CN101565678B - Metarhizium sp. SC0924 strain and method for preparing beta-m-dihydroxybenzoic acid macrolide derivatives by utilizing Metarhizium sp. SC0924 strain - Google Patents
Metarhizium sp. SC0924 strain and method for preparing beta-m-dihydroxybenzoic acid macrolide derivatives by utilizing Metarhizium sp. SC0924 strain Download PDFInfo
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Abstract
The invention relates to Paecilomyces sp. SC0924 CGMCC No.2900. The invention also relates to a method for preparing a compound in a general formula (1) from the Paecilomyces sp. SC0924 CGMCC No.2900, which is characterized in that: firstly, the Paecilomyces sp. SC0924 strain is fermented by a wheat solid medium or a YMG liquid medium to obtain fermentation culture; secondly, an organic solvent is used to extract the fermentation culture to obtain extract; and thirdly, the extract is separated by silicon gel column chromatography or other methods to obtain the compound expressed by the general formula (1).
Description
Technical field
The present invention relates to a kind of fungi, relate in particular to a kind of Paecilomyces varioti Paecilomyces sp.SC0924 fungal bacterial strain, also relate to the method that this bacterial strain of a kind of usefulness prepares β-m-dihydroxy-benzoic acid macrocyclic lactone derivative in addition.
β-m-dihydroxy-benzoic acid macrocyclic lactone (β-Resorcylic acid lactones, RALs) be 14 yuan of rings of the very unique benzo of class formation macrolide, since nineteen fifty-three Delmotte etc. separate first obtain radicicol since, people have altogether to separate from natural product and obtain nearly 30 RALs, they all contain 1,2,4,6 quaternary phenyl ring basically, 1 of phenyl ring is connected with an ester group, 6 link to each other with a long carbochain, and by carbochain 10 ' is connected to form 14 yuan of ring macrolides with 1 carbonyl.9 ' in long carbochain is methylene radical, and 10 ' is connected with methyl, and other position then is connected with oxygen-containing substituents such as hydroxyl, ketone group, epoxy group(ing), or carbon-carbon double bond etc.
In research green muscardine fungus Metarhizium sp.SC0924 meta-bolites and the mould active process of anti-lichee frost epidemic disease thereof, we have also found seven kinds of β-m-dihydroxy-benzoic acid macrocyclic lactone derivatives, be disclosed in the doctorate paper of Xu Liangxiong (seeing ID 10001200418010715012), (this compound has been disclosed in Isaka to the aigialomycin B that is represented by formula (1)-1 comprising structure, et al.J.Org.Chem., 2002,67 (5): 1561-1566):
Formula (1)-1
Structure is by his rhzomorph A (metarhizimycin A) of the wheat of formula (1)-2 expression:
Formula (1)-2
Structure is by his rhzomorph C (metarhizimycin C) of the wheat of formula (1)-3 expression:
Formula (1)-3
Structure is by his rhzomorph D (metarhizimycin D) of the wheat of formula (1)-4 expression:
Formula (1)-4
Structure is by his rhzomorph E (metarhizimycin E) of the wheat of formula (1)-5 expression:
Formula (1)-5
The zeaenol that structure is represented by formula (1)-6 (this compound has been disclosed in Sugawara, et al.Phytochemistry, and 1992,31 (6): 1987-1990):
Formula (1)-6
The aigialomycin D that structure is represented by formula (1)-7 (this compound has been disclosed in Isaka, et al.J.Org.Chem., and 2002,67 (5): 1561-1566):
Formula (1)-7
Summary of the invention
The objective of the invention is to develop a kind of new paecilomyces fungi Paecilomyces varioti Paecilomyces sp.SC0924, also develop the method for preparing these β-m-dihydroxy-benzoic acid macrocyclic lactone derivatives with this fungi in addition.
We separate from the pedotheque of national natural reserves, Dinghu Hill and obtain a kind of Paecilomyces varioti Paecilomyces sp.SC0924 bacterial strain; in addition by this bacterial strain is obtained fermenting culture with wheat solid medium or the fermentation of YMG liquid nutrient medium; then fermenting culture is obtained extract with organic solvent extraction; again extract is separated obtaining β-m-dihydroxy-benzoic acid macrocyclic lactone derivative with methods such as silica gel column chromatographies, thereby realized purpose of the present invention.
