CN110698537B - Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof - Google Patents
Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 32
- 238000000855 fermentation Methods 0.000 title claims abstract description 26
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- 238000000605 extraction Methods 0.000 title abstract description 6
- 230000000694 effects Effects 0.000 claims abstract description 13
- 241001148471 unidentified anaerobic bacterium Species 0.000 claims abstract description 10
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims abstract description 8
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 8
- 229960003085 meticillin Drugs 0.000 claims abstract description 8
- 241000194032 Enterococcus faecalis Species 0.000 claims abstract description 6
- 108010059993 Vancomycin Proteins 0.000 claims abstract description 6
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- ABSPRNADVQNDOU-UHFFFAOYSA-N Menaquinone 1 Natural products C1=CC=C2C(=O)C(CC=C(C)C)=C(C)C(=O)C2=C1 ABSPRNADVQNDOU-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
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- 229940124350 antibacterial drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- SNVLJLYUUXKWOJ-UHFFFAOYSA-N methylidenecarbene Chemical compound C=[C] SNVLJLYUUXKWOJ-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 description 1
- 235000019175 phylloquinone Nutrition 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin B1-2 and a fermentation extraction method thereof. The invention separates the new Rakicidin component of Rakicidin B1-2 from the marine micromonospora fermentation broth, and compared with the known Rakicidin B1, the structure of the compound is characterized in that 11 and 14 open loops are formed, and ethoxy is connected to the 11 position. The natural compound Rakicidin B1-2 has a certain in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 cultured under normoxic and hypoxic conditions, has a certain inhibition activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile and vancomycin-resistant enterococcus faecalis, is a secondary metabolite with novel structure and better activity, and has higher medicinal value.
Description
Technical Field
The invention belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin B1-2 and a fermentation extraction method thereof.
Background
At present, hundreds of bioactive substances have been separated from metabolites of marine microorganisms, wherein part of the bioactive substances are antibiotics with antitumor activity or antibacterial activity which may have clinical application value, rakicidins compounds are one of important compounds, and a series of Rakicidins compounds with antitumor activity or antibacterial activity have been reported to be discovered from Micromonospora and Streptomyces.
In recent years, the literature has reported that marine Micromonospora sp.FIM 02-523 is capable of producing a range of lipopeptides having antitumor activity, including Rakicidin A, B, B1, and the like. In 2006, the subject group first reported the isolation of compounds Rakicidin a and B from this strain of micromonospora at home. The research shows that Rakicidin A has excellent activity of anti-tumor cells with hypoxia selectivity, and the activity of anti-colon cancer HCT-8 cells under the hypoxia condition is 17.5 times that under the normoxic condition, and is considered by a plurality of peer lines as an anti-hypoxia tumor cell and an anti-CSC drug with very rich development prospect; in-vitro antitumor activity examination of Rakicidin B also shows that the Rakicidin B has obvious inhibition effect on the growth of tumor cell strains K562 and L929. The subject group obtains a new compound Rakicidin B1 in 2016, experiments test the in vivo tumor inhibition activity of Rakicidin A and B, B1 by using a transplanted human colon cancer HCT-8 tumor zebra fish model, and the results show that both Rakicidin A and B, B1 have inhibition activity on HCT-8 cell transplantation tumor in zebra fish, and preliminary experiments show that the anti-hypoxia tumor cells can effectively inhibit the expression of HIF-1 (hypoxia inducible factor-1). Previous studies have further determined the cytotoxic activity of Rakicidin a, B, B1 compounds against five human tumor cell lines HCT-8, MGC803, a549, a375, hepG2 and CASKI, and the results show that the three isolated compounds have significant inhibitory activity against these tumor cell lines. In 2018, the subject group is separated and purified from metabolites of marine Micromonospora sp.FIM 02-523 strain to three other novel compounds of Rakicidin G and H, I, and the activity experiment results show that the Rakicidin G and H, I compounds have strong cytotoxic activity on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 and are stronger under the condition of hypoxia, and the three compounds have good inhibitory activity on various gram-positive anaerobic bacteria. It can be seen that the Rakicidins compounds have excellent clinical application prospects.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a natural Rakicidins compound Rakicidin B1-2 and further disclose a fermentation extraction method thereof.
