Enhanced Mitochondrial Respiratory Metabolism in UVB-Damaged HDF cells Based on Metabolomics Analysis of Fermented Galla rhois gallnut Extract

Enhanced Mitochondrial Respiratory Metabolism in UVB-Damaged HDF cells Based on Metabolomics Analysis of Fermented Galla rhois gallnut Extract | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced Mitochondrial Respiratory Metabolism in UVB-Damaged HDF cells Based on Metabolomics Analysis of Fermented Galla rhois gallnut Extract Xiang Li, Jiaying Wu, Ning Wang, Denghui Chen, Yi Zhang, Jingxia Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8653896/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Both natural aging and photoaging of the skin can lead to disruption of mitochondrial homeostasis, which in turn exacerbates skin aging. This study developed a novel fermented Galla rhois gallnut extract (FGRE) using Lactiplantibacillus plantarum with mitochondrial respiratory homeostasis amelioration. The results of biochemical experiments showed that FGRE had better antioxidant and anti-elastase activity compared to unfermented extract. Seahorse XF Cell Mito Stress Test demonstrated that 1% FGRE significantly enhanced mitochondrial respiratory function in UVB-irradiated human dermal fibroblasts (HDFs) compared to unfermented extract (21% increase in basal respiration and spare respiratory capacity, 26% increase in ATP production, and 30%increase in maximal respiration). The non-targeted metabolomics analysis revealed differential metabolites predominantly enriched in amino acid metabolic pathways, and obviously increased metabolites were leucinic acid, Val-Asn, Arg-Val-Phe, Urolithin A (UroA), Urolithin M5 (UroM5) and other flavonoids. The quantitative detection showed that the levels of UroA (0.49 ng/mL) and its derivative UroM5 (8.35 ng/mL) were quite low, while the concentration of leucinic acid was about 97.20 μg/mL. And cell experiments suggested that 1 μg/mL leucinic acid (equivalent to concentration in 1% FGRE) ameliorated UVB-induced mitochondrial respiratory dysfunction in HDFs, that determine leucine as primary bioactive constituent. These results indicated that FGRE had potential anti-aging capacity by enhancing mitochondrial function, which also provided a novel candidate for dermo-cosmetic applications. Biological sciences/Biochemistry Health sciences/Diseases Biological sciences/Physiology Galla rhois Fermentation Mitochondrial Respiratory Metabolism Metabolomics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Skin aging is a complex biological process, which is intuitively presented as increased wrinkles, decreased elasticity, laxity, enlarged pores and pigmentation of skin 1 . In addition to endogenous aging, external environmental factors, especially ultraviolet (UV) radiation, accelerate the aging process 2 . Recent years, mitochondrial homeostasis is considered crucial for skin barrier and skin health 3 . Both natural aging and photoaging cause the decline of mitochondrial function, the accumulation of mitochondrial DNA deletions, and the increase of reactive oxygen species production 4 . That leads to oxidative stress and insufficient energy metabolism in skin cells, thus aggravating the skin aging 5 . Therefore, improving cellular mitochondrial function is considered as a good method to alleviate skin aging. Galla rhois , a kind of traditional Chinese medicine, is the gall caused by the Chinese aphid, Schlechtendalia chinensis (Bell), on the leaves of Rhus chinensis 6 . Some studies reported that Galla rhois had anti-inflammatory, hemostatic and analgesic properties, that could be used to promote clotting following traumatic injuries, to treat burns and skin infections 7 . A study showed that Galla rhois extract could inhibit the elastase and tyrosinase activity in vitro, and reduced the expression of MMP-1 in UVA-induced fibroblasts 8 . Fermentation is the main technology for the food and plant ingredient processing. Lactobacillaceae are major fermenting organisms frequently found in traditional fermentations, which can transform large molecules into more easily absorbed and bioactive small molecules 9 . Lactiplantibacillus plantarum fermented Rhodiola rosea can activate the Nrf2/Keap1 signaling pathway and protect fibroblasts from oxidative stress caused by UVA, and the effect is significantly better than unfermented Rhodiola rosea 10 . In addition, lactic acid bacteria own metabolites or their extracts also have beneficial effect on skin. For example, extracellular vesicles, produced by Lactiplantibacillus plantarum , reduced wrinkle formation, improve skin elasticity, increase skin moisture content, improve skin density, as well as inhibit skin pigmentation caused by aging in clinical trial 11 . Therefore, it is a good way to enhance the bioactivity of Galla rhois by lactic acid bacteria fermentation. In this study, we developed a kind of fermented ingredient Galla rhois gallnut extract (GRE) fermented by Lactiplantibacillus plantarum . The FGRE were was systematically evaluated for its modulatory effects on mitochondrial function in UVB-irradiated human dermal fibroblasts (HDFs). The non-targeted and targeted metabolomics were used to analyze the metabolites of FGRE, and combined with cell experiments to determine the key active compounds. Our results indicated that FGRE could improve the mitochondrial function and ameliorate UVB-indeced photoaging, which provided a novel ingredient for skin care. Methods Chemicals and reagents GRE was purchased from Focusherb Co. Ltd (Shanxi, China). Lactiplantibacillus plantarum PIAS240228 isolated from fermented food by our laboratory. Fermented nutrient salt NS01 was obtained from AngelYeast Co., Ltd. Leucine acid and leucinic acid were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Urolithin C (UroC, purity of 95%), urolithin A (UroA, purity of 98%) were obtained from Sigma (Shanghai, China). Urolithin M5 (UroM5, purity of 96%) and urolithin M6 (UroM6, purity of 92%) were purchased from NatureStandard Shanghai Standard Technology Co., Ltd. Human dermal fibroblasts (HDFs) purchased from Guangdong BioCell Biotech Co., Ltd (Guangdong, China). All other chemicals of analytical grade were obtained from Sigma (Shanghai, China), unless otherwise noted. Fermentation Galla rhois gallnut extract The strain Lactiplantibacillus plantarum PIAS240228 was inoculated into MRS medium for 2 ~ 3 times activation. Then, Lactiplantibacillus plantarum PIAS was inoculated in GRE medium with 2% inoculation and fermented for 36 ~ 48h with constant pH = 5.5. The GRE medium contained 100 mg/L GRE, 10g/L glucose and 10g/L fermented nutrient salt NS01 dissolved in 0.03 M potassium phosphate buffer (PBS, pH = 7.2). The fermentation broth was homogenized and broken by high pressure homogenization. Then, the final FGRE samples was obtained by centrifugating at 11000×g for 10 min and concentrating 3 ~ 4 times. GRE samples for follow-up experiments was also obtained by centrifugating at 11000×g for 10 min and concentrating 3 ~ 4 times from GRE medium. GRE and FGRE were further explored the effect and compound composition. Biochemical Experiments The antioxidant activity of FGRE was determined by DPPH radical scavenging assay according to the method described by Lee et al 12 . Briefly, the samples were mixed with an equal amount of DPPH solution and left to stand for 30 min at room temperature in the dark. And then determine the absorbance of the resulting solution at 517 nm. Water was taken as control, while 12.5 µg/mL ascorbic acid (Vc) was taken as positive control. The scavenging ability was defined as: Scavenging activity (%) = (A control −A sample )/A control × 100%. The anti-elastase activity was measured according to Shirzad’s research with slight modification 13 . 420 µL 0.05 M Tris-HCl (pH = 8), 180 µL of samples and 150 mL elastase enzyme (600 mU/mL) were mixed together with 15 min incubation in room temperature (25°C). 150 µL N-succinyl-Ala-Ala-Ala-Val-p-nitroanilide substrate (1.015 mmol/L) was added to each tube. After 20 min incubation in room temperature (25°C), absorbance was read at 410 nm. The epigallocatechin gallate (EGCG, 500 µg/mL) was used as positive control. The percentage inhibition was calculated as follows: Inhibition (%) = [(A control –A sample )/A control ] ×100%. Cellular mitochondrial energy metabolism assessment by Seahorse XF Cell Mito Stress Test HDFs were uesd to explore the effcet of FGRE on mitochondrial. First, Determine the toxicity of different samples on HDF cells using the CCK-8 experimental method. Then, the experimental groups and treatment were performed as following. First day, all HDF cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 5% CO 2 and 37℃ for 24h. Then, the cells in control group (NC) and UVB group were still cultured in DMEM medium supplemented with 10% fetal bovine serum for 24h. And GRE group and FGRE group were cultured in 1% GRE and 1% FGRE for 24 h, respectively. At third day, the cells in UVB, GRE and FGRE groups were washed 2 ~ 3 times with PBS and exposed to UVB (0.30mW/cm 2 ) in PBS for 10 min, while NC group with no UVB exposure. The oxygen consumption rate (OCR) in HDF cells was determined by Seahorse Extracellular Flux (XF) analyzer (Seahorse Bioscience, USA) and analyzed the basal respiration, ATP production, maximal respiration and spare respiratory capacity according to Wang’s research 14 . Further, 0.3 µg/mL leucine, UroM5 and UroA were also explored the effect on mitochondrial function by the methods as mentioned above. Non-targeted metabolomics analysis The GR and FGR were analyzed for non-target metabolites by Majorbio Bio-Pharm Technology (Shanghai, China). Briefly, the samples were analyzed using a Thermo UHPLC-Q Exactive HF-X system (Thermo, USA), which was fitted with an ACQUITY HSS T3 column (Waters, USA). The raw data was imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) and ultimately resulting in a data matrix. Subsequently, the software was used for feature peak library identification, matching MS and MS/MS mass spectrometry information with metabolic databases. The MS mass error was set to be less than 10 ppm, and metabolites were identified based on the matching scores from the secondary mass spectrometry. The main databases used include mainstream public databases such as HMDB ( http://www.hmdb.ca/ ) and METLIN ( https://metlin.scripps.edu/ ), KEGG ( http://www.kegg.jp/kegg/kegg1.html ) and Majorbio database. KEGG pathway enrichment analysis of metabolites was performed through the Majorbio cloud platform ( https://www.majorbio.com ) 15 , 16 .The heatmap of differential metabolites was performed by R-3.3.2. Quantitative analysis of metabolite concentration in FGRE The leucinic acid concentration was analyzed by HPLC. The samples were analyzed using an ultraviolet (UV) detector (λ = 205 nm) in an Agilent 1260 HPLC system equipped with an XDB-C18 column (5 µm × 4.6 mm × 150 mm, with a column temperature of 30°C). The mobile phase consisted of 0.1% (NH 4 ) 2 HPO 4 (A) and acetonitrile (B), delivered at a flow rate of 1 mL/min. The gradient program was as follows: 0–15 min, 10% B; 15–20 min, 10%-20% B; 15-50min, 20% B. The samples of GRE and FGRE were pre-processed before determing the concentration of UM5, UM6, UC and UA. A 200 µL aliquot of each sample was taken and mixed with 600 µL of acetonitrile to precipitate the proteins. After centrifugation, the supernatant was collected and evaporated to dryness. The dried samples were then redissolved in 200 µL of a 50% methanol solution and centrifuged at 12,000 rpm for 10 min. The resulting supernatant was collected for analysis using an Triple Quadrupole Liquid Chromatography-Mass Spectrometer (Agilent 6470, USA) equipped with Waters Acquity Premier CSH Phenyl-Hexyl (1.7 µm, 2.1×100 mm) (Waters, USA). The temperatures of the column and autosampler were maintained at 35 ℃. The mobile phase consisted of water with 0.1% (v/v) formic acid (A) and acetonitrile with 0.1% (v/v) formic acid (B), delivered at a flow rate of 0.3 mL/min. The gradient program was as follows: 0–7 min, 5%-60% B; 7–9 min, 60%-95% B; 9–12 min, 95% B; 12-12.5min, 95%-5% B; and 12.5 − 16.5 min, 5% B. An ion source used heated electrospray ionization (ESI) and was set as follows: dry gas flow rate, 5 L/min; dry gas temperature 325 ℃;sheath gas flow rate, 11 L/min; sheath gas temperature, 350 ℃ and capillary pressure, 3500V. The detection used the negative ion scanning mode, with a monitoring mode of Multiple Reaction Monitoring (MRM). Standard UM5, UM6, UC and UA were used for qualitative and quantitative analysis. Molecular Docking and Molecular Dynamics Simulation The structure model of human D-lactate dehydrogenase (hLDHD) was predicted using AlphaFold3, while the structure model of human L-lactate dehydrogenase A (hLDHA) was derived from crystal structure (PDB ID: 4JNK). Preprocessing of both protein structures involved structure optimization, hydrogen atom addition, and charge correction. D-leucinic acid and L-leucinic acid, as the docking ligands, was preprocessed using AutoDock Tools. The docking site for hLDHD was defined based on the position of the D-leucinic acid in the crystal structure of mouse D-lactate dehydrogenase (mLDHD) (PDB ID: 8JDQ). The center coordinates of the docking site were (-2.55, 9.18, 0.21), with a docking radius of 10 Å. For hLDHA, the docking site was defined according to the binding position of the pyruvate in the crystal structure. The center coordinates of the docking site were (52.12, -10.63, 39.63), with a docking radius of 10 Å. Molecular docking procedures were performed using the Autodock Vina. The optimal docking conformation was subjected to molecular dynamics simulation using the Gromacs 2019 on the MaxFlow platform. The AMBER14SB force field was selected for standard residues, while the GAFF2 force field was applied for non-standard residues. The TIP3P model was employed as the water molecule. Solvation was performed to simulate an aqueous environment using explicit water molecules. Sodium (Na⁺) and chloride (Cl⁻) ions were added to neutralize the system charge. The geometry and energy of the system were optimized using the steepest descent algorithm to reach a local energy minimum. Temperature equilibration was subsequently performed in the NVT ensemble at 298 K for 0.5 ns. Finally, a 50 ns molecular dynamic simulation was performed using the NPT ensemble, with the temperature coupling method set to the Berendsen thermostat and the pressure coupling method to the Berendsen barostat. MD simulation analysis was also performed using the MaxFlow platform. Structural analysis and figure preparation were carried out using chimera X. Statistical Analysis The data were analyzed using one-way analysis of variance (ANOVA) with SPSS 22.0 and Origin 9.0 software. Results are presented as the mean ± standard error of the mean (SEM). The Tukey's post hoc test was applied following one-way ANOVA to determine statistically significant differences ( p < 0.05) among different groups. Results Assessment of antioxidant activity and anti-elastase activity of fermented galla rhois gallnut extract The biochemical experiments were used to explore the effect of FGRE on antioxidant and anti-elastase activity (Fig. 1 ). The results demonstrated DPPH radical scavenging activity of 75.74% and elastase inhibition of 58.12% for FGRE. Notably, FGRE exhibited significantly stronger antioxidant and anti-elastase effects compared to GRE ( p < 0.05), suggesting that fermentation enhances the bioactivity of GRE. Effect of fermented galla rhois gallnut extract on cellar mitochondrial energy metabolism The results of CCK-8 showed that different concentration (1%~10%) of GRE and FGRE were no harmful but beneficial to HDF cells survival (Fig. 2 a). To investigate the effects of FGRE on mitochondrial function of HDF cells using Seahorse XF-96. In Fig. 2 b, the OCR of NC group, GRE group and FGRE group were obviously higher than that of UVB group( p < 0.05). Compared with NC group, the level of basal respiration, ATP production and maximal respiration in UVB group were markedly decreased ( p < 0.05), indicated that UVB caused damage to cellular mitochondria (Fig. 2 cde). In addition, the level of spare respiratory capacity in NC and UVB group were no significance (Fig. 2 f). According to S1, 1%~10% FGRE significantly improved the level of cellular basal respiration, ATP production, maximal respiration and spare respiratory capacity compared to UVB group ( p < 0.05). This meaned that 1% FGRE was sufficient to mitigate cellular mitochondrial function disorder by UVB irradiation. Consequently, compared the difference in effect at the same concentration (1%) between FGRE and GRE. The results indicated that GRE and FGRE intervention both effectively enhanced the indicators related to mitochondrial function compared to model group ( p < 0.05). Particularly, FGRE group was significantly more effective than GRE group on cellular basal respiration, ATP production, maximal respiration ( p < 0.05). These results suggested that FGRE could ameliorate the UVB-induced impairment of cellular mitochondrial function better. Analysis of differential metabolites in fermented Galla rhois gallnut extract based on non-targeted metabolomics In order to explore the key active compounds in FGRE, the non-targeted metabolomics analysis of GER and FGRE were performed. The principal component analysis (PCA) of GER and FGRE was presented in Fig. 3 A. The contribution of the first principal component is 68.30%, while the contribution of the second principal component is 4.23%. And FGRE was far from GRE in the first principal component, indicating that the metabolites of FGER were significantly different from GRE. In the volcano plot (Fig. 3 b), there were 236 significant up-regulation metabolites and 451 significant down-regulation metabolites by comparing FGRE and GRE. According to fold change (FC) of metabolites, tripeptide Leu-Ser-Ile content downgraded the most, while Val-Asn content increased most obviously. Comparison based on p -value, the concentration of dipeptide Ala-Leu decreased sharply and the concentration of Cucurbitacin B most significantly up-regulated. For further research, the compounds classification and KEGG enrichment of differential metabolites between FGRE and GRE were analyzed (Fig. 3 cd). The compounds were divided into 15 categories, and most compounds concentrated on amino acids and carboxylic acids. Furthermore, KEGG enrichment analysis suggested that all differential metabolites were enrich in 20 metabolic pathways, and most were enriched in ABC transporters and biosynthesis of cofactors. A few differential metabolites concentrated on metabolic pathways including aminoacyl-tRNA biosynthesis, nucleotide metabolism, D-amino acid metabolism and purine metabolism. The rich factor of valine, leucine and isoleucine biosynthesis was the most in all pathways, indicating that changes of valine, leucine and isoleucine biosynthesis maybe caused by fermentation. In addition, differential metabolites were screened by FC > 2 or < 0.05, and p < 0.05, and the heatmap presented the top 30 metabolites (Fig. 3 e). The obvious reduction metabolites of FGRE were mainly amino acids and small molecule peptides, such as some tripeptides (Leu-Ser-Ile, Ile-Asn-Val and His-Gly-Glu), some dipeptides (Ala-Ile, Ser-Ile, Ala-Leu and Thr-Ile), and some amino acids (L-arogenate and L-aspartic acid). The composition of up-regulation metabolites in FGRE was quite complex. Cyclokievitone, O-desmethylangolensin, and 6-geranylnaringenin belong to flavans and isoflavans, that significantly increased in FGRE group. And potential plant-derived compounds periplocin and cucurbitacin A were promoted the abundance. Some amino acids and peptides were increased after fermentation, such as leucinic acid, Val-Asn, and Arg-Val-Phe. In addition, UroA and UroM5 were also obviusly increased in FGRE, this might be the special metabolites in Galla rhois fermented by Lactiplantibacillus plantarum . Quantitative analysis of metabolite concentration in FGRE Since UroA, UroM5 and leucinic acid were characteristic differential metabolites, the concentration of UroA, its Derivatives and leucinic acid was determined in FGRE (Table 2 ). Although we try to quantify the UroA and its derivatives as much as possible, the UroC and UroM6 were no detection in GER and FGRE. And the results showed that GRE contained 0.01 ng/mL UroM5 and 26.96 µg/mL leucinic acid, while no UroA was detected. Differently, the levels of UroA, UroM5 and leucinic acid all increased in FGRE after fermentation, increased to 0.49 ng/mL,8.35 ng/mL, 97.20 µg/mL. These results indicated that fermentation process involved the transformation of substances. Table 1 This is a table. Tables should be placed in the main text near to the first time they are cited. Sample UroM5 (ng/mL) Uro A (ng/mL) Leucinic acid (µg/mL) GRE 0.01 ± 0.01 - 26.96 ± 0.60 FGRE 8.35 ± 0.93 0.49 ± 0.06 97.20 ± 1.88 Effect of leucinic acid on cellular mitochondrial energy metabolism Related to HPLC-MS and non-targeted metabolomics analysis, leucinic acid, the differential compound in FGRE, was explored the effect on cellular mitochondrial function (Fig. 4 ). The results of CCK-8 presented that the different levels of leucinic acid below 30 µg/mL were non-toxic to HDF cell s, and 0.3 ~ 3 µg/mL leucinic acid obviously increased the cell viability (Fig. 4 a). The effect of different concentration of leucinic acid on mitochondrial respiratory capacity in UVB-induced HDF cells was dose-dependent. The effect of 0.2 µg/mL leucinic acid was no significance with UVB group. Differently, 1 ~ 5 µg/mL leucinic acid obviously increased the OCR of cellular basal respiration, ATP production, and maximal respiration compared to UVB group ( p < 0.05). These results indicated that 1 ~ 5 µg/mL leucinic acid could improve the mitochondrial respiratory metabolism in UVB-induced HDF cells. In addition, 1% FGRE contained about 1 µg/mL leucinic acid, suggesting that leucinic acid was likely to be the key active compounds in FGRE. Lactate dehydrogenase potentially participates in the conversion of leucinic acid Studies have reported that mouse D-lactate dehydrogenase (mLDHD) catalyzes the conversion of D-leucinic acid 17 . Moreover, mLDHD exhibits over 80% sequence similarity with human D-lactate dehydrogenase (hLDHD) as predicted by AlphaFold3, and their active site structures are essentially conserved (Fig. 5 a). Consequently, computational simulation was performed between hLDHD and D-leucinic acid. The docking score was − 8.76, with the binding free energy of -52.16 kcal/mol (Table 2 ). The binding conformation indicated that subsite, which accommodates the glycolate group of the substrate, comprises Arg370, His421, and Glu465, while subsite, which binds the hydrophobic moiety of the substrate, includes Leu101, Trp374, Ser388, and Ile430 (Fig. 5 b). A hydrogen bond was formed between Arg370 and the substrate. The binding conformation of D-leucinic acid is similar for hLDHD and mLDHD. These results suggest that hLDHD may plays a potential role in catalyzing the conversion of D-leucinic acid. It was further hypothesized that the conversion of L-leucinic acid might also be mediated by L-lactate dehydrogenase. In skin, L-lactate dehydrogenase primarily exists as the LDH-5 18 , a tetrameric protein encoded by the ldha gene. Therefore, computational simulations were performed using LDHA subunit (hLDHA) with L-leucinic acid. The docking score was − 7.33, with the binding free energy of -128.29 kcal/mol (Table 2 ), indicating the strong affinity between L-leucinic acid and hLDHA. The binding conformation revealed that Arg105 and Arg168 formed hydrogen bonds with the substrate (Fig. 5 c), with Arg105 potentially playing a crucial role in stabilizing the transition state of the hydride transfer reaction 19 . These results suggest that hLDHA may potentially participates in the conversion of L-leucinic acid. Table 2 Molecular docking and molecular dynamics simulation results of leucinic acid with LDH. Substrate Protein Docking score Binding free energy (kcal/mol) D-leucinic acid hLDHD -8.76 -52.16 L-leucinic acid hLDHA -7.33 -128.29 Discussion Galla rhios is primarily applied externally in clinical practice for burn management and tissue repair facilitation, in addition to its internal administration as a traditional Chinese medicinal agent. Although microbial fermentation is widely employed in modifying traditional Chinese medicinal materials, the application of Lactobacillus strains for biotransformation of GRE to potentiate their bioactive potential remains underexplored in pharmacological research. Thus, we fermented and obtained FGRE by Lactiplantibacillus plantarum . Experimental analysis demonstrated that FGRE had higher antioxidant and anti-aging activity than GRE in vitro , and significantly enhanced the mitochondrial respiratory capacity in UVB-induced HDF cells. Compositional profiling identified leucinic acid as the primary bioactive constituent responsible for this effect, along with trace amounts of UroM5 and UroA. As a high-turnover organ, the skin's continuous cellular renewal and proliferation depend on mitochondrial respiration to meet its energy demands. However, both chronological aging (time-induced) and extrinsic aging, which are caused by environmental stressors including UV radiation, cigarette smoke, and air pollution, impair mitochondrial bioenergetic function 20 . Mitochondrial dysfunction is specified by decreased diminished per-mitochondrion respiratory capacity and membrane depolarization along with elevated oxidant stress, which is recognized as both a cause and consequence of cellular senescence 21 . This means that cellular senescence progressively exacerbates mitochondrial dysfunction, while the accumulation of functionally impaired mitochondria further contributes to skin aging. In this study, we confirmed the bioactivity of FGRE on DPPH radical scavenging and elastase inhibition by biochemical experiments in vitro , which was also obviously better than that of GRE. And FGRE was considered as having the ability of anti-oxidant and antiaging, and the effect of anti-aging was possibly related to mitochondrial function. Thus, we employed the Seahorse XF analyzer to dynamically monitor cellular energetics in UVB-irradiated HDFs, assessing the effect of FGRE on mitochondrial bioenergetics. During the experiments, mitochondrial inhibitors (Oligomycin, FCCP, Rotenone / antimycin A) were sequentially added to obtain the key bioenergetic metrics including basal respiration, ATP production, maximal respiration and spare respiratory capacity 22 . Basal respiration showed the energetic demand of cells under basal conditions, and oxygen consumption of basal respiration is mainly used to meet ATP synthesis. Thus, the oligomycin-induced OCR attenuation specifically corresponded to ATP-linked respiratory flux, with this parameter quantitatively reflecting mitochondrial ATP production capacity 23 . Maximal respiration represented the maximum capacity that the electron respiratory chain can achieve, and Spare respiratory capacity defined as the difference between maximal and basal respiration. These metrics reflected the capability of the cells to respond to changes in energetic demand and indicates the fitness of the cells 24 . Significant alterations in these bioenergetic parameters emerge following mitochondrial damage induced by oxidative stress or UV irradiation. Zheng’s research showed that the OCR of basal respiration, maximal respiration and ATP production in UVB-induced HaCaT keratinocytes was obviously lower than that in untreated group 25 . In our study, similar results were also found in HDFs. In addition, FGRE and GRE both significantly improved the mitochondrial bioenergetic metrics compared to UVB group, indicated the improvement on mitochondrial respiratory metabolism. Specially, FGRE presented the better effect on mitochondrial respiration compared to unfermented GRE, which also indicated the enhancement of bioactivity by fermentation. According to non-targeted metabolomics analysis, the main distinguishing metabolites between FGRE and GRE, generated through fermentation by Lactiplantibacillus plantarum PIAS240228, primarily comprise short peptides and free-form amino acids. Studies demonstrated that Lactiplantibacillus plantarum fermentation of milk or milk protein generated differential metabolites, also predominantly short peptides and bioactive amino acids 26 . Meanwhile, the increased concentration of amino acids not only promotes strain growth but also enhances nutritional value and functional efficacy 27 . KEGG enrichment analysis revealed that fermentation processes significantly modulated the branched-chain amino acids (BCAAs, including valine, leucine and isoleucine) biosynthesis pathway. The supplementation of BCAAs is often beneficial to energy expenditure 28 . This occurs because acetyl-CoA, the catabolic products of BCAAs, drives tricarboxylic acid (TCA) cycle and oxidative phosphorylation to provide cellular energy 29 . Leucinic acid is considered as the end metabolite of leucine, which can be converted from leucine to α-ketoiosocaparoic acid (α-KIC) and further converted from α-KIC (Fig. 6 a). This transformantion can be conducted by Lactococcus with aminotransferase and hydroxyacid dehydrogenase 30 . Thus, the increase of leucinic acid in FGRE is due to the fermentation by Lactiplantibacillus plantarum . Some research suggested that the reaction between leucinic acid and α-KIC was reversible 31 . Our results of molecular docking showed that human lactate dehydrogenase may can transform leucinic acid into α-KIC. Then, α-KIC further converted into acetyl-CoA, and participated in TCA cycle to regulate cellular energy metabolism 29 . According to our results, leucinic acid also has been proven to enhance mitochondrial energy metabolism in UVB-induced HDF cells. What’s more, 1 µg/mL leucinic acid (equivalent to the concentration in 1% FGRE) ameliorated the mitochondrial respiratory dysfunction in HDF cells, suggesting that leucinic acid was considered as the principal bioactive compounds in FGRE. In addition, the relative abundance of BCAA-rich tripeptides and dipeptides (e.g., Leu-Ser-Ile, Ile-Asn-Val, Ala-Leu, Ala-Ile) in FGRE showed marked reduction. This decrease correlated with elevated levels of Leu, Val-Asn and Arg-Val-Phe. Val-Asn functions as a dipeptidyl peptidase IV inhibitor demonstrating hypoglycemic activity 32 . Arg-Val-Phe was demonstrated to protects SH-SY5Y cells from cell damage by reducing ROS accumulation, and thereby maintaining membrane integrity, Ca 2+ homeostasis, and changing the mitochondrial transmembrane potential 33 . These compounds have the potential to maintain mitochondrial homeostasis. A study indicated GRE significantly enhanced collagen biosynthesis pathways, thereby accelerating wound closure processes due to its rich tannins and flavonoids 34 . However, its potential modulatory effects on mitochondrial dynamics (including biogenesis and energy metabolism) remain poorly characterized in extant literature. Differently, UroA, a gut microbiota-derived metabolite of ellagic acid, has been reported to induce mitophagy and stimulate mitochondrial biogenesis 35 . Lactiplantibacillus plantarum , Bifidobacterium pseudocatenulatum , Streptococcus thermophilus , and related gut microbiota have been shown to transform either ellagitannins or EA into UroA through microbial fermentation 36 . Thus, we utilized Lactiplantibacillus plantarum to ferment Galla rhois , and the presence of UroM5 and UroA was found after fermentation. This indicated that the ellagic acid was biotransformed into UroM5, and further produce UroA by a series of dehydroxylation reaction (Fig. 6 b) 37 . The undetectable levels of UroM6 and UroC likely reflect sub-threshold metabolite concentrations rather than non-occurrence of the reaction. Notably, the limited yields of UroA and UroM5 in FGRE are potentially attributed to their limited aqueous solubility, as evidenced by Hu’s research 38 . While UroA has been well-characterized as a mitochondrial modulator with demonstrated regulatory effects, the bioactivity profile of its structural analog UroM5 remains largely unexplored in mitochondrial contexts 39 . Li’s study showed that UroM5 demonstrated superior antioxidant capacity and α-glucosidase inhibition potency compared to UroA at concentrations 40 . Despite the notably low concentrations of UroA and UroM5 in FGRE, their antioxidant activity might still be effective. Our study demonstrated that relative equivalent of UroA (0.005 ng/mL) and UroM5 (0.08 ng/mL) in 1%FGRE could significantly inhibit the ROS production in UVB-induced HaCaT cells (S2), suggesting that they take effect by inhibiting ROS production and reducing oxidative stress. Therefore, although the actual effect of UroA and UroM5 on mitochondrial function may be not better than leucinic acid because of concentration limit in FGRE, they still were the effective compounds in FGRE. Conclusion In this study, we fermented GRE by using Lactiplantibacillus plantarum , and found that FGRE had better antioxidant and anti-elastase activity than GRE by biochemical experiments. The obtained FGRE was also proven to enhance mitochondrial respiratory metabolism in UVB-irradiated HDFs. Combined with metabolomics analysis, FGRE was found to contain leucinic acid, UroM5 and UroA. And the cellular experiments confirmed the function of leucinic acid on mitochondrial respiratory improvement in UVB-induced HDF cells, which was identified as the predominant bioactive compound in FGRE. Therefore, FGRE with bioactive constituents (leucine and urolithins), as a novel nutricosmetic ingredient, establishes a novel solution for mitochondrial dysfunction in skin aging. Declarations Conflict of Interest Statement There are no conflicts of interest to declare. Funding No Funding. Author Contribution J.W., N. W. D.C, Y.Z and X.Z.(Xiao Zhang) finished the experiments and data curation. J.W. and X.Z.(Xiao Zhang) wrote the main manuscript text andprepared figures and tables. J.S, G.L. were responsible for validation. X.L. was responsible for project administration. H. H. and X.Z. (Xiangna Zhang) were responsible for supervision. All authors reviewed the manuscript. Acknowledgement This work was supported by a grant from Proya International Academy of Sciences. The authors would like to thank PROYA Cosmetics Co. Ltd for excellent technical and financial support. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Gu, Y., Han, J., Jiang, C. & Zhang, Y. Biomarkers, oxidative stress and autophagy in skin aging. Ageing Res. Rev. 59 , 101036. https://doi.org/10.1016/j.arr.2020.101036 (2020). Rittié, L. & Fisher, G. J. Natural and sun-induced aging of human skin. Cold Spring Harb Perspect. Med. 5 , a015370 (2015). Martic, I., Papaccio, F., Bellei, B. & Cavinato, M. Mitochondrial dynamics and metabolism across skin cells: implications for skin homeostasis and aging. Front. Physiol. 14 , 1284410 (2023). Sreedhar, A., Aguilera-Aguirre, L. & Singh, K. K. Mitochondria in skin health, aging, and disease. Cell Death Dis. 11 , 444 (2020). Stout, R. & Birch-Machin, M. Mitochondria's Role in Skin Ageing. 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Zheng, Q. et al. Autophagy-Enhancing Properties of Hedyotis diffusa Extracts in HaCaT Keratinocytes: Potential as an Anti-Photoaging Cosmetic Ingredient. Molecules 30 10.3390/molecules30020261 (2025). Mazzei, P. & Piccolo, A. 1H HRMAS-NMR metabolomic to assess quality and traceability of mozzarella cheese from Campania buffalo milk. Food Chem. 132 , 1620–1627. https://doi.org/10.1016/j.foodchem.2011.11.142 (2012). Wang, Y. et al. Metabolites profile analysis of fermented milk with Lactobacillus plantarum P-8 based on ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS). Science Technol. Food Industry , 152–160 (2019). Yoneshiro, T. et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature 572 , 614–619. 10.1038/s41586-019-1503-x (2019). Sivanand, S. & Vander Heiden, M. G. Emerging Roles for Branched-Chain Amino Acid Metabolism in Cancer. Cancer Cell. 37 , 147–156. 10.1016/j.ccell.2019.12.011 (2020). Chambellon, E. et al. The D-2-hydroxyacid dehydrogenase incorrectly annotated PanE is the sole reduction system for branched-chain 2-keto acids in Lactococcus lactis. J. Bacteriol. 191 , 873–881. 10.1128/jb.01114-08 (2009). Blanchard, M. & Green, D. E. L-Hydroxy acid oxidase. J. Biol. Chem. 163 , 137–144 (1946). Lan, V. T. T. et al. Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor. Food Chem. 175 , 66–73. https://doi.org/10.1016/j.foodchem.2014.11.131 (2015). Cheng, Y. et al. Protective effects of a wheat germ peptide (RVF) against H2O2-induced oxidative stress in human neuroblastoma cells. Biotechnol. Lett. 36 , 1615–1622. 10.1007/s10529-014-1521-6 (2014). Park, H. H. et al. Potential Wound Healing Activities of Galla Rhois in Human Fibroblasts and Keratinocytes. Am. J. Chin. Med. 43 , 1625–1636. 10.1142/s0192415x15500925 (2015). Faitg, J., D’Amico, D., Rinsch, C. & Singh, A. Mitophagy Activation by Urolithin A to Target Muscle Aging. Calcif. Tissue Int. 114 , 53–59. 10.1007/s00223-023-01145-5 (2024). Zhang, M. et al. Ellagic acid and intestinal microflora metabolite urolithin A: A review on its sources, metabolic distribution, health benefits, and biotransformation. Crit. Rev. Food Sci. Nutr. 63 , 6900–6922. 10.1080/10408398.2022.2036693 (2023). Banc, R., Rusu, M. E., Filip, L. & Popa, D. S. The Impact of Ellagitannins and Their Metabolites through Gut Microbiome on the Gut Health and Brain Wellness within the Gut–Brain Axis. Foods 12 , 270 (2023). Hu, Y. et al. Liposomes encapsulation by pH driven improves the stability, bioaccessibility and bioavailability of urolithin A: A comparative study. Int. J. Biol. Macromol. 253 , 127554. https://doi.org/10.1016/j.ijbiomac.2023.127554 (2023). D’Amico, D. et al. Impact of the Natural Compound Urolithin A on Health, Disease, and Aging. Trends Mol. Med. 27 , 687–699. https://doi.org/10.1016/j.molmed.2021.04.009 (2021). Li, Z. R. et al. Comparative Study on the Antioxidative Effects and α-Glucosidase Inhibitory Potential In Vitro among Ellagic Acid and Its Metabolites Urolithins. J. Agric. Food Chem. 72 , 26711–26721. 10.1021/acs.jafc.4c06542 (2024). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials10.31.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Mar, 2026 Reviews received at journal 01 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 21 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 16 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 02 Feb, 2026 Editor invited by journal 28 Jan, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 23 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Ltd","correspondingAuthor":true,"prefix":"","firstName":"Hu","middleName":"","lastName":"Huang","suffix":""},{"id":584883002,"identity":"fd918990-d0c8-4aab-abe9-b62d53305492","order_by":9,"name":"Xiangnan Zhang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiangnan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-21 01:23:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8653896/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8653896/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101869544,"identity":"fbff329d-f1ca-4b0d-8aa4-cd48b0258c1d","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148750,"visible":true,"origin":"","legend":"\u003cp\u003eThe bioactivity of FGRE determined by biochemical experiments. (a) DPPH radical scavenging assay; (b) Anti-elastase activity.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/76714cf48c7d273f1db326fa.png"},{"id":101869548,"identity":"6f656bed-1297-4bdf-b954-df35b5753f42","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334033,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of fermented Galla rhois gallnut extract on cellular mitochondrial function. (a) Cell viability; (b) Cellular OCR curve; (c) Basal respiration; (d) ATP production; (e) Maximal respiration; (f) Spare respiration capacity.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/f00d4629c416063f7acd8694.png"},{"id":101869545,"identity":"6f371d57-7926-4905-b3de-f5f5f70e7dcb","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":710657,"visible":true,"origin":"","legend":"\u003cp\u003eThe non-targeted metabolomics analysis of fermented Galla rhois gallnut extract. (A) Principal component analysis; (B) Volcano plot; (C) Compounds classification; (D) KEGG enrichment analysis; (E) Heatmap.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/d7e05dccb5a705c6cb2519eb.png"},{"id":101881777,"identity":"283e7878-73d7-455d-8cd9-e3c3381a7ed4","added_by":"auto","created_at":"2026-02-04 15:16:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":356601,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of leucinic acid on cellular mitochondrial function. (a) Cell viability; (b) Cellular OCR curve; (c) Basal respiration; (d) ATP production; (e) Maximal respiration; (f) Spare respiration capacity.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/22beee7e44d33dec5baad471.png"},{"id":101869546,"identity":"31a216c8-f7ae-4f48-8bfd-eb1de4e72c48","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":307082,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking results of leucinic acid with LDH: (a) Comparison of the active pockets between hLDHD (blue) and mLDHD (yellow); (b) Docking conformation of hLDHD (gray) with D-leucinic acid (pink); (c) Docking conformation of hLDHA (gray) with L-leucinic acid (pink). The yellow sticks represent the cofactor.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/5f4904e8043abf9a5295df11.png"},{"id":101869547,"identity":"28db0b14-fd1f-4c80-b98b-8d0785fd3d78","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":166093,"visible":true,"origin":"","legend":"\u003cp\u003eThe potential metabolic pathway of differential metabolites: (a) Leucinic acid; (b) UroAand its metabolites.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/9a00534be1f3bca7b4c5577e.png"},{"id":102397215,"identity":"7534df75-c970-4bd1-a822-981adccbc41b","added_by":"auto","created_at":"2026-02-11 10:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2741722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/d6f7225f-7d5a-4674-8c1e-d545740d9667.pdf"},{"id":101869550,"identity":"d398d9df-8b03-44ee-ace4-4d91e3c46af6","added_by":"auto","created_at":"2026-02-04 13:05:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":313432,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials10.31.docx","url":"https://assets-eu.researchsquare.com/files/rs-8653896/v1/9b6e1069d1a254a9a5666670.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Mitochondrial Respiratory Metabolism in UVB-Damaged HDF cells Based on Metabolomics Analysis of Fermented Galla rhois gallnut Extract","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin aging is a complex biological process, which is intuitively presented as increased wrinkles, decreased elasticity, laxity, enlarged pores and pigmentation of skin \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In addition to endogenous aging, external environmental factors, especially ultraviolet (UV) radiation, accelerate the aging process \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Recent years, mitochondrial homeostasis is considered crucial for skin barrier and skin health \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Both natural aging and photoaging cause the decline of mitochondrial function, the accumulation of mitochondrial DNA deletions, and the increase of reactive oxygen species production \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. That leads to oxidative stress and insufficient energy metabolism in skin cells, thus aggravating the skin aging \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, improving cellular mitochondrial function is considered as a good method to alleviate skin aging.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGalla rhois\u003c/em\u003e, a kind of traditional Chinese medicine, is the gall caused by the Chinese aphid, \u003cem\u003eSchlechtendalia chinensis\u003c/em\u003e (Bell), on the leaves of Rhus chinensis \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Some studies reported that \u003cem\u003eGalla rhois\u003c/em\u003e had anti-inflammatory, hemostatic and analgesic properties, that could be used to promote clotting following traumatic injuries, to treat burns and skin infections \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A study showed that \u003cem\u003eGalla rhois\u003c/em\u003e extract could inhibit the elastase and tyrosinase activity in vitro, and reduced the expression of MMP-1 in UVA-induced fibroblasts \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFermentation is the main technology for the food and plant ingredient processing. \u003cem\u003eLactobacillaceae\u003c/em\u003e are major fermenting organisms frequently found in traditional fermentations, which can transform large molecules into more easily absorbed and bioactive small molecules \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e fermented \u003cem\u003eRhodiola rosea\u003c/em\u003e can activate the Nrf2/Keap1 signaling pathway and protect fibroblasts from oxidative stress caused by UVA, and the effect is significantly better than unfermented \u003cem\u003eRhodiola rosea\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In addition, lactic acid bacteria own metabolites or their extracts also have beneficial effect on skin. For example, extracellular vesicles, produced by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, reduced wrinkle formation, improve skin elasticity, increase skin moisture content, improve skin density, as well as inhibit skin pigmentation caused by aging in clinical trial \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, it is a good way to enhance the bioactivity of \u003cem\u003eGalla rhois\u003c/em\u003e by lactic acid bacteria fermentation.\u003c/p\u003e \u003cp\u003eIn this study, we developed a kind of fermented ingredient \u003cem\u003eGalla rhois\u003c/em\u003e gallnut extract (GRE) fermented by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e. The FGRE were was systematically evaluated for its modulatory effects on mitochondrial function in UVB-irradiated human dermal fibroblasts (HDFs). The non-targeted and targeted metabolomics were used to analyze the metabolites of FGRE, and combined with cell experiments to determine the key active compounds. Our results indicated that FGRE could improve the mitochondrial function and ameliorate UVB-indeced photoaging, which provided a novel ingredient for skin care.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eGRE was purchased from Focusherb Co. Ltd (Shanxi, China). \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e PIAS240228 isolated from fermented food by our laboratory. Fermented nutrient salt NS01 was obtained from AngelYeast Co., Ltd. Leucine acid and leucinic acid were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Urolithin C (UroC, purity of 95%), urolithin A (UroA, purity of 98%) were obtained from Sigma (Shanghai, China). Urolithin M5 (UroM5, purity of 96%) and urolithin M6 (UroM6, purity of 92%) were purchased from NatureStandard Shanghai Standard Technology Co., Ltd. Human dermal fibroblasts (HDFs) purchased from Guangdong BioCell Biotech Co., Ltd (Guangdong, China). All other chemicals of analytical grade were obtained from Sigma (Shanghai, China), unless otherwise noted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFermentation Galla rhois gallnut extract\u003c/h3\u003e\n\u003cp\u003eThe strain \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e PIAS240228 was inoculated into MRS medium for 2\u0026thinsp;~\u0026thinsp;3 times activation. Then, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e PIAS was inoculated in GRE medium with 2% inoculation and fermented for 36\u0026thinsp;~\u0026thinsp;48h with constant pH\u0026thinsp;=\u0026thinsp;5.5. The GRE medium contained 100 mg/L GRE, 10g/L glucose and 10g/L fermented nutrient salt NS01 dissolved in 0.03 M potassium phosphate buffer (PBS, pH\u0026thinsp;=\u0026thinsp;7.2). The fermentation broth was homogenized and broken by high pressure homogenization. Then, the final FGRE samples was obtained by centrifugating at 11000\u0026times;g for 10 min and concentrating 3\u0026thinsp;~\u0026thinsp;4 times. GRE samples for follow-up experiments was also obtained by centrifugating at 11000\u0026times;g for 10 min and concentrating 3\u0026thinsp;~\u0026thinsp;4 times from GRE medium. GRE and FGRE were further explored the effect and compound composition.\u003c/p\u003e\n\u003ch3\u003eBiochemical Experiments\u003c/h3\u003e\n\u003cp\u003eThe antioxidant activity of FGRE was determined by DPPH radical scavenging assay according to the method described by Lee et al \u003csup\u003e12\u003c/sup\u003e. Briefly, the samples were mixed with an equal amount of DPPH solution and left to stand for 30 min at room temperature in the dark. And then determine the absorbance of the resulting solution at 517 nm. Water was taken as control, while 12.5 \u0026micro;g/mL ascorbic acid (Vc) was taken as positive control. The scavenging ability was defined as: Scavenging activity (%) = (A\u003csub\u003econtrol\u003c/sub\u003e\u0026minus;A\u003csub\u003esample\u003c/sub\u003e)/A\u003csub\u003econtrol\u003c/sub\u003e \u0026times; 100%.\u003c/p\u003e \u003cp\u003eThe anti-elastase activity was measured according to Shirzad\u0026rsquo;s research with slight modification \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. 420 \u0026micro;L 0.05 M Tris-HCl (pH\u0026thinsp;=\u0026thinsp;8), 180 \u0026micro;L of samples and 150 mL elastase enzyme (600 mU/mL) were mixed together with 15 min incubation in room temperature (25\u0026deg;C). 150 \u0026micro;L N-succinyl-Ala-Ala-Ala-Val-p-nitroanilide substrate (1.015 mmol/L) was added to each tube. After 20 min incubation in room temperature (25\u0026deg;C), absorbance was read at 410 nm. The epigallocatechin gallate (EGCG, 500 \u0026micro;g/mL) was used as positive control. The percentage inhibition was calculated as follows: Inhibition (%) = [(A\u003csub\u003econtrol\u003c/sub\u003e\u0026ndash;A\u003csub\u003esample\u003c/sub\u003e)/A\u003csub\u003econtrol\u003c/sub\u003e] \u0026times;100%.\u003c/p\u003e\n\u003ch3\u003eCellular mitochondrial energy metabolism assessment by Seahorse XF Cell Mito Stress Test\u003c/h3\u003e\n\u003cp\u003eHDFs were uesd to explore the effcet of FGRE on mitochondrial. First, Determine the toxicity of different samples on HDF cells using the CCK-8 experimental method. Then, the experimental groups and treatment were performed as following. First day, all HDF cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37℃ for 24h. Then, the cells in control group (NC) and UVB group were still cultured in DMEM medium supplemented with 10% fetal bovine serum for 24h. And GRE group and FGRE group were cultured in 1% GRE and 1% FGRE for 24 h, respectively. At third day, the cells in UVB, GRE and FGRE groups were washed 2\u0026thinsp;~\u0026thinsp;3 times with PBS and exposed to UVB (0.30mW/cm\u003csup\u003e2\u003c/sup\u003e) in PBS for 10 min, while NC group with no UVB exposure. The oxygen consumption rate (OCR) in HDF cells was determined by Seahorse Extracellular Flux (XF) analyzer (Seahorse Bioscience, USA) and analyzed the basal respiration, ATP production, maximal respiration and spare respiratory capacity according to Wang\u0026rsquo;s research\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Further, 0.3 \u0026micro;g/mL leucine, UroM5 and UroA were also explored the effect on mitochondrial function by the methods as mentioned above.\u003c/p\u003e\n\u003ch3\u003eNon-targeted metabolomics analysis\u003c/h3\u003e\n\u003cp\u003eThe GR and FGR were analyzed for non-target metabolites by Majorbio Bio-Pharm Technology (Shanghai, China). Briefly, the samples were analyzed using a Thermo UHPLC-Q Exactive HF-X system (Thermo, USA), which was fitted with an ACQUITY HSS T3 column (Waters, USA). The raw data was imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) and ultimately resulting in a data matrix. Subsequently, the software was used for feature peak library identification, matching MS and MS/MS mass spectrometry information with metabolic databases. The MS mass error was set to be less than 10 ppm, and metabolites were identified based on the matching scores from the secondary mass spectrometry. The main databases used include mainstream public databases such as HMDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hmdb.ca/\u003c/span\u003e\u003cspan address=\"http://www.hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and METLIN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metlin.scripps.edu/\u003c/span\u003e\u003cspan address=\"https://metlin.scripps.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp/kegg/kegg1.html\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp/kegg/kegg1.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Majorbio database. KEGG pathway enrichment analysis of metabolites was performed through the Majorbio cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.majorbio.com\u003c/span\u003e\u003cspan address=\"https://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.The heatmap of differential metabolites was performed by R-3.3.2.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative analysis of metabolite concentration in FGRE\u003c/h2\u003e \u003cp\u003eThe leucinic acid concentration was analyzed by HPLC. The samples were analyzed using an ultraviolet (UV) detector (λ\u0026thinsp;=\u0026thinsp;205 nm) in an Agilent 1260 HPLC system equipped with an XDB-C18 column (5 \u0026micro;m \u0026times; 4.6 mm \u0026times; 150 mm, with a column temperature of 30\u0026deg;C). The mobile phase consisted of 0.1% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (A) and acetonitrile (B), delivered at a flow rate of 1 mL/min. The gradient program was as follows: 0\u0026ndash;15 min, 10% B; 15\u0026ndash;20 min, 10%-20% B; 15-50min, 20% B.\u003c/p\u003e \u003cp\u003eThe samples of GRE and FGRE were pre-processed before determing the concentration of UM5, UM6, UC and UA. A 200 \u0026micro;L aliquot of each sample was taken and mixed with 600 \u0026micro;L of acetonitrile to precipitate the proteins. After centrifugation, the supernatant was collected and evaporated to dryness. The dried samples were then redissolved in 200 \u0026micro;L of a 50% methanol solution and centrifuged at 12,000 rpm for 10 min. The resulting supernatant was collected for analysis using an Triple Quadrupole Liquid Chromatography-Mass Spectrometer (Agilent 6470, USA) equipped with Waters Acquity Premier CSH Phenyl-Hexyl (1.7 \u0026micro;m, 2.1\u0026times;100 mm) (Waters, USA). The temperatures of the column and autosampler were maintained at 35 ℃. The mobile phase consisted of water with 0.1% (v/v) formic acid (A) and acetonitrile with 0.1% (v/v) formic acid (B), delivered at a flow rate of 0.3 mL/min. The gradient program was as follows: 0\u0026ndash;7 min, 5%-60% B; 7\u0026ndash;9 min, 60%-95% B; 9\u0026ndash;12 min, 95% B; 12-12.5min, 95%-5% B; and 12.5\u0026thinsp;\u0026minus;\u0026thinsp;16.5 min, 5% B. An ion source used heated electrospray ionization (ESI) and was set as follows: dry gas flow rate, 5 L/min; dry gas temperature 325 ℃;sheath gas flow rate, 11 L/min; sheath gas temperature, 350 ℃ and capillary pressure, 3500V. The detection used the negative ion scanning mode, with a monitoring mode of Multiple Reaction Monitoring (MRM). Standard UM5, UM6, UC and UA were used for qualitative and quantitative analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Docking and Molecular Dynamics Simulation\u003c/h3\u003e\n\u003cp\u003eThe structure model of human D-lactate dehydrogenase (hLDHD) was predicted using AlphaFold3, while the structure model of human L-lactate dehydrogenase A (hLDHA) was derived from crystal structure (PDB ID: 4JNK). Preprocessing of both protein structures involved structure optimization, hydrogen atom addition, and charge correction. D-leucinic acid and L-leucinic acid, as the docking ligands, was preprocessed using AutoDock Tools. The docking site for hLDHD was defined based on the position of the D-leucinic acid in the crystal structure of mouse D-lactate dehydrogenase (mLDHD) (PDB ID: 8JDQ). The center coordinates of the docking site were (-2.55, 9.18, 0.21), with a docking radius of 10 \u0026Aring;. For hLDHA, the docking site was defined according to the binding position of the pyruvate in the crystal structure. The center coordinates of the docking site were (52.12, -10.63, 39.63), with a docking radius of 10 \u0026Aring;. Molecular docking procedures were performed using the Autodock Vina.\u003c/p\u003e \u003cp\u003eThe optimal docking conformation was subjected to molecular dynamics simulation using the Gromacs 2019 on the MaxFlow platform. The AMBER14SB force field was selected for standard residues, while the GAFF2 force field was applied for non-standard residues. The TIP3P model was employed as the water molecule. Solvation was performed to simulate an aqueous environment using explicit water molecules. Sodium (Na⁺) and chloride (Cl⁻) ions were added to neutralize the system charge. The geometry and energy of the system were optimized using the steepest descent algorithm to reach a local energy minimum. Temperature equilibration was subsequently performed in the NVT ensemble at 298 K for 0.5 ns. Finally, a 50 ns molecular dynamic simulation was performed using the NPT ensemble, with the temperature coupling method set to the Berendsen thermostat and the pressure coupling method to the Berendsen barostat. MD simulation analysis was also performed using the MaxFlow platform. Structural analysis and figure preparation were carried out using chimera X.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using one-way analysis of variance (ANOVA) with SPSS 22.0 and Origin 9.0 software. Results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The Tukey's post hoc test was applied following one-way ANOVA to determine statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among different groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of antioxidant activity and anti-elastase activity of fermented galla rhois gallnut extract\u003c/h2\u003e \u003cp\u003eThe biochemical experiments were used to explore the effect of FGRE on antioxidant and anti-elastase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results demonstrated DPPH radical scavenging activity of 75.74% and elastase inhibition of 58.12% for FGRE. Notably, FGRE exhibited significantly stronger antioxidant and anti-elastase effects compared to GRE (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that fermentation enhances the bioactivity of GRE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of fermented galla rhois gallnut extract on cellar mitochondrial energy metabolism\u003c/h2\u003e \u003cp\u003eThe results of CCK-8 showed that different concentration (1%~10%) of GRE and FGRE were no harmful but beneficial to HDF cells survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To investigate the effects of FGRE on mitochondrial function of HDF cells using Seahorse XF-96. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the OCR of NC group, GRE group and FGRE group were obviously higher than that of UVB group(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with NC group, the level of basal respiration, ATP production and maximal respiration in UVB group were markedly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicated that UVB caused damage to cellular mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e cde). In addition, the level of spare respiratory capacity in NC and UVB group were no significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eAccording to S1, 1%~10% FGRE significantly improved the level of cellular basal respiration, ATP production, maximal respiration and spare respiratory capacity compared to UVB group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This meaned that 1% FGRE was sufficient to mitigate cellular mitochondrial function disorder by UVB irradiation. Consequently, compared the difference in effect at the same concentration (1%) between FGRE and GRE. The results indicated that GRE and FGRE intervention both effectively enhanced the indicators related to mitochondrial function compared to model group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Particularly, FGRE group was significantly more effective than GRE group on cellular basal respiration, ATP production, maximal respiration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggested that FGRE could ameliorate the UVB-induced impairment of cellular mitochondrial function better.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of differential metabolites in fermented\u003c/b\u003e \u003cb\u003eGalla rhois\u003c/b\u003e \u003cb\u003egallnut extract based on non-targeted metabolomics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to explore the key active compounds in FGRE, the non-targeted metabolomics analysis of GER and FGRE were performed. The principal component analysis (PCA) of GER and FGRE was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The contribution of the first principal component is 68.30%, while the contribution of the second principal component is 4.23%. And FGRE was far from GRE in the first principal component, indicating that the metabolites of FGER were significantly different from GRE. In the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), there were 236 significant up-regulation metabolites and 451 significant down-regulation metabolites by comparing FGRE and GRE. According to fold change (FC) of metabolites, tripeptide Leu-Ser-Ile content downgraded the most, while Val-Asn content increased most obviously. Comparison based on \u003cem\u003ep\u003c/em\u003e-value, the concentration of dipeptide Ala-Leu decreased sharply and the concentration of Cucurbitacin B most significantly up-regulated.\u003c/p\u003e \u003cp\u003eFor further research, the compounds classification and KEGG enrichment of differential metabolites between FGRE and GRE were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e cd). The compounds were divided into 15 categories, and most compounds concentrated on amino acids and carboxylic acids. Furthermore, KEGG enrichment analysis suggested that all differential metabolites were enrich in 20 metabolic pathways, and most were enriched in ABC transporters and biosynthesis of cofactors. A few differential metabolites concentrated on metabolic pathways including aminoacyl-tRNA biosynthesis, nucleotide metabolism, D-amino acid metabolism and purine metabolism. The rich factor of valine, leucine and isoleucine biosynthesis was the most in all pathways, indicating that changes of valine, leucine and isoleucine biosynthesis maybe caused by fermentation.\u003c/p\u003e \u003cp\u003eIn addition, differential metabolites were screened by FC\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.05, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and the heatmap presented the top 30 metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The obvious reduction metabolites of FGRE were mainly amino acids and small molecule peptides, such as some tripeptides (Leu-Ser-Ile, Ile-Asn-Val and His-Gly-Glu), some dipeptides (Ala-Ile, Ser-Ile, Ala-Leu and Thr-Ile), and some amino acids (L-arogenate and L-aspartic acid). The composition of up-regulation metabolites in FGRE was quite complex. Cyclokievitone, O-desmethylangolensin, and 6-geranylnaringenin belong to flavans and isoflavans, that significantly increased in FGRE group. And potential plant-derived compounds periplocin and cucurbitacin A were promoted the abundance. Some amino acids and peptides were increased after fermentation, such as leucinic acid, Val-Asn, and Arg-Val-Phe. In addition, UroA and UroM5 were also obviusly increased in FGRE, this might be the special metabolites in \u003cem\u003eGalla rhois\u003c/em\u003e fermented by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative analysis of metabolite concentration in FGRE\u003c/h2\u003e \u003cp\u003eSince UroA, UroM5 and leucinic acid were characteristic differential metabolites, the concentration of UroA, its Derivatives and leucinic acid was determined in FGRE (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although we try to quantify the UroA and its derivatives as much as possible, the UroC and UroM6 were no detection in GER and FGRE. And the results showed that GRE contained 0.01 ng/mL UroM5 and 26.96 \u0026micro;g/mL leucinic acid, while no UroA was detected. Differently, the levels of UroA, UroM5 and leucinic acid all increased in FGRE after fermentation, increased to 0.49 ng/mL,8.35 ng/mL, 97.20 \u0026micro;g/mL. These results indicated that fermentation process involved the transformation of substances.\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\u003eThis is a table. Tables should be placed in the main text near to the first time they are cited.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUroM5 (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUro A (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeucinic acid (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e97.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of leucinic acid on cellular mitochondrial energy metabolism\u003c/h2\u003e \u003cp\u003eRelated to HPLC-MS and non-targeted metabolomics analysis, leucinic acid, the differential compound in FGRE, was explored the effect on cellular mitochondrial function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results of CCK-8 presented that the different levels of leucinic acid below 30 \u0026micro;g/mL were non-toxic to HDF cell s, and 0.3\u0026thinsp;~\u0026thinsp;3 \u0026micro;g/mL leucinic acid obviously increased the cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The effect of different concentration of leucinic acid on mitochondrial respiratory capacity in UVB-induced HDF cells was dose-dependent. The effect of 0.2 \u0026micro;g/mL leucinic acid was no significance with UVB group. Differently, 1\u0026thinsp;~\u0026thinsp;5 \u0026micro;g/mL leucinic acid obviously increased the OCR of cellular basal respiration, ATP production, and maximal respiration compared to UVB group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicated that 1\u0026thinsp;~\u0026thinsp;5 \u0026micro;g/mL leucinic acid could improve the mitochondrial respiratory metabolism in UVB-induced HDF cells. In addition, 1% FGRE contained about 1 \u0026micro;g/mL leucinic acid, suggesting that leucinic acid was likely to be the key active compounds in FGRE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLactate dehydrogenase potentially participates in the conversion of leucinic acid\u003c/h2\u003e \u003cp\u003eStudies have reported that mouse D-lactate dehydrogenase (mLDHD) catalyzes the conversion of D-leucinic acid\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, mLDHD exhibits over 80% sequence similarity with human D-lactate dehydrogenase (hLDHD) as predicted by AlphaFold3, and their active site structures are essentially conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Consequently, computational simulation was performed between hLDHD and D-leucinic acid. The docking score was \u0026minus;\u0026thinsp;8.76, with the binding free energy of -52.16 kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The binding conformation indicated that subsite, which accommodates the glycolate group of the substrate, comprises Arg370, His421, and Glu465, while subsite, which binds the hydrophobic moiety of the substrate, includes Leu101, Trp374, Ser388, and Ile430 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). A hydrogen bond was formed between Arg370 and the substrate. The binding conformation of D-leucinic acid is similar for hLDHD and mLDHD. These results suggest that hLDHD may plays a potential role in catalyzing the conversion of D-leucinic acid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was further hypothesized that the conversion of L-leucinic acid might also be mediated by L-lactate dehydrogenase. In skin, L-lactate dehydrogenase primarily exists as the LDH-5\u003csup\u003e18\u003c/sup\u003e, a tetrameric protein encoded by the \u003cem\u003eldha\u003c/em\u003e gene. Therefore, computational simulations were performed using LDHA subunit (hLDHA) with L-leucinic acid. The docking score was \u0026minus;\u0026thinsp;7.33, with the binding free energy of -128.29 kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating the strong affinity between L-leucinic acid and hLDHA. The binding conformation revealed that Arg105 and Arg168 formed hydrogen bonds with the substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), with Arg105 potentially playing a crucial role in stabilizing the transition state of the hydride transfer reaction\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These results suggest that hLDHA may potentially participates in the conversion of L-leucinic acid.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular docking and molecular dynamics simulation results of leucinic acid with LDH.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDocking\u0026nbsp;score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBinding\u0026nbsp;free\u0026nbsp;energy\u0026nbsp;(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-leucinic\u0026nbsp;acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehLDHD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-52.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-leucinic\u0026nbsp;acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehLDHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-128.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eGalla rhios\u003c/em\u003e is primarily applied externally in clinical practice for burn management and tissue repair facilitation, in addition to its internal administration as a traditional Chinese medicinal agent. Although microbial fermentation is widely employed in modifying traditional Chinese medicinal materials, the application of \u003cem\u003eLactobacillus\u003c/em\u003e strains for biotransformation of GRE to potentiate their bioactive potential remains underexplored in pharmacological research. Thus, we fermented and obtained FGRE by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e. Experimental analysis demonstrated that FGRE had higher antioxidant and anti-aging activity than GRE \u003cem\u003ein vitro\u003c/em\u003e, and significantly enhanced the mitochondrial respiratory capacity in UVB-induced HDF cells. Compositional profiling identified leucinic acid as the primary bioactive constituent responsible for this effect, along with trace amounts of UroM5 and UroA.\u003c/p\u003e \u003cp\u003eAs a high-turnover organ, the skin's continuous cellular renewal and proliferation depend on mitochondrial respiration to meet its energy demands. However, both chronological aging (time-induced) and extrinsic aging, which are caused by environmental stressors including UV radiation, cigarette smoke, and air pollution, impair mitochondrial bioenergetic function \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Mitochondrial dysfunction is specified by decreased diminished per-mitochondrion respiratory capacity and membrane depolarization along with elevated oxidant stress, which is recognized as both a cause and consequence of cellular senescence \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This means that cellular senescence progressively exacerbates mitochondrial dysfunction, while the accumulation of functionally impaired mitochondria further contributes to skin aging. In this study, we confirmed the bioactivity of FGRE on DPPH radical scavenging and elastase inhibition by biochemical experiments \u003cem\u003ein vitro\u003c/em\u003e, which was also obviously better than that of GRE. And FGRE was considered as having the ability of anti-oxidant and antiaging, and the effect of anti-aging was possibly related to mitochondrial function. Thus, we employed the Seahorse XF analyzer to dynamically monitor cellular energetics in UVB-irradiated HDFs, assessing the effect of FGRE on mitochondrial bioenergetics. During the experiments, mitochondrial inhibitors (Oligomycin, FCCP, Rotenone / antimycin A) were sequentially added to obtain the key bioenergetic metrics including basal respiration, ATP production, maximal respiration and spare respiratory capacity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Basal respiration showed the energetic demand of cells under basal conditions, and oxygen consumption of basal respiration is mainly used to meet ATP synthesis. Thus, the oligomycin-induced OCR attenuation specifically corresponded to ATP-linked respiratory flux, with this parameter quantitatively reflecting mitochondrial ATP production capacity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Maximal respiration represented the maximum capacity that the electron respiratory chain can achieve, and Spare respiratory capacity defined as the difference between maximal and basal respiration. These metrics reflected the capability of the cells to respond to changes in energetic demand and indicates the fitness of the cells \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Significant alterations in these bioenergetic parameters emerge following mitochondrial damage induced by oxidative stress or UV irradiation. Zheng\u0026rsquo;s research showed that the OCR of basal respiration, maximal respiration and ATP production in UVB-induced HaCaT keratinocytes was obviously lower than that in untreated group \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In our study, similar results were also found in HDFs. In addition, FGRE and GRE both significantly improved the mitochondrial bioenergetic metrics compared to UVB group, indicated the improvement on mitochondrial respiratory metabolism. Specially, FGRE presented the better effect on mitochondrial respiration compared to unfermented GRE, which also indicated the enhancement of bioactivity by fermentation.\u003c/p\u003e \u003cp\u003eAccording to non-targeted metabolomics analysis, the main distinguishing metabolites between FGRE and GRE, generated through fermentation by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e PIAS240228, primarily comprise short peptides and free-form amino acids. Studies demonstrated that \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e fermentation of milk or milk protein generated differential metabolites, also predominantly short peptides and bioactive amino acids \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Meanwhile, the increased concentration of amino acids not only promotes strain growth but also enhances nutritional value and functional efficacy \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. KEGG enrichment analysis revealed that fermentation processes significantly modulated the branched-chain amino acids (BCAAs, including valine, leucine and isoleucine) biosynthesis pathway. The supplementation of BCAAs is often beneficial to energy expenditure \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This occurs because acetyl-CoA, the catabolic products of BCAAs, drives tricarboxylic acid (TCA) cycle and oxidative phosphorylation to provide cellular energy \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Leucinic acid is considered as the end metabolite of leucine, which can be converted from leucine to α-ketoiosocaparoic acid (α-KIC) and further converted from α-KIC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This transformantion can be conducted by \u003cem\u003eLactococcus\u003c/em\u003e with aminotransferase and hydroxyacid dehydrogenase \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Thus, the increase of leucinic acid in FGRE is due to the fermentation by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e. Some research suggested that the reaction between leucinic acid and α-KIC was reversible \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our results of molecular docking showed that human lactate dehydrogenase may can transform leucinic acid into α-KIC. Then, α-KIC further converted into acetyl-CoA, and participated in TCA cycle to regulate cellular energy metabolism \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. According to our results, leucinic acid also has been proven to enhance mitochondrial energy metabolism in UVB-induced HDF cells. What\u0026rsquo;s more, 1 \u0026micro;g/mL leucinic acid (equivalent to the concentration in 1% FGRE) ameliorated the mitochondrial respiratory dysfunction in HDF cells, suggesting that leucinic acid was considered as the principal bioactive compounds in FGRE. In addition, the relative abundance of BCAA-rich tripeptides and dipeptides (e.g., Leu-Ser-Ile, Ile-Asn-Val, Ala-Leu, Ala-Ile) in FGRE showed marked reduction. This decrease correlated with elevated levels of Leu, Val-Asn and Arg-Val-Phe. Val-Asn functions as a dipeptidyl peptidase IV inhibitor demonstrating hypoglycemic activity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Arg-Val-Phe was demonstrated to protects SH-SY5Y cells from cell damage by reducing ROS accumulation, and thereby maintaining membrane integrity, Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, and changing the mitochondrial transmembrane potential \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These compounds have the potential to maintain mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA study indicated GRE significantly enhanced collagen biosynthesis pathways, thereby accelerating wound closure processes due to its rich tannins and flavonoids \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, its potential modulatory effects on mitochondrial dynamics (including biogenesis and energy metabolism) remain poorly characterized in extant literature. Differently, UroA, a gut microbiota-derived metabolite of ellagic acid, has been reported to induce mitophagy and stimulate mitochondrial biogenesis \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, \u003cem\u003eBifidobacterium pseudocatenulatum\u003c/em\u003e, \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e, and related gut microbiota have been shown to transform either ellagitannins or EA into UroA through microbial fermentation \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Thus, we utilized \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e to ferment \u003cem\u003eGalla rhois\u003c/em\u003e, and the presence of UroM5 and UroA was found after fermentation. This indicated that the ellagic acid was biotransformed into UroM5, and further produce UroA by a series of dehydroxylation reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) \u003csup\u003e37\u003c/sup\u003e. The undetectable levels of UroM6 and UroC likely reflect sub-threshold metabolite concentrations rather than non-occurrence of the reaction. Notably, the limited yields of UroA and UroM5 in FGRE are potentially attributed to their limited aqueous solubility, as evidenced by Hu\u0026rsquo;s research \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. While UroA has been well-characterized as a mitochondrial modulator with demonstrated regulatory effects, the bioactivity profile of its structural analog UroM5 remains largely unexplored in mitochondrial contexts \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Li\u0026rsquo;s study showed that UroM5 demonstrated superior antioxidant capacity and α-glucosidase inhibition potency compared to UroA at concentrations \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Despite the notably low concentrations of UroA and UroM5 in FGRE, their antioxidant activity might still be effective. Our study demonstrated that relative equivalent of UroA (0.005 ng/mL) and UroM5 (0.08 ng/mL) in 1%FGRE could significantly inhibit the ROS production in UVB-induced HaCaT cells (S2), suggesting that they take effect by inhibiting ROS production and reducing oxidative stress. Therefore, although the actual effect of UroA and UroM5 on mitochondrial function may be not better than leucinic acid because of concentration limit in FGRE, they still were the effective compounds in FGRE.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we fermented GRE by using \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, and found that FGRE had better antioxidant and anti-elastase activity than GRE by biochemical experiments. The obtained FGRE was also proven to enhance mitochondrial respiratory metabolism in UVB-irradiated HDFs. Combined with metabolomics analysis, FGRE was found to contain leucinic acid, UroM5 and UroA. And the cellular experiments confirmed the function of leucinic acid on mitochondrial respiratory improvement in UVB-induced HDF cells, which was identified as the predominant bioactive compound in FGRE. Therefore, FGRE with bioactive constituents (leucine and urolithins), as a novel nutricosmetic ingredient, establishes a novel solution for mitochondrial dysfunction in skin aging.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo Funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.W., N. W. D.C, Y.Z and X.Z.(Xiao Zhang) finished the experiments and data curation. J.W. and X.Z.(Xiao Zhang) wrote the main manuscript text andprepared figures and tables. J.S, G.L. were responsible for validation. X.L. was responsible for project administration. H. H. and X.Z. (Xiangna Zhang) were responsible for supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by a grant from Proya International Academy of Sciences. The authors would like to thank PROYA Cosmetics Co. Ltd for excellent technical and financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGu, Y., Han, J., Jiang, C. \u0026amp; Zhang, Y. Biomarkers, oxidative stress and autophagy in skin aging. \u003cem\u003eAgeing Res. 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Comparative Study on the Antioxidative Effects and α-Glucosidase Inhibitory Potential In Vitro among Ellagic Acid and Its Metabolites Urolithins. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, 26711\u0026ndash;26721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.jafc.4c06542\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.4c06542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Galla rhois, Fermentation, Mitochondrial Respiratory Metabolism, Metabolomics analysis","lastPublishedDoi":"10.21203/rs.3.rs-8653896/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8653896/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBoth natural aging and photoaging of the skin can lead to disruption of mitochondrial homeostasis, which in turn exacerbates skin aging. This study developed a novel fermented \u003cem\u003eGalla rhois\u003c/em\u003e gallnut extract (FGRE) using \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e with mitochondrial respiratory homeostasis amelioration. The results of biochemical experiments showed that FGRE had better antioxidant and anti-elastase activity compared to unfermented extract. Seahorse XF Cell Mito Stress Test demonstrated that 1% FGRE significantly enhanced mitochondrial respiratory function in UVB-irradiated human dermal fibroblasts (HDFs) compared to unfermented extract (21% increase in basal respiration and spare respiratory capacity, 26% increase in ATP production, and 30%increase in maximal respiration). The non-targeted metabolomics analysis revealed differential metabolites predominantly enriched in amino acid metabolic pathways, and obviously increased metabolites were leucinic acid, Val-Asn, Arg-Val-Phe, Urolithin A (UroA), Urolithin M5 (UroM5) and other flavonoids. The quantitative detection showed that the levels of UroA (0.49 ng/mL) and its derivative UroM5 (8.35 ng/mL) were quite low, while the concentration of leucinic acid was about 97.20 μg/mL. And cell experiments suggested that 1 μg/mL leucinic acid (equivalent to concentration in 1% FGRE) ameliorated UVB-induced mitochondrial respiratory dysfunction in HDFs, that determine leucine as primary bioactive constituent. These results indicated that FGRE had potential anti-aging capacity by enhancing mitochondrial function, which also provided a novel candidate for dermo-cosmetic applications.\u003c/p\u003e","manuscriptTitle":"Enhanced Mitochondrial Respiratory Metabolism in UVB-Damaged HDF cells Based on Metabolomics Analysis of Fermented Galla rhois gallnut Extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 13:05:35","doi":"10.21203/rs.3.rs-8653896/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T15:36:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-01T08:45:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T07:45:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120249397918159331049524417359066356447","date":"2026-02-21T09:03:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316866070899706629119243229737279942140","date":"2026-02-19T02:14:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169623949883386656746985902566376918958","date":"2026-02-17T04:51:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322986799676099013441611284907771830042","date":"2026-02-02T19:43:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T15:44:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-02T15:25:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-28T09:56:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-23T07:54:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-23T07:32:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a9e81308-aa98-4038-9777-0b4150119379","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62221943,"name":"Biological sciences/Biochemistry"},{"id":62221944,"name":"Health sciences/Diseases"},{"id":62221945,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-04T11:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 13:05:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8653896","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8653896","identity":"rs-8653896","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}