CN112457424A - Preparation method and application of liquorice polysaccharide effective part - Google Patents
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Abstract
The invention relates to a preparation method and application of anti-inflammatory effective part of licorice, the method dries the licorice rhizome naturally, pulverize mechanically, and sieve and separate the impurity, petroleum ether degreases, anhydrous alcohol decolourizes the pigment, dry naturally and then extract with the hot water, centrifugate, concentrate, the absolute alcohol is precipitated, centrifugate again, dissolve the polysaccharide precipitate with water and remove the protein, dialyse, the vacuum freeze-drying, get water-soluble licorice polysaccharide, purify by ion exchange resin and sephadex chromatographic column and get the licorice polysaccharide effective part; the polysaccharide effective component has total sugar content of 94.87%, uronic acid content of 8.76%, and protein content of 3.14%. The glycyrrhiza polysaccharide effective part obtained by the method has high purity and wide activity; proved by verification, has the functions of resisting inflammation, reducing blood sugar and promoting the growth of probiotics. And physicochemical property analysis and biological activity evaluation are carried out on the effective part of the glycyrrhiza polysaccharide, and reference can be provided for the application and development of the glycyrrhiza polysaccharide in functional food, medicine and cosmetics.
Description
Technical Field
The invention relates to the technical field of biological activity, in particular to a preparation method of a glycyrrhiza polysaccharide effective part and application of the glycyrrhiza polysaccharide effective part in research and development of functional foods and medicines such as anti-inflammation, blood sugar reduction, prebiotics and the like.
Background
The polysaccharide is a natural polymer formed by connecting monosaccharides through glycosidic bonds, widely exists in plants, microorganisms, algae and animals, and is an indispensable important component of life bodies. A great amount of polysaccharides and polysaccharide substances are extracted and separated from animals, plants and microorganisms and are used as therapeutic or adjuvant therapeutic drugs. Polysaccharides play an extremely important role in cell-to-cell information exchange, cell adsorption, and molecular recognition of the immune system. Polysaccharides have become important components in the research and development of natural medicines and health care products, and most of foreign pharmaceutical companies are engaged in the research and development of sugar medicines. Researches show that the polysaccharide has multiple functions of oxidation resistance, tumor resistance, anticoagulation, immunoregulation and the like. The polysaccharide is one of the most important active ingredients of medicinal plants, and the plant polysaccharide has wide application prospect in the fields of medicines, foods, cosmetics, environmental management and the like due to wide treatment effect and extremely low cytotoxicity. Some polysaccharides extracted from natural resources have attracted great attention in the fields of biochemistry and pharmacology, and especially traditional Chinese medicine polysaccharides are more concerned due to the unique action of traditional Chinese medicines and the deep research on the action mechanism of traditional Chinese medicines.
Licorice (Glycyrrhiza L.) belongs to leguminous, perennial grasses and is mainly distributed in the continental europe and asia. Licorice is intensively distributed in the three north areas (northeast, North China and northwest provinces) in China, and Sinkiang, inner Mongolia, Ningxia and Gansu are central production areas. Most of the glycyrrhiza species are very narrow in distribution, some are endangered and extinct, and only a few are wide in distribution. The liquorice has the effect of harmonizing the traditional Chinese medicines and is the herbal medicine with the largest dosage in the compatibility of the traditional Chinese medicines, so the liquorice wins the reputations of king Chinese herbal medicines, the aged and the like by listing the liquorice as a hundred medicines in the compendium of materia medica. The liquorice not only occupies a place in the traditional Chinese medicine, but also is an important raw material in the modern pharmaceutical industry, and has very important functions in a plurality of industries such as food, health care