In this work, leaf transcriptomic changes were evaluated in the diploid C. canephora and its polyploid C. arabica, grown at 25 °C and at two supra-optimal temperatures , under ambient (aCO2) or elevated air CO2 (eCO2). Both species expressed fewer genes as temperature rose, although a high number of differentially expressed genes (DEGs) were observed, especially at 42 °C. An enrichment analysis revealed that the two species reacted differently to the high temperatures but with an overall up-regulation of the photosynthetic machinery until 37 °C. Although eCO2 helped to release stress, 42 °C had a severe impact on both species. A total of 667 photosynthetic and biochemical related-DEGs were altered with high temperatures and eCO2, which may be used as key probe genes in future studies. This was mostly felt in C. arabica, where genes related to ribulose-bisphosphate carboxylase (RuBisCO) activity, chlorophyll a-b binding, and the reaction centres of photosystems I and II were down-regulated, especially under 42°C, regardless of CO2. Transcriptomic changes showed that both species were strongly affected by the highest temperature, although they can endure higher temperatures (37 °C) than previously assumed.Understanding the effect of extreme temperatures and elevated air (CO Froehner , with the former contributing around 60–65% of world production [Coffea arabica is thought to be originated from a single natural polyploidization event between C. canephora and C. eugenioides that occurred in very recent evolutionary times .Coffee is among the most important agricultural products . It is poduction . Coffea ears ago ). The quhis crop . The preanephora , with imanephora ,22. In fanephora ,24,25).  quality . Continu quality . A relat quality . Neverthf Brazil ,29. Furtat 24 °C . Also, sat 24 °C  or even at 24 °C ,30. The  arabica . For C. to 26 °C , or fromto 26 °C , but higto 26 °C . In thisCoffea, eCO2 promotes a high plant vigor [2 causes significant changes in the transcriptomic responses of C. arabica and C. canephora, with differentially expressed genes (DEGs) being much more abundant in the diploid than in C. arabica [2, C. canephora expressed DEGs associated to general biological processes and to a lower extent to abiotic stress responses while C. arabica showed an upregulation of genes linked to plant tolerance, oxidative stress control, aquaporins, reorganization of the lipid matrix membrane, chloroplast/thylakoid organization, and PS II repair. This suggested a protective role of eCO2 under stressful conditions.In the last 20 years, a considerable amount of research has been devoted to environmental coffee physiology focusing on water relations and drought tolerance mechanisms, but little is still known on the mechanisms of tolerance to unfavorable temperatures, namely transcriptional and metabolic differences in response to warmer temperatures (see ). For innt vigor  and evennt vigor , while int vigor ,38,39 annt vigor ,37,40,41nt vigor . We prev arabica . FunctioC. arabica (cv. Icatu) and C. canephora (cv. Conilon Clone 153) leaves for a thorough understanding of how coffee plants regulate transcriptomic responses to elevated temperatures, and how eCO2 can mitigate some deleterious effects at the physiological and metabolic level. For this, we have used a comparative approach using two coffee genotypes grown under a control temperature of 25/20 °C (day/night) and then gradually exposed to increased temperatures up to 42/30 °C in combination with ambient CO2 (aCO2: 380 μL L−1) or elevated CO2 (eCO2: 700 μL L−1) air. This analysis is complementary to (and advances) previous studies, in which the plants from these two genotypes showed a temperature tolerance up to 37/28 °C [2 [In the present study we explored the transcriptome of 28 °C [2 , which mStringent quality assessment and data filtering generated an average of 26.3 million clean reads, from an average 28.3 million raw reads. Overall, a high proportion of reads were aligned to the reference genome since only an average of 3.17% of reads were not mapped . Statist−1 aCO2 or 700 μL L−1 eCO2. Overall, fewer genes were expressed as temperatures increased, especially in combination with eCO2 where the lowest number of expressed genes was observed (The number of expressed genes varied widely between the control temperature at 25/20 °C (25 °C) and the two different supra-optimal temperatures of 37/28 °C (37 °C) and 42/30 °C (42 °C), either under 380 μL Lobserved .2 condition and the species involved. PC1 was able to separate and group all Conilon Clone 153 (CL153) plants under 25 °C and 37 °C in the lower right part of this axis, whereas Icatu plants showed a wider variation. However, it seems remarkable that at the highest temperature, the CO2 condition become determinant with PC1 separating the plants under aCO2 (left upper quadrant) from those under eCO2 (right upper quadrant).The principal coordinate analysis (PCoA) revealed a stronger transcriptomic response in the two species as a result of the highest temperature B. In fac2) to 13,134  in Icatu and from 8240  to 12,115  in CL153, being consistently higher at the highest temperature in both species, especially when combined with eCO2  to 19.9% (aCO2) of DEGs were specific of 37 °C, while 30.0% (aCO2) to 37.9% (eCO2) were specific of 42 °C (2 where the highest number of DEGs were found (4980). This reflects a very specific response to the highest temperature that was further amplified by eCO2  and eCO2 (3735), as temperatures rose its percentage was lower under eCO2 than under aCO2 (31% vs. 35%)  C,D.2 where DEGs were predominantly up-regulated in Icatu  terms using the functional annotation of the reference genome of anephora . Tempera2 conditions  was related to molecular functions  and was found only at 42 °C in eCO2. The remaining were less enriched categories with a predominance of GO terms related to photosynthesis (GO:0015979), thylakoid (GO:0009579), photosystem (GO:0009521), photosynthesis, light reaction (GO:0019684), and chlorophyll binding (GO:0016168) found only at 37 °C. Up-regulated DEGs in 42 °C Icatu plants were also enriched for heat shock protein binding (GO:0031072) but only under aCO2. In fact, at 42 °C in eCO2 there was an enrichment in the folding and binding of proteins.Up-regulated DEGs at 37 °C showed more enriched GO terms than at 42 °C, under both COnditions . Within 2 while under eCO2, Icatu and especially CL153 showed an enrichment in general activities as for instance, oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen (GO:0016705). At 42 °C in aCO2, down-regulated DEGs linked with microtubule motor activity (GO:0003777) and microtubule binding (GO:0008017) were highly enriched in Icatu while CL153 showed an enrichment in the molecular functions related to transporter activity. In this supra-optimal temperature, eCO2 triggered less enriched categories: Icatu plants were mostly enriched in two biological processes, the secondary metabolic process (GO:0019748), and the lignin catabolic process (GO:0046274), while CL153 plants showed an enrichment in the molecular functions linked to calcium ion binding (GO:0005509), xyloglucan:xyloglucosyl transferase activity (GO:0016762), sulfotransferase activity (GO:0008146), and oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as an acceptor (GO:0016682).Down-regulated DEGs featured 37 enriched categories (17 in Icatu and 20 in CL153), although all with a very small number of gene counts . At 37 °A total of 667 responsive DEGs associated with photosynthesis and other important metabolic functions in coffee leaves  were found in the two genotypes: 180 directly related with the photosynthetic pathway , 73 to antioxidant activities, 123 to lipid metabolism, and 291 directly related with the respiratory pathway  . Fold ch2, while in CL153 they were always up-regulated  I and II were down-regulated in Icatu, contrary to CL153 where they were up-regulated. Under these extreme conditions, Icatu also repressed genes involved in chlorophyll a-b binding and most PsbQ and PsbP genes, contrary to CL153 where they were generally up-regulated  and malate dehydrogenase (MDH) activity (involved in glycolysis) were mostly down-regulated in all treatments, except for MDH in 37 °C-CL153 plants under aCO2.The remaining categories were mostly down-regulated, especially at 42 °C under eCOenotypes A,B. For y of CO2 A,B. OverC. arabica cv. Icatu expressed more genes than the diploid C. canephora cv. Conilon Clone 153. However, both showed a decrease in the number of responsive genes to the rising temperatures, especially under the highest one (42 °C)  A. A marknd 37 °C B. Sample pathway ,30,41.C. arabica (cv. Acauã and Catuaí) where the number of DEGs decreased as a response to elevated temperatures . Nevert30/26 °C  vs. 10 m30/26 °C ,30.2, where a higher up-regulation was seen in Icatu than in CL153 . An important point of our experiments is that coffee plants were gradually exposed to supra-optimal temperatures, allowing them to express their acclimation capabilities under 37 °C (and partly 39 °C). This intrinsic capability to acquire thermotolerance would contribute to metabolic homeostasis, even under fluctuating field environments, explaining why these genotypes perform better under elevated temperatures than expected in classical reports [2 allowed the plant to maintain a greater metabolic activity than under aCO2, particularly at 42 °C. In fact, at that temperature, we found less enriched categories in down-regulated DEGs under eCO2 than aCO2. At 42 °C in aCO2, down-regulated DEGs in Icatu were enriched in microtubule motor activity (GO:0003777) and microtubule binding (GO:0008017), while in CL153 they showed an enrichment in transporter activity, being possibility involved in the response to stress. Although mechanisms are still being studied, several studies suggest that microtubules act as sensors of abiotic stressors, changing cellular transport, division, and the formation of the cell wall in response to stresses such as drought and temperature [2 seem to alleviate some stress effects and only six categories were enriched , particularly at a PSs level ) [2), especially in Icatu, when compared with the numbers at 37 °C ) . Therefoat 37 °C , suggest2 [2 [2 conditions  [2, which had the lowest number of up-regulated DEGs at 42 °C  component of the photosynthetic apparatus in contrast with  PS components ,30, and 2 . These e2 [2 . Therefoer eCO2) ,30. The er eCO2) ,30, espeat 42 °C  togetherPsbQ and PsbP) exhibited a reduced expression in Icatu in response to supra-optimal temperatures contrary to CL153 where they were generally up-regulated  and its progenitors (C. canephora cv. Nemaya and C. eugenioides) when grown under a constant temperature of 24 °C [2 [Several genes coding for the photosynthetic machinery . Several antioxidant genes such as peroxidase 4, or thioredoxin and FAD-related genes such as Reticuline oxidase  that wa regions ,77, whil regions , despite impacts ,38,40, e impacts  and bean impacts . Therefo impacts ,37 to guC. arabica and C. canephora genotypes can acclimate to projected future climate changes by exploring the effects of elevated temperatures alone, and in combination with eCO2. Both Icatu and CL153 revealed an intrinsic tolerance until 37 °C, which is well above what is usually assumed, but both were clearly impacted at 42 °C [2 by the extreme temperature of 42 °C than the polyploid Icatu, although both genotypes presented severe impacts in the leaf metabolism including the photosynthetic level [2 of the negative impacts imposed by this high temperature was not entirely reflected in the up-regulation of DEGs associated with photosynthetic genes (particularly in Icatu), suggesting that the maintenance of photosynthetic activity was preferentially supported by protective mechanisms rather than de novo protein synthesis. Still, the identification of key genes related with photosynthesis can provide clues to the temperature tolerance/sensitivity of coffee genotypes. Taken together with previous physiological and biochemical studies, this RNAseq study provides evidence of acclimation responses that underpin key processes for coffee acclimation to extreme temperatures.In this study, we provide new clues as how at 42 °C ,30. Chanic level ,30,41. NC. canephora Pierre ex A. Froehner cv. Conilon Clone 153 (CL153) and C. arabica L. cv. Icatu Vermelho (Icatu); the latter an introgressed variety resulting from a cross of C. canephora and C. arabica cv. Bourbon Vermelho, then further crossed with C. arabica cv. Mundo Novo). The experimental conditions were implemented as previously described [−1), photoperiod (12 h), and either at ambient (aCO2: 380 μL L−1) or elevated (eCO2: 700 μL L−1) CO2. Thereafter, temperature was raised at a rate of 0.5 °C day−1  from 25/20 °C to 42/30 °C, with 7 days of stabilization at 31/25, 37/28, and 42/30 °C to allow for programmed evaluations [2 or eCO2 conditions. The value for the eCO2 treatment was chosen considering the predictions for the second half of the current century [In this study, we used 1.5 years old potted plants from two cultivated Brazilian genotypes, escribed . Brieflyluations ,41. The operiod 1 h, and e century  and fert century .2 treatments × three temperatures × three biological replicates) using the RNeasy Plant Mini Kit  according to the manufacturer’s instructions. All RNA samples were individually analyzed for the possible presence of DNA contamination by standard PCR reactions (35 cycles) using primers designed for ubiquitin (UBQ) gene, in the absence of cDNA synthesis [Leaves from plagiotropic and orthotropic branches were collected after ca. 2–3 h of illumination (between 10:00–11:00) from the two top pairs of recently matured leaves in the upper (illuminated) part of each plant (nine per treatment), flash frozen in liquid nitrogen and stored at −80 °C until analysis. Total RNA was extracted from 36 samples  and adapter databases .High-quality reads were obtained after several steps of quality checks which included trimming and the removal of adaptor/primer and low-quality reads using FastQC version 0.11.8  and TrimC. canephora genome downloaded from the Coffee Genome Hub  [The filtered high-quality reads were mapped to the reference genome of the ly 2019)  using STly 2019)  with defly 2019)  was usedly 2019)  and gffrly 2019)  were use2 treatments/temperature treatments were estimated using DESeq2 v1.28.1 [2 fold change expression and an FDR < 0.01 were defined as differentially expressed. Python’s matplotlib library was used to plot Venn diagrams and barplots [Gene expression normalization of all the samples was estimated in FPKM (fragments per kilobase of exon per million fragments mapped). The changes in the relative abundance of the genes between the different genotypes/CO v1.28.1  and edge v1.28.1 . Only th v1.28.1 ). Genes barplots . Principbarplots .C. canephora, downloaded from the Coffee Genome as described above. Gene ontology (GO) enrichment analyses were applied to understand the functional classification of responsive DEGs through an over-representation analysis (ORA) using gProfiler [2 fold change was scaled by gene across treatments (row Z-score).DEGs followed the functional annotation of the reference genome Profiler  under FDProfiler  by removProfiler . This sa


Cymbopogon winterianus (EOCW) is a natural product with antioxidant, anti-inflammatory, and antifibrotic properties. We studied the effect of EOCW in the progression of histological changes of pulmonary fibrosis (PF) in a rodent model. The oil was obtained by hydrodistillation and characterized using gas chromatography–mass spectrometry. Intratracheal instillation of bleomycin was performed in 30 rats to induce PF, while Sham animals were subjected to instillation of saline solution. The treatment was performed using daily oral administration of distilled water, EOCW at 50, 100, and 200 mg/kg, and deflazacort (DFC). After 28 days, hemogram and bronchoalveolar lavage fluid (BALF), tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were assayed. Histological grading of PF, immunohistochemical expression of α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) were also analyzed. The EOCW major compounds were found to be citronellal, geraniol, and citronellol. EOCW significantly reduced inflammation in BALF, reduced MDA levels, and increased SOD activity. EOCW attenuated histological grading of PF and reduced immunohistochemical expression of α-SMA and TGF-β in a dose-dependent way, likely due to the reduction of oxidative stress, inflammation, and TGF-β-induced myofibroblast differentiation.The essential oil of  Idiopathic pulmonary fibrosis (PF) is a chronic interstitial lung disease that affects over 3 million individuals worldwide, with a mean survival time of about 3 years, characterized by progressive deposition of fibrotic tissue in the lungs and overall poor prognosis ,2. StudiThe main pathologic changes that play a critical role in the pathogenesis of idiopathic PF are oxidative stress, inflammation, and collagen deposition, but the comprehensive understanding of the pathobiology of the disease remains elusive . HoweverTransforming growth factor beta (TGF-β) is a cytokine that plays a key role in the pathogenesis of idiopathic PF . IncreasThe multifactorial etiopathogenetic factors involving continuous oxidative stress, unresolved inflammation, and tissue scarring have made the management of the idiopathic PF so challenging that few therapeutics have succeeded in the clinic, and they have failed to improve patient survival . As mostCymbopogon winterianus Jowitt is an aromatic grass cultivated in India and Brazil. The essential oil obtained from its leaves has demonstrated anti-inflammatory and antioxidant properties [operties  and low operties . These boperties . In factoperties , whereasoperties . Citroneoperties .Cichorium glandulosum Boiss et Huet, whose major chemical constituent is citronellol (74.060%), reduces the extracellular fibrillar proteins deposition, such as collagen and elastic fibers, on carbon tetrachloride-induced liver fibrosis in rats [There is also evidence that the EOCW chemical constituents modulate the fibrosis process in different tissues. Geraniol attenuates fibrosis, prevents atherogenesis, and exerts anti-inflammatory effects by the NF-κB signaling pathway on atherogenic diet induced fibrosis in hamsters . Further in rats .Cymbopogon winterianus, and its chemical composition, we hypothesize that it may prevent or attenuate the progression of histological changes of pulmonary fibrosis in a bleomycin-induced murine model.Considering the antioxidant, anti-inflammatory, and antifibrotic properties of the essential oil of Cymbopogon Winterianus were obtained from the Brazilian Agricultural Research Corporation (EMBRAPA), who collected the samples in July of 2018 in Aracaju (coordinates: 10°57′02.4” S 37°03′07.4” W), Sergipe, Brazil. The specimens were deposited in the herbarium of the Department of Botany nf the Tiradentes University with the registration no. 0844. Fresh leaves of Cymbopogon Winterianus were dried at 60 ± 1 °C for four days in a drying oven . Hydrodistillation was used to obtain the essential oil of the leaves in a Clevenger-type apparatus. The obtained essential oil was separated from the aqueous phase and stored in an amber bottle in a freezer (at −4 °C) until further used. The chemical analysis of its components was performed by a GC/MS  equipped with an AOC-20i autoinjector , following a previously described method [®-5MS Restek fused silica capillary column (5% diphenyl-95% dimethylpolysiloxane) with a 0.25 mm internal diameter and 0.25 mm film thickness. Helium 5.0 was used as the carrier gas at a flow rate of 1.0 mL/min. The injection temperature was 280 °C. The volume of 1.0 μL (10 mg/mL) of sample was injected at a split ratio of 1:30. The oven temperature was programmed isothermally at 50 °C for 1.5 min, followed by a rate increase of 4 °C/min until reaching 200 °C, and then at 10 °C/min up to 300 °C, which was kept for 5 min. For the GC/MS, the ionic capture detector impact energy was 70 eV. The fragments were analyzed by a quadrupolar system programmed to filter fragments/ions with m/z from 40 to 500 Da and detected by an electron multiplier. The data were processed with the aid of GCMS Postrun Analysis software . The components were identified by a comparison of their retention times with those available in the literature [The fresh leaves of d method , with soterature , for a hRattus norvegicus albinus) weighing 225 ± 25 g were randomly assigned into six experimental groups . Bleomycin-induced lung fibrosis was performed according to Bahri et al. (2017) [®, Rhobifarma Indústria Farmacêutica Ltda, Hortolândia, São Paulo, Brazil) and 0.25 mL/100 mg of xylazine . Briefly, induction of fibrosis was carried out by intratracheal instillation of 5.0 mg/kg body weight (bw) of bleomycin sulfate  dissolved in 0.5 mL of sterile saline. The same procedures were performed in the control group (Sham), using saline solution instead of bleomycin. The treatment of the animals proposed for the different experimental groups started three days after these procedures. Deflazacort , soybean oil, and EOCW were administered daily by gavage, for 28 days. The animals were euthanized by cardiac puncture exsanguination, and the lungs were surgically removed. The left lung was fixed in 10% phosphate-buffered formalin (pH 7.4) for further histological procedures. The right accessory pulmonary lobes were frozen (−80 °C) for further colorimetric assays.The animal experiments were approved by the Ethics Committee on Animal Research of the Tiradentes University (CEUA/UNIT) through Opinion No. #020917. Ethics principles Use of Laboratory Animals of the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA) were followed together with the 3R principles of the EU Directive 2010/63/EU transferred to the national Decreto-Lei 113/2013 , the 2001/83/EC and 86/609/EEC , and the Amsterdam protocol on animal protection and welfare of 1997 FP7 Decision number 1982/2006EC. Thirty-six adult male Wistar rats .3 of peripheral blood.Hematologic analysis was performed using the automatic hematologic analyzer Sysmex Xs1000i . Differential leukocyte counting was performed with an optical microscopy after staining with Pappenheim’s method for each case. Data were expressed as absolute number of cells per mmAfter cardiac puncture exsanguination, 1 mL of sodium phosphate buffer was slowly injected into the lungs through a catheter and then immediately aspirated. Samples were centrifuged, supernatant was stored at –80 °C, and the pellet was resuspended in 250 μL of sodium phosphate buffer. The total number of cells was assessed by direct counting in a hemocytometer using the Trypan blue exclusion method. For cell differentiation analysis, 200 μL of cell suspension was added in a cytospin slide chamber , spun at 800 rpm for 5 min in a Cytospin 4  and stained with Stain Set Diff-Quik . Percentages of macrophages, neutrophils, and lymphocytes were obtained and adjusted by total cell number.® brand commercial kit.Lipid peroxidation was determined based on the detection of thiobarbituric acid (TBA) reactive products . A reactw/v) with a solution containing KCl, phenylmethylsulfonylfluoride (PMSF 100 m/mol), and Triton solution (10%). Homogenates were centrifuged at 3000× g for 10 min at 4 °C, and the supernatant was stored at −80 °C for the determination of oxidative stress markers (TBARS and SH). Superoxide dismutase (SOD) activity was based on the inhibition of the auto-oxidation of epinephrine to adenochrome in the presence of SOD (pH 10.2) [The organs that were removed were washed thrice in potassium chloride solution (1.15% KCl), and then homogenized (1:5 pH 10.2) . Briefly2O2 in 0.019 M, 0.05 M phosphate buffer (pH 7), and 0.03 mL of lung sample. CAT activity was expressed as µmole of H2O2 consumed/min/mg of protein.In the samples prepared as previously described, CAT activity was assessed using the method previously reported by Bahri et al. (2020)  at 240 n2) of each section  were analyzed and classified as described in For histological analysis, the formalin-fixed lung samples were dehydrated in ethanol, diaphanized in xylene, and embedded in paraffin. Then, 20 serial histological sections were obtained from the lung sample. Four of them were stained in hematoxylin/eosin (HE) and six in Masson’s trichrome (5.0 μm thick). The other 10 histological sections (3.0 μm thick) were used for the immunohistochemical study. All of the histological analysis was blindly conducted by two examiners previously calibrated. In order to assess the severity and the extension of lung fibrosis, a semi-quantitative grading system described by Ashcroft et al. (1988)  and modi2O2, in five cycles of 5 min each. Subsequently, the sections were incubated with primary antibodies for 2 h. Primary antibodies included: anti-α-SMA  and anti-TGF-β . Immunohistochemical staining was performed with Advance , following the manufacturer’s instructions. Slides were then exposed to diaminobenzidine tetrahydrochloride  and counterstained with Carazzi’s hematoxylin. Twenty histological fields , 10 from the upper and 10 from the lower halves of the lung histological sections, were randomly selected and recorded, and positive cells (identified by cytoplasmic brown stain) were counted. Data were expressed as mean number of positive cells/histological field (0.0625 mm2).Both of the immunohistochemical reactions were performed on five 3.0-µm-thick sections of paraffin-embedded tissues. Antigen retrieval was performed with citrate buffer solution (pH 6.0) for 15 min in an electric pressure cooker. Endogenous peroxidase activity was suppressed with 10% Hp < 0.05).Statistical analysis was performed in Graph Pad Prism software, version 7.0 . Analysis of the normality of distribution of data was performed using the Shapiro–Wilk test. Gaussian data (expressed as mean ± standard mean error) were analyzed using analysis of variance (ANOVA) and Tukey’s multiple comparisons test. Non-gaussian data (expressed as median and interquartile amplitude) were analyzed using the Kruskal–Wallis test and Dunn’s multiple comparisons test. The significance level adopted for all of the tests was 5% . On the other hand, the Vehicle group and all of the other treated groups  showed statistically similar percentages of body weight loss (p > 0.05). p < 0.001). Although a significant decrease in the leukocytes counts was determined by the administration of EOCW at 50 and 100 mg/kg (p < 0.001), the leukocyte counts remained significantly above the Sham group (p < 0.001). However, the treatment with EOCW at 200 mg/kg and deflazacort not only promoted a significant decrease in the BALF leukocyte counts in comparison with groups Vehicle, EOCW50, and EOCW100 (p < 0.001), but also reduced them to values statistically comparable to the Sham group (p > 0.05). Differential analysis showed increase in neutrophils, lymphocytes, and macrophages counts in Vehicle (p < 0.001). All of the treatment protocols tested in this study similarly promoted significant reduction of neutrophils and macrophages to the basal ranges observed in Sham (p < 0.001), and there was no significant difference between them (p > 0.05). Regarding lymphocytes, however, although EOCW at 50 and 100 mg/kg determined a significant decrease in the cell counts (p < 0.001), only the administration of EOCW at 200 mg/kg and deflazacort promoted a reduction intense enough to bring the values to the basal ranges seen in Sham (p > 0.05).As demonstrated in p < 0.001). Significant reduction of MDA tissue levels in comparison with group Vehicle was observed in groups EOCW 50 , EOCW100 , EOCW200 , and DFC . In addition, the MDA levels observed in groups EOCW100 and EOCW200 were statistically comparable to group Sham (p > 0.05). A significant decrease in SOD activity was observed in Vehicle , EOCW50 , and DFC  groups in comparison with that of Sham (75.5 ± 1.5 U/min/mg). However, the SOD activity in groups EOCW100 (94.8 ± 0.4 U/min/mg) and EOCW200 (97.7 ± 4.8 U/min/mg) was statistically comparable to Sham (p > 0.05) (p > 0.05), they all significantly decreased in comparison with the Sham group (36.4 ± 1.2 µMol/min/mg)  in samples of lung tissue from animals with bleomycin-induced pulmonary fibrosis A. Increa > 0.05) B. Althoup < 0.001) and EOCW at 100  and 200 mg/kg  but not with 50 mg/kg . Furthermore, only the dose of 200 mg/kg of the essential oil promoted histological grading attenuation of the pulmonary damage comparable to deflazacort (p > 0.05).BLM induced marked morphologic changes of the lung tissue in the Vehicle group, compared with the Sham group, including: (i) intense inflammatory infiltration, with lymphocytes and alveolar macrophages in the lung interstitium; (ii) severe thickening of the alveolar septa due to intense fibrosis, leading to partial or total obliteration of the pulmonary alveoli ; (iii) intense peribronchial and peribronchiolar fibrosis; (iv) formation of air bubbles in the middle of the obliterated parenchyma, resembling the honeycomb aspect of hives ; and (v) hyperemia and hemorrhage, associated with areas of marked interstitial edema. The treatment with EOCW attenuated BLM-induced lung damage as follows: (i) reduced inflammatory/phagocytic infiltration; (ii) fewer damaged alveoli, including less alveolar obliteration and honeycombing; and (iii) reduced alveolar thickening and peribronchial and peribronchiolar fibrosis. In addition, as observed in DFC (deflazacort-treated group), the pulmonary changes observed in groups EOCW100 and EOCW200 were markedly attenuated to almost normal levels, suggesting that the doses of 100 and 200 mg/kg were more effective in inhibiting PF at a histological level A. As dem2). Although strong positivity was observed in peribronchiolar and perivascular areas, these positive cells were interpreted as smooth muscle cells or eventual pericytes instead of myofibroblasts. No positivity was observed in the alveolar epithelial cells. Group Vehicle exhibited significantly increased α-SMA expression in interstitial cells and type II pneumocytes lining alveolar spaces . Significant reduction of α-SMA expression was observed in all of the groups undergone treatment with C. winterianus essential oil and deflazacort in comparison with the Vehicle group (p < 0.001). No significant difference was observed between the groups treated with EOCW 100 (2.9 ± 0.2 cells/0.0625 µm2), EOCW 200 (2.7 ± 0.2 cells/0.0625 µm2), and DFC (2.3 ± 0.3 cells/25 µm2) (p > 0.01).2; p < 0.01). The groups Vehicle (3.17 ± 0.21 cells/0.0625 µm2) and EOCW 50 (2.661 ± 0.25 cells/0.0625 µm2) presented the greatest counts of positive cells, which were both greater than in group EOCW 200 (1.70 ± 0.22 cells/0.0625 µm2) (p < 0.001 and p < 0.05) and DFC (1.40 ± 0.18 cells/0.0625 µm2) (p < 0.001 and p < 0.001). Although the intermediate dose of the essential oil of C. winterianus (100 mg/kg) promoted a significant decrease in the immunoexpression of TGF-β (2.44 ± 0.19 cells/0.0625 µm2) in comparison with group Vehicle (p < 0.05), it was still significantly greater than in 200 mg/kg of the essential oil (p < 0.05) and DFC (p < 0.001).In the Sham group, cytoplasmic immunohistochemical expression of TGF-β was observed in alveolar macrophages, endothelial cells, and interstitial fibroblasts. However, in the groups subjected to bleomycin-induced pulmonary fibrosis, positivity was also seen in bronchiolar epithelium and type I/II pneumocytes. As expected, the immunohistochemical expression of TGF-β in all of the BLM groups was greater than in the Sham group  and geraniol (26.32%), promotes moderate inhibition of MDA production and increase in SOD activity in injured brain tissue [To investigate the role of the antioxidant properties of EOCW in attenuating bleomycin-induced pulmonary fibrosis, we assessed the levels of MDA produced by lipid peroxidation in situ, as well as SOD and CAT activity. We found increased levels of MDA and decreased activity of SOD and CAT in animals subjected to bleomycin-induced pulmonary fibrosis treated with Vehicle only in comparison to Sham, as also observed in previous investigations ,60. Bleon tissue . So, altThe occurrence of severe pathological alterations in the lung tissue in response to bleomycin intratracheal instillation is well established , and, foThere are significant parallels between inflammatory response and myofibroblast differentiation in fibrosis of different tissues , and myoAlthough the precise origin of myofibroblasts in pulmonary fibrosis is controversial, studies have suggested that they likely derive from TGF-β activated preexisting peribronchial and perivascular adventitial fibroblasts and pericytes, migration of smooth muscle cells from adjacent areas, as well as EMT of type II pneumocytes and endothelial cells ,78. As aMyofibroblast differentiation was reduced in response to the treatment with EOCW and deflazacort. The precise mechanisms underlying the negative modulation of myofibroblast differentiation by EOCW is not fully clear. Inhibition of in vitro and in vivo myofibroblastic differentiation potential through blockage of TGF-β1 expression exerted by corticosteroids in palmar fibromatosis-derived stem cells has been previously reported . As the As far as we know, no previous study has focused on the inhibitory effects of the major chemical compounds of EOCW on myofibroblastic differentiation, but we hypothesized that it might be related to their anti-inflammatory activity and suppression of TGF-β expression—just as demonstrated with corticosteroids. To prove this theory right, the immunohistochemical expression of TGF-β was assessed in histological sections of lungs with BLM-induced pulmonary fibrosis. As expected, untreated animals presented the greatest counts of TGF-β-expressing cells, which agrees with other studies previously reported ,82,83. TOral administration of EOCW attenuates the histological changes associated to the progression and severity of bleomycin-induced pulmonary fibrosis in rodents, likely due to inhibition of TGF-β immunohistochemical expression and the consequent decrease in the myofibroblast differentiation. We also provide evidence that this antifibrotic activity could be associated with the anti-inflammatory and antioxidant properties of the essential oil. Therefore, EOCW is a potential candidate to be used as a phytotherapeutic in further clinical trials for the treatment of pulmonary fibrosis.


Kd = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry ( SkM and cardiac muscle myosin promote hemostasis and thrombotic events in murine model studies , 14, andvia its Apple (A) 3 domain and enhances FXI activation by thrombin. These findings are the first to reveal and establish the novel concept that the procoagulant mechanisms of SkM include enhancement of FXI activation by thrombin.To advance knowledge about procoagulant mechanisms for SkM, here, we report studies leading to the discoveries that coagulation FXI is required for the prothrombotic activity of SkMs in fresh flowing human blood and that, in purified protein reaction mixtures, SkM binds FXI with high affinity ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-FXI monoclonal antibody (mAb) (1A6) (ex vivo procoagulant response of blood during its exposure to immobilized SkM independent of the TF pathway. This raises the question of whether the contribution of FXI to the procoagulant activity of SkM involves direct and/or indirect effects of SkM on FXI (New data from b) (1A6) , but notb) (1A6) , indicatM on FXI .Kdapp = 1.7 nM) (A). The endogenous FXI in plasma also bound to immobilized SkM  (B). This implies that the exposed SkM can capture endogenous plasma FXI as well as purified FXI in model systems. Activation occurs on SkM.To test the direct binding of FXI to SkM, SkM was immobilized onto microtiter plates for binding assays that employed purified FXI and endogenous FXI in plasma. In this assay system, purified FXI bound immobilized SkM ( 1.7 nM) A. The en nM FXI) B. This iKd of 0.20 nM based on ratio of koff/kon (kon = 3.87 × 106 M−1 s−1 and koff = 7.95 × 10−4 s−1) (A). In contrast, 25 nM prekallikrein (PK), which is a homolog of FXI but with distinct functions, did not detectably bind to SkM under the same conditions (A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb 1A6, which binds the A3 domain of FXI (B), implying SkM binds to the A3 domain. Thus, BLI shows that FXI binds reversibly with a high affinity to SkM, and that the SkM-binding site is likely, at least in part, on the A3 domain of FXI.To characterize in more detail, the direct interactions between SkM and FXI, biolayer interferometry (BLI) (Octet Red System) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI binds to SkM with a 0−4 s−1) A. In connditions A, indican of FXI , potentln of FXI B, implyiA and B). As expected, two different PolyPs enhanced FXI activation by thrombin (C) (D). Thus, the activity of SkM is independent of potential PolyP contamination of SkM, and it is, in general, not susceptible to ALP. Small unilamellar phospholipid vesicles (20% phosphatidylserine [PS]/80% phosphatidylcholine [PC]) did not affect FXI activation by thrombin under the conditions employed (E). Multiple reagents that neutralize procoagulant anionic phospholipids, including lactadherin, annexin V, and phospholipase A2 (E). Thus, this activity of SkM is not because of anionic phospholipids linked to SkM , namely a ∼100-mer and a ∼700-mer, that are known to enhance FXI activation , 20, we thrombin C  D. Thus, employed E. Multipipase A2 , 17, didipase A2 E. Thus, d to SkM . These dA). Recombinant FXI (rFXI) proteins in which the four A domains of the FXI heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK were prepared. The chimeras were used in studies of FXI activation by thrombin in the presence of SkM. The FXI chimera with the substitution of the PKA1 domain (FXI/PKA1) was not significantly activated by thrombin with or without SkM, which is consistent with the known importance of the FXI A1 domain for interactions with thrombin (F) (F). As noted previously (3B), the anti-FXI mAb 1A6, which recognizes the A3 domain of FXIs . T. TA). Rembin (F) F. As noteviously  and 3B,  of FXIs , inhibit of FXIs . It is pG) or FXI activation by alpha-FXIIa in the absence or the presence of high molecular weight kininogen . This further emphasizes that the enhancement by SkMs of FXI activation by thrombin is not because of any PolyP-like compounds and that enhancement of FXI activation by thrombin is a unique procoagulant property of SkM. In terms of broad implications, this indicates that the procoagulant activities of SkM, inter alia, include thrombin activation of FXI, which is a critical element of the intrinsic coagulation pathway's remarkable thrombin-positive feedback loops wherein thrombin activates FXI, FVIII, and FV.The effects of SkM on FXI autoactivation and FXI activation by alpha-FXIIa were also evaluated. As is well known, PolyP and some other anionic reagents, for example, nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by alpha-FXIIa , 21, 23.Fig. 3FXI is a component of the intrinsic coagulation pathway, and thrombin-mediated feedback activation of FXI may contribute to hemostasis by amplification of thrombin generation through this pathway. Excessive thrombin generation is linked to thrombosis, whereas inadequate thrombin generation is linked to bleeding. Accordingly, tissue damage that exposes blood and FXI to procoagulant SkM may contribute either to hemostasis or to thrombosis, depending on many factors and circumstances. When damaged tissue enables FXI binding to SkM, it also enables FXI to colocalize with other coagulation factors that bind to SkM, such as factor VIII/von Willebrand factor complex, factor Xa, and the prothrombinase complex , 2, therIn vitro assays involving both clotting and fibrinolytic components may be more useful for predicting bleeding tendencies in FXI-deficient subjects than are routine clinical tests  was from Calbiochem. L-α-PS and L-α-PC (each from porcine brain) were from Avanti Polar Lipids. ALP (MB grade) was from Roche Diagnostics.Rabbit SkM and phospholipase A2 from honeybee venom were purchased from Sigma–Aldrich. Human purified FXI, thrombin, bovine lactadherin, and anti-FXI mAb (AHXI-5061) were from Haematologic Technologies. Factor X and PK were from Enzyme Research Laboratories, Inc. Factor XIIaα was from Aniara Diagnostica LLC. Horseradish peroxidase–conjugated goat antimouse antibody was from Thermo Fisher Scientific. KPL SureBlue TMB 1-Component Microwell Peroxidase Substrate was from SeraCare Life Science, Inc. The chromogenic substrate for FXIa, PyrGlu-Pro-Arg-p-nitroanilide (S-2366), and H-D-Lys(Cbo)-Pro-Arg-pNA.2AcOH (Pefachrome PCa) was purchased from Diapharma and DSM Nutritional Products Ltd Branch Pentapharm, respectively. Recombinant human annexin V was from Biovision. Long-chain (p700) and medium-chain (p100) versions of PolyPs were obtained from the laboratory of James H. Morrissey, University of Michigan g for 1 min). Then SkM aliquots were stored at −80 °C until being used.SkM was dialyzed against buffer containing 600 mmol/l NaCl, 50 mmol/l Tris, pH 7.4, and after dialysis, some particles causing turbidity were removed by high-speed centrifugation  or rFXI/PK A domain chimeras  were produced and purified as described (rFXI (wt)  in sodium bicarbonate buffer, pH 9.3, was immobilized to the microtiter plate for 1 h at room temperature, followed by blocking with 0.5% of BSA solution. Then, various concentrations of purified FXI (0–10 nM) or plasma with various dilutions were added to SkM-immobilized microtiter plate for 2 h at room temperature. Then, the bound FXI was incubated with anti-FXI mAb (AHXI-5061). The SkM-FXI–anti-FXI mAb complex was detected by the horseradish peroxidase–conjugated goat antimouse polyclonal antibodies with KPL SureBlue TMB 1-Component Microwell Peroxidase Substrate.Binding studies using BLI were done with purified FXI or PK that were in 30 mM Hepes, pH 7.4, 0.01% Tween-20, and 0.1% PEG, and 50 mM NaCl at 30 °C. SkM or BSA was immobilized onto the Octet Red Amine Reactive Second-Generation Biosensor surface prior to addition of FXI or PK.2, and 0.1% BSA. The reaction was quenched at varying times using hirudin  to inhibit thrombin, and the amount of FXIa generated was measured using an FXIa chromogenic substrate .FXI  and thrombin  were mixed with varying concentrations of SkM in 30 mM Hepes buffer, pH 7.4, containing 50 mM NaCl, 25 μM ZnCl2, and 0.1% BSA. The amount of FXIa activity was measured as described previously.FXI  or rFXI or rFXI/PK chimeras and thrombin  were mixed with 50 nM SkM in 30 mM Hepes buffer, pH 7.4, containing 50 mM NaCl, 25 μM ZnClFXI  was incubated at 37 °C with SkM  or with PolyP (100-mer) (4 μM) in 30 mM Hepes, pH 7.4, buffer containing 50 mM NaCl, and 0.1% BSA. After 30 min, polybrene  was added to neutralize PolyP (100-mer), and FXIa amidolytic activity was measured.Small unilamellar vesicles of 80% PC:20% PS (w/v) in the buffer containing 150 mmol/l NaCl, 50 mmol/l Tris, and pH 7.4 (TBS) were prepared by sonication under a flow of nitrogen using a microtip sonicator.FXI  was incubated with alpha-factor XIIa (5 nM) with SkM (100 nM) or PolyP (100 mer) (4 μM) in 30 mM Hepes, pH 7.4, buffer containing 50 mM NaCl, and 0.1% BSA in the presence or the absence of high–molecular weight kininogen (100 nM) as indicated. After 30 min at 37 °C, corn trypsin inhibitor  was added to neutralize FXIIa enzyme activity, and then FXIa amidolytic activity was measured using a chromogenic substrate.All data are contained within the article and supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Cichorium intybus L. or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being harnessed. We developed and optimized a new strategy for SL extraction that can contribute to the conversion of chicory root waste into valuable products to be used in human health-promoting applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical CO2. A response surface methodology was used to optimize the process parameters  for the extraction performance. The best operating conditions were achieved at 350 bar, 40 °C, and 10% EtOH as a co-solvent in a 15 g/min flow rate for 120 min. The extraction with supercritical CO2 revealed to be more selective for the SLs than the conventional solid–liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene lactones , which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at different concentrations for their toxic profile and anti-inflammatory potential towards a human calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin inhibited the activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. These results reveal a possible exploitable green technology to recover potential anti-inflammatory compounds from chicory roots waste after inulin extraction. Cichorium intybus L., Asteraceae family) contains several health-related compounds such as inulin, flavonoids, vitamins, and sesquiterpene lactones (SLs) + of 263.12811, corresponds to the elemental formula C15H18O4, characteristic of 11β,13-dihydro-8-deoxylactucin (at 1.23 ppm mass accuracy). Both compounds were previously described in chicory extracts [Fraction 1 could not be identified with the available SL standards, and so it was then analyzed using LC-MS . The anaextracts ,33.50 of 38.81 ± 0.32 µg/mL (2), Crz1 dephosphorylates from calcineurin and migrates towards the nucleus, thus promoting the expression of calcineurin dependent response element (CDRE) regulated genes similarly to human NFAT. SFE extract is rich in SLs, which have been already demonstrated to possess anti-inflammatory potential in a previous study [The SFE extracts were then tested for their potential for anti-inflammatory activity. Cytotoxicity evaluation on yeast cells showed a toxicity for concentrations higher than 500 µg/mL A. The po32 µg/mL B. Both ius study . HoweverAs mentioned above, other pure SLs present in the SFE extract, such as lactucin, lactucopicrin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin, all of which are commercially available, were already investigated for their anti-inflammatory potential in our previous study . Therefo50 of 7.2 ± 3.15 µg/mL. The SFE purified fraction’s ability to reduce the activation of calcineurin/Crz1 was tested as an indicator of an anti-inflammatory potential. The fraction presents its maximum activity at 10 µg/mL, promoting a reduction in Crz1 activation of 53.38% ± 3.9 B. This rThe SL 8-deoxylactucin contains an α-methylene-γ-lactone ring in its structure. This moiety was described to be the most bioactive group present in SLs with respect to anti-inflammatory activity by interacting with proteins that contain exposed sulfhydryl groups via Michael-type addition . This coIn a previous study, chicory SL 11β,13-dihydrolactucin, lacking the α-methylene-γ-lactone ring, was able to modulate the activation of calcineurin/Crz1 pathway due to the presence of α,β-unsaturated carbonyl groups . In addi50 of the fraction (7.2 µg/mL). The Crz1 signal is dispersed throughout the cells in the control condition, challenging cells with MnCl2 triggered Crz1 nuclear accumulation (48.7%), which was inhibited by SFE purified fraction treatment (14.6%) in a similar manner to that of the positive control FK506, known to inhibit calcineurin activity and thereby preventing the translocation of Crz1 to the nucleus  and EtOH (96%) were used for high pressure extraction experiments. Solvents used for conventional extractions purification steps and chromatographic analysis included ethyl acetate , methanol , dichloromethane , distilled water, and ultrapure water purified with a Milli-Q water purification system . For the inflammation assays, the inflammatory stimulus was carried out by the usage of MnCl2 , and the anti-inflammatory positive control was carried by FK506 . Cellular lysis was preformed using Yeast Protein Extraction Reagent . To quantify the expression of reporter gene lacZ, we employed a solution buffer composed of Na2HPO4 , NaH2PO4·H2O , KCl , and MgSO4·7H2O , containing o-nitrophenyl β-D-galactopyranoside . Nuclear staining was carried out using nuclear dye Hoechst 33342 .COChicory roots were obtained from a processing industry , which were harvested in November 2018. For sample preparation, roots were received, cleaned, and cut into pieces (1 cm) for freeze-drying . The samples were further milled, and the particle size was determined using an AS 200 basic vertical vibratory sieve shaker , with a measuring range between 250 and 1000 µm. The milled roots were stored and protected from light while kept at room temperature in a desiccator until the day of the experiments.2 was fed into the extraction vessel using a Thar SFC P-50 high pressure pump . The extraction vessel was filled with 10 g of freeze-dried, milled chicory roots and glass beads in order to decrease solvent consumption. EtOH used as a co-solvent was pumped to the extraction vessel using a Thar SFC P-50 high pressure pump . The vessel was pressurized with CO2 until reaching the required pressure (100–550 bar), which was maintained by an automated back pressure regulator . CO2 was expanded into the first fraction collector, and the extracts were recovered in a flask placed in an ice bath.High-pressure extractions were carried out in an SFE system  . Liquid Response surface methodology, a collection of statistical and mathematical techniques for improvement and optimize processes, is proposed to model the effect of different parameters such as pressure, temperature, co-solvent percentage, and flow rate on the extraction performance by SFE ,42,43.2) and adjusted R2 (Radj2).In this study, response surface methodology was used to model the recovery of SLs from chicory roots and to optimize the SFE process conditions. The extraction of SL was carried out following two different central composite face-centered (CCFC) designs as a function of pressure (100–550 bar), temperature (25–80 °C), flow rate (10–30 g/min), and co-solvent percentage (10–40%) to optimize the mass and SL extraction yield. After the selection of independent variables and their ranges, experiments were established based on a CCFC design, and each one was coded at three levels, i.e., −1, 0, and +1 . The resCichorium intybus L. roots were extracted with either ethyl acetate or water ) for 1 h at 60 °C with mechanic stirring  at 900 rpm. Then, the extract was filtered  and centrifuged (Eppendorf Centrifuge 5810 R) to remove any remaining solid part of the unsolved raw material. The extract was dried in a rotary evaporator under reduced pressure at 40 °C. The experiments were performed in triplicate.The solid–liquid extraction, a process implemented by an industrial project partner , was performed for comparison with the SFE results. Briefly, the freeze-dried and milled Purification of the crude extract obtained from SFE consisted of dry loading approximately 1.5 g of the sample into a chromatographic column (30 × 400 mm) packed with silica gel 60 . The eluent for the purification was dichloromethane:methanol (9.5:0.5), which was used with an isocratic flow. Thin-layer chromatography (TLC) was conducted with TLC silica gel 60 F 254 and visualized under UV light to follow the purification, using the same eluent system. The pure fractions were collected, and the solvent was evaporated in a rotary evaporator under reduced pressure at 40 °C, for further analysis.® 100 RP-18, 250 × 4 mm; 5 mm; Merck®) at 35 °C. Elution was carried out in gradient mode employing the following solvent system: mobile phase A, methanol/water 14/86 (v/v); mobile phase B, methanol/water 64/36 (v/v). The gradient program used was 0 to 20 min, 100–58% A; 20 to 30 min, 58% A; 30 to 45 min, 58–0% A; 45 to 50 min, 0% A; 50 to 52 min, 0–100% A; 52 to 62 min, 100% A. The flow rate used was 0.5 mL/min, and the injection volume was 20 µL. A photodiode array detector was used to scan the wavelength absorption from 210 to 600 nm. The software used was Thermo Scientific™ Dionex™ Chromeleon™ (version 7.2 SR4) processed data.HPLC analyses were carried out on a Thermo HPLC Dionex Ultimate 3000, equipped with a quaternary pump, solvent degasser, autosampler, and column oven, coupled to a Photodiode Array Detector Thermo Dionex DAD-3000. The separation was performed on a reversed-phase column (LiCrospherv/v)) and eluent B ) were used at a flow rate of 0.19 mL min−1. A linear gradient from 5 to 75% acetonitrile (v/v) in 45 min was applied, which was followed by 15 min of washing and equilibration. FTMS full scans (m/z 90.00–1350.00) were recorded with a resolution of 60,000.For LC-MS analysis purified fraction 1 was dissolved in 70% methanol containing formic acid (0.1%) at the concentration of 10 μg/mL. LC-MS analysis was performed using the LC-PDA-LTQ-Orbitrap FTMS system (Thermo Scientific), which consists of an Acquity UPLC (H-Class) with Acquity eLambda photodiode array detector (220–600 nm) connected to a LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionizator (ESI). The injection volume was 5 μL. Chromatographic separation was conducted on a reversed phase column Luna C18 column  at 40 °C. Degassed eluent A  and YAA5 (aur1::AUR1-C-4xCDRE-lacZ) [w/v) CSM , 0.67% (w/v) YNB , and 2% (w/v) glucose), and cultures were incubated overnight at 30 °C under orbital shaking. Cultures were diluted in a fresh medium and incubated under the same conditions until the optical density at 600 nm (OD600) reached 0.5 ± 0.05. Readings were performed in a 96-well microtiter plate using a Biotek Power Wave XS plate spectrophotometer .The RE-lacZ) . The groRE-lacZ) . Briefly® Instruments, Winooski, VT, USA). Doses of chicory SFE extracts ranging from 100 to 1000 µg/mL, and purified fractions containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin in the range of 12.5–100 µg/mL were tested in the viability assays. Readings were performed using a Biotek Power Wave XS plate spectrophotometer .Cell viability was monitored as previously described . BrieflyMeasurement of β-galactosidase activity was performed as previously described . BrieflyThe monitoring of Crz1 subcellular localization was performed as previously described . Briefly50 value for the SFE total extract and the SFE purified fraction was also calculated using the GraphPad Prims 8.4.2 software.Results for the toxicity and bioactivity of SFE extract and fraction are the averages of three independent experiments and are reported as mean ± SD. Differences between the controls and the experimental concentrations were assessed by an analysis of variance with Dunnett’s multiple comparison tests (α = 0.05), utilizing GraphPad Prims 8.4.2 software. The IC2 was explored for the first time to recover SLs from Cichorium intybus L. roots. The best operating conditions to obtain sesquiterpene-rich extracts were achieved at 350 bar and 40 °C in a 15 g/min flow rate (10% EtOH). SFE proved to be an efficient process for extracting SL (53 µg/mg extract) while reducing the quantity of organic solvents consumed. The SFE method produced an extract rich in SLs, compounds that had been previously described as anti-inflammatory in S. cerevisiae model. Moreover, a purified fraction containing a mixture of noncommercially available 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was obtained. The SFE extract was then tested for its anti-inflammatory potential, where it was able to decrease the activity of the calcineurin/NFAT orthologue Crz1 in a S. cerevisiae model with an IC50 of 38.81 ± 8.32 µg/mL, indicating the possibility of exploiting this extraction method to produce a possible anti-inflammatory extract using green technology with reduced costs. The purified fraction’s anti-inflammatory capacity was also measured, resulting in an inhibition of the pathway with an IC50 of 7.2 ± 3.15 µg/mL, which are results similar to those of the immunosuppressant pharmaceutical drug FK506. We also verified the ability of this purified fraction to inhibit the translocation of the transcription factor Crz1 to the nucleus by cellular localization, observed by fluorescence microscopy. Calcineurin is highly conserved among eukaryotes, and the docking of the NFAT sequence recognized by calcineurin is highly similar to Crz1. This makes the yeast model suitable for studying the interaction of compounds with the mammalian equivalent calcineurin-NFAT pathway. However, further studies are required in order to validate this anti-inflammatory potential achieved through the inhibition of NFAT using other advanced mammalian cellular models and to address the molecular targets of the purified fraction. NFAT nuclear translocation and target genes expression can confirm the potential previously observed. A final validation in animal models subjected to an inflammatory insult is ultimately required for a deeper understanding of the mechanisms, doses, and role of the compounds on inflammatory biomarkers.In this study, a green extraction approach using supercritical COCichorium intybus L. with potential human health-promoting applications.Based on these results, we can consider pressurized fluid extraction technology as a good alternative to traditional solvent extraction methods for the development of high-value products from


Supercapacitors have gained e wide attention because of high power density, fast charging and discharging, as well as good cycle performance. Recently, expanded graphite (EG) has been widely investigated as an effective electrode material for supercapacitors owing to its excellent physical, chemical, electrical, and mechanical properties. Based on charge storage mechanism, supercapacitors have been divided into symmetric, asymmetric, and hybrid supercapacitors. Here, we review the study progress of EG-based materials to be electrode materials. Furthermore, we discuss the application prospects and challenges of EG-based materials in supercapacitors. Howperation ,12,13. Hpacitors ,16,17,18Generally, supercapacitors are categorized into two groups by charge storage mechanism: electric double layer capacitors (EDLCs) and pseudocapacitors a,b. CharElectrode material is vital in supercapacitors because it determines the capacitance, cycle, and rate performances of the supercapacitor . ExpandeEG uses graphite as a raw material and intercalates suitable chemical substances between graphite layers in different ways to obtain expandable graphite a. In 184EG can be prepared in several ways. Using different methods results in differences in the structure and surface chemical state of EG, which greatly affect its electrochemical behavior and performance. Therefore, choosing a suitable EG synthesis method according to the purpose is vital in developing supercapacitor materials.EG has a long-range ordered layered structure, adjustable interlayer distance, abundant honeycomb microstructures, and turbine-layered ordered structures ,26. ThesSymmetrical supercapacitors combine the same material with the same capacitance on the anode and cathode or a device with a working electrode of an electric double layer or pseudocapacitance level based on the working mechanism of the electrode material . Symmetr−1 under 1.0 A g−1. A two-electrode symmetric supercapacitor showed the high energy density of 6.83 W h kg−1 under the power density of 146 W kg−1 and remained 76.3% after 2000 cycles with 1.0 A g−1 (−1) and remarkable cycling stability (90.6% after 5000 cycles at 5.0 mA cm−2) (−2 with 0.5 mA cm−2), excellent rate capability, together with good cycling life (94.1% of capacitance retention within 5000 cycles). Zhou et al. forwarded an easy electrochemical method to prepare EG and prepared a polyaniline/carbon nanotube composite electrode with EG as a substrate (PANI-CNT/EG) through the one-step co-electrodeposition method [−1. The as-assembled flexible solid-state supercapacitor, on the basis of PANI-CNT/EG composite, showed good flexibility and satisfying rate performance, high energy/power performance (7.1 kW kg−1 with the energy density of 12.0 W h kg−1), together with excellent cycling retention (77.6% capacitance retention for 3000 cycles)  produced upon EG (S-PEDOT/EG) with 3D hierarchical nanostructures through a surfactant-free in situ chemical oxidation polymerization . As-prep.0 A g−1 a,b. ThesmA cm−2) c,d. Zhou cycles) e,f.2) hybrids with a 3D structure were prepared via the self-limiting redox reaction of MEGO and potassium permanganate [2 nanosheets attached to MEGO surface. Because of the short ion diffusion path, the conductivity was increased, improving the utilization efficiency of MnO2. The energy density of the symmetrical supercapacitor, based on the MEGO-MnO2 mixed material containing 24.5 wt% MnO2, was 14 W h kg−1 (13.6 W h L−1), and the power density was 250 W kg−1 (243 W L−1), when the energy density was 5.46 W h kg−1 (5.3 W h L−1), the power density was 7.67 kW kg−1 (7.44 kW L−1) at a voltage of 2 V within 1 M sodium sulfate electrolyte (2 and PANI) composite material for supercapacitor electrodes by one-step electrochemical plug-electrode deposition of MnO2 or PANI into 3D RTEG [−1 (~4 F cm−2) and 700 F g−1 (~6 F cm−2) were achieved for 3D RTEG–MnO2 and RTEG–PANI, respectively. Furthermore, both materials showed good energy efficiencies of 65–70% and 75–78%, respectively. Additionally, constructed supercapacitors with the 3D RTEG–MnO2 and RTEG–PANI hybrids exhibited good energy densities of 50.12 W h kg−1 (451.08 μW h cm−2) and 61.23 W h kg−1 (551.07 μW h cm−2) while retaining satisfying power densities of 15.26 W h kg−1 (137.34 mW cm−2) together with 20.15 kW kg−1 (181.35 mW cm−2), respectively –manganese dioxide  due to the synergistic effects between Ni–S and Co–S.Metal sulfide/EG composites can also enhance EG electrochemical performance. Wei et al. successfully prepared the Ni–Co–S/EG composite electrode with a 3D nanosheet structure through the facile and green electrodeposition method . Ni–Co–S−2), satisfying rate capability (maintained 80% with the current density of 20 mA cm−2), and excellent capacity retention . Further, the flexible symmetric supercapacitor was fabricated with two electrodes and a sulfuric acid–polyvinyl alcohol gel to be a solid-state electrolyte. The symmetric supercapacitor delivered excellent areal capacitance (30.5 mF cm−2), high-rate performance (76% capacitance retained when the current density was increased from 1 to 20 mA cm−2). Additionally, the device also exhibited good capacity retention . Moreover, the device characteristics didn’t exhibit any apparent fluctuate when it was bent 180°, indicating good mechanical flexibility 2/EG hybrid within an N, N-dimethylformamide-water system through in-situ electrodeposition [−1 under 1 A g−1 and good rate performance (1181.3 F g−1 under 10 A g−1) resulting from high capacitive characteristics chiefly derived from the synergistic impact in the layered Ni(OH)2/EG composite. Moreover, the constructed active carbon (AC)//Ni(OH)2/EG device exhibited a high energy density (32.3 W h kg−1 at a power density of 504.7 W kg−1) togrther with a long cyclic stability (retaining 79% capacitance within 1000 cycles of 5 A g−1). Similarly, Ni(OH)2/EG hybrid was prepared by the green microwave-assisted approach, while Ni(OH)2 particles were evenly distributed on the EG layers’ surface, helped to obtain the high specific capacitance with 1569 F g−1 under 1 A g−1 [2/EG asymmetric supercapacitors exhibited an energy density of 37.7 W h kg−1 at a power density of 490.9 W kg−1, 26.1 W h kg−1 even at a high-power density of 10.1 kW kg−1. Furthermore, when the current is 0.5 A g−1 and the voltage window is 1.6 V, it can get the maximum specific capacitance with 86.4 F g−1. When the current density is 5 A g−1 after 1000 cycles, specific capacitance remains at 80.1%. Ndiaye et al. synthesized vanadium dioxide/activated EG (VO2/AEG) hybrid material and carbon–vanadium–oxynitride (C–V2NO) porous network structure using chemical vapor deposition [2/AEG//C–V2NO) was measured in a two-electrode asymmetric device by VO2/AEG composite to be anode and C–V2NO to be cathode with a 6 M KOH electrolyte. The asymmetric device delivered a specific energy of 41.6 W h kg−1 with a specific power of 904 W kg−1 under 1 A g−1 specific current and a high operating voltage of 1.8 V. Specific energy of 9 W h kg−1 was retained under an amplified specific current of 20 A g−1 with the specific power of 18 kW kg−1. The supercapacitor showed a 93% capacity retention after 10,000 constant gravimetric current cycles life with the specific current of 10 A g−1 and an excellent rate capability. It maintained significant device stability without any failure after voltage-floating tests in 100 h or above  composites with the easy hydrothermal method [−1, its maximum specific capacitance of three-electrode test has been 185.5 F g−1. Under 5 A g−1, the half-cell obtained 99.7% efficiency in 2000 cycles. The assembled device with the α-MnO2/AEG hybrid and AC-PVA composite as anode and cathode, respectively, showed good capacitive properties with a specific energy of 33 W h kg−1 under specific power of 999 W kg−1 at 1 A g−1 within 2.0 V cell potential. It showed with the 5 A g−1 specific current, after more than 10,000 cycles, 97.8% of high cycle life was obtained. This device cycle stability has been further evaluated through carrying out the voltage keeping for more than 70 h and remained 70% of initial capacitance with 5 A g−1 was maintained. Wang et al. prepared partially exfoliated graphite paper (EGP) through the cathode electrochemical method of tetrabutylammonium cation intercalation [−2 with 0.5 A cm−2 as well as maintained 1.38 C cm−2 even at 60 mA cm−2. The constructed NiCo–CH@EGP//AC asymmetric supercapacitor exhibited an excellent energy density of 0.30 mW h cm−2 with a power density of 0.92 mW cm−2 together with a satisfying cycle life, retaining 78.1 % after 10,000 cycles at 20 mA cm−2  and a high rate of characteristics (70% capacitance retention rate from 1 to 10 A g−1). The as-assembled MoO3/C//EG asymmetric supercapacitor delivered a specific capacitance (88 F g−1 at 1 A g−1) and a satisfying specific energy density (31.3 W h kg−1 with a power density of 838.4 W kg−1) in 0–1.6 V voltage range and excellent capacity retention properties (86.5% capacity retention after 5000 cycles with 1 A g−1)  the formation of a sandwich-type hybrid nanostructure by molybdenum trioxide and the embedded carbon layer, which facilitates the penetration and diffusion of electrolyte ions. Using a fast and energy-saving microwave heating approach, Ni2CoS4/EG hybrids were prepared within a mixed solvent of ethylene glycol and water [2CoS4/EG hybrids could reach up to 2056.8 F g−1 under 5 A g−1, and specific capacitance would be 1923.3 F g−1 even under the 30 A g−1 current density; therefore, 92.5% of rate performance was obtained with the increase of current density from 5 to 30 A g−1. The composite also exhibited good stability of 94.4% when cycling with a current density of 30 A g−1 for 2000 cycles. It showed good initial capacitance, high-rate performance, together with excellent cycle life. Moreover, the constructed AC//Ni2CoS4/EG asymmetric supercapacitor exhibited an excellent specific capacitance of 120.3 F g−1 under 0.5 A g−1, a good cycle stability (91% under 5 A g−1 for 5000 cycles), and a high energy density of 52 W h kg−1 under 477 W kg−1  a–c. The nd water . The spe7 W kg−1 d–f. The Li-ion capacitors (LICs) are made up of a capacitor-type cathode, one battery-type anode, and one appropriate electrolyte ,72,73,744)/EG (LFP/EG) composites through in situ sol–gel method [−1 was 326.23 F g−1. The optimized LIC suggested a high specific capacitance at 200 mA g−1 was 53.31 F g−1. The LFP/EG composite and LIC maintained 84.8 % and 84.6 % of their initial specific capacitance when having 100 cycles, respectively. Qin et al. impregnated lithium dihydrogen phosphate and ferric citrate precursor solutions in a vacuum through an in situ sol–gel process and calcined them to form LFP/EG nanohybrids [−1. Furthermore, after 500 cycles, the LFP/EG hybrids-based LIC retained 100% of its initial capacitance  as the anode was fabricated for effective energy storage [−1, excellent cycling life of 94.4% when having 200 cycles with a current density of 30 A g−1, and energy densities of 252–222.6 W h kg−1 with power densities of 215–5240 W kg−1 , drew extensive attention within various electrochemical energy storage devices for its outstanding power density, extraordinarily quick charging time, good low-temperature characteristics and long cycle life. EG has excellent electrical conductivity, distinct physical and chemical characteristics, and excellent electrical and mechanical properties; thus, it has broad application prospects in the field of supercapacitors. In the past seven years, the electrochemical performance and applications of EG as a supercapacitor electrode material have been rapidly developed.This article reviews the research progress of EG-based materials as an electrode material  and theiGreen, efficient, and controllable syntheses with interlayer spacing are prerequisites for obtaining advanced EG-based materials. However, the current preparation methods for EG are mainly restricted by the excessively long expansion time, severe environmental pollution, and the difficulty to control the size of the interlayer spacing, and these limit the application of EG. Developing a low-cost and green preparation process with a high yield and an adjustable number of layers has been a difficult problem in EG research.Choosing other suitable materials to compound with EG is of great significance for constructing high-performance supercapacitor electrodes. EG exhibits excellent electrochemical performance, thus, is an excellent candidate material to practical supercapacitors. Nevertheless, its low energy and power densities do not conform with the requirements for practical applications. One of the shortcomings can be overcome by compounding other pseudocapacitive materials. Therefore, choosing the right composite material is crucial to improving the energy and power densities of EG-based supercapacitors.To control the interface reaction between an EG-based electrode and electrolyte, thorough comprehension for the energy storage mechanism is necessary. There is also a need for further simulations and modeling to reveal potential electrochemical mechanisms in the nanoscale. Many theoretical and computational researches on EDLCs have been published. However, due to surface redox and ion intercalation pseudocapacitances have been more complex and arduous for simulation, the pseudocapacitances theoretical understanding is limited. What is more, the latest in situ microscopy and spectroscopy techniques provide direct experimental proof for them.Apart from energy and power densities, the self-discharge and high and low temperature of EG-based supercapacitors should be noted. Supercapacitors have good high- and low-temperature performance, but they exhibit rapid self-discharge. Therefore, observing the self-discharge and high- and low-temperature performance of EG-based supercapacitors in future research is important. Furthermore, the reliability of devices based on EG-based supercapacitors such as high temperature storage, damp heat test, rapid temperature change, vibration, safety test  should also be concern.The rapid popularization of smart electronic products requires the continuous development of stimulus–response integrated smart power supplies. To gather more functions into an electrochemical energy storage device has been an interesting challenge. With more in-depth study, novel supercapacitors will take a vital role in providing lightweight, flexible, and wearable supercapacitors in the future.


A metabolomics-flavoromics approach was conducted to assess the micromolecules of ‘Nam Dok Mai Si Thong’ and ‘Nam Dok Mai No. 4’ mango cultivars from two seasons. During ripening, FAMEs, FFAs, fatty alcohols, sterols, and organic acids were dominant at 0–2 days, whereas amino acids, sugars, and volatile organic compounds, including esters, alcohols, ketones, aldehydes, and terpenes, were at higher levels at 4–8 days. Nine metabolites  and two volatile organic compounds (ethyl octanoate/decanoate) were related to ripening-associated changes within eight days. During ripening, sucrose at 6–8 days, citric/malic acid at 0–2 days, glycine and leucine at 4 days, and ethyl octanoate and ethyl decanoate at 8 days could be used as quality biomarkers for Nam Dok Mai Si Thong; palmitic/linoleic/linolenic acids at 0 days and β-sitosterol at 0–4 days could be used as quality biomarkers for Nam Dok Mai No. 4. Mangifera indica L.) is one of the famous tropical fruits and is widely cultivated and consumed globally. Among the tropical climacteric fruits produced worldwide, mango production levels are second only to those of bananas [Mango , 8 sugars , and 15 acids (organic and amino acids). Whereas volatile organic compounds with high vapor pressure were investigated using GC-ToF-MS with 48 total peaks including 35 identified compounds and 13 unidentified compounds comprising 8 esters, 13 alcohols, 5 ketones and aldehydes, 5 terpenes, 1 furan, 2 volatile acids, and 1 lactone. In the present study, micromolecules including lipophilics, hydrophilics, and high vapor pressure volatile organic compounds were analyzed by GC-FID and GC-Tof-MS. Previous studies evaluated in the way of cross-detector analysis by online coupling of gas chromatography (GC)-mass spectrometry (MS) and flame ionization detector (FID). GC-FID is useful for quantitative description and estimation of sample composition as well as molar ratios of different metabolites. On the other hand, GC-ToF-MS also provides reliable structural information with superior sensitivity. As a result, both detectors support a resilient tool for exploratory studies by supporting together with a powerful data-processing algorithm and could appear to be useful in metabolic profiling study . HoweverA heat plot of two replicates normalized and standardized with an internal standard was also used to investigate more specific compounds during different ripening stages of the mango cultivars. Among the 13 FAMEs, 16:0 , 16:1 , 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), and 18:3 (linolenic acid) were selected because they showed dynamic fluctuations among the various lipid classes during days 0–8 of ripening. In addition, 16:0-OH, 22:0-OH, 18:2 free fatty acids (FFA), and 18:0 FFA were also found as primary fatty alcohols at 0 days of ripening. Their relative concentration gradually decreased as the fruit reached 8 days of ripening. Comparatively, the total lipid classes at 0 days were one-fold higher than those detected at 8 days of ripening in both cultivars and seasons. These fatty alcohols might be formed by reducing fatty acyl-CoAs or fatty acyl-ACPs catalyzed by a fatty acyl reductase . AdditioTo perform robustness and continuous injection, polar metabolites via derivatization reaction were also investigated using GC-FID. Changes in sugars and amino and acids were largely detected at the 4-, 6-, and 8-day ripening stages. Starch stored in the pulp during the early ripening stages of ‘Nam Dok Mai’ mango fruits might be due to the carbon source required for the soluble sugars synthesis. Consequently, the level of sugars substantially increased at 6 and 8 days of ripening. According to Sweetman et al., the metabolic pathway of gluconeogenesis, which results in the generation of glucose from phosphoenolpyruvate, appears mainly during fruit ripening when sugars compile promptly . AdditioAmino acid content was significantly higher at day 4 of ripening in both cultivars. However, the amino acid content from 4 to 6 days of ripening varied only slightly. Both the ‘Nam Dok Mai’ cultivars had uniform acid content, although they were harvested in different seasons. The most prominent acids were glycine, leucine, and succinic acid, followed by threonine, β-aminoisobutyric acid, pyroglutamic acid, GABA, threonic acid, and fumaric acid. Glycine and leucine were significantly higher (one-fold increase) at the 4- and 6-day stages relative to their levels at the 0-, 2- and 8-day stages in both dry and rainy season mango cultivars. Consistently, succinic, fumaric, and α-ketoglutaric acid, which are the intermediates of the TCA cycle, also decreased as the ripening day increased. In contrast, as previously mentioned by Batista-Silva et al., the interrelation between organic acid and sugar metabolism during ripening seems to be a commonly associated aspect that contributes to the improvement of quality and flavor during fruit ripening . ConversCitric and malic acids increased at 0 and 2 days of ripening by nearly six-fold (17.8% vs. 2.8%) and three-fold (2.8% vs. 1.06%), respectively, compared with their levels at 8 days of ripening. Comparing cultivars, the citric and malic acid levels were slightly higher in ‘Nam Dok Mai Si Thong’ than they were in ‘Nam Dok Mai No. 4’. Additionally, the dry season ‘Nam Dok Mai’ cultivars had slightly higher levels of citric and malic acids than were detected in the rainy season cultivars. Understanding the accumulation of these acids in fruit is of predominant to the priority of fruit quality. Citric acid and malic acid are generally recognized as safe-listed compounds that are commonly used in industry as preservatives, acidulants, or flavoring agents ; they arEven distribution of volatile organic compounds contents was observed in both dry and rainy season ‘Nam Dok Mai’ mango cultivars. Esters play an essential fraction of volatile organic compounds in many fruits . At variIn Thailand, the mango season occurs around mid-March to mid-June, which is usually regarded as the dry season. The rainy season is assumed to be from mid-July to mid-November. Dry season mangoes are usually harvested around March, whereas rainy season mangoes are usually picked around the end of June. Another critical factor to consider is that mangoes are produced all year round in Thailand. In the present study, mangoes harvested in the dry season showed slightly higher volatiles content. Comparing total volatile organic compounds between cultivars, dry season ‘Nam Dok Mai Si Thong’ was a rich source of esters, terpenes, aldehydes, and ketones, whereas dry season ‘Nam Dok Mai No. 4’ had slightly more total volatile alcohols than did ‘Nam Dok Mai Si Thong’. Considering the vital role played by these metabolites and aroma volatiles in imparting the characteristics of mango cultivars, ripe fruits of ‘Nam Dok Mai Si Thong’ and ‘Nam Dok Mai No. 4’ cultivars seem to possess differentiation and unique qualities with an abundance of metabolites and volatile organic compounds.The identified and obtained metabolites and volatiles data in dry and rainy season ‘Nam Dok Mai’ mango cultivars were clearly explored, and find the relationships between the samples by using PCA. Additionally, PCA is a multivariate data analysis method that can show the variance of samples using metabolites as descriptive data . All metBoth ‘Nam Dok Mai’ cultivars were shifted along PC1 according to their ripening stage. Both dry and rainy season mango genotypes were typically distributed along PC2, depending on their quantity of metabolites. Their metabolite content also showed minor variation between seasons. Although there was a clear differentiation between the two genotypes along PC1, the metabolite compounds had similar concentrations in both ‘Nam Dok Mai’ cultivars. Therefore, the two ‘Nam Dok Mai’ mango cultivars had strong genetic and cultivar effects according to the quantitative abundance of their metabolites. This suggests that the major variability in metabolite content is given by the ripening status of the fruit, then by the genotype. The PCA loading plot was supported by observing the variance of metabolites, including the polar, non-polar, and unknown compounds analyzed in this study .Interestingly, most volatile organic compounds were equally variated in both PC1 and PC2, which represented 83% of the total variability . In PC2,The metabolites and organic volatile compounds showed four distinct clusters in a PCA biplot . The 0-dAccording to AHC analysis, a dendrogram was produced that contained four groups . The firPairwise correlation between metabolites was used to assess metabolic relationships among metabolites and the coordination of metabolic changes during the ripening of mangoes . AccordiAs previously described by Wattanakul et al., amino acids play as the substrates for the formation of higher levels of alcohols and volatile acids as the result of the Ehrlich pathway . Most FASolvents and reagents, which were all of HPLC and analytical grade, were purchased from Thermo Fisher Scientific . All standard chemicals, which were reagent grade, were purchased from Sigma-Aldrich . For the identification of metabolites, lauric acid methyl ester (12:0 FAME), myristic acid methyl ester (14:0 FAME), pentadecanoic acid methyl ester (15:0 FAME), pentadecenoic acid methyl ester 15:1 FAME, palmitic acid methyl ester (16:0 FAME), palmitoleic acid methyl ester (16:1 FAME), steric acid methyl ester (18:0 FAME), oleic acid methyl ester (18:1 FAME), linoleic acid methyl ester (18:2 FAME), linolenic acid methyl ester (18:3 FAME), erucic acid methyl ester (22:1 FAME), tricosanoic acid methyl ester (23:0 FAME), nonanoic acid (9:0 FFA), lauric acid (12:0 FFA), methyl-p-hydroxy cinnamate, methyl ferulate, hexadecanol (16:0-OH), steric acid (18:0 FFA), oleic acid (18:1 FFA), linoleic acid (18:2 FFA), linolenic acid (18:3 FFA), arachidyl alcohol (20:0-OH), linoleyl alcohol , behenyl alcohol (22:0-OH), 1-octacosanol (28:0-OH), campesterol, β-sitosterol, sitostanol, stigmasterol, gramisterol, Δ7-avenasterol, citrostradienol, glycerol, fructose, glucose, mannitol, sorbitol, myo-inositol, sucrose, trehalose, glycine, 4-hydroxybutyric acid, leucine, isoleucine, proline, succinic acid, fumaric acid, threonine, β-aminoisobutyric acid, malic acid, pyroglutamic acid, γ- aminobutyric acid, threonic acid, glutamine, citric acid, histidine, and tyrosine were used as the authentic metabolite standards.The mango samples were acquired from a Saraburi commercial orchard. Dry and rainy season mango samples were collected at 90–100 days after the fruit set and immediately transported to the laboratory at Kasetsart University, Bangkok, Thailand. Dry season samples were collected in mid-March, and rainy season samples were collected at the end of June. The fruits were selected according to their homogeneity in weight (400–420 g), length (14–16 cm), and width (7–9 cm). To control the ripening index, the uniform densities of the fruits were applied using 3% NaCl solution and sinkage in a 1% NaCl solution . The selAfter collecting the mango samples according to their days of ripening, the samples were washed, peeled, cut into 1 cm cubes, and freeze-dried in a Gamma 2-16 LSC freeze-drying machine . Freeze-dried samples were mill into a powder with an RS 300 rotor mill . This powder was stored at −20 °C till it was analyzed.TM, C18-LP (Large Pore) Solid-phase extraction, Vertical Chromatography Co. Ltd.; Nonthaburi, Thailand), the ratio for fatty acid methyl esters was 100:2 v/v and 70:20 v/v for polar lipids. A parallel evaporator was used to evaporate all of the eluents at 50 °C. The fatty acid methyl ester (FAME) residue from the dried fraction was re-dissolved in 300 µL of hexane and kept in a glass amber vial. At 70 °C for 15 min, the dried fraction polar lipids were silylated with 250 µL pyridine and 50 µL N-trimethylsilyl-N-methyl trifluoroacetamide. Lipid fractions were stored at −20 °C until using gas chromatography-flame ionized detection (GC-FID)  analysis.Extraction and fractionation of the samples were performed as previously described by Na Jom et al.  with a sThe 500 µL of polar extracts were evaporated till dry for the sugar fractionation. With 300 µL of pyridine and 100 µL of trimethylsilylimidazole, the polar residue was silylated. Silylation was carried out for 15 min in a water bath at 70 °C. Then, for the layer separation process, 300 µL of hexane and 300 µL of deionized water were added to separate the silylated sugars and sugar alcohol from the sample. The solution was then diluted with 300 µL of hexane, and the acid compounds were selectively hydrolyzed with 300 µL of deionized water. The GC-FID analysis was performed on the top layer of hexane, which contains silylated sugar compounds. The 1 mL of acids fractions was evaporated until dry with a parallel evaporator, then oximated with 300 µL of hydroxyl ammonium chloride in pyridine (2 mg/mL). After 30 min of oximation at 70 °C, silylation was performed by using 100 µL of N-methyl-N-(trimethylsilyl) fluoroacetamide to the reaction at 70 °C for 15 min. The 500 µL of hexane and 300 µL of deionized water were used for selective hydrolysis. The silylated sugars in the upper phase were eliminated. By adding 150 µL of acetonitrile and 100 µL of MSTFA at 70 °C for 60 min, the bottom phase, which contained acid compounds was re-dissolved and silylated. For GC-FID analysis, the sample was immediately stored at −20 °C. All polar and lipid extracts obtained were analyzed by gas chromatography with flame ionization detection (GC-FID).GC-FID analysis was performed by using gas chromatography (GC) conjoined with a flame ionization detector . The DB-1 capillary column (60 m × 0.32 mm × 0.25 μm film thicknesses) with a 100% dimethylpolysiloxane stationary phase  was used. Subsequently, 1 μL of each sample was injected into the GC flame ionization detector in splitless mode. As the carrier gas, helium was used at 1.8 mL per min with a constant flow rate. The inlet temperature was 280 °C; the oven temperature program started at 100 °C and then ramped up to 320 °C at 4 °C per min before being held at 320 °C for 25 min; the detector temperature was 320 °C.−1 of NaCl added to the samples. The samples were incubated at 50 °C for 5 min with constant magnetic stirring (60× g) before the SPME extraction process. The fiber was desorbed into the injection port of a gas chromatograph (260 °C) for 10 min. An inlet liner (78.5 × OD 6.5 × ID 0.75 mm) was used.Volatile organic compounds analysis followed the method described by Charve et al. . A solidAnalyses were conducted using a 6890N gas chromatograph arrayed with an HP-5MS capillary column  and paired with a time-of-flight mass spectrometer . The operating conditions were as follows: GC: split 5:1; oven temperature: initially 40 °C increasing by 5 °C per min to 150 °C and then 40 °C per min to 250 °C; TOF-MS: 40–280 u, centroid mode, 0.1 s scan time, 0.02 s inter-scan time, 2750 V and −70 eV. The GC chromatograms were compared using retention times, mass spectra of reference compounds, and mass spectra of library entries.p ≤ 0.05 using the correlation/association test mode were performed using XLSTAT-base version 2020.5 .Chromatographic areas of each metabolite were obtained and assimilated using the HP-ChemStation A.06.03 program . Flavor compounds were handled using ChromaTOF-GC Software v4.50.8.0 . Identification involved using an analytical authentic standard comparison technique, and the process was semi-quantified using a comparative investigation with an internal standard area for metabolites and volatile organic compounds. Principal component analysis (PCA), agglomerative hierarchical clustering analysis (AHC), and Spearman’s rank correlations with a significance level of Here, a comparative investigation of combined metabolomics-flavoromics was conducted that provided new insights into the different ripening stages of two ‘Nam Dok Mai’ cultivars across two seasons. Mangoes from the dry and rainy seasons were highly similar with approximately equal loads of various micromolecules. Sucrose at 6–8 days, citric and malic acid at 0–2 days, glycine and leucine at 4 days, and ethyl octanoate and ethyl decanoate at 8 days of ripening could be used as quality biomarkers for ‘Nam Dok Mai Si Thong’. In contrast, palmitic, linoleic, and linolenic acid at 0 days and β-sitosterol at 0–4 days of ripening could be used as quality biomarkers for ‘Nam Dok Mai No. 4’. In conclusion, ‘Nam Dok Mai Si Thong’ and ‘Nam Dok Mai No. 4’ cultivars could show substantial genetic viability across cultivars and seasons by demonstrating an equivalent distribution of metabolomics-flavoromics constituents at various ripening stages. The integration of combined metabolomics-flavoromics approach provided a useful tool applied for characterizing the remarkable alterations pointing out biomarker compounds that occur in ‘Nam Dok Mai’ mango cultivars during different ripening stages within the dry and rainy seasons.