CN104829738B - 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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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, is related to a kind of new blade of grass sargassan extract,
And its application in the metabolic diseases such as diabetes is prevented and treated intestinal microbial population is improved.
Background technology
Alga Sgrgassi Enervess (Sargassum) are Phaeophyta Pelvetia siliquosa Tseng et C. F. Chang mesh Sargassaceae plants.Blade of grass Alga Sgrgassi Enervess
(Sargassum.graminifolium.turn) belong to one kind of Sargassum, be grown on the cay of low tide band and subtidal zone
On, frond green or brown are high about 50 centimetres, are distributed in Zhoushan Of Zhejiang Province, Xiangshan, Fujian, Guangdong and Macao in China coastal, money
Source very abundant.
Alga Sgrgassi Enervess with other classes Sargassum it is the same also containing abundant polysaccharide, brown algae polyphenols, gamma-Linolenic acid, aminoacid and
The various bioactivators such as trace element；Wherein, sargassan be extract from Alga Sgrgassi Enervess it is a kind of soluble in water,
The higher polysaccharides compound of viscosity, even more receives much concern because of its higher medical value.Sargassum polysaccharides are that Sargassum institute is peculiar
A class polysaccharide, with extensive biological activity, including anticoagulant, antioxidation, antiviral, antitumor and immunoregulatory activity.So
And concentrate on extraction separation, structural analyses and antitumor, antiallergic at present both at home and abroad to the research majority of sargassan and prevent
In terms of controlling renal calculuss.
In recent years, importance of the increasing focus of attention to intestinal microbial population to human health.The mankind are with microorganism altogether
With having evolved millions of years, human body intestinal canal forms a large amount of normal floras in the process, and these floras are in intestinal by specific
Mode form the physiological entity interacted with host, play nutrition, immunity, a series of physiological actions such as metabolism.People
Some diseases of class are caused due to microbial imbalances, rather than due to the presence of certain pathogenic microorganism, such as anaphylaxis
The generation development of the diseases such as disease, obesity, inflammatory bowel.Dysbacteriosiss can cause superinfection or superinfection.Flora loses
Tune is more common in using antibiotic and Chronic consumptionss etc..
At this stage, the relevant disease preventions such as fat and diabetes caused by the high sugar of high fat and treatment there is no very good
Method, and diabetes prevalence is high, it has also become threaten the Social Events of health of people.In recent years numerous studies table
Bright, the quantity of Bacterial community, flora ratio and flora in diabeticss intestinal has different with normal person, intestinal microbial population knot
Structure imbalance is likely to result in endotoxin and enters blood, induces chronic inflammatory disease, so as to destroy Insulin receptor INSR, injured blood vessel, interference
Lipid metabolism process, ultimately results in the metabolic diseases such as obesity, diabetes.With intestinal microbial population as target spot research metabolic disease into
For the important research field of prevention and treatment of chronic diseases, but there are the medicine and natural product of precise effects actually rare at present.
At present with regard to sargassan scalable intestinal microbial population, metabolic disease is further prevented and treated (such as diabetes, obesity
Deng) in terms of purposes have not been reported.
The content of the invention
The purpose of the present invention is for deficiency of the prior art, there is provided a kind of blade of grass sargassan extract and its
Improve the application in intestinal microbial population preventing and treating metabolic disease.
