Dereplication of tropane alkaloids from four Erythroxylum species using liquid chromatography coupled with ESI-MSn and HRESIMS.

RATIONALE: Tropane alkaloids represent an important class of secondary metabolites, but many of these compounds are already described in the scientific literature, so the use of guided identification and isolation strategies, such as dereplication, represent a fast and safe alternative. METHODS: For the annotation of the tropane alkaloids the chloroform phases of the four Erythroxylum species were analyzed by high-performance liquid chromatography coupled to mass spectrometry with positive-mode electrospray ionization, then the ions of their protonated molecules, molecular formulas and fragmentation patterns were observed and a comparison of the obtained data with those present in the scientific literature was performed. The compounds not fully annotated were isolated and characterized by 1 H and 13 C nuclear magnetic resonance spectroscopy. RESULTS: The annotation of 29 tropane alkaloids was performed, some being described for the first time in the family Erythroxylaceae. The chemical profiles of these secondary metabolites in the four Erythroxylum species analyzed were traced and compared. Isolation of three compounds whose mass spectral data were not sufficient for their full annotation was performed. They were 6-(benzoyloxy)-3-(3,5-dimethoxy-4-hydroxybenzoyloxy)tropane, 6-(benzoyloxy)-3-(3,4,5-trimethoxybenzoyloxy)tropane and 6-(benzoyloxy)-3-(3,4,5-trimethoxycinamoyloxy)tropane, first reported in the species Erythroxylum revolutum Mart. CONCLUSIONS: This work contributes to the phytochemical knowledge of the genus Erythroxylum, and demonstrates the efficiency and importance of using guided isolation methodologies of secondary metabolites in natural products research. Since safe results were presented in the annotation of the compounds evidenced, employing small quantities of organic solvents, when compared to classical methodologies, besides promoting an optimization in the research time.

Anti-inflammatory kaurane diterpenoids of Erythroxylum bezerrae.

Five unusual kaurane diterpenes, designated as bezerraditerpenes A-E (1-5), along with six known ones (6-11), were isolated from the hexane extract of the stems of Erythroxylum bezerrae. Their structures were elucidated based on the interpretation of the NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis. The anti-inflammatory potential of the diterpenes 1-11 was screened through cellular viability and lipopolysaccharide (LPS)-induced nitric oxide (NO) production on murine macrophage-like cells RAW 264.7. Diterpene 6 (cauren-6ß-ol) showed potent cytotoxicity and increased ability to inhibit NO production. Diterpenes 1 (bezerraditerpene A), 2 (bezerraditerpene B), and 8 (ent-kaur-16-ene-3ß,15ß-diol) exhibited the same significant anti-inflammatory activity with NO CI50 inhibition (3.21-3.76 µM) without cytotoxicity, in addition to decreasing the levels of pro-inflammatory cytokines TNF-α and IL-6 in LPS-induced RAW264.7 cells.

Variation in pollen sterility and gender specialization: an investigation with distylous species of Erythroxylum (Erythroxylaceae).

Differences in pollen sterility between morphs in distylous populations may represent the first step in the evolution of gender specialization. Theoretically, for partially or completely male sterile individuals to persist they must have some fitness advantage that compensates for the loss of male function. Gender specialization is considered a widespread process in Erythroxylum, but male sterility and the resource reallocation to female function have been investigated in few species and populations. In 18 populations of four distylous species of Erythroxylum, we quantified the levels of male sterility, estimated through pollen sterility, in short- and long-styled flowers to test if sterility is morph-biased. In one population per species, we also described the frequency of floral visitors, the production of flowers and fruits, and the quality of fruits and seeds of short- and long-styled plants to evaluate the expression of trade-offs in allocation to male and female function. In some populations of E. campestre and E. deciduum, short-styled flowers possessed higher levels of pollen sterility than long-styled flowers. Although most flowers of E. suberosum and E. tortuosum also expressed pollen sterility, the frequency of sterility was similar between morphs in all populations. Differences in reproductive output between morphs occurred only in populations of species with morph-biased sterility, but none of the variations reflect resource allocation to female fitness of short-styled plants. Differences in the level of sterility between morphs indicates the potential for gender specialization in populations of E. campestre and E. deciduum, despite the apparent lack of a trade-off in allocation.

Dereplication of phenolic derivatives of three Erythroxylum species using liquid chromatography coupled with ESI-MSn and HRESIMS.

INTRODUCTION: Given the diversity of secondary metabolites produced by species of the genus Erythroxylum, in addition to the many methods that have already been described in the literature, modern screening and identification methodologies, such as dereplication, represent an efficient and quick strategy compared to the classic techniques linked to natural product research. OBJECTIVE: The objective of the present study was to determine the phenolic profiles obtained from three species of Erythroxylum (Erythroxylum pauferrense Plowman, Erythroxylum pulchrum A.St.-Hil. and Erythroxylum simonis Plowman) by dereplication using liquid chromatography coupled with ESI-MSn and HRESIMS. MATERIAL AND METHODS: Ethyl acetate and n-butanolic fractions from crude ethanolic extract of Erythroxylum species were analyzed by HPLC-ESI-MSn and HPLC-HRESIMS, in order to identify its corresponding compounds. Experiments were performed in negative ionization mode, and the metabolites were provisionally identified based on deprotonated molecules, molecular formulas, fragmentation patterns and literature data. The corresponding isolated compounds were characterized by 1 H and 13 C NMR spectroscopy. RESULTS: According to the dereplication method, it was possible to establish and compare the phenolic profile of the corresponding species by the assignment of 55 compounds, most of which were first described in these species and among which some were also new to the Erytroxylum genus. Additionally, nine compounds were isolated, including biphenyl-3,3',4,4'-tetraol, where the mass spectral data were not sufficient for their identification, and reported for the first time in the Erythroxylaceae family. CONCLUSION: This research contributes to the phytochemical knowledge of the Erythroxylum genus and demonstrates the importance of the dereplication method regarding the investigation of natural products, enabling accurate identification of the metabolites while avoiding the efforts and material expenses involved in the isolation of known compounds.

Cytotoxic Activity of Tropane Alkaloides of Species of Erythroxylum.

Erythroxylaceae is a family composed of four genera, with Erythroxylum being the only one represented in the Neotropical region. Chemical studies indicate the presence of alkaloids, terpenes, flavonoids, and phenolic compounds as main compounds. The incorporation of cytotoxic activity assays of natural products using cell cultures assists in the selection of potential chemotherapeutic agents. In this work, we describe a revision of the cytotoxicity evaluation studies performed with extracts or pure substances obtained from Erythroxylum species through an integrative review. We found studies that evaluated the cytotoxic activity of 21 species of Erythroxylum against 45 different cell lines. The analysis of the chemical composition of these species shows that the metabolites present in each species influence their cytotoxic potential, especially the presence of disubstituted tropane alkaloid species with the highest cytotoxic potential. MTT and Sulforrodamine B assays were the main in vitro tests used for the evaluation of the cytotoxic activities. From the total species, less than 10% of the Erythroxylum species have already been evaluated for cytotoxic activity. Four of them showed high cytotoxic activity according to the criteria of the NCI plant screening program. Thus, this genus represents a potential source of natural products with antitumor activity.

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.

The Origins of Coca: Museum Genomics Reveals Multiple Independent Domestications from Progenitor Erythroxylum gracilipes.

Coca is the natural source of cocaine as well as a sacred and medicinal plant farmed by South American Amerindians and mestizos. The coca crop comprises four closely related varieties classified into two species (Amazonian and Huánuco varieties within Erythroxylum coca Lam., and Colombian and Trujillo varieties within Erythroxylum novogranatense (D. Morris) Hieron.) but our understanding of the domestication and evolutionary history of these taxa is nominal. In this study, we use genomic data from natural history collections to estimate the geographic origins and genetic diversity of this economically and culturally important crop in the context of its wild relatives. Our phylogeographic analyses clearly demonstrate the four varieties of coca comprise two or three exclusive groups nested within the diverse lineages of the widespread, wild species Erythroxylum gracilipes; establishing a new and robust hypothesis of domestication wherein coca originated two or three times from this wild progenitor. The Colombian and Trujillo coca varieties are descended from a single, ancient domestication event in northwestern South America. Huánuco coca was domesticated more recently, possibly in southeastern Peru. Amazonian coca either shares a common domesticated ancestor with Huánuco coca, or it was the product of a third and most recent independent domestication event in the western Amazon basin. This chronology of coca domestication reveals different Holocene peoples in South America were able to independently transform the same natural resource to serve their needs; in this case, a workaday stimulant. [Erythroxylum; Erythroxylaceae; Holocene; Museomics; Neotropics; phylogeography; plant domestication; target-sequence capture.].

Tropane alkaloids from the stem bark of Erythroxylum bezerrae.

Six previously undescribed tropane alkaloids, designated as erythrobezerrines A-F, were isolated from the EtOH extract from the stem bark of Erythroxylum bezerrae Plowman. Their structures were elucidated based on the interpretation of the NMR and MS data and in some instances, confirmed by X-ray diffraction analysis. The cytotoxicity of the isolated compounds was evaluated against the cancer cell lines L929, PC-3, HCT-116, SNB-19 and NCI-H460, but only erythrobezerrine C showed moderate activity with IC50 values of 3.38 and 5.43 µM for HCT-116 and NCI-H460, respectively.

Estimation of leaf area of Erythroxylum citrifolium from linear leaf dimensions / Estimativa da área foliar de Erythroxylum citrifolium a partir de dimensões lineares da folha

Erythroxylum citrifolium is a neotropical plant species recorded in all regions of Brazil. Determining leaf area is of fundamental importance to studies related to plant propagation and growth. The objective was to obtain an equation to estimate the leaf area of E. citrifolium from linear dimensions of the leaf blade (length and width). A total of 200 leaf blades were collected in Parque Estadual Mata do Pau-Ferro in the municipality of Areia, state of Paraíba, Northeast Brazil. The models evaluated were: linear, linear without intercept, quadratic, cubic, power and exponential. The best model was determined by the criteria of: high coefficient of determination (R²), low root mean square error (RMSE), low Akaike information criterion (AIC), high Willmott concordance index (d) and a BIAS index close to zero. All of the models constructed satisfactorily estimated the leaf area of E. citrifolium, with coefficients of determination above 0.9050, but the power model using the product between length and width (L*W) y = 0.5966 * LW1.0181 was the best, with the highest values of R² and d, low values of RMSE and AIC, and a BIAS index closest to zero.

Erythroxylum citrifolium é uma espécie de planta neotropical com registros em todas as regiões do Brasil. A determinação da área foliar é de fundamental importância em estudos relacionados a propagação e crescimento vegetal. O objetivo foi obter uma equação que permita estimar a área foliar de E. citrifolium a partir de dimensões lineares do limbo foliar (comprimento e largura). Foram coletados 200 limbos foliares no Parque Estadual Mata do Pau-Ferro, Areia, Paraíba, Nordeste do Brasil. Os modelos empregados foram: linear, linear sem intercepto, quadrático, cúbico, potencial e exponencial. Os critérios utilizados para escolher o melhor modelo, teve como base o maior coeficiente de determinação (R²), menor raiz do quadrado médio do erro (RMSE), menor critério de informação de Akaike (AIC), maior índice de concordância de Willmott (d) e índice BIAS mais próximo de zero. Todos os modelos construídos podem estimar satisfatoriamente a área foliar de E. citrifolium, com coeficientes determinação acima de 0,9050, porém o modelo potencial utilizando o produto entre comprimento e largura (L*W) y = 0,5966 * LW1,0181 é o mais indicado, com os maiores valores de R² e d, menores valores de RMSE e AIC, e índice BIAS mais próximo de zero.

Vallissiana universitaria (Lepidoptera: Gracillariidae): a new genus and species of leaf-mining moth associated with Erythroxylum (Erythroxylaceae) in the Atlantic Forest of Brazil.

Vallissiana universitaria Pereira Arévalo, a new genus and species of leaf-miner moth (Gracillariidae: Gracillariinae) is described and illustrated with the aid of optical and scanning electron microscopy, including adults, larva, pupa and the mine. Its monophyletic status is confirmed within the subfamily based on a DNA barcode CoI tree. The immature stages are associated with Erythroxylum argentinum O. E. Schulz (Erythroxylaceae) and four larval instars are found, all forming a round blotch mine from the beginning of ontogeny. The first two instars are sap-feeders, using only the epidermal cells, whereas the last two are tissue-feeders, mining the parenchyma cells. Pupation occurs inside the leaf mine within a flimsy, silk-made cocoon. This is the third endemic genus of gracillariid moths described from the Atlantic Forest of Brazil and the first associated with Erythroxylum P. Browne. Characteristics found on the forewing and in the last abdominal segments of the adult were determinant for the proposition of the new genus. The CoI tree indicated that it is closely related to Aspilapteryx, while this genus was recovered as polyphyletic in the analyses. Morphological evidence supports this polyphyly. Consequently, Sabulopteryx Triberti, 1985, stat. nov. is considered a valid genus.

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production.

Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.

Phylogenetic inference in section Archerythroxylum informs taxonomy, biogeography, and the domestication of coca (Erythroxylum species).

PREMISE OF THE STUDY: This investigation establishes the first DNA-sequence-based phylogenetic hypothesis of species relationships in the coca family (Erythroxylaceae) and presents its implications for the intrageneric taxonomy and neotropical biogeography of Erythroxylum. We also identify the closest wild relatives and evolutionary relationships of the cultivated coca taxa. METHODS: We focused our phylogenomic inference on the largest taxonomic section in the genus Erythroxylum (Archerythroxylum O.E.Schulz) using concatenation and gene tree reconciliation methods from hybridization-based target capture of 427 genes. KEY RESULTS: We show that neotropical Erythroxylum are monophyletic within the paleotropical lineages, yet Archerythroxylum and all of the other taxonomic sections from which we sampled multiple species lack monophyly. We mapped phytogeographic states onto the tree and found some concordance between these regions and clades. The wild species E. gracilipes and E. cataractarum are most closely related to the cultivated E. coca and E. novogranatense, but relationships within this "coca" clade remain equivocal. CONCLUSIONS: Our results point to the difficulty of morphology-based intrageneric classification in this clade and highlight the importance of integrative taxonomy in future systematic revisions. We can confidently identify E. gracilipes and E. cataractarum as the closest wild relatives of the coca taxa, but understanding the domestication history of this crop will require more thorough phylogeographic analysis.

Temperatures and substrates for germination and vigor ofErythroxylum pauferrense Plowman seeds

Erythroxylum pauferrense is an endemic understory plant species of the Northeast Region of Brazil. The species is of great importance to the region and so ecophysiological studies are needed for its preservation. The objective of the present study was to determine the best substrates and temperatures for testing germination and seed vigor of E. pauferrense. An experiment was performed comprising a completely randomized design in a 5 x 4 factorial scheme, with five temperature regimes (20, 25, 30, 35°C constant and 20-30°C alternating) and four types of substrates (paper, vermiculite, sand and commercial substrate). The following characteristics were evaluated: germination percentage, first germination count, germination speed index, mean germination time, seedling length and dry mass (root and shoot). Paper and vermiculite substrates combined with constant temperatures of 20, 25, 30 and alternated between 20-30°C, provide greater seed germination and vigor while 35°C reduces seed physiological quality.

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.

Asociación entre la masticación de la hoja de coca y la prevención de la caries dental en los pobladores del caserio de Buenos Aires, Jaen ­ 2017 / Association between chewing coca leaf and prevention of dental caries in the villagers of Buenos Aires, Jaen - 2017

El objetivo del presente estudio fue "Determinar la asociación entre la masticación de la hoja de coca y la prevención de la caries dental en los Pobladores del Caserío de Buenos Aires, Jaén ­ 2017". Se realizó con el enfoque cuantitativo; de nivel relacional de asociación de riesgo con un diseño de Cohortes; fue de tipo: observacional; prospectivo; longitudinal y analítico. La muestra fue de 33 pobladores donde 16 fueron los casos y 17 los controles. La recolección de datos se realizó mediante la lista de cotejo y la ficha del odontograma. Para contrastar la hipótesis se utilizó Riesgo Relativo (RR). Los resultados evidencian: antes del inicio de la investigación del 100% el 12.1 % que si mastica la hoja de la coca presentó caries dental mientras que el 18.2% que no mastican la hoja de la coca si presentó caries dental así mismo el 36.4% que mastican la hoja de la coca no presentaron caries dental mientras que el 33.3% que no mastican la hoja de la coca no presentaron caries dental; el valor del RR = 0.71, lo cual significa que la masticación de la hoja de coca es un factor protector de la caries dental. Después del estudio el 3.03% que si mastica la hoja de la coca presentó caries dental mientras que el 12.1% que no mastican si presentó caries dental, así mismo el 45.5% que mastican la hoja de la coca no presentó caries dental mientras que el 39.4% que no masticaron la hoja de coca no presentaron caries dental, el valor del RR = 0.27 significa que la masticación de la hoja de coca es un factor protector de la caries dental. En conclusión, la masticación de la hoja de coca es considerada como una medida preventiva de la caries dental

Revisión sobre el uso del mate de hoja de coca en la prevención del mal agudo de montaña / Review on the use of coca tea in prevention of acute mountain sickness

El número de turistas que viajan a zonas de gran altitud en países andinos se está  incrementando en los últimos años, con aumento concomitante del riesgo de desarrollar el mal agudo de montaña. En las zonas alto andinas el mate de hoja de coca es usado frecuentemente para la prevención de este problema, pero las evidencias no soportan esta medida; se estima que, por el contrario, podría incrementar los síntomas del cuadro clínico que se intenta controlar por lo que se sugiere la conveniencia de no incentivar su uso.

In recent years, an increasing number of tourists visiting high altitude zones and places in Andean countries are, in turn, making a concomitantgreater risk of developing acute mountain sickness. In the Andean region, coca tea is frequently used for the prevention of this condition. However, existing evidence does not seem to support such use and, on the contrary, it is estimated that it may increase the symptoms. The convenience of not to encourage the use of coca tea forthe prevention of acute mountain sickness, is suggested.

Enantiomeric high-performance liquid chromatography resolution and absolute configuration of 6ß-benzoyloxy-3α-tropanol.

The absolute configuration of the naturally occurring isomers of 6ß-benzoyloxy-3α-tropanol (1) has been established by the combined use of chiral high-performance liquid chromatography with electronic circular dichroism detection and optical rotation detection. For this purpose (±)-1, prepared in two steps from racemic 6-hydroxytropinone (4), was subjected to chiral high-performance liquid chromatography with electronic circular dichroism and optical rotation detection allowing the online measurement of both chiroptical properties for each enantiomer, which in turn were compared with the corresponding values obtained from density functional theory calculations. In an independent approach, preparative high-performance liquid chromatography separation using an automatic fraction collector, yielded an enantiopure sample of OR (+)-1 whose vibrational circular dichroism spectrum allowed its absolute configuration assignment when the bands in the 1100-950 cm(-1) region were compared with those of the enantiomers of esters derived from 3α,6ß-tropanediol. In addition, an enantiomerically enriched sample of 4, instead of OR (±)-4, was used for the same transformation sequence, whose high-performance liquid chromatography follow-up allowed their spectroscopic correlation. All evidences lead to the OR (+)-(1S,3R,5S,6R) and OR (-)-(1R,3S,5R,6S) absolute configurations, from where it follows that samples of 1 isolated from Knightia strobilina and Erythroxylum zambesiacum have the OR (+)-(1S,3R,5S,6R) absolute configuration, while the sample obtained from E. rotundifolium has the OR (-)-(1R,3S,5R,6S) absolute configuration.

Cytotoxic Effect of Erythroxylum suberosum Combined with Radiotherapy in Head and Neck Cancer Cell Lines.

The mouth and oropharynx cancer is the 6th most common type of cancer in the world. The treatment may involve surgery, chemotherapy and radiotherapy. More than 50% of drugs against cancer were isolated from natural sources, such as Catharanthus roseus and epipodophyllotoxin, isolated from Podophyllum. The biggest challenge is to maximize the control of the disease, while minimizing morbidity and toxicity to the surrounding normal tissues. The Erythroxylum suberosum is a common plant in the Brazilian Cerrado biome and is popularly known as "cabelo-de-negro". The objective of this study was to evaluate the cytotoxic activity of Erythroxylum suberosum plant extracts of the Brazilian Cerrado biome associated with radiotherapy in human cell lines of oral and hypopharynx carcinomas. Cells were treated with aqueous, ethanolic and hexanic extracts of Erythroxylum suberosum and irradiated at 4 Gy, 6 Gy and 8 Gy. Cytotoxicity was evaluated by MTT assay and the absorbance was measured at 570 nm in a Beckman Counter reader. Cisplatin, standard chemotherapy, was used as positive control. The use of Erythroxylum suberosum extracts showed a possible radiosensitizing effect in vitro for head and neck cancer. The cytotoxicity effect in the cell lines was not selective and it is very similar to the effect of standard chemotherapy. The aqueous extract of Erythroxylum suberosum, combined with radiotherapy was the most cytotoxic extract to oral and hypopharynx carcinomas.

A new tropane alkaloid from the leaves of Erythroxylum subsessile isolated by pH-zone-refining counter-current chromatography.

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time- and solvent-consuming procedure. In this work we present the isolation of a novel alkaloid, 6ß,7ß-dibenzoyloxytropan-3α-ol, together with the two known 3α-benzoyloxynortropan-6ß-ol and 3α,6ß-dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH-zone-refining counter-current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.