CN109320628A - Safflower petal total starches and its extracting method and application - Google Patents
Safflower petal total starches and its extracting method and application Download PDFInfo
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- CN109320628A CN109320628A CN201811188946.XA CN201811188946A CN109320628A CN 109320628 A CN109320628 A CN 109320628A CN 201811188946 A CN201811188946 A CN 201811188946A CN 109320628 A CN109320628 A CN 109320628A
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- safflower petal
- safflower
- petal
- total starches
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Obesity (AREA)
- Sustainable Development (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Birds (AREA)
- Dermatology (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention provides the extracting methods of safflower petal total starches, belong to safflower active constituent extractive technique field, the following steps are included: by the filter residue air-dried after safflower petal alcohol extracting and water with 1g:(15~25) ratio of ml mixes after being placed in 95~105 DEG C of extractions 3h, alcohol precipitation acquisition safflower petal total starches crude product；The safflower petal total starches crude product is redissolved and obtains safflower petal total starches by chromatographic column chromatographic purifying.The method of the invention safflower petal total starches yield is high, and activity is high, shows higher DPPH free radical and OH Scavenging activity, and acute toxicity is evaluated as practical nontoxic, can reduce TC, TG content in liver to a certain extent.This shows that safflower petal polysaccharide can reduce blood lipid level, effectively prevention blood lipid level, effectively prevents hyperlipidemia caused by long term high-fat diet.
Description
Technical field
The invention belongs to safflower active constituent extractive technique field more particularly to safflower petal total starches and its extractions
Methods and applications.
Background technique
Safflower (Saffron, Crocus sativus L.) also known as safron, west safflower, up to husband indigo plant, Sa Fa are that is, be
Iridaceae crocus herbaceos perennial is recorded according to " Chinese name republic pharmacopeia ", and medicinal part is dry flower
Column, safflower is as its effect of traditional Chinese medicine are as follows: activating microcirculation and removing stasis medicinal, removing pattogenic heat from the blood and toxic material from the body, resolving stagnation for tranquilization.For Jing Bi Disorder lump in the abdomen, the postpartum stasis of blood
Resistance, febrile virulent maculae, melancholy ruffian is bored, and palpitation with fear is gone mad.In addition to medicinal, it can be used as food additives, edible pigment and advanced perfume (or spice)
Material.Show principle active component-saffron glucoside in its style for cancer, coronary disease the pharmacological research of safflower in recent years
A variety of diseases such as disease, atherosclerosis, hyperlipemia have preferable preventive and therapeutic effect.Its style has simultaneously because yield is extremely low
Magical drug effect and sealed by common people with the title of " plant gold ", meanwhile, a large amount of dis-medicinal part-petal is in safflower
Become byproduct in pharmaceuticals industry, causes the great wasting of resources.
For the prior art to the active constituent extraction process of safflower petal, recovery rate is low, and the Ingredients Active for extracting acquisition is low.
Summary of the invention
In view of this, the present invention provides, a kind of recovery rate is high, the extracting method of the high safflower petal total starches of activity and
The application of safflower petal total starches.
To achieve the goals above, the present invention provides following technical schemes: the extracting method of safflower petal total starches,
The following steps are included: 1) by the filter residue and water that are air-dried after safflower petal powder alcohol extracting with 1g:(15~25) ratio of ml mixes
It is separated by solid-liquid separation after being placed in 95~105 DEG C of 2.5~3.5h of extraction, collects liquid phase component, obtain safflower petal Aqueous extracts；2) by institute
Safflower petal Aqueous extracts alcohol precipitation is stated, solid phase components is collected and obtains safflower petal water extract solid powder；3) by the safranine
Flower petal water extract solid powder redissolve, after dialysis, purify with anion exchange chromatography, gel filtration chromatography purify acquisition safranine
Flower petal total starches.
Preferably, the ratio of the filter residue and water is 1g:20ml, and the temperature of the extraction is 98~102 DEG C.
Preferably, the time of the extraction is 2.7~3.3h.
Preferably, the alcohol precipitation be ethyl alcohol is mixed with the safflower petal Aqueous extracts, until in mixed liquor ethyl alcohol body
Product concentration is 85~95%.
Preferably, the molecular cut off of the dialysis is 2500~3500D；The total time of the dialysis is 42~54h, often
It is primary that 6~8h changes water.
Preferably, the anion-exchange column is DEAE-Sephadex A-50 column.
Preferably, the sample concentration of the anion exchange chromatography is 30~40mg/ml；The anion-exchange column layer
Analysis uses the NaCl solution gradient elution of 0.05~0.6mol/L, and the elution speed is 0.8~1.2ml/min.
Preferably, the gel filtration chromatography uses Sephadex G-200, and the eluent of the gel filtration chromatography is water, washes
Separation of flow speed is 0.08~0.12ml/min.