Paecilomyces varioti Paecilomyces sp.SC0924 of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 12nd, 2009 and (abbreviates CGMCC as, the address: No. 1 garden Institute of Microorganism, Academia Sinica in BeiChen West Road, Chaoyang District, BeiJing City), preserving number is CGMCC No.2900.This bacterial strain separates acquisition from one pedotheque of national natural reserves, Dinghu Hill; separation method is a dilution-plate method: will gather good pedotheque 1g and change in the aseptic triangular flask; add and to make soil suspension after the 100mL sterilized water stirs 1h, get 1mL suspension be transferred in the test tube that contains the 9mL sterilized water 10
-1Diluent, dilute successively 10
-2, 10
-3, 10
-4Diluent.Draw 1mL 10
-4The soil diluent is to the MEA media surface, with aseptic triangle rod evenly with the coating of soil diluent, put to 25 ℃ of incubators cultivation 7d, with aseptic toothpick picking colony edge, and be transferred to be further purified on the new MEA substratum white filamentous fungus, described MEA substratum contains wort agar extract 33.6g for every liter, and all the other are water, and pH 4.5～4.9.Wide 2～3 μ m of the mycelia of this bacterial strain; Spore is subsphaeroidal, and size is 2.4～4.9 * 2.5～3.7 μ m to oval, and the form of mycelia and spore as depicted in figs. 1 and 2.Mycelium extracts with the CTAB method of classics and obtains DNA, and by with ITS4 (5 '-3 '): TCC TCC GCT TAT TGA TAT GC and ITS5 (5 '-3 '): GGA AGT AAA AGT CGT AAC AAG G is a primer, at 10 * PCR buffer, 5 μ L, Mg
2+(25mM) 4 μ L, dNTPS (10mM) 1 μ L, Primers (10 μ M/each) 2 μ L, Taq enzyme (5U/ μ L) 0.3 μ L, template DNA (10ng/ μ L) 5 μ L, ddH
2In the system of O 32.7 μ L, at 94 ℃ of 5min, 94 ℃ of 40s, 57-52 ℃ of 1min, 72 ℃ of 1min, carry out pcr amplification under 35,72 ℃ of 6min programs of cycles after, see appendix 1:SEQ ID NO.1 through the dna sequence dna table that order-checking obtains.Sequencing result is retrieved with homology with Blast at the Genbank database and is compared, the homology of its ITS sequence homology and Metarhizium flavoviride var.novazealandicum is the highest, be 95%, can only preliminary evaluation be the kind that Metarhizium belongs in conjunction with its preliminary morphological specificity again, tentative green muscardine fungus Metarhizium sp.SC0924 by name.
The present invention is by the preparation method of the β-m-dihydroxy-benzoic acid macrocyclic lactone derivative of following general formula (1) expression,
Wherein compound 1 is aigialomycin B:R
1=OH, R
2=H, R
3=CH
3,
Be singly-bound, R
4And R
5Merge into a Sauerstoffatom ,====be two keys, R
6=OH, R
7=OH; Or compound 2 is his rhzomorph A:R of wheat
1=H, R
2=OH, R
3=CH
3,
Be singly-bound, R
4And R
5Merge into a Sauerstoffatom ,====be two keys, R
6=OH, R
7=OH; Or compound 3 is his rhzomorph C:R of wheat
1=OH, R
2=H, R
3=CH
3,
Be singly-bound, R
4And R
7Merge into a Sauerstoffatom ,====be two keys, R
5=OH, R
6=OH; Or compound 4 is his rhzomorph D:R of wheat
1=OH, R
2=H, R
3=CH
3,
Be two keys, R
4And R
5Do not exist ,====be singly-bound, R
6=OH, R
7=OH; Or compound 5 is his rhzomorph E:R of wheat
1=H, R
2=OH, R
3=CH
3,
Be two keys, R
4And R
5Do not exist ,====be singly-bound, R
6=OH, R
7=OH; Or compound 6 is zeaenol:R
1=OH, R
2=H, R
3=CH
3,
Be singly-bound, R
4And R
5Do not exist ,====be two keys, R
6=OH, R
7=OH; Or compound 7 is aigialomycin D:R
1=H, R
2=OH, R
3=H,
Be two keys, R
4And R
5Do not exist ,====be two keys, R
6=H, R
7=OH, its preparation method comprises the steps:
(1) with Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 on the wheat solid medium, leave standstill cultivation in 24～28 ℃ in the dark, obtain solid fermentation culture, described wheat solid medium is by mass parts, comprise 1 part of wheat, 1.8 parts of YMG liquid nutrient mediums; Or with Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 on the YMG liquid nutrient medium, cultivate in 24～28 ℃ of shaking tables in the dark, obtain the liquid fermentation and culture thing;
