Traditional Chinese medicine extract for treating nephritis as well as preparation method and medical application thereof

Abstract

The invention relates to a traditional Chinese medicine clerodendranthus spicatus extract for treating nephritis. The extract contains tanshinol (content gt; 0.07%), protocatechuic acid (content gt; 0.12%), rosmarinic acid (content gt; 9%), alkannic acid (content gt; 1.2%), helelisters C (the content of gt; 4.0%), salvianolic acid B (content gt; 2.0%), salvianolic acid A (content gt; 2.0%). The invention further provides a pharmaceutical composition containing the extract provided by the invention, and the pharmaceutical composition is selected from any dosage form capable of being taken. The dosage form is selected from tablets, capsules, oral liquid, granules, pills, ointment, dripping pills and injections.

Description

Technical Field

[0001] The invention aims to provide a Chinese herbal medicine kidney tea extract for treating nephritis and a composition thereof, as well as a preparation method, medical use and content determination method thereof. The extract is characterized in that it contains danshensu, protocatechuic acid, rosmarinic acid, lithospermic acid, helisterculins C, salvianolic acid B and salvianolic acid A, and can be used to treat acute nephritis, chronic nephritis, renal injury and other nephritis diseases, including renal fibrosis, and can shorten the recovery period and improve symptoms. Background Art

[0002] Nephrosis (NS) broadly refers to various diseases that occur in the kidneys. According to the course of the disease, it can be divided into acute kidney injury (AKI) and chronic kidney disease (CKD). Kidney disease is a serious disease that endangers human health in recent years, and the number of patients has increased year by year. Finding targeted drugs to treat kidney disease is one of the current research hotspots in the medical field.

[0003] Kidney tea (Clerodendranthus spicatus (Thunb.) CYWu) is a Dai ethnic medicine with pharmacological effects such as regulating renal function, regulating immunity, lowering uric acid, and anti-inflammatory. It is clinically used in acute and chronic nephritis, urinary tract infection, rheumatoid arthritis, etc. Kidney tea contains a variety of flavonoids, a variety of phenolic acid chemical components (such as rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, lithospermic acid, salvianolic acid A, salvianolic acid B, etc.), polyterpenoids (including diterpenes, triterpenes, sesquiterpenes and norstaminane, among which the diterpenes mainly have isopimarane skeleton, staminane skeleton, secoisopimarane skeleton and norstaminane skeleton; triterpenoid components isolated from kidney tea: orthosiphonoic acid, betulinic acid, ursolic acid, tormentic acid, euscaphic acid, oleanolic acid, maslinic acid and 2α,3α-dihydroxyolean-12-en-28-oic acid. It is not clear which type of components are the effective components of kidney tea for kidney protection.

[0004] The present invention has conducted an in-depth study on kidney tea, prepared a variety of kidney tea extracts, tested their effects on treating nephritis, and unexpectedly found that only the phenolic acid components in kidney tea (SC-CqB extracts obtained using the detection method of the present invention) have the effect of protecting kidney damage, while other types of extracts or extracts prepared by other methods do not have the effect of treating nephritis.

[0005] The inventors used the unilateral renal pedicle clamp and contralateral ligation method to simulate clinical renal ischemia-reperfusion injury (RIRI) to establish an AKI rat model and a 5 / 6 nephrectomy rat renal injury CKD rat model, and found the pharmacodynamic effect of SC-CqB on AKI and the inhibitory effect on the transformation process of AKI to CKD, and discovered the mechanism by which SC-CqB exerts a renal protective effect. Summary of the invention

[0006] The invention aims to provide a Chinese medicinal kidney tea extract for treating nephritis and a composition thereof, as well as a preparation method, medical use and content determination method thereof.

[0007] To this end, the present invention provides a kidney tea extract (SC-CqB) for treating nephritis, which contains danshensu (content>0.07%), protocatechuic acid (content>0.12%), rosmarinic acid (content>9%), lithospermic acid (content>1.2%), helisterculins C (content>4.0%), salvianolic acid B (content>2.0%), and salvianolic acid A (content>2.0%).

[0008] Preferably, the kidney tea extract of the present invention contains danshensu (content>0.1%), protocatechuic acid (content>0.15%), rosmarinic acid (content>12%), lithospermic acid (content>1.8%), helisterculins C (content>6.0%), salvianolic acid B (content>4.0%), and salvianolic acid A (content>4.0%); the optimal extract characteristic chemical component content is danshensu (content>0.14%), protocatechuic acid (content>0.22%), rosmarinic acid (content>16%), lithospermic acid (content>2.2%), helisterculins C (content>8.0%), salvianolic acid B (content>5.0%), and salvianolic acid A (content>5.0%).

[0009] The present invention further provides a pharmaceutical composition containing the Herba Spatholobi extract of the present invention. The pharmaceutical composition is selected from any edible dosage form, which is selected from tablets, capsules, oral liquids, granules, pills, pastes, pellets, and injections.

[0010] The present invention further provides the Chinese medicinal herb kidney tea extract, and the pharmaceutical composition is used in the preparation of medicines for treating acute nephritis, chronic nephritis, renal injury and other nephritis diseases, including renal fibrosis, which can shorten the recovery period and improve symptoms.

[0011] The present invention further provides a method for preparing the kidney tea extract, wherein the method comprises the following steps:

[0012] The kidney tea is extracted with water or aqueous ethanol, the extract is concentrated, the solvent is recovered to a small volume, diluted with water, and then the pH value is adjusted to acidity with an organic acid or an inorganic acid, and extracted with an organic solvent to obtain an organic solvent layer and an acid water layer; pure water is added to the organic solvent layer, and the water layer is adjusted to alkaline (pH value is between 7-14) with an appropriate amount of organic base or inorganic base, and then extracted to obtain an alkaline water layer and an organic solvent layer; the alkaline water layer is adjusted to acidity with an organic acid or an inorganic acid, and extracted with an organic solvent to obtain an organic solvent layer and an acid water layer, and after the organic solvent layer is washed with water to neutrality, the organic solvent is recovered and dried to obtain a Chinese medicine kidney tea extract for treating nephritis, wherein:

[0013] The organic solvent for extraction is selected from: ethyl acetate, n-butanol, isopropanol, chloroform, dichloromethane or a combination of these solvents, preferably ethyl acetate, chloroform, dichloromethane or a combination of these solvents, and most preferably ethyl acetate;

[0014] The organic acid or inorganic acid is selected from: formic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates, etc., preferably formic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof, and most preferably hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates and a combination thereof;

[0015] The organic base or inorganic base is selected from: ammonia water, diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, alkaline phosphates and combinations thereof, preferably ammonia water, sodium hydroxide, sodium carbonate, sodium bicarbonate, alkaline phosphates and combinations thereof; and most preferably sodium hydroxide, sodium carbonate, sodium bicarbonate, alkaline phosphates and combinations thereof;

[0016] The first extraction was adjusted to an acidic pH value of <6, optimized to a pH value of <4, and optimally to a pH value of <2;

[0017] The second extraction was adjusted to an alkaline pH value > 7.5, optimized to a pH value > 8, and the optimal pH value was pH = 8;

[0018] The third extraction adjusts the acidic pH value to <6, optimizes it to pH <4, and optimally to pH <2; 7) The extraction solvent of the kidney tea medicinal material is water or hydrous ethanol, optimized to water or hydrous ethanol with an ethanol content of <70%; the optimal solvent is hydrous ethanol with an ethanol content of <70%.

