Application of C21 steroid compound in preparation of anti-inflammatory product

Abstract

The invention discloses an application of a C21 steroid compound in preparation of an anti-inflammatory product. The C21 steroid compound comprises a compound as shown in a formula I which is described in the specification. The C21 steroid compound has an excellent anti-inflammatory effect, and can reduce expression of inflammatory proteins such as TNF-alpha, COX-2, iNOS, IL-1beta, TLR4, p38, ERK and JNK by inhibiting activation of signaling pathway related factors such as TLR4 / NF-kappa B and MAPK, so that generation and release of inflammatory factors are reduced to play a role.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a C 21 Application of steroidal compounds in the preparation of anti-inflammatory products. Background Technology

[0002] Inflammation is a common self-defense mechanism against infection and tissue damage. While the inflammatory response is intended to activate the body's immune system to defend against disease, an excessive inflammatory response is also a significant contributing factor to many diseases, such as sepsis, systemic reaction syndrome, and septic shock. Therefore, anti-inflammatory drugs play a crucial role in the prevention and treatment of diseases.

[0003] C 21 Steroids (C 21 Pregnane steroids (C-steroids) are a class of steroidal derivatives with a parent nucleus containing 21 carbon atoms. They are derivatives of pregnane or its isomers and possess a wide range of pharmacological activities, including antitumor, anti-inflammatory, analgesic, antifertility, antidepressant, and immunomodulatory effects. They have significant clinical application value and have become widely recognized natural products. Highly effective and low-toxicity C-steroids have been discovered in traditional Chinese medicine and natural drugs. 21 Steroids, and exploring their pharmacological activities and mechanisms of action, have become an important research direction in medicinal chemistry. Natural C 21 Steroids are widely distributed in the plant kingdom, and are abundant in plants of families such as Asclepiadaceae, Apocynaceae, Scrophulariaceae, Ranunculaceae, Dioscoreaceae, Gentianaceae, and Solanaceae. 21 Steroidal compounds have complex and diverse skeletal structures, with nine common types of parent carbon skeletons.

[0004] *Tylophora floribunda* Miq., a perennial twining vine belonging to the genus *Tylophora* in the family Asclepiadaceae, contains latex and its fibrous roots are pale yellow. In the Shaoguan area of ​​northern Guangdong, this plant is commonly known as "liver-nourishing herb" or "liver-protecting golden herb." Traditionally, its roots are used to make soup, which is clear, fragrant, and not greasy, and is believed to have the effects of aiding digestion, strengthening the spleen and stomach, and clearing liver heat. It is worth noting that plants in the Asclepiadaceae family are a natural source of vitamin C. 21 One of the main sources of steroid compounds, its derived C 21 Steroids (C 21 Pregnanes (steroids) with pregnane or its isomers as the parent nucleus structure have attracted much attention due to their multiple pharmacological activities, including antitumor, anti-inflammatory, and immunomodulatory effects. However, there are currently few reports on *Schefflera heptaphylla*, a plant in the genus *Schefflera* of the family Asclepiadaceae, and studies on its chemical composition and biological activities are rare, requiring further investigation. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art described above. To this end, the present invention proposes a C 21 The application of steroidal compounds in the preparation of anti-inflammatory products, this C 21 Steroid compounds have excellent anti-inflammatory effects. They can reduce the production and release of inflammatory factors by inhibiting the activation of signaling pathway-related factors such as TLR4 / NF-κB and MAPK, downregulating the expression of inflammatory proteins such as TNF-α, COX-2, iNOS, IL-1β, TLR4, p38, ERK and JNK.

[0006] This invention also proposes an anti-inflammatory product.

[0007] According to a first aspect of the invention, a C is proposed. 21 The application of steroidal compounds in the preparation of anti-inflammatory products, wherein C 21 Steroidal compounds include those represented by Formula I:

[0008]

[0009] In some embodiments of the present invention, the C 21 The effective in vitro concentration of steroidal compounds is 1–30 μmol / L.

[0010] In some embodiments of the present invention, the C 21 The effective in vitro concentration of steroidal compounds is 1.25–20 μmol / L.

