CN104829738A - Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes - Google Patents
Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes Download PDFInfo
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- CN104829738A CN104829738A CN201510237905.5A CN201510237905A CN104829738A CN 104829738 A CN104829738 A CN 104829738A CN 201510237905 A CN201510237905 A CN 201510237905A CN 104829738 A CN104829738 A CN 104829738A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to an application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes. Concretely, the invention provides a sargassum graminifolium polysaccharide extract. A preparation method of the sargassum graminifolium polysaccharide extract comprises the following steps: drying, and crushing, so that sargassum graminifolium dry powder is obtained; carrying out ethanol reflux extraction, so that dry and defatted sargassum graminifolium powder is obtained; extracting the dry and defatted sargassum graminifolium powder by utilizing microwave and taking water as a solvent; separating and purifying extracting solution with a polyethyleneglycol/inorganic salt or ethanol/inorganic salt aqueous two-phase system; and collecting in inorganic salt bottom phase, filtering and concentrating by adopting an ultrafiltration membrane, and drying, so that the sargassum graminifolium polysaccharide extract is obtained. The sargassum graminifolium polysaccharide extract has high purity and good biological activity, and animal experiments verify that the intestinal flora can be obviously improved, and immunity of mouse can be obviously improved, so that the sargassum graminifolium polysaccharide extract can be used for preparing a medicine, health product or food which can be used for improving the intestinal flora and preventing and treating metabolic diseases such as diabetes.
Description
Technical field
The present invention relates to sargassan extract, specifically, relate to a kind of new blade of grass sargassan extract, and prevent and treat application in the metabolic diseases such as diabetes improving intestinal microflora.
Background technology
Sargassun (Sargassum) is phaeophyta siliquosa Pelvetia order Sargassaceae plant.Blade of grass sargassun (Sargassum.graminifolium.turn) belongs to the one of Sargassum, be grown on the cay of low tide band and subtidal zone, frond green or brown, height about 50 centimetres, be distributed in Zhoushan Of Zhejiang Province, Xiangshan, Fujian, Guangdong and Macao in China coastal, resource is very abundant.
Sargassun is the same also containing various bioactivators such as abundant polysaccharide, brown algae polyphenols, gamma-linolenic acid, amino acid and trace elements with the marine alga of other classes; Wherein, sargassan is that the one extracted from sargassun is soluble in water, the more much higher saccharide complex of viscosity, receives much concern especially because of its higher pharmaceutical use.Sargassum polysaccharides is a class polysaccharide specific to marine alga, has biological activity widely, comprises anti-freezing, anti-oxidant, antiviral, antitumor and immunoregulatory activity.But the domestic and international research majority to sargassan concentrates on extraction and isolation, structural analysis and antitumor, antianaphylaxis and prevents and treats urinary stone disease aspect at present.
In recent years, increasing focus of attention is to the importance of intestinal microflora to human health.The mankind and microorganism common evolutionary millions of year, human intestinal forms a large amount of normal microflora in the process, these floras form the interactional physiological entity with host by specific mode in enteron aisle, play a series of physiological actions such as nutrition, immunity, metabolism.Some disease of the mankind causes because microorganism is unbalance, instead of due to the existence of certain pathogenic microorganism, developing of the diseases such as such as anaphylactic disease, obesity, inflammatory bowel.Flora imbalance can cause superinfection or superinfection.Being more common in of flora imbalance uses microbiotic and chronic wasting disease etc.
Present stage, the relative disease Prevention and Curation such as the obesity cause high fat height sugar and diabetes there is no very good method, and diabetes prevalence remains high, and have become the Social Events threatening health of people.In recent years large quantity research shows, the quantity of the Bacterial community in diabetic subject's enteron aisle, flora ratio and flora all has different with normal people, intestinal microflora imbalance may cause intracellular toxin to enter blood, bring out chronic inflammatory diseases, thus destroy insulin receptor, injured blood vessel, interference lipid metabolism process, finally to cause fat, the metabolic disease such as diabetes.Be that target spot research metabolic disease has become the important research field of prevention and treatment of chronic diseases with intestinal microflora, but have at present the medicine of precise effects and natural product actually rare.
