Gallic Acid–Rich Ephedra alata: Integrated Phytochemical Profiling, Aromatase Docking, and Optimized Extraction via Box–Behnken Design

Nadjat Chekroun‐Bechlaghem; Nabila Belyagoubi‐Benhammou; Zineb Kaid; Amel Zitouni; Imad Abdelhamid El Haci; Abdelghani Bouchama; Ahmed Djafri

doi:10.1002/cbdv.71618

. 2026 Aug 26;23(8):e71618. doi: 10.1002/cbdv.71618

Gallic Acid–Rich Ephedra alata: Integrated Phytochemical Profiling, Aromatase Docking, and Optimized Extraction via Box–Behnken Design

Nadjat Chekroun‐Bechlaghem ¹, Nabila Belyagoubi‐Benhammou ^2,^✉, Zineb Kaid ^3,⁴, Amel Zitouni ^2,⁵, Imad Abdelhamid El Haci ^2,⁶, Abdelghani Bouchama ^6,⁷, Ahmed Djafri ^6,⁸

PMCID: PMC13510113 PMID: 42644827

ABSTRACT

This study investigated the phenolic composition and antioxidant potential of E. alata using an integrated approach combining phytochemical profiling, molecular docking, and Box–Behnken optimization of extraction conditions. Stem extracts were prepared by maceration, sonication, and ultrasound‐assisted extraction using dichloromethane, acetone, ethyl acetate, methanol, and methanol/water. Solvent polarity markedly affected extraction efficiency, with methanol and aqueous methanol yielding the highest extraction rates (up to 26.11 ± 0.01%), total phenolics (58.09 ± 1.07 mg GAE/g DW), flavonoids (9.81 ± 0.11 mg CE/g DW), condensed tannins (635.23 ± 3.05 mg CE/g DW), antioxidant capacity (500.46 ± 55.10 mg AAE/g DW), and the lowest DPPH IC₅₀ (0.005 ± 0.001 mg/mL). RP‐HPLC‐PDA identified nine phenolic compounds, with gallic acid as the predominant constituent. Molecular docking of gallic acid against human aromatase (CYP19A1) revealed favorable binding (ΔG = −5.88 kcal/mol; Ki = 49.38 µM), supported by hydrogen bonding and hydrophobic interactions. Box–Behnken response surface analysis generated significant quadratic models (R² > 0.95), enabling optimization of extraction conditions for maximum phenolic recovery and antioxidant activity. These findings identify E. alata as a promising natural source of gallic acid and bioactive phenolic antioxidants.

Keywords: antioxidant activity, Ephedra alata, extraction method, molecular docking, phytochemical analysis, response surface methodology

An integrated phytochemical, computational, and statistical approach identifies gallic acid as the predominant phenolic compound in E. alata. RP‐HPLC‐PDA profiling, aromatase molecular docking, and Box–Behnken optimization collectively demonstrate the plant's antioxidant potential and establish optimal extraction conditions for maximizing bioactive phenolic compounds.

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Abbreviations

DPPH: 2, 2‐diphenyl‐1‐picrylhydrazyl
IC₅₀: Inhibitory concentration
RP‐HPLC‐PDA: High‐performance liquid chromatography system
TAC: Total Antioxidant Capacity
TFC: Total Flavonoid Content
TPC: Total Phenolic Content
V/V: volume/volume

1. Introduction

Oxidative stress, defined as the imbalance between reactive oxygen species (ROS) and the body's antioxidant defenses, is increasingly recognized as a central mechanism driving the development and progression of numerous chronic degenerative disorders, including neurodegenerative diseases, cardiovascular diseases, cancer, and diabetes, by damaging DNA, proteins, and lipids [1, 2, 3]. Medicinal plants have long served as a rich source of therapeutic agents and continue to provide new natural products or their derivatives with significant biological activities [4]. For centuries, traditional remedies have guided scientists in the discovery of new drugs to maintain and promote human and animal health [5]. Consequently, increasing research efforts have focused on identifying plant‐derived antioxidants capable of mitigating oxidative stress and reducing the burden of chronic diseases.

Algeria possesses a rich and diverse flora estimated at around 3000 species, of which 15% are endemic and belong to various botanical families [6, 7]. Many of these plants remain underexplored despite their potential pharmacological applications. One such species is E. alata. This plant is a perennial member of the Ephedraceae family, characterized as a small, dioecious, densely branched shrub, 50–100 cm in height, with light green stems, a strong pine‐like odor, and an astringent taste [8]. This species is widely distributed in arid regions of Iran, Palestine, Saudi Arabia, Algeria, Morocco, Egypt, Tunisia, Libya, Lebanon, Jordan, and Iraq, where it grows on rocky, sandy, and clay soils, often near shifting dunes [9]. The genus Ephedra is native to temperate and subtropical zones of Europe, Asia, and the Americas, with notable populations in China, India, Spain, and the United States, particularly along the Rocky Mountains [10]. Traditionally, Ephedra species have been valued for their antipyretic, diaphoretic, circulatory stimulant, and cough‐suppressing properties. In Chinese medicine, they have been used to treat asthma, allergies, colds, flu, and headaches, while in Russia, they were applied against respiratory disorders and rheumatism. Indigenous peoples of the southwestern United States also employed Ephedra preparations for venereal diseases [11, 12]. In Algeria, E. alata is commonly consumed as an herbal tea to treat respiratory ailments such as flu and colds [13]. Beyond its traditional uses, recent phytochemical and pharmacological investigations have renewed interest in E. alata owing to its richness in phenolic compounds and its antioxidant, antimicrobial and enzyme‐modulating activities, highlighting its potential for pharmaceutical and nutraceutical applications [14, 15, 16].

Phytochemical investigations of Ephedra species have revealed a wide range of bioactive constituents, including alkaloids, tannins, saponins, proanthocyanidins, phenolic acids, and flavonoids, which contribute significantly to their antioxidant and antimicrobial properties [17, 18, 19, 20]. Recent studies further demonstrated that extraction conditions strongly influence both the qualitative and quantitative composition of these phenolic metabolites and, consequently, their biological activities. Polar extraction solvents generally enhance the recovery of phenolic acids and flavonoids, leading to improved antioxidant performance [14, 21, 22]. The genus is particularly rich in alkaloids such as ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine [23]. In addition, Ephedra species contain nonalkaloid constituents, including volatile oils, sterols, triterpenes, carbohydrates, and flavonoids, which require appropriate extraction techniques and solvents to be efficiently recovered [24]. Despite their therapeutic value, Ephedra alkaloids, especially ephedrine and pseudoephedrine, are associated with toxicological effects. At high doses, they may induce hepatotoxicity characterized by massive necrosis [25], as well as neurological and cardiovascular side effects such as insomnia, palpitations, headaches, dizziness, and nausea [26, 27]. These findings have shifted current research from alkaloid‐centered investigations toward the characterization of nonalkaloid phenolic constituents, which may provide safer natural antioxidants with promising pharmaceutical potential [15, 16].

Aromatase, a cytochrome P450 enzyme (CYP19A1), is essential for the biosynthesis of steroid hormones. It catalyzes the conversion of androgens (testosterone and androstenedione) into estrogens (estradiol and estrone) through aromatization [28]. This enzyme is present in both men and women and plays a critical role in reproductive function and hormonal regulation. In medicine, aromatase inhibition is an established therapeutic strategy for treating hormone‐dependent cancers, particularly breast cancer in postmenopausal women, by lowering estrogen production [29]. Consequently, naturally occurring phenolic compounds capable of interacting with aromatase have attracted increasing attention as potential lead molecules for future drug development, although computational predictions require subsequent experimental validation.

Despite the growing body of literature describing the phytochemical composition and biological activities of E. alata, comprehensive studies integrating extraction optimization, detailed phenolic profiling, and molecular modeling remain scarce. Therefore, the present study aimed to optimize the extraction of phenolic compounds from E. alata stems using different extraction techniques and solvent systems combined with a Box–Behnken experimental design. The optimized extracts were evaluated for total phenolic, flavonoid, and condensed tannin contents as well as antioxidant activity using phosphomolybdenum and DPPH assays. RP‐HPLC‐PDA analysis was subsequently performed to characterize the major phenolic constituents, whereas molecular docking was employed as an exploratory computational approach to investigate the theoretical interaction between the predominant phenolic compound and human aromatase (CYP19A1). By integrating extraction optimization, phytochemical characterization, antioxidant evaluation, and in silico molecular analysis within a single multidisciplinary framework, this study provides new insights into the valorization of E. alata as a promising natural source of bioactive compounds.

2. Results and Discussion

2.1. Yields and Quantification of Polyphenolic Compounds

Different factors, including the extraction method, solvent type, and the chemical nature of phytochemicals, influence extraction efficiency. In the present study, the extraction yield varied considerably according to both the extraction technique and the solvent employed. The extraction yields obtained under the different experimental conditions are presented in Figure 1 and Table S1.