products, cosmetics, tobacco, light industry, petroleum, fire fighting and the like. The liquorice is a common traditional Chinese medicinal material and has wide application, and can be used as an immunomodulator, a feed additive, an antiviral preparation, an auxiliary medicament for treating tumors, an antioxidant and the like. Modern researches find that liquorice contains various active ingredients, mainly triterpenes, flavonoids and glycyrrhiza polysaccharides, and in addition, coumarins, amino acids, alkaloids, volatile oils, various trace elements and the like. Research shows that the glycyrrhiza polysaccharide mainly comprises rhamnose, glucan, arabinose and galactose. Glycyrrhiza polysaccharide is one of important active ingredients of liquorice, and has physiological functions of inhibiting tumor, resisting virus, controlling blood sugar, reducing cholesterol, reducing blood fat and the like. The health of the intestinal function is vital to the health of an organism, the intestinal tract has the function of digesting food and is a main way for the organism to obtain nutrition, and meanwhile, a large number of lymphocytes are distributed in the epithelial tissue of the intestinal tract and are the first barrier for the organism to deal with the infection of external pathogenic microorganisms, so the intestinal health is vital to the individual health, and the intestinal dysfunction can cause various health problems. The polysaccharide is difficult to digest and absorb, but can be used as a carbon source of intestinal flora to promote the growth of beneficial bacteria, regulate the intestinal flora through metabolites of the polysaccharide, change the diversity of intestinal microecology and play a role of prebiotics. In recent research, the influence of polysaccharide on the diversity and composition of intestinal flora is determined, specific bacteria and functional bacteria are searched, the relation of polysaccharide-intestinal flora-disease (health) is established, and the probiotic mechanism of polysaccharide is determined. The existing research shows that the regulation and protection effects of polysaccharide on intestinal flora are various. In the structure of the intestinal tract, the polysaccharide can improve the integrity of the intestinal tract and relieve the damage of the intestinal mucosa; in terms of flora composition, the polysaccharide can promote the proliferation of beneficial bacteria and inhibit the proliferation of harmful bacteria, so that intestinal flora is regulated to form a more balanced flora structure; in terms of flora function, polysaccharides can up-regulate the expression of carbohydrate-active enzyme (CAZymes) genes, improve the activity of CAZymes, increase the production of short chain fatty acids, reduce metabolic endotoxins, reduce the expression of inflammatory factors and increase the expression of tight junction proteins.
Disclosure of Invention
The invention aims to provide a preparation method and application of an effective part of glycyrrhiza polysaccharide, wherein the method comprises the steps of naturally drying glycyrrhiza rhizome, mechanically crushing, sieving to separate impurities, degreasing with petroleum ether, decoloring with absolute ethyl alcohol, naturally drying in the air, extracting with hot water, centrifuging, concentrating, precipitating with absolute ethyl alcohol, centrifuging again, dissolving polysaccharide precipitate with water to remove protein, dialyzing, and freeze-drying in vacuum to obtain the water-soluble glycyrrhiza polysaccharide (the yield is 2.73%). Purifying with ion exchange resin and Sephadex column chromatography to obtain a 24.96 KDa-molecular-weight effective fraction of Glycyrrhiza polysaccharide (yield of 0.21%); the polysaccharide effective component has total sugar content of 94.87%, uronic acid content of 8.76%, and protein content of 3.14%. The effective part of the polysaccharide consists of rhamnose, arabinose, xylose, mannose, glucose and galactose (relative molar ratio is 0.65:0.48:0.11:0.09:1: 0.67). The glycyrrhiza polysaccharide effective part obtained by the method has high purity and wide activity; proved by verification, has the functions of resisting inflammation, reducing blood sugar and promoting the growth of probiotics. The invention carries out physical and chemical property analysis and biological activity evaluation aiming at the effective part of the glycyrrhiza polysaccharide, and can provide reference for the application and development of the glycyrrhiza polysaccharide in functional food, medicine and cosmetics.
The preparation method and the application of the anti-inflammatory effective part of the liquorice are carried out according to the following steps:
pretreatment:
a. mechanically pulverizing Glycyrrhrizae radix for 120 s, sieving with 60 mesh sieve, separating impurities to obtain Glycyrrhrizae radix powder, adding petroleum ether into Glycyrrhrizae radix powder at a feed liquid weight ratio of 1:5, stirring at room temperature for 8hr, repeatedly extracting for 3 times until no oil is extracted, filtering, naturally drying in the shade, adding anhydrous ethanol into the filtrate at a feed liquid weight ratio of 1:5, stirring and refluxing for 4 hr, repeatedly extracting for 3 times until no pigment is extracted, filtering, and naturally drying until no petroleum ether and ethanol solvent remain to obtain defatted and decolorized Glycyrrhrizae radix powder;
hot water extraction:
b. b, adding water into the licorice powder obtained in the step a according to the weight ratio of 1:30, stirring and extracting for 3 times at the temperature of 100 ℃, extracting for 3 hours each time, combining the extracting solutions, centrifuging for 10min at the temperature of 4 ℃ at 7000rpm, and concentrating to obtain polysaccharide concentrated solution for later use;
alcohol precipitation:
c. c, precipitating the polysaccharide concentrated solution obtained in the step b by using absolute ethyl alcohol, and centrifuging for 10min at the temperature of 4 ℃ overnight at 8000rpm and 4 ℃ to obtain polysaccharide precipitate, namely crude licorice polysaccharide;
deproteinization:
d. d, adding distilled water into the polysaccharide precipitate obtained in the step c for redissolving, adding 1/3 volumes of chloroform-n-butanol mixed solution with the volume ratio of 4:1 for deproteinization, magnetically stirring for 30min, centrifuging at 3000rpm for 5min, and repeating the operation for 5 times;
and (3) dialysis:
e. d, dialyzing the polysaccharide solution after deproteinization in the step d for 48h by using a dialysis bag with the cut-off of 3500Da, dialyzing for 24h by using tap water and distilled water, concentrating, and performing vacuum freeze drying to obtain crude licorice polysaccharide;
DEAE-52 column chromatography:
f. preparing the crude licorice polysaccharide obtained in the step e into polysaccharide solution with the concentration of 10mg/ml by using ultrapure water, loading the solution into a DEAE-cellulose 52 ion exchange column, carrying out gradient elution by using the ultrapure water, 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution, detecting the absorption value at 490nm of each collecting pipe by using a phenol-sulfuric acid method to obtain neutral polysaccharide and two acidic polysaccharide parts, collecting the absorption peak of the acidic polysaccharide part eluted by 0.3M NaCl solution, dialyzing the eluent by using distilled water for 48h by using a dialysis bag with the interception amount of 3500Da, concentrating, and freeze-drying to obtain crude pure licorice acidic polysaccharide;
sephadex G-100 column chromatography:
g. and f, further purifying the crude purified glycyrrhiza acid polysaccharide obtained in the step f by Sephadex G-100 column chromatography, dissolving the glycyrrhiza acid polysaccharide to the concentration of 10mg/ml by using ultrapure water, then loading the glycyrrhiza acid polysaccharide, using the ultrapure water as an eluent, detecting the absorption value at 490nm by using a phenol-sulfuric acid method, collecting the eluent of an absorption peak, concentrating under reduced pressure, and freeze-drying to obtain the purified high-purity glycyrrhiza polysaccharide effective part.
The application of the glycyrrhiza polysaccharide effective part obtained by the method in preparing anti-inflammatory drugs.
The application of the glycyrrhiza polysaccharide effective part obtained by the method in preparing hypoglycemic drugs.
The application of the liquorice polysaccharide effective part obtained by the method in preparation of probiotics for promoting the growth of bifidobacterium adolescentis, lactobacillus delbrueckii subsp bulgaricus and enterococcus faecalis.
Compared with the prior art, the invention provides a preparation method and application of the glycyrrhiza polysaccharide effective part, and the preparation method has the following beneficial effects:
1. the method provided by the invention has the advantages that the liquorice is subjected to hot water extraction, alcohol precipitation, deproteinization and subsequent separation and purification, the extraction process is simple and feasible, the liquorice polysaccharide is high in yield, high in purity and low in protein residual rate, and the physicochemical properties of the effective parts of the liquorice polysaccharide are analyzed.
2. The licorice polysaccharide effective part obtained by the method has obvious effects of resisting inflammation, reducing blood sugar and promoting the growth of probiotics.
The preparation method and the application of the anti-inflammatory effective part of the liquorice provided by the invention have the advantages of reasonable design, simple process, convenience in operation, high product extraction rate, high purity, low preparation cost, easiness in popularization and application and the like. The effective part of the glycyrrhiza polysaccharide as a prebiotic is not decomposed, absorbed and utilized by human bodies, and after the effective part reaches the colon through the alimentary canal, some effective parts can be decomposed and utilized by colon flora to promote the growth of the colon flora, thereby having important significance in improving the intestinal microecology and promoting the metabolism of lipid, protein and minerals, and being increasingly and widely applied to the fields of food, feed and the like.
Drawings
FIG. 1 is a DEAE-52 column chromatography separation and elution graph of crude licorice polysaccharide of the present invention;
FIG. 2 is a Sephadex G-100 column chromatography purification elution graph of the effective part of the glycyrrhiza acidic polysaccharide;
FIG. 3 is a GC-MS diagram of the monosaccharide analysis of the glycyrrhiza acid polysaccharide effective part of the invention.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the use and purpose of these exemplary embodiments are merely to exemplify the present invention, and do not set forth any limitation on the actual scope of the present invention in any form, and the scope of the present invention is not limited thereto.
Examples
Pretreatment:
a. mechanically pulverizing Glycyrrhrizae radix for 120 s, sieving with 60 mesh sieve, separating impurities to obtain Glycyrrhrizae radix powder, adding petroleum ether into Glycyrrhrizae radix powder at a feed liquid weight ratio of 1:5, stirring at room temperature for 8hr, repeatedly extracting for 3 times until no oil is extracted, filtering, naturally drying in the shade, adding anhydrous ethanol into the filtrate at a feed liquid weight ratio of 1:5, stirring and refluxing for 4 hr, repeatedly extracting for 3 times until no pigment is extracted, filtering, and naturally drying until no petroleum ether and ethanol solvent remain to obtain defatted and decolorized Glycyrrhrizae radix powder;
hot water extraction:
b. b, adding water into the licorice powder obtained in the step a according to the weight ratio of 1:30, stirring and extracting for 3 times at the temperature of 100 ℃, extracting for 3 hours each time, combining the extracting solutions, centrifuging for 10min at the temperature of 4 ℃ at 7000rpm, and concentrating to obtain polysaccharide concentrated solution for later use;
alcohol precipitation:
c. c, precipitating the polysaccharide concentrated solution obtained in the step b by using absolute ethyl alcohol, and centrifuging for 10min at the temperature of 4 ℃ overnight at 8000rpm and 4 ℃ to obtain polysaccharide precipitate, namely crude licorice polysaccharide;
deproteinization:
d. d, adding distilled water into the polysaccharide precipitate obtained in the step c for redissolving, adding 1/3 volumes of chloroform-n-butanol mixed solution with the volume ratio of 4:1 for deproteinization, magnetically stirring for 30min, centrifuging at 3000rpm for 5min, and repeating the operation for 5 times;
and (3) dialysis:
e. d, dialyzing the polysaccharide solution after deproteinization in the step d for 48h by using a dialysis bag with the cut-off of 3500Da, dialyzing for 24h by using tap water and distilled water, concentrating, and performing vacuum freeze drying to obtain crude licorice polysaccharide;
DEAE-52 column chromatography:
f. preparing the crude licorice polysaccharide obtained in the step e into polysaccharide solution with the concentration of 10mg/ml by using ultrapure water, loading the solution into a DEAE-cellulose 52 ion exchange column, carrying out gradient elution by using the ultrapure water, 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution, detecting the absorption value at 490nm of each collecting pipe by using a phenol-sulfuric acid method to obtain neutral polysaccharide and two acidic polysaccharide parts, collecting the absorption peak of the acidic polysaccharide part eluted by 0.3M NaCl solution, dialyzing the eluent by using distilled water for 48h by using a dialysis bag with the interception amount of 3500Da, concentrating, and freeze-drying to obtain crude pure licorice acidic polysaccharide;
sephadex G-100 column chromatography:
g. and f, further purifying the crude purified glycyrrhiza acid polysaccharide obtained in the step f by Sephadex G-100 column chromatography, dissolving the glycyrrhiza acid polysaccharide to the concentration of 10mg/ml by using ultrapure water, then loading the glycyrrhiza acid polysaccharide, using the ultrapure water as an eluent, detecting the absorption value at 490nm by using a phenol-sulfuric acid method, collecting the eluent of an absorption peak, concentrating under reduced pressure, and freeze-drying to obtain the effective part of the purified glycyrrhiza polysaccharide (figure 2).
The yield of the effective part of the glycyrrhiza polysaccharide obtained by the invention is equal to the mass of the effective part of the glycyrrhiza polysaccharide obtained by Sephadex G-100 column chromatography/the weight of the glycyrrhiza powder obtained after the pretreatment of the medicinal materials is multiplied by 100 percent. The yield of the effective part of the purified glycyrrhiza polysaccharide is 0.21 percent by calculation.
Example 2
Purity and molecular weight determination by HPLC:
sigma chromatographic purity 5KDa, 25KDa, 50KDa, 80KDa,0.2M K use of 150kDa, 410kDa glucan and blue glucan2HPO4(pH 6.6) solution was prepared to a concentration of 2mg/mL polysaccharide molecular weight standards, filtered through a 0.45 μm filter and injected, blue dextran retention time V was used0Calibrating pore volume, plotting standard curve with logarithm of molecular weight (1gMw) as abscissa and retention time as ordinate, and obtaining regression equation of standard curve as y-158.83 x3+583.76x2-714.16x +295.24, R2 ═ 0.956(n ═ 5); simultaneously, sampling the effective part of the glycyrrhiza polysaccharide, and calculating the relative molecular mass according to the retention time, wherein the chromatographic conditions are as follows: TSK-G3000 PWXL (7.8X 300mm), mobile phase 0.2M K2HPO4(PH 6.6) solution, the flow rate is 0.6mL/min, the temperature of the column incubator is 25 ℃, and the detector is a differential detector; according to HPLC analysis, the high-purity glycyrrhiza polysaccharide effective part is prepared by extraction and purification, and the relative molecular weight of the glycyrrhiza acidic polysaccharide effective part is 24.96KDa according to the retention time calculation;
determination of total sugar, uronic acid and protein content:
and (3) total sugar content determination:
measuring the total sugar content by adopting a phenol-sulfuric acid method, namely accurately weighing 10mg of a glucose standard substance, adding distilled water for dissolving, fixing the volume to 50mL, and accurately sucking 0, 0.4, 0.8, 1.2, 1.6 and 2.0mL of the standard substance into different test tubes respectively, wherein the final concentration of the standard substance is 0.2 mg/mL; adding distilled water to make each tube volume reach 2 mL; respectively and accurately sucking 50uL of the concentrated sulfuric acid into a 96-pore plate, quickly adding 150uL of concentrated sulfuric acid into each tested pore, adding 30uL of 5% phenol into each pore, shaking uniformly, standing at room temperature for 20min, standing at room temperature for 10min, and measuring absorbance at 490nm by using an enzyme-linked immunosorbent assay (ELIAS); taking the absorbance as an abscissa, taking the mass concentration of the standard solution as an ordinate, and taking distilled water as a blank control to prepare a standard curve; and (3) sample determination: preparing a glycyrrhiza polysaccharide effective part solution with the concentration of 200 mug/mL, determining the absorbance by the standard curve operation method, calculating the total sugar content, and calculating to obtain the total sugar content of 94.87%;
measurement of uronic acid content:
drawing a standard curve: accurately weighing 10.00mg of galacturonic acid standard, fully dissolving the galacturonic acid standard in distilled water, fixing the volume in a 10mL volumetric flask, preparing 1mg/mL solution, diluting to 0.2mg/mL standard solution, respectively taking 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL, placing the solution in a 15mL dry glass test tube with a plug, adding water to 1mL, sequentially adding 6.0mL sodium tetraborate sulfuric acid solution, mixing uniformly, placing in a boiling water bath, heating for 15min, taking out, cooling to room temperature, adding 80 mu L of 0.15% m-hydroxybiphenyl solution, mixing uniformly, standing for 30min at room temperature, and measuring the absorbance at 525nm by using an ultraviolet spectrophotometer; taking the mass concentration of the standard solution as an abscissa, taking the absorbance as an ordinate, and taking distilled water as a blank control to prepare a standard curve; accurately weighing 10.00mg of the glycyrrhiza polysaccharide effective part sample, fully dissolving the glycyrrhiza polysaccharide effective part sample in distilled water, fixing the volume to a 10mL volumetric flask, measuring the absorbance by the same standard curve operation method, and calculating to obtain the content of uronic acid in the glycyrrhiza polysaccharide to be 8.76%;
protein content determination:
adopting a BCA method: accurately measuring a 2mg/mL standard bovine serum albumin solution, and preparing a 25-2000 mu g/mL standard solution; preparing a working solution according to the operation of the BCA protein measurement kit specification, wherein the BCA reagent is divided into a reagent A and a reagent B, and the reagent B with the volume of 1 time is added into the reagent A with the volume of 50 times, and is fully shaken up for standby; measuring 25uL of standard solutions with different concentrations in a 96-well plate, respectively adding 175uL of BCA working solution, slightly oscillating, fully mixing, keeping the temperature in an incubator at 37 ℃ for 30min, and measuring the absorbance of the solution at 562nm by using an enzyme-linked immunosorbent assay; drawing a standard curve by taking the absorbance of the standard substance as an abscissa and taking the mass concentration of bovine serum albumin as an ordinate; accurately weighing 2.0mg of the glycyrrhiza polysaccharide effective part sample, adding 2mL of distilled water for dissolving, taking 25 mu L of sample solution to a 96-well plate, determining the absorbance value of the sample solution by the standard curve operation method, and calculating to obtain the protein content in the glycyrrhiza polysaccharide of 3.14%;
analysis of monosaccharide composition:
hydrolysis: taking 10mg of a purified glycyrrhiza polysaccharide effective part sample, adding 4mL of 2mol/L trifluoroacetic acid into a headspace bottle, sealing, hydrolyzing at 110 ℃ for 6h at constant temperature, adding a proper amount of methanol, evaporating to dryness under reduced pressure, and repeating for three times; acetylated derivatives: to the hydrolysate was added 8mg glycolic acid, 1mL pyridine and 1mL acetic anhydride at 90 deg.CHeating for 1h, cooling N2Blow-drying, diluting the acetylated monosaccharide alcohol with chloroform, and then carrying out gas chromatography-mass spectrometry (GS) analysis; GC analysis (FIG. 3) shows that the effective part of Glycyrrhiza polysaccharide consists of rhamnose, arabinose, xylose, mannose, glucose and galactose (relative molar ratio is 0.65:0.48:0.11:0.09:1: 0.67).
Example 3
The external anti-inflammatory action of the effective part of the glycyrrhiza polysaccharide is as follows:
the capacity of the effective part of the glycyrrhiza polysaccharide for inhibiting cyclooxygenase activity COX-2 is detected in vitro: briefly, 176. mu.L of buffer and 5. mu.L of COX-2 enzyme (0.5U) were added sequentially to the plate, then 4. mu.L of test sample (5 mg/mL in DMSO) or 4. mu.L of DMSO was added to the plate, allowed to stand at room temperature for 5min, then 5. mu.L of arachidonic acid AA (10. mu.M) was added, allowed to stand at 37 ℃ for 20min, 10. mu.L of 10% formic acid was added to the plate, and the next test was performed according to the procedures described in the PGE2 kit; the absorbance of the microplate at 450nm was obtained using Spectra Max MD5 (molecular devices, USA); calculating a formula according to the inhibition ratio: the inhibition rate is 1- (drug group-drug control/enzyme active group-enzyme active control) multiplied by 100 percent, and the COX-2 inhibition rate of the sample is obtained; when the concentration of the sample is 100 mug/ml, the inhibition rate of the licorice polysaccharide effective part on COX-2 enzyme is 60.3%.
Example 4
The licorice acidic polysaccharide has the function of reducing blood sugar in vitro:
PTP1B activity was assayed by hydrolyzing phosphate groups of pNPP with PTP1B using disodium p-nitrophenylphosphate (pNPP) as a substrate, producing a color reaction according to PTP1B, adding 1. mu.L (0.115mg/mL) of PTP1B protein solution to a 96-well plate in a buffer (50mM HEPES, pH 7.3,100mM NaCl, 0.1% BSA, and 1mM dithiothreitol), mixing 1. mu.L (sample concentration gradient) or 1. mu.L of dimethyl sulfoxide (DMSO), adding 35mM disodium p-nitrophenylphosphate (pNPP) after 10min, incubating at 25 ℃ in the dark for 30min, adding 10. mu.L 3M NaOH per well to terminate the reaction, and determining the absorbance at 405nm using a SpectraMmax 5 microplate reader (Molecular Devices, USA) in a system without an enzyme solution;
inhibition rate (I%) [ enzyme active group- (drug group)Drug control group)/(enzyme active group-enzyme active control group)]X 100%, calculation of inhibition ratio IC50Concentration, inhibitory rate IC of glycyrrhizic acid polysaccharide effective part on PTP1B enzyme50The value was 11.03. + -. 1.82. mu.g/mL (PTP1B Inhibitor 1.46. + -. 0.40. mu.g/mL).
Example 5
Experiment on the probiotic growth promoting effect of the liquorice acid polysaccharide:
the effect test of the effective part of the glycyrrhiza polysaccharide on the external growth of three probiotics is as follows: respectively inoculating lactobacillus delbrueckii subsp bulgaricus, bifidobacterium adolescentis and streptococcus faecalis liquid into MARS, TPY and KEA culture media, respectively adding samples with different concentrations into a basic culture medium, culturing for 16-18hrs, detecting the number of bacteria by using a cell counter, and repeating the steps for three times in each sample, wherein the number of the bacteria is shown in table 1;
TABLE 1 growth promoting effect of glycyrrhizic acid polysaccharide on probiotics
As can be seen from Table 1, the effective parts of the glycyrrhiza polysaccharide with different concentrations can significantly promote the growth of probiotic bifidobacterium adolescentis, lactobacillus delbrueckii subsp bulgaricus and enterococcus faecalis, so that the effective parts of the glycyrrhiza polysaccharide have the function of promoting the growth of probiotic bacteria, and have great reference values for the application of the glycyrrhiza polysaccharide in the research and development of medicines and health-care foods and the maximum development and utilization of the glycyrrhiza polysaccharide.
The preparation method and the application of the glycyrrhiza polysaccharide effective part provided by the invention have the advantages that the yield of the high-purity glycyrrhiza polysaccharide effective part obtained by the preparation method of glycyrrhiza rhizome through pretreatment, hot water extraction, alcohol precipitation, deproteinization, dialysis, ion exchange, sephadex chromatographic column chromatography and the like is 0.28%; the effective part of the polysaccharide has a molecular weight of 24.96KDa, and consists of rhamnose, arabinose, xylose, mannose, glucose and galactose (relative molar ratio is 0.65:0.48:0.11:0.09:1: 0.67). The effective part of the purified glycyrrhiza polysaccharide is verified to have the effects of resisting inflammation, reducing blood sugar and benefiting prebiotics. Can be used as a potential immunomodulator and prebiotics, and has wide application prospect in the fields of food, medicine and health care products.
The above embodiments are illustrative, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. A preparation method and application of a glycyrrhiza polysaccharide effective part are characterized by comprising the following steps:
pretreatment:
a. mechanically pulverizing Glycyrrhrizae radix for 120 s, sieving with 60 mesh sieve, separating impurities to obtain Glycyrrhrizae radix powder, adding petroleum ether into Glycyrrhrizae radix powder at a feed liquid weight ratio of 1:5, stirring at room temperature for 8hr, repeatedly extracting for 3 times until no oil is extracted, filtering, naturally drying in the shade, adding anhydrous ethanol into the filtrate at a feed liquid weight ratio of 1:5, stirring and refluxing for 4 hr, repeatedly extracting for 3 times until no pigment is extracted, filtering, and naturally drying until no petroleum ether and ethanol solvent remain to obtain defatted and decolorized Glycyrrhrizae radix powder;
hot water extraction:
b. b, adding water into the licorice powder obtained in the step a according to the weight ratio of 1:30, stirring and extracting for 3 times at the temperature of 100 ℃, extracting for 3 hours each time, combining the extracting solutions, centrifuging for 10min at the temperature of 4 ℃ at 7000rpm, and concentrating to obtain polysaccharide concentrated solution for later use;
alcohol precipitation:
c. c, precipitating the polysaccharide concentrated solution obtained in the step b by using absolute ethyl alcohol, and centrifuging for 10min at the temperature of 4 ℃ overnight at 8000rpm and 4 ℃ to obtain polysaccharide precipitate, namely crude licorice polysaccharide;
deproteinization:
d. d, adding distilled water into the polysaccharide precipitate obtained in the step c for redissolving, adding 1/3 volumes of chloroform-n-butanol mixed solution with the volume ratio of 4:1 for deproteinization, magnetically stirring for 30min, centrifuging at 3000rpm for 5min, and repeating the operation for 5 times;
and (3) dialysis:
e. d, dialyzing the polysaccharide solution after deproteinization in the step d for 48h by using a dialysis bag with the cut-off of 3500Da, dialyzing for 24h by using tap water and distilled water, concentrating, and performing vacuum freeze drying to obtain crude licorice polysaccharide;
DEAE-52 column chromatography:
f. preparing the crude licorice polysaccharide obtained in the step e into polysaccharide solution with the concentration of 10mg/ml by using ultrapure water, loading the solution into a DEAE-cellulose 52 ion exchange column, carrying out gradient elution by using the ultrapure water, 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution, detecting the absorption value at 490nm of each collecting pipe by using a phenol-sulfuric acid method to obtain neutral polysaccharide and two acidic polysaccharide parts, collecting the absorption peak of the acidic polysaccharide part eluted by 0.3M NaCl solution, dialyzing the eluent by using distilled water for 48h by using a dialysis bag with the interception amount of 3500Da, concentrating, and freeze-drying to obtain crude pure licorice acidic polysaccharide; polysaccharide solutions
Sephadex G-100 column chromatography:
g. and f, further purifying the crude purified glycyrrhiza acid polysaccharide obtained in the step f by Sephadex G-100 column chromatography, dissolving the glycyrrhiza acid polysaccharide to the concentration of 10mg/ml by using ultrapure water, then loading the glycyrrhiza acid polysaccharide, using the ultrapure water as an eluent, detecting the absorption value at 490nm by using a phenol-sulfuric acid method, collecting the eluent of an absorption peak, concentrating under reduced pressure, and freeze-drying to obtain the purified high-purity glycyrrhiza polysaccharide effective part.
2. Use of the glycyrrhiza polysaccharide effective fraction obtained by the method of claim 1 in the preparation of an anti-inflammatory agent.
3. Use of the glycyrrhiza polysaccharide effective fraction obtained by the method of claim 1 in the preparation of hypoglycemic drugs.
4. Use of the glycyrrhiza polysaccharide fraction obtained by the method according to claim 1 for the preparation of a medicament for promoting the growth of probiotic bacteria of bifidobacterium adolescentis, lactobacillus delbrueckii subsp bulgaricus and enterococcus faecalis.
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