At present about the adjustable intestinal microflora of sargassan, the purposes of preventing and treating metabolic disease (as diabetes, obesity etc.) aspect further have not been reported.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of blade of grass sargassan extract is provided and is improving the application in intestinal microflora control metabolic disease.
The invention provides a kind of blade of grass sargassan extract, it prepares by the following method:
A (), by raw material blade of grass sargassun crushed after being dried, is crossed 10-40 mesh sieve and is obtained blade of grass sargassun dry powder;
B () adds refluxing extraction 2-4 hour after the ethanol of volume fraction 70%-90% in blade of grass sargassun dry powder, material liquid volume is than being 1:5-10, and backflow terminates rear removing ethanol, dries, obtains the blade of grass sargassun powder of drying defatted;
C () selects power to be that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 500-1000 watt, material liquid volume is than being 1:10-30, and each microwave treatment time is 15-30 minute, altogether microwave treatment 1-4 time;
D () merges all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol/inorganic salt or ethanol/inorganic salt aqueous two-phase system separation and purification, is placed in constant temperature oscillator and vibrates at 40-50 DEG C 25-35 minute, leaves standstill; Described polyoxyethylene glycol or the mass concentration of ethanol are 10%-40%, and described inorganic salt are selected from ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate or sodium-chlor, and mass concentration is 10%-25%;
Phase under (e) collection inorganic salt, employing ultrafiltration membrance filter concentrates, described ultra-filtration membrane operating pressure is 0.1-0.6MPa, the molecular weight cut-off of ultra-filtration membrane is 3000-20000Da, ultrafiltration time 20-40min, ultrafiltrate temperature 30-40 DEG C, the trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains described blade of grass sargassan extract.
Preferably, the aqueous two-phase system described in step (d) is polyoxyethylene glycol/inorganic salt, and described inorganic salt are ammonium sulfate.
More preferably, described molecular weight polyethylene glycol is 6000, and mass concentration is 10%-20%; Described Inorganic salt is 20%-25%.
Preferably, in step (d), temperature is 50 DEG C.
Preferably, in described blade of grass sargassan extract, sulfate radical content is 19.6%-25.6%.
Present invention also offers described blade of grass sargassan extract and prepare the application in medicine, healthcare products or the food improving intestinal microflora.
As a kind of concrete embodiment, described improvement intestinal microflora refers to be increased intestinal bifidobacteria and lactobacillus quantity and reduces enteron aisle intestinal bacteria and suis quantity.
Present invention also offers described blade of grass sargassan extract and prepare the application in medicine, healthcare products or the food preventing and treating diabetes or obesity.
Present invention also offers described blade of grass sargassan extract and prepare the application in medicine, healthcare products or the food improving immunizing power.
Blade of grass sargassan extract of the present invention can join in the food such as milk-product, bread, noodles such as tap water, functional beverage, drinks, all kinds of seasonings, Yoghourt, improves the sanatory effect of intestinal microflora to give full play to.
When blade of grass sargassan extract of the present invention is for the preparation of medicine, healthcare products or food, can be made into various forms of dosage form, comprise tablet, capsule, granule or oral liquid and can pre-conceivable nanometer formulation and targeting drug administration preparation etc.Described preparation is preferably that the blade of grass sargassan extract of 1%-99% and the pharmaceutical excipient of 99%-1% or additives form by weight percent.
The invention has the advantages that: the invention provides a kind of blade of grass sargassan extract, its purity is high, biological activity good, confirm that this blade of grass sargassan extract significantly can improve intestinal microflora through experimentation on animals, and significantly improve the immunizing power of mouse, therefore can be used for preparing the medicine, healthcare products or the food that improve intestinal microflora, for preventing and treating the metabolic disease such as diabetes, obesity.
Embodiment
Below embodiment provided by the invention is elaborated.
