CN112624955B - Separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide - Google Patents
Separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 22
- IPWFJLQDVFKJDU-UHFFFAOYSA-N pentanamide Chemical compound CCCCC(N)=O IPWFJLQDVFKJDU-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 229950001902 dimevamide Drugs 0.000 title claims abstract description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- 239000003208 petroleum Substances 0.000 claims abstract description 28
- 239000002904 solvent Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000004440 column chromatography Methods 0.000 claims abstract description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 241000607757 Xenorhabdus Species 0.000 claims abstract description 14
- 238000000605 extraction Methods 0.000 claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 78
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 36
- 241000607735 Xenorhabdus nematophila Species 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 23
- 238000012258 culturing Methods 0.000 claims description 23
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- 230000001580 bacterial effect Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 7
- -1 3-indolylethyl Chemical group 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000002955 isolation Methods 0.000 claims description 2
- NIQUZFQMNMNAMD-NSHDSACASA-N (3s)-n-[2-(1h-indol-3-yl)ethyl]-3-methyl-2-oxopentanamide Chemical compound C1=CC=C2C(CCNC(=O)C(=O)[C@@H](C)CC)=CNC2=C1 NIQUZFQMNMNAMD-NSHDSACASA-N 0.000 abstract description 27
- NIQUZFQMNMNAMD-UHFFFAOYSA-N nematophin Natural products C1=CC=C2C(CCNC(=O)C(=O)C(C)CC)=CNC2=C1 NIQUZFQMNMNAMD-UHFFFAOYSA-N 0.000 abstract description 27
- 239000000575 pesticide Substances 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 26
- 239000000243 solution Substances 0.000 description 19
- 150000001875 compounds Chemical class 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 9
- 241000244206 Nematoda Species 0.000 description 8
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 8
- 230000000967 entomopathogenic effect Effects 0.000 description 7
- 244000052616 bacterial pathogen Species 0.000 description 6
- 230000005526 G1 to G0 transition Effects 0.000 description 5
- 229960001701 chloroform Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241001148062 Photorhabdus Species 0.000 description 2
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- 239000012507 Sephadex™ Substances 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 2
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- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- WWDWYVXPTRYQOQ-QRPNPIFTSA-N CN.N[C@@H](CC1=CC=CC=C1)C(=O)O Chemical class CN.N[C@@H](CC1=CC=CC=C1)C(=O)O WWDWYVXPTRYQOQ-QRPNPIFTSA-N 0.000 description 1
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- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241001480238 Steinernema Species 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
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- 229940088710 antibiotic agent Drugs 0.000 description 1
- TXVHTIQJNYSSKO-UHFFFAOYSA-N benzo[e]pyrene Chemical class C1=CC=C2C3=CC=CC=C3C3=CC=CC4=CC=C1C2=C34 TXVHTIQJNYSSKO-UHFFFAOYSA-N 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
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- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
- ARQRPTNYUOLOGH-UHFFFAOYSA-N chcl3 chloroform Chemical compound ClC(Cl)Cl.ClC(Cl)Cl ARQRPTNYUOLOGH-UHFFFAOYSA-N 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 1
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- 150000002475 indoles Chemical class 0.000 description 1
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- OHRURASPPZQGQM-GCCNXGTGSA-N romidepsin Chemical class O1C(=O)[C@H](C(C)C)NC(=O)C(=C/C)/NC(=O)[C@H]2CSSCC\C=C\[C@@H]1CC(=O)N[C@H](C(C)C)C(=O)N2 OHRURASPPZQGQM-GCCNXGTGSA-N 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical class C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of pesticides, and in particular relates to a separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide (Nematophin), which comprises the following steps: subjecting the fermentation broth of the bacteria of the genus Xenorhabdus to column chromatography, collecting the fraction containing 3-indoloethyl (3 '-methyl-2' -keto) pentanamide; the components containing 3-indoloethyl (3 '-methyl-2' -ketone) valeramide are sequentially concentrated under reduced pressure and extracted by solvent to obtain the product; the solvent is petroleum ether, and the volume ratio of petroleum ether to the extraction object is 1:1. The method has simple separation steps and easily obtained separation materials, and provides a new way for developing microbial source pesticides.
Description
Technical Field
The invention belongs to the field of pesticides, and particularly relates to a separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide (Nematophin).
Background
Entomopathogenic nematode symbiotic bacteria are a model organism for studying symbiotic relationships, and are now becoming a development hotspot in the field of biopharmaceutical research. Entomopathogenic nematode symbiotic bacteria are gram-negative bacteria of the enterobacteriaceae family, which are parasitic in the intestinal tract of entomopathogenic nematodes, including the genera Xenorhabdus (Xenorhabdus) and Photorhabdus (Photorhabdus), and which are symbiotic with, respectively, the nematodes stinerenema (Steinernema) and heterorhabdis (Heterhabditis). Symbiotic entomopathogenic nematodes can be directly used as a biological agent, and the insecticidal effect is achieved mainly by improving the plant immunity, so that the symbiotic entomopathogenic nematodes are successfully applied to the field of plant protection. Besides insect-proof effect, the secondary metabolite of symbiotic bacteria is a rich source of antibacterial, nematicidal, antiviral, cytotoxin and other compounds. Therefore, the secondary metabolite of the entomopathogenic nematode symbiotic bacteria has become a novel biological resource with development potential and application prospect.
At present, a great deal of research is carried out on chemical components of metabolites of symbiotic bacteria of entomopathogenic nematodes at home and abroad, and hundreds of components are separated from the metabolites of symbiotic bacteria so far, and are mainly indole compounds, amide compounds, isonitrile compounds, benzylidene ketone compounds, benzopyrene compounds, phenylalanine methylamine compounds, dithiopyrrole antibacterial compounds, furanone compounds, cyclic peptides and depsipeptide compounds, soluble proteins, catechol electrophilic compounds, anthraquinone compounds, stilbene derivatives, carbapenem antibiotics and other compounds. The method for extracting active ingredients in Xenorhabdus comprises ultrasonic extraction, solvent extraction, etc., and the extraction solvent is water, ethanol, methanol, chloroform, ethyl acetate, etc. The method for separating active compounds from Xenorhabdus comprises silica gel column chromatography, sephadex column chromatography, supercritical fluid extraction, high-speed countercurrent chromatography, etc.
Nematophin has the structural formula:
the 3-indoloethyl (3 '-methyl-2' -keto) pentanamide (Nematophin) compound is a major active compound in xenorhabdus nematophilus. The compound has better activity on some plant diseases in agriculture, and also has higher activity (MIC, 0.57 mg/L) on staphylococcus aureus in medicine. In addition, the compound also has better anti-tumor activity. The compound was first isolated and identified from x.nematophila BC1 by Li et al. Firstly, extracting the BC1 strain fermentation liquor by ethyl acetate, then performing silica gel column chromatography, and eluting by adopting 100% chloroform to obtain Nematophin. Xu Peng et al also separated Nematophin from ethyl acetate extract of x.nematophila YL001 fermentation broth, but the separation procedure was somewhat different from Li et al, which first extracted the x.nematophila YL001 strain fermentation broth with ethyl acetate, followed by a second silica gel column chromatography, eluting with a petroleum ether-ethyl acetate system, and repeatedly crystallizing the V Stone :V Acetic acid ethyl ester =2:1 components to obtain compound Nematophin.
Disclosure of Invention
The invention aims to provide a separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide, which comprises the following specific scheme:
A process for the isolation of 3-indoloethyl (3 '-methyl-2' -one) pentanamide comprising: subjecting a fermentation broth of a bacterium of the genus Xenorhabdus (Xenorhabdus) to column chromatography, and collecting a fraction containing 3-indoloethyl (3 '-methyl-2' -ketone) pentanamide; the components containing 3-indoloethyl (3 '-methyl-2' -ketone) valeramide are sequentially concentrated under reduced pressure and extracted by solvent to obtain the product; the solvent is petroleum ether, and the volume ratio of petroleum ether to the extraction object is 1:1.
