CN1951907A - Compound and its preparation method and its application in pharmacy - Google Patents
Compound and its preparation method and its application in pharmacy Download PDFInfo
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- CN1951907A CN1951907A CN 200610096784 CN200610096784A CN1951907A CN 1951907 A CN1951907 A CN 1951907A CN 200610096784 CN200610096784 CN 200610096784 CN 200610096784 A CN200610096784 A CN 200610096784A CN 1951907 A CN1951907 A CN 1951907A
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Abstract
The invention discloses a compound and preparing method and application in the drug, which is 2, 3-diamino-6-hydroxy-benzene carbonic acid-2-ethyl group-hexaester fermented by M 206095 strain with excellent antimycotic action to prepare antimycotic drug.
Description
Technical field
The present invention relates to a kind of compound, the invention still further relates to the preparation method of this compound and the application in pharmacy with antifungic action.
Background technology
The root of langdu is the root of malicious platymiscium thymelaeceae Stellera chamaejasme L. (Stellera chamaejasm eL.) or Euphorbiaceae euphorbia fischeriana (Euphorbia fischeriana Stued.), euphorbia ebiacteolata Hayata (Euphorbia ebracteolata Hayata.), having relieves oedema or abdominal distension through diuresis or purgation eliminates the phlegm, the effect of dissipating bind desinsection, is conventional Chinese medicine.Discovering in recent years, root of langdu extract has stronger inhibition activity to tumour cell, and antibiotic in addition, antiviral in addition and insecticidal action is an ideal natural drug exploitation kind.In euphorbia fischeriana (Euphorbia fischeriana Stued.) plant, some endogenetic fungus are arranged, but at present these root of langdu plant endogenesis epiphytes and anti-mattress effective constituent thereof are not seen relevant report.
Summary of the invention
The objective of the invention is provides a kind of compound at the problems referred to above, and this compound anti-mycotic efficiency is better, can adopt euphorbia fischeriana plant endogenesis epiphyte fermentative preparation to obtain.
Another object of the present invention provides the preparation method of this compound.
A further object of the invention provides the application of this compound in pharmacy.
The objective of the invention is to realize by following technical measures:
A kind of compound is 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester (2,3-Diamino-6-hydroxy-benzoic acid 2-ethyl-hexyl ester).
This compound is the white plates crystallization, is dissolved in organic solvents such as acetone, ethanol, ethyl acetate, and is also water-soluble.Its molecular formula C
15H
24N
2O
3, molecular weight M=280.Its structural formula is suc as formula shown in (I).
Be used to prepare the bacterial strain of described compound, its called after aspergillus niger HX839251 (Aspergillus nigerHX839251) that classifies has been preserved in Chinese typical culture collection center (being called for short CCTCC), and deposit number is CCTCCNO:M206095.Preservation date is on September 18th, 2006.
The preparation method of described compound, this preparation method comprises the following steps:
A. fermentation strain employing deposit number is the bacterial strain of CCTCC NO:M206095:
B. strain culturing: fermentation strain is seeded on the solid medium, cultivated 4～8 days for 25～30 ℃, collect the spore of bacterial strain; The spore of bacterial strain is inserted seed culture medium, 150～300rpm, 25～30 ℃, cultivated 1～3 day, get seed liquor; Seed liquor is inoculated in fermention medium by the section of connecing of fermention medium volume 1～20% amount, 150～300rpm, 25～30 ℃ of fermentation culture 5～8 days, collect fermented liquid:
C. tunning extracts: add ethanol in the fermented liquid or ethyl acetate is soaked 12～48h, and centrifugal or remove by filter thalline, supernatant or filtrate are concentrated into an amount of volume, an amount of ethyl acetate extraction 1～3 time, extraction liquid concentrate and promptly get fermented product extract; Perhaps
Fermented liquid is centrifugal or filter thalline is separated, and thalline adds an amount of ethanol or ethyl acetate is soaked 12～48h, soak solution; Supernatant or filtrate add an amount of ethyl acetate extraction 1～3 time, get extraction liquid; Soak solution is centrifugal or remove by filter thalline, merge with extraction liquid, vacuum concentration promptly gets fermented product extract;
D. target product purifying: column chromatography on the fermented product extract, carry out wash-out with chloroform and methanol mixed solution, collect elutriant; The elutriant crystallization purifying, promptly.