(2) the concentrated extractum A that obtains in back is filtered in the solid fermentation culture ethanol lixiviate that step (1) is obtained, or with the liquid fermentation and culture thing alcohol immersion that step (1) obtains, concentrates after filtering and obtain medicinal extract B;
(3) with described extractum A of step (2) or medicinal extract B dissolve with ethanol, with dissolve with ethanol thing petroleum ether extraction, the abandonment petroleum ether extract, after remaining aqueous ethanolic solution removed ethanol and water and refill original volume, with aqueous solution chloroform extraction, isolate chloroform extract and remainder water solution, chloroform extract is concentrated obtain chloroform extract, with remainder water solution ethyl acetate extraction, with the concentrated ethyl acetate extract that obtains of the acetic acid ethyl ester extract that obtains;
(4) silicagel column on the chloroform extract that step (3) is obtained, carry out gradient elution with 95: 5～80: 20 chloroform-methanol mixed solvent of volume ratio, detect the similar stream part of merging with thin-layer chromatography, stream part B of Rf value 0.4～0.7 when the chloroform-methanol volume ratio was launched at 90: 10 on stream part A of Rf value 0.6～0.8 or the thin layer plate when chloroform-methanol volume ratio was launched at 95: 5 on the collection thin layer plate;
To flow a part A and carry out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 85: 15～60: 40 of volume ratio, stream part merging back of collecting Rf value 0.4～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate is the methyl alcohol separation and purification of volume fraction 66% with moving phase at high performance liquid chromatography, obtain the compound 4 of general formula (1) expression, or compound 5; Or
To flow a part B and carry out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 85: 15～60: 40 of volume ratio, the stream part of collecting Rf value 0.3～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate merges the back obtains general formula (1) expression for the solvent recrystallization with methyl alcohol compound 2; Or
With normal pressure reversed-phase column on the methanol mother liquor behind the recrystallization, carry out gradient elution with 2: 1～4: 1 methanol-water mixed solvent of volume ratio, is the methyl alcohol purifying of volume fraction 66% with stream part with moving phase at high performance liquid chromatography, obtain the compound 1 of general formula (1) expression, the compound 7 of the compound 6 of general formula (1) expression or general formula (1) expression; Or
Silicagel column on the ethyl acetate extract that step (3) is obtained, carried out gradient elution in 98: 2～50: 50 with chloroform-methanol mixed solvent volume ratio, detect the similar stream part of merging with thin-layer chromatography, the stream part C that collects Rf value 0.3～0.5 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate carries out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 80: 20～50: 50 of volume ratio, stream part merging back of collecting Rf value 0.3～0.4 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate is the methyl alcohol purifying of volume fraction 34% in high performance liquid chromatography moving phase, obtains the compound 3 of general formula (1) expression.
YMG liquid nutrient medium described in the step (1) is a collective media, pH 5.3～5.7, contain glucose 4g in every liter of substratum, malt extract 10g, yeast extract 4g, all the other are water, the described incubation time that leaves standstill can be 6～12d, and culture temperature can be 24～28 ℃, and the rotating speed that described shaking table is cultivated can be 100～120r/min, culture temperature can be 24～28 ℃, and incubation time can be 3～6d.
The described lixiviate of step (2) preferably 3 times, each 48h, described soak time is 48h preferably.
The described petroleum ether extraction of step (3), the inferior number average of chloroform extraction and ethyl acetate extraction preferably 3 times.
