INTRODUCTION:

Medicinal plants have been the traditional source of raw materials for medicine wich contain many natural products and that presents antimicrobial, antioxidant, anti-inflammatory, anticancer, analgesic, antiseptic and insecticide properties use in the prevention and treatment¹.

An estimate suggests that about 13,000 plant species worldwide are known to have use as drugs. The trend of using natural products has increased and the active plant extracts are frequently for new drug discoveries and for the presence of antimicrobials. In recent years one of the areas which attracted a great deal of attention is the possible therapeutic potential of antioxidants in controlling degenerative diseases associated with marked oxidative damage². Several plant extracts and different classes of phytochemicals have been found to have quite prominent antioxidant activity. The term “oxidative stress” means an increase of species which contains more reactive oxygen in them. These species are produced as by products during many metabolic processes. Among these hydroxyl radicals is most reactive oxidant than others because of its high reduction potential and relatively high velocity constants. These reactive oxygen species are extremely reactive. They have one or more unpaired electrons. They can cause oxidative damage to cells and propagate through the membrane as well. Structures and functions of many biomolecules are damaged which further cause many diseases like progressive nervous system disorder (Parkinson's disease) and Alzheimer disease³. The importance of antioxidants in food: If intake of lipid peroxides exceeds in the body, it may lead to adverse health effects. In order to reduce oxidative stress in the body there is a need of antioxidant defense system to stop over production of free radicals.

Argania spinosa L. is a xerothermophilic, endemic to Nord-Ouest Africa⁴ and Maroc⁵ In Algeria, on the beach in the northwest of the Hamada du Drâa de Tindouf (Sahara occidental Algeria)⁴, Argania spinosa is a day earlier. This plant used in traditional medicine by mouth for treated cardiovascular for the treatment of diabetes, gastritis, ulcers and microbial diseases. It is also possible to use an injection to prevent dysentery, migraines and illness⁶, rich in acids and antioxidants. The argan tree are used by local populations: the wood and the lignified shell, Fruit for heating, fruit almond for argan oil production, the foliage, fruit pulp, and the oil cake (argan oil production residue) for cattle^7,8. The fruit pulp of the argan tree is naturally consumed in the argan plantations by goats⁹. The chemical composition of the derivatives of Argan fruit pulp was investigated with the objective of identifying new metabolites that could enhance the industrial and commercial value of Argan fruit pulp¹⁰. Furthermore, it is essential to use supplementary studies to confirm scientific findings on Santé Humaine¹¹. More studios on the territory of the Argan tree are designed to remove the maladies that tell the diabetics and the maladies of the cardiovasculature. A new study is carried out on the territory of the country. ‘Argane for better anti-plaque effects and antithrombotics¹². The phytochemical components of various parts of the argan tree have been identified¹³, According to¹⁴, argan oil from fruits might improve plasmatic lipid profiles, paraoxonas activity, and LDL peroxidation in healthy Moroccan males while also protecting against atherosclerogenesis and cancer. Therefore, the aim of this study was to examine the phytochemical screening, total polyphenol, flavonoids and tannin contents, as well as in vitro antioxidant activity of several extracts of Argania spinosa L., as well as to discover possible therapeutic uses and areas for further investigation. To the best of our knowledge, there are few research has been done on these plant extracts for the treatment of oxidative stress. These experiments were also carried out to describe the antioxidant effects to determine whether traditional medicine's use for inflammation is warranted.

MATERIALS AND METHODS:

Plant material:

The ripe fruits of Argania spinosa L. were collected from two origins (when they fell to the ground): Argan tree located in Tindouf in the South-West of Algeria: 27°39’57.8” N 8°06’44.8”W (Oued-Djezz nursery and the Argan trees from Stidia (Mostaganem) wich are located on the coast (Mediterranean area), with a geographical position of 35°48’11.6”N 0°03’59.5”W, both regions characterized by a Mediterranean bioclimatic regime (Figure 1).

Figure 1. Regins of argan trees: endemic in Stidia (a) and Tindouf (b) in the Algerian Sahara.

