CN101492356A - Method for separating and preparing compound 2, 4-dihydroxy-5-methyl-acetophenone by using fermentation liquor of Polyporus fuscus - Google Patents
Method for separating and preparing compound 2, 4-dihydroxy-5-methyl-acetophenone by using fermentation liquor of Polyporus fuscus Download PDFInfo
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- CN101492356A CN101492356A CNA2009100209637A CN200910020963A CN101492356A CN 101492356 A CN101492356 A CN 101492356A CN A2009100209637 A CNA2009100209637 A CN A2009100209637A CN 200910020963 A CN200910020963 A CN 200910020963A CN 101492356 A CN101492356 A CN 101492356A
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- acetophenone
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- dihydroxyl
- silica gel
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Abstract
The invention relates to a method for separating and preparing a compound 2, 4-dihydroxy-5-methyl-acetophenone by utilizing fermentation liquor of Polyporus umbellatus (Polyporus picipes), which adopts a method of liquid culture in a plate rotating bottle, and firstly, peeled potatoes and grapes are usedSugar, distilled water, pH7.2, sterilizing to obtain culture medium; then culturing and fermenting to obtain fermentation liquor, extracting the fermentation liquor with ethyl acetate to obtain extract, separating by column chromatography, and performing gradient elution with chloroform-methanol system; performing multiple normal phase silica gel column chromatography and reverse phase C18Separating with Sephadex LH-20 gel column chromatography and recrystallization method to obtain pure compound 2, 4-dihydroxy-5-methyl-acetophenone (II). The compound 2, 4-dihydroxy-5-methyl-acetophenone (II) can be developed into a raw pesticide of the anti-pathogenic fungus antibiotic, the problem of raw material source of the 2, 4-dihydroxy-5-methyl-acetophenone is solved, and the preparation method has the advantages of mild culture conditions, simple and easy culture medium preparation, short fermentation time and the like.
Description
Technical field
The present invention relates to a kind of preparation method of compound, particularly a kind of fermented liquid of brown pore fungus that utilizes separates preparation compound 2, the method for 4-dihydroxyl-5-methyl-acetophenone (II).The compound 2 of the present invention's preparation, 4-dihydroxyl-5-methyl-acetophenone (II) is a new natural product, can suppress the growth of various plants pathogenic fungi, and to cotton-wilt fusarium, corn is bent the spore germ, and apple anthrax bacteria has the good restraining effect.
Background technology
Higher fungi comprises the part kind and Basidiomycetes (Basidiomycetes) fungi of Ascomycetes (Ascomycetes).Because long-term coevolution effect, a lot of higher fungies have formed own distinctive chemical defence system.In case injured is that the start-up system Secretases is converted into activated material with secondary metabolite.An interesting phenomenon is: the sporophore of a lot of macro fungis is never by insect infestations.One of them typical example is: be converted at once after the impaired reaction of stearoyl velutinal of non-activity in the Lactarius and have very strong active microbiotic isovelleral.In addition, people contrast injured and unscathed sporophore by experiment, find sporophore a large amount of secondary metabolite of very fast generation after injured, various active such as that these products have is antimycotic, desinsection.In view of this, higher fungi also is the valuable source of biogenic pesticide research and development.
From the higher fungi fermented product, seek the very big concern that the lead compound with killing pests and suppressing bacteria effect has caused Chinese scholars.Be because from macro fungi, constantly find novel structure and compound on the one hand, and structural changes is big with notable biological activity.This structure diversity is significant for the discovery of medicine (or agricultural chemicals).On the other hand, macro fungi not only can directly be adopted and be used as research material usefulness, and can pass through mycelium or spore fermentation culture more conveniently, its advantage be can carry out the industrialization continuous production, scale is big, output is high, fermentation period is short, productivity effect is high.This provides the resource assurance for possible from now on suitability for industrialized production, and this is a potential advantage with respect to the restriction botanical pesticide that is subjected to resource.Improving the condition in the fermenting process, promote fermenting process to carry out towards the direction that improves the purpose product, improve the product quality, reduce fermentation costs, make input-output ratio reach minimum, is a direction of liquid fermenting research.Medicinal fungi in the liquid fermenting process, can a large amount of propagation except that mycelia or spore, also can in fermented liquid, produce polysaccharide, polypeptide, alkaloid, terpenoid, sterol, plant hormone and have all cpds of microbiotic effect.These materials have the effect of preventing and curing diseases to human organs such as cardiovascular, liver, neural system, sexual organs respectively, and effects such as anticancer, anti-inflammatory, antibiotic, anti-ageing, antiulcer agent are arranged.People are to the liquid fermenting of medicinal fungi, and except that obtaining mycelium, also above-mentioned to have active secondary metabolite be purpose to obtain.