By structural analysis, confirm that also product of the present invention is respectively a structure by the aigialomycin B of general formula (1) expression, his rhzomorph A of wheat, his rhzomorph C of wheat, his rhzomorph D of wheat, his rhzomorph E, zeaenol and aigialomycin D of wheat.Aigialomycin B wherein, colourless needle (MeOH), molecular formula is C
19H
24O
8, 186～189 ℃ of mp, [α]
24D-3.7 ° (c 0.27, MeOH); UV (MeOH) λ
Max(log ε) 220 (4.28), 265 (3.98), 306 (3.65) nm; Positive ion ESIMS m/z 381[M+H]
+, 403[M+Na]
+, 363,345,327,309,265,253,235,207,135; Negative ion ESIMSm/z 379[M-H]
-, 415[M+Cl]
-, 275,231,205,187,179,164,151,135,95,85.
1H NMR and
13(solvent: data deuterated pyridine) see Table 1 to C NMR.His rhzomorph A of wheat, (MeOH: THF=4: 1), molecular formula is C to colourless column crystallization
19H
24O
8, 184～186 ℃ of mp, [α]
24 D-58.5 ° (c 0.27, MeOH); UV (MeOH) λ
Max(log ε) 222 (4.31), 265 (4.05), 306 (3.71) nm; Positive ion ESIMS m/z 381[M+H]
+, 403[M+Na]
+, 363,345,327,309,265,253,235,207,135; Negative ion ESIMS m/z 379[M-H]
-, 415[M+Cl]
-, 335,275,205,187,179,164,151,135.
1H NMR and
13C NMR (solvent: DMSO-d
6) data see Table 2.His rhzomorph C of wheat, pale brown look solid, molecular formula is C
19H
24O
8, [α]
24 D+ 40.4 ° (c0.27, MeOH); UV (MeOH) λ
Max(log ε) 216 (4.04), 283 (3.34) nm; Positive ion ESIMS m/z 403[M+Na]
+Negative ion ESIMS m/z 379[M-H]
-, 415[M+Cl]
-, 759[2M-H]
-, 239,205,189,179,163,151,148,137,122,95,59.
1H NMR and
13(solvent: data deuterated pyridine) see Table 3 to C NMR.His rhzomorph D of wheat, white powder, molecular formula is C
19H
26O
7, 104～106 ℃ of mp, [α]
20 D-96.4 ° (c 0.28, MeOH); UV (MeOH) λ
Max(log ε) 233 (4.31), 271 (3.95), 311 (3.64) nm; Positive ion ESIMS m/z367[M+H]
+, 389[M+Na]
+, 349,331,313,295,219,191,99,81; Negative ion ESIMS m/z365[M-H]
-, 401[M+Cl]
-, 305,277,207,189,187,174,163,148,146,59.
1H and
13(solvent: data deuterochloroform) see Table 4 to C NMR.His rhzomorph E of wheat, white powder, molecular formula is C
19H
26O
7, 175～178 ℃ of mp, [α]
20 D-106.4 ° (c 0.28, MeOH); UV (MeOH) λ
Max(log ε) 235 (4.36), 271 (4.01), 314 (3.70) nm; Positive ion ESIMS m/z 367[M+H]
+, 389[M+Na]
+Negative ion ESIMS m/z 365[M-H]
-, 401[M+Cl]
-, 731[2M-H]
-, 305,277,207,189,187,174,163,148,146,59.
1H NMR and
13(solvent: data deuterochloroform) see Table 5 to C NMR.Zeaenol, colourless needle (MeOH), molecular formula is C
19H
24O
7, 192～195 ℃ of mp, [α]
24D-72.6 ° (c 0.27, MeOH); UV (MeOH) λ
Max(log ε) 235 (4.33), 273 (3.95), 314 (3.63) nm; Positive ion ESIMS m/z 365[M+H]
+, 387[M+Na]
+, 729[2M+H]
+, 751[2M+Na]
+, 347,329,311,283,269,237,229,219,191,175,163; Negative ion ESIMS m/z 363[M-H]
-, 399[M+Cl]
-, 727[2M-H]
-, 763[2M+Cl]
-, 249,216,207,189,174,163,148,122,95,59.
1H NMR and
13(solvent: data deuterated pyridine) see Table 6 to C NMR.Aigialomycin D, colourless needle (MeOH), molecular formula is C
18H
22O
6, 148～152 ℃ of mp, [α]
20 D-26.7 ° (c 0.27, MeOH); UV (MeOH) λ
Max(log ε) 235 (4.37), 274 (4.17), 314 (3.83) nm; Positive ion ESIMS m/z 335[M+H]
+, 357[M+Na]
+Negative ion ESIMS m/z 333[M-H]
-, 369[M+Cl]
-, 315,271,253,217,189,175,161,145,130.