The present invention also provides the safflower petal total starches that the extracting method prepares.
The present invention also provides the safflower petal total starches to prepare answering in drug, health care product and cosmetics
With.
Preferably, the drug includes the drug of anti-curing hyperlipemia.
Beneficial effects of the present invention: the extracting method of heretofore described safflower petal total starches, by by safflower
The filter residue and water air-dried after petal powder alcohol extracting is with 1g:(15~25) mixing of the ratio of ml be placed in 95~105 DEG C extract 2.5~
It is separated by solid-liquid separation after 3.5h, alcohol precipitation, the safflower petal total starches that dialysis, anion exchange chromatography and gel filtration chromatography obtain obtain
Rate is high, yield 32.10%, purity 88.10%.Antioxidation in vitro is the results show that as safflower petal polysaccharide quality is dense
The increase of degree gradually increases the scavenging effect of DPPH and OH free radical.When concentration is 16mg/mL, to the clear of DPPH free radical
Removing solid capacity is 29.46%, and concentration is more than half to OH free radical scavenging activity when being 20mg/mL, is 63.86%, shows higher body
Outer oxidation resistance.
Detailed description of the invention
Fig. 1 is clearance rates for trial result of the PPC solution to DPPH free radical of various concentration；
Fig. 2 is clearance rates for trial result of the PPC solution to OH free radical of various concentration；
Fig. 3~8 are influence of the safflower petal polysaccharide to hyperlipemia in mice organ coefficient；
Fig. 9~11 be safflower petal polysaccharide to hyperlipemia in mice serum glucose, high in fat group of serum TC, TG shadow
It rings.
Specific embodiment
The present invention provides the extracting methods of safflower petal total starches, comprising the following steps: 1) by safflower petal powder
The filter residue and water air-dried after last alcohol extracting is with 1g:(15~25) mixing of the ratio of ml consolidates after being placed in 95~105 DEG C of 2.5~3.5h of extractions
Liquid separation, collects liquid phase component, obtains safflower petal Aqueous extracts；2) it by the safflower petal Aqueous extracts alcohol precipitation, collects solid
Phase component obtains safflower petal water extract solid powder；3) the safflower petal water extract solid powder is redissolved, dialysis
Afterwards, purified with anion exchange chromatography, gel filtration chromatography purify obtain safflower petal total starches.
In the present invention, using safflower petal as raw material, the safflower petal is preferably to remove doing for safflower style
Dry petal.In the present invention, the safflower petal is preferred crush after drying to constant weight and obtains safflower petal powder；Institute
The granularity for stating safflower petal powder is preferably less than 30 mesh.Heretofore described safflower petal powder preferably uses petroleum
After ether degreasing, it is dried for standby.The temperature of heretofore described drying is preferably 50~60 DEG C, and more preferably 56 DEG C.In the present invention,
The safflower petal powder, which preferably seals, to be kept in dark place.Heretofore described alcohol extracting is preferred the following steps are included: will
The ethyl alcohol that above-mentioned safflower petal powder and volume fraction are 55~65% is with 1g:(15~25) the ratio mixing of ml is placed in 65~
It is separated by solid-liquid separation after 75 DEG C of 1.5~2.5h of extraction, collects after solid phase components air-dry and obtain the filter residue.
The present invention is by the filter residue and water with 1g:(15~25) mixing of the ratio of ml be placed in 95~105 DEG C extract 2.5~
It is separated by solid-liquid separation after 3.5h, collects liquid phase component, obtain safflower petal Aqueous extracts.In the present invention, the ratio of the filter residue and water
Example is preferably 1g:(18~22) ml, more preferably 1g:20ml.The temperature of heretofore described extraction is preferably 98~102 DEG C,
More preferably 100 DEG C；The time of the extraction is preferably 2.7~3.3h, more preferably 3h.The present invention carries out after the extraction
It is separated by solid-liquid separation, collects liquid phase component；In the present invention, the method for the separation of solid and liquid is preferably filtered, and the present invention is to the filtering
There is no particular/special requirement, is filtered using the filtered through gauze of this field routine, filter paper filtering or film.
The present invention is after obtaining the safflower petal Aqueous extracts, by the safflower petal Aqueous extracts alcohol precipitation.The present invention
Described in alcohol precipitation be ethyl alcohol is mixed with the safflower petal Aqueous extracts, until in mixed liquor the volumetric concentration of ethyl alcohol for 85~
95%, preferably 90%.In the present invention, the temperature of the alcohol precipitation is preferably 4 DEG C, and the time of the alcohol precipitation is preferably 10~16h,
More preferably 12~14h；The present invention is separated by solid-liquid separation after the alcohol precipitation, collects solid phase components.
