CN112480282A - Cyclocarya paliurus polysaccharide component and preparation method thereof - Google Patents
Cyclocarya paliurus polysaccharide component and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cyclocarya paliurus polysaccharide component, wherein monosaccharides of the cyclocarya paliurus polysaccharide component are in the following weight ratio: glc (20.96%), GalUA (14.71%), Gal (23.75%), Ara (17.9%), Man (7.8%), Rha (6.88%), GlcUA (4.43%), Xyl (1.67%), Fuc (1.9%) with an average molecular weight of 700D for the cyclocarya polysaccharide fraction. The preparation method mainly comprises five steps of water extraction, alcohol precipitation, dialysis, fractionation and drying. The cyclocarya paliurus polysaccharide component is applied to preparing the hypoglycemic drug.
Description
Technical Field
The invention relates to a cyclocarya paliurus polysaccharide component and a preparation method thereof.
Background
Cyclocarya paliurus (Batal) Iljinskaja is a plant of Cyclocarya of Juglandaceae, also called sweet tea tree, Qingqian plum, and Cyclocarya paliurus, and is widely distributed in Jiangxi, Guizhou, Hunan and other places in China. Cyclocarya paliurus is a specific medicinal plant in China, has the effects of clearing heat and removing toxicity, promoting fluid production to quench thirst, relieving summer heat, relieving pain and the like, and can be used for treating intractable tinea. Cyclocarya paliurus contains various chemical components, mainly including flavonoid, polysaccharide, triterpenes, organic acids, inorganic elements and the like. Cyclocarya paliurus polysaccharide is a bioactive component with multiple physiological functions in cyclocarya paliurus leaves. The study shows that the cyclocarya paliurus polysaccharide compound has various biological activities of reducing blood sugar, resisting tumors, reducing blood pressure, resisting oxidation, improving human immunity and the like.
The method for separating and purifying polysaccharide mainly comprises ethanol precipitation, ion exchange resin method, gel filtration column chromatography, affinity chromatography, and membrane separation. Chenxylon et al used different types of resin to separate and purify cyclocarya paliurus polysaccharide, preliminarily determined that D301R resin has a good purification effect, xie et al used membrane separation combined with gel permeation chromatography to separate cyclocarya paliurus polysaccharide into four components.
The alpha-glucosidase inhibitor can effectively control blood sugar and has exact treatment effect on I, II type diabetes. Alpha-glucosidase is an important enzyme involved in sugar metabolism in human bodies, and when carbohydrates such as starch, sucrose and the like enter gastrointestinal tracts, the carbohydrates are degraded into glucose by the alpha-glucosidase and can be absorbed by mucous membranes to enter metabolic cycle. The action mechanism of the alpha-glucosidase inhibitor is to generate competitive inhibition to alpha-glucosidase and reduce the generation rate of glucose, so that the blood sugar of a diabetic patient is stable and maintained at a certain level. Foreign studies have confirmed that acarbose is a representative drug for α -glucosidase inhibitors. At present, the research on the inhibition of alpha-glucosidase by cyclocarya paliurus polysaccharide mainly focuses on the research on the activities of total polysaccharide and ethanol precipitation products with different concentrations, and the research on the pharmacological activity of obtaining uniform polysaccharide components through a DEAE-cellulose ion exchange column does not exist.
Disclosure of Invention
One of the purposes of the invention is to provide a cyclocarya paliurus polysaccharide component, the inhibition rate of the component on alpha-glucosidase is higher than that of total polysaccharide of cyclocarya paliurus, the problems of complex components, large molecular weight, undefined sugar-reducing part and the like of the total polysaccharide of cyclocarya paliurus are solved, and the obtained component has a stable structure.
The polysaccharide component of the cyclocarya paliurus comprises the following monosaccharides in percentage by weight:
the average molecular weight is 700D.
The invention also aims to provide the application of the cyclocarya paliurus polysaccharide component in preparing the hypoglycemic drug. Pharmacological results show that the polysaccharide component of the cyclocarya paliurus can obviously inhibit a-glucosidase, and the effect of the polysaccharide component is better than that of acarbose and total polysaccharide of the cyclocarya paliurus.
The invention also aims to provide a preparation method of the cyclocarya paliurus polysaccharide component, which comprises the following steps:
1. water extraction: taking a proper amount of cyclocarya paliurus stem, branch and leaf, adding 15-25 times of water, preferably 25 times of water, carrying out reflux heating extraction at 100 ℃, wherein the extraction time is 1-3 hours, preferably 1 hour, the extraction frequency is 1-3 times, preferably 3 times, and after the extraction is finished, combining all water extract to obtain cyclocarya paliurus water extract; centrifuging for 15min at 4000 r/min;
2. alcohol precipitation and drying: concentrating the cyclocarya paliurus water extract to obtain cyclocarya paliurus concentrated solution, adding 95% ethanol for alcohol precipitation, wherein the concentration of the ethanol for alcohol precipitation is 60% -80%, preferably 70%, standing in the shade overnight, removing supernatant, and drying the precipitate under reduced pressure to obtain cyclocarya paliurus total polysaccharide;
3. and (3) dialysis: dissolving the obtained total cyclocarya paliurus polysaccharides, centrifuging, taking supernate, dialyzing by a dialysis bag with the density of 1KD to obtain a cyclocarya paliurus polysaccharide component 1 solution in the dialysis bag and a cyclocarya paliurus polysaccharide component 2 solution outside the dialysis bag, concentrating, freeze-drying, and respectively obtaining a cyclocarya paliurus polysaccharide component 1 and a cyclocarya paliurus polysaccharide component 2;
4. and (3) fractional separation: dissolving the cyclocarya paliurus polysaccharide component 1, performing DEAE-cellulose column chromatography, eluting with pure water and 0.2-0.5 mol/L NaCl in sequence, preferably 0.5mol/L to obtain cyclocarya paliurus polysaccharide component 3 solution and cyclocarya paliurus polysaccharide component 4 solution;
5. and (3) drying: and (3) concentrating and drying the obtained polysaccharide component 3 solution under reduced pressure to obtain the cyclocarya paliurus polysaccharide component.
The method for separating and purifying the cyclocarya paliurus polysaccharide component is simple, the monosaccharide content and the molecular weight range of the polysaccharide component are clear, the structure is stable, and pharmacological results show that the cyclocarya paliurus polysaccharide component can obviously inhibit a-glucosidase, and the effect of the polysaccharide component is better than that of acarbose and total polysaccharide of cyclocarya paliurus.
Drawings
FIG. 1 shows the molecular weight ranges of different polysaccharide components of cyclocarya paliurus.
Detailed Description
Example 1 cyclocarya paliurus polysaccharide fraction preparation.
1. The polysaccharide component of the cyclocarya paliurus is separated and purified by the following steps: taking a proper amount of cyclocarya paliurus stems and branches, adding 15-25 times of water, carrying out reflux heating extraction at 100 ℃, wherein the extraction time is 1-3 hours, the extraction times are 1-3 times, and combining all water extracts after the extraction is finished; concentrating the cyclocarya paliurus extract, adding 95% ethanol for alcohol precipitation, keeping the alcohol concentration of the alcohol precipitation to be 60% -80%, standing overnight in a shade place, removing the supernatant, taking the precipitate, and drying under reduced pressure to obtain 50.61g of cyclocarya paliurus total polysaccharide. Dissolving cyclocarya paliurus total polysaccharide in 10 times of distilled water, stirring overnight for full dissolution, 4000r/min, and centrifuging for 15min to obtain precipitate (impurity 6.9g) and supernatant (dissolved cyclocarya paliurus total polysaccharide). And (3) dialyzing the obtained supernatant (cyclocarya paliurus total polysaccharide) by a dialysis bag with the KD of 1KD to obtain a cyclocarya paliurus polysaccharide component 1 in the bag and a cyclocarya paliurus polysaccharide component 2 solution outside the bag. And (3) concentrating and drying the cyclocarya paliurus polysaccharide component 1 solution and the cyclocarya paliurus polysaccharide component 2 solution under reduced pressure to obtain a cyclocarya paliurus polysaccharide component 1(23.4g) and a cyclocarya paliurus polysaccharide component 2(14.8 g). Dissolving the cyclocarya paliurus polysaccharide component 1(18g), performing DEAE-cellulose column chromatography, and eluting with pure water and 0.2-0.5 Mol/L NaCl in sequence to obtain cyclocarya paliurus polysaccharide component 3 solution and cyclocarya paliurus polysaccharide component 4 solution; concentrating all polysaccharide component solutions under reduced pressure, and drying to obtain cyclocarya paliurus polysaccharide component 3(7.6g) and cyclocarya paliurus polysaccharide component 4(3.4 g).
2. And identifying the total polysaccharide of cyclocarya paliurus and the component structures of each polysaccharide of cyclocarya paliurus.
(1) And (3) analyzing the composition content: analyzing the sugar component content of the prepared cyclocarya paliurus total polysaccharide and cyclocarya paliurus polysaccharide component 1, cyclocarya paliurus polysaccharide component 2, cyclocarya paliurus polysaccharide component 3 and cyclocarya paliurus polysaccharide component 4 in the step 1, specifically determining the sugar content by adopting a phenol-sulfuric acid method, determining the uronic acid content by adopting an m-hydroxybiphenyl method, and determining the protein content by adopting a Coomassie brilliant blue method, wherein the specific results are as follows:
TABLE 1 Total polysaccharide of cyclocarya paliurus and the composition content of each polysaccharide component of cyclocarya paliurus
(2) And (3) monosaccharide composition determination: and (2) performing monosaccharide composition analysis on the prepared cyclocarya paliurus total polysaccharide in the step 1, the cyclocarya paliurus polysaccharide component 2, the cyclocarya paliurus polysaccharide component 3 and the cyclocarya paliurus polysaccharide component 4, and determining by adopting high performance liquid chromatography. The method comprises the following specific steps: using a Shimadzu HPLC system (LC-10ATvp pump and SPD-10AVD UV detector), Compass C18 chromatography column (4.6X 150mm), PBS (0.1M, pH 7.0-acetonitrile 81:19(v/v), flow rate of 1.0mL/min, sample size of 10. mu.L, detection wavelength of 245 nm.
TABLE 2 Total polysaccharide of cyclocarya paliurus and the polysaccharide composition and content in each of the polysaccharide components of cyclocarya paliurus
(3) And (3) measuring the molecular weight: the molecular weights of the cyclocarya paliurus total polysaccharide prepared in the step 1, the cyclocarya paliurus polysaccharide component 2, the cyclocarya paliurus polysaccharide component 3 and the cyclocarya paliurus polysaccharide component 4 are determined by adopting a high-efficiency gel permeation chromatography, and the specific method comprises the following steps: an LC-10Avp high performance liquid chromatograph system manufactured by Shimadzu corporation of Japan, an RID-10A parallax refraction detector, a Shimadzu CLASS-Vp workstation, a TSK-gel G-3000PWXL stainless steel chromatographic column (7.8X 300mm) with a column temperature of 40 ℃ are adopted. A cyclocarya paliurus polysaccharide sample is dissolved in a 0.2M NaCl aqueous solution, the concentration is 2mg/mL, and the loading amount is 20 mu L. The mobile phase was 0.2M aqueous NaCl at a flow rate of 0.6 mL/min. The molecular weight distribution results of the fractions are shown in FIG. 1.
Example 2: cyclocarya paliurus polysaccharide a-glucosidase inhibition experiment
Experimental materials and preparation of (i) phosphate buffer (PH 6.8): precision measuring K2HPO4(0.2mol/L) solution and KH2PO4(0.2mol/L) solution, and uniformly mixing; preparing a test solution: precisely weighing 0.5g of sample, and preparing into solution with concentration of 5.0, 10.0, 20.0, 40.0, 80.0, 160.0 μ g/ml with phosphate buffer solution; preparing an alpha-glucosidase solution: accurately weighing alpha-glucosidase powder, and preparing the alpha-glucosidase powder into 0.04U/ml alpha-glucosidase solution by using a phosphate buffer solution; preparation of substrate solution: precisely weighing 7.52g of PNPG powder, and preparing the PNPG powder into a solution with the concentration of 0.5mM by using a phosphate buffer solution; preparing a positive medicine acarbose solution: precisely weighing acarbose, and preparing into a solution with the concentration of 2mg/ml by using a phosphate buffer solution; sixth of ten2CO3Preparation of the solution: precisely weighing Na2CO3The powder was added with distilled water to prepare a solution having a concentration of 0.1mol/L (PH 9.8).
The experimental steps are as follows: firstly, precisely sucking 40 mul of test solution with different concentrations into a 96-well plate, adding 40 mul of alpha-glucosidase solution, incubating for 5min in a constant temperature box at 37 ℃, respectively adding 20 mul of PNPG solution, and shaking to be uniform to serve as the test solution (six times in parallel). Three groups are used as test groups, and three groups are used as test article background groups. Precisely pipette 40. mu.l of acarbose into a 96-well plate (six replicates), three groups were used as experimental groups and three groups were used as positive drug background groups. The test group is added with 40 mul of alpha-glucosidase solution, incubated for 5min at constant temperature of 37 ℃, added with 20 mul of PNPG solution and shaken evenly. Precisely sucking 40 mu l of buffer solution into a 96-well plate, adding 40 mu l of alpha-glucosidase solution, incubating at constant temperature of 37 ℃ for 5min, adding 20 mu l of PNPG solution, shaking uniformly to serve as negative control (six times in parallel), using three groups as negative control groups, and using three groups as negative background groups. Placing the sample in a constant temperature and humidity incubator, incubating at 37 deg.C for 40min, adding Na2CO3The reaction was stopped with 100. mu.l of the solution. Detecting the sample to be detected with enzyme linked immunosorbent assay (ELISA) detector with detection wavelength of 405nm, and recording OAnd D value.
Subject: respectively are cyclocarya paliurus total polysaccharides; cyclocarya paliurus polysaccharide component 1; cyclocarya paliurus polysaccharide component 2; cyclocarya paliurus polysaccharide component 3; cyclocarya paliurus polysaccharide component 4
TABLE 3 comparison of the inhibitory Activity of the total polysaccharide of cyclocarya paliurus and the polysaccharide component a-glucosidase of cyclocarya paliurus
And (4) conclusion: the activity test shows that the cyclocarya paliurus total polysaccharide and the cyclocarya paliurus component have alpha-glucosidase inhibition activity, but the cyclocarya paliurus component 3 has the strongest activity, and the activity is stronger than that of a control drug acarbose and cyclocarya paliurus total polysaccharide.
Claims (5)
1. The polysaccharide component of the cyclocarya paliurus is characterized by comprising the following monosaccharides in percentage by weight:
the average molecular weight is 700D.
2. Use of the cyclocarya paliurus polysaccharide fraction of claim 1 in the preparation of a hypoglycemic medicament.
3. A method of preparing a cyclocarya paliurus polysaccharide fraction as claimed in claim 1 or 2, comprising the steps of:
(1) water extraction: extracting stem, branch and leaf of cyclocarya paliurus medicinal material with water to obtain cyclocarya paliurus water extract;
(2) alcohol precipitation and drying: concentrating cyclocarya paliurus water extract, precipitating with ethanol, standing overnight, centrifuging to obtain precipitate, and vacuum drying to obtain cyclocarya paliurus total polysaccharide;
(3) and (3) dialysis: dissolving the obtained cyclocarya paliurus total polysaccharide, centrifuging, taking supernate, dialyzing by a dialysis bag with the density of 1KD to obtain cyclocarya paliurus polysaccharide component 1 solution in the dialysis bag and cyclocarya paliurus polysaccharide component 2 solution outside the dialysis bag, concentrating, freeze-drying and respectively obtaining cyclocarya paliurus polysaccharide component 1 and cyclocarya paliurus polysaccharide component 2;
(4) and (3) fractional separation: dissolving the cyclocarya paliurus polysaccharide component 1, performing DEAE-cellulose column chromatography, and eluting with pure water and 0.2-0.5 mol/L NaCl in sequence to obtain cyclocarya paliurus polysaccharide component 3 solution and cyclocarya paliurus polysaccharide component 4 solution;
(5) and (3) drying: and (3) drying the cyclocarya paliurus polysaccharide component 3 solution under reduced pressure to obtain the cyclocarya paliurus polysaccharide component.
4. The method of preparing a cyclocarya paliurus polysaccharide component of claim 3, wherein: the water extraction step in the step (1) is as follows: taking a proper amount of cyclocarya paliurus stems, branches and leaves, crushing, adding 15-25 times of water, carrying out reflux heating extraction at 100 ℃, wherein the extraction time is 1-3 hours, the extraction times are 1-3 times, and after the extraction is finished, combining all water extracts to obtain a cyclocarya paliurus water extract;
and (3) centrifuging for 15min under the centrifugation condition of 4000r/min in the step (2).
5. A method of preparing a cyclocarya paliurus polysaccharide component of claim 3 or claim 4, wherein:
(1) water extraction: taking a proper amount of cyclocarya paliurus stems, branches and leaves, crushing, adding 25 times of water, carrying out reflux heating extraction at 100 ℃, wherein the extraction time is 1h, the extraction times are 3 times, and after the extraction is finished, combining all water extracts to obtain a cyclocarya paliurus water extract;
(2) alcohol precipitation and drying: concentrating cyclocarya paliurus water extract, precipitating with ethanol, standing overnight, centrifuging to obtain precipitate, and vacuum drying to obtain cyclocarya paliurus total polysaccharide;
(3) and (3) dialysis: dissolving the obtained cyclocarya paliurus total polysaccharide, centrifuging, taking supernate, dialyzing by a dialysis bag with the density of 1KD to obtain cyclocarya paliurus polysaccharide component 1 solution in the dialysis bag and cyclocarya paliurus polysaccharide component 2 solution outside the dialysis bag, concentrating, freeze-drying and respectively obtaining cyclocarya paliurus polysaccharide component 1 and cyclocarya paliurus polysaccharide component 2;
(4) and (3) fractional separation: dissolving the cyclocarya paliurus polysaccharide component 1, performing DEAE-cellulose column chromatography, and eluting with pure water and 0.5mol/L NaCl in sequence to obtain cyclocarya paliurus polysaccharide component 3 solution and cyclocarya paliurus polysaccharide component 4 solution;
(5) and (3) drying: and (3) drying the cyclocarya paliurus polysaccharide component 3 solution under reduced pressure to obtain the cyclocarya paliurus polysaccharide component.
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