo lipoid compositions
1. experimental strain, raw material
1.1 for the examination strain
Salmonella ATCC14028, staphylococcus aureus ATCC25923 and Aspergillus niger ATCC16404
1.2 testing sample
The samples such as gained compound 1～8 and polyprenol in embodiment 1
2 experimental techniques
2.1 the bacteriostatic activity of filter paper method working sample
(1) preparation of culture medium
Heat of solution, cooling after, regulate pH to 7.0～7.2, this is fluid medium, adds therein the 2.0g agar powder, this is solid medium, 121 ℃ of sterilizing 20min.
(2) fluid medium inoculation and activated spawn
Open the ultraviolet sterilization lamp of clean work station, after the half an hour that sterilizes, open the console switch of clean bench, getting a ring strain with the inoculating loop after sterilizing dissolves in fluid medium, the conical flask that rubs gently wall, allow bacterium colony separate with on inoculating loop, takes out inoculating loop, seal conical flask, the shaken cultivation base, be uniformly dispersed strain gently, is put in biochemical cultivation case, 37 ℃ (Salmonella and staphylococcus aureus), 28 ℃ (Aspergillus niger) lower activation 24h.
(3) test the sterilizing of required instrument
The culture dish that is down flat plate culture dish used and the 6mm filter paper is housed is wrapped with kraft paper, has filled in the test tube of plug, the box that liquid transfer gun head is housed is wrapped with kraft paper respectively, and the normal saline, the solid medium that prepare are sealed with gauze and kraft paper respectively.By it at 121 ℃, autoclaving 20min under 100Pa.
(4) regulate bacterium colony concentration
Pour the solid medium dissolved into the 20mL left and right in culture dish, be placed on clean bench, Self-leveling is cooling.
Get 7 test tubes, the bacteria suspension that activate of dilution successively, the bacteria suspension that No. 1 test tube is stock solution after 10 times of normal saline dilutions, the bacteria suspension that No. 2 test tubes are 100 times of dilutions, after the dilution, No. seven test tube is to dilute 10 successively
7bacteria suspension doubly.
Pipette the bacteria suspension applying solid culture medium in 100 μ L1～No. 7 test tube, standing it is permeated in culture medium, rear inversion culture dish, in biochemical cultivation case 37 ℃ (Salmonella and staphylococcus aureus), 28 ℃ (Aspergillus niger) cultivates 24h.
According to the bacterium colony computing rule: colony forming units cfu * extension rate=clump count, count denumerable bacterium colony plate, calculate every ware clump count, selecting the bacterium colony coating concentration is 10
5cfu/mL.
(5) preparation of specimen
Respectively sample preparation is become to 500 μ g/mL solution with normal hexane, will be dipped in through the filter paper of sterilizing in sample solution, measure its bacteriostatic activity after half an hour.
(6) the filter paper method is measured the bacteriostatic activity of test sample
Pipette 100 μ L bacteria suspensions with pipettor and coat fluid medium, after placing a period of time, until bacterium liquid, permeate after culture medium, with the filter paper that is soaked with testing sample gently docile in ware, note not overexerting, it is sunk in solid medium, 3 filter papers are equidistantly even, with two ware contrast parallel tests, 37 ℃ (Salmonella and staphylococcus aureus), 28 ℃ (Aspergillus niger) the lower biochemical 24h of cultivation, measuring samples antibacterial circle diameter to Salmonella, staphylococcus aureus and Aspergillus niger be inverted.
2.2 the calculating of the mensuration of sample MIC (minimal inhibitory concentration), MBC (minimum is killed bacterial concentration) and MFC (minimal fungicidal concentration) value and FIC classification Mlc index
(1) by upper method preparation culture medium, fluid medium inoculation and activated spawn, regulate bacterium colony concentration and preparation specimen, sample is configured to variable concentrations 250,125,62.5,31.3,15.6,7.8, the solution of 3.9 μ g/mL.
(2) mensuration of the MIC of each sample, MBC and MFC value
The culture medium of having mixed sample is down flat to ware, the Self-leveling cooled and solidified, take Salmonella, staphylococcus aureus and Aspergillus niger as detecting strain, spread plate, place a period of time and treat that bacteria suspension infiltrates, 37 ℃ (Salmonella and staphylococcus aureus), 28 ℃ (Aspergillus niger) lower biochemistry of being inverted is cultivated 24h.Observe the least concentration plate of asepsis growth, being designated as this concentration is the MIC value of sample for this strain; The maximum concentration of 100% asepsis growth is MBC and MFC.
