CN102697800B - Herba stellariae mediae polysaccharide composition and the application in preparation antiviral drugs thereof - Google Patents
Herba stellariae mediae polysaccharide composition and the application in preparation antiviral drugs thereof Download PDFInfo
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Abstract
The present invention relates to Herba stellariae mediae polysaccharide composition and the application in preparation antiviral drugs thereof.Herba stellariae mediae polysaccharide composition disclosed by the invention is mixed by a certain percentage by 5 kinds of Herba stellariae mediae polysaccharide (A, B, C, D, E), it is characterized in that said composition is brown floccule, soluble in water, be insoluble to the ethanol of high concentration, acetone, ether, n-butyl alcohol, isopropyl alcohol; Containing the Herba stellariae mediae polysaccharide of 5 kinds of molecular weight distribution between 5000 ~ 30000 in polysaccharide composition, be made up of by certain mol ratio rhamnose, arabinose, xylose, mannose, glucose and galactose.5 kinds of polysaccharide, by after specific proportioning mix homogeneously, utilize its mutual synergism, can strengthen its antiviral activity.The present invention also discloses the purposes of Herba stellariae mediae polysaccharide composition for the preparation of antiviral drugs as hepatitis B virus, HIV (human immunodeficiency virus), herpesvirus, influenza virus, enterovirus and adenovirus etc., and can be made into multi-medicament preparation.Said composition treatment good effect, and have no toxicity, have broad prospects in new antiviral drug exploitation.
Description
Technical field
The present invention relates to the application of Herba stellariae mediae polysaccharide composition in preparation antiviral drugs, particularly a kind of press after active ingredient of Chinese herbs separation and purification again by certain proportion mix homogeneously Herba stellariae mediae polysaccharide composition and preparing the application in antiviral drugs; The invention still further relates to the preparation containing Herba stellariae mediae polysaccharide composition and health product, belong to field of medicaments.
Background technology
Virus is minimum pathogenic microorganism, that a class does not have a cellularity, the microorganism of the vital signs such as there is heredity, copy, its core is ribonucleic acid or DNA (deoxyribonucleic acid) (DNA), the propagation of virus is the widest, is one of infectious disease that sickness rate is higher in the world at present.The disease caused by virus common are infectious hepatitis, herpes, encephalitis, viral respiratory tract infection, influenza and herpetic keratitis etc.Disease more is clinically relevant with viral infection, according to foreign statistic document announcement, the infectious disease of nearly 3/4 is caused by virus, 18 kinds are had to belong to Causative virus in 55 kinds of viruses that international virus taxis association (ICTV) in 1984 has been announced, different viral infection causes different illness, needs different antiviral treatments.
The approach of viral infection resisting is a lot, as directly suppression or kill virus, viral interference absorption, stops virus to penetrate cell, the synthesis of suppression viral organism, the release of suppression virus or enhancing host anti-virus ability etc.Although current antiviral drugs is clinically more, but be mostly nucleoside derivate, its Main Function target spot is viral dna polymerase, compete with the form of enzyme reaction substrate analog and normal substrates, directly the activity of reduction or blocking dna polymerase, shows preferably antiviral activity, but it has and easily produces drug resistance, antiviral narrow range, the shortcomings such as toxic and side effects is large, expensive.In recent years, many Chinese herbal medicine are used for the concern that the control of viral disease causes people, as Cacumen Securinegae Suffruticosae, Rhizoma Polygoni Cuspidati, Flos Lonicerae, Radix Arnebiae (Radix Lithospermi), Rhizoma Osmundae, Radix Et Rhizoma Rhei etc. obtain significant curative effect for the control of some disease of viral infection.
Herba stellariae mediae (Stellaria media (L.) Villar.) is Caryophyllaceae Stellaria 1 year or 2 years raw herbaceous plant, has effect of heat-clearing and toxic substances removing, benefiting QI and nourishing blood, tonifying speen and tonifying kidney; Cure mainly dysentery, carbuncle pyogenic infections from tumour or sore, acute mastitis, acute appendicitis, furuncle and phyma, traumatic injury, the stagnant stomachache of the stasis of blood in puerperal etc.Root it is documented nearly 120 kinds of the whole world, and be widely distributed in temperature to refrigerant latitudes, China produces 63 kind of 15 mutation and 2 modification, and each province all produces it.Herba stellariae mediae, as folk medicine, is widely used.Herba stellariae mediae is rich in the bioactive ingredients such as flavone, phenolic acid, polysaccharide, saponins, organic acid, alkaloid, and research at present shows that Herba stellariae mediae has the antivirus action of wide spectrum.
Polysaccharide (polysaccharide) is formed by multiple monosaccharide molecule condensation, dehydration, is molecule mechanism complexity and huge glucide.All meet macromolecular compound concept carbohydrate and derivant be all called polysaccharide.Such material is particularly important in natural plant, and research shows that polysaccharide is at biological activitys such as immunomodulating, antiviral and anticancer, blood sugar lowering, treatment hepatorenal disease and anti-inflammatory analgesics.The research of current Herba stellariae mediae polysaccharide mainly rests on crude polysaccharides level, mainly the research of the aspect such as extraction and biological activity of crude polysaccharides.Herba stellariae mediae polysaccharide described in China Patent Publication No. CN1521193A and the preparation method of compositions thereof, point out that Herba stellariae mediae polysaccharide composition is that the total flavones of 80 ~ 90% and the crude polysaccharides of 10 ~ 20% mix, there is certain AIDS virus resisting and the effect of herpesvirus, this is only the extraction preparation of Herba stellariae mediae crude polysaccharides, the polysaccharide obtained contains other compositions such as protein, pigment, have no the relevant report of homogeneity Herba stellariae mediae polysaccharide, more have no the report being mixed into Herba stellariae mediae polysaccharide composition by a certain percentage by Herba stellariae mediae polysaccharide and being applied in preparation antiviral drugs.
Summary of the invention
The object of the invention is to study the active component of Herba stellariae mediae, a kind of new Herba stellariae mediae polysaccharide composition with medical value is provided, and disclose it in preparation antiviral drugs and antiviral application, exploitation antiviral related preparations and health product.
Herba stellariae mediae polysaccharide composition of the present invention, through certain processing mode separation and purification by the fresh herb of Herba stellariae mediae Stellaria media (L.) Villar., again by the homogeneous Herba stellariae mediae polysaccharide of 5 kinds of molecular weight of separation and purification by a certain percentage scope mix, 5 kinds of Herba stellariae mediae polysaccharide adopt modern analytical technique, analyze its molecular weight, molecular weight distribution, mol ratio and monosaccharide thereof composition, have no relevant report at present, it extracts and separation and purification flow process is shown in accompanying drawing 1.The present invention institute experimentally fast, accurately, said composition does not have bibliographical information to have the effect of broad-spectrum antiviral.
Herba stellariae mediae polysaccharide in Herba stellariae mediae polysaccharide composition of the present invention can extract from following plants, and these plants comprise plant Herba stellariae mediae or Stellaria plant, and subordinate's species mainly contain Alishan Herba stellariae mediae Stellaria arisanensisvar.Leptophylla Hayata, short sunken Herba stellariae mediae Stellaria depressa, white hair Herba stellariae mediae Stellaria patens, embrace stem mountain valley with clumps of trees and bamboo aunt grass Stellaria vestita var.amplexicaulis, hide southern Herba stellariae mediae Stellaria zangnanensis, Herba Stellariae dichotomae Stellaria dichotoma, Herba Stellariae dichotomae (former mutation), long lobe Herba stellariae mediae Stellaria dichotoma, long lobe Herba stellariae mediae (former mutation), length splits Herba stellariae mediae Stellaria martjanovii, become mildewed mountain valley with clumps of trees and bamboo aunt grass Stellaria pilosa, come into leaves Herba stellariae mediae Stellaria longifolia, come into leaves Herba stellariae mediae (former mutation), evergreen standing grain leaf Herba stellariae mediae Stellaria graminea var.viridescens, Cuona County Herba stellariae mediae Stellaria decumbens var.arenarioides, Caryo phyllaceae Stellariadelavayi, list Herba stellariae mediae, cushion is laid down sleeping Herba stellariae mediae Stellaria decumbens var.pulvinata, tundra Herba stellariae mediae Stellaria irrigua, only son's Herba stellariae mediae Stellaria monosperma, only son's Herba stellariae mediae (former mutation), short lobe Herba stellariae mediae Stellaria brachypetala, blunt calyx Herba stellariae mediae Stellaria amblyosepala, spend more Herba stellariae mediae Stellarianipponica, spend more the sleeping Herba stellariae mediae Stellaria decumbens var.polyantha that lays down, high eyebrow Herba stellariae mediae Stellariaomeiensis, two post Herba stellariae mediae Stellaria bistyla, turn over white Herba stellariae mediae Stellaria discolor, Herba stellariae mediae Stellariamedia, Herba stellariae mediae (former mutation), cover a watt Herba stellariae mediae Stellaria imbricata, high mountain Stellaria alsine Grim. Stellaria uliginosavar.alpina, standing grain leaf Herba stellariae mediae Stellaria graminea, standing grain leaf Herba stellariae mediae (former mutation), Helan Mountain Herba stellariae mediae Stellariaalaschanica, Stellaria henryi williams Stellaria henryi, Intestinum Gallus domesticus Herba stellariae mediae Stellaria neglecta, Jilong Herba stellariae mediae Stellaria gyirongensis, Jiangzi's Herba stellariae mediae Stellaria gyangtseensis, eyelashes come into leaves Herba stellariae mediae Stellariagyangtseensis, Kangding Herba stellariae mediae Stellaria souliei, woods Herba stellariae mediae Stellaria bungeana var.stubendorfii, Folium Salicis Babylonicae Herba stellariae mediae Stellaria salicifolia, ovum leaf Herba stellariae mediae Stellaria ovatifolia, Mao Heye Herba stellariae mediae Stellaria graminea var.pilosula, certain kind of berries tongue shape Herba stellariae mediae Stellaria oxycoccoides, Milin Herba stellariae mediae Stellaria mainlingensis, close colored Herba stellariae mediae Stellaria congestiflora, close pubescence Herba stellariae mediae Stellariayunnanensis f.villosa, continuous handle Herba stellariae mediae Stellaria lanipes, MIAOMAO Herba stellariae mediae Stellaria lanata, inflection Herba stellariae mediae Stellaria infracta, Nepal Herba stellariae mediae Stellaria nepalensis, Pamir Herba stellariae mediae Stellariawinkleri, Radix Stellariae Yunnanensis Stellaria yunnanensis, Radix Stellariae Yunnanensis (former mutation), mountain valley with clumps of trees and bamboo aunt grass Stellariavestita, mountain valley with clumps of trees and bamboo aunt grass (former mutation), Stellaria alsine Grim. Stellaria uliginosa, Stellaria alsine Grim. (former mutation), umbrella flower Herba stellariae mediae Stellaria umbellata, husky raw Herba stellariae mediae Stellaria arenaria, wetland Herba stellariae mediae Stellaria uda, the raw Herba stellariae mediae of stone, Stellaria dianthifolia Williams Stellaria dianthifolia, Tassel lobe Herba stellariae mediae Stellaria radians, Herba Stellariae Alsines, net arteries and veins Herba stellariae mediae Stellaria reticulivena, Stellaria wushanensis Williams Stellaria wushanensis, without lobe Herba stellariae mediae Stellaria apetala, without luxuriant Herba stellariae mediae Stellaria ebracteatav, Tibet Herba stellariae mediae Stellaria tibetica, thin handle Herba stellariae mediae Stellariapetiolaris, spire Herba stellariae mediae Stellaria filicaulis, linear leaf bud Herba stellariae mediae Stellaria crassifolia Ehrh.var.linearis, line leaf Herba stellariae mediae Stellaria dichotoma var.linearis, glandular hair Herba stellariae mediae Stellaria nemorum, blinks Stellaria pusilla, Stellaria micrantha Hayata Stellaria media var.micrantha, umbellule flower Herba stellariae mediae Stellaria parviumbellata, Xingan Herba stellariae mediae Stellaria cherleriae, sub-umbrella flower Herba stellariae mediae Stellariasubumbellata, Piper pulicatulum C. D C. Herba stellariae mediae Stellaria petraea, lay down sleeping Herba stellariae mediae Stellaria decumbens, to lay down sleeping Herba stellariae mediae (former mutation), leaf bud Herba stellariae mediae Stellaria crassifolia Ehrh., leaf bud Herba stellariae mediae (former mutation), Radix Stellariae Stellariadichotoma var.lanceolata, circle calyx Herba stellariae mediae Stellaria strongylosepala, the raw Herba stellariae mediae Stellariapalustris in natural pond, China Herba stellariae mediae Stellaria chinensis, China standing grain leaf Herba stellariae mediae Stellaria graminea var.chinensis, wrinkle leaf Herba stellariae mediae Stellaria monosperma var.japonica, cone flower Herba stellariae mediae Stellariamonosperma var.paniculata, Zhunger Basin Herba stellariae mediae Stellaria soongorica.
Herba stellariae mediae polysaccharide composition of the present invention, containing 1% ~ 95% Herba stellariae mediae Polysaccharide A, 5% ~ 90% Herba stellariae mediae Polysaccharide B, 11% ~ 54% Herba stellariae mediae polysaccharide C and D, 1% ~ 30% Herba stellariae mediae polysaccharide E; Employing High Performance Gel Permeation Chromatography detects, and result shows, Herba stellariae mediae Polysaccharide A molecular weight distribution, between 15000 ~ 30000Da, mainly concentrates on about 20000; Herba stellariae mediae Polysaccharide B, C, D molecular weight distribution, between 6000 ~ 20000Da, mainly concentrate on 13000,12000 and about 10000; Herba stellariae mediae polysaccharide E weight average molecular weight is distributed in 5000 ~ 10000Da, and wherein 5 kinds of polysaccharide molecular weight profile exponents are all less than 2, see accompanying drawing 2.
Show through gas chromatography analysis (see accompanying drawing 3) result, Herba stellariae mediae Polysaccharide A, B, C, D, E is by rhamnose, arabinose, xylose, mannose, glucose and galactose 6 kinds of monosaccharide residue compositions, ratio is respectively (1 ~ 5: 1 ~ 9: 1: 8 ~ 17: 3 ~ 68: 11 ~ 95), (1 ~ 10: 4 ~ 6: 1: 17 ~ 96: 5 ~ 13: 24 ~ 64), (6 ~ 33: 7 ~ 25: 1 ~ 12: 1: 11 ~ 45: 20 ~ 73), (1 ~ 5: 1: 0 ~ 0.5: 1 ~ 9: 2 ~ 15: 1 ~ 66), (1 ~ 14: 25 ~ 64: 0 ~ 1: 1 ~ 18: 1: 26 ~ 40).SDS-PAGE method is analyzed in known said composition without glycopeptide.
Another technical problem to be solved by this invention is the openly application of above-mentioned Herba stellariae mediae polysaccharide composition in preparation antiviral drugs.Virus of the present invention comprises hepatitis B virus, HIV (human immunodeficiency virus), herpesvirus, influenza virus, enterovirus and adenovirus, and wherein herpesvirus comprises HSV-1, HSV-2 and varicella zoster virus.
The present composition is prepared antiviral drugs and is used with the form of conventional galenic preparations, and described compositions can meet different administering modes with pharmaceutically acceptable form, as with solid, solution.The form such as gel, liposome gives.Containing compositions of the present invention oneself can be the form being applicable to orally using, as tablet, pill, granule, capsule, emulsion etc., according to known method any in pharmaceutical formulation production field can prepare orally use needed for preparation, for clinical antiviral drugs.
Auxiliary substance, stabilizing agent, wetting agent and other conventional additive can be contained, as lactose, sucrose, corn starch, citric acid, tartaric acid, magnesium stearate, Gypsum Fibrosum powder, Pulvis Talci, gelatin, mannitol, ethylene glycol, olive oil, Oleum Arachidis hypogaeae semen, ascorbic acid etc. in above-mentioned preparation.
Above-mentioned preparation can be made according to the preparation technology of various preparation routine.
Advantage of the present invention is: 1, Herba stellariae mediae polysaccharide composition has the antiviral activity of wide spectrum, and said composition and antiviral activity thereof are reported first.Its great advantage of this medicine is natural product, and compared with existing antiviral agents, toxicity is little, has no adverse reaction, can life-time service without bounce-back, be expected to be developed as novel antiviral drug.2, compared with the Chinese herbal medicine directly used, Herba stellariae mediae polysaccharide composition is as the material of concrete chemical constituent directly as antiviral active ingredient, and consumption is few, and reduce toxic and side effects to the injury of body, clinical application is simple; 3, Herba stellariae mediae polysaccharide composition combines relevant preparation technique, and Development volue is high, convenient for production, and technique is simple, has broad application prospects.
Accompanying drawing explanation
Fig. 1 Herba stellariae mediae Polyose extraction, separation and purification flow chart
Fig. 2 Herba stellariae mediae polysaccharide molecule quantitative determination HGPC schemes
Wherein A, B, C, D, E are respectively the HGPC collection of illustrative plates of Herba stellariae mediae Polysaccharide A, B, C, D, E
HGPC chromatographic condition: gel chromatographic columns is TSK-GEL3000 SW series, mobile phase is 0.71% metabisulfite solution (including 0.02% sodium azide), column temperature: 35 DEG C, differential refraction detector (detector temperature 35 DEG C), flow velocity 0.5mlmin
-1, get need testing solution and each 20 μ l injection liquid chromatographies of reference substance solution.
Fig. 3 Herba stellariae mediae polysaccharide monosaccharide composition GC analysis chart
Wherein A, B, C, D, E are respectively Herba stellariae mediae Polysaccharide A, B, C, D, E Monosaccharide analysis GC schemes
Chromatographic column: HP-5 capillary chromatographic column (30mm × 0.32nm × 0.25 μm); Temperature programming: 100 DEG C (5 DEG C/min) → 190 DEG C (4 DEG C/min) → 240 DEG C (5min); Carrier gas: high pure nitrogen; Injector temperature: 200 DEG C; Injection port adopts shunt mode, split ratio: 150: 1; Detector: FID; Vaporizer temperature: 260 DEG C; Nitrogen volume flow: 30mL/min; Hydrogen volume flow: 40mL/min; Volume of air flow: 400mL/min; Sample size: 1 μ L.
Fig. 4 Herba stellariae mediae polysaccharide composition is to the suppression ratio of Hepatitis B Surface (HBsAg)
Fig. 5 Herba stellariae mediae polysaccharide composition is to the suppression ratio of HBeAg (HBeAg)
Fig. 6 Herba stellariae mediae polysaccharide composition is to the suppression ratio of HBV DNA
Detailed description of the invention
Below in conjunction with example, the present invention is described in detail:
The extraction of common vegetable polysaccharides usually adopts water, salt, acid, alkali and enzyme to extract, the extracting solution obtained is reclaimed by the alcohol settling of high concentration, this process part that can be removed impurity, then obtain the higher refined polysaccharide of purity through decolouring, deproteinization, different materials column chromatography for separation.The present invention adopts fresh Herba stellariae mediae to squeeze the juice or warm water lixiviate etc. obtains Aqueous extracts, combines classical separation of polysaccharides method finally obtain Herba stellariae mediae refined polysaccharide according to certain processing mode, and it mixes according to a specific ratio and obtains Herba stellariae mediae polysaccharide composition.
Embodiment 1 Herba stellariae mediae polysaccharide composition effect on hepatitics B virus in vitro is studied
This experiment adopts classical effect on hepatitics B virus in vitro medicaments sifting model HepG2.2.15 cell model, Herba stellariae mediae polysaccharide composition administration HepG2.2.15 cell a couple of days of variable concentrations, get its cell conditioned medium liquid, measure its hepatitis B surface antigen (HBsAg) respectively, e antigen (HBeAg) and HBV DNA secrete situation, judge that whether medicine is effective, adopt mtt assay to measure the cytotoxicity of different Herba stellariae mediae polysaccharide composition in experimentation simultaneously.Comprehensive above experimental design, Herba stellariae mediae polysaccharide composition is carried out to the pharmacodynamic study of effect on hepatitics B virus in vitro, and to apply the lamivudine (3TC) that is confirmed clinically as positive control medicine, evaluate the anti-hepatitis virus effect of Herba stellariae mediae compositions with this.
Cytotoxicity when HepG2.2.15 cell proliferation to a certain extent and antigen secretion stable after, carry out cell counting, then prepare 5 × 10
4cell suspension cultivate in 96 well culture plates, every hole 100 μ L, after cultivating 24h, absorb cell conditioned medium, the Herba stellariae mediae polysaccharide composition adding variable concentrations carries out cultivation 6d or 9d, within every 3 days, change a pastille culture medium, collecting cell supernatant is standby in-70 DEG C of refrigerators to be deposited, for the detection of antigen and HBVDNA.Dye with MTT after collecting supernatant, every hole adds the MTT serum-free medium 100 μ L of 1mg/mL, hatches 4 hours, abandons supernatant, add 100 μ L in incubator, vibration 10min, by colorimetric determination OD value under 550nm wavelength, and experiment repetition 3 times.
Herba stellariae mediae polysaccharide composition adopts ELISA method to measure the secretion situation of HBsAg and HBeAg in cell conditioned medium liquid on the impact that HBsAg and HBeAg secretes, get the HepG2.2.15 cell conditioned medium of positive drug 3TC and the administration of Herba stellariae mediae polysaccharide composition variable concentrations, operate according to test kit operating instruction, measure the OD value of each sample cell conditioned medium, calculate its suppression ratio to HBsAg and HBeAg.
Herba stellariae mediae polysaccharide composition is further illustrate the effect that Herba stellariae mediae polysaccharide composition has certain anti-hepatitis virus to the inhibitory action of HBV DNA, adopts FQ-PCR method to measure the level of HepG2.2.15 emiocytosis HBV DNA.Detection kit is purchased from Xiamen Amplly Bio-Tech Inc..The all operations such as extraction, amplification of HBV DNA is all undertaken by test kit description.
Interpretation
MTT cytotoxicity analysis is for the analysis of Herba stellariae mediae polysaccharide composition anti-hepatitis virus, experiment adopts multiple concentration to analyze, the Herba stellariae mediae polysaccharide composition getting 3,10,30,60,100,200,400 μ g/mL acts on HepG2.2.15 cell, every 3 days collecting cell supernatants also renew fresh pastille culture medium, collect all cells supernatant in 6d or 9d, adopt mtt assay to analyze sample variable concentrations to Cytotoxic impact.The result display of MTT, when the concentration of Herba stellariae mediae polysaccharide composition is less than or equal to 200 μ g/mL, cell survival rate > 95%.3TC positive control is the anti-hepatitis virus preferably concentration of being screened by many experiments, and data analysis only adopts 100 μ g/mL 3TC and the relative analysis of Herba stellariae mediae polysaccharide composition.The Herba stellariae mediae polysaccharide composition of variable concentrations and the cytotoxic assay of 3TC the results are shown in Table 1.
Herba stellariae mediae polysaccharide composition is to the inhibitory action of HepG2.2.15 emiocytosis HBsAg and HBeAg
The Herba stellariae mediae polysaccharide composition of variable concentrations to the inhibitory action of HepG2.2.15 emiocytosis HBsAg and HBeAg in table 2 and table 3.Along with the increase of concentration, Herba stellariae mediae polysaccharide composition increases the suppression ratio of HBsAg and HBeAg, and when concentration is 200 μ g/mL, within the 6th day, it is respectively 24.28% and 28.94% to the suppression ratio of HBsAg and HBeAg; And the 9th day, suppression ratio reaches 25.56% and 30.25% (see accompanying drawing 4 and Fig. 5) respectively, analyzes thus, and Herba stellariae mediae compositions has inhibitory action to HBsAg and HBeAg, and along with the increase of concentration, suppression ratio increases; Along with the increase of time under same concentration, suppression ratio also increases.6d or 9d to the suppression of two kinds of antigens under comparable sodium with lamivudine there was no significant difference (P > 0.05).
Table 1 Herba stellariae mediae polysaccharide composition is to HepG2.2.15 cytotoxicity
Table 2 Herba stellariae mediae polysaccharide composition is to the inhibitory action of HepG2.2.15 emiocytosis HBsAg
Note: compared with normal group, * p < 0.05, * * p < 0.01
Table 3 Herba stellariae mediae polysaccharide composition is to the inhibitory action of HepG2.2.15 emiocytosis HBeAg
Note: compared with normal group, * p < 0.05, * * p < 0.01
Herba stellariae mediae polysaccharide composition is further illustrate the effect that Herba stellariae mediae polysaccharide composition has certain anti-hepatitis virus to the inhibitory action of HBV DNA, this experiment adopts FQ-PCR method to measure the level of HepG2.2.15 emiocytosis HBV DNA, result shows, 100, 200 μ g/mL Herba stellariae mediae polysaccharide compositions are to HepG2.2.15 cytosis 6d, all can the secretion of suppression HBV DNA in various degree after 9d, significant difference (p < 0.01) is had with the measurer of HBVDNA contained by normal group, and with positive drug lamivudine there was no significant difference in the corresponding time.The content of HBVDNA in cell conditioned medium liquid after variable concentrations Herba stellariae mediae polysaccharide composition effect HepG2.2.15 cell, as shown in Figure 6.
