Oral capsule for in-situ hydrogen production in intestinal tract as well as preparation method and application of oral capsule

Abstract

The invention discloses an oral capsule for in-situ hydrogen production in intestinal tracts as well as a preparation method and application of the oral capsule. According to the invention, an industrial catalytic hydrogen production technology is miniaturized into an oral capsule, so that hydrogen can be controllably, continuously and safely produced in vivo. The interior of the capsule is composed of a catalyst Ag / Pd nano-particles and a hydrogen carrier formic acid, and a capsule shell is composed of a medical material polydimethylsiloxane which is breathable, waterproof and resistant to gastric acid and digestive enzymes, so that the high efficiency of catalytic hydrogen production in the capsule, the stability of hydrogen release and the safety of complete metabolism of excrement are ensured. In mechanism, hydrogen regenerates ferroptosis related antioxidants CoQ10H2, VKH2 and BH4 through hydrogenation reduction, ferroptosis is effectively inhibited, and intestinal diseases are treated.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an oral capsule for in-situ hydrogen production in the intestine, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a major intestinal disease characterized by systemic inflammatory disorders of the gut (such as radiation enteritis and bacterial enteritis), causing numerous deaths worldwide and posing a significant challenge to global public health. Ferraphobia, an iron-dependent programmed cell death mechanism characterized by lipid peroxidation, is a key pathological mechanism mediating IBD. Cells possess a defense system against ferrophobia, primarily relying on the glutathione peroxidase 4 (GPX4) pathway and the parallel free radical scavenging antioxidant (RTA) pathway. The core molecules of the RTA pathway include coenzyme Q. 10 CoQ 10 Vitamins B, B, and C contain free radicals such as ferroptamine (BH2), reduced form of vitamin K (VKH2), and tetrahydrobiopterin (BH4). However, under inflammatory conditions, these free radical scavenging antioxidants are easily oxidized and depleted, thereby compromising the cell's own defense capabilities. Notably, methods of exogenously supplementing these antioxidants directly are greatly limited by their susceptibility to oxidation and chemical instability. Therefore, there is an urgent need for a strategy that can regenerate these substances in situ to inhibit ferroptosis and treat intestinal diseases.

[0003] Hydrogen, as an emerging therapeutic gas, is considered an ideal candidate for treating inflammatory bowel disease due to its selective antioxidant properties, high tissue penetration, and excellent biocompatibility. It also holds potential for in-situ regeneration of these free radical antioxidants. Current clinical hydrogen delivery methods, such as inhalation, oral administration of hydrogen-rich water, or injection of hydrogen-rich saline, share a common drawback: hydrogen has extremely low solubility in the body and is prone to rapid escape. The short half-life of hydrogen release makes it impossible to form and maintain a sufficiently high and sustained therapeutic concentration at the target intestinal lesion site, resulting in very limited efficacy. To address these issues, several technologies aiming for in-situ hydrogen generation have emerged, such as those based on photocatalysis, magnesium-based metals, or battery materials. While these technologies show great promise, they often suffer from unstable hydrogen production rates, difficulty in precise control, and unknown long-term biocompatibility in complex physiological environments, making them unsuitable for clinical applications requiring precise and long-term treatment. Therefore, the precise, stable, and safe release of hydrogen is crucial for the effective treatment of intestinal diseases and is a pressing technical challenge in the clinical application of gas medicine.

[0004] Therefore, a key unmet need remains in existing technologies: the need for a product that can safely, controllably, and continuously deliver therapeutically effective concentrations of hydrogen locally in the intestine. Furthermore, there is an urgent need for a novel therapeutic strategy that can effectively utilize hydrogen to combat ferroptosis, particularly by regenerating the endogenous antioxidant system to repair radiation damage. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an oral capsule for in-situ hydrogen production in the intestine, along with its preparation method and applications. The oral capsule of this invention can safely, controllably, and continuously generate therapeutic hydrogen gas in the intestine, achieving a therapeutic effect on intestinal diseases by inhibiting the biological mechanism of ferroptosis from a chemo-biological reduction perspective.

[0006] In a first aspect, the present invention provides an oral capsule for in-situ hydrogen production in the intestine, which is achieved through the following technical solution.

[0007] An oral capsule for in situ hydrogen production in the intestine, comprising: (a) A medical silicone rubber shell that is gas-permeable and liquid-impermeable; (b) A catalyst supported inside the shell, said catalyst being capable of catalyzing the decomposition of a hydrogen carrier to produce hydrogen under physiological conditions; (c) One or more hydrogen carriers contained within the shell.

