CN102697800A - Chickweed polysaccharide composition and applications thereof in preparation of antiviral medicine - Google Patents
Chickweed polysaccharide composition and applications thereof in preparation of antiviral medicine Download PDFInfo
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Abstract
The invention relates to a chickweed polysaccharide composition and applications thereof in preparation of an antiviral medicine. The chickweed polysaccharide composition disclosed by the invention is prepared by mixing five kinds of chickweed polysaccharides (A, B, C, D and E) according to a certain proportion and is characterized in that the composition is of brown floccule which is easily dissolved in water and is not dissolved in ethanol, acetone, diethyl ether, n-butanol and isopropanol with high concentration; the polysaccharide composition contains five kinds of chickweed polysaccharides with molecular weights ranging from 5000 to 30000 and is formed by rhamnose, arabinose, xylose, mannose, glucose and galactose according to certain molar ratio. After being mixed uniformly in a special proportion, the five kinds of the polysaccharides can be used for strengthening antiviral activities by utilizing mutual synergy. The invention further discloses a use of the chickweed polysaccharide composition in preparation of the antiviral medicine, which is used for treating the diseases such as hepatitis B virus, HIV (human immunodeficiency virus), herpes virus, influenza virus, enterovirus and adenovirus and the like and can be prepared into various pharmaceutic preparations. The composition has a good therapeutic effect, no toxicity and wide prospect in development of new antiviral medicines.
Description
Technical field
The present invention relates to the Herba stellariae mediae polysaccharide composition in the application of preparation in the antiviral drugs, particularly a kind of by after the active ingredient of Chinese herbs separation and purification again by the Herba stellariae mediae polysaccharide composition and the application in the preparation antiviral drugs thereof of certain proportion mix homogeneously; The invention still further relates to the preparation and the health product that contain the Herba stellariae mediae polysaccharide composition, belong to field of medicaments.
Background technology
Virus is minimum pathogenic microorganism; Be one type and do not have a cellularity, the microorganism of vital signs such as have heredity, duplicate, its core is ribonucleic acid or DNA (DNA); The propagation of virus is the widest, is one of higher infectious disease of present sickness rate in the world.The common hepatitis that is infectious, herpes, encephalitis, viral respiratory tract infection, influenza and the herpetic keratitis etc. of disease that cause by virus.More clinically disease is relevant with viral infection; According to the foreign statistic document announcement; Nearly 3/4 infectious disease is caused by virus; Have 18 kinds to belong to Causative virus in 55 kinds of viruses having announced of international virus taxis association (ICTV) in 1984, different viral infection causes different illness, needs different antiviral treatments.
The approach of viral infection resisting is a lot, as direct inhibition or kill virus, viral interference absorption, stop virus penetrate cell, suppress viral organism synthetic, suppress virus and discharge or strengthen host anti-virus ability etc.Though the antiviral drugs of usefulness is more clinically at present, is nucleoside derivate mostly, its main action target spot is a viral dna polymerase; With the form and normal substrate competition of enzyme reaction substrate analog, directly the activity of reduction or blocking dna polymerase shows preferable antiviral activity; But it has the generation of being prone to drug resistance; The antiviral narrow range, toxic and side effects is big, and shortcoming such as cost an arm and a leg.In recent years, the control that many Chinese herbal medicine are used for viral disease causes people's attention, and the control that is used for some disease of viral infection like Cacumen Securinegae Suffruticosae, Rhizoma Polygoni Cuspidati, Flos Lonicerae, Radix Arnebiae (Radix Lithospermi), Rhizoma Osmundae, Radix Et Rhizoma Rhei etc. obtains significant curative effect.
Herba stellariae mediae (Stellaria media (L.) Villar.) is Caryophyllaceae Stellaria 1 year or 2 years these plants of SHENGCAO, has the 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.According to nearly 120 kinds of the document record whole world, be widely distributed in temperature to refrigerant latitudes, 63 kind of 15 mutation of China's product and 2 modification, the each province all produces it.Herba stellariae mediae is widely used as folk medicine.Herba stellariae mediae is rich in bioactive ingredients such as flavone, phenolic acid, polysaccharide, saponins, organic acid, alkaloid, and research at present shows that Herba stellariae mediae has the broad-spectrum antivirus action.
Polysaccharide (polysaccharide) is to be formed by the condensation of a plurality of monosaccharide molecule, dehydration, is the complicated and huge glucide of molecule mechanism.All carbohydrate and derivants thereof that meets the macromolecular compound notion all are 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.At present the Herba stellariae mediae STUDY ON POLYSACHAROSE mainly rests on the crude polysaccharides level, mainly is the research of the aspects such as extraction and biological activity of crude polysaccharides.Described Herba stellariae mediae polysaccharide of Chinese patent publication number CN1521193A and preparation of compositions method thereof; Point out that the Herba stellariae mediae polysaccharide composition is that 80～90% total flavones and 10～20% crude polysaccharides mix; Effect with certain AIDS virus resisting and herpesvirus; This is the just extraction of Herba stellariae mediae crude polysaccharides preparation only; Resulting polysaccharide contains other compositions such as protein, pigment, and the relevant report of not seeing homogeneity Herba stellariae mediae polysaccharide is not more seen the report that is mixed into the Herba stellariae mediae polysaccharide composition by a certain percentage and in the preparation antiviral drugs, is used by the Herba stellariae mediae polysaccharide.
Summary of the invention
The objective of the invention is to the active component of Herba stellariae mediae is studied, 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; Be through certain processing mode separation and purification by the fresh herb of Herba stellariae mediae Stellaria media (L.) Villar.; Again by the Herba stellariae mediae polysaccharide of 5 kinds of molecular weight homogeneous of separation and purification by a certain percentage scope mix, 5 kinds of Herba stellariae mediae polysaccharide adopt modern analytical techniques, analyze its molecular weight, molecular weight distribution, mol ratio and monosaccharide thereof and form; Do not see relevant report at present, its extraction and separation and purification flow process are seen accompanying drawing 1.The used experimental technique of the present invention is quick, accurate, and said composition does not have bibliographical information to have the effect of broad-spectrum antiviral.
The invention described chickweed polysaccharide composition chickweed polysaccharide extracted from the following plants, these plants include plants or chickweed chickweed species, under the species mainly Alishan chickweed Stellaria? Arisanensis? Var.Leptophylla ? Hayata, dwarf trap chickweed Stellaria? depressa, Solanum Chickweed Stellaria? patens, amplexicaul Qing Kou grass Stellaria? vestita? var.amplexicaulis, southern Tibet Chickweed Stellaria? zangnanensis, fork manifold chickweed Stellaria? dichotoma, fork discrimination chickweed (original variety), long flap Chickweed Stellaria? dichotoma, long flap chickweed (original variety), a long crack Chickweed Stellaria? martjanovii, hairy Qing Kou grass Stellaria? pilosa, leaves Chickweed Stellaria? longifolia, Long Leaf fan strand (original variety), evergreen Wo Ye chickweed Stellaria? graminea? var.viridescens, Cona Chickweed Stellaria? decumbens? var.arenarioides, large leaves Chickweed Stellaria? delavayi, list chickweed, chickweed lying cushion Yan Stellaria? decumbens? var.pulvinata, tundra chickweed Stellaria? irrigua, only son Chickweed Stellaria? monosperma, only son chickweed (original variety), short flap Chickweed Stellaria? brachypetala, blunt calyx Chickweed Stellaria? amblyosepala, spend a Fan Strand Stellaria? nipponica, spend Yan lying chickweed Stellaria? decumbens? var.polyantha, Emei chickweed Stellaria? omeiensis, two columns chickweed Stellaria? bistyla, discolor chickweed Stellaria? discolor, chickweed Stellaria? media, chickweed (original variety), imbricate Chickweed Stellaria? imbricata, Buxus alpine grass Stellaria? uliginosa? var.alpina, Wo Ye Chickweed Stellaria? graminea, Wo Ye chickweed (original variety), Helan Mountain Chickweed Stellaria? alaschanica, Hubei Chickweed Stellaria? henryi, Intestinal Chickweed Stellaria? neglecta, Kuala Chickweed Stellaria? gyirongensis, Gyantse Chickweed Stellaria? gyangtseensis, long eyelashes leaves chickweed Stellaria? gyangtseensis, Kangding Chickweed Stellaria? souliei, Lin chickweed Stellaria? bungeana ? var.stubendorfii, willow Chickweed Stellaria? salicifolia, egg leaves chickweed Stellaria? ovatifolia, hair Wo Ye chickweed Stellaria? graminea? var.pilosula, cranberry-like Chickweed Stellaria? oxycoccoides, Millington Chickweed Stellaria? mainlingensis , dense flowers Chickweed Stellaria? congestiflora, densely pubescent Chickweed Stellaria? yunnanensis? f.villosa, cotton handle Chickweed Stellaria? lanipes, woolly Chickweed Stellaria? lanata, recurved Chickweed Stellaria? infracta, Nepal Chickweed Stellaria ? nepalensis, Pamir Chickweed Stellaria? winkleri, Thousand Needles million lines grass Stellaria? yunnanensis, Thousand Needles million lines grass (original variety), Qing Gu grass Stellaria? vestita, Qing Gu grass (original variety), Buxus grass Stellaria? uliginosa, Buxus Grass (original variety), parachutes Chickweed Stellaria? umbellata, sandy chickweed Stellaria? arenaria, wetlands Chickweed Stellaria? uda, chickweed raw stone, stone bamboo chickweed Stellaria? dianthifolia, tassels flap Chickweed Stellaria ? radians, canopy grass, stockwork chickweed Stellaria? reticulivena, Wushan Chickweed Stellaria? wushanensis, no flap Chickweed Stellaria? apetala, no bud Chickweed Stellaria? ebracteatav, Tibet Chickweed Stellaria? tibetica, shank, chickweed Stellaria ? petiolaris, Egeria Chickweed Stellaria? filicaulis, linear leaf bud Chickweed Stellaria? crassifolia? Ehrh.var.linearis, line leaves chickweed Stellaria? dichotoma? var.linearis, glandular Chickweed Stellaria? nemorum, small chickweed Stellaria ? pusilla, flowers chickweed Stellaria? media? var.micrantha, small parachutes Chickweed Stellaria? parviumbellata, Larix Chickweed Stellaria? cherleriae, Asia parachutes Chickweed Stellaria? subumbellata, rocks Chickweed Stellaria? petraea, Yan lying chickweed Stellaria? decumbens, Yan lying chickweed (original variety), leaf bud Chickweed Stellaria? crassifolia? Ehrh., leaf bud chickweed (original variety), silver Bupleurum Stellaria? dichotoma? var.lanceolata, round calyx chickweed Stellaria? strongylosepala, marsh Chickweed Stellaria? palustris, China Chickweed Stellaria? chinensis, China Wo Ye Chickweed Stellaria? graminea? var.chinensis, wrinkled leaves Chickweed Stellaria? monosperma? var.japonica, cone flowers Chickweed Stellaria? monosperma? var.paniculata, Junggar Chickweed Stellaria? soongorica.
Herba stellariae mediae polysaccharide composition of the present invention contains 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; Adopt the efficient gel permeation chromatography to detect, the result shows that Herba stellariae mediae Polysaccharide A molecular weight distribution mainly concentrates on about 20000 between 15000～30000Da; Herba stellariae mediae Polysaccharide B, C, D molecular weight distribution mainly concentrate on about 13000,12000 and 10000 between 6000～20000Da; 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 seen accompanying drawing 2 all less than 2.
The result shows through gc analysis (seeing accompanying drawing 3); 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) Herba stellariae mediae Polysaccharide A, B, C, D, E are made up of rhamnose, arabinose, xylose, mannose, glucose and 6 kinds of monosaccharide residues of galactose, and ratio is respectively (1～5: 1～9:.The analysis of SDS-PAGE method can be known does not have glycopeptide in the said composition.
Another technical problem to be solved by this invention is to disclose the application of above-mentioned Herba stellariae mediae polysaccharide composition in the preparation antiviral drugs.Virus of the present invention comprises hepatitis B virus, HIV, herpesvirus, influenza virus, enterovirus and adenovirus, and wherein herpesvirus comprises HSV-1, HSV-2 and varicella zoster virus.
The present composition prepares antiviral drugs to be used with the form of conventional pharmaceutical formulation, and said compositions can satisfy different administering modes with pharmaceutically acceptable form, as with solid, solution.Form such as gel, liposome gives.What contain compositions of the present invention oneself can be the form that is fit to orally use; Like tablet, pill, granule, capsule, emulsion etc.; Can prepare the required preparation that orally uses according to any known method in the pharmaceutical formulation production field, be used for clinical antiviral drugs.
Can contain auxiliary substance, stabilizing agent, wetting agent and other additive commonly used in the above-mentioned preparation, like 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.
Above-mentioned preparation can be processed according to the conventional preparation technology of various preparations.
Advantage of the present invention is: 1, the Herba stellariae mediae polysaccharide composition has the broad-spectrum antiviral activity, and said composition and antiviral activity thereof are reported first.Its great advantage of this medicine is a natural product, compares with existing antiviral agents, and toxicity is little, have no adverse reaction, but life-time service does not have bounce-back, is expected to be developed as novel antiviral drug.2, compare with the Chinese herbal medicine of direct use, the material of the concrete chemical constituent of Herba stellariae mediae polysaccharide composition conduct is directly as antiviral active ingredient, and consumption is few, reduces the injury of toxic and side effects to body, and clinical application is simple; 3, the Herba stellariae mediae polysaccharide composition combines relevant preparation technique, and exploitation is worth high, and convenient for production, technology is simple, has broad application prospects.
Description of drawings
Fig. 1 Herba stellariae mediae polysaccharide extracts, the separation and purification flow chart
Fig. 2 Herba stellariae mediae polysaccharide molecule quantitative determination HGPC figure
Wherein A, B, C, D, E are respectively the HGPC collection of illustrative plates of Herba stellariae mediae Polysaccharide A, B, C, D, E
The HGPC chromatographic condition: gel chromatographic columns is a TSK-GEL3000SW series, and mobile phase is 0.71% metabisulfite solution (including 0.02% sodium azide), column temperature: 35 ℃, and differential refraction detector (35 ℃ of detector temperatures), flow velocity 0.5mlmin
-1, get each 20 μ l of need testing solution and reference substance solution and inject chromatograph of liquid.
Fig. 3 Herba stellariae mediae polysaccharide monosaccharide is formed the GC analysis chart
Wherein A, B, C, D, E are respectively Herba stellariae mediae Polysaccharide A, B, C, D, E monosaccharide analysis GC figure
Chromatographic column: HP-5 capillary chromatographic column (30mm * 0.32nm * 0.25 μ m); Temperature programming: 100 ℃ (5 ℃/min) → 190 ℃ (4 ℃/min) → 240 ℃ (5min); Carrier gas: high pure nitrogen; Injector temperature: 200 ℃; Injection port adopts shunt mode, split ratio: 150: 1; Detector: FID; Vaporizer temperature: 260 ℃; 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 on hepatitis B virus surface (HBsAg)
Fig. 5 Herba stellariae mediae polysaccharide composition is to the suppression ratio of hepatitis B virus e antigen (HBeAg)
Fig. 6 Herba stellariae mediae polysaccharide composition is to the suppression ratio of HBV DNA
The specific embodiment
Below in conjunction with instance the present invention is elaborated:
The extraction of common vegetable polysaccharides usually adopts water, salt, acid, alkali and enzyme to extract; The extracting solution that obtains reclaims through the ethanol precipitation of high concentration; This process part that can be removed impurity obtains the higher refining polysaccharide of purity through decolouring, deproteinization, different materials column chromatography for separation again.The present invention adopts fresh Herba stellariae mediae to squeeze the juice or warm water lixiviate etc. obtains the water extract, combines classical separation of polysaccharides method finally to obtain the refining polysaccharide of Herba stellariae mediae according to certain processing mode, and it obtains the Herba stellariae mediae polysaccharide composition by specific mixed.
The external anti-hepatitis virus research of embodiment 1 Herba stellariae mediae polysaccharide composition
Classical external anti-hepatic-B virus medicine screening model HepG2.2.15 cell model is adopted in this experiment; 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 secretion situation; Judge whether medicine is effective, adopt mtt assay to measure the cytotoxicity of different Herba stellariae mediae polysaccharide compositions simultaneously in the experimentation.Comprehensive above experimental design carries out the pharmacodynamic study of external anti-hepatitis virus to the Herba stellariae mediae polysaccharide composition, and the lamivudine (3TC) that is confirmed with application is clinically estimated the anti-hepatitis virus effect of Herba stellariae mediae compositions as the positive control medicine with this.
Cytotoxicity when the HepG2.2.15 cell proliferation to a certain degree and antigen secretion stable after, carry out cell counting, prepare 5 * 10 then
4Cell suspension cultivate in 96 well culture plates; Every hole 100 μ L behind the cultivation 24h, absorb cell conditioned medium; The Herba stellariae mediae polysaccharide composition that adds variable concentrations is cultivated 6d or 9d; Changed a pastille culture medium in per 3 days, the collecting cell supernatant is deposited in-70 ℃ of refrigerators fully, is used 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, in incubator, hatches 4 hours, abandons supernatant, adds 100 μ L, vibration 10min, and with colorimetric determination OD value under the 550nm wavelength, experiment repetition 3 times.
