CN112321738B - Preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase - Google Patents
Preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase Download PDFInfo
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Abstract
The invention provides a preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, belonging to the technical field of biological medicine. The preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase comprises sun drying cyclocarya paliurus leaves, pulverizing, sequentially defatting leaf powder, oven drying, soaking in water, and microwave extracting; centrifuging and filtering the extract to remove impurities, concentrating, performing two-aqueous phase extraction, taking aqueous phase for dialysis to remove salt, concentrating again, and performing fractional precipitation with ethanol to obtain polysaccharide precipitate; dissolving the polysaccharide precipitate in water, loading onto DEAE-52 cellulose column, eluting with NaCl solution, dialyzing to remove salt, concentrating to a certain concentration, performing ultrasonic degradation, concentrating, and freeze drying to obtain the final product. The preparation method has the advantages of low cost, high polysaccharide extraction rate, less impurities and higher alpha-glucosidase inhibition rate of the degraded polysaccharide.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase.
Technical Field
Diabetes is a common endocrine metabolism disease, and sugar metabolism dysfunction is caused by disorder of the endocrine system of a human body, so that the blood sugar value is higher than a normal level. In recent years, the prevalence rate of diabetes in China is gradually rising, and survey data shows that the prevalence rate of diabetes in China is 9.7%. In 2019, the number of Chinese diabetic patients is about 1.16 hundred million, and the Chinese diabetic patients become the countries with the largest number of diabetic patients all over the world.
Cyclocarya Paliurus (Cyclocarya Paliurus), also known as Cyclocarya Paliurus, prunus salicina, christina loosestrife, a bunch of cash and the like, belongs to the dicotyledonous plant juglans family, is only stored in China, is scattered in the south of China, is mixed in a wet evergreen forest with a mountain height of 500-2500 m, is a rare tree species which survives in the glacier century, and belongs to the national secondary protection plant. Cyclocarya paliurus branches and leaves are sweet in taste, have the effects of clearing heat, reducing swelling and relieving pain, people can make the branches and leaves of cyclocarya paliurus into tea products, commonly called as 'sweet tea', have remarkable curative effects on hyperglycemia which is a second killer of human beings, are called as a third tree of medical community after being praised as a first tree-willow and a second tree-Chinese yew of medical community by people, and are also praised as pandas of plant community. According to records in the book of Chinese materia medica resource, cyclocarya paliurus bark and leaves have the functions of clearing heat, reducing swelling and relieving pain. Modern medical research results show that cyclocarya paliurus has multiple obvious effects of reducing blood sugar, blood pressure and blood fat, enhancing immunity, resisting aging and the like, and is called as natural insulin by the medical field.
Tests show that the cyclocarya paliurus polysaccharide has a remarkable blood sugar reducing effect, Glu, Ins, C peptide and Gln of mice in a model group can be remarkably reduced in different dosage groups, and the effect of a high-dosage group is better; meanwhile, cyclocarya paliurus polysaccharide can remarkably improve the sugar tolerance of mice to glucose and starch, and has no remarkable influence on sucrose; HE staining is reported in documents to show that cyclocarya paliurus polysaccharide has a certain protection effect on islet tissue injury caused by alloxan. The blood sugar reducing mechanism of the traditional Chinese medicine is mainly realized in the aspects of improving insulin content, increasing insulin sensitivity, inhibiting glucose absorption, influencing receptor glucose metabolism, improving the free radical scavenging capacity of an organism, resisting lipid peroxidation and the like. Tests show that the cyclocarya paliurus polysaccharide has a remarkable inhibition effect on the activity of alpha-glucosidase, belongs to mixed non-competitive inhibition, and is a high-efficiency alpha-glucosidase inhibitor. The alpha-glucosidase inhibitor is a novel hypoglycemic drug, and regulates and controls the absorption of carbohydrate in the intestinal tract by inhibiting the activity of the alpha-glucosidase on the brush edge of the small intestine, thereby achieving the purposes of preventing and treating postprandial hyperglycemia and relieving hyperinsulinemia.
Researches find that the biological activity of the polysaccharide is related to the relative molecular weight of the polysaccharide, and the larger the relative molecular weight is, the larger the volume is, the more unfavorable the polysaccharide crosses multiple cell membrane barriers and enters into organisms to exert the biological activity; meanwhile, when the cyclocarya paliurus polysaccharide is used as a biological macromolecule to be combined with enzyme, only part of structural parts can be combined with the enzyme, and the structural parts are core parts for the polysaccharide to play a role. The core part of the polysaccharide is reserved by a proper physicochemical method, and some non-functional parts are degraded and cut, so that the function of the polysaccharide is favorably exerted, and the activity of the polysaccharide is improved. Compared with other methods for degrading polysaccharide, the ultrasonic degradation method has the advantages of low cost, energy and time saving, simple and convenient operation procedure, no need of using toxic and harmful reagents, no waste generation basically and wide industrial prospect. After Tang and the like adopt ultrasonic degradation to treat cyclocarya paliurus leaf polysaccharide, compared with undegraded polysaccharide, the sugar content, the uronic acid content and the protein content in the polysaccharide after ultrasonic degradation are not obviously changed, the composition proportion of monosaccharide is changed, and the polysaccharide after degradation has better DPPH (dehydroepiandrosterone) removal activity and hydroxyl radical removal activity. Therefore, the cyclocarya paliurus polysaccharide is feasible to be degraded by ultrasonic waves, the molecular weight of the cyclocarya paliurus polysaccharide can be reduced, and the biological activity of the cyclocarya paliurus polysaccharide is improved. Therefore, the production process for degrading cyclocarya paliurus polysaccharide by using ultrasonic is explored through a large number of experiments, the process can obviously improve the inhibitory activity of the cyclocarya paliurus polysaccharide on alpha-glucosaccharase, the product has a good blood sugar reducing effect, and the patent has a good application prospect and production practicability.
Disclosure of Invention
The invention provides a preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, which comprises the steps of obtaining relatively pure cyclocarya paliurus polysaccharide through microwave extraction and double aqueous phase extraction, carrying out fractional precipitation on cyclocarya paliurus polysaccharide ethanol, carrying out DEAE-52 cellulose column chromatography to obtain a component with relatively high activity of inhibiting the alpha-glucosidase, and then carrying out ultrasonic degradation to further improve the activity of inhibiting the alpha-glucosidase by the polysaccharide. The invention obtains the cyclocarya paliurus polysaccharide with higher activity of inhibiting alpha-glucosidase by combining multiple processes.
The invention discloses a preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, which comprises the following steps:
(1) sun drying cyclocarya paliurus leaves, pulverizing, sequentially defatting leaf powder, oven drying, soaking in water, and microwave extracting;
(2) centrifuging and filtering the extracting solution to remove impurities, and concentrating to obtain a concentrated solution I;
(3) carrying out double-water phase extraction on the concentrated solution I, and obtaining a polysaccharide solution I after water phase dialysis and desalination;
(4) concentrating the polysaccharide solution I again to obtain a concentrated solution II, and performing fractional precipitation on the concentrated solution II by using ethanol to obtain polysaccharide precipitate;
(5) dissolving the polysaccharide precipitate in water, loading onto DEAE-52 cellulose column, eluting with NaCl solution, dialyzing to remove salt to obtain polysaccharide solution II;
(6) and (3) concentrating the polysaccharide solution II to a certain concentration, performing ultrasonic degradation, concentrating, and freeze-drying to obtain a target product.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, the cyclocarya paliurus leaves are middle leaves, are picked in 9 th ten days, dried in the sun and then crushed, the leaf powder is sieved by a 40-mesh sieve, soaked in 80% ethanol for 5 hours to be degreased, and then dried at 60 ℃.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (1), the microwave extraction is microwave constant-temperature extraction, the used equipment is ORW1.5S-3E type microwave extraction equipment, the microwave power density is 200-600W/L, the extraction solvent is distilled water, the material-liquid ratio is 1: 10-30, the extraction temperature is 60-100 ℃, and the extraction time is 10-40 min.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (2), the extracting solution is centrifuged for 5-15 min by using a disc centrifuge under the condition of 6000-8000 r/min, and the supernatant is filtered by a plate-and-frame filter and then concentrated to 1/20-1/100 of the original volume.
