Leixihaokang, preparation method therefor, and use thereof

Abstract

The present invention belongs to the field of traditional Chinese medicines, and particularly relates to Leixihaokang, a preparation method therefor, and use thereof. The Leixihaokang of the present invention is prepared from the following active pharmaceutical ingredients in parts by weight: 10 parts of Tripterygium glycosides and 0.1-1.3 parts of Artemisia annua extract. According to the present invention, animal experiments prove that the Leixihaokang can ameliorate renal damage in nephrotic syndrome caused by adriamycin, reduce the occurrence of proteinuria, and reduce the apoptosis of renal tissue cells. Meanwhile, the Leixihaokang of the present invention has a good inhibitory effect on renal, hepatic, splenic, and cardiac toxicity in the treatment process of nephrotic syndrome. According to the present invention, animal experiments also prove that the Leixihaokang can ameliorate skin injury in imiquimod-induced psoriasis, reduce inflammatory infiltration and alleviate skin damage. Meanwhile, the Leixihaokang has a certain degree of inhibitory effect on the hepatic and reproductive system toxicity in the treatment process of psoriasis.

Description

Leixihaokang, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to Leishihaokang, a preparation method and application thereof. Background Art

[0002] Tripterygium glycosides (TG) are a mixture of highly polar, fat-soluble components extracted and purified from the dried, peeled root core of Tripterygium wilfordii (Tripterygium wilfordii Hook.f.), a plant of the Celastraceae family. Its physiological activity is synergistically produced by diterpenoids such as triptolide and triptolide ketone, triterpenoids such as triptolide and triptolide A, and alkaloids such as triptolide and triptolide. TG, known as a "Chinese herbal hormone," is currently used clinically as a nonsteroidal immunosuppressant for the treatment of rheumatic and autoimmune diseases such as nephrotic syndrome, Behçet's disease, systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis. It also has promising clinical therapeutic effects on a variety of inflammatory diseases, including chronic gastritis, ankylosing spondylitis, optic neuritis, and urinary tract disorders.

[0003] However, tripterygium wilfordii polyglycosides also exhibit significant toxic side effects, which seriously damage the reproductive system, digestive system, liver, kidneys, hematopoietic system, immune system, nervous system and urinary system. Therefore, how to reduce its toxic side effects is a problem that needs to be solved in the clinical application of tripterygium wilfordii polyglycosides. Currently, combined medication is often used in clinical practice to reduce the toxicity of tripterygium wilfordii polyglycosides, which greatly reduces the damage of tripterygium wilfordii polyglycosides to the liver. However, when using tripterygium wilfordii polyglycosides in combination, in addition to reducing toxicity, maintaining the efficacy or even enhancing the efficacy is also an issue that needs to be focused on.

[0004] Summary of the Invention

[0005] According to a first aspect of the present invention, there is provided Leixihaokang, the raw materials and the weight proportions of which are: 10 parts of Tripterygium wilfordii polyglycosides and 0.1-1.3 parts of Artemisia annua extract.

[0006] In some embodiments, the mass ratio of artemisinin to artemisinin in the Artemisia annua extract is (0.8-1.2):(0.8-1.2), and the mass ratio of artemisinin to artemisinin in the Artemisia annua extract is 85-95%.

[0007] In some embodiments, the mass ratio of artemisinin to artemisinin in the Artemisia annua extract is 1:1.

[0008] In some embodiments, the Artemisia annua extract is obtained by mixing a first Artemisia annua extract with an artemisinin purity of 85-98% and a second Artemisia annua extract with an artemisinin purity of 85-98% in a mass ratio of (0.8-1.2):(0.8-1.2).

[0009] In some embodiments, the method for preparing the first Artemisia annua extract comprises the following steps:

[0010] (1) Weigh Artemisia annua medicinal material, crush it, add 10-15 times the amount of petroleum ether, soak it for 1-2 hours, heat it to 60-65°C, reflux it for 1-2 hours, extract it 2-3 times, combine the extracts, and evaporate the solvent under reduced pressure to obtain Artemisia annua extract;

[0011] (2) Mixing the Artemisia annua extract with 1-2 times the amount of 200-300 mesh silica gel, dry-packing the column with 10-20 times the amount of 200-300 mesh silica gel, eluting with petroleum ether-ethyl acetate, collecting the artemisinin-containing fractions, combining them, and recovering the solvent by vacuum rotary evaporation to obtain a mixture containing artemisinin;

[0012] (3) Repeat the operation of step (2) 2 to 3 times on the obtained mixture to obtain a first Artemisia annua extract with an artemisinin purity of 85 to 95%.

[0013] In some embodiments, the second Artemisia annua extract is prepared according to the method for preparing artemisinin disclosed in Structure-activity relationships of the antimalarial agent artemisinin.8.design, synthesis, and CoMFA studies toward the development of artemisinin-based drugs against leishmaniasis and malaria (doi:10.1021 / jm030181q) published by Avery MA, et al.

[0014] According to a second aspect of the present invention, a method for preparing Leishihaokang is provided, which comprises mixing the raw materials according to a ratio.

[0015] According to a third aspect of the present invention, there is provided use of Leishihaokang in preparing a medicament for treating nephrotic syndrome.

[0016] According to a fourth aspect of the present invention, there is provided a use of Leishihaokang in the preparation of a medicament for treating an immunosuppressive disease, particularly psoriasis.

[0017] The beneficial effects of the present invention include:

[0018] (1) The present invention has demonstrated through animal experiments that Leifenhaokang can improve the kidney damage of nephrotic syndrome caused by doxorubicin, reduce the production of proteinuria, and reduce the apoptosis of renal tissue cells. Among them, the effect of Leifenhaokang of the present invention on reducing the urine protein / creatinine ratio is better than that of Tripterygium wilfordii polyglycosides, and the effect of Leifenhaokang of the present invention on alleviating the kidney tissue damage caused by doxorubicin is equivalent to that of Tripterygium wilfordii polyglycosides, and the effect of improving the serum urea nitrogen value and serum total cholesterol is better than that of Tripterygium wilfordii polyglycosides. At the same time, compared with Tripterygium wilfordii polyglycosides, Leifenhaokang of the present invention has a better inhibitory effect on the kidney, liver, spleen and heart toxicity that occurs during the treatment of nephrotic syndrome.

[0019] (2) The present invention has demonstrated through animal experiments that Reirenhaokang can improve the skin damage of psoriasis induced by imiquimod, reduce inflammatory infiltration, and relieve skin lesions. Among them, the effects of Reirenhaokang and TG in improving the skin lesions of psoriasis mice are equivalent; the effects of Reirenhaokang of the present invention in improving the scales, thickness, erythema and cumulative PASI score of the skin lesions of psoriasis mice are better than those of Tripterygium wilfordii polyglycosides; the pharmacological effects of Reirenhaokang of the present invention and TG in reducing the thickness of the skin of psoriasis mice are consistent; the pharmacological effects of Reirenhaokang of the present invention and TG in reducing the pathological changes of the skin of psoriasis mice are consistent; the effects of Reirenhaokang of the present invention and TG in reducing the expression of PCNA protein are equivalent. At the same time, compared with Tripterygium wilfordii polyglycosides, Reirenhaokang of the present invention has a certain degree of inhibitory effect on the liver and reproductive system toxicity that occurs during the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is the fitted standard curve and linear equation obtained from the liquid phase data of pure artemisinin.

