The biological activity of Jinzhen Oral Liquid through in vitro interaction with Lactobacillus johnsonii

The biological activity of Jinzhen Oral Liquid through in vitro interaction with Lactobacillus johnsonii | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 30 October 2025 V1 Latest version Share on The biological activity of Jinzhen Oral Liquid through in vitro interaction with Lactobacillus johnsonii Authors : Ying Zhang , Huifang Gao , Hongyu Peng , Yizhao Tang , Yuanjing Ma , Xia Gao , Xialin Chen [email protected] , Liang Cao , Zhenzhong Wang , and Wei Xiao 0000-0001-8809-9137 Authors Info & Affiliations https://doi.org/10.22541/au.176181861.11775736/v1 204 views 143 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Jinzhen Oral Liquid (JZOL), a Traditional Chinese Medicine for respiratory diseases, exhibits gut lactobacillus-promoting activity, but its active compounds remain unclear. This study aimed to identify JZOL’s core efficacious ingredients via microbial metabolism. Lactobacillus was screened from healthy rat feces using selective MRS medium and identified as Lactobacillus johnsonii via whole-genome sequencing. Ultra-high performance liquid chromatography with Orbitrap tandem mass spectrometry (UPLC-Orbitrap-MS/MS) detected metabolites from its co-culture with 5 candidates (emodin, wogonoside, baicalin, aloe-emodin, glycyrrhetinic acid). Extracted Supernatant from Co-incubation (ESC) treated IL-1β-induced A549 cells; cytotoxicity (CCK-8 assay) and inflammatory factor mRNA levels (TNF-α, IL-6, IL-1β; qRT-PCR) were assessed. Results revealed 5 compounds and 9 metabolites; only aloe-emodin altered butyric acid metabolism. JZOL and ESC reduced inflammatory factor activity; aloe-emodin’s metabolite chrysophanic acid was abundant. Conclusion: L. johnsonii metabolizes JZOL, with aloe-emodin as the key. A rapid strategy for detecting compound-bacterial effects was established. The biological activity of Jinzhen Oral Liquid through in vitro interaction with Lactobacillus johnsonii Ying Zhang a, b, 1 , Huifang Gao a, b, 1 , Hongyu Peng a, b , Yizhao Tang a, b , Yuanjing Ma a, b , Xia Gao a, b, c , Xialin Chen a, b, c * , Liang Cao a, b, c , Zhenzhong Wang a, b, c , Wei Xiao a, b, c * a State Key Laboratory of Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, Jiangsu Kanion Pharmaceutical Co., Ltd. Lianyungang, 222001, China b Jiangsu Kanion Pharmaceutical Co., Ltd., Nanjing, 211100, China c School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China * Corresponding author: Fax: +86-518-81152367. E-mail addresses: [email protected] (W. Xiao) ; [email protected] (Xialin Chen) State Key Laboratory of Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, Jiangsu Kanion Pharmaceutical Co., Ltd. Lianyungang, 222001, China. 1 These two authors contributed equally to this work. Abstract Jinzhen Oral Liquid (JZOL), a Traditional Chinese Medicine for respiratory diseases, exhibits gut lactobacillus-promoting activity, but its active compounds remain unclear. This study aimed to identify JZOL’s core efficacious ingredients via microbial metabolism. Lactobacillus was screened from healthy rat feces using selective MRS medium and identified as Lactobacillus johnsonii via whole-genome sequencing. Ultra-high performance liquid chromatography with Orbitrap tandem mass spectrometry (UPLC-Orbitrap-MS/MS) detected metabolites from its co-culture with 5 candidates (emodin, wogonoside, baicalin, aloe-emodin, glycyrrhetinic acid). Extracted Supernatant from Co-incubation (ESC) treated IL-1β-induced A549 cells; cytotoxicity (CCK-8 assay) and inflammatory factor mRNA levels (TNF-α, IL-6, IL-1β; qRT-PCR) were assessed. Results revealed 5 compounds and 9 metabolites; only aloe-emodin altered butyric acid metabolism. JZOL and ESC reduced inflammatory factor activity; aloe-emodin’s metabolite chrysophanic acid was abundant. Conclusion: L. johnsonii metabolizes JZOL, with aloe-emodin as the key. A rapid strategy for detecting compound-bacterial effects was established. Keywords : Jinzhen Oral Liquid; Lactobacillus johnsonii; Untargeted metabolomics; Anti-inflammatory activity；microbial metabolism Abbreviations: ESI + Positive ion mode ESI − Negative ion mode FC Fold change JZOL Jinzhen Oral Liquid PCR Polymerase chain reaction PCA Principle component analysis PCoA Principal coordinate analysis PLS-DA Partial least squares-discriminant analysis QC Quality