The preparation (one) of embodiment 1 blade of grass sargassan of the present invention extract
1, method steps
The preparation of 1.1 blade of grass sargassan extracts
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 30 DEG C of dryings 20 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 20 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 3 hours after the ethanol of volume fraction 80%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:8.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 30 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 800 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:20, each microwave treatment time is 20 minutes, altogether microwave treatment 3 times.
(4) merge all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, and mass concentration is 10%; Inorganic salt are ammonium sulfate, and mass concentration is 20%.Be placed in constant temperature oscillator to vibrate 30 minutes at 50 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.15MPa, and the molecular weight cut-off of film is 10000Da, ultrafiltration time 30min, ultrafiltrate temperature 35 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.The mensuration of polysaccharide content, polysaccharide extract rate in 1.2 Crude polysaccharides yield, crude product
(1) collect the microwave extraction liquid of above-mentioned steps (3), be condensed into medicinal extract, dry and obtain Crude polysaccharides.Crude polysaccharides yield %=(the total Crude polysaccharides quality/raw materials quality of gained) × 100%.
(2) phenol-sulfuric acid and colorimetric method is adopted to measure the content of polysaccharide in crude product.With glucose as a standard product, accurately take standard glucose 20mg in 500ml volumetric flask, add water to scale, draw 0 respectively, 0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6ml solution is in scale test tube, again each with distilled water mend to 2.0ml, then 6% phenol 1.0ml and vitriol oil 5.0ml is added, shake up cooling, room temperature is placed after 20 minutes and is measured absorbance in 490nm, X-coordinate is glucose concn μ g/ml, ordinate zou is absorbance, and drawing standard curve, obtains equation of linear regression.Crude polysaccharides 2mg is got under similarity condition, be settled in 50ml volumetric flask, draw in 1.0ml liquid to be measured to scale test tube and use distilled water polishing to 2.0ml, then add 6% phenol 1.0ml and vitriol oil 5.0ml, shake up cooling, room temperature is placed after 20 minutes and is measured absorbance in 490nm.The concentration C of liquid to be measured is calculated according to typical curve before.Polysaccharide content %=[Crude polysaccharides quality is got by (C × 1000 × 50)/institute] × 100% in crude product.
(3) polysaccharide extract rate %=polysaccharide quality/raw materials quality=(in crude product the total Crude polysaccharides quality of polysaccharide content % × gained)/raw materials quality.
The mensuration of 1.3 blade of grass sargassan purity
Purity=(polysaccharide content in blade of grass sargassan extract/blade of grass sargassan extract weight) × 100% of blade of grass sargassan.
The mensuration of sulfate radical content in 1.4 blade of grass sargassan extracts
Barium sulfate turbidimetry is adopted to measure sulfate content.
(1) experimental principle
Polysaccharide, by hydrochloric acid hydrolysis, discharges sulfate.Sulfate and bariumchloride generate barium sulfate.360nm place measure its absorbancy, sulfate content and its absorbancy linear.General potassium sulfate, as standard substance, calculates the content of sulfate.
(2) configuration of experiment reagent
1. the preparation of potassium sulfate standardized solution: accurately take the potassium sulfate 200mg being dried to constant weight through 105 DEG C, be settled to 200mL with the hydrochloric acid soln of lmoL/L, obtain sulfate standard reserving solution.
2. the configuration of 1moL/L HCl: 36%-38% concentrated hydrochloric acid 83.333mL is settled to 1L volumetric flask.
3. 0.5% gelatin: 1.25g gelatin is dissolved in 200mL water at 60-70 DEG C, is settled to 250mL volumetric flask after cooling.
4. l% bariumchloride-gelatin: 1g bariumchloride is dissolved in 100mL gelatin solution.
5. trichoroacetic acid(TCA): 3g trichoroacetic acid(TCA) is dissolved in 100mL distilled water.
(3) making of typical curve
Accurate absorption 0,0.04,0.08,0.12,0.16,0.2mL standard sulfate solution mends to 0.2mL in test tube with hydrochloric acid soln, using 0.2mL HCl solution as blank, add solution of trichloroacetic acid 3.8mL and bariumchloride-gelatin solution 1.0mL, shake up, room temperature leaves standstill 15min, survey optical density A1 in 360nm, separately replace bariumchloride-gelatin solution by same standardized solution series with gelatin test solution, obtain A2 using 0.2mL HCl solution as blank.With sulfate milligram number for X-coordinate, ordinate zou is that optical density (A1-A2) makes typical curve.
(4) process of blade of grass sargassan extract and mensuration
Precision took blade of grass sargassan extract 5mg and puts into the test tube filling 3mL 1mol/L HCl, sealing, in 100 DEG C of hydrolysis 6 hours.Get hydrolyzed solution 0.1mL and put into test tubes, add cumulative volume to 0.2mL with 1mol/L HCl solution, repeat operation during production standard curve, measure its light absorption value at 360nm place.In triplicate, its mean value is got.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 15.30%,
41.08％、6.285％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 91.53%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 25.6%.
The preparation (two) of embodiment 2 blade of grass sargassan of the present invention extract
1, method steps
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 20 DEG C of dryings 24 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 40 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 2 hours after the ethanol of volume fraction 90%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:10.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 20 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 500 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:10, each microwave treatment time is 30 minutes, altogether microwave treatment 1 time.
(4) merge all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, and mass concentration is 20%; Inorganic salt are sodium sulfate, and mass concentration is 25%.Be placed in constant temperature oscillator to vibrate 35 minutes at 40 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.6MPa, and the molecular weight cut-off of film is 20000Da, ultrafiltration time 20min, ultrafiltrate temperature 40 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.
Polysaccharide content, polysaccharide extract rate, blade of grass sargassan purity in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1, and sulfate radical content in blade of grass sargassan extract.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 13.36%,
38.24％、5.109％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 89.63%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 24.8%.
The preparation (three) of embodiment 3 blade of grass sargassan of the present invention extract
1, method steps
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 40 DEG C of dryings 12 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 10 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 4 hours after the ethanol of volume fraction 70%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:5.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 40 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 1000 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:30, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solutions, filter, filtrate utilizes ethanol/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, and in two phase aqueous extraction system, ethanol mass concentration is 20%; Inorganic salt are dipotassium hydrogen phosphate, and mass concentration is 10%.Be placed in constant temperature oscillator to vibrate 25 minutes at 45 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.1MPa, and the molecular weight cut-off of film is 3000Da, ultrafiltration time 40min, ultrafiltrate temperature 20 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.
Polysaccharide content, polysaccharide extract rate, blade of grass sargassan purity in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1, and sulfate radical content in blade of grass sargassan extract.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 12.42%,
35.07％、4.356％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 79.65%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 20.3%.
The preparation (four) of embodiment 4 blade of grass sargassan of the present invention extract
1, method steps
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 30 DEG C of dryings 12 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 20 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 3 hours after the ethanol of volume fraction 80%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:8.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 30 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 800 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:20, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, and in two phase aqueous extraction system, molecular weight polyethylene glycol is 6000, and mass concentration is 10%; Inorganic salt are sodium-chlor, and mass concentration is 20%.Be placed in constant temperature oscillator to vibrate 30 minutes at 40 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.15MPa, and the molecular weight cut-off of film is 10000Da, ultrafiltration time 20min, ultrafiltrate temperature 30 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.
Polysaccharide content, polysaccharide extract rate, blade of grass sargassan purity in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1, and sulfate radical content in blade of grass sargassan extract.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 12.28%,
35.14％、4.315％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 75.85%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 19.6%.
The preparation (five) of embodiment 5 blade of grass sargassan of the present invention extract
1, method steps
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 40 DEG C of dryings 20 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 40 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 2 hours after the ethanol of volume fraction 80%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:10.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 40 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 500 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:20, each microwave treatment time is 30 minutes, altogether microwave treatment 2 times.
(4) merge all extracting solutions, filter, filtrate utilizes ethanol/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, and in two phase aqueous extraction system, ethanol mass concentration is 40%; Inorganic salt are sodium sulfate, and mass concentration is 20%.Be placed in constant temperature oscillator to vibrate 30 minutes at 40 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.15MPa, and the molecular weight cut-off of film is 10000Da, ultrafiltration time 20min, ultrafiltrate temperature 30 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.
Polysaccharide content, polysaccharide extract rate, blade of grass sargassan purity in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1, and sulfate radical content in blade of grass sargassan extract.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 13.59%,
38.83％、5.277％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 75.19%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 20.7%.
Comparative example 1
(1) raw material blade of grass sargassun removed impurity and wash, clean blade of grass sargassun is put into drying baker, 30 DEG C of dryings 20 are little of constant weight, then dried blade of grass sargassun is obtained powder in crusher for crushing, cross 20 mesh sieves and obtain blade of grass sargassun dry powder, freezing is for subsequent use.
(2) in blade of grass sargassun dry powder, add refluxing extraction 3 hours after the ethanol of volume fraction 80%, solid-liquid ratio (blade of grass sargassun dry powder volume: ethanol contend) is 1:8.Backflow terminates rear elimination ethanol, is then positioned in stink cupboard by blade of grass sargassun powder and ethanol is volatilized completely, and finally that blade of grass sargassun powder is dry in drying baker, drying temperature is 30 DEG C, is dried to constant weight, for subsequent use.
(3) the blade of grass sargassun powder microwave radiation exaraction of drying defatted is got: selected frequency is 2450MHz, power is that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 800 watts, solid-liquid ratio (the blade of grass sargassun powder volume of drying defatted: volume of water) is 1:15, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, and mass concentration is 10%; Inorganic salt are zinc sulfate, and mass concentration is 25%.Be placed in constant temperature oscillator to vibrate 30 minutes at 50 DEG C.Be divided into two-phase after leaving standstill, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt obtained, adopt ultrafiltration membrance filter to concentrate, ultra-filtration membrane operating pressure is 0.15MPa, and the molecular weight cut-off of film is 10000Da, ultrafiltration time 30min, ultrafiltrate temperature 35 DEG C.Trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains blade of grass sargassan extract.
Polysaccharide content, polysaccharide extract rate, blade of grass sargassan purity in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1, and sulfate radical content in blade of grass sargassan extract.
2, result
Polysaccharide content, polysaccharide extract rate in 2.1 Crude polysaccharides yield, crude product
Polysaccharide content in Crude polysaccharides yield, crude product, polysaccharide extract rate measurement result be respectively 14.04%,
39.16％、5.498％。
2.2 blade of grass sargassan purity
The measurement result of blade of grass sargassan purity is 72.50%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 12.8%.
Embodiment 6 blade of grass sargassan of the present invention extract improves intestinal microflora experiment
1, method
The foundation of 1.1 mouse intestinal flora imbalance models and administration
The male c57BL/6J mouse of cleaning grade, 8 weeks ages of mouse, body weight 18-20g.
Modeling method: after mouse adaptability raises one week, feeding high lipid food.The formula of high lipid food reference literature report: normal diet 60%, lard 12%, sucrose 5%, milk powder 5%, peanut 5%, egg 10%, sesame oil 2%, salt 1%.During modeling, all mouse all freely ingest, drink water, and feed and water change 1 time every day, weigh Mouse Weight 1 time weekly.All mouse continue nursing 8 weeks.Rejecting body weight is the animal more than 10% lower than 30g and the gavage body weight range of decrease.
Successful for induction obese model mouse is divided into model control group and embodiment 1 extract high, medium and low dosed administration group and the high, medium and low dosed administration group of comparative example 1 extract at random by body weight and blood sugar.Then model control group mouse gives oral distilled water, other groups give corresponding blade of grass sargassan extract, once a day, each according to table 1 administration, continuous 90 days, wherein, for the embodiment 1 extract high, medium and low dosed administration group in table 1 and the high, medium and low dosed administration group of comparative example 1 extract, described dosage refers to blade of grass sargassan dosage pure in extract.After last administration, put to death mouse, get its rectum section content and carry out microbial culture.
Table 1 dosage and time
1.2 intestinal microfloras detect
Every cage gets about 0.2g mouse fresh excreta with aseptic procedure, is placed in sterile centrifugation tube, adds appropriate physiological saline by 1:10, vortex 15min, make it homogenize, and then carries out 10 times of serial dilutions to 10
-1-10
-7.Adopt suitable extent of dilution according to the substratum selected, drip kind with liquid getting device, carry out aerobic and Anaerobic culturel respectively.Intestinal bacteria: adopt coliform EMB substratum, 37 DEG C, the aerobic cultivation of 24h; Bifidus bacillus: adopt the Medium of Bifidobacterium BBL that Qingdao Hai Bo Bioisystech Co., Ltd provides, 37 DEG C, 72h Anaerobic culturel; Suis: adopt suis selective medium (TATAC), 37 DEG C, the aerobic or amphimicrobian of 24-72h is cultivated; Lactobacillus: adopt MRS substratum, 37 DEG C, 72h Anaerobic culturel.Finally count the colony number of each ware.
Bacterial count (CFU/g)=colony number × extension rate (10 of every gram of ight soil
nheavy (the g) × saline volume (ml) of) × 20+ sample.Result represents with the logarithmic value log CFU/g of the bacteria colony count in every gram of ight soil.
In this experiment, within every two weeks, collect mouse fresh excreta, observe pharmacological agent to the impact of intestinal microflora.Lactobacillus and bifidus bacillus as the probiotic bacterium in mouse intestinal, by reducing gut pH or suppressing the field planting of harmful bacteria in enteron aisle and growth by the shielding Competition in space, nutrition.
2, result
The results are shown in Table 2.At experimental session, the intestinal bacteria quantity of each dosage group of blade of grass sargassan extract is lower than model control group, and bifidus bacillus and lactobacillus quantity are higher than model control group.And having dependency with dosage, dosage is higher, and the value changing intestinal microflora is more obvious.These results show, and blade of grass sargassan extract can promote the propagation of probiotics milk-acid bacteria, bifidus bacillus in enteron aisle, and can suppress or reduce the growth of pathogenic bacteria and intestinal bacteria etc., are conducive to the balance and the state of health that keep intestinal environment.From embodiment 1 and comparative example 1 corresponding dosage group comparative result, blade of grass sargassan extract of the present invention is all significantly better than blade of grass sargassan extract prepared by comparative example 1 to the increasing action of bifidus bacillus and lactobacillus quantity and to the reducing effect of intestinal bacteria and suis quantity, shows that its biological activity is better.
Table 2 blade of grass sargassan extract is on the impact of obese model mouse intestinal flora
Note: with model control group ratio, * P < 0.05; With comparative example 1 extract corresponding dosage group ratio, #P < 0.05.Embodiment 7 blade of grass sargassan of the present invention extract is on the impact of immunologic function
1, method
The detection of 1.1 immune organ quality, body weight
The mouse that embodiment 6 is respectively organized after 90 days at successive administration, after mouse claims quality, is put to death and dissects, take out spleen and thymus gland, claim quality after blotting bloodstain with filter paper.Measure the ratio of internal organs/weight.
1.2 lymphocyte transformation experiments
The mouse of each group of embodiment 6 at successive administration after 90 days, asepticly gets spleen, is placed in the little plate filling appropriate aseptic Hank's liquid, makes cell suspension, living cell counting number, be inoculated in 24 well culture plates with RPMI RPMI-1640 adjustment cell concn.Each sample establishes ConA liquid and control wells, puts 5% carbonic acid gas, cultivates 72h for 37 DEG C.Cultivation terminates front 4h, and every hole sucks supernatant liquor 0.7mL gently, adds not containing the former RPMI1640 nutrient solution of calf serum, adds MTT (5mg/mL) simultaneously and measures absorbance in 550nm wavelength.Lymphocytic multiplication capacity deducts with the absorbance adding ConA hole the absorbance not adding ConA hole and represents.
