Comparing and integrating TMT-SPS-MS3 and label-free quantitative approaches for proteomics scrutiny in recalcitrant Mango (Mangifera indica L.) peel tissue during postharvest period.

Despite substantial advances in the use of proteomic technologies, their widespread application in fruit tissues of non-model and recalcitrant species remains limited. This hampers the understanding of critical molecular events during the postharvest period of fleshy tropical fruits. Therefore, we evaluated label-free quantitation (LFQ) and TMT-SPS-MS3 (TMT) approaches to analyse changes in the protein profile of mango peels during postharvest period. We compared two extraction methods (phenol and chloroform/methanol) and two peptide fractionation schemes (SCX and HPRP). We accurately identified 3065 proteins, of which, 1492 were differentially accumulated over at 6 days after harvesting (DAH). Both LFQ and TMT approaches share 210 differential proteins including cell wall proteins associated with fruit softening, as well as aroma and flavour-related proteins, which were increased during postharvest period. The phenolic protein extraction and the high-pH reverse-phase peptide fractionation was the most effective pipeline for relative quantification. Nevertheless, the information provided by the other tested strategies was significantly complementary. Besides, LFQ spectra allowed us to track down intact N-glycopeptides corroborating N-glycosylations on the surface of a desiccation-related protein. This work represents the largest proteomic comparison of mango peels during postharvest period made so far, shedding light on the molecular foundation of edible fruit during ripening.

Antifungal and Insecticidal Activities of Selected Plant Species from Cloud Forest of Veracruz, Mexico: A Contribution to the Search of Novel Control Agents against Ambrosia Pest Complexes.

The antifungal and insecticidal activities of 34 extracts from 27 plant species were evaluated against fungal phytopathogens of the genus Fusarium and Xyleborus Scolytine ambrosia beetles involved in Fusarium dieback (FD) and laurel wilt (LW) diseases. Sixteen extracts caused mycelial growth inhibition (MGI) above 23 % at 2 mg mL-1 against F. solani, those from S. nudum and M. argyrophylla exhibited the highest MGI (57 % and 49 %, respectively). Thirteen extracts displayed significant antifungal activity against F. kuroshium, those from C. nocturnum and M. argyrophylla exhibited the highest MGI (100 % and 54.9 %, respectively). Additionally, ten plants extracts caused mortality in at least one of the beetle species tested, mainly from Solanaceae species. In the most active species, 39 phenolics were identified that may have contributed to their biological effects. This study is one of the first to report the potential of plant-derived natural products against the causative agents of FD and LW.

Morphological and Physio-Chemical Responses to PEG-Induced Water Stress in Vanilla planifolia and V. pompona Hybrids.

Vanilla planifolia is an orchid of cultural and economic value. However, its cultivation in many tropical countries is threatened by water stress. In contrast, V. pompona is a species that is tolerant of prolonged periods of drought. Due to the need for plants' resistant to water stress, the use of hybrids of these two species is considered. Therefore, the objective of this study was to evaluate the morphological and physio-chemical responses of in vitro vanilla seedlings of the parental genotype V. planifolia, and the hybrids V. planifolia × V. pompona and V. pompona × V. planifolia, which were then exposed over five weeks to polyethylene glycol-induced water stress (-0.49 mPa). Stem and root length, relative growth rate, number of leaves and roots, stomatal conductance, specific leaf area, and leaf water content were determined. Metabolites potentially associated with the response to water stress were identified in leaves, through untargeted and targeted metabolomics. Both hybrids exhibited a smaller decrease in the morphophysiological responses compared to V. planifolia and exhibited an enrichment of metabolites such as carbohydrates, amino acids, purines, phenols, and organic acids. Hybrids of these two species are considered as a potential alternative to the traditional cultivation of vanilla to face drought in a global warming scenario.

Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens.

Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.

In vitro Evaluation of Anti-Inflammatory Activity of "Habanero" Chili Pepper (Capsicum chinense) Seeds Extracts Pretreated with Cellulase.

The aim of this study was to explore the effect of capsaicin and particular phenolic compounds profile from cellulase assisted extracts of Habanero (Capsicum chinense) chili pepper seeds (CPS) on the concentration of cytokines (IL-2, IL-6, TNF-α, IL-1ß) in murine macrophages (RAW 264.7) stimulated with lipopolysaccharides (LPS). Capsaicin was quantified by HPLC-DAD, and the phenolic profile was determined by UPLC-MS-QqQ. Anti-inflammatory activity was evaluated by Mouse Cytokine/Chemokine Magnetic Bead Panel 96-well plate assay. Among the 15 different phenolics found in CPS extracts obtained at 120 or 150 min of maceration with 2,500 UI/L at 30 ºC or 45 ºC in a 1:15 (w:v) proportion, the most abundant was vanillic acid (7.97-12.66 µg/g). The extract obtained at 30 ºC and 120 min, showed similar effects than the observed for synthetic anti-inflammatory drugs indomethacin and dexamethasone, and capsaicin standard. Beyond capsaicin, salicylic, protocatechuic and trans-cinnamic acids as well as vanillin in CPS extracts were correlated with the anti-inflammatory effect. On the other hand, capsaicin and chlorogenic acid contents were potential immunostimulants whose concentration varied depending on the cellulase treatment time.

The use of ecological analytical tools as an unconventional approach for untargeted metabolomics data analysis: the case of Cecropia obtusifolia and its adaptive responses to nitrate starvation.

Plant metabolomics studies haves revealed new bioactive compounds. However, like other omics disciplines, the generated data are not fully exploited, mainly because the commonly performed analyses focus on elucidating the presence/absence of distinctive metabolites (and/or their precursors) and not on providing a holistic view of metabolomic changes and their participation in organismal adaptation to biotic and abiotic stress conditions. Therefore, spectral libraries generated from Cecropia obtusifolia cell suspension cultures in a previous study were considered as a case study and were reanalyzed herein. These libraries were obtained from a time-course experiment under nitrate starvation conditions using both electrospray ionization modes. The applied methodology included the use of ecological analytical tools in a systematic four-step process, including a population analysis of metabolite α diversity, richness, and evenness (i); a chemometrics analysis to identify discriminant groups (ii); differential metabolic marker identification (iii); and enrichment analyses and annotation of active metabolic pathways enriched by differential metabolites (iv). Our species α diversity results referring to the diversity of metabolites represented by mass-to-charge ratio (m/z) values detected at a specific retention time (rt) (an uncommon way to analyze untargeted metabolomic data) suggest that the metabolome is dynamic and is modulated by abiotic stress. A total of 147 and 371 m/z_rt pairs was identified as differential markers responsive to nitrate starvation in ESI- and ESI+ modes, respectively. Subsequent enrichment analysis showed a high degree of completeness of biosynthetic pathways such as those of brassinosteroids, flavonoids, and phenylpropanoids.

Identification of Antifungal Compounds from Avocado Rhizobacteria (Bacillus spp.) against Fusarium spp., by a Bioassay-Guided Fractionation Approach.

Antimicrobial compounds produced by bacteria have been increasingly acknowledged as an important resource for the control of phytopathogens. We used a bioassay-guided fractionation approach to identify antifungal metabolites produced by two avocado rhizobacteria (INECOL-4742 and INECOL-5927), both members of the Bacillus subtilis/B. amyloliquefaciens species complex, against Fusarium solani and F. kuroshium, causal agent of Fusarium dieback in avocado and other hosts. The butanol (BuOH) organic extract from INECOL-4742 (B1-Bu) exhibited the highest percentage of inhibition (PI) against F. solani (78.76 %), also inhibiting F. kuroshium by up to 44.30 %. Primary fractions, Bu-F3, Bu-F12 and Bu-F15, obtained by silica gel open column chromatography, exhibited the highest PI against F. solani (28.57 % to 33.50 %) and F. kuroshium (38.78 % to 45.00 %). The presence of cyclic lipopeptides from the iturin, surfactin and fengycin families in B1-Bu extracts and primary fractions was determined by UPLC-ESI-HRMS. The Confocal Laser Microscopy analysis revealed deformations in the hyphae of F. kuroshium exposed to extracts, primary fractions and C-13 surfactin chemical standard. These results emphasize the potential of natural products from Bacillus for the control of the emerging phytopathogenic fungus F. kuroshium.

Isolation and Identification of Pennogenin Tetraglycoside from Cestrum nocturnum (Solanaceae) and Its Antifungal Activity against Fusarium kuroshium, Causal Agent of Fusarium Dieback.

Antifungal assay-guided fractionation of the methanolic crude extract of Cestrum nocturnum (Solanaceae), popular known as 'lady of the night', led the isolation and identification of the steroidal saponin named pennogenin tetraglycoside, which was identified for the first time in this plant species by spectroscopic means. The crude extract, fractions and pennogenin tetraglycoside exhibited mycelial growth inhibition of Fusarium solani and F. kuroshium. F. solani is a cosmopolitan fungal phytopathogen that affects several economically important crops. However, we highlight the antifungal activity displayed by pennogenin tetraglycoside against F. kuroshium, since it is the first plant natural product identified as active for this phytopathogen. This fungus along with its insect symbiont known as Kuroshio shot hole borer (Euwallacea kuroshio) are the causal agents of the plant disease Fusarium dieback that affects more than 300 plant species including avocado (Persea americana) among others of ecological relevance. Scanning electron microscopy showed morphological alterations of the fungal hyphae after exposure with the active fractions and 12 phenolic compounds were also identified by mass spectrometry dereplication as part of potential active molecules present in C. nocturnum leaves.

Contribution of Bioactive Compounds to the Antioxidant Capacity of the Edible Mushroom Neolentinus lepideus.

Neolentinus lepideus is a fungus consumed by rural communities in Central America and Asia due to its rich flavor; however, little information on its chemical composition is available. With this in mind, the objective of this work was to determine the content of vitamin E and C, ergosterol, and phenolic compounds of this fungus, as well as its antioxidant capacity. The quantified bioactive compounds were two isoforms of vitamin E, highlighting α-tocopherol (3370.35 mg/100 g dry weight, DW) and ergosterol (11.70 mg/100 g DW). The total phenolic content was 164.80 mg gallic acid equivalents/100 g, and nine phenolic compounds were identified (protocatechuic, p-hydroxybenzoic, caffeic, vanillic, ferulic, salicylic, p-anisic, trans-cinnamic acids, and scopoletin). The highest antioxidant capacity was detected in the lipophilic extract with TEAC (27688 µmoles Trolox equivalents/100 g). These results suggest that lipophilic compounds are among the main bioactive compounds in N. lepideus, and they might exhibit the highest radical scavenging properties in non-polar extracts.

Chitosan coatings reduce fruit fly (Anastrepha obliqua) infestation and development of the fungus Colletotrichum gloeosporioides in Manila mangoes.

BACKGROUND: Mangoes are tropical fruits appreciated worldwide but are extremely perishable, being susceptible to decay, pest infestation and fungal diseases. Using the flavorful and highly valued 'Manila' cultivar, we examined the effect of second-generation chitosan coatings on shelf-life, phenolic compound variation, phytohormones, pest infestation by fruit flies (Anastrepha obliqua) and anthracnose disease caused by the fungus Colletotrichum gloeosporioides. RESULTS: We observed almost total elimination of A. obliqua eggs with 10 and 20 g L-1 chitosan in diluted acetic acid and a five- to sixfold reduction in anthracnose damage. Treatment with 20 g L-1 chitosan also extended the shelf-life. External (skin) and internal (pulp) discoloration processes were delayed. Fruit firmness was higher when compared with control and acetic acid treatments, and total soluble solids were lower in chitosan-treated fruit. Targeted and non-targeted metabolomics analyses on chitosan-coated fruit identified some phenolic compounds related to the tannin pathway. In addition, abscisic acid and jasmonic acid in the peel were downregulated in chitosan-coated mango peels. Both phytohormones and phenolic content may explain the reduced susceptibility of mangoes to anthracnose development and A. obliqua egg eclosion or larval development. CONCLUSIONS: We conclude that chitosan coatings represent an effective postharvest treatment that significantly reduces anthracnose disease, inhibits A. obliqua egg eclosion and significantly extends 'Manila' mango shelf-life, a key factor currently inhibiting large-scale commercialization of this valuable fruit. © 2020 Society of Chemical Industry.

Insights into the Interaction between the Monophagous Tephritid Fly Anastrepha acris and its Highly Toxic Host Hippomane mancinella (Euphorbiaceae).

Despite their enormous economic importance and the fact that there are almost 5000 tephritid (Diptera) species, fruit fly - host plant interactions are poorly understood from a chemical perspective. We analyzed the interactions among Anastrepha acris (a little studied monophagous tephritid) and its highly toxic host plant Hippomane mancinella from chemical, ecological and experimental perspectives, and also searched for toxicants from H. mancinella in the larval-pupal endoparasitoid Doryctobracon areolatus. We identified 18 phenolic compounds from H. mancinella pulp belonging to different chemical groups including phenylpropanoids, flavonoids, chalcones and coumarins. No traces of Hippomanin A were detected in larvae, pupae or A. acris adults, or in D. areolatus adults, implying that A. acris larvae can metabolize this toxicant, that as a result does not reach the third trophic level. We tested the "behavioral preference - lack of larval specialization-hypothesis" via feeding experiments with a larval rearing medium containing H. mancinella fruit (skin + pulp or pulp alone). The high toxicity of H. mancinella was confirmed as only two (out of 2520 in three experiments) A. ludens larvae (a polyphagous pest species that preferentially feeds on plants within the Rutaceae) survived without reaching the adult stage when fed on media containing H. mancinella, whereas A. acris larvae developed well and produced healthy adults. Together, these findings open a window of opportunity to study the detoxification mechanisms used by tephritid fruit flies.

Integrated Analysis of the Transcriptome and Metabolome of Cecropia obtusifolia: A Plant with High Chlorogenic Acid Content Traditionally Used to Treat Diabetes Mellitus.

This investigation cultured Cecropia obtusifolia cells in suspension to evaluate the effect of nitrate deficiency on the growth and production of chlorogenic acid (CGA), a secondary metabolite with hypoglycemic and hypolipidemic activity that acts directly on type 2 diabetes mellitus. Using cell cultures in suspension, a kinetics time course was established with six time points and four total nitrate concentrations. The metabolites of interest were quantified by high-performance liquid chromatography (HPLC), and the metabolome was analyzed using directed and nondirected approaches. Finally, using RNA-seq methodology, the first transcript collection for C. obtusifolia was generated. HPLC analysis detected CGA at all sampling points, while metabolomic analysis confirmed the identity of CGA and of precursors involved in its biosynthesis. Transcriptome analysis identified differentially expressed genes and enzymes involved in the biosynthetic pathway of CGA. C. obtusifolia probably expresses a key enzyme with bifunctional activity, the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HQT/HCT), which recognizes shikimic acid or quinic acid as a substrate and incorporates either into one of the two routes responsible for CGA biosynthesis.

Phenylpropanoids Are Connected to Cell Wall Fortification and Stress Tolerance in Avocado Somatic Embryogenesis.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including p-coumaric acid and t-ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.

A First Glimpse of the Mexican Fruit Fly Anastrepha ludens (Diptera: Tephritidae) Antenna Morphology and Proteome in Response to a Proteinaceous Attractant.

Anastrepha ludens is a key pest of mangoes and citrus from Texas to Costa Rica but the mechanisms of odorant perception in this species are poorly understood. Detection of volatiles in insects occurs mainly in the antenna, where molecules penetrate sensillum pores and link to soluble proteins in the hemolymph until reaching specific odor receptors that trigger signal transduction and lead to behavioral responses. Scrutinizing the molecular foundation of odorant perception in A. ludens is necessary to improve biorational management strategies against this pest. After exposing adults of three maturity stages to a proteinaceous attractant, we studied antennal morphology and comparative proteomic profiles using nano-LC-MS/MS with tandem mass tags combined with synchronous precursor selection (SPS)-MS3. Antennas from newly emerged flies exhibited dense agglomerations of olfactory sensory neurons. We discovered 4618 unique proteins in the antennas of A. ludens and identified some associated with odor signaling, including odorant-binding and calcium signaling related proteins, the odorant receptor co-receptor (Orco), and putative odorant-degrading enzymes. Antennas of sexually immature flies exhibited the most upregulation of odor perception proteins compared to mature flies exposed to the attractant. This is the first report where critical molecular players are linked to the odor perception mechanism of A. ludens.

Novel findings to the biosynthetic pathway of magnoflorine and taspine through transcriptomic and metabolomic analysis of Croton draco (Euphorbiaceae).

BACKGROUND: Croton draco is an arboreal species and its latex as well as some other parts of the plant, are traditionally used in the treatment of a wide range of ailments and diseases. Alkaloids, such as magnoflorine, prevent early atherosclerosis progression while taspine, an abundant constituent of latex, has been described as a wound-healer and antitumor-agent. Despite the great interest for these and other secondary metabolites, no omics resources existed for the species and the biosynthetic pathways of these alkaloids remain largely unknown. RESULTS: To gain insights into the pathways involved in magnoflorine and taspine biosynthesis by C. draco and identify the key enzymes in these processes, we performed an integrated analysis of the transcriptome and metabolome in the major organs (roots, stem, leaves, inflorescences, and flowers) of this species. Transcript profiles were generated through high-throughput RNA-sequencing analysis while targeted and high resolution untargeted metabolomic profiling was also performed. The biosynthesis of these compounds appears to occur in the plant organs examined, but intermediaries may be translocated from the cells in which they are produced to other cells in which they accumulate. CONCLUSIONS: Our results provide a framework to better understand magnoflorine and taspine biosynthesis in C. draco. In addition, we demonstrate the potential of multi-omics approaches to identify candidate genes involved in the biosynthetic pathways of interest.

Plant origin authentication of Sonoran Desert propolis: an antiproliferative propolis from a semi-arid region.

The main chemical composition of Sonoran propolis (SP), as well as its antiproliferative activity on cancer cells through apoptosis induction, has been reported. The chemical constitution of SP remained qualitatively similar throughout the year, whereas the antiproliferative effect on cancer cells exhibited significant differences amongst seasonal samples. The main goal of this study was to provide phytochemical and pharmacological evidence for the botanical source of SP and its antiproliferative constituents. A chemical comparative analysis of SP and plant resins of species found in the surrounding areas of the beehives was carried out by HPLC-UV-DAD, as well as by 1H NMR experiments. The antiproliferative activity on cancerous (M12.C3.F6, HeLa, A549, PC-3) and normal cell lines (L-929; ARPE-19) was assessed through MTT assays. Here, the main polyphenolic profile of SP resulted to be qualitatively similar to Populus fremontii resins (PFR). However, the antiproliferative activity of PFR on cancer cells did not consistently match that exhibited by SP throughout the year. Additionally, SP induced morphological modifications on treated cells characterised by elongation, similar to those induced by colchicine, and different to those observed with PFR treatment. These results suggest that P. fremontii is the main botanical source of SP along the year. Nevertheless, the antiproliferative constituents of SP that induce that characteristic morphological elongation on treated cells are not obtained from PFR. Moreover, the presence of kaempferol-3-methyl-ether in SP could point Ambrosia ambrosioides as a secondary plant source. In conclusion, SP is a bioactive poplar-type propolis from semi-arid zones, in which chemical compounds derived from other semi-arid plant sources than poplar contribute to its antiproliferative activity.

Identification of Antibacterial Phenolics in Selected Plant Species from Mexican Cloud Forest by Mass Spectrometry Dereplication.

Fifteen plant species from a protected cloud forest (CF) in Veracruz, Mexico, were screened for their in vitro capacity to inhibit the growth of the phytopathogenic bacteria Chryseobacterium sp., Pseudomonas cichorii, Pectobacterium carotovorum and Pantoea stewartii, causal agents of damage to crops like 'chayote', lettuce, potato and corn. As a result, the bioactivity of Turpinia insignis and Leandra cornoides is reported for the first time against Chryseobacterium sp. and P. cichorii. In addition, 24 and 18 compounds not described for these species were dereplicated by an UPLC/MS-MS method, respectively. The identified compounds included simple phenols, hydroxycinnamic acids, flavonoids and coumarins. The antibacterial assay of 12 of them demonstrated the bacteriostatic effect of vanillin, trans-cinnamic acid, scopoletin and umbelliferone against Chryseobacterium sp. These findings confirm for the first time the value of the CF plants from Veracruz as sources of bioactive natural products with antimicrobial properties against phytopathogenic bacteria.

Developmental transcriptional regulation by SUMOylation, an evolving field.

SUMOylation is a reversible post-translational protein modification that affects the intracellular localization, stability, activity, and interactions of its protein targets. The SUMOylation pathway influences several nuclear and cytoplasmic processes. The expression of many genes, in particular those involved in development is finely tuned in space and time by several groups of proteins. There is growing evidence that transcriptional regulation mechanisms involve direct SUMOylation of transcriptional-related proteins such as initiation and elongation factors, and subunits of chromatin modifier and remodeling complexes originally described as members of the trithorax and Polycomb groups in Drosophila. Therefore, it is being unveiled that SUMOylation has a role in both, gene silencing and gene activation mechanisms. The goal of this review is to discuss the information on how SUMO modification in components of these multi-subunit complexes may have an effect in genome architecture and function and, therefore, in the regulation of gene expression in time and space.

Integrating Proteomics and Metabolomics Approaches to Elucidate the Mechanism of Responses to Combined Stress in the Bell Pepper (Capsicum annuum).

Bell pepper plants are sensitive to environmental changes and are significantly affected by abiotic factors such as UV-B radiation and cold, which reduce their yield and production. Various approaches, including omics data integration, have been employed to understand the mechanisms by which this crop copes with abiotic stress. This study aimed to find metabolic changes in bell pepper stems caused by UV-B radiation and cold by integrating omic data. Proteome and metabolome profiles were generated using liquid chromatography coupled with mass spectrometry, and data integration was performed in the plant metabolic pathway database. The combined stress of UV-B and cold induced the accumulation of proteins related to photosynthesis, mitochondrial electron transport, and a response to a stimulus. Further, the production of flavonoids and their glycosides, as well as affecting carbon metabolism, tetrapyrrole, and scopolamine pathways, were identified. We have made the first metabolic regulatory network map showing how bell pepper stems respond to cold and UV-B stress. We did this by looking at changes in proteins and metabolites that help with respiration, photosynthesis, and the buildup of photoprotective and antioxidant compounds.

Bioassay-guided identification of antithrombotic compounds from Cnidoscolus aconitifolius (Mill.) I. M. Jhonst.: molecular docking, bioavailability, and toxicity prediction.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Conventional antithrombotic therapy has reported hemorrhagic accidents. Ethnobotanical and scientific reports point to Cnidoscolus aconitifolius as an antithrombotic adjuvant. Previously, C. aconitifolius leaves ethanolic extract displayed antiplatelet, anticoagulant, and fibrinolytic activities. This work aimed to identify compounds from C. aconitifolius with in vitro antithrombotic activity through a bioassay-guided study. Antiplatelet, anticoagulant, and fibrinolytic tests guided the fractionation. Ethanolic extract was subjected to a liquid-liquid partitioning, followed by vacuum liquid, and size exclusion chromatography to obtain the bioactive JP10B fraction. The compounds were identified through UHPLC-QTOF-MS, and their molecular docking, bioavailability, and toxicological parameters were determined computationally. Kaempferol-3-O-glucorhamnoside and 15(S)-HPETE were identified; both showed affinity for antithrombotic targets, low absorption, and safety for human consumption. Further in vitro and in vivo evaluations will better understand their antithrombotic mechanism. This bioassay-guided fractionation demonstrated that C. aconitifolius ethanolic extract has antithrombotic compounds.Communicated by Ramaswamy H. Sarma.