In order to solve the technical problems, the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof disclosed by the invention have the structure shown in the following formula (I):
the invention also discloses a method for preparing the natural Rakicidins compound Rakicidin B1-2, wherein the Rakicidin B1-2 is obtained by fermenting and separating a marine Micromonospora sp.FIM-R160609 strain with a preservation number of CGMCC No.14823.
In particular, the Micromonospora sp.FIM-R160609 strain of the marine Micromonospora is preserved in China general microbiological culture Collection center, and has the preservation address: the preservation number is CGMCC No.14823 and the preservation date is 2017, 10 months and 16 days.
Preferably, the method for preparing the natural Rakicidins Rakicidin B1-2 comprises the following steps of:
(1) Fermenting the preserved marine Micromonospora sp.FIM-R160609 strain, and collecting fermentation liquor for solid-liquid separation to obtain mycelium; soaking the mycelium with methanol or ethanol, and collecting soaking solution;
(2) Carrying out chromatography by using a D3502 macroporous resin adsorption column, carrying out gradient elution by using 67% and 75% ethanol-water solutions respectively, and collecting 75% ethanol-water eluent components;
(3) Subjecting the collected eluate to HZ816 resin adsorption column chromatography, and gradient eluting with 66%, 69% and 73% ethanol-water solution, respectively, to collect 73% ethanol-water eluate component containing Rakicidin B1-2;
(4) Extracting the obtained eluent with ethyl acetate or dichloromethane, concentrating to obtain a crude product, dissolving with methanol, performing semi-preparative liquid chromatography, and performing volume ratio 710: eluting with 290 acetonitrile-water, and collecting in segments.
Specifically, in the step (2), the diameter-to-height ratio of the D3502 macroporous resin adsorption column is 1:5-1:10, the column loading volume is 2.5-4.0L, and the adsorption flow rate is 30-45ml/min.
Specifically, in the step (3), the HZ816 resin adsorption column diameter-to-height ratio is 1:5-1:8, the packed volume is 2.2-3L, and the adsorption flow rate is 25-35ml/min.
Specifically, in the step (4), the semi-preparative liquid chromatograph is Welch Material,5 μm,250mm×10mm, and the eluent flow rate is controlled to 8ml/min.
The invention also discloses application of the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof in preparing medicines with anti-tumor activity and anti-clinical pathogenic anaerobic bacteria activity.
Specifically, the tumor comprises human colon cancer and human pancreas cancer; the pathogenic anaerobic bacteria comprise methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus faecalis and clostridium difficile.
Specifically, the daily dosage of the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof is 1-5000 mg/day, and the dosage can be out of the range according to different dosage forms and disease severity.
The invention also discloses a medicine for treating pathogenic anaerobic bacteria infection diseases, which takes the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof as active ingredients and adds pharmaceutically acceptable carriers.
Specifically, the medicine can be in the form of common tablet or capsule, slow-release tablet or capsule, controlled-release tablet or capsule, oral liquid, injection and other conventional preparations.
The invention also discloses application of the marine Micromonospora sp.FIM-R160609 strain to preparation of natural Rakicidins compound Rakicidin B1-2 by fermentation, wherein the marine Micromonospora sp.FIM-R160609 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 14823.
The invention separates the new Rakicidin component of Rakicidin B1-2 from the marine micromonospora fermentation broth, and compared with the known Rakicidin B1, the structure of the compound is characterized in that 11 and 14 open loops are formed, and ethoxy is connected to the 11 position. The natural compound Rakicidin B1-2 has a certain in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 cultured under normoxic and hypoxic conditions, has a certain inhibition activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile and vancomycin-resistant enterococcus faecalis, is a secondary metabolite with novel structure and better activity, and has higher medicinal value.
