In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (2025)

1 Oxidation of lipids is a big concern for the food industry and consumers; it leads to the development of unpleasant aromas, discoloration of fish flesh, fetid smells, and to potentially toxic reaction products [1]. The role of free radicals in many disease conditions has been well established. Oxidative stress due to the production of free radicals such as the hydroxyl radical, peroxide radical, and superoxide radical is the major cause of a variety of pathological conditions including reperfusion injury, drug toxicity, diabetes, and carcinogenesis such as Parkinson and Alzheimer diseases [2]. To avoid serious illnesses and food deterioration, it is better to inhibit lipid peroxidation in the living body and foodstuffs. Antioxidants are used to protect food products from oxidation that causes discoloration and deterioration [3]. An antioxidant is a molecule that slows or prevents the oxidation of chemical substances. Several synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene, are used as food additives to reduce deterioration, but they cause side effects; they have also been proven as carcinogenic products [4]. These synthetic antioxidants show stronger antioxidant activity than natural antioxidants such as ascorbic acid and α-tocopherol, but there is a concern about their safety with regard to health. Currently, the development of natural antioxidants as an alternative to syntheticones is of great interest among researchers. Phenolic compounds (including flavonoids and phenolic acid), α-tocopherol, and vitamin C, which are present naturally in vegetables, seeds, and fruits, possess the ability to reduce oxidative damage associated with many diseases [5]. Thus, antioxidants, especially those of plant origin, have great appeal to researchers. Over the past decade, there have been several studies on plant extracts and different types of phytochemicals, in particular polyphenols and secondary plant metabolites [6]. It was found that most natural phenolic compounds are able to protect themselves against damage caused by reactive oxygen species.

2 Pistacia is a genus of the Anacardiaceae family [7]. It includes many species widely distributed in the Mediterranean and Middle Eastern regions [8]. Eleven species are used as ornamental plants; some of them are regarded as fruit trees. This species is the key resource of immense economic (in the form of oleoresins) and pharmaceutical importance. Species of the genus Pistacia have occupied an important place in traditional and pharmaceutical medicine since antiquity and have attracted the attention of researchers because of their antioxidant potential besides their antimicrobial and anti-inflammatory activities, particularly due to the presence of flavonoids and other phenolic compounds [9]. The antioxidant activity of Pistacia species is due to the presence of flavones[10], gallotannins [11], and simple phenols such as gallic acid and ρ-coumaric acid, [12] oleoresin, and other essential oils [13]. A study showed the richness of this plant in phenolic compounds and identified a new antioxidant [14]. Another study [15] identified a new anti-Plasmodium falciparum substance, the flavone 3-methoxycarpachromene in the ethyl acetate extract of Pistaciaatlantica. Consequently, many studies have analyzed the antioxidant and free radical scavenging activities of phenolic compounds in Pistacia genus [16–18]. Indeed, their role as natural antioxidants allows the body to fight against a large number of diseases, which arouses more and more interest in the use of this plant in the prevention and treatment of cancer [19], cardiovascular [20], and neurodegenerative diseases [21].

3 The aim of this study is to evaluate the antioxidant activity of Pistaciaatlantica by different methods (in vitro) such as determining its scavenging ability of 2,2-diphenyl-1-picrylhydrazyl, its reducing power, and using phosphomolybdenum and cyclic voltammetry methods. It also aims to identify the phenolic compounds present in Pistaciaatlantica by high performance liquid chromatography (HPLC).

4 The leaves of Pistaciaatlantica Desf were collected in the plant's natural habitat in the month of May 2016 (Fig. 1a). The harvest was made in the Middle Atlas in the Khenifra region (Morocco). Only the aerial parts (leaves) were collected and dried for 15days in the shade at ambient temperature (Fig. 1b). The botanical identification of the plant was made in the Department of Botany and Ecological Plants of the Scientific Institute of Rabat.

Fig. 1

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (1)

5 Phytochemical screening is the main way to highlight the secondary metabolites in the studied plant extracts. This screening has focused either on the formation of insoluble complexes using precipitation reactions or the formation of a colored complex using colored reactions. The characterization of different chemical groups were done according to experimental protocols [22]. The most commonly used operations are: maceration, infusion, and decoction.

6 The moisture content of the Pistaciaatlantica Desf plant was determined by the drying process at 50°C. 5g of the fresh matter was dried for one day; three repetitions were performed for each test, and the average moisture rate (%M) calculated by the following relationship.

