CN101239982B - Alkaloids compounds with hepatitis virus resisting activity - Google Patents
Alkaloids compounds with hepatitis virus resisting activity Download PDFInfo
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- CN101239982B CN101239982B CN2007100567318A CN200710056731A CN101239982B CN 101239982 B CN101239982 B CN 101239982B CN 2007100567318 A CN2007100567318 A CN 2007100567318A CN 200710056731 A CN200710056731 A CN 200710056731A CN 101239982 B CN101239982 B CN 101239982B
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Abstract
The invention relates to a novel alkaloid compound (I) with anti-hepatitis activity, preparation and usage thereof. The novel compound (I) related to in the invention is a alkaloid compound metabolized by mixed culture with Aspergllus niger fungi which uses swertiamarin as raw material. As the pharmacological test indicated, the inventive compound has a prominence anti-inflammatory effect, and can be used in preparation of medicament for treatment of hepatitis.
Description
Technical field
The present invention relates to medical technical field, being specifically related to the swertiamarin is raw material, and by the new compound that the metabolism with fungi Aspergillus niger mixed culture generates, its skeleton belongs to alkaloids.The present invention also relates to the preparation method of this compound; The invention still further relates to the purposes of this compound aspect the preparation medicine, particularly in fields such as preparation Antihepatitis medicaments.
Background technology
Hepatitis is great in the world communicable disease, is ascendant trend gradually in recent years.China is hepatitis big country, and there were 3.5 hundred million hepatitis B virus carrierss in the whole world in 2006, in state-owned nearly 1.2 hundred million; Hepatitis C virus carrier 1.7 hundred million, China also have nearly 2,000 ten thousand.The whole world had 2,000,000 people to die from hepatitis B cancer and the end-age cirrhosis relevant with virus approximately in 2000, and also there are nearly 300,000 people's death in China.ZANGYINCHEN derives from Gentianaceae (Gentianaceac) Swertia (Swertia) plant, and this genus plurality of Chinese, ethnic drug and medicine among the people all have the clinical settings of long-term treatment hepatitis.ZANGYINCHEN has all obtained good effect in treatment posthepatitic hyperbilirubinemia, acute icterohepatitis hepatitis and chronic hepatitis.Swertiamarin is a main effective constituent in the ZANGYINCHEN.To the metabolism of swertiamarin research existing report since 20th century the eighties, the separated evaluations of compound such as erythrocentaurin, erythricine have been arranged successively.We are separating in the metabolism research in fungi Aspergillus niger at swertiamarin and are identifying that it serves as that the main preparation of forming has good anti-inflammatory action that a new water miscible compound, pharmacodynamic study show with this compound.At this compound, be determined by experiment rational technology of preparing, utilize the product of this technology preparation can make the content of this compound be higher than 70%.
Summary of the invention
The purpose of this invention is to provide a kind of is raw material with the swertiamarin, prepares a new Compound I by metabolism, separation:
The Compound I called after (4-(1-Methylaminpmethyl-vinyl)-5,6-dihydropyran-2-one).Molecular composition: C
10H
12O
3N; Molecular weight: 195; UV spectrum UV: λ max:279.2nm.High resolution mass spec: ESI-MS (int.rel): 194.2165 (100, M+), 178.1823 (81, [M+-CH3]), 150.2017 (62, [M+-CO2]), 132.1857 (12, [M+-CO2-H2O]), 104.1323 (51), 90.105 (52).Proton nmr spectra
1H NMR (400MHz, CDCl
3) δ: 3.41 (2H, t, J=6.0Hz, 6-H2), 4.36 (2H, t, J=6.0Hz, 7-H2), 7.42 (1H, t, J=7.7Hz, 10-H), 7.87 (1H, dd, J=7.7Hz, J=1.3Hz, 8-H), 8.23 (1H, dd, J=7.7Hz, J=1.3Hz, 3-H), 10.3 (1H, s, 1-H).Carbon-13 nmr spectra
13C NMR (CDCl
3, 100MHz) δ: 24.2 (C-6), 66.6 (C-7), 127.1 (C-5), 127.8 (C-10), 132.6 (C-4), 135.8 (C-3), 138.2 (C-8), 141.0 (C-9), 164.7 (C-11), 191.5 (C-1).
According to above-mentioned physico-chemical property, in conjunction with two dimensional NMR spectrum HMBC, HSQC and COSY, the structure of proving conclusively this compound is as follows, i.e. formula (I).
