Exploration of the Antiviral Mechanism of Gypsum-Licorice Compatibility Pairing in Ma- Xing-Shi-Gan Decoction from the Perspective of Metal-Organic Supramolecular Interactions

Exploration of the Antiviral Mechanism of Gypsum-Licorice Compatibility Pairing in Ma- Xing-Shi-Gan Decoction from the Perspective of Metal-Organic Supramolecular Interactions | 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 Research Article Exploration of the Antiviral Mechanism of Gypsum-Licorice Compatibility Pairing in Ma- Xing-Shi-Gan Decoction from the Perspective of Metal-Organic Supramolecular Interactions Chang Lu, Ying Ma, Haoyu Zhao, Yu Zheng, Qun Zhao, Lili Lin, XiuZhen Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6643243/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The efficacy of gypsum-licorice (SG-GC) pair was evaluated by evaluating its in vivo and in vitro anti-respiratory syncytial virus(RSV) efficacy. The results showed that SG-GC had significant efficacy against RSV infection in mice, which was close to that of the whole formula, and could significantly reduce the viral load in the lungs, improve the symptoms of lung injury, and reduce the inflammatory cell infiltration in the pathological sites. The decoction of single-flavored gypsum showed some anti-RSV efficacy, but not significant; when combined with sub-inhibitory concentrations of GA, it showed a significantly enhanced anti-RSV effect. The experimental results of this study suggest that gypsum may be a potential key antiviral substance, but its efficacy needs to be complemented by licorice; the significant enhancement of antiviral efficacy of SG-GC may be related to the formation of metal-organic supramolecules after the interaction of trace metal ions in gypsum and GA in licorice. licorice gypsum antiviral compatibility metal-organic supramolecule Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Ma-Xing-Shi-Gan Decoction (MXSGT) is well-known for its straightforward compatibility and impressive therapeutic effects. The scientific implications of its compatibility have consistently attracted the attention of the traditional Chinese medicine (TCM) community. MXSGT originates from the "Treatise on Febrile and Miscellaneous Diseases," authored by Zhang Zhongjing during the Eastern Han Dynasty. This decoction comprises whole Ephedra ( Ephedra sinica herb, Sovereign, 9 g), gypsum (Sovereign, 24 g), bitter almond ( Prunus armeniaca seeds, minister, 9 g) and licorice ( Glycyrrhiza uralensis roots, courier, 6 g). With its four key ingredients, MXSGT is recognized for its capacity to release heat, cool the body, clear the lungs, and alleviate asthma(Guo et al., 2021 ). In the clinical practice of TCM, MXSGT and its modified formulations are extensively employed to address pneumonia caused by various viral infections, including respiratory syncytial virus, influenza A, and COVID-19 in pe, diatric patients. Throughout the COVID-19 pandemic, the "three prescriptions and three medicines" have demonstrated efficacy in combating viral infections and alleviating symptoms in numerous clinical cases. Notably, Qingfei Paidu Decoction, Huashi Baidu Decoction, Xuanfei Baidu Decoction, Lianhua Qingwen Capsules and Jinhua Qinggan Granules are all derived from MXSGT, further substantiating its effectiveness as an antiviral prescription(Li et al., 2020 ). The medicinal pair of SG-GC within MXSGT exemplifies a classic sovereign-courier compatibility, evaluated by Mr. Zhang Xichun, one of the "Four Great Doctors" in the medical field, who noted that "the combination of the two can drive away evil without harming good". A review of the "Dictionary of Traditional Chinese Medicine Prescriptions" and the "Treatise on Febrile and Miscellaneous Diseases" indicates that licorice is the most frequently paired Chinese medicine with gypsum. Utilizing the sweet and soothing properties of licorice can help reduce phlegm and relieve coughs, while also moderating the cold and cooling effects of gypsum. This combination is primarily employed to treat cough and asthma associated with lung heat(Yang, Kong, & Yue, 2009 ). Consequently, exploring the scientific basis of SG-GC compatibility is of great significance for elucidating the antiviral efficacy of MXSGT. Since the Han and Tang Dynasties, physicians have frequently utilized gypsum in medicine to clear heat, purge fire, and alleviate heat-related ailments. Analyzing the formulation of MXSGT reveals a significant reliance on gypsum, particularly when symptoms such as sweating, asthma, and lung heat are pronounced; in these instances, the quantity of gypsum may reach five times that of ephedra(Jia et al., 2011 ). The primary component of gypsum is calcium sulfate (CaSO 4 ), an inorganic salt that exhibits slight solubility in water. This solubility results in a rapid attainment of saturation in aqueous decoctions, with the calcium concentration remaining constant despite increases in the quantity of gypsum. Additionally, gypsum contains various trace elements, including iron, zinc, and copper (Lu et al., 1997). Increasing the amount of gypsum can significantly enhance the concentration of trace elements in the soup, suggesting that trace metal ions may play a crucial role in the functional properties of gypsum. Similarly, some studies have indicated that the use of pure CaSO 4 , as opposed to gypsum, results in a lack of antipyretic effect, which may be attributed to the differences in trace metal content. Specific trace elements found in gypsum have been shown to possess antiviral properties. For instance, zinc has the ability to inhibit RNA viruses, including the dengue virus and the novel coronavirus(Faghfouri et al., 2022 ; Kar et al., 2019 ); manganese is involved in the activation of innate immunity, which helps to inhibit the activity of DNA viruses(S. Sun et al., 2023 ); and copper can generate reactive oxygen species, utilizing redox signaling mechanisms to eliminate viruses(Albalawi et al., 2024 ). These findings underscore the importance of considering trace elements in the study of gypsum and its compound formulas. They may provide insights into the differences in the medicinal properties of gypsum compared to CaSO 4 , or they may offer a novel approach to understanding the antiviral efficacy mechanisms of MXSGT. Licorice is a well-documented antiviral agent, with its principal component, glycyrrhizinic acid (GA), demonstrating inhibitory effects against various DNA and RNA viruses(Huan et al., 2021 ). GA has been shown to inhibit the novel coronavirus (SARS-CoV-2), hepatitis B virus (HBV), and human papillomavirus (HPV), primarily by blocking viral attachment or enhancing host cell activity(Bravo, Serrano, Duque, Ferragud, & Coronado, 2023 ; Z. G. Sun, Zhao, Lu, Yang, & Zhu, 2019 ; Yu et al., 2021 ). The carboxyl and hydroxyl groups present in the GA structure readily form coordination bonds with metal ions, leading to the creation of metal-organic supramolecular complexes. A study utilizing high-performance liquid chromatography (HPLC) indicated that the addition of gypsum affects the concentrations of other key components in MXSGT, notably resulting in a decrease in GA content. This decrease is hypothesized to arise from the formation of numerous metal-GA supramolecular complexes during the co-cooking process. The formation of these metal-organic supramolecules may enhance the activity of the original organic components or generate new pharmacological properties, particularly in antiviral applications. For instance, polyphenol-zinc complexes have been shown to significantly reduce genome replication levels across various respiratory RNA virus groups, including human coronavirus OC43 (HCoV-OC43), influenza A virus (IAV), and human metapneumovirus (hMPV)(Kreiser et al., 2022 ); similarly, copper complexes derived from pine resin ligands can effectively inactivate the novel coronavirus (SARS-CoV-2)(Correa et al., 2023 ). Therefore, it is imperative to conduct comprehensive research on the antiviral efficacy of metal-GA supramolecules in MXSGT. This study utilizes the SG-GC medicinal pair as a case study to investigate the antiviral principles underlying the compatibility of medicinal pairs through the lens of metal-organic supramolecular chemistry. It aims to provide novel insights into the antiviral mechanisms of MXSGT while also serving as a reference for identifying candidate drugs against Respiratory Syncytial Virus (RSV) within the framework of TCM. RSV is a globally prevalent respiratory virus that primarily affects infants, the elderly, and individuals with compromised immune systems, and it is a leading cause of pneumonia in young children. Currently, there is a scarcity of effective RSV vaccines(Lin, Liao, & Li, 2024 ), and no specific antiviral treatments are available. Severe cases of RSV can be life-threatening. Ribavirin and palivizumab are the only RSV treatments approved by the US FDA(Zou, Cao, Gao, Yie, & Wu, 2024 ); however, ribavirin, a broad-spectrum antiviral, has uncertain efficacy against RSV and is associated with numerous adverse effects. In contrast, palivizumab is costly, intended solely for high-risk infants to prevent RSV, and offers limited therapeutic benefits(Resch, 2017 ). TCM posits that RSV infection is an "external evil," with pneumonia resulting from RSV classified as "pneumonia and cough." Most viral pneumonia syndromes in infants and young children are characterized by phlegm-heat obstructing the lungs. Consequently, MXSGT, recognized for its heat-clearing, detoxifying, blood-cooling, and fire-purging properties, is frequently employed in clinical settings to combat RSV infections, demonstrating significant efficacy. Therefore, the exploration of drugs derived from TCM prescriptions for the treatment of RSV is supported by extensive clinical experience and holds considerable promise(Xi et al., 2020 ). This research will investigate the antiviral mechanisms of the SG-GC drug pair and explore the potential of metal-organic supramolecular drugs. 2 Materials and Methods 2.1 Drugs and Materials Ephedra, gypsum, bitter almond, and licorice (200812, 201138, 191108001, 210214) were sourced from Beijing Tongrentang Nanjing Pharmacy. Reference substances, including glycyrrhizic acid, ribavirin, calcium sulfate, zinc sulfate, copper sulfate, and iron sulfate (A12HS191107, 026GS165579, S05HS193206, S24260, S70004), were obtained from Shanghai Yuanye Biotechnology Co., Ltd. Magnesium sulfate (20141231) was acquired from Shanghai Lingfeng Chemical Reagent Co., Ltd. The EDTA standard solution (M10IR16124A) was also procured from Shanghai Yuanye Biotechnology Co., Ltd. Fetal bovine serum (7E721D3) was purchased from Nanjing Novezan Biotechnology Co., Ltd. Concentrated nitric acid (20200930) was obtained from Sinopharm Chemical Reagent, while the H2O2 solution (20220601) was acquired from Tianjin Oriental Guangcheng Pharmaceutical Chemical Co., Ltd. Additionally, DMEM high-glucose culture medium and PBS buffer were sourced from Nanjing Senbeiga Biotechnology Co., Ltd. 2.2 Instruments CO 2 cell culture incubator (Formascientific, USA), enzyme label spectrophotometric plate reader (Spectra Max 190, USA), ultra-clean workbench (Suzhou Purification Equipment Factory), BX20 inverted microscope (Olympus, Japan), digital display constant temperature water bath Pot (Qun'an Instrument Laboratory Co., Ltd.), fluorescence quantitative PCR instrument (Shanghai Roche Diagnostic Products Co., Ltd.), small animal living Micro CT imaging system (PerkinElmer, USA), ultra-trace spectrophotometer (Thermo Fisher Scientific, USA), inductively coupled plasma mass spectrometer (Agilent, USA), microwave digestion instrument (TANK40, Shanghai Xinyi Microwave Chemical Technology Co., Ltd., Shanghai, China). 2.3 Cells, viruses and experimental animals Laryngeal epidermoid carcinoma cells Hep-2 were purchased from Wuhan Shangen Biotechnology Co., Ltd., and human non-small cell lung cancer cells A549 were purchased from Hunan Fenghui Biotechnology Co., Ltd. The human respiratory syncytial virus type A2 long strain was a kind gift from the Jiangsu Provincial Key Laboratory of Pediatric Respiratory Diseases (Traditional Chinese Medicine). 48 BALB/c mice, female, 5 weeks old, weighing 15–18 g, were adaptively reared for 1 week before starting the experiment, purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.; ethics number: 202404A065; animal license number: SCXK (Shanghai) 2023-0009. 2.4 Determination of RSV virulence Hep-2 cells were seeded in a 96-well cell plate. The following day, when the cell density reached 80–90%, the RSV virus liquid was diluted tenfold in a gradient of DMEM medium containing 2% fetal calf serum. Dilutions of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , and 10 9 were inoculated into the Hep-2 cells, with each dilution replicated in eight wells and a blank control group included. After inoculation, the cells were incubated at 37°C with 5% CO 2 for 2 hours. The virus liquid in the 96-well plate was then aspirated, and the wells were washed twice with PBS solution. DMEM medium containing 2% fetal calf serum was added, and the plate was placed back at 37°C with 5% CO 2 for an additional 3–4 days. After this incubation period, the plate was examined under a microscope to observe and record the number of wells exhibiting cytopathic effects (CPE) in Hep-2 cells at each dilution. The highest dilution at which no new lesions appeared was noted. Hep-2 cells infected with serially diluted viruses displayed cytopathic effects 3–4 days later. The Reed-Muench method was employed to calculate the 50% tissue culture infectious dose (TCID 50 ) of respiratory syncytial virus (RSV). Notably, syncytial formation of RSV was observed on the third day of culture. The TCID 50 value for RSV virulence calculated using the Reed-Muench formula was 10 − 8 /0.1 mL. 2.5 Sample preparation Preparation of MXSGT: Weigh 9 g of ephedra (MH), 24 g of gypsum(SG), 9 g of bitter almond, and 6 g of licorice, and place them in a decoction bag within a 500 mL round-bottomed flask. Add 8 times the amount of deionized water, bringing it to room temperature. Allow the mixture to stand for 30 minutes, then apply strong heat to boil the liquid. Reduce to a simmer and let the mixture boil gently for 30 minutes, concentrating it to 40 mL. Filter the liquid through gauze while it is still hot. Preparation of SG-GC Co-Decoction: Weigh 24 g of gypsum and 6 g of licorice, and place them in a decoction bag within a round-bottomed flask. Add deionized water in a quantity that is eight times the weight of the solids, and allow the mixture to stand at room temperature for 30 minutes. Following this, apply heat to the decoction. Bring the liquid to a boil, then reduce the heat to maintain a gentle simmer for 30 minutes, concentrating the solution to a final volume of 40 mL. While the liquid is still hot, filter it through gauze. Preparation of Calcium Sulfate-Licorice (CaSO 4 -GC) Co-Decoction: Weigh 24 g of anhydrous calcium sulfate and 6 g of licorice, placing them in a decoction bag within a round-bottomed flask. Add 8 times the amount of deionized water. Decoct, concentrate, and filter as described above to obtain the calcium sulfate-licorice co-decoction. Preparation of Licorice(GC) Decoction: Weigh 6 g of licorice, placing it in a decoction bag within a round-bottomed flask. Add 8 times the amount of deionized water, then boil, concentrate, and filter as previously outlined to obtain the licorice single decoction. Preparation of gypsum (SG) decoction: Weigh 20, 24, 30, and 60 g of gypsum into separate decoction bags and place them in three round-bottomed flasks. Add eight times the amount of deionized water, then decoct as previously described and concentrate the solution to 40 mL. Filter the mixture while hot to obtain gypsum single decoction with crude drug concentrations of 0.5, 0.6, 0.75, and 1.5 g/mL. Preparation of Ribavirin Positive Drug Solution: Weigh 25 mg of ribavirin into 10 mL of ultrapure water to achieve a concentration of 2.5 mg/mL ribavirin solution. The aforementioned drugs are stored at 4°C for future use. 2.6 The detection of calcium ion content in gypsum decoction was performed using the ethylenediamine tetraacetic acid complex titration method Weigh 5.4 g of ammonium chloride and dissolve it in 20 mL of distilled water. Subsequently, add 35 mL of concentrated ammonia solution and dilute the mixture with distilled water to a final volume of 100 mL to prepare the NH 3 ·H 2 O-NH 4 Cl buffer. Accurately pipette 0.1 mL of gypsum decoctions at varying concentrations (0.5, 0.75, and 1.5 g/mL) into 250 mL Erlenmeyer flasks. To each flask, add 10 mL of the NH 3 ·H 2 O-NH 4 Cl buffer along with a suitable amount of chrome black. Introduce an appropriate quantity of T indicator and titrate with a 1 mol/L EDTA standard solution until the solution transitions from wine red to pure blue. This determination should be performed in triplicate. Calculation formula: 2.7 Detection of trace metal ion content in gypsum water decoction by inductively coupled plasma mass spectrometry Pipette precisely 0.1 mL of gypsum decoctions at varying concentrations (0.5, 0.75, and 1.5 g/mL of crude drug) into microwave digestion tanks. Add 0.8 mL of concentrated nitric acid and 0.1 mL of H 2 O 2 solution, then place the tanks in the microwave digestion instrument. Follow the digestion procedure as outlined in Table S1. After digestion, adjust the volume to 10 mL in a volumetric flask, shake well to prepare the test solution, and measure the concentrations of the four metal ions (Mg, Al, Fe, and Zn) using ICP-MS, ensuring that each measurement is conducted in triplicate. 