A novel ginsenoside sulfur-containing derivative, and a preparation method and application thereof

Abstract

The application discloses a novel ginsenoside sulfur-containing derivative and a preparation method and application thereof, and belongs to the technical field of medicines. The ginsenoside sulfur-containing derivative is synthesized by using oktillone type ginsenoside and alpha-lipoic acid as raw materials, selecting DCC and EDCI as condensing agents, and selecting DMAP as a catalyst, and a protection effect on liver injury is researched. In-vivo and in-vitro experimental results show that the effect of the ginsenoside sulfur-containing derivative is significantly better than that of oktillone and other ginsenosides and other drugs, and the ginsenoside sulfur-containing derivative opens up a new direction for the development of anti-liver injury drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a novel ginsenoside sulfur-containing derivative and a preparation method and application thereof. BACKGROUND

[0002] Panax ginseng, a perennial herb of the Araliaceae Panax genus, is known as the "King of Herbs" and has a long history in traditional medicine. Its medicinal value is mainly derived from its rich active ingredients, including ginsenosides, polysaccharides, volatile oils, etc., among which ginsenosides are considered the core pharmacological substances. The growth of ginseng is strictly dependent on the environment, usually distributed in shady and humid forest valleys, and ecological factors such as light, temperature, and soil significantly affect its morphology and chemical composition.

[0003] Ginsenosides are the main active ingredients of ginseng, with the effects of enhancing physical fitness, regulating the nervous system, and delaying aging. Therefore, developing drugs with good efficacy and low side effects around ginsenosides has broad prospects. Ginsenosides are a class of triterpenoid saponins, which can be divided into three major categories based on the structure of the aglycone: PPD type (protopanaxadiol type): such as Rb1, Rb2, Rc, which accounts for the majority of root saponins. PPT type (protopanaxatriol type): such as Rg1, Re, which is higher in leaves and fruits. C17 SCV type (C17 side chain variation type): such as Rg3, which is higher in flowers and flower buds. As of 2019, 170 types of ginsenosides have been identified from ginseng, of which 69 types have isomerism in their molecular formula.

[0004] Among them, ocotillol-type ginsenosides (OC) are a class of dammarane-type tetracyclic triterpenoid saponins containing a tetrahydrofuran ring in the side chain, mainly found in Panax plants. Unlike common dammarane or oleanane saponins. This type of saponin has a low content in ginseng, but due to its unique pharmacological activities (such as anti-inflammatory, anti-tumor, immune regulation, etc.), it has attracted attention.

[0005] The present application found that the combination of ocotillol-type ginsenosides and thioctic acid has a synergistic protective effect on drug-induced liver injury (as shown in Figure 1 Therefore, we designed and synthesized ocotillol-type ginsenoside hydrogen sulfide donor complexes and studied their protective effect on drug-induced liver injury, laying a foundation for the development of related drugs. SUMMARY

[0006] The present application provides a novel ginsenoside sulfur-containing derivative and a preparation method and application thereof to solve the problems in the prior art.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a novel ginsenoside sulfur derivative, wherein the ginsenoside sulfur derivative is a ginsenoside sulfur derivative generated by a reaction between a ginsenoside and thioctic acid, the ginsenoside is an oleanolic-type ginsenoside, and the novel ginsenoside sulfur derivative is OCLA ((20S, 24R)-oleanolic acid-thioctic acid derivative), wherein the OCLA has a molecular formula of C 38 H 64 O6S2, and a structural formula as shown in the following formula (I):

[0009] .

[0010] Further, the OCLA is synthesized by using an oleanolic-type ginsenoside and alpha-thioctic acid as raw materials, selecting DCC (dicyclohexyl carbodiimide) and EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide) as condensing agents, DMAP (4-dimethylaminopyridine) as a catalyst, and stirring at 30°C to synthesize the target product (20S, 24R)-oleanolic acid-thioctic acid derivative.

[0011] Further, the mass ratio of the oleanolic-type ginsenoside to the alpha-thioctic acid is (454-1362):584.

[0012] Further, the target product is purified by using dichloromethane:methanol=160:1-100:1 as an eluent to obtain a purified compound.

