CN101480422A - Tibetan oriental wormwood extract as well as preparation method and use thereof - Google Patents
Tibetan oriental wormwood extract as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a Tibetan herba artemisiae capillaris extract which at least contains general flavone. Taking the total weight of the Tibetan herba artemisiae capillaris extract as 100 percent, and the general flavone accounts for 20-75 percent of the Tibetan herba artemisiae capillaris extract. The Tibetan Herba Artemisiae Capillaris extract has the activity of treating diseases in the liver and the gallbladder.
Description
Technical field
The present invention relates to a kind of extract that derives from the long stalk of the distinctive Gentianaceae of Qinghai-Tibet alpine condition (Gentianacese) larynx hair flower plant larynx hair flowering plant and preparation method thereof, this plant is used as the extensive use of Tibetan medicine ZANGYINCHEN in Tibetan medicine and pharmacology, this extract has the pharmaceutically active of significant treatment liver and gall diseases.
Background technology
ZANGYINCHEN is the traditional Tibetan medicine that is grown in extremely frigid zones, and for one of Tibetan medicine eight delicacies, in China, Tibetan medicine and pharmacology is a few ethnomedicine with own unique theoretical system, and its abundant in content, system is a complete ethnomedicine system that is only second to Chinese medicine.Tibetan medicine and pharmacology has unique diagnosis and treatment method and proved recipe to multiple acute and chronic diseases such as liver and gall diseases and difficult diseases, has accumulated and has enriched clinical experience and square medicine, and wherein ZANGYINCHEN is one of medicine of the clinical treatment liver and gall diseases of using always the most of Tibetan medicine.
ZANGYINCHEN derives from the distinctive a few class plants of Qinghai-Tibet alpine condition, its former plant comprises that mainly Gentianaceae Swertia, Herba Haleniae Corniculatae genus, larynx hair Pittosporum and Herba Gentianopsis Paludosae belong to about twenties kind of plant such as several plants and Saxifragaceae, mainly are distributed in the area of 3000～4800 meters of height above sea level such as Tibet, Qinghai, Sichuan in China.ZANGYINCHEN is all on the books in ancient books and records such as Tibetan medicine and pharmacology classical works " brilliant pearl book on Chinese herbal medicine ", " the brilliant mirror book on Chinese herbal medicine of Tibetan medicine ", and the Tibetan medicine name is called " DIDA ".The Tibetan medicine thinks that ZANGYINCHEN has the function of heat clearing and inflammation relieving, promoting the function of the gallbladder to alleviate jaundice, cures mainly various calentura, especially the excessive heat of liver and gallbladder disease is had unique curative effect.Compiled the successive dynasties in " encyclopaedical Tibetan medicine's bundling " with the famous prescription of ZANGYINCHEN name side surplus in the of totally ten, wherein eight flavor ZANGYINCHEN are loose, the 25-component ZANGYINCHEN looses and all belong to the clinical commonly used and very significant prescription of curative effect.Be that primary raw material has the safe relieving capsule of brilliant pearl liver, DIDA ball, ZANGYINCHEN sheet/capsule, anxious proheparin/YIGANNING etc. through the Tibetan medicine new drug of modern crafts processing and preparing in the last few years with the ZANGYINCHEN, all be applied to clinically mainly in order to treatment acute, chronic hepatitis, cholecystitis etc., curative effect is very remarkable.
In the past, because this medical herbs mainly is ethnic groups medication among the people, not fully aware of to effective ingredient, the mechanism of action and safety, this has just influenced the further application clinically of this medicine.From natural drug, national medicine, seek and develop the focus that effective medicine becomes domestic and international research work in recent years, the Tibetan medicine ZANGYINCHEN is with secular medicinal basis among the people and the definite extremely concern of Chinese scholars of clinical efficacy, a series of researchs have all been launched in aspects such as its chemistry, pharmacology, clinical practice and artificial culture, obtained a lot of progress.Especially preliminary experimentation has been carried out on the hepatoprotective effect and the chemical substance basis thereof of this Tibetan medicine, for the development Tibetan medicine provides the research basis.
Summary of the invention
At ZANGYINCHEN Study on plants present situation, purpose of the present invention is exactly a Journal of Sex Research by experiment, illustrates effective ingredient, the mechanism of action and the safety of this medical material, so that clinical better use.
ZANGYINCHEN derives from the long stalk of Gentianaceae (Gentianacese) larynx hair flower plant larynx hair flower Comastoma Pedunlulatum[Rogle ex D.Dou in this research] the dry herb of Holub, adopt Gonghe County, through being accredited as the long stalk of Gentianaceae larynx hair flower plant larynx hair flower Comastoma Pedunlulatum[Rogleex D.Dou in Qinghai Province] Holub.
The inventor passes through great deal of experimental, from ZANGYINCHEN, extract the ZANGYINCHEN effective site (extract) that has obtained containing total flavones, this effective site is called ZANGYINCHEN extract, this extract is that the ZANGYINCHEN crude extract makes through making with extra care, effective ingredient wherein is clear and definite, the total flavonoid chemical composition content is higher, reached clear and definite ZANGYINCHEN treatment aspect the liver and gall diseases its relatively clear and definite composition of effective site and the purpose of content, thereby improved bioavailability.
The object of the present invention is to provide a kind of ZANGYINCHEN extract, this extract has the activity of obvious treatment liver and gall diseases.
The present invention also aims to provide the preparation method of above-mentioned ZANGYINCHEN extract, this method comprises the process of extraction and refining purification, especially adopt the ethanol extraction of specific concentrations and eluting with enrichment of carrying out effective ingredient (position) etc., make that the effective ingredient that has the effect of treatment liver and gall diseases in the ZANGYINCHEN extract is clear and definite, content is high, impurity is few, more help its application on the preparation of treatment liver and gall diseases.
The invention provides a kind of ZANGYINCHEN extract, wherein contain total flavones, is 100% in this ZANGYINCHEN extract weight, and this total flavones accounts at least 20%.
This ZANGYINCHEN extract has the activity of treatment liver and gall diseases.Wherein, the total flavones in the ZANGYINCHEN extract of the present invention accounts for the 20-75% of this ZANGYINCHEN extract weight.
Above-mentioned ZANGYINCHEN extract provided by the invention, its total flavones that contains is topmost active site, representative compounds is 1-O-β-D-Glucopyranose .-3 in this total flavones, 8-dihydroxy-7-methoxyl group
Ketone and oleanolic acid, its separately content in this total flavones is 0.1%-10%wt, preferred 0.5%-2%wt.
That is, the invention provides a kind of ZANGYINCHEN extract, wherein contain total flavones, is 100% in this ZANGYINCHEN extract weight, and this total flavones accounts at least 20%; Contain 1-O-β-D-Glucopyranose .-3 in this total flavones, 8-dihydroxy-7-methoxyl group
Ketone and oleanolic acid, this 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Every kind of ketone and oleanolic acid all account for 0.1%-10%wt at this total flavones, preferred 0.5%-2%wt.
According to the difference of purification step, total flavones (or being called the total flavonoid chemical compound) generally can reach the 20-75% of ZANGYINCHEN extract weight, and in the preferred embodiment of the present invention, total flavones accounts for 30-65%; Wherein, comprise 1-O-β-D-Glucopyranose .-3 in the above-mentioned total flavones, 8-dihydroxy-7-methoxyl group
Ketone (larynx lanatoside), oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1-hydroxyl-3, the 7-dimethoxy
Ketone, 1-hydroxyl-3,7, the 8-trimethoxy
Ketone, 7,22-bean steroid diene 3 alcohol, hexadecylic acid, 1,8-dihydroxy-3,7-dimethoxy
Ketone and swertisin, above-mentioned these chemical compounds account for separately described total flavones weight at least 0.1%, be generally 1% to 20%, preferred 1%-15%; Usually above-mentioned these chemical compounds can account for the 0.1-15% of ZANGYINCHEN extract gross weight, preferred 0.5-10%; In a preferred embodiment, 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Total content of ketone above-claimed cpds such as (larynx lanatosides) is the 20-75% of ZANGYINCHEN extract, preferred 25-65%, more preferably 30-60%.
