CN108530557A - Laminarin extraction and purification process and application - Google Patents
Laminarin extraction and purification process and application Download PDFInfo
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- CN108530557A CN108530557A CN201810283516.XA CN201810283516A CN108530557A CN 108530557 A CN108530557 A CN 108530557A CN 201810283516 A CN201810283516 A CN 201810283516A CN 108530557 A CN108530557 A CN 108530557A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a kind of laminarin extraction and purification process and applications, specially：Laminaria powder is quantitatively weighed, 60 ~ 120 mesh screens are crossed；It is added 5 ~ 10 times 90% of ethyl alcohol, 60 ~ 90 DEG C of heating water bath degreasings 1 ~ 3 time, each 2h；It filters, takes out and closely do, then add the sodium carbonate liquor of 30 ~ 120ml 0.5 ~ 2%, digest 2h in 40 ~ 70 DEG C of water-bath, stand overnight；Centrifugal filtration takes filtrate, crosses the NF membrane or Ultra filtration membrane of different molecular weight, takes concentrate；5% hydrochloric acid solution is added to pH value 1 ~ 5 in concentrate, stands centrifugation, obtains sediment；It is dissolved again with 1 ~ 2% aqueous sodium carbonate, the ethyl alcohol of 1 ~ 3 times of amount of solution is added in solution, obtains sediment；Sediment is centrifuged into drying.Extraction and purification process of the present invention is simple to operation, and gained laminarin significantly inhibits colon cancer cell, and the application further to expand laminarin provides foundation.
Description
Technical field
The present invention relates to technical field of polysaccharide extraction, specially a kind of laminarin extraction and purification process and application.
Background technology
Kelp（Ecklonia kurome）For Laminariaceae plant sea-tangle or Alariaceae plant kelp（Goose slaps dish）Dried leaf
Shape body.It is distributed in the east area of the Liao River, coextensive with eastern and southern Liaoning Province, Shandong, Zhejiang, Fujian more, it can be edible and medicinal.Kelp is a kind of with very high medical value
Seaweed.《Chinese Pharmacopoeia》It records, kelp gas raw meat, taste is salty；Return liver, stomach, kidney channel；There are softening and resolving hard mass, dissolving phlegm, the function of sharp water.Face
On bed for goitre, lymphonodi cervicales is swollen, bronchitis, pulmonary tuberculosis, cough and cataract of old people etc., also try out in treatment
Cancer.
Laminarin refers to the macromolecular polysaccharide substance in kelp, it is primarily present in kelp, therefore is laminarin.
There is laminarin extensive biological activity, academia to confirm：Laminarin can reduce Urine proteins, purify the urine of blood
Acid improves serum lipid concentrations, has good preventive and therapeutic effect to gout, hyperlipidemia nephrosis, early metaphase kidney failure.Laminarin is a kind of
Natural ingredient.It is extremely rare in the content of nature, is primarily present in brown algae plant（Such as kelp）In cell wall.According to point
It analyses, in 1000g kelps, the content of laminarin is less than 1g.Different from the primary nourishing class health food such as cordyceps sinensis, kelp is more
Sugar is highly purified healthy food.The purity of laminarin has direct relationship with its medical effect, at present the kelp in China
Polysaccharide extracting process level is relatively low, and international most advanced level has not yet been reached.
Kelp and kelp belong to Laminariaceae plant, lot of domestic and international scholar by studies have shown that the two polysaccharide extraction work
Skill has similar place, and on the whole, the extracting method of laminarin is also classified into sour formulation, enzyme formulation and alkaline extraction.But it is pervious
Spininess is studied to the extracting method of laminarin, the association of purifying and curative effect to laminarin is research and few.For many years
Inventor has not only carried out numerous studies to laminarin extraction and purifying process, and to laminarin after purification the effect of into
It has gone detection, it is found that the product of different molecular weight fraction has inhibition to Colon Cancer Cells.
Invention content
In view of the deficiencies of the prior art, the present invention provides a kind of laminarin extraction and purification process and applications, further carry
The high purifying process of laminarin adds the pure of over-molecular sieve by the optimization and improvement to laminarin extraction process
Chemical industry skill, obtained laminarin have preferable inhibition to Colon Cancer Cells, for colon cancer treatment provide according to
According to.
The technical proposal of the invention is realized in this way：
Laminarin extraction and purification process, includes the following steps：
（1）Laminaria powder is quantitatively weighed, 60 ~ 120 mesh screens are crossed, it is spare.
（2）It is added the ethyl alcohol that 5 ~ 10 times of volume fractions are 90%, 60 ~ 90 DEG C of heating water bath degreasings 1 ~ 3 time, each 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 30 ~ 120ml weight concentrations 0.5 ~ 2%, in 40 ~ 70 DEG C of water
2h is digested in bath, is stood overnight.
（4）Centrifugal filtration, rotating speed are 2000 ~ 5000r/min；Take filtrate, cross 200 ~ 100000 molecular weight NF membrane or
Ultra filtration membrane takes concentrate.
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 1 ~ 5 in concentrate, stands centrifugation, obtains sediment A；The volume
5% hydrochloric acid solution of concentration is to be added in 100 milliliters of distilled water to dilute by 5 milliliter 36 ~ 38% of concentrated hydrochloric acid to obtain.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1 ~ 2% to dissolve again, and the body of 1 ~ 3 times of amount of solution is added in solution
The ethyl alcohol that fraction is 75 ~ 95%, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
The laminarin that the extraction and purification process obtains is preparing the application in treating colon cancer drug, and inventor is logical
The laminarin to being obtained under the conditions of different molecular weight NF membrane or ultrafiltration membrane is crossed, the inhibition of colon cancer cell is done as follows
Test：
1, experiment material：
Given the test agent：The laminarin obtained by 200,5000,10,000,50,000,100,000 molecular weight NF membranes or ultrafiltration membrane is crossed respectively
Colon cancer cell：HCT116
2, experiment reagent and consumptive material：Fetal calf serum, CCK8 kits, McCoy5A Medium, 0.25%Trypsin, Pen-
Strep（It is dual anti-））, 1XPBS, 100mm Tissue Culture Dish, 96 orifice plates, 0.22um Filter（BD）, 0.1 ~ 10ul rifles point, 10 ~
200ul rifles point, 100 ~ 1000ul rifles point, 50ml sterile centrifugation tubes, the sterile independent packaging pipettes of 5ml, 1.5ml sterile centrifugations
Pipe.
3, experimental method：
1. HCT116 cell culture
2. experiment packet：
Control group：It is not added with the experimental group of polysaccharide given the test agent, as Normal group.In addition one is set again is free of cell and polysaccharide
Blank control group, the background data as detection.
Experimental group：Be separately added into 200,5000,10000,50000,100,000 5 kind of seperation film obtain different kelps
Polysaccharide, the final concentration of each different given the test agent be set to 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml,
The different gradients of 10mg/ml five.
3. by the colon cancer cell HCT-116 of exponential phase, a concentration of 3 × 10 are made into after being digested with pancreatin4~ 1×105
The cell suspension of a/ml（Optimum cell number needs to optimize）, 96 orifice plates are inoculated in, adds 100ul cell suspensions per hole, tablet is set
In 37 DEG C, 5%CO2, after cultivating 24 hours under conditions of 95% humidity, it is added and contains 0.625mg/ml, 1.25mg/ml, 2.5g/
The culture solution 100ul of five different fractions of laminarin of the different final concentrations of ml, 5mg/ml, 10mg/ml five, control group add not
Culture solution 100ul containing given the test agent, every group sets 5 multiple holes, is changed the liquid once every 24 hours.After being incubated 72h, it is added to every hole
The CCK8 solution of 10ul is placed in incubator and is incubated 3 hours, and the absorbance at 450nm is measured with microplate reader.
4. experimental result statistical analysis：The survival of cell is calculated according to the absorbance value of blank group, control group, experimental group
Rate.
4, experimental result
Different molecular weight NF membrane and ultrafiltration membrane are obtained by measuring the 1st ~ 5 day i.e. D1, D2, D3, the absorbance value of D4, D5
（200,5000,10000,50000 and 100000）Laminarin obtained by retaining, the influence to HCT-116 cell Proliferations, specifically
For：
The test discovery for retaining laminarin to HCT-116 cell-proliferation activities by comparing different molecular weight ultrafiltration membrane, it is different
The laminarin of molecular weight retention has preferable inhibition to colon cancer cell, and concentration is higher, and inhibiting effect is stronger.
Meanwhile molecular weight it is bigger ultrafiltration membrane retention laminarin to HCT-116 cell inhibitory effects effect gradually increase.
The beneficial effects of the present invention are：
Laminarin extraction and purification process of the present invention is rationally effective, and required raw material are easy to get, no complicated technology.Using not in technique
Ultrafiltration membrane and NF membrane with molecular weight 200 ~ 100000 retain laminarin, obtain purified product, are kelp extraction purification work
Skill has started new approach, and the method for extraction and purification to improve kelp provides thinking.Meanwhile being obtained by experimental verification, this
The laminarin for inventing extraction has stronger inhibition to colon cancer cell, has expanded the purposes of laminarin, is into one
Step exploitation laminarin provides foundation.
Specific implementation mode
1 laminarin extraction and purification process 1 of embodiment
（1）Laminaria powder is quantitatively weighed, 60 mesh screens are crossed, it is spare.
（2）It is added the ethyl alcohol that 5 times of volume fractions are 90%, 60 DEG C of heating water bath degreasings 1 time, time 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 30ml weight concentrations 0.5%, digested in 40 DEG C of water-bath
2h is stood overnight.
（4）Centrifugal filtration, rotating speed 2000r/min；Filtrate is taken, the NF membrane or Ultra filtration membrane of 200 molecular weight is crossed, takes
Concentrate.
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 1 in concentrate, stands centrifugation, obtains sediment A.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1% to dissolve again, and the volume of 1 ~ 3 times of amount of solution is added in solution
The ethyl alcohol that score is 75%, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
2 laminarin extraction and purification process 2 of embodiment
（1）Laminaria powder is quantitatively weighed, 120 mesh screens are crossed, it is spare.
（2）It is added the ethyl alcohol that 10 times of volume fractions are 90%, 90 DEG C of heating water bath degreasings 3 times, each 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 120ml weight concentrations 2%, digested in 70 DEG C of water-bath
2h is stood overnight.
（4）Centrifugal filtration, rotating speed 5000r/min；Filtrate is taken, the NF membrane or ultrafiltration membrane point of 100000 molecular weight are crossed
From taking concentrate.
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 5 in concentrate, stands centrifugation, obtains sediment A.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 2% to dissolve again, and the volume point of 3 times of amount of solution is added in solution
Number is 95% ethyl alcohol, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
3 laminarin extraction and purification process 3 of embodiment
（1）Laminaria powder is quantitatively weighed, 80 mesh screens are crossed, it is spare.
（2）It is added the ethyl alcohol that 6 times of volume fractions are 90%, 70 DEG C of heating water bath degreasings 2 times, each 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 50ml weight concentrations 1%, digested in 50 DEG C of water-bath
2h is stood overnight.
（4）Centrifugal filtration, rotating speed 3000r/min；Filtrate is taken, the NF membrane or Ultra filtration membrane of 5000 molecular weight are crossed,
Take concentrate.
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 2 in concentrate, stands centrifugation, obtains sediment A.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1.5% to dissolve again, and the volume of 2 times of amount of solution is added in solution
The ethyl alcohol that score is 90%, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
4 laminarin extraction and purification process 4 of embodiment
（1）Laminaria powder is quantitatively weighed, net is sieved with 100 mesh sieve, it is spare.
（2）It is added the ethyl alcohol that 8 times of volume fractions are 90%, 80 DEG C of heating water bath degreasings 3 times, each 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 60ml 2%, digest 2h in 60 DEG C of water-bath, stood
Night.
（4）Centrifugal filtration, rotating speed 4000r/min；Filtrate is taken, the NF membrane or Ultra filtration membrane of 10000 molecular weight are crossed,
Take concentrate.
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 3 in concentrate, stands centrifugation, obtains sediment A.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1.5% to dissolve again, and the volume of 2 times of amount of solution is added in solution
The ethyl alcohol that score is 80%, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
5 laminarin extraction and purification process 5 of embodiment
（1）Laminaria powder is quantitatively weighed, 90 mesh screens are crossed, it is spare.
（2）It is added the ethyl alcohol that 8 times of volume fractions are 90%, 70 DEG C of heating water bath degreasings 2 times, each 2h.
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 90ml weight concentrations 1%, digested in 60 DEG C of water-bath
2h is stood overnight.
（4）Centrifugal filtration, rotating speed 3000r/min；Filtrate is taken, the NF membrane or Ultra filtration membrane of 50000 molecular weight are crossed,
Take concentrate.
（5）5% hydrochloric acid solution of weight concentration is added to pH value 3 in concentrate, stands centrifugation, obtains sediment A.
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1.5% to dissolve again, and the volume of 3 times of amount of solution is added in solution
The ethyl alcohol that score is 85%, obtains sediment B.
（7）By sediment B centrifugation drying to get laminarin.
Claims (4)
1. laminarin extraction and purification process, it is characterised in that：Include the following steps：
（1）Laminaria powder is quantitatively weighed, 60 ~ 120 mesh screens are crossed, it is spare；
（2）It is added the ethyl alcohol that 5 ~ 10 times of volume fractions are 90%, 60 ~ 90 DEG C of heating water bath degreasings 1 ~ 3 time, each 2h；
（3）It filters, takes out and closely do, then add the sodium carbonate liquor of 30 ~ 120ml weight concentrations 0.5 ~ 2%, in 40 ~ 70 DEG C of water-bath
Middle digestion 2h, stands overnight；
（4）Centrifugal filtration takes filtrate, crosses the NF membrane or Ultra filtration membrane of different molecular weight, takes concentrate；
（5）5% hydrochloric acid solution of volumetric concentration is added to pH value 1 ~ 5 in concentrate, stands centrifugation, obtains sediment A；
（6）Sediment A uses the aqueous sodium carbonate of weight concentration 1 ~ 2% to dissolve again, and the volume point of 1 ~ 3 times of amount of solution is added in solution
Number is 75 ~ 95% ethyl alcohol, obtains sediment B；
（7）By sediment B centrifugation drying to get laminarin.
2. laminarin extraction and purification process according to claim 1, it is characterised in that：Step（4）The centrifugal filtration,
Rotating speed is 2000 ~ 5000r/min.
3. laminarin extraction and purification process according to claim 1, it is characterised in that：Step（4）The NF membrane or
Ultrafiltration membrane molecular weight is 200 ~ 100000 molecular weight.
4. the laminarin that extraction and purification process described in claim 1 obtains is preparing the application in treating colon cancer drug.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023112454A1 (en) * | 2021-12-16 | 2023-06-22 | 学校法人東京理科大学 | Intestinal tumor suppressor, pge2 production inhibitor, and il-22bp production promoter |
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| CN101585890A (en) * | 2008-06-20 | 2009-11-25 | 青岛聚大洋海藻工业有限公司 | Method for preparing sodium alginate from kelp |
| CN101891904A (en) * | 2010-06-23 | 2010-11-24 | 中国农业科学院植物保护研究所 | Kelp oligosaccharide and preparation method and application thereof |
| CN102153669A (en) * | 2010-05-12 | 2011-08-17 | 北京雷力联合海洋生物科技有限公司 | Preparation method of low-molecule brown seaweed glucan |
| CN104610463A (en) * | 2015-02-04 | 2015-05-13 | 山东洁晶集团股份有限公司 | Method for industrial production of sodium alginate by adopting South Africa great kelps |
| CN104804108A (en) * | 2014-01-25 | 2015-07-29 | 青岛海之林生物科技开发有限公司 | Ultra-high viscosity algin production process |
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2018
- 2018-04-02 CN CN201810283516.XA patent/CN108530557B/en active Active
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| CN101585890A (en) * | 2008-06-20 | 2009-11-25 | 青岛聚大洋海藻工业有限公司 | Method for preparing sodium alginate from kelp |
| CN102153669A (en) * | 2010-05-12 | 2011-08-17 | 北京雷力联合海洋生物科技有限公司 | Preparation method of low-molecule brown seaweed glucan |
| CN101891904A (en) * | 2010-06-23 | 2010-11-24 | 中国农业科学院植物保护研究所 | Kelp oligosaccharide and preparation method and application thereof |
| CN104804108A (en) * | 2014-01-25 | 2015-07-29 | 青岛海之林生物科技开发有限公司 | Ultra-high viscosity algin production process |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023112454A1 (en) * | 2021-12-16 | 2023-06-22 | 学校法人東京理科大学 | Intestinal tumor suppressor, pge2 production inhibitor, and il-22bp production promoter |
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