Available online on www.ijppr.com International Journal of Pharmacognosy and Phytochemical Research 2016; 8(7); 1121-1131 ISSN: 0975-4873 Research Article Hepatoprotection and Antioxidant Activity of Gazania longiscapa and G. rigens with the Isolation and Quantitative Analysis of Bioactive Metabolites Samar Y Desoukey1*, Wafaa M El Kady2, Abeer A A Salama3, Eman G Hagag4, Siham M El-Shenawy3, M A El-Shanawany5 1Pharmacognosy Department, Faculty of Pharmacy, Minia University, Minia, Egypt 2Pharmacognosy Department, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University in Egypt, New Cairo, Egypt. 3Pharmacology Department, National Research Centre, Giza, Egypt. 4Pharmacognosy Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt. 5Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt. Available Online:12th July, 2016 ABSTRACT Gazania longiscapa and G. rigens are two species belonging to family Asteraceae. The present study aimed the isolation of the main active constituents from the methanol extracts using different chromatographic methods and their identification using different spectroscopic techniques, beside the quantitation of some biologically important active constituent as rutin using HPLC technique, together with estimation of total polyphenolic content calculated as gallic acid and estimation of total flavonoid content calculated as rutin using UV technique. Concomitantly the determination of the antioxidant and hepatoprotective activity of the total methanol extracts of the aerial parts of G. longiscapa and G. rigens. This work resulted in the isolation of 4 flavonoids (Apigenin, Luteolin, Luteolin 7-O-β-D-glucopyranosid, Apigenin 7-O-β-D- glucopyranosid), 3 phenolic acids (Caffeic acid, Chlorogenic acid and 3,5- di- O-caffeoylquinic acid) from G. longiscapa for the first time; these 3 phenolic acids were also isolated from G. rigens, together with one flavonoid (rutin), The quantitative determination of the methanol extracts showed that G. longiscapa is a richer source of phenolic acids than G. rigens and both Gazania species are valuable sources of rutin beside having hepatoprotective and antioxidant activity. Keywords: Gazania, flavonoids, phenolic acids, hepatoprotective, antioxidant. INTRODUCTION MATERIALS Genus Gazania belongs to family Asteraceae, a rich family Plant material with valuable medicinal plants, and revealed a rich harvest G. longiscapa and G. rigens aerial and underground parts of biologically active principles1. Gazania longiscapa DC. were collected from the plantation of the Ministry of and G. rigens L. are native to South Africa, perennial or Agriculture located in the Fifth Settlement, New Cairo, rarely annual and cultivated for their ornamental value2. Egypt in June 2013 during the flowering stage. Few reports were traced on the chemical constituents and Authentication of the plant was performed by Prof. Dr. the biological activities of Gazania speciesdemonstrating Abd Alsalam El Noiehy, Prof. of Plant Taxonomy, Botany the presence of flavonoids, coumarins, polyphenolic acids department, Faculty of Science, Ain Shams University, as well as terpenes and sterols3-6. From the biological point Cairo, Egypt. Voucher specimens for each species were of view, it was found that other species as G. nivea have kept in the department of Pharmacognosy, Faculty of hepatoprotective and antioxidant activities7, while Pharmacy, Future University in Egypt (FUE). antimicrobial activity was observed in G. rigens8. This Materials for chromatographic study: prompted us to investigate the bioactive constitutes of both Column chromatography; microcrystalline cellulose G. longiscapa and G. rigens; beside the quantitation of (Merck, Darmstadt, Germany), polyamide (Fluka, some important biologically active constituent as rutin Switzerland), sephadex LH-20 (Fluka, Switzerland), silica using HPLC technique and estimation of total phenolic and gel for column chromatography (CC, E. Merck, flavonoid using UV technique; together with the Darmstadt, Germany). For paper chromatography; determination of the hepatoprotective and antioxidant Whatman no. 1 and Whatman no. 3 sheets (Whatman Ltd, activity of the total aqueous methanol extracts of the aerial Maidstone, Kent, England). For thin layer parts (without the flower heads) of both Gazania species. chromatography; pre-coated cellulose plates (20 × 20 cm) E. Merck, Darmstadt, Germany. The pure compounds *Author for Correspondence: [email protected] Samar et al. / Hepatoprotection and Antioxidant… were visualized by spraying with Naturstoff reagent9 9:00 and 15:00 h. Animal procedures were performed in [(a)1% diphenyl boryloxyethanolamine in ethanol, (b) 5% accordance with the Ethics Committee of the National polyethylene glycol 400 in ethanol, heating the dry Research Centre and followed the recommendations of the chromatogram at 120°C for 10 min. and visualizing under National Institutes of Health Guide for Care and Use of UV light (365 nm)] and FeCl3 (1% in ethanol). Solvent Laboratory Animals. systems used were S1 (15% aqueous HOAc), S2 (n-BuOH: Drugs, Chemicals and Diagnostic kits: HOAc: H2O (4:1:5)). The NMR spectra were recorded at Paracetamol (EIPICO, Egypt) 300, 400 and 500 (1H) and 75, 100 and 125 (13C) MHz, on Silymarin (SEDICO, pharmaceutical Co., 6 October City- a Varian Mercury 300, a JEOL GX-400 and a Bruker Top Egypt). Spin 3.0 Software on the Upgraded 500 MHz DPPH [2, 2-Diphenyl-1-picrylhydrazyl radical] (Sigma, Spectrometer. The results were reported as δ ppm values USA). relative to TMS in the convenient solvents. ESI-MS Diagnostic kits ALT (alanine aminotransferase) and AST analyses were run on LCQ (Finnigan MAT, Bremen, (aspartate aminotransferase) according to Reitman and Germany) and LCQ-FT-MS mass spectrometers (Thermo Frankel[11] All kits depend on colorimetric method and Electron 400, Waltham, USA). UV analyses for pure were obtained from Biodiagnostic Co., Egypt. samples were recorded on MeOH solutions and with different diagnostic UV shift reagents on a (Shimadzu METHODS 1800, Germany) spectrophotometer. Methods are done after approval of the research ethics Materials for quantitation of rutin using HPLC committee; the approval form has serial no. REC-FPSPI- High performance (pressure) liquid chromatography 2/7. [HPLC] apparatus Isolation and identification KNAUER Smart Line High-performance liquid Extraction of aerial parts of G. longiscapa & G. rigens chromatography (HPLC) including: Smart line pump 100 The air-dried powdered aerial parts of G. longiscapa (1 V5010, an ultraviolet detectorV2139, Injection switching Kg) and G. rigens (0.6 Kg) were separately exhaustively valves V7452, Smart line degasser V7620, Smart line extracted with 80% aqueous methanol. The residues left column oven 4050 V7335, Smart line RI detector V7607 after evaporation of the solvent were (250 g) & (140 g) (KNAUER, Germany). respectively, successively pre-purified with CHCl3 under Methanol HPLC grade (Scharlau, Spain). reflux (2 L x 2, 50°C) and solvent evaporated under Acetonitrile HPLC grade (Scharlau, Spain). vacuum, leaving 80 g of CHCl3 extract and 160 g of Water HPLC grade (Scharlau, Spain). methanol extract from G. longiscapa and 45 g of CHCl3 Materials for estimation of total polyphenolics and extract and 90 g of methanol extract from G. rigens. The flavonoid content methanol extracts were precipitated from H2O with excess UV- visible spectrophotometer (Shimadzu 1800, methanol (1:10) for desalting. The desalted residues Germany). obtained from the filtrates (115 g) & (75 g) respectively, Reference compound for authenticated standard (gallic were each suspended in water and chromatographed on acid & rutin) were obtained from E. Merck, Darmstadt, polyamide S column (650 g, 120 x 5 cm) using a step- Germany; were used to construct linear standard curves gradient of H2O-MeOH. Aluminum Chloride reagent for assay of flavonoids (Peach Fractionation of G. longiscapa methanol extract and Tracey, 1955); 0.1 M Aluminum Chloride prepared by Five major collective fractions (I - V) were obtained from dissolving 2.4 g/100 ml distilled water. the polyamide S column, monitored by comparative paper Folin Ciocalteu reagent [10] obtained from LOBA chemie chromatography, TLC cellulose and UV-light. Fraction I PVT, Ltd. India. was found to be polyphenolic-free (NH3vapour and FeCl3 Materials for pharmacological study spray reagent/PC). Fraction II; was chromatographed on a Extract preparation consecutive microcrystalline cellulose column using Aerial parts of the two Gazania species (without the flower gradient aqueous MeOH to give compound Gl (1) (15 mg). heads) were separately, air dried, powdered and extracted Purification of fraction III was achieved by sephadex LH- with 80% methanol till exhaustion. The obtained methanol 20 column using gradient aqueous MeOH to afford Gl (2) extracts were evaporated under reduced pressure and the (25 mg) and Gl (3) (18 mg). Fraction IV has 2 major obtained residues were exhaustively extracted with CHCl3. separate spots which were best viewed on pre-coated The remaining methanol fractions were used for biological cellulose plates using S1 as developer, where preparative investigations. Whatmann No. 3 was used with S1 as developer for Animals separation and isolation of Gl (4) (5 mg) and Gl (5) (7 mg). Wiser male rats, weighing from 125-150 g were used Fraction V was chromatographed on silica columns using throughout the experiments. Rats used for the CH2Cl2/MeOH with gradual