Another object of the present invention provides the preparation method of above-claimed cpd, and preparation method of the present invention can make the content of this compound be higher than 90%, may further comprise the steps:
1, swertiamarin solution is added in the fungi Aspergillus niger nutrient solution, the ultimate density that makes swertiamarin is 0.5mg/ml, and at 37 ℃, 160rpm cultivated 5 days down.Nutrient solution collection, filtration, concentrating under reduced pressure;
2, above-mentioned concentrated solution is centrifugal, supernatant liquor with petroleum ether extraction after, with trichloromethane or ethyl acetate extraction 3 times, extraction liquid merges, and reclaims solvent to doing again;
3, residue is soluble in water, filter supernatant liquor, this supernatant liquor is used to prepare liquid phase separation, the preparation liquid-phase condition be: moving phase: A (0.04% formic acid/acetonitrile solution) and B (the 0.04% formic acid/aqueous solution); Gradient condition is: 0～10min 15%A, 10～30min 15%A～24%A, 30～40min 24%A～100%A.Flow velocity is 20ml/min.The preparation liquid phase separation receives liquid concentrating under reduced pressure, lyophilize, and through once preparing liquid phase production, the preparation liquid-phase condition is dry thing: the 15%A constant gradient again.Flow velocity is 20ml/min.Receive liquid concentrating under reduced pressure, lyophilize, dry purity is 98% compound (I).
The content of the compound (I) that aforesaid method obtains is greater than 98%.
The method for preparing content and be more than 70% is as follows:
1, swertiamarin solution is added among the fungi Aspergillus niger, the ultimate density that makes swertiamarin is 0.5mg/ml, and at 37 ℃, 160rpm cultivated 5 days down.Nutrient solution collection, filtration, concentrating under reduced pressure;
2, above-mentioned concentrated solution is centrifugal, supernatant liquor with petroleum ether extraction after, with trichloromethane or ethyl acetate extraction 3 times, extraction liquid merges, and reclaims solvent to doing again;
3, residue is soluble in water, filter supernatant liquor, this supernatant liquor is with macroporous resin adsorption, earlier with the water elution of 2 times column volume, again with the ethanol elution of 5 times of column volumes, 50 ℃ of concentrating under reduced pressure.
4, with dextrane gel Sephades G-25 on the concentrated solution, with the methanol-eluted fractions of 2 times of column volumes, collect corresponding colour band, 50 ℃ of drying under reduced pressure promptly get content greater than 70% compound (I).
Above-mentioned preparation method be a kind of indefiniteness for example, the present invention includes any compound (I) content that makes greater than 70% metabolism, method for separating and preparing.
Another object of the present invention provides the application of compound (I) in Antihepatitis medicament.
In order to have observed this medicine better at the action effect aspect the anti-hepatitis, the present invention has designed following pharmacodynamics test.
(1) experiment material
1, medicine and reagent The compounds of this invention, white powder, self-control.
2, laboratory animal Wistar kind rat, animal housing provides by Tianjin Inst. of Materia Medica.
(2) experimental technique and result
Medicine p-Xylol induced mice auricle edema of the present invention influence the anti-inflammatory pharmacodynamic experiment:
Get body weight and be 60 of the Wistar kind rats of 100～140g, male and female and usefulness are divided into 6 groups at random, 10 every group.Control group is a distilled water, is the preparation that contains 50% The compounds of this invention for reagent.Each organizes rat 4 days gastric infusions in advance, and once a day, dosage is 30mg/kg.In the later half h of last administration, only inject left side foot with 10% fresh albumen 0.05ml/ and cause inflammation, cause scorching 0.5h, 1h, 2h, 3h, 4h, measure the ankle joint circumference with the arrowband chi, doing contrast with right side foot, is the swelling degree with ankle joint circumference before and after the administration, respectively organizes swelling degree difference.
Table 1 The compounds of this invention is to the influence of rat egg white pedal swelling
＊P＜0.05；
＊＊P＜0.001
As can be seen from Table 1, cause in the scorching back 0.5h, the difference of the swelling degree of administration group and control group is (P＜0.001) extremely significantly; The difference of two groups of swelling degree also significantly (P＜0.05) this shows The compounds of this invention under the dosage of 30mg/kg in from 1h to 4h, and Wistar kind rat egg white pedal swelling is had significant effect amount.
Description of drawings
Fig. 1 is the HMBC spectrogram of Compound I;
Fig. 2 is the COSY spectrogram of Compound I;
Fig. 3 is the hsqc spectrum figure of Compound I.
Embodiment
The present invention is further illustrated with the test example by the following examples, but the present invention is not subject to these embodiment.
Swertiamarin (90%) is dissolved in is made into the swertiamarin solution that concentration is 50mg/ml in the methyl alcohol, standby.Aspergillus niger bacterial classification inoculation is in nutrient solution, and the composition of nutrient solution is as follows: glucose 8g/L, peptone 2.58g/L, yeast extract paste 2.58g/L, MgSO
47H
2O 18g/L, MnSO
44H
2O 18g/L, K
2HPO
418g/L, NaCl 2.58g/L, pH 6.0.37 ℃, 160rpm cultivated 2 days, and the amount with 2% adds swertiamarin solution, continued to cultivate 5 days, and cultivation finishes the back and collects nutrient solution, filtration, and filtrate is evaporated to 1/10th of original volume under 50 ℃ of conditions.Concentrated solution filters, and supernatant liquor is used sherwood oil, chloroform extraction 3 times successively.Chloroform extraction liquid merges, and merges evaporated under reduced pressure, and residue is dissolved in 15% the acetonitrile solution, is used to prepare liquid phase separation.Preparation liquid phase separation condition is: moving phase: A (0.04% formic acid/acetonitrile solution) and B (the 0.04% formic acid/aqueous solution); Gradient condition is: 0～10min 15%A, 10～30min, 15%～24%A, 30～40min, 24%～100%A.Flow velocity is 20ml/min.The preparation liquid phase separation receives liquid concentrating under reduced pressure, lyophilize, and through once preparing liquid phase production, the preparation liquid-phase condition is dry thing: the 15%A constant gradient again.Flow velocity is 20ml/min.Receive liquid concentrating under reduced pressure, lyophilize, dry purity is 98% compound (I).
Swertiamarin (50%) is dissolved in is made into the swertiamarin solution that concentration is 50mg/ml in the methyl alcohol, standby.The Aspergillusniger bacterial classification inoculation is in nutrient solution, and the composition of nutrient solution is as follows: glucose 8g/L, peptone 2.58g/L, yeast extract paste 2.58g/L, MgSO
47H
2O 18g/L, MnSO
44H
2O 18g/L, K
2HPO
418g/L, NaCl 2.58g/L, pH 6.0.37 ℃, 160rpm cultivated 2 days, and the amount with 2% adds swertiamarin solution, continued to cultivate 5 days, and cultivation finishes the back and collects nutrient solution, filtration, and filtrate is evaporated to 1/10th of original volume under 50 ℃ of conditions.Concentrated solution filters, and supernatant liquor is used sherwood oil, chloroform extraction 3 times successively.Chloroform extraction liquid merges, merge evaporated under reduced pressure, in the molten thing water of residue, filter supernatant liquor, this supernatant liquor injects the macroporous resin column of having handled (ratio of medicinal mixture and resin is 1: 20), earlier with the water elution of 2 times column volume, again with the ethanol elution of 5 times of column volumes, 50 ℃ of concentrating under reduced pressure of ethanol.With dextrane gel Sephades G-25 on the concentrated solution, with the methanol-eluted fractions of 2 times of column volumes, collect corresponding colour band, 50 ℃ of drying under reduced pressure promptly get content greater than 70% compound (I).
Claims (7)
1. compound, its chemical structure is as follows:
2. the preparation method of the compound of claim 1 may further comprise the steps:
A, be raw material with the swertiamarin, through with fungi Aspergillus niger nutrient solution mixed culture 5 days, nutrient solution collection, filtration, concentrating under reduced pressure;
B, above-mentioned concentrated solution is centrifugal, supernatant liquor with petroleum ether extraction after, with trichloromethane or ethyl acetate extraction 3 times, extraction liquid merges, and reclaims solvent to doing again;
C, residue is soluble in water, filter supernatant liquor, this supernatant liquor further separates with column chromatography, the preparation liquid phase separation receives liquid concentrating under reduced pressure, lyophilize, and to get purity be 98% compound (I).
3. the application of the compound of claim 1 in the antiphlogistic medicine of preparation.
4. the pharmaceutical composition that contains the compound of the claim 1 for the treatment of significant quantity.
5. according to the pharmaceutical composition of claim 4, wherein the content of compound (I) is greater than 50%.
6. according to the pharmaceutical composition of claim 5, wherein the content of compound (I) is greater than 70%.
7. according to the pharmaceutical composition of claim 6, wherein the content of compound (I) is greater than 90%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100567318A CN101239982B (en) | 2007-02-06 | 2007-02-06 | Alkaloids compounds with hepatitis virus resisting activity |
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|---|---|---|---|
| CN2007100567318A CN101239982B (en) | 2007-02-06 | 2007-02-06 | Alkaloids compounds with hepatitis virus resisting activity |
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| CN101239982A CN101239982A (en) | 2008-08-13 |
| CN101239982B true CN101239982B (en) | 2010-08-11 |
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| CN2007100567318A Expired - Fee Related CN101239982B (en) | 2007-02-06 | 2007-02-06 | Alkaloids compounds with hepatitis virus resisting activity |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1704086A (en) * | 2004-06-03 | 2005-12-07 | 昆明聚智达医药技术有限公司 | Method for preparing drug material of swertiamarin |
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2007
- 2007-02-06 CN CN2007100567318A patent/CN101239982B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1704086A (en) * | 2004-06-03 | 2005-12-07 | 昆明聚智达医药技术有限公司 | Method for preparing drug material of swertiamarin |
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