[0011] In some embodiments of the present invention, the C 21 The half-maximal inhibitory concentration (WMC) for steroidal compounds is 2.9–7.5 μmol / L.

[0012] In some embodiments of the present invention, the C 21 The preparation method of steroidal compounds includes the following steps:

[0013] S1: Use ethanol to extract the powder of the rhizome of the seven-layered building. Each extraction cycle is 5 to 9 days. Combine the extracts and evaporate under reduced pressure to obtain the total extract.

[0014] S2: Disperse the total extract obtained in step S1 with water, and then perform gradient extraction with petroleum ether and ethyl acetate in sequence to obtain a petroleum ether layer and an ethyl acetate layer.

[0015] S3: The ethyl acetate layer obtained in step S2 was eluted and separated by column chromatography to obtain the C 21 Steroidal compounds.

[0016] In some embodiments of the present invention, the extraction in step S1 is performed 2 to 5 times.

[0017] In some embodiments of the present invention, the ethanol in step S1 is a 90% to 98% aqueous ethanol solution.

[0018] According to a second aspect of the present invention, an anti-inflammatory product is provided, the anti-inflammatory product comprising C as described in the first aspect of the present invention. 21 Steroidal compounds and pharmaceutically acceptable excipients.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of binders, disintegrants, lubricants, coating agents, suspending agents, thickeners, and surfactants.

[0020] In some embodiments of the present invention, the adhesive is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, or polyvinylpyrrolidone.

[0021] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, crosporovinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosporovinyl cellulose sodium, carboxymethyl cellulose, calcium carboxymethyl cellulose, or alginate.

[0022] In some embodiments of the present invention, the lubricant is selected from at least one of micronized silica gel, magnesium stearate, calcium stearate, stearic acid, talc, or anhydrous silica gel.

[0023] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, polyvinyl alcohol phthalate, ethyl cellulose, and cellulose acetate.

[0024] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel.

[0025] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate.

[0026] In some embodiments of the present invention, the administration method of the anti-inflammatory product includes at least one of oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retroocular, retinal, choroidal, and intrathecal injection.

[0027] In some embodiments of the present invention, the dosage form of the anti-inflammatory product includes at least one of tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

[0028] The present invention has at least the following beneficial effects:

[0029] This invention provides a C 21 The application of steroidal compounds in the preparation of anti-inflammatory products, this C 21 Steroid compounds possess excellent anti-inflammatory effects, and C... 21 Treatment of lipopolysaccharide-induced inflammatory cell models with steroidal compounds can reduce the production and release of inflammatory factors by inhibiting the activation of signaling pathway-related factors such as TLR4 / NF-κB and MAPK, and downregulating the expression of inflammatory proteins such as TNF-α, COX-2, iNOS, IL-1β, TLR4, p38, ERK, and JNK. This invention provides C 21 Steroid compounds can be used to prepare anti-inflammatory products, providing a theoretical basis and research direction for the development of related drugs. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 The compound Disformone in Example 1 of this invention 1 1H NMR spectrum (in CDCl3);

[0032] Figure 2 The compound Disformone in Example 1 of this invention 13 C NMR spectrum (in CDCl3);

[0033] Figure 3 The DEPT spectrum (in CDCl3) of compound Disformone in Example 1 of this invention;

[0034] Figure 4 The HSQC spectrum (in CDCl3) of compound Disformone in Example 1 of this invention;

[0035] Figure 5 The HMBC spectrum (in CDCl3) of compound Disformone in Example 1 of this invention;

[0036] Figure 6 The compound Disformone in Example 1 of this invention 1 H- 1H COSY spectrum (in CDCl3);

[0037] Figure 7 The HR-ESI-MS spectrum (in MeOD) of the compound Disformone in Example 1 of this invention;

[0038] Figure 8 This is a single crystal image of the compound Disformone used in Example 1 of this invention;

[0039] Figure 9 The figure shows the detection results of the effect of Disformone on the viability of RAW264.7 cells in Example 2 of the present invention;

[0040] Figure 10 The image shows the detection results of NO release in LPS-induced RAW264.7 cells treated with Disformone in Example 2 of this invention; where, compared with the Control group, #### represents P<0.0001; compared with the Model group, *** represents P<0.001, **** represents P<0.0001, and dexamethasone was used as a positive control in the cell experiments;

[0041] Figure 11 The image shows the detection results of NO release in RAW264.7 cells induced by LPS treated with different concentrations of Disformone in Example 2 of this invention; where, compared with the Model group, #### represents P<0.0001; compared with the Model group, *** represents P<0.001, **** represents P<0.0001, and dexamethasone was used as a positive control in the cell experiments;

[0042] Figure 12 This is a graph showing the detection results of the effect of Disformone on the expression levels of iNOS and COX-2 in LPS-induced RAW264.7 cells in Example 2 of the present invention; where, compared with the Control group, #### represents P<0.0001; compared with the Model group, ** represents P<0.01;

[0043] Figure 13 The figure shows the detection results of the effect of compound Disformone on the phosphorylation levels of p38, JNK, ERK and NF-κB proteins in LPS-induced RAW264.7 cells in Example 2 of this invention; where, compared with the Model group, ### represents P<0.001; compared with the Model group, * represents P<0.1, ** represents P<0.01, and *** represents P<0.001. Detailed Implementation

[0044] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0045] Example 1C 21 Isolation, purification and structural identification of the steroid compound Disformone

[0046] In this embodiment, C was obtained by extracting, separating, and purifying dried rhizome material from *Seven-Story Building*. 21 The specific preparation method and structural identification results of the steroidal compound Disformone are as follows:

[0047] 1. Preparation method:

[0048] 19.8 kg of dried rhizome of *Seven-layered Tree* was processed using a traditional Chinese medicine pulverizing equipment and then subjected to a traditional solvent-based staged extraction process. Three extractions were performed at room temperature using 95% ethanol (approximately 20 L / time), with each extraction cycle lasting 7 days. The combined extracts were then concentrated by rotary evaporation under reduced pressure at approximately 45°C to obtain 1360 g of dark brown total extract. The total extract was dispersed in water and then subjected to a staged gradient extraction using three solvents of different polarities: petroleum ether, ethyl acetate, and n-butanol. This yielded 149 g of petroleum ether extract (PE layer), 593 g of ethyl acetate extract (EA layer), and 502 g of n-butanol extract (n-BuOH layer), respectively. Through research on plants of the same family in the literature (①Shi Hui, Cui Jiongmo, Guan Jian, et al. Study on chemical constituents of Cynanchum paniculatum[J]. Chinese Traditional and Herbal Drugs, 2008, 39(7):970-972. ②HAO D,FENG BM,CHEN SF, et al. C21 steroidal glycosides from the roots of Cynanchum paniculatum[J].Fitoterapia, 2016, 113:51-57. ③CHEN G,XU N,LI ZF, et al. Steroidal glycosides with anti-tumor activity from the roots of Cynanchum wallichii Wight[J].Journal of Asian Natural Products Research, 2010, 12(6):453-457.), it was learned that the C21 steroidal glycosides of Cynanchum paniculatum plants are... 21Steroid compounds are mainly concentrated in the ethyl acetate layer, so the ethyl acetate layer extract was selected for coarse fractionation.

[0049] A glass chromatography column (5 kg sample loading, silica gel 200-300 mesh) was dry-packed. After packing, an air pump was used to continuously remove air bubbles from the silica gel layer for 12 hours. The EA layer (593 g) of the sample to be separated was dry-mixed with 100-200 mesh silica gel (800 g) and then uniformly loaded onto the column.

[0050] The elution procedure employed a gradient solvent system: first, elution was performed using a petroleum ether-ethyl acetate mixture (volume ratio 50:1 → 10:1 → 5:1 → 2:1 → pure ethyl acetate), followed by a switch to a dichloromethane-methanol system (volume ratio 15:1 → 10:1 → 5:1 → 1:1 → pure methanol). The eluent was collected in fractions using 2000 mL Erlenmeyer flasks, yielding a total of 312 fractions.

