CN100584345C - Distillage of Ardisia chinensis Benth of possessing function of antivirus, extraction method and application - Google Patents
Distillage of Ardisia chinensis Benth of possessing function of antivirus, extraction method and application Download PDFInfo
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Abstract
An antiviral extract of Ardisia chinensis Benth is prepared from the dried plant of Ardisia chinensis Benth through extracting in water and purifying. It can be used to prepare the medicine, health-care product, or food for preventing and treating the diseases caused by HBV and CoxB3 virus.
Description
Technical field
The present invention relates to derive from medicinal extract and the extracting method and the purposes of vegetable material, specifically, is extract and extracting method and the purposes of little Herba Ardisiae Japonicae plant.
Background technology
Human body is owing to the disease that viral infection causes is a lot, and hazardness is also very big, can cause chronic viral infection disease as the chronic hepatitis that is caused by hepatitis B virus (HBV), swollen illness of lymph and chronic kidney obstacle.At present, 200,000,000 populations of having an appointment in the world are HBV virus carriers, wherein 80% concentrate on the Asian-Pacific area, occur hepatitis B virus surface antigen (HBsAg) positive in most of blood.Though the existing medicine of having developed some treatment hepatitis B viruss does not all have satisfied medicine, synthetic drug such as ethyl phosphine formates, suramin, 2-deoxyguanosine etc., side effect is big, and is prone to drug resistance.Seek new, anti-hepatic-B virus medicine is necessary safely and effectively.In addition, coxsackie B 3 type viruses (CoxB3) are the main paathogenic factors of viral myocarditis, and virus can continue to exist in vivo, causes DCM (dilated cardiomyopathy), cause that patients acuity is overworked dead, and other has report to point out that CoxB3 can cause respiratory tract infection and enteritis.But clinically Coxsackie virus infection is still lacked effective Therapeutic Method and medicine at present.Seeking effective antiviral drugs from plant is present research focus.
Myrsinacea (Myrsinaceae) Ardisa (Ardisia) the plant whole world has 300 kinds approximately, is distributed in tropical America, Austronesia, east, the peninsula, the Indian Ocean and south, and minority is distributed in the Pacific Ocean; Ardisa plant resources in China is abundant, there are 68 kinds approximately, the composition that the Herba Ardisiae Japonicae plant not of the same race that produces in different regions contains is not quite similar, and physiologically active also can have different, it is reported that the Ardisa plant mainly contains Coumarins, phenols, flavonoid and triterpene saponin constituents.We find that at Chinese North Guangdong Yao Xiang a kind of locals who is grown in small stream limit, remote mountains calls it plant of " Herba Dichondrae Repentis ", after expert testimony, " Herba Dichondrae Repentis " has for the little Herba Ardisiae Japonicae of Myrsinacea Ardisa (Ardisia chinensis Benth) different name; Lionet, Radix osteomelis schwerinais sunshade, little sunshade.According to the literature, the character hardship of this plant, flat.Effect is hemostasis, pain relieving.Cure mainly pulmonary tuberculosis, spitting of blood, spit blood traumatic injury, dysmenorrhea etc.According to the locality is precious jade born of the same parents introductions, and this medical herbs is the secret prescription handed down in the family from generation to generation that is used for curing local commonly encountered diseases icterohepatitis, good effect, and side effect is little.But by retrieval, do not see the systematic study of the chemical constituent and the bioactive aspect thereof of little Herba Ardisiae Japonicae, thereby cause these medical herbs development and use to lag behind.
Summary of the invention
The objective of the invention is from the little Herba Ardisiae Japonicae extract of plant amedica, to seek the composition of tool antivirus action and its extracting method is provided, and its purposes on preparation antiviral drugs, health product and food further is provided.
For achieving the above object, the little Herba Ardisiae Japonicae extract of tool antivirus action of the present invention contains little Herba Ardisiae Japonicae polysaccharide.
The content of described little Herba Ardisiae Japonicae polysaccharide is no less than 20%, and the content of total polyphenols is for being not more than 30%.
Described little Herba Ardisiae Japonicae polysaccharide is a neutral polysaccharide, and molecular weight is 100～10KDa, and the D-glucose content is no less than 50% in this polysaccharide; Described polyphenol is gallic acid and polyhydroxy benzenes formic acid derivates.
Extracting method of the present invention is: adopt the dry Herb of little Herba Ardisiae Japonicae (the Ardisia chinensis Benth) plant of Myrsinacea (Myrsinaceae) Ardisa (Ardisia SW) that is grown in the North Guangdong Yao nationality, obtain with water extraction.During extraction, the little Herba Ardisiae Japonicae water that dries in the shade, shreds can be decocted secondary, each 1-2 hour, filter extracting solution, be condensed into extractum, dry or be spray dried to brown powder in vacuum drying oven again, be little Herba Ardisiae Japonicae water extract.Pharmacological evaluation shows that little Herba Ardisiae Japonicae water extract has antivirus action.
Higher in order from the water extract of little Herba Ardisiae Japonicae plant, further to obtain purity, the product of better efficacy, little Herba Ardisiae Japonicae extracting solution with gained, concentrating under reduced pressure, centrifugalize gets supernatant, and it is 60～80% that supernatant transfers to concentration of alcohol with 95% ethanol, stir, staticly settle centrifugalize precipitate, dry little Herba Ardisiae Japonicae crude polysaccharides dry product; Calculate with extract dry product weight, little Herba Ardisiae Japonicae polyoses content is 60～80%, total polyphenols content is 5-10%.Pharmacological evaluation shows, the enrichment of little Herba Ardisiae Japonicae crude polysaccharides have a composition of antivirus action.
Get the little Herba Ardisiae Japonicae crude polysaccharides of gained dry product and be dissolved in the distilled water, behind the ultrasonic degas, cross ion exchange column or macroporous resin column, be eluted to eluent with distilled water and do not develop the color with phenolsulfuric acid reagent, eluent concentrates, dry must little Herba Ardisiae Japonicae polysaccharide dry product; Contain little Herba Ardisiae Japonicae polysaccharide 85～95%, total polyphenols 1～3% with the calculating of extract dry product weight.Pharmacological evaluation shows that little Herba Ardisiae Japonicae polysaccharide is the main active of little Herba Ardisiae Japonicae plant virus resistance.
The total sugar content assay method of little each extract of Herba Ardisiae Japonicae:
Total sugar content is done standard with anthrone-sulphuric acid colorimetric method for determining with galactose.The preparation of D-galactose standard curve: accurately take by weighing a certain amount of D-galactose, be made into the 100mL storing solution with distilled water, get 1,2,3,4 respectively, the 5mL storing solution is made into 100mL solution again, promptly obtains the standard galactose solution of variable concentrations.Drawing 1mL variable concentrations standard sugar solution respectively moves in the tool plug test tube, simultaneously make blank with the 1mL distilled water, first cooling number minute in ice-water bath, slowly add 5mL anthrone reagent (the 0.2g anthrone is dissolved in 100mL80% sulphuric acid) along test tube wall, jolt the back and in 80 ℃ of water-baths, react 20min, take out and be cooled to room temperature, at wavelength is to measure absorbance under the 640nm, draw the absorbance of variable concentrations standard galactose, make standard curve, obtain the standard curve equation.Algoscopy: accurately take by weighing a certain amount of each little Herba Ardisiae Japonicae extract, use the distilled water heating for dissolving, be made into 100mL solution.Get of the operation of this solution of 1mL respectively, record the absorbance of each sample, check in total sugar content (or standard curve equation calculate) sample from standard curve by above-mentioned standard sugar.
The polyphenol content assay method of little each extract of Herba Ardisiae Japonicae
Total polyphenols content Folin-Ciocalteu ' s reagent colorimetric method for determining is done standard with gallic acid.The preparation of gallic acid standard curve: accurately take by weighing a certain amount of gallic acid, be made into the 100mL storing solution with distilled water, get 1,2,3,4 respectively, the 5mL storing solution is made into 100mL solution again, promptly obtains the standard gallic acid solution of variable concentrations.The standard gallic acid solution of drawing the 8ml variable concentrations respectively moves in the tool plug test tube, makes blank with the 8ml distilled water simultaneously.Folin-Ciocalteu ' the s reagent (available from Sigma company) that in each test tube, adds 0.5ml, behind the mix homogeneously, 20% the sodium carbonate liquor that adds 1.5ml again, remix is even, place after 1 hour, with the standard gallic acid absorbance of ultraviolet spectrophotometer at 750nm mensuration variable concentrations, make standard curve, obtain the standard curve equation.Algoscopy: accurately take by weighing a certain amount of each little Herba Ardisiae Japonicae extract, be dissolved in the distilled water of 8ml, Folin-Ciocalteu ' the s reagent (available from Sigma company) that adds 0.5ml, behind the mix homogeneously, 20% the sodium carbonate liquor that adds 1.5ml again, remix is even, places after 1 hour, measures absorption value with ultraviolet spectrophotometer at 750nm.Check in total polyphenols content (or standard curve equation calculate) sample from standard curve.
Gallic acid qualitative analysis in each extract of little Herba Ardisiae Japonicae
With the gallic acid in each extract of method qualitative detection of thin layer chromatography (TLC) analysis, each extract of little Herba Ardisiae Japonicae (comprising water extract, crude polysaccharides and polysaccharide) and gallic acid standard specimen (available from Chinese medicine and biological products assay institute) are dissolved in respectively in a certain amount of methanol, put in silica gel thin-layer GF
254In the plate (Haiyang Chemical Plant, Qingdao), use chloroform: methanol=9: 1 (or 8: 2, or 7: 3) launch, FeCl used
3/ EtOH reagent colour development is done qualitative analysis according to the size of Rf value.In addition, we separate by silica gel column chromatography, isolate gallic acid and polyhydroxy benzenes formic acid derivates from little Herba Ardisiae Japonicae water extract, and the structure of separating obtained chemical compound is identified the method that adopts nuclear magnetic resonance, NMR H spectrum, C spectrum and mass spectral analysis.
Little Herba Ardisiae Japonicae water extract, crude polysaccharides, determination of polysaccharide result:
(1) in the little Herba Ardisiae Japonicae water extract dry product, in the D-galactose, polyoses content should be 30～20%; In gallic acid, polyphenol content should be 20～30%.
(2) in the little Herba Ardisiae Japonicae crude polysaccharides dry product, in the D-galactose, polyoses content should be 80～60%, and in gallic acid, polyphenol content should be 5～10%.
(3) in the little Herba Ardisiae Japonicae polysaccharide dry product, in the D-galactose, polyoses content should be 85～95%, and in gallic acid, polyphenol content should be 1～3%.
(4) polyphenol in the little Herba Ardisiae Japonicae extract is gallic acid and polyhydroxy benzenes formic acid derivates.
The monosaccharide of little Herba Ardisiae Japonicae polysaccharide is formed and content:
Use the trifluoroacetic acid of 2mol/L earlier, in 110 ℃ of following hydrolyzation sample 1.5h, evaporated under reduced pressure then.Add 10mg oxammonium hydrochloride., 0.5ml pyridine in the sample after hydrolysis, oscillating reactions 30min in 90 ℃ of water-baths, take out postcooling to room temperature, add the 0.5ml acetic anhydride, in 90 ℃ of water-baths, continue reaction 30min, make it to change saccharogenesis nitrile acetic ester derivative, gas chromatographic analysis is carried out in the product direct sample.It is that (carrier gas is high-purity helium to the DB-5 post for 0.2mm * 35m), the thick 0.25 μ m of liquid film that gas chromatographic column is adopted in this experiment.Chromatographic condition: adopt temperature programming, initial temperature is 60 ℃, keeps that the speed with 30 ℃/min rises to 120 ℃ behind the 2min, keeps 1min, rises to 250 ℃ and keep 10min with the speed of 2.5 ℃/min again.280 ℃ of injector temperatures adopt not shunt mode sample introduction, and the post flow is 2ml/min.Mass spectrum condition: adopt electron bombard (EI) ion source, ion energy 70eV.
The result: from Fig. 1 as seen, the D-glucose content surpasses 50% in the little Herba Ardisiae Japonicae polysaccharide.
The quantitative determination of little Herba Ardisiae Japonicae polysaccharide molecule
Adopt sephadex column to measure little Herba Ardisiae Japonicae polysaccharide molecular weight.The preparation of glucosan (Dextran 500,400,76.9,42,10KDa) standard curve: the method for pressing earlier product description is handled polydextran gel, adorns post, balance then.Be dissolved in the standard glucosan of an amount of different molecular weight (Mr) in the distilled water respectively, add in the pillar, NaCl eluting pillar with 0.05mol/ml, phenolsulfuric acid reagent is monitored the terminal point that washes out of polysaccharide, record elution volume (Ve) is a vertical coordinate with Ve, and lgMr is an abscissa, make standard curve, obtain the standard curve equation.Algoscopy: the samely get an amount of little Herba Ardisiae Japonicae polysaccharide sample and be dissolved in the distilled water, upper prop, eluting, the Ve when surveying it and flowing out from pillar checks in (or standard curve equation calculate) little Herba Ardisiae Japonicae polysaccharide molecular weight from standard curve.
The result: little Herba Ardisiae Japonicae polysaccharide molecular weight is 100～10KDa.
With each extract of the present invention is that active component adds the adjuvant that pharmacy is allowed, and preparation process routinely can be formed various oral formulations.
The extract of above-mentioned different parts is carried out the antiviral in vitro tests to confirm its purposes.
One, coxsackie B 3 type virus (CoxB3) activity determination methods of each extract:
1, sample vitro cytotoxicity experiment: adopt the cytotoxicity of tetrazolium salts colorimetry (MTT colorimetric method) working sample, the used cell of this experiment is the Hella cell.Experimentation is summarized as follows: the Hella cell inoculation on 96 porocyte culture plates, is put 37 ℃, CO
2(5%) cultivated in the incubator 2 days, treat that cell grows up to monolayer after, discard culture fluid.Test liquid behind the two-fold dilution is joined in the cell monolayer of 96 orifice plates cultivation, establish normal cell matched group (only add and keep liquid) simultaneously.96 orifice plates behind the application of sample place 37 ℃, 5%CO
2Cultivate 72h in the incubator,, write down each concentration porocyte toxicity situation with the optical microscope cytotoxicity that every day, observation sample caused.Discard culture fluid in the hole behind the 72h, the MTT solution that adds 10 μ L 5mg/ml, place incubator to cultivate 5h, add the DMSO dissolving again, measure the OD value in each hole with microplate reader, the measurement wavelength is 570nm, and reference wavelength is 630nm, calculates the half lethal toxicity concentration (CC50) of polysaccharide sample pair cell.
2, the external anti-CoxB3 activity experiment of sample: the extracorporeal antivirus effect activity that adopts cytopathic-effect inhibition assay (CPE reduction assay) working sample.In this experiment, the Hela cell is the host cell of CoxB3, and the positive control drug of anti-CoxB3 is a ribavirin.Experimentation is summarized as follows: the Hela cell inoculation on 96 porocyte culture plates, is put in 37 ℃, CO2 (5%) incubator and was cultivated 2 days, treat that cell grows up to monolayer after, discard culture fluid.Test liquid behind the two-fold dilution is joined in the cell monolayer of 96 orifice plates cultivation.Cell contrast and ribavirin that not dosing is set are simultaneously made positive control drug.Again isopyknic viral suspension (100TCID50/ml) is added in the cell monolayer.Place 37 ℃, CO2 (5%) incubator to cultivate 2～5 days in cell, observe cytopathy (CPE) situation that virus causes every day with optical microscope.CPE kept the score (make 0=0%CPE, 1=0～25%CPE, 2=25～50%CPE, 3=50～75%CPE, 4=75～100%CPE).Half-inhibition concentration (IC50) is observed the concentration that cytopathy is 50% o'clock test liquid after being meant and adding test liquid and virus under light microscopic.Selectivity index SI=CC
50/ IC
50, the SI value is high more, and the activity of expression sample is high, toxicity is little.
