CN102690366B - Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application - Google Patents
Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application Download PDFInfo
- Publication number
- CN102690366B CN102690366B CN201210191422.2A CN201210191422A CN102690366B CN 102690366 B CN102690366 B CN 102690366B CN 201210191422 A CN201210191422 A CN 201210191422A CN 102690366 B CN102690366 B CN 102690366B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- radix chuanminshen
- chuanminshen
- preparation
- dialysis
- 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.)
- Expired - Fee Related
Links
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 96
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 96
- -1 polysaccharide sulfate Chemical class 0.000 title claims abstract description 47
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 230000002155 anti-virotic effect Effects 0.000 title abstract 2
- 150000004676 glycans Chemical class 0.000 claims abstract description 49
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 19
- 241000196324 Embryophyta Species 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 7
- 239000003674 animal food additive Substances 0.000 claims abstract description 6
- 244000144972 livestock Species 0.000 claims abstract description 6
- 201000010099 disease Diseases 0.000 claims abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 4
- 241000685179 Chuanminshen Species 0.000 claims description 76
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 47
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 35
- 238000000502 dialysis Methods 0.000 claims description 35
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000010410 layer Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 229960001701 chloroform Drugs 0.000 claims description 14
- 229960004756 ethanol Drugs 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 14
- XTHPWXDJESJLNJ-UHFFFAOYSA-N sulfurochloridic acid Chemical compound OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 claims description 14
- 238000001291 vacuum drying Methods 0.000 claims description 14
- 230000000840 anti-viral effect Effects 0.000 claims description 13
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000005238 degreasing Methods 0.000 claims description 7
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000012044 organic layer Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 244000144977 poultry Species 0.000 claims description 5
- 241001492342 Anatid alphaherpesvirus 1 Species 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 241000685176 Chuanminshen violaceum Species 0.000 abstract 4
- 241000711404 Avian avulavirus 1 Species 0.000 abstract 1
- 230000032050 esterification Effects 0.000 abstract 1
- 238000005886 esterification reaction Methods 0.000 abstract 1
- 238000007670 refining Methods 0.000 abstract 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 10
- 241000700605 Viruses Species 0.000 description 8
- 241000287828 Gallus gallus Species 0.000 description 7
- 238000011160 research Methods 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 5
- 208000010359 Newcastle Disease Diseases 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 206010035148 Plague Diseases 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 241000271566 Aves Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 206010014599 encephalitis Diseases 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a plant polysaccharide sulfate with immune enhanced activity and anti-virus effect and a preparation method thereof. The preparation method comprises the following steps of: extracting and refining polysaccharide from a Chinese medicament Chuanminshen violaceum; uniformly distributing Chuanminshen violaceum polysaccharide in anhydrous formamide; adding an esterification reagent; and preparing the Chuanminshen violaceum polysaccharide sulfate at a certain temperature. The prepared Chuanminshen violaceum polysaccharide sulfate has immune enhanced activity, in-vitro activity resistant to newcastle disease virus and duck plague virus, and can be used as a biological feed additive which resists diseases, promotes growth and does not have residues in livestock.
Description
Technical field
The present invention relates to a kind of preparation method and the application with the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, be specifically related to a kind of have strengthen immunocompetence, the biology feed additive Radix Chuanminshen Polysaccharide sulfate and preparation method thereof of antiviral activity and application.
Background technology
Medicated feed additive promotes the effect such as growth of animals or poultry and preventing disease because it has, and improves productivity and raises economic benefit, playing an important role in China's livestock and poultry breeding industry.But along with the life-time service of medicated feed additive, there is the problem such as resistance, drug residue, environmental pollution in especially microbiotic, the chemical synthetic drug abuse in feed.This not only lowers the quality of livestock product, also can have influence on human health and the eubiosis by biologic chain.Along with the paces of world market after China joined WTO are accelerated, and the raising that people require quality of life, develop nuisanceless livestock industry, produce the top priority that safe animal products has become herding competent authorities of the Chinese government, find green, safety, efficiently the nuisanceless green fodder additives of fodder additives substitute antibiotics, chemicals etc. become the task of top priority accelerating China's poultry industry Sustainable development.
Herbal polysaccharide is one of main active ingredient of Chinese medicine, its biological activity is subject to extensive concern in recent years, research shows that it both had aboundresources, safety and low toxicity, pollution-free, the advantages such as noresidue, have again antitumor, anti-oxidant, anti-diabetic, antiviral, the function such as radioprotective, immunity moderation, Adjust-blood lipid.But vegetable polysaccharides self does not have some biological activity or biological activity is very weak, thus limit its application.Existing scientific and technical means transformation polysaccharide how is utilized to become a major issue to improve its biological activity.
Summary of the invention
In view of the above-mentioned deficiency of vegetable polysaccharides, the present invention, by a large amount of research and practices, provides a kind of preparation method and the application with the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy.
In order to improve the biological activity of vegetable polysaccharides, present invention employs following technical scheme:
There is a preparation method for the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, polysaccharide in purifying Chinese medicine Radix Chuanminshen, in Radix Chuanminshen polysaccharide, then add esterifying reagent be prepared into Radix Chuanminshen Polysaccharide sulfate.The preparation method of described Radix Chuanminshen polysaccharide is as follows: Radix Chuanminshen dried powder is placed in cable type extractor according, and with ethanol, the ether mixed solution reflux degreasing 2 ~ 6h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature.Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 10 ~ 20 (g/ml), 80 ~ 100 DEG C are extracted 3 times, each 1 ~ 3h, united extraction liquid, be concentrated into minimum volume, with distill water dialysis 12 ~ 24h after flowing water dialysis 24 ~ 48h, the trichloromethane that volume is extracting solution 1/5 is added in dialysis tubing, add the propyl carbinol that volume is trichloromethane 1/5 subsequently again, violent jolting 10 ~ 20min, the denatured protein of centrifugal segregation water layer and organic layer intersection, water layer still removing protein 3 ~ 5 times repeatedly in this way.1 ~ 3 times amount 95% ethanol (V/V) hold over night is added after concentrated.Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 30 ~ 40 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide.The preparation method of described Radix Chuanminshen Polysaccharide sulfate is as follows:
(1) preparation of esterifying reagent: by chlorsulfonic acid and pyridine by according to 1: 6 ~ 1: 10 volume ratio, in cryosel bath and under constantly stirring, dropwise join in the 50mL anhydrous pyridine of precooling, wherein chlorsulfonic acid should dropwise in 20 ~ 30min;
(2) be uniformly distributed in the preparation of the Radix Chuanminshen polysaccharide of dry formamide: join in 10 ~ 20mL dry formamide while stirring by 100 ~ 300mg Radix Chuanminshen polysaccharide, continue stirring 20 ~ 30min and Radix Chuanminshen polysaccharide is uniformly distributed in dry formamide;
(3) esterifying reagent is joined in the dry formamide solution containing Radix Chuanminshen polysaccharide, stirring reaction 1 ~ 2h under 40 ~ 60 DEG C of bath temperatures.After completion of the reaction, be cooled to room temperature, then add 15%NaOH solution adjust pH to 7.0 ~ 8.0, distill water dialysis 12 ~ 24h after flowing water dialysis 48 ~ 72h.Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 12 ~ 24h.
The described application with the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, the biology feed additive as a kind of disease-resistant, growth promotion and noresidue is used for livestock and poultry.
Polysaccharide sulfate in the present invention measures its sulfuric acid substitution value mainly through bariumchloride-gelatin method.The present invention shows through pharmacodynamic study, and Radix Chuanminshen Polysaccharide sulfate has enhancing immunocompetence and antiviral activity.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 300g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 6h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature.Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 20 (g/ml), 100 DEG C are extracted 3 times, each 3h, united extraction liquid, is concentrated into small volume, with distill water dialysis 24h after flowing water dialysis 48h, in dialysis tubing, liquid amasss the trichloromethane into extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 subsequently again, violent jolting 20min, the denatured protein of centrifugal removing water layer and organic layer intersection, water layer still removing protein 5 times repeatedly in this way.3 times amount 95% ethanol (V/V) hold over night are added after concentrated.Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 40 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide.
Prepare esterifying reagent: the volume ratio (V/V) of chlorsulfonic acid and pyridine 1: 6, in cryosel bath and under constantly stirring, chlorsulfonic acid is dropwise joined in the 50mL anhydrous pyridine of precooling;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures.After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h.Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.Measuring its sulfuric acid substitution value through bariumchloride-gelatin method is 1.37.
Embodiment 2
The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 200g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 4h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature.Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 15 (g/ml), 90 DEG C are extracted 2 times, each 2h, united extraction liquid, is concentrated into small volume, with distill water dialysis 18h after flowing water dialysis 36h, in dialysis tubing, liquid adds the trichloromethane that volume is extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 more thereupon, violent jolting 15min, the denatured protein of centrifugal removing water layer and organic layer intersection, water layer still removing protein 3 times repeatedly in this way.2 times amount 95% ethanol (V/V) hold over night are added after concentrated.Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 35 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide.
Prepare esterifying reagent: chlorsulfonic acid, by the volume ratio (V/V) with pyridine 1: 8, in cryosel bath and under constantly stirring, dropwise joins in the 50mL anhydrous pyridine of precooling by chlorsulfonic acid;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures.After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h.Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.Measuring its sulfuric acid substitution value through bariumchloride-gelatin method is 0.95.
Embodiment 3
The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 100g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 4h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature.Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 10,100 DEG C are extracted 2 times, each 1.5h, united extraction liquid, is concentrated into small volume, with distill water dialysis 12h after flowing water dialysis 24h, in dialysis tubing, liquid adds the trichloromethane that volume is extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 more thereupon, violent jolting 10min, the denatured protein of centrifugal branch vibration layer and organic layer intersection, water layer still removing protein 4 times repeatedly in this way.1.5 times amount 95% ethanol (V/V) hold over night are added after concentrated.Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 40 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide.
Prepare esterifying reagent: chlorsulfonic acid, by the volume ratio (V/V) with pyridine 1: 10, in cryosel bath and under constantly stirring, dropwise joins in the 50mL anhydrous pyridine of precooling by chlorsulfonic acid;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures.After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h.Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.Measuring its sulfuric acid substitution value through bariumchloride-gelatin method is 0.69.
Embodiment 4
Radix Chuanminshen Polysaccharide sulfate is studied the immune-enhancing activity of ND-HI titer chick
This test employing 1 age in days Yellow Chicken 200 plumage, stochastic averagina is divided into four groups, comprise the high, medium and low dosage group of Radix Chuanminshen Polysaccharide sulfate and control group, the test chicken of each group when 7 age in days (HI lower than 3log2, mean body weight 120g) carries out live Newcastle disease vaccine immunity by eye droppings collunarium.The chicken of high, medium and low dosage group gives 0.5ml Radix Chuanminshen Polysaccharide sulfate according to the oral dose of 100mg/kg.d, 50mg/kg.d, 25mg/kg.d respectively, the physiological saline of control group then oral 0.5ml, continuous 5d.In 14,21,28 age in days time measure the mensuration of its serum antibody titer, lymphopoiesis, red corpuscle rosettes rate and red blood cell immune complexes rosett.Result shows: Radix Chuanminshen Polysaccharide sulfate significantly can strengthen the level of chick Serum Antibody and can maintain the long period (see table 1); Significantly improve the lymphocytic propagation of T (see table 2); Significantly improve chicken red blood cell rosettes rate (see table 3), improve the speed (see table 4) that it removes immunocomplex.
Table 1 Radix Chuanminshen Polysaccharide sulfate is on the impact of chicken serum antibody
Note: when comparing between high, medium and low dosage group and control group, letter is different represents significant difference (P < 0.05)
The impact that table 2 Radix Chuanminshen Polysaccharide sulfate is bred chicken periphery lymphocyte
Note: when comparing between high, medium and low dosage group and control group, letter is different represents significant difference (P < 0.05)
Table 3 Radix Chuanminshen Polysaccharide sulfate is on the impact of chicken red blood cell rosettes rate
Note: when comparing between high, medium and low dosage group and control group, letter is different represents significant difference (P < 0.05)
Table 4 Radix Chuanminshen Polysaccharide sulfate is on the impact of chicken red blood cell RBC immune complex flower rate rate
Note: when comparing between high, medium and low dosage group and control group, letter is different represents significant difference (P < 0.05)
The above results shows, the Radix Chuanminshen Polysaccharide sulfate prepared by the present invention significantly can strengthen humoral immune function, cellular immune function and the hematid immunity function of ND-HI titer chick.
The activity research of embodiment 5 Radix Chuanminshen Polysaccharide sulfate In Vitro Anti duck plague virus
After DEF grows up to individual layer on 24 orifice plates, add the 50PFU duck plague virus of 100 μ L, add the Radix Chuanminshen Polysaccharide sulfate (sulfuric acid substitution value is respectively 1.37,095 and 0.69) of equivalent volumes different concns, 5%CO simultaneously
2cultivate 24h for 37 DEG C in incubator, then wash out liquid, after PBS cleans 3 times, add the maintenance medium (methylcellulose gum containing 1%) of the Radix Chuanminshen Polysaccharide sulfate containing different concns, 5%CO
2after continuing cultivation 37 DEG C cultivation 72h in incubator, count plaque after violet staining, calculate medium effective concentration (IC according to Reed-Muech method
50).The Radix Chuanminshen Sulfate of polysaccharide of result display different degree of substitution has good anti-duck plague virus activity, and medium effective concentration is 77.12 ~ 104.81 μ g/mL, and substitution value height person antiviral effect is good.The cytotoxic assay of Radix Chuanminshen Sulfate of polysaccharide shows that it has minimum toxicity to cell, half cytotoxic concentration (CC
50) be greater than 2000 μ g/mL.Selectivity index (the CC of the anti-duck plague virus activity of Radix Chuanminshen Polysaccharide sulfate
50/ IC
50) being greater than 19, concrete data are in table 5.
The anti-duck plague virus activity research of table 5 Radix Chuanminshen Polysaccharide sulfate
The activity research of embodiment 6 Radix Chuanminshen Polysaccharide sulfate In Vitro Anti Avian pneumo-encephalitis virus
After hamster kidney cell (BHK-21) grows up to individual layer on 24 orifice plates, add the 50PFU Avian pneumo-encephalitis virus of 100 μ L, add the Radix Chuanminshen Polysaccharide sulfate (sulfuric acid substitution value is respectively 1.37,095 and 0.69) of equivalent volumes different concns, 5%CO simultaneously
2cultivate 24h for 37 DEG C in incubator, then wash out liquid, after PBS cleans 3 times, add the maintenance medium (methylcellulose gum containing 1%) of the Radix Chuanminshen Polysaccharide sulfate containing different concns, 5%CO
2after continuing cultivation 37 DEG C cultivation 72h in incubator, count plaque after violet staining, calculate medium effective concentration (IC according to Reed-Muech method
50).The Radix Chuanminshen Sulfate of polysaccharide of result display different degree of substitution has good anti-new castle disease virus activity, and medium effective concentration is 86.43 ~ 113.97 μ g/mL, and substitution value height person antiviral effect is good.The cytotoxic assay of Radix Chuanminshen Sulfate of polysaccharide shows that it has minimum toxicity to cell, half cytotoxic concentration (CC
50) be greater than 2000 μ g/mL.Selectivity index (the CC of Radix Chuanminshen Polysaccharide sulfate anti-new castle disease virus activity
50/ IC
50) being greater than 17, concrete data are in table 6.
Table 6 Radix Chuanminshen Polysaccharide sulfate anti-new castle disease virus activity research
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (4)
1. one kind has the preparation method of the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, it is characterized in that: polysaccharide in purifying Chinese medicine Radix Chuanminshen, in Radix Chuanminshen polysaccharide, then add esterifying reagent be prepared into Radix Chuanminshen Polysaccharide sulfate; The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 300g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 6h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature; Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 20 (g/ml), 100 DEG C are extracted 3 times, each 3h, united extraction liquid, is concentrated into small volume, with distill water dialysis 24h after flowing water dialysis 48h, in dialysis tubing, liquid amasss the trichloromethane into extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 subsequently again, violent jolting 20min, the denatured protein of centrifugal removing water layer and organic layer intersection, water layer still removing protein 5 times repeatedly in this way; 3 times amount 95% ethanol (V/V) hold over night are added after concentrated; Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 40 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide;
Prepare esterifying reagent: the volume ratio (V/V) of chlorsulfonic acid and pyridine 1: 6, in cryosel bath and under constantly stirring, chlorsulfonic acid is dropwise joined in the 50mL anhydrous pyridine of precooling;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures; After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h; Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.
2. one kind has the preparation method of the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, it is characterized in that: polysaccharide in purifying Chinese medicine Radix Chuanminshen, in Radix Chuanminshen polysaccharide, then add esterifying reagent be prepared into Radix Chuanminshen Polysaccharide sulfate; The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 200g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 4h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature; Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 15 (g/ml), 90 DEG C are extracted 2 times, each 2h, united extraction liquid, is concentrated into small volume, with distill water dialysis 18h after flowing water dialysis 36h, in dialysis tubing, liquid adds the trichloromethane that volume is extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 more thereupon, violent jolting 15min, the denatured protein of centrifugal removing water layer and organic layer intersection, water layer still removing protein 3 times repeatedly in this way; 2 times amount 95% ethanol (V/V) hold over night are added after concentrated; Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 35 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide;
Prepare esterifying reagent: chlorsulfonic acid, by the volume ratio (V/V) with pyridine 1: 8, in cryosel bath and under constantly stirring, dropwise joins in the 50mL anhydrous pyridine of precooling by chlorsulfonic acid;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures; After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h; Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.
3. one kind has the preparation method of the Plant polysaccharide sulfate compound of immune-enhancing activity and antiviral efficacy, it is characterized in that: polysaccharide in purifying Chinese medicine Radix Chuanminshen, in Radix Chuanminshen polysaccharide, then add esterifying reagent be prepared into Radix Chuanminshen Polysaccharide sulfate; The preparation of Radix Chuanminshen polysaccharide: the Radix Chuanminshen dried powder taking 100g is placed in cable type extractor according, with ethanol, the ether mixed solution reflux degreasing 4h of 1: 1 (V/V), after filtering, the dregs of a decoction are dry under being placed in room temperature; Distilled water is added in the dregs of a decoction by solid-liquid ratio 1: 10,100 DEG C are extracted 2 times, each 1.5h, united extraction liquid, is concentrated into small volume, with distill water dialysis 12h after flowing water dialysis 24h, in dialysis tubing, liquid adds the trichloromethane that volume is extracting solution 1/5, adds the propyl carbinol that volume is trichloromethane 1/5 more thereupon, violent jolting 10min, the denatured protein of centrifugal branch vibration layer and organic layer intersection, water layer still removing protein 4 times repeatedly in this way; 1.5 times amount 95% ethanol (V/V) hold over night are added after concentrated; Centrifugal collecting precipitation, after using dehydrated alcohol, washing with acetone successively, centrifugation, throw out is placed in 40 DEG C of vacuum-dryings and obtains Radix Chuanminshen polysaccharide;
Prepare esterifying reagent: chlorsulfonic acid, by the volume ratio (V/V) with pyridine 1: 10, in cryosel bath and under constantly stirring, dropwise joins in the 50mL anhydrous pyridine of precooling by chlorsulfonic acid;
300mg Radix Chuanminshen polysaccharide is joined in 20mL dry formamide while stirring, continues to stir 30min and polysaccharide is evenly distributed in dry formamide; Then esterifying reagent is added, stirring reaction 2h under 60 DEG C of bath temperatures; After completion of the reaction, be cooled to room temperature, then add mass percentage concentration be 15%NaOH solution adjust pH to 7.0-8.0, flowing water dialysis 72h after distill water dialysis 24h; Alcohol settling, acetone, anhydrous diethyl ether wash respectively, obtain Radix Chuanminshen Polysaccharide sulfate after vacuum-drying 24h.
4. the application of Plant polysaccharide sulfate compound prepared by the method according to any one of claim 1-3, the biology feed additive as a kind of disease-resistant, growth promotion and noresidue is used for livestock and poultry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210191422.2A CN102690366B (en) | 2012-06-11 | 2012-06-11 | Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210191422.2A CN102690366B (en) | 2012-06-11 | 2012-06-11 | Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102690366A CN102690366A (en) | 2012-09-26 |
| CN102690366B true CN102690366B (en) | 2015-01-21 |
Family
ID=46856117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210191422.2A Expired - Fee Related CN102690366B (en) | 2012-06-11 | 2012-06-11 | Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102690366B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103520201B (en) * | 2013-07-08 | 2015-09-30 | 南京农业大学 | A kind of have the polygonatum polysaccharide of anti-new castle disease virus effect and the compositions of sulphation Radix Codonopsis polysaccharide |
| CN103554297B (en) * | 2013-10-21 | 2016-09-07 | 兰州大学 | A kind of preparation method and applications of high-sulfation-degree radix hedysari polysaccharide sulfate |
| CN103739733B (en) * | 2014-01-09 | 2016-03-09 | 南京农业大学 | A kind of anti-duck viral hepatitis Radix Sophorae Tonkinensis polysaccharide and molecular modification thing thereof |
| CN104177470B (en) * | 2014-03-18 | 2017-03-08 | 四川农业大学 | A kind of effective ways extracting protein with Radix Chuanminshen slag |
| CN104177483B (en) * | 2014-06-20 | 2017-03-08 | 四川农业大学 | A kind of method that synchronous high-efficiency prepares Radix Chuanminshen albumen and polysaccharide |
| CN108203473A (en) * | 2017-05-03 | 2018-06-26 | 南昌大学 | A kind of new antitumoral compounds obtained by sulphation |
| CN110776579A (en) * | 2019-11-14 | 2020-02-11 | 济宁医学院 | Process for optimizing sulfuric acid esterification of radix scrophulariae polysaccharide by using response surface method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781372A (en) * | 2010-03-11 | 2010-07-21 | 南京农业大学 | Sulphation modification method of codonopsis pilosula polysaccharide |
-
2012
- 2012-06-11 CN CN201210191422.2A patent/CN102690366B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781372A (en) * | 2010-03-11 | 2010-07-21 | 南京农业大学 | Sulphation modification method of codonopsis pilosula polysaccharide |
Non-Patent Citations (2)
| Title |
|---|
| 中药多糖在兽医临床的应用与展望;马士龙;《养殖技术顾问》;20120305;第224和275页 * |
| 川明参多糖的提取工艺;张梅等;《华西药学杂志》;20070620;第22卷(第3期);第361-362页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102690366A (en) | 2012-09-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102690366B (en) | Preparation method of plant polysaccharide sulfate with immune enhanced activity and anti-virus effect, as well as application | |
| Wei et al. | Effects of Taishan Pinus massoniana pollen polysaccharide on immune response of rabbit haemorrhagic disease tissue inactivated vaccine and on production performance of Rex rabbits | |
| CN103262943B (en) | A kind of Herba Lophatheri extract and application thereof | |
| CN105722974A (en) | Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method | |
| CN102225146B (en) | Formula and production technology of traditional Chinese medicine injection for controlling bird flu | |
| CN101342266A (en) | Anti-newcastle disease virus traditional Chinese medicine oral liquid and preparation method thereof | |
| CN101209258B (en) | Immunopotentiator and antiviral composition for animal | |
| CN103720932B (en) | One boar Chinese medicine composition and its preparation method and application | |
| CN100586454C (en) | Chinese medicine composition for birds, preparing method and application of the same | |
| CN102872307A (en) | Traditional Chinese medicine oral liquid for preventing and treating virus diseases of poultry and method for preparing traditional Chinese medicine oral liquid | |
| CN104083433A (en) | External veterinary medicine composition for treating animal scabies, and application and preparation method thereof | |
| CN104435023A (en) | Traditional Chinese medicine extract and application thereof | |
| CN101670005B (en) | Anticoccidiosis pharmaceutical composition and preparation method and use thereof | |
| CN102670694A (en) | Traditional Chinese medicine combination for treating porcine pseudorabies and preparation method and application thereof | |
| CN102100730B (en) | Traditional Chinese medicine for improving poultry immunity | |
| CN102727579A (en) | Pharmaceutical composition for treatment of oral ulcers | |
| CN103610738A (en) | Traditional Chinese medicine particle for preventing and treating avian influenza and improving chicken flock immunity and preparation method thereof | |
| CN106798924A (en) | String is preparing the application of prevention chicken coccidiasis medicine | |
| CN103463628B (en) | A kind ofly to be used for the treatment of or the pharmaceutical composition of prophylactic treatment senile dementia and preparation thereof | |
| CN101549011B (en) | Application of tassel herbamoslae in preparing medicaments for treating infectious diseases of beasts and birds | |
| CN103143012B (en) | Traditional Chinese medicine composition and applications in preparation of porcine pseudorabies vaccine immunopotentiator | |
| CN101940613A (en) | Codonopsis pilosula and radix astragali oral solution as well as preparation method and application thereof on aspect of enhancing immunologic functions of livestock and poultry | |
| CN104758340B (en) | Coffee mesitoyl quinine acid extract and its preparation method and application in a kind of folium lonicerae | |
| CN104352826B (en) | Membranous milkvetch stem and leaf is the herbal medicine and preparation method of main ingredient | |
| CN104744448B (en) | Isoflavone compounds, its pharmaceutical composition and its production and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160330 Address after: 611130, Chengdu District, Sichuan City, Wenjiang province cross-strait science and Technology Park, Baili Road Patentee after: KUNHONG ANIMAL PHARMACEUTICAL CO.,LTD. Address before: 610064 Sichuan city of Chengdu province Wuhou District Guo Jia Qiao North Street 5 No. 14 Building 12 floor No. 7 Patentee before: Yin Zhongqiong |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150121 |