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
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- -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
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- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 19
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- 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
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- 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
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- 241001492342 Anatid alphaherpesvirus 1 Species 0.000 abstract description 3
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- 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.
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