CN101822372A - Medical compound for curing hepatitis B and health food for inhibiting hepatitis B virus - Google Patents

Medical compound for curing hepatitis B and health food for inhibiting hepatitis B virus Download PDF

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CN101822372A
CN101822372A CN200910126370A CN200910126370A CN101822372A CN 101822372 A CN101822372 A CN 101822372A CN 200910126370 A CN200910126370 A CN 200910126370A CN 200910126370 A CN200910126370 A CN 200910126370A CN 101822372 A CN101822372 A CN 101822372A
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opc
polymer
health food
monomer
pharmaceutical compositions
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李连滋
张秀凤
林家伃
吴慧眼
陈淑丰
叶俊邦
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Industrial Technology Research Institute ITRI
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Abstract

The invention provides a chemical compound for curing hepatitis B, which comprises an effective amount of proanthocyanidin polymer and a pharmaceutically acceptable carrier or salt, wherein the monomer molecular formula of the proanthocyanidin polymer is as follows: wherein R1 is OCH3, R2 is OH and R3 is H, or R1 is OH and R2 and R3 are H, or R1 and R2 are OH and R3 is H, or R1, R2 and R3 are OH, and R4 is 3-(Alpha)-OH, 3-(Beta)-OH, 3-(Alpha)-O-sugar or 3-(Beta)-O-sugar, the monomer connection of the proanthocyanidin polymer is C4 and C8 carbon bonds, C4 and C6 carbon bonds or C2 and C7 oxygen bonds and the isomeric compounds thereof, and the polymerization degree of the proanthocyanidin is 2 to 30. The invention also relates to a health food for inhibiting hepatitis B virus.

Description

Pharmaceutical compositions and the health food that suppresses hepatitis B virus in order to the treatment viral hepatitis type b
Technical field
The present invention relates to a kind of composition for the treatment of viral hepatitis type b, and is particularly related to a composition with OPC, and it can suppress the hepatitis B virus activity.
Background technology
The viral hepatitis type b carrier whole world has 3.5 hundred million people approximately now, and the whole world has 300,000 people to die from the relevant disease that viral hepatitis type b causes every year approximately again, and wherein Taiwan promptly has above 8,000 people every year.Yet, still do not have effective medicine at present and can treat viral hepatitis type b, have curative effect person at present and have only how two kinds of medicines of interferon and liver, but it fails the hepatitis or the carrier of all models are done fully effectively treatment, and have very big side effect.
Therefore the relevant new drug of studying viral hepatitis type b is the very big development space of tool still.Then is one of good method with natural plants as the object that development suppresses the medicine of viral hepatitis type b.
Known crude drug mountain ramie (Boehmeria nivea (L.) Gaud) has the effect of strong liver, anti-inflammatory, and after taking human body is had no side effect, yet, there is no the report of using it for the treatment viral hepatitis type b at present, why do not understand its active component yet.
The PCA that extracts again from natural plants has splendid antioxidation, often by as health food.The nutriment that previous document (the patent announcement I274551 of TaiWan, China) was once reported the PCA that comprises in taurine, beta-carrotene, the grapestone extract, vitamin E, vitamin C etc. has the effect of improving chronic hepatitis.Yet, do not learn clearly yet at present whether PCA can effectively suppress viral hepatitis type b yet.
Summary of the invention
The object of the present invention is to provide a kind of pharmaceutical compositions in order to the treatment viral hepatitis type b.
Another object of the present invention is to provide a kind of health food with the effect that suppresses hepatitis B virus.
The invention provides a kind of pharmaceutical compositions, comprising: an effective dose OPC polymer in order to the treatment viral hepatitis type b; And a pharmaceutically acceptable carrier or a salt, wherein the monomer molecule formula of this OPC polymer is as follows:
Figure B2009101263709D0000021
R wherein 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH; And R 4Be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar, and the monomer of this OPC polymer be connected to C4, C8 carbon bond, C4, C6 carbon bond or C2, C7 oxygen key and isomeric compound thereof, the degree of polymerization of this OPC is 2-30 again.
The present invention provides a kind of health food in addition, and it has the effect that suppresses hepatitis B virus, comprising: an effective dose OPC polymer, and the monomer molecule formula of this OPC polymer is as follows:
Figure B2009101263709D0000022
Wherein, R 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH; And R 4Be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar, and the monomer of this OPC polymer be connected to C4, C8 carbon bond, C4, C6 carbon bond or C2, C7 oxygen key and isomeric compound thereof, the degree of polymerization of this OPC is 2-30 again.
