1. INTRODUCTION:

The Oxidative stress is a condition happens when the production of reactive oxygen species (ROS) exceeds the capacity of the body's antioxidant defense systems to neutralize them¹. This imbalance leads to the accumulation of free radicals, which can damage biomolecules such as lipids, proteins, and DNA². Over time, this oxidative damage leads to the onset and progression of various chronic and degenerative diseases, including cardiovascular disorders, diabetes, cancer, and neurodegenerative conditions²^,³.

To combat oxidative stress, there has been growing interest in identifying natural sources of antioxidants, particularly from medicinal plants⁴^,⁵. These plants often synthesize a wide array of secondary metabolites, such as phenolic compounds, flavonoids, and tannins that are capable of scavenging free radicals and enhancing cellular antioxidant defenses⁶^,⁷. The exploration of such natural antioxidants not only supports the development of safer therapeutic agents but also aligns with the global trend toward phytotherapy and functional foods.

Medicinal plants continue to play a significant role in the discovery of bioactive compounds due to their pharmacological diversity and accessibility⁴^,⁸. Many plant species that grow in harsh environmental conditions, such as salinity or drought, often accumulate high levels of stress-responsive secondary metabolites⁹. Halophytic plants, in particular, are promising candidates for anti-oxidant studies owing to their unique biochemical adaptations¹⁰. The genus Atriplex (family Chenopodiaceae) comprises over 300 species of halophytes and xerophytes, commonly found in saline soils and distributed in arid and semi-arid regions worldwide¹¹^,¹². Many species of this genus have been traditionally utilized for their nutritional, medicinal, and ecological significance, including their use in treating diabetes, urinary tract infections, bowel disorders, skin rashes, and cancer, in addition to their use in soil stabilization and as fodder plants¹². Despite this, many Atriplex species remain underexplored in terms of their phytochemical composition and biological activities.

Atriplex micrantha, commonly known as two-scale saltbush, is one such species that has received little to no attention in scientific literature. Native to saline and disturbed soils, this halophytic plant may possess adaptive biochemical mechanisms that contribute to its resilience. These mechanisms often involve the production of secondary metabolites such as phenolics and flavonoids, which are known for their anti-oxidant effects and a plenty of biological properties. To our knowledge, no comprehensive phytochemical or antioxidant analysis of this species has been previously reported.

This research was targeted to explore the phytochemical composition and anti-oxidant potency of Atriplex micrantha for the first time. A qualitative phytochemical detection was done on ethanolic and ethyl acetate extracts of the aerial parts of the plant to detect the existence of polyphenols, flavonoids, tannins, saponins, alkaloids, and anthraquinones. Furthermore, the total phenols content (TPC), total flavonoids content (TFC), and free radical scavenging activity using the DPPH assay were evaluated. The antioxidant activity was expressed as RSA% and the half maximal inhibitory concentration (IC₅₀), with gallic acid used as a reference standard. This study provides baseline data on the chemical and biological profile of A. micrantha, contributing to the expanding knowledge of underexplored medicinal plants and their potential health benefits.

2. MATERIALS AND METHODS:

2.1. Chemical reagents:

A variety of analytical-grade reagents were utilized, including absolute ethanol and quercetin (Sigma-Aldrich, Germany), absolute methanol (Panreac, Spain), gallic acid and anhydrous sodium carbonate (AvonChem, UK), Folin-Ciocalteu reagent (Merck, Germany), sodium acetate, chloroform (99%), aluminium chloride, and ferric chloride (Riedel-de Haen, Germany), concentrated hydrochloric acid (Shamlab, Syria), and 2,2-diphenyl-1-picrylhydrazyl (DPPH, Tokyo Chemical Industry, Japan).

2.2. Botanical Source:

Specimens of Atriplex micrantha were gathered in December 2024, during their fruiting stage, from Ain Mneen, located in the Damascus countryside in Syria (altitude: 1200 meters, coordinates: 33°38′32″N, 36°52″E). The plant's aerial parts were separated and subjected to shade drying at ambient temperature for 20 days prior to grinding into fine powder for extraction.

2.3. Extraction procedure:

A sample of 30grams of dried plant powder was subjected to extraction with 300mL of 95% ethanol in an ultrasonic bath at 60°C for half an hour. The residue from this process was then macerated for an additional 24hours and filtered. This extraction cycle was repeated three times for completeness. All combined filtrates were concentrated using a rotary evaporator at 40°C. Yields were determined using:

Extraction yield (%) = (mass of dried extract / mass of initial plant material) × 100

The final extracts were kept at 4°C until further analysis.

2.4. Preliminary Phytochemical Evaluation:

Each extract underwent qualitative screening for phytoconstituents such as flavonoids, anthraquinones, tannins, coumarins, saponins, and alkaloids. Protocols followed were adapted from established literature.¹³^,¹⁴.

2.5. Quantification of total Phenols content (TPC) of A. micrantha:

Total phenolic compounds were quantified via the Folin-Ciocalteu assay according to AlKhoury and AlKhatib¹³. Results were standardized against gallic acid, with calculations based on the calibration curve (y = 0.0024x + 0.0487, R² = 0.9953), and expressed as milligrams gallic acid equivalents (GAE) per gram of dried extract.

2.6. Quantification of the total Flavonoids content (TFC) of A. micrantha:

The aluminium chloride colorimetric approach, as per AlKhoury and AlKhatib ¹³ was employed for total flavonoid determination. Standardization was achieved using Quercetin, applying the calibration curve (y = 0.0274x + 0.0154, R² = 0.9951). TFC values are reported as milligrams quercetin equivalents (QE) per gram dried extract.

2.7. Evaluation of DPPH free radicals scavenging activity:

Antioxidant capacity was assessed via the DPPH free radical scavenging assay. Ethanolic solutions of DPPH (4.5mg per 100mL) were prepared. Plant extracts were tested at five concentrations (100–1000mg/L), alongside a dilution series (0.5–10mg/L) of gallic acid. Assays were conducted following Alassaf and Khazem¹⁵. Radical scavenging activity (RSA%) was calculated:

RSA (%) = 100 × (A₀ – A₁) / A₀

where A₀ is control absorbance and A₁ is sample absorbance.

IC₅₀ values (mg/L), indicating the concentration for 50% inhibition of DPPH, were derived from plots of sample concentration versus RSA%.

2.8. Statistical Analysis:

All experiments were performed in triplicate, and data are presented as mean values±standard deviation, analyzed using Microsoft Excel (version 2019).

3. RESULTS AND DISCUSSION:

In this study, the extracts from aerial parts of A. micrantha were analysed for the first time. The approach involved investigating the presence/absence of some secondary metabolites, determining the total content of phenols, flavonoids, and evaluating the anti-oxidant activity using DPPH assay.

The extraction of the aerial parts resulted in two crude extracts, ethanolic extract and ethyl acetate extract. The extraction yield is presented in Table 1. The 95% ethanolic extract produced a higher yield (10.17%) compared to the ethyl acetate extract (4.43 %). This suggests that the extracted substances are of a high or moderate polarity and may present in the form of salts or glycosides more than the free form.

3.1 Preliminary Phytochemical Screening:

The results of the preliminary investigation for secondary metabolites are shown in Table 1. The (+) mark indicates a positive test result, while the (-) mark indicates a negative result.

Table 1: The results of preliminary investigation tests for secondary metabolites in A. micrantha extracts

Secondary metabolites	Test name	Ethanolic extract	Ethyl acetate extract
Polyphenols	Ferric chloride	+	+
Flavonoids	Aluminium chloride	+	+
	Shinoda	+	+
	Wilson-Taubock	+	+
Anthraquinones	Borntrager	-	-
Anthraquinones	Modified Borntrager	-	-
Tannins	Lead acetate	+	+
Tannins	Gelatine	+	-
Coumarins	Fluorescence	+	+
Saponins	Foam	+	-
Alkaloids	Dragendorff	-	-
	Mayer	-	-
	Hager	-	-
	Wagner	-	-

Preliminary phytochemical screening reactions revealed that the studied extracts of A. micrantha are rich in various secondary metabolites. Both extracts tested positive for polyphenols, flavonoids, and coumarins. The presence of flavonoids was further confirmed through positive Shinoda and Wilson–Tauböck tests¹⁶, suggesting the occurrence of flavones and 3-hydroxyflavones¹⁶. Tannins were indicated by a positive lead acetate test in both extracts; however, since this test can also react with general phenolics, further specificity was provided by the gelatin test, which was positive only for the ethanolic extract, confirming tannins in that extract. The foam test for saponins yielded a positive result only in the ethanolic extract. This can be explained by the fact that saponins are water-soluble and thus more readily extracted by polar solvents such as ethanol.

Alkaloids and anthraquinones in their free and glycoside forms were not detected in either extract. But many previous studies reported the presence of alkaloids in other species and determined its content. Further detection could be done to confirm this result.

3.2 Total Phenolic content of A. micrantha extracts:

The TPC of A. micrantha extracts are shown in table 2. The ethanolic extract exhibited the highest TPC 73.59± 0.74 GAE/g DE and the lowest was for the ethyl acetate extract 45.12±1.07 GAE/g DE. This result confirms that ethanol is more efficient than ethyl acetate in extracting phenolic compounds, suggesting that the nature phenolic compounds in A. micrantha aerial parts are of polar or intermediately polar nature and tend to be better soluble in ethanol.

To our knowledge, this is the first report on the TPC of Atriplex micrantha. Comparisons can be made with other Atriplex species. For example, a study by Chaouche et al.¹⁷, the methanol-acetone (60:40) extract of A. halimus yielded a significantly lower phenolic content. Similarly, an aqueous extract of A. halimus leaves prepared by hot water stirring (24h) yielded a TPC closer to our ethyl acetate extract but significantly lower than the ethanolic extract¹⁸. Such comparisons may not be scientifically accurate due to variations in extraction methods, solvents, plant part used, and species identity¹⁹. These differences affect the extractable phenolic content and can lead to discrepancies in reported values²⁰.

3.3. Total Flavonoids content in A. micrantha extracts:

The TFC of the extracts is shown in Table 2. The ethanolic extract exhibited the highest flavonoid content (17.52±0.057mg QE/g DE), whereas the ethyl acetate extract had a lower yield (9.61±0.065mg QE/g DE). The results can be interpreted as suggesting that the flavonoids present in the plant may be in their glycosidic (non-free) form, which is more susceptible to ethanol solubility than ethyl acetate.

A comparative study on A. lasiantha in Pakistan reported a much higher TFC²¹, in contrast, a study from Algeria on A. halimus reported a lower TFC¹⁷. As with phenolic content, it is important to note that direct comparison with other published values is not methodologically significant due to substantial differences in extraction conditions, solvent polarity, plant species, and analytical procedures¹⁹^,²².

3.4 DPPH free radical scavenging activity of A. micrantha extracts:

This study is the first to investigate the free radical scavenging activity of A. micrantha. The antioxidant activity measured using the DPPH radical scavenging activity (RSA%) (figure1) and IC₅₀(table 2) for each extract and gallic acid. The result showed that RSA% increased in a concentration-dependent manner for all samples. All extracts exhibited free radical scavenging activity, the ethanolic extract demonstrated the strongest activity, with an IC₅₀ of 81.0±2.13mg/L. In contrast, the ethyl acetate extract exhibited an IC₅₀ of 154.7±7.5 mg/L.

Figure 1: DPPH free radical scavenging activity (RSA%) of A. micrantha extracts and gallic acid

Table 2: TPC, TFC, and DPPH free radical scavenging activity of A. micrantha ethanolic extracts:

Extract	TPC (mg GAE/g DE)	TFC (mg QE/g DE)	IC₅₀ mg/L
95% Ethanol	73.59 ± 0.74	17.52 ± 0.057	81.0 ± 2.13
Ethyl acetate	45.12 ± 1.07	9.61 ± 0.065	154.7 ± 7.5
Gallic acid			0.78 ± 0.007

These results represent the average of 3 triplicates ± standard deviation (STD)

Accordingly, the ethanolic extract of A. micrantha is classified as having strong antioxidant activity, while the ethyl acetate extract shows moderate activity²³^,²⁴. These classifications reinforce the observed trend in total phenolic and flavonoid contents, indicating that constituents are likely contributing significantly to the antioxidant potential of this species.

These results highlight the potential of A. micrantha as a valuable natural source of antioxidants, especially in the ethanolic extract, and rich in Phenols and flavonoids which are potent secondary metabolites known for their strong antioxidant and other bioactivities. Therefore, further investigation into its bioactive constituents and isolation of chemical compounds responsible for the activity are highly recommended in addition to evaluating other potential activities of this promising plant.

4. CONCLUSIONS:

This research represents the inaugural examination of the phytochemistry and antioxidant properties of the halophytic species Atriplex micrantha. The investigation revealed that extracts prepared with ethanol and ethyl acetate from the plant’s aerial portions contain a diverse spectrum of secondary metabolites, such as polyphenols, flavonoids, tannins, saponins, and coumarins. Among the tested extracts, those obtained with ethanol yielded a higher quantity and demonstrated superior bioactive capabilities.

Quantitative assessments indicated that the ethanol-derived extract contained elevated concentrations of total phenolics and flavonoids compared to the ethyl acetate extract. Moreover, the ethanol extract displayed a more robust antioxidant potential in the DPPH scavenging assay, implicating the significant presence of active antioxidant molecules. The study identified a direct relationship between phenolic-flavonoid levels and free radical scavenging activity, underlining the medicinal value of this plant.

Collectively, these results point to Atriplex micrantha as a promising candidate for natural antioxidant sources. Future studies are warranted for isolation and identification of the specific compounds responsible for biological effects, as well as evaluating other possible activities, including antimicrobial, cytotoxic, and anti-inflammatory potential. Given its adaptability and chemical diversity, this halophyte may hold promise for the development of pharmaceutical or nutraceutical products.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGEMENTS:

Authors are grateful to Ms. Abeer Mahfoud, Ms. Rahaf Srour, and Ms. Dania Hadla for their contribution in the phytochemical screening study.

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Received on 11.08.2025 Revised on 16.11.2025

Accepted on 05.02.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2160-2164.

DOI: 10.52711/0974-360X.2026.00311

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.