2.8 Grouping of mice, modeling and drug administration Feeding of Mice: The temperature is maintained at 25 ± 5°C, the relative humidity is kept at 60 ± 5%, and the light cycle is set to 12 hours. Mice are provided with mouse-specific feed and have free access to food and water. Mice Grouping and Modeling: A total of 48 five-week-old BALB/c mice were acclimatized for one week and subsequently randomized into eight groups: control group (Control), model group (Model), gypsum group (SG), licorice group (GC), SG-GC group (SG-GC), calcium sulfate-licorice group (CaSO 4 -GC), MXSGT group (MXSGT), and positive drug group (Ribavirin), with six animals in each group. The mice were anesthetized using isoflurane gas. Mice in the model and administration groups were intranasally infected with 80 µL of RSV virus at a titer of 10 6 TCID50 per mouse, while those in the normal group received 80 µL of normal saline via the same route. Administration to Mice: Mice in each group were administered intragastrically two hours post-modeling and continued for three days. The same volume of normal saline was administered to both the blank and model groups. Dosages were calculated based on the conversion ratio of adult (60 kg) drug dosage to the body surface area of mice (20 g). The dosages for each group were consistent with that of the MXSGT group. The body weight of mice in each group was recorded daily. The dosages assigned to the mice in each experimental group were as follows: Mice in the Ribavirin group received a dosage of 46 mg/kg, a value corroborated by the data from reference. The MXSGT-treated mice were administered a dosage of 18 g/kg, as previously reported in reference. In both the SG-GC and CaSO₄-GC groups, the dosages were set at 11 g/kg each. The SG group received a dosage of 9 g/kg, while the GC group was subjected to a lower dosage of 2.25 g/kg. These precisely determined dosages are critical for comprehensively evaluating the specific impacts of each treatment regimen on the experimental outcomes. 2.10 Micro-computed tomography of mouse lungs On the second day, eight groups of mice underwent CT imaging experiments, utilizing high-resolution X-ray imaging technology. Prior to scanning, isoflurane is administered to anesthetize the mouse. The mouse is then swiftly positioned on the scanning bed, and a mask filled with isoflurane gas is placed over its face. The scanning position is fine-tuned using a micro-adjuster to ensure that the lungs are oriented at the optimal angle for a 360° scan. 2.11 Mouse Material Collection The mice were fasted and deprived of water following the last administration. On the third day of modeling, six mice from each group were euthanized by dislocation. After blunt separation of the skin, fresh lung tissue was photographed and examined, and the right lungs of the mice were harvested. The middle lobe was utilized for H&E section staining to assess lung pathology, while the remaining lung tissue was frozen at -80°C for future analysis. 2.12 Pathological examination of mouse lung tissue The middle lobe of the right lung from each group of mice was placed in 1 mL of 4% paraformaldehyde within a centrifuge tube, fixed at 4°C for 24 hours, dehydrated, embedded in paraffin, and sectioned onto a glass slide. During hematoxylin and eosin (H&E) staining, hematoxylin stains the cell nuclei blue, while eosin stains the cytoplasm and connective tissue a light pink, facilitating the observation of pathological conditions, which were subsequently examined and photographed using a microscope. 2.13 Real-time quantitative PCR method to detect viral load Sample pretreatment and extraction of RNA: Weigh 50 mg of mouse lung tissue into a sterile, enzyme-free 1.5 mL centrifuge tube. Add 500 µL of FreeZol Reagent lysis solution and homogenize using a ball mill for 10 minutes. Allow the mixture to stand at room temperature for an additional 10 minutes. Next, add 100 µL of dilution buffer solution, shake to mix, and centrifuge at 11,200 rpm for 15 minutes. Transfer 400 µL of the supernatant into a new sterile, enzyme-free centrifuge tube. Add an equal volume of isopropyl alcohol, mix by inverting, and let it stand at room temperature for 10 minutes before centrifuging at 11,200 rpm for 10 minutes. Carefully discard the supernatant. Wash the precipitate by adding 1 mL of 75% ethanol, followed by centrifugation at 9,100 rpm for 3 minutes at room temperature. Discard the supernatant, repeat the washing process twice, and subsequently discard the supernatant. Allow the precipitate to dry at room temperature, then add 20 µL of enzyme-free water to dissolve the precipitate, vortexing for 3 minutes to ensure complete dissolution. Store the solution at -80°C. Determination of RNA concentration: Take 1 µL of the total RNA sample from each tube and measure the RNA concentration and the A 260 /A 280 ratio using an ultra-micro-volume spectrophotometer. A ratio in the range of 1.8-2.0 indicates that the RNA is of sufficient quality. Use enzyme-free water to dilute each sample to a total RNA concentration of 200 ng/µL. Reverse transcription of RNA into cDNA: Prepare the following mixture in a sterile, enzyme-free PCR tube: x µL RNA (1 µg), 4 µL of 4×gDNA Wiper Mix, and (16-x) µL of RNase-free ddH 2 O. Gently mix by pipetting and heat at 42°C for 2 minutes. Directly add 4 µL of 5×Hiscript II qRT SuperMix II to the reaction tube from the previous step, and gently mix with a pipette. Heat at 50°C for 15 minutes, then at 85°C for 5 seconds, and finally hold at 4°C. store at -20°C for long-term use. qPCR detection: quantitative PCR was performed using GAPDH as the internal reference. The primer sequences utilized are detailed in Table S2. The reaction conditions utilized are detailed in Table S3. Prepare 20 µL reaction system in a qPCR tube. The quantified viral RNA levels were presented as a percentage relative to the control. 2.14 detects the effect of drugs on cell survival rate Sample preparation: Dissolve 13.2 mg of GA in 10 mL of DMEM high-sugar medium to prepare a 1600 µM GA solution. Pass the solution through a 0.22 µm sterile filter to eliminate impurities and ensure sterilization. Subsequently, dilute the solution with DMEM to achieve final concentrations of 25, 50, 100, 200, 400, 600, and 800 µM. Weigh and dissolve standards of calcium sulfate, zinc sulfate, copper sulfate, iron sulfate, and magnesium sulfate in DMEM, diluting each to a concentration of 50 µM. Additionally, dilute gypsum decoctions of 500, 750, and 1500 mg/mL with DMEM to final concentrations of 250, 375, and 750 mg/mL, respectively. CCK-8 detection: Add 100 µL of A549 cell suspension at a density of 2×10 4 cells/mL to each well of a 96-well plate. Pre-incubate the culture plate in an incubator for 24 hours at 37°C with 5% CO 2 . After removing the culture plate from the culture bottle, discard the cell supernatant from each well and wash the wells with PBS solution. Next, inoculate 200 µL of the diluted sample into each well of the 96-well plate, ensuring that there are 6 replicate wells for each concentration. Additionally, set up control wells for the cells. Once all wells are prepared, label them on the 96-well plate and place the plate in a 37°C incubator with 5% CO 2 for culture. After 24 hours, terminate the culture by removing the 96-well plate from the incubator, discarding the supernatant from each well, and washing the wells twice with PBS solution. Prepare a mixture of CCK-8 and medium in the appropriate proportion, then add 100 µL of this mixture to each well. Incubate the culture plate in the incubator for 1 hour. Finally, use a microplate reader to measure the absorbance at 450 nm. Calculation formula: 2.15 in vitro anti-RSV experiment Add 0.8 mL of A549 cell suspension, with a density of 10^5 cells/mL, to each well of a 24-well plate. Incubate the plate in a 37°C, 5% CO 2 environment for 24 hours. Prepare the gypsum decoction by diluting it with DMEM containing 2% fetal calf serum to achieve crude drug concentrations of 0.25, 0.375, and 0.75 g/mL. Additionally, prepare GA solutions at varying concentrations (10, 40, 80, 100, and 200 µM), along with 50 µM solutions of calcium sulfate, zinc sulfate, copper sulfate, iron sulfate, and magnesium sulfate for administration. Once the cells have formed a monolayer, add each drug and incubate the cells for 1 hour. Concurrently, incubate 100 TCID50 of RSV virus along with the drugs for 1 hour. After this incubation, aspirate the liquid and wash the cells with PBS. Subsequently, add the RSV virus solution to infect the cells for 2 hours. Following this, aspirate the virus solution, wash the cells three times with PBS to remove any unadsorbed virus, and then add the drugs to the cells. Incubate the cells for an additional 24 hours in a 37°C, 5% CO 2 incubator. Finally, lyse the cells using lysis buffer and detect the viral load via qRT-PCR, following the same methodology as described above. 2 Results 3.1. Evaluation of the anti-RSV efficacy of SG-GC in vivo 3.1.1 Detection of viral load Perform relevant operations through specific methods, as shown in Fig. 1 a. The RT-qPCR method was employed to quantify the viral load in lung of mice across all experimental groups. As illustrated in Fig. 1 b, the virus inhibition rates for the positive control drugs Ribavirin, MXSGT, and the SG-GC pair were 75.0%, 68.4%, and 68.7%, respectively, all demonstrating significant inhibitory effects (p < 0.001). In contrast, the inhibition rates for single-flavored gypsum (SG) and single-flavored licorice (GC) were not significant. Notably, the antiviral effect of the SG-GC group was markedly superior to that of the SG group (p < 0.001) and the GC group (p < 0.01), highlighting the characteristics of compatibility and synergy. Given that the inhibition rate of the calcium sulfate-licorice group (CaSO4-GC) was only 33%, it is preliminarily suggested that the enhanced inhibition rate observed in the SG-GC group (p < 0.05) may be attributed to the trace elements present in gypsum. 3.1.2 Observation of Lung Damage Lung damage was assessed through examination of fresh lung tissue. Figure 2 a illustrates that the fresh lung tissue from the model group, SG group, and GC group exhibited dark red congestion and edema that were visible to the naked eye (indicated by the yellow circle). In contrast, the fresh lung tissue of the CaSO 4 -GC group displayed dark red lesions at the junction of the lung lobes (marked by the white circle), although the extent and severity of damage were reduced compared to the model group. As illustrated in Fig. 2 b, CT scans revealed a "white lung" phenomenon (highlighted by the red circle), lung consolidation (indicated by the yellow arrow), and bronchiectasis (shown by the white arrow), characterized by flocculent and ground-glass opacities diffusely distributed throughout both lungs. The lung tissue in the ribavirin group, MXSGT group, and SG-GC group appeared normal and was light pink in color, with no significant pulmonary capillary congestion, edema, or hemorrhage observed. The lung tissue damage was minimal, and no notable pathological findings were evident on CT scans. These results indicate that mice treated with the "SG-GC" drug pair not only reduce viral load but also exhibit effects comparable to those of the full prescription in alleviating symptoms of lung infection. 3.1.3 Pathological Changes in Lung Tissue The H&E staining results of mouse lung tissue sections from each group are presented in Fig. 2 c. The model group, SG group, GC group and CaSO 4 -GC group exhibited alveolar wall rupture and congestion, accompanied by significant inflammatory cell infiltration between the alveoli (indicated by the blue arrow). After treatment, the ribavirin group, MXSGT group, and SG-GC group showed pathological conditions of the lung tissue that were comparable to those of the blank group. This suggests a marked improvement in inflammatory cell infiltration, with the alveolar wall structure remaining largely intact. These findings indicate that the SG-GC drug treatment not only reduced viral load but also exhibited effects similar to those of the full prescription in alleviating symptoms of pulmonary infection. In contrast, the CaSO 4 -GC group did not demonstrate these characteristics. Therefore, it is speculated that Ca 2+ is not the primary active substance of the SG-GC drug against RSV, and the observed effects may be related to trace metal elements. 3.1.4 Changes in Mouse Body Weight Mice may experience a decreased appetite and subsequent weight loss during viral infections. As illustrated in Fig. 3 , the mice in the blank group exhibited a gradual increase in weight, while those in the other groups experienced a progressive decrease in weight following the onset of the viral infection (day 3). Notably, the body weight of treated mice in the ribavirin, MXSGT, and SG-GC groups showed a slight increase by the 6th day. 3.2 Evaluation of the in vitro anti-RSV efficacy of SG-GC on the main component 3.2.1 Effect of glycyrrhizic acid on the anti-RSV efficacy of gypsum Glycyrrhizic acid (GA), the principal component of licorice, interacts with various metal ions present in the gypsum decoction. This interaction leads to the formation of diverse metal-organic supramolecular complexes, which may alter pharmacological activity. Initially, the cytotoxic concentration range of each substance on host cells was assessed using CCK-8 assays. As illustrated in Fig. 4 a, GA exhibited no cytotoxicity within the concentration range of 25-1600 µM, while SG demonstrated similar safety between 250–750 mg/mL. Subsequently, the viral load in the cells was measured through RT-qPCR experiments. As depicted in Fig. 4 b, GA did not show any inhibitory effect on RSV at concentrations up to 100 µM; however, at a concentration of 200 µM, GA displayed a notable inhibitory effect on RSV, achieving an inhibition rate of up to 75%. SG exhibited some anti-RSV activity, although this effect was not statistically significant, with inhibition rates for RSV decoctions containing varying amounts of crude drug remaining below 25%. However, the anti-RSV effect was significantly enhanced when GA was combined with SG at a sub-inhibitory concentration of 50 µM. At SG crude drug concentrations of 0.25 g/mL, 0.375 g/mL, and 0.75 g/mL, the inhibition rates increased to 56%, 69%, and 77%, respectively, which is more than double that observed at the same concentration, indicating a certain concentration dependence. This phenomenon is analogous to the synergistic properties of the SG-GC drug pair. Importantly, this synergy does not appear to be directly attributed to GA; rather, it may result from the interaction of the metal ion complex with the gypsum decoction. 