Brucea javanica A derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an acetylated derivative of brucein A as well as a preparation method and application of the acetylated derivative. The molecular formula of an acetylated derivative P1 of brucein A is C32H40O14, and the molecular formula of the acetylated derivative P1 is C32H40O14. And the molecular weight of the polymer is 648.65800. By accurately controlling the reaction conditions of bruceine A and acetic anhydride, acetylation of hydroxyl groups at C-3, C-11 and C-12 sites in bruceine A molecules is successfully realized, and an acetylated derivative P1 of bruceine A is generated. According to the chemical modification process, the in-vivo toxicity of bruceine A is reduced, meanwhile, the original biological activity of bruceine A is reserved, and the safe administration dosage range of bruceine A is widened. The brucein A acetylated derivative P1 is used for treating chronic nephritis, has remarkable anti-inflammatory and anti-fibrosis effects, and effectively reduces urine protein of chronic nephritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an acetylated derivative of brucein A and a preparation method and application thereof. Background Art

[0002] Chronic nephritis is one of the most common chronic kidney diseases in the world. It has a certain incidence in both developed and developing countries, and the incidence has been on the rise in recent years. Due to the insidious onset of chronic nephritis, some patients have no obvious symptoms in the early stages of the disease, and the actual incidence data may be underestimated. Although renal replacement therapy can save lives, the aging of society has brought an unbearable economic burden to families, especially in middle- and low-income countries, where patients often find it difficult to afford the expensive treatment costs. At present, the main clinical treatment drugs for chronic nephritis include angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor antagonists (ARB), which can lower blood pressure and reduce urine protein. Glucocorticoids and immunosuppressants can inhibit immune responses. There are also diuretics used to reduce edema. These drugs are used in combination to control the disease, but these treatments have different shortcomings and adverse reactions, and cannot completely prevent chronic nephritis from developing into end-stage renal disease. Therefore, it is of great clinical value to discover new effective means of treating chronic nephritis.

[0003] Bruceine A (BA), also known as bruceine A, is a quassinolide compound extracted and separated from the fruit of the plant Brucea brucea. It is reported that bruceine A can be used to treat chronic nephritis, but its high biological toxicity has hindered its widespread clinical therapeutic development.

[0004] Bruceine A has a high biological toxicity, which limits its application and further research and development in organisms. It has been reported that the median lethal concentration (LC50) of Bruceine A in goldfish is 5.17 mg / L. At this concentration, half of the goldfish are expected to die within 48 hours.

[0005] In the literature on the use of brucein A to treat chronic nephritis, the dosage is 2 mg / kg, administered intraperitoneally every two days, and it takes more than 8 weeks of administration to see a significant decrease in urine protein. Due to the toxicity of the drug, the dosing interval is long and the treatment time is long. Therefore, the development of a low-toxic brucein A derivative P1 is necessary and has broad application value. Summary of the invention

[0006] The present invention aims at an acetylated derivative of brucein A and a preparation method and application thereof.

[0007] An acetylated derivative P1 of brucein A has a structure shown in Formula I:

[0008]

[0009] A method for preparing the acetylated derivative P1 of brucein A, the method comprising the following steps:

[0010] S1. Weigh brucein A and DMAP in a reaction flask, add dichloromethane, dropwise pyridine and acetic anhydride, and stir;

[0011] S2, after the reaction in the reaction bottle of S1 is completed, dilute the solution with ethyl acetate, and extract with 1M HCl, saturated NaHCO3, and saturated NaCl in sequence;

[0012] S3. After the extraction of S2 is completed, the mixture is dried with a desiccant, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain the acetylated derivative P1 of brucein A.

[0013] In step S3, the desiccant is Na2SO4

[0014] In step S1, the mass ratio of brucein A to the catalytic amount of DMAP is 11:5.

[0015] Preferably, the amount of DMAP used is 15 mg, 0.12 mmol; the amount of pyridine used is 0.5 mL; and the amount of acetic anhydride used is 0.5 mL, 5.0 mmol.

[0016] Preferably, the reaction time is 20 h.

[0017] Preferably, the reaction temperature is between 15°C and 30°C.

