In Vitro Assessment of Total Phenolic and Flavonoid Contents, Antioxidant and Photoprotective Activities of Crude Methanolic Extract of Aerial Parts of Capnophyllum peregrinum (L.) Lange (Apiaceae) Growing in Algeria

Background: Capnophyllum peregrinum (L.) Lange (Apiaceae) is the unique taxon of capnophyllum genus in Algerian flora. It has never been investigated in regards to its total phenolic and flavonoid contents and antioxidant and photoprotective activities. Methods: C. peregrinum aerial parts extracted with absolute methanol. The total flavonoid and phenolic contents of the extract were evaluated to determine their correlation with the antioxidant and photoprotective activities of the extract. Results: The methanolic extract demonstrated a significant amount of phenolics and flavonoids (74.06 ± 1.23 mg GAE/g, 44.09 ± 2.13 mg QE/g, respectively) and exhibited good antioxidant activity in different systems, especially in 1,1-Diphenyl-2-picrylhydrazyl (DPPH), reducing power and total antioxidant capacity assays. Furthermore the extract showed high photoprotective activity with the sun protection factor (SPF) value = 35.21 ± 0.18. Conclusions: The results of the present study show, that the methanolic extract could be used as a natural sunscreen in pharmaceutics or cosmetic formulations and as a valuable source of natural antioxidants.


Introduction
Since ancient times, medicinal plants have been used for their medicinal virtues and nutriment values. Presently, the crude extracts from both medicinal and aromatic plants have attracted keen interest in the development and preparation of alternative traditional medicine and food additives [1].
Apiaceae is one of the best-known families of flowering plants, which comprises of about 300-455 genera and 3000-3750 species worldwide [2]. In Algerian flora this family is represented by 55 genera and 130 species [3]. Several Apiaceae members are aromatic plants that are popularly

Determination of Total Phenolic Content (TPC)
Total phenolic content of crude methanolic extract of C. peregrinum was determined by the Folin-Ciocalteu method adopted by Tanguy [14]. A volume of 300 µL of extract solution (1 mg/mL in methanol) was mixed with 1500 µL of Folin-Ciocalteu reagent (diluted 10 fold). After 4 min, 1200 µL of Na 2 CO 3 (75 g/L) was added. The mixture was incubated at room temperature in the dark for 2 h. The absorbance of the reaction mixture was measured at 765 nm with UV/VIS spectrophotometer. Gallic acid was used as a standard for calibration curve and results were expressed as Gallic acid equivalents (µg GAE/mg).

Determination of Total Flavonoid Content (TFC)
Total flavonoid content of crude methanolic extract of C. peregrinum was performed by aluminium chloride colorimetric method adopted by Djeridane et al. [15]. A volume of 1 mL of 2% AlCl 3 ethanol solution was mixed with 1 mL of sample solution (1 mg/mL). After incubation for 10 min at room temperature, the absorbance was measured at 415 nm with UV/VIS spectrophotometer (Thermo, Waltham, MA, USA). Quercetin was used as a standard for calibration curve and the results were expressed as Quercetin equivalents (µg QE/mg).

DPPH Radical Scavenging Assay
Free radical scavenging activity of crude methanolic extract of C. peregrinum was determined spectrophotometrically according to the modified Blois methods [16]. Forty microliters of crude methanolic extract and standards (BHT, BHA) at various concentrations (12.5-800 µg/mL) were added to 160 µL of a methanol solution of DPPH (0.4 M) in a 96 well plate. The mixture was vigorously shaken and then incubated at room temperature for 30 min in the dark. The absorbance of the mixture was measured at 517 nm with UV/VIS spectrophotometer. The scavenging activity on the DPPH radical was expressed as inhibition percentage using the following equation: where A control is the absorbance of the control reaction (containing all reagents except the test extract or standard), and A sample is the absorbance of the test extract or standard.

ABTS Radical Scavenging Assay
The ABTS radical scavenging activity was determined according to the method adopted by Re et al. [17]. The stock solution which was allowed to stand in the dark for 12 h at room temperature contained equal volumes of 7 mM ABTS salt and 2.4 mM potassium persulfate. The resultant ABTS + solution was diluted with ethanol until an absorbance of 0.708 ± 0.025 at 734 nm was obtained. Forty microliters of varying concentrations (12.5-800 µg/mL) of the crude methanolic extract and standards (BHT, BHA) were allowed to react with 160 µL of the ABTS + for 10 min. The absorbance of mixture was recorded at 734 nm with UV/VIS spectrophotometer.
The scavenging activity on the ABTS + radical was expressed as inhibition percentage using the following equation: where A control is the absorbance of the control reaction (containing all reagents except the test extract or standard), and A sample is the absorbance of the extract or standard.

Reducing Power Assay
This assay was performed following the method of Oyaizu [18]. A volume of 1 mL in different concentrations of crude methanolic extract and standards (BHA, α-tocopherol) at various concentrations (3.125-200 µg/mL) and 0.75 mL of distilled water were mixed with 1 mL of 0.2 M sodium phosphate buffer (pH 6.6) and, a volume of 1 mL of potassium ferricyanide (1%) was added to the mixture. Following incubation at 50 • C for 20 min, the reaction mixture was acidified with 1 mL of trichloroacetic acid (10%) and 0.25 mL of FeCl 3 (0.1%) was added to this solution. The absorbance was measured at 700 nm with UV/VIS spectrophotometer.

Total Antioxidant Capacity by Phosphomolybdenum Method
Total antioxidant capacity (TAC) of the crude methanolic extract was evaluated by the phosphomolybdenum method according to the procedure described by Prieto et al. [19]. An aliquot 300 µL (2 mg/mL) of crude methanolic extract was added in 3 mL of reagent solution containing (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixture solution was subjected to incubation in a water bath at 95 • C for 90 min. The absorbance was measured at 695 nm with UV/VIS spectrophotometer. The total antioxidant capacity was expressed as Ascorbic acid equivalents (µg AAE/mg).

Photoprotective Activity
The photoprotective activity of crude methanolic extract was measured by determination in vitro of sun protection factor (SPF), which is considered to be one of the most frequently used indicators for the classification of protection levels afforded by sunscreen products against sunburn due mainly to harmful UV-B radiation [20]. In order to perform the photoprotective activity of C. peregrinum aerial parts, the crude methanolic extract was diluted in absolute methanol to obtain a concentration of 2 mg/mL. Spectrophotometric scanning was performed at wavelengths between 290 and 320 nm, with intervals of 5 nm with UV/VIS spectrophotometer. The readings were performed using 1 cm quartz cell and methanol used as a blank. SPF value was obtained according to the equation developed by Mansur et al. [21]: where EE(λ) is the erythemal effect spectrum; I(λ) is the solar intensity spectrum; Abs(λ) is the absorbance; and CF is the correction factor (=10).

Statistical Analysis
All data were expressed as means ± SD for at least three replications for each prepared sample. Statistical analysis was performed using one-sample t-test. The results are considered to be significant when p < 0.05.

Total Phenolic and Flavonoid Content Evaluation
The total phenolic content of the crude methanolic extract of C. peregrinum was estimated by Folin-Ciocalteu reagent and expressed in gallic acid equivalents (GAE) and it was calculated from the linear regression equation of standard curve (y = 0.0143x + 0.2567, R 2 = 0.9929). The results showed that the crude methanolic extract was found to contain a high amount of phenols (74.06 ± 1.23 µg GAE/mg).
The total flavonoid content of crude methanolic extract was determined via aluminum chloride colorimetric method and it was calculated from the linear regression equation of standard curve of quercetin (y = 0.0225x + 0.0078, R 2 = 0.9965) and expressed as quercetin equivalent per gram of plant extract. The tested extract was found to contain high amounts of flavonoids (44.09 ± 2.13 µg QE/mg).

Antioxidant Activity
The antioxidant activity is a complex process, usually happening via several mechanisms. Thus evaluation of plant extracts antioxidant activity must be carried out by more than one test [23]. DPPH free radical scavenging, ABTS free radical scavenging, ferric-reducing power and total antioxidant capacity assays were used to evaluate the in vitro antioxidant effect of crude methanolic extract of C. peregrinum.

DPPH Radical Scavenging Assay
DPPH free radical scavenging activity of crude methanolic extract is shown in ( Table 2). The results reveal that the percentage inhibition of DPPH radical was found to be increased with concentration. The methanolic extract showed maximum activity of 85.21 ± 0.67% at 100 µg/mL; whereas positive standards, BHT and BHA exhibited 94.00 ± 0.31% and 84.18 ± 0.10% inhibition respectively. The IC 50 value of the extract was determined, and which is defined as the concentration of the extract required to scavenge 50% of DPPH free radical. Low IC 50 value represents high antioxidant activity. In this study the IC 50 values of extract and standards (BHT, BHA) were: 48.68 ± 1.71 µg/mL, 12.99 ± 0.41 µg/mL and 6.14 ± 0.41 µg/mL. The results showed that the methanolic extract has a good DPPH radical scavenging effect.

ABTS Radical Scavenging Assay
In case of ABTS assay the experimental data (Table 3) reveal that the crude methanolic extract was found to be effective in scavenging the ABTS + radical. The percentage inhibition of ABTS + radical was concentration-dependent. The scavenging effect was 91.91 ± 0.28% at higher concentrations 800 µg/mL; whereas positive standards BHT and BHA exhibited 96.68 ± 0.39%, 95.86 ± 0.10% inhibition respectively. The IC 50 values of the extract and the standards BHT and BHA were found to be: 63.62 ± 0.66, 1.29 ± 0.30 and 1.81 ± 0.10 µg/mL, respectively, this suggests that the tested extract has a weak ABTS + free radical scavenging activity when compared with standards.

Reducing Power Assay
The reducing power of the crude methanolic extract was evaluated using ferric to ferrous reducing activity as determined spectrophotometrically from the formation of Perl's Prussian blue colour complex [24]. In our study, the reducing power of the methanolic extract was compared with α-Tocopherol and BHA. The results are shown in (Table 4). The results showed that the reducing power activity of the crude methanolic extract increases with the increase in concentrations of the extract solutions. The methanolic extract exhibited (0.94 ± 0.02) average absorbance at 700 nm in 200 µg/mL concentration; whereas the positive standards α-Tocopherol and BHA exhibited (1.81 ± 0.09) (1.86 ± 0.46) average absorbance in the same concentrations.

Total Antioxidant Capacity by Phosphomolybdenum Method
Total antioxidant capacity of crude methanolic extract of C. peregrinum was evaluated by the phosphomolybdenum method and was expressed as ascorbic acid equivalent per mg of plant extract. The results reveal that the crude methanolic extract was found to possess strong total antioxidant capacity (130.91 ± 4.40 µg AAE/mg).

Photoprotective Activity
The photoprotective activity of the crude methanolic extract was evaluated by calculation of SPF; the SPF parameter was determined by the spectrophotometric method developed by Mansur et al. [21], using the UV-B region, considered to be the region of greatest incidence during the day in which people are exposed for longer [10], in this way it has been reported that in SPF ratings, SPF values 2-12, 12-30 and ≥30 are considered as having respectively minimum, moderate and high sun protective activity [25]. As shown in experimental data (Table 5) the extract showed high absorbance values ranged between 3.45 and 3.58 at 290-320 nm, and the SPF value was found to be 35.21 ± 0.18. The results indicate that the methanolic extract have displayed high photoprotective activity.

Discussion
In the present study, phenolic and flavonoid composition as well as antioxidant and photoprotective activities of Capnophyllum peregrinum were investigated the first time in an attempt to unravel new possible uses of this plant. Concerning total phenolic and flavonoid contents, the results suggest that phenolics and flavonoids are important components of the crude methanolic extract. These results are in accordance with previous findings reported in other Apiaceae species extracted with methanol such as Prangos ferulacea Lindl, Prangos meliocarpoides Boiss and Prangos uechtritzii Boiss. & Hausskn [26], Pituranthos chloranthus Benth and Hook [13]. Because of polyphenols are linked to the cell-wall matrix through a glycosidic/ester linkage, their water solubility may be reduced [27]. Thus several authors displayed that alcohols are suitable solvents for the extraction of phenolic and flavonoids compounds. For example, methanol or ethanol are mostly used for the separation of this class of natural products [28] according to their best solubility in alcohols, particularly in methanol [13]. Also, it has been observed that methanolic extracts of medicinal plants exhibited high phenolic and flavonoids contents as well as antioxidant activities more than water, ethyl acetate, chloroform and acetone extracts [13,29,30]. From literature, it has been well noted that medicinal plants with high amounts of phenols and flavonoids have potent antioxidant actions [31][32][33][34]. This may explain the antioxidant activity of C. peregrinum. In term of DPPH-scavenging potential of methanolic extract, our results are in accordance with those of previous works, indicating close relationship between DPPH radical scavenging effect and total phenolic and flavonoid contents of Apiaceae species such as Ammi majus L. [35], Ferula gummosa Boiss [36], Daucus carota Linn [37], Sesili libanotis (L.) Koch, Sesili libanotis (L.) Koch [38], Coriandrum sativum [39], Foeniculum vulgare, Anethum graveolens [40]. Concerning the reducing power and the total antioxidant capacity, the effectiveness of the methanolic extract may be explained by its high phenol and flavonoid contents. In this context, the results of our investigation are in accordance with those published earlier, which mentioned that containing flavonoids and polyphenols may play an important role in reducing power [7] and they contribute significantly to the total antioxidant activity of many fruits such as red grape [41], Star Apple [42] and medicinal plants [43].
In case of photoprotective activity, it has been well mentioned that flavonoids have significant photoprotective effects because of their UV absorbing capacity and their antioxidants effects [44]. Thus, some flavonoids such as quercetin, kaempferol, galanin and apigenin have been evaluated for their photoprotective activity [9]. Recently, it is well reported that plant extracts rich in flavonoids are capable of absorbing ultraviolet light in the UV-B and UV-A regions [11]. In this context, several authors have associated the photoprotective activity of plant extracts with their flavonoids content [45][46][47]. Thus the photoprotective potential of the methanolic extract of C. peregrinum could be explained by its flavonoids content. Indeed, it has been reported that UV-B rays absorbed by the skin led to the production of aggressive free radicals such as (O 2 , 1 O 2 , HO 2 , OH, ROO) [48]. Hence, the incorporation of antioxidants is now widely recommended in sunscreens. In this context, it also has been reported that the evaluation of most effective extracts for the antioxidant activity would be important for the development of more effective sunscreens [49]. Since, the methanolic extract exhibited antioxidant effects, is having a possibility to be used as sunscreen in pharmaceutics or cosmetics formulations.

Conclusions
In conclusion, our study evaluates, for the first time, total phenolic and flavonoid contents in addition to antioxidant and photoprotective activities of aerial parts of Capnophyllum peregrinum (L.) Lange (Apiaceae) growing in Algeria, our findings revealed that the methanolic extract was found to have a high phenolic and flavonoid contents as well as antioxidant and photoprotective activities. Therefore, it appears to be used a sunscreen in pharmaceutical or cosmetic preparations and as a natural source of antioxidant. Also, it can be speculated that the findings of our work could make the background for further investigation of this species, especially research concerning individual phenolic compounds.