3.2.2 The Effect of Glycyrrhizic Acid on the Anti-RSV Efficacy of Various Metal Ions in Gypsum To further investigate the compatibility and synergistic effects of metal ions present in the gypsum decoction with glycyrrhizic acid (GA), we selected Ca 2+ , Cu 2+ , Mg 2+ , Fe 3+ , and Zn 2+ for anti-RSV experiments. As illustrated in Fig. 5 a, at a metal ion concentration of 50 µM, host cell safety was satisfactory; however, the anti-RSV effects varied (Fig. 5 b). Ca 2+ and Fe 3+ exhibited negligible direct anti-RSV activity, while Cu 2+ and Mg 2+ demonstrated inhibition rates of 63% and 47%, respectively. This suggests that these ions possess some inherent anti-RSV properties, but their effectiveness remains largely unchanged when combined with sub-inhibitory concentrations of GA (50 µM). Notably, the inhibition rate of 50 µM Zn 2+ against RSV was 27%, which increased to 66% when combined with 50 µM GA, representing a 2.4-fold enhancement compared to the inhibition rate observed with Zn 2+ alone. These findings indicate that the gypsum decoction itself exhibits a certain antiviral effect, likely attributable to trace metal elements such as copper, magnesium, and zinc. However, only the combination of Zn 2+ and glycyrrhizic acid displayed synergistic properties akin to those of the SG-GC pair, suggesting that the Zn-GA supermolecule formed through the coordination of Zn 2+ and GA may be a critical factor in the antiviral mechanism of the SG-GC pair. 3.2.3 Determination of Metal Element Content in Gypsum Water Decoction with Varying Crude Drug Concentrations The determination of metal element content in gypsum water decoction involves titrating the Ca 2+ concentration with an EDTA standard solution, while trace elements are quantified using ICP-MS. Results presented in Fig. 6 a indicate that as the amount of gypsum increases (simulating the reuse of gypsum in the recipe), the Ca 2+ concentration in the decoction remains relatively constant at 3.8 g/L, suggesting that the CaSO 4 in the decoction has reached saturation. Additionally, Fig. 6 b demonstrates that with an increase in gypsum quantity, the concentrations of trace elements Cu and Fe in the decoction exhibit a slight increase, whereas the levels of Mg, Al, and Zn increase significantly.Specifically, when the amount of gypsum increased threefold, the concentration of Cu 2+ rose from 183 ng/mL to 262 ng/mL, representing an increase of 1.4 times. Similarly, the concentration of Fe 3+ increased from 1335 ng/mL to 2467 ng/mL, reflecting an increase of 1.8 times. The Mg 2+ concentration showed a notable increase from 1960 ng/mL to 4480 ng/mL, a rise of 2.3 times. The concentration of Al 3+ increased significantly from 120 ng/mL to 1172 ng/mL, which is an increase of 9.7 times. Particularly noteworthy is the surge in Zn 2+ concentration, which escalated from 166 ng/mL to 2180 ng/mL, indicating a remarkable increase of 13.1 times, the highest among the trace elements. This observation suggests that as the amount of gypsum increases, the content of trace elements generally rises. Consequently, the anti-RSV effect of varying amounts of SG combined with GA is dependent on concentration (Fig. 4 b). Furthermore, while the trace elements may contribute to gypsum's anti-RSV efficacy, its compatibility with licorice is also a crucial factor in enhancing this efficacy. 3 Discussion This study investigates the Junshi compound SG-GC within the MXSGT as the primary research focus. Using a mouse model of RSV infection, the findings demonstrate that both MXSGT and the SG-GC drug pair exhibit significant anti-RSV effects. Specifically, both treatments resulted in reduced viral loads in the lungs of mice, alleviated lung injury symptoms, and decreased inflammatory cell infiltration. In contrast, the antiviral effects of the individual components, gypsum or licorice, were found to be minimal, suggesting that gypsum must be used in conjunction with licorice to effectively exert its antiviral properties. A comparative analysis between the SG-GC medicinal pair and the CaSO 4 -GC group indicated that the antiviral efficacy may derive from trace metal elements present in gypsum. To further investigate the compatibility principle of gypsum and licorice, an RSV-infected cell model was utilized to examine the interactions between mineral and organic components. After confirming the safety of each component on host cells, it was observed that gypsum alone exhibited limited inhibitory potential against RSV, with an inhibition rate ranging from 16.2–28.4%. However, when combined with GA at a sub-inhibitory concentration (50 µM), the anti-RSV effect was significantly enhanced, improving by 2.7 to 4.3 times. Subsequent studies revealed that the inhibitory effects of metal ions (Ca 2+ , Fe 3+ , Cu 2+ , Mg 2+ , Zn 2+ ) in the gypsum decoction varied. At equivalent concentrations, Cu 2+ demonstrated the most effective inhibition, although no enhancement was noted when combined with GA. In contrast, the combination of Zn 2+ with GA resulted in an increased inhibitory effect of up to 2.4 times, showcasing a compatibility enhancement similar to that observed with the SG-GC drug pair. In addition, ICP-MS analysis confirmed that as the amount of gypsum increases, the concentration of trace elements also rises, with Zn 2+ exhibiting the most pronounced increase. This finding suggests, from another perspective, that one potential purpose of reusing gypsum may be to enhance the content of trace elements. While Zn 2+ has been extensively documented for its antiviral properties, this study is the first to reveal a significantly enhanced antiviral effect when it is combined with GA. From the perspective of metal-organic supramolecular chemistry, GA contains carboxyl, hydroxyl, and other functional groups that can coordinate with metal ions. However, the spatial arrangement of the three carboxyl groups in GA is such that they are too distant from one another to form a multiradical ligand group, resulting in lower stability compared to cyclic structures formed by chelating agents like ethylenediamine and ethylenediaminetetraacetic acid (EDTA). Consequently, dissociation is more likely to occur. Conversely, this characteristic also facilitates the release of metal ions from the GA complex. GA molecules possess a hydrophobic triterpene skeleton and hydrophilic sugar groups, resulting in an amphiphilic structure that exhibits effective cell membrane penetration. The hydrophobic component can integrate into the phospholipid bilayer of the cell membrane, disrupting its structure and thereby enhancing drug permeability. This study posits that GA molecules may facilitate the translocation of Zn 2+ across the cell membrane, subsequently releasing Zn 2+ within the intracellular environment. This function resembles that of an ionophore, as illustrated in Fig. 7 . Metal-organic supramolecules can be classified into chelating agents (such as EDTA), metal carboxyls (like porphyrin derivatives), and ionophores based on the type of ligands(Lee, Janisse, & Heffern, 2023 ; Steinbrueck et al., 2020 ), as detailed in Table 1 . Notably, only ionophores are capable of repeatedly transporting metal ions across the membrane, thereby enhancing the intracellular concentration of these ions. In this study, it was observed that the anti-RSV efficacy was augmented when GA was combined with Zn 2+ , potentially linked to the ionophore-like properties of GA. However, whether GA functions as a specific zinc ionophore warrants further investigation. Table 1 Classification of metal-organic complexes according to ligand type Type of ligand Function of ligand Physiological activity of metal ions Metal chelator Non-selective reduction of metal ion concentration inside and outside the cell Reduced drug efficacy Metal shuttle May help extracellular metal ions cross the membrane, but cannot release free metal ions No change Metal Ionophores May help extracellular metal ions cross the membrane and release free metal ions after entering the cell Enhanced drug efficacy Gypsum, a mineral medicine, serves as a rich source of metal ions. When utilized in medicinal applications, gypsum readily forms various metal-organic supramolecular complexes with herbal medicines. Notably, the metal-GA supramolecular complex generated from the combination of gypsum and licorice has been the subject of prior studies(Zhang et al., 2025 ). However, existing research has yet to investigate the synergistic, antagonistic, or novel bioactive properties that may arise from the formation of metal-organic supramolecular structures due to the interaction between metal ions and organic components in gypsum. This study focuses on the supramolecular assembly phenomenon previously observed in research concerning the antiviral efficacy of MXSGT. It represents the first exploration of the antiviral compatibility mechanism of the SG-GC drug pair through the lens of metal-organic supramolecular substances, aiming to elucidate the antiviral compatibility mechanism of this pairing. The supramolecular interactions between trace metal ions and licorice, particularly the coordination and transmembrane transport of Zn 2+ and GA, are pivotal for achieving compatibility and synergy. Furthermore, this study posits that research on the material basis of gypsum should not be conducted in isolation, but rather in conjunction with its compatible medicinal pairs or prescriptions. The metal-organic supramolecular theory marks a significant advancement in compatibility research. Although this study found that SG-GC and MXSGT exhibit certain equivalence in direct antiviral efficacy, this does not imply that ephedra and bitter almond can be entirely excluded from the prescription. These medicinal components may play a significant role in alleviating coughs, asthma, and enhancing symptoms associated with viral infections. The precise compatibility of the four medicinal materials contributes to the effectiveness of the antiviral prescription MXSGT. Classic TCM prescriptions possess both enduring traditional advantages and unique clinical experiences in managing antiviral infections. In contemporary times, there is a need for scientific interpretations of their compatibility theories and pharmacodynamic mechanisms to facilitate the advancement of these ancient classic prescriptions. This study, based on the unique perspective of "organic-metal supermolecules," offers a preliminary scientific explanation of the compatibility theory underlying MXSGT in TCM. It is anticipated that this will inspire new ideas for fundamental research in TCM and provide novel antiviral strategies. The application of innovative TCM opens up new possibilities. Declarations Author contributions Chang Lu: Data curation, Investigation, Formal analysis, Validation, Writing-original draft; Ying Ma: Formal analysis; HaoYu Zhao: Visualization; Yu Zhen: Investigation, Validation; Qun Zhao: Methodology; Lili Lin: Resources; XiuZhen Chen: Investigation; Linwei Chen: Supervision; Gang Li: Resources; Rui Chen: Conceptualization, Supervision, Funding acquisition, Writing-review & editing. Conflicts of interest All authors declare that they have no conflicts of interest. Data availability Data is available on request to the authors. Acknowledgements We acknowledge the support of Jiangsu Key Laboratory of Pediatric Respiratory Disease at Nanjing University of Chinese Medicine and Clinical Research Center of the Second Hospital of Nanjing affiliated to Nanjing University of Chinese Medicine for virus experimental support; Experiment center for science and technology at Nanjing University of Chinese Medicine for technical support; Jiangsu key laboratory of Chinese Medicine Processing for experimental support. Funding Supported by National Natural Science Foundation of China (Grant No. 82474195), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. SJCX24_0897), Youth medical Innovation research project of China（P24021887623）, Nanjing Medical University (TZKY20230104, 2024KF0292). References Albalawi, S. A., Albalawi, R. A., Albalawi, A. A., Alanazi, R. F., Almahlawi, R. M., Alhwity, B. S., . . . Abdel-Maksoud, M. S. (2024). The Possible Mechanisms of Cu and Zn in the Treatment and Prevention of HIV and COVID-19 Viral Infection. Biol Trace Elem Res, 202 (4), 1524-1538. doi:10.1007/s12011-023-03788-9 Bravo, V., Serrano, M., Duque, A., Ferragud, J., & Coronado, P. J. (2023). Glycyrrhizinic Acid as an Antiviral and Anticancer Agent in the Treatment of Human Papillomavirus. J Pers Med, 13 (12). doi:10.3390/jpm13121639 Correa, J. S., Primo, J. O., Balaba, N., Pratsch, C., Werner, S., Toma, H. E., . . . Bittencourt, C. (2023). Copper(II) and Cobalt(II) Complexes Based on Abietate Ligands from Pinus Resin: Synthesis, Characterization and Their Antibacterial and Antiviral Activity against SARS-CoV-2. Nanomaterials (Basel), 13 (7). doi:10.3390/nano13071202 Faghfouri, A. H., Khabbazi, A., Baradaran, B., Khajebishak, Y., Baghbani, E., Noorolyai, S., . . . Alipour, B. (2022). Immunomodulatory and clinical responses to zinc gluconate supplementation in patients with Behçet's disease: A double-blind, randomized placebo-controlled clinical trial. Clin Nutr, 41 (5), 1083-1092. doi:10.1016/j.clnu.2022.03.019 Guo, T., Guo, Y., Liu, Q., Xu, Y., Wei, L., Wang, Z., . . . Sun, L. (2021). The TCM prescription Ma-xing-shi-gan-tang inhibits Streptococcus pneumoniae pathogenesis by targeting pneumolysin. J Ethnopharmacol, 275 , 114133. doi:10.1016/j.jep.2021.114133 Huan, C., Xu, Y., Zhang, W., Guo, T., Pan, H., & Gao, S. (2021). Research Progress on the Antiviral Activity of Glycyrrhizin and its Derivatives in Liquorice. Front Pharmacol, 12 , 680674. doi:10.3389/fphar.2021.680674 Jia, L., Zhao, Y. L., Wang, J. B., Zou, W. J., Li, R. S., Yang, H. B., . . . Xiao, X. H. (2011). Study on the complex prescription compatibility law of the cold and hot nature of Mahuang Decoction and its categorized formulae based on the cold-hot pad differentiating assay. Chin J Integr Med, 17 (4), 290-295. doi:10.1007/s11655-011-0694-2 Kar, M., Khan, N. A., Panwar, A., Bais, S. S., Basak, S., Goel, R., . . . Medigeshi, G. R. (2019). Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells. Front Immunol, 10 , 2347. doi:10.3389/fimmu.2019.02347 Kreiser, T., Zaguri, D., Sachdeva, S., Zamostiano, R., Mograbi, J., Segal, D., . . . Gazit, E. (2022). Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal Ions. Pharmaceuticals (Basel), 15 (3). doi:10.3390/ph15030377 Lee, V. J., Janisse, S. E., & Heffern, M. C. (2023). Plant-derived chelators and ionophores as potential therapeutics for metabolic diseases. Chem Soc Rev, 52 (11), 3927-3945. doi:10.1039/d3cs00167a Li, Q., Bai, C., Yang, R., Xing, W., Pang, X., Wu, S., . . . Gu, X. (2020). Deciphering the Pharmacological Mechanisms of Ma Xing Shi Gan Decoction against COVID-19 through Integrating Network Pharmacology and Experimental Exploration. Front Pharmacol, 11 , 581691. doi:10.3389/fphar.2020.581691 Lin, L., Liao, Z., & Li, C. (2024). Exploring Ribosomal Genes as Potential Biomarkers of the Immune Microenvironment in Respiratory Syncytial Virus Infection. Biochem Genet . doi:10.1007/s10528-024-10778-6 Resch, B. (2017). Product review on the monoclonal antibody palivizumab for prevention of respiratory syncytial virus infection. Hum Vaccin Immunother, 13 (9), 2138-2149. doi:10.1080/21645515.2017.1337614 Steinbrueck, A., Sedgwick, A. C., Brewster, J. T., 2nd, Yan, K. C., Shang, Y., Knoll, D. M., . . . Sessler, J. L. (2020). Transition metal chelators, pro-chelators, and ionophores as small molecule cancer chemotherapeutic agents. Chem Soc Rev, 49 (12), 3726-3747. doi:10.1039/c9cs00373h Sun, S., Xu, Y., Qiu, M., Jiang, S., Cao, Q., Luo, J., . . . Zhu, J. (2023). Manganese Mediates Its Antiviral Functions in a cGAS-STING Pathway Independent Manner. Viruses, 15 (3). doi:10.3390/v15030646 Sun, Z. G., Zhao, T. T., Lu, N., Yang, Y. A., & Zhu, H. L. (2019). Research Progress of Glycyrrhizic Acid on Antiviral Activity. Mini Rev Med Chem, 19 (10), 826-832. doi:10.2174/1389557519666190119111125 Xi, S., Li, Y., Yue, L., Gong, Y., Qian, L., Liang, T., & Ye, Y. (2020). Role of Traditional Chinese Medicine in the Management of Viral Pneumonia. Front Pharmacol, 11 , 582322. doi:10.3389/fphar.2020.582322 Yang, X., Kong, X. L., & Yue, X. Q. (2009). [Dose-response patterns of Radix Glycyrrhizae in Shanghan Lun]. Zhong Xi Yi Jie He Xue Bao, 7 (3), 268-272. doi:10.3736/jcim20090313 Yu, S., Zhu, Y., Xu, J., Yao, G., Zhang, P., Wang, M., . . . Zhang, J. (2021). Glycyrrhizic acid exerts inhibitory activity against the spike protein of SARS-CoV-2. Phytomedicine, 85 , 153364. doi:10.1016/j.phymed.2020.153364 Zhang, Y. Z., Pi, W. M., Tan, X. R., Xu, R., Wang, X., Xu, M. Y., . . . Wang, P. L. (2025). [Exploration of differences in decoction phase state, material form, and crystal form between Glycyrrhizae Radix et Rhizoma-Gypsum Fibrosum and Glycyrrhizae Radix et Rhizoma-CaSO_4·2H_2O based on supramolecules of traditional Chinese medicine]. Zhongguo Zhong Yao Za Zhi, 50 (2), 412-421. doi:10.19540/j.cnki.cjcmm.20240923.301 Zou, G., Cao, S., Gao, Z., Yie, J., & Wu, J. Z. (2024). Current state and challenges in respiratory syncytial virus drug discovery and development. Antiviral Res, 221 , 105791. doi:10.1016/j.antiviral.2023.105791 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6643243","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455108435,"identity":"5fb1c9c0-f3ae-429a-bea5-76e015252380","order_by":0,"name":"Chang Lu","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Lu","suffix":""},{"id":455108439,"identity":"08620297-b983-4a84-b972-d7f4e35a7dd2","order_by":1,"name":"Ying Ma","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Ma","suffix":""},{"id":455108440,"identity":"b38b6f2f-896e-4dc0-bb7f-77ebed835106","order_by":2,"name":"Haoyu Zhao","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haoyu","middleName":"","lastName":"Zhao","suffix":""},{"id":455108442,"identity":"d2edabd6-b139-4d0f-bc37-713ce704e281","order_by":3,"name":"Yu Zheng","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zheng","suffix":""},{"id":455108443,"identity":"199149ee-60e2-471d-a0bb-39fa31959e21","order_by":4,"name":"Qun Zhao","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qun","middleName":"","lastName":"Zhao","suffix":""},{"id":455108444,"identity":"2661bb33-3dae-4429-ba0e-6fced9da3838","order_by":5,"name":"Lili Lin","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Lin","suffix":""},{"id":455108445,"identity":"7868cff1-3007-4663-ab0f-770fb512bdfb","order_by":6,"name":"XiuZhen Chen","email":"","orcid":"","institution":"The Second Hospital of Nanjing, Affiliated to Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"XiuZhen","middleName":"","lastName":"Chen","suffix":""},{"id":455108446,"identity":"f33c1faf-e79a-4358-87a0-fc4a1cb4915c","order_by":7,"name":"Linwei Chen","email":"","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linwei","middleName":"","lastName":"Chen","suffix":""},{"id":455108448,"identity":"0bc04b79-c3bf-4f77-a3fc-8c7367cf95f9","order_by":8,"name":"Gang Li","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Li","suffix":""},{"id":455108453,"identity":"67622735-39d7-4563-9572-0eed595df4c9","order_by":9,"name":"Rui Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCQgpx8BwAMxibCBWizHJWhgSYSoJa+Gf3XzscWWbRfp2xjOGn24w2MhuOMD87AFeS+4cSzc82yaRu7PhjLF0DkOa8YYDbOYG+LQYSOSYSTYCtWw4cMaMOYfhcOKGAzxsEvi15H8DaUk3gGj5T4yWHDaQlgSolgOEtUjcSDOTbDgnYbjhwLFi6RyDZOOZh9nM8Grhn5H8TLKhrE7e4MbhjZ9zKuxk+443P8OrBcm+AyB3AjEzcepB9jUQrXQUjIJRMApGGAAAkPdHcZn81YcAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Rui","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-12 06:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6643243/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6643243/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82594008,"identity":"96413897-3f62-475d-896b-8a06f9768b29","added_by":"auto","created_at":"2025-05-13 08:28:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71338,"visible":true,"origin":"","legend":"\u003cp\u003eStudy on the \u003cem\u003ein vivo\u003c/em\u003e anti-RSV effect of drugs in each group. (a) Schematic diagram of mouse modeling and drug administration process. (b) RSV virus load in lung of mice in each group (n=5). *\u003cem\u003e p\u003c/em\u003e＜0.05，**\u003cem\u003e p\u003c/em\u003e＜0.01，***\u003cem\u003e p\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/bc49a64b41adc21dd2816085.jpg"},{"id":82592659,"identity":"78b93446-7a63-44f7-b65f-5d6f2e400741","added_by":"auto","created_at":"2025-05-13 08:20:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":242415,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of lung injury of mice in each group. (a) Fresh lung tissue morphology. (b) Lung micro-CT scanning image. (c) Pathological changes of lung tissue in mice in each group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/6f6c7bee0d4d865b032b2512.jpg"},{"id":82592657,"identity":"34a8d99c-4b01-49aa-aa2f-31d185a5c083","added_by":"auto","created_at":"2025-05-13 08:20:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75087,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/19c5f12f651d245173e6d54c.jpg"},{"id":82592677,"identity":"fbb10ac5-4237-4717-ab53-0170da116bd2","added_by":"auto","created_at":"2025-05-13 08:20:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antiviral effect of GA and gypsum decoction (a) on cell viability (b) on RSV viral load. *\u003cem\u003e p\u003c/em\u003e＜0.05，**\u003cem\u003e p\u003c/em\u003e＜0.01，***\u003cem\u003e p\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/1cd92d7788898c9f246f35fd.jpg"},{"id":82592667,"identity":"bd99bf1a-a2d4-43f5-8c27-b71ae48cb021","added_by":"auto","created_at":"2025-05-13 08:20:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53794,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different metal ions (50 μM) (a) on cell viability and (b) on RSV viral load in the presence of 50 μM GA. *\u003cem\u003e p\u003c/em\u003e＜0.05，**\u003cem\u003e p\u003c/em\u003e＜0.01，***\u003cem\u003e p\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/2ff864648a4cfc82b9ed73bb.jpg"},{"id":82594009,"identity":"e58d1e69-c750-429b-bb29-ec1a734608ea","added_by":"auto","created_at":"2025-05-13 08:28:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37882,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of metal ion concentration in gypsum decoction. (a) Determination of Ca\u003csup\u003e2+\u003c/sup\u003e in gypsum decoction by EDTA complexation titration; (b) Determination of Cu, Mg, Al, Fe and Zn in gypsum decoction by ICP-MS.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/ec98653ed3d4fa63b36ab66d.jpg"},{"id":82592660,"identity":"5e32188d-3dc1-4e35-99f3-18a8705a71da","added_by":"auto","created_at":"2025-05-13 08:20:12","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18207,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of ionophore assisting metal ions to enter cells\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/89b9bbe0185c83afcecf6767.jpg"},{"id":83594532,"identity":"beeca29b-459d-42f6-933b-dc66f6f7b607","added_by":"auto","created_at":"2025-05-29 07:24:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1696230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6643243/v1/eeba6feb-99d4-466c-9384-5734a000cd0f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploration of the Antiviral Mechanism of Gypsum-Licorice Compatibility Pairing in Ma- Xing-Shi-Gan Decoction from the Perspective of Metal-Organic Supramolecular Interactions","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMa-Xing-Shi-Gan Decoction (MXSGT) is well-known for its straightforward compatibility and impressive therapeutic effects. The scientific implications of its compatibility have consistently attracted the attention of the traditional Chinese medicine (TCM) community. MXSGT originates from the \"Treatise on Febrile and Miscellaneous Diseases,\" authored by Zhang Zhongjing during the Eastern Han Dynasty. This decoction comprises whole Ephedra (\u003cem\u003eEphedra sinica\u003c/em\u003e herb, Sovereign, 9 g), gypsum (Sovereign, 24 g), bitter almond (\u003cem\u003ePrunus armeniaca\u003c/em\u003e seeds, minister, 9 g) and licorice (\u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e roots, courier, 6 g).\u003c/p\u003e \u003cp\u003eWith its four key ingredients, MXSGT is recognized for its capacity to release heat, cool the body, clear the lungs, and alleviate asthma(Guo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the clinical practice of TCM, MXSGT and its modified formulations are extensively employed to address pneumonia caused by various viral infections, including respiratory syncytial virus, influenza A, and COVID-19 in pe, diatric patients. Throughout the COVID-19 pandemic, the \"three prescriptions and three medicines\" have demonstrated efficacy in combating viral infections and alleviating symptoms in numerous clinical cases. Notably, Qingfei Paidu Decoction, Huashi Baidu Decoction, Xuanfei Baidu Decoction, Lianhua Qingwen Capsules and Jinhua Qinggan Granules are all derived from MXSGT, further substantiating its effectiveness as an antiviral prescription(Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The medicinal pair of SG-GC within MXSGT exemplifies a classic sovereign-courier compatibility, evaluated by Mr. Zhang Xichun, one of the \"Four Great Doctors\" in the medical field, who noted that \"the combination of the two can drive away evil without harming good\". A review of the \"Dictionary of Traditional Chinese Medicine Prescriptions\" and the \"Treatise on Febrile and Miscellaneous Diseases\" indicates that licorice is the most frequently paired Chinese medicine with gypsum. Utilizing the sweet and soothing properties of licorice can help reduce phlegm and relieve coughs, while also moderating the cold and cooling effects of gypsum. This combination is primarily employed to treat cough and asthma associated with lung heat(Yang, Kong, \u0026amp; Yue, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Consequently, exploring the scientific basis of SG-GC compatibility is of great significance for elucidating the antiviral efficacy of MXSGT.\u003c/p\u003e \u003cp\u003eSince the Han and Tang Dynasties, physicians have frequently utilized gypsum in medicine to clear heat, purge fire, and alleviate heat-related ailments. Analyzing the formulation of MXSGT reveals a significant reliance on gypsum, particularly when symptoms such as sweating, asthma, and lung heat are pronounced; in these instances, the quantity of gypsum may reach five times that of ephedra(Jia et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The primary component of gypsum is calcium sulfate (CaSO\u003csub\u003e4\u003c/sub\u003e), an inorganic salt that exhibits slight solubility in water. This solubility results in a rapid attainment of saturation in aqueous decoctions, with the calcium concentration remaining constant despite increases in the quantity of gypsum. Additionally, gypsum contains various trace elements, including iron, zinc, and copper (Lu et al., 1997).\u003c/p\u003e \u003cp\u003eIncreasing the amount of gypsum can significantly enhance the concentration of trace elements in the soup, suggesting that trace metal ions may play a crucial role in the functional properties of gypsum. Similarly, some studies have indicated that the use of pure CaSO\u003csub\u003e4\u003c/sub\u003e, as opposed to gypsum, results in a lack of antipyretic effect, which may be attributed to the differences in trace metal content. Specific trace elements found in gypsum have been shown to possess antiviral properties. For instance, zinc has the ability to inhibit RNA viruses, including the dengue virus and the novel coronavirus(Faghfouri et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); manganese is involved in the activation of innate immunity, which helps to inhibit the activity of DNA viruses(S. Sun et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); and copper can generate reactive oxygen species, utilizing redox signaling mechanisms to eliminate viruses(Albalawi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings underscore the importance of considering trace elements in the study of gypsum and its compound formulas. They may provide insights into the differences in the medicinal properties of gypsum compared to CaSO\u003csub\u003e4\u003c/sub\u003e, or they may offer a novel approach to understanding the antiviral efficacy mechanisms of MXSGT.\u003c/p\u003e \u003cp\u003eLicorice is a well-documented antiviral agent, with its principal component, glycyrrhizinic acid (GA), demonstrating inhibitory effects against various DNA and RNA viruses(Huan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). GA has been shown to inhibit the novel coronavirus (SARS-CoV-2), hepatitis B virus (HBV), and human papillomavirus (HPV), primarily by blocking viral attachment or enhancing host cell activity(Bravo, Serrano, Duque, Ferragud, \u0026amp; Coronado, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Z. G. Sun, Zhao, Lu, Yang, \u0026amp; Zhu, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The carboxyl and hydroxyl groups present in the GA structure readily form coordination bonds with metal ions, leading to the creation of metal-organic supramolecular complexes. A study utilizing high-performance liquid chromatography (HPLC) indicated that the addition of gypsum affects the concentrations of other key components in MXSGT, notably resulting in a decrease in GA content. This decrease is hypothesized to arise from the formation of numerous metal-GA supramolecular complexes during the co-cooking process. The formation of these metal-organic supramolecules may enhance the activity of the original organic components or generate new pharmacological properties, particularly in antiviral applications. For instance, polyphenol-zinc complexes have been shown to significantly reduce genome replication levels across various respiratory RNA virus groups, including human coronavirus OC43 (HCoV-OC43), influenza A virus (IAV), and human metapneumovirus (hMPV)(Kreiser et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); similarly, copper complexes derived from pine resin ligands can effectively inactivate the novel coronavirus (SARS-CoV-2)(Correa et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, it is imperative to conduct comprehensive research on the antiviral efficacy of metal-GA supramolecules in MXSGT.\u003c/p\u003e \u003cp\u003eThis study utilizes the SG-GC medicinal pair as a case study to investigate the antiviral principles underlying the compatibility of medicinal pairs through the lens of metal-organic supramolecular chemistry. It aims to provide novel insights into the antiviral mechanisms of MXSGT while also serving as a reference for identifying candidate drugs against Respiratory Syncytial Virus (RSV) within the framework of TCM. RSV is a globally prevalent respiratory virus that primarily affects infants, the elderly, and individuals with compromised immune systems, and it is a leading cause of pneumonia in young children. Currently, there is a scarcity of effective RSV vaccines(Lin, Liao, \u0026amp; Li, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and no specific antiviral treatments are available. Severe cases of RSV can be life-threatening. Ribavirin and palivizumab are the only RSV treatments approved by the US FDA(Zou, Cao, Gao, Yie, \u0026amp; Wu, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); however, ribavirin, a broad-spectrum antiviral, has uncertain efficacy against RSV and is associated with numerous adverse effects. In contrast, palivizumab is costly, intended solely for high-risk infants to prevent RSV, and offers limited therapeutic benefits(Resch, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). TCM posits that RSV infection is an \"external evil,\" with pneumonia resulting from RSV classified as \"pneumonia and cough.\" Most viral pneumonia syndromes in infants and young children are characterized by phlegm-heat obstructing the lungs. Consequently, MXSGT, recognized for its heat-clearing, detoxifying, blood-cooling, and fire-purging properties, is frequently employed in clinical settings to combat RSV infections, demonstrating significant efficacy. Therefore, the exploration of drugs derived from TCM prescriptions for the treatment of RSV is supported by extensive clinical experience and holds considerable promise(Xi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This research will investigate the antiviral mechanisms of the SG-GC drug pair and explore the potential of metal-organic supramolecular drugs.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Drugs and Materials\u003c/h2\u003e\n \u003cp\u003eEphedra, gypsum, bitter almond, and licorice (200812, 201138, 191108001, 210214) were sourced from Beijing Tongrentang Nanjing Pharmacy. Reference substances, including glycyrrhizic acid, ribavirin, calcium sulfate, zinc sulfate, copper sulfate, and iron sulfate (A12HS191107, 026GS165579, S05HS193206, S24260, S70004), were obtained from Shanghai Yuanye Biotechnology Co., Ltd. Magnesium sulfate (20141231) was acquired from Shanghai Lingfeng Chemical Reagent Co., Ltd. The EDTA standard solution (M10IR16124A) was also procured from Shanghai Yuanye Biotechnology Co., Ltd. Fetal bovine serum (7E721D3) was purchased from Nanjing Novezan Biotechnology Co., Ltd. Concentrated nitric acid (20200930) was obtained from Sinopharm Chemical Reagent, while the H2O2 solution (20220601) was acquired from Tianjin Oriental Guangcheng Pharmaceutical Chemical Co., Ltd. Additionally, DMEM high-glucose culture medium and PBS buffer were sourced from Nanjing Senbeiga Biotechnology Co., Ltd.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Instruments\u003c/h2\u003e\n \u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e cell culture incubator (Formascientific, USA), enzyme label spectrophotometric plate reader (Spectra Max 190, USA), ultra-clean workbench (Suzhou Purification Equipment Factory), BX20 inverted microscope (Olympus, Japan), digital display constant temperature water bath Pot (Qun\u0026apos;an Instrument Laboratory Co., Ltd.), fluorescence quantitative PCR instrument (Shanghai Roche Diagnostic Products Co., Ltd.), small animal living Micro CT imaging system (PerkinElmer, USA), ultra-trace spectrophotometer (Thermo Fisher Scientific, USA), inductively coupled plasma mass spectrometer (Agilent, USA), microwave digestion instrument (TANK40, Shanghai Xinyi Microwave Chemical Technology Co., Ltd., Shanghai, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Cells, viruses and experimental animals\u003c/h2\u003e\n \u003cp\u003eLaryngeal epidermoid carcinoma cells Hep-2 were purchased from Wuhan Shangen Biotechnology Co., Ltd., and human non-small cell lung cancer cells A549 were purchased from Hunan Fenghui Biotechnology Co., Ltd. The human respiratory syncytial virus type A2 long strain was a kind gift from the Jiangsu Provincial Key Laboratory of Pediatric Respiratory Diseases (Traditional Chinese Medicine). 48 BALB/c mice, female, 5 weeks old, weighing 15\u0026ndash;18 g, were adaptively reared for 1 week before starting the experiment, purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.; ethics number: 202404A065; animal license number: SCXK (Shanghai) 2023-0009.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Determination of RSV virulence\u003c/h2\u003e\n \u003cp\u003eHep-2 cells were seeded in a 96-well cell plate. The following day, when the cell density reached 80\u0026ndash;90%, the RSV virus liquid was diluted tenfold in a gradient of DMEM medium containing 2% fetal calf serum. Dilutions of 10\u003csup\u003e1\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e were inoculated into the Hep-2 cells, with each dilution replicated in eight wells and a blank control group included. After inoculation, the cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 hours. The virus liquid in the 96-well plate was then aspirated, and the wells were washed twice with PBS solution. DMEM medium containing 2% fetal calf serum was added, and the plate was placed back at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for an additional 3\u0026ndash;4 days. After this incubation period, the plate was examined under a microscope to observe and record the number of wells exhibiting cytopathic effects (CPE) in Hep-2 cells at each dilution. The highest dilution at which no new lesions appeared was noted. Hep-2 cells infected with serially diluted viruses displayed cytopathic effects 3\u0026ndash;4 days later. The Reed-Muench method was employed to calculate the 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) of respiratory syncytial virus (RSV). Notably, syncytial formation of RSV was observed on the third day of culture. The TCID\u003csub\u003e50\u003c/sub\u003e value for RSV virulence calculated using the Reed-Muench formula was 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e/0.1 mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Sample preparation\u003c/h2\u003e\n \u003cp\u003ePreparation of MXSGT: Weigh 9 g of ephedra (MH), 24 g of gypsum(SG), 9 g of bitter almond, and 6 g of licorice, and place them in a decoction bag within a 500 mL round-bottomed flask. Add 8 times the amount of deionized water, bringing it to room temperature. Allow the mixture to stand for 30 minutes, then apply strong heat to boil the liquid. Reduce to a simmer and let the mixture boil gently for 30 minutes, concentrating it to 40 mL. Filter the liquid through gauze while it is still hot.\u003c/p\u003e\n \u003cp\u003ePreparation of SG-GC Co-Decoction: Weigh 24 g of gypsum and 6 g of licorice, and place them in a decoction bag within a round-bottomed flask. Add deionized water in a quantity that is eight times the weight of the solids, and allow the mixture to stand at room temperature for 30 minutes. Following this, apply heat to the decoction. Bring the liquid to a boil, then reduce the heat to maintain a gentle simmer for 30 minutes, concentrating the solution to a final volume of 40 mL. While the liquid is still hot, filter it through gauze.\u003c/p\u003e\n \u003cp\u003ePreparation of Calcium Sulfate-Licorice (CaSO\u003csub\u003e4\u003c/sub\u003e-GC) Co-Decoction: Weigh 24 g of anhydrous calcium sulfate and 6 g of licorice, placing them in a decoction bag within a round-bottomed flask. Add 8 times the amount of deionized water. Decoct, concentrate, and filter as described above to obtain the calcium sulfate-licorice co-decoction.\u003c/p\u003e\n \u003cp\u003ePreparation of Licorice(GC) Decoction: Weigh 6 g of licorice, placing it in a decoction bag within a round-bottomed flask. Add 8 times the amount of deionized water, then boil, concentrate, and filter as previously outlined to obtain the licorice single decoction.\u003c/p\u003e\n \u003cp\u003ePreparation of gypsum (SG) decoction: Weigh 20, 24, 30, and 60 g of gypsum into separate decoction bags and place them in three round-bottomed flasks. Add eight times the amount of deionized water, then decoct as previously described and concentrate the solution to 40 mL. Filter the mixture while hot to obtain gypsum single decoction with crude drug concentrations of 0.5, 0.6, 0.75, and 1.5 g/mL. Preparation of Ribavirin Positive Drug Solution: Weigh 25 mg of ribavirin into 10 mL of ultrapure water to achieve a concentration of 2.5 mg/mL ribavirin solution.\u003c/p\u003e\n \u003cp\u003eThe aforementioned drugs are stored at 4\u0026deg;C for future use.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.6 The detection of calcium ion content in gypsum decoction was performed using the ethylenediamine tetraacetic acid complex titration method\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWeigh 5.4 g of ammonium chloride and dissolve it in 20 mL of distilled water. Subsequently, add 35 mL of concentrated ammonia solution and dilute the mixture with distilled water to a final volume of 100 mL to prepare the NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO-NH\u003csub\u003e4\u003c/sub\u003eCl buffer. Accurately pipette 0.1 mL of gypsum decoctions at varying concentrations (0.5, 0.75, and 1.5 g/mL) into 250 mL Erlenmeyer flasks. To each flask, add 10 mL of the NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO-NH\u003csub\u003e4\u003c/sub\u003eCl buffer along with a suitable amount of chrome black. Introduce an appropriate quantity of T indicator and titrate with a 1 mol/L EDTA standard solution until the solution transitions from wine red to pure blue. This determination should be performed in triplicate.\u003c/p\u003e\n \u003cp\u003eCalculation formula:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 805px; height: 204.556px;\" width=\"805\" height=\"204.556\"\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.7 Detection of trace metal ion content in gypsum water decoction by inductively coupled plasma mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePipette precisely 0.1 mL of gypsum decoctions at varying concentrations (0.5, 0.75, and 1.5 g/mL of crude drug) into microwave digestion tanks. Add 0.8 mL of concentrated nitric acid and 0.1 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, then place the tanks in the microwave digestion instrument. Follow the digestion procedure as outlined in Table S1. After digestion, adjust the volume to 10 mL in a volumetric flask, shake well to prepare the test solution, and measure the concentrations of the four metal ions (Mg, Al, Fe, and Zn) using ICP-MS, ensuring that each measurement is conducted in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Grouping of mice, modeling and drug administration\u003c/h2\u003e\n \u003cp\u003eFeeding of Mice: The temperature is maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C, the relative humidity is kept at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and the light cycle is set to 12 hours. Mice are provided with mouse-specific feed and have free access to food and water.\u003c/p\u003e\n \u003cp\u003eMice Grouping and Modeling: A total of 48 five-week-old BALB/c mice were acclimatized for one week and subsequently randomized into eight groups: control group (Control), model group (Model), gypsum group (SG), licorice group (GC), SG-GC group (SG-GC), calcium sulfate-licorice group (CaSO\u003csub\u003e4\u003c/sub\u003e-GC), MXSGT group (MXSGT), and positive drug group (Ribavirin), with six animals in each group. The mice were anesthetized using isoflurane gas. Mice in the model and administration groups were intranasally infected with 80 \u0026micro;L of RSV virus at a titer of 10\u003csup\u003e6\u003c/sup\u003e TCID50 per mouse, while those in the normal group received 80 \u0026micro;L of normal saline via the same route.\u003c/p\u003e\n \u003cp\u003eAdministration to Mice: Mice in each group were administered intragastrically two hours post-modeling and continued for three days. The same volume of normal saline was administered to both the blank and model groups. Dosages were calculated based on the conversion ratio of adult (60 kg) drug dosage to the body surface area of mice (20 g). The dosages for each group were consistent with that of the MXSGT group. The body weight of mice in each group was recorded daily.\u003c/p\u003e\n \u003cp\u003eThe dosages assigned to the mice in each experimental group were as follows: Mice in the Ribavirin group received a dosage of 46 mg/kg, a value corroborated by the data from reference. The MXSGT-treated mice were administered a dosage of 18 g/kg, as previously reported in reference. In both the SG-GC and CaSO₄-GC groups, the dosages were set at 11 g/kg each. The SG group received a dosage of 9 g/kg, while the GC group was subjected to a lower dosage of 2.25 g/kg. These precisely determined dosages are critical for comprehensively evaluating the specific impacts of each treatment regimen on the experimental outcomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Micro-computed tomography of mouse lungs\u003c/h2\u003e\n \u003cp\u003eOn the second day, eight groups of mice underwent CT imaging experiments, utilizing high-resolution X-ray imaging technology. Prior to scanning, isoflurane is administered to anesthetize the mouse. The mouse is then swiftly positioned on the scanning bed, and a mask filled with isoflurane gas is placed over its face. The scanning position is fine-tuned using a micro-adjuster to ensure that the lungs are oriented at the optimal angle for a 360\u0026deg; scan.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Mouse Material Collection\u003c/h2\u003e\n \u003cp\u003eThe mice were fasted and deprived of water following the last administration. On the third day of modeling, six mice from each group were euthanized by dislocation. After blunt separation of the skin, fresh lung tissue was photographed and examined, and the right lungs of the mice were harvested. The middle lobe was utilized for H\u0026amp;E section staining to assess lung pathology, while the remaining lung tissue was frozen at -80\u0026deg;C for future analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Pathological examination of mouse lung tissue\u003c/h2\u003e\n \u003cp\u003eThe middle lobe of the right lung from each group of mice was placed in 1 mL of 4% paraformaldehyde within a centrifuge tube, fixed at 4\u0026deg;C for 24 hours, dehydrated, embedded in paraffin, and sectioned onto a glass slide. During hematoxylin and eosin (H\u0026amp;E) staining, hematoxylin stains the cell nuclei blue, while eosin stains the cytoplasm and connective tissue a light pink, facilitating the observation of pathological conditions, which were subsequently examined and photographed using a microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.13 Real-time quantitative PCR method to detect viral load\u003c/h2\u003e\n \u003cp\u003eSample pretreatment and extraction of RNA: Weigh 50 mg of mouse lung tissue into a sterile, enzyme-free 1.5 mL centrifuge tube. Add 500 \u0026micro;L of FreeZol Reagent lysis solution and homogenize using a ball mill for 10 minutes. Allow the mixture to stand at room temperature for an additional 10 minutes. Next, add 100 \u0026micro;L of dilution buffer solution, shake to mix, and centrifuge at 11,200 rpm for 15 minutes. Transfer 400 \u0026micro;L of the supernatant into a new sterile, enzyme-free centrifuge tube. Add an equal volume of isopropyl alcohol, mix by inverting, and let it stand at room temperature for 10 minutes before centrifuging at 11,200 rpm for 10 minutes. Carefully discard the supernatant. Wash the precipitate by adding 1 mL of 75% ethanol, followed by centrifugation at 9,100 rpm for 3 minutes at room temperature. Discard the supernatant, repeat the washing process twice, and subsequently discard the supernatant. Allow the precipitate to dry at room temperature, then add 20 \u0026micro;L of enzyme-free water to dissolve the precipitate, vortexing for 3 minutes to ensure complete dissolution. Store the solution at -80\u0026deg;C.\u003c/p\u003e\n \u003cp\u003eDetermination of RNA concentration: Take 1 \u0026micro;L of the total RNA sample from each tube and measure the RNA concentration and the A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e ratio using an ultra-micro-volume spectrophotometer. A ratio in the range of 1.8-2.0 indicates that the RNA is of sufficient quality. Use enzyme-free water to dilute each sample to a total RNA concentration of 200 ng/\u0026micro;L.\u003c/p\u003e\n \u003cp\u003eReverse transcription of RNA into cDNA: Prepare the following mixture in a sterile, enzyme-free PCR tube: x \u0026micro;L RNA (1 \u0026micro;g), 4 \u0026micro;L of 4\u0026times;gDNA Wiper Mix, and (16-x) \u0026micro;L of RNase-free ddH\u003csub\u003e2\u003c/sub\u003eO. Gently mix by pipetting and heat at 42\u0026deg;C for 2 minutes. Directly add 4 \u0026micro;L of 5\u0026times;Hiscript II qRT SuperMix II to the reaction tube from the previous step, and gently mix with a pipette. Heat at 50\u0026deg;C for 15 minutes, then at 85\u0026deg;C for 5 seconds, and finally hold at 4\u0026deg;C. store at -20\u0026deg;C for long-term use.\u003c/p\u003e\n \u003cp\u003eqPCR detection: quantitative PCR was performed using GAPDH as the internal reference. The primer sequences utilized are detailed in Table S2. The reaction conditions utilized are detailed in Table S3. Prepare 20 \u0026micro;L reaction system in a qPCR tube. The quantified viral RNA levels were presented as a percentage relative to the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.14 detects the effect of drugs on cell survival rate\u003c/h2\u003e\n \u003cp\u003eSample preparation: Dissolve 13.2 mg of GA in 10 mL of DMEM high-sugar medium to prepare a 1600 \u0026micro;M GA solution. Pass the solution through a 0.22 \u0026micro;m sterile filter to eliminate impurities and ensure sterilization. Subsequently, dilute the solution with DMEM to achieve final concentrations of 25, 50, 100, 200, 400, 600, and 800 \u0026micro;M. Weigh and dissolve standards of calcium sulfate, zinc sulfate, copper sulfate, iron sulfate, and magnesium sulfate in DMEM, diluting each to a concentration of 50 \u0026micro;M. Additionally, dilute gypsum decoctions of 500, 750, and 1500 mg/mL with DMEM to final concentrations of 250, 375, and 750 mg/mL, respectively.\u003c/p\u003e\n \u003cp\u003eCCK-8 detection: Add 100 \u0026micro;L of A549 cell suspension at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL to each well of a 96-well plate. Pre-incubate the culture plate in an incubator for 24 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After removing the culture plate from the culture bottle, discard the cell supernatant from each well and wash the wells with PBS solution. Next, inoculate 200 \u0026micro;L of the diluted sample into each well of the 96-well plate, ensuring that there are 6 replicate wells for each concentration. Additionally, set up control wells for the cells. Once all wells are prepared, label them on the 96-well plate and place the plate in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for culture. After 24 hours, terminate the culture by removing the 96-well plate from the incubator, discarding the supernatant from each well, and washing the wells twice with PBS solution. Prepare a mixture of CCK-8 and medium in the appropriate proportion, then add 100 \u0026micro;L of this mixture to each well. Incubate the culture plate in the incubator for 1 hour. Finally, use a microplate reader to measure the absorbance at 450 nm.\u003c/p\u003e\n \u003cp\u003eCalculation formula:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cimg 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v7yYlpaW8O2332L37t0AgFKphG+//Za/xhDFDBcxREREFFlPTw9mZ2erN8a///77LX2TfKFQwNdff43Hjx8DAC5cuIDvfe97vg8HIKLWw0UMERERRdbf34+uri585zvfQSKRwO7du9HZ2SmTtYyFhQWMjo7izJkzSCQSKBQK+Oijj2QyImpxfDoZERERERHFCn+JISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWuIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWuIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWuIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYiXWi5jh4WEMDg6iXC7Ll56aVoihVXzyySfYtm0bEokEBgcHUSwWZRLyIceS3G6mYrGI7du3Y3h4WL60KZpZl6cd+9NmayvbvjiIa9xb4Vkf1xSM8yWaZs+dZufXquI23srlMgYHB2v2OS1i9IGJRALj4+Py5U0RVGaxWMTs7CzS6TSSyaR8+alohRhaRT6fx09/+lNkMhncvXsXb775Jtra2mQy8iDHktxutgsXLgAALl26VLO/XC5jaGgIo6OjTTuxNbsua2tr2NjYwJEjR4BNinmr2NrKti8O4hr3VjHHddzHdLlcxgcffICenh4kEgkkEgm8/PLLWFpaCpVmeHgY27dvr/lCTH822LZtG/L5fHV/FOYXb9u2bcONGzdkkqem2fMl7mMoDPme0Khm59eK4jjekskk0ul0Tf5Oi5iHDx9iZmYGADA/Py9f3hRBZepB1tvbK196alohhlYxMTGB9vZ2/OpXv8Ibb7yBv/qrv2rKxHheyLEkt5spn89jenoaIyMj6OzsrHltaWmpOu+a1X/NrkuhUAAApNNpYJNi3iq2trLti4O4xr1VzHEd5zFdLBaxd+9enDt3Dj/4wQ9w9+5dXLlyBRsbG/izP/sz5PN55zSzs7Po6uqqfiFWLpdx+vRpzMzM4J/+6Z+wa9cuWbyzXC6Hn/70p/j1r38NpRS++uorXLx40fql6dPQ7PkS5zEUlnxPaFSz82tFcR1vvb29KJVK1e3ARUy5XMbExAQOHz6MgYEBFAqFTVtlaS5lFgoFrK+vo6enp2b/09QKMbQC/WazZ8+eug/FYWzmT7ibmXczyLEkt5ulXC7j/PnzaG9vx7Fjx+TL2LVrF7755pumvpE3uy737t1De3s7Ojo6gE2KeavY2sq2Lw5k3K0+B7eaOa7jOqbL5TLeeustLC8vY25uDh9//DHeeOMNvPPOO3j8+DEOHz6MZDIZmKajo6P6IWv//v1IJpPVBczk5CSmpqZw9OhRWbwz/RnjwIED1YVQd3c3RkZGrF+aPg1yvrjymldxHUNRyPeERjU7v1YU5/G2trZW/X/gIubatWt48uQJ/vZv/xbpdBorKys1q6DN4FLmwsICUqnUlq6UWyGGVtCsFX2z8rHZzLybQY4lud0sS0tLePLkifVXmM3SzLqUy2UUCoWab2efJba2su2LAxl3q8/BrfSsjOtr165hZmYGN2/erPuVJJlM4uOPP8Znn30WmKazs7PuQ9bp06cxPT2NqakpHDx4sOa4qGxfkG4VOV9cPe/zqtlzp9n5taq4jre6RZfysbq6qjKZjMpms0oppcbGxlQqlVJzc3MyaaCpqSmVSqWqeXlxLTObzaqBgQE1Nzen9u3bpwCoVCqlrl+/XpNubm5O9fX1eb4+PT1dfR2A6uvrs5aVzWbVnTt3VCqVqsbjGoNSSl2/fl2lUikFQO3YsUPdvXtXDQwMVOupy1hcXPSNVylVjcMrjazTG2+8oRYXF2vSuJJ5me1TKpXUwMBA9TX9L6iPJa98BgYGVKlUUsoSh6yTVz+75O0im82qTCajVldXq/vkWNWmpqYUADU1NVXdFxS/Hks6JrntVb+wbPUwyXKbMS5lnrpPZB/Y2tOcN319fWp6eroujcw/TMwy/9/85jc18zIsrzbQwo4D275mjQU/XvUI03c67mKx6DkH9Wuu+WUt52Hl0K5hBI0fs236+vrqynGJRY47Oa5ln7u2e6OxN0LHImM0uaTRzPf+bDarUqlUzTm1UWNjYwqAGhsbU0optbi4qIaGhjzPjX6C5qQcE30enzN0u7j0t05jzikY721yDKkQcfiNobDMsW777KNClhk0d8JyyU/HZ5579HuFbGM5JzVZTiPvNUHjTTn0tRwfQWPu8OHDocebcozDte9V5fOVebzvIkYuIPSkj3IikScMLy5l6sbesWOHSqVS6sSJE+ru3buqr6+v5ti5uTmVSqXU4cOH1dLSkrpz547asWNHXd779u1Td+/eVVeuXKkOUJ1Gl/XKK6+oVCqlrly5oiYmJtTi4qJTDEopde7cOQVAnThxQn3++efqxIkT1Q6dmpqqK0PnlclknPKytdfPf/5ztbS0pKanp1UqlbIOriAu7fOb3/ymGsP169fV559/HvqNsVQq1eRz9+7dmnyC6uTXz0F5u5LjUu8zJ7Eyxou5UAiKXx+jT2Zy269+YXidXDVZbjPGpe1E7RWHrqee62aed+/erX5RYJ4PmhXz3bt36+ZlWF5t4Do3ZV2UpX7NGgt+/Orh2ndm3H5zMGx+8jy8uroa2K5hyHL02EilUiqTyajBwUG1Y8cOdeXKFfXzn/9cQXxp4xKLHHdyXMs+V45zptHYG6W/vPF7j3dJo2UrX7gMDQ3VjIVm0e0FQA0NDakrV66EHi/KYU66vI/KPnftb695JfNTIeOQY8h2/nRhO5eYY12FLDNo7oTlkp+MT5975ubmAvtIk+XY2sFV0HhTDn1tGx9BY+7OnTuhxpsKEYdL35t5mvs9FzE6cDOoMCcgm2KxKHfVcC1Tp5MDQKYdGxtTGY9vnWVar/26LNmgrjHIbW3M+FDslZeZRvnkVSwWValU8nxdvxnY2sGLV162/VHyt8laVvK28nRaXaZfP2u2vMOQfaEnPMQiRvdlUP+b8ctj5LZL/VzoWMwxZpLlNmNc/uY3v6nJU1nK0aYqv9bOzc155inLlnk1GrNM58orvzBzU9ZFWerXrLHgxStOXQ8Zj2b2nbLErTzmoC2d8slP9o1XvFHPSX7jR851+abvEotXGnPc2drEtk+Jdmok9mbIVn4t8Zs7LmmUEZ/+klCOm0aVSiV148YN9Ytf/KL67a/ZZmH4zUmv/pb7Zf/KbfM42X4u80qWp8n9fmNIlhtE5q3JvFzLdM3PlWt+Oj5ZhmxjTfaRazmuxnzGm/Ipz9xvi922Tx9nxuky3vRxrnEE9b1JH6d53hMzMTEBADh79mx1XzqdRiqVinzjW9BNUq5l6mtlx8bGfK+N7enpwfLyMt566626RztOTEwgk8lYb2426bLee++9mut3XWO4d++etZz5+fnqjWNeeZl19ou5o6MDyWQS9+7dw8DAAE6dOlU95v3338fk5GSom+79ypL09aNh8rfxetyfS528+lnzyjuMnp4erK+vo1B5asnt27cBAKOjozX3bH355Zc1N827xC+v/ZbbQfVzFXQNrCy3GeNyY2Oj7uZBWY5m3kzpMm9gycslZjjOS1dBbeA6N2VdYKlfs8aCjUs9ZDyavBFWpvOagzKd5pWfPA+7tGsYfuMnlUrh8uXLdecQfW24Sywu487WJrZ9EO3USOyN0u8DfnPHJY2mr7s/efIk3nvvPczMzODhw4cyWY1yuYz79+/X/LPNkWKxiCNHjuCll17Cz372M3z55ZcYGBjAmTNnah7l7MprTvrNJ0n2r9zW5LxwmVdR4rCNobBcxjpClOmanyvX/LzOPa595FqOK6/xhhBjzha7bR9EfVzGGyLEEdT3pvn5+Zo2sy5i9CNYM5kMlpaWqieEx48fA5WCy02+GS5MmfrD2N69e2vyWFhYAIwbfw4ePIjr16/jyZMn2LFjR/X51aVSCSsrK75vcjoPv7K89qNyvNdTu/TJXN84ZstLp9m5cye6u7sDY9ZlzczMoK2tDYlEAm1tbfjHf/xHXLlyBR999JE8xFNQWTDaR6dt9M3QdrOYa528+tkv77D0YnphYQHFYhFXr17FyMgI+vv7sbGxgbW1tep+3W6u8cvFhdwOqp+roJOmLLcZ41LmqfdlMhns3r27us+cE+Vy2Wne6LzCxuw6L10FtUHUcWDb16yxYBNUDzj0nVe/eM3BsPmZ/erarmHYypHjR9N1Ms/1frG4jjvZdnBsp6ixtyL94aanpwfHjh1DJpPBxMSE71h/+PAhfvjDH9b8++yzz2rSlCtPTzt9+nT1A2kymcTly5exsbFR/bwRhtecdJlP8nOGeR4L6m8EzCudX5Q4XMaQH9exDscyw+TnIkx+tvj0/qA+ClOOK6/xBsdzeE9PT914g2N9XMYbIsTh1/cm/ZrZZnWLmHLlEazr6+v4+uuvMTQ0VD0hnDhxAuvr6/KQhoUtc35+3lrB+fn5uk54++238fvf/x4///nPceXKFVy7ds2zI2CsmnUefmV57dfHe5Wj/waOXs3a8tKDQKfxykvTr9+5cwdLS0tYWlqCUgrffPMN3nnnnbpv4Pz4lSXb5/Hjx1heXm74zdB809LC1MnWz5ot77A6OjrQ3t4OGL/CyG8Y9H79ByRd45f9L7cRUD8X8mRkI8uV24gwLm15zM/P18VhzolyuWzNU84bWPKX2wgRsy1/F175aVHHgde+RseCl6B6wKHvvOriNQej5ocQ7RqGrRw9fvSjfrUvv/wSqHzB4RKLV/vK+tpicGkn23EusTfTRuULHT8uacwPRZ2dnRgZGcHMzIzvWD948CAql8hX/42OjtakWVpawh//+Me6euvz+0LlS8iwbHPSq79heR+VfefS3wiYVzq/RuKA5fzpwqtMWx1cygyTn4sw+dni0/uD+ihMOWHYxht86gXR17Y6udTHZbyhwThk35uWKk9XNffXLWJ00GNjY3UnhFKphIGBAaxYHnkcxO+kFaZM/WFMyuVymJycrD461iwvmUzi5MmTyGQyNcfMi5+sdB569ajLko3pGoNmllMsFnHmzBkAqP5lZlsZfoPAtLa2Vl2Fo1LXF198ES+++GJNuiiC2geWFXhUfvn41cmln/3yDuvf//3fcf/+/Wof6zxzuRwuXrxY1/cIiF/2v9x2qZ8LvxMDHOLQwozLtbW1ujxsc0fOCc1v3qAJMQfl36gwc9MWu9znOhbK5bLv+TYsXQ/XvpNxw2MONpKfya9dw/Aqx+uLmnnLL5susfiNO1sMLu1kOw4hY0cDYyeZTGL//v1YX1+vLpBM4+PjuHbtWmAa/TcmZHz615irV6+iGOGSryD6HCHbKYjLnAx6H5V959Lfmt+8kmMhbBya1/nThd9Yh0+sXmUG5RdWUH5e8YXpIziU48plvCGgr9va2urq5FqfMOMNEeKAT9+j8uWL/FED5g0z+kkBfjcNud6Yp+k8vY4JW6Z5I1BfX1/NEw/0zUY6T/36XfHUMP06LE/N8LtJW3OJQRk3J6YqT7TQafSTGMz6yDLkzVQyLx2zbjeznfXrN27cUPv27VPnzp2ryTuIa/uoBm6glcaMp/rcuHFDTU9PB9YpqJ/98g7LbBOzvrpfbG0TFL+yjDFz27V+LoJu5PWLwxRmXOonM5l5yDYx54T5RDmvNGbZMka5rQXF7JW/K5lflLlpiz3KWNDposzJsPWw9Z3yqIttDjaSn7KMJVu7huFVzpjlJlNdtp7zLrHINLZxZ4vB7zjdTrbjlGPscn+UsaPEuVA/jejGjRs1N86HSSPPVV43Codh1l0/oVI/aVSWFyRoTurXg95HZd+59Ldmm1de+YWNQ5PnTxfyXGIb6ypEmX5tEjY2FSI/r/j8jjf7yLUdXASNNzONX1/b6uRaH5fx1kgcytL3ms4zk8nU7K9ZxOgAzacESPpEotPoY7yOMxvedmIMW6auoHz2tP5Qq5nPnEblhGk+VndxcbHaqKg8l/qKeMSirKu53yUGVZkEOs2OHTvUnTt31JjxdAmvMmyLAzMvXSfzA/ni4mLNIwJTqVRdvV25tI/u27Anfxv5jHA9gIPqFNTPMo1tcrjKZrMK4k3UPEnZ8g2KX/a/3HapnwuvDwaaLFdua2HGpVce+uQFjzkh8/RKI/OX21pQzDp/WzpXXm2ghR0Htn0uYyGon4ME1cOl72TcymcORs1PC2rXMLzKsY0L3c7mucAlFtu4c3k/CGonr+NcYzf3Rx07qvKeIN83Tpw4UdMGQWl0XWV85oeYKHNUK5VK1Ufe6vK9/iZFkKA5uejwPmrru6D+1mzzypZf1DiUxxhyETTWVcgyXfILwyU/r/j08S59ZCvHVj8XQeNNp/Hra686udTHdbzptFHi8Gobr/NCQimlan+bcZfP53Hp0iX88z//c93PSPTsKxaL2LNnD0ZGRuquPabWUi6X8dprryGdTuPWrVvy5eeebh8A+PTTT2N7PhseHsb09DR+/etf1/01dCI/HDtEm+9Zea952oaHhzE7O4vZ2dmaS/br7okJo6OjA//1X/+F06dPy5foOeB1vTW1nmQyiXQ6jcImPFnwWaBvGJQ3QMdJLpfjh1CKhGOH6Ol4Ft5rnjb99GLbPccNLWI6Ozvx3//93+jt7cXg4CA/HD0nHj16hA8++ADHjx/HwMAA+vv7ZRJqQb29vZEeyvGsyeVy6OnpwQcffID79+/jgw8+wNGjR9Fu/H2fuCmXy/jlL3+Jmzdv8kMohcKxQ7Q5nsX3mq0wMTHh2WYNXU6m8bKy54e+hGx5eRl9fX34l3/5l7pHD1JryuVyOH78+HP/bevS0hL+5m/+Bg8ePKjuO3HiBM6fP8+xTERETcH3msbpzy03b960/mH5yIsYfV3fzMwMAGBqaspaABERERERUTNFXsQQERERERFthYbuiSEiIiIiInrauIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWuIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWuIghIiIiIqJY4SKGiIiIiIhihYsYIiIiIiKKFS5iiIiIiIgoVriIISIiIiKiWOEihoiIiIiIYoWLGCIiIiIiihUuYoiIiIiIKFa4iCEiIiIioljhIoaIiIiIiGKFixgiIiIiIooVLmKIiIiIiChWYruIKZfLGBwcxPDwsHzpqdjq8lvJ8PAwEokEEokExsfH5cvPvFYdCzIuud1sxWIR27dvrxkDuVwOiUQCuVyuJm0jml2P8fFxbN++HcViEdikmLeCbCe5HRdxjXsrmGP5WRnHFKyZc4TjprntiZjNS/l+2Ap0f9g+XzotYvL5PLZt2/ZUKxZU5tLSEp48eYLe3l750lOx1eW3ivHxcczOzmJ1dRVKKYyOjsokz7xWHQsyLrndbBcuXEBXVxdOnTpV3Xfv3j2kUimk0+matI1odj3m5+fR1dWFtrY2YJNi3gqyneR2XMQ17q1gjuU4j2P9oUV/OZZIJOo+CwSl0V+qyA+i+kOk3B/WjRs3sG3bNiQSCWzbtg03btyQSZ6aZs6ROI+bZmlmeyJm81K+H7aCZDKJdDqN+fl5+ZLbImZiYgLr6+vY2NjA2tqafHlTBJVZKBQAAHv37pUvPRVbXX4rKBaLuHr1Kvbs2YPOzk75ckvyemNrRKuOBRmX3G6mXC6HyclJnD17Fslksrr/1q1b+Oabb7Br166a9I1oZj2KxSJmZ2eRTqercW9GzFtBtpPcjgsZ92bM4WeBHMtxHce5XA5tbW1Ip9NQSlX/jYyM4Pbt285p1tbWsLGxUfNBNJfL4dChQ8hms7h161Z1f1jj4+O4ePEivvrqKyil8NVXX+Hjjz+2flP8NMg54sJrHsV13DRTlPb0Eqd5KWNtJb29vZidna37USNwEaM/nPziF78AjM7dTC5lLiwsoL29HR0dHfKlp2Kry28F+k3iyJEj8qWWtRkxt+pYkHHJ7WYpl8uYmJhANpvFwYMH5ctN18x62D7oPCtkO8ntuJBxb8YcfhY8C2M5n8/j+PHj1kXG6OgoRkdHndKg8rlhfX0dPT09QBMXMOVyGffv38fIyAi6u7sBAN3d3RgaGrJ+U/w0yDnigvPIW5T29BKnednqsdp+1PBdxJgfTt5++220t7djYWFBJnMyPj7udB2ga5n6J6/f/e531Z90Zf76Z2PbazBisv0UrQ0PD2N4eLiaVqdxKd/Mwywjn89j+/bt1bS6DH0Jnf55Op/Pe+ZjK0vWV37DEoZf2+if8V966SWsr6/j0KFD1njCCKqbXzz6eL829IpZt5FXP7uUHfXn1+Hh4bq8dPzyGz3bdapB/S3jktvyeNnmrh4+fIiZmRnrm+Hw8DAGBwdRLper2379JI+1xSfrofvWLAeV+sm8zTwHBwer5xbzG7dmxTw4OIhHjx6hp6fHmjaIV/012X9h+x+WPGQZzWCrR5g+03HrNrXN4TD56f60zXXZHrJNXdnGjC5HXgYl5zoc45BjTY5lcxxHaZ+osUdVLpdx/vx5tLe349KlS/JlwDGNtrCwUL1sJ9ekBYzp/v37NW05Pz8f6cOf7Gs5B4PefyDmdlBfP3r0yHMewXL+04LisI0bObaaRbaZOT+GLe+r4+PjdbGY80e2e5j2lPULMy/1tmu7ybwbeX+BJT9brMOWc2WYNpFl2GKW/SnnAIDqlxF1lI+xsTGVSqXU3NycKpVKamBgQGWzWZnMSTabreblx6XM1dVVlclk1CuvvKIGBgZUqVRSqlJGJpNRq6urampqSgFQU1NTSiml5ubm1BtvvFGT1oxHl6WPl+WYMbiU75WnjkuXbctL79Nl2vIJqs/c3JxKpVJqbGxMhSXzspWv05n1j8KWd1Dd5DEubaiNjY3V1cOrn1WIsuVxLmyxZLNZBcA63sx9Mi7Z3/IYuR00P1zp9rCNA1mmaz/JNlZGfIuLi3XpdR5yrJvta8tzbGxMAajZJ+NxjVlZ5r8tfxe2WGX/NNr/qoljwItfPXQ/+vWZ8ohbpjHTuebnMtdlm7ryGzODg4Oqr6+vWoYcM3qfXxy2dpVjTbZblPaJEnsj9HiUMZpc0mjZyvvTnTt36s6pjdJxZLNZVSwW1ZUrV9T169dlskBBc1COBVvfR+1rua0seWlBcfiNG5lXo2Qscn7IbdnGMnZ9jG53Gbdre9rydZ2XLu0m55vMO4wwsdrOla5t4hKz7B85B7QxY21g8lzE2BpSnxRk5s3iWqYepLLzzMaQjWeSjea1X5cTpXy9bWt0s6NteekBpsv16jxNlqt8BlkQW162/TpGObjDilI3ud+lDb32KZ9+DlN22HZWlvGh84J4w5VxyG1l6W8Zl9z2mx9hyHz9XnPtJ78xIfM095ntIfOVbW1LY+YVNmZb/srj/BXEr/6qSf2vmjgGvPjVw6XPzHQ6bluaKPnJ413a1JXfmJFljIk3fZc4bGNN1lW2W9j2iRJ7o7IOX3S6pFFGmwGoO582Q6lUUocPH1aI+CFS85uDtrFg2x+lr+W2JvNSlvJs+/3GjSyjEbZY5PxQxnnX9qWX33lJWdrApT1Vg/MyqN1seauI7y/KIz9Zro7Nlr9Lm9jKUJaY/eaAyavfPC8nu337NjY2NnD27Nnqvt7eXqysrKBUKtWkbRbXMvW1rh9++KHnDeW9vb1YXl7GhQsXavaXK5erDQwMoL+/v+Y1SZcjb1Z2KV+Xs3Pnzup1s3r//fv3qzfD++Wln15x//59HDhwwHojmK0+5XIZb731FjY2NkLdmGbLy0upVMLKykqkn8813RZh6mYT1Ia673TM+/fvt/an2c9hykYDNwCur69X89Dj5fDhwygUCihXLgMx45Db8OhvGZfc9pofYX355ZeAR/1lmS79FDQmZJ7mPvNpL7qv9T7bXDTT6H6X+bvGbMu/GOEmyaD6N6v/0cQxYBNUj6A+8+oPvzkMx/xc5rqtTV3ZxoyORd43Zt5D4RKH11iTdZXtFrZ9wsbeqHK5jEKh4HsfgksaTV/bPzo6ikwmg0LlfNoMN27cwPXr13Hr1i3cuXOnoTnkNQdtY8FLlL4Omkc6L9c4bONGC3P+82OLRc4P7ciRI5iZmcGOHTvQ1dWFjz76qJre77wESxu4tGcj8zKo3bzyjvL+AqMdZX62WOW5UgtqE3i859pi9poD0vz8vHXuWxcx+XweFy9exHvvvVfT0T09PdYba5ohTJkLCwvIZDLYvXt3dZ/er6+BHR0dRTabxeTkJBLGtbtek1cer7e9yvHar4/3Kkfv1x/+bXmZaYIe9adfn5mZQVtbGxKJRPWa99///veeE9XGK2ZY2ka/SXhep+ggqG6u8QS1oeYVs9/xLmXbJpaLdDqNVCoFGJP77NmzeOWVV6pp9P0m+kTi2t8yLrntNT/C8jqxwFKmXzvrfgoaEzJPVB4JKveZN6169aXtxlaZv0vMQfl71cUmqP7N6n80cQzYBNUjqM80GbfXHA6Tn+xP1zZ1ZSvDFov+UK6/0HKJI2is6fxluzXSPrZ0MvZG6XrJe7ZMLmk0/QGsv78fIyMjmJmZwcOHD2WyGvK6fNu1+aOjo9jY2MA777yDZDKJo0ePYmxsDNPT05HuS/Cag179DPH+o7fD9rXXPJJ5ucZhGzfyPNkol/mh7d69G5lMBqlUCpcvX67GHnRegqUNXNrTq51kOpm3S7sF5e1XF5ug/MxYZWxaUJsElWHG7DUHTPp8Y5v71kXMROXxxu+++27NhD506FDNN8fNFKZM2w2qeoVtrvxu3boFpRSy2SwuXryIfD5fbUQb+YHMVo7Xflm+VzmPHz/G8vJy9eRhy8tMo0/G8mQjTU1N1Txq8osvvqg76QTxihmWttHfwJsr8bCC6uYaT1AbarZvD+DRB42U7aqjowPtlQdX3L59G11dXejv70dPTw9WVlZQLBbrvnnSgvpbxiW34TE/wvA7scBSptyGpZ+CxoTMwysGc3x69aVtDMv85TYsMQflH+ab/KD6a83ofzRhDHjxq4dLn2kybtscbiQ/U1CburKVYYtbfljR/OIIGms6fzOGRtsnTOxRtbW1oaurCys+V3q4pNHMD2CnTp3CwMAAzpw5U3dDvOngwYM17a6UqvnlKZ/P49GjRzh27FjNcceOHUN7e3u1PcOyzUGvfoZ4/9HbYfva1qew9L9rHPI4WM6TzeI3P2D8OrO8vIz19fWafvE7L2lR2tOrnWQ62U5yG5Z2C8o7zPsLHPLzilVzaZOgMmTMtjlg0otPuSiCbRGTqzzeeGxsrG5Cr66uIpPJYMHytLBGhCnT9nMUAip55MiRuoWQlM/nMT09jZGREXR2dnqW47Xfr3ytWCzizJkz1W8vvPLyWwFvBdk2qAxw+VPh02LGA8C5DW3fHnj1gReXsl3pN+UHDx7g6tWrNT/bbmxs4Gc/+xmePHlS802SC1knuS25zA8/tnxlmXJbs/WTF1se+sOUuU//qiv72lSs/I0jM43MX25rLjHb8n9aZNxy26bRMRCGa5/Z4rbN4UbyazavMuQ3sPD5NjwsOdZkDI22z2bGriUrf8xuw+dKD5c0mvkBLJlM4uzZs1heXq7+DZko9AdgL422hcsclO/Fss9c+9o2j2ReflzeB13Ok5vh9OnTWFlZwerqKrLZLK5evYqiz+LVJNvAtT1tgual3NZc2k3m3SiZn1dsCHG+lWQZNl5zwGvxA7mI0dfKDQwM1PzVbWk+xLWw5cqj2OQj78zXw5SpT56Tk5PVn3qLxSJef/117Ny5Ez/+8Y8xNDRUXcmZ+ff396O7uxs7d+6seTxiPp/Hq6++igMHDlSfMa/LMX9CN/d7la/r0FH5ll3HXSwWsWfPHnR0dNT9WiPLME/A+mfRiYmJary5XK76SEFdzr1796rHl8vlmjZw5do2eiVuG+BhBNXNJR6XNvTjdXyYshv9NvKLL75AV+VXGBjfhNy9e7fu8kqX/pZxmdsyrZwfzSLbVW5rsp/8xoSsF4yFYKFyzbueiz09PdV8/eai7VvHsDHb8v/Rj36EF154wWkMmvzqjyb0vy291xgoB5y7/fjVw6XPYInbS9j8ZH+6tKkrWxnlyq/0cizIbz5d4rCNNTmWZQyNtI9r7Dpt1PECoHov7Ouvv15zfC6Xqz7K1SVN0fIB7ODBg57f8rrSHyrHxsaqY3ppaQlvvfUW9uzZE+pvZMl+lXPQ5f0Hljni2tc2tv53icN2HCznSTQ4Rlzmx/j4OCYnJ6v3mBw5cgTLy8t4/PgxEHBeQgPtGWVeym1Ntpst76jvL/DILyhWk0ub2MqQMcu+k3NAKwfdx2Te5W978oNJPn3AhX5yhNcxYcucmppSmUxGzc3NeT59RD85Qb8my5ZPLrGV7/VkBZfyzbT6dZ1XNputprWVoetr5mfmY6uPrK9XPC5c2sb2NJCoguoWFI9rGyrRTvppGLbjtShlh5WtPFLZzNfvqSDm62ZccryYccltebxXOUG82llZypTbyud4rzFhy0Om1/1qzjNbmsXFxbqyZf5yWznGbJ4fZDoXXvXXZP8hZP/b8pBlKIdzdxC/esj28uozv7j1cY3kp8n2kG3qylaGbkeZX9byZCGXOGRd5Vi2xSCPcW2fMLE3Ol6UxznX7GeXNLoN5fuT3i/rEkapVFInTpyolptKpdSVK1ci1Vf2tWw3Wz2nHD6nuPS1bR7Z8lIOcdiO8zpPNjpGZJvBmB+63ma/6zi8zj2y3W11cWlPW7qgeSm3lU+7ybznGnh/UZb8gmKV5PG2NpFpbDHL/rSNi6BYahYxYZlBRm1Mih898MwTGT2fbB9m6NmYI0FvHkQmjhcKwjHSHHF8f4kSs9eizlR3T4yrYrGIDz/8EKurq1BKNe2v4FLrm6g8Oq+Zlx9RPPVaHoH+vDEvZUHl52/9V8X9rmtuZcXK/Xs3b960/4RPZOB4oSAcI9HE8f2lWTFfu3YNKysruHTpknypKvIi5vHjx9i3b19THq9IrU9fy5pIJDA7O4tf/vKXDd0PQ8+GvXv34oUXXsBawI22z7KDBw8inU6jTTz287e//W1sz48XLlzAyMhIqOv86fnF8UJBOEaiieP7SzNizlceFmD7GzqmhFJKyZ0u9LOc9Q1mRERERERET0PkX2L27t2LixcvIpFIRHrSBBERERERURSRf4khIiIiIiLaCpF/iSEiIiIiItoKXMQQEREREVGscBFDRERERESxwkUMERERERHFChcxREREREQUK1zEEBERERFRrHARQ0REREREscJFDBERERERxQoXMUREREREFCtcxBARERERUaxwEUNERERERLHCRQwREREREcUKFzFERERERBQrXMQQEREREVGscBFDRERERESxwkUMERERERHFChcxREREREQUK1zEEBERERFRrHARQ0REREREscJFDBERERERxQoXMUREREREFCtcxBARERERUaxwEUNERERERLHCRQwREREREcUKFzFERERERBQrXMQQEREREVGscBFDRERERESxwkUMERERERHFChcxREREREQUK1zEEBERERFRrHARQ0REREREscJFDBERERERxQoXMUREREREFCtcxBARERERUaxwEUNERERERLHCRQwREREREcUKFzFERERERBQrsVzElMtlDA4OYnh4WL606bay7FYwPDyMRCKBRCKB8fFx+XIgl/ZzSRPFZuXbLDI+ud1MxWIR27dvj9SHWyWXyyGRSCCXy8mXfEU9rpVs5lh4FoyPj2P79u0oFovPRH9TvWbOged1jDSzDZ+1OSfbRm7HgdknjWpmXs2g+0N+ZnFaxOTzeWzbtu2pVSiovKWlJTx58gS9vb3ypU23lWVvtfHxcczOzmJ1dRVKKYyOjsokgVzazyVNFJuVb7PI+OR2M124cAFdXV04deqUfKll3bt3D6lUCul0Wr7kK+pxrWQzx8KzYH5+Hl1dXWhra4tlf+s3aP0FUSKRqHv/80ujv5SQH7j0h0u5P6wbN25g27ZtSCQS2LZtG27cuCGTbLpmzoE4jpFmaGYbxn3OSbJt5HYcmH3SqGbm1QzJZBLpdBrz8/M1+50WMRMTE1hfX8fGxgbW1tbky00XVF6hUAAA7N27V7606bay7K1ULBZx9epV7NmzB52dnfJlZy7t55Imis3Kt1lkfHK7WXK5HCYnJ3H27Fkkk0n58qby+rDl4tatW/jmm2+wa9cu+ZKvqMe1EjkWGmnHZ02xWMTs7CzS6TSSyWTs+juXy6GtrQ3pdBpKqeq/kZER3L592ynN2toaNjY2aj5w5XI5HDp0CNlsFrdu3TJKDGd8fBwXL17EV199BaUUvvrqK3z88cd134huNjkHXHjNk7iNkWaJ0oY2cZ9zNrJt5Hark33SiGbm1Uy9vb2YnZ2t2Re4iNEfeH7xi18ARsduFpfyFhYW0N7ejo6ODvnSptvKsreSfpM8cuSIfCkUl/ZzSRPFZuXbLDI+ud0M5XIZExMTyGazOHjwoHx50zVrHD1v5FhgO/7/bB/g4yKfz+P48ePWhcbo6ChGR0ed0hQKBayvr6Onpwdo4gKmXC7j/v37GBkZQXd3NwCgu7sbQ0NDdd+IbjY5B1xwntSK0oY2cZ5zXmTbyO1W18w+aWZezbaxsVGz7buIMT/wvP3222hvb8fCwoJMFmh8fNzpeknX8vTPXL/73e+qP3HL/PXP6LbXYMQkf5bXhoeHMTw8XE2nX3cpWx9v5p3P57F9+/ZqOp2/vnRO/0yfz+c987GVI+spv3Fy5dUe+hKGl156Cevr6zh06JA1Dlcu7Wf7GbMZ7Snz1WkHBwdRLper6fwMDw/XjRWdj/xmUl5TGtRXMj65LY+P0gcPHz7EzMyM55u633jzGiPmsV594DWOzDZw6WPZV35lmmnM41yO0el0PIODg3j06BF6enrq0rmwtatuE1mnXC5XF48eCzoWWzu65qfrL89tOq0Zp9k/rmztK88nOn85Z1zKl/2i3yP0N6Zmf4dtkygxR1Uul3H+/Hm0t7fj0qVL8mXAMQ0qH7j05Ty5Ji1gTPfv369pv/n5+dAfcGTfyvNX0PnFPB8G9eujR4885wks5wQ4lG8bI3KeNoNsJ3MODFvef8bHx+vGslc7h2lDeQ7V+QXNOb3t2lYy72afZzW/doXl/VZuy+Pl+HVlxuj1PufSdrLdZJ+E4ZKXjst831haWoo8hmQ/u7Sv/pKmhvIxNjamUqmUmpubU6VSSQ0MDKhsNiuTBcpms9V8/LiUt7q6qjKZjHrllVfUwMCAKpVKSlXKyGQyanV1VU1NTSkAampqSiml1NzcnHrjjTdq0prx6LL08WYZZvkuZcu8lFLVeHSZtnz0Pl2eLZ+geszNzalUKqXGxsYqEbuR+djKzmazNXWOwlZvJdrPpR2itKfc1nnI8RVkbGysJhZViV/mJcuTbSz7SqaX20Fj2oVuS1s/2trZLEPGL9O79IHyaD+Zl/LpY1sb+5Xpte13jBJjUlXiBlAXexBb3XS7Li4uqkwmUzdfZRvZ4pNpzHR++Zn1l2Nf9rEcoy782ndwcFD19fVV85dtHFS+rS1lv8i2CtsmYWNuhB7jMjaTSxplnJ/v3LlTdy5qlHmuLBaL6sqVK+r69esyma+g85fse9nXUfrVtq085pNr+bYx0sy2lnHIOSC3zXaVMev0up1lvC5taMvTdc65tJWcTzJvV7Y4zboHtauMT24HjV8Xthi93uf82s6WTzPbzZaXGZfZh3q/3xhSDv3s2r5jlTWCyXMRIxtONemDrBfX8vTgk51lNoJsMJNsLNt+XUaUsqempmomi2Z2qi0fPZh0mbqzZD6arR5eA8qPLR+5X8cmT0Bh2eqtPNrePEnb2iFse5r56vLCtJMm49H5QnxwkGNCtrHsK1lvue03pl3JPE1+480Wv9zv0gdy28zHVratj83YXcqUx7kc4xWP7XwUxK9ddSxmu8pYzHS6DrY0rvnpNPJYWx/LMerCr31l/mb/upRv6xev+sn+dmmTsDE3Sn6wsnFJo9sJQN15qBlKpZI6fPiwQoQPSZrf+cvW93J/lH6V25rMK0z5tjEi84/KFoecA8o4D+kvQXR/+51rlKXeLm3YyJwLaitb3moTzrMu7epVB73tN35dedXXPKe4tJ0tH5nGlWteOi6Zf9QxpEQ/u7av7meT5+Vkt2/fxsbGBs6ePVvd19vbi5WVFZRKpZq0zeBanr7298MPP/S8wby3txfLy8u4cOFCzf5y5XK1gYEB9Pf317xm0mXIG5+Dytb579y5s3r9sN5///796k3xfvnop3vcv38fBw4csN4oZ6tHuVzGW2+9hY2NDeefE2352JRKJaysrIS+fEDyq7dm3kzXrPZMJpPVfL/++mscOnQIY2NjkZ6uBgDr6+vV/HR8hw8fRqFQQLlyCYtu1z/90z+ta2NbX8mbCOW215gO48svvwQsPzfr9rSNN9cx4tIHehzt37+/Oq/C9DFE7C5lyuOCjoHRp2Y8UW509GtXGH1sPtFHt5FZjqyDrR3NdH756fqb5zZbH9vGqAtb++oY5H1Y+p4Kl/K9xomtfrDMI5c2CRNzo8rlMgqFgu819y5pYFy/Pjo6ikwmg0LlPNQMN27cwPXr13Hr1i3cuXMn8jnI6/xl63ubKP0aNE/MceVSvhwjWphzghdbHHIOaEeOHMHMzAx27NiBrq4ufPTRR4HnGkRoQ3icC2U72/INaiuv+dzs86xru9rqYG57jV9XXvXVsbt+loFjn7jwismWl47L9pkYEcaQ7GfX9p2fn0d7e3vNPusiJp/P4+LFi3jvvfdqBkVPTw82PJ4Y1ogw5S0sLCCTyWD37t3VfXq/viZ4dHQU2WwWk5OTSBjXMHud1OTxfmV47U+lUvg//+f/WPPX5epFgC0fM03Qo/306zMzM2hra0Mikahet/n73/++biJ7cW0P/SZpvR4xBFu99X6z7fWbtld8YdtTp0FlrKVSqVAfzEzpdBqpVAowJuLZs2fxyiuvVNPo+07Onj2LYrHo1FdmvW3bXmM6DH0CkB+I/MabVx/A0m9BfWAbR175y2Nle+h9QWXK44KO8Yonyo2Ofu2KymNeZZ1sNyLLOtjaEY752erfrPMJPPKXMcD4gL5nzx6Uy+XA8oP6xayf2QZR20SmgYhZfsAIS9dH3vtnckkD4wNGf38/RkZGMDMzg4cPH8pkNeT157Zr0EdHR7GxsYF33nkHyWQSR48exdjYGKanp+uuzw/idf7y6ldYzi9h+9Vrnph5hSlfjhF5rmlEmDm4e/duZDIZpFIpXL58GclkMvBcgwhzw6ttZDvLfF3aKihvv3pIfnV3bVdbHZr5/utVX9kuQW3nlY/sExdh8rLFhSaMIV1vl/bV59+urq6a/dZFzETlEcfvvvtuzUnu0KFDNd9CN0uY8uTNVjBWs+Zq79atW1BKIZvN4uLFi8jn89WGszE/4NnK0GnkfrPstrY2a/6PHz/G8vJy9WRqy8dMo9+U5MlXmpqaqnnc5hdffFF3Ivbj2h76G3xztR2Frd6y78w0XvGFbU+dZufOncjlcmhvb8f58+dRjvBtZUdHB9orD5y4ffs2urq60N/fj56eHqysrKBYLNZ98wOHvpJ1kNvwGNOuzBOAHNd+482rD+AwZ2Qf2L618cpfHmvL37Yv6Di5LY/xisfrVyw/fu3q1R+2uSZjtrWja34yL1PQGHVhy98Wr3zzRkD5Qf1iq18jbeIac1RtbW3o6urCis+VDS5pID5gnDp1CgMDAzhz5kzNzd/SwYMHa9paKVXzi1M+n8ejR49w7NixmuOOHTuG9vb2ahuGYTt/efUrfM4vrv1q60OI/o5SvibPNc3gNwdg/IqwvLyM9fX1ap39zjVa2Db0ahvZzrJt5DYsbRWUd7POs1pQu8qY5TY8xq8rr/rKdrGVG+b9SY51P2HyssXVjDFk9nNQ++oF6f79+2v21y1icpVHHI+NjdWd5FZXV5HJZLBgeWJYVGHKkz9BaWbl5JvukSNH6hZCUj6fx/T0NEZGRgDAWkaUslE57syZM9VvdLzy8VrpbgWzPTo7OzFfWQCYPweG5VVvs/1KpZI1jSlKe5ppuru7nb+ttNEfLB48eICrV6/W/Ly6sbGBn/3sZ3jy5En1GzIXsg5yW3IZ01688gzLZc7IMW371sbGpY9t++DT737tKuOUipW/keQSuyv9gdiMRf8ibZZji9nWji752fJqJq/85bea8PmWPAzZL7L8Rtpks2LWkpU/3Lbhc2WDSxqIDxjJZBJnz57F8vJy9W/MRKE/GHpppA1czl9+5xeXfoXHPPHqb8mvfC3ovLEZTp8+jZWVFayuriKbzeLq1aso+ixWNVlv1zaUguac3NZc2krm/bTImOW25DJ+XRQd3ufg0HbNbDdbXl5xNWsMSV7ta1v4QC5iysb1g35/yXu+cl1wLpez/uyjlSuP8JOPBDRfD1OefhOZnJys/vRdLBbx+uuvY+fOnfjxj3+MoaGh6grOzL+/vx/d3d3YuXNnzeMi8/k8Xn31VRw4cACjo6PVMuTPckFlnzp1Ch2Vb+l1vMViEXv27EFHR0d1EeCVv/lGpH8unpiYqMaZy+WqjwTU5dy7d696fLlcrqm7C5f20KttOYDDcmk/nUZ/09ms9pT5Hjt2rK59w/riiy/QVfkVBsa3Fnfv3q25LNKlr2R85rZMK8d0M/iNN5cx4tIHXqL2sW0fPPpdp5HbtmNs8fzoRz/CCy+8EFgXya9d9WK4ULl/Qc+Fnp6emnLk2PDikp9X/V3GqAtb/uXKL62y7cxv61zKt/WLHide9YvaJi4xm2n93uP86Ps/X3/99Zpjc7lc9ZGlQWmWlpbqPmAcPHjQ89tMV/oD09jYWHXsLi0t4a233sKePXuc/8aU7Ed5/gpzftFzwKVfvcj+DlO+nDe281vU8eAyB8bHxzE5OVm9X+LIkSNYXl7G48ePfc81sJxHXNowypyT25psK1vem3GedWlX2Tbmtkwrx68rW31d3ufg8P4k+8SVa15ecUUdQ2Y/JxIJp/Yt+9z3BPMuf9tTHEzyqQNjlSc9edFPgJBPNNDCljc1NaUymYyam5vzfBqLflqCfk2WLZ/kIsv3epKCS9k6nX5N55PNZqvpbPnrepp5mfnY6iHraYvFRVB7yKd4ROXSfra2aUZ72tLofKPUK1t5pLLZTl5P7zBf8+orGZ/clsfbyggi20TyG29BY0TGqzzKM+thPokkSh/b9skyZRq5bTtGp9PxmGPWq+38+LWrLGd1dbWm3jqNjNmlHW352fLS5BhDhPOJLX89dmReWfEEIpfyZf0WFxd9+9t2jEubuMZsppX7XdnmltmnQWl0u8nzmN4v6xBGqVRSJ06cqJaZSqXUlStXQtdT9q1sK1v9pgLOL0H9qjzmiS2vKOXbzhuqwfEg2wnGHND1NftZx6DLMttEtrOtDi5tKNMEzTm5rXzaSuY9t0nnWb921cf61UEeH6VvlYhR5x90LrK1nWw32SdhuORli8t8zTzeZQzJfnZpX78YEkopVbuscVMul3Hu3DmsrKzgiy++QCqVwq9//ev6VRLFmv5W6ubNm87fvlFrGh4eRqFQwKefftrQr2rPG84B8pLL5XD8+HG+9xHA8dAQnmefD2H7uVwu47XXXkM6nbb+8d66e2JclUolzMzM4MyZM1BK4ebNm5iYmJDJKOYmKo/HC/PTKbWmXssjy6mWeSkPKidQ/RfTva5JpudTsXJN+82bN/mBlTgeQuB59vnQjH6+du0aVlZWcOnSJfkS0MgiZm1tDa+99lp1JbWwsBB43TbFQ7lyXW8ikcDs7Cx++ctf8pv7Z8DevXvxwgsvYM3nJuHn3cGDB5FOp9EmHsf529/+tuHH6tKz5cKFCxgZGXH6NpGefRwP7niefT402s/5yoMCbH83R4t8OVkul8PCwgJGR0erN+tcu3aN30AQEREREdGmauiXmI8//hiJRALf+c538Hd/93dcwBARERER0aaL/EsMERERERHRVoj8SwwREREREdFW+L84crya2GnWBwAAAABJRU5ErkJggg==\" style=\"width: 783px; height: 216.595px;\" width=\"783\" height=\"216.595\"\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.15 in vitro anti-RSV experiment\u003c/h2\u003e\n \u003cp\u003eAdd 0.8 mL of A549 cell suspension, with a density of 10^5 cells/mL, to each well of a 24-well plate. Incubate the plate in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e environment for 24 hours. Prepare the gypsum decoction by diluting it with DMEM containing 2% fetal calf serum to achieve crude drug concentrations of 0.25, 0.375, and 0.75 g/mL. Additionally, prepare GA solutions at varying concentrations (10, 40, 80, 100, and 200 \u0026micro;M), along with 50 \u0026micro;M solutions of calcium sulfate, zinc sulfate, copper sulfate, iron sulfate, and magnesium sulfate for administration. Once the cells have formed a monolayer, add each drug and incubate the cells for 1 hour. Concurrently, incubate 100 TCID50 of RSV virus along with the drugs for 1 hour. After this incubation, aspirate the liquid and wash the cells with PBS. Subsequently, add the RSV virus solution to infect the cells for 2 hours. Following this, aspirate the virus solution, wash the cells three times with PBS to remove any unadsorbed virus, and then add the drugs to the cells. Incubate the cells for an additional 24 hours in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Finally, lyse the cells using lysis buffer and detect the viral load via qRT-PCR, following the same methodology as described above.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Evaluation of the anti-RSV efficacy of SG-GC in vivo\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Detection of viral load\u003c/h2\u003e \u003cp\u003ePerform relevant operations through specific methods, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. The RT-qPCR method was employed to quantify the viral load in lung of mice across all experimental groups. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the virus inhibition rates for the positive control drugs Ribavirin, MXSGT, and the SG-GC pair were 75.0%, 68.4%, and 68.7%, respectively, all demonstrating significant inhibitory effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, the inhibition rates for single-flavored gypsum (SG) and single-flavored licorice (GC) were not significant. Notably, the antiviral effect of the SG-GC group was markedly superior to that of the SG group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the GC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), highlighting the characteristics of compatibility and synergy. Given that the inhibition rate of the calcium sulfate-licorice group (CaSO4-GC) was only 33%, it is preliminarily suggested that the enhanced inhibition rate observed in the SG-GC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) may be attributed to the trace elements present in gypsum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Observation of Lung Damage\u003c/h2\u003e \u003cp\u003eLung damage was assessed through examination of fresh lung tissue. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea illustrates that the fresh lung tissue from the model group, SG group, and GC group exhibited dark red congestion and edema that were visible to the naked eye (indicated by the yellow circle). In contrast, the fresh lung tissue of the CaSO\u003csub\u003e4\u003c/sub\u003e-GC group displayed dark red lesions at the junction of the lung lobes (marked by the white circle), although the extent and severity of damage were reduced compared to the model group. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, CT scans revealed a \"white lung\" phenomenon (highlighted by the red circle), lung consolidation (indicated by the yellow arrow), and bronchiectasis (shown by the white arrow), characterized by flocculent and ground-glass opacities diffusely distributed throughout both lungs. The lung tissue in the ribavirin group, MXSGT group, and SG-GC group appeared normal and was light pink in color, with no significant pulmonary capillary congestion, edema, or hemorrhage observed. The lung tissue damage was minimal, and no notable pathological findings were evident on CT scans. These results indicate that mice treated with the \"SG-GC\" drug pair not only reduce viral load but also exhibit effects comparable to those of the full prescription in alleviating symptoms of lung infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Pathological Changes in Lung Tissue\u003c/h2\u003e \u003cp\u003eThe H\u0026amp;E staining results of mouse lung tissue sections from each group are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. The model group, SG group, GC group and CaSO\u003csub\u003e4\u003c/sub\u003e-GC group exhibited alveolar wall rupture and congestion, accompanied by significant inflammatory cell infiltration between the alveoli (indicated by the blue arrow). After treatment, the ribavirin group, MXSGT group, and SG-GC group showed pathological conditions of the lung tissue that were comparable to those of the blank group. This suggests a marked improvement in inflammatory cell infiltration, with the alveolar wall structure remaining largely intact. These findings indicate that the SG-GC drug treatment not only reduced viral load but also exhibited effects similar to those of the full prescription in alleviating symptoms of pulmonary infection. In contrast, the CaSO\u003csub\u003e4\u003c/sub\u003e-GC group did not demonstrate these characteristics. Therefore, it is speculated that Ca\u003csup\u003e2+\u003c/sup\u003e is not the primary active substance of the SG-GC drug against RSV, and the observed effects may be related to trace metal elements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Changes in Mouse Body Weight\u003c/h2\u003e \u003cp\u003eMice may experience a decreased appetite and subsequent weight loss during viral infections. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the mice in the blank group exhibited a gradual increase in weight, while those in the other groups experienced a progressive decrease in weight following the onset of the viral infection (day 3). Notably, the body weight of treated mice in the ribavirin, MXSGT, and SG-GC groups showed a slight increase by the 6th day.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Evaluation of the in vitro anti-RSV efficacy of SG-GC on the main component\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Effect of glycyrrhizic acid on the anti-RSV efficacy of gypsum\u003c/h2\u003e \u003cp\u003eGlycyrrhizic acid (GA), the principal component of licorice, interacts with various metal ions present in the gypsum decoction. This interaction leads to the formation of diverse metal-organic supramolecular complexes, which may alter pharmacological activity. Initially, the cytotoxic concentration range of each substance on host cells was assessed using CCK-8 assays. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, GA exhibited no cytotoxicity within the concentration range of 25-1600 \u0026micro;M, while SG demonstrated similar safety between 250\u0026ndash;750 mg/mL. Subsequently, the viral load in the cells was measured through RT-qPCR experiments. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, GA did not show any inhibitory effect on RSV at concentrations up to 100 \u0026micro;M; however, at a concentration of 200 \u0026micro;M, GA displayed a notable inhibitory effect on RSV, achieving an inhibition rate of up to 75%. SG exhibited some anti-RSV activity, although this effect was not statistically significant, with inhibition rates for RSV decoctions containing varying amounts of crude drug remaining below 25%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, the anti-RSV effect was significantly enhanced when GA was combined with SG at a sub-inhibitory concentration of 50 \u0026micro;M. At SG crude drug concentrations of 0.25 g/mL, 0.375 g/mL, and 0.75 g/mL, the inhibition rates increased to 56%, 69%, and 77%, respectively, which is more than double that observed at the same concentration, indicating a certain concentration dependence. This phenomenon is analogous to the synergistic properties of the SG-GC drug pair. Importantly, this synergy does not appear to be directly attributed to GA; rather, it may result from the interaction of the metal ion complex with the gypsum decoction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 The Effect of Glycyrrhizic Acid on the Anti-RSV Efficacy of Various Metal Ions in Gypsum\u003c/h2\u003e \u003cp\u003eTo further investigate the compatibility and synergistic effects of metal ions present in the gypsum decoction with glycyrrhizic acid (GA), we selected Ca\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, and Zn\u003csup\u003e2+\u003c/sup\u003e for anti-RSV experiments. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, at a metal ion concentration of 50 \u0026micro;M, host cell safety was satisfactory; however, the anti-RSV effects varied (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Ca\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e exhibited negligible direct anti-RSV activity, while Cu\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e demonstrated inhibition rates of 63% and 47%, respectively. This suggests that these ions possess some inherent anti-RSV properties, but their effectiveness remains largely unchanged when combined with sub-inhibitory concentrations of GA (50 \u0026micro;M). Notably, the inhibition rate of 50 \u0026micro;M Zn\u003csup\u003e2+\u003c/sup\u003e against RSV was 27%, which increased to 66% when combined with 50 \u0026micro;M GA, representing a 2.4-fold enhancement compared to the inhibition rate observed with Zn\u003csup\u003e2+\u003c/sup\u003e alone. These findings indicate that the gypsum decoction itself exhibits a certain antiviral effect, likely attributable to trace metal elements such as copper, magnesium, and zinc. However, only the combination of Zn\u003csup\u003e2+\u003c/sup\u003e and glycyrrhizic acid displayed synergistic properties akin to those of the SG-GC pair, suggesting that the Zn-GA supermolecule formed through the coordination of Zn\u003csup\u003e2+\u003c/sup\u003e and GA may be a critical factor in the antiviral mechanism of the SG-GC pair.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Determination of Metal Element Content in Gypsum Water Decoction with Varying Crude Drug Concentrations\u003c/h2\u003e \u003cp\u003eThe determination of metal element content in gypsum water decoction involves titrating the Ca\u003csup\u003e2+\u003c/sup\u003e concentration with an EDTA standard solution, while trace elements are quantified using ICP-MS. Results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea indicate that as the amount of gypsum increases (simulating the reuse of gypsum in the recipe), the Ca\u003csup\u003e2+\u003c/sup\u003e concentration in the decoction remains relatively constant at 3.8 g/L, suggesting that the CaSO\u003csub\u003e4\u003c/sub\u003e in the decoction has reached saturation. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb demonstrates that with an increase in gypsum quantity, the concentrations of trace elements Cu and Fe in the decoction exhibit a slight increase, whereas the levels of Mg, Al, and Zn increase significantly.Specifically, when the amount of gypsum increased threefold, the concentration of Cu\u003csup\u003e2+\u003c/sup\u003e rose from 183 ng/mL to 262 ng/mL, representing an increase of 1.4 times. Similarly, the concentration of Fe\u003csup\u003e3+\u003c/sup\u003e increased from 1335 ng/mL to 2467 ng/mL, reflecting an increase of 1.8 times. The Mg\u003csup\u003e2+\u003c/sup\u003e concentration showed a notable increase from 1960 ng/mL to 4480 ng/mL, a rise of 2.3 times. The concentration of Al\u003csup\u003e3+\u003c/sup\u003e increased significantly from 120 ng/mL to 1172 ng/mL, which is an increase of 9.7 times. Particularly noteworthy is the surge in Zn\u003csup\u003e2+\u003c/sup\u003e concentration, which escalated from 166 ng/mL to 2180 ng/mL, indicating a remarkable increase of 13.1 times, the highest among the trace elements. This observation suggests that as the amount of gypsum increases, the content of trace elements generally rises. Consequently, the anti-RSV effect of varying amounts of SG combined with GA is dependent on concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Furthermore, while the trace elements may contribute to gypsum's anti-RSV efficacy, its compatibility with licorice is also a crucial factor in enhancing this efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eThis study investigates the Junshi compound SG-GC within the MXSGT as the primary research focus. Using a mouse model of RSV infection, the findings demonstrate that both MXSGT and the SG-GC drug pair exhibit significant anti-RSV effects. Specifically, both treatments resulted in reduced viral loads in the lungs of mice, alleviated lung injury symptoms, and decreased inflammatory cell infiltration. In contrast, the antiviral effects of the individual components, gypsum or licorice, were found to be minimal, suggesting that gypsum must be used in conjunction with licorice to effectively exert its antiviral properties. A comparative analysis between the SG-GC medicinal pair and the CaSO\u003csub\u003e4\u003c/sub\u003e-GC group indicated that the antiviral efficacy may derive from trace metal elements present in gypsum. To further investigate the compatibility principle of gypsum and licorice, an RSV-infected cell model was utilized to examine the interactions between mineral and organic components. After confirming the safety of each component on host cells, it was observed that gypsum alone exhibited limited inhibitory potential against RSV, with an inhibition rate ranging from 16.2\u0026ndash;28.4%. However, when combined with GA at a sub-inhibitory concentration (50 \u0026micro;M), the anti-RSV effect was significantly enhanced, improving by 2.7 to 4.3 times. Subsequent studies revealed that the inhibitory effects of metal ions (Ca\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) in the gypsum decoction varied. At equivalent concentrations, Cu\u003csup\u003e2+\u003c/sup\u003e demonstrated the most effective inhibition, although no enhancement was noted when combined with GA. In contrast, the combination of Zn\u003csup\u003e2+\u003c/sup\u003e with GA resulted in an increased inhibitory effect of up to 2.4 times, showcasing a compatibility enhancement similar to that observed with the SG-GC drug pair. In addition, ICP-MS analysis confirmed that as the amount of gypsum increases, the concentration of trace elements also rises, with Zn\u003csup\u003e2+\u003c/sup\u003e exhibiting the most pronounced increase. This finding suggests, from another perspective, that one potential purpose of reusing gypsum may be to enhance the content of trace elements. While Zn\u003csup\u003e2+\u003c/sup\u003e has been extensively documented for its antiviral properties, this study is the first to reveal a significantly enhanced antiviral effect when it is combined with GA.\u003c/p\u003e \u003cp\u003eFrom the perspective of metal-organic supramolecular chemistry, GA contains carboxyl, hydroxyl, and other functional groups that can coordinate with metal ions. However, the spatial arrangement of the three carboxyl groups in GA is such that they are too distant from one another to form a multiradical ligand group, resulting in lower stability compared to cyclic structures formed by chelating agents like ethylenediamine and ethylenediaminetetraacetic acid (EDTA). Consequently, dissociation is more likely to occur. Conversely, this characteristic also facilitates the release of metal ions from the GA complex. GA molecules possess a hydrophobic triterpene skeleton and hydrophilic sugar groups, resulting in an amphiphilic structure that exhibits effective cell membrane penetration. The hydrophobic component can integrate into the phospholipid bilayer of the cell membrane, disrupting its structure and thereby enhancing drug permeability. This study posits that GA molecules may facilitate the translocation of Zn\u003csup\u003e2+\u003c/sup\u003e across the cell membrane, subsequently releasing Zn\u003csup\u003e2+\u003c/sup\u003e within the intracellular environment. This function resembles that of an ionophore, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Metal-organic supramolecules can be classified into chelating agents (such as EDTA), metal carboxyls (like porphyrin derivatives), and ionophores based on the type of ligands(Lee, Janisse, \u0026amp; Heffern, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Steinbrueck et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Notably, only ionophores are capable of repeatedly transporting metal ions across the membrane, thereby enhancing the intracellular concentration of these ions. In this study, it was observed that the anti-RSV efficacy was augmented when GA was combined with Zn\u003csup\u003e2+\u003c/sup\u003e, potentially linked to the ionophore-like properties of GA. However, whether GA functions as a specific zinc ionophore warrants further investigation.\u003c/p\u003e \u003cp\u003e \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\u003eClassification of metal-organic complexes according to ligand type\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of ligand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFunction of ligand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhysiological activity of metal ions\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetal chelator\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon-selective reduction of metal ion concentration inside and outside the cell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReduced drug efficacy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetal shuttle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay help extracellular metal ions cross the membrane, but cannot release free metal ions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo change\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetal Ionophores\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay help extracellular metal ions cross the membrane and release free metal ions after entering the cell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnhanced drug efficacy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGypsum, a mineral medicine, serves as a rich source of metal ions. When utilized in medicinal applications, gypsum readily forms various metal-organic supramolecular complexes with herbal medicines. Notably, the metal-GA supramolecular complex generated from the combination of gypsum and licorice has been the subject of prior studies(Zhang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, existing research has yet to investigate the synergistic, antagonistic, or novel bioactive properties that may arise from the formation of metal-organic supramolecular structures due to the interaction between metal ions and organic components in gypsum. This study focuses on the supramolecular assembly phenomenon previously observed in research concerning the antiviral efficacy of MXSGT. It represents the first exploration of the antiviral compatibility mechanism of the SG-GC drug pair through the lens of metal-organic supramolecular substances, aiming to elucidate the antiviral compatibility mechanism of this pairing. The supramolecular interactions between trace metal ions and licorice, particularly the coordination and transmembrane transport of Zn\u003csup\u003e2+\u003c/sup\u003e and GA, are pivotal for achieving compatibility and synergy. Furthermore, this study posits that research on the material basis of gypsum should not be conducted in isolation, but rather in conjunction with its compatible medicinal pairs or prescriptions. The metal-organic supramolecular theory marks a significant advancement in compatibility research.\u003c/p\u003e \u003cp\u003eAlthough this study found that SG-GC and MXSGT exhibit certain equivalence in direct antiviral efficacy, this does not imply that ephedra and bitter almond can be entirely excluded from the prescription. These medicinal components may play a significant role in alleviating coughs, asthma, and enhancing symptoms associated with viral infections. The precise compatibility of the four medicinal materials contributes to the effectiveness of the antiviral prescription MXSGT. Classic TCM prescriptions possess both enduring traditional advantages and unique clinical experiences in managing antiviral infections. In contemporary times, there is a need for scientific interpretations of their compatibility theories and pharmacodynamic mechanisms to facilitate the advancement of these ancient classic prescriptions. This study, based on the unique perspective of \"organic-metal supermolecules,\" offers a preliminary scientific explanation of the compatibility theory underlying MXSGT in TCM. It is anticipated that this will inspire new ideas for fundamental research in TCM and provide novel antiviral strategies. The application of innovative TCM opens up new possibilities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChang Lu: Data curation, Investigation, Formal analysis, Validation, Writing-original draft; Ying Ma: Formal analysis; HaoYu Zhao: Visualization; Yu Zhen: Investigation, Validation; Qun Zhao: Methodology; Lili Lin: Resources; XiuZhen Chen: Investigation; Linwei Chen: Supervision; Gang Li: Resources; Rui Chen: Conceptualization, Supervision, Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available on request to the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the support of Jiangsu Key Laboratory of Pediatric Respiratory Disease at Nanjing University of Chinese Medicine and Clinical Research Center of the Second Hospital of Nanjing affiliated to Nanjing University of Chinese Medicine for virus experimental support; Experiment center for science and technology at Nanjing University of Chinese Medicine for technical support; Jiangsu key laboratory of Chinese Medicine Processing for experimental support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by National Natural Science Foundation of China (Grant No. 82474195), Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (No. SJCX24_0897), Youth medical Innovation research project of China（P24021887623）, Nanjing Medical University (TZKY20230104, 2024KF0292).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlbalawi, S. A., Albalawi, R. A., Albalawi, A. A., Alanazi, R. F., Almahlawi, R. M., Alhwity, B. S., . . . Abdel-Maksoud, M. S. (2024). The Possible Mechanisms of Cu and Zn in the Treatment and Prevention of HIV and COVID-19 Viral Infection. \u003cem\u003eBiol Trace Elem Res, 202\u003c/em\u003e(4), 1524-1538. doi:10.1007/s12011-023-03788-9\u003c/li\u003e\n\u003cli\u003eBravo, V., Serrano, M., Duque, A., Ferragud, J., \u0026amp; Coronado, P. J. (2023). Glycyrrhizinic Acid as an Antiviral and Anticancer Agent in the Treatment of Human Papillomavirus. \u003cem\u003eJ Pers Med, 13\u003c/em\u003e(12). doi:10.3390/jpm13121639\u003c/li\u003e\n\u003cli\u003eCorrea, J. S., Primo, J. O., Balaba, N., Pratsch, C., Werner, S., Toma, H. E., . . . Bittencourt, C. (2023). Copper(II) and Cobalt(II) Complexes Based on Abietate Ligands from Pinus Resin: Synthesis, Characterization and Their Antibacterial and Antiviral Activity against SARS-CoV-2. \u003cem\u003eNanomaterials (Basel), 13\u003c/em\u003e(7). doi:10.3390/nano13071202\u003c/li\u003e\n\u003cli\u003eFaghfouri, A. H., Khabbazi, A., Baradaran, B., Khajebishak, Y., Baghbani, E., Noorolyai, S., . . . Alipour, B. (2022). Immunomodulatory and clinical responses to zinc gluconate supplementation in patients with Beh\u0026ccedil;et\u0026apos;s disease: A double-blind, randomized placebo-controlled clinical trial. \u003cem\u003eClin Nutr, 41\u003c/em\u003e(5), 1083-1092. doi:10.1016/j.clnu.2022.03.019\u003c/li\u003e\n\u003cli\u003eGuo, T., Guo, Y., Liu, Q., Xu, Y., Wei, L., Wang, Z., . . . Sun, L. (2021). The TCM prescription Ma-xing-shi-gan-tang inhibits Streptococcus pneumoniae pathogenesis by targeting pneumolysin. \u003cem\u003eJ Ethnopharmacol, 275\u003c/em\u003e, 114133. doi:10.1016/j.jep.2021.114133\u003c/li\u003e\n\u003cli\u003eHuan, C., Xu, Y., Zhang, W., Guo, T., Pan, H., \u0026amp; Gao, S. (2021). Research Progress on the Antiviral Activity of Glycyrrhizin and its Derivatives in Liquorice. \u003cem\u003eFront Pharmacol, 12\u003c/em\u003e, 680674. doi:10.3389/fphar.2021.680674\u003c/li\u003e\n\u003cli\u003eJia, L., Zhao, Y. L., Wang, J. B., Zou, W. J., Li, R. S., Yang, H. B., . . . Xiao, X. H. (2011). Study on the complex prescription compatibility law of the cold and hot nature of Mahuang Decoction and its categorized formulae based on the cold-hot pad differentiating assay. \u003cem\u003eChin J Integr Med, 17\u003c/em\u003e(4), 290-295. doi:10.1007/s11655-011-0694-2\u003c/li\u003e\n\u003cli\u003eKar, M., Khan, N. A., Panwar, A., Bais, S. S., Basak, S., Goel, R., . . . Medigeshi, G. R. (2019). Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-\u0026kappa;B and Induction of Antiviral Response in Epithelial Cells. \u003cem\u003eFront Immunol, 10\u003c/em\u003e, 2347. doi:10.3389/fimmu.2019.02347\u003c/li\u003e\n\u003cli\u003eKreiser, T., Zaguri, D., Sachdeva, S., Zamostiano, R., Mograbi, J., Segal, D., . . . Gazit, E. (2022). Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal Ions. \u003cem\u003ePharmaceuticals (Basel), 15\u003c/em\u003e(3). doi:10.3390/ph15030377\u003c/li\u003e\n\u003cli\u003eLee, V. J., Janisse, S. E., \u0026amp; Heffern, M. C. (2023). Plant-derived chelators and ionophores as potential therapeutics for metabolic diseases. \u003cem\u003eChem Soc Rev, 52\u003c/em\u003e(11), 3927-3945. doi:10.1039/d3cs00167a\u003c/li\u003e\n\u003cli\u003eLi, Q., Bai, C., Yang, R., Xing, W., Pang, X., Wu, S., . . . Gu, X. (2020). Deciphering the Pharmacological Mechanisms of Ma Xing Shi Gan Decoction against COVID-19 through Integrating Network Pharmacology and Experimental Exploration. \u003cem\u003eFront Pharmacol, 11\u003c/em\u003e, 581691. doi:10.3389/fphar.2020.581691\u003c/li\u003e\n\u003cli\u003eLin, L., Liao, Z., \u0026amp; Li, C. (2024). Exploring Ribosomal Genes as Potential Biomarkers of the Immune Microenvironment in Respiratory Syncytial Virus Infection. \u003cem\u003eBiochem Genet\u003c/em\u003e. doi:10.1007/s10528-024-10778-6\u003c/li\u003e\n\u003cli\u003eResch, B. (2017). Product review on the monoclonal antibody palivizumab for prevention of respiratory syncytial virus infection. \u003cem\u003eHum Vaccin Immunother, 13\u003c/em\u003e(9), 2138-2149. doi:10.1080/21645515.2017.1337614\u003c/li\u003e\n\u003cli\u003eSteinbrueck, A., Sedgwick, A. C., Brewster, J. T., 2nd, Yan, K. C., Shang, Y., Knoll, D. M., . . . Sessler, J. L. (2020). Transition metal chelators, pro-chelators, and ionophores as small molecule cancer chemotherapeutic agents. \u003cem\u003eChem Soc Rev, 49\u003c/em\u003e(12), 3726-3747. doi:10.1039/c9cs00373h\u003c/li\u003e\n\u003cli\u003eSun, S., Xu, Y., Qiu, M., Jiang, S., Cao, Q., Luo, J., . . . Zhu, J. (2023). Manganese Mediates Its Antiviral Functions in a cGAS-STING Pathway Independent Manner. \u003cem\u003eViruses, 15\u003c/em\u003e(3). doi:10.3390/v15030646\u003c/li\u003e\n\u003cli\u003eSun, Z. G., Zhao, T. T., Lu, N., Yang, Y. A., \u0026amp; Zhu, H. L. (2019). Research Progress of Glycyrrhizic Acid on Antiviral Activity. \u003cem\u003eMini Rev Med Chem, 19\u003c/em\u003e(10), 826-832. doi:10.2174/1389557519666190119111125\u003c/li\u003e\n\u003cli\u003eXi, S., Li, Y., Yue, L., Gong, Y., Qian, L., Liang, T., \u0026amp; Ye, Y. (2020). Role of Traditional Chinese Medicine in the Management of Viral Pneumonia. \u003cem\u003eFront Pharmacol, 11\u003c/em\u003e, 582322. doi:10.3389/fphar.2020.582322\u003c/li\u003e\n\u003cli\u003eYang, X., Kong, X. L., \u0026amp; Yue, X. Q. (2009). [Dose-response patterns of Radix Glycyrrhizae in Shanghan Lun]. \u003cem\u003eZhong Xi Yi Jie He Xue Bao, 7\u003c/em\u003e(3), 268-272. doi:10.3736/jcim20090313\u003c/li\u003e\n\u003cli\u003eYu, S., Zhu, Y., Xu, J., Yao, G., Zhang, P., Wang, M., . . . Zhang, J. (2021). Glycyrrhizic acid exerts inhibitory activity against the spike protein of SARS-CoV-2. \u003cem\u003ePhytomedicine, 85\u003c/em\u003e, 153364. doi:10.1016/j.phymed.2020.153364\u003c/li\u003e\n\u003cli\u003eZhang, Y. Z., Pi, W. M., Tan, X. R., Xu, R., Wang, X., Xu, M. Y., . . . Wang, P. L. (2025). [Exploration of differences in decoction phase state, material form, and crystal form between Glycyrrhizae Radix et Rhizoma-Gypsum Fibrosum and Glycyrrhizae Radix et Rhizoma-CaSO_4\u0026middot;2H_2O based on supramolecules of traditional Chinese medicine]. \u003cem\u003eZhongguo Zhong Yao Za Zhi, 50\u003c/em\u003e(2), 412-421. doi:10.19540/j.cnki.cjcmm.20240923.301\u003c/li\u003e\n\u003cli\u003eZou, G., Cao, S., Gao, Z., Yie, J., \u0026amp; Wu, J. Z. (2024). Current state and challenges in respiratory syncytial virus drug discovery and development. \u003cem\u003eAntiviral Res, 221\u003c/em\u003e, 105791. doi:10.1016/j.antiviral.2023.105791\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"licorice, gypsum, antiviral, compatibility, metal-organic supramolecule","lastPublishedDoi":"10.21203/rs.3.rs-6643243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6643243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe efficacy of gypsum-licorice (SG-GC) pair was evaluated by evaluating its in vivo and in vitro anti-respiratory syncytial virus(RSV) efficacy. The results showed that SG-GC had significant efficacy against RSV infection in mice, which was close to that of the whole formula, and could significantly reduce the viral load in the lungs, improve the symptoms of lung injury, and reduce the inflammatory cell infiltration in the pathological sites. The decoction of single-flavored gypsum showed some anti-RSV efficacy, but not significant; when combined with sub-inhibitory concentrations of GA, it showed a significantly enhanced anti-RSV effect. The experimental results of this study suggest that gypsum may be a potential key antiviral substance, but its efficacy needs to be complemented by licorice; the significant enhancement of antiviral efficacy of SG-GC may be related to the formation of metal-organic supramolecules after the interaction of trace metal ions in gypsum and GA in licorice.\u003c/p\u003e","manuscriptTitle":"Exploration of the Antiviral Mechanism of Gypsum-Licorice Compatibility Pairing in Ma- Xing-Shi-Gan Decoction from the Perspective of Metal-Organic Supramolecular Interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 08:20:07","doi":"10.21203/rs.3.rs-6643243/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69f2cbcf-d4d3-44ee-84bd-c01a9e7a0307","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-29T07:23:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 08:20:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6643243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6643243","identity":"rs-6643243","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}