CN107163157B - Schisandra chinensis acidic polysaccharose and its preparation method and application - Google Patents
Schisandra chinensis acidic polysaccharose and its preparation method and application Download PDFInfo
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- CN107163157B CN107163157B CN201710413275.1A CN201710413275A CN107163157B CN 107163157 B CN107163157 B CN 107163157B CN 201710413275 A CN201710413275 A CN 201710413275A CN 107163157 B CN107163157 B CN 107163157B
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- schisandra chinensis
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- acidic polysaccharose
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- water
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- 230000036542 oxidative stress Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 230000004203 pancreatic function Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000007348 radical reaction Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 229960004586 rosiglitazone Drugs 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 229960004499 scopolamine hydrobromide Drugs 0.000 description 1
- WTGQALLALWYDJH-MOUKNHLCSA-N scopolamine hydrobromide (anhydrous) Chemical compound Br.C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 WTGQALLALWYDJH-MOUKNHLCSA-N 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000001624 sedative effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a kind of Schisandra chinensis acidic polysaccharoses and its preparation method and application, belong to natural product active ingredient technical field.The Schisandra chinensis acidic polysaccharose is prepared by the following method to obtain: Fructus Schisandrae Polysaccharide extracts: taking Schisandra chinensis, water is added as Extraction solvent, extracts 1-8hr at 80-100 DEG C, obtains extracting solution, the extracting solution is concentrated into the 1/3-1/7 of additional amount, removal precipitating, obtains supernatant, ethyl alcohol is added in Yu Suoshu supernatant, make the concentration expressed in percentage by volume 70-80% of ethyl alcohol in supernatant, it stands, collects precipitating to get Fructus Schisandrae Polysaccharide；Schisandra chinensis acidic polysaccharose extract: take the Fructus Schisandrae Polysaccharide, using DEAE- cellulose as stationary phase, using concentration for 0-1mol/L sodium-chloride water solution as mobile phase, eluted, isolate and purify to obtain Schisandra chinensis acidic polysaccharose.The Schisandra chinensis acidic polysaccharose being prepared by the above method has the function of anti-oxidative damage, improves learning and memory, hypoglycemic and treatment hepatic injury.
Description
Technical field
The present invention relates to natural product active ingredient technical fields, more particularly to a kind of Schisandra chinensis acidic polysaccharose and its system
Preparation Method and application.
Background technique
The ripening fruits of Schisandra chinensis system magnoliaceae schisandra is that the five of national modernization of Chinese medicine scientific and technological industry base are big
One of kind, especially fructus schisandrae are the superfine product in Schisandra chinensis, have astringing lung-QI enriching yin, antidiarrheal of promoting the production of body fluid, the function such as antitoxic heart-soothing and sedative
Effect, has been put into the list of National Pharmacopeia and health food useful raw materials.
Redox reaction is the important life process of body response internal and external environment.Free radical and radical reaction spread in
All life systems, generate, be quenched, using and damaging action be the opposition system almost carried out simultaneously during vital movement
One process.Comprehensive work of the interior environment of body by various oxidizing substances and antioxidant and its in relation to enzyme system
With making to be maintained at a relative constant redox state between oxidative damage-anti-oxidative defense.At Antioxidative Defense System
, cannot be clean by radicals scavenging when non-equilibrium state, free radical is constantly accumulated in vivo, and excessive free radical will attack
Intracorporal large biological molecule is hit, permanent damage is caused to cell, tissue and organ, makes its structure and function that aging occur,
Cause infection and various degenerative diseases.And brain tissue is high oxygen consumption organ, and more activity is generated in brain cell metabolic process
Oxygen radical, with the accumulation of time, oxidation product increases, and purge mechanism is limited, leads to brain aging, and oxidative stress is caused to damage
Wound, and then cause nerve cell death, eventually lead to brain degenerative change.
In recent years, China gradually steps into aging society, and the disease incidence of neurodegenerative disease caused by body oxidation is also
In trend is risen year by year, although the Therapy study and clinical experience to the disease gradually increase, but there is no at present ideal anti-
Drug is controlled, therefore finding has the drug for improving antioxidant ability of organism significant.
Human mind obstacle is an important medicine and Psychologic Problems.This phenomenon has in mankind's all age group
Occur, including teenager's memory disorders and elderly dementia's disease etc..Especially it is worth noting that China has stepped into aging
National ranks, elderly population subject the pain of hypomnesia mostly, using memory disorders as the Alzheimer disease of important feature
Have become the fourth-largest fatal disease after elderly population relaying heart disease, tumour and apoplexy.Therefore, develop safety,
Effectively there is the Chinese medicine and health food for improving memory function to be of great significance.
Also, in recent years, in the case where social economy rapidly develops, one side people's living standard is continuously improved, dietary structure
It changes, the absorption ratio of high-fat High cholesterol diet is continuously increased；On the other hand, people's rhythm of life constantly becomes faster,
Daily schedule is more and more irregular, is negligent of moving, and the illness rate of China's diabetes is caused to improve year by year.Diabetes are as a kind of slow
Venereal disease, treatment cycle is long, is easy the features such as causing multiple complications and Relapse rate.Diabetes are divided into Type I diabetes, II type sugar
Urine disease.Existing hypoglycemic drug, such as Rosiglitazone, acarbose, insulin, glibenclamide etc. all have in various degree
Side effect, and cannot fundamentally improve pancreas function.
Meanwhile with the continuous improvement of people's living standards, the national capacity for liquor of intake per capita is also increasing.Long-term alcohol is easy
Cause alcoholic liver disease, seriously endangers health.Alcoholic liver injury is the liver diseases due to caused by heavy drinking, hair
Sick rate is high, and consequence is serious, and influence of the alcoholism to nervous system, reproductive system, has become alcoholic liver injury in addition
A kind of disease seriously endangering people's health.China is with the change of living condition in recent years, and excessive drinking is in increasing trend, by alcohol
The disease incidence of caused hepatic lesion is also in rise year by year trend, although the Therapy study and clinical experience to the disease gradually increase
It is more, but there is no ideal protective agents in addition to abstinence from alcohol and supportive treatment at present, therefore finding has protection to make alcoholic liver injury
Drug is significant.
Summary of the invention
Based on this, it is necessary to which, in view of the above-mentioned problems, providing a kind of Schisandra chinensis acidic polysaccharose, which has
Anti-oxidative damage improves learning and memory, hypoglycemic and treatment hepatic injury effect.
A kind of preparation method of Schisandra chinensis acidic polysaccharose, comprising the following steps:
Fructus Schisandrae Polysaccharide extracts: taking Schisandra chinensis, water is added as Extraction solvent, extracts 1-8hr at 80-100 DEG C, must mention
Liquid is taken, the extracting solution is concentrated into the 1/3-1/7 of additional amount, removal precipitating obtains supernatant, second is added in Yu Suoshu supernatant
Alcohol makes the concentration expressed in percentage by volume 70-80% of ethyl alcohol in supernatant, stands, and collects precipitating to get Fructus Schisandrae Polysaccharide；
Schisandra chinensis acidic polysaccharose extracts: taking the Fructus Schisandrae Polysaccharide, is 0- with concentration using DEAE- cellulose as stationary phase
The sodium-chloride water solution of 1mol/L is mobile phase, is eluted, isolates and purifies to obtain Schisandra chinensis acidic polysaccharose.
There is anti-oxidative damage by the Schisandra chinensis acidic polysaccharose that the above method is prepared, improve learning and memory, drop blood
The effect of sugar and treatment hepatic injury.
In one of the embodiments, in the Schisandra chinensis acidic polysaccharose extraction step, isolate and purify to obtain Schisandra chinensis acid
Property polysaccharide method particularly includes: the Fructus Schisandrae Polysaccharide is taken, it is soluble in water, it is splined on Cl- type DEAE- cellulose ion switching layer
Column is analysed, first with distillation water elution, eluent, then the NaCl aqueous solution gradient elution for being 0.05-1mol/L with concentration is discarded, collects
Eluent, recycling design is to get Schisandra chinensis acidic polysaccharose SCP-A.The Schisandra chinensis acidic polysaccharose being in the above way prepared, tool
There is preferable activity.
The preparation method further includes Schisandra chinensis acidic polysaccharose purification procedures in one of the embodiments, and described five
Taste acidic polysaccharose purification procedures are as follows: take the Schisandra chinensis acidic polysaccharose, using Cl- type DEAE- cellulose as stationary phase, first
With distillation water elution, discard eluent, then respectively with 0.08-0.12mol/L, 0.18-0.22mol/L, 0.28-0.32mol/L,
The sodium-chloride water solution of 0.48-0.52mol/L is that mobile phase is eluted, and Schisandra chinensis acidic polysaccharose SCP-A- is prepared respectively
1, Schisandra chinensis acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4.Using above-mentioned
Method is finely divided purifying to Schisandra chinensis acidic polysaccharose, for different applications, improves the validity of the Fructus Schisandrae Polysaccharide.
In one of the embodiments, in the Schisandra chinensis acidic polysaccharose purification procedures, take Schisandra chinensis described in 5mg sour
Property sugar, be dissolved in 1mL distilled water, upper column volume balance for 20mL Cl- type DEAE- cellulose ion-exchange chromatography column,
Water elution first is distilled with 40mL, discards eluent, then is used respectively with 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.5mol/L
Each 20mL of sodium-chloride water solution NaCl aqueous solution carries out linear gradient elution, and flow velocity 1mL/min collects eluent respectively, recycles
Schisandra chinensis acidic polysaccharose SCP-A-1, Schisandra chinensis acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose is prepared in solvent respectively
SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4.
In one embodiment, the Fructus Schisandrae Polysaccharide takes Schisandra chinensis in extracting, and 5-8ml water is added according to every gram of Schisandra chinensis
Amount be added Extraction solvent water, extract 4-6hr at 90-100 DEG C, obtain extracting solution, the extracting solution is concentrated at 70-90 DEG C
To the 1/4-1/6 of additional amount, removal precipitating obtains supernatant, and it is 90-95%'s that concentration expressed in percentage by volume is added in Yu Suoshu supernatant
Ethanol solution makes the concentration expressed in percentage by volume 70-80% of ethyl alcohol in supernatant, stands, and collects precipitating, and the precipitating successively uses body
The ethanol solution that product percentage concentration is 95%, 100% washs, dry to get Fructus Schisandrae Polysaccharide；
The Schisandra chinensis acidic polysaccharose that the bright preparation method for also disclosing above-mentioned Schisandra chinensis acidic polysaccharose of this law is prepared.
The Schisandra chinensis acidic polysaccharose has the function of anti-oxidative damage, improves learning and memory, hypoglycemic and treatment hepatic injury.
This law is bright also disclose above-mentioned Schisandra chinensis acidic polysaccharose preparation for it is anti-oxidant, improve memory, hypoglycemic
And/or anti-liver injury drug or the application in health care product.
Schisandra chinensis acidic polysaccharose SCP-A-1, the five tastes are prepared with above-mentioned preparation method in one of the embodiments,
Sub- acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4；
Wherein, when the Schisandra chinensis acidic polysaccharose is when preparation is for oxidation resistant drug or health care product, the Schisandra chinensis
Acidic polysaccharose is acidic polysaccharose SCP-A-1；
When the Schisandra chinensis acidic polysaccharose is when preparing the drug or health care product for improving memory, the Schisandra chinensis is sour
Property polysaccharide be acidic polysaccharose SCP-A-2；
When the Schisandra chinensis acidic polysaccharose is when preparation is for hypoglycemic drug or health care product, the Schisandra chinensis is acid more
Sugar is acidic polysaccharose SCP-A-3；
When the Schisandra chinensis acidic polysaccharose is when preparation is used for the drug or health care product of anti-liver injury, the Schisandra chinensis is acid
Polysaccharide is acidic polysaccharose SCP-A-1.
For unused application, the Schisandra chinensis acidic polysaccharose of different subdivision ingredients, more specific aim are selected, is had preferable
Effect.
In one of the embodiments, the dosage form of the drug or health care product be tablet, granule, hard capsule, soft capsule,
Oral solution, pulvis, mixture, pill or dripping pill.
Customary adjuvant can be used in above-mentioned dosage form and preparation process is prepared.
This law is bright to also disclose a kind of pharmaceutical composition, including the above-mentioned Schisandra chinensis acidic polysaccharose as active constituent,
And pharmaceutically acceptable carrier or auxiliary material.
Compared with prior art, the invention has the following advantages:
The preparation method of Schisandra chinensis acidic polysaccharose of the invention has the characteristics that step simplification, simple process, can use workmanship
Industry produces and uses.And the Schisandra chinensis acidic polysaccharose being prepared using the preparation method, there is anti-oxidative damage, improve study note
Recall, hypoglycemic and treatment hepatic injury effect.
Also, the preparation method has also carried out further refinement, isolates and purifies Schisandra chinensis acidic polysaccharose to obtain the five tastes
Sub- acidic polysaccharose SCP-A-1, Schisandra chinensis acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose SCP-A-3 and Schisandra chinensis acidic polysaccharose
SCP-A-4 more targetedly, has optimal effect for unused application.
Detailed description of the invention
Fig. 1 is Schisandra chinensis acidic polysaccharoses different in embodiment 2 to linoleic acid autoxidation system suppression result schematic diagram；
Fig. 2 is the reducing power schematic diagram of different Schisandra chinensis acidic polysaccharoses in embodiment 2.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
Embodiment 1
Schisandra chinensis acidic polysaccharose is prepared by the following method to obtain:
One, Fructus Schisandrae Polysaccharide extracts.
Schisandra chinensis 1.5kg is taken, distilled water 10L (i.e. solid-liquid ratio is 1:6.7) is added, soaked overnight boils (about 100 DEG C)
5h obtains extracting solution, and extracting solution is concentrated into 2L at 80 DEG C, is centrifuged (4500rpm, 15min), discards precipitating, obtain supernatant.Upwards
The ethanol solution that concentration expressed in percentage by volume is 95% is added in clear liquid, makes ethyl alcohol final concentration of 75% in supernatant, staticly settles overnight,
It is centrifuged (4500rpm, 15min), collects precipitating.Precipitating is successively washed with 95% ethyl alcohol and dehydrated alcohol, and conventional drying obtains powder
Shape Fructus Schisandrae Polysaccharide.
Two, Schisandra chinensis acidic polysaccharose.
1, it isolates and purifies.
The Fructus Schisandrae Polysaccharide is taken, with DEAE- cellulose (being purchased from: Whatman company model DE-52) for stationary phase, with
The sodium-chloride water solution that concentration is 0-1mol/L is mobile phase, is eluted, and the specific method is as follows:
5mg Fructus Schisandrae Polysaccharide is taken, is dissolved in 1mL distilled water, the upper DEAE- cellulose ion-exchange chromatography column balanced
(20mL, Cl- type), first with 40mL distillation water elution to get Schisandra chinensis neutral sugar SCP-N.The sodium chloride water of 0.5mol/L again
Solution 80mL is eluted, flow velocity 1mL/min, collects eluent, Schisandra chinensis acidic polysaccharose SCP- is prepared in recycling design
A.With sugared content in phend-sulphuric acid detection eluent, xenol method measures glucuronic acid content in eluent.
The results show that under distillation water energy elutes most of Schisandra chinensis neutral polysaccharide SCP-N from DEAE- cellulose column
Come, 0.5M NaCl can elute most of Schisandra chinensis acidic polysaccharose SCP-A.
So can largely prepare Schisandra chinensis neutral sugar SCP-N and acid sugar SCP-A by ion-exchange chromatography.
2, constituent analysis.
2.1, phend-sulphuric acid measures sugared content.
Sugared content measurement is carried out using phend-sulphuric acid.
Standard curve preparation: with pipette measure 0.1g/L standard Glc (glucose) solution 0mL, 0.2mL, 0.4mL,
0.6mL, 0.8mL, 1.0mL turn in teat glass, add distilled water to mend to 1.0mL, each concentration in triplicate, respectively to every
6% phenol reagent 0.5mL is added in test tube, concentrated sulfuric acid 2.5mL, rapid oscillation is uniform, is cooled to room temperature, is at 490nm in λ
Measure absorbance A.Using trap A as ordinate, sugared content C is abscissa, obtains standard curve A=k C.
Sugared content measures in sample: taking the sample solution 1mL of concentration 0.1g/L or so, phenol reagent 0.5mL, dense sulphur is added
Sour 2.5mL measures trap by the operation of standard curve preparation method.Content is calculated according to standard curve and sample light absorption value.
2.2, xenol method measurement glucuronic acid content measurement.
Between xenol reagent: weigh xenol between 30mg, be dissolved in 0.5% sodium hydrate aqueous solution, and be settled to
10mL, 4 DEG C are kept in dark place.
Saturation potassium hydroxide: weighing 5g potassium hydroxide, and 2mL distilled water is added, and dissolution is sufficiently stirred, and upper clear supernate is full
And potassium hydroxide.
Sulfamic acid reagent: weighing 3.9g sulfamic acid, adds distilled water 5mL, saturation potassium hydroxide aqueous solution is added dropwise, sufficiently
Oscillation is completely dissolved to sulfamic acid, is cooled to room temperature, and is further continued for being added dropwise saturation potassium hydroxide aqueous solution to pH 2.5, with distillation
Volume is supplemented to 10mL by water, and room temperature preservation is spare.
0.1g/L D-GalA standard solution: weighing 10mg D-GalA (D- galacturonic), be dissolved in distilled water, and fixed
Hold to 100mL.
Standard curve preparation: pipettor measure respectively 0 μ L of 0.1g/L D-GalA standard solution, 50 μ L, 100 μ L, 200 μ L,
300 μ L, 400 μ L turn in teat glass, add distilled water to mend to 400 μ L, each concentration repeats three samples.Into every test tube
40 μ L of sulfamic acid reagent is added, shakes up, then concentrated sulfuric acid 2.5mL, shaken well is added to each pipe, boiling water bath boils 20min.It is cold
But to after room temperature, 40 μ L of xenol reagent between being added into each pipe shakes up, is placed at room temperature for 15min.It measures and inhales at λ 525nm
Luminosity A.Using trap A as ordinate, D- galacturonic content (μ g) C is abscissa, obtains standard curve A=k C.
Glucuronic acid content measures in sample: sample solution is taken, by the operation of standard curve preparation method, measures trap,
Glucuronic acid content therein is calculated according to standard curve and sample concentration.
2.3, Coomassie Brilliant Blue measures protein.
Coomassie brilliant blue reagent: weighing Coomassie brilliant blue 10mg, is dissolved in 95% ethyl alcohol of 5mL, is added 10mL's
85% phosphoric acid is diluted to 100mL with distilled water, and filter paper filtering is spare.Contain 0.01% Coomassie brilliant G-250 in final reagent
(w/v), 4.7% ethyl alcohol (w/v).
Protein standard solution: stock solution 5mg/mL bovine serum albumin(BSA), used time are diluted with water to 50 μ g/mL.
Standard curve preparation: pipette measure respectively 50 μ g/mL protein standard solution 0mL, 0.2mL, 0.4mL,
0.6mL, 0.8mL, 1.0mL are transferred in teat glass, and distilled water is added to mend to 1.0mL, three repetitions of each concentration, respectively to every
Coomassie brilliant blue reagent 4mL is added in test tube, rapid oscillation is uniform, and absorbance A is measured at λ 595nm after standing 5min.To inhale
Receipts degree A is ordinate, and bovine serum albumin content C (μ g) is abscissa, obtains standard curve A=k C.
The measurement of protein content in sample: the sample solution 1mL of concentration 0.1g/L or so is taken, by standard curve preparation side
The operation of method measures trap, calculates protein content therein according to standard curve and sample concentration.
2.4, monosaccharide composition analysis.
Polysaccharide sample 2mg is weighed, the absolute methanol solution 1mL containing 1M hydrochloric acid is added, fills N2Tube sealing, 80 DEG C of hydrolysis 16 are small
When, after air pump drying, 2M trifluoroacetic acid 1mL is added, 120 DEG C hydrolyze 1 hour, and a small amount of ethyl alcohol, 60 DEG C of drying with water baths, weight is added
It is 3~5 times multiple, trifluoroacetic acid is evaporated off completely.
PMP reagent (1-phenyl-3-methyl-5-pyrazolones ketone) is added in the drying sample obtained after to complete sour water solution
The NaOH solution 0.5mL of 0.5mL and 0.3M takes 0.1mL therein in small centrifuge tube after completely dissolution to sample, 70 DEG C of water-baths
30min.After 10000rpm is centrifuged 5min, 0.3M hydrochloric acid solution 0.05mL and distilled water 0.05mL is added, mixes well.It is added
1mL chloroform extracts remaining PMP reagent after mixing, suck chloroform layer, retains water layer.With 0.22 μm of membrane filtration
Afterwards, it after appropriate distilled water dilution is added, is detected to HPLC.
Using Shimadzu HPLC system (LC-10ATvp pump and SPD-10AVD UV detector),
DIKMAInertsil ODS-3 chromatographic column (4.6 × 150mm), mobile phase are PBS (0.1M, p H 7.0- acetonitrile 82:18 (v/
V), flow velocity 1.0mL/min, sample volume are 20 μ L, Detection wavelength 245nm.
3, composition analysis result.
The Schisandra chinensis acidic polysaccharose being prepared is analyzed using above-mentioned analysis method, by analysis of physical and chemical property and
Monosaccharide composition analysis, as a result as shown in the table.
1. monosaccharide composition analysis of table
* yield refers to ratio of the quality of gained SCP-N or SCP-A relative to chromatography loading quality.
Schisandra chinensis neutral polysaccharide SCP-N and five is obtained as can be seen that separating from Fructus Schisandrae Polysaccharide in from the above
Taste acidic polysaccharose SCP-A, wherein the yield of Schisandra chinensis neutral polysaccharide SCP-N is 47.00%, and the yield of acid sugar SCP-A is
27.00%；And neutral sugar is mainly made of Glc, acid sugar is mainly made of GalA and Clc.
Three, Schisandra chinensis acidic polysaccharose isolates and purifies.
The Schisandra chinensis acidic polysaccharose SCP-A is taken, using DEAE- cellulose as stationary phase, be respectively 0.1mol/L with concentration,
The sodium-chloride water solution of 0.2mol/L, 0.3mol/L and 0.5mol/L are mobile phase, are eluted, the specific method is as follows:
5mg Fructus Schisandrae Polysaccharide is taken, is dissolved in 1mL distilled water, the upper DEAE- cellulose ion-exchange chromatography column balanced
(20mL, Cl- type) distills water elution with 40mL first, then uses 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.5mol/L respectively
Each 20mL of sodium-chloride water solution NaCl aqueous solution carry out gradient elution, flow velocity 1mL/min collects eluent respectively, recycles molten
Schisandra chinensis acidic polysaccharose SCP-A-1, Schisandra chinensis acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose is prepared in agent respectively
SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4, phend-sulphuric acid detect sugared content in eluent, xenol method measurement
Glucuronic acid content in eluent.
The results show that acid sugar SCP-A can be divided with 0.1M NaCl, 0.2M NaCl, 0.3M NaCl and 0.5M NaCl
It is eluted for four fractions.Illustrate can by ion-exchange chromatography, largely prepare Schisandra chinensis acid sugar SCP-A-1,
Tetra- fractions of SCP-A-2, SCP-A-3 and SCP-A-4.Schisandra chinensis acidic polysaccharose SCP-A-1, Schisandra chinensis acid are prepared respectively
Property polysaccharide SCP-A-2, Schisandra chinensis acidic polysaccharose SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4.
2, constituent analysis.
Four kinds of Schisandra chinensis acidic polysaccharoses being prepared are analyzed using above-mentioned analysis method, by physicochemical property point
Analysis and monosaccharide composition analysis, as a result as shown in the table.
The constituent analysis of 2 four kinds of acidic polysaccharoses of table
* yield refers to ratio of the quality of gained heterogeneity relative to chromatography loading quality.
Embodiment 2
Antioxidation in vitro experiment.
1, research of the Fructus Schisandrae Polysaccharide to the rejection ability of lipid peroxidation.
1.1, method.
According to linoleic acid-thiocyanate-ferric, Schisandra chinensis acidic polysaccharose SCP-A-1, SCP-A-2 and SCP-A-3 are resisted
The research of oxidation activity, specific as follows:
Configure the 50ml linoleic acid emulsion (phosphorus of 310 μ l linoleic acid, 350mg polysorbas20 and 0.04mol.L-1.PH=7.0
Phthalate buffer is configured to 50ml).Toward the 200 configured 5mM of μ l different Fructus Schisandrae Polysaccharides (SCP-A-1, SCP-A-2 and
SCP-A-3) and the positive control VE (vitamin E) of comparable sodium, in blank control distilled water, 2.5ml linoleic acid cream is added
The mixing of shape liquid.
Be placed in 37 DEG C of incubators and cultivate 72h.It is sampled every all group of test liquid of the 12h to culture, often
Secondary 200 μ l, is added in the ethyl alcohol of 2.5ml (75%), and the ammonium thiocyanate (30%) of 300 μ l is added later, after mixing well, is added
The solution of ferrous chloride of 300 μ l (2g frerrous chloride is dissolved in the 3.5%HCl of 50ml).Reaction 3min after at 500nm measure
Its absorbance value.
1.2, result.
Experimental result is as shown in Fig. 1 and table 3.
3 Fructus Schisandrae Polysaccharide of table is to linoleic acid autoxidation system suppression result (N=3)
By the absorbance curve of above-mentioned blank control group it is known that the autoxidation of linoleic acid system is very rapid,
3 kinds of Fructus Schisandrae Polysaccharides and vitamin E are added in system can effectively inhibit linoleic autoxidation.WSCPA-1 is to linoleic acid
The inhibition of oxidation system is significantly stronger than vitamin E, and the light absorption value of positive control vitamin E is less than WSCPA-1, illustrates WSCPA-
The oxidizing and depressing rate of 1 pair of linoleic acid system is better than vitamin E.
2, Fructus Schisandrae Polysaccharide reduction activation is tested.
2.1, method.
The survey of the reduction activation of Schisandra chinensis acidic polysaccharose SCP-A-1, SCP-A-2 and SCP-A-3 is carried out using Oyaizu method
It is fixed, configured different Fructus Schisandrae Polysaccharide (SCP-A-1, the SCP-A-2 and SCP-A-3) prepare liquids of step are taken, 0.2mol.L is added-1, pH value 6.6 sodium phosphate buffer 1.0ml and 1% potassium ferricyanide 1.0ml, in 50 DEG C of water-baths react 20min after rapidly
It is cooling；The trichloroacetic acid 1.0ml, 3 000r.min-1 for being added 10% are centrifuged 10min；Supernatant 2ml is taken, distilled water 2ml is added,
0.1% liquor ferri trichloridi 0.5ml；Its light absorption value is measured in 700nm after 10min.
2.2, result.
Experimental result is as shown in Fig. 2 and table 4.
The reducing power (N=3) of 4 Schisandra chinensis acidic polysaccharose of table
As can be seen that 3 kinds of polysaccharide all have reducing power in from the above, absorbance has with the increase of polysaccharide concentration
It is significant to increase, and the reducing power of WSCPA-1 is better than vitamin E, shows that WSCPA-1 has optimal oxidation resistance.
Embodiment 3
Improve learning and memory experiment.
1, step-through test.
1.1, experimental animal.
Healthy ICR male mice, 20 ± 2g of weight are provided by Jilin University's Animal Experimental Study center, and credit number is
SCXK- (Ji) 2016-0005, divides after cage that be put in quiet, temperature and humidity be suitable for, in draughty environment that free water is rationally taken the photograph
Food starts to test after adapting to local environment 7 days.
1.2, experimental method.
ICR male mice is grouped at random, Normal group (distilled water is given in CON, stomach-filling), model group (MOD, stomach-filling
Give distilled water, hyoscine 5mg/kg be injected intraperitoneally), Schisandra chinensis acidic polysaccharose group (SCP-A, 10mg/kg), Schisandra chinensis it is acid
Polysaccharide SCP-A-1 group (SCP-A-1,10mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-2 group (SCP-A-2,10mg/kg), the five tastes
Sub- acidic polysaccharose SCP-A-3 group (SCP-A-3,10mg/kg), every group 15.Dosage is 0.1ml/10g, once a day.It is small
Mouse carries out continuous gavage administration in 30 days by a definite date, and Behaviors survey is carried out after last dose 30min.
Started to carry out keeping away dark test in stomach-filling the 29th day and the 30th day.Start to train within 29th day, 30min is carried out before training
Gastric infusion, after gastric infusion 20min, blank control group mouse intraperitoneal injection of saline, model group and other each group mouse
5mg/kg scopolamine hydrobromide is injected intraperitoneally.After 10min is injected intraperitoneally, mouse is put into and keeps away dark instrument septum reset and is put backwards to hole
Enter bright room, while starting timer.It is latent with the time that mouse enters timer record when hole reaches darkroom by mistake
Fu Qi.This experiment is recorded in the errors number of mouse in 5min to mouse training 5min.The was carried out in gastric infusion the 30th day
Second trial, medication was the same as the 29th day.The errors number for recording the incubation period of every mouse and occurring in 5min.
1.3, experimental result
The experimental results are shown inthe following table.
5. each group mouse of table keep away dark test result (N=15)
Note: compared with blank group, P < 0.01 * P < 0.05, * *；Compared with model group, #P < 0.05, ##P < 0.01；
In from the above as can be seen that compared with blank group, model group mouse incubation period is obviously shortened (P < 0.01), wrong
Accidentally number obviously increases (P < 0.05), shows modeling success.Compared with model group, it is obvious that SCP-A-2 group mouse keeps away dark incubation period
Extend (P < 0.01), the mouse wrong times of SCP-A-2 group significantly reduce (P < 0.05), prompt SCP-A-2 group to have optimal
Improve mouse memory effect.
2, water maze laboratory.
2.1, experimental animal.
Healthy ICR male mice, 20 ± 2g of weight are provided by Jilin University's Animal Experimental Study center, and credit number is
SCXK- (Ji) 2016-0005, divides after cage that be put in quiet, temperature and humidity be suitable for, in draughty environment that free water is rationally taken the photograph
Food starts to test after adapting to local environment 7 days.
2.2, experimental method.
ICR male mice is randomly divided into 5 groups, (MOD is filled for Normal group (distilled water is given in CON, stomach-filling), model group
Stomach gives distilled water, and hyoscine 5mg/kg is injected intraperitoneally), Schisandra chinensis acidic polysaccharose group (SCP-A, 10mg/kg), Schisandra chinensis acid
Property polysaccharide SCP-A-1 group (SCP-A-1,10mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-2 group (SCP-A-2,10mg/kg), five
Taste acidic polysaccharose SCP-A-3 group (SCP-A-3,10mg/kg), every group 15.Dosage is 0.1ml/10g, once a day.
Mouse carries out continuous gavage administration in 30 days by a definite date, and Behaviors survey is carried out after last dose 30min.
In the 25th day progress water maze test of gastric infusion, 30min carries out gastric infusion, gastric infusion before training
After 20min, blank control group mouse intraperitoneal injection of saline to model group and other each group mouse carries out that eastern Liang is injected intraperitoneally
Henbane alkali 5mg/kg.After 10min is injected intraperitoneally, mouse is placed in Morris water maze video tracking test macro WMT-100, if
The training time for setting mouse is set as 120s, and the mouse that terminal is not up in 120s is recorded by 120s.Before testing for the first time
Mouse is placed near platform, climbs up it automatically 3 times.Mouse is placed near platform before training every time later, keeps it autonomous
It climbs up primary to platform.It is trained 1 time every for 24 hours, continues to be administered during training, medication was the same as the 25th day.Acquisition administration the 29th
It experimental data.It is administered the 30th day and platform is removed statistics mouse spatial search capability, medication was the same as the 25th day.And it records
The number experimental result of mouse spanning platform time.
2.3, experimental result.
The experimental results are shown inthe following table.
6. each group water maze test in mice result of table (N=15)
Note: compared with blank group, P < 0.01 * *；Compared with model group, #P < 0.05, ##P < 0.01；
As can be seen that compared to the blank group, model group mouse finds plateau time and extends (P < 0.01) in from the above,
(P < 0.01) is reduced by platform number, prompts modeling success.Compared with model group, SCP-A-2 group mouse finds plateau time
It is obviously shortened (P < 0.05), is obviously increased (P < 0.01) by platform number, prompting SCP-A-2 group to have improves mouse memory work
With.
Embodiment 4
Hypoglycemic experiment.
1, experimental animal.
With embodiment 3.
2, method.
Mouse is taken, takes 10 to be only used as blank control group at random, the chain urea of 40mg/kg is injected intraperitoneally in remaining mouse empty stomach 12h
It helps rhzomorph (STZ) solution (citrate buffer of 0.1mol), continuously injects 5d, fasting 12h is needed before per injection.1 week
Afterwards, the intraocular corner of the eyes takes blood, the fasting blood sugar after measuring fasting 12h, take mouse of the blood glucose value greater than 8mmol/L be both modeling at
Function.
The successful mouse of modeling is grouped at random, every group 10, one group is used as model control group, remaining group is respectively the five tastes
Sub- acidic polysaccharose group (SCP-A, 40mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-1 group (SCP-A-1,40mg/kg), Schisandra chinensis acid
Property polysaccharide SCP-A-2 group (SCP-A-2,40mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-3 group (SCP-A-3,40mg/kg), it is empty
Distilled water is given in white control group and model control group stomach-filling, the daily gastric infusion of remaining administration group 1 time, continuous 15d, last dose
Afterwards by each group mouse empty stomach 12h, eyeball takes blood, separates serum, determination of glucose oxidase blood-sugar content.
2, result.
The experimental results are shown inthe following table.
7. each group mouse fasting blood-glucose content of table (N=6)
Note: compared with blank group, P < 0.001 * * *；Compared with model group, #P < 0.05, ###P < 0.001；
As can be seen that compared to the blank group in from the above, model group mouse blood sugar is obviously increased, prompt modeling at
Function.
Compared with model group, SCP-A-2 and SCP-A-3 group mouse blood sugar level be remarkably decreased (P < 0.05 or P <
0.001), and SCP-A-3 is capable of the extremely significant hyperglycemia for reducing mouse, and normal value can be reached；Illustrate that SCP-A-3 has drop
The effect of sugar.
Embodiment 5
Anti-liver injury experiment.
1, experimental animal.
With embodiment 3.
2, method.
Mouse is taken, random to be grouped, every group 10, respectively Normal group (Control), model group (Model), the five tastes
Sub- acidic polysaccharose group (SCP-A, 20mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-1 group (SCP-A-1,20mg/kg), Schisandra chinensis acid
Property polysaccharide SCP-A-2 group (SCP-A-2,20mg/kg), Schisandra chinensis acidic polysaccharose SCP-A-3 group (SCP-A-3,20mg/kg).Often
Advise forage feed, free water.Administration group gives Schisandra chinensis acidic polysaccharose and carries out stomach-filling, and Normal group and model group are given together
Volume distilled water stomach-filling, once a day, continuous 15 days.After last dose 1 hour, model group and administration group mouse give 50% second
Alcohol 12mL/kg stomach-filling, Normal group mouse give same volume distilled water stomach-filling, are deprived of food but not water after 12h.It puts to death, detection is each
Item index.
The modeling method of the model group are as follows: give mouse 50% ethyl alcohol, according to 12mL/kg dosage stomach-filling, using enzyme process
AST, ALT in the kit measurement serum of Bioengineering Research Institute are built up with Nanjing.
2, result.
The experimental results are shown inthe following table.
ALT and AST content in 8. each group mice serum of table (N=7)
Note: compared with blank group, P < 0.01 * *；Compared with model group, #P < 0.05, ##P < 0.01；
As can be seen that compared to the blank group, model group mice serum AST and ALT level significantly increases in from the above
High (P < 0.01) illustrates that disposable 50% alcohol of stomach-filling of mouse can cause acetsminophen, prompts modeling success.
Compared with model group, ALT, AST level are remarkably decreased (P < 0.05 in SCP-A-1 and SCP-A-2 group mice serum
Or P < 0.01), prompt SCP-A-1 and SCP-A-2 to prompt it that there is certain protective effect to alcohol-induced mouse liver injury.
And SCP-A and SCP-A-3 all has no significant effect ALT and AST level in mice serum；Illustrate the liver protection effect of SCP-A-1 most
It is good.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (2)
1. a kind of Schisandra chinensis acidic polysaccharose is preparing the application in the drug for improving memory；The Schisandra chinensis acidic polysaccharose
For acidic polysaccharose SCP-A-2；
The acidic polysaccharose SCP-A-2 is prepared by the following method to obtain:
Fructus Schisandrae Polysaccharide extracts: taking Schisandra chinensis, Extraction solvent water is added according to the amount that 5-8ml water is added in every gram of Schisandra chinensis, in 90-
4-6hr is extracted at 100 DEG C, obtains extracting solution, the extracting solution is concentrated into the 1/4-1/6 of additional amount at 70-90 DEG C, removal is heavy
It forms sediment, obtains supernatant, the ethanol solution that concentration expressed in percentage by volume is 90-95% is added in Yu Suoshu supernatant, makes ethyl alcohol in supernatant
Concentration expressed in percentage by volume be 70-80%, stand, collect precipitating, it is described precipitating successively with concentration expressed in percentage by volume be 95%, 100%
Ethanol solution washing, it is dry to get Fructus Schisandrae Polysaccharide；
Schisandra chinensis acidic polysaccharose extracts: the Fructus Schisandrae Polysaccharide is taken, it is soluble in water, and it is splined on the friendship of Cl- type DEAE- cellulose ion
Chromatographic column is changed, first with distillation water elution, discards eluent, then the NaCl aqueous solution for being 0.5mol/L with concentration elutes, collection is washed
De- liquid, recycling design is to get Schisandra chinensis acidic polysaccharose SCP-A；
Schisandra chinensis acidic polysaccharose isolates and purifies: Schisandra chinensis acid sugar SCP-A described in 5mg taken, is dissolved in 1mL distilled water, upper balance
Good column volume is the Cl- type DEAE- cellulose ion-exchange chromatography column of 20mL, first distills water elution with 40mL, discards elution
Liquid, then each 20mL of sodium-chloride water solution NaCl aqueous solution of 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.5mol/L are used respectively
Gradient elution is carried out, flow velocity 1mL/min collects eluent respectively, Schisandra chinensis acidic polysaccharose is prepared in recycling design respectively
SCP-A-1, Schisandra chinensis acidic polysaccharose SCP-A-2, Schisandra chinensis acidic polysaccharose SCP-A-3 and Schisandra chinensis acidic polysaccharose SCP-A-4.
2. application according to claim 1, it is characterised in that: the dosage form of the drug be tablet, granule, hard capsule,
Soft capsule, oral solution, pulvis, mixture, pill or dripping pill.
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