2, result
2.1 immune organ quality, body weight detected result
After medication, each group mouse immune organ weight compares in table 3.Result shows, and blade of grass sargassan extract has the effect increasing index and spleen index, and the effect of blade of grass sargassan extract of the present invention is more remarkable.
Table 3 respectively group mouse immune organ weight compares
| Group | Dosage (mg/kg) | Index and spleen index | Thymus index |
| Model control group | 0 | 3.065±0.052 | 3.215±0.067 |
| Embodiment 1 extract low dose group | 50 | 3.518±0.031 | 3.626±0.303 |
| Dosage group in embodiment 2 extract | 100 | 4.557±0.056＊ | 3.754±0.148 |
| Embodiment 3 extract high dose group | 200 | 5.162±0.027＊＊# | 3.952±0.072 |
| Comparative example 1 extract low dose group | 50 | 3.319±0.032 | 3.521±0.105 |
| Dosage group in comparative example 2 extract | 100 | 3.766±0.041 | 3.598±0.163 |
| Comparative example 3 extract high dose group | 200 | 4.283±0.039＊ | 3.687±0.085 |
Note: with model control group ratio, * * P < 0.01, * P < 0.05; With comparative example 1 extract corresponding dosage group ratio, #P < 0.05.
2.2 lymphocyte transformation experimental results
Lymphocyte transformation experimental result is in table 4.
Table 4 lymphocyte transformation experimental result
| Group | Dosage (mg/kg) | Splenic vein hemodynamics rate (%) |
| Model control group | 0 | 36±2 |
| Embodiment 1 extract low dose group | 50 | 39±3 |
| Dosage group in embodiment 2 extract | 100 | 43±4＊ |
| Embodiment 3 extract high dose group | 200 | 49±3＊＊# |
| Comparative example 1 extract low dose group | 50 | 38±2 |
| Dosage group in comparative example 2 extract | 100 | 39±2 |
| Comparative example 3 extract high dose group | 200 | 42±3＊ |
Note: with model control group ratio, * * P < 0.01, * P < 0.05, with comparative example 1 extract corresponding dosage group ratio, #P < 0.05.
In sum, blade of grass sargassan extract of the present invention can promote the propagation of lactobacillus, bifidus bacillus in diabetic mice enteron aisle, suppress intestinal bacteria and streptococcic growth simultaneously, recover the normal eubiosis of enteron aisle, and obviously can increase index and spleen index and increase cellular immune function.Above result shows, blade of grass sargassan extract tool of the present invention is significantly improved intestinal microflora and improves the effect of immunizing power.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (9)
1. a blade of grass sargassan extract, is characterized in that, it prepares by the following method:
A (), by raw material blade of grass sargassun crushed after being dried, is crossed 10-40 mesh sieve and is obtained blade of grass sargassun dry powder;
B () adds refluxing extraction 2-4 hour after the ethanol of volume fraction 70%-90% in blade of grass sargassun dry powder, material liquid volume is than being 1:5-10, and backflow terminates rear removing ethanol, dries, obtains the blade of grass sargassun powder of drying defatted;
C () selects power to be that solvent extract to the blade of grass sargassun powder of drying defatted with water at the microwave of 500-1000 watt, material liquid volume is than being 1:10-30, and each microwave treatment time is 15-30 minute, altogether microwave treatment 1-4 time;
D () merges all extracting solutions, filter, filtrate utilizes polyoxyethylene glycol/inorganic salt or ethanol/inorganic salt aqueous two-phase system separation and purification, is placed in constant temperature oscillator and vibrates at 40-50 DEG C 25-35 minute, leaves standstill; Described polyoxyethylene glycol or the mass concentration of ethanol are 10%-40%, and described inorganic salt are selected from ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate or sodium-chlor, and mass concentration is 10%-25%;
Phase under (e) collection inorganic salt, employing ultrafiltration membrance filter concentrates, described ultra-filtration membrane operating pressure is 0.1-0.6MPa, the molecular weight cut-off of ultra-filtration membrane is 3000-20000Da, ultrafiltration time 20-40min, ultrafiltrate temperature 30-40 DEG C, the trapped fluid after concentrating ultrafiltration membrance filter carries out lyophilize, obtains described blade of grass sargassan extract.
2. blade of grass sargassan extract according to claim 1, is characterized in that, the aqueous two-phase system described in step (d) is polyoxyethylene glycol/inorganic salt, and described inorganic salt are ammonium sulfate.
3. blade of grass sargassan extract according to claim 2, is characterized in that, described molecular weight polyethylene glycol is 6000, and mass concentration is 10%-20%; Described Inorganic salt is 20%-25%.
4. blade of grass sargassan extract according to claim 1, is characterized in that, in step (d), temperature is 50 DEG C.
5. blade of grass sargassan extract according to claim 1, is characterized in that, in described blade of grass sargassan extract, sulfate radical content is 19.6%-25.6%.
6. the arbitrary described blade of grass sargassan extract of claim 1-5 is preparing the application in medicine, healthcare products or the food improving intestinal microflora.
7. application according to claim 6, is characterized in that, described improvement intestinal microflora refers to be increased intestinal bifidobacteria and lactobacillus quantity and reduce enteron aisle intestinal bacteria and suis quantity.
8. the arbitrary described blade of grass sargassan extract of claim 1-5 is preparing the application in medicine, healthcare products or the food preventing and treating diabetes or obesity.
9. the arbitrary described blade of grass sargassan extract of claim 1-5 is preparing the application in medicine, healthcare products or the food improving immunizing power.
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| CN104829740A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium |
| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
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| CN104829740A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium |
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| CN102793715A (en) * | 2012-08-14 | 2012-11-28 | 华东理工大学 | Sargassum polysaccharide and application of Sargassum polysaccharide in medicine preparation used for treating kidney injury |
| CN104829740A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium |
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| CN105483183B (en) * | 2016-01-07 | 2019-05-10 | 福建农林大学 | A kind of preparation method of sargassum oligosaccharides and its application in hypoglycemic drug |
| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
| CN106071985A (en) * | 2016-06-21 | 2016-11-09 | 锦州医科大学 | A kind of method improving pig flesh flavor and application |
| CN106343076A (en) * | 2016-11-04 | 2017-01-25 | 广东海洋大学 | Processing method of sargassum polysaccharide tea bag |
| CN106509895A (en) * | 2016-11-08 | 2017-03-22 | 全椒先奇医药科技有限公司 | Probiotic health-care product and preparation method thereof |
| CN106892996A (en) * | 2017-04-01 | 2017-06-27 | 林婷 | One kind lifting immunity polysaccharide extract and its manufacture method |
| KR20200046398A (en) * | 2018-10-24 | 2020-05-07 | 목포대학교산학협력단 | Composition for improving intestinal health containing extract of Sargassum thunbergii or Sargassum fusiforme |
| KR102225351B1 (en) | 2018-10-24 | 2021-03-09 | 목포대학교산학협력단 | Composition for improving intestinal health containing extract of Sargassum thunbergii |
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| CN112920024A (en) * | 2021-01-30 | 2021-06-08 | 海南大学 | Sargassum stolonifera phenolic substance and extraction method and application thereof |
| CN112961259A (en) * | 2021-03-16 | 2021-06-15 | 盐城工学院 | Preparation method of taurolimus polysaccharide and application of taurolimus polysaccharide in field of improving intestinal function |
| CN112778435A (en) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | Method for separating and purifying dendrobium officinale polysaccharide by aqueous two-phase extraction |
| CN114957500A (en) * | 2021-12-24 | 2022-08-30 | 济宁医学院 | Selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof |
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