[0008] Furthermore, the medical silicone rubber shell is a capsule shell formed by wrapping a medical silicone rubber polydimethylsiloxane (PDMS) base material around the surface of a gelatin capsule shell.

[0009] By adopting the above technical solution, the medical silicone rubber shell of this application allows hydrogen molecules generated internally to permeate, while the internal catalyst and hydrogen carrier remain inside the capsule. Furthermore, the capsule can block gastric acid and intestinal fluid, ensuring the integrity of capsule metabolism and exhibiting good biocompatibility.

[0010] Furthermore, the catalyst is selected from nanomaterials containing noble metals, specifically silver (Ag) and palladium (Pd).

[0011] Furthermore, the hydrogen support is selected from industrial catalytic hydrogen supports, including formic acid, benzoic acid, formate, and potassium diformate. Formic acid is preferred as the hydrogen support.

[0012] Furthermore, the mass ratio of the catalyst to the hydrogen support is (1-200):(40-3000). Preferably, the mass ratio of the catalyst to the hydrogen support is 1:46.

[0013] Secondly, the present invention provides a method for preparing an oral capsule for in-situ hydrogen production in the intestine, which is achieved through the following technical solution.

[0014] A method for preparing the above-mentioned oral capsule for in-situ hydrogen production in the intestine includes the following steps: S1. Capsule shell preparation: Polydimethylsiloxane substrate material and methyl vinyl cyclosiloxane crosslinking agent are stirred evenly in a mass ratio of 5:1-10:1 to form a mixture. The gelatin capsule shell is then fully coated with the mixture and cured at 40-120 ℃ for 0.5-24 h. After cooling, a polydimethylsiloxane-coated capsule shell is obtained. S2. Catalyst Synthesis: a. Dissolve silver nitrate and polyvinylpyrrolidone in ethylene glycol to form a homogeneous mixed solution; b. React the mixed solution at 100-150 ℃ for 20-40 min under an argon atmosphere, and then rapidly cool to room temperature; c. Add an ethylene glycol solution containing palladium nitrate and polyvinylpyrrolidone and mix thoroughly; wherein the mass ratio of silver nitrate to palladium nitrate is (2-3):(4-6); d. Then, under continuous argon protection, the reaction was carried out at 90-120 °C for 1.5-3 h. The synthesized nanoparticles were precipitated with acetone and washed, and finally dried at 40-80 °C under nitrogen atmosphere to obtain Ag / Pd nanoparticles. S3. Capsule construction: Ag / Pd nanoparticles are mixed with water and sonicated for 5-30 min; then the hydrogen carrier is dissolved in water, and the Ag / Pd nanoparticle solution and hydrogen carrier solution are injected into the capsule shell prepared in step S1 using a syringe. The injection volume is 50-90% of the capsule shell volume, and the mass ratio of Ag / Pd nanoparticles to hydrogen carrier in the capsule shell is (1-200):(40-3000), thus obtaining an oral capsule for in-situ hydrogen production in the intestine.

[0015] Furthermore, in step S2a, the mass ratio of silver nitrate to polyvinylpyrrolidone is (2-3):(10-20); in step S2c, the mass ratio of palladium nitrate to polyvinylpyrrolidone is (2-3):(5-10).

[0016] Preferably, in step S3, the injection volume is 80-90% of the capsule shell volume. Specifically, a 14 mm capsule is used. 3 When using capsule shells, the injection volume is 85.7% of the capsule shell volume; when using 140 mm... 3 When using capsule shells, the injection volume is 85.7% of the capsule shell volume; when using 680 mm... 3 When using capsule shells, the injection volume is 88.2% of the capsule shell volume.

[0017] Preferably, in step S3, the mass ratio of Ag / Pd nanoparticles to hydrogen support is 1:46.

[0018] Thirdly, the present invention provides the use of an oral capsule for in-situ hydrogen production in the intestine, which is achieved through the following technical solution.

[0019] The application of the above-mentioned oral capsule for in-situ hydrogen production in the intestine in the preparation of products for treating intestinal diseases.

[0020] Furthermore, intestinal diseases include radiation enteritis and bacterial enteritis.

[0021] Specifically, radiation enteritis refers to enteritis induced by radiation doses of 8-15 Gy; bacterial enteritis refers to enteritis caused by Escherichia coli at doses of 10 Gy. 9 -10 12 CFU / ml-induced intestinal inflammation.

