CN102743424A - Flos chrysanthemi indici effective ingredient and application thereof - Google Patents
Flos chrysanthemi indici effective ingredient and application thereof Download PDFInfo
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- CN102743424A CN102743424A CN2012102386248A CN201210238624A CN102743424A CN 102743424 A CN102743424 A CN 102743424A CN 2012102386248 A CN2012102386248 A CN 2012102386248A CN 201210238624 A CN201210238624 A CN 201210238624A CN 102743424 A CN102743424 A CN 102743424A
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Abstract
The invention discloses a flos chrysanthemi indici effective ingredient, which is extracted by a method comprising the following steps of: 1) taking flos chrysanthemi indici, adding 60-90% ethanol which is 5-10 times of the weight of the flos chrysanthemi indici to extract, filtering the extracting solution, and concentrating the filtrate into extract; and 2) adding water into the extract to prepare suspension, adding extract liquor to extract, and recovering the extract liquor to obtain the flos chrysanthemi indici effective ingredient. Experiments show that the flos chrysanthemi indici effective ingredient can more obviously inhibit hepatitis B virus, can also inhibit the glutamic-oxalacetic transaminease (AST) and glutamic-pyruvic transaminease (ALT) indexes of liver serum of the damaged liver, can reduce the tumor necrosis factor (TNF) content, liver indexes and malondialdehyde content of the liver serum of the damaged liver, and has good liver protection function. The flos chrysanthemi indici effective ingredient can be used as the active ingredient, and can be prepared into anti-hepatitis B virus medicaments or compositions with the liver protection function, such as food, drinks, health care products, drugs and the like, and the medicaments or compositions can be prepared into tablet, capsule, powder, injections and any other medically acceptable formulations.
Description
Technical field
The present invention relates to a kind of Flos Chrysanthemi Indici anti-hepatitis virus regulating liver-QI protective effect effective site, relate to the application of this effective site simultaneously, belong to the biological medicine technology field.
Background technology
Hepatitis B is to be continued to duplicate and a kind of infectiousness hepatic disease of causing by hepatitis B virus (HBV), is a kind of viral disease that present infectiousness is the strongest, sickness rate is the highest, harm is maximum.China is " hepatitis B " big country; The HBVer surpasses 1.2 hundred million people, and the liver cirrhosis that is caused by hepatitis B, the case fatality rate of hepatocarcinoma occupy the 5th in China, are called as " reticent killer "; But hepatitis B is still lacked effective control device and solution completely both at home and abroad so far; Do not have specific drug yet, therefore, important social meaning and realistic meaning are arranged the development and the exploitation of effective treatment hepatitis B medicine.Chinese medicine is because of it is obvious to several diseases viral disease whole structure, antivirus action is machine-processed various and be not easy to produce focus and the focus that drug resistance becomes international concern.
Flos Chrysanthemi Indici is Compositae (Compositae, Asteraceae) head inflorescence of Chrysanthemum (Dendranthema) herbaceos perennial Herba Dendranthematis indici (Chrysanthemum indicum L.), HERBA CHRYSANTHEMI LAVANDULAEFOLII (Chrysanthemum boreale Mak.) or lavandulaleaf dendranthema herb (Chrysanthemum lavandulaefolium (Fisch.) Mak.).Ancient Chinese medicine doctor thinks that Flos Chrysanthemi Indici " goes into Liver Channel "; Effect with " heat-clearing and toxic substances removing, soothing the liver making eye bright "; Clinical application is extensive; Much more especially to be used to treat various inflammation, viral disease, cardiovascular disease etc., to comprise chronic pharyngitis, prostatitis, pelvic inflammatory disease, molluscum contagiosum, viral hepatitis, hypertension etc.Chinese scholars finds that its crude extract has multiple pharmacological effect such as anti-inflammation is analgesic, antiviral, blood pressure lowering, immunomodulating, antioxidation.And we have the effect that suppresses hepatitis B virus more significantly through the ethanol extract of discovering Flos Chrysanthemi Indici, and through optimizing Study on extraction, obtain an active site with the protective effect of anti-hepatitis virus regulating liver-QI, and not seeing as yet both at home and abroad at present has report.
Summary of the invention
The purpose of this invention is to provide a kind of Wild chrysanthemum effective component, have the effect of anti-hepatitis virus regulating liver-QI protection.