Described preparation method, wherein the substratum among the step b is: each component of substratum volume ratio (g/ml) meter by weight in substratum, carbon source 0.5～20%, nitrogenous source 0.5～10%, inorganic salt 0-1%, wherein solid medium need add the agar of 1.5-4%, and all the other are water.Better, each component of substratum volume ratio (g/ml) meter by weight in substratum, carbon source 2～8%, nitrogenous source 0.5～6%, inorganic salt 0-1%, wherein solid medium need add the agar of 1.5-4%, and all the other are water.
Described preparation method, wherein said carbon source are selected from one or more in amylum hydrolysate of the sugar, molasses, glucose, sucrose, starch based, murphy juice, wort, fermented bean drink, potato leaching powder, the oat; Described nitrogenous source is selected from one or more in soybean cake powder, corn steep liquor, peptone, fish peptone, silkworm chrysalis hydrolyzed solution, urea, ammonium sulfate, yeast extract, analysis for soybean powder, cotton seed meal, ammoniacal liquor, the nitrate; Described inorganic salt are selected from one or more in sodium-chlor, magnesium chloride, Manganous chloride tetrahydrate, Repone K, calcium chloride, potassium primary phosphate, ferrous sulfate, the manganous sulfate.
Described preparation method, column chromatography adopts the silica gel G post in its steps d; Crystallization purifying adopts methyl alcohol or acetone recrystallization.
Described preparation method, in its steps d in chloroform and the methanol mixed solution volume ratio of chloroform and methyl alcohol be 80-95%: 20-5%.
A kind of pharmaceutical composition, this pharmaceutical composition is made up of with auxiliary material the described compound (2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester) of treatment significant quantity.
The application of described compound in the preparation antifungal drug.
Beneficial effect of the present invention:
Compound of the present invention is to have antifungic action, show through test the various skin pathomycete, not only Candida albicans (Candida albicans) is had outside the excellent antibiotic activity, other common skin pathomycetes also there is better anti-bacterial effect, its antimicrobial spectrum and effect are better than several external-applied ointments commonly used at present (clotrimazole solution, golden Monistat IV ketoconazole cream, miconazole emulsifiable paste, the upright health emulsifiable paste of Mei Teke skin), can be used for preparing antifungal drug.
Preparation method's cost of the present invention is low, the target compound purity that obtains is higher, HPLC measures purity 〉=98%, by ultimate analysis, infrared spectra, mass spectrum and nuclear-magnetism C spectrum and H spectrum, determine that the gained crystalline compounds is 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester (2,3-Diamino-6-hydroxy-benzoic acid 2-ethyl-hexyl ester), its molecular formula is C
15H
24N
2O
3, molecular weight: M=280.
One, the evaluation of The compounds of this invention:
1. high performance liquid chromatography (HPLC) detects: amino analytical column (phenomenex NH
2Post, 4.6mm * 250mm, 5 μ m); Moving phase: second eyeball: methyl alcohol: 0.05% ammonium acetate solution=15: 5: 80 (volume ratio), flow velocity: 1ml/min, column temperature are room temperature, detect wavelength: λ 207, sample size: 20 μ l.As seen maximum absorption band the results are shown in Figure 1.
2. UV scanning the results are shown in Figure 2.
3. infrared scan: an amount of crystal prototype KBr compressing tablet, determination of infrared spectroscopy, sweep limit 400cm
-1～4000cm
-1Infrared scan the results are shown in Figure 3.
4, mass spectrum: EI-MS (70eV) m/z (%): 279[M-1]
+(10), 167[M-C
8H
17]
+(35), 149[M-C
8H
17-H
2O]
+(100), 113[C
8H
17]
+(9), 104 (5), 83 (5), 71 (17), 57 (18).Collection of illustrative plates is seen Fig. 4.
5, nuclear-magnetism C spectrum and H spectrum: hydrogen spectrum and carbon are composed data and are seen Table 1 and Fig. 5-11:
Table 1 The compounds of this invention
1H and
13C NMR determination data (500MHz, in CD
3OD)
| | C | ||
| 1 2 3 4 5 6 7 | 7.63(1H，dd，J＝5.8，3.4Hz) 7.72(1H，dd，J＝5.7，3.3Hz) | 132.2s 159.0s 158.2s 131.0d 128.5d 159.6s 168.0s |
| 8 9 10 11 12 13 14 15 | 4.22(2H，m) 1.68(1H，m) 1.38(2H，overlapped) 1.35(2H，overlapped) 1.34(2H，overlapped) 0.92(3H，t，J＝7.2Hz) 1.44(2H，dq，J＝6.6，3.4Hz) 0.95(3H，t，J＝7.5Hz) | 67.8t 38.8d 30.2t 28.7t 22.6t 13.0q 23.6t 10.0q |
Annotate: BrukerAM-400 type NMR spectrometer with superconducting magnet, TMS is interior mark.
Two, the anti-microbial effect of The compounds of this invention:
1). the skin pathomycete:
YM2005: Candida albicans Candida albicans;
YM3092: star trichophyton gypseum (sycosis trichophyton) Trichophyton gypseum asteroides;
YM3080: microsporon gypseum Microsporum gypseum;
YM3096: Trichophyton verrucosum Trichophyton verrucosum.
Above skin pathomycete provides by Chinese yunnan province institute of microbiology.
2). prepare for test agent:
The compounds of this invention, appearance character white crystals body, concentration 5mg/ml is used in test, dissolves with sterile distilled water.
Positive control drug: restrain mould side ointment (the accurate word H44023465 of traditional Chinese medicines), content: 15mg/ml; Guangzhou Hejigong Pharmaceutical Factory produces.Gold Monistat IV ketoconazole cream (the accurate word H20043171 of traditional Chinese medicines) content: 20mg/g; Xian-Janssen Pharmaceutical Ltd. produces.Miconazole emulsifiable paste (the accurate word H31022627 of traditional Chinese medicines) content: 20mg/g; Shanghai General Pharmaceutical Co., ltd. produces.The Mei Teke skin founds health emulsifiable paste (cloud is defended and disappeared accurate word (2001) 53-0063 numbers), 20mg/g, (Chinese Kunming Alice's chemical industry articles for use development company produce).Test is 5mg/ml (by main component cubage preparation) with concentration, dissolves with sterile distilled water.
3). experimental technique:
According to national new medicament screen method, antifungal drug also will examine measured matter and whether stop conidia germination except that adopting paper disk method or dilution method to measure drug effect, measure the inhibition zone scope, suppresses the mycelial growth diffusion, or causes mycelia deformity etc.Pathomycete is made bacteria suspension, be added to the sabouraud's agar plate (substratum that pathogenic bacterium are cultivated: glucose 4%, peptone 1%, agar 1.5%, pH 5.6, and is standby after autoclaving or the filtration sterilization) middle coating is evenly, compound is added in the plate that contains indicator, cultivated 24-96 hour for 37 ℃, observation is also measured antibacterial circle diameter (mm) respectively, the results are shown in Table 1.
4). the result
The result of the anti-skin pathomycete of table 1. The compounds of this invention
| Sample | Concentration (mg/ml) | Antibacterial circle diameter (mm) | |||
| YM2005 | YM3096 | YM3080 | YM3092 | ||
| The compounds of this |
5 | 21，23 | 22，20 | 35，27 | 40，40 |
| Clotrimazole ointment | 5 | - | * | 23，21 | 45，37 |
| Ketoconazole |
5 | 17，16 | * | 30，32 | 40，38 |
| The miconazole |
5 | 20，21 | * | 21，17 | 22，22 |
| The Mei Teke skin founds the health emulsifiable paste | 5 | - | * | 31，30 | 37，32 |
Annotate: separating each data with comma in the table is minimum and maximum antibacterial circle diameter (mm) scope."-" expression does not have anti-microbial effect; " * " expression anti-microbial effect is not obvious.
The result shows that the antimicrobial spectrum of The compounds of this invention and effect are better than 4 positive control drugs.Particularly to the inhibition growth effect of Candida albicans (also claiming Candida albicans) Candida albicans, The compounds of this invention obviously is better than the upright health breast of clotrimazole ointment, golden Monistat IV ketoconazole cream, miconazole emulsifiable paste and Mei Teke skin cream drug.
Three, deposit number is the classification evaluation of the fermentation strain of CCTCC NO:M206095
1.1 cultural characteristic
With protecting the good bacterial strain (to call CCTCC NO:M206095 bacterial strain in the following text) that is numbered CCTCC NO:M206095, inoculate PDA liquid nutrient medium (100ml PDA liquid nutrient medium is housed in the 500ml triangular flask) shake flask fermentation with PDA slant medium activation 5 days.The fermentation culture feature: cultivated the 1st day, bacterial classification has odd white hypha point; Cultivated the 2nd day, tiny white bacterium ball is arranged; Cultivated the 3rd day, fermented liquid bacterium ball increases, the mycelium pellet look that is creamy white; Cultivated the 4th day, mycelium pellet is intensive, and fermented liquid is limpid faint yellow, cultivated 5-7 days, and the intensive increase of fermented liquid mycelium pellet, light yellow, the bottle mural margin has black aleurioconidium (as shown in figure 12).
1.2CCTCC the microscopic morphology feature of NO:M206095 bacterial strain.
CCTCC NO:M206095 bacterial strain conidiophore is similar to aspergillus niger Aspergillus niger with conidium microscopic morphology feature, sees Figure 13 and Figure 14.
1.3 molecular biological characteristics
The 18SrDNA series of CCTCC NO:M206095 bacterial strain is made up of 1750 bases (bp), MolecularWeight (kDa): ssDNA:542.12, and the dsDNA sequence is seen SEQ ID No.1.
Based on the 18SrDNA sequential analysis, CCTCC NO:M206095 bacterial strain and aspergillus niger in phylogeny (Aspergillu niger) should be in same branches, and homology is 98.55%.
CCTCC NO:M206095 bacterial strain belongs to (facultative) aerobic small-sized (thread) fungi.Under natural situation, static cultivation can be grown (cultivating as slant strains), and in the situation of aerobic, growth is better, and such as shaking bottle or fermentation (stirring-type, bubbling style, hoisting type etc.), according to cultivating needs, air flow can reach 1: 1.5 (v/v).
Description of drawings
Fig. 1 is the HPLC figure of The compounds of this invention.
Fig. 2 is the UV scanning figure of The compounds of this invention.
Fig. 3 is the infrared scan figure of The compounds of this invention.
Fig. 4 is mass spectrum (ESI-MS) collection of illustrative plates of The compounds of this invention.
Fig. 5 is the nucleus magnetic resonance of The compounds of this invention
1H (
1H-NMR) collection of illustrative plates.
Fig. 6 is the nucleus magnetic resonance of The compounds of this invention
13C (
13C-NMR) collection of illustrative plates.
Fig. 7 is the nucleus magnetic resonance of The compounds of this invention
13C two-dimensional spectrum collection of illustrative plates.
Fig. 8 is the nucleus magnetic resonance of The compounds of this invention
13C two-dimensional spectrum collection of illustrative plates.
Fig. 9 is the nucleus magnetic resonance of The compounds of this invention
13C two-dimensional spectrum collection of illustrative plates.
Figure 10 be The compounds of this invention the nucleus magnetic resonance two-dimensional spectrum (
1H-1HCOSY) collection of illustrative plates.
Figure 11 be The compounds of this invention the nucleus magnetic resonance two-dimensional spectrum (
1H-1HCOSY) collection of illustrative plates.
Figure 12 is a YM38124 cultural characteristic photo.
Figure 13 is a YM38124 bacterial strain conidium optical microscope photograph.
Figure 14 is a YM38124 bacterial strain conidiophore optical microscope photograph.
Bacterial strain preservation situation: be preserved in Chinese typical culture collection center (being called for short CCTCC), deposit number is CCTCC NO:M206095, classification called after aspergillus niger HX839251 (Aspergillus niger HX839251), preservation date is on September 18th, 2006.
Embodiment
The invention will be further elaborated by the following examples, but do not limit the present invention.
Of the present invention 20% murphy juice is meant: get the 200g potato, use 1000ml water, its filtrate is got in well-done filtration, is settled to 1000ml.
1, bacterial strain: it is the bacterial strain of CCTCC NO:M206095 that fermentation strain adopts deposit number.
2, substratum:
Slant medium: 20% murphy juice adds glucose 20g, ammonium nitrate 10g, agar 15g, sodium-chlor 0.1g, natural pH value;
Seed culture medium: 20% murphy juice adds glucose 25g, ammonium nitrate 12g, sodium-chlor 0.1g, natural pH value;
Fermention medium: acetyl starch 20g, glucose 20g, corn steep liquor 15g (solid content is 40%), sodium-chlor 0.1g adds an amount of tap water dissolving, adopts 10%NaOH solution to regulate pH to 6.8-7.0, adds tap water and is settled to 1000ml;
Substratum all adopts 121 ℃ of sterilization 30min.
