CN104829740A - Method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium - Google Patents
Method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium Download PDFInfo
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- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 94
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 54
- 150000004676 glycans Chemical class 0.000 title abstract description 81
- 239000005017 polysaccharide Substances 0.000 title abstract description 81
- 229920001282 polysaccharide Polymers 0.000 title abstract description 81
- 241000195474 Sargassum Species 0.000 title abstract description 28
- 238000000605 extraction Methods 0.000 claims abstract description 54
- 238000000926 separation method Methods 0.000 claims abstract description 22
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 244000025254 Cannabis sativa Species 0.000 claims description 274
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 198
- 239000000284 extract Substances 0.000 claims description 98
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- 229960004756 ethanol Drugs 0.000 claims description 84
- 238000001035 drying Methods 0.000 claims description 55
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 53
- 238000000108 ultra-filtration Methods 0.000 claims description 50
- 239000007788 liquid Substances 0.000 claims description 46
- -1 polyoxyethylene Polymers 0.000 claims description 36
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 26
- 239000000463 material Substances 0.000 claims description 20
- 238000000746 purification Methods 0.000 claims description 20
- 239000002994 raw material Substances 0.000 claims description 19
- 239000000706 filtrate Substances 0.000 claims description 18
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 17
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 17
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 17
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 13
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 12
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 12
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 12
- 235000011152 sodium sulphate Nutrition 0.000 claims description 12
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 11
- 239000012141 concentrate Substances 0.000 claims description 11
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 11
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- 238000002360 preparation method Methods 0.000 claims description 6
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- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
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- 238000000874 microwave-assisted extraction Methods 0.000 abstract description 3
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- 238000005259 measurement Methods 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 11
- 241000199919 Phaeophyceae Species 0.000 description 8
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 8
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- 239000008103 glucose Substances 0.000 description 3
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 3
- 229910052939 potassium sulfate Inorganic materials 0.000 description 3
- 235000011151 potassium sulphates Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003809 water extraction Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000006286 aqueous extract Substances 0.000 description 2
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 2
- 229940045511 barium chloride Drugs 0.000 description 2
- 229910001626 barium chloride Inorganic materials 0.000 description 2
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- 241000512259 Ascophyllum nodosum Species 0.000 description 1
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- 241000212297 Pelvetia Species 0.000 description 1
- JPYHHZQJCSQRJY-UHFFFAOYSA-N Phloroglucinol Natural products CCC=CCC=CCC=CCC=CCCCCC(=O)C1=C(O)C=C(O)C=C1O JPYHHZQJCSQRJY-UHFFFAOYSA-N 0.000 description 1
- 241000195475 Sargassaceae Species 0.000 description 1
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- 102000011759 adducin Human genes 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
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- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
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- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
- 229960002733 gamolenic acid Drugs 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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- 239000000178 monomer Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 description 1
- 229960001553 phloroglucinol Drugs 0.000 description 1
- 229920001339 phlorotannin Polymers 0.000 description 1
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a method for synchronously extracting sargassum graminifolium polysaccharide and sargassum graminifolium polyphenol from sargassum graminifolium. The method for synchronously extracting the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol from the sargassum graminifolium is characterized in that microwave-assisted extraction, two aqueous phase extraction and membrane separation techniques are applied for extracting the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol from the sargassum graminifolium at the same time. The invention also provides the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol which are obtained by adopting the method provided by the invention. The method for synchronously extracting the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol from the sargassum graminifolium has the advantages that sargassum graminifolium polysaccharide extraction ratio is high, biological activity loss is low, and the purities of the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol are high; meanwhile, time is short, handing capacity is high, continuous operation can be realized, cost is low, and clean environment can be realized, so that the method for synchronously extracting the sargassum graminifolium polysaccharide and the sargassum graminifolium polyphenol from the sargassum graminifolium is especially applicable to industrial application.
Description
Technical field
The present invention relates to sargassan and the sargassun polyphenol of medicine and field of health care products, specifically, relate to a kind of method of simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun.
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.