[0022] The mechanism of action of the oral capsules in this application is as follows: The hydrogen released from the oral capsule is activated into active hydrogen atoms by an endogenous biocatalyst (such as iron porphyrin). Endogenous free radicals oxidized by radiation or inflammation are captured and reduced to their biologically active reduced forms by antioxidants. The regenerated antioxidants effectively scavenge lipid peroxidation free radicals, thereby interrupting and inhibiting ferroptosis, ultimately protecting intestinal epithelial cells and repairing the intestinal barrier function. Free radical scavenging antioxidants include reduced forms of the Coenzyme Q series (CoQ0, CoQ1, CoQ2, CoQ3, CoQ4, CoQ5, CoQ6, CoQ7, CoQ8, CoQ9, CoQ1). 10 Vitamin K, hydroquinone, tetrahydrobiopterin, and also include the oxidative effects of radiation, including ultraviolet, X-ray and gamma-ray, with a radiation dose of 8-40 Gy.

[0023] The present invention achieves the following beneficial effects.

[0024] (1) Controllable, continuous and long-lasting delivery of hydrogen is achieved: The oral delivery system provided by this invention can continuously produce hydrogen in the intestine for more than 20 hours, which is far superior to traditional hydrogen-rich water and other delivery methods, ensuring the continuous therapeutic concentration at the lesion site. Its hydrogen production performance can also be customized as needed by adjusting the components. (2) High biocompatibility: The present invention uses medical-grade PDMS packaging, which effectively isolates the catalytic core from the digestive tract environment, avoids material leakage and digestive enzyme degradation, and exhibits excellent biocompatibility and has great potential for clinical translation. (3) A novel mechanism of action was revealed and applied: This invention is the first to demonstrate and apply the therapeutic mechanism of hydrogen regeneration endogenous antioxidant to inhibit ferroptosis, fundamentally blocking the pathological cascade reaction of the disease and providing a novel approach for radiation protection and treatment of related diseases. Attached Figure Description

[0025] Figure 1 The images show actual capsule shells obtained from the preparation process (the left side shows commercially available gelatin capsule shells, and the right side shows capsule shells encapsulated with PDMS). Figure 2 This is a graph showing the results of the in vitro and in vivo hydrogen production performance evaluation of the capsules in this application (wherein, A. the cumulative hydrogen production at different silver / palladium nanoparticle concentrations determined by gas chromatography; B. the cumulative hydrogen production at different capsule sizes determined by gas chromatography; C. X-ray images of rats at different time points; D. a comparison of gray values ​​(relative hydrogen levels) of X-ray images of rats at different time points). Figure 3 This is a graph showing the results of the evaluation of the release of catalyst and hydrogen carrier inside the capsule and its resistance to gastric acid (where A. formic acid release from the capsule; B. silver / palladium nanoparticle release; C. capsule dissolution at different time points in simulated gastric juice (SGF) and simulated intestinal juice (SIF)). Figure 4 This is a diagram showing the in vivo safety and metabolic assessment results of the capsules in this application (where A. Capsule metabolism in vivo; B. Capsule metabolism in rat feces). Figure 5 This application is for capsules that are effective against CoQ. 10 Redox assessment results of VK and BH2 (where A. ionizing radiation or capsule effect on CoQ) 10 A schematic diagram of the redox remodeling of H2, VKH2, and BH4; B. UV absorption and mass spectra of the three substances reduced by the capsule. Figure 6 This is a diagram showing the evaluation results of the capsule's inhibitory effect on ferroptosis in this application (where A. electron micrograph of the capsule's inhibition of ferroptosis in MODE-K cells; B. image of the capsule's inhibition of ferroptosis in MODE-K cells using CoQ). 10 Flow cytometry plots of lipid peroxidation in cells co-cultured with capsules after treatment with enzyme inhibitors corresponding to H2, VKH2, and BH4; C. Calculations using CoQ 10 (Graph showing cell survival rates of cells treated with H2, VKH2, and BH4 enzyme inhibitors after co-culturing with capsules). Figure 7 The following are the treatment results of the capsules in this application on a radiation enteritis model (where A. Schematic diagram of the therapeutic effect of the capsules on radiation enteritis; B and C. H&E staining analysis diagrams after treatment of radiation enteritis with the capsules; D. Evaluation results of MPO and Nrf2 after treatment of radiation enteritis with the capsules). Figure 8The images show the treatment results of the capsules in the Escherichia coli-induced enteritis model (where A is a schematic diagram of the therapeutic effect of the capsules in Escherichia coli-induced enteritis; B is a diagram of colon length after treatment with the capsules for Escherichia coli-induced enteritis; and C is a diagram of H&E staining analysis after treatment with the capsules for Escherichia coli-induced enteritis). Figure 9 This is a schematic diagram of the catalytic hydrogen production oral delivery capsule of this application and its treatment of intestinal diseases by inhibiting ferroptosis. Detailed Implementation

