A chemical component for treating nephritis, its preparation process, medical use and composition

Abstract

The present invention relates to a chemical component for treating nephritis, a preparation process, medical use and a composition thereof, wherein the chemical component comprises a compound having a structure of formula I or formula II or a racemate thereof, a cardamom volatile oil containing the compound of formula I and formula II or a cardamom extract containing the compound; the medical use thereof is to treat kidney damage such as acute and chronic nephritis and diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to a new menthane-type monopeptide chemical component (1R, 3R, 4S)-3-methoxy-menthol-1, 3, 7-triol and (1S, 3S, 4R)-3-methoxy-menthol-1, 3, 7-triol having the function of treating nephritis, and volatile oil containing the component, extract, preparation process, medical use and composition thereof. Background Art

[0002] The kidney is an important organ for drug metabolism and excretion, and is the "blood sieve" of the human body. Acute Kidney Injury (AKI), also known as acute renal failure, is a clinical syndrome caused by a rapid decline in renal function due to various causes. It has an acute onset, rapid progression, and a high mortality rate, and seriously threatens human health. AKI can cause serious internal environmental disorders and endanger life in the short term, and can also cause irreversible renal damage and chronic renal failure in the long term, which requires great attention. How to effectively prevent and alleviate acute kidney injury, prevent further deterioration and development of renal injury, find specific therapeutic targets, and develop effective preventive and therapeutic drugs are particularly urgent and important.

[0003] Epidemiological data show that about 13.3 million people suffer from AKI each year, and about 1.7 million people die due to delayed diagnosis and treatment. About 20% to 34% of patients with acute renal failure are related to the use of nephrotoxic drugs. Due to the wide variety of drugs and the serious problem of drug abuse, the number of acute and chronic renal failure caused by drugs is increasing. Drug-induced kidney injury has seriously threatened human health and life. Protecting kidney health has become an urgent task before us.

[0004] Modern medicine does not have specific drugs to treat kidney damage. It mostly uses treatments such as removing the cause, maintaining a stable internal environment, nutritional support, dealing with complications, and blood purification. Currently, the use of herbal medicines and their preparations to treat liver diseases is quite common in my country. Modern pharmacological studies have shown that many Chinese medicines (including compound prescriptions, single herbs, and monomer components) can effectively treat or improve kidney damage. Chinese medicines, with their multi-component, multi-pathway, and multi-target combined effects, have great advantages and broad clinical application prospects in the treatment of acute kidney injury.

[0005] White cardamom is the dried mature fruit of Amomum KraVanh Pjerre ex Gagne P., a plant of the ginger family. White cardamom is an aromatic dehumidifying medicine with the effects of dehumidifying, promoting qi circulation, warming the middle and stopping vomiting. It is mainly used to treat dampness stagnation, spleen and stomach disharmony, abdominal distension, loss of appetite, initial damp-heat, chest tightness without hunger, stomach cold vomiting, and indigestion. The main components of white cardamom volatile oil include 19 compounds such as eucalyptol, α-humulene, β-pinene, α-pinene, α-cuberene, and thymol. Modern pharmacological studies have shown that white cardamom can increase gastric mucosal blood flow, increase serum gastrin levels, and enhance gastric mucosal tissue's resistance to free radical damage, antioxidant effects, mosquito repellent, antioxidant, anti-insomnia, and antibacterial effects. White cardamom is one of the most commonly used Mongolian and Chinese medicines in clinical practice. Mongolian medicine is mainly used for kidney "He Yi", chronic kidney disease, kidney "Dari Gan", qi stagnation in the palace, insomnia, indigestion, urinary retention, wandering pain, kidney deficiency and low back pain.

[0006] However, there are no reports on the research of white cardamom in the treatment of acute kidney injury at home and abroad. Summary of the invention

[0007] The purpose of the present invention is to provide a new menthane-type monopeptide chemical component 3-methoxy-p-menth-1,3,7-triol {(3-methoxy-menth-1,3,7-triol} with the effect of treating nephritis and a volatile oil containing the component, as well as a preparation process, medical use and composition thereof. The medical use thereof is to treat kidney damage such as acute and chronic nephritis and diabetic nephropathy.

[0008] The present invention provides a compound having a structure of formula I or formula II or a racemate thereof having a therapeutic effect on nephritis, wherein the chemical name of the compound of formula I is (1R, 3R, 4S)-3-methoxy-menthol-1, 3, 7-triol; the chemical name of the compound of formula II is (1S, 3S, 4R)-3-methoxy-menthol-1, 3, 7-triol

[0009]

[0010] The present invention also provides white cardamom volatile oil containing compounds of formula I and formula II or their racemates, which is characterized in that the content of 3-methoxy-mentha-1,3,7-triol is 0.1%-99.8%.

[0011] The present invention also provides a white cardamom extract containing compounds of formula I and formula II or their racemates, characterized in that the content of 3-methoxy-mentha-1,3,7-triol in the extract is 0.1%-99.8%.

[0012] The present invention also provides a pharmaceutical composition containing the compounds of formula I and formula II or their racemates, or containing white cardamom volatile oil, or containing white cardamom extract, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, pills, powders, ointments, oral liquids, gels and injections.

[0013] The white cardamom volatile oil involved in the present application comprises a preparation method comprising extracting the oil using water or an organic solvent, wherein the organic solvent is ethanol, ethyl acetate, and ethanol is the most preferred; the volume percentage concentration of ethanol used is 10%-100%;

[0014] The preparation method of the volatile oil is characterized by steam distillation or supercritical CO2 extraction. The optimal extraction process of steam distillation is preferably an extraction time of 5 hours, a solid-liquid ratio of 1:15, and a soaking time of 8 hours; the optimal extraction process conditions of supercritical CO2 extraction are an extraction pressure of 35MPa, an extraction temperature of 35°C, and a CO2 flow rate of 20Lh -1 .

[0015] The optimal extraction process conditions for supercritical CO2 extraction are extraction pressure of 30-35MPa, extraction temperature of 15-50℃, and CO2 flow rate of 5-100Lh -1 The optimal parameters are: supercritical CO2 extraction The optimal extraction process conditions are extraction pressure of 35MPa, extraction temperature of 30℃, CO2 flow rate of 20Lh -1 The best preparation process for supercritical CO2 distillation is a distillation column with a silica gel filler of 150-250 μM particle size, a temperature distribution of 56-72°C, a CO2 flow rate of 2.5 kg / h, and collection of volatile oils at 15 MPa.

[0016] The preparation method of the white cardamom extract is characterized in that the extract is extracted with water or an organic solvent, refined with a macroporous resin, precipitated, and prepared, wherein the organic solvent for extraction is ethanol, ethyl acetate, and ethanol is the best; the volume percentage concentration of ethanol used is 10%-100%; the macroporous resin process is characterized in that 2-20 times the amount of macroporous resin, the white cardamom extract is wet-loaded by water suspension or dry-mixed, water and low-concentration ethanol are used to elute and remove impurities, and then high-concentration ethanol is used to elute and obtain the extract, wherein the eluent for removing impurities is 0%-50% ethanol, and the best is 5-15% ethanol; the high-concentration eluent is 40%-100% ethanol, optimized to be 60%-95% ethanol, and the best is 65%-95% ethanol.

[0017] The preparation method of 3-methoxy-mentha-1,3,7-triol monomer compound is characterized in that: the prepared white cardamom volatile oil is used as raw material, and is refined by macroporous resin, silica gel column chromatography, ODS column chromatography, and preparative high performance liquid chromatography. It is characterized in that: the silica gel column is separated, and the silica gel column after loading is first eluted with a petroleum ether-ethyl acetate mixed solvent (petroleum ether-ethyl acetate, 100:2, volume ratio) for 5 column volumes, and then eluted with a petroleum ether-ethyl acetate mixed solvent (petroleum ether-ethyl acetate, 100:8, volume ratio) for 10 column volumes, and the petroleum ether-ethyl acetate 100:8 eluent is dried after recovering the organic solvent. Then, it is separated by an ODS chromatographic column, first eluted with 60% methanol water for 4 column volumes, and then eluted with 90% methanol water for 6 column volumes, and the 90% methanol water eluent is dried after recovering the organic solvent, and then the pure product is prepared by preparative liquid chromatography with 88% isocratic methanol water.

[0018] The medical use of the white cardamom volatile oil is characterized by its role in treating acute renal injury, chronic renal injury, and diabetic nephropathy. It is characterized by being able to improve renal injury, regulate inflammatory mediators, reduce serum creatinine, serum urea nitrogen, and urine protein concentrations, inhibit renal tissue cell apoptosis, and improve abnormal lipid peroxidation and oxidative stress reactions in renal tissue caused by nephritis.