(3) calculating of FIC index: FIC index=MIC first medicine coupling/MIC first prescription use+MIC second medicine coupling/MIC second prescription is used
2.3 the investigation of sample inhibition zone
(1) preparation of culture medium
Prepare on request Salmonella and staphylococcus aureus culture medium; Aspergillus niger according to the preparation of Cha Shi culture medium, is 100mL water by culture medium, 3.0g sucrose, 2.0g agar, 0.2g NaNO
3, 0.1g K
2hPO
4, 0.05g KCl, 0.05g MgSO
47H
2o, 0.001g FeSO
4; Above culture medium all is placed in 121 ℃ of lower autoclaving 20min.
(2) the filter paper method is measured antimicrobial spectrum
With normal hexane, sample preparation is become to the solution of 500 μ g/mL, measure its antibacterial circle diameter for Salmonella, staphylococcus aureus and Aspergillus niger.
2.4 antibacterial experiment result
The compound 1～8 of gained, be followed successively by different vegetable alcohol (inhibition zone scope 9.9-13.4 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 62.5-125 μ g/mL, 125 μ g/mL), nerolidol (inhibition zone scope 16.2-20.1 μ g/mL, MIC, MBC and MFC value are followed successively by 3.9-15.6 μ g/mL, 31.3-5-62.5 μ g/mL, 62.5 μ g/mL), linalool (inhibition zone scope 14.9-17.9 μ g/mL, MIC, MBC and MFC value are followed successively by 7.8-31.3 μ g/mL, 62.5 μ g/mL, 31.3 μ g/mL), cupreol acetas (inhibition zone scope 10.9-11.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 250 μ g/mL), cupreol (inhibition zone scope 12.1-14.1 μ g/mL, MIC and MBC value are followed successively by 31.3 μ g/mL, 125 μ g/mL), stigmasterol (inhibition zone scope 7.7-9.2 μ g/mL, MIC and MBC value are followed successively by 125 μ g/mL,>250 μ g/mL), ergosterol (inhibition zone scope 8.1-10.0 μ g/mL, MIC and MBC value are followed successively by 62.5-125 μ g/mL, >=250 μ g/mL) and daucosterol (inhibition zone scope 11.1-12.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125-250 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.Compound 1 and 5 and Polyprenols From Ginkgo Biloba L collaborative bacteriostasis is arranged.Compound 1 (different vegetable alcohol) shows as inhibition zone scope 14.1-17.1mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 7.8-15.6 μ g/mL, and the FIC index range is 0.25-0.5; Compound 5 (cupreol) shows as 10.4-16.3mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 15.6-31.3 μ g/mL, and the FIC index is 0.5.The total non-saponifiable matter of Folium Ginkgo lipoid (inhibition zone scope 14.4-16.9 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3 μ g/mL, 62.5 μ g/mL, 125 μ g/mL), lipoid crystal (inhibition zone scope 12.3-12.8 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125 μ g/mL), lipoid light fraction (inhibition zone scope 12.9-14.8 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 125 μ g/mL, 125 μ g/mL), lipoid heavy distillat (inhibition zone scope 15.0-16.8 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3 μ g/mL, 62.5 μ g/mL, 62.5 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.Refer to form 1,2 and 3.
The size of the different sample inhibition zone of form 1. value is (Tukey checks P=0.05) relatively.
S1: the total non-saponifiable matter of Folium Ginkgo lipoid; S2: lipoid crystal; S3: lipoid light fraction; S4: lipoid heavy distillat; GBP: Polyprenols From Ginkgo Biloba L; C1～C8: compound 1～8; MN: miconazole nitrate (positive control 1); GS: gentamycin sulfate (positive control 2).△ inhibition zone value theory add and be worth (compound 1～8 respectively with polyprenol in mass ratio 1: 1 theory add and meansigma methods).* add and compared significant difference (Tukey check, p<0.05) with value with theory of correspondences.In same perpendicular hurdle, identical lower case means there is no significant difference (Tukey check, p>0.05).▲ all samples is investigated under 500 μ g/mL total concentrations.
The MIC of form 2. different samples, MBC and MFC value
* MBC value; * MFC value.S1: the total non-saponifiable matter of Folium Ginkgo lipoid; S2: lipoid crystal; S3: lipoid light fraction; S4: lipoid heavy distillat; GBP: Polyprenols From Ginkgo Biloba L; C1～C8: compound 1～8; The total concentration of all biased sample groups of △.
Form 3.FIC index is determined the drug combination effect
C1～C8: compound 1～8; GBP: Polyprenols From Ginkgo Biloba L.* synergism (0<FIC index≤0.5); △ addition (0.5<FIC index≤1) and ▲ irrelevant effect (1<FIC index≤4).