Experimental example
1. Antitumor Activity test
The extracted Rakicidin B1-2 samples were dissolved in DMSO to a solubility of 1ug/ml, and diluted to a final concentration of 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 0.03125ug/ml, 0.015625ug/ml, respectively.
Common culture: taking human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 in exponential growth phase, respectively planting in 96-well plates (cell concentration is HCT-8 5.0X10) 4 Each ml, PANC-14.0X10 4 100 ul/well), culturing for 24hr to adhere, adding 100 ul/well fresh culture medium with medicine, eachThe concentration was set at 3 duplicate wells and a blank control well (medium only) was set as negative control, as were 3 duplicate wells. Culturing was continued for 48hr, and the culture was terminated.
The Rakicidin B1-2 samples were dissolved in DMSO to a solubility of 1ug/ml, respectively, and diluted to final concentrations of 0.4444ug/ml, 0.148148ug/ml, 0.0493827ug/ml, 0.0164609ug/ml, 0.00548697ug/ml, 0.00182899ug/ml, respectively.
Anaerobic culture: taking human intestinal cancer cells HCT-8 and human pancreatic cancer cells PANC-1 in exponential growth phase, respectively planting in 96-well plates (cell concentration is HCT-8 5.0X10) 4 Each ml, PANC-14.0X10 4 100 ul/well), ventilation valve was closed, placed in incubator 37℃and incubated for 24hr to adhere to the wall, 3 duplicate wells were added for each concentration, and a blank control well (medium only) was used as negative control, and 3 duplicate wells were also used. Ventilation valve is closed after ventilation for 30 min, and the culture is continued at 37deg.C for 48hr in incubator, and the culture is terminated.
MTT method detection: the cells which were terminated in culture were added with CCK-810ul per well, cultured in an incubator for 2 hours, the supernatant was discarded, 150ul of DMSO solution was added per well, gently mixed by shaking table for 10 minutes, OD value per well was read out by an ELISA reader at a wavelength of 450nm, and the inhibition ratio was calculated. Calculation of IC by conversion of inhibition Rate Using SPSS software 50 Values, results are shown in tables 1 and 2 below. Inhibition (%) = (negative control OD value-experimental OD value)/negative control OD value x 100%.
TABLE 1 inhibitory Activity of Rakicidin B1-2 against human colon cancer cell HCT-8 in the hypoxic state
TABLE 2 inhibitory Activity of Rakicidin B1-2 against human pancreatic cancer cells PANC-1 in the hypoxic state
The results show that the Rakicidin B1-2 has certain anti-tumor activity under normoxic and hypoxic states.
2. Test of anti-pathogenic anaerobic bacteria Activity
(1) In vitro antibacterial test method for staphylococcus and enterococcus
Sample preparation: dissolving a compound Rakicidin B1-2 in DMSO to obtain a final concentration: 16. 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008ug/ml.
Preparation of inoculum: preparing a suspension with a concentration equivalent to 0.5 McCondition standard, diluting with broth (staphylococcus: MH broth; enterococcus: brain heart infusion broth), adding 100 μl of the test bacterial liquid (200 μl per well volume) into each well, and final concentration of bacterial liquid is about 10 5 CFU/ml. Sealing, and culturing in 35-37deg.C incubator for 18-24 hr.
MIC determination: the lowest inhibitory concentration was taken as the lowest drug concentration that completely inhibited bacterial growth in the wells, and the test results are shown in table 3 below.
(2) Method for testing in-vitro antibacterial activity of clostridium difficile
Inoculum preparation and inoculation: preparation of a suspension having a concentration equivalent to 0.5 McMeter Standard turbidimeter, aspiration of the prepared bacterial solution (1-2. Mu.l) with a multipoint inoculator [ use of Brookfield Medium supplemented with 5% (V/V) defibrinated sheep blood, hemin (5 mg/L) and vitamin K1 (1 mg/L)]Inoculating on the surface of agar plate (adding the antibacterial drugs with different concentrations diluted by multiple ratio into the reinforced Broth agar with high pressure dissolution and sterilization, and balancing in water bath at 60deg.C, mixing thoroughly, pouring the agar plate, and preparing agar plate with agar thickness of 3-4mm according to 1:9 ratio, and setting 0.008-16 mg/L12 drug concentration gradients), inoculating bacteria amount at each point of about 10 5 CFU, forming plaque with a diameter of 5-8 mm. After inoculation, the cells were incubated in an anaerobic environment at 35℃for 48 hours, and the results were observed.
MIC determination: the plate was placed on a dark, non-reflective object surface to determine the end point of the test, to give the MIC as the lowest drug concentration that inhibited bacterial growth. The lowest drug concentration that inhibited bacterial growth by more than 80% as compared to the growth control was used as the endpoint concentration and the test results are shown in table 3 below.
TABLE 3 antibacterial Activity of Rakicidin B1-2
The antibacterial test results show that the compound Rakicidin B1-2 has certain inhibitory activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile and vancomycin-resistant enterococcus faecalis.
Detailed Description
Example 1 preparation of Rakicidin B1-2
Taking a stored marine Micromonospora sp.FIM-R160609 strain (with a preservation number of CGMCC No. 14823) for fermentation, wherein the specific fermentation conditions can be referred to the fermentation method disclosed in Chinese patent CN108300672B, and the method specifically comprises the following steps: scraping the freshly cultured marine micromonospora FIM-R160609 oblique spores to prepare bacterial suspension, inoculating the bacterial suspension into shake flask seed culture, culturing at 32 ℃ for 48 hours at 250rpm, inoculating the bacterial suspension into shake flask fermentation medium according to 5% inoculum size, culturing at 30 ℃ for 120 hours at 250rpm, placing the culture flask, and measuring a fermentation product by HPLC.
Seed culture medium formula (mass fraction): maltodextrin 2.0%, glucose 1.0%, soybean cake powder 1.0%, yeast powder 2.0%, mgSO 4 ·7H 2 O 0.05％，KH 2 PO 4 0.05％，CaCO 3 0.3 percent of tap water, and the pH value is regulated to 7.0 to 7.5.
Fermentation medium formula (mass fraction): maltodextrin 5.0%, glucose 1.0%, soybean cake powder 2.0%, yeast powder 1.5%, ammonium sulfate 0.8%, mgSO 4 ·7H 2 O 0.05％，NaCl 0.5％，CaCO 3 0.5 percent of tap water, and the pH value is adjusted to 7.5.
After fermentation, collecting fermentation liquor and centrifuging at 4500rpm for 15min to obtain mycelium residues, soaking the mycelium residues with 2 times of 90% ethanol overnight for 2 times, centrifuging the mycelium residues containing ethanol at 4500rpm for 15min again, and mixing the supernatants to obtain fermentation extract.
45L of the fermentation extract was diluted to 55% ethanol concentration, then subjected to D3502 macroporous resin adsorption column chromatography (diameter-to-height ratio: 1:8, column loading volume: 3.0L) at an adsorption flow rate of 33ml/min, eluting with 67% and 75% ethanol water for 3BV and 8.5BV (column doubling volume), respectively, and subjecting to HPLC (YMCODS C) 18 The column, 5 μm,250mm×4.6 mm) detects the eluate (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm) and collection of ethanol eluent (23L) containing 75% of Rakicidin B1-2.
