CN109206531A - Polysaccharide P2 and its isolation and purification method and preparing the application in blood lipid-lowering medicine - Google Patents
Polysaccharide P2 and its isolation and purification method and preparing the application in blood lipid-lowering medicine Download PDFInfo
- Publication number
- CN109206531A CN109206531A CN201810987144.9A CN201810987144A CN109206531A CN 109206531 A CN109206531 A CN 109206531A CN 201810987144 A CN201810987144 A CN 201810987144A CN 109206531 A CN109206531 A CN 109206531A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- hill gooseberry
- fruit
- solution
- isolation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- 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
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Epidemiology (AREA)
- Sustainable Development (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of hill gooseberry's polysaccharide P2 and its isolation and purification method and preparing the application in blood lipid-lowering medicine, the polysaccharide includes the monosaccharide of following molar percentage: ribose 8.25%, rhamnose 2.10%, arabinose 53.75%, xylose 5.16%, mannose 8.22%, glucose 12.01%, galactolipin 10.51%.It is of the invention the experimental results showed that; hill gooseberry's polysaccharide P2 has certain external conjucated bile acids salt ability; using Cholestyramine as positive control; it is 100% calculating by Percentage bound of the Cholestyramine to each cholate, converts out hill gooseberry's polysaccharide P2 and 29.87%, 47.49%, 53.69% is respectively as follows: to the opposite Percentage bound of natrium taurocholicum, NaGC, sodium taurocholate.
Description
Technical field
The invention belongs to natural products (drug) fields, and in particular to a kind of hill gooseberry's polysaccharide P2 and its isolation and purification method
With preparing the application in blood lipid-lowering medicine.
Background technique
(Guangdong) is harvested on hill gooseberry (Rhodomyrtus tomentosa) also known as hilllock, beans harvest dry (Guangxi), stone all spills (" this
Grass is picked up any lost article from the road "), mangosteen (" ridge table record different "), the viscous son (" Soviet Union Shen Liangfang ") etc. that falls, be distributed widely in south subtropics region；Normal
The green broad-leaf forest ecosystem is sociales, and coverage 30~60% is in prodophytium；It is distributed mainly on the south of the Five Ridges in China, is me
The main understory species in state southern area, resource is very rich, is born in hilly upland, wilderness, roadside, and wild peach gold mostly
The distribution area of ma is extensive, but lower to its utilization rate at present, and processing and utilization level is relatively low, and Myrtus is in one
Kind not yet carries out the potential new resource for food of scale development and utilization.
The fruit of hill gooseberry is berry, and pericarp is in atropurpureus when mature, and pulp is then in aubergine, meat succulence, and nutrition is rich
It is rich.Some researches show that when, wild hill gooseberry's fruit maturation, total sugar content, which accounts for 8.06%, reduced sugar and accounts for 7.72%, pectin, to be accounted for
1.01%, total acid, which accounts for 0.38%, protein and accounts for 1.30%, fat and account for 0.32%, starch and account for 3.08%, crude fibre, accounts for 34.97%,
Vitamin C content is 5.48mg/kg.
Hill gooseberry's fruit contains a variety of active ingredients such as polysaccharide, flavonoids, saponins, Polyphenols, Anthraquinones, has anti-
The bioactivity such as oxidation, anti-aging, anti-inflammatory, antibacterial, liver protection.As a kind of wild plant resource, hill gooseberry is in food processing, guarantor
Strong product exploitation and medical medicine research etc. have wide development prospect.
Summary of the invention
The primary purpose of the present invention is that providing a kind of hill gooseberry's polysaccharide P2, the purity of polysaccharide is high, has bioactivity.
Another object of the present invention is to provide the isolation and purification method of above-mentioned polysaccharide P2, this method is to utilize water extract-alcohol precipitation
Method carries out Polyose extraction to hill gooseberry's fruit, and is isolated and purified by ion-exchange chromatography method to polysaccharide.
A further object of the present invention is to provide above-mentioned polysaccharide P2 to prepare the application in blood lipid-lowering medicine.
