CN102786603B - Method for preparing chang date antineoplastic active polysaccharides by separation and purification - Google Patents

Abstract

The invention relates to a preparation method of chang date antineoplastic active polysaccharides by separation and purification, characterized by using Ningxia Lingwu chang date as a raw material. The separation and purification process is characterized by 1) preparing a DEAE-52 cellulose packed column: conducting alkali washing, pickling, alkali washing, ultrapure water washing respectively by using 0.5-1mol/L sodium hydroxide solution, ultrapure water, and 0.5-1mol/L hydrochloric acid solution, and filling in a packed column; 2) letting an extract processed by decoloring and dialyzing pass through the cellulose packed column, conducting gradient elution respectively by using double distilled water and 0.1-0.5mol/L NaCl solution, determining the absorbance to no effusion of sugar by a spectro photometer, and collecting ingredients according to the peak condition, wherein the flow velocity of each tube is 3-4min, and 5ml is collected in each tube; 3) dialyzing with a dialysis bag to desalt, condensing, then conducting alcohol precipitation, centrifugation and freeze drying; and 4) purifying by a DEAE-52 gel column to obtain classified and purified chang date single-component polysaccharides(LJU-0, LJU-1, LJU-2, LJU-3, LJU-4, LJU-5).

Description

A kind of preparation method for separating and purifying of long jujube anti-tumor activity polysaccharide

Technical field

The present invention relates to a kind of preparation method of polysaccharide, particularly relate to a kind of preparation method for separating and purifying of long jujube anti-tumor activity polysaccharide.

Background technology

Polysaccharide is the isolating protein, the another bioactive macromolecule outside nucleic acid that exist in natural product, phenotype and the antigenicity structure of some cells and molecule is determined as informational molecule, controlling cell fission or differentiation, the growth of adjustment cell and aging, is a kind of immunopotentiating agent of acellular poison.The a series of physiology of animal body and pathologic process and sugar chain participate in intercellular identification direct relation.Just there are 500,000 glycoprotein molecules on an erythrocyte surface, and in human body on immunoglobulin IgG sugar chain monose composition and the change a little of sugared shape will cause a certain disease, as will similar rheumatism be produced when semi-lactosi in sugar chain lower than during normal people.Therefore, in recent years, both at home and abroad the research of natural product polysaccharide is attached great importance to.Natural polysaccharide, because of the function of its uniqueness and very low toxicity, obtains development and utilization in antiviral drug, the medicines that delay senility etc. such as antitumour drug, treatment acquired immune deficiency syndrome (AIDS) are several.In the pharmacology activity research of jujube polysaccharide, the research of Yamada etc. shows that the polysaccharide that separation and purification obtains from jujube has immunocompetence; The research of friend Tian Zhengsi shows that Japanese jujube polysaccharide has antineoplastic pharmacologically active; The research of ZhihuiZhao etc. shows, a kind of neutral polysaccharide in jujube fruit and 4 kinds of acidic polysaccharoses show good anti-tumor activity.Domestic much research shows, jujube polysaccharide can strengthen the activity of Turnover of Mouse Peritoneal Macrophages; Mouse spleen cell proliferation can be promoted; And there is oxyradical in purged body, antidotal effect, and take part in cell signaling.

Patent report about jujube Polyose extraction mainly contains in recent years: Shen Lin and Sheng Jiping " utilize red date to produce method and the jujube polysaccharide of jujube polysaccharide " (application number CN201010606048.9), big and the model Liu Ping of lijin " a kind of Golden jujube acidic polysaccharose extracts and structural characterization " (application number CN201010580105.0), Liu Dongfeng, Guo Qin and Yang Chengdong " a kind of preparation method of hovenia polysaccharides " (application number CN201010558105.0), Liu Yulin, Sun Shuguang and Wang Zhongshan " method for refining jujube polysaccharide " (application number CN201010102224.5), Wang Qingning, Zhao Qiuping, Lv Xinglian, Zhang Fei dragon, Li Lan and Wang Zhengmin " extracting method of narrow-leaved oleaster polysaccharide " (application number CN200910021709.9), Zhao Yuying " fructus choerospondiatis polysaccharide and reparation technology thereof " (application number CN200710117623.7), Liu Jinfu, Zhang Pingping and Li Tuoping " extracting method of polysaccharide from winter jujub " (application number CN200610014444.6).These patent reports have greatly enriched long jujube Polyose extraction means of purification, but do not study for the distinctive Dominant variety in this Ningxia of long jujube.

Summary of the invention

The object of the invention is to the deficiency overcoming prior art, a kind of preparation method for separating and purifying of long jujube anti-tumor activity polysaccharide is provided.

The present invention is achieved in that its processing step is:

A preparation method for separating and purifying for long jujube anti-tumor activity polysaccharide, with Ningxia produce Lingwu Long Jujube for raw material, its technological process comprise pulverize and sieve, water-bath lixiviate, decolouring, separation and purification, lyophilize, its separation and purification process is:

1) prepare DEAE-52 Mierocrystalline cellulose packed column: DEAE-52 Mierocrystalline cellulose carries out alkali cleaning-pickling-alkali cleaning-ultrapure washing process with the sodium hydroxide solution of 0.5 ~ 1mol/L, ultrapure water, 0.5 ~ 1mol/L hydrochloric acid soln respectively, load packed column;

2) by through decolouring and dialysis after extracting solution by being equipped with cellulosic packed column, gradient elution is carried out respectively by the NaCl solution of distilled water and 0.1 ~ 0.5mol/L, often pipe flow velocity is 3 ~ 4min, often 5ml collected by pipe, measure its absorbancy with spectrophotometer to flow out to sugar-free, press out peak situation and collect component;

3) dialysis tubing dialysis desalination, alcohol precipitation after concentrated, centrifugal freeze-drying;

4) cross DEAE-52 gel column and carry out purifying, obtain the long jujube one-component polysaccharide (LJU-0, LJU-1, LJU-2, LJU-3, LJU-4, LJU-5) of grading purification;

Above-mentioned pulverizing and sieving is carried out on high speed disintegrator, by raw material through 50 ~ 60 DEG C of dryings, dry in the air cool after, use high speed disintegrator by raw material pulverizing, the raw material crushed is crossed 80 mesh sieves, sieve upper part return repeat pulverize, sieve;

The temperature of above-mentioned water-bath lixiviate is 80 ~ 100 DEG C;

Above-mentioned lyophilize is that isolated polysaccharide liquid is concentrated into about 1/3 of original volume in vacuum decker, then carries out vacuum lyophilization;

Above-mentioned decolouring is repeatedly decoloured through active carbon powder core post by extracting solution, no longer changes to liquid color.Add again dehydrated alcohol settle out after freeze-drying stand-by.

Characteristic of the present invention is to reach in long jujube polysaccharide extracting process and improves the object such as extraction yield, shortening extraction time, minimizing energy consumption, the effectively long jujube polysaccharide biological activity of preservation, and purifying obtains an one-component polysaccharide (LJU-3), the present invention compensate for the content of long jujube polysaccharide and the unclear deficiency of anti-tumor activity.

The product that application the present invention obtains has carried out inhibition tumor cell proliferation experiment (mtt assay):

The inoculum size of cancer cells is 180 μ L/ holes, constant temperature culture exhausts after about 24 hours nutrient solution in micropore the testing compound nutrient solution added containing different concns, positive controls adds 20 μ L5-Fluracils makes its final concentration be 100mg/L, negative control adds 20 μ L RPMI1640 complete culture solutions, continues constant temperature culture.Then add in 20 μ L MTT (5mg/Ml) to each micropore, cultivate 4 hours at 37 DEG C.Incline liquid all in clean micropore, and air natural is dry, and every hole adds the acid primary isoamyl alcohol (200 μ L primary isoamyl alcohol add 0.05MHCl) of 200 μ L, and on plate shaker, shake 30min, 490nm enzyme-linked immunosorbent assay instrument measures light absorption value.Test design often processes repetition six times, and result is the mean value of six groups of data.

In the present invention, as follows to long jujube anti-tumor activity of polysaccharides experimental result:

(1) to LJU-3, in cell culture fluid, LJU-3 can significantly suppress human hepatoma cell strain Bel7402 with the level of 198 ~ 400mg/L, and 178 ~ 400mg/L can significantly suppress human gastric carcinoma cell line BGC 823,167 ~ 400mg/L significantly can suppress human nasopharyngeal carcinoma cell line KB.

(2) within the scope of 50 ~ 400mg/L, LJU-0, LJU-1, LJU-2, LJU-4 and LJU-5 inhibiting rate to Bel7402, BGC823 and KB tri-kinds of tumour cells is all less than 50%, illustrates that they are insensitive to these three kinds of tumours.

Result shows, long jujube polysaccharide energy anticancer propagation in vitro, the inhibiting rate of propagation means the suppression to cancer, experiment proves that long jujube polysaccharide can significantly suppress human hepatoma cell strain Bel7402, human gastric carcinoma cell line BGC 823 and human nasopharyngeal carcinoma cell line KB, long jujube polysaccharide has antitumour activity in other words.

Technique effect of the present invention is: overcome the deficiency that long jujube anti-tumor activity of polysaccharides is unclear, to improve long jujube yield, effectively preserves long jujube polysaccharide biological activity.

Embodiment

Choose dry Ningxia Lingwu Long Jujube, by raw material through 50 ~ 60 DEG C of dryings, dry in the air cool after, use high speed disintegrator by raw material pulverizing, the raw material crushed crossed 80 mesh sieves, the bits on sieve top is reentered in pulverizer and again pulverizes.By the jujube powder sample of certain mass with solid-to-liquid ratio 1:4 at 80 ~ 100 DEG C of water-bath lixiviate 2 hours.After centrifuging, filter residue is re-started water-bath lixiviate, repeatedly carry out 3 times.

The clear liquid obtained is decoloured through active carbon powder core post repeatedly, no longer changes to liquid color.Add dehydrated alcohol settle out after freeze-drying stand-by.

DEAE-52 Mierocrystalline cellulose is carried out alkali cleaning-pickling-alkali cleaning-ultrapure washing process with the sodium hydroxide solution of 0.5 ~ 1mol/L, ultrapure water, 0.5 ~ 1mol/L hydrochloric acid soln respectively.By decolouring and the polysaccharide liquid of dialyse by being equipped with cellulosic packed column, film used of dialysing is Mierocrystalline cellulose semi-permeable membranes, utilizes reverse osmosis theory, water and small molecules is gone out; Carry out gradient elution by the NaCl solution of distilled water and 0.1 ~ 0.5mol/L respectively, flow velocity is 1 pipe/7 minute, and often 10ml collected by pipe, measures its absorbancy flow out to sugar-free with spectrophotometer, presses out peak situation and collects component.Dialysis tubing dialysis desalination, alcohol precipitation after concentrated, centrifugal freeze-drying.Finally cross DEAE-52 gel column and carry out purifying, obtain the long jujube one-component polysaccharide of grading purification.

Isolated polysaccharide sample is carried out being concentrated into about 1/3 of original volume in vacuum decker, the liquid glucose concentrated is carried out vacuum lyophilization.

Claims (1)

1. a preparation method for separating and purifying for long jujube anti-tumor activity polysaccharide, with Ningxia produce Lingwu Long Jujube for raw material, its technological process comprise pulverize and sieve, water-bath lixiviate, decolouring, separation and purification, lyophilize, it is characterized in that separation and purification process is:

1) preparation is by DEAE-52 Mierocrystalline cellulose packed column: DEAE-52 Mierocrystalline cellulose uses the sodium hydroxide solution of 0.5 ~ 1mol/L, ultrapure water, 0.5 ~ 1mol/L hydrochloric acid soln to carry out alkali cleaning-pickling-alkali cleaning-ultrapure washing process respectively, loads packed column;

2) by through decolouring and dialysis after extracting solution by being equipped with cellulosic packed column, the NaCl solution of distilled water and 0.1 ~ 0.5mol/L is used to carry out gradient elution respectively, often pipe flow velocity is 3 ~ 4min, often 5ml collected by pipe, measure its absorbancy with spectrophotometer to flow out to sugar-free, press out peak situation and collect component;

3) dialysis tubing dialysis desalination, alcohol precipitation after concentrated, centrifugal freeze-drying;

4) cross DEAE-52 gel column and carry out purifying, obtain the long jujube one-component polysaccharide LJU-0 of grading purification, LJU-1, LJU-2, LJU-3, LJU-4, LJU-5;

Above-mentioned pulverizing and sieving is carried out on high speed disintegrator, by raw material through 50 ~ 60 DEG C of dryings, dry in the air cool after, use high speed disintegrator by raw material pulverizing, the raw material crushed is crossed 80 mesh sieves, sieve upper part return repeat pulverize, sieve;

The temperature of above-mentioned water-bath lixiviate is 80 ~ 100 DEG C;

Above-mentioned lyophilize is that isolated polysaccharide liquid is concentrated into about 1/3 of original volume in vacuum decker, then carries out vacuum lyophilization;

Above-mentioned decolouring is repeatedly decoloured through active carbon powder core post by extracting solution, no longer changes to liquid color, then add dehydrated alcohol settle out after freeze-drying stand-by.