Antitumoral activity of different Amaryllidaceae alkaloids: In vitro and in silico assays.

ETHNOPHARMACOLOGY RELEVANCE: The plants of Amaryllidaceae family, such as Amaryllis belladonna L., have been used as herbal remedies for thousands of years to address various disorders, including diseases that might today be identified as cancer. AIM OF THE STUDY: The objective of this work was to evaluate the potential of three Amaryllidaceae alkaloids against four cancer cell lines. MATERIAL AND METHODS: The alkaloids lycorine, 1-O-acetylcaranine, and montanine were evaluated in vitro against colon adenocarcinoma cell line (HCT-116) and breast carcinoma cell lines (MCF-7, MDAMB231, and Hs578T). Computational experiments (target prediction and molecular docking) were conducted to gain a deeper comprehension of possible interactions between these alkaloids and potential targets associated with these tumor cells. RESULTS: Montanine presented the best results against HCT-116, MDAMB231, and Hs578T cell lines, while lycorine was the most active against MCF-7. In alignment with the target prediction outcomes and existing literature, four potential targets were chosen for the molecular docking analysis: CDK8, EGFR, ER-alpha, and dCK. The docking scores revealed two potential targets for the alkaloids with scores similar to co-crystallized inhibitors and substrates: CDK8 and dCK. A visual analysis of the optimal docked configurations indicates that the alkaloids may interact with some key residues in contrast to the other docked compounds. This observation implies their potential to bind effectively to both targets. CONCLUSIONS: In vitro and in silico results corroborate with data literature suggesting the Amaryllidaceae alkaloids as interesting molecules with antitumoral properties, especially montanine, which showed the best in vitro results against colorectal and breast carcinoma. More studies are necessary to confirm the targets and pharmaceutical potential of montanine against these cancer cell lines.

Acetylcholinesterase Inhibition Activity of Hippeastrum papilio (Ravenna) Van Scheepen (Amaryllidaceae) Using Zebrafish Brain Homogenates.

The Amaryllidaceae family constitutes an interesting source of exclusive alkaloids with a broad spectrum of biological activity. Galanthamine, the most relevant one, has been commercialized for the palliative treatment of Alzheimer's disease symptoms since 2001 due to its potential as an acetylcholinesterase (AChE) inhibitor. In vitro screenings against AChE by applying different Amaryllidaceae species and alkaloids have been reported in the literature; however, they are usually carried out using purified market enzymes. The main goal of this work is to evaluate the AChE inhibitory potential of Hippeastrum papilio (Amaryllidaceae) extracts using zebrafish brain homogenates. The biological assays show that the H. papilio bulb extracts present an interesting AChE inhibitory activity in comparison with the positive reference control galanthamine (IC50 values of 1.20 ± 0.10 and 0.79 ± 0.15 µg/mL, respectively). The chemical profile of H. papilio shows that this species has a high amount of galanthamine, which may contribute to the inhibitory effect on AChE activity of zebrafish brains. Computational experiments were used to build the model for zebrafish AChE and to evaluate the interactions between galanthamine and the enzymic active site. This work suggests that zebrafish could represent an important model in the search for bioactive molecules from the Amaryllidaceae family for the treatment of Alzheimer's disease.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

Structural Diversity and Biological Potential of Alkaloids from the Genus Hippeastrum, Amaryllidaceae: an Update.

The subfamily Amaryllidoideae, Amaryllidaceae, presents an exclusive group of structures known as Amaryllidaceae alkaloids, which have a broad spectrum of biological activities. These plants are classified into 59 genera, including Hippeastrum Herb., which comprises approximately 60 species distributed mainly in South America, being widely used as ornamental plants due to the beauty of its flowers. This review presents an update about the alkaloid profiling of Hippeastrum extracts published between 2012 and 2021, as well as an approach to the biological potential of these compounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43450-021-00211-z.

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves.

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets for modulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga.

Erythroxylum pungens Tropane Alkaloids: GC-MS Analysis and the Bioactive Potential of 3-(2-methylbutyryloxy)tropan-6,7-diol in Zebrafish (Danio rerio).

Tropane alkaloids are specialized plant metabolites mostly found in the Erythroxylaceae and Solanaceae families. Although tropane alkaloids have a high degree of structural similarity because of the tropane ring, their pharmacological actions are quite distinct. Brazil is one of the main hotspots of Erythroxylum spp. diversity with 123 species (almost 66% of the species catalogued in tropical America). Erythroxylum pungens occurs in the Caatinga, a promising biome that provides bioactive compounds, including tropane alkaloids. As part of our efforts to investigate this species, 15 alkaloids in specimens harvested under different environmental conditions are presented herein. The occurrence of 3-(2-methylbutyryloxy)tropan-6,7-diol in the stem bark of plants growing in their natural habitat, greenhouse controlled conditions, and after a period of water restriction, suggests that it is a potential chemical marker for the species. This alkaloid was evaluated for several parameters in zebrafish (Danio rerio) as a model organism. Regarding toxicity, teratogenic effects were observed at 19.5 µM and the lethal dose for embryos was 18.4 µM. No mortality was observed in adults, but a behavioral screen showed psychostimulatory action at 116.7 µM. Overall, the alkaloid was able to cause zebrafish behavioral changes, prompting further investigation of its potential as a new molecule in the treatment of depression-like symptoms. In silico, targets involved in antidepressant pathways were identified by docking.

Isoquinoline alkaloids reduce beta-amyloid peptide toxicity in Caenorhabditis elegans.

Alzheimer's disease (AD) is a multifactorial health problem widespread over the world. Regarding the historical importance of the alkaloids in the central nervous system pharmacology they remain as promising drug candidates against AD. Seven alkaloids from Amaryllidaceae and Fabaceae were evaluated in vivo, in vitro and in silico targets related to the AD pathophysiology. Erythraline and erysodine showed the greatest potential compared to Memantine, a drug currently used in AD therapy, by delaying the Aß1-42-induced paralysis in the transgenic strain CL2006 Caenorhabditis elegans, an alternative model to assess the impairment of beta-amyloid peptide deposition. The in vitro inhibition of the acetylcholinesterase was observed for the first time for Erythrina alkaloids; however Lycorine was the most active. Docking simulation contributed to comprehend this potential by showing a hydrophobic interaction between acetylcholinesterase and Lycorine in the amino acid residue TRP 84 as well as hydrogen bonds with TRY 121 and ASP 72.

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

Hepatoprotective activity of Verbena litoralis, Verbena montevidensis and their main iridoid, brasoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Verbena montevidensis and Verbena litoralis are plants that present morphological similarities. They are both known as "gervão" and "fel-da-terra", among other popular names, and are used in folk medicine to treat diseases related to the liver and stomach. AIMS OF THE STUDY: The aim of the current investigation was to determine the chemical composition and evaluate the hepatoprotective properties and cytotoxicity of the methanolic and aqueous extracts of V. montevidensis, V. litoralis and their main iridoid in HepG2 cells. MATERIALS AND METHODS: Aqueous and methanolic extracts from the dried aerial parts of V. montevidensis and V. litoralis were obtained. The methanolic extract of V. montevidensis afforded an iridoid as the main compound. The extracts and isolated compound were examined for the hepatoprotective effect and cytotoxicity in human hepatoblastoma HepG2 cells by MTT reduction and neutral red uptake methods. RESULTS: The methanolic and aqueous extracts of both species showed the presence of iridoid and phenylethanoids as the main compounds. The iridoid brasoside was isolated and identified by spectroscopic methods. The phenylethanoid was characterized by HPLC, comparing the UV profile and retention time with an authentic sample. The results of the biological assays indicate that both aqueous and methanolic extracts of V. montevidensis and V. litoralis as well as brasoside were hepatoprotective against ethanol-induced damage in HepG2 cells. The effect can be attributed to the main compounds present since both classes are recognized for this activity. CONCLUSIONS: Our results contribute towards validation of the traditional use of V. montevidensis and V. litoralis in the treatment of liver disorders.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

OBJECTIVES: The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. RESULTS: We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days. CONCLUSION: This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Trifolium pratense: Friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.

Isolation, spectral characterization, molecular docking, and cytotoxic activity of alkaloids from Erythroxylum pungens O. E. Shulz.

Stem bark, root bark, and leaf extracts of Erythroxylum pungens were subjected to phytochemical analysis. N,N-dimethyltryptamine (DMT) was isolated and characterized from E. pungens roots. This unprecedented result is remarkable since no indole alkaloid has been previously reported from Erythroxylaceae so far. Eleven known tropane alkaloids were identified by their mass spectra and 3-(2-methylbutyryloxy)tropan-6,7-diol as well as 3-(2-methylbutyryloxy)nortropan-6,7-diol were isolated and characterized based on mass spectrometry, 1H, 13C, COSY, and NOESY NMR analysis. The complete NMR data are reported for the first time. Inverse Structure-based and Ligand-Based virtual screening were carried out to identify possible targets for 3-(2-methylbutyryloxy)tropan-6,7-diol. The level of cytotoxicity of this tropane alkaloid aliphatic ester was discrete with potencies on the order of 0.3-1.0 mg/mL and better results against HeLa (50% cell viability reduction). Otherwise, atropine (0.3 mg/mL), a Solanaceae tropane alkaloid, and DMT (0.5 mg/mL) from E. pungens roots impaired at 50% the cell viability against HeLa, SiHa, PC3, and 786-0. This study stimulates scientific investigation of the impact of edaphoclimatic features in a semi-arid environment on tropane alkaloid biosynthesis.

N-oxide alkaloids from Crinum amabile (Amaryllidaceae).

Natural products play an important role in the development of new drugs. In this context, the Amaryllidaceae alkaloids have attracted considerable attention in view of their unique structural features and various biological activities. In this study, twenty-three alkaloids were identified from Crinum amabile by GC-MS and two new structures (augustine N-oxide and buphanisine N-oxide) were structurally elucidated by NMR. Anti-parasitic and cholinesterase (AChE and BuChE) inhibitory activities of six alkaloids isolated from this species, including the two new compounds, are described herein. None of the alkaloids isolated from C. amabile gave better results than the reference drugs, so it was possible to conclude that the N-oxide group does not increase their therapeutic potential.

Hydroxypropyl-ß-cyclodextrin-containing hydrogel enhances skin formononetin permeation/retention.

OBJECTIVES: This study was aimed to investigate the in vitro permeation potential of hydrogel formulations containing the isoflavones formononetin and biochanin A and cyclodextrins in different combinations. METHODS: The permeation assay was performed using porcine skin discs on Franz diffusion cells model. The isoflavone contents of the formulations were quantified in the different layers of the skin using a validated HPLC-PDA method. KEY FINDINGS: The isoflavones individually incorporated into the formulations showed high permeation potential, especially formononetin, after the incorporation of hydroxypropyl-ß-cyclodextrin that enhanced its permeation in the epidermis and dermis. Biochanin A showed 2.7 times of permeation capacity in the epidermis and dermis mainly after incorporation of cyclodextrins in the formulations. Formononetin showed reduction in its permeation when incorporated in the formulations together to biochanin A, showing the absence of synergism. CONCLUSIONS: Our results indicated a noticeable skin permeation promoting effect of HPßCD in formononetin formulation. Furthermore, formononetin and biochanin A can permeate the skin being mostly retained in the epidermis and dermis, revealing its potential use in cosmetic preparations intended to prevent skin aging.

Forebrain glutamate uptake and behavioral parameters are altered in adult zebrafish after the induction of Status Epilepticus by kainic acid.

The development of new antiepileptic drugs is a high-risk/high-cost research field, which is made even riskier if the behavioral epileptic seizure profile is the unique approach on which the development is based. In order to increase the effectiveness of the screening conducted in the zebrafish model of status epilepticus (SE), the evaluation of neurochemical markers of SE would be of great relevance. Epilepsy is associated with changes in the glutamatergic system, and glutamate uptake is one of the critical parameters of this process. Therefore, we evaluated the levels of glutamate uptake in the zebrafish brain and analyzed its correlation with the progression of behavioral changes in zebrafish at different times after the administration of kainic acid (5 mg/kg). The results showed that the zebrafish suffered with lethargy while swimming for up to 72 h after SE, had reduced levels of GFAP cells 12 h after SE, reduced levels of S100B up to 72 h after SE, and reduced levels of glutamate uptake in the forebrain between 3 h and 12 h after SE. The forebrain region of adult zebrafish after SE present similar changes to the neurochemical limbic alterations that are seen in rodent models of SE. This study demonstrated that there is a time window in which to use the KA zebrafish model of SE to explore some of the known neurochemical alterations that have been observed in rodent models of epilepsy and epileptic human patients.

Hippeastrum reticulatum (Amaryllidaceae): Alkaloid Profiling, Biological Activities and Molecular Docking.

The Amaryllidaceae family has proven to be a rich source of active compounds, which are characterized by unique skeleton arrangements and a broad spectrum of biological activities. The aim of this work was to perform the first detailed study of the alkaloid constituents of Hippeastrum reticulatum (Amaryllidaceae) and to determine the anti-parasitological and cholinesterase (AChE and BuChE) inhibitory activities of the epimers (6α-hydroxymaritidine and 6ß-hydroxymaritidine). Twelve alkaloids were identified in H. reticulatum: eight known alkaloids by GC-MS and four unknown (6α-hydroxymaritidine, 6ß-hydroxymaritidine, reticulinine and isoreticulinine) by NMR. The epimer mixture (6α-hydroxymaritidine and 6ß-hydroxymaritidine) showed low activity against all protozoan parasites tested and weak AChE-inhibitory activity. Finally, a molecular docking analysis of AChE and BuChE proteins showed that isoreticulinine may be classified as a potential inhibitory molecule since it can be stabilized in the active site through hydrogen bonds, π-π stacking and hydrophobic interactions.

Alkaloid Constituents of the Amaryllidaceae Plant Amaryllis belladonna L.

The plant family Amaryllidaceae is well-known for its unique alkaloid constituents, which exhibit a wide range of biological activities. Its representative, Amaryllis belladonna, has a geographical distribution covering mainly southern Africa, where it has significant usage in the traditional medicine of the native people. In this study, A. belladonna samples collected in Brazil were examined for alkaloid content. Alkaloid profiles of A. belladonna bulbs were generated by a combination of chromatographic, spectroscopic and spectrometric methods, including GC-MS and 2D NMR. In vitro screening against four different parasitic protozoa (Trypanosoma cruzi, T. brucei rhodesiense, Leishmania donovani and Plasmodium falciparum) was carried out using the A. belladonna crude methanol extract, as well as three of its alkaloid isolates. Twenty-six different Amaryllidaceae alkaloids were identified in the A. belladonna bulb samples, and three of them were isolated. Evidence for their respective biosynthetic pathways was afforded via their mass-spectral fragmentation data. Improved data for 1-O-acetylcaranine was provided by 2D NMR experiments, together with new ¹H-NMR data for buphanamine. The crude extract and 3-O-acetylhamayne exhibited good antiprotozoal activity in vitro, although both with a high cytotoxic index.

Toxicity of Glandularia selloi (Spreng.) Tronc. leave extract by MTT and neutral red assays: influence of the test medium procedure.

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of plant extracts with potential phytotherapeutic properties. This study compared three methods to prepare culture media for the exposure of Vero cells to plant extracts. Leaves of Glandularia selloi (Spreng.) Tronc. were used to prepare culture medium with aqueous extract, extract in culture medium and methanol extract. Toxicity was assessed using the MTT and neutral red (NR) assays. In general, alterations in the cellular functions were found in all extracts and assays. Cytotoxic effect occurred at lower doses in aqueous extract and the range of effect of the methanol extract was small. The procedure of preparing the test medium has an effect on the outcome of the assay. Cytotoxicity of plant extract can be assessed by MTT and NR assays. Aqueous extract added to the culture medium presented the best profile to assess cytotoxicity.

New lycosinine derivative from Hippeastrum breviflorum

AbstractA new lycosinine derivative, 9-O-demethyllycosinine B, was isolated from the native Brazilian Hippeastrum breviflorumHerb., Amaryllidaceae, along with the well-known alkaloids lycosinine B and lycorine. The structure of the new compound was established by physical and spectroscopic methods. 9-O-demethyllycosinine B is the third lycosinine variant identified in the Amaryllidaceae family.

Study of the effects of Psidium cattleyanum on gene expression from senescent mouse hippocampus / Perfil de expresión génica en hipocampo de ratones idosos tratados con Psidium cattleyanum

In this work, we report a characterization of gene expression profiles of mice hippocampus by use microarray after treatment with Psidium cattleyanum fruit (1000 mg/kg oral). After eight months, no toxic effects were detected with the supplementation. Genes differentially expressed include genes involved in a wide range of physiological functions, such as metabolism, transport, signal transduction and a group of genes whose function has not yet been identified. These results suggest that global analysis of gene expression might be useful to elucidate the mechanisms of beneficial phytochemical action and may also help to identify potential targets for further investigation.

El objetivo de éste trabajo fue evaluar el efecto de la administración oral prolongada (1000 mg/kg, 8 meses) del extracto de Psidium cattleyanum Sabine (Myrtaceae) en el perfil de la expresión génica en hipocampo de ratones idosos (Swiss). Después de 8 meses de suplementacion, no se detectaron efectos tóxicos en los animales tratados con relación al grupo control. Los genes con expresión diferencial incluyen, genes que codifican proteínas relacionadas con procesos de señalización, transcripción, metabolismo, así como genes con función desconocida. Los resultados demuestran la importancia de los microarray como herramienta para el estudio del mecanismo de acción de los compuestos fitoquímicos.