CN108586380A - A kind of natural Rakicidins classes compound R akicidin H and its extracting method - Google Patents
A kind of natural Rakicidins classes compound R akicidin H and its extracting method Download PDFInfo
- Publication number
- CN108586380A CN108586380A CN201810256080.5A CN201810256080A CN108586380A CN 108586380 A CN108586380 A CN 108586380A CN 201810256080 A CN201810256080 A CN 201810256080A CN 108586380 A CN108586380 A CN 108586380A
- Authority
- CN
- China
- Prior art keywords
- akicidin
- alcohol
- compound
- rakicidins
- rakicidin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to native compound technical fields, and in particular to a kind of natural Rakicidins classes compound R akicidin H and its fermentation extraction method.The present invention isolated new Rakicidins component Rakicidin H from sea micromonoad zymotic fluid, native compound Rakicidin H of the present invention have preferable In-vitro Inhibitory Effect to human colon cancer cell HCT 8 and human pancreatic cancer cell PANC 1, it is 7.89 times and 18.73 times strong to the difference of the 1 more normal oxygen of activity of tumour cell HCT 8 and PANC of weary oxygen culture, fight the anaerobic bacterias good activities such as clostridium difficile, it is a kind of secondary metabolite of structure novel good activity, there is higher medical value.
Description
Technical field
The invention belongs to native compound technical fields, and in particular to a kind of natural Rakicidins classes compound
Rakicidin H and its fermentation extraction method.
Background technology
Currently, having isolated hundreds of kinds of bioactive substances from the metabolite of marine microorganism, which part is can
It is the important compound of one type that can have the antitumor antibiotics of clinical value, Rakicidins compounds, at present
It has reported and has been found that a series of Rakicidinsization with antitumor activity or bacteriostatic activity from micromonospora and streptomycete
Close object：Such as nineteen ninety-five, Kimberly D.Mcbrien et al. are found that in Micromonospora sp.R385-2
Rakicidin A and B；2000, Hu Jin-Feng et al. were found that from Streptomyces sp.GT61042
Rakicidin C；2010, Yasuhiro Igarashi etc. were found that from Streptomyces sp.MWW064
Rakicidin D；2014, Naoya Oku et al. were found that Rakicidin in Micromonospora sp.TP-A0860
E；2017, Shigeru Kitani et al. were found that Rakicidin F in Streptomyces sp.GKU220.
During screening novel anti-tumor bioactive substance from sea micromonoad, it has been found that ocean
Micromonospora sp.FIM 02-523 can generate a series of with antitumor activity including Rakicidin A
Lipopeptide compound, research in recent years is it has also been found that there is Rakicidin A remarkable weary oxygen selective antitumor cell to live
Property, inhibitor against colon carcinoma cells HCT-8 cell activity is 17.5 times under normoxic condition under hypoxic condition.Takeuchi (2011) is reported for the first time
Road Rakicidin A can induce the apoptosis of marrow chronic leukemia stem cell under hypoxic condition, although the compound resists weary oxygen
Tumour cell and resisting tumour stem cells (CSC) though mechanism of action it is unclear, Rakicidin A oneself by it is many colleague be considered
One anti-hypoxic tumor cells for being rich in development prospect and anti-CSC drugs.
Inventor seminar reported first at home in 2006 is separated to compound from the micromonospora of marine source
Rakicidin A and B, and the anti tumor activity in vitro of Rakicidin B is investigated, find it to tumor cell line
The growth of K562, L929 have obvious inhibiting effect；Isolated Rakicidin A, B and a kind of new again in 2016
Rakicidins compounds -- Rakicidin B1 can have apparent body under normal oxygen, anoxia state to tumour cell
Outer inhibiting effect；The internal tumor suppression of this 3 compounds is tested using transplanting human colon carcinoma HCT-8 tumour zebra fish models simultaneously
Activity, the results showed that, Rakicidin A, B, B1 have inhibitory activity to the HCT-8 cell transplantation tumors in zebra fish body, and
Preliminary test shows that its anti-hypoxic tumor cells is the expression that can effectively inhibit HIF-1 (Hypoxia inducible factor-1) based on it.It grinds
Study carefully and further determines Rakicidin A, B, B1 compound to HCT-8, MGC803, A549, A375, Hep G2 and CASKI five
As a result the cytotoxic activity of kind human tumor cell line shows that 3 compounds all have significant inhibition to these tumor cell lines
Activity.As it can be seen that
Rakicidins compounds have fabulous potential applicability in clinical practice.