1H NMR and
13(solvent: deuterated acetone) data see Table 7 to C NMR.
Table 1 Aigialomycin B's
1H and
13C NMR data
His rhzomorph A's of table 2 wheat
1H and
13C NMR data
His rhzomorph C's of table 3 wheat
1H and
13C NMR data
His rhzomorph D's of table 4 wheat
1H and
13C NMR data
His rhzomorph E's of table 5 wheat
1H and
13C NMR data
Table 6 Zeaenol's
1H and
13C NMR data
Table 7Aigialomycin D's
1H and
13C NMR data
Description of drawings
Fig. 1: Paecilomyces varioti Paecilomyces sp.SC0924 spore shape.
Fig. 2: Paecilomyces varioti Paecilomyces sp.SC0924 mycelia form.
Embodiment
Following embodiment further specifies of the present invention, is not that the present invention is limited.
Embodiment 1:
Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 bacterial strain on wheat solid medium (1 part of wheat, 1.8 parts of above-mentioned YMG liquid nutrient mediums), is left standstill in 24 ℃ in the dark and cultivates 12d, obtain solid fermentation culture.
With solid fermentation culture (25L) with isopyknic volume fraction 95% alcohol immersion three times, each 48h, be evaporated to medicinal extract shape (630g) after the filtration, with aqueous ethanolic solution (4L) dissolving of medicinal extract with volume fraction 80%, with the dissolve with ethanol thing with isopyknic petroleum ether extraction 3 times, the petroleum ether extract abandonment, ethanol is removed in remaining aqueous ethanolic solution merging, water refills original volume then, with this aqueous solution with the chloroform extraction of equivalent 3 times, isolate chloroform extract and remainder water solution, chloroform extract is merged and the concentrated chloroform extract 19.23g that obtains, with equivalent ethyl acetate extraction 3 times of remainder water solution, the acetic acid ethyl ester extract that obtains is merged and the concentrated ethyl acetate extract 39.31g that obtains.
Chloroform extract 19.00g is gone up silicagel column, and (silica gel is 100～200 orders, 300g), with the chloroform-methanol mixed solvent as eluent, carry out wash-out with 95: 5～80: 20 gradient of volume ratio, detect the similar stream part of merging with thin-layer chromatography (TLC), collect stream part A of Rf value 0.6～0.8 when the chloroform-methanol volume ratio is launched at 95: 5 on the thin layer plate, distillation concentrate 3.40g, stream part B of Rf value 0.4～0.7 when launching at 90: 10 with chloroform-methanol volume ratio on the thin layer plate, distillation concentrate 6.83g, to flow a part A and carry out silica gel column chromatography, (silica gel is 100～200 orders to silica gel, 70g), with sherwood oil-acetone mixed solvent as eluent, carried out gradient elution in 85: 15～60: 40 in volume ratio, the stream part of collecting Rf value 0.4～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate merges the back with high performance liquid chromatography (LC-6AD type half preparative high-performance liquid chromatographic instrument, the RID-10A detector, Japan Shimadzu company produces, moving phase is the methyl alcohol of volume fraction 66%) separation and purification, obtain two compounds of white powder 0.060g and 0.025g, the former is the compound 4 of general formula (1) expression by the said structure analytical proof, promptly be his rhzomorph D of wheat, the latter is a compound 5, promptly is his rhzomorph E of wheat.To flow a part B carries out silica gel column chromatography (silica gel is 100～200 orders, 150g), with sherwood oil-acetone mixed solvent as eluent, at 85: 15～60: 40 gradient elutions of volume ratio, the stream part of collecting Rf value 0.3～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate merges the back gets colourless column crystallization for the solvent recrystallization with methyl alcohol compound 0.320g, by the said structure analytical proof is the compound 2 of general formula (1) expression, promptly is his rhzomorph A of wheat.Normal pressure reversed-phase column on the methanol mother liquor behind the recrystallization, with the methanol-water mixed solvent as eluent, carried out gradient elution in 2: 1～4: 1 in volume ratio, (instrument is the same with high performance liquid chromatography will to flow part, moving phase is the methyl alcohol of volume fraction 66%) purifying, obtain needle crystal first 0.110g, needle crystal second 0.060g and three compounds of needle crystal third 0.091g, by said structure analytical proof needle crystal first is the compound 1 of general formula (1) expression, promptly be aigialomycin B, needle crystal second is the compound 6 of general formula (1) expression, promptly is zeaenol, needle crystal third is the compound 7 of general formula (1) expression, promptly is aigialomycin D.