Effect of extraction method and solvent polarity on extraction yield (A), TPC (B), TFC (C), and condensed tannin content (D) of *E. alata* extracts.

The evaluation of extraction yields obtained using the different extraction methods revealed that the highest recoveries were achieved with aqueous methanol and methanol (Figure 1A). The highest extraction yield was obtained with the methanolic extract prepared by sonification (26.11% ± 0.00%), followed by the aqueous methanolic extract obtained by maceration (22.20% ± 0.00%). These solvents are the most suitable for extracting bioactive compounds from E. alata, regardless of the extraction method, although sonification proved to be the most efficient technique. These results are consistent with those reported by Bourgou et al. [30] for ethyl acetate extracts and are generally comparable to those reported by Boussena et al. [31], who obtained extraction yields of approximately 15.01% and 12.92% for aqueous methanol and methanol extracts, respectively. The differences observed among extraction solvents may be attributed to variations in solvent polarity, diffusion capacity, and the structural characteristics of the extracted phytochemicals, all of which strongly influence the solubility and recovery of bioactive constituents [32].

Recent investigations have similarly demonstrated that solvent polarity remains the principal factor governing the extraction efficiency of phenolic compounds from Ephedra species. Al Jaafreh [33] reported significantly higher recoveries of phenolics using polar alcoholic solvents than less polar solvents, while recent studies on E. alata also confirmed that methanol‐based extraction systems maximize the recovery of antioxidant constituents through enhanced hydrogen‐bond interactions with phenolic hydroxyl groups. These observations further support the extraction strategy adopted in the present study.

The quantitative determination of total phenolic content (TPC), total flavonoid content (TFC), and condensed tannins is presented in Figure 1B–D (Table S1). The solvent exerted a pronounced influence on the recovery of these phytochemicals. Methanol consistently produced the highest phenolic concentrations, with TPC values ranging from 50.89 to 57.04 mg GAE/g DW, while aqueous methanol combined with ultrasound‐assisted extraction yielded the highest TPC value (58.09 mg GAE/g DW), but ethyl acetate proved to be the least effective solvent, producing TPC values ranging from only 6.28 to 9.29 mg GAE/g DW.

The results clearly demonstrate that solvent polarity is the principal determinant governing phenolic extraction efficiency. Polar solvents such as methanol effectively solubilize phenolic compounds because their hydroxyl groups promote strong hydrogen‐bond interactions with phenolic molecules [34, 35]. Conversely, the lower polarity of ethyl acetate and dichloromethane limits their ability to extract highly polar phenolic constituents, resulting in substantially lower TPC and tannin contents [36]. Methanol was particularly efficient for condensed tannin extraction, producing concentrations ranging from 525.40 to 635.22 mg CE/g DW, which agrees with previous reports demonstrating the superior ability of methanol to dissolve high‐molecular‐weight tannins [37]. Mohammed et al. [38] demonstrated that methanolic extracts of medicinal plants exhibited significantly higher total phenolic concentrations and antioxidant activities, which reinforce the hypothesis that solvent polarity is the dominant factor controlling polyphenol recovery, irrespective of the extraction technique employed.

The extraction technique also affected phytochemical recovery. Sonification generally enhanced the extraction of phenolics and flavonoids compared with maceration. For example, methanol extracts prepared by sonification yielded 57.04 ± 0.08 mg GAE/g DW, slightly exceeding those obtained by maceration (55.21 mg GAE/g DW) and ultrasound‐assisted extraction (50.89 mg GAE/g DW). This is consistent with previous studies showing that ultrasound‐assisted extraction enhances cell wall disruption, thereby facilitating solvent penetration and improving the release of intracellular phytochemicals [39]. However, prolonged ultrasonic treatment may generate localized heating and reactive radicals capable of partially degrading high‐molecular‐weight tannins [40]. This phenomenon may explain the slightly lower tannin concentration observed in ultrasonic methanol extracts (525.40 mg CE/g DW) compared with sonified methanol extracts (635.22 mg CE/g DW).

Flavonoid levels also varied according to both the extraction solvent and the extraction method. Methanolic extracts consistently exhibited the highest flavonoid concentrations (7.88 – 9.81 mg CE/g DW), whereas less polar solvents (dichloromethane, acetone, and ethyl acetate) yielded considerably lower concentrations (< 2 mg CE/g DW). Sonification generally enhanced flavonoid extraction, although ultrasound‐assisted extraction produced the highest flavonoid content in methanol (9.81 mg CE/g DW). The relatively higher recovery observed in acetone extracts (up to 1.74 mg CE/g DW) may be attributed to its intermediate polarity, which enables the extraction of both flavonoid glycosides and aglycones [41, 42].

Mohammed et al. [38] reported that methanolic extracts of E. alata exhibited the highest flavonoid content and antioxidant activity. Similarly, Mufti et al. [43] identified flavonoids and phenolic acids as the predominant metabolites in E. alata, confirming the suitability of polar solvents for recovering bioactive phytochemicals.

Condensed tannin recovery followed a similar polarity‐dependent trend, with methanolic extracts exhibiting the highest concentrations (525.40–635.22 mg CE/g DW). Dichloromethane extracted only negligible amounts (4.39–5.02 mg CE/g DW). Sonification was again the most efficient extraction method for tannin recovery in most solvent systems. Statistical analysis revealed significant differences among solvents (F = 4.89, p < 0.001), whereas extraction methods exerted a comparatively smaller effect (F = 6.36, p = 0.01), confirming that solvent polarity is the primary factor governing tannin extraction efficiency.

Al Jaafreh [33] demonstrated that polar alcoholic solvents significantly enhanced the extraction of polyphenols and tannins from E. alata, resulting in improved antioxidant potential.

In addition, Boussena et al. [31] reported considerably higher total phenolic (214.92 mg GAE/g extract) and flavonoid (30.74 mg CE/g extract) contents in methanolic extracts. Whereas ethanolic leaf extracts of E. alata contained 157.54 ± 0.05 mg GAE/g DM and 48.7 ± 0.9 mg/g flavonoids [15]. Similarly, Tunisian E. alata extracts contained elevated levels of TPC (125.73 mg GAE/g DW), TFC (27.89 mg QE/g DW), and tannins (5.55 mg tannic acid equivalent/g DW) [44]. In contrast, Algerian E. altissima exhibited lower and more variable polyphenol contents depending on the extraction solvent [45]. More recently, Mohammed et al. [38] demonstrated that methanolic extracts of E. alata collected under saline environmental conditions exhibited enhanced phenolic and flavonoid contents together with remarkable antioxidant activity. Mufti et al. [43] identified gallic acid, flavonoids, and other phenolic derivatives as the predominant bioactive constituents of E. alata using LC–MS analysis, whereas Al Jaafreh [33] confirmed that solvent polarity markedly influences the phytochemical composition and antioxidant potential of E. alata extracts. The discrepancies are most likely attributable to differences in geographical origin, climatic and environmental conditions, plant organ, harvesting period, extraction procedure, solvent system, and analytical methodology, all known to influence the biosynthesis and accumulation of secondary metabolites. The evidence supporting E. alata as a promising natural source of polyphenolic compounds with considerable phytopharmaceutical potential is highly supported.

2.2. Evaluation of Antioxidant Activity

The results of the antioxidant activity evaluated by the phosphomolybdenum (TAC) and DPPH assays are presented in Figure 2 and Table S2. The aqueous methanolic extracts of E. alata stems exhibited the highest total antioxidant capacity (TAC) (408.84–500.46 mg AAE/g DW), followed by methanolic extracts (197.57–210.84 mg AAE/g DW), whereas the ethyl acetate fractions displayed only weak activity (2.46–4.88 mg AAE/g DW). This trend closely paralleled the distribution of total phenolics and flavonoids among the extracts and was further supported by the significant positive correlations observed between phenolic constituents and TAC, confirming that extracts richer in polyphenols possessed greater reducing capacity.

Total antioxidant capacity (TAC, mg AAE/g DW) by the phosphomolybdate method (A) and DPPH radical scavenging activity (IC₅₀, mg/mL) (B) for E. alata extracts.

The DPPH assay further demonstrated the remarkable radical‐scavenging capacity of the extracts, with IC₅₀ values ranging from < 0.01 to 0.46 mg/mL depending on the solvent and extraction procedure. The sonicated and ultrasonicated methanolic extracts exhibited the strongest activity (IC₅₀< 0.01 mg/mL), surpassing even the reference antioxidant ascorbic acid (IC₅₀ = 0.12 mg/mL). These results agree with recent investigations demonstrating that polar extracts of E. alata possess superior antioxidant properties. For example, methanolic seed extracts from Tunisian E. alata showed DPPH IC₅₀ values close to 0.01 mg/mL, confirming the effectiveness of polar solvents for recovering phenolic antioxidants [14]. Aqueous extracts of Algerian aerial parts exhibited moderate scavenging activity (IC₅₀ = 0.74 mg/mL) [16], whereas methanolic fruit extracts collected in Palestine yielded IC₅₀ values of approximately 1.07 mg/Ml [8]. Both studies reported lower antioxidant activity than that obtained for the optimized stem extracts in the present work, highlighting the influence of plant organ, geographical origin, extraction solvent, and extraction technique on antioxidant performance.

The strong antioxidant performance observed in this study is consistent with the correlation analysis, which revealed significant positive relationships between total phenolics, flavonoids, condensed tannins, and TAC, together with significant negative correlations with DPPH IC₅₀ values, indicating that increasing concentrations of these phytochemicals are associated with enhanced radical‐scavenging efficiency. These findings reinforce the widely accepted role of phenolic acids, flavonoids, and tannins as the principal contributors to the antioxidant activity of E. alata [46].

The results reinforce the evidence that the antioxidant activity of E. alata is primarily governed by its phenolic composition and is strongly influenced by extraction conditions. Recent investigations have similarly demonstrated that polar solvents maximize the recovery of phenolic acids and flavonoids, resulting in enhanced antioxidant, anti‐inflammatory, and other biological activities [33, 38, 43]. The correlations observed in the present work between phenolic constituents, TAC, and DPPH radical‐scavenging activity confirm that phenolic compounds are the principal contributors to the antioxidant potential of E. alata. Furthermore, the superior performance of the sonicated and ultrasonicated extracts highlights the importance of ultrasound‐assisted extraction in improving cell wall disruption and mass transfer, thereby increasing the recovery of bioactive compounds, supporting their potential application as natural antioxidants for nutraceutical and pharmaceutical development.

2.3. RP‐HPLC‐PDA Analysis

In this study, the methanolic extract of E. alata exhibited the highest antioxidant activity. This extract was subsequently analyzed by RP‐HPLC‐PDA, and the chromatographic profile is presented in Figure 3.

HPLC chromatogram of methanol extract at 280 nm.

Nine phenolic compounds were identified, comprising four phenolic acids (gallic, chlorogenic, caffeic, and p‐coumaric acids) and five flavonoids (resorcinol, catechin, rutin, naringenin, and quercetin). Among these compounds, gallic acid was detected at a particularly high concentration, distinguishing the phytochemical profile of the investigated extract from those previously reported for E. alata. These findings differ from those of Ziani et al. [9] who reported that the aqueous ethanolic extract of Algerian E. alata was dominated by flavonoids, particularly hydroxypuerarin isomer 1 and myricetin derivatives. Likewise, Sioud et al. [47] demonstrated that Tunisian methanolic extracts contained mainly quercetin and myricetin derivatives together with other phenolic acids and proanthocyanidins. Similarly, Al‐Rimawi et al. [48] identified luteolin‐7‐O‐glucuronide and myricetin‐3‐rhamnoside as the major flavonoid glycosides in Palestinian E. alata, whereas gallic acid was not reported as the predominant constituent. In contrast, Mahmoudi et al. [21] identified eleven phenolic compounds in E. altissima seed extracts, including seven flavonoids and four phenolic acids, with gallic acid and quercetrin as the principal constituents, indicating that gallic acid accumulation may vary among Ephedra species. Comparative studies from Algeria, Tunisia, and Saudi Arabia have further demonstrated that the qualitative and quantitative composition of phenolic compounds in Ephedra species is strongly influenced by geographical origin, environmental conditions, plant organ, and extraction solvent [22]. These observations are in agreement with recent studies highlighting that optimized extraction conditions and appropriate solvent selection markedly improve the recovery of phenolic constituents and consequently influence the biological activity of medicinal plant extracts [33, 38, 43, 49, 50, 51]. Collectively, these findings indicate that the phytochemical composition of E. alata is highly dependent on extraction conditions and environmental factors, and they support the high gallic acid content and antioxidant potential observed in the present methanolic extract.

2.4. Molecular Docking Results

The molecular docking results are summarized in Table 1 and Figure 4. Gallic acid exhibited a favorable binding affinity toward aromatase, with a predicted binding energy (ΔG) of −5.88 kcal/mol and an estimated inhibition constant (Ki) of 49.38 µM, suggesting a moderate interaction with the enzyme. Analysis of the best docking pose showed that gallic acid was stabilized within the active site primarily through multiple hydrogen bonds involving Arg115, Ile132, Ile133, Trp141, Arg145, and Arg435. Additional stabilization was provided by van der Waals interactions with Gly439 and π‐alkyl interactions involving Phe134 and Ala438. Together, these noncovalent interactions contributed to the stable accommodation of gallic acid within the aromatase binding pocket. The predominance of hydrogen bonding is consistent with the chemical structure of gallic acid, which possesses three hydroxyl groups and one carboxyl group capable of acting as hydrogen bond donors and acceptors. These functional groups facilitate favorable polar interactions with residues lining the binding cavity, whereas hydrophobic contacts further contribute to ligand stabilization. Although the calculated binding affinity is moderate compared with potent synthetic aromatase inhibitors, the docking results suggest that gallic acid has the potential to interact with aromatase and may contribute, at least in part, to the biological activity of E. alata extracts. It should be noted that molecular docking provides a computational prediction of ligand–protein interactions and does not constitute experimental evidence of enzyme inhibition. Therefore, the present findings should be considered preliminary and warrant further validation using enzymatic inhibition assays and additional computational approaches, such as docking protocol validation and molecular dynamics simulations.

