Carbon and Oxygen Isotopic Ratio Analysis by FT ICR MS for Natural Vanillin Authentication.

Vanillin is the main component of vanilla flavor and is naturally produced from an orchid. However, due to the high cost and time-intensive nature of cultivating natural vanilla pods, most of the vanillin is mainly artificially manufactured. Existing methodologies, such as isotope ratio mass spectrometry (IRMS) and site-specific natural isotopic fractionation by nuclear magnetic resonance (SNIF-NMR), are employed to differentiate natural vanillin from other sources based on carbon and hydrogen isotope measurements. Nevertheless, these methods have limitations, as the carbon isotopic ratio can be counterfeited by adding commercially available enriched vanillin. For this research, we purified 1 mg of vanillin from pods from various geographical and botanical sources. We developed a novel method for analyzing 13C/12C and 18O/16O isotopic ratios of vanillin using direct injection analysis coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). This innovative approach enables the examination of bulk vanillin carbon and oxygen isotopic ratios, as well as specific molecular fragments. By analyzing a characteristic vanillin fragment that provides site-specific 18O/16O isotopic ratio data, we achieved superior clustering and discrimination of samples based on their botanical source and geographical origin. Our proposed method holds significant potential for vanillin authentication and can be performed using a mere 20 µg of pure vanillin in just 10 min of analysis time. Subsequent research should focus on acquiring additional vanillin samples from diverse botanical, geographical, and biosynthetic origins while exploring various isotopic ratios to further enhance the reproducibility and reliability of this methodology.

Gene Functional Networks from Time Expression Profiles: A Constructive Approach Demonstrated in Chili Pepper (Capsicum annuum L.).

Gene co-expression networks are powerful tools to understand functional interactions between genes. However, large co-expression networks are difficult to interpret and do not guarantee that the relations found will be true for different genotypes. Statistically verified time expression profiles give information about significant changes in expressions through time, and genes with highly correlated time expression profiles, which are annotated in the same biological process, are likely to be functionally connected. A method to obtain robust networks of functionally related genes will be useful to understand the complexity of the transcriptome, leading to biologically relevant insights. We present an algorithm to construct gene functional networks for genes annotated in a given biological process or other aspects of interest. We assume that there are genome-wide time expression profiles for a set of representative genotypes of the species of interest. The method is based on the correlation of time expression profiles, bound by a set of thresholds that assure both, a given false discovery rate, and the discard of correlation outliers. The novelty of the method consists in that a gene expression relation must be repeatedly found in a given set of independent genotypes to be considered valid. This automatically discards relations particular to specific genotypes, assuring a network robustness, which can be set a priori. Additionally, we present an algorithm to find transcription factors candidates for regulating hub genes within a network. The algorithms are demonstrated with data from a large experiment studying gene expression during the development of the fruit in a diverse set of chili pepper genotypes. The algorithm is implemented and demonstrated in a new version of the publicly available R package "Salsa" (version 1.0).

Systemic whitefly-induced metabolic responses in newly developed distal leaves of husk tomato plants (Physalis philadelphica) impairs whiteflies development.

BACKGROUND: Metabolic reconfiguration in plants is a hallmark response to insect herbivory that occurs in the attack site and systemically in undamaged tissues. Metabolomic systemic responses can occur rapidly while the herbivore is still present and may persist in newly developed tissue to counterattack future herbivore attacks. This study analyzed the metabolic profile of local and newly developed distal (systemic) leaves of husk tomato (Physalis philadelphica) plants after whitefly Trialeurodes vaporariorum infestation. In addition, the effect of these metabolomic adjustments on whitefly oviposition and development was evaluated. RESULTS: Our results indicate that T. vaporariorum infestation induced significant changes in husk tomato metabolic profiles, not only locally in infested leaves, but also systemically in distal leaves that developed after infestation. The distinctive metabolic profile produced in newly developed leaves affected whitefly nymphal development but did not affect female oviposition, suggesting that changes driven by whitefly herbivory persist in the young leaves that developed after the infestation event to avoid future herbivore attacks. CONCLUSIONS: This report contributes to further understanding the plant responses to sucking insects by describing the metabolic reconfiguration in newly developed, undamaged systemic leaf tissues of husk tomato plants after whitefly infestation. © 2022 Society of Chemical Industry.

Metabolomic Analysis Identifies Differences Between Wild and Domesticated Chili Pepper Fruits During Development (Capsicum annuum L.).

Capsicum spp. members are a rich source of specialized compounds due to their secondary metabolism. Some metabolic pathways have suffered modifications during the domestication process and improvement of agricultural traits. Here, we compared non-targeted LC-MS profiles from several areas: wild accessions (C. annuum L. var. glabriusculum), domesticated cultivars (C. annuum L.), and the F1 progeny of a domesticated, and a wild accession cross (in both directions) throughout seven stages of fruit development of chili pepper fruits. The main detected differences were in glycerophospholipid metabolism, flavone and flavonol biosynthesis, sphingolipid metabolism, and cutin biosynthesis. The domesticated group exhibited a higher abundance in 12'-apo-ß-carotenal, among others capsorubin, and ß-tocopherol. Palmitic acid and derivates, terpenoids, and quercitrin were prevalent in the wild accessions. F1 progeny showed a higher abundance of capsaicin, glycol stearate, and soyacerebroside I. This work supports evidence of the side-affectation of trait selection over the metabolism of chili pepper fruit development. Furthermore, it was also observed that there was a possible heterosis effect over the secondary metabolism in the F1 progeny.

Structure characterization of an arabinogalactan from Cynanchum atratum and its immune stimulatory activity on RAW264.7 cells.

In the present study, a water-soluble neutral polysaccharide (CAPW-1) with an average molecular weight of 64 kDa was purified from the root of Cynanchum atratum Bunge (Apocynaceae). The monosaccharide residue analysis revealed that CAPW-1 was composed of arabinose and galactose with a relative molar ratio of 7: 3. The backbone of CAPW-1 was consisted of 1,3-Galp and 1,3,6-Galp, the branches were attached to the O-6 of 1,3-Galp, and the side chains contained 1,6-Galp, 1,3,6-Galp, 1,5-linked, 1,3-linked, 1,3,5-linked, and terminal-Araf, which was attached to the O-3 of side 1,6-Galp. The bioactivity study indicated CAPW-1 could stimulate the proliferation of RAW264.7 cells and promote the secretion of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with no cytotoxicity. The results suggested a potential application of CAPW-1 as an immunostimulant for the treatment of diseases such as infection and tumor.

Natural or light-induced pigment accumulation in grain amaranths coincides with enhanced resistance against insect herbivory.

MAIN CONCLUSION: Increased resistance to insect herbivory in grain amaranth plants is associated with increased betalain pigmentation, either naturally acquired or accumulated in response to blue-red light irradiation. Betalains are water-soluble pigments characteristic of plants of the Caryophyllales order. Their abiotic stress-induced accumulation is believed to protect against oxidative damage, while their defensive function against biotic aggressors is scarce. A previous observation of induced betalain-biosynthetic gene expression in stressed grain amaranth plants led to the proposal that these pigments play a defensive role against insect herbivory. This study provided further support for this premise. First, a comparison of "green" and "red" Amaranthus cruentus phenotypes showed that the latter suffered less insect herbivory damage. Coincidentally, growth and vitality of Manduca sexta larvae were more severely affected when fed on red-leafed A. cruentus plants or on an artificial diet supplemented with red-leaf pigment extracts. Second, the exposure of A. cruentus and A. caudatus plants, having contrasting pigmentation phenotypes, to light enriched in the blue and red wavelength spectra led to pigment accumulation throughout the plant and to increased resistance to insect herbivory. These events were accompanied by the induced expression of known betalain-biosynthetic genes, including uncharacterized DODA genes believed to participate in this biosynthetic pathway in a still undefined way. Finally, transient co-expression of different combinations of betalain-biosynthetic genes in Nicotiana benthamiana led to detectable accumulation of betalamic acid and betanidin. This outcome supported the participation of certain AhDODA and other genes in the grain amaranth betalain-biosynthetic pathway.

Antineoplastic Activity of Rhus trilobata Nutt. (Anacardiaceae) against Ovarian Cancer and Identification of Active Metabolites in This Pathology.

Rhus trilobata (RHTR) is a medicinal plant with cytotoxic activity in different cancer cell lines. However, the active compounds in this plant against ovarian cancer are unknown. In this study, we aimed to evaluate the antineoplastic activity of RHTR and identify its active metabolites against ovarian cancer. The aqueous extract (AE) and an active fraction (AF02) purified on C18-cartridges/ethyl acetate decreased the viability of SKOV-3 cells at 50 and 38 µg/mL, respectively, compared with CHO-K1 (>50 µg/mL) in MTT assays and generated changes in the cell morphology with apoptosis induction in Hemacolor® and TUNEL assays (p ≤ 0.05, ANOVA). The metabolite profile of AF02 showed a higher abundance of flavonoid and lipid compounds compared with AE by UPLC-MSE. Gallic acid and myricetin were the most active compounds in RHTR against SKOV-3 cells at 50 and 166 µg/mL, respectively (p ≤ 0.05, ANOVA). Antineoplastic studies in Nu/Nu female mice with subcutaneous SKOV-3 cells xenotransplant revealed that 200 mg/kg/i.p. of AE and AF02 inhibited ovarian tumor lesions from 37.6% to 49% after 28 days (p ≤ 0.05, ANOVA). In conclusion, RHTR has antineoplastic activity against ovarian cancer through a cytostatic effect related to gallic acid and myricetin. Therefore, RHTR could be a complementary treatment for this pathology.

Metabolic response to larval herbivory in three Physalis species.

The Physalis genus includes species of commercial importance due to their ornamental, edible and medicinal properties. These qualities stem from their variety of biologically active compounds. We performed a metabolomic analysis of three Physalis species, i.e., P. angulata, P. grisea, and P. philadelphica, differing in domestication stage and cultivation practices, to determine the degree of inter-species metabolite variation and to test the hypothesis that these related species mount a common metabolomic response to foliar damage caused by Trichoplusia ni larvae. The results indicated that the metabolomic differences detected in the leaves of these species were species-specific and remained even after T. ni herbivory. They also show that each Physalis species displayed a unique response to insect herbivory. This study highlighted the metabolite variation present in Physalis spp. and the persistence of this variability when faced with biotic stressors. Furthermore, it sets an experimental precedent from which highly species-specific metabolites could be identified and subsequently used for plant breeding programs designed to increase insect resistance in Physalis and related plant species.

Effects of the Developmental Regulator BOLITA on the Plant Metabolome.

Transcription factors are important regulators of gene expression. They can orchestrate the activation or repression of hundreds or thousands of genes and control diverse processes in a coordinated way. This work explores the effect of a master regulator of plant development, BOLITA (BOL), in plant metabolism, with a special focus on specialized metabolism. For this, we used an Arabidopsis thaliana line in which the transcription factor activity can be induced. Fingerprinting metabolomic analyses of whole plantlets were performed at different times after induction. After 96 h, all induced replicas clustered as a single group, in contrast with all controls which did not cluster. Metabolomic analyses of shoot and root tissues enabled the putative identification of differentially accumulated metabolites in each tissue. Finally, the analysis of global gene expression in induced vs. non-induced root samples, together with enrichment analyses, allowed the identification of enriched metabolic pathways among the differentially expressed genes and accumulated metabolites after the induction. We concluded that the induction of BOL activity can modify the Arabidopsis metabolome. Future work should investigate whether its action is direct or indirect, and the implications of the metabolic changes for development regulation and bioprospection.

Inhibition of the intestinal postprandial glucose transport by gallic acid and gallic acid derivatives.

Inhibition of glucose uptake in the intestine through sodium-dependent glucose transporter 1 (SGLT1) or glucose transporter 2 (GLUT2) may be beneficial in controlling postprandial blood glucose levels. Gallic acid and ten of its derivatives were identified in the active fractions of Terminalia chebula Retz. fructus immaturus, a popular edible plant fruit which has previously been associated with the inhibition of glucose uptake. Gallic acid derivatives (methyl gallate, ethyl gallate, pentyl gallate, 3,4,6-tri-O-galloyl-ß-d-glucose, and corilagin) showed good glucose transport inhibition with inhibitory rates of 72.1 ± 1.6%, 71.5 ± 1.4%, 79.9 ± 1.2%, 44.7 ± 1.2%, and 75.0 ± 0.7% at 5 mM d-glucose and/or 56.3 ± 2.3, 52.1 ± 3.2%, 70.2 ± 1.7%, 15.6 ± 1.6%, and 37.1 ± 0.8% at 25 mM d-glucose. However, only 3,4,6-tri-O-galloyl-ß-d-glucose and corilagin were confirmed GLUT2-specific inhibitors. Whilst some tea flavonoids demonstrated minimal glucose transport inhibition, their gallic acid derivatives strongly inhibited transport effect with GLUT2 specificity. This suggests that gallic acid structures are crucial for glucose transport inhibition. Plants, such as T. chebula, which contain high levels of gallic acid and its derivatives, show promise as natural functional ingredients for inclusion in foods and drinks designed to control postprandial glucose levels.

Transcriptome Analyses Throughout Chili Pepper Fruit Development Reveal Novel Insights into the Domestication Process.

Chili pepper (Capsicum spp.) is an important crop, as well as a model for fruit development studies and domestication. Here, we performed a time-course experiment to estimate standardized gene expression profiles with respect to fruit development for six domesticated and four wild chili pepper ancestors. We sampled the transcriptomes every 10 days from flowering to fruit maturity, and found that the mean standardized expression profiles for domesticated and wild accessions significantly differed. The mean standardized expression was higher and peaked earlier for domesticated vs. wild genotypes, particularly for genes involved in the cell cycle that ultimately control fruit size. We postulate that these gene expression changes are driven by selection pressures during domestication and show a robust network of cell cycle genes with a time shift in expression, which explains some of the differences between domesticated and wild phenotypes.

Mass spectrometry-based quantification and spatial localization of small organic acid exudates in plant roots under phosphorus deficiency and aluminum toxicity.

Low-molecular-weight organic acid (OA) extrusion by plant roots is critical for plant nutrition, tolerance to cations toxicity, and plant-microbe interactions. Therefore, methodologies for the rapid and precise quantification of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial location of root exudates is also important to understand the molecular mechanisms directing OA production and release into the rhizosphere. Here, we report the development of two complementary methodologies for OA determination, which were employed to evaluate the effect of inorganic ortho-phosphate (Pi) deficiency and aluminum toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al tolerance. Using targeted ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS), we achieved the quantification of six OAs in root exudates at sub-micromolar detection limits with an analysis time of less than 5 min per sample. We also employed targeted (MS/MS) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OA exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpressing genes involved in OA excretion. Finally, we show the transferability of the MALDI-MSI method by analyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.

Growth Inhibition of Phytopathogenic Fungi and Oomycetes by Basidiomycete Irpex lacteus and Identification of its Antimicrobial Extracellular Metabolites.

In dual culture confrontation assays, basidiomycete Irpex lacteus efficiently antagonized Fusarium spp., Colletotrichum spp., and Phytophthora spp. phytopathogenic strains, with growth inhibition percentages between 16.7-46.3%. Antibiosis assays evaluating the inhibitory effect of soluble extracellular metabolites indicated I. lacteus strain inhibited phytopathogens growth between 32.0-86.7%. Metabolites in the extracellular broth filtrate, identified by UPLC-QTOF mass spectrometer, included nine terpenes, two aldehydes, and derivatives of a polyketide, a quinazoline, and a xanthone, several of which had antifungal activity. I. lacteus strain and its extracellular metabolites might be valuable tools for phytopathogenic fungi and oomycete biocontrol of agricultural relevance.

Functional Characterization of the Lin28/let-7 Circuit During Forelimb Regeneration in Ambystoma mexicanum and Its Influence on Metabolic Reprogramming.

The axolotl (Ambystoma mexicanum) is a caudate amphibian, which has an extraordinary ability to restore a wide variety of damaged structures by a process denominated epimorphosis. While the origin and potentiality of progenitor cells that take part during epimorphic regeneration are known to some extent, the metabolic changes experienced and their associated implications, remain unexplored. However, a circuit with a potential role as a modulator of cellular metabolism along regeneration is that formed by Lin28/let-7. In this study, we report two Lin28 paralogs and eight mature let-7 microRNAs encoded in the axolotl genome. Particularly, in the proliferative blastema stage amxLin28B is more abundant in the nuclei of blastemal cells, while the microRNAs amx-let-7c and amx-let-7a are most downregulated. Functional inhibition of Lin28 factors increase the levels of most mature let-7 microRNAs, consistent with an increment of intermediary metabolites of the Krebs cycle, and phenotypic alterations in the outgrowth of the blastema. In summary, we describe the primary components of the Lin28/let-7 circuit and their function during axolotl regeneration, acting upstream of metabolic reprogramming events.

Structural characterization of a heteropolysaccharide from fruit of Chaenomelese speciosa (Sweet) Nakai and its antitumor activity.

The present study aimed at investigating the structural features and antitumor properties of a novel heteropolysaccharide (CSP-W-2) obtained from the fruit of Chaenomeles Speciosa (Sweet) Nakai. CSP-W-2 demonstrate that they mainly contain glucose, galactose, arabinose, mannose, xylose in a ratio of 3.7: 3.2: 1.7: 0.9: 0.4, with the molecular weight of 8.7 kDa. Its backbone is predominantly composed of 1,4 linked ß-D-Galp, 1,4 linked α-D-Glcp, 1,4 linked ß-D-Glcp, and 1,4,6-ß-D-Glcp, additionally some branches contained 1,5 linked α-L-Araf, 1,4 linked ß-D-Glcp, 1,3 linked α-L-Araf, and T linked ß-D-Manp according to the results of partial acid hydrolysis analysis, methylation analysis, IR and NMR spectra. The antitumor properties study results demonstrated that CSP-W-2 had an inhibitory effect on HepG2 growth by enhancing the nucleus shrinkage and apoptosis. These findings indicate that CSP-W-2 had antitumor potential in the treatment of human liver tumor.

Localization and Composition of Fructans in Stem and Rhizome of Agave tequilana Weber var. azul.

Methodology combining mass spectrometry imaging (MSI) with ion mobility separation (IMS) has emerged as a biological imaging technique due to its versatility, sensitivity and label-free approach. This technique has been shown to separate isomeric compounds such as lipids, amino acids, carboxylic acids and carbohydrates. This report describes mass spectrometry imaging in combination with traveling-wave ion mobility separation and matrix-assisted laser desorption/ionization (MALDI). Positive ionization mode was used to locate fructans on tissue printed sections of Agave rhizome and stem tissue and distinguished fructan isoforms. Here we show the location of fructans ranging from DP3 to DP17 to be differentially abundant across the stem tissue and for the first time, experimental collision cross sections of endogenous fructan structures have been collected, revealing at least two isoforms for fructans of DP4, DP5, DP6, DP7, DP8, DP10, and DP11. This demonstrates that complex fructans such as agavins can be located and their isoforms resolved using a combination of MALDI, IMS, and MSI, without the need for extraction or derivatization. Use of this methodology uncovered patterns of fructan localization consistent with functional differences where higher DP fructans are found toward the central section of the stem supporting a role in long term carbohydrate storage whereas lower DP fructans are concentrated in the highly vascularized central core of rhizomes supporting a role in mobilization of carbohydrates from the mother plant to developing offsets. Tissue specific patterns of expression of genes encoding enzymes involved in fructan metabolism are consistent with fructan structures and localization.

Seasonal Changes in the Metabolic Profiles and Biological Activity in Leaves of Diospyros digyna and D. rekoi "Zapote" Trees.

Leaves of semi-domesticated Diospyros digyna and wild D. rekoi trees, sampled seasonally in Mexico in 2014, were analyzed. Metabolic fingerprints revealed higher metabolite diversity in D. rekoi leaves. The TLC bands characteristic of glycosylated flavonoids, predominant in this species, matched the detection of quercetin and quercetin 3-O-glucuronides by liquid chromatography (UPLC-MS) of spring leaf extracts (LEs). Further gas chromatography (GC-MS) analysis revealed abundant fatty acids, organic acids, and secondary metabolites including trigonelline, p-coumaric, and ferulic and nicotinic acids. Phenolic-like compounds prevailed in D. digyna LEs, while unidentified triterpenoids and dihydroxylated coumarins were detected by UPLC-MS and GC-MS. A paucity of leaf metabolites in leaves of this species, compared to D. rekoi, was evident. Higher antioxidant capacity (AOC) was detected in D. digyna LEs. The AOC was season-independent in D. digyna but not in D. rekoi. The AOC in both species was concentrated in distinct TLC single bands, although seasonal variation in band intensity was observed among trees sampled. The AOC in D. digyna LEs could be ascribed to the coumarin esculetin. The LEs moderately inhibited phytopathogenic bacteria but not fungi. Leaf chemistry differences in these Mesoamerican Diospyros species substantiated previous variability reported in tree physiology and fruit physical chemistry, postulated to result from domestication and seasonality.

Metabolomic Markers for the Early Selection of Coffea canephora Plants with Desirable Cup Quality Traits.

Genetic improvement of coffee plants represents a great challenge for breeders. Conventional breeding takes a too long time for responding timely to market demands, climatic variations and new biological threads. The correlation of genetic markers with the plant phenotype and final product quality is usually poor. Additionally, the creation and use of genetically modified organisms (GMOs) are often legally restricted and rejected by customers that demand natural products. Therefore, we developed a non-targeted metabolomics approach to accelerate conventional breeding. Our main idea was to identify highly heritable metabolites in Coffea canephora seedlings, which are linked to coffee cup quality. We employed a maternal half-sibs approach to estimate the metabolites heritability in open-pollinated plants in both leaves and fruits at an early plant development stage. We evaluated the cup quality of roasted beans and correlated highly heritable metabolites with sensory quality traits of the coffee beverage. Our results provide new insights about the heritability of metabolites of C. canephora plants. Furthermore, we found strong correlations between highly heritable metabolites and sensory traits of coffee beverage. We revealed metabolites that serve as predictive metabolite markers at an early development stage of coffee plants. Informed decisions can be made on plants of six months old, compared to 3.5 to 5 years using conventional selection methods. The metabolome-wide association study (MWAS) drastically accelerates the selection of C. canephora plants with desirable characteristics and represents a novel approach for the focused breeding of crops.

Placenta, Pericarp, and Seeds of Tabasco Chili Pepper Fruits Show a Contrasting Diversity of Bioactive Metabolites.

Chili pepper (Capsicum spp.) is one of the most important horticultural crops worldwide, and its unique organoleptic properties and health benefits have been established for centuries. However, there is little knowledge about how metabolites are distributed throughout fruit parts. This work focuses on the use of liquid chromatography coupled with high resolution mass spectrometry (UHPLC-ESI-HRMS) to estimate the global metabolite profiles of the pericarp, placenta, and seeds of Tabasco pepper fruits (Capsicum frutescens L.) at the red mature stage of ripening. Our main results putatively identified 60 differential compounds between these tissues and seeds. Firstly, we found that pericarp has a higher content of glycosides, showing on average a fold change of 5 and a fold change of 14 for terpenoids when compared with other parts of the fruit. While placenta was the richest tissue in capsaicinoid-related compounds, alkaloids, and tocopherols, with a 35, 3, and 7 fold change, respectively. However, the seeds were richer in fatty acids and saponins with fold changes of 86 and 224, respectively. Therefore, our study demonstrates that a non-targeted metabolomic approach may help to improve our understanding of unexplored areas of plant metabolism and also may be the starting point for a detailed analysis in complex plant parts, such as fruits.

Biological and toxicological evaluation of Rhus trilobata Nutt. (Anacardiaceae) used traditionally in mexico against cancer.

BACKGROUND: Rhus trilobata Nutt. (Anacardiaceae) (RHTR) is a plant of Mexico that is traditionally used as an alternative treatment for several types of cancer. However, the phytochemical composition and potential toxicity of this plant have not been evaluated to support its therapeutic use. Therefore, this study aimed to evaluate the biological activity of RHTR against colorectal adenocarcinoma cells, determine its possible acute toxicity, and analyze its phytochemical composition. METHODS: The traditional preparation was performed by decoction of stems in distilled water (aqueous extract, AE), and flavonoids were concentrated with C18-cartridges and ethyl acetate (flavonoid fraction, FF). The biological activity was evaluated by MTT viability curves and the TUNEL assay in colorectal adenocarcinoma (CACO-2), ovarian epithelium (CHO-K1) and lung/bronchus epithelium (BEAS-2B) cells. The toxicological effect was determined in female BALB/c mice after 24 h and 14 days of intraperitoneal administration of 200 mg/kg AE and FF, respectively. Later, the animals were sacrificed for histopathological observation of organs and sera obtained by retro-orbital bleeding for biochemical marker analysis. Finally, the phytochemical characterization of AE and FF was conducted by UPLC-MSE. RESULTS: In the MTT assays, AE and FF at 5 and 18 µg/mL decreased the viability of CACO-2 cells compared with cells treated with vehicle or normal cells (p ≤ 0.05, ANOVA), with changes in cell morphology and the induction of apoptosis. Anatomical and histological analysis of organs did not reveal important pathological lesions at the time of assessment. Additionally, biochemical markers remained normal and showed no differences from those of the control group after 24 h and 14 days of treatment (p ≤ 0.05, ANOVA). Finally, UPLC-MSE analysis revealed 173 compounds in AE-RHTR, primarily flavonoids, fatty acids and phenolic acids. The most abundant compounds in AE and FF were quercetin and myricetin derivates (glycosides), methyl gallate, epigallocatechin-3-cinnamate, ß-PGG, fisetin and margaric acid, which might be related to the anticancer properties of RHTR. CONCLUSION: RHTR exhibits biological activity against cancer cells and does not present adverse toxicological effects during its in vivo administration, supporting its traditional use.