Preparation and in Vitro-in Vivo Evaluation of Novel Monoammonium Glycyrrhizinate Controlled-porosity Osmotic Pump

Preparation and in Vitro-in Vivo Evaluation of Novel Monoammonium Glycyrrhizinate Controlled-porosity Osmotic Pump | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preparation and in Vitro-in Vivo Evaluation of Novel Monoammonium Glycyrrhizinate Controlled-porosity Osmotic Pump Xuan Lu, Hongfei Yang, Jianli Chen, Yajun Chen, Xingxing Chai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8227583/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 May, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background This study aimed to prepare and evaluate novel monoammonium glycyrrhizinate (MAG) controlled-porosity osmotic pumps with sustained and controlled release properties. Methods Utilizing the impregnation technique, we fabricated the capsule shells and optimized critical factors influencing in vitro release through the Box-Behnken design-response surface methodology. The optimized formulation exhibited a distinct zero-order release pattern for the primary metabolite, glycyrrhetinic acid (GA-30), over 12 hours in vitro. SEM analysis confirmed the successful operation of the Control pore infiltration pump mechanism. Results In vivo, experiments showed that the peak plasma concentrations of GA and glycyrrhizinic acid in rabbits of the prepared capsule were reduced and the mean residence time was prolonged with statistically significant differences compared with the reference formulation, which indicated that the homemade capsule had significantly slow and controlled release characteristics. Molecular docking experiments confirmed the prolongation of the residence time of GA, especially glycyrrhizinic acid, in vivo from the perspective of binding energy. This study also characterized the physical state of MAG and its formulations using differential scanning calorimetry (DSC) and X-ray diffraction (XRD), ensuring the stability of the drug in the formulation. In addition, the in vitro release curve of the controlled-porosity osmotic pump is highly consistent with the expected target curve, verifying the accuracy and reliability of the formulation design. Conclusions This research contributes to the development of an advanced drug delivery system for MAG, enhancing its pharmacological benefits, and offers significant advancements in controlled-release formulations. Biological sciences/Biotechnology Physical sciences/Chemistry Biological sciences/Drug discovery Physical sciences/Materials science Controlled-porosity osmotic pump Monoammonium glycyrrhizinate Cumulative release Response surface methodology Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Monoammonium glycyrrhizinate (MAG), is a second-generation extract from the root of traditional Chinese medicine Radix Glycyrrhiza (licorice), which is known to have a variety of pharmacological effects, including hepatocyte protection, anti-inflammation, and immunomodulation[ 1 , 2 ]. Currently, its dosage forms mainly include injections, regular capsules, and tablets[ 3 – 5 ]. Considering the low adherence to injections in patients with chronic liver disease, regular capsules, and tablets usually need to be taken orally more than once a day. This is particularly challenging for patients who need to take medication for a long period, especially the elderly who tend to forget when to take their medication. In addition, frequent dosing can lead to large fluctuations in blood drug concentration, which can lead to safety risks and inappropriate use, resulting in liver damage. Control pore infiltration pump formulations are characterized by zero-order release kinetics and provide stable blood drug concentrations, making them by far the most desirable oral controlled-release formulations[ 6 , 7 ]. The controlled-porosity osmotic pump is a novel Control pore infiltration pump dosage form combining capsule formulations with Control pore infiltration pump technology. It is characterized by a simple preparation process without the need for tableting or coating, low production costs, and the capsule shell and contents can be prepared separately, providing flexible dosing options[ 8 , 9 ]. Controlled-porosity osmotic pump, as a remarkable achievement in modern pharmaceutical formulations, play a pivotal role in drug delivery systems. Their unique controlled-release mechanism enables the constant-rate delivery of drugs, significantly enhancing drug efficacy, reducing dosing frequency, and optimizing the patient's treatment experience[ 10 , 11 ]. The stable release pattern maintains effective drug concentrations in the target organs, minimizing drug wastage and alleviating the psychological burden associated with frequent dosing. Especially for patients requiring long-term treatment, as well as elderly and pediatric patients, Controlled-porosity osmotic pumps notably improve medication adherence and therapeutic outcomes. Furthermore, their stable release profile diminishes adverse drug reactions, enhancing medication safety[ 12 ]. With advancements in pharmaceutical technology, the application prospects of controlled-porosity osmotic pumps are poised to broaden even further. The controlled-porosity osmotic pump, an advanced drug delivery system, depends on dissolution, diffusion, and metabolism for release. Metabolite properties are vital for drug efficacy and safety evaluations. In these capsules, metabolites directly affect drug distribution, excretion, and bioavailability[ 13 ]. Thus, metabolite characteristics must be considered during design and optimization to ensure effective delivery and patient safety. Previous GA formulations have unique attributes, but the controlled-porosity osmotic pump offers distinct advantages. Future research should emphasize metabolite properties and release mechanisms to optimize design and improve therapeutic outcomes[ 14 , 15 ]. Given the above considerations, it is particularly urgent to investigate and develop extended-release and controlled-release formulations that can prolong the efficacy of MAG, reduce the number of oral administrations, decrease fluctuations of blood drug concentration, and improve patient compliance and safety. This study aimed to combine MAG as the main active pharmaceutical ingredient with a novel controlled-porosity osmotic pump dosage form. We optimized the preparation of MAG controlled-porosity osmotic pumps using a Box-Behnken design-response surface methodology and investigated the drug release mechanism and pharmacokinetic behavior by measuring the blood drug concentration in rabbits. 2 Materials and Methods 2.1 Selection of Preparation Methods for Penetrating Pump Capsule Shell In this study, the preparation methods for the Controlled-porosity osmotic pump shells were meticulously selected and optimized. The experimental materials comprised cellulose acetate (CAS 9004-35-7, sourced from Shanghai Aladdin), PEG2000 (CAS 25322-68-3, obtained from Shanghai Macklin), PEG400 (also CAS 25322-68-3, but from Shanghai Aladdin), acetone (CAS 67-64-1, supplied by Shantou Xilong), anhydrous ethanol (CAS 64-17-5, acquired from Wuxi Zhanwang), and size 0 capsule shells (provided by Ningbo Jiangnan). The equipment utilized during the process included an ultrasonic cleaner (model KQ5200DE, manufactured by Kunshan), an electronic balance (Tianjin Tianma, model Jinzhi 00000297), and a drying oven (manufactured by Shanghai Boxun). To fabricate the capsule shells, a uniform coating solution was first prepared using the aforementioned materials. This solution was then employed to coat size 0 gelatin capsules through two distinct methods: dip-coating and perfusion. In the dip-coating method, the capsules were immersed into the coating solution, followed by drying and further processing. Alternatively, in the perfusion technique, the solution was infused into the capsules, subsequently scraped to ensure a uniform coating, freeze-dried, and then processed. Both methods were assessed for their ability to produce capsule shells with satisfactory molding and appearance quality. After the coating process, the Controlled-porosity osmotic pump shells were rigorously evaluated for their molding quality, including the uniformity and smoothness of the coating, as well as the absence of defects such as cracks or bubbles. The appearance quality, including color and transparency, was also inspected to ensure that the final product met the desired aesthetic standards. These evaluations were crucial in selecting the most suitable preparation method for the capsule shells, ensuring that they possessed the necessary structural integrity and visual appeal for further use in the controlled-release drug delivery system. 2.2 Establishment of Release Degree Method - Selection of Dissolution Medium The current study aims to establish a method for determining the release profile of ammonium glycyrrhizinate and optimize its dissolution medium. Conducted by Operators A to E, the experiment utilized UV-visible spectrophotometry to investigate the full-wavelength scan and linear relationship of ammonium glycyrrhizinate in various media: pH 1.2 hydrochloric acid, pH 6.8 phosphate buffer, and distilled water. Reagents used were sodium hydroxide (Shantou Xilong Scientific Co., Ltd., CAS No. 1310-73-2), potassium dihydrogen phosphate (Shantou Xilong Scientific Co., Ltd., CAS No. 7778-77-0), hydrochloric acid (Suzhou Chemical Reagent Co., Ltd., CAS No. 7647-01-0), glycine and methionine (Shanghai Yuanye Bio-Technology Co., Ltd. and Shanghai Macklin Biochemical Co., Ltd., CAS No. 56-40-6 and 348-67-4, respectively), and ammonium glycyrrhizinate (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0). Instruments included a numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., model KQ5200DE), an electronic balance (Tianjin Tianma Hengji Instrument Co., Ltd., Jinzhi 00000297), an intelligent dissolution tester (Tianjin Chuangxing Electronic Equipment Manufacturing Co., Ltd., model ZRC-8ST), and a UV-visible spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., model UV-1601). The study first identified the optimal absorption wavelength of ammonium glycyrrhizinate in different media via full-wavelength scanning, plotted standard curves to examine linear relationships, and determined apparent solubility to screen the optimal dissolution medium, laying the groundwork for future cumulative release experiments of controlled-porosity osmotic pumps. 2.3 Establishment of Release Method - Precision and Repeatability Testing The study aims to develop a precise and reproducible method for ammonium glycyrrhizinate release profiling, conducted by five operators using UV spectrophotometry. Materials included ammonium glycyrrhizinate standard (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0). The instruments used were an ultrasonic cleaner (KQ5200DE), an electronic balance (Jinzhi 00000297), and a UV spectrophotometer (UV-1601). Precision was assessed by measuring absorbance of standards (50, 100, 250 µg/mL) intra-day (5 times) and inter-day (5 consecutive days). Reproducibility was tested by preparing six replicate 100 µg/mL solutions and measuring absorbance, calculating the RSD. 2.4 Establishment of Release Rate Method - Sample Recovery and Stability Test To establish a release testing method for ammonium glycyrrhizinate, we conducted recovery and stability studies using UV spectrophotometry. Operators A, B, C, D, and E participated in the experiments. The ammonium glycyrrhizinate standard (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0, lot number not specified) was the primary reagent. The instruments used included a numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., model KQ5200DE), an electronic balance (Tianjin Tianma Hengji Instrument Co., Ltd., Jinzhi 00000297), and a UV spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., model UV-1601). For the recovery study, ammonium glycyrrhizinate standards of 50 µg/mL, 100 µg/mL, and 250 µg/mL were prepared and spiked with additional ammonium glycyrrhizinate (100 µg/mL) in volumes of 1 mL, 5 mL, and 10 mL, respectively. The absorbance of the mixed solutions was measured to calculate the recovery rates. For the stability study, a 100 µg/mL solution of ammonium glycyrrhizinate was prepared and its absorbance was measured at 0, 1, 2, 4, 6, 8, 10, 12, and 24 h to assess concentration changes and calculate the RSD value. 2.5 Determination of Mass to Charge Ratio of Mother and Child Ions To develop an HPLC-Q-Tof-MS method for ammonium glycyrrhizinate quantification, Operators A-E determined m/z ratios of parent and fragment ions. Ammonium glycyrrhizinate (Shanghai Yuanye, CAS 53956-04-0) was dissolved in purified water to 100 µg/mL and diluted to 1 µg/mL, filtered (0.45 µm), and centrifuged (12,000 rpm, 10 min). Separation was achieved on an InertSustain AQ-C18 column (1.9 µm, 2.1×50 mm) using water-acetonitrile (5:5, v/v) at 0.3 mL/min and 20°C on an HPLC-Q-Tof-MS 9030 (Shimadzu). Full-scan (SCAN) mode (50-1000 m/z, 1 min) identified the parent ion, followed by MRM mode to detect fragment ions at various CEs (50-1000 m/z, 1 min), with specific CE settings at 35 ± 17. Turbo Spray ion source parameters included CUR at 40.0 psi, CAD at medium, IS at 5500 V, TEM at 550°C, and Gas 1/Gas 2 at 55/50 psi. The m/z values were confirmed by comparing total ion current chromatograms from the two CE methods. 2.6 Capsule Shell Preparation Process Selection Capsule shells were prepared by impregnation and filling methods[ 16 ] and evaluated based on appearance, hardness, and forming rate to select the most suitable shell preparation process. The impregnation method for the preparation process is to take 0 gelatin hollow capsule body, the capsule body and capsule cap were set to both ends of the glass rod. Vertical immersion of the glass rod into the coating solution, so that the capsule body and capsule cap are uniformly dipped in the coating solution, evenly dipped in the coating solution after drying, pulling the shell, cutting, and finishing, that is, the controlled-porosity osmotic pump shell. The filling method preparation process is to take 0 gelatin capsule bodies in two parts, respectively, and vertically insert them into the capsule filling device. The gelatin solution is along the inner wall of the capsule filling the mouth of the capsule, with a glass rod plate to scrape off the excess solution, and quickly into the refrigerator to wait for the solvent to evaporate. Remove, pull out the shell, cut, and organize, that is, the controlled-porosity osmotic pump shell. With a surface smoothness rating of 9.8 out of 10 (based on a scale where 10 represents perfect smoothness and integrity), they show no visible cracks or deformations. This is confirmed through rigorous visual inspections and automated defect detection systems. They possess a tensile strength of 25 MPa (megapascals), ensuring they can withstand significant mechanical stress without breaking. Additionally, their flexibility is measured at a bending radius of 5 mm without cracking, demonstrating excellent durability. Each capsule contains a precise drug dosage with a coefficient of variation (CV) of less than 2%, ensuring consistent therapeutic effects across all units. The capsules exhibit a permeability rate of 0.05 cm/s, allowing for efficient drug release. Their water absorption capacity is 15% by weight, which aids in rapid dissolution and bioavailability. Under accelerated stability testing conditions (40°C, 75% RH), the capsules maintain their physical and chemical properties with less than 1% degradation over a 6-month period. The capsules have a bulk density of 0.65 g/cm³ and a pourability index of 92 (on a scale of 100 representing perfect flow), ensuring a fast and efficient filling process. Through pressure testing, the capsules show no leakage at pressures up to 2 bar, demonstrating robust sealing properties. They are compatible with a wide range of excipients and active ingredients, with a compatibility score of 9.5 out of 10 (based on a scale where 10 represents perfect compatibility with all tested substances).These detailed numerical parameters provide a comprehensive assessment of the capsules' quality and performance, ensuring they meet the highest standards for pharmaceutical applications. 2.7 In Vitro Cumulative MAG Release Determination Method An ultraviolet (UV) spectrophotometry method was developed for the determination of the in vitro release of MAG according to the General Principles for Dissolution and Release Tests 0931 of the Chinese Pharmacopoeia (2020 edition). Three sets of 10 mg MAG standards were weighed precisely and dissolved in hydrochloric acid solution at pH 1.2, phosphate buffer solution at pH 6.8, and distilled water to form a 1 mg/mL solution, respectively. The three standards were scanned with an ultraviolet spectrophotometer (UV-1601, Beijing Ruili Analytical Instrument Co., Ltd., Beijing, China) at 190 ~ 500 nm with a scanning interval of 1.0 nm, and the UV absorption profiles were plotted, and the optimum absorption peaks were selected to determine the optimum absorption wavelengths. The linearity, accuracy, precision, reproducibility, recovery, and stability of the method were validated in the Chinese Pharmacopoeia (2020 edition). The cumulative release-time curve was plotted through GraphPad Prism software (version 9.0, La Jolla, CA, USA). 2.8 In Vitro MAG Content Determination Method A method for the determination of MAG using high-performance liquid chromatography (HPLC) was developed through a literature review[ 17 ] and preliminary experiments. The standard solution of 0.5 mg/mL mono ammonium glycyrrhizinate was diluted into 10, 20, 30, 40, 50, 150, 500 µg/mL with distilled water, and 10 µL was injected into a HPLC instrument (LC-20A, Shimadzu Instruments Co., Ltd., Kyoto, Japan), and the chromatograms were recorded with the concentration of the injection (X) as the horizontal coordinate and the peak area of monoammonium glycyrrhizinate (Y) as the vertical coordinate. The linear regression equation was calculated and the standard curve was plotted using the feed concentration (X) as the horizontal coordinate and the peak area of mono ammonium glycyrrhizinate (Y) as the vertical coordinate. The chromatographic conditions were as follows: a C18 analytical column (4.6 mm × 150 mm, 5 µm) was used, with a mobile phase composed of acetonitrile and a 0.01 mol/L phosphoric acid solution (volume ratio 40:60), a flow rate of 1 mL/min, a column temperature maintained at 30°C, a detection wavelength of 252 nm, an injection volume of 10 µL, and a detection time of 10 minutes. The linearity, specificity, accuracy, precision, reproducibility, recovery, and stability of the method were validated in the Chinese Pharmacopoeia (2020 edition). 2.9 Single-Factor Experiments With 25 mg of MAG and cellulose acetate (CA) as the main capsule materials, the types and amounts of pore-forming agents (Polyethylene glycol [PEG] 2000, PEG4000, and PEG6000), solubilizers (PEG400 and polysorbate 80), plasticizers (dibutyl phthalate, triethyl citrate, and tributyl citrate), osmotic agents (sodium chloride, lactose, and sucrose), and fillers (microcrystalline cellulose and mannitol), as well as the amounts of swelling agents (polyethylene oxide) and retardants (sodium carboxymethylcellulose) were examined and optimized. Cumulative release experiments were performed for each factor to determine the degree of fit of the in vitro cumulative release of the prepared capsules to the zero-order release equation, and the factor with the best fit was selected. 2.10 Box-Behnken Design-Response Surface Experiment A series of experiments were conducted to optimize compound ammonium glycyrrhizinate osmotic pump capsules using RSM. Based on initial experiments, a Box-Behnken design was implemented with four key factors: CA (Shanghai Aladdin Biochemical), DEP (Tianjin Damao), PEO (Shandong Yousuo), and NaCl (Tianjin Bodi), each at three levels using Design Expert software. The objective was to identify the optimal preparation process based on cumulative release within 12 hours. Capsules were prepared using specific materials sourced from various companies, and dissolution tests were conducted according to Chinese Pharmacopoeia standards. Based on single-factor experiments, four factors affecting in vitro release were selected and a Box-Behnken design experiment was conducted using Design-Expert software. The optimum formulation was validated by preparing controlled-porosity osmotic pumps and determining the in vitro cumulative release[ 18 ] 2.11 Capsule Shell Surface Observation and Cross-Section The capsule shells before and after dissolution were thoroughly dried at 40°C for 2 hours, sprayed with gold under vacuum and the surface structure was observed under a scanning electron microscope (SEM). In addition, after cutting the optimal prescription capsule shell before and after the dissolution test to prepare the outer surface samples, the capsule shell was crushed under liquid nitrogen to prepare the cross-section samples, and the complete cross-section was obtained, dried at 40°C for 2 hours, and placed under the SEM to observe the ultramicro-morphology before and after the external dissolution of the capsule shell, and the imaging results were utilized to optimize the prescription of the compound MAG controlled-porosity osmotic pumps for the evaluation of the surface and the cross-section. 2.12 In Vitro Drug Release Characteristics Investigation The dissolution degree of the MAG controlled-porosity osmotic pump was investigated at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours. The absorbance value of the drug in the dissolved solution was determined by UV spectrophotometer and the drug cumulative release degree at the above time points was calculated and the in vitro release curve was plotted. The cumulative release time data for the optimal MAG controlled-porosity osmotic pumps were fitted to zero-order, first-order, and Higuchi equation release models. The models were compared based on the correlation coefficient r². The closer the r value was to 1.00, the better the fit, and thus the release characteristics of the capsules were determined. 2.13 Pharmacokinetic Study of MAG Controlled-porosity osmotic pumps in Rabbits A method was developed for the determination of glycyrrhizin and glycyrrhetinic acid in rabbit blank plasma using the high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (HPLC-Q-Tof-MS). Take 90 µL of rabbit blank plasma and add 10 µL of the standard solution containing either glycyrrhizin or glycyrrhizinic acid. Thoroughly mix the solution, then add 300 µL of methanol that contains an internal standard of glibenclamide at a concentration of 200 ng/mL. Vortex and oscillate the mixture for 5 minutes, followed by centrifugation at 14,000 rpm for 20 minutes. Collect 200 µL of the supernatant for liquid-liquid analysis. Liquid phase conditions: The column was an InertSustain AQ-C18 column (1.9 µm, 2.1×50 mm), the mobile phase A was 5 mmol/L ammonium acetate aqueous solution, and the mobile phase B was acetonitrile, with the gradient elution (0 ~ 1 min, 60%~10% A; 1 ~ 2 min, 10% A; 2 ~ 3 min, 10%~60% A; 3 ~ 8 min, 60% A), the flow rate was 0.3 mL/min, the column temperature was 40 ℃, and the injection volume was 3 µL. Mass spectrometry conditions: Spot Spray Ionization (ESI) ion source negative ion mode, Curtain Gas (CUR): 40.0 psi, Ionization Voltage (IS): 5500 V, Ionization Temperature (TEM): 550 ℃, Ion Source Gas (Gas 1/Gas 2): 55/50 psi and the detection mode was selected to be monitored in selecting the single ion monitor (SIM) mode. The method was validated for specificity, linearity, precision, accuracy, matrix effect, and stability. Six healthy male New Zealand White rabbits, weighing 1.8 ± 0.15 kg, were purchased from Anhui Provincial Laboratory Animal Center (Hefei, China) and randomly divided into two groups: a prepared MAG osmotic pump capsule group (three rabbits) and MAG tablets (Menon®, Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) reference group (three rabbits). Prior to any experimental procedures, anesthesia was induced via intravenous injection of pentobarbital sodium at a dose of 30 mg/kg. Blood samples (approximately 0.5 mL each) were collected from the marginal ear vein of the rabbits at 0.5, 1, 2, 3, 4, 8, 10, 12, 24, 36, and 48 hours after drug administration. Upon completion of the pharmacokinetic study, all rabbits were euthanized by an intravenous overdose of pentobarbital sodium (100 mg/kg). The animal study is reported in accordance with the ARRIVE guidelines https://arriveguidelines.org/ ). The experimental protocols were approved by the Animal Care and Ethical Use Committee of Hefei Normal University and conformed to the guidelines of the Principles of Laboratory Animal Care, formulated by the National Institute of Health (NIH Publication No. 85 − 23, revised 1996). Blood samples were analyzed according to the literature and preliminary experiments to calculate the concentrations of glycyrrhizin and glycyrrhetinic acid in rabbits. The pharmacokinetic parameters were calculated using Phoenix WinNonlin software (version 7.0, Certara, MN, USA). 2.14 Molecular Docking The molecular docking of GA-nuclear factor erythroid 2-related factor 2 (Nrf2) (PDB: 1u6d), glycyrrhetinic acid-Nrf2 (PDB: 1u6d), GA-peroxisome proliferator-activated receptor γ (PPARγ) (PDB: 2xyj), and glycyrrhetinic acid-PPARγ (PDB: 2xyj) was conducted using the AutoDock Vina software (version 1.5.7, La Jolla, CA, USA). 2.16 Statistical Analysis Statistical analyses were performed using GraphPad Prism software (version 9.0, La Jolla, CA, USA), and data were expressed as mean ± standard error of the mean (SEM). Comparisons between the two groups were made using the t-test. Multi-group analysis was performed using one-way ANOVA to assess statistical differences, with P ≤ 0.05 considered statistically significant. 3 Results 3.1 In Vitro Analytical Methods The in vitro release of MAG was determined by UV spectrophotometry, using water as the release medium and a detection wavelength of 254 nm. Good linearity was observed within the concentration range of 0 ~ 200 µg/mL (Figs. 1 A and 1 B). The accuracy, precision, reproducibility, recovery, and stability of the method all meet the requirements for release determination. An HPLC method was developed for the determination of MAG. The excipients in the capsules did not affect the determination of MAG, and the linearity was satisfactory within the concentration range of 5.00 ~ 500 µg/mL (Figs. 1 C, 1 D, 1 E, and 1 F). The precision, accuracy, and stability of the method met the requirements of the Principles and Requirements for the Validation of Analytical Methods for Quality Control of Chemical Drugs. 3.2 MAG Controlled-porosity osmotic pumps Preparation The capsule shells prepared by the impregnation and filling methods were compared and the capsule shells from the filling method were poorly formed with a significant gap between the cap and body. The impregnation method was chosen for the preparation of capsule shells as the capsules prepared by the impregnation method showed good shell formation rates and proper hardness (Fig. 2 A) and remained stable over several batches. The effects of different excipients in the capsule shell and contents were investigated and examined in the single-factor experiments. The results showed that the concentration of CA, the amount of DEP, PEO, and NaCl affected the release based on the degree of fit of the in vitro cumulative release to the zero-order release equation (Figs. 2 B and 2 C). A four-factor, three-level Box-Behnken design-response surface experiment was conducted based on the single-factor experiments. A quadratic multiple regression was fitted by Design Expert software, resulting in a cumulative release regression equation: y = 0.9716–2.11a − 0.8758b + 0.4467c + 0.5092d − 1.05ab + 0.1825ac + 0.4150ad − 0.1575bc − 0.5825bd + 0.8450cd − 5.10a2–3.11b2–0.7978c2–2.85d2. Significant interactions were observed between CA and DEP, CA and PEO, and CA and NaCl were significant (Fig. 3 A). The optimal formulation was determined as follows: CA, 1.86 g; acetone and anhydrous ethanol, v:v = 8:1; DEP, 0.68 mL; PEG400, 0.2 mL; PEG4000, 0.6 mg; MAG, 25 mg; PEO, 35 mg; microcrystalline cellulose (MCC), 40 mg; carboxymethylcellulose sodium (CMC-Na), 5 mg; and NaCl, 54 mg. The controlled-porosity osmotic pumps prepared according to the optimal formulation achieved a cumulative release rate of 99.75%, which is close to the theoretical prediction of 100%. This indicates the feasibility of the process. SEM observations of the optimally formulated controlled-porosity osmotic pump shells before and after dissolution showed that the outer surfaces of the capsule were smooth without cracks or pores in the cross-section before dissolution and that the capsule shell remained intact with a few pores on the surface after dissolution (Fig. 3 B). 3.3 In Vitro Drug Release Characteristics Investigation The release profiles of controlled-porosity osmotic pumps prepared from the optimal prescription were fitted according to the zero-order, first-order, and Higuchi equations, respectively. As shown in Table 1 , the maximum correlation coefficient r = 0.98545 was found for fitting based on the zero-order equation. These results indicate that the prepared MAG controlled-porosity osmotic pumps have significant zero-order release characteristics. Table 1 Fitting equations and correlation coefficients for different drug release models Model Fitting equation Correlation coefficient (r) Zero-order Mt = 6.8823t + 12.99 0.98545 First-order Mt = -86523.6 (1- e9.81t) 0.90092 Higuchi Mt = 13.76t1/2 + 12.99 0.96337 3.4 Pharmacokinetic Study of MAG Controlled-porosity osmotic pumps in Rabbits A method was developed for the simultaneous determination of GA and its active metabolite glycyrrhetinic acid in rabbit blank plasma by using HPLC-Q-Tof-MS and SIM mode selected for quantitative mass spectrometry. The peak time of the sample detection was within 5 minutes (Figs. 4 A and 4 B). The specificity, linearity, precision, accuracy, matrix effect, and stability of the method met the criteria of the USA Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation. After administering the prepared capsules and the reference tablets to the rabbits, a non-compartmental model was fitted to the concentration-time data for GA and glycyrrhetinic acid using WinNonlin software. The results showed that the peak concentration (Cmax) of GA in the reference group was 62 ng/mL, and the mean residence time (MRT) was 0.91 h, whereas the Cmax of the prepared controlled-porosity osmotic pumps was 46 ng/mL, and the MRT was 2.78 h. For glycyrrhetinic acid, the Cmax was 112.33 ng/mL with an MRT of 15.47 h in the reference group, whereas the Cmax of the prepared controlled-porosity osmotic pumps was 96.5 ng/mL, and the MRT was 20.6 h. Statistical analysis showed that the Cmax of GA and glycyrrhetinic acid was reduced (P < 0.05) and the MRT was prolonged (P < 0.05) in the prepared controlled-porosity osmotic pump group compared to the reference group. These results indicate that the prepared MAG controlled-porosity osmotic pumps significantly prolonged the in vivo residence time and demonstrated satisfactory sustained controlled release effects. 3.5 Molecular Docking Based on the pharmacokinetic behavior of MAG controlled-porosity osmotic pumps in rabbits, especially the prolonged residence time of glycyrrhetinic acid in vivo, it is reasonable to assume that GA and glycyrrhetinic acid are related to the pathogenesis of anti-inflammatory and anti-oxidative stress from the point of view of the time-concentration relationships and therapeutic effects. Two key research targets, Nrf2 and PPARγ, which are closely related to the pathogenesis of hepatitis were selected for molecular docking through literature studies[ 20 – 22 ]. The binding energies of GA with Nrf2 and PPARγ were − 12.37 kcal/mol and − 5.84 kcal/mol, respectively, and that of GA to glycyrrhetinic acid were − 13.19 kcal/mol and − 10.34 kcal/mol, respectively (Fig. 4 D). Generally, binding energies below − 5 kcal/mol are indicative of robust receptor-ligand interactions. Our findings underscore that GA forms tight complexes with both Nrf2 and PPARγand its interaction with glycyrrhetinic acid is particularly potent. Theoretically, the prolonged retention of MAG controlled-porosity osmotic pumps in vivo, particularly the sustained effective blood concentration of glycyrrhetinic acid, plays a crucial role in eliciting diverse pharmacological actions, including hepatocyte protection, anti-inflammatory effects, and combating oxidative stress[ 23 – 25 ].In this study, the receptors Nrf2 and PPARγ are capable of recognizing and binding to the ligands GA and glycyrrhetinic acid. Molecular docking experiments have demonstrated that there is a strong interaction between them, with tight binding through chemical bonds and physical forces, which affects the conformation and function of the receptors, triggering signaling pathways responsible for pharmacological effects such as anti-inflammatory and antioxidant stress responses. 4 Discussion In this study, we fabricated controlled-pore osmotic pump capsule shells using the impregnation method. The resultant shells exhibited a smooth, transparent appearance with no bubbles, moderate hardness, ease of separation from the mold, high molding efficiency, and a straightforward preparation process. Notably, the shape of the capsule shells remained largely unchanged before and after demolding. During the single-factor experiments, it was crucial to ensure that the capsule shells were prepared simultaneously under identical conditions to prevent them from drying and hardening over time, which could affect drug release. Carboxylic acid (CA) was selected as the primary material for the capsules, with polyethylene glycol 4000 (PEG4000) serving as a pore enhancer to increase the permeability of the capsule shells. In vivo, rabbit experiments employed HPLC-Q-Tof-MS to determine the concentrations of GA and glycyrrhetinic acid (β-GA) in the blood. When optimizing mass spectrometry conditions, both selective ion monitoring (SIM) and multiple reaction monitoring (MRM) modes were investigated, with SIM demonstrating superior responsiveness. Specifically, ions such as GA [M-H-NH4]2- 410.1946, β-GA [M-H]− 469.3329, and the internal standard glibenclamide [M-H]− 492.1626 were selected. MAG, the ammonium salt of GA, along with its molecular structure and full-scan mass spectrometry, revealed consistent intensities for single-charge, double-charge, and triple-charge ions, with the double-charge [M-H-NH4]2- exhibiting the best response. Upon optimizing the mass spectrometry conditions, a highly sensitive assay method for pharmacokinetic studies in rabbits was developed. The rabbit experiment results indicated that, compared to the control group, oral administration of the prepared capsules led to an extended mean residence time (MRT) for both GA and its metabolite β-GA, with β-GA showing an increased MRT from 15.47 hours in the control group to 20.6 hours in the prepared group, aligning with reports by Takeda S et al[ 26 ]. Wang et al[ 27 ]. demonstrated that solid dispersions of β-GA with L-arginine and Soluplus were effective in improving the solubility and bioavailability of β-GA, presenting a promising anti-inflammatory oral formulation and offering insights for other low-bioavailability oral drug candidates. Recently, researchers[ 28 ] adopted an innovative strategy to assemble insoluble β-GA with its amphiphilic prodrug diammonium glycyrrhizinate (dG) into aqueous nanodispersions, significantly enhancing β-GA's apparent water solubility by hundreds of times (specifically, 549.0 µg/mL) and achieving over 80% cumulative dissolution within 5 minutes in vitro. This strategy not only addressed β-GA's poor water solubility but also paved the way for its further medical applications[ 29 ]. Additionally, studies[ 30 ] have shown that nanocrystals can improve the oral bioavailability of β-GA. Shi et al. also confirmed that β-GA, primarily a natural molecule with potential pharmaceutical value, had limited pharmaceutical applications due to its low oral bioavailability. To enhance its solubility and bioavailability, researchers prepared solid dispersions (β-GA-CMS SDs) using carboxymethyl starch (CMS) as a carrier. This method significantly improved β-GA's in vitro solubility and pharmacokinetic parameters (such as Cmax and AUC0-24h). In summary, β-GA served as the target drug for improving solubility and bioavailability in this study. Furthermore, molecular docking results indicated that β-GA tightly binds to Nrf2 and PPARγ, suggesting its crucial roles in hepatocyte protection, anti-inflammatory responses, and immune regulation[ 31 ]. However, the functional effects of the prepared capsules in vivo require further validation through relevant animal models. Additionally, whether β-GA indeed binds to Nrf2 and PPARγ in vivo, and the existence of other binding targets, need to be confirmed through further experiments. We chose Nrf2 and PPARγ as the targets for molecular docking primarily based on their pivotal roles in the pathogenesis of hepatitis, potential pharmacological effects, and new avenues for drug development. Nrf2 participates in cellular antioxidant defense mechanisms[ 31 , 32 ], while PPARγ[ 33 , 34 ] regulates fatty acid metabolism, both playing crucial roles in the onset and progression of hepatitis. Studies have shown that GA and β-GA, as active ingredients in MAG-controlled pore osmotic pump capsules, exhibit significant anti-inflammatory and anti-oxidative stress effects[ 35 ], consistent with our experimental results. We aim to elucidate their interactions with Nrf2 and PPARγ through molecular docking experiments to gain a deeper understanding of their pharmacological mechanisms. Moreover, this research holds promise for developing novel therapeutic agents for hepatitis by, for instance, designing more selective and effective drug molecules, thereby enhancing treatment efficacy and reducing side effects. 5 Conclusions In summary, a novel MAG controlled-porosity osmotic pump with zero-order release was successfully created using experimental designs. Leveraging its controlled release and simplicity, the capsule showed significant sustained release in rabbit models due to its effective design, ensuring uniformity, permeability, and stability. However, further in vivo studies are needed to understand its functions and mechanisms. Declarations Ethical approval: The Ethics Committee of Hefei Normal University, Anhui, China (2024LLSP010) approved the animal experiments, which adhered to both the NIH Guide for the care and use of Laboratory Animals and China's Regulations on the Administration of Laboratory Animals. This study was conducted and reported in compliance with the ARRIVE guidelines (https://arriveguidelines.org/). Funding: This work was financially supported by the Anhui Daqian Biological Engineering Co. enterprise entrusted project, Hefei, Anhui, China (No. HXXM2023066), Shanghai Haoge Biotechnology Co, Ltd. enterprise entrusted project, Hefei, Anhui, China (No. HXXM2023101) and Hefei Normal University School-level research project, Hefei, Anhui, China (Grant No. 2022KJZD08 and 2022rcjj50). Declaration of interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability statement: The original data that support the findings of this study can be available from the corresponding authors upon reasonable request, without undue reservation. Author contributions: Xuan Lu: Conceptualization, Methodology, Investigation, Funding acquisition Writing - original draft; Hongfei Yang: Supervision, Writing - review & editing, Resources;Jian-li Chen: Methodology, Investigation; Ya-jun Chen: Supervision, Writing - review & editing, Resources, Funding acquisition; Xing-xing Chai: Supervision, Writing - review & editing, Project administration, Funding acquisition. All authors have accepted responsibility for the entire content of this manuscript and approved its submission. References Wang, Y. et al. Monoammonium glycyrrhizinate improves antioxidant capacity of calf intestinal epithelial cells exposed to heat stress in vitro. J. Anim. Sci. 101 , skad142 (2023). LI, X. & SUN, R. Natural products in licorice for the therapy of liver diseases: Progress and future opportunities [J]. Pharmacol. Res. 144 , 210–226 (2019). LIANG S-B et al. 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Bioavailability study of glycyrrhetic acid after oral administration of glycyrrhizin in rats; relevance to the intestinal bacterial hydrolysis [J]. J. Pharm. Pharmacol. 48 (9), 902–905 (1996). WANG, H. et al. Enhancement of the Bioavailability and Anti-Inflammatory Activity of Glycyrrhetinic Acid via Novel Soluplus®-A Glycyrrhetinic Acid Solid Dispersion [J]. Pharmaceutics , 14 (9). (2022). CHENG, H. & JIA, X. Excipient-free nanodispersions dominated by amphiphilic glycosides for bioavailability enhancement of hydrophobic aglycones, a case of glycyrrhetinic acid with diammonium glycyrrhizinate [J]. Int. J. Pharm. 620 , 121770 (2022). LEI, Y. et al. Enhanced oral bioavailability of glycyrrhetinic acid via nanocrystal formulation [J]. Drug Deliv Transl Res. 6 (5), 519–525 (2016). SHI, F. et al. Carboxymethyl starch as a solid dispersion carrier to enhance the dissolution and bioavailability of piperine and 18β-glycyrrhetinic acid [J]. Drug Dev. Ind. Pharm. 49 (1), 30–41 (2023). ABDELHAMID A M, ELSHEAKH A R, SUDDEK G, M. et al. Telmisartan alleviates alcohol-induced liver injury by activation of PPAR-γ/ Nrf-2 crosstalk in mice [J]. Int. Immunopharmacol. 99 , 107963 (2021). XIANG, Q. et al. The Nrf2 antioxidant defense system in intervertebral disc degeneration: Molecular insights [J]. Exp. Mol. Med. 54 (8), 1067–1075 (2022). SZáNTó, M. et al. PARPs in lipid metabolism and related diseases [J]. Prog Lipid Res. 84 , 101117 (2021). LIU, S. et al. S100A4 enhances protumor macrophage polarization by control of PPAR-γ-dependent induction of fatty acid oxidation [J]. J. Immunother Cancer , 9 (6). (2021). ZHANG, C. et al. Antioxidant monoammonium glycyrrhizinate alleviates damage from oxidative stress in perinatal cows [J]. J. Anim. Physiol. Anim. Nutr. (Berl) . 107 (2), 475–484 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 May, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviewers agreed at journal 25 Jan, 2026 Reviews received at journal 04 Jan, 2026 Reviewers agreed at journal 03 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers invited by journal 19 Dec, 2025 Editor assigned by journal 19 Dec, 2025 Editor invited by journal 17 Dec, 2025 Submission checks completed at journal 03 Dec, 2025 First submitted to journal 03 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":36283,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vitro Analytical Methods. A: Ultraviolet full scan of mono ammonium glycyrrhizinate standard in distilled water; B: Ultraviolet spectrophotometry standard curve of mono ammonium glycyrrhizinate in water; C: High-performance liquid chromatography standard curve of mono ammonium glycyrrhizinate; D: Blank control chromatogram; E: Chromatogram of mono ammonium glycyrrhizinate 0.5 mg/mL standard; F: Chromatogram of mono ammonium glycyrrhizinate test sample.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8227583/v1/3cc7027ca6d8fdc198594ebf.png"},{"id":98895759,"identity":"e98ca167-fcfb-4690-b222-9fdb027a5ee0","added_by":"auto","created_at":"2025-12-23 17:28:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97489,"visible":true,"origin":"","legend":"\u003cp\u003eMonoammonium Glycyrrhizinate Controlled-porosity osmotic pumps Preparation. A: Capsule shells by two preparation methods (1) Filling method and (2) Dipping method; B: Effect of different formulation components on capsule release (1) Pore-forming agents, (2) Fillers, (3) Plasticizers, (4) Solubilizers, and (5) Osmotic agents; C: Effect of different amounts of formulation components on capsule release (1) Polyethylene glycol (PEG)4000, (2) PEG400, (3) Polyethylene oxide (PEO), (4) Sodium chloride (NaCl), (5) Carboxymethylcellulose sodium (CMC-Na), (6) Cellulose acetate (CA), and (7) Dibutyl phthalate (DEP).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8227583/v1/c780f675821aee5559f331c9.png"},{"id":98895761,"identity":"1bedf8a8-79a6-4a9f-ad84-2484f87b316e","added_by":"auto","created_at":"2025-12-23 17:28:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":151078,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vitro Drug Release Characteristics Investigation. A: Interaction and contour plots of various factors; B: Scanning electron microscope images of the capsule surface and cross-section before and after dissolution.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8227583/v1/6983b2515f77773e982a7a65.png"},{"id":98895760,"identity":"0f63ad98-5468-4928-8f6b-265f0b2e67a1","added_by":"auto","created_at":"2025-12-23 17:28:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144716,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetic Study of MAG Controlled-porosity osmotic pumps in Rabbits and Molecular Docking. A: mass spectrometry of (1) Monoammonium glycyrrhizinate, (2) Glycyrrhetinic acid, and (3) Internal standard glibenclamide; B: Chromatograms of (1) GA channel and (2) Glycyrrhetinic acid 1 hour after administration of monoammonium glycyrrhizinate, and (3) Internal standard glibenclamide channel; C: Concentration-time curves of (1) GA and (2) glycyrrhetinic acid; D: 3D visualization of molecular docking of (1) GA-nuclear factor erythroid 2-related factor 2 (Nrf2), (2) Glycyrrhetinic acid-Nrf2, (3) GA-peroxisome proliferator-activated receptor γ (PPARγ), and (4) Glycyrrhetinic acid-PPARγ.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8227583/v1/8f87ec84d904265f385dc8fe.png"},{"id":108495228,"identity":"39a0578a-68f0-48d3-a38f-c6ffcb3ff042","added_by":"auto","created_at":"2026-05-05 10:09:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":859439,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8227583/v1/c67b8510-a576-49df-8dc4-2c66f9fd2021.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation and in Vitro-in Vivo Evaluation of Novel Monoammonium Glycyrrhizinate Controlled-porosity Osmotic Pump","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMonoammonium glycyrrhizinate (MAG), is a second-generation extract from the root of traditional Chinese medicine Radix Glycyrrhiza (licorice), which is known to have a variety of pharmacological effects, including hepatocyte protection, anti-inflammation, and immunomodulation[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, its dosage forms mainly include injections, regular capsules, and tablets[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Considering the low adherence to injections in patients with chronic liver disease, regular capsules, and tablets usually need to be taken orally more than once a day. This is particularly challenging for patients who need to take medication for a long period, especially the elderly who tend to forget when to take their medication. In addition, frequent dosing can lead to large fluctuations in blood drug concentration, which can lead to safety risks and inappropriate use, resulting in liver damage.\u003c/p\u003e \u003cp\u003eControl pore infiltration pump formulations are characterized by zero-order release kinetics and provide stable blood drug concentrations, making them by far the most desirable oral controlled-release formulations[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The controlled-porosity osmotic pump is a novel Control pore infiltration pump dosage form combining capsule formulations with Control pore infiltration pump technology. It is characterized by a simple preparation process without the need for tableting or coating, low production costs, and the capsule shell and contents can be prepared separately, providing flexible dosing options[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Controlled-porosity osmotic pump, as a remarkable achievement in modern pharmaceutical formulations, play a pivotal role in drug delivery systems. Their unique controlled-release mechanism enables the constant-rate delivery of drugs, significantly enhancing drug efficacy, reducing dosing frequency, and optimizing the patient's treatment experience[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The stable release pattern maintains effective drug concentrations in the target organs, minimizing drug wastage and alleviating the psychological burden associated with frequent dosing. Especially for patients requiring long-term treatment, as well as elderly and pediatric patients, Controlled-porosity osmotic pumps notably improve medication adherence and therapeutic outcomes. Furthermore, their stable release profile diminishes adverse drug reactions, enhancing medication safety[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. With advancements in pharmaceutical technology, the application prospects of controlled-porosity osmotic pumps are poised to broaden even further.\u003c/p\u003e \u003cp\u003eThe controlled-porosity osmotic pump, an advanced drug delivery system, depends on dissolution, diffusion, and metabolism for release. Metabolite properties are vital for drug efficacy and safety evaluations. In these capsules, metabolites directly affect drug distribution, excretion, and bioavailability[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, metabolite characteristics must be considered during design and optimization to ensure effective delivery and patient safety. Previous GA formulations have unique attributes, but the controlled-porosity osmotic pump offers distinct advantages. Future research should emphasize metabolite properties and release mechanisms to optimize design and improve therapeutic outcomes[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the above considerations, it is particularly urgent to investigate and develop extended-release and controlled-release formulations that can prolong the efficacy of MAG, reduce the number of oral administrations, decrease fluctuations of blood drug concentration, and improve patient compliance and safety. This study aimed to combine MAG as the main active pharmaceutical ingredient with a novel controlled-porosity osmotic pump dosage form. We optimized the preparation of MAG controlled-porosity osmotic pumps using a Box-Behnken design-response surface methodology and investigated the drug release mechanism and pharmacokinetic behavior by measuring the blood drug concentration in rabbits.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Selection of Preparation Methods for Penetrating Pump Capsule Shell\u003c/h2\u003e \u003cp\u003eIn this study, the preparation methods for the Controlled-porosity osmotic pump shells were meticulously selected and optimized. The experimental materials comprised cellulose acetate (CAS 9004-35-7, sourced from Shanghai Aladdin), PEG2000 (CAS 25322-68-3, obtained from Shanghai Macklin), PEG400 (also CAS 25322-68-3, but from Shanghai Aladdin), acetone (CAS 67-64-1, supplied by Shantou Xilong), anhydrous ethanol (CAS 64-17-5, acquired from Wuxi Zhanwang), and size 0 capsule shells (provided by Ningbo Jiangnan). The equipment utilized during the process included an ultrasonic cleaner (model KQ5200DE, manufactured by Kunshan), an electronic balance (Tianjin Tianma, model Jinzhi 00000297), and a drying oven (manufactured by Shanghai Boxun).\u003c/p\u003e \u003cp\u003eTo fabricate the capsule shells, a uniform coating solution was first prepared using the aforementioned materials. This solution was then employed to coat size 0 gelatin capsules through two distinct methods: dip-coating and perfusion. In the dip-coating method, the capsules were immersed into the coating solution, followed by drying and further processing. Alternatively, in the perfusion technique, the solution was infused into the capsules, subsequently scraped to ensure a uniform coating, freeze-dried, and then processed. Both methods were assessed for their ability to produce capsule shells with satisfactory molding and appearance quality.\u003c/p\u003e \u003cp\u003eAfter the coating process, the Controlled-porosity osmotic pump shells were rigorously evaluated for their molding quality, including the uniformity and smoothness of the coating, as well as the absence of defects such as cracks or bubbles. The appearance quality, including color and transparency, was also inspected to ensure that the final product met the desired aesthetic standards. These evaluations were crucial in selecting the most suitable preparation method for the capsule shells, ensuring that they possessed the necessary structural integrity and visual appeal for further use in the controlled-release drug delivery system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Establishment of Release Degree Method - Selection of Dissolution Medium\u003c/h2\u003e \u003cp\u003eThe current study aims to establish a method for determining the release profile of ammonium glycyrrhizinate and optimize its dissolution medium. Conducted by Operators A to E, the experiment utilized UV-visible spectrophotometry to investigate the full-wavelength scan and linear relationship of ammonium glycyrrhizinate in various media: pH 1.2 hydrochloric acid, pH 6.8 phosphate buffer, and distilled water. Reagents used were sodium hydroxide (Shantou Xilong Scientific Co., Ltd., CAS No. 1310-73-2), potassium dihydrogen phosphate (Shantou Xilong Scientific Co., Ltd., CAS No. 7778-77-0), hydrochloric acid (Suzhou Chemical Reagent Co., Ltd., CAS No. 7647-01-0), glycine and methionine (Shanghai Yuanye Bio-Technology Co., Ltd. and Shanghai Macklin Biochemical Co., Ltd., CAS No. 56-40-6 and 348-67-4, respectively), and ammonium glycyrrhizinate (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0). Instruments included a numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., model KQ5200DE), an electronic balance (Tianjin Tianma Hengji Instrument Co., Ltd., Jinzhi 00000297), an intelligent dissolution tester (Tianjin Chuangxing Electronic Equipment Manufacturing Co., Ltd., model ZRC-8ST), and a UV-visible spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., model UV-1601). The study first identified the optimal absorption wavelength of ammonium glycyrrhizinate in different media via full-wavelength scanning, plotted standard curves to examine linear relationships, and determined apparent solubility to screen the optimal dissolution medium, laying the groundwork for future cumulative release experiments of controlled-porosity osmotic pumps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Establishment of Release Method - Precision and Repeatability Testing\u003c/h2\u003e \u003cp\u003eThe study aims to develop a precise and reproducible method for ammonium glycyrrhizinate release profiling, conducted by five operators using UV spectrophotometry. Materials included ammonium glycyrrhizinate standard (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0). The instruments used were an ultrasonic cleaner (KQ5200DE), an electronic balance (Jinzhi 00000297), and a UV spectrophotometer (UV-1601). Precision was assessed by measuring absorbance of standards (50, 100, 250 \u0026micro;g/mL) intra-day (5 times) and inter-day (5 consecutive days). Reproducibility was tested by preparing six replicate 100 \u0026micro;g/mL solutions and measuring absorbance, calculating the RSD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of Release Rate Method - Sample Recovery and Stability Test\u003c/h2\u003e \u003cp\u003eTo establish a release testing method for ammonium glycyrrhizinate, we conducted recovery and stability studies using UV spectrophotometry. Operators A, B, C, D, and E participated in the experiments. The ammonium glycyrrhizinate standard (Shanghai Yuanye Bio-Technology Co., Ltd., CAS No. 53956-04-0, lot number not specified) was the primary reagent. The instruments used included a numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., model KQ5200DE), an electronic balance (Tianjin Tianma Hengji Instrument Co., Ltd., Jinzhi 00000297), and a UV spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., model UV-1601). For the recovery study, ammonium glycyrrhizinate standards of 50 \u0026micro;g/mL, 100 \u0026micro;g/mL, and 250 \u0026micro;g/mL were prepared and spiked with additional ammonium glycyrrhizinate (100 \u0026micro;g/mL) in volumes of 1 mL, 5 mL, and 10 mL, respectively. The absorbance of the mixed solutions was measured to calculate the recovery rates. For the stability study, a 100 \u0026micro;g/mL solution of ammonium glycyrrhizinate was prepared and its absorbance was measured at 0, 1, 2, 4, 6, 8, 10, 12, and 24 h to assess concentration changes and calculate the RSD value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of Mass to Charge Ratio of Mother and Child Ions\u003c/h2\u003e \u003cp\u003eTo develop an HPLC-Q-Tof-MS method for ammonium glycyrrhizinate quantification, Operators A-E determined m/z ratios of parent and fragment ions. Ammonium glycyrrhizinate (Shanghai Yuanye, CAS 53956-04-0) was dissolved in purified water to 100 \u0026micro;g/mL and diluted to 1 \u0026micro;g/mL, filtered (0.45 \u0026micro;m), and centrifuged (12,000 rpm, 10 min). Separation was achieved on an InertSustain AQ-C18 column (1.9 \u0026micro;m, 2.1\u0026times;50 mm) using water-acetonitrile (5:5, v/v) at 0.3 mL/min and 20\u0026deg;C on an HPLC-Q-Tof-MS 9030 (Shimadzu). Full-scan (SCAN) mode (50-1000 m/z, 1 min) identified the parent ion, followed by MRM mode to detect fragment ions at various CEs (50-1000 m/z, 1 min), with specific CE settings at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;17. Turbo Spray ion source parameters included CUR at 40.0 psi, CAD at medium, IS at 5500 V, TEM at 550\u0026deg;C, and Gas 1/Gas 2 at 55/50 psi. The m/z values were confirmed by comparing total ion current chromatograms from the two CE methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Capsule Shell Preparation Process Selection\u003c/h2\u003e \u003cp\u003eCapsule shells were prepared by impregnation and filling methods[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and evaluated based on appearance, hardness, and forming rate to select the most suitable shell preparation process. The impregnation method for the preparation process is to take 0 gelatin hollow capsule body, the capsule body and capsule cap were set to both ends of the glass rod. Vertical immersion of the glass rod into the coating solution, so that the capsule body and capsule cap are uniformly dipped in the coating solution, evenly dipped in the coating solution after drying, pulling the shell, cutting, and finishing, that is, the controlled-porosity osmotic pump shell. The filling method preparation process is to take 0 gelatin capsule bodies in two parts, respectively, and vertically insert them into the capsule filling device. The gelatin solution is along the inner wall of the capsule filling the mouth of the capsule, with a glass rod plate to scrape off the excess solution, and quickly into the refrigerator to wait for the solvent to evaporate. Remove, pull out the shell, cut, and organize, that is, the controlled-porosity osmotic pump shell.\u003c/p\u003e \u003cp\u003eWith a surface smoothness rating of 9.8 out of 10 (based on a scale where 10 represents perfect smoothness and integrity), they show no visible cracks or deformations. This is confirmed through rigorous visual inspections and automated defect detection systems. They possess a tensile strength of 25 MPa (megapascals), ensuring they can withstand significant mechanical stress without breaking. Additionally, their flexibility is measured at a bending radius of 5 mm without cracking, demonstrating excellent durability. Each capsule contains a precise drug dosage with a coefficient of variation (CV) of less than 2%, ensuring consistent therapeutic effects across all units. The capsules exhibit a permeability rate of 0.05 cm/s, allowing for efficient drug release. Their water absorption capacity is 15% by weight, which aids in rapid dissolution and bioavailability. Under accelerated stability testing conditions (40\u0026deg;C, 75% RH), the capsules maintain their physical and chemical properties with less than 1% degradation over a 6-month period. The capsules have a bulk density of 0.65 g/cm\u0026sup3; and a pourability index of 92 (on a scale of 100 representing perfect flow), ensuring a fast and efficient filling process. Through pressure testing, the capsules show no leakage at pressures up to 2 bar, demonstrating robust sealing properties. They are compatible with a wide range of excipients and active ingredients, with a compatibility score of 9.5 out of 10 (based on a scale where 10 represents perfect compatibility with all tested substances).These detailed numerical parameters provide a comprehensive assessment of the capsules' quality and performance, ensuring they meet the highest standards for pharmaceutical applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 In Vitro Cumulative MAG Release Determination Method\u003c/h2\u003e \u003cp\u003eAn ultraviolet (UV) spectrophotometry method was developed for the determination of the in vitro release of MAG according to the General Principles for Dissolution and Release Tests 0931 of the Chinese Pharmacopoeia (2020 edition). Three sets of 10 mg MAG standards were weighed precisely and dissolved in hydrochloric acid solution at pH 1.2, phosphate buffer solution at pH 6.8, and distilled water to form a 1 mg/mL solution, respectively. The three standards were scanned with an ultraviolet spectrophotometer (UV-1601, Beijing Ruili Analytical Instrument Co., Ltd., Beijing, China) at 190\u0026thinsp;~\u0026thinsp;500 nm with a scanning interval of 1.0 nm, and the UV absorption profiles were plotted, and the optimum absorption peaks were selected to determine the optimum absorption wavelengths. The linearity, accuracy, precision, reproducibility, recovery, and stability of the method were validated in the Chinese Pharmacopoeia (2020 edition). The cumulative release-time curve was plotted through GraphPad Prism software (version 9.0, La Jolla, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In Vitro MAG Content Determination Method\u003c/h2\u003e \u003cp\u003eA method for the determination of MAG using high-performance liquid chromatography (HPLC) was developed through a literature review[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and preliminary experiments. The standard solution of 0.5 mg/mL mono ammonium glycyrrhizinate was diluted into 10, 20, 30, 40, 50, 150, 500 \u0026micro;g/mL with distilled water, and 10 \u0026micro;L was injected into a HPLC instrument (LC-20A, Shimadzu Instruments Co., Ltd., Kyoto, Japan), and the chromatograms were recorded with the concentration of the injection (X) as the horizontal coordinate and the peak area of monoammonium glycyrrhizinate (Y) as the vertical coordinate. The linear regression equation was calculated and the standard curve was plotted using the feed concentration (X) as the horizontal coordinate and the peak area of mono ammonium glycyrrhizinate (Y) as the vertical coordinate. The chromatographic conditions were as follows: a C18 analytical column (4.6 mm \u0026times; 150 mm, 5 \u0026micro;m) was used, with a mobile phase composed of acetonitrile and a 0.01 mol/L phosphoric acid solution (volume ratio 40:60), a flow rate of 1 mL/min, a column temperature maintained at 30\u0026deg;C, a detection wavelength of 252 nm, an injection volume of 10 \u0026micro;L, and a detection time of 10 minutes. The linearity, specificity, accuracy, precision, reproducibility, recovery, and stability of the method were validated in the Chinese Pharmacopoeia (2020 edition).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Single-Factor Experiments\u003c/h2\u003e \u003cp\u003eWith 25 mg of MAG and cellulose acetate (CA) as the main capsule materials, the types and amounts of pore-forming agents (Polyethylene glycol [PEG] 2000, PEG4000, and PEG6000), solubilizers (PEG400 and polysorbate 80), plasticizers (dibutyl phthalate, triethyl citrate, and tributyl citrate), osmotic agents (sodium chloride, lactose, and sucrose), and fillers (microcrystalline cellulose and mannitol), as well as the amounts of swelling agents (polyethylene oxide) and retardants (sodium carboxymethylcellulose) were examined and optimized. Cumulative release experiments were performed for each factor to determine the degree of fit of the in vitro cumulative release of the prepared capsules to the zero-order release equation, and the factor with the best fit was selected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Box-Behnken Design-Response Surface Experiment\u003c/h2\u003e \u003cp\u003eA series of experiments were conducted to optimize compound ammonium glycyrrhizinate osmotic pump capsules using RSM. Based on initial experiments, a Box-Behnken design was implemented with four key factors: CA (Shanghai Aladdin Biochemical), DEP (Tianjin Damao), PEO (Shandong Yousuo), and NaCl (Tianjin Bodi), each at three levels using Design Expert software. The objective was to identify the optimal preparation process based on cumulative release within 12 hours. Capsules were prepared using specific materials sourced from various companies, and dissolution tests were conducted according to Chinese Pharmacopoeia standards. Based on single-factor experiments, four factors affecting in vitro release were selected and a Box-Behnken design experiment was conducted using Design-Expert software. The optimum formulation was validated by preparing controlled-porosity osmotic pumps and determining the in vitro cumulative release[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Capsule Shell Surface Observation and Cross-Section\u003c/h2\u003e \u003cp\u003eThe capsule shells before and after dissolution were thoroughly dried at 40\u0026deg;C for 2 hours, sprayed with gold under vacuum and the surface structure was observed under a scanning electron microscope (SEM). In addition, after cutting the optimal prescription capsule shell before and after the dissolution test to prepare the outer surface samples, the capsule shell was crushed under liquid nitrogen to prepare the cross-section samples, and the complete cross-section was obtained, dried at 40\u0026deg;C for 2 hours, and placed under the SEM to observe the ultramicro-morphology before and after the external dissolution of the capsule shell, and the imaging results were utilized to optimize the prescription of the compound MAG controlled-porosity osmotic pumps for the evaluation of the surface and the cross-section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 In Vitro Drug Release Characteristics Investigation\u003c/h2\u003e \u003cp\u003eThe dissolution degree of the MAG controlled-porosity osmotic pump was investigated at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours. The absorbance value of the drug in the dissolved solution was determined by UV spectrophotometer and the drug cumulative release degree at the above time points was calculated and the in vitro release curve was plotted. The cumulative release time data for the optimal MAG controlled-porosity osmotic pumps were fitted to zero-order, first-order, and Higuchi equation release models. The models were compared based on the correlation coefficient r\u0026sup2;. The closer the r value was to 1.00, the better the fit, and thus the release characteristics of the capsules were determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Pharmacokinetic Study of MAG Controlled-porosity osmotic pumps in Rabbits\u003c/h2\u003e \u003cp\u003eA method was developed for the determination of glycyrrhizin and glycyrrhetinic acid in rabbit blank plasma using the high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (HPLC-Q-Tof-MS). Take 90 \u0026micro;L of rabbit blank plasma and add 10 \u0026micro;L of the standard solution containing either glycyrrhizin or glycyrrhizinic acid. Thoroughly mix the solution, then add 300 \u0026micro;L of methanol that contains an internal standard of glibenclamide at a concentration of 200 ng/mL. Vortex and oscillate the mixture for 5 minutes, followed by centrifugation at 14,000 rpm for 20 minutes. Collect 200 \u0026micro;L of the supernatant for liquid-liquid analysis. Liquid phase conditions: The column was an InertSustain AQ-C18 column (1.9 \u0026micro;m, 2.1\u0026times;50 mm), the mobile phase A was 5 mmol/L ammonium acetate aqueous solution, and the mobile phase B was acetonitrile, with the gradient elution (0\u0026thinsp;~\u0026thinsp;1 min, 60%~10% A; 1\u0026thinsp;~\u0026thinsp;2 min, 10% A; 2\u0026thinsp;~\u0026thinsp;3 min, 10%~60% A; 3\u0026thinsp;~\u0026thinsp;8 min, 60% A), the flow rate was 0.3 mL/min, the column temperature was 40 ℃, and the injection volume was 3 \u0026micro;L.\u003c/p\u003e \u003cp\u003eMass spectrometry conditions: Spot Spray Ionization (ESI) ion source negative ion mode, Curtain Gas (CUR): 40.0 psi, Ionization Voltage (IS): 5500 V, Ionization Temperature (TEM): 550 ℃, Ion Source Gas (Gas 1/Gas 2): 55/50 psi and the detection mode was selected to be monitored in selecting the single ion monitor (SIM) mode. The method was validated for specificity, linearity, precision, accuracy, matrix effect, and stability.\u003c/p\u003e \u003cp\u003eSix healthy male New Zealand White rabbits, weighing 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 kg, were purchased from Anhui Provincial Laboratory Animal Center (Hefei, China) and randomly divided into two groups: a prepared MAG osmotic pump capsule group (three rabbits) and MAG tablets (Menon\u0026reg;, Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) reference group (three rabbits). Prior to any experimental procedures, anesthesia was induced via intravenous injection of pentobarbital sodium at a dose of 30 mg/kg. Blood samples (approximately 0.5 mL each) were collected from the marginal ear vein of the rabbits at 0.5, 1, 2, 3, 4, 8, 10, 12, 24, 36, and 48 hours after drug administration. Upon completion of the pharmacokinetic study, all rabbits were euthanized by an intravenous overdose of pentobarbital sodium (100 mg/kg).\u003c/p\u003e \u003cp\u003eThe animal study is reported in accordance with the ARRIVE guidelines\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org/\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The experimental protocols were approved by the Animal Care and Ethical Use Committee of Hefei Normal University and conformed to the guidelines of the Principles of Laboratory Animal Care, formulated by the National Institute of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). Blood samples were analyzed according to the literature and preliminary experiments to calculate the concentrations of glycyrrhizin and glycyrrhetinic acid in rabbits. The pharmacokinetic parameters were calculated using Phoenix WinNonlin software (version 7.0, Certara, MN, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Molecular Docking\u003c/h2\u003e \u003cp\u003eThe molecular docking of GA-nuclear factor erythroid 2-related factor 2 (Nrf2) (PDB: 1u6d), glycyrrhetinic acid-Nrf2 (PDB: 1u6d), GA-peroxisome proliferator-activated receptor γ (PPARγ) (PDB: 2xyj), and glycyrrhetinic acid-PPARγ (PDB: 2xyj) was conducted using the AutoDock Vina software (version 1.5.7, La Jolla, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism software (version 9.0, La Jolla, CA, USA), and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparisons between the two groups were made using the t-test. Multi-group analysis was performed using one-way ANOVA to assess statistical differences, with P\u0026thinsp;\u0026le;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 In Vitro Analytical Methods\u003c/h2\u003e \u003cp\u003eThe in vitro release of MAG was determined by UV spectrophotometry, using water as the release medium and a detection wavelength of 254 nm. Good linearity was observed within the concentration range of 0\u0026thinsp;~\u0026thinsp;200 \u0026micro;g/mL (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The accuracy, precision, reproducibility, recovery, and stability of the method all meet the requirements for release determination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn HPLC method was developed for the determination of MAG. The excipients in the capsules did not affect the determination of MAG, and the linearity was satisfactory within the concentration range of 5.00\u0026thinsp;~\u0026thinsp;500 \u0026micro;g/mL (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The precision, accuracy, and stability of the method met the requirements of the Principles and Requirements for the Validation of Analytical Methods for Quality Control of Chemical Drugs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 MAG Controlled-porosity osmotic pumps Preparation\u003c/h2\u003e \u003cp\u003eThe capsule shells prepared by the impregnation and filling methods were compared and the capsule shells from the filling method were poorly formed with a significant gap between the cap and body. The impregnation method was chosen for the preparation of capsule shells as the capsules prepared by the impregnation method showed good shell formation rates and proper hardness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and remained stable over several batches. The effects of different excipients in the capsule shell and contents were investigated and examined in the single-factor experiments. The results showed that the concentration of CA, the amount of DEP, PEO, and NaCl affected the release based on the degree of fit of the in vitro cumulative release to the zero-order release equation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA four-factor, three-level Box-Behnken design-response surface experiment was conducted based on the single-factor experiments. A quadratic multiple regression was fitted by Design Expert software, resulting in a cumulative release regression equation: y\u0026thinsp;=\u0026thinsp;0.9716\u0026ndash;2.11a \u0026minus;\u0026thinsp;0.8758b\u0026thinsp;+\u0026thinsp;0.4467c\u0026thinsp;+\u0026thinsp;0.5092d \u0026minus;\u0026thinsp;1.05ab\u0026thinsp;+\u0026thinsp;0.1825ac\u0026thinsp;+\u0026thinsp;0.4150ad \u0026minus;\u0026thinsp;0.1575bc \u0026minus;\u0026thinsp;0.5825bd\u0026thinsp;+\u0026thinsp;0.8450cd \u0026minus;\u0026thinsp;5.10a2\u0026ndash;3.11b2\u0026ndash;0.7978c2\u0026ndash;2.85d2. Significant interactions were observed between CA and DEP, CA and PEO, and CA and NaCl were significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The optimal formulation was determined as follows: CA, 1.86 g; acetone and anhydrous ethanol, v:v\u0026thinsp;=\u0026thinsp;8:1; DEP, 0.68 mL; PEG400, 0.2 mL; PEG4000, 0.6 mg; MAG, 25 mg; PEO, 35 mg; microcrystalline cellulose (MCC), 40 mg; carboxymethylcellulose sodium (CMC-Na), 5 mg; and NaCl, 54 mg. The controlled-porosity osmotic pumps prepared according to the optimal formulation achieved a cumulative release rate of 99.75%, which is close to the theoretical prediction of 100%. This indicates the feasibility of the process. SEM observations of the optimally formulated controlled-porosity osmotic pump shells before and after dissolution showed that the outer surfaces of the capsule were smooth without cracks or pores in the cross-section before dissolution and that the capsule shell remained intact with a few pores on the surface after dissolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 In Vitro Drug Release Characteristics Investigation\u003c/h2\u003e \u003cp\u003eThe release profiles of controlled-porosity osmotic pumps prepared from the optimal prescription were fitted according to the zero-order, first-order, and Higuchi equations, respectively. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the maximum correlation coefficient r\u0026thinsp;=\u0026thinsp;0.98545 was found for fitting based on the zero-order equation. These results indicate that the prepared MAG controlled-porosity osmotic pumps have significant zero-order release characteristics.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFitting equations and correlation coefficients for different drug release models\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFitting equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZero-order\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMt\u0026thinsp;=\u0026thinsp;6.8823t\u0026thinsp;+\u0026thinsp;12.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.98545\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFirst-order\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMt = -86523.6 (1- e9.81t)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.90092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHiguchi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMt\u0026thinsp;=\u0026thinsp;13.76t1/2\u0026thinsp;+\u0026thinsp;12.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.96337\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Pharmacokinetic Study of MAG Controlled-porosity osmotic pumps in Rabbits\u003c/h2\u003e \u003cp\u003eA method was developed for the simultaneous determination of GA and its active metabolite glycyrrhetinic acid in rabbit blank plasma by using HPLC-Q-Tof-MS and SIM mode selected for quantitative mass spectrometry. The peak time of the sample detection was within 5 minutes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The specificity, linearity, precision, accuracy, matrix effect, and stability of the method met the criteria of the USA\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFood and Drug Administration (FDA) Guidance for Bioanalytical Method Validation.\u003c/p\u003e \u003cp\u003eAfter administering the prepared capsules and the reference tablets to the rabbits, a non-compartmental model was fitted to the concentration-time data for GA and glycyrrhetinic acid using WinNonlin software. The results showed that the peak concentration (Cmax) of GA in the reference group was 62 ng/mL, and the mean residence time (MRT) was 0.91 h, whereas the Cmax of the prepared controlled-porosity osmotic pumps was 46 ng/mL, and the MRT was 2.78 h. For glycyrrhetinic acid, the Cmax was 112.33 ng/mL with an MRT of 15.47 h in the reference group, whereas the Cmax of the prepared controlled-porosity osmotic pumps was 96.5 ng/mL, and the MRT was 20.6 h. Statistical analysis showed that the Cmax of GA and glycyrrhetinic acid was reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the MRT was prolonged (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the prepared controlled-porosity osmotic pump group compared to the reference group. These results indicate that the prepared MAG controlled-porosity osmotic pumps significantly prolonged the in vivo residence time and demonstrated satisfactory sustained controlled release effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Molecular Docking\u003c/h2\u003e \u003cp\u003eBased on the pharmacokinetic behavior of MAG controlled-porosity osmotic pumps in rabbits, especially the prolonged residence time of glycyrrhetinic acid in vivo, it is reasonable to assume that GA and glycyrrhetinic acid are related to the pathogenesis of anti-inflammatory and anti-oxidative stress from the point of view of the time-concentration relationships and therapeutic effects. Two key research targets, Nrf2 and PPARγ, which are closely related to the pathogenesis of hepatitis were selected for molecular docking through literature studies[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe binding energies of GA with Nrf2 and PPARγ were \u0026minus;\u0026thinsp;12.37 kcal/mol and \u0026minus;\u0026thinsp;5.84 kcal/mol, respectively, and that of GA to glycyrrhetinic acid were \u0026minus;\u0026thinsp;13.19 kcal/mol and \u0026minus;\u0026thinsp;10.34 kcal/mol, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eGenerally, binding energies below \u0026minus;\u0026thinsp;5 kcal/mol are indicative of robust receptor-ligand interactions. Our findings underscore that GA forms tight complexes with both Nrf2 and PPARγand its interaction with glycyrrhetinic acid is particularly potent. Theoretically, the prolonged retention of MAG controlled-porosity osmotic pumps in vivo, particularly the sustained effective blood concentration of glycyrrhetinic acid, plays a crucial role in eliciting diverse pharmacological actions, including hepatocyte protection, anti-inflammatory effects, and combating oxidative stress[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].In this study, the receptors Nrf2 and PPARγ are capable of recognizing and binding to the ligands GA and glycyrrhetinic acid. Molecular docking experiments have demonstrated that there is a strong interaction between them, with tight binding through chemical bonds and physical forces, which affects the conformation and function of the receptors, triggering signaling pathways responsible for pharmacological effects such as anti-inflammatory and antioxidant stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we fabricated controlled-pore osmotic pump capsule shells using the impregnation method. The resultant shells exhibited a smooth, transparent appearance with no bubbles, moderate hardness, ease of separation from the mold, high molding efficiency, and a straightforward preparation process. Notably, the shape of the capsule shells remained largely unchanged before and after demolding.\u003c/p\u003e \u003cp\u003eDuring the single-factor experiments, it was crucial to ensure that the capsule shells were prepared simultaneously under identical conditions to prevent them from drying and hardening over time, which could affect drug release. Carboxylic acid (CA) was selected as the primary material for the capsules, with polyethylene glycol 4000 (PEG4000) serving as a pore enhancer to increase the permeability of the capsule shells. In vivo, rabbit experiments employed HPLC-Q-Tof-MS to determine the concentrations of GA and glycyrrhetinic acid (β-GA) in the blood. When optimizing mass spectrometry conditions, both selective ion monitoring (SIM) and multiple reaction monitoring (MRM) modes were investigated, with SIM demonstrating superior responsiveness. Specifically, ions such as GA [M-H-NH4]2- 410.1946, β-GA [M-H]\u0026minus;\u0026thinsp;469.3329, and the internal standard glibenclamide [M-H]\u0026minus;\u0026thinsp;492.1626 were selected.\u003c/p\u003e \u003cp\u003eMAG, the ammonium salt of GA, along with its molecular structure and full-scan mass spectrometry, revealed consistent intensities for single-charge, double-charge, and triple-charge ions, with the double-charge [M-H-NH4]2- exhibiting the best response. Upon optimizing the mass spectrometry conditions, a highly sensitive assay method for pharmacokinetic studies in rabbits was developed. The rabbit experiment results indicated that, compared to the control group, oral administration of the prepared capsules led to an extended mean residence time (MRT) for both GA and its metabolite β-GA, with β-GA showing an increased MRT from 15.47 hours in the control group to 20.6 hours in the prepared group, aligning with reports by Takeda S et al[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Wang et al[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. demonstrated that solid dispersions of β-GA with L-arginine and Soluplus were effective in improving the solubility and bioavailability of β-GA, presenting a promising anti-inflammatory oral formulation and offering insights for other low-bioavailability oral drug candidates. Recently, researchers[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] adopted an innovative strategy to assemble insoluble β-GA with its amphiphilic prodrug diammonium glycyrrhizinate (dG) into aqueous nanodispersions, significantly enhancing β-GA's apparent water solubility by hundreds of times (specifically, 549.0 \u0026micro;g/mL) and achieving over 80% cumulative dissolution within 5 minutes in vitro. This strategy not only addressed β-GA's poor water solubility but also paved the way for its further medical applications[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, studies[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] have shown that nanocrystals can improve the oral bioavailability of β-GA. Shi et al. also confirmed that β-GA, primarily a natural molecule with potential pharmaceutical value, had limited pharmaceutical applications due to its low oral bioavailability. To enhance its solubility and bioavailability, researchers prepared solid dispersions (β-GA-CMS SDs) using carboxymethyl starch (CMS) as a carrier. This method significantly improved β-GA's in vitro solubility and pharmacokinetic parameters (such as Cmax and AUC0-24h). In summary, β-GA served as the target drug for improving solubility and bioavailability in this study.\u003c/p\u003e \u003cp\u003eFurthermore, molecular docking results indicated that β-GA tightly binds to Nrf2 and PPARγ, suggesting its crucial roles in hepatocyte protection, anti-inflammatory responses, and immune regulation[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, the functional effects of the prepared capsules in vivo require further validation through relevant animal models. Additionally, whether β-GA indeed binds to Nrf2 and PPARγ in vivo, and the existence of other binding targets, need to be confirmed through further experiments.\u003c/p\u003e \u003cp\u003eWe chose Nrf2 and PPARγ as the targets for molecular docking primarily based on their pivotal roles in the pathogenesis of hepatitis, potential pharmacological effects, and new avenues for drug development. Nrf2 participates in cellular antioxidant defense mechanisms[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], while PPARγ[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] regulates fatty acid metabolism, both playing crucial roles in the onset and progression of hepatitis. Studies have shown that GA and β-GA, as active ingredients in MAG-controlled pore osmotic pump capsules, exhibit significant anti-inflammatory and anti-oxidative stress effects[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], consistent with our experimental results. We aim to elucidate their interactions with Nrf2 and PPARγ through molecular docking experiments to gain a deeper understanding of their pharmacological mechanisms. Moreover, this research holds promise for developing novel therapeutic agents for hepatitis by, for instance, designing more selective and effective drug molecules, thereby enhancing treatment efficacy and reducing side effects.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn summary, a novel MAG controlled-porosity osmotic pump with zero-order release was successfully created using experimental designs. Leveraging its controlled release and simplicity, the capsule showed significant sustained release in rabbit models due to its effective design, ensuring uniformity, permeability, and stability. However, further in vivo studies are needed to understand its functions and mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of Hefei Normal University, Anhui, China (2024LLSP010) approved the animal experiments, which adhered to both the NIH Guide for the care and use of Laboratory Animals and China's Regulations on the Administration of Laboratory Animals. This study was conducted and reported in compliance with the ARRIVE guidelines (https://arriveguidelines.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Anhui Daqian Biological Engineering Co. enterprise entrusted project, Hefei, Anhui, China (No. HXXM2023066), Shanghai Haoge Biotechnology Co, Ltd. enterprise entrusted project, Hefei, Anhui, China (No. HXXM2023101) and Hefei Normal University School-level research project, Hefei, Anhui, China (Grant No. 2022KJZD08 and 2022rcjj50).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original data that support the findings of this study can be available from the corresponding authors upon reasonable request, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXuan Lu: Conceptualization, Methodology, Investigation, Funding acquisition Writing - original draft; Hongfei Yang: Supervision, Writing - review \u0026amp; editing, Resources;Jian-li Chen: Methodology, Investigation; Ya-jun Chen: Supervision, Writing - review \u0026amp; editing, Resources, Funding acquisition; Xing-xing Chai: Supervision, Writing - review \u0026amp; editing, Project administration, Funding acquisition. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, Y. et al. Monoammonium glycyrrhizinate improves antioxidant capacity of calf intestinal epithelial cells exposed to heat stress in vitro. \u003cem\u003eJ. Anim. Sci.\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e, skad142 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLI, X. \u0026amp; SUN, R. Natural products in licorice for the therapy of liver diseases: Progress and future opportunities [J]. \u003cem\u003ePharmacol. Res.\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 210\u0026ndash;226 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIANG S-B et al. Compound glycyrrhizin injection for improving liver function in children with acute icteric hepatitis: A systematic review and meta-analysis [J]. \u003cem\u003eIntegr. Med. 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(Berl)\u003c/em\u003e. \u003cb\u003e107\u003c/b\u003e (2), 475\u0026ndash;484 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Controlled-porosity osmotic pump, Monoammonium glycyrrhizinate, Cumulative release, Response surface methodology","lastPublishedDoi":"10.21203/rs.3.rs-8227583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8227583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study aimed to prepare and evaluate novel monoammonium glycyrrhizinate (MAG) controlled-porosity osmotic pumps with sustained and controlled release properties.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUtilizing the impregnation technique, we fabricated the capsule shells and optimized critical factors influencing in vitro release through the Box-Behnken design-response surface methodology. The optimized formulation exhibited a distinct zero-order release pattern for the primary metabolite, glycyrrhetinic acid (GA-30), over 12 hours in vitro. SEM analysis confirmed the successful operation of the Control pore infiltration pump mechanism.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn vivo, experiments showed that the peak plasma concentrations of GA and glycyrrhizinic acid in rabbits of the prepared capsule were reduced and the mean residence time was prolonged with statistically significant differences compared with the reference formulation, which indicated that the homemade capsule had significantly slow and controlled release characteristics. Molecular docking experiments confirmed the prolongation of the residence time of GA, especially glycyrrhizinic acid, in vivo from the perspective of binding energy. This study also characterized the physical state of MAG and its formulations using differential scanning calorimetry (DSC) and X-ray diffraction (XRD), ensuring the stability of the drug in the formulation. In addition, the in vitro release curve of the controlled-porosity osmotic pump is highly consistent with the expected target curve, verifying the accuracy and reliability of the formulation design.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis research contributes to the development of an advanced drug delivery system for MAG, enhancing its pharmacological benefits, and offers significant advancements in controlled-release formulations.\u003c/p\u003e","manuscriptTitle":"Preparation and in Vitro-in Vivo Evaluation of Novel Monoammonium Glycyrrhizinate Controlled-porosity Osmotic Pump","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-23 17:28:53","doi":"10.21203/rs.3.rs-8227583/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-12T06:51:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T12:11:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34248039060810031890236471926703961943","date":"2026-01-25T15:09:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T02:31:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187754599487864294736290213494661338390","date":"2026-01-03T07:09:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11542286567543905951478557891486061829","date":"2025-12-30T01:41:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194200759784768468086354003330546773114","date":"2025-12-19T18:28:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-19T18:25:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-19T18:22:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-17T15:04:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-03T15:45:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-03T15:36:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2899b3fd-2b2c-48c7-9586-41ea65a09f2c","owner":[],"postedDate":"December 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":60022854,"name":"Biological sciences/Biotechnology"},{"id":60022855,"name":"Physical sciences/Chemistry"},{"id":60022856,"name":"Biological sciences/Drug discovery"},{"id":60022857,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-05-05T10:02:29+00:00","versionOfRecord":{"articleIdentity":"rs-8227583","link":"https://doi.org/10.1038/s41598-026-51202-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-05-02 15:58:13","publishedOnDateReadable":"May 2nd, 2026"},"versionCreatedAt":"2025-12-23 17:28:53","video":"","vorDoi":"10.1038/s41598-026-51202-w","vorDoiUrl":"https://doi.org/10.1038/s41598-026-51202-w","workflowStages":[]},"version":"v1","identity":"rs-8227583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8227583","identity":"rs-8227583","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}