Claims (7)
1. the preparation method that has the Folium Ginkgo lipoid composition of bacteriostatic activity, its feature comprises preparation and the bacteriostatic activity of the total non-saponifiable matter of Folium Ginkgo lipoid, lipoid crystal, lipoid light fraction and lipoid heavy distillat and compound 1～8 (being followed successively by different vegetable alcohol, nerolidol, linalool, cupreol acetas, cupreol, stigmasterol, ergosterol, daucosterol) and polyprenol lipoid composition.
2. the preparation method that has according to claim 1 the Folium Ginkgo lipoid composition of bacteriostatic activity is characterized in that compound 1～8 preparation method comprises lower several step:
The first step, the preparation of Folium Ginkgo class fat ointment: get drying and crushing ginkgo leaf powder (diameter is below 0.25～5 millimeter), add solvent orange 2 A 1, ginkgo leaf powder is 1: 5～50 with the solvent quality ratio; 30～75 ℃ of heating and refluxing extraction, time 2～8h, repeat 3 times or above extraction; Merge extractive liquid,, reclaim solvent, obtains Folium Ginkgo liposoluble constituent ointment; Add 1～15% solution B 1, (kg: L), adopt 30～75 ℃ of hot refluxs to stir, mixing speed is 20～200r/min to ointment and ratio of solvent 1: 2～15, and time 0.5～5h, obtain saponification liquor; Add solvent C 1, saponification liquor and ratio of solvent 1: 2～10 (V/V), merge extractive liquid,, reclaim solvent, thereby obtain the total non-saponifiable matter of Folium Ginkgo lipoid.
Second step, the molecular distillation separation method of Folium Ginkgo lipoid composition: get the total non-saponifiable matter of Folium Ginkgo lipoid, be put in 0～-40 ℃ of lower freezing and crystallizing 1～5h, after taking out, rapid sucking filtration, obtain crystal; The grease filtered separates by the wiped film molecular distillation device, knifing rotating speed 100～300r/min, and 120～250 ℃ of vapo(u)rizing temperatures, under high vacuum condition, (vacuum is more than or equal to 1.0 * 10
5), light component flies to straight interconderser and condenses into liquid with gaseous state, enters the light fraction catcher, obtains light fraction; Heavy constituent enters the heavy distillat catcher along cylinder inboard wall, obtains heavy distillat.
The 3rd step, the chromatography separating method of Folium Ginkgo lipoid composition: the crystal of gained in second step and filler A fully are mixed and finely ground in mass ratio at 1: 1, upper filler A post, with eluant chloroform/methanol system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor; 100% chloroform fraction, volatilize after solvent with solvent B under 5～-20 ℃, and recrystallization obtains compound 4 (cupreol acetas); Fraction chloroform/methanol (99%: 1%, V/V), go up the C chromatographic column after volatilizing solvent, with eluant chloroform/methanol system eluting, volatilize after solvent with solvent D under 0～-20 ℃, recrystallization obtains compound 5 (cupreol), residue uses solvent E under 5～-10 ℃ after volatilizing solvent, recrystallization obtains compound 6 (stigmasterol), and residue uses solvent F under 0～-20 ℃ after volatilizing solvent, and recrystallization obtains compound 7 (ergosterol); Chloroform/methanol (90%: 10%, V/V) fraction, volatilize after solvent with solvent G under 0～-20 ℃, and recrystallization obtains compound 8 (daucosterol); In second step, the light fraction of gained and filler A fully are mixed and finely ground in mass ratio at 1: 1, upper filler A post (particle diameter 0.04～0.1mm), and with eluant petrol ether/ethyl acetate system eluting, the fraction of collection is followed the tracks of with the colour developing of thin layer chromatography iodine vapor; Petrol ether/ethyl acetate (95%: 5%, V/V) fraction, volatilize after solvent and prepare the plate preparation with filler H thin layer, and Rf value, at the I place, obtains compound 1 (different vegetable alcohol); Petrol ether/ethyl acetate (92%: 8%, V/V) fraction, volatilize upper C chromatographic column after solvent, obtains compound 2 (nerolidol); Petrol ether/ethyl acetate (90%: 10%, V/V) fraction, volatilize after solvent and prepare the plate preparation with filler H thin layer, and Rf value, at the J place, obtains compound 3 (linalool); The heavy distillat of gained in second step and filler A fully are mixed and finely ground in mass ratio at 1: 1, upper filler A post, with eluant petrol ether/ethyl acetate system eluting, the fraction of collecting is followed the tracks of with the colour developing of thin layer chromatography iodine vapor, get petrol ether/ethyl acetate (99%～97%: 1～3%, V/V) fraction, volatilize after solvent that to add mass ratio be 1: 5～15 solvent K, and stirring at room is dissolved; Then by itself and adsorbent L, by the solid-liquid mass ratio, be 1: 10～100 to mix, at-40 ℃～30 ℃, stir 4～20 hours, 1000～10000 rev/mins of centrifugal rear filtrations, supernatant reclaims after solvent the polyprenol lipoid be after purification.
3. the preparation method that has according to claim 2 the Folium Ginkgo lipoid composition of bacteriostatic activity, is characterized in that in the first step, solvent orange 2 A 1 is acetone or petroleum ether (60～90 ℃) or ethyl acetate or dehydrated alcohol; Methanol or alcoholic solution that solution B 1 is NaOH or KOH; Solvent C 1 is acetone or petroleum ether (60～90 ℃) or ethyl acetate.
4. the preparation method that has according to claim 2 the Folium Ginkgo lipoid composition of bacteriostatic activity, is characterized in that in the 3rd step, filler A is silica gel G, and solvent B is isopropyl alcohol or dehydrated alcohol or acetonitrile; The C chromatographic column is Sephadex LH-20 filler; Solvent D is Ketohexamethylene or acetone; Solvent E is n-amyl alcohol or n-butyl alcohol; Solvent F is ether or normal hexane; Solvent G is chloroform or dehydrated alcohol; Filler H is fluorescence (GF
254) silica gel; Rf value I is in 0.50～0.65; Rf value J is in 0.40～0.55; Solvent K is acetone or ethyl acetate; Adsorbent L is active carbon and the kieselguhr that mix mass ratio 1: 1～5.
5. Folium Ginkgo lipoid composition according to claim 1 has bacteriostatic activity, the compound 1～8 that it is characterized in that gained, be followed successively by different vegetable alcohol (inhibition zone scope 9.9-13.4 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 62.5-125 μ g/mL, 125 μ g/mL), nerolidol (inhibition zone scope 16.2-20.1 μ g/mL, MIC, MBC and MFC value are followed successively by 3.9-15.6 μ g/mL, 31.3-5-62.5 μ g/mL, 62.5 μ g/mL), linalool (inhibition zone scope 14.9-17.9 μ g/mL, MIC, MBC and MFC value are followed successively by 7.8-31.3 μ g/mL, 62.5 μ g/mL, 31.3 μ g/mL), cupreol acetas (inhibition zone scope 10.9-11.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 250 μ g/mL), cupreol (inhibition zone scope 12.1-14.1 μ g/mL, MIC and MBC value are followed successively by 31.3 μ g/mL, 125 μ g/mL), stigmasterol (inhibition zone scope 7.7-9.2 μ g/mL, MIC and MBC value are followed successively by 125 μ g/mL,>250 μ g/mL), ergosterol (inhibition zone scope 8.1-10.0 μ g/mL, MIC and MBC value are followed successively by 62.5-125 μ g/mL, >=250 μ g/mL) and daucosterol (inhibition zone scope 11.1-12.7 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125-250 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.
6. Folium Ginkgo lipoid composition according to claim 1 has bacteriostatic activity, also show compound 1 and 5 and Polyprenols From Ginkgo Biloba L collaborative bacteriostasis is arranged; Compound 1 (different vegetable alcohol) shows as inhibition zone scope 14.1-17.1mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 7.8-15.6 μ g/mL, and the FIC index range is 0.25-0.5; Compound 5 (cupreol) shows as 10.4-16.3mm with the collaborative bacteriostasis of Polyprenols From Ginkgo Biloba L, and the MIC value is 15.6-31.3 μ g/mL, and the FIC index is 0.5.
7. Folium Ginkgo lipoid composition according to claim 1 has bacteriostatic activity, also show the total non-saponifiable matter of Folium Ginkgo lipoid (inhibition zone scope 14.4-16.9 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3ug/mL, 62.5 μ g/mL, 125 μ g/mL), lipoid crystal (inhibition zone scope 12.3-12.8 μ g/mL, MIC and MBC value are followed successively by 62.5 μ g/mL, 125 μ g/mL), lipoid light fraction (inhibition zone scope 12.9-14.8 μ g/mL, MIC, MBC and MFC value are followed successively by 31.3 μ g/mL, 125 μ g/mL, 125 μ g/mL), lipoid heavy distillat (inhibition zone scope 15.0-16.8 μ g/mL, MIC, MBC and MFC value are followed successively by 15.6-31.3 μ g/mL, 62.5 μ g/mL, 62.5 μ g/mL) for Salmonella, staphylococcus aureus and Aspergillus niger are inhibited.
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