Optionally, concentrating under reduced pressure, redissolving in water, and extracting with solvent to obtain 3-indoloethyl (3 '-methyl-2' -ketone) valeramide;
the solvent is petroleum ether, and the volume ratio of petroleum ether to water redissolved substance is 1:1.
Optionally, the column chromatography separation process includes:
The fermentation liquor of the nematophila bacteria is statically adsorbed for 12 hours by macroporous resin, 5 times of column ponding, 1 time of column volume of methanol with the volume concentration of 50 percent, 1 time of column volume of methanol with the volume concentration of 80 percent and 3 times of column volume of methanol are sequentially used for eluting, and methanol eluent is collected.
Optionally, the macroporous resin is selected from XAD-11 macroporous resin, XAD-12 macroporous resin, porapak S macroporous resin, HPD-600 macroporous resin, XAD-9 macroporous resin, XAD-10 macroporous resin, XAD-7HP macroporous resin, LSA-10 macroporous resin, LX-28 macroporous resin, AB-8 macroporous resin, DA macroporous resin, X-5 macroporous resin, D320 macroporous resin, D101 macroporous resin, CAD-40 macroporous resin, GDX-105 macroporous resin, D macroporous resin and DM2 macroporous resin.
Optionally, the solvent extraction comprises:
mixing solvent and the object to be extracted once every 30min, repeating for 3-4 times, and collecting petroleum ether extract.
Optionally, the preparation of the fermentation broth comprises:
① Seed liquid preparation: culturing strain in LB medium at 28deg.C and shaking table rotation speed of 180r/min for 48 hr at room temperature on NBTA plate by streaking method, selecting blue single colony in LB medium, culturing at 28deg.C and rotation speed of 180r/min, and culturing for use in logarithmic phase;
② Fermentation culture: seed solution was inoculated into TSB medium at 10% (V/V) and cultured at 28℃and 180r/min with shaking.
Optionally, concentrating the supernatant of the xenorhabdus nematophilus fermentation broth under reduced pressure;
the specific process is as follows: collecting supernatant of fermentation broth, and concentrating at 40deg.C and 0.1Mpa to 1/5 of original volume.
Alternatively, the xenorhabdus nematophilus (Xenorhabdus nematophila) bacterium is selected from one or more than two of X.nematophila YL001, X.nematophila ALL, X.nematophila AN6 and X.nematophila A24.
Optionally, re-dissolving the concentrated solution of the pathogenic bacteria of nematophila by using 1.5-2 times of water, statically absorbing the concentrated solution for 12h by using macroporous resin, sequentially eluting macroporous resin chromatographic columns of the bacterial fermentation solution of the nematophila by using acetone-water (the volume ratio of the acetone-water is sequentially 0:100, 50:50, 80:20 and 100:0), ethanol-water (the volume ratio of the ethanol-water is sequentially 0:100, 50:50, 80:20 and 100:0), acetone-water-0.01N hydrochloric acid (the volume ratio of the acetone-water-0.01N hydrochloric acid is sequentially 0:100 and 30:70) or methanol-water-0.01N hydrochloric acid (the volume ratio of the methanol-water-0.01N hydrochloric acid is sequentially 0:100 and 30:70), sequentially eluting each eluting section by using 1-5 times of column volume, and collecting methanol eluent, acetone eluent, ethanol eluent, 30% acetone-0.01N hydrochloric acid eluent or 30% methanol-0.01N hydrochloric acid as fractions;
Concentrating the fraction under reduced pressure at 40deg.C and 0.1Mpa, adding 5-7 times of water for redissolution, and extracting with solvent; the solvent is petroleum ether, the volume ratio of petroleum ether to water redissolution is 1:1, the solvent and the object to be extracted are uniformly mixed once every 30min, and petroleum ether extract is collected after repeating for 3 times.