The processing and utilization scope of current sargassun is narrow, only has small portion to be used as the raw material of feed, phycocolloid, and part is edible, and major part is not yet comprehensively developed.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.Brown algae polyphenols is that the structure be made up of as monomer Phloroglucinol extracted from brown alga is different, general name containing Polyhydroxy phenol, have excellent anti-oxidant function, research in the past shows that kelp belongs to and in Sargassum, Content of Phlorotannins belongs to high compared with other brown algas.
Carry out Separation Research to the polysaccharide in the middle of blade of grass sargassun and polyphenol to contribute to making full use of blade of grass sargassun resource, development is blue economical, provides foundation for researching and developing brown algae polyphenols and sargassan class active substance or other health care goods and lays the foundation.
At present, Sargassum polysaccharides preparation technology generally adopts hot water extraction, ultrasonic wave or microwave radiation exaraction, method of purification many employings alcohol settling or membrane sepn; The extracting method of brown algae polyphenols has the methods such as alcohol extracting, microwave digestion, enzymolysis, supercutical fluid.Wherein alcohol extracting yield is high, product anti-microbial activity is good, it is little to pollute, but solvent load is large; Microwave radiation exaraction is considered to the most efficient extracting method, energy-saving and environmental protection.
Chinese periodical " food research and development ", 2006.Vol.27.No.7, the paper " optimization of sargassan extraction process " published, the extraction conditions of sargassan is optimized by Responds Surface Methodology, be studied the optimum level scope of key influence factor and interaction thereof, reach a conclusion: be 469s when the ultrasonic disruption time, Extracting temperature is 90.9 DEG C, extraction time is 4.66 hours, and the theoretical yield of polysaccharide is 2.48% to the maximum.
Chinese periodical " Agriculture of Anhui science " the 37th volume the 17th phase in 2009, paper " the extraction of sargassun polyphenol published, separation and purification and antibacterial activity test ", adopt the method for test, to the extraction of polyphenol in Beihai Fisheries Base Guangxi Province sargassun, separation and purification, qualitative, quantitative analysis and antibacterial activity assay are studied, investigate different alcohol concn, volume number and extraction time are on the impact of brown algae polyphenols extracted amount, result: the extraction of 40ml65% extraction using alcohol 2h on brown algae polyphenols has remarkably influenced, the content recording brown algae polyphenols in sargassun is 7.777mg/g, conclusion: the concentration of ethanol, volume number and extraction time all can affect the extraction of sargassun polyphenol.
But, yet there are no and utilize synchronous from same marine alga, the efficient extraction and isolation polysaccharide of microwave radiation exaraction, Bi-aqueous extraction and membrane separation technique and these two kinds of active substances of polyphenol, and be suitable for the method for industrial application.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of synchronous from blade of grass sargassun, method of extracting blade of grass sargassan and blade of grass sargassun polyphenol is efficiently provided, and extract the blade of grass sargassan extract, the blade of grass sargassun polyphenol extract that obtain.
The invention provides a kind of method of simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun, comprise the following steps:
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%; 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 blade of grass sargassan extract; Phase in collection, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains blade of grass sargassun polyphenol 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.
Present invention also offers 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.
Preferably, in described blade of grass sargassan extract, sulfate radical content is 19.6%-25.6%.
Present invention also offers a kind of preparation method of blade of grass sargassan extract, comprise the following steps:
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.
Present invention also offers a kind of blade of grass sargassun polyphenol 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%;
E () collects upper phase, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains described blade of grass sargassun polyphenol extract.
Preferably, the aqueous two-phase system described in step (d) is polyoxyethylene glycol/inorganic salt, and described inorganic salt are ammonium sulfate.
Present invention also offers a kind of preparation method of blade of grass sargassun polyphenol extract, comprise the following steps:
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%;
E () collects upper phase, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains described blade of grass sargassun polyphenol extract.
The invention has the advantages that:
1, applied microwave assisted extraction of the present invention, aqueous two-phase extraction and membrane separation technique simultaneous extraction can obtain blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun, and step and optimum configurations appropriate, make blade of grass sargassan extraction yield high, biological activity loss is little, and the purity of blade of grass sargassan and blade of grass sargassun polyphenol is all very high;
2, method of the present invention has that time short, treatment capacity is large, operation can serialization, advantage with low cost, can separation and purification blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun fast and efficiently;
3, the energy of present invention process, solvent consumption comparatively common process compare and belong to typical clean environment production technique;
4, the blade of grass sargassan extract purity for preparing of method of the present invention is high, and biological activity is good, can well for the exploitation of sargassan medicine, healthcare products and food;
Known in sum, method of the present invention is very applicable to industrial application.