[0018] The acetylated derivative P1 of brucein A is used for treating chronic nephritis.

[0019] The acetylated derivative P1 of brucein A is used for reducing urine protein in chronic nephritis.

[0020] The acetylated derivative P1 of brucein A has significant anti-inflammatory and anti-fibrosis effects. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. An acetylated derivative P1 of brucein A. P1 is chemically modified to acetylate the hydroxyl groups at the C-3, C-11, and C-12 positions in the brucein A molecule, significantly reducing its in vivo toxicity. This enables patients to better tolerate the drug during treatment, reduces adverse reactions caused by drug toxicity, and improves the safety and compliance of treatment. At the same time, P1 retains the original biological activity of brucein A.

[0022] 2. The acetylated derivative P1 of brucein A can be used to treat chronic nephritis. The acetylated derivative P1 of brucein A has a significant anti-inflammatory effect. It can effectively inhibit the inflammatory response of the kidney, reduce the infiltration of inflammatory cells and the release of inflammatory mediators, thereby protecting the kidney tissue from inflammatory damage. In addition, P1 also exhibits excellent anti-fibrotic effects, which can inhibit the excessive deposition of extracellular matrix in the process of renal fibrosis, slow down the progression of renal fibrosis, and effectively maintain the normal structure and function of the kidney. The acetylated derivative P1 of brucein A can effectively reduce the urine protein of chronic nephritis. It has a positive protective effect on the renal function of patients with chronic nephritis, can reduce the excretion of proteinuria, delay the progression of kidney disease, and improve the quality of life and prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 or the description of the prior art 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.

[0024] Figure 1 is the chemical structural formula of brucein A derivative P1;

[0025] Figure 2 is the chemical reaction formula for the preparation of brucein A derivative P1;

[0026] Figure 3 HNMR spectrum of brucein A derivative P1;

[0027] Figure 4 HNMR spectrum of brucein A derivative P1;

[0028] Figure 5 The effect of brucein A derivative P1 on the mRNA expression of inflammatory factors such as IL-6 and Ccl2 and fibrosis-related factors such as Fn1, Col4a1, and Tgf-β in mesangial cells induced by high glucose;

[0029] Figure 6 HE staining pictures of the heart, spleen, lung and kidney in the acute toxicity experiment of brucein A derivative P1 in vivo;

[0030] Figure 7 The effect of brucein A derivative P1 on urinary protein in mice treated for 0, 2, and 4 weeks;

[0031] Figure 8 PAS staining of the effect of brucein A derivative P1 on renal pathology in mice treated for 4 weeks;

[0032] Fig. 9 This is an electron micrograph of the effect of brucein A derivative P1 on renal pathology in mice after administration for 4 weeks. DETAILED DESCRIPTION

[0033] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0034] The following is combined with Figure 1 To Attachment Fig. 9 And specific embodiments, the present invention is described in detail:

[0035] like Figures 1 to 9 As shown, the object of the present invention is to provide an acetylated derivative of brucein A and a preparation method and application thereof.

[0036] The purpose of the present invention is achieved through the following technical solutions:

[0037] An acetylated derivative P1 of brucein A has a structure shown in Formula I:

[0038]

[0039] Among them, the molecular formula of the acetylated derivative P1 of brucein A is: 32 H 40 O 14 ; Molecular weight: 648.65800.

[0040] Embodiment 1:

[0041] A method for preparing a brucein A derivative P1:

[0042] Bruceine A (33 mg, 0.063 mmol) and a catalytic amount of DMAP (15 mg, 0.12 mmol) were weighed into a reaction flask, 1 mL of dichloromethane was added as a solvent, pyridine (0.5 mL) and acetic anhydride (0.5 mL, 5.0 mmol) were added dropwise, and stirred at room temperature for 20 h. The reaction temperature was between 15°C and 30°C, and the optimal temperature was 25°C. After the reaction was completed, the solution was diluted with 30 mL of ethyl acetate, and extracted with 1 M HCl (20 mL × 3), saturated NaHCO3 (20 mL × 3), and saturated NaCl (20 mL × 3) in sequence. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography.

