CN102219865A - Preparation method of cherokee rose polysaccharide derivatives with antitumor activity - Google Patents
Preparation method of cherokee rose polysaccharide derivatives with antitumor activity Download PDFInfo
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Abstract
The invention discloses a preparation method of cherokee rose polysaccharide derivatives with an antitumor activity. The preparation method comprises the following steps: firstly smashing cherokee rose fruit, degreasing, removing oligosaccharide, lixiviating with hot water twice, then mixing extracts, filtrating, concentrating, precipitating and drying so as to obtain crude cherokee rose polysaccharide; adding diethylin ethyl amino ethyl (DEAE)-cellulose 52 to carry out static absorption for 30 minutes; washing with distilled water, filtering, and collecting filtrate so as to obtain the decolored crude cherokee rose polysaccharide; carrying out protein removal on a crude polysaccharide solution by using an enzyme method and a Sevag method; further separating the crude cherokee rose polysaccharide with the DEAE-cellulose 52, collecting, dialyzing, and freeze-drying so as to obtain cherokee rose polysaccharide; and finally, carrying out sulphating, carboxymethylation and hydrochloric acid degradation on the cherokee rose polysaccharide, and carrying out carboxymethylation on degradation products so as to obtain a plurality of cherokee rose polysaccharide derivatives. The plurality of cherokee rose polysaccharide derivatives have in vitro antitumor characteristics, and can be used for inhibiting the growth of three tumor cells, namely, ovarian cancer cells, liver cancer cells and rectal cancer cells.
Description
Technical field
The invention belongs to the bioactive polysaccharide technical field, relates generally to vegetable polysaccharides chemical modification and antitumor drug preparation method.
Background technology
Carbohydrate is an isolating protein and a nucleic acid class important biomolecule molecule in addition in the organism, and especially the important informational molecule of a class has diversified biological function.Wherein, polysaccharide is the abundant biological polymers of occurring in nature reserves, and it is the polymer that is linked together by glycosidic link by aldose or ketose, extensively is present in animal cell membrane, plant and the microorganism wall.The active polysaccharide of having found at present has the hundreds of kind, and fungus polysaccharide, higher plant polysaccharide, algae lichenstarch, animal polysaccharide, bacterial polysaccharides are arranged.Polysaccharide has many-sided biological functions such as energy storage, structural support, defense reaction and antigen is decisive.
Vegetable polysaccharides, especially the water-soluble polysaccharide that extracts from Chinese medicine is particularly important, found that the polysaccharide compound in the plurality of Chinese has colourful biological activity, as antitumor, immunomodulatory, hypoglycemic, antiviral, reducing blood-fat, anticoagulation and promote the wound healing isoreactivity, this class polysaccharide does not have cytotoxicity, and drug quality controls easily by chemical means, become one of developing direction of current new drug.
Fruit of Cherokee Rose (Rosa laevigata Michx), another name hemiplelea davidii, thorn pear, golden small-mouthed jar, sugared warbler, Chinese bush cherry ball, sugar bowl, candy, yellow bur, honeybee sugar bowl, golden tall bottle with spout, balaustine, sugared orange etc.Be evergreen climbing shrub, barb-like prickle of the close life of stem branch and seta, the subsphaeroidal or obovate of rose-hip, puce, the outside is close by seta.The florescence 4-6 month, the fruit phase 7-11 month.Be born in hill, limit, field, the small stream side shrubbery on the sunny side of height above sea level 100-1600m.Be distributed in ground such as East China, south China, southwest and Henan, Shaanxi, Taiwan, Hubei, Hunan.
Total sugar content reaches 24% in the Fruit of Cherokee Rose fruit, and wherein polysaccharide content is about 8.73% of Fruit of Cherokee Rose fruit.Fruit of Cherokee Rose fruit nutritive ingredient is abundant, have antibiotic, improve multiple biological activity such as immunity, particularly the content of its zinc, selenium is respectively 20.140 * 10
-6G/100g and 0.072 * 10
-6G/100g.These two kinds of elements are the trace element of specific health care of having of needed by human and anti-cancer effect.These provide theoretical basis for the Fructus Rosae Laevigatae polysaccharides activity research.
At present Fructus Rosae Laevigatae polysaccharides (RLP) has been carried out preliminary research, and obtain some achievements in research, mainly concentrate on antioxygenation, press down the fat effect, aspects such as immunocompetence, antibacterial and anti-inflammatory action, but do not carry out as yet for the structural modification of Fructus Rosae Laevigatae polysaccharides and the research of anti-tumor biological thereof.
Summary of the invention
The objective of the invention is to overcome prior art, at first prepare the higher Fructus Rosae Laevigatae polysaccharides of purity, by Fructus Rosae Laevigatae polysaccharides is degraded, then, the degraded product of Fructus Rosae Laevigatae polysaccharides is carried out sulphating and carboxymethylation modification, can significantly improve Fructus Rosae Laevigatae polysaccharides derivative after the modification to the restraining effect of growth of tumour cell, for making full use of the distinctive plant resources of China, development of new natural radioactivity medicine and healthcare products are significant.
Purpose of the present invention is achieved through the following technical solutions
Several preparation methods with Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity may further comprise the steps:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of controlling sulfate polyose derivative: 50mLN, dinethylformamide place 0 ~ 4 ℃ of ice bath; Stir and slowly to drip the 15mL chlorsulfonic acid down, the control rate of addition makes temperature of reaction system be lower than 10 ℃, continues to be stirred to obtain settled solution and be esterifying agent; Take by weighing Fructus Rosae Laevigatae polysaccharides 0.5g, add 15mLN, dinethylformamide, 4 ℃ of refrigerators are placed and are spent the night, and add esterifying agent, behind 50 ℃ of shaking table reaction 3h, cooling, alcohol precipitation, centrifugal, with resolution of precipitate, water-soluble 3 times of alcohol precipitation repeatedly adds 0.1mol/L NaOH solution and regulates pH to 7 again, concentrates, drying obtains the controlling sulfate polyose derivative.
The present invention can also carry out purifying with DEAE-52 with the controlling sulfate polyose derivative, is further purified through DEAE-Sepharose FF ion-exchanger again, and elution peak is single symmetrical peak.Dialysis, lyophilize obtains the controlling sulfate polyose behind the purifying.
Two, have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, further comprising the steps of:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative: take by weighing the 5g Fructus Rosae Laevigatae polysaccharides and add the 76mL Virahol, stir 30min, the adding massfraction is 10% NaOH solution 25mL, and 30 ℃ are soaked 1h; The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, join in the above-mentioned soak solution, the adding massfraction is 10% NaOH solution 25mL, behind 70 ℃ of reaction 2.5h, outwell upper strata alcohol liquid, with the product water dissolution, HCl is neutralized to neutral back alcohol precipitation, adds the less water dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative.
The present invention can also carry out purifying with DEAE-52 with carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative, is further purified through DEAE-Sepharose FF ion-exchanger, and the elution peak peak shape is symmetry comparatively.Collect the main peak polysaccharide, dialysis, lyophilize obtains purifying carboxymethylation polysaccharide.
Three, have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, further comprising the steps of:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative: with Fructus Rosae Laevigatae polysaccharides and massfraction is that 0.6 ~ 1.0% hydrochloric acid soln mixes, 1 ~ 2.5h degrades in 70 ℃ ~ 80 ℃ water-baths, regulate pH to 7.0 with NaOH solution, alcohol precipitation, centrifugal, the water dissolution precipitation, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative.
Salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative of the present invention can also carry out purifying with Sephadex G-100, collects the main peak polysaccharide, dialysis, and lyophilize obtains purifying Fructus Rosae Laevigatae polysaccharides degraded product.
Four, have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, further comprising the steps of:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative: with Fructus Rosae Laevigatae polysaccharides and massfraction is that 0.6 ~ 1.0% hydrochloric acid soln mixes, 1 ~ 2.5h degrades in 70 ℃ ~ 80 ℃ water-baths, regulate pH to 7.0 with NaOH solution, alcohol precipitation, centrifugal, the water dissolution precipitation, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative;
(6) preparation of degraded-carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative: take by weighing 5g salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative, add the 76mL Virahol, stir 30min, the adding massfraction is 10% NaOH solution 25mL, and 30 ℃ are soaked 1h; The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, adds in the soak solution, the adding massfraction is 10% NaOH solution 25mL, behind 70 ℃ of reaction 2.5h, outwell upper strata alcohol liquid, with the product water dissolution, after HCl is neutralized to neutrality, alcohol precipitation, dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrates, drying obtains the carboxymethylation product of Fruit of Cherokee Rose degradation of polysaccharide.
The carboxymethylation product of Fruit of Cherokee Rose degradation of polysaccharide of the present invention can also carry out purifying with Sephadex G-100, collects the main peak polysaccharide, dialysis, and lyophilize obtains the carboxymethylation product of purifying Fruit of Cherokee Rose degradation of polysaccharide.
The preferred processing condition of step of the present invention (3) are: the consumption of described papoid is the 1%w/v of concentration of substrate, and bath temperature is 60 ℃, degraded 3h, and pH is 6.0, repeating step (3) 5 times.
Degreasing of the present invention is with the tangible 50 ℃ of sherwood oil backflow 1h of Fructus Fructus Rosae Laevigatae after pulverizing.
Removal oligose of the present invention is that to add volume fraction in the Fruit of Cherokee Rose fruit after degreasing be that 80% ethanol refluxes twice at 96 ℃, each 2h.
Simmer down to membrane concentration of the present invention, usefulness be that to hold back molecular weight cut-off be 10,000 daltonian ultra-filtration membranes.
The damping fluid preferably phosphoric acid salt buffer of pH7.6 of the present invention.
0.1 the damping fluid of ~ 0.5mo1/LNaC1 pH7.6 is the buffered soln that replaces the pH7.6 that water joined with the NaC1 of different concns.
Described usefulness 0.1 ~ 0.5mol/LNaCl solution gradient wash-out be use 0.1,0.2,0.3,0.4 successively, the 0.5mol/LNaCl eluant solution.
The present invention is with respect to advantage and beneficial effect that prior art had
(1) the DEAE-52 decoloring method that adopts of the present invention decolours to the Fruit of Cherokee Rose Crude polysaccharides, and not only percent of decolourization is greater than 95%, and the polysaccharide content before and after the decolouring, and purity of polysaccharide is brought up to more than 80% by 60%.
(2) enzyme process and the Sevag method bonded method for removing protein effect of the present invention's employing are fine, and have optimized Deproteinated method by experiment.The result shows: top condition is, enzyme dosage is 1% of a concentration of substrate, and temperature is 60 ℃, and pH is 6.0 o'clock, and water-bath 3h adds chloroform-propyl carbinol (5:1) solution of its volume 1/5 again, vibration 20min, and deproteinated repeats 5 times.
(3) the present invention adopts DEAE-Sepharose FF gel filtration chromatography method that the Fruit of Cherokee Rose Crude polysaccharides is carried out purifying, and preparation Fructus Rosae Laevigatae polysaccharides (RLP) identifies that by polyacrylamide gel electrophoresis and gel chromatography RLP has higher degree.The weight-average molecular weight Mw of gel permeation chromatography RLP is 227kDa.
(4) the present invention has carried out chemical structures modifications such as sulfation, carboxymethylation, degraded first to Fructus Rosae Laevigatae polysaccharides, obtains the carboxymethylation product (CMDG-RLP) of sulfation Fructus Rosae Laevigatae polysaccharides (SF-RLP), carboxymethylation Fructus Rosae Laevigatae polysaccharides (CM-RLP), Fruit of Cherokee Rose degradation of polysaccharide (DG-RLP) and degradation of polysaccharide.
(5) the Fructus Rosae Laevigatae polysaccharides derivative for preparing of the present invention, restraining effect to three kinds of growth of tumour cell such as ovarian cancer cell, liver cancer cell and rectum cancer cells, when the Fructus Rosae Laevigatae polysaccharides derivatives concentration was 50ug/mL, SF-RLP, CM-RLP, DG-RLP and CM-DG-RLP were respectively 66.4%, 37.5%, 79.6% and 72.5% to the inhibiting rate of SKVO cell growth; Under identical administration concentration, these four kinds of derivatives all can suppress the HepG2 growth extremely significantly, and its inhibiting rate is respectively 55.22%, 87.79%, and 73.2% and 63.1%; SF-RLP and CMDG-RLP are obvious dose-dependence to the inhibiting rate and the drug level of the growth of LoVo cell, and both are to the IC of LoVo cell
50Value is respectively 49.32ug/mL and 35.92ug/mL.
(6) the present invention has promoted the comprehensive utilization of Fruit of Cherokee Rose resource, and the novel polysaccharide derivative that has anti-tumor activity for preparation provides new way, has promoted application and the development of Fructus Rosae Laevigatae polysaccharides in food and medicine.
Description of drawings
Fig. 1 is the technical process of embodiment 1;
Fig. 2 is a Fructus Rosae Laevigatae polysaccharides DEAE-52 elution curve;
Fig. 3 is a Fructus Rosae Laevigatae polysaccharides DEAE-Sepharose FF elution curve;
Fig. 4 is a Sephadex G-100 elution curve;
Fig. 5 is the GPC color atlas of RLP;
Fig. 6 is the GPC color atlas of DG-RLP;
Fig. 7 is Fructus Rosae Laevigatae polysaccharides and modified product thereof the inhibiting rate to SKVO cell proliferation;
Fig. 8 is Fructus Rosae Laevigatae polysaccharides and modified product thereof the inhibiting rate to HepG 2 cell proliferations;
Fig. 9 is Fructus Rosae Laevigatae polysaccharides and modified product thereof the inhibiting rate to LoVo cell proliferation.
Embodiment
The preparation of embodiment 1 Fructus Rosae Laevigatae polysaccharides
(1) earlier the Fruit of Cherokee Rose fruit is pulverized as Fig. 1; Use sherwood oil in 50 ℃ of backflow 1h degreasing then; Reflux twice with 80%wt ethanol in 96 ℃ again, each 2h, adopt 80 ℃ of hot water lixiviate 2h after, united extraction liquid, again it is carried out filtered through gauze and B suction filtration; Concentrate, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in 10mLH respectively
2O, 0.1 and 0.2mol/LNaCl solution adds DEAE-Mierocrystalline cellulose 52(filler) Static Adsorption 30min, flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring; Detect the percent of decolourization that DEAE-Mierocrystalline cellulose 52 adsorbs in different solvents, as shown in table 1;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 3h degrades in 60 ℃ of water-baths, regulate pH6.0, the centrifuging and taking supernatant liquor adds the chloroform-butanol solution that accounts for centrifugate volume 1/5, and 20 min vibrate, separatory is removed organic phase, centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated for 5 times; The mass volume ratio of described papoid and substrate (substrate is exactly the Crude polysaccharides solution after the decolouring) is 1% g/l
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/L NaCl eluant solution, and mean flow rate 1.0 mL/min; Elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again,, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 more earlier with the buffer solution elution of pH7.6, mean flow rate 1.0 mL/min, collect the elutriant of main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides RLP;
Effect analysis:
As can be seen from Table 1, step (2) adopts the percent of decolourization of polysaccharide soln of different solvents all greater than 95%, and the polysaccharide content before and after the Crude polysaccharides decolouring, the polysaccharide content of the polysaccharide content after the decolouring before greater than decolouring.A large amount of carbonyls and sulfate are arranged on the polysaccharide glycosyl, and easy and DEAE-Mierocrystalline cellulose adsorbs.Therefore, can be with DEAE-cellulose adsorption polysaccharide, the purifying that the change by elution requirement reaches polysaccharide separates with fraction.So DEAE-Mierocrystalline cellulose 52 has not only been removed the pigment in the Fruit of Cherokee Rose Crude polysaccharides, has also played the effect of purified polysaccharide.
Table 1 DEAE-52 decolorizing effect
The deproteinated of Fruit of Cherokee Rose Crude polysaccharides
Crude polysaccharides solution adds papoid in the step (3), degrades in the water-bath, and the centrifuging and taking supernatant liquor adds chloroform-propyl carbinol (5:1) solution that accounts for its volume 1/5,20 min that vibrate, and separatory is removed organic phase, and is centrifugal, gets supernatant liquor, repeats aforesaid operations for several times.
After handling by papoid, because most free proteins and part are hydrolyzed with polysaccharide bonded protein, Protein content reduces greatly in the polysaccharide precipitation, only needs on a small quantity Sevag method several times, can reach and remove proteic requirement, reduce polysaccharide loss.And in enzymolysis process, part is decomposed with polysaccharide bonded protein, and the protein removal rate is improved, and the polyose-protein content behind the deproteinated is lower.By experimental result as seen, it is different that enzyme process and Sevag method are handled in the Fructus Rosae Laevigatae polysaccharides degree of polysaccharide and protein loss, the trend that tapers off basically, and the deproteinated effect mainly concentrates on preceding 5 times, preceding 5 albumen decreasing ratiies reach 88.06%, and the polysaccharide loss rate is 21.78%.And the deproteinated that is lost in of polysaccharide tends towards stability later on for 5 times, thus Fructus Rosae Laevigatae polysaccharides to adopt enzyme process and Sevag method deproteinated be for 5 times the best.
The gel filtration chromatography purifying of Fruit of Cherokee Rose Crude polysaccharides
Step (4) adopts DEAE-52 that the Fruit of Cherokee Rose Crude polysaccharides is further separated, and chromatographic column Ф 1.6 * 30, take by weighing the 100mg polysaccharide and are dissolved in H
2Upper prop among the O is used 0.1 ~ 0.5mol/L NaCl eluant solution successively, and mean flow rate 1.0 mL/min, every 5min collect a pipe, follow the tracks of detection with sulfuric acid-phynol method 490nm, with light absorption value elution volume are mapped, and draw elution curve, collect.With the solution concentration of collecting, be further purified through DEAE-Sepharose FF anionite again,, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 more earlier with the buffer solution elution of pH7.6, mean flow rate 1.0 mL/min, per 5 min collect a pipe, follow the tracks of with sulfuric acid-phynol method 490 nm and detect, and with light absorption value elution volume are mapped, draw elution curve, collect, dialysis, freeze-drying obtain Fructus Rosae Laevigatae polysaccharides be called RLP (
Rosa laevigata MichxPolysaccharide).
The Fruit of Cherokee Rose Crude polysaccharides is carried out purifying with DEAE-52, elution curve such as Fig. 2.A main peak occurs at 17 pipes to 29 pipe, a small peak appears in the next door, and the rate of recovery at two peaks is respectively 71.5% and 15.2%.Collect main polysaccharide (main peak part).
Be further purified through DEAE-Sepharose FF ion-exchanger, elution curve such as Fig. 3, elution peak is single symmetrical peak, obtains simple relatively polysaccharide.
The purity of Fructus Rosae Laevigatae polysaccharides is identified
Fruit of Cherokee Rose Crude polysaccharides and refining polysaccharide RLP detect with Sephadex G-100, obtain elution curve Fig. 4, and visible elution peak is a simple spike among the figure, and peak shape is symmetrical, illustrates that this polysaccharide is an one-component.
Sephadex G-100 gel chromatography qualification result shows that RLP has higher degree.
Through the GPC canonical plotting of Breeze GPC software processes gained dextran Dextran series standard product, obtain the molecular weight and the retention time graph of a relation of dextran Dextran series standard product.With the logarithm logMw of standard dextran molecule amount to elution volume return the GPC canonical plotting of dextran Dextran series standard product.Fig. 5 is the GPC color atlas of RLP, and the Mw that is tried to achieve RLP by typical curve is 227kDa.
The preparation of embodiment 2 Fruit of Cherokee Rose sulfated polysaccharides
(1) earlier the Fruit of Cherokee Rose fruit is pulverized as Fig. 1; Use sherwood oil in 50 ℃ of backflow 1h degreasing then; Reflux twice with 80%wt ethanol in 96 ℃ again, each 2h, adopt 80 ℃ of hot water lixiviate 2h after, united extraction liquid, again it is carried out filtered through gauze and B suction filtration; Concentrate, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in 10mLH respectively
2O adds DEAE-Mierocrystalline cellulose 52(filler) Static Adsorption 30min, flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 3h degrades in 60 ℃ of water-baths, regulate pH6.0, the centrifuging and taking supernatant liquor adds the chloroform-butanol solution that accounts for centrifugate volume 1/5, and 20 min vibrate, separatory is removed organic phase, centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated for 5 times; The mass volume ratio of described papoid and substrate (substrate is exactly the Crude polysaccharides solution after the decolouring) is 1% g/l
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/L NaCl eluant solution, and mean flow rate 1.0 mL/min; Elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again,, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 more earlier with the buffer solution elution of pH7.6, mean flow rate 1.0 mL/min, collect the elutriant of main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides RLP;
(5) preparation of controlling sulfate polyose derivative: 50mLN, dinethylformamide place 4 ℃ of ice baths; Stir and slowly to drip the 15mL chlorsulfonic acid down, the control rate of addition makes temperature of reaction system be lower than 10 ℃, continues to be stirred to obtain settled solution and be esterifying agent; Take by weighing Fructus Rosae Laevigatae polysaccharides 0.5g, add 15mLN, dinethylformamide, 4 ℃ of refrigerators are placed and are spent the night, and add esterifying agent, behind 50 ℃ of shaking table reaction 3h, cooling, alcohol precipitation, centrifugal, with resolution of precipitate, water-soluble 3 times of alcohol precipitation repeatedly adds 0.1mol/L NaOH solution and regulates pH to 7 again, concentrates, drying obtains the controlling sulfate polyose derivative.
The Fructus Rosae Laevigatae polysaccharides sulfation product is carried out purifying with DEAE-52, be further purified through DEAE-Sepharose FF ion-exchanger, elution peak is single symmetrical peak.Dialysis, lyophilize obtains the controlling sulfate polyose behind the purifying, is designated as SF-RLP.
The preparation of embodiment 3 Fruit of Cherokee Rose carboxymethylation polysaccharide
Take by weighing the 5g Fructus Rosae Laevigatae polysaccharides that embodiment 2 makes and place reaction flask, add the 76mL Virahol, stir and soak 30min, add 10%NaOH solution 25mL, 30 ℃ are soaked 1h.The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, join in the above-mentioned soak solution, add 10%NaOH solution 25mL, 70 ℃ of reaction 2.5h, after reaction finishes to going out upper strata alcohol liquid, with the product water dissolution, HCl is neutralized to neutral back and adds the long-pending 95% ethanol sedimentation polysaccharide of tetraploid, adds the less water dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains product carboxymethylation polysaccharide.
Fructus Rosae Laevigatae polysaccharides carboxymethylation product is carried out purifying with DEAE-52, be further purified through DEAE-Sepharose FF ion-exchanger, the elution peak peak shape is symmetry comparatively.Collect the main peak polysaccharide, dialysis, lyophilize obtains purifying carboxymethylation polysaccharide, is designated as CM-RLP.
The preparation of embodiment 4 Fruit of Cherokee Rose degradation of polysaccharide
The Fructus Rosae Laevigatae polysaccharides that embodiment 2 is made and massfraction are that 0.6% hydrochloric acid soln mixes, 2.5h degrades in 70 ℃ of water-baths, degradation solution is regulated pH to 7.0 with 2mol/LNaOH solution, adding 95% ethanol sedimentation separates, the water dissolution precipitation, alcohol precipitation is water-soluble 3 times repeatedly, concentrates, drying obtains salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative.
Fructus Rosae Laevigatae polysaccharides hydrochloric acid degraded product is carried out purifying with Sephadex G-100, collect the main peak polysaccharide, dialysis, lyophilize obtains purifying Fructus Rosae Laevigatae polysaccharides degraded product, is designated as DG-RLP.
Fig. 6 is the GPC color atlas of DG-RLP, and the Mw that is tried to achieve DG-RLP by typical curve is 38KDa.This shows that through the HCl degraded, the molecular weight of Fructus Rosae Laevigatae polysaccharides is reduced to 38KDa by original 227KDa.
The preparation of embodiment 5 Fruit of Cherokee Rose degraded-carboxymethylation polysaccharide
Take by weighing the DG-RLP that 5g embodiment 4 makes and place reaction flask, add the 76mL Virahol, stir and soak 30min, add 10%wtNaOH solution 25mL, 30 ℃ of alkaline purification 1h.The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, join in the above-mentioned soak solution, add 10%wtNaOH solution 25mL, 70 ℃ of reaction 2.5h, after finishing, reaction pours out upper strata alcohol liquid, with the product water dissolution, HCl is neutralized to neutral back and adds the long-pending 95%(volume fraction of tetraploid) the ethanol sedimentation polysaccharide, add the less water dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains the carboxymethylation product of Fruit of Cherokee Rose degradation of polysaccharide.
The carboxymethylation product of Fruit of Cherokee Rose degradation of polysaccharide is carried out purifying with Sephadex G-100, collect the main peak polysaccharide, dialysis, lyophilize obtains the carboxymethylation product of purifying Fruit of Cherokee Rose degradation of polysaccharide, is designated as CMDG-RLP.
With the SKVO that has just thawed, HepG2 and LoVo cell strain, increment is cultivated in 1640 type substratum, and culturing bottle is placed under 37 ℃, contains 5% CO
2Incubator in, change nutrient solution one time every 24h, the SKVO that takes the logarithm respectively vegetative period, HepG2 and LoVo cell are with 3 * 10
4ML
-1Cell concn inoculation 100mL in every hole on 96 orifice plates, allow behind the cell attachment growth 24h, drug level is 50 μ gmL
-1, 40 μ gmL
-1, 30 μ gmL
-1, 20 μ gmL
-1, 10 μ gmL
-1Serum free medium acts on cell, and control group is a serum free medium, every hole 200uL, and every group of concentration is established four multiple holes, cultivates after 48 hours, gets microplate reader at 570nm place photometry density value, calculates inhibiting rate.
Inhibitory rate of cell growth=
(1) Fructus Rosae Laevigatae polysaccharides and modified product thereof are to the inhibition effect of ovarian cancer (SKVO) cell proliferation
With concentration is the RLP(embodiment 2 of 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL), SF-RLP, CM-RLP, DG-RLP and CMDG-RLP act on the SKVO cell, the results are shown in Figure 7, as can be seen from Figure 7, RLP when drug level 10 ~ 50 μ g/mL to the SKVO cell inhibitory effect effect there were significant differences (P＜0.05), under same concentrations, SF-RLP, CM-RLP, DG-RLP and CMDG-RLP all are higher than the restraining effect of RLP to the restraining effect of SKVO cell proliferation, have utmost point significant difference (P＜0.01).SF-RLP, CM-RLP, DG-RLP and CMDG-RLP are significant concn dependence effect to SKVO cell inhibiting effect, and the restraining effect of cell growth is the most remarkable when drug level is 50 μ g/mL, and its inhibiting rate is respectively 66.4%, 37.5%, 79.6% and 72.5%.Relatively SF-RLP, CM-RLP, DG-RLP, CMDG-RLP and RLP are to the restraining effect of SKVO cell proliferation as can be seen, under 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 each concentration of μ g/mL, the restraining effect of modified polysaccharide and RLP pair cell has utmost point significant difference (P＜0.01), when drug level was 50 μ g/mL, SF-RLP, CM-RLP, DG-RLP and CMDG-RLP be higher by 48.1% respectively than RLP to the inhibiting rate of SKVO cell proliferation, 19.2%, 61.3% and 54.2%.This shows that above-mentioned several modifications all can improve the restraining effect of Fructus Rosae Laevigatae polysaccharides to SKVO cell proliferation by the utmost point significantly.
(2) Fructus Rosae Laevigatae polysaccharides and modified product thereof are to the inhibition effect of lung cancer (HepG2) cell proliferation
With concentration is that RLP, SF-RLP, CM-RLP, DG-RLP and the CMDG-RLP of 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL acts on HepG 2 cells, the results are shown in Figure 8, as can be seen from Figure 8, compare with control group, there were significant differences (P＜0.05) to the restraining effect of HepG 2 cell proliferations when drug level 10 ~ 50 μ g/mL for RLP, under same concentrations, SF-RLP, CM-RLP, DG-RLP and CMDG-RLP all are higher than the restraining effect of RLP to the restraining effect of HepG 2 cell proliferations, have utmost point significant difference (P＜0.01).SF-RLP, CM-RLP, DG-RLP and CMDG-RLP are significant concn dependence effect to HepG 2 cell inhibiting effects, and the restraining effect of cell growth is the most remarkable when drug level is 50 μ g/mL, and its inhibiting rate is respectively 55.2%, 87.8%, 73.2% and 63.1%.Relatively SF-RLP, CM-RLP, DG-RLP, CMDG-RLP and RLP are to the restraining effect of HepG 2 cell proliferations as can be seen, difference under 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 each concentration of μ g/mL between the restraining effect of modified polysaccharide and RLP pair cell is (P＜0.01) extremely significantly, when drug level was 50 μ g/mL, SF-RLP, CM-RLP, DG-RLP and CMDG-RLP exceeded 42.6%, 75.2%, 60.6% and 50.5% to the inhibiting rate of HepG 2 cell proliferations respectively than RLP.This shows that above-mentioned several modifications all can improve the restraining effect of Fructus Rosae Laevigatae polysaccharides to HepG2 cell proliferation by the utmost point significantly.
(3) Fructus Rosae Laevigatae polysaccharides and modified product thereof are to the inhibition effect of the rectum cancer (LoVo) cell proliferation
With concentration is that RLP, SF-RLP, DG-RLP and the CMDG-RLP of 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL acts on the LoVo cell.The results are shown in Figure 9, as can be seen from Figure 9, compare with control group, there were significant differences (P＜0.05) to the restraining effect of LoVo cell proliferation when drug level 10 ~ 50 μ g/mL for RLP, and also there is significant difference (P＜0.05) between the different pharmaceutical concentration, under identical administration concentration, SF-RLP, DG-RLP and CMDG-RLP all are higher than the restraining effect of RLP to the restraining effect of LoVo cell proliferation, have utmost point significant difference (P＜0.01).SF-RLP and CMDG-RLP have linear relationship to LoVo cell inhibiting rate and drug level, and the restraining effect of cell growth is the most remarkable when drug level is 50 μ g/mL, and its inhibiting rate is respectively 56.8% and 52.7%.Relatively SF-RLP, DG-RLP, CMDG-RLP and RLP are to the restraining effect of LoVo cell proliferation as can be seen, when drug level is low (10 μ g/mL ~ 30 μ g/mL), inhibiting rate and the RLP of CMDG-RLP do not have significant difference, increase with concentration and to show utmost point significant difference (P＜0.01), SF-RLP and DG-RLP at concentration 10 μ g/mL ~ 50 μ g/mL and RLP to the difference between the effect of LoVo cell inhibiting extremely remarkable (P＜0.01).When drug level was 50 μ g/mL, SF-RLP, DG-RLP and CMDG-RLP exceeded 31.6%, 14.0% and 27.5% to the inhibiting rate of LoVo cell proliferation respectively than RLP.This shows that above-mentioned several modifications all can improve the restraining effect of Fructus Rosae Laevigatae polysaccharides to LoVo cell proliferation by the utmost point significantly.
Claims (6)
1. have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, it is characterized in that, may further comprise the steps:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of controlling sulfate polyose derivative: 50mLN, dinethylformamide place 0 ~ 4 ℃ of ice bath; Stir and slowly to drip the 15mL chlorsulfonic acid down, the control rate of addition makes temperature of reaction system be lower than 10 ℃, continues to be stirred to obtain settled solution and be esterifying agent; Take by weighing Fructus Rosae Laevigatae polysaccharides 0.5g, add 15mLN, dinethylformamide, 4 ℃ of refrigerators are placed and are spent the night, and add esterifying agent, behind 50 ℃ of shaking table reaction 3h, cooling, alcohol precipitation, centrifugal, with resolution of precipitate, water-soluble 3 times of alcohol precipitation repeatedly adds 0.1mol/L NaOH solution and regulates pH to 7 again, concentrates, drying obtains the controlling sulfate polyose derivative.
2. have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, it is characterized in that, may further comprise the steps:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative: take by weighing the 5g Fructus Rosae Laevigatae polysaccharides and add the 76mL Virahol, stir 30min, the adding massfraction is 10% NaOH solution 25mL, and 30 ℃ are soaked 1h; The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, join in the above-mentioned soak solution, the adding massfraction is 10% NaOH solution 25mL, behind 70 ℃ of reaction 2.5h, outwell upper strata alcohol liquid, with the product water dissolution, HCl is neutralized to neutral back alcohol precipitation, adds the less water dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative.
3. have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, it is characterized in that, may further comprise the steps:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative: with Fructus Rosae Laevigatae polysaccharides and massfraction is that 0.6 ~ 1.0% hydrochloric acid soln mixes, 1 ~ 2.5h degrades in 70 ℃ ~ 80 ℃ water-baths, regulate pH to 7.0 with NaOH solution, alcohol precipitation, centrifugal, the water dissolution precipitation, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative.
4. have the preparation method of the Fructus Rosae Laevigatae polysaccharides derivative of anti-tumor activity, it is characterized in that, may further comprise the steps:
(1) the Fruit of Cherokee Rose fruit degreasing after will pulverizing, remove adopt 80 ℃ of lixiviate 2h of hot water lixiviate after the oligose after, united extraction liquid, filtration, concentrate the back and add the dehydrated alcohol precipitation, obtain the Fruit of Cherokee Rose Crude polysaccharides after will precipitating drying again;
(2) the Fruit of Cherokee Rose Crude polysaccharides is dissolved in H
2O or 0.1～0.2mol/LNaCl solution add DEAE-Mierocrystalline cellulose 52 Static Adsorption 30min, and flushing DEAE-Mierocrystalline cellulose 52 filters elutriant, collects the Crude polysaccharides solution after filtrate is decolouring;
(3) the Crude polysaccharides solution after the decolouring adds papoid, 2～4h degrades in 45～65 ℃ of water-baths, the centrifuging and taking supernatant liquor, add the chloroform-butanol solution that accounts for supernatant liquor volume 1/5,20 min that vibrate, separatory is removed organic phase, and is centrifugal, get supernatant liquor, repeating step (3) operation obtains the Crude polysaccharides solution behind the deproteinated;
(4) the Crude polysaccharides solution behind the deproteinated is through DEAE-Mierocrystalline cellulose 52 ion-exchange chromatographies, with 0.1 ~ 0.5mol/LNaCl solution gradient wash-out, mean flow rate 1.0 mL/min, elutriant is followed the tracks of with sulfuric acid-phynol method and is detected, collect the elutriant of main peak, the elutriant of collecting is concentrated, pass through DEAE-Sepharose FF anionite again, use the buffer solution elution of pH7.6 earlier, use the buffer solution for gradient elution of 0.1 ~ 0.5mo1/LNaC1 pH7.6 again, mean flow rate 1.0 mL/min, the elutriant of collection main peak, dialysis, freeze-drying obtains Fructus Rosae Laevigatae polysaccharides;
(5) preparation of salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative: with Fructus Rosae Laevigatae polysaccharides and massfraction is that 0.6 ~ 1.0% hydrochloric acid soln mixes, 1 ~ 2.5h degrades in 70 ℃ ~ 80 ℃ water-baths, regulate pH to 7.0 with NaOH solution, alcohol precipitation, centrifugal, the water dissolution precipitation, alcohol precipitation is water-soluble 3 times repeatedly, concentrate, drying obtains salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative;
(6) preparation of degraded-carboxymethylation Fructus Rosae Laevigatae polysaccharides derivative: take by weighing 5g salt acid degradation Fructus Rosae Laevigatae polysaccharides derivative, add the 76mL Virahol, stir 30min, the adding massfraction is 10% NaOH solution 25mL, and 30 ℃ are soaked 1h; The 4g Mono Chloro Acetic Acid is dissolved in the 10mL Virahol, adds in the soak solution, the adding massfraction is 10% NaOH solution 25mL, behind 70 ℃ of reaction 2.5h, outwell upper strata alcohol liquid, with the product water dissolution, after HCl is neutralized to neutrality, alcohol precipitation, dissolving again, alcohol precipitation is water-soluble 3 times repeatedly, concentrates, drying obtains the carboxymethylation product of Fruit of Cherokee Rose degradation of polysaccharide.
5. according to the described preparation method of one of claim 1-4, it is characterized in that the mass volume ratio of the Crude polysaccharides solution after described papoid of step (3) and the decolouring is 1% g/l, bath temperature is 60 ℃, degraded 3h, and pH is 6.0, repeating step (3) 5 times.
6. according to the described preparation method of one of claim 1-4, it is characterized in that, described usefulness 0.1 ~ 0.5mol/LNaCl solution gradient wash-out be use 0.1,0.2,0.3,0.4 successively, the 0.5mol/LNaCl eluant solution.
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| CN110742901B (en) * | 2019-11-28 | 2023-03-10 | 郑州大学第一附属医院 | Metabolism check point FBP1 agonist and application thereof in anti-tumor aspect |
| CN112759661A (en) * | 2021-01-15 | 2021-05-07 | 南开大学 | Preparation method, identification method and application of cherokee rose fruit polysaccharide |
| CN112759661B (en) * | 2021-01-15 | 2023-04-25 | 南开大学 | Cherokee rose fruit polysaccharide preparation method, identification method and application |
| CN113754787A (en) * | 2021-08-10 | 2021-12-07 | 华南理工大学 | Selenized cherokee rose fruit polysaccharide for enhancing immunoregulation and antioxidant activity, and preparation method and application thereof |
| CN114507296A (en) * | 2022-02-25 | 2022-05-17 | 南京农业大学 | Lichenin and its preparation method and application |
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