Diaporthe sp. AC1 originated from Artemisia argyi was executed to investigate its secondary metabolites. It was fermented in a culture medium containing 5-hydroxytryptophan (5-HTP), 1-methyl-L-tryptophan (1-MT), and tryptamine (TA), respectively. The antibacterial activities of crude extracts against pathogenic bacteria and pathogenic fungi were determined by using the Oxford cup method, while the cytotoxicity of crude extracts against cancer cells was determined by using the MTT method. The results showed that the secondary metabolites of Diaporthe sp. AC1 induced by 1-MT exhibited optimal antibacterial activity and tumor cytotoxicity. The induction conditions of 1-MT were optimized, and the antibacterial activities and tumor cytotoxicity of crude extracts under different induction conditions were investigated. As indicated, the optimal moment for 1-MT addition was before inoculation and its optimal concentration was 0.25 mM. Under these conditions, Diaporthe sp. AC1 was fermented and approximately 12 g of crude extracts was obtained. The crude extracts were then separated and purified to acquire nine monomer compounds, including three new compounds (1–3) and six known compounds (4–9). The antibacterial activities of the compounds against pathogenic bacteria and pathogenic fungi were investigated by using the microdilution method, while their cytotoxicity against cancer cells was analyzed by using the MTT method. The results demonstrated that Compound 1 exhibited moderate antibacterial activities against Verticillium dahlia, Fusarium graminearum, and Botrytis cinerea, as well as a low inhibitory activity against Listeria monocytogenes. Nevertheless, Compound 1 showed significant cytotoxicity against five cancer cells, with IC50 ranging from 12.26 to 52.52 μM. Compounds 2 and 3 exhibited negligible biological activity, while other compounds showed detectable inhibitory activities against pathogenic bacteria and cancer cells.Small molecule-induced fermentation of the endophytic fungus  By tunilkaloids . Additio culture .Diaporthe is typically isolated as endophytic fungi and secondary metabolites produced by this genus have been thoroughly investigated ] were added into the fermented culture of this strain, with the aim of generation of new fungal metabolites. Herein, 1-MT was screened as the optimal inducer, and three new compounds (1–3) as well as six known ones were produced by the endophytic fungus Diaporthe sp. AC1 after adding 1-MT into media for fungal cultivation.stigated . The endlkaloids . The str2.2.1.The media used for the experiments included the potato dextrose agar (PDA) medium , the malt extract (ME) liquid medium , and the Luria-Bertani (LB) agar medium .The organic solvent used for chromatographic separation was purchased from Tianjin Kaitong Chemical Reagent Co., Ltd., China. The silica gel (200–300 mesh) used for column chromatography was purchased from Qingdao Ocean Chemical Co., Ltd., China. Sephadex LH-20 gel was purchased from GE Healthcare. Thin-layer chromatography silica gel plates (GF254) were purchased from Qingdao Ocean Chemical Co., Ltd., China. 5-HTP, 1-MT, and tryptamine (TA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China.2.2.Diaporthe sp. AC1 was isolated from Artemisia argyi and identified by following a previously reported protocol . Pathogenic fungi Fusarium graminearum, F. moniliforme, Botrytis cinerea and Verticillium dahlia were isolated, identified and preserved by our laboratory (Pathogenic bacteria boratory .2.3.Diaporthe sp. AC1 was inoculated on PDA medium and incubated at 28°C for 5 days. Five disks (diameter = 1 cm) were then inoculated in 100 mL of ME liquid medium and shaken under 180 rpm at 28°C for 2 days. Afterwards, 5-HTP, 1-MT, and TA were separately added to the fungal cultures at 4 × 12 h intervals until a final concentration of 1 mM ( of 1 mM . After tWhole culture broths (including mycelia) were extracted with equal volume of ethyl acetate for two times. Meanwhile, organic solvents were evaporated under reduced pressure to obtain crude extracts. Additionally, the antibacterial activities and cytotoxicity of the crude extracts were evaluated.2.4.According to the methods described before  with som2.5.Diaporthe sp. AC1 was fermented to obtain crude extracts. Briefly, Diaporthe sp. AC1 was inoculated in the PDA medium and incubated at 28°C for 5 days. Subsequently, five disks (diameter = 1 cm) were inoculated in 100 mL of ME liquid medium and shaken under 180 rpm at 28°C for 5 days to obtain the seed solution, which was then transferred to 2.5 L of ME medium for induced fermentation. Herein, 50 L of culture (200 bottles of 2.5 L medium) was fermented. After fermentation, the crude extract was extracted and concentrated by using the method described above. Additionally, antibacterial activities and cytotoxicity of the crude extracts were evaluated.Under optimized induction conditions, 2.6.7 CFU/mL) and 15 mL of medium  were poured into the Petri dish. After solidification, the Oxford cups were removed, resulting in generation of four small wells in the upper medium.As described in our previous study , the antIn the experimental group, 50 μL of the crude extract solution  was added to each well. Ampicillin sodium (20 mg/mL) and amphotericin B (20 mg/mL) were used as the positive control of antagonistic bacteria and fungi, respectively. Then, 50 μL of methanol was added to the negative control wells. The plates inoculated with bacteria were cultured at 37°C for 24 h, and those containing fungi were cultured at 28°C for 5~7 days. Additionally, the diameters of inhibition zones were measured.2.7.4 cell/mL in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS, 100 μL/well) and incubated at 37°C (7% CO2) for 20 h. Then, 100 μL of the cell culture media containing compounds of different concentrations  was added into different wells; the well added with 100 μL of the cell culture medium with no compound was used as the negative control, while the blank control group contained culture medium but no cells. After 48 h, the plate was rinsed once with PBS buffer (200 μL/well) and MTT solution  was added, followed by another four-hour incubation. After that, the solution was removed and DMSO (150 μL/well) was added to dissolve the formazan crystals generated during incubation. After 10 min of shaking, the absorbance was measured at 570 nm. Each group had three replicates. The inhibition rates of the compounds were calculated by:The cytotoxicity of the crude extracts against five cancer cells  was analyzed by using the MTT method . Specifi0, A1, and A2 refer to the absorbance of the blank control, the experimental group, and the negative control, respectively.where A2.8.As shown in 3 or acetone-d6, and identified by nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), and/or two-dimensional nuclear magnetic resonance (2D-NMR). 1H NMR (600 MHz) and 13C NMR (151 MHz) spectra of the samples were obtained by using the NMR spectrometer  with tetramethylsilane (TMS) as the internal standard. Additionally, HR-MS spectra of the samples were obtained by using an AB SCIEX X500R QTOF mass spectrometer.The compounds were dissolved with CDCl2.9.600 value reached to 0.2. The culture was further diluted 300 times to obtain a bacterial suspension in which the concentration was 1 ~ 5 × 105 CFU/mL.Preparation of bacterial suspension: Pathogenic bacteria were inoculated in LB liquid medium, and incubated at 37°C 200 rpm for 12 h. The bacterial culture was then diluted with LB liquid medium so that its OD5 CFU/mL with PDB medium.Preparation of fungal suspension: Pathogenic fungi were incubated at 28°C on PDA medium for 3 ~ 7 days, the dishes were then rinsed with sterile water to prepare spore suspension. The spores were counted using the Hemocytometer, and the concentration of spores was diluted to 1 ~ 5 × 105 CFU/mL bacteria or fungal spores) was added to each well of a 96-well plate. Then, 100 μL of the culture medium (LB medium for bacteria and PDB medium for fungi) containing compounds of different concentrations  was added into each well. The well added with 100 μL of culture medium but no compound was used as the negative control, while the blank control contained culture medium but no pathogenic bacteria or compounds. Each group had three replicates. The 96-well plate inoculated with bacteria was incubated at 37°C for 24 h, while the plate containing fungi was incubated at 28°C for 48 h. The absorbance was measured at 600 nm. The inhibition rates of compounds were calculated by:Modified microdilution method  was used0, A1, and A2 refer to the absorbance of the blank control, the experimental group, and the negative control, respectively.where A2.10.The cytotoxicity of the compounds against five cancer cells was determined by using the MTT method as described above. The final concentrations of the compounds ranged from 200 to 12.5 μM with two-fold serial dilution.3.3.1.Chaetomium globosum 1C51 and generate novel active compounds , respectively.As indicated by previous studies, tryptophan analogs 1-MT could change the metabolic pathway of ompounds , and addlkaloids . In ordeThe results in L. monocytogenes and S. aureus, and those induced by TA exhibited enhanced inhibitory activity against E. coli, S. enteritidis, and P. aeruginosa. In particular, crude extracts induced by 1-MT exhibited optimized activity, as reflected by enhanced inhibitory activity against all of the five pathogenic bacteria involved.The antibacterial activities of the crude extracts are summarized in C. albicans, F. moniliform, and B. cinerea.According to The cytotoxicity of crude extracts is summarized in In summary, crude extracts induced by 1-MT exhibited the optimal antimicrobial and inhibitory performance toward cancer cells. Hence, 1-MT was employed as the inducer for subsequent experiments.3.2.The yields and characteristics of crude extracts induced by 1-MT are shown in L. monocytogenes, S. enteritidis, S. aureus, F. moniliforme, and B. cinerea being above 20 mm. Moreover, its antibacterial activities against E. coli, C. albicans, V. dahlia, and F. graminearum were also considerably high, with the diameters of inhibition zones ranging from 15.93 to 18.47 mm. The inhibition performance of 1-MT-4 was also rational, with the diameters of the inhibition zones for pathogenic bacteria and pathogenic fungi ranging from 14.83 to 19.03 mm and from 12.03 to 17.80 mm, respectively. Additionally, 1-MT-1 and 1-MT-3 exhibited poorer antimicrobial activities against pathogenic bacteria and fungi. Therefore, it can be deduced that direct addition of excessive 1-MT has negative impacts on the yield of active compounds.The inhibition activities of the crude extracts fermented under four induction conditions against pathogenic bacteria and pathogenic fungi are shown in Crude extracts fermented under four induction conditions showed different inhibitory activities against the five cancer cells . 1-MT-1,Overall, the optimal induction conditions were the addition of 0.25 mM 1-MT and mycelium before fermentation.3.3.2 (4.0 mg), 3 (3.6 mg), 4 (69.3 mg), and 5 (4.4 mg) were isolated from Fr.C; Compounds 7 (7.4 mg) and 8 (66.6 mg) were isolated from Fr.D; and Compounds 1 (62.5 mg), 6 (10.0 mg), and 9 (6.9 mg) were isolated from Fr.E.As demonstrated in 1 is reddish-brown oil with a molecular formula of C10H10O4, as deduced from HR-ESI-MS ion peak at m/z 195.0652 [M + H]+ , 114.57 (3), 104.35 (5)), and two quaternary carbons (δC166.41 (6), 162.93 (2)). The data in 1H NMR spectrum displayed singlets for two methylene , 2.65 ) as well as a methyl ), and that in 13C NMR indicated the presence of two carbonyl (δC 198.52 (3′), 198.14 (4′)). Key HMBC correlations  , 7.61 , 7.11 , 7.23 , 7.29  and 3.76  were ascribed to a 1-methylindole moiety. The 13C NMR and HSQC data displayed singlets for a methyl ester (δC 172.33 (14), 51.85 (15)), an ethyl ester (δC 24.66 (8), 65.05 (9), 172.82 (11)) and two methylene (δC 29.20 (12), 28.92 (13)). Key HMBC correlations (2.Compound  + Na]+)  and was NMR data . The NMRelations  from H-13 is yellow oil with a molecular formula of C22H22N2O2, as deduced from HR-ESI-MS at m/z 369.1573 [M + Na]+ (3 and fusariumindole A (3 had two extra methyl groups (δC 32.69 and δC 32.56). 1H-1H COSY and HMBC spectra demonstrated that the two imino groups in fusariumindole A were methylated  (5) (N-methyl-3a-hydroxyfuroindoline (6) (7) (8) (H)-one (9) (4–9 are shown in Based on the NMR data of compounds phol (4) , 3-(2-ac (4) (5) , N-methyline (6) , 3,5-dim (6) (7) , p-hydro (7) (8) , 3-phe-n-one (9) . The strDiaporthe sp. AC1 led to identification of eight compounds with no indole alkaloids (2–6) were obtained. Researches have demonstrated that 1-MT not only could elicit the production of Pictet-Spengler reaction-based alkaloids  against L. monocytogenes, while Compounds 2 and 3 exhibited low inhibitory activities against pathogenic bacteria. For the known compounds, Compounds 4 and 9 exhibited good antibacterial activities against five pathogenic bacteria with all inhibition rates above 80%, and Compound 8 showed promising inhibitory performance on P. aeruginosa, with an inhibition rate > 90%. As reported before, Compound 9 showed good inhibitory activity against S. aureus, L. monocytogenes, and S. typhimurium  was few reported, but the antifungal and antibacterial activities of tryptophol have been proved -6-(4-hydroxy-3-oxopent-1-en-1-yl)-2H-pyran-2-one  against A549 and MDA-MB-231 was 84.5 and 64.3 μM, respectively -6-(4-hydroxy-3-oxopent-1-en-1-yl)-2H-pyran-2-one changed into a carbonyl group, demonstrating that the carbonyl group of C-4′ in compound 1 was extremely important for the enhanced cytotoxicity.As shown in ectively , while i4 showed moderate cytotoxicity against Hela (IC50 = 29.74 μM). Among the derivatives of 1-MT (Compounds 2–6), only Compound 4 showed relatively good inhibitory activity against the cancer cells, further demonstrating the key role of the hydroxyl group at C11 in Compound 4.Compound 4.Diaporthe sp. AC1 to increase the diversity of metabolites, three novel compounds (1–3), together with six known ones were isolated and characterized. Most of the compounds were the biotransformation products of 1-MT. In the bioactivity assay, Compound 1 showed moderate antibacterial activity against V. dahlia, F. graminearum, and B. cinerea, with MIC values being 256, 128, and 256 μg/mL, respectively. It also showed high cytotoxicity against five cancer cells, with IC50 ranging from 12.26 to 52.52 μM. Other compounds also exhibited certain antimicrobial and cytotoxic activities. Under the severe situation that antibiotic resistance is a growing public threat, this work provides an efficient and eco-friendly methodology for the development of structurally undescribe compounds, from which lead molecules can be recognized and optimized for new drugs discovery.By feeding 1-MT into cultures of the endophytic fungus The original contributions presented in the study are included in the article/SZ: Writing – original draft, Investigation. QX: Writing – original draft, Investigation. CJ: Writing – original draft, Investigation. XH: Investigation, Writing – original draft. YZ: Investigation, Writing – original draft. CL: Investigation, Writing – original draft. LM: Investigation, Writing – original draft. WS: Investigation, Writing – original draft. YL: Writing – original draft, Formal analysis. ZY: Formal analysis, Writing – review & editing. FZ: Writing – review & editing, Project administration. YT: Project administration, Writing – original draft.


Caesalpinia mimosoides Lam. are used ethnomedically by the traditional healers of Uttara Kannada district, Karnataka (India) for the treatment of wounds. The current study was aimed at exploring phenol-enriched fraction (PEF) of crude ethanol extract of tender shoots to isolate and characterize the most active bio-constituent through bioassay-guided fractionation procedure. The successive fractionation and sub-fractionation of PEF, followed by in vitro scratch wound, antimicrobial, and antioxidant activities, yielded a highly active natural antioxidant compound ethyl gallate (EG). In vitro wound healing potentiality of EG was evidenced by a significantly higher percentage of cell migration in L929 fibroblast cells (97.98 ± 0.46% at 3.81 μg/ml concentration) compared to a positive control group (98.44 ± 0.36%) at the 48th hour of incubation. A significantly higher rate of wound contraction (98.72 ± 0.41%), an elevated tensile strength of the incised wound , and increased quantity of connective tissue elements were observed in the granulation tissues of the 1% EG ointment treated animal group on the 15th post-wounding day. The accelerated wound healing activity of 1% EG was also exhibited by histopathological examinations through Hematoxylin and Eosin, Masson’s trichome, and Toluidine blue-stained sections. Significant up-regulation of enzymatic and non-enzymatic antioxidant contents  and down-regulation of oxidative stress marker (lipid peroxidation) clearly indicates the effective granular antioxidant activity of 1% EG in preventing oxidative damage to the skin tissues. Further, in vitro antimicrobial and antioxidant activities of EG supports the positive correlation with its enhanced wound-healing activity. Moreover, molecular docking and dynamics for 100 ns revealed the stable binding of EG with cyclooxygenase-2  and matrix metalloproteinase-9  and unstable binding with tumor necrosis factor-α , suggesting the potential applicability of EG in inflammation and wound treatment.The tender shoots of  The plant was authentically identified using standard regional floras and the voucher specimen (PB/GRH-111) was deposited in the Herbarium, Karnatak University, Dharwad for future reference. The freshly collected tender shoots of the plant (2.5 kg) were shade dried and coarsely powdered with an electrical blender. The powdered material [1 kg dry weight (d.w.)] was sequentially extracted with the solvents  of increasing polarity and dielectric constants through the hot percolation method using Soxhlet apparatus , and MeOH as mobile phase to obtain 86 primary fractions. A thin-layer chromatography  profile of all the collected fractions was performed using the combinations of CHCl3:MeOH as mobile phase and the bands were viewed under UV lights of 254 and 366 nm wavelengths. Fractions with similar retention factor (Rf) of bands were mixed and finally grouped into 24 fractions (Fraction 1–Fraction 24). The percent yield of all the fractions was calculated and screened for in vitro wound-healing activity through scratch wound assay against L929 cell lines, followed by antimicrobial activity against bacterial and fungal skin pathogens.A flow chart representing a brief methodology of bioassay-guided fractionation, isolation, and purification of the biologically active compound EG is given in 3, CHCl3:MeOH  and MeOH as eluents to obtain 47 sub-fractions. All the sub-fractions were collectively included into 15 groups based on their Rf values on the TLC plates (Sub-fraction 9.1– Sub-fraction 15). The percent yield of all the sub-fractions was calculated and screened for in vitro wound-healing and antimicrobial activities. The bioactive sub-fraction 9.13 with a highly significant percentage of cell migration and antimicrobial activities yielded a major compound. It was dissolved in MeOH with activated charcoal to obtain purified off-white, non-crystalline powder after drying.The bioactive fraction 9, which showed a significantly higher percentage of cell migration during scratch wound assay with potential antimicrobial activity, was further subjected to column chromatography (weight of the fraction nine taken for separation: 1.006 g) using CHClThe structure of the isolated compound was determined through various spectroscopic techniques such as Fourier transform infra red (FT-IR), Nuclear magnetic resonance (NMR), Gas chromatography-mass spectrometry (GC-MS), and Ultraviolet-visible spectroscopy (UV-Vis).1H-NMR and 13C-NMR spectra were recorded on a Bruker 400 and 100 MHz spectrometer respectively, using DMSO-d6 as the eluting solvent and TMS as an internal standard. The chemical shifts were expressed as δ (ppm) and the mass spectra were recorded using the Agilent-single Quartz GC-MS instrument. UV spectra were recorded on U-3310 UV-Vis spectrophotometer and structure of the isolated compound was determined as ethyl gallate (EG) (structure elucidation results provided in IR spectra of the isolated compound were recorded on a Nicolet-5700 FT-IR spectrophotometer (KBr disc). 2 T-Flask (Nunc EasYFlask) containing 7 ml complete growth medium  and maintained at 37°C, 5% CO2 in a humidified incubator. The fresh medium was supplied a day after plating and for every 2–3 days following until cells reached 70%–80% confluence before subculture , Pune, India. The cells were immediately thawed in a water bath at 37°C, and transferred to 25 cmbculture .TMMicrowellTM96-well microplates, ThermoFisher Scientific) at 1 × 104 cells/well density, containing a complete growth medium, and incubated for 24 h. After incubation, the medium was replaced by fresh serum-free medium with various concentrations  of samples and the plate was incubated for a further 48 h. The cells under treatment with serum-free medium without any samples were considered as control. A serum-free medium containing 25 µL of MTT (5 mg/ml concentration) was added to each well. The medium was carefully removed from the wells followed by complete drying after 4 h of incubation. Dimethyl sulfoxide solution  was put in each well with intermittent shaking of the plate for 10 min to suspend the purple-colored residual formazan crystals. Absorbance of the solution was measured spectrophotometrically with a 96-well microplate reader (Thermo Scientific Multiscan Go Version 1.00.40) at 540 nm wavelength. The percentage cell viability and toxicity (IC50) of all the samples were determined using ED50 plus v1.0 software.Cell viability and toxicity of samples  was analyzed through MTT assay  . Cells wth, 24th, and 48th hours of treatment using an inverted microscope (Olympus CKX41) attached to a camera (Magcam DC5). The ×100 magnification images were photographed to evaluate the percentage migration of cells towards the scratch wound. The area between wound edges was precisely calculated using NIH ImageJ software, and the percentage wound contraction was estimated.The cell migration activity of all the samples against L929 cells was performed through scratch wound assay as described in our previous study . The celStaphylococcus aureus (MTCC737), Pseudomonas aeruginosa (MTCC 1688), Proteus vulgaris (MTCC 1771), Klebsiella pneumoniae (MTCC 109), Micrococcus luteus (NCIM 2103), Salmonella typhimurium (NCIM 2501), and Micrococcus flavus (NCIM 2379) were the procured bacterial strains. Trichophyton rubrum (MTCC 296), Microsporum canis (MTCC 2820), Microsporum gypseum (MTCC 2819), Trichophyton mentagrophytes (MTCC 7687), Malassezia furfur (MTCC 1374), Epidermophyton floccosum (MTCC 7880), and Candida albicans (MTCC 183) were the fungal strains procured for antimicrobial evaluation. The media used for subculturing the strains were Sabouraud Dextrose Agar (SDA), Nutrient Agar (NA), and Potato Dextrose Agar (PDA). Peptone water was used to prepare the bacterial and fungal suspension cultures and they were incubated for 18–24 h at 37°C. The turbidity of suspension cultures was set to 1.5 × 106 CFU/ml and the relative antimicrobial activity was compared with the positive standards viz. streptomycin , nystatin, and voriconazole . The stock solutions of samples and the positive standards were dissolved in 1% DMSO to make two-fold serial dilutions ranging between 1,000–0.97 μg/ml concentrations. The tubes containing inocula were incubated for 24–48 h at 37°C and the lowest concentration of samples/standards, which inhibits the growth of microbial strains, was considered as MIC.The antimicrobial property of all the samples was performed using tube dilution assay against bacterial and fungal pathogens causing wound infections . The staDPPH activity of bioactive samples  was measured as per the method described by ABTS radical scavenging activity of bioactive samples  was estimated as per the method of 50 of samples and corresponding standards against DPPH and ABTS radicals were calculated using the ED50 plus v1.0 software program.ICin vivo wound-healing activity . The fur from the dorsal surface of the animals was removed using an electric shaver. The test was performed in control and treatment groups, each with five male and five female rats, respectively, and they were topically applied once with ointment base and EG ointment. The animals were observed for 14 days of the study period for the toxic synptoms on rat skin, viz. itching, irritation, redness, swelling, and other alterations in behavior, if any.Acute dermal toxicity of the ointment containing EG was performed as per the Organization for Economic Co-operation and Development guidelines (OECD Guideline-402) to determine the suitable doses for evaluating activity . Wistar in vivo wound-healing activity of EG. The approval for experimental protocols and the use of animals was given by Animal Ethics Committee, S.E.T. College of Pharmacy, Sangolli Rayanna Nagar, Dharwad, India (SETCP/IAEC/2012-13/519). Animals of corresponding treatment groups were managed individually in clean separate cages for 7 days of acclimatization with standard diet and water ad libitum, as described earlier. Wound-healing property of EG evaluated using circular excision and linear incision wound models was as described in our previous study (n = 6 (either sex in each group). The animals for circular excision and linear incision wound models were categorized into the following groups:Wistar rats (200–280 g) of both sexes were used to evaluate us study . Differeus study . The samus study  and the Group 1- Ointment treated with EG 0.5% w/w.Group 2- Ointment treated with EG 1% w/w.Group 3- Treated with standard ointment Group 4- Treated with Ointment base I.P. (Ointment base group).Group 5- No treatment (Negative control group).2 diameter size was created with a surgical scissor on the shaved dorsal region of the rats. The wounds were topically applied with approximately 1 G of test ointments, standard Povidone-Iodine, and the ointment base to the respective group of animals once a day up to the 15th post-wounding day. The raw wound was traced every alternate day up to the end of the study (15th post-wounding day) using transparent polythene sheets sterilized with 70% ethanol and a permanent marker. The area of traced wounds was measured using millimeter scale graph sheets, and the wound progression images of all the experimental animals were taken using a Nikon D5600 camera. The percentage wound contraction in all the experimental animals was determined through the following equation:n = wound area on every alternate day (from 1st to 15th post wounding days). The study also includes Complete epithelialization period (CEP) in all the animals of corresponding groups.day “0”–Wound area on day “n”/Wound area on day “0” ×100.The experimental rats were anesthetized through the open mask method before wound creation . A day p2).A longitudinal incised wound (size: 6 cm length) was created on the shaved dorsal region of animals of corresponding groups as per standard procedure . The inc® C5-JENOPTIK) attached to Carl Zeiss Axio Imager M2 compound microscope and the images were analyzed at  per unit area of images at ×100 magnification were performed using NIH ImageJ software , followed by staining with hematoxylin and eosin (H&E), Masson’s trichome, and Toluidine blue stains as per standard procedures . The micsoftware .Quantitative biochemical analysis of granulation tissues such as hydroxyproline, hexosamine, superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and lipid peroxidase (LPO) were carried out as per the method described in our previous publication , using shttps://pubchem.ncbi.nlm.nih.gov). Using the conjugate gradients approach, the “uff” forcefield was used to minimize ligand structure. By including the gasteigers charges and polar hydrogens, the ligands were changed into “.pdbqt” format.(i) Preparation of the ligand: Three-dimensional (3D) structure of EG was obtained in “.sdf” format from the PubChem small molecule database (https://www.rcsb.org/). The computed atlas of surface topography of proteins  (https://prankweb.cz/) ((ii) Preparation of macromolecules: The 3D x-ray crystallographic structure of COX-2 (5IKT) , MMP-9 , and TNFml?1bxw)  and P2Raweb.cz/)  server whttps://autodock.scripps.edu/) was used to perform molecular docking via executing through the POAP pipeline  was used for visualizing protein-ligand intermolecular interaction and molecules with the lowest binding energy.(iii) Docking: AutoDock vina , matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-alpha (TNF-α), were prioritized. Molecular docking was performed to infer the intermolecular interaction of EG with COX-2, MMP-9, and TNF-α. Three stages of molecular docking were carried out: 1) preparation of the ligand, 2) preparation of macromolecules (targets), and 3) identification of the active site and docking the ligand with the target.https://www.gromacs.org) software package (https://ambermd.org/AmberTools.php) was used to prepare the topological properties of the ligands and the whole complex. Further, an antechamber with a “bcc” charge model was used to calculate the partial charges of small molecules. The salvation of the prepared systems in a rectangular box were performed using the three-site water (TIP3P) model, with 10.0 Å boundary conditions from the protein’s boundaries in all directions. The required number of counter ions were added to neutralize the charges on the prepared systems. To establish the near-global state least energy conformations, the steepest descent and conjugate gradient energy minimization methods were used. Canonical (NVT and NPT) and isobaric (NPT) ensembles were employed for system equilibration for 1 ns. A modified Berendsen thermostat approach was adopted (300 K) to maintain the volume and temperature constant during NVT equilibration. A Parrinello-Rahman barostat was employed during NPT equilibration to keep 1 bar pressure constant. To determine van der Waals, coulomb, and long-range electrostatic interactions, the Particle Mesh Ewald approximation was utilized with 1 nm cut-off value. Similarly, to constrain the bond length, the Linear Constraint Solver approach was used. Each complex went through a 100-ns production run, with coordinates recorded every 2 fs. In-built gromacs tools were used to analyze the generated trajectories and the additional software was used for further specialized investigations, wherever appropriate.All-atom molecular dynamics (MD) simulation was carried out with the docked conformations of three distinct targets for 100 ns in an explicit solvent to investigate their intermolecular interactions and structural stabilities. The execution of MD simulations with the Amber ff99SB-ildn force field was employed with the GROMACS ver. 2021.3  with Tukey Among 24 fractions collected during column chromatography, the percent yield of all the fractions was in the range of 0.30%–20.21%. The highest yield was exhibited by fraction 9 , followed by fraction 12 , fraction 13 , and fraction 16 . Among 15 sub-fractions and EG, the percent yields ranged between 1.52% and 74.29%. In comparison, EG exhibited the highest yield , followed by sub-fraction 9.13 , 9.15 , 9.14 , and 9.8  .-13452 (-OH), 1707 (Ester C=O); UV λmax (EtOH): 270 nm; 1H-NMR  δ (ppm): 9.13 , 6.90 , 4.16 , 1.23 ; 13C-NMR  δ (ppm): 14.21, 59.92, 108.42, 119.54, 138.28, 145.50, and 165.77; EI-MS m/z198.Ethyl 3, 4, 5-trihydroxybenzoate  IR (KBr) cm−1; a band at 1707 cm−1 exhibits typical ester stretching. In 1H-NMR, the ethoxy protons resonated in triplet-quartet pattern at 1.23 ppm and 4.16 ppm with J-value of 7.2 Hz. The two symmetrical aromatic protons resonated at 6.90 ppm as a singlet. A broad singlet observed at 9.13 ppm corresponds to three hydroxyl protons. Further, in 13C-NMR, the methyl carbon resonated at 14.21 ppm. A signal at 59.92 ppm corresponds to methylene carbon. The carbonyl carbon of the ester was observed in the most downfield region at 165.77 ppm. The signal at 119 ppm is attributed to the methine carbon attached to the carbonyl carbon. The methine carbon ortho to hydroxyl group resonated at 108.42 ppm, whereas the two carbons meta and para to the ester functionality were observed at 145.50 and 138.28 ppm, respectively. The molecular ion peak at m/z 198 confirms the compound ethyl gallate  of fractions, sub-fractions, and EG for 24 h and the cytotoxicity was analyzed through MTT assay. The percentage viability of L929 cells showed a gradual increase with increasing concentration of all the samples  up to certain limits . It later decreased rapidly with increased concentrations. It was found that all the fractions (1–24) and sub-fractions (9.1–9.15) showed ≥100% cell viability at 15.62–7.81 μg/ml concentrations . HoweverAmong all the fractions, the highest percentage of cell viability was achieved by fraction 2 (108.27 ± 0.16%), followed by fraction 9 (106.21 ± 0.77%), fraction 6 (104.48 ± 1.10%), and fraction 4 (103.74 ± 0.96%) at 15.62 μg/ml concentration . While a50 of cell toxicity was found to be 323.75 ± 2.19, 312.01 ± 2.14, 307.51 ± 4.60, and 302.87 ± 6.49 μg/ml for fraction 6, fraction 15, fraction 9, and fraction 7 respectively  compared to the negative control group (60.55 ± 1.14%) at the incubation period of the 48th hour. In the positive control group, the migration rate was increased to 98.44 ± 0.36% at the 48th hour of the incubation period, which was comparable to the effect of fraction 9 , 15 sub-fractions (7.81 μg/ml), and EG (3.90 μg/ml and 1.95 μg/ml) were chosen to carry out action 9 . TherefoDuring the second phase of the chromatographic separation process, 15 sub-fractions (sub-fractions 9.1–9.15) were obtained and were evaluated for scratch wound assay. Among all sub-fractions, sub-fraction 9.13 exhibited a significantly higher percentage of cell migration (94.62 ± 0.17%) at the 48th hour of the incubation period compared to the negative control group (60.55 ± 1.14%) . The higP. aeruginosa, M. luteus, and M. flavus with the least MICs (7.81 μg/ml each), followed by P. vulgaris and S. aureus with 15.62 μg/ml MICs respectively. In the case of sub-fractions, the sub-fraction 9.13 was highly active with the least MIC (3.90 μg/ml) against P. aeruginosa, followed by P. vulgaris, M. luteus, and M. flavus (7.81 μg/ml each). The isolated compound EG was found efficient against P. aeruginosa, S. aureus, and M. luteus with the least MICs (3.90 μg/ml each), followed by P. vulgaris and M. flavus (7.81 μg/ml each), which were comparable to the standard streptomycin.Among all the samples tested, only fraction 9, fraction 12–fraction 17, fraction 19, sub-fractions 9.12–9.15, and EG exhibited considerably effective antibacterial activity with MIC<1,000 μg/ml . Other tT. rubrum (7.81 μg/ml), followed by M. furfur (15.62 μg/ml), M. gypseum, and C. albicans (31.25 μg/ml each). Sub-fraction 9.13 effectively inhibited the growth of M. gypseum and C. albicans with the lowest MICs (7.81 μg/ml each). Similar results were also observed in EG against M. gypseum (3.90 μg/ml), T. rubrum, M. furfur, and C. albicans with 7.81 μg/ml MICs each, which were comparable to Nystatin and Voriconazole standards.Similarly, fraction 9, fraction 12, fraction 13, fraction 16, sub-fraction 9.13, and EG exhibited considerably effective antifungal activity with MIC<1,000 μg/ml . The oth50 results with 8.33 ± 0.41 and 8.83 ± 0.71 μg/g respectively, which revealed the potential antioxidant activity of EG , 28.38 ± 0.47% to 91.22 ± 0.47% (sub-fraction 9.13), 23.25 ± 0.79% to 98.42 ± 0.20% (EG), and 27.20 ± 0.34% to 98.87 ± 0.20%  at 1.95 and 125 μg/g concentrations respectively . Interesty of EG .50 values with significantly similar results (15.99 ± 0.33 and 15.12 ± 0.42 μg/g respectively) , 89.54 ± 0.55% (sub-fraction 9.13), and 98.68 ± 0.29% (EG) at the same concentration . From thctively) .viz. changes in mucous membranes, fur, locomotory organs, eyes, or on other body parts of the animals were not reported during the study period. Any adverse effects of EG on animal skin viz. itching, swelling, redness, irritation, other behavioral patterns, and mortality of the animals were also not noticed during the toxicity study.Acute dermal toxicity of isolated compound EG was evaluated and no indications of any toxic symptoms on experimental animals were detected at 2000 mg/kg limited dose. Toxic signs p ≤ 0.01 and p ≤ 0.001) compared to the ointment base, the negative control, and even the positive control groups from the third day up to the 11th post-wounding day  was observed from the 5th to 11th post-wounding day compared to all three treatment groups  and later on it was limited to only ointment base and negative control groups. Interestingly, on the 15th post-wounding day, 0.5% and 1% EG ointment-treated groups showed relatively higher percentages of wound contraction (98.10 ± 0.20% and 98.72 ± 0.41% respectively) when compared to the positive control group (96.84 ± 1.27%). Effective wound-healing activity of 1% EG ointment, positive control, and 0.5% EG ointment could be noticed by significantly less CEP in all three treatment groups  in comparison with the ointment base and negative control (21.67 ± 0.33 and 22.00 ± 0.58 days) groups respectively. The photographic images of wound contraction in different treatment groups of excision wound models between the 0th to15th post-wounding days are given in In the circular excision wound model, 1% EG ointment-treated group started showing a significantly higher rate of wound contraction (ding day . Thereaf2 respectively at p ≤ 0.001) compared to a positive control (814.02 ± 3.59 g/mm2), ointment base (536.78 ± 14.78 g/mm2), and negative control (524.30 ± 11.34 g/mm2) groups  groups .epidermis containing 2-3 layers of epithelial cells, stratified keratinization over the epidermal layer and formation of hair follicles. Differential cell count results in positive control and 1% EG groups illustrated a greater number of newly formed blood vessels (10.33 ± 0.88 and 7.67 ± 0.88), thickly populated myofibroblasts (20.33 ± 1.20 and 19.33 ± 0.88), and mast cells (9.67 ± 0.88 and 7.67 ± 0.88) respectively. Significant reduction in the number of inflammatory cells, neutrophils, and macrophages established the effectiveness of 1% EG and positive control groups in the successful regeneration and remodeling of wounded skin tissues  of hydroxyproline content was achieved by positive control (49.82 ± 0.44 mg/g), followed by 1% EG (42.92 ± 2.12 mg/g) and 0.5% EG (37.97 ± 0.88 mg/g) ointment treated groups, compared to ointment base (16.87 ± 0.07 mg/g) and negative control (14.97 ± 0.92 mg/g) groups  such as hydroxyproline and hexosamine in the granulation tissues of all the treatment groups were measured on the 15th post-wound day. A significantly higher quantity  groups . Whereas) groups .p ≤ 0.001) in 1% EG (16.31 ± 0.15 and 45.83 ± 4.76 U/mg respectively), positive control (16.30 ± 0.24 and 51.72 ± 4.65 U/mg), and 0.5% EG (15.60 ± 0.15 and 40.97 ± 3.39 U/mg) ointment treated groups, in comparison with ointment base and negative control groups , 1% EG (4.10 ± 0.06 μg/g), and 0.5% EG (4.05 ± 0.09 μg/g) ointment-treated groups, in comparison with ointment base and negative control groups, with 2.58 ± 0.06 and 2.38 ± 0.09 μg/g respectively .A significant downregulation of LPO, the oxidative stress-mediated indicator, was reported in the positive control and 1% and 0.5% EG ointment-treated groups with 2.91 ± 0.16, 3.16 ± 0.18, and 3.40 ± 0.11 nM/mg compared to ointment base (7.03 ± 0.16 nM/mg) and negative control (7.20 ± 0.14 nM/mg) groups . DownregThe active site residues of COX-2 (PDB ID: 5IKT) were His90, Val116, Arg120, Phe205, Phe209, Val228, Tyr348, Val349, Leu352, Ser353, Tyr355, Leu359, Asn375, Ile377, Phe381, Leu384, Tyr385, Trp387, Phe518, Met522, Val523, Gly526, Ala527, Phe529, Ser530, Leu531, Gly533, and Leu534. In MMP-9 (PDB ID: 4HMA) the active site residues were Tyr179, Pro180, Gly186, Leu187, Leu188, Ala189, His190, Ala191, Phe192, Pro193, Leu222, Val223, His226, Gln227, His230, Gly233, Leu234, Asp235, His236, Ser237, Ser238, Pro240, Ala242, Leu243, Tyr245, Pro246, Met247, and Tyr248. Similarly, the active site residues in TNF-α (PDB ID: 2AZ5) were His15, Leu57, Ile58, Tyr59, Ser60, Tyr119, Leu120, Gly121, Gly122, Gln149, Val150, Tyr151, and Ile155.EG scored the lowest binding energy (BE) of -6.2 kcal/mol with COX-2 through the formation of five non-hydrogen bond interactions with Ala527 (2), Tyr355, Val523, and Leu352. The lowest BE of −4.6 kcal/mol was scored by EG with TNF-α by forming one hydrogen bond interaction with Ser60 and four non-hydrogen bond interactions with Leu57, Ile155, and Tyr59 (2). Likewise, the binding energy of EG with MMP-9 was predicted to be −7.2 kcal/mol through the formation of one hydrogen bond interaction with Ala242 and seven non-hydrogen bond interactions with Arg249, Leu222, Leu243, His226 (2), Val223, and Leu188. 2 and 255 nm2. Interestingly, a similar trend in the surface area was observed to that of Rg and which indicates a decreased surface area of the binding pocket and the formation of a stable ligand-protein complex. The complex formed three H-bonds, of which one H-bond was consistent throughout the simulation. The EG-COX-2 complex exhibited stable dynamics after an equilibration period of 35 ns. Initially, the backbone and the complex root mean square deviation (RMSD) values gradually increased from ∼1 to 4 Å respectively and further showed steady decrease in the RMSD up to 73 ns and showed stable RMSD throughout 100 ns MD simulation. The N-terminal residues showed larger fluctuations up to ∼8 Å and the residues Ala527, Tyr355, Val523, and Leu352 showed relatively fewer fluctuations (∼2.0 Å), as they participated in stable non-bonded interactions . The com2. The complex formed four H-bonds, of which two H-bonds were consistent throughout the simulation. The EG-MMP-9 complex showed stability during 100 ns of stimulation after a 40 ns equilibration period. Initially the complex RMSD and backbone values gradually increased from 4.0 to 5.3 Å and from ∼1.0 to 3.5 Å, respectively. The N terminal residue showed the highest fluctuation up to 8Å and the residues Ala242, Arg249, Leu243, His226, and Val223 showed relatively fewer fluctuations (∼1.8 Å), as they participated in stable non-bonded interactions with MMP-9, while the residue Leu222 and Leu188 showed higher fluctuations (∼3.5 Å) . In EG-MThe EG-TNF-α complex showed higher fluctuations during 100 ns of stimulation as compared to the backbone. The backbone showed stability throughout the stimulation process of 100 ns. The RMSD of the backbone was observed to be from 0 to 4. However, an increase in the RMSD value of the complex was observed from 0 to 25 Å. EG was found to be unstable with TNF-α as it moved out of the binding pocket . The N tWound healing is an important physiological process to restore the anatomical and functional integrity of injured skin tissue. This is regulated by various complex cellular and molecular pathways to induce proliferation, migration, and differentiation of cells and thereby affected tissue renovates at the earliest to its normal status . Even thC. mimosoides is one of the most important ethnomedicinal plants being utilized by the herbal healers of Thailand and India to treat multiple diseases, chiefly wounds and other skin-related ailments. We have previously evaluated the wound-healing activity of crude ethanol extract and its PEF (3:MeOH) in different ratios, producing 24 fractions. Of them all, fraction nine exhibited a significantly higher percentage of cell migration in scratch wound assay with effective antimicrobial and antioxidant activities. Owing to its potential activities, fraction nine was re-fractionated to get sub-fractions which were again analyzed for scratch wound assay, followed by an antimicrobial activity. Among all the 15 sub-fractions, the sub-fraction 9.13 was the most active in promoting a higher percentage of cell migration followed by significant antimicrobial and antioxidant activities. The sub-fraction 9.13, containing only one dominant compound, was directly analyzed by various spectroscopic studies and further was confirmed as EG. its PEF , demonstAcacia nilotica (L.) Delile  Pers. (Libidibia ferrea (Mart. ex Tul.) L.P.Queiroz  Wight & Arn.  Delile , Acalyphacalypha , Caesalpiosa DC. , Dimocargan Lour , Euphorbna Steud , Geraniuianum L. , Lagerst.) Pers. , Libidib.Queiroz , Phyllannaria L. , Pistaci Brandis , Terminat & Arn. , and Terula Retz . We repoectively . Previouectively  and it sectively . It was ectively , antimicectively , muscle ectively , and antectively  activitiC. mimosoides through bioassay-guided fractionation and isolation procedure, followed by its wound-healing activity for the first time. More than 100% viability of cells was recorded at ≤7.81 μg/ml dose and the cell toxicity (IC50) was determined at 104.08 ± 1.19 μg/ml concentration. This is predominantly within the recommended threshold (toxicity level: IC50 ≤ 30 μg/ml) set by the American National Cancer Institute (ANCI) . The stimulation of cell migration at 3.90 μg/ml dose of EG was nearly similar to that of the positive control (EGF). The study revealed that in vitro wound-healing efficacy of EG was comparatively superior over PEF (the fraction containing EG), in which the cell migration rate in PEF was 89.16 ± 1.98% and 92.59 ± 1.53% in L929 cells, at 3.90 and 7.81 μg/ml doses respectively at the 48th hour of incubation period , and the activity was decreased with increased concentrations of the sample (Boesenbergia kingii Mood and L.M. Prince enhanced the migration of L929 cells to 60%–76% at 10 μM concentrations on the second day of incubation (Pistacia vera L. at concentrations higher than 200 μg/ml.Here we reported the isolation and characterization of EG in e (ANCI) , which pn period . Previoun period . In the e sample . Similarcubation . On the O-rhamnoside, quercetin-3-O-rhamnoside, and rutin   . NeverthHydroxyproline and hexosamine are vital components of collagen fiber and hence the measurement of these two contents in granular tissue is a key indicator for collagen turnover . In the 2 signaling pathways (in silico studies. The docking parameter was performed for EG against the protein targets of inflammatory markers such as COX-2, MMP-9, and TNF-α. The mode of binding predicted by the docking studies established a connection with the affinity of binding and the biological activity of the constituent. Previous studies carried out by D. longan fruits in cell lines and experimental animals might be attributed to the activation of COX-1 and COX-2 peroxidase active sites stimulated by EG. The molecular dynamic studies elucidated its competitive binding mode and stability of the complex with COX-2. EG at C-3-OH or C-4-OH site markedly shortened the distance between Fe4+ and the respective O−and thereby increased the binding affinity. Further, κB p-65 and Bcl-2/Bax. Our finding is in line with previous reports and EG was found to bind stably with the active site residues of MMP-9. However, on looking into TNF-α, EG was unstable throughout 100 ns MD simulation, which supports EG as a potent inhibitor for inflammatory markers and playing a crucial role in the wound-healing process.It is a notable fact that normal levels of pro-inflammatory cytokines prevent infection and accelerate the normal wound-healing process. However, excessive production of inflammatory markers is detrimental to successful wound healing as it leads to prolonged inflammatory conditions . Thus, cpathways . In our C. mimosoides. The compound displayed a remarkable wound-healing activity with its enhanced antimicrobial and anti-inflammatory properties. The study highlighted the role of EG, which could facilitate the proliferation of extracellular matrix components such as fibroblasts, keratinocytes, epithelial cells, blood vessels, and collagen fibers, which are known to contribute to successful re-epithelialization of wounded tissues. Additionally, the significant up-regulation of enzymatic and non-enzymatic cellular antioxidant elements and significant down-regulation of oxidative stress-mediated markers endorsed the potentiality of EG in the wound-healing process. Molecular docking analysis of EG against different molecular targets predicted the possible mechanism of action in wound-healing activity. Our study successfully validated the wound-healing efficacy of EG and supports the traditional applications of C. mimosoides plant in wound treatments. Further, these findings pave the way for molecular mechanistic studies and clinical investigations that could enable the use of EG as a promising natural remedy for the treatment of cutaneous wounds.Using a bioassay-guided fractionation procedure, we successfully isolated an active natural antioxidant compound, EG, from PEF of


Compared to the control group, the synergistic approach bolstered enzymatic responses linked to disease resistance and ROS-scavenge system, consistently preserving total phenolics, flavonoids, ascorbic acid, and glutathione levels. Concurrently, the accumulation of hydrogen peroxide and malondialdehyde was significantly diminished post-treatment. These results show that there is good synergistic effect between 1-MCP and ε-PL, which could effectively maintain the quality of flat peach fruit by modulating cell wall metabolism and enhancing the resistance.Flat peach, a predominant fruit consumed in China, is highly susceptible to softening and perishable. The impact of 1-methylcycloproene (1-MCP) fumigation combined with ε-poly-L-lysine (ε-PL) on softening and postharvest reactive oxygen species (ROS) and phenylpropanoid pathway metabolisms in peaches and its relationship to disease resistance were investigated. Findings revealed that a combination of 1 µL L Prunus persica L. Batsch. var. Compressa Bean), a variant of the peach (Prunus persica (L.) Batsch), belongs to the Rosaceae family [Flat peach  and in turn can help preserve cell wall components, such as cellulose, pectin, and hemicellulose [−1 of 1-MCP, the decay rate of strawberries and oranges increased [Penicillium digitatum did not change after 1-MCP fumigation [Currently, cold storage stands as the predominant strategy to retard postharvest peach softening, yet its utilization risks inducing chilling injuries. Consequently, it often synergizes with alternative treatments . Empiricellulose ,9. Howevellulose ,11. Treancreased ,13. Moremigation . On the migation . Thus, sε-Poly-L-lysine (ε-PL) is recognized as an innate antagonist against plant pathogens . Its broTo date, no literature elucidates the cooperative modulation of 1-MCP and ε-PL in governing postharvest physiology and pathogenic susceptibility in peaches. This investigation pivots on the merger of 1-MCP fumigation and ε-PL atomization for postharvest peach conservation. The ability to maintain fruit quality was studied by analyzing the cell wall metabolism, reactive oxygen species (ROS) metabolism and disease resistance. The aim of this study is to lay the foundation for further application of physiological and disease control preservatives in the postharvest disease control and quality maintenance of fruits.Prunus persica L. Batsch. var. Compressa Bean) were cultivated in an orchard in Shihezi, Xinjiang, China , with harvest executed in August 2021. The mature fruit exhibited the following characteristics: firmness of 14.97 ± 0.84 N, soluble solids content of 12.7 ± 0.53%, and titratable  acidity of 0.15 ± 0.006%. Experimental treatment according to the described process: fruit selection → 1-MCP Fumigation → transport → ε-PL soaking → precooling (Including drying) → packaging→ storage at 0 °C. The specific parameters are described below. Only morphologically consistent fruits, devoid of visible defects or ailments, were selected. These peach samples underwent 1-MCP fumigation in a hermetically sealed 1.0 m3 polyethylene tent (0.12 mm thickness) at 16 ± 0.5 °C, where a preformulated solution released 1 µL L−1 1-MCP vapor [−1 under 0 ± 0.5 °C for a 30-day period, discerned the 300 mg L−1 ε-PL concentration as most efficacious against peach decay. The flat peaches, pre-treated with or without 1-MCP, were randomly immersed in ε-PL solution (300 mg L−1) (as ε-PL+1-MCP or ε-PL) or distilled water (as 1-MCP or the control) for 5 min. The fruit were placed in a 0 °C cold storage for pre-cooling for 24 h before proceeding with the next step of processing. Once air-dried, fruits were packaged in 0.05 mm polyethylene bags  and stored at 0 °C with 90–95% relative humidity for 30 d.“Yinggeer” flat peaches . This device, fitted with a 3.5 mm-diameter probe, was inserted at three equidistant points around the fruit’s equatorial zone . FirmnesMicrostructures of the peach pulp specimens from both initial and 30 d were analyzed with a scanning electron microscope , in alignment with the procedures outlined by Jia et al. . For preβ-Gal, and cellulase were ascertained using previous methods [w/v), 1 M NaCl, and mercaptoethanol . The resulting mixture was centrifuged at 10,000× g for 30 min, after which the separated supernatants (yielding crude enzyme extracts) were employed for enzymatic activity assessments.The activities of PG,  methods ,30. Specw/v) and 0.2 mL sodium acetate buffer . This solution underwent incubation at 37 °C for 30 min. Following the addition of 1 mL of 3, 5-dinitrosalicylic acid , the reaction was stopped with a 5 min boiling bath. PG activity’s absorbance was quantified at 540 nm, with D-galacturonic acid as a reference. For β-Gal activity, 0.2 mL of the crude enzyme extract combined with 0.5 mL of 50 mM sodium acetate buffer, and 10 mM p-nitrobenzene-β-D-Galactoside 0.2 mL. After incubating the solution at 37 °C for 1 h, 2 mL of sodium carbonate  was introduced. β-GAL activity’s absorbance was determined at 400 nm using p-nitrophenol as a standard. For cellulase activity, the mixture consists of 0.1 mL of crude enzyme, 0.5 mL of 0.1 M sodium acetate buffer, and 0.4 mL of 1% carboxymethyl cellulose, with subsequent steps aligning with those for PG activity. PME activity was determined as per Zhao et al. [w/v) solution in an ice bath, agitated for 4 h, and centrifuged at 10,000× g for 20 min at 4 °C. The supernatant was curated as the enzyme extract. This extract (0.1 mL) was combined with 0.75 mL distilled water, 2 mL pectin solution , and 0.15 mL bromothymol blue . PME activity’s absorbance was evaluated at 620 nm, with results indicated as U kg−1.For PG activity, 0.1 mL of the crude enzyme combined with 0.3 mL polygalacturonic acid  at 4 °C for 15 min. Subsequent fixation, dehydration, and embedding steps were conducted as per Wang et al. [Random peach flesh samples, sized 1 mmg et al. . MicrogrDecay rates were measured using a prior visual evaluation technique . Peaches2O2 concentrations were quantified following the protocol of Li et al. [−1 fresh weight (FW). For the determination of malondialdehyde (MDA), the thiobarbituric-acid-reactive substance methodology [−1) and centrifuged at 16,000× g for 15 min. The assay mixture, comprising 2.0 mL supernatant and 2.0 mL of 0.5% thiobarbituric acid (TBA), underwent a boiling treatment, subsequent cooling, and then centrifugation at 10,000× g for 10 min. Absorbance was read at wavelengths of 450, 532, and 600 nm. MDA concentrations are denoted as μmol kg−1 FW.The Hi et al.  and are hodology  was empl−1 FW.The quantification of total phenolics in nectarines was executed using the previous methodology . Absorba3, and 2 mL of sodium acetate buffer (pH 5.5) were mixed evenly. The optical density was measured at 510 nm, and findings are presented as mg rutin equivalents per kg−1 FW.For flavonoid content determination, an adapted protocol from Toor and Savage  was foll−1 FW.The ascorbic acid (AsA) content was quantified using a sample size of three replicates, with each replicate consisting of five fruits. Assays utilized the 2,6-dichlorophenol-indophenol dye titration technique as delineated previously . Results−1 FW.Lastly, the determination of total GSH content was performed according to the Castillo and Greppin protocol . An extr−1), 2.0 mL phosphate buffers (100 mmol L−1), 0.5 mL 0.5 mmol L−1 H2O2, 0.2 mL enzyme extract, and 0.5 mmol L−1 EDTA. For GR, the mixture contained 3.0 mL of 100 mmol L−1 phosphate buffers (pH 7.5), 100 μL of 5.0 mmol L−1 oxidized glutathione, 0.2 mL enzyme extract, and 30 μL of 300 μmol L−1 NADPH. Optical densities for APX and GR mixtures were noted at 290 nm and 340 nm, respectively, with activities denoted in U g−1 FW. The activities of POD, CAT, and SOD were determined with the respective kits. Specifically, a 0.1 g flat peach sample was processed in 1 mL extraction buffer, centrifuged at 10,000× g, 4 °C for 10 min, and the resultant supernatant was isolated. Following the kit protocols, reagents were incorporated in sequence, with absorbance readings at 470 nm, 240 nm, and 560 nm post specific reaction intervals. Activity measurements were uniformly represented in U g−1 FW.APX and GR enzymatic activities were quantified employing a refined method from Ma et al. . The APXThe enzyme activity of PAL and 4CL were determined employing Zhang et al.’s method . Absorbap = 0.05. Data are reported as mean ± standard deviation.Three experiments were conducted systematically for each treatment. Data analysis utilized SPSS 22.0. A one-way ANOVA assessed the data with post hoc comparisons using Duncan’s multiple range tests, significant at p < 0.05) during the 6–30 d storage period. The firmness in ε-PL group was 13.53% or 12.07% higher than untreated group at 12 d or 18 d, respectively. Furthermore, from 0–24 d, no discernible difference in firmness was found between ε-PL+1-MCP and 1-MCP treated fruits. The ethylene production in peaches from four groups during storage showed a fluctuating tendency (tendency C. Ethyletendency D. In thep < 0.05), compared to the control group ε-PL, on the 18th day of storage. Conversely, PME activities on the 30th day were noticeably reduced (p < 0.05) in the ε-PL+1-MCP compared to the 1-MCP.The PG activities of flat peaches in all treatments increased with storage time A. On dayβ-Gal activity in control flat peaches rose slowly from 0–12 d, rose sharply during storage (12–18 d), and enhanced slowly from 18–30 d  between 18 to 30 d of storage. In addition, from 24 d to 30 d, ε-PL+1-MCP showed significantly lower (p < 0.05) β-Gal activity when compared to ε-PL or 1-MCP alone.  18–30 d C. HoweveTEM imaging detailed the microstructure of cell walls within flat peach pulp across storage A–F. At ip < 0.05) curtailed the spoilage rates over the storage duration  surpassing those in ε-PL, 1-MCP, and ε-PL+1-MCP treatments. However, ε-PL and the combined ε-PL+1-MCP efficaciously curbed MDA accumulation, registering 1.51 ± 0.10 μmol kg−1 and 1.45 ± 0.09 μmol kg−1, respectively.Relative to controls, treatments with ε-PL, 1-MCP, and ε-PL+1-MCP on the 30th day led to a diminished Hat order B. MDA leat order C. By the−1 in total flavonoids was reached on day 18 in peaches treated with the combined ε-PL+1-MCP, overshadowing all alternative groups.A discernible amplification in total phenolic contents was noted in the peaches, succeeded by a minor decrement as storage persisted D. In rel−1 and 220.81 ± 9.52 mg kg−1, respectively  POD activity from 24–30 d when compared to ε-PL or 1-MCP alone. The CAT activity of the control group and 1-MCP group reached its maximum on the 18th and 24th days of storage, respectively. Both values were less than of the combined treatments (p < 0.05)  D. Compar < 0.05) E. Over dp < 0.05). The peak of PAL activity for flat peaches with ε-PL+1-MCP treatment occurred on the 24th day of storage, registering an increase of 1.85 times compared to the control. For all groups, the pulp C4H activity in flat peach rose initially, then dropped (p < 0.05).Throughout the storage period, PAL activity in flat peach tissues exhibited an initial rise, followed by a decline A. In the dropped B. Althou dropped C. The 4Cβ-Gal, and cellulase activities (p ≤ 0.05). The decay rate shows a positive correlation trend with H2O2 and MDA, while H2O2 is positively correlated with MDA, GSH, POD, and CAT. In addition, total phenolics was positively correlated with the GSH, POD, CAT, SOD, PAL, and C4H, while total flavonoids was significantly positive correlated with the APX, GR, CAT, SOD, PAL, C4H, and 4CL activities.Pearson’s correlation analysis was used to evaluate the potential relations between firmness and cell-wall-degrading enzymes as well as between decay incidence, reactive oxygen species, and phenylpropanoid metabolism indexes of peach fruit . During The softening of fruit is a major aspect that affects transportability, storage, and shelf life. One of the most direct characteristics of quality loss during storage is the fast decrease in peach fruit firmness. In this study, the firmness of peach fruit showed a significant downward trend during the storage cycle. However, 1-MCP treatment maintained the firmness of flat peaches in cold storage. Similar results have also been obtained in other fruit, such as cheri-moyas , nectariβ-GAL, and cellulase [β-Gal, a critical enzyme in pectin debranching, partakes in the hydrolysis of β-1, 4-galactan bonds. Moreover, β-Gal, as a critical enzyme of pectin-debranching, is involved in the hydrolysis of β-1, 4-galactan bonds. Cellulase can cause cellulose degradation in the cell wall [β-GAL and cellulase activities compared to the control fruit. Win et al. [β-galactosidase activity in apples, contributing to a delay in the degradation of pectin polysaccharides. Agreeing with our result, Xiong et al. [β-Gal, and cellulase activities (p ≤ 0.05). Our results showed that ε-PL+1-MCP was effective in delaying flat peach softening by suppressing the above enzymatic activities. Furthermore, the results from ultrastructural observation again provided direct evidence that flat peach fruit softening was accompanied by disruption of the cell wall structure. Importantly, ε-PL+1-MCP treatment delayed cell wall degradation, and this served to maintain the integrity of the cell wall structure and further delay the softening process of cold-stored peach fruit, which was in line with previous reports demonstrated in nectarine treated with hot air and 1-MCP [Fruit softening during ripening associates with the polysaccharide compositions degradation in cell wall, which is modulated by several enzymes such as PG, PME, ellulase . Specifiellulase . In the ellulase . Moreoveell wall . During n et al.  found thg et al.  found thnd 1-MCP  and aprind 1-MCP  treated 2•−) under the action of nicotinamide adenine dinucleotide phosphate oxidase, which is disproportionated to H2O2 by the SOD in the plant cells [2O2 into H2O and O2 to prevent excessive ROS accumulation in plant cells, while APX and GR also participate in ROS elimination through the AsA–GSH cycle. In this work, ε-PL application raised AsA and GSH contents and the activities of APX, GR, POD, CAT, and SOD and decreased H2O2 content in flat peaches during storage, strengthening resistance against pathogens. A study revealed that ε-PL upregulated the relative gene expression of MdAPX, MdPOD, MdCAT, and MdSOD to control Alternaria rot of postharvest apples [2O2 and MDA. These results showed that ε-PL combined with 1-MCP inhibited H2O2 and MDA accumulation and induced fruit disease resistance.Enormous economic losses in the flat peaches during storage, transportation, and retail are caused by pathogen infection . ε-PL hant cells . As antit apples . The flaSecondary metabolites generated from the phenylpropanoid pathway maintain integrity of plant cell walls and protect plants against pathogenic invasion . PAL, C4−1 1-MCP fumigation and 300 mg L−1 ε-PL not only delayed the softening but also maintained a lower decay rate of flat peaches stored at 0 °C for 30 days after harvest. The composite treatment has a good effect on maintaining fruit firmness due to the inhibited PG, PME, β-Gal, and cellulase activities retarding the degradation of cell wall structure in peaches. The reactive oxygen species (ROS) and phenylpropanoid pathway metabolisms were regulated by ε-PL and 1-MCP. Thus, the postharvest decay of peaches was reduced. These results imply that a synergistic application of ε-PL and 1-MCP presents a promising strategy for improving the quality of peach fruit.In summary, the combination of 1 µL L


Rodgersia podophylla A. Gray (R. podophylla) is a traditional Chinese medicine with various pharmacological effects. However, its antioxidant and anti-hyperuricemia components and mechanisms of action have not been explored yet. In this study, we first assessed the antioxidant potential of R. podophylla with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric ion reducing antioxidant power (FRAP) assays. The results suggested that the ethyl acetate (EA) fraction of R. podophylla not only exhibited the strongest DPPH, ABTS radical scavenging and ferric-reducing activities, but also possessed the highest total phenolic and total flavonoid contents among the five fractions. After that, the potential superoxide dismutase (SOD) and xanthine oxidase (XOD) ligands from the EA fraction were quickly screened and identified through the bio-affinity ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS). Accordingly, norbergenin, catechin, procyanidin B2, 4-O-galloylbergenin, 11-O-galloylbergenin, and gallic acid were considered to be potential SOD ligands, while gallic acid, 11-O-galloylbergenin, catechin, bergenin, and procyanidin B2 were recognized as potential XOD ligands, respectively. Moreover, these six ligands effectively interacted with SOD in molecular docking simulation, with binding energies (BEs) ranging from −6.85 to −4.67 kcal/mol, and the inhibition constants (Ki) from 9.51 to 379.44 μM, which were better than the positive controls. Particularly, catechin exhibited a robust binding affinity towards XOD, with a BE value of −8.54 kcal/mol and Ki value of 0.55 μM, which surpassed the positive controls. In conclusion, our study revealed that R. podophylla possessed remarkable antioxidant and anti-hyperuricemia activities and that the UF-LC-MS method is suitable for screening potential ligands for SOD and XOD from medicinal plants. Rodgersia podophylla A. Gray (R. podophylla), a member of the Saxifragaceae family, is primarily found in the Hubei, Sichuan, Yunnan, and Tibet of China. It grows in shady and wet places such as undergrowth, shrubland, meadows, and rock crevices, at altitudes range of 1,100–3,400 m , and non-free radical species, such as hydrogen peroxide (2•− to H2O2 and oxygen (O2) in the presence of transition metal ions, while H2O2 can be further decomposed into hydroxyl radicals (·OH) and hydroxide ions (OH−) (2•−) from xanthines and hypoxanthines (2•− produced by XOD metabolism are closely associated with hyperuricemia (Reactive oxygen species (ROS) are cellular metabolites, and are normally classified into two categories: free radicals, such as superoxide anion radicals  . XOD is anthines . Excess uricemia , gout , ABTS ), and FRAP (ferric-reducing antioxidant power). The extracts included crude extract (CE), n-hexane (n-Hex), dichloromethane (DCM), ethyl acetate (EA), and water (H2O) fractions. At the same time, the total phenolic (TPC) and total flavonoid content (TFC) in different fractions of R. podophylla were detected to reveal their correlations with antioxidant activity. Then, the potential SOD and XOD ligands within the EA fraction were fast fished out with the UF-LC-MS method. Finally, molecular docking, along with the XOD inhibition assays in vitro, were employed to explain the interactions between the active components and the target enzymes. Consequently, this work provided a valuable reference for the development and utilization of R. podophylla as a natural antioxidant and anti-hyperuricemia drug or health supplement.In this case, due to the complex and diverse chemical compositions of R. podophylla was collected in Xintang Township, Enshi City, Hubei Province, China, and was identified by Guangwan Hu, a senior taxonomist from the Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture , Chinese Academy of Sciences. The voucher specimen (No. 20220705) was preserved in the herbarium of the Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture.The rhizome of R. podophylla . 2 g of CE was dissolved in 100 mL of water and extracted consecutively with n-hexane , dichloromethane , and ethyl acetate  to obtain n-Hex (24.3 mg), DCM (15.10 mg), EA (398.40 mg), and water  fractions, respectively. The obtained samples were stored in a sealed container and kept in a refrigerator at 4 °C for future use.The dry, crushed rhizome (20 g) was soaked overnight in 200 mL of 50% ethanol at room temperature, then extracted using ultrasound for 50 min. This process was repeated three times, and the filtrate was collected. The filtrate was then concentrated using a rotary evaporator, and dried using a vacuum freeze dryer to obtain the crude extract of The chemical standards of gallic acid, bergenin, procyanidin B2 and catechin (Purity ≥98.0%) were bought from Chengdu Alfa Biotechnology Co., Ltd . Rutin was purchased from J&K Scientific Ltd. , Folin-Ciocalteu reagent, 1,3,5-tri (2-pyridyl)-2,4,6-triazine (TPTZ), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and ascorbic acid  were purchased from Sigma-Aldrich Corp . The acetonitrile (ACN) and methanol of HPLC grade were supplied by TEDIA Company Inc . All other analytical solvents and chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. . Superoxide dismutase (SOD) and xanthine oxidase (XOD) were bought from Shanghai Yuanye Bio-Technology Co., Ltd. . Ultrafiltration membranes  were purchased from Millipore Co. Ltd. . Water (ultrapure grade) for HPLC and HPLC-UV-ESI-MS/MS analyses was prepared with EPED .HPLC-UV/ESI-MS/MS was conducted with a Thermo Accela 600 series HPLC system coupled with a TSQ Quantum Access MAX mass spectrometer . As for the HPLC analysis, an Agilent 1220 LC  with a RP-C18 column  was applied, and the UV absorbance was recorded by UV/VIS Spectrophotometer . Centrifugation of samples was carried out by low-temperature high-speed centrifuge .R. podophylla samples was determined based on a previously reported study by Muema et al. . The ABTS+ solution was then diluted with methanol to ensure that its absorbance at 734 nm was approximately 0.700 ± 0.100. Next, 190 μL of the ABTS+ solution was mixed with 10 μL of the sample and the absorbance was measured at 734 nm after incubating in the dark for 30 min. Methanol and Vc  were used as blank and positive controls, respectively. The calculation of ABTS scavenging activity results followed the previous format in DPPH part.The ABTS free radical scavenging activity of different n et al. . An ABTS3+-TPTZ solution) containing 20 mM FeCl3.6H2O, 10 mM TPTZ, and 300 mM acetate buffer  was prepared in a ratio of 1:1:10 (v/v/v) and stored at 37°C. Next, 10 μL of appropriately diluted sample, 30 μL of ultrapure water, and 260 μL of fresh Fe3+-TPTZ solution were added to a 96-well plate and incubated at 37°C for 10 min. The absorbance of the mixture at 593 nm was then measured using the multifunctional microplate reader, with three replicates per sample. FeSO4.7H2O  was used as a standard to establish a calibration curve, and FRAP activity was expressed as the equivalent mM Fe2+ per Gram of sample (mM Fe2+/g sample).The determination of ferric-iron-reducing antioxidant power (FRAP) was performed using a slightly modified method described by Xu et al. . The FRA2CO3, 1 M), and 20 μL of ultrapure water. The reaction was allowed to proceed for 1 h at room temperature in the dark. The absorbance was then recorded at 760 nm. Gallic acid solution (5–40 μg/mL) was used as a standard to establish a calibration curve. Results for TPC were expressed as milligrams of gallic acid equivalents per Gram of dry sample (mg GAE/g sample).TPC was measured using the Folin-Ciocalteu method reported by Fan et al. . The reaR. podophylla ethanol extract and different fractions was determined using a slightly modified method described by Chen et al.  and incubated at room temperature for 6 min. Next, 12 μL of aluminum nitrate (Al(NO3)3, 10%, w/v) was added and incubated for an additional 6 min. Finally, 120 μL of sodium hydroxide solution  and 58 μL of methanol solution were added, and the reaction was allowed to proceed for 15 min before measuring the absorbance at 510 nm. Rutin was used as the standard, and the results for TFC are expressed as milligrams of rutin equivalents per Gram of dry sample (mg RE/g sample).The TFC of n et al. . A 96-weR. podophylla EA fraction was dissolved in 1 mL of Tris-HCl buffer solution (pH = 7.8) and sonicated for 30 min. Next, 180 μL of the sample solution was mixed with 20 μL of SOD  or XOD  and incubated at 37°C for 1 h. The mixed solutions were then transferred to a 30 kD ultrafiltration tube and centrifuged at 10,000 rpm for 10 min. The tube was washed three times with the appropriate PBS buffer to remove non-specifically bound components. Next, 200 μL of methanol  was added and incubated for 20 min to release the enzyme-bound compounds, followed by centrifugation at 10,000 rpm for 10 min (n = 3). Finally, the ultrafiltrate was collected, dried using a termovap sample concentrator, and redissolved in 50 μL of 90% methanol for further analysis. As a negative control, an equal amount of enzyme was incubated in boiling water (100°C) for 10 min and treated in the same manner as the active enzyme group.Based on previous studies , the UF-R. podophylla EA fraction were currently analyzed and identified with a Thermo Accela 600 HPLC system in combination with a TSQ Quantum Access MAX MS equipped with an ESI interface. Separation was performed using a Waters Sunfire RP-C18 column . The mobile phases for HPLC elution were 0.1% (v/v) formic acid aqueous solution (A) and ACN (B). The elution conditions were as follows: 1%–5% (B) in 0–5 min, 5%–14% (B) in 5–25 min, and 14%–20% (B) in 25–60 min. The injection volume was 10 μL, the flow rate was 0.8 mL/min, and the wavelength was set at 310 nm (2) and axu gas  was 40 psi and 10 psi, respectively; the source voltage was 3000 V and the cone voltage and collision energy were 40 V and 10 V, respectively; mass range (m/z) was from 150 to 1,500, spray voltage was 3 kV. MS data  was obtained in the full-scan mode. The preliminary identification of compounds in the EA fraction of R. podophylla was carried out by comparing the parent ions, MS fragments, and retention times with the references.The chemical compositions of the t 310 nm . The ESIwww.rcsb.org), while the 3D structure of the ligand was downloaded from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (old.tcmsp-e.com). Next, water molecules and ligand fractions were removed using the PyMOL. Hydrogen atoms were added to proteins and ligands using AutoDock Tools, which was also used to calculate charges and perform other processing of their 3D structures. The coordinates of the active sites of SOD and XOD were  and   , respectSample, ABlank and ABackground are defined as the absorbance of the test sample (with the enzyme), the blank (the test sample without the enzyme) and the 100% enzyme activity (only the solvent with the enzyme and the substrate), respectively. The extent of inhibition was expressed as the concentration of sample needed to inhibit XOD activity by 50% (IC50).The XOD inhibition experiment was performed using a modified method previously reported by Wang et al. . In a 9650 values were calculated by plotting the percentages of scavenging activities or inhibition rates against the sample concentrations (six different concentration gradients in triplicate). Statistical analysis was performed using software of SPSS 25.0 , Origin 2021  and GraphPad Prism 9.0 .All data in this work were measured in triplicate and were expressed as mean ± standard deviation (SD). The ICR. podophylla extracts . This deviated slightly from the findings presented by Zhang (R. aesculifolia (the Rodgersia genus), and exhibited a moderately higher FRAP values compared to Vc. Among the three antioxidant capacity assays, the CE fraction also showed better antioxidant activity, followed by the H2O fraction, while the other extracts (DCM and n-Hex) showed moderate antioxidant capacities. Considering that the EA fraction showed the strongest antioxidant capacity compared to other fractions, this result aligned with Yan’s findings  was also the highest among the five samples, followed by the CE fraction (595.326 ± 4.14 mg RE/g). The lowest TFC was 77.65 ± 0.13 mg RE/g at the HR. podophylla and its TPC and TFC. The results are presented in R2) between DPPH with ABTS and FRAP were notably high at 0.998 (p < 0.01) and −0.838 (p < 0.05), respectively. Furthermore, the R2 value between ABTS and FRAP was −0.812 (p < 0.05). These findings suggested that these three methodologies were not only reliable but also interchangeable. On the other hand, there were also strong correlations between antioxidant activities and chemical compositions. The FRAP values showed an closely positive correlation with the TPC and TFC at 0.960 (p < 0.01) and 0.991 (p < 0.01), respectively. While the DPPH and ABTS values were highly negative correlated with TPC  and TFC , respectively. The R2 value between theTPC and TFC was found to be 0.956 (p < 0.01), indicating an exceptionally robust correlation. Synthesizing these findings, it was discerned that polyphenols and flavonoids could potentially be the primary contributors to the antioxidant activity exhibited by R. podophylla.A comprehensive correlation analysis was executed to thoroughly assess the correlation between the antioxidant activity  of R. podophylla through its various extracts , deprotonated molecular ion [M-H]-, with characteristic fragment ions at m/z 125 and 97. Based on its exact mass, fragment ions, and retention time, as well as comparison with the standard, it was identified as gallic acid , followed by the removal of a portion of water to form the fragment at m/z 407 [C22H17O9-H2O]-. Another characteristic fragment, at m/z 289, corresponded to the cleavage of m/z 577 [M-H]- into the catechin characteristic fragment at m/z 289 [C15H13O6]-. Additionally, m/z 577 also underwent C-ring cleavage to produce the characteristic fragment at m/z 125 [C6H5O3]-, which was also present in the fragment ion of peak 10. Through comparison with standards, peak 10 was ultimately determined as procyanidin B2  from m/z 289, which then further lost -C2H2O to generate m/z 203 [M-H-CO2-C2H2O]-. Namely, it could be inferred that peak 11 represented either catechin or epicatechin. Additionally, the fragment ion at m/z 151 [M-H-C6H6O2-CO]- corresponded to the characteristic fragment ions generated by the cleavage of catechin or epicatechin via C-ring RDA in negative ion mode. Through comparison with standards, peak 11 was ultimately determined to be catechin  (Peak 11 presented a deprotonated molecular ion at m/z 289.18 [M-H]catechin . Previou1 μg/mL) .-, indicating that they were isomers with the formula C21H20O13. The fragment ion at m/z 327 was characteristic of bergenins, while the fragment ions at m/z 169 [C7H5O5]- and m/z 125 [C6H5O3]- were characteristic of gallic acid. Thus, it can be inferred that peaks 14 and 15 represented monogalloyl bergenin. The fragment ion at m/z 327 represented [M-H-galloyl]-, while the fragment ion at m/z 312 represented [M-H-galloyl-CH3]-, m/z 207 represented [M-H-galloyl-C4H8O4]-, and m/z 193 represented [M-H-galloyl-C5H10O4]-. In light of the available literature, it can be speculated that the phytochemical composition of genus Rodgersia may be galloylbergenin at the 3, 4 or 11 position. Based on the different retention times, peak 14 and peak 15 were inferred to be 11-O-galloylbergenin and 4-O-galloylbergenin, respectively , which indicated that these compounds exhibited strong interactions with SOD. Among them, peak 11  displayed the strongest affinity with SOD, followed by peak 15 , peak 14 , peak 2 , peak 1 , and peak 10 . These results were generally consistent with those obtained from bioaffinity ultrafiltration . ThroughFor XOD, peak 11 (catechin) exhibited the highest interaction with XOD, with the lowest BE value of −8.54 kcal/mol and the Ki value of 0.55 μM. In summary, peaks 11, 15, 14, 2, 1, and 10 for SOD and peaks 11, 10, 14, 6 and 1 for XOD were identified as potential ligands that warranted further investigation. Their structures were shown in R. aesculifolia extract achieved an XOD inhibition rate of 72.79%. This rate increased to an impressive 85.18% when the concentration was elevated to 1.25 mg/mL. Meanwhile, the XOD inhibitory activity in vitro of the compounds, identified through UF-LC-MS screening, was further confirmed. As shown in 50 value of 0.680 ± 0.061 mM, which was comparable with the positive control drug, allopurinol (IC50 = 0.156 ± 0.005 mM) (p > 0.05). Compound 11 (catechin) and compound 1  also showed attractive inhibitory effects, with IC50 values of 1.208 ± 0.015 mM and 2.949 ± 0.016 mM, respectively. To this end, the present results not only confirmed the significant XOD inhibitory activity of the compounds screened through bio-affinity ultrafiltration, but also provided a foundation for further investigation into the anti-hyperuricemia properties of R. podophylla.The XOD inhibition assay was conducted with a slightly modified methodology previously described by Wang et al. . At the R. podophylla extracts were firstly assessed using DPPH, ABTS, and FRAP assays. The EA fraction of R. podophylla exhibited the highest radical scavenging and ferric reduction abilities, with IC50 values of 3.863 ± 0.34 μg/mL and 5.537 ± 0.38 μg/mL for DPPH and ABTS, and the FRAP value of 10.655 ± 0.77 mM Fe2+/g, respectively. Additionally, the EA fraction exhibited the highest total phenolic and flavonoid contents among all fractions, with values of 70.984 ± 3.49 mg GAE/g dw and 1,026.096 ± 8.11 mg RE/g dw, respectively. A bio-affinity ultrafiltration coupled with LC-MS/MS strategy with SOD and XOD as target enzymes was employed to screen potential antioxidant and anti-hyperuricemic compounds from the EA fraction. Based on the binding capacities, norbergenin (peak 2), catechin (peak 11), procyanidin B2 (peak 10), 4-O-galloylbergenin (peak 15), 11-O-galloylbergenin (peak 14), and gallic acid (peak 1) were identified as potential SOD ligands, while gallic acid (peak 1), 11-O-galloylbergenin (peak 14), catechin (peak 11), bergenin (peak 6), and procyanidin B2 (peak 10) were identified as potential XOD ligands, respectively. Furthermore, molecular docking revealed that these seven compounds exhibited favorable interactions with SOD and XOD through hydrogen bonding and other types of intermolecular forces. Notably, their binding affinities even surpassed those of the positive controls (DTC and ALL). Thereafter, the XOD inhibition assays in vitro discerned that all these molecular entities demonstrated strong XOD inhibition, especially bergenin and catechin. In summary, this study represented the first application of the UF-LC-MS method for the rapid screening of bioactive compounds in R. podophylla extracts, specifically targeting their antioxidant and anti-hyperuricemia properties. The research not only provided novel evidence to support the antioxidant and anti-hyperuricemic properties of R. podophylla, but also clarified the potential bioactive ligands for its further research and application. These bioactive compounds hold promise for future development as health supplements or natural medicines for the prevention and treatment of oxidative stress and hyperuricemia-associated diseases.In this study, the antioxidant capacities of


Introduction: The genus Trissolcus includes a number of egg parasitoids that are known to contribute to the control of Halyomorpha halys. The number of progenies, particularly females, is important for the efficient mass rearing of species used in augmentative biological control programs. Cold storage is an important technique for extending the shelf life of natural enemies used in such programs.Methods: We assessed how fecundity, sex ratio, lifespan, and the number of hosts parasitized within 24 h were affected by host density for T. japonicus and T. cultratus when offered fresh H. halys eggs and how these parameters were affected if adult parasitoids were first placed in cold storage (11°C in the dark) for 19 weeks before being used for propagation.Results: The fecundity were 110.2 and 84.2 offspring emerged at 25°C, for parasitoids not placed in cold storage; among the offspring that emerged, 82.6% and 85.6% were female for T. japonicus and T. cultratus, respectively. If first placed in cold storage, T. japonicus and T. cultratus produced 35.1 and 24.6 offspring per female, respectively, although cold storage significantly extended the shelf life. The survival rates of parasitoids that were placed in cold storage were 90.3% and 81.3% for females, and 3.2% and 0.9% for males of T. japonicus and T. cultratus, respectively. The number of hosts parasitized within 24 h was not shown to be density dependent, but it was significantly lower after cold storage.Discussion: This information can be used to estimate the likely production for augmented rearing colonies for use in biological control programs. Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is an invasive and polyphagous pest native to China, Japan, and Korea , Eupelmidae (Anastatus spp.), and Encyrtidae (Ooencyrtus spp.)   . The relhabitats . TrissolH. halys . Adventid States , Italy  . HoweverH. halys . Anyhow,ong-term . A recen similar . This fiT. japonicus can be reared on previously frozen (−80°C) H. halys egg masses  of H. halys laboratory colony was established using adults collected from an organic kiwifruit orchard located at the Northwest Agriculture and Forestry University kiwifruit experimental field station  in Mei County, Baoji city, Shaanxi Province, China. The colony was continuously reared on green beans (Phaseolus vulgaris L.) and corn (Zea mays L.) in gauze cages (60 × 60 × 60 cm3) and kept in the laboratory at a constant climatic condition 25°C ± 1°C, 60% ± 1% RH, and a 16:8 h L:D cycle. Food was changed every 3 to 4 days. Newly laid eggs were collected from the cages daily and maintained under the same conditions in separate rearing cages or provided to parasitoids.The Trissolcus japonicus and T. cultratus colonies were started from field collections of parasitized H. halys eggs conducted in the same kiwifruit orchard where the H. halys were collected in Mei County, Baoji city, Shaanxi Province. Both populations were reared separately in transparent acrylic rearing cages (25 × 25 × 25 cm3). Pure honey soaked into cotton wicks was provided weekly for nutrition. For colony production, adult females of either T. japonicus or T. cultratus were provided with fresh H. halys egg masses days old , and parT. japonicus and 563 (28 tubes) T. cultratus for cold storage.All the mated  3-day-old female and male parasitoids were grouped based on each egg mass and placed in a 10-mL tube (16 × 81 mm), and stored in an incubator  at 11°C ± 1°C, 60% ± 1% RH, and continuous darkness for 19 weeks (133 days). All groups were checked weekly for mortality, and honey droplets in tubes were renewed weekly. The same procedure was carried out for each parasitoid species. The total number was 582 (30 tubes) H. halys egg masses (each egg mass of around 28 eggs) were provided for parasitoid oviposition. Egg masses were replaced every 2 days: three egg masses for the first three consecutive times, then one egg mass was provided from the fourth time to refresh host eggs until the female wasp died. Exposed egg masses were placed and reared individually in Petri dishes, under laboratory conditions (as described above) until all eggs produced either parasitoid progeny or stink bug nymphs. In total, 23 replicates  were conducted for T. japonicus and 26 for T. cultratus. The numbers of emerging parasitoids or H. halys nymphs were recorded. Unhatched eggs were dissected under a stereomicroscope and classified as dead immature parasitoids, dead H. halys nymphs, or failed eggs . The proportion of adult parasitoid progeny that were female was calculated, and the average oviposition period and longevity (from parasitoids emergence to death) at 25°C ± 1°C, 60% ± 5% RH, and 16 h light conditions were also recorded. The parasitism rate was defined as (number of emerged parasitoids + number of dead parasitoids within host egg)/total number of H. halys eggs × 100. The rate of nymph was defined as (number of emerged nymphs + number of dead nymphs within host egg)/total number of H. halys eggs × 100. The rate of egg mortality was defined as number of dead eggs/total number of H. halys eggs × 100.Fecundity trials were conducted with parasitoids under ambient (non-cold storage) rearing conditions . Three-day-old mated female parasitoids were randomly selected and placed individually in Petri dishes (d = 5 cm). Honey was provided for adult nutrition, and T. japonicus and T. cultratus.Parasitoid fecundity after cold storage was assessed using the same methods as described for wasps not subject to cold storage. Wasps tested were female parasitoids that had survived 19 weeks of cold storage period. These females were allowed 2 days to adapt to warm ambient conditions before testing. At least ten replicates  were run for T. japonicus and T. cultratus were offered different numbers of H. halys egg masses . Females  were allowed to oviposit for 24 h at 25°C and were then removed from Petri dishes (d = 5 cm). Exposed egg masses were held and reared individually in Petri dishes, under laboratory conditions (as described above), until all eggs produced either parasitoid progeny, live stink bug nymphs, or failed. The numbers of emerging parasitoids or H. halys nymphs were recorded. Failed eggs were dissected under a stereomicroscope and classified as immature parasitoids, dead H. halys nymphs, or failed eggs . Ten replicates were conducted for each treatment. The per-female rate of oviposition was defined as the number of emerged parasitoids + immature parasitoids.Three-day-old mated females of Trissolcus japonicus and T. cultratus, after cold storage for 19 weeks, were also parallely tested, and the female rate of oviposition depends on five host densities. The method was the same as that mentioned previously. Three to nine replicates were conducted for each treatment.T. japonicus and T. cultratus), and treatments (control wasps and one subjected to cold storage) were analyzed using GLM with Poisson distribution followed by LSD post-hoc tests. All statistical analyses were carried out using SPSS 21.0 statistical software. All figures were made using Origin 2022 software.Differences in lifetime fecundity , the duration of the oviposition period and the parasitoid lifespan, the female proportion of parasitoid offspring, parasitism rate, rate of nymph, rate of egg mortality, and female oviposition rate were compared between the two parasitoids species (T. japonicus (n = 23) and T. cultratus (n = 26), respectively, to assess the number of parasitoids produced over their lifetime. An average of 555.5 ± 12.4 or 427.8 ± 7.5 fresh H. halys eggs were offered to each T. japonicus and T. cultratus female, respectively. The average number of parasitoids that emerged from T. japonicus per parental female was 110.2 ± 6.6, while for T. cultratus, it was 84.2 ± 3.2. The former was significantly higher  (In tests conducted with parasitoids reared under ambient temperature conditions (25°C), a total of 12,776 and 11,122 host eggs were provided to < 0.001) .T. japonicus (n = 10) and T. cultratus (n = 10), respectively. The number of parasitoids produced per parental females (surviving cold storage) of T. japonicus was 35.1 ± 7.4, which was a significant decrease of 68.1% compared to the control . Similarly, the number of parasitoids produced per parental female of T. cultratus was 24.6 ± 3.6, which was a significant decrease of 70.8% compared to the control  . There were significant differences in the number of parasitoids produced by T. japonicus versus T. cultratus after cold storage  , a total of 7,171 and 7,327 host eggs were offered to < 0.001) .T. japonicus and T. cultratus in the parasitism rate , rate of nymphal , or the rate of egg mortality  (At ambient conditions (25°C), there was no significant difference between = 0.057) .T. japonicus GLM χ2 = 92.361, df = 1, p < 0.001; T. cultratus GLM χ2 = 193.876, df = 1, p < 0.001) and the egg mortality rate  compared to those in the ambient condition (25°C). Conversely, the nymph rate increased significantly for both parasitoid species after cold storage   .T. japonicus and T. cultratus after cold storage, there were no significant differences in the parasitism rate , the nymph rate , and egg mortality rate  (Between = 0.532) .T. japonicus and T. cultratus, respectively. After cold storage, the proportion of females parasitoids was 78.0% ± 3.9% of T. japonicus, which was not significantly different from wasps reared under ambient conditions . However, the percentage of females produced by T. cultratus in the offspring was 72.4% ± 4.2%, which is significantly lower after cold storage than wasps reared under ambient conditions   .T. japonicus, which was significantly different  from that for T. cultratus (12.8 ± 0.40 days). The number of ovipositions decreased with female age, and the highest level of oviposition was on the first day of access of new parasitoids to hosts when there were 29.5 ± 2.1 eggs/female/day for T. japonicus and 25.1 ± 1.4 eggs for T. cultratus (At ambient rearing conditions (25°C), the mean oviposition duration was 21.5 ± 0.9 days for ultratus .T. japonicus and 17.8 ± 1.9 days for T. cultratus. Oviposition occurred from the first day that parasitoids were exposed to host eggs. The oviposition duration was significantly different between the two species after cold storage   .T. japonicus and T. cultratus females under ambient rearing conditions (25°C) were 30.7 ± 0.7 and 18.9 ± 0.5 days, respectively, which differed significantly between the two species . Adults of both parasitoids were still alive and fertile after 19 weeks of cold storage. The lifespan (period from parasitoid emergence to death) showed no significant difference between the two parasitoid species after cold storage   .T. japonicus GLM χ2 = 883.283, df = 1, p < 0.001; T. cultratus GLM χ2 = 612.088, df = 1, p < 0.001), with mean female survival rates of 90.3% ± 1.6% and 81.3% ± 3.1% for T. japonicus and T. cultratus after 19 weeks of cold storage, respectively. Male survival rates, in contrast, were very low after cold storage, being 3.2% ± 2.5% and 0.9% ± 0.9% for T. japonicus and T. cultratus, respectively    . The ovi= 0.921) .T. japonicus were 14.1 ± 3.01, 12.7 ± 2.37, 7.8 ± 1.69, 5.7 ± 3.60, and 9.0 ± 3.11 eggs for the five host densities, which did not differ significantly   (The oviposition rate significantly decreased after cold storage for both species of parasitoids. The oviposition rates of = 0.096) . The ovi= 0.509) .T. japonicus was significantly higher than T. cultratus at low host egg densities, one egg mass , and two egg masses . No significant differences were found between the two species of parasitoids when provided with three, four, and five host egg masses.Comparisons for the number of oviposition within 24 h between two species after cold storage show that the number of oviposition by Trissolcus are well known as control agents of H. halys, especially T. japonicus, whose release has been widely recommended to control H. halys in field crops, both in its native and adventive ranges. For example, T. japonicus and Anastatus sp. for suppression of H. halys in kiwifruit orchards in China. T. japonicus was intentionally released across four eco-regions in Oregon (United States) for investigation of its dispersal capacity  and T. cultratus (25.1). This suggests that one egg mass of H. halys (about 28 eggs) per day would be sufficient to support the daily oviposition of T. japonicus or T. cultratus. Subsequently, daily oviposition declined with female age. Age-related declines in parasitoid fecundity were similarly noted T. japonicus and T. mitsukurii.The numbers of ovipositions on the first day of life after access to hosts were the highest for both T. japonicus are ideally reared on fresh newly laid unfertilized H. halys eggs, which leads to the highest parasitoid development rate and fecundity  could not tolerate storage temperature at either 7 or 11°C. Storage of pupae was feasible, but storage of adult females was optimal, with adult survival being 90% (T. japonicus production with release needs (T. japonicus at 8°C and short photoperiod (8L:14D) resulted in lower realized fertility of stored females and fewer female progeny than those in storage at 13°C and 18°C . The lifespan of both species was significantly extended by cold storage at 11°C for 19 weeks. Both parasitoid species remained fertile after cold storage, although the lifetime fecundity was significantly reduced to around one-third of that of wasps not subjected to cold storage. Under ambient conditions (no cold storage), both wasp species laid about 28 eggs within 24 h of contact with hosts, but this value was much lower (less than 14.1) after cold storage.Lifetime fecundity of