[0026] The invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0027] The 14 mm used in the following embodiments of the present invention 3 Gelatin capsules (2 mm (diameter) × 4.5 mm (length)) were purchased from Shanghai Yuyan Scientific Instruments Co., Ltd., item number: YG00-2; 140 mm diameter capsules were used. 3 Gelatin capsules (4.9 mm (diameter) × 11.1 mm (length)) were purchased from Qingdao Yiqing Biotechnology Co., Ltd., item number: 5#; 680 mm gelatin capsules were used. 3 Gelatin capsules (7.6mm (diameter) × 21.7mm (length)) were purchased from Qingdao Yiqing Biotechnology Co., Ltd., item number: 0#; The polydimethylsiloxane and methyl vinylcyclosiloxane crosslinking agent used in the following embodiments of the present invention were purchased from Shenzhen Handa Import & Export Trading Co., Ltd., item number: Dow Corning DC184; The polyvinylpyrrolidone used in the following embodiments of the present invention was purchased from Aladdin, catalog number: P110611.

[0028] Example A method for preparing an orally delivered capsule for catalytic hydrogen production includes the following steps: (1) Capsule shell preparation: To make the capsules breathable but not water-permeable, PDMS was coated on the outer layer of the gelatin capsules. 20 g of PDMS base material and 2 g of methyl vinyl cyclosiloxane crosslinking agent were stirred evenly to form a mixture. The gelatin capsule shells (2 mm (diameter) × 4.5 mm (length), 4.9 mm (diameter) × 11.1 mm (length), and 7.6 mm (diameter) × 21.7 mm (length)) were then fully coated with the mixture and cured at 65 ℃ for 1 h. The final PDMS-coated capsule shells had dimensions of 2.8 mm (diameter) × 5.4 mm (length), 5.7 mm (diameter) × 12 mm (length), and 8.4 mm (diameter) × 22.5 mm (length), corresponding to internal volumes of 14, 140, and 680 mm², respectively. 3 ,like Figure 1 As shown.

[0029] (2) Catalyst Synthesis: In a 100 mL round-bottom flask, 25 mg of silver nitrate and 120 mg of polyvinylpyrrolidone (molecular weight 10000) were dissolved in 30 mL of ethylene glycol to form a homogeneous mixture. The mixture was reacted at 120 °C for 30 min under an argon atmosphere and then rapidly cooled to room temperature. Subsequently, 56 mg of palladium nitrate and 120 mg of polyvinylpyrrolidone (molecular weight 10000) were weighed and dissolved in 30 mL of ethylene glycol and thoroughly mixed. This mixture was added to the cooled reaction solution. The reaction was then carried out at 100 °C for 2 h under continuous argon protection. The synthesized silver / palladium nanoparticles were precipitated with acetone, washed three times with acetone, and dried at 60 °C under a nitrogen atmosphere.

[0030] (3) Capsule construction: First, prepare 5 mL of 2 mg / mL silver / palladium nanoparticle solution and sonicate for 5 min. Then, prepare 5 mL of 2 M formic acid solution. Finally, use an insulin syringe to inject the silver / palladium nanoparticle solution and formic acid solution (for an internal volume of 14 mm) into the PDMS-encapsulated capsule shell. 3 Capsule shell: Injected with 6 μL of silver / palladium nanoparticle solution and 6 μL of formic acid solution; for an internal volume of 140 mm². 3 Capsule shell: Injected with 60 μL of silver / palladium nanoparticle solution and 60 μL of formic acid solution; for an internal volume of 680 mm². 3 Capsule shell: Inject 300 μL of silver / palladium nanoparticle solution and 300 μL of formic acid solution, so that the final concentration of catalyst and hydrogen carrier in the capsule is 1 mg / mL silver / palladium nanoparticles and 1 M formic acid, to obtain an orally delivered capsule for catalytic hydrogen production (Hydrogen-Producing Capsule, HPC).

[0031] Performance Evaluation a. In vitro hydrogen production performance evaluation of the capsule: Qualitative and quantitative analysis of H2 produced by the capsule within 24 h was performed using gas chromatography. First, 300 μL of 2M formic acid and 300 μL of 0.5, 1, and 2 mg / mL Ag / Pd nanoparticles were injected into a 680 mm... 3 Within the capsule, 200 μL gas samples were extracted at time points of 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h and analyzed using gas chromatography (Thermo Fisher Trace 1600) (this procedure was repeated three times to ensure reproducibility). In addition, different volumes of 6, 60, and 300 μL of 2M formic acid and 6, 60, and 300 μL of 2 mg / mL Ag / Pd nanomaterial solution were injected into 14, 140, and 680 mm ablation sites, respectively. 3 Gas samples of 200 μL were extracted from the capsule at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and analyzed by gas chromatography (Thermo Fisher Trace 1600) (this process was repeated three times to ensure reproducibility).

[0032] like Figure 2 As shown in Figures A and 2B, gas chromatography analysis indicates that, within the linear range, the hydrogen generation rate increases with increasing catalyst concentration. Specifically, at silver / palladium nanoparticle loadings of 0.5, 1, and 2 mg / mL, the corresponding catalytic reaction rates were 0.24, 0.53, and 1.19 mM / h, respectively. At a Ag / Pd nanoparticle concentration of 2 mg / mL, the monitored gas yield was 10.3 mM, close to the theoretical hydrogen yield of 12 mM. The capsule maintained hydrogen release for approximately 20 hours at an Ag / Pd nanoparticle concentration of 0.5 mg / mL, significantly exceeding the approximately 30 min duration of hydrogen-rich water. Furthermore, different sizes (14, 140, and 680 mm) were also observed. 3 The capsules also demonstrated dose-dependent hydrogen generation, producing 0.8 mM, 4.9 mM, and 7 mM of hydrogen, respectively. These results indicate that the capsules can efficiently and controllably generate and continuously release hydrogen.

[0033] b. Evaluation of hydrogen production performance in the capsule: X-ray imaging was used to detect the gas. On the X-ray image, gas accumulation showed a low-density signal (manifested as a dark area), while in ultrasonic detection it showed a high-echo signal (manifested as a bright area). Specifically, 14 mm 3The capsule contained 2 μL of iohexol (300 mg iodine / mL), 6 μL of 2M formic acid, and 6 μL of 2 mg / mL Ag / Pd nanoparticles. The capsule was orally administered to rats via a rodent drug delivery system. X-ray imaging was performed at 0, 8, and 16 h post-administration, and the imaging data were then quantitatively analyzed using ImageJ software. Figure 2 As shown in C and 2D, the gray values ​​of the capsules increased significantly at 8 and 16 hours, indicating that hydrogen gas was produced inside the capsules.

[0034] c. Assessment of catalyst and hydrogen carrier release from inside the capsule: A 30 mL solution of simulated intestinal fluid (SIF) was mixed with 680 mm... 3 Capsules (300 μL of 2 M formic acid and 300 μL of 2 mg / mL Ag / Pd nanoparticles) were co-incubated, and 1 mL solutions were collected at 0, 3, 6, 12, 24, and 36 h. Each 1 mL solution was divided into two equal portions. 0.5 mL of the first portion was digested with aqua regia at 80 °C for 2 h, and the Ag ion concentration was quantitatively determined by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmerNexION 2000). The other 0.5 mL was analyzed by high-performance liquid chromatography (HPLC, ThermoUltimate 3000) using a ZORBAX SB-Aq 5 μm RP column (4.6 mm × 250 mm; column temperature 30 °C). The mobile phase was isocratic elution (95% acetonitrile; 5% ammonium acetate solution (50 mM)) at a flow rate of 1.0 mL / min. The experimental results are as follows: Figure 3 As shown in A and 3B, the maximum concentration of formic acid detected by HPLC was 2 μM, which is far below 5% of the endogenous short-chain fatty acid (SCFA) level and is within the safe range for human use. ICP-MS analysis confirmed the absence of Ag / Pd nanoparticle release from HPC.

[0035] d. Assessment of capsule resistance to gastric acid: Commercially available gelatin capsules and HPC were immersed in 10 mL of simulated gastric juice (SGF) and simulated intestinal juice (SIF), respectively. The mixture was stirred at 60 rpm, and the integrity of the capsules was visually assessed using digital photography at 0.5, 12, 24, and 48 h. Figure 3 As shown in Figure C, at 0.5 h, commercially available gelatin capsules dissolved rapidly in SGF and showed obvious damage and leakage in SIF, while HPC capsules maintained capsule integrity within 48 h without leakage or structural damage.

[0036] e. In vivo safety and metabolic assessment of the capsule: X-ray imaging was used to detect the specific location of the capsule in the rat intestine at different time points. Specifically, the PDMS-encapsulated capsule of this invention contained only 12 μL of iohexol (300 mg iodine / mL), ensuring sufficient imaging under X-ray to observe the capsule's metabolism. The capsule was orally administered to rats via a rodent drug delivery device. X-ray imaging was performed at 0, 2, 8, 16, and 24 h after administration. Subsequently, rats were administered the capsule via gavage every other day, and feces were collected every 1 h to examine fecal excretion. Figure 4 As shown, the feces were metabolized well in the intestines without any intestinal retention, and the capsules remained intact in the feces, indicating that the capsules have good biosafety.

[0037] f. Capsules for CoQ 10 Redox activity of VK and BH2: First, under argon protection, 2 mg VKH2 was dissolved in a mixture of 5 mL anhydrous ethanol and 400 μL deionized water, and 5.0 mg CoQ was added. 10 H2 was dissolved in 10 mL of ethanol solution containing 200 μL of dichloromethane, and 1.0 mg of BH4 was dissolved in 5 mL of water. Subsequently, the solutions were oxidized to CoQ by irradiation with 10 Gy X-ray. 10 VK and BH2 were used to record time-dependent violet-visible absorption spectra in the 200-600 nm range at specified time intervals (0, 3, 6, 12, and 24 h) using a violet-visible spectrophotometer. After 24 h, 1 mL of the mixed solution was taken for mass spectrometry analysis. Subsequently, iron porphyrin (Hematin, 100 μL, 5 mg / mL) was added, followed by the addition of 5 oral delivery capsules (140 mm³) for catalytic hydrogen production. 3 The capsule contained 60 μL of 2M formic acid and 60 μL of 2 mg / mL Ag / Pd nanoparticles. The reduction of VK to VKH2 and CoQ was monitored by recording the ultraviolet-visible absorption spectrum of the mixed solution. 10 Restore to CoQ 10 The process of reducing H2 and BH2 to BH4 was analyzed by taking 1 mL of the mixed solution for mass spectrometry.

[0038] like Figure 5 As shown, VKH2 and CoQ after radiation (IR) 10 H2 and BH4 were oxidized, and after treatment with capsules / Hematin, VK showed a characteristic disappearance of UV-Vis absorption at 265 nm. A mass-to-charge ratio peak of 453.3725 was also observed in the mass spectrometry results, further confirming the transformation of VK to VKH2; for CoQ... 10 After treatment with capsules / Hematin, the results of CoQ assays were confirmed by UV-Vis absorption spectroscopy and mass spectrometry.10 Restore to CoQ 10 H2; the same hydrogenation reduction effect was obtained for BH4. These results collectively indicate that the hydrogen gas generated in the capsule can effectively reduce VK and CoQ under the action of a biocatalyst (iron porphyrin). 10 BH2 is VKH2, CoQ 10 H2 and BH4.

[0039] g. Orally delivered capsules for catalytic hydrogen production via the hydrogenation reduction of CoQ 10 Evaluation of the inhibitory effects of VK and BH2 mechanisms on ferroptosis 1.5×10 5 MODE-K cells (1.5 × 10⁶ cells per well) 5 Cells were seeded into 6-well plates and cultured for 24 h in an incubator with 95% relative humidity and 5% carbon dioxide. Then, the cells were treated for 12 h with the following reagents: IR, 0.3 μM iFSP1 (FSP1 inhibitor, MCE), 1 μM Vidofludimus (VFD, DHODH inhibitor, MCE), and 1 μM methotrexate (MTX, dihydrofolate reductase inhibitor, MCE). The HPC group was cultured in 140 mm... 3 (60 μL 2M formic acid and 60 μL 2 mg / mL Ag / Pd nanoparticles) capsules were added to fresh culture medium. Hydrogen-rich water (HRW) group: Cells were cultured in hydrogen-rich cups (Rongtian, 3000 ppb R8 hydrogen cups) to obtain 1.6 ppm hydrogen-rich medium for co-culturing with cells. After 24 h, cells were treated with trypsin, collected, and resuspended in 150 μL of Ellerley balanced salt solution (EBSS) containing 5 μM C11-BODIPY581 / 591 for lipid peroxidation staining. After incubation at 37°C for 15 min, cells were analyzed by flow cytometry (Celesta, BD Biosciences, USA). Fluorescence intensity was quantified using FlowJo software (v10.8.1), with 2 × 10⁻⁶ cells recorded per sample. 4 Single-cell event.

[0040] like Figure 6 As shown, after IR treatment, mitochondrial cristae decreased or disappeared, and significant shrinkage occurred, which is a typical characteristic of ferroptosis. However, HPC treatment could significantly reverse mitochondrial damage and restore mitochondrial size and cristae structure. Figure 6A). Therefore, the mechanism by which HPC inhibits ferroptosis was further investigated. For the FSP1-VK axis, MODE-K cells were treated with the FSP1 inhibitor iFSP1 (200 nM). This blockade significantly exacerbated IR-induced lipid peroxidation and ferroptosis sensitivity, confirming the key role of FSP1-mediated VK reduction. Crucially, HPC treatment effectively eliminated this susceptibility, indicating that HPC bypasses the inhibited FSP1 and directly promotes the regeneration of reduced VKH2. Next, in order to induce CoQ... 10 In response to the systemic collapse of the defense system, this application combined iFSP1 with the mitochondrial DHODH inhibitor Vidofludimus (VFD, 1 μM). This dual inhibition enhanced oxidative stress, leading to a 1.5-fold increase in lipid peroxidation levels and a 20% decrease in cell viability. Surprisingly, HPC successfully reversed this phenotype, reducing lipid peroxidation levels to near baseline. These results demonstrate that HPC can replenish CoQ independently of endogenous reductase enzymes. 10 The H2 pool effectively neutralizes PL-PUFA-OO•. Finally, this application evaluated the BH4 pathway using methotrexate (MTX, 1 μM), a specific inhibitor of DHFR. Although MTX induced severe lipid peroxidation and cell death (survival rate decreased to 57%), HPC treatment significantly reduced lipid peroxidation to 16.9% and restored survival rate to >93%. These findings provide strong evidence that HPC possesses “enzyme mimicry” activity and can effectively replace DHFR in promoting the conversion of BH2 to BH4 (Figure 6B, C). It is evident that the HPC group showed significantly better therapeutic effects than the clinical dosing regimen (hydrogen-rich water, HRW group), which could not maintain the therapeutic concentration of hydrogen for a prolonged period, resulting in its limited therapeutic effect.

[0041] h. Application of orally delivered capsules for catalytic hydrogen production in intestinal inflammation models Model establishment and treatment of radiation enteritis: SD rats weighing 280 g ± 10 g were randomly divided into five groups: control group (PBS), radiation group (10 Gy X-ray irradiation), radiation + HRW (1.6 ppm) group, radiation + HPC (14 mm) group, and radiation + HPC (14 mm) group. 3 The study included two groups: a group containing 6 μL of 2M formic acid and 6 μL of 2 mg / mL Ag / Pd nanoparticles, and a group receiving radiation plus amifostine (AMF, 200 mg / kg) (a clinically approved radiation protection drug). For the radiation plus HPC group, 14 mm of amifostine was administered orally by gavage before radiation exposure. 3HPC was administered once, followed by twice-daily administration for four consecutive days after irradiation. The irradiation + HRW and irradiation + AMF groups received a single intraperitoneal administration before irradiation, followed by twice-daily administration for four consecutive days after irradiation. Small intestine was then collected for analysis at the end of the treatment period.

[0042] like Figure 7 As shown, H&E staining analysis revealed that radiation-induced mice exhibited villus loss accompanied by hemorrhage, structural damage, local glandular loss, and extensive inflammatory cell infiltration, characteristic of radiation-induced enteritis. The IR+HRW group showed muscle layer thickening, structural damage, and inflammatory cell infiltration. The IR+AMF group showed muscle layer structural damage and hemorrhage, villus shortening, and inflammatory cell infiltration. In contrast, the IR+HPC group preserved villus structure, reduced mucosal damage, and maintained almost intact villi and orderly glands. Furthermore, villus length and gland depth were restored to the control group levels. These results indicate that HPC has a superior intestinal protective effect against radiation-induced enteritis compared to the hydrogen-rich water group and the amifostine group. Figure 7 B, C). Subsequently, the antioxidant capacity of HPC was assessed by immunofluorescence staining of small intestinal tissue for MPO and Nrf2. The fluorescence intensity of myeloperoxidase (MPO) in the HPC group was significantly lower than that in the hydrogen-rich water group and the amifostine group, indicating that it has a significant anti-inflammatory effect. As a central regulator of antioxidant defense, nuclear factor erythrocyte-associated factor 2 (Nrf2) translocates to the nucleus and activates the transcription of cytoprotective genes that can neutralize reactive oxygen species. The expression of nuclear Nrf2 was further increased in the HPC group, indicating that the endogenous antioxidant system was activated to resist oxidative damage. Figure 7 D).

[0043] Construction and treatment of Escherichia coli-induced enteritis model: First, SD rats weighing 280 g ± 10 g were administered 10 mg of Escherichia coli-induced enteritis model via oral gavage. 9 To establish an E. coli-induced enteritis model, rats were administered a CFU / mL E. coli (CICC 10411) solution twice daily for 7 consecutive days. These rats were then randomly divided into two groups: an E. coli group and an E. coli + HPC group. The treatment group received 14 ml of the solution orally via gavage daily. 3 HPC was performed twice, and at the end of the experiment, the small intestine was collected for analysis.

[0044] like Figure 8 As shown, HPC treatment significantly reduced weight loss and colon shortening. HE staining revealed that E. coli infection caused villus structure destruction, muscle layer hemorrhage, and inflammatory cell infiltration. In contrast, animals receiving HPC treatment maintained normal epithelial structure, with villus length and crypt depth being the same as the control group.

[0045] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An oral capsule for in situ hydrogen production in the intestinal tract, characterized in that: Comprising (a) a medical silicone rubber shell with gas permeability and liquid impermeability; (b) a catalyst loaded inside the shell, which is capable of catalyzing the decomposition of hydrogen carriers to produce hydrogen gas under physiological conditions; (c) one or more hydrogen carriers contained inside the shell.

2. An oral capsule for in situ hydrogen production in the intestinal tract according to claim 1, characterized in that: The medical silicone rubber shell is a medical silicone rubber polydimethylsiloxane base material wrapped in a capsule shell formed on the surface of a gelatin capsule shell.

3. The oral capsule for in situ hydrogen production in the intestinal tract according to claim 1, characterized in that: The catalyst is selected from nanomaterials containing noble metals, and the noble metals are selected from silver and palladium.

4. The oral capsule for in situ hydrogen production in the intestinal tract according to claim 1, characterized in that: The hydrogen carrier is selected from industrial catalytic hydrogen carriers, which include formic acid, benzoic acid, formate, and potassium dicarboxylate.

5. The oral capsule for in situ hydrogen production in the intestinal tract according to claim 1, characterized in that: The mass ratio of catalyst to hydrogen carrier is (1-200):(40-3000).

6. A process for the preparation of an oral capsule for in situ hydrogen production in the intestinal tract as claimed in any one of claims 1 to 5, characterized by: Comprising the following steps: S1. Capsule shell preparation: uniformly stir polydimethylsiloxane base material and methylvinylcyclosiloxane crosslinking agent in a mass ratio of 5:1-10:1 to form a mixed solution, then fully wrap the gelatin capsule shell with the mixed solution, and then solidify at 40-120 ℃ for 0.5-24 h, and obtain a polydimethylsiloxane wrapped capsule shell after cooling; S2. Catalyst synthesis: a. Dissolve silver nitrate and polyvinylpyrrolidone in ethylene glycol to form a homogeneous mixed solution; b. React the mixed solution under argon atmosphere at 100-150 ℃ for 20-40 min, and cool quickly to room temperature; c. Add an ethylene glycol solution containing palladium nitrate and polyvinylpyrrolidone and mix thoroughly; wherein the mass ratio of silver nitrate to palladium nitrate is (2-3):(4-6); d. Then react at 90-120 ℃ for 1.5-3 h under continuous protection of argon, and the synthesized nanoparticles are precipitated with acetone and washed, and finally dried under nitrogen atmosphere at 40-80 ℃ to obtain Ag / Pd nanoparticles; S3. Capsule construction: mix Ag / Pd nanoparticles with water and ultrasonic for 5-30 min; then dissolve the hydrogen carrier in water, and then use a syringe to inject the Ag / Pd nanoparticle solution and the hydrogen carrier solution into the capsule shell prepared in step S1, the injection volume is 50-90% of the volume of the capsule shell, and the mass ratio of Ag / Pd nanoparticles to hydrogen carrier in the capsule shell is (1-200):(40-3000), to obtain an oral capsule for in situ hydrogen production in the intestinal tract.

7. A process for the preparation of an oral capsule for in situ hydrogen production in the intestinal tract according to claim 6, characterized in that: In step S2a, the mass ratio of silver nitrate to polyvinylpyrrolidone is (2-3):(10-20); and in step S2c, the mass ratio of palladium nitrate to polyvinylpyrrolidone is (2-3):(5-10).

8. Use of the oral capsule for in situ hydrogen production in the intestinal tract according to any one of claims 1-5 in the preparation of a product for treating intestinal diseases.

9. Use according to claim 8, characterized in that: Intestinal diseases include radiation enteritis and bacterial enteritis.

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