TABLE 1.

Binding energy, inhibition constant, and intermolecular interactions formed between the residues of the investigated enzyme and gallic acid.

Ligands	Receptor	ΔG (Kcal/mol)	Ki (µM)	Amino acids form hydrogen bonds
Gallic acid	Aromatase	−5.88	49.38	ILE:132; ILE:133; ARG: 115; ARG: 435; TRP: 141; ARG: 145

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Docked pose of gallic acid compound with aromatase enzyme. 3D and 2D views of the interaction types of ligands with surrounding amino acids in the receptor active site.

2.4.1. Factor Significance

Across all evaluated responses (yield, TPC, TFC, tannins, TAC, and DPPH), the two‐way ANOVA demonstrated that the type of solvent exerted the most pronounced influence on the extraction performance (p < 0.001 for all responses), followed by the extraction method (p < 0.05 in most cases). Furthermore, the interaction between solvent and extraction method was highly significant for all measured parameters (p < 0.01), indicating that extraction efficiency is governed by a synergistic interplay between the physicochemical properties of the solvent and the mechanical energy applied during extraction. Crucially, the interaction between solvent and extraction method was highly significant for all measured parameters (p < 0.01), specifically yield, TPC, tannins, and DPPH (p < 0.001, ***); TFC (p = 0.0022, **); and TAC (p = 0.0090, **). All measured responses (Table 2).

TABLE 2.

Two‐way ANOVA results for the effects of solvent type, extraction method, and their interaction on phytochemical yields and antioxidant activities of E. alata extracts.

Response variables	Source of Variation	DF	Sum of Squares	F‐value	p‐value	Significance
Yield (%)	Solvent	4	2307.47	838.965	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	16.22	11.795	< 2.2×10⁻ ¹ ⁶	^a
	Solvent × Method	8	110.25	20.043	< 2.2×10⁻ ¹ ⁶	^a
TPC (mg GAE/g DW)	Solvent	4	7856.4	5719.59	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	195.8	285.12	1.16×10⁻ ¹ ²	^a
	Solvent × Method	8	1445.8	526.29	< 2.2×10⁻ ¹ ⁶	^a
TFC (mg CE/g DW)	Solvent	4	302.417	754.75	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	2.072	10.34	0.00150	^b
	Solvent × Method	8	4.445	5.55	0.00220	^b
Tannins (mg CE/g DW)	Solvent	4	1.436.646	2246.32	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	1932	6.04	0.0119	^c
	Solvent × Method	8	15.357	12.01	2.91×10⁻ ⁵	^a
TAC (mg AAE/g DW)	Solvent	4	980.317	1031.80	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	2720	5.73	0.0142	^c
	Solvent × Method	8	7804	4.11	0.0090	^b
DPPH IC₅₀ (mg/mL)	Solvent	4	0.156	25.914	< 2.2×10⁻ ¹ ⁶	^a
	Method	2	0.062	20.637	< 2.2×10⁻ ¹ ⁶	^a
	Solvent × Method	7	0.185	17.567	< 2.2×10⁻ ¹ ⁶	^a

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Significance codes:

^{^a}

p < 0.001.

^{^b}

p < 0.01.

^{^c}

p < 0.05. DF = degrees of freedom.

This observation confirms that the optimal extraction conditions cannot be deduced from the main effects independently. Based on Tukey's HSD post‐hoc analysis applied to the interaction means, the methanol–ultrasound combination yielded the highest overall extraction performance across phenolic, flavonoid, tannin, and antioxidant parameters. This selection reflects the high polarity and solvent power of methanol for phenolic compounds, coupled with the enhanced mass transfer efficiency provided by UAE. Consequently, the methanol–ultrasound conditions were selected and fixed as the categorical factors in the subsequent Box–Behnken design (BBD) optimization phase.

2.5. Effect of Extraction Parameters on Polyphenol Content and Antioxidant Activity

Subsequent to the screening phase, the Box‐Behnken Design (BBD) was implemented to establish the optimum operational window for the most influential continuous process variables. Drawing upon established literature for UAE optimization, specifically the work by Slimani et al. [52], three continuous factors were systematically investigated at three distinct coded levels (‐1, 0, and +1) (see Table 3).

TABLE 3.

Three extraction factors and their actual values used at each level and the experimental design matrix of ultrasonic‐assisted extraction (Box‐Behnken Design).

Exp. N	Material/Solvent (mg·mL⁻ ¹)	Temperature (°C)	Time (min)	Coded X₁	Coded X₂	Coded X₃
Factor Level Definition
Lower (−1)	40/10	20	15	−1	−1	−1
Middle (0)	40/20	35	30	0	0	0
Upper (+1)	40/30	50	45	+1	+1	+1
BBD Experimental Runs
1	40/10	20	30	−1	−1	0
2	40/30	20	30	+1	−1	0
3	40/10	50	30	−1	+1	0
4	40/30	50	30	+1	+1	0
5	40/10	35	15	−1	0	−1
6	40/30	35	15	+1	0	−1
7	40/10	35	45	−1	0	+1
8	40/30	35	45	+1	0	+1
9	40/20	20	15	0	−1	−1
10	40/20	50	15	0	+1	−1
11	40/20	20	45	0	−1	+1
12	40/20	50	45	0	+1	+1
13 (Center)	40/20	35	30	0	0	0
14 (Center)	40/20	35	30	0	0	0
15 (Center)	40/20	35	30	0	0	0

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2.6. Effects of Extraction Parameters

The Box–Behnken Design evaluated the influence of the solid–solvent ratio (X₁), extraction temperature (X₂), and time (X₃) on the extraction response. As shown in Table 4. The second‐order model demonstrated strong predictive ability, with a high coefficient of determination and a statistically significant overall regression for most responses. All linear terms were positive and significant, confirming that increasing the solvent concentration, temperature, or extraction time improves the extraction efficiency within the studied range. The most influential factor was the solid–solvent ratio (X₁), followed by extraction time (X₃) and temperature (X₂). In contrast, the quadratic and interaction terms showed no significant effects (p > 0.05), indicating that the factor–response relationship is predominantly linear under the tested operating domain.

TABLE 4.

Regression coefficients for TPC, total flavonoid, tannins, TAC and IC₅₀ from DPPH.

Responses	Quadratic equation	R²	Adjusted R²
Yield	20.49 ‐ 1.39×1 + 1.37×2 + 2.26×3 + 1.22×1×2 + 0.97×1×3 – 6.80×2×3 ‐ 14.83×12 ‐ 1.14×22 – 2.70×32	0.8933	0.7011
TPC	35.39 ‐ 0.80×1 + 4.38×2 + 3.63×3 – 2.01×1×2 + 1.01×1×3 – 13.77×2×3 ‐ 21.99×12 + 7.30×22 ‐ 0.33×32	0.6593	0.0461
TFC	3.82 + 0.24×1 + 1.04×2 + 1.04×3 + 0.26X₁ X2 + 0.36×1×3 – 1.67×2×3‐ 4.43×12 + 1.40×22 + 1.81×32	0.8331	0.5327
Tannins	275.35 + 2.08×1 + 58.78×2 + 60.19×3 + 6.45×1×2 + 3.72×1×3 – 165.99×2×3 ‐ 330.62×12 + 66.47×22 + 102.44×32	0.9038	0.7306
TAC	467.17 + 0.12×1 + 23.83×2 + 24.55×3 + 1.02×1×2 + 1.81×1×3– 51.40×2×3 ‐ 301.42×12‐ 157.67×22 ‐ 156.69×32	0.9687	0.9123
DPPH	0.05 ‐ 0.10×1+ 0.04×2 ‐ 0.04×3 – 0.08×1×2 – 0.02×1×3 – 0.02×2×3 + 0.10×12+ 0.05×22 ‐ 0.01×32	0.8815	0.6148

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Response surface contour plots (Figures 5 and 6) revealed that extraction efficiency was strongly dependent on the interaction between material/solvent ratio, temperature, and time. Optimal extraction conditions for maximizing total phenolic content (TPC: 35–45 mg GAE/g DW), flavonoids (TFC: 4–5 mg CE/g DW), condensed tannins (250–400 mg CE/g DW), and antioxidant capacity (TAC: 150–200 mg AAE/g DW; DPPH IC₅₀: 0.05‐0.15 mg/mL) were achieved in the central region of the design space, specifically at material/solvent ratios of 15–25 mg/mL, temperatures of 30°C–40°C, and extraction times of 20–35 min. Higher temperatures (>45°C) and prolonged extraction times (>40 min) resulted in decreased yields, likely due to thermal degradation of heat‐sensitive phenolic compounds and oxidative stress induced by extended ultrasound exposure. Conversely, lower temperatures (20°C) and insufficient extraction times (<20 min) led to incomplete cell wall disruption and poor mass transfer, resulting in suboptimal bioactive compound recovery. These findings confirm that moderate conditions provide the best compromise between extraction efficiency and preservation of bioactive compound integrity, with the optimal extraction window identified at 20 mg/mL ratio, 35°C, and 30 min extraction time.

Response surface contour plots showing the predicted effect of material/solvent ratio (X₁, mg/mL) and temperature (X₂,°C) on each extraction response (yield, TPC, TFC, tannins, TAC, DPPH IC₅₀), with extraction time held at its center‐point value (X₃ = 30 min). Plots were generated in MATLAB from the fitted quadratic Box–Behnken models; color scale represents the predicted response value for each panel, and black dots indicate the design points.

Response surface contour plots showing the predicted effect of material/solvent ratio (X₁, mg/mL) and extraction time (X₃, min) on each extraction response (yield, TPC, TFC, tannins, TAC, DPPH IC₅₀), with temperature held at its center‐point value (X₂ = 35°C). Plots were generated in MATLAB from the fitted quadratic Box–Behnken models; color scale represents the predicted response value for each panel, and black dots indicate the design points.

The optimum conditions predicted by the Box–Behnken response surface model were obtained from the fitted quadratic model. Experimental validation of these predicted optimum conditions is currently underway. Future work will compare the predicted and experimentally obtained responses and evaluate the model performance through the percentage prediction error, thereby providing additional confirmation of the model's predictive capability.

2.7. Correlation Analysis Between Phytochemical Content and Antioxidant Activity

Correlation coefficients are presented in Figure 7, whereas the corresponding 95% confidence intervals, coefficients of determination (r²), and Bonferroni‐adjusted p‐values are summarized in Tables S3, S4, and S5. Significant positive correlations were observed among the phytochemical constituents and TAC. The strongest associations were found between TFC and tannin content (r = 0.936), followed by TFC and TAC (r = 0.832), tannin content and TAC (r = 0.743), and TPC and TAC (r = 0.714). All of these relationships remained significant after Bonferroni correction, indicating a robust association between phenolic composition and antioxidant capacity (Figure 7).

Spearman correlation matrix illustrating the relationships between bioactive compounds and antioxidant activities of *E. alata* extracts. The color intensity represents the Spearman correlation coefficient r_s, where dark red indicates a strong positive correlation and dark blue indicates a strong negative correlation.

Conversely, DPPH IC₅₀ values were negatively correlated with TFC (r = −0.653), tannin content (r = −0.596), and TAC (r = −0.574), whereas the correlation with TPC was weaker (r = −0.429, p = 0.126). Although these inverse relationships were consistent with the expected decrease in IC₅₀ values as antioxidant constituents increased, they did not remain statistically significant after Bonferroni correction, suggesting that these trends should be interpreted cautiously because of the limited sample size and the conservative nature of the multiple‐testing adjustment.

Biologically, these findings indicate that the antioxidant potential of E. alata is more closely associated with its flavonoid and condensed tannin contents than with TPC alone. The strong positive correlations between TFC, tannin content, and TAC suggest that these phenolic subclasses are major contributors to the antioxidant capacity of the extracts. Similar observations have been reported in recent studies, which demonstrated that flavonoid‐rich extracts consistently exhibit superior antioxidant activity owing to the high redox potential and radical‐scavenging properties of these compounds [20, 33]. The comparatively weaker association between TPC and antioxidant activity further indicates that the antioxidant efficacy of E. alata depends not only on the total amount of phenolics but also on their chemical composition and possible synergistic interactions among individual bioactive constituents. This interpretation is supported by the RP‐HPLC–PDA analysis, which identified several antioxidant phenolic acids and flavonoids, including gallic acid, catechin, rutin, quercetin, and naringenin, suggesting that the combined action of these compounds underlies the remarkable antioxidant potential of E. alata.

3. Conclusions

This study demonstrates that the stems of E. alata are a promising source of bioactive phenolic compounds with significant antioxidant potential. Ultrasound‐assisted extraction optimised using the Box–Behnken design efficiently enhanced the recovery of phenolic constituents and antioