A cyclohexaneethanol compound, its preparation method, application, and pharmaceutical composition.

Abstract

This invention provides a cyclohexaneethanol compound, its preparation method, applications, and pharmaceutical compositions, belonging to the field of biomedical technology. This invention is the first to prepare a cyclohexaneethanol compound from *Clerodendrum trichotomum*, a plant in the Verbenaceae family. This cyclohexaneethanol compound significantly inhibits the mRNA expression of iNOS, IL-1β, IFN-γ, TNF-α, and IL-6 in LPS-induced RAW264.7 cells, and also inhibits NO production in LPS-induced RAW264.7 cells, indicating that the cyclohexaneethanol compound has a significant anti-inflammatory effect. Furthermore, this cyclohexaneethanol compound can effectively improve the survival rate and body weight of septic mice, reduce lung and kidney damage, and decrease serum TNF-α, IFN-γ, and IL-6 levels, thereby exerting an anti-septic effect. This invention provides a new option for clinical anti-inflammatory and / or anti-septic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to a cyclohexaneethanol compound, its preparation method, application, and pharmaceutical composition. Background Technology

[0002] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, characterized by high morbidity, high mortality, and high medical costs. The most effective treatments for sepsis include aggressive systemic anti-inflammatory therapy, immunomodulatory therapy, and supportive care to improve organ damage. Despite significant advancements in anti-infective therapy and organ support techniques in recent years, the mortality rate of sepsis remains as high as 30%–70%. Therefore, existing drugs for treating sepsis still cannot meet clinical needs.

[0003] Natural products often possess low toxicity and multiple target characteristics, gradually attracting researchers' attention in the development of antiseptic drugs. Clerodendrum trichotomum, a plant belonging to the genus Clerodendrum in the Verbenaceae family, has stems and leaves with blood-activating, stasis-removing, swelling-reducing, and detoxifying effects, and can be used to treat inflammatory diseases such as mastitis and arthritis. It contains chemical components such as diterpenes, flavonoids, phenolic acids, phenylethanol derivatives, and cyclohexylethanol derivatives, exhibiting various activities including cytotoxicity, anti-complement, anti-inflammatory, glycosidase, and angiotensin-converting enzyme inhibition. However, the effects of its chemical components on sepsis have not yet been reported in the literature. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a cyclohexaneethanol compound, its preparation method, application and pharmaceutical composition thereof, wherein the cyclohexaneethanol compound has significant anti-inflammatory and antiseptic effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a cyclohexaneethanol compound and its pharmaceutically acceptable salt, the structural formula of which is as follows:

[0007]

[0008] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the above-described cyclohexaneethanol compounds or pharmaceutically acceptable salts thereof.

[0009] The present invention also provides a method for preparing the above-mentioned cyclohexaneethanol compounds, comprising the following steps:

[0010] The branches and leaves of Clerodendrum tigrinum were mixed with anhydrous methanol and refluxed for extraction. The extract was collected, and after removing the anhydrous methanol, a methanol extract was obtained.

[0011] The methanol extract was mixed with water to obtain a suspension. The suspension was extracted with ethyl acetate until the upper layer of solution became clear. The ethyl acetate layer was collected, and the ethyl acetate was removed to obtain the extract.

[0012] The extract was separated by silica gel column chromatography, eluted sequentially with petroleum ether:acetone mixed solvents at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30. Eluents a at volume ratios of 100:20 and 100:30 were collected, and the petroleum ether:acetone mixed solvent was removed from eluent a to obtain the separated product.

[0013] The separated molecule was dissolved in anhydrous methanol and separated by medium-pressure liquid chromatography. It was eluted with 75% methanol aqueous solution. After 32-33 min, 500 mL of eluent b was collected and the 75% methanol aqueous solution was removed to obtain subfraction I.

[0014] Subfraction I was dissolved in anhydrous methanol, separated and purified by high performance liquid chromatography, eluted with 55% methanol aqueous solution, and the eluent with a retention time of 9.5 to 10.5 min was collected to obtain cyclohexaneethanol compounds.

[0015] Preferably, the mass-to-volume ratio of the stinking peony branches and leaves to anhydrous methanol is 1:6-8 kg / L; the extraction temperature is 60-65℃; the number of extractions is 2-4 times; and the extraction time for each extraction is 1-3 hours.

[0016] Preferably, the volume ratio of the suspension to ethyl acetate is 1:1; when eluting with a petroleum ether:acetone mixed solvent, the volume of each mixed solvent used for elution is 17-19 L.

[0017] Preferably, when performing medium-pressure liquid chromatography separation, the medium-pressure liquid chromatography conditions are as follows:

[0018] Column: DN 40×400; Packing material: medium-pressure column packing material ODS; Column temperature: room temperature; Mobile phase: 75% methanol aqueous solution, flow rate: 9.0 mL / min, elution mode: isocratic elution;

[0019] During high-performance liquid chromatography (HPLC) separation and purification, the HPLC conditions are as follows:

[0020] Column: YMC-Pack ODS-AM; Column temperature: room temperature; Mobile phase: 55% methanol aqueous solution; Flow rate: 5.5 mL / min; Injection volume: 200 μL; Elution mode: isocratic elution.

[0021] The present invention also provides a cyclohexaneethanol compound prepared by the above preparation method.

[0022] The present invention also provides the use of the above-mentioned cyclohexaneethanol compounds or pharmaceutical compositions in the preparation of drugs for treating inflammation-related diseases.

[0023] Preferably, the inflammation-related disease includes at least one of sepsis, sepsis-related acute kidney injury, and sepsis-related acute lung injury.

[0024] The present invention also provides the use of the above-mentioned cyclohexaneethanol compounds or pharmaceutical compositions in the preparation of drugs for treating sepsis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention provides a cyclohexaneethanol compound, its preparation method, applications, and pharmaceutical compositions. This invention is the first to prepare a cyclohexaneethanol compound from *Clerodendrum trichotomum*, a plant in the Verbenaceae family. This cyclohexaneethanol compound significantly inhibits the mRNA expression of iNOS, IL-1β, IFN-γ, TNF-α, and IL-6 in LPS-induced RAW264.7 cells, and also significantly inhibits NO production in LPS-induced RAW264.7 cells, indicating a significant anti-inflammatory effect. Furthermore, the cyclohexaneethanol compound can effectively improve the survival rate and body weight of septic mice, reduce lung and kidney damage, and decrease serum TNF-α, IFN-γ, and IL-6 levels, thus exerting an anti-septic effect. This invention provides a new option for clinical anti-inflammatory and / or anti-septic drugs. Attached Figure Description

[0027] Figure 1 Cyclohexaneethanol compounds 1 H NMR spectrum;

[0028] Figure 2 Cyclohexaneethanol compounds 13 C NMR spectrum;

[0029] Figure 3 The spectrum of DEPT135 for cyclohexaneethanol compounds;

[0030] Figure 4 HMQC spectra of cyclohexaneethanol compounds;

[0031] Figure 5 HMBC spectrum of cyclohexaneethanol compounds;

[0032] Figure 6 Cyclohexaneethanol compounds 1 H- 1 H COSY spectrum;

[0033] Figure 7NOESY spectrum of cyclohexaneethanol compounds (δppm in DMSO-d6);

[0034] Figure 8 HRESIMS spectra of cyclohexaneethanol compounds;

[0035] Figure 9 The structural formula is that of a cyclohexaneethanol-like compound;

[0036] Figure 10 The effects of cyclohexaneethanol compounds on the cytotoxicity of RAW264.7 cells;

[0037] Figure 11 The effect of cyclohexaneethanol compounds on LPS-induced nitric oxide production levels in RAW264.7 cells;

[0038] Figure 12 The effect of cyclohexaneethanol compounds on the mRNA levels of inflammatory factors in LPS-induced RAW264.7 cells;

[0039] Figure 13 The effect of cyclohexaneethanol compounds on the LPS-induced NF-κB pathway in RAW264.7 cells;

[0040] Figure 14 The effect of cyclohexaneethanol compounds on the survival rate of septic mice;

[0041] Figure 15 The effect of cyclohexylethanol compounds on body weight in septic mice;

[0042] Figure 16 The effect of cyclohexylethanol compounds on serum inflammatory factor TNF-α in septic mice;

[0043] Figure 17 The effect of cyclohexaneethanol compounds on the serum inflammatory factor IL-6 in septic mice;

[0044] Figure 18 The effect of cyclohexaneethanol compounds on the serum inflammatory factor IFN-γ in septic mice;

[0045] Figure 19 Images showing HE staining of the lungs and kidneys of septic mice with cyclohexylethanol compounds. Detailed Implementation

[0046] This invention provides a cyclohexaneethanol compound and its pharmaceutically acceptable salt, the structural formula of which is as follows:

[0047]

[0048] The cyclohexylethanol compounds of this invention are extracted from Clerodendrum bungei, a plant belonging to the genus Clerodendrum in the family Verbenaceae. In a preferred embodiment, the cyclohexylethanol compounds are isolated from methanol extracts of Clerodendrum bungei branches and leaves.

[0049] The present invention also provides a method for preparing the above-mentioned cyclohexaneethanol compounds, comprising the following steps:

[0050] The branches and leaves of Clerodendrum tigrinum were mixed with anhydrous methanol and refluxed for extraction. The extract was collected, and after removing the anhydrous methanol, a methanol extract was obtained.

[0051] The methanol extract was mixed with water to obtain a suspension. The suspension was extracted with ethyl acetate until the upper layer of solution became clear. The ethyl acetate layer was collected, and the ethyl acetate was removed to obtain the extract.

[0052] The extract was separated by silica gel column chromatography, eluted sequentially with petroleum ether:acetone mixed solvents at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30. Eluents a at volume ratios of 100:20 and 100:30 were collected, and the petroleum ether:acetone mixed solvent was removed from eluent a to obtain the separated product.

[0053] The separated molecule was dissolved in anhydrous methanol and separated by medium-pressure liquid chromatography. It was eluted with 75% methanol aqueous solution. After 32-33 min, 500 mL of eluent b was collected and the 75% methanol aqueous solution was removed to obtain subfraction I.

[0054] Subfraction I was dissolved in anhydrous methanol, separated and purified by high performance liquid chromatography, eluted with 55% methanol aqueous solution, and the eluent with a retention time of 9.5 to 10.5 min was collected to obtain cyclohexaneethanol compounds.

[0055] In this invention, the branches and leaves of *Clerodendrum trichotomum* are mixed with anhydrous methanol and refluxed for extraction. The extract is collected, and after removing the anhydrous methanol, a methanol extract is obtained. The preferred mass-to-volume ratio of *Clerodendrum trichotomum* branches and leaves to anhydrous methanol is 1:6–8 kg / L, more preferably 1:7 kg / L. The extraction temperature is 60–65°C, the extraction is preferably performed 2–4 times, more preferably 3 times, and the extraction time for each extraction is preferably 1–3 hours, more preferably 1.5–2.5 hours. The anhydrous methanol is removed by concentration, such as by vacuum concentration, meaning the methanol extract of this invention contains no methanol.

[0056] In this invention, after obtaining the methanol extract, the methanol extract is mixed with water to obtain a suspension. The suspension is extracted with ethyl acetate until the supernatant is clear. The ethyl acetate layer is collected, and the ethyl acetate is removed to obtain the extract. The preferred volume ratio of methanol extract to water is 1:1.8–2.2 g / mL, and the preferred volume ratio of the suspension to ethyl acetate is 1:1. The ethyl acetate layer is the supernatant. The ethyl acetate is removed by concentration, such as by vacuum concentration, meaning that the extract of this invention contains no ethyl acetate.

[0057] In this invention, the extract is separated by silica gel column chromatography, eluted sequentially with petroleum ether:acetone mixed solvents at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30. Eluents a at volume ratios of 100:20 and 100:30 are collected, and the petroleum ether:acetone mixed solvent in eluent a is removed to obtain the separated product. The preferred mass ratio of the extract to the silica gel packing material is 0.035–0.037:1. The preferred elution volume for each petroleum ether:acetone mixed solvent is 17–19 L. The petroleum ether:acetone mixed solvent in eluent a is removed by distillation, such as distillation drying at 90–100 °C, ensuring that the separated product contains no petroleum ether:acetone mixed solvent.

[0058] In this invention, the extract is dissolved in anhydrous methanol and separated by medium-pressure liquid chromatography (MPLC). Elution is performed with a 75% methanol-water solution. After 32-33 min, 500 mL of the eluent is collected, and the 75% methanol-water solution is removed to obtain subfraction I. The preferred MPLC chromatographic conditions for medium-pressure liquid chromatography are: column: DN 40×400; packing material: medium-pressure column chromatographic packing material ODS; column temperature: room temperature; mobile phase: 75% methanol-water solution; flow rate: 9.0 mL / min; elution program: isocratic elution. The mass-to-volume ratio of the extract to anhydrous methanol is 4.5:40 g / mL. The 75% methanol-water solution refers to a 75% (v / v) methanol-water solution. The preparation method of the 75% methanol-water solution is not particularly limited and can be prepared using conventional methods in the art. The DN 40×400 column was purchased from Rissui Technology (Suzhou) Co., Ltd., and the ODS packing material with a particle size of 50 μm was purchased from YMC Corporation, Japan. The detection wavelength for the medium-pressure liquid chromatography separation is 210 nm. The method for removing 75% methanol aqueous solution is concentration, such as vacuum concentration, i.e., subcomponent I of the present invention does not contain 75% methanol aqueous solution.

[0059] In this invention, subcomponent I is dissolved in anhydrous methanol, separated and purified by high-performance liquid chromatography (HPLC), eluted with 55% methanol aqueous solution, and the eluent with a retention time of 9.5–10.5 min is collected to obtain cyclohexaneethanol compounds. The HPLC conditions for separation and purification are as follows: column: YMC-Pack ODS-AM; column temperature: room temperature; mobile phase: 55% methanol aqueous solution; flow rate: 5.5 mL / min; injection volume: 200 μL; elution method: isocratic elution. The YMC-Pack ODS-AM has a size of 250 mm × 20 mm. As a preferred embodiment, the eluent with retention times of 9.5 min, 9.6 min, 9.7 min, 9.8 min, 9.9 min, 9.99 min, 10 min, 10.1 min, 10.2 min, 10.3 min, 10.4 min, or 10.5 min is collected. The 55% methanol aqueous solution refers to a methanol aqueous solution with a volume percentage of 55%. There are no particular limitations on the preparation method of the 55% methanol aqueous solution; any conventional method in the art can be used. The detection wavelength of the high-performance liquid chromatography is 210 nm.

[0060] The present invention also provides a cyclohexaneethanol compound prepared by the above preparation method.

[0061] This invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient comprises the aforementioned cyclohexaneethanol compounds or their pharmaceutically acceptable salts. The active ingredient constitutes 20% to 80% of the drug by mass. The pharmaceutical excipients include one or more of flavoring agents, excipients, binders, and diluents. In this invention, "pharmaceutically acceptable" excipients or salts are substances suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The drug may be in the form of granules, tablets, lyophilized powder, or capsules. The drug may be administered orally, intravenously, or intramuscularly.

[0062] The present invention also provides the use of the above-mentioned cyclohexaneethanol compounds or pharmaceutical compositions in the preparation of drugs for treating inflammation-related diseases.

[0063] In this invention, the inflammation-related diseases preferably include at least one of sepsis, sepsis-related acute kidney injury, and sepsis-related acute lung injury.

[0064] The present invention also provides the use of the above-mentioned cyclohexaneethanol compounds or pharmaceutical compositions in the preparation of drugs for treating sepsis.

[0065] In the above-described applications of this invention, the cyclohexaneethanol compound constitutes 20% to 80% of the drug by mass. The drug may also include pharmaceutically acceptable excipients, which include one or more of flavoring agents, excipients, binders, and diluents. In this invention, "pharmaceutically acceptable" excipients or salts are substances suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The drug may be in the form of granules, tablets, lyophilized powder, or capsules. The drug may be administered orally, intravenously, intraperitoneally, or intramuscularly.

[0066] The effective anti-inflammatory dose range of the cyclohexylethanol compounds in this invention is 5–80 μM, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 μM. The effective antiseptic dose range of the cyclohexylethanol compounds in this invention is 5–20 mg / kg, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mg / kg. In this invention, "effective dose" refers to the amount of therapeutic drug used to treat, alleviate, or prevent inflammation and / or sepsis.

[0067] Unless otherwise specified, all reagents and materials involved in this invention are commercially available products well known to those skilled in the art.

[0068] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0069] In the following embodiments, the statistical methods used were as follows: Data analysis was performed using Graph Pad Prism 7.0. A t-test was used to measure the significance of differences between two groups. One-way ANOVA was used to detect significant differences among multiple groups. A p-value < 0.05 was considered statistically significant.

[0070] Example 1

[0071] A method for preparing a cyclohexaneethanol derivative compound, comprising the following steps:

[0072] (1) Take 5.0 kg of stinking peony branches and leaves, add 35 L of anhydrous methanol and soak for 30 min, then place on a constant temperature heating mantle and heat at 65 °C for reflux extraction for 2 h, filter and collect filtrate 1 and filter residue 1, add 35 L of anhydrous methanol to filter residue 1 and heat at 65 °C for reflux extraction for 1.5 h, filter and collect filtrate 2 and filter residue 2, add 35 L of anhydrous methanol to filter residue 2 and heat at 65 °C for reflux extraction for 1.5 h, filter and collect filtrate 3, combine filtrate 1 to 3 to obtain extract, concentrate the extract under reduced pressure and evaporate to dryness, remove anhydrous methanol to obtain 0.775 kg of methanol extract;

[0073] (2) 0.775 kg of methanol extract was suspended in pure water and the volume was adjusted to 1500 mL to obtain a suspension. 1500 mL of ethyl acetate was added to the suspension and extracted until the upper layer of solution was clear. The ethyl acetate layer was collected and concentrated under reduced pressure until no ethyl acetate was obtained to obtain 120 g of extract.

[0074] (3) 36.9 g of extract was added to a chromatographic column packed with 1 kg silica gel for separation and purification. Then, it was eluted sequentially with petroleum ether:acetone mixed solvents at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30, with an elution volume of 18 L for each solvent gradient. The eluents a with volume ratios of 100:20 and 100:30 were collected and dried by distillation at 98 °C to remove the petroleum ether:acetone mixed solvent from eluent a, thus obtaining the separated product.

[0075] (4) Dissolve 4.5 g of the isolate in 40 mL of anhydrous methanol and perform MPLC chromatography separation:

[0076] The MPLC chromatographic conditions were as follows: column: DN40×400 (purchased from Rissui Technology (Suzhou) Co., Ltd.); packing material: medium-pressure column packing material ODS (particle size 50μm, YMC Corporation, Japan); column temperature: room temperature; mobile phase: 75% methanol aqueous solution; flow rate: 9.0 mL / min; after 32.7 min, 500 mL of eluent was collected, concentrated under reduced pressure, and the 75% methanol aqueous solution was removed to obtain subfraction I;

[0077] (5) The sub-fraction I obtained in step (4) was dissolved in 45 mL of anhydrous methanol and purified by high performance liquid chromatography. The high performance liquid chromatography conditions were as follows: column: YMC-Pack ODS-AM (250 mm × 20 mm); column temperature: room temperature; mobile phase: 55% methanol aqueous solution; flow rate: 5.5 mL / min; injection volume: 200 μL; elution mode: isocratic elution; the eluent with a retention time of 9.99 min was collected to obtain 161.8 mg of cyclohexaneethanol compounds.

[0078] The above compounds were subjected to 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 spectrum, HMQC spectrum, HMBC spectrum, 1 H- 1 A comprehensive analysis of the H COSY, NOESY, and HRESIMS spectra, with detection results shown in Tables 1 and 2, respectively. Figures 1-8 The structure of the compound was determined.

[0079] The structural identification data of this compound are as follows: reddish-brown oil; CD(CH3CN)204(Δε+1.15)nm,219(Δε+0.79)nm; IR(KBr)ν max 2959,2924,2856,1713,1682,1455,1378,1260,1092,1065,1018,799,704,662,589cm -1 ; 1 H NMR (400MHz, MeOD) and 13 C NMR (100MHz, MeOD) data are shown in Table 1; HRESIMS m / z 333.1312 [M+Na] + (calcd for C 16 H 22 O6Na3133.1314).

[0080] Table 1 Cyclohexaneethanol compounds 1 H and 13 CNMR data (δppm in MeOD).

[0081]

[0082] Note: "-" indicates no hydrogen signal.

[0083] According to Table 1 and Figures 1-8 The results showed that this compound was a new compound, named Clerodenone B, and structurally belonged to the cyclohexaneethanol derivative category. The structure of this compound is shown below. Figure 9 As shown.

[0084] Example 2 investigates the anti-inflammatory effect of the cyclohexaneethanol compound (Clerodenone B) prepared in Example 1 on RAW264.7 cells.

[0085] 1. Experimental Methods

[0086] 1.1. Detection of the effect of cyclohexaneethanol compounds on the cytotoxicity of RAW264.7 cells.

[0087] The CCK-8 assay kit was used to determine the effect of compounds on cell viability. RAW264.7 cells were seeded at 5000 cells / well and cultured in 96-well plates for 24 h. Cells were then treated with 0, 2, 5, 10, 20, 40, 60, and 80 μM cyclohexaneethanol compounds for 12, 24, and 48 h, with three replicates for each concentration. 10 μL of CCK-8 solution was added and the cells were incubated in the dark for 1 h. The absorbance at 540 nm was measured using a microplate reader.

[0088] like Figure 10 As shown, cyclohexaneethanol compounds have no toxic effect on RAW264.7 cells at 0, 2, 5, 10, 20, 40, 60, and 80 μM.

[0089] 1.2. Detection of the inhibitory effect of Clerodenone B on nitric oxide

[0090] RAW264.7 cells were cultured in medium containing 10% FBS at 37°C and 5% CO2. Cells were seeded at 5000 cells / well in 96-well plates and cultured overnight. Cells were then treated with cyclohexaneethanol compounds at concentrations of 0, 2, 5, 10, 20, 40, 60, and 80 μM for 1 h (triple replicates for each concentration). Cells were then stimulated with 1 μg / mL lipopolysaccharide (LPS) and cultured for another 24 h. 50 μL of cell supernatant was collected and mixed with an equal volume of Griess I and Griess II at room temperature for 5 min. The absorbance at 540 nm was measured using a microplate reader. Cells were treated with 4 μg / mL Bay 11-7082 (Beyotime, S1523, China) for 24 h, followed by stimulation with 1 μg / mL lipopolysaccharide (LPS) as a positive control. Cells without any treatment served as a blank control. Cells stimulated with only 1 μg / mL lipopolysaccharide (LPS) served as a negative control. Each control group had three replicates. 2- The concentration was determined using a standard sodium nitrite curve.

[0091] like Figure 11 As shown, compared with the LPS group, Clerodenone B at concentrations of 2, 5, 10, 20, 40, 60, and 80 μM significantly inhibited NO production.

[0092] 1.3. Effects of Clerodenone B on the levels of iNOS, IL-1β, IFN-γ, TNF-α, and IL-6 mRNA in RAW 264.7 cells.

[0093] RAW 264.7 cells were treated with 0, 5, 10, and 20 μM cyclohexaneethanol compounds for 1 h, followed by culturing with 1 μg / mL LPS for 24 h. Each concentration was tested in triplicate. Untreated cells served as a blank control, and cells stimulated with only 1 μg / mL lipopolysaccharide (LPS) served as a negative control, with three replicates for each control group. Total RNA was extracted from the cells using Triquick Reagent and quantified using a nanodroplet 8000 spectrophotometer. Then, [the following was performed using...]. II. cDNA was obtained by reverse transcription using the 1st Strand cDNA Synthesis SuperMix. qRT-PCR was performed using a SYBR GreenER qPCR SuperMix Universal. 2 -ΔΔCt The method uses an endogenous internal reference gene (GAPDH) as an internal reference to quantify gene expression and determine its effect on target mRNA.

[0094] like Figure 12 As shown, compared with the LPS group, Clerodenone B significantly inhibited the production of iNOS, IL-1β, IFN-γ, TNF-α and IL-6 mRNA.

[0095] 1.4 Inhibitory effect of Clerodenone B on NF-κB signaling pathway expression in RAW264.7 cells

[0096] RAW 264.7 cells were treated with 0, 5, 10, and 20 μM cyclohexaneethanol compounds for 1 h, followed by culturing with 1 μg / mL LPS for 30 min. Each concentration was tested in triplicate. Untreated cells served as a blank control, and cells stimulated with only 1 μg / mL lipopolysaccharide (LPS) served as a negative control, with three replicates for each control group. Total protein was extracted from the cells, and the content of NF-κB pathway proteins was analyzed by Western blot. RAW 264.7 macrophages were washed three times with PBS and lysed using RIPA lysis buffer containing protease inhibitors. Protein content was determined using a BCA assay kit. Proteins from the extracts were separated using 10% SDS-PAGE and transferred to a nitrocellulose membrane. Cells were incubated with primary antibodies against NF-κB p65, p-IKB, NF-κBp-p65, IKB, and Tubulin. After washing with Tris-buffered saline and Tween 80, HRP-labeled secondary antibody was added. Hybridization was then performed using detection reagents.

[0097] Figure 13 The results showed that, compared with the LPS group, Clerodenone B significantly inhibited the expression of the NF-κB signaling pathway in RAW264.7 cells in a concentration-dependent manner.

[0098] Example 3: Effect of Clerodenone B (compound 1) prepared in Example 1 on cecal ligation-induced sepsis in mice.

[0099] 1. Experimental Methods

[0100] 1.1 Model Establishment

[0101] Male C57bl / 6 mice (18-20g) were randomly divided into 5 groups (8 mice per group): (1) Sham group; (2) CLP group (induced by cecal ligation and puncture); (3) CS group (cefpirome sulfate, 200mg / kg); (4) low-dose Clerodenone B group (5mg / kg); (5) high-dose Clerodenone B group (15mg / kg). In this group, mice in the Sham group underwent only sham surgery, mice in the CLP group underwent cecal ligation and puncture surgery, mice in the CS group underwent cecal ligation and puncture surgery and were then treated with an intraperitoneal injection of 200 mg / kg of CS, mice in the low-dose Clerodenone B group underwent cecal ligation and puncture surgery and were then treated with an intraperitoneal injection of 5 mg / kg of the compound, and mice in the high-dose Clerodenone B group underwent cecal ligation and puncture surgery and were then treated with an intraperitoneal injection of 15 mg / kg of the compound. Survival rate and body weight were observed at 0, 12, 24, 36, and 48 hours postoperatively. 48 hours postoperatively, mice were anesthetized and blood was collected from the eyeballs. Serum was obtained after centrifugation. Mice were then sacrificed and tissues were collected.

[0102] 1.2 Detection Indicators

[0103] 1.2.1 Mouse survival rate and body weight detection

[0104] The survival status of CLP model mice was observed and their weight was measured every 12 hours, up to a total of 48 hours.

[0105] Figure 14 The results showed that Clerodenone B significantly improved the survival rate of septic mice.

[0106] Figure 15 The results showed that, compared with the sham control group, the CLP group mice had a lower body weight, while Clerodenone B significantly improved body weight.

[0107] 1.2.2 Detection of pro-inflammatory factors in serum

[0108] The levels of IFN-γ, TNF-α, and IL-6 in serum were detected using an ELISA kit.

[0109] Figures 16-18 The results showed that, compared with the sham control group, the serum concentration of pro-inflammatory factors in the CLP group was significantly increased, while the high-dose Clerodenone B group (15 mg / kg) significantly reduced the levels of IFN-γ, TNF-α and IL-6.

[0110] 1.2.3 Pathological changes in mouse lung and kidney tissues

[0111] Lung and kidney tissues were fixed in 10% formalin solution and embedded in paraffin. Then, sections with a thickness of 5 micrometers were stained with hematoxylin and eosin (H&E) to observe the infiltration of inflammatory cells.

[0112] like Figure 19 As shown, H&E staining revealed that CLP induced inflammatory cell infiltration in mouse lung and kidney tissues; however, the low-dose Clerodenone B group, the high-dose Clerodenone B group, and the CS group significantly restored this inflammatory change.

[0113] In summary, the cyclohexaneethanol compounds prepared in this invention have a significant inhibitory effect on the mRNA expression of iNOS, IL-1β, IFN-γ, TNF-α and IL-6 in LPS-induced RAW264.7 cells. The mechanism of action study found that they exert their anti-inflammatory effect by inhibiting the NF-κB signaling pathway, and the cyclohexaneethanol compounds have a significant anti-inflammatory effect.

[0114] The cyclohexaneethanol compounds prepared by this invention can effectively improve the survival rate and body weight of septic mice, reduce lung and kidney organ damage, and reduce the levels of TNF-α, IFN-γ and IL-6 in serum, thereby playing an anti-septic role and effectively treating sepsis.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cyclohexaneethanol compound and a pharmaceutically acceptable salt thereof, characterized by, The cyclohexane ethanol compound has the following structural formula: 。 2. A process for the preparation of cyclohexane ethanol compounds as claimed in claim 1, wherein, The method comprises the following steps: The branches and leaves of B. officinalis are mixed with anhydrous methanol to extract by refluxing, and the extract is collected to obtain a methanol extract after removing the anhydrous methanol; The methanol extract is mixed with water to obtain a suspension, and the suspension is extracted with ethyl acetate until the upper solution is clear, and the ethyl acetate layer is collected to obtain an extract after removing the ethyl acetate; The extract is separated by silica gel column chromatography, and the eluent is eluted with petroleum ether:acetone mixed solvents with volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20 and 100:30, respectively, and the eluent a with volume ratios of 100:20 and 100:30 is collected, and the petroleum ether:acetone mixed solvents in the eluent a are removed to obtain a separation product; The separation product is dissolved in anhydrous methanol, and is separated by medium-pressure liquid chromatography, and is eluted with 75% methanol water solution, and the eluent b is collected 500 mL after 32-33 min, and 75% methanol water solution is removed to obtain a sub-component I; The sub-component I is dissolved in anhydrous methanol, and is separated and purified by high-performance liquid chromatography, and is eluted with 55% methanol water solution, and the eluent with a retention time of 9.5-10.5 min is collected to obtain the cyclohexane ethanol compound; When the petroleum ether:acetone mixed solvents are used for elution, the volume of each mixed solvent is 17-19 L; When the medium-pressure liquid chromatography is performed, the medium-pressure liquid chromatography conditions are as follows: The column: DN 40*400; the chromatographic packing: medium-pressure column chromatographic packing ODS; the column temperature: room temperature; the mobile phase: 75% methanol water solution, the flow rate: 9.0 mL / min, and the elution mode is isocratic elution; When the high-performance liquid chromatography is performed, the high-performance liquid chromatography conditions are as follows: The column: YMC-Pack ODS-AM, 250*20 mm; the column temperature: room temperature; the mobile phase: 55% methanol water solution, the flow rate: 5.5 mL / min; the injection amount: 200 μL; and the elution mode is isocratic elution.

3. The preparation method according to claim 2, characterized in that, The mass-volume ratio of the branches and leaves of B. officinalis to anhydrous methanol is 1:6-8 kg / L, the extraction temperature is 60-65 °C, and the extraction times are 2-4 times, and the extraction time of each time is 1-3 h.

4. The production method according to claim 2, characterized by, The volume ratio of the suspension to ethyl acetate is 1:

1.

5. The cyclohexane ethanol compound of claim 1 or the cyclohexane ethanol compound prepared by the preparation method of any one of claims 2-4 is used for preparing a medicine for treating an inflammation-related disease.

6. Use according to claim 5, characterized in that, The inflammation-related disease comprises at least one of sepsis, sepsis-related acute kidney injury and sepsis-related acute lung injury.

7. The cyclohexane ethanol compound of claim 1 or the cyclohexane ethanol compound prepared by the preparation method of any one of claims 2-4 is used for preparing a medicine for treating sepsis.

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