CN110092790B - Alkaloid compound and preparation method and application thereof - Google Patents
Alkaloid compound and preparation method and application thereof Download PDFInfo
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- CN110092790B CN110092790B CN201910501587.7A CN201910501587A CN110092790B CN 110092790 B CN110092790 B CN 110092790B CN 201910501587 A CN201910501587 A CN 201910501587A CN 110092790 B CN110092790 B CN 110092790B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/10—Spiro-condensed systems
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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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
Abstract
The invention discloses an alkaloid compound and a preparation method and application thereof, and relates to a compound and a preparation method and application thereof, wherein the alkaloid compound has a chemical structural formula shown as a formula I:
Description
Technical Field
The invention relates to a compound, a preparation method and application thereof, in particular to an alkaloid compound, and a preparation method and application thereof.
Background
Viral diseases are highly transmissible and seriously harm human health and life. AIDS caused by human immunodeficiency disease (HIV) discovered in the 80 th of the 20 th century is an infectious disease with great harmfulness and high mortality rate. In 2003, a new coronavirus causing Severe Acute Respiratory Syndrome (SARS) was discovered, which is highly contagious and highly lethal. At present, there is no drug which can effectively treat the disease. Therefore, the research of antiviral drugs has become one of the hot spots of the current international research. The medicines commonly used in clinic at present mainly comprise the following medicines: anti-influenza virus and respiratory virus drugs: anti-herpes virus drugs: anti-cytomegalovirus drugs; anti-hepatitis virus drugs: anti-HIV drugs. Antiviral drugs have been treated for nearly 30 years, and since viruses enter the body and replicate and propagate in host cells by mimicking the biochemical mechanism of the host, drugs that inhibit virus propagation have different degrees of damage to the host cells or the body, making the development of antiviral drugs slower than antibiotics. To date, there are no fully satisfactory antiviral agents.
Plant viruses are an important pathogen which harms crops, and are commonly called plant cancers because of great harm and difficulty in prevention and treatment. Because the absolute parasitism of plant viruses to plant cells, substances, energy places and the like required by virus replication are completely provided by hosts, the viruses can stress the biochemical mechanism of the host cells and are entangled with the biochemical mechanism of the viruses to be difficult to identify, so that the drugs are difficult to selectively attack the viruses without damaging the host cells, and the research of high-selectivity chemical antiviral preparations faces great difficulty, and still has no major breakthrough.
In recent years, the research on natural antiviral drugs at home and abroad is increasing, and the screening of the active ingredients from the natural antiviral drugs becomes a hot spot in the research and development of new antiviral drugs at present, and the research and development of new antiviral drugs are greatly advanced. At present, the research on antiviral natural medicines mainly focuses on plant active ingredients, but the research on the antiviral metabolites of microorganisms is very little, so that the microorganisms have great potential as natural treasury to be discovered and researched.
Disclosure of Invention
In order to improve the current situation, the invention provides a novel alkaloid compound obtained from Streptomyces gamamensis and a preparation method and application thereof.
Firstly, the invention provides an alkaloid compound, the chemical structural formula of which is shown as formula I:
secondly, the present invention provides a method for preparing the above alkaloid compounds, comprising the steps of:
(1) fermenting and culturing streptomyces (streptomyces gamamensis) to prepare fermentation liquor;
(2) centrifuging the fermentation liquor obtained in the step (1) to obtain thalli and supernatant, and adsorbing and eluting the supernatant by resin to obtain organic solvent eluent;
(3) concentrating the organic solvent eluent obtained in the step (2), and carrying out silica gel column chromatography and gel column chromatography to obtain a fraction sample containing the alkaloid compound;
(4) and (4) carrying out reversed-phase preparative column chromatography on the fraction sample obtained in the step (3) to obtain the alkaloid compound.
Performing fermentation culture in the step (1), wherein a carbon source in a culture medium comprises one or more than two of glucose, soluble starch, corn starch, dextrin, industrial molasses, glycerol, sucrose, sorbitol, mannitol, lactose, maltose or xylan; the nitrogen source in the culture medium comprises one or more of yeast powder, yeast extract, soybean cake powder, soybean powder, peptone, beef extract, yeast extract, corn steep liquor dry powder, bran, gluten powder, urea or ammonium salt.
Preferably, the fermentation culture in the step (1) is carried out, and the carbon source in the culture medium is a mixture of glucose and soluble starch or a mixture of glucose and corn starch; the nitrogen source in the culture medium is a mixture of yeast powder and soybean cake powder.
The 16S rRNA of the strain involved in the step (1) is registered with the Genbank number of KT963951, which is provided by the university of northeast agriculture and chemical laboratories and is deposited in the China general microbiological culture Collection center with the preservation number of CGMCC No.4.7304 (the strain is disclosed in the journal: Antonie Van L eewenhoek.2017; 110(4):471-477.Streptomyces gammahensis. nov., alpha. actinomycete with immobilized cellulose isolated from Gama, Chad. Shanghan Zha, L an Ye, Chongxi L iu, Aducouba, Weijiang Acagan, Weiiang Zhai, Shanghan Xgang, Shanghan Xi).
The resin in the step (2) is macroporous adsorption resin, preferably HP-20 macroporous adsorption resin; the organic solvent used for elution comprises one or two or three of acetone, methanol and ethanol.
And (3) performing silica gel column chromatography by using silica gel with the particle size of 100-200 meshes and using a solution of acetone and n-hexane (V/V) in a ratio of 0:100-50:50 as an elution solution, wherein the gel column chromatography is performed by using a gel column Sephadex L H-20 and using a solution of chloroform and methanol in a ratio of 1: 1 as an elution solution.
And (4) performing reverse-phase preparative column chromatography by using a C18 reverse-phase filler, wherein the used elution solvent is acetonitrile aqueous solution, methanol aqueous solution or a mixed solution of methanol, acetonitrile and water.
Thirdly, the invention also provides the application of the alkaloid compound in the preparation of antiviral compositions or antiviral drugs.
The virus is tobacco mosaic virus, cucumber mosaic virus and the like.
Advantageous effects
Compared with commercial antiviral drug Ningnanmycin, the novel alkaloid compound HX117A obtained by the invention shows equivalent or stronger antiviral activity to cucumber mosaic virus and tobacco mosaic virus, and can be used for developing antiviral medicaments.
Detailed Description
The fermentation culture of the alkaloid compound producing strain Streptomyces gamamensis and the extraction, separation and activity experiments of the alkaloid compound are shown in the following specific examples. The following examples are further illustrative of the present invention but do not limit the invention.
Example 1
(1) Fermenting and culturing streptomyces (Streptomyces gamamensis), preparing fermentation liquor:
1) fermentation strain: the fermentation strain is streptomyces gamamensis.
2) Slant culture: sterilizing ISP2 culture medium (yeast extract 4.0g, wort 10.0g, glucose 4.0g, agar 20.0g, distilled water 1000ml, pH 7.0-7.2) at 121 deg.C for 20min, inoculating, and culturing at 28 deg.C for 6-8 days.
3) Seed culture, wherein the seed culture medium comprises glucose 4.0 g/L, malt extract powder 10.0 g/L, and yeast powder 4.0 g/L30.2 g/L, 1000ml of distilled water, pH 7.0, 250ml per bottle in 1000ml triangular flask, washing off the spores of Streptomyces on the slant with 12ml of sterile water and making into spore suspension with concentration of 1 × 107-1×108Per bottle/m L, 2ml of spore suspension is added into each bottle, and the mixture is put on a shaker, the rotating speed is 250r/min, and the mixture is cultured for 24 hours at the temperature of 28 ℃.
4) Fermentation culture, wherein the fermentation culture medium comprises yeast powder 0.5 g/L, corn starch 3.0 g/L, glucose 1.0 g/L, soybean cake powder 2.0 g/L0.1.1 g/L, and K2HPO40.2g/L，MgSO4·7H2O 0.1g/L，CaCO30.2 g/L, pH 7.2-7.4, preparing with distilled water, sterilizing at 121 deg.C for 20min, inoculating seed solution into 50L fermenter (30L volume) at 28 deg.C, stirring at 100r/min, and ventilating at 120m3H, culturing for 6-7 days.
(2) And (2) centrifuging the fermentation liquor obtained in the step (1) to obtain thalli and supernatant, adsorbing the supernatant by using HP-20 macroporous resin, washing by using water, and eluting by using 80% ethanol to obtain ethanol eluent.
(3) Concentrating the organic solvent eluent obtained in the step (2), performing silica gel column chromatography and gel column chromatography to obtain a fraction sample containing the alkaloid compound, concentrating the collected ethanol eluent at 50 ℃ under reduced pressure to remove an ethanol phase until the ethanol phase is dried to obtain 69g of oily substance, performing column chromatography on the obtained oily substance on a silica gel column (the particle size is 100-200 meshes), eluting with n-hexane and acetone (50: 50(V/V), detecting by T L C to obtain 3 components (1-3), and performing gel L H-20 column chromatography on the component 2 (chloroform/methanol (1: 1, V/V)) to obtain the components 2-2.
(4) Subjecting the fraction obtained in step (3) to reverse-phase preparative column chromatography to obtain the alkaloid compound of claim 1:
the conditions were as follows:
liquid phase system: the Agilent 1100 semi-preparative high pressure liquid chromatograph;
chromatographic column SepaxAlmethyl C18-H (4.6mm × 250 mm);
eluent acetonitrile/water (18: 82(V/V), flow rate (1.0 m L/min);
detection wavelength: λ 220 nm;
collecting the peak with retention time of 9.9min to obtain the alkaloid compound, which is named as compound HX 117A.
Structural identification of compound HX117A
The structure of compound HX117A was determined by 1D and 2D NMR, MS etc. spectroscopy as follows:
the physicochemical properties of compound HX117A are as follows:
the characteristics are as follows: white powder
Solubility: easily soluble in methanol and acetone, slightly soluble in water, and insoluble in petroleum ether
The molecular formula is as follows: c9H13N5O4
Specific rotation degree:
high resolution mass spectrometry (HRESI-MS) 278.0874[ M + Na [ ]]+(calcd for C9H13N5O4Na 278.0870)
Ultraviolet absorption spectrum (UV absorbance spectrum) lambdamax(EtOH)nm(log)：209(4.72)
Infrared absorption Spectrum (IR absorption Spectrum) Vmaxcm-1：3409，3222，2953，1730，1459，1391，1329，1025，911
Melting Point (. degree. C.): 189-192
Of compound HX117A1H and13C NMR(CDCl3) The data are shown in Table 1.
TABLE 1 Nuclear magnetic data of Compound HX117A (hydrogen spectrum: 400 MHz; carbon spectrum: 100MHz)
Example 2
Antiviral Activity of Compound HX117A
Selecting 4-6 leaf period heart leaf tobacco as a dead spot host of tobacco mosaic virus TMV or cucumber mosaic virus CMV, inoculating whole leaf of the heart leaf tobacco by a conventional juice rubbing method, carrying out HX117A liquid medicine spraying treatment 2 days after inoculation (the treatment process of a control group is the same), checking the incidence condition 14 days later, calculating the inhibition activity of a compound HX117A on the Tobacco Mosaic Virus (TMV), and taking tiadinil (TD L), Benzothiadiazole (BTH) and Ningnanmycin (Ningnanmycin) as control groups, wherein the results are shown in a table (table 2) and the inhibition effects of the compound HX117A in the table 2 on the Tobacco Mosaic Virus (TMV) and the Cucumber Mosaic Virus (CMV) are shown in the table 2
From the results, it was found that compound HX117A exhibited a very strong inhibitory activity against cucumber mosaic virus at a concentration of 100 mg/L, with an inhibition rate of 72.34%, which was higher than the antiviral activity of ningnanmycin at the same concentration (inhibition rate of 57.11%), and that compound HX117A exhibited a very strong inhibitory activity against tobacco mosaic virus at a concentration of 400 mg/L, with an inhibition rate of 80.06%, but at a concentration of 100 mg/L, the inhibition rate against tobacco mosaic virus (62.09%) was slightly lower than the antiviral activity of ningnanmycin at the same concentration (67.56%).
Claims (10)
1. An alkaloid compound characterized by: the chemical structural formula is shown as the formula
Shown in the figure:
formula (II)
。
2. A process for the preparation of alkaloid compounds according to claim 1, characterized in that: the method comprises the following steps:
(1) fermentation culture of Streptomyces (Streptomyces gamaensis) Preparing fermentation liquor;
(2) centrifuging the fermentation liquor obtained in the step (1) to obtain thalli and supernatant, and adsorbing and eluting the supernatant by resin to obtain organic solvent eluent;
(3) concentrating the organic solvent eluent obtained in the step (2), and carrying out silica gel column chromatography and gel column chromatography to obtain a fraction sample containing the alkaloid compound;
(4) subjecting the fraction obtained in step (3) to reverse-phase preparative column chromatography to obtain the alkaloid compound of claim 1.
3. The process for producing alkaloid compounds according to claim 2, wherein: performing fermentation culture in the step (1), wherein a carbon source in a culture medium comprises one or more than two of glucose, soluble starch, corn starch, dextrin, industrial molasses, glycerol, sucrose, sorbitol, mannitol, lactose, maltose or xylan; the nitrogen source in the culture medium comprises one or more of yeast powder, yeast extract, soybean cake powder, soybean powder, peptone, beef extract, yeast extract, corn steep liquor dry powder, bran, gluten powder, urea or ammonium salt.
4. The process for producing alkaloid compounds according to claim 2, wherein: performing fermentation culture in the step (1), wherein a carbon source in a culture medium is a mixture of glucose and soluble starch or a mixture of glucose and corn starch; the nitrogen source in the culture medium is a mixture of yeast powder and soybean cake powder.
5. The process for producing alkaloid compounds according to claim 2, wherein: the resin in the step (2) is HP-20 macroporous adsorption resin; the organic solvent used for elution comprises one or two or three of acetone, methanol and ethanol.
6. The process for producing alkaloid compounds according to claim 2, wherein: the silica gel column chromatography in the step (3) uses silica gel with the particle size of 100-200 meshes, and uses a solution of acetone-n-hexane (V/V) =100-50:50 as an elution solution for elution.
7. The process for producing alkaloid compounds according to claim 2, wherein the gel column chromatography in step (3) is performed using Sephadex L H-20, and a solution of chloroform-methanol (V/V) = 1: 1 is used as an elution solution.
8. The process for producing alkaloid compounds according to claim 2, wherein: and (4) performing reverse-phase preparative column chromatography by using a C18 reverse-phase filler, wherein the used elution solvent is acetonitrile aqueous solution, methanol aqueous solution or a mixed solution of methanol, acetonitrile and water.
9. Use of the alkaloid compounds of claim 1 for the preparation of an antiviral composition or a medicament.
10. Use of the alkaloid compounds according to claim 9 for the preparation of antiviral compositions or antiviral medicaments, characterized in that: the virus is one or two of tobacco mosaic virus and cucumber mosaic virus.
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