INTRODUCTION
⌅The Cuban flora have a wealth and diversity of the native species (around 7500) and a high percentage of the endemism (51.4%) (CITMA, 2014CITMA (Ministerio de Ciencia Tecnología y Medio Ambiente de la República de Cuba). 2014. V Informe Nacional al Convenio sobre la Diversidad Biológica. La Habana.). Inside this high vegetable diversity, it stands out the family Euphorbiaceae, no only to be one the most numerous, but also for the quantity and diversity of the chemical elements and the applications it possesses (Jimenez, 1992Jimenez LC, Bernal HY. 1992. “El Inchi”. Especies Vegetales Promisorias de los Países del Convenio Andrés Bello (Programa de Recursos Vegetales del Convenio Andrés Bello: PREVECAB). SECAB, Santa Fé de Bogotá.). In Cuba, the mentioned family is integrated by 37 genera, among which Croton stands out so much for the number of species that compose it (54), of them 38 endemics that represent the 70.4% (Greuter y Rankin, 2022Greuter W, Rankin R. 2022. Vascular Plants of Cuba A Checklist. Third, updated edition of The Spermatophyta of Cuba. Botanischer Garten und Botanisches Museum Berlin. https://doi.org/10.3372/cubalist.2022.1.), like for the reports as for the uses in traditional medicine and other fines (Greinge, 1988Greinge M, Ahmed S. 1988. Handbook of Plants with Pest- Control Properties. John Wiley & Sons, New York.; Jimenez, 1992Jimenez LC, Bernal HY. 1992. “El Inchi”. Especies Vegetales Promisorias de los Países del Convenio Andrés Bello (Programa de Recursos Vegetales del Convenio Andrés Bello: PREVECAB). SECAB, Santa Fé de Bogotá.; Napralert Database, 2013Napralert Database. 2013. University of Illinois: Chicago. Disponible en: http://stneasy.cas.org (consultado 15 de marzo de 2013). ). To mention some examples, they can be related its effectiveness like diuretic, diaphoretic, detersive, spasmolytic, antimalarial, antimicrobial, insecticide, molusquicide and nematicide.
The alkaloids and essential oils constitute important groups of the secondary metabolites of the plants for the varied physiologic activity that unchain, but less interesting has been composed of the hexanics extracts of several species of the family, being included of the genus Croton, among them Croton campestres A.St-Hil. (Matias, 2010Matias EFF, Santos KKA, Costa JGMH, Coutinho DM. 2010. Light enhanced antibiotic activity of Brazilian medical plants (Croton campestris A., Ocimum gratissimum L. and Cordia verbenaceae DC.. Asian Biomedicine. 4: 183-186.) and Croton scabiosus Bebb. (Sarojini, 2011Sarojini DN, Mani J, Venkata RRR. 2011. In vitro antimicrobial activity and phytochemical constituents of Croton scabiosus Bedd. International Journal of Pharmaceutical Sciences and Research. 3: 149-154.) to exhibit antimicrobial activity in front of Staphylococcus aereus Rosenbach, Pseudomonas aeruginosa (Schoroeter) Migula and Escherichia coli (Migula) Castellani & Chalmers, among other pathogenic microorganisms. Herein, we report for the first time the composition of the hexanic extract from the leaves and stems of the Cuban endemic species potentially medicinal Croton micradenus Urb. and its antimicrobial activity in front of the references strains S. aureus, E. coli, P. aeuroginosa and Candida albicans (Robin) Berkhout.
MATERIAL AND METHODS
⌅General Procedures
⌅Gas Chromatography-Mass Spectra (GC-MS), Gas Chromatograph 6890N to a selective detector mass 5975 B inert (Agilent, USA) with a computation system and a capillary column HP-5 Ms (30 m x 0.25 mm Inner Diameter and 0.25 m thickness, Agilent, USA). The identification was carried out by comparison of the spectra obtained with the libraries NIST 2011 and Wiley-275, as well as with substances commercial references available and the literature. The retention times (Rt) were also compared with substances commercial of reference. The determination of the relative percentage was based on the internal normalization method (n=3).
Plant material
⌅The leaves and stems of individual adults sterile of the specie C. micradenus, were recollected by Ricardo Rosa Angulo in May 2012 in Mine Yeso, Baitiquirí, San Antonio del Sur, Guantánamo, Cuba, and identified by Ramona Oviedo Prieto. A voucher specimen (number HAC 41947) is retained in the Herbarium of the Institute, CITMA, Havana, Cuba.