3, cultural method:
Slant culture: will-70 ℃ the bacterial classification sterilized water dissolved dilution streak inoculation that is CCTCC NO:M206095 of the frozen deposit numbers of low temperature on slant medium, cultivated 7 days for 26 ℃, be that 30% aseptic glycerine washes spore with concentration ,-70 ℃ of preservations are standby.
Seed liquor preparation: will-70 ℃ the frozen bacterial strain spore inoculating of low temperature in seed culture medium (the 100ml seed culture medium is housed in the 500ml triangular flask), 220rpm, 26 ℃, cultivated 2 days, must seed liquor.
Fermentation culture: the 25L fermention medium of packing in the 50L fermentor tank, the seed liquor that accounts for fermention medium volume 10% is inoculated in fermention medium, 220rpm, 26 ℃, cultivated 6～7 days, adopt vegetables oil to carry out froth breaking in the fermenting process, adopt and collect fermented liquid.
4, extraction and purifying:
Add alcohol immersion 48h in the fermented liquid, the centrifugal thalline of removing to an amount of volume, adds ethyl acetate extraction 3 time by 1: 1 volume with supernatant concentration, and extraction liquid suitably concentrates and promptly gets fermented product extract.Silica gel G column chromatography on the fermented product extract, chloroform: methyl alcohol=8: 2 (v/v) wash-out, collect elutriant; Elutriant is evaporated to dried, has needle-like crystal to separate out, and with the repeatedly crystallization of acetone-water system, the gained crystal is purpose product 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester.HPLC measures purity 〉=98%.
Embodiment 2:
1, bacterial strain: it is the bacterial strain of CCTCC NO:M206095 that fermentation strain adopts deposit number;
2, substratum:
Slant medium: glucose 120g, ammonium sulfate 20g, agar 15g adds tap water to 1000ml, natural pH value;
Plant in substratum: glucose 130g, ammonium sulfate 20g adds tap water to 1000ml, natural pH value;
Fermention medium: sucrose 100g, solid content is 40% corn steep liquor 5g, peptone 5g adds tap water, adopts 10%NaOH solution to regulate pH to 6.8-7.0, is settled to 1000ml;
Substratum all adopts 121 ℃ of sterilization 30min.
3, cultural method:
Slant culture: will-70 ℃ the bacterial classification sterilized water dissolved dilution streak inoculation that is CCTCC NO:M206095 of the frozen deposit numbers of low temperature on slant medium, cultivated 5 days for 28 ℃, be that 30% aseptic glycerine washes spore with concentration ,-70 ℃ of preservations are standby.
Seed liquor preparation: will-70 ℃ the frozen bacterial strain spore inoculating of low temperature in seed culture medium (the 100ml seed culture medium is housed in the 500ml triangular flask), 200rpm, 27 ℃, cultivated 2 days, must seed liquor.
Fermentation culture: fermention medium is packed into and is shaken bottle, and the seed liquor that accounts for fermention medium volume 10% is inoculated in fermention medium, and 28 ℃, 250rpm cultivated 6 days, adopts and collects fermented liquid.
4, extraction and purifying:
Add isopyknic 95% ethanol or dehydrated alcohol in the fermented liquid, 24h is soaked in the 200rpm vibration, removes by filter thalline, and filtrate is concentrated into an amount of volume, adds ethyl acetate extraction 3 times by 1: 1 volume, and extraction liquid suitably concentrates and promptly gets fermented product extract.Silica gel G post on the fermented product extract, chloroform: methyl alcohol=95: 5 (v/v) wash-out, collect component of mixture, go up the silica gel G post again, chloroform: methyl alcohol=80: 20 (v/v) wash-out, collect elutriant, recrystallizing methanol 3 times are used in crystallization, and the gained crystal is purpose product 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester, HPLC measures purity 〉=98%.
Embodiment 3:
1, bacterial strain: it is the bacterial strain of CCTCC NO:M206095 that fermentation strain adopts deposit number;
2, substratum:
Slant medium: molasses 40g, soya-bean cake 80g, agar 15g, ferrous sulfate 0.05g, adding distil water be to 1000ml, natural pH value;
Seed culture medium: molasses 40g, soya-bean cake 80g, ferrous sulfate 0.05g, adding distil water be to 1000ml, natural pH value;
Fermention medium: molasses 60g, fish peptone 5g, soya-bean cake 20g, ferrous sulfate 0.05g, adding distil water be to 1000ml, natural pH value;
Substratum all adopts 121 ℃ of sterilization 30min.
