The high nutritional value of vegetables is well recognized, but their short shelf life and seasonal nature result in massive losses and wastes. Vegetable’s byproducts are an opportunity to develop value-added ingredients, increasing food system efficiency and environmental sustainability. In the present work, pulps and powders of byproducts from rocket and spinach leaves and watercress were developed and stored for six months under freezing and vacuum conditions, respectively. After processing and storage, microbiological quality, bioactive compounds , antioxidant capacity, and pulps viscosity were analyzed. Generally, the developed vegetable’s pulps and powders were considered microbiologically safe. Although some variations after processing and storage were verified, the antioxidant activity was preserved or improved. A rich phenolic composition was also registered and maintained. During freezing, the quantitative carotenoid profile was significantly improved (mainly in rocket and spinach), while after drying, there was a significant decrease. A positive effect was verified in the vitamin E level. Both processing and storage conditions resulted in products with relevant phenolics, carotenoids and tocopherol levels, contributing to the antioxidant activity registered. Thus, this study demonstrates the potential of vegetable byproducts valorization through developing these functional ingredients bringing economic and environmental value into the food chain. Vegetables’ and fruits’ short shelf life and seasonal nature result in massive losses and wastes ,2. The lIn the last years, the consumer demand for healthier products rich in bioactive compounds, minimally processed and without synthetic additives, increased exponentially ,9,10. InRocket, spinach and watercress are among the most consumed green leafy vegetables, with an exceptional bioactive composition . SeveralFreezing is one of the most used processing technology for the long-term preservation of plant foods by industrial companies and households ,14. The In contrast, drying is one of the oldest procedures for food processing and preservation, increasing its popularity in the last years ,8,13. ThStudies generally lack the understanding of the impact of both processing and storage time on bioactive and biological properties. From a reducing food losses and waste perspective, it is of utmost importance to increase circular economy studies and contribute to the development of functional products . Due to Sodium hypochlorite (11–15% available chlorine) and HPLC-grade methanol (≥99.9%) were supplied by Honeywell Riedel-de Haën AG, Seelze, Germany. The microbiological media used were purchased from Biokar, namely, plate count agar (PCA), rose bengal chloramphenicol agar  and Bacillus cereus selective agar  as well as from VWR specifically, violet red bile glucose agar (VRBGA) and trypticase soy agar (TSA).Regarding antioxidant and total phenolic compounds (TPC) assays, Folin–Ciocalteu were supplied by Merck , while sodium carbonate, ABTS diammonium salt ), 2,2-diphenyl-1-picrylhydrazyl, fluorescein, 2,2′-azo-bis-(2-methylpropionamidine)-dihydrochloride , butylated hydroxytoluene  and 6-hydroxy-2,5,7,8-tetramethylbroman-2-carboxylic acid  by Sigma-Aldrich . Solvents used for HPLC analysis were also purchased from Sigma-Aldrich: acetonitrile, dichloromethane, formic acid, hexane and 2-propanol. Other reagents used during extracts preparation, carotenoids, and vitamin E extraction were also purchased from Sigma-Aldrich . Sigma-Aldrich also supplied the chemical standards: β- and α-carotene (≥95%), α-tocopherol (≥95%), β-tocopherol (≥95%), γ-tocopherol (≥95%), δ-tocopherol (≥95%), gallic acid (≥99%), rutin (≥95%), ferulic acid (≥98%), isoferulic acid (≥98%), trans-ferulic acid (≥99%), chlorogenic acid (≥99%), p-coumaric acid (≥98%) quercetin-7-O-glucoside (≥98%), sinapic acid (≥98%), myricetin (≥99%) and caffeic acid (≥98%). The remaining standards were purchased Extrasynthese : protocatechuic acid (≥99%), lutein (≥95%), lycopene (≥98%), ferulic (≥98%), isoferulic (≥98%), naringenin-7-glycoside (≥99%), naringenin (≥99%), lutein (≥99%) and zeaxanthin (≥98%).−1 was applied. The byproducts were rewashed with water abundance and centrifuged to remove wash water. Two different processing methods were used: pulps preparation and subsequent freezing and drying and vacuum storage. Vitacress Portugal S.A. kindly provided the byproducts present in this study. Rocket and spinach leaves and watercress that did not comply with commercial standards were used. The washing and disinfection step was carried out for 15 min with sodium hypochlorite solution (150 ppm), where a proportion of solution vegetable of 5 L kgFor pulps development, rocket leaves, spinach leaves and watercress were grounded with a water ratio of 4:1 (vegetable: water), using a vertical slicer blender (Hällde VCB-61) for 2 min. After processing, the pulps were frozen at −20 °C and stored for six months. The sampling points were fresh, M0 corresponded to 24 h after the freezing storage, and M1, M2, M3, M4, M5, M6 corresponding to months one, two, three, four, five and six of the freezing storage, respectively.−1 of air velocity. The byproducts were then milled and stored for six months under vacuum conditions. In this specific case, the time points were performed every two months due to the increased known stability of dried products. DM0 corresponded to the beginning of the dry experiment (day 0 of storage) and DM2, DM4 and DM6 corresponded to the following sampling times at months two, four and six of the storage period, respectively.Regarding the drying processes, the vegetables were dried in a hot air cabinet, using a 50 °C temperature and 0.65 m sRegarding the microbiological analysis of nonwashed, washed vegetables or pulp, 1 g of each fraction was added to 9 mL of sterile 0.1% peptone solution. Each test sample was homogenized in a stomacher  for 1 min. Dilutions were then prepared and spread in the specific culture media. Total aerobic bacteria (TAB) were analyzed according to the international standard (ISO) 4833-1, being this microbial group enumerated in PCA media after incubation for 72 h at 30 °C. Enterobacteriaceae were grown in VRBGA at 37 °C for 24 h. Yeasts and molds were grown and counted on supplemented BCSAB at 30 °C for 3 to 5 days while the Bacillus cereus group was incubated on mannitol egg polymyxin agar at 30 °C for 24 h. −1).For main microbial groups enumeration in dry vegetables, the sample homogenization was also performed in a stomacher with 10 g of sample and 90 mL of buffered peptone water. Concerning TAB enumeration, the dilutions were then prepared and spread onto PCA and incubated as previously described. The method described by Ijabadeniyi et al.  was foll−1) at a speed of 250 rpm for 30 s. The measurements were carried out five times for each sample at room temperature and expressed in mPa s−1.To monitor the viscosity of pulps, a rotational springless viscometer B-one plus  was used, with an R-2 rotor disc  at 12,000 rpm for 30 s. The mixture was left under continuous stirring (300 rpm) at room temperature for 2 h. The resulting mixture was then centrifuged at 5000 rpm at 4 °C for 10 min. Then, the supernatant was filtered with Whatman filter paper (no. 1). A rotary evaporator  was used to remove the methanol from the extracts under 40 °C and 175 mbar conditions. The resultant fraction was resuspended in deionized water with 2% of maltodextrin and lyophilized for further analysis. All extracts were prepared in duplicate.Total phenolic compounds, polyphenolic profile and antioxidant activity were evaluated through the preparation of methanolic extracts. For the preparation of the extracts, 5 g of each homogenized fraction  were used. The homogenization was carried out with 50 mL of 80% methanol (methanol:water) in an Ultra-turraxv/v) and 100 µL of sodium carbonate (7.4% m/v) in a 96-well microplate. A standard curve of gallic acid was prepared with concentrations ranging between 0.015 and 0.225 mg/mL, where the same mixture was used, but the standard replaced the extract. The mixture was incubated for 1 h in the dark at room temperature. Subsequently, the absorbance at 750 nm was measured with a multidetection plate reader , operated using the Gen5 Biotek software version 3.04. TPC present in extracts was expressed as milligrams of gallic acid equivalent (GAE) per 100 g of dry matter (DM).The Folin–Ciocalteau colourimetric method measured the total phenolic content (TPC) present in extracts, as described by Ainsworth et al. , with mi•+ (7 mM) in ultra-pure water. The reaction was stirred in the dark for 16 h at room temperature. Daily, the stock solution was filtered with a 0.45 µm syringe filter and the absorbance was adjusted to 0.70 ± 0.02 with up water to prepare the ABTS•+ working solution. The final reaction volume was 200 µL (20 µL of extract sample and 180 µL of ABTS•+ working solution), being the reaction carried out in a 96-well microplate. The control sample consisted of 20 µL of up water and 180 µL of ABTS•+ working solution, while in the standard curve (with concentrations between 25 and 175 µM) or sample, the same proportion was used, replacing up water by Trolox or by extract, respectively. The mixture was mixed and incubated in the dark for 5 min. After the reaction period, the absorbance was recorded at 734 nm with also a multidetection plate reader. The extracts were analyzed in triplicate and through the calculation of regression equations, the Trolox concentration in extracts was expressed in mg Trolox equivalent (TE) 100 g−1 DM.For 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) scavenging assay, the method described by Gonçalves et al.  was foll−1 DM.The antioxidant capacity was also evaluated by ORAC assay in a multidetection plate reader, as described by Coscueta et al. . Succinc−1 of DM.To quantify the hydrophilic antioxidant compounds, the DPPH assay was performed, following the method described by Schaich et al. , with so® C18, Waters, Milford, MA, USA; 100 Å, 5 µm, 4.6 mm × 150 mm) was used. The mobile phases used were phase A (92.5% water: 5% methanol: 2.5% formic acid) and phase B (92.5% methanol: 5% water: 2.5% formic acid), for 59 min of the HPLC run in a continuous flow of 0.5 mL min−1. The HPLC conditions started with mobile phase A at 100% and, after 50 min ended with mobile phase B at 55%. Until 55 min, the mobile phase A returns to 100% and remains until the end of the HPLC run. The volume of injection was 50 µL. The detection was performed at wavelengths between 200 and 600 nm with a diode array detector , measured at 2 nm intervals. At 280 nm were identified catechins or procyanidins, 320 nm phenolic acids and 350 nm flavonoids by comparing retention time and spectra of pure standards. Each extract replicate was injected in duplicate.Concerning the phenolic profile of methanolic extracts, the method described by Campos et al.  was carr−1 KOH , mixing using vortex and shaking water bath (200 rpm) at 85 °C. After 10 min, the tubes were cooled and maintained on ice for the following extraction procedures. Then, 3 mL of 1 mol L−1 NaCl was added to the tubes and they were gently mixed. The extraction was performed with 3 mL of 25 μg mL−1 BHT solution in n-hexane followed by vigorous vortexing and centrifuging samples . The organic phase was collected and the extraction was repeated. The new phase was collected again and joined to the first. For each sample, this procedure was performed in triplicate.Total carotenoids and vitamin E isomers  were extracted according to the method described by Araújo-Rodrigues et al. . Brieflyv/v/v/v/w). A Kromasil 100-5-C18 4.6 mm ID, 250 mm column was used at 30 °C. The injection volume was 50 μL of sample extract (in duplicate), the flow rate was constant at 1 mL min−1 and the run time was 20 min. The diode-array detector was set at 454 nm to identify the different carotenoids . The identification was performed by comparing pure standards retention time and the quantification was performed through standard calibration curves, being expressed as mg 100 g−1 DM of byproduct.The carotenoid profile was identified and quantified according to the method described by Oliveira et al. , with sl® coupled to a Waters™ 474 Scanning Fluorescence Detector  with a normal-phase silica column  was used. The mobile phase was 1% v/v isopropanol in n-hexane. Then, 20 µL (in duplicate) was injected, the flow rate was constant at 1 mL min−1 and the run time was 20 min. Pure standards of the four tocopherols isomers  were used to quantify the compounds by using calibration curves. Results were expressed as mg 100 g−1 DM.Tocopherols isomers were analyzed according to the HPLC method described by Savlin et al. , with sop < 0.05. This statistical analysis was only carried out by analyzing viscosity, TPC, antioxidant capacity, polyphenols, carotenoids and vitamin E. In the microbiological monitoring, only two independent analyses were carried out. In the first phase, data were compared statistically using ANOVA to understand the significance and confirm a normal data distribution. Post hoc multiple comparisons were carried out using Tukey’s test (p < 0.05).Statistical analysis was performed recurring to the SPSS statistical software (27.0) via a one-way analysis of variance (ANOVA), at a degree of significance of In the present study, based on a circular economy approach, rocket and spinach leaves and watercress byproducts were used to study the impact of two processing methods and storage periods on several bioactive properties. Byproducts that do not comply with commercial standards were used to prepare value-added pulps and powders. After the grinding process, the pulps of each vegetable were frozen at −20 °C and all analyses were performed monthly. In parallel, byproducts were also subjected to hot air drying and stored under vacuum conditions. Due to the higher stability of dry byproducts, all analyses were performed every two months, except the monitoring of microbial counts only performed in the initial and final storage period. −1) in the rocket, spinach and watercress byproducts, respectively. After washing and sodium hypochlorite disinfection, the microbial load of all byproducts significantly decreased. The Portuguese microbiological guidelines classified ready-to-eat vegetables and fruits as satisfactory when the microbial counts of TAB are lower than 6 log (CFU g−1), Enterobacteriaceae and yeasts lower than 5 log (CFU g−1) and Bacillus cereus spp. lower than 3 log (CFU g−1) [The main microbial groups were monitored in nonwashed and washed vegetables and pulp and powder forms . The anaCFU g−1) . Thus, aCFU g−1) . −1); in spinach from 4.60 to 4.72 log (CFU g−1); and in watercress from 4.94 to 5.00 log (CFU g−1)  . Thus, nRegarding microbial monitorization in dry byproducts, the results globally showed good stability in microbial counts , except Pulps viscosity was monitored from fresh samples until M6 of freezing, and the results are presented in During the application of both approaches and storage periods, TPC and antioxidant activity by ABTS, ORAC and DPPH were monitored through the preparation of methanolic extracts, presented in −1 DM; spinach leaves byproducts were 474.14 ± 8.19 mg GAE 100 g−1 DM; and watercress byproducts were 629.60 ± 24.94 mg GAE 100 g−1 DM. The results indicated a significant improvement in TPC values during the first months of freezing. Higher TPC values were registered in M2 of rocket and spinach leaves with 1186.65 ± 34.44 and 1065.35 ± 50.68 mg GAE 100 g−1 DM, respectively—and in M4 of watercress with TPC value of 1262.13 ± 51.13 mg GAE 100 g−1 DM. After this period, the TPC values significantly declined in all frozen byproducts. In the case of the rocket, the TPC values decreased significantly until M6 to 363.10 ± 4.71 mg GAE 100 g−1 DM. TPC values of spinach and watercress in M5 and M6 were approximately 310.00 and 740.00 mg GAE 100 g−1 DM, respectively, with no key alterations in this final phase of storage. Comparing the TPC results of fresh pulps with pulps at the end of the freezing period, watercress showed slightly higher TPC values in M6, while rocket and spinach presented slightly lower TPC values than fresh forms. The frozen process promotes cell breakages and loss of cellular integrity, which may positively affect TPC and antioxidant capacity due to the decompartmentalization of bioactive compounds. The possible release of bound phenolic acids and anthocyanins during the frozen storage is also suggested by some authors, resulting in the improvement of the functional properties [The TPC results of fresh rocket leaves byproducts were 469.51 ± 34.14 mg GAE 100 goperties . However−1 DM during drying, respectively. The TPC values significantly increase during rocket drying until 504.16 ± 17.59 mg GAE 100 g−1 DM. During drying storage of all byproducts, the results suggested significant decreases until 354.47 ± 30.33 mg GAE 100 g−1 DM in the rocket; 169.96 ± 8.17 mg GAE 100 g−1 DM in spinach; and 294.32 ± 11.23 mg GAE 100 g−1 DM in watercress in DM6. As previously pointed, several drying technologies are available for vegetables and fruit drying. However, hot air drying is one of the most widely used methodologies at the industrial level, allowing the production of large amounts in a faster and more economical way. However, hot air drying can lead to physical, biological and chemical changes in the overall nutritional profile. These main nutritional variations are associated with the susceptibility of phytochemicals to temperature and consequent significant losses in vitamins, antioxidants, carotenoids and other bioactive compounds [Concerning spinach and watercress, the TPC values declined significantly to 279.90 ± 21.14 and 409.42 ± 20.13 mg GAE 100 gDifferent methods were used to evaluate the antioxidant capacity of different time points under study. The ABTS and ORAC assays were used to evaluate the contributions of hydrophilic and amphipathic compounds to the total antioxidant activity. On the other hand, the DPPH assay evaluated the lipophilic compound’s contribution . −1 DM; spinach leaves byproducts was 2212.57 ± 48.62 mg TE 100 g−1 DM; while watercress byproducts was 938.56 ± 41.03 mg TE 100 g−1 DM. As suggested by the TPC results, the data of the ABTS assay indicated significant increases in antioxidant capacity during the first months of frozen storage. The highest antioxidant capacity was registered in spinach and rocket in M2, with ABTS values of 3285.07 ± 42.16 and 2889.67 ± 31.52 mg TE 100 g−1 DM, respectively. Subsequently, the antioxidant activity decreased until M4 in the rocket (around 1270 mg TE 100 g−1 DM), stabilizing until the final storage period. The antioxidant capacity of spinach pulps also decreased after M2, with significant variations until the last storage period, showing an antioxidant capacity of 1575.99 ± 16.22 mg TE 100 g−1 DM in M6. Although the spinach and rocket results suggested some decreases in antioxidant capacity in M6 compared to fresh forms, a key improvement in functional characteristics was verified during the initial phase of freezing. Concerning watercress, the highest antioxidant capacity was only registered in M4 (2587.12 ± 82.84 mg TE 100 g−1 DM). In M5, a significant decline was verified, but no significant variations were found until M6, with antioxidant capacities around 1750 mg TE 100 g−1 DM. Thus, in watercress, the antioxidant capacity by ABTS assay significantly improved with the freezing storage.The antioxidant capacity of fresh rocket leaves byproducts was 2228.46 ± 76.95 mg TE 100 g−1 DM in spinach, 952.29 ± 46.99 mg TE 100 g−1 DM in rocket and 747.87 ± 29.50 mg TE 100 g−1 DM in watercress. This decline was less pronounced in watercress, where some reductions were registered during the first two months of drying storage until approximately 640.45 mg TE 100 g−1 DM, with no critical alterations until DM6. Relating to spinach, some key changes were verified until DM4, when the antioxidant capacity was around 457.61 mg TE 100 g−1 DM. The results of rocket drying storage suggested significant variations during all storage periods with antioxidant capacity in DM6 of 481.79 ± 3.41 mg TE 100 g−1 DM.In all byproducts, the results suggested that the hot air dry significantly reduced the antioxidant capacity to 1032.69 ± 73.71 mg TE 100 g−1 DM; rocket leaves byproducts of 2565.10 ± 28.38 mg TE 100 g−1 DM; and watercress byproducts 3064.98 ± 303.46 mg TE 100 g−1 DM. Following the ABTS results, a positive impact of freezing on antioxidant activity was registered, with ORAC values in M1 in spinach (10089.90 ± 325.79 mg TE 100 g−1 DM) in M2 in the rocket (7789.00 ± 147.18 mg TE 100 g−1 DM) and in M4 in watercress (6038.52 ± 720.05 mg TE 100 g−1 DM). In watercress, no significant variations were registered in ORAC antioxidant capacity after M4. After M2, the results indicated key reductions until the final freezing phase but with no significant variations compared with fresh form. The antioxidant capacity of spinach pulps decreased after M1 but no key variations until M4 were verified, with ORAC antioxidant capacity around 3200 mg TE 100 g−1 DM.Regarding ORAC results of fresh byproducts, spinach leaves byproducts exhibited an antioxidant capacity of 5208.2 ± 130.92 mg TE 100 g−1 DM in DM0. In contrast, only a slight decline in antioxidant capacity was verified in the rocket compared to fresh leaves byproducts.No key alterations were registered for antioxidant activity determined by ORAC assay in all byproducts studied during all drying storage. No influence of processing was verified for watercress by comparing the fresh and dry results. In spinach, significant reductions were verified after the drying process, with an antioxidant capacity of 2379.74 ± 191.26 mg TE 100 gAlthough both ABTS and ORAC assays measure the contribution of hydrophilic and amphipathic compounds to antioxidant capacity, the variations between antioxidant capacities found resulted from the distinct reaction mechanisms underlying. In the case of ABTS assay, the radical used possesses a higher molecular weight than the transfer mechanism molecule of hydrogen atom used in ORAC assay. Thus, in ABTS a steric block of active centers may occur, resulting in a decrease in its reaction rate. As suggested by Campos et al., this reason makes this method considered more accurate . This fa−1 DM, respectively. The same increasing profile followed by a significant decline was registered in DPPH assay, with the higher antioxidant values in rocket and spinach in M2  and in watercress in M4 (1811.05 ± 18.14 mg TE 100 g−1 DM). An extremely positive effect in antioxidant capacity was registered in watercress; even with a significant reduction after M4, it showed higher values than the fresh byproduct, namely, 943.08 ± 48.21 mg TE 100 g−1 DM. Contrary to the other antioxidant assays, the results suggested that watercress byproducts possessed considerably higher DPPH values than other byproducts during both freezing and drying storage. A possible explanation to these results is the high presence of isothiocyanates in watercress. These very reactive compounds are hydrophobic [−1 DM, higher than fresh form. Concerning rocket byproducts, no significant variations were found compared to the fresh byproduct and M6 frozen pulp. As previously stated, the DPPH method evaluates the contribution of lipophilic compounds to the total antioxidant capacity, requiring the use of organic solvents . Generalrophobic  and cons−1 DM, with no significant variations during all storage phase. The same tendency was suggested (DM0: 274.97 ± 10.36 mg TE 100 g−1 DM) in rocket, but with a significant reduction until 215.52 ± 18.85 mg TE 100 g−1 DM in DM2, stabilizing in the remaining storage period. Finally, in watercress, the results indicated a considerable increase in antioxidant capacity after drying, with DPPH values of 825.50 ± 29.71 mg TE 100 g−1 DM in DM0 and a decline until approximately 714.00 mg TE 100 g−1 DM in DM4, coupled with maintenance until the final period of storage. Thus, spinach and watercress showed higher antioxidant capacity values in all drying storage period rocket than fresh byproducts, showing a positive impact of hot air drying in DPPH antioxidant capacity. In this study, lipophilic compounds such as carotenoids and tocopherols were quantified , germination and maturation stages as well as post-harvest factors, for instance, storage conditions, processing technologies ,30,31,32As explained in the discussion of the TPC results, the increase in antioxidant capacity in the first months of frozen storage could be promoted by the loss of cellular membrane integrity, resulting in bioactive compounds release and an increase in antioxidant activity ,15. To tMost of the literature data found using blanching before freezing to inactivate enzymes involved in deterioration during frozen storage. Thus, no literature studies were found to directly compare the present results during freezing storage. For example, other authors studied the effect of spinach freezing but with the previous application of blanching, showing significant reductions in TPC and ORAC antioxidant capacity compared with fresh spinach. The authors suggested that this considerable decrease may be associated with the mild heat treatment used in the blanching process . Ninfali−1 on some nutritional parameters of watercress leaves. The results suggested that all nutritional parameters evaluated were negatively correlated with the increase of drying temperature, including TPC and ABTS antioxidant activity. Other work studied the effect of convective oven dry at 65–70 °C on spinach leaves and stems’ TPC and antioxidant capacity. The results suggested decreased TPC and antioxidant capacity by ORAC and DPPH assays by dry weight after dehydration [Generally, the literature data focus on the impact of green leafy vegetables drying on TPC and antioxidant activity, aligned with the present study results. As suggested by the work of Pickmony et al. , using aydration , aligninydration . Polyphenolic chromatograms of fresh rocket byproducts, during pulps freezing and dry storage, are present in The polyphenolic chromatograms of fresh, freezing and drying spinach byproducts are represented in The chromatograms of polyphenols of watercress byproducts are present in Quantitative profiles of polyphenolic compounds present in rocket and spinach leaves and watercress byproducts during all experimental periods were also determined by HPLC and are summarized in During freezing storage, some significant variations were observed. Chlorogenic, p-coumaric, ferulic and transferulic acids, apigenin, rutin and quercetin-3-glucoside declined significantly after freezing. However, after this period, the concentration of these phenolic compounds increased significantly, followed by a new decrease phase. In chlorogenic and transferulic acids and rutin, this period was followed by a new increase and decrease. Concerning quercetin derivatives, the freezing improved the concentration of these antioxidant compounds, followed by a final stage of concentration decrease. Some losses were reported compared fresh forms with M6, mainly in the case of chlorogenic, p-coumaric, ferulic and transferulic acids and rutin and quercetin-3-glucoside. However, a positive effect of freezing was also verified in quercetin derivatives and apigenin concentration. Thus, the results suggested that spinach pulps possessed a very interesting polyphenolic profile during freezing and after six months of storage.The drying process significantly decreased the chlorogenic acid and rutin concentrations, but no significant variations during storage were verified. No significant variations in p-coumaric concentration were verified after the drying process and during storage. In ferulic acid, no key variations were registered with processing; however, a significant decline in its concentration was visible after DM4. In contrast, the concentration of quercetin and apigenin derivatives increased significantly after drying, while the concentration of transferulic acid and quercetin-3-glucoside declined significantly. The monitorization of these antioxidant compounds suggested some significant decreases during drying storage. The antioxidants variations during freezing and drying processes were previously discussed in the “Monitorization of TPC and antioxidant activity” section.p-coumaric acid and caffeic acid derivative), flavones (luteolin and rutin derivatives) and flavanols  families. The most prevalent antioxidant was luteolin derivative in fresh, frozen pulps and dry byproducts. Other works focusing on fresh spinach’s phenolic profile also suggested the hydroxycinnamic acid group as the most prevalent phenolic group in spinach [The main phenolic compounds detected in spinach were from hydroxycinnamic acid . In the remaining phenolic compounds detected, the results indicated a significant increase, with the maximum concentration in M4 in the case of lutein derivative 1, while other lutin derivative and rutin derivatives in M4 decreased significantly in all cases until M6. However, in all cases, a significant improvement of these compounds was verified comparatively with fresh forms. Kamiloglu [The results suggested significant reductions in phenolic compounds in M0, namely epigallocatechin gallate and caffeic acid derivatives. During freezing storage, the concentration of these antioxidants increased significantly until M6. In caffeic acid derivative, a significant reduction was verified from M4 to M5, followed by a new improvement until M6. However, in both cases, the concentration was significantly higher than in fresh spinach. Regarding amiloglu  studied p-coumaric acid and derivatives of epigallocatechin gallate, rutin, and luteolin. No variations were verified during storage in luteolin derivative 1. Some declines were detected in epigallocatechin gallate derivative, p-coumaric, luteolin derivative 2 and rutin derivatives. However, key improvements comparatively with fresh spinach byproducts were registered, suggesting that the drying approach may be a good approach to improve the spinach polyphenolic profile.The drying processing negatively affected caffeic acid derivatives, with no variations registered during the storage period. On the other hand, after drying processing, the results suggested a positive impact in p-coumaric acid and derivatives). Recently, some authors also studied the phenolic profile of fresh watercress, being flavonols and hydroxycinnamic acid groups the most representative [p-coumaric acid, caffeic acid and caffeic acid derivatives, were detected by these authors. As previously mentioned, other work focused on the phenolic composition of 12 cruciferous vegetables, including rocket and watercress. This study suggested hydroxycinnamic acid as the most incident group in watercress, followed by the flavonoids group [p-coumaric acids, quercetin-3-glucoside, quercetin and luteolin derivatives.Concerning watercress byproducts, the results suggested a higher concentration of phenolic compounds in all forms analyzed. The most prevalent was the hydroxycinnamic acid group . Regarding p-coumaric acid and ferulic acid, no key variations were registered with processing. In contrast, the quercetin-3-glucoside and luteolin derivative were improved with the preparation of pulps and freezing processing, where significant increases were registered. During freezing storage, a first period with some significant declines was registered in the quercetin derivative, quercetin-3-glucoside, p-coumaric acid derivatives and ferulic acid until M3. In the chlorogenic acid derivative, p-coumaric acid, caffeic acid and luteolin derivatives, significant declines were verified until M4. After this period, the results suggested a significant improvement until the final stage of freezing. p-coumaric acid derivatives, quercetin-3-glucoside and quercetin derivatives showed higher concentrations in M6 than fresh forms, indicating a positive effect of freezing storage in the concentration of these antioxidants. The pulps processing and freezing negatively impacted chlorogenic, p-coumaric acid, ferulic acid, p-coumaric derivatives and quercetin derivatives, but no significant variations were registered during all storage periods. The caffeic acid and luteolin derivatives results also suggested a significant decline after drying, with minor variations during the storage period. On the contrary, quercetin-3-glucoside concentration increased after the drying process, and some variations during storage were also verified. Accordingly, the results of phenolic compounds identified and quantified in watercress suggest that freezing may be the most effective way to preserve or enhance phenolic compounds in watercress byproducts. The drying processing negatively affected the chlorogenic acid derivative, Generally, the results aligned with TPC results obtained by Folin–Ciocalteu. However, some variations between HPLC quantifications and TPC results were registered, mainly due to the detection limit of phenolic antioxidants. Other reason may be the interferent molecules existent in prepared extracts . These can react with Folin–Ciocalteu reagent, resulting in overestimation ,41. Tabl−1 DM in the fresh rocket, 69.75 ± 5.66 and 74.03 ± 9.91 mg 100 g−1 DM in the fresh spinach, and 100.74 ± 19.67 and 87.86 ± 1.21 mg 100 g−1 DM in the fresh watercress. These results confirmed previous results, which also found lutein and β-carotene as the main carotenoids in leaf vegetables [The carotenoid profile was also monitored and is represented in getables ,43,44. Pgetables . This fagetables . Moreovegetables .−1 DM, respectively, while in spinach in M4 at a concentration of 231.55 ± 5.92 mg 100 g−1 DM. After this period, the β-carotene concentration decreased, with some significant variations. In the case of the rocket, a significant decrease was registered after M3 with no key variations after M5, with β-carotene concentrations around 97.00 mg 100 g−1 DM. Regarding watercress, after M3, the β-carotene concentrations reduced considerably until 118.24 ± 7.74 mg 100 g−1 DM in M5 and until 102.06 ± 11.49 mg 100 g−1 DM in M6. After M4 of freezing, the results indicated that β-carotene in spinach pulps declined drastically in M5, with a slight decrease until 80.56 ± 9.12 mg 100 g−1 DM in M6. Lutein content followed the same tendency, with the higher values in M4 in all byproducts, decreasing until approximately 68.00, 83.00 and 78.22 mg 100 g−1 DM of the rocket, spinach and watercress, respectively, with no key alterations from M5 to M6. Although in lower concentrations, α-carotene concentration increased during all freezing period in spinach, with no significant variations from the M3. In the rocket, α-carotene concentration improved until M5, with no key concentration changes from M2 until the final phase of freezing storage. Previous works also demonstrated an increased carotenoids content after freezing [In all byproducts, a significant increase in carotenoid content was registered during the freezing period. The higher concentrations were registered in rocket and watercress in M3 at 258.54 ± 6.56 and 224.89 ± 17.22 mg 100 gfreezing . The incfreezing . To the −1 DM, respectively. Previous studies have demonstrated that the milling process promotes the liberation of antioxidant molecules associated with fiber, which could explain the increase in carotenoids content because the free form of the carotenoids is evaluated in samples [−1 DM), with a slight increase in DM2, while a negative effect in β-carotene concentration was verified (DM0: 47.38 ± 4.50 mg 100 g−1 DM), with no variations during six months of storage. Finally, the watercress drying process had a positive impact on β-carotene concentration, where its concentration was improved until 206.18 ± 9.24 mg 100 g−1 DM. In contrast, drying may negatively impact lutein concentrations, which showed approximately 72.00 mg 100 g−1 DM in DM0 and DM2 and after DM4 concentrations around 58.07 mg 100 g−1 DM.During drying of rocket leaves, the α-, β-carotene and lutein concentrations significantly increase compared with fresh form, with concentrations of 50.03 ± 5.02, 68.16 ± 0.78 and 76.47 ± 2.68 mg 100 g samples ,50. The  samples . Howeverα-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol (isomers of vitamin E) content was also assessed during all experiments . These b−1 DM. β-tocopherol content of 0.64 ± 0.02 mg 100 g−1 DM, and the γ-tocopherol content was 0.22 ± 0.01 mg 100 g−1 DM. Fresh spinach presented a content of α-, β- and γ-tocopherols of 1.78 ± 0.03, 0.54 ± 0.01 and 0.82 ± 0.05 mg 100 g−1 DM, respectively. Watercress tocopherols content were 5.31 ± 1.49, 0.20 ± 0.08, 0.10 ± 0.02 and 0.11 ± 0.05 mg 100 g−1 DM for α-, β-, γ- and δ-tocopherols, respectively. Other works found the α-, and γ-tocopherol in the rocket, spinach and watercress where α-tocopherol was in higher content than the γ-tocopherol [All four tocopherols were present in the watercress, but in both rocket and spinach vegetables, only the α-, β- and γ-tocopherol were detected. For all vegetables, α-tocopherol was the major tocopherol found in fresh byproducts, pulps and powders. Fresh rocket presented an α-tocopherol content of 2.68 ± 0.08 mg 100 gcopherol ,53,54,55−1 DM), with no significant variations until M5. After this period, a decline in the α-tocopherol concentration from 5.64 ± 0.53 to 4.74 ± 0.70 mg 100 g−1 DM was verified. A continuous increase in β-tocopherol until M2 was also registered (4.94 ± 0.32 mg 100 g−1 DM) and a significant decrease until M3 was verified (3.64 ± 0.24 mg 100 g−1 DM), followed by stabilization until M6. No key variations were seen in γ-tocopherol content in the rocket, ranging its concentration between 0.28 ± 0.11 and 0.99 ± 0.05 mg 100 g−1 DM. During freezing storage of spinach pulps, there is an increase in tocopherol content until M2, in the case of α-tocopherol and γ-tocopherol, namely 4.40 ± 0.49 and 2.79 ± 0.30 mg 100 g−1 DM, while in the case of β-tocopherol, until M3 (1.70 ± 0.05 mg 100 g−1 DM). Posteriorly, α-tocopherol concentration in spinach declines significantly until M5 and then stabilizes, with concentrations around 2.69 mg 100 g−1 DM. Concerning β-tocopherol and γ-tocopherol concentrations in spinach, the concentration decreases significantly until M6, namely 0.79 ± 0.16 and 1.36 ± 0.16 mg 100 g−1 DM, respectively. In watercress pulps, α, β- and γ-tocopherols content increases until M2 of storage  and δ-tocopherol until M3 (0.63 ± 0.09 mg 100 g−1 DM), but start decreasing after this period. β-tocopherol and α-tocopherol concentrations decline significantly until M3 , in the first case stabilizing until the final phase of storage. In contrast, in the α-tocopherol profile, minor variations were verified. Concerning γ-tocopherol concentration, the results suggested a decline until 0.08 ± 0.02 mg 100 g−1 DM (M6). Finally, δ-tocopherol concentration in the watercress decline until 0.30 ± 0.02 mg 100 g−1 DM (M4), and after this period, it was not detected. Except for spinach, the pulps elaboration induced liberation of tocopherols, significantly increasing their concentration . This fa−1 DM of α-, β- and γ-tocopherols, respectively (DM0), while spinach powders presented contents of α-, β- and γ-tocopherols of 5.86 ± 0.50, 2.18 ± 0.16 and 3.22 ± 0.52 mg 100 g−1 DM, respectively. Regarding watercress powders, these had tocopherols contents of 48.98 ± 2.76, 7.45 ± 1.07, 9.21 ± 1.44 and 0.18 ± 0.01 mg 100 g−1 DM for α-, β-, γ- and δ-tocopherols, respectively. The increase of these compounds in powders was also associated with the same reasons explained by carotenoids increase. Milling induces liberation of bioactive compounds bounded to fiber [−1 DM. The results also indicated a decline in watercress δ-tocopherol concentration during dry storage, and from DM4, this isomer was not detected. As previously appointed for carotenoids, some chemical and enzymatic reactions can lead to some degradations after releasing tocopherols into the free form [The powder’s elaboration significantly increased the tocopherols content to a more extended amount than carotenoids. Previous works demonstrated that tocopherols are more stable than carotenoids, especially β-carotene . Rocket to fiber ,50. Generee form .The present work suggested that the green leafy vegetables, namely rocket and spinach leaves and watercress byproducts, can be valorized by processing into two types of food ingredients: pulps and powders. The results corroborated that these added-value ingredients are rich in bioactive compounds such as phenolic compounds, carotenoids and vitamin E, and presented great antioxidant activity. The TPC and antioxidant capacity typically increase during the first months of freezing and decrease after this period. Although some losses were registered, the frozen pulps of rocket and spinach generally showed values close to the fresh byproduct, presenting a high antioxidant capacity after six months of freezing storage. The results suggested a higher TPC and antioxidant capacity in M6 than in fresh form in watercress pulp. The TPC and antioxidant capacity in the powders were often lower than fresh forms but still demonstrating a high TPC and antioxidant capacity. On the contrary, the antioxidant capacity of watercress evaluated by the DPPH assay suggested a significant increase with drying, while no significant variations observed by ORAC assay.Concerning phenolic composition, the results indicated a rich composition, where bioactive compounds belonging to hydroxycinnamic acid, flavonols, flavones and flavanols families were identified and quantified in the three byproducts studied. Some variations after processing and storage were verified depending on the antioxidant metabolite, with some improvements and declines. However, generally, a rich phenolic composition was found during all storage periods. The carotenoid profile was significantly improved with freezing (mainly in rocket and spinach), while a more significant positive impact was verified in the vitamin E profile during drying. However, freezing and drying processing, followed by storage, resulted in products with very interesting carotenoid and tocopherol profiles.Generally, both ingredients were considered safe in microbial counts and pre-served great antioxidant activity and bioactive compounds during storage or, in the case of watercress, improved the functional profile of fresh byproducts. Thus, this study demonstrates the high economic and environmental potential of recovery of these functional ingredients into the food chain.


The studied system is CTS in its cubic polymorph, produced in powder form via a bottom-up approach based on high-energy reactive milling. The as-milled powder was sintered in two batches with different synthesis conditions to produce bulk CTS samples: manual cold pressing followed by traditional sintering (TS), or open die pressing (ODP). Despite the significant differences in densities, ~75% and ~90% of the theoretical density for TS and ODP, respectively, we observed no significant difference in electrical transport. The stable, best performing TS samples reached zT ~0.45, above 700 K, whereas zT reached ~0.34 for the best performing ODP in the same conditions. The higher zT of the TS sintered sample is due to the ultra-low thermal conductivity (κ ~0.3–0.2 W/mK), three-fold lower than ODP in the entire measured temperature range. The effect of porosity and production conditions on the transport properties is highlighted, which could pave the way to produce high-performing TE materials.The thermoelectric behavior and stability of Cu Thus, a high zT TE material requires a high power factor (S2/ρ) and low κ, which consists of lattice (lκ) and electronic (eκ) contributions. That being said, it is not the only important parameter as the stability and durability of TE materials are also vital for producing reliable TE devices.Thermoelectric (TE) materials are a class of functional materials employed in solid-state energy generators and coolers, without the use of fossil fuels and combustion processes, and with no moving parts, exploiting only the temperature gradients. For this reason, TE materials were first successfully utilized in deep-space probes in the second half of the last century . In recep-type and n-type semiconductors ,,κl <0.5 TS by In , Ni 19]κl <0.5 WTS by In , Co 21]κl <0.5 WTS by In  substituSn2S7 [2 , Cu4Sn7S[4Sn7S16 , and Cu2 [2SnSe3  also shozT > 1, and 2–4 fold lower lκ in hollow nanostructured Be2Te2.5Se0.5 than in the existing literature. Hong et al. [Manipulation of porosity in materials is a less-explored subject for TE applications. Many articles have put forward a crucial need to investigate porous TE materials ,27,28,29g et al.  showed ig et al.  investigg et al. . This mag et al. . In partHerein, we stabilized the disordered CTS polymorph using different production conditions and sintering techniques. CTS samples from two batches (three samples each) were produced, and their TE properties and thermal stability were systematically investigated by repeated Seebeck and resistivity measurements (heating and cooling) in the temperature range of 323–723 K. Pellets of samples were produced using manual cold pressing followed by sintering in a traditional tubular furnace (TS batch), or via open die pressing (ODP batch) . Informa2 and H2O < 10 ppm). All the samples were sintered in Ar flux. Additionally, sample TS3 was sintered in the presence of sulfur powder (1 g).CTS samples were produced from the elemental powders using a high-energy reactive ball-mill , according to a well-assessed procedure with details discussed elsewhere . Three dThe other three samples  were produced in a controlled environment with the above-mentioned preconditions. Differently, the sintering was performed via ODP using an iron die. To prevent possible reactions between the die and the powders, a boron nitride layer was used to cover the internal surface of the die. Such a layer also prevents sticking phenomena, allowing for an easier extraction of the sintered bulk. Sample ODP1 was sintered at 400 °C for 30 min, whereas samples ODP2 and ODP3 were sintered at 500 °C for 10 and 30 min, respectively. During all the sintering processes, a ~100 MPa axial pressure was applied.It should be emphasized that ODP was performed on a large quantity of the as-milled powder, namely ~30–40 g. The as-sintered samples from ODP are usually large bulk samples, which are cut into the required sample sizes for various measurements (large as-sintered ODP samples are shown in Kα (λ = 1.5406 Å) source and a scintillation counter detector. XRD data on the ODP samples were collected in Bragg–Brentano geometry using a Bruker D8  diffractometer equipped with a Co Kα (λ = 1.7889 Å) source and area linear (1D) detector. After the phase identification, Rietveld refinement [The structural characterization of TS samples was performed in Bragg–Brentano geometry using a Rigaku PMG  powder diffractometer, equipped with a Cu finement  was perffinement , as implfinement  software−3 bar).Simultaneous Seebeck coefficient and resistivity measurements were performed in four contact set-up using LSR-3 , at a temperature ranging from 323–723 K. Thermal diffusivity (D) was measured using a LFA-500 , equipped with a xenon-flash lamp over a temperature range of 323–723 K. Seebeck and resistivity measurements were performed under an He atmosphere (0.1 bar). However, the diffusivity was measured under a vacuum  were collected on powder produced by crushing the sample pellets, and EDX spectroscopy was performed using transmission electron microscopy . Moreover, morphological images and EDX data were collected on pellet samples to investigate the microstructure and bulk chemistry using a Jeol IT300 scanning electron microscope (SEM) equipped with a tungsten source .2θ~28.5°, 47.3°, and 56.0°, and 2θ ~33.2°, 55.5°, and 66.3° using Cu Kα and Co Kα X-ray sources, respectively. These Bragg peaks represent planes (1 1 1), (2 2 0), and (3 1 1), respectively, confirming the disordered cubic CTS structure, which is derived from the zinc-blende structure (SG: F-43m). A small amount of WC (SG: P-6m2) is present in all of the samples due to the use of WC vials for milling. Additionally, a weak peak (2θ ~27°) of SnO2 (SG: P42mnm) was observed in the pattern of sample TS1  of SnO2, which is possibly formed due to open environment processing. We did not observe the formation of secondary phase oxides in samples TS2 and TS3, produced in a strictly controlled environment. ODP, like spark plasma sintering (SPS), is a fast-sintering technique needing a short sintering time and applying external pressure. The applied pressure during ODP decreases the sintering temperature and time, simultaneously improving the densification kinetics. Thus, the crystalline domains show limited growth, while reaching full densification. The average domain sizes for the ODP1, ODP2, and ODP3 samples are 42 ± 10, 54 ± 10, and 64 ± 10 nm, respectively. In our recent work, we showed that SPS on as-milled CTS powder can constrain the grain growth below 50 nm [Different crystallographic phases were identified, and their respective weight fractions were obtained by the Rietveld method ,39. Info2. Samples TS2 and TS3 showed the formation of a new secondary phase, SnO (SG: Pnm21), possibly due to an oxygen-deficient production environment. We also observed the presence of SnS (SG: Pbmn) in sample TS1, confirming the chemical instability of the sample prepared in an open environment. Samples sintered using ODP were less prone to growth in the secondary phases. We did not observe any grain growth or increase in the weight fraction of secondary phases for ODP1 and ODP2. The applied pressure during sintering seems to better stabilize the samples than traditional sintering. Nevertheless, an increased amount of SnO2 was observed for ODP3, due to its extended sintering time in comparison to ODP2. The densities of ODP3 and ODP2 samples are similar, in spite of the different sintering times of 30 and 10 min, respectively. Therefore, the densification was complete within ~10 min of sintering. Furthermore, extended sintering mainly promotes phase degradation, leading to the formation of SnO2.After the repeated TE measurements (discussed in the next section), the XRD data were collected for the second time. All TS samples showed an increase in the weight fraction of SnOAs discussed in the Introduction, the loss of nanostructure has adverse effects on the TE properties of the material, which could drive it towards instability. The average crystalline size for the TS1 sample increased from 40 ± 10 to 75 ± 10 nm after repeated TE measurements, whereas samples TS2 and TS3 did not lose their nanostructure, maintaining their average crystallite size of 75 ± 10 and 70 ± 10 nm, respectively. Similarly, the ODP sintered samples maintained their average domain size of 42 ± 10, 54 ± 10, and 68 ± 10 nm for ODP1, ODP2, and ODP3 samples, respectively, after repeated TE measurements.SEM micrographs at different magnifications collected on TS and ODP sintered sample surfaces are shown in 2 grains, as we observed from the XRD. The evolution of SnO2 grains makes the sample unstable during TE measurements by unbalancing the stoichiometry. For samples TS2 and TS3, no SnO2/SnO grains appeared in the micrographs, although a weak peak for oxygen could be observed by EDX. The chemical composition for the TS2 sample was copper-rich, whereas, for the sulfurized sample (TS3), the stoichiometry was close to the theoretical  of ball-milled powder, producing large samples see . The SEM g of balp-type semiconducting nature. The trend of resistivity for sample TS1 is increasing with the temperature, whereas it is decreasing for all other samples, typical of degenerate and non-degenerate semiconductors, respectively.Repeated Seebeck and resistivity measurements are shown in S (40–100 µV/K) and ρ (0.01–0.03 mΩ-m). However, these measurements are not reproducible during the second and third measurement cycles. The low Seebeck and resistivity with a non-degenerate semiconducting trend suggest the presence of a higher carrier concentration in sample TS1. This could be due to the alteration in chemical composition, as we observed a large amount of SnO2 using TEM-EDX and XRD. The XRD on the same sample after repeated TE measurement cycles revealed the segregation of SnS  and TS3 , the TE measurements were repeatable during the measurement cycles, and the values of S and ρ were in agreement with the literature [2 and the formation of a new SnO phase, segregation of SnS was not observed. Similar values of S and ρ during several measurement cycles confirmed that the CTS samples prepared in a strictly controlled environment were stable. It is known that sulfurization leads to the formation of stoichiometric CTS [For samples TS2  and ρ (0.55–0.50 mΩ-m). ODP2 and ODP3 samples sintered at 500 °C for 10 min and 30 min showed increasing values of S and ρ. The Seebeck coefficients and resistivities for the ODP2 and ODP3 samples were S ~400–550 µV/K and ρ ~2.2–1.0 mΩ-mm, and S ~550–650 µV/K and ρ ~4.0–1.7 mΩ-m, respectively. The increase in S and ρ have a clear relation with the increased average domain size of the samples. These results are in agreement with our recent work showing the effects of grain growth on SPS sintered CTS samples, owing to the conduction based on the metallic nature of surfaces due to dangling bonds [2SnSe3.The repeated TE measurements on ODP sintered samples were reproducible, especially after the first measurement cycle, and for the second and third measurements cycles, the ng bonds  showed aS in comparison with the ODP samples. This could be related to the overall chemical composition of the TS samples, where we noticed a large chemical fluctuation in TEM-EDX. Some chemical fluctuations were also present in the ODP samples, but the overall chemistry of ODP samples was close to the theoretical, as observed by means of the chemical maps.However, it is important to notice here that two different sintering were used for the sample preparation, resulting in significantly different densities, even if we did not observe a significant difference in the resistivity of the same samples. Thus, we put forward that electrical transport is less likely affected by porosity in these systems. However, we noticed that the TS sintered samples had a lower D) was measured using a xenon flash instrument, and the thermal conductivity (κ) was calculated as, κ = D × d × pC, where, d and pC are the density of the sample and specific heat capacity, respectively. The density was measured using the Archimedes method  than ODP2 (0.8–0.6 W/mK).All the samples showed a decreasing trend of thermal conductivity with temperature, implying that the phonon–phonon  is dominating the phonon transport see . Here, i for CTS . However systems ,18. A cllymorphs ,12,18. TPF = 2S/ρ, and is shown in PF ~3.5 µW/K2 cm, above 700 K, which is continuously decreasing with successive measurements. Samples TS2 and TS3 showed PF ~1.25 µW/K2 cm and ~1 µW/K2 cm, respectively, above 700 K, but they are stable over repeated measurements. Due to the reproducibility of S and ρ, ODP sintered samples showed reproducible PF. Among all the ODP sintered samples, ODP2 has the highest PF ~2.75 µW/K2 cm, above 700 K, because of moderate S and ρ. However, lower S and very high ρ for samples ODP1 and ODP3, respectively, have an adverse effect on each other, resulting in a comparatively lower PF ~1.75 µW/K2 cm and ~2.25 µW/K2 cm, respectively.The power factor was calculated as zT ~0.50, 0.45, and 0.27, respectively, above 700 K , which blocked the thermal transport effectively. Impotently, the many folds reduction of κ due to the pores does not show a significant increase in the resistivity of the TS samples. Furthermore, the porous CTS samples also showed reproducible results during many measurements cycles. Total elimination of secondary phase oxides is not easy due to the low partial pressure of their formations, but a small amount of tin oxides does not seem to affect the TE properties during repeated TE measurements.Concerning the figure of merit, samples TS1, TS2, and TS3 show ve 700 K . Althougκ ~0.3–0.2 W/mK) showed ~3 times lower thermal conductivity than the high density or ODP sintered samples (κ ~0.8–0.6 W/mK). However, we did not observe any significant difference in electrical transport properties between the TS and ODP samples. The best performing stable TS samples present zT~0.45, whereas the best performing ODP sample showed zT of ~0.34, around 700 K, a result clearly due to the ultra-low thermal conductivity of the traditionally sintered, porous samples.The present work shows that porosity leads to a significant suppression of thermal conductivity, and CTS pellets can be stabilized with different fractions of porosity. The low density or TS samples (Due to the low partial pressure of tin oxides formation, it is difficult to produce pure CTS and similar chalcogenides. However, the continuous evolution of secondary phase oxides can be eliminated by using a highly controlled environment, and a small fraction of secondary phase oxides seem to have little effect on the TE properties and stability of the samples. The results presented in this work give a general overview of the effects of different experimental conditions and porosity on the stability and TE performance of CTS samples. Similar considerations should hold for other Cu-Sn/Fe-S/Se-based systems, chalcogenides, colusites, and chalcopyrite, etc., used for various applications, ranging from photovoltaics to thermoelectricity and LED production.


Neither DMB nor gallic acid or chlorogenic acid reduced TMA production through cytotoxic effects, indicating mechanisms such as altered TMA-lyase activity or expression.Choline is metabolized by the gut microbiota into trimethylamine (TMA), the precursor of pro-atherosclerotic molecule trimethylamine N-oxide (TMAO). A reduction in TMA formation has shown cardioprotective effects, and some phytochemicals may reduce TMA formation. This study aimed to develop an optimized, high-throughput anaerobic fermentation methodology to study the inhibition of choline microbial metabolism into TMA by phenolic compounds with healthy human fecal starter. Optimal fermentation conditions were: 20% fecal slurry (1:10 in PBS), 100 µM choline, and 12 h fermentation. Additionally, 10 mM of 3,3-dimethyl-1-butanol (DMB) was defined as a positive TMA production inhibitor, achieving a ~50% reduction in TMA production. Gallic acid and chlorogenic acid reported higher TMA inhibitory potential (maximum of 80–90% TMA production inhibition), with IC The World Health Organization (WHO) reports that cardiovascular disease (CVD) is the leading cause of death worldwide , with an estimate of 17.3 million deaths a year , and sevTMAO is formed due to consecutive gut bacterial and host metabolism of substrates such as choline and L-carnitine. The first metabolic step occurs in the gut, where bacteria oxidize these substrates into trimethylamine (TMA) through TMA-lyase enzymes. Specifically, TMA is formed from choline mainly through CutC/D enzyme complex activity , and froin silico approach alone does not take into account the diversity of bacteria in the gut . Additionally, no cytotoxic tests are reported, which poses the question of whether the results are due to a TMA-lyase inhibitory effect of phytochemicals or a cytotoxic one. Thus, there still exists a lack of in vitro studies that screen the potential of phytochemicals as TMAO-reducing agents, and phytochemicals are currently being tested in vivo one at a time in a non-systematic fashion. To date, Bresciani et al. [It is believed that the reported cardioprotective effects of fruit and vegetal consumption are attributed, at least to some extent, to their bioactive phytochemicals ,13. Whili et al.  are the i et al. . A high-Thus, the aims of this study were to optimize the experimental conditions for a high-throughput, low-volume fecal fermentation method capable of screening the TMA-lowering potential of phytochemicals, and demonstrate the efficacy of selected test compounds. Optimized conditions included fecal slurry to growth media ratio, fermentation time, choline concentration, positive TMA-lyase inhibitor (DMB) concentration, and phytochemical  range of concentrations.2HPO4, KH2PO4, MgSO4 × 7H2O, CaCl2 × 6H2O, ZnSO4 × 7H2O, NaHCO3, ammonium formate, hemin, bile salts, Tween 80, vitamin K1, resazurin, L-cysteine, choline, choline-d9, L-carnitine, betaine, γ-butyrobetaine, TMAO, TMA, 3,3-dimethyl-1-butanol (DMB), gallic acid, chlorogenic acid, 3--2,5-diphenyl-tetrazolium bromide, ammonia and ethyl bromoacetate were purchased from Sigma-Aldrich/Millipore . Choline-d9, L-carnitine-d9, betaine-d9, TMAO-d9 and TMA-d9  were also purchased from Sigma-Aldrich. Acetonitrile and water (LC-MS grade) as well as dimethyl sulfoxide  were purchased from VWR International . Fecal samples from two different healthy donors (ID: 0105-0003-11 and 0128-0001-01) were obtained from OpenBiome . OpenBiome collects samples from rigorously screened donors, for which the health histories, clinical data, pathogen screen results and 16S rDNA sequences are available. Samples are processed in sterile 12.5% glycerol and 0.9% saline buffer at 2.5 mL of buffer per gram of stool, and filtered through a 330 µm filter to remove large particulate matter, and frozen at −80 °C until use.Glucose, peptone, yeast extract, KCl, NaCl, Na2HPO4, 40 mg KH2PO4, 10 mg MgSO4·7H2O, 10 mg CaCl2·6H2O, 2 g NaHCO3, 50 mg hemin, 0.5 g bile salts, 2 mL Tween 80, 10 µL vitamin K1, 1 mg resazurin, and 0.5 g L-cysteine. Two different 500 mL solutions were prepared at 2X concentration. Solution A included all components except for resazurin and L-cysteine, which were included in Solution B. Both solutions were filter-sterilized separately through a 0.22-µm sterile filtering system , and pH was adjusted to 6.8. Solution B was then boiled for 10 min. Thereafter, both solutions were sparged overnight (minimum of 8 h) with N2 (g) under agitation and were then combined in the anaerobic chamber (O2 < 15 ppm) to 1X concentration. This growth media mixture was used to grow bacteria in fecal slurries under different experimental conditions. The PBS 1X solution was filter-sterilized  and sparged overnight with N2 to maintain sterile and anaerobic working conditions. Fecal samples were thawed, pooled (1 mL each from2 different donors) and diluted 10X with PBS immediately prior to use. This is referred to as “fecal slurry”. Fermentation media composition and preparation were adapted from Alqurashi et al. . The com2, 5% CO2 and 90% N2 provided by Airgas . H2  and O2  levels were monitored with a CAM-12 anaerobic monitor from Coy Lab Products . Temperature was set at 37 °C, and it was maintained constant  throughout the fermentation procedure. Humidity ranged between 40 and 50% during all experiments.All fermentations were carried out inside a 4-glove 855-ACB anaerobic chamber from Plas-Labs . The anaerobic chamber was filled with a mixed gas composed of 5% Hn = 3). Choline concentration (5–100 µM) and fecal slurry percentage (5–45%) in the final fermentation mixtures with media were studied in various combinations to select the optimal fermentation conditions. Range selection was based on the study of Bresciani et al. . In 1.1 n = 6).In order to validate the results of our study, the known TMA-lyase inhibitor DMB was used as a positive control . DMB conn = 6). Then, samples were treated as in previous steps to quantify TMA-related compounds. In order to evaluate the inhibitory effect of two representative phytochemicals  on microbial oxidation of choline to TMA, these two compounds were tested in optimal fermentation conditions  in a concentration range within 0.1–10 mM (concentrations chosen for comparison with DMB). A choline-free treatment and a DMB (10 mM) treatment were included as controls. A volume of 50 µL of sample was drawn at 0, 8 and 12 h, and the optical density was read at 600 nm , 8 µL concentrated ammonia and 120 µL of ethyl bromoacetate (20 mg/mL), and left to sit for 30 min. Then, 120 µL of 50% acetonitrile/0.025% formic acid in distilled water were added. TMA samples were filtered through AcroprepAdv 0.2 µm WWPTFE 96-well filtering plates  by centrifugation , collected in a new 96-well collection plate and frozen at −80 °C until UHPLC-MS/MS analysis. No more than 48 h passed between TMA/choline extraction and their analysis. To extract the other compounds, 25 µL of sample were mixed with 10 µL of ZnSO4 (5% w/v in water) and 100 µL of a mixture of internal standard compounds  at a concentration of 0.25 µM (except 1.20 µM for L-carnitine-d9) in 96-well plates. After sonication for 5 min in a water bath, samples were filtered and stored as described above. Choline, L-carnitine, betaine, γ-butyrobetaine, TMAO and TMA were extracted according to the previously reported methodology ,23. TMA 2) were set at 800 and 20 L/h, respectively. Electrospray ionization (ESI) was operated in the positive mode, and data were acquired using the multiple reaction monitoring (MRM) mode. Multi-reaction monitoring (MRM) fragmentation conditions of choline, L-carnitine, betaine, γ-butyrobetaine, TMAO and TMA, as well as IS compounds can be found in TMA was analyzed separately from the other compounds, but with the same UHPLC-ESI-MS/MS method. Briefly, compound separation was achieved on a Waters Acquity UPLC system  with an ACQUITY BEH HILIC column  coupled to an ACQUITY BEH HILIC pre-column  (Waters). Mobile phases consisted of 15 mM ammonium formate in water (pH 3.5) (A) and acetonitrile (B). The gradient was set to isocratic at 80% B for 3 min, with a flow rate of 0.65 mL/min. Column temperature was set at 30 °C, and autosampler at 10 °C. Quantification was achieved by coupling the above system with a Waters Acquity triple quadrupole mass spectrometer. Source and capillary temperatures were set at 150 and 400 °C, respectively. Capillary voltage was set at 0.60 kV, and desolvation and cone gas flow (both N9 (IS) peak abundance ratios in the standard curves . Data acquisition was carried out using Masslynx software . For choline, the concentration of choline at each time point found in the 0 µM choline treatment in each fecal slurry percentage (5–45%) from the experimental samples was subtracted from the same time point and same fecal slurry percentage, to account for background choline in the fecal samples. For TMA, the concentration of TMA reported at 0 h from 0 µM choline in each fecal slurry percentage (5–45%) from the experimental samples was subtracted from any other time point with the same fecal slurry concentration to account for background TMA in the fecal samples at baseline. Method sensitivity was determined by limit of detection (LOD) and limit of quantification (LOQ), respectively defined as the concentration of analyte corresponding to 3 and 10 times the signal/noise ratio. Method detection (MDL) and quantification (MQL) limits were calculated for the analysis of 25 µL of non-diluted fecal fermentation media samples. Method quality parameters can be found in For sample quantification, 45% growth media in PBS 1X was spiked with 7 different concentrations each of choline, L-carnitine, betaine, γ-butyrobetaine, TMAO and TMA standards to obtain external calibration curves in a relevant background matrix. Samples were quantified by interpolating the analyte/analyte-dTo determine the background (endogenous) levels of TMA and related compounds in experimental conditions, fecal slurry 1:10 in PBS 1X and fermentation media (45% growth media + 55% PBS 1X) with fecal slurry (1:10 in PBS 1X) at a final concentration of 20% were analyzed as previously stated. Given the presence of TMA-related compounds in the background, these samples were quantified using water to construct the calibration curves. n = 3 or 6).The number of cells present in the fermentation media was approximated by reading the optical density at 600 nm in a SpectraMax iD3 plate reader . Cell count was performed in 100 µL of sample drawn at the last timepoint of the fermentation (12–36 h), which depended on experimental conditions. Results are expressed as percentages of change versus choline-free conditions ± SEM (m/v) and 10 µL of 3--2,5-diphenyl-tetrazolium bromide at a concentration of 25 mg/mL in PBS 1X [2 < 15 ppm), and the resulting formazan crystals were resuspended to a final volume of 1 mL with DMSO by stirring for 30 min. An aliquot of 100 µL was read at 560 nm in a SpectraMax iD3 plate reader . All reagents were prepared with overnight-sparged PBS 1X and filter-sterilized 22 µm sterile filtering system, Corning) before their use. An MTT test was performed at the last timepoint of the fermentation (12–24 h), which depended on experimental conditions. Results are expressed as percentages of change versus choline-free conditions ± SEM (n = 3 or 6). A mitochondrial toxicity test (MTT) was used to estimate cell viability. Briefly, 10 µL of fermentation mixture was mixed with 80 µL of pre-heated (37 °C) PBS 1X with glucose 0.2%  or overall treatment effect (1-way) were first detected. In all cases, statistical significance was stablished a priori as p < 0.05. IC50 for gallic acid and chlorogenic acid were calculated using a 4-factor sigmoidal fitting with interpolation of concentrations producing a 50% of maximum TMA levels (mean TMA of n = 6 levels were used). Prism 8.0  was used for statistical analyses and graph creation purposes. Two-way ANOVA  was used to estimate differences in choline and TMA kinetic curves . One-way ANOVA (Tukey post hoc test) or Student’s To monitor the presence of endogenous TMA and TMA-containing substrates, TMA, choline, L-carnitine, betaine, γ-butyrobetaine and TMAO levels were quantified in fecal slurry 1:10 in PBS 1X and a 20% dilution of this mixture in fermentation background (representing everything added to fermentation experiments except exogenous choline substrate: 45% growth media + 55% PBS 1X) . Due to Choline consumption and TMA formation were monitored in fermentations with 5–45% of fecal slurry (1:10 in PBS) and exogenous choline concentrations between 0 and 100 µM (above background) in various combinations . RegardlSeveral controls to validate the utility of this model were employed. First, we wished to verify that the observed conversion of exogenous choline and production of TMA was primarily due to bacterial metabolic activity, as opposed to spontaneous degradation. In microbiota-free  and anerobic conditions A, cholinA second set of experiments was then carried out to study the best DMB concentration (0.2–10 mM) to inhibit TMA production , in ordeApparent choline concentrations at initial conditions (0 h) were different between DMB-free and DMB 5 mM A. CholinWe next sought to ascertain whether inhibition of TMA formation by DMB was due to reduced bacterial cell numbers (due to cell death or inhibition of cell growth), or by another mechanism. Cell number, measured as optical density at 600 nm, and mitochondrial respiration (MTT) were used to assess any toxic or growth-inhibition effect of DMB treatment at 24 h  on the microbial conversion of choline to TMA . FermentWe then evaluated the % inhibition of gallic acid and chlorogenic acid in choline use and TMA production out to 12 h . No chan50 for gallic acid and chlorogenic acid both at 8 and 12 h by analyzing the levels of TMA production  were flagged as “ambiguous” and may not be valid. IC50 for gallic acid at 8 h was of 8.75 mM. Chlorogenic acid reported lower IC50s than gallic acid, both at 8 and 12 h .We then calculated the ICoduction . Althoug9 peak area ratio over time, and these relative from gallic acid 10 mM and 0.1 mM were statistically different from choline 100 µM, while gallic acid 1 mM, 2 mM and 5 mM were not  were used to assess any toxic effect of gallic acid (0.1–10 mM) and chlorogenic acid (0.1–10 mM) treatments at 12 h . This tip < 0.0001) from PBS 1X (one-way ANOVA with Dunnett’s post hoc test). Choline 100 µM and DMB 10 mM (optimized conditions) did not alter formazan production when compared to the vehicle.The MTT results indicated a large dose-dependent increase in mitochondrial respiration rate for gallic acid, which was not mirrored in cell count. To determine whether gallic acid and chlorogenic acid were possibly interfering in the MTT reaction due to their reducing properties, the MTT reaction was conducted by emulating a regular MTT assay (concentration and volumes of reagents), with some key differences: in all cases, PBS 1X was used (filter-sterilized and pre-heated at 37 °C), and no fermentation media or fecal slurries were added. Results indicate that both gallic acid and chlorogenic acid increase the absorbance at 560 nm compared to the vehicle (PBS 1X) above 120% in a dose-dependent way, demonstrating that these compounds reduce 3--2,5-diphenyl-tetrazolium bromide to insoluble formazan , mimickiElevated TMAO circulating levels have been associated with CVD and atherosclerosis development ,5,6, andTo reach our goal, we first studied choline (0–100 µM) utilization and TMA production in fermentations containing 5–45% fecal slurry (1:10 in PBS 1X) for 36 h . CholineTo study the inhibition of choline microbial conversion to TMA in an in vitro assay, TMA production should be significantly different from the background in the assay. Choline concentrations of 5 and 10 µM were discarded because of their lack of statistical difference in kinetic curve and AUCs compared to the background. Although choline concentrations of 25 and 50 µM generated statistically different kinetic curves and AUCs against the background, the absolute differences in TMA production in at 36 h, and at time to reach TMA plateau were low. For example, in fecal slurry 10%, at 20 h (time to reach plateau in TMA) and at 36 h, the fold-change in TMA production between choline 25 and 50 µM and the background ranged between 1.1 and 1.3. However, this fold-change ranged between 1.4 and 1.6 in choline 100 µM. Due to the fact that the 5% fecal slurry percentage did not report a TMA concentration plateau, and that fecal slurry 45% required a high proportion of fecal slurry and reported the fastest kinetics, both conditions were not selected for further experiments. Fast kinetics might involve practical issues (missing relevant sample collection timepoints), and slower kinetics are also inconvenient due to longer experimental conditions. For these reasons, fecal slurry 20% was preferred over 10%. Thus, we selected 20% fecal slurry (1:10 in PBS 1X) and choline 100 µM for further experiments. Additionally, based on the observed TMA plateau profile, fermentation time was shortened to 24 h in follow-up experiments. These experimental conditions are in line with the ones used in similar fermentation experiments ,20.The first set of experiments was used to select optimal choline and fecal slurry fermentation conditions. Some questions still remained. For example, does non-microbial conversion of choline contribute significantly to TMA formation in this model? To study this contribution, choline (100 µM) was incubated in fermentation media (45% growth media and 55% PBS 1X) for 24 h under anaerobic conditions in the absence of fecal slurry, which was substituted by PBS 1X . OverallE. coli cells transfected with D. alaskensis CutC/D genes [A range of DMB concentrations between 0.2 and 10 mM were tested to optimize the concentrations of a known TMA-lyase inhibitor . This ra/D genes . Of noteThe odd behavior of DMB 5 mM compared to 2 and 10 mM could be attributed to the higher initial measured choline concentration in that condition A, or to P. mirabillis, P. penneri and E. fergusonii) under ~8 mM DMB was not compromised. We analyzed the cell content and mitochondrial respiration rate of our fermentations with or without DMB (0.2–10 mM) at the end (24 H) of the fermentations , MTT will not be a generally valid approach to assess cellular viability when these compounds are present. Other approaches of assessing cell viability will need to be employed.Some phenolic compounds have shown their ability to reduce TMAO levels in different models . Thus, w9 peak areas. Of note, no differences were reported at time 0 h between treatment groups, but relative betaine levels versus choline-free (0 µM) increased over time in gallic acid- and chlorogenic acid-supplemented groups. This effect was most relevant for chlorogenic acid treatments, especially at 8 h  and at 12 h for 5 mM. Choline is primarily metabolized to TMA through its direct oxidation mediated by the bacterial CutC/D enzyme complex [To further study the effect of gallic acid and chlorogenic acid on TMA production inhibition, other potential sources of TMA were studied. Besides choline, L-carnitine, γ-butyrobetaine and TMAO were not detected or not quantified at 0 h in all samples. However, betaine was present in fermentation media. Betaine levels were monitored in this third experiment, and showed interesting results. Given the high concentration of betaine in fermentation media, its quantification through the addition of standard betaine would provide inaccurate values. Rather, we monitored the relative changes of betaine in fermentation conditions relativized to choline 100 µM fermentation conditions. We also plotted relativized betaine/betaine-d complex . Alterna complex . Our dat complex , and in  complex . However50 for chlorogenic acid is higher at 12 h than at 8 h could indicate that chlorogenic acid is being degraded to a more potent inhibitory metabolite. Moreover, these compounds could also inhibit choline bacterial uptake. Future studies using cell lysates and the analysis of choline and TMA within bacterial cell should shed some light onto those mechanisms. Furthermore, the mechanisms of action may be to reduce the abundance of bacterial species that express the TMA-lyase gene. Further studies are necessary in order to investigate this possibility.Overall, gallic acid and chlorogenic acid reduce TMA formation, either through a direct TMA-lyase inhibitory effect or another non-toxic one. In fact, the data presented herein do not prove that gallic acid and/or chlorogenic acid reduce TMA production through direct TMA-lyase inhibitory effects. For example, in the study of Bresciani et al. , the mosConcentrations of gallic acid and chlorogenic acid at 5–10 mM have herein been demonstrated to be efficient at inhibiting the microbial conversion of choline into TMA in vitro, although the mechanisms by which this occurs are still unknown. Although these doses can be perceived as high for phenolic compounds, in the context of TMA production inhibition and selected phenolic compounds, those doses are likely relevant. First, the poor bioavailability and extensive phase-II metabolism of phenolics, which is a main limiting factor in systemic bioavailability and effects in peripheral organs and tissues such as the liver and adipose tissue of these compounds , are notOverall, we have reported that: (1) DMB 10 mM can be used as a control substance in TMA production inhibition studies; (2) gallic acid and chlorogenic acid (10 mM) are phenolic compounds able to reduce TMA formation at a higher proportion than DMB 10 mM; and (3) the effects reported by DMB, gallic acid and chlorogenic acid (10 mM) are due to non-toxic effects within a period of 12 h. These results open the door to using this developed methodology to study other phenolic compounds in a high-throughput way. However, the main limitation of this study is the fecal slurry itself. Further studies using fecal starters from high TMAO and/or TMA producers should be selected to study the inhibition potential of phenolic compounds, or any other treatments for that matter. Additionally, the source of the fecal slurry can have a great impact on other parameters, such as capacity to metabolize phenolic compounds such as chlorogenic acid ,50, whicThe method presented herein facilitates efficient, rapid screening of large compounds libraries for potential TMA-reducing activity. This will allow rational selection of particularly effective lead compounds for focused animal studies, potentially leading to the development of strategies to benefit specific population groups with elevated TMAO circulating levels. This approach should prove much more effective at identifying lead compounds for human trials, as opposed to the scattered approach that is currently being employed.In this study, fecal slurry (20%), choline concentrations (100 µM), reference TMA-lyase inhibitor concentrations (DMB 10 mM), and fermentation time (12 h) have been optimized to study the inhibition of TMA production by phenolic compounds. Gallic acid and chlorogenic acid were identified as TMA production inhibitors at concentrations of ~5 mM, which demonstrates that this methodology can be applied to screen for lead phenolic compounds with TMA production inhibition properties. Given the 96-well plate format (fermentation volume of 0.9 mL), this can be used to screen for compounds in a high-throughput manner.


Currently, few effective drugs are available for IPF. Hence, we sought to explore the role of mefunidone (MFD), a newly synthesized drug developed by our team, in lung fibrosis. In this study, MFD was found to attenuate bleomycin (BLM) -induced lung fibrosis and inflammation in mice according to Ashcroft and alveolitis scoring. The protein contents and total cell counts in bronchoalveolar lavage fluids of BLM-treated mice were also lowered by MFD. Moreover, the elevation of TGF-β/Smad2 and phosphorylation of MAPK pathways was repressed by MFD. Additionally, MFD attenuated the swelling and vacuolization of mitochondria, lowered the ratio of apoptotic cells, restored the mitochondrial membrane potential, and reversed the expression of cleaved-caspase 3, Bcl-2 and Bax. Meanwhile, the level of epithelial marker, E-cadherin, was restored by MFD, while the levels of mesenchymal markers such as Snail and vimentin were down-regulated by MFD. Besides, MFD inhibited the expression of fibronectin and α-smooth muscle actin in TGF-β treated normal human lung fibroblasts. Thus, our findings suggested that MFD could ameliorate lung fibrosis, cell apoptosis and EMT potentially  Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with increasing morbidity and mortality worldwide . The etiApart from lung transplantation, only two drugs were approved by Food and Drug Administration (FDA) for the treatment of IPF, including pirfenidone (PFD) and nintedanib. Although previous studies have demonstrated that these two drugs could slow the decline of lung function , there iBased on AKFPD, we further improved its structure and found mefunidone (MFD). MFD [1-(4-((3-(4-methylpiperazin-1-yl) propyl)amino)benzyl)-5-(trifluoromethyl) pyridin-2(1H)-one], is a newly synthesized compound with higher water solubility and lower toxicity compared to AKFPD and PFD . AccordiHence, we aimed to investigate the effects of MFD on bleomycin (BLM)-induced lung fibrosis and elucidate its potential mechanism. Our data demonstrated that MFD could ameliorate BLM-induced pulmonary fibrosis, abate mitochondria-dependent cell apoptosis, and inhibit EMT via suppression of TGF-β/Smad2 and MAPK pathways.All the animal studies were approved by the Laboratory Animal Welfare Ethics Committee of Central South University. 8-week-old ICR mice were purchased from Silaike Company , housed at Central South University, and given ad libitum access to food and water.All the 40 mice were randomly divided into the following five groups with eight mice per group: saline group (Control group), BLM model group (BLM group), MFD-treated group (50 mg/kg/d) (MFD group), PFD-treated group (500 mg/kg/d) (PFD group), and AKFPD group (500 mg/kg/d) (AKFPD group). BLM  was solved in saline and given to mice via a single injection of tail vein at day 1 (10 mg/kg). MFD, PFD and AKFPD were solved in saline, 0.5% CMCNa, and 0.5% CMCNa respectively, and administered daily by gavage for 28 days. All the mice were sacrificed at day 28. Lung tissues and bronchoalveolar lavage fluids (BALFs) were collected and stored for subsequent experiments.in situ with cold saline before removal. The right lungs were snap-frozen in liquid nitrogen. The left lungs were fixed in 10% formaline buffer, embedded in paraffin, and sectioned at 4 µm. Sections were fully deparaffinized and rehydrated. Hematoxylin and Eosin (H and E) staining and Masson’s trichrome staining were performed following the manufacturers’ protocols. Alveolitis score  , α-smooth muscle actin (α-SMA)  and collagen Ⅰ . Slides were further stained with secondary antibodies and DAB the following day. Images were obtained at Central South University on a Nikon microscope, and quantified using Image-Pro Plus 7.0.2. When the cells grow to 70–80% of confluence, they were incubated with serum-free medium overnight. TGF-β , BLM  and MFD were filtered with 0.22 µm filters before use. Each experiment was replicated for at least three times.The murine alveolar epithelial type Ⅱ cells (MLE-12) were obtained from Zhongqiao Xinzhou Biotechnology Company . Normal human lung fibroblasts (NHLF) were primarily cultured from normal human lungs as previously reported . Cells were grown in DMEM/F12 or DMEM medium  supplemented with 10% FBS  and 1% Penicillin-Streptomycin Liquid , and maintained at 37°C with 5% COTransmission electron microscope was used to observe the morphology of mitochondria. Right lungs were collected and immediately fixed in 2.5% glutaraldehyde, dehydrated with ethanol and acetone, embedded in resin, and cut into sections of ∼60 nm. Images were acquired on a Leica TEM at Central South University.A terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) kit  was used to detect cell apoptosis. Lung sections were fully deparaffined and rehydrated, permeablized with protease K, incubated with TUNEL reaction solution followed by DAB solution. Images were acquired on a fluorescent microscope.BALFs were collected by intratracheal instillation of 3 ml of sterile saline solution with gentle aspiration. The recovery of the lavage fluids ranged from 2 to 2.6 ml. Total cell numbers in BALFs were counted using hemocytometer with 0.4% trypan blue . The protein in BALFs was measured using the BCA Protein Assay Kit . The TGF-β level in BALFs was detected using a TGF-β enzyme-linked immunosorbent assay kit . The assay was performed following the manufacturer’s protocol. The results were presented as the mean of duplicates in pictograms per ml.p-ERK1/2 , ERK1/2 , p-Smad2 , α-tubulin , GAPDH , and TGF-β . The bands were visualized with ECL detection reagents  and quantified using ImageJ software.Whole proteins from lung tissues or cells were extracted using RIPA buffer containing protease inhibitors. Collected lysates were boiled at 95°C for 5 min, separated on 8–12% SDS-PAGE gels, and transferred to PVDF membranes. Membranes were blocked and then incubated overnight at 4°C with primary antibodies from the following sources: Fn , α-SMA , collagen-Ⅰ , Bax , Bcl-2 , cleaved-caspase 3 , E-cadherin , Snail , vimentin , Total RNA was extracted from lung tissues with Trizol reagent  according to the manufacturer’s instructions. Reverse transcription reactions were carried out with 1 μg of total RNA using a cDNA synthesis kit . The synthesized cDNA was further used for PCR where β-actin was used as loading control. SYBR Green gene expression assay  was performed in a CFX96 Real-Time System  to measure the mRNA expression. The specific primers were designed from the GenBank sequences and synthesized by Sangon Biotec . The forward primer of β-actin was 5′-GGCCAACCGTGAAAAGATGA-3′, and the reverse primer was 5′-GACCAGAGGCATACAGGGACAA-3’. The forward primer of TGF-β was 5′-GCAACATGTGGAACTCTACCAGA-3′, and the reverse primer was 5′-GACGTCAAAAGACAGCCACTCA-3’.A cell counting kit-8 (CCK-8) was purchased from APExBIO Technology LLC  to detect the effects of MFD on the viability of MLE-12 cells. It was performed following the manufacturer’s protocol. An aliquot of 100 µl of cell suspension  was inoculated in each well of the 96-well plate, and pre-incubated in a humidified incubator for 24 h. After that, cells were treated with different concentrations of MFD from 0 to 320 μg/ml for 24 h or 48 h, followed by incubation with CCK-8 solution for 2 h at 37°C. The absorbance of each well was detected at 450 nm using a microplate reader. The number of living cells was directly proportional to the absorbance.−/PI−), early apoptotic (FITC+/PI−), and later apoptotic (FITC+/PI+) cells. Data were obtained with an Aurora-10 (Cytek) flow cytometer and analyzed with FlowJo 10.1.MLE-12 cells were inoculated in each well of 6-well plates with DMEM/F12 medium containing 10% fetal bovine serum (FBS) for 24 h. Each plate contained three groups: control, BLM, and BLM plus MFD group. After incubation in medium with 2% FBS for 12 h, MFD group was pretreated with MFD (40 μg/ml) for 2 h. Afterwards, BLM group and MFD group were incubated with BLM (400 μg/ml) for 24 h to induce cell death. Cells were then collected, and stained with the FITC Annexin V and PI staining kit  according to the manufacturer’s instructions. This assay discriminates between intact  was used to measure mitochondrial membrane potential (MMP). It was performed following the manufacturer’s protocol. Briefly, MLE12 cells were seeded in 6-well plates with DMEM/F12 medium for 24 h. Each plate contained three groups: control, BLM, and BLM plus MFD group. After incubation in medium with 2% FBS for 12 h, MFD group was pretreated with MFD (40 μg/ml) for 2 h. Afterwards, both BLM group and MFD group were incubated with BLM (400 μg/ml) for 24 h to induce cell death. Diluted TMRM reagent was added, incubated for 30 min, and then exchanged with live cell imaging buffer. TMRM was detected at 575 nm. Data were acquired with an Aurora-10 (Cytek) flow cytometer.p values were calculated through one-way analysis of variance. p < 0.05 was considered significantly different. All analyses were performed using SPSS 22.0 and GraphPad Prism 8.0.All data were expressed as the mean ± SD. All experiments were conducted with more than three independent replications. BLM-induced lung fibrosis in mice has been the most widely used experimental model of IPF. At day 28, H&E staining  and MassAs one of the most important features in the initiation and progression of lung fibrosis, the up-regulation of TGF-β has been largely reported. In our study, an increase of TGF-β level in BALF was also observed after BLM treatment . We furtPrevious studies have demonstrated that cell apoptosis plays an important role in the pathogenesis of IPF . In our in vitro. We adopted MLE-12 cells, a cell line of mouse type Ⅱ alveolar epithelial cell, and firstly tested the effects of different concentration and time duration of MFD on the viability of MLE-12 cells. According to the results of CCK-8 assay . As shown in in vivo. In summary, MFD could suppress the apoptosis of MLE-12 cells, potentially through a mitochondria-dependent way.Primarily, flow cytometry was used to detect the apoptosis of MLE-12 cells. As shown in in vitro. The levels of E-cadherin, Snail and vimentin in BLM-treated MLE-12 cells were tested using western blotting. Consistent with what found in vivo, MFD significantly increased the expression of E-cadherin, and decreased that of Snail and vimentin  to testify that. According to As reported, excessive accumulation of extracellular matrix is intrinsically linked to the development of lung fibrosis . To furtIPF is a devastating disease with poor prognosis which requires more effective therapies. Currently, there are only two drugs available for IPF, including PFD and nintedanib. AKFPD and MFD are newly synthesized and orally available compounds that are developed by our team. Our previous data indicated that AKFPD and MFD inhibited renal interstitial fibrosis and collagen accumulation in rat/murine models of unilateral ureteral obstruction and diabetic kidney diseases. Still, no data was available concerning the effects of MFD on lung fibrosis. The present study demonstrated that MFD could ameliorate BLM-induced pulmonary fibrosis and inflammatory injuries in mice. Meanwhile, EMT and mitochondria-associated cell apoptosis were suppressed by MFD. Furthermore, MFD might decrease TGF-β levels and phosphorylation of Smad2 and ERK1/2. Collectively, these findings suggest the possibility that MFD might attenuate pulmonary fibrosis, inhibit cell apoptosis, and suppress EMT via down-regulation of TGF-β/Smad2 and MAPK pathways.BLM has been extensively used as an agent for lung fibrosis model in mice. In this study, we conducted a single injection of BLM via tail vein, and found massive fibrosis and inflammation in the lung at day 28. The treatment of MFD strongly inhibited lung fibrosis and collagen accumulation according to histopathology scoring and IHC staining. Meanwhile, MFD inhibited the infiltration of inflammatory cells in airways and lowered protein concentration in BALFs. Thus, these evidence supported that MFD could exert anti-fibrotic effects and ameliorate BLM-induced inflammation, which is in agreement with MFD’s anti-fibrotic role in renal fibrosis . Notablyin vivo and in vitro, suggesting that MFD inhibited cell apoptosis potentially via a mitochondria-dependent way.As is previously reported, cell apoptosis is one of the hallmarks of senescent phenotype present in fibrotic lungs . Mitochoin vivo and in vitro, namely inhibit EMT.There is abundant evidence that EMT plays a vital role in the development of IPF . EMT is Although the mechanism underlying MFD’s anti-fibrotic role has not been comprehensively determined, we identified several possibilities. TGF-β is one of the key signal cascades in the pathogenesis of IPF which might work as a master switch to trigger sequential interconnected events . ConvincTo our knowledge, this is the first study to investigate the role of MFD in lung fibrosis. In summary, we demonstrated that MFD could attenuate BLM-induced lung fibrosis, reduce cell apoptosis and EMT, and suppress TGF-β/Smad2 and phosphorylation of MAPK pathway. Therefore, considering its high solubility and comparatively lower toxicity, MFD, as a multi-target compound, might be a promising candidate for pulmonary fibrosis.