The invention provides a kind of blade of grass sargassan extract, it is prepared by the following method and obtains：
A raw material blade of grass Alga Sgrgassi Enervess crushed after being dried is crossed 10-40 mesh sieves and obtains blade of grass Alga Sgrgassi Enervess dry powder by ()；
(b) in blade of grass Alga Sgrgassi Enervess dry powder add volume fraction 70%-90% ethanol after reflux, extract, 2-4 hour, material
Liquid volume ratio is 1:5-10, backflow remove ethanol after terminating, and dry, obtain the blade of grass Alga Sgrgassi Enervess powder of drying defatted；
C the microwave of () from power at 500-1000 watt is carried out by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted
Extract, material liquid volume ratio is 1:10-30, each microwave treatment time are 15-30 minutes, altogether microwave treatment 1-4 time；
D () merges all extracting solution, filter, and filtrate utilizes Polyethylene Glycol/inorganic salt or ethanol/inorganic salt aqueous two-phase system
Isolate and purify, be placed in constant temperature oscillator the vibration 25-35 minutes at 40-50 DEG C, stand；Described Polyethylene Glycol or ethanol
Mass concentration is 10%-40%, and described inorganic salt is selected from ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate or Sodium Chloride, mass concentration
For 10%-25%；
E () collects phase under inorganic salt, concentrated using ultrafiltration membrance filter, and described ultrafilter membrane operating pressure is 0.1-
0.6MPa, the molecular cut off of ultrafilter membrane is 3000-20000Da, ultrafiltration time 20-40min, 30-40 DEG C of ultrafiltrate temperature, to super
Trapped fluid after membrane filtration concentration carries out lyophilization, obtains described blade of grass sargassan extract.
Preferably, the aqueous two-phase system described in step (d) is Polyethylene Glycol/inorganic salt, and described inorganic salt is sulphuric acid
Ammonium.
It is highly preferred that described molecular weight polyethylene glycol is 6000, mass concentration is 10%-20%；Described inorganic salt
Mass concentration 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 improves medicine, the health care of intestinal microbial population in preparation
Application in product or food.
Used as a kind of specific embodiment, described improvement intestinal microbial population refers to increase intestinal bifidobacteria and lactic acid bar
Bacterium number amount simultaneously reduces intestinal escherichia coli and streptococcus quantity.
Present invention also offers described blade of grass sargassan extract prepare prevent and treat diabetes or obesity medicine,
Application in health product or food.
Present invention also offers described blade of grass sargassan extract is preparing medicine, the health product of enhance immunity
Or the application in food.
The present invention blade of grass sargassan extract can be added to drinking water, drinks, drinks, all kinds of flavoring agent,
In the food such as the milk product such as Yoghourt, bread, noodles, improve the sanatory effect of intestinal microbial population to give full play to.
When the blade of grass sargassan extract of the present invention is used to prepare medicine, health product or food, can be made into various
The dosage form of form, including tablet, capsule, granule or oral liquid and it is envisioned that the nanometer formulation that obtains and targeting
Drug-delivery preparation etc..Described preparation is preferably by the blade of grass sargassan extract and 99%- that percentage by weight is 1%-99%
1% pharmaceutic adjuvant or additives composition.
The invention has the advantages that：The invention provides a kind of blade of grass sargassan extract, its purity is high, biological activity
It is good, animal experiment proves that the blade of grass sargassan extract can significantly improve intestinal microbial population, and significantly improve mice
Immunity, therefore can be used to prepare the medicine for improving intestinal microbial population, health product or food, for preventing and treating diabetes, obesity etc.
Metabolic disease.
Specific embodiment
Below the specific embodiment that the present invention is provided is elaborated.
The preparation (one) of the blade of grass sargassan extract of 1 present invention of embodiment
1st, method and step
The preparation of 1.1 blade of grass sargassan extracts
(1) raw material blade of grass Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 30 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 20 hours, cross 20 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 3 hours or so after the ethanol of volume fraction 80%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:8.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 30 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
800 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (blade of grass of drying defatted
Alga Sgrgassi Enervess powder volume:Water volume) for 1:20, each microwave treatment time is 20 minutes, altogether microwave treatment 3 times.
(4) merge all extracting solution, filter, filtrate is isolated and purified using Polyethylene Glycol (PEG)/inorganic salt aqueous two-phase system
Blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, mass concentration are 10%；Inorganic salt is sulphuric acid
Ammonium, mass concentration are 20%.It is placed in constant temperature oscillator and vibrates 30 minutes at 50 DEG C.It is divided into biphase after standing, blade of grass horse hair
Polysaccharides enter phase under inorganic salt.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.15MPa, film cut
Molecular weight is stayed for 10000Da, ultrafiltration time 30min, 35 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.Polyoses content, the measure of polysaccharide extract rate in 1.2 crude polysaccharides yield, crude product
(1) the microwave extraction liquid of above-mentioned steps (3) is collected, extractum, drying crude polysaccharides are condensed into.Crude polysaccharides yield %
=(the total crude polysaccharides quality/raw materials quality of gained) × 100%.
(2) content of polysaccharide in crude product is determined using phenol-sulfuric acid and colorimetric method.With glucose as a standard product, accurately weigh
Standard glucose 20mg adds water to scale in 500ml volumetric flasks, respectively draw 0,0.2,0.4,0.6,0.8,1.0,1.2,
1.4th, 1.6ml solution is mended to 2.0ml with distilled water into scale test tube, then respectively, is subsequently adding 6% phenol 1.0ml and concentrated sulphuric acid
5.0ml, shakes up cooling, and room temperature places 20 minutes after 490nm mensuration absorbance values, and abscissa is concentration of glucose μ g/ml, is indulged
Coordinate is absorbance, draws standard curve, obtains equation of linear regression.Crude polysaccharides 2mg is taken under similarity condition, 50ml is settled to
In volumetric flask, draw 1.0ml prepare liquids into scale test tube, use distilled water polishing to 2.0ml, be subsequently adding 6% phenol 1.0ml
And concentrated sulphuric acid 5.0ml, cooling is shaken up, room temperature places 20 minutes after 490nm mensuration absorbance values.According to standard curve before
Calculate the concentration C of prepare liquid.Polyoses content %=[(C × 1000 × 50)/taken crude polysaccharides quality] × 100% in crude product.
(3) polysaccharide extract rate %=polysaccharide quality/raw materials quality=(total crude polysaccharides matter of polyoses content % × gained in crude product
Amount)/raw materials quality.
The measure of 1.3 blade of grass sargassan purity
The purity of blade of grass sargassan=(polyoses content/blade of grass sargassan in blade of grass sargassan extract
Extract weight) × 100%.
The measure of sulfate radical content in 1.4 blade of grass sargassan extracts
Sulfate content is determined using barium sulfate turbidimetry.
(1) experimental principle
Polysaccharide is hydrolyzed by hydrochloric acid, discharges sulfate.Sulfate generates barium sulfate with barium chloride.Which is determined at 360nm
Absorbance, sulfate content are linear with its absorbance.Typically the containing of sulfate is calculated as reference material with potassium sulfate
Amount.
(2) configuration of experiment reagent
1. the preparation of potassium sulfate standard solution：The potassium sulfate 200mg of 105 DEG C of dryings of Jing to constant weight is weighed accurately, with lmoL/
The hydrochloric acid solution of L is settled to 200mL, obtains final product sulfate standard reserving solution.
2. the configuration of 1moL/L HCl：36%-38% concentrated hydrochloric acid 83.333mL are settled to 1L volumetric flasks.
3. 0.5% gelatin：1.25g gelatin is dissolved at 60-70 DEG C in 200mL water, and 250mL capacity is settled to after cooling
Bottle.
4. l% barium chlorides-gelatin：1g barium chlorides are dissolved in 100mL gelatin solutions.
5. trichloroacetic acid：3g trichloroacetic acids are dissolved in 100mL distilled water.
(3) making of standard curve
It is accurate draw 0,0.04,0.08,0.12,0.16,0.2mL standard sulphuric acid based sols in test tube with hydrochloric acid solution mend to
0.2mL, using 0.2mL HCl solutions as blank, adds solution of trichloroacetic acid 3.8mL and barium chloride-gelatin solution 1.0mL, shakes
It is even, be stored at room temperature 15min, in 360nm survey trap A1, separately with same standard liquid series with gelatin test solution replace barium chloride-
Gelatin solution, obtains A2 as blank using 0.2mL HCl solutions.With sulfate milligram number as abscissa, vertical coordinate is absorption
Degree (A1-A2) makes standard curve.