[0013] In a second aspect, the present application further provides a preparation method of the novel ginsenoside sulfur derivative, comprising the following steps:

[0014] Alpha-thioctic acid is weighed and dissolved in dichloromethane by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide, 4-dimethylaminopyridine and dicyclohexyl carbodiimide as condensing agents, and stirring at 30°C for 30 min, then adding dichloromethane, continuing to stir for 30 min, then adding oleanolic acid, and detecting the reaction progress in real time by TLC until the raw material disappears, then extracting with saturated sodium chloride solution for three times, combining the water phases, adding anhydrous sodium sulfate to the organic phase, concentrating in a vacuum to obtain a crude product, pouring the obtained crude product into a chromatography column, eluting with pure dichloromethane, receiving the eluent, and qualitatively analyzing by thin layer chromatography, then eluting with dichloromethane:methanol=200:1, 160:1, 150:1 and 100:1 in sequence to completely separate the target product, then concentrating and rotary evaporating, dissolving the target product in ethyl acetate, and finally obtaining a pure product by freeze-drying.

[0015] In a third aspect, the present application further provides an application of the novel ginsenoside sulfur derivative in any of the above aspects in the preparation of a liver damage resisting drug.

[0016] Compared with the prior art, the present application has the beneficial effects of:

[0017] The novel ginsenoside sulfur-containing derivative provided by the present application is synthesized successfully by using the oktillone type ginsenoside and alpha-lipoic acid as raw materials, DCC and EDCI as condensing agents, and DMAP as a catalyst, and the structure-activity relationship of the anti-liver injury is studied, and the in vitro and in vivo experimental results show that the effect is significantly better than other ginsenosides and other drugs, which opens up a new direction for the development of anti-liver injury drugs. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure for drug synergy results of oktillone type ginsenoside and lipoic acid. Figure 1 In the figure, A is the survival rate of HepG2 cells (human liver cancer cells) under the action of APAP (paracetamol) measured by CCK8 method; B is the synergistic effect of OC and lipoic acid combination dose response evaluated by LOEWE model.

[0019] Figure 2 Figure for OCLA synthesis line.

[0020] Figure 3 Figure for high-resolution spectrum results of OCLA.

[0021] Figure 4 Figure for hydrogen spectrum results of OCLA.

[0022] Figure 5 Figure for carbon spectrum results of OCLA.

[0023] Figure 6 Figure for molecular structure formula of OCLA.

[0024] Figure 7 Figure for in vitro activity results of OCLA on the protective effect of drug-induced liver injury. Figure 7 In the figure, A is the effect of OC and OCLA on the survival rate of HepG2 cells compared by CCK8 experiment; B is the effect of different concentrations of OC and OCLA on the viability of HepG2 cells after APAP (10 mM) treatment for 24 h evaluated by CCK8 experiment.

[0025] Figure 8 Figure for in vivo activity results of OCLA on the protective effect of drug-induced liver injury. Figure 8 In the figure, A is a schematic diagram of mouse experiment; B is the level of ALT (glutamic-pyruvic transaminase) and AST (glutamic-oxaloacetic transaminase) in the serum of each group of mice; C is the histological analysis of liver tissue H&E staining (hematoxylin-eosin staining) of each group of mice.

[0026] Figure 9This is a figure showing the results of RNA transcriptome sequencing analysis of mouse liver tissue. Figure 9 In the diagram, A shows the volcano plot of differentially expressed genes between the APAP group and the control group; B shows the volcano plot of differentially expressed genes between the OCLA group and the APAP group; C shows the differentially expressed genes downregulated between the OCLA group and the APAP group, and the differentially expressed genes upregulated between the APAP group and the control group; D shows the differentially expressed genes upregulated between the OCLA group and the APAP group, and the differentially expressed genes downregulated between the APAP group and the control group; E shows the GO functional analysis results of differentially expressed genes activated by APAP but suppressed by OCLA; F shows the GO functional analysis results of differentially expressed genes suppressed by APAP but activated by OCLA; G shows the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment results of genes activated by APAP but suppressed by OCLA; and H shows the KEGG pathway enrichment results of genes suppressed by APAP but activated by OCLA.

[0027] Figure 10 This is a heatmap of DAMs (differentially expressed metabolites) obtained from the comparative analysis of the APAP group and the control group.

[0028] Figure 11 This is a heatmap of DAMs (differentially expressed metabolites) obtained from the comparative analysis of the OCLA and APAP groups.

[0029] Figure 12 This study presents a KEGG enrichment analysis of DAMs obtained from comparative analyses between the APAP group and the control group, and between the OCLA group and the APAP group.

[0030] Figure 13 A subset of overlapping DAMs: DAMs that were downregulated in the OCLA group compared to the APAP group but upregulated in the APAP group compared to the control group, and DAMs that were upregulated in the OCLA group compared to the APAP group but downregulated in the APAP group compared to the control group.

[0031] Figure 14 Heatmap of DAMs activated by OCLA but suppressed by APAP.

[0032] Figure 15 Heatmap of DAMs that are suppressed by APAP but activated by OCLA.

[0033] Figure 16 The figure shows the results of molecular docking and target network interaction analysis. Figure 16In the table, A shows the results of the STRING database (gene function association database) analysis, revealing the interactions between HIF1α (hypoxia-inducible factor-1α) and other glycolysis-related proteins (HK1 / 2 (hexokinase 1 / 2), ENO1 (α-enolase), PFKL (hepatic phosphofructokinase), Pfkfb3 (6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase 3)), inflammatory mediators (TNF (tumor necrosis factor), p65 (nuclear transcription factor protein 65), IL-6 (interleukin-6)) and apoptosis-related proteins (p21); B is a schematic diagram of the HIF1α signaling pathway (KEGG analysis); and C shows the molecular docking results between HIF1α and OCLA.

[0034] Figure 17 The image shows the results of real-time quantitative PCR and immunoblotting verification. Figure 17 In the table, A shows the mRNA expression results of HIF1α, glycolysis-related proteins (HK1 / 2, ENO1, PFKL, Pfkfb3), inflammatory mediators (TNF, p65, IL-6), and apoptosis-related proteins (p21) in mouse liver tissue; B shows the protein expression results of pp65 (phosphoprotein 65), p65, Bcl2 (B-cell lymphoma / leukemia-2), Bax (apoptosis regulator), and p21 in mouse liver tissue; C shows the semi-quantitative results of the protein expression levels of pp65, p65, Bcl2, Bax, and p21. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. The embodiments described below are merely specific embodiments exemplified in this application to illustrate the technical solutions of this application, and are not intended to limit them. The scope of protection of this application is not limited thereto.

[0036] Example 1

[0037] (1) Synthesis of OCLA

[0038] Starting with α-lipoic acid, an esterification reaction was carried out with octylene under stirring at 30°C in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI), dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) to generate an octylene-lipoic acid derivative. The synthetic route is as follows: Figure 2 As shown, the specific method is as follows:

[0039] Weigh 0.4540 g of α-lipoic acid and add it to 0.2530 g of EDCI, 0.0500 g of DMAP, and 0.4310 g of DCC. Dissolve these in 50 ml of pre-dried dichloromethane. Stir at 30 °C for 30 min. After stirring, a small amount of white flocculent matter was observed in the flask. Add another 10 ml of dichloromethane and continue stirring for 30 min. Then add 0.5840 g of octyl ether (dissolved in a small amount of dichloromethane and added dropwise to the flask). Simultaneously, monitor the reaction progress in real time using TLC (developing solvent: dichloromethane:methanol = 20:1, 10% sulfuric acid ethanol for color development). After the starting material disappears, perform three extractions with an appropriate amount of saturated sodium chloride solution. Combine the aqueous phases, add anhydrous sodium sulfate to the organic phase, seal the flask with plastic wrap, let it stand for 24 hours, and then concentrate under vacuum to obtain the crude product.

[0040] (2) Separation of OCLA

[0041] Place a dry, clean chromatography column on a shelf, pour in silica gel, and add dichloromethane until the silica gel is completely submerged. Remove air bubbles to ensure the silica gel is tightly packed. Pour the obtained crude product into the column, and place a wad of cotton at the top for buffering. Elute with pure dichloromethane, collecting the required eluent. Develop and qualitatively analyze the eluent using thin-layer chromatography (TLC) every 10 ml collected, constantly monitoring the fluorescence of the eluent. Develop with petroleum ether:ethyl acetate:methanol:water = 100:100:4:1 as the developing solvent, and perform color development with 10% sulfuric acid in ethanol, observing for fluorescence. If fluorescence is observed, change the collection tube until the collected liquid shows no fluorescence. Perform TLC on the fluorescent liquid to determine its specific components. After a period of time, if no fluorescence is observed after elution with 300 ml of dichloromethane, gradually increase the polarity of the eluent (dichloromethane:methanol = 200:1 / 160:1 / 150:1 / 100:1). During the experiment, 300 ml of a dichloromethane:methanol ratio of 200:1 was used as an eluent. TLC analysis showed fluorescence but no color development. Therefore, 120 ml of a dichloromethane:methanol ratio of 160:1 was used, which resulted in color development. The polarity was then increased to 150:1 and 100:1 to completely separate the target product. The product was concentrated and evaporated to dryness using a rotary evaporator, and finally dissolved in ethyl acetate and lyophilized to obtain the pure product.