3, cultural method:
Slant culture: will-70 ℃ the frozen deposit numbers of low temperature be the streak inoculation of CCTCC NO:M206095 bacterial classification sterilized water dissolved dilution on slant medium, cultivated 5 days for 28 ℃, be that 30% aseptic glycerine washes spore with concentration ,-70 ℃ of preservations are standby.
Seed liquor preparation: will-70 ℃ the frozen bacterial strain spore inoculating of low temperature in seed culture medium (the 100ml seed culture medium is housed in the 500ml triangular flask), 200rpm, 27 ℃, cultivated 2 days, must seed liquor.
Fermentation culture: fermention medium is packed into and is shaken bottle, and the seed liquor that accounts for fermention medium volume 10% is inoculated in fermention medium, and 28 ℃, 250rpm cultivated 6 days, adopts and collects fermented liquid.
4, extraction and purifying:
Filtering fermentation liquor separates thalline, and thalline adds an amount of ethyl acetate and soaks 12～48h, gets soak solution; Filtrate adds an amount of ethyl acetate extraction 1～3 time, gets extraction liquid; Soak solution is removed by filter thalline, merge with extraction liquid, vacuum concentration promptly gets fermented product extract; Silica gel G post on the fermented product extract, chloroform: methyl alcohol=95: 5 (v/v) wash-out, collect component of mixture, go up the silica gel G post again, chloroform: methyl alcohol=80: 20 (v/v) wash-out, collect elutriant, recrystallizing methanol 3 times are used in crystallization, get target product 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester, HPLC measures purity 〉=98%.
Embodiment 4:
1, bacterial strain: it is CCTCC NO:M206095 bacterial strain that fermentation strain adopts deposit number;
2, substratum:
Slant medium: sucrose 200g, soya-bean cake 50g, agar 15g, magnesium chloride 0.01g, calcium chloride 0.01g, potassium primary phosphate 0.05g adds tap water to 1000ml, natural pH value;
Seed culture medium: sucrose 200g, soya-bean cake 50g, magnesium chloride 0.01g, calcium chloride 0.01g, potassium primary phosphate 0.05g adds tap water to 1000ml, natural pH value;
Fermention medium: potato leaches powder 50g, sucrose 30g, and glucose 20g, peptone 40g, magnesium chloride 0.01g, calcium chloride 0.01g, potassium primary phosphate 0.05g adds tap water, adopts 10%NaOH solution to regulate pH to 6.8-7.0, is settled to 1000ml;
Substratum all adopts 121 ℃ of sterilization 30min.
3, cultural method:
Slant culture: will-70 ℃ the frozen deposit numbers of low temperature be the streak inoculation of CCTCC NO:M206095 bacterial classification sterilized water dissolved dilution on slant medium, cultivated 4 days for 30 ℃, be that 30% aseptic glycerine washes spore with concentration ,-70 ℃ of preservations are standby.
Seed liquor preparation: will-70 ℃ the frozen bacterial strain spore inoculating of low temperature in seed culture medium (the 100ml seed culture medium is housed in the 500ml triangular flask), 250rpm, 28 ℃, cultivated 1 day, must seed liquor.
Fermentation culture: fermention medium is packed into and is shaken bottle, and the seed liquor that accounts for fermention medium volume 20% is inoculated in fermention medium, and 30 ℃, 280rpm cultivated 4 days, adopts and collects fermented liquid.
4, extraction and purifying:
Fermented liquid centrifugation thalline, thalline add an amount of ethyl acetate and soak 24h, get soak solution; Supernatant adds an amount of ethyl acetate (volume ratio 1: 1) extraction 2 times, gets extraction liquid; With the centrifugal thalline of removing of soak solution, merge soak solution and extraction liquid, vacuum concentration is to proper volume, last silica gel G post, chloroform: methyl alcohol=95: 5 (v/v) wash-out, collect component of mixture, go up the silica gel G post again, chloroform: methyl alcohol=8: 2 (v/v) wash-out, collect elutriant, crystallization, with acetone recrystallization 3 times, get target compound 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester, HPLC measures purity 〉=98%.