(4) process of blade of grass sargassan extract and measure
Precision weighs blade of grass sargassan extract 5mg and is put in the test tube for filling 3mL 1mol/L HCl, sealing, in
100 DEG C hydrolyze 6 hours.Take hydrolyzed solution 0.1mL to be put in test tubes, cumulative volume is added to 0.2mL with 1mol/L HCl solutions,
Repeat to make operation during standard curve, determine its light absorption value at 360nm.In triplicate, take its meansigma methods.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 15.30%,
41.08%th, 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 the blade of grass sargassan extract of 2 present invention of embodiment
1st, method and step
(1) raw material blade of grass Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 20 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 24 hours, cross 40 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 2 hours or so after the ethanol of volume fraction 90%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:10.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 20 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
500 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (blade of grass of drying defatted
Alga Sgrgassi Enervess powder volume:Water volume) for 1:10, each microwave treatment time is 30 minutes, altogether microwave treatment 1 time.
(4) merge all extracting solution, filter, filtrate is isolated and purified using Polyethylene Glycol (PEG)/inorganic salt aqueous two-phase system
Blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, mass concentration are 20%；Inorganic salt is sulphuric acid
Sodium, mass concentration are 25%.It is placed in constant temperature oscillator and vibrates 35 minutes at 40 DEG C.It is divided into biphase after standing, blade of grass horse hair
Polysaccharides enter phase under inorganic salt.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.6MPa, film cut
Molecular weight is stayed for 20000Da, ultrafiltration time 20min, 40 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.
Method according to embodiment 1 determines polyoses content, polysaccharide extract rate, blade of grass Alga Sgrgassi Enervess in crude polysaccharides yield, crude product
Sulfate radical content in purity of polysaccharide, and blade of grass sargassan extract.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 13.36%,
38.24%th, 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 the blade of grass sargassan extract of 3 present invention of embodiment
1st, method and step
(1) raw material blade of grass Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 40 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 12 hours, cross 10 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 4 hours or so after the ethanol of volume fraction 70%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:5.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 40 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
1000 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (grass of drying defatted
Leaf Alga Sgrgassi Enervess powder volume:Water volume) for 1:30, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solution, filter, filtrate isolates and purifies blade of grass horse hair using ethanol/inorganic salt aqueous two-phase system
Polysaccharides, in two phase aqueous extraction system, ethanol mass concentration is 20%；Inorganic salt is dipotassium hydrogen phosphate, and mass concentration is 10%.
It is placed in constant temperature oscillator and vibrates 25 minutes at 45 DEG C.It is divided into biphase after standing, blade of grass sargassan is entered under inorganic salt
Phase.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.1MPa, film cut
Molecular weight is stayed for 3000Da, ultrafiltration time 40min, 20 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.
Method according to embodiment 1 determines polyoses content, polysaccharide extract rate, blade of grass Alga Sgrgassi Enervess in crude polysaccharides yield, crude product
Sulfate radical content in purity of polysaccharide, and blade of grass sargassan extract.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 12.42%,
35.07%th, 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 the blade of grass sargassan extract of 4 present invention of embodiment
1st, method and step
(1) raw material blade of grass Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 30 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 12 hours, cross 20 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 3 hours or so after the ethanol of volume fraction 80%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:8.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 30 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
800 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (blade of grass of drying defatted
Alga Sgrgassi Enervess powder volume:Water volume) for 1:20, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solution, filter, filtrate is isolated and purified using Polyethylene Glycol (PEG)/inorganic salt aqueous two-phase system
Blade of grass sargassan, in two phase aqueous extraction system, molecular weight polyethylene glycol is 6000, and mass concentration is 10%；Inorganic salt is chlorine
Change sodium, mass concentration is 20%.It is placed in constant temperature oscillator and vibrates 30 minutes at 40 DEG C.It is divided into biphase after standing, blade of grass horse
Tail polysaccharides enter phase under inorganic salt.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.15MPa, film cut
Molecular weight is stayed for 10000Da, ultrafiltration time 20min, 30 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.
Method according to embodiment 1 determines polyoses content, polysaccharide extract rate, blade of grass Alga Sgrgassi Enervess in crude polysaccharides yield, crude product
Sulfate radical content in purity of polysaccharide, and blade of grass sargassan extract.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 12.28%,
35.14%th, 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 the blade of grass sargassan extract of 5 present invention of embodiment
1st, method and step
(1) raw material blade of grass Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 40 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 20 hours, cross 40 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 2 hours or so after the ethanol of volume fraction 80%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:10.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 40 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
500 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (blade of grass of drying defatted
Alga Sgrgassi Enervess powder volume:Water volume) for 1:20, each microwave treatment time is 30 minutes, altogether microwave treatment 2 times.
(4) merge all extracting solution, filter, filtrate isolates and purifies blade of grass horse hair using ethanol/inorganic salt aqueous two-phase system
Polysaccharides, in two phase aqueous extraction system, ethanol mass concentration is 40%；Inorganic salt is sodium sulfate, and mass concentration is 20%.It is placed in
Vibrate 30 minutes at 40 DEG C in constant temperature oscillator.It is divided into biphase after standing, blade of grass sargassan enters phase under inorganic salt.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.15MPa, film cut
Molecular weight is stayed for 10000Da, ultrafiltration time 20min, 30 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.
Method according to embodiment 1 determines polyoses content, polysaccharide extract rate, blade of grass Alga Sgrgassi Enervess in crude polysaccharides yield, crude product
Sulfate radical content in purity of polysaccharide, and blade of grass sargassan extract.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 13.59%,
38.83%th, 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 Alga Sgrgassi Enervess are gone the removal of impurity and are washed, the blade of grass Alga Sgrgassi Enervess cleaned are put in drying baker,
Then 30 DEG C of dryings obtain powder in crusher for crushing dried blade of grass Alga Sgrgassi Enervess to constant weight in 20 hours, cross 20 mesh sieves
Blade of grass Alga Sgrgassi Enervess dry powder is obtained, freezing is standby.
(2) reflux, extract, 3 hours or so after the ethanol of volume fraction 80%, solid-liquid ratio are added in blade of grass Alga Sgrgassi Enervess dry powder
(blade of grass Alga Sgrgassi Enervess dry powder volume:Ethanol volume) for 1:8.Backflow filters off ethanol after terminating, and then puts blade of grass Alga Sgrgassi Enervess powder
Being placed in fume hood makes ethanol volatilize completely, and finally blade of grass Alga Sgrgassi Enervess powder is dried in drying baker, and baking temperature is 30 DEG C,
It is dried to constant weight, it is standby.
(3) take the blade of grass Alga Sgrgassi Enervess powder microwave radiation exaraction of drying defatted：Selected frequency is 2450MHz, power exists
800 watts of microwave is extracted by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted, the solid-liquid ratio (blade of grass of drying defatted
Alga Sgrgassi Enervess powder volume:Water volume) for 1:15, each microwave treatment time is 15 minutes, altogether microwave treatment 4 times.
(4) merge all extracting solution, filter, filtrate is isolated and purified using Polyethylene Glycol (PEG)/inorganic salt aqueous two-phase system
Blade of grass sargassan, molecular weight polyethylene glycol 6000 in two phase aqueous extraction system, mass concentration are 10%；Inorganic salt is sulphuric acid
Zinc, mass concentration are 25%.It is placed in constant temperature oscillator and vibrates 30 minutes at 50 DEG C.It is divided into biphase after standing, blade of grass horse hair
Polysaccharides enter phase under inorganic salt.
(5) phase under the inorganic salt for obtaining, using ultrafiltration membrance filter concentrate, ultrafilter membrane operating pressure be 0.15MPa, film cut
Molecular weight is stayed for 10000Da, ultrafiltration time 30min, 35 DEG C of ultrafiltrate temperature.Trapped fluid after ultrafiltration membrance filter concentration is carried out cold
Lyophilizing is dry, obtains blade of grass sargassan extract.
Method according to embodiment 1 determines polyoses content, polysaccharide extract rate, blade of grass Alga Sgrgassi Enervess in crude polysaccharides yield, crude product
Sulfate radical content in purity of polysaccharide, and blade of grass sargassan extract.
2nd, result
Polyoses content, polysaccharide extract rate in 2.1 crude polysaccharides yield, crude product
In crude polysaccharides yield, crude product polyoses content, polysaccharide extract rate measurement result be respectively 14.04%,
39.16%th, 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%.
The blade of grass sargassan extract of 6 present invention of embodiment improves intestinal microbial population experiment
1st, method
The foundation and administration of 1.1 mouse intestinal floras imbalance model
Cleaning grade male c57BL/6J mices, 8 weeks Mus ages, body weight 18-20g.
Modeling method：After mice adaptability is raised one week, feeding high lipid food.What high lipid food reference literature was reported matches somebody with somebody
Side：Normal diet 60%, Adeps Sus domestica 12%, sucrose 5%, milk powder 5%, Semen arachidis hypogaeae 5%, egg 10%, Oleum Sesami 2%, Sal 1%.Make
During mould, all mices freely ingest, drink water, and feedstuff and water are changed 1 time daily, weigh weekly Mouse Weight 1 time.It is all little
Mus continue to feed 8 weeks.Reject body weight and be less than the animal of 30g and the gavage body weight range of decrease more than 10%.
Induction successful obese model mice is randomly divided into into model control group by body weight and blood glucose and embodiment 1 is extracted
The high, medium and low dosed administration group of thing and the high, medium and low dosed administration group of 1 extract of comparative example.Then model control group mice gives
Oral distilled water, other groups give corresponding blade of grass sargassan extract, once a day, are administered according to table 1 every time, continuously
90 days, wherein, for the high, medium and low dosed administration group of 1 extract of embodiment and high, medium and low dose of 1 extract of comparative example in table 1
Amount administration group, described dosage refer to pure blade of grass sargassan dosage in extract.After last dose, mice is put to death, is taken
Its rectum section content carries out antibacterial culturing.
1 dosage of table and time
1.2 intestinal microbial populations are detected
About 0.2g mice fresh excretas are taken with aseptic procedure per cage, is placed in sterile centrifugation tube, by 1:10 add appropriate life
Then reason saline, vortex 15min so as to homogenize carry out 10 times and are serially diluted to 10-1-10-7.Culture medium according to selecting is adopted
With suitable dilution factor, with liquid getting device drop kind, aerobic and Anaerobic culturel is carried out respectively.Escherichia coli：Trained using coliform EMB
Foster base, 37 DEG C, the aerobic cultures of 24h；Bacillus bifiduss：The bacillus bifiduss culture provided using Qingdao Hai Bo Bioisystech Co., Ltd
Base BBL, 37 DEG C, 72h Anaerobic culturels；Streptococcus：Using streptococcus selective medium (TATAC), 37 DEG C, 24-72h it is aerobic or
Amphimicrobian culture；Lactobacilluss：Using MRS culture medium, 37 DEG C, 72h Anaerobic culturels.The clump count of each ware is counted finally.
Bacterial population (CFU/g)=clump count × extension rate (10 of every gram of fecesn) × 20+ samples weight (g) × physiology salt
The water yield (ml).As a result represented with the logarithm value log CFU/g of the bacteria colony count in every gram of feces.
In this experiment, mice fresh excreta is collected every two weeks, impact of the Drug therapy to intestinal microbial population is observed.Lactobacilluss
With bacillus bifiduss as the probiotic bacteria in mouse intestinal, by reducing gut pH or competing by the shielding in space, nutrition
Strive the field planting and growth for acting on suppressing harmful bacteria in intestinal.
2nd, result
The results are shown in Table 2.During testing, the escherichia coli quantity of each dosage group of blade of grass sargassan extract is less than mould
Type matched group, and bacillus bifiduss and lactobacilluss quantity are higher than model control group.And have dependency with dosage, dosage is higher,
The value for changing intestinal microbial population is more obvious.These results show that blade of grass sargassan extract can promote probioticss in intestinal
The propagation of lactic acid bacteria, bacillus bifiduss, and can suppress or reduce the growth of pathogen and escherichia coli etc., be conducive to keeping in intestinal
The balance and health status of environment.From the point of view of 1 corresponding dosage group comparative result of embodiment 1 and comparative example, the blade of grass horse of the present invention
Tail polysaccharides extract is to the increasing action of bacillus bifiduss and lactobacilluss quantity and to escherichia coli and streptococcus quantity
Reduction effect is all significantly better than the blade of grass sargassan extract of the preparation of comparative example 1, shows that its biological activity is more preferable.
Impact of the 2 blade of grass sargassan extract of table to obese model mouse intestinal flora
Note：With model control group ratio, * P ＜ 0.05；With 1 extract corresponding dosage group ratio of comparative example, #P ＜ 0.05.Implement
Impact of the blade of grass sargassan extract of 7 present invention of example to immunologic function
1st, method
1.1 immune organ quality, the detection of body weight
The mice of 6 each group of embodiment after successive administration 90 days after mice claims quality, is put to death and dissects, take out spleen and breast
Gland, is blotted with filter paper and claim after bloodstain quality.Determine the ratio of internal organs/weight.
1.2 lymphocyte transformations are tested
The mice of 6 each group of embodiment is after successive administration 90 days, aseptic to take spleen, is placed in and fills appropriate aseptic Hank's liquid
In little plate, cell suspension is made, living cell counting number is inoculated in the training of 24 holes with RPMI RPMI-1640s adjustment cell concentration
In foster plate.Each sample sets ConA liquid and control wells, puts 5% carbon dioxide, 37 DEG C of culture 72h.Culture terminates front 4h, light per hole
Supernatant 0.7mL is gently sucked, is added without calf serum original RPMI1640 culture fluid, while MTT (5mg/mL) is added in 550nm
Wavelength mensuration absorbance value.The multiplication capacity of lymphocyte deducts the absorbance for being not added with ConA holes with the absorbance for adding ConA holes
Value is represented.
2nd, result
2.1 immune organ quality, body weight testing result
After medication, each group mouse immune organ weight is relatively shown in Table 3.As a result show, blade of grass sargassan extract has increasing
Plus the effect of index and spleen index, and the effect of the blade of grass sargassan extract of the present invention is more notable.
Table 3 each group mouse immune organ weight compare
| Group | Dosage (mg/kg) | Index and spleen index | Thymus index |
| Model control group | 0 | 3.065±0.052 | 3.215±0.067 |
| 1 extract low dose group of embodiment | 50 | 3.518±0.031 | 3.626±0.303 |
| 2 extract middle dose group of embodiment | 100 | 4.557 ± 0.056* | 3.754±0.148 |
| 3 extract high dose group of embodiment | 200 | 5.162 ± 0.027**# | 3.952±0.072 |
| 1 extract low dose group of comparative example | 50 | 3.319±0.032 | 3.521±0.105 |
| 2 extract middle dose group of comparative example | 100 | 3.766±0.041 | 3.598±0.163 |
| 3 extract high dose group of comparative example | 200 | 4.283 ± 0.039* | 3.687±0.085 |
Note：With model control group ratio, * * P ＜ 0.01, * P ＜ 0.05；With 1 extract corresponding dosage group ratio of comparative example, #
P ＜ 0.05.
2.2 lymphocyte transformations experimental results
Lymphocyte transformations experimental result is shown in Table 4.
4 lymphocyte transformations experimental result of table
| Group | Dosage (mg/kg) | Splenic vein hemodynamics rate (%) |
| Model control group | 0 | 36±2 |
| 1 extract low dose group of embodiment | 50 | 39±3 |
| 2 extract middle dose group of embodiment | 100 | 43 ± 4* |
| 3 extract high dose group of embodiment | 200 | 49 ± 3**# |
| 1 extract low dose group of comparative example | 50 | 38±2 |
| 2 extract middle dose group of comparative example | 100 | 39±2 |
| 3 extract high dose group of comparative example | 200 | 42 ± 3* |
Note：With model control group ratio, * * P ＜ 0.01, * P ＜ 0.05, with 1 extract corresponding dosage group ratio of comparative example, #
P ＜ 0.05.
In sum, blade of grass sargassan extract of the invention can promote lactic acid bar in diabetic mice intestinal
Bacterium, the propagation of bacillus bifiduss, while suppressing escherichia coli and streptococcic growth, recover the normal ecological balance of intestinal, and energy
Substantially increase index and spleen index and increase cellular immune function.Result above shows, the blade of grass sargassan extract of the present invention
Tool is significantly improved the effect of intestinal microbial population and enhance immunity.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of without departing from the inventive method, can also make some improvement and supplement, and these improve and supplement also should be regarded as
Protection scope of the present invention.
Claims (8)
1. a kind of blade of grass sargassan extract, it is characterised in that it is prepared by the following method and obtains：
A raw material blade of grass Alga Sgrgassi Enervess crushed after being dried is crossed 10-40 mesh sieves and obtains blade of grass Alga Sgrgassi Enervess dry powder by ()；
(b) in blade of grass Alga Sgrgassi Enervess dry powder add volume fraction 70%-90% ethanol after reflux, extract, 2-4 hour, feed liquid body
Product is than being 1:5-10, backflow remove ethanol after terminating, and dry, obtain the blade of grass Alga Sgrgassi Enervess powder of drying defatted；
C the microwave of () from power at 500-1000 watt is carried by solvent of water to the blade of grass Alga Sgrgassi Enervess powder of drying defatted
Take, material liquid volume ratio is 1:10-30, each microwave treatment time are 15-30 minutes, altogether microwave treatment 1-4 time；
D () merges all extracting solution, filter, and filtrate utilizes Polyethylene Glycol/inorganic salt or ethanol/inorganic salt aqueous two-phase system to separate
Purification, is placed in constant temperature oscillator the vibration 25-35 minutes at 40-50 DEG C, stands；The quality of described Polyethylene Glycol or ethanol
Concentration is 10%-40%, and described inorganic salt is selected from ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate or Sodium Chloride, and mass concentration is
10%-25%；
E () collects phase under inorganic salt, concentrated using ultrafiltration membrance filter, and described ultrafilter membrane operating pressure is 0.1-0.6MPa, is surpassed
The molecular cut off of filter membrane be 3000-20000Da, ultrafiltration time 20-40min, 30-40 DEG C of ultrafiltrate temperature, to ultrafiltration membrance filter
Trapped fluid after concentration carries out lyophilization, obtains described blade of grass sargassan extract.
2. blade of grass sargassan extract according to claim 1, it is characterised in that the double water described in step (d)
Phase system is Polyethylene Glycol/inorganic salt, and described inorganic salt is ammonium sulfate.
3. blade of grass sargassan extract according to claim 2, it is characterised in that described molecular weight polyethylene glycol
For 6000, mass concentration is 10%-20%；Described Inorganic salt is 20%-25%.
4. blade of grass sargassan extract according to claim 1, it is characterised in that temperature is 50 DEG C in step (d).
5. blade of grass sargassan extract according to claim 1, it is characterised in that described blade of grass sargassan
In extract, sulfate radical content is 19.6%-25.6%.
6. the arbitrary described blade of grass sargassan extract of claim 1-5 improves medicine, the health care of intestinal microbial population in preparation
Application in product or food.
7. application according to claim 6, it is characterised in that described improvement intestinal microbial population refers to increase intestinal bifid bar
Bacterium and lactobacilluss quantity simultaneously reduce intestinal escherichia coli and streptococcus quantity.
8. the arbitrary described blade of grass sargassan extract of claim 1-5 is preparing medicine, the health product of enhance immunity
Or the application in food.
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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 |
| 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 |
| KR102225351B1 (en) * | 2018-10-24 | 2021-03-09 | 목포대학교산학협력단 | Composition for improving intestinal health containing extract of Sargassum thunbergii |
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| CN112961259B (en) * | 2021-03-16 | 2022-08-16 | 盐城工学院 | Preparation method of taurolimus polysaccharide and application of taurolimus polysaccharide in field of improving intestinal function |
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