The experiment proved that pharmaceutical compositions of the present invention contains hepatitis B virus surface antigen (HBs) and the active ingredient that hepatitis B virus e antigen (HBe) generation suppresses, and can be applied to prepare in the medicine for the treatment of viral hepatitis type b.Health food of the present invention contains the active ingredient that hepatitis B virus surface antigen (HBs) and hepatitis B virus e antigen (HBe) generation are suppressed, thereby has the effect that suppresses hepatitis B virus.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and cooperate appended accompanying drawing, be described in detail below:
Description of drawings
Fig. 1 a-f shows 3-flavanols, 3 respectively, 4-flavanols, catechin ((2R, 3S) ((2S is 3S) with the molecular formula of (2R, 3R)) with (2S, 3R)) and epicatechin;
Fig. 2 a and Fig. 2 b-e show the mass spectrogram and the structural analysis of OPC polymer respectively;
Fig. 3 shows the infrared Absorption spectrum analysis of the OPC sample of purifying;
Fig. 4 a and Fig. 4 b show purifying OPC high performance liquid chroma-tography mass spectrum just/negative mass spectrogram;
The OPC sample that Fig. 5 a-c shows purifying with carbon 13 ( 13C NMR) result of nuclear magnetic resonance apparatus detection;
Fig. 6 a and Fig. 6 b show that the high molecular monomer of OPC links;
The meson assisted laser desorption ionization mass spectrometry results of the OPC of Fig. 7 a-c display part purifying;
Fig. 8 a and Fig. 8 b suppress the hepatitis B virus activity analysis after showing not equal plant extraction OPC polymer;
Fig. 9 a and Fig. 9 b show that the OPC polymer suppresses the hepatitis B virus activity analysis.
The specific embodiment
The present invention treats viral hepatitis type b (and/or relevant disease that viral hepatitis type b was caused) and the activity that suppresses hepatitis B virus with the OPC polymer as the active ingredient of pharmaceutical compositions or health food.
Can go out the OPC polymer from a plant extraction, it has the effect that suppresses hepatitis B virus.In one embodiment, plant can comprise the plant of Du's pulse family (Leguminosae), Crassulaceae (Crassulaceae), combretaceae (Combretaceae), trailing plants mushroom section (Asclepiadaceae), the rose family (Rosaceae), Ericaceae (Ericaceae), Pinaceae (Pinaceae), Labiatae (Lamiaceae), polygonaceae (Polygonaceae), Vitaceae (Vitaceae) or Urticaceae (Urticaceae), wherein is preferably the mountain ramie of Urticaceae (Urticaceae).And the extraction of plant part can comprise root, stem, leaf and/or fruit.
And in the present invention, can general known method carry out the extraction of plant.In one embodiment for cutting into slices after root, stem, leaf and/or the fruit drying with plant or grind, and then come this plant is extracted with extraction solution.In one embodiment, root and/or the stem with the mountain ramie extracts.
Extraction solution can be selected water, or the solution of water and opposed polarity solvent.The opposed polarity solvent can comprise alcohol, acetone, methyl alcohol and ethyl acetate.And these solvents can use separately, mix and use, and perhaps mix use with water.The ratio of extraction solution and plant there is no specific restriction, and the ratio of extraction solution and plant is 1: 10 (W/W) in one embodiment.
Again, extraction temperature can some changes along with the extraction solution difference.Can utilize soaking at room temperature in one embodiment, and also can be heated to the reflux temperature (60-100 ℃) of different solutions in another embodiment.The time of extraction is about 2 hours to 7 days, decides on the extraction temperature of operation.In extracting operation, can optionally for example sodium chloride, diluted mineral acid (as watery hydrochloric acid) or organic acid (as Vitamin C, tartaric acid) be added in the extraction solution to regulate the pH value of extraction solution.
After concentrating, the extract that will contain the active component of OPC polymer afterwards carries out drying.Perhaps can optionally further carry out partial purification or complete purifying to above-mentioned extract.In one embodiment, partially purified method be extract with drying with 95% alcohol and/or methanol aqueous solution Hui Rong, then with the solvent extraction of opposed polarity to remove partial impurities.For example remove lipid and apolar substance with nonpolar solvent (as normal hexane) earlier, again with chloroform and/or ethyl acetate, little molecular phenolics compound etc. is removed in extraction, to carry out obtaining the part OPC material of purifying more behind the concentrate drying through these solvent-extracted water layers afterwards.
