Chemical characterization and antioxidant activity of Froriepia subpinnata and Eryngium campestre from Iran Hassan nikkhah-kouchaksaraei1* and Mojtaba Ranjbar2 1-Faculty member of Department of Agronomy, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran [email protected] 2-Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran [email protected] Abstract This study is outlined to define the chemical composition, total phenols, flavonoids, flavonols, Reducing power and in vitro antioxidant activity of the extracts of Froriepia subpinnata and Er- yngium campestre from Iran. The chemical compositions of F. subpinnata and E. campestre were analysed by GC–MS. Sixteen and ten compounds were detected in the leaves essential oils of F. subpinnata and E. campestre at the first vegetative phase. The major compound of the essential oils of F.subpinnata and E. campestre were terpinolene(34%) and limonene(62.73%), respectively. Total flavonoid and flavonol contents were calculated as quercetin equivalents from a calibration curve. The highest total phenolic and flavonoid contents were observed in F. subpinnata in the second vegetative phase. Antioxidant activity was determined by DPPH assay. The highest radical scavenging activity was observed in F.subpinnata -2(86.1±0.89% inhibition), followed by E. campestre-2(83.61±0.73%), F. subpinnata-1(77.41±0.85%) and E. campestre-1(76.72±0.93%). Keywords: Froriepia subpinnata, Eryngium campestre, DPPH, antioxidant activity, Monoterpene hydrocarbons. Introduction Free radicals are produced while cells use oxygen to generate energy and they are named Reactive Oxygen Species(ROS) such as singlet oxygen, superoxide ion, hydroxyl ion and hydrogen peroxide that are result of cellular redox process. ROS illustrates beneficial effects on cellular responses and immune function at low or moderate concentrations. But high levels of ROS can cause damage to proteins, lipids, enzymes and DNA and have also been linked to pathogenesis of 1 oxidative diseases(Singh et al. 2009: 642–645 and Halliwell 1997: 44-52). Normally, alive cells possess an excellent scavenging mechanism to avoid excess ROS-induced cellular injury; however, with ageing and under influence of external stresses, these mechanisms become inefficient leading to metabolic distress such as heart diseases, acquired immunodeficiency syndrome, diabetes mellitus, arthritis, cancer, aging, liver disorder etc. Hence, dietary supplementation with synthetic antioxidants is required(Eshwarappa1 et al. 2014: 101-107). In recent years, due to toxicological concerns associated with the use of synthetic substances in food and increasing awareness about natural foods, there has been an increased interest in the use of natural substances as food preservatives and antioxidants. In this context, plants have been valuable source of natural products for maintaining human health(Bhattacharjee et al. 2006: 645-648). According to World Health Organization(WHO), medicinal plants would be the best source to obtain a variety of drugs and the World Health Organization estimated that 80% of the population of developing countries rely on traditional medicines, mostly plant drugs, for their primary health care needs. Also, modern pharmacopoeia still contains at least 25% drugs derived from plants and many others which are synthetic analogues built on prototype compounds isolated from plants(Gossell-Williams et al. 2006: 217–218 and Ahmad et al. 2012: 629-631). Phenols and polyphenolic compounds are secondary metabolites present in plants and they have physiologic properties including anti allergic, anti-microbial, anti-coagulant, anti-inflammation and conservation effect. Phenol compounds also have a beneficial role for coronary disease management as well as cancer and neurodegenerative diseases prevention(Asgharpour et al. 2013: 169- 176; Kay et al. 2002: 389-98 and Morton et al. 2000: 152-159). Studies have demonstrated the beneficial effects of phenolic compounds in human health due to their antioxidant activity(Walter et al. 2011: 371- 377; Manach et al. 2005: 77-84). Globally, plant extracts are employed for their antibacterial, antifungal, antiviral and antioxidant activities(Singh et al. 2009: 642–645; Gossell-Williams et al. 2006: 217–218; Nebija et al. 2009: 22 – 32; Bajpai 2009: 1127–1133 and Karamoddini et al. 2011: 63-68). The Froriepia subpinnata and Eryngium campestre belonging are two endemic species of Apiaceae family, that have been distributed in the northern parts of Iran(Gilan, Golestan and Mazandaran Provinces of Iran). Their young leaves are used as a cooked vegetable and for flavoring in preparation of few local foods (Akhani 2003: 369-85; Hashemabadi and Kaviani 2011: 693 – 698; Khoshbakht et al. 2007: 445-448). In recent years, there are some reports regarding the main effects of two endemic species(Hashemabad and Kaviani 2011: 693 – 698; Morteza-Semnani et al. 2009: 127-128; Rustaiyan et al. 2001: 405-406; Nabavi et al. 2008: 19-25; Nabavi et al. 2012: 81-87). According to previous studies, secondary metabolites are highly variable and depend on several factors, such as climatic conditions, growing stage in harvesting, plant genotype and plant chemotype( Hashemabadi and Kaviani 2011: 693 – 698; Morteza-Semnani et al. 2003: 43-48). The aim of the present study was the determine of essential oils content and antioxidant properties of F. subpinnata 2 and E. campestre in first and second vegetative phases, in suburb of Qaemshahr city, Mazandaran province, north of Iran. Materials and methods Plant materials The leaves of E. Campestre and F. subpinnata were collected during the first and second vegetative phases(April and August 2013, respectively), from the suburb of Qaemshahr city, Mazandaran province, North of Iran, in 2013. Isolation of essential oil In first vegetative phase, the samples were dried at room temperature and the dried leaves(50g) were hydro distillated for 3 hours by using a Clevenger-type apparatus. The extracted oils were dehydrated, using sodium sulfate, then were stored at 4ºC until future use(Baydar et al. 2004: 169–172). Gas chromatography-mass spectrometry The gas chromatography analysis was carried out on an Agilent789, a gas chromatography with 5975C mass selective detector and a HP5M5 column(30m×0.25mm,film thickness 0.25µm). The operating conditions were as follows: a Helium carrier gas with a flow rate of 1ml/min with split ratio 1:40. The GC analysis was carried out in the oven, while temperature was held at 60ºC for 3min then programmed at 3ºC min-1 to 150ºC(held for 1min), after that programmed at 3ºC min-1 to 260ºC(held for 3min). The injector and the temperature of detector were at 230 and 250ºC, respectively. The components of oil were identified by their retention indices relative C8-C25 n- alkanes and commercial library(Willey)( Joulain and König 1998: 658; Thiem et al. 2011: 7115-7124). Preparation of plant extracts At the first and second vegetative phases, the extracts of both leaves were prepared base on the Ghimire et al.(2011) method. A two gram of powdered samples were extracted in 25ml of 80% methanol(V/V) and were kept for 1 day on a shaker at room temperature. The methanolic extracts were filtered using a Whatman N. 1 filter paper and rotary evaporator in a water bath at 40ºC. The yield of evaporated dried extracts was calculated based on dry weight basis from the following equation(Stanojevic et al. 2009: 5702-5714) Yield (g/100 g of dry plant material) = (W1 × 100) / W2 (1) 3 Where W1 is the weight of the extract after the solvent evaporation and W2 is the weight of the dry plant material. Determination of Total Phenolic Compounds Total phenolic constituents were determined by the Folin-Ciocalteanu method(Kim et al. 2007: 443– 450). The extract samples(1mg/ml,1000µl) were mixed with 50µl of 1m Folin-Ciocalteau reagent. A futher 1.85 ml of distilled deionized water were added to the mixture and mixed by vortexing. After 3 min, amount of 100µl of 20% Na2Co3 were added with mixing. The solutions were then immediately diluted to a volume of 4 ml with distilled deionized water and mixed thoroughly, after incubation for 90 min in the dark at room temperature. The absorbance was measured at 760nm and results were expressed as gallic acid equivalents. Determination of flavonoid and flavonol Contents Total flavonoids in the plant extracts were estimated using the method of Moreno et al.(2000) and Ghimire et al.(2011). Briefly, 500µl of each extract(1mg/ml) was mixed with 100µl of 10% aluminum chloride, 100ml of 1M potassium acetate, and 4.3ml of 80% ethanol, then the mixture was vortexed and allowed to stand for 40 min for reaction at room temperature. The absorbance of the reaction mixture was measured at 415nm and total flavonoid contents were calculated as quercetin from a calibration curve(Moreno et al. 2000: 109-114; Ghimire et al. 2011: 1884-1891) Total flavonols were estimated as previously described(Loziene et al., 2007). To 2000µl of extract samples, 2000µl of 2% aluminum chloride and 6ml(50ml/L sodium acetate solutions were added. The absorbance was reed at 440nm after 2.5h at 20ºC and total flavonol contents were calculated as quercetin from a calibration curve(Lopez-Ochoa et al. 2007: 4397–4406). DPPH Radical-Scavenging Activity DDPH assay was performed following the procedure described by Sing et al.(2009) with minor modifications in different concentrations of extracted(150-800μg/ml), where the 1000µl amount was mixed with solution of 1000µl of 300µM DPPH in methanol, then the final volume was made to 4ml by methanol. The mixture was shaken and left for few minutes at room temperature in the dark then the absorbance of the solutions was measured at 517nm(Singh et al. 2009: 642–645). Reducing Power Determination 4 The reducing power was determined according to the method of Yen and Chen(1995: 27-32) 2500µl of different concentration of extracted(200,400 and 600μg/ml) were mixed with 2500µl of 0.2M phosphate buffer(pH=6.6) and 2500µl of 1% potassium ferricyanide.