[0019] The present invention discloses for the first time that white cardamom volatile oil has a significant protective effect on acute kidney injury in rats induced by gentamicin and 5 / 6 nephrectomy. This study has not been reported at home and abroad, and its results have great practical significance for the development of AKI prevention and treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Gas chromatogram of essential oil from white cardamom prepared by steam distillation

[0021] Figure 2 Gas chromatogram of volatile oil from white cardamom prepared by supercritical CO2 extraction

[0022] Figure 3 Gas chromatogram of volatile oil from white cardamom prepared by supercritical CO2 distillation

[0023] Figure 4 Thin layer chromatogram of monomeric compounds

[0024] Figure 5 The structure and main HMBC relationship of compound 3-methoxy-menthol-1,3,7-triol (“→”: HMBC correlation)

[0025] Figure 6 1 H-NMR nuclear magnetic resonance spectrum

[0026] Figure 713 C-NMR nuclear magnetic resonance spectroscopy

[0027] Figure 8 DEPT NMR spectroscopy

[0028] Fig. 9 HMQC NMR spectroscopy

[0029] Fig.10 HMBC NMR spectroscopy

[0030] Fig.11 1 H- 1 H COSY NMR spectrum

[0031] Fig.12 The effect of white cardamom volatile oil on the kidney appearance morphology of rats. The figure shows A blank control group, B model group, C low-dose volatile oil group, D medium-dose volatile oil group, E high-dose volatile oil group, and F verapamil positive drug group.

[0032] Fig.13 HE staining of rat kidney tissue (200×), A blank control group, B model group, C low-dose volatile oil group, D medium-dose volatile oil group, E high-dose volatile oil group, F verapamil positive drug group.

[0033] Fig.14 Results of MDA content determination in rat renal tissue. **p<0.01, *p<0.05 compared with the model group; #p<0.05 compared with the normal group.

[0034] Fig.15 Results of SOD activity determination in rat kidney tissue. Compared with the model group, *p<0.05; compared with the normal group, #p<0.05.

[0035] Fig.16 Results of GSH-PX activity determination in rat kidney tissue. Compared with the model group *p<0.05; compared with the normal group ##P<0.01.

[0036] Fig.17 Results of NOS activity determination in rat renal tissue. **p<0.01, *p<0.05 compared with the model group; ##P<0.01 compared with the normal group.

[0037] Fig.18 Results of determination of NO content in rat renal tissue. Compared with the model group, *p<0.05; compared with the normal group, #p<0.05.

[0038] Fig.19Effects of white cardamom volatile oil on the expression of NF-кB p65, Caspase-3, Bax, and Bcl-2. In the figure, A is the expression diagram of NF-кB p65, Caspase-3, Bax, and Bcl-2 proteins, B is the quantitative analysis of NF-кB p65 protein expression, C is the quantitative analysis of Caspase-3 protein expression, D is the quantitative analysis of Bcl-2 protein expression, and E is the quantitative analysis of Bax protein expression.

[0039] Fig. 20 Effects of essential oil of cardamom on urine protein, serum creatinine and urea nitrogen levels after 5 / 6 nephrectomy

[0040] Fig.21 Renal histopathology

[0041] Fig. 22 Expression of MCP-1, α-SMA and TGF-β1 in the renal cortex

[0042] Fig.23 Expression of MCP-1, α-SMA and TGF-β1 in renal medulla

[0043] Fig.24 White cardamom can reduce the expression of proteins related to p38MAPK / NF-κB pathway in CKD rats DETAILED DESCRIPTION

[0044] The present invention will be further described below by way of embodiments only in conjunction with the accompanying drawings.

[0045] Example 1 Study on the process of preparing white cardamom volatile oil by steam distillation

[0046] 1. Experimental Methods

[0047] 1.1 Extraction of volatile oil from white cardamom by steam distillation

[0048] Crush the white cardamom with a grinder, weigh 50 g of the crushed white cardamom, put it in a 2000 mL round-bottom flask, add a certain amount of water, connect it to a steam distillation device, continue distillation for a corresponding time, and extract the volatile oil of white cardamom.

[0049] 1.2 Orthogonal experiment

[0050] Taking the yield of volatile oil from white cardamom as an indicator, the extraction time, solid-liquid ratio and soaking time were subjected to three-factor and three-level L9 (3 3 ) Orthogonal experiment was performed to optimize the extraction process. The level of each factor is shown in Table 1.

[0051] Table 1 Orthogonal experimental factor level table

[0052]

[0053] 2. Results and Discussion

[0054] 2.1 Orthogonal experimental results

[0055] According to the orthogonal experiment in 1.2, the extraction process of white cardamom volatile oil was optimized, and the average value and range of each factor level under the investigation index were calculated. The results of the orthogonal experiment are shown in Table 2.

[0056] Table 2 Orthogonal experiment results analysis table

[0057]

[0058] According to the results of the range analysis in Table 2, the soaking time has the greatest impact on the extraction results of the volatile oil of white cardamom, followed by the extraction time, and the solid-liquid ratio has the least impact on the extraction results. The optimal combination obtained through intuitive analysis is A3B2C3, that is, the optimal extraction process conditions for the volatile oil of white cardamom are extraction time of 5h, solid-liquid ratio of 1:15, and soaking time of 8h.

[0059] 2.2 Verification Experiment

[0060] In order to verify the accuracy of the orthogonal experiment, the optimal combination A3B2C3 obtained by the orthogonal experiment was compared with the No. 5 combination A2B2C3 with the highest yield of white cardamom volatile oil in the orthogonal experiment for verification. The verification results are shown in Table 3.

[0061] Table 3 Verification results analysis table

[0062]

[0063] Through comparative verification experiments, it was found that the yield of volatile oil from the optimal combination A3B2C3 obtained by the orthogonal experiment was higher than that of the No. 5 combination A2B2C3 with the highest volatile oil yield in the orthogonal table. This shows that the condition combination A3B2C3 selected by the orthogonal experiment is the optimal extraction condition. Therefore, the optimal extraction process conditions for white cardamom volatile oil are extraction time of 5h, solid-liquid ratio of 1:15, and soaking time of 8h; its gas chromatogram is shown in the figure below. Figure 1 As shown, the monomer concentration of 3-methoxy-menthol-1,3,7-triol was 0.118 mg / ml.

[0064] Example 2 Study on the process of preparing white cardamom volatile oil by supercritical CO2 method

[0065] 1. Experimental Methods

[0066] 1.1 Extraction of volatile oil from white cardamom by supercritical CO2 extraction

[0067] The white cardamom was crushed with a grinder, 100 g of the crushed white cardamom was weighed, and loaded into an extraction kettle for supercritical CO2 extraction, and relevant extraction parameters such as pressure, temperature, and CO2 flow rate during the extraction process were controlled. After the extraction was completed, the white cardamom volatile oil was collected from the collection port at the lower end of the separation kettle, placed in a brown sample bottle, and stored at 4°C.

[0068] 1.2 Orthogonal experiment

[0069] Taking the yield of volatile oil from white cardamom as an indicator, the extraction pressure, extraction temperature and CO2 flow rate were adjusted by three factors and three levels. 3 ) Orthogonal experiment was performed to optimize the extraction process. The level of each factor is shown in Table 1.

[0070] Table 4 Orthogonal experimental factor level table

[0071]

[0072] 2. Results and Discussion

[0073] 2.1 Orthogonal experimental results

[0074] According to the orthogonal experiment in 1.2, the extraction process of white cardamom volatile oil was optimized, and the average value and range of each factor level under the investigation index were calculated. The results of the orthogonal experiment are shown in Table 5.

[0075] Table 5 Orthogonal experiment results analysis table

[0076]

[0077]

[0078] According to the results of the range analysis, the extraction pressure has the greatest impact on the extraction results of the essential oil of white cardamom, followed by the CO2 flow rate, and the extraction temperature has the least impact on the extraction results. The optimal combination obtained through intuitive analysis is A3B2C3, that is, the optimal extraction process conditions for the essential oil of white cardamom are extraction pressure of 35MPa, extraction temperature of 35℃, and CO2 flow rate of 20L·h -1 .

[0079] 2.2 Verification Experiment

[0080] In order to verify the accuracy of the orthogonal experiment, the optimal combination A3B2C3 obtained from the orthogonal experiment was compared with the No. 9 combination A3B3C2 with the highest yield of white cardamom volatile oil in the orthogonal experiment. The verification results are shown in Table 6.

[0081] Table 6 Verification results analysis table

[0082]

[0083] Through comparative verification experiments, it was found that the volatile oil yield of the optimal combination A3B2C3 obtained by the orthogonal experiment was higher than that of the No. 9 combination A3B3C2 with the highest volatile oil yield in the orthogonal table. This shows that the condition combination A3B2C3 screened by the orthogonal experiment is the optimal extraction condition. Therefore, the optimal extraction process conditions for white cardamom volatile oil are extraction pressure of 35MPa, extraction temperature of 35℃, and CO2 flow rate of 20L·h -1 ; Gas chromatogram as Fig. 9 Its gas chromatogram is shown as Figure 1 As shown, the monomer concentration of 3-methoxy-menthol-1,3,7-triol was 0.207 mg / ml.

[0084] Example 3 Preparation of white cardamom volatile oil by supercritical CO2 distillation

[0085] First, add 50g of white cardamom extract into the extraction kettle, set the temperature of the extraction kettle to 50℃ and the temperature range of the distillation column to 56-72℃. After the temperature reaches the set temperature, use a high-pressure pump to press CO2 into the system, maintain the CO2 flow rate at 2.5kg / h, and adjust the separation pressure to 15MPa. Supercritical CO2 flows into the extraction kettle from the bottom to dissolve the raw materials, and carries the raw materials through the distillation column from bottom to top. The separated fraction flows out from the top of the column into the separation kettle, the pressure is relieved, the essential oil components are analyzed, and CO2 is recycled. The gas chromatogram of the prepared white cardamom essential oil is shown as follows: Fig.10 As shown, the monomer concentration of 3-methoxy-menthol-1,3,7-triol was 0.975 mg / ml.

[0086] Example 4 Characteristic Components of White Cardamom Volatile Oil Neomenthane-type Monopeptide Compounds

[0087] Preparation and Characterization of 3-methoxy-p-menth-1,3,7-triol

[0088] The white cardamom was crushed with a grinder, and 100 g of the crushed white cardamom was weighed and loaded into an extraction kettle for supercritical CO2 extraction. The optimal extraction process conditions were extraction pressure of 35 MPa, extraction temperature of 35 °C, and CO2 flow rate of 20 L·h -1. . After the extraction is completed, the white cardamom volatile oil obtained by extraction is separated under reduced pressure from the collecting port at the lower end of the separation kettle, placed in a brown sample bottle, and stored at 4°C. Then the volatile oil is separated by a silica gel column. The silica gel column after loading is first eluted with a petroleum ether-ethyl acetate mixed solvent (petroleum ether-ethyl acetate, 100:2, volume ratio) for 5 column volumes, and then eluted with a petroleum ether-ethyl acetate mixed solvent (petroleum ether-ethyl acetate, 100:8, volume ratio) for 10 column volumes. The petroleum ether-ethyl acetate 100:8 eluate is dried after recovering the organic solvent. Then it is separated by an ODS chromatographic column, first eluted with 60% methanol water for 4 column volumes, and then eluted with 90% methanol water for 6 column volumes. The 90% methanol water eluate is dried after recovering the organic solvent, and then the pure product is prepared by preparative liquid chromatography with 88% isocratic methanol water.

[0089] The monomer component is a light yellow oily substance with no dark spots under ultraviolet light (254nm). Molecular formula: C 11 H 22 O4; GF254 silica gel analytical thin layer, petroleum ether: ethyl acetate = 8:1 development, Rf = 6.1, 10% vanillin-concentrated sulfuric acid showed dark green ( Figure 4 ), 1 H-NMR (Chloroform-d, 400 MHz) and 13 C-NMR (Chloroform-d, 100MHz) data and results are as follows ( Figure 5-11 ):

[0090] 1 H-NMR and 13 C-NMR data showed the following characteristic signals: 4 methyl proton signals: [δH3.36(s,3H),1.24(s,6H),1.05(s,3H);δC,30.9(C-8),28.9(C-9),27.6(C-10),55.1(OCH3)],[1.24(s,6H);30.9(C-8),28.9(C-9)] signals showed that the two methyl groups may be in the same chemical environment; [δH 3.36(s,3H),55.1(OCH3)] signal showed that the methyl group was connected to the oxygen atom; three oxygen-connected quaternary carbon signals [δC,69.8(C-1),97.5(C-3),73.6(C-6)]. Combined with the above data, it was preliminarily inferred that the compound was a menthane-type monoterpene compound. In addition, HMBC results showed that: H-5 / C-7; H-8, H-9 / C-7; H-10 / C-1, C-2, C-6; OCH3 / C-3 were correlated, confirming that the compound was a new menthane-type monoterpene compound: 3-methoxy-p-menth-1,3,7-triol. After chiral column chromatography, it was found that the compound was a partial racemate. -1.6 (c 1.7, CH3OH), including the corresponding isomers of (1R,3R,4S)-3-methoxy-p-menth-1,3,7-triol {(1R,3R,4S)-3-methoxy-menth-1,3,7-triol} and (1S,3S,4R)--3-methoxy-p-menth-1,3,7-triol {(1S,3S,4R)-3-methoxy-menth-1,3,7-triol}, whose optical rotation values ​​are respectively

[0091] Table 7. 3-methoxy-p-menth-1,3,7-triol 1 H-NMR and 13 C-NMR data (400 MHz, 100 MHz) (Chloroform-d, δ ppm, J in Hz)

[0092]

[0093] Example 5 Effect of white cardamom volatile oil on renal function in gentamicin-induced acute kidney injury

[0094] 1 Materials, reagents and instruments

[0095] 1.1 Animals

[0096] Male clean-grade SD rats, weighing 200-250 g, were purchased from Xinjiang Medical University. Before the experiment, all animals were adaptively fed for one week and kept in an animal room with good temperature, humidity and ventilation, with no restriction on drinking water and food.

[0097] 1.2 Main materials and reagents

[0098] White cardamom volatile oil (prepared according to Example 3); gentamicin sulfate injection (batch number: 1804164): Henan Runhong Pharmaceutical Co., Ltd.; verapamil (batch number: 180201): Tianjin Central Pharmaceutical Co., Ltd.; nitric oxide synthase (NOS) (batch number: 20190226), creatinine (Cr) (batch number: 20190307), nitric oxide (NO) (batch number: 20190307), urea nitrogen (BUN) (batch number: 20190308) , superoxide dismutase (SOD) (batch number: 20190308), malondialdehyde (MDA) (batch number: 20190308), glutathione peroxidase (GSH-PX) (batch number: 20190308), urine protein (batch number: 20190311) test kit: Nanjing Jiancheng Bioengineering Institute; Caspase3 antibody (batch number: 0911412Da9488120), Bax antibody (batch number: 13CM400A), NF-кB p65 antibody (batch number: BM3940), Bcl-2 antibody (batch number: 196841): Boster Company; β-actin antibody (batch number: 16A00205), goat anti-rabbit antibody (batch number: 129256), goat anti-mouse antibody (batch number: 127655): Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0099] 1.3 Main instruments

[0100] Legend Micro 21R high-speed refrigerated centrifuge (Thermo Fisher), GT200 vibrating ball mill (Shanghai Jingxin), power wave XS2 microplate reader (BIO-RAD, USA), DYCZ-24DN gel casting apparatus (Beijing Liuyi Biotechnology Co., Ltd.), Axio Imager 2 optical microscope (ZEISS), CO2 incubator (Thermo Scientific), DYCZ-40D electrophoresis apparatus (Beijing Liuyi Biotechnology Co., Ltd.).

[0101] 2 Experimental methods

[0102] The essential oil of white cardamom was prepared according to Example 3. When administering, an appropriate amount of the essential oil was diluted with a 0.5% CMC-Na solution and a 2% Tween-80 solution in a ratio of 5:1 for use.

[0103] SD male rats were randomly divided into 7 groups, 10 in each group, including blank control group (volatile oil diluent), model group (volatile oil diluent), low, medium and high dose groups of white cardamom volatile oil (low: 62.5 mg / kg / d; medium: 125 mg / kg / d; high: 250 mg / kg / d), white cardamom extract group (25 g / kg / d), and verapamil positive drug group (verapamil: 15 mg / kg / d). Except for the blank control group rats injected with normal saline, the other groups were intraperitoneally injected with gentamicin (140 mg / kg / d) once a day for 7 consecutive days to prepare the acute renal failure model. From the 8th day, the blank group and the model group were given solvent, the low, medium and high dose groups of white cardamom volatile oil were given corresponding doses of white cardamom volatile oil, and the positive drug group was given verapamil solution for 28 consecutive days.

[0104] On the 7th day of the experiment, rats were fasted but not watered for 24 hours, and urine was collected from rats. On the 8th day, blood was collected (orbital blood) 24 hours after administration. On the 35th day of the experiment, rats were fasted but not watered for 24 hours, and urine was collected from rats. On the 36th day, blood was collected from the abdominal aorta 24 hours after administration. The blood was centrifuged at 4000r / min at 4°C for 10 minutes, and serum was collected for later use. After blood collection, the rats were killed, and the whole kidneys on both sides were quickly taken and weighed. The right kidney was fixed in 10% neutral formalin for later use. All urine, serum and left kidney samples were frozen in a -80°C refrigerator for inspection.

[0105] 3 indicators detection

[0106] Urine and blood samples were collected to detect urine protein, Scr, and Bun contents; right kidney tissue was stained with hematoxylin-eosin (HE) and observed under an optical microscope; left kidney tissue samples were collected to prepare 10% renal cortex homogenate, and the MDA and NO contents and SOD, NOS, and GSH-Px activities were detected; left kidney tissue samples were collected to prepare renal tissue homogenate, protein was extracted, and protein was quantified by the BCA method. The Western Blot method was used to detect the expression of NF-кB p65, Caspase-3, Bax, and Bcl-2 proteins in renal tissue homogenate, with β-actin as the internal reference, Ecl color development, and photography. ImageJ image analysis software was used to determine the optical density of the bands, and the grayscale value was calculated.

[0107] 4. Statistical analysis The data of each group were expressed as mean ± standard deviation, and SPSS 17.0 statistical software was used to analyze and process the data. One-way analysis of variance was used for inter-group comparison, and P < 0.05 was considered to be statistically significant.

[0108] 5 Experimental results

[0109] 5.1 Effect of essential oil of white cardamom on renal body index of rats injured by gentamicin

[0110] Compared with the blank control group, the renal body index of rats in the model group was significantly increased (p<0.05), indicating kidney damage; compared with the model group, the renal body index of rats in the verapamil positive drug group was significantly decreased, indicating that verapamil can effectively alleviate the acute kidney injury caused by gentamicin; like verapamil, the renal body index of rats in the medium and high dose groups of white cardamom volatile oil and white cardamom extract groups was significantly decreased (p<0.05), indicating that white cardamom volatile oil and white cardamom extract have anti-acute kidney injury activity. The results of renal body index of each group are shown in Table 8 and Fig.13 .

[0111] Table 8 Effect of white cardamom volatile oil on renal index of rats

[0112]

[0113] Note: *p<0.01 compared with the model group; ##p<0.05 compared with the blank control group.

[0114] 5.2 Analysis of the effect of cardamom volatile oil on renal tissue pathology in rats injured by gentamicin

[0115] HE staining showed that compared with the blank control group, the right kidney tissue of the model group rats had obvious lesions, the renal tubular epithelial cells had obvious lesions, the renal tubules were highly dilated, and the necrosis was severe; compared with the model group, except for the low-dose volatile oil, the renal tubular damage in the other groups was alleviated, and the high-dose volatile oil group had the best alleviating effect. Fig.13 .

[0116] 5.3 Effect of white cardamom volatile oil on urine protein, serum Scr and BUN in rats with gentamicin injury

[0117] Compared with the blank control group, the urine protein, serum urea nitrogen and serum creatinine content of the rats in the model group were significantly increased (p<0.05), and the model was successfully established; compared with the model group, the serum urea nitrogen and creatinine of the rats in the verapamil positive drug group were significantly reduced, indicating that verapamil can effectively alleviate the acute kidney injury caused by gentamicin; like verapamil, compared with the model group, the serum urea nitrogen and serum creatinine content of the rats in the medium and high dose groups of white cardamom volatile oil and white cardamom extract groups were significantly reduced (p<0.05), indicating that white cardamom volatile oil and white cardamom extract have anti-acute kidney injury activity. The results of serum urine protein, urea nitrogen and serum creatinine of rats in each group are shown in Table 9.

[0118] Table 9 Effects of white cardamom essential oil on urine protein, serum urea nitrogen and serum creatinine in rats

[0119]

[0120] Note: #p<0.05 compared with the blank control group; *p<0.05 compared with the model group.

[0121] 5.4 Effect of white cardamom volatile oil on the MDA and NO contents and the activities of GSH-PX, SOD and NOS in the right kidney tissue of rats injured by gentamicin.

[0122] Compared with the blank control group, the MDA content in the model group was significantly increased (P<0.05), while the SOD activity was significantly decreased (P<0.05). The medium and high dose groups of white cardamom volatile oil, the white cardamom extract group and the verapamil positive drug group significantly reduced the increase in the MDA content in the rat renal tissue, and also had a significant recovery effect on the decrease in SOD activity. Compared with the blank control group, the GSH-PX activity in the renal tissue of the model group was significantly decreased (P<0.05). The medium and high dose groups of white cardamom volatile oil, the white cardamom extract group and the verapamil positive drug group had a significant increase in the decrease in GSH-PX activity caused by gentamicin, which was significantly higher than the model group (P<0.05). Compared with the control group, the NO content and NOS activity in the renal tissue of the model group increased significantly (P<0.05). The rats in the medium and high dose groups of white cardamom volatile oil, the white cardamom extract group and the verapamil positive drug group had a significant reduction effect on the increase of NO content caused by gentamicin, which was significantly different from the model group (P<0.05); the rats in the low, medium and high dose groups of white cardamom volatile oil, the white cardamom extract group and the verapamil positive drug group had a significant reduction effect on the increase of NOS activity caused by gentamicin, which was significantly different from the model group (P<0.05), indicating that white cardamom volatile oil can protect renal tissue by reducing lipid peroxidation and scavenging free radicals. The results of the determination of MDA, NO content and SOD, GSH-PX and NOS activity in each group are shown in Tables 10 and Figure 14-18 .

[0123] Table 10 Effects of white cardamom volatile oil on MDA, NO content and SOD, GSH-PX, NOS activity in kidney tissue

[0124]

[0125] Note: **p<0.01, *p<0.05 compared with the model group; #p<0.05 compared with the blank control; ##P<0.01. 5.5 White cardamom volatile oil can inhibit apoptosis of renal tissue cells induced by gentamicin

[0126] Compared with the blank control group, the expression of NF-кB p65, Caspase-3, and Bax proteins in the renal tissue of rats in the model group was significantly increased (p<0.05), and the expression of Bcl-2 protein was significantly decreased (p<0.05). Compared with the model group, the expression of NF-кB p65, Caspase-3, and Bax proteins in the medium and high dose groups of white cardamom volatile oil and the positive drug group was significantly decreased (p<0.05), and the expression of Bcl-2 protein was significantly increased (p<0.05), indicating that white cardamom volatile oil inhibited the expression of NF-кB p65, caspase-3, and Bax proteins, and promoted the expression of Bcl-2 protein, indicating that white cardamom volatile oil can alleviate gentamicin-induced renal injury and protect renal function by inhibiting renal tissue apoptosis. The expression levels of NF-кB p65, caspase-3, and Bax proteins in each group are shown in Fig.19 .

[0127] In summary, white cardamom volatile oil has a significant renal protective effect, which can significantly inhibit the decline of renal function caused by gentamicin, reduce renal tissue pathological damage, inhibit renal tissue cell apoptosis, reduce lipid peroxidation, and scavenge free radicals to protect renal tissue. Therefore, white cardamom volatile oil is expected to become a preventive and therapeutic drug for clinical AKI patients.

[0128] Example 6 Effect of white cardamom volatile oil on renal function in 5 / 6 nephrectomized rat model

[0129] 5.1 Test methods

[0130] CKD rats (weight 200-250g) were used in this study and given standard rodent chow and tap water ad libitum under a 12-hour light-dark cycle. The rats were randomly divided into 5 groups: Group 1 was the 5 / 6Nx group (n=10), in which the upper and lower poles of the left kidney were removed in addition to the right kidney; Groups 2-4 were the 5 / 6Nx group treated with white cardamom volatile oil (WC group), which were intraperitoneally injected with 2.5, 5, and 10 mg / kg of white cardamom volatile oil (prepared according to Example 3) (5 / 6Nx+2.5, 5, and 10 mg / kg; n=10); Group 5 was the control group (n=10), which underwent sham surgery. After 4 weeks of continuous treatment, the experiment was terminated and the surgery was performed under chloral hydrate solution anesthesia (350 mg / kg, ip). Pathological tissues and other biochemical indicators were detected.

[0131] 5.2 Effects of white cardamom volatile oil on renal function indicators

[0132] We tested the kidney function indicators urine protein, serum creatinine, and urea nitrogen. The results showed that white cardamom volatile oil can significantly reduce the increase in urine protein, serum creatinine, and urea nitrogen caused by 5 / 6 nephrectomy ( Fig. 20), which indicates that white cardamom volatile oil can effectively protect renal function damage caused by 5 / 6 nephrectomy.

[0133] 5.3 Renal histopathological examination

[0134] Furthermore, we conducted pathological examinations on kidney tissues. After HE staining, we found that the glomeruli of CKD rats would swell or atrophy, and the lumen of the renal tubules would expand. White cardamom volatile oil could slightly improve these pathological changes ( Fig.21 ). This indicates that white cardamom volatile oil can have a certain protective function on kidney tissue.

[0135] 5.4 Expression of MCP-1, α-SMA and TGF-β1 in the renal cortex and medulla

[0136] The expression of MCP-1, α-SMA and TGF-β1 in the cortex and medulla of the kidney was further analyzed. The results of tissue immunohistochemistry showed that the inflammatory factor α-SMA and the fibrotic factors MCP-1 and TGF-β1 in the model group were significantly increased, indicating the occurrence of inflammation and fibrosis. In the group treated with white cardamom volatile oil, the expression of related factors was inhibited in a dose-dependent manner ( Fig. 22 , 23), suggesting that white cardamom volatile oil can improve inflammation and fibrosis in rats caused by 5 / 6 kidney.

[0137] 5.5 Cardamom volatile oil can reduce the expression of p38MAPK / NF-κB pathway-related proteins in CKD rats

[0138] In order to further clarify the potential molecular mechanism of action of cardamom volatile oil, we used Western blotting to detect the relevant signaling pathways. The results showed that the treatment of cardamom volatile oil could significantly inhibit the expression of inflammation-related proteins in the p38MAPK / NF-κB pathway ( Fig.24 ), the results suggested that white cardamom volatile oil had a good inhibitory effect on inflammation caused by 5 / 6 nephrectomy in rats, and played a renal protective role by inhibiting inflammation of the p38MAPK / NF-κB pathway.

Claims

1. A compound having a structure of formula I or formula II or a racemate thereof for treating nephritis, wherein the chemical name of the compound of formula I is (1R, 3R, 4S)-3-methoxy-menthol-1, 3, 7-triol; the chemical name of the compound of formula II is (1S, 3S, 4R)-3-methoxy-menthol-1, 3, 7-triol 2. A pharmaceutical composition comprising the compounds of formula I and formula II or their racemates as claimed in claim 1, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, pills, powders, ointments, oral liquids, gels and injections.

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