CN108530557B - Laminarin extraction and purification process and application - Google Patents
Laminarin extraction and purification process and application Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a laminarin extraction and purification process and application, which specifically comprises the following steps: quantitatively weighing kelp powder, and sieving with a 60-120 mesh sieve; adding 90% ethanol of which the amount is 5-10 times that of the raw materials, heating in a water bath at the temperature of 60-90 ℃ for degreasing for 1-3 times, and each time for 2 hours; carrying out suction filtration, nearly drying, adding 30-120 ml of 0.5-2% sodium carbonate solution, digesting for 2h in a water bath at 40-70 ℃, and standing overnight; centrifuging, collecting filtrate, separating with nanofiltration membrane or ultrafiltration membrane with different molecular weights, and collecting concentrated solution; adding a 5% hydrochloric acid solution into the concentrated solution until the pH value is 1-5, standing and centrifuging to obtain a precipitate; dissolving the precipitate by using 1-2% sodium carbonate aqueous solution, and adding 1-3 times of ethanol into the solution to obtain a precipitate; and (4) centrifuging and drying the precipitate to obtain the compound. The extraction and purification process is simple and easy to operate, and the obtained laminarin has an obvious inhibiting effect on colon cancer cells, so that a basis is provided for further expanding the application of the laminarin.
Description
Technical Field
The invention relates to the technical field of polysaccharide extraction, in particular to a laminarin extraction and purification process and application.
Background
Thallus laminariae (Ecklonia kurome) is dried thallus of Laminaria japonica of Laminariaceae or thallus laminariae (Caryophyllum Crassirhizome) of Alariaceae. It is widely distributed in Liaodong, Shandong, Zhejiang and Fujian, and can be used for food and medicine. Thallus laminariae is a seaweed with high medicinal value. The kelp is fishy in smell and salty in taste recorded in Chinese pharmacopoeia; entering liver, stomach and kidney meridians; has the functions of softening and resolving hard mass, eliminating phlegm and promoting diuresis. The traditional Chinese medicine composition is clinically used for treating goiter, cervical lymph node swelling, bronchitis, pulmonary tuberculosis, cough, senile cataract and the like, and is also used for treating cancer.
Laminarin refers to a macromolecular polysaccharide substance in kelp, which is mainly present in kelp, and thus is called laminarin. Laminarin has a wide range of biological activities, as confirmed by academia: the laminarin can reduce urine protein, purify uric acid in blood, improve blood lipid concentration, and has good prevention and treatment effects on gout, hyperlipidemia nephropathy, early and middle stage renal failure. Laminarin is a natural ingredient. It is present in an extremely rare amount in nature, and is mainly present in the cell wall of brown algae (such as kelp). According to analysis, 1000g of thallus laminariae contains laminarin less than 1 g. Unlike primary tonic health foods such as Cordyceps, laminarin is a highly purified health food. The purity of laminarin has a direct relation with the medical effect, and the extraction process level of the laminarin in China is lower at present and does not reach the international advanced level yet.
Researches of many scholars at home and abroad show that the extraction processes of laminarin of the laminarin and the kelp belong to the same genus Laminariaceae are similar, and the extraction methods of laminarin are also divided into an acid extraction method, an enzyme extraction method and an alkali extraction method on the whole. However, the previous researches are directed to extraction methods of laminarin, and the researches are not much about the purification and curative effect of laminarin. The inventor not only has carried out a great deal of research on the extraction and purification process of laminarin for many years, but also has tested the curative effect of purified laminarin, and finds that products with different molecular weight fractions have the inhibiting effect on colon cancer cell proliferation.
Disclosure of Invention
The invention provides a laminarin extracting and purifying process and application aiming at the defects of the prior art, further improves the laminarin purifying process, obtains laminarin by optimizing and improving the laminarin extracting process and adding a molecular sieve purifying process, has better inhibiting effect on colon cancer cell proliferation, and provides basis for the treatment of colon cancer.
The technical scheme of the invention is realized as follows:
the laminarin extracting and purifying process includes the following steps:
(1) quantitatively weighing the kelp powder, and sieving the kelp powder with a 60-120 mesh sieve for later use.
(2) Adding ethanol with volume fraction of 90% which is 5-10 times of that of the mixture, heating in water bath at 60-90 ℃ for degreasing for 1-3 times, and each time for 2 hours.
(3) And (3) carrying out suction filtration, nearly drying, adding 30-120 ml of 0.5-2% sodium carbonate solution, digesting for 2 hours in a water bath at the temperature of 40-70 ℃, and standing overnight.
(4) Performing centrifugal filtration at the rotating speed of 2000-5000 r/min; and (3) taking the filtrate, separating the filtrate by using a nanofiltration membrane or an ultrafiltration membrane with the molecular weight of 200-100000, and taking a concentrated solution.
(5) Adding a hydrochloric acid solution with the volume concentration of 5% into the concentrated solution until the pH value is 1-5, standing and centrifuging to obtain a precipitate A; the hydrochloric acid solution with the volume concentration of 5% is obtained by adding 5ml of 36-38% concentrated hydrochloric acid into 100 ml of distilled water for dilution.
(6) Dissolving the precipitate A by using a sodium carbonate aqueous solution with the weight concentration of 1-2%, and adding ethanol with the volume fraction of 75-95% in an amount which is 1-3 times that of the solution into the solution to obtain a precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
According to the application of the laminarin obtained by the extraction and purification process in preparing the medicine for treating colon cancer, the inventor tests the inhibiting effect of laminarin on colon cancer cells by using nanofiltration membranes or ultrafiltration membranes with different molecular weights as follows:
1. experimental materials:
and (3) testing a sample: respectively passing through nanofiltration membrane or ultrafiltration membrane with molecular weight of 200, 5000, 1 ten thousand, 5 ten thousand, and 10 ten thousand to obtain laminarin
Colon cancer cells: HCT116
2. Experimental reagents and consumables: fetal bovine serum, a CCK8 kit, McCoy5A Medium, 0.25% Trypsin, Pen-Strep (double antibody)), 1XPBS, a 100mm cell culture dish, a 96-pore plate, 0.22um Filter (BD), 0.1-10 ul of gun tip, 10-200 ul of gun tip, 100-1000 ul of gun tip, 50ml of sterile centrifuge tube, 5ml of sterile independently packaged pipette and 1.5ml of sterile centrifuge tube.
3. The experimental method comprises the following steps:
(ii) HCT116 cell culture
Experiment grouping:
control group: the experimental group without the polysaccharide test sample was used as a normal control group. In addition, a blank control group without cells and polysaccharide is set as background data of detection.
Experimental groups: adding 200, 5000, 10000, 50000 and 100000 separation membranes to obtain different laminarin, wherein the final concentration of each sample is set to 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml and 10 mg/ml.
③ digesting colon cancer cells HCT-116 in logarithmic growth phase with pancreatin to prepare the colon cancer cells with the concentration of 3 multiplied by 104~ 1×105Cell suspension of one/ml (optimal cell number needs to be optimized), and seeded in 96-well wellsPlates, 100ul of cell suspension per well, plates at 37 ℃ in 5% CO2After 24 hours of incubation at 95% humidity, 100ul of the culture medium containing five different fractions of laminarin at different final concentrations, 0.625mg/ml, 1.25mg/ml, 2.5g/ml, 5mg/ml, 10mg/ml, was added to the control group, 100ul of the culture medium containing no test sample was added, 5 replicate wells per group, and the medium was changed every 24 hours. After 72h incubation, 10ul of CCK8 solution was added to each well, incubated in an incubator for 3 hours, and absorbance at 450nm was measured using a microplate reader.
Fourthly, statistical analysis of experimental results: and calculating the survival rate of the cells according to the absorbance values of the blank group, the control group and the experimental group.
4. Results of the experiment
Measuring absorbance values of D1, D2, D3, D4 and D5 at 1-5 days to obtain laminarin intercepted by nanofiltration membranes and ultrafiltration membranes (200, 5000, 10000, 50000 and 100000) with different molecular weights, wherein the influence on HCT-116 cell proliferation is specifically as follows:
by comparing the test of the proliferation activity of HCT-116 cells by the laminarin intercepted by the ultrafiltration membranes with different molecular weights, the laminarin intercepted with different molecular weights has better inhibition effect on colon cancer cells, and the higher the concentration is, the stronger the inhibition effect is.
Meanwhile, the larger the molecular weight, the stronger the inhibitory effect of laminarin on HCT-116 cell proliferation is gradually retained by the ultrafiltration membrane.
The invention has the beneficial effects that:
the extraction and purification process of laminarin is reasonable and effective, the required raw materials are easy to obtain, and no complex process is needed. In the process, the laminarin is trapped by adopting ultrafiltration membranes and nanofiltration membranes with different molecular weights of 200-100000 to obtain a purified product, a new way is created for the kelp extraction and purification process, and an idea is provided for improving the extraction and purification method of kelp. Meanwhile, experiments prove that the laminarin extracted by the method has a strong inhibition effect on colon cancer cells, the application of the laminarin is expanded, and a basis is provided for further developing the laminarin.
Detailed Description
Example 1 laminarin extraction purification Process 1
(1) Weighing thallus laminariae powder quantitatively, and sieving with 60 mesh sieve.
(2) Adding 90% ethanol 5 times volume fraction, heating in water bath at 60 deg.C for defatting for 1 time for 2 hr.
(3) Suction filtration, suction drying, then adding 30ml sodium carbonate solution with weight concentration of 0.5%, digesting in water bath at 40 ℃ for 2h, standing overnight.
(4) Centrifuging and filtering at 2000 r/min; filtering the filtrate with nanofiltration membrane or ultrafiltration membrane of 200 molecular weight, and collecting the concentrated solution.
(5) Adding hydrochloric acid solution with volume concentration of 5% into the concentrated solution until the pH value is 1, standing and centrifuging to obtain a precipitate A.
(6) And dissolving the precipitate A by using a sodium carbonate aqueous solution with the weight concentration of 1%, and adding ethanol with the volume fraction of 75% in an amount which is 1-3 times that of the solution into the solution to obtain a precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
Example 2 laminarin extraction purification Process 2
(1) Weighing thallus laminariae powder, and sieving with 120 mesh sieve.
(2) Adding 90% ethanol 10 times volume fraction, heating in 90 deg.C water bath for defatting for 3 times, each time for 2 hr.
(3) Suction filtration, suction drying, then adding 120ml of 2% by weight sodium carbonate solution, digesting in a water bath at 70 ℃ for 2h, standing overnight.
(4) Centrifuging and filtering at the rotating speed of 5000 r/min; separating the filtrate with nanofiltration membrane or ultrafiltration membrane of 100000 molecular weight, and collecting the concentrated solution.
(5) Adding hydrochloric acid solution with volume concentration of 5% into the concentrated solution until the pH value is 5, standing and centrifuging to obtain a precipitate A.
(6) Dissolving the precipitate A with 2 wt% sodium carbonate water solution, and adding 95 vol% ethanol 3 times the solution to obtain precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
Example 3 laminarin extraction purification Process 3
(1) Weighing thallus laminariae powder, and sieving with 80 mesh sieve.
(2) Adding 90% ethanol 6 times volume fraction, heating in 70 deg.C water bath for 2 times, and defatting for 2 hr each time.
(3) Suction filtration, suction drying, then adding 50ml of 1% by weight sodium carbonate solution, digesting in a water bath at 50 ℃ for 2h, standing overnight.
(4) Centrifuging and filtering at 3000 r/min; separating the filtrate with nanofiltration membrane or ultrafiltration membrane of 5000 molecular weight to obtain concentrated solution.
(5) Adding hydrochloric acid solution with volume concentration of 5% into the concentrated solution until the pH value is 2, standing and centrifuging to obtain a precipitate A.
(6) Dissolving the precipitate A with 1.5 wt% sodium carbonate water solution, and adding 90% ethanol 2 times the volume of the solution to obtain precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
Example 4 laminarin extraction purification Process 4
(1) Weighing thallus laminariae powder, and sieving with 100 mesh sieve.
(2) Adding 90% ethanol 8 times volume fraction, heating in 80 deg.C water bath for defatting for 3 times, each time for 2 hr.
(3) Suction filtration, suction drying, then adding 60ml 2% sodium carbonate solution, digesting in water bath at 60 ℃ for 2h, standing overnight.
(4) Centrifuging and filtering at the rotating speed of 4000 r/min; filtering the filtrate with nanofiltration membrane or ultrafiltration membrane of 10000 molecular weight, and collecting the concentrated solution.
(5) Adding hydrochloric acid solution with volume concentration of 5% into the concentrated solution until the pH value is 3, standing and centrifuging to obtain a precipitate A.
(6) Dissolving the precipitate A with 1.5 wt% sodium carbonate water solution, and adding 80 vol% ethanol 2 times the solution to obtain precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
Example 5 Laminarin extraction and purification Process 5
(1) Weighing thallus laminariae powder, and sieving with 90 mesh sieve.
(2) Adding 90% ethanol 8 times volume fraction, heating in 70 deg.C water bath for 2 times, and defatting for 2 hr each time.
(3) Suction filtration, suction drying, then adding 90ml of 1% by weight sodium carbonate solution, digesting in a water bath at 60 ℃ for 2h, standing overnight.
(4) Centrifuging and filtering at 3000 r/min; separating the filtrate with nanofiltration membrane or ultrafiltration membrane of 50000 molecular weight, and collecting the concentrated solution.
(5) Adding a hydrochloric acid solution with the weight concentration of 5% into the concentrated solution until the pH value is 3, standing and centrifuging to obtain a precipitate A.
(6) Dissolving the precipitate A with 1.5 wt% sodium carbonate water solution, and adding 85% ethanol in 3 times of the solution to obtain precipitate B.
(7) And (4) centrifuging and drying the precipitate B to obtain laminarin.
Claims (2)
1. The application of laminarin in preparing the medicine for inhibiting the proliferation of colon cancer cells, wherein the extraction and purification process of the laminarin comprises the following steps:
(1) quantitatively weighing kelp powder, and sieving with a 60-120 mesh sieve for later use;
(2) adding ethanol with volume fraction of 90% which is 5-10 times of that of the mixture, heating in water bath at 60-90 ℃ for degreasing for 1-3 times, and each time for 2 hours;
(3) carrying out suction filtration, nearly drying, adding 30-120 mL of 0.5-2% sodium carbonate solution by weight, digesting for 2h in a water bath at 40-70 ℃, and standing overnight;
(4) centrifuging, collecting filtrate, separating with nanofiltration membrane, and collecting concentrated solution;
(5) adding a hydrochloric acid solution with the volume concentration of 5% into the concentrated solution until the pH value is 1-5, standing and centrifuging to obtain a precipitate A;
(6) dissolving the precipitate A by using a sodium carbonate aqueous solution with the weight concentration of 1-2%, and adding ethanol with the volume fraction of 75-95% in an amount which is 1-3 times that of the solution into the solution to obtain a precipitate B;
(7) centrifuging and drying the precipitate B to obtain laminarin;
the molecular weight of the nanofiltration membrane in the step (4) is 50000-100000.
2. Use according to claim 1, characterized in that: and (4) performing centrifugal filtration at a rotating speed of 2000-5000 r/min.
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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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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
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