In the preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (3), the aqueous two phases in the aqueous two-phase extraction are ethanol and (NH)4)2SO4The double water phases are formed, wherein the mass fraction of ethanol is 18-26 percent (NH)4)2SO4The mass fraction is 21-30%.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (4), ethanol is added into the concentrated solution for fractional precipitation by using ethanol until the concentration reaches 50%, 60%, 70% and 80% in sequence, and the concentrated solution is centrifuged for 10min at 3000r/min under each concentration, and polysaccharide precipitate is collected.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (5), the polysaccharide which is obtained by dissolving the polysaccharide precipitate in water is preferably polysaccharide which is obtained by precipitating and collecting the polysaccharide in 70% ethanol concentration, and is dissolved in distilled water, wherein the mass ratio of the polysaccharide to the water is 1: 10-50.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (5), the loading amount of the DEAE-52 cellulose column loading sample is 4mg/mL (polysaccharide mass/column volume), the NaCl solution elution is sequentially performed by using 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution elution, the elution part with the preferable concentration of 0.2mol/L NaCl is collected, and the polysaccharide solution II is obtained after desalting through semipermeable membrane dialysis.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (6), the polysaccharide solution II is concentrated to a certain concentration and then is subjected to ultrasonic degradation, wherein the polysaccharide solution II is concentrated to a concentration of 0.2-1.8 mg/mL; during ultrasonic degradation, the ultrasonic frequency is 20-40 KHz, the temperature is 30-90 ℃, the power density is 45-105W/L, and the working time is 10-50 min.
In the preparation method of the cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase, in the step (6), the degradation solution is concentrated and freeze-dried to obtain the polysaccharide solution subjected to ultrasonic degradation, the polysaccharide solution is concentrated to 1/20-1/100 of the original volume by rotary evaporation, and the concentrated solution is freeze-dried to obtain the target product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the cyclocarya paliurus middle leaf is used as the raw material after being dried in the sun, the cyclocarya paliurus leaf has the highest polysaccharide content in the middle leaf stage, and the extraction rate can be improved by using the leaf in the middle leaf stage as the raw material; compared with the existing drying, the sun-drying saves labor and energy, has lower cost, and simultaneously has lower sun-drying temperature, thereby avoiding the change of the properties of the polysaccharide in the leaves.
(2) The preparation method adopts microwave constant-temperature extraction, can avoid the damage of uncontrollable high temperature to the polysaccharide structure in the common microwave extraction, fully exerts the special non-thermal effect of the microwave, and has short extraction time and high efficiency; the polysaccharide of the cyclocarya paliurus is extracted by adopting a double aqueous phase, the pigment is dissolved in an ethanol phase, and the protein exists between the aqueous phase and the ethanol phase, so that the processes of activated carbon decolorization and organic reagent deproteinization are omitted, the polysaccharide extraction rate is high, and the impurities are few; the aqueous two-phase extraction system is easy to be amplified, is easy to be industrially produced and has lower cost.
(3) The preparation method comprises the steps of grading the polysaccharide by using the ethanol concentration, finding out the position where the highest-activity component exists, carrying out ultrasonic degradation by using the highest-activity component, cutting off some parts influencing function exertion in the structure, and reserving the core structure with biological activity, wherein the degraded polysaccharide has higher inhibition rate on alpha-glucosidase, and is a high-efficiency alpha-glucosidase inhibitor.
Drawings
FIG. 1 shows the development stages of cyclocarya paliurus leaves.
FIG. 2 shows ethanol/(NH) in the present invention4)2SO4Aqueous two phase diagram.
Detailed Description
In order to ensure the accuracy of the test and ensure the consistency of the test raw materials, the raw materials used in the embodiment and the parameter optimization test of the invention are provided by Yuanyang agriculture development Co., Ltd, Yunqiu, Zhejiang, Tanshui, a natural forest of 10-year-old Qingnqiu in Tuchangchang, Tanshui, 9 months later, leaves are collected, naturally dried in the sun, crushed by a hammer crusher, and sieved by a 40-mesh sieve for later use.
The method for measuring the polysaccharide content and the alpha-glucosidase inhibition rate in the embodiment and the parameter optimization experiment part of the invention comprises the following steps:
(1) method for measuring polysaccharide content by sulfuric acid phenol method
a. Preparing a glucose standard solution: accurately weighing 0.1000g of glucose dried at 96 +/-2 ℃ for 2h, dissolving the glucose in distilled water, adding 5mL of hydrochloric acid, and diluting the solution to 1000mL by using water to obtain a glucose standard solution.
b.preparation of 80% phenol solution: 80g of phenol (distilled in a sand bath, 0.1g of aluminum sheet and 0.05g of sodium carbonate are added to 100g of phenol, and 180 to 182 ℃ fractions are collected) is dissolved in 20mL of distilled water and stored at 4 ℃ in the dark.
c.5% working solution of phenol: it is prepared with 80% phenol before use.
0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0mL of glucose standard solution were taken out of the test tube, and the volume was made up to 1mL with water. Adding 1mL of phenol solution and 5mL of concentrated sulfuric acid, standing at room temperature for 30min, adjusting to zero with 1mL of distilled water as blank control instead of glucose standard solution, and measuring standard solutionAbsorbance A490And regressing the absorbance value by taking the glucose concentration as an abscissa, and solving a regression equation of the standard curve as follows: y is 11.06x +0.0328 (R)2＝0.9999)。
Taking 1mL polysaccharide extract to be tested, adding 1mL phenol solution and 5mL concentrated sulfuric acid, standing at room temperature for 30min, using 1mL distilled water to replace polysaccharide extract as blank control for zero adjustment, and testing absorbance A of polysaccharide extract490. Substituting the absorbance value into the regression equation of the standard curve to calculate polysaccharide concentration c (mg/mL) in the polysaccharide extract, so that the cyclocarya paliurus polysaccharide content (%) is (Vc/W) multiplied by 100%. Wherein V is the volume (mL) of the polysaccharide extract; w is the mass (mg) of cyclocarya paliurus leaf powder.
(2) Alpha-glucosidase inhibition assay
480. mu.L of the polysaccharide solution was placed in a 1mL EP tube, heated to a constant temperature in a 37 ℃ water bath, and then 20. mu.L of 0.25U/mL alpha-glucosidase and 500. mu.L of 0.1mmol/L pNPG which had been thermostated to 37 ℃ were added, and immediately after shaking, transferred to a 1cm cuvette to measure the absorbance at 400nm as a function of time. 480 μ L of water was used as a control sample. The absorbance is plotted as ordinate and the time as abscissa, and the slope of the straight line is the absorbance change rate per minute.
Inhibition rate ═ [ (control sample slope-test sample slope)/control sample slope ] × 100%
Parameter optimization test
(1) Leaf development period selection
The development period of cyclocarya paliurus leaves is divided into 4 periods according to reference documents, wherein the 4 periods are respectively as follows: the young (F1) stage is the smallest fully expanded leaf, while the large (F4) stage is the one that reaches the maximum leaf width and thickness, the new (F2) and middle (F3) refer to the leaves at a stage of development between F1 and F4 (fig. 1). The test results show that the polysaccharide contents of F1, F2, F3 and F4 are respectively 3.96%, 5.07%, 5.58% and 5.13%, and show a trend of ascending first and then descending, wherein the polysaccharide content of the leaves in the F3 period is the highest, so that the leaves are taken as raw materials in picking (F3).
(2) Optimization of microwave extraction process
Microwaves are high-frequency electromagnetic waves and have high energy. The microwave-assisted extraction method is based on the characteristic of strong microwave penetration capability, and utilizes the difference of microwave absorption capability of substances in a microwave field to ensure that certain areas or certain components of matrix substances are selectively dissolved. Therefore, the microwave-assisted extraction method has the advantages of short extraction time, high extraction rate and the like.
The microwave extraction equipment used in the invention is ORW1.5S-3E type microwave extraction equipment designed and produced by Nanjing Australian microwave science and technology Limited company, the microwave working frequency is 2450MHz, the microwave output power is 0-1500W and can be continuously adjusted, the extraction time and temperature can be set, the equipment can realize microwave constant-temperature extraction in the real sense, and under the condition of rated microwave output, a water cooling mode is adopted to ensure that the extraction temperature is kept unchanged, and the specific non-thermal effect of the microwave can be fully exerted. The microwave extraction process optimization test is as follows: accurately weighing 10g of cyclocarya paliurus powder, adding distilled water for extraction, wherein the material-liquid ratio is respectively as follows: 1:10, 1:15, 1:20, 1:25, 1: 30; the microwave power density is respectively as follows: 200. 300, 400, 500, 600W/L; the extraction temperature is respectively as follows: 60. 70, 80, 90 and 100 ℃; the extraction time is 10, 20, 30, 40, 50min respectively, and the absorbance value A of the extractive solution is measured by sulphuric acid phenol method after extraction490High absorbance values indicate high polysaccharide extraction. Selecting 3 levels for each factor to carry out a 4-factor 3-level orthogonal test, wherein the optimal extraction conditions of the test result are as follows: microwave power density of 500W/L, material-liquid ratio of 1:20, extraction temperature of 90 deg.C, and extraction time of 30 min. Under the condition, the extraction rate of polysaccharide is higher and is 5.14%.
(3) Optimization of two-aqueous phase system
The aqueous two-phase system refers to 2 immiscible aqueous phase systems formed spontaneously after aqueous solutions of 2 hydrophilic compounds are mixed at a certain mass fraction. The double-aqueous-phase extraction is a novel liquid-liquid extraction separation technology, has the advantages of simple operation, high separation efficiency, mild separation conditions and easy process amplification, has the prospect of industrially separating natural products, is similar to the conventional solvent extraction principle, namely the selective distribution of target substances between two phases is possibly related to factors such as intermolecular hydrogen bond action, van der waals force, hydrophobic action, interfacial properties and the like. At present, the existing extraction systems comprise high polymer-water, high polymer-salt-water, ionic liquid-salt-water, small molecular organic solvent-salt-water and the like.
The two-aqueous phase system optimization test is as follows: weighing 20g (NH)4)2SO4The solid was dissolved in 30g of distilled water to obtain a clear solution (NH)4)2SO4And stirring the solution, dropwise adding absolute ethyl alcohol until the solution is turbid, then adding 1g of distilled water until the system is clear, continuously dropwise adding absolute ethyl alcohol until the solution is turbid, and repeating the operation. Recording the mass of ethanol added at each cloud point and the total mass of the liquid, and calculating the sum of ethanol concentration (NH)4)2SO4Concentration, ethanol/(NH)4)2SO4The double water phase system phase diagram has single phase area in the lower left part and double phase area in the upper right part (FIG. 2).
Adding ethanol (NH) with certain mass4)2SO4The cyclocarya paliurus polysaccharide extracting solution and distilled water are placed in a 50mL centrifuge tube to prepare a double water phase system with the total mass of 30g, the double water phase system is fully vibrated, the centrifugal operation is carried out for 10min at 4000R/min after the double water phase system is kept stand for 1h to ensure complete phase separation, the volume of an upper phase and a lower phase and the mass concentration of the polysaccharide in each phase are respectively measured, and the ratio (R) and the polysaccharide distribution coefficient (K) of the double water phase system are calculated.
R＝Vb/Vt
K＝Cb/Ct
Y＝VbCb/(VtCt+VbCb)×100＝RK/(1+RK)×100％
In the formula: r is the volume ratio of the lower phase to the upper phase of the system; vtAnd VbVolume of upper and lower phases (mL) respectively; k is the distribution coefficient of polysaccharide in the system; ctAnd CbThe mass concentrations (mg/mL) of the polysaccharides in the upper and lower phases, respectively; y is the extraction rate of the polysaccharide in the lower phase.
After the phase separation of the system, the upper phase is ethanol phase, the color is darker due to the enriched pigment, and the lower phase is (NH)4)2SO4The phase, polysaccharide is concentrated in the lower phase, probably because polysaccharide is poorly soluble in high concentrations of ethanol. The fixed ethanol mass fraction is 24%, and 22%, 24%, 26%, 28%, 30% (NH) is selected between the longest line and the binodal line4)2SO4The mass fraction and the polysaccharide extraction rate are respectively as follows: 68.27%, 75.13%, 78.78%, 85.64%, 82.38%, preferably (NH)4)2SO4The concentration is 28%; fixation (NH)4)2SO4The mass fraction is 28%, and 5 different ethanol mass fractions are selected between the longest line and the double line respectively: 18%, 20%, 22%, 24% and 26%, and the polysaccharide extraction rates are respectively as follows: 82.86%, 90.23%, 88.54%, 88.01%, 87.36%, preferably 20% ethanol mass fraction.
(4) Polysaccharide fractional precipitation ethanol concentration optimization
Adding ethanol into the polysaccharide concentrated solution II until the concentration is 50%, 60%, 70% and 80% in sequence, centrifuging at each concentration to collect polysaccharide precipitate, freeze-drying to prepare a solution of 50 μ g/mL, wherein the inhibition ratios to alpha-glucosidase are respectively as follows: 76.47%, 67.59%, 82.14%, 75.17%, preferably 70% ethanol concentration.
(5) Sample loading elution solution concentration optimization
Loading DEAE-52 cellulose column with loading amount of 4mg/mL (polysaccharide mass/column volume), eluting with 0.1, 0.2, 0.3, 0.4mol/L NaCl solution in sequence, dialyzing each eluent to remove salt, lyophilizing to obtain polysaccharide powder, dissolving in water to obtain 20 μ g/mL solution, and determining the inhibition rate of each polysaccharide component to alpha-glucosidase: 76.47%, 82.14%, 67.59%, 75.17%, preferably 0.2mol/L NaCl solution concentration.
(6) Ultrasonic degradation process optimization
The ultrasonic wave degrades the polysaccharide through mechanical bond breaking and cavitation effects. The current research shows that the influence of ultrasonic waves on the physicochemical properties and the structure of polysaccharide mainly focuses on rheological characteristics, monosaccharide composition ratio, molar molecular weight, spatial micro-size, spatial conformation and the like, and the polysaccharide degrading effect is closely related to ultrasonic power, ultrasonic treatment time, ultrasonic temperature and polysaccharide concentration. The ultrasonic degradation process test is as follows:
preparing 0.2, 0.6, 1.0, 1.4 and 1.8mg/mL polysaccharide solution, performing ultrasonic degradation for 10min at constant temperature of 30 ℃ by using power density of 28KHz and 60W/L, diluting the polysaccharide solution to 10 mu g/mL, and measuring the inhibition rates of alpha-glucosidase, wherein the inhibition rates are respectively as follows: 46.83%, 52.34%, 48.27%, 50.72%, 49.21%; preparing 0.6mg/mL polysaccharide solution, performing ultrasonic degradation for 10min at constant temperature of 30, 45, 60, 75 and 90 ℃ by using power density of 28KHz and 60W/L, diluting the polysaccharide solution to 10 mu g/mL, and respectively measuring the inhibition rates of alpha-glucosidase as follows: 52.34%, 50.42%, 58.27%, 60.72%, 49.21%; preparing 0.6mg/mL polysaccharide solution, performing ultrasonic degradation for 10min at constant temperature of 75 ℃ by using frequencies of 20KHz, 28KHz and 40KHz and a power density of 60W/L, diluting the polysaccharide solution to 10 mu g/mL, and measuring the alpha-glucosidase inhibition rates as follows: 78.27%, 62.34%, 60.72%; preparing 0.6mg/mL polysaccharide solution, performing ultrasonic degradation for 10min at the constant temperature of 75 ℃ by using 20KHz of frequency and 45, 60, 75, 90 and 105W/L of power density, diluting the polysaccharide solution to 10 mu g/mL, and respectively measuring the inhibition rates of alpha-glucosidase as follows: 56.83%, 78.27%, 68.27%, 80.72%, 69.21%; preparing 0.6mg/mL polysaccharide solution, performing ultrasonic degradation for 10min, 20min, 30min, 40min and 50min at the constant temperature of 75 ℃ by using power density of 20KHz and 90W/L, diluting the polysaccharide solution to 10 mu g/mL, and respectively measuring the inhibition rates of alpha-glucosidase: 80.72%, 89.34%, 78.27%, 60.72% and 59.21%.
According to the conclusion of the above parameter optimization test, the technical solution of the present invention is further described in detail below with reference to specific examples, but the examples do not limit the scope of the present invention, and the specific examples described herein are only illustrative for the spirit of the present invention.
Example 1:
picking central leaves of cyclocarya paliurus in 9 months, drying in the sun, sieving with a 40-mesh sieve, soaking in 80% ethanol for 5h for degreasing, and drying at 60 ℃; putting 200g into a microwave extractor, adding distilled water according to a ratio of 1:20, and performing microwave extraction for 30min at 90 ℃ by using a power density of 500W/L; centrifuging the extractive solution with disk centrifuge at 6000r/min for 15min, filtering the supernatant with plate and frame filter, and concentrating to 1/60 of original volume to obtain concentrated solution I. Adding ethanol and (NH) into the concentrated solution I4)2SO4Wherein the mass fraction of ethanol is 20%, (NH)4)2SO4The mass fraction is 28%, stirring, centrifuging at 4000r/min for 10min to form aqueous two phase, and taking offRemoval by phase dialysis (NH)4)2SO4Polysaccharide solution I. Concentrating the polysaccharide solution I to obtain a concentrated solution II, carrying out fractional precipitation by using ethanol, collecting polysaccharide precipitate with the concentration of 70 percent, dissolving the polysaccharide precipitate in distilled water, loading the sample on a DEAE-52 cellulose column, wherein the loading amount is 4mg/mL, eluting by using a NaCl solution, collecting a 0.2mol/L NaCl solution, eluting a part of semipermeable membrane, and dialyzing to remove salt to obtain the polysaccharide solution II. And concentrating the polysaccharide solution II to 0.6mg/mL, and performing ultrasonic degradation at an ultrasonic frequency of 20KHz and a power density of 90W/L at a temperature of 75 ℃ for 20 min. And (3) performing rotary evaporation and concentration on the polysaccharide solution subjected to ultrasonic degradation, and performing freeze drying on the concentrated solution by using a freeze dryer to obtain a cyclocarya paliurus polysaccharide product. The polysaccharide product is dissolved in water, diluted to 10 mu g/mL, and the alpha-glucosidase inhibition rate is measured to be 90.25%.
Example 2:
picking central leaves of cyclocarya paliurus in 9 months, drying in the sun, sieving with a 40-mesh sieve, soaking in 80% ethanol for 5h for degreasing, and drying at 60 ℃; putting 200g into a microwave extractor, adding distilled water according to a ratio of 1:15, and performing microwave extraction at 85 ℃ for 20min by using a power density of 600W/L. Centrifuging the extractive solution with disc centrifuge at 7000r/min for 10min, filtering the supernatant with plate and frame filter, and concentrating to 1/50 of original volume to obtain concentrated solution I. Adding ethanol and (NH) into the concentrated solution I4)2SO4Wherein the mass fraction of ethanol is 18%, (NH)4)2SO4Stirring and mixing at 26 wt%, centrifuging at 4000r/min for 10min to form aqueous two phase, removing the aqueous two phase by dialysis (NH)4)2SO4Polysaccharide solution I. Concentrating the polysaccharide solution I to obtain a concentrated solution II, carrying out fractional precipitation by using ethanol, collecting polysaccharide precipitate with the concentration of 70 percent, dissolving the polysaccharide precipitate in distilled water, loading the sample on a DEAE-52 cellulose column, wherein the loading amount is 4mg/mL, eluting by using a NaCl solution, collecting a 0.2mol/L NaCl solution, eluting a part of semipermeable membrane, and dialyzing to remove salt to obtain the polysaccharide solution II. And concentrating the polysaccharide solution II to 0.8mg/mL, and performing ultrasonic degradation at 20KHz ultrasonic frequency, 70 deg.C power density of 95W/L for 15 min. And (3) performing rotary evaporation and concentration on the polysaccharide solution subjected to ultrasonic degradation, and performing freeze drying on the concentrated solution by using a freeze dryer to obtain a cyclocarya paliurus polysaccharide product. Dissolving polysaccharide product in water, and diluting to 10 μg/mL, the alpha-glucosidase inhibition was found to be 88.43%.
Example 3:
picking the middle leaves of cyclocarya paliurus in 9 months, drying in the sun, sieving with a 40-mesh sieve, soaking in 80% ethanol for 5h for degreasing, and drying at 60 ℃. Putting 200g into a microwave extractor, adding distilled water according to a ratio of 1:25, and performing microwave extraction at 90 ℃ for 30min by using a power density of 500W/L. Centrifuging the extractive solution with disk centrifuge at 6000r/min for 15min, filtering the supernatant with plate and frame filter, and concentrating to 1/70 of original volume to obtain concentrated solution I. Adding ethanol and (NH) into the concentrated solution I4)2SO4Wherein the mass fraction of ethanol is 22%, (NH)4)2SO4The mass fraction is 28%, stirring, centrifuging at 4000r/min for 10min to form aqueous two phase, removing the aqueous two phase by dialysis (NH)4)2SO4Polysaccharide solution I. Concentrating the polysaccharide solution I to obtain a concentrated solution II, carrying out fractional precipitation by using ethanol, collecting polysaccharide precipitate with the concentration of 70 percent, dissolving the polysaccharide precipitate in distilled water, loading the sample on a DEAE-52 cellulose column, wherein the loading amount is 4mg/mL, eluting by using a NaCl solution, collecting a 0.2mol/L NaCl solution, eluting a part of semipermeable membrane, and dialyzing to remove salt to obtain the polysaccharide solution II. And concentrating the polysaccharide solution II to 0.6mg/mL, and performing ultrasonic degradation at an ultrasonic frequency of 20KHz and a temperature of 80 ℃ and a power density of 85W/L for 25 min. And (3) performing rotary evaporation and concentration on the polysaccharide solution subjected to ultrasonic degradation, and performing freeze drying on the concentrated solution by using a freeze dryer to obtain a cyclocarya paliurus polysaccharide product. The polysaccharide product is dissolved in water, diluted to 10 mu g/mL, and the alpha-glucosidase inhibition rate is 87.64 percent.
Example 4:
picking central leaves of cyclocarya paliurus in 9 months, drying in the sun, sieving with a 40-mesh sieve, soaking in 80% ethanol for 5h for degreasing, and drying at 60 ℃; putting 200g into a microwave extractor, adding distilled water according to a ratio of 1:20, and performing microwave extraction at 85 ℃ for 20min by using a power density of 600W/L; centrifuging the extractive solution with disc centrifuge at 7000r/min for 10min, filtering the supernatant with plate and frame filter, and concentrating to 1/70 of original volume to obtain concentrated solution I. Adding ethanol and (NH) into the concentrated solution I4)2SO4Wherein the mass fraction of ethanol is 20%, (NH)4)2SO4Stirring and mixing at 26 wt%, centrifuging at 4000r/min for 10min to form aqueous two phase, removing the aqueous two phase by dialysis (NH)4)2SO4Polysaccharide solution I. Concentrating the polysaccharide solution I to obtain a concentrated solution II, carrying out fractional precipitation by using ethanol, collecting polysaccharide precipitate with the concentration of 70 percent, dissolving the polysaccharide precipitate in distilled water, loading the sample on a DEAE-52 cellulose column, wherein the loading amount is 4mg/mL, eluting by using a NaCl solution, collecting a 0.2mol/L NaCl solution, eluting a part of semipermeable membrane, and dialyzing to remove salt to obtain the polysaccharide solution II. And concentrating the polysaccharide solution II to 0.8mg/mL, and performing ultrasonic degradation at an ultrasonic frequency of 20KHz and a temperature of 75 ℃ and a power density of 95W/L for 18 min. And (3) performing rotary evaporation and concentration on the polysaccharide solution subjected to ultrasonic degradation, and performing freeze drying on the concentrated solution by using a freeze dryer to obtain a cyclocarya paliurus polysaccharide product. The polysaccharide product is dissolved in water and diluted to 10 mu g/mL, and the measured alpha-glucosidase inhibition rate is 89.47%.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (2)
1. A preparation method of cyclocarya paliurus polysaccharide for inhibiting alpha-glucosidase is characterized by comprising the following steps:
(1) the cyclocarya paliurus leaves are middle leaves, the leaves are dried in the sun and then crushed, the leaf powder is sieved by a 40-mesh sieve, 80% ethanol is soaked for 5 hours for degreasing, then the leaves are dried at 60 ℃, water is added for soaking, and then microwave extraction is carried out, wherein the microwave extraction is microwave constant-temperature extraction, the microwave power density is 200-600W/L, the material-liquid ratio is 1: 10-30, the extraction temperature is 60-100 ℃, and the extraction time is 10-40 min;
(2) centrifuging and filtering the extracting solution to remove impurities, and concentrating to obtain a concentrated solution I;
(3) performing aqueous two-phase extraction on the concentrated solution I, wherein the aqueous two phases in the aqueous two-phase extraction are ethanol and (NH)4)2SO4The double water phases are formed, wherein the mass fraction of ethanol is 18-26 percent (NH)4)2SO4The mass fraction is 21-30%, and the polysaccharide solution I is obtained after water phase dialysis and desalination;
(4) concentrating the polysaccharide solution I again to obtain a concentrated solution II, carrying out fractional precipitation on the concentrated solution II by using ethanol to obtain polysaccharide precipitates, adding ethanol into the concentrated solution II in the fractional precipitation of the concentrated solution II by using ethanol until the concentration reaches 50%, 60%, 70% and 80% in sequence, and centrifuging to collect polysaccharide precipitates precipitated by using 70% ethanol concentration;
(5) dissolving the polysaccharide precipitate in water, loading the polysaccharide precipitate on a DEAE-52 cellulose column, eluting the polysaccharide precipitate by using 0.1 mol/L, 0.2mol/L, 0.3 mol/L and 0.4mol/L NaCl solutions in sequence, collecting an eluted part at the concentration of 0.2mol/L NaCl, and dialyzing and desalting the eluted part by using a semipermeable membrane to obtain a polysaccharide solution II;
(6) and concentrating the polysaccharide solution II to a certain concentration, then carrying out ultrasonic degradation, wherein the ultrasonic frequency is 20KHz, the temperature is 70-80 ℃, the power density is 85-95W/L, and the working time is 15-25 min, and then concentrating, freezing and drying to obtain the target product.
2. The method for preparing cyclocarya paliurus polysaccharide inhibiting alpha-glucosidase, according to the claim 1, wherein in the step (6), the polysaccharide solution II is concentrated to a certain concentration and then is subjected to ultrasonic degradation, wherein the concentration of the polysaccharide solution II is 0.2-1.8 mg/mL.
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