Application of selenium-containing Chinese herbal medicine polysaccharide in preparation of preparations for preventing and treating calculus

Abstract

The application discloses application of seleniumized Chinese herbal medicine polysaccharide in preparation of preparations for preventing and treating calculus. The seleniumized corn silk polysaccharide and the seleniumized desmodium polysaccharide are obtained by extracting corn silk polysaccharide and desmodium polysaccharide and seleniumizing the same. It is found through detection that the seleniumization of the corn silk polysaccharide and the desmodium polysaccharide improves the protection of the corn silk polysaccharide and the desmodium polysaccharide on renal epithelial cells, so that the corn silk polysaccharide and the desmodium polysaccharide can not only effectively inhibit the oxidative damage of renal epithelial cells induced by oxalic acid, but also effectively inhibit the adhesion of calcium oxalate crystals on the renal epithelial cells, and the generation of calculus can be reduced from two aspects of protecting cells and inhibiting the adhesion of calcium oxalate crystals. In addition, it is also found that the combination of the seleniumized corn silk polysaccharide and the seleniumized desmodium polysaccharide can further improve the effect of preventing and treating calculus, has obvious synergistic effect, and is helpful to the prevention and treatment of calculus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of Chinese herbal medicine polysaccharides. More particularly, it relates to the application of seleniumized Chinese herbal medicine polysaccharides in the preparation of preparations for preventing and treating calculi. BACKGROUND

[0002] Kidney stones are a common and frequently-occurring disease of the urinary system, and its occurrence is closely related to the precipitation and deposition of calcium oxalate crystals in the kidney from urine. The deposition of calcium oxalate crystals can induce a series of toxic reactions in renal epithelial cells, including oxidative stress, organelle damage, etc., thereby causing damage or even death of renal epithelial cells and inducing the formation of kidney stones.

[0003] Polysaccharides are a class of natural high-molecular polymers formed by the connection of aldehyde sugars or ketones through glycosidic bonds, are important biological macromolecules in the body, and are one of the basic substances for maintaining normal operation of life activities. Chinese herbal medicine polysaccharides are an important source of polysaccharides, and have various biological activities, such as regulating immunity, reducing blood sugar, and inhibiting cancer cell proliferation. Although there have been reports of Chinese herbal medicine polysaccharides with anti-kidney stone effects, such as astragalus polysaccharides and polysaccharides from the heart shield winged fig, etc. However, such polysaccharides only disclose their ability to inhibit the formation and adhesion of calcium oxalate, and the effects on cell oxidative stress and cell damage caused by calcium oxalate crystal deposition are unknown, and their effects on preventing and treating kidney stones may be limited. It is necessary to continuously explore Chinese herbal medicine polysaccharides with effects on preventing and treating kidney stones.

[0004] In addition, it has been found that when polysaccharides reach a certain concentration, their biological activities, such as the effect on repairing cell damage, will not be improved with the increase of the concentration of polysaccharides, and the effect achieved is still not ideal. Therefore, it is necessary to further improve the biological activity of polysaccharides in order to achieve better effects. Chemical modification of polysaccharides can improve their biological activity, and common modification methods include sulfation, carboxylation, and seleniumization, etc. However, the modification method for improving the biological activity of polysaccharides of different types or sources is not the same. At the same time, the effect of modified polysaccharides on preventing kidney stones is also limited. The effect of modified polysaccharides will not increase with the increase of their concentration, and the number of groups that can be increased by existing chemical modification methods is also limited. In order to achieve the ideal effect of preventing kidney stones, in addition to chemical modification, how to further improve the effect of polysaccharides or chemically modified polysaccharides on preventing kidney stones still needs to be explored. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing Chinese herbal medicine polysaccharides with limited effects on preventing and treating kidney stones and low activity, and to provide the application of seleniumized Chinese herbal medicine polysaccharides in the preparation of preparations for preventing and treating kidney stones.

[0006] The above-mentioned object of the present application is achieved by the following technical solutions:

[0007] The present application extracts corn silk polysaccharide and polysaccharide of desmodium styracifolium and seleniumizes the same to obtain seleniumized corn silk polysaccharide and seleniumized polysaccharide of desmodium styracifolium. It is found through detection that seleniumization of corn silk polysaccharide and polysaccharide of desmodium styracifolium improves the protection of corn silk polysaccharide and polysaccharide of desmodium styracifolium on renal epithelial cells, so that it can not only effectively inhibit the oxidative damage of renal epithelial cells induced by oxalate, but also effectively inhibit the adhesion of calcium oxalate crystals on renal epithelial cells, and reduce the generation of stones from the aspects of protecting cells and inhibiting the adhesion of calcium oxalate crystals.

[0008] Therefore, the present application claims the use of seleniumized Chinese herbal medicine polysaccharide in the preparation of preparations for preventing and treating stones. Specifically, the seleniumized Chinese herbal medicine polysaccharide is seleniumized corn silk polysaccharide and / or seleniumized polysaccharide of desmodium styracifolium.

[0009] Specifically, the main component of the stone is calcium salt crystals.

[0010] More specifically, the calcium salt is calcium oxalate.

[0011] Specifically, the selenium content in the seleniumized corn silk polysaccharide is 1.5-2 mg / g.

[0012] More specifically, the selenium content in the seleniumized corn silk polysaccharide is 1.75 mg / g.

[0013] Specifically, the preparation method of the seleniumized corn silk polysaccharide is as follows: corn silk polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted in a water bath at 65-75°C for 4-8 h, and the pH value of the solution is adjusted to 5-6; an excess of Na2SO4 is added to remove Ba2+ ions in the solution; the supernatant is first dialyzed with running water for 48-72 h, and then dialyzed with distilled water for 12-24 h; a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped when there is no red color; the solution obtained by dialysis is alcohol precipitated, and the obtained precipitate is dried to obtain seleniumized corn silk polysaccharide. 2+ ion; the supernatant is first dialyzed with running water for 48-72 h, and then dialyzed with distilled water for 12-24 h; a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped when there is no red color; the solution obtained by dialysis is alcohol precipitated, and the obtained precipitate is dried to obtain seleniumized corn silk polysaccharide.

[0014] More specifically, the HNO3 is 5% HNO3.

[0015] More specifically, 50 mL of 5% HNO3 is added per 500 mg, and after dissolution, 400 mg of Na2SeO3 and 1.0 g of BaCl2 are added, and the solution is reacted in a water bath at 70°C for 6 h.

[0016] More specifically, the reagent used for alcohol precipitation is 95% ethanol, and the precipitation is carried out at room temperature for 12 h.

[0017] Specifically, the preparation method of the corn silk polysaccharide comprises the following steps:

[0018] S1. Extraction of corn silk crude polysaccharide: dry or fresh corn silk is crushed and soaked in distilled water overnight, and then heated for extraction. The filtrate obtained by extraction is alcohol precipitated, and the obtained precipitate is dried to obtain corn silk crude polysaccharide. The purity of the obtained corn silk crude polysaccharide should reach 20%.

[0019] S2. Degradation of corn silk crude polysaccharide: the extracted corn silk crude polysaccharide is prepared into a solution, and the solution is subjected to ultrasonic degradation under the condition of an ultrasonic frequency of 400-800 W and a frequency of 30-50 kHz. The degradation solution is collected after ultrasonic degradation for 10-60 min, and the degradation solution is alcohol precipitated. The obtained precipitate is dried to obtain corn silk polysaccharide.

