inal chem ic is d t Babili et al., Med Chem (Los Angeles) 2017, 7:10 e r M y Medicinal chemistry DOI: 10.4172/2161-0444.1000468 ISSN: 2161-0444 t Research Article Open Access Pharmacognostic, Phytochemical and Antioxidant Studies of Hydnophytum formicarum L. Fatiha EL Babili1*, Amandine Guillouty2, Halova-Lajoie Barbora3, Caroline Vincent4 and Nathalie Sejalon-Delmas1,5,6 1Jardin Botanique Henri Gaussen, Université Paul Sabatier, Toulouse III – 2 rue Lamarck 31400 Toulouse, France 2Faculté des Sciences Pharmaceutiques – UPS Toulouse III – 35 Chemin des Maraîchers 31062 Toulouse, France 3Laboratoire de Chimie Bioinorganique Medicale LU 07, Universite Paul Sabatier, 118 Route de Narbonne Bat. 3SC, 31062 Toulouse, France 4Ecole Prératoire de Chimie, Lycée d'Enseignement Général 26, bd Déodat de Séverac - 31076 Toulouse, France 5Laboratoire de Recherche en Sciences Végétales (LRSV), Université de Toulouse, UPS, CNRS, 31326 Castanet-Tolosan, France 6ECOLAB, Université de Toulouse, CNRS, INPT, UPS, France Abstract The leaves of Hydnophytum formicarum Jack belonging to the family Rubiaceae, a tropical tree commonly found in eastern and southern Thailand, South Asia, Papua New Guinea and the Pacific Islands, were studied for these antioxidant compounds. Sterols, flavonoids and phenolic compounds appear to be good markers for this species. Chromatographic and histochemical techniques were used to analyze its secondary metabolites and to localize these molecules in the leaves of H. formicarum and its petiole. With histochemistry, we can locate the phenols in situ by an iron chloride reagent, which gives a black color, especially in fibers, hypodermis and xylem. Flavonoids are detected histochemically using a potassium hydroxide reagent to obtain a yellow coloration under visible light. Histochemical studies of H. formicarum showed a high concentration of natural antioxidants in the central cylinder, particularly in the vascular bundle, whereas condensed catechic tannins were mainly detected in collenchyma, fibers and also in xylem. The high levels of phenolic compounds and their localization in the conductive tissues allow us to explain the traditional medicinal use carried out in Thailand with this tree because, very often, compounds with antioxidant properties prove to be good anti-cancer agents. Keywords: Hydnophytum formicarum; Phenols; Antioxidants; flowers evolve in a few weeks in fleshy, orange fruits, containing only a Histolocalization few seeds that can germinate quickly. Introduction Materials and Methods After the immense infatuation that modern allopathy has Plant material succeeded, a strong return of interest for traditional medicine is noted. Leaves were collected from the investigated plant species grown in People increasingly want to know something about the plants that the tropical greenhouse of the botanical garden Henri Gaussen. Study surround them and especially their ancient medicinal uses. of morphology and anatomy foliar were identified by C. Chatelain and On the other hand, the rising concern for the preservation of our F. EL Babili and voucher specimen were deposited at the herbarium of environment and the rejection of drugs, having too many undesirable the botanical garden Henri Gaussen. effects, pushes plant remedies to the front of the stage. Plant drugs are Preparing slides gradually regaining their important role, through food supplements, in preserving health in both underdeveloped and industrialized Observations are based on microscopic studies of sectioned countries. The plant is indeed a true chemical factory of both primary and stained material of tissues transverse sections are prepared and secondary metabolites. It is these latter who are often involved with a sliding microtome (MSE) and stained in alun carmine-green in the biological properties of plants that man has always known to combination or Mirande reagent [1] during 2 to 3 minutes then washed exploit in health service, in other things. Screenings of the aqueous or with water. Following staining, the transverse sections are mounted on alcoholic drugs extracts are systematically carried out for the plants glass slides using glycerine gel. Powder observations were made using used traditionally in order to confirm and corroborate or invalidate Chloral hydrate solution R [2]. Observations were made with a LEICA the traditional uses described. From this, a phytochemical study can Microsystems DMLB microscope, and pictures were taken with Digital be initiated in order to locate the most active fraction of this anticancer Camera Power Shot S40 CANON photo-micrographic system. For the plant and its chemical profile. To carry out this work, a precise botanical description we have used some help books [3-5]. identification (morphological and anatomical) is necessary in order to avoid any mistake that could have serious consequences, as was the case in losing products. For this, our work focuses on the botanical, *Corresponding author: Fatiha EL Babili, Jardin Botanique Henri Gaussen, phytochemical and biological study of the leaves of a medicinal Université Paul Sabatier, Toulouse III–2 rue Lamarck 31400 Toulouse, France, Tel: +33561556611; E-mail: [email protected] myrmecophyte called Hydnophytum formicarum. Received October 07, 2016; Accepted October 17, 2017; Published October 23, Hydnophytum formicarum (Jack) is part of the epiphytic Rubiaceae 2017 family, found in Southeast Asia (Borneo, Java, Malaysia). It can reach Citation: Babili FE, Guillouty A, Barbora HL, Vincent C, Sejalon-Delmas N (2017) 50 cm and is provided with its stem base bulge also called “caudex”, Pharmacognostic, Phytochemical and Antioxidant Studies of Hydnophytum which is wide and has orifices that can house ants. formicarum L. Med Chem (Los Angeles) 7: 276-284. doi: 10.4172/2161- 0444.1000468 Its leaves, few and much spaced on the stem, are supple and of a Copyright: © 2017 Babili FE, et al. This is an open-access article distributed under light green colour. The odourless flowers, blooming in spring, are small the terms of the Creative Commons Attribution License, which permits unrestricted (<1 cm), white, sessile, generally grouped in the axils of the leaves. The use, distribution, and reproduction in any medium, provided the original author and source are credited. Med Chem (Los Angeles), an open access journal Volume 7(10): 276-284 (2017) - 276 ISSN: 2161-0444 Citation: Babili FE, Guillouty A, Barbora HL, Vincent C, Sejalon-Delmas N (2017) Pharmacognostic, Phytochemical and Antioxidant Studies of Hydnophytum formicarum L. Med Chem (Los Angeles) 7: 276-284. doi: 10.4172/2161-0444.1000468 Anatomical analysis Sugars: The leaf sections are immersed in sulfuric acid (95% VWR Prolabo) thymol reagent for 5 min with heating and then placed The dry materials were divided into appropriate sizes. Samples between slide and cover slip. They were observed using a microscope. of leaf were sectioned on a microtome in slices thick. Tissues were The sugars are pink colour. stained by alun carmine-green combination or Mirande reagent and finally mounted on glass slides using glycerine gel for observation. Phenols: Phenols are detected by iron chloride reagent (0.2M Some sections were not stained so that idioblasts and other deposits in 2-methyltetrahydrofuran – Sigma Aldrich). The sections were would not be destroyed or otherwise altered during processing. The placed for 5 min in this reagent. The tannins are coloured blue-black, samples of crude drug were powdered. Ten different slides from the the phenols are coloured green-black, the flavonoids are green and same powder were observed. Microchemical reactions were applied pyrogallol is coloured red or blue. with lactic reagent or Gazet reagent [6]. To study powders in order Starch: Starch is detected by iodine or Lugol reagent (Jeulin). The to reveal lignified elements such as wood fibres, sclerenchyma and sections were placed for 2 min in this reagent. The starches are coloured calcium oxalate crystals. All representative microscopic features were blue-black. recorded by colour photography. Flavonoids: The leaf sections are immersed in potassium hydroxide TLC analysis reagent (HKO Sigma Aldrich) for 5 min and then placed between slide An aliquot of methanol extract of Hydnophytum formicarum (40 and cover slip. They were observed using a microscope. The flavonoids μL) was directly deposited (as spots or bands) onto the TLC plates. are yellow. TLC plates were developed in a pre-saturated solvent chamber with Coumarins: Coumarins are detected by ammoniac or potassium toluene-ethyl acetate-formic acid-methylene chloride (10:12:6:12) hydroxide reagent (HKO was purchased from Sigma–Aldrich). The as developing reagents until the solvent front reached 1 cm from the sections were placed for 5 min in these reagents. Coumarins are top of plates. The developed TLC plates were then removed from the coloured bright yellow. chamber, and allowed to air-dry for 30 min. Each TLC plate was then monitored under visible and UV light at 254 and 366 nm. Catechic tannins: Tannins are detected by hydrochloric acid reagent. The sections were placed for 10 min in this reagent. Tannins Histolocalisation of metabolite studied on the leaves section are coloured red brown. On the basis of histochemical reactions due to the interaction Alkaloids: For alkaloids, transverse sections were immersed (15 between specific reagents and cellular metabolites, it is possible to carry min) in Dragendorff’s reagent (spray solution was purchased from out in situ