CN106008483A - Preparation and application of isovitexin - Google Patents
Preparation and application of isovitexin Download PDFInfo
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- CN106008483A CN106008483A CN201610405166.0A CN201610405166A CN106008483A CN 106008483 A CN106008483 A CN 106008483A CN 201610405166 A CN201610405166 A CN 201610405166A CN 106008483 A CN106008483 A CN 106008483A
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- alcohol
- isovitexin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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Abstract
The invention specifically relates to a preparation method and novel application of a C-glycosylflavone compound, which belongs to the fields of medicine and chemistry of natural products. According to the invention, normal-phase (silica gel) column chromatography is employed for separation at first and inversed-phase (open ODS) column chromatography for separation next, and an isovitexin crystal is naturally precipitated. A dry-process sample introduction way is employed during separation via silica gel column chromatography, while a wet-process sample introduction way is employed during separation via open ODS column chromatography. The invention also provides application of isovitexin to myocardial preservation.
Description
Technical field
The invention belongs to medicine and field of natural product chemistry, be specifically related to the preparation method of a kind of flavone c-glycosides and new
Purposes.
Background technology
Isovitexin is a typical flavone c-glycoside, universally present in veterinary antibiotics and Chinese herbal medicine, such as Verbenaceae
The leaf of shore Herba Viticis Cannabifoliae (Vitex littoralis A.Cunn.), leguminous plant Fructus Gleditsia (Gleditsia sinensis Lam.) and Herba Lespedezae Cuneatae (Lespedeza
Cuneata G.Don) leaf, and the seed etc. of Semen phaseoli radiati (Vigna radiate L.).Numerous studies show, isovitexin has anti-
Oxidation, antibacterial, antipathogen and blood sugar lowering isoreactivity, but the myocardium protecting action of isovitexin at present there is not been reported.
In recent years, the filler such as silica gel and ODS is reformed at extracting and developing purification, the preparation process of Natural Medicine Chemistry composition, is made
The aspects such as agent quality analysis have had wide applied research, and are explicitly shown out the effect of its uniqueness.Utilize silica gel and ODS's
The similar compatibility principle of contrary adsorption function and material can from Chinese medicine extraction liquid separation and purification effective ingredient or effective site,
Discard the dross and select the essential to limits, promote the development of modernization of cmm.
Give isoproterenol (isoprenaline, ISO) can induced rat generation Damage of Myocardial Ischemia, its electrocardiogram,
The change of the aspect such as Myocardial Enzymologic and histopathology is similar to the characteristics of lesion of acute human myocardial ischemia, it has also become cardiovascular disease
A kind of important animal model of sick research field.
Summary of the invention
It is an object of the present invention to provide the preparation method of isovitexin.Specifically comprise the following steps that
(1) take Semen phaseoli radiati appropriate, with ethanol or methanol extraction, filter, merging filtrate, obtain alcohol extract；By alcohol extract recovered under reduced pressure
Alcohol, to without alcohol taste, obtains crude extract；
(2) crude extract obtained extracts with opposed polarity organic solvent successively, discards extract, then with n-butanol extraction, closes
And butanol extraction liquid, recovered under reduced pressure n-butyl alcohol, obtain Semen phaseoli radiati extract；
(3) Semen phaseoli radiati extract of gained, disperses by proper amount of methanol, adds proper silica gel, stirs, and loads and anticipates
Silicagel column in, carry out gradient elution with the mixed organic solvents (100:0～0:100) of two kinds of organic solvents mixing, collect mixing
Organic solvent (50:50～30:70) eluent, is concentrated into thick, adds ethanol in proper amount or methanol dispersion；Upper open ODS post,
With alcohol-water (0:100～100:0), preferably alcohol-water (10:90～40:60), carry out gradient elution, collect alcohol-water (35:65～40:60)
Eluent, stands overnight precipitation yellow crystal, is isovitexin, and purity is more than 95%.
In the present invention, the concentration of the ethanol described in step (1) or methanol is 30%～100%.
In the present invention, the extraction described in step (1) is 30%～100% ethanol by 5～20 times amount or methanol homogenate extraction 1～3
Secondary, and united extraction liquid.
In the present invention, the opposed polarity organic solvent described in step (2) include but not limited to: petroleum ether, hexamethylene,
Dichloromethane, chloroform, ethyl acetate.
In the present invention, the opposed polarity organic solvent described in step (2), the consumption of n-butyl alcohol are 0.5～2 times of volume, extraction
Number of times is 1～5 time.
In the present invention, the mixed organic solvents described in step (3) is methylene chloride-methanol, or chloroform-methanol, or dichloromethane
Alkane-ethanol, or chloroform-ethanol.
In the present invention, the alcohol-water described in step (3) is methanol-water or alcohol-water.
In the present invention, first use positive (silica gel) pillar layer separation, then after using anti-phase (open ODS) pillar layer separation,
The most naturally isovitexin crystallization is separated out.
In the present invention, when using silica gel column chromatography to separate, use a dry method on a sample；When using open ODS pillar layer separation, use
Wet method loading.
Further object is that the myocardial preservation purposes that isovitexin is provided.
Accompanying drawing explanation
Fig. 1 is the hydrogen spectrogram of isovitexin.
Fig. 2 is the carbon spectrogram of isovitexin.
Fig. 3 is that isovitexin extracts separation process figure.
Fig. 4 is that isovitexin causes the morphologic impact of rats with myocardial ischemia myocardial histopathology (HE dyeing) to ISO.
Detailed description of the invention
Prepared by embodiment one, the separation of isovitexin
1. vegetable material: the seed of leguminous plant Semen phaseoli radiati (Vigna radiate L.).
2. method for separating and preparing: mung bean seed 50kg, with 95% alcohol reflux 3 times of 10,10,10 times amount, filters,
Merging filtrate, decompression recycling ethanol, to without alcohol taste, obtains concentrated solution about 5L.Respectively by petroleum ether 5L, dichloromethane 5L, positive fourth
Alcohol 5L respectively extracts 3 times, merges butanol extraction liquid, recovered under reduced pressure n-butyl alcohol, obtains Semen phaseoli radiati extract.By Semen phaseoli radiati extract with suitable
Amount methanol dissolves, and takes proper silica gel and mix sample.The sample mixed is loaded in silicagel column, uses methylene chloride-methanol
(100:0～0:100) gradient elution, obtains 5 flow points, collects flow point 4 (45:55～35:65), the open ODS post of loading,
With methanol-water (10:90～40:60) gradient elution, collect methanol-water (40:60) eluent, place, separate out yellow solid,
It is isovitexin.
Embodiment two, the myocardial preservation activity test of isovitexin
1. laboratory animal: SD rat, body weight 200～220g, Shenyang Pharmaceutical University's animal center provides；The quality certification number: SCXK
(distant) 2015-0001.
2. reagent: ISO injection (Shanghai Hefeng Pharmaceutical Co., Ltd., lot number: 41150402), urethane (Chinese Medicine (collection
Group) Solution on Chemical Reagents in Shanghai company), creatine kinase (CK), lactic acid dehydrogenase (LDH), glutamic oxaloacetic transaminase, GOT (AST), malonaldehyde (MDA),
Superoxide dismutase (SOD) test kit (Bioengineering Research Institute is built up in Nanjing).
Isovitexin used by experiment is that inventor makes by oneself, and its chemical constitution is composed through hydrogen, carbon spectrum determines errorless, and HPLC purity exists
More than 98%.
3. experimental technique: take SD rat 18, is randomly divided into three groups: blank group, model group and isovitexin group, often organizes 6
Only.Blank group and 7 days normal saline of the continuous gavage of model group, 7 days isovitexin solution (30 of the continuous gavage of isovitexin group
Mg/kg), model group is administered latter 5 minutes at the 6th day and the 7th day with isovitexin group, and abdominal cavity gives ISO 2mg/kg respectively,
Within 7th day, giving after ISO 2 hours, orbital venous plexus takes blood, separates serum, measures serum creatine kinase (CK), lactic acid takes off
Hydrogen enzyme (LDH), the activity of glutamic oxaloacetic transaminase, GOT (AST).Put to death rat after taking blood, win rapidly heart.Take part of heart
Tissue homogenate, centrifugal (3500r/min, 15min), take out myocardial homogenates supernatant, measure SOD, MDA in myocardial homogenates
Content, experimental result Excel statistical disposition, through t test and judge significance.Separately take part of heart tissue and be placed in the Fu Er of 10%
Fixing in Malin's solution, conventional dehydration, paraffin embedding, section, hematoxylin-eosin (HE) dyes, tissues observed under light microscopic
Pathological change.
4. result of the test: as shown in table 2, contrasts with blank group, and CK, LDH in model group rats serum are the brightest with AST
Aobvious raising, show that the three MDA in increases, and model group rats cardiac muscular tissue that all leaks outside from cell dramatically increases, SOD is aobvious
Writing and decline, above index all shows that the model of ISO induced rat myocardial ischemia is set up.Isovitexin group contrasts with model group, different
CK, LDH in vitexin group serum are all decreased obviously with AST, it is suppressed that three's cell leaks outside, and isovitexin group rat
In cardiac muscular tissue, MDA is remarkably decreased, and SOD significantly rises, and prompting isovitexin can be by strengthening oxidation resistance to the ischemia heart
Flesh produces protective effect.
Table 2. isovitexin causes the impact of rats with myocardial ischemia Enzyme Activities to ISO
Optical microphotograph Microscopic observation, blank group rat heart muscle has no fragmentation of myocardium seen from pathomorphology change model group rats, the heart
Flesh interstitial edema, inflammatory cell infiltration is obvious.The infringement of isovitexin group rat heart muscle Histopathologic changes relatively model group and has subtracted
Gently, have no cardiac interstitium edema, seldom see inflammatory cell infiltration.Result is shown in that Fig. 4, prompting isovitexin can substantially alleviate ISO
The degree of the myocardial ischemia in rats damage of induction.
This invention result of the test shows, isovitexin has myocardial preservation activity, therefore this compound has good applicating and exploitation
Prospect.
Claims (10)
1. the preparation method of isovitexin, comprises the following steps:
(1) preparation of Semen phaseoli radiati crude extract: take Semen phaseoli radiati appropriate, ethanol or methanol eddy extract, and filter, merging filtrate, obtain alcohol extract, by alcohol extract recovered under reduced pressure alcohol to without alcohol taste, obtain crude extract；
(2) preparation of Semen phaseoli radiati extract: Semen phaseoli radiati crude extract, extracts with opposed polarity organic solvent successively, discards extract, then with n-butanol extraction, merges butanol extraction liquid, recovered under reduced pressure n-butyl alcohol, obtain Mung Bean Plant extract；
(3) isovitexin is refined: the Semen phaseoli radiati extract of gained, disperse by proper amount of methanol, add proper silica gel, stir, load in the silicagel column anticipated, carry out gradient elution with the mixed organic solvents (100:0～0:100) of two kinds of organic solvent mixing, collect mixed organic solvents (50:50～30:70) eluent, it is concentrated into thick, adds ethanol in proper amount or methanol dispersion；Upper open ODS post, with alcohol-water (0:100～100:0), preferably alcohol-water (10:90～40:60), carry out gradient elution, collect alcohol-water (35:65～40:60) eluent, stand overnight precipitation yellow crystal, be isovitexin.
2. preparation method as claimed in claim 1, it is characterized in that, ethanol described in step (1) or methanol concentration are 30%～100%, middle 30%～100% ethanol using 5～20 times amount of step (1) or methanol extraction 1～3 times, and united extraction liquid.
3. preparation method as claimed in claim 1, it is characterized in that, after step (2) first extracts 1～5 time with 0.5～2 times of volume petroleum ether or hexamethylene, again with 0.5～2 times of volumes methylene chloride or chloroform or ethyl acetate extraction 1～5 times, finally with the n-butanol extraction 1 of 0.5～2 times of volume～5 times.
4. preparation method as claimed in claim 1, it is characterised in that the mixed organic solvents gradient elution described in step (3), its mixed organic solvents is methylene chloride-methanol, or chloroform-methanol, or dichloromethane-ethanol, or chloroform-ethanol.
5. preparation method as claimed in claim 1, it is characterised in that the alcohol-water described in step (3) is methanol-water or alcohol-water.
6. a pharmaceutical composition, comprises isovitexin and pharmaceutically acceptable carrier prepared by the preparation method described in claim 1-5 any one.
7. isovitexin purposes in preparation is used for myocardial preservation medicine.
8. purposes as claimed in claim 7, it is characterised in that described myocardial preservation is the protective effect of the myocardial ischemia to isoproterenol induction.
9. purposes as claimed in claim 7, it is characterised in that described myocardial preservation medicine is the angina pectoris prevention that causes of heart disease, especially myocardial ischemia and the medicine of remission.
10. purposes as claimed in claim 9, it is characterised in that described medicine is tablet, capsule, granule, oral administration solution, injectable sterile powder or injection.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610405166.0A CN106008483A (en) | 2016-06-08 | 2016-06-08 | Preparation and application of isovitexin |
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| CN201610405166.0A CN106008483A (en) | 2016-06-08 | 2016-06-08 | Preparation and application of isovitexin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020043182A1 (en) * | 2018-08-30 | 2020-03-05 | 中国科学院上海生命科学研究院 | Application of flavonoid c-glycoside monomer compound |
| CN114805321A (en) * | 2022-04-20 | 2022-07-29 | 广西医科大学 | Preparation method and application of isovitexin |
Citations (3)
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| CN1820743A (en) * | 2005-01-05 | 2006-08-23 | 胡幼圃 | Inhibitor or promoter of uridinediphosphate glucuronosyltransferase 2B (UGT2B) |
| CN101390921A (en) * | 2007-09-21 | 2009-03-25 | 朱耕新 | Composition of starwort total glycopeptides and total flavone and preparation method and uses thereof |
| US20140141082A1 (en) * | 2012-11-16 | 2014-05-22 | Song Gao | Compositions Containing Enriched Natural Crocin and/or Crocetin, and Their Therapeutic or Nutraceutical Uses |
-
2016
- 2016-06-08 CN CN201610405166.0A patent/CN106008483A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820743A (en) * | 2005-01-05 | 2006-08-23 | 胡幼圃 | Inhibitor or promoter of uridinediphosphate glucuronosyltransferase 2B (UGT2B) |
| CN101390921A (en) * | 2007-09-21 | 2009-03-25 | 朱耕新 | Composition of starwort total glycopeptides and total flavone and preparation method and uses thereof |
| US20140141082A1 (en) * | 2012-11-16 | 2014-05-22 | Song Gao | Compositions Containing Enriched Natural Crocin and/or Crocetin, and Their Therapeutic or Nutraceutical Uses |
Non-Patent Citations (6)
| Title |
|---|
| JEONG SEI JOON ET AL.: "Flavonoids from the seeds of Phaseolus radiatus", 《KOREAN JOURNAL OF PHARMACOGNOSY》 * |
| LIU-YI DONG ET AL.: "Cardioprotection of Vitexin on Myocardial Ischemia/Reperfusion Injury in Rat via Regulating Inflammatory Cytokines and MAPK Pathway", 《THE AMERICAN JOURNAL OF CHINESE MEDICINE》 * |
| YAN BAI ET AL.: "Antioxidant and Myocardial Preservation Activities of Natural Phytochemicals from Mung Bean (Vigna radiata L.) Seeds", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 刘定梅 等: "绿豆化学成分及生物活性研究进展", 《黔南民族医专学报》 * |
| 董六一 等: "牡荆素对大鼠实验性心肌缺血损伤的保护作用及其机制", 《中草药》 * |
| 邵莹 等: "碳苷黄酮保护心肌缺血损伤作用的研究进展", 《中草药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020043182A1 (en) * | 2018-08-30 | 2020-03-05 | 中国科学院上海生命科学研究院 | Application of flavonoid c-glycoside monomer compound |
| CN114805321A (en) * | 2022-04-20 | 2022-07-29 | 广西医科大学 | Preparation method and application of isovitexin |
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