Extraction and obtaining of chromatograms
⌅The dried (40°C) and ground leaves and stems of C. micradenus (100 g) were extracted with n-hexane to reflux at 100°C (4 hours) of residue upon evaporation in vacuum. For GC-MS analysis, 4 mg of the hexanic extract from the leaves and stems was dissolved in 0.2 mL of chloroform and derivatized with 100 µL of N-metil-N-trimetilsilil trifluoro acetamida (MSTFA) to 70ºC during 30 min in a dry thermostat.
Biological assay
⌅The antimicrobial activity of the hexanic extract from the leaves and stems was determinate for microdilution plating method (Wayne, 2000Wayne P. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards. Pennsylvania.) for determining the Minimum Inhibitory Concentration (MIC) according the National Committee for Clinical Laboratory Standard. The microorganisms S. aereus (ATCC 6538), P. aeruginosa (ATCC 9027), E. coli (ATCC 10536) and C. albicans (ATCC 10231) were adjusted with the turbidity standard McFarland 0.5. It was used as control the antibiotic chloramphenicol to a concentration of 5 mg/mL. The MIC is the lowest concentration capable of inhibiting bacterial growth.
RESULTS AND DISCUSSION
⌅Through GC-MS analysis, we have identified around 62 compounds in hexanic extract from the leaves and stems of C. micradenus. These compounds were identified for the first time in this specie, representing an important contribution to the chemo-taxonomical characterization of the same one. Alcohols (32.1%) and aldehyde (21.9%) are the main constituents of this organic fraction. Some of these compounds were also found in the other specie of the same genus (Mukherjee, 1984Mukherjee R, Axt EM. 1984. Cyclitols from Croton celtidifolius. Phytochemistry. 23: 2682-2684.; Peres, 1997Peres MTLP, Delle Monache F, Bella Cruz A, Pizzolatti MG, Yunes RA. 1997. Chemical composition and antimicrobial activity of Croton urucurana Baillon (Euphorbiaceae). Journal of Ethnopharmacology. 56: 223. DOI: 10.1016/s0378-8741(97)00039-1.; 1998Peres MTLP, Delle Monache F, Pizzolatti MG, Santos ARS, Beirith A, Calixto JB, et al. 1998. Analgesic compounds of Croton urucurana Baillon. Pharmaco-chemical criteria used in their isolation. Phytotherapy Research. 12: 209-211.; Maciel, 1998Maciel MAM, Pinto AC, Brabo SN, Silva MN. 1998. Terpenoids from Croton cajucara. Phytochemistry. 49, 823-828.; Santos, 1999Dos Santos Pereira A, Fernandes do Amaral AC, Barnes RA, Cardoso Jarí N, De Aquino Neto FR. 1999. Identification of isoquinoline alkaloids in crude extracts by high temperature gas chromatography-mass spectrometry. Phytochemistry Anales. 10: 254-258.; Maciel, 2000Maciel MAM, Pinto AC, Arruda AC, Pamplona SGSR, Vanderlinde FA, Lapa AJ, et al. 2000. Ethnopharmacology, phytochemistry and pharmacology: a successful combination in the study of Croton cajucara. Journal of Ethnopharmacology. 70: 5541-5543.; Murillo, 2001Murillo RM, Jakupovic J, Rivera V, Castro VH. 2001. Diterpenes and other constituents from Croton draco (Euphorbiaceae). Revista de Biología Tropical. 49: 259-264.; Dos Santos, 2002Dos Santos Pereira A, Albero Carbonell S, De Aquino Neto FR, Fernandes do Amaral AC, Barnes RA. 2002. High-temperature gas chromatography-mass spectrometry with glass capillary columns for the screening of natural products. Journal of Chromatography. A. 947: 255-265.; Catalán, 2003Catalán CAN, Heluani CS, Kotowicz C, Gedris TE, Herz W. 2003. A linear sesterterpene, two squalene derivatives and two peptide derivatives from Croton hieronymi. Phytochemistry. 64: 625-632.; Barbosa, 2003Barbosa PR, Fascio M, Martins D, Guedes S, Roque NF. 2003. Triterpenes of Croton betulaster (Euphorbiaceae). Biochemestry Systematic and Ecology. 31: 307-308.; 2004Barbosa PR, Fascio M, Martins D, Roque NF. 2004. Benzoyl-methylpolyols from Croton species (Euphorbiaceae). Arkivoc. 6: 95-102.).
In the Figure 1 is shown the chromatogram of the extract. The identification of the compounds is given in Appendix 1, and it was corroborated when comparing with the libraries spectra of the equipment, available patterns and for that described in the literature. Of the 65 compounds found in the extract, 62 were identified what represents 95.4%. The majority components were 1-octacosanol (C28OH) (12.5%), triacontanal (C30Al) (9.11%), octacosanal (C28Al) (7.67%), heptacosanal (C27Al) (6.58%), 1-hexacosanol (C26OH) (6.58%), 5,5-dimetil-1-etil 1,3-cyclopentadiene (5.70%), triacontanol (C30OH) (4.62%), nonacosane (C29) (3.46%), triacontanoic acid (C30:0) (3.44%), palmitic acid (C16:0) (3.36%), 1-tetracosanol (C24OH) (2.90%) and 1-nonacosanol (C29OH) (2.24%), which represent 68.2% of the total.
The hexanic extract from the leaves and stems of C. micradenus showed antimicrobial activity of 3.1 mg/mL MIC in front of S. aureus and E. coli with regard to the control Chloramphenicol (5 mg/mL), not being this way in front of the bacteria P. aeruginosa and to the mushroom C. albicans that turn out to be negative. Despite being of another species of another family and genus, similar result to those reported by Heng et al. (2020)Heng YW, Ban JJ, Khoo KS, Sit NW. 2020. Biological activities and phytochemical content of the rhizome hairs of Cibotium barometz (L.) J.Sm. (Cibotiaceae). Industrial Crops & Products. 153. https://doi.org/10.1016/j.indcrop.2020.112612. for the hexanic, extract from the rhizome hairs of Cibotium barometz (L.) J. Sm. (Cibotiaceae) which shows antimicrobial activity of 2.50 mg/mL MIC in from of S. aureus.
Sengupta et al. (2018)Sengupta S, Nandi I, Bhattacharyya DK, Ghosh M. 2018. Anti-Oxidant and anti-bacterial properties of 1-octacosanol isolated from rice bran wax. Journal Plant Biochemistry Physiology. 6: 1-8. reported that the octacosanol also showed anti-bacterial activities against E. coli to a concentrations of 1 mg/mL and 2 mg/mL. At 2 mg/mL, octacosanol was observed to inhibit microbial activity by gram-negative bacteria E. coli. They were compared with the activity of a standard antibiotic, tetracycline 500 using an isopropanol control. According to Mustapha et al. (2016)Mustapha N, Abubakar, Majinda RRT. 2016. GC-MS Analysis and Preliminary Antimicrobial Activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines. 3, 3. DOI: 10.3390/medicines3010003., the preliminary antimicrobial assay of the hexane extract from the heartwood of Albizia adianthifolia (Schumach) (Fabaceae), showed activity against E. coli with MIC of 1 µg. Among of constituents identified and characterized include palmitic acid, oleic acid, stigmasterol and 1-octacosanol present in the hexanic extract of C. micradenus, some majority. On the other hand, Dambatta et al. (2017)Dambatta MB, Ndukwe GL, Amupitan AO. 2017. Stigmasterol as antimicrobial agents from root extracts of Cola millenii. International Journal of Science for Global Sustainability. 4: 47-53. reported that the hexanic extract of root part of Cola millenii K. Schum (Malvaceae) showed antimicrobial activity of 7.5 mg/mL MIC in from of E. coli. Taking into account the presence of octacosanol (12.5%) (Marrero, 2013Marrero D, Cora M, Laguna A, González VL. 2013. Caracterización espectroscópica del D-003 obtenido de la cera de caña de azúcar (Saccharum officinarum L.). Revista Cubana de Farmacia. 47: 389-399.), palmitic acid (3.36%), oleic acid (1.48%) (Saxton, 1979Saxton JE. 1979. Review of the literature Published between July 1977 and June 1978. The Royal Society of Chemistry. Burlington House, London.) and estigmasterol (0.65%) (Marrero, 2013Marrero D, Rodríguez EA, González VL, Morales CL. 2013.Determinación de esteroles en la fracción insaponificable del aceite de los frutos de la palma real cubana (Roystonea regia). Revista Cubana de Farmacia. 47: 110-116.) in the hexanic extract of leaves and stems of C. micradenus, they confer him responsibility on the antimicrobial activity expressed by the sample.
CONCLUSIONS
⌅The chemical composition and antimicrobial activity against E. coli and S. aureus was determined for the first time in the hexanic extract from the leaves and stems of Cuban endemism C. micradenus. This, is one of the species that characterizes to the phytogeographic district of Coastal Area Maisí-Guantánamo for its representativeness in the most part of the xerophytic vegetation the south of the oriental counties mainly, compare these results whit those of others of endemics species that integrate the main populations in this Oriental region of Cuba, will be interesting.