Invention content
For this purpose, technical problem to be solved by the present invention lies in provide a kind of natural Rakicidins classes compound
Rakicidin H, and its fermentation extraction method is further disclosed.
In order to solve the above technical problems, a kind of natural Rakicidins classes compound R akicidin H of the present invention
And its pharmaceutically acceptable salt, the compound R akicidin H have the structure as shown in following formula (I)：
The invention also discloses a kind of method preparing the natural Rakicidins classes compound R akicidin H, institutes
State Rakicidin H be by deposit number be CGMCC No.12132 micromonospora bacterial strain FIM02-523 it is fermented and separation
It obtains.
Micromonospora bacterial strain FIM02-523 of the present invention is to screen to obtain from the marine clay of Fujian Province Putian, in
The preserving number of state's Microbiological Culture Collection administration committee common micro-organisms center is：CGMCC No.12132.Preservation address：North
The institute 3 of the Chaoyang Districts Jing Shi North Star West Road 1, preservation day：On 2 18th, 2016.
The method for preparing the natural Rakicidins classes compound R akicidin H, includes the following steps：
(1) the micromonospora bacterial strain FIM02-523 deposited that goes bail for ferments, and collects zymotic fluid and is separated by solid-liquid separation acquisition mycelia
Body；Gained mycelium methanol or ethyl alcohol are impregnated, soak is collected；
(2) gained soak is chromatographed with HP20 macroporous resin adsorption columns, and respectively with 60% and 68% alcohol-water
Gradient elution is carried out, 68% alcohol-water eluent is collected；
(3) eluent of collection is chromatographed with D3502 resin absorbing columns, and respectively with 62% and 67% alcohol-water
Gradient elution is carried out, 67% alcohol-water eluent is collected；
(4) eluent of collection is subjected to Image processing with HZ816 resin absorbing columns, respectively with 63% and 66% ethyl alcohol-
Water carries out gradient elution, collects the 66% alcohol-water eluent containing Rakicidin H；
(5) eluent is extracted with ethyl acetate or dichloromethane, concentration obtains crude product and dissolved with methanol, carries out half preparation solution
Phase chromatographic isolation, and with volume ratio 650：350 acetonitrile-water is eluted, Fractional Collections to get.
In the step (2), the blade diameter length ratio of the HP20 macroporous resin adsorptions column is 1：5-1：10, packed column volume 2.5-
4.0L, adsorption flow rate 30-45ml/min.
In the step (3), the D3502 resin absorbing columns blade diameter length ratio is 1：5-1：8, packed column volume 2.2-3L, absorption
Flow velocity is 25-35ml/min.
In the step (4), the blade diameter length ratio of the HZ816 resin absorbing columns is 1：6-1：10, packed column volume 1.5-2.2L,
Adsorption flow rate is 15-20ml/min.
In the step (5), the semi-preparative liquid chromatography is Welch Material, 5 μm, 250mm × 10mm, is controlled
Eluent flow rate 8ml/min.
The invention also discloses natural Rakicidins classes compound R akicidin H and its pharmaceutically acceptable
Salt is used to prepare the purposes with antitumor activity and anti-clinical pathogenic anaerobic bacteria active medicine.
The tumour includes human colon carcinoma, human pancreas cancer；The pathogenic anaerobic bacteria include clostridium difficile, peptostreptococcus,
Porphyromonas gingivalis and propionibacterium acnes.
The day taking dose of the natural Rakicidins classes compound R akicidin H and its pharmaceutically acceptable salt
It is 1-5000mg/ days, also can exceeds the range according to the difference and disease severity of dosage form, dosage.
The invention also discloses a kind of drugs for treating pathogenic anaerobic infection disease, with the natural Rakicidins classes
Compound R akicidin H and its pharmaceutically acceptable salt are active constituent, and add pharmaceutically acceptable carrier.
The drug can be conventional tablet or capsule, sustained-release tablet or capsule, Dospan or capsule, oral solution, note
Penetrate dosage form conventional on the galenic pharmacies such as agent.
The present invention isolated new Rakicidins component Rakicidin H from sea micromonoad zymotic fluid, should
Compound structure is only different in lipopeptid side chain compared with Rakicidin A, and the side chain terminal of Rakicidin H is propyl, and
Rakicidin A are isopropyl, and native compound Rakicidin H of the present invention are to human colon cancer cell HCT-8 and human pancreas
Cancer cell PANC-1 has preferable In-vitro Inhibitory Effect, to the strong of the more normal oxygen of tumour cell HCT-8 activity of weary oxygen culture
7.89 times, 18.73 times strong, the anaerobic bacterias work such as confrontation clostridium difficile to the more normal oxygen of tumour cell PANC-1 activity of weary oxygen culture
Property it is excellent, be a kind of secondary metabolite of structure novel good activity, have higher medical value.
Experimental example
One, antitumor activity is tested
Rakicidin H samples are dissolved in DMSO respectively so that solubility reaches 4ug/ml, then dilution makes eventually respectively
A concentration of 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 0.03125ug/ml,
0.015625ug/ml。
Nostoc commune Vanch：Human colon cancer cell HCT-8, human pancreatic cancer cell PANC-1 in Exponential growth stage is taken to plant respectively
(cell concentration is HCT-8 5.0 × 10 in 96 orifice plates4A/ml, PANC-14.0 × 104The hole a/ml, 100ul/), culture
R keeps its adherent for 24 hours, and 100ul/ pore area medicine fresh cultures, each concentration is added to set 3 multiple holes；And it sets blank control wells and (only plus trains
Support base) it is used as negative control, equally set 3 multiple holes；The Rakicidin A and Rakicidin equally being had found with the prior art
B compounds equally set 3 multiple holes as effect reference.Continue culture to 72hr, terminates culture.
Rakicidin H samples are dissolved in DMSO respectively so that solubility reaches 4ug/ml, then dilution makes eventually respectively
A concentration of 1.3333ug/ml, 0.4444ug/ml, 0.148148ug/ml, 0.0493827ug/ml, 0.0164609ug/ml,
0.00548697ug/ml、0.00182899ug/ml、0.000609663ug/ml；Rakicidin A and Rakicidin B are same
Processing.
Weary oxygen culture：People's colon-cancer cell HCT-8, human pancreatic cancer cell PANC-1 in Exponential growth stage is taken to plant respectively
(cell concentration is HCT-8 5.0 × 10 in 96 orifice plates4A/ml, PANC-14.0 × 104The hole a/ml, 100ul/), weary oxygen ventilation
30 minutes, gas valve to be closed, is put into 37 DEG C of incubator, r keeps its adherent for 24 hours for culture, adds 100ul/ pore area medicine fresh cultures,
Each concentration sets 3 multiple holes, and sets blank control wells (only plus culture medium) and be used as negative control, equally sets 3 multiple holes.Weary oxygen is logical
Gas 30 minutes closes gas valve, is put into 37 DEG C of incubator, continues culture to 72hr, terminates culture.
Mtt assay detects：The cell of culture will be terminated, the MTT 20ul of 5mg/ml are added per hole, it is small to continue culture 4 in incubator
When, supernatant is abandoned, is added 150ul DMSO solutions per hole, the lightly mixing of shaking table 10 minutes, microplate reader is read under 490nm wavelength
OD values per hole, and calculate inhibiting rate.By the conversion to inhibiting rate, IC is calculated using SPSS softwares50Value, as a result see the table below
1 and table 2.
Inhibiting rate (%)=(negative control group OD values-experimental group OD values)/negative control group OD value × 100%.
To the inhibitory activity of human colon cancer cell HCT-8 under 1 Rakicidin H anoxia states of table
To the inhibitory activity of human pancreatic cancer cell PANC-1 under 2 Rakicidin H anoxia states of table
The above results show that there is the compounds of this invention Rakicidin H potent antitumor activity especially to resist weary oxygen swollen
Oncocyte activity, to the 7.89 times strong of the more normal oxygen of tumour cell HCT-8 activity of weary oxygen culture, to the tumour cell of weary oxygen culture
The more normal oxygen of PANC-1 activity it is 18.73 times strong.
Two, resist pathogenic anaerobic bacteria active testing
Compound R akicidin H are taken to be dissolved into 0.64mg/ml in DMSO.Its test concentrations is：128,64,32,16,
8,4,2,1,0.5,0.25,0.125ug/ml.
To be positioned over by testing remaining solution by -20 DEG C, and antibiotic metronidazole is as reference compound.
The preparation of 2 × solution：Maximum concentration is 256ug/ml, and then dilute by twice of 10 times in 96 deep-well plates
It releases, obtains 2 required × solution.L00ril is dispensed to 96 hole round bottom plates with work station, and the 12nd row are negative controls, are contained only identical
The culture medium of volume.
The preparation of bacterial inoculum：It is inoculated on accordingly growth tablet within 2-3 days in advance, culture medium includes：Brucella
Broth supplemented with hemin (5g/mL), Vitamin K1 (1g/mL), and lysed horse blood
(5%v/v).Bacterial concentration is transferred to about (1-2) × 10 by experimental day6Then CFU/ml shifts the holes 100ul to 96- round bottom plate
In (96 orifice plates added with compound of above-mentioned preparation).
MIC is measured：The holes 96- round bottom plate derived above is positioned over 37 DEG C, under 85% damp condition, is trained under anaerobic condition
It supports 46-48 hours.The concentration point that bacterial growth is completely or significantly inhibited will be defined as the MIC of the compound, as a result see below
Table 3.
3 Rakicidin H antibacterial activities of table
Above-mentioned anti-bacteria test result shows that Rakicidin H have anti-clostridium difficile, peptostreptococcus, porphyromonas list
The clinical pathogenic anaerobic bacteria activity such as born of the same parents bacterium and propionibacterium acnes.
Specific implementation mode
The preparation of embodiment 1Rakicidin H
The preparation of compound R akicidin H, includes the following steps described in the present embodiment：
The micromonospora bacterial strain FIM02-523 (deposit number is CGMCC No.12132) deposited that goes bail for ferments, and ferments
Condition of culture reference literature (Jiang Hong, Lin Ru, Zheng Wei, the lipopeptid class chemical combination for waiting the oceans Micromonospora chalcea FIM02-523 to generate
The separation discriminating of object FW523 and biological activity [J] China antibiotic magazine, 2006,31 (5)：267-270), it specifically includes：
Taking platinum loop micromonospora bacterial strain FIM02-523 Gao Shi asparagine agar slant cultures access seed culture medium, (solubility is formed sediment
Powder 1.5%, glucose 0.5%, peptone 0.5%, yeast powder 0.5%, (NH4)2SO40.05%, K2HPO4·3H2O
0.05%, NaCl 0.05%, MgSO4·7H2O 0.05%, CaCO30.1%, tap water prepare, pH7.5), in 32 DEG C,
240r/min shake cultures 48h；Subsequent 5% culture transferring amount is transferred to fermentation medium (soluble starch 2%, sucrose 0.5%, cheese
Element 1.0%, corn steep liquor 0.5%, MgSO4·7H2O 0.04%, FeSO4·7H2O 0.005%, CuSO4·5H2O 0.005%,
CoCl2·6H2O 0.0005%, CaCO30.3%, tap water is prepared, pH7.5), in 32 DEG C, 240r/min shake cultures 96-
120h collects zymotic fluid and after 4500rpm centrifuges 15min, obtains mycelia slag, 2 times of volumes of the mycelia slag of acquisition
90% ethyl alcohol impregnates 2 times overnight, spirituous mycelia slag will be contained again with 4500rpm centrifuge 15min after supernatant is merged,
Obtain fermentation extracting solution；
To ferment extracting solution 45L, and it is 55% to be diluted to concentration of alcohol, then carries out layer with HP20 macroporous resin adsorption columns
Analyse (blade diameter length ratio 1：8, packed column volume 3.0L), adsorption flow rate 35ml/min is carried out with 60% and 68% alcohol-water respectively
2.5BV, 9BV (times column volume) are eluted, with HPLC (YMC ODS C18Column, 5 μm, 250mm × 4.6mm) detection eluent (acetonitrile-
Water 720：280,40 DEG C of column temperature, flow velocity 1.0mL/min, wavelength 262nm), collect 68% ethyl alcohol-containing Rakicidin H
Water elution (26L)；
It is 60% that the eluent of above-mentioned collection, which is diluted to concentration of alcohol, and (diameter height is chromatographed with D3502 resin absorbing columns
Than being 1：6, packed column volume 2.5L), adsorption flow rate 30ml/min carries out elution 3BV with 62% and 67% alcohol-water respectively
And 8BV, HPLC (YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280, column temperature 40
DEG C, flow velocity 1.0mL/min, wavelength 262nm), collect the 67% alcohol-water eluent (16.5L) containing Rakicidin H；
It is 55% that above-mentioned eluent, which is diluted to concentration of alcohol, and (blade diameter length ratio 1 is chromatographed with HZ816 resin absorbing columns：
8, packed column volume 1.8L), adsorption flow rate 16ml/min elutes 3.5BV, 6BV respectively with the alcohol-water of 63% and 66%,
HPLC(YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280,40 DEG C of column temperature, stream
Fast 1.0mL/min, wavelength 262nm), collect 66% ethanol water (8.5L) containing Rakicidin H；
The eluent of collection is concentrated into about 1/3 volume, is extracted 3 times with isometric dichloromethane, is concentrated under reduced pressure, obtains slightly
Product, and by gained crude product, dissolved with methanol, progress semi-preparative liquid chromatography (5 μm, 250mm × 10mm, Welch
Material), with volume ratio 650：350 acetonitrile-water is eluted, coutroi velocity 8ml/min, HPLC (YMC ODS C18Column,
5 μm, 250mm × 4.6mm) detection collection liquid, obtain sample Rakicidin H.
