1. Introduction

The genus Pulicaria, family Asteraceae, is represented 100 species widespread all around the world, more than 10 of which grow wild in Yemen. Chemically, this genus is not homogenous. As pointed out previously some species contain monoterpenes, diterpenes, sesquiterpene lactones ^[1] and caryophyllane derivatives ^[2]. Also the literature reports that Pulicaria species afforded different flavonoid profiles ^[3]. The Pulicaria species proved various activities such as antiinflammatory, antilukemic, Potential cancer chemopreventive and cytotoxic agents ^[4]. The Pulicaria crisps indigenous toYemen, locally known as Anssif, is traditionally used as diuretic, pyritic conditions in urogenetic organs, and to cure fever. The flowers of Pulicaria inuloides was also used as spice and to make various delicious foods. Some investigation reported that this species reveal antimicrobial, antifungal, antimalaria and insecticides properties ^[5]. P. arabica is used medicinally to treat painful swellings and boils. The whole plant is strongly aromatic and sprigs were often plucked and rubbed between the hands and then over the body as a deodorant. It grows abundantly in drier areas, where the water levels lie close to the surface, and is a reliable indicator for the proximity of water ^[6]. The biological activities of medicinal plants are mainly due to the presence of their active constituents or secondary metabolites, which are present almost in higher plants, usually in a high structural diversity. As a rule, a single group of secondary metabolites dominates within a given taxon. A few major compounds are often accompanied by several derivatives and minor components ^[7]. In this respect, since up to the present, there is no study on the chemical or the antioxidative activity of Pulicaria inuloides. The aim of this study was to investigate the antioxidative activity of different extracts (methanol, ethanol and diethyl ether extracts) of the leaves of Pulicaria inuloides as new potential source of natural antioxidants.

2. Materials and Methods

The leaves of P. inuloides were collected from natural sources in August, 2013 from Sana'a area in Yemen. Plant was from natural sources in Yemen: Pulicaria inuloides Identification was carried out by Agricultural Research Corporation (Dammar).

The aerial part essential oils of P. inuloides (3kg) were ground in blender. The essential oils were obtained by hydro distillation at 100°C with a clevenger type apparatus (Full description of model with the city and province (Sana’a) for 4 h with 3 L of distilled water. Started oils were dried extract with sodium sulphate (Na₂O₄S), and stored at 4°C until use.

The components of the essential oils were identified by GC-MS analysis ^[8]. Gas chromatography-mass spectrometry (Varian 1200L) was incorporated with a relatively no polar capillary column (DB-5, 30 m length, 0.25 mm film thickness, 0.25 internal diameters). The injection port and interface were held at 220 and 260°C, respectively. The temperature was programmed from 50°C to 220°C at 15°C per min and a hold at 220°C for 25 min with helium as the carrier gas. Mass spectra: electronic impact, ionisation potential 70 e V, ion source temperature 200°C and mass range 35 – 500 Da. Most of the compounds were identified according Toes Kovats indexes in reference to n –alkanes(NIST, 2010) and mass spectra (authentic chemicals and Wiley spectral library collection).

Twenty grams leaves from plant was individually extracted with 600 ml of 90% methanol, Ethanol and diethyl ether (separately) in an ultrasonic device at room temperature, The extracts were filtered and the residues were re-percolated for three times and was eliminated from the solvent using a rotary evaporator and sample was preserved at 4°C until used.

Total phenolics of P. inuloides extracts were determined using the method of Singleton ^[10] with some modification. 200 μl portions of diluted extracts were introduced into test tubes followed by addition of 1000 μl of Folin-Cio- calteu reagent (1:10). After Thirty seconds and just prior to 8 min, 800 μl of Na₂CO₃ (7.5%) was added to extracts in tubes. The reaction mixtures were incubated at 24°C for 1 h prior to recording the absorbance at 765 nm against blank. Total phenolic were calculated from standard gallic acid solutions used under the same conditions, and concentrations were expressed as mg gallic acid equivalents (GAE) per g extract.

Total flavonoid content was determined by the AlCl₃ ^[10], with slight modifications. Avolume of 0.5 ml of 2 % AlCl3 ethanol solution was added to 0.5 ml of sample solution. After one hour at room temperature, the absorbance was measured at 430 nm. A yellow color indicated the presence of flavonoids. Extract samples were evaluated at a final concentration of 0.2 mg/ml. Total flavonoids were calculated as quercetin (mg/g) using the calibration curve. Results were expressed as mg quercetin equivalents (QE) / g extract.

The tannins were determined by Folin and Ciocalteu method ^[10]. 0.1 ml of the sample extract was added with 7.5 ml of distilled water and adds 0.5 ml of Folin Phenol reagent, 1 ml of 35% Na₂CO₃ solution and dilute to 10 ml with distilled water. The mixture was shaken well, kept at room temperature for 30 min and absorbance was measured at 725 nm. Blank was prepared with water instead of the sample. A set of standard solutions of gallic acid is treated in the same manner as described earlier and read against a blank.

Total anthocyanin content of diluted P. inuloides extracts were estimated by the pH differential method ^[11]. Absorbance was measured with a UV visible spectrophotometer at 510 nm and 700 nm at pH 1 and 4.5 where A = (A510 - A700) pH 1 - (A510 - A700) pH 4.5. Data were calculated with the extinction coefficient for cyanidin-3-glucoside (29 600) and expressed as mg cyanidin 100 g-1 fresh weight. All analyzes were performed in triplicate.

The ability to scavenge the stable free radical 1, 1- diphenyl-2-picrylhydrazyl (DPPH) was determined based on the method ^[12]. A solution of 0.2 mM DPPH in methanol was prepared and 1 ml of this solution was mixed with 1 ml of extract in methanol (5 to 150 µg/ml).The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. A control sample containing the same volume of solvent in place of extract was used to measure the maximum DPPH absorbance. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid and quercetin were used as references. Results were expressed as percentage of inhibition of the DPPH radical according to the following equation:

Antioxidant activity was determined also using the B-carotene bleaching test with some modifications 14. 1 ml of β-carotene (0.2mg/ml chloroform) was mixed with linoleic acid. Following 90 min, the absorbance was read spectrophotometrically at 725 nm. The standard calibration (0.01 – 0.05 mg/ml) curve of Gallic acid in 80% methanol curve was plotted. Results were expressed as small cap gallic acid equivalents GAE) in mg per 100 g sample extracts.

The chelation of ferrous ions by extracts was estimated by the method of ^[13]. The reaction mixture contained 0.5 mL of various concentrations of the extracts (0.2, 0.4, 0.6, 0.8, and 1 mg/mL), 1.6 mL of deionized water and 0.05 mL of 2 mM of FeCl²⁺ solution. After 30 Second, 0.1 mL of 5 mM ferrozine solution was added. Fe2⁺-Ferrozine magenta complex was very soluble and stable in water. After 10 min at room temperature, the absorbance at 562 nm was measured. The percentage inhibition of ferrozine-Fe²⁺ complex formation was calculated as:

Ascorbic acid and TBHQ were used as positive control.

Data are means of triplicate experiments, each in duplicate. The analysis of variance (ANOVA, one-way) was used to separate means at a 0.05 significant level (SPSS Version 13.0 for Windows, SPSS Inc., Chicago, IL).

3. Results

The composition of the essential oils obtained by hydrodistillation from the flowers and leaves of P. inuloides was analyzed by GC/MS, and show in Table 1 and Forty-two components were identified accounting for 98.955% of leave essential oil of P. inuloides included 2-Cyclohexen-1-one, 2-methyl-5-(1-methyl (56.8 %), Benzene, methyl- (21.7%), Z-Citral (2.00 %), Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)- (1.734 %), alpha.-Farnesene (1.68 %), 7 Oxabicyclo [4.1.0] heptane, 1-methyl- (C (1.538 %), Thymohydroquinone dimethyl ether 1.511 %), Eugenol (1.16%) and Zingiberene (1.094 %) (Table 1).

Table 1. The main compounds of the essential oil of Pulicaria Inuloides

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In Table 2, the total phenolic content of methanolic, ethanolic and diethyl ether extracts of P. inuloides measured by Foline Ciocalteau reagent in terms of gallic acid equivalent was 91.2, 75, 89.9 ± 0.61 and 64.9 ± 07 mg/g respectively. The flavonoid content of the plant sample calculated as catechol equivalent was 75 ± 85.7, 56.4 ± 2.7 and 37.9 ± 0.67 mg/g respectively. The tannin content was 0.66 ± 0.07, 0.51 ± 0.01, and 08.5 ± 11.5 mg/g respectively and total anthocyanins was 0.44 ± 0.03, 0.63 ± 0.02 and 0.02 ± 0.01 mg/ g respectively. It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable. The results strongly suggest that the phenolics are important components of this plant, and some of the pharmacological effect could be attributed to the presence of this invaluable component.

Table 2. Total phenol, flavonoid, tannin and anthocyanin contents of the P. inuloides extrects

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The extract exhibited concentration-dependent radical scavenging activity, that is, the higher the concentration, the scavenging potential. The DPPH radical scavenging activity of methanolic extract of P. inuloides is shown in the Figure 1.

Figure 1. DPPH radical scavenging activity of P. inuloides extracts. Data are expressed as mean ± SD (standard deviation) (n = 3). Ascorbic acid and TBHQ were used as positive control

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Table 3. Means of antioxidant activity of selected extracts for plant and standard assayed by β-carotene linoleate bleaching

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The antioxidant activity of P. inuloides was also studied using the β -carotene bleaching method, which is based on oxidative decomposition of b-carotene in the presence of linoleic acid. As seen in Table 3.

The antioxidant activity of P. inuloides was also studied using the Metal chelating method. As seen in Figure 2.

Figure 2. Metal chelating effect on ferrous ions in extracts of Pulicaria inuloides

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4. Discussion

The percentage and retention time of components analysed by GC/MS are shown in Table 1, in P. inuloides oil, 52 components represented about 100% of the total oil, were identified. 2-Cyclohexen-1-one, 2-methyl-5-(1-methyl (55.1%), Benzene, methyl- (20.6%) and eugenol (1.16%) were the major component in P. inuloides essential oil. As it is shown in Table 2, the largest part of P. inuloides essential oil was formed by Bicyclo [3.1.1] hept-2-ene, 2, (22.369%) and Naphthalene, 1,2,3,4,4a,5,6 (19.327%). Essential oil extracted from P. inuloides was light yellow in colour and with a perfumery odor.

The plant is important source of potential compounds for the development of new therapeutic agents. Plants phenolics are widely distributed in the tissues of plants as well as play a vital role in the highly effective free radical scavengers and antioxidant activity. It has been reported that the antioxidant activity of phenol is mainly due to their redox properties, hydrogen donors and singlet oxygen quenchers ^[15]. Typical phenolics that possess antioxidant activity are known to be mainly phenolic acid and flavonoids. These compounds possess diverse biological activities, such as anti-inflammatory, anti-carcinogenic and anti-atherosclerotic activities. These activities might be related to their antioxidant activity ^[16]. The antioxidant activity of flavonoids is due to their ability to reduce free radical formation and to scavenge free radicals. Phenolic compounds are important plant antioxidants which exhibited reduce free radical formation and to scavenge free radicals. Thus antioxidant capacity of a sample can be attributed mainly to its phenolic compounds ^[17].

The result showed the control had a substantial and rapid oxidation of β-carotene. Accordingly, the absorbance decreased rapidly in sample without antioxidant, while the sample extracts with the presence of antioxidant retained their color and also absorbance for a longer time. Table 3 show the mean antioxidant activity based on the β-carotene bleaching rate of the extracts of different plant of the P. inuloides. The extract with the lowest β-carotene degradation rate exhibit the highest antioxidant activity. All extracts had lower antioxidant activities than had standards (V.C and TBHQ). The highest antioxidant activity among the samples was observed in methanol extract whereas diethyl ether had the lowest antioxidant activity. The highest antioxidant activity among the samples was observed in methanol extract whereas diethyl ether had the lowest antioxidant activity. Result showed that there was considerably variation in the antioxidant activities where it ranges from the lowest of 58% to the highest of 96% where the orders of the antioxidant activity are as follow: TBHQ, Essential oil, Ascorbic acid, Methanol, Ethanol and Diethyl ether.

In this study, radical scavenging activity was determined for the selected extracts of P. inuloides plant. Being a stable free radical, the DPPH assay is a simple and rapid method frequently used to evaluate the ability of antioxidants to scavenge free radicals. As shown in Figure 1, all the sample extracts exhibited significant dose dependent inhibition of DPPH activity that rapidly increase from 1 to 0.2 mg/ml. Scavenging effect increases as the concentration of the sample increased until reached a plateau at 0.2 mg/ml. This showed that the young leaves exhibit a strong scavenging activity and it has been reported that phytochemicals especially plant phenolics constitute a major group of compounds that act as primary antioxidant ^[13]. Their protection mechanisms are through the reaction with the oxygen radicals, superoxide anion radicals and lipid peroxyl radicals.

This study showed that the selected extracts of Pulicaria inuloides plant varied significantly. The phenolics ranged from 91.2 ± 09.5 to 64.9 ± 07.1 mg GAE/ 100 respectively (Table 2). The result also indicates that the methanol extract of plant contained high phenolic content that may provide good sources of dietary antioxidant. For this reason, it is obvious that TPC present in the sample have strong effects against the scavenging activity rather than discoloration of β-carotene. However, ^[18] found that the radicals scavenging activity is not only due to the phenolic content itself, but with other various antioxidant compounds. They respond differently depending on the number of phenolic groups that they have ^[19]. More to the point, TPC does not incorporate necessarily to all the antioxidants that may present in the extracts. Therefore, sometimes there is a vague correlation between TPC and antioxidant activity of several plant species ^[20]. Other than that, TFC of the extracts in terms of rutin equivalent/ 100 g dry weight (standard curve equation: y = 3.021x + 0.0831, R² = 0.9975) were between 64.9 and 91.2. mg rutin equivalent/ 100 g dry weight as shown in Table 2.

In recent years, studies have shown that papaya fruit contains not only vitamins and other nutrients but also contains biologically flavonoids ^[21].

From the data presented in Table (2) it was apparent that tannin content varied significantly in the some plants. Extract where Tannin was low in Z. vulgaris (0.48 mg/g) and high in C. canadensis (0.99 mg/g) which also had the highest level of total phenolics. the results of P. granatum extract agreed with the CSMD study ^[22] Anthocyanins are water-soluble pigments responsible for the red and blue colors in some plants.In the present study Found that there were significant differences evident between study samples at (P ≤ 0.05). The highest Anthocyanins content ranged between 0.97 and 0.63 mg/100 g for C. canadensis and P. granatum respectively, where the smallest values of total anthocyanins 0.02 mg/100 g in R. graveolens due to their low total and colored anthocyanins content, have weakly expressed in color.

Antioxidant properties, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and biological systems ^[18]. DPPH molecule that contains a stable free radical has been widely used to evaluate the radical scavenging ability of antioxidant. The free radical scavenging activities of the three extracts, methanol, ethanol and diethyl ether were also assayed by using DPPH. In this study results showed that DPPH radical scavenging abilities of the extracts were low than those of ascorbic acid and TBHQ (Figure 1). According to ^[23], methanolic extracts may include phenolic and hydrox- phenolic compounds with acid, alcohol, sugar or glycoside and part of the antioxidative activity may be due to these components or flavonoids. In other studies on the DPPH scavenging abilities of extracts were that are different from each other and also from ours. These differences might have been derived from local, climatic and seasonal factors ^[24].

Iron is the most important lipid oxidation Pro-oxidant among the transition metals. Lipid oxidation is accelerated by breaking down of hydrogen peroxide and lipid peroxides by ferrous state of iron to reactive free radicals through the reaction: Fe²⁺ +H₂O₂→ F³⁺ +^-OH+^-OH, known as the Fenton reaction. Radicals from peroxides are also produced by F³⁺ ion. However the rate is one tenth of Fe^{2+ [25]}. In the metal chelating assay, ferrozine can quantitatively form complexes with Fe²⁺ In the presence of other chelating agents, the complex formation is disrupted with consequent decrease in the intensity of the red color of the complex ^[25]. As shown in results (Figure 2), the formation of the red colored complex was inhabited in the presence of methanol and ethanol extracts, indicating chelating activity. However, the color did not change much in the presence of ethanol suggesting weak chelating activity. The reducing power results of various extracts of leaves of P. inuloides and standard references which are presented in Figure 2, followed the order: TBHQ, ascorbic acid, methanol, ethanol and diethyl ether. The results indicated that both methanol and ethanol extracts of P. inuloides interfered with the formation of activity and captured ferrous ions before ferrozine. Asit can be deduced from Figure 2, these two extracts, methanol (64.67 %) and ethanol extract (55.65 %) had the highest ferrous ion chelating activity at a concentration 1 mg/ml. Although the diethyl ether extract did not show ion chelator activity even at the highest concentration, the antioxidant compounds contained in methanol and ethanol extracts of P. inuloides probably inhibited interaction between metal and lipid through formation of insoluble complexes with ferrous ion ^[23].

5. Conclusion

The results obtained in this study clearly showed that both methanol and ethanol extracts from the leaves of P. inuloides possess antioxidant activity. The methanol extract exhibited a strong antioxidant activity and had the most potent scavenging abilities of various radicals which may be caused by the high content of polyphenols and flavonoids. The very strong antioxidant activity of the P. inuloides suggests that the extracts obtained by polar solvents from the leaves could be used as an effective natural source of antioxidant and food additives.

Acknowledgment

This work is supported by National Science and Technology program (no. 2014BAD33B05). The authors express their thanks to the students at the Nutrition Laboratory of School of Food Science and Technology at Jiangnan University for their technical assistance.

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