Through the pharmacological testing test, the extract that contains mentioned component of the present invention has the pharmacologically active of treatment liver and gall diseases, especially hepatic injury is had significant inhibitory effect.
Therefore, the present invention also provides above-mentioned ZANGYINCHEN extract, it is for containing the ZANGYINCHEN extract of the total flavones that accounts for this extract weight 20% at least, this extract is generally the ZANGYINCHEN medical material and obtains through alcohol reflux, this ZANGYINCHEN extract also contains 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone (larynx lanatoside), oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1,8-dihydroxy-3,7-dimethoxy
Ketone and swertisin, these chemical compounds respectively account at least 0.1% of ZANGYINCHEN extract gross weight, can account for extract gross weight 0.5-10% usually.
The present invention also provides above-mentioned ZANGYINCHEN extract, it is for containing the ZANGYINCHEN extract of the total flavones that accounts for this extract weight 20% at least, this extract is preferably the ZANGYINCHEN medical material and obtains through alcohol reflux, the total flavones that this ZANGYINCHEN extract contains is comprising containing 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone, oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1-hydroxyl-3, the 7-dimethoxy
Ketone, 1-hydroxyl-3,7, the 8-trimethoxy
Ketone, 7,22-bean steroid diene 3 alcohol, hexadecylic acid, 1,8-dihydroxy-3,7-dimethoxy
Ketone and swertisin, above-mentioned these chemical compounds account at least 0.1% of ZANGYINCHEN extract gross weight of the present invention separately, can account for extract gross weight 0.5-10% usually, in the preferred embodiment of the present invention, 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
The total content (weight) of ketone above-claimed cpds such as (larynx lanatosides) is the 30-60% of ZANGYINCHEN extract gross weight of the present invention.
The present invention also provides the preparation method of above-mentioned ZANGYINCHEN extract, and the step of this preparation method comprises:
With the pure heating and refluxing extraction of ZANGYINCHEN medical material with the 55-95% (wt%) of at least 2 times of amounts (weight), preferred same method repeats to extract merge extractive liquid, at least 1 time again, reclaim solvent, obtain the alcohol extract of ZANGYINCHEN, that is, and ZANGYINCHEN crude extract of the present invention.The yield of ZANGYINCHEN crude extract is about 19%.
The invention provides a kind of preparation (extraction) method of ZANGYINCHEN crude extract, step is:
With the pure heating and refluxing extraction of ZANGYINCHEN medical material with the 55-95% (wt%) of at least 2 times of amounts (weight), extracting solution reclaims solvent, obtains the alcohol extract of ZANGYINCHEN; Wherein extract in the above-mentioned steps and use pure preferred alcohol, mass concentration is generally 55-95%, more preferably 60-90%, and 65-85% most preferably, in order to save cost and comprehensive consideration, 70-80% most preferably obtains the crude extract of ZANGYINCHEN;
Above-mentioned ZANGYINCHEN crude extract adopts petroleum ether, ethyl acetate and n-butanol extraction successively, collect acetic acid ethyl acetate extract, obtain acetic acid ethyl ester extract TE after reclaiming solvent, and collection butanol extraction liquid, obtain n-butyl alcohol extract TN after reclaiming solvent, merge TE and TN, get ZANGYINCHEN extract of the present invention.
In a preferred embodiment of the invention, the preparation method of ZANGYINCHEN crude extract of the present invention comprises:
The dried plant herb extracted 1-3 hour with the alcohol heating reflux of the 60-90% (wt%) of 2～10 times of amounts (weight), arbitrary time in 2 hours or 1.5～5 hours for example, same method is extracted 2 times again, merge extractive liquid,, reclaim etoh solvent, obtain the ethanol extraction of ZANGYINCHEN, be concentrated into about 0.95kg, obtain ZANGYINCHEN crude extract or ZANGYINCHEN total extract extractum ZYCT;
ZANGYINCHEN crude extract extractum adds aqueous suspension, add petroleum ether extraction, obtain petroleum ether extraction liquid and water layer A, water layer A adds ethyl acetate (about 1:1) volume extraction at least 1 time, obtain acetic acid ethyl acetate extract and water layer B, acetic acid ethyl acetate extract obtains acetic acid ethyl ester extract TE (95/950g) after reclaiming solvent, water layer B adds n-butyl alcohol (about 1:1) volume extraction at least 1 time, obtain butanol extraction liquid and water layer, butanol extraction liquid obtains n-butyl alcohol extract TN (240/950g) after reclaiming solvent, merge TE and TN, promptly get ZANGYINCHEN extract of the present invention.
In order to obtain having the effective ingredient and the active site of anti-liver and gall damage more exactly, in another preferred embodiment, the preparation method of ZANGYINCHEN extract of the present invention comprises:
Adopt macroporous absorption resin chromatography to carry out crude separation respectively above-mentioned n-butyl alcohol extract that obtains (TN) and acetic acid ethyl ester extract (TE), every kind of extract all adopts the ethanol of 40%, 60%, 80% and 95% mass concentration to carry out gradient elution, comprising:
Macroporous adsorptive resins is also through behind the ethanol gradient elution on the acetic acid ethyl ester extract, obtain 40% ethanol elution, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution successively, collection also keeps 60% ethanol elution (ES) and 80% ethanol elution (EE), reclaim solvent, the ethyl acetate extraction that obtains ZANGYINCHEN is again through the purification thing Y of 60%～80% ethanol elution, it accounts for the 2/5-3/5 of the weight of acetic acid ethyl ester extract, accounts for the 1%-10% of ZANGYINCHEN total extract extractum ZYCT weight greatly;
Macroporous adsorptive resins is also through behind the ethanol gradient elution on the n-butyl alcohol extract, obtain 40% ethanol elution, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution successively, collection also keeps 60% ethanol elution (BF) and 80% ethanol elution (BS), reclaim solvent, the n-butanol extraction that obtains ZANGYINCHEN is again through the purification thing Z of 60%～80% ethanol elution, it accounts for the 3/10-3/5 of the weight of n-butyl alcohol extract greatly, accounts for the 6%-16% of ZANGYINCHEN total extract extractum ZYCT weight greatly;
Merge above-mentioned purification thing Y and purification thing Z, obtain the active ZANGYINCHEN extract of anti-liver and gall that has of the present invention, the 30-55% that it accounts for organic solvent (ethyl acetate and n-butyl alcohol) the extract weight of ZANGYINCHEN accounts for the 10%-20% of ZANGYINCHEN total extract extractum ZYCT weight.
The polar fraction that rough segmentation obtains through the adsorbent resin chromatograph, get essence through remove impurity, what obtain has an active ZANGYINCHEN extract of anti-liver and gall, and (the chromatographic solvent system is that (the chromatographic solvent system is that (the chromatographic solvent system is MeOH-H for CHCl3-MeOH 10:1～3:1) and HPLC (preparation high performance liquid chromatography) for CHCl3-MeOH 20:1～3:1), Sephadex LH-20 (gel column chromatography) to adopt Si-gel (silica gel column chromatography) then
2The further separation and purification of method of O 65:35～80:20), the monomeric compound that separation is obtained be through determination of physical constant, use UV, MS,
1H-NMR and
13Modern spectral method such as C-NMR and technology identify its structure, separates to obtain D16 compound structure and be respectively: cupreol (β-sitosterol, EN1), daucosterol (daucosterol, EN2), 1,5,8-trihydroxy-3-methoxyl group
Ketone (1,5,8-trihydroxy-3-methoxyxanthone, EN3), 1,8-dihydroxy-3,7-dimethoxy
Ketone (methylswertiamin, EN4), the 1-hexacosyl alcohol (1-hexacosanol, EN5), the octacosanoic acid methyl ester (Methyl Octacosanoate, EN6), ursolic acid (Ursolic acid, EE1), 1,3,8-trihydroxy-7-methoxyl group
Ketone (1,3,8-tridroxy-7-methoxyxanthone, EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (1-droxy-3,7-dimethoxy xanthone, EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (1-droxy-3,7,8-trimethoxy xanthone, EE4), 7,22-bean steroid diene 3 alcohol (stigmasta-7,22-3-dienol, EE5), hexadecylic acid (hexadecanoic acid, EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (1,8-diidroxy-3,7-dimethoxy xanthone, ES1), swertisin (Swertisin, ES2), 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside, Comastomaside, BS1), (Oleanolic Acid BS2) waits chemical compound to oleanolic acid.
Wherein, the most representative pharmacodynamics active component is a total flavones in the ZANGYINCHEN extract, is 100% to be benchmark with this ZANGYINCHEN extract weight, and such chemical constituent accounts at least 20% of ZANGYINCHEN extract weight, preferred 40%-60%.
The present invention is through a large amount of craft screening experiments, utilize solvent method that ZANGYINCHEN is extracted, and its main effective site carried out the purification enrichment technical study, obtain mainly containing effective component content higher effective position, and isolation identification monomer chemical constituent wherein, as above-mentioned 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside Comastomaside), oleanolic acid chemical compounds such as (Oleanolic Acid), has therefore obtained having the extract of the pharmacologically active of obvious treatment liver and gall diseases, screening experiment cut select as follows:
1, the selection of extraction conditions
Employing ethanol is extraction solvent, extracts 3 kinds of methods with supersound extraction, reflux, extract,, water-bath earlier and carries out methodological study, determines that reflux, extract, method extraction ratio is higher.
2, the preferred extraction process of orthonormal design of experiments
(1) sample treatment: sample extracts by the factor level of orthogonal design after waiting pre-treatment, and extracting solution carries out through processing such as defats, must supply test agent.
(2) methodological study:
With the rutin is index components, the regression equation of standard curve: Y=12.968x-0.0189, R2=0.9992, linear good, press spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measuring general flavone content at the 510nm place with ultraviolet spectrophotometry, is evaluation criterion with the general flavone content.
(3) technology is preferred: according to medical material character and the actual requirement of production, draft 3 investigation factors that influence extraction ratio, the liquid material is set up 3 levels respectively than (A), concentration of alcohol (B) and extraction time (C), and establishment factor level table carries out the optimal process experiment; Carry out orthogonal experiment by methodology in (2) item, respectively content of total flavone in the determination experiment sample;
Experimentation shows that concentration of alcohol has the significance influence to it, and the influence degree to total flavones is followed successively by: concentration of alcohol〉liquid material ratio〉extraction time, draw the optimum process condition (subordinate list 1,2 and 3) that the ZANGYINCHEN total flavones extracts by orthogonal experiments intuitive analysis and variance analysis.
(4) experimental result: experimentation shows that concentration of ethanol has the significance influence to experimental result, and each factor to the size order of the influence of index is: B〉A〉C, be that concentration of alcohol has the greatest impact to experimental result, solid-liquid ratio takes second place, and extraction time is to the minimum that influences of this technology.
From the result of variance analysis also as can be seen, best extraction process is A1B3C3.By intuitive analysis as can be seen, the productive rate of total flavones is all higher in the experimental group 3,6,9, and be more or less the same, analysis-by-synthesis is respectively organized experiment condition, takes all factors into consideration solvent extraction impurity situation, and considers from the angle of economy, environmental protection, the Financial cost that long extraction can bring, to the pollution of environment, select technology A1B3C2 for use so take all factors into consideration, promptly adopted 6 times of amount 50% ethanol extractions 2 hours.
(5) checking row experiment: after craft screening is finished, for investigating the stability of above-mentioned optimised process, carry out repeated demonstration test 3 times by these process conditions and method, measure larynx hair flower content of total flavone respectively, extraction ratio is respectively 1.510%, 1.742% and 1.720%.
Result of the test generally is better than the result of the test of orthogonal design table, and favorable reproducibility proves that process stabilizing is feasible.
Table 1 factor level table
Table 2 Orthogonal experiment results
| Row number | A | B | C | Total flavones extraction ratio % |
| 1 | 1 | 1 | 1 | 0.659 |
| 2 | 1 | 2 | 2 | 1.156 |
| 3 | 1 | 3 | 3 | 1.543 |
| 4 | 2 | 1 | 2 | 0.815 |
| 5 | 2 | 2 | 3 | 1.024 |
| 6 | 2 | 3 | 1 | 1.518 |
| 7 | 3 | 1 | 3 | 0.897 |
| 8 | 3 | 2 | 1 | 1.400 |
| 9 | 3 | 3 | 2 | 1.465 |
| k 1 | 1.119 | 0.790 | 1.192 | |
| k 2 | 1.119 | 1.193 | 1.145 | |
| k 3 | 1.254 | 1.509 | 1.155 | |
| R | 0.135 | 0.719 | 0.047 |
Table 3 The results of analysis of variance
Annotate:
*Be significant factors
Adopt conventional etoh solvent extraction method to extract the extract of ZANGYINCHEN, through a large amount of screening experiment checkings, the alcoholic solution of about 50% (wt%) concentration is excessive as extracting solvent polarity, may increase the content of extract Semi-polarity impurity, the alcoholic solution of using 75% (wt%) concentration instead extracts the ethanol extraction of the ZANGYINCHEN that obtains, through experimental verification repeatedly, wherein the content of polar impurity obviously reduces.Therefore formed the preferred version of extracting method of the present invention.
Preferred extracting method is specific as follows: dried plant herb 5kg, pulverize through rough, doubly measure the soak with ethanol of the 70-80% (wt%) of (weight) with 4-8, the more preferably soak with ethanol 2h of about 75% (wt%) of 6 times of amounts (weight), then heating and refluxing extraction at least 1 hour or 2 hours or longer time, preferred same method is extracted merge extractive liquid, again 2 times, reclaim solvent, obtain the ethanol extraction of ZANGYINCHEN.
The present invention has also carried out the separation of chemical constituent and a large amount of experiments of evaluation aspect in the ZANGYINCHEN, and is as follows:
1, ZANGYINCHEN crude extract is extracted flow process as mentioned above.
2, effective site enrichment and purification
Extract the total extract extractum ZYCT (950g) of preparation through solvent method, progressively separate obtaining four opposed polarity positions again through systematic solvent extraction.
Separation process is as follows: the ethanol extraction of above-mentioned resulting ZANGYINCHEN or it is concentrated the ZANGYINCHEN total extract extractum (ZYCT) obtain, add aqueous suspension, add petroleum ether extraction, obtain petroleum ether extraction liquid and water layer A, wherein petroleum ether extraction liquid obtains petroleum ether extract TP (25/950g) after reclaiming solvent, water layer A adds ethyl acetate (about 1:1) volume extraction at least 1 time, obtain acetic acid ethyl acetate extract and water layer B, acetic acid ethyl acetate extract obtains acetic acid ethyl ester extract TE (95/950g) after reclaiming solvent, water layer B adds n-butyl alcohol (about 1:1) volume extraction at least 1 time, obtain butanol extraction liquid and water layer, butanol extraction liquid obtains n-butyl alcohol extract TN (240/950g) after reclaiming solvent.
On screening active ingredients result of the test basis, carry out enriching and purifying at the chemical constituent that has in active 2 effective site n-butyl alcohol extracts of anti-liver injury (TN) and the acetic acid ethyl ester extract (TE).Its enriching and purifying flow process is as follows:
After acetic acid ethyl ester extract (90g) dissolving, the dissolve with ethanol of preferred 20%wt, last AB-8 macroporous adsorptive resins adsorbs the crude separation of impurity, adopt the ethanol of 40%, 60%, 80% and 95% mass concentration to carry out the gradient ethanol elution, obtain successively 40% ethanol elution (EF, 8g), 60% ethanol elution (ES, 12g), 80% ethanol elution (EE, 29g), 95% ethanol elution (EN, 40g).
In a preferred embodiment, the acetic acid ethyl ester extract ethanol ultrasonic dissolution of 20%wt, better effects if.
The dissolve with ethanol of n-butyl alcohol extract (230g) the dissolving preferred 20%wt in back, last AB-8 macroporous adsorptive resins adsorbs the crude separation of impurity, adopt the ethanol of 40%, 60%, 80% and 95% mass concentration to carry out the gradient ethanol elution, obtain 40% ethanol elution (BT successively, 42g), 60% ethanol elution (BF, 45g), 80% ethanol elution (BS, 52g), 95% ethanol elution (BN, 31g).
In another preferred embodiment, the n-butyl alcohol extract ethanol ultrasonic dissolution of 20%wt, better effects if.
The position that behind above-mentioned enriching and purifying, obtains, wherein ES, EE position and BF, BS position are main effective ingredient enrichment positions in the ZANGYINCHEN, therefore, these 4 positions are merged, the ZANGYINCHEN effective site that obtains after the merging is called ZANGYINCHEN extract in the present invention.
3. the screening test of active site
Adopt carbon tetrachloride to cause 30 of the laboratory animals of acute liver damage mice, investigate ZANGYINCHEN crude extract (ZYCT), Petroleum ether extraction position (TP), ethyl acetate extraction position (TE) and n-butanol extraction position (TN) influence, tentatively investigate its anti-liver injury effect experimental animal serum alt, AST.Result of the test shows: administration was compared CCl with the blank group after 7 days
4Model group mice serum ALT, AST content significantly raise; Crude extract (ZYCT), n-butyl alcohol extract (TN) and acetic acid ethyl ester extract (TE) all have the effect of ALT and AST level in the experimental hepatic injury animal serum of tangible reduction, and petroleum ether extract (TP) does not have obviously and changes.The experimental result prompting, the ZANGYINCHEN crude extract has the anti-liver injury effect, and wherein TN and TE position are the active site of its anti-liver injury effect.
Through separating and identifying; experimental results show that; the eluent that ethanol below 60% and 95% above ethanol elution obtain; though may comprise certain type the chemical compound that pharmacologically active is arranged; but these compounds more are that almost active or these compositions of nonreactive liver and gall diseases play and are unfavorable for treating liver and gall diseases even deleterious on the contrary side effect; n-butyl alcohol extract and acetic acid ethyl ester extract adopt concentration of alcohol below 60% and the composition that comprises of the eluent that obtains of 95% above eluting and be not suitable for the purpose for the treatment of liver and gall diseases safely and effectively that the present invention desires to reach; that is, not the present invention's ZANGYINCHEN extract required for protection.
Therefore, the extracting method of ZANGYINCHEN extract provided by the invention is:
(1) the ZANGYINCHEN medical material being used the mass concentration of at least 2 times of weight is the alcohol reflux extraction of 55-95%, and extracting solution reclaims solvent, obtains the crude extract of ZANGYINCHEN;
(2) crude extract of the ZANGYINCHEN that obtains of above-mentioned steps (1) adopts petroleum ether, ethyl acetate and n-butanol extraction successively, collects acetic acid ethyl acetate extract, obtains acetic acid ethyl ester extract after reclaiming solvent; Collect butanol extraction liquid, obtain n-butyl alcohol extract behind the recovery solvent;
(3) combined ethyl acetate extract and n-butyl alcohol extract;
Process for purification after extracting, i.e. enrichment and purification method is preferably:
Adopt macroporous absorption resin chromatography to carry out the alcoholic acid gradient elution that mass concentration is 40-95% respectively acetic acid ethyl ester extract and n-butyl alcohol extract, collect this acetic acid ethyl ester extract and n-butyl alcohol extract 40-95% ethanol elution separately, reclaim solvent, merge, obtain ZANGYINCHEN extract.
Above-mentioned enrichment and purification method is more preferably:
On the acetic acid ethyl ester extract macroporous adsorptive resins and be 40%, 60%, 80% and 95% ethanol gradient elution through mass concentration after, obtain 40% ethanol elution, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution successively, collection also keeps 60% ethanol elution and 80% ethanol elution, reclaim solvent, the ethyl acetate extraction that obtains ZANGYINCHEN is again through the purification thing Y of 60%～80% ethanol elution;
Macroporous adsorptive resins and be behind 40%, 60%, 80% and 95% the gradient elution through mass concentration on the n-butyl alcohol extract, obtain 40% ethanol elution, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution successively, collection also keeps 60% ethanol elution and 80% ethanol elution, reclaim solvent, the n-butanol extraction that obtains ZANGYINCHEN is again through the purification thing Z of 60%～80% ethanol elution;
Merge above-mentioned purification thing Y and purification thing Z, obtain ZANGYINCHEN extract of the present invention.
In a preferred embodiment of the invention, the concrete steps of preparation method with ZANGYINCHEN extract of active function are:
1. the extraction of effective site
ZANGYINCHEN dried plant herb, pulverize through rough, doubly measure the soak with ethanol of the 70-80% (wt%) of (weight) with 4-8, the more preferably soak with ethanol 2h of about 75% (wt%) of 6 times of amounts (weight), heating and refluxing extraction at least 1 hour or 2 hours or longer time then, same method is extracted 2 times, merge extractive liquid,, reclaim solvent, obtain the ethanol extraction of ZANGYINCHEN, or it is condensed into ZANGYINCHEN crude extract or the ZANGYINCHEN total extract extractum ZYCT (950g/5kg) of the 1/6-1/4 that is about as much as exsiccant raw medicinal material weight.
2. the enrichment of effective site
Above-mentioned resulting ZANGYINCHEN crude extract or ZANGYINCHEN total extract extractum (ZYCT), add water (mass ratio of water and total extract extractum is about 2:1) and make its suspension, add petroleum ether extraction (volume ratio of petroleum ether and water is about 2:1), obtain petroleum ether extraction liquid and water layer A, discard petroleum ether extraction liquid or reclaim solvent and do his in addition and use, in water layer A, add ethyl acetate (volume of water and the about 1:1 of the volume of ethyl acetate) extraction at least 1 time, obtain acetic acid ethyl acetate extract and water layer B, water layer B adds n-butyl alcohol (volume of water and the about 1:1 of the volume of n-butyl alcohol) extraction at least 1 time, obtain butanol extraction liquid and water layer, discard water layer; Obtain acetic acid ethyl ester extract TE after acetic acid ethyl acetate extract reclaimed solvent, it is approximately 1/10 (95/950g) of the quality of ZANGYINCHEN total extract extractum ZYCT, obtain n-butyl alcohol extract TN after butanol extraction liquid reclaimed solvent, it is approximately 1/4 (240/950g) of the quality of ZANGYINCHEN total extract extractum ZYCT;
The experimental result prompting, ZANGYINCHEN extract has the anti-liver injury effect, wherein above-mentioned TN that obtains and TE position also are ZANGYINCHEN extract for the active site of its anti-liver injury effect, and this active site accounts for the 1/2-1/3 (335/950) of ZANGYINCHEN total extract extractum ZYCT weight.
3. the purification of effective site
Adopt macroporous absorption resin chromatography to carry out rough segmentation respectively n-butyl alcohol extract (TN) and acetic acid ethyl ester extract (TE), every kind of extract all adopts the ethanol of 40%, 60%, 80% and 95% mass concentration to carry out gradient elution, wherein:
After acetic acid ethyl ester extract (90g) is gone up macroporous adsorptive resins and process ethanol gradient elution, obtain 40% ethanol elution successively, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution, collection also keeps 60% ethanol elution (ES) and 80% ethanol elution (EE), reclaim solvent, the ethyl acetate extraction that obtains ZANGYINCHEN is again through the purification thing Y of 60%～80% ethanol elution (about 41g), it accounts for the 2/5-3/5 (41/90) of the weight of acetic acid ethyl ester extract greatly, account for the 1%-10% (41/950) of ZANGYINCHEN total extract extractum ZYCT weight greatly, comprising the purification thing (about 12g) of 60% ethanol elution and the purification thing (about 29g) of 80% ethanol elution;
After n-butyl alcohol extract (230g) is gone up macroporous adsorptive resins and process ethanol gradient elution, obtain 40% ethanol elution successively, 60% ethanol elution, 80% ethanol elution, 95% ethanol elution, collection also keeps 60% ethanol elution (BF, 45g) with 80% ethanol elution (BS, 52g), reclaim solvent, the n-butanol extraction that obtains ZANGYINCHEN is again through the purification thing Z of 60%～80% ethanol elution (about 97g), it accounts for the 3/10-3/5 (97/230) of the weight of n-butyl alcohol extract greatly, account for the 6%-16% (97/950) of ZANGYINCHEN total extract extractum ZYCT weight greatly, comprising the purification thing (about 45g) of 60% ethanol elution and the purification thing (about 52g) of 80% ethanol elution;
Merge above-mentioned purification thing Y and purification thing Z, obtain the active ZANGYINCHEN extract of liver and gall that has of the present invention, the 30-55% (138/320) that it accounts for organic solvent (ethyl acetate and n-butyl alcohol) the extract weight of ZANGYINCHEN accounts for the 10%-20% ([138 * " 335/320 "]/950) of ZANGYINCHEN total extract extractum ZYCT weight.
The above-mentioned polar fraction that rough segmentation obtains through the adsorbent resin chromatograph, get essence through remove impurity, what obtain has an active ZANGYINCHEN extract of anti-liver and gall, again further through adopting Si-gel (silica gel column chromatography) repeatedly, the method of Sephadex LH-20 (gel column chromatography) and HPLC (prepare and use high performance liquid chromatography) is carried out further separation and purification, therefrom obtain effective ingredient and active site that several chemical compounds are formed, promptly, the chemical compound that ZANGYINCHEN extract of the present invention contained comprises: 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group mouth diphenylene ketone oxide, oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1-hydroxyl-3, the 7-dimethoxy
Ketone, 1-hydroxyl-3,7, the 8-trimethoxy
Ketone, 7,22-bean steroid diene 3 alcohol, hexadecylic acid, 1,8-dihydroxy-3,7-dimethoxy
Ketone, swertisin.
Therefore, ZANGYINCHEN active site of the present invention, it is ZANGYINCHEN extract of the present invention, this extract is for through the purification thing Y that obtains of above-mentioned preparation method (preparation process that comprises extraction, separation, purification) and the mixture of purification thing Z, with the gross weight of this extract is 100% as calculating benchmark, containing total flavones in this extract, is 100% in this ZANGYINCHEN extract gross weight, and this total flavones accounts for 40-60%.Wherein, comprise 1-O-β-D-Glucopyranose .-3 in this total flavones, 8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside, BS1), oleanolic acid (BS2), ursolic acid (EE1), 1,3,8-trihydroxy-7-methoxyl group
Ketone (EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (EE4), 7,22-bean steroid diene 3 alcohol (EE5), hexadecylic acid (EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (ES1) and swertisin (ES2).Wherein, the representative compounds of topmost active site is 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
(the larynx lanatoside, BS1) and oleanolic acid (BS2), its content (wt%) separately is 0.1%-10% to ketone, preferred 0.5%-2%.
Sephadex LH-20 is a kind of of gel chromatography, and kind is sephadex (Sephadex), and according to different solvent systems, Sephadex LH-20 is applicable to the chemical constituent of separating opposed polarity.(10:1～3:1) carries out the column chromatography of chemical constituent to be separated the CHCl3-MeOH of solvent-applied system among the present invention, obtains above-mentioned chemical constituent 1,3,8-trihydroxy-7-methoxyl group
Ketone (EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (EE4), 7,22-bean steroid diene 3 alcohol (EE5), hexadecylic acid (EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (ES1) and swertisin (ES2).
(high performance liquid chromatography HPLC) also is high pressure liquid chromatography, high-speed liquid chromatography etc. to high performance liquid chromatography.(65:35～80:20) carry out the chemical constituent separation and purification obtains above-mentioned chemical constituent 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group to the solvent-applied MeOH-H2O of system of the present invention
Ketone (the larynx lanatoside, BS1), oleanolic acid (BS2) and ursolic acid (EE1).
Because the effective ingredient that is contained in the extract of the present invention is a mixture, the clear and definite after testing chemical constituent that it contained is mainly flavone compound, concrete chemical compound composition comprises 1-O-β-D-Glucopyranose .-3 in the effective site, 8-dihydroxy-7-methoxyl group
Ketone (larynx lanatoside), oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1-hydroxyl-3, the 7-dimethoxy
Ketone, 1-hydroxyl-3,7, the 8-trimethoxy
Ketone, 7,22-bean steroid diene 3 alcohol, hexadecylic acid, 1,8-dihydroxy-3,7-dimethoxy
Ketone, swertisin etc.Up to now, there is not document to report that openly ZANGYINCHEN specifically comprises those compositions at the effective ingredient of aspects such as treatment liver and gall diseases, it is rationally formed is what, how many preferred therapeutic doses is, certainly will cause occurring dose-effect relationship like this and be not easy defective characteristics such as assurance, curative effect instability, especially for the form of Chinese drug of treatment jaundice, hepatitis etc., constituent is definite, content accurately can impel and is easier to hold the quality of the pharmaceutical preparations, is the key of preparation and clinical application.The applicant finds that in the extract that preparation method of the present invention obtains, the most representative pharmacodynamics composition is a total flavones, experiment showed, that its therapeutic effect of ZANGYINCHEN extract of the present invention is best.
Definite effective ingredient is based on the flavone compound of treatment effective dose, and get rid of cupreol (β-sitosterol, EN1), daucosterol (daucosterol, EN2), 1,5,8-trihydroxy-3-methoxyl group
Ketone (1,5,8-trihydroxy-3-methoxyxanthone, EN3), 1,8-dihydroxy-3,7-dimethoxy
Ketone (methylswertiamin, EN4), the 1-hexacosyl alcohol (1-hexacosanol, EN5), octacosanoic acid methyl ester (Methyl Octacosanoate, the impurity constituents of EN6) etc. invalid or nearly unavailable.
The present invention has also carried out a large amount of experiment and has detected, and the result confirms, in the ZANGYINCHEN extract with 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
(the larynx lanatoside is main for the total flavonoid chemical compound of representative Comastomaside) to ketone.The amount that contains total flavones in the ZANGYINCHEN extract of the present invention is generally at 30-60%.
By follow-up study, the ZANGYINCHEN plant of the different collecting seasons of different batches, through above-mentioned same extraction preparation, content of total flavone can reach this ZANGYINCHEN extract gross weight 20% in the ZANGYINCHEN extract that obtains, even the higher crude drug of quality is through after repeating that said extracted is refining and separating the remove impurity process, content of total flavone can reach 65% of ZANGYINCHEN extract gross weight in the ZANGYINCHEN extract, generally can reach 30-60%.
The present invention also provides a kind of pharmaceutical composition, and it contains the above-mentioned ZANGYINCHEN extract and the excipient substance of clinical treatment effective dose.
Aforementioned pharmaceutical compositions comprises peroral dosage form and injection type.Oral formulations comprises tablet (ordinary tablet, dispersible tablet, slow/controlled release sheet, effervescent tablet, oral cavity disintegration tablet), capsule, granule, drop pill, oral liquid, suppository.
The present invention also provides the application of described ZANGYINCHEN extract in preparation treatment liver and gall diseases medicine.
And, contain the application of compositions in preparation treatment liver and gall diseases medicine of ZANGYINCHEN extract.
The ZANGYINCHEN extract test of pesticide effectiveness of the present invention and result are as follows:
Following " the ZANGYINCHEN crude extract " of the present invention is meant the ethanol extraction of Tibetan medicine material ZANGYINCHEN, or the ethanol extraction of ZANGYINCHEN is condensed into the ethanol extraction extractum of the ZANGYINCHEN of the 1/6-1/4 that is about as much as medicinal raw material weight, abbreviates ZANGYINCHEN crude extract or ZANGYINCHEN total extract extractum ZYCT as.
One. adopt carbon tetrachloride to cause 30 of the laboratory animals of acute liver damage mice, investigate ZANGYINCHEN crude extract (ZYCT), Petroleum ether extraction position (TP), ethyl acetate extraction position (TE) and n-butanol extraction position (TN) influence, the effect of investigation anti-liver injury to experimental animal serum alt, AST.Result of the test: administration was compared CCl with the blank group after 7 days
4Model group Serum ALT, AST content significantly raise; Crude extract (TZYC), n-butyl alcohol extract (TN) and acetic acid ethyl ester extract (TE) all obviously reduce ALT and AST level in the experimental hepatic injury animal serum, and petroleum ether extract (TP) does not have obviously change.
The experimental result prompting, the ZANGYINCHEN crude extract has the anti-liver injury effect, wherein ethyl acetate extraction and n-butanol extraction partly are the active sites of its anti-liver injury effect, and the petroleum ether extract part in the ZANGYINCHEN crude extract does not then have the active function of anti-liver injury.
Two. use classical experimental liver damage animal model (CCl4 causes the acute liver damage mice), 60 mices are divided into 6 groups, are respectively normal group, model group, bifendate matched group, the high, medium and low dosage group of ZANGYINCHEN extract (ZTG).Except that normal group, all the other are respectively organized with 50% carbon tetrachloride lumbar injection modeling.Wherein 4 treated animals are distinguished high, medium and low dosage of administration ZANGYINCHEN extract and positive control drug bifendate, and normal and model group only gives the equivalent normal saline, 1 time/day.Each treated animal is got blood system from serum after the 12nd week, detects serum liver functional biochemistry index, adopts the intracardiac perfusion fixation to get rat liver simultaneously and does paraffin section, observes the morphologic change of hepatic pathology.
Experimental result:
1. ZANGYINCHEN extract causes the influence of acute liver damage mice serum ALT, AST to CCl4
The result shows (seeing Table 1), with the blank group relatively, CCl4 model group mice serum ALT, AST content significantly raise (P＜0.01); Compare with model group, ZANGYINCHEN extract 300, two dosage groups of 200mg/kg and bifendate group ALT, AST content obviously reduce (P＜0.05 or P＜0.01), and ZANGYINCHEN extract 100mg/kg dosage group is to the reduction not statistically significant of ALT, AST content.
Table 1 ZANGYINCHEN extract causes the influence of acute liver damage mice serum ALT, AST to CCl4
(n＝10)
Compare with normal group, ▲ P＜0.05, ▲ ▲ * P＜0.05 is compared, * * P＜0.01 in P＜0.01 with model group
2. hepatic tissue HE dyeing is observed
The hepatic tissue specimen carries out observing under the optical microscope after HE dyeing, finds that respectively organizing rat liver except that the normal control group has all shown tangible histology's pathological change.
(1) normal control group
The no abnormal phenomenon of rats in normal control group hepatic tissue.As seen om observation should organize hepatic cords marshalling in the hepatic tissue, and the lobules of liver clear in structure is complete, and no proliferation of fibrous tissue and portal area enlarge, and also do not have hepatic cell fattydegeneration, necrosis, no cell infiltration.
(2) model group
Model group liver tissues of rats pathological lesion is serious.The hepatic cords arrangement disorder, the part sinus hepaticus disappears, the lobules of liver structural deterioration, collagen hypertrophy connective tissue sinks to hepatic tissue to be cut apart and holds hepatocyte, be separated out the pseudolobuli that differs in size in a large number, see that a small amount of downright bad hepatocyte is arranged around the obvious swelling of hepatocyte in the pseudolobuli, the degeneration, central vein, and with inflammatory cell infiltration.
(3) administration group
Big or middle dosage group of ZANGYINCHEN extract and bifendate group therapeutic effect are better, and it is clear substantially that hepatic cords is arranged, and pseudolobuli does not appear in the light damage of lobules of liver structure, a small amount of cell infiltration, and hepatic cell fattydegeneration is lighter; ZANGYINCHEN extract small dose group therapeutic effect is more not obvious, and visible pseudolobuli forms, and hepatic cell fattydegeneration, necrosis and cellular swelling are more serious.
The extract that relates in the pharmacological evaluation of the present invention makes according to the method for embodiment 1.
Utilize ZANGYINCHEN extract of the present invention to the research that experimentizes of experimental liver damage animal model, by observing ZANGYINCHEN extract to experimental hepatic injury animal serum liver function index and the microstructural influence of hepatic tissue, ZANGYINCHEN extract of the present invention as can be seen has the pharmacologically active of anti-liver and gall damage.
The specific embodiment
Following examples are in order to illustrate in greater detail the present invention, are not that the present invention is construed as limiting.
Embodiment 1 ZANGYINCHEN crude extract
Dried plant herb 5kg, pulverize through rough, with the soak with ethanol of 75% (wt%) of 6 times of amounts (weight) 2 hours, heating and refluxing extraction 1-3 hour then, arbitrary time in 2 hours or 1.5～5 hours for example, same method is extracted 2 times again, merge extractive liquid, reclaims etoh solvent, obtains the ZANGYINCHEN ethanol extraction, be concentrated into about 0.95kg, obtain ZANGYINCHEN crude extract or ZANGYINCHEN total extract extractum ZYCT.
The yield of ZANGYINCHEN crude extract is about 19%; Wherein content of total flavone is the 20%wt of ZANGYINCHEN extract gross weight in the ZANGYINCHEN extract.
Through detecting and the pharmacological evaluation checking, this ZANGYINCHEN crude extract has the pharmacological action of anti-liver and gall damage.
Detection method comprises employing organic solvent (comprising petroleum ether, ethyl acetate and n-butyl alcohol) extraction earlier, adopt macroporous absorption resin chromatography to carry out ethanol gradient elution and separate again, adopt the method for Si-gel (silica gel column chromatography), Sephadex LH-20 (gel column chromatography) and HPLC (prepare and use high performance liquid chromatography) to carry out further separation and purification then, use at last UV, MS,
1H-NMR and
13Modern spectral method such as C-NMR and technology are identified its structure, separate obtaining with 1-O-β-D-Glucopyranose .-3 8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside, Comastomaside, BS1), oleanolic acid (Oleanolic Acid, BS2), ursolic acid (Ursolic acid, EE1), 1,3,8-trihydroxy-7-methoxyl group
Ketone (1,3,8-tridroxy-7-methoxyxanthone, EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (1-droxy-3,7-dimethoxy xanthone, EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (1-droxy-3,7,8-trimethoxy xanthone, EE4), 7,22-bean steroid diene 3 alcohol (stigmasta-7,22-3-dienol, EE5), hexadecylic acid (hexadecanoic acid, EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (1,8-diidroxy-3,7-dimethoxy xanthone, ES1), (Swertisin ES2) is the chemical compound of representative to swertisin.
The preparation of the effective site of embodiment 2 ZANGYINCHEN extract of the present invention
The crude extract of the ZANGYINCHEN that embodiment 1 obtains or ZANGYINCHEN total extract extractum (ZYCT) are (950g), add water (mass ratio of water and total extract extractum is about 2:1) and make its suspension, add petroleum ether extraction (volume ratio of petroleum ether and water is about 2:1) extraction, obtain petroleum ether extraction liquid and water layer A, water layer A adds approximately and the isopyknic ethyl acetate of water layer A (extracts 2～3 times, obtain acetic acid ethyl acetate extract and water layer B, acetic acid ethyl acetate extract obtains acetic acid ethyl ester extract TE (approximately being 95g) after reclaiming solvent, water layer B adding pact and the isopyknic n-butanol extraction of water layer B 2～3 times, obtain butanol extraction liquid and water layer, butanol extraction liquid obtains n-butyl alcohol extract TN (approximately 240g) after reclaiming solvent, collect TE and TN, get ZANGYINCHEN extract.ZANGYINCHEN extract yield (accounting for exsiccant ZANGYINCHEN medical material) is about 6.7% (335/5000g); Wherein content of total flavone is 40% of this ZANGYINCHEN extract gross weight.
Adopt carbon tetrachloride to cause 30 of the laboratory animals of acute liver damage mice, investigate ZANGYINCHEN crude extract (ZYCT), Petroleum ether extraction position (TP), ethyl acetate extraction position (TE) and n-butanol extraction position (TN) influence, investigate its anti-liver injury effect experimental animal serum alt, AST.Result of the test shows: administration was compared CCl with the blank group after 7 days
4Model group mice serum ALT, AST content significantly raise; Crude extract (ZYCT), n-butyl alcohol extract (TN) and acetic acid ethyl ester extract (TE) all have the effect of ALT and AST level in the experimental hepatic injury animal serum of tangible reduction, and petroleum ether extract (TP) does not have obviously and changes.
Experimental result prompting and proof, the ZANGYINCHEN crude extract has the anti-liver injury effect, wherein ethyl acetate extraction and n-butanol extraction partly are the active sites of its anti-liver injury effect, and the petroleum ether extract part in the ZANGYINCHEN crude extract does not then have the active function of anti-liver and gall damage.
Through detecting and the pharmacological evaluation checking, detection method is with embodiment 1, and this ZANGYINCHEN extract (that is the effective site of ZANGYINCHEN) has the pharmacological action of anti-liver and gall damage.
The separation and the evaluation of the chemical constituent of embodiment 3. ZANGYINCHEN extract (ZANGYINCHEN effective site or ZANGYINCHEN active site).
(1) system of chemical constituent separates
The TE that embodiment 2 obtains, promptly, acetic acid ethyl ester extract (90g), ethanol ultrasonic dissolution with 20%wt, last AB-8 macroporous adsorptive resins adsorbs the crude separation of impurity, adopt 40% successively, 60%, the ethanol of 80% and 95% mass concentration carries out the gradient ethanol elution, obtain 40% ethanol elution (EF successively, 8g), 60% ethanol elution (ES, 12g), 80% ethanol elution (EE, 29g), 95% ethanol elution (EN, 40g), four polar fractions that obtain for the rough segmentation of adsorbent resin chromatograph, further adopt Si-gel again, the method of SephadexLH-20 and HPLC is carried out separation and purification, obtain compd E N1,2,3,4,5,6, compd E E1,2,3,4,5,6, and compd E S1,2,3.
The TN that embodiment 2 obtains, promptly, n-butyl alcohol extract (230g) the ethanol ultrasonic dissolution of 20%wt, last AB-8 macroporous adsorptive resins adsorbs the crude separation of impurity, adopt 40% successively, 60%, the ethanol of 80% and 95% mass concentration carries out the gradient ethanol elution, obtain 40% ethanol elution (BT successively, 42g), 60% ethanol elution (BF, 45g), 80% ethanol elution (BS, 52g), 95% ethanol elution (BN, 31g), four polar fractions that obtain for the rough segmentation of adsorbent resin chromatograph, further adopt Si-gel again, the method of SephadexLH-20 and HPLC is carried out separation and purification, obtains compd B S1 and BS2.
(2) evaluation of chemical constituent
The monomeric compound that separation is obtained is through determination of physical constant, use UV, MS,
1H-NMR and
13Modern spectral method such as C-NMR and technology identify its structure, and the structure of separating 16 chemical compounds that obtain is respectively: cupreol (β-sitosterol, EN1), daucosterol (daucosterol, EN2), 1,5,8-trihydroxy-3-methoxyl group
Ketone (1,5,8-trihydroxy-3-methoxyxanthone, EN3), 1,8-dihydroxy-3,7-dimethoxy
Ketone (methylswertiamin, EN4), the 1-hexacosyl alcohol (1-hexacosanol, EN5), the octacosanoic acid methyl ester (Methyl Octacosanoate, EN6), ursolic acid (Ursolic acid, EE1), 1,3,8-trihydroxy-7-methoxyl group
Ketone (1,3,8-tridroxy-7-methoxy xanthone, EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (1-droxy-3,7-dimethoxy xanthone, EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (1-droxy-3,7,8-trimethoxy xanthone, EE4), 7,22-bean steroid diene 3 alcohol (stigmasta-7,22-3-dienol, EE5), hexadecylic acid (hexadecanoic acid, EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (1,8-diidroxy-3,7-dimethoxy xanthone, ES1), swertisin (Swertisin, ES2), 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside, Comastomaside, BS1), oleanolic acid (Oleanolic Acid, BS2).
Above-mentioned isolated compound is in the ZANGYINCHEN extract gross weight, and the content of every kind of chemical compound (wt%) generally is distributed between the 0.1%-10%, concentrates between the 0.5%-5%.
Above-claimed cpd has been formed the total flavones of ZANGYINCHEN extract of the present invention, and often the ormal weight method is measured, and content of total flavone is 40% of a ZANGYINCHEN extract gross weight in this ZANGYINCHEN extract.
Through pharmacological evaluation checking, in this ZANGYINCHEN extract contain cupreol (β-sitosterol, EN1), daucosterol (daucosterol, EN2), 1,5,8-trihydroxy-3-methoxyl group
Ketone (1,5,8-trihydroxy-3-methoxyxanthone, EN3), 1,8-dihydroxy-3,7-dimethoxy
Ketone (methylswertiamin, EN4), the 1-hexacosyl alcohol (1-hexacosanol, EN5), the octacosanoic acid methyl ester (Methyl Octacosanoate, EN6) position of above-claimed cpd does not have the pharmacological action of anti-liver and gall damage; And contain 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone (the larynx lanatoside, Comastomaside, BS1), oleanolic acid (Oleanolic Acid, BS2), ursolic acid (Ursolic acid, EE1), 1,3,8-trihydroxy-7-methoxyl group
Ketone (1,3,8-tridroxy-7-methoxy xanthone, EE2), 1-hydroxyl-3, the 7-dimethoxy
Ketone (1-droxy-3,7-dimethoxy xanthone, EE3), 1-hydroxyl-3,7, the 8-trimethoxy
Ketone (1-droxy-3,7,8-trimethoxy xanthone, EE4), 7,22-bean steroid diene 3 alcohol (stigmasta-7,22-3-dienol, EE5), hexadecylic acid (hexadecanoic acid, EE6), 1,8-dihydroxy-3,7-dimethoxy
Ketone (1,8-diidroxy-3,7-dimethoxy xanthone, ES1), (Swertisin, ES2) position of above-claimed cpd then has the pharmacologically active of anti-liver and gall damage to swertisin.
By follow-up study, the ZANGYINCHEN plant of the different collecting seasons of different batches, through above-mentioned same extraction preparation, content of total flavone can reach 20% of this ZANGYINCHEN extract gross weight in the ZANGYINCHEN extract that obtains, even the higher crude drug of quality is through after repeating that said extracted is refining and separating the remove impurity process, content of total flavone can reach 65% of ZANGYINCHEN extract gross weight in the ZANGYINCHEN extract, the general equal 30-60% that reaches.
Above-mentioned ZANGYINCHEN extract and reference substance ZANGYINCHEN extract A and B are carried out the simple effects contrast experiment, wherein ZANGYINCHEN extract A is that prior art (literature method) is extracted the ZANGYINCHEN extract that obtains, ZANGYINCHEN extract B contains total flavones, but the not enough 20%wt of general flavone content wherein, and this total flavones does not contain or trace contains (content is lower than 0.1%wt) 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
The ZANGYINCHEN extract of ketone and oleanolic acid; Through containing 1-O-β-D-Glucopyranose .-3 of 0.1%wt at least, 8-dihydroxy-7-methoxyl group in the total flavones of tentatively relatively finding, have only to contain in the ZANGYINCHEN extract 20%wt at least and its this total flavones
The group of ketone and oleanolic acid just demonstrates the activity of significant anti-liver and gall damage.The repeated experiments of embodiment 4 ZANGYINCHEN extract
Repeat the present invention and extract purge process, the experiment of different batches obtains the ZANGYINCHEN extract of different batches, and wherein content of total flavone is respectively 28%, 33%, 41%, 45%, 46%55%, 61% etc. of ZANGYINCHEN extract gross weight.Through scientific method statistics, total flavones occupies weight percentage data and shows in ZANGYINCHEN extract, and more multidata concentrates on total flavones and accounts between the ZANGYINCHEN extract 30-60% (weight).
Embodiment 5 contains the preparation of the compositions (capsule formulation) of ZANGYINCHEN extract
Get the ZANGYINCHEN extract 250g that the method for embodiment 2 makes, starch 100g is that binding agent is granulated with 20% ethanol, and is encapsulated, promptly.
Embodiment 6 contains the preparation of the compositions (tablet formulation) of ZANGYINCHEN extract
Get the ZANGYINCHEN extract 25g that the method for embodiment 2 makes, add starch 50g, mixing is a binding agent with the starch slurry, granulate, and oven dry, granulate, tabletting, promptly.
Embodiment 7 contains the preparation of the compositions (effervescent tablet preparation formulation) of ZANGYINCHEN extract
Prescription: citric acid 60g, sodium bicarbonate 50g, the ZANGYINCHEN extract 500g that the method for embodiment 2 makes, starch 400g makes 1000 altogether.
Preparation technology: the citric acid of recipe quantity is added ZANGYINCHEN extract and the appropriate amount of starch that the method for getting embodiment 2 makes granulate, sodium bicarbonate adds remaining starch granulates, and after the drying, mixes respectively, and tabletting promptly.
Embodiment 8 contains the preparation of the compositions (granule) of ZANGYINCHEN extract
Get the extract 250g that the method for embodiment 2 makes, glucose 100g, dextrin 200g is that binding agent is granulated with rare gelatinized corn starch, packing, promptly.
Claims (10)
1, a kind of ZANGYINCHEN extract, wherein, this ZANGYINCHEN extract contains total flavones, is 100% in this ZANGYINCHEN extract gross weight, and this total flavones accounts for the 20-75% of this ZANGYINCHEN extract.
2, ZANGYINCHEN extract as claimed in claim 1, wherein, total flavones described in this ZANGYINCHEN extract comprises 1-O-β-D-Glucopyranose .-3,8-dihydroxy-7-methoxyl group
Ketone, oleanolic acid, ursolic acid, 1,3,8-trihydroxy-7-methoxyl group
Ketone, 1-hydroxyl-3, the 7-dimethoxy
Ketone, 1-hydroxyl-3,7, the 8-trimethoxy
Ketone, 7,22-bean steroid diene 3 alcohol, hexadecylic acid, 1,8-dihydroxy-3,7-dimethoxy
Ketone, swertisin, above-claimed cpd respectively account for the 0.1%-10% of the weight of described total flavones.
3, ZANGYINCHEN extract as claimed in claim 1, it is made by following method:
(1) the ZANGYINCHEN medical material being used the mass concentration of at least 2 times of weight is the alcohol reflux extraction of 55-95%, and extracting solution reclaims solvent, obtains the crude extract of ZANGYINCHEN;
(2) crude extract of the ZANGYINCHEN that obtains of above-mentioned steps (1) adopts petroleum ether, ethyl acetate and n-butanol extraction successively, collects acetic acid ethyl acetate extract, obtains acetic acid ethyl ester extract after reclaiming solvent; Collect butanol extraction liquid, obtain n-butyl alcohol extract behind the recovery solvent;
(3) combined ethyl acetate extract and n-butyl alcohol extract obtain described ZANGYINCHEN extract.
4, ZANGYINCHEN extract as claimed in claim 3, wherein, step (2) comprising:
ZANGYINCHEN extract adds water, add petroleum ether extraction then, obtain petroleum ether extraction liquid and water layer A, water layer A adds ethyl acetate extraction at least 1 time, obtain acetic acid ethyl acetate extract and water layer B, acetic acid ethyl acetate extract obtains acetic acid ethyl ester extract after reclaiming solvent, and water layer B adds n-butanol extraction at least 1 time, obtain butanol extraction liquid and water layer, butanol extraction liquid obtains n-butyl alcohol extract after reclaiming solvent.
5, ZANGYINCHEN extract as claimed in claim 3, wherein, step (2) also comprises afterwards:
Adopt macroporous absorption resin chromatography to carry out the alcoholic acid gradient elution that mass concentration is 40-95% respectively acetic acid ethyl ester extract and n-butyl alcohol extract, collect this acetic acid ethyl ester extract and n-butyl alcohol extract 40-95% ethanol elution separately, reclaim solvent, merge, obtain ZANGYINCHEN extract.
6, the preparation method of the described ZANGYINCHEN extract of claim 1, this method comprises:
(1) the ZANGYINCHEN medical material being used the mass concentration of at least 2 times of weight is the alcohol reflux extraction of 55-95%, and extracting solution reclaims solvent, obtains the crude extract of ZANGYINCHEN;
(2) crude extract of the ZANGYINCHEN that obtains of above-mentioned steps (1) adopts petroleum ether, ethyl acetate and n-butanol extraction successively, collects acetic acid ethyl acetate extract, obtains acetic acid ethyl ester extract after reclaiming solvent; Collect butanol extraction liquid, obtain n-butyl alcohol extract behind the recovery solvent;
(3) combined ethyl acetate extract and n-butyl alcohol extract obtain described ZANGYINCHEN extract.
7, preparation method as claimed in claim 6, wherein, described step (2) also comprises afterwards:
Adopt macroporous absorption resin chromatography to carry out the alcoholic acid gradient elution that mass concentration is 40-95% respectively acetic acid ethyl ester extract and n-butyl alcohol extract, collect this acetic acid ethyl ester extract and n-butyl alcohol extract 40-95% ethanol elution separately, reclaim solvent, merge, obtain ZANGYINCHEN extract.
8, the Pharmaceutical composition that contains the described ZANGYINCHEN extract of claim 1, wherein, this Pharmaceutical composition contains the ZANGYINCHEN extract and the pharmacy adjuvant of pharmacy effective dose.
9, the application of the described ZANGYINCHEN extract of claim 1 in the medicine of preparation treatment liver and gall diseases.
10, the described application of Pharmaceutical composition in the medicine of preparation treatment liver and gall diseases that contains ZANGYINCHEN extract of claim 8.
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| CNA2009100785788A CN101480422A (en) | 2009-02-27 | 2009-02-27 | Tibetan oriental wormwood extract as well as preparation method and use thereof |
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| CN101845037A (en) * | 2010-05-19 | 2010-09-29 | 重庆市中药研究院 | Method for separating xanthione chemical component in Swertia mussoti |
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| CN101653475B (en) * | 2009-09-14 | 2011-06-08 | 成都力思特制药股份有限公司 | Novel application of plant extract in preparing medicament for treating light type alcoholic hepatic disease |
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| CN108096320A (en) * | 2018-01-04 | 2018-06-01 | 青岛科技大学 | A kind of Tibetan Herba Schizonepetae extract with hypoglycemic effect and preparation method thereof |
| CN114191529A (en) * | 2021-12-28 | 2022-03-18 | 殷万年 | A Chinese medicinal composition for treating hepatitis B in combination with vinegar |
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