[0042] In this embodiment, octylene-type ginsenosides and α-lipoic acid were used as raw materials. DCC and EDCI were selected as condensing agents, and DMAP was used as a catalyst to successfully synthesize the target product, octylene-lipoic acid derivative. The pure compound was obtained by silica gel column purification. After separation by silica gel column, the separated substance, when monitored by TLC (thin-layer chromatography), showed both fluorescence and color development, indicating that it was the target product.

[0043] (3) Identification of OCLA

[0044] The obtained compound was preliminarily identified by high-resolution mass spectrometry, and the final product was dissolved in pyridine for further identification by 1H-NMR and 13C-NMR. The compound, octylene-lipoic acid derivative, is a pale yellow solid, readily soluble in dichloromethane and dimethyl sulfoxide, sparingly soluble in water, with the molecular formula C2. 38 H 64 O6S2, high-resolution spectral results [M+H] + 681.3856 (e.g.) Figure 3 (As shown).

[0045] Hydrogen spectrum analysis (e.g.) Figure 4 ): 1H NMR (600 MHz, Pyr) δ 5.81 (s, 1H), 5.43 (s, 1H), 4.95 (s, 1H), 4.86 (s, 1H), 4.82 (m, 1H), 4.37 (s, 1H), 3.96 (dd, 1H), 3.72(td, 1H), 3.53 (dd, 1H), 3.09 (m, 1H), 3.03 (m, 1H), 2.49 (m, 1H), 2.44 (m,2H), 2.27 (m, 2H), 2.16 (m, 1H), 2.02 (dd, 1H), 1.92-1.83 (m, 5H), 1.82-1.72(m, 5H), 1.71 (s, 1H), 1.69 (s, 3H), 1.63-1.58 (m, 4H), 1.56-1.53 ​​(m, 3H), 1.49 (s, 3H), 1.44 (m, 1H), 1.35 (s, 3H), 1.28 (s, 3H), 1.27 (s, 3H), 1.25 (s, 1H), 1.20 (d, 1H), 1.10 (s, 3H), 1.05 (m, 1H), 0.94 (s, 3H), 0.93 (s, 3H).

[0046] Carbon spectrum analysis (e.g.) Figure 5): 13C NMR (151 MHz, Pyr) δ 173.62, 87.07, 86.03, 81.38,71.44, 70.69, 67.69, 61.93, 57.19, 52.46, 50.59, 49.81, 48.73, 47.69, 41.40,40.81, 39.36, 39.28, 39.12, 38.92, 35.23, 35.08, 33.17, 32.79, 32.10, 31.72,29.46, 29.14, 28.07, 27.55, 27.34, 25.86, 25.63, 24.30, 18.68, 18.03, 17.46, 17.41. The molecular structure of OCLA is as follows: Figure 6 As shown.

[0047] Example 2

[0048] (1) Synthesis of OCLA

[0049] Starting with α-lipoic acid, an esterification reaction was carried out with octylene under stirring at 30°C in the presence of EDCI, DCC, and DMAP to generate an octylene-lipoic acid derivative. The synthetic route is as follows: Figure 2 As shown, the specific method is as follows:

[0050] 0.9080 g of α-lipoic acid was weighed and added to 0.4540 g of EDCI, 0.2530 g of DMAP, and 0.0500 g of DCC, dissolved in 50 ml of pre-dried dichloromethane. The mixture was stirred at 30 °C for 30 min. A small amount of white flocculent matter was observed in the flask. 10 ml of dichloromethane was then added, and stirring continued for another 30 min. 0.5840 g of octyl ether (dissolved in a small amount of dichloromethane and added dropwise to the flask) was then added. The reaction was monitored in real-time by TLC (using dichloromethane:methanol = 20:1 as the developing solvent, and 10% sulfuric acid ethanol for color development). After the starting material disappeared, the mixture was extracted three times with a suitable amount of saturated sodium chloride solution. The aqueous phases were combined, and anhydrous sodium sulfate was added to the organic phase. The flask was sealed with plastic wrap and allowed to stand for 24 hours. The mixture was then concentrated under vacuum to obtain the crude product.

