Evaluation of morpho-anatomical and chemical differences between varieties of the medicinal plant Casearia sylvestris Swartz

JOSIANE C. CLAUDINO LUIS V.S. DO SACRAMENTO INGRID KOCH HELEN A. SANTOS ALBERTO J. CAVALHEIRO ARISTEU G. TININIS ANDRé G. DOS SANTOS About the authors

Abstracts

Casearia sylvestris Swartz (Salicaceae) has been used in traditional medicine and its leaf extracts have been exhibited important pharmacological activities. The species presents morphological, chemical and genetic variation. Two varieties are considered due external morphological differences: C. sylvestris var. sylvestris and var. lingua. There are difficulties in definition of these varieties. The objective of this work is to evaluate chemical and morpho-anatomical differences between C. sylvestris varieties that can be applied in their distinction for pharmaceutical or botanical purposes. Transverse and paradermic sections of leaves were prepared for morpho-anatomical, histochemical and quantitative microscopy (stomatal and palisade index) analyses. Diterpene profiles of the specimens were obtained by HPLC-DAD and TLC. Morpho-anatomical analyses demonstrated significant differences between the varieties only in paradermic sections: var.sylvestris - polygonal epidermic cell walls and hypostomatic; var. lingua - rounded epidermic cell walls and amphistomatic. No differences were observed for stomatal index; palisade index was found 2.8 for var. lingua and 3.9 for var.sylvestris. Chromatographic analyses confirmed previous results demonstrating that diterpene profile in varieties differs, with predominance of these metabolites in var. sylvestris. In conclusion, this work indicates that chromatographic analysis besides morpho-anatomical analysis can be applied in distinction of C. sylvestris varieties.

Casearia sylvestris ; chromatography; diterpenes; morpho-anatomy


Casearia sylvestris Swartz (Salicaceae) é uma planta utilizada na medicina tradicional, cujos extratos de folhas demonstraram importantes ações farmacológicas. A espécie apresenta variação morfológica, genética e química. Duas variedades são consideradas devido a diferenças morfológicas: C. sylvestris var.sylvestris e var. lingua. Há dificuldades na definição destas variedades. O objetivo deste trabalho é avaliar diferenças morfo-anatômicas e químicas entre as variedades de C. sylvestris que permitam sua diferenciação com aplicação farmacêutica ou botânica. Seções transversais e paradérmicas de folhas foram preparadas para análises morfo-anatômicas, histoquímicas e microscopia quantitativa (indices de estômatos e paliçada). Análises cromatográficas (CLAE-DAD e CCD) foram realizadas para obter o perfil de diterpenos clerodânicos. Os resultados das análises morfo-anatômicas demonstraram diferenças significativas entre as variedades somente em cortes paradérmicos: var.sylvestris - paredes celulares epidérmicas poligonais e hipoestomática, var. lingua - paredes celulares epidérmicas arredondadas e anfiestomática. Os índices de estômatos não revelaram diferenças; os valores dos índices de paliçada foram de 2,8 para var.lingua e 3,9 para var. sylvestris. As análises cromatográficas confirmaram resultados prévios, demonstrando predomínio de diterpenos na var.sylvestris. Este trabalho sugere que análises cromatográficas e morfo-anatômicas podem ser ferramentas aplicáveis na distinção das variedades da espécie.

Casearia sylvestris ; cromatografia; diterpenos; morfo-anatomia


INTRODUCTION

Casearia sylvestris Swartz (Salicaceae), known as “guaçatonga” or “erva-de-bugre”, is a plant species distributed throughout South America that occurs in 22 states of Brazil, especially within the Atlantic and Amazon forests and the Cerrado biomes (Marquete 2001Marquete R. 2001. Reserva ecológica do IBGE (Brasília-DF): Flacourtiaceae. Rodriguésia 52: 5-16., Sleumer 1980Sleumer HO. 1980. Flacourtiaceae: flora neotropica; monograph number 22. New York: The New York Botanic Garden, 499 p.). The plant has been widely used in traditional medicine for the treatment of snake bites and in wound healing, and also as an antiulcer and topical antiseptic (Hoehne 1939Hoehne FC. 1939. Plantas e substâncias vegetais tóxicas e medicinais. São Paulo: Graphicars, 355 p., Lorenzi and Matos 2002Lorenzi H and Matos FJA. 2002. Plantas medicinais no Brasil nativas e exóticas. Nova Odessa: Instituto Plantarum, 512 p.). Pharmacological studies on its leaf extracts have demonstrated antiulcerogenic, anti-inflammatory, antivenom, and cytotoxic (tumor cell lines) activities. Moreover, no significant toxicological effects of its ethanolic or hydroalcoholic extracts have been observed in vitro or following oral administration in animals (Basile et al. 1990Basile AC, Sertie JAA, Pannizza S, Oshiro TT and Azzolini CA. 1990. Pharmacological assay of Casearia sylvestris. I: Preventive anti-ulcer activity and toxicity of the leaf crude extract. J Ethnopharmacol 30: 185-197., Borges et al. 2000Borges MH, Soares AM, Rodrigues VM, Andrião-Escarso SH, Diniz H, Hamaguchi A, Quintero A, Lizano S, Gutiérrez JM, Giglio JR and Homsi-Brandeburgo MI. 2000. Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2. Comp Biochem Physiol Part B: Biochem Mol Biol 127: 21-30., Ferreira et al. 2010Ferreira PMP, Santos AG, Tininis AG, Costa PM, Cavalheiro AJ, Bolzani V Da S, Moraes MO, Costa-Lotufo LV, Montenegro RC and Pessoa C. 2010. Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis. Chem-Biol Interact 188: 497-504., Maistro et al. 2004Maistro EL, Carvalho JC and Mantovani MS. 2004. Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay. Toxicol In Vitro 18: 337-342., Santos et al. 2010Santos AG, Ferreira PMP, Vieira Júnior GM, Perez CC, Tininis AG, Silva Gh, Bolzani VS, Costa-Lotufo LV, Pessoa C Do Ó and Cavalheiro AJ. 2010. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodiversity 7: 205-215., Sertiè et al. 2000Sertiè JAA, Carvalho JCT and Panizza S. 2000. Antiulcer activity of the crude extract from the leaves of Casearia sylvestris. Pharmaceutical Biology 38: 112-119., Silva et al. 2004Silva FB, Almeida JM and Sousa SMG. 2004. Natural medicaments in endodontics - a comparative study of the anti-inflammatory action. Braz Oral Res 18: 174-179.). The Brazilian Agency of Medicines (Agência Nacional de Vigilância Sanitária - ANVISA) recognizes the importance of the species that was included in a positive list of medicinal plants for sale in pharmacies (Brasil 2010Brasil. 2010. Agência Nacional de Vigilância Sanitária. Resolução n. 10, de 9 de março de 2010. Dispõe sobre a notificação de drogas vegetais junto à Agência Nacional de Vigilância Sanitária (ANVISA) e dá outras providências. Diário Oficial da União, Brasília, 11 mar.).

Secondary metabolites isolated-identified from C. sylvestris include monoterpenes and sesquiterpenes (Sousa et al. 2007Sousa FG, Schneider FZ, Mendes CE, Moura NF, Denardin RBN, Matuo R and Mantovani MS. 2007. Clastogenic and anticlastogenic effect of the essential oil from Casearia sylvestris Swart. J Essent Oil Res 19: 376-378.),nor-isoprenoids (Santos 2008Santos AG. 2008. Identificação dos princípios ativos antiulcerogênicos das folhas de Casearia sylvestris: contribuição para o desenvolvimento de um fitoterápico. Tese (Doutorado em Química) – Instituto de Química, Universidade Estadual Paulista, Araraquara, 361 p. (Unpublished)., Wang et al. 2009aWang W, Li XC, Ali Z and Khan IA. 2009a. Two new C13 nor-isoprenoids from the leaves of Casearia sylvestris. Chem Pharm Bull 57: 636-638.), triterpenes, lapachol, cafeic, chlorogenic and vanillic acids, flavonoids (Raslan et al. 2002Raslan DS, Jamal CM, Duarte DS, Borges MH and De Lima ME. 2002. Anti-PLA2 action test of Casearia sylvestris Sw. Boll Chim Farmac 141: 457-460.), neolignans (Wang et al. 2010Wang W, Ali Z, Li XC and Khan IA. 2010. Neolignans from the Leaves of Casearia sylvestris Swartz. Helv Chim Acta 93: 139-146.), ellagic and gallic acids derivatives (Silva et al. 2008aSilva SL, Calgarotto AK, Chaar JS and Marangoni S. 2008a. Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris Sw aqueous extract with anti-PLA2 activity. Toxicon 52: 655-666., Silva et al. 2008bSilva SL, Chaar JS, Damico DC, Figueiredo PMS and Yano T. 2008b. Antimicrobial activity of ethanol extract from leaves of Casearia sylvestris. Pharmaceutical Biology 46: 347-351.). Additionally, phytochemical studies of Caseariaspecies have revealed the presence of typical oxygenated tricycliccis-clerodane diterpenes in which the tetrahydrofuran ring bears two acyloxy groups at C18 and C19 (Chen and Wiemer 1991Chen TB and Wiemer DF. 1991. Corymbotins A-I: highly oxidized kolovane derivatives from Casearia corymbosa. J Nat Prod 54: 1612-1618., Guittet et al. 1988Guittet E, Stoven V, Lallemand J, Ramiandrasoa F and Kunesch G. 1988. Pitumbin, a novel kolavene acylal from Casearia pitumba Pleumer. Tetrahedron 44: 2893-2901., Kanokmedhakul et al. 2005Kanokmedhakul S, Kanokmedhakul K, Kanarsa T and Buayairaksa M. 2005. New bioactive clerodane diterpenoids from the bark of Casearia grewiifolia. J Nat Prod 68: 183-188.,Oberlies et al. 2002Oberlies NH, Burgess JP, Navarro HA, Pinos RE, Fairchild CR, Peterson RW, Soejarto DD, Farnsworth NR, Kinghorn AD, Wani MC and Wall ME. 2002. Novel bioactive clerodane diterpenoids from the leaves and twigs of Casearia sylvestris. J Nat Prod 65: 95-99., Santos et al. 2010Santos AG, Ferreira PMP, Vieira Júnior GM, Perez CC, Tininis AG, Silva Gh, Bolzani VS, Costa-Lotufo LV, Pessoa C Do Ó and Cavalheiro AJ. 2010. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodiversity 7: 205-215.). Diterpenes of this group were isolated from C. sylvestris: casearins A-X and caseargrewiin F - leaves (Carvalho et al. 1998Carvalho PRF, Furlan M, Young MCM, Kingston DGI and Bolzani V Da S. 1998. Acetylated DNA-damage clerodane diterpenes from Casearia sylvestris. Phytochemistry 49: 1659-1662., Itokawa et al. 1990Itokawa H, Totsuka N, Morita H, Takeya K, Iitaka Y, Schenkel EP and Motidome M. 1990. Antitumor principles from Casearia sylvestris Sw. (Flacourtiaceae), structure elucidation of new clerodane diterpenes by 2-D NMR spectroscopy. Chem Pharm Bull 38: 3384-3388., Morita et al. 1991Morita H, Nakayama M, Kojima H, Takeya K, Itokawa H, Schenkel EP and Motidome M. 1991. Structures and cytotoxic activity relationship of casearins, new clerodane diterpenes from Casearia sylvestris Sw. Chem Pharm Bull 39: 693-697., Santos et al. 2010Santos AG, Ferreira PMP, Vieira Júnior GM, Perez CC, Tininis AG, Silva Gh, Bolzani VS, Costa-Lotufo LV, Pessoa C Do Ó and Cavalheiro AJ. 2010. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodiversity 7: 205-215., Wang et al. 2009bWang W, Zhao J, Wang YH, Smillie TA, Li XC and Khan IA. 2009b. Diterpenoids from Casearia sylvestris. Planta Med 75: 1476-1481.); casearvestrins A-C - stems and leaves (Oberlies et al. 2002Oberlies NH, Burgess JP, Navarro HA, Pinos RE, Fairchild CR, Peterson RW, Soejarto DD, Farnsworth NR, Kinghorn AD, Wani MC and Wall ME. 2002. Novel bioactive clerodane diterpenoids from the leaves and twigs of Casearia sylvestris. J Nat Prod 65: 95-99.); and roots ofrel-19S-acetoxy-18R-butanoyloxy-18,19-epoxy-6S-hydroxy-2R-(2-methylbutanoyloxy)-5S,8R,9R,10S-cleroda-3,13(16),14-triene (Espíndola et al. 2004Espíndola LS, Vasconcelos Junior JR, De Mesquita ML, Marquie P, de Paula JE, Mambu L and Santana JM. 2004. Trypanocidal activity of a new diterpene from Casearia sylvestris var. lingua. Planta Med 70: 1093-1095.). These compounds exhibited some important biological activities as anti-fungal, antiulcer, trypanocidal, and cytotoxicity against human tumor cell lines (Espíndola et al. 2004Espíndola LS, Vasconcelos Junior JR, De Mesquita ML, Marquie P, de Paula JE, Mambu L and Santana JM. 2004. Trypanocidal activity of a new diterpene from Casearia sylvestris var. lingua. Planta Med 70: 1093-1095., Itokawa et al. 1990Itokawa H, Totsuka N, Morita H, Takeya K, Iitaka Y, Schenkel EP and Motidome M. 1990. Antitumor principles from Casearia sylvestris Sw. (Flacourtiaceae), structure elucidation of new clerodane diterpenes by 2-D NMR spectroscopy. Chem Pharm Bull 38: 3384-3388., Oberlies et al. 2002Oberlies NH, Burgess JP, Navarro HA, Pinos RE, Fairchild CR, Peterson RW, Soejarto DD, Farnsworth NR, Kinghorn AD, Wani MC and Wall ME. 2002. Novel bioactive clerodane diterpenoids from the leaves and twigs of Casearia sylvestris. J Nat Prod 65: 95-99., Santos 2008Santos AG. 2008. Identificação dos princípios ativos antiulcerogênicos das folhas de Casearia sylvestris: contribuição para o desenvolvimento de um fitoterápico. Tese (Doutorado em Química) – Instituto de Química, Universidade Estadual Paulista, Araraquara, 361 p. (Unpublished)., Santos et al. 2010Santos AG, Ferreira PMP, Vieira Júnior GM, Perez CC, Tininis AG, Silva Gh, Bolzani VS, Costa-Lotufo LV, Pessoa C Do Ó and Cavalheiro AJ. 2010. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodiversity 7: 205-215.).

Casearia sylvestris was first described by Swartz in Flora Indiae Occidentalis (1797) and Eichler and Martius (1871)Eichler AG and Martius CFP. 1871. Bixaceae. Flora Brasiliensis 1: 421-488. included it inFlora Brasiliensis of Carl Friedrich Philipp von Martius, classified in Bixaceae family. Later it was classified inFlacourtiaceae family (Absy and Scavone 1973Absy ML and Scavone O. 1973. Sobre a morfologia e anatomia da Casearia sylvestris Swartz. Bol Zool Biol Mar 30: 641-676., Sleumer 1980Sleumer HO. 1980. Flacourtiaceae: flora neotropica; monograph number 22. New York: The New York Botanic Garden, 499 p.). The Angiosperm Phylogeny Group(APG) updated the classification of Angiosperms in 2003 and some genera ofFlacourtiaceae (e.g. Casearia Jacq.) were classified as Salicaceae. C. sylvestris Swartz (Crateria Bentahm section) is classified inSamydeae tribe, Salicaceae family andMalpighiales order (Chase et al. 2002Chase MW, Zmartzty S, Lledó MD, Wurdack KJ, Swensen SM and Fay MF. 2002. When in doubt, put it in Flacourtiaceae: a molecular phylogenetic analysis based on plastid rbcL DNA sequences. Kew Bull 57: 141-181., The Angiosperm Phylogeny Group 2003The Angiosperm Phylogeny Group. 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Bot J Linnean Soc 141: 399-436.). The macromorphology of the species is described in the work of Torres and Yamamoto (1986)Torres RB and Yamamoto K. 1986. Taxonomia das espécies de Casearia Jacq. (Flacourtiaceae) do estado de São Paulo. Revta Bras Bot 9: 239-258., based on descriptions of Eichler and Martius (1871)Eichler AG and Martius CFP. 1871. Bixaceae. Flora Brasiliensis 1: 421-488. and Sleumer (1980)Sleumer HO. 1980. Flacourtiaceae: flora neotropica; monograph number 22. New York: The New York Botanic Garden, 499 p.. The morpho-anatomical characters of the leaves were described by Absy and Scavone (1973)Absy ML and Scavone O. 1973. Sobre a morfologia e anatomia da Casearia sylvestris Swartz. Bol Zool Biol Mar 30: 641-676. and Alquini and Takemori (2000)Alquini Y and Takemori NK. 2000. Organização estrutural de espécies vegetais de interesse farmacológico. Curitiba: Herbarium Laboratório Botânico, 80 p..

C. sylvestris occurs in different biomes showing great morphological variability: the size, shape, texture and consistency of the leaves, the pubescence of the branches and inflorescences, the number of flowers per inflorescence, the length of peduncle, and its height - shrubs to trees (Marquete 2001Marquete R. 2001. Reserva ecológica do IBGE (Brasília-DF): Flacourtiaceae. Rodriguésia 52: 5-16., Torres and Yamamoto 1986Torres RB and Yamamoto K. 1986. Taxonomia das espécies de Casearia Jacq. (Flacourtiaceae) do estado de São Paulo. Revta Bras Bot 9: 239-258.). Sleumer (1980)Sleumer HO. 1980. Flacourtiaceae: flora neotropica; monograph number 22. New York: The New York Botanic Garden, 499 p. considered two varieties to the species due external morphological differences, connected by intermediate forms that are difficult to distinguish: C. sylvestris var. sylvestris andC. sylvestris var. lingua(Cambess.). Silva et al. (2006)Silva MAS, Ming LC, Pereira MAS, Bertoni BW, Batistini AP and Pereira PS. 2006. Phytochemical and genetic variability of Casearia sylvestris Sw. From São Paulo State Atlantic Forest and Cerrado populations. Rev Bras Pl Med 8: 159-166. reported differences observed during collection between varieties. In Cerrado biome the specimens were shrubs, with coriaceous lighter green leaves with smaller width and length, corresponding to C. sylvestris var. lingua; unlike, specimens of Atlantic Forest were trees with different heights (always higher than 2 m), green dark leaves larger in width and length, corresponding to C. sylvestris var. sylvestris. The differences between varieties were summarized by Klein and Sleumer (1984)Klein RM and Sleumer HO. 1984. Flora ilustrada catarinense: flacourtiáceas. Itajaí: FLAC, 96 p..

Besides the macromorphological differences between the varieties, metabolism variability analyses have suggested a minor expression of diterpenes and a greater expression of flavonoids (as rutin) in C. sylvestris var. lingua. Studies of genetic variability also indicated distinct genetic profiles for the varieties (Cavallari 2008Cavallari MM. 2008. Variabilidade genética e química entre e dentro de populações de Casearia sylvestris Sw. (Salicaceae) no Estado de São Paulo. Tese (Doutorado em Ciências Biológicas) – Instituto de Biociências, Universidade Estadual Paulista, Botucatu, 115 p. (Unpublished)., Silva et al. 2006Silva MAS, Ming LC, Pereira MAS, Bertoni BW, Batistini AP and Pereira PS. 2006. Phytochemical and genetic variability of Casearia sylvestris Sw. From São Paulo State Atlantic Forest and Cerrado populations. Rev Bras Pl Med 8: 159-166.).

Considering the importance of C. sylvestris as medicinal plant, the chemical variability observed in previous works, mainly for the bioactive diterpenes and rutin, and the difficulties to distinguish the varieties ofC. sylvestris by macromorphological analyses in the present article, we report on the evaluation of chemical and morpho-anatomical differences between C. sylvestris varieties that can be applied in their distinction for botanical purposes or pharmaceutical applications.

MATERIALS AND METHODS

Materials

Leaves of C. sylvestris were collected at Araraquara, São Paulo State, in February 16, 2011 (in the morning).C. sylvestris var. lingua(Estação Experimental de Araraquara, Instituto Florestal, São Paulo): AGS38 (S 21.73619, W 48.17987), AGS43 (S 21.73605, W 48.17610), AGS44 (S 21.73600, W 48.17603), AGS45 (S 21.73596, W 48.17605) and AGS51 (S 21.71112, W 48.17339).C. sylvestris var. sylvestris(Universidade Estadual Paulista, UNESP Araraquara; except AGSDER): AGS101 (S 21.81466, W 48.20215), AGS102 (S 21.81466, W 48.20215), AGS103 (S 21.81631, W 4819838), AGS104 (S 21.81430, W 48.20160) and AGSDER (S 21.73596, W 4818850). Voucher specimens of AGS38, AGS43, AGS44, AGS45 AGS51, AGS101 and AGS102 were deposited with the Herbarium “Maria Eneida P. Kaufmann” of Instituto Botânico do Estado de São Paulo. AGS103 e AGS104 are clones (plant cutting) of specimens Cs400 and Cs78 that were identified asC. sylvestris var. sylvestris (Cavallari 2008Cavallari MM. 2008. Variabilidade genética e química entre e dentro de populações de Casearia sylvestris Sw. (Salicaceae) no Estado de São Paulo. Tese (Doutorado em Ciências Biológicas) – Instituto de Biociências, Universidade Estadual Paulista, Botucatu, 115 p. (Unpublished).).

The solvents used for extraction and TLC analyses were analytical grade (Synth®, Diadema, São Paulo, Brazil) whilst those used in HPLC analyses and solid phase extraction (SPE) were HPLC-UV grade (J. T. Baker®, Xalostoc, Mexico). Water was purified (18.1 MΩ.cm) with a Milli Q plus system (Millipore®, São Paulo, Brazil) immediately prior to use. SPE was performed using as stationary phase LiChroprep RP18 (40-63 µm; Merck®, Darmstadt, Germany). Analytical reversed-phase HPLC-DAD was performed using a Shimadzu® system (Kyoto, Japan) comprising a model Prominence® LC-20AT pump, SIL-20A autosampler, DGU-20A5 degasser, CTO-20A column oven, SPD-M20A photodiode array detector and CBM-20A communication bus module, fitted with Hypersil Gold® C18 (Thermo® Scientific, USA) column (250 × 4.6 mm, 5 µm), with control and data handling managed by LCsolution® multi-PDA software. TLC was developed in silica gel plate aluminum backed (20 × 20 cm; 200 µm) from Sorbent® Technologies. The chromatographic standards of clerodane diterpenes (caseargrewiin F, casearin B and X) were purified from leaves extracts of C. sylvestris and identified as published (Santos et al. 2010Santos AG, Ferreira PMP, Vieira Júnior GM, Perez CC, Tininis AG, Silva Gh, Bolzani VS, Costa-Lotufo LV, Pessoa C Do Ó and Cavalheiro AJ. 2010. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodiversity 7: 205-215.). Optical microscopy was developed in a Carl Zeiss® Primo Star microscope. Reagents used in histochemical analysis were prepared as described in literature (Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p., Ganter and Jollés 1969-1970, Johansen 1940Johansen DA. 1940. Plant Microtechnique. New York: McGraw-Hill, 523 p., Valette et al. 1998Valette C, Andary C, Geiger JP. Sarah JL and Nicole M. 1998. Histochemical and cytochemical investigations of phenols in roots of banana infected by the burrowing nematode Radopholus similes. Phytopathology 88: 1141-1148.).

Morpho-Anatomical Analyses

Anatomical freehand sections (paradermic and transverse) of the middle third of fresh leaves from C. sylvestris were obtained with a blade. The sections were clarified with sodium hypochlorite commercial solution and wash with deionized water before stain with different reagents (Delafield hematoxylin, Astra blue, iodine green and toluidine blue) for selection of better cell wall staining and visualization of tissue structures. The anatomical descriptions were realized based on microscope visualization (40 x) of the sections on covered glass sheet and comparison with literature data (Absy and Scavone 1973Absy ML and Scavone O. 1973. Sobre a morfologia e anatomia da Casearia sylvestris Swartz. Bol Zool Biol Mar 30: 641-676., Alquini and Takemori 2000Alquini Y and Takemori NK. 2000. Organização estrutural de espécies vegetais de interesse farmacológico. Curitiba: Herbarium Laboratório Botânico, 80 p.).

Histochemical Investigations

The identification and localization of secondary metabolites in the leaf transverse sections were realized using the follow reagents: Sudan III (Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p., Johansen 1940Johansen DA. 1940. Plant Microtechnique. New York: McGraw-Hill, 523 p.) and 2.4-dinitrophenylhydrazine (Ganter and Jollés 1969-1970) for terpenes; ferric chloride (Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p., Johansen 1940Johansen DA. 1940. Plant Microtechnique. New York: McGraw-Hill, 523 p.) and vanillin-hydrochloric acid (Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p., Valette et al. 1998Valette C, Andary C, Geiger JP. Sarah JL and Nicole M. 1998. Histochemical and cytochemical investigations of phenols in roots of banana infected by the burrowing nematode Radopholus similes. Phytopathology 88: 1141-1148.) for phenolic compounds.

Quantitative Microscopy

Palisade index: the average number of palisade cells beneath each epidermal cell is termed the palisade index. The paradermic sections obtained and clarified as described in Morpho-Anatomical Analyses section were mounted on covered glass sheet. Five groups of four epidermal cells were selected in each section. The palisade cells lying beneath each group were counted, being included in the count wich are more than half-covered by the epidermal cells (Evans 2002Evans WC. 2002. Trease and Evans Pharmacognosy, 15th ed., London: WB Saunders, 585 p., Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p.).

Stomatal index: the percentage proportion of the ultimate divisions of the epidermis of a leaf which has been converted into stomata is termed the stomatal index (I):

I = [(S x 100)/(E = S)] x 100

Where S is the number of stomata per unit area and E is the number of ordinary epidermal cells in the same unit area. The paradermic sections (upper and lower surfaces), that were obtained and clarified as described in Morpho-Anatomical Analyses section, were stained with Astra blue and mounted on covered glass sheet (Evans 2002Evans WC. 2002. Trease and Evans Pharmacognosy, 15th ed., London: WB Saunders, 585 p.,Costa 2001Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p.).

Chromatographic Analyses

The following methods were developed and validated to evaluate the diterpene chromatographic profile of C. sylvestris leaf samples in the laboratories of Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Instituto de Química, UNESP, Araraquara-SP (data not published).

Extraction: Dried (40° C, 7 days) and powdered leaves (1.0 g) from the specimens of C. sylvestriswere extracted twice in a test tube with 10 mL of ethyl acetate: hexane: isopropanol 91:08:01 (v-v) by sonication for 1 h. The liquid extracts were filtered, dried at room temperature and at desiccator with silica gel under reduced pressure.

High Performance Liquid Chromatography: 20.0 mg of dried extracts were dissolved in 1.0 mL of methanol: water (98:02, v-v) and submitted to further clean-up by SPE. Laboratory-prepared SPE columns (20 × 7 mm i.d.), dry packed with ca. 1.5 g of LicChroprep RP18, were activated with methanol and conditioned with methanol: water (98:02, v-v). Following application of samples, columns were eluted with 4.0 mL of methanol: water (98:02, v-v). Each eluated was dried in a desiccator with silica gel under reduced pressure, dissolved in methanol (1.0 mL), and filtered through PVDF membranes (0.45 µm) prior to HPLC analysis. The standards of clerodane diterpenes (1.0 mg) were dissolved in methanol HPLC grade (1.0 mL) and filtered through PVDF membranes (0.45 µm). Aliquots of 20 µL were injected onto Hypersil Gold® C18 column (250 × 4.6 mm, 5 µm), which were eluted with a mixture of methanol, acetonitrile and water, initially at 22:44:34 (v-v), changing by linear gradient to 47:53:00 over 42 min, isocratically with 47:53:00 for 5 min, and finally isocratically with 22:44:34 for 10 min (equilibration). The solvent flow rate was 0.8 mL-min. Detection was at 200-700 nm for all samples. Caseargrewiin F, casearin B and X (1.0 mg-mL; methanol) were used as standards.

Casearin-like diterpenes present two different patterns of conjugated double bond in their lateral chain (C11-C16) - C12(Z orE)-C14 or C13(16)-C14 - and their UV spectra present one band and λmax= 231-238 or 221-228 nm, respectively (Carvalho et al. 2009Carvalho ES, Santos AG and Cavalheiro AJ. 2009. Identificação de diterpenos clerodânicos em diferentes órgãos de Casearia sylvestris Swartz. Rev Ciênc Farm Básica Apl 30: 277-284.,Espíndola et al. 2004Espíndola LS, Vasconcelos Junior JR, De Mesquita ML, Marquie P, de Paula JE, Mambu L and Santana JM. 2004. Trypanocidal activity of a new diterpene from Casearia sylvestris var. lingua. Planta Med 70: 1093-1095., Itokawa et al. 1990Itokawa H, Totsuka N, Morita H, Takeya K, Iitaka Y, Schenkel EP and Motidome M. 1990. Antitumor principles from Casearia sylvestris Sw. (Flacourtiaceae), structure elucidation of new clerodane diterpenes by 2-D NMR spectroscopy. Chem Pharm Bull 38: 3384-3388.). Thus, we suggested that peaks with UV spectra with one band and λmax= 221-228 or 231-238 nm were relative to the casearin-like diterpenes. Carvalho et al. (2009)Carvalho ES, Santos AG and Cavalheiro AJ. 2009. Identificação de diterpenos clerodânicos em diferentes órgãos de Casearia sylvestris Swartz. Rev Ciênc Farm Básica Apl 30: 277-284. analyzed different organs of C. sylvestris with similar approach and results from HPLC-DAD analyses were confirmed by 1H NMR and TLC data. Moreover, five casearin-like diterpenes isolated from leaves of C. sylvestris were identified in its extract and presented peaks with tR in the range of 10-30 min in the same HPLC conditions (Santos 2008Santos AG. 2008. Identificação dos princípios ativos antiulcerogênicos das folhas de Casearia sylvestris: contribuição para o desenvolvimento de um fitoterápico. Tese (Doutorado em Química) – Instituto de Química, Universidade Estadual Paulista, Araraquara, 361 p. (Unpublished).). Additionally, as observed by Carvalho et al. (2009)Carvalho ES, Santos AG and Cavalheiro AJ. 2009. Identificação de diterpenos clerodânicos em diferentes órgãos de Casearia sylvestris Swartz. Rev Ciênc Farm Básica Apl 30: 277-284., peaks with a second band in the UV spectra with λmax of c.a. 280 nm, indicate the presence of clerodane diterpenes with a aromatic ester substituent, as reported in the literature (Beutler et al. 2000Beutler JA, McCall KL, Herbert K, Herald DL, Pettit GR, Johnson T, Shoemaker RH and Boyd MR. 2000. Novel cytotoxic diterpenes from Casearia arborea. J Nat Prod 63: 657-661.). Thus, clerodane diterpenes were identified in the extract chromatograms on the basis of UV spectra of the peaks. Additionally, the comparison of retention time (tR) and UV spectra of peaks from standard and extract chromatograms was realized.

Thin Layer Chromatography: 8.0 mg of dried extracts and 1.0 mg of caseargrewiin F and casearin B were dissolved in 1.0 mL of ethyl acetate. TLC was developed in silica gel plate aluminum backed (20 × 20 cm; 200 µm) using hexane: ethyl acetate: isopropanol 70:28:02 (v-v) as eluent and sulfuric anisaldehyde as spray reagent.

RESULTS

The macromorphological differences between the varieties of C. sylvestris reported were evident during the collection and the specimens were classified as C. sylvestris var.lingua or C. sylvestris var.sylvestris as described by Sleumer (1980)Sleumer HO. 1980. Flacourtiaceae: flora neotropica; monograph number 22. New York: The New York Botanic Garden, 499 p. and Klein and Sleumer (1984)Klein RM and Sleumer HO. 1984. Flora ilustrada catarinense: flacourtiáceas. Itajaí: FLAC, 96 p..

In the morpho-anatomical study, the best visualization in transverse sections was achieved with dual staining: Delafield hematoxylin followed by green iodine. No significant anatomical differences were observed in transverse sections for the varieties. Morpho-anatomical characters of the leaves were in agreement with literature (Absy and Scavone 1973Absy ML and Scavone O. 1973. Sobre a morfologia e anatomia da Casearia sylvestris Swartz. Bol Zool Biol Mar 30: 641-676., Alquini and Takemori 2000Alquini Y and Takemori NK. 2000. Organização estrutural de espécies vegetais de interesse farmacológico. Curitiba: Herbarium Laboratório Botânico, 80 p.). In the midvein region of leaf sections we observed typical cells of xylem, phloem, collenchyma and parenchyma, in addition to fibers associated with vascular bundles (Figure 1a). Both upper and lower epidermises were uniseriate, presenting a lower number of simple nongladular trichomes. The mesophyll is asymmetrical and heterogeneous with two (rarely three) layers of palisade parenchyma and several layers of spongy parenchyma with irregular cells in shape and size. The air spaces (meatus) occur in small proportion in spongy parenchyma (Figure 1b). We also noted the presence of spiral vessels, oil glands, druses and prismatic crystals (calcium oxalate) (Figure 2).

Figure 1
Tranverse section of the midvein region from the leaf of C. sylvestris var. lingua (a) and tranverse section of the region between the midvein and the margin from the leaf of C. sylvestris var. sylvestris (b).

Figure 2
Transverse section of the region between the midvein and the margin from the leaf of C. sylvestris var.lingua.

For the paradermic sections, the best stain reagent was Astra blue. In this case we observed significant differences between the varieties. The epidermic cell walls are polygonal in C. sylvestris var.sylvestris and rounded in C. sylvestris var.lingua (Figure 3). Moreover, C. sylvestris var.sylvestris is hypostomatic and C. sylvestrisvar. lingua is amphistomatic. The stomata are paracytic in both varieties.

Figure 3
Paradermic sections (lower epidermis) from the leaf ofC. sylvestris var. sylvestris (a) and var. lingua (b).

Histochemical reagents did not differentiate the varieties and the results were positive for both terpenes and phenolics (all reagents tested).

The results of quantitative microscopy are in Tables I and II. The values of stomatal index for specimens of C. sylvestris var.sylvestris presented great variation: 11.9 to 20.7 % (standard deviation = 3.4). In the case of C. sylvestris var. lingua specimens these values were more homogeneous (standard deviation = 0.2). The mean value of stomatal index for both varieties was similar: 15.9 (var.sylvestris) and 16.4 % (var.lingua). On the other hand, palisade index was found 2.8 for var. lingua and 3.9 for var. sylvestris, demonstrating that this index should be useful in differentiation of C. sylvestris varieties.

TABLE I
Stomatal index for specimens of C. sylvestris.
TABLE II
Palisade index for specimens of C. sylvestris.

TLC analyses provided information about clerodane diterpenes profile of the extracts. Caseargrewiin F (Rf = 0.23) was identified by TLC (Figure 4) in the extracts of specimens AGS101, AGS102, AGS103, AGS104 (var.sylvestris), AGS44, AGS45 and AGS51 (var.lingua), while casearin B (Rf = 0.26) was not identified in any extract. Moreover, TLC profiles of var.sylvestris specimens are similar, except for AGSDER. Unlike, specimens of var. lingua presented different TLC profiles, except for AGS44 and AGS45. These specimens have TLC profile similar to specimens of var.sylvestris.

Figure 4
Thin layer chromatographic profile (silica; hexane: ethyl acetate: isopropanol 70:28:02; sulfuric anisaldehyde) of extracts from leaves of C. sylvestris. Samples (from left to right): AGS38, AGS51, AGS43, AGS45, AGS44 (var.lingua); AGSDER, AGS102, AGS101, AGS103, AGS104 (var. sylvestris); caseargrewiin F, casearin B (clerodane diterpenes).

Table III presents results of HPLC-DAD analyses for the ten specimens considering number of peaks and area of selected peaks based on UV spectra-λmax. Figure 5 shows representative chromatograms of both varieties and the structures of diterpene standards. As observed in the example of Figure 5, there is a greater total area of peaks with tR< 5 min in the chromatograms of specimens of C. sylvestris var. lingua and AGSDER (var.sylvestris) than in chromatograms of AGS101-104 (var.sylvestris). On the other hand, the total area of peaks with λmax= 221-228 or 231-238 nm is greater in chromatograms of AGS101-104 (Table III).

TABLE III
HPLC-DAD analyses data of leaf extracts from different specimens ofC. sylvestris. The number of peaks and total area of selected peaks based on UV spectra-λmax are presented.

Figure 5
Representative high performance liquid chromatography (C18; methanol: acetonitrile: water gradient; 235 nm) profile of extracts from leaves of C. sylvestris - specimens AGS38 var.lingua (top) and AGS101 var.sylvestris (bottom). Structures of diterpenes standards are presented: caseargrewiin F (cas F), casearins B and X (cas B and cas X).

We classified the specimens in three groups on the basis of the observed common main peaks (peaks with larger area), identification of clerodane diterpenes (caseargrewiin F, casearin B and X) and predominance of peaks with UV spectrum with λmax= 221-228 or 231-238 nm. The diterpene standards were identified in the extracts by comparison of their tR and UV spectra. Tables IV-VI present data from chromatograms of the three groups: group 1 - AGS38, AGS44-45 (var.lingua); group 2 - AGS43 and AGS51 (var.lingua); group 3 - AGS101-104 (var.sylvestris). AGSDER demonstrated a chromatographic profile with characteristics of different groups.

TABLE IV
Data from HPLC-DAD analyses of specimens AGS38, AGS44 and AGS45. Retention time, area and λmax of selected peaks (tR > 10 min; area > 106 in at least one specimen of the group) are presented.
TABLE V
Data from HPLC-DAD analyses of specimens AGS43 and AGS51. Retention time, area and λmax of selected peaks (tR> 10 min; area >106 in at least one specimen of the group) are presented.
TABLE VI
Data from HPLC analyses of specimens AGS101-104. Retention time, area and λmax of selected peaks (tR > 10 min; area >107 in at least one specimen of the group) are presented.

DISCUSSION

The morpho-anatomical comparison between the varieties revealed differences only in the paradermic sections. The shape of epidermic cell walls (polygonal in var. sylvestris and rounded in var.lingua) and stomata distribution (var.sylvestris is hypostomatic and var. lingua is amphistomatic) are characteristics that can be applied in the differentiation of the varieties as well as the palisade index. According Costa (2001)Costa AF. 2001. Farmacognosia: farmacognosia experimental, 3rd ed., v.3. Lisboa: Fundação Calouste Gulbekian, 999 p. the palisade index is less affected by environmental factors than stomatal index. These results are interesting for the pharmaceutical quality control application because the analysis of the powdered drug will lead to the identification of the varieties. Obviously, the assays will be applied to more representative number of specimens from different environments or regions to be validated.

Data from HPLC-DAD analyses (tR and UV spectrum) allowed to classify the specimens in three groups. In the first group - AGS38, AGS44 and AGS45 (var. lingua) - we found the following common characteristics: identification only of caseargrewiin F, predominance of peaks with λmax= 221-228 nm (22-23 peaks and total peak area of 22-55 × 106), and seven common main peaks with area > 106 (Tables III andIV). In TLC analyses caseargrewiin F was not identified in AGS38 probably due its minor concentration as demonstrated by HPLC analysis (small peak area).

In the second group - AGS43 and AGS51 (var.lingua) - caseargrewiin F, casearins B and X were identified. In the TLC analyses, diterpenes were not identified as discussed above. The peaks with λmax= 231-238 nm have greater area (16-17 peaks and total peak area of 21-53 × 106) than peaks with λmax= 221-228 nm (6-8 peaks and total peak area of 0.8-2.4 × 106) (Table III). Finally, there are five common main peaks with area > 106 (Table V) in their chromatograms.

The third group includes AGS101-104 (var.sylvestris). In this group the peaks with λmax= 231-238 nm predominate notably in number of peaks (24-33) and total peak area (142-242 × 106) in comparison with all specimens of var. lingua (Table III). The three diterpenes were identified in these specimens and they have six common main peaks with area > 4 × 106 (Table VI). In the TLC analysis, casearin B was not identified as discussed above.

The specimen AGSDER presented characteristics of different groups. Its chromatogram demonstrated that peaks with λmax= 221-228 nm predominate in number (13) but no differences were observed for peak total area (8 × 106) considering the two ranges of λmax. Casearin B and caseargrewiin F were identified in HPLC-DAD analysis.

The results obtained suggest the predominance of clerodane diterpenes, especially with λmax= 231-238 nm, in C. sylvestris var. sylvestris. Moreover, there is a greater total area of peaks with tR< 5 min in the chromatograms of specimens of C. sylvestris var. lingua and AGSDER (var. sylvestris) than in chromatograms of AGS101-104 (var. sylvestris) indicating greater concentration of polar compounds (e.g. phenolics) as demonstrated in previous work (Cavallari 2008Cavallari MM. 2008. Variabilidade genética e química entre e dentro de populações de Casearia sylvestris Sw. (Salicaceae) no Estado de São Paulo. Tese (Doutorado em Ciências Biológicas) – Instituto de Biociências, Universidade Estadual Paulista, Botucatu, 115 p. (Unpublished)., Silva et al. 2006Silva MAS, Ming LC, Pereira MAS, Bertoni BW, Batistini AP and Pereira PS. 2006. Phytochemical and genetic variability of Casearia sylvestris Sw. From São Paulo State Atlantic Forest and Cerrado populations. Rev Bras Pl Med 8: 159-166.). AGSDER was in an urban area subjected to pollutants and it may be correlated with its different chemical pattern.

As morpho-anatomical analyses, the chemical analyses by HPLC-DAD have to be applied to greater number of specimens from different environments or regions to give more conclusive results.

In conclusion, this work indicates that chemical analyses, mainly HPLC-DAD, besides morpho-anatomical analyses of paradermic sections and palisade index can be applied in distinction of C. sylvestris varieties for botanical or pharmaceutical purposes.

The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação para o Desenvolvimento da UNESP (FUNDUNESP), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Instituto Florestal da Secretaria do Meio Ambiente do Estado de São Paulo for support of the project.

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Publication Dates

  • Publication in this collection
    10 Nov 2013
  • Date of issue
    2013

History

  • Received
    19 Jan 2012
  • Accepted
    27 Feb 2013
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