[0020] More specifically, the dry or fresh corn silk is washed and cleaned, crushed, and then sieved through a 60-mesh sieve to obtain corn silk powder.

[0021] More specifically, the corn silk powder is soaked in distilled water at a ratio of 1:20 overnight.

[0022] More specifically, the extraction is performed under boiling conditions (100°C) for 2 h.

[0023] More specifically, after the filtrate is concentrated, 3 times the volume of 100% ethanol is added, and alcohol precipitation is performed at 4°C for 24 h.

[0024] More specifically, the obtained precipitate is dried by vacuum drying.

[0025] The corn silk polysaccharide prepared by the above method has an average molecular weight of 6000 Da, an apparent viscosity of 2.29 mL / g, and a uronic acid content of 31.3%. The structure mainly consists of α-D-glucose, α-L-arabinose, α-L-rhamnose, β-D-galactose, β-D-mannose, and β-D-xylose.

[0026] Specifically, the selenium content in the seleniumized broadleaf money grass polysaccharide is 1.5-3.2 mg / g.

[0027] Specifically, the preparation method of the seleniumized broadleaf money grass polysaccharide is as follows: the broadleaf money grass polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted at 65°C-75°C for 4-8 h. The pH value of the solution is adjusted to 7-8. Excess Na2SO4 is added to remove Ba 2+ ions in the solution; the supernatant is dialyzed with distilled water, a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped until there is no red color. The dialyzed solution is alcohol precipitated, and the obtained precipitate is dried to obtain the seleniumized broadleaf money grass polysaccharide.

[0028] More specifically, 1 g of the prepared polygonum multiflorum polysaccharide is dissolved in 100 mL of 4.5%-5% HNO3, heated and stirred, and then 0.8 g of Na2SeO3 and 2.0 g of BaCl2 are added, and the reaction is carried out at 60-70°C in a water bath for 6 h.

[0029] Preferably, 100 mL of 5% HNO3 is added per 1 g of polygonum multiflorum polysaccharide, and after dissolution, 0.8 g of Na2SeO3 and 2 g of BaCl2 are added, and the reaction is carried out at 70°C in a water bath for 6 h.

[0030] More specifically, the supernatant is first dialyzed with running water for 48 h, and then dialyzed with distilled water for 24 h.

[0031] More specifically, the reagent used for alcohol precipitation is anhydrous ethanol.

[0032] Specifically, the preparation method of the polygonum multiflorum polysaccharide comprises the following steps:

[0033] S1. Extraction of polygonum multiflorum crude polysaccharide: polygonum multiflorum medicinal material powder is extracted with boiling water for 2-3 times, 1-2 h each time, and the obtained filtrate is concentrated, centrifuged, and the supernatant is taken, and the obtained supernatant is alcohol precipitated, and the obtained precipitate is freeze-dried to obtain polygonum multiflorum crude polysaccharide;

[0034] S2. Degradation of polygonum multiflorum crude polysaccharide: the extracted polygonum multiflorum crude polysaccharide is prepared into a solution, heated to a temperature of 88-92°C, and hydrogen peroxide is added to make the concentration of hydrogen peroxide in the degradation system 4-6%, and after degradation for 1-2 h, it is cooled to room temperature, the pH of the solution is adjusted to 6.8-7.2, and alcohol precipitation is carried out, and the obtained precipitate is dried to obtain polygonum multiflorum polysaccharide.

[0035] More specifically, the polygonum multiflorum medicinal material powder is extracted with boiling water at a solid-liquid ratio of 1:40 (unit g / mL) for 3 times, 1 h each time.

[0036] More specifically, when alcohol precipitation, ethanol is added to the supernatant to a content of 80% (v / v), and alcohol precipitation is carried out overnight at 4°C.

[0037] The application also provides a preparation for preventing and treating stones, which contains seleniumized corn silk polysaccharide and seleniumized polygonum multiflorum polysaccharide.

[0038] Specifically, the selenium content in the seleniumized corn silk polysaccharide is 1.5-2 mg / g, and the selenium content in the seleniumized polygonum multiflorum polysaccharide is 1.5-3.2 mg / g.

[0039] More specifically, the selenium content in the seleniumized corn silk polysaccharide is 1.75 mg / g, and the selenium content in the seleniumized polygonum multiflorum polysaccharide is 3.2 mg / g.

[0040] The application has the following beneficial effects:

[0041] The present application extracts corn silk polysaccharide and polysaccharide of desmodium styracifolium, and seleniumizes the same, to obtain seleniumized corn silk polysaccharide and seleniumized polysaccharide of desmodium styracifolium. It is found through detection that seleniumization of corn silk polysaccharide and polysaccharide of desmodium styracifolium improves the protection of corn silk polysaccharide and polysaccharide of desmodium styracifolium on renal epithelial cells, so that it can not only effectively inhibit the oxidative damage of renal epithelial cells induced by oxalate, but also effectively inhibit the adhesion of calcium oxalate crystals on renal epithelial cells, and reduce the generation of stones from the aspects of protecting cells and inhibiting the adhesion of calcium oxalate crystals.

[0042] In addition, it is also found in the present application that the combination of seleniumized corn silk polysaccharide and polysaccharide of desmodium styracifolium can further improve the effect of preventing and treating stones, has obvious synergistic effect, and is helpful for the prevention and treatment of stones. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 is the cytotoxicity detection results of CSP and Se-CSP and the repair effect on the oxidative damage of cells induced by oxalate; A in the figure is the cytotoxicity detection results of CSP and Se-CSP; B in the figure is the repair effect on the oxidative damage of cells induced by oxalate.

[0044] Figure 2 Fig. 2 is the PS outflowing situation of damaged renal epithelial (HK-2) cells before and after being repaired by CSP and Se-CSP of different dosages; A in the figure is the flow quantitative analysis diagram of the PS outflowing situation of HK-2 cells before and after being repaired by CSP and Se-CSP; B in the figure is the corresponding quantitative columnar chart.

[0045] Figure 3 Fig. 3 is the adhesion situation of COD crystals with a size of about 100 nm to HK-2 cells before and after being repaired by CSP and Se-CSP; the COD crystal concentration is 200 μg / mL, and the adhesion time is 1 h.

[0046] Figure 4 Fig. 4 is the quantitative detection results of CSP and Se-CSP inhibiting COD adhesion; A in the figure is the adhesion amount of FITC fluorescent labeled COD crystals; B in the figure is the corresponding quantitative columnar chart; the COD crystal concentration is 200 μg / mL, and the adhesion time is 1 h.

[0047] Figure 5 Fig. 5 is the difference of endocytosis of nano COD crystals in HK-2 cells before and after being repaired by CSP and Se-CSP; A in the figure is the flow quantitative analysis diagram of endocytosis of nano COD crystals in HK-2 cells before and after being repaired by CSP and Se-CSP; B in the figure is the corresponding quantitative columnar chart.

[0048] Figure 6 Fig. 6 is the Zeta potential of Se-DSPs.

[0049] Figure 7 Microscopic surface morphology of DSP and Se-DSPs; A in the figure is the microscopic surface morphology of DSP; B in the figure is the microscopic surface morphology of Se-DSP1; C in the figure is the microscopic surface morphology of Se-DSP2.