By solid component as 30~50 DEG C, 10~14 hours are dried in vacuo to get safflower petal water extract solid powder
End.In the present invention, the mode of the separation of solid and liquid is preferably centrifuged or filtering；Specific item of the present invention to the centrifugation or filtering
Part is not particularly limited, as long as can be realized separation of solid and liquid；In the present invention, the drying temperature is preferably 40 DEG C, dry
Time is 12 hours.
The present invention dialyses after obtaining the safflower petal water extract solid powder and redissolving.In the present invention, institute
Redissolution is stated preferably to be carried out with distilled water.The molecular cut off of the dialysis is preferably 2500~3500D in the present invention, more excellent
It is selected as 3000D；The total time of the dialysis is preferably 42~54h, more preferably 46~50h, most preferably 48h；The present invention exists
It is primary to change water by preferred every 6~8h in the dialysis procedure.
The present invention is concentrated again after the dialysis, dry (method is identical as safflower petal water extract drying means)
Up to safflower petal Thick many candies.
After obtaining safflower petal Thick many candies, 100mg sample is taken, ultrapure water redissolves, purified with anion exchange chromatography.
In the present invention, the anion-exchange column is preferably DEAE-Sephadex A-50 column.In the present invention, the DEAE-
The preparation of Sephadex A-50 column is carried out using the means of this field routine, without other particular/special requirements.It is heretofore described yin from
The sample concentration of sub- exchange column chromatography is preferably 30~40mg/ml, more preferably 31~35mg/ml；It is heretofore described yin from
Sub- exchange column chromatography uses the NaCl solution gradient elution of 0.05~0.6mol/L, and in the present invention, the gradient elution NaCl is molten
The concentration gradient of liquid is preferably 0.05,0.1,0.2,0.3,0.4,0.5,0.6mol/L；The elution speed is preferably 0.8~
1.2ml/min, more preferably 1.0ml/min.In specific implementation process of the present invention, automatic fraction collector collects eluent,
4min/ pipe.Sugared content in every pipe is measured with Phenol-sulphate acid method.The component for collecting sugar enrichment is further purified.
The present invention the anion exchange chromatography after purification, by the sugared enriched composition being collected into carry out gel column layer
Analysis.Heretofore described gel column is preferably Sephadex G-200.In the present invention, the system of the Sephadex G-200 column
The standby means using this field routine carry out, without other particular/special requirements.In the present invention, the eluent of the gel filtration chromatography is
Water, the elution flow rate are preferably 0.08~0.12ml/min, more preferably 0.1ml/min.In specific implementation process of the present invention
In, automatic fraction collector collects the liquid eluted, and 20mL/ pipe surveys sugared content, the component that sugar reaction is positive is collected,
Concentrated, dry (method is identical as safflower petal water extract drying means) obtains safflower petal total starches.
The present invention provides said extracted methods to prepare safflower petal total starches, and wherein total starches content is 86.0
~91.0%, the content of reduced sugar is 2.5~3.5%, and glucuronic acid content is 38.5~43.5%, protein content is 2.0~
2.2%.
The present invention also provides the safflower petal total starches to prepare answering in drug, health care product and cosmetics
With.
In the present invention, the safflower petal total starches have the function of removing free radical, have to hyperlipidemia
Preventive and therapeutic effect, safflower petal total starches physiological-toxicity-free of the present invention, can be applied to drug, health care product and cosmetics
Preparation.The present invention does not have special limit to content of the safflower petal total starches in the drug, health care product and cosmetics
It is fixed；A kind of described this active constituent of safflower petal total starches can be only included in the drug, health care product and cosmetics, it can also
To include the safflower petal total starches and other active components；The present invention is in the drug, health care product and cosmetics
Auxiliary material is not particularly limited, using conventional acceptable auxiliary material.In the present invention, the drug includes anti-curing hyperlipemia
Drug.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
The safflower petal powder air-dried after 3 parts of alcohol extractings is accurately weighed, every part of 2g is put in 3 similar triangular flasks respectively
In, it is separately added into 40mL distilled water in the ratio of material-water ratio 1:20, mixing is sufficiently stirred with glass bar.Plastic fresh-keeping membrane sealing with
Afterwards, it is placed in 100 DEG C of water-bath and extracts 3h.Merge the filtrate filtered through Buchner funnel and repeat suction filtration once, later in rotation
Turn to be concentrated under reduced pressure into minimum volume in evaporimeter, ethyl alcohol, which is added, makes ethyl alcohol final volume about reach 90%, and 4 DEG C of ice are put into after stirring
Case is stood overnight.Gained sediment is collected after being filtered with 200 mesh nylon leaching nets, and dry 12h, obtains in 40 DEG C of vacuum ovens
Safflower petal water extract powder.
Safflower petal water extract powder is dissolved with ultrapure water, is transferred in the bag filter of D3000, both ends are pressed from both sides with bag filter
Sub solid clamping, is put into ultrapure water, 4 DEG C of dialysis 48h change a water every 6h.It is concentrated, is put into true with Rotary Evaporators after dialysis
Dry 12h, obtains safflower petal Thick many candies in empty drying box.