Utilize the precedent of the existing business success of higher fungi exploitation agricultural chemicals: have a liking for from higher fungi as German scholar and found to have the active lead compound strobilurin of antifungal the cone umbrella (Strobilurus tenacellus), but this project is interrupted narrowly, because the activity that this compound shows in field experiment is not strong.From the angle of chemistry, strobilurin contains conjugated double bond, and instability is easily oxidized.About 10000 compounds have been synthesized for parent according to this, class novel pesticide Kresoxim-methyl (the trade(brand)name Brio that develops of BASF Aktiengesellschaft (BASF) therefrom, Allegro, Diskus, Juwel, fungicides such as Mentor), the Zeneca of another company also similarly studies always, the said firm has successfully synthesized Compound I CIA5504 at last, also has extraordinary activity, and commodity are called Amistar.Now the sales volume in this compounds whole world every year has reached more than 500,000,000 dollar.This compounds can suppress mitochondrial breathing and cause effect, wide scope pathogenic bacteria such as ascomycetes, basidiomycetes, imperfect fungi and ovum guiding principle bacterium had sterilization effect, except that paddy rice rice blast, also rice diseases such as rice sheath blight disease, fringe rot, leaf blight had the inhibition effect.
Brown pore fungus (Polyporus picipes Fr) is a Basidiomycetes, Aphyllophorales, and polyporaceae belongs to wood-decay fungi, is born on the rotten wood of deciduous tree, also is born on the softwood tree sometimes, causes the xylem of birch, linden, Cortex Fraxini mandshuricae, maple or fir to form white rot.Sporophore is big.Lid diameter 4-16cm, thick 2-3.5mm, fan-shaped, kidney shape, subcircular are to circular, and it is protruding slightly that base portion is often recessed to open and flat, badius, the middle part look darker, and the surface is chocolate entirely sometimes, and is smooth, and thin edge and sharp is wavyly split to lobe.Stem adnation or wilfully, long 2-5mm, thick 0.3-1.3cm, black or base portion black polish behind the initial stage tool fine hair.Bacterial context white or near-white, thick 0.5-2mm.Tube prolongs life, long 0.5-1.5mm, with the bacterial context form and aspect seemingly, be light powder grey after doing.Mouth of pipe dihedral is to subcircular, and every millimeter 5-7 is individual.Mycelium water white transparency in the thalamium, the thick 1.2-2 μ of mycelia m.The spore ellipse is to oblong, and an end point is narrow, and water white transparency is level and smooth, 5.8-7.5 μ m * 2.8-3.5 μ m.It is distributed in ground such as Liaoning, Jilin, Heilungkiang, Hebei, Gansu, Jiangsu, Anhui, Zhejiang, Jiangxi, Guangxi, Fujian, Sichuan, Yunnan, Guizhou, Tibet.It can endocrine regulation function, vessel softening, blood pressure regulation dissolves thrombus, prevents and treats myocardial infarction and cerebral thrombosis, and has prevention and delay heart aging, strengthens immune function of human body, regulates the cerebral nerve function, anti-ageing effect.The cardiovascular and cerebrovascular series decoction of brown pore fungus prescription can be treated high worry, coronary heart disease, rheumatic heart disease, pulmonary heart disease, myocarditis, myocardial strain, myocardial infarction and cerebral infarction, hydrocephalus, cerebral concussion sequela etc.The brown pore fungus of Daxing'an Mountainrange virgin forest and serial decoction thereof are intended to improve or recover the vigor that the cardiovascular and cerebrovascular intrinsic is supplied with mechanism and metabolic mechanism, thrombolysis and recovery collagen protein, make triglyceride in the blood, cholesterol etc. recover normally determined curative effect.
