Revista Brasileira de Farmacognosia 29 (2019) 11–16 ww w.elsevier.com/locate/bjp Original Article Anatomy, histochemistry and oxalic acid content of the leaflets of Averrhoa bilimbi and Averrhoa carambola a a,b a c c Rafaela D. Sá , Alex L. Vasconcelos , Andréa V. Santos , Rafael J.R. Padilha , Luiz C. Alves , a a,∗ Luiz A.L. Soares , Karina Perrelli Randau a Laboratório de Farmacognosia, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil b Faculdade Pernambucana de Saúde, Recife, PE, Brazil c Setor de Microscopia Eletrônica, Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Recife, PE, Brazil a b s t r a c t a r t i c l e i n f o Article history: Averrhoa bilimbi L. and A. carambola L., Oxalidaceae, are the only two species of the genus Averrhoa L. Their Received 5 June 2018 leaves are widely used in folk medicine as an adjuvant in the treatment of diabetes. Some species may Accepted 19 September 2018 contain, for example, calcium oxalate crystals, which may lead to risk in its use when there is predispo- Available online 11 October 2018 sition of individuals with reduced renal activity. Therefore, there are still few studies on the content of oxalic acid present in them, highlighting the importance of this investigation. The objective of this work Keywords: was to conduct a comparative anatomical and histochemical study between the species and determining Bilimbi its content of oxalic acid. Semipermanent histological slides were prepared, following common plant Carambola anatomy procedures, for analysis of the leaflets in optical microscopy, polarization and scanning elec- Polarization microscopy tron microscopy coupled with energy-dispersive X-ray spectrometry. To determine the total, soluble and Oxalic acid insoluble oxalate content was used titration with potassium permanganate. The anatomical characteri- zation allowed identifying the characters useful in the differentiation of the species. The histochemistry revealed the location of the metabolites. Chemical microanalyses demonstrated that the crystals are of calcium oxalate. A. carambola presented the highest levels of total oxalate and soluble oxalate. The study assists in the identification and quality control of A. bilimbi and A. carambola and brings new data on its oxalic acid content, which are important, in view of the medicinal use of the species. © 2019 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction The leaves of these species are widely used in folk medicine for diabetes (Messias et al., 2015), disorders of the nervous system, The Oxalidaceae R. Br. family comprises eight genera and about colic (Rodrigues and Andrade, 2014), against urinary, kidney and 601 species (The Plant List, 2013), distributed in the tropical and liver diseases (Albuquerque et al., 2007; Agra et al., 2008). Scientific subtropical regions of the world (Souza and Lorenzi, 2012). In Brazil, studies have proven the antidiabetic potential of leaf extracts from the family is represented by the genera Averrhoa L., Biophytum these plants (Tan et al., 2005; Ferreira et al., 2008; Daud et al., 2013; DC. and Oxalis L., comprising 103 species found in all regions of Putra et al., 2017). the country. The genus Oxalis is the most numerous, while the The use of medicinal plants is commonly reported in the litera- genus Averrhoa has only two species in Brazil flora, A. bilimbi L. ture as an adjuvant in the treatment of diabetes (Santos et al., 2012). and A. carambola L. (Abreu and Fiaschi, 2015). A. bilimbi is a peren- However, its popular use is not always done correctly in relation nial tree of 5–9 m in height, with little dense and low crown. In to the quality of the vegetable raw material and may still pose a Brazil, it is popularly known as “azedinho”, “bilimbi”, “biri-biri” and serious risk to the health of the population, because they are com- “limão-caiena”. A. carambola is also a perennial tree, but its crown is posed of complex mixtures of substances (Leal and Tellis, 2015). elongated and dense, with 5–10 m in height. It is popularly known Some species may contain, for example, calcium oxalate crystals as carambola and “fruta-estrela” (Lorenzi et al., 2015). (Franceschi and Horner, 1980) and there are still few studies on the content of oxalic acid present in them, which may lead to risk in its use when there is predisposition of individuals with reduced renal activity (Nakata, 2012). Some studies have shown that the absorp- ∗ tion and excretion of a rich diet in oxalate can be considered as an Corresponding author. E-mail: [email protected] (K.P. Randau). https://doi.org/10.1016/j.bjp.2018.09.005 0102-695X/© 2019 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 12 R.D. Sá et al. / Revista Brasileira de Farmacognosia 29 (2019) 11–16 important factor in the development of kidney stones (Siener et al., digital camera (Leica ICC50W) coupled to an optical and polarized 2003; Holmes and Assimos, 2004). microscope (Leica DM750M). Therefore, for the determination of the quality parameters For the anatomical characterization in Scanning Electron related to these species and to increase the knowledge about the Microscopy (SEM), samples of fresh leaflets were fixed in 2.5% chemical compounds present in them, the objective of the study glutaraldehyde (buffered with 0.1 M sodium cacodylate) and post- was to perform an anatomical and histochemical study of the fixed using 2% osmium tetroxide solution (buffered with 0.1 M leaflets of A. bilimbi and A. carambola, besides determining its oxalic sodium cacodylate). The material was submitted to dehydration acid content. in ethanol series and to critical point drying (Bal-Tec CPD 030), mounted onto SEM stubs, using double-sided adhesive tape and sputter-coated with gold (Leica EM SCD 500) (Haddad et al., 1998). Materials and methods The samples were examined with a scanning electron microscope (Quanta 200 FEG) in the Centro de Tecnologias Estratégicas do Plant material Nordeste (CETENE). The botanical material of Averrhoa bilimbi L., and A. carambola L., Oxalidaceae, was collected in February 2017, by Rafaela Dam- asceno Sá and Alex Lucena de Vasconcelos, in the metropolitan ◦ ◦ Histochemical characterization region of Recife, Pernambuco, Brazil (8 04 03 S, 34 55 00 W). After drying according to standard herbarium techniques (Bridson and Histochemical tests were made on cross-sections of fresh Forman, 1999), vouchers were deposited in the Herbarium Dár- leaflets obtained by hand, using a common razor blade (Johansen, dano de Andrade Lima of the Instituto Agronômico de Pernambuco, 1940). The specific reagents used were: potassium dichromate 10% under registration number 89750 for A. bilimbi and 89332 for A. for phenolic compounds (Gabe, 1968), vanillin chloridric for tan- carambola. nins (Mace and Howell, 1974), antimony trichloride for triterpenes and steroids (Mace et al., 1974), Dragendorff’s reagent for detect- ing alkaloids (Yoder and Mahlberg, 1976), Sudan III for lipophilic Anatomical characterization substances (Sass, 1951), phloroglucinol for lignin (Johansen, 1940), Lugol’s iodine reagent for starch (Johansen, 1940) and hydrochlo- For the anatomical characterization in optical microscopy and ric acid 10% to establish the nature of the crystals (Jensen, 1962). polarized light microscopy were used mature leaflets from the Controls were performed in parallel with the tests. Semipermanent third to five node from three specimens of each species, fixed in histological slides were prepared containing the cross-sections and FAA (formaldehyde, acetic acid and ethyl alcohol 50%) (Johansen, were analyzed in optical microscope (Alltion microscope). 1940). Cross-sections were obtained by hand, using a common razor blade, at the middle region of the leaflets. Paradermal sec- tions were also performed on the adaxial and abaxial faces. The sections were subjected to decolorization with sodium hypochlo- rite solution 50% (Kraus and Arduin, 1997). Cross-sections were Analysis of the elemental composition of crystals stained with safranin and astra blue (Bukatsch, 1972) and parader- mal sections were stained with methylene blue 1% (Krauter, 1985). Cross-sections of leaflets were processed following the same Semipermanent histological slides were prepared containing the methodology described for the analysis in SEM (Haddad et al., sections, following common plant anatomy procedures (Johansen, 1998). The chemical microanalyses by Energy Dispersive Spec- 1940; Sass, 1951). The analysis of the semipermanent histological troscopy (EDS) were done with X-ray detector attached to the slides were conducted on images in software (LAS EZ), obtained by Zeiss-EVO-LS15 scanning electron microscope. Fig. 1. Photomicrographs of the leaflets surface view of Averrhoa bilimbi and A. carambola. A, C, E, G, I, K: Averrhoa bilimbi. B, D, F, H, J, L: Averrhoa carambola. A–D: optical microscopy. E–L: scanning electron microscopy. A,B,E,F,I,J: surface view of the abaxial face. C, D, G, H, K, L: surface view of the adaxial side. ew: epicuticular wax; st: stomata; tr: trichome. Bars: A, B, C, D: 50 ␮m; E, F, G, H: 10 ␮m; I, J, K: 100 ␮m; L: 25 ␮m. R.D. Sá et al. / Revista Brasileira de Farmacognosia 29 (2019) 11–16 13 Determination of oxalic acid ◦ Leaflets were oven dried at 60 C for 48 h and powdered in a blender. For the determination of total oxalate and soluble oxalate, 0.5 g of the powder of the leaflets were weighed into 100 ml erlen- meyer flasks and 50 ml of 2 N hydrochloric acid (total oxalate) or distilled water (soluble oxalate) were added. The flasks were ◦ AB placed in a shaking water bath at 80 C for 30 min.