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (2)

7 Mf: Fresh plant weight

8 Ms: Dry matter weight

9 Dry matter (DM) is obtained as follows: DM% = 100 – %M

10 The plant Pistaciaatlantica was dried in a dry and ventilated place, protected from sunlight. It was crushed completely and then weighed (M = 30g).The plant material obtained was extracted by solvent using the Soxhlet method in a hydro-alcoholic mixture (ethanol/water and methanol/water: 70/30), for 6hours. The extracts were filtered on Whatman filter paper and evaporated to dry the filtrate using the rotavapor. The extract was suspended in distilled water and partitions were made with increasing polarity of solvents by ethyl acetate and butanol. The extract and all fractions were dried using the rotavapor, and preserved (Fig. 2). Then each fraction was evaluated for its amount of total polyphenols, total flavonoids, and condensed tannins to measure antioxidant activity.

Fig. 2

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (3)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions obtained byethanolic extraction respectively

11 The total polyphenolic content was determined by the Folin–Ciocalteu method. This method was originally described by Singleton and Rossi [23] to determine the total polyphenols content in a given sample. 1.5ml of Folin–Ciocalteu reagent (10%) was mixed with 5 μl of fractions. The solution was heated to 25°C for 10min and mixed with 1.5ml of Na2CO3 solution. Finally, the mixture was diluted with distilled water. Before taking the absorbance at 725nm, the mixture was held for 2hours at 25°C. A calibration curve was plotted for the standard of gallic acid. Total phenolics were calculated asmg of gallic acid equivalent (GAE) per gram of extract (mg GAE/g).

12 Total flavonoid contents of Pistaciaatlantica was estimated according to the method described by Atanasova [24]. 10μl of each fraction was mixed with 0.1ml of chloride aluminum 10%, followed by 20ml of distilled water and completed with absolute methanol to 50ml. The mixture was held for 2hours at 25°C. The absorbance was measured at 433nm. Quercetine was used to plot the calibration curve. Total flavonoids contents were calculated asmg quercetine equivalents per gram of extract (mg QE/g).

13 The amount of condensed tannins are estimated using the vanillin acid method [25]. 20μl of each fraction was added to 3ml of the vanillin/methanol solution (4%, m/v). Then, 1.5ml of concentrated hydrochloric acid was added and allowed to react at room temperature for 20minutes. The absorbance at 499nm was measured against a blank. The concentration of tannins was estimated in milligrams (mg) catechin equivalents per gram (g) of the extract (mg CE/g) from the calibration curve.

14 The phenolic analysis was carried out using an HPLC-PDA system as previously described by Skendi etal. (2017) [26] using a Nucleodur 100C18 core-shell reversed-phase column (250mm × 4.6mm i.d., 5μm particle size, Germany). The mobile phase consists of three solvents: (A) 1% of acetic acid in water, (B) acetonitrile, and (C) methanol. The following gradient program was performed: at 0min, the A:B:C proportion was 90:0:0; at 10min, 80:4:16; at 25min, 75:5:20; at 30min, 65:5:30; at 31min, 40:0:60; at 37min, 35:20:45; and at 50min, 20:80:0. The system was allowed to run for another 5min at 100% of B in order to clean the column before re-equilibrating it at initial conditions. The flow rate of the mobile phase was 1.3ml/min. Injection volume was 50μl of methanolic and ethanolic extracts and the double wavelengths of detection were set at 260and 330nm. The identification of phenolic compounds was made by comparing the retention times and spectra obtained with those of commercially available standard compounds.

15 DPPH solution is prepared in advance by solubilizing 2.4mg of DPPH in 100ml of absolute ethanol. The series of dilutions of the Pistaciaatlantica Desf extracts was prepared. 200μl of each fraction at different concentrations are added to 2.8ml of DPPH. The reference of antioxidant solutions (ascorbic acid) was also prepared under the same conditions. The control consisted only of 2.8ml of DPPH and 200μl of ethanol. The mixture was left in the dark for 30min until discoloration occurred. The readings were performed by spectrophotometry at a wavelength of 515nm. The results were expressed in the following equation as a percentage of reduction of DPPH (PI%) [27].

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (4)

16 With:

17 Acontrol: Absorbance of the solution containing only DPPH radical solution

18 Asample: Absorbance of the sample solution to be tested in the presence of DPPH

19 The values of concentrations to inhibit or reduce 50% of the initial concentration of DPPH (IC50) were determined graphically by linear regression.

