Total alkaloids from the root of Torchwort and extraction method and application thereof

Abstract

The invention discloses the use of total alkaloids of the root of torch flower in the preparation of medicines for treating rheumatoid arthritis and membranous nephropathy. The method for extracting total alkaloids of the root of torch flower comprises the following steps: S1, crushing the root of torch flower medicinal material, adding water to perform decoction extraction, and concentrating the filtrate into torch flower root clear paste; S2, adding ethanol to the torch flower root clear paste, stirring, standing, filtering and concentrating; S3, extracting with chloroform and n-butanol, and performing decompression recovery treatment on the extract to obtain extracts of chloroform part and n-butanol part; S4, treating the chloroform part extract with a certain concentration of hydrochloric acid to submit the pH value, extracting with an equal volume of ethyl acetate, removing impurities, decompressing, concentrating and drying to obtain a crude product; S5, treating the crude product with neutral alumina wet column loading and wet loading technology to obtain an eluent; S6, decompressing, concentrating and vacuum drying the eluent to obtain total alkaloids of the root of torch flower. The total alkaloid active ingredients of the root of Torchwort provided by the invention can produce significant improvement effects on rheumatoid arthritis and membranous nephropathy.

Description

Technical Field

[0001] The invention relates to the field of natural extracts from the roots of Torchwood, and in particular to a method for extracting total alkaloid active ingredients from the roots of Torchwood and application of the total alkaloids from the roots of Torchwood in preparing medicines for treating rheumatoid arthritis and membranous nephropathy. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic disease with unknown etiology, characterized by inflammatory synovitis. It has a high morbidity, disability rate and difficulty in treatment. It is listed as one of the 10 major diseases that endanger people's health in my country. It is characterized by polyarticular, symmetrical and invasive joint inflammation of the small joints of the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor, which can lead to joint deformity and loss of function. Studies have shown that in rheumatoid arthritis, tumor necrosis factor (TNF), interleukin-6 (IL-6), interleukin-1β (IL-1β), etc. are important cytokines that mediate inflammatory responses. The autoantigen-antibody complex produced by the autoimmune response may play an important stimulating factor in synovitis.

[0003] Membranous nephropathy (MN) is a disease in which immune complexes (formed by the combination of antibodies and antigens) are deposited in the basement membrane of glomerular capillaries, leading to basement membrane lesions. MPC-5 cells are the target cells of MN. The complement membrane attack complex C5b-9 formed in MN will damage its target cells, thus manifesting as nephrotic syndrome. Its incidence rate is 8-10 cases per 1 million people, with no significant racial and gender differences.

[0004] The root of the torch flower is the peeled dried root core of Tripterygium hypoglaucum (Levl.) Hutch, a plant of the Celastraceae family. It can be harvested throughout the year, with impurities removed, the root bark removed, crushed, and dried. It tastes bitter and warm; it is toxic. It enters the spleen and kidney meridians. It has the effects of dispelling wind and dampness, relaxing muscles and tendons, clearing away heat and detoxifying, and is mainly used for rheumatic arthritis (rheumatoid arthritis, rheumatoid arthritis), chronic nephritis, lupus erythematosus, traumatic injuries, skin itching, etc. The root of the torch flower contains diterpenes, triterpenes, etc., such as triptolide, triptolide, etc., but these ingredients are active and relatively toxic. In addition, total alkaloids are also one of the active ingredients in the root of the torch flower, but their specific pharmacological activities have not been reported so far. In previous studies, we found that the activity of total alkaloids from the root of torch flower is different from the pharmacological activity of triptolide and celastrol. Triptolide and celastrol have obvious hepatotoxicity, while the toxicity of total alkaloids from the root of torch flower is much lower than both, but its pharmacological activity is not worse than either of them.

[0005] At present, the country encourages the research and development and innovation of new Chinese medicines. In the past five years, five Class 1.2 new drugs (extract parts) have been approved for production, and many companies are conducting the research and development and application of Class 1.2 new drugs. Studies have found that the total alkaloids of the root of torch flower have good pharmacological activity for rheumatoid arthritis and membranous nephropathy. Therefore, the development of new drugs based on extracts related to the root of torch flower has great research significance.

[0006] Patent application number 202310294769.8 discloses a method for extracting total flavonoids from the root of Torch Flower and its application in the preparation of membranous nephropathy drugs. In this method, extracts of chloroform and n-butanol parts are obtained, the n-butanol extract is added to an ethanol solution, and eluted with ethanol to extract the total flavonoids from the root of Torch Flower.

[0007] Currently, no effective solution has been proposed for the problems in the related technologies. Summary of the invention

[0008] In the previous study, we found that the total alkaloids are mainly in the chloroform part, and there are many other fat-soluble impurities in the chloroform part. The existing total alkaloid extraction method results in more total alkaloid impurities, which are not easy to separate and purify. In view of the problems in the related art, the present invention proposes a method for extracting the total alkaloid active ingredients in the root of Torch Flower and its application in the preparation of drugs for treating rheumatoid arthritis and membranous nephropathy, so as to overcome the above technical problems existing in the existing related technology.

[0009] The present invention uses acid water to convert alkaloids into salt solution, then adjusts the pH value to alkaline, releases the alkaloids, and then extracts with an organic solvent, thereby increasing the yield of total alkaloids; the extraction rate can reach more than 90%. Then, an alumina column is used for separation and purification to remove other impurities, thereby obtaining purer total alkaloids.

[0010] To this end, the specific technical solution adopted by the present invention is as follows:

[0011] In one aspect, the present invention provides a method for extracting total alkaloid active components from the roots of Psoralea corylifolia, the method comprising the following steps:

[0012] S1, crushing the root of Torch Flower and adding water to perform decoction and extraction, and filtering and concentrating the obtained extract to obtain Torch Flower Root Paste;

[0013] S2, adding the pre-prepared ethanol to the torch flower root paste, and stirring, standing, filtering and concentrating the torch flower root paste with ethanol to obtain a concentrated solution without ethanol taste;

[0014] S3, extracting the concentrated solution three times with equal volumes of chloroform and n-butanol in sequence, combining the extracts respectively, and performing a decompression recovery treatment on the combined extracts to obtain extracts of the chloroform part and the n-butanol part;

[0015] S4. Add hydrochloric acid to the chloroform extract, repeatedly squeeze and dissolve, filter, repeat 3 times, combine the filtrate, adjust the pH to 9-10 with ammonia water, let stand for 24 hours, extract 3 times with an equal amount of ethyl acetate, combine the ethyl acetate layers, wash twice with a small amount of water, and concentrate to dryness under reduced pressure to obtain the crude total alkaloids of the root of Torch Flower;

[0016] S5, dissolving the crude total alkaloids from the root of Torch Flower in ethyl acetate, applying the solution to a neutral alumina column, wet-packing the column, eluting with ethyl acetate, and combining the eluates to obtain an ethyl acetate eluate;

[0017] S6. The ethyl acetate eluate is concentrated under reduced pressure and dried in vacuo to obtain total alkaloids from the root of Psoralea corylifolia.

[0018] Furthermore, the water decoction is performed three times in step S1, the first water decoction takes 1.5 hours, and the second and third water decoctions take 1 hour each; the density of the obtained torch flower root paste is 1.05-1.07.

[0019] Furthermore, the alcohol content of the torch flower root paste added with ethanol in step S2 is 65% to 85%.

[0020] Furthermore, the amount of hydrochloric acid added in step S4 is 5 to 10 times the amount, and the concentration of hydrochloric acid is 3% to 10%.

[0021] Furthermore, the neutral alumina in step S5 is 100-300 mesh, and the amount of the wet column loading is 10-30 times.

[0022] Furthermore, in step S5, the column volume of ethyl acetate elution is 5 to 20 column volumes.

[0023] Another aspect of the present invention provides a total alkaloid from the root of the hollyhock tree, which is extracted by the aforementioned extraction method.

[0024] In another aspect, the present invention provides the use of the total alkaloids from the roots of Psoralea corylifolia in the preparation of medicines for treating rheumatoid arthritis.

[0025] Furthermore, the total alkaloids from the roots of the hollyhock tree are used to reduce the levels of IL-1β and IL-6 in the supernatant of MH7A cells.

[0026] In another aspect, the present invention provides the use of the total alkaloids from the roots of Psoralea corylifolia in the preparation of a medicine for treating membranous nephropathy.

[0027] Furthermore, the total alkaloids from the roots of the hollyhock flower are used to increase the F-actin protein content in the MPC-5 cells induced by C5b-9.

[0028] Furthermore, the total alkaloids from the root of Torchwood are used to increase the expression levels of Nephrin and Potocin proteins in MPC-5 cells induced by C5b-9.

[0029] Furthermore, the total alkaloids from the roots of the hollyhock flower are used to enhance the expression level of skeleton proteins in MPC-5 cells induced by C5b-9.

[0030] The invention has the following beneficial effects: the invention uses acid water to generate a salt solution from alkaloids, then adjusts the pH value to be alkaline, releases the alkaloids, and then extracts with an organic solvent, thereby improving the yield of total alkaloids; the advantage is that after the salt solution is generated, the polarity of the alkaloids increases, and the alkaloids can be dissolved in water and separated from chloroform, so that the alkaloids can be separated from other components in the chloroform, thereby achieving the purpose of purification; after adjusting the alkalinity with ammonia water, the alkaloids can be released, and then extracted with an organic solvent, and finally passed through an alumina column, impurities can be greatly reduced, so that the extraction rate and purity of the alkaloids can be greatly improved, so that the extraction rate can reach more than 90%, and the purity can reach more than 95%; if the alkaloids are directly extracted with an organic solvent and then directly passed through a column without acid-base action, the purity of the total alkaloids will be relatively low (about 60%), and the extraction rate can only reach 70%.

[0031] The total alkaloids of the root of Torch Flower extracted from the Torch Flower root medicinal material provided by the present invention are found in pharmacological activity studies to be able to reduce the contents of IL-1β and IL-6 in the supernatant of MH7A cells, and have a significant improvement effect on rheumatoid arthritis; in addition, the compounds can also increase the content of F-actin and the expression levels of Nephrin and Potocin proteins in MPC-5 cells induced by C5b-9, and increase the expression level of skeleton proteins in MPC-5 cells induced by C5b-9, and have a significant improvement effect on membranous nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is a flow chart of a method for extracting total alkaloid active components from the roots of Torch Flower according to an embodiment of the present invention;

[0034] Figure 2 This is a diagram of the separation and purification process of total alkaloids from the roots of Torch Flower in a method for extracting total alkaloid active components from the roots of Torch Flower according to an embodiment of the present invention;

[0035] Figure 3 It is a schematic diagram of the effects of triptolide, celastrol, total alkaloids from the root of Torch Flower, and a positive drug group (methotrexate) on the contents of IL-1β and IL-6 in the supernatant of MH7A cells in the application of total alkaloids from the root of Torch Flower in the preparation of a drug for treating rheumatoid arthritis according to an embodiment of the present invention;

[0036] Figure 4 It is a schematic diagram of the effects of triptolide, celastrol, total alkaloids from the root of pyrocalyx ulmoides, and a positive drug group (prednisone) on the expression of F-actin protein and skeletal protein in MPC-5 cells in the application of total alkaloids from the root of pyrocalyx ulmoides in the preparation of a drug for membranous nephropathy according to an embodiment of the present invention;

[0037] Figure 5 This is a schematic diagram of the effects of triptolide, celastrol, total alkaloids from the root of torch flower, and a positive drug group (prednisone and tacrolimus) on the expression of nephrin and podocin proteins in MPC-5 cells in the application of total alkaloids from the root of torch flower in the preparation of a drug for membranous nephropathy according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] To further illustrate each embodiment, the present invention provides drawings, which are part of the disclosure of the present invention and are mainly used to illustrate the embodiments. They can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these contents, ordinary technicians in this field should be able to understand other possible implementation methods and advantages of the present invention.

[0039] According to an embodiment of the present invention, a method for extracting total alkaloid active components from the root of Psoralea corylifolia and application of the method in preparing a drug for treating rheumatoid arthritis and membranous nephropathy are provided.

[0040] The present invention is further described with reference to the accompanying drawings and specific embodiments. Figure 1 As shown, according to the method for extracting total alkaloid active components from the root of Torch Flower according to an embodiment of the present invention, the method comprises the following steps:

[0041] S1, crushing the root of Torch Flower and adding water to perform decoction and extraction, and filtering and concentrating the obtained extract to obtain Torch Flower Root Paste;

[0042] S2, adding the pre-prepared ethanol to the torch flower root paste, and stirring the torch flower root paste with ethanol, standing and filtering it to obtain a concentrated solution without ethanol taste;

[0043] S3, extracting the concentrated solution with chloroform and n-butanol in turn, combining the extracts respectively, and performing a decompression recovery treatment on the combined extracts to obtain extracts of the chloroform part and the n-butanol part;

[0044] S4. Add hydrochloric acid to the chloroform extract, repeatedly squeeze and dissolve, filter, repeat 3 times, combine the filtrate, adjust the pH to 9-10 with ammonia water, let stand for 24 hours, extract 3 times with an equal amount of ethyl acetate, combine the ethyl acetate layers, wash twice with a small amount of water, and concentrate to dryness under reduced pressure to obtain the crude total alkaloids of the root of Torch Flower;

[0045] S5, dissolving the crude total alkaloids from the root of Torch Flower in ethyl acetate, applying the solution to a neutral alumina column, wet-packing the column, eluting with ethyl acetate, and combining the eluates to obtain an ethyl acetate eluate;

[0046] S6. The ethyl acetate eluate is concentrated under reduced pressure and dried in vacuo to obtain total alkaloids from the root of Psoralea corylifolia.

[0047] The method comprises the following steps: the crushed torch flower root medicinal material is added with water for decoction three times, the first decoction time is 1.5 hours, and the second and third decoction times are both 1 hour.

[0048] Wherein, the density of the torch flower root paste is 1.05-1.07 (60° C.).

[0049] The alcohol content of the torch flower root paste added with ethanol is 65% to 85%.

[0050] Specifically, add a proper amount of ethanol to the torch flower root paste to make the alcohol content reach 65% to 85%, and let it stand for 12 to 24 hours.

[0051] Wherein, the amount of hydrochloric acid added is 5 to 10 times, and the concentration of hydrochloric acid is 3% to 10%.

[0052] Wherein, the neutral alumina is 100-300 meshes, and the amount of the wet column packing is 10-30 times.

[0053] Wherein, the column volume of ethyl acetate elution in the elution is 5 to 20 column volumes.

[0054] Specifically, the neutral alumina chromatography column is eluted with ethyl acetate, and the ethyl acetate eluate is collected.

[0055] According to another embodiment of the present invention, there is provided an application of total alkaloids from the roots of Pyrola japonica in the preparation of a drug for rheumatoid arthritis, wherein the total alkaloids from the roots of Pyrola japonica are used to reduce the content of IL-1β and IL-6 in the supernatant of MH7A cells.

[0056] According to another embodiment of the present invention, there is provided an application of total alkaloids from the roots of Pyrola japonica in the preparation of a drug for membranous nephropathy, wherein the total alkaloids from the roots of Pyrola japonica are used to increase the content of F-actin protein in MPC-5 cells induced by C5b-9.

[0057] The total alkaloids from the roots of the torch flower are used to increase the expression levels of Nephrin and Potocin proteins in MPC-5 cells induced by C5b-9.

[0058] The total alkaloids from the roots of the torch flower are used to enhance the expression level of skeleton proteins in MPC-5 cells induced by C5b-9.

[0059] Specifically, the total alkaloids from the roots of the torch flower can reduce the levels of IL-1β and IL-6 in the supernatant of MH7A cells, and have a significant improvement effect on rheumatoid arthritis; in addition, this type of compound can also increase the content of F-actin and the expression levels of Nephrin and Potocin proteins in MPC-5 cells induced by C5b-9, and increase the expression levels of skeleton proteins in MPC-5 cells induced by C5b-9, and have a significant improvement effect on membranous nephropathy.

[0060] The following is a description of the method for extracting the total alkaloid active components from the root of the jasmine flower and its application in preparing drugs for treating rheumatoid arthritis and membranous nephropathy in conjunction with specific examples:

[0061] Embodiment 1

[0062] like Figure 2 As shown, the extraction, separation and analysis of total alkaloids: 10 kg of torch flower root medicinal materials were crushed, decocted with water three times, the first time for 1.5 hours, the second and third times for 1 hour each, the decoctions were combined, filtered, and the filtrate was concentrated to a clear paste with a relative density of 1.05-1.07 (60°C), ethanol was added to make the alcohol content reach 85%, stirred evenly, left to stand for 12 hours, filtered, and the filtrate was concentrated until there was no ethanol taste. It was extracted with equal volumes of chloroform and n-butanol in turn, and each solvent was extracted 3 times. The extracts were combined and recovered to dryness under reduced pressure to obtain the chloroform part and the n-butanol part. Take the chloroform part extract, add 8 times the amount of 5% hydrochloric acid, repeatedly squeeze and dissolve, filter, repeat 3 times, combine the filtrate, adjust the pH to 9-10 with ammonia water, let stand for 24 hours, extract 3 times with an equal amount of ethyl acetate, combine the ethyl acetate layer, wash twice with a small amount of water, and concentrate to dryness under reduced pressure to obtain the crude total alkaloids. The column was loaded with 10 times amount of neutral alumina, the crude product was dissolved in ethyl acetate and then loaded, eluted with ethyl acetate, the eluate was combined, concentrated to dryness under reduced pressure, and dried in vacuum to obtain total alkaloids.

[0063] Embodiment 2

[0064] like Figure 3As shown, the expression of inflammatory factors in MH7A cell culture medium was detected based on ELISA method, and the in vitro efficacy of total alkaloids from the roots of Torchwood and the mechanism of action in improving rheumatoid arthritis were observed.

[0065] Experimental steps:

[0066] (1) MH7A cell culture: cultured in a medium containing 10% FBS and 100 U mL -1 Penicillin and 100 mg mL -1 RPMI 1640 medium containing streptomycin.

[0067] (2) Modeling: MH7A cells were divided into normal group and model group (15 ng mL -1 TNF-α), triptolide group, triptolide group, total alkaloids from the root of flavonoids, and positive control group (methotrexate). After 24 h of intervention, the supernatant of each group of MH7A cells was aspirated and centrifuged at 4°C for 2500 r·min. -1 After 10 minutes, take the supernatant for testing.

[0068] (3) ELISA method to detect the expression of inflammatory factors in MH7A cell culture medium: After 24 h of intervention, the supernatant of each group of MH7A cells was aspirated and centrifuged at 2500 r / min at 4°C. -1 After 10 minutes, the supernatant was taken for testing. The ELISA kit was used to detect the IL-1β and IL-6 content in the supernatant of MH7A cells according to the instructions.

[0069] (4) Statistical analysis: All experiments in this study were repeated three times. GraphPad Prism7 was used for statistical analysis. The data were expressed as mean ± standard deviation and then tested with one-way ANOVA. P < 0.05 was considered significant.

[0070] Experimental results:

[0071] Compared with the normal group, the -1 After TNF-α intervention, the levels of IL-1β and IL-6 in the supernatant of MH7A cells increased significantly (P < 0.001). Triptolide, celastrol and total alkaloids from the root of radix pyrophorae could significantly reduce the levels of IL-1β and IL-6 in the supernatant of cells in the model group (P < 0.05, P < 0.001). Figure 3 The total alkaloids from the root of Torch Flower had the best effect, and the efficacy was equivalent to that of the positive control group of methotrexate. Therefore, the total alkaloids from the root of Torch Flower can better improve the levels of inflammatory factors IL-1β and IL-6 caused by rheumatoid arthritis.

[0072] Embodiment 3

[0073] like Figure 4 As shown, based on immunofluorescence detection of F-actin protein expression in MPC-5 cells, the in vitro efficacy of total alkaloids from the roots of Pleurotus eryngii and the mechanism of action in improving membranous nephropathy were observed.

[0074] Experimental steps:

[0075] (1) MPC-5 cell culture: MPC-5 cells were cultured in RPMI1640 medium containing 10% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin. MPC-5 cells were first cultured at 33°C in a 5% CO2 environment and treated with 10 U / ml IFN-γ to induce podocyte proliferation.

[0076] (2) Modeling: Differentiated MPC-5 cells were inoculated in a 6-well plate and cultured at 37°C for 12 h to allow the cells to adhere to the wall. After rinsing with serum-free medium, 2 ml of rabbit anti-mouse IgG serum was added and incubated at 37°C for 2 h. After washing with serum-free medium, medium containing 2% normal human serum was added, and 8 μg / mL C5b-6 complex was added and incubated at 37°C for 2 h. MPC-5 cells were divided into the following groups: normal group, model group (8 μg / mL C5b-6 complex), triptolide group, triptolide group, total alkaloids from the root of radix pyrophorae group, and positive drug group (prednisone) and treated for 24 h.

[0077] (3) After drug treatment, the culture medium was removed, and the sections were rinsed with PBS three times. After adding 4% paraformaldehyde to fix for 20 minutes, the sections were rinsed with PBS three times, incubated with 0.3% Triton X-100 solution for 20 minutes, and then rinsed with PBS three times. A 5% BSA solution prepared with PBS was added and incubated at room temperature for 30 minutes. FITC-labeled phalloidin working solution (1:200) diluted in PBS was added and incubated at 4°C overnight in the dark. After the sections were taken out and returned to room temperature, they were washed with PBS in the dark for three times, each time for 10 minutes. DAPI working solution was added and incubated at room temperature in the dark for 4 minutes. The sections were washed with PBS three times. After the sections were slightly dried, they were sealed with an anti-fluorescence quencher. The cell luminescence was observed under a fluorescence microscope and photographed.

[0078] (4) Statistical analysis: All experiments in this study were repeated three times. GraphPad Prism7 was used for statistical analysis. The data were expressed as mean ± standard deviation and then tested with one-way ANOVA. P < 0.05 was considered significant.

[0079] Experimental results:

[0080] Compared with the normal group, the expression level of F-actin protein in MPC-5 cells after C5b-6 intervention was significantly decreased (P<0.001). Compared with the model group, triptolide, triptolide, and total alkaloids from the root of pyrocalyx could significantly increase the expression levels of F-actin protein and skeleton protein (P<0.001), among which the total alkaloids from the root of pyrocalyx had the best effect. Therefore, the total alkaloids from the root of pyrocalyx can better improve the damage of podocytes (MPC-5) caused by complement membrane attack complex C5b-9 in membranous nephropathy.

[0081] Embodiment 4

[0082] like Figure 5 As shown, the expression of nephrin and podocin proteins in MPC-5 cells was detected by Western blot, and the in vitro efficacy and mechanism of the total alkaloids from the roots of Pleurotus eryngii in improving membranous nephropathy were observed.

[0083] Experimental steps:

[0084] (1) MPC-5 cell culture: cultured in RPMI1640 medium containing 10% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin. MPC-5 were first cultured at 33°C in a 5% CO2 environment and treated with 10 U / ml IFN-γ to induce podocyte proliferation.

[0085] (2) Modeling: Differentiated MPC-5 cells were inoculated in a 6-well plate and cultured at 37°C for 12 h to allow the cells to adhere to the wall. After rinsing with serum-free medium, 2 ml of rabbit anti-mouse IgG serum was added and incubated at 37°C for 2 h. After washing with serum-free medium, medium containing 2% normal human serum was added, and 8 μg / mL of C5b-6 complex was added at the same time and incubated at 37°C for 2 h. MPC-5 cells were divided into the following groups: normal group, model group (8 μg / mL of C5b-6 complex), triptolide, triptolide, total alkaloids of radix pyrophorae group, positive drug: prednisone group (PED80 μg / mL), tacrolimus group (TAC40 μg / mL) and treated for 24 h.

[0086] (3) Western blot analysis of nephrin and podocin protein expression in MPC-5 cells: After drug treatment, the culture medium was discarded and the cells were washed three times with PBS. RIPA cell lysis buffer containing phosphatase inhibitors was added and the cells were lysed on ice for 30 min before scraping the cells and the solution. The cells were centrifuged at 20,000 r / min for 10 min at 4°C and the supernatant was obtained. The total protein concentration was quantitatively determined by the BCA method. The protein samples were separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked in 5% BSA blocking solution at room temperature for 1.5 h, incubated with primary antibody at 4°C overnight, washed with TBST three times for 10 min each time, incubated with corresponding secondary antibody at room temperature for 3 h, washed with TBST three times for 10 min each time, and analyzed with Image J software after development.

[0087] (4) Statistical analysis: All experiments in this study were repeated three times. GraphPad Prism 7 was used for statistical analysis. The data were expressed as mean ± standard deviation and then subjected to LSD test using one-way ANOVA. P < 0.05 was considered significant.

[0088] Experimental results:

[0089] Compared with the normal group, the expression levels of nephrin and podocin proteins in MPC-5 cells after C5b-6 intervention were significantly decreased (P < 0.01), and triptolide, triptolide, and total alkaloids from the root of pyrocalyx could significantly increase the expression levels of nephrin and podocin proteins in MPC-5 cells in the model group (P < 0.01), which was equivalent to the positive drugs. Therefore, total alkaloids from the root of pyrocalyx can better improve the damage of podocytes (MPC-5) caused by complement membrane attack complex C5b-9 in membranous nephropathy.

[0090] Embodiment 5

[0091] Preparation Examples:

[0092] The total alkaloid active ingredients of the root of Torch Flower are prepared into tablets according to the following method, each tablet consisting of the following components:

[0093] 100 mg of total alkaloids from the root of Torch Flower, appropriate amount of starch, appropriate amount of talcum powder, appropriate amount of magnesium stearate, and the above components are made into tablets according to a conventional tablet making method.

[0094] Embodiment 6

[0095] Preparation Examples:

[0096] The total alkaloid active ingredients of the root of Torch Flower are made into granules according to the following method. Each bag of granules consists of the following components:

[0097] 100 mg total alkaloids from the root of Torch Flower, appropriate amount of sucrose, appropriate amount of dextrin, the above components are made into granules according to a conventional granule making method.

[0098] Embodiment 7

[0099] Preparation Examples:

[0100] The total alkaloid active ingredients of the root of Torch Flower are prepared into capsules according to the following method. Each capsule is composed of the following components:

[0101] 100 mg of total alkaloids from the root of Torch Flower, appropriate amount of starch, appropriate amount of talcum powder, appropriate amount of magnesium stearate, and the above components are made into capsules according to a conventional capsule making method.

[0102] In summary, with the aid of the above-mentioned technical scheme of the present invention, the total alkaloids of the root of the torch flower extracted from the root of the torch flower provided by the present invention were found in the pharmacological activity study to be able to reduce the content of IL-1β and IL-6 in the supernatant of MH7A cells, and have a significant improvement effect on rheumatoid arthritis; in addition, the compounds can also increase the content of F-actin and the expression levels of Nephrin and Potocin proteins in MPC-5 cells after C5b-9 induction, and increase the expression levels of skeleton proteins in MPC-5 cells after C5b-9 induction, and have a significant improvement effect on membranous nephropathy.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of total alkaloids from the root of Torchwort in the preparation of a drug for treating membranous nephropathy, The total alkaloids from the root of Torch Flower are extracted by the following method: S1, crushing the root of Torch Flower and adding water to perform decoction and extraction, and filtering and concentrating the obtained extract to obtain Torch Flower Root Paste; S2, adding the pre-prepared ethanol to the torch flower root paste, and stirring the torch flower root paste with ethanol, standing, filtering and concentrating the filtrate to obtain a concentrated solution without ethanol taste; S3, extracting the concentrated solution three times with equal volumes of chloroform and n-butanol in sequence, combining the extracts respectively, and performing a decompression recovery treatment on the combined extracts to obtain extracts of the chloroform part and the n-butanol part; S4. Add 5-10 times the amount of hydrochloric acid to the chloroform extract, with a hydrochloric acid concentration of 3% to 10%, repeatedly knead and dissolve, filter, repeat 3 times, combine the filtrate, adjust the pH to 9-10 with ammonia water, let stand for 24 hours, extract 3 times with an equal amount of ethyl acetate, combine the ethyl acetate layers, wash twice with a small amount of water, and concentrate to dryness under reduced pressure to obtain the crude total alkaloids of the root of Torch Flower; S5, dissolving the crude total alkaloids from the root of Torch Flower in ethyl acetate, applying the solution to a neutral alumina column, wet-packing the column, eluting with ethyl acetate, and combining the eluates to obtain an ethyl acetate eluate; S6, concentrating the ethyl acetate eluate under reduced pressure and performing vacuum drying to obtain total alkaloids from the root of Pleurotus eryngii; The alcohol content of the torch flower root paste added with ethanol in step S2 is 65% to 85%; the column volume of ethyl acetate elution in step S5 is 5 to 20 column volumes.

2. The use according to claim 1, characterized in that: The water decoction in step S1 is performed three times, the first decoction takes 1.5 hours, and the second and third decoctions take 1 hour each; the density of the obtained torch flower root paste is 1.05-1.

07.

3. The use according to claim 1, characterized in that: The neutral alumina in step S5 is 100-300 mesh, and the amount of the wet column loading is 10-30 times.

Images