[0051] Preliminary component merging was performed based on the colorimetric characteristics of thin-layer chromatography (TLC): fractions 1–26 were merged into EA1, 27–84 into EA2, 85–109 into EA3, 110–130 into EA4, 131–214 into EA5, and 215–312 into EA6, resulting in six major components, EA1–EA6. Further analysis by ultra-high performance liquid chromatography (UPLC) showed a significant correlation in the chromatographic behavior of components EA2–EA4. These three components (total mass 40.2 g) were redissolved and merged to obtain the target component G2. G2 was then separated by forward column chromatography using gradient elution with mobile phases of different polarities (PE:EA volume ratio = 50:1 → 30:1 → 20:1 → 10:1 → 5:1 → 2:1 → 1:1 → pure ethyl acetate → pure methanol). The fractions were merged based on TLC spotting and UPLC analysis results. The components were named in sequence as G2A1 (1193mg), G2A2 (183.7mg), G2A3 (354.2mg), G2A4 (483.3mg), G2A5 (276.4mg), G2A6 (247.6mg), G2A7 (228.2mg), G2A8 (3410.2mg), G2A9 (1120mg), and G2A10 (3.2g).

[0052] Two fractions, G2A4 (483.3 mg) and G2A5 (276.4 mg), were separated by gel column chromatography. Both showed deep red spots on a TLC plate. By comparing the spots on the same thin-layer silica gel plate, it was determined that they were the same component, and they were combined and named G2A45B2 (46 mg). This combined fraction was further eluted by normal column chromatography (300-400 mesh) with a gradient elution. The mobile phase was in ascending polarity order: n-hexane-dichloromethane (25:1→15:1→10:1→5:1→2:1→1:1→pure acetone), and the fraction collection volume was 10 mL / tube. TLC tracking (developing solvent: n-hexane-dichloromethane = 3:1) showed that the target deep red spot was enriched in fractions 37-43. After combining, G2A45B2C1 (28 mg) was obtained. To further remove impurities, the subfraction was purified by secondary gel electrophoresis (eluent: CH2Cl2-MeOH = 1:1), and tubes 21–27 were collected to obtain preliminary enrichments. Ultra-high performance liquid chromatography (UHPLC) analysis (mobile phase: methanol-water gradient) showed that the target component still exhibited co-elution peaks. Therefore, semi-preparative HPLC was used to further optimize the separation, with conditions set as isocratic elution of methanol-water (3:7, v / v), a flow rate of 3 mL / min, and detection wavelengths of 254 nm and 280 nm. Baseline separation was achieved at 24 min, and the fraction was collected, dried under reduced pressure, and a colorless solid, Disformone (14 mg), was obtained.

[0053] The structure of Disformone is as follows:

[0054]

[0055] 2. Structural characterization of Disformone:

[0056] 1 H NMR(500MHz,Chloroform-d)δ:7.27(1H,s,H1-15),6.59(1H,dd,J=9.9,2.2Hz,H1-7),6.27(1H,dd,J=9.9,2.7Hz,H1-6),6.22(1H, d,J=5.9Hz,H1-16),5.69(1H,s,H1-4),5.29(1H,s,H1-17),2.65(1H,d,J=11.7Hz,H1-8),1.13(3H,s,H3-19),1.09(3H,s,H3-18). That 1 H NMR spectrum as follows Figure 1 As shown.

[0057] 13C NMR(126MHz,Chloroform-d)δ:210.99(C-17),199.65(C-3),161.88(C-5), 160.70(C-15),137.11(C-16),133.39(C-4),129.60(C-7),124.21(C-4),8 2.88(C-14),52.36(C-13),43.04(C-9),41.55(C-8),36.31(C-10),33.75( C-1),33.34(C-2),30.01(C-12),19.68(C-11),19.26(C-19),16.12(C-18). That 13 C NMR spectrum as follows Figure 2 As shown.

[0058] After comparison with literature data, the spectral data of the compound Disformone extracted and separated in this embodiment are basically consistent with the literature, including its DEPT spectrum, HSQC spectrum, HMBC spectrum, and... 1 H- 1 The H COSY spectra are as follows: Figures 3-6 As shown.

