Stigmasterol hydrogen sulfide derivative and application thereof in anti-inflammation, anti-oxidation and anti-fibrosis

Abstract

The invention belongs to the technical field of drug development, and particularly relates to stigmasterol hydrogen sulfide derivatives and application thereof in anti-inflammation, anti-oxidation and anti-fibrosis. The molecular formula of the stigmasterol hydrogen sulfide derivative is C42H56O4S3. The stigmasterol hydrogen sulfide derivative disclosed by the invention can be used as an anti-inflammatory, anti-oxidation and anti-fibrosis drug, and shows an ear swelling inhibition effect equivalent to that of a positive drug diclofenac in acute inflammation; the pain threshold is obviously improved when the compound is used as an analgesic drug. The compound has good safety, and CCK-8 experiments show that the compound has no significant influence on the survival rate of macrophages under the treatment concentration (2-16 [mu] M), and has low cytotoxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of drug development, and particularly relates to stigmasterol hydrogen sulfide derivatives and applications thereof in anti-inflammatory, anti-oxidation and anti-fibrosis. Background Art

[0002] Inflammation is a complex physiological response to injury, infection, or irritation. It aims to protect the body, promote healing, and restore tissue homeostasis, playing a critical role in maintaining health and defending against external threats. However, when the inflammatory response becomes dysregulated or persists for too long, it can lead to a range of pathological conditions. Persistent infection can develop into systemic inflammatory response syndrome, which in severe cases can lead to multiple organ failure and be life-threatening. Therefore, regulating the inflammatory response to maintain normal immune balance is crucial.

[0003] Oxidative stress is a pathological condition in which the body, under harmful stimuli, generates excessive free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), exceeding the clearance capacity of the endogenous antioxidant system, leading to damage to biomolecules such as lipids, proteins, and DNA. Oxidative stress is a key factor in the development of inflammatory diseases.

[0004] Fibrosis is a pathological process in which normal tissue is replaced by abnormally proliferating fibrous connective tissue due to chronic injury or inflammation, leading to organ structural destruction and functional failure. Fibrosis is a major cause of disability and mortality in many diseases. According to statistics from the United States, nearly 45% of deaths from various illnesses in the country can be attributed to fibrotic disorders. Fibrosis plays a significant role in the development and progression of diseases affecting all major organs in the human body.

[0005] Currently, commonly used anti-inflammatory drugs fall into two main categories: steroids and nonsteroidal anti-inflammatory drugs (NSAIDs), with dexamethasone, aspirin, and diclofenac being common. Antioxidants include N-acetylcysteine, glutathione, and vitamin C. Antifibrotic drugs include pirfenidone and nintedanib. These anti-inflammatory drugs still have significant drawbacks. Steroids can cause serious side effects such as edema, hypertension, diabetes, muscle atrophy, and adrenal insufficiency. Long-term use can also lead to dependence and immune decline, increasing the risk of infection. Furthermore, children are more restricted in their use of steroids to avoid disrupting their growth and development. While NSAIDs offer lower risks of dependency and growth, they are commonly associated with gastrointestinal side effects, cardiovascular risks, and potential for iron deficiency anemia and platelet dysfunction. These issues severely limit the use of current anti-inflammatory drugs, making enhancing the safety of classic anti-inflammatory drugs an urgent issue.

[0006] Currently, commonly used antioxidant drugs in clinical practice have problems such as low bioavailability, a single mechanism of action, and some drugs may interfere with hemoglobin synthesis, causing anemia. These problems make it difficult to cope with complex disease conditions.

[0007] Currently, common adverse reactions of commonly used anti-fibrosis drugs in clinical practice include photosensitivity, gastrointestinal reactions (nausea, anorexia), etc. There are also problems such as low bioavailability and the possibility of abnormal liver function.

[0008] In recent years, the anti-inflammatory activity of plant sterols has garnered considerable research interest. Stigmasterol, in particular, exerts anti-inflammatory and antioxidant effects by regulating inflammatory factors and scavenging oxygen free radicals, showing potential therapeutic benefits for a variety of inflammatory-related diseases. Hydrogen sulfide, a third gaseous signaling molecule discovered in recent years, has positive effects on cardiovascular and nervous system function, as well as immune regulation. Furthermore, hydrogen sulfide can provide significant gastrointestinal protection by improving gastrointestinal mucosal blood supply and protecting its integrity. It can also protect pancreatic beta cells and regulate sugar metabolism in various organs, potentially preventing the onset of diseases such as diabetes.

[0009] From a pharmaceutical perspective, stigmasterol, as a plant sterol, is highly safe. As a natural plant extract, long-term use is less likely to produce serious side effects. Furthermore, modern pharmacological research has shown that stigmasterol not only has anti-inflammatory effects but also possesses multiple pharmacological activities, including antioxidant and immunomodulatory properties. This multi-target effect gives it potential advantages in treating complex diseases. Its mechanism of action involves multiple targets, including regulating cholesterol metabolism and intervening in cell cycle progression.

[0010] In terms of acquisition channels, stigmasterol is widely present in oil crops such as soybeans and rapeseed oil, and medicinal plants such as traditional Chinese medicine astragalus and ginseng. It is relatively easy to obtain. Stigmasterol has the potential to become a new anti-inflammatory drug.

[0011] Stigmasterol is a white crystalline powder with strong lipid solubility. Its water solubility at 25°C is only 0.002 mg / mL. Its oral bioavailability is less than 5%, and it has defects such as poor metabolic stability and insufficient tissue distribution specificity. It has poor drugability and is difficult to prepare into a stable pharmaceutical preparation.

[0012] Patent CN117567539A provides a method for the synthesis and application of a stigmasterol hydrogen sulfide derivative. The application of the drug is only mentioned for acute inflammation, without studying other inflammation-related diseases or its antioxidant and anti-fibrotic therapeutic effects on complex diseases. Summary of the Invention

[0013] The purpose of the present invention is to provide a stigmasterol hydrogen sulfide derivative with anti-inflammatory activity and its application in anti-inflammatory, anti-oxidation and anti-fibrosis.

[0014] In order to solve the problems existing in the prior art, the present invention adopts the following technical solutions: Stigmasterol hydrogen sulfide derivatives, wherein the molecular formula of the stigmasterol hydrogen sulfide derivatives is C 42 H 56 O4S3.

[0015] The above-mentioned stigmasterol hydrogen sulfide derivative has stigmasterol as its parent body and includes a hydrogen sulfide-releasing group.

[0016] Use of stigmasterol hydrogen sulfide derivatives in the preparation of anti-inflammatory drugs.

[0017] Use of stigmasterol hydrogen sulfide derivatives in the preparation of antioxidant drugs.

[0018] Use of stigmasterol hydrogen sulfide derivatives in the preparation of anti-fibrosis drugs.

[0019] The stigmasterol hydrogen sulfide derivatives of this invention can be used as anti-inflammatory, antioxidant, and anti-fibrotic drugs. They exhibit comparable anti-ear swelling inhibition effects to the active drug diclofenac in acute inflammation and significantly increase pain thresholds when used as an analgesic. They also have a favorable safety profile. CCK-8 assays have shown no significant effect on macrophage survival at therapeutic concentrations (2-16 μM) and low cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the effect of DZCD-1 on xylene-induced ear swelling in mice; Figure 2 This figure shows the effect of DZCD-1 on the degree of fibrosis, the release of inflammatory factors and the level of oxidative stress in the isoproterenol-induced myocardial fibrosis model in mice; Figure 3 This is a graph showing the analgesic activity of DZCD-1 of the present invention against acetic acid-induced painful writhing in mice; Figure 4 This figure shows the effect of DZCD-1 on the degree of liver damage, the release of inflammatory factors and the level of oxidative stress in mice in an acute liver injury model induced by LPS combined with D-GalN; Figure 5 This figure shows the effect of different concentrations of DZCD-1 on the survival rate of macrophages treated for 24 hours; Figure 6 This is a graph showing the effect of DZCD-1 of the present invention on the oxidative stress level of zebrafish induced by LPS. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0022] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0023] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0024] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0025] Example 1

[0026] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56 O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0027] This example was applied to the xylene-induced ear swelling test in mice, and the dosage range for mice was 15-100 mg / kg.

[0028] Male Kunming mice, weighing 18-22 g, were housed in a room at a temperature of 20-24°C and a humidity of 65-70%. They were fed with standard feed. The bedding and food were changed daily and the cages were kept clean.

[0029] The test compound was prepared as a suspension in 0.5% CMC-Na solution and administered at a dose of 45.3 mg / kg. The positive control drug, diclofenac, was administered at a dose of 20 mg / kg, and eugenol at a dose of 25.9 mg / kg. The molar doses of the three drugs were comparable. Six mice were enrolled in each group and fasted for 12 hours before dosing, with free access to water. The test and positive drugs were administered orally at a volume of 0.2 mL per 10 g body weight. One and a half hours after dosing, 20 μL of xylene was evenly applied to the inner and outer sides of the right auricle using a microinjector to induce inflammation. The left auricle served as a control. 0.5 hour after inflammation, the mice were sacrificed by cervical dislocation. Both ears were removed along the base of the auricle. A 7 mm diameter ear piece was removed from each ear at the same location and weighed using an electronic balance. The degree of swelling was calculated by subtracting the weight of the control ear piece from the weight of the inflamed ear piece. The swelling degrees of the model and treatment groups were statistically analyzed.

[0030] The anti-inflammatory activity of DZCD-1 was evaluated using the xylene-induced ear swelling model in mice. Figure 1 .

[0031] from Figure 1 It can be seen that DZCD-1 exhibits a strong ear swelling inhibitory effect, which is significantly different from stigmasterol and is comparable to the positive drug diclofenac, indicating that the anti-inflammatory activity of stigmasterol is significantly enhanced after the hydrogen sulfide-releasing group is connected.

[0032] The results of Example 1 show that DZCD-1 has a high anti-inflammatory activity against acute inflammation.

[0033] Example 2

[0034] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56 O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0035] This example was applied to an isoproterenol-induced myocardial fibrosis test in mice.

[0036] Male Kunming mice, weighing 18-22 g, were housed at a temperature of 20-24°C and a humidity of 65-70%. They were fed with a standard diet, and the bedding and food were changed daily to keep the cages clean.

[0037] The test compound was prepared as a suspension in 0.5% CMC-Na solution and administered at low (15 mg / kg), medium (30 mg / kg), and high (60 mg / kg) doses. The positive drug captopril was prepared in saline at a dose of 25 mg / kg. ISO was dissolved in saline to prepare a 50 mg / mL working solution.

[0038] Mice were divided into six groups: a control group, a model group, a captopril group, and groups with different concentrations of DZCD-1, with six mice per group. The DZCD-1 treatment groups received the corresponding doses of drug daily via oral gavage. The captopril group received the positive drug captopril. The control and model groups received equal amounts of vehicle. Three days later, each group, except the control group, received a subcutaneous injection of isoproterenol working solution into the back at a volume of 0.1 mL / kg. After the modeling period, the mice were sacrificed, and serum and heart tissue were collected.

[0039] The inflammatory factor IL-1β was measured in mouse serum using a kit. An appropriate amount of heart tissue was ground and assayed for SOD and MDA levels, as well as the fibrosis marker hydroxyproline, using a kit. The data were statistically analyzed.

[0040] Figure 2The results showed that the test drug DZCD-1 significantly reduced IL-1β levels in mouse serum and hydroxyproline levels in heart tissue, revealing that it effectively inhibited chronic inflammation and alleviated myocardial fibrosis in mice in a dose-dependent manner. Furthermore, the test drug DZCD-1 significantly reduced oxidative stress levels in mouse heart tissue.

[0041] The results of Example 2 show that DZCD-1 also has a high inhibitory and alleviating effect on chronic inflammation and fibrosis and has strong antioxidant activity.

[0042] Example 3

[0043] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56 O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0044] This example was applied to the acetic acid-induced painful writhing test in mice.

[0045] Male KM mice, weighing 18-22 g, were housed at a temperature of 20-24°C and a humidity of 65-70%. They were fed with a standard diet. The bedding and food were changed daily and the cages were kept clean.

[0046] The test compound was prepared as a suspension in 0.5% CMC-Na solution and administered at a dose of 45.3 mg / kg. The positive control drug, diclofenac, was administered at a dose of 20 mg / kg, and eugenol at a dose of 25.9 mg / kg. The molar doses of the three drugs were comparable. Six mice were enrolled in each group and fasted for 12 hours before dosing with free access to water. The test and positive drugs were administered orally at a volume of 0.2 mL / 10 g body weight. Approximately 1 hour after dosing, 0.6% acetic acid solution was injected intraperitoneally at a volume of 0.1 mL / 10 g. The test temperature was 28 ± 1°C. The onset of the first writhing was recorded and designated as the threshold. The number of writhings in the following 15 minutes was measured (a complete writhing response was defined as a behavioral response characterized by abdominal indentation, extension of the trunk and hind limbs, and elevation of the hips). The threshold and number of writhings in the model and treatment groups were statistically analyzed.

[0047] Figure 3 The results showed that compared with the model group, the threshold and the number of writhing times of mice within 15 minutes in the DZCD-1 group were significantly reduced (P<0.01), indicating that the test drug DZCD-1 has a strong analgesic effect. Its analgesic effect is not statistically different from that of the positive drug diclofenac, and its analgesic effect is stronger than that of stigmasterol.

[0048] The results of Example 3 show that DZCD-1 has analgesic effect on pain caused by inflammation.

[0049] Example 4

[0050] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56 O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0051] This example was applied to the LPS combined with D-GalN-induced acute liver injury test in mice.

[0052] Male Kunming mice, weighing 18-22 g, were housed at a temperature of 20-24°C and a humidity of 65-70%. They were fed with a standard diet, and the bedding and food were changed daily to keep the cages clean.

[0053] The test compounds were prepared as suspensions in 0.5% CMC-Na solution and administered at low (25 mg / kg), medium (50 mg / kg), and high (100 mg / kg) doses. The positive drug, dexamethasone, was prepared in 5% hydroxypropyl-β-cyclodextrin solution and administered at a dose of 10 mg / kg. Mice were divided into six groups: control, model, dexamethasone, and different DZCD-1 concentration groups, with six mice in each group. They were fasted for 12 hours before administration and had free access to water. The test and positive drugs were administered orally at a volume of 0.2 mL / 10 g body weight. One hour after administration, mice in all groups except the control group received an intraperitoneal injection of the modeling working solution (0.1 mL / 10 g); the control group received an equal volume of normal saline. Six hours later, the mice were sacrificed and serum collected.

[0054] Kits were used to measure serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the inflammatory factor IL-1β. Appropriate amounts of liver tissue were minced and assayed for levels of superoxide dismutase (SOD) and malondialdehyde (MDA) using kits. The data were statistically analyzed.

[0055] Figure 4 The results showed that the test drug DZCD-1 significantly reduced the enzyme activities of alanine aminotransferase and aspartate aminotransferase in the mouse serum, and also significantly reduced the level of IL-1β in the serum, suggesting that it protects against acute liver injury in mice by inhibiting inflammation. In addition, DZCD-1 significantly reduced the level of oxidative stress in the mouse liver tissue.

[0056] The results of Example 4 show that DZCD-1 also has an antioxidant effect.

[0057] Example 5

[0058] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0059] This example was applied to a cytotoxicity test to detect the effects of different concentrations of DZCD-1 on cell viability.

[0060] Human macrophage THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0061] The test compounds were prepared in DMSO to a concentration of 2-8 mM and diluted in culture medium to the desired concentration.

[0062] Cell culture: The cell lines were inoculated into RPMI-1640 medium containing 10% fetal bovine serum and cultured in an incubator with 5% CO2 and saturated humidity at 37°C. The cells were passaged and the medium was replaced in time.

[0063] Take THP-1 cells in the logarithmic growth phase, discard the culture medium in the dish, blow off the cells with 1 mL of fresh culture medium, gently pipette to mix, and then inoculate into 96-well plates, with 10 μL of cell suspension per well, and add serum-containing RPMI-1640 culture medium to 100 μL, and continue to culture in the incubator.

[0064] When the cell density reaches 80%, discard the culture medium from the plate and add 100 μL of culture medium containing various concentrations of DZCD-1 (1, 2, 4, 8, and 16 μM). A control group containing 0.1% DMSO and a blank group without cells should be set up, with six replicates per group. Continue incubation for 24 hours. Aspirate the culture medium, wash the cells with sterile PBS, and add 110 μL of culture medium containing 10% CCK-8 reagent to each well. After incubation at 37°C in the dark for approximately 1 hour, use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm. Calculate the cell viability of each group according to the following formula: Survival rate (%) = (mean absorbance of the test group - mean absorbance of the blank group) / (mean absorbance of the control group - mean absorbance of the blank group) × 100% Figure 5 The results showed that the survival rates of the groups at concentrations of 2-16 μM were all greater than 95% and there was no significant difference.

[0065] Example 5 shows that DZCD-1 has no obvious toxicity to cells at therapeutic concentrations.

[0066] Example 6

[0067] This embodiment discloses a stigmasterol hydrogen sulfide derivative, the molecular formula of which is C 42 H 56O4S3, the stigmasterol hydrogen sulfide derivative is based on stigmasterol and includes a hydrogen sulfide-releasing group, which continuously releases H2S gas in vivo and in vitro.

[0068] This example is applied to zebrafish oxidative stress test.

[0069] The wild-type AB strain zebrafish used in the experiment were purchased from Shanghai Feixi Biological The test compounds were prepared as suspensions in 0.5% CMC-Na solution and administered at low (1.5 μg / mL), medium (3 μg / mL), and high (6 μg / mL) doses. The positive drug dexamethasone was prepared in 5% hydroxypropyl-β-cyclodextrin solution and administered at a dose of 5 μg / mL. Zebrafish were divided into six groups: control group, model group, dexamethasone group, and groups with different concentrations of DZCD-1.

[0070] All groups except the control group were treated with LPS working solution to a final concentration of 10 μg / mL, to which an equal volume of solvent was added. Each drug-treated group received the corresponding drug concentration, and the model group received an equal volume of solvent. Cultures were continued for 48 hours, with two-thirds of the culture medium replaced every 24 hours. After model construction, DCFH-DA working solution or DPPP probe was added to each group. The cells were incubated in a dark incubator for 1 hour and then observed and photographed using an EVOS fluorescence microscope.

[0071] Figure 6 The results showed that the levels of reactive oxygen species and lipid peroxides in zebrafish of different groups were positively correlated with the fluorescence intensity.

[0072] Example 6 shows that DZCD-1 has strong antioxidant activity.

[0073] The present invention demonstrates DZCD-1's potent anti-inflammatory activity across a broad range using acute inflammation models (ear swelling test and liver injury test) and chronic inflammation models (myocardial fibrosis test). Furthermore, the acetic acid writhing test and the aforementioned myocardial fibrosis test demonstrate that DZCD-1 also exhibits significant analgesic and anti-fibrotic effects. Furthermore, DZCD-1's excellent antioxidant activity has been demonstrated in liver injury and myocardial fibrosis tests, as well as in a zebrafish oxidative stress model, effectively reducing oxidative stress levels in vivo.

[0074] The present invention verifies through cytotoxicity tests that DZCD-1 has no obvious toxicity to cells at therapeutic concentrations and has good safety.

[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A stigmasterol hydrogen sulfide derivative, characterized in that: The molecular formula of the stigmasterol hydrogen sulfide derivative is C 42 H 56 O4S3.

2. The stigmasterol hydrogen sulfide derivative according to claim 1, characterized in that The stigmasterol hydrogen sulfide derivative takes stigmasterol as a parent body and includes a hydrogen sulfide-releasing group.

3. Use of the stigmasterol hydrogen sulfide derivative according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

4. Use of the stigmasterol hydrogen sulfide derivative according to claim 1 or 2 in the preparation of antioxidant drugs.

5. Use of the stigmasterol hydrogen sulfide derivative according to claim 1 or 2 in the preparation of anti-fibrosis drugs.

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