Pertinent literature [A.A.Morais, R.B.Fo, S.V.Fraiz Jr.Synthesis of threenatural 1,3-diarylpropanes:Two revised structures, Phytochemistry, 1989,28 (1): 239-242] and [Cai Mengshen, Wang Lanming. synthesizing of the plain analogue of the different black soya bean of carbon glycosides research-XXVI., the chemistry journal, 1990,48,1191-1198] only reported 2,4-dihydroxyl-5-methyl-acetophenone is as the intermediate of synthetic; But do not see its relevant report as natural product and anti-fungal activity of plant pathogenic.The applicant once reported first the bacteriostatic activity preliminary study of brown pore fungus fermentating metabolism thing (Dong Yanhong opens saddle spirit, Ma Huini, Li Xiaoming, Gao Jinming, Xibei Forest College's journal, 2007,22 (2): 105-108), still, antibacterial active compounds it be unclear that in the fermented liquid.
Summary of the invention
The objective of the invention is to, provide a kind of fermented liquid of brown pore fungus that utilizes to separate preparation compound 2, the method for 4-dihydroxyl-5-methyl-acetophenone (II).
In order to realize above-mentioned task, the present invention adopts following technical solution:
A kind of fermented liquid of brown pore fungus that utilizes separates preparation compound 2, the method of 4-dihydroxyl-5-methyl-acetophenone (II), it is characterized in that, this method adopts the method for plate rolling bottle liquid culture, at first with peeling potato, glucose, distilled water, make substratum through sterilization, the pH value of liquid nutrient medium is 7.2; Cultivation and fermentation gets fermented liquid then, obtains medicinal extract with the ethyl acetate extraction fermented liquid; By the organic solvent lixiviate,, use CHCl through pressurization purification on normal-phase silica gel chromatography
3/ MeOH gradient elution, normal pressure column chromatography adopt wet method dress post and silica gel mixed sample upper prop, select the eluting solvent system according to the thin-layer developing situation, through repeatedly purification on normal-phase silica gel column chromatography, anti-phase C
18Post, Sephadex LH-20 gel filtration chromatography and thin layer preparation separate with recrystallization method, obtain compound 2, the 4-dihydroxyl-pure product of 5-methyl-acetophenone (II).
Specifically undertaken by following step:
1) fermentation of bacterial strain and fermented liquid concentrates
From activated solid plate bacterial classification (28 ℃ are cultivated 5～6d) and get 8～10mm
22～3 accesses of bacterium cake of size are with peeling potato 200g/L, glucose 20g/L, distilled water 1000mL, about pH7.2, in 121 ℃ of liquid PDA substratum (liquid amount is the 50mL/150mL triangular flask) of making of sterilization 30min, in 28 ℃ of rotary shakers (150r/min) cultivate formed the bacterium ball in 3～4 days after with 10% inoculum size transfer in fermentation flask (liquid amount is the 150mL/500mL triangular flask), fermentation 11～13d, obtain fermented liquid 50L altogether, the 50L fermented liquid is reduced by half in 55 ℃ of following concentrating under reduced pressure;
2) preparation of crude extract
Fermented product concentrated solution ethyl acetate extraction, concentrating under reduced pressure obtain ethyl acetate medicinal extract 16g;
3) choosing ethyl acetate medicinal extract is material, by the organic solvent lixiviate, through pressurization purification on normal-phase silica gel chromatography, uses CHCl
3/ MeOH gradient elution, normal pressure column chromatography adopt wet method dress post and silica gel mixed sample upper prop, select the eluting solvent system according to the thin-layer developing situation, through repeatedly purification on normal-phase silica gel column chromatography, anti-phase C
18Post, Sephadex LH-20 gel filtration chromatography and thin layer preparation separate with recrystallization method, obtain pure compound 2,4-dihydroxyl-5-methyl-acetophenone (II).
