CN103483462A - Preparation method for phascolosoma esculenta polysaccharide and application of phascolosoma esculenta polysaccharide in treating septicemia - Google Patents

Abstract

The invention discloses a preparation method for a phascolosoma esculenta polysaccharide and application of the phascolosoma esculenta polysaccharide. Endogenous enzyme killing is firstly performed on phascolosoma esculenta power, then enzymolysis is performed on the phascolosoma esculenta power through a combined enzyme formed by subtilisin and Alacase protease, and after tri-filtering on the enzymatic hydrolysate, the phascolosoma esculenta polysaccharide is obtained through vacuum concentration and drying. The preparation method has the advantages that the preparation is simple, the deproteinization process and the ethyl alcohol precipitation process are eliminated, the production period is shortened, energy conservation, emission reduction and environment protection are facilitated, the enzymolysis effect is still good, and the content of polysaccharide in the phascolosoma esculenta polysaccharide can reach 50 to 70 percent. The phascolosoma esculenta polysaccharide has the effect of resisting septicemia.

Description

A kind of preparation method of Phascolosoma polysaccharide and antisepsis application thereof

Technical field

The present invention relates to the making method of siphon-worm polysaccharide, be specifically related to the application at antisepsis of a kind of preparation method of Phascolosoma polysaccharide and Phascolosoma polysaccharide.

Background technology

Phascolosoma ( phascolosoma esculenta), be under the jurisdiction of the sipunculan door, be China's native species, be distributed widely in the coastland on the south entrance of Changjiang River, output is abundant.Phascolosoma is the marine organisms of high protein and low fat.Having put down in writing its edible and pharmaceutical use in the multiple book on Chinese herbal medicine of China, is China's folk tradition medicine food excellent tonic product, and some area replaces Chinese medicine Cordyceps sinensis with it.The various active materials such as Phascolosoma rich in proteins and polysaccharide, can regulate the multiple function of body.For a long time, Phascolosoma is mainly used in eating, but the application for a patent for invention of publication number CN102153666A, the preparation method of ocean siphon-worm polysaccharide and the application in anti-fatigue effect are disclosed, its preparation method is the pancreatin solution added in the siphon-worm raw material under alkaline solution or alkalescence, then heating is extracted and is obtained Crude polysaccharides, also disclose in this application for a patent for invention with trichoroacetic acid(TCA) and sloughed siphon-worm albumen and ethanol alcohol precipitation process (generally need spend the night or standing 12h), after deproteinated, the siphon-worm polysaccharide content can reach more than 80%.This application for a patent for invention discloses the effect that the siphon-worm polysaccharide has antifatigue, specifically can increase mouse swimming time etc.

Summary of the invention

The purpose of this invention is to provide a kind of Phascolosoma polyoses producing method, the method has been save deproteinated technique and ethanol alcohol precipitation process, makes simply, and in the Phascolosoma polysaccharide obtained, polysaccharide content still reaches 50～70%.The invention also discloses the effect that the Phascolosoma polysaccharide has antisepsis.

Technical problem to be solved by this invention is a kind of preparation method of Phascolosoma polysaccharide, and step is as follows:

A, by impurity elimination, after going the Phascolosoma of internal organ to clean, pulverize, the endogenous enzyme 10min that goes out at 100 ℃ of temperature, obtain the Phascolosoma powder;

B, add distilled water and combination enzyme in the Phascolosoma powder, stir as Phascolosoma solution, Phascolosoma solution is enzymolysis 1.5～2.5h under 45～55 ℃ of temperature condition, enzyme 15min goes out after enzymolysis under 85 ℃, go out and filter and obtain enzymolysis solution with 80～100 orders after enzyme, wherein in Phascolosoma solution, the weight ratio of Phascolosoma powder and distilled water is 1:5～10, in Phascolosoma solution, the weight percent final concentration of combination enzyme is 0.1～0.5%, the combination enzyme is formulated by subtilisin and the Alacase proteolytic enzyme of weight ratio 1:1,

C, by aperture 0.45mm filtering with microporous membrane for above-mentioned enzymolysis solution, the ultra-filtration membrane ultrafiltration that filtrate is 5000 ~ 10000 Da with molecular weight cut-off again obtains ultrafiltrated, ultrafiltrated vacuum concentration drying obtains the Phascolosoma polysaccharide.

The application of Phascolosoma polysaccharide in preparing the antisepsis medicine.Every day, oral dosage can be 100～500mg/day kg BW etc.

Compared with prior art the invention has the advantages that a kind of preparation method of Phascolosoma polysaccharide and the application of Phascolosoma polysaccharide, the Phascolosoma powder endogenous enzyme of first going out, the combination enzyme enzymolysis formed by subtilisin and Alacase proteolytic enzyme again, after the enzymolysis solution three-stage filtration, the vacuum concentration drying obtains the Phascolosoma polysaccharide, this preparation method makes simply, deproteinated technique and ethanol alcohol precipitation process have been save, shorten the production cycle, be conducive to reduce discharging energy-conservation and protection of the environment, hydrolysis result is still better, in the Phascolosoma polysaccharide obtained, polysaccharide content still reaches 50～70%.This Phascolosoma polysaccharide has the effect of antisepsis.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

Embodiment 1

Phascolosoma is removed to the impurity such as silt, internal organ, grinds after cleaning, the endogenous enzyme 10min that goes out at 100 ℃ of temperature, obtain 10 kilograms, Phascolosoma powder after cooling; Add 75 kilograms of distilled water and 0.2 kilogram of combination enzyme (subtilisin of weight ratio 1:1 and the preparation of Alacase proteolytic enzyme) in the Phascolosoma powder, stir as Phascolosoma solution, Phascolosoma solution is enzymolysis 1.5h under 45～55 ℃ of temperature condition, the enzyme 15min that goes out under 85 ℃ after enzymolysis, filter and obtain enzymolysis solution with 80～100 orders after the enzyme that goes out; By aperture 0.45mm filtering with microporous membrane for enzymolysis solution, the ultra-filtration membrane ultrafiltration that filtrate is 5000 ~ 10000 Da with molecular weight cut-off again obtains ultrafiltrated, ultrafiltrated vacuum concentration drying obtains the Phascolosoma polysaccharide, and measuring polysaccharide content in the Phascolosoma polysaccharide is 62%.

Embodiment 2

Substantially the same manner as Example 1, difference be distilled water be 50 kilograms, the combination enzyme be 0.07 kilogram, enzymolysis time is 2.5h, in the Phascolosoma polysaccharide, polysaccharide content is 50%.

Embodiment 3

Substantially the same manner as Example 1, difference be distilled water be 100 kilograms, the combination enzyme be 0.54 kilogram, enzymolysis time is 2.0h, in the Phascolosoma polysaccharide, polysaccharide content is 70%.

Application examples

300 mouse are divided into to siphon-worm polysaccharide group and control group at random, every group each 150, the continuous gavage of Phascolosoma polysaccharide of siphon-worm polysaccharide group use above-described embodiment 1 30 days, (200mg/day kg BW), 200 mg/day kg BW continuous normal saline gavage 30 days for control group, gavage finishes the vetanarcol anesthesia with 80 mg/kg by mouse, cut abdominal cavity and take out caecum, use the cotton thread ligation under ileocaecal sphineter, twice of No. 22 pinprick of caecum employing of ligation part, prepare ripe mouse septicemia model, caecum is put back to abdominal cavity, sew up abdominal cavity, obtain the septicemia mouse model.1, the septicemia mouse model to setting up, get each 50 of siphon-worm polysaccharide group and control groups, observes survival condition the record of a mouse every day, continues 8 days; After 8 days, control group septicemia mouse model is entirely dead, and 18 survivals of siphon-worm polysaccharide group septicemia mouse model illustrate that the siphon-worm polysaccharide can improve the survival rate of suffering from the septicemia mouse, and survival rate reaches 42%.2, to the septicemia mouse model of setting up, get each 50 of siphon-worm polysaccharide group and control groups, observe 3 days mouse of survival afterwards, get the mouse whole blood and win Mouse Liver and the spleen tissue, respectively to liver, after each tissue homogenate of spleen and blood, spread plate, 37 ° of C cultivate 18 h, calculate the colony number of each sample, the mouse colony number of siphon-worm polysaccharide group septicemia mouse model as a result: 17500 ± 531/milligram tissues of hepatic tissue colony number average out to, spleen is organized 51000 ± 648/milligram tissues of colony number average out to, 171000 ± 862/milligram tissues of haemal tissue colony number average out to, the mouse colony number of control group septicemia mouse model: 43200 ± 963/milligram tissues of hepatic tissue colony number average out to, spleen is organized 84600 ± 1854/milligram tissues of colony number average out to, 697000 ± 3731/milligram tissues of haemal tissue colony number average out to, illustrate that the Phascolosoma polysaccharide can reduce the pathogenic bacterium number of septicemia mouse.3, the septicemia mouse model to setting up, get each 50 of siphon-worm polysaccharide group and control groups, get the mouse whole blood lived after observing 24 h, add antithrombotics in blood, centrifugal 15 min of 3500 rpm, get blood plasma, adopt Enzyme Linked Immunoadsorbent Assay to detect proinflammatory inflammation factor interleukin-11 β and tumor necrosis factor alpha, the expression of anti-inflammatory factor IL-10, as a result in the mice plasma of siphon-worm polysaccharide group septicemia mouse model: interleukin-11 β average out to 665 ± 31pg/ml, tumor necrosis factor alpha average out to 574 ± 47pg/ml, IL-10 average out to 951 ± 62pg/ml; In the mice plasma of control group septicemia mouse model: interleukin-11 β average out to 1131 ± 91pg/ml, tumor necrosis factor alpha average out to 1263 ± 89pg/ml, IL-10 average out to 1013 ± 93pg/ml; Illustrate that the Phascolosoma polysaccharide can obviously reduce proinflammatory inflammation factor interleukin-11 β and the expression of tumor necrosis factor alpha in blood plasma in the septicemia mouse, and do not affect the expression of anti-inflammatory factor IL-10.

Toxicology detects

The Phascolosoma polysaccharide that adopts 1000 mg/day kg BW and two kinds of dosage of 10000 mg/day kg BW is continuously to mouse stomach 30 days, the mouse of 1000 mg/day kg BW dosage gavages does not demonstrate obvious toxicity sign as a result, and death does not occur the mouse of 10000 mg/day kg BW dosage gavages.

Claims (2)

1. the preparation method of a Phascolosoma polysaccharide is characterized in that step is as follows:

A, by impurity elimination, after going the Phascolosoma of internal organ to clean, pulverize, the endogenous enzyme 10min that goes out at 100 ℃ of temperature, obtain the Phascolosoma powder;

B, add distilled water and combination enzyme in the Phascolosoma powder, stir as Phascolosoma solution, Phascolosoma solution is enzymolysis 1.5～2.5h under 45～55 ℃ of temperature condition, enzyme 15min goes out after enzymolysis under 85 ℃, go out and filter and obtain enzymolysis solution with 80～100 orders after enzyme, wherein in Phascolosoma solution, the weight ratio of Phascolosoma powder and distilled water is 1:5～10, in Phascolosoma solution, the weight percent final concentration of combination enzyme is 0.1～0.5%, the combination enzyme is formulated by subtilisin and the Alacase proteolytic enzyme of weight ratio 1:1,

C, by aperture 0.45mm filtering with microporous membrane for above-mentioned enzymolysis solution, the ultra-filtration membrane ultrafiltration that filtrate is 5000 ~ 10000 Da with molecular weight cut-off again obtains ultrafiltrated, ultrafiltrated vacuum concentration drying obtains the Phascolosoma polysaccharide.

2. the application of Phascolosoma polysaccharide in preparing the antisepsis medicine.