The purpose of the invention is achieved by the following technical solution:
A kind of hill gooseberry's polysaccharide P2, the monosaccharide comprising following molar percentage: ribose 8.25%, rhamnose 2.10%, Ah
Draw uncle's sugar 53.75%, xylose 5.16%, mannose 8.22%, glucose 12.01%, galactolipin 10.51%；The composition of monosaccharide
It is to be calculated by GC-MS area normalization method, is that all peak areas that goes out are regarded as 100%, calculate master in addition to solvent peak
Peak area accounts for the percentage of the gross area；
The number-average molecular weight (Mn) of above-mentioned hill gooseberry's polysaccharide P2 is 2.57 × 104Da, weight average molecular weight (Mw) be 6.75 ×
104Da。
The isolation and purification method of above-mentioned hill gooseberry's polysaccharide P2, comprising the following steps:
(1) it extracts hill gooseberry's fruit polysaccharide: hill gooseberry's fruit is dried and crushed, its dry powder and distilled water is taken to fill in reflux
Extraction is boiled in setting for several times, combined extract is simultaneously concentrated under reduced pressure, 95% (V/V) ethyl alcohol of several times volume is added in concentrate,
Being stirred continuously makes polysaccharide homogeneous precipitation, and 12h or more is then stood at 4 DEG C, precipitating is collected by centrifugation, and hill gooseberry's fruit is obtained after drying
Thick many candies；
Extraction, preferred 4h of each extraction time are boiled described in step (1)；
The preferred 5000r/min of centrifugation described in step (1) is centrifuged 15min；
(2)H2O2Method decoloration: taking hill gooseberry's fruit Thick many candies that distilled water is added to dissolve, and adjusts pH value to 8.0, then drips on one side
Add 30%H2O2Solution stirs on one side, until solution colour shoals, then keeps the temperature 2h under 50 DEG C of water-baths；
The preferred 0.2g/mL of concentration that Thick many candies described in step (2) add distilled water to dissolve；
(3) Sevag method takes off albumen: the Sevag reagent of 1/5 volume of solution being added in the polysaccharide solution after decoloration, shakes
Culture 20min is swung, is centrifuged after standing 10min, discards what lower layer's organic phase and intermediate layer protein matter were generated with chloroform and n-butanol
Jello, repeat the centrifugation and discard organic phase, jello operation several times；After last time operates, revolving removes solution
In remaining Sevag reagent, hill gooseberry's fruit polysaccharide solution after obtaining de- albumen collected by the alcohol deposition method of step (1) and precipitated,
Hill gooseberry's fruit polysaccharide of preliminary purification is obtained after drying；
Sevag reagent described in step (3), by chloroform and n-butanol, 5:1 is formed by volume；
Shaken cultivation described in step (3), the preferred 150r/min of revolving speed；
The preferred 4000r/min of centrifugation described in step (3) is centrifuged 10min；
(4) DEAE-Sepharose fast flow ion-exchange chromatography separates hill gooseberry's fruit polysaccharide: DEAE- agar
Sugared gel filler fills column, mistake after hill gooseberry's fruit polysaccharide deionized water dissolving of the preliminary purification of step (3) after pretreatment
0.45 μm of miillpore filter, then upper prop；First with water elution is distilled, every 0.1g hill gooseberry's fruit polysaccharide distills water elution with 150mL,
Do not collect flow point；Then it is eluted with the NaCl solution that concentration is 0.1mol/L, every 5mL collects a pipe eluent, uses phenolsulfuric acid
Method detects eluent in the absorbance value that Detection wavelength is 490nm, until stopping collecting when absorbance value is close to zero, merges stream
Point, concentration, dialysis, freeze-drying obtain polysaccharide P2；
Filler pre-treatment described in step (4) the following steps are included: filler filter and with distilled water flushing, by
In bubble can be generated during the rinsing process, it is de-gassed bubble with ultrasonic wave later, is stood；
Dress column described in step (4) the following steps are included: filler is slowly drained to chromatographic column (column specification be 1.5cm ×
In 20cm), bed volume is about 20mL；Distilled water flushing chromatographic column 1h, then the NaCl solution for being 1mol/L with concentration is first used to rush
1h is washed, distilled water flushing 2h is finally used, that is, is ready for loading；
Hill gooseberry's fruit polysaccharide deionized water dissolving of preliminary purification described in step (4), the preferred 50mg/ of concentration
mL；
Loading described in step (4) and the preferred 0.5mL/min of elution flow rate.
Dialysis described in step (4) is that the flow point after concentration is transferred in bag filter (3000Da) in 4 DEG C of dialysis 48h.
Hill gooseberry's polysaccharide P2 of the invention has effect for reducing blood fat, can be used for preparing blood lipid-lowering medicine；
The drug also includes acceptable auxiliary material and other play the effective component of compatibility synergistic effect；
The drug can be various dosage forms, such as tablet, granule, capsule, dripping pill, sustained release agent, oral solution, injection
Deng.
It is of the invention the experimental results showed that, hill gooseberry's polysaccharide P2 has certain external conjucated bile acids salt ability, is come with examining
Enamine (a kind of blood lipid-lowering medicine, reducing blood lipid mechanism are conjucated bile acids salt) is used as positive control, by Cholestyramine to each gallbladder
The Percentage bound of hydrochlorate is 100% calculating, converts out hill gooseberry's polysaccharide P2 to the phase of natrium taurocholicum, NaGC, sodium taurocholate
29.87%, 47.49%, 53.69% is respectively as follows: to Percentage bound.It is certain that the experimental result tentatively shows that hill gooseberry's polysaccharide P2 has
The effect for reducing blood fat of effect.
The present invention obtains hill gooseberry's fruit Thick many candies by water extraction and alcohol precipitation method, using H2O2Method decoloration, Sevag method take off albumen,
And isolated and purified using DEAE-Sepharose fast flow ion exchange column, have compared with the existing technology such as
Under advantage and effect:
(1) present invention establishes complete feasible hill gooseberry's fruit Polyose extraction, isolates and purifies, structure feature and life
The technology path of object activity research provides technology and refers to for the extraction separation and purification of wild plant resource hill gooseberry's fruit polysaccharide
It leads.
(2) water extraction and alcohol precipitation method used in the present invention can complete a large amount of Polyose extraction operation, and cost is relatively low, repeatability
Good, yield is high, is suitable for industrialization large-scale production.
(3) the DEAE-Sepharose fast flow physical and chemical stability used in the present invention can be more preferable with mechanical performance, hands over
It is big to change capacity, can be varied less with incumbent firms, bed volume with the ionic strength of pH value, since flow velocity and carrying capacity are high, is suitable for
Carry out the purification work of a large amount of crude products.
(4) present invention is creatively by the water extract-alcohol precipitation extracting method of polysaccharide and ion-exchange chromatography isolation and purification method knot
It is used for the research of hill gooseberry's fruit polysaccharide altogether, relatively and preferably technological parameter is obtained, for mentioning for hill gooseberry's fruit polysaccharide
It takes to isolate and purify and technological guidance and new approaches is provided.
Detailed description of the invention
Fig. 1 is the elution curve of hill gooseberry's fruit polysaccharide.
Fig. 2 is the ultraviolet spectrogram of polysaccharide P2.
Fig. 3 is the outer conjucated bile acids salt ability of hill gooseberry's fruit polysaccharide body.
Fig. 4 is the infrared spectrogram of polysaccharide P2.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
In embodiment, the analysis of obtained hill gooseberry's fruit polysaccharide component P2 by China Guangzhou Analysis &. Test Center into
Row, report number distinguish 2017011500-3.