[0050] Figure 8 Antioxidant capacity determination results of DSP and Se-DSPs; A in the figure is the clearance rate of DSP and Se-DSPs on hydroxyl radicals; B in the figure is the clearance rate of DSP and Se-DSPs on DPPH radicals; C in the figure is the clearance rate of DSP and Se-DSPs on ABTS radicals.

[0051] Figure 9 Cell toxicity and protective effect on HK-2 cells of DSP and Se-DSPs; A in the figure is the cell toxicity test results of DSP and Se-DSPs; B in the figure is the test results of the protective effect of DSP and Se-DSPs on HK-2 cells; NC: normal control group; DC: nano COM injury group; nano COM concentration is 200 μg / mL; injury time is 24 h; polysaccharide protection time is 24 h.

[0052] Figure 10 The results of the effect of DSP and Se-DSPs on PS exocytosis; A in the figure is the histogram of PS exocytosis after DSP and Se-DSPs treatment detected by flow cytometry; B in the figure is the corresponding quantitative column chart; polysaccharide concentration is 80 μg / mL; oxalic acid concentration is 2.8 mM; injury time is 3 h; repair time is 12 h.

[0053] Figure 11 The results of the effect of DSP and Se-DSPs on intracellular NO level; A in the figure is the NO fluorescence photo; B in the figure is the relative fluorescence intensity column chart; polysaccharide concentration is 80 μg / mL; oxalic acid concentration is 2.8 mM; repair time is 12 h; the scale is 200 μm.

[0054] Figure 12 Repair results of DSP and Se-DSPs on cell morphology; NC: normal control group; DC: nano COM injury group; nano COM concentration is 200 μg / mL, injury time is 24 h; polysaccharide concentration is 60 μg / mL, polysaccharide protection time is 24 h.

[0055] Note: *P<0.05; **P<0.01 in the figure. DETAILED DESCRIPTION

[0056] The present application is further described below in conjunction with the accompanying drawings and specific examples, which do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art.

[0057] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0058] Example 1 Preparation of corn silk polysaccharide and selenized corn silk polysaccharide

[0059] 1. Preparation of corn silk polysaccharide (CSP)

[0060] (1) Extraction of corn silk crude polysaccharide

[0061] Dry or fresh corn silk (the style of corn) was washed to remove impurities, crushed and passed through a 60-mesh sieve, soaked in distilled water (the ratio of corn silk powder to distilled water was 1:20) overnight, and extracted under boiling conditions (100°C) for 2 hours. The filtrate was concentrated to 1 / 3 of the original volume, 3 volumes of 100% ethanol were added, and alcohol precipitation was performed at 4°C for 24 hours. The precipitate was obtained by centrifugation and vacuum drying, and the corn silk crude polysaccharide was obtained. The purity of the corn silk crude polysaccharide should reach 20%, and it was stored at 4°C for later use.

[0062] (2) Degradation of corn silk crude polysaccharide

[0063] The extracted corn silk crude polysaccharide was prepared into a solution with a concentration of 5 mg / mL, and ultrasonic degradation was performed under the conditions of an ultrasonic frequency of 600 W and a frequency of 40 kHz. After 20 minutes of ultrasonic degradation, the degradation solution was collected, concentrated at 60°C, and alcohol precipitation was performed overnight. The precipitate was obtained by centrifugation and vacuum drying, and the corn silk polysaccharide (CSP) was obtained.

[0064] The average molecular weight of the corn silk polysaccharide prepared by the present application was 6000 Da, the apparent viscosity was 2.29 mL / g, and the uronic acid content was 31.3%. NMR, FT-IR and other tests showed that the structure was mainly composed of α-D-glucose, α-L-arabinose, α-L-rhamnose, β-D-galactose, β-D-mannose and β-D-xylose.

[0065] 2. Preparation of selenized corn silk polysaccharide (Se-CSP)

[0066] CSP 500 mg prepared was added to 50 mL of 5% HNO3, heated and stirred to completely dissolve, then 400 mg of Na2SeO3 and 1.0 g of BaCl2 were added, and the reaction was carried out at 70°C for 6 hours. The pH value of the solution was adjusted to 5-6 with 15 mol / L NaOH, and excess Na2SO4 was added to remove Ba 2+The supernatant was centrifuged, and the supernatant was dialyzed with running water for 48 h and distilled water for 24 h. A small amount of dialysate was taken every 6 h, and ascorbic acid was added until the solution was free of red color (i.e., free Na2SeO3 in the solution was removed). The solution obtained after dialysis was evaporated to about 3 mL, 10 mL of 95% ethanol was added, and the solution was alcohol precipitated at room temperature for 12 h. The precipitate was centrifuged and vacuum dried to obtain Se-CSP.

[0067] The ICP-AES detection results show that the selenium content in the prepared Se-CSP is 1.75 mg / g, and the carboxyl content in the polysaccharide before and after selenium treatment (4.23% to 4.34%) does not change significantly.

[0068] Example 2: Detection of the stone prevention effect of CSP and Se-CSP at the cell level

[0069] 1. Repair of oxalate-induced cell oxidative damage and cell toxicity detection of CSP and Se-CSP

[0070] (1) Cell toxicity detection

[0071] HK-2 cells were cultured in a culture box with DMEM-F12 culture solution containing 10% fetal bovine serum under the following conditions: 37°C, 5% CO2, and saturated humidity. The cells were subcultured every 2 days using a trypsin digestion method. When the cell confluence was 80-90%, the cells were washed with PBS buffer, moderately digested with trypsin, and then terminated with 10% fetal bovine serum culture solution. The cells were dispersed into a single cell suspension by blowing.

[0072] The prepared single cell suspension was inoculated into 6, 12, or 96 well culture plates at a concentration of 1.0 x 10 5 cells / mL. After incubation for 24 h, the culture solution was removed, and the cells were washed twice with PBS. 200 μL of CSP or Se-CSP at concentrations of 30, 60, 90, and 120 μg / mL (prepared with serum-free culture medium) was added to the cells, and the control group was added with an equal amount of serum-free culture medium. Five replicate wells were set for each concentration. After 24 h of culture, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark for 1.5 h. The absorbance (A) was measured at 450 nm using a microplate reader. The average value of the A values of the five replicate wells was calculated, and the survival rate-time curve and the survival rate-concentration curve were plotted.

[0073] Cell survival rate (%) = A (experimental group) / A (control group) x 100%

[0074] (2) Repair of oxalate-induced cell oxidative damage

[0075] The single cell suspension was prepared as in (1), and the prepared single cell suspension was inoculated in 6, 12 or 96 well culture plates at a concentration of 1.0 x 105 cells / mL, and after incubation for 24 h, the culture solution was removed, the cells were washed twice with PBS, and subsequent experiments were performed; the experimental model was divided into three groups: ① control group (NC): adding serum-free medium; ② damage group (DC): adding 2.8 mM oxalic acid for 3.5 h; ③ polysaccharide repair group: after oxalic acid damage for 3.5 h, the cells were repaired with serum-free medium containing 30, 60, 90 and 120 μg / mL CSP or Se-CSP for 10 h, respectively.

[0076] After the repair time was reached, 10 μL of CCK-8 reagent was added to each well of the 96 well plate, and incubation was performed for 4 h, and the absorbance (A) was measured at 450 nm by an enzyme marker, and the average value of the A values of three replicate wells was calculated.

[0077] Cell viability (%) = A (experimental group) / A (control group) x 100; wherein the experimental group is the damage group or the polysaccharide repair group.

[0078] The results of the toxicity detection of DCSP and Se-DCSP on HK-2 cells are shown in A of Figure 1 As can be seen from the figure, after HK-2 cells were incubated with 30-120 μg / mL of CSP or Se-CSP for 24 h, the cell viability was more than 100%, indicating that DCSP and Se-DCSP had no toxic effect on HK-2 cells, and promoted the growth of cells.

[0079] The repair effect of CSP and Se-CSP on oxalic acid-induced cell oxidative damage is shown in B of Figure 1 As can be seen from the figure, the viability of the cells in the oxalic acid-induced damage group (DC) was 64.1%, and after repair with different concentrations of CSP or Se-CSP, the cell viability gradually increased. Among them, the cell viability value of the Se-CSP group was proportional to the concentration of polysaccharide, and when the polysaccharide concentration in the CSP group exceeded 60 μg / mL, the cell viability did not increase significantly, and when the concentration reached 120 μg / mL, the cell viability of the Se-CSP and CSP groups was 88.2% and 77.2%, respectively, i.e. under the same concentration, the repair effect of Se-CSP was better than that of CSP.

[0080] 2, Effect of CSP and Se-CSP on the amount of phosphatidylserine (PS) eversion

[0081] Renal tubular epithelial cell damage is one of the main risk factors of kidney stone formation. After injury by high concentration of oxalate or other factors, the structure of renal tubular epithelial cell membrane will change. For example, the negatively charged PS in the cell membrane will turn outwards, and CD44 and HA will be expressed on the cell surface, which provides an effective location for the nucleation and growth of urinary microcrystals, promotes the formation of early urinary microcrystals, and also enhances the adhesion between the cell membrane and the urinary microcrystals, thereby accelerating the formation of kidney stones. The PS of normal cells is generally located in the inner leaflet of the plasma membrane, but in apoptotic cells, PS will transfer from the inner leaflet to the outer leaflet of the plasma membrane, thereby being exposed to the extracellular environment. The present application detects the anti-stone effect of CSP and Se-CSP by determining the influence of CSP and Se-CSP on the amount of PS outturning.

[0082] HK-2 cells were cultured and treated as above, and were repaired with serum-free culture medium containing 30, 60 μg / mL CSP or Se-CSP; after reaching the repair time, the culture solution was aspirated, washed twice with PBS buffer, 100 μL Binding Buffer and FITC-labeled Annexin-V (20 μg / mL) 10 μL were added, and incubated at room temperature for 30 min in the dark, and then quantitatively detected by flow cytometry.

[0083] The influence of CSP and Se-CSP on the amount of phosphatidylserine (PS) outturning is shown in Figure 2 Figure 2 A in Figure 2 is a flow cytometric quantitative analysis chart of PS outturning of HK-2 cells; Figure 2 It can be seen from that the amount of PS outturning of HK-2 cells in the control group is very small, only 2.91%. But when the cells are induced to be damaged by 2.8 mmol / L of oxalate, the amount of PS outturning increases sharply to 25.1%. After repair by CSP and Se-CSP, the PS outturning decreases to different degrees, and the repair effect of 60 μg / mL Se-CSP is the most obvious, and the amount of PS outturning decreases to 4.28%.

[0084] 3、The influence of CSP and Se-CSP on the adhesion of calcium oxalate crystals on the cell membrane

[0085] HK-2 cells were cultured and treated as above, and were repaired with serum-free medium containing 30, 60 μg / mL CSP or Se-CSP; after the repair time was reached, the 12-well plates were transferred to a 4°C environment to inhibit the endocytosis of the cells; 0.5 h later, 200 μg / mL of freshly prepared calcium oxalate dihydrate (COD) was added, and the cells were continuously cultured at 4°C for 1 h; after the culture time was reached, the cells were washed twice with pre-cooled PBS, fixed with 2.5% glutaraldehyde at 4°C for 24 h, and then dehydrated with 50%, 70%, 90%, and 100% ethanol; after being fixed with isoamyl acetate, the samples were CO2-critical dried, were treated with gold spraying, and were observed for the adhesion of calcium oxalate crystals on the cell membranes by scanning electron microscopy (SEM).

[0086] The effects of CSP and Se-CSP on the adhesion of calcium oxalate crystals (about 100 nm in size) on the cell membranes are shown in Figure 3 Figure 3 It can be seen that the morphology of the control group cells is normal, and the amount of crystals adhered to the surface is very small. The morphology of the oxalate injury group cells is contracted and rounded, and a large amount of crystals is adhered to the surface. After being repaired by CSP and Se-CSP, the morphology of the cells gradually returns to normal, and the amount of crystals adhered is significantly reduced compared with the injury group cells, and the amount of crystals adhered is further reduced with the increase of the polysaccharide concentration. At the same dose, the amount of crystals adhered to the surface of the cells repaired by Se-CSP is less than that of the cells repaired by CSP. In addition to the difference in the amount of crystals adhered, the aggregation degree of the crystals on the surface of the cells in the repair group is also lower than that in the injury group.

[0087] 4. Quantitative detection of the inhibition of the adhesion of calcium oxalate crystals by CSP and Se-CSP

[0088] HK-2 cells were cultured and treated as above, and were repaired with serum-free medium containing 30, 60 μg / mL CSP or Se-CSP; after the repair time was reached, the 12-well plates were transferred to a 4°C environment to inhibit the endocytosis of the cells; 0.5 h later, 200 μg / mL of freshly prepared calcium oxalate dihydrate (COD) was added, and the cells were continuously cultured at 4°C for 1 h; after the culture time was reached, the cells were washed twice with pre-cooled PBS, were resuspended with PBS after being trypsinized, and were detected for the proportion of cells with adhered crystals by flow cytometry. The cells with FITC signal can be regarded as cells with adhered crystals.

[0089] The results of the quantitative detection of the inhibition of the adhesion of nano-COD by CSP and Se-CSP are shown in Figure 4 Figure 4 A in the figure is the adhesion amount of the FITC fluorescently labeled nano-COD crystals; Figure 4 B in the figure is the corresponding quantitative column chart. It can be seen that Figure 4 ​​It was found that the percentage of cells adhering COD crystals was 6.13% in the normal group, while the percentage of cells adhering COD crystals increased to 46.63% in the oxalate injury group. After the repair by different concentrations of CSP and Se-CSP, the percentage of cells adhering crystals gradually decreased, indicating that the repaired cells could inhibit the adhesion of crystals, and the ability of cells in the Se-CSP repair group to inhibit the adhesion of crystals was stronger than that in the CSP repair group.

[0090] 5. Effects of CSP and Se-CSP on the endocytosis of calcium oxalate crystals

[0091] HK-2 cells were cultured and treated as described above, and were repaired with serum-free medium containing 30 and 60 μg / mL of CSP or Se-CSP. After the repair time was reached, 200 μg / mL of FITC fluorescently labeled nano-COD crystals were added for incubation at 37°C for 6 h. The cells were washed twice, and were treated with 5 mM EDTA for 5 min to complex the adhered crystals. After the cells were digested and resuspended, the proportion of cells with fluorescence was detected by flow cytometry.

[0092] The results of the effects of CSP and Se-CSP on the endocytosis of calcium oxalate crystals are shown in Figure 5 Figure 5 A in Figure 5 is a flow cytometry quantitative analysis chart of the endocytosis of nano-COD crystals by HK-2 cells; and Figure 5 B is a corresponding quantitative column chart. It was found that, compared with the proportion of cells endocytosing COD crystals (16.1%) in the normal group, the proportion of cells endocytosing crystals decreased to 4.2% (P<0.01) in the oxalate-induced injury group. After the repair by different concentrations of CSP and Se-CSP, the endocytosis of COD crystals by the cells gradually increased, and the proportion of cells endocytosing crystals increased to 14.7% after the repair by 60 μg / mL of Se-CSP.

[0093] Example 3. Preparation of polysaccharides from Desmodium styracifolium and seleniumized polysaccharides from Desmodium styracifolium

[0094] 1. Preparation of polysaccharides from Desmodium styracifolium (DSP)

[0095] (1) Extraction of crude polysaccharides from Desmodium styracifolium

[0096] The crude polysaccharides from Desmodium styracifolium were extracted by water extraction and alcohol precipitation. An appropriate amount of Desmodium styracifolium medicinal material sample was weighed, crushed, and 5 g of powder was taken. The powder was extracted with 200 mL of boiling water by reflux extraction for 3 times, 1 h each time. The filtrate was filtered while hot, and was concentrated. The supernatant was obtained by centrifugation at 5000 r / min for 10 min. The supernatant was added with an appropriate amount of ethanol to a content of 80% (v / v), and was placed in a refrigerator at 4°C overnight. The precipitate was collected by centrifugation at 5000 r / min for 5 min, and was freeze-dried to obtain the crude polysaccharides from Desmodium styracifolium.

[0097] ​(2) Degradation of crude polysaccharides from Desmodium styracifolium

[0098] Accurately weigh 1.20 g of the extracted crude polysaccharides from Desmodium styracifolium, dissolve in 20 mL of 70°C distilled water, when the temperature rises to 90°C, quickly add 5 mL of 30% hydrogen peroxide, so that the concentration of hydrogen peroxide in the degradation system is 5%; after 2 h of degradation at 90°C, cool to room temperature, adjust the pH to 7.0 with 2 mol / L NaOH solution, then concentrate to 1 / 3 of the original volume at 60°C under reduced pressure, add 3 volumes of anhydrous ethanol to precipitate the polysaccharides, centrifuge after overnight standing, and dry to obtain Desmodium styracifolium polysaccharides (DSP).

[0099] 2. Preparation of selenized Desmodium styracifolium polysaccharides (Se-DSPs)

[0100] Take 1 g of the prepared Desmodium styracifolium polysaccharides, dissolve in 100 mL of 4.5% to 5% HNO3, heat and stir, then add 0.8 g of Na2SeO3 and 2.0 g of BaCl2, react at 60°C to 70°C water bath for 6 h, adjust the pH of the solution to 7 to 8 with NaOH; add excess Na2SO4 to remove Ba 2+ ions in the solution, centrifuge; sequentially dialyze the supernatant against running water for 48 h and distilled water for 24 h, take a small amount of dialysate every 6 h, add ascorbic acid for detection, until there is no red color (all free Na2SeO3 in the solution is removed) to stop dialysis; evaporate the dialyzed solution to reduce the volume, evaporate to about 3 to 5 mL, precipitate the polysaccharides with anhydrous ethanol, dry the precipitate to obtain Se-DSPs.

[0101] The specific selenization reaction conditions are shown in Table 1.

[0102] Table 1. Synthesis conditions of selenized Desmodium styracifolium polysaccharides and selenium content

[0103]

[0104] 3. Zeta potential of Se-DSPs

[0105] Dissolve the prepared DSP, Se-DSP1, and Se-DSP2 in deionized water to prepare polysaccharide solutions with a concentration of 0.20 mg / mL, detect the Zeta potential of the polysaccharides at a constant temperature of 25°C using a nanoparticle size analyzer. The Zeta potentials of DSP, Se-DSP1, and Se-DSP2 are shown in Table 2. Figure 6 As can be seen from Table 2, Figure 6 after selenization of DSP, the Zeta potential value decreases from -18.1 mV before selenization to -32.3 mV, the analysis reason is that the selenization of polysaccharides increases the negative charge of selenite, so the Zeta potential becomes negative, i.e. the absolute value of Zeta potential increases.

[0106] 4. Microscopic surface morphology observation of DSP and Se-DSPs

[0107] 1 mg of each of the prepared DSP, Se-DSP1 and Se-DSP2 was weighed, dispersed in 10 mL of anhydrous ethanol, spotted on a 10 mm x 10 mm glass slide, naturally air-dried, and observed with SEM after gold spraying of the sample.

[0108] The microscopic surface morphologies of DSP, Se-DSP1 and Se-DSP2 are shown in Figure 7 ; Figure 7 A in the above figure is the microscopic surface morphology of DSP; Figure 7 B in the above figure is the microscopic surface morphology of Se-DSP1; Figure 7 C in the above figure is the microscopic surface morphology of Se-DSP2. It can be seen from the above figures that the DSP before seleniumization appears in the form of a block with multiple pores; and the DSP after seleniumization becomes spherical granular, with significantly reduced size and uniform distribution, which is conducive to increasing the solubility and biological activity thereof. Figure 7

[0109] Example 4. Detection of the anti-stone effect of DSP and Se-DSPs at the cell level

[0110] Free radicals, such as superoxide anion (O2·), hydroxyl radical (·OH) and peroxide radical (ROO·), are considered to be related to the damage of renal tubular epithelial cells. Free radicals can destroy important biological macromolecules such as DNA, protein, carbohydrate and lipid in the cell nucleus and cell membrane, leading to cell damage and disruption of homeostasis. In a healthy state, there are abundant antioxidants in the kidney tissue, including glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD), etc., which can effectively scavenge free radicals. However, in pathological conditions (such as cell damage), the amount of free radicals produced in the body will exceed the cell clearance capacity. Excessive free radicals will damage kidney tissue cells, cause oxidation of unsaturated fatty acids in the cell membrane, thereby changing the fluid state, fluidity and permeability of the cell membrane, and leading to membrane dysfunction, and finally forming kidney stones. And studies have shown that kidney tissue damaged due to peroxide stress is more likely to produce CaOx crystal retention. Therefore, the free radical scavenging capacity of polysaccharides represents the anti-stone effect of polysaccharides. The H2O2 / Fe system method was used to detect the antioxidant capacity of DSP and Se-DSPs.

[0111] 1. Antioxidant capacity determination of DSP and Se-DSPs

[0112] (1) ·OH clearance rate

[0113] ​In the test tube, add 1 mL of 2.5 mmol / L FeSO4 solution and 1 mL of 2.5 mmol / L o-dinitrogen phenol solution, then add 1 mL of 20 mmol / L PBS (pH 7.4) and 1 mL of 20 mmol / L H2O2 in turn, and finally take 1 mL of different concentrations of polysaccharide samples (DSP, Se-DSP-1, Se-DSP-2) (concentrations are 0.15, 0.5, 0.8, 1.0, 2.0, 3.0 mg / mL) into the test tube; mix well, incubate at 37℃ for 90 min, detect the absorbance at 536 nm by ultraviolet spectrophotometer, repeat twice, take the average absorbance, and record it as A3; among them, the non-injury group (control group) does not add sugar solution and hydrogen peroxide, and the volume is made up with distilled water, and the absorbance is recorded as A2; the injury group does not add sugar solution, and the volume is made up with distilled water, and the absorbance is recorded as A1; Vc is used as the positive control group.

[0114] ·OH clearance rate (%) = (A3-A1) / (A2-A1) x 100%

[0115] (2) DPPH free radical clearance rate

[0116] 0.4 mmol / L DPPH is prepared with anhydrous ethanol, 3 mL of different concentrations of polysaccharide samples (DSP, Se-DSP-1, Se-DSP-2) (0.15, 0.5, 0.8, 1.0, 2.0, 3.0 mg / mL) are mixed with DPPH solution (0.4 mmol / L, 1 mL) in a test tube, and after reaction in the dark at 25℃ for 30 min, the absorbance is detected at 517 nm, and Vc is used as the positive control.

[0117] DPPH clearance rate (%) = [1-(A2-A1) / A0] x 100%

[0118] In the formula: A2 is the absorbance of 3 mL of sample solution + 1 mL of DPPH reagent; A1 is the absorbance of 3 mL of sample solution + 1 mL of anhydrous ethanol; A0 is the absorbance of 1 mL of DPPH solution + 3 mL of water.

[0119] (3) ABTS free radical clearance rate

[0120] 7 mmol / L of ABTS reagent and 2.45 mmol / L of potassium persulfate reagent are mixed in a ratio of 1:1 by volume, and reacted in the dark for 12-16 h; 3 mL of the reacted reagent is taken, mixed with 1 mL of different concentrations of polysaccharide samples (DSP, Se-DSP-1, Se-DSP-2) (0.15, 0.5, 0.8, 1.0, 2.0, 3.0 mg / mL) in a test tube, and reacted at 25℃ for 6 min, and the absorbance is measured at 734 nm.

[0121] ABTS scavenging rate (%) = [1-(A2-A1) / A0] x 100%

[0122] In the formula: A0 is the absorbance of the control group, which does not contain polysaccharides; A1 is the absorbance of the test group; A2 is the absorbance of the reagent-free group (here, the absorbance A2 of the polysaccharide sample solution is 0).

[0123] (4) Results

[0124] The results of the determination of the antioxidant capacity of DSP, Se-DSP-1, and Se-DSP-2 are shown in Table 1. Figure 8 A in Table 1 is the scavenging rate of DSP, Se-DSP-1, and Se-DSP-2 on hydroxyl radicals; Figure 8 B in Table 1 is the scavenging rate of DSP, Se-DSP-1, and Se-DSP-2 on DPPH radicals; Figure 8 C in Table 1 is the scavenging rate of DSP, Se-DSP-1, and Se-DSP-2 on ABTS radicals. Figure 8

[0125] As can be seen from A in Table 1, within the tested dosage range, the scavenging rate of DSP, Se-DSP-1, and Se-DSP-2 on hydroxyl radicals increases with increasing concentration. At the same concentration (3 mg / mL), the scavenging rates of DSP, Se-DSP-1, and Se-DSP-2 reach 26%, 42%, and 56%, respectively, indicating that Se-DSPs have stronger hydroxyl radical scavenging capacity than DSP. Figure 8 DPPH is a stable non-lipophilic nitrogen-centered free radical, which is scavenged by antioxidants by providing a proton to form a stable DPPH-H molecule. As can be seen from B in Table 1, the DPPH scavenging capacity of DSP, Se-DSP-1, and Se-DSP-2 increases in a concentration-dependent manner, and Se-DSPs exhibit strong antioxidant activity.

[0126] Figure 8 As can be seen from C in Table 1, the ABTS radical scavenging capacity of DSP, Se-DSP-1, and Se-DSP-2 exhibits a dose-dependent manner within the concentration range of 0.25-3 mg / mL. When the concentration increases from 0.25 mg / mL to 3 mg / mL, the ABTS scavenging capacity of DSP, Se-DSP-1, and Se-DSP-2 increases from 26%, 33%, and 45% to 86%, 91%, and 95%, respectively. It can be seen that Se-DSPs have stronger antioxidant capacity than DSP.

[0127] Figure 8

[0128] ​​​​The above results show that the DSP after selenization, the scavenging ability of ·OH free radicals, DPPH free radicals, ABTS free radicals is stronger, which represents the ability to prevent kidney stones is stronger, and the ability to prevent stones is gradually increased with the increase of selenium content in polysaccharide, that is, Se-DSP2>Se-DSP1>DSP.

[0129] 2. Cell toxicity detection of DSP and Se-DSPs and protective effect on cells

[0130] (1) Cell toxicity detection

[0131] HK-2 cells were cultured in a culture box with DMEM-F12 culture solution containing 10% fetal bovine serum, and the culture conditions were 37°C, 5% CO2, and saturated humidity; the cells were subcultured every 2 days using trypsin digestion method; when the cell confluence was 80-90%, the cells were washed with PBS buffer, and the digestion was stopped by adding 10% fetal bovine serum culture solution after moderate trypsin digestion, and the cells were dispersed to form a single cell suspension by blowing. The single cell suspension with a concentration of 1.0×10 5 cells / mL was inoculated into a 96 or 6-well culture plate, and after 24h of incubation, the culture solution was removed, 200μL of DSP or Se-DSPs with concentrations of 30, 60, 90 and 120μg / mL respectively (prepared with serum-free culture medium) was added, and the control group was added with the same amount of serum-free culture medium; 5 replicate wells were set for each concentration, and after 24h of culture, 10μL of CCK-8 reagent was added to each well, and incubated in the dark for 1.5h; the absorbance (A) was measured at 450nm by an enzyme marker, and the average value of the A values of the 5 replicate wells was obtained, and the survival rate-time curve and the survival rate-concentration curve were drawn.

[0132] Cell survival rate (%) = A (experimental group) / A (control group) x 100%

[0133] (2) Protective effect on cells

[0134] The cells were divided into 4 groups: ① control group: adding serum-free culture medium; ② injury group: adding nano COM crystals with a concentration of 200μg / mL; ③ DSP protection group: adding DSP with concentrations of 30, 60, 90 and 120μg / mL and 200μg / mL of nano COM crystals; ④ Se-DSP protection group: adding Se-DSPs (Se-DSP2 or Se-DSP1) with concentrations of 30, 60, 90 and 120μg / mL and 200μg / mL of nano COM crystals; after 12h of incubation of the above groups, the cell viability was detected by CCK-8 reagent.

[0135] (3) Results

[0136] The results of toxicity detection of DSP, Se-DSP1 and Se-DSP2 on HK-2 cells are shown in A of Figure 9 From A of Figure 9 , it can be seen that the cell viability of HK-2 cells is above 100% after incubation with 30-120 μg / mL polysaccharides for 24 h, indicating that DSP, Se-DSP1 and Se-DSP2 have no toxicity on HK-2 cells, and even have a promoting effect on cell growth. The protective effect of DSP, Se-DSP1 and Se-DSP2 on HK-2 cells is shown in B of Figure 9 From B of Figure 9 , it can be seen that the cell viability of HK-2 cells decreases from 100% to 62% after incubation with 200 μg / mL of calcium oxalate monohydrate (nano-COM crystal) with a size of about 100 nm for 24 h, indicating that nano-COM crystal has a significant damaging effect on HK-2 cells. When DSP or Se-DSP1, Se-DSP2 with a concentration of 30, 60, 90, 120 g / L is added to the cells together with nano-COM crystal, the cell viability is increased to different degrees, indicating that the addition of polysaccharides can reduce the cell damage caused by nano-COM crystal, and the protective effect is best at 60 μg / mL.

[0137] The above results show that the polysaccharides of Desmodium styracifolium treated with selenium not only have no toxicity on cells, but also have a greater increase in cell viability than the polysaccharides without selenium treatment, and the protective ability on damaged cells is increased with the increase of selenium content, i.e. Se-DSP2>Se-DSP1>DSP.

[0138] 3. Effect of DSP and Se-DSPs on the amount of phosphatidylserine (PS) flipping

[0139] HK-2 cells were cultured and treated as in Example 4, and were repaired with serum-free medium containing 80 μg / mL of DSP and Se-DSPs; after the repair time, the effect of DSP, Se-DSP1 and Se-DSP2 on the amount of PS flipping was detected by the same method as in Example 2, and the results are shown in Figure 10 ; A of Figure 10 is a flow quantitative analysis diagram of PS flipping of HK-2 cells; and B of Figure 10 is a corresponding quantitative column chart. From Figure 10 , it can be seen that the amount of PS flipping of the damaged cells (DC) (27.5%) is much greater than that of the control cells (2.80%). After the damaged cells are repaired by DSP, Se-DSP1 and Se-DSP2, the amount of PS flipping is reduced (5.07%-18.3%); among them, the amount of PS flipping after repair by Se-DSP2 (5.07%) is close to that of the control cells, and the repair effect is the best.

[0140] 4. Effects of DSP and Se-DSPs on intracellular nitric oxide (NO) level

[0141] HK-2 cells were cultured and treated as in Example 4, and were repaired with serum-free medium containing 80 μg / mL DSP and Se-DSPs; after the repair time was reached, the supernatant in the 6-well plate was aspirated, 1 mL of DAF-FM diluent was added to each well, and incubation was carried out in a 37°C incubator for 30 min in the dark; the cells were washed with serum-free medium for 3 times, and the 6-well plate was observed under an inverted fluorescence microscope.

[0142] The effects of DSP and Se-DSPs on intracellular NO level are shown in Figure 11 Figure 11 A in Figure 11 is a NO fluorescence photograph; B in Figure 11 is a column chart of relative fluorescence intensity. It can be seen from that under the fluorescence microscope observation, the green fluorescence of normal cells is weak, indicating that the intracellular NO is less. However, the green fluorescence of the cells after oxalic acid damage is significantly enhanced, i.e. oxalic acid causes severe oxidative damage to the cells, resulting in an increase in intracellular NO, which will increase the oxidative stress level of the cells and further aggravate the cell damage. After the damaged cells are repaired by DSP and Se-DSPs, the green fluorescence of the cells gradually weakens, i.e. the NO level decreases, and the Se-DSP2 group is closest to the normal group.

[0143] 5. Observation of the repair of cell morphology by DSP and Se-DSPs by hematoxylin and eosin (HE) staining

[0144] The nucleus is composed of negatively charged acidic substances, and has strong affinity with positively charged basic dye hematein oxide hematein red; while the cytoplasm is opposite, and has strong affinity with negatively charged acidic dye eosin due to containing positively charged basic substances. Therefore, after the cells are stained by HE, the nucleus is stained blue-violet, and the cytoplasm is stained red, pink or orange-red.

[0145] Figure 12 HK-2 cells were cultured and treated as in Example 4, and were repaired with serum-free medium containing 60 μg / mL DSP and Se-DSPs; after the repair time was reached, the cells were washed with PBS to remove the culture medium, and were fixed with 4% paraformaldehyde at room temperature for 15 min; hematoxylin and eosin staining solution was used for staining for 15 min and 5 min, respectively; the unbound dye was washed away with tap water, and the morphology of the cells was observed under a microscope. The repair results of cell morphology by DSP and Se-DSPs are shown in Figure 12 ​It can be seen that the normal cell morphology is full and the cell number is more without oxalic acid damage. The cell morphology is shriveled and disordered, the cell membrane is ruptured, and the cell number is obviously less in the oxalic acid damage group. After the repair by DSP, Se-DSP1 and Se-DSP2, the cell morphology is gradually perfect, the cell number is gradually increased, the number of cells with ruptured cell membrane is reduced, and especially the cell state after the repair by DSP2 is close to that of normal cells.

[0146] The present application obtains two kinds of selenium polysaccharides Se-DSP1 and Se-DSP2 with selenium contents of 1.5 mg / g and 3.2 mg / g respectively by seleniumizing DSP. The in vitro antioxidant activity and the ability of repairing damaged cells of the selenium polysaccharides are obviously improved. The seleniumized polysaccharides of Desmodium styracifolium can enhance the activity and self-healing ability of damaged HK-2 cells and reduce PS exocytosis.

[0147] Example 5: Test of the anti-stone effect of different selenium polysaccharides and selenium polysaccharide combinations

[0148] The present application detects the effect of different selenium polysaccharides and selenium polysaccharide combinations in preventing stones by constructing a calcium oxalate kidney stone model of rats. The experimental process is as follows:

[0149] 1. Experimental animals and grouping

[0150] The rats used in the experiment are 7-week-old SPF male SD rats with a body weight of 180-220 g, which are purchased from the Guangdong Medical Experimental Animal Center. All animal studies comply with the National "Regulations on the Management of Experimental Animals" and "Detailed Rules for the Implementation of the Regulations on the Management of Experimental Animals in Guangdong Province", and are approved by the Ethics Committee. The animal feeding and experimental procedures during the animal experiment comply with all applicable guidelines for the care and use of experimental animals. The rats are raised at a temperature of 23±2℃, a relative humidity of about 55%, and a 12h cycle of illumination, and are free to eat and drink.

[0151] Experimental grouping: Before the start of the animal experiment, the animals are adapted to the environment around the feeding room for one week. The rats are randomly divided into 8 groups (n=6), all of which are fed with standard granular feed and free to drink sterile water. The body weight changes of the rats in each group are detected every day, and the injection amount of acetaldehyde and the gavage amount of polysaccharides are reasonably and appropriately changed. The modeling process lasts for 4 weeks, and the intraperitoneal injection and gavage are performed 5 times a week (Monday to Friday).

[0152] Group 1 (normal rat group): 1 mL of normal saline intraperitoneal injection, 2 mL of normal saline gavage.

[0153] Group 2 (kidney stone modeling group): 1 mL of 60 mg / kg acetaldehyde intraperitoneal injection, 2 mL of normal saline gavage, continuously inducing the deposition of microcrystals in the kidney;

[0154] Group 3 (Se-CSP treatment group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 2 mL of 200 mg / kg Se-CSP gavage,

[0155] Group 4 (Se-DSP1 treatment group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 2 mL of 200 mg / kg Se-DSP1 gavage;

[0156] Group 5 (Se-DSP2 treatment group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 2 mL of 200 mg / kg Se-DSP2 gavage;

[0157] Group 6 (Se-CSP+Se-DSP2 treatment group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 1 mL of 100 mg / kg Se-CSP gavage, 1 mL of 100 mg / kg Se-DSP2 gavage;

[0158] Group 7 (Se-CSP+Se-Astragalus polysaccharide treatment group, comparison group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 1 mL of 100 mg / kg Se-CSP gavage, 1 mL of 100 mg / kg Se-Astragalus polysaccharide gavage;

[0159] Group 8 (Se-DSP2+Se-Astragalus polysaccharide treatment group, comparison group): 1 mL of 60 mg / kg glyoxylic acid intraperitoneal injection, 1 mL of 100 mg / kg Se-CSP2 gavage, 1 mL of 100 mg / kg Se-Astragalus polysaccharide gavage.

[0160] The preparation of the Se-Astragalus polysaccharide can refer to the literature [A1].

[0161] [A1] Huang F, Sun X-Y, Ouyang J-M. Preparation and characterization of selenized Astragalus polysaccharide and its inhibitory effect on kidney stones. Mater. Sci. Eng. C, 2020, 110: 110732.

[0162] 2. Sample collection

[0163] One day before the end of animal modeling, the urine of rats in each group was collected by metabolic cages for 24 hours, and the urine biochemical indicators were detected.

[0164] 3. Detection index and result

[0165] The detection indexes include the concentrations of creatinine, citric acid, calcium ion and oxalic acid in urine, and the results are shown in Table 2. Among them, the concentrations of urine creatinine and urine calcium ion are detected by using a full-automatic biochemical analyzer; the concentrations of urine citric acid and urine oxalic acid are detected by using an ion chromatograph.

[0166] Table 2: Determination results of urine biochemical indexes of rats in each group for 24 hours

[0167]

[0168] As can be seen from the results in Table 2, the combination of Se-CSP and Se-DSP2 (i.e. group 6) can further improve the effect of preventing calculus of the seleniumized polysaccharide, which is not only superior to the Se-CSP group (group 3) and the Se-DSP2 group (group 5) alone, but also shows a significant synergistic effect compared with group 7 and group 8, indicating that the specific combination of seleniumized corn silk polysaccharide (Se-CSP) and seleniumized broad money grass polysaccharide (Se-DSPs) plays a significant synergistic effect.

[0169] The basis for evaluating the effect of preventing calculus is that the decrease of the concentration of creatinine in urine indicates the decrease of kidney damage; citric acid is an inhibiting factor of kidney calculus, and the increase of the concentration of urine citric acid indicates that the formation of kidney calculus is inhibited, and the greater the concentration, the better the inhibiting effect; calcium and oxalic acid are promoting factors of kidney calculus, and the decrease of the concentrations of urine calcium and urine oxalic acid indicates that the formation of kidney calculus is inhibited, and the smaller the concentration, the better the inhibiting effect.

[0170] 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 and simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. Use of selenium-containing Chinese herbal medicine polysaccharide in the preparation of a preparation for preventing and treating calculus, characterized in that, The selenium-containing Chinese herbal medicine polysaccharide is selenium-containing corn silk polysaccharide and / or selenium-containing desmodium styracifolium polysaccharide. The selenium content of the selenium corn silk polysaccharide is 1.5-2 mg / g, and the preparation method is as follows: corn silk polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted in water bath at 65-75 DEG C for 4-8 h, the pH value of the solution is adjusted to 5-6; excess Na2SO4 is added to remove Ba 2+ Ions; the supernatant is first dialyzed with running water for 48-72 h, and then dialyzed with distilled water for 12-24 h; a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped until there is no red color; the dialysate is alcohol precipitated, and the obtained precipitate is dried to obtain selenium corn silk polysaccharide; The selenium content of the selenium-containing psuedostellaria heterophylla polysaccharide is 1.5-3.2 mg / g, and the preparation method is as follows: the psuedostellaria heterophylla polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted at 65-75 DEG C for 4-8 h, the pH value of the solution is adjusted to 7-8; excessive Na2SO4 is added to remove Ba 2+ Ions; the supernatant is dialyzed with distilled water, a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped until there is no red color; the solution obtained by dialysis is alcohol precipitated, and the obtained precipitate is dried to obtain selenium-containing psuedostellaria heterophylla polysaccharide.

2. Use according to claim 1, characterized in that, The component of the calculus is calcium salt crystal.

3. Use according to claim 2, characterized in that, The calcium salt is calcium oxalate.

4. The use according to claim 1, characterized in that, The preparation method of the corn silk polysaccharide comprises the following steps: S1. Extraction of corn silk crude polysaccharide: dry or fresh corn silk is crushed and soaked in distilled water overnight, and then heated for extraction. The filtrate obtained by extraction is subjected to alcohol precipitation, and the obtained precipitate is dried to obtain corn silk crude polysaccharide; S2. Degradation of corn silk crude polysaccharide: the extracted corn silk crude polysaccharide is prepared into a solution, and is subjected to ultrasonic degradation under the condition that the ultrasonic frequency is 400-800 W and the ultrasonic frequency is 30-50 kHz. The degradation solution is collected after ultrasonic degradation for 10-60 min, and is subjected to alcohol precipitation. The obtained precipitate is dried to obtain corn silk polysaccharide.

5. The use according to claim 1, characterized in that, The preparation method of the desmodium styracifolium polysaccharide comprises the following steps: S1. Extraction of desmodium styracifolium crude polysaccharide: the desmodium styracifolium medicinal material powder is extracted with boiling water for 2-3 times, each time for 1-2 h. The obtained filtrate is concentrated and centrifuged, and the supernatant is taken. The obtained supernatant is subjected to alcohol precipitation, and the obtained precipitate is freeze-dried to obtain desmodium styracifolium crude polysaccharide; S2. Degradation of desmodium styracifolium crude polysaccharide: the extracted desmodium styracifolium crude polysaccharide is prepared into a solution, heated to a temperature of 88-92 DEG C, and then hydrogen peroxide is added to make the concentration of hydrogen peroxide in the degradation system 4-6%. After degradation for 1-2 h, the solution is cooled to room temperature, the pH of the solution is adjusted to 6.8-7.2, and then subjected to alcohol precipitation. The obtained precipitate is dried to obtain desmodium styracifolium polysaccharide.

6. A preparation for preventing and treating calculus, characterized by, The selenium-containing corn silk polysaccharide and the selenium-containing desmodium styracifolium polysaccharide. The selenium content of the selenium corn silk polysaccharide is 1.5-2 mg / g, and the preparation method is as follows: corn silk polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted in water bath at 65-75 DEG C for 4-8 h, the pH value of the solution is adjusted to 5-6; excess Na2SO4 is added to remove Ba 2+ Ions; the supernatant is first dialyzed with running water for 48-72 h, and then dialyzed with distilled water for 12-24 h; a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped until there is no red color; the dialysate is alcohol precipitated, and the obtained precipitate is dried to obtain selenium corn silk polysaccharide; The selenium content of the selenium-containing psuedostellaria heterophylla polysaccharide is 1.5-3.2 mg / g, and the preparation method is as follows: the psuedostellaria heterophylla polysaccharide is dissolved in HNO3, Na2SeO3 and BaCl2 are added, and the solution is reacted at 65-75 DEG C for 4-8 h, the pH value of the solution is adjusted to 7-8; excessive Na2SO4 is added to remove Ba 2+ Ions; the supernatant is dialyzed with distilled water, a small amount of dialysate is taken every 6-8 h, ascorbic acid is added, and the dialysis is stopped until there is no red color; the solution obtained by dialysis is alcohol precipitated, and the obtained precipitate is dried to obtain selenium-containing psuedostellaria heterophylla polysaccharide.

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