Anion exchange chromatography
It takes 8g DEAE-Sephadex A-50 powder in 500mL beaker, 300mL ultrapure water is added, is swollen in boiling water bath
3~4h.The particle for removing the floating on upper layer after swelling impregnates 1h with 0.5mol/L NaOH, pours into sand core funnel with ultrapure washing
To neutrality；It is transferred in beaker and impregnates 0.5h with 0.5mol/L HCl, be washed till neutrality, then with being washed till neutrality after alkali cleaning.Add
0.05mol/L NaCl stirring stands half an hour hypsokinesis and removes upper layer particle, is put into vacuum pump and vacuumizes 2h.
Pillar is rinsed with 0.05mol/L NaCl, outlet pipe is clamped with tweezers, retains about 1/5 volume buffer, will be swollen
Good gel disposably fills pillar, connects constant flow pump, adjusts the flow velocity of 1mL/min, maintain this flow velocity rinse 5 times of column volumes into
Row balance, until column bed height is stablized.100mg safflower petal Thick many candies are dissolved in 3mL ultrapure water loading, with 0.05,0.1,
0.2,0.3,0.4,0.5,0.6mol/L NaCl is eluted, and flow velocity 1ml/min, automatic fraction collector collects eluent,
4min/ pipe.Sugared content in every pipe is measured with Phenol-sulphate acid method.It collects sugar and reacts part of being positive, concentration, dry (method and hiding
Safflower petal water extract drying means is identical), sugared enriched composition is obtained, waiting is further purified.Gel filtration chromatography
Sephadex G-200 pre-treating method is similar with DEAE-Sephadex A-50, adds after alkali-Acid-Base is washed after swelling
Ultrapure water evacuation fills column, balance.100mg sugar enriched composition 3ml ultrapure water is taken to dissolve, loading.With ultrapure washing after loading
De-, flow velocity 0.1mL/min, 20mL/ pipe, automatic fraction collector is collected, and is collected the component that sugar reaction is positive, is obtained after dry
Obtain safflower petal total starches.
Wherein total reducing sugar be 88.6%, reduced sugar 3.26%, uronic acid 42.03%, protein 2.14%.To its list
Sugar composition carries out thin-layer chromatographic analysis, thus it is speculated that the monosaccharide group of safflower petal polysaccharide (may also as glucuronic acid, glucose
For galactolipin), arabinose, xylose, rhamnose, there are one unknown monosaccharide.
Embodiment 2
The safflower petal powder air-dried after 3 parts of alcohol extractings is accurately weighed, every part of 5g is put in 3 similar triangular flasks respectively
In, it is separately added into 100mL distilled water in the ratio of material-water ratio 1:20, mixing is sufficiently stirred with glass bar.Plastic fresh-keeping membrane sealing
After, it is placed in 98 DEG C of water-bath and extracts 2.8h.Merge the filtrate filtered through Buchner funnel and repeats suction filtration once, Zhi Houyu
Minimum volume is concentrated under reduced pressure into Rotary Evaporators, ethyl alcohol, which is added, makes ethyl alcohol final volume reach 88%, and 4 DEG C of ice are put into after stirring
Case is stood overnight.Gained sediment is collected after being filtered with 200 mesh nylon leaching nets, and dry 12h, obtains in 40 DEG C of vacuum ovens
Safflower petal water extract powder.
Safflower petal water extract powder is dissolved with ultrapure water, is transferred in the bag filter of D3000, both ends are pressed from both sides with bag filter
Sub solid clamping, is put into ultrapure water, 4 DEG C of dialysis 48h change a water every 6h.It is concentrated, is put into true with Rotary Evaporators after dialysis
Dry 12h, obtains safflower petal Thick many candies in empty drying box.
Anion exchange chromatography
It takes 8g DEAE-Sephadex A-50 powder in 500mL beaker, 300mL ultrapure water is added, is swollen in boiling water bath
3~4h.The particle for removing the floating on upper layer after swelling impregnates 1h with 0.5mol/L NaOH, pours into sand core funnel with ultrapure washing
To neutrality；It is transferred in beaker and impregnates 0.5h with 0.5mol/L HCl, be washed till neutrality, then with being washed till neutrality after alkali cleaning.Add
0.05mol/L NaCl stirring stands half an hour hypsokinesis and removes upper layer particle, is put into vacuum pump and vacuumizes 2h.
Pillar is rinsed with 0.05mol/L NaCl, outlet pipe is clamped with tweezers, retains about 1/5 volume buffer, will be swollen
Good gel disposably fills pillar, connects constant flow pump, adjusts the flow velocity of 1mL/min, maintain this flow velocity rinse 5 times of column volumes into
Row balance, until column bed height is stablized.100mg safflower petal Thick many candies are dissolved in 3mL ultrapure water loading, with 0.05,0.1,
0.2,0.3,0.4,0.5,0.6mol/L NaCl is eluted, and flow velocity 1ml/min, automatic fraction collector collects eluent,
4min/ pipe.Sugared content in every pipe is measured with Phenol-sulphate acid method.It collects sugar and reacts part of being positive, concentration obtains sugar after dry
Enriched composition, waiting are further purified.Gel filtration chromatography
Sephadex G-200 pre-treating method is similar with DEAE-Sephadex A-50, adds after alkali-Acid-Base is washed after swelling
Ultrapure water evacuation fills column, balance.100mg sugar enriched composition 3ml ultrapure water is taken to dissolve, loading.With ultrapure washing after loading
De-, flow velocity 0.1mL/min, 20mL/ pipe, automatic fraction collector is collected, and is collected the component that sugar reaction is positive, is obtained after dry
Obtain safflower petal total starches.
Wherein total reducing sugar be 87.5%, reduced sugar 3.21%, uronic acid 42.13%, protein 2.10%.To its list
Sugar composition carries out thin-layer chromatographic analysis, thus it is speculated that the monosaccharide group of safflower petal polysaccharide (may also as glucuronic acid, glucose
For galactolipin), arabinose, xylose, rhamnose, there are one unknown monosaccharide.
Embodiment 3
The safflower petal total starches obtained in detection embodiment 1 are to DPPH Scavenging activity
Polysaccharide is configured to the solution that concentration is 1,2,4,8,16mg/mL, follows these steps measurement DPPH Scavenging activity, often
A concentration three parallel, is averaged.
Experimental setup of the 1 safflower petal total starches (PPC) of table to DPPH Scavenging activity
Absorbance is measured under room temperature avoid light place 30min, 517nm
Each pipe average value marks A1 A2 A0
Clearance rate (%)=[1- (A1-A2)/A0] × 100%
By the PPC solution of 6 various concentrations to the clearance rates for trial the result is shown in Figure 1 of DPPH free radical, we can be seen
Out with the increase of PPC mass concentration, the scavenging effect of DPPH free radical is gradually increased, there is certain dose dependent.
Polysaccharide starts to show the Scavenging activity to DPPH free radical when being 1mg/mL, when concentration is 16mg/mL, to the clear of DPPH free radical
Removing solid capacity is 29.46%, shows higher DPPH free radical scavenging activity.
Safflower petal total starches are to OH Scavenging activity
Polysaccharide is configured to the solution that concentration is 2.5,5,10,20,30mg/mL, measurement OH is followed these steps and removes energy
Power, each concentration three parallel, is averaged.
Experimental setup of the 2 safflower petal total starches (PPC) of table to OH Scavenging activity
It mixes, 37 DEG C of water-baths 30min, 12000r/min are centrifuged 6min, and 510nm measures absorbance value.
Each pipe average value mark A1 A2 A0
Note
Clearance rate (%)=[1- (A1-A2)/A0] × 100%
The PPC solution of 6 various concentrations is as shown in Figure 2 to the clearance rate of OH.Increase with PPC concentration, scavenging effect is got over
It is more obvious, when PPC concentration is 10mg/mL, clearance rate 30.93%；Clearance rate is more than half when concentration is 20mg/mL, is
63.86%.Concentration continues growing, and clearance rate increases but effect is disproportionate.PPC concentration shows higher when being 20mg/mL
OH Scavenging activity.
Embodiment 4
The evaluation of safflower petal total starches Mouse Acute Toxicity
It is SPF grades ICR mouse 50, male and female 25,18 ± 2g of weight, limited purchased from the western Poole-Bi Kai experimental animal in Shanghai
Company, animal quality certification number: 2008001636633.
The mouse purchased is randomly divided into five groups, every group 10, half male and half female is numbered respectively, and adaptive feeding is prohibited after 3 days
Food 12 hours.First group is control group, the isometric solvent, that is, distilled water of stomach-filling；Other four groups of experimental groups successively press 1720mg/kg,
2450mg/kg, 3500mg/kg, 5000mg/kg BW (Bodyweight) stomach-filling safflower petal total starches (PPC) solution, group
Between the ratio between dosage be 1:0.7；The stomach-filling in such a way that not isoconcentration is isometric has filled for one day in three times.Continue two weeks, observes phase
It closes index and records death toll；Three days weighings weight, amount of drinking water and food rations, cervical dislocation puts to death mouse after two weeks, weighs the heart
The organ weights such as dirty, liver, spleen, kidney and fat pad, and calculate organ coefficient (organ coefficient=organ weights/weight ×
100%)
Physical signs observes and records
Dead mouse situation and death time, the state of mind, behavioral activity, Respiratory behavior, meal situation are observed after administration
And skin, hair, eye ear mouth and nose, four limbs etc..All data are all made of 17.0 software of SPSS and carry out variance analysis, test institute
The form for obtaining data mean+SD indicates that comparison among groups use ANOVA (one-way analysis of variance), and p < 0.05 is aobvious
Difference is write, p < 0.01 is extremely significant difference.