Another object of the present invention is to provide application and the application in preparation liver-protecting combination of a kind of Wild chrysanthemum effective component in the preparation anti-hepatic-B virus medicine.
In order to realize above purpose, the technical scheme that Wild chrysanthemum effective component of the present invention adopted is: adopt the method for following steps to extract the Wild chrysanthemum effective component that forms:
1) gets Flos Chrysanthemi Indici, add 60～90% ethanol extractions of 5～10 times of weight, behind the extracting liquid filtering filtrating is concentrated into extractum;
2) extractum is added water and process suspension; Adding extract extracts; Reclaim extract and obtain Wild chrysanthemum effective component; Wherein extract adopts two or three in petroleum ether, ethyl acetate, the ethanol, and the volume ratio of extraction process PetroChina Company Limited. ether, ethyl acetate, amount of ethanol is 40～10:5～1:1～0.
Before the said ethanol extraction of step 1) Flos Chrysanthemi Indici is carried out the physics fragmentation.
The said extraction time of step 1) is three times, and said extracting solution is three amalgamation liquids that extract.
Step 2) number of times of said extraction is more than three times.
Through experiment showed, that Wild chrysanthemum effective component of the present invention has the effect of anti-hepatitis virus regulating liver-QI protection.Active site mainly comprises sesquiterpenoids and flavone compound.Wherein, total sesquiterpene content >=45%, general flavone content >=35%; Linarin >=0.65%, luteolin >=0.55%, ambrosin A (cumambrinA) >=2.90%; Apigenin >=0.50%, acacetin >=0.10%, Radix Angelicae Sinensis acyl ambrosin B (angeloylcumambrinB) >=0.50%.
The present invention utilizes the HepG2.2.15 cell as platform, has studied the external anti-HBV effect of Wild chrysanthemum effective component of the present invention (CIC).Adopt mtt assay detection cytotoxicity and calculate TC50; Adopt the ELISA method to detect the influence of HBsAg in the pair cell supernatant, HBeAg expression.The result shows that Wild chrysanthemum effective component of the present invention has comparatively obvious suppression effect to hepatitis B virus.
The present invention through experiment confirm Wild chrysanthemum effective component of the present invention, can suppress AST, ALT index in the liver serum of hepatic injury, reduce TNF content, liver exponential sum MDA content in the liver serum of hepatic injury, have good liver protective effect.
The present invention also provides two kinds of application of Wild chrysanthemum effective component, the application that one of which is a Wild chrysanthemum effective component in the preparation anti-hepatic-B virus medicine, and it two be that Wild chrysanthemum effective component is protected the application in the compositions the preparation liver.Wild chrysanthemum effective component of the present invention can be used as active component; Be prepared into medicine with anti-hepatitis virus; The compositions that perhaps has liver protection effect; Like food, beverage, health product, medicine etc., what relate to medicine can adopt medically acceptable any dosage forms such as tablet, capsule, powder, injection.
The specific embodiment
Embodiment 1
Wild chrysanthemum effective component of the present invention adopts following steps to extract and forms:
1) get Flos Chrysanthemi Indici, 90% ethanol extraction of 5 times of weight of adding three times, merge extractive liquid, is concentrated into extractum with filtrating after the filtration;
2) get extractum and add water and process suspension, add extract and extract three times, extract adopts petroleum ether-ethyl acetate, its volume ratio 40:1, and three extraction backs merge carries out decompression and solvent recovery, promptly obtains effective site.
Embodiment 2
Wild chrysanthemum effective component of the present invention adopts following steps to extract and forms:
1) get Flos Chrysanthemi Indici, 75% ethanol extraction of 8 times of weight of adding three times, merge extractive liquid, is concentrated into extractum with filtrating after the filtration;
2) get extractum and add water and process suspension, add extract and extract three times, extract adopts petroleum ether-ethyl acetate-ethanol, its volume ratio 30:3:0.5, and three extraction backs merge carries out decompression and solvent recovery, promptly obtains effective site.
Embodiment 3
Wild chrysanthemum effective component of the present invention adopts following steps to extract and forms:
1) get Flos Chrysanthemi Indici, 60% ethanol extraction of 10 times of weight of adding three times, merge extractive liquid, is concentrated into extractum with filtrating after the filtration;
2) get extractum and add water and process suspension, add extract and extract five times, extract adopts petroleum ether-ethyl acetate-ethanol, its volume ratio 10:5:1, and five extraction backs merge carries out decompression and solvent recovery, promptly obtains effective site.
Experimental example
(1) anti-HBV effect
Experimental technique: utilize the HepG2.2.15 cell as platform, studied the external anti-HBV effect of effective site (CIC).Adopt mtt assay detection cytotoxicity and calculate TC50; Adopt the ELISA method to detect the influence of HBsAg in the pair cell supernatant, HBeAg expression.
The result: active site has comparatively obvious suppression effect (effectively low toxicity) to hepatitis B virus, and has dose-effect relationship.
Table 1 is 9 days HBsAg expressions of results, and table 2 is 9 days HBeAg expressions of results
The judgement of experimental result effectiveness and explanation:
TC50: expression median lethal concentration, the μ g/ml of unit
IC50: expression suppresses antigen medium effective concentration, the μ g/ml of unit
TI: the expression therapeutic index, computing formula is: TC50/IC50
TI < 1.0: poisonous invalid
1.0≤TI≤2.0: low toxicity is effective
2.0 < TI < 10.0: effective low toxicity
TI >=10.0: high-efficiency low-toxicity
Table 19 day HBsAg expression of results
Table 29 day HBeAg expression of results
(2) liver protective effect
Utilize con A (Con-A) to cause the test of mouse immune liver damage, studied the function for protecting liver and reducing enzyme activity of effective site (CIC position) hepatic injury.Research shows that the effective site position is different to the influence of the ALT in the liver serum, AST, IFN, TNF, liver index, hepatic tissue MDA, and pathological section shows that effective site can help hepatic injury to recover preferably.The result sees table 3,4,5,6.
The effective site preparation: the effective site that takes by weighing embodiment 1～3 extraction is an amount of, with propylene glycol and the dissolving of 1% sodium carboxymethyl cellulose.
Effective site group dosage range: 5～45%, concrete embodiment 1 is 5%, and embodiment 2 is 25%, and embodiment 3 is 45%.
Method for establishing model: experimental animal model adopts Balb/c mice iv.ConA to make the immunologic liver injury model, detects AST, ALT level in the mice serum, the MDA content in cytokine IFN-γ, TNF-α and the hepatic tissue; Detect index and all obviously raise, observe the hepatic pathology section, hepatocyte has vacuolar degeneration and necrosis; Being dispersivity distributes; Inflammatory cell infiltration is arranged, and the cell arrangement disorder, the modeling success.
Test method: the mouse stomach administration, continuous 7 days, each administration of the morning and afternoon first day and the next afternoon 1 time, all the other are all once a day.After the last administration, water 4h is can't help in fasting, and every mice is with 20mg/kg dosage tail vein injection ConA (blank control group injection PBS), and water 12h is can't help in fasting, to make the immunologic liver injury model.
Mice is plucked the eyeball blood sampling, and separation of serum is to be measured, is stored in-20 ℃ of refrigerators.Mice takes off neck puts to death, and dissects immediately, gets liver and weighs, and chooses diseased region, fixing in 4% formaldehyde, the preparation hepatic tissue section, and remainder is stored in-20 ℃ of refrigerators, is used for hepatic tissue MDA content detection.Each biochemical indicator detects and is all undertaken by the test kit operation instruction, and hepatic tissue is made pathology section examination.
Table 3 different pharmaceutical dosage is to the influence of immunologic liver injury mice serum ALT, AST
Compare * P＜0.05 with model group
Result: CIC group can suppress increasing of AST, ALT in the immunologic liver injury mice serum.
Table 4 different pharmaceutical dosage is to the influence of immunologic liver injury mice serum IFN, TNF
Compare * P＜0.05 with model group
Result: CIC group does not have obvious influence to IFN content in the immunologic liver injury mice serum, and the content of TNF in the immunologic liver injury mice serum is had obvious reduction effect.
Table 5 different pharmaceutical dosage is to the influence of immunologic liver injury liver index and hepatic tissue MDA
Compare * P＜0.05 with model group
The result: compare with model group, the mouse liver exponential sum MDA content of CIC group all reduces.
Table 6 pathology section examination result
The result: pathology section examination shows that CIC group hepatic injury tissue recovers basically.
Claims (9)
1. Wild chrysanthemum effective component is characterized in that: adopt the method for following steps to extract and form:
1) gets Flos Chrysanthemi Indici, add 60～90% ethanol extractions of 5～10 times of weight, behind the extracting liquid filtering filtrating is concentrated into extractum;
2) extractum is added water and process suspension; Adding extract extracts; Reclaim extract and obtain Wild chrysanthemum effective component; Wherein extract adopts two or three in petroleum ether, ethyl acetate, the ethanol, and the volume ratio of extraction process PetroChina Company Limited. ether, ethyl acetate, amount of ethanol is 40～10:5～1:1～0.
2. Wild chrysanthemum effective component according to claim 1 is characterized in that: the said extraction time of step 1) is three times, and said extracting solution is three amalgamation liquids that extract.
3. Wild chrysanthemum effective component according to claim 1 is characterized in that: step 2) number of times of said extraction is more than three times.
4. the application of Wild chrysanthemum effective component in the preparation anti-hepatic-B virus medicine according to claim 1.
5. the application of Wild chrysanthemum effective component in the compositions of preparation liver protection effect according to claim 1.
6. according to the said application of claim 5, it is characterized in that: said hepatoprotective is for suppressing the liver serum alt of hepatic injury or increasing of AST.
7. according to the said application of claim 5, it is characterized in that: said hepatoprotective is TNF content in the liver serum that reduces hepatic injury.
8. according to the said application of claim 5, it is characterized in that: said hepatoprotective is liver index or MDA content in the liver serum that reduces hepatic injury.
9. according to each said application of claim 5-8, it is characterized in that: said compositions is food, health product, medicine or beverage.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961427A (en) * | 2012-12-14 | 2013-03-13 | 安徽老炊食品有限公司 | Wild chrysanthemum extraction process and measuring method for total flavonoids in wild chrysanthemums |
| CN104628801A (en) * | 2015-01-21 | 2015-05-20 | 郑州大学 | Process for extracting and separating linarin from wild chrysanthemum |
| CN105920073A (en) * | 2016-06-03 | 2016-09-07 | 郑州大学 | Preparation method for wild chrysanthemum flower liver-protection soft capsule |
| WO2017097012A1 (en) * | 2015-12-11 | 2017-06-15 | 深圳市贝沃德克生物技术研究院有限公司 | Pure natural functional beverage suitable for chronic hepatitis b patients and preparation method thereof |
| CN114209728A (en) * | 2022-02-14 | 2022-03-22 | 郑州大学 | Wild chrysanthemum active site with functions of regulating and controlling epigenetic expression balance and resisting liver cancer, and preparation method and application thereof |
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| CN101721455A (en) * | 2009-12-28 | 2010-06-09 | 贵州大学 | Manyflower tickclove herb extract, and preparation method and application thereof |
| CN102429933A (en) * | 2011-12-22 | 2012-05-02 | 武汉大学 | Application of tuckahoe triterpenes to preparation of liver-protecting medicine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961427A (en) * | 2012-12-14 | 2013-03-13 | 安徽老炊食品有限公司 | Wild chrysanthemum extraction process and measuring method for total flavonoids in wild chrysanthemums |
| CN104628801A (en) * | 2015-01-21 | 2015-05-20 | 郑州大学 | Process for extracting and separating linarin from wild chrysanthemum |
| WO2017097012A1 (en) * | 2015-12-11 | 2017-06-15 | 深圳市贝沃德克生物技术研究院有限公司 | Pure natural functional beverage suitable for chronic hepatitis b patients and preparation method thereof |
| CN105920073A (en) * | 2016-06-03 | 2016-09-07 | 郑州大学 | Preparation method for wild chrysanthemum flower liver-protection soft capsule |
| CN114209728A (en) * | 2022-02-14 | 2022-03-22 | 郑州大学 | Wild chrysanthemum active site with functions of regulating and controlling epigenetic expression balance and resisting liver cancer, and preparation method and application thereof |
| CN114209728B (en) * | 2022-02-14 | 2023-08-25 | 郑州大学 | Wild chrysanthemum active site with function of regulating epigenetic expression balance and resisting liver cancer as well as preparation method and application thereof |
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Application publication date: 20121024 |