[0059] In this embodiment, the quasi-molecular ion peak m / z 321.1461 [M+Na] was also obtained by high-resolution mass spectrometry (HR-ESI-MS). + (calcd for 315.1464), HR-ESI-MS spectrum as follows Figure 7 As shown; and its structure was completely confirmed by X-ray single-crystal diffraction experiments as 14-hydroxyandrosta-4,6,15-triene-3,17-dione, i.e., Disformone, and its single-crystal image is shown below. Figure 8 As shown.

[0060] Example 2: Anti-inflammatory activity test of Disformone

[0061] This embodiment investigated the anti-inflammatory activity of Disformone by detecting multiple indicators. The specific experimental methods and results are as follows:

[0062] 1. Cell culture:

[0063] 1) Cell resuscitation: Mouse macrophages (RAW264.7) were removed from the liquid nitrogen tank and then placed in a 37°C water bath for rapid shaking to dissolve them. The cell suspension was then transferred to a centrifuge tube with an appropriate amount of culture medium and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant in the centrifuge tube was removed with a pipette. 1 mL of culture medium was added and the cells were dispersed by gently blowing on the centrifuge tube wall. The cells were then transferred to a cell culture flask and 6 mL of complete culture medium (DMEM + 10% FBS + 1% antibiotics) was added. The culture flask was then placed in a 37°C CO2 incubator for static culture.

[0064] 2) Cell passage: Prepare new cell culture flasks. Take out the flasks where cells have reached a density of 80%–90%, remove the old culture medium with a pipette, and then wash 1–2 times with PBS buffer. Next, add trypsin and digest the cells at 37°C to promote detachment. Gently shake the flasks to facilitate detachment. After partial detachment, add complete culture medium (DMEM + 10% FBS + 1% antibiotics) to stop the digestion process. Then, gently blow the contents of the culture flask with a pipette. After mixing the cells with the culture medium, transfer them to centrifuge tubes and centrifuge at 1000 rpm for 3 min. Add an appropriate amount of culture medium, mix well, and distribute evenly into each new culture flask. Place the flasks in a CO2 incubator for static incubation.

[0065] 3) Cell plating: Calculate the amount of cell suspension needed according to the required plating density, and make up the difference with culture medium; after mixing, add the cell suspension evenly to the 96-well plate, and finally put it in an incubator for 24 hours.

[0066] 2. Cytotoxicity test:

[0067] The cytotoxicity of the compound Disformone against RAW264.7 cells was evaluated using the CCK-8 assay.

[0068] The growth density of RAW264.7 cells was observed. When the density reached approximately 80%, the cells were digested with trypsin, then centrifuged, resuspended, and counted. RAW264.7 cells were then seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 4 Cells were cultured in complete culture medium at 37°C and 5% CO2 for 24 hours before drug administration. Three groups were formed: a blank group (culture medium only), a control group (culture medium + cells), and an experimental group (culture medium + cells + 20 μM Disformone). After drug administration, the cells were incubated for another 24 hours. Then, 10 μL of CCK-8 reagent was added to each well of each of the three groups, and incubation continued for 2 hours. OD values ​​were measured at 450 nm using a microplate reader. Cell viability was calculated using the OD values: Cell viability = [(Experimental group - Blank group) / (Control group - Blank group)] × 100%. The results are shown below. Figure 9 As shown.

[0069] Depend on Figure 9 It can be seen that at a concentration of 20 μM, the cell survival rate after treatment with the compound Disformone was not significantly different from that of the control group, indicating that Disformone has no significant toxicity to cell growth at this concentration.

[0070] 3. Detection of the inhibitory effect on nitric oxide (NO) production:

[0071] The inhibitory effect of the compound Disformone on NO production was tested by constructing a lipopolysaccharide (LPS)-induced inflammatory cell model.

[0072] RAW264.7 cells were grown at a rate of 6 × 10⁻⁶. 5 Seeds were inoculated at a density of [number] cells / mL into 96-well plates and cultured for 24 h to allow adhesion. Dexamethasone was used as a positive control. 100 μL of culture medium was added to each well in the control group; LPS was added to each well in the model group (to a final concentration of 0.2 μg / mL); and a mixture of 10 μM dexamethasone and 0.2 μg / mL LPS was added to each well in the experimental groups. After drug administration, the plates were incubated in a standard incubator for 24 h. 50 μL of supernatant was aspirated from each well, and 50 μL each of Griess Reagent I and II NO detection kits were added. After shaking for 10 min, the OD value was measured at 540 nm. The results are shown below. Figure 10 As shown.

[0073] Depend on Figure 10 It can be seen that 20 μM Disformone can significantly inhibit the formation of NO, and the effect is remarkable.

[0074] Next, based on the concentration of 20 μM, different concentration gradients were set up to detect the Disformone IC. 50 The results are as follows Figure 11 As shown.

[0075] Depend on Figure 11 It can be seen that Disformone's IC 50 The concentration was 5.2±2.3 μM, and the inhibitory effect on NO was significantly better than that of the positive control group when the concentration was 1.25~2.5 μM.

[0076] 4. Detection of the effect of Disformone on the expression levels of COX-2 and iNOS enzymes:

[0077] COX-2 (cyclooxygenase-2) is a core mediator of the inflammatory response. Its upregulation leads to excessive production of prostaglandins (such as PGE2), triggering vasodilation, increased permeability, and pain signal transduction. iNOS (inducible nitric oxide synthase) is typically induced by cytokines (such as LPS) in inflammatory responses, catalyzing the conversion of L-arginine to NO and citrulline. Therefore, NO release in cells often serves as a signaling molecule and a standard for assessing inflammation. In chronic inflammation (such as arthritis and colitis), COX-2 and iNOS often synergistically amplify the inflammatory response through the NF-κB and MAPK pathways. Therefore, this embodiment utilizes Western blot to detect changes in the protein expression levels of COX-2 and iNOS.

[0078] Four groups were set up: a control group, a model group, and two groups treated with different concentrations of Disformone. RAW264.7 cells were seeded in 6-well plates (1×10⁻⁶ cells / well). 6 Cells were cultured in 6-well plates for 24 hours, then the old medium was removed, and fresh medium containing different concentrations of Disformone (5 and 10 μM) was added accordingly. After 2 hours of culture, 0.2 μg / mL LPS was added to each well except for the Control group. Protein was extracted after 1 hour. The 6-well plates were removed, the medium was removed, and the cells were washed three times with 3 mL of cold PBS and transferred to EP tubes. 300 μL of RIPA buffer was added to lyse the cells, and the plates were incubated on ice for 30 minutes. The cells were then centrifuged at 13000 rpm for 15 minutes at 4 °C. The protein concentration was detected using a BCA protein assay kit. The protein was separated by SDS-PAGE gel and transferred to a PVDF membrane. The PVDF membrane was incubated with 5% skim milk for 2 hours, the milk was poured off, and the membrane was washed three times with TBST for 5 minutes each time. The diluted primary antibody was then added to the PVDF membrane and incubated overnight at 4 °C. The primary antibody was recovered, and the membrane was washed three times with TBST for 5 minutes each time. After washing, the secondary antibody was added, and the membrane was reacted at room temperature for 90 minutes. The secondary antibody was recovered, and the membrane was washed three times with TBST for 5 minutes each time. The working concentration developer was prepared according to the instructions in the ECL reagent package. The band grayscale values ​​were analyzed using ImageJ software, and the results are as follows: Figure 11 As shown in Table 1, the WB antibody information used in this embodiment is as follows.

[0079] Depend on Figure 11 It was found that, compared with the control group, the expression of COX-2 and iNOS proteins was significantly increased in the model group. However, treatment with the compound Disformone significantly inhibited the expression of these two proteins, and the degree of inhibition was positively correlated with the concentration of Disformone. Therefore, further research will be conducted on the effects of Disformone on the NF-κB and MAPK pathways.

[0080] Table 1 Antibody Information

[0081]

[0082]

[0083] 5. Detection of the effect of Disformone on the expression levels of COX-2 and iNOS enzymes:

[0084] The MAPK signaling pathway is a crucial regulatory system for cellular responses to external stimuli (such as cytokines, stress signals, and pathogen-associated molecular patterns). It primarily consists of three branches: ERK, JNK, and p38. In inflammatory states, the activation of these kinase cascades regulates the expression of inflammation-related genes through phosphorylation of transcription factors (e.g., NF-κB, AP-1), promoting the production of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6), thereby exacerbating local or systemic inflammatory responses.

[0085] This embodiment uses Western blot to detect the expression of MAPK inflammation pathway-related signal transduction proteins in cells, further revealing the effect of the compound Disformone on the MAPK pathway. The results are as follows: Figure 13 A~ Figure 13 As shown in C.

[0086] Depend on Figure 13 A~ Figure 13 As shown in Figure C, compared to the Control group, the phosphorylation levels of p38(A), JNK(B), and ERK(C) in the Model group were significantly increased, indicating successful modeling. However, compared to the Model group, the phosphorylation levels of ERK, JNK, and p38 were significantly decreased at different concentrations of the compound Disformone, exhibiting a dose-dependent effect. This suggests that the compound Disformone exerts its anti-inflammatory effect by inhibiting the phosphorylation of key proteins in the MAPK inflammatory signaling pathway.

[0087] NF-κB, or nuclear factor κB, is a transcription factor that plays a crucial role in regulating key biological processes such as immune inflammatory responses, cell proliferation, and apoptosis. As a central element in inflammatory signaling, it mediates the body's inflammatory response by regulating the expression of various pro-inflammatory genes. Its abnormal activation is often closely related to the development and progression of chronic inflammation and related diseases. The expression of proteins related to the NF-κB inflammatory pathway in cells was detected using Western blot, and the results are as follows: Figure 13 As shown in D.

[0088] Depend on Figure 13As shown in D, compared with the Model group, treatment with the compound Disformone significantly inhibited the activation of the NF-κB signaling pathway in a dose-dependent manner, and at 10 μM, it significantly reduced the phosphorylation level of NF-κB. This indicates that the compound Disformone may exert its anti-inflammatory effect by inhibiting protein phosphorylation in the NF-κB inflammatory signaling pathway.

[0089] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a C 21 steroid for the preparation of an anti-inflammatory product, characterized in that, The C 21 Steroids include compounds of Formula I:

2. Use according to claim 1, characterized in that, The C 21 The effective concentration of the steroid in vitro is 1-30 μmol / L.

3. Use according to claim 1, characterized in that, The C 21 The half-inhibitory concentration of the steroid is 2.9-7.5 μmol / L.

4. Use according to claim 1, characterized in that, The C 21 The method of preparing a steroid compound comprises the steps of: S1: seven-layer root rhizome powder is extracted by using ethanol, each extraction cycle is 5-9 days, the extraction solution is combined and rotary evaporated under reduced pressure to obtain total extract; S2: the total extract obtained in step S1 is dispersed with water, and then gradient extraction is carried out by using petroleum ether and ethyl acetate in sequence to obtain petroleum ether layer and ethyl acetate layer; S3: using column chromatography to elute and separate the ethyl acetate layer obtained in step S2 to obtain the C 21 steroids.

5. Use according to claim 4, characterized in that, The number of times of extraction in step S1 is 2-5 times.

6. Use according to claim 4, characterized in that, The ethanol in step S1 is 90%-98% ethanol aqueous solution.

7. An anti-inflammatory product, characterized in that, The anti-inflammatory product comprises C according to any one of claims 1 to 6 21 a steroid and a pharmaceutically acceptable excipient.

8. Anti-inflammatory product according to claim 7, characterized in that, The pharmaceutically acceptable adjuvant includes at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

9. The anti-inflammatory product of claim 7, wherein, The administration mode of the anti-inflammatory product includes at least one of oral administration, enteric administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbit administration, retrobulbar administration, retinal administration, choroidal administration and intrathecal injection.

10. The anti-inflammatory product of claim 7, wherein, The dosage form of the anti-inflammatory product includes at least one of tablet, capsule, pill, injection, inhalation, lozenge, suppository, emulsion, microemulsion, submicroemulsion, nanoparticle, gel, powder, suspoemulsion, cream, jelly, spray.

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