[0021] FIG2A is a HPLC spectrum of the first Artemisia annua extract according to Example 1 of the present invention.

[0022] FIG2B is a HPLC spectrum of the Artemisia annua extract of Example 1 of the present invention.

[0023] FIG3 is a graph showing the changes in body weight of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0024] FIG4 is a diagram showing changes in urine protein / creatinine ratios in SD rats with adriamycin-induced nephrotic syndrome in various groups of the animal experiment of the present invention.

[0025] FIG5 is a graph showing the renal index of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0026] FIG6 is a graph showing liver indexes of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0027] FIG7 is a graph showing the spleen index of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0028] FIG8 is a graph showing cardiac index of SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0029] FIG9 is a diagram showing renal tissue pathology of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention (200X).

[0030] FIG10 is a statistical diagram of changes in serum urea nitrogen in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0031] FIG11 is a statistical diagram of changes in serum creatinine in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0032] FIG12 is a statistical diagram of changes in serum albumin in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0033] FIG13 is a statistical diagram of changes in serum total cholesterol in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0034] FIG14 is a statistical diagram of changes in serum triglycerides in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0035] 15 is a western blot diagram showing changes in Cleave-caspase3 and pro-apoptotic protein Bax protein in kidney tissue apoptosis-related in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0036] FIG16 is a graph showing daily body weight changes of imiquimod-induced psoriasis BalB / c mice in each group in the animal experiment of the present invention.

[0037] FIG17 is a statistical graph of spleen index of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0038] FIG18 is a diagram showing skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0039] FIG19 is a statistical graph showing the scores of scales at the skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0040] FIG20 is a statistical graph showing the thickness of skin lesions in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0041] FIG21 is a statistical graph showing the erythema scores of the skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0042] FIG22 is a statistical graph showing the cumulative PASI scores of skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0043] FIG23 is a statistical graph showing the average skin thickness at the lesion site of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0044] FIG24 is a diagram showing the distribution of subcutaneous blood vessels in the lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0045] FIG25 is a diagram showing the pathological changes of skin lesions on the back of BalB / c mice induced by imiquimod in each group of the animal experiment of the present invention.

[0046] FIG26 is a graph showing the PCNA protein expression in the skin tissue of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0047] FIG27 is a graph showing changes in serum alanine aminotransferase (ALT) levels in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0048] FIG28 is a graph showing changes in serum aspartate aminotransferase (AST) levels in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0049] FIG29 is a graph showing changes in liver index of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0050] FIG30 is a graph showing changes in renal index of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0051] FIG31 is a graph showing changes in reproductive organ (ovary + fallopian tube + uterus) indexes of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0052] FIG32 is a diagram showing the morphological changes of the ovaries and uteri in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to specific examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the raw materials and reagents used in the present invention are all purchased from the market.

[0054] The raw materials used in the following examples are from:

[0055] Tripterygium wilfordii glycosides: purchased from Hunan Qianjin Xieli Pharmaceutical Co., Ltd.

[0056] Artemisia annua: A traditional Chinese medicine, the dried aerial parts of Artemisia annua L., a plant of the Asteraceae family. It originates from Fengshun County, Meizhou City, Guangdong Province. It was harvested after flowering in 2018 and air-dried for later use.

[0057] Artemisinin standard sample: Artemisinin purity>98%, purchased from Chongqing Kerui Pharmaceutical Co., Ltd., Lot: P2160403, CAS: 63968-64-9.

[0058] Example 1

[0059] The Artemisia annua extract of this embodiment is obtained by mixing a first Artemisia annua extract and a second Artemisia annua extract, and the preparation method thereof specifically comprises the following steps:

[0060] First, the first Artemisia annua extract is prepared by the following method:

[0061] (1) Weigh 10 kg of Artemisia annua medicinal material, crush it, add 10 times the amount of petroleum ether (II) and soak it for 1 hour, heat it to 60°C, reflux it for 1.5 hours, and extract it 3 times. Combine the extracts and evaporate the solvent under reduced pressure to obtain 540 g of Artemisia annua extract with an extract yield of 5.4%.

[0062] (2) 535 g of Artemisia annua extract was mixed with 1 times the amount of 200-300 mesh silica gel, and 10 times the amount of 200-300 mesh silica gel was dry-loaded on the column. The mixture was eluted with petroleum ether-ethyl acetate (100:0-80:20) and monitored by TLC. The fractions containing artemisinin were collected, combined, and the solvent was recovered by vacuum rotary evaporation to obtain a mixture containing artemisinin. The artemisinin content was detected by ultra-high performance liquid chromatography and increased to about 30%.

[0063] (3) 84.8 g of the mixture obtained in step (2) was mixed with 2 times the amount of 200-300 mesh silica gel, and 20 times the amount of 200-300 mesh silica gel was dry-loaded onto a column. The mixture was eluted with petroleum ether-ethyl acetate (100:0-80:20) and monitored by TLC. The fractions containing artemisinin were collected, combined, and the solvent was recovered by vacuum rotary evaporation to obtain 22.7 g of a component containing artemisinin.

[0064] (4) 5.0 g of the component obtained in step (3) was taken, mixed with 2 times the amount of 200-300 mesh silica gel, and dry-packed on a column with 20 times the amount of 200-300 mesh silica gel. The mixture was eluted with petroleum ether-ethyl acetate (100:0-80:20), monitored by TLC, and the artemisinin-containing fractions were collected, combined, and the solvent was recovered by vacuum rotary evaporation to obtain 1.9 g of a high-content artemisinin fraction. The content of artemisinin in the fraction was about 85% by ultra-high performance liquid chromatography, thereby obtaining the first Artemisia annua extract.

[0065] In order to accurately determine the content of artemisinin in the first Artemisia annua extract, high performance liquid chromatography was used for determination. The operation method is as follows:

[0066] (1) Preparation of artemisinin acetonitrile standard solution: 307 mg of artemisinin standard sample (artemisinin purity > 98%, purchased from Chongqing Kerui Pharmaceutical Co., Ltd.; Lot: P2160403; CAS: 63968-64-9) was dissolved in 3 mL of acetonitrile to prepare an artemisinin acetonitrile standard solution with a concentration of 102.33 mg / mL. The above solution was further diluted with chromatographic acetonitrile to artemisinin acetonitrile standard solutions with concentrations of 40.93 mg / mL, 51.57 mg / mL, 61.40 mg / mL, and 81.87 mg / mL, respectively. The standard solutions filtered through a 0.22 μm organic microporous filter membrane were placed in a sample tray in ascending order of concentration and injected sequentially. The HPLC detection conditions were as follows:

[0067] Instrument: HPLC (Agilent 1200).

[0068] Detector: Evaporative light scattering detector (Agilent, 1260 Infinity ELSD).

[0069] ELSD setting conditions: temperature 70 °C, nitrogen pressure 2.0 Bar.

[0070] Chromatographic column: 250 mm × 4.6 mm, 5 μm, C18 packing.

[0071] Column temperature: 30°C.

[0072] Mobile phase conditions: acetonitrile-water system 0~20min 40%→90%; 20~25min 90%→40%; 25~30min 40% acetonitrile.

[0073] Injection volume: 10 μL.

