CN104945525B - Yellow bur reality polysaccharide and its preparation method and application - Google Patents
Yellow bur reality polysaccharide and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of yellow bur reality polysaccharide, the polysaccharide is the extract of the straight fringe barberry Berberis dasystachya Maxim. fruits of Berberidaceae plant, and polyoses content is 60~95%w/w in extract.The present invention is the study found that yellow bur reality polysaccharide can increase amount of insulin, be to corresponding ROS metabolizing enzymes activity with good function of blood sugar reduction：Superoxide dismutase, catalase, activity of glutathione peroxidase have a degree of adjustment effect.Simultaneously, it has also been found that, the effect of yellow bur reality high dose group is suitable with the effect of glibenclamide, though and glibenclamide be treat diabetes a line chemicals, take glibenclamide in large quantities for a long time, it will ultimately result in the hypoglycemia and nephrosis of patient, death is even resulted in, conversely, real this of yellow bur is edible berry, good security is more suitable for treating and preventing food, health products or the drug of diabetes.
Description
Technical field
The invention belongs to field of health care products, and in particular to yellow bur reality polysaccharide and its preparation method and application.
Technical background
Huang thorn, the straight fringe barberry Berberis dasystachya Maxim. of Berberidaceae plant are (black with thorn, sea-buckthorn in vain
Thorn) equally it is the medicinal and edible traditional wild acinus of the ethnic groups such as illiteracy, Tibetan, Uygur, it is referred to as " thorn of Qinghai three ".Mesh
Before, for Bai Ci and Hei Ci, research and application are very thorough, and Huang is pierced, composition Study and function application still compared with
For blank.
《Often use Chinese herbal medicine handbook in Qinghai》In Cortex berberidis dasystachyae (i.e. root skin) is documented：Cortex berberidis dasystachyae is dicotyledon
The stem skin of the straight fringe barberry of medicine Berberidaceae plant.Brief description has also been made in its fruit medicine function, has treatment abdominal pain, digestion
It is bad, abdominal distension, the functions such as dysentery.
It yet there are no the report of the research to yellow bur reality polysaccharide.
Invention content
The purpose of the present invention is to provide yellow bur reality polysaccharide and its preparation method and application.
Specifically, the present invention provides a kind of yellow bur reality polysaccharide, the polysaccharide is the straight fringe barberry of Berberidaceae plant
The extract of Berberis dasystachya Maxim. fruits, polysaccharide is main component in extract.
It is measured in the specific embodiment of the invention and shows that polyoses content is 93 ± 2%w/w, the purity of polysaccharide in extract
It is higher, biological activity test is carried out with the polysaccharide under this purity, result can really reflect the life of yellow bur reality polysaccharide
Object activity, efficiently avoids the interference that other compositions measure active polysaccharide.On the basis of known polysaccharide bioactivity, i.e.,
To reduce content of the polysaccharide in extract, as long as suitably adjusting extract dosage so that polysaccharide reach work accordingly it is dense
Degree, can equally play same or analogous bioactivity.Therefore, the present invention limits, and polyoses content should be 60 in extract
~95%w/w.
Further, polyoses content is 80~95%w/w in extract, is further 90~95%w/w.
The present invention also provides the preparation method of yellow bur reality polysaccharide, the polysaccharide is prepared by water extraction and alcohol precipitation method.
Further, in alcohol precipitation step, ethyl alcohol final concentration reaches 60~85%v/v, is further 70~80%
V/v, preferably 75 ± 2%v/v.
Further, the water extraction and alcohol precipitation method concrete operations are as follows：After yellow bur reality degreasing, removing monosaccharide, extracting in water,
Aqueous extracts decoloration, removing protein, dialysis are stood except after small molecule, ethyl alcohol (ethyl alcohol final concentration is made to reach 60~85%v/v) is added,
It is to be precipitated to take precipitation completely, it dries to get to yellow bur reality polysaccharide.
Further, dialyse removing small molecule molecular weight in 3500Da or less.
Further, degreasing concrete operations are as follows：By yellow bur in fact crush after, sieving, use petroleum ether or ether for
Extraction solvent, the residue filter after extraction, volatilizes solvent, obtains degreasing Huang bur reality residue.
Further, go the concrete operations of monosaccharide as follows：Gained degreasing Huang bur reality residue, with ethyl alcohol (70~
It 85%v/v) extracts, the residue after extraction volatilizes solvent to get monosaccharide Huang bur reality residue has been removed；Preferably concentration of alcohol is
80%v/v.
Further, dialysis is except ethyl alcohol is added after small molecule to alcohol content to 70~80%v/v, preferably 75 ± 2%
v/v。
By above-mentioned purification step, the purity of yellow bur reality polysaccharide can effectively improve.Certainly, if it is only necessary to prepare
The lower yellow bur reality polysaccharide of purity, can also omit one or more in other purification steps in addition to alcohol precipitation.
In addition, the decolorization for polysaccharide, usually uses ion-exchange (such as DEAE- celluloses), oxidizing process (such as
H2O2), absorption method (such as activated carbon), in actual use can according to self-demand, in conjunction with later stage polysaccharide yield and
Purity selectes suitable discoloration method, and H is selected in a specific implementation mode of the invention2O2Decoloration, but this is not limited to this hair
It is bright to use such decoloration mode.
For removing protein, common method has trichloroacetic acid method, hydrochloric acid method, Sevage methods, and (also on the books is Sevag
Method) etc., in actual use removing of being suitble to can be selected in conjunction with the yield and purity of later stage polysaccharide according to self-demand
Protein process selects Sevage methods in a specific implementation mode of the invention, but this is not limited to the present invention and can only use
Such removing protein mode.
The present invention also provides use of the above-mentioned yellow bur reality polysaccharide in preparing oxidation resistant food, drug or health products
On the way.
Wherein, the food, drug or health products are food, drug or the health products for removing free radical.
Further, the free radical is DPPH, ABTS, ultra-oxygen anion free radical or hydroxy radical.
The present invention also provides a kind of pharmaceutical composition, it be by above-mentioned yellow bur reality polysaccharide with it is pharmaceutically acceptable auxiliary
Material or the complementary pharmaceutical preparation prepared at subassembly.
Pharmaceutically acceptable auxiliary material of the present invention includes but are not limited to filler (diluent), lubricant (helps stream
Agent or antitack agent), dispersant, wetting agent, adhesive, conditioning agent, solubilizer, antioxidant, bacteriostatic agent, emulsifier, disintegrant
Deng.Adhesive include syrup, Arabic gum, gelatin, sorbierite, tragacanth, cellulose and its derivates (such as microcrystalline cellulose,
Sodium carboxymethylcellulose, ethyl cellulose or hypromellose etc.), gelatine size, syrup, starch slurry or polyvinylpyrrolidine
Ketone etc.；Filler includes lactose, Icing Sugar, dextrin, starch and its derivative, cellulose and its derivates, inorganic calcium salt (such as sulfuric acid
Calcium, calcium phosphate, calcium monohydrogen phosphate, precipitated calcium carbonate etc.), sorbierite or glycine etc.；Lubricant includes superfine silica gel powder, stearic acid
Magnesium, talcum powder, aluminium hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol etc.；Disintegrant includes starch and its derivative (such as carboxylic first
Base sodium starch, Explotab, pregelatinized starch, modified starch, hydroxypropul starch, cornstarch etc.), polyvinylpyrrolidine
Ketone or microcrystalline cellulose etc.；Wetting agent includes lauryl sodium sulfate, water or alcohol etc.；Antioxidant packages are containing sodium sulfite, sulfurous acid
Hydrogen sodium, sodium pyrosulfite, dibutyl benzoic acid etc.；Bacteriostatic agent includes 0.5% phenol, 0.3% cresols, 0.5% anesin etc.；
Conditioning agent includes hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffer (including phosphoric acid dioxy sodium and phosphoric acid hydrogen two
Sodium) etc.；Emulsifier includes Tween-80, does not have that sour sorb is smooth, pluronic gram F-68, lecithin, Fabaceous Lecithin etc.；Solubilizer
Including Tween-80, bile, glycerine etc..
Heretofore described pharmaceutically acceptable complementary ingredient, it have certain physiological activity, but the ingredient plus
Enter the leading position that will not change aforementioned pharmaceutical compositions in treatment of diseases, and only play auxiliary effect, these are auxiliary
Assist effect is only the utilization to the ingredient known activity, is the usual adjuvant treatment modality of field of medicaments.If by above-mentioned auxiliary
Property ingredient is used cooperatively with pharmaceutical composition of the present invention, still should belong to the scope of protection of the invention.
Further, the pharmaceutical preparation is through gastrointestinal administration preparation.
Wherein, described to be selected from common dosage form in the market through gastrointestinal administration preparation, use peroral dosage form more,
Such as granule, powder, pill, tablet, capsule, oral solution.
The present invention also provides aforementioned pharmaceutical compositions prepare it is anti-oxidant, hypoglycemic, antitumor or improve lipid metaboli it is disorderly
Purposes in random drug.
Wherein, the drug can be used for the treatment of I types or type-2 diabetes mellitus, can also be used to treat gastric cancer, liver cancer, breast
The malignant tumours such as gland cancer are especially suitable for the treatment to liver cancer.
Wherein, the improvement disorders of lipid metabolism refers to improving diabetic's disorders of lipid metabolism.
Further, the improvement disorders of lipid metabolism refers to reducing diabetic cholesterol, triglyceride or/and low close
Spend lipoprotein, increasing high density lipoprotein.
Further, the diabetic is the normal or impaired patient of liver function.The present invention is the study found that dative arranges
Unlike the chemical drugs such as this urea, yellow bur reality polysaccharide is harmless to liver function, therefore, can yellow bur reality polysaccharide be used for liver function
The treatment of energy impaired subjects.
Beneficial effects of the present invention are as follows：
(1) present invention has carried out preparation and the research of bioactivity to yellow bur reality polysaccharide for the first time, as a result, it has been found that yellow bur
Real polysaccharide has the effects that good anti-oxidant, hypoglycemic, antitumor.
(2) present invention can increase amount of insulin the study found that yellow bur reality polysaccharide has good function of blood sugar reduction, right
ROS metabolizing enzyme activity is accordingly：Superoxide dismutase, catalase, activity of glutathione peroxidase have
A degree of adjustment effect.
Meanwhile it has also been found that, the effect of yellow bur reality high dose group is suitable with the effect of glibenclamide, and Ge Lieben
Though urea is the line chemicals for treating diabetes, takes glibenclamide in large quantities for a long time, will ultimately result in the hypoglycemia of patient
And nephrosis, death is even resulted in, conversely, real this of yellow bur is edible berry, good security is more suitable for treatment and pre-
Food, health products or the drug of anti-diabetes.
(3) through antioxidation in vitro it is demonstrated experimentally that yellow bur reality polysaccharide has very strong antioxidant activity：The product is to DPPH
Free radical, ABTS free radicals, ultra-oxygen anion free radical, hydroxy radical, reducing power etc. have apparent scavenging effect；Through external
Anti-tumor experiment proves that yellow bur reality polysaccharide is to five kinds of cell human gastric cancer cells BGC-823s, breast cancer in exponential phase
Cell MCF-7 and MDA-MB-231, hepatocellular carcinoma H22 and SMMC-7721 have preferable antitumor activity, can be by yellow bur
Real polysaccharide is applied to anti-oxidant, antitumor action food, health products or drug.
(4) present invention can reduce cholesterol studies have shown that yellow bur reality polysaccharide has the function of improvement disorders of lipid metabolism
(TC), triglycerides (TG), low-density lipoprotein (LDL), increasing high density lipoprotein (HDL)；And lipid metaboli phase can be increased
The enzymatic activity of pass increases lipoproteinesterase and liver esterase activity, while can reduce glutamic-oxalacetic transaminease and glutamic-pyruvic transaminase work
Property, it is not damaged to hepatic tissue, it can be as drug, health products or the food of the improvement disorders of lipid metabolism of good security
Description of the drawings：
Influence of Fig. 1 Huangs bur reality polysaccharide to the weight of the STZ diabetes rats induced
Scavenging activities of Fig. 2 Huangs bur reality Polysaccharide B DP and Vc to ABTS
Scavenging activities of Fig. 3 Huangs bur reality Polysaccharide B DP and Vc to DPPH
The reducing power of Fig. 4 Huang bur reality Polysaccharide Bs DP and Vc
Scavenging activities of Fig. 5 Huangs bur reality Polysaccharide B DP and Vc to ultra-oxygen anion free radical
Scavenging activities of Fig. 6 Huangs bur reality Polysaccharide B DP and Vc to hydroxy radical
Specific implementation mode
Embodiment 1：The preparation of yellow bur reality polysaccharide
1) yellow bur reality degreasing：Take the fruit 400g drying of ripe yellow thorn (i.e. straight fringe barberry), grinding and sieving；Take Huang
Bur mixes with petroleum ether in fact, and ultrasonic extraction after mixing is centrifuged off extracting solution, and residue filter volatilizes petroleum ether, obtains grease removal
Yellow bur reality residue；
2) yellow bur removes monosaccharide in fact：Grease removal Huang bur reality residue is mixed with ethyl alcohol, and the concentration of alcohol is 70~85%
V/v refluxing extractions are centrifuged off extracting solution, ethyl alcohol are volatilized after residue filter, obtain and have removed monosaccharide Huang bur reality residue；
3) yellow thorn Thick many candies extraction：It is mixed monosaccharide Huang bur reality residue has been removed obtained by step 2) with pure water, microwave radiation technology
Extraction, centrifugation obtain extracting solution, are discarded after residue filter；Extracting solution is concentrated to give Thick many candies extracting solution；
4) purifying of yellow thorn Thick many candies：H is used in Thick many candies extracting solution successively2O2, decolourized, Savege methods remove egg
White matter, then dialyse and remove 3500Da or less small-molecule substances, the polysaccharide Aqueous extracts purified；
5) preparation of yellow bur reality polysaccharide：It is molten that ethyl alcohol is added in purified polysaccharide Aqueous extracts concentrate obtained by step 4)
Liquid makes ethyl alcohol final concentration reach 75%v/v, amounts to 2.0 6g after gained precipitation is dry to get to yellow bur reality Polysaccharide B DP.
Embodiment 2：The preparation of yellow bur reality polysaccharide
1) yellow bur reality degreasing：Take the fruit 400g drying of ripe yellow thorn (i.e. straight fringe barberry), grinding and sieving；Take Huang
Bur mixes with petroleum ether in fact, and refluxing extraction is centrifuged off extracting solution, and residue filter volatilizes petroleum ether, obtains grease removal Huang bur
Real residue；
2) yellow bur removes monosaccharide in fact：Grease removal Huang bur reality residue is mixed with ethyl alcohol, and the concentration of alcohol is 70~85%
V/v, ultrasonic extraction are centrifuged off extracting solution, ethyl alcohol are volatilized after residue filter, obtain and have removed monosaccharide Huang bur reality residue；
3) yellow thorn Thick many candies extraction：It being mixed monosaccharide Huang bur reality residue has been removed obtained by step 2) with pure water, temperature extraction takes,
Centrifugation obtains extracting solution, is discarded after residue filter；Extracting solution is concentrated to give Thick many candies extracting solution；
4) purifying of yellow thorn Thick many candies：H is used in Thick many candies extracting solution successively2O2, decolourized, Savege methods remove egg
White matter, then dialyse and remove 3500Da or less small-molecule substances, the polysaccharide Aqueous extracts purified；
5) preparation of yellow bur reality polysaccharide：It is molten that ethyl alcohol is added in purified polysaccharide Aqueous extracts concentrate obtained by step 4)
Liquid makes ethyl alcohol final concentration reach 80%v/v, and gained precipitation is dry to amount to 1.355g to get to yellow bur reality Polysaccharide B DP.
Embodiment 3：The preparation of yellow bur reality polysaccharide
1) yellow bur reality degreasing：Take the fruit 400g drying of ripe yellow thorn (i.e. straight fringe barberry), grinding and sieving；Take Huang
Bur mixes with petroleum ether in fact, it is ultrasonic after refluxing extraction again, be centrifuged off extracting solution, residue filter volatilizes petroleum ether, obtains and has removed
Fat Huang bur reality residue；
2) yellow bur removes monosaccharide in fact：Grease removal Huang bur reality residue is mixed with ethyl alcohol, and the concentration of alcohol is 70~85%
V/v, ultrasonic extraction are centrifuged off extracting solution, ethyl alcohol are volatilized after residue filter, obtain and have removed monosaccharide Huang bur reality residue；
3) yellow thorn Thick many candies extraction：It is mixed monosaccharide Huang bur reality residue has been removed obtained by step 2) with pure water, refluxing extraction,
Centrifugation obtains extracting solution, is discarded after residue filter；Extracting solution is concentrated to give Thick many candies extracting solution；
4) purifying of yellow thorn Thick many candies：H is used in Thick many candies extracting solution successively2O2, decolourized, Savege methods remove egg
White matter, then dialyse and remove 3500Da or less small-molecule substances, the polysaccharide Aqueous extracts purified；
5) preparation of yellow bur reality polysaccharide：It is molten that ethyl alcohol is added in purified polysaccharide Aqueous extracts concentrate obtained by step 4)
Liquid makes ethyl alcohol final concentration reach 70%v/v, and gained precipitation is washed 3 times with 20mL absolute ethyl alcohols, and the sediment after washing carries out
Spray drying so that moisture content of material is down to 10% and amounts to 1.67g hereinafter, obtaining yellow bur reality Polysaccharide B DP.
Anthrone-sulfuricacid method measurement is carried out to yellow bur reality polysaccharide prepared by the method for the present invention, wherein polyoses content is 93%
±2w/w。
In experiment provided in an embodiment of the present invention, the dosage of yellow bur reality polysaccharide is with the Polyose extraction substance of preparation
Amount calculates.
The yellow bur reality polysaccharide of embodiment 4 improves disorders of lipid metabolism effect experiment
1) experimental drug：Yellow bur reality polysaccharide prepares gained, as white powder solid for the present invention.Dosage is by big
Mouse batheroom scale, gavage low dosage, middle dosage, high dose are respectively 75mg/kg, 150mg/kg, 300mg/kg,
2) animal：Four week old Kunming rats, male, regular grade, 135g are provided by Gansu Chinese of Traditional Chinese Medicine.In in laboratory
It is placed in raise in mouse cage and be tested after a week (per 8, cage, 22 ± 1 DEG C of temperature, humidity 42% ± 2%).It is divided into six groups：Point
Not Wei blank group (intravenous injection the non-modeling of citrate buffer solution, ultra-pure water gavage), (tail vein injection STZ modelings surpass model group
Pure water gavage), positive drug group (tail vein injection STZ modelings, glibenclamide 20mg/kg gavages), yellow bur reality polysaccharide low dosage
Group (tail vein injection STZ modelings, yellow bur reality polysaccharide gavage 75mg/kg), yellow bur reality polysaccharide middle dose group (tail vein injection
STZ modelings, yellow bur reality polysaccharide gavage 150mg/kg), yellow bur reality polysaccharide high dose group (tail vein injection STZ modelings, Huang thorn
Fruit polysaccharide gavage 300mg/kg).Modeling method：Fasting 16h in advance then carries out it STZ (citrate buffer solution preparation)
Tail vein injection 60mg/kg after injection, surveys the indexs such as its blood glucose, blood fat, testing model success or not for 72 hours.
3) experiment reagent：95% medicinal alcohol, Nanjing Ning Shi chemical reagent Co., Ltd；Physiological saline, Shandong all medicine together
Industry Co., Ltd；Cholesterol, triglyceride, high-density lipoprotein, density lipoprotein, glutamic-oxalacetic transaminease, glutamic-pyruvic transaminase, fat
Albumen esterase, liver esterase kit, bio tech ltd is built up in Nanjing.
(4) laboratory apparatus：Bio-Rad680 type microplate reader, Bio Rad Laboratories；DS-1 type tissue Syrup-homogenizing instruments, Shanghai are just intelligent
Trade Co., Ltd.；TGL-16G-A type tube centrifuges, Anting Scientific Instrument Factory, Shanghai；The visible uv-spectrophotometrics of UV-722N
Meter, Shanghai Ni Ke Co., Ltds；HD type thermostat water baths, Constant Temp. Instrument Factory, Liaoyang.
(5) experimentation：Healthy SD rat 48 is taken, 6 groups is randomly divided by weight, is divided into blank group, model group, the positive
Medicine group and low, in, high dose group.Blank group and the daily gavage of model group give distilled water；The daily gavage of positive drug group gives lattice
Arrange this urea 1 time；Yellow bur reality polysaccharide is given to the daily gavage of test medicine group 1 time, and six groups are continuously given 28d；Last is given
After tested material, after anesthesia, blood, acquired blood centrifuging and taking supernatant is taken to turn for detecting blood glucose, blood lipids index and millet straw through arteria carotis
Ammonia enzyme, glutamic-pyruvic transaminase.To its death, rat liver tissue is taken, surface blood stains is washed away with physiological saline, is blotted with filter paper,
Tissue Syrup-homogenizing instrument is homogenized, and is collected in homogenate to centrifuge tube, centrifuges 10min with 3000rpm, Aspirate supernatant carries out fat
Albumen esterase and liver esterase determination of activity.
(6) yellow bur reality polysaccharide reduces the blood glucose suffered from diabetes rat body, lipid determination result
The diabetes of STZ inductions can so that karyopycnosis and hyaloid lesion occur for rat Islet cells, lipid metaboli with
Get muddled.The lipid metaboli of rat gets muddled simultaneously, and related blood lipids index and the relevant enzymatic activity of lipid metaboli also occur disorderly
Disorderly.The present invention is simultaneously determined blood fat and blood glucose, as a result 1,2 and Fig. 1 of part table.
Influence (Mean ± SD) of the yellow bur reality polysaccharide of table 1 to the STZ rat blood sugars induced
Note：Compared with normal groupaP ＜ 0.05；Compared at modulebP ＜ 0.05；
Rat Cholesterol TC that the yellow bur reality polysaccharide component of table 2 induces STZ, triglycerides TG, high-density lipoprotein
The influence (Mean ± SD) of HDL, low-density lipoprotein LDL
Note：Compared with normal groupaP ＜ 0.05；Compared at modulebP ＜ 0.05
By table 1,2 can and Fig. 1 know, in above-mentioned experiment STZ induce rat blood sugar, blood fat increase, show that above-mentioned experiment is made
Mould success.Huang each dosage group of bur reality polysaccharide of the invention has apparent reduction to act in the blood glucose of STZ induced diabetic rats, together
When, there is the reduction effect that adjusts to the cholesterol, triglycerides, low-density lipoprotein of the disorders of lipid metabolism rat of STZ inductions, it is right
High-density lipoprotein has adjusting effect of increasing, and is in dose-effect relationship, high dose group and the significant difference of model group (p ＜
0.05).Meanwhile experiment shows that the effect of high dose group is suitable with the effect of glibenclamide, and glibenclamide is as chemical synthesis
Drug, have certain side effect to body, therefore, it is suggested that can be replaced using the yellow bur reality polysaccharide extracted in natural plants
It is used for drug.
(7) to the influence of relevant enzyme
Liver esterase HL is that have phosphorus A1 and triglyceride hydrolytic enzyme activities by one kind that hepatic parenchymal cells synthesize, to plasma adiponectin
Matter transports the extracellular protein to play an important role, is primarily involved in the generation of the reconstruct and chylomicron remains, low-density lipoprotein of HDL-C
It thanks and the antiport of cholesterol.HL vigor is abnormal to have higher correlation with atherosclerosis and diabetes.Lipoprotein
Esterase LPL is the synthesis of the parenchymas such as adipocyte, cardiac muscle cell, Skeletal Muscle Cell, mammary glandular cell and macrophage and divides
A kind of glycoprotein secreted.Active LPL exists in the form of homodimer, passes through electrostatic attraction and capillary endothelial cell surface
Polysaccharide combine, heparin can promote the LPL of this combining form to be released into blood, and its activity can be improved.LPL is lipoprotein generation
The key enzyme thanked is key enzyme of the hydrolysis rich in triglyceride in three casein of glycerine.Its major function is hydrolysis CM and VLDL
In triglyceride be glycerine and aliphatic acid.Lipoproteinesterase LPL and liver esterase HL can decompose triglycerides TG and decompose
For glycerine and free fatty FFA, fat can be calculated separately out by measuring the free fatty that its decomposition generates using copper reagent
The activity of albumen esterase and liver esterase.
For AST with ALT contents under the stimulation of some drugs, internal hepatic tissue is invaded profit by adipocyte, influences correlation
Hepatocyte enzyme activity, and under normal circumstances, as Simvastatin and glibenclamide etc. can all have a certain impact to hepatic tissue.
Rat lipoproteinesterase LPL, liver esterase HL, the glutamic-oxalacetic transaminease that the yellow bur reality polysaccharide component of table 3 induces STZ
The influence (Mean ± SD) of AST, glutamic-pyruvic transaminase ALT
Note：Compared with normal groupaP ＜ 0.05；Compared at modulebP ＜ 0.05；Compared with normal groupcP ＜ 0.05.
Yellow bur reality polysaccharide can be such that HL the and LPL activity of experimental rat increases, and LPL is to remove the limit rich in TG lipoprotein
Fast enzyme, to play its important function in lipoprotein cycle.The HL activity of diabetes rat can be made to increase simultaneously, to
It is used as ligand by HL, promotes liver cell intake cholesterol and chylomicron remnant, reduce total cholesterol and glycerine three in blood plasma
Vinegar concentration, and then have the function that adjust body disorders of lipid metabolism.
And glibenclamide positive drug group is compared for AST with ALT activity, there is different degrees of reduction, illustrates positive drug
Group has damage liver, and yellow bur reality polysaccharide to liver then without damaging action, and there are apparent differences.
Experiment conclusion：Blood lipids index and the esterase active of diabetes rat through STZ inductions are it is demonstrated experimentally that yellow bur is real more
Sugar has very strong improvement disorders of lipid metabolism activity.The diabetes that Huang each dosage group of bur reality polysaccharide of the invention induces STZ are big
Cholesterol, triglycerides, the low-density lipoprotein of mouse, which have, adjusts reduction effect, is made by adjusting to rise to high-density lipoprotein
With.And it is in dose-effect relationship, high dose group and the significant difference of model group (p ＜ 0.05)；Yellow bur reality polysaccharide can make
HL the and LPL activity for suffering from the liver of diabetes rat increases, and is in dose-effect relationship, high dose group and the significant difference of model group
Different (p ＜ 0.05)；Meanwhile it is demonstrated experimentally that AST with ALT activity compares glibenclamide positive drug group, there is different degrees of drop
It is low, illustrate positive drug group for liver by damaging action, and yellow bur reality polysaccharide to liver then without damaging action.Therefore, yellow
Bur reality polysaccharide can be applied to improve food, health products or the drug of disorders of lipid metabolism effect.
The yellow bur reality polysaccharide antioxidation activity in vitro experiment of embodiment 5
(1) material：The yellow bur reality polysaccharide of prepared gained of the invention.
(2) laboratory apparatus and reagent：DS-1 type tissue Syrup-homogenizing instruments, Shanghai Zheng Hui Trade Co., Ltd.s；TGL-16G-A type pipes
Formula centrifuge, Anting Scientific Instrument Factory, Shanghai；UV-722N visible ultraviolet spectrophotometers, Shanghai Ni Ke Co., Ltds；HD types are permanent
Warm water bath, Constant Temp. Instrument Factory, Liaoyang.DPPH, ABTS are purchased from Shanghai Jing Chun biochemical technologies limited liability company (Aladdin)；
The reagents such as potassium peroxydisulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, trichloroacetic acid are purchased from the limited public affairs of Shandong king's Yu industry
Department；Superoxide anion kit, hydroxy radical kit are purchased from Nanjing and build up bio tech ltd.
(3) ABTS Scavenging activities：By ABTS dissolvings (+H2O) to final concentration of 7mmol/L, potassium peroxydisulfate is configured to
2.45mmol/L mixes the two solution, is positioned over dark, stirs 16h under room temperature；Then ABTS mixed solutions are used
It is 0.70 ± 0.02 that PBS solution (PH=7.0), which is diluted to its light absorption value (being surveyed at 734nm),；Yellow bur reality polysaccharide ultra-pure water
It is diluted to various concentration after dissolving, 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.40mg/mL, 0.80mg/mL,
1.60mg/mL 3.20mg/mL；Finally the sample of above-mentioned various concentration is added in ABTS mixed solutions, sample and ABTS
Mixed solution ratio is 1: 20, is incubated 6min after mixing at room temperature, and light absorption value is measured at 734nm.
Calculation formula：ABTS clearance rates (%)=(1-Asample/Acontrol) × 100%
Wherein AcontrolFor the light absorption value of ABTS mixed solutions；AsampleTo be measured after addition sample in ABTS mixed solutions
Light absorption value.
As shown in Figure 2, yellow thorn seed oil has the stronger ability for removing ABTS, minimum concentration, that is, 0.05mg/mL polysaccharide
Clearance rate to ABTS is 31.83%, and clearance rate increases in the increase with yellow bur reality polysaccharide concentration, and
The yellow bur reality polysaccharide of 3.20mg/mL is up to 96.72% to the clearance rate of ABTS, close to the clearance rate of VC.Therefore, institute is prepared
Obtaining yellow bur reality polysaccharide has the stronger ability for removing ABTS.
(4) DPPH Scavenging activities：The preparation of DPPH reagents and sample various concentration is carried out first：DPPH reagent ethyl alcohol is molten
Liquid is prepared, its final concentration is made to reach 0.2mmol/L；It is diluted to various concentration after the ultrapure water dissolution of yellow bur reality polysaccharide,
0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.40mg/mL, 0.80mg/mL, 1.60mg/mL, 3.20mg/mL；Secondly will
Various concentration Huang bur reality polysaccharide is added into 2mL DPPH reagents, reacts 60min at ambient temperature；Finally, reaction solution in
Its light absorption value is detected at 517nm.
Calculation formula：DPPH clearance rates (%)=(1-Asample/Acontrol) × 100%
Wherein AcontrolFor the light absorption value of DPPH solution；AsampleFor the suction measured after example reaction is added in DPPH solution
Light value.
From the figure 3, it may be seen that yellow bur reality polysaccharide has a stronger ability for removing DPPH, clearance rate with polysaccharide it is dense
The increase of degree and increase, and the yellow bur reality polysaccharide of 3.20mg/mL is up to 92.12% to the clearance rate of DPPH.Therefore, yellow bur
Real polysaccharide has the stronger ability for removing DPPH.
(5) measurement of reducing power：First, by 0.2M, the phosphate buffer of pH=6.6
The 1%KOH solution of 1.0mL mixes, and various concentration is diluted to after the ultrapure water dissolution of yellow bur reality polysaccharide,
0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.40mg/mL, 0.80mg/mL, 1.60mg/mL, 3.20mg/mL；Above-mentioned
1.0mL samples are added in mixed solution, make its water-bath 20min at 50 DEG C；Then it is added in above-mentioned reaction solution
10mL10% trichloroacetic acids, after mixing, 5000r/min centrifuge 15min；Supernatant 5mL is taken to be dissolved in 0.1% ferric trichloride
In, its light absorption value is measured at 700nm.
Calculation formula：Reducing power (%)=(1-Asample/Acontrol) × 100%
Wherein AcontrolTo be not added with the light absorption value of example reaction liquid；AsampleFor the light absorption value of example reaction liquid is added.
As shown in Figure 4, yellow bur reality polysaccharide has the stronger ability for removing iron ion, and clearance rate is with polysaccharide
The increase of concentration and increase, and the yellow bur reality polysaccharide of 3.20mg/mL is up to 95.04% to the clearance rate of iron, close to VC to iron
The Scavenging activity 95.92% of ion.Therefore, yellow bur reality polysaccharide has the stronger ability for removing reducing power.
(6) ultra-oxygen anion free radical Scavenging activity and hydroxy radical are all made of Nanjing and build up kit detection.Yellow thorn
It is diluted to various concentration after the ultrapure water dissolution of fruit polysaccharide, 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.40mg/mL,
0.80mg/mL, 1.60mg/mL, 3.20mg/mL；As a result see Fig. 5 and Fig. 6
As shown in Figure 5, yellow bur reality polysaccharide has the stronger ability for removing ultra-oxygen anion free radical, clearance rate equal
Increase in the increase with polysaccharide concentration, and the yellow bur reality polysaccharide of 3.20mg/mL is to ultra-oxygen anion free radical clearance rate
Up to 89.36%.Therefore, yellow bur reality polysaccharide has the stronger ability for removing ultra-oxygen anion free radical.
It will be appreciated from fig. 6 that yellow bur reality polysaccharide has the ability of stronger scavenging hydroxyl, clearance rate is with more
The increase of sugared concentration and increase, and the yellow bur reality polysaccharide of 3.20mg/mL is up to 95.890% to Scavenging action to hydroxyl free radical.Cause
This, yellow bur reality polysaccharide has the ability of stronger scavenging hydroxyl.
The yellow bur reality polysaccharide anticancer experiment in vitro of embodiment 6
(1) experimental drug：Yellow bur reality polysaccharide prepares gained, as white powder solid for the present invention.The concentration of setting
For 521 μ g/mL, 256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL；
(2) reagent and instrument：DMEM culture mediums, dimethyl sulfoxide (DMSO) DMSO, penicillin, streptomysin (Sigma Co., USA),
Calf serum (FCS) (U.S. Hyclone, South Logan, UT), 3- (4,5- dimethyl -2- thiazolyls) -2,5- diphenyl -
2H- tetrazoles base (MTT) (U.S. Amresco0793)；CO2 constant incubators；Bio-Rad680 type microplate reader, U.S. Bole are public
Department；Five kinds of cell human gastric cancer cells BGC-823s of exponential phase, breast cancer cell MCF-7 and MDA-MB-231, liver cancer cells
HepG2 and SMMC-7721 (Shanghai life science institute cell resource center).
(3) experimentation：Take the cell of five kinds of exponential phases by 1*105Cell density is inoculated in 96 orifice plates, culture
Overnight, serum free medium is replaced；The yellow bur reality polysaccharide for preparing gained is diluted to various concentration (521 μ g/mL, 256 μ g/
ML, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL)；It is added in the cell being incubated overnight, while setting up blank
Control group；It shakes uniformly mixed, is placed in the CO2 constant incubators of 37 DEG C of 5% concentration and cultivates, after 48 hours, MTT is added per hole
(5mg/mL) 20 μ L, are placed in the CO of 37 DEG C of 5% concentration2Continue culture 4 hours in constant incubator, abandons supernatant (culture solution), often
The DMSO of 100 μ L is added in hole, and sample shakes 15min, measures light absorption value (OD) at 570nm with microplate reader；Experiment is in triplicate.
Cell inhibitory rate (Cell Vability) (%)=(OD control group-OD experimental groups)/(OD control group-OD blank groups) ×
100%.
(4) anticancer experiment in vitro result (Averge ± S)
Inhibiting rate (%) of the yellow bur reality polysaccharide component of table 4 to stomach cancer cell, liver cancer cells, breast cancer cell
As shown in Table 4, after the yellow bur reality polysaccharide various concentration of the present invention acts on three classes tumour cell 48 hours, tumour
The growth of cell is inhibited by different degrees of, also, with the increase of yellow bur reality polysaccharide concentration, inhibitory rate of cell growth
Also it gradually increases, shows preferable dose-effect relationship, especially the inhibiting rate of liver cancer cells is reached in 512 μ g/mL
40% or more.Therefore, yellow bur reality polysaccharide is to five kinds of cell human gastric cancer cells BGC-823s, breast cancer in exponential phase
Cell MCF-7 and MDA-MB-231, hepatocellular carcinoma H22 and SMMC-7721 have preferable antitumor activity.
Claims (10)
1. a kind of Huang bur reality polysaccharide, it is characterised in that：The polysaccharide is the straight fringe barberry Berberis of Berberidaceae plant
The extract of dasystachya Maxim. fruits, polyoses content is 80~95%w/w in extract；The polysaccharide is carried by water
Alcohol deposition method is prepared, and concrete operations are as follows：After yellow bur reality degreasing, removing monosaccharide, extracting in water, Aqueous extracts decoloration removes egg
In vain, dialysis is to be precipitated complete except after small molecule, addition ethyl alcohol to ethyl alcohol final concentration reaches 60~85%v/v, stands, and takes precipitation,
It is dry to get to yellow bur reality polysaccharide.
2. Huang bur reality polysaccharide according to claim 1, it is characterised in that：Polyoses content is 90~95%w/ in extract
w。
3. the preparation method of yellow bur reality polysaccharide described in claim 1, it is characterised in that：The small molecule molecular weight that dialysis removes exists
3500Da or less.
4. preparation method according to claim 3, it is characterised in that：Degreasing concrete operations are as follows：Yellow bur is crushed in fact
Afterwards, it is sieved, uses petroleum ether or ether for Extraction solvent, the residue filter after extraction volatilizes solvent, and it is real to obtain degreasing Huang bur
Residue；
Go the concrete operations of monosaccharide as follows：Gained degreasing Huang bur reality residue is extracted with 70~85%v/v ethyl alcohol, after extraction
Residue volatilize solvent to get monosaccharide Huang bur reality residue has been removed.
5. preparation method according to claim 4, it is characterised in that：Gained degreasing Huang bur reality residue, with 80%v/v
Ethyl alcohol extracts.
6. preparation method according to claim 3, it is characterised in that：Dialysis is except ethyl alcohol being added after small molecule to alcohol content extremely
70~80%v/v.
7. preparation method according to claim 6, it is characterised in that：Dialysis is except ethyl alcohol being added after small molecule to alcohol content extremely
75 ± 2%v/v.
8. a kind of pharmaceutical composition, it is characterised in that：It is by yellow bur reality polysaccharide described in claims 1 or 2 and pharmaceutically may be used
The auxiliary material of receiving or the complementary pharmaceutical preparation prepared at subassembly.
9. pharmaceutical composition according to claim 8, it is characterised in that：The pharmaceutical preparation is through gastrointestinal administration system
Agent.
10. claim 8 or 9 described pharmaceutical compositions are in preparing drug that is hypoglycemic, antitumor or improving disorders of lipid metabolism
Purposes；Wherein, the tumour is selected from gastric cancer, liver cancer or breast cancer.
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