The step of complete purifying can comprise with alcohol or methanol-water dissolves through partially purified extract, it is inserted in the molecular sieve tubing string again, dashes with different solutions and/or mixed solution then and carries, and carries out the purifies and separates of OPC.In one embodiment, different solutions dashes the order of putting forward and is: 95% alcohol, 95% alcohol/methyl alcohol (1: 1, v/v), 50% methyl alcohol and 50% aqueous acetone solution.With each different solutions, the solution that puts forward that dashes all carries out Fractional Collections, and with the 280nm absorption value of LC instrument, the purified former cyanine in the solution that puts forward is dashed in detection then.And collect the solution part of being dashed proposition by different solutions, can obtain containing the solution of the OPC polymer of different molecular weight distribution.Difference dashed the solution put forward part and concentrate with the temperature that is lower than 40 ℃, and freeze drying.Obtain the OPC polymer of purifying.The molecular sieve tubing string is Sephadex LH-20 tubing string (GE, a peace agate West Asia limited company) in one embodiment.
And through above-mentioned purified OPC polymer, the molecular formula of monomer whose is as follows.
Figure B2009101263709D0000051
In one embodiment, R 1Be OCH 3, R 2Be OH and R 3Be H.In another embodiment, R 1Be OH and R 2With R 3All be H.R 1With R 2All be OH and R 3Be H.Perhaps R 1, R 2With R 3All be OH.And R 4Can be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar.
Again, above-mentioned monomer can be included in the R or the S optical isomeric compound of C2, C3 or C4 position.
In addition, above-mentioned monomer can comprise flavone compound.And flavone compound can comprise catechin (catechin), epicatechin (epicatechin), epiafzelechin (epiafzelechin), gallic acid catechin (gallocatechin), gallic acid epicatechin (galloepicatechin), epigallocatechin (epigallocatechin), gallate (gallates), flavonols (flavonols), flavandiols (flavandiols), leucocyanidin (leucocyanidins) or anthocyanidin (procynidins).In one embodiment, the monomer of OPC polymer can comprise flavan-3-alcohol or flavan derivatives.3-flavanols (flavanol), 3 wherein, and the 4-flavanols (3,4-flavanol), catechin (catechin) ((2R, 3S) with (2S, 3R)) with epicatechin (epicatechin) ((2S, 3S) with the molecular formula of (2R, 3R)) respectively shown in Fig. 1 a-f.
And the degree of polymerization of purified OPC polymer is about 2-30, is preferably 3-20.Bond comprises C4, C8 carbon bond, C4, C6 carbon bond or C2, C7 oxygen key between two monomers that link to each other of OPC polymer.OPC polymer weight average molecular weight is about 600-10000.
In one embodiment, purified OPC oligomerization compound is the OPC polymer of the single degree of polymerization, and in another embodiment, purified OPC polymer then comprises the OPC mixture of polymers of different polymerization degree.
In one embodiment, the R of the monomer of OPC polymer 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH, monomer contain above different R 1The OPC polymer to suppressing 50% (IC 50) dosage of B hepatovirus surface antigen (HBs) is between about 127-164 μ g/ml, inhibition 50% (IC 50) dosage of B hepatovirus e antigen (HBe) is between about 44-84 μ g/ml, shows the different R1 of OPC polymer monomer, can not influence the biologically active of its inhibition hepatitis B virus.
And in the present invention, OPC polymer that can above-mentioned extraction is made a pharmaceutical compositions in order to the treatment viral hepatitis type b, and it can comprise OPC polymer and a pharmaceutically acceptable carrier or a salt.
Pharmaceutically acceptable carrier can include but not limited to that solvent, dispersion medium (dispersion medium), mantle (coating), antibiotic and antifungal agents and an isosmoticity and absorption delay (absorption delaying) reagent etc. are thrown with pharmacy and give compatible person.For different administering modes, can utilize conventional method that pharmaceutical compositions is configured to formulation (dosage form).
The pharmaceutically acceptable salt class can include but not limited to salt, described salt comprises inorganic cation, for example, and alkaline metal salt (as sodium, potassium or amine salt), alkaline earth gold family's salt (as magnesium, calcium salt) contains the salt (as zinc, aluminium or zirconates) of divalence or quadrivalent cation.In addition, described salt is organic salt also, as the bicyclohexylamine salt, and methyl D-aminoglucose, amidates (as arginine, from propylhomoserin, histidine, bran propylhomoserin acid amides).