[0074] The HPLC mobile phase composition and gradient setting are shown in Table 1:

[0075] Table 1 HPLC mobile phase composition and gradient setting

[0076] (2) Draw the standard curve of artemisinin: According to the peak area of ​​artemisinin standard solution, the concentration was plotted and the standard curve and linear equation were fitted, as shown in Figure 1. According to the corresponding relationship between the peak area and the concentration of artemisinin reference solution, the binary regression equation was fitted: y = 37.029x-1010, R 2 =0.9953.

[0077] (3) Determination of artemisinin content in the first Artemisia annua extract: A 65 mg / mL acetonitrile solution of the first Artemisia annua extract was prepared, filtered through a 0.22 μm organic microporous filter membrane, and then placed in a sample tray for detection. The HPLC detection conditions were:

[0078] Instrument: HPLC (Agilent 1200).

[0079] Detector: Evaporative light scattering detector (Agilent, 1260 Infinity ELSD).

[0080] ELSD setting conditions: temperature 70 °C, nitrogen pressure 2.0 Bar.

[0081] Chromatographic column: 250 mm × 4.6 mm, 5 μm, C18 packing.

[0082] Column temperature: 30°C.

[0083] Mobile phase conditions: acetonitrile-water system 0~20min 40%→90%; 20~25min 90%→40%; 25~30min 40% acetonitrile.

[0084] Injection volume: 10 μL.

[0085] The HPLC mobile phase composition and gradient setting are shown in Table 2:

[0086] Table 2 HPLC mobile phase composition and gradient setting

[0087] The retention time of artemisinin in the first Artemisia annua extract was determined based on the retention time of the reference substance, and the artemisinin content was calculated based on its peak area and a binary regression equation. The HPLC spectrum is shown in Figure 2A. Substituting the artemisinin peak area in the first Artemisia annua extract into the linear equation y = 37.029x - 1010, the artemisinin content was calculated to be 56.90 mg / mL, representing 87.54% of the total components in the first Artemisia annua extract.

[0088] Second, the second Artemisia annua extract was prepared according to the method for preparing artemisinin disclosed in Avery MA, et al. (Structure-activity relationships of the antimalarial agent artemisinin.8. Design, synthesis, and CoMFA studies toward the development of artemisinin-based drugs against leishmaniasis and malaria) (doi:10.1021 / jm030181q). Testing revealed that the artemisinin in the second Artemisia annua extract had a purity of 98%.

[0089] Third, the Artemisia annua extract is prepared by the following steps:

[0090] 65.13 mg of the first Artemisia annua extract with an artemisinin purity of 87.54% was weighed, and 58.18 mg of the second Artemisia annua extract with an artemisinin purity of 98% was added thereto, and the mixture was mixed evenly to obtain the Artemisia annua extract.

[0091] The components of the Artemisia annua extract prepared in Example 1 were determined by high performance liquid chromatography. The operation method was as follows: 62.57 mg of the Artemisia annua extract was dissolved in 1 mL of acetonitrile, filtered through a 0.22 μm organic microporous filter membrane, and then the content of each index component was detected by HPLC.

[0092] HPLC detection conditions are:

[0093] Instrument: HPLC (Agilent 1200).

[0094] Detector: Evaporative light scattering detector (Agilent, 1260 Infinity ELSD).

[0095] ELSD setting conditions: temperature 70 °C, nitrogen pressure 2.0 Bar.

[0096] Chromatographic column: 250 mm × 4.6 mm, 5 μm, C18 packing.

[0097] Column temperature: 30°C.

[0098] Mobile phase conditions: acetonitrile-water system 0~20min 40%→90%; 20~25min 90%→40%; 25~30min 40% acetonitrile.

[0099] Injection volume: 10 μL.

[0100] The HPLC mobile phase composition and gradient setting are shown in Table 3:

[0101] Table 3 HPLC mobile phase composition and gradient setting

[0102] The detection results are shown in Figure 2B. In Figure 2B, the peak with a retention time of 13.617 min represents artemisinin, the peak with a retention time of 14.974 min represents artemisinin, and the rest are impurity peaks. Due to the sensitivity problem of the ELSD detector, some impurity peaks are not displayed.

[0103] The artemisia annua extract of the present invention is mixed with tripterygium wilfordii polyglycosides to obtain tripterygium wilfordii glycosides.

[0104] Example 2

[0105] The active ingredients of the Leixihaokang of this embodiment are tripterygium wilfordii polyglycosides and the Artemisia annua extract prepared in Example 1, wherein the mass of tripterygium wilfordii polyglycosides is 10 g, and the mass of the Artemisia annua extract is 0.106 g.

[0106] Example 3

[0107] The active ingredients of the Leixihaokang of this embodiment are tripterygium wilfordii polyglycosides and the Artemisia annua extract prepared in Example 1, wherein the mass of tripterygium wilfordii polyglycosides is 10 g, and the mass of the Artemisia annua extract is 0.213 g.

[0108] Example 4

[0109] The active ingredients of the Leienhaokang of this embodiment are tripterygium wilfordii polyglycosides and the Artemisia annua extract prepared in Example 1, wherein the mass of tripterygium wilfordii polyglycosides is 10 g, and the mass of the Artemisia annua extract is 0.426 g.

[0110] Example 5

[0111] The active ingredients of the Leixihaokang of this embodiment are tripterygium wilfordii polyglycosides and the Artemisia annua extract prepared in Example 1, wherein the mass of tripterygium wilfordii polyglycosides is 9.1 g, and the mass of the Artemisia annua extract is 1.1 g.

[0112] Example 6

[0113] The active ingredients of the Leixihaokang of this embodiment are tripterygium wilfordii polyglycosides and the Artemisia annua extract prepared in Example 1, wherein the mass of tripterygium wilfordii polyglycosides is 8.19 g, and the mass of the Artemisia annua extract is 1 g.

[0114] Next, in order to explore the sustained effect of the Leishihaokang of the present invention on nephrotic syndrome, a pharmacodynamic animal experiment on Leishihaokang in treating doxorubicin-induced nephrotic syndrome was conducted.

[0115] 1. Experimental Animals

[0116] The experimental animals used in the experiment were SPF - level healthy male SD rats, purchased from the Guangdong Provincial Medical Experimental Animal Center, with the license numbers: SCXK(Yue)2013 - 0002, SCXK(Yue)2018 - 0002. The feeding environment for the experimental animals was as follows: maintained in a 12 - hour cycle under light / dark conditions (lights on: 07:00 - 19:00), controlling the temperature (20 ± 2 °C), humidity (50 ± 10%), ventilation, with more than 10 air changes per hour (all fresh air systems), and allowing free access to standard rat food and tap water. All animal experimental protocols were carried out in accordance with the "Regulations on the Administration of Experimental Animals" promulgated by the National Science and Technology Commission and approved by the Experimental Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine; the experimental animal ethics number: 2020004.

[0117] II. Drug Preparation

[0118] Tripterygium glycosides were dissolved in pure water to prepare a 2 - mg / mL tripterygium glycosides solution for standby.

[0119] The Artemisia annua extract prepared in Example 1 was added to pure water and ultrasonically suspended to obtain an Artemisia annua extract solution for standby.

[0120] Respectively, take the 2 - mg / mL tripterygium glycosides solution, and add appropriate amounts of the above - mentioned Artemisia annua extract solution to the tripterygium glycosides solution according to the concentrations of Artemisia annua extract in the solution being 0.021 mg / ml, 0.042 mg / ml, and 0.085 mg / ml, to obtain low - dose Leixikang solution, medium - dose Leixikang solution, and high - dose Leixikang solution.

[0121] Prednisone: The dosing dose for rats was 6.3 mg / kg / day, and it was configured into a 1.26 - mg / mL prednisone solution using pure water.

[0122] III. Grouping, Modeling and Drug Administration Methods

[0123] After 3 days of adaptive feeding, SPF - level healthy male SD rats were randomly divided into 7 groups: blank group (Control), model group (Model), tripterygium glycosides group (TG), low - dose Leixikang group (LXHK - L), medium - dose Leixikang group (LXHK - M), high - dose Leixikang group (LXHK - H), and prednisone group (Prednisone), with 6 rats in each group.

[0124] Except for the blank group, each group of rats was injected with adriamycin at a dose of 6 mg / kg via the tail vein once. Three weeks later, the model - making groups started drug administration according to the urine protein / creatinine ratio according to the grouping, and the drug administration continued for 5 weeks. The drug administration situation for each group was as follows:

[0125] Blank group (Control): Administered an equal volume of pure water by gavage daily according to body weight;

[0126] Model group (Model): Administer an equal amount of pure water by gavage daily according to body weight;

[0127] Tripterygium wilfordii polyglycosides group (TG): 10 mg / kg / day of Tripterygium wilfordii polyglycosides solution was given orally;

[0128] Low-dose Leixiehaokang group (LXHK-L): The patients were given low-dose Leixiehaokang solution orally at the dose of 10 mg / kg / day of Tripterygium wilfordii polyglycosides and 0.106 mg / kg / day of Artemisia annua extract;

[0129] The medium-dose Leixihaokang group (LXHK-M) was given a medium-dose Leixihaokang solution by oral gavage at the rate of 10 mg / kg / day of Tripterygium wilfordii polyglycosides and 0.213 mg / kg / day of Artemisia annua extract.

[0130] The high-dose Leixiehaokang group (LXHK-H) was given high-dose Leixiehaokang solution by oral gavage at the rate of 10 mg / kg / day of Tripterygium wilfordii polyglycosides and 0.426 mg / kg / day of Artemisia annua extract.

[0131] Positive drug prednisone group (Prednisone): Prednisone solution was administered orally at a dose of 6.3 mg / kg / day.

[0132] 4. Statistical Methods

[0133] GraphPad Prism 8.0.2 was used for statistical analysis and processing of data. The measurement data were first tested for normality, and the data that met the normal distribution were expressed as mean ± standard deviation. One-way ANOVA was used to compare means for multiple independent groups of data that met normal distribution. When statistically significant results were obtained through one-way ANOVA, pairwise comparisons of means between groups were performed. When variances were equal, the least significant difference t-test (LSD-t-test) was used to compare and analyze mean differences between groups. When variances were unequal, Dunnett's T3 test was used for statistical analysis. When variances did not meet normal distribution, the Kruskal-Wallis nonparametric test was used. P < 0.05 was considered statistically significant. Graphs were created using GraphPad Prism 6, Microsoft Office 365, and Photoshop CC 2019.

[0134] 5. Observation indicators

[0135] 1. General

[0136] The clinical manifestations of nephrotic syndrome include hyperlipidemia, severe edema, hyperproteinuria, hypoproteinemia, etc. When observing the SD rats with nephrotic syndrome induced by adriamycin, it is important to observe changes in the animal's mental state, body weight, food intake, water intake, coat color, and blood biochemistry.

[0137] 2. Urine protein / creatinine ratio

[0138] When collecting urine from SD rats, rat metabolic cages were used to collect the urine. The feces and urine of the rats were collected separately, and the changes in the 24-hour urine protein / creatinine ratio were detected.

[0139] 3. Organ Index

[0140] When the animals were collected, the liver and kidneys were removed and the excess water was absorbed with filter paper. The wet weight was then weighed to calculate the organ index (organ index = organ weight (g) / rat body weight (g) × 100%).

[0141] 4. Kidney wax staining

[0142] One-half of the kidney was fixed with 4% paraformaldehyde for 24-48 hours, then dehydrated in an automatic dehydrator and embedded in paraffin. Each kidney tissue was finally cut into 3 μm wax slices for subsequent experiments as follows:

[0143] H&E staining: Before staining, sections were baked at 65°C for 1.5 h to secure the tissue attachment. H&E staining was then performed as follows: dewaxing: xylene I for 10 min, xylene II for 10 min, and xylene III for 10 min; rehydration: 100% ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, 50% ethanol for 5 min, 20% ethanol for 5 min, and H2O for 5 min.

[0144] Hematoxylin staining: stain with hematoxylin for 15 minutes; color separation: separate with hydrochloric acid alcohol (0.1% hydrochloric acid / 75% ethanol) for a few seconds to make the cell nucleus turn blue, and immediately rinse with running water for a few minutes to wash off the excess stain.

[0145] Eosin staining: stain the cytoplasm with eosin solution for 5 minutes; dehydration and transparentization: 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, 95% ethanol for 2 minutes, 100% ethanol for 2 minutes, 100% ethanol:xylene (1:1) for 2 minutes, xylene for 2 minutes; sealing and microscopic examination: sealing with neutral gum and microscopic examination.

[0146] 5. Blood biochemical index detection

[0147] When the animals were collected, rats were anesthetized by intraperitoneal injection, and blood was collected from the abdominal aorta using a coagulant tube. The collected blood was centrifuged at 3000 r / min for 15 min, and the serum was aspirated and stored at -80°C for later use.

[0148] Biochemical indicators BUN, Cr, ALB, ALT, and AST were measured using Nanjing Jiancheng kits according to the manufacturer's instructions. TC and TG were measured using a fully automatic biochemical indicator analyzer.

[0149] 6. WB experiment:

[0150] Animal Tissue Protein Extraction: 30 mg of kidney tissue was placed in a tissue homogenizer tube and 400 μL of tissue lysis buffer containing protease and phosphatase inhibitors was added (Roche tablets should be used at 1 tablet / 10 mL). After high-speed homogenization, centrifuge at 12,000 rpm for 15 minutes. The supernatant was collected and the protein concentration of the sample was determined by the BCA assay. After adjusting the concentration of each sample to a consistent level, 5x protein loading buffer was added and the sample was denatured at 100°C for 10 minutes. The sample was then stored at -80°C.

[0151] Use Yazyme premix to prepare polyacrylamide gel, load 20-30ug of sample, run the sample through the stacking gel at 80V, then adjust the voltage to 100V to run the sample through the entire gel to complete the SDS-PAGE gel electrophoresis operation; secondly, perform the membrane transfer operation (PVDF membrane, pre-wetted in methanol for 5 minutes in advance), the transfer conditions are: 300mA, 60min; after the transfer is completed, block with 5% skim milk for 2h, and incubate the primary antibody in a refrigerator at 4℃ overnight according to the corresponding band; the next day, wash away the unbound primary antibody with TBST washing solution (wash 3 times, 5min each time); incubate with secondary antibody at 37℃ for 2h, and wash away the unbound secondary antibody with TBST washing solution (wash 3 times, 5min each time); ECL luminescent liquid is used as a substrate to expose the target band, and the results are analyzed, recorded and statistically analyzed.

[0152] 6. Experimental Results

[0153] FIG3 is a graph showing the changes in body weight of SD rats with nephrotic syndrome induced by adriamycin in each group.

[0154] As can be seen from Figure 3, compared with the blank group, the body weight of the rats in the model group was significantly reduced, but there was no significant difference between the drug-treated group and the model group.

[0155] FIG4 is a graph showing changes in urine protein / creatinine ratio in SD rats with adriamycin-induced nephrotic syndrome in each group.

[0156] As can be seen from Figure 4, after the tail vein disposable injection adriamycin modeling three weeks, each group, compared with the blank group, urine protein / creatinine ratio all increased, and compared with the blank group, all had significant differences (P < 0.01).Modeling started administration three weeks later, compared with the model group, gavage four weeks from LXHK-L, LXHK-M, LXHK-H groups and positive drug group urine protein / creatinine ratio significantly decreased, and the difference had statistical significance (P < 0.01), while TG group gavage five weeks urine protein / creatinine ratio just significantly decreased (P < 0.05).LXHK-L, LXHK-M, LXHK-H groups reduce the onset time of urine protein / creatinine ratio earlier than TG group, show that the effect of the urine protein / creatinine ratio of Lei En Hao Kang of the present invention is better than Tripterygium wilfordii polyglycosides.

[0157] FIG5 is a graph showing the kidney index of each group of SD rats with nephrotic syndrome induced by adriamycin; FIG6 is a graph showing the liver index of each group of SD rats with nephrotic syndrome induced by adriamycin.

[0158] As shown in Figure 5, the renal index of the model group was significantly increased compared with the blank group, while the renal index of each drug-treated group was decreased compared with the model group, and the differences were significant (P<0.01).

[0159] As shown in Figure 6, the liver index of the model group was significantly increased compared with the blank group, while the liver index of the TG group was decreased compared with the model group, and the difference was significant (P<0.05). The liver index of the LXHK-M and LXHK-H groups was decreased compared with the model group, and the difference was extremely significant (P<0.01).

[0160] FIG7 is a graph showing the spleen index of each group of SD rats with nephrotic syndrome induced by adriamycin; FIG8 is a graph showing the heart index of each group of SD rats with nephrotic syndrome induced by adriamycin.

[0161] As can be seen from Figure 7, compared with the blank group, the spleen index of the model group decreased to a certain extent; compared with the model group, the spleen index of the TG group recovered to a certain extent after treatment, and the recovery effect of the spleen index in the LXHK-L group, LXHK-M group, and LXHK-H group was more obvious.

[0162] As shown in Figure 8, compared with the blank group, the cardiac index of the model group increased to a certain extent; compared with the model group, the cardiac index of the TG group was further increased after medication, while the cardiac index of the LXHK-L group and the LXHK-M group was further decreased after medication.

[0163] FIG9 is a diagram showing the renal tissue pathology of SD rats with adriamycin-induced nephrotic syndrome in each group (200X).

[0164] As shown in Figure 9, compared with the blank group, the model group showed severe renal tubular damage, including tubular dilatation with granular degeneration, partial vacuolar degeneration of renal tubular epithelial cells, brush border shedding, multiple protein casts and partial inflammatory cell infiltration, and varying degrees of atrophy and deformation of the glomeruli. After administration, the renal tissues of rats in the LXHK-L, LXHK-M, LXHK-H and TG groups were relieved to varying degrees, indicating that the present invention's Leishenhaokang has a certain effect in alleviating renal tissue damage caused by doxorubicin, and its effect is comparable to that of TG.

[0165] Figures 10-12 are statistical graphs showing changes in renal function and serum biochemistry of SD rats induced by adriamycin in each group, wherein Figure 10 is a statistical graph showing changes in serum urea nitrogen, Figure 11 is a statistical graph showing changes in serum creatinine, and Figure 12 is a statistical graph showing changes in serum albumin.

[0166] As shown in Figure 10, after modeling, serum urea nitrogen increased significantly, and after drug administration, the serum urea nitrogen values ​​of each drug administration group decreased significantly (P < 0.01), among which the decrease in serum urea nitrogen values ​​of the LXHK-M group and the LXHK-H group was better than that of the TG group.

[0167] As shown in Figure 11, after modeling, the serum creatinine value increased, while the serum creatinine value of each drug-treated group was significantly reduced after administration, and the difference was statistically significant.

[0168] As shown in Figure 12, after modeling, the serum albumin level in the model group decreased significantly (P<0.01), and after administration of the TG group and the LXHK-M and LXHK-H groups, the serum albumin levels recovered to some extent (P<0.05).

[0169] Figures 13-14 are statistical charts showing changes in serum total cholesterol (TC) and blood lipid index triglyceride (TG).

[0170] As shown in Figure 13, after modeling, the serum total cholesterol of the model group was significantly increased compared with the blank group, and after administration, the serum total cholesterol of the TG group was significantly decreased compared with the model group, and the difference was significant (P < 0.05). The serum total cholesterol of the LXHK-L, LXHK-M, and LXHK-H groups was significantly decreased compared with the model group, and the difference was extremely significant (P < 0.01), indicating that the intervention of the present invention can significantly reduce the content of serum total cholesterol, and its effect is better than that of tripterygium wilfordii polyglycosides.

[0171] As shown in FIG14 , after modeling, the triglyceride content in the model group increased significantly, while the triglyceride content in each drug-treated group was significantly reduced.

[0172] FIG15 is a Western blot diagram showing the changes in Cleave-caspase3 and Bax proteins in the renal tissues of rats with nephrotic syndrome induced by adriamycin in each group.

[0173] As can be seen from Figure 15, the total apoptosis-executing protein Cleave-caspase3 and pro-apoptotic protein Bax in the model group were significantly increased, while the expression levels of Cleave-caspase3 and Bax in each drug administration group decreased, indicating that both TG and the artemisia scoparia kang of the present invention can reduce the production of apoptosis proteins Cleave-caspase3 and Bax in renal tissue cells. The protective effects of TG and the artemisia scoparia kang of the present invention on renal tissue may act by reducing apoptosis.

[0174] In summary, for adriamycin-induced nephrotic syndrome rats, the artemisia scoparia kang of the present invention can reduce proteinuria, improve renal function, lower blood lipid, reduce apoptosis, and reduce renal pathological damage. Among them, the artemisia scoparia kang of the present invention has a better effect on reducing the urine protein / creatinine ratio than tripterygium glycosides. The effect of the artemisia scoparia kang of the present invention in reducing adriamycin-induced renal tissue damage is equivalent to that of tripterygium glycosides, and the improvement effect on serum urea nitrogen value and serum total cholesterol is better than that of tripterygium glycosides. At the same time, compared with tripterygium glycosides, the artemisia scoparia kang of the present invention has a better inhibitory effect on the toxicity of kidneys, livers, spleens and hearts that occur during the treatment of nephrotic syndrome.

[0175] In addition, in order to explore the pharmacodynamic effect of the artemisia scoparia kang of the present invention on psoriasis, a pharmacodynamic animal experiment of artemisia scoparia kang in the treatment of psoriasis was also carried out.

[0176] I. Experimental animals

[0177] The experimental animals used in the experiment were SPF-grade healthy male BalB / c mice, purchased from the Guangdong Provincial Center for Medical Experimental Animals, with the license numbers: SCXK(Yue)2013-0002, SCXK(Yue)2018-0002. The feeding environment of the experimental animals was: maintained in a 12-hour cycle under light / dark conditions (lights on: 07:00-19:00), controlling the temperature (20±2°C), humidity (50±10%), ventilation, with more than 10 air changes per hour (all fresh air systems), and allowing free access to standard mouse food and tap water. All animal experiment protocols were carried out in accordance with the "Regulations on the Administration of Experimental Animals" promulgated by the National Science and Technology Commission and approved by the Experimental Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (Experimental Animal Ethics Number: 2021018).

[0178] II. Drug preparation

[0179] Appropriate amounts of tripterygium glycosides were respectively suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) solution to prepare tripterygium glycoside solutions of 0.91mg / mL and 8.19mg / mL for standby.

[0180] The Artemisia annua extract was added to the above-mentioned Tripterygium wilfordii polyglycosides solution to prepare a Leixihaokang solution, which was ultrasonically suspended and set aside. Specifically, according to the concentration of the Artemisia annua extract in the solution being 0.11 mg / ml, an appropriate amount of the Artemisia annua extract prepared in Example 1 was added to the 0.91 mg / mL Tripterygium wilfordii polyglycosides solution to prepare a low-dose Leixihaokang solution; according to the concentration of the Artemisia annua extract in the solution being 1 mg / ml, an appropriate amount of the Artemisia annua extract prepared in Example 1 was added to the 8.19 mg / mL Tripterygium wilfordii polyglycosides solution to prepare a high-dose Leixihaokang solution.

[0181] Mixed solution of tripterygium wilfordii polyglycosides and artemisinin: Weigh an appropriate amount of artemisinin and suspend it in 8.19 mg / mL tripterygium wilfordii polyglycosides solution to obtain a mixed solution containing 0.5 mg / mL artemisinin.

[0182] Mixed solution of tripterygium wilfordii polyglycosides and artemisinin: weigh an appropriate amount of artemisinin and suspend it in 8.19 mg / mL tripterygium wilfordii polyglycosides solution to obtain a mixed solution containing 0.5 mg / mL artemisinin.

[0183] Methotrexate: The dose for mice is 1 mg / kg / day, prepared in 0.5% CMC-Na solution to a 0.1 mg / mL methotrexate solution.

[0184] 3. Grouping, Modeling and Dosage Methods

[0185] SPF healthy female BalB / c mice were adaptively fed for 3 days and then randomly divided into 8 groups: blank group (control), model group (Model), tripterygium wilfordii polyglycosides group (TG), tripterygium wilfordii polyglycosides + artemisinin group (TG+ART), tripterygium wilfordii polyglycosides + artemisinin group (TG+AT), low-dose of artemisinin-containing group (LXHK-L), high-dose of artemisinin-containing group (LXHK-H) and methotrexate group (MTX), with 4 mice in each group.

[0186] Except for the blank group, mice in each group received 62.5 mg of imiquimod cream (IMQ) applied to their backs at the same time daily for 8 consecutive days. The drug-treated group was also given the drug by gavage once daily for 8 consecutive days. The dosing schedule for each group is as follows:

[0187] Blank group (control): The mice were given an equal amount of CMC-Na solution by gavage according to their body weight every day;

[0188] Model group: The mice were given an equal amount of CMC-Na solution by gavage every day according to their body weight;

[0189] Tripterygium wilfordii polyglycosides group (TG): 81.9 mg / kg / day of Tripterygium wilfordii polyglycosides solution was given orally;

[0190] Tripterygium wilfordii polyglycosides + artemisinin group (TG+ART): 81.9 mg / kg / day of tripterygium wilfordii polyglycosides (9 times the adult clinical dose) and 5 mg / kg / day of artemisinin were administered by oral gavage.

[0191] Tripterygium wilfordii glycosides + artemisinin group (TG+AT): Administer a mixed solution of tripterygium wilfordii glycosides and artemisinin by oral gavage at a dose of 81.9 mg / kg / day of tripterygium wilfordii glycosides and 5 mg / kg / day of artemisinin.

[0192] Low-dose Leixiehaokang group (LXHK-L): 9.1 mg / kg / day of tripterygium wilfordii polyglycosides (1 times the adult clinical dose) and 1.1 mg / kg / day of Artemisia annua extract were administered orally.

[0193] High-dose Leixiehaokang group (LXHK-H): Administered high-dose Leixiehaokang solution orally at 81.9 mg / kg / day of Tripterygium wilfordii polyglycosides and 10 mg / kg / day of Artemisia annua extract;

[0194] Methotrexate group (MTX): methotrexate solution was administered orally at a dose of 1 mg / kg / day (calculated based on the adult clinical oral dose).

[0195] 4. Statistical Methods

[0196] GraphPad Prism 8.0.2 was used for statistical analysis and processing of data. The measurement data were first tested for normality, and the data that met the normal distribution were expressed as mean ± standard deviation. One-way ANOVA was used to compare means for multiple independent groups of data that met normal distribution. When statistically significant results were obtained through one-way ANOVA, pairwise comparisons of group means were performed. When variances were equal, the least significant difference t-test (LSD-t-test) was used to compare and analyze differences in means between groups. When variances were unequal, Dunnett's T3 test was used for statistical analysis. When variances did not meet normal distribution, the Kruskal-Wallis nonparametric test was used. P < 0.05 was considered statistically significant. Graphs were created using GraphPad Prism 8.0.2, Microsoft Office 365, and Photoshop CC 2019.

[0197] 5. Observation indicators

[0198] 1. General

[0199] The mental state of the mice was observed and their body weight was recorded.

[0200] 2. Liver function test

[0201] The eyeballs of mice were removed for blood collection. The blood was collected in yellow coagulant tubes, kept at 4°C for 2 h, and centrifuged at 3000 rpm for 15 min at room temperature. The supernatant was collected and serum ALT and AST were detected according to the method indicated in the kit instructions.

[0202] 3. Organ index

[0203] After the experiment, the spleen, kidneys, liver, ovaries, and uterus of the mice were removed and excess water was removed using filter paper. The wet weight was then weighed and the organ index was calculated: organ index = organ weight (g) / mouse body weight (g) × 100%.

[0204] 4. Skin lesions

[0205] The skin lesions of the mice were observed for epidermal scales, lesion texture, lesion thickness, erythema and punctate hemorrhage.

[0206] 5. Assessment of skin lesion severity

[0207] The Psoriasis Area and Severity Index (PASI) was used to assess the severity of psoriasis lesions in each group of mice before and at the end of treatment. The PASI score includes erythema, epidermal scaling, and skin thickness at the lesion site. The PASI scoring criteria are shown in Table 4. The total score is the sum of erythema, epidermal scaling, and skin thickness, based on the lesion severity score (0 to 4 points).

[0208] Table 4 PASI scoring criteria

[0209] 6. Measure the thickness of the skin lesions

[0210] Use Image-Pro Plus software to calibrate the ruler and click Measure Distances. Randomly select 5 locations in each photo to measure the epidermal thickness (the distance from the stratum corneum to the basement membrane zone). The average of the 5 measurements represents the visual field measurement data for subsequent statistical analysis.

[0211] 7. Observe the distribution of skin blood vessels at the lesion site

[0212] After the experiment, the back skin was cut off to observe the distribution of subcutaneous blood vessels in the lesion area of ​​the mice.

[0213] 8. HE staining of skin tissue

[0214] The mouse skin lesion tissue was separated and 0.5 cm 2 Skin tissue was fixed with 4% paraformaldehyde for 24-48 h, dehydrated, embedded in paraffin, cut into 4 μm wax slices, and the pathological changes of skin lesion tissue were observed after HE staining.

[0215] HE staining process:

[0216] ① Before staining, bake the sections at 65°C for 1 hour to secure the tissue attachment. Then proceed with the H&E staining procedure: dewaxing: xylene I for 10 minutes, xylene II for 10 minutes; rehydration: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and H2O for 3 minutes.

[0217] ②Hematoxylin staining: stain with hematoxylin for 5 minutes; color separation: separate with hydrochloric acid alcohol (0.1% hydrochloric acid / 75% ethanol) for a few seconds to make the cell nucleus turn blue, and immediately rinse with running water for a few minutes to wash off the excess stain.

[0218] ③ Eosin staining: stain the cytoplasm with eosin solution for 3 min; dehydration and clearing: 80% ethanol for 20 s, 90% ethanol for 20 s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, 100% ethanol I for 2 min, 100% ethanol II for 2 min, xylene I for 2 min, xylene II for 2 min; mounting and microscopic examination: mounting with neutral gum and microscopic examination.

[0219] 9. Immunohistochemical staining to observe the expression of PCNA in the skin of the lesion

[0220] Immunohistochemistry staining process:

[0221] ① Dewaxing and hydration: Bake the sections in a 60°C oven for approximately 1 hour. Immediately soak in xylene for 10 minutes twice. Then, soak in a gradient of alcohol (100% ethanol → 100% ethanol → 95% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → 50% ethanol → 20% ethanol) for 5 minutes each. Remove the sections and rinse with distilled water before placing them in PBS buffer.

[0222] ② Antigen retrieval: Soak the tissue sections in a retrieval box and inject the diluted citric acid antigen retrieval solution. Heat in a microwave oven at medium-high heat (80-90°C) for 8 minutes, then stop heating for 8 minutes. Heat again at medium-low heat (50-60°C) for 8 minutes. Remove from the heat retrieval box, open the lid, and cool to room temperature. Rinse with PBS buffer three times for 5 minutes.

[0223] ③ Block endogenous peroxidase: wipe off excess PBS buffer on the slice, place it in an incubation box, add 100 μl of 3% H2O2 solution, let it stand at 37°C for 10 minutes, and rinse with PBS buffer 3 times for 3 minutes.

[0224] ④ Serum blocking: Add normal goat serum working solution for blocking into the histochemical circle and incubate at room temperature for 15 minutes.

[0225] ⑤ Primary antibody incubation: Gently shake off the blocking solution, do not wash, add an appropriate amount of prepared primary antibody solution (Note: the primary antibody solution is 5% bovine serum albumin solution with PCNA anti-rabbit primary antibody added at a ratio of 1:4000), place in a moisturizing box, and incubate at 4°C overnight.

[0226] ⑥ Secondary antibody incubation: The next day, warm the sections in a 37°C incubator for approximately 30 minutes. Rinse with PBS buffer three times for 5 minutes. Add commercial anti-rabbit secondary antibody solution and incubate at room temperature for 15 minutes. Rinse with PBS buffer three times for 3 minutes.

[0227] ⑦Add an appropriate amount of commercial HRP horseradish enzyme working solution, incubate at room temperature for 15 minutes, and rinse with PBS buffer 3 times for 3 minutes.

[0228] DAB color development: Add the prepared DAB color development solution (freshly prepared and used) on the tissue, control the color development time under a microscope, and when brown-yellow color appears, rinse with tap water to stop the color development.

[0229] ⑧Restain the cell nucleus: add hematoxylin solution for about 1 minute, rinse with tap water, turn blue in 0.1% ammonia solution for about 60 seconds, and rinse with double distilled water.

[0230] ⑨ After dehydration, transparency, and sealing, observe under an optical microscope, collect images, analyze and evaluate.

[0231] 6. Experimental Results

[0232] FIG16 is a graph showing daily changes in body weight of mice in each group.

[0233] As can be seen from FIG16 , compared with the blank group, the body weight of the mice in the model group was significantly reduced, but there was no significant change in body weight between the drug-treated groups and the model group.

[0234] FIG17 shows the spleen index of mice in each group.

[0235] As can be seen from Figure 17, compared with the blank group, the spleen index of the model group mice was significantly increased, proving that its pathogenesis is related to immunity; compared with the model group, the spleen index of the LXHK-H group showed a downward trend.

[0236] Figure 18 shows the skin lesions of mice in each group.

[0237] As can be seen from Figure 18, the skin of normal mice did not change during the entire experiment; the model group mice began to develop mild erythema and skin infiltration and hypertrophy on the second day. Over time, the erythema deepened, the infiltration and hypertrophy were obvious, and flaky scales appeared. The skin lesions were most severe on the 6th to 7th day. The erythema, scales, and hypertrophy in the LXHK-H and LXHK-L groups were significantly reduced, and the degree of skin lesions was alleviated. Among them, the improvement effect of the LXHK-H group was more obvious than that of the LXHK-L group. At the same time, compared with the model group, the degree of skin lesions in the LXHK-L and LXHK-H groups was comparable to that of the TG group, indicating that the effects of the present invention's Leifenhaokang and TG on improving the skin lesions of psoriasis mice are comparable.

[0238] Figures 19, 20, 21, and 22 show the scales, thickness, erythema, and cumulative PASI scores of the skin lesions in each group of mice, respectively.

[0239] As can be seen from Figures 19-22, except for the blank group, the PASI scores of mice in each group increased, and the score increase in the model group was the most significant. Among them, the scale score of the LXHK-H group showed a downward trend from the 7th day, and the scale score of the MTX group showed a downward trend from the 8th day, and the scale score of the LXHK-H group was lower than that of the MTX group from the 7th day; the erythema score of the LXHK-H group and the MTX group showed a downward trend from the 8th day, and the erythema score of the LXHK-H group was lower than that of the MTX group from the 7th day; the cumulative score of the LXHK-H group and the MTX group showed a downward trend from the 8th day, and the cumulative score of the LXHK-H group was lower than that of the MTX group from the 6th day; the scores of the other groups continued to increase, and the scale, thickness, erythema and cumulative PASI score of the LXHK-H group were lower than those of the TG group from the 8th day, indicating that the effect of the present invention's Leirenhaokang on improving scales, thickness, erythema and cumulative PASI scores of skin lesions in psoriasis mice is better than that of tripterygium wilfordii polyglycosides.

[0240] FIG23 shows the average thickness of the skin at the lesion site of mice in each group.

[0241] As can be seen from Figure 23, the epidermal thickness of the LXHK-H, LXHK-L and TG groups was at the same level, and there was a statistical difference compared with the model group (P < 0.01), indicating that the pharmacological effect of the present invention, Leifenhaokang, and TG in reducing the skin thickness of psoriasis mice was consistent.

[0242] FIG24 shows the distribution of subcutaneous blood vessels in the lesion area of ​​mice in each group.

[0243] As can be seen from Figure 24, compared with the blank group mice, the number of blood vessels in the skin tissue of the model group increased significantly, and the vascular tortuosity increased; compared with the model group, except for the MTX group with a larger number of blood vessels and tortuosity, the number of blood vessels in the mice of each drug-treated group decreased, and the tortuosity was significantly improved.

[0244] FIG25 shows the pathological changes of skin lesions on the back of mice in each group (HE staining, 200X).

[0245] As shown in Figure 25, the blank group showed no significant epidermal hyperplasia, a thin stratum corneum, flat epidermal projections, a small amount of chronic inflammatory cell infiltration in the dermis, and no significant vasodilation. The model group showed epidermal hyperplasia and hyperkeratosis, with numerous neutrophil infiltrations in the epidermis and stratum corneum, loss or thinning of the stratum granulosum, dilated blood vessels in the superficial dermis, and scattered or focal infiltration of chronic inflammatory cells (Note: In the figure, green arrows indicate microabscesses formed by aggregated neutrophils in the stratum corneum; yellow arrows indicate hyperkeratosis; orange arrows indicate epidermal thickening; blue arrows indicate acanthosis; black arrows indicate loss of the stratum granulosum; red arrows indicate mononuclear or multinuclear cell infiltration in the dermis; and gray arrows indicate capillary dilation). Compared with the model group, the degree of epidermal hyperplasia and parakeratosis in all treatment groups was significantly reduced, with a decrease in the number of neutrophils in the epidermis and stratum corneum, and inflammatory cell infiltration in the superficial dermis, as well as a decrease in dilated blood vessels in the dermis. Compared with the model group, the degree of lesion reduction in the LXHK-H and LXHK-L groups was comparable to that in the TG group, indicating that the pharmacological effects of the present invention and TG in reducing pathological changes in the skin of psoriasis mice were consistent.

[0246] FIG26 shows the expression of PCNA in the skin tissues of mice in each group.

[0247] As can be seen from Figure 26, compared with the blank group, the PCNA level in the model group was significantly increased; compared with the model group, the PCNA expression levels in each drug-treated group were decreased, among which the expression decreases were most obvious in the TG group and the LXHK-H group.

[0248] Figures 27, 28 and 29 respectively show the changes in liver function and liver index of mice in each group.

[0249] As can be seen from Figure 27, compared with the blank group, the ALT level in the model group showed an upward trend; compared with the model group, the ALT in the TG group and the LXHK-L group decreased; the ALT in the TG+ART group, the TG+AT group and the LXHK-H group increased, and the ALT level in the MTX group increased significantly, indicating obvious hepatotoxicity.

[0250] As can be seen from Figure 28, compared with the blank group, the AST level in the model group showed an upward trend; compared with the model group, the AST in the TG group showed no significant change, but the AST in the LXHK-L group showed a downward trend after administration.

[0251] As can be seen from Figure 29, compared with the blank group, the liver index of the model group increased significantly, which was statistically significant; compared with the model group, except for the liver index of the MTX group which was equivalent to the model group, the liver index of each drug-treated group showed a downward trend.

[0252] FIG30 shows the changes in kidney index of mice in each group.

[0253] As can be seen from Figure 30, compared with the blank group, the renal index of the model group increased significantly, which was statistically significant; compared with the model group, except for the renal index of the MTX group which was equivalent to the model group, the renal index of each drug-treated group showed a downward trend.

[0254] Figures 31 and 32 show the organ indexes and morphological changes of the ovaries and uteri of mice in each group.

[0255] As can be seen from Figures 31 and 32, compared with the blank group, the organ indexes of the ovary and uterus in the model group were significantly reduced, the volume of the ovary and uterus became smaller, and the morphology was atrophied; compared with the model group, the ovary and uterine indexes of the TG group were even lower, while the ovary and uterine indexes of the TG+ART, TG+AT, LXHK-L, LXHK-H and MTX groups recovered to a certain extent after administration, and the morphology of the ovary and uterus also recovered significantly.

[0256] In summary, for imiquimod-induced psoriasis mice, the present invention's Leirenhaokang can, to a certain extent, inhibit the body's immune response, reduce inflammatory cell infiltration, reduce PCNA protein expression, alleviate the production of scales and skin keratinization in the lesion area, reduce erythema, and reduce subcutaneous angiogenesis. Among them, the effects of the present invention's Leirenhaokang and TG in improving the skin lesions of psoriasis mice are comparable; the effect of the present invention's Leirenhaokang in improving the scales, thickness, erythema and cumulative PASI score of the skin lesions of psoriasis mice is better than that of tripterygium wilfordii polyglycosides; the present invention's Leirenhaokang and TG have the same pharmacodynamic effect of reducing the thickness of the skin of psoriasis mice; the present invention's Leirenhaokang and TG have the same pharmacodynamic effect of reducing pathological changes in the skin of psoriasis mice; the present invention's Leirenhaokang and TG have the same effect of reducing the expression of PCNA protein. At the same time, compared with tripterygium wilfordii polyglycosides, the present invention's Leirenhaokang has a certain degree of inhibitory effect on the liver and reproductive system toxicity that occurs during the treatment of psoriasis.

Claims

1. Artemisia selengensis, characterized in that The raw materials and their weight proportions are: 10 parts of tripterygium wilfordii polyglycosides and 0.1-1.3 parts of artemisia annua extract.

2. The Artemisia selengensis according to claim 1, characterized in that The mass ratio of artemisinin to artemisinin in the Artemisia annua extract is (0.8-1.2):(0.8-1.2), and the mass ratio of artemisinin to artemisinin in the Artemisia annua extract is 85-95%.

3. The Artemisia selengensis according to claim 2, characterized in that The mass ratio of artemisinin to artemisinin in the Artemisia annua extract is 1:

1.

4. The artemisia selengensis according to any one of claims 1 to 3, characterized in that The artemisia annua extract is obtained by mixing a first artemisia annua extract with an artemisinin purity of 85-98% and a second artemisia annua extract with an artemisinin purity of 85-98% in a mass ratio of (0.8-1.2):(0.8-1.2).

5. The Artemisia selengensis according to claim 4, characterized in that The preparation method of the first Artemisia annua extract comprises the following steps: (1) Weigh Artemisia annua medicinal material, crush it, add 10-15 times the amount of petroleum ether, soak it for 1-2 hours, heat it to 60-65°C, reflux it for 1-2 hours, extract it 2-3 times, combine the extracts, and evaporate the solvent under reduced pressure to obtain Artemisia annua extract; (2) Mixing the Artemisia annua extract with 1-2 times the amount of 200-300 mesh silica gel, dry-packing the column with 10-20 times the amount of 200-300 mesh silica gel, eluting with petroleum ether-ethyl acetate, collecting the artemisinin-containing fractions, combining them, and recovering the solvent by vacuum rotary evaporation to obtain a mixture containing artemisinin; (3) Repeat the operation of step (2) 2 to 3 times on the obtained mixture to obtain a first Artemisia annua extract with an artemisinin purity of 85 to 95%.

6. The method for preparing the Leishihaokang according to any one of claims 1 to 5, characterized in that: Just mix the raw materials according to the ratio.

7. Use of the Leixihaokang according to any one of claims 1 to 5 in the preparation of a drug for treating nephrotic syndrome.

8. Use of the Leixihaokang according to any one of claims 1 to 5 in the preparation of drugs for treating immunosuppressive diseases.

9. The use according to claim 8, characterized in that The immunosuppressive disease is psoriasis.

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