Steady-state macro-quantity preparation method and application of lycium barbarum polysaccharide

Abstract

The invention discloses a lycium barbarum polysaccharide steady-state macro-quantity preparation method which comprises the following steps: step 1, crushing a lycium barbarum raw material to obtain lycium barbarum dry powder, and performing subcritical extraction on the lycium barbarum dry powder to obtain lycium barbarum fruit oil and lycium barbarum residues; 2, taking the Chinese wolfberry fruit residues in the step 1, and performing high-speed shearing and stirring extraction to obtain an LBPs extracting solution; 3, separating the LBPs extracting solution in the step 2 by using an ultrafiltration membrane, and collecting an LBPs concentrated solution; step 4, purifying the LBPs concentrated solution by using macroporous resin to obtain eluent, and collecting adsorption raffinate at the same time; and 5, combining the residual liquid and the eluent, carrying out secondary concentration by using an ultrafiltration membrane, and freeze-drying to obtain the product LBPs. Compared with the prior art, the invention has the advantages that a novel technology for steady-state macro preparation of lycium barbarum polysaccharide is provided, and the steady-state macro preparation method and application of lycium barbarum polysaccharide are used for researching the structural characteristics and in-vivo and in-vitro hypoglycemic activity of the obtained LBPs.

Description

Technical Field

[0001] The present invention relates to the technical field of Lycium barbarum polysaccharide extraction, and specifically refers to a method for the steady-state macroscale preparation of Lycium barbarum polysaccharide and its application. Background Art

[0002] Diabetes is one of the common metabolic diseases, and its main characteristics are elevated blood glucose and abnormal metabolic function. Prediabetes is an intermediate stage in the development of diabetes, and the population in this stage is at high risk of developing into diabetic patients.

[0004] Reasonable intervention in the prediabetes population to block, delay or even reverse abnormal glucose metabolism has become one of the important measures for preventing the occurrence of diabetes.

[0005] Both traditional Chinese medicine theory and modern pharmacology have found that some Chinese medicine polysaccharides have significant hypoglycemic effects. For example, Dendrobium officinale polysaccharide can reduce fasting blood glucose levels, regulate blood lipid levels, and thus improve insulin sensitivity; Coix lacryma-jobi polysaccharide can reduce blood glucose levels, maintain the viability of pancreatic islet β cells, and act as an insulin secretagogue; Codonopsis pilosula neutral polysaccharide has an obvious hypoglycemic effect on type 2 diabetic mice, can reduce oxidative stress, increase the activity of glycolytic enzymes and improve lipid metabolism.

[0006] Lycii Fructus is the mature and dried fruit of Lycium barbarum L., and it is also one of the first batch of traditional Chinese medicines included in the list of "medicinal and edible homologous" substances. Many ancient medical books such as "Shennong Ben Cao Jing" and "Newly Revised Materia Medica" record that it has the effect of "quenching thirst", that is, the hypoglycemic effect in modern medicine.

[0007] In view of the deficiencies of the current methods for the isolation and purification of LBPs, the present invention has developed a new technology for the steady-state macroscale preparation of LBPs by subcritical pretreatment - high-speed shear-assisted extraction - ultrafiltration membrane separation - macroporous resin purification, characterized the structure of the obtained LBPs, and evaluated the hypoglycemic activities of LBPs in vitro and in vivo, which is expected to provide intervention measures for the prediabetes population. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the above technical defects and provide a new technology for the steady-state macroscale preparation of Lycium barbarum polysaccharide, and a method for the steady-state macroscale preparation of Lycium barbarum polysaccharide and its application for studying the structural characteristics and hypoglycemic activities in vitro and in vivo of the obtained LBPs.

[0009] To solve the above technical problem, the technical solution provided by the present invention is as follows: A method for the steady-state macroscale preparation of Lycium barbarum polysaccharide, comprising the following steps:

[0010] Step 1: Take Lycium barbarum raw materials and crush them to obtain dried Lycium barbarum powder. Subject the dried Lycium barbarum powder to subcritical extraction to obtain Lycium barbarum fruit oil and Lycium barbarum residue.

[0011] Step 2: Take the Lycium barbarum residue in Step 1 and extract it through high-speed shearing and stirring to obtain an LBP extract.

[0012] Step 3: Use an ultrafiltration membrane to separate and collect the LBP concentrate from the LBP extract in Step 2.

[0013] Step 4: Purify the LBP concentrate using macroporous resin to obtain an eluate, and collect the adsorption residue liquid at the same time.

[0014] Step 5: Combine the adsorption residue liquid and the eluate, and use an ultrafiltration membrane for secondary concentration. Then freeze-dry to obtain the product LBP.