[0051] (2) Separation of OCLA

[0052] The OCLA separation method is the same as in Example 1, and the thin-layer monitoring results are similar to those in Example 1.

[0053] (3) Identification of OCLA

[0054] The identification method for OCLA is the same as in Example 1, and the mass spectrometry identification results are the same as in Example 1. The molecular structural formula of OCLA is as follows:Figure 6 As shown.

[0055] Example 3

[0056] (1) Synthesis of OCLA

[0057] Starting with α-lipoic acid, an esterification reaction was carried out with octylene under stirring at 30°C in the presence of EDCI, DCC, and DMAP to generate an octylene-lipoic acid derivative. The synthetic route is as follows: Figure 2 As shown, the specific method is as follows:

[0058] 1.362 g of α-lipoic acid was weighed and added to 0.4540 g of EDCI, 0.2530 g of DMAP, and 0.0500 g of DCC, dissolved in 50 ml of pre-dried dichloromethane. The mixture was stirred at 30 °C for 30 min. A small amount of white flocculent matter was observed in the flask. 10 ml of dichloromethane was then added, and stirring continued for another 30 min. 0.5840 g of octyl ether (dissolved in a small amount of dichloromethane and added dropwise to the flask) was then added. The reaction was monitored in real-time by TLC (using dichloromethane:methanol = 20:1 as the developing solvent, and 10% sulfuric acid in ethanol for color development). After the starting material disappeared, the mixture was extracted three times with a suitable amount of saturated sodium chloride solution. The aqueous phases were combined, and anhydrous sodium sulfate was added to the organic phase. The flask was sealed with plastic wrap and allowed to stand for 24 hours. The mixture was then concentrated under vacuum to obtain the crude product.

[0059] (2) Separation of OCLA

[0060] The OCLA separation method is the same as in Example 1, and the thin-layer monitoring results are similar to those in Example 1.

[0061] (3) Identification of OCLA

[0062] The identification method for OCLA is the same as in Example 1, and the mass spectrometry identification results are the same as in Example 1. The molecular structural formula of OCLA is as follows: Figure 6 As shown.

[0063] Example of effect 1

[0064] 1. In vitro activity assay

[0065] 1.1 Experimental Objective

[0066] Study on the protective effect of ginsenoside sulfur-containing derivatives against acetaminophen (APAP)-induced liver injury.

[0067] 1.2 Experimental Samples and Materials

[0068] 1.2.1 Test drugs: ginsenoside sulfur derivatives (OCLA), octyl ginsenosides (OC), and acetaminophen (APAP) prepared in Example 1.

[0069] 1.2.2 Test cells: HepG2 human liver tumor cells (gifted by Tianjin Institute of Pharmaceutical Research).

[0070] 1.3 Experimental Methods:

[0071] (1) HepG2 hepatocyte culture

[0072] 1) Preparation of complete culture medium: Measure 450 mL of high glucose DMEM medium, add 50 mL of fetal bovine serum and 5 mL of antibiotics to prepare a complete culture medium containing 10% fetal bovine serum, mix well and store at 4℃; Cell cryopreservation solution: Take 9 mL of complete culture medium, add 1 mL of DMSO to prepare a cryopreservation solution containing 10% DMSO, mix well and store at 4℃.

[0073] 2) Cell resuscitation: Remove HepG2 cells from the liquid nitrogen container and rapidly shake the cryovial in a 37°C water bath to quickly thaw them and reduce ice crystal formation. After the ice in the cryovial has melted, transfer the cryopreservation solution to a centrifuge tube containing culture medium to dilute the cryopreservation solution. Centrifuge at 200×g for 5 min at room temperature and discard the supernatant. Resuspend the cells thoroughly in complete culture medium, add fresh culture medium, mix well, transfer to a culture dish, and incubate in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Change the culture medium every two days.

[0074] 3) Cell passage and seeding: When HepG2 cells reach a confluence of 80-90%, discard the culture medium, wash three times with sterile PBS, add 1 mL of 0.25% trypsin, and digest in a 37°C incubator for 3 min. When cells begin to detach from the bottom of the dish, add fresh complete culture medium to stop digestion, gently pipette the bottom of the dish, collect the cells, centrifuge at 200×g at room temperature for 5 min, and discard the culture medium. Resuspend the cells in 1 mL of complete culture medium, gently pipette until dispersed into single cells, count and dilute, and seed in well plates or culture dishes.

[0075] 4) Cell cryopreservation: Select cells in the logarithmic growth phase, digest them with 0.25% trypsin, collect the cells, gently pipette them with cryopreservation solution to resuspend them, and transfer them to cryovials. Place the cryovials in a programmed cooling box and put the box in a -80°C freezer overnight. Then transfer the cells to liquid nitrogen for storage.

[0076] (2) Determination of HepG2 cell viability

[0077] HepG2 cells were seeded into 96-well plates and cultured for 24 h. The culture medium was discarded, and gradients of ginsenoside sulfur derivatives and octylene-type ginsenosides (0 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM) were added, followed by 24 h of further culture. The culture medium was then discarded, and the cells were washed with PBS. 90 μL of DMEM medium and 10 μL of LCK-8 reagent were added to each well, and the cells were incubated in the dark for 2 h. The optical density (OD) value was measured using a microplate reader. Furthermore, the effects of different concentrations (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 50 μM, 100 μM) of OC and OCLA on the survival rate of HepG2 cells after 24 h of APAP (10 mM) treatment were evaluated using the above experimental methods.

[0078] 1.4 Experimental Results

[0079] The results showed that when the concentrations of OC and OCLA were less than or equal to 25 μM, neither had a significant effect on the survival rate of HepG2 cells; however, when the concentration was greater than 25 μM, the cell survival rate in the OCLA group was significantly higher than that in the OC group. Furthermore, compared with the OC group, OCLA had a stronger protective effect against APAP-induced HepG2 cells, meaning that the cell survival rate in the OCLA group was higher than that in the OC group (see results below). Figure 7 Show).

[0080] Example 2

[0081] 2. In vivo activity assay

[0082] 2.1 Experimental Objective

[0083] Study on the protective effect of OCLA against liver injury in mice

[0084] 2.2 Experimental Samples and Materials

[0085] 2.2.1 Test drugs: ginsenoside sulfur-containing derivatives, octyltrione-type ginsenosides, acetaminophen, and silybin prepared in Example 1.

[0086] 2.2.2 Test animals: 40 male ICR mice (Changchun Yisi Laboratory Animal Technology Co., Ltd.), weighing 20±2 g.

[0087] 2.3 Experimental Methods

[0088] Male BALB / c mice were randomly divided into 5 groups: a blank control group (CON), a model group (APAP, 350 mg / kg), a positive control group (silymarin, 16 mg / kg), a low-dose OCLA group (16 mg / kg), and a high-dose OCLA group (32 mg / kg). All mice were administered the drugs via gavage for 7 consecutive days. Sixteen hours after the last administration, except for the CON group, each group received an intraperitoneal injection of APAP to establish a liver injury model. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured. Histopathological changes in the mouse liver were observed by HE staining.

[0089] 2.4 Experimental Results

[0090] The results are as follows Figure 8 As shown, the results indicated that administration of 350 mg / kg acetaminophen (APAP) to mice led to a significant increase in serum ALT and AST levels. Compared with the model group, treatment with OCLA significantly reduced ALT and AST levels, especially in the high-dose group. Histological examination revealed no significant pathological changes in liver sections of the group treated with 32 mg / kg OCLA compared to the control group; conversely, the APAP-treated group showed significant pathological changes. These results confirm that OCLA can effectively alleviate acetaminophen-induced liver injury in vivo.

[0091] Example 3

[0092] 3. Study on the protective mechanism of OCLA against APAP-induced liver injury

[0093] 3.1 RNA transcriptome sequencing (RNA-seq) analysis

[0094] 3.1.1 Experimental Methods:

[0095] Total RNA was extracted from liver tissues of mice in the control group, APAP group, and OCLA group and sequenced on the Illumina NovaSeq platform; clean reads were obtained using FASTP software (v0.19.5) and aligned to the reference genome; GO and KEGG enrichment analyses were performed on the Majorbio Cloud platform.

[0096] 3.1.2 Experimental Results

[0097] The results are as follows Figure 9The results showed significant transcriptomic differences between the APAP group and the control group. Specifically, 3923 differentially expressed genes (DEGs) were identified between the APAP and control groups: 2383 genes were significantly upregulated in the APAP group, while 1540 genes were significantly downregulated. Comparing the OCLA group with the APAP group, 3795 differentially expressed genes (DEGs) were detected. Enrichment analysis of the GO and KEGG pathways showed that the differentially expressed genes upregulated by APAP but downregulated by OCLA were significantly associated with multiple pathways related to inflammation, apoptosis, and anaerobic metabolism, including the IL-17 signaling pathway, TNF signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, and NF-κB signaling pathway. These results indicate that OCLA can effectively alleviate APAP-induced liver inflammation and apoptosis. Furthermore, those differentially expressed genes that were decreased in the APAP group but increased in the OCLA group were significantly enriched in pathways related to "drug metabolism - other enzymes" and "drug metabolism - cytochrome P450". These findings suggest that OCLA may alleviate APAP-induced liver injury by regulating drug metabolism processes.

[0098] 3.2 Metabolomics Analysis

[0099] 3.2.1 Experimental Methods

[0100] 80 mg of liver tissue was taken, 200 μL of water was added, and then 800 μL of methanol:acetonitrile = 1:1 (volume ratio) solution was added. The mixture was then subjected to low-temperature sonication. Protein precipitate was obtained by centrifugation at 4℃ for 20 min, and the supernatant was freeze-dried. Metabolite analysis was performed using a UHPLC-Q-TOF / MS (ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry) system, and GO and KEGG enrichment analysis was performed on the Majorbio Cloud platform.

[0101] 3.2.2 Experimental Results

[0102] The results are as follows Figures 10-15As shown, through metabolomics analysis and metabolite identification using HMDB (Human Metabolomics Database), we identified a total of 991 metabolites. KEGG analysis results indicated that, compared to the control group, the differentially expressed metabolites produced after APAP administration were mainly concentrated in pathways related to alanine, aspartate, and glutamate metabolism, riboflavin metabolism, retinol metabolism, and tryptophan metabolism. On the other hand, the differentially expressed metabolites (DAMs) between the APAP and OCLA groups were mainly concentrated in linoleic acid, arachidonic acid, and riboflavin metabolism pathways. Furthermore, 19 differentially expressed metabolites (DAMs) showed increased expression during APAP administration, decreased expression during OCLA administration, and increased expression again after OCLA administration. These results indicate significant differences in the metabolographic profiles between the APAP and control groups, and between the OCLA and APAP groups, suggesting that OCLA can reverse APAP-induced metabolic disorders.

[0103] 3.3 Target network interaction analysis and molecular docking

[0104] 3.3.1 Experimental Methods

[0105] The interaction between the target and downstream effector molecules was analyzed using the STRING and KEGG databases. The HIF1α structure (PDB ID: 4H6j) was obtained from the protein database, and the .pdb file of HIF1α and the .mol ​​file of OCLA were submitted to the online molecular docking platform CB-DOCK2 for analysis.

[0106] 3.3.2 Experimental Results

[0107] like Figure 16 As shown, analysis using the STRING database revealed interactions between HIF1 and glycolysis-related proteins (HK1 / 2, ENO1, PFKL, Pfkfb3), inflammatory mediators (TNF, p65, IL-6), and apoptosis-related proteins (p21). KEGG analysis showed that HIF1α expression was significantly upregulated in the APAP group compared to the control group, while it was significantly downregulated in the OCLA group. Furthermore, glycolysis-related genes, including GLUT1, HK1 / 2, ENO1, PFKL, and PFK2, were significantly upregulated in the APAP group, but significantly downregulated in the OCLA group. Molecular docking results showed that OCLA could effectively bind to HIF1α protein, with a binding fraction of -7.3. These results indicate that OCLA affects glycolytic metabolism by regulating HIF1α expression, which in turn may inhibit inflammation progression and provide protection against liver damage.

[0108] 3.4 qRT-PCR (quantitative real-time PCR) and Western Blot (immunoblotting) validation

[0109] 3.4.1 Experimental Objective

[0110] The mRNA expression levels of HIF1α, glycolysis-related genes (HK1, PFKL, ENO1, PFKFB3), inflammatory factors (IL-6, Rela, TNF-α), and apoptosis-related gene (CDKN1A) in liver tissue were detected by qRT-PCR; the expression levels of p65, BAX, BCL2, and p21 proteins in liver tissue were detected by Western blotting.

[0111] 3.4.2 Experimental Methods

[0112] qRT-PCR: Mouse liver tissue was treated with Trizol reagent. 1 mL of Trizol was added to every 50–100 mg of mouse liver tissue, followed by 0.2 mL of chloroform. The mixture was shaken vigorously for 15 seconds. The sample was then centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected. An equal volume of isopropanol was added to the supernatant, and the mixture was allowed to stand at room temperature for 10 min, followed by centrifugation at 7500 rpm for 10 min. Next, 1 mL of 75% ethanol was added to the sample, and the mixture was centrifuged again at 7500 rpm for 5 min. The resulting RNA was dissolved in 30–100 mL of RNase-free deionized water. cDNA synthesis was performed using a cDNA synthesis kit and PCR instrument. mRNA expression was detected using the SYBR Green Supermix kit, followed by quantification using a QuantStudio 3 real-time PCR instrument. Relative gene expression levels were measured using a 2-1 ⁻ΔΔCt Method calculation.

[0113] Western Blot: Mouse liver tissue was cryopreserved in liquid nitrogen and then ground into powder. The tissue was then treated with RIPA separation buffer containing 1% protease inhibitor for 1 h. The supernatant was obtained by centrifugation (12000 rpm, 15 min). Protein concentration was determined using a BCA protein assay kit. Electrophoresis (60-120 V), membrane transfer, and blocking were then performed. The membrane was incubated overnight at 4°C with the primary antibody. Subsequently, a secondary antibody was labeled with HRP (horseradish peroxidase) at a 1:10000 concentration and incubated at room temperature for 1 h. Finally, the membrane was treated with ECL solution, and immunoreactive proteins were detected using an ImageQuant 800 system.

[0114] 3.4.3 Experimental Results

[0115] The results are as follows Figure 17 The results showed that, compared with the control group, the expression of mRNAs of HIF1α, HK1, PFKL, ENO1, PFKFB3 (PFK2), CDKN1A (p21), IL-6, Rela (p65), and TNF-α was significantly increased in the APAP group. Conversely, OCLA treatment led to a significant decrease in the expression of these genes. Furthermore, Western blot results showed that APAP significantly increased the expression of the inflammatory marker p65 and the apoptosis proteins BAX / BCL2 and p21; however, OCLA treatment significantly decreased the levels of these proteins. These results suggest that OCLA may inhibit HIF1α transcription through interaction with HIF1α, thereby suppressing HIF1α-mediated glycolysis and reducing the expression of inflammatory factors, ultimately providing protection against APAP-induced liver injury.

[0116] The embodiments described above are merely specific implementations of this application, used to illustrate the technical solutions of this application, and not to limit them. The protection scope of this application is not limited thereto. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in this application, or make equivalent substitutions for some of the specific technologies; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application. All should be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the protection scope of the claims.

Claims

1. A ginsenoside sulfur-containing derivative, characterized in that, The structural formula of the sulfur-containing ginsenoside derivative is as follows: 。 2. The method for preparing ginsenoside sulfur-containing derivatives according to claim 1, characterized in that, Includes the following steps: α-Lipoic acid was weighed and added to 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide, which were dissolved in dichloromethane. The mixture was stirred at 30°C for 30 min, followed by the addition of dichloromethane and stirring for another 30 min. Then, octylene-type ginsenosides were added, and the reaction process was monitored in real time by TLC until the raw materials disappeared. The mixture was then extracted three times with saturated sodium chloride solution. The aqueous phases were combined, and anhydrous sodium sulfate was added to the organic phase. The mixture was concentrated under vacuum to obtain the crude product. The crude product was then poured into a chromatography column and eluted with pure dichloromethane. The eluent was collected and developed for qualitative analysis by thin-layer chromatography. The product was then eluted sequentially with dichloromethane:methanol at ratios of 200:1, 160:1, 150:1, and 100:1 to completely separate the target product. The product was then concentrated and dried by rotary evaporation. Finally, the product was dissolved in ethyl acetate and lyophilized to obtain the pure product.

3. The application of the ginsenoside sulfur-containing derivatives according to claim 1 in the preparation of drugs for treating liver damage.

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