Take by weighing oil phase stearyl alcohol 20g, white vaseline 22g, whiteruss 15g, container are heated to 75-85 ℃ of thawing, are oil phase; Other gets the The compounds of this invention 0.26g by embodiment 1 preparation, with 85 ℃ of left and right sides hot water 200ml dissolvings, adds sodium laurylsulfate 2g, ethyl p-hydroxybenzoate 0.2g, and glycerine 1g, constant temperature stirs evenly for 80 ℃, is the pastille water; The pastille water is added in the oil phase with the thread shape, and the limit edged is stirred to the emulsifiable paste shape, promptly.
Embodiment 6 gelifying agents
Get Carbopol-940 (acrylate copolymer) 3g and be scattered in the 70g50 ℃ of water, after the placement swelling, transfer PH6.5-7.5, get gel with trolamine; Other gets about 85 ℃ of water 10g with 0.01g raw material (The compounds of this invention of embodiment 2 preparations) dissolving, and with ethyl p-hydroxybenzoate 0.8g, propylene glycol 15g, azone 3g adds in the gel jointly, and mixing and stirring promptly.
Sequence table
＜110〉Hangzhou Xiayang Bioengineering Co., Ltd.
＜120〉a kind of compound and preparation method thereof and the application in pharmacy
<160>1
<210>1
<211>1750
<212>DNA
＜213〉native sequences comes from aspergillus niger (Aspergillu niger)
<400>1
ctggttgatt ctgccagtag tcatatgctt gtctcaaaga ttaagccatg catgtctaag 60
tataagcact ttatactgtg aaactgcgaa tggctcatta aatcagttat cgtttatttg 120
atagtacctt actacatgga tacctgtggt aattctagag ctaatacatg ctgaaaacct 180
cgacttcgga aggggtgtat ttattagata aaaaaccaat gcccttcggg gctccttggt 240
gaatcataat aacttaacga atcgcatggc cttgcgccgg cgatggttca ttcaaatttc 300
tgccctatca actttcgatg gtaggatagt ggcctaccat ggtggcaacg ggtaacgggg 360
aattagggtt cgattccgga gagggagcct gagaaacggc taccacatcc aaggaaggca 420
gcaggcgcgc aaattaccca atcccgacac ggggaggtag tgacaataaa tactgatacg 480
gggctctttt gggtctcgta attggaatga gtacaatcta aatcccttaa cgaggatcaa 540
ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag cgtatattaa 600
agttgttgca gttaaaaagc tcgtagttga accttgggtc tggctggccg gtccgcctca 660
ccgcgagtac tggtccggct ggacctttcc ttctggggaa tctcatggcc ttcactggct 720
gtggggggaa ccaggacttt tactgtgaaa aattagagtg ttcaaagcag gcctttgctc 780
gaatacatta gcatgcatat agatacgacg tgcggttcta ttttgttggt ttctaggacg 840
cgtatgatat aggatagtcg ggggcgtcag tattcagctg tcagaggtga aattctagat 900
ttgctgaaga ctaactactg cgaaagcatt cgccaaggat gttttcatta atcagggaac 960
gaaagttagg ggatcgaaga cgatcagata ccgtcgtagt cttaaccata aactatgccg 1020
actagggatc ggacggtgtt tctattatga cccgttcggc accttacgag aaatcaaagt 1080
ttttgggttc tggggggagt atggtcgcaa ggctgaaact taaagaaatt gacggaaggg 1140
caccaccagg cgtggagcct gcggcttaat ttgactcaac acggggaaac tcaccaggtc 1200
cagacaaaat aaggattgac agattgagag ctctttcttg atcttttgga tggtggtgca 1260
tggccgttct tagttggtgg agtgatttgt ctgcttaatt gcgataacga acgagacctc 1320
ggcccttaaa tagcccggtc cgcatttgcg ggccgctggc ttcttagggg gactatcggc 1380
tcaagccgat ggaagtgcgc ggcaataaca ggtctgtgat gcccttagat gttctgggcc 1440
gcacgcgcgc tacactgaca gggccagcga gtacatcacc ttggccgaga ggtctgggta 1500
atcttgttaa accctgtcgt gctggggata gagcattgca attattgctc ttcaacgagg 1560
aatgcctagt aggcacgagt catcagctcg tgccgattac gtccctgccc tttgtacaca 1620
ccgcccgtcg ctactaccga ttgaatggct cggtgaggcc ttcggactgg ctcaggaggg 1680
ttggcaacga ccccccagag ccggaaagtt ggtcaaaccc ggtcatttag aggaagtaaa 1740
agtcgtaaca 1750
Claims (10)
1, a kind of compound is 2,3-diamino-6-hydroxy-benzoic acid-2-ethyl-own ester.
2, be used to prepare the bacterial strain of the described compound of claim 1, its called after aspergillus niger HX839251 (Aspergillus niger HX839251) that classifies has been preserved in Chinese typical culture collection center, and deposit number is CCTCCNO:M 206095.
3, the preparation method of the described compound of claim 1 is characterized in that this preparation method comprises the following steps:
A. fermentation strain employing deposit number is the bacterial strain of CCTCC NO:M 206095;
B. strain culturing: fermentation strain is seeded on the solid medium, cultivated 4～8 days for 25～30 ℃, collect the spore of bacterial strain; The spore of bacterial strain is inserted seed culture medium, 150～300rpm, 25～30 ℃, cultivated 1～3 day, get seed liquor; Seed liquor is inoculated in fermention medium by the inoculum size of fermention medium volume 1～20%, 150～300rpm, 25～30 ℃ of fermentation culture 5～8 days are collected fermented liquid;
C. tunning extracts: add ethanol in the fermented liquid or ethyl acetate is soaked 12～48h, and centrifugal or remove by filter thalline, supernatant or filtrate are concentrated into an amount of volume, an amount of ethyl acetate extraction 1～3 time, extraction liquid concentrate and promptly get fermented product extract; Perhaps
Fermented liquid is centrifugal or filter thalline is separated, and thalline adds an amount of ethanol or ethyl acetate is soaked 12～48h, soak solution; Supernatant or filtrate add an amount of ethyl acetate extraction 1～3 time, get extraction liquid; Soak solution is centrifugal or remove by filter thalline, merge with extraction liquid, vacuum concentration promptly gets fermented product extract;
D. target product purifying: column chromatography on the fermented product extract, carry out wash-out with chloroform and methanol mixed solution, collect elutriant; The elutriant crystallization purifying, promptly.
4, preparation method according to claim 3, it is characterized in that the substratum among the step b is: each component of substratum volume ratio (g/ml) meter by weight in substratum, carbon source 0.5～20%, nitrogenous source 0.5～10%, inorganic salt 0-1%, wherein solid medium need add the agar of 1.5-4%, and all the other are water.
5. preparation method according to claim 4 is characterized by each component of substratum volume ratio (g/ml) meter by weight in substratum, carbon source 2～8%, nitrogenous source 0.5～6%, inorganic salt 0-1%, wherein solid medium need add the agar of 1.5-4%, and all the other are water.
6. according to claim 4 or 5 described preparation methods, it is characterized in that described carbon source is selected from one or more in amylum hydrolysate of the sugar, molasses, glucose, sucrose, starch based, murphy juice, wort, fermented bean drink, potato leaching powder, the oat; Described nitrogenous source is selected from one or more in soybean cake powder, corn steep liquor, peptone, fish peptone, silkworm chrysalis hydrolyzed solution, urea, ammonium sulfate, yeast extract, analysis for soybean powder, cotton seed meal, ammoniacal liquor, the nitrate; Described inorganic salt are selected from one or more in sodium-chlor, magnesium chloride, Manganous chloride tetrahydrate, Repone K, calcium chloride, potassium primary phosphate, ferrous sulfate, the manganous sulfate.
7. preparation method according to claim 3 is characterized in that column chromatography adopts the silica gel G post in the steps d; Crystallization purifying adopts methyl alcohol or acetone recrystallization.
8, preparation method according to claim 3 is characterized in that in the steps d that the volume ratio of chloroform and methyl alcohol is 80-95%: 20-5% in the chloroform and methanol mixed solution.
9, a kind of pharmaceutical composition, it is characterized in that this pharmaceutical composition by the treatment significant quantity described compound of claim 1 and auxiliary material form.
10, the application of the described compound of claim 1 in the preparation antifungal drug.
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| CN112521296A (en) * | 2021-02-05 | 2021-03-19 | 武汉柔显科技股份有限公司 | Diamine compound, heat-resistant resin or heat-resistant resin precursor using same, photosensitive resin composition, cured film, and display device |
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| CN112521296A (en) * | 2021-02-05 | 2021-03-19 | 武汉柔显科技股份有限公司 | Diamine compound, heat-resistant resin or heat-resistant resin precursor using same, photosensitive resin composition, cured film, and display device |
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