Total allium tuberosum saponins, preparation method and application thereof in preparation of medicine for treating nephrotic syndrome

Abstract

The application provides a preparation method of total saponins of Radix Fritillariae Thunbergii, which comprises the following steps: adding Radix Fritillariae Thunbergii into ethanol to extract, and then concentrating under reduced pressure to obtain a crude extract; dissolving the crude extract in water, and then adsorbing the water on macroporous adsorption resin, and then eluting the macroporous adsorption resin with water and ethanol; collecting the ethanol eluate, and then concentrating under reduced pressure to obtain component 1; dissolving the component 1 in water, and then extracting with an organic solvent; and drying the water phase obtained by extraction under reduced pressure to obtain the total saponins of Radix Fritillariae Thunbergii. The application optimizes the extraction and purification method of the total saponins of Radix Fritillariae Thunbergii to a certain extent, shortens the preparation time of the total saponins, improves the content of the total saponins, and proves through experimental research that the total saponins of Radix Fritillariae Thunbergii can treat the nephrotic syndrome caused by adriamycin (ADR), improve the glomerular filtration rate, reduce the glomerular inflammation, and has a significant protective effect on the kidney.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a total saponin from leek seeds, a preparation and purification method, and its new applications in the preparation of drugs and health products for treating nephrotic syndrome. Background Technology

[0002] Entering the 21st century, due to lifestyle habits and dietary preferences, the acquired incidence of kidney diseases has been increasing year by year, especially nephrotic syndrome, which seriously endangers human health and quality of life. Nephrotic syndrome is characterized by severe proteinuria, hyperlipidemia, hypoproteinemia, and external skin edema, the so-called "three highs and one low," along with other metabolic disorders, forming a group of clinical symptoms. Nephrotic syndrome is usually caused by glomerular damage. When blood passes through the kidneys, the glomeruli filter the blood, separating what the body needs from what it doesn't. Healthy glomeruli maintain the body's required protein levels; when the glomeruli are damaged, excessive blood protein is allowed to leak into the urine, leading to nephrotic syndrome. Massive proteinuria is the most prominent clinical manifestation and the most fundamental pathophysiological mechanism of nephrotic syndrome. Nephrotic syndrome is clinically common and difficult to treat. Patients may experience platelet abnormalities, hemoconcentration, and significant loss of anticoagulants, which can lead to complications such as thrombosis, seriously threatening human health. Currently, clinical treatment for this disease mainly involves glucocorticoids and diuretics. However, the therapeutic effects and side effects of these drugs vary widely among different patient groups. Therefore, exploring new, potentially effective drugs for the treatment of nephrotic syndrome is of great significance. Currently, there are no literature reports on the use of leek seeds in the treatment of nephrotic syndrome.

[0003] Leek seeds (Semen Allii Tuberose) are the dried, mature seeds of the perennial plant *Alliium tuberosum* Rottl. ex Spreng., belonging to the genus *Alliium* of the Liliaceae family. The medicinal history of leek seeds can be traced back to the Spring and Autumn and Warring States periods. Records of leek seeds first appeared in the *Mingyi Bielu* (Records of Famous Physicians), where they were listed as a medium-grade herb, possessing the functions of warming the kidneys and strengthening yang, and consolidating essence. They are commonly used for symptoms of exhaustion such as seminal emission, frequent urination, enuresis, and other signs of weakness. Studies have shown that the main chemical components of leek seeds include steroidal saponins, alkaloids, sulfides, and flavonoids. Modern pharmacological research has demonstrated that leek seeds have aphrodisiac, immune-enhancing, antioxidant, anti-aging, and antibacterial effects. Due to the widespread cultivation of leeks throughout China, leek seeds, as a byproduct of leek production, are in abundant supply, keeping their price stable and reasonable. This is of great significance for developing an inexpensive and effective drug or health product for the treatment of nephrotic syndrome. Based on this, this application was developed. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the prior art and provide a total saponin from leek seeds, which can be used to treat nephrotic syndrome with significant efficacy.

[0005] To optimize the extraction and purification method of total saponins from leek seeds and increase their content, this invention provides an extraction and purification process for total saponins from leek seeds optimized using single-factor experiments and response surface methodology.

[0006] This invention also provides a new application of the above-mentioned total saponins from leek seeds in the preparation of drugs and health products for treating nephrotic syndrome.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing total saponins from leek seeds involves extracting leek seeds with ethanol, concentrating the extract under reduced pressure to obtain a crude extract, dissolving the crude extract in water, adsorbing it onto a macroporous adsorption resin, eluting it with water and ethanol, collecting the ethanol eluent, and concentrating it under reduced pressure to obtain component 1, dissolving component 1 in water, extracting it with an organic solvent, and concentrating and drying the aqueous phase obtained from the extraction under reduced pressure to obtain total saponins from leek seeds.

[0009] Specifically, leek seeds can be extracted by adding ethanol with a volume concentration of 5-90%, 1-5 times, with an extraction time of 60-120 seconds. Furthermore, the preferred ethanol volume concentration is 10-40%.

[0010] Specifically, after dissolving the crude extract in water, it is adsorbed onto HPD100 macroporous adsorption resin for 8-15 hours.

[0011] Specifically, 60-80% ethanol can be used for elution. More preferably, 2-4 column volumes of 65-75% ethanol are used for elution at a rate of 0.4-1 BV / h.

[0012] Specifically, the organic solvent used in the extraction can be ethyl acetate, etc.

[0013] This invention provides total saponins from leek seeds prepared using the above-described preparation method.

[0014] This invention also provides the application of the above-mentioned total saponins from leek seeds in the preparation of drugs or health products for treating nephrotic syndrome, especially in the preparation of drugs or health products for treating nephrotic syndrome caused by doxorubicin.

[0015] Furthermore, the present invention also provides the application of the above-mentioned total saponins from leek seeds in the preparation of drugs or health products that protect against oxidative damage to kidney tissue.

[0016] In this invention, a material-to-liquid ratio of 1g:10mL was used as a fixed factor for flash extraction. Single-factor experiments were conducted to determine the effects of the required ethanol concentration, extraction time, and number of extractions on the quality of the total saponins extracted from leek seeds. The steps were as follows: In the single-factor experiments, 0%-95% ethanol was added to the pulverized leek seeds at a material-to-liquid ratio of 1:10 for flash extraction, with 1-5 extractions and an extraction time of 60-120s.

[0017] The preferred single-factor experimental optimization conditions for flash extraction of total saponins from leek seeds are: 20% ethanol, 3 extractions, and 105s extraction time.

[0018] In the response surface methodology (RSM) study of total saponins from leek seeds, a three-factor, three-level RSM design was used, with the optimal extraction conditions optimized by a single factor as the center value and the total saponin content of leek seeds as the response value. The quadratic multiple regression equation model established in the RSM is as follows:

[0019] Y=7.79-0.3869A-0.2020B-0.4004C-0.0373AB-0.3449AC+0.2258BC-1.49A 2 -1.27B 2 -1.19C 2 ,

[0020] In the formula, Y is the saponin mass (mg), A is the ethanol concentration, B is the number of extractions, and C is the flash extraction time.

[0021] The extract obtained from the optimal response surface methodology was eluted with 2-3 BV column volumes of 70% ethanol at a rate of 0.5 BV / h using a macroporous resin (preferably HPD100 with a loading concentration of 12 mg / mL). The fractions were combined, concentrated under reduced pressure to recover the solvent, dried, reconstituted with water, and then extracted with ethyl acetate at a ratio of 1:1 (volume:volume). The resulting aqueous phase was concentrated and dried again to obtain the final total saponins from leek seeds.

[0022] The optimal extraction process response surface methodology was: ethanol concentration of 19.052%, 2.97 extractions, and extraction time of 104.569 s. For ease of practical operation, the optimal conditions were adjusted to: ethanol concentration of 20%, 3 extractions, and extraction time of 105 s, resulting in a total saponin content of 1.95% in the crude extract. The crude extract was purified using HPD100 macroporous resin, eluted with 70% ethanol, and extracted with ethyl acetate to obtain the final total saponins from leek seeds, with a total saponin content ranging from 54% to 58%.

[0023] This application discloses an optimized extraction and purification process for total saponins from leek seeds. The extraction method was optimized using single-factor and response surface methodology, followed by purification. The pharmacological activity of the final product in treating nephrotic syndrome was then studied. The results showed that total saponins from leek seeds could alleviate proteinuria in a mouse model of nephrotic syndrome, increase serum albumin levels, and reduce serum low-density lipoprotein (LDL-C), serum creatinine (CR), total cholesterol (T-CHO), triglycerides (TG), blood urea nitrogen (BUN) levels, as well as renal podocin protein expression, thus improving renal tissue pathological changes. Both medium and high doses of total saponins from leek seeds significantly reduced MDA content in the kidneys of mice after doxorubicin administration and significantly increased T-SOD enzyme activity.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1) This invention provides total saponins from leek seeds prepared by the above method, which can greatly shorten the extraction time of total saponins from leek seeds. It is an economical and efficient method for preparing total saponins from leek seeds, wherein the content of total steroidal saponin compounds is >54%.

[0026] 2) The present invention also provides the application of the above-mentioned total saponins of leek seeds in the treatment of nephrotic syndrome, especially in the treatment of nephrotic syndrome caused by doxorubicin.

[0027] 3) Experiments conducted according to this invention have shown that total saponins from leek seeds have a significant therapeutic effect on doxorubicin-induced nephropathy in mice, and its protective mechanism may be related to enhancing the antioxidant function of mouse kidneys. Therefore, this invention also provides the application of the above-mentioned total saponins from leek seeds in the preparation of drugs or health products for treating nephrotic syndrome.

[0028] 4) This invention extracts total steroidal saponins from leek seeds and uses them to induce a mouse model of nephrotic syndrome via tail vein injection with doxorubicin. Experimental studies have shown that total saponins from leek seeds can alleviate doxorubicin-induced kidney damage in mice and have a certain protective effect, manifested in reducing glomerular sclerosis, alleviating mesangial cell proliferation and diffuse mesangial matrix hyperplasia, and reducing the degree of fibrosis in peripheral glomerular vessels. It also reduces urinary protein and serum levels of BUN, CR, T-CHO, LDL-C, and TG, and decreases MDA content in mouse kidneys; it increases SOD, podocin, and serum albumin levels in the kidneys. This indicates that the total saponins from leek seeds of this invention can alleviate kidney damage in mice with nephrotic syndrome, enhance renal function, and improve antioxidant capacity. Furthermore, the preparation method of the total saponins from leek seeds in this invention is simple, the extract has high purity, and leek seeds are widely available and easily accessible, thus expanding the application of total saponins from leek seeds as a drug or health product for treating nephrotic syndrome. Attached Figure Description

[0029] Figure 1 The standard curve for protodiosgenin;

[0030] Figure 2 The effects of ethanol concentration (left), number of extractions (middle), and extraction time (right) on the quality of total saponins from leek seeds;

[0031] Figure 3 The following are response surface plots for the extraction process of total saponins from leek seeds: 3D response surface plot of ethanol concentration (top left), ethanol concentration response surface contour plot (bottom left); 3D response surface plot of extraction times (top center), extraction times response surface contour plot (bottom center); 3D response surface plot of extraction time (top right), extraction time response surface contour plot (bottom right);

[0032] Figure 4 Liquid chromatography-mass spectra for the identification of total saponins in leek seeds: positive ion spectrum (top) and negative ion spectrum (bottom);

[0033] Figure 5 The effect of total saponins from leek seeds on urinary protein content was as follows: compared with the control group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0034] Figure 6 The effects of total saponins from leek seeds on serum albumin (left), low-density lipoprotein (middle), and serum creatinine (right) levels were compared with the control group; ###P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0035] Figure 7 The effects of total saponins from leek seeds on total cholesterol (left), triglyceride (middle), and blood urea nitrogen (right) levels were compared with the control group; ###P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0036] Figure 8 PAS-stained and HE-stained pathological sections of the kidney;

[0037] Figure 9 To show the effect of total saponins from leek seeds on the expression of Podocin protein in the kidney tissue of mice with nephrotic syndrome, the image shows the Podocin protein band (left) and the quantitative analysis of Podocin protein (right). Compared with the control group, ###P<0.001; compared with the model group, *P<0.05, ***P<0.001.

[0038] Figure 10The effects of total saponins from leek seeds on MDA levels (left) and T-SOD enzyme activity (right) in the kidney tissue of mice with nephrotic syndrome were compared with those of the normal group. The results showed that, compared with the normal group, ##P<0.01 and ###P<0.001, and compared with the model group, *P<0.05, **P<0.01, and ***P<0.001. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0040] Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products that can be purchased directly or can be prepared using conventional techniques in the art.

[0041] Room temperature refers to 25±5℃.

[0042] Example 1

[0043] An optimized extraction process for total saponins from leek seeds

[0044] 1. Construction of the standard curve of protodiosgenin

[0045] The total saponins in leek seeds contain protodioscin, and the content is not low (see Table 5 and...). Figure 4 Since its structure is similar to other saponins, protodiosgenin was used as a reference standard. Protodiosgenin solutions of 0.05 mg / mL, 0.20 mg / mL, 0.35 mg / mL, 0.50 mg / mL, and 0.65 mg / mL were accurately pipetted to prepare 1 mL each, placed in clean 25 mL round-bottom flasks. The solutions were then evaporated to dryness under vacuum at 70 °C. Immediately, 0.8 mL of perchloric acid and 0.2 mL of 5% vanillin-glacial acetic acid (0.5 g vanillin diluted to 10 mL with glacial acetic acid in a 10 mL volumetric flask) were accurately added using a pipette. A blank sample was prepared simultaneously. All flasks were placed in a 60 °C water bath for 15 min for color development, and then rapidly cooled with running water for 5 min. 5 mL of glacial acetic acid was added, and the solution was shaken well. The absorbance at 454 nm was immediately measured and the data recorded. A standard curve was plotted with absorbance as the ordinate and concentration as the abscissa. Figure 1 As shown: y = 1.609x - 0.0436, R 2 =0.9995.

[0046] 2. Determination of total saponins in leek seeds

[0047] The crude extract obtained under the optimal single-factor extraction conditions was prepared into a 50 mL extract solution with a concentration of 20 mg / mL. 1 mL of this solution was subjected to a colorimetric reaction using the method described in Example 1, and the absorbance of the crude leek seed saponin extract solution was determined colorimetrically. (Triple replicates) The total saponin concentration in the corresponding crude extract was calculated using a standard curve. Substituting these values ​​into formula ① yielded the saponin mass, and formula ② yielded the saponin content. The formulas are as follows:

[0048] M = CVn ①

[0049]

[0050] In the formula: M represents the mass of saponin (mg / g); Y represents the content of saponin (%); C represents the concentration of total saponins in the extract (mg / mL); V represents the volume of the test solution (mL); n represents the dilution factor from the test solution to the test solution; and m represents the mass of the crude extract powder weighed (mg).

[0051] 3. Single-factor experimental methods and results

[0052] Single-factor experiments were conducted to select ethanol concentration, extraction time, and extraction times to obtain the relationship between each factor and the quality of total saponins in leek seeds.

[0053] (1) Effect of ethanol concentration in extract on saponin quality

[0054] With a fixed material-to-liquid ratio of 1g:10mL, extraction was performed three times for 90s. The total saponin mass was determined at ethanol concentrations of 0%, 20%, 35%, 50%, 65%, 80%, and 95% (v / v). The results are shown below. Figure 2 .from Figure 2 It can be seen that the saponin content increases continuously during water extraction and 20% ethanol extraction, and then gradually decreases. Therefore, it is believed that the extraction is optimal when the volume fraction of ethanol is 20%.

[0055] (2) Effect of extraction time on saponin quality

[0056] With a fixed ethanol concentration of 20% and a solid-liquid ratio of 1g:10mL, extraction was performed three times. The total saponin content in the solution was measured at extraction times of 60s, 75s, 90s, 105s, and 120s. The results are shown in the figure. Figure 2 .from Figure 2 It can be seen that the quality of saponins increases continuously with the extension of extraction time. The highest quality of saponins is obtained when the extraction time is 105s, and it gradually decreases from 105s to 120s. Therefore, 105s is taken as the optimal extraction time.

[0057] (3) Effect of extraction times on saponin quality

[0058] With a fixed ethanol concentration of 20%, a solid-liquid ratio of 1g:10mL, and an extraction time of 105s, the total saponin content in the solution was determined at extractions 1, 2, 3, 4, and 5. The results are shown below. Figure 2 . Figure 2 The results showed that the extraction effect was best when the number of extractions was 3.

[0059] 4. Optimize extraction conditions using response surface methodology

[0060] (1) Response surface methodology

[0061] Based on the results of the single-factor experiments, a response surface methodology optimization experiment was further designed. The factor level with the best total saponin extraction effect was selected, with ethanol concentration, extraction times, and extraction time as independent variables, and the extracted saponin mass as the response value. A 3-factor, 3-level Box-Behnken Design experiment was adopted, with each experiment repeated in triplicate. Specific implementation methods are shown in Tables 1 and 2.

[0062] Table 1. Factors and levels in a single-factor experiment.

[0063]

[0064] Table 2 Box-Behnken experimental protocol and total saponin content of leek seeds

[0065]

[0066]

[0067] (2) Regression model establishment and analysis of variance

[0068] The results of the Box-Behnken response surface design are shown in the table above. Design-Expert 13.0 software was used to perform quadratic multiple regression fitting on each factor, resulting in a binomial multiple regression model equation for saponin quality (Y):

[0069] Y=7.79-0.3869A-0.2020B-0.4004C-0.0373AB-0.3449AC+0.2258BC-1.49A 2 -1.27B 2 -1.19C 2 ,

[0070] R 2 =0.9985, R 2 adj=0.9967, meaning this equation can explain 99.67% of the experimental data variation. The regression model P < 0.05, indicating a significant difference. The difference in the level of the lack-of-fit term was not significant (P > 0.05), indicating that the model has a good fit. Factors A, B, C, BC, and AC have significant effects and can be analyzed using a mathematical model. In the formula: Y is the saponin mass, A is the ethanol concentration, B is the number of extractions, and C is the extraction time.

[0071] 3D response surface methodology for the extraction process of total saponins from leek seeds, as shown in... Figure 3 As shown. The response surface plots drawn based on the quadratic regression equation are all convex surfaces opening downwards, indicating the existence of a maximum response value within the scope of the study. The shape of the contour lines reflects the strength of the interaction between influencing factors; the closer the contour lines are to circles, the weaker the interaction between the two factors; when the contour lines are elliptical, the interaction between the two factors is strong. From... Figure 3 It can be seen that ethanol concentration, extraction times, and extraction time all have a certain impact on the extraction of total saponins from leek seeds. Ethanol concentration and extraction time, extraction time and extraction times reflect a certain degree of interaction, while the interaction between ethanol concentration and extraction times is relatively weak.

[0072] Table 3. Analysis of Variance for Regression Models

[0073]

[0074]

[0075] Note: P>0.05 indicates no significant difference; P<0.05 indicates a significant difference; P<0.01 indicates an extremely significant difference.

[0076] (3) Verification test

[0077] Based on the optimal process conditions and specific experimental steps predicted by response surface methodology, the optimized process parameters were adjusted to: ethanol concentration 20%, extraction times 3 times, and extraction time 105 s. Three parallel experiments were conducted under these optimal conditions, with 10 g of leek seeds weighed for each group for verification. The results are shown in Table 4 below. The results of the three parallel experiments are close to the model predictions, indicating that the model is suitable for the extraction of total saponins from leek seeds.

[0078] Table 4. Verification Test Results

[0079]

[0080] Example 2

[0081] A process for extracting and purifying total saponins from leek seeds, the specific steps of which are as follows:

[0082] 1 kg of leek seeds were extracted under the following conditions: 70% ethanol was added at a material-to-liquid ratio of 1 g:10 mL, and flash extraction was performed at room temperature for 3 times, each time for 105 s. After the flash extraction, the three extracts were combined and concentrated under reduced pressure to obtain 121.9 g of crude extract. The crude extract was dissolved in water to prepare a 12 mg / mL aqueous solution, which was then adsorbed onto HPD100 macroporous adsorption resin overnight (12 h). The solution was then washed with water until the eluent was colorless (discarded). Elution was then performed with 2.5 BV column volumes of 70% ethanol at a rate of 1 BV / h. The collected eluents were combined, concentrated under reduced pressure to recover the solvent, and dried to obtain 12.3 g of fraction 1. Fraction 1 was dissolved in water and extracted with ethyl acetate at a 1:1 (v / v) ratio. The aqueous phase obtained from the extraction was concentrated under reduced pressure and dried to obtain 5.5 g of total leek seed saponins. The total leek seed saponin content was determined to be 54.42% by spectrophotometry.

[0083] The total ion chromatogram of total saponins from leek seeds is shown below. Figure 4 Positive ion chromatograms detected 22 saponin components, while negative ion chromatograms detected 10 saponin components. Specific compound names, molecular formulas, retention times, and mass spectrometry information are shown in Tables 5 and 6. A total of 32 saponin components were identified.

[0084] Table 5. Identification of Total Saponins from Leek Seeds by Positive Ions

[0085]

[0086]

[0087] Table 6. Identification of Total Saponins and Negative Ions in Leek Seeds

[0088]

[0089]

[0090] Example 3

[0091] A process for extracting and purifying total saponins from leek seeds, the specific steps of which are as follows:

[0092] Take 10 g of semen allii tuberosi, and the extraction conditions are as follows: Add 70% ethanol at a solid-liquid ratio of 1 g:10 mL and perform flash extraction at room temperature for 3 times, 105 s each time. After the flash extraction, combine the three extracts and concentrate under reduced pressure to obtain 1.08 g of crude extract. Dissolve the crude extract in water to prepare an aqueous solution with a concentration of 12 mg / mL, adsorb it on HPD100 macroporous adsorption resin overnight (12 h), and then wash it with water until the effluent is colorless (discarded); then elute it with 3 column volumes (BV) of 70% ethanol at a speed of 0.5 BV / h, combine the collected eluates, recover the solvent by concentration under reduced pressure, and dry to obtain 0.17 g of Component 1. Dissolve Component 1 in water and extract it with ethyl acetate at a volume ratio of 1:1. The aqueous phase obtained by extraction is concentrated under reduced pressure and dried to obtain 76.8 mg of total saponins from semen allii tuberosi. The content of total saponins from semen allii tuberosi is measured to be 57.43% by spectrophotometry.

[0093] Effect Experiment 1: Therapeutic effect of total saponins from semen allii tuberosi on adriamycin-induced nephrotic syndrome in mice.

[0094] Experimental raw materials: The total saponins from semen allii tuberosi prepared in Example 2 are used as experimental raw materials.

[0095] Experimental animals: Balb / c mice, weighing 18 - 22 g, SPF grade, provided by Henan Experimental Animal Center, production batch number: SCXK(Yu)2020 - 0005.

[0096] Experimental reagents: See Table 7 below.

[0097] Experimental instruments: See Table 8 below.

[0098] Table 7. Experimental reagents

[0099]

[0100]

[0101] Table 8. Experimental instruments

[0102]

[0103]

[0104] Experimental method:

[0105] Male Balb / c mice were acclimatized for one week, weighed, photographed, and their coat color observed. Then, the mice were placed in metabolic cages to collect 24-hour urine (fasting but with free access to water), and the volume was accurately recorded. The urine was centrifuged at 3500 rpm for 15 minutes at 4°C, and the supernatant was collected. The 24-hour urinary protein content was detected using a urine protein detection kit to confirm that all mice were normal. All mice were divided into a control group (n=15) and a model group (n=75). Except for the control group, all mice in the model group underwent a single tail vein injection of doxorubicin 10 mg / kg to establish an doxorubicin nephropathy model. The control group received a single tail vein injection of the same volume of physiological saline as a control. Seven days after doxorubicin injection, the mice in both the control and model groups were weighed, photographed, and their coat color observed. Then, 24-hour urine was collected from both groups in metabolic cages (fasting but with free access to water), and the volume was accurately recorded. The urine was centrifuged at 3500 rpm for 15 minutes at 4°C, and the supernatant was collected. The 24-hour urinary protein content was detected using a kit. Mice with a urinary protein content ≥50mg / kg in the model group were included in subsequent experiments.

[0106] Mice in the control group and those successfully modeled were divided into four groups: control group (n=15), model group (n=15), positive control group (n=15), low-dose group (n=15), medium-dose group (n=15), and high-dose group (n=15). After grouping, each group was administered oral gavage daily. The control group and model group received 10g / 0.1mL of physiological saline via gavage daily, while the positive control group received 10mg / kg prednisolone acetate in physiological saline solution via gavage. The low, medium, and high-dose groups received 20mg / kg, 40mg / kg, and 80mg / kg total saline solutions of leek seed saline solution, respectively. Urine analysis: Urine protein in each group was measured using the Elabscience urine protein colorimetric assay kit on days 7, 14, 21, 28, 35, and 42 of the experiment, strictly following the kit instructions. Blood tests: Serum samples were analyzed for albumin, low-density lipoprotein, blood urea nitrogen, serum creatinine, total cholesterol, and triglycerides. All tests were performed using Nanjing Jiancheng Biochemical Detection Kits, and the procedures were strictly followed according to the kit instructions. Statistical analysis was performed using Graphpad Prism 6 software. Results are presented as bar charts. A p-value < 0.05 was considered statistically significant. See attached table. Figure 4 , 5 And 6.

[0107] After blood collection, mice were euthanized by cervical dislocation. Both kidneys and livers were dissected and rinsed with physiological saline. The right kidney tissue was then preserved in 4% paraformaldehyde solution for 20-24 hours. Under aseptic conditions, the tissue was trimmed to a thickness of approximately 5 mm and placed sequentially in a dehydration chamber for dehydration, embedding, sectioning, baking, PAS staining, and HE staining. Microscopic examination and image acquisition and analysis were then performed. Results are shown below. Figure 7 .

[0108] Kidneys from mice in the blank group, model group, medium-dose group, and high-dose group were weighed and added to 1 mL of pre-prepared high-efficiency RIPA lysis buffer (containing 1% PMSF) and 0.01 mL of protein phosphatase inhibitor. The mixture was then homogenized at low temperature. The homogenate was placed on ice and allowed to stand for 30 min. It was then centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was collected. Protein quantification was performed using a BCA protein assay kit (specific procedures followed the instructions). An appropriate amount of protein loading buffer (the buffer provided with the BCA protein assay kit) was added to the supernatant and mixed well. The mixture was boiled for denaturation for 10 min, cooled in an ice bath, and aliquoted. A suitable amount of protein sample was then taken for SDS-PAGE gel electrophoresis. The stacking gel was kept at 80V for approximately 30-40 minutes; the separating gel was kept at 120V until the pre-stained protein marker (bromophenol blue) reached the bottom, at which point electrophoresis was stopped. The gel was then transferred to a PVDF membrane, blocked with 5% skim milk powder blocking buffer, and placed on a shaker at 80 rpm / min at room temperature for 2 h. Podocin and GAPDH (1:1000, v / v) primary antibody were incubated overnight at 4°C for approximately 13-15 hours. Goat anti-rabbit IgG (H+L)HRP (1:30000) was incubated at room temperature for 2 hours. 100 μL of ECL ultrasensitive luminescent solution was added, and the mixture was developed using a Western blot imaging system. ImageJ was used for grayscale analysis of the protein bars. Results are shown below. Figure 8 .

[0109] The experimental procedures and conditions not mentioned or recorded in detail in the above experiments can be carried out using conventional techniques in this field. Since they are not the innovation of this application, they will not be described in detail here.

[0110] Experimental results:

[0111] Figure 5 , 6 Figures 7 and 8 show the changes in biochemical indicators of total saponins from leek seeds in mice with doxorubicin-induced nephrotic syndrome. Figure 5 This refers to the amount of protein in urine. Figure 6 These include serum albumin levels, low-density lipoprotein levels, and serum creatinine levels. Figure 7 These are the total cholesterol content, triglyceride content, and blood urea nitrogen content. (From...) Figure 5 , 6As shown in Figures 7 and 8, total saponins from leek seeds can significantly alleviate proteinuria in mice, increase serum albumin levels, and reduce serum low-density lipoprotein (LDL-C), serum creatinine (CR), total cholesterol (T-CHO), triglycerides (TG), and blood urea nitrogen (BUN). These results indicate that both the medium and high dose groups of total saponins from leek seeds in this invention have a significant therapeutic effect on doxorubicin-induced nephrotic syndrome in mice, with the medium dose group showing the best therapeutic effect.

[0112] Figure 8 This invention relates to the effect of total saponins from leek seeds on the pathological changes of renal tissue in mice with doxorubicin-induced nephrotic syndrome (PRS staining and HE staining of kidney pathological sections). Figure 7 It can be seen that: in the blank group, the glomeruli were morphologically intact, without mesangial proliferation or glomerular sclerosis, and without pathological features such as glomerular adhesions or inflammatory cell infiltration. The cytoplasm was uniform, the glomerular capillary lumen was open, and the overall cell morphology was normal. In contrast, the model group showed obvious glomerular sclerosis, widening of the mesangial area, and extracellular matrix deposition in the mesangial area, with localized glomerular adhesions and inflammatory cell infiltration. In the medium and high dose groups of the total saponins from leek seeds of this invention, the glomeruli were generally morphologically normal, without pathological phenomena such as glomerular sclerosis, glomerular adhesions, mesangial cell proliferation, diffuse mesangial matrix proliferation, etc. The glomerular capillary lumen was clearly visible, with little or no inflammatory cell infiltration, and the cytoplasm was uniform.

[0113] Figure 9 This study describes the effect of total saponins from leek seeds on the expression of podocin protein in the kidney tissue of mice with nephrotic syndrome. Podocin is a transmembrane protein of glomerular podocytes. Alterations in podocin affect changes in the foot processes of podocytes and the integrity of the slit septum, thus impacting glomerular filtration function. Nephrotic damage is closely related to this podocyte podocin protein. Figure 8 It can be seen that the model group had significantly lower podocin levels compared to the blank group. The medium and high doses of the total saponins from leek seeds in this invention can significantly increase the podocin levels in the kidneys, indicating that the treatment with total saponins from leek seeds has a certain protective effect on glomerular podocytes and can alleviate kidney podocyte damage caused by ADR.

[0114] Experiment 2: Antioxidant Test of Total Saponins from Leek Seeds

[0115] Experimental materials: Same as in Experiment 1.

[0116] Laboratory animals: Same as Experiment 1.

[0117] Experimental reagents: see Table 9 below.

[0118] Experimental apparatus: see Table 10 below.

[0119] Table 9 Experimental Reagents

[0120]

[0121] Table 10 Experimental Instruments

[0122]

[0123] Experimental methods: The animal experiments were the same as in Experiment 1. The reagent kit procedures were performed according to the kit instructions. Kidney tissue was homogenized with physiological saline, then centrifuged at 4℃, 3500 rpm for 10 min. The supernatant was used for the experiments. The MDA content and T-SOD enzyme activity of mouse kidney tissue were measured.

[0124] Figure 10 The effects of total saponins from leek seeds on MDA and T-SOD enzymes in the kidney tissue of mice with nephrotic syndrome were presented. Figure 9 The experimental results showed that, compared with the model group, the low, medium, and high dose groups of the total saponins from leek seeds of this invention significantly reduced the MDA content in the kidneys of mice after doxorubicin administration, and the medium and high dose groups significantly increased the T-SOD enzyme activity in the kidneys. The results indicate that the total saponins from leek seeds of this invention have a protective effect against oxidative damage to mouse kidney tissue, enhancing the antioxidant function of the mouse kidneys and reducing free radical damage to the kidneys after administration.

Claims

1. The application of total saponins from leek seeds in the preparation of a drug for treating doxorubicin-induced nephrotic syndrome, characterized in that, The total saponins from leek seeds were prepared by the following steps: Leek seeds were extracted with ethanol, and the crude extract was concentrated under reduced pressure after extraction. The crude extract was dissolved in water and then adsorbed onto a macroporous adsorption resin. It was then eluted with water and ethanol, and the ethanol eluent was collected and concentrated under reduced pressure to obtain component 1. Component 1 was dissolved in water and extracted with an organic solvent. The aqueous phase obtained from the extraction was concentrated under reduced pressure and dried to obtain the final product. Eluent with a volume concentration of 60-80% was used for elution; The organic solvent used in the extraction was ethyl acetate.

2. The application as described in claim 1, characterized in that, Add leek seeds to 5-90% ethanol for flash extraction, extract 1-5 times, for 60-120 seconds each time.

3. The application as described in claim 1, characterized in that, After dissolving the crude extract in water, it was adsorbed onto HPD100 macroporous adsorption resin for 8-15 hours.

4. The application as described in claim 1, characterized in that, Elution was performed using 2-4 column volumes of 65-75% ethanol at a rate of 0.4-1 BV / h.

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