[0019] The present invention further provides another preparation method of the extract of the present invention, wherein the steps are as follows: extracting kidney tea with water or aqueous ethanol, concentrating the extract, recovering the solvent to a small volume, diluting with water, adjusting the pH value with an acid to be acidic, loading the sample onto a macroporous adsorption resin column, first eluting with water and low-concentration aqueous ethanol, then eluting with high-concentration aqueous ethanol, recovering the high-concentration aqueous ethanol eluate, concentrating, and drying, wherein the acidic pH value is adjusted to <6 before loading, optimized to a pH value <4, and optimally to a pH value <2;

[0020] The acid used for adjusting the acidity before loading is an organic acid or an inorganic base, which can be various water-soluble acids, such as formic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates, etc., preferably formic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof, and most preferably hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof;

[0021] The macroporous resin can be various styrene-type macroporous resins, optimized as non-polar, low-polar and medium-polar macroporous resins, and the best is medium-polar macroporous resin (such as HPD-500); 4) The solvent for eluting impurities for the first time is water and low-concentration aqueous ethanol (ethanol concentration <15%), optimized as water and low-concentration aqueous ethanol (ethanol concentration <10%), and the best is water and low-concentration aqueous ethanol (ethanol concentration <5%); 5) The solvent for eluting the effective extract for the second time is high-concentration aqueous ethanol (ethanol concentration>20%), optimized as high-concentration aqueous ethanol (ethanol concentration>30%), and the best is high-concentration aqueous ethanol (ethanol concentration>60%).

[0022] The present invention further provides a detection method for the kidney tea extract of the present invention, and the method steps are as follows: preparation of a test solution: accurately weigh the extract (or kidney tea medicinal material) sample powder, place it in a stoppered triangular flask, accurately add 20 mL of 50% methanol, accurately weigh the mass, ultrasonically treat it at room temperature for 0.5 h, take it out and cool it to room temperature, make up the reduced mass with 50% methanol, shake it well and let it stand, take the supernatant and centrifuge it, take 5 mL of the supernatant after centrifugation to a 10 mL volumetric flask, dilute it to the scale with water, shake it well, take 10 mL and apply it to the pretreated ODS-SPE column, first wash 10 mL with water and discard it, then elute about 5 mL to a 5 mL volumetric flask with 100% MeOH, dilute it to the scale with 50% MeOH, shake it well, and pass it through a 0.22 μm filter membrane to obtain the test solution. Preparation of reference solution: Accurately weigh an appropriate amount of each reference compound and use methanol to prepare mixed standard solutions with concentrations of protocatechuic acid (190ug / mL), rosmarinic acid (1.2mg / mL), lithospermic acid (1.2mg / mL), helisterculins C (2.4mg / mL), salvianolic acid B (2mg / mL), salvianolic acid A (170ug / mL), and sodium danshensu (110ug / mL).

[0023] Detection: Inject the test solution and the reference solution into a high performance liquid chromatograph to obtain a chromatogram, and calculate the content of each component based on the chromatogram.

[0024] The HPLC conditions are as follows

[0025] The chromatographic column is an ODS chromatographic column (4.6×150 mm, 5 μm), and acetonitrile (A)-0.3% phosphoric acid aqueous solution (B) is used as the mobile phase for gradient elution (0-10 minutes, 5% A, 10-15 minutes, 5%-15% A, 15-30 minutes, 15%-17% A, 30-35 minutes, 17%-22% A, 35-50 minutes, 22%-23% A, 50-60 minutes, 23%-90% A), the flow rate is 1.0 mL / min; the column temperature is 20° C.; the detection wavelength is 190-400 nm; and the injection volume is 10 μL.

[0026] The most preferred extraction method of the present invention is shown in the Examples of the present invention.

[0027] The following test results are the results of multiple batches of tests on the extracts obtained by the preparation method in the embodiment of the present invention:

[0028] name CFX-1 CFX-2 CFX-3 CFX-4 CFX-5 CFX-6 Mean RSD Danshensu Sodium 0.159 0.156 0.162 0.163 0.161 0.162 0.161 1.596 Protocatechuic acid 0.239 0.249 0.247 0.249 0.249 0.253 0.248 1.925 Rosmarinic acid 19.343 20.029 19.375 20.198 20.035 19.964 19.824 1.858 Lithospermic acid 2.734 2.701 2.776 2.683 2.714 2.672 2.713 1.387 helisterculins C 10.631 10.837 10.675 10.962 11.033 10.868 10.834 1.451 Dan B 5.621 5.749 5.660 5.505 5.581 5.690 5.634 1.515 Dan A 5.223 5.443 5.284 5.459 5.409 5.412 5.371 1.775

[0029] The beneficial effects of the present invention are demonstrated by experimental data as follows.

[0030] 1. Protective effect of SC-CqB on rats with acute renal injury induced by ischemia-reperfusion

[0031] 1.1 Effects of SC-CqB on body weight and renal coefficient in rats with acute renal injury induced by ischemia-reperfusion

[0032] The experimental process and RIRI modeling process are shown in the figure ( Figure 2-1 AB), the left kidney of the rat was clamped by an artery clamp. The kidney was slightly pale in the initial stage. After the kidney was clamped for 45 minutes, it turned obvious purple-red, or even purple-black. After the artery clamp was removed, the ischemic kidney returned to bright red within 5 minutes.

[0033] The body weight and kidney coefficient of each group of experimental animals are shown in the figure ( Figure 2-1 CE). As shown in Figures C and D, after the first day of RIRI modeling, the body weight of rats in each modeling group was significantly reduced compared with the Control group, and there was no significant difference in the 1 / 2Nx group. Ten days after administration, compared with the RIRI group, there was no significant change in the body weight of rats in the SC-CqB dose groups and the Dex group. As shown in Figure E, ten days after administration, compared with the Control group, the kidney coefficient of rats in the 1 / 2Nx group and each RIRI modeling group was significantly increased; compared with the RIRI group, the kidney coefficient of each SC-CqB dose group was reduced, but it was not statistically significant, and the kidney coefficient of the Dex group was significantly reduced. This suggests that SC-CqB has no significant effect on the body weight of rats in the RIRI-induced AKI model.

[0034] Figure 2-1.Effects of SC CqB on body weight and renal index of rats with AKI induced by I / R injury (A) Renal ischemia-reperfusion injury protocol of SD rats. (B) RIRI surgery. (C) The modeled body weight was measured 1 day and 3 days, 5 days, 7 days and 10 days after administration. n=10-11. (D) The body weight gain after 10 days of administration was calculated. n=10-11. (E) The renal index after 10 days of administration was calculated. n=6. All data are shown as mean ± SEM. a is the Dex group; b is the SC-CqB-L group; c is the SC-CqB-H group; RIRI group compared with the control group ## p<0.01, ### p<0.001; RIRI group compared with 1 / 2Nx group & p<0.05; 1 / 2Nx group compared with control group $$$ p<0.001; compared with RIRI group * p<0.05, ** p<0.01.

[0035] 1.2 Effects of SC-CqB on serum creatinine and urea nitrogen in rats with acute renal injury induced by ischemia-reperfusion

[0036] Increased SCr and BUN are one of the characteristics of patients with renal insufficiency

[103] Clinically, only an increase in SCr or only an increase in BUN cannot be used as an indicator of renal insufficiency. The SCr and BUN contents of each group of experimental animals are shown in the figure ( Figure 2-2 ). As shown in Figure A, compared with the Control group, the SCr content of rats in the 1 / 2Nx group and the RIRI group was significantly increased; compared with the 1 / 2Nx group, the SCr content of rats in the RIRI group was significantly increased; compared with the RIRI group, the SCr content of rats in the SC-CqB medium-dose, high-dose group and Dex group was significantly decreased. As shown in Figure B, compared with the Control group, the BUN content of rats in the 1 / 2Nx group and the RIRI group was significantly increased; compared with the 1 / 2Nx group, the BUN content of rats in the RIRI group was significantly increased; compared with the RIRI group, the BUN content of rats in the SC-CqB low-dose, medium-dose, high-dose group and Dex group was significantly decreased. This suggests that SC-CqB can significantly improve the renal function of rats with RIRI-induced AKI model.

[0037] Figure 2-2.Effects of SC-CqB on SCr and BUN in rats with AKI induced by I / R injury. (A) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC CqB on SCr. (B) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SCCqB on BUN. All data are shown as mean ± SEM, n = 4. Compared with the control group, RIRI group ###p<0.001; compared with the 1 / 2Nx group, RIRI group &p<0.05; compared with the control group, 1 / 2Nx group $p<0.05, $$p<0.01; compared with the RIRI group *p<0.05

[0038] 1.3 Effect of SC-CqB on uric acid in rats with acute renal injury induced by ischemia-reperfusion

[0039] The UA content of each group of experimental animals is shown in the figure ( Figure 2-3 ). As shown in the figure, compared with the Control group, the UA content of rats in the 1 / 2Nx group and the RIRI group increased significantly; compared with the 1 / 2Nx group, the UA content of rats in the RIRI group increased significantly; compared with the RIRI group, the UA content of rats in the low, medium, and high dose groups of SC-CqB and the Dex group decreased significantly. This indicates that SC-CqB can significantly reduce the serum UA content of rats in the RIRI-induced AKI model and alleviate renal damage.

[0040] Figure 2-3 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC CqB on UA ​​in rats with AKI induced by I / R injury. All data are shown as mean ± SEM, n = 4. Compared with the control group, the RIRI group ### p<0.001; RIRI group compared with 1 / 2Nx group & p<0.05; compared with the control group, 1 / 2Nx group $ p<0.05; compared with RIRI group ** p<0.01.

[0041] 1.4 Effects of SC-CqB on body weight and renal coefficient in rats with chronic renal injury induced by ischemia-reperfusion

[0042] The experimental process is shown in Figure ( Figure 2-4 The body weight and kidney coefficient of each group of experimental animals are shown in ( Figure 2-4). As shown in Figures A and B, after the first day of RIRI modeling, the body weight of rats in the 1 / 2Nx group and the RIRI group was significantly reduced compared with the Control group. After 28 days of administration, there was no significant change in the body weight of rats in the RIRI group compared with the 1 / 2Nx group; compared with the RIRI group, there was no significant change in the body weight of rats in the low, medium, and high dose groups of SC-CqB and the Dex group. As shown in Figure C, after 28 days of administration, compared with the Control group, the renal coefficient of rats in the 1 / 2Nx group and the RIRI group was significantly increased; compared with the 1 / 2Nx group, there was no significant change in the renal coefficient of rats in the RIRI group; compared with the RIRI group, the renal coefficient of rats in the medium dose group of SC-CqB and the UCG group was significantly reduced. This suggests that SC-CqB has no significant effect on the body weight of RIRI-induced CKD rats.

[0043] Figure 2-4 Effects of SC-CqB on body weight and renal index of CKD rats induced by I / R injury (A) The body weight of the model was measured on the 1st day, 3rd day, 5th day, 1st week, 2nd week, 3rd week, and 4th week after administration, n=10-11. (B) The body weight gain was calculated after 4 weeks of administration, n=10-11. (C) The renal index was calculated after 4 weeks of administration, n=6. All data are expressed as mean ± SEM. a, SC-CqB-L group; b, SC-CqB-M group. Comparison between RIRI group and control group # p<0.05, ## p<0.01, ### p<0.001; $ p<0.05, comparison between 1 / 2Nx group and control group $$$ p<0.001; compared with the RIRI group, * p<0.05, ** p<0.01.

[0044] Effects of 1.5SC-CqB on serum creatinine and urea nitrogen levels in rats with chronic renal injury induced by ischemia-reperfusion

[0045] The SCr and BUN contents of experimental animals in each group are shown in the figure ( Figure 2-5). As shown in Figure A, compared with the Control group, the SCr content of rats in the 1 / 2Nx group and the RIRI group increased significantly; compared with the 1 / 2Nx group, the SCr content of rats in the RIRI group increased, but it was not statistically significant; compared with the RIRI group, the SCr content of rats in the medium and high dose groups of SC-CqB decreased significantly. As shown in Figure B, compared with the Control group, the BUN content of rats in the RIRI group increased significantly, and the BUN content of rats in the 1 / 2Nx group increased but was not statistically significant; compared with the 1 / 2Nx group, the BUN content of rats in the RIRI group increased significantly; compared with the RIRI group, the BUN content of rats in the low, medium, high dose groups of SC-CqB and the UCG group decreased significantly. This suggests that SC-CqB can significantly improve the renal function of rats with RIRI-induced CKD model.

[0046] Figure 2-5 Effects of SC-CqB on SCr and BUN in CKD rats induced by I / R injury (A) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on SCr. (B) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on BUN. All data are expressed as mean ± SEM, n = 4. Comparison between RIRI group and control group ### p<0.001; RIRI group compared with 1 / 2Nx group ** p<0.05; 1 / 2Nx group compared with control group *** p<0.01; compared with RIRI group * p<0.05.

[0047] 1.6 Effect of SC-CqB on uric acid content in rats with chronic renal injury induced by ischemia-reperfusion

[0048] The UA content of each group of experimental animals is shown in the figure ( Figure 2-6 ). As shown in the figure, compared with the Control group, the UA content of rats in the RIRI group increased significantly, and the UA content of rats in the 1 / 2Nx group increased but was not statistically significant; compared with the 1 / 2Nx group, the UA content of rats in the RIRI group increased significantly; compared with the RIRI group, the UA content of rats in the SC-CqB medium-dose, high-dose and UCG groups decreased significantly. This suggests that SC-CqB can significantly reduce the UA content in the serum of RIRI-induced CKD model rats and alleviate renal damage.

[0049] Figure 2-6 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on UA ​​in rats with CKD induced by I / R injury. All data are presented as mean ± SEM, n = 4. RIRI group compared with Control group ###p<0.001; RIRI group compared with 1 / 2Nx group &&& p<0.001; compared with the RIRI group, ** p<0.01, *** p<0.001.

[0050] 1.7 Effect of SC-CqB on renal pathological structure in rats with acute and chronic renal injury induced by ischemia-reperfusion

[0051] The effect of SC-CqB on the pathological structural changes of kidneys after renal injury in AKI and CKD rats was investigated by using renal section histopathological examination. The HE staining results of experimental animals in the RIRI-AKI and RIRI-CKD groups are shown in the figure ( Figure 2-7 ).

[0052] HE staining results showed that the kidneys of rats in the RIRI-AKI Control group presented normal physiological structure, while the kidneys of rats in the RIRI-AKI 1 / 2Nx group showed mild lesions with a small amount of tubular dilatation, inflammatory cell infiltration, interstitial edema, and vacuolation of tubular epithelial cells, while the kidneys of rats in the RIRI-AKI RIRI group showed obvious pathological changes such as tubular dilatation, inflammatory cell infiltration, interstitial edema, and vacuolation and granulation of tubular epithelial cells. Administration of SC-CqB significantly improved the renal pathological damage of rats in the RIRI-AKI group.

[0053] The renal pathological changes of rats in each group of RIRI-CKD experiment were consistent with the results of RIRI-AKI. Compared with the RIRI-CKD Control group, the RIRI-CKD 1 / 2Nx group had almost no lesions or slight lesions, and the RIRI-CKD 1 / 2Nx group had no significant changes compared with the RIRI-AKI 1 / 2Nx group. The 28-day RIRI model group showed more obvious pathological changes in the kidneys of the renal tubular lumen dilatation, inflammatory cell infiltration, interstitial edema, and tubular epithelial cell vacuolation and granulation than the 10-day RIRI model group. After administration of SC-CqB, the renal pathological damage of rats in the RIRI-CKD group was significantly improved. This suggests that SC-CqB can significantly reduce the changes in the renal tissue pathological structure of RIRI-induced AKI and CKD model rats and alleviate renal function damage.

[0054] Figure 2-7Effects of SC-CqB on renal histopathological changes in rats with I / R-induced AKI or CKD. Pathological changes in renal tissue were observed by hematoxylin and eosin staining; scale bar = 50 μm (200×), 20 μm (400×). Blue arrows indicate inflammatory cell infiltration. Red arrows indicate the shedding of tubular epithelial cells, showing a bare basement membrane state. Green arrows indicate tubular regeneration, a large number of cells, and black nuclei. Yellow arrows indicate renal interstitial edema. Black arrows indicate cavitation and granular changes in tubular epithelial cells. Gray arrows indicate dilated tubular lumens. Contraction arrows indicate glomerular thrombosis. White dot arrows indicate shedding necrotic tubular epithelial cells.

[0055] 1.8 Effect of SC-CqB on renal fibrosis in rats with acute and chronic renal injury induced by ischemia-reperfusion

[0056] The effect of SC-CqB on renal fibrosis in AKI and CKD rats was investigated by histopathological examination of kidney sections. The results of Masson staining of experimental animals in the RIRI-AKI and RIRI-CKD groups are shown in the figure ( Figure 2-8 A).

[0057] Masson staining results showed that the kidneys of rats in the RIRI-AKI Control group presented normal physiological structure, the kidneys of rats in the RIRI-AKI 1 / 2Nx group presented mild fibrotic tissue, the glomeruli were basically normal, and a small amount of collagen fibers were expressed in the renal tissues. However, the kidneys of rats in the RIRI-AKI RIRI group presented obvious enlargement of glomerular volume, infiltration of interstitial inflammatory cells, and significantly increased expression of renal collagen fibers. After administration of SC-CqB, the expression of renal collagen fibers in the rats in the RIRI-AKI group was significantly reduced, the area of ​​collagen fibers was significantly reduced, and the lesions were alleviated.

[0058] The renal fibrosis of rats in each group of RIRI-CKD experiment was consistent with the results of RIRI-AKI. Compared with the RIRI-CKD Control group, the RIRI-CKD 1 / 2Nx group had almost no lesions or mild lesions. There was no significant change in the RIRI-CKD 1 / 2Nx group compared with the RIRI-AKI1 / 2Nx group. The kidneys of rats in the RIRI model group for 28 days showed more obvious enlargement of glomerular volume, interstitial inflammatory cell infiltration, and a significant increase in collagen fiber area compared with the 10-day RIRI model group. After administration of SC-CqB, the expression of collagen fibers in the kidneys of rats in the RIRI-CKD group could be significantly improved, the collagen fiber area was significantly reduced, and the lesions were alleviated.

[0059] The expression results of TGF-β1 and α-SMA in experimental animals of RIRI-AKI and RIRI-CKD groups are shown in the figure (Fig.2-8, B). Consistent with the results of Masson staining, 10 days after modeling, the expression of TGF-β1 and α-SMA in the RIRI group was significantly increased compared with the 1 / 2Nx group, and the expression of TGF-β1 and α-SMA in the Dex group and the medium and high dose groups of SC-CqB was significantly decreased; 28 days after modeling, the expression of TGF-β1 in the RIRI group was significantly increased compared with the 1 / 2Nx group, and the expression of α-SMA increased but was not statistically significant, the expression of α-SMA in the UCG group and the medium and high dose groups of SC-CqB was significantly decreased, and the expression of TGF-β1 in the UCG group and the high dose group of SC-CqB was significantly decreased.

[0060] These results suggest that SC-CqB can significantly reduce the degree of renal tissue fibrosis in rats with RIRI-induced AKI and CKD models, inhibit the progressive fibrosis process, and inhibit the transformation of RIRI-AKI to RIRI-CKD. Figure 2-8 Effects of SC-CqB on tubulointerstitial fibrosis in rats with I / R-induced AKI or CKD. (A) Masson staining to observe the pathological changes of renal tissues; scale bar = 50 μm (200×), 20 μm (400×). (B) Protein expression of α-SMA and TGF-β1 in renal tissues of rats with AKI and CKD, n = 3. All data are shown as mean ± SEM. Comparison between RIRI group and Control group # p<0.05, ## p<0.01, ### p<0.001; RIRI group compared with 1 / 2Nx group & p<0.05, &&& p<0.001; compared with the RIRI group, * p<0.05, ** p<0.01, *** p<0.001.

[0061] 2. Protective effect of SC-CqB on renal injury in rats with 5 / 6 nephrectomy 2.1 Effect of SC-CqB on body weight and renal coefficient in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0062] A total of 100 rats were included in this experiment. The experimental process is shown in the figure ( Figure 3-1A). During the 5 / 6 nephrectomy, one rat died of overdose of anesthesia, three rats died of excessive bleeding due to continuous bleeding one day after the operation, and four rats died of hematuria, urinary stones and urine retention one day after the operation. The surgical survival rate reached 90.70%. After random grouping, rats died in the 5 / 6Nx group, UCG group, SC-Cq-L group and SC-CqB-H group in the first week of administration; rats died in the 5 / 6Nx group and UCG group in the second week of administration. After adaptive feeding, the rats had smooth fur, soft texture, thick fur that was not easy to fall off, and normal urination and defecation. After surgery, the fur of the shaved area of ​​the rats in the Sham group grew rapidly, and there was no obvious difference in the hair on the whole body from before the operation. At the end of the experiment, it had grown almost to the preoperative appearance, and the rats were free to move and had normal urination and defecation. After surgery, the hair growth in the shaved area of ​​the rats in the 5 / 6Nx group was slow, the hair on the whole body was quite different from that before surgery, the hair was dry, hard and easy to fall off, the vitality was slightly reduced compared with that before surgery, the rats were free to move, and the amount of urine and feces increased. After 4 weeks of medication, the symptoms of the rats in each medication group were significantly improved compared with those in the 5 / 6Nx group.

[0063] The body weight and kidney coefficient of each group of experimental animals are shown in the figure ( Figure 3-1 BD). As shown in Figures B and C, after four weeks of administration, compared with the Sham group, the body weight of rats in the 5 / 6Nx group was significantly reduced, and the weight gain was significantly reduced; compared with the 5 / 6Nx group, there was no significant change in the body weight of rats in the SC-CqB dose groups and the UCG group. As shown in Figure D, after four weeks of administration, compared with the Sham group, the kidney coefficient of the 5 / 6Nx group was significantly increased; compared with the 5 / 6Nx group, the kidney coefficient of the SC-CqB medium and high dose groups and the UCG group was significantly reduced. This suggests that SC-CqB has no significant effect on the body weight of rats in the CKD model induced by 5 / 6Nx.

[0064] Figure 3-1 Effects of SC-CqB on body weight and renal index of CKD rats induced by 5 / 6 nephrectomy. (A) 5 / 6 nephrectomy scheme in rats (B) The body weight of the model was measured at 1d, 3d, 5d, 1 week, 2 weeks, 3 weeks, and 4 weeks after administration, n=13-16. (C) The body weight gain was calculated after 4 weeks of administration. (D) The renal index was calculated after 4 weeks of administration, n=8. All data are shown as mean ± SEM. Compared with the Sham group, # p<0.05, ### p<0.001; compared with 5 / 6Nx group. * p<0.05

[0065] Effect of SC-CqB on urine flow in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0066] In patients with renal insufficiency, renal tubular function is impaired, reabsorption dysfunction occurs, primary urine absorption is reduced, and nocturia increases

[116] The 24-hour urine flow of each group of experimental animals is shown in the figure ( Figure 3-2 ). After four weeks of administration, the urine flow of rats in the 5 / 6Nx group increased significantly compared with the Sham group; compared with the 5 / 6Nx group, the urine flow of rats in the UCG group and the medium- and high-dose groups of SC-CqB decreased significantly. This suggests that SC-CqB improves the renal reabsorption function of CKD model rats induced by 5 / 6Nx.

[0067] Figure 3-2 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on urine flow in CKD rats after 5 / 6 nephrectomy. Figure 3-2 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on urine flow in CKD rats after 5 / 6 nephrectomy. All data are shown as mean ± SEM, n = 8. Compared with the Sham group ## p<0.01; compared with 5 / 6Nx group * p<0.05.

[0068] 2.3 Effect of SC-CqB on creatinine and urea nitrogen in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0069] The UCr and SCr contents of experimental animals in each group are shown in the figure ( Figure 3-3 AB). After four weeks of administration, compared with the Sham group, the UCr and SCr of the rats in the 5 / 6Nx group were significantly increased; compared with the 5 / 6Nx group, the UCr of the rats in the UCG group and the medium- and high-dose groups of SC-CqB were significantly decreased, and the SCr of the rats in the medium- and high-dose groups of SC-CqB were significantly decreased.

[0070] The BUN content of experimental animals in each group is shown in the figure (Fig. 3-3C). After four weeks of administration, compared with the Sham group, the BUN of rats in the 5 / 6Nx group increased significantly; compared with the 5 / 6Nx group, the BUN of rats in the UCG group and the SC-CqB high-dose group decreased significantly. This suggests that SC-CqB can significantly improve the renal function of CKD model rats induced by 5 / 6Nx.

[0071] Figure 3-3 Effects of SC-CqB on Cr and BUN in 5 / 6 nephrectomized rats. (a, b) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on urinary and serum Crin (C) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on BUN. All data are expressed as mean ± SEM, n = 4. Compared with the Sham group ### p<0.001; compared with 5 / 6Nx group, * p<0.05, ** p<0.01.

[0072] Effect of SC-CqB on Glomerular Filtration Rate in Rats with Chronic Renal Injury Induced by 5 / 6 Nephrectomy

[0073] The GFR of experimental animals in each group is shown in the figure ( Figure 3-4 ). After four weeks of administration, the GFR of rats in the 5 / 6Nx group increased significantly compared with the Sham group; compared with the 5 / 6Nx group, the GFR of rats in the UCG group and the medium- and high-dose groups of SC-CqB decreased significantly. This suggests that SC-CqB improves the renal filtration function of 5 / 6Nx-induced CKD model rats.

[0074] Figure 3-4 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on CKD in rats after 5 / 6 nephrectomy. All data are shown as mean ± SEM, n = 4. Compared with the Sham group ### p<0.001; compared with 5 / 6Nx group ** p<0.01.

[0075] Effect of 2.5SC-CqB on uric acid and urine protein in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0076] The UA and 24h Up of experimental animals in each group are shown in the figure ( Figure 3-5 ). After four weeks of administration, compared with the Sham group, the UA and 24hUp of the rats in the 5 / 6Nx group were significantly increased; compared with the 5 / 6Nx group, the UA and 24hUp of the rats in the UCG group and the medium- and high-dose groups of SC-CqB were significantly decreased. This suggests that SC-CqB can significantly improve the renal excretion function of CKD model rats induced by 5 / 6Nx.

[0077] Figure 3-5 Effects of SC-CqB on UA ​​and Up in CKD rats after 5 / 6 nephrectomy. (A) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on UA ​​(B) Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on 24-hour Up. All data are shown as mean ± SEM. n = 4. Compared with the Sham group ## p<0.01; compared with 5 / 6Nx group, * p<0.05, ** p<0.01.

[0078] Effect of SC-CqB on renal pathological structure in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0079] The effect of SC-CqB on renal injury in CKD rats was investigated by histopathological examination of kidney sections. The HE staining results of experimental animals in each group of CKD are shown in the figure ( Figure 3-6 ). HE staining results showed that the kidneys of rats in the sham group showed normal physiological structure, while the kidneys of rats in the 5 / 6Nx group showed obvious pathological changes such as tubular dilatation, inflammatory cell infiltration, interstitial edema, and vacuolization and granulation of tubular epithelial cells; after administration of SC-CqB, the renal pathological damage of rats in the 5 / 6Nx group was significantly improved. This indicates that SC-CqB can significantly reduce the changes in the pathological structure of renal tissue in 5 / 6Nx-induced CKD model rats and alleviate renal function damage, which is consistent with the results of 2.5.7. This indicates that SC-CqB can significantly improve the renal structural changes in 5 / 6Nx-induced CKD model rats.

[0080] Figure 3-6 Effects of SC-CqB on renal histopathological changes in CKD rats induced by 5 / 6 nephrectomy injury. Pathological changes in renal tissues were observed by he staining; scale bar = 50 μm (200×), 20 μm (400×). Blue arrows indicate inflammatory cell infiltration. Red arrows indicate the shedding of tubular epithelial cells, showing a bare basement membrane state. Green arrows indicate tubular regeneration, a large number of cells, and black nuclei. Yellow arrows indicate renal interstitial edema. Black arrows indicate cavitation and granular changes in tubular epithelial cells. Gray arrows indicate dilated tubular lumens. Contraction arrows indicate glomerular thrombosis. White dot arrows indicate shedding necrotic tubular epithelial cells.

[0081] Effect of SC-CqB on renal fibrosis in rats with chronic renal injury induced by 5 / 6 nephrectomy

[0082] The effect of SC-CqB on renal injury in CKD rats was investigated by histological examination of kidney sections. The results of Masson staining of experimental animals in each group of CKD are shown in the figure (Figure 3-7A). The results of Masson staining showed that the kidneys of rats in the Sham group showed normal physiological structure, while the kidneys of rats in the 5 / 6Nx group showed obvious pathological changes such as inflammatory cell infiltration and increased renal collagen fibers; after administration of SC-CqB, the collagen fiber staining of rats in the 5 / 6Nx group could be significantly reduced. This suggests that SC-CqB can significantly reduce renal fibrosis in CKD model rats induced by 5 / 6Nx, which is consistent with the results of 2.5.8A.

[0083] The results of TGF-β1 and α-SMA expression in experimental animals of each group of CKD are shown in the figure (Figure 3-7B). 28 days after modeling, compared with the Sham group, the expression of TGF-β1 and α-SMA in rats in the 5 / 6Nx group was significantly reduced; compared with the 5 / 6Nx group, after treatment with medium and high doses of UCG and SC-CqB, the expression of TGF-β1 and α-SMA in rats was significantly increased, and the expression of TGF-β1 and α-SMA in rats in the low-dose SC-CqB group was increased, but there was no statistical significance.

[0084] The above results suggest that SC-CqB can significantly improve renal fibrosis in 5 / 6Nx-induced CKD model rats.

[0085] Figure 3-7 Effect of SC-CqB on tubulointerstitial fibrosis in CKD rats induced by 5 / 6 nephrectomy injury. (A) Masson staining to observe renal histopathological changes; scale bar = 50 μm (200×), 20 μm (400×). (B) Protein expression of α-SMA and TGF-β1 in the kidneys of CKD rats induced by 5 / 6 nephrectomy injury, n = 3. All data are shown as mean ± SEM. Compared with the Sham group, # p<0.05, ## p<0.01; compared with 5 / 6Nx group, * p<0.05, ** p<0.01, *** p<0.001. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 . Flow chart of extraction of Chinese herbal medicine kidney tea extract for treating nephritis

[0087] Figure 2-1 Effects of SC CqB on body weight and renal indexes in rats with AKI induced by I / R injury

[0088] Figure 2-2 .Effects of SC-CqB on SCr and BUN in rats with AKI induced by I / R injury.

[0089] Figure 2-3 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC CqB on UA ​​in rats with AKI induced by I / R injury.

[0090] Figure 2-4 Effects of SC-CqB on body weight and renal indexes in CKD rats induced by I / R injury

[0091] Figure 2-5 Effects of SC-CqB on SCr and BUN in CKD rats induced by I / R injury

[0092] Figure 2-6 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on UA ​​in CKD rats induced by I / R injury

[0093] Figure 2-7 Effects of SC-CqB on renal histopathological changes in rats with AKI or CKD induced by I / R

[0094] Figure 2-8 Effects of SC-CqB on tubulointerstitial fibrosis in rats with I / R-induced AKI or CKD

[0095] Figure 3-1 Effects of SC-CqB on body weight and renal indexes in CKD rats induced by 5 / 6 nephrectomy

[0096] Figure 3-2 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on urine flow in CKD rats after 5 / 6 nephrectomy

[0097] Figure 3-3 Effects of SC-CqB on Cr and BUN in 5 / 6 nephrectomized rats

[0098] Figure 3-4 Effects of different concentrations (20 mg / kg, 40 mg / kg, 80 mg / kg) of SC-CqB on CKD in rats after 5 / 6 nephrectomy

[0099] Figure 3-5 Effects of SC-CqB on UA ​​and Up in CKD rats after 5 / 6 nephrectomy

[0100] Figure 3-6 Effects of SC-CqB on renal histopathological changes in CKD rats induced by 5 / 6 nephrectomy

[0101] Figure 4-1 Sodium carbonate pH extraction flow chart

[0102] Figure 4-2 PBS extraction flow chart

[0103] Figure 4-3 PBS pH investigation flow chart

[0104] Figure 4-4 Extraction scale-up test

[0105] Figure 4-5 Comparison of two purification methods

[0106] Figure 4-6 Investigation of different pH values ​​of PBS

[0107] Figure 4-7Extraction process flow chart

[0108] Figure 5-1 Chromatograms of sample solutions from different macroporous resins

[0109] Figure 5-2 Chromatograms of ADS eluents with different solvents

[0110] Figure 5-3 Elution volume investigation

[0111] Figure 5-4 Sample HPLC chromatogram

[0112] Specific implementation methods (including but not limited to the following embodiments)

[0113] Example 1 Preparation of total phenolic acid extract SC-CqB from scutellaria baicalensis and investigation of extraction process of total phenolic acid from scutellaria baicalensis

[0114] 1 Investigation on pH adjustment with sodium carbonate

[0115] Weigh 10g of kidney tea extract accurately, dissolve it in 100mL of water, adjust the pH to less than 2 with HCl, extract it with 100mL of ethyl acetate to obtain ethyl acetate layer and water layer, test the water layer by high performance liquid chromatography, if the extraction is complete, discard the water layer, adjust the pH of the ethyl acetate layer to 8 with an appropriate amount of sodium carbonate, add 100mL of pure water to extract, obtain water layer and ethyl acetate layer, adjust the pH of the water layer to less than 2 with HCl, extract it with 100mL of ethyl acetate to obtain ethyl acetate layer and water layer, discard the water layer, wash the ethyl acetate layer with water 3 times, recover the solvent to obtain a dry sample, weigh it and take an appropriate amount for analysis by high performance liquid chromatography. The process flow is as follows Figure 4-1 shown.

[0116] 2 Investigation of pH adjustment of phosphate buffer

[0117] Weigh 10g of kidney tea extract accurately, dissolve it with 100mL water, adjust pH to less than 2 with HCl, extract with 100mL ethyl acetate to obtain ethyl acetate layer and water layer, test the water layer with HPLC, discard the water layer if the extraction is complete, extract the ethyl acetate layer with 100mL phosphate buffer (PBS) (pH=8) to obtain PBS layer and ethyl acetate layer, adjust pH of PBS layer with HCl to less than 2, extract with 100mL ethyl acetate to obtain ethyl acetate layer and water layer, discard the water layer, wash the ethyl acetate layer with 100mL water 3 times, recover the solvent to obtain a dry sample, weigh and take an appropriate amount for analysis by HPLC. The process flow is as follows: Figure 4-2 shown.

[0118] 3. Phosphate buffer pH investigation

[0119] Accurately weigh 10g of kidney tea extract, dissolve it fully with 100mL of water, and then adjust the pH to <2 with HCl. After adjusting the pH, extract it with 100mL of ethyl acetate to obtain an ethyl acetate layer and an aqueous layer. After testing the aqueous layer by HPLC, if the extraction is complete, discard the aqueous layer. Extract the ethyl acetate layer with 100mL of phosphate buffer (PBS) (pH=6, 7, 8) to obtain a PBS layer and an ethyl acetate layer. Adjust the pH of the PBS layer with HCl to <2, extract it with 100mL of ethyl acetate to obtain an ethyl acetate layer and an aqueous layer, discard the aqueous layer, wash the ethyl acetate layer with 100mL of water 3 times, recover the solvent to obtain a dry sample, weigh it, and take an appropriate amount for analysis by HPLC. The process flow is as follows: Figure 4-3 shown.

[0120] 4. Scale-up experiment on the extraction process of total phenolic acids from spatholobi

[0121] Weigh 300g of kidney tea extract accurately, dissolve it with 3000mL water, adjust pH to <2 with HCl, extract with 3000mL ethyl acetate to obtain ethyl acetate layer and water layer, test the water layer with HPLC, discard the water layer if the extraction is complete, extract the ethyl acetate layer with 3000mL phosphate buffer (PBS) (pH=7) to obtain PBS layer and ethyl acetate layer, adjust pH of PBS layer with HCl to <2, extract with 3000mL ethyl acetate to obtain ethyl acetate layer and water layer, discard the water layer, wash the ethyl acetate layer with 3000mL water 3 times, recover the solvent to obtain a dry sample, weigh and take an appropriate amount for analysis by HPLC. The process flow is as follows: Figure 4-4 shown.

[0122] 5 Results of pH adjustment with sodium carbonate and phosphate buffer

[0123] After comparison, compared with adjusting pH with sodium carbonate, the sample obtained by adjusting pH with phosphate buffer (PBS=8) had a higher content of total phenolic acid and less impurities, so phosphate buffer was selected for pH adjustment for extraction. Figure 4-5 shown.

[0124] 6 Phosphate buffer pH investigation results

[0125] like Figure 4-6 As shown, when phosphate buffer saline (PBS) is used to adjust the pH, the extraction effect is better when the pH of PBS is 8. After the PBS pH adjusted extraction component is dried, it weighs 0.5331 g, and the yield is calculated to be 5.331%.

[0126] 7 Final extraction process determination

[0127] After investigation, the final extraction process of kidney tea was determined as follows Figure 4-7As shown, the final sample was named total phenolic acid of kidney tea (SC-CqB) for subsequent experiments (Note: The process method of the sample used in the experiment of the present invention is Figure 4-7 in the method).

[0128] 8 Extraction process scale-up test

[0129] Weigh 100g of nephrodisiac extract accurately, and conduct three parallel experiments according to the nephrodisiac extraction process determined under "4.4.3". The experimental results are shown in Table 1: The average yield of the nephrodisiac total phenolic acid dry sample finally obtained is 5.884%, and the RSD is 1.71%, which proves that the process has good stability.

[0130] Table-1 Extraction process scale-up test

[0131]

[0132] Example 2 Determination of the content of total phenolic acid extract of kidney tea Preparation of macroporous resin

[0133] 1 Investigation of macroporous resin materials

[0134] Depend on Figure 5-1 It can be seen that the macroporous resins D001, AB-8, ADS-17, HPD-200A, HPD-950, and D301 have poor adsorption properties. Figure 5-2 It can be seen that the macroporous resin ADS-7 is difficult to elute after the components are adsorbed. After comparison, the macroporous resin HPD-500 was finally selected as the filler for macroporous resin elution. The eluents of the remaining macroporous resin columns of different materials were analyzed by high performance liquid chromatography, and HPD-500 was finally selected as the filler for separation and purification of kidney tea extract.

[0135] 2 Investigation of elution volume of macroporous resin column

[0136] like Figure 5-3 As shown, after the Herba Cynanchum sphaerocarpa extract was loaded onto the macroporous resin column, it could be completely eluted by eluting with 60% ethanol for 10 column volumes.

[0137] Example 3 Determination of the content of total phenolic acid extract of kidney tea

[0138] Preparation of test solution

[0139] Accurately weigh 36 mg of total phenolic acid powder of nephroticum ulmoides, place it in a 10 mL volumetric flask, add an appropriate amount of methanol to dissolve it by ultrasonication, and then dilute it to the mark with methanol. After fully mixing, take the supernatant through a 0.22 μm filter membrane and put it in an automatic injection bottle, marked as SC-CQ-BH. Pipette 1 ml of SC-CQ-BH into a 10 mL volumetric flask, dilute it to the mark with methanol, and after fully mixing, take the supernatant through a 0.22 μm filter membrane and put it in an automatic injection bottle.

[0140] Preparation of reference solution

[0141] Weigh an appropriate amount of protocatechuic acid reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 190μg / mL. Weigh an appropriate amount of rosmarinic acid reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 1.2mg / mL. Weigh an appropriate amount of lithospermic acid reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 1.2mg / mL. Weigh an appropriate amount of helisterculins C reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 2.4mg / mL. Weigh an appropriate amount of salvianolic acid B reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 2mg / mL. Weigh an appropriate amount of salvianolic acid A reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 170μg / mL. Weigh an appropriate amount of sodium danshensu reference substance accurately by weight loss method, dissolve it in methanol, and prepare a reference solution of 110μg / mL.

[0142] Accurately pipette 0.2mL protocatechuic acid reference stock solution (190ug / mL, 20220907), 0.21mL rosmarinic acid reference stock solution (1.2mg / mL, 20220907), 0.21mL lithospermic acid reference stock solution (1.2mg / mL, 20220907), 0.25mL helisterculins C reference stock solution (2.4mg / mL, 20220907), 0.087mL salvianolic acid B reference stock solution (2mg / mL, 20220907), 0.5mL Dan A reference stock solution (170ug / mL, 20220907), 0.75mL danshensu sodium reference stock solution (110ug / mL, 20220907) into a 5mL volumetric flask, add methanol in small amounts several times, and dilute to 1mL. The concentrations of each component in the mixed standard solution are shown in the table below.

[0143] Table-2 Concentration of each component in the mixed standard

[0144] Table-1 Concentration of each component of the mixed standard product

[0145]

[0146] HPLC chromatographic conditions

[0147] The chromatographic column was YMC-Pack ODS-A (4.6×150 mm, 5 μm), and acetonitrile (A)-0.3% phosphoric acid aqueous solution (B) was used as the mobile phase for gradient elution (0-10 minutes, 5% A, 10-15 minutes, 5% to 15% A, 15-30 minutes, 15% to 17% A, 30-35 minutes, 17% to 22% A, 35-50 minutes, 22% to 23% A, 50-60 minutes, 23% to 90% A), the flow rate was 1.0 mL / min; the column temperature was 20° C.; the detection wavelength was 190 to 400 nm; and the injection volume was 10 μL.

Claims

1. A Chinese herbal medicine kidney tea extract for treating nephritis, characterized by: Contains danshensu (content>0.07%), protocatechuic acid (content>0.12%), rosmarinic acid (content>9%), lithospermic acid (content>1.2%), helisterculins C (content>4.0%), salvianolic acid B (content>2.0%), and salvianolic acid A (content>2.0%).

2. The Herba Lysimachiae extract according to claim 1, characterized in that: It contains danshensu (content > 0.1%), protocatechuic acid (content > 0.15%), rosmarinic acid (content > 12%), lithospermic acid (content > 1.8%), helisterculins C (content > 6.0%), salvianolic acid B (content > 4.0%), and salvianolic acid A (content > 4.0%); the optimal extract characteristic chemical components are danshensu (content > 0.14%), protocatechuic acid (content > 0.22%), rosmarinic acid (content > 16%), lithospermic acid (content > 2.2%), helisterculins C (content > 8.0%), salvianolic acid B (content > 5.0%), and salvianolic acid A (content > 5.0%).

3. A pharmaceutical composition comprising the extract of any one of claims 1 or 2.

4. The pharmaceutical composition according to claim 3, selected from any edible dosage form.

5. The pharmaceutical composition according to claim 4, wherein the dosage form is selected from tablets, capsules, oral liquids, granules, pills, ointments, pellets, and injections.

6. The Chinese medicinal herb tea extract as claimed in any one of claims 1 to 5 is used as a pharmaceutical composition for the preparation of a medicine for treating acute nephritis, chronic nephritis, renal injury and other nephritis diseases, including renal fibrosis, which can shorten the recovery period and improve symptoms.

7. The method for preparing the nephrolepis chinensis extract according to claim 1, characterized in that: The kidney tea is extracted with water or aqueous ethanol, the extract is concentrated, the solvent is recovered to a small volume, diluted with water, and then the pH value is adjusted to acidity with an organic acid or an inorganic acid, and extracted with an organic solvent to obtain an organic solvent layer and an acid water layer; pure water is added to the organic solvent layer, and the water layer is adjusted to alkaline (pH value is between 7-14) with an appropriate amount of organic base or inorganic base, and then extracted to obtain an alkaline water layer and an organic solvent layer; the alkaline water layer is adjusted to acidity with an organic acid or an inorganic acid, and extracted with an organic solvent to obtain an organic solvent layer and an acid water layer, and after the organic solvent layer is washed with water to neutrality, the organic solvent is recovered and dried to obtain a Chinese medicine kidney tea extract for treating nephritis, wherein: The organic solvent for extraction is selected from: ethyl acetate, n-butanol, isopropanol, chloroform, dichloromethane or a combination of these solvents, preferably ethyl acetate, chloroform, dichloromethane or a combination of these solvents, and most preferably ethyl acetate; The organic acid or inorganic acid is selected from: formic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates, etc., preferably formic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof, and most preferably hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates and a combination thereof; The organic base or inorganic base is selected from: ammonia water, diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, alkaline phosphates and combinations thereof, preferably ammonia water, sodium hydroxide, sodium carbonate, sodium bicarbonate, alkaline phosphates and combinations thereof; and most preferably sodium hydroxide, sodium carbonate, sodium bicarbonate, alkaline phosphates and combinations thereof; The first extraction was adjusted to an acidic pH value of <6, optimized to a pH value of <4, and optimally to a pH value of <2; The second extraction was adjusted to an alkaline pH value > 7.5, optimized to a pH value > 8, and the optimal pH value was pH = 8; The third extraction adjusts the acidic pH value to <6, optimizes it to pH <4, and optimally to pH <2; 7) The extraction solvent of the kidney tea medicinal material is water or hydrous ethanol, optimized to water or hydrous ethanol with an ethanol content of <70%; the optimal solvent is hydrous ethanol with an ethanol content of <70%.

8. The method for preparing the nephrolepis chinensis extract according to claim 1, characterized in that: Kidney tea is extracted with water or aqueous ethanol, the extract is concentrated, the solvent is recovered to a small volume, diluted with water, the pH value is adjusted to acidic with acid, the sample is loaded onto a macroporous adsorption resin column, first eluted with water and low-concentration aqueous ethanol, then eluted with high-concentration aqueous ethanol, the high-concentration aqueous ethanol eluate is recovered, concentrated, and dried to obtain, wherein, Adjust the acidic pH value to <6 before loading, optimize to pH <4, and the best value is pH <2; The acid used for adjusting the acidity before loading is an organic acid or an inorganic base, which can be various water-soluble acids, such as formic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, acid phosphates, etc., preferably formic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof, and most preferably hydrochloric acid, sulfuric acid, phosphoric acid and a combination thereof; The macroporous resin can be various styrene-type macroporous resins, optimized as non-polar, low-polar and medium-polar macroporous resins, and the best is medium-polar macroporous resin (such as HPD-500); 4) The solvent for eluting impurities for the first time is water and low-concentration aqueous ethanol (ethanol concentration <15%), optimized as water and low-concentration aqueous ethanol (ethanol concentration <10%), and the best is water and low-concentration aqueous ethanol (ethanol concentration <5%); 5) The solvent for eluting the effective extract for the second time is high-concentration aqueous ethanol (ethanol concentration>20%), optimized as high-concentration aqueous ethanol (ethanol concentration>30%), and the best is high-concentration aqueous ethanol (ethanol concentration>60%).

9. The preparation method according to claim 7, characterized in that: The following steps are performed: 10 g of kidney tea extract is weighed, fully dissolved with 100 mL of water, and then adjusted to pH < 2 with HCl, extracted with 100 mL of ethyl acetate after the pH is adjusted to obtain an ethyl acetate layer and a water layer, the water layer is tested by high performance liquid chromatography, and if the extraction is complete, the water layer is discarded, the ethyl acetate layer is adjusted to pH = 8 with an appropriate amount of sodium carbonate, and then 100 mL of pure water is added to extract to obtain a water layer and an ethyl acetate layer, the water layer is adjusted to pH < 2 with HCl, and extracted with 100 mL of ethyl acetate to obtain an ethyl acetate layer and a water layer, the water layer is discarded, the ethyl acetate layer is washed with water 3 times, and the solvent is recovered to obtain a dry sample.

10. The method for detecting the Chinese herbal medicine Herba Spatholobi extract according to claim 1, characterized in that: The method steps are as follows: Preparation of test solution: Accurately weigh the extract (or kidney tea medicinal material) sample powder, place it in a stoppered conical flask, accurately add 20 mL of 50% methanol, accurately weigh the mass, ultrasonically treat at room temperature for 0.5 h, take out and cool to room temperature, make up the reduced mass with 50% methanol, shake well and let it stand, take the supernatant and centrifuge, after centrifugation, take 5 mL of the supernatant to a 10 mL volumetric flask, dilute to the scale with water, shake well, take 10 mL and load it onto the pretreated ODS-SPE column, first wash 10 mL with water and discard it, then elute about 5 mL with 100% MeOH to a 5 mL volumetric flask, dilute to the scale with 50% MeOH, shake well, and pass through a 0.22 μm filter membrane to obtain the test solution. Preparation of reference solution: Accurately weigh an appropriate amount of each reference compound and use methanol to prepare mixed standard solutions with concentrations of protocatechuic acid (190ug / mL), rosmarinic acid (1.2mg / mL), lithospermic acid (1.2mg / mL), helisterculins C (2.4mg / mL), salvianolic acid B (2mg / mL), salvianolic acid A (170ug / mL), and sodium danshensu (110ug / mL). Detection: Inject the test solution and the reference solution into a high performance liquid chromatograph to obtain a chromatogram, and calculate the content of each component based on the chromatogram. The HPLC conditions are as follows The chromatographic column is an ODS chromatographic column (4.6×150 mm, 5 μm), and acetonitrile (A)-0.3% phosphoric acid aqueous solution (B) is used as the mobile phase for gradient elution (0-10 minutes, 5% A, 10-15 minutes, 5%-15% A, 15-30 minutes, 15%-17% A, 30-35 minutes, 17%-22% A, 35-50 minutes, 22%-23% A, 50-60 minutes, 23%-90% A), the flow rate is 1.0 mL / min; the column temperature is 20° C.; the detection wavelength is 190-400 nm; and the injection volume is 10 μL.

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