And administration is can be oral, non-oral, via sucking spraying (inhalation spray) or by implanting the mode of reservoir (implanted reservoir).Non-oral (intraleaional) injection and the perfusion technique in (intraarticular) artery (intraarterial) in subcutaneous (subcutaneous), intracutaneous (intracutaneous) intravenous (intravenous), muscle interior (intramuscular), the joint, synovial bursa (chamber) interior (intrasynovial), interior (intrastemal) subarachnoid space (intrathecal) of breastbone, the disease location that comprise.
The form of oral composition can including but not limited to, lozenge, capsule, emulsion (emulsions), waterborne suspension (aqueous suspensions), dispersion liquid (dispersions) or solution.
Perhaps, the present invention also can provide a health food, comprises aforesaid OPC polymer, and has the effect that suppresses hepatitis B virus.
[embodiment]
The monomer structure of embodiment 1, OPC polymer is measured
The structure of procyanidin monomers is to detect (Gas Chrimatograph-Mass Spectrometry) with thermal decomposition gas chromatography mass spectrograph.The method that detects is the purifying OPC (sample of purifying voluntarily) with solid, directly place the thermal decomposition gas chromatograph, heat gradually or moment heats in the setting operation mode of segmentation temperature (50 ℃ to 500 ℃) or single temperature, the sample that will add thermal decomposition separates through the specific metal tubing string of thermal decomposition instrument, through the matter collection of illustrative plates that the mass spectrograph detector is produced, determine the monomer structure of OPC polymer.The mass spectrogram of OPC polymer and structural analysis are shown in Fig. 2 a and Fig. 2 b-e respectively.Wherein the left side among Fig. 2 b-e is the m/z value and the chemical constitution thereof of Fig. 2 a crest, and right-hand part is the monomer parsing of left side crest.The monomer structure molecular formula of the OPC polymer that determines is as follows:
Wherein, R 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH.
And, therefore infer R because of the measured mass spectrum of thermal decomposition shows the peak (peak) that contains glycoside body signal 4Composition may be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar.
Embodiment 2, contain the mountain ramie medicinal material extract preparation of OPC
Method 1
The root of mountain ramie medicinal material is clean to wash with the stem that is connected root, be positioned under the natural environment dry.The section of dried medicinal material is to about 5mm thickness, storage be 4 ℃ standby.Get the mountain ramie medicinal material that stores for future use, grind with mill, get the back powder less than mesh 20 orders (20mesh) that sieves, (1: 10,95% alcohol w/w) added hot reflux and boils 2 hours (boiling secondary altogether), leaves standstill cooling and rises again to add 10 times of weight.Collect and boiled the extract of rising again, pour in the centrifugal bag with centrifuge centrifugal filtration.Filtered fluid concentrates in being lower than 40 ℃ decompression thickener, utilizes freeze drier with the extract drying.This dry extract has the medicine that suppresses the hepatitis B virus activity for containing the OPC composition.
Method 2
Get 4 ℃ the dry medicinal material of being stored in of said method 1, grind, get the powder of back that sieve less than mesh 20 orders (20mesh) with mill, added 10 times of weight (1: 10, w/w) water (RO water) is handled in reverse osmosis, adds hot reflux and boils 2 hours (boiling secondary altogether), leaves standstill cooling and rises again.Collect and boiled the extract of rising again, add alcohol (95% to the 50%) aqueous solution, leave standstill cooling after the mixing and wait to precipitate, upper strata liquid is poured in the centrifugal bag with centrifuge centrifugal filtration.Filtered fluid concentrates in being lower than 40 ℃ decompression thickener, utilizes freeze drier with the extract drying.This dry extract has the medicine that suppresses hepatitis B virus for containing the OPC component composition.
The partial purification preparation of the OPC of embodiment 3, mountain ramie medicinal material extract
Method 1Solvent extraction-1
The mountain ramie dry extract that will contain OPC, and the adding normal hexane (1: 10, w/v) in apparatus,Soxhlet's (Soxhelt apparatus), added hot reflux 6 hours, remove the lipid in the extract.Solids is with the dissolving of 70% methanol aqueous solution and/or the 0.3wt% Vitamin C aqueous solution, concentrates in being lower than 40 ℃ decompression thickener and removes solvent, add chloroform (1: 1, chloroform: condensation product, v/v), with oscillator shake 30 minutes (repeatedly extraction).Water intaking layer adding ethyl acetate (1: 1, ethyl acetate: water layer, v/v), vibrate 30 minutes (repeatedly extraction).Water intaking layer concentrates in being lower than 40 ℃ decompression thickener, utilizes freeze drier with the extract drying that concentrates.
Method 2Solvent extraction-2
The mountain ramie dry extract that will contain the OPC polymer, add entry/alcohol dissolve (1: 10, w/v), afterwards and add normal hexane (1: 10, v/v), with oscillator shake 30 minutes (repeatedly extraction) remove lipid in extract.Water intaking layer adding ethyl acetate (1: 1, ethyl acetate: water layer, v/v), vibrated 30 fens, repeatedly extraction.Afterwards again water intaking layer add a n-butanol (1: 10, v/v), shook 30 fens, repeatedly extraction with oscillator.Water intaking layer concentrates in being lower than 40 ℃ decompression thickener, utilizes freeze drier with the extract drying that concentrates.
Method 3Molecular sieve tubing string chromatography (gel permeation chromatography)
The partially purified composition that contains OPC by said method 1 gained, utilize molecular sieve tubing string chromatography (Gel Permeation Chromatography, 4em diameter x 45em length Sephadex LH-20), dashes with the solution of opposed polarity ratio and to carry, remove impurity wherein again.2.5 the part pure material of gram with the dissolving of 0.5mL 95% alcohol, is inserted in the molecular sieve tubing string.Continuously towards carrying, the collection different solvents dashes the flowing lotion after carrying with a series of solvent.Towards extract be respectively 95% alcoholic solution of 300mL, 95% alcohol/methyl alcohol of 300mL (1/1, v/v), 300ml methyl alcohol, 50% methanol aqueous solution of 300mL and 50% aqueous acetone solution of 300mL.Except that the collection liquid of the 95% alcohol flowing lotion of 300mL, the Fractional Collections liquid of other each several part all concentrates in being lower than 40 ℃ decompression thickener, utilizes freeze drier with the extract drying, dried matter be stored in-20 ℃ standby.Get the contained partial purification of the dry mountain ramie of collection and or the OPC compound of purifying, check analysis and resolve its physical and chemical performance.The stream washing matter of this drying is the OPC composition that contains partial purification or purifying, for having the medicine that suppresses hepatitis B virus.
Embodiment 4, infrared Absorption spectrum analysis
With the OPC sample and the potassium chloride mixed pressuring plate of purifying, detect with the penetration infrared spectrum, the result as shown in Figure 3, wherein stronger absorption crest is 3412.38nm, 1610.57nm, 1521.40nm, 1441.14nm, 1284.86nm, 1100.88nm.
Embodiment 5, high performance liquid chroma-tography mass-spectrogram are analyzed
OPC sample with purifying, (EFI is just spilling/is bearing mass spectrograph to analyse mass spectrograph with efficient liquid layer, HPLC/ESI+, HPLC/ESI-) (Micromass Quattro/Waters 2690) detects, detect the monomer and the polymer of the OPC degree of polymerization 1 to 6, and contain 164 glycoside body (being that the monomer molecule amount adds a glycoside molecular weight 164).The high performance liquid chroma-tography of the OPC of purifying mass spectrum just/negative mass spectrogram is shown in Fig. 4 a and Fig. 4 b.
Embodiment 6, nuclear magnetic resonance carbon 13 ( 13C NMR) and hydrogen ( 1H NMR) atlas analysis
The OPC sample of purifying with carbon 13 ( 13C NMR) and hydrogen ( 1H NMR) nuclear magnetic resonance apparatus detects, carbon 13 ( 13C NMR) result is shown in Fig. 5 a-c.Wherein at 142-145.7ppm except that the crest that shows doublet-doublet, do not have other crest, displaying monomer has anthocyanidin (cyanidin), and does not have delphinidin (delphindin), be that B ring has 3-OH substituting group person, identical with EGA/MS institute analysis result herein.And in Fig. 5 b R 1=H or OH and R 2=H or OH or OCH.