Method of the present invention has advantages such as culture condition gentleness, the substratum preparation is simple, fermentation time is short.Compound 2 with the present invention's preparation, 4-dihydroxyl-5-methyl-acetophenone (II) is developed to the antibiotic former medicine of anti-plant pathogenic fungi, can suppress the mycelial growth of various plants pathogenic fungi, to cotton-wilt fusarium, corn is bent the spore germ, apple anthrax bacteria has significant inhibitory effect, can solve 2, the raw material sources problem of 4-dihydroxyl-5-methyl-acetophenone.
Description of drawings
Fig. 1 is that brown pore fungus fermented liquid separates preparation compound 2, the schema of 4-dihydroxyl-5-methyl-acetophenone (II);
Fig. 2～Figure 11 is a compound 2, and the structure of 4-dihydroxyl-5-methyl-acetophenone is identified figure, and wherein, Fig. 2 is a Compound I I infrared spectrogram; Fig. 3 is a Compound I I ultraviolet spectrogram; Fig. 4 is Compound I I
1HNMR; Fig. 5 is Compound I I
13CNMR; Fig. 6 is the HMBC of Compound I I; Fig. 7 is the H-HCOSY of Compound I I; Fig. 8 is the HSQC of Compound I I; Fig. 9 is the NOESY of Compound I I; The HPLC color atlas of Compound I I in Figure 10 fermenation raw liquid; The antibacterial graphic representation of Figure 11 Compound I I.
Below in conjunction with accompanying drawing the present invention is described in further detail.
Embodiment
The applicant separates from brown pore fungus (Polyporus picipes) fermented liquid first and has prepared compound 2, and 4-dihydroxyl-5-methyl-acetophenone (II) has also been measured its disease-resistant fungal pathogens activity.EC
50The value proof, this natural product can suppress the mycelial growth of various plants pathogenic fungi, and to cotton-wilt fusarium, corn is bent the spore germ, and apple anthrax bacteria has significant inhibitory effect.
Compound known 2, the chemical structure of 4-dihydroxyl-5-methyl-acetophenone is as follows;
In view of this compound in the fine activity that shows aspect the disease-resistant fungal pathogens, with 2,4-dihydroxyl-5-methyl-acetophenone is developed as the value that the antibiotic bulk drug of disease-resistant fungal pathogens has further investigation.
The applicant gropes to have optimized substratum, the fermentation condition of brown pore fungus (Polyporus picipes) under study for action and separates preparation process, thereby make in this strain fermentating liquid 2,4-dihydroxyl-5-methyl-acetophenone component content improves, and fermentation time shortens (11～13 days).
The fermented liquid of brown pore fungus that utilizes of the present invention separates preparation compound 2, the method for 4-dihydroxyl-5-methyl-acetophenone (II), and the microorganism strains that is utilized is:
Brown pore fungus (Polyporus picipes) derives from Kunming Inst. of Botany, Chinese Academy of Sciences.
Cotton-wilt fusarium (Fusarium oxysporium f.sp.uasinfectum), withered germ of water-melon (Fusarium oxysporium f.sp.niveum), botrytis cinerea (Botrytiscintrea), fusarium graminearum (Gibberella zeae), dry rot of potato bacterium (Fusarium oxysporium), Exserohilum turcicum (Exserohilum turcicum), apple anthrax bacteria (Colletotrichumg loeosporioides), tomato early blight bacterium (Alternaria solani), withered germ of water-melon (Fusarium bulbigenum) is provided by Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology microbe research center.Mentioned microorganism is disclosed biomaterial.
The reagent that the present invention mainly utilizes is:
Amphotericin B available from the gloomy Bioisystech Co., Ltd in Wal, Xi'an, produces article No.: AB0004, the place of production: Amresco.
Sherwood oil (60～90), chloroform, ethyl acetate, acetone, methyl alcohol, formic acid, acetate reagent is analytical pure; Acetonitrile, methanol reagent are chromatographically pure.
Preparation embodiment 1:
This embodiment adopts the method for plate rolling bottle liquid culture, and its concrete steps are as follows:
1) fermentation of bacterial strain and fermented liquid concentrates
From activated solid plate bacterial classification (28 ℃ are cultivated 5～6d) and get 8～10mm
22～3 accesses of bacterium cake of size are with peeling potato 200g/L, glucose 20g/L, distilled water 1000mL, about pH7.2, in 121 ℃ of liquid PDA substratum (liquid amount is the triangular flask of 50mL/150mL) of making of sterilization 30min, in 28 ℃ of rotary shakers (rotating speed: 150r/min), cultivate behind 3～4 days formation bacterium balls with 10% inoculum size transfer in fermentation flask (liquid amount is the 150mL/500mL triangular flask), fermentation 13d obtains fermented liquid 50L altogether.The 50L fermented liquid is reduced by half in 55 ℃ of following concentrating under reduced pressure;
2) preparation of crude extract
Fermented product concentrated solution ethyl acetate extraction, concentrating under reduced pressure obtain ethyl acetate medicinal extract 16g;
3) separation and purification of compound
Referring to Fig. 1, choosing acetic acid ethyl ester extract is material, by organic solvent lixiviate ethyl acetate medicinal extract, through pressurization purification on normal-phase silica gel chromatography, uses CHCl
3/ MeOH gradient elution, normal pressure column chromatography adopt wet method dress post and silica gel mixed sample upper prop, select the eluting solvent system according to the thin-layer developing situation, through repeatedly purification on normal-phase silica gel column chromatography, anti-phase C
18Post, Sephadex LH-20 gel filtration chromatography and recrystallization method separate, and get the crystallization of compound at last.
Silica gel column chromatography separates No. 4 samples.No. 4 sample: dry weight 4.38g;
1. number silicagel column: column chromatography silica gel 120g (200-300 order) mixes sample silica gel 6g (100-200 order).Use chloroform/methanol=98: 2, chloroform/methanol=95: 5 wash-outs successively; Elutriant is that unit receives with 120ml, and underpressure distillation concentrates; Through GF
254The thin-layer silicon offset plate detects, similar merging; Altogether 7 components.Wherein 2,3 components similar crystallization all occurs and merge about 500mg, go up the further separation and purification of Sephadex LH-20 (chloroform/methanol=1: 1) gel then.
2. number gel column: Sephadex LH-20 (chloroform/methanol=1: 1) gel; With chloroform/methanol=1: 1 sample dissolution, sample on the wet method.Chloroform/methanol=1: 1 wash-out is through GF
254Offset plate detects, iodine powder and 5% sulfuric acid ethanol colour developing, similar merging; About 250mg.Behind acetone solution, on the 3. further separation and purification of number silicagel column.
3. number silicagel column: column chromatography silica gel 15g (200-300 order) mixes sample silica gel 0.6g (100-200 order).With chloroform/acetone=100: 1 wash-outs, through GF
254The thin-layer silicon offset plate detects, iodine powder and 5% sulfuric acid ethanol colour developing, similar merging; About 230mg.Behind acetone solution, on the 4. further separation and purification of number silicagel column.Number silicagel column: column chromatography silica gel 15g (200-300 order) mixes sample silica gel 1g (100-200 order).With chloroform/methanol=98: 2 wash-outs, detect iodine powder and 5% sulfuric acid ethanol colour developing, similar merging through GF254 thin-layer silicon offset plate; About 180mg.At last the 180mg crystal is carried out repeatedly recrystallization with chloroform/acetone and obtain white needle-like crystals pure compound 2,4-dihydroxyl-5-methyl-acetophenone (II).
Preparation embodiment 2:
Present embodiment adopts the method for plate rolling bottle liquid culture, and its concrete steps are as follows:
1) fermentation of bacterial strain and fermented liquid concentrates
From activated solid plate bacterial classification (25 ℃ are cultivated 6～7d) and get 6～8mm
23～4 accesses of bacterium cake of size are with peeling potato 180g/L, glucose 18g/L, distilled water 1000mL, about pH7.2, in 121 ℃ of liquid PDA substratum (liquid amount is the triangular flask of 50mL/150mL) of making of sterilization 30min, (rotating speed: 130r/min) cultivation was transferred in fermentation flask (liquid amount is the triangular flask of 130mL/500mL) with 10% inoculum size after forming the bacterium ball in 4～5 days, and fermentation 14d obtains fermented liquid 10L altogether in 25 ℃ of rotary shakers.The 10L fermented liquid is reduced by half in 55 ℃ of following concentrating under reduced pressure;
2) preparation of crude extract
Fermented product concentrated solution ethyl acetate extraction, concentrating under reduced pressure obtain ethyl acetate medicinal extract 2.78g;
3) separation and purification of compound
Silicagel column on the ethyl acetate medicinal extract is separated, with the CHCl of volume ratio 100-85: 0-15
3/ MeOH system gradient elution, every 30ml are a stream part; At CHCl
3/ MeOH (volume ratio 92: 8) wash-out part obtains containing 2, one group of mixture of 4-dihydroxyl-5-methyl-acetophenone through column chromatography repeatedly; This mixture adopts sherwood oil-acetone (volume ratio 10: 1) wash-out to separate, and with the thin layer preparation, uses Sephadex LH-20 gel filtration chromatography (acetone) at last again, obtains compound 2, the 4-dihydroxyl-pure product of 5-methyl-acetophenone (II).
Preparation embodiment 3:
Present embodiment adopts the method for plate rolling bottle liquid culture, and its concrete steps are as follows:
1) fermentation of bacterial strain and fermented liquid concentrates
From activated solid plate bacterial classification (30 ℃ are cultivated 4～5d) and get 6～8mm
22～3 accesses of bacterium cake of size are with peeling potato 250g/L, glucose 25g/L, distilled water 1000mL, about pH7.2,121 ℃ of liquid PDA substratum interior (liquid amount is the triangular flask of 50mL/150mL) that sterilization 30min makes, transfer in fermentation flask (liquid amount is the triangular flask of 170mL/500mL) with 10% inoculum size after 34 ℃ of rotary shakers (170r/min) cultivation formed the bacterium ball in 3～4 days, fermentation 11d obtains fermented liquid 15L altogether.The 15L fermented liquid is reduced by half in 55 ℃ of following concentrating under reduced pressure;
2) preparation of crude extract
Fermented product concentrated solution ethyl acetate extraction, concentrating under reduced pressure obtain ethyl acetate medicinal extract 4.37g;
3) separation and purification of compound
Silicagel column on the ethyl acetate medicinal extract is separated, with the CHCl of volume ratio 100-85: 0-15
3/ MeOH system gradient elution, every 60ml are a stream part; At CHCl
3/ MeOH (volume ratio 93: 7) wash-out part obtains containing 2, one group of mixture of 4-dihydroxyl-5-methyl-acetophenone through column chromatography repeatedly; This mixture adopts chloroform-acetone (volume ratio 99: 1), sherwood oil-acetone (volume ratio 95: 5) wash-out separates, use Sephadex LH-20 gel filtration chromatography (acetone) again, at last with chloroform-acetone repeatedly recrystallization obtain compound 2, the 4-dihydroxyl-pure product of 5-methyl-acetophenone (II).
To gained compound 2 in the example 1,2,3,4-dihydroxyl-pure product of 5-methyl-acetophenone (II) carry out physics and chemistry and POP data analysis respectively, and the result is as follows:
White, needle-shaped crystals (CHCl
3), fusing point 167-168 ℃; EI-MS m/z (%): 166 (54, [M]
+), 167 (11.5), 151 (100), 95 (4), 77 (4), 69 (4.4).Positive TOF-MS-MS-ES:167.0700 (calculated value is 166.0621); Its molecular formula is defined as C
9H
10O
3 1H-NMR (acetone-d
6Ppm/ δ; 500MHz) compose: δ 2.20 (3H, s, H-9); 2.50 (3H, s, H-8); 6.25 (1H, s, H-3); 7.64 (1H, s, H-6); 9.4 (4-OH); 12.6 (2-OH).
13C NMR (ppm/ δ 125 MHz; Acetone-d
6) spectrum demonstration 112.8 (s, C-1), 163.1 (s, C-2), 102.0 (d, C-3), 162.7 (s, C-4), 117.2 (s, C-5), 134.12 (d, C-6), 202.5 (s, C-7), 26.0 (q, C-8), 15.2 (q, C-9).Above data and document [Phytochemistry, 1989,28 (1): 239-242; The chemistry journal, 1990,48,1191-1198] middle record unanimity.Chinese is 2,4-dihydroxyl-5-methyl-acetophenone (II), and structural formula is:
This structure is looked into newly through database, can confirm that this compound is a kind of new natural product.