Preferably, a separation method of 3-indoloethyl (3 '-methyl-2' -ketone) valeramide, and concentrating the supernatant of X.nematophila YL001 fermentation liquor under reduced pressure to obtain concentrated liquor;
Re-dissolving the concentrated solution with 1.5-2 times of water, then statically adsorbing for 12h by macroporous resin, eluting with 5 times of column water, 1 time of column volume of 50% methanol, 1 time of column volume of 80% methanol and 3 times of column volume of methanol in sequence, and collecting methanol eluent;
Concentrating under reduced pressure at 40deg.C and 0.1Mpa, and re-dissolving with 5-7 times of water and extracting with solvent; the solvent is petroleum ether, the volume ratio of petroleum ether to water redissolution is 1:1, the solvent and the object to be extracted are uniformly mixed once every 30min, and petroleum ether extract is collected after repeating for 3-4 times.
The method for extracting Nematophin from the pathogenic bacteria of the genus xenorhabdus in the invention has simple equipment and method, namely a conventional column chromatography and solvent extraction method are used, and expensive reagents or instruments such as silica gel, sephadex, a supercritical fluid extractor, a high-speed countercurrent chromatograph and the like are not needed. The method greatly simplifies Nematophin separation and purification equipment, is simple and easy to operate, has low cost, can reach more than 95 percent of product purity, and is easy to popularize and use.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a HPLC chart of Nematophin isolated in example 1;
FIG. 2 is an HPLC plot of Nematophin standard.
Detailed Description
Unless otherwise specified, all reagents used in the experiments are commercially available, all liquid ratios in the experiments are volume ratios, and all experimental methods which are not described in detail are conventional experimental methods in the field.
The invention provides a method for extracting Nematophin from pathogenic bacteria of nematophila, which comprises the following steps:
(1) Test bacterial material: fermenting and culturing the pathogenic bacteria of nematophila, centrifuging, taking supernatant, and concentrating under reduced pressure to obtain solution A for later use. The specific flow is as follows:
① Seed liquid preparation: culturing strain stored in a strain tube in LB medium at constant temperature of 28deg.C at rotation speed of 180r/min until logarithmic phase, culturing at room temperature on NBTA plate by streaking method for 48 hr, selecting blue single colony in LB medium (culture temperature: 28deg.C, rotation speed: 180 r/min), and culturing until logarithmic phase;
② Fermentation culture: inoculating 10% (V/V) seed solution into TSB culture medium, and culturing at 28deg.C and shaking table rotation speed 180r/min for 3d;
③ Preparing supernatant: centrifuging the obtained fermentation broth at 4deg.C and 10000r/min for 15min to obtain supernatant, concentrating the supernatant at 40deg.C and 0.1Mpa to 1/5 times of the original volume, and preserving at 4deg.C.
(2) And (3) column chromatography separation and purification:
① Re-dissolving with distilled water (pH=7.0) 1.5-2 times of solution A, statically adsorbing with macroporous resin column D101 for 12 hr, and eluting with distilled water (pH=7.0) 5 times of column volume, methanol 50% 1 time of column volume, methanol 80% 1 time of column volume, and methanol 3 times of column volume sequentially. Collecting methanol eluent, concentrating under reduced pressure at 40deg.C and 0.1Mpa to obtain solution B;
② Re-dissolving the solution B with distilled water (pH=7.0) 5-7 times, and extracting with petroleum ether (AR) at the ratio of petroleum ether (AR): the water redissolution=1:1, fully and uniformly mixed once every 30min, repeated for 3-4 times, and then petroleum ether (AR) extract is collected, and concentrated under reduced pressure at 40 ℃ and 0.1Mpa to prepare Nematophin.
Nematophin structural identification: nematophin: pale yellow needle crystals ,1H NMR(500MHz,CDCl3)δ8.13((1H,s,NH),),7.66(1H,dd,J＝9.8Hz,0.75,Hz,H-4),7.28(1H,dd,J＝10.1Hz,1.5Hz,H-5),7.18(1H,dd,J＝9.9Hz,1.3Hz,H-6),7.11(1H,bd,J＝3.0Hz,1-NH),7.07(1H,bd,J＝3.0Hz,H-2),3.70(2H,d,J＝5.0Hz),3.55(1H,t,J＝4.9Hz,H-3'),3.07(2H,t,J＝5.1Hz,H-10),1.791.79(1H,m,H-4'),1.46(1H,m,H-4');13C NMR(125MHz,CDCl3)δ202.39(C-1'),160.08(C-2'),136.47(C-9),127.19(C-8),122.34(C-5),119.62(C-6),118.68(C-4),112.58(C-2),111.29(C-7),40.40(C-3'),39.55(C-11),25.46(C-10),25.20(C-4'),15.17(3'-CH3),11.50(C-5'). have spectral data consistent with Nematophin reported in the literature (Canadian Journal of Microbiology 1997, 43 (8): 770-773).
The pathogenic bacteria of the nematophila in the invention are selected from one or more strains of X.nematophila YL001, X.nematophila ALL, X.nematophila AN6, X.nematophila A24 and the like. Pathogenic bacteria nematophilus used in the present invention refer to the paper: pathogenic nematophila YL001 antibacterial active ingredient research, xu Peng, 2016, shuoshi treatises.
The column packing used in the column chromatography separation of the present invention is selected from the group consisting of XAD-11 macroporous resin, XAD-12 macroporous resin, porapak S macroporous resin, HPD-600 macroporous resin, XAD-9 macroporous resin, XAD-10 macroporous resin, XAD-7HP macroporous resin, LSA-10 macroporous resin, LX-28 macroporous resin, AB-8 macroporous resin, DA macroporous resin, X-5 macroporous resin, D320 macroporous resin, D101 macroporous resin, CAD-40 macroporous resin, GDX-105 macroporous resin, D macroporous resin and DM2 macroporous resin.
The invention is described in further detail below by way of specific examples of separation methods.
For a better understanding of the present invention, an example of a separation of Nematophin will now be given, including but not limited to this separation method.
Example 1:
seed liquid preparation: culturing X.nematophila ALL strain stored in a fungus tube in LB culture medium at constant temperature of 28deg.C at rotation speed of 180r/min until logarithmic phase, culturing on NBTA plate by streaking method at room temperature for 48 hr, selecting blue single colony in LB culture medium (culture temperature: 28deg.C, rotation speed: 180 r/min), and culturing until logarithmic phase;
Fermentation culture: inoculating 10% (V/V) seed solution into TSB culture medium, culturing at 28deg.C and shaking table rotation speed 180r/min for 3d, enriching fermentation broth 20L, centrifuging fermentation broth at 9000r/min for 15min, collecting supernatant, and concentrating under reduced pressure at 40deg.C and 0.1Mpa to 4L.
The concentrate obtained above was reconstituted to 6L with distilled water (ph=7.0) and subjected to column chromatography. Macroporous resin (D101) is used as a stationary phase (the column loading amount is 2 kg), fermentation liquor is slowly injected into a chromatographic column (10 multiplied by 200 cm) at a flow rate of 20mL/min, the flow rate of the chromatographic column is 10mL/min, the fermentation liquor to be concentrated is totally adsorbed, a column chromatography valve is closed, and static adsorption is carried out for 12h. Sequentially eluting with 5 times of column volume distilled water (pH=7.0), 1 time of column volume 50% methanol, 1 time of column volume 80% methanol and 3 times of column volume methanol, collecting methanol eluent, concentrating under reduced pressure at 40deg.C and 0.1Mpa, redissolving with 5-7 times of concentrated solution volume distilled water (pH=7.0), adding petroleum ether at volume ratio of 1:1, extracting for three times at 2h at room temperature to obtain Nematophin with purity not less than 90%.
Detection result:
HPLC detection patterns are shown in figures 1 and 2;
The nuclear magnetic data and physical properties of Nematophin prepared in this example are: pale yellow needle-like crystals ,1H NMR(500MHz,CDCl3)δ8.13((1H,s,NH),),7.66(1H,dd,J＝9.8Hz,0.75,Hz,H-4),7.28(1H,dd,J＝10.1Hz,1.5Hz,H-5),7.18(1H,dd,J＝9.9Hz,1.3Hz,H-6),7.11(1H,bd,J＝3.0Hz,1-NH),7.07(1H,bd,J＝3.0Hz,H-2),3.70(2H,d,J＝5.0Hz),3.55(1H,t,J＝4.9Hz,H-3'),3.07(2H,t,J＝5.1Hz,H-10),1.791.79(1H,m,H-4'),1.46(1H,m,H-4');13C NMR(125MHz,CDCl3)δ202.39(C-1'),160.08(C-2'),136.47(C-9),127.19(C-8),122.34(C-5),119.62(C-6),118.68(C-4),112.58(C-2),111.29(C-7),40.40(C-3'),39.55(C-11),25.46(C-10),25.20(C-4'),15.17(3'-CH3),11.50(C-5').
Example 2:
Fermenting and culturing X.nematophila YL001, concentrating the enriched fermentation liquid 20L, centrifuging at 9000r/min for 15min, and concentrating under reduced pressure at 40deg.C and 0.1 Mpa.
The concentrate obtained above was reconstituted with 1.5-2 times distilled water (ph=7.0), and then subjected to column chromatography separation. Macroporous resin (X-5) is used as a stationary phase, distilled water (pH=7.0) with 5 times of column volume, acetone with 1 time of column volume and 50%, acetone with 1 time of column volume and acetone with 3 times of column volume are sequentially used for eluting, acetone eluent is collected, reduced pressure concentration is carried out at 40 ℃, distilled water (pH=7.0) with 5-7 times of concentrated solution volume is used for re-dissolving, petroleum ether is added for extraction for three times according to the volume ratio of 1:1, and Nematophin with the purity of more than or equal to 90% is obtained.
Example 3:
Fermenting and culturing X.nematophila AN6, concentrating the enriched fermentation liquor 20L under reduced pressure at 40 ℃ and 0.1Mpa after centrifugation for 15min at 9000 r/min.
The concentrate obtained above was reconstituted with 1.5-2 times distilled water (ph=7.0). Macroporous resin (XAD-11) is used as a stationary phase, 5 times of column water, 1 time of column volume of 50% ethanol, 1 time of column volume of 80% ethanol and 3 times of column volume of ethanol are sequentially used for eluting, ethanol eluent is collected, reduced pressure concentration is carried out at 40 ℃ and 0.1Mpa, and redissolution is carried out by using distilled water (pH=7.0) with 5-7 times of concentrated solution volume according to the volume ratio of 1:1 adding petroleum ether for extraction for three times to obtain Nematophin with purity more than or equal to 90%.
Example 4:
Fermenting and culturing X.nematophila A24, centrifuging enriched fermentation liquor 20L at 9000r/min for 15min, and concentrating under reduced pressure at 40deg.C and 0.1 Mpa.
Re-dissolving the obtained concentrated solution with 1.5-2 times of distilled water (pH=7.0), adopting macroporous resin (XAD-11) as a stationary phase, eluting with 5 times of column water-0.01 n hydrochloric acid and 3 times of column water-30% acetone-0.01 n hydrochloric acid in sequence, collecting 30% acetone-0.01 n hydrochloric acid section, concentrating under reduced pressure at 40 ℃ and 0.1Mpa, re-dissolving with 5-7 times of distilled water (pH=7.0) of concentrated solution volume, and mixing according to the volume ratio of 1:1 adding petroleum ether for extraction for three times to obtain Nematophin with purity more than or equal to 90%.
Example 5:
Fermenting and culturing X.nematophila YL001, concentrating the enriched fermentation liquid 20L under reduced pressure at 40 ℃ and 0.1Mpa after centrifugation for 15min at 9000 r/min.
Re-dissolving the concentrated solution with 1.5-2 times distilled water (pH=7.0), adopting macroporous resin (AB-8 macroporous resin) as stationary phase, eluting with 5 times column water-0.01 n hydrochloric acid and 3 times column water-30% methanol-0.01 n hydrochloric acid in sequence, collecting 30% methanol-0.01 n hydrochloric acid section, concentrating under reduced pressure at 40 ℃ and 0.1Mpa, re-dissolving with 5-7 times distilled water (pH=7.0) of concentrated solution volume, adding petroleum ether according to volume ratio of 1:1, and extracting for three times to obtain Nematophin with purity not less than 90%.
Example 6:
Fermenting and culturing X.nematophilyl 001, concentrating the enriched fermentation liquid 50L under reduced pressure at 40 ℃ and 0.1Mpa after centrifugation for 15min at 9000 r/min.
Referring to Xu Peng (2016), adding 1.5-2 times distilled water (pH=7.0) into the concentrated fermentation broth obtained above for re-dissolution, extracting with ethyl acetate four times to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography (silica gel 312g, 200-300 mesh; chromatographic column: 100 x 5 cm), respectively using petroleum ether: ethyl acetate system (V petroleum ether ：V Acetic acid ethyl ester = 100:1, 80:1, 60:1, 40:1, 20:3) and chloroform: methanol system (V Chloroform (chloroform) ：V Methanol =80:1, 60:1, 40:1, 20:1, 10:1, methanol) is used as eluent, the fractions are collected in equal volume (400 mL), HPLC detection (detection wavelength: 245nm; mobile phase: 40% acetonitrile; flow rate: 1mL/min; sample injection amount: 10 mu L; column specification: C18 reverse column (5 mu m,4.6mm x 25 cm) is used for finding out fraction sections containing Nematophin by comparison with standard substances, petroleum ether-ethyl acetate system (V petroleum ether ：V Acetic acid ethyl ester =20:1, 10:3, 2:1) is used for eluting, V petroleum ether ：V Acetic acid ethyl ester =2:1 fraction is collected, repeated recrystallization is carried out to obtain compound Nematophin, and the purity is detected to be more than 90%.
Example 7:
Fermenting and culturing X.nematophila YL001, enriching 5L of fermentation liquor, 12000 Xg, centrifuging for 20min at 4 ℃ to obtain supernatant.
Referring to the separation method of the compound Nematophin by Li et al (1997), the above concentrate was extracted four times with ethyl acetate, and the extract was concentrated under reduced pressure at 30℃and 0.1MPa after removing water by anhydrous sodium sulfate. The concentrated fractions were then subjected to column chromatography on silica gel (200 g silica gel, column 40 x 5 cm) with an eluent of chloroform-methanol system (V Trichloromethane ：V Methanol = 100:0, 80:20, 50:50, 20:80, 0:100), 100% chloroform eluted fractions were collected by reference to the method of Li et al (1997), and the detection results showed that Nematophin had a purity of less than 50%.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (3)
1. A process for the isolation of 3-indoloethyl (3 '-methyl-2' -one) pentanamide comprising: separating bacterial concentrate of Xenorhabdus bacteria (Xenorhabdus) by column chromatography, collecting eluent containing 3-indolylethyl (3 '-methyl-2' -ketone) valeramide, concentrating the eluent under reduced pressure and extracting with solvent to obtain solvent extract containing 3-indolylethyl (3 '-methyl-2' -ketone) valeramide;
when the Xenopharacterium bacteria (Xenorhabdus) are Xenopharacterium nematophilum X. nematophila ALL, the column chromatography separation process is as follows: re-dissolving the bacterial concentrate of the xenorhabdus nematophilus X. nematophila ALL with 1.5-2 times of water, statically adsorbing for 12h by macroporous resin, eluting the statically adsorbed macroporous resin chromatographic column with 5 times of column volume distilled water, 1 time of column volume 50% methanol, 1 time of column volume 80% methanol and 3 times of column volume methanol in sequence, and collecting methanol eluent;
When the Xenopharacterium bacteria (Xenorhabdus) is Xenopharacterium nematophilum X. nematophila YL001, the column chromatography separation process is as follows: re-dissolving the bacterial concentrate of the xenorhabdus nematophilus X. nematophila YL001 with 1.5-2 times of water, statically adsorbing for 12h by macroporous resin, eluting the statically adsorbed macroporous resin chromatographic column with 5 times of column volume distilled water, 1 time of column volume of 50% acetone, 1 time of column volume of 80% acetone and 3 times of column volume acetone in sequence, and collecting acetone eluent; or sequentially eluting with 5 times of column water-0.01 n hydrochloric acid and 3 times of column volume 30% methanol-0.01 n hydrochloric acid to obtain macroporous resin chromatographic column, and collecting 30% methanol-0.01 n hydrochloric acid section eluate;
when the Xenopharacterium bacteria (Xenorhabdus) are Xenopharacterium nematophilum X. nematophila AN6, the column chromatography separation process is as follows: re-dissolving the bacterial concentrate of the xenorhabdus nematophilus X. nematophila AN6 with 1.5-2 times of water, statically adsorbing for 12h by macroporous resin, eluting the statically adsorbed macroporous resin chromatographic column with 5 times of column volume distilled water, 1 time of column volume 50% ethanol, 1 time of column volume 80% ethanol and 3 times of column volume ethanol in sequence, and collecting ethanol eluent;
when the Xenopharacterium bacteria (Xenorhabdus) are Xenopharacterium nematophilum X. nematophila A24, the column chromatography separation process is as follows: re-dissolving the bacterial concentrate of the xenorhabdus nematophilus X. nematophila A24 with 1.5-2 times of water, statically adsorbing for 12h by macroporous resin, eluting the statically adsorbed macroporous resin chromatographic column by 5 times of column water-0.01 n hydrochloric acid and 3 times of column volume of 30% acetone-0.01 n hydrochloric acid in sequence, and collecting 30% acetone-0.01 n hydrochloric acid section eluent;
the eluent is concentrated under reduced pressure and extracted by solvent: concentrating the eluate obtained after column chromatography under reduced pressure at 40deg.C and 0.1Mpa, and redissolving with distilled water of 5-7 times of the volume of the concentrate to obtain extract; adding solvent into an extraction object for three times, wherein the solvent is petroleum ether, the volume ratio of the solvent to the extraction object is 1:1, the extraction time is 2 hours each time, the solvent and the extraction object are uniformly mixed every 30 min, and the solvent extract is collected after the extraction is finished.
2. The method for separating 3-indoloethyl (3 '-methyl-2' -ketone) valeramide according to claim 1, wherein said macroporous resin is selected from the group consisting of XAD-11 macroporous resin, XAD-12 macroporous resin, porapak S macroporous resin, HPD-600 macroporous resin, XAD-9 macroporous resin, XAD-10 macroporous resin, XAD-7HP macroporous resin, LSA-10 macroporous resin, LX-28 macroporous resin, AB-8 macroporous resin, DA macroporous resin, X-5 macroporous resin, D320 macroporous resin, D101 macroporous resin, CAD-40 macroporous resin, GDX-105 macroporous resin, D macroporous resin and DM2 macroporous resin.
3. The method for separating 3-indoloethyl (3 '-methyl-2' -keto) pentanamide of claim 1, wherein the preparation of the bacterial concentrate comprises:
① Seed liquid preparation: culturing strain in LB medium at 28deg.C and shaking table rotation speed of 180 r/min to logarithmic phase, culturing at room temperature on NBTA plate by streaking method for 48: 48 h, selecting blue single colony in LB medium, culturing at 28deg.C and rotation speed of 180 r/min, and culturing to logarithmic phase;
② Fermentation culture: inoculating 10% (V/V) seed solution into TSB culture medium, and culturing at 28deg.C and shaking table rotation speed of 180 r/min for 3d to obtain fermentation broth; centrifuging the fermentation broth at 9000 r/min for 15: 15min, collecting supernatant, and concentrating under reduced pressure at 40deg.C and 0.1Mpa to obtain bacterial concentrate.
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