CN103623056A - Traditional Chinese medicine extract for treating hepatitis B, and preparation method and application thereof - Google Patents
Traditional Chinese medicine extract for treating hepatitis B, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an extract of the traditional Chinese medicine chinaberry for treating hepatitis B and a preparing method and application thereof. The traditional Chinese medicine chinaberry extract comprises more than 50% of total phenolic compounds and more than 20% of chinaberry phenolic compounds. The preparation method for the traditional Chinese medicine chinaberry extract comprises the following steps: subjecting a chinaberry raw material to hot extraction or cold extraction so as to obtain a crude chinaberry extract; and then carrying out purification by using a methacrylic synthetic adsorbent, an aromatic (styrene and didivinyl benzene) synthetic adsorbent or a gel synthetic absorbent so as to obtain the traditional Chinese medicine chinaberry extract. The invention also discloses application of the traditional Chinese medicine extract in preparation of medicines used for treating hepatitis B.
Description
Technical field
The present invention relates to medical technical field, be specifically related to a kind of Chinese medicine chinaberry extract for the treatment of hepatitis B and its production and use.
Background technology
Virosis serious harm people's life is healthy, is the main infectious disease of the mankind.Propagate very extensively, sickness rate is high, and viral species is various, in cell, breeds, and can infect different tissues cell and cause disease.According to incompletely statistics, in infectious diseases, viral disease is up to 60 ~ 65%.According to incidence type, can be divided into: 1) acute epidemic disease, causes viral encephalitis that acute hepatitis, encephalitis b virus cause etc. as influenza, hepatitis A or hepatitis E virus; 2) chronic infectious disease, as acquired immune deficiency syndrome (AIDS), second, hepatitis C patient cause liver cirrhosis, hepatocarcinoma with virus for a long time; 3) latent infection, as herpes simplex, herpes zoster, epstein-barr virus and cytomegalovirus infection; 4) viral infection relevant with tumor, as second, infection with hepatitis C virus and hepatocarcinoma, epstein-barr virus infect and nasopharyngeal carcinoma; 5) slow virus infection, the nerve injury and the disease that cause as JC virus.
Since Blumberg in 1963 etc. find hepatitis B virus (HBV) in human serum, HBV is wide-scale distribution in worldwide.According to World Health Organization's data, show, the people that hepatitis B is suffered from the whole world is at present over 300,000,000, and wherein 25% patient faces the threat of liver cirrhosis and hepatocarcinoma.China is the district occurred frequently of hepatitis B virus infection, and hepatitis B surface antigen (HBsAg) carrying rate is 10%, and chronic viral hepatitis B patient approximately 3,000 ten thousand, this type of conditions of patients protracted course of disease, and part can develop into liver cirrhosis and hepatocarcinoma, very big to human health risk.HB vaccination prevention hepatitis B is effective measures, once but morbidity, must positive treatment.
The therapy of domestic generally acknowledged treatment hepatitis B is Comprehensive Treatment at present, is specially: 1) remove virus (Clear Virus), the poison that gets rid of illness is the basic treatment measure of hepatitis B, only has the virus of removing, and old complaint just can be gone, that is the traditional Chinese medical science said " effecting a permanent cure ".In recent years both at home and abroad generally acknowledged anti-hepatitis virus active drug is interferon, but with rear HBV-DNA negative conversion rate only 30 ~ 50%, after drug withdrawal, easily bounces.Recently research discovery, peace body Wei Kang, lamivudine, famciclovir, cinobufacin have anti-HBV effect, and several drugs coupling can make curative effect lasting.But long-term taking lamivudine therapy causes hepatitis B virus variation; 2) adjust (Modulate), adjust immunity, improve the effective measures that T cell function is also treatment hepatitis B.Medicine is adjusted in conventional immunity DNA vaccination, gene adjuvant, Zadaxin, thymosin, interleukin polysaccharide, Ganoderma amine polysaccharide, levamisole (smears) etc., the normal and antiviral agents use in conjunction of this class medicine; 3) protect (Protect), the liver protecting and ALT lowering jaundice eliminating subcutaneous ulcer is a kind of measure for the treatment of hepatitis B it " mark ".Conventional medicine has liver power treasured, tiopronin, silymarin, bifendate etc.; 4) resistance (Prevent), prevention and prevention hepatitis develop into liver cirrhosis and hepatocarcinoma, and conventional medicine has caterpillar fungus chemical fiber capsule, colchicine, Beracilline, compound recipe 861 etc.
Although said medicine has certain effect for the treatment tool of hepatitis B, still exist the problems such as curative effect is not obvious, toxic and side effects is larger.Therefore, find natural, safety, the clear and definite new treatment hepatitis B medicine of mechanism of action has important scientific meaning and economic worth.
Meliaceae (Meliaceae) Melia (Melia) plant has very large medical value.Nearly 20 kinds of whole world Meliaceae Melia plant, is distributed widely in subtropical and tropical zones, and minority is distributed in Temperate Region in China.Wherein study the more Melia azedarach L. Melia azedarach Linn.(M.A. that mainly contains China), Fructus Toosendan Melia toosendan Sieb.et Zucc.(M.T.) and Melia dubia Cav. Melia dubia Cav.(M.D.); The day chinaberry Melia azedarach Linn.Var.Japonica Makino(M.A.J. of Japan); The Azadirachta Indica A. Azadirachta indica A.Juss(A.I. of India); East Africa chinaberry Melia volkensii Gurke(M.V. in East Africa); Also has in addition Australian Australia chinaberry Melia azedarach Linn.Var.Australasica(M.A.A.).
At China's root bark with Melia azedarach L. among the people, dried bark or fruit, make lumbricide on the books already, and use with a long history.China's book on Chinese herbal medicine is recorded Cortex Meliae and be can be used for controlling ascariasis, ancylostomiasis, dysentery, acute eczema, urticaria, fistula skin ulcer, carious pain, scabies wind worm, intractable exudative dermatitis; Folium Meliae is used for controlling skin pruritus, eczema, chronic eczema, exudative dermatitis, tinea corporis, hernia and enters into scrotum pain, toxic swelling, Scolopendra honeybee wound, malaria; Melia azedarach L. can be treated miliaria pruritus, tinea capitis; Fructus Toosendan be used for the treatment of stomachache, depression of liver-QI costalgia of chest, colic, stomachache, abdominal distention, hernia, menstrual pain, ascariasis, tinea alba, tinea capitis, head sore, let out that blood is more than, hemorrhoid; Fructus Toosendan can be controlled epigastric pain with cold limbs due to adverseness of heat, intercostal neuralgia, pregnant motive pain, bladder hernia, colic of cold type pain, children's ascarid, all infantile malnutritions, trichomonas vaginitis, ear malignant boil, chilblain.Azadirachta Indica A. has strong defecating feces excretion, the anti-calculus of leaf energy, diuresis, regulating menstruation; Micromicro is as parasite killing and analeptic, spasmolytic; Root bark is for ascarifuge; Flower, for tonneau medicine, can be subsided a swelling, stimulate the menstrual flow; Fruit cold high heat, retention of urine, pelycalgia ， Saudi Arabia high hot, the dermatosis of typhoid fever and the rheumatism of being used for the treatment of among the people that can be used for curing the wound; Azadirachta Indica A. seed oil can be used as analeptic and analeptic, and Azadirachta Indica A. water extract can also alleviate the intensity of attack of asthma.
Therefore recent domestic is also a lot of to the research of Melia plant, to its root bark, dried bark, piece, leaf, core etc. all carried out the researchs such as chemistry, pharmacology, clinical, production technology, quality standard, and therefrom separated and driven ascarid effective ingredient, the limonin of locust refusing to eat and antiinflammatory, anticancer active substance, and because Melia plant has the growth feature such as rapid, of many uses, caused many research workers' broad interest.And the effect that has treatment hepatitis B for active component in chinaberry is also less than report.
Summary of the invention
Object of the present invention be just to provide a kind of treat hepatitis B Chinese medicine chinaberry extract.
Another object of the present invention is to provide the preparation method of Chinese medicine chinaberry extract.
A further object of the present invention is to provide the application of this Chinese medicine chinaberry extract on preparation treatment hepatitis B medicament.
The present invention has in the Chinese medicine chinaberry extract for the treatment of hepatitis B effect: total content of phenolic compounds is greater than 50%, and the chinaberry content of phenolic compounds shown in formula (1) is greater than 20%.
Wherein:
In formula, 7', 8', 9', 7 ＂, 8 ＂, 9 ＂ positions, can be independently R type or S type.
R
1to R
8can be independently H, OH, OR
a(R
afor C1 ~ 5 alkyl, alkanoyl), NH
2, NHR
b(R
bfor C
1 ~ 5alkyl, alkanoyl), NR
cr
d(R
c, R
dbe C independently of one another
1 ~ 5alkyl, alkanoyl), SH, SR
e(R
efor C
1 ~ 5alkyl, alkanoyl), NO
2, halogen, carboxyl, carboxylate.
" C
1 ~ 5alkyl " represent saturated or unsaturated, substituted or non-substituted straight chain, branched alkane hydrocarbon chain containing 1 ~ 5 carbon atom, can enumerate as methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group, isopentyl, neopentyl, tertiary pentyl etc. particularly.In these groups, the alkyl that the carbon numbers such as methyl, ethyl, propyl group, isopropyl, butyl of take are 1 ~ 4, as good, for better, be take methyl, ethyl as best with methyl, ethyl and propyl group.
" alkanoyl " represents, containing saturated or unsaturated, substituted or non-substituted straight chain, the acyl group of branched alkane hydrocarbon chain, can enumerate particularly as formoxyl, acetyl group, propiono, isopropyl acyl group, bytyry, isobutyryl, secondary bytyry, tertiary bytyry, valeryl, isovaleryl, valeryl, pivaloyl etc.
In Chinese medicine chinaberry extract of the present invention, total phenolic compound, except chinaberry phenolic compound, also comprises the polyphenol compounds such as catechin, epicatechin class and their dimer, trimer, phenol glycoside compound.
Extracting method to chinaberry extract, has hot lifting manipulation and cold extraction method;
Hot lifting manipulation 1: water reflux, extract, chinaberry raw material, filter, concentrating under reduced pressure, precipitate with ethanol, collects alcoholic solution, and concentrating under reduced pressure, obtains chinaberry crude extract.
Hot lifting manipulation 2: with the aqueous solution reflux, extract, chinaberry raw material of methanol, ethanol, propanol or acetone, filter, concentrating under reduced pressure, obtains chinaberry crude extract.
Cold extraction method: be solvent with the aqueous solution of methanol, ethanol, propanol or acetone, the lixiviate chinaberry raw material that spends the night, filters, and concentrating under reduced pressure, obtains chinaberry crude extract.
Chinaberry raw material of the present invention comprises part or all of the chinaberry plants such as the skin, fruit, leaf, seed, flower of chinaberry.
The purification process of chinaberry extract of the present invention is, chinaberry crude extract is dissolved in the aqueous alcohol of water or suitable determining alcohol, being adsorbed on metering system is synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is synthetic adsorbent, or on gel-like synthetic adsorbent, first wash remove impurity with water, then with 10% ~ 80% aqueous alcohol or acetone gradient elution, collect and merge the component that contains phenolic compound.In the chinaberry extract that purification obtains: total content of phenolic compounds is greater than 50%, and chinaberry content of phenolic compounds is greater than 20%.
Metering system system used in the present invention and aromatic series (styrene, divinylbenzene) are that two kinds of synthetic adsorbents are not quite similar according to different manufacturing firms, have various trade names.As the synthetic adsorbent that Mitsubishi KCC produces, what belong to metering system and be commodity has HP10, a HP2MG; As fragrant family commodity, there are HP20, HP21, HP30, a HP40; What as aromatic series, add hydrophobic group commodity has SP205, SP206, a SP207; The commodity of processing through high surface as aromatic series have SP800, SP825, SP850, SP875; The precision separation less as particle diameter has HP20SS, SP20SS with commodity.Hangzhou China win honour for resin company limited SD300, SD301, SD302 etc. for aromatic series be synthetic adsorbent.HZ802, the HZ803 etc. of Yadong, Shanghai Nuclear grade resin company limited for aromatic series be synthetic adsorbent.The HZ801 of Shanghai Huazhen Science and Technology Co., Ltd., HZ816, HZ818 etc. for aromatic series be synthetic adsorbent.
Gel-like synthetic adsorbent used in the present invention includes but not limited to Sephadex series and TSK gel Toyopearl series.Sephadex series is by glucosan and glyceryl by the crosslinked cancellated hydrophilic gel of porous forming of ether bridge, and the model that in this series, the micromolecule in natural product is had to a good separating effect is the Sephadex LH-20 being transformed by Sephadex G-25.TSK gel Toyopearl series is a kind of porous polyethylene high polymer containing hydrophilic hydroxyl, acid and alkali-resistance and mycete, granule is thin, hardness is large, longitudinal diffusion is little, go out peak and concentrate, in chromatography behavior roughly the same with Sephadex LH-20.Common are TSK gel Toyopearl HW-40F, HW-40C, HW-65 etc.TSK gel Phenyl-Toyopearl 650 and TSK gel Butyl-Toyopearl 650 are these serial modified models.
The another kind of purification process of chinaberry extract of the present invention is, chinaberry crude extract is dissolved in the aqueous alcohol of water or suitable determining alcohol, being adsorbed on metering system is synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is on synthetic adsorbent, first wash remove impurity with water, then with 10% ~ 80% aqueous alcohol gradient elution, collect and merge the component that contains phenolic compound; The chinaberry extract of this preliminary purification is dissolved in the aqueous alcohol of water or suitable determining alcohol, is adsorbed on gel-like synthetic adsorbent, first washes remove impurity with water, then with 10% ~ 80% aqueous alcohol or acetone gradient elution, collects and merges the component that contains phenolic compound; In the chinaberry extract being further purified: total content of phenolic compounds is greater than 80%, and chinaberry content of phenolic compounds is greater than 50%.
Another purification process of chinaberry extract of the present invention is, chinaberry crude extract is dissolved in the aqueous alcohol of water or suitable determining alcohol, be adsorbed on gel-like synthetic adsorbent, first wash remove impurity with water, then with 10% ~ 80% aqueous alcohol or acetone gradient elution, collect and merge the component that contains phenolic compound; The chinaberry extract of this preliminary purification is dissolved in the aqueous alcohol of water or suitable determining alcohol, being adsorbed on metering system is synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is on synthetic adsorbent, first wash remove impurity with water, then with 10% ~ 80% aqueous alcohol gradient elution, collect and merge the component that contains phenolic compound.In the chinaberry extract being further purified: total content of phenolic compounds is greater than 80%, and chinaberry content of phenolic compounds is greater than 50%.
The present invention further provides that total content of phenolic compounds is greater than 50%, chinaberry content of phenolic compounds is greater than the application on preparation treatment hepatitis B medicament of 20% Chinese medicine chinaberry extract.
In vitro and in vivo pharmacological research shows that Chinese medicine chinaberry extract of the present invention has obvious inhibition HBsAg and HBeAg effect and good dose-dependence.And to because of CC1
4due to immunologic liver injury due to experimental acute and chronic liver injury and bacillus calmette-guerin vaccine, gavage this medicine and can make the abnormal ALT raising, AST vigor decline, and alleviate fatty degeneration of liver, edema, downright bad scope and degree.
One, the external activity testing experiment of chinaberry extract anti-hepatitis B virus (HBV)
(1) test objective
Sample anti-hepatitis B virus (HBV) screening active ingredients.Test comprises: in the test of virus-cellular level, detect cytotoxicity, the secretion to hepatitis B virus surface and cAg of sample, and the influence of the levels of replication of viral nucleic acid (DNA).
(2) test method
1) mtt assay detects the toxicity of sample to HepG 2.2.15 cell
HepG 2.2.15 cell is cultivated after 3 days in 96 well culture plates, added variable concentrations pastille culture fluid, continue to cultivate 9 days (changing liquid once in every 3 days), collection culture supernatant is to be measured.In 96 well culture plates, add MTT, after 4 hours, add 150 μ L DMSO, concussion 10min, crystal is fully dissolved, insert in enzyme connection detector and measure each hole absorbance value (OD value) with 570nm wavelength, repeat 3 times, according to Reed2Muench method, calculate the toxic action of medicine to HepG 2.2.15 cell, affect the situation of Growth of Cells, cause the required concentration (TC of half cell death amount
50) and maximal non-toxic concentration (TC
0).
2) detection of HBsAg and HBeAg content (ELISA method) in culture supernatant
With HBsAg and the HBeAg diagnostic kit of Shanghai industry Ke Hua biotech company, measure, add sample, and add the enzyme mark conjugate of equivalent in the stripe board being coated with, 37 ℃ of reactions were washed plate after 1 hour, repeated 5 times.Add nitrite ion A and B, cessation reaction after 15 minutes, measures OD
450/630, and according to OD value calculation sample the suppression ratio to HBV antigen.
3) detect sample to the inhibitory action of HBV DNA by HepG 2.2.15 cell at 12cm
2in culture bottle, cultivate after 3 days, add pastille culture fluid, continue to cultivate 9 days (changing liquid once in every 3 days), extract intracellular DNA, by the high primed DNA labelling of digoxin and detection kit (Roche company), through the hybridization of Southern transferring film, detect the inhibitory action of sample to HBV DNA.
(3) result of the test
Result of the test shows: chinaberry extract is better than lamivudine to the inhibition of HBsAg and HBeAg.Chinaberry extract has HBV DNA in cell is had to inhibitory action simultaneously.
The inhibition of table 1 chinaberry extract to HBsAg and HBeAg
The inhibition of table 2 chinaberry extract to HBV DNA in cell
Two, chinaberry extract therapeutic effect to duck hepatitis B virus infection in duck body
(1) test objective
For hepatitis virus resisting effect in checking medicine body of the present invention, this test adopts the oral medicine of the present invention of duck hepatitis B virus infection duckling to treat, observe its impact on DHB DNA level, and compare with acycloguanosine (ACV), observe the toxicity of the oral medicine of the present invention of duck.
(2) test method
1) duck hepatitis B virus infection
1 age in days Beijing duck is through intravenous injection Shanghai sheldrake DNA strong positive serum, and every 0.2mL gets blood after infection, separation of serum, and-70 ℃ of preservations are to be checked.
2) Drug therapy test
DHBV infect duckling after 7 days random packet carry out.Drug therapy test, 5 ~ 7 every group, 3 dosage groups of chinaberry extract administration component, minutes 100,200 and 400mg/kg organize oral 1 day 2 times, 10 days.If virus control group, with physiologic saline for substitute medicine.Acycloguanosine for positive drug (ACV), oral administration 100mg/kg, 1 day 2 times, 10 days; After infection, the 7th day is (T before administration
0), the 5th day (T of administration
5), the 10th day (T of administration
10) and drug withdrawal after the 3rd day (P
3), from duck shin venous blood sampling, separation of serum ,-70 ℃ of preservations are to be checked.
3) detection method
Get above-mentioned Sanguis Anas domestica to be checked clear, every batch with time point film, measure Sanguis Anas domestica clear in DHBV-DNA level dynamically.Press nick translation test kit description method, with P labelling DHBV-DNA probe, and do the clear dot blot hybridization of Sanguis Anas domestica, autoradiography diaphragm speckle, on enzyme mark detector, measure OD value (490nm), calculate DHBV-DNA density in serum, using hybridization spot OD value as specimen DHBV-DNA level value.
(3) result of the test
Result of the test shows: during the middle high dose group of chinaberry extract administration group the 5th day, administration the 10th day after administration is clear with the Sanguis Anas domestica of the 3rd day after drug withdrawal the OD value of DHBV-DNA with before administration, compared remarkable inhibition.And acycloguanosine after administration the 5th day with the OD value of DHBV-DNA during the administration Sanguis Anas domestica of the 10th day is clear with before administration, compared highly significant inhibition, but after drug withdrawal, have knock-on.
Three, the protective effect of chinaberry extract to mouse carbon tetrachloride acute liver damage
60 of Kunming kind white mice, body weight 20 ± 2g, male and female dual-purpose, is divided into 6 groups at random.1 group of normal control, 2 groups gavage biological saline, and 3,4,5 groups gavage respectively medicine 0.4g/kg of the present invention, 0.2g/kg, the aqueous solution of 0.1g/kg, 6 groups of aqueous suspensions that gavage bifendate 0.2g/kg, once a day, give 6 continuously.After last administration 1 hour, rear 5 groups of lumbar injection 0.2%CC1 respectively
4oil solution 10ml/kg, fasting be can't help water after 20 hours, and each organizes mouse orbit venous blood collection, measures ALT (ALT), and win liver and do pathomorphology inspection by the reitman-frankel method of improvement.
The effect of reducing enzyme levels of table 3 chinaberry extract to mouse carbon tetrachloride acute liver damage
From result, the ALT that each administration group all can make pathologic raise obviously reduces (P<0.01), sees that lobules of liver structure is substantially clear under microscope, the rarely seen slight cloudy swelling of hepatocyte, and sinus hepaticus is slightly narrow, is dispersed in spotty necrosis kitchen range, CCl
4model group is the most of vacuolar degeneration of hepatocyte or extensive cloudy swelling, and majority is dispersed in a lamellar necrosis region, and indivedual blood vessels form large lamellar around and infiltrate, and portal area cell infiltration is obvious.
Four, the protective effect of chinaberry extract to immunological liver injury
Get 60 of female BALB/C mice, body weight 22 ± 2g, is divided into 6 groups at random.Except Normal group gives same volume normal saline, give every caudal vein injection lyophilized bacillus calmette-guerin vaccine 2.5mg, be dissolved as 0.2ml for all the other 5 groups.Gavage respectively normal saline or medicinal liquid, every day 1 time, totally 12 days simultaneously.After last administration, give every Mus intravenous injection antibacterial endomycin 5000Eu(0.2ml), Normal group injecting normal saline.After 16 hours, the blood sampling of eye socket venous plexus, separation of serum, surveys ALT and AST.Cut open and get liver and fix, carry out pathologic finding.
The effect of reducing enzyme levels of table 4 chinaberry extract to immunological liver injury
Result demonstration, chinaberry extract senior middle school dosage all can reduce ALT and the AST of abnormal rising, illustrates immunologic liver injury is also had to same protective effect.The demonstration of histopathology result, medicine of the present invention can reduce hepatocyte point, lamellar necrosis, edema, hypertrophy and inflammatory cell infiltration in various degree, and its effect is similar to positive control drug bifendate.Therefore chinaberry extract has certain protective effect to immunological liver injury.
Five, the protective effect of chinaberry extract to rat carbon tetrachloride chronic hepatic injury
60 of rat, body weight 150-200g, male and female have concurrently.Be divided at random 6 groups, i.e. Normal group, normal saline, the high, medium and low dosage of chinaberry extract (0.3g/kg, 0.15g/kg, 0.075g/kg) and positive control drug YIGANNING CHONGJI (1.7g/kg).Every day, gastric infusion was 1 time, totally 8 weeks (56 days).Except Normal group, during all the other 5 groups of administrations every 3 days in every Mus nape subcutaneous injection 40%CCl
4oil solution 3ml/kg, initial dose is 5ml/kg, and last administration is after 24 hours, and eye socket venous plexus is taken a blood sample, and separation of serum is surveyed total protein, albumin content, and ALT, AST are active.Sacrificed by decapitation, gets the hepatic tissue of every same leaf site of rat, fixing, send check pathological section.
Result shows, chinaberry extract senior middle school dosage can make abnormal ALT and the AST vigor raising have obvious reduction, though total protein and albumin are had to the trend of increase, with matched group there was no significant difference (P>0.05) relatively.The discovery of hepatic tissue section microscopy, with model group comparison, the hepatic cell fattydegeneration of administration group, edema and downright bad obviously less or lighter.Therefore chinaberry extract has certain protective effect to rat carbon tetrachloride chronic hepatic injury.
Therefore, Chinese medicine chinaberry extract of the present invention can be made into the various preparations that comprise safe and effective amount chinaberry extract and pharmaceutical carrier.
" safe and effective amount " refers to: the amount of medicine is enough to obviously improve the state of an illness, and is unlikely to produce serious side effect.Safe and effective amount is according to determining the age for the treatment of target, the state of an illness, the course for the treatment of etc.
" pharmaceutical carrier " refers to: one or more compatibility solids or liquid filler or gelatinous mass, they are suitable for people uses, and must have enough purity and enough low toxicity." compatibility " referred to herein as each component energy and compound of the present invention and blending mutually between them in compositions, and the drug effect of not obvious reduction compound.Pharmaceutically acceptable carrier part example has sugar (as glucose, sucrose, lactose etc.), starch (as corn starch, potato starch etc.), cellulose and derivant thereof (as sodium carboxymethyl cellulose, ethyl cellulose sodium, cellulose ethanoate etc.), gelatin, Talcum, kollag (as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (as Oleum Glycines, Oleum sesami, Oleum Arachidis hypogaeae semen, Fructus Canarii albi wet goods), polyhydric alcohol (as propylene glycol, glycerol, mannitol, sorbitol etc.), emulsifying agent is (as tween
), wetting agent (as sodium lauryl sulphate), coloring agent, flavoring agent, stabilizing agent, antioxidant, antiseptic, apirogen water etc.
The specific embodiment
Below in conjunction with embodiment, the present invention is further elaborated, but these embodiment are never any limitation of the invention.
Embodiment 1
Get Cortex Meliae 50kg, by 400L deionized water heating and refluxing extraction 3 times, extraction time is respectively 2, 2, 1 hour, water extraction liquid merges, filter, 60 ℃ are evaporated to relative density 1.05, in concentrated solution, add ethanol, to concentration of alcohol be 80%, precipitation is spent the night, filter, filtrate merges, decompression and solvent recovery to concentration is about 2.0g crude drug/ml, filter, macroporous adsorptive resins HZ816 on filtrate, resin demand is 50kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, successively use 200L deionized water, 150L 10% ethanol elution remove impurity, then use 150L 70% ethanol elution, collect alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 240g, total content of phenolic compounds is 52%, chinaberry content of phenolic compounds is 20.3%.
Embodiment 2
Get Fructus Toosendan 15kg, use 90L deionized water heating and refluxing extraction 3 times, extraction time is respectively 2,2,1.5 hours, and water extraction liquid merges, and filters, and 60 ℃ are evaporated to relative density 1.05; In concentrated solution, add ethanol, to concentration of alcohol be 90%, precipitation is spent the night, and filters, and filtrate merges, decompression and solvent recovery to concentration is about 2.0g crude drug/ml, filter, resin column HP10 on filtrate, resin demand is 15kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, successively, with 30L deionized water, 30L 10% ethanol elution remove impurity, then use 40L 80% ethanol elution, collect alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverizes, and obtains chinaberry extract 80g, total content of phenolic compounds is 53%, and chinaberry content of phenolic compounds is 21.7%.
Embodiment 3
Get Melia dubia Cav. leaf 150kg, with 900L 80% alcohol heating reflux, extract 2 times, extraction time is respectively 3 and 2 hours, alcohol extract merges, filter, 60 ℃ of concentrating under reduced pressure, reclaim solvent to concentration and be about 2.0g crude drug/ml, filter, macroporous adsorptive resins HZ801 on filtrate, resin demand is 120kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, successively use 300L deionized water, 250L 10% ethanol elution remove impurity, then use 250L 60% ethanol elution, collect alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 906g, total content of phenolic compounds is 57%, chinaberry content of phenolic compounds is 24.5%.
Embodiment 4
Get Fructus Toosendan skin 50kg, with 350L 60% alcohol heating reflux, extract 3 times, extraction time is respectively 2, 2, 1 hour, alcohol extract merges, filter, 60 ℃ of concentrating under reduced pressure, reclaim solvent to concentration and be about 2.0g crude drug/ml, filter, macroporous adsorptive resins HZ801 on filtrate, resin demand is 50kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, successively use 200L deionized water, 150L 10% ethanol elution remove impurity, then use 150L 60% ethanol elution, collect alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 280g, total content of phenolic compounds is 60%, chinaberry content of phenolic compounds is 28.6%.
Embodiment 5
Get Cortex Meliae 50kg, with 400L 60% acetone extraction 3 times, extraction time is respectively 48, 48, 24 hours, acetone extract merges, filter, decompression and solvent recovery to concentration is about 2.0g crude drug/ml, filter, macroporous adsorptive resins XAD-2 on filtrate, resin demand is 50kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 200L deionized water eluting, then use 100L 20% ethanol elution remove impurity, then use 150L 60% ethanol elution, collect 60% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 290g, total content of phenolic compounds is 67%, chinaberry content of phenolic compounds is 25.4%.
Embodiment 6
Get embodiment 5 chinaberry extract 290g, add deionized water dissolving, filter, gel column Sephadex LH-20 on filtrate, resin demand is 2kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 10L deionized water eluting, then use 30L 80% ethanol elution, collect 80% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverizes, chinaberry extract 140g after must refining, total content of phenolic compounds is 81%, and chinaberry content of phenolic compounds is 51%.
Embodiment 7
Get Cortex Meliae 50kg, with 300L 70% alcohol steep 3 times, extraction time is respectively 72, 72, 48 hours, alcohol extract merges, filter, decompression and solvent recovery to concentration is about 2.0g crude drug/ml, filter, macroporous adsorptive resins AB-8 on filtrate, resin demand is 50kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 200L deionized water eluting, then use 100L 20% ethanol elution remove impurity, then use 150L70% ethanol elution, collect 70% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 310g, total content of phenolic compounds is 65%, chinaberry content of phenolic compounds is 27.2%.
Embodiment 8
Get embodiment 7 chinaberry extract 310g, add deionized water dissolving, filter, gel column TSK gel Toyopearl HW-40F on filtrate, resin demand is 5kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 10L deionized water eluting, then use 30L 80% ethanol elution, collect 80% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverizes, chinaberry extract 150g after must refining, total content of phenolic compounds is 86%, and chinaberry content of phenolic compounds is 53%.
Embodiment 9
Get Azadirachta Indica A. skin 10kg, with 50L 70% methanol lixiviate 3 times, extraction time is respectively 72, 72, 48 hours, alcohol extract merges, filter, decompression and solvent recovery to concentration is about 2.0g crude drug/ml, filter, gel column Sephadex LH-20 on filtrate, resin demand is 10kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 30L deionized water eluting, then use 30L 80% ethanol elution, collect 80% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, obtain chinaberry extract 75g, total content of phenolic compounds is 55%, chinaberry content of phenolic compounds is 34.1%.
Embodiment 10
Get embodiment 9 chinaberry extract 65g, add deionized water dissolving, filter, macroporous adsorptive resins X-5 on filtrate, resin demand is 1kg, coutroi velocity is 0.2 ~ 0.5 times of column volume/h, first use the remove impurity of 2L deionized water eluting, then use 2L 20% ethanol elution remove impurity, then use 3L 70% ethanol elution, collect 70% alcohol eluen, concentrating under reduced pressure, vacuum drying, pulverize, must refine rear chinaberry extract 37g, total content of phenolic compounds is 81%, and chinaberry content of phenolic compounds is 61%.
Claims (9)
1. a Chinese medicine chinaberry extract for the treatment of hepatitis B, is characterized in that, in described chinaberry extract: total content of phenolic compounds is greater than 50%, and chinaberry content of phenolic compounds is greater than 20%.
2. total phenolic compound in a kind of Chinese medicine chinaberry extract for the treatment of hepatitis B as claimed in claim 1, it is characterized in that, described total phenolic compound comprises polyphenol compound, phenol glycoside compound and the chinaberry phenolic compounds such as catechin, epicatechin class and their dimer, trimer.
3. a kind of preparation method for the treatment of the Chinese medicine chinaberry extract of hepatitis B as claimed in claim 1, is characterized in that comprising the steps: that chinaberry raw material obtains chinaberry crude extract by hot lifting manipulation or cold extraction method; Then with synthetic adsorbent, purify and obtain Chinese medicine chinaberry extract of the present invention.
4. chinaberry raw material as claimed in claim 3 obtains chinaberry crude extract by hot lifting manipulation or cold extraction method, it is characterized in that, described hot lifting manipulation is water reflux, extract, chinaberry raw material, filter concentrating under reduced pressure, precipitate with ethanol, collect alcoholic solution, concentrating under reduced pressure, obtains crude extract; Or with the aqueous solution reflux, extract, chinaberry raw material of methanol, ethanol, propanol or acetone, filter, concentrating under reduced pressure, obtains crude extract.
5. chinaberry raw material as claimed in claim 3 obtains chinaberry crude extract by hot lifting manipulation or cold extraction method, it is characterized in that, described cold extraction method is to be solvent with the aqueous solution of methanol, ethanol, propanol or acetone, lixiviate chinaberry raw material spends the night, filter, concentrating under reduced pressure, obtains crude extract.
6. as claimed in claim 3ly with synthetic adsorbent, purify and obtain Chinese medicine chinaberry extract of the present invention, it is characterized in that, described synthetic adsorbent is that metering system is synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is synthetic adsorbent, or gel-like synthetic adsorbent.
7. as claimed in claim 3ly with synthetic adsorbent, purify and obtain Chinese medicine chinaberry extract of the present invention, it is characterized in that, described purification process is that to use separately metering system be synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is synthetic adsorbent, or gel-like synthetic adsorbent; Or these synthetic adsorbents are combined to use.
8. as claimed in claim 7 these synthetic adsorbents are combined to use, it is characterized in that, described combine that to use be that first to use metering system be synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is synthetic adsorbent preliminary purification, re-uses gel-like synthetic adsorbent and be further purified; Or first use gel-like synthetic adsorbent preliminary purification, re-using metering system is synthetic adsorbent, or aromatic series (styrene, divinylbenzene) is that synthetic adsorbent is further purified.
9. the application of chinaberry extract on preparation treatment hepatitis B medicament as claimed in claim 1.
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Application publication date: 20140312 |