Experimental result sees Table 1:
The external anti-CoxB3 activity of the little Herba Ardisiae Japonicae extract of table 1
| Sample | Polyoses content | CC 50(μg/ml) | IC 50(μg/ml) | |
| Water extract | ||||
| 30～20％ | ＞1000 | 31.25 | ＞32 | |
| |
80～60％ | ＞1000 | 10 | ＞100 |
| Little Herba Ardisiae Japonicae |
85～98％ | ＞1000 | 3.9 | ＞265 |
| Virazole | 62.5 | 12.5 | 5 |
Data show in the table, and little Herba Ardisiae Japonicae water extract, crude polysaccharides and polysaccharide all have the anti-enterovirus activity of good in vitro, their IC
50Be respectively 31.25 μ g/ml, 10 μ g/ml and 3.9 μ g/ml, their SI value is respectively greater than 32,100 with greater than 265, their external anti-enterovirus specific activity virazole (IC
50Be 12.5 μ g/ml, the SI value is 5) much better.Along with the increase of polyoses content in the extract, their external anti-enterovirus is active to be increased, and especially little Herba Ardisiae Japonicae polysaccharide has excellent anti-enterovirus activity.
Two, the external anti-hepatitis B virus of sample (HBV) activity experiment method
Adopt dhbv dna (DHBV) model:
1, the cultivation of primary duck hepatocyte (PDH)
1 age in days Guangzhou sheldrake intravenous injection 0.2ml dhbv dna (DHBV) positive serum.Get the positive duck of 7 age in days DHBV, inject and cut open the belly after 5% pentobarbital sodium 0.4ml anaesthetizes, behind the venous perfusion perfusion buffer 200ml, perfusion digestion buffer 50ml.Carry out for guaranteeing to be poured under 37 ℃, all buffer are preheated to 37 ℃ earlier, and liver covers with gauze.After perfusion finishes, take off liver, liver is placed bottle, shred, add the L-15 growth-promoting media, the piping and druming cell dispersion.Obtained cell suspension, centrifugal after the cell breakdown sieve filters (50 * g, 4min), sedimentation cell is washed 3 times with the L-15 growth-promoting media.Cell is behind the hemocytometer counting, with being inoculated in 24 orifice plates (1 * 10 after the dilution of L-15 growth-promoting media
6Cell/ml/ hole), place 37 ℃ to cultivate 24h down.Dilute each extract of little Herba Ardisiae Japonicae with the L-15 growth-promoting media, add in 24 orifice plates, each concentration do 3 parallel, other establishes the virus control group, with the L-15 growth-promoting media replace medicine, each concentration also do 3 parallel.Getting supernatant behind the 48h, centrifugal (6000 * g, 10min) ,-70 ℃ down frozen, detects for dot blot hybridization and use.
2, dot blot hybridization detects
Get the above-mentioned supernatant point of 750 μ l on nitrocellulose filter, room temperature is dried back degeneration 10min under 0.5M NaOH effect, and in reuse 1M tris-HCl-0.6M NaCl (pH7.5) solution and 15min, 60 ℃ are toasted the 4h fixed dnas.
32The P-DHBV-DNA probe mark is undertaken by nick translation test kit description method.With the nitrocellulose filter of preparation in above-mentioned successively at 60 ℃, prehybridization 20min and 60 ℃ in 1% sodium lauryl sulphate (SDS) and 3 * SSC (pH7.0) solution, 1% sodium lauryl sulphate (SDS), 100 μ g/ml degeneration salmon sperm dnas, in 10 * Denhardt ' s solution behind the prehybridization 4h, again film is placed 50% Methanamide, 5 * SSC, in 3 * Denhardt ' s solution and the 100 μ g/ml degeneration salmon olein dna solutions, hybridize 8h in 42 ℃ of hybridization casees, the hybridization solution that inclines, with 1% sodium lauryl sulphate (SDS) and 3 * SSC solution respectively at room temperature, 42 ℃, 60 ℃ of each rinsing 10min.Film is done and is used the X-ray film, places-70 ℃ of autoradiography.Survey the OD value of formed speckle with microplate reader.HBV-DNA with known content handles as stated above, measures the OD value of each speckle, makes standard curve.Obtain the DHBV-DNA content of each testing sample by standard curve.(x ± s) expression adopts Student ' s T-test to carry out the non-matching statistical analysis to the standard test data, in P＜0.05 level, judges whether difference has statistical significance with meansigma methods ± standard deviation.And be calculated as follows the suppression ratio of DHBV DNA in the medicine pair cell: the DHBV-DNA content of the DHBV dna content-sample of suppression ratio %=[virus control]/DHBV dna content * 100% of virus control.With the drug level is abscissa, and suppression ratio is the vertical coordinate curve plotting, obtains the DHBV suppression ratio and be 50% o'clock drug level, i.e. IC
50
The little Herba Ardisiae Japonicae extract of variable concentrations sees Table 2 to DHBV DNA inhibition situation:
Each extract of the little Herba Ardisiae Japonicae of table 2 is to the suppression ratio (%) of DHBV DNA
* each group compares P＜0.05, * * P＜0.01 with the virus control group.
From table 2 data as seen: experiment higher concentration group (〉=200 μ g/ml) relatively has significant difference with the virus control group, illustrate that each extract of little Herba Ardisiae Japonicae all can suppress duplicating of DHBV DNA, and each extract is when experiment maximum concentration 1000 μ g/ml, and the suppression ratio of DHBV DNA is all reached more than 90%.
The IC that little Herba Ardisiae Japonicae water extract suppresses DHBV DNA
50Be the IC that 273 μ g/ml, crude polysaccharides suppress DHBV DNA
50Be the IC that 200 μ g/ml, little Herba Ardisiae Japonicae polysaccharide suppress DHBV DNA
50Less than 200 μ g/ml.
Three, anti-hepatitis B virus (HBV) activity experiment method in the sample body
Adopt dhbv dna (DHBV) model, concrete grammar is as follows: 1 age in days Guangzhou sheldrake intravenous injection 0.2ml dhbv dna (DHBV) positive serum.Get the positive duck of 13 age in days DHBV, get blood, centrifugalize serum ,-70 ℃ are frozen down.In addition, the positive duck random packet of 13 age in days DHBV is carried out the Drug therapy experiment, 10～7 every group are not waited.The heavy dose of group of the little Herba Ardisiae Japonicae water extract of administration component 10gkg
-1D
-1, middle dosage group (5gkg
-1D
-1) and small dose group (1gkg
-1D
-1), all oral, 2 times/d, 10 days is a course of treatment.Other establishes the virus control group, and with the physiologic saline for substitute medicine, positive control is oral with acycloguanosine, administration 10mgkg
-1D
-1, 2 times/d, 10 days is a course of treatment.The 13rd day (before the administration), administration 5 days after infection, administration 10 days and drug withdrawal 3 days are got blood from duck lower limb vein, separation of serum, and-70 ℃ are frozen down.
It is clear to get above-mentioned Sanguis Anas domestica, every batch with time point on nitrocellulose filter, according to aforesaid dot blot hybridization method, do the clear dot blot hybridization of Sanguis Anas domestica, autoradiography diaphragm speckle, on microplate reader, measure the OD value, calculate serum DHBV-DNA content, (x ± s) expression adopts Student ' sT-te st to carry out the non-matching statistical analysis to experimental data with meansigma methods ± standard deviation, in P＜0.05 level, judge whether difference has statistical significance.And be calculated as follows the suppression ratio of DHBV-DNA in the medicine pair cell:
Suppression ratio %=(OD
Before the administration-OD
After the administration/ OD
After the administration) * 100%
Zoopery the results are shown in Table 3:
Table 3, little Herba Ardisiae Japonicae water extract in the duck body to the suppression ratio of DHBV-DNA
* each group compares P＜0.05, * * P＜0.01 with the virus control group.
As seen: medication is after 10 days, and each dosage group of little Herba Ardisiae Japonicae water extract all has certain inhibition to DHBV-DNA content in the serum, and heavy dose of and middle dosage group reaches 50% to suppressing in the HBV-DNA body, and significant difference is arranged.In addition, little Herba Ardisiae Japonicae water extract still keeps suppressing preferably to DHBV-DNA after the drug withdrawal, and the rise of DHBV-DNA does not have positive control drug obvious.Be that little Herba Ardisiae Japonicae water extract is at 5gkg
-1D
-1And 10gkg
-1D
-1Dosage the time, hepatitis B virus is had preferably suppress in vivo.
Though little Herba Ardisiae Japonicae is used for curing the disease icterohepatitis in Yao nationality area as secret prescription handed down in the family from generation to generation, in long-term practice, prove good effect, side effect is little.But never there is modern scientific research to confirm its curative effect, research its effective site of report and composition are not more arranged, thereby caused the development and use of this traditional herbal medicine to lag behind.The present invention confirms that first the water extract of little Herba Ardisiae Japonicae has the activity of resistance of hepatitis B and anti-Coxsackie virus, and active tracking separates and shows that its antiviral activity composition is little Herba Ardisiae Japonicae polysaccharide.On basis of the present invention, can prepare medicine, health product and the food of the clinical symptoms that treatment and prevention hepatitis B virus (HBV) and coxsackie B 3 type viruses (CoxB3) cause.
Description of drawings
Fig. 1 is monosaccharide is formed in the little Herba Ardisiae Japonicae polysaccharide of the present invention analysis (GC-MS analysis) figure as a result.
The specific embodiment
The present invention can be elaborated by following examples, but is not limited only to following examples.
Embodiment one: the preparation of little Herba Ardisiae Japonicae water extract
Get the little Herba Ardisiae Japonicae Herb after drying in the shade, shredding, with 10 times of water gagings, in 100 ℃ of reflux, extract, secondaries, each 1～2 hour, filter extracting solution, filtrate is condensed into extractum with Rotary Evaporators, dry or be spray dried to brown powder in vacuum drying oven, be little Herba Ardisiae Japonicae water extract.Contain little Herba Ardisiae Japonicae polysaccharide 30～20%, total polyphenols 20～30% with the calculating of extract dry product weight.
Embodiment two: the preparation of little Herba Ardisiae Japonicae crude polysaccharides
Get the little Herba Ardisiae Japonicae Herb after drying in the shade, shredding, with 10 times of water gagings, in 100 ℃ of reflux, extract, secondaries, each 1～2 hour, filter extracting solution, it is 1.05-1.15 (60 ℃) that filtrate is concentrated into relative density with Rotary Evaporators, and centrifugalize gets supernatant, and adding 95% alcoholic solution concentration of alcohol to the system is 60～80%, stirring is spent the night precipitation is separated out fully, the centrifugalize precipitate, the dry dry product that gets is little Herba Ardisiae Japonicae crude polysaccharides.Calculate with extract dry product weight, little Herba Ardisiae Japonicae polyoses content is 60～80%, total polyphenols content is 5-10%.
Embodiment three: the preparation method 1 of little Herba Ardisiae Japonicae polysaccharide
Take by weighing DEAE Cellulose-52 ion exchange resin 100g, adding distil water 500mL stirs, the water floatation is removed a spot of cellulose monomer or fragment, at room temperature places 24h then, treat the abundant swelling of resin after, resin is drained, by specification require activated resin.With activatory resin, adding distil water 300mL uses ultrasonic degas, after stirring, carefully is poured into lentamente in the post along glass rod, knocks post jamb gently simultaneously, makes resin settled evenly, closely, then with the abundant drip washing 48h of distilled water, places the balance pillar that spends the night.
Take by weighing little Herba Ardisiae Japonicae crude polysaccharides dry product 200mg, heating is dissolved in the 1mL distilled water, behind the ultrasonic degas, sample is added in the ion exchange column that above-mentioned activation and balance cross, with distilled water eluting pillar, use the HL-2 constant flow pump, the control flow velocity is 1mL/min, and automatic collection instrument is collected eluent, every pipe is collected 10mL, collecting liquid with the colour developing of phenolsulfuric acid method, is to measure absorbance under the 490nm at wavelength, draws elution curve, the eluent that merges absworption peak, concentrating under reduced pressure, lyophilization obtains little Herba Ardisiae Japonicae polysaccharide.Contain little Herba Ardisiae Japonicae polysaccharide 90～95%, total polyphenols 1-3% with the calculating of extract dry product weight.
Embodiment four: the preparation method 2 of little Herba Ardisiae Japonicae polysaccharide
Take by weighing macroporous adsorbent resin (AB-8 or D101) 500g, adding distil water 800mL stirs, the water floatation is removed a spot of resin monomer or fragment, at room temperature places 24h then, treat the abundant swelling of resin after, resin is drained, by specification require process resin.With the resin of having handled, adding distil water 800mL after stirring, is added in the post, knocks post jamb gently simultaneously, makes resin settled evenly, closely, then with the abundant drip washing 48h of distilled water, places the balance pillar that spends the night.
Take by weighing little Herba Ardisiae Japonicae crude polysaccharides dry product 30g, be dissolved in the 70mL distilled water, behind the ultrasonic degas, sample is added in the macroporous resin column that above-mentioned processing and balance cross, with " embodiment three " method, with distilled water eluting pillar, collect liquid with phenolsulfuric acid method color developing detection, merge the eluent that develops the color, concentrating under reduced pressure, lyophilization obtains little Herba Ardisiae Japonicae polysaccharide.Contain little Herba Ardisiae Japonicae polysaccharide 85～95%, total polyphenols 1-3% with the calculating of extract dry product weight.
Embodiment five: little Herba Ardisiae Japonicae capsule preparation
Take by weighing the little Herba Ardisiae Japonicae extract of 85-90g embodiment two gained, add medicinal starch 15-10g, mix homogeneously, with suitable quantity of water or 95% alcohol granulation, cross 12 mesh sieves extruding system wet granular, at 60 ℃ of following vacuum dryings, with 20 mesh sieve granulate, add the 1-5g micropowder silica gel simultaneously, mix homogeneously, granule is filled into capsule and gets final product.Oral capsule can prevent and treat the clinical symptoms that hepatitis B virus (HBV) and coxsackie B 3 type viruses (CoxB3) cause.
Certainly, also can preparation process routinely make other peroral dosage form as tablet, oral liquid etc.
Claims (4)
1, a kind of extracting method of little Herba Ardisiae Japonicae extract of tool antivirus action is characterized in that: the back water is dried in the shade, shredded to the dry Herb of the little Herba Ardisiae Japonicae plant of Myrsinacea Ardisa decoct secondary, each 1～2 hour, filter extracting solution; With the extracting solution concentrating under reduced pressure that obtains after the described filtration, centrifugalize gets supernatant, and transferring to the supernatant concentration of alcohol with ethanol is 60～80%, staticly settles, the dry crude polysaccharides dry product that gets; Calculate with extract dry product weight, little Herba Ardisiae Japonicae polyoses content is 60～80%, total polyphenols content is 5～10%;
Described little Herba Ardisiae Japonicae polysaccharide is a neutral polysaccharide, and molecular weight is 100～10KDa, and the D-glucose content is no less than 50% in this polysaccharide; Described polyphenol is gallic acid and polyhydroxy benzenes formic acid derivates.
2, extracting method according to claim 1, it is characterized in that: gained crude polysaccharides dry product is dissolved in the distilled water, behind the ultrasonic degas, cross ion exchange column or macroporous resin column, be eluted to eluent with distilled water and do not develop the color with phenolsulfuric acid reagent, eluent concentrates, dry little Herba Ardisiae Japonicae polysaccharide dry product; Contain little Herba Ardisiae Japonicae polysaccharide 85～95%, total polyphenols 1～3% with the calculating of extract dry product weight.
3, the little Herba Ardisiae Japonicae extract that is made by claim 1 or 2 described extracting method is used for the purposes of medicine, health product or the food of the clinical symptoms that preparation treatment and prevention hepatitis B virus (HBV) and coxsackie B 3 type viruses (CoxB3) cause.
4, purposes according to claim 3 is characterized in that: with claim 1 or 2 prepared little Herba Ardisiae Japonicae extracts is that active component adds the adjuvant that pharmacy is allowed, and preparation process is routinely made various oral formulations.
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| CN104042647B (en) * | 2014-06-16 | 2016-08-31 | 桂林医学院 | The application in preparing the liver protecting medicine of the Herba Ardisiae Japonicae total flavones |
| CN110187029A (en) * | 2019-06-06 | 2019-08-30 | 重庆医科大学 | The method for building up and its finger-print of 'Chenxiang Huaqi piece gas-phase fingerprint pattern |
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