Aforementioned ethyl acetate extract 39.31g is gone up silicagel column, and (silica gel is 100～200 orders, 800g), with the chloroform-methanol mixed solvent as eluent, carry out wash-out in 98: 2～50: 50 gradient of volume ratio, detect the similar stream part of merging with thin-layer chromatography, collect stream part C of Rf value 0.3～0.5 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate, distillation concentrates to such an extent that 5.56g carries out silica gel column chromatography (silica gel is 100～200 orders, 80g), with sherwood oil-acetone mixed solvent as eluent, carry out wash-out in 80: 20～50: 50 gradient of volume ratio, the stream part of collecting Rf value 0.3～0.4 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate merges the back, and (instrument is the same with high performance liquid chromatography, moving phase is the methyl alcohol of volume fraction 34%) purifying, obtain compound 0.056g, by the said structure analytical proof is the compound 3 of general formula (1) expression, promptly is his rhzomorph C of wheat.
Embodiment 2:
Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 bacterial strain on wheat solid medium (1 part of wheat, 1.8 parts of above-mentioned YMG liquid nutrient mediums), is left standstill in 28 ℃ in the dark and cultivates 6d, obtain solid fermentation culture.
With solid fermentation culture (25L) with isopyknic volume fraction 95% alcohol immersion three times, each 48h, be evaporated to medicinal extract shape (500g) after the filtration, with aqueous ethanolic solution (4L) dissolving of medicinal extract with volume fraction 80%, with the dissolve with ethanol thing with isopyknic petroleum ether extraction 3 times, the petroleum ether extract abandonment, ethanol is removed in remaining aqueous ethanolic solution merging, water refills original volume then, with this aqueous solution with the chloroform extraction of equivalent 3 times, isolate chloroform extract and remainder water solution, chloroform extract is merged and the concentrated chloroform extract 28.60g that obtains, with equivalent ethyl acetate extraction 3 times of remainder water solution, the acetic acid ethyl ester extract that obtains is merged and the concentrated ethyl acetate extract 44.50g that obtains.
Chloroform extract 28.00g is obtained compound 4 (wheat he the rhzomorph D) 0.080g of general formula (1) expression with embodiment 1 same procedure separation and purification, compound 5 (wheat he rhzomorph E) 0.016g, compound 2 (wheat he rhzomorph A) 1.280g, compound 1 (aigialomycin B) 0.180g, compound 6 (zeaenol) 0.076g, compound 7 (aigialomycin D) 0.420g.
Aforesaid ethyl acetate extract 44.00g is obtained compound 3 (wheat he the rhzomorph C) 0.080g of general formula (1) expression with embodiment 1 same procedure separation and purification.
Embodiment 3:
(pH 5.7 with the YMG liquid nutrient medium with Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 bacterial strain, every liter contains glucose 4g, malt extract 10g, yeast extract 4g, all the other are water) place 120r/min on the shaking table, 24 ℃, dark condition is cultivated 6d down, obtains the liquid fermentation and culture thing.
With liquid fermentation and culture thing (20L) with isopyknic volume fraction 95% alcohol immersion 48h, filtrate filtered is evaporated to medicinal extract shape (180g), with aqueous ethanolic solution (1L) dissolving of medicinal extract with volume fraction 80%, with the dissolve with ethanol thing with isopyknic petroleum ether extraction 3 times, the petroleum ether extract abandonment, ethanol is removed in remaining aqueous ethanolic solution merging, water refills original volume then, with this aqueous solution with the chloroform extraction of equivalent 3 times, isolate chloroform extract and remainder water solution, chloroform extract is merged and the concentrated chloroform extract 8.30g that obtains, with equivalent ethyl acetate extraction 3 times of remainder water solution, the acetic acid ethyl ester extract that obtains is merged and the concentrated ethyl acetate extract 14.20g that obtains.
Chloroform extract 8.00g is obtained compound 4 (wheat he the rhzomorph D) 0.080g of general formula (1) expression with embodiment 1 same procedure separation and purification, compound 5 (wheat he rhzomorph E) 0.015g, compound 2 (wheat he rhzomorph A) 0.660g, compound 1 (aigialomycin B) 0.060g, compound 6 (zeaenol) 0.020g, compound 7 (aigialomycin D) 0.031g.
Aforesaid ethyl acetate extract 14.00g is obtained compound 3 (wheat he the rhzomorph C) 0.016g of general formula (1) expression with embodiment 1 same procedure separation and purification.
Embodiment 4:
(pH 5.3 with the YMG liquid nutrient medium with Paecilomyces varioti Paecilomyces sp.SC0924 CGMCC No.2900 bacterial strain, every liter contains glucose 4g, malt extract 10g, yeast extract 4g, all the other are water) place 100r/min on the shaking table, 28 ℃, dark condition is cultivated 3d down, obtains the liquid fermentation and culture thing.
With liquid fermentation and culture thing (20L) with isopyknic volume fraction 95% alcohol immersion 48h, filtrate filtered is evaporated to medicinal extract shape (140g), with aqueous ethanolic solution (1L) dissolving of medicinal extract with volume fraction 80%, with the dissolve with ethanol thing with isopyknic petroleum ether extraction 3 times, the petroleum ether extract abandonment, ethanol is removed in remaining aqueous ethanolic solution merging, water refills original volume then, with this aqueous solution with the chloroform extraction of equivalent 3 times, isolate chloroform extract and remainder water solution, chloroform extract is merged and the concentrated chloroform extract 6.80g that obtains, with equivalent ethyl acetate extraction 3 times of remainder water solution, the acetic acid ethyl ester extract that obtains is merged and the concentrated ethyl acetate extract 10.20g that obtains.
Chloroform extract 6.50g is obtained compound 4 (wheat he the rhzomorph D) 0.040g of general formula (1) expression with embodiment 1 same procedure separation and purification, compound 5 (wheat he rhzomorph E) 0.006g, compound 2 (wheat he rhzomorph A) 0.480g, compound 1 (aigialomycin B) 0.060g, compound 6 (zeaenol) 0.010g, compound 7 (aigialomycin D) 0.020g.
The extract 10.00g of ethyl acetate branch is obtained compound 3 (wheat he the rhzomorph C) 0.010g of general formula (1) expression with embodiment 1 same procedure separation and purification.
Sequence table
＜110〉South China Botanical Garden Chinese Academy of Sciences
＜120〉Paecilomyces varioti SC0924 bacterial strain and utilize it to prepare the method for β-m-dihydroxy-benzoic acid macrocyclic lactone derivative
<160>1
<210>1
<211>585
<212>DNA
＜213〉Paecilomyces varioti Paecilomyces sp.
<400>1
GGATCCTACC?TGATTCGAGG?TCACTTCTAA?AAAAGTTGGG?CGTTTTACGG?CAGTGGCCGC 60
GTCGCGCTCC?TGTTGCGAGG?TTGTGCTACT?ACGCAGAGGA?GGCCGCGACG?AGACCGCCAA 120
TTCATTTCGG?GGGCGGCGCA?ACGCTGCCGA?GGCAGCGAGG?ATCGCCGGTC?CCCAACACCA 180
AACCACGGGG?GCTTGAGGGT?TGAAATGACG?CTCGAACAGG?CATGCCCGCC?AGAATACTGG 240
CGGGCGCAAT?GTGCGTTCAA?AGATTCGATG?ATTCACTGAA?TTCTGCAATT?CACATTACTT 300
ATCGCATTTC?GCTGCGTTCT?TCATCGATGC?CAGAACCAAG?AGATCCGTTG?TTGAAAGTTT 360
TGATTCATTT?GATATGATTC?CACTCAGACA?TGTAAATGTA?AAAAATACAA?GAGTTTAGGT 420
CCCCCGGCGG?GCGCCTGGTT?CCGGGCAGGA?AGAACCTGTT?CCGGGGCGAG?AACCCGCCGA 480
AGCAACGGTA?AAAGGTATAA?GTTCACAGGG?GTTTGGGAGT?TGTAAACTCG?GTAATGATCC 540
CTCCGCTGGT?TCACCAACGG?AGACCTTGTT?ACGCTTTTTA?CTTCA 585
Claims (2)
1. Paecilomyces varioti Paecilomyces sp.SC0924CGMCC No.2900.
2. one kind prepares following formula 1-1 by the described Paecilomyces varioti SC0924 of claim 1, formula 1-2, and formula 1-3, formula 1-4, formula 1-5, the method for the compound that formula 1-6 or formula 1-7 represent,
Formula 1-1 formula 1-2 formula 1-3
Formula 1-4 formula 1-5
Formula 1-6 formula 1-7
, its preparation method comprises the steps:
(1) with Paecilomyces varioti Paecilomyces sp.SC0924CGMCC No.2900 on the wheat solid medium, leave standstill cultivation in 24～28 ℃ in the dark, obtain solid fermentation culture, described wheat solid medium is by mass parts, comprise 1 part of wheat, 1.8 parts of YMG liquid nutrient mediums; Or with Paecilomyces varioti Paecilomyces sp.SC0924CGMCC No.2900 on the YMG liquid nutrient medium, cultivate in 24～28 ℃ of shaking tables in the dark, obtain the liquid fermentation and culture thing; Described YMG liquid nutrient medium is pH 5.3～5.7, contains glucose 4g in every liter of substratum, malt extract 10g, and yeast extract 4g, all the other are water;
(2) the concentrated extractum A that obtains in back is filtered in the solid fermentation culture ethanol lixiviate that step (1) is obtained, or with the liquid fermentation and culture thing alcohol immersion that step (1) obtains, concentrates after filtering and obtain medicinal extract B;
(3) with described extractum A of step (2) or medicinal extract B dissolve with ethanol, with dissolve with ethanol thing petroleum ether extraction, the abandonment petroleum ether extract, after remaining aqueous ethanolic solution removed ethanol and water and refill original volume, with aqueous solution chloroform extraction, isolate chloroform extract and remainder water solution, chloroform extract is concentrated obtain chloroform extract, with remainder water solution ethyl acetate extraction, with the concentrated ethyl acetate extract that obtains of the acetic acid ethyl ester extract that obtains;
(4) silicagel column on the chloroform extract that step (3) is obtained, carry out gradient elution with 95: 5～80: 20 chloroform-methanol mixed solvent of volume ratio, detect the similar stream part of merging with thin-layer chromatography, stream part B of Rf value 0.4～0.7 when the chloroform-methanol volume ratio was launched at 90: 10 on stream part A of Rf value 0.6～0.8 or the thin layer plate when chloroform-methanol volume ratio was launched at 95: 5 on the collection thin layer plate;
To flow a part A and carry out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 85: 15～60: 40 of volume ratio, stream part merging back of collecting Rf value 0.4～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate is the methyl alcohol separation and purification of volume fraction 66% with moving phase at high performance liquid chromatography, the compound that the formula 1-4 of obtaining represents, or the compound represented of formula 1-5; Or
To flow a part B and carry out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 85: 15～60: 40 of volume ratio, stream part merging back of collecting Rf value 0.3～0.5 when sherwood oil-acetone launches at 50: 50 on the thin layer plate obtains the compound that formula 1-2 represents with methyl alcohol for the solvent recrystallization; Or
With normal pressure reversed-phase column on the methanol mother liquor behind the recrystallization, carry out gradient elution with 2: 1～4: 1 methanol-water mixed solvent of volume ratio, is the methyl alcohol purifying of volume fraction 66% with stream part with moving phase at high performance liquid chromatography, the compound that compound that the compound that the formula 1-1 of obtaining represents, formula 1-6 are represented or formula 1-7 represent; Or
Silicagel column on the ethyl acetate extract that step (3) is obtained, carried out gradient elution in 98: 2～50: 50 with chloroform-methanol mixed solvent volume ratio, detect the similar stream part of merging with thin-layer chromatography, the stream part C that collects Rf value 0.3～0.5 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate carries out silica gel column chromatography, carry out gradient elution with sherwood oil-acetone mixed solvent of 80: 20～50: 50 of volume ratio, stream part merging back of collecting Rf value 0.3～0.4 when the chloroform-methanol volume ratio is launched at 90: 10 on the thin layer plate is the methyl alcohol purifying of volume fraction 34% with moving phase at high performance liquid chromatography, the compound that the formula 1-3 of obtaining represents.
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