Diluting the above collected eluate to 60% ethanol concentration, subjecting to chromatography with HZ816 resin adsorption column (diameter-to-height ratio of 1:6, column loading volume of 2.5L), controlling adsorption flow rate to 30ml/min, eluting with 66%, 69% and 73% ethanol-water solution, eluting with 3BV, 2.5BV and 7BV, respectively, and subjecting to HPLC (YMC ODS C 18 The column, 5 μm,250mm x 4.6 mm) was followed for detection of the eluate (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm), and 73% aqueous ethanol (13.8L) containing RakicidinB1-2 were collected.
The collected eluate was concentrated to about 1/3 volume, extracted three times with an equal volume of dichloromethane, concentrated under reduced pressure to obtain a crude product, and the obtained crude product was dissolved with methanol, and subjected to semi-preparative liquid chromatography (5 μm,250 mm. Times. 10mm,Welch Material) at a volume ratio of 710:290 in acetonitrile-water, controlling the flow rate to 8ml/min, and detecting the collected liquid by HPLC to obtain the sample Rakicidin B1-2.
Through observation and detection, the compound Rakicidin B1-2 prepared in the embodiment is white amorphous powder, is soluble in chloroform, methanol and DMSO, and is insoluble in water.
The detection result of high-resolution mass spectrum (HR-ESI-MS) shows that the molecular ion peak [ M+H ] is] + 669.4433, [ M+Na ]] + 691.4248, the molecular formula is deduced to be C 34 H 60 N 4 O 9 The unsaturation was 7.
1 HNMR detection results show that the compound has 2 double bond protons [ delta ] H 7.44(1H,d,J＝15.5Hz),6.57(1H,d,J＝15.5Hz)]6 exchangeable protons, including [ delta ] H 7.40(1H,d,J＝9.0Hz),6.84(1H,s),6.81(1H,s),5.79(1H,s)]6 methyl protons [ delta ] H 3.04(3H,s),1.25(3H,t,J＝6.0Hz),0.99(3H,d,J＝7.0Hz),0.85(3H,t,J＝6.9Hz),0.83(3H,d,J＝6.6Hz),0.82(3H,d,J＝6.6Hz)]。
13 CNMR and DEPT135 detection showed that the molecule contained 34 carbon signals, including 6 quaternary carbons (delta C 176.1,169.6,169.4,168.5,165.3,164.7), 2 double bond carbons (delta) C 134.9,130.6), 6 sp 3 Methine carbon [ comprising four oxygen-linked carbon atoms (delta) C 78.6,71.6,55.4,42.0)]14 sp 3 Methylene carbon and 6 methyl carbon atoms (delta) C 36.6,19.6,14.8,14.5,13.8,11.7)。
All hydrogen protons pass through HSQC spectrum 1 H- 13 C-correlation is assigned. 1 H- 1 The coupling constants of the H COSY correlation spectrum and protons show that the compound contains 4 independent spin-coupling systems: NH-2-C-2-C-3-OH-3, C-9-C-10, C-32-C-15-C-16-C-17 (C-33) -C-18-C-19 and C-28-C-29 (C-34) -C-30-C-31. Bonding of 1 H- 1 H COSY and HMBC are known to be C-1/C-5, H-3 to C-4, OH-3 to C-2/C-3, ha-6 to C-5/C-7, H-7 to C-6/C-8, H-9 to C-8/C-11, H-10 to C-8/C-11, H-12 to C-11/C-13, H-16 to C-1, H-32 to C-14/C-15/C-16, H-33 to C-16/C-17/C-18, and H-31 to C-29/C-30, H-34 to C-29/C-30, and the chemical shifts of hydrogen and carbon are listed in Table 4 by analysis of the nuclear magnetic data, unsaturation, molecular formula, and chemical shift described above.
TABLE 4 chemical shifts of hydrogen and carbon
The chemical structure of the compound 1 is shown as a formula (I) through analysis of the nuclear magnetic data, the unsaturation degree, the molecular formula and the chemical shift. Therefore, the compound Rakicidin B1-2 prepared by fermentation and extraction in the embodiment has correct structure.
Example 2 preparation of Rakicidin B1-2
Fermenting Micromonospora sp.FIM-R160609, collecting fermentation broth, centrifuging at 4500rpm for 15min to obtain mycelium residue, soaking the mycelium residue in 90% ethanol 2 times volume overnight for 2 times, centrifuging at 4500rpm for 15min again, and mixing the supernatants to obtain fermentation extract.
30L of the fermentation extract was diluted to an ethanol concentration of 50%, followed by subjecting to D3502 macroporous resin adsorption column chromatography (diameter-to-height ratio: 1:5, packed column volume: 2.5L) at an adsorption flow rate of 45ml/min, eluting with 67% and 75% ethanol water respectively at 2.5BV and 6BV, eluting with HPLC (YMC ODS C 18 The column, 5 μm,250mm×4.6 mm) detects the eluate (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm) and collection of ethanol eluent (12.5L) containing 75% of RakicidinB 1-2.
Diluting the above collected eluate to 55% ethanol concentration, subjecting to chromatography with HZ816 resin adsorption column (diameter-to-height ratio of 1:8, column loading volume of 3.0L), controlling adsorption flow rate to 35ml/min, eluting with 66%, 69% and 73% ethanol-water solution, eluting with 3BV, 2BV and 4.5BV, respectively, and subjecting to HPLC (YMC ODS C 18 The column, 5 μm,250mm x 4.6 mm) was followed for detection of the eluate (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm), and 73% aqueous ethanol solution (12L) containing RakicidinB1-2 were collected.
The collected eluate was concentrated to about 1/3 volume, extracted three times with an equal volume of ethyl acetate, concentrated under reduced pressure to give a crude product, and the obtained crude product was dissolved with methanol and prepared using a semi-preparative liquid phase (5 μm,250mm× 10mm,Welch Material) in a volume ratio of 710:290 acetonitrile-water elution, control of flow rate 8ml/min, HPLC (YMC ODS C 18 The column is provided with a plurality of holes,5 μm,250 mm. Times.4.6 mm) and the collected liquid was examined to obtain a sample of Rakicidin B1-2.
The product prepared by the scheme of the embodiment has correct structure through detection.
Example 3 preparation of Rakicidin B1-2
Fermenting Micromonospora sp.FIM-R160609, collecting fermentation broth, centrifuging at 4500rpm for 15min to obtain mycelium residue, soaking the mycelium residue in 90% ethanol 2 times volume overnight for 2 times, centrifuging at 4500rpm for 15min again, and mixing the supernatants to obtain fermentation extract.
Taking 55L of fermented extract, diluting to ethanol concentration of 60%, subjecting to D3502 macroporous resin adsorption column chromatography (diameter-to-height ratio of 1:8, column loading volume of 4.0L) at adsorption flow rate of 30ml/min, eluting with 67% and 75% ethanol water for 3.5BV and 8BV, respectively, detecting by HPLC (YMC ODSC 18 Column, 5 μm,250mm×4.6 mm) eluent (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm) and collection of ethanol eluent (29.4L) containing 75% of Rakicidin B1-2.
Diluting the above collected eluate to 60% ethanol concentration, subjecting to chromatography with HZ816 resin adsorption column (diameter-to-height ratio of 1:5, column loading volume of 2.2L), controlling adsorption flow rate to 25ml/min, eluting with 66%, 69% and 73% ethanol-water solution, eluting with 2.5BV, 3BV and 6BV, respectively, and subjecting to HPLC (YMC ODS C 18 The column, 5 μm,250mm x 4.6 mm) was followed for detection of the eluate (acetonitrile-water 700:300, column temperature 40 ℃, flow rate 1.0mL/min, wavelength 262 nm) and 73% aqueous ethanol solution (11.5L) containing RakicidinB1-2 were collected.
The collected eluate was concentrated to about 1/3 volume, extracted three times with an equal volume of dichloromethane, concentrated under reduced pressure to give a crude product, and the obtained crude product was dissolved with methanol and prepared using a semi-preparative liquid phase (5 μm,250mm× 10mm,Welch Material) in a volume ratio of 710:290 acetonitrile-water elution, control of flow rate 8ml/min, HPLC (YMC ODS C 18 Column, 5 μm,250 mm. Times.4.6 mm) and the collection liquid was examined to obtain a sample of Rakicidin B1-2.
The product prepared by the scheme of the embodiment has correct structure through detection.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A natural Rakicidin compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof are characterized in that the structure of the Rakicidin B1-2 is shown as the following formula (I):
2. a method for preparing the natural Rakicidins B1-2 of claim 1, wherein the Rakicidins B1-2 are obtained by fermenting and separating a marine Micromonospora sp.FIM-R160609 strain with a preservation number of CGMCC No.14823.
3. A process for the preparation of said natural Rakicidins, rakicidin B1-2, as claimed in claim 2, comprising the steps of:
(1) Fermenting the preserved marine Micromonospora sp.FIM-R160609 strain, and collecting fermentation liquor for solid-liquid separation to obtain mycelium; soaking the mycelium with methanol or ethanol, and collecting soaking solution;
(2) Carrying out chromatography by using a D3502 macroporous resin adsorption column, carrying out gradient elution by using 67% and 75% ethanol-water solutions respectively, and collecting 75% ethanol-water eluent components;
(3) Subjecting the collected eluate to HZ816 resin adsorption column chromatography, and gradient eluting with 66%, 69% and 73% ethanol-water solution, respectively, to collect 73% ethanol-water eluate component containing Rakicidin B1-2;
(4) Extracting the obtained eluent with ethyl acetate or dichloromethane, concentrating to obtain a crude product, dissolving with methanol, performing semi-preparative liquid chromatography, and performing volume ratio 710: eluting with 290 acetonitrile-water, and collecting in segments.
4. A process for the preparation of said natural Rakicidins group compound Rakicidin B1-2 as claimed in claim 3 wherein in step (2) the D3502 macroporous resin adsorption column has an aspect ratio of 1:5-1:10, the column loading volume is 2.5-4.0L, and the adsorption flow rate is 30-40ml/min.
5. The method for preparing Rakicidin B1-2 as claimed in claim 3 or 4, wherein in step (3), the HZ816 resin column aspect ratio is 1:5-1:8, the packed volume is 2.2-3L, and the adsorption flow rate is 25-35ml/min.
6. The method for preparing Rakicidin B1-2 as claimed in claim 3 or 4, wherein in step (4), the semi-preparative liquid chromatography is Welch Material,5 μm,250mm×10mm, and eluent flow rate is controlled to 8ml/min.
7. The use of the natural Rakicidins of claim 1, rakicidin B1-2, and pharmaceutically acceptable salts thereof, for the preparation of a medicament having activity against clinically pathogenic anaerobic bacteria; wherein,,
the anti-clinical pathogenic anaerobic bacteria are methicillin-resistant staphylococcus aureus MRSA15-1, methicillin-resistant staphylococcus aureus MRSA15-2, methicillin-sensitive staphylococcus aureus MSSA15-1, methicillin-sensitive staphylococcus aureus MSSA15-2, vancomycin-resistant enterococcus faecalis ATCC51575 and vancomycin-resistant enterococcus faecalis ATCC700802.
8. A medicament for treating pathogenic anaerobic bacterial infection diseases, which is characterized in that natural Rakicidins B1-2 and pharmaceutically acceptable salts thereof as active ingredients in claim 1 are added with pharmaceutically acceptable carriers.
9. The use of a marine Micromonospora sp.fim-R160609 strain for the fermentative preparation of natural Rakicidins of type Rakicidin B1-2 according to claim 1, characterized in that said marine Micromonospora sp.fim-R160609 strain has been deposited in the China general microbiological culture collection center with the accession number CGMCC No.14823.
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