The HPLC method is measured the content analysis of Compound I I in the fermenation raw liquid of brown pore fungus (Polyporus picipes):
1.1 chromatographic condition
Chromatographic column: employing Agilent zorbax extend C18 (4.6mm * 200mm, 5um); Moving phase: the acetonitrile solution-water of 0.3% formic acid (the acetonitrile solution 50%-100% of 0.3% formic acid, water 50%-0%), gradient elution 1h; Flow velocity: 0.4mL min
-118 ℃ of column temperatures; Detect wavelength: 254nm.
1.2 the preparation of reference substance solution
It is an amount of to get the Compound I I that is dried to constant weight, and accurate the title decides, and is mixed with 100 μ g/mL successively with methyl alcohol, 80 μ g/mL, and 50 μ g/mL, 25 μ g/mL, 10 μ g/mL isoconcentrations are standby.
1.3 the preparation of need testing solution
Fermenation raw liquid is got 50mL be concentrated into driedly, 2mL methyl alcohol ultrasonic dissolution, millipore filtration (0.45 μ m) filter, and then dilute 5 times standby.
1.4 linear relationship is investigated
1.5 compound 2 in the fermented liquid, 4-dihydroxyl-5-methyl-acetophenone (II) assay
Get trial-product, by 1.3 preparation sample test liquids, by 1.1 chromatographic conditions, difference sample introduction reference substance and need testing solution, calculate Compound I I content in the fermented liquid by external standard method, compound 2 in the fermented liquid as a result, 4-dihydroxyl-5-methyl-acetophenone (II) content is 4.696 μ g/mL.
Growth velocity inhibition method is measured compound 2, the virulence and the EC of 4-dihydroxyl-5-methyl-acetophenone (II)
50(μ g/mL)
Table 1. compound 2,4-dihydroxyl-5-methyl-acetophenone is to the inhibiting rate of 9 kinds of pathogenic fungies
Table 2. compound 2, the virulence equation and the EC of 4-dihydroxyl-5-methyl-acetophenone
50(μ g/mL)
| Strain name | Virulence regression equation | EC 50 | EC 90 | R | 95% fiducial limit |
| Cotton-wilt fusarium) F.oxysporium f.sp. uasinfectum) | Y＝1.92+0.6911x | 86.2 | 551 | 0.996343 | 28.234019 |
| Corn is bent the spore germ) Curvularia lunata) Walk.) Boed) | Y＝1.9582+0.7357x | 62.4 | 356 | 0.967523 | 28.234019 |
| Apple anthrax bacteria) Colletotrichumg loeosporioides) | Y＝0.7316+1.0285x | 63.4 | 221 | 0.934719 | 28.234019 |
In sum, the compound 2 of method preparation of the present invention, 4-dihydroxyl-5-methyl-acetophenone (II) can be used in the antibiotic medicinal application of preparation plant pathogenic fungi.
Claims (4)
1, a kind of fermented liquid of brown pore fungus that utilizes separates preparation compound 2, the method of 4-dihydroxyl-5-methyl-acetophenone (II), it is characterized in that, this method adopts the method for plate rolling bottle liquid culture, at first with peeling potato, glucose, distilled water, make substratum through sterilization, the pH value of liquid nutrient medium is 7.2; Cultivation and fermentation gets fermented liquid then, obtains medicinal extract with the ethyl acetate extraction fermented liquid; By the organic solvent lixiviate,, use CHCl through pressurization purification on normal-phase silica gel chromatography
3/ MeOH gradient elution, normal pressure column chromatography adopt wet method dress post and silica gel mixed sample upper prop, select the eluting solvent system according to the thin-layer developing situation, through repeatedly purification on normal-phase silica gel column chromatography, anti-phase C
18Post, Sephadex LH-20 gel filtration chromatography and thin layer preparation separate with recrystallization method, obtain pure compound 2,4-dihydroxyl-5-methyl-acetophenone (II).
2, the method for claim 1 is characterized in that, described compound 2, and the preparation of 4-dihydroxyl-5-methyl-acetophenone (II) is undertaken by following step:
1) fermentation of bacterial strain and fermented liquid concentrates
Get 8～10mm from activated solid plate bacterial classification
22～3 accesses of bacterium cake of size are with peeling potato 200g/L, glucose 20g/L, distilled water 1000mL, pH is 7.2, in the liquid PDA substratum that 121 ℃ of sterilization 30min make, after forming the bacterium ball in 3～4 days, 28 ℃ of rotary shakers cultivations transfer in fermentation flask fermentation 11～13d with 10% inoculum size, obtain fermented liquid 50L altogether, the 50L fermented liquid is reduced by half in 55 ℃ of following concentrating under reduced pressure;
2) preparation of crude extract
Fermented product concentrated solution ethyl acetate extraction, concentrating under reduced pressure obtain ethyl acetate medicinal extract 16g;
3) be material with ethyl acetate medicinal extract,,, use CHCl through pressurization purification on normal-phase silica gel chromatography by the organic solvent lixiviate
3/ MeOH gradient elution, normal pressure column chromatography adopt wet method dress post and silica gel mixed sample upper prop, select the eluting solvent system according to the thin-layer developing situation, through repeatedly purification on normal-phase silica gel column chromatography, anti-phase C
18Post, Sephadex LH-20 gel filtration chromatography and thin layer preparation separate with recrystallization method, obtain compound 2, the 4-dihydroxyl-pure product of 5-methyl-acetophenone (II).
3, method as claimed in claim 1 or 2 is characterized in that, described compound 2, and 4-dihydroxyl-5-methyl-acetophenone (II) is new natural product, its molecular formula is C
9H
10O
3, its chemical structure is as follows:
4, claim 1 or 2 or 3 described compounds 2,4-dihydroxyl-5-methyl-acetophenone (II) is used to prepare the antibiotic medicinal application of plant pathogenic fungi.
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| CN103014078A (en) * | 2012-11-16 | 2013-04-03 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
| CN103203117A (en) * | 2013-03-26 | 2013-07-17 | 国家海洋局第三海洋研究所 | Method for extracting substances in microbial fermenting cultures by pressurized solvent |
| CN103980102A (en) * | 2014-04-29 | 2014-08-13 | 西北农林科技大学 | Synthesis of 2,4-dihydroxy-5-methyl acetophenone and derivative thereof and application of 2,4-dihydroxy-5-methyl acetophenone and derivative thereof as bactericides |
| CN108018323A (en) * | 2018-01-12 | 2018-05-11 | 中国医学科学院药用植物研究所 | A kind of preparation method of adenylosuccinic acid or salt |
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|---|---|---|---|---|
| CN103014078A (en) * | 2012-11-16 | 2013-04-03 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
| CN103014078B (en) * | 2012-11-16 | 2014-09-10 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
| CN103203117A (en) * | 2013-03-26 | 2013-07-17 | 国家海洋局第三海洋研究所 | Method for extracting substances in microbial fermenting cultures by pressurized solvent |
| CN103203117B (en) * | 2013-03-26 | 2015-11-18 | 国家海洋局第三海洋研究所 | A kind of method of material in pressurized liquid extraction fermentable culture |
| CN103980102A (en) * | 2014-04-29 | 2014-08-13 | 西北农林科技大学 | Synthesis of 2,4-dihydroxy-5-methyl acetophenone and derivative thereof and application of 2,4-dihydroxy-5-methyl acetophenone and derivative thereof as bactericides |
| CN103980102B (en) * | 2014-04-29 | 2016-01-27 | 西北农林科技大学 | The synthesis of 2,4-dihydroxyl-5-methyl acetophenone and derivative thereof and the application as sterilant |
| CN108018323A (en) * | 2018-01-12 | 2018-05-11 | 中国医学科学院药用植物研究所 | A kind of preparation method of adenylosuccinic acid or salt |
| CN108018323B (en) * | 2018-01-12 | 2021-04-02 | 中国医学科学院药用植物研究所 | Preparation method of adenylyl succinic acid or salt |
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