CN100406027C - Combination of Chinese traditional medicine for treating disease of liver and preparation method - Google Patents
Combination of Chinese traditional medicine for treating disease of liver and preparation method Download PDFInfo
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- CN100406027C CN100406027C CNB2004100992681A CN200410099268A CN100406027C CN 100406027 C CN100406027 C CN 100406027C CN B2004100992681 A CNB2004100992681 A CN B2004100992681A CN 200410099268 A CN200410099268 A CN 200410099268A CN 100406027 C CN100406027 C CN 100406027C
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Abstract
The present invention belongs to the technical field of medicine, particularly to a combination of Chinese traditional medicine for treating diseases of liver and a preparation method thereof. The effective parts of the combination of Chinese traditional medicine is extracted and purified from herba hyperici japonici of traditional Chinese medicine, wherein the content of flavonoid component is from 80 to 99.5%. The flavonoid components mainly comprise quercitrin, isoquercitrin, herba hyperici japonici bigenicide C and herba hyperici japonici bigenicide D. The preparation method of the composition uses a medicinal material of herba hyperici japonici as a raw material, and the effective parts of the total flavonoids are obtained through ethanol extraction, condensation, regulation of a pH value and ethyl acetate extraction. The present invention has the advantages of simple manufacturing process and high transfer rate of the total flavonoids. The combination of Chinese traditional medicine of the present invention can be used for treating liver diseases, such as acute and chronic viral hepatitis, hepatic fibrosis and hepatic injury.
Description
Technical field
The present invention relates to medical technical field, relate to a kind of Chinese medicine composition that is used for the treatment of hepatic disease and preparation method thereof.
Background technology
Hepatitis is to endanger one of the most serious infectious disease at present in the world, and takes place closely related with hepatocarcinoma again.According to the statistics of World Health Organization (WHO), at present, there are 3.5 hundred million people in the whole world approximately for chronic hepatitis patient or be symptomless virus the infected.The patient of these chronic hepatitiss has the liver cirrhosis of transferring to, and the high risk of hepatocarcinoma has more than 100 ten thousand people to die from and hepatitis, liver cirrhosis and hepatocarcinoma diseases associated every year.China is the sick hotspot of hepatitis, according to statistics, 90% background that hepatites virus infections arranged is arranged in the liver cancer patient, and hepatocarcinoma has accounted for second of tumor fatality rate at present.
Herba Hyperici Japonici Herba Hyperici Japonici, this product beginning is stated from " the SHENGCAO property of medicine is wanted fully ", and " Zhiwu Mingshi Tukao " claims Herba Hyperici Japonici.Be Guttiferae plant Herba Hyperici Japonici herb.Former plant Herba Hyperici Japonici Hypericum japonicum Thumb.ex Murray.Be born in more moistening place, field.Be widely distributed in the Yangtze river basin extremely on the south various places.The Herba Hyperici Japonici main product in Jiangxi, ground such as Fujian, Guangdong, Guangxi, Sichuan, Guizhou.Nearly domestic and international chemists of more than ten years are to extraction, separation, the evaluation of its chemical constituent; Pharmacological action; Clinical practices etc. have been done dark step research and have been obtained huge progress.On chemical constituent, extract and determined nearly 20 kinds new compound structure.Many-sided developmental research has been carried out in the pharmacology aspect, as the bacteriostasis of Herba Hyperici Japonici; Hepatoprotective effect; Function of tumor inhibition; To cardiovascular system effect etc.In modern clinical research, the Herba Hyperici Japonici refinement is made injection and is used for the acute and chronic hepatitis of clinical treatment etc.
At present, form and other drug matching with compound recipe more than the clinical practice of Herba Hyperici Japonici are used, and surplus the Chinese patent relevant with the clinical practice of Herba Hyperici Japonici has 20 at present, mostly are compound recipe and use; Chinese patent 200310104504.X discloses a kind of preparation method of Herba Hyperici Japonici total flavones injectable powder.
In the research to Herba Hyperici Japonici medical material and Herba Hyperici Japonici injection, Shang Weijian carries out the research report and the patent application of liver disease therapy aspect screening active ingredients about separation and purification effective site and monomer from Herba Hyperici Japonici at present; The present invention be directed to the liver-protecting activity of Herba Hyperici Japonici, under the tracking instruction of pharmacologically active screening, from Herba Hyperici Japonici separation and purification go out to have protect the liver, effective site---the Herba Hyperici Japonici total flavones of anti-hepatitis, anti-hepatic fibrosis, and therefrom isolation identification four main monomeric compounds: two xanthone C of Quercitroside, isoquercitrin, Herba Hyperici Japonici and the two xanthone D of Herba Hyperici Japonici, wherein two xanthone C of Herba Hyperici Japonici and the two xanthone D of Herba Hyperici Japonici be the noval chemical compound of discovery first.
Summary of the invention
The objective of the invention is to propose a kind of from the medical material Herba Hyperici Japonici, extract can treat active ingredient composition of hepatic disease and preparation method thereof, and the application in the medicine of acute and chronic viral hepatitis, hepatic injury in treatment.
The present invention extracts from the Chinese medicine Herba Hyperici Japonici has treatment hepatic disease effect preparation of compositions method, and step is as follows:
1, extracts: adopt solvent extraction method, extract solvent and can select organic solvent or its several mixed solvents such as water, methanol, ethanol, propanol, butanols or ethyl acetate for use, extracting method is that supersound extraction, percolation extract or heating and refluxing extraction, and extraction time can be for 1-3 time.
2, extraction: adopt solvent extraction, can be with the extract aqueous dispersion of step 1 gained, the reuse organic solvent extraction, organic solvent can be selected chloroform, dichloromethane, ether, ethyl acetate or butanols etc. for use, ethyl acetate, extraction times can be for 1-4 time.
3, concentrate drying: step 2 gained extract is crossed 0.22 μ m filter membrane, and filtrate decompression is concentrated into dried, obtains active ingredient Herba Hyperici Japonici total flavones compositions.
The flavonid composition that adopts above processing step to make, wherein the flavonoid weight content is 80～99.5%, and main chemical compositions is clear and definite in the compositions, and content is fixed, and it is succinct to have technology, the advantage that production cost is low.
The pharmaceutical composition of hepatic disease such as the acute and chronic viral hepatitis of treatment provided by the invention, hepatic injury, hepatic fibrosis, wherein contain the above-mentioned active ingredient Herba Hyperici Japonici total flavones that obtains from Chinese medicine Herba Hyperici Japonici extraction purification, the weight content of flavones ingredient is 80%-99.5% in this total flavones.
Chemical constituent and pharmacologically active screening
To above resulting Herba Hyperici Japonici total flavones compositions; use the chromatography technology; further therefrom isolation identification 21 kinds of flavone compounds; filter out 4 chemical compounds through the pharmacologically active tracking with liver protection and anti-hepatic fibrosis; be respectively Quercitroside, isoquercitrin, the two xanthone C of Herba Hyperici Japonici and the two xanthone D of Herba Hyperici Japonici, they are the Main Ingredients and Appearance of total flavones.
Its structural formula is as follows respectively:
Quercitroside structural formula isoquercitrin structural formula
The two xanthone C-structure formulas of Herba Hyperici Japonici
The two xanthone D structural formulas of Herba Hyperici Japonici
The pharmacologically active screening of above-mentioned composition is as follows:
One, Herba Hyperici Japonici total flavones, to the animal checking of the protective effect of tentative hepatic injury:
(1) thioacetamide causes the chmice acute liver injury model
1, experimental animal: Kunming mouse, male or female, body weight 19～22g, ad libitum access and drinking-water, room temperature (23 ± 2 ℃), natural lighting.
2, test method: mice is divided into 7 groups at random, except that the normal control group, all the other are respectively organized all with thioacetamide (TAA) 30mg/Kg, i.p, and back 3h, 6h, 9h poison, successive administration 3 times, after the last administration 24 hours, got blood, and measured following index in promptly second day, claim liver heavy simultaneously, and carry out pathological examination.The results are shown in Table 1.
Table 1 respectively organize reagent to thioacetamide cause each index of chmice acute liver injury model influence (X ± SD, n=20)
*Compare with the NS matched group:
P＜0.05;
P＜0.01
The result: compare with the NS matched group, Herba Hyperici Japonici total flavones can significantly reduce thioacetamide and cause the hepatic injury that the chmice acute liver injury model causes.
(2) carbon tetrachloride causes rat acute liver poisoning model
1, experimental animal: adult SD rats, male or female, body weight are about 250～350g, and feed plain particles feedstuff is arbitrarily drunk water.
2, test method: rat is divided into 6 groups at random, except that the normal control group, all the other each groups are all with 50% carbon tetrachloride 0.5ml/100g, and subcutaneous injection is poisoned, and is administered once simultaneously, 4h, 8h respectively are administered once after poisoning, after the last administration 12 hours, get hematometry ALT, the variation of indexs such as AST, put to death animal and claim liver heavy, and carry out pathological examination.The results are shown in Table 2.
Table 2 respectively organize reagent to carbon tetrachloride cause each index of rat acute liver injury model influence (X ± SD, n=20)
*Compare with the NS matched group:
P＜0.05;
P＜0.01
The result: compare with the NS matched group, Herba Hyperici Japonici total flavones can significantly reduce carbon tetrachloride and cause the hepatic injury that rat acute liver poisoning model causes.
(3) D-Gal causes the rats'liver poisoning model
1, experimental animal: adult SD rats, male or female, body weight are about 250～350g, and feed plain particles feedstuff is arbitrarily drunk water.
2, test method: rat is divided into 4 groups at random, except that the normal control group, all the other each groups are all with D-Gal 800mg/Kg, lumbar injection is poisoned, and is administered once after the administration 12 hours simultaneously, get hematometry SGPT, the variation of indexs such as TBIL is put to death animal and is claimed liver heavy, and carries out pathological examination.The results are shown in Table 3.
Table 3 respectively organize reagent to D-Gal cause each index of rats'liver poisoning model influence (X ± SD, n=20)
*Compare with the NS matched group:
P＜0.05;
P＜0.01
The result: compare with the NS matched group, Herba Hyperici Japonici total flavones can significantly reduce D-Gal and cause the hepatic injury that the rats'liver poisoning model causes.
(4) tetrachloro-methane induction Liver Fibrosis Model
1, experimental animal: male Wistar rat, body weight 100～150g during on-test raises 22 ℃ of room temperatures, in the periodicity of illumination 12h:12h environment, ad libitum access and drinking-water.
2, test method: male Wistar rat, subcutaneous injection 0.3ml/100g body weight CCl4 (be dissolved in the Oleum Arachidis hypogaeae semen, the CCl4 volume ratio is 40%), 2 times weekly, 12 weeks of injection form hepatic fibrosis, are the IV phase.Subsequently hepatic fibrosis rats is divided at random following each group: normal saline matched group, each sample sets, positive controls.Intramuscular injection every day sample 1 time, totally 8 weeks.Normal saline matched group intramuscular injection every day 0.2ml normal saline carried out for 8 weeks altogether.After 8 weeks of treatment, make biochemical measurement, put to death animal then, get the lobus dexter hepatic tissue, fix, be used for histological examination with 10% neutral formalin solution from the postcava blood sampling.The results are shown in Table 4:
Table 4 is respectively organized reagent causes each index of rat chronic liver poisoning model to carbon tetrachloride influence (X ± SD, n=20) (after continuous 11 weeks)
| Group | ALT | AST | The liver hydroxyproline |
| The total flavones low dose group | 2136.28±552.86 | 2561.81±826.55 | 0.182±0.011 |
| Dosage group in the total flavones | 1727.54±338.21 | 1682.83±417.28 | 0.177±0.017 |
| The total flavones high dose group | 1611.26±351.28 | 1528.33±401.89 | 0.168±0.012 |
| The NS matched group | 2284.2±273.6 | 2949.9±1572.4 | 0.195±0.024 |
| Positive controls | 1644.7±158.3 | 1834.6±836.4 | 0.217±0.040 |
| The normal control group | 989.6±180.8 | 1085.3±437.7 | 0.169±0.018 |
*Compare with the NS matched group:
P＜0.05;
P＜0.01
The result: compare with the NS matched group, Herba Hyperici Japonici total flavones can significantly reduce carbon tetrachloride and cause the hepatic injury that rat chronic liver poisoning model causes.
(2) two xanthone C of Herba Hyperici Japonici and the two xanthone D of Herba Hyperici Japonici are to the influence of Liver Fibrosis Model
It is 10mg/mL that medicine preparation: a. is mixed with concentration according to drug solubility with the DMSO dilution; B.0.45um filter membrane filters; C.4 ℃ preservation
Recovery HSC-T6 cell strain, its phenotype are activatory HSC
Changed liquid in every 2-3 days, go down to posterity, used culture fluid is DMEM+FCS (10% calf of deactivation)+PS (penicillin of 100u/mL and streptomycin)
Test method:
Trophophase HSC5 * 103/ hole of taking the logarithm is inoculated in 96 orifice plates, and 37 ℃, 5%CO2,24 hours
Culture fluid is changed into the DMEM of serum-free, 37 ℃, 5%CO2,24 hours
The medicine that adds variable concentrations makes its final concentration be (3.125,6.25,12.5,25,50,100ug/mL), 37 ℃, 5%CO2,48 hours
Add developer KIT-810uL/ hole, 37 ℃, 5%,CO2 60 minutes, measures the OD value
Result such as table 5,6,7,8:
The two xanthone C different time variable concentrations of table 5 Herba Hyperici Japonici act on HSC cell OD value
| Time-concentration (ug/ml) | 12 hours | 24 hours | 48 hours |
| 0 | 1.4847± 0.0756 | 1.775±0.107 | 1.665±0.1195 |
| The DMSO contrast | 1.44±0.097 | 1.769±0.109 | 1.82±0.06 |
| 3.125 | 1.54±0.06 | 1.749±0.148 | 1.9125±0.1 |
| 6.25 | 1.46±0.134 | 1.578±0.078 | 1.698±0.049 |
| 12.5 | 1.456±0.1464 | 1.681±0.088 | 1.67±0.08 |
| 25 | 1.39±0.044 | 1.538±0.1 | 1.474±0.15 |
| 50 | 1.443±0.055 | 1.562±0.161 | 1.21±0.34 |
The two xanthone C different time variable concentrations of table 6 Herba Hyperici Japonici act on L02 cell OD value
| Time-concentration | 12 hours | 24 hours | 48 hours | 60 hours |
| 0 | 0.876±0.048 | 1.208±0.0897 | 1.3363±0.038 | 1.51167± 0.128 |
| The DMSO contrast | 0.762± 0.026395 | 0.9697± 0.0423 | 1.112±0.063 | 1.1698± 0.0979 |
| 3.125 | 0.7767±0.0928 | 1.127±0.121 | 1.301±0.1925 | 1.50225± 0.1318 |
| 6.25 | 0.9263±0.138 | 1.13±0.0181 | 1.37425± 0.0509 | 1.52375± 0.0607 |
| 12.5 | 1.307±0.8004 | 1.1715± 0.02786 | 1.3035±0.1275 | 1.234± 0.09893 |
| 25 | 0.8267± 0.03164 | 1.04575± 0.0631 | 1.31775± 0.0516 | 1.54175± 0.09155 |
| 50 | 0.774±0.0603 | 0.8775±0.122 | 0.86125± 0.2273 | 1.21875± 0.1886 |
| 100 | 0.54467± 0.05426 | 0.44125± 0.1135 | 0.31425± 0.07675 | 0.311±0.0615 |
The two xanthone D different time variable concentrations of table 7 Herba Hyperici Japonici act on the OD value of HSC cell
| Time-concentration (ug/ml) | 12 hours | 24 hours | 48 hours |
| 0 | 1.4847±0.0756 | 1.775±0.107 | 1.665±0.1195 |
| The DMSO contrast | 1.44±0.097 | 1.769±0.109 | 1.82±0.06 |
| 3.125 | 1.39±0.159 | 1.6945±0.123 | 1.55±0.22 |
| 6.25 | 1.4375±0.094 | 1.7275±0.11 | 1.326±0.126 |
| 12.5 | 1.354±0.124 | 1.6555±0.19 | 1.478±0.167 |
| 25 | 1.21±0.054 | 1.453±0.089 | 1.254±0.225 |
| 50 | 1.364±0.14 | 1.064±0.1 | 0.35±0.065 |
The two xanthone D different time variable concentrations of table 8 Herba Hyperici Japonici act on the OD value of L02 cell
| Time-concentration (ug/ml) | 12 hours | 24 hours | 48 hours | 60 hours |
| 0 | 0.876±0.048 | 1.208± 0.0897 | 13363±0.038 | 1.51167±0.128 |
| The DMSO contrast | 0.762± 0.026395 | 0.9697± 0.0423 | 1.112±0.063 | 1.1698±0.0979 |
| 3.125 | 1.0853±0.1912 | 1.02125± 0.0756 | 1.2905±0.1506 | 1.3665±0.078 |
| 6.25 | 1.0133±0.1961 | 1.17375±0.04 | 1.3435±0.0543 | 1.4065±0.1337 |
| 12.5 | 1.307±0.8004 | 1.1715± 0.02786 | 1.2615±0.128 | 1.44225±0.1816 |
| 25 | 0.97067± 0.08937 | 1.03225± 0.036 | 1.26375±0.156 | 1.48125±0.1883 |
| 50 | 0.9733±0.09026 | 0.973±0.0646 | 1.10675± 0.1556 | 1.277±0.949 |
| 100 | 0.71567± 0.03086 | 0.8405±0.064 | 0.65225±0.319 | 0.6695±0.3828 |
Pharmaceutics research
Pharmaceutical composition of the present invention contains the Herba Hyperici Japonici total flavones for the treatment of effective dose, and contains one or more pharmaceutically acceptable carriers.
Pharmaceutical composition of the present invention can be used for preparing the medicine of the acute and chronic viral hepatitis of treatment, hepatic injury, hepatic fibrosis.
Pharmaceutically acceptable carrier mentioned above is meant the pharmaceutical carrier of pharmaceutical field routine, for example: diluent, excipient such as water etc., filler such as starch, sucrose etc.; Binding agent such as cellulose derivative, alginate, gelatin and polyvinylpyrrolidone; Wetting agent such as glycerol; Disintegrating agent such as agar, calcium carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Surfactant such as hexadecanol; Absorption carrier such as Kaolin and soap clay; Lubricant such as Pulvis Talci, calcium stearate and magnesium and Polyethylene Glycol etc.Can also in compositions, add other adjuvant such as flavouring agent, sweeting agent etc. in addition.
The present composition can by oral, snuffing is gone into, the mode of rectum or parenteral is applied to the patient who needs this treatment.Be used for when oral, can be made into conventional solid preparation such as tablet, powder, granule, capsule etc., make liquid preparation such as water or oil-suspending agent or other liquid preparation such as syrup, elixir etc.; When being used for parenteral, can be made into solution, water or the oiliness suspending agent etc. of injection.Preferred form is tablet, coated tablet, capsule, suppository, nasal spray and injection, freeze-dried powder injection, capsule, drop pill etc.
The various dosage forms of pharmaceutical composition of the present invention can be according to the conventional production method preparation of pharmaceutical field.Active component is mixed with one or more carriers, be made into required dosage form then.
Containing weight ratio in the drug combination preparation of the present invention is the total flavones active component of 0.1%-99.5%, and preferably containing weight ratio is the total flavones active component of 0.5%-95%.
The specific embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1: the dry medical material 5.0Kg of Herba Hyperici Japonici, and with 50 liters of reflux, extract, of 60% alcoholic solution 2 times, each 2 hours, merge extracted twice liquid, concentrating under reduced pressure obtains extractum 620g, after disperseing with 2 premium on currency, regulates pH value to 5～6, with three extractions of 30 liters of branches of ethyl acetate, merge three times extract, behind the adding anhydrous sodium sulfate dehydration, cross 0.45 μ m and filter, filtrate decompression is concentrated into dried, obtains Herba Hyperici Japonici total flavones 120 grams.
Embodiment 2: get above-mentioned Herba Hyperici Japonici total flavones compositions 10g, mix with 50g silica gel and to mix sample, be added to the silicagel column top that has installed, with chloroform: methanol system gradient elution, TLC follows the tracks of, the difference combined chloroform: methanol (15: 1,5: 1) part obtains the two xanthone C 0.4g of Herba Hyperici Japonici, Herba Hyperici Japonici two xanthone D 0.7g, Quercitroside 3.6g and isoquercitrin 4.7g behind column chromatography purification.
Embodiment 3:
Tablet: Herba Hyperici Japonici total flavones 60g
Lactose 187g
Corn starch 50g
Magnesium stearate 3g
Preparation method: Herba Hyperici Japonici total flavones, lactose and starch are mixed, and water is evenly moistening, the mixture after moistening is sieved and drying, after sieve, adds magnesium stearate, then with the mixture tabletting, and every heavy 300mg, Herba Hyperici Japonici total flavones content is 60mg.
Embodiment 4:
Ampulla: Herba Hyperici Japonici total flavones 10g
Sodium chloride 9g
Preparation method: Herba Hyperici Japonici total flavones and sodium chloride are dissolved in the proper amount of water for injection, filter gained solution, in the ampoule bottle of packing under aseptic condition, every contains Herba Hyperici Japonici total flavones 10mg
Embodiment 5:
Freeze-dried powder injection: Herba Hyperici Japonici total flavones 10g
Mannitol 20g
Preparation method: Herba Hyperici Japonici total flavones and mannitol are dissolved in the proper amount of water for injection, filter gained solution, in the cillin bottle of under aseptic condition, packing into, lyophilizing, every contains Herba Hyperici Japonici total flavones 10mg.
Claims (4)
1. Chinese medicine composition that is used for the treatment of hepatic disease is characterized in that: contain from the effective site Herba Hyperici Japonici total flavones that the Chinese medicine Herba Hyperici Japonici extracts, purification obtains, the weight content of flavones ingredient is 80～99.5% in the total flavones; Wherein, described total flavones composition is mainly Quercitroside, isoquercitrin, the two xanthone C of Herba Hyperici Japonici and the two xanthone D of Herba Hyperici Japonici; Described extraction, purification step are:
(1) extract: adopt solvent extraction method, extract solvent and select methanol, ethanol, acetone or butanols for use, or its several mixed solvent, extracting method is that supersound extraction, percolation extract or heating and refluxing extraction, extraction time is 1-3 time;
(2) extraction: adopt solvent extraction, with the extract aqueous dispersion of step (1) gained, reuse organic solvent extraction, organic solvent are selected chloroform, dichloromethane, ether or ethyl acetate for use, and extraction times can be for 1-4 time;
(3) concentrate drying: step (2) gained extract is crossed 0.22 μ m filter membrane, and filtrate decompression is concentrated into dried, obtains active ingredient Herba Hyperici Japonici total flavones compositions.
2. preparation of drug combination method as claimed in claim 1 is characterized in that adopting following steps:
(1) extract: adopt solvent extraction method, extract solvent and select methanol, ethanol, acetone or butanols for use, or its several mixed solvent, extracting method is that supersound extraction, percolation extract or heating and refluxing extraction, extraction time is 1-3 time;
(2) extraction: adopt solvent extraction, with the extract aqueous dispersion of step (1) gained, reuse organic solvent extraction, organic solvent are selected chloroform, dichloromethane, ether or ethyl acetate for use, and extraction times can be for 1-4 time;
(3) concentrate drying: step (2) gained extract is crossed 0.22 μ m filter membrane, and filtrate decompression is concentrated into dried, obtains active ingredient Herba Hyperici Japonici total flavones compositions.
3. pharmaceutical composition according to claim 1 is characterized in that: also contain one or more pharmaceutically acceptable vehicle group and become tablet, injection, freeze-dried powder injection, capsule, drop pill.
4. pharmaceutical composition according to claim 3, the weight content that it is characterized in that the total flavones active ingredient is 01%-99.5%.
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Non-Patent Citations (4)
| Title |
|---|
| 田基黄及其注射液的研究进展. 陈丽云.中药材，第25卷第7期. 2002 |
| 田基黄及其注射液的研究进展. 陈丽云.中药材，第25卷第7期. 2002 * |
| 田基黄黄酮类化学成分的研究. 傅芃.中国天然药物，第2卷第5期. 2004 |
| 田基黄黄酮类化学成分的研究. 傅芃.中国天然药物，第2卷第5期. 2004 * |
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