Purification of sophora japonica polysaccharide, and preparation method and application thereof

Abstract

The application belongs to the field of plant polysaccharides, and discloses a purified sophora japonica polysaccharide and a preparation method and application thereof. The purified sophora japonica polysaccharide SJP-VI is a heteropolysaccharide composed of galactose, rhamnose, arabinose and glucose, and the molar ratio of galactose, rhamnose, arabinose and glucose is 38.20:33.54:22.92:5.34. The weight average molecular weight of the purified sophora japonica polysaccharide SJP-VI is 10000-18000 Da. Experimental results show that the purified polysaccharide has good antioxidant, hypoglycemic and immune activation activities, has application potential for preparing functional food and medicine, and has a prospect of high-value utilization of sophora japonica.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant polysaccharides, and particularly relates to a purified sophora japonica flower polysaccharide, a preparation method thereof and an application thereof. BACKGROUND

[0002] Sophora japonica is the flower bud of Sophora japonica, which is a kind of medicinal and edible plant, and is rich in flavonoids, saponins, volatile oils, polysaccharides and other active ingredients.

[0003] Plant polysaccharides are natural macromolecular compounds, which are reported to have antioxidant, antitumor, hypoglycemic, anti-inflammatory, lipid-lowering and uric acid-lowering biological activities. Among them, the polysaccharides extracted from medicinal and edible plants also have the characteristics of non-toxic and no residue, which has attracted widespread attention in the fields of functional food and biological medicine.

[0004] Polysaccharides have very complex structural composition, and the biological activity of polysaccharides is closely related to their structure. Polysaccharides have four levels of structure, and the primary structure mainly includes the monosaccharide composition, connection mode, branching situation and substituent site of polysaccharides. The primary structure of polysaccharides determines the higher structure of polysaccharides, so the analysis of the primary structure of polysaccharides is of great significance in the study of the activity of polysaccharides.

[0005] Li Zhuoyue et al. found in "Study on the antioxidant and immune-enhancing effects of active polysaccharides from Sophora japonica flowers" (Shandong Animal Husbandry and Veterinary Medicine, 2018, 39(6): 1-3.) that Sophora japonica crude polysaccharides can effectively scavenge DPPH free radicals and H2O2, and show good antioxidant activity. Han Lingling et al. found in "Therapeutic effect of Sophora japonica alcohol extract on type 2 diabetes mellitus combined with hyperuricemia in mice" (Chinese Herbal Medicine, 2017, 40(11): 2697-2700.) that Sophora japonica polysaccharides have good hypoglycemic activity. By constructing a type II diabetes mouse model and feeding with Sophora japonica extract, it was found that the glucose utilization rate of diabetic mice fed with Sophora japonica polysaccharides was significantly improved, and the blood glucose concentration was reduced to a certain extent. The above experiments show that Sophora japonica polysaccharides have potential antioxidant activity and hypoglycemic activity, but the specific mechanism is not clear.

[0006] Zhao Qingyou in "the extraction of the Tai Shan Huaihua polysaccharide and the research of the immune enhancement of chicken rabbit" (Shandong Agricultural University, 2012.) With the chicken, the rabbit as the research object, found that the Huaihua polysaccharide solution can enhance the immune function of chicken and rabbit, can promote the development of chicken and rabbit immune organs. Chen Zhongjie et al. in "the test of the immune regulation of Huaihua polysaccharide on mice" (Chinese Journal of Veterinary Medicine, 2016, 52 (03): 115-117.) With different concentrations of Huaihua polysaccharide injection, found that it can improve the activity of mouse peritoneal macrophages, and promote the proliferation of mouse peripheral blood and spleen lymphocytes. Li Rongqiao et al. in "the influence of Huaihua polysaccharide on immune function of immunosuppressed mice" (Food Research and Development, 2016, (24): 155-159.) The establishment of the mouse model of immunosuppression, found that the Huaihua polysaccharide can improve the content of immune factors in the serum of immunosuppressed mice, and enhance the immunity of mice. The above experiments show that Huaihua polysaccharide has potential immune activation activity, but the specific mechanism is not clear.

[0007] It can be seen that Huaihua polysaccharide shows great potential in blood glucose lowering activity and immune activation activity, but the current research on Huaihua polysaccharide mainly takes the crude extract of Huaihua polysaccharide as the object, lacks the analysis of the fine structure of the purified polysaccharide, and has not carried out systematic and in-depth research on its biological activity. SUMMARY

[0008] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a purified Huaihua polysaccharide.

[0009] Another purpose of the present application is to provide a preparation method of the above-mentioned purified Huaihua polysaccharide.

[0010] Still another purpose of the present application is to provide the application of the above-mentioned purified Huaihua polysaccharide.

[0011] The purpose of the present application is achieved by the following scheme:

[0012] A purified Huaihua polysaccharide SJP-VI, which is a heteropolysaccharide composed of galactose, rhamnose, arabinose and glucose, the molar ratio of the monosaccharide composition galactose: rhamnose: arabinose: glucose is 38.20: 33.54: 22.92: 5.34.

[0013] Further, the weight average molecular weight of the above-mentioned purified Huaihua polysaccharide SJP-VI is 10000-18000 Da; preferably 10542 Da.

[0014] A preparation method of the above-mentioned purified Huaihua polysaccharide SJP-VI, which specifically comprises the following steps:

[0015] (1) taking the Sophora japonica L. powder, and extracting by ultrasonic-assisted hot water extraction method, and obtaining the water extract by concentration, decolorization, deproteinization, and alcohol precipitation to obtain the crude polysaccharide;

[0016] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange chromatography column, and then eluting, and collecting the eluate;

[0017] (3) subjecting the eluate obtained in step (2) to dialysis bag dialysis and concentration;

[0018] (4) freeze-drying the concentrated solution after step (3) to obtain the purified Sophora japonica L. polysaccharide.

[0019] Further, in step (1), the ultrasonic-assisted hot water extraction method refers to ultrasonic treatment followed by hot water extraction, wherein the ultrasonic treatment time is 5-20 min (such as 5, 10, 15, 20 min), and preferably 10 min, and the ultrasonic treatment power is 300 W; the hot water extraction temperature can be 80-100 ℃, and the hot water extraction time is 1-5 h; preferably, the hot water extraction temperature is 85 ℃, and the extraction time is 2 h.

[0020] Further, in step (1), the Sophora japonica L. powder and water have a solid-liquid ratio (W / V, g / mL) of 1:10-1:40 (such as 1:10, 1:20, 1:30, 1:40); in an embodiment of the present application, the solid-liquid ratio is preferably 1:25.

[0021] Further, in step (1), the ultrasonic-assisted hot water extraction method is performed for 1-5 times, and preferably 3 times.

[0022] Further, in the decolorization step of step (1), the macroporous resin D354FD is used, and the macroporous resin D354FD is mixed with the concentrated solution at a volume ratio of 1:1, and stirred at 55 ℃ for 3 h for decolorization.

[0023] Further, in the deproteinization step of step (1), the Sevage method is used.

[0024] Further, in the alcohol precipitation step of step (1), the volume ratio of alcohol to the concentrated solution after deproteinization is 1-10:1 (such as 1:1, 3:1, 5:1, 10:1); preferably 4:1; and the alcohol is 95% ethanol.

[0025] In an embodiment of the present application, step (1) comprises: taking the Sophora japonica L. powder, ultrasonic-assisted hot water extraction, collecting the supernatant, and concentrating; decolorizing by using the macroporous resin D354FD, recovering the concentrated solution after removing the resin, deproteinizing by using the Sevage method, collecting the supernatant, and concentrating; alcohol precipitation overnight, collecting the polysaccharide, and freeze-drying to obtain the crude polysaccharide.

[0026] Further, in step (2), the ion exchange column is a cellulose ion chromatography column, and the filler of the cellulose column is DEAE-52.

[0027] Further, in step (2), gradient elution is used, and the eluent used in the gradient elution is a NaCl solution with a concentration of 0-0.3 mol / L.

[0028] In one embodiment of the present application, step (2) comprises: passing the aqueous solution of the crude polysaccharide obtained in step (1) through a DEAE-52 cellulose ion exchange column, eluting with eluents with different concentrations, collecting the eluents, and concentrating; and preferentially collecting the obtained solution eluted by the 0.25 mol / L NaCl eluent for concentration.

[0029] Further, in step (3), the molecular weight cut-off of the dialysis bag is 6000-10000 Da (such as 6000, 7000, 8000, 9000, 10000 Da); in one embodiment of the present application, the molecular weight cut-off is 8000 Da.

[0030] In one embodiment of the present application, step (3) comprises: placing the eluent obtained in step (2) in a dialysis bag, dialyzing with running water for 48 h, and then dialyzing with deionized water for 48 h. After dialysis, the solution is concentrated, freeze-dried, and weighed, to obtain the purified polysaccharide.

[0031] The purified sophora japonica polysaccharide SJP-VI described above can be applied in the fields of preparing antioxidant functional food and medicine, blood glucose-lowering functional food and medicine, and immune-enhancing functional food and medicine.

[0032] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0033] The present application uses sophora japonica as a raw material to extract and separate a purified polysaccharide SJP-VI, and analyzes and identifies the molecular weight, monosaccharide composition, and chemical structure of the purified polysaccharide SJP-VI, and determines the weight average molecular weight and structural composition of the purified polysaccharide SJP-VI. In vitro experiments show that the purified polysaccharide SJP-VI has significant antioxidant capacity; can play a blood glucose-lowering activity by inhibiting gluconeogenesis and promoting glycogen synthesis; and can enhance immune activity by regulating the expression of immune-related factors and increasing the accumulation of cytokines. Based on this, the purified polysaccharide SJP-VI has good antioxidant, blood glucose-lowering, and immune activation activities, has application potential for preparing functional food and medicine, and has a prospect for high-value utilization of sophora japonica. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 FIG. 4 is a GPC spectrum of SJP-VI in Example 2.

[0035] Figure 2 FIG. 6 is an infrared spectrum of SJP-VI in Example 2.

[0036] Figure 3 Gas chromatogram of the standard.

[0037] Figure 4 Gas chromatogram of SJP-VI in Example 2.

[0038] Figure 5 Total ion current chromatogram of SJP-VI in Example 2.

[0039] Figure 6 Mass spectrum of SJP-VI in Example 2, wherein a) T-GalpNAc, b) T-Rhap, c) T-GlcpNAc, d) 1,4-Arap, e) 1,2-Arap, f) 1,6-Galp, g) 1,2,3,4-Rhap.

[0040] Figure 7 Graph of the hypoglycemic effect of SJP-VI at low, medium and high concentrations on IR-HepG2 cell model.

[0041] Figure 8 Graph of the effect of SJP-VI at low, medium and high concentrations on IR-HepG2 cell hexokinase (HK).

[0042] Figure 9 Graph of the effect of SJP-VI at low, medium and high concentrations on IR-HepG2 cell pyruvate kinase (PK).

[0043] Figure 10 Graph of the effect of SJP-VI at low, medium and high concentrations on IR-HepG2 cell glucose-6-phosphatase (G-6-Pase).

[0044] Figure 11 Graph of the effect of SJP-VI on reactive oxygen species (ROS) in IR-HepG2 cells.

[0045] Figure 12 Graph of the regulatory effect of SJP-VI on the hypoglycemic pathway in IR-HepG2 cells.

[0046] Figure 13 Graph of the toxic effect of SJP-VI on macrophage RAW264.7 cells.

[0047] Figure 14 Graph of the effect of SJP-VI on the phagocytosis of neutral red by RAW264.7 cells.

[0048] Figure 15 Graph of the effect of SJP-VI on the release of cytokines IL-6 and TNF-α by macrophages.

[0049] Figure 16Figure 1 shows the effect of SJP-VI on the mRNA expression of IL-6 and TNF-α produced by macrophages. DETAILED DESCRIPTION

[0050] The present application is described in further detail below with reference to Examples and the accompanying drawings, but the embodiments of the present application are not limited thereto. In the Examples, unless otherwise specified, the procedures were carried out under conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used were conventional products that can be obtained commercially, unless otherwise specified.

[0051] The reagents used in the Examples were conventional products that can be obtained commercially, unless otherwise specified.

[0052] In the present application, "Sophora japonica" is the flower bud of Sophora japonica, which is a plant in the family Leguminosae, and is a food and medicine dual-purpose plant.

[0053] Example 1: Isolation and extraction of purified Sophora japonica polysaccharide SJP-VI

[0054] 1. Extraction of Sophora japonica crude polysaccharide by ultrasonic-assisted hot water extraction

[0055] A dried Sophora japonica 100 g was crushed, and mixed with distilled water in a beaker at a ratio of 1:25 (W / V, g / ml). The mixture was subjected to ultrasonication for 10 min using an ultrasonic instrument (ultrasonic power 300 W) and then water-bathed at 85°C for 2 h. The supernatant was collected and concentrated, and the ultrasonic treatment and hot water extraction were repeated three times. The total supernatant was concentrated to 200 mL. Activated macroporous resin D354FD was mixed with the concentrated solution at a ratio of 1:1, and stirred at 55°C for 3 h to decolorize. After decolorization, the macroporous resin was removed, and the Sevage method was used to remove protein. After protein removal, the supernatant was collected, concentrated to 200 mL, and four times the volume of 95% ethanol was added. The polysaccharide sample was collected and dried after alcohol precipitation overnight to obtain Sophora japonica crude polysaccharide.

[0056] 2. Isolation and purification of Sophora japonica crude polysaccharide by DEAE-52 cellulose column chromatography

[0057] The DEAE-52 ion chromatography column filler was soaked overnight with deionized water, and then sequentially soaked with 0.5 mol / L HCl and 0.5 mol / L NaOH solution for 1.5 h each time. After each soaking, the filler was washed with deionized water until neutral. The filler was degassed without heating using a rotary evaporator for half an hour. The treated filler was loaded into a glass chromatography column, and the constant flow pump speed was adjusted.

[0058] The alcohol precipitated crude polysaccharide SJP was dissolved (dissolved with ultrapure water) into a solution of 10 mg / mL, and then was loaded onto a DEAE-52 ion chromatography column system after stabilization. Elution was performed with deionized water, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L NaCl solution in sequence. The sample amount was 100 mg, the flow rate was 1 mL / min, and one tube was collected every 5 min. Each component was collected for 100 tubes. The absorbance value of each tube was determined by the phenol-sulfuric acid method. The elution curve was plotted according to the determination results.

[0059] The eluate eluted by 0.25 mol / L NaCl solution was collected and concentrated under reduced pressure. The concentrated solution was placed in a dialysis membrane with a molecular weight cut-off of 8000 Da for dialysis. First, water dialysis was performed for 48 h, and then deionized water dialysis was performed for 48 h. The solution after dialysis was concentrated, freeze-dried and weighed to obtain the sophora japonica polysaccharide component eluted by 0.25 mol / L NaCl eluate, named SJP-VI.

[0060] Example 2: Structure identification of purified sophora japonica polysaccharide SJP-VI

[0061] The primary structure of the purified sophora japonica polysaccharide SJP-VI was preliminarily analyzed by infrared spectrum analysis, monosaccharide composition analysis and methylation analysis.

[0062] 1. Determination of molecular weight

[0063] 2 mL of purified sophora japonica polysaccharide SJP-VI was dissolved in 0.02 mol / L potassium dihydrogen phosphate solution and then passed through a water-soluble filter membrane (0.45 μm) for GPC analysis.

[0064] 2. Infrared spectrum analysis of purified sophora japonica polysaccharide SJP-VI

[0065] 2 mg of SJP-VI was weighed and pressed into a transparent wafer using the tabletting method. Infrared scanning was performed in the range of 4000-500 cm -1

[0066] 3. Monosaccharide composition analysis of purified sophora japonica polysaccharide SJP-VI

[0067] After SJP-VI was hydrolyzed into monosaccharides with 2 mol / L trifluoroacetic acid solution (TFA), it was derivatized and passed through a 0.22 μm organic membrane for gas phase analysis. Rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose were set as standard samples. 10 mg of each of the 7 standard samples was accurately weighed, mixed and derivatized to prepare a mixed standard sample, which was passed through a 0.22 um organic membrane for gas phase analysis.

[0068] 4. Methylation analysis of purified sophora japonica polysaccharide SJP-VI​

[0069] Take 20 mg SJP-VI sample into centrifuge tube, dissolve with 7.5 mL dimethyl sulfoxide (DMSO), ultrasonic 1 h to completely dissolve the sample. After adding 200 mg sodium hydroxide, oscillate in 55 °C water bath for 6 h. In ice water bath environment, slowly add 5 mL iodomethane, ultrasonic 30 min until the solution turns light yellow, then add 5 mL deionized water to terminate the reaction, and after water dialysis for 24 h with 3500 Da dialysis bag, transfer the solution to a chicken heart bottle and dry with a vacuum rotary evaporator at 55 °C. Repeat the above steps three times to complete the methylation. After dialysis of the third methylation, transfer the dialysis product into a separatory funnel, extract with chloroform three times, collect the organic phase into a chicken heart bottle, and rotary evaporate at 55 °C to dryness. Add 5 mL 2 mol / L trifluoroacetic acid to the chicken heart bottle to completely dissolve the dried methylation product, transfer into a hydrolysis tube, and acid hydrolyze in an oven at 110 °C for 8 h. Transfer the acid-hydrolyzed sample into a chicken heart bottle, rotary evaporate at 50 °C to dryness, then add 1-2 mL methanol to dissolve the sample and rotary evaporate again to dryness, repeat three times to completely remove residual trifluoroacetic acid. Add 1 mL pyridine and 1 mL acetic anhydride solution to the above rotary dried sample, boil in a water bath for 30 min, wash repeatedly with methanol three times to obtain a rotary dried sample, dissolve the sample with 2 mL CH2Cl2, filter through a 0.22 μm organic filter membrane into a gas phase bottle for GC-MS analysis.

[0070] 5、Results

[0071] 5.1, Molecular weight of purified sophora flower polysaccharide SJP-VI

[0072] The GPC spectrum of SJP-VI is shown in Figure 1 , which shows that the weight average molecular weight of SJP-VI is 10542 Da.

[0073] 5.2, Infrared spectrum analysis of purified sophora flower polysaccharide SJP-VI

[0074] As shown in Figure 2 , 3750-3000 cm -1 is the O-H compression vibration zone, SJP-VI (3423 cm -1 ) has an absorption peak in this range, which is the characteristic absorption peak of O-H in the polysaccharide molecule; 3000-2700 cm -1 is the aldehyde C-H bond and the C-H stretching vibration of saturated carbon, SJP-VI (2940 cm -1 ) has an absorption peak in this range; the absorption peak of 1200-1400 cm -1 is related to the deformation vibration of C-O and O-H, SJP-VI (1326 cm -1There are absorption peaks in this range, which are characteristic absorption peaks of polysaccharides. Thus, it can be verified that SJP-VI is a polysaccharide substance.

[0075] 5.3, Analysis of monosaccharide composition of purified sophora japonica polysaccharide SJP-VI

[0076] Figure 3 The gas chromatogram of the standard, Figure 4 The gas chromatogram of SJP-VI. By comparison, it can be seen that the carbon skeleton of SJP-VI is mainly composed of galactose, rhamnose and arabinose, and also contains a small amount of glucose. Among them, the molar ratio of galactose, rhamnose, arabinose and glucose is 38.20:33.54:22.92:5.34.

[0077] 5.4, Methylation analysis of purified sophora japonica polysaccharide SJP-VI

[0078] After methylation and derivatization treatment of SJP-VI, the corresponding PMAAs are obtained, and the PMAAs are detected by gas chromatography-mass spectrometry (GC-MS). The total ion chromatogram of SJP-VI is shown in Figure 5 The mass spectrum is shown in Figure 6 The results are shown in Table 1.

[0079] Table 1: Methylation analysis results of SJP-VI

[0080]

[0081] Example 3: Study on hypoglycemic activity of purified sophora japonica polysaccharide SJP-VI

[0082] Reagents: DMEM high-sugar medium (produced by American Gibco Company), FBS (produced by American Gibco Company), double antibody (produced by American Gibco Company), mix DMEM high-sugar medium and fetal bovine serum according to the ratio of 9:1 (w / v, (mg / mL)), add 1% (herein, volume ratio) double antibody, and mix thoroughly to obtain DMEM complete medium.

[0083] Instruments: cell incubator, enzyme marker, low-speed centrifuge, etc.

[0084] By determining the influence of SJP-VI on glucose consumption of IR-HepG2 cells (HB-8065), key enzymes of cell glycometabolism, oxidative stress state and its regulation on hypoglycemic pathway, the hypoglycemic activity of SJP-VI is evaluated.

[0085] 1. Hypoglycemic effect of SJP-VI on IR-HepG2 cell model

[0086] IR-HepG2 cells were cultured to logarithmic growth phase, and cell counting was performed. The cell suspension with a density of 5 x 10 4 -7 mol / L insulin for 48 h to induce insulin resistance. Subsequently, the model group was added with complete medium, the positive group was treated with metformin hydrochloride, and the sample group was treated with SJP-VI sample. Three concentration gradients (200, 400, and 800 μg / mL) were set up. After 24 h of culture, the glucose consumption was determined. That is, the blank control group: DMEM complete medium was used to culture HepG2 cells; the model group: 10 -7 mol / L insulin was used to induce HepG2 cells for 48 h, and then complete medium was added; the positive group: 10 -7 mol / L insulin was used to induce HepG2 cells for 48 h, and then 200 μg / mL metformin hydrochloride was added for 24 h of culture of IR-HepG2 cells; the sample group: 10 -7 mol / L insulin was used to induce HepG2 cells for 48 h, and then SJP-VI sample was used to treat IR-HepG2 cells for 24 h. Three concentration gradients were set up, i.e., low concentration 200 μg / mL, medium concentration 400 μg / mL, and high concentration 800 μg / mL.

[0087] 2. Effect of SJP-VI on HK of IR-HepG2 cells

[0088] HepG2 cells in logarithmic growth phase were digested and diluted with complete medium to 2 x 10 5 4 ​​(1): Add the extraction solution (the extraction solution provided in the kit) at a ratio of 500:1 (mL). Disrupt the cells in an ice-water bath using an ultrasonic disruptor (3 seconds of sonication, 10 seconds interval, 200W power, 30 cycles). Centrifuge at 8000 rpm and 4°C for 10 minutes. After centrifugation, take the supernatant and perform the assay according to the instructions of the hexokinase (HK) assay kit.

[0089] 3. Effects of SJP-VI on pyruvate kinase (PK) in IR-HepG2 cells

[0090] With a density of 2×10 5 HepG2 cells were seeded at a density of 300,000 cells / mL in six-well plates. After 48 hours of cell modeling and 24 hours of drug administration as described above, the cells were removed and placed on ice. The old culture medium was discarded, and the cells were washed twice with pre-chilled PBS. The cells were then digested with trypsin and collected into 1.5 mL centrifuge tubes. After centrifugation, the cells were resuspended in 300 μL of PBS and counted. Cells were then counted according to their number (102). 4 Cells were extracted at a ratio of 500:1 (the extraction solution provided in the kit was added to the extract). The cells were then disrupted in an ice-water bath using an ultrasonic disruptor (3 seconds of sonication, 10 seconds interval, 200W power, 30 cycles). After centrifugation, the supernatant was collected and measured according to the instructions of the pyruvate kinase (PK) detection kit.

[0091] 4. Effects of SJP-VI on glucose-6-phosphatase (G-6-Pase) in IR-HepG2 cells

[0092] With a density of 2×10 5 HepG2 cells were seeded at a rate of 300,000 cells / mL in six-well plates. Cells were subjected to 48 h of cell modeling and 24 h of drug administration as described above. Cells were then collected by trypsin digestion into 1.5 mL centrifuge tubes, centrifuged, resuspended in 300 μL PBS, and counted. Cell counts were determined according to cell number (102). 4 (1): Add the extraction solution (containing the extract in the kit) at a ratio of 500:1. Sonicate the cells under ice bath conditions, centrifuge, and take the supernatant to determine the glucose-6-phosphatase (G-6-Pase) detection kit according to the product instructions.

[0093] 5. Effects of SJP-VI on reactive oxygen species (ROS) in IR-HepG2 cells

[0094] With a density of 2×10 5HepG2 cells were seeded in 24-well plates at a density of 300,000 cells per well. The cells were modeled for 48 h and dosed for 24 h according to the above method. The IR-HepG2 cell model was treated with different concentrations of SJP-VI (200, 400, and 800 μg / mL) and a metformin solution as a positive control. After 24 h of treatment, the cells were washed twice with PBS, and the probe was loaded in situ according to the product instructions of the reactive oxygen species detection kit. After incubation at 37 °C for 20 min in a cell incubator, the cells were washed three times with serum-free cell culture medium, and then observed under a fluorescence microscope to obtain fluorescence images.

[0095] 6. Regulation of the glucose-lowering pathway in IR-HepG2 cells by SJP-VI

[0096] After the HepG2 cells were modeled for 48 h and dosed for 24 h according to the above method, the culture medium was discarded, and the cells were gently washed twice with PBS along the inner wall. Lysis solution (500 μL) from an RNA extraction kit was added, and the cells were shaken at 120 r / min for 1 min at room temperature. Anhydrous ethanol was then added to obtain a cell lysate. The cell lysate was transferred to a separation column, centrifuged, treated with DNase, and then washed with Wash Buffer. Finally, the RNA in the column was eluted with Elution Buffer to obtain total RNA. The RNA was reverse transcribed according to the Color Reverse Transcription Kit instructions (42 °C for 15 min; 95 °C for 30 s). The reaction product was cDNA, which was stored at -80 °C. The PCR reaction system was prepared according to the Dy-Namo Flash SYRB Green qPCR Kit instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and amplification was performed using a fluorescence quantitative PCR instrument. The relative expression of the target gene mRNA was calculated using the ΔΔCt method.

[0097] 7. Results

[0098] 7.1. Glucose-lowering effect of SJP-VI on the IR-HepG2 cell model

[0099] Figure 7 The results showed that the glucose consumption of the IR-HepG2 cell model treated with low-, medium-, and high-concentration SJP-VI was higher than that of the model group, and the difference was statistically significant, indicating that SJP-VI has a certain glucose-lowering activity.

[0100] 7.2. Effect of SJP-VI on the hexokinase (HK) of IR-HepG2 cells

[0101] Figure 8The results show that the hexokinase activity of the low, medium and high concentration SJP-VI treated groups is higher than that of the model group, and the difference is statistically significant, showing a certain blood glucose lowering activity.

[0102] 7.3, Effect of SJP-VI on pyruvate kinase (PK) in IR-HepG2 cells

[0103] Figure 9 The results show that the pyruvate kinase activity of the low, medium and high concentration SJP-VI treated groups is higher than that of the model group, and the difference is statistically significant, showing a certain blood glucose lowering activity.

[0104] 7.4, Effect of SJP-VI on glucose-6-phosphatase (G-6-Pase) in IR-HepG2 cells

[0105] Figure 10 The results show that the pyruvate kinase activity of the low, medium and high concentration SJP-VI treated groups is higher than that of the model group, and the difference is statistically significant, showing a certain blood glucose lowering activity.

[0106] 7.5, Effect of SJP-VI on reactive oxygen species (ROS) in IR-HepG2 cells

[0107] Figure 11 The results show that the green fluorescence of the SJP-VI group is significantly weaker than that of the model group, indicating that it has a significant inhibitory effect on the generation of ROS. Therefore, different concentrations of SJP-VI can reduce the intracellular ROS level to different degrees.

[0108] 7.6, Regulation of SJP-VI on the blood glucose lowering pathway in IR-HepG2 cells

[0109] Figure 12 The results show that the mRNA expression of the AMPK gene in the SJP-VI group is increased to a certain extent compared with the model group, promoting the absorption and utilization of glucose; the mRNA expression of the G-6-Pase gene in the sample group is significantly down-regulated compared with the model group, indicating that the sample can inhibit gluconeogenesis and promote glycogen synthesis by inhibiting the G-6-Pase gene.

[0110] Example 4: Immune activity study of purified sophora japonica polysaccharide SJP-VI

[0111] The immune activity of SJP-VI was evaluated by determining the phagocytic ability of SJP-VI on mononuclear macrophages RAW264.7, the accumulation amount of RAW264.7 cell factors (NO, TNF-α and IL-6) and the mRNA expression amount thereof.

[0112] 1. Toxicity of SJP-VI on macrophage RAW264.7

[0113] The mouse peritoneal macrophage RAW264.7 was cultured, and when the cells grew to the logarithmic phase, the cell density was adjusted to 5 x 10 4 The cells were inoculated in a 96-well plate at 100 μL per well, and the outermost circle was not inoculated with cells. 100 μL of PBS buffer was added to avoid edge effects. The plate was incubated in a cell incubator at 5% CO2 and 37°C for 24 h. After incubation, the original culture medium was discarded, and 100 μL of SJP-VI solution at different concentrations (100, 200, 400, 800, and 1200 μg / mL) was added. The blank control was DMEM complete medium, and three parallel samples were set for each group. The plate was incubated in the cell incubator for another 24 h. The relative proliferation rate of the cells was calculated by the MTT method to evaluate the toxicity of SJP-VI on RAW264.7. A relative proliferation rate of more than 80% was considered to be within the acceptable range. The relative proliferation rate was calculated according to the following formula:

[0114] Relative proliferation rate (%) = (A sample / A blank control) x 100%

[0115] Relative proliferation rate (%) = (A sample / A blank control) x 100%

[0116] 2. Effect of SJP-VI on the phagocytic ability of macrophages

[0117] The neutral red was used to determine the effect of SJP-VI on the phagocytic ability of macrophages. The RAW264.7 cells cultured to the logarithmic phase were plated as described above, and incubated for 24 h. Different concentrations of SJP-VI solution (non-toxic concentration range) were set for the sample group, and DMEM complete medium was used as the blank control. LPS (1 μg / mL) was used as the positive control, and three parallel samples were set for each group. After incubation for another 24 h, the culture medium was discarded, and 150 μL of 0.1% neutral red solution (DMEM complete medium) was added. The plate was incubated in the cell incubator for 1 h for staining. The supernatant was aspirated and washed twice with PBS. 150 μL of lysis solution (50% acetic acid + 50% ethanol) was added to each well, and the plate was incubated at 37°C in the dark for 2 h. The absorbance at 550 nm was measured by a microplate reader. The phagocytic ability was proportional to the absorbance value.

[0118] 3. Effect of SJP-VI on the production of cytokines IL-6 and TNF-α by macrophages

[0119] The density of the RAW264.7 cells was adjusted to 5 x 10 6RAW264.7 cells were seeded at a rate of 1 mL / well in six-well plates. After cell adhesion, the cells were treated with different concentrations (100, 200, 400, 800, 1200 μg / mL) of SJP-VI for 24 h. LPS (1 μg / mL) served as a positive control, and each group was divided into three replicates. After another 24 h of incubation, the supernatant was collected, and the accumulation of TNF-α and IL-6 in the cell supernatant was measured using an ELISA kit, strictly following the kit instructions.

[0120] 4. Effects of SJP-VI on RAW264.7 cytokine mRNA expression

[0121] RAW264.7 cells cultured to the logarithmic growth phase were injected into each well at a rate of 1.5 mL (4 × 10⁻⁶) per well. 6 Cells (cells / mL) were seeded in six-well plates and incubated in a cell culture incubator for 24 hours. The original culture medium was then removed, and the sample groups were prepared using DMEM complete medium containing different concentrations of SJP-VI. The blank control was DMEM complete medium, and the positive control was 1 μg / mL LPS. Each group was prepared in triplicate, and incubation continued for another 24 hours. 500 μL of lysis buffer from the RNA extraction kit was added, and the mixture was shaken at 120 rpm for 1 min at room temperature, followed by the addition of anhydrous ethanol to obtain cell lysis buffer. The cell lysis buffer was transferred to a separation column, centrifuged, treated with DNase, washed thoroughly with Wash Buffer, and finally eluted with elution buffer to obtain total cellular RNA. Reverse transcription was performed according to the Color Reverse Transcription Kit instructions using three different methods (42℃, 15 min; 95℃, 30 s). The resulting cDNA was stored at -80℃. The PCR reaction system was then prepared according to the instructions of the Dy-Namo Flash SYRB Green qPCR Kit. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and amplification was performed using a real-time PCR instrument. The relative expression level of the target gene mRNA was calculated using the ΔΔCt method.

[0122] 5. Results

[0123] 5.1. The toxic effect of SJP-VI on RAW264.7 macrophages

[0124] like Figure 13 Within the range of 100-1600 μg / mL, the survival rate of RAW264.7 cells treated with SJP-VI was above 90%, indicating that the sample was non-toxic to RAW264.7 cells. Therefore, three concentration gradients of 400, 800, and 1600 μg / mL were ultimately selected for the study of immunomodulatory activity.

[0125] 5.2, Effect of SJP-VI on phagocytosis of macrophage RAW264.7

[0126] Figure 14 For the effect of SJP-VI on phagocytosis of neutral red of RAW264.7 cells, the results show that SJP-VI can significantly enhance the phagocytosis of RAW264.7 cells, and compared with the blank group, there is a statistical difference.

[0127] 5.3, Effect of SJP-VI on production of cytokines IL-6 and TNF-α by macrophages

[0128] Figure 15 and Figure 16 The results show that SJP-VI can significantly increase the release amount of IL-6 and TNF-α by regulating the expression of mRNA, and enhance the immune activity.

[0129] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A purified sophora japonica polysaccharide SJP-VI, characterized in that A heteropolysaccharide composed of galactose, rhamnose, arabinose and glucose, and the molar ratio of the monosaccharides is 38.20:33.54:22.92:5.34; The preparation method of the purified sophora japonica polysaccharide SJP-VI comprises the following steps: (1) taking sophora japonica powder, extracting by ultrasonic-assisted hot water extraction method, concentrating, decolorizing, deproteinizing, and alcohol precipitating to obtain crude polysaccharide; (2) collecting the eluate by ion exchange chromatography column and elution; (3) concentrating the eluate by dialysis bag; (4) freeze-drying the concentrated solution to obtain the purified sophora japonica polysaccharide; In step (2), gradient elution is used, and the eluent is a NaCl solution with a concentration of 0-0.3 mol / L, and the eluate obtained by elution with a 0.25 mol / L NaCl solution is collected.

2. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein the weight average molecular weight of the purified sophora japonica polysaccharide SJP-VI is 10,000-18,000 Da.

3. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein the weight average molecular weight of the purified sophora japonica polysaccharide SJP-VI is 10,542 Da.

4. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in step (1), the ultrasonic-assisted hot water extraction method refers to ultrasonic treatment followed by hot water extraction, wherein the ultrasonic treatment time is 5-20 min, the ultrasonic treatment power is 300 W, the hot water extraction temperature is 80-100 ℃, and the hot water extraction time is 1-5 h.

5. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in step (1), the solid-liquid ratio of sophora japonica powder to water is 1:10-1:40, g / mL.

6. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in the decolorizing step of step (1), a macroporous resin D354FD is used; 7. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in the deproteinizing step of step (1), the Sevage method is used; 8. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in the alcohol precipitation step of step (1), the volume ratio of alcohol to the deproteinized concentrated solution is 1-10:1, and the alcohol is 95% ethanol.

7. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in step (2), the ion exchange column is a cellulose ion chromatography column, and the filler of the cellulose column is DEAE-52.

8. The purified sophora japonica polysaccharide SJP-VI of claim 1, wherein in step (3), the molecular weight cut-off of the dialysis bag is 6,000-10,000 Da.

9. Use of the purified sophora japonica polysaccharide SJP-VI of any one of claims 1-3 in the preparation of antioxidant functional food, antioxidant functional medicine, blood glucose-lowering functional food, blood glucose-lowering functional medicine, immune-enhancing functional food, and immune-enhancing functional medicine. ​ ​ ​ ​ ​ ​ ​

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