It is observed that and detection, compound R akicidin H made from the present embodiment are white amorphous powder, dissolve in chlorine
Imitative, methanol, DMSO, it is not soluble in water.
High resolution mass spectrum (HR-ESI-MS) shows its molecular ion peak [M+Na]+It is 629.3890, infers that its molecular formula is
C32H54N4O7, degree of unsaturation 8.1HNMR shows that the compound has 4 double bond proton [δH6.86 (1H, d, J=15.0Hz),
6.24 (1H, d, J=15.0Hz), 5.42 (1H, s), 5.33 (1H, s)], and 5 exchangeable protons [δ H 8.95 (1H, s), 8.06
(1H, d, J=10.0Hz), 7.30 (1H, s), 7.28 (1H, s), 5.72 (1H, d, J=6.0Hz)], 4 methyl proton [δH
2.95 (3H, s), 1.04 (3H, d, J=6.9Hz), 0.93 (3H, d, J=6.8Hz), 0.84 (3H, t, J=7.0Hz)].
13CNMR and DEPT135 shows that the molecule contains 32 carbon signals, including 5 quaternary carbons (δ C172.7,172.5,
169.1,167.6,166.0), 4 double key carbons [include a sp2 mesomethylene carbons (δC117.0), two sp2Methine carbon (δ C
137.9,119.1)], 6 sp3Methine carbon [including two company's oxygen carbon atoms (δ C 78.2,72.4)], 14 sp3Mesomethylene carbon
With 4 methine carbon atom (δC 36.5,15.3,14.0,13.2)。
All Hydrogen Protons pass through hsqc spectrum1H-13C correlations are pointed out.1H-1The coupling of H COSY Correlated Spectroscopies and proton is normal
The compound of digital display showing contains 4 independent spin coupling systems：NH-2-C-2-C-3-OH-3, C-9-C-10, C-31-C-14-
C-15-C-16 (C-32)-C-17-C-18 and C-28-C-29-C-30.In conjunction with1H-1H COSY and HMBC is it is found that H-2 and C-1/C-
5 is related, and H-3 is related to C-4, and OH-3 is related to C-2/C-3, and Ha-6 is closed with the cities C-5/C-7 mesh, and H-7 is closed with the cities C-6/C-8 mesh,
H-9 is related to C-8/C-11, and H-10 is related to C-8/C-11, and Hb-12 is related to C-10/C-11, and H-15 is related to C-1, H-31
Related to C-13/C-14/C-15, H-32 is related to C-15/C-16/C-17 and H-30 and C-28/C-29 is related, hydrogen and carbon
Chemical shift be listed in the table below 4.
The chemical shift of table 4 hydrogen and carbon
As it can be seen that the obtained compound R akicidin H structures of the present embodiment are correct.
The preparation of embodiment 2Rakicidin H
The preparation of compound R akicidin H, includes the following steps described in the present embodiment：
The micromonospora bacterial strain FIM02-523 deposited that goes bail for ferments, and fermentation process collects zymotic fluid simultaneously with embodiment 1
After 4500rpm centrifuges 15min, mycelia slag is obtained, the mycelia slag of acquisition is impregnated 2 overnight with 90% ethyl alcohol of 2 times of volumes
It is secondary, spirituous mycelia slag will be contained again to merge supernatant after 4500rpm centrifugations 15min, obtain fermentation extracting solution；
To ferment extracting solution 30L, and it is 50% to be diluted to concentration of alcohol, is then chromatographed with HP20 macroporous resin adsorption columns
(blade diameter length ratio 1：10, packed column volume 4.0L), adsorption flow rate 30ml/min is carried out with 60% and 68% alcohol-water respectively
3.5BV, 10BV are eluted, with HPLC (YMC ODSC18Column, 5 μm, 250mm × 4.6mm) detection eluent (acetonitrile-water 720：280,
40 DEG C of column temperature, flow velocity 1.0mL/min, wavelength 262nm), collect 68% ethanol eluate (34L) containing Rakicidin H；
It is 55% that the eluent of above-mentioned collection, which is diluted to concentration of alcohol, and (diameter height is chromatographed with D3502 resin absorbing columns
Than being 1：5, packed column volume 2.2L), adsorption flow rate 35ml/min, with 62% and 67% alcohol-water elute respectively 3BV,
8BV, HPLC (YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280, column temperature 40
DEG C, flow velocity 1.0mL/min, wavelength 262nm), collect 67% ethanol water (15L) containing Rakicidin H；
It is 50% that above-mentioned eluent, which is diluted to concentration of alcohol, and (blade diameter length ratio 1 is chromatographed with HZ816 resin absorbing columns：
10, packed column volume 2.2L), adsorption flow rate 20ml/min elutes 3.5BV, 6BV respectively with the alcohol-water of 63% and 66%,
HPLC(YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280,40 DEG C of column temperature, stream
Fast 1.0mL/min, wavelength 262nm), collect 66% ethanol water (10.5L) containing Rakicidin H；
The eluent of collection is concentrated into about 1/3 volume, is extracted 3 times with isometric ethyl acetate, is concentrated under reduced pressure, obtains slightly
Product, and by gained crude product, dissolved with methanol, progress semi-preparative liquid chromatography (5 μm, 250mm × 10mm, Welch
Material), with volume ratio 650：350 acetonitrile-water is eluted, coutroi velocity 8ml/min, HPLC (YMC ODS C18Column,
5 μm, 250mm × 4.6mm) detection collection liquid, obtain sample Rakicidin H.After testing, the structure of products therefrom is correct.
The preparation of embodiment 3Rakicidin H
The preparation of compound R akicidin H, includes the following steps described in the present embodiment：
The micromonospora bacterial strain FIM02-523 deposited that goes bail for ferments, and fermentation process collects zymotic fluid simultaneously with embodiment 1
After 4500rpm centrifuges 15min, mycelia slag is obtained, the mycelia slag of acquisition is impregnated 2 overnight with 90% ethyl alcohol of 2 times of volumes
It is secondary, spirituous mycelia slag will be contained again to merge supernatant after 4500rpm centrifugations 15min, obtain fermentation extracting solution；
To ferment extracting solution 55L, and it is 60% to be diluted to concentration of alcohol, is then chromatographed with HP20 macroporous resin adsorption columns
(blade diameter length ratio 1：5, packed column volume 2.5L), adsorption flow rate 45ml/min is eluted with 60% and 68% alcohol-water respectively
3.5BV, 8BV, with HPLC (YMC ODS C18Column, 5 μm, 250mm × 4.6mm) detection eluent (acetonitrile-water 720：280, column temperature
40 DEG C, flow velocity 1.0mL/min, wavelength 262nm), collect the 68% alcohol-water eluent (19L) containing Rakicidin H；
It is 60% that the eluent of above-mentioned collection, which is diluted to concentration of alcohol, and (diameter height is chromatographed with D3502 resin absorbing columns
Than being 1：8, packed column volume 3L), adsorption flow rate 25ml/min elutes 2BV, 7BV respectively with the alcohol-water of 62% and 67%,
HPLC(YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280,40 DEG C of column temperature, stream
Fast 1.0mL/min, wavelength 262nm), collect the 67% alcohol-water eluent (17.5L) containing Rakicidin H；
It is 55% that above-mentioned eluent, which is diluted to concentration of alcohol, and (blade diameter length ratio 1 is chromatographed with HZ816 resin absorbing columns：
6, packed column volume 1.5L), adsorption flow rate 15ml/min elutes 3.5BV, 6BV respectively with the alcohol-water of 63% and 66%,
HPLC(YMC ODS C18Column, 5 μm, 250mm × 4.6mm) tracing detection eluent (acetonitrile-water 720：280,40 DEG C of column temperature, stream
Fast 1.0mL/min, wavelength 262nm), collect 66% ethanol water (7L) containing Rakicidin H；
The eluent of collection is concentrated into about 1/3 volume, is extracted 3 times with isometric dichloromethane, is concentrated under reduced pressure, obtains slightly
Product, and by gained crude product, dissolved with methanol, progress semi-preparative liquid chromatography (5 μm, 250mm × 10mm, Welch
Material), with volume ratio 650：350 acetonitrile-water is eluted, coutroi velocity 8ml/min, HPLC (YMC ODS C18Column,
5 μm, 250mm × 4.6mm) detection collection liquid, obtain sample Rakicidin H.After testing, the structure of products therefrom is correct.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of natural Rakicidins classes compound R akicidin H and its pharmaceutically acceptable salt, which is characterized in that institute
Stating compound R akicidin H has the structure as shown in following formula (I)：
2. a kind of method preparing natural Rakicidins class compound R akicidin H described in claim 1, feature exist
In, Rakicidin H be by micromonospora bacterial strain FIM02-523 that deposit number is CGMCC No.12132 it is fermented and
Separation obtains.
3. the method according to claim 2 for preparing the natural Rakicidins classes compound R akicidin H, special
Sign is, includes the following steps：
(1) the micromonospora bacterial strain FIM02-523 deposited that goes bail for ferments, and collects zymotic fluid and is separated by solid-liquid separation acquisition mycelium；It will
Gained mycelium methanol or ethyl alcohol impregnate, and collect soak；
(2) gained soak is chromatographed with HP20 macroporous resin adsorption columns, and respectively with 60% and 68% alcohol-water into
Row gradient elution collects 68% alcohol-water eluent；
(3) eluent of collection is chromatographed with D3502 resin absorbing columns, and respectively with 62% and 67% alcohol-water into
Row gradient elution collects 67% alcohol-water eluent；
(4) by the eluent of collection with HZ816 resin absorbing columns carry out Image processing, respectively with 63% and 66% alcohol-water into
Row gradient elution collects the 66% alcohol-water eluent containing Rakicidin H；
(5) eluent is extracted with ethyl acetate or dichloromethane, concentration obtains crude product and dissolved with methanol, carries out half and prepares liquid phase color
Spectrum separation, and with volume ratio 650：350 acetonitrile-water is eluted, Fractional Collections to get.
4. the method according to claim 3 for preparing the natural Rakicidins classes compound R akicidin H, special
Sign is, in the step (2), the blade diameter length ratio of the HP20 macroporous resin adsorptions column is 1：5-1：10, packed column volume 2.5-
4.0L, adsorption flow rate 30-45ml/min.
5. the method according to claim 3 or 4 for preparing the natural Rakicidins classes compound R akicidin H,
It is characterized in that, in the step (3), the D3502 resin absorbing columns blade diameter length ratio is 1：5-1：8, packed column volume 2.2-3L inhale
Attached flow velocity is 25-35ml/min.
6. preparing the natural Rakicidins classes compound R akicidin H's according to claim 3-5 any one of them
Method, which is characterized in that in the step (4), the blade diameter length ratio of the HZ816 resin absorbing columns is 1：6-1：10, packed column volume
1.5-2.2L, adsorption flow rate 15-20ml/min.
7. preparing the natural Rakicidins classes compound R akicidin H's according to claim 3-6 any one of them
Method, which is characterized in that in the step (5), the semi-preparative liquid chromatography is Welch Material, 5 μm, 250mm ×
10mm, control eluent flow rate 8ml/min.
8. natural Rakicidins classes compound R akicidin H and its pharmaceutically acceptable salt described in claim 1 are used for
Prepare the purposes with antitumor activity and anti-clinical pathogenic anaerobic bacteria active medicine.
9. purposes according to claim 8, which is characterized in that the tumour includes human colon carcinoma, human pancreas cancer；The cause
Sick anaerobic bacteria includes clostridium difficile, peptostreptococcus, porphyromonas gingivalis and propionibacterium acnes.
10. a kind of drug for treating pathogenic anaerobic infection disease, which is characterized in that with natural described in claim 1
Rakicidins classes compound R akicidin H and its pharmaceutically acceptable salt are active constituent, and addition can pharmaceutically connect
The carrier received.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256080.5A CN108586380B (en) | 2018-03-27 | 2018-03-27 | Natural Rakicidins compound Rakicidin H and extraction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256080.5A CN108586380B (en) | 2018-03-27 | 2018-03-27 | Natural Rakicidins compound Rakicidin H and extraction method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108586380A true CN108586380A (en) | 2018-09-28 |
| CN108586380B CN108586380B (en) | 2021-08-31 |
Family
ID=63624717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810256080.5A Active CN108586380B (en) | 2018-03-27 | 2018-03-27 | Natural Rakicidins compound Rakicidin H and extraction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108586380B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066745A (en) * | 2019-03-08 | 2019-07-30 | 福建省微生物研究所 | A kind of the sea micromonoad strain and its application of the high yield Rakicidin H that ferments |
| CN110437314A (en) * | 2019-08-12 | 2019-11-12 | 福建省微生物研究所 | A kind of natural Rakicidins class compound R akicidin B1-1 and its fermentation extraction method |
| CN110698541A (en) * | 2019-08-12 | 2020-01-17 | 福建省微生物研究所 | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105504025A (en) * | 2014-10-16 | 2016-04-20 | 南开大学 | Rakicidin a and preparation method thereof |
| CN105753936A (en) * | 2016-03-28 | 2016-07-13 | 福建省微生物研究所 | Rakicidins compounds Rakicidin B1 and preparation method thereof |
-
2018
- 2018-03-27 CN CN201810256080.5A patent/CN108586380B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105504025A (en) * | 2014-10-16 | 2016-04-20 | 南开大学 | Rakicidin a and preparation method thereof |
| CN105753936A (en) * | 2016-03-28 | 2016-07-13 | 福建省微生物研究所 | Rakicidins compounds Rakicidin B1 and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| CHEN LI等: "Rakicidin G-I,Three New Cyclic Depsipeptides from Marine Micromonospora sp.", 《第十三届全国抗生素学术会议论文集》 * |
| SHIGERU KITANI ET AL.: "Rakicidin F, a new antibacterial cyclic depsipeptide from a marine sponge-derived Streptomyces sp.", 《THE JOURNAL OF ANTIBIOTICS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066745A (en) * | 2019-03-08 | 2019-07-30 | 福建省微生物研究所 | A kind of the sea micromonoad strain and its application of the high yield Rakicidin H that ferments |
| CN110066745B (en) * | 2019-03-08 | 2021-03-30 | 福建省微生物研究所 | Marine micromonospora strain for fermenting high-yield Rakicidin H and application thereof |
| CN110437314A (en) * | 2019-08-12 | 2019-11-12 | 福建省微生物研究所 | A kind of natural Rakicidins class compound R akicidin B1-1 and its fermentation extraction method |
| CN110698541A (en) * | 2019-08-12 | 2020-01-17 | 福建省微生物研究所 | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof |
| CN110437314B (en) * | 2019-08-12 | 2021-03-26 | 福建省微生物研究所 | Natural Rakicidins compound Rakicidin B1-1 and fermentation extraction method thereof |
| CN110698541B (en) * | 2019-08-12 | 2023-04-14 | 福建省微生物研究所 | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108586380B (en) | 2021-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105753936B (en) | A kind of Rakicidins class compound R akicidin B1 and preparation method thereof | |
| CN108329280A (en) | A kind of natural Rakicidins classes compound R akicidin I and its extracting method | |
| CN105709205B (en) | Purposes of the Rakicidins class compound for anti-clinical pathogenic anaerobic bacteria | |
| CN106834160B (en) | Streptomyces erythropolis for producing keratin compound | |
| CN108586380A (en) | A kind of natural Rakicidins classes compound R akicidin H and its extracting method | |
| CN108530379A (en) | A kind of natural Rakicidins classes compound R akicidin G and its extracting method | |
| CN103172507B (en) | Ophiobollin sesterterpine compound as well as preparation method and application thereof | |
| CN111548954A (en) | Four anthraquinone compounds and preparation method and application thereof | |
| CN111778172B (en) | Streptomyces for producing antibacterial active compound and separation method and application thereof | |
| CN111909858A (en) | Mangrove endophytic fungus TGM112 secondary metabolite and application thereof | |
| CN109810919B (en) | Ansha all-carbon cyclic polyketone antibiotics and application thereof in preparation of antibacterial drugs or antitumor drugs | |
| CN102965300B (en) | Micromonospora strain, its preparation method and application | |
| CN108660093B (en) | Marine streptomycete and tunicamycin compound and preparation method thereof | |
| CN109988129A (en) | Compound and its preparing the application in antituberculotic | |
| CN114149445B (en) | Preparation method of xanthone compound and application of xanthone compound in resisting drug-resistant bacteria | |
| CN115109023A (en) | Macrolide compound FWYZ52-A, and fermentation strain, fermentation method and application thereof | |
| CN107954839A (en) | A kind of antiinflammatory active compound peniroquesine A and its preparation method and application | |
| CN108949610B (en) | Streptomyces and angucycline compound generated by streptomyces as well as preparation and application of angucycline compound | |
| CN111808112A (en) | Pratensilin D compound and preparation and application thereof | |
| CN110437314B (en) | Natural Rakicidins compound Rakicidin B1-1 and fermentation extraction method thereof | |
| JP3719689B2 (en) | Novel substance triprostatin, process for producing the same, cell cycle inhibitor and antitumor agent | |
| CN115536616B (en) | Rearranged steroid compound derived from coral coccus and preparation method and application thereof | |
| CN110698541B (en) | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof | |
| CN110698537B (en) | Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof | |
| CN105837590A (en) | Compound with anti-Candida albicans activity, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |