CN104277136A - Yellow river beach date polysaccharides, and extraction and refining method and application thereof - Google Patents
Yellow river beach date polysaccharides, and extraction and refining method and application thereof Download PDFInfo
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- CN104277136A CN104277136A CN201410495370.7A CN201410495370A CN104277136A CN 104277136 A CN104277136 A CN 104277136A CN 201410495370 A CN201410495370 A CN 201410495370A CN 104277136 A CN104277136 A CN 104277136A
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Abstract
The invention discloses yellow river beach date polysaccharides and an extraction and refining method and application thereof. The extraction and refining method comprises the following steps: performing pitting treatment on dates to obtain date powder, performing defatting, water extraction and alcohol sedimentation to obtain date crude polysaccharides, and performing deproteinization and decolorization to obtain refined date polysaccharides. The process for extracting and refining the date polysaccharides, disclosed by the invention, is advanced, the method is simple, the energy consumption is relatively low, and convenient conditions are provided for deep processing of the dates. The invention further provides the application of the yellow river beach date polysaccharides, and pharmacological experiments show that the date polysaccharides prepared by the method disclosed by the invention have the effects of resisting oxidation, clearing free radicals, resisting cancers, improving immunity and protecting the liver, and can be used for preparing medicines, foods and/or health care products for preventing liver injuries.
Description
Technical field
The present invention relates to beach, a kind of the Yellow River jujube polysaccharide, its Hydrolysis kinetics method and application, belong to medical art.
Background technology
Date is the fruit of Rhamnaceae jujube jujube tree, and main product, in China, has the cultivation history of more than 4000 year so far.Nowadays in East Asia, all there is distribution in South Asia, Europe, Africa and Australia.The fruit of date has high nutritive value, is often used as food in Asia, foodstuff additive and seasonings.According to the literature, date, except containing except moisture, Mierocrystalline cellulose and multivitamin, also contains triterpenic acid, Flavonoid substances, amino acid, phenolic acid, trace quantity mineral substance element and polysaccharide.Date also has medical care effect, is traditional Chinese medicine simply, is used for the treatment of poor appetite, tired weak and loose stool.
Even to this day, had been found that more than 7000 kind of jujube class in China, cultivated area is more than 1500000 hectares.Only 2013 1 year, the output of fresh jujube more than 400,000 ton.Beach, the Yellow River jujube formal name used at school wood jujube, in the Yellow River, littoral extensively plantation, is characterized in that particle is very large, cylindrical, and look is as black jade, and the sweet soft profit of pulp is fragrant.Because the pharmacy value of beach, the Yellow River jujube has a high potential, the development along with science and technology and the concern to natural product, also go deep into gradually to the research of beach, the Yellow River jujube.
Liver injury is the invasion that liver is subject to extraneous factor, thus the liver damage caused.Liver injury mainly comprises violence nature liver injury, pathologic liver injury and chemical damage etc.Wherein, violence nature liver injury multilist is now obviously quick and serious; And pathologic liver injury and chemical damage more common, the patients with liver deficiency caused because of factors such as pathogenic agent, chemicals or excessive consumptions of alcohol gets more and more, the patients with liver deficiency of current China exceeded ten million, the serious threat life security of the people and the raising of quality of life.Modern medicine does not also have specific medicine in the treatment intervening liver injury, and wherein, the treatment of chemicals to liver injury adopts interferon therapy more, but have that dosage is large, toxic side effect is large, the easy shortcoming such as recurrence after drug withdrawal; The treatment of Chinese medicine to liver injury adopts the Chinese patent medicine of compound decoction of medicinal ingredients or compound more, but its flavour of a drug are numerous, causes difficult quality to control.Therefore, be badly in need of exploitation one and come from single plant, the efficient part of determined curative effect or effective constituent, for the prevention and therapy of liver injury.
Vegetable polysaccharides is from plant, extract the effective kind part or effective constituent that obtain, and comprise starch, Mierocrystalline cellulose, dextran, Polylevulosan etc., vegetable polysaccharides has multiple physiologically active, has therapeutic action to the various diseases such as diabetes, stomach ulcer.
In recent years, along with improving constantly of standard of living, the health care consciousness of people also strengthens gradually, and be that the protective foods of main component occurs in a large number with polysaccharide, go deep into along with to the bioactive research of polysaccharide, the Application Areas of polysaccharide also can more be widened.
Jujube polysaccharide has immunomodulatory, the multiple physiologically active such as antitumor, but due to the Crude polysaccharides complicated component of beach, the Yellow River jujube, difficult quality controls, less for this research with the kind of larger pharmaceutical use of beach, the Yellow River jujube at present.
Summary of the invention
The object of this invention is to provide beach, a kind of the Yellow River jujube Polyose extraction process for purification.
Another object of the present invention is to provide the application of beach, the Yellow River jujube polysaccharide in preparation prevention hepatic, healthcare products and/or food.
For achieving the above object, the present invention adopts following technical proposals:
A kind of beach, the Yellow River jujube Polyose extraction process for purification, comprises the following steps:
(1) by the jujube stoning of beach, the Yellow River, dry, pulverize, obtain jujube powder, obtain degreasing jujube powder with organic solvent backflow degreasing;
(2) being 1g:(15-30 by degreasing jujube powder and water by mass volume ratio) mL mixes, supersound extraction 15-30min, filter, filtrate is concentrated into the 1/3-1/2 of former filtrate volume, and in concentrated solution, add volumetric concentration is 80%-100% ethanol, to mixed solution, the volumetric concentration of ethanol is 75-85%, alcohol precipitation spends the night, washing precipitation, dry, obtain Crude polysaccharides, the mass yield of Crude polysaccharides is 6.93%-9.82%;
(3) by water-soluble for the Crude polysaccharides that step (2) is obtained, making the Crude polysaccharides solution of 1-3g/L, is 1:(2.5-3.5 by Sevag reagent and Crude polysaccharides solution by volume) mix, jolting, stratification, discard lower floor's organic layer and middle white denatured protein layer, upper strata polysaccharide soln is evaporated to the 1/8-1/6 of former Crude polysaccharides liquor capacity, alcohol precipitation spends the night, filter, washing precipitation, dry, obtain Deproteinated jujube polysaccharide;
(4) by water-soluble for the Deproteinated jujube polysaccharide that step (3) is obtained, make the polysaccharide soln of 4-6g/L, the pH of regulator solution is 8.5-9, in polysaccharide soln, add H
2o
2, H to polysaccharide soln
2o
2volume fraction be 40%, heating in water bath decolours, and alcohol precipitation spends the night, and filters, washing precipitation, and dry, obtain the jujube polysaccharide after purifying, the polysaccharide after purifying is 32%-40% (with glucose meter) relative to the mass yield of Crude polysaccharides.
In step (1), the method for backflow degreasing was: jujube powder being added to volumetric concentration is in 95% ethanol, and the mass volume ratio of jujube powder and ethanol is 1g:5mL, 45 DEG C of backflow degreasings 3 hours;
In step (2), the temperature of supersound extraction is 50 DEG C;
In step (3), described Sevag reagent is the solution of trichloromethane and propyl carbinol 5:1 mixing by volume;
In step (3), after Sevag reagent is mixed with Crude polysaccharides solution, violent jolting 20min, layering after standing 30min, discards lower floor's organic layer and middle white denatured protein layer, repetitive operation 10 times;
In step (4), regulator solution pH reagent used is the NaOH solution of 0.1M;
In step (4), the temperature of water-bath decolouring is 40 DEG C, and the time is 4h;
In step (2), (3), (4), washing precipitation solvent used is acetone, is deposited in 40 DEG C of vacuum-dryings 12 hours after washing.
Beach, the Yellow River jujube polysaccharide prepared by the present invention has good application preparing in the medicine of preventing liver injury, healthcare products and/or food.
Beneficial effect of the present invention:
Beach, the Yellow River of the present invention jujube polysaccharide can make prevention hepatic, and has anti-oxidant, antitumor, to improve immunizing power effect, and anxious malicious experimental result shows that it is nontoxic, can meet prevention and protect the liver the demand with treatment and health, have a extensive future.Liver is one of indispensable internal organs of human body, based on metabolic function.The major function of liver comprises secretion of bile, stored animal starch, the metabolism etc. of Function protein matter, fat and carbohydrate.Also have removing toxic substances, hematopoiesis and Blood clotting.In modern times allegro life, people work busy, overwork and easily cause irritability on the weak side.It is reported, long working order makes health each organ blood demand greatly increase, and vim and vigour consumption increases, and liver is the store blood organ in body, tired will be impaired in work, so for overwork gang, what need maintenance at first is exactly liver.The present invention establishes chemical damage and Drug damages two kinds of liver injury models, and has carried out preventing and treating the research of two aspects to it.Wherein the liver injury of chemical is the liver injury caused by chemical Hepatoxic substance.Cause the externalities of chemical damage to have a lot, particularly city, air passage rates is slow, and various industrial waste, vehicle exhaust etc. make in air and be filled with various chemical toxicant, liver injury and healthy.The injury of drug administration to liver is inestimable in addition, can cause drug induced hepatic injury time serious, even can crisis life.If administration time is long or the consumption of medicine is excessive, can damage liver.Share two or more medicine lack of standardizationly, part of hepatocytes also can be caused downright bad.Cardinal symptom shows: appetite stimulator, nauseating, weak, insomnia etc.The jujube polysaccharide that the present invention extracts all has good effect for prevention and therapy two kinds of liver injury models, and especially for the prevention of liver injury, effect is more remarkable.
Accompanying drawing explanation
Fig. 1 is the HPLC figure of mixing monose standard substance and beach, the Yellow River jujube polysaccharide hydrolysis sample, and wherein, (A) is mixing monose standard substance, and (B) is beach, the Yellow River jujube polysaccharide hydrolysis sample; In figure, 1-seminose, 2-rhamnosyl, 3-galacturonic acid, 4-glucose, 5-semi-lactosi, 6-pectinose;
Beach, Fig. 2 the Yellow River jujube polysaccharide radical scavenging activity measurement result;
Beach, Fig. 3 the Yellow River jujube polysaccharide reducing power measurement result;
Beach, Fig. 4 the Yellow River jujube polysaccharide component is to the restraining effect measurement result of HepG2 cell;
Beach, Fig. 5 a the Yellow River jujube polysaccharide is on the impact of normal mouse spleen lymphocyte metabolic activity;
Beach, Fig. 5 b the Yellow River jujube polysaccharide is on the impact of splenocyte secrete cytokines;
Beach, Fig. 6 a the Yellow River jujube polysaccharide is on the impact of normal Peritoneal Cells of Mice metabolic activity;
Beach, Fig. 6 b the Yellow River jujube polysaccharide is on the impact of normal Peritoneal Cells of Mice secrete cytokines TNF-α;
Fig. 7 CCl
4beach, the Yellow River jujube polysaccharide therapeutic action dose-dependant Journal of Sex Research after induced liver injury;
Fig. 8 CCl
4beach, the Yellow River jujube polysaccharide therapeutic action time-dependent Journal of Sex Research after induced liver injury;
Beach, Fig. 9 the Yellow River jujube polysaccharide is to CCl
4induced liver injury prophylactic effect is studied;
Beach, the Yellow River jujube polysaccharide therapeutic action dose-dependant Journal of Sex Research after Figure 10 APAP induced liver injury;
Beach, the Yellow River jujube polysaccharide therapeutic action time-dependent Journal of Sex Research after Figure 11 APAP induced liver injury;
Beach, Figure 12 the Yellow River jujube polysaccharide is studied APAP induced liver injury prophylactic effect.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1: the extraction of beach, the Yellow River jujube, purifying and preparation
(1) beach, the Yellow River jujube is clean, stoning, 45 DEG C of oven dry, pulverizes, obtains jujube powder, be added in 100mL95% ethanol by 20g jujube powder, within 3 hours, obtains degreasing jujube powder 45 DEG C of backflow degreasings;
(2) 10g degreasing jujube powder and 250mL water are mixed, in 50 DEG C of supersound extraction 15min, be cooled to room temperature, suction filtration, gets filtrate, is evaporated to 50mL at 60 DEG C, in concentrated solution, slowly add the ethanol that volumetric concentration is 90% while stirring, to mixed solution, the volumetric concentration of ethanol is that 80%, 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Crude polysaccharides, the mass yield of Crude polysaccharides is 9.82%;
(3) by water-soluble for the Crude polysaccharides that step (2) is obtained, make the Crude polysaccharides solution of 1g/L, Sevag reagent is mixed than for 1:3 with Crude polysaccharides liquor capacity, violent jolting 20min, leaves standstill 30min layering, discards lower floor's organic layer and middle white denatured protein layer, repetitive operation 10 times, polysaccharide soln is evaporated to 1/6 of original volume, 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Deproteinated jujube polysaccharide;
(4) by water-soluble for the Deproteinated jujube polysaccharide that step (3) is obtained, make the polysaccharide soln of 5g/L, be 8.8 with the pH of the NaOH solution regulator solution of 0.1M, in polysaccharide soln, add H
2o
2, H to polysaccharide soln
2o
2volume fraction be 40%, 40 DEG C of waters bath with thermostatic control decolouring 4h, alcohol precipitation spends the night, and filters, and by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtains the jujube polysaccharide after purifying.
Jujube polysaccharide after purifying is hydrolyzed, the composition after hydrolysis is analyzed, the results are shown in Table 1:
The composition of the jujube polysaccharide after table 1 purifying
Polysaccharide after purifying is 40% (with glucose meter) relative to the mass yield of Crude polysaccharides.
Embodiment 2: the extraction of beach, the Yellow River jujube, purifying and preparation
(1) beach, the Yellow River jujube is clean, stoning, 45 DEG C of oven dry, pulverizes, obtains jujube powder, be added in 200mL95% ethanol by 40g jujube powder, within 3 hours, obtains degreasing jujube powder 45 DEG C of backflow degreasings;
(2) degreasing jujube powder 30g and 750mL water are mixed, in 50 DEG C of supersound extraction 30min, be cooled to room temperature, suction filtration, gets filtrate, is evaporated to 100mL at 60 DEG C, in concentrated solution, slowly add dehydrated alcohol while stirring, the volumetric concentration to ethanol is that 80%, 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Crude polysaccharides, the mass yield of Crude polysaccharides is 6.93%;
(3) by water-soluble for the Crude polysaccharides that step (2) is obtained, make the Crude polysaccharides solution of 2g/L, Sevag reagent is mixed than for 1:3 with Crude polysaccharides liquor capacity, violent jolting 20min, leaves standstill 30min layering, discards lower floor's organic layer and middle white denatured protein layer, repetitive operation 10 times, polysaccharide soln is evaporated to 200mL, and 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Deproteinated jujube polysaccharide;
(4) by water-soluble for the Deproteinated jujube polysaccharide that step (3) is obtained, make the polysaccharide soln of 6g/L, be 9.0 with the pH of the NaOH solution regulator solution of 0.1M, in polysaccharide soln, add H
2o
2, H to polysaccharide soln
2o
2volume fraction be 40%, 40 DEG C of waters bath with thermostatic control decolouring 4h, alcohol precipitation spends the night, and filters, and by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtains the jujube polysaccharide after purifying.Polysaccharide after purifying is 32% (with glucose meter) relative to the mass yield of Crude polysaccharides.
Embodiment 3: the extraction of beach, the Yellow River jujube, purifying and preparation
(1) beach, the Yellow River jujube is clean, stoning, 45 DEG C of oven dry, pulverize, obtain jujube powder, it is in 95% ethanol that 1kg jujube powder is added to 5L volumetric concentration, within 3 hours, obtains degreasing jujube powder 45 DEG C of backflow degreasings;
(2) 1kg degreasing jujube powder and 15L water are mixed, in 50 DEG C of supersound extraction 30min, be cooled to room temperature, suction filtration, gets filtrate, is evaporated to 2.5L at 60 DEG C, in concentrated solution, slowly add dehydrated alcohol while stirring, the volumetric concentration to ethanol is that 80%, 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Crude polysaccharides, the mass yield of Crude polysaccharides is 8.59%;
(3) by water-soluble for the Crude polysaccharides that step (2) is obtained, make the Crude polysaccharides solution of 3g/L, Sevag reagent is mixed than for 1:3 with Crude polysaccharides liquor capacity, violent jolting 20min, leaves standstill 30min layering, discards lower floor's organic layer and middle white denatured protein layer, repetitive operation 10 times, polysaccharide soln is evaporated to 1/6 of original volume, 4 DEG C of alcohol precipitations spend the night, suction filtration, get precipitation, by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtain Deproteinated jujube polysaccharide;
(4) by water-soluble for the Deproteinated jujube polysaccharide that step (3) is obtained, make the polysaccharide soln of 4g/L, be 8.5 with the pH of the NaOH solution regulator solution of 0.1M, in polysaccharide soln, add H
2o
2, H to polysaccharide soln
2o
2volume fraction be 40%, 40 DEG C of waters bath with thermostatic control decolouring 4h, alcohol precipitation spends the night, and filters, and by washing with acetone precipitation, at 40 DEG C of vacuum-drying 12h, obtains the jujube polysaccharide after purifying.Polysaccharide after purifying is 36% (with glucose meter) relative to the mass yield of Crude polysaccharides.
Embodiment 4: jujube polysaccharide antioxidation activity in vitro is evaluated
One. material
1,1-hexichol-2-picryl hydrazine (DPPH, I
shanghai Jing Chun biochemical technology limited-liability company)
Dehydrated alcohol (analytical pure, Tianjin Fu Yu Fine Chemical Co., Ltd)
The Tripotassium iron hexacyanide (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
Trichoroacetic acid(TCA) (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
Iron trichloride (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
Potassium primary phosphate (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
NaOH (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
Vortex (IKA MS basic)
Low speed desk centrifuge (Anting Scientific Instrument Factory, Shanghai)
TU-1900 twin-beam ultraviolet-visible pectrophotometer (Beijing Puxi General Instrument Co., Ltd)
Two, method
1.DPPH radical scavenging activity measures
DPPH method was suggested in 1958, was widely used in the resistance of oxidation of quantitative assay biological material and food.DPPH is the stable free radical centered by nitrogen, and its stability, mainly from Resonance Stabilization action and the spatial obstacle of three phenyl ring, makes azygous electronics on the nitrogen-atoms that is clipped in the middle can not play its due electronics and acts in pairs.As a kind of stable free radical, DPPH can catch other free radical.Because DPPH free radical has strong absorption at place centered by 517nm, therefore present intense violet color in the solution, and to become colorless after being neutralized or light yellow.When there being free-radical scavengers to exist, make it absorb owing to matching with its single electron and fade away, its fading extent becomes quantitative relationship with the electron amount of its acceptance, thus can carry out quantitative analysis fast with spectrophotometer.
Jujube polysaccharide obtained for the embodiment of the present invention 1 is configured to the solution of 0.31,0.62,1.25,2.50,5.00mg/mL.Get the polysaccharide sample solution of each concentration of 2mL and ethanolic soln (0.1mM) vortex mixed of 2mL DPPH.Room temperature lucifuge reaction 30min, measures absorbance at 517nm place.Absorbancy is lower shows that the radical scavenging activity of sample is higher.DPPH radical scavenging activity calculation formula is as follows:
In formula: Abs
0for the DPPH solution not containing polysaccharide sample, Abs
samplethe absorbancy of example reaction liquid, Abs
groundfor the absorbancy of polysaccharide soln.
As shown in Figure 2, radical scavenging activity and the polysaccharide concentration of jujube polysaccharide are proportionate, and concentration is larger, and Scavenging activity is stronger.When concentration is 5mg/mL, DPPH clearance rate is 88.08%.Therefore, jujube polysaccharide has certain radical scavenging activity, can be used as antioxidant.
2. polysaccharide reducing power measures
Antioxidant can make the ferric iron back of the Tripotassium iron hexacyanide become ferrous iron (yellow prussiate of potash), and ferrous iron (yellow prussiate of potash) generates Prussian blue (Fe with iron trichloride further
4[Fe (CN)
6]
3), there is maximum absorbance at 700nm place.Therefore, can be reflected the size of antioxidant reducing power by the absorption measuring 700nm place, absorbancy is larger, and reducing power is stronger, and namely resistance of oxidation is stronger.
Respectively jujube polysaccharide obtained for the embodiment of the present invention 2 is made into the solution of 0.31,0.62,1.25,2.50,5.00mg/mL.2.5mL phosphate buffered saline buffer (pH 6.6), 2.5mL 1%K
3fe (CN)
6the polysaccharide sample solution vortex mixed of solution and each concentration of 1.0mL.After 50 DEG C of water-bath 20min, add 10%TCA solution 2.5mL termination reaction.After the centrifugal 10min of 3000r/min, get 2.5mL supernatant and 2.5mL distilled water and 0.5mL 0.1%FeCl
3solution mixes.The absorption of each sample at 700nm place is measured in 5min.Absorb and illustrate that more by force reducing power is stronger.As shown in Figure 3, the reducing power of jujube polysaccharide and polysaccharide concentration are dose-dependence, and concentration is larger, and reducing power is stronger.DPPH radical scavenging activity and reducing power experiment, prove that jujube polysaccharide has obvious oxidation-resistance.
Embodiment 5: jujube polysaccharide anti tumor activity in vitro is evaluated
One. material
1. test medicine: beach, the Yellow River jujube polysaccharide prepared by the embodiment of the present invention 1, is prepared into the polysaccharide soln (3.5-5.0mg/mL) of a series of concentration.
Blank:
2. hepatoma cell line HepG2 (being provided by Shandong University's pharmaceutical preparation and medicine releasing system research centre)
3. reagent:
DMEM high glucose medium (
sai Mo flies generation that biological chemistry Products Co., Ltd)
Tetrazolium bromide (MTT, Beijing Suo Laibao Science and Technology Ltd.)
Dual anti-(penicillin 100U/mL and Streptomycin sulphate 100 μ g/mL,
life Technologies Corporation)
0.25% pancreas enzyme-EDTA Digestive system (Mai Chen Science and Technology Ltd.)
Foetal calf serum (Tian Hang bio tech ltd, Zhejiang)
DMSO (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)
Low speed desk centrifuge (Anting Scientific Instrument Factory, Shanghai)
CO2gas incubator (HERAcell 150i, Thermo Scientific)
Microplate reader (Infinite 200 Pro, Tecan Austria GmbH)
Two, method
1. clone and cell cultures
The hepatoma cell line HepG2 choosing people is cell model.Cell cultures is in the DMEM high glucose medium containing 10% foetal calf serum and 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates (37 DEG C, 5%CO2, saturated humidity).Change liquid every day once, every 2-3 days goes down to posterity 1 time.The HepG2 cell of taking the logarithm vegetative period is inoculated in 96 well culture plates, carries out cytotoxicity experiment after cultivating 12h.
2. cell toxicity test
The HepG2 cell of taking the logarithm vegetative period, be seeded in 96 well culture plates with the density of 2000cells/well, every pore volume is 100 μ L, by cell 37 DEG C, under 5%CO2 and saturated humidity condition after overnight incubation, draw substratum, PBS washes twice, add 200 μ L containing polysaccharide nutrient solution (3.5,4.0,4.5,5.0mg/mL) cell continues to cultivate under conventional culture conditions afterwards, blank: cell culture fluid (not containing polysaccharide).Incubation time terminates the MTT liquid 20 μ L that rear every hole adds 5mg/mL, and after 4h is cultivated in 37 DEG C of continuation, stop cultivating, supernatant liquor is abandoned in careful suction, and every hole adds 150 μ LDMSO, and lucifuge vibration makes blue formazan crystallization fully dissolve.The absorbance (Abs) of solution at 570nm place is detected, by formulae discovery cells survival rate (Cell viability rate) by microplate reader.Cells survival rate (%)=Abs treated/Abs control.
3.MTT method measures the restraining effect of jujube polysaccharide HepG2 cell
The polysaccharide soln (3.5-5.0mg/mL) of HepG2 cell and a series of concentration hatches 48,72 and 96 hours respectively, and result as shown in Figure 4.As shown in Figure 4, compared with blank group, jujube polysaccharide significantly suppress the growth (P<0.05) of HepG2 liver cancer cell, and is concentration dependent and time-dependent manner to the restraining effect of HepG2, shows the activity of stronger inhibition tumor cell growth.
Embodiment 6: jujube polysaccharide is on the impact of normal mouse boosting cell metabolic activity and secrete cytokines TNF-α
One. material
1. test medicine: beach, the Yellow River jujube polysaccharide prepared by the embodiment of the present invention 1, is prepared into the polysaccharide soln (1mg/mL, 0.8mg/mL, 0.5mg/mL) of a series of concentration.
Positive control: lipopolysaccharides (LPS, Sigma company)
Concanavalin A (ConA, Sigma company)
2. laboratory animal:
Kunming mouse (Center for New Drugs Evaluation of Shandong University provides)
3. reagent:
DMEM high glucose medium (
sai Mo flies generation that biological chemistry Products Co., Ltd)
CCK-8 (Japanese Dojindo company)
Dual anti-(penicillin 100U/mL and Streptomycin sulphate 100 μ g/mL,
life Technologies Corporation)
0.25% pancreas enzyme-EDTA Digestive system (Mai Chen Science and Technology Ltd.)
Foetal calf serum (Tian Hang bio tech ltd, Zhejiang)
Erythrocyte cracked liquid (Beijing Suo Laibao Science and Technology Ltd.)
Mouse TNF-α test kit (eBioscience company of the U.S.)
Low speed desk centrifuge (Anting Scientific Instrument Factory, Shanghai)
CO2gas incubator (HERAcell 150i, Thermo Scientific)
Microplate reader (Infinite 200 Pro, Tecan Austria GmbH)
Two, method
Get healthy Kunming mouse 6, cervical dislocation puts to death mouse, steep 75% alcohol 5min, aseptic taking-up spleen is put into and is filled the culture dish of 5mL containing dual anti-PBS buffer salt solution, fatty tissue is cut with eye scissors, put into and fill 5mL in addition containing lower several minutes of dual anti-PBS buffer salt solution bubble, be placed in the 25cm containing 1mL DMEM in high glucose substratum
2in culture dish, be ground to spleen with the inject cores of 20mL syringe and become white, then add 1mL nutrient solution, 200 order nylon net filters, move in centrifuge tube, add erythrocyte cracked liquid 500 μ L, place 2min, to destroy red corpuscle wherein, the centrifugal 5min of filtrate 1000rpm, abandons supernatant, adds PBS buffered soln resuspended, collecting cell, 5%C0
2static gas wave refrigerator 2h in incubator under 37 DEG C of conditions, to remove attached cell, collect not adherent cell suspension, centrifugal 5min, washes three times with cell culture fluid, counting, be made into 2 × 106 cell/mL, in 96 orifice plates having added medicine or contrast agents, every hole adds above-mentioned cell suspension 100 μ L.
With entirely training, jujube polysaccharide is mixed with high, medium and low three concentration, after sterile filtration, in 96 orifice plates, every hole adds 100 μ L, make final concentration be 1,0.8,0.5mg/mL; The every hole of Normal group adds full training 100 μ L; The ConA 100 μ L of ConA positive controls every Kong Jiaquan training preparation, final concentration is 5 μ g/mL.Every hole adds mouse boosting cell 100 μ L.When measuring mouse boosting cell metabolic activity, do positive control with ConA.Often group establishes 6 multiple holes.96 orifice plates are placed in incubator, at saturated humidity, 5%CO
2, under 37 DEG C of conditions, cultivate the corresponding time as required.24h is cultivated when measuring cellular metabolism vigor.When measuring splenocyte TNF secretion-α, after cultivating 24h, collect culture supernatant for corresponding mensuration.
4h before cell cultures terminates, adds 20 μ L CCK-8 solution in 96 orifice plates in every hole, then continues to cultivate 4h, fully after vibration mixing 60s, detects the absorbance at its 450nm place by full-automatic microplate reader.After spleen cell cultures 24h, 50 μ L cell culture supernatant are got in every hole respectively, detect spleen cell cultures supernatant TNF-alpha content respectively by ELISA method.
Measure the impact of taking on mouse spleen lymphocyte metabolic activity and TNF secretion-α after jujube polysaccharide, lipopolysaccharides (LPS) is as positive control.As Fig. 5 a, the vigor of mouse spleen lymphocyte metabolism CCK-8 strengthens, and within the specific limits in dose-dependant trend, prompting jujube polysaccharide has immuno-potentiation.As Fig. 5 b, measure jujube polysaccharide to the impact of splenocyte secrete cytokines, find that jujube polysaccharide obviously can promote the secretion of TNF-α.After jujube polysaccharide process, the vigor of mouse spleen lymphocyte metabolism CCK-8 strengthens, and within the specific limits in dose-dependant trend, prompting jujube polysaccharide has immuno-potentiation.Then observe the impact of jujube polysaccharide on splenocyte secrete cytokines, we find that jujube polysaccharide obviously can promote the secretion of TNF-α.Because TNF-α is primarily of macrophages secrete, it is a kind of important immune-regulating factor, can not only directly suppress or killing tumor cell, and there is activating macrophage and functions of neutrophils and promote the effect such as Antigen-activated T, B lymphocyte proliferation, differentiation, promote mononuclear macrophage and other cell cytokine production IL-l, IL-6, IL-8, IFN-γ and TNF-α itself, play important immunoregulation effect.
Embodiment 7: jujube polysaccharide is on the impact of normal Peritoneal Cells of Mice metabolic activity and secrete cytokines TNF-α
One. material
1. test medicine: beach, the Yellow River jujube polysaccharide prepared by the embodiment of the present invention 1, is prepared into the polysaccharide soln (1mg/mL, 0.8mg/mL, 0.5mg/mL) of a series of concentration.
Positive control: lipopolysaccharides (LPS, Sigma company)
2. laboratory animal: Kunming mouse (Center for New Drugs Evaluation of Shandong University)
3. reagent:
DMEM high glucose medium (
sai Mo flies generation that biological chemistry Products Co., Ltd)
CCK-8 (Japanese Dojindo company)
Dual anti-(penicillin 100 U/mL and Streptomycin sulphate 100 μ g/mL,
life Technologies Corporation)
0.25% pancreas enzyme-EDTA Digestive system (Mai Chen Science and Technology Ltd.)
Foetal calf serum (Tian Hang bio tech ltd, Zhejiang)
Erythrocyte cracked liquid (Beijing Suo Laibao Science and Technology Ltd.)
Mouse TNF-α test kit (eBioscience company of the U.S.)
Low speed desk centrifuge (Anting Scientific Instrument Factory, Shanghai)
CO2gas incubator (HERAcell 150i, Thermo Scientific)
Microplate reader (Infinite 200 Pro, Tecan Austria GmbH)
Two, method
Get healthy Kunming mouse 6, de-the saying of cervical vertebra puts to death mouse, 75% alcohol disinfecting skin of abdomen, cuts an osculum with scissors, tears skin and fully exposes stomach wall, the aseptic PBS solution of 5mL is injected to intraperitoneal with syringe, gently stomach wall is rubbed, then pumpback irrigating solution, centrifugal collecting cell with finger, concentrate the peritoneal exudate cells of every 2 mouse, PBS liquid washes 3 times.Add DMEM nutrient solution 1mL to count, then training adjustment cell concn completely with DMEM is 2 × 10
6individual cell/mL, in 96 orifice plates that oneself has added medicine or contrast agents, every hole adds above-mentioned cell suspension 100 μ L.
With entirely training, jujube polysaccharide is mixed with high, medium and low three concentration, after sterile filtration, in 96 orifice plates, every hole adds 100 μ L, make final concentration be 1,0.8,0.5mg/mL; The every hole of Normal group adds full training 100 μ L; The LPS 100 μ L of LPS (lipopolysaccharides) positive controls every Kong Jiaquan training preparation, final concentration is 10 μ g/mL; Mouse peritoneal emigrated cell suspension 100 μ L is added in every hole.Measure mouse peritoneal emigrated cell metabolic activity and TNF secretion-α, do positive control with LPS.Often group establishes 6 multiple holes.96 orifice plates are placed in incubator, at saturated humidity, 5%CO
2, under 37 DEG C of conditions, cultivate the corresponding time as required.24h is cultivated when measuring cellular metabolism vigor.When measuring peritoneal exudate cells TNF secretion-α, collect culture supernatant after cultivating 10h for measuring, observation and comparison difference group cellular form under inverted microscope before collection supernatant liquor.
4h before cell cultures terminates, adds 20 μ L CCK-8 solution in 96 orifice plates in every hole, then continues to cultivate 4h, fully after vibration mixing 60s, detects the absorbance at its 450nm place by full-automatic microplate reader.
After peritoneal exudate cells cultivates 10h, 50 μ L cell culture supernatant are got in every hole respectively, detect peritoneal exudate cells culture supernatant TNF-alpha content respectively by ELISA method.On the impact of mouse peritoneal emigrated cell (being mainly scavenger cell) metabolic activity and TNF secretion-α after mensuration takes jujube polysaccharide, lipopolysaccharides (LPS) is as positive control.As Fig. 6 a with as Fig. 6 b, jujube polysaccharide significantly can strengthen peritoneal exudate cells metabolism CCK-8 vigor, promotes peritoneal exudate cells TNF secretion-α, proves that jujube polysaccharide can activate and strengthens mononuclear macrophage function and promote TNF secretion-α.Jujube polysaccharide is on the impact of mouse peritoneal emigrated cell (being mainly scavenger cell) metabolic activity and TNF secretion-α.Result shows, jujube polysaccharide significantly can strengthen peritoneal exudate cells metabolism CCK-8 vigor, promote peritoneal exudate cells TNF secretion-α, prompting jujube polysaccharide may be by activating and strengthening mononuclear macrophage function and promote TNF secretion these two approach of-α and play immunoregulation effect.
Embodiment 8: jujube polysaccharide protect the liver experimental study
One. material
1. test medicine: beach, the Yellow River jujube polysaccharide prepared by the embodiment of the present invention 2, is prepared into the polysaccharide soln (100mg/Kg, 200mg/Kg, 400mg/Kg) of a series of concentration.
Positive control: silibinin (Tasly Pharmaceutical Group Co., Ltd.)
2. laboratory animal: Kunming mouse (Center for New Drugs Evaluation of Shandong University)
3. reagent:
Tetracol phenixin (medicine company limited-liability company of traditional Chinese medicines group)
Vegetables oil (Lu Hua group)
Gpt test kit (Science and Technology Ltd. is built up in Nanjing)
Glutamic-oxal(o)acetic transaminase test kit (Science and Technology Ltd. is built up in Nanjing)
HPMC, PVP K30 and F68 (sigma company of the U.S.)
Paracetamol (sigma company of the U.S.)
Two, method
After Kunming mouse conforms 1 week, be divided into 8 often groups at random.First, tetracol phenixin carries out mouse peritoneal injection according to the dosage of 0.4% (volume fraction, v/v, tetracol phenixin/vegetables oil) (solvent is vegetables oil), often only injects 0.3mL.Naive mice only injects vegetables oil in contrast.After liver injury 1h, date and silibinin intraperitoneal injection respectively.For jujube polysaccharide group, inject the jujube polysaccharide solution of basic, normal, high (100,200,400mg/kg) Three doses respectively.For silibinin group, the silibinin solution of injection 150mg/kg.Now, the physiological saline of blank group and model group only abdominal injection same volume.After administration, water is can't help in the equal fasting of all mouse.After liver injury 24h, carry out mouse orbit and get blood.Blood is placed under room temperature after 1h, the centrifugal 15min of 4000r/min, takes out supernatant liquor (serum) and preserves at 4 DEG C.As Fig. 7, CCl
4jujube polysaccharide therapeutic action dose-dependant Journal of Sex Research after induced liver injury.CCl
41h after liver injury, the jujube polysaccharide (100,200, or 400mg/kg) of the basic, normal, high three kinds of concentration of mouse peritoneal injection and positive control drug silibinin (150mg/kg), and the activity of transaminase is measured.Often organize 8 data, all adopt mean+SD to represent.Compare blank group, ##p<0.01; Relatively CCl
4modeling group, * * p<0.01.By the treatment of jujube polysaccharide, the enzyme that obviously can reduce AST and ALT is lived, when especially jujube polysaccharide dosage is middle and high dosage (200mg/kg, 400mg/kg) (p<0.01).Illustrate that jujube polysaccharide significantly can improve CCl
4the liver injury caused.
In order to verify the therapeutic action of jujube polysaccharide further, this experiment have also been made research for time factor.Namely at CCl
4after damage, respectively at 1h, 3h and 6h jujube polysaccharide solution to separate groups of mice abdominal injection same concentration (400mg/kg).Meanwhile, blank group and model group only injecting normal saline.After liver injury 24h, serum takes out and preserves at 4 DEG C.As Fig. 8, CCl
4jujube polysaccharide therapeutic action time-dependent Journal of Sex Research after induced liver injury.After liver injury after 1h, 3h or 6h, abdominal injection jujube polysaccharide (400mg/kg); Positive controls is 1h pneumoretroperitoneum injection silibinin (150mg/kg), and measures the activity of transaminase.Often organize 8 data, all adopt " mean+SD " to represent.Compare blank group, ##p<0.01; Relatively CCl
4modeling group, * * p<0.01.After liver injury after 1h, 3h or 6h, abdominal injection jujube polysaccharide, all has unusual effect, AST and ALT activation recovering can be made to normal value.
At CCl
4before carrying out liver injury, jujube polysaccharide group mouse continuous 5 days abdominal injection jujube polysaccharide solution (100 or 400mg/kg), once a day.Silibinin group mouse adopts same method injection silibinin (150mg/kg).Naive mice and model group mouse only injecting normal saline.On 6th, the CCl of equal abdominal injection 0.4% except naive mice
4solution (vegetables oil makees solvent) 0.3mL carrys out induced liver injury.After liver injury, water is can't help in the equal fasting of all mouse, takes out serum and preserve at 4 DEG C after 24h.As Fig. 9, jujube polysaccharide is to CCl
4induced liver injury prophylactic effect is studied.CCl
4before liver injury continuous 5 days, once a day to the jujube polysaccharide of low, the high two kinds of concentration of mouse peritoneal injection (100 or 400mg/kg) and positive control drug silibinin (150mg/kg), and the activity of transaminase is measured.Often organize 8 data, all adopt " mean+SD " to represent.Compare blank group, #p<0.05, ##p<0.01; Relatively CCl
4modeling group, * p<0.05, * * p<0.01.The prophylactic effect of jujube polysaccharide reduces AST and ALT activity value.Especially when jujube polysaccharide dosage is 400mg/kg, close with blank group.Proved by the above results, jujube polysaccharide can to CCl
4prophylactic effect is played in the liver injury of induction.
As Figure 10, jujube polysaccharide therapeutic action dose-dependant Journal of Sex Research after paracetamol (APAP) induced liver injury.
Because paracetamol is insoluble in water, adopt HPMC, PVP K30 and F68 to carry out solubilising, mass ratio is APAP:HPMC:PVP K30:F68=8:5:5:5.By its random packet after mouse conforms, the APAP solution 0.5mL of every abdominal injection 400mg/kg except blank group.After liver injury 1h, date and silibinin intraperitoneal injection respectively.For jujube polysaccharide group mouse, inject the jujube polysaccharide solution of basic, normal, high (100,200,400mg/kg) Three doses respectively.For silibinin group mouse, the silibinin solution of injection 150mg/kg.Now, the physiological saline of blank group and model group only abdominal injection same volume.Water is can't help in the equal fasting of all mouse.After liver injury 24h, carry out mouse orbit and get blood.Blood is placed under room temperature after 1h, the centrifugal 15min of 4000r/min.Take out supernatant liquor (serum) to preserve at 4 DEG C.1h after APAP liver injury, the jujube polysaccharide (100,200, or 400mg/kg) of the basic, normal, high three kinds of concentration of mouse peritoneal injection and positive control drug silibinin (150mg/kg), and the activity of transaminase is measured.Often organize 8 data, all adopt " mean+SD " to represent.Compare blank group, ##p<0.01; Relatively APAP modeling group, * * p<0.01.After APAP modeling, the activity of model group AST and ALT significantly improves (p<0.01).From AST and ALT assessment of levels, after jujube polysaccharide abdominal injection, significantly liver injury can be reduced.
In order to verify the therapeutic action of jujube polysaccharide further, this experiment have also been made research for time factor.Namely after APAP damage, respectively at 1h, 3h and 6h jujube polysaccharide solution to separate groups of mice abdominal injection same concentration (400mg/kg).Meanwhile, blank group and model group only injecting normal saline.After liver injury 24h, serum takes out and preserves at 4 DEG C.As jujube polysaccharide therapeutic action time-dependent Journal of Sex Research after Figure 11, APAP induced liver injury.After liver injury after 1h, 3h or 6h, abdominal injection jujube polysaccharide (400mg/kg); Positive controls is 1h pneumoretroperitoneum injection silibinin (150mg/kg), and measures the activity of transaminase.Often organize 8 data, all adopt " mean+SD " to represent.Compare blank group, ##p<0.01; Relatively APAP modeling group, * p<0.05, * * p<0.01.After APAP modeling, after jujube polysaccharide abdominal injection, significantly can reduce liver injury.
Before APAP carries out liver injury, jujube polysaccharide group mouse continuous 5 days abdominal injection jujube polysaccharide solution (100 or 400mg/kg), once a day.Silibinin group mouse adopts same method injection silibinin (150mg/kg) as positive control.Naive mice and model group mouse only injecting normal saline.The 6th day time, equal abdominal injection 400mg/kg APAP induced liver injury except naive mice.After liver injury, water is can't help in the equal fasting of all mouse, takes out serum and preserve at 4 DEG C after 24h.As Figure 12, jujube polysaccharide is studied APAP induced liver injury prophylactic effect.Before APAP liver injury continuous 5 days, once a day to jujube polysaccharide (100 or 400mg/kg) and the positive control drug silibinin (150mg/kg) of low, the high two kinds of concentration of mouse peritoneal injection, and the activity of transaminase is measured.Often organize 8 data, all adopt " mean+SD " to represent.Compare blank group, ##p<0.01; Relatively APAP modeling group, * p<0.05, * * p<0.01.Jujube polysaccharide, when high dosage (400mg/kg) prevents administration, can reduce the vigor of AST and ALT significantly, and jujube polysaccharide can play preventive effect clearly.
Jujube polysaccharide prepared by embodiment of the present invention 1-3, can it can be used as effective constituent, makes in conventional manner for the medicine of preventing liver injury, food or healthcare products.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (10)
1. beach, a Yellow River jujube Polyose extraction process for purification, is characterized in that, comprise the following steps:
(1) by the jujube stoning of beach, the Yellow River, dry, pulverize, obtain jujube powder, obtain degreasing jujube powder with organic solvent backflow degreasing;
(2) being 1g:(15-30 by degreasing jujube powder and water by mass volume ratio) mL mixes, supersound extraction 15-30min, filter, filtrate is concentrated into the 1/3-1/2 of former filtrate volume, and in concentrated solution, add volumetric concentration is 80%-100% ethanol, and to mixed solution, the volumetric concentration of ethanol is 75-85%, alcohol precipitation spends the night, washing precipitation, dry, obtain Crude polysaccharides;
(3) by water-soluble for the Crude polysaccharides that step (2) is obtained, making the Crude polysaccharides solution of 1-3g/L, is 1:(2.5-3.5 by Sevag reagent and Crude polysaccharides solution by volume) mix, jolting, stratification, discard lower floor's organic layer and middle white denatured protein layer, upper strata polysaccharide soln is evaporated to the 1/8-1/6 of former Crude polysaccharides liquor capacity, alcohol precipitation spends the night, filter, washing precipitation, dry, obtain Deproteinated jujube polysaccharide;
(4) by water-soluble for the Deproteinated jujube polysaccharide that step (3) is obtained, make the polysaccharide soln of 4-6g/L, the pH of regulator solution is 8.5-9, in polysaccharide soln, add H
2o
2, H to polysaccharide soln
2o
2volume fraction be 40%, heating in water bath decolours, and alcohol precipitation spends the night, and filters, washing precipitation, dry, obtains the jujube polysaccharide after purifying.
2. beach, a kind of the Yellow River as claimed in claim 1 jujube Polyose extraction process for purification, it is characterized in that, in step (1), the method of backflow degreasing is: jujube powder being added to volumetric concentration is in 95% ethanol, the mass volume ratio of jujube powder and ethanol was 1g:5mL, 45 DEG C of backflow degreasings 3 hours.
3. beach, a kind of the Yellow River as claimed in claim 1 jujube Polyose extraction process for purification, is characterized in that, in step (2), the temperature of supersound extraction is 50 DEG C, and the time of supersound extraction is 15min.
4. beach, a kind of the Yellow River as claimed in claim 1 jujube Polyose extraction process for purification, it is characterized in that, in step (3), after Sevag reagent is mixed with Crude polysaccharides solution, violent jolting 20min, layering after standing 30min, discards lower floor's organic layer and middle white denatured protein layer, repetitive operation 10 times; Described Sevag reagent is the solution of trichloromethane and propyl carbinol 5:1 mixing by volume.
5. beach, a kind of the Yellow River as claimed in claim 1 jujube Polyose extraction process for purification, is characterized in that, in step (4), regulator solution pH reagent used is the NaOH solution of 0.1M.
6. beach, a kind of the Yellow River according to claim 1 jujube Polyose extraction process for purification, is characterized in that, in step (4), the temperature of water-bath decolouring is 40 DEG C, and the time is 4h.
7. beach, the Yellow River jujube polysaccharide prepared by the Hydrolysis kinetics method as described in any one of claim 1 to 6.
8. beach, the Yellow River according to claim 7 jujube polysaccharide is for the preparation of the application in the medicine of preventing liver injury, food and/or healthcare products.
9. for a pharmaceutical composition for preventing liver injury, it is characterized in that, containing beach, the Yellow River according to claim 7 jujube polysaccharide in described pharmaceutical composition.
10. there is a food for prevention liver-protecting efficacy, it is characterized in that, containing beach, the Yellow River according to claim 7 jujube polysaccharide in described food.
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