CN104147042A - Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament - Google Patents
Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament Download PDFInfo
- Publication number
- CN104147042A CN104147042A CN201410441135.1A CN201410441135A CN104147042A CN 104147042 A CN104147042 A CN 104147042A CN 201410441135 A CN201410441135 A CN 201410441135A CN 104147042 A CN104147042 A CN 104147042A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- radix codonopsis
- pilosul
- codonopsis
- sulfated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses application of sulfated codonopsis pilosul polysaccharide to preparation of a herpes simplex virus type I resistance medicament and a preparation method of the herpes simplex virus type I resistance medicament. According to the invention, a sulphamic acid method is adopted to carry out sulphating modification on codonopsis pilosul polysaccharide, a modification method is optimized and an excellent prevention and treatment effect of codonopsis pilosul polysaccharide on the herpes simplex virus type I is found. Meanwhile, the sulfated codonopsis pilosul polysaccharide (the substituted ratio is 0.763) with the best activity is also screened out; no matter prevention administration or treatment administration, the cell survival rate of the sulfated codonopsis pilosul polysaccharide is higher than that of a virus control group (P is smaller than 0.01), the prevention effect of the sulfated codonopsis pilosul polysaccharide is equivalent to that of acyclovir (P is greater than 0.05) and the treatment effect of the sulfated codonopsis pilosul polysaccharide is superior to that of acyclovir (P is smaller than 0.05). The effect that the antiviral activity of the sulfated codonopsis pilosul polysaccharide is the highest is shown; the sulfated codonopsis pilosul polysaccharide can be used as a material for developing the novel HSV-I ( herpes simplex virus type I) resistance medicament and the material and theoretical foundation are provided for development of the novel HSV-I resistance medicament.
Description
Technical field
The present invention relates to a kind of pharmaceutical field, especially a kind of Radix Codonopsis polysaccharide sulfuric ester is preparing application in resisting I-type herpes simplex virus medicament and preparation method thereof.
Background technology
Herpes simplex virus I-form (herpes simplex virus type I, HSV-I) belong to herpesvirus subfamily, mainly cause the infection of genitals skin, mucosa (oral mucosa) and organ (brain) in addition, can cause the above mucosa of people's waist and nervous system infection, associated diseases mainly contains keratitis, herpes simplex encephalitis, herpes labialis and gingivostomatitis etc., and can form latent infection.People is unique natural reservoir (of bird flu viruses) of herpes simplex virus.Virus enters in body through respiratory tract, oral cavity, genitals mucosa and damaged skin, dives and occupy in human normal mucosa, blood, saliva and sensory nerve ganglion cell.It is recessive that primary infection mostly is, and mostly without clinical symptoms or be subclinical performance, only has minority can occur clinical symptoms.After primary infection occurs, virus can be hidden in body for a long time.In normal population, approximately have more than 50% is this viral carrier.HSV does not produce permanent immunity in human body, and in the time that Abwehrkraft des Koepers declines, during as heating, gastrointestinal dysfunction, menstruation, gestation, focal infection and mood change, the HSV hiding in body is activated and falls ill.Studies have shown that, recurrent herpes simplex patient can have cellular immunity deficiency.HSV-I is ubiquitous and contagious, and in the time of patient's generation and releasing virus, herpes simplex virus will be propagated, and endangers very large.General propagate very quick and extensive of HSV-I, the time of the latent infection to people is very long.
It is large that herpes simplex virus I-type has host's excursion, and in cell culture, breeding and spread speed are fast, sets up the feature of latent infection in destructible infection cell and the neuroganglion of being everlasting.After infection human body, can invade central nervous system and cause herpes simplex virus encephalitis, meningitis, causes viral brain injury, can infect human internal organ tissue and cause organ lesion (as non-Hepadna Virus hepatitis), threat to life.Can also cause herpes labialis, keratitis, internal organs infection etc.Virus is made up of peplos, tunicle, nucleocapsid and the core that contains viral DNA, because the gene of HSV is double-stranded DNA, and easily with host DNA generation integration, or formation latency, limit development and the effect of viral vaccine.Therefore, the novel antiviral medicine of development high-efficiency low-toxicity is still one of current antiviral study focus.
At present both at home and abroad the medicine for the treatment of herpes simplex virus is taking acyclovir and derivant thereof as many, but such medicine exists, toxic and side effects is large, easy many deficiencies such as drug resistance, in clinical practice, is restricted.Therefore lot of domestic and foreign scholar is constantly seeking the newtype drug of anti-HSV-I safely and effectively.
Polysaccharide (Polysaccharide) is one of Chinese medicine important activity composition, be polymerized by glycosidic bond by 10 above monosaccharide molecules, in vivo as energy matter, be extensively formed in the bodies of aminal and plant and microorganism wall in, participate in the various physiological activities of cell.Polysaccharide can resist various virus as bioactive substance, as immunodeficiency virus, hepatitis virus, influenza virus, herpes simplex virus, cytomegalovirus etc. have inhibitory action, there is no cytotoxicity and quality by advantages such as the easy controls of chemical means, oneself causes the great attention of the world of medicine, has shown wide prospect in medicine.
But the drug effect of natural drug is relatively low, the activity of polysaccharide is subject to the impact of its molecular structure directly or indirectly, and elementary with it particularly three dimensions conformation is closely related with higher structure, and relevant with physicochemical properties such as molecular weight, dissolubility, viscosity.Take certain method to modify polysaccharide molecule structure, by changing space structure, molecular weight and substituent group kind, number and the position of polysaccharide, can improve or give biological activity that polysaccharide is new, reduce its toxic and side effects.The most common method of modifying has at present: sulphation, phosphorylation, acetylation, alkylation, carboxy methylation etc.No matter natural or chemosynthesis polysaccharide its antiviral activity after sulphation modification in various degree has obvious variation, more especially after the polysaccharide sulfated modification without antivirus action, has had antiviral activity.If though lentinan itself is without HIV (human immunodeficiency virus)-resistant activity, the cytopathy can HIV (human immunodeficiency virus) inhibiting HIV after sulphation modification producing.Polysaccharide sulfated antiviral activity research becomes the focus of polyose modification research field, has become an important directions in Polysaccharide Modification.
Polysaccharide sulfate (polysaccharides sulfate, PSS), also claim sulfated polysaccharide (sulfated potysaccharides, SPS), being that in polysaccharide macro-molecular chain, the hydroxyl on monosaccharide molecule is replaced by sulfate radical and the natural and semisynthetic acidic polysaccharose that forms, is polyanionic compound sugar.There is biological property widely, comprise antiviral, antitumor, to immune system effect and anticoagulating active.There is the multiple polysaccharide sulfate of bibliographical information to there is antivirus action.There is research data to show, the significant antiviral activity of polysaccharide sulfate may be that the negative charge of sulfate group is combined by electrostatic interaction with the positive charge of the upper albumen of virus, and then stop the absorption of virus to cell, sealed the position that enveloped virus is combined with cell receptor, its sulphuric acid polyanion carries out the stronger antiviral activity of inhibitory action performance to virus infected cell.In addition the Antiviral mechanism of polysaccharide sulfate is also given the credit to the inhibition to cell absorption dependent interaction to virus, a certain step copying after directly suppressing cell entry cell or entering cell.Study from existing polysaccharide sulfate, be to complete adsorption process by the molecule affinity between virus and host cell mostly, thereby reach antiviral effect.In addition introducing the variation of caused polysaccharide stereochemical structure after sulfate group is also the major reason that sulfated polysaccharides produces biologic activity.
Radix Codonopsis is Campanulaceae Radix Codonopsis
codorwpsis pilosula(Franch.) Nannf., element flower Radix Codonopsis
codonopsis pilosulanannf. var. modesta (Nannf.) L. T. Shen or radix codonpsis tangshen
codonopsis tang shenoliv. dry root, eats or is used as medicine with fleshy tap root.Radix Codonopsis has spleen invigorating lung benefiting, the effect of nourishing blood to promote the production of body fluid.Be usually used in deficiency of both the splenic and pulmonary QI, lack of appetite asthenia, cough dyspnea due to deficiency, the diseases such as insufficiency of vital energy and blood.Radix Codonopsis main component has Radix Codonopsis polysaccharide, Radix Codonopsis saponin, sterols, triterpenes, alkaloid, lactone, Coumarins etc.Radix Codonopsis polysaccharide (
codonopsis tangshenoliv.polysaccharides) be from the dry root of Radix Codonopsis, to extract to separate the water solublity heteropolysaccharide obtaining, pharmacological testing proves that Radix Codonopsis polysaccharide has the pharmacological action of clear and definite enhancing immunity, antitumor, anti-stress, the anti-ageing many aspects of waiting for a long time.But the Gou Peng wheel blade Radix Codonopsis extract of having reported for work is only shown in by the document of Radix Codonopsis antiviral study, Canine Parvovirus is had to certain inhibitory action, and Radix Codonopsis polysaccharide sulfuric ester there is inhibitory action to newcastle disease virus (NDV).
Summary of the invention
The object of the invention is: the new purposes that a kind of Radix Codonopsis polysaccharide sulfuric ester is provided.
Also provide a kind of new system of Radix Codonopsis polysaccharide sulfuric ester for scheme.
The present invention is achieved in that Radix Codonopsis polysaccharide sulfuric ester is in the application of preparing in resisting I-type herpes simplex virus medicament.
The preparation method of Radix Codonopsis polysaccharide sulfuric ester, first extraction, purification Radix Codonopsis polysaccharide, carries out sulphation modification by sulfamic acid method to Radix Codonopsis polysaccharide, and modification condition is that Radix Codonopsis polysaccharide is 1:0.04 g/mol with the molal weight ratio of sulfamic acid, response time 15 min.
The Radix Codonopsis polysaccharide sulfuric ester that acquisition substitution value is 0.763.
In order to verify technique effect of the present invention, inventor has carried out following experiment:
1, the extraction of Radix Codonopsis polysaccharide
By the heavy method of decocting-ol.Get Radix Codonopsis decoction pieces 1000 g, pulverize, in apparatus,Soxhlet's, with 3 defats of 80% alcohol heating reflux, medicinal residues add 10 times of water gagings and decoct 3 times, the 1st 3 h, the 2nd 2 h, the 3rd 1 h; Sucking filtration medicinal residues, merge after 3 filtrates concentrated.It is 80% to ethanol content that concentrated solution adds 95% ethanol, in 4 DEG C of refrigerators, leaves standstill after 12 h, and 3000 r/min are centrifugal, collecting precipitation part, after vacuum drying, redissolve, after adding 95% ethanol and being 80% to ethanol content, centrifugal, collecting precipitation, lyophilizing, dry sediment, obtains dark-brown Radix Codonopsis crude polysaccharides.The optimum extraction process of Radix Codonopsis polysaccharide is 100 DEG C of temperature, and solid-liquid ratio is 16 times, and extraction time is 3h, and extraction time is 2 times, and with this understanding, the extraction ratio of Radix Codonopsis raw sugar is 26% left and right.
2, the purification of Radix Codonopsis polysaccharide
(1) in crude polysaccharides, remove deproteinize: after Radix Codonopsis crude polysaccharides is dissolved in enough distilled water according to 1:100 (g/mL), the solution that separately takes a morsel after adding distil water dilution, scans to 200nm wavelength from 800nm under ultraviolet spectrophotometer.To being dissolved with in the solution of crude polysaccharides, in every 1000mL, add 1g pepsin, and at 37 DEG C, carry out after water bath with thermostatic control 12h, the chloroform of 200mL and n-butyl alcohol mixture are added and contained in pepsic crude polysaccharides, fully concussion, separated and collected lower floor organic facies after standing 12h, discard medial degeneration albumin layer, collect the aqueous solution that Radix Codonopsis polysaccharide is contained on upper strata, and do full wavelength scanner under ultraviolet spectrophotometer, repeatedly for several times, until the protein free characteristic absorption peak 260 ~ 280nm of aqueous solution place is during without protein specificity absworption peak, albumen in proof polysaccharide has been removed totally.
(2) Radix Codonopsis polysaccharide depigmentation processing
After the Radix Codonopsis polysaccharide solution of Deproteinization is concentrated, add 5 times of amount 95% ethanol precipitate with ethanol, centrifugal collection precipitate with ethanol polysaccharide, after redissolution, precipitate with ethanol processing again, after several, after precipitate with ethanol supernatant fluid color bleach, proves that the pigment in crude polysaccharides is divested.The Radix Codonopsis polysaccharide lyophilizing of collecting repeatedly precipitate with ethanol, obtains canescence Radix Codonopsis polysaccharide, proves that the pigment in polysaccharide is removed, and the Radix Codonopsis polysaccharide Collection and conservation after removing is for subsequent use in 4 DEG C.
(3) membrane separation purification Radix Codonopsis polysaccharide
By the Radix Codonopsis crude polysaccharides 10g of Deproteinization, the depigmentation, after dissolving, this solution is centrifugal under the speed of 3500r/min, discard after insoluble matter, supernatant is transferred to and in volumetric flask, is settled to 500ml, making Radix Codonopsis crude polysaccharides solution concentration is 20mg/mL, obtain Radix Codonopsis polysaccharide COP-1 component, under 4 DEG C of conditions, preserve and wait to dialyse.
COP-1 polysaccharide after centrifugal is packed in the bag filter of different interception sizes and dialyse, collect respectively dialysis solution in concentrated bag filter, after lyophilizing, obtain COP-2, COP-3, tri-parts of polysaccharide samples of COP-4.COP-2 component sugars molecular weight is between 3500 ~ 10000; COP-3 polysaccharide molecular weight is greater than 10000; COP-4 polysaccharide molecular weight is between 3500 ~ 7000, and after bag filter is separated, polysaccharide lyophilized products all saves backup under 4 DEG C of conditions.
(4) DEAE-52 anion exchange chromatography separates Radix Codonopsis polysaccharide
Wet method dress DEAE-52 anion-exchange chromatography post, by the DEAE-52 powder by immersion treatment in HCl, NaOH solution, repeatedly rinse to neutrality with a large amount of deionized waters, balance, spend the night, get membrane separation purification Radix Codonopsis polysaccharide COP-2 after treatment solution loading, open constant flow pump, with the NaCl eluting of 0 ~ 0.3mol/L, constant flow pump flow velocity is set to 0.2mL/min, collect eluent, 5 mL/pipe, detects polyoses content according to phenolsulfuric acid method after each pipe sampling, draws polysaccharide elution curve, merge and collect sugary pipe eluent, further separation and purification.
Radix Codonopsis polysaccharide is through DEAE-52 cellulose gel to after separating, and taking pipe number as abscissa, absorbance is vertical coordinate, get eluent in each pipe, adopt phenolsulfuric acid method to do elution curve as shown in Figure 1, elution curve shows in the 25th pipe-28 pipes larger absworption peak, collects 25 ~ 28 pipes and merges.Collect after component 1, use the NaCl eluant solution DEAE gel of 0.1mol/L instead, after obtain component 2.Component 1 content is more, to collect merge after component 1 repeatedly cross after column purification, merge after concentrated freeze-dried and obtain first separation component, this component journey white powder, without special odor.
(5) glucosan G-25 gel filtration chromatography separates Radix Codonopsis polysaccharide
Wet method dress glucosan G-25 gel chromatography column, by 0.9% NaCl flushing, balance, spend the night, the Radix Codonopsis polysaccharide component 1 solution loading that the DEAE-52 anion exchange chromatography of learning from else's experience separates, opens constant flow pump, carry out eluting with deionized water, constant flow pump flow velocity is set to 0.2mL/min, collects eluent, 5 mL/pipe, after each pipe sampling, detect polyoses content according to phenolsulfuric acid method, draw polysaccharide elution curve, merge and collect sugary pipe eluent more further separation and purification.
Adopt G-25 polydextran gel, the Radix Codonopsis polysaccharide component 1 after DEAE-52 gel is separated is further done desalting processing.Component 1 part that DEAE gel separates is shown in Fig. 2 through G-25 gel separating resulting: under this gel, elution curve way is identical with way under DEAE-52.Due in G-25 gel, the material that last elution fraction is molecular weight, is not that experiment separates the target polysaccharide of setting, and after therefore G-25 gel being separated, only collects component 1 part.
(6) glucosan G-100 gel filtration chromatography separates Radix Codonopsis polysaccharide
Wet method dress glucosan G-100 gel chromatography column, with the NaCl flushing of 0.15mol/L, balance, spend the night, Radix Codonopsis polysaccharide solution loading after the G-25 gel of learning from else's experience separates, open constant flow pump, eluent is the NaCl solution of 0 ~ 0.3mol/L, constant flow pump flow velocity is set to 0.2mL/min, collect eluent, 5 mL/pipe, after each pipe sampling, detect polyoses content according to phenolsulfuric acid method, draw polysaccharide elution curve, G-25 gel is separated to component 1 part merging, after glucosan G-100 gel separating for several times, carry out purification again, elution fraction is done after elution curve, collect and merge 21st ~ 27 pipe components, merge concentrated rear lyophilizing, separation obtains Radix Codonopsis polysaccharide COP-5(and sees Fig. 3) component, adopt phenolsulfuric acid method to measure its polyoses content, sugar content reaches 98.61%, glucuronic acid content is 4.72%.
(7) molecular weight determination of Radix Codonopsis polysaccharide
Radix Codonopsis polysaccharide molecular weight HPLC method condition determination: instrument: Agilent 1100 type highly effective liquid phase chromatographic systems; Chromatographic column is: chromatographic column: Ultrahydrogel
tM2000 and Ultrahydrogel
tM500 two, post series connection (25 cm × 0.75 cm; The NaCl solution of mobile phase: 0.2mol/mL; Flow velocity: 0.8mL/min, sampling volume 40 μ l, 40 DEG C of column temperatures, detector: differential detector, reference is liquid flow phase, under above-mentioned chromatographic condition, Radix Codonopsis polysaccharide is carried out to molecular weight determination.
Assay method: precision takes the pulullan polysaccharide standard reference material of known relative molecular weight, with distilled water dissolve after, preparation 1mg/ml solution, reference substance relative molecular weight be respectively into: 2.0 × 10
6, 7.88 × 10
5, 4.04 × 10
5, 2.12 × 10
5, 1.12 × 10
4, 4.73 × 10
3, according to method determining molecular weight standard curve under 3.5.1.
Precision takes after the Radix Codonopsis polysaccharide COP-5 sample of 1mg, joins in the mobile phase of 1mL, and after fully vibration is dissolved, centrifugal 10min removes after insoluble matter, gets supernatant, obtains after polysaccharide test sample, after sample introduction, different polysaccharide molecular weight analytes is measured.
Get the sugar of known molecular amount, adopt after SEC chromatography determination molecular weight standard curve using molecular weight as vertical coordinate, time is as abscissa, after adopting method of least square to date processing, obtain the first regression equation Y=-0.2291X+9.453(r=0.998 of molecular weight-time graph), molecular weight is 4.73 × 10
3~ 2.0 × 10
6internal linear relation is good.
To separating several Radix Codonopsis polysaccharide molecular weight that obtain under different condition, carry out gel exclusion method and carry out molecular weight, purity testing, concrete data are as shown in table 1, through DEAE-52 gel, G-25, polysaccharide COP-5 component after G-100 gel chromatography column combined separation, it is 1.42 that gel exclusion chromatography is measured its purity D value, show that sugared homogeneity is good, 90% molecular weight and number-average molecular weight, many average molecular weights, breadth coefficient data show, these 4 indexs of the Radix Codonopsis polysaccharide of COP-5 component approach, also shown after gel separating polyose, obtain good control for the molecular weight of polysaccharide.
Note: Mn is the equal relative molecular mass of number, and Mw is average weight-molecular mass, and Mz is wherein breadth coefficient of z average molecular weight, and D is (Mn/Mw), and 90% place's relative molecular weight is labeled as M90, D value reflection purity of polysaccharide situation.
3, the preparation of Radix Codonopsis polysaccharide sulfuric ester (sulphation modification of Radix Codonopsis polysaccharide)
(1) method of polysaccharide sulfate, adopt sulfamic acid method to carry out Sulfation research both at home and abroad, rare report, it is that Sulfation reagent is prepared sulfuric ester that the present invention adopts sulfamic acid, select sulfamic acid gentle under room temperature as esterifying reagent, in test in design, selected uniform design to optimize the sulfuric acid esterification of polysaccharide.
(2) sulphation modification condition optimizing due to reaction temperature (A), response time (B), rate of charge (polysaccharide quality/sulfamic acid mole) (C)) be the major influence factors of polysaccharide sulfate, on the basis of experiment of single factor, above-mentioned 3 factors are selected in nature according to polysaccharide and Sulfation reagent, 4 levels of each selecting factors, and adopt uniform Design pseudo level method, uniform design U
8(4
3) experiment arrangement, to obtain the experiment condition of best sulphation modification.
(4) in 250mL three-neck flask, add precision to take Radix Codonopsis polysaccharide (the Codonopsis pilosul polysaccharide of purification, CPPS) 1 g, stir 6 h Radix Codonopsis polysaccharide is dissolved in the dimethyl sulfoxide (DMSO) of 50 mL completely, reaction temperature, response time and the rate of charge (g/mol) of setting by table 1 be stirring reaction respectively.After reaction finishes, reactant is poured in beaker, be neutralized to pH=7.0～8.0 cessation reaction with 10% NaOH solution, then add the dehydrated alcohol of triplication volume, hold over night at 4 DEG C, centrifugal collecting precipitation, after the bag filter dialysis 72h that is 3500 with interception after redissolving, collect concentrate, after centrifugal, concentrated also lyophilization, obtains Radix Codonopsis polysaccharide sulfuric ester (sulfated Codonopsis pilosul polysaccharide, SCPPS), obtain altogether 8 kinds of SCPPS, be labeled as respectively SCPPS
1, SCPPS
2, SCPPS
3, SCPPS
4, SCPPS
5, SCPPS
6, SCPPS
7, SCPPS
8.
(6) data analysis
Uniform Design data (table 3) data acquisition carries out obtaining fit equation after numerical simulation with uniform design process software DPS7.0 standard edition: Y=22.794021-0.0428288958X
1-0.776477473X
2
+ 0.1434399243X
3 2+ 0.00805862119X
1x
2-0.0538186794X
1x
3+ 0.0354103763X
2x
3; In fit equation, Y is sulfate radical content (%), X
1for reaction temperature (DEG C); X
2for response time (min); X
3for rate of charge (ml/mol).
P=0.0402 in fit equation.Regression equation significance; Surplus standard deviation S=0.1070392,
Coefficient R a=0.998393 after adjustment.By each coefficient in fit equation, can show that rate of charge is positively related effect to the content of sulfate radical, time and temperature are negative effect accordingly.Thereby should reduce temperature in reaction system, Reaction time shorten, increases rate of charge, reaches successively maximum sulfuric ester rate.
According to regression equation, show that optimum combination is 60 DEG C of reaction temperatures, response time 15min, ((polysaccharide quality/sulfamic acid mole) is 1:0.04 (g/mol) to rate of charge.
(7) demonstration test
60 DEG C of reaction temperatures, response time 15min, it is under the condition of 0.04mol that every 50ml adds sulfamic acid, take the three parts of each 1.0g of Radix Codonopsis polysaccharide that are dried to constant weight, carry out 3 esterifications operation, obtain three parts of sulphation Radix Codonopsis polysaccharides, adopt barium chloride-gelatin method to measure sulfate radical content, measure its substitution value and be respectively 0.7630,0739,0.754, not remarkable with the difference of Uniform Design result, the Radix Codonopsis polysaccharide Sulfation process of card description of test uniform design screening, stable to polysaccharide sulfated replacement, prove that this condition is optimum condition.
(8) polysaccharide sulfated rear discriminating
Precision takes reacted sulfated polysaccharides SCPPS250mg, is dissolved in 25mL distilled water, gets after 5mL, drips the BaCl of 1mol/mL
2after solution number drips, observe phenomena; Separately get after 5mL, add the dehydrated alcohol precipitation of 20mL, and centrifugal, then the BaCl of dropping 1mol/mL
2after solution number drips, observe phenomena.
Negate should add after KBr tabletting by front polysaccharide, carries out infrared scan, and after contrast sulphation, the INFRARED ABSORPTION of polysaccharide, resolves its characteristic peak.
In SCPPS solution (10mg/mL), drip the BaCl of 1mol/mL
2after solution, there is white precipitate; When add 20mL ethanol precipitation and centrifugal after, drip the BaCl of 1mol/mL
2, there is not white precipitate in solution, result shows, sulfate radical is combined well with Radix Codonopsis polysaccharide.
Radix Codonopsis polysaccharide is carried out to infrared scan, can see that in conjunction with former figure relevant range wavelength absorption changes: 3436cm-1 place-OH hydroxyl absorption minimizing, show to have occurred esterification, the C=O of 1632cm-1 place carbonyl absorption peak increases, show that the acetylamino structure in polysaccharide increases after sulphation, strong absworption peak before and after 1250cm-1 produces, show to have produced in sulfated polysaccharides-C-SO2, the stretching vibration of-S=O molecular link, have at 1057cm-1 place the characteristic absorption that is similar to chondroitin sulfates medicine in addition, the absorption of vibrations of be absorbed as-C-S of 799cm-1 place key.Two kinds of materials before and after esterification simultaneously approach on waveform, show that sulphation occurs in (Fig. 4) on polysaccharide skeleton.
(9) sulfate radical content analytical method
Sulfate radical standard curve: precision takes the anhydrous Na that is dried to constant weight at 120 DEG C
2sO
450mg, with after deionized water dissolving, standardize solution is to 100ml, i.e. and preparation obtains the Na that concentration is 0.5mg/ml
2sO
4standard solution is for subsequent use.Accurate standard solution 100 μ L, 200 μ L, the 400 μ L of drawing, 600 μ L, 800 μ L, 1000 μ L, 1200 μ L, be placed in tool plug test tube, separately get a pipe adding distil water 5ml as blank tube, all the other add water and are settled to 5ml, add the 3% trichloroacetic acid 3.8ml having prepared, barium chloride-gelatin 1.2ml measures absorbance, record data after 25 DEG C of water-baths are placed 18 minutes in 420nm place after shaking up; Separately get one group of tool plug test tube, application of sample is identical with last group, adds water after standardize solution, adds 3.8ml trichloroacetic acid, adds gelatin solution, places after 18 minutes in 25 DEG C of water-baths, measure after absorbance, and taking liquid volume added as abscissa, absorbance (A
gelatin-barium chloride-A
gelatin) be vertical coordinate drawing standard curve (Fig. 5).
Sample determination: get synthetic Radix Codonopsis polysaccharide sulfuric ester 70mg, join in 50ml volumetric flask, add the HCL10ml of 1mol/L, at 100 DEG C, react 3h cessation reaction, be settled to after 50ml with the HCL of 1mol/L, shake up, after centrifugal, get supernatant, measure containing sulfate radicals content.According to standard curve, calculate gained sample SO
4 2-content.The sulfate radical substitution value computing formula of Radix Codonopsis polysaccharide is: DS=1.62S%/(32-1.02S%).Sulfate radical content standard curve: Y=1.3156x+0.0159, R=0.9957, the better (see figure 5) of curvilinear correlation.
4, the anti-HSV-1 virus activity comparison of Radix Codonopsis polysaccharide sulfuric ester
On the result basis of trial test, select SCPPS
6for object of study, with the CPPS that do not carry out sulphation modification in contrast, their safe concentrations to African green monkey kidney cell (Vero cell) are first measured, then with the relatively impact of their HSV-1 infection cell abilities of mtt assay.
(1) mensuration of the virus titer Vero cell of trophophase of taking the logarithm, by 4.0 × 10
3the cell number in individual/hole is inoculated in 96 well culture plates, puts 37 DEG C, 5%CO
2incubator is cultivated, and after cell attachment, exhausts supernatant, adds different dilution virus liquids (to use containing the maintenance medium of 2% hyclone HSV-I virus is become to 10 with 10 times of dilutions
-5, 10
-6, 10
-7, 10
-8, 10
-9), each dilution factor is established 6 multiple holes, establish viral negative control group, every hole 100 μ l, put incubator and cultivate simultaneously, daily check viral growth situation, observe the cytopathic hole count of characteristic, after 72h, record as follows result: note "+" below 25%, 26%～50% note " ++ ", 51%～75% note " +++ ", 76%～100% note " ++++"; According to Reed-Muench formula, calculate viral half cell infection amount (TCID50).
The measurement result of viral infection titre is compared with normal Vero cell, and the cell that HSV-I infects occurs the cellular swelling, becomes circle, from the typical phenomenon (Fig. 6) of the cytopathic effecies (CPE) such as bottle wall comes off.Calculate the TCID of HSV-1 according to Reed-Muench formula
50be 10
-8/ 0.1mL.
(2) cell toxicity test of sulphation Radix Codonopsis polysaccharide is inoculated in Vero cell in 96 well culture plates according to the method described above, put 37 DEG C, 5%CO2 incubator is cultivated, after cell attachment, exhaust supernatant, to vulcanize by maintenance medium that Radix Codonopsis polysaccharide dilutes is respectively 6 concentration such as 0,10,20,50,100,200 μ g/ml, and each concentration medicine is added in cell with every hole 100 μ l, and each concentration arranges 6 multiple holes; In incubator, cultivate after 72h, every hole adds the MTT dye liquor 10 μ l of 5mg/ml, continue to cultivate after 4h, abandon supernatant liquid, add DMSO 100 μ l/ holes, vibration 10min, until the first a ceremonial jade-ladle, used in libation crystallization forming is all dissolved, measure absorbance (A) value at microplate reader 490nm place, calculate the maximal non-toxic concentration of medicine.
Radix Codonopsis polysaccharide (CPPS) and sulphation Radix Codonopsis polysaccharide (SCPPS
6) to Cytotoxic mensuration along with CPPS and SCPPS
6the increase of working concentration, the corresponding minimizing of living cells quantity.In the time that concentration reaches 200 μ g/ml, the cell survival rate of CPPS group have compared with matched group significant difference (
p< 0.05), and SCPPS
6group without statistics difference (
p>0.05).Therefore, CPPS and SCPPS
6vero cell maximal non-toxic concentration is defined as respectively to 100 μ g/ml and 200 μ g/ml(tables 4).
(3) the anti-HSV-I experiment of sulphation Radix Codonopsis polysaccharide will be inoculated in after Vero cell counting in 96 well culture plates, put 37 DEG C, in 5% carbon dioxide incubator, cultivate, after growing up to monolayer, cell is divided into following several groups: cell matched group, virus control group, positive drug matched group, variable concentrations medicine group.Establish 6 multiple holes for every group, experiment repeats 3 times.
1) preventive effect of medicine adds the medicine of variable concentrations in cell monolayer, after 24h, abandon supernatant, with the virus liquid attack cells 1h of 100 × TCID50, then PBS cleans to remove unconjugated virus, add again the drug effect 72h of variable concentrations, Microscopic observation cytopathy situation, and record.
2) therapeutical effect of medicine attacks with the virus liquid of 100 × TCID50 the cell 1h that has grown up to monolayer, and then PBS cleans, then adds the drug effect 72h of variable concentrations, Microscopic observation cytopathy situation, and record.
3) statistical analysis method adopts SPSS14.0 software, and measurement data is carried out to one factor analysis of variance, relatively adopts t inspection between two groups, experimental data with mean ± standard deviation (
± s) represent,
p<0.05 is for there being significant difference.
CPPS and SCPPS
6anti-HSV-I effect in the time of prevention and treatment administration as can be seen from Figure 7, without the CPPS-prevention of sulphation modification and the cell survival rate for the treatment of administration group all higher than virus control group, but only prevention group has a significant difference, compared with positive drug acyclovir (ACV), the cell survival rate of prevention group and treatment group is all lower, the effect that CPPS only has certain prevention HSV-I to infect is described, and this effect is weaker than ACV; And SCPPS after sulphation modification
6present obvious prevention and therapeutic effect (compared with virus control group,
p<0.01), its preventive effect and ACV quite (compared with ACV prevention group,
p>0.05), therapeutic effect is better than ACV(compared with ACV treatment group,
p<0.05).
Radix Codonopsis polysaccharide is carried out to sulphation modification and qualification, and the effect of the Radix Codonopsis polysaccharide In Vitro Anti HSV-1 before and after sulphation modification has been studied.Prevention administration group is to give virus attack after first adding medicine pretreatment 24h again, and its mode is the prevent disease administration of simulating clinically, its objective is that can observe medicine affect the absorption phase in viral biocycle; Treatment administration group is first to give virus attack to add medicine again, simulation metainfective treatment administration clinically, its objective is that can observe medicine play a role to the virus that enters host cell, this is mainly the after-stage that penetrates in viral biocycle, comprises shelling, biosynthesis, assembling, maturation and discharges.Experimental result shows: the Radix Codonopsis polysaccharide after sulphation modification, its prevention and therapeutic effect to HSV-I obviously strengthens, this may be that variation has occurred for polysaccharide space conformation after sulphation modification, occupy the site of virus and host cell receptor combination, cause viruses adsorption to reduce, show as obvious preventive effect; And that the impact polysaccharide that may change with conformation that absorption after-stage is suppressed has affected the function of some key enzymes in virus replication cycle is relevant, the further research of still needing of its related mechanism and action target spot thereof.
Prove according to early-stage Study, sulphation modification can significantly improve the biological activity of Radix Codonopsis polysaccharide, especially antiviral activity, the present invention adopts sulfamic acid method to carry out Sulfation modification to Radix Codonopsis polysaccharide, optimize method of modifying, and found that Radix Codonopsis polysaccharide sulfuric ester has stronger prevention and therapeutic effect to herpes simplex virus I-form.No matter also filter out active best Radix Codonopsis polysaccharide sulfuric ester (substitution value is 0.763), be prevention administration or treatment administration simultaneously, cell survival rate all higher than virus control group (
p<0.01), and its preventive effect and acyclovir quite (
p>0.05), therapeutic effect be better than acyclovir (
p<0.05).Show that its antiviral activity is the strongest, can be used as the material of the anti-HSV-I of development of new virus drugs, for the anti-HSV-I of development of new virus drugs provides material and theoretical foundation.
Brief description of the drawings
The DEAE-52 gel detach Spline that accompanying drawing 1 is Radix Codonopsis polysaccharide;
The G-25 gel column chromatography that accompanying drawing 2 is Radix Codonopsis polysaccharide separates;
The G-25 gel column chromatography that accompanying drawing 3 is Radix Codonopsis polysaccharide separates;
The G-25 gel column chromatography that accompanying drawing 4 is Radix Codonopsis polysaccharide separates;
Accompanying drawing 5 is sulfate radical content standard curve;
Accompanying drawing 6 is the Vero cell before and after the infection of HSV-I;
Accompanying drawing 7 is CPPS-1 and the anti-HSV-1 effect (mtt assay) of SCPPS-1 in the time of prevention and treatment administration;
* representative in accompanying drawing 7
p<0.05, * * representative
p<0.01; With the comparison of virus control group: Δ representative
p<0.05, the representative of Δ Δ
p<0.01; Represent with ACV prevention group comparison: #
p<0.05, ## representative
p<0.01; Represent with ACV treatment group comparison :@
p<0.05 ,@@representative
p<0.01.
Detailed description of the invention
Embodiments of the invention 1: the preparation method of Radix Codonopsis polysaccharide sulfuric ester, specific as follows:
1) extraction of Radix Codonopsis polysaccharide
By the heavy method of decocting-ol.Get Radix Codonopsis decoction pieces 1000 g, pulverize, in apparatus,Soxhlet's, with 3 defats of 80% alcohol heating reflux, medicinal residues add 10 times of water gagings and decoct 3 times, the 1st 3 h, the 2nd 2 h, the 3rd 1 h; Sucking filtration medicinal residues, merge after 3 filtrates concentrated.It is 80% to ethanol content that concentrated solution adds 95% ethanol, in 4 DEG C of refrigerators, leaves standstill after 12 h, and 3000 r/min are centrifugal, collecting precipitation part, after vacuum drying, redissolve, after adding 95% ethanol and being 80% to ethanol content, centrifugal, collecting precipitation, lyophilizing, dry sediment, obtains dark-brown Radix Codonopsis crude polysaccharides.The optimum extraction process of Radix Codonopsis polysaccharide is 100 DEG C of temperature, and solid-liquid ratio is 16 times, and extraction time is 3h, and extraction time is 2 times, and with this understanding, the extraction ratio of Radix Codonopsis raw sugar is 26% left and right;
2) purification of Radix Codonopsis polysaccharide
(1) in crude polysaccharides, remove deproteinize: after Radix Codonopsis crude polysaccharides is dissolved in enough distilled water according to 1:100 (g/mL), the solution that separately takes a morsel after adding distil water dilution, scans to 200nm wavelength from 800nm under ultraviolet spectrophotometer, to being dissolved with in the solution of crude polysaccharides, in every 1000mL, add 1g pepsin, and at 37 DEG C, carry out after water bath with thermostatic control 12h, the chloroform of 200mL and n-butyl alcohol mixture are added and contained in pepsic crude polysaccharides, fully concussion, separated and collected lower floor organic facies after standing 12h, discard medial degeneration albumin layer, collect the aqueous solution that Radix Codonopsis polysaccharide is contained on upper strata, and do full wavelength scanner under ultraviolet spectrophotometer, repeatedly for several times, until the protein free characteristic absorption peak 260 ~ 280nm of aqueous solution place is during without protein specificity absworption peak, albumen in proof polysaccharide has been removed totally,
(2) Radix Codonopsis polysaccharide depigmentation processing
After the Radix Codonopsis polysaccharide solution of Deproteinization is concentrated, add 5 times of amount 95% ethanol precipitate with ethanol, centrifugal collection precipitate with ethanol polysaccharide, after redissolution, precipitate with ethanol processing again, after several, after precipitate with ethanol supernatant fluid color bleach, proves that the pigment in crude polysaccharides is divested; The Radix Codonopsis polysaccharide lyophilizing of collecting repeatedly precipitate with ethanol, obtains canescence Radix Codonopsis polysaccharide, proves that the pigment in polysaccharide is removed, and the Radix Codonopsis polysaccharide Collection and conservation after removing is for subsequent use in 4 DEG C;
(3) membrane separation purification Radix Codonopsis polysaccharide
By the Radix Codonopsis crude polysaccharides 10g of Deproteinization, the depigmentation, after dissolving, this solution is centrifugal under the speed of 3500r/min, discard after insoluble matter, supernatant is transferred to and in volumetric flask, is settled to 500ml, making Radix Codonopsis crude polysaccharides solution concentration is 20mg/mL, obtain Radix Codonopsis polysaccharide COP-1 component, under 4 DEG C of conditions, preserve and wait to dialyse;
COP-1 polysaccharide after centrifugal is packed in the bag filter of different interception sizes and dialyse, collect respectively dialysis solution in concentrated bag filter, after lyophilizing, obtain COP-2, COP-3, tri-parts of polysaccharide samples of COP-4.COP-2 component sugars molecular weight is between 3500 ~ 10000; COP-3 polysaccharide molecular weight is greater than 10000; COP-4 polysaccharide molecular weight is between 3500 ~ 7000, and after bag filter is separated, polysaccharide lyophilized products all saves backup under 4 DEG C of conditions;
3) by sulfamic acid method, Radix Codonopsis polysaccharide is carried out to sulphation modification
In 250mL three-neck flask, add precision to take Radix Codonopsis polysaccharide (the Codonopsis pilosul polysaccharide of purification, CPPS) 1 g, stirring 6 h is dissolved in the dimethyl sulfoxide (DMSO) of 50 mL Radix Codonopsis polysaccharide completely, modification condition is that Radix Codonopsis polysaccharide is 1:0.04 g/mol with the molal weight ratio of sulfamic acid, response time is 15 min, stirring reaction, after reaction finishes, reactant is poured in beaker, be neutralized to pH=7.0～8.0 cessation reaction with 10% NaOH solution, then add the dehydrated alcohol of triplication volume, hold over night at 4 DEG C, centrifugal collecting precipitation, after the bag filter dialysis 72h that is 3500 with interception after redissolving, collect concentrate, after centrifugal, concentrated also lyophilization, the Radix Codonopsis polysaccharide sulfuric ester that acquisition substitution value is 0.763.
Embodiments of the invention 2:
get Radix Codonopsis polysaccharide sulfuric ester 3g, medical starch 75 g, microcrystalline Cellulose 20 g, medical starch is first dry, cross 120 mesh sieves, then with
radix Codonopsis polysaccharide sulfuric ester, microcrystalline Cellulose mix, cross twice 120 mesh sieves, insert in hard capsule, make 1000 capsules of the present invention.Every hard capsule is containing principal agent composition
0.3mg.
Embodiments of the invention 3:
get Radix Codonopsis polysaccharide sulfuric ester 3g, hypromellose 6g, carboxymethylstach sodium 10g, microcrystalline Cellulose 8g, lactose 115g, starch 50g, magnesium stearate 1g; Principal agent and adjuvant are fully mixed in rear input homogenizer, and spraying adds water appropriate, granulate, and moisture Control is 3 ~ 4%, and then tabletting, makes 1000, film coating.Every containing principal agent composition
0.3mg.
Embodiments of the invention 4
: get Radix Codonopsis polysaccharide sulfuric ester3g, adds 400 ml Macrogol 200s to dissolve, then adds appropriate distilled water to dilute, and then adds appropriate sucrose and adjust volume to 1000 ml, stirs evenly, and filters, and fill becomes every of 10 ml or 20 ml, sterilization packaging.
Claims (3)
1. a Radix Codonopsis polysaccharide sulfuric ester is in the application of preparing in resisting I-type herpes simplex virus medicament.
2. the preparation method of a Radix Codonopsis polysaccharide sulfuric ester, it is characterized in that: first extraction, purification Radix Codonopsis polysaccharide, by sulfamic acid method, Radix Codonopsis polysaccharide is carried out to sulphation modification again, modification condition is that Radix Codonopsis polysaccharide is 1:0.04 g/mol with the molal weight ratio of sulfamic acid, response time 15 min.
3. the preparation method of Radix Codonopsis polysaccharide sulfuric ester claimed in claim 2, is characterized in that: the Radix Codonopsis polysaccharide sulfuric ester that acquisition substitution value is 0.763.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410441135.1A CN104147042A (en) | 2014-09-02 | 2014-09-02 | Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410441135.1A CN104147042A (en) | 2014-09-02 | 2014-09-02 | Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104147042A true CN104147042A (en) | 2014-11-19 |
Family
ID=51872807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410441135.1A Pending CN104147042A (en) | 2014-09-02 | 2014-09-02 | Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104147042A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105906734A (en) * | 2016-05-09 | 2016-08-31 | 遵义医学院 | Radix codonopsis homogeneous polysaccharide COP-2, and preparation method and application thereof |
| CN105906735A (en) * | 2016-05-09 | 2016-08-31 | 遵义医学院 | Radix codonopsis homogeneous polysaccharide COP-1, and preparation method and application thereof |
| CN107033255A (en) * | 2017-05-25 | 2017-08-11 | 遵义医学院 | Codonopsis pilosula polysaccharide COP W1 and preparation method and application |
| CN107556399A (en) * | 2017-09-27 | 2018-01-09 | 遵义医学院 | A kind of Radix Codonopsis homogeneous polysaccharide COP W2 and preparation method and application |
| CN108324730A (en) * | 2018-03-09 | 2018-07-27 | 华南农业大学 | Sulphation Codonopsis pilosula polysaccharide is preparing the application in preventing and/or treating irritability lesions of liver and kidney drug |
| CN115286721A (en) * | 2022-07-28 | 2022-11-04 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application of active polysaccharide composition in preparation of product with effect of preventing or treating gastric injury |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058610A (en) * | 2006-09-21 | 2007-10-24 | 云南中医学院 | Codonopsisbulleyana polysaccharide sulfate and use thereof |
-
2014
- 2014-09-02 CN CN201410441135.1A patent/CN104147042A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058610A (en) * | 2006-09-21 | 2007-10-24 | 云南中医学院 | Codonopsisbulleyana polysaccharide sulfate and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张驰等: "中国板党中硒多糖提取分离技术研究", 《食品科学》 * |
| 王毅等: "均匀设计优化党参多糖硫酸酯合成工艺", 《食品科技》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105906734A (en) * | 2016-05-09 | 2016-08-31 | 遵义医学院 | Radix codonopsis homogeneous polysaccharide COP-2, and preparation method and application thereof |
| CN105906735A (en) * | 2016-05-09 | 2016-08-31 | 遵义医学院 | Radix codonopsis homogeneous polysaccharide COP-1, and preparation method and application thereof |
| CN107033255A (en) * | 2017-05-25 | 2017-08-11 | 遵义医学院 | Codonopsis pilosula polysaccharide COP W1 and preparation method and application |
| CN107556399A (en) * | 2017-09-27 | 2018-01-09 | 遵义医学院 | A kind of Radix Codonopsis homogeneous polysaccharide COP W2 and preparation method and application |
| CN108324730A (en) * | 2018-03-09 | 2018-07-27 | 华南农业大学 | Sulphation Codonopsis pilosula polysaccharide is preparing the application in preventing and/or treating irritability lesions of liver and kidney drug |
| CN115286721A (en) * | 2022-07-28 | 2022-11-04 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application of active polysaccharide composition in preparation of product with effect of preventing or treating gastric injury |
| CN115286721B (en) * | 2022-07-28 | 2023-08-25 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application thereof in preparation of products with effect of preventing or treating gastric injury |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104147042A (en) | Application of sulfated codonopsis pilosul polysaccharide to preparation of herpes simplex virus type I resistance medicament and preparation method of herpes simplex virus type I resistance medicament | |
| Lu et al. | Recent advances in antiviral activities and potential mechanisms of sulfated polysaccharides | |
| CN102114044B (en) | Artificially processed bear bile powder and preparation method thereof | |
| CN104710538B (en) | A kind of sanchi flower arabogalactan and its production and use | |
| CN101455736B (en) | Use of wild jujube seeds extract | |
| CN101129473B (en) | Radix fici simplicissimatis extractive and method of producing the same and use thereof | |
| CN105859903B (en) | radix glehniae polysaccharide and preparation method and application thereof | |
| CN105906734A (en) | Radix codonopsis homogeneous polysaccharide COP-2, and preparation method and application thereof | |
| CN102653568A (en) | Herba taraxaci homogeneous polysaccharide and preparation method and application thereof | |
| CN106974943A (en) | A kind of purposes and preparation method of trametes robinioplila alcohol extract | |
| CN105985451A (en) | Chinese angelica acidic polysaccharide component, and preparation method and application thereof | |
| CN104083427A (en) | Compound astragalus polysaccharide particle for preventing and treating avian influenza | |
| CN1290867C (en) | Polysaccharide of echinacea angustifolia | |
| CN105906735A (en) | Radix codonopsis homogeneous polysaccharide COP-1, and preparation method and application thereof | |
| CN106692255A (en) | Vaccaria segetalis polysaccharide hydrolysate and medical application thereof | |
| CN105943523A (en) | Application of dendrophenol in preparing medicines for inhibiting proliferation of cervical carcinoma cells and for inducing autophagic apoptosis of cervical carcinoma cells | |
| CN108752491A (en) | Ultrasound-microwave radiation technology water extraction extracts folium isatidis active polysaccharide technique | |
| CN103118686B (en) | The purposes of Banlangen Polysaccharide in the medicine preparing resisiting influenza virus | |
| CN100362022C (en) | Cuttlebone polysaccharide CPS-1 and its preparation and use | |
| CN100493522C (en) | Medicinal composition of oxymatrine and polysaccharide | |
| CN105753998A (en) | Peach gum polysaccharide degradation product PGP-1 and preparation method and application thereof | |
| CN1256349C (en) | Prepn and use of curcuma oligosaccharide sulfate derivative | |
| CN113546090A (en) | Application of sea urchin polysaccharide in medicine for resisting novel coronavirus or SARS virus infection | |
| CN103040857B (en) | Application of phyllanthus urinaria polysaccharide component in preparing drug for resisting hepatitis B virus | |
| CN101011411A (en) | Usage of algal polysaccharide sulfate in preparation of medicament for treating hepatic disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141119 |
|
| RJ01 | Rejection of invention patent application after publication |