Anatomical analysis and phytochemical screening of Frangula rupestris (Scop.) Schur (Rhamnaceae)

42 (2): (2018) 231-239 Original Scientific Paper Anatomical analysis and phytochemical screening of Frangula rupestris (Scop.) Schur (Rhamnaceae) Jelena Arsenijević1✳, Milica Drobac1, Violeta Slavkovska2, Nada Kovačević1 and Branislava Lakušić2 1 University of Belgrade, Faculty of Pharmacy, Department of Pharmacognosy, Vojvode Stepe 450, 11221 Belgrade, Serbia 2 University of Belgrade, Faculty of Pharmacy, Department of Botany, Vojvode Stepe 450, 11221 Belgrade, Serbia ABSTRACT: Frangula rupestris and F. alnus are the only two species of the genus Frangula in the flora of the Balkan Peninsula. Frangula alnus is well-known for anthranoid content, and its stem bark and fruits are widely used as laxatives. Data on anatomy, plant metabolites, and potential use of F. rupestris are scarce. In this work we analysed anatomy of the stem and leaves and performed phytochemical screening of the bark and leaves of F. rupestris. Specific anatomical characteristics of the stem include the presence of large mucilage cavities in the bark and pith, as well as numerous parenchyma cells containing solitary or cluster crystals of calcium oxalate. The majority of leaf epidermal cells are filled with mucilage. In the main leaf vein there is parenchyma with numerous mucilage cavities and solitary or cluster crystals of calcium oxalate. The levels of flavonoids, total phenolics, and tannins in bark and leaves of plants from two localities were determined by spectrophotometric methods, and the results were compared with those obtained for bark of F. alnus. Bark and leaves of F. rupestris contained 2.68-3.03% and 2.22-3.77% total phenolics, 1.70-2.10% and 0.57-1.54% tannins, and 0.12-0.36% and 0.57-0.99% flavonoids, respectively. The conducted HPLC and LC-MS analyses of hydromethanol extracts of bark and leaves of F. rupestris revealed the presence of flavonols. The dominant compound in all extracts was hyperoside and its content, determined by HPLC, ranged from 30.40 to 82.03 mg/g. Swelling indices determined for 0.5 g of plant material of F. rupestris bark and leaves (5.8-11.4 and 5.8-13.8) were higher than that of the bark of F. alnus (4.4) and greater than those of the mucilaginous drugs Althaeae folium and Althaeae radix (4.7-4.8). The high swelling indices of F. rupestris bark and (especially) leaves suggest their potential use as bulk-forming laxatives. In addition, differences in the content of metabolites were observed in plants from different localities. Keywords: Frangula rupestris, stem, bark, leaves, anatomy, metabolites Received: 17 January 2018 Revision accepted: 19 March 2018 UDC: 582.782.1:581.192: 581.4 DOI: 10.5281/zenodo.1468339 INTRODUCTION The family Rhamnaceae comprises more than 500 species belonging to around 50 genera distributed throughout the world (Jovanović 1973). Several species of the genera Rhamnus L. (R. cathartica L., R. falax Boiss., R. purshiana DC.) and Frangula L. (F. alnus Mill.) are wellknown for anthranoid content, and their parts, i.e., stem correspondence: ✳ bark and fruits, are widely used as laxatives (Kovačević 2004; Hänsel & Sticher 2010). Frangula rupestris (Scop.) Schur (syn. Rhamnus rupestris Scop, R. wulfenii Hoppe, Frangula wulfenii Rchb.) and F. alnus are the only two species of the genus Frangula in the flora of the Balkan Peninsula (Tutin 1968). Frangula rupestris is an ascending or procumbent shrub up to 100 cm high. The young shoots are light-brown © 2018 Institute of Botany and Botanical Garden Jevremovac, Belgrade 232 vol. 42 (2) with inconspicuous lenticels. Leaves are 2-5 cm long, elliptical to suborbicular, with dentate or sometimes entire margins, pubescent on veins of the lower side. The flowers are small, light-green or yellowish, in umbellate inflorescences. The fruit is a round drupe, red initially, becoming purple to black during ripening. Frangula rupestris grows in warm, dry, limestone places, mainly in mountainous areas. It is found in the central and western regions of the Balkan Peninsula, spreading to northeastern Italy (Tutin 1968; Jovanović 1973). Frangula alnus bark (Frangulae cortex) is a wellknown herbal drug with longstanding use as an anthranoid laxative, and its anatomy and chemical composition have been thoroughly analysed (EMEA/ HMPC/76306/2006; Hänsel & Stitcher 2010). On the other hand, available data on metabolites of F. rupestris are limited to several reports, mainly treating anthranoid constituents of the stem bark and leaves (Sajc et al. 1999; Kovačević & Grubišić 2005; Kremer et al. 2012). Frangula rupestris bark from Serbia contained 0.03% of total anthranoid aglycons, determined by HPLC after acid hydrolysis, with aloe-emodin as the most abundant, followed by chrysophanol and emodin (Sajc et al. 1999). Low contents of anthranoids were also found in bark samples from Croatia, i.e., 0.26-0.54% of total glucofrangulins as determined by the spectrophotometric method (Maleš et al. 2010). In contrast to the bark of plants from Serbia, physcion was the principle anthranoid aglycon, emodin and chrysophanol were present in lower quantities, and aloe-emodin was below the limit of quantitation in the bark sample from Croatia, analysed by HPLC after acid hydrolysis (Kremer et al. 2012). The amount of anthranoids in F. rupestris leaves from Serbia was even lower than in the corresponding bark (0.01% of total aglycons, determined by HPLC after hydrolysis). Aloe-emodin was the dominant aglycon, and chrysophanol and emodin were less abundant (Sajc et al. 1999). There are sporadic data on the content of flavonoids in the bark of F. rupestris (Maleš et al. 2010; Kremer et al. 2012), but composition of the flavonoid fraction of neither bark nor leaves was investigated. Furthermore, data on the potential use and pharmacological effects of this species are lacking. With these facts in mind, we set out to investigate F. rupestris with the aim of establishing its potential as a herbal drug source. In that connection, we examined anatomy of stem and leaves and performed phytochemical screening that included HPLC and LC-MS analyses of flavonoids; spectrophotometric determination of flavonoids, total phenolics, and tannins; and determination of the swelling indices of bark and leaves. MATERIALS AND METHODS Plant material. Shoots of F. rupestris were collected from two localities in Montenegro, Luštica and Njeguši, Table 1. Plant material used in the study. Species Locality Luštica Frangula rupestris Njeguši Frangula alnus Goč Plant part Stem Leaves Stem Leaves Bark Voucher number 42962 BEOU 42970 BEOU 1465 HFF in August of 2015, whereas F. alnus shoots were collected in Serbia on Mt. Goč in June of 2005. Voucher specimens were deposited in the herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade – Faculty of Biology (BEOU); and in that of the Department of Botany, University of Belgrade – Faculty of Pharmacy (HFF) (Table 1). Analysis of stem, petiole, and leaf anat (...truncated)