Preparation of hydro-methanol extract and sub-fractions:

The extraction was done by fractionation using liquid- liquid method ¹⁵. Approximately 100 g of powdered plant materials (ripe fruits of Argania spinosa L.) was soaked separately in a methanol-water mixture (80 %, v/v) for 24 h at room temperature with occasional stirring. The mixtures were filtered separately and residues were extracted with two additional 1L portions of methanol: water (1:1) for 4 h. Then the solutions were filtered through muslin cloth, and the solvents were evaporated under reduced pressure to produce an initial hydro-methanol extract (HME). HME was successively extracted with different solvents of increasing polarity: hexane for defatting, ethyl acetate for glycoside flavonoids and butanol. Each fraction was evaporated to dryness under reduced pressure to produce ethyle acetate extract (EAE) and n-butanol extract (BuE) for Tindouf and Stidia regions, respectively. All the solvents weres eliminated by evaporation under reduced pressure and the resulting sub-fractions were stored at 4°C for further pharmacological studies.

Phytochemical Screening:

The hydro-methanol and sub-fractions Argania spinosa L. were subjected to screening to detect the presence of potential phytochemical constituents such as alkaloids, flavonoids, polyphenols, hydrolysable tannins, condensed tannins, free quinones, anthraquinones, proteins, coumarins, terpenoids, saponins according to published procedures¹⁵. These are qualitative analyses based on coloring and/or precipitation reactions.

Determination of total phenolic contents:

The total amount of phenolic compounds in the extracts was evaluated using the folin-ciocalteu method ¹⁶.^.Briefly, 100μL of different extracts were mixed with 500μL of folin-ciocalteu reagent for 4 minutes, then 400 μL of 7.5% aqueous Na₂CO₃solution was added. Subsequently, the absorbance of the solution at 765nm was measured after 2hours of incubation. The amount of polyphenols was measured as gallic acid equivalent (GAE)/mg dry extract (DE). The total concentration of polyphenols in various extracts was calculated using a gallic acid standard curve ranging from 0.00 to 160 μg/mL. All analyses were performed in triplicate (n = 3).

Determination of total flavonoids contents:

Total flavonoids were assessed using the aluminum chloride method¹⁷. According to this approach, 1 mL of extract was added to 1mL of aluminum chloride solution (2%). After 10min of incubation, the absorbance of the mixture at 430nm was measured against a methanol blank. Total flavonoid concentrations were reported as μg quercetin equivalent (QE) per mg dry extract (DE). Total flavonoid concentration in various extracts was calculated using a quercetin standard curve ranging from 0.00 to 40μg/mL.

Determination of condensed tannins contents:

Condensed tannins tend to bind to vanillin and form a red complex when bound to phenolic rings according to vanillin method¹⁸. Briefly, 750μL of 4% vanillin solution was added to each extract. Then, 374μL of concentrated hydrochloric acid was added, and the mixture was incubated for 20 minutes at room temperature. Absorbance readings were recorded at a wavelength of 765nm. A standard calibration curve of varying catechin concentration was used to determine the amount of condensed tannins.

In vitro antioxidant capacity:

DPPH radical scavenging assay:

The 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay was used to evaluate the free radical scavenging ability of the extracts by measuring the reduction in the maximum absorbance of DPPH at 517nm¹⁹. In this method, 50μL of different concentrations of the extracts/standard were mixed with 1.25mL of DPPH solution in methanol (0.004%). After 30min of incubation in the dark at room temperature, the absorbance of the sample at 517nm was measured. Butylated hydroxytoluene (BHT) was used as a positive control. The antioxidant capacity was calculated using equation : I% = (A_blank - A_test / A_blank) × 100. Where A_blank= absorbance of the solution excluding the test sample, and A_test = absorbance of the test sample.

Statistical analysis:

Results are represented as the mean ± standard deviation (SD) and all measurements were conducted in three determinations (n=3). The statistical interpretation was directed by the help of Student’s t-test or by one-way analysis of variance (ANOVA) for significance with the aid of GraphPad Prism-5.03; differences were examined significant at p ≤ 0.05.