Each group mouse does not occur the phenomena of mortality, and activity is normal after 3 administrations of the administration same day, the secretion of eyes no abnormality seen
Object, skin, hair are normal, and stool and urine is normal, and the state of mind is normal.Diet drinking-water is normal after giving food.
Experimental result is shown: since each group does not occur death, not measuring safflower petal polysaccharide LD50.When stomach-filling agent
When amount reaches 5000mg/kg weight, any toxic reaction is had no, all kinds of internal organs of visual inspection are no abnormal existing after putting to death dissection
As no significant difference compared with the control group, it is inferred that safflower petal polysaccharide median lethal dose is greater than 5000mg/kg weight.
The variation of weight before and after 3 intragastric administration on mice of table
Note: p < 0.01 * p < 0.05 compared with the control group, * *
Each group mouse weight obviously increases, and compared with the control group, each dosage group is without significant difference (p > 0.05).Therefore it pushes away
Safflower petal polysaccharide does not impact the change of mouse weight during surveying stomach-filling.
Table 4 and table 5 show the organ coefficient of each dosage group mouse heart, liver, spleen, kidney, thymus gland and fat pad.
By chart it is found that compared with the control group, the organ coefficient of each dosage group mouse there are no significant difference (p > 0.05).Thus speculate
Each internal organs of stomach-filling safflower petal Polysaccharides on Mice will not impact.
Table 4 is the organ coefficient of each dosage group mouse heart, liver, spleen
Note: p < 0.01 * p < 0.05 compared with the control group, * *
The organ coefficient of each dosage group mouse kidney of table 5, thymus gland and fat pad
Note: p < 0.01 * p < 0.05 compared with the control group, * *
6 mice serum TC, TG content of table
Note: p < 0.01 * p < 0.05 compared with the control group, * * is not it is found that each dosage group mouse TG has compared with the control group
Significant difference (p>0.05), but each group mouse TC content has decline compared with the control group, and difference is extremely significant (p<0.01),
High dose group is significantly reduced (p < 0.05) compared to middle low dose group.Speculated by above data, to intragastric administration on mice safflower
Petal polysaccharide may will will affect Normal Mouse Serum TC content, but not influence TG content.
Embodiment 5
Prevention effect of the safflower petal total starches (PPC) to hyperlipemia
Experimental animal
SPF grades C57BL/6 male mice 50,18 ± 2g of weight, it is purchased from Shanghai Ling Chang Biotechnology Co., Ltd, animal
Quality certification number: 2013001801015.
Chow diet is purchased from Shanghai Slac Experimental Animal Co., Ltd., irradiation sterilization processing；45% fat energy supply
Feed is customized by above-mentioned company, irradiation sterilization processing, freezen protective.Two kinds of feed formulas are as follows:
Chow diet formula such as table 7,45% fatty feed formula is as shown in table 8.
7 chow diet formula of table
The fatty feed formula of table 845%
Experimental design
50 SPF grades of C57BL/6 male mices were randomly divided into five groups, often in animal experimental center adaptable fed three days
Group 10: normal group, high in fat group, low dose group, middle dose group, high dose group.It is numbered after grouping, feeds corresponding feed respectively,
The drug of stomach-filling corresponding dosage.Normal group feeding normal mice material, while stomach-filling distilled water；The fatty function of high in fat group of feeding 45%
High lipid food, stomach-filling distilled water；Low, middle and high dose groups give high lipid food, at the same respectively stomach-filling 200mg/kg BW,
The polysaccharide solution of 400mg/kg BW, 800mg/kg BW.
Using not isoconcentration stomach-filling in equal volume, daily 9:00am stomach-filling, free diet drinking-water, weight of one week title, food
Weight, water weight.It is administered continuously after 12 weeks, every group of 8 mouse fasting 12h.Every group of other two non-fasting.The next morning eye socket takes
Mouse cervical dislocation after blood.The internal organs such as liver,kidney,spleen, the heart, thymus gland, fat pad are taken out in dissection, weigh its weight in wet base, calculate internal organs system
Number；And number cryopreservation tube packing after liver and the rinsing of adipose tissue physiological saline, it is transferred to -80 DEG C of refrigerators after liquid nitrogen flash freezer and protects
It deposits to be measured.
1. Biochemical Indices In Serum is analyzed
Blood places 2h in 4 DEG C of refrigerators, and 4 DEG C of refrigerated centrifuge 10000r/min are centrifuged 10min, separate serum.In serum
The measurement of TC, TG, NO, ALT, AST, glucose (Glu), HDL-C are carried out by purchased kit specification step, low close in serum
Spend lipoprotein (Low density lipoprotein cholesterol, LDL-C) and atherosclerosis index (AI) press with
Lower formula calculates LDL-C=TC- (HDL-C)-TG/2.2；AI=LDL-C/HDL-C.
2. liver biochemical indexes are analyzed
After the cold physiological saline rinsing of liver organization block, filter paper is blotted, and weighing by weight/volume ratio adds 9 times of physiology
Salt water, tissue homogenizer are ground into homogenate, and 2500r/min is centrifuged 10min, take supernatant for 10% hepatic homogenate, at -20 DEG C
It saves to be measured.According to TC, TG, NO, MDA, SOD content in kit specification measurement tissue homogenate.
The variation of weight before and after 9 mouse test of table
Note: a indicates that p < 0.05, aa indicate p < 0.01
The variation of mouse weight is as shown in table 9 before and after this experiment.When initial random grouping, each group mouse weight does not have difference
(p > 0.05).After 12 weeks feeding different feeds, drug treatment, the weight of five groups of mouse has extremely significant compared with when initial
Increase (p < 0.01), and high in fat group of mouse weight increase is more obvious.Compared with high in fat group, stomach-filling safflower petal polysaccharide is molten
Each experimental group weight of liquid is slightly different the decline of degree, but without significant difference (p > 0.05).
The variation of table 10 mouse food ration, amount of drinking water high in fat
Note: a indicates that p < 0.05, aa indicate p < 0.01
Food weight and water weight are weighed during administration weekly, detects to obtain each group mouse food ration and amount of drinking water such as table 10.Normal group
Mouse is slightly above each group of remaining feeding high lipid food because of feeding chow diet food ration, but there was no significant difference (p > 0.05).
Compared with normal group, remaining each group amount of drinking water is by (p < 0.05) is decreased obviously, wherein low dose group amount of drinking water and normally group phase
Than there is extremely significant decline (p < 0.01), but without significant difference between feeding high lipid food each group.Compare food ration and weight gain
Amount, high in fat group fewer than normal group food intake dose, and body weight evolution is more.3 safflower administration group food intake doses with high in fat group
Than being not much different, but weight is obviously reduced.
Influence of the safflower petal polysaccharide to hyperlipemia in mice organ coefficient is as shown in Fig. 3~8.Compared with normal group,
The internal organs such as the heart, spleen, liver, thymus gland do not have significant changes (p > 0.05), and Spleen coefficient standard deviation is bigger, it may be possible to due to a
Caused by body difference；High in fat group of kidney, which has, obviously dramatically increases (p < 0.05)；High in fat group and each safflower administration group fat pad system
Number dramatically increases (respectively p < 0.05, p < 0.01, p < 0.01, p < 0.01).It is aobvious to feed high lipid food group fat pad coefficient
It writes and is higher than chow diet group, illustrate that modeling success, high fat diet increase hoarding for mouse body fat.
Compared with high in fat group, the internal organs such as the heart, spleen, kidney, fat pad do not have significant changes (p > 0.05)；Each safflower administration
Group liver coefficient dramatically increases (respectively p < 0.05, p < 0.01, p < 0.01)；High dose group thymus gland coefficient is significant in safflower
Increase (respectively p < 0.05, p < 0.01).High in fat group of liver, thymus gland coefficient be less than normal group, high fat diet do not cause liver,
Thymus gland enlargement increases to a certain degree though each administration group liver coefficient of safflower and thymus gland coefficient have, not significant with normal group
Difference.
Influence such as Fig. 9~11 of the safflower petal polysaccharide to hyperlipemia in mice serum glucose, high in fat group of serum TC, TG
It is shown；Compared with normal group, high in fat group of serum glucose (blood glucose) has extremely significant increase (p < 0.01), each administration group of safflower
Serum glucose dramatically increases (p < 0.05)；With high in fat group of ratio, each administration group blood glucose of safranine has decreasing trend, but without aobvious
It writes sex differernce (p > 0.05).
Compared with normal group, high in fat group of serum TC, TG have significant raising (respectively p < 0.01, p < 0.05), explanation
Long-time high fat diet causes blood lipid raising, hyperlipemia in mice modeling success.Compared with high in fat group, each administration group of safflower
TC, TG level have solution reduction, wherein low dose group safflower petal polysaccharide can significantly reduce TC, TG level (p <
0.05), high dose group, which can reduce, significantly reduces TG level (p < 0.05).
Table 11 mice serum HDL-C, LDL-C and AI high in fat are horizontal
Note: p < 0.01 a p < 0.05, aa compared with normal group；P < 0.01 b p < 0.05 compared with high in fat, bb；N=10.
After testing 12 weeks, compared with normal group, the horizontal extremely significant raising (p < 0.01) of high in fat group of LDL-C；But HDL-C water
Flat also significant to increase, this may be (p < 0.01) is significantly increased due to its TC concentration caused by, therefore calculate HDL-C and TC ratio, height
The HDL-C/TC ratio of rouge group declines, but without statistical discrepancy compared with normal group.Compared with normal group, extremely significant raising (the p < of AI
0.01), feeding high lipid food can dramatically increase the disease incidence of atherosclerosis.
Compared with high in fat group, 3 dosage group LDL-C, LDL-C/TC levels have decline after giving drug, and low dosage can
It is horizontal (p < 0.05) to significantly reduce LDL-C, LDL-C/TC.HDL-C/TC level has raising trend, but no statistical significance.With height
Rouge group ratio, each administration group AI has different degrees of reduction, and low dose group AI value has significant difference (p < 0.05).
Compared with high in fat group, low dose group safflower petal polysaccharide can significantly reduce TC, TG level, and high dose group can reduce
It is horizontal to significantly reduce TG；3 dosage group LDL-C, LDL-C/TC levels have decline after giving drug, and low dosage can significantly drop
Low LDL-C, LDL-C/TC are horizontal.HDL-C/TC level has raising trend, but no statistical significance；Each administration group AI has different journeys
Degree reduces, and low dose group AI value has significant difference.In addition, safflower petal polysaccharide can reduce liver to a certain extent
Middle TC, TG content.This shows that safflower petal total starches can reduce blood lipid level, effectively prevents height caused by long term high-fat diet
Pionemia.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the extracting method of safflower petal total starches, comprising the following steps:
1) filter residue after safflower petal powder alcohol extracting is air-dried, obtains air-drying filter residue and water with 1g:(15~25) ratio of ml
Mixing is separated by solid-liquid separation after being placed in 95~105 DEG C of 2.5~3.5h of extraction, collects liquid phase component, obtains safflower petal Aqueous extracts；
2) by the safflower petal Aqueous extracts alcohol precipitation, solid phase components is collected and obtain safflower petal water extract solid powder；
3) the safflower petal water extract solid powder is redissolved, after dialysis, is purified with anion exchange chromatography, gel column
Chromatographic purifying obtains safflower petal total starches.
2. extracting method according to claim 1, which is characterized in that the ratio of the filter residue and water is 1g:20ml, described
The temperature of extraction is 98~102 DEG C, and the time of the extraction is 2.7~3.3h.
3. extracting method according to claim 1, which is characterized in that the alcohol precipitation is by ethyl alcohol and the safflower petal
Aqueous extracts mixing, until the volumetric concentration of ethyl alcohol is 85~95% in mixed liquor.
4. extracting method according to claim 1, which is characterized in that the molecular cut off of the dialysis be 2500~
3500D；The total time of the dialysis is 42~54h, and it is primary that every 6~8h changes water.
5. extracting method according to claim 1, which is characterized in that the anion-exchange column is DEAE-Sephadex
A-50 column.
6. extracting method according to claim 1 or 5, which is characterized in that the loading of the anion exchange chromatography is dense
Degree is 30~40mg/ml；The anion exchange chromatography uses the NaCl solution gradient elution of 0.05~0.6mol/L, described
Elution speed is 0.8~1.2ml/min.
7. extracting method according to claim 6, which is characterized in that the gel filtration chromatography uses Sephadex G-
200, the eluent of the gel filtration chromatography is water, and elution flow rate is 0.08~0.12ml/min.
8. extracting method described in claim 1~7 any one extracts the safflower petal total starches obtained.
9. safflower petal total starches according to any one of claims 8 are preparing the application in drug, health care product and cosmetics.
10. application according to claim 9, which is characterized in that the drug includes the drug of anti-curing hyperlipemia.
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| CN112552420A (en) * | 2020-11-05 | 2021-03-26 | 浙江大学 | Polysaccharide with immunologic adjuvant effect, and preparation method and application thereof |
| CN113304206A (en) * | 2021-05-25 | 2021-08-27 | 上海中医药大学附属岳阳中西医结合医院崇明分院(上海市崇明区第三人民医院) | Refined alcohol extract of saffron petals and preparation method and application thereof |
| CN114751995A (en) * | 2022-02-28 | 2022-07-15 | 浙江中医药大学 | Stigma croci Sativi petal polysaccharide CSP1 with anti-inflammatory effect, and its preparation method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113304206A (en) * | 2021-05-25 | 2021-08-27 | 上海中医药大学附属岳阳中西医结合医院崇明分院(上海市崇明区第三人民医院) | Refined alcohol extract of saffron petals and preparation method and application thereof |
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| EP4374867A1 (en) | 2022-11-22 | 2024-05-29 | Lietuvos sveikatos mokslu universitetas | Water soluble polysacharide extraction method from saffron corms and its application |
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