control SCFAs Short-Chain Fatty Acids TCM Traditional Chinese Medicine TNF-α Tumor necrosis factor alpha IL-6 Interleukin- 6 IL-1β Interleukin-1β UPLC-Orbitrap-MS/MS Ultra-high performance liquid chromatography with Orbitrap tandem mass spectrometry ESC Extracted Supernatant from Co-incubation HDAC histone deacetylase Introduction Jinzhen Oral Liquid (JZOL) is a Traditional Chinese Medicine (TCM) compound preparation derived from the folk prescription ”Lingyang Qingfei Powder”. Clinical evidence reported the significant efficacy in treating a variety of respiratory diseases, including viral infections, infantile bronchiolitis, pneumonia, bronchitis, and other respiratory disorders [1, 2]. These diseases typically present with clinical symptoms such as fever, headache, fatigue, and cough, and mainly characterized by respiratory epithelial damage and bronchial mucosal inflammation caused by pathogens like viruses and bacteria [3, 4]. The formulation of JZOL follows the ”monarch-minister-adjuvant-messenger” principle: Artificial Calculus Bovis, Astragalus membranaceus, Antelope Horn, and Gypsum Fibrosum serve as ”monarch herbs” with the effects of clearing heat and detoxifying; Melanterite (with a porous structure) and Anemarrhena asphodeloides act as ”minister herbs” to clear lung heat and reduce fire. The synergistic effect of all compounds has been strengthened in clearing heat, detoxifying, moistening the lung, and relieving cough. Except for Fritillaria thunbergii, Fritillaria ussuriensis, and Fritillaria cirrhosa, the other five plant compounds have been used in various medical systems in Europe, Japan, Russia, and the Americas. JZOL has been reported to inhibit respiratory inflammation by regulating the NF-κB/MAPK pathway and immune homeostasis [5-9]. Scutellaria baicalensis, Glycyrrhiza uralensis, and Rheum palmatum all exert strong anti-inflammatory effects. Baicalin derived from Scutellaria baicalensis Georgi regulating the TLR4/NF-κB signaling pathway by inhibiting the polarization of macrophages to pro-inflammatory M1 type and promote the polarization to anti-inflammatory M2 type, thereby reducing the release of pro-inflammatory factors [10]. Meanwhile, TCM compounds containing Scutellaria baicalensis (e.g., the combination of Rheum palmatum, Scutellaria baicalensis, and Poria cocos) have an effective rate of 94% in treating upper respiratory tract infections and bronchial asthma, and closely related to correcting the Th1/Th2 immune imbalance [11]. Glycyrrhizic acid derived from Glycyrrhiza uralensis reduces airway hyperresponsiveness by inhibiting the release of histamine and leukotrienes from mast cells. The combination of Glycyrrhiza uralensis and Ephedra sinica can decrease the levels of Th2-type cytokines in bronchoalveolar lavage fluid (BALF) of asthmatic mice and reduce eosinophil infiltration. Glycyrrhetinic acid reduces dry cough by acting on the cough center and promotes the secretion of respiratory mucus [12]. Glycyrrhiza uralensis effectively alleviate lung-heat cough by inhibiting the release of inflammatory mediators and relaxing airway smooth muscle [13]. Researchers have found that the blood-borne compounds of JZOL are mainly flavonoids, saponins, and anthraquinones, including baicalin and glycyrrhizic acid [14-20]. As a key hub for maintaining host homeostasis and regulating drug metabolism, gut microbiota has become a core regulatory factor for the efficacy of TCM, with a complex ”bidirectional interaction”. TCM compounds sometimes easily regulate the function of the intestinal microenvironment by remodeling the gut microbiota structure and regulating the microbial metabolite profile [12]; meanwhile, gut microbiota can convert low-activity precursors into high-activity derivatives through biotransformation (deglycosylation, hydrogenation), significantly enhancing efficacy [13]. Lactobacillus , a core functional group of gut microbiota genus, not only regulate the effects of drugs through multiple mechanisms but also maintains intestinal microenvironment homeostasis. Previous studies have confirmed the key role of Lactobacillus , Rhubarb and Moutan Decoction increase the number of L. johnsonii during treatment for patients with Henoch-Schönlein purpura [14], Gegen Qinlian Decoction selectively enriches Lactobacillus in the intestines of mice with diarrhea due to intestinal damp-heat syndrome, while regulating the level of intestinal short-chain fatty acids (SCFAs) [21]. Astragalus polysaccharides can promote Lactobacillus crispatus proliferation through prebiotic effects and inhibit opportunistic pathogens [22]. Significantly, Lactobacillus enhances TCM activity through enzymatic hydrolysis and fermentation [23]. In addition, Lactobacillus can also synergize with other gut microbiota to maintain the balance of the microbiota structure [24], for example, organic acids secreted by Lactobacillus provide a growth microenvironment for Bifidobacterium , while oligosaccharides produced by Bifidobacterium can promote the proliferation of Lactobacillus , forming a symbiotic cycle [25-27]. Based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) combined with different separation techniques, various separation methods have been developed, therefore, untargeted mass spectrometry technology were widely used [28]. New research ideas in untargeted metabolomics and gut microbiomics also provide promising application prospects. Calprotectin, C-reactive protein (CRP), and fetal hemoglobin (FHB) have already been adopted as biomarkers in feces and blood for evaluating the therapeutic response to inflammatory bowel disease [29, 30]. Currently, untargeted metabolomics research on gut microbiota-related metabolic analysis has been widely used. However, its research progress in the mutual transformation between gut microbiota and herbal compounds remains relatively slow [31], and the complexity of bacterial metabolomics requires further exploration. The advantages of UPLC-MS/MS are beneficial for detecting metabolites after the interaction between compounds and Lactobacillus. In this study, we added multiple active compounds from JZOL to the cultured medium of separated L. johnsonii and tested whether these compounds could be metabolized. In turn, untargeted metabolomics on L. johnsonii was performed to know the affected metabolic pathways. In addition, the extracted of supernatant from co incubation (ESC) committed to anti-inflammatory factor effects by acting on inflammatory A549 cells. Through the above research, we simulated some reactions between JZOL and L. johnsonii after oral administration in vitro , in order to understand the multi-target and synergistic effects of multiple compounds in TCM. Materials and methods 2.1 Experimental reagents Emodin (batch no. 110756-201512, purity 99.0%), Aloe-emodin (batch no. 110795-201710, purity 98.3%), Baicalin (batch no. 110715-201821, purity 94.2%), Wogonoside (batch no. 112002-201702, purity 98.5%), Glycyrrhizic acid (batch no. 110723-201715, purity 99.6%) were obtained from the China National Institute for Food and Drug Control (Beijing, China). Jinzhen oral liquid (batch no. 221201) were provided by Jiangsu Kanion Pharmaceutical Co., Ltd. (Lianyungang, China). Ultra-pure water was prepared with a Milli-Q water purification system (Shanghai Millipore Co. Ltd, China). Acetonitrile and methanol were of MS grade from Merck (Darmstadt, Germany). Formic acid (HPLC grade) was obtained from ACS (American). Other reagents were of analytical grade. 2.2 Isolation and culture of Lactobacillus Preparation of fecal microbiota solution: Suspend fresh feces in 0.9% sodium chloride solution. Diluted 300 mg of feces to a volume of 3 mL and stirred until no obvious large particles remain. And centrifuge at 12,000 rpm for 10 minutes under the condition of 4°C. Remove the fecal bacterial solution, and discard the fecal sediment simultaneously. If excessive precipitation occurred or the feces were not stirred evenly, separation was performed to obtain a homogeneous fecal suspension for subsequent experiments. Single bacterium isolation: Taken the rat fecal fluid, with an inoculum of 3%, into the liquid MRS broth medium at 37 ℃ under anaerobic conditions (5% CO₂ + 95% N₂, Don Whitley Scientific. Britain), enrichment cultured 48 h to the logarithmic growth period. The solution was sequentially diluted 10 1 to 10 7 times gradient respectively and spread on an MRS solid plate contained 0.75% CaCO₃, the target colony was purified more than three times until pure bacteria. Single colony were selected for gram-staining microscopic examination. Whole genome sequencing of strains: The pure Lactobacillus strain was used to whole genome sequencing to identify the species. Illumina NovaSeq sequencing platform (Shanghai Meiji Biotechnology Co., Ltd.) was used for sequencing. 2.3 Strain Growth Curve Determination Activated strains were inoculated with MRS liquid medium for 24 h, inoculated in new MRS liquid medium at 5% (v / v), and their OD 600 values were measured at 0, 2, 4, 6, 8…24 h respectively. 2.4 Untargeted metabolomics analysis The co incubation solution was set to 20 μL compound (10 mg/mL, baicalin, wogonoside, glycyrrhizic acid, aloe-emodin and emodin dissolved in alcohol reagent) added to 3.98 mL enterobacteria culture solution separately. Under the same conditions, the control group included drug free (methanol + bacterial culture medium) and bacterial free (medicinal solution + physiological saline + anaerobic culture medium), parallel six times for each group. The incubation time was 4, 7 and 24 h. Sample preparation: Added an equal amount of iced cold acetonitrile solution to the incubation sample solution, mixed and centrifuged at 12000 r/min for 10 min, then taken out the supernatant and added twice the amount (based on the incubation liquid volume) of ethyl acetate for extraction, vortex and mixed, centrifuged at 8000 r/min for 5 min, collected the ethyl acetate layer, and dry under nitrogen gas, finally, added 50% acetonitrile for substrate reconstitution. QC samples: Mixed the solutions of each group in equal proportions. UPLC-Orbitrap-MS/MS conditions: The characterization of the chemical components was analyzed using a UPLC-Orbitrap-MS/MS system (Thermo Fisher Scientific Inc., USA). Agilent ZORBAX Eclipse Plus C 18 column. (2.1 × 100 mm, 1.8 μm) was selected for separation. The mobile phase consisted of eluent A (0.1% aqueous formic acid, v/v) and eluent B (acetonitrile) and at a flow rate of 0.4 mL/min. The gradient elution program was set as follows: 0-1 min, 2% B; 1−5 min, 46% B; 5−8 min, 50% B; 8−13 min, 60% B; 13−17 min, 76% B; 17−25 min, 90% B; 26−27 min, 100% B; 27−29 min, 2% B. The temperature of the column was maintained at 30 °C. The sample injection volume was 2 μL. The heated electrospray ionization mode was used in both positive (HESI + ) and negative (HESI - ), with a scanning range of m/z 50−1200 and collision energies of 10, 20, and 40 V. Ionization parameters were spray voltage 3500 V and 2500 V, temperature 350 ℃, sheath gas flow rate of 50 L/min, and auxiliary gas flow rate of 10 L/min. The scanning modes were Full MS/dd-MS 2 . 2.5 Effect of compounds on the growth of Lactobacillus 100 μL of the co-incubation solution from each group was transferred to a 96-well plate, and then put them into the enzyme labeling instrument (Agilent Technologies (Shanghai) China Co., Ltd.) to detect OD 600 value every hour within 24 h. 2.6 CCK-8 assay The cells were cultured in 1640 complete medium containing 10% fetal bovine serum and 1% penicillin (100 U/mL) and streptomycin (100 μg/mL) mixed solution and maintained in 37 ℃ incubator containing 5% CO 2 . The A549 cell was seeded in a 96-well plate, a total of 1× 10 5 cells were plated and cultured in normal growth conditions for 24 h. The cells were divided into control group, administration group (containing JZOL 43.09, 21.55, 10.77, …, 0.34 mg/mL medium, converted to the concentration of the crude drug), ESC group (added to medium with concentrations of 0.001, 0.0005, …., .6.25× 10 -5 of emodin, glycyrrhizic acid, baicalein, baicalin, and aloe-emodin components with L. johnsonii extract), and the blank group. The cells were then treated with 100 μL fresh serum-free 1640 solution containing 10% CCK-8. After incubation for 2 h, OD value was detected at 490 nm. To ensure accuracy, conducted three parallel experiments. 2.7 IL-1β‑induced inflammatory The inflammatory model was constructed by inducing A549 cells with IL-1β. Cultivated total of 2.5x10 5 cells seeded in a 6-well plate and maintained in normal growth conditions for 24 h. After cells were treated with 1 mM IL-1β for 24 h. JZOL and ESC of 5 compounds solution were separately acted on A549 cell for an additional 24 h and a control group (no drug) was set. Collected cells for qPCR experiment and cell supernatant for identification of compounds by UPLC-Orbitrap-MS/MS, used the same qualitative method as in 2.4. 2.8 Determination of mRNA levels using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) Total RNA was extracted from cultured A549 cells using the TaKaRa MiniBEST Universal RNA Extraction Kit (Takara Biomedical Technology (Beijing) Co., Ltd.), cDNA was synthesized using the Takara PrimeScript™ RT reagent Kit, qPCR was performed with Takara TB Green Premix Ex Taq Master Mix. The fold-change for mRNA relative to β-actin was determined using the 2 -∆∆ C t . The all primers were synthesized by GenScript (Nanjing GenScript Biotechnology Co., Ltd). The primer names and their sequences are shown in Table 1. 2.9 Data analysis Preliminary chemical identification of the compounds was carried out using Thermo Compound Doscover 3.3 (Thermo Fisher, USA), and the differences between two groups were compared using the t-test of SPSS 26.0; ANOVA (Tukey post hoc test) was used to compare the differences among the various groups. P < 0.05 was considered a statistically significant difference. Differential metabolites were screened using the MetaboAnalyst website at P < 0.05, FC ≥ 2. All bar graphs were plotted using GraphPad Prism 8.0.2(Dotmatics, USA). Results 3.1. Isolation and identification of Lactobacillus After strains were anaerobically cultured on MRS plates for 48 h, the colonies formed were white and round with smooth and moist surfaces, ranging from 0.1−2.5 mm, neat edges and protruding centers. The microscopic examination became positive for Gram staining, and the organisms were mostly rod-shaped; most of them were arranged in chains of different lengths, and some of them were arranged in a single dispersed manner [32]. It was preliminarily determined to belong to the Lactobacillus genus. The phylogenetic tree was constructed by the NJ (Neighbor-Joining) method based on the sequences of the housekeeping genes using MEGA 6.0 software. The sequences were compared with the known reference strains in the NCBI database and identified as L. johnsonii with a 99.5% similarity. The OD 600 absorbance value displayed that L. johnsonii growth slowly at 0−2 h for the delayed period, into the logarithmic growth period at 2−7 h and reached the peak of the growth at 6−8 h. In addition, the logarithmic phase of L. johnsonii was selected for intervention, emodin, glycyrrhizic acid, baicalein, baicalin, and aloe-emodin did not show significant inhibitory effect on L. johnsonii growth. These results are shown in Figure 1. 3.2. Characterization of the main compounds after the interaction Based on the preliminary experiments, emodin, wogonoside, baicalin, aloe-emodin, glycyrrhizic acid, glycyrrhetinic acid, peimisine, peimine and peiminine were important material basis for JZOL to exert its medicinal effects. JZOL and its candidate compounds were co-incubated with L. johnsonii respectively. The metabolites mainly include flavonoids, anthraquinones and alkaloids. Flavonoids main metabolites were Oroxindin, Genkwanin, Naringenin, and Kaempferol; The metabolites of anthraquinones main were Chrysophanic acid, Emodin-3- O -glucuronide, and Emodin-1- O -sulfate. Flavonoid cleavage pathways mainly include glycosidic bond breaking, RDA rearrangement, removal of neutral fragments, loss of carbonyl group, glycan ring cleavage and cross ring excision [33]. Intestinal microbial enzymes metabolize different flavonoids, including deglycosylation reaction, demethylation reaction, dehydroxylation reaction, reduction reaction and ring fission reaction. The negative ion mode of anthraquinones is superior to the positive ion mode. The molecular ion peak characterized the mass spectra as the base peak, and the free quinone was sequentially stripped of 2 CO to obtain the strong peaks of M-CO and M-2CO and their double-charged peaks [34]. The metabolic conversion of rhub anthraquinones by intestinal flora is mainly hydrolysis, supplemented by reduction and oxidation. The corresponding hydrolases, oxidoreductases and lyases are produced by intestinal flora, which are metabolized to active substances that are more easily absorbed and play drug effects in the body. Alkaloid metabolism mainly involves oxidation reaction, reduction reaction, hydrolysis reaction and glucuronic acid binding. Among them, we focused on the secondary fragmentation of flavonoid and anthraquinone metabolites. The information on the prototypical components and metabolites of JZOL is shown in Table 1. 3.2.1 Analysis of flavonoids The mass spectrometry lysis rule was analyzed by taking baicalin as an example. The excimer ion peak of baicalin is m/z 447.0923 [M+H] + . The molecular formula fitted by software is C 21 H 18 O 11 . The secondary fragment is methylated and protonated in the negative ion mode, with a molecular peak of m/z 461.10815 [M+H] + , and one molecule of C 6 H 8 O 6 is removed. Generate fragment ions m/z 285.07574 [M+H- C 6 H 8 O 6 ] + ; One molecule of CH 3 was removed from m/z 285 to obtain the ionic peak of m/z 270.05240 [M+H-C 6 H 8 O 6 -CH 3 ]. The cleavage rule pathway of the secondary fragment ions is shown in Figure 2A. Compared with the reference substance, it was determined to be baicalin. 3.2.2 Analysis of anthraquinones A total of four anthraquinone glycosides, including rhein, emodin, aloe-emodin, and chrysophanol, which were all derived from rhubarb. Aloe-emodin was taken as an example to analyze its cleavage pattern, and the cleavage pathway of its fragment ions is shown in Figure 2B. The highest abundance of m/z 211 in the secondary mass spectrum, 183.04517 C 12 H 7 O 2 , was obtained for 211.04001 CO. The cleavage pathway of its fragment ions is shown in Figure 2C, and it was identified as aloe-emodin by comparison with the reference substance. 3.3. The metabolic effects of compound on L. johnsonii The data were analyzed using the MetaboAnalyst website. The principal component analysis (PCA) showed that the within group samples were relatively aggregated and the inter group samples significantly separated, QC samples had a high degree indicating that the assay process was stable and reliable. As seen from Figure 3A, on the first principal compound, aloe-emodin was completely separated from other four administration groups. On the second principal compound, aloe-emodin, emodin, and wogonoside groups could be completely distinguished from the blank group. Therefore, the aloe-emodin group has a strong effect on the metabolism of L. johnsonii than other four groups. The supervised partial least squares discriminant analysis (PLS-DA) (Figure 3B) showed that aloe-emodin was significantly separated from the rest of the four administration groups. The identified compounds were analyzed by cluster analysis (Figure 3C), where erucamide showed a high correlation between emodin and 2-aminophenol, and a low correlation between paracetamol and glycurrhizic acid. 3.4. Metabolic pathway analysis of differential metabolites After calibrating the metabolic data, metabolites related to L. johnsonii were screened according to P < 0.05 and FC ≥ 2.15 potential metabolites between aloe-emodin and control group were obtained and confirmed by matching the secondary fragmentation from the database (Table 2). The differential metabolites were imported into MetaboAnalyst for metabolic pathway analysis. Following median normalization, -log10 transformation, and mean-centered standard deviation scaling, candidate pathways were screened using thresholds of pathway impact value > 0.1 or -log10 (p) > 1.33. Butyrate metabolism pathway was enriched with succinate as the involved differential metabolite. As shown in Table 3. 3.5. CCK-8 results The highest test concentration with a mean cell survival rate > 95% was taken as the maximum non-toxic concentration of JZOL and ESC of 5 compounds for A549 cells. The test results ( Figure 4A) showed that after dilution of JZOL by 6.25, 12.5, and 25 times, the survival rates of A549 cells were (85.2 ± 3.5) %, (90.5 ± 2.8) %, and (93.1 ± 2.1) %, respectively. When diluted to 50 times (crude drug concentration ≤ 5.39 mg/mL), the cell survival rate > 95%. The maximum non-toxic concentrations of the ESC of 5 compounds were respectively: emodin group was ≤ 3.125 × 10 -5 mg/mL (diluted 8 times), the glycyrrhizic acid group and the wogonoside group were both ≤ 1.25 × 10 -4 mg/mL (diluted 4 times), and the baicalin group and the aloe-emodin group were both ≤ 1.5625 × 10 -5 mg/mL (diluted 10 times). 3.6. Identification of metabolites in the supernatant of A549 cells The relevant compounds were characterized based on the accurate molecular weight, MS/MS product ions and the information reported in the literature (Table 4). The metabolites of ESC of aloe-emodin included chrysophanic acid, aloe-emodin-8- O -β-D-glucopyranoside, and emodin-8- O -methyl ether. 3 anthraquinone and 2 flavonoids compounds in these metabolites. T he endogenous metabolic products of A549 cells mainly included amino acids and their derivatives (6 kinds), organic acids (3 kinds), vitamins (2 kinds). 3.7. qRT-PCR results Compared with the control group, the mRNA expressions of IL-6, IL-1β and TNF-α in model group were significantly upregulated ( P < 0.01), indicating that the inflammation model was successfully constructed. Compared with the model group, intervention with JZOL and ESC showed that high-dose JZOL significantly downregulated the mRNA expression of IL-6 and IL-1β ( P < 0.01), but no significant effect on the expression of TNF-α; low dose of JZOL also significantly downregulated the mRNA expressions of IL-6 and IL-1β ( P < 0.01), and the effect of anti-inflammatory factors was dose-dependent; except for ESC of emodin, all significantly downregulated the mRNA expression of IL-6 ( P < 0.01). The five ESC compounds significantly downregulated the mRNA expression of IL-1β ( P < 0.01). However, only ESC of aloe-emodin could significantly downregulate the TNF-α mRNA expression ( P < 0.05). 4 Discussion The main pathological features of respiratory diseases (such as infantile bronchiolitis and pneumonia) include pathogen-induced respiratory epithelial damage, bronchial mucosal inflammation, and excessive release of pro-inflammatory cytokines (e.g., IL-6, IL-1β, TNF-α) [4]. Clinical studies have confirmed that JZOL can effectively alleviate such inflammatory symptoms, but whether its efficacy depends on gut microbiota regulation remains unclear[3]. Therefore, this study combined untargeted metabolomics with cytological experiments to reveal the interaction mechanism between the candidate compounds of JZOL and L. johnsonii . Isolated and identified from the feces of healthy rats, L. johnsonii is a dominant Lactobacillus strain in the gut, and it exhibited stable metabolic transformation ability in co-culture system with JZOL candidate components. This finding is consistent with the core role of the genus Lactobacillus in TCM metabolism, Lactobacillus species can transform flavonoids and anthraquinones via enzyme systems such as β-glucosidase, thereby enhancing the bioactivity of these compounds [35]. A key finding of this study is that L. johnsonii exhibits high efficiency in the biotransformation of active compounds in JZOL, which is consistent with the well-documented role of Lactobacillus species in the metabolism of TCM. Specifically, flavonoids (baicalein, puerarin) undergo glycosidic bond cleavage and demethylation to generate chrysin and naringenin—reactions that conform to the classical metabolic patterns of flavonoids mediated by intestinal microbiota [35]. This observation suggests that L. johnsonii may secrete specific glycosidases to facilitate the conversion of flavonoid glycosides to their corresponding aglycones, a process that has been validated to enhance the bioavailability and anti-inflammatory bioactivity of flavonoids [38]. For anthraquinones, aloe-emodin undergoes dehydroxylation to form chrysophanol; this metabolite possesses higher lipid solubility, which favors transmembrane transport [39]. This structural modification not only explains the presence of chrysophanol in both the co-culture supernatant (ESC) and A549 cell supernatant, but also highlights the pivotal role of L. johnsonii in ”activating” inactive or low-activity precursors in JZOL into potent anti-inflammatory agents. Metabolomic profiling further revealed that aloe-emodin modulates the butyrate biosynthesis pathway of L. johnsonii , with succinic acid identified as a key differential metabolite. Butyrate, a major short-chain fatty acid (SCFA), exerts anti-inflammatory effects by activating G protein-coupled receptors (FFAR2/3) or inhibiting histone deacetylases (HDACs); additionally, it maintains immune homeostasis by inducing the differentiation of regulatory T cells (Tregs) [36, 37]. As a precursor of butyrate, fluctuations in succinic acid levels directly regulate the efficiency of butyrate biosynthesis. Combined with the observation that JZOL downregulates IL-6 and IL-1β expression, our findings demonstrated that JZOL modulates the metabolic profile of L. johnsonii to promote the conversion of succinic acid to butyrate, thereby alleviating lung inflammatory injury via the ”lung-gut axis”—a mechanism that aligns with the TCM doctrine of ”the exterior-interior relationship between the lung and large intestine” [42, 43]. Furthermore, this study complements previous research on the anti-inflammatory effects of JZOL via the NF-κB/MAPK signaling pathways [10, 40], and identifies intestinal microbiome metabolism as an upstream regulatory node. In vitro anti-inflammatory assays further elucidated the synergistic nature of the multi-compound and multi-target actions of JZOL. Both JZOL and ESC fractions inhibit the expression of pro-inflammatory cytokines, yet their target specificities are distinct: the ESC fraction derived from chrysophanol is the only component capable of downregulating TNF-α expression (aloe-emodin reduces TNF-α mRNA levels by specifically inhibiting aloe-emodin acetylase [ESC]), while ESC fractions derived from baicalin and wogonoside primarily target IL-6, and all ESC fractions suppress IL-1β. This specificity may originate from structural differences in the compounds and their interactions with metabolic enzymes of L. johnsonii . For instance, the anthraquinone scaffold of aloe-emodin may be specifically modified by redox enzymes derived from L. johnsonii to form chrysophanol, which then targets TNF-α via the TLR4/NF-κB pathway [39]; in contrast, the flavone scaffold of baicalein is more readily hydrolyzed to its aglycone by glycosidases, thereby regulating IL-6 through the same pathway [41]. This ”parent compound-metabolite” synergy explains why JZOL exerts a broader anti-inflammatory spectrum than individual compounds—a inherent advantage of TCM compound prescriptions (Chinese herbal compound, composite formulations). Notably, none of the five candidate compounds inhibited the proliferation of L. johnsonii ; instead, all exerted a proliferation-promoting effect. This observation, combined with our previous finding that JZOL increases the abundance of L. johnsonii in vitro [44], indicates the existence of a beneficial feedback loop: JZOL promotes the growth of L. johnsonii , which in turn enhances the conversion of compounds in JZOL to active metabolites, ultimately potentiating the bioactivity of JZOL. This loop underscores the critical role of the intestinal microbiome in sustaining the efficacy of TCM. This study also has limitations: the focus on a single bacterial strain ( L. johnsonii ) may fail to account for the synergistic or competitive interactions of others intestinal microbiota with JZOL metabolism. Future studies should extend to multi-strain co-culture systems and animal models to more comprehensively simulate the complex intestinal microenvironment. 5 Conclusion In this study, L. johnsonii was isolated, purified, and identified from healthy rat feces. In vitro co-culture showed this strain efficiently biotransforms five JZOL’s candidate compounds (emodin, wogonoside, baicalin, aloe-emodin, glycyrrhetinic acid) into nine bioactive metabolites (flavonoids, anthraquinones, alkaloids); notably, aloe-emodin-derived chrysophanic acid was abundant in both ESC and IL-1β-induced inflammatory A549 cell supernatant, implying it mediates JZOL’s anti-inflammatory effects, and aloe-emodin may regulates L. johnsonii’s butyric acid pathway. 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International journal of biological macromolecules 2023, 249:126038. 44. Feng J, Gao X, Chen X, et al. Mechanism of Jinzhen Oral Liquid against influenza-induced lung injury based on metabonomics and gut microbiome. J Ethnopharmacol. 2023;303:115977. doi:10.1016/j.jep.2022.115977 Supplementary Material File (figure.docx) Download 878.05 KB File (table.docx) Download 35.72 KB Information & Authors Information Version history V1 Version 1 30 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords lactobacillus johnsonii anti-inflammatory activity；microbial metabolism jinzhen oral liquid untargeted metabolomics Authors Affiliations Ying Zhang Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Huifang Gao Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Hongyu Peng Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Yizhao Tang Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Yuanjing Ma Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Xia Gao Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Xialin Chen [email protected] Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Liang Cao Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Zhenzhong Wang Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Wei Xiao 0000-0001-8809-9137 Jiangsu Kanion Pharmaceutical Co Ltd View all articles by this author Metrics & Citations Metrics Article Usage 204 views 143 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Ying Zhang, Huifang Gao, Hongyu Peng, et al. 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