[0015] Preferably, in Step 1, it includes passing through a 20-mesh sieve after crushing to obtain the dried Lycium barbarum powder.

[0016] Preferably, the subcritical extraction includes placing the Lycium barbarum powder into an extraction bag, mixing it with the extraction agent butane at a material-solvent ratio of 1:2 (kg / L) for extraction. After completion, extract the liquid butane solvent and evaporate and desolventize the extraction material and the extraction solvent respectively to obtain Lycium barbarum residue and Lycium barbarum fruit oil.

[0017] Among them, the subcritical extraction is carried out twice for 1 h, and the extraction temperature is 40 °C and the pressure is 0.4 MPa.

[0018] Preferably, in Step 2, it includes feeding the Lycium barbarum residue into a high-speed shearing and stirring device with a rotation speed of 12 Hz and a water temperature of 60 °C, extracting for 1.5 h, filtering through a 200-mesh filter cloth to obtain a filtrate, and centrifuging the filtrate through a three-foot centrifuge and a tubular centrifuge to obtain the LBP extract.

[0019] The rotation speed of the three-foot centrifuge is 1400 rpm, and the rotation speed of the tubular centrifuge is 16000 rpm.

[0020] Preferably, in Step 3, it includes subjecting the LBP extract to nanofiltration using a membrane element with a cut-off molecular weight of 3500 Da. When the volume of the LBP extract Ⅰ is less than 40 - 50 L, pump in 100 L of purified water to continue nanofiltration. When the solution volume is less than 20 L, stop nanofiltration and collect the LBP concentrate.

[0021] Preferably, Step 4 includes wet-loading resin LX-360 into a resin column with a diameter-height ratio of 1:10, pumping the LBP concentrate into the resin column at a flow rate of 10 - 15 BV / h, collecting the adsorption residue liquid, and adding pure water for elution at a flow rate of 15 - 20 BV / h, and collecting the eluate.

[0022] The combined adsorption residue liquid and eluent are subjected to nanofiltration through a membrane element with a molecular weight cut-off of 5000 Da. When the liquid volume is less than 40 - 50 L, 100 L of purified water is pumped in to continue nanofiltration. When the liquid volume is less than 20 L, nanofiltration is stopped, and the resulting solution is freeze-dried to obtain the product LBPs.

[0023] On the other hand, the present invention discloses a method for the steady-state macroscale preparation of wolfberry polysaccharides to obtain wolfberry polysaccharides.

[0024] On the other hand, the present invention discloses the application of wolfberry polysaccharides in blood sugar lowering.

[0025] Preferably, it is applied in blood sugar lowering health foods or blood sugar lowering drugs.

[0026] The advantages of the present invention compared with the prior art are as follows: An integrated technology of subcritical pretreatment - high-speed shearing-assisted extraction - ultrafiltration membrane separation - macroporous resin purification is used to prepare LBPs, improving the existing separation and purification methods of hypoglycemic active components in wolfberries;

[0027] At the same time, in the present invention, the subcritical extraction technology is used to obtain wolfberry fruit oil rich in wolfberry pigments, especially rich in zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate, which can be used in the food and cosmetics industries. The wolfberries treated by subcritical extraction are removed of pigments, laying a foundation for the purification of LBPs;

[0028] The high-speed shearing technology enables the wolfberry residues to be subjected to cavitation, mechanical, and shearing effects, efficiently and rapidly releasing LBPs into the surrounding solvent at room temperature; The ultrafiltration membrane separation technology relies on the principle of mechanical sieving, retaining LBPs and removing water-soluble components such as monosaccharides and oligosaccharides; While the macroporous adsorption resin purification technology removes small molecules such as pigments in LBPs, further improving the purity of LBPs;

[0029] The present invention is easy to achieve seamless technical docking from laboratory small-scale tests to pilot-scale amplification and then to industrialization, and maximally retains the structure and activity of LBPs. Using the newly constructed technology, the polysaccharide yield of the prepared LBPs is 5.71%, the total sugar content is 73.75%, the protein content is 17.50%, and the uronic acid content is 8.36%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a flow chart for the steady-state macroscale preparation of LBPs.

[0031] Figure 2 It is the HPSEC (A) and HPLC (B) chromatograms of LBPs.

[0032] Figure 3 It is the TG (A), DTG, and DSC (B) curves of LBPs.

[0033] Figure 4 It is the atomic force microscope scanning image of LBPs.

[0034] Figure 5 They are the inhibition rates of LBPs against α-glucosidase (A) and α-amylase (B). Specific implementation manners

[0035] The present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Materials and instruments in the present invention:

[0037] Kunming mice, SPF grade, with body weight of 18 - 22 g, half male and half female, production license number: SCXK (Chuan) 2023 - 0019, provided by the Animal Experiment Center of Sichuan Academy of Traditional Chinese Medicine; Kunming mice, SPF grade, with body weight of 18 - 22 g and 22 - 24 g, all male, production license number: SCXK (Chuan) 2018 - 026, provided by the Experimental Animal Center of Sichuan University; Chinese wolfberry provided by Ningxia Zhongqi Wolfberry Trading Group Co., Ltd.; reference substances of D-glucose, D-galactose, D-mannose, L-arabinose, L-rhamnose, L-fucose, D-galacturonic acid, D-glucuronic acid with purity ≥ 99%, from the National Institutes for Food and Drug Control; dichloromethane, potassium dihydrogen phosphate, analytical grade, from Tianjin Damao Co., Ltd.; trifluoroacetic acid, analytical grade, from Chengdu Kelong Chemical Co., Ltd.; hydrochloric acid, analytical grade, from Xilong Chemical Co., Ltd.; sodium hydroxide, analytical grade, from Lian Anlong Bohua Pharmaceutical Chemistry Co., Ltd.; acetonitrile, chromatographic grade, PMP with purity ≥ 99%, from Shanghai Aladdin Biochemical Technology Co., Ltd.; BCA protein concentration determination kit from Beijing Lanbolide Biotechnology Co., Ltd.; alloxan from Melen Biotechnology Co., Ltd.; soluble starch with purity ≥ 99%, from Sigma Co., Ltd.; α-glucosidase 50 units / mg protein yeast from Shanghai Yuanye Bio-Technology Co., Ltd.; α-amylase ≥ 10 u / mg solid from Shanghai Yuanye Bio-Technology Co., Ltd.; carboxymethyl cellulose sodium from Chengdu Kelong Chemical Reagent Factory; glibenclamide tablets 2.5 mg × 100 tablets from Tianjin Pacific Pharmaceutical Co., Ltd.; protamine zinc insulin injection 3 mL: 300 U from Novo Nordisk.

[0038] STA449F3 type synchronous thermal analyzer from NETZSCH Company, Germany; STA449C thermogravimetric analyzer from Mettler Toledo Columbus OH, USA; atomic force microscope from Bruker Company, Germany; Agilent 1290 liquid chromatograph from Agilent Technologies, USA; Varioskan Flash type microplate reader from Thermo Fisher Scientific, Germany; blood glucose meter from Roche Diabetes Care, Switzerland.

[0039] New method for the steady-state macroscale separation and preparation of LBPs:

[0040] Pretreatment of Lycium barbarum fruits:

[0041] The steady-state macroscale preparation process of LBPs is as Figure 1 shown. Crush 25 kg of Lycium barbarum fruits with a pulverizer and pass through a 20-mesh sieve to obtain dry powder of Lycium barbarum fruits. Place the powder of Lycium barbarum fruits into the extraction bag of a subcritical extraction device, mix it with the extractant butane at a material-solvent ratio of 1:2 (kg / L), and carry out subcritical extraction twice under the conditions of an extraction temperature of 40 °C, an extraction pressure of 0.4 MPa, and in the dark, with each extraction lasting 1 h. After the subcritical extraction is completed, pump out the liquid butane solvent, and perform evaporation and solvent removal on the extraction material and the extraction solvent respectively. After the solvent removal of the extraction solvent, Lycium barbarum fruit oil is obtained. Take out the residue of Lycium barbarum fruits from the storage tank for standby;

[0042] Pump 240 L of purified water into a 400 L extraction tank, turn on the high-speed shear stirring system, set the rotation speed to 12 Hz, turn on the power supply of the water circulation temperature control machine, and set the temperature to 60 °C. After heating to 60 °C, add 20 kg of the residue of Lycium barbarum fruits and extract for 1.5 h. At the same time, install a 200-mesh wet filter cloth on a three-foot centrifuge and wait for filtration. After the extraction is completed, pump the LBPs extract into the three-foot centrifuge, set the rotation speed to 1400 rpm, and collect the filtrate into the feed liquid storage tank. Open the feed valve and discharge valve of the tubular centrifuge, centrifuge at a fixed rotation speed of 16000 rpm, collect the LBPs extract I, and pump it into the feed liquid bucket of the ultrafiltration membrane system. Install a membrane element with a cut-off molecular weight of 3500 Da for nanofiltration. When the volume of the LBPs extract I in the feed liquid bucket is less than 40 - 50 L, pump 100 L of purified water into the feed liquid bucket to continue nanofiltration. When the volume of the solution in the feed liquid bucket is less than 20 L, stop nanofiltration and collect the LBPs concentrated solution I;

[0043] Wet-load the resin LX-360 into a resin column with a diameter-height ratio of 1:10. After pretreatment, pump about 20 L of the LBPs concentrated solution I into the resin column at a flow rate of 10 - 15 BV / h (1 BV is 1 resin bed volume), collect the adsorption residual liquid, add pure water for elution at a flow rate of 15 - 20 BV / h, and collect the eluate. Combine the adsorption residual liquid and the eluate to obtain the LBPs extract II, and pump it into the feed liquid bucket of the ultrafiltration membrane system. Install a membrane element with a cut-off molecular weight of 5000 Da for nanofiltration. When the volume of the LBPs extract II in the feed liquid bucket is less than 40 - 50 L, pump 100 L of purified water into the feed liquid bucket to continue nanofiltration. When the volume of the solution in the feed liquid bucket is less than 20 L, stop nanofiltration and collect the LBPs concentrated solution II. Freeze-dry to obtain LBPs.

[0044] Chemical composition analysis and primary structure characterization of LBPs:

[0045] The total sugar content, uronic acid content, and protein content in LBPs were determined by the phenol-sulfuric acid method, sulfuric acid-carbazole method, and BCA kit, respectively, using glucose, galacturonic acid, and bovine serum albumin (BSA) as standards;

[0046] The molecular weight of the polysaccharide was determined by HPSEC-MALLS-RI. The LBPs were prepared into a 10 mg / mL sample solution with 0.2 mol / L NaCl, and after passing through a 0.22 μm microporous filter membrane, it was injected for analysis. The chromatographic conditions were as follows: the detectors were a differential refractive index detector (RID-G7162A) and a DAWN laser light scattering detector (Wyatt Technology Co., USA); the chromatographic columns were OHpakSB-806HQ and OHpak SB-804HQ (7.9 mm I.D.×300 mm, Shodex, Japan) used in series; the mobile phase was 0.2 mol / L NaCl, and the flow rate was 1.0 mL / min;

[0047] The monosaccharide composition of LBPs was determined by pre-column derivatization high performance liquid chromatography with PMP. Weighed 5 mg of polysaccharide powder, hydrolyzed the polysaccharide with 1 mL of 3 mol / L TFA at 110 °C for 8 h, used N 2 Dried it, removed the excess TFA, and added 300 μL of distilled water to dissolve it again. Then, 100 μL of the standard sample and 100 μL of the polysaccharide hydrolysate were taken into a test tube, 100 μL of 0.5 mol / L PMP solution and 100 μL of 0.3 mol / L NaOH solution were added, and after reacting in the dark at 70 °C for 1 h, 100 μL of 0.3 mol / L HCl solution was added to terminate the reaction. Then, 700 μL of dichloromethane was added, and the residual PMP was extracted 3 times to remove it. Centrifuged at 14000 rpm for 10 min, took out the supernatant, and after passing through a 0.22 μm microporous filter membrane, it was analyzed by an Agilent 1260 chromatograph. The chromatographic conditions were as follows: SinoChrom ODS-BP chromatographic column (5 μm, 4.6 mm I.D.×250 mm), the column temperature was 30 °C, the mobile phase was composed of 83% phosphate buffer solution (50 mmol / L, pH 6.7) and 17% acetonitrile, the flow rate was 1.2 mL / min, and the wavelength of the DAD detector was 245 nm;

[0048] The thermal properties of the LBPs powder were determined by a thermogravimetric analyzer (STA449F3, NETZSCH Co., Germany). The sample (2 - 3 mg) was sealed in an aluminum crucible, and then under a nitrogen flow rate of 50 mL / min, it was heated from 25 °C to 800 °C at a rate of 10 °C / min, and an empty aluminum crucible was used as a control;

[0049] The microstructure of LBPs was analyzed by atomic force microscope (AFM). LBPs were formulated into a 10 μg / mL sample solution with ultrapure water. 10 μL of the sample solution was dropped onto a silicon wafer soaked in absolute ethanol and left to dry naturally at room temperature. After drying, measurements were taken by AFM in Tapping mode, using Si 3 N 4 as a probe for scanning, and NanoScope Analysis software was used for image processing;

[0050] In vitro hypoglycemic activity assay:

[0051] Using the α-glucosidase inhibition rate as an evaluation index and making slight modifications to the experimental method, the in vitro hypoglycemic activity of LBPs was evaluated. The specific experimental procedure was as follows: 50 μL of LBPs solutions with different final concentrations (2 - 10 mg / mL) and 50 μL of α-glucosidase solution were added to a 96-well plate. After incubation at 37 °C for 15 min, 50 μL of 1 mmol / L p-NPG solution was added as the reaction substrate and incubation continued for 15 min. Finally, 50 μL of 0.2 mol / L Na 2 CO 3 solution was added to terminate the reaction, and the absorbance of the reaction solution was measured at 405 nm. PBS buffer was used instead of α-glucosidase solution as a blank, and acarbose was used as a positive control. The formula for calculating the α-glucosidase inhibitory activity is as follows:

[0052]

[0053] Where: A 1 represents the absorbance of the negative control; A 2 represents the absorbance of the negative blank; A 3 represents the absorbance of the sample control; A 4 represents the absorbance of the sample blank.

[0054] Animal experiments:

[0055] Acute toxicity test of mice orally administered LBPs:

[0056] Ten Kunming mice, weighing 18 - 22 g, half male and half female, were taken. After 2 days of adaptive feeding, they were randomly divided into different concentration LBPs groups (1320, 2650, 3970, and 5300 mg / kg) according to body weight and fasted for 16 h before dosing. The dosing dose, dosing time, poisoning symptoms, and death time were recorded. If no mice died, a maximum tolerated dose determination test was required;

[0057] Determination of the maximum tolerated dose of mice orally administered LBPs:

[0058] Forty Kunming mice, weighing 18 - 22 g, with half males and half females, were selected. After 2 days of adaptive feeding, they were randomly divided into a control group and an LBPs group according to body weight, and fasted for 16 h before administration. Record the dosage, administration time, poisoning symptoms and death time. On the 14th day after administration, dissect the mice to observe the appearance of the main organs and calculate the organ index;

[0059] Effect of LBPs on glucose tolerance in normal mice:

[0060] Sixty-three male Kunming mice, weighing 18 - 22 g, were randomly divided into a control group, a model group, a glibenclamide (2 mg / kg) group, and low, medium, and high dose (20, 100, and 500 mg / kg) LBPs groups, a total of 6 groups. Administer the drug at a dose of 0.1 mL / 10 g of body weight. Among them, the control group and the model group were given the same volume of 0.5% CMC-Na solution, administered for 7 days, once a day, and fasted for 16 h before the last administration. Measure the basal blood glucose of the mice. After administration, except for the control group, immediately orally administer 2 g / kg of soluble starch by gavage (administration volume 0.2 mL / 10 g). The control group was given an equal volume of 0.5% CMC-Na solution. Measure and record the changes in blood glucose values at 0.5, 1, 1.5, and 2 h. The calculation formula for the area under the blood glucose curve is as follows:

[0061] S = 0.25×(G 0 +G 2 ) + 0.5×(G 0.5 +G 1 +G 1.5 )

[0062] Where: S is the area under the curve, in mmol / L﹡h; G 0 is the basal blood glucose, in mmol / L; G 0.5 is the blood glucose at 0.5 h, G 1 is the blood glucose at 1 h, G 1.5 is the blood glucose at 1.5 h, G 2 is the blood glucose at 2 h, in mmol / L.

[0063] Effect of LBPs on blood glucose levels in alloxan-induced diabetic mice:

[0064] Eighty-four male Kunming mice, weighing 22-24 g, were randomly divided into a control group (12 mice) and a model group (72 mice) according to body weight. After 2 days of adaptive feeding, they were allowed to eat freely but not drink water for 20 h. The model group was intraperitoneally injected with 200 mg / kg alloxan, and the injection volume was 0.1 mL / 10 g. The control group was injected with the same volume of normal saline. The blood glucose level was measured 72 h after injection. Animals with a random blood glucose level > 16.7 mmol / L in the model group were considered successfully modeled. The successfully modeled animals were randomly divided into a model group, an insulin (200 U / kg) group, and low, medium, and high-dose LBP (20, 100, and 500 mg / kg) groups, for a total of 5 groups. The drugs were administered at a volume of 0.1 mL / 10 g. The control group and the model group were given a 0.5% CMC-Na solution, and the insulin group was subcutaneously injected in the neck and back. After 7 days of drug administration, the blood glucose level was measured before the last drug administration, and the changes in blood glucose levels in the mice were measured 1 h and 2 h after drug administration.

[0065] The experiment was repeated 3 times under the same conditions. SPSS 26.0 software was used for significance analysis. P < 0.05 indicated a significant difference. The results were expressed as the mean ± standard deviation, and Origin 2021 software was used for plotting.

[0066] Result analysis:

[0067] Ethanol precipitation is a common method for preparing Lycium barbarum polysaccharides (LBPs) in laboratories and industries. In recent years, it has been found that when organic lower alcohols are added to the polysaccharide solution, due to the enhancement of intramolecular hydrogen bonds, the polysaccharide molecules begin to dehydrate, and then conformational transformation and reassembly occur, which brings obstacles to the research on the structure, activity and structure-activity relationship of polysaccharides themselves. At the same time, the large-scale preparation of LBPs by ethanol precipitation method involves explosion-proof requirements in the production workshop and ethanol recovery, etc., significantly increasing the production cost. In this study, a new steady-state macroscale preparation technology of LBPs was constructed, which included subcritical pretreatment - high-speed shearing-assisted extraction - ultrafiltration membrane separation - macroporous resin purification. The subcritical extraction technology was used to obtain Lycium barbarum fruit oil rich in Lycium barbarum pigments, especially rich in zeaxanthin, zeaxanthin monopalmitate and zeaxanthin dipalmitate, which can be used in the food and cosmetic industries. The Lycium barbarum fruits pretreated by subcritical extraction were removed of pigments, laying a foundation for the purification of LBPs; the high-speed shearing technology made the Lycium barbarum residues subjected to cavitation, mechanical and shearing effects, and efficiently and rapidly released LBPs into the surrounding solvent at room temperature; the ultrafiltration membrane separation technology relied on the principle of mechanical sieving, retained LBPs, and removed water-soluble components such as monosaccharides and oligosaccharides; the macroporous adsorption resin purification technology removed small molecules such as pigments in LBPs, further improving the purity of LBPs. The integration of the above technologies not only easily realized seamless technical docking from laboratory small-scale test to pilot-scale amplification and then to industrialization, but also maximally retained the structure and activity of LBPs. Using the newly constructed technology, the polysaccharide yield of the prepared LBPs was 5.71%, the total sugar content was 73.75%, the protein content was 17.50%, and the uronic acid content was 8.36%;

[0068] Molecular weight (Mw) distribution and monosaccharide composition are key factors affecting the biological activity of polysaccharides. The Mw distribution results of LBPs are as Figure 2 shown in Figure A, presenting three different chromatographic peaks MwⅠ, MwⅡ and MwⅢ, with molecular weights of 480.10 kDa (24.7%), 33.31 kDa (54.8%) and 28.90 kDa (20.5%) respectively, indicating that the Mw distribution of LBPs has dispersion and inhomogeneity.

[0069] As Figure 2 shown in Figure B, the monosaccharide composition of LBPs is mannose (Man): rhamnose (Rha): glucuronic acid (GlcA): galacturonic acid (GalA): glucose (Glc): galactose (Gal): arabinose (Ara) = 2.67: 2.61: 1.83: 2.46: 31.20: 23.50: 35.73. Among them, the relative molar ratios of glucose, galactose and arabinose are significantly higher than those of other monosaccharides, accounting for more than 90% of the total monosaccharides, and are the main monosaccharide units constituting the backbone of LBPs;

[0070] Thermal stability analysis:

[0071] Thermogravimetric analysis is the main method for determining the thermal stability of polysaccharides and exploring the degradation temperature of macromolecules, which is crucial for evaluating the applications of polysaccharides in food processing and the pharmaceutical industry. The thermogravimetry (TG), derivative thermogravimetry (DTG), and differential scanning calorimetry (DSC) curves of LBPs are shown in Figure 3 Figures A and 3B. As can be seen from the figures, the mass loss of LBPs is mainly divided into three stages. The first stage occurs at 35°C - 155°C, and the mass loss in this stage is 6.73%. Moreover, in this temperature range, the heat flow change of the DSC curve is negative, and an exothermic peak appears downward at 52.17°C, indicating that an exothermic reaction occurs in the sample in this stage, which is related to the evaporation of free water and bound water in the polysaccharide; the second stage occurs at 240°C - 500°C, and the mass loss of the polysaccharide is the most severe in this stage, being 56.31%, and an endothermic peak appears upward in the DSC curve at 460.07°C, attributed to the depolymerization of the sugar chain and the destruction of the higher structure of the polysaccharide induced by high temperature in this stage, including the breakage of C-C and C-O bonds; the third stage occurs at 500°C - 800°C, and the weight change of the polysaccharide sample tends to be gentle in this stage, forming a solid residue mainly composed of minerals. Finally, when the temperature reaches 800°C, the residual mass of LBPs is 25.40%. The above results show that LBPs have relatively good thermal stability below 240°C, and a certain degree of heat treatment will not cause the thermal decomposition of the polysaccharide.

[0072] AFM analysis:

[0073] AFM is used to observe the apparent morphology and molecular characteristics of polysaccharides. The 2D and 3D AFM images of LBPs are shown in Figure 4 the figures. It can be observed from the figures that LBPs show a large number of irregular block and chain structures in aqueous solution. Among them, the height of some sugar chains in the 3D figure is between 1.0 - 5.0 nm. Generally, the height of a single sugar chain is between 0.1 - 1.0 nm. This result indicates that LBPs will aggregate in aqueous solution and do not all exist as single sugar chain molecules, which is due to the aggregation and entanglement of polysaccharide chains through hydrogen bonds and van der Waals forces, forming irregular aggregates.

[0074] Analysis of the inhibitory activities of Lycium barbarum polysaccharides against α-glucosidase and α-amylase:

[0075] Inhibiting the activities of α-glucosidase and α-amylase is an effective method to delay the digestion of carbohydrates in the gastrointestinal tract and cause a postprandial blood glucose increase.Figure 5 A and 5B respectively show the inhibitory effects of LBPs on α-glucosidase and α-amylase with acarbose as the positive control. The increase in the concentration of LBPs shows a concentration-dependent relationship with the inhibition rate of α-glucosidase. When the concentration of LBPs is 10 mg / mL, the inhibition rate of α-glucosidase is as high as 86.72 ± 1.51%, indicating that LBPs can exert good hypoglycemic activity through competitive inhibition of α-glucosidase. With the increase in the concentration of LBPs (1.4 - 22.5 mg / mL), the inhibitory effect on α-amylase also increases significantly. When the concentration of LBPs is 22.5 mg / mL, the inhibition rate of α-amylase is as high as 80.39 ± 1.42%. The above results show that LBPs have strong inhibitory activities on both α-glucosidase and α-amylase, but the concentration of LBPs required to inhibit α-amylase is significantly higher than that required to inhibit α-glucosidase.

[0076] Safety evaluation of orally administered LBPs in mice:

[0077] The results of the acute toxicity test of LBPs are shown in Table 1. No death occurred in any group of mice. The mental state, appetite, water intake, etc. of all mice were normal, and there were no obvious symptoms of poisoning. The results indicate that due to the limitations of the mass concentration and volume of the test drug, the median lethal dose of the test drug could not be measured, suggesting that LBPs has relatively high safety. To further accurately evaluate its safety, after single-dose administration with the maximum solubility concentration of the test drug and the maximum tolerable volume of mice, continuous observation was carried out for 14 days. As shown in Table 2, no death occurred in the mice, and compared with the blank control group, there were no significant differences in the body weight and organ indices (heart, liver, spleen, lung, kidney) of the mice, indicating that the maximum tolerable dose of LBPs for mice is 5318 mg / kg.

[0078] Table 1 Acute toxicity test of orally administered LBPs in mice

[0079]

[0080] Note: N is the number of test animals.

[0081] Table 2 Effect of LBPs on body weight and Organ Index in mice (mg / 10g, N = 20)

[0082] Table 2 Effect of LBPs on body weight and Organ Index in mice (mg / 10g,

[0083] N = 20)

[0084]

[0085] Note: N is the number of test subjects.

[0086] Effect of LBPs on blood glucose changes in mice:

[0087] The glucose tolerance level is an important means to evaluate the body's glucose metabolism ability. As shown in Table 3, compared with the control group, after intragastric administration of soluble starch at 0.5, 1, 1.5, and 2 h, the blood glucose values and the area under the blood glucose curve of the mice in the model group were significantly increased (P<0.01), indicating successful modeling; compared with the model group, the blood glucose values of the mice in the medium-dose group and high-dose group of LBPs were significantly decreased at 1.5 h after intragastric administration of soluble starch (P<0.05, P<0.01). Among them, the high-dose group of LBPs also significantly decreased the blood glucose values and the area under the blood glucose curve of the mice at 1 and 2 h after intragastric administration of soluble starch (P<0.01), suggesting that LBPs at a certain concentration can significantly improve the glucose tolerance ability of mice, and then promote the recovery of blood glucose to normal.

[0088] Table 3 Effect of LBPs on glucose tolerance in normal mice

[0089]

[0090]

[0091] Note: N is the number of test subjects; * indicates P<0.05 compared with the model group, and ** indicates P<0.01 compared with the model group.

[0092] The present invention comprehensively evaluated the hypoglycemic activity of LBPs by using α-glucosidase and α-amylase activity inhibition experiments, as well as alloxan-induced diabetic mouse models and oral glucose tolerance tests in normal mice.

[0093] The inhibition rates of LBPs on α-glucosidase and α-amylase activities were 86.72±1.51% and 80.39±1.42% respectively. Although they were not as good as the inhibition activity of acarbose, they had reached 86.85% and 83.92% of its maximum inhibition rate. Alloxan is one of the main chemicals for inducing diabetic models. It can lead to selective necrosis of β cells by inducing the formation of reactive oxygen species, thus forming insulin-dependent diabetes.

[0094] Therefore, alloxan-induced diabetic mice generally have the morphological characteristics of hyperglycemia and hypoinsulinemia. LBPs have a significant effect on the fasting blood glucose (FBG) of alloxan-induced diabetic mice. Compared with the model group, the FBG in the medium- and high-dose groups of LBPs decreased by 22.73% and 33.00% respectively, indicating that LBPs have potential hypoglycemic activity. Meanwhile, after the diabetic mice were given LBPs, the secretion of insulin and blood glucose control were improved, thus preventing the loss of tissue protein and the consumption of fat, and significantly slowing down the weight loss of the mice. In addition, the oral glucose tolerance test is a key indicator to measure the ability of cells to utilize glucose (the main energy source of the human body). Through the oral glucose tolerance test of normal mice in this study, it was proved that LBPs can improve the utilization rate of glucose and the glucose tolerance of mice, and thus play a good role in lowering blood sugar.

[0095] The above describes the present invention and its implementation manners, and such description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments to this technical solution without creative efforts without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a steady-state macro-scale of Lycium barbarum polysaccharide, characterized in that: The following steps are involved: Step 1: crushing wolfberry raw materials to obtain wolfberry dry powder, and subjecting the wolfberry dry powder to subcritical extraction to obtain wolfberry fruit oil and wolfberry residue; Step 2: extracting the wolfberry residue in step 1 by high-speed shearing and stirring to obtain LBPs extract; Step 3: Using an ultrafiltration membrane to separate the LBPs extract in step 2 to collect the LBPs concentrate; Step 4: Purify the LBPs concentrate using a macroporous resin to obtain an eluate, and collect the adsorption residual liquid; Step 5: Combine the residual liquid and the eluate, use an ultrafiltration membrane for secondary concentration, and freeze-dry to obtain the product LBPs.

2. The method for preparing a steady-state macro-quantity of Lycium barbarum polysaccharide according to claim 1, characterized in that: The step 1 includes crushing and then passing through a 20-mesh sieve to obtain wolfberry dry powder.

3. A method for preparing a steady-state macro-quantity of Lycium barbarum polysaccharide according to claim 2, characterized in that: The subcritical extraction comprises placing wolfberry powder into an extraction bag, mixing it with an extractant butane at a material-solvent ratio of 1:2 (kg / L) for extraction, and after the extraction, extracting the liquid butane solvent to evaporate and desolventize the extraction material and the extraction solvent to obtain wolfberry residue and wolfberry oil; Subcritical extraction was performed twice, lasting 1 h, with extraction temperature of 40°C and pressure of 0.4 MPa.

4. The method for preparing a steady-state macro-quantity of Lycium barbarum polysaccharide according to claim 2, characterized in that: The step 2 includes feeding the wolfberry residue into a high-speed shear stirring device with a rotation speed of 12 Hz and a water temperature of 60° C., extracting for 1.5 hours, filtering through a 200-mesh filter cloth to obtain a filtrate, and centrifuging the filtrate through a tripod centrifuge and a tubular centrifuge to obtain an LBPs extract; The rotation speed of the tripod centrifuge is 1400 rpm, and the rotation speed of the tubular centrifuge is 16000 rpm.

5. A method for preparing a steady-state macro-quantity of Lycium barbarum polysaccharide according to claim 3 or 4, characterized in that: The step 3 includes nanofiltration of the LBPs extract using a membrane element with a molecular weight cutoff of 3500Da. When the volume of the LBPs extract I is less than 40-50L, 100L of purified water is pumped in to continue nanofiltration. When the volume of the solution is less than 20L, the nanofiltration is stopped and the LBPs concentrate is collected.

6. A method for preparing a steady-state macro-quantity of Lycium barbarum polysaccharide according to claim 5, characterized in that: The step 4 comprises using resin LX-360 to wet-load a resin column with a diameter-to-height ratio of 1:10, pumping the LBPs concentrate into the resin column at a flow rate of 10 to 15 BV / h, collecting the adsorption residue, adding pure water at a flow rate of 15 to 20 BV / h for elution, and collecting the eluate; The adsorption residual liquid and the eluate were combined and nanofiltered through a membrane element with a molecular weight cutoff of 5000Da. When the liquid volume was lower than 40-50L, 100L of purified water was pumped in to continue nanofiltration. When the liquid volume was less than 20L, the nanofiltration was stopped and the obtained solution was freeze-dried to obtain the product LBPs.

7. Lycium barbarum polysaccharide is obtained by a steady-state macro-preparation method of Lycium barbarum polysaccharide as described in any one of claims 1 to 6.

8. Use of Lycium barbarum polysaccharide as claimed in claim 7 in lowering blood sugar.

9. The use of Lycium barbarum polysaccharide in reducing blood sugar as claimed in claim 8, characterized in that: Application in blood sugar lowering health food or blood sugar lowering medicine.

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