CN108033877B - MRSA-resistant naphthalene compound and preparation method thereof - Google Patents

MRSA-resistant naphthalene compound and preparation method thereof Download PDF

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CN108033877B
CN108033877B CN201711067146.8A CN201711067146A CN108033877B CN 108033877 B CN108033877 B CN 108033877B CN 201711067146 A CN201711067146 A CN 201711067146A CN 108033877 B CN108033877 B CN 108033877B
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杨海英
杜刚
胡秋芬
胡秋月
刘赟
詹梦涛
陆小凯
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Yunnan Minzu University
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Abstract

The invention discloses an MRSA resistant naphthalene compound and a preparation method thereof. The molecular formula of the naphthalene compound is C18H18O4This compound was named naphallide B. The preparation method is to take a rice solid fermentation product of the phomopsis strain CGMCC M2017632 as a raw material, and separate the raw material by the steps of extract extraction, silica gel column chromatography, high pressure liquid chromatography and the like. The application is the application of the naphalde B in preparing the methicillin-resistant staphylococcus aureus resistant medicament. Determining the activity and MIC of naphthalide B on staphylococcus aureus standard strain ATCC25923 and methicillin-resistant staphylococcus aureus standard strain ATCC43300 by a broth microdilution method50Respectively reach 4 mug/ml and 16 mug/ml. The compound has simple structure and good activity, can be used as a lead compound for resisting methicillin-resistant staphylococcus aureus, and has good application prospect.

Description

MRSA-resistant naphthalene compound and preparation method thereof
Technical Field
The invention belongs to the technical field of microbial secondary metabolites, and particularly relates to an MRSA (methicillin resistant Staphylococcus) resistant naphthalene compound and a preparation method and application thereof.
Background
With the long-term, non-standard use of antibiotics, drug-resistant bacteria have become a global problem; the most serious of them is methicillin-resistant staphylococcus aureus (MRSA), which is a growing nosocomial infection; klevens et al reported that more than 9 million people were expected to be infected with this deadly super pathogen in the United states each year in 2005, with nearly 1.9 million people dying, with a 2-fold increase in this data over the U.S. centers for disease control and prevention (CDC) report in 2001. MRSA has great treatment difficulty and high fatality rate, is combined with hepatitis B and AIDS as three infectious diseases in the world, and becomes one of the global public health problems. China is one of the most serious countries in the world with antibiotics abuse, in 1978, 200 strains of staphylococcus aureus are spot-tested in Shanghai by medical staff, and the isolated MRSA is less than 5%. A sampling survey of hospitalized patients in 2009 showed that methicillin-resistant staphylococcus aureus (MRSA) was over 60%, increasing by 40% compared to 2000. The DNA of the intestinal flora of Chinese, Denmark and Spain was sequenced by Zhu Bao Li and co-workers at the institute of microbiology, university of Chinese medical science, and the study showed that: chinese intestinal flora has more drug-resistant genes. Therefore, some experts believe that once the true sense of "superbacteria" outbreak, china will likely become a serious disaster area for "superbacteria". Therefore, the research and development of new strategies and new drugs capable of effectively controlling drug-resistant bacterial infection become a hotspot of antibiotic research.
In recent years, the focus of research has turned back to the search for new secondary metabolites of the skeleton from microorganisms with different antibacterial mechanisms, and endophytic fungal metabolites are the most attractive hot spots in this field, among which the groups most frequently producing antibacterial active compounds are Phomopsis (Phomopsis), Phoma (Phoma) and Fusarium (Fusarium). The phomopsis is a large genus of fungi of Deuteromycotina and coelomycetes, and the strain can produce rich secondary metabolite, and Weber and the like are separated from erythrina cristataPhomopsis spThe obtained lactone compound phomol has good antibacterial activity on 24 bacteria and fungi. Horn et al isolated from Salix integraPhomopsis sp.The strain and the separated cytochalasin alkaloid phomopsichalasin can inhibit staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, staphylococcus aureus and escherichia coli.
The invention relates to a phomopsis (A) and B)Phomopsis sp.) In the fermentation productA new naphthalene compound naphthalide B is obtained by separation, and the compound has remarkable MRSA (methicillin resistant Staphylococcus aureus) resistance activity.
Disclosure of Invention
The first purpose of the invention is to provide a naphthalide B; the second aim is to provide a fermentation method of the used patent strain; the third purpose is to provide a preparation method of the naphthalide B; the fourth purpose is to provide the application of the naphthalide B in preparing the anti-gram-positive bacteria medicament, in particular the application in resisting methicillin-resistant staphylococcus aureus.
The first purpose of the invention is realized by that the naphthalene compound is separated from phomopsis, named naphthalide B, and the molecular formula of the naphthalene compound is C18H18O4Has the following structure:
Figure RE-DEST_PATH_IMAGE002
the second purpose of the invention is realized in such a way that the fermented microorganism provided by the invention is a Phomopsis Fukushii (Phomopsis fukushii) strain preserved by the common microorganism center of China Committee for culture Collection of microorganisms, CCTCC M2017632, and the preservation date is 2017, 10 and 23 days.
The molecular identification characteristics of the phomopsis Fuji strain CCTCC M2017632 are as follows: the sequencing result is subjected to homology comparison in GenBank, a sequence with the similarity of more than 98 percent is extracted, MEGA6.0 software is utilized, a phylogenetic tree, a bacterial strain and phomopsis viticola are constructed by a domain joining method (A), (B), (CPhomopsis viticola) The homology rates were all 97.2%, and were identified as Phomopsis fukushii. The fermentation by using the Phomopsis fulvidrosis (Phomopsis fukushii) strain CCTCC M2017632 comprises the following steps:
(1) placing Phomopsis fukushii strain CCTCC M2017632 in a slant solid culture medium for storage, wherein the slant solid culture medium comprises 200 g/L g of potato, 20 g/L g of glucose and 20 g/L g of agar, the pH value is 6.5, the slant solid culture medium is sterilized at 121 ℃ for 25min, and the slant solid culture medium is cooled to 60 ℃ for storage;
(2) transferring a stored Phomopsis fusco (Phomopsis fukushii) strain CCTCC M2017632 into a liquid seed culture medium, and culturing at 28 ℃ for 24h on a shaker at the rotating speed of 180r/min to obtain a Phomopsis fusco seed liquid; the liquid seed culture medium is as follows: NaNO32g/L，K2HPO41g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H2O0.01 g/L, glucose 20 g/L, pH 6.5, placing into a 250m L conical flask, 100m L/flask, sterilizing at 121 deg.C for 25 min;
(3) and (3) inoculating the phomopsis longipes seed liquid obtained in the step (2) into a rice solid culture medium according to the mass ratio of 2% of the culture medium for solid fermentation under the conditions of 25-32 ℃ and 30-60 days of fermentation, wherein the rice culture medium comprises 100g of rice, 20g of perlite and 100ml of distilled water, putting into a 600m L tissue culture bottle, and sterilizing at 121 ℃ for 25min for later use.
The third purpose of the invention is realized in such a way that the preparation method of the naphthalide B comprises the steps of fermentation product treatment, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation and the like, and specifically comprises the following steps:
(1) organic solvent extraction: ultrasonically extracting for 2-4 times with 60-80% ethanol as solvent for 4-8 h each time, mixing the extractive solutions, filtering, and concentrating under reduced pressure to obtain extract;
(2) performing silica gel column chromatography, namely dissolving the extract by using pure methanol with the weight ratio of 1.5-3 times, mixing the extract by using 200-mesh silica gel with the weight ratio of 1-3 times of the extract, loading the mixture on a silica gel column, performing gradient washing by using a chloroform-methanol solution with the volume ratio of 1: 0-0: 1, respectively collecting and concentrating the eluates of each part, and monitoring and combining the same parts by using T L C;
(3) high pressure liquid chromatography separation, wherein the eluent in the 7:3 part in the step (2) is taken, 40-50% methanol is used as a mobile phase, and the specification is 20mm × 250mm and 5 mu m C18The preparation column is a stationary phase, the flow rate is 20ml/min, the detection wavelength of an ultraviolet detector is 238nm, 200 mu L is injected each time, eluent is collected, and the naphthalide B is obtained after multiple accumulation and evaporation to dryness.
The structure of naphthalide B prepared by the above method was determined by the following method:
the compounds of the invention are red gums; ultraviolet spectrum (the solvent is methanol),λ max(log) 348 (3.68),235 (3.22), 205 (3.96) nm; infrared spectrum (Potassium bromide tablet)ν max: 3357, 3079, 2931, 2749,1697, 1682, 1586, 1536, 1428, 1183, 1076, 859, 768cm-1(ii) a HRESIMS shows the molecular ion peak M/z 321.1108 [ M + Na ] of the compound of the invention plus sodium]+（C18H18NaO4Calculated 321.1103), combined1H and13the CNMR spectra (FIGS. 2 and 3, data attribution in Table-1) give the molecular formula C18H18O4。
Process for preparing compounds1H and13c NMR spectrum shows that the compound has 18 carbon signals and 17 hydrogen signals, including 1,2,5,7-4 substituted naphthalene ring signals [ C-1 (C-1)C126.9 s），C-2（C146.7 s），C-3（C125.3 d），C-4（C128.2 d），C-5（C155.5 s），C-6（C108.9 d），C-7（C138.2 s），C-8（C116.5 d），C-9（C131.5 s），C-10（C124.3 s）; H-3（H7.42 d，J=8.2）， H-4（H8.33 d，J=8.2），H-6（H6.87 d，J=1.6），H-8（H8.50 d，J=1.6）]A formaldehyde group substitution signal: (C191.5 d;H10.01s), a methoxy signal (A) ((II)C56.9 q;H3.82 s), a hydroxymethyl signal: (C63.6 t;H4.63 s), a 3-methyl-2-carbonyl-3-butenyl group [ C-3 '(C-3')C43.2 t），C-4'（C201.9 s），C-5'（C144.7 s），C-6'（C123.1 t），C-7'（C16.7 q）；H2-3'（H4.49 s），H2-6'（H6.12, 5.86 s），H3-7'（H2.09 s）]. The infrared spectrum shows a carbonyl group (1697, 1682 cm)-1) And phenyl (1586, 1536, 1428 cm-1) The absorption peak of (1). The UV spectrum absorbs at 348, 235 and 205 nm indicating the presence of an extended chromophore and aromatic rings. H-3 in the HMBC spectrum (7.42 d,J=8.2) and C-1 (C126.9 s）/ C-2（C146.7 s）/C-4（C128.2 d）/C-10（C124.3 s），H-4（8.33 d，J=8.2) and C-2 (C146.7 s）/C-3（C125.3 d）/C-6（C108.9 d）/C-7（C138.2 s）/C-8（C116.5 d）/C-10（C124.3 s）, H-6（6.87 d，J= 1.6) and C-5 (C-5: (C-5) (C-1)C155.5 s）/C-7（C138.2 s）/C-8（C116.5 d）/C-10（C124.3s）, H-8（8.50 d，J= 1.6) and C-1 (C: (C-1)C126.9 s）/C-6（C108.9 d）/C-7（C138.2 s）/C-9（C131.5 s）/C-10（C124.3 s), the structure of the 4-membered substituted naphthalene ring in the compound is confirmed. H-1' ()H10.01) and C-1 (C126.9)/C-2(C146.7)/ C-9 (C131.5) correlation, confirming the attachment of the aldehyde group at the C-1 position, H2-2′(H4.63) and C-1 (C: (1:)C126.9)/C-2 (C146.7)/C-3 (C125.3) confirming the attachment of the hydroxymethyl group in the C-2 position, methoxy OMe (C) ((iii)H3.82) and C-5 (C155.5) correlation, confirming that the methoxy group is attached at the C-5 position, H2-3′ (H4.49) and C-6 (C108.9)/C-7 (C138.2)/ C-8 (C116.5)，H-6 (H6.87) and C-3' ((III)C43.7)，H-8 (H8.50) and C-3' ((II)C43.2) correlation, confirming the 3-methyl-2-carbonyl-3-butenyl group attached at the C-7 position. The structure of the compound was thus confirmed.
TABLE-1H NMR and 13C NMR data for the compound (solvent C5D5N)
Atom(s) δ C（mult） δ H(mult, J, Hz)
1 126.9 s
2 146.7 s
3 125.3 d 7.42 d (8.2)
4 128.2 d 8.33 d (8.2)
5 155.5 s
6 108.9 d 6.87 d (1.6)
7 138.2 s
8 116.5 d 8.50 d (1.6)
9 131.5 s
10 124.3 s
1′ 191.5 d 10.01 s
2′ 63.6 t 4.63 s
3′ 43.2 t 4.49 s
4′ 201.9 s
5′ 144.7 s
6′ 123.1 t 6.12, 5.86s
7′ 16.7 q 2.09 s
-OMe 56.9 q 3.82 s
The fourth aim of the invention is achieved by the use of said naphthalide B for the preparation of a medicament against gram-positive bacteria, in particular against methicillin-resistant Staphylococcus aureus.
The naphthalide B is separated for the first time, and the molecular formula and the structure of the naphthalide B are determined through nuclear magnetic resonance, mass spectrum, infrared spectrum and ultraviolet spectrum data. A trace broth dilution method is used for testing the anti-staphylococcus aureus standard strain ATCC25923 and the methicillin-resistant staphylococcus aureus standard strain ATCC43300 and the activity of the naphthotide A, the MIC of the anti-staphylococcus aureus standard strain ATCC25923 of the naphthotide A is 8 mu g/ml, and the MIC of the anti-methicillin-resistant staphylococcus aureus standard strain ATCC43300 is 16 mu g/ml, so that the compound has better anti-methicillin-resistant staphylococcus aureus activity and can be used as a lead compound for resisting methicillin-resistant staphylococcus aureus.
Drawings
FIG. 1 shows the NMR spectra of the compounds of the present invention: (1H NMR) pattern;
FIG. 2 shows NMR spectra of compounds of the present invention: (13C NMR) pattern;
FIG. 3 is a graph relating to HMBC of the compounds of the present invention;
FIG. 4 shows the chemical structure of the compound naphallide B of the present invention.
Description of preservation of biological Material
The strain of the invention is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 10 months and 23 days, wherein the center addresses are as follows: in Wuhan university school of eight-channel 299 # in Wuchang district of Wuhan city, Hubei province of China, the strain is classified and named as: phomopsis Fukushii (Phomopsis fukushii) with the preservation number of CCTCC M2017632.
Detailed Description
The present invention is further illustrated by the following figures and examples, but is not limited thereto in any way, and any variations or modifications based on the teachings of the present invention are within the scope of the present invention.
The naphthalide B is separated from a fermentation product of phomopsis CCTCC M2017632, and the molecular formula of the naphthalide B is C18H18O4，
Figure RE-270214DEST_PATH_IMAGE002
The compound was named: naphthaldehyde B (5-Methoxy-2-methyl-7- (3-methyl-2-oxobout-3-enyl) -1-naphthaldehyde). The preparation method of the naphthalene compound naphthalide B comprises the steps of solid fermentation of Phomopsis fukushii strain, ultrasonic extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:
(1) placing a Phomopsis fuskushii strain CCTCC M2017632 in a slant solid culture medium for storage and standby, transferring the stored standby strain into a liquid seed culture medium, and culturing for 24h at 28 ℃ on a shaker at the rotating speed of 180r/min to obtain a Phomopsis seed solution; and (3) taking the phomopsis seed liquid, inoculating the phomopsis seed liquid into a rice solid culture medium according to the mass ratio of 2-5% of the culture medium, and fermenting for 30-60 days at the temperature of 25-35 ℃ to obtain a fermentation product.
(2) Ultrasonic extraction: ultrasonically extracting for 2-4 times with 60-80% ethanol as solvent for 4-8 h each time, mixing the extractive solutions, filtering, and concentrating under reduced pressure to obtain extract;
(3) silica gel column chromatography, namely dissolving the extract by using pure methanol with the weight ratio of 1.5-3 times, mixing the extract by using 80-160-mesh silica gel with the weight ratio of 1-3 times, loading the mixture on a silica gel column, performing gradient washing by using chloroform-methanol solutions with the volume ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0:1, respectively collecting and concentrating eluates of each part, and monitoring and combining the same parts by using T L C;
(4) high pressure liquid chromatography comprises eluting with methanol-water at volume ratio of 7:3, 40-50% methanol as mobile phase, and C with specification of 20mm × 250mm and 5 μm18The preparation column is a stationary phase, the flow rate is 20ml/min, the detection wavelength of an ultraviolet detector is 238nm, 200 mu L is injected each time, eluent is collected, and the naphthalide B is obtained after multiple accumulation and evaporation to dryness.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail by examples and experimental data. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention.
Example 1
(1) Inoculating a Phomopsis Fukushii (Phomopsis fukushii) strain CCTCCM 2017632 preserved in a slant solid culture medium into a plate of the slant solid culture medium for activation, and inoculating the strain CCTCCM 2017632 into the slant solid culture medium after purification for storage, wherein the slant solid culture medium is prepared from 200 g/L of potatoes, 20 g/L of glucose, 20 g/L of agar, 6.5 of pH value, 121 ℃, 25min for sterilization, and cooling to 60 ℃ to pour the plate and slant for later use;
(2) the stored Phomopsis fumosus (Phomopsis fukushii) strain CCTCC M2017632 is transferred into a liquid seed culture medium, and is cultured for 24 hours at 28 ℃ on a shaking bed at the rotating speed of 180r/min to obtain the Phomopsis fumaria seed liquid. The liquid seed culture medium is as follows: NaNO32g/L，K2HPO41g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H2O0.01 g/L, glucose 20 g/L, pH 6.5, placing into a 250m L conical flask, 100m L/flask, sterilizing at 121 deg.C for 25 min;
(3) and (3) inoculating the phomopsis longipes seed liquid obtained in the step (2) into a rice solid culture medium according to the mass ratio of 2% of the culture medium to perform solid fermentation at 25 ℃, completing the fermentation within 45 days, wherein the rice culture medium comprises 100g of rice, 20g of perlite and 100ml of distilled water, filling the rice culture medium into a 600m L tissue culture bottle, and sterilizing at 121 ℃ for 25min for later use.
Example 2
Example 1 was repeated with the following differences:
(3) the fermentation temperature is 28 ℃, and the fermentation is completed in 40 days.
Example 3
(3) The fermentation temperature is 30 ℃, and the fermentation is completed in 30 days.
Example 4
(3) And inoculating the phomopsis longipes seed liquid into a rice solid culture medium according to the mass ratio of 5% of the culture medium for solid fermentation, wherein the fermentation temperature is 28 ℃, and the fermentation is completed within 40 days.
Example 5
(1) Taking 10kg of fermentation product of Phomopsis fukushii strain CCTCC M2017632, ultrasonically extracting with 80% ethanol for 3 times, 4h each time, mixing the extractive solutions, filtering, and concentrating under reduced pressure to obtain extract A1220 g; adding equal volume of ethyl acetate into the extract A for extraction for 5 times, combining the extract phases, and concentrating under reduced pressure to 153g of extract B.
(2) Dissolving the extract B with methanol, mixing with 200g of 80-mesh silica gel, loading into a 160-mesh silica gel column 1500g column, performing chromatographic separation, performing gradient washing with chloroform-methanol solution at volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, 5:5, collecting eluate of each part, concentrating, and monitoring with T L C to combine the same parts.
(3) Collecting 11.3g of the chloroform-methanol 7:3 eluate of (2), separating with high performance liquid chromatography, and collecting the eluate with 44% methanol mobile phase, C18Preparing a chromatographic column (20 × 250mm,20m L/min), taking a semi-preparative column as a stationary phase, detecting ultraviolet with the wavelength of 238nm, collecting a chromatographic peak for 17.8min, accumulating for multiple times, and evaporating to dryness to obtain the novel naphthalide B.
Example 6
Example 5 was repeated with the following differences:
(1) ultrasonically extracting with 70% ethanol for 3 times, each time for 5 hr; the extract A obtained by vacuum concentration is 1180 g; and (5) 146g of extract B obtained by vacuum concentration.
(2) And mixing the extract B with 200g of 160-mesh silica gel.
(3) The chloroform-methanol 7:3 elution part is 10.6g, and high performance liquid chromatography separation is carried out by taking 45% methanol as a mobile phase, and a chromatographic peak of 16.2min is collected.
Example 7
Example 5 was repeated with the following differences:
(1) ultrasonic extracting with 75% ethanol for 3 times; the extract A after decompression concentration is 1310 g; the extract B obtained by concentration under reduced pressure was 163 g.
(3) The chloroform-methanol 7:3 elution fraction was 11.3g, and the chromatographic peak was collected for 12.5min with 50% methanol as the mobile phase.
Example 8
Example 5 was repeated with the following differences:
(2) and mixing the extract B with 200g of 100-mesh silica gel.
(3) The chloroform-methanol 7:3 eluate fraction was 11.6g, and high performance liquid chromatography was performed using 45% methanol as the mobile phase to collect the chromatographic peak at 16.2 min.
Example 9
The MIC (ug/m L) value of naphalide B was determined using the broth dilution method.
(1) Preparing an antibacterial agent and a culture medium: dissolving the naphthalene compound to be detected in DMSO to prepare a mother solution with a concentration (2560 mu g/ml), and filtering and sterilizing the mother solution for later use. The prepared MH broth culture medium is sterilized for 30min at 121 ℃ for standby.
(2) The MRSA bacterial colony cultured for 24h is prepared into bacterial suspension with 0.5 McLeod's ratio turbidity standard, and the bacterial suspension is diluted 1: 100 with MH broth to obtain about 1 × 106And (5) preparing a bacterial solution of CFU/m L for later use.
(3) Preparing diluted antibacterial drugs and inoculating bacterial liquid, taking 13 sterile test tubes (13 × 100 mm), adding 1ml of MH broth into each tube except the 1 st tube, adding 0.4ml of antibacterial drug stock solution (such as 1280 mu g/ml) into each tube, uniformly mixing, sucking 1ml to the 2 nd tube, sucking 1ml to the 3 rd tube after uniform mixing, diluting to the 11 th tube in a continuous multiple ratio, sucking 1ml from the 11 th tube, discarding, adding 1ml of prepared inoculum into each tube, taking the 1 st tube to the 11 th tube as a growth control without drugs, taking the drug concentration of each tube as 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 mu g/ml, adding 1ml of prepared inoculum into each tube, taking the 1 st tube to the 11 th tube as a drug concentration of 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125 mu g/ml of diluted inoculum into each tube, taking 96 samples of vancomycin as a culture control, taking three positive samples and taking DMSO as a control sample, and transferring the three samples into a culture plate in parallel.
(4) Incubation the inoculated 96-well plate was incubated in a 37 ℃ incubator for 24 h.
(5) And (5) judging a result: the lowest drug concentration that completely inhibited bacterial growth in the wells was the MIC, while there was no significant inhibition of bacteria in the solvent control.
The strains used in the broth microdilution method include Staphylococcus aureus standard strain ATCC25923, methicillin-resistant Staphylococcus aureus standard strain ATCC 43300. The result shows that the naphthaldehyde B can inhibit ATCC25923 and ATCC43300, the MIC of a staphylococcus aureus standard strain ATCC25923 is 4 mu g/ml, the MIC of a methicillin-resistant staphylococcus aureus standard strain ATCC43300 is 8 mu g/ml, and the naphthaldehyde B can be used as a lead compound for developing methicillin-resistant staphylococcus aureus medicaments.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and changes made within the spirit and principles of the present invention are intended to be included therein.

Claims (2)

1. A preparation method of an MRSA resistant naphthalene compound has a structure shown as the following formula:
Figure DEST_PATH_IMAGE001
named naphallide B; the preparation method is that phomopsis formosana (A) and (B)Phomopsis fukushii) The fermentation product is taken as a raw material and is obtained by the steps of organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation; said phomopsis formosana (A), (B), (C), (Phomopsis fukushii) The strain preservation number is CCTCC M2017632, and the preservation date is 10 and 23 days in 2017;
A. phomopsis Fuji (A. Fuji)Phomopsis fukushii) The fermentation method of the strain comprises the steps of slope, seed liquid preparation and solid amplification fermentation, and comprises the following specific steps:
(1) the slant solid culture medium is prepared from potato 200 g/L, glucose 20 g/L, agar 20 g/L, pH 6.5, 121 deg.C, sterilizing for 25min, cooling to 60 deg.C, and pouring onto slant;
(2) the liquid seed culture medium is as follows: NaNO32g/L，K2HPO41g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H2O0.01 g/L, glucose 20 g/L and pH value 6.5, putting into a 250M L conical flask, 100M L/flask, sterilizing at 121 ℃ for 25min for later use, transferring the stored phomopsis Fuzicola strain CCTCC M2017632 inclined plane into a liquid seed culture medium, and culturing at 28 ℃ for 24h on a shaking bed at the rotating speed of 180r/min to obtain phomopsis seed liquid;
(3) the rice solid culture medium is prepared by putting 100g of rice, 20g of perlite and 100ml of distilled water into a 600m L tissue culture bottle, sterilizing at 121 ℃ for 25min for later use, inoculating 2% of the phomopsis mycete seed liquid obtained in the step (2) into the rice solid culture medium according to the mass ratio of the culture medium to perform solid fermentation under the fermentation condition of 25-32 ℃, and fermenting for 30-60 days to obtain a fermentation product;
B. the organic solvent extraction, silica gel column chromatography and high pressure liquid chromatography separation steps are as follows:
(1) organic solvent extraction: ultrasonically extracting for 2-4 times with 60-80% ethanol as solvent for 4-8 h each time, mixing the extractive solutions, filtering, and concentrating under reduced pressure to obtain extract;
(2) performing silica gel column chromatography, namely dissolving the extract by using pure methanol with the weight ratio of 1.5-3 times, mixing the extract by using 200-mesh silica gel with the weight ratio of 1-3 times of the extract, loading the mixture on a silica gel column, performing gradient washing by using a chloroform-methanol solution with the volume ratio of 1: 0-0: 1, respectively collecting and concentrating the eluates of each part, and monitoring and combining the same parts by using T L C;
(3) high-pressure liquid chromatography separation: taking chloroform-methanol solution eluent with the volume ratio of 7:3, and adding 40-50% methanolThe mobile phase is C with specification of 20mm × 250mm and 5 μm18The preparation column is a stationary phase, the flow rate is 20ml/min, the wavelength detected by an ultraviolet detector is 238nm, 50-200 mu L is injected each time, chromatographic peaks of 10-20 min are collected, and the chromatographic peaks are evaporated to dryness after multiple accumulation to obtain the naphthalide B.
2. The method according to claim 1, wherein the chloroform-methanol solution in step B and step 2 is at a volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0: 1.
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