[0043] The Bruceine A (BA) described in the present invention is a quassin compound extracted from the dried fruit of Brucea javanica, CAS: 25514-31-2, with a molecular formula of C 26 H 34 O 11 , molecular weight 522.53, contains an oxygen-containing heterocycle and a lactone ring, and the chemical structure is shown in the figure. The present invention has no special limitation on the sources of brucein and other chemical reagents, and conventional commercial materials in the art can be used.

[0044] The reaction formula of the above preparation process is as follows Figure 2 shown.

[0045] Among them, the C-3, C-11, and C-12 hydroxyl groups of brucein A were acetylated by acetic anhydride under the catalysis of DMAP, DCM, and pyridine. The specific structure is shown below:

[0046]

[0047] The HNMR spectrum of brucein A derivative P1 is shown in the attached figure. Figure 3 As shown, the NMR data are as follows:

[0048] 1 HNMR (600MHz, CDC l3 )δ6.14(s,1H),5.32(s,1H),5.21(d,J=3.4Hz,1H),4.83(s,1H),4.75(d,J=7.9Hz,1H),3.83(d, J=8.0Hz,1H),3.75(s,3H),3.06(d,J=12.7Hz,1H),2.63(d,J=16.0Hz,1H),2.39(t,J=16.6Hz,2 H),2.29(s,1H),2.25(s,3H),2.23(d,J=7.0Hz,1H),2.17(dd,J=15.1,7.2Hz,1H),2.12(s,3H), 2.06(m,1H),2.04(s,3H),1.81(m,1H),1.80(d,J=1.6Hz,3H)1.30(s,3H),0.98(t,J=6.4Hz,6H).

[0049] The HNMR spectrum of brucein A derivative P1 is shown in the attached figure. Figure 4 As shown, the NMR data are as follows:

[0050] 13 CNMR(151MHz,CDC l3)δ187.95,171.48,168.87,168.84,168.69,167.78,166.50,144.91,142.43,82.35,80.00,73.63,71.05,69.16,65. 83,53.03,51.38,50.27,45.13,42.99,42.85,41.36,40.37,28.80,25.57,22.55,21.59,20.82,20.33,15.78,14.62.

[0051] As one of the main causes of chronic nephritis in China, diabetic nephropathy poses a serious threat to the health of patients and also places a heavy burden on the medical system. Therefore, exploring effective treatment methods has important clinical significance and social value. This study took diabetic nephropathy (diabetic kidney) as the research object and conducted animal experiments to deeply explore the therapeutic effect of the drug brucein A derivative P1 on chronic nephritis and provide support for its clinical application.

[0052] Example 2

[0053] Anti-inflammatory and anti-fibrotic effects of brucein A derivative P1 in the treatment of chronic kidney disease cell models.

[0054] IL-6 (interleukin-6): is a multifunctional cytokine that plays a key role in inflammatory responses. It can be produced by a variety of cells, such as macrophages, T cells, B cells, etc. Elevated IL-6 is usually associated with acute and chronic inflammation, and is often elevated in some autoimmune diseases, infectious diseases and tumors.

[0055] Ccl2 (chemokine ligand 2): mainly involved in the recruitment and migration of inflammatory cells. It can attract inflammatory cells such as monocytes and macrophages to the inflammatory site, thereby promoting the development of inflammatory response.

[0056] Tgf-β (transforming growth factor-β): plays a core role in the process of fibrosis. It can promote the synthesis of extracellular matrix and inhibit the activity of matrix-degrading enzymes, thus leading to the accumulation of extracellular matrix and the occurrence of fibrosis.

[0057] Fn1 (fibronectin 1): It is an important component of the extracellular matrix and plays an important role in tissue repair and fibrosis. It can promote cell adhesion, migration and proliferation, and can interact with other extracellular matrix components to form a stable fiber network.

[0058] Col4a1 (type IV collagen α1 chain): is one of the main components of the basement membrane and plays an important role in maintaining the structure and function of tissues. During fibrosis, the expression of Col4a1 usually increases, leading to basement membrane thickening and tissue fibrosis.

[0059] Celastrol is a natural triterpenoid compound with multiple biological activities. Celastrol can inhibit the production and release of multiple inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), thereby reducing inflammatory responses.

[0060] (1) Experimental plan: Rat mesangial cells HBZY-1 were purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences. The cells were seeded in a six-well plate at 1.5*105 cells / well. After the cells adhered to the wall, they were synchronized with 5.6mM glucose and serum for 24 hours and then divided into a normal control group, a model group (35mM glucose), a positive drug control group (100nM Celastrol, Cel), 100nM Bruceine A (BA), and a low, medium, and high concentration P1 administration group (5μM, 10μM, and 20μM, respectively). After 2 hours of pre-administration, the cells were administered with 35mM glucose for 48 hours of stimulation and intervention. Inflammatory fibrosis-related indicators such as IL-6, Ccl2, Tgf-β, Fn1, and Col4a1 were detected by RT-qPCR.

[0061] (2) Experimental results: P1 can inhibit the mRNA expression of inflammatory factors such as IL-6 and Ccl2 and fibrosis-related factors such as Fn1, Col4a1, and Tgf-β in vitro experiments ( Figure 5 ).

[0062] Experimental results:

[0063] Effects of brucein A derivative P1 on the mRNA expression of inflammatory factors such as IL-6 and Ccl2 and fibrosis-related factors such as Fn1, Col4a1, and Tgf-β in mesangial cells induced by high glucose (HG) Figure 5 As shown:

[0064] In the RT-qPCR detection of Ccl2 inflammatory fibrosis related indicators, the expression level of Ccl2 in the normal control group was the lowest, and the expression level of Ccl2 in the model group (35mM glucose) was the highest. The control group (Celastrol, Cel) had the best anti-inflammatory effect, followed by Bruceine A (BA). The three groups of low, medium and high concentrations of bruceine A derivative P1, 5μM, 10μM and 20μM, all had anti-inflammatory effects. As the concentration of bruceine A derivative P1 increased, its anti-inflammatory effect became better. When used at a concentration of 20μM, its anti-inflammatory effect was equivalent to that of 100nM Bruceine A (BA).

[0065] In the RT-qPCR detection of IL-6 inflammatory fibrosis related indicators, the expression level of IL-6 in the normal control group was the lowest, and the expression level of IL-6 in the model group (35mM glucose) was the highest. Bruceine A (BA) had a good anti-inflammatory effect, and the control group (Celastrol, Cel) also had a certain anti-inflammatory effect. The three groups of low, medium and high concentrations of bruceine A derivative P1 were 5μM, 10μM, and 20μM, all of which had anti-inflammatory effects. As the concentration of bruceine A derivative P1 increased, its anti-inflammatory effect became better. When used at a concentration of 20μM, its anti-inflammatory effect was better than that of bruceine A (BA) 100nM, and 20μM bruceine A derivative P1 had the best anti-inflammatory effect.

[0066] In the RT-qPCR detection of Tgf-β inflammatory fibrosis related indicators, the normal control group had the lowest expression level of Tgf-β, and the model group (35mM glucose) had the highest expression level of Tgf-β. The control group (Celastrol, Cel) and Bruceine A (BA) had equivalent anti-inflammatory effects. The three groups of low, medium and high concentrations of bruceine A derivative P1, 5μM, 10μM and 20μM, all had anti-inflammatory effects. As the concentration of bruceine A derivative P1 increased, its anti-inflammatory effect became better. At a concentration of 20μM, its anti-inflammatory effect was slightly worse than that of Bruceine A (BA) 100nM.

[0067] In the RT-qPCR detection of Fn1 inflammatory fibrosis related indicators, the expression level of Fn1 in the normal control group was the lowest, and the expression level of Fn1 in the model group (35mM glucose) was the highest. Bruceine A (BA) had the best anti-inflammatory effect, followed by the anti-inflammatory effect. The low and high concentrations of 5μM, 10μM, and 20μM of the three groups of bruceine A derivative P1 all had anti-inflammatory effects. As the concentration of bruceine A derivative P1 increased, its anti-inflammatory effect became better. When used at a concentration of 20μM, its anti-inflammatory effect was slightly better than that of the control group (Celastrol, Cel).

[0068] In the RT-qPCR detection of Col4a1 inflammatory fibrosis related indicators, the expression level of Col4a1 in the normal control group was the lowest, and the expression level of Col4a1 in the model group (35mM glucose) was the highest. Bruceine A (BA) had the best anti-inflammatory effect, followed by Bruceine A derivative P1. The three groups of low, medium and high concentrations of 5μM, 10μM and 20μM all had anti-inflammatory effects. The anti-inflammatory effects of Bruceine A derivative P1 were equivalent when the concentrations were 5μM and 10μM, and the anti-inflammatory effect was best when the concentration was 20μM, and its anti-inflammatory effect was slightly better than that of the control group (Celastrol, Cel).

[0069] In summary, the brucein A derivative P1 showed obvious anti-inflammatory effects in multiple inflammatory fibrosis-related indicators, and at higher concentrations (20 μM), the anti-inflammatory effects in different indicators were outstanding, showing obvious anti-inflammatory effects.

[0070] Embodiment 3:

[0071] In vivo acute toxicity test of brucein A derivative P1:

[0072] 1. Animal husbandry and grouping

[0073] This experiment used 7 to 9 weeks old C57 mice, all of which were SPF grade. These mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd. The experimental mice were placed in the experimental animal barrier system of the Guangdong Provincial Institute of Traditional Chinese Medicine for breeding. During the breeding process, the mice were provided with ordinary feed, and they were allowed to eat and drink freely. In addition, the breeding environment maintained 12 hours of alternating lighting every day to create a suitable life rhythm for the mice. This experiment has been successfully approved by the Animal Experiment Ethics Committee, which fully reflects the scientificity and standardization of the experiment.

[0074] A total of 20 C57 mice participated in the experiment, half of them were male and half were female. Before the formal experiment began, these mice were fed adaptively for 7 days. Subsequently, the mice were randomly divided into 4 groups of 5 mice each according to gender and weight. The specific groups were solvent female control group, P1-administered female group, solvent male control group and P1-administered male group.

[0075] 2. Dosage regimen

[0076] The mice in the control group were intraperitoneally injected with the corresponding volume of 10% DMSO + 40% PEG400 + 5% Tween80 + 45% saline. The mice in the drug administration group were given 300 mg / kg of P1 by a single intraperitoneal injection. After administration, the mice were observed for 14 consecutive days to fully evaluate the effects of P1 on the mice.

[0077] 3. Experimental Results

[0078] After administration, the mice in the experimental group showed a series of normal physiological characteristics. First, breathing was normal and the body color did not change. Secondly, although the movement was reduced, it returned to normal after about 10 minutes, and there was no abnormal movement. In addition, there was no convulsion, reflexes were normal, eyelid indications were normal, saliva secretion was normal, and there was no erection of hair. At the same time, defecation was normal, and there were no adverse symptoms such as vomiting, hematuria, and urinary incontinence. The mice in the control group also showed reduced movement after being given the solvent, but returned to normal after about 4 minutes, and there were no abnormal changes.

[0079] Attached Figure 6 This is a HE staining picture of the heart, liver, lung, spleen, and kidney in the acute toxicity experiment of the brucein A derivative P1 in vivo. There is no significant difference in the cells of the heart, liver, lung, spleen, and kidney between the C57+Veh and C57+P1 groups, indicating that there is no significant difference in the effects of the P1 and Veh groups.

[0080] The experimental results fully show that no death occurred in C57 mice when the drug was administered at a concentration of 300 mg / kg. In addition, HE staining images of the heart, liver, lung, spleen, and kidney of C57 mice were analyzed (see Appendix). Figure 6 ), it can be seen that P1 has no obvious damage to these organs and no obvious toxic side effects.

[0081] Embodiment 4:

[0082] In vivo efficacy study of Brucea brucea A derivative P1 in the treatment of chronic nephritis

[0083] (1) Animal husbandry and grouping:

[0084] This experiment used 7 to 8-week-old male db / db spontaneous diabetic nephropathy mice and db / m normal control mice, all of which were SPF grade and purchased from Changzhou Cavens Experimental Animal Co., Ltd. The experimental mice were housed in the experimental animal barrier system of the Guangdong Provincial Hospital of Traditional Chinese Medicine. During the feeding process, the mice were provided with high-fat feed, while ensuring that they could eat and drink freely. In addition, the breeding environment maintained 12 hours of alternating lighting every day to create a suitable life rhythm for the mice. This experiment has been successfully approved by the Animal Experiment Ethics Committee, which fully reflects the scientificity and standardization of the experiment.

[0085] A total of 36 male db / db mice and 6 db / m normal control mice participated in the experiment. Before the formal experiment began, these mice were adaptively fed for 7 days. Subsequently, the db / db mice were randomly divided into 6 groups according to their body weight, with 6 mice in each group. The specific groups were model group, brucein A control group, tripterygium wilfordii positive control group, brucein A derivative P1 low-dose administration group, brucein A derivative P1 medium-dose administration group and brucein A derivative P1 high-dose administration group.

[0086] (2) Dosage regimen:

[0087] The mice in the normal control group and the model group were intraperitoneally injected with the corresponding volume of normal saline. The mice in the low, medium and high dose groups of brucein A derivative P1 were intraperitoneally injected with 10 mg / kg, 20 mg / kg and 40 mg / kg of P1 solution, respectively, once every other day for 4 consecutive weeks. The mice in the brucein A control group were intraperitoneally injected with 2 mg / kg of brucein A solution, once every 2 days, for 4 consecutive weeks. The mice in the tripterygium wilfordii positive control group were intraperitoneally injected with 0.5 mg / kg of tripterygium wilfordii, which was administered every day for 4 consecutive weeks.

[0088] (3) Testing indicators:

[0089] Urine microalbumin to creatinine ratio:

[0090] Urine microalbumin-to-creatinine ratio is an important medical test indicator. Urine microalbumin refers to a small amount of albumin that appears in the urine. Under normal circumstances, the albumin content in urine is extremely low. When the kidneys are damaged or diseased, the filtration function of the glomeruli is impaired, which will lead to an increase in the content of microalbumin in the urine. Creatinine is a product of muscle metabolism and is usually excreted by the kidneys. Since the generation of creatinine is relatively stable and almost all of it is excreted by the kidneys, the concentration of urine creatinine can reflect the excretion function of the kidneys. Urine microalbumin-to-creatinine ratio (UACR) is the ratio of microalbumin to creatinine in urine. By detecting this ratio, early kidney damage can be more accurately assessed. Compared with the detection of urine microalbumin or urine creatinine alone, UACR can reduce the influence of individual differences and other factors and improve the accuracy and reliability of the test.

[0091] Mouse metabolic cages were used to collect 8-hour urine before administration, 2 weeks after administration, and 4 weeks after administration. The mice were placed in metabolic cages for 8 hours, during which they were fasted but not watered. After collecting urine for 8 hours, the urine volume was recorded. Subsequently, a fully automatic biochemical instrument was used to detect changes in the urine microalbumin-creatinine ratio. This test indicator can more accurately reflect the changes in the renal function of mice, which is of great significance for evaluating the therapeutic effect of the drug.

[0092] Renal Pathology:

[0093] PAS staining and electron microscopy were used to observe the improvement of mouse kidney pathology

[0094] (4) Experimental results

[0095] Attached Figure 7 The effect of brucein A derivative P1 on urinary protein in mice administered for 0, 2, and 4 weeks. Among them, db / m is a normal control, db / db is a model control group, db / db+cel is a tripterygium positive control group (tripterygium 0.5 mg / kg), db / db+BA is a brucein A control group (brucein A 2 mg / kg), db / db+P1 L is a brucein A derivative P1 low-dose group (brucein A derivative P1 10 mg / kg), db / db+P1 L is a brucein A derivative P1 medium-dose group (brucein A derivative P1 20 mg / kg), and db / db+P1 L is a brucein A derivative P1 high-dose group (brucein A derivative P1 40 mg / kg).

[0096] Starting from the second week of administration, the urine protein in the tripterygium wilfordii positive control group (db / db+cel), brucei brucei A derivative P1 low-dose group (db / db+P1 L), P1 medium-dose group (db / db+P1 M), and P1 high-dose group (db / db+P1 H) was significantly reduced compared with the Model group (db / db). There were dose-effect differences in the P1 low-, medium-, and high-dose groups, and the therapeutic effect on reducing urine protein increased with the increase in administration time.

[0097] Attached Figure 8 PAS staining of the effect of brucein A derivative P1 on renal pathology in mice after 4 weeks of administration:

[0098] After careful observation, it was found that in the PAS staining results of pathological evaluation 4 weeks after administration, the model group showed obvious proliferation of mesangial cells and matrix, and the opening of capillary loops was poor. However, after the administration of brucein A derivative P1, the proliferation of mesangial cells and matrix was significantly improved.

[0099] Attached Fig. 9 Electron micrograph of the effect of brucein A derivative P1 on renal pathology in mice after 4 weeks of administration:

[0100] The electron microscope showed that the model group had problems such as proliferation of mesangial cells and matrix, poor opening of capillary loops, segmental fusion of podocyte foot processes, and irregular segmental thickening of a few basement membranes. At the same time, the mitochondria of renal tubular epithelial cells were neatly arranged but slightly swollen. After the administration of brucein A derivative P1, the proliferation of mesangial cells and matrix was significantly improved, the opening of capillary loops was acceptable, most of the podocyte foot processes were well preserved, and only a few showed staged fusion. Most of the mitochondria of renal tubular epithelial cells were neatly arranged or slightly less neat, and no swelling was observed.

[0101] In summary, the experimental results fully demonstrate that brucein A derivative P1 has many important functions. It can effectively protect the kidneys, reduce urine protein levels, significantly inhibit the proliferation of mesangial cells and matrix, greatly improve the opening of capillary loops, and also has a good protective effect on podocytes.

[0102] According to Example 2, Example 3 and Example 4, it is concluded that the acetylated derivative of brucein A P1 has good anti-inflammatory and anti-fibrosis effects, has no obvious toxic side effects in vivo, effectively reduces urine protein, and improves kidney pathology, indicating that the brucein A derivative P1 has broad prospects in the application of treating chronic nephritis.

[0103] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only optional examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An acetylated derivative P1 of brucein A, characterized in that: It has the structure shown in formula I:

2. A method for preparing the acetylated derivative P1 of brucein A according to claim 1, characterized in that: The method comprises the following steps: S1. Weigh brucein A and DMAP in a reaction flask, add dichloromethane, dropwise pyridine and acetic anhydride, and stir; S2, after the reaction in the reaction bottle of S1 is completed, dilute the solution with ethyl acetate, and extract with HCl, saturated NaHCO3, and saturated NaCl in sequence; S3. After the extraction of S2 is completed, the mixture is dried with a desiccant, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain the acetylated derivative P1 of brucein A.

3. The method for preparing the acetylated derivative P1 of brucein A according to claim 2, characterized in that: In step S3, The desiccant is Na2SO4.

4. The method for preparing the acetylated derivative P1 of brucein A according to claim 2, characterized in that: In step S1, The mass ratio of brucein A to catalytic amount of DMAP was 11:

5.

5. The method for preparing the acetylated derivative P1 of brucein A according to claim 2, characterized in that: The amount of DMAP used is 15 mg, 0.12 mmol; the amount of pyridine used is 0.5 mL; the amount of acetic anhydride used is 0.5 mL, 5.0 mmol.

6. The method for preparing the acetylated derivative P1 of brucein A according to claim 2, characterized in that: The reaction time is 20h.

7. The method for preparing the acetylated derivative P1 of brucein A according to claim 2, characterized in that: The reaction temperature is between 15°C and 30°C.

8. An acetylated derivative P1 of brucein A as claimed in claim 1 for use in treating chronic nephritis.

9. An acetylated derivative P1 of brucein A as claimed in claim 1 for reducing urine protein in chronic nephritis.

10. An acetylated derivative P1 of brucein A as claimed in claim 1 for use in anti-inflammatory and anti-fibrosis activities.

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