Embodiment
Below embodiment provided by the invention is elaborated.
The method (one) of embodiment 1 simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun
1, method steps
1.1 simultaneous extraction blade of grass sargassans and blade of grass sargassun polyphenol
(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) all extracting solutions are merged, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan and blade of grass sargassun polyphenol, 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 sargassun polyphenol enters phase on ethanol, and blade of grass sargassan enters phase under inorganic salt.
(5) purifying
A. because of sargassun polyphenol easy oxidation discoloration, therefore the upper phase obtained, ethanol is boiled off with vacuum rotary evaporator in 40 DEG C, then dehydrated alcohol is added by solid-to-liquid ratio (g:ml) 1:20, elimination wherein solid after remaining inorganic salt are separated out completely, boil off ethanol in 40 DEG C with vacuum rotary evaporator again, lyophilize 3 days, obtain blade of grass sargassun polyphenol extract.
B. 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 sargassans and blade of grass sargassun polyphenol purity
(1) purity=(in the blade of grass sargassan extract that above-mentioned steps (5) obtains polysaccharide content/blade of grass sargassan extract weight) × 100% of blade of grass sargassan.
(2) purity=(polyphenol content in the blade of grass sargassun polyphenol extract that above-mentioned steps (5) obtains/blade of grass sargassun polyphenol extract weight) × 100% of blade of grass sargassun polyphenol.
Folin-Ciocalteu method is adopted to measure blade of grass sargassun polyphenol content.Get respectively gallic acid standardized solution 0,0.1,0.2,0.3,0.4,0.5ml is placed in the brown volumetric flask of 10ml, add ultrapure water and be diluted to lml.Add 2ml Folin-Ciocalteu working fluid and shake up, after 2min, adding the Na of 12mol/L
2cO
3solution 6ml, after mixing fully, dark place leaves standstill 60min, measures absorbancy in 765nm place.According to polyphenol content in said determination method Detection and Extraction thing, calculate polyphenol content (heavily representing with milligram gallic acid equivalant (GAE) every gram of dry powder) in blade of grass sargassun extract.
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 HCI 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 the blade of grass sargassan extract 5mg that above-mentioned steps (5) obtains and puts into the test tube filling 3mL1mol/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
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 15.30%, 41.08%, 6.285%.
2.2 blade of grass sargassans and blade of grass sargassun polyphenol purity
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 91.53%, 72.37%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 25.6%.
The method (two) of embodiment 2 simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun of the present invention
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) all extracting solutions are merged, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan and blade of grass sargassun polyphenol, 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 sargassun polyphenol enters phase on ethanol, and blade of grass sargassan enters phase under inorganic salt.
(5) purifying
A. because of sargassun polyphenol easy oxidation discoloration, therefore the upper phase obtained, ethanol is boiled off with vacuum rotary evaporator in 35 DEG C, then dehydrated alcohol is added by solid-to-liquid ratio (g:ml) 1:15, elimination wherein solid after remaining inorganic salt are separated out completely, boil off ethanol in 35 DEG C with vacuum rotary evaporator again, lyophilize 4 days, obtain blade of grass sargassun polyphenol extract.
B. 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 and blade of grass sargassun polyphenol 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
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 13.36%, 38.24%, 5.109%.
2.2 blade of grass sargassans and blade of grass sargassun polyphenol purity
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 89.63%, 69.18%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 24.8%.
The method (three) of embodiment 3 simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun of the present invention
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 blade of grass sargassun polyphenol, 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 sargassun polyphenol enters phase on ethanol, and blade of grass sargassan enters phase under inorganic salt.
(5) purifying
A. because of sargassun polyphenol easy oxidation discoloration, therefore the upper phase obtained, ethanol is boiled off with vacuum rotary evaporator in 45 DEG C, then dehydrated alcohol is added by solid-to-liquid ratio (g:ml) 1:25, elimination wherein solid after remaining inorganic salt are separated out completely, boil off ethanol in 45 DEG C with vacuum rotary evaporator again, lyophilize 2 days, obtain blade of grass sargassun polyphenol extract.
B. 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 and blade of grass sargassun polyphenol 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
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 12.42%, 35.07%, 4.356%.
2.2 blade of grass sargassans and blade of grass sargassun polyphenol purity
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 79.65%, 60.17%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 20.3%.
The method (four) of embodiment 4 simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun of the present invention
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) all extracting solutions are merged, filter, filtrate utilizes polyoxyethylene glycol (PEG)/inorganic salt aqueous two-phase system separation and purification blade of grass sargassan and blade of grass sargassun polyphenol, 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 sargassun polyphenol enters phase on ethanol, and blade of grass sargassan enters phase under inorganic salt.
(5) purifying
A. because of sargassun polyphenol easy oxidation discoloration, therefore the upper phase obtained, ethanol is boiled off with vacuum rotary evaporator in 50 DEG C, then dehydrated alcohol is added by solid-to-liquid ratio (g:ml) 1:20, elimination wherein solid after remaining inorganic salt are separated out completely, boil off ethanol in 50 DEG C with vacuum rotary evaporator again, lyophilize 3 days, obtain blade of grass sargassun polyphenol extract.
B. 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 and blade of grass sargassun polyphenol 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
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 12.28%, 35.14%, 4.315%.
2.2 blade of grass sargassans and blade of grass sargassun polyphenol purity
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 75.85%, 56.33%.
Sulfate radical content in 2.3 blade of grass sargassan extracts
In blade of grass sargassan extract, sulfate radical content is 19.6%.
The method (five) of embodiment 5 simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun of the present invention
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 blade of grass sargassun polyphenol, 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 sargassun polyphenol enters phase on ethanol, and blade of grass sargassan enters phase under inorganic salt.
(5) purifying
A. because of sargassun polyphenol easy oxidation discoloration, therefore the upper phase obtained, ethanol is boiled off with vacuum rotary evaporator in 50 DEG C, then dehydrated alcohol is added by solid-to-liquid ratio (g:ml) 1:20, elimination wherein solid after remaining inorganic salt are separated out completely, boil off ethanol in 50 DEG C with vacuum rotary evaporator again, lyophilize 3 days, obtain blade of grass sargassun polyphenol extract.
B. 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 and blade of grass sargassun polyphenol 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
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 13.59%, 38.83%, 5.277%.
2.2 blade of grass sargassans and blade of grass sargassun polyphenol purity
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 75.19%, 57.43%.
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 Hot water extraction extracts blade of grass sargassun Crude polysaccharides from blade of grass sargassun
1, method steps
Take a certain amount of pretreated blade of grass sargassun dry powder and be placed in flask, add distilled water 10-15 times amount, flask is placed in electric mantle and carries out heating extraction, each extraction 2-4 hour, collecting by filtration filtrate, repeats extraction 2 times by filter residue, merged by 3 Aqueous extracts.Get liquid 4000r/min collected by centrifugation supernatant liquor, supernatant concentration becomes medicinal extract, and dry 24-48 hour after 85 DEG C in baking oven, just can obtain blade of grass sargassun Crude polysaccharides.
Polysaccharide content, polysaccharide extract rate in Crude polysaccharides yield, crude product is measured according to the method for embodiment 1.
2, result
In Crude polysaccharides yield, crude product, polysaccharide content, polysaccharide extract rate measurement result are respectively 11.95%, 33.07%, 3.951%.
Comparative example 2 water extraction and alcohol precipitation method extracts purification blade of grass sargassan, blade of grass sargassun polyphenol from blade of grass sargassun
1, method steps
(1) take a certain amount of pretreated blade of grass sargassun dry powder and be placed in flask, add distilled water 10-15 times amount, flask is placed in electric mantle and carries out heating extraction, each extraction 2 hours, collecting by filtration filtrate, repeats extraction 2 times by filter residue, merged by 3 Aqueous extracts.
(2) filtrate in step (1) is evaporated to 1/6 of original volume with Rotary Evaporators, add 95% ethanol, make the total concn of ethanol reach 80%, hold over night, the centrifugal 10min of 8000r/min, the precipitation of gained is blade of grass sargassun Crude polysaccharides.Use dehydrated alcohol, washing with acetone respectively, lyophilize, namely obtain sargassan.
(3) by the ethanol supernatant liquid Rotary Evaporators concentrating under reduced pressure in step (2) until remove ethanol completely, the medicinal extract in flask is the extract containing blade of grass sargassun polyphenol.
The purity of blade of grass sargassan and blade of grass sargassun polyphenol is measured according to the method for embodiment 1.
2, result
The measurement result of blade of grass sargassan and blade of grass sargassun polyphenol purity is respectively 34.82%, 15.26%.
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 (10)
1. the method for simultaneous extraction blade of grass sargassan and blade of grass sargassun polyphenol from blade of grass sargassun, is characterized in that, comprise the following steps:
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%; 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 blade of grass sargassan extract; Phase in collection, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains blade of grass sargassun polyphenol extract.
2. method 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. method 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. method according to claim 1, is characterized in that, in step (d), temperature is 50 DEG C.
5. 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.
6. blade of grass sargassan extract according to claim 5, 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.
7. a preparation method for blade of grass sargassan extract, is characterized in that, comprises the following steps:
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.
8. a blade of grass sargassun polyphenol 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%;
E () collects upper phase, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains described blade of grass sargassun polyphenol extract.
9. blade of grass sargassun polyphenol extract according to claim 8, 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.
10. a preparation method for blade of grass sargassun polyphenol extract, is characterized in that, comprises the following steps:
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%;
E () collects upper phase, ethanol is boiled off with vacuum rotary evaporator in 35-45 DEG C, then by solid-to-liquid ratio 1g:(15-25) ml adds dehydrated alcohol, elimination wherein solid after remaining inorganic salt are separated out completely, ethanol is boiled off with vacuum rotary evaporator again in 35-45 DEG C, lyophilize, obtains described blade of grass sargassun polyphenol extract.
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| CN104829738A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes |
| CN107652370A (en) * | 2017-09-15 | 2018-02-02 | 刘锐 | Leaf lettuce polysaccharide and its application in mouth disease medicine or health products are prepared |
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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 |
| CN104829738A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes |
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Patent Citations (2)
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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 |
| CN104829738A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes |
Cited By (11)
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| CN104829738A (en) * | 2015-05-12 | 2015-08-12 | 上海海洋大学 | Application of sargassum graminifolium polysaccharide extract in improvement of intestinal flora and prevention and treatment of diabetes |
| CN107652370A (en) * | 2017-09-15 | 2018-02-02 | 刘锐 | Leaf lettuce polysaccharide and its application in mouth disease medicine or health products are prepared |
| CN111669976A (en) * | 2018-01-25 | 2020-09-15 | 赫马斯有限公司 | Composition for animal feed containing brown algae polyphenol as active ingredient and animal product |
| CN109943893A (en) * | 2019-02-27 | 2019-06-28 | 唐卫兵 | A kind of preparation method of the warming natural nano bamboo fibre/cotton fiber composite material of long acting antibiotic |
| CN111249321A (en) * | 2020-03-27 | 2020-06-09 | 东北农业大学 | Method for simultaneously preparing black currant polyphenol and polysaccharide |
| CN111249321B (en) * | 2020-03-27 | 2022-01-18 | 东北农业大学 | Method for simultaneously preparing black currant polyphenol and polysaccharide |
| CN112920024A (en) * | 2021-01-30 | 2021-06-08 | 海南大学 | Sargassum stolonifera phenolic substance and extraction method and application thereof |
| CN114957502A (en) * | 2022-06-22 | 2022-08-30 | 山东中医药大学 | Synchronous extraction optimization process of artichoke polysaccharide and polyphenol |
| CN115403574A (en) * | 2022-08-03 | 2022-11-29 | 李玉山 | Preparation method of rauwolfine hydrochloride |
| CN116003648A (en) * | 2023-01-31 | 2023-04-25 | 广东药科大学 | Extraction method of active ingredients in gulfweed |
| CN117122059A (en) * | 2023-08-29 | 2023-11-28 | 中国海洋大学 | Preparation method and application of antiallergic and anti-inflammatory seaweed polyphenol |
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