RESULTS AND DISCUSSION:

The yield:

The yields of Tindouf and Stidia extracts were determined from the weight of dry plant matter. According to the results presented in Table 1. In the methanol extract: Tindouf: 16.53%, Stidia: 19.86% The yield of Stidia in methanol is also higher, but the difference is less marked According to our results, the extraction rate was significantly higher than that obtained by²⁰. This could be explained by the solvent and the extraction method used., the ethyl acetate extract: Tindouf: 8.49%, Stidia: 10.75% Stidia also has a higher Ethyl Acetate rate; while for the Butanol extract: Tindouf: 2.83%, Stidia: 1.19% Tindouf has a higher butanol rate. In addition, A. spinosa samples collected from the Stidia site showed higher yields than those from the Tindouf site. The pulp yielded a yellow-orange extract with a slightly acidic fruity odor. Several factors influence yield variations between Tindouf and Stidia, such as climatic conditions and soil, botanical and genetic variations of the plants, and cultivation and extraction techniques. These factors impact the quantity of phytochemicals and the quality of the plant material. In addition, yield variation is also influenced by ecological interactions and environmental pressures.

Table 1. Yields of the extraction of A. spinosa pulp (Tindouf, Stidia)

Study region	Yields (%)
Study region	HME	EAE	BuE
Tindouf	16.53	8.49	2.83
Stidia	19.86	10.75	1.19

Phytochemical Screening:

Qualitative phytochemical studies were performed on extracts using suitable chemicals and reagents to confirm the presence of alkaloids, flavonoids, polyphenols, free quinones, tannins, coumarins, anthraquinones, proteins and terpenoids. Our results revealed the presence of alkaloids, polyphenols, free quinone, anthraquinones, tannins, terpenoids and coumarins, while the proteins were absence in all extracts. The saponins were detected only in the sub-fractions of A. spinosa L. from Tindouf. The phytochemical screening results of the extracts are reported in table 2. However, the absence of some phytochemicals in plant extracts does not mean that these compounds are not present. The extraction methods used in the studies might not be efficient enough to extract desired metabolites. Phytochemical analysis reported by Zhar et al.²¹on the A. spinosa extracts revealed the presence of tannins, flavonoids, saponins, unsaturated sterols, terpenes, steroids and sterols, which does agree with our results. It is possible to conclude that the maturity of the fruit, the region and the morphotypic variations have a significant impact on the variations of these phytochemical compounds in the pulp of the argan fruit^22,23,24.

Table 2: Phytochemical screening of different extracts from A. spinosa L. of two regions (Tindouf and Stidia)

Phytochemical compounds	Tindouf			Stidia
Phytochemical compounds	HME	EAE	BuE	HME	EAE	BuE
Alkaloids	+	+	+	+	+	+
Polyphenols	+	+	+	+	+	+
Flavonoids	+	+	+	+	+	+
Free quinones	+	+	+	+	+	+
Anthraquinones	+	+	+	+	+	+
Coumarins	+	+	+	+	+	+
Proteins	-	-	-	-	-	-
Hydrolysable tannins	+	+	+	+	+	+
Condensed tannins	+	+	+	+	+	+
Terpenoids	+	+	+	+	+	+
Saponin	+	+	+	-	-	-

Key: += presence, -=absence.

Total phenolic, flavonoids and condensed tannins contents:

Polyphenols, flavonoids and condensed tannins were measured using spectrometric techniques. Different extracts of Tindouf and Stidia pulp of A. spinosa were analyzed to evaluate total phenolic concentrations (TPC), total flavonoid concentrations (TFC) and tannin concentrations (TC) using Folin-Ciocalteu reagent, aluminum chloride and vanillin methods, respectively. Quantitative tests were performed to measure the concentration of phytochemicals present in the extracts. Table 3 presents the results, indicating that TPC, TFC, TC. When comparing the extracts of Tindouf and Stidia, significant variations in polyphenol content are noted. In both regions, the methanol extract present the highest content of polyphenols with a value of 172.55±1.46 and 151.60±0.85μg gallic acid equivalent (GAE)/mg of the dry extract in Stidia and Tindouf, respectively. In flavonoids analysis, butanol and methanol extracts present a high amount of flavonoids with a values of 14.75±0.21 and 13.46±0.76μg quercetin equivalent /mg dry extract in Tindouf and Stidia, respectively. Whereas, in tannin analysis, methanol extract showed the high amount of condensed tannins with avalue of 31.04±0.75 μg QE/mg of extract, in Stidia region (Table 3).