The Herba stellariae mediae polysaccharide composition is to the secretion situation of HBsAg and HBeAg in HBsAg and the excretory influence employing of the HBeAg ELISA method mensuration cell conditioned medium liquid; 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 the test kit operating instruction; Measure the OD value of each sample cell conditioned medium, calculate its suppression ratio HBsAg and HBeAg.
The Herba stellariae mediae polysaccharide composition is to further specify the Herba stellariae mediae polysaccharide composition to have certain function of resisting hepatitis B virus to the inhibitory action of HBV DNA, adopts the FQ-PCR method to measure the level of HepG2.2.15 emiocytosis HBV DNA.Detection kit is available from Xiamen Amplly Bio-Tech Inc..All operations such as the extraction of HBV DNA, amplification are all undertaken by the test kit description.
Interpretation
The MTT cytotoxicity analysis is for the analysis of Herba stellariae mediae polysaccharide composition anti-hepatitis virus; Experiment adopts a plurality of concentration to analyze; The Herba stellariae mediae polysaccharide composition of getting 3,10,30,60,100,200,400 μ g/mL acts on the HepG2.2.15 cell; Per 3 days collecting cell supernatants also renew bright pastille culture medium, collect all cells supernatant in 6d or 9d, adopt the toxic influence of mtt assay analytic sample variable concentrations pair cell.The result of MTT shows, the concentration of Herba stellariae mediae polysaccharide composition is during smaller or equal to 200 μ g/mL, cell survival rate＞95%.The 3TC positive control is that data analysis only adopts 100 μ g/mL 3TC and the relative analysis of Herba stellariae mediae polysaccharide composition through the preferable concentration of anti-hepatitis virus of experiment screening repeatedly.The Herba stellariae mediae polysaccharide composition of variable concentrations and the CTA result of 3TC see table 1.
The Herba stellariae mediae polysaccharide composition is seen table 2 and table 3 to the Herba stellariae mediae polysaccharide composition of the inhibitory action variable concentrations of HepG2.2.15 emiocytosis HBsAg and HBeAg to the inhibitory action of HepG2.2.15 emiocytosis HBsAg and HBeAg.Along with the increase of concentration, the Herba stellariae mediae polysaccharide composition increases the suppression ratio of HBsAg and HBeAg, and when concentration was 200 μ g/mL, its suppression ratio to HBsAg and HBeAg was respectively 24.28% and 28.94% in the 6th day; And the 9th day, suppression ratio has reached 25.56% and 30.25% (seeing accompanying drawing 4 and Fig. 5) respectively, analyzes thus, and the Herba stellariae mediae compositions has inhibitory action to HBsAg and HBeAg, and along with the increase of concentration, suppression ratio increase; Along with the increase of time, suppression ratio also increases under the same concentration.6d or 9d are to two kinds of antigenic being suppressed at isoconcentration down and lamivudine there was no significant difference (P＞0.05).
Table 1 Herba stellariae mediae polysaccharide composition is to the HepG2.2.15 cytotoxicity
Table 2 Herba stellariae mediae polysaccharide composition is to the inhibitory action of HepG2.2.15 emiocytosis HBsAg
Annotate: with compared with normal,
*P＜0.05,
*P＜0.01
Table 3 Herba stellariae mediae polysaccharide composition is to the inhibitory action of HepG2.2.15 emiocytosis HBeAg
Annotate: with compared with normal,
*P＜0.05,
*P＜0.01
The Herba stellariae mediae polysaccharide composition is to further specify the Herba stellariae mediae polysaccharide composition to have certain function of resisting hepatitis B virus to the inhibitory action of HBV DNA; This experiment adopts the FQ-PCR method to measure the level of HepG2.2.15 emiocytosis HBV DNA; The result shows; 100, the secretion of the inhibition HBV DNA that 200 μ g/mL Herba stellariae mediae polysaccharide compositions all can be in various degree after to HepG2.2.15 cytosis 6d, 9d; With the measurer of the contained HBVDNA of normal group significant difference (p＜0.01) is arranged, and with the corresponding time in positive drug lamivudine there was no significant difference.The content of HBVDNA in the cell conditioned medium liquid behind the variable concentrations Herba stellariae mediae polysaccharide composition effect HepG2.2.15 cell is shown in accompanying drawing 6.
The external anti-herpesvirus research of embodiment 2 Herba stellariae mediae polysaccharide compositions
5 kinds of Herba stellariae mediae polysaccharide are respectively with 1%, 40%, 20%, 17%, 22% content ratio mix homogeneously; Obtain the Herba stellariae mediae polysaccharide composition; Adopt the cell model HepG2 cell model among the embodiment 1; Different modes inoculation herpesvirus HSV-1HS-1 and HSV-2333 Strain are investigated its external herpesvirus resisting activity.With the Vero cell is model; Directly be added on the monolayer Vero cell 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 viral dilution liquid (100TCID) equal-volume mixed; Place 37 ℃, 5%CO2 cell culture incubator to cultivate 48h, observation of cell pathological changes effect (CPE) under the light microscopic.(table can be known by analysis of experimental data; The Vero cell and the normal group of 60 μ g/mL and following concentration experimental group do not have significant difference; When concentration was higher than 100 μ g/mL, the experimental group cell had cellular morphologies such as tangible change circle dwindles, death comes off to change, and explains to have cytotoxicity.
Foretell Cultivation of Vero at 24 orifice plates; Discard growth-promoting media after covering with into monolayer, Herba stellariae mediae polysaccharide composition medicinal liquid and each the 50 μ l of viral dilution liquid with variable concentrations mix in the back adding cell respectively, and each concentration is established 3 multiple holes; If virus control group and normal cell matched group, 37 ℃, 5%CO
2Cultivate 2h, abandon supernatant, add and contain 0.5% carboxymethyl cellulose cell maintenance medium (covering liquid) 1mL; After observing 72h, 10% formaldehyde fixed 30min, 1% violet staining 30min; The clear water rinsing, dry, the method plaque counting with reference to Nitta calculates the plaque suppression ratio.Plaque suppression ratio (%)=(virus control group plaque number-drug treating group plaque number)/virus control group plaque number * 100%.
Test data shows that said composition has significantly the infection directly killed, block, suppresses proliferation function two kinds of Strain, and its drug effect and positive drug ACV comparison there was no significant difference.Along with the increase of Herba stellariae mediae polysaccharide composition dosage, HSV-1, HSV-2 plaque suppression ratio also increase thereupon, and are directly proportional with 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 suppresses average plaque number and the plaque suppression ratio (
n=3) of HSV-1 and HSV-2
Annotate: compare with the virus control group,
*P＜0.05,
*P＜0.01
The external anti-Coxsackie virus research of embodiment 3 Herba stellariae mediae polysaccharide compositions
5 kinds of Herba stellariae mediae polysaccharide with 43%, 5%, 11%, 11%, 30% content ratio mix homogeneously, are investigated the direct deactivation of Herba stellariae mediae polysaccharide composition to Coxsackie virus (CVB3), on 24 orifice plates respectively; 24h is cultivated in the myocardial cell 1mL/ hole that adds 0.5 * 104/mL concentration, removes the CVB3 1mL that the every hole of normal group adds 100TCID; After hatching 1h, abandoning supernatant, PBS flushing 3 times; The Herba stellariae mediae polysaccharide composition (3,10,30,60,100 μ g/mL) that adds variable concentrations, positive controls adds virazole 1mmol/mL, every group 4 hole; Add cell maintenance medium; The 5%CO2 incubator is cultivated 72h, and the mtt assay of observation of cell pathological changes effect (CPE), and employing day by day detects cell survival rate.
Experimental result shows, behind the virus infected cell, the CPE due to the virus group is characterized as cell shrinkage, become justify, come off, cracked.Virazole groups of cells cell shrinkage, come off, arhythmicity fluctuation, and Herba stellariae mediae polysaccharide composition administration group still is rhythmic pulsation, the part cell shrinkage is proportionate to suppression ratio and its concentration of CVB3.Hence one can see that, and the Herba stellariae mediae polysaccharide composition has direct deactivation to CVB3, and dose-effect relationship is seen table 6.
Table 6 Herba stellariae mediae polysaccharide composition is to the inhibitory action of CVB3
Resisiting influenza virus effect in the embodiment 4 Herba stellariae mediae polysaccharide composition bodies
5 kinds of Herba stellariae mediae polysaccharide with 22%, 5%, 11%, 54%, 8% content ratio mix homogeneously, obtain the Herba stellariae mediae polysaccharide composition respectively.Death has protective effect to this polysaccharide composition to the influenza induced mice, and 72 of 2-3 kunming mices in age in week are got in experiment, body weight 13-15g, and male and female half and half are divided into 6 groups at random, 12 every group.The 1st group is that normal control group, the 2nd group are the virus control group, and the 3-5 group is high, medium and low 3 dosage of Herba stellariae mediae polysaccharide composition, and the 6th group is the ribavirin matched group.Each treated animal infects a few days ago gastric infusion, and every day 1 time is until infecting back 5d, administration 7d altogether; The 1st, 2 group of normal saline of irritating stomach equivalent, the 3-5 group is irritated stomach Herba stellariae mediae polysaccharide composition 1.50 respectively; 1.20 0.80g/kg irritates stomach ribavirin (virazole) 0.20g/kg for the 6th group.After administration the 3rd day, with mice (except the 1st group) the slight anesthesia of ether down 8 times of LD50 of inoculation influenza virus 0.1mL/ only, inoculate normal saline 0.1mL with method for the 1st group.From infecting, observe 14d continuously, record dead mouse number (dead mice is not counted in the death toll in the virus inoculation 24h) calculates mortality rate, dead protective rate, mean survival time.Normal group does not have dead mouse as a result, and all the other each groups all have dead mouse, and the model group dead mouse is maximum, with other 5 groups of comparing differences remarkable (p＜0.05); 3 dose groups mouse death rates of Herba stellariae mediae polysaccharide composition are starkly lower than model group (p＜0.05); The mean survival time of model group mice is the shortest, with other each groups significant difference (table 7) is arranged more all.
The Herba stellariae mediae polysaccharide composition can play preventive and therapeutic effect to influenza virus induced mice virus bronchopneumonia, and 72 of 2-3 Kunming mouses in age in week are got in experiment, body weight 13-15g, and male and female half and half are divided into 6 groups at random, 12 every group.The 1st group is that normal group, the 2nd group are the virus model group, and the 3-5 group is respectively Herba stellariae mediae polysaccharide composition 1.50,1.20,0.80gkg
-1, 3 dose groups, the 6th group is the ribavirin group.Except that the 1st group, all the other respectively organize mice 8 times of LD of inoculation under the slight anesthesia of ether
50Influenza virus, 0.1mL/ only inoculates normal saline 0.1mL for the 1st group by the same method.Begin administration in infecting back 24h, irritate stomach for the 1st, 2 group and wait the capacity normal saline, 3-5 irritates stomach Herba stellariae mediae polysaccharide composition 1.50,1.20,0.80gkg respectively
-1, irritate stomach ribavirin 0.20gkg for the 6th group
-1, successive administration 4d, water 8h is prohibited in fasting in the 5th day, and mice is weighed; Cut off the carotid artery blood-letting and cause death, take out full lung, clean, and sop up residual brine with clean filter paper with normal saline; Weigh, calculate lung index and lung index suppression ratio, and the Mus lung is done histopathologic examination.There were significant differences (P＜0.01) for the lung of each administration group and normal group and virus model group comparison as a result index; Each dose groups lung index of Herba stellariae mediae polysaccharide composition and ribavirin group do not have obviously poor (table 8).
Table 7 Herba stellariae mediae polysaccharide composition is to the protective effect (
n=12) of influenza virus infecting mouse
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Table 8 Herba stellariae mediae polysaccharide composition is to the influence (
n=12) of influenza virus Lung Index of mice infected by Influenza virus
Annotate: compare with model group,
*P＜0.01
Influenza virus infecting mouse lung inner virus blood clotting is dripped and ID 50, median infective dose (EID
50) influence after as above method is taken out mouse lung, the Mus lung suspension of preparation 10%.Get suspension 0.1mL for every group, measure its blood clotting titre, all the other inoculate instar chicken embryo on the 10th with 8 dilution factors of 10 times of serial dilutions of normal saline, measure EID.。The EID of normal group as a result
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 one magnitude, wherein Herba stellariae mediae polysaccharide composition low dose group and ribavirin group EID
50Reduce at most, surpass 3 one magnitude.Show the effect (table 9) that the Herba stellariae mediae polysaccharide composition has therapeutical effect and resisiting influenza virus preferably to infect to the influenza virus infecting mouse.
Table 9 is respectively organized lung suspension EID50 (
n=12)
Annotate: the inoculated into chick embryo number is 32 pieces
10% lung suspension blood clotting titer determination result: the blood clotting titre of virus control group is 1: 640, and the blood clotting titre of 3 dose groups of Herba stellariae mediae polysaccharide composition is 1: 80, and the blood clotting phenomenon does not appear in the ribavirin group.Explain that the Herba stellariae mediae polysaccharide composition has inhibitory action to the growth of influenza virus in the infecting mouse lung.
Except that the instantiation of the Herba stellariae mediae polysaccharide composition inside and outside test of pesticide effectiveness, aspect the antiviral drugs preparation, mainly contain following instance:
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 950mg, 1200mg, 950mg, 900mg, 1000mg respectively; Add an amount of adjuvant, carry out tabletting, process 500 tablets of tablets by the common process of galenic pharmacy tablet; Every contains the 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 of treatment.
Embodiment 6
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2100mg, 2400mg, 1800mg, 2000mg, 1700mg respectively, add an amount of amylodextrin and process granule by the common process of galenic pharmacy capsule, insert 1000 capsules, every contains the 10mg active component.Usage and dosage: oral, each 1,2 times on the one, children's is cut down according to the circumstance, and 15 days is a course of treatment.
Embodiment 7
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2000mg, 2400mg, 1600mg, 1900mg, 2100mg respectively, add suitable amount of sucrose and dextrin, prepare by the common process of galenic pharmacy granule, every bag heavy 0.5g, every bag contains 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 of treatment.
Embodiment 8
Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2200mg, 2300mg, 1800mg, 1800mg, 2100mg respectively, add an amount of sucrose, thin up is to 1000mL, and every bottle of 10mL, 1mL contain Herba stellariae mediae polysaccharide composition 10mg.Usage and dosage: oral, each 1 bottle, 2 times on the one, 15 days courses of treatment.
Embodiment 9
Pharmaceutical formulation Herba stellariae mediae polysaccharide composition is prepared into injection; To produce 1000 of injection products is example; Get Herba stellariae mediae Polysaccharide A, B, C, D, E 2100mg, 2300mg, 1900mg, 2000mg, 1700mg respectively; Add an amount of injection mannitol, undertaken by the common process of galenic pharmacy injection, every contains Herba stellariae mediae polysaccharide composition 10mg.Usage and dosage: be dissolved in the 100mL injection, 1 time on the one, children's is cut down according to the circumstance, 30 days courses of treatment.
Claims (7)
1. a Herba stellariae mediae polysaccharide composition is characterized in that said composition is to be formed by 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 uniform mixing.
2. Herba stellariae mediae polysaccharide composition according to claim 1 is characterized in that wherein said Herba stellariae mediae Polysaccharide A weight average molecular weight is distributed between 15000～30000Da, and profile exponent D is between 1～1.5; Herba stellariae mediae Polysaccharide B, C, D weight average molecular weight are distributed between 6000～20000Da, and profile exponent D is between 1～2; Herba stellariae mediae polysaccharide E weight average molecular weight is distributed in 5000～10000Da, and profile exponent D is between 1.5～2, and 5 kinds of polysaccharide molecular weight profile exponents are all less than 2.
3. Herba stellariae mediae polysaccharide composition according to claim 1; 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) it is characterized in that wherein said Herba stellariae mediae Polysaccharide A, B, C, D, E are made up of rhamnose, arabinose, xylose, mannose, glucose and 6 kinds of monosaccharide residues of galactose, ratio is respectively (1～5: 1～9:.
4. the application of Herba stellariae mediae polysaccharide composition in the preparation antiviral drugs, its purposes is to prepare anti-virus formulation and health product.
5. application according to claim 4, wherein said virus comprises hepatitis B virus, HIV, herpesvirus, influenza virus, enterovirus and adenovirus etc.
6. application according to claim 5, wherein said herpesvirus comprise herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) and varicella zoster virus.
According to the said compositions of claim 4 application, the adjuvant that it is characterized in that utilizing galenic pharmacy to allow prepares the formulation art related dose forms, like injection, injectable powder, tablet, capsule, granule, oral liquid, micropill, spray, Emulsion etc.
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| CN115819634A (en) * | 2022-11-23 | 2023-03-21 | 宁夏大学 | Stellaria polysaccharide, preparation method and application thereof |
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| CN114057907A (en) * | 2021-12-21 | 2022-02-18 | 中国药科大学 | Method for extracting, separating and purifying red ginseng polysaccharide |
| CN114057907B (en) * | 2021-12-21 | 2023-03-17 | 中国药科大学 | Method for extracting, separating and purifying red ginseng polysaccharide |
| CN115819634A (en) * | 2022-11-23 | 2023-03-21 | 宁夏大学 | Stellaria polysaccharide, preparation method and application thereof |
| CN115819634B (en) * | 2022-11-23 | 2024-01-26 | 宁夏大学 | Radix bupleuri polysaccharide and preparation method and application thereof |
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