Embodiment 2 Herba stellariae mediae polysaccharide composition In Vitro Anti herpesvirus is studied
5 kinds of Herba stellariae mediae polysaccharide respectively with 1%, 40%, 20%, 17%, 22% content ratio mix homogeneously, obtain Herba stellariae mediae polysaccharide composition, adopt the cell model HepG2 cell model in embodiment 1, different modes inoculation herpesvirus HSV-1 HS-1 and HSV-2 333 Strain, investigates its external herpesvirus resisting activity.With Vero cell for model, directly be added to after the medicinal liquid of the Herba stellariae mediae polysaccharide composition (1,3,10,30,60,100,200 μ g/mL) of variable concentrations and the mixing of viral dilution liquid (100TCID) equal-volume on Vero cell monolayer, be placed in 37 DEG C, 5%CO2 cell culture incubator cultivates 48h, observation of cell pathological changes effect (CPE) under light microscopic.(shown from analysis of experimental data, the Vero cell of 60 μ g/mL and following concentration experiment group and normal group are without significant difference, when concentration is higher than 100 μ g/mL, experimental group cell has the significantly cellular morphologies such as change circle reduces, death comes off to change, and illustrates to there is cytotoxicity.
Cultivation of Vero is foretold at 24 orifice plates, discard growth-promoting media after covering with into monolayer, add in cell after the Herba stellariae mediae polysaccharide composition medicinal liquid of variable concentrations and each 50 μ l of viral dilution liquid being mixed respectively, each concentration establishes 3 multiple holes, if virus control group and normal cell controls group, 37 DEG C, 5%CO
2cultivate 2h, abandon supernatant, add containing 0.5% carboxymethyl cellulose cell maintenance medium (covering liquid) 1mL, after observing 72h, 10% formaldehyde fixes 30min, 1% violet staining 30min, clear water rinsing, dry, the method plaque with reference to Nitta counts, and calculates Plaque reduction ratio.Plaque reduction ratio (%)=(virus control group plaque number-drug treating group plaque number)/virus control group plaque number × 100%.
Test data shows, said composition has two-strain strain significantly directly to be killed, block infection, Inhibit proliferaton effect, and its drug effect compares there was no significant difference with positive drug ACV.Along with the increase of Herba stellariae mediae polysaccharide composition dosage, HSV-1, HSV-2 Plaque reduction ratio also increases thereupon, and is directly proportional to the increase of its dosage.Experimental data slightly.
Table 4 Herba stellariae mediae polysaccharide composition suppresses the pathogenic change effect of HSV-1 and HSV-2
Table 5 Herba stellariae mediae polysaccharide composition suppress the average plaque number of HSV-1 and HSV-2 and Plaque reduction ratio (
n=3)
Note: compare with virus control group, * p < 0.05, * * p < 0.01
The research of embodiment 3 Herba stellariae mediae polysaccharide composition In Vitro Anti Coxsackie virus
5 kinds of Herba stellariae mediae polysaccharide are respectively with 43%, 5%, 11%, 11%, the content ratio mix homogeneously of 30%, investigate Herba stellariae mediae polysaccharide composition to the direct deactivation of Coxsackie virus (CVB3), on 24 orifice plates, add the myocardial cell 1mL/ hole of 0.5 × 104/mL concentration, cultivate 24h, except the every hole of normal group adds the CVB31mL of 100TCID, after hatching 1h, abandoning supernatant, PBS rinses 3 times, add the Herba stellariae mediae polysaccharide composition (3 of variable concentrations, 10, 30, 60, 100 μ g/mL), positive controls adds virazole 1mmol/mL, often organize 4 holes, add cell maintenance medium, 5%CO2 incubator cultivates 72h, day by day observation of cell pathological changes effect (CPE), and adopt mtt assay to detect cell survival rate.
Experimental result shows, after virus infected cell, the CPE caused by viral group is characterized as cell shrinkage, become justify, come off, cracked.Virazole groups of cells cell shrinkage, come off, arhythmicity fluctuates, and Herba stellariae mediae polysaccharide composition administration group is still in rhythmic pulsation, part cell shrinkage, is proportionate to the suppression ratio of CVB3 and its concentration.It can thus be appreciated that Herba stellariae mediae polysaccharide composition has direct deactivation to CVB3, and dose-effect relationship is in table 6.
Table 6 Herba stellariae mediae polysaccharide composition is to the inhibitory action of CVB3
Resisiting influenza virus effect in embodiment 4 Herba stellariae mediae polysaccharide composition body
5 kinds of Herba stellariae mediae polysaccharide respectively with 22%, 5%, 11%, 54%, 8% content ratio mix homogeneously, obtain Herba stellariae mediae polysaccharide composition.This polysaccharide composition has protective effect to the death of influenza induced mice, and 2-3 kunming mice in age in week 72 is got in experiment, and body weight 13-15g, male and female half and half, are divided into 6 groups at random, often organize 12.1st group be Normal group, the 2nd group be virus control group, 3-5 group is high, medium and low 3 dosage of Herba stellariae mediae polysaccharide composition, and the 6th group is ribavirin matched group.Each treated animal infects a few days ago gastric infusion, every day 1 time, until 5d after infecting, altogether administration 7d, 1st, the normal saline of 2 groups of gavage equivalent, 3-5 group is gavage Herba stellariae mediae polysaccharide composition 1.50 respectively, 1.20,0.80g/kg, the 6th group of gavage ribavirin (virazole) 0.20g/kg.After administration the 3rd day, only mice (except the 1st group) is inoculated under ether light anesthesia the influenza virus 0.1mL/ of 8 times of LD50, the 1st group with method inoculation normal saline 0.1mL.From infection, Continuous Observation 14d, record dead mouse number (mice dead in virus inoculation 24h is not counted in death toll), calculates mortality rate, Death prevention rate, mean survival time.Result normal group is without dead mouse, and all the other each group all has dead mouse, and model group dead mouse is maximum, with other 5 groups of comparing differences remarkable (p < 0.05); Herba stellariae mediae polysaccharide composition 3 dosage group mouse death rates are starkly lower than model group (p < 0.05); The mean survival time of model group mice is the shortest, and respectively organizing with other more all has significant difference (table 7).
Herba stellariae mediae polysaccharide composition infected by influenza induced mice virus bronchopneumonia can play preventive and therapeutic effect, and 2-3 Kunming mouse in age in week 72 is got in experiment, and body weight 13-15g, male and female half and half, are divided into 6 groups at random, often organize 12.1st group be normal group, the 2nd group be virus model group, 3-5 group is respectively Herba stellariae mediae polysaccharide composition 1.50,1.20,0.80gkg
-1, 3 dosage groups, the 6th group is ribavirin group.Except the 1st group, all the other are respectively organized mice and inoculate 8 times of LD under ether light anesthesia
50influenza virus, 0.1mL/ only, inoculates normal saline 0.1mL for the 1st group by the same method.After infection, 24h starts administration, the 1st, and 2 groups of gavages give to wait capacity normal saline, and 3-5 is gavage Herba stellariae mediae polysaccharide composition 1.50,1.20,0.80gkg respectively
-1, the 6th group of gavage ribavirin 0.20gkg
-1, successive administration 4d, water 8h is prohibited in fasting in the 5th day, mouse weights, cut off carotid artery blood-letting lethal, take out full lung, clean with normal saline, and sop up residual brine with clean filter paper, weigh, calculate Lung Exponent and lung index, and to Shu Feizuo histopathologic examination.The each administration group of result and normal group compare Lung Exponent, and there were significant differences (P < 0.01) with virus model group; Herba stellariae mediae polysaccharide composition each dosage group Lung Exponent and ribavirin group are without obviously poor (table 8).
The protective effect of table 7 Herba stellariae mediae polysaccharide composition influenza virus infected (
n=12)
Note: compare with model group, * p < 0.05, * * p < 0.01
The impact of table 8 Herba stellariae mediae polysaccharide composition influenza virus infected Lung Exponent (
n=12)
Note: compare with model group, * * p < 0.01
Influenza virus infected lung inner virus blood clotting drips and median infective dose (EID
50) impact as above method take out after mouse lung, the Mus lung suspension of preparation 10%.Often group gets suspension 0.1mL, measures its Hemagglutination titer, and all the other, with normal saline 10 times of serial dilutions 8 dilution factors, inoculate instar chicken embryo on the 10th, measures EID.。The EID of result normal group
50be 0, influenza infection mice EID
50maximum, the lung suspension EID of ribavirin and Herba stellariae mediae polysaccharide composition various dose group
50all low than influenza infection group, reduce more than at least 2 orders of magnitude, wherein Herba stellariae mediae polysaccharide composition low dose group and ribavirin group EID
50reduce at most, more than 3 orders of magnitude.Show that Herba stellariae mediae polysaccharide composition influenza virus infected has the effect (table 9) of good therapeutical effect and resisiting influenza virus infection.
Table 9 each group lung suspension EID50 (
n=12)
Note: inoculated into chick embryo number is 32 pieces
10% lung suspension Hemagglutination titer measurement result: the Hemagglutination titer of virus control group is 1: 640, and the Hemagglutination titer of Herba stellariae mediae polysaccharide composition 3 dosage groups is 1: 80, and blood clotting phenomenon does not appear in ribavirin group.Illustrate that the growth of Herba stellariae mediae polysaccharide composition infected by influenza in infecting mouse lung has inhibitory action.
Except the instantiation of the Herba stellariae mediae polysaccharide composition inside and outside test of pesticide effectiveness, in antiviral drugs preparation, mainly contain following instance:
Embodiment 5
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 950mg, 1200mg, 950mg, 900mg, 1000mg respectively, add appropriate adjuvant, carry out tabletting by the common process of galenic pharmacy tablet, make 500 tablets of tablets, every sheet contains 10mg active component, as anti-virus formulation or health product.Usage and dosage: oral, each 1,2 times on the one, children's is cut down according to the circumstance, 15 days courses for the treatment of.
Embodiment 6
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2100mg, 2400mg, 1800mg, 2000mg, 1700mg respectively, add appropriate amylodextrin and make granule by the common process of galenic pharmacy capsule, insert 1000 capsules, every containing 10mg active component.Usage and dosage: oral, each 1,2 times on the one, children's is cut down according to the circumstance, and within 15 days, is a course for the treatment of.
Embodiment 7
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2000mg, 2400mg, 1600mg, 1900mg, 2100mg respectively, add appropriate sucrose and dextrin, be prepared by the common process of galenic pharmacy granule, every bag heavy 0.5g, every bag containing Herba stellariae mediae polysaccharide composition 20mg.Usage and dosage: oral, each 1 bag, 1 time on the one, children's is cut down according to the circumstance, 15 days courses for the treatment of.
Embodiment 8
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2200mg, 2300mg, 1800mg, 1800mg, 2100mg respectively, add suitable amount of sucrose, thin up is to 1000mL, and every bottle of 10mL, 1mL are containing Herba stellariae mediae polysaccharide composition 10mg.Usage and dosage: oral, each 1 bottle, 2 times on the one, 15 days courses for the treatment of.
Embodiment 9
Pharmaceutical formulation Herba stellariae mediae polysaccharide composition is prepared into injection, to produce injection product 1000, get Herba stellariae mediae Polysaccharide A, B, C, D, E 2100mg, 2300mg, 1900mg, 2000mg, 1700mg respectively, add appropriate injection mannitol, undertaken by the common process of galenic pharmacy injection, often prop up containing Herba stellariae mediae polysaccharide composition 10mg.Usage and dosage: be dissolved in 100mL injection, 1 time on the one, children's is cut down according to the circumstance, 30 days courses for the treatment of.
Claims (4)
1. a Herba stellariae mediae polysaccharide composition, it is characterized in that said composition Herba stellariae mediae Polysaccharide A, B, C, D, E composition, each polysaccharide is made up of rhamnose, arabinose, xylose, mannose, glucose and galactose 6 kinds of monosaccharide residues respectively, wherein said Herba stellariae mediae Polysaccharide A weight average molecular weight is distributed in about 20000Da, profile exponent D is between 1 ~ 1.5, molecular weight determination High Performance Gel Permeation Chromatography and HGPC retention time are 16.163 minutes, and monosaccharide residue proportion of composing is 1 ~ 5: 1 ~ 9: 1: 8 ~ 17: 3 ~ 68: 11 ~ 95; Described Herba stellariae mediae Polysaccharide B weight average molecular weight is distributed in about 13000Da, and profile exponent D is between 1 ~ 2, and molecular weight determination HGPC retention time is 16.772 minutes, and monosaccharide residue proportion of composing is 1 ~ 10: 4 ~ 6: 1: 17 ~ 96: 5 ~ 13: 24 ~ 64; Described Herba stellariae mediae polysaccharide C molecular weight distribution is at about 12000Da, and profile exponent D is between 1 ~ 2, and molecular weight determination HGPC retention time is 16.848 minutes, and monosaccharide residue proportion of composing is 6 ~ 33: 7 ~ 25: 1 ~ 12: 1: 11 ~ 45: 20 ~ 73; Described Herba stellariae mediae polysaccharide D molecular weight distribution is at about 10000Da, and profile exponent D is between 1 ~ 2, and molecular weight determination HGPC retention time is 16.928 minutes, and monosaccharide residue proportion of composing is 1 ~ 5: 1: 0 ~ 0.5: 1 ~ 9: 2 ~ 15: 1 ~ 66; Herba stellariae mediae polysaccharide E weight average molecular weight is distributed in 5000 ~ 10000Da, and profile exponent D is between 1.5 ~ 2, and molecular weight determination HGPC retention time is 16.975 minutes, and monosaccharide residue proportion of composing is 1 ~ 14: 25 ~ 64: 0 ~ 1: 1 ~ 18: 1: 26 ~ 40; Described HGPC chromatographic condition is: gel chromatographic columns is TSK-GEL3000SW series, and mobile phase is 0.71% metabisulfite solution, includes 0.02% sodium azide, column temperature: 35 DEG C, differential refraction detector, detector temperature 35 DEG C, flow velocity 0.5mlmin
-1; 5 kinds of described polysaccharide molecular weight profile exponents are all less than 2; In Herba stellariae mediae polysaccharide composition, the proportion of composing of Herba stellariae mediae Polysaccharide A, B, C, D, E is 1% ~ 95% Herba stellariae mediae Polysaccharide A, 5% ~ 90% Herba stellariae mediae Polysaccharide B, 11% ~ 54% Herba stellariae mediae polysaccharide C and D, 1% ~ 30% Herba stellariae mediae polysaccharide E, and above component sum is no more than 100%.
2. a kind of Herba stellariae mediae polysaccharide composition according to claim 1, its purposes is to prepare hepatitis B virus, influenza virus, enterovirus, adenovirus anti-virus formulation and health product.
3. a kind of Herba stellariae mediae polysaccharide composition according to claim 2, is characterized in that utilizing the corresponding pharmaceutical dosage form of formulation art.
4. a kind of Herba stellariae mediae polysaccharide composition according to claim 3, is characterized in that concrete dosage form is injection, tablet, capsule, granule, oral liquid, micropill, spray, Emulsion.
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| CN101108200A (en) * | 2006-04-21 | 2008-01-23 | 朱耕新 | Composition of starwort sulphonic acid or vitriolic acid polyoses ester total phenolic glycoside and method of preparing the same and antiviral application |
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| CN101108200A (en) * | 2006-04-21 | 2008-01-23 | 朱耕新 | Composition of starwort sulphonic acid or vitriolic acid polyoses ester total phenolic glycoside and method of preparing the same and antiviral application |
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| Title |
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| 王统一,等.多糖免疫调节和抗肿瘤研究进展.《过程工程学报》.2006,第6卷(第4期),674-682. * |
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