20 The ability of the extracts of the plants to reduce iron (III) was determined according to the method described by Olorunjuwon etal. and Bougatef etal. [28,29]. 1ml of each fraction at different concentrations was mixed with 2.5ml of 0.2M phosphate buffer (pH6.6) and 2.5ml of 1% potassium ferricyanide (K3Fe(CN)6). The mixtures were incubated at 50 °C for 20min, followed by addition of 2.5ml of 10% (w/v) trichloroacetic acid. The mixture was then centrifuged at 3000rpm for 10min. Finally, 2.5ml of the supernatant solution were mixed with 2.5ml of distilled water and 0.5ml of 0.1% (w/v) ferric chloride. After 10min reaction, the absorbance of the resulting solution was measured at 700nm. Increased absorbance of the reaction mixture indicated increased reducing power.

21 The total antioxidant capacity (CAT) of the extracts was evaluated by the phosphomolybdenum method described by Olorunjuwon etal. [28]. This assay is based on the reduction of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green phosphate–Mo (V) complex at acidic pH. 20μl of each extract was mixed with 1ml of reagent solution (0.6M sulfuric acid, 28mm sodium phosphate and 4mm ammonium molybdate). The tubes were capped and incubated at 90°C for 90min. After that, each sample was allowed to cool to room temperature and the absorbance was measured at 695nm against a control. The control solution consisted of 1ml of reagent solution and 1ml of distilled water. The total antioxidant capacity of each extract was calculated from the equation of regression of the calibration range with gallic acid (y = 0.902x + 0.036), with adjusted coefficient of determination R2 = 0.991.

22 Solutions of the extracts dissolved in 0.1M sodium acetate–acetic acid buffer at pH3.6 (used as supporting electrolyte), were prepared for the analysis. Calibration curves were constructed with gallic acid, the solutions of which were also dissolved in 0.1M sodium acetate–acetic acid buffer at pH3.6. The total antioxidant capacity obtained by electrochemical methods was expressed in terms of equivalent of gallic acid. The cyclic voltammograms scans were made from zero to 700mV at a scanning rate of 25mV/s. All measurements were carried out at room temperature using a conventional three electrode system consisting of a glassy carbon working electrode (3-mm diameter), platinum auxiliary electrode, and Ag/AgCl reference electrode. Prior to the measurements, the electrolyte solutions were de-aerated with an inert gas (N2).

23 The phytochemical tests carried out on the Pistaciaatlantica plant revealed the presence of the following secondary metabolism compounds: tannins (gallic and catechic), flavonoids, terpenes, and sterols and the absence of anthocyanins, leucoanthocyanins, saponosides, and alkaloids. Other studies on the same species have indicated the presence of flavonoids and flavones, [9,10] gallotannins [11,30], and simple phenols such as gallic acid and p-coumaric acid. [12]

24 The result of the moisture assay of Pistaciaatlantica Desf leaves is illustrated in figure3. The result shows that the plant contains a moisture content of 5.2%, which confirms the non-water richness of the plant.

Fig. 3

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (5)

25 The liquid–liquid extraction, using various organic solvents, was followed by polyphenol assays, which allowed us to estimate the concentration of polyphenols, total flavonoids, and condensed tannins of the plant Pistaciaatlantica. In fact, two different solvents with different polarities (methanol and ethanol) were used for extraction. They showed a considerable difference in the content of the chemical composition of the plant. The methanolic (F0) and ethanolic (F0′) extracts were found to be similar in condensed total tannin content (TTC) with values of 29.13 and 28.32mg CE/g of extract, respectively (Fig. 4c). Additionally, the ethanolic extract (F0′) was richer in total phenol content (TPC) than the methanolic extract (F0), with values of 260and 108mg GAE/g of extract, respectively (Fig. 4a). We also noticed that the ethanolic extract (F0′) was rich in flavonoids content (TFC) with a value of 129.15mg QE/g of extracts (Fig. 4b). According to the results, the total phenol, flavonoids, and condensed tannins content presented very significant levels in the ethanol extract. However, these results could be due to the polyphenolic content in the ethanolic extract more than in the methanolic extract. It could also have been caused by the possible complex training of some phenolic compounds in the extract which are more soluble in ethanol than methanol. The liquid–liquid fraction showed that the total phenol content (TPC) was more abundant in the ethyl acetate fraction (F0ʹ) with a value of 205mg GAE/g of extract; however, the butanolic fraction (F2) was rich in total flavonoid content (TFC) with a value of 174mg QE/g of extract (Fig. 4ab). Our results can be compared to the work done by other authors working on the same species of Pistaciaatlantica. Yousfi etal. [14] have determined that the total phenol content in the extract of the leaves of Pistaciaatlantica Desf is equal to 117mg GAE/g of extract. On the other hand, Rhouma etal. [31] has found that the total phenol content of the methanolic extract of the leaves of Pistaciaatlantica Desf reaches about 25mg GAE/g of extract. The work carried out by Koffi etal. [32] confirm our results by stating that ethanol in combination with water allow better extraction of total polyphenols. The addition of water to organic solvents increases the solubility of polyphenols by modulating the polarity of the organic solvent [33]. This increase may be due to the weakening of the hydrogen bonds in the aqueous solutions. It could also be due to the increase in the basicity and ionization of polyphenols in such solutions [34]. The solubility of the polyphenols depend mainly on the number of hydroxyl groups, the molecular weight, and the length of the carbon chain of the basic skeleton of the compound [33].

Fig. 4

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (6)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions obtained byethanolic extraction respectively

26 HPLC-PDA (photo diode array) has been used to determine the phenolic compounds in the methanolic and ethanolic extracts of the leaves of Pistaciaatlantica Desf. Figure 5, presents the chromatograms of Pistaciaatlantica Desf. According to this figure, we have identified the molecules that have been used as standard molecules with different wavelengths of 260and 330nm. The phenolic compounds have been identified according to their retention time of each extract; these compounds were compared with the standard molecules. In 260and 330nm wavelengths, we identified the following molecules in methanolic and ethanolic extracts: ascorbic acid, gallic acid, 4-hydroxybenzoic acid, tannic acid, rutin, and quercetin, with high intensity in gallic acid, 4-hydroxybenzoic acid, and rutin in the methanolic extract at 260nm because of the high polarity of methanol comparing to ethanol. Furthermore, we observed that gallic acid presented more intense than the other molecules to be identified in the ethanolic extract in 260nm. In addition, the methanolic and ethanolic extracts showed a lower amount of the quercetin molecule with less intense area at 330nm. The extraction of the phenolic compounds that were identified by HPLC depends on the types of solvents used and their polarity.

Fig. 5

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (7)

27 During this study, a range of concentrations from 0.05 to 1mg/ml was used. The values of the optical density obtained showed a proportional increase in the percentage of inhibition as a function of the concentrations of extracts of the plant and ascorbic acid, which was used as a positive control because of its strong antioxidant activity. The obtained results are illustrated in the form of curves exponential with an R2 between 0.959 and 0.996 (Fig. 6). From Fig. 4, we have seen that the methanolic extract (F0) and the ethyl acetate (F1) and butanolic (F2) fractions of Pistaciaatlantica Desf have a significant capacity to trap the DPPH radical at a concentration of 0.2mg/ml with percentages of inhibition values 94.93%, 91.72%, and 78.33% respectively. The ethanol extract (F0ʹ) and the ethyl acetate (F1ʹ) and butanol (F2ʹ) fractions show percentages of inhibition less than other extracts; they recorded values: 39.94%, 62.01% and 50.9% at concentration 0.2mg/ml, respectively (Fig. 6).

Fig. 6

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (8)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic andaqueous fractions are obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions are obtained byethanolic extraction respectively

28 From the results in table1, we found that the ethyl acetate fraction (F2) presents a very significant activity with a value IC50 = 0.04mg/ml followed by that of the butanolic (F2) fraction with a value IC50 = 0.08mg/ml and the methanolic extract (F0) with a value IC50 = 0.081mg/ml compared with that of ascorbic acid equal to 0.05mg/ml. A low value of IC50 indicates strong antioxidant activity.

Table 1

Extraction byMethanolExtraction byEthanolControl
FractionsF0F1F2F3F0ʹF1ʹF2ʹF3ʹAscorbic acid
IC50 (mg/ml)0.0810.040.080.040.170.130.230.320.05

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (9)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions obtained byethanolic extraction respectively

29 The results indicate that the ethyl acetate fraction (F2) of the Pistaciaatlantica Desf plant is rich in phenolic and flavonoids, compounds extracted by ethyl acetate; they are also responsible for trapping free radicals of DPPH. The results show that all the fractions of Pistaciaatlantica Desf act as antioxidants because they have donor properties of a hydrogen atom. These values could be compared to those obtained from other species of Pistacia [35]. The concentration values for inhibition at 50% (IC50) for the Pistaciachinensis ethanolic and methanolic extracts were 39.4 and 54.4μg/ml, respectively. According to the obtained results, the Pistaciaatlantica Desf extract is endowed with significant antioxidant potential. This high antioxidizing activity of the extracts is attributed to their richness in phenolic compounds, which constituted the highest percentage in content of the extract (polyphenols, flavonoids). Astudy carried out by Kang etal. [36], suggested that polar molecules present in plant extracts contribute to increased anti-free radical activity.

30 The reducing power assay is often used to evaluate the ability of antioxidants to donate electrons [37]. Different studies have reported that there is a direct correlation between antioxidative activities and the reducing power of some bioactive compounds. In this study, the ability of the extract and fractions to reduce Fe3+ to Fe2+ was determined. For the reducing power assay, the presence of reductants (antioxidants) in the tested samples resulted in the reduction of Fe3+/ferricyanide complex to its ferrous form. Figure7shows the reducing power activities of the different extracts and fraction compared with ascorbic acid. Reducing power of all extracts and fractions increased with increasing concentrations (0.1 to 4.6mg/ml). Several works also reported that the reducing power increased with increasing amount of samples [38]. The reducing power of the methanolic extract (F0), the butanolic (F2) and ethyl acetate (F1) fractions of the Pistaciaatlantica Desf leaves were 2.215, 2.667, and 2.006 at a concentration of 1.16mg/ml; it was 2.183, 2.667, and 2.157 at a concentration of 2.32mg/ml respectively. According to these results, we found that the butanolic fraction (F2) has a high antioxidant activity. However, the ethanolic extract (F0ʹ), and the butanolic (F2ʹ) and ethyl acetate (F1ʹ) fractions recorded lower values than other fractions (F0), (F1), and (F2) at the same concentration of 1.16 and 2.32mg/ml (Fig. 7).

Fig. 7

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (10)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions are obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions are obtained byethanolic extraction respectively

31 The EC50 concentration was determined to compare the reductive activity of the extracts and fractions. The results are summarized in table2. In table2, the antioxidant capacity of the extracts and fractions were expressed by the determination of their effective concentrations (EC50) which corresponds to an absorbance equal to 0.5. According to the results, it can be seen that the lowest EC50 concentrations were reported in the methanolic extract (F0), the ethyl acetate (F1) and butanolic (F2) fractions and ascorbic acid. They recorded EC50 values of 0.07, 0.03, 0.02, and 0.052mg/ml, respectively (Table 2). Indeed, the highest EC50 concentration was recorded in the ethanolic extract (F0ʹ) and the ethyl acetate (F1ʹ) and butanolic (F2ʹ) fractions equal to the values 0.189, 0.145, and 0.129, respectively. Furthermore, these results enabled us to conclude that the butanolic fraction (F2) of Pistaciaatlantica Desf leaves had better antioxidant activity compared to ascorbic acid and all extracts.

Table 2

Extraction byMethanolExtraction byEthanolControl
FractionsF0F1F2F3F0ʹF1ʹF2ʹF3ʹAscorbic acid
EC50(mg/ml)0.070.030.020.1530.1890.1450.1290.6610.052

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (11)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions obtained byethanolic extraction respectively

32 All studies have shown that there exists a correlation between the polyphenol concentration and the antioxidant activity, which confirms that polyphenols are potent antioxidants capable of inhibiting the formation of free radicals and of opposing the anti-oxidation of macromolecules. These results are also consistent with that of several authors who have reported similar positive correlation between total phenolic content and antioxidant activity [12,38].

33 The total antioxidant capacity of the methanolic and ethanolic extracts and fractions of Pistaciaatlantica Desf were measured by spectrophotometry using a method known as the phosphomolybdenum method. This method is based on the reduction of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green phosphate–Mo (V) complex with a maximum absorption at 695nm. The results are expressed in milligrams of gallic acid equivalents per gram of the extract (mg GAE/g of extract).

34 Table 3shows the total antioxidant capacity (TAC) of the fractions from the leaves of Pistaciaatlantica Desf in the following order: the aqueous fraction (F3ʹ) > butanolic fraction (F2ʹ) > ethyl acetate fraction (F1ʹ) > methanolic fraction (F0). The aqueous fraction (F3ʹ) showed a total antioxidant activity greater than the other extracts or fractions with a value of 99.84mg GAE/g of extract.

Table 3

Extraction byMethanolExtraction byEthanol
FractionsF0F1F2F3F0ʹF1ʹF2ʹF3ʹ
TAC (mg GAE/g ofextract)70.80963.61879.8464.4861.21964.07283.67899.584

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (12)

F0: Methanolic extract; F0ʹ: Ethanolic extract; F1, F2, andF3: Ethyl acetate, butanolic, andaqueous fractions obtained bymethanolic extraction respectively; F1ʹ, F2ʹ, andF3ʹ: Ethyl acetate, butanolic, andaqueous fractions obtained byethanolic extraction respectively

35 This antioxidant power of the aqueous fraction (F3ʹ) is probably due to the high solubility of the reductive bioactive molecules in this fraction compared to the other fractions. The fraction contains more reducing-effect compounds and has different affinities for molybdate Mo (IV). This effect is probably attributed to the fraction's richness in phenolic compounds and flavonoids. The antioxidant activity of a phenolic compound depends on its structure, in particular, the number, the positions of the hydroxyl groups and the nature of the substitutions on the aromatic rings.

36 Among the solvents tested, sodium acetate is the best in solubility. However, there is not much work that has been done on the solvents used in antioxidant activity by electrochemical methods. The electrochemical technique that is used in our study is cyclic voltammetry. In this technique, currents of the different peaks such as the peak anodic current (Ipa) and correspondent potential, which is the peak anodic potential (Epa) observed by each voltammogram were measured. Figure 6show the cyclic voltammograms of gallic acid at different concentrations; oxidation peaks in the region of 400–500mV can be observed. There was linearity of the gallic acid concentration with the anodic current measured by the voltammograms, giving the relationship (y = 32.97x + 0.536) in the gallic acid concentration interval from 0.007 to 0.06mg/ml, with R2 = 0.95.The use of cyclic voltammetry led to better linearity of the calibration curves and higher intervals of linearity.

37 The calibration of the electrochemical method, to find the ideal concentration of the extract to be used, was also carried out with ethanolic and methanolic extracts of the Pistaciaatlantica Desf leaves. Cyclic voltammograms of the ethanolic and methanolic extracts were obtained at concentration 0.1mg/ml (Fig. 8ab). In the cyclic voltammograms, one oxidation peak was observed between 0.501 and 0.436V in the ethanolic and methanolic extracts respectively. The oxidation peak potential depends on the structure of the antioxidant molecule. Phenolics with meta-diphenols and isolated phenols, such as t-resveratrol, ferulic acid, malvin, vanillic acid, and p-coumaric acid, are typically found to have high oxidation peak potentials than compounds with ortho- or para-diphenol groups, such as gallic acid, morin, and tannic acid [39,40]. The calculation of TAA (total antioxidant activity) of the extract at a specific concentration was carried out with the previously obtained calibration curves, using gallic acid as standard. The anodic current values represent the oxidation of the phenolic compounds contained in the ethanolic and methanolic extracts of the plant. These results are shown in table4. All ethanolic and methanolic extracts of Pistaciaatlantica Desf have been observed to have total antioxidant activity total (TAA). The ethanol extract showed a greater value of TAA than that of the methanolic extract. These extracts had values of 184.4 and 119.6mg GAE/g of extract respectively.

Fig. 8

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (13)

Table 4

Conc (mg/ml)Electrochemical parametersEthanolic extractMethanol extract
0.1Peak anodic potential (Epa) (V)0.5010.436
Peak anodic current (Ipa) (i/μA cm−2)15.7510.41
Conc (mg GAE/ml)0.4610.299
TAA (mg GAE/g)184.4119.6

In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (14)

38 Based on these results, we concluded that the methanolic and ethanolic extracts and their fractions of Pistaciaatlantica Desf have a total content of phenols, flavonoids, and condensed tannins, as well as significant antioxidant activity by different methods namely: free radical trapping activity, iron reduction, total antioxidant capacity (or phosphomolybdate) and cyclic voltammetry. The plant can be used as a natural antioxidant in the food industry (but after a toxicological review) to preserve lipid peroxidation, and in the pharmaceutical industry to prevent and treat various human diseases. This study will provide a base for future studies. It would be interesting to continue investigating the constituents of the various phenolic compounds of this plant in order to identify the individual components (or the combination of components) that are responsible for this biological activity.

the authors have no conflicts of interests to declare.

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In vitro Evaluation of Antioxidant Activity of the Methanol and Ethanol Extracts of Pistacia atlantica Desf from Morocco (2025)
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