Dalia gh. Alrekabi et al /J. Pharm. Sci. & Res. Vol. 10(9), 2018, 2242-2248 Phytochemical Investigation of Sonchus Oleraceus (Family:Asteraceae) Cultivated in Iraq, Isolation and Identification of Quercetin And Apigenin Dalia gh. Alrekabi1 , Maha N. Hamad2, 1Department of Pharmacognosy and Medicinal Plants , Baghdad College of Pharmacy, Baghdad, Iraq. 2Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Objective:The aim of our study was to investigate chemical constituents of leaves of Sonchus oleraceus grown naturally in Iraq because in Iraq no phytochemical investigation had been previously made in Iraq. Methods: Leaves of S . oleraceus were macerated in absolute methanol for 2 days, filtered and fractionated by petroleum ether, chloroform, ethyl acetate, and n-butanol. The ethyl acetate fraction was analyzed by high-performance liquid chromatography (HPLC) and high- performance thin-layer chromatography (HPTLC) for its phenolic acid contents and phenolic acid were isolated from this fraction and identified by gas chromatography/mass spectrometry, infrared, ultraviolet, HPLC, and HPTLC. Results: The different chromatographic and spectroscopic results revealed the presence of flavnoids. Conclusion: The results of the current study showed the presence of quarcetin in the ethyl acetate fractionand and apigenin in n-butanol fraction of S. oleraceus. Keywords: Sonchus oleraceus, flavnoids , High-performance liquid chromatography, High-performance thin-layer chromatography. INTRODUCTION flavonoids isolated from S. oleraceus showed antioxidant activity Herbal products are complex mixtures of organic chemicals that [15]. can come from any raw or processed part of a plant, including Flavonoids are naturally occurring compounds as secondary leaves, stems, flowers, roots and seeds [1]. they usually contain a metabolites in plants functioning as a plant's physiological number of pharmacologically active compounds. survival by protecting against fungal infections and UV radiation The plant Sonchus oleraceus belongs to the daisy family [16,17]. (Asteraceae)[2] is an upright, annual herb with simple branches. A Apigenin: A Flavones are the most common substances in the special feature of these sow thistle is that the largest part of the flavonoid group with antitumor activity.[18], anxiolytic plant is smooth and bare, without ant hair or bristles. The stems properties[19], anti- inflammatory[20,21], antioxidant, and are hollowed out and have a milky sap and their lower part gets a anticarcinogenic properties[22,23], antiviral, antibacterial, purple color later in the spring. The leaves vary according to age, insecticidal, apoptotic, anti- aging, and antioxidant the old leaves are long-stalked and deeply lobed. The color of the properties[24].Apigenins play an important role in human cancer: leaves varies from light green to greenish blue and may have a breast cancer[25], Cervical Cancer[26], Colon Cancer[27], jagged outline, but on spikes or hairs. The fruits are simple Hematologic Cancer[28], Lung Cancer[29]. achenes, brownish in color and oval / oblong in shape. Plant has Quercetin is one of the important bioflavonoids which has anti- various uses such as cancer, digestion, laxative, emollient, blood inflammatory[30], antihypertensive[31], anticarcinogenic purifier and also of strain of Rajsthan used as liver tonic[3]. properties[32], antibacterial[33] and antiviral activity[34] S. oleraceus is native to Europe, North Africa and West Asia. It vasodilator effects, antiobesity, antihypercholesterolemic and has spread to North and South America, India, China, southern antiatherosclerotic activities.[35,36]. Australia[4]. In New Zealand[5], S. oleraceus is estimated as a green vegetable that is usually cooked with meat. The genus Sonchus comprises about 60 species, and three of them have METHODS become common weeds around the world. These are S. arvensis, Collection of plant materials perennial Saw thistle and the two annual species S. oleraceus, The plant was collected from Almasyab -Babil - Iraq in May- common Saw thistle and S. asper, prickly Saw thistle. The main 2017. The plant was identified and authenticated by Dr .Ibrahim constituents of Sonchus L. were terpenes, steroids, flavones, Saleh Abbas. coumarins , etc. It has hepatoprotective activity, antitumor effect, Equipment and chemical cardiovascular therapy, etc.[6]. The instruments used were rotary evaporator (BȔCHI Rotavapor Ten compounds were isolated and elucidated as luteolin(Ⅰ R-205, Swiss), sonicator (Baranson Sonifier, USA), high- ),luteolin-7-O-β-D-glucoside(Ⅱ),apigenin(Ⅲ),apigenin-7-O-β-D- performance liquid chromatography (HPLC) (Sykam, Germany), glucuronide methyl ester(Ⅳ),apigenin-7-O-β-D-glucuronide ethyl and high-performance thin-layer chromatography (HPTLC) (Eike Reich/CAMAG-Laboratory, Switzerland). All chemicals and ester(Ⅴ),apigenin-7-O-β-D-glucopyranuronide(Ⅵ),germanicyl solvents used were of analytical grade and obtained from Riedel- Ⅶ Ⅷ acetate( ),3β-hydroxy-6β,7α,11β-H-eudesm-4-en-6,12-olide( de Haen, Germany, except trifluoroacetic acid, and methanol ),oleanolic acid(Ⅸ) and 1-cerotol(Ⅹ) from Sonchus oleraceus.[7]. which are HPLC grade purchased from Sigma-Aldrich, Germany. Caffeic acid is a type of polyphenol, -7-glucoside and apigenin-7- The standard quercetin and apigenin were purchased from glucoside. In addition, flavonoids (luteolin, apigenin, kaempferol, Chengdu Biopurify Phytochemicals, China (purity >97). Thin- and quercetin) and their glucoside derivatives were identified layer chromatography (TLC) aluminum plates pre-coated with from whole plant extracts Recently, caftaric acid has also been silica gel 60 F 254 (100 mm×100 mm, 0.2 mm thick) used were identified from leaf extracts [8]. S.oleraceus L. (family obtained from E. Merck Ltd., India. Asteraceae) is known for its high content of antioxidants and Extraction antioxidant activity [9,10,11,12,13].The amount of polyphenol Leaves of S. oleraceus were thoroughly washed and dried in the and the antioxidant activity in plants depend on environmental shade. The dried plant was pulverized in a mechanical grinder. factors such as the growing season and location [14]. Some 500g S. oleraceus leaf powder were macerated in methanol for 3 2242 Dalia gh. Alrekabi et al /J. Pharm. Sci. & Res. Vol. 10(9), 2018, 2242-2248 days and filtered, and the filtrate was evaporated to dryness using model:S2100 Quaternary Gradient pump ,Column Oven a rotary evaporator under vacuum. 15 g of the residue was modl:S4115,Fraction Collector Model=Frac-950. suspended in water and then fractionated by partitioning with petroleum ether, chloroform, ethyl acetate and n-butanol (100 ml HPTLC analysis x 3) for each fraction. The first three organic layers were dried The ethyl acetate fraction was also analyzed for its flavonoid and over anhydrous sodium sulfate, filtered and evaporated to dryness. phenolic acid contents using HPTLC (Eike Reich / CAMAG Laboratory, Switzerland) using silica gel GF254 plates prepared Preparations of standards and samples for analysis in a mobile phase from chloroform: acetone: formic acid (75 Standard solutions for the quarcetin and apigenin HPLC were :16.5: 8.5) at 280 and 366 nm wavelengths. prepared by dissolving 0.04 mg in 1 ml HPLC HPLC grade. Dried samples were prepared for HPLC analysis by dissolving in Identification of isolated flavonoids derivatives methanol and sonicating at 60 ° C for 25 minutes at 60 ° C, The isolated flavonoids derivatives were identified by different followed by centrifugation at 7500 rpm for 15 minutes. The clear spectroscopic and chromatographic techniques listed below: supernatant of each sample was evaporated in vacuum. Residues • Gas chromatography/mass spectrometry (GC/MS): The GC/MS were individually resuspended in 1 ml HPLC grade, homogenized instrument used was GC/MS-QP 2010 ultra: SHIMADZU/GC GC using a vortex mixer and passed through a 2.5 μm disposable filter 2010 Plus. and stored at 4 ° C for further analysis. 20 μl of the sample was • Conditions used for GC/MS analysis are the followings: injected into the HPLC system for analysis. • Column temperature: 50.0°C Standards used for HPTLC analysis (quercetin and apigenin) were • Injection temperature: 280.0°C prepared by dissolving 1 mg of each standard in 1 ml of methanol • Injection mode: Split less while preparing samples by dissolving a few milligrams of each • Sampling time: 1 minute sample in 1 ml of methanol. • Flow control mode: Pressure • Pressure: 100.0 kPa Preliminary phytochemical investigation • Column flow: 1.2 ml/min Test for flavonoids • Linear velocity: 45.4 cm/s Few milligrams of the ethyl acetate fraction were suspended in • Purge flow: 3.0 ml/min. ethanol and few drops of 5% ethanolic KOH were added, and • Infrared (IR): IR spectra for quercetin and apigenin was then, few drops of 5% HCl were added. The changes in colors recorded in KBr disk , the range of scanning 4000-400 cm−1 were recorded. • UV: Few milligrams of quercetin and apigenin were dissolved in Test for phenols 1 ml absolute methanol, and its UV absorbance was scanned from Few milligrams of methanol plant extract were treated with few 200 to 400 nm drops of 1% FeCl3. Formation of dark greenish-blue color • HPLC: As listed before indicates the presence of phenols. • HPTLC: As listed before. Isolation of flavonoids derivative by preparative TLC RESULTS A phenolic derivative was isolated by preparative TLC, utilizing Preliminary examination of the ethyl acetate