Table 3: Total phenolic, flavonoids and condensed tannins contents of extracts.

Regions	Extracts	TPC (µg GAE/ mg dry extract)	TFC (µg QE/ mg dry extract)	TC (µg CE/ mg dry extract)
Tindouf	HME	151.60 ± 0.85	1.88 ± 0.17	11.43 ± 0.23
	EAE	12.08 ± 2.80	7.87 ± 0.58	14.82 ± 0.83
	BuE	108.06 ± 4.51	14.75 ± 0.21	10.26 ± 1.47
Stidia	HME	172.55 ± 1.46	13.46 ± 0.76	31.04 ± 0.75
	EAE	89.01 ± 1.09	8.13 ± 0.04	18.85 ± 5.30
	BuE	147.63 ± 2.07	11.61 ± 0.02	29.65 ± 0.11

The data represent the mean ± SD of three determinants.

TPC: total phenols content (µg GAE/mg dry extract);

TFC: total flavonoids content (µg QE/mg dry extract);

TC: total condensed tannins (µg CE/mg dry extract). Data are presented as the mean ± SD (n = 3).

Phenolic compounds, especially flavonoids, play a crucial role as secondary metabolites in plants, supporting various physiological processes and acting as stress biomarkers, enabling plants to survive under various environmental conditions²⁵. This difference in the polyphenolic profiles of the two stations highlights the impact of geographical and environmental conditions on the chemical composition of plants, especially for polyphenols and flavonoids, which are sensitive to growing conditions. There may be a slight or a large variation in the composition, depending on various factors, including the extraction process²⁶. The present results corroborated with the research of^27,28,29, which also reported high levels of phenolic compounds in extracts of similar plants. On the other hand, other studies^11,19 reported lower values in polyphenols and tannins which was disagree with this study. According to³⁰ the presence of phenolic compounds can be affected by plant maturity at harvest, climatic conditions and geographical location, plant ecotypes, time and extraction methods. Phenolic compounds are present in almost all plants and have physiological and morphological importance due to their antioxidant potentials. It is therefore essential to quantify phenolic contents and evaluate their contribution against oxidative stress³¹ .

Antioxidant activity:

Many plants extracts exhibit efficient antioxidant properties due to their phytoconstituents, including phenolic acids and flavonoids. In the present study, reduction of DPPH^• radicals can be significantly observed at 517nm by the extracts of A. spinosa L. Measured by DPPH^• method (Figure 3) the free radical scavenging activity (IC₅₀) of A. spinosa extracts are ranged from 05.83±0.00 to 58.39±0.00µg/mL, respectively, compared to BHT as standard (87.26 µg/mL). The free radical scavenging activity of the HMSE, HMTE and BuSE showed a high activity with an IC₅₀= 05.83±0.00, 08.40±0.00 and 10.57±0.00 µg/mL, respectively. Furthermore, EATE, EASE and BuTE present an important antioxidant activity with an IC₅₀= 25.93±0.00, 28.35±0.00 and 58.39±0.00µg/mL, respectively. This is due in part to the presence of phenolic compounds and flavonoids. This radical scavenging activity of extract could be related to the nature of phenolic, thus contributing to their electron transfer or hydrogen donating ability.

Figure 1: Antioxidant activities of extracts measured by scavenging DPPH radical. Data are presented as the mean ± SD (n = 3). ***: p < 0.001.

The present work was the subject of a comparative study between Tindouf and Stidia extracts antioxidant activity under the same experimental conditions. In vitro antioxidant activity estimation showed that both regions had significant antioxidant potential. The antioxidant capacity of Stidia extracts were found to be significantly higher than that of Tindouf extracts. The different analysed extract fractions of both regions revealed the presence of high content of phenolic compounds responsible for the observed antioxidant activities. Several researches have demonstrated a significant correlation between the amount of phenolic compounds and the antioxidant activity of the body. Phenolics form an important category of antioxidants due to their ability to scavenge free radicals³¹. In comparison, the study conducted by Benaouf et al.¹¹ in the Mostaganem region revealed an IC₅₀ of 386.56±1.65mg/ml, which suggests a significantly lower antioxidant activity. Similarly, El Manfalouti et al.³²reporteda very low IC₅₀ with a value of 0.37±0.07mg/mL, which does not corroborate with our results for all extracts. In addition, Alaoui et al. ²⁷reported the maximum IC₅₀ value of 1.57μg/mL for the shell precipitate, followed by the leaves (2.63μg/mL), the branch (11.23μg/mL), the pulp (90.74μg/mL) and the seed (372.87μg/mL). The shell, leaves and branch have IC₅₀ values very similar to those of ascorbic acid (1.92μg/mL) compared to those of the pulp and seed. The shell, leaves and branch have a higher antioxidant activity than the pulp and seed which does corroborate with our results. In addition, the study of Chernane et al. ²⁸present an IC₅₀= 9.73±0.21μg/mL in the methanol extract is in agreement with our results. These significant differences can be explained by fluctuations in climatic conditions, extraction techniques and particular compositions of phytoconstituents. These phenolic compounds, their derivatives and various subgroups of phenolic compounds contribute their major role in exhibiting antioxidant activity. Phenolic compounds exhibit their antioxidant activity by rapidly donating their electrons or a hydrogen atom to free radical or molecule that is oxidized and get paired. They chelate metal ions that speed up the lipid peroxidation process. In this way, they terminate free radicals or making it less harmful and prevent them to cause lipid peroxidation. Compounds of coumarin exhibit properties against inflammation, hypertension, blood coagulation, tuberculosis, convulsion, obesity, reactive oxygen species, hyperglycemia, neural disorders, tumors, bacteria, fungal and viral species³⁰.

Argan and olive leaves can be considered as having significant antioxidant potential, expanding their ability to replace synthetic antioxidants in the food industry³³. On the other hand, different phenolic compounds such as ferulic, syrin, p-hydroxybenzoic, protocatechuic, vanillic and caffeic acids, quercetin, myricitrin, epicatechin, rutin, hyperoside and isoquercetin have been identified in seeds oil, leaves and fruit pulp³⁴.

CONCLUSION:

In conclusion, it is highlighted that the Stidia region of Algeria contains a total amount of phenolic compounds and antioxidant properties. In addition, the Stidia region present the highest amounts of total phenols, flavonoids and tannins, as well as significant antioxidant capacities. This could explain the use of this medicinal plant in traditional medicine. In addition, these results show that the phenolic fractions of Argania spinosa fruit pulp has promising prospects for the treatment or prevention of various diseases related to oxidative damage. Although the composition of the phenolic fraction of fruits can evolve over the years, they deserve a better valuation in the pharmacological, cosmetic, and agro-food fields because of their antioxidant properties. Nevertheless, these results provide further research that seeks to study the pharmacological effects and potential uses of argan fruit extracts in the nutrition and medicine sectors. Studies on improving extraction techniques and characterization of the various bioactive compounds of this plant that are responsible for the antioxidant properties and other activities. However, further research is needed to fully understand the clinical implications and molecular mechanisms behind the effects of argan extracts on health.

ACKNOWLEDGEMENT:

This work is supported by DGRSDT, Algeria. We would like to thank the Laboratory of Applied Biochemistry, Faculty of Nature and Life Sciences, Setif-1, University Ferhat Abbas, Algeria, for providing laboratory resources and valuable assistance during the experiments.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 22.02.2025 Revised on 06.06.2025

Accepted on 18.08.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):186-192.

DOI: 10.52711/0974-360X.2026.00028

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