Anales de Biología 43: 93-99, 2021 ARTICLE http://dx.doi.org/10.6018/analesbio.43.09 ISSN/eISSN 1138-3399/1989-2128 Microscopic, histochemical and preliminary phytochemical characterization of leaves of Trema micrantha (L.) Blume Cledson dos Santos Magalhães1, Rafaela Damasceno Sá1, Solma Lúcia Souto Maior de Araújo Baltar2 & Karina Perrelli Randau1 1 Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, 50740-321, Recife, Pernambuco, Brazil. 2 Universidade Federal de Alagoas, 57309-005, Arapiraca, Alagoas, Brazil. Resumen Correspondence Caracterización microscópica, histoquímica y fitoquímica preilmi- K.P. Randau nar de las hojas de Trema micrantha (L.) Blume E-mail: [email protected] Para enriquecer el enriquecer el conocimiento sobre Trema mi- Received: 22 November 2020 crantha (L.) Blume, esta investigación tuvo como objetivo realizar Accepted: 15 April 2021 la caracterización anatómica, histoquímica y fitoquímica de las ho- Published on-line: 30 May 2021 jas de la especie. Se realizaron cortes transversales del pecíolo y limbo, así como cortes paradérmicos del limbo, analizados en mi- croscopía óptica y polarizada. Se utilizaron diferentes reactivos para el análisis histoquímico. Se han descrito estructuras anatómi- cas que proporcionan un diagnóstico detallado de las especies es- tudiadas. La histoquímica mostró la presencia de metabolitos es- enciales (flavonoides, taninos, entre otros) para la especie y me- diante análisis SEM-EDS se confirmó que los cristales están com- puestos por oxalato de calcio. El análisis fitoquímico permitió la identificación de mono y sesquiterpenos, triterpenos y esteroides, entre otros. El estudio proporcionó datos sin precedentes sobre la especie, ampliando la información científica de T. micrantha. Palabras clave: Cannabaceae; Microscopía; Farmacobotánica. Abstract In order to enrich the knowledge about Trema micrantha (L.) Blume, this research aims to perform the anatomical, histochemical and phytochemical characterization of the leaves of this species. Cross sections of the petiole and limbus were made, as well as paradermic sections of the limbus, analyzed in optical and polar- ized microscopy. Different reagents were used for histochemical analysis. Anatomical structures have been described and provide a detailed diagnosis of the species studied. Histochemistry showed the presence of essential metabolites (flavonoids, tannins, among others) for the species and through SEM-EDS analysis it was con- firmed that the crystals are composed of calcium oxalate. Phyto- chemical analysis allowed the identification of mono and sesquiter- penes, triterpenes and steroids, among others. The study provided unprecedented data on the species, expanding the scientific in- formation of T. micrantha. Key words: Cannabaceae; Microscopy; Pharmacobotany. 94 dos Santos Magalhães et al. Anales de Biología 43, 2021 Introduction the exact localisation of its compounds. Materials and methods Trema micrantha (L.) Blume, popularly known as candiúba, candiúva, crindiúva, grandiúva, pau- Expanded leaves of adult specimens of T. micran- pólvora, pau-de-bugre, among others, is a tree of tha, located between the third and fifth node, were the family Cannabaceae (Moressi et al. 2014). collected in the city of Janga, in the community of The description of this family had been quite Tururu, Pernambuco, Brazil. Voucher specimen nº questionable, since traditionally their genera were 88149 was deposited in the Herbarium Dárdano included in Urticaceae or Moraceae, or it was con- de Andrade Lima, of the Instituto Agronômico de fused with the complex Ulmaceae-Celtidaceae- Pernambuco (IPA). Cannabaceae (Martins & Pirani 2009). However, For the anatomical characterization, the mate- due to the accumulation of studies in phylogeny rial was fixed in FAA50 (formaldehyde, acetic (Yang et al. 2013), the previously used Canna- acid and ethyl alcohol 50%, 1:1:18 v/v) (Johansen baceae classification was expanded, and the gen- 1940). Cross-sections of petiole and leaf blade era Celtis L. and Trema Lour. were included in the were obtained by the classical sectioning method family. Before that, these genera belonged to the using a common razor blade. For the leaf blade, Ulmaceae family (Martins & Pirani 2009). Thus, paradermic sections were also performed on the the family Cannabaceae presents 10 genera and adaxial and abaxial faces. Subsequently, the sec- estimates include about 110 species (APG IV tions were submitted to the treatment with a solu- 2016). In Brazil, only the genera Celtis and Trema tion of sodium hypochlorite (50%) for clarifica- occur, with five and one species, respectively tion (Kraus & Arduin 1997). After washing in dis- (Machado & Silva 2020). tilled water, the cross-sections were stained Native from Brazil, T. micrantha can reach according to the technique described by Bukatsch 1.5-10 m in height (Viana & Gil 2018), inhabiting (Bukatsch 1972), with safranin and Astra blue; the different vegetation communities (seasonal paradermic sections were stained with methylene forests, riparian forests, cerrado and carrasco, as blue (Krauter 1985). Thereafter, all sections were well as in xerophytic vegetation, such as Caa- mounted on semipermanent slides, following tinga) (Martins & Pirani 2009, Machado & Silva usual procedures in plant anatomy (Johansen 2020). 1940, Krauter 1985). A light and polarization In Brazil, T. micrantha is commonly used in microscope (Leica DM750M), coupled with a dig- the recovery of degraded or burned soils, as well ital camera (Leica ICC50W), were used to analyse as in traditional medicine (Lorenzi 2000, Moressi the slides, through which were obtained images et al. 2014, Oliveira et al. 2018). Its leaves are tra- processed by software (LAS EZ). ditionally used in teas indicated for the treatment Histochemical tests were performed in cross- of syphilis, rheumatism (Lorenzi 2000), diabetes sections of fresh leaf blades, obtained by the same (Schoenfelder et al. 2006) and to combat poison- method used for anatomical characterization. The ing from snakebite (Thomas 2001). Extracts from following reagents were used to indicate the pres- T. micrantha leaves showed analgesic, antiinflam- ence of the metabolites: potassium dichromate matory (Barbera et al. 1996) and hypoglycemic (10%) for phenolic compounds (Johansen 1940); activities in animals (Schoenfelder et al. 2006). Dragendorff for alkaloids (Sass 1951), vanillin Taken all this into consideration, T. micrantha chloridric for tannins (Yoder & Mahlberg 1976); has great potential for medicinal exploitation. Sudan III for lipophilic compounds (Mace & However, there is still few information in the liter- Howell 1974); antimony trichloride for triterpenes ature on its anatomical characteristics and its and steroids (Mace et al. 1974); Nadi reagent for chemical compounds. A recent research has essential oils (David & Carde 1964); Lugol for focused primarily on histolocalization tests on T. starch (Johansen 1940); phloroglucinol for lignin micrantha trichomes (Nascimento 2017). There- (Johansen 1940) and hydrochloric acid (10%) to fore, this study aimed to expand the scientific establish the nature of the crystals (Jensen 1962). information on the species, through the anatomi- Controls were performed in parallel to the histo- cal, histochemical and phytochemical characteri- chemical tests and semipermanent slides were zation of its leaves. Thus, the data obtained assist prepared containing cross-sections (Johansen in the correct identification of T. micrantha and on 1940, Mace & Howell 1974). The analysis of the Anales de Biología 43, 2021 Microscopic and chemical traits of Trema micrantha 95 slides was conducted on images, using Toup View bundle, arranged in an open arch shape turned fac- Image software, obtained by a digital camera cou- ing the adaxial face (Fig. 1A). Druses are found in pled to a light microscope (Alltion). the parenchyma and phloem (Figs. 1C and 1D). For analysis of the elemental composition of The leaf blade, in frontal view, presents cells the crystals, cross-sections of fresh leaf blades of straight contour in the adaxial face (Fig. 2A) were fixed in Karnovsky solution for at least 2 and of slightly sinuous contour in the abaxial face hours. Subsequently, samples were washed three (Fig. 2B). It is classified as hypostomatic, with times with 1M sodium buffer, post-fixed with anomocytic stomata (Fig. 2B). Non-glandular and 0.5% osmium tetroxide and washed again with glandular trichomes are observed in both faces 1M cacodylate buffer and with distilled water. (Figs. 2A and 2B). They are similar to those found After dehydration in ethanol series, the material in the petiole. was submitted to critical point drying (Bal-Tec In cross-section, the midrib shows a plan-con- CPD 030), mounted on stubbs and sputter-coated vex contour, presenting uniseriate epidermis, cov- with gold (Haddad et al. 1998). Chemical micro- ered by a slightly thick cuticle (Fig. 2C). Subepi- analyses by Energy Dispersive Spectroscopy dermal angular collenchyma has about four layers (EDS) were performed with an X-ray detector (Fig. 2D). As in the petiole, the vascular bundle is coupled to a scanning electron microscope (TES- collateral, closed and arranged in the shape of an CAN – Mira 3 SEM) at the Laboratório de Micro- arch (Fig. 2D). Druses are found in the scopia Eletrônica of the Departamento de Física parenchyma and phloem (Figs. 2E and 2F). of the Universidade Federal de Pernambuco The mesophyll is dorsiventral, with palisade (UFPE).