Embodiment 1
A method of it is extracted from hill gooseberry's fruit and isolates and purifies polysaccharide, comprising the following steps:
(1) extract hill gooseberry's fruit polysaccharide: hill gooseberry's fruit after weighing 110g drying crushes, and weighs after crossing 40 meshes
100g dry powder adds 1000mL distilled water, boils 4h in reflux unit, after suction filtration, filter residue is taken to add 1000mL distilled water, in
4h is boiled in reflux unit, merges supernatant, is concentrated under reduced pressure.95% (V/V) second of 4 times of volumes is slowly added in concentrate
Alcohol, and constantly make polysaccharide homogeneous precipitation with glass bar stirring, it is then placed in 4 DEG C of refrigerators and stands 12h, 5000r/min is centrifuged later
15min collects precipitating, and hill gooseberry's fruit Thick many candies are obtained after drying；
(2)H2O2Method decoloration: weighing 20g hill gooseberry's fruit Thick many candies, adds 100mL distilled water to dissolve, with 1%NaOH solution
Polysaccharide solution pH value is adjusted to 8.0,30%H is then added dropwise on one side2O2Solution stirs on one side, until color gradually becomes shallower as, then
2h is kept the temperature under 50 DEG C of water-baths；
(3) Sevag method takes off albumen: the Sevag reagent of 1/5 times of volume of the solution being added in the polysaccharide solution after decoloration
(5:1 is formulated by volume for chloroform and n-butanol) vibrates 20min with 150r/min revolving speed on shaking table, after standing 10min
Centrifugation, discards the jello that lower layer's organic phase and intermediate layer protein matter and chloroform and n-butanol generate, and 5 times repeatedly.For the last time
Remaining Sevag reagent in solution is evaporated off in 4000r/min centrifugation 10min back spin, and hill gooseberry's fruit after obtaining de- albumen is more
Sugar juice collects precipitating by the alcohol deposition method of step (1), hill gooseberry's fruit polysaccharide of preliminary purification is obtained after drying；
(4) DEAE-Sepharose fast flow ion-exchange chromatography separates hill gooseberry's fruit polysaccharide:
4.1 filler pre-treatments: the protection liquid in order to remove 20% ethyl alcohol, when suction filtration, are rinsed with distilled water.Due to
Bubble can be generated in flushing process, is de-gassed bubble with ultrasonic wave later, stood；
4.2 dress columns: condiment is slowly drained in chromatographic column (column specification is 1.5cm × 20cm), bed volume is about
20mL.It first uses distilled water flushing chromatographic column 1h, then the NaCl solution for being 1mol/L with concentration to rinse 1h, finally uses distilled water flushing
2h is ready for loading；
4.3 loadings: taking polysaccharide sample 0.1g, is dissolved with 2mL distilled water, is configured to the solution that concentration is 50mg/mL, mistake
After 0.45 μm of miillpore filter, with 0.5mL/min flow velocity loading；
4.4 elutions: first distilling water elution with 150mL, is then eluted with the NaCl solution that concentration is 0.1mol/L, every 5mL
A pipe eluent is collected, the polyoses content of every pipe is measured with Phenol sulfuric acid procedure, the detection of no polysaccharide is eluted to and is then considered as collection and finish.
Using received pipe number as abscissa, using the light absorption value at wavelength 490nm as ordinate, elution curve, i.e. Fig. 1 are drawn；
Phend-sulphuric acid detection polyoses content concrete operations are as follows: it is molten accurately to pipette 0.1mg/mL anhydrous grape Standard for Sugars
Liquid 0,0.2,0.4,0.6,0.8,1.0mL, are sequentially placed into 6 test tubes, then sequentially add distilled water 1.0,0.8,0.6,
0.4,0.2,0mL, sequentially add 6% phenol solution 0.5mL, concentrated sulfuric acid 2.5mL is placed at room temperature for 20min after mixing,
Absorbance is measured at wavelength 490nm, using concentration of glucose as abscissa, absorbance is ordinate, draws standard curve.It measures
Sample solution 1mL measures corresponding absorbance by standard curve operating method, calculates total starches content in sample solution.
4.5 dialysis: collecting each component eluted respectively, is transferred to after concentration in bag filter (3000Da) and dialyses in 4 DEG C
48h is freeze-dried after reduced pressure, obtains hill gooseberry's fruit polysaccharide P2.
Embodiment 2
Ultraviolet spectral analysis is carried out to the hill gooseberry fruit polysaccharide P2 that embodiment 1 obtains, 1mg polysaccharide sample is weighed, prepares
1mg/mL polysaccharide solution, scanning uv-spectrogram within the scope of 200-500nm.
Fig. 2 is the ultraviolet spectrogram of P2, the results show that the component is at 260nm, 280nm without apparent absorption peak, table
It is bright to be free of protein and nucleic acid substances.
Embodiment 3
Polysaccharide molecular weight analysis is carried out to the hill gooseberry fruit polysaccharide P2 that embodiment 1 obtains, specific experimental method is as follows:
Molecular weight is measured using gel permeation chromatography (GPC).Polysaccharide sample 2mg is taken, 0.02mol/L phosphate buffer is added
Dissolution, is configured to 2.0mg/mL solution, is filtered with 0.22 μm of sterilised membrane filter, took cleaner liquid spare.Chromatographic condition: column temperature 35
DEG C, using 0.02mol/L phosphate buffer as mobile phase, flow velocity 0.6mL/min, 20 μ L, Waters 2410 of sample volume shows difference
Refraction detector detection.Prepare a series of dextran solution (700,400,200,100,50,30,10,5kD) of different molecular weights
As standard items, standard curve is drawn, molecular weight analyte is then calculated according to its corresponding elution volume reference standard curve.
The molecular weight for measuring hill gooseberry's fruit polysaccharide P2 is as shown in table 1 below:
The molecular weight of 1 hill gooseberry's fruit polysaccharide P2 of table
Embodiment 4
Monosaccharide composition analysis is carried out to the hill gooseberry fruit polysaccharide P2 that embodiment 1 obtains, the specific method is as follows:
Polysaccharide sample 10mg is weighed, 4mol/L trifluoroacetic acid 5mL is added, in 100 DEG C of hydrolysis 2h, hydrolyzate is blown with nitrogen evaporator
It is dry, it is washed 3 times with Chromatographic Pure Methanol, obtains polysaccharide hydrolysis object.Hydroxylamine hydrochloride 10mg, internal standard flesh are sequentially added in polysaccharide hydrolysis object
Acetic anhydride 2mL is added after alcohol 1mg and pyridine 2mL, 90 DEG C of water-bath 30min, distilled water 2mL is added after 90 DEG C of water-bath 30min and terminates
Reaction.It is extracted 2 times with methylene chloride 2mL, merges methylene chloride phase, anhydrous sodium sulfate drying is added, crosses 0.22 μm of organic micropore
Filter membrane, it is spare.
Gas chromatographic detection condition: analytical column is HP-5MS (30m × 0.25mm × 0.25 μm)；Sampling volume is 1 μ L；Into
Sample mouth temperature is 250 DEG C；Constant voltage mode is 20PSI；Temperature programming: 100 DEG C of initial column temperature, keep 0.5min, then with 20 DEG C/
Min rises to 140 DEG C, keeps 5min, then rises to 160 DEG C with 3 DEG C/min speed, then be raised to 250 DEG C with 10 DEG C/min speed, protects
Hold 5min；Split ratio is 10:1, and mobile phase is helium, flow velocity 1mL/min.
Monosaccharide standard detection: ribose, rhamnose, arabinose, xylose, mannose, glucose, gala Standard for Sugars are taken
Product are handled and are detected according to above-mentioned same steps, as standard control.
Measure monosaccharide composition result such as the following table 2 of hill gooseberry's fruit polysaccharide P2:
The monosaccharide of 2 hill gooseberry's fruit polysaccharide P2 of table forms
Embodiment 5
FTIR spectrum analysis is carried out to the hill gooseberry fruit polysaccharide P2 that embodiment 1 obtains:
2mg polysaccharide sample is weighed, it is mixed into grinding with the KBr (potassium bromide) after drying in mortar, through tablet press machine pressure
In flakes, using Fourier Transform Infrared Spectrometer, in 400-4000cm-1Wave-number range in scanning.
Functional group has characteristic absorption peak in infrared spectrogram, and therefore, infrared spectrogram can preferably analyze polysaccharide
Structure.Fig. 4 is the infrared spectrogram of hill gooseberry's fruit polysaccharide purification component P2, in 3402cm-1The absorption peak at place is stretched by O-H
Vibration generates, 2930cm-1The absorption peak at place is generated by C-H stretching vibration, 1385cm-1The absorption peak at place is by the flexible vibration of C-O
Movable property is raw, these peaks are all the characteristic peaks of polysaccharide, it can be determined that P2 belongs to polysaccharose substance.
In the infrared spectrogram (Fig. 4) of P2, in 1152cm-1The absorption peak at place, illustrating P2, there are pyranose rings；?
1049cm-1The absorption peak at place illustrates the presence of glucose unit.
Embodiment 6
External conjucated bile acids salt ability measurement is carried out to the hill gooseberry fruit polysaccharide P2 that embodiment 1 obtains, with Cholestyramine
For positive control, preliminary characterization reducing blood lipid ability.Measuring method is as follows:
The drafting of cholate standard curve: 0.3mmol/L is prepared respectively with 0.1mol/L, pH=6.3 phosphate buffer solution
Natrium taurocholicum, NaGC, sodium taurocholate.Take respectively above-mentioned solution 0,0.1,0.5,1.0,1.5,2.0,2.5mL in
In 10mL tool plug test tube, 0.1mol/L, pH=6.3 phosphate buffer solution is added to 2.5mL, it is 60% that mass concentration, which is then added,
Sulfuric acid solution 7.5mL is put into ice bath 5min in 70 DEG C of water-bath 20min after taking-up, survey absorbance at 387nm wavelength.With cholate
Concentration is abscissa, and absorbance is ordinate, draws the standard curve of various cholates.
Sample simulates human gastrointestinal tract environment: taking sample solution 1mL in 10mL tool plug test tube, 0.01mol/L salt is added
Acid solution 1mL, constant temperature oscillation digests 1h at 37 DEG C, then adjusts pH value to 6.3 with the sodium hydroxide solution of 0.1mol/L, with
10mg/mL pancreatin is added afterwards and (prepares) 4mL with the 0.1mol/l phosphate buffer of pH value 6.3, constant temperature oscillation digests at 37 DEG C
30min。
The experiment of external conjucated bile acids salt: it is added respectively into the sample liquid after simulating human gastrointestinal tract environmental treatment
Various cholates (natrium taurocholicum, NaGC, sodium taurocholate) solution 4mL, the constant temperature oscillation 1h at 37 DEG C of 0.3mmol/L
After be transferred to centrifuge tube, with 4000r/min be centrifuged 20min.Cholic acid salt content in supernatant is analyzed: taking supernatant respectively
For 2.5ml in tool plug test tube, addition mass concentration is 60% sulfuric acid solution 7.5ml, in 70 DEG C of water-bath 20min, takes out ice bath
5min measures absorbance at 387nm.The concentration that cholate in sample liquid is acquired by calibration curve equation, with the cholate of addition
It is the cholic acid salinity combined, the content of unit of account quality conjucated bile acids salt that concentration, which subtracts this concentration,.
Fig. 3 is the external conjucated bile acids salt ability of hill gooseberry fruit purified polysaccharide component P2, the results showed that, with Cholestyramine
(a kind of blood lipid-lowering medicine, reducing blood lipid mechanism are conjucated bile acids salt) is used as positive control, by Cholestyramine to each cholate
Percentage bound be 100% to calculate, convert out hill gooseberry's polysaccharide P2 to natrium taurocholicum, NaGC, sodium taurocholate opposite knot
Conjunction rate is respectively as follows: 29.87%, 47.49%, 53.69%.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of hill gooseberry's polysaccharide P2, it is characterised in that the monosaccharide comprising following molar percentage: ribose 8.25%, rhamnose
2.10%, arabinose 53.75%, xylose 5.16%, mannose 8.22%, glucose 12.01%, galactolipin 10.51%.
2. hill gooseberry's polysaccharide P2 according to claim 1, it is characterised in that: number-average molecular weight is 2.57 × 104Da, weight are equal
Molecular weight is 6.75 × 104Da。
3. the isolation and purification method of hill gooseberry's polysaccharide P2 of any of claims 1 or 2, it is characterised in that the following steps are included:
(1) by hill gooseberry's fruit refluxing extraction, hill gooseberry's fruit Thick many candies are obtained；
(2) 30%H is added in hill gooseberry's fruit Thick many candies2O2Solution decoloration；
(3) Sevag reagent deproteinized is added in the polysaccharide after decolourizing, and obtains hill gooseberry's fruit polysaccharide of preliminary purification；
(4) DEAE- Ago-Gel filler fills column after pretreatment, and hill gooseberry's fruit polysaccharide of the preliminary purification of step (3) is used
0.45 μm of miillpore filter is crossed after deionized water dissolving, then upper prop；First with distillation water elution, every 0.1g hill gooseberry's fruit polysaccharide use
150mL distills water elution, does not collect flow point；Then it is eluted with the NaCl solution that concentration is 0.1mol/L, every 5mL collects a pipe and washes
De- liquid, the absorbance value for being 490nm in Detection wavelength with phend-sulphuric acid detection eluent, until when absorbance value is close to zero
Stop collecting, merge flow point, concentration, dialysis, freeze-drying obtain polysaccharide P2.
4. isolation and purification method according to claim 3, it is characterised in that: the step (1) includes following operation: will
Hill gooseberry's fruit is dried and is crushed, and takes its dry powder and distilled water to boil extraction in reflux unit for several times, combined extract simultaneously subtracts
Pressure concentration, is added 95% (V/V) ethyl alcohol of several times volume, being stirred continuously makes polysaccharide homogeneous precipitation, then at 4 DEG C in concentrate
Lower standing 12h or more, is collected by centrifugation precipitating, and hill gooseberry's fruit Thick many candies are obtained after drying.
5. isolation and purification method according to claim 3, it is characterised in that: the step (2) includes following operation: being taken
Hill gooseberry's fruit Thick many candies add distilled water to dissolve, and adjust pH value to 8.0,30%H is then added dropwise on one side2O2Solution stirs on one side,
Until solution colour shoals, 2h then is kept the temperature under 50 DEG C of water-baths.
6. isolation and purification method according to claim 3, it is characterised in that: the step (3) includes following operation:
The Sevag reagent of 1/5 volume of solution is added in polysaccharide solution after decoloration, shaken cultivation 20min is centrifuged after standing 10min,
The jello that lower layer's organic phase and intermediate layer protein matter and chloroform and n-butanol generate is discarded, the centrifugation is repeated and is discarded organic
Phase, jello operation several times；After last time operates, revolving removes remaining Sevag reagent in solution, obtains de- albumen
Hill gooseberry's fruit polysaccharide solution afterwards collects precipitating by the alcohol deposition method of step (1), hill gooseberry's fruit of preliminary purification is obtained after drying
Polysaccharide.
7. isolation and purification method according to claim 3, it is characterised in that: loading and elution flow rate described in step (4)
For 0.5mL/min.
8. hill gooseberry's polysaccharide P2 of any of claims 1 or 2 is preparing the application in blood lipid-lowering medicine.
9. application according to claim 8, it is characterised in that: the drug includes acceptable auxiliary material and other rise and match
The effective component of 5 synergistic effects.
10. application according to claim 8, it is characterised in that: the drug be tablet, granule, capsule, dripping pill,
Sustained release agent, oral solution, injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810987144.9A CN109206531B (en) | 2018-08-28 | 2018-08-28 | Polysaccharide P2, separation and purification method thereof and application thereof in preparation of hypolipidemic drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810987144.9A CN109206531B (en) | 2018-08-28 | 2018-08-28 | Polysaccharide P2, separation and purification method thereof and application thereof in preparation of hypolipidemic drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109206531A true CN109206531A (en) | 2019-01-15 |
| CN109206531B CN109206531B (en) | 2021-06-04 |
Family
ID=64985649
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810987144.9A Active CN109206531B (en) | 2018-08-28 | 2018-08-28 | Polysaccharide P2, separation and purification method thereof and application thereof in preparation of hypolipidemic drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109206531B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319185A (en) * | 2007-06-05 | 2008-12-10 | 王晓明 | Colorful white spirit and preparation method thereof |
| CN108324759A (en) * | 2018-03-18 | 2018-07-27 | 启东创绿绿化工程有限公司 | The preparation method of component for reducing blood fat in a kind of downy rosemyrtle fruit |
-
2018
- 2018-08-28 CN CN201810987144.9A patent/CN109206531B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319185A (en) * | 2007-06-05 | 2008-12-10 | 王晓明 | Colorful white spirit and preparation method thereof |
| CN108324759A (en) * | 2018-03-18 | 2018-07-27 | 启东创绿绿化工程有限公司 | The preparation method of component for reducing blood fat in a kind of downy rosemyrtle fruit |
Non-Patent Citations (3)
| Title |
|---|
| 王汝聪等: "《生物有机化学专论（第1版）》", 31 May 1990, 山东科学技术出版社 * |
| 隋亚君等: "桃金娘多糖的分离纯化及其化学结构研究", 《中国优秀博硕士学位论文全文数据库（硕士）工程科技I辑》 * |
| 颜真等: "《蛋白质研究技术（第1版）》", 31 January 2007, 第四军医大学出版社 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109206531B (en) | 2021-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105037577B (en) | Procoagulant blackberry seed polysaccharide, and extraction separation method and application thereof | |
| CN107011453B (en) | A kind of dimension medicine just ancient polysaccharide of fiber crops and its extracting method and application | |
| CN103239467B (en) | Agaricus blazei polysaccharide and preparation method thereof | |
| CN105524183B (en) | A kind of extracting method of water chestnut polysaccharide | |
| CN107012184B (en) | Angelica dahurica polysaccharide extracted by enzyme method, preparation method and application thereof | |
| CN112457422A (en) | Preparation method of phlebopus portentosus polysaccharide | |
| CN106749729B (en) | A kind of Smilacina japonica polysaccharide and its preparation method and application | |
| CN108265092A (en) | A kind of mushroom oligosaccharides and preparation method with excellent antioxidant activity | |
| CN102276754B (en) | Organosulfate glucan in hedysarum polybotys saccharide as well as preparation method and application thereof | |
| CN108948223A (en) | Hill gooseberry's polysaccharide P1 and its separation method and preparing the application in blood lipid-lowering medicine | |
| CN104877037B (en) | Separation and purification method, products and application of Christia vespertilionis polysaccharides | |
| CN109206532A (en) | A method of it is extracted from hill gooseberry's fruit and isolates and purifies polysaccharide | |
| CN109206531A (en) | Polysaccharide P2 and its isolation and purification method and preparing the application in blood lipid-lowering medicine | |
| CN114957497B (en) | Gentiana rigescens acidic polysaccharide and preparation method and application thereof | |
| CN109134679A (en) | A kind of hill gooseberry's polysaccharide P3 and its separation method and the purposes in blood lipid-lowering medicine | |
| CN109134678A (en) | A kind of polysaccharide P4 and its isolation and purification method and the purposes in blood lipid-lowering medicine | |
| CN104119427B (en) | A kind of Huai Er albumen and its production and use | |
| CN112159484B (en) | Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof | |
| CN112898445B (en) | Separation and extraction method and application of urtica macrorrhiza polysaccharide | |
| CN108314745A (en) | A method of preparing Fuscoporia obliqua polysaccharide | |
| CN104292351B (en) | A kind of Fructus Camelliae sinensis Phellinus polysaccharide and its preparation method and application | |
| CN104606297B (en) | Method for extracting active ingredients in cistanche tubulosa | |
| CN112076175A (en) | Cordyceps militaris polysaccharide effervescent tablet, and preparation method and application thereof | |
| CN106986949B (en) | Dogbane flower polysaccharide, extracting method and its application | |
| CN104119428B (en) | A kind of Huai Er albumen and its production and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |