INTRODUCTION:

Free radicals play a pivotal role in human health by causing several chronic diseases, such as diabetes, cancer, atherosclerosis, hypertension, heart attacks and other degenerative diseases^[1]. Free radicals are generated during body metabolism. Exogenous intake of antioxidants plays an important role in health care to prevent and scavenge free radicals. Nowadays, there is a noticeable interest in antioxidants, especially in those which can prevent the presumed deleterious effects of free radicals in the human body, and to prevent the deterioration of fats and other constituents of foodstuffs. In both cases, there is a preference for antioxidants from natural rather than from synthetic sources^[2].

At present, most of the antioxidants are manufactured synthetically. The main disadvantage with the synthetic antioxidants is the side effects in vivo^[3]. Previous studies reported that butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) accumulate in the body and result in liver damage and carcinogenesis^[4]. Thus, investigation of new and safe antioxidants from natural sources has become very important for food and medicinal functions^[5].

Atractylis flava Desf. (Asteraceae family) is a kind of endemic plant of North Africa^[6,7]. In Algeria, this plant is quite rare and its geographic range covers a short territory in the south-west of the country^[7]. The Asteraceae family (divided into 11 subfamilies and 35 tribes comprises about 1400 genera and more than 25.000 species of herbaceous plants, shrubs, and trees spread throughout the world^{[8, 9]}. Atractylis flava Desf. (Syn. Atractylis carduus (Forsk.) locally called “assenan aouragh”^[7]. Atractylis plants have been used in folk medicine for treatment of various diseases such as cholelithiasis, ulcer, tumor and circulatory disorders, hepatitis, intestinal parasites and snake-bite poisoning when flava Desf is particularly known in traditional Algerian medicine for its diuretic effects^[10,11,12].

the objective of this study was to assess the phenolic and flavonoids compounds content, and in vitro antioxidant activity of the of different extracts (PE, DCM, EtAOc and n-BuOH) obtained from Atractylis flava Desf grown in Algeria using several methods in vitro.

MATERIAL AND METHODS:

Plant material:

The whole plant Atractylis flava Desf was collected in the flowering season at Biskra region (Algeria) in May 2015. The plant material was authenticated by Pr. Bachir Oudjehih (Agronomic Institute of Batna1 University, Algeria). A voucher specimen (660/LCCE) was deposited in the Herbarium of the mentioned department.

Extraction:

The collected whole plant Atractylis flava was air-dried and powdered. 500 g powder was macerated with MeOH–H₂O (80:20). After filtration, the filtrate was concentrated under vacuum at room temperature the hydro alcoholic extract was submitted to liquid–liquid fractioning using solvents with increasing polarities (petroleum ether, dichloromethane, ethyl

acetate, and n-butanol). The n-butanol fraction was used for the investigation of the acute toxicity study, anti-inflammatory and antipyretic effects ^[13].

Total phenolic content:

The extracts total phenol content was determined spectrophotometrically using Folin–Ciocalteu method ^[14]. Two hundred microliters of each extract were added to 1 mL of Folin–Ciolcalteu reagent (10%). After 4 min, a volume of eight hundred microliters of sodium carbonate solution (75 g/L) was added. The mixture was allowed to react for 2 h in darkness at room temperature. After incubation, the absorbance was recorded at 765 nm using UV–Vis spectrophotometer (VIS-7220G). Gallic acid (5–200 μg/mL) was used as reference to establish the calibration curve from which the concentration of polyphenols was calculated and the results were expressed in microgram equivalents of gallic acid per milligram of extract (μg GA/mg of extract).

Total flavonoid content:

The total flavonoid content of each extract was t was performed by the trichloroaluminum method ^[15]. 1 mL of AlCl3 (2%) solution was added to 1 mL of the plant extracts. The mixture was vigorously stirred and incubated for 10 min at room temperature, and then the absorbance of each sample was read at 430 nm. Quercetin (1.25–25 μg/mL) was used to realize the calibration curve to estimate the concentration of flavonoids found in the crude extracts and the results have been given in micrograms equivalent of quercetin per milligram of extract (μg EQ/mg of extract).

ANTIOXIDANT ACTIVITIES:

DPPH radical scavenging activity:

The antioxidant activity of our extracts was measured by the free radical DPPH^[16]. 40 µL of different dilutions of the extracts (PE, DCM, EtOAc and n-BuOH) or standard (BHT) were added to 160 µL of DPPH prepared in methanol. The obtained mixture was incubated for 30 min in obscurity. Then, the absorbances were measured using a UV–VIS spectrophotometer at 517 nm and the percentage of DPPH.

radical-scavenging activity of each extract was calculated as follows:

DPPH scavenging efect (%)

= [(A_Control−A_Sample) ∕A_Control] × 100

A_control is the absorbance of blank and A_Sample is the absorbance of positive control or sample

ABTS radical cation scavenging activity:

This assay was performed according to the procedure described by^[17]. The ABTS•+ was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Before usage, the ABTS•+ solution was diluted to get an absorbance of 0.708 ± 0.025 at 734 nm with methanol. Methanol was used as negative control, while BHA and BHT were used as positive controls. All the tests were conducted in triplicate and the IC₅₀ were calculated from the graph of the ABTS•+ scavenging effect percentage against the sample concentration.the percentage of inhibition was calculated using the following equation:

ABTS efect (%) = [(A_Control−A_Sample) ∕A_Control] × 100

A_control is the absorbance of blank and A_Sampleis the absorbance of positive control or sample.

β-Carotene bleaching test:

β-carotene bleaching assay was done according to the method of^[18]. BHA was used as positive control. For the preparation of β-carotene emulsion 0.5 mg of β-carotene was mixed initially with two hundred mg of Tween 40 and 25 μL of linoleic acid prepared in 1 mL of chloroform (CHCl3). After the evaporation of the chloroform under vacuum, 100 mL of distilled water saturated with oxygen were then added to the mixture with vigorous stirring. 160 μL of emulsion were added to 40 μL of samples (extracts or standards) at different concentrations. The mixtures were incubated at 50 °C for 2 h and then the absorbances were measured at 470 nm and the percentage of the antioxidant activity (AA) was calculated using the following formula:

the percentage of the antioxidant activity (AA) was calculated using the following formula:

AA%= (Abs of 𝛽-carotene content after 2 h∕ Abs of initial 𝛽-carotene content) ×100

Reducing power assay:

This assay was performed according to the procedure described according to the previous method^[19]. 100 μL of sample solution (extracts or references) prepared at different concentrations were added to 2.5 mL of phosphate buffer (0.2 M pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was incubated at 50°C for 20 min. Then 2.5 mL of CCl3COOH (10%) was added to the mixture, which was centrifuged (10 min/3000 rpm). A volume of 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 500μL of FeCl3 (1%) freshly prepared. The absorptions were read at 700 nm and the results were calculated as A0.5 (μg/mL), which indicates the concentration corresponding to the absorption at 0.50 nm. The reducing power of the various extracts was compared with those of BHA, BHT and quercetin as standard.

Cupric reducing antioxidant capacity (CUPRAC) :

The cupric reducing antioxidant capacity was determined according to the method of^[20]. Fifty microliters of a solution of copper (II) chloride (10 mM) were added to 50 μL of the neocuprine solution (7.5 mM) and 60 μL of ammonium acetate buffer solution (1 M, pH = 7.0). Different concentrations of extracts and standards were added to the initial mixture to make a final volume of 200 μL. The samples were shielded from light and the absorbance was measured at 450 nm after 1 h of incubation. The results were calculated as A0.5 (μg/mL) and the reduction capacity of the extracts was compared with those of BHA and BHT as standards.

Chelation of the metal ions:

This assay was done according to Decker and Welch ^[21], the chelation of metal ions was assessed. 40 μL of prepared sample (extracts and EDTA) at different concentrations were added to 40 μL of FeCl2 (0.2 mM) and 40 μL of ethanol. The reaction was initiated by the addition of 80 μL of ferene solution (0.5 mM). After a vigorous stirring, the obtained samples were incubated for 10 min at room temperature and then their absorbances were measured at 562 nm. The results were given as a percentage of inhibition using the following formula:

Activity (%) = ((A_Control−A_Sample) ∕A_Control) × 100.

Statistical analysis:

Statistical analysis Total phenolic contents and free radical scavenging activity were measured in 3 replicates (n=3). All values were expressed as mean±S.D.

RESULTS AND DISCUSSION:

Total phenolic and Total Flavonoids contents:

Total phenolic contents (Table 1) were calculated from standard curve of gallic acid. It is found that n-BuOH extract has the highest total phenolic content (122,74±0,78 µg GAE/mg extracts), followed by ethyl acetate extract (118,76±0,27µg GAE/mg extracts).

Phenolic compounds are one of the major chemical classes of plants’ secondary metabolites. They play a crucial role in the defense of plants against pathogens, diseases, parasites, and predators^[22]. They involve in a number of physiological mechanisms such as antioxidant activity^[23]. The amount of phenolics depends on several factors such as temperature, UV-light, nutrition available to the plant, and genetic factors ^[24].

The amounts of total flavonoids obtained from whole plant extracts of Atractylis flava Desf are presented in Table 1. Total Flavonoids Contents Flavonoids have the ability to repress free radicals, reduce their levels in the body, and increase antioxidant defense activity ^[22]. Total ﬂavonoids in A. flava Desf. ranged from 13,10±1,13 to 66,75±6,12 µg QE/mg. Among all extracts, a signiﬁcantly high amount was found in EtAOc and n-BuOH extracts with values 63,32 and 66,75 µg QE/mg, respectively; while PE and DCM extracts of A. flava Desf had the lowest amount.

Table 01.Total phenolic contents and Flavonoids content of Atractylis flava Desf. Extracts

Extraits	Total phenolic compounds content (µg GAE/mg)	Flavonoïdes content (µg EQ/mg)
PE	31,89±0,56	13,10±1,13
DCM	46,18±0,33	18,22±3,37
EtAOc	118,76±0,27	63,32±3,60
n-BuOH	122,74±0,78	66,75±6,12

Antioxidant capacity of atractylis flava desf.

The antioxidant capacity of A. flava Desf was evaluated in different extracts (PE, DCM, EtAOc and n-BuOH) using DPPH, ABTS, β-carotene, chelating metal ions, CUPRAC and reducing power methods. The results are expressed as IC₅₀ and A_0.50values which are given in Table 2 and Figure 01.

Table 2. Antioxidant activities of Atractylis flava Desf.

EXTRACTS and standards	DPPH ^a	ABTS ^a	β-carotene ^a	chelating metal ions ^a	CUPRAC^a	reducing power ^a
EXTRACTS and standards	IC₅₀ (µg/mL)	IC₅₀ (µg/mL)	IC₅₀(µg/mL)	IC₅₀ (µg/mL)	A_0.50 (µg/mL)	A0.50 (µg/mL)
PE	>800	326,41±2,82	39,12±2,93	711,70±13,57	171, 39±1,44	165,28±5,42
DCM	625,26±5,25	77,62±4,39	37,52±0,92	165,61±3,14	86,59±0,55	197,06±1,34
EtAOc	158,41±3,24	15,57±2,93	16,52±1,87	78,11±4,18	28,58±0,65	7,49±0,54
n-BuOH	427,53±4,69	34,28±1,12	35,98±3,04	176,52±8,54	62,17±0,27	19,24±2,27
BHA ^b	6.14±0.41	16,91±0,1	0,90±0,02	NT	5,35±0,71	9,29±0,22
BHT ^b	NT	NT	NT	NT	8.97±3.94	NT
EDTA ^b	NT	NT	NT	48,11±0,32	NT	NT
QUERSITIN ^b	NT	NT	NT	NT	NT	4,31±0,64

^a Values expressed are mean ± SD of three measurements, b Reference compounds, NT not tested.

EtAOc extract possessed the best capacity for preventing lipid peroxidation (IC₅₀ = 16,52±1,87 μg/mL) and CUPRAC assay (A_0.50 = 28,58±0,65 μg/mL). The results of ferric reducing power ability of all the tested samples were affected in a dose-dependent manner. The results obtained show that the tested extracts have an important capacity to scavenger free radical DPPH, ABTS and a strong activity to inhibit oxidation of β-carotene. Given the richness of Atractylis Flava Desf extracts in polyphenols and flavonoids, however, the presence of flavonoids could effectively capture the free radical DPPH, ABTS and trap linoleic acid radicals thus preventing the bleaching of β-carotene ^[25]. Phenolic compounds such as flavonoids are known by their ability to trap free radicals and therefore delay lipid auto-oxidation^{[26,27,28,29]}. It has been reported that these compounds serve as good donors of electrons and hydrogen that are derived primarily from their B ring ^[14].

In addition, the antioxidant activity was evaluated using CUPRAC, iron reduction and chelation of ferrous ions, which are significant indicators of the antioxidant power of plants. The results show that our extracts contain compounds that can reduce copper and ferric iron is competing with ferrozine to chelate ferrous iron (Fe+2). The reducing power of pure compounds or in the form of crude extract can be measured by spectrophotometric detection.

The results of this study show that our extracts have a reducing effect, which could be due to its richness in polyphenolic compounds. Indeed, the chelating capacity is very important because it reduces the concentration of metal peroxidation catalysts. however, iron can stimulate lipid oxidation by the Fenton reaction, and also accelerates this oxidation by decomposing hydroperoxides into peroxyl and alkoxyl radicals which, in turn, can maintain the chain reaction ^[30].

Overall, the results of the antioxidant activity of the extracts evaluated by these different tests indicate that these extracts possess important antioxidant properties. This activity appears to be attributed to phenolic compounds. The plant Atractylis Flava Desf is characterized by the existence of Vicenin 3, Schaftoside, Isorhamnetine 3-O-robinobioside, Ladanein, Stigmasterol, quercetin Chrysin, Apigenin and oleanolic acid^[31]. which possess strong antioxidant and radical scavenging activities (DPPH, ABTS+) and reduction power^{[32,33,34,35,36]}. These compounds therefore seem to be the main contributors to the antioxidant capacity of our extracts.

It seems important to stress that ethyl acetate extract is the most active. The work of^[37]. showed that the ethyl acetate fraction of this plant has a good capacity to trap the DPPH radical in Atractylis Flava Desf, quercetin, stigmasterol, Chrysin, Apigenin and oleolic acid are the main compounds isolated in the ethyl acetate extract of this plant^[37]. These constituents have a strong antioxidant activity. These properties of our extract are probably due to these compounds. Indeed, the antioxidant effect is simply that expected from the content of the most effective components. Moreover, antioxidant activity depends not only on the concentration, but also on the structure and nature of antioxidants^[38]. We can therefore say that the overall performance as an antioxidant is the result of the interaction of a single component or complex between components, one can expect a synergistic or antagonistic effect.

CONCLUSION:

This study indicated that the EtAOc and n-BuOH extracts prepared from whole plant A. flava Desf contain high amounts of phenolic and flavonoid compounds and exhibit strong antioxidant activities. The use of A. flava Desf as a natural antioxidant source appears to be an alternative to synthetic antioxidants at a low cost. This study was the first report about the antioxidant activities of A. flava Desf. however, further studies are necessary to determine the chemical composition of the extracts responsible for the majority of antioxidant activities of Atractylis flava Desf.

ACKNOWLEDGEMENT:

The authors wish to express thanks to the general directorate for scientific research and technological development (DGRSDT) of the Algerian Minister of Higher Education and Scientific Research for providing a research grant.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.07.2018 Modified on 16.09.2018

Research J. Pharm. and Tech 2018; 11(12): 5221-5226.

DOI: 10.5958/0974-360X.2018.00952.6