Foundation 1HNMR reaches 13The check collection of illustrative plates of CNMR shows that the high molecular monomer of the OPC of purifying of the present invention links based on 4-8.The binding unit of 4-8 and 4-6 is respectively shown in Fig. 6 a and Fig. 6 b.
(Matrix Assisted Laser DesorptionIonization Time-of-Flight MALDI-TOF) analyzes for embodiment 7, meson assisted laser desorption ionization mass spectrum
Partially purified OPC molecular weight distribution is to measure with meson assisted laser desorption ionization mass spectrograph.The result is shown in Fig. 7 a-c.The result who detects shows that the molecular weight distribution through partially purified OPC is 500-5000, by the testing result demonstration of molecular weight distribution, infers the high molecular degree of polymerization and is about 2-18.
Suppress the hepatitis B virus activity analysis behind embodiment 8, the not equal plant extraction OPC polymer
Belong to not equal plant and just come together, will carry out the inhibiting analysis of B hepatovirus after its collection fluid freeze-drying through 95% alcohol.To behind transfection B hepatovirus plastid, can produce the human liver cancer cell line HepG2.2.15 or the 1.3ES8 of hepatitis B virus, with 1 * 10 5The density in individual cell/100 μ l/ holes (well) is inoculated in the 96 porocyte culture plates, places cell culture incubator to carry out cultivation overnight.Every other day sample is mixed with 100mg/ml with 10%DMSO and 90% sterilized water, dilute the concentration of test again with cell culture fluid, original fluid in 96 porose discs is absorbed with vavuum pump, must note in the process being drawn onto cell, add 100 μ l/ holes again and be dissolved with the nutrient solution that contains through the diluent substrate concentration, hepatitis B virus HBs and HBe antigen suppress test: be chosen in and cultivate the 4th day pair cell in back altogether with extract and do not have the co-culture of cells liquid of toxicity (being cell survival rate＞85%) to analyze.With SURASE B-96 (TMB) (GENERALBIOLOGICALS) the cover group measure surface antigen, with EASE BN-96 (TMB) (GENERALBIOLOGICALS) the cover group measure e antigen.Each extract co-culture of cells liquid with 50 μ l adds in the micropore dish of the antibody that covers (coating) anti-surface antigen (Anti-HBs) and anti-e antigen (Anti-HBe), under 40 ℃, leave standstill, HBs measure to place two hours and HBe measures to place and goes after overnight, clean with cleaning cushioning liquid (washing buffer) afterwards and add anti-surface antigen secondary antibodies (Anti-HBs.peroxidase) 50 μ l or anti-e antigen secondary antibodies (Anti-HBe.peroxidase) the 100 μ l that engage with peroxidase after six times respectively again, after leaving standstill one hour under 40 ℃, go, clean six times to clean cushioning liquid.Every hole adds 100 μ l TMB again, and lucifuge made its colour generation in 30 minutes under room temperature, add the 2M sulfuric acid cessation reaction of 100 μ l at last after, in continuous wavelength micropore dish analytical system, survey extinction with the wavelength of 450nm.Control group extinction with not dosing is worked as denominator, and the difference of experimental group and control group light absorption value is a molecule, calculates its percentage, as extract to hepatitis B virus antigen inhibiting rate.
Extract the plant that 11 kinds of plants contain OPC altogether, comprise a kind of Du's pulse family (Leguminosae), 1 kind of Crassulaceae (Crassulaceae), 2 kinds of combretaceaes (Combretaceae), 1 kind of trailing plants mushroom section (Asclepiadacea), 1 kind of rose family (Rosaceae), 1 kind of Labiatae (Lamiaceae), the plant of a kind of Vitaceae (Vitaceae) and 3 kinds of polygonaceaes (Polygonaceae), its (222 or 74  g/ml or 25 μ g/ml) under the suitable concentration respectively testing drug and co-culture of cells 2 days with 4 days after the ability of its inhibition B hepatovirus surface antigen (HBs) and e antigen, the result is respectively shown in Fig. 8 a and Fig. 8 b.
The result shows that the contained OPC polymer of above 11 kinds of plants all can suppress the generation of B hepatovirus antigen, and because of its former extract Central Plains proanthocyanidin polymer content difference, so different restraints is arranged.
Embodiment 9, OPC polymer suppress the hepatitis B virus activity analysis
The mountain ramie sample 7.0110g grinding back adding 300ml normal hexane of 95% alcoholic extract was added hot reflux 6 hours in apparatus,Soxhlet's (Soxhelt apparatus), get solid portion (liquid removal) afterwards and add acetone (70/30) and extract afterwards, after water intaking layer rotation volatilization removes acetone, carry out drying after the extracted residues of adding chloroform (chloroform) back and obtain sample 1.Sample 1 with 2.3116g dashes extract continuously towards carrying with molecular sieve tubing string chromatography (Sephadex LH-20) with different afterwards, be respectively 95% alcoholic solution of 300mL, 95% alcohol of 300mL/methyl alcohol (1/1 towards extract, v/v), 50% methanol aqueous solution and 50% aqueous acetone solution of 300mL and the aqueous acetone solution of 300mL of 300ml methyl alcohol, 300mL, obtain sample 2-6 (thing of going out of the aqueous acetone solution of 50% methanol aqueous solution of 300mL and 50% aqueous acetone solution of 300mL and 300mL is combined into sample 6) then respectively.Sample 1-7 is carried out the inhibition B capable hepatitis viruse activity analysis identical with embodiment 8.Positive control group then contains the CPB of 50 μ g/ml for nutrient solution, then for not containing the nutrient solution of any extract, each concentration of each extract must be done two repetitions to control group.
Can find out from Fig. 9 a, in the time of the 2nd day, can find out except sample 2, promptly hepatitis B virus e antigen (HBe) be produced and suppress.And Fig. 9 b is when being presented at the 4th day, and except that sample 2, all samples all produce with hepatitis B virus e antigen (HBe) hepatitis B virus surface antigen (HBs) and suppress.
Embodiment 10, suppress the hepatitis B virus activity analysis after containing the OPC polymer of different R1 monomers
With different sample separation A and B through the analysis of the embodiment 1-7 R of its OPC polymer monomer as can be known 1The structure difference, this two sample is carried out OPC polymer poly compound suppress 50%B Hepatitis virus activity (IC50) test: after testing the human liver cancer cell line that can produce hepatitis B virus with it and cultivate altogether with the variable concentrations sample, according to as method specimen as described in the embodiment 8 to the inhibiting rate of hepatitis B virus surface antigen (HBs) with hepatitis B virus e antigen (HBe), through after serial dilution measures the inhibition percentage of its variable concentrations then, the sample concentration (IC of sample when calculating this sample and suppress viral efficient 50% with grafit5 software 50Value), result such as following table 1.
Table 1
Figure B2009101263709D0000121
* OPC sample A: contain general flavone alcohol (flavonols) 99.3%, OPC 86.5%.
* OPC sample B: the polymer that contains OPC structure through identifying.
The result shows that OPC sample A and OPC sample are respectively 127.1 ± 13.4 μ g/ml and 158.8 ± 15.5 μ g/ml in the concentration of the 2nd day inhibition 50%B hepatovirus surface antigen; The concentration that suppressed 50%B hepatovirus e antigen at the 2nd day is respectively 84.1 ± 38.4 μ g/ml and 46.3 ± 11.2 μ g/ml, at the 4th day then was 47.4 ± 3.9 μ g/ml and 44.1 ± 6.3 μ g/ml, and this result shows that the difference of procyanidin monomers R1 is similar to the inhibition ability of hepatitis B virus.
Though the present invention with preferred embodiment openly as above; right its is not in order to limit the present invention; any person skilled in the art; without departing from the spirit and scope of the present invention; when can doing a little change and retouching, so protection scope of the present invention is as the criterion when looking the scope that the accompanying Claim book defined.

Claims (20)

  1. One kind in order to the treatment viral hepatitis type b pharmaceutical compositions, comprising:
    One effective dose OPC polymer, the monomer molecule formula of described OPC polymer is as follows:
    Figure F2009101263709C0000011
    Wherein, R 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH; And R 4Be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar; And
    One pharmaceutically acceptable carrier or salt.
  2. 2. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, bond is C4, C8 carbon bond, C4, C6 carbon bond or C2, C7 oxygen key between two monomers that link to each other of wherein said OPC polymer.
  3. 3. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, the degree of polymerization of wherein said OPC polymer is 2-30.
  4. 4. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, wherein said monomer is in the R of C2, C3 or C4 position or S optical isomeric compound.
  5. 5. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, the monomer of wherein said OPC polymer is a flavone compound.
  6. 6. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 5, wherein said flavone compound is catechin, epicatechin, epiafzelechin, gallic acid catechin, gallic acid epicatechin, epigallocatechin, gallate, flavonols, flavandiols, leucocyanidin or anthocyanidin.
  7. 7. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, the monomer of wherein said OPC polymer is a flavan-3-alcohol.
  8. 8. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 1, wherein said OPC polymer extraction is from a plant.
  9. 9. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 8, wherein said plant is the plant of Ericaceae Ericaceae, rose family Rosaceae, Pinaceae Pinaceae, Vitaceae Vitaceae or Urticaceae Urticaceae.
  10. 10. the pharmaceutical compositions in order to the treatment viral hepatitis type b according to claim 9, the plant of wherein said Urticaceae Urticaceae is the mountain ramie.
  11. 11. a health food, it has the effect that suppresses hepatitis B virus, comprising:
    One effective dose OPC polymer, the monomer molecule formula of described OPC polymer is as follows:
    Figure F2009101263709C0000021
    Wherein, R 1Be OCH 3, R 2Be OH and R 3Be H, or R 1Be OH and R 2With R 3All be H, or R 1With R 2All be OH and R 3Be H, or R 1, R 2With R 3All be OH; And R 4Be 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar or 3-(β)-O-sugar.
  12. 12. health food according to claim 11, bond is C4, C8 carbon bond, C4, C6 carbon bond or C2, C7 oxygen key between two monomers that link to each other between wherein said OPC polymer.
  13. 13. health food according to claim 11, the degree of polymerization of wherein said OPC polymer are 2-30.
  14. 14. health food according to claim 11, wherein said monomer are in the R of C2, C3 or C4 position or S optical isomeric compound.
  15. 15. health food according to claim 11, the monomer of wherein said OPC polymer are flavone compound.
  16. 16. health food according to claim 15, wherein said flavone compound are catechin, epicatechin, epiafzelechin, gallic acid catechin, gallic acid epicatechin, epigallocatechin, gallate, flavonols, flavandiols, leucocyanidin or anthocyanidin.
  17. 17. health food according to claim 11, the monomer of wherein said OPC polymer are flavan-3-alcohol.
  18. 18. health food according to claim 11, wherein said OPC polymer extraction is from a plant.
  19. 19. health food according to claim 18, wherein this plant is the plant of Ericaceae Ericaceae, rose family Rosaceae, Pinaceae Pinaceae, Vitaceae Vitaceae or Urticaceae Urticaceae.
  20. 20. health food according to claim 19, the plant of wherein said Urticaceae Urticaceae are the mountain ramie.
CN200910126370A 2009-03-05 2009-03-05 Medical compound for curing hepatitis B and health food for inhibiting hepatitis B virus Pending CN101822372A (en)

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
US9474735B2 (en) 2009-12-30 2016-10-25 Industrial Technology Research Institute (Itri) Liver function improvement and treatment of liver disease
WO2012126178A3 (en) * 2011-03-22 2012-11-22 财团法人工业技术研究院 Pharmaceutical composition for treating hepatic disease
CN103442709A (en) * 2011-03-22 2013-12-11 财团法人工业技术研究院 Pharmaceutical composition for treating hepatic disease
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AU2011362905B2 (en) * 2011-03-22 2015-11-12 BELX Bio-Pharmaceutical (Taiwan) Corporation Pharmaceutical composition for treating hepatic disease
CN103442709B (en) * 2011-03-22 2018-10-19 财团法人工业技术研究院 Slow down liver cancer to deteriorate, improve liver function, improve liver fibrosis, improve hepatic sclerosis, improve liver inflammation and promote the pharmaceutical composition of damaged liver regeneration
WO2014101366A1 (en) * 2012-12-26 2014-07-03 Industrial Technology Research Institute Methods for inhibition of shc-1/p66 to combat aging-related diseases
CN104487069A (en) * 2012-12-26 2015-04-01 财团法人工业技术研究院 Methods for inhibition of shc-1/p66 to combat aging-related diseases
WO2014121590A1 (en) * 2013-02-07 2014-08-14 上海正基医药科技有限公司 Flavonoids or isoflavonoids compounds and uses thereof
WO2015067108A1 (en) * 2013-11-05 2015-05-14 上海唐润医药科技有限公司 Ringlike flavone or isoflavone compound, and application of same
WO2022205137A1 (en) * 2021-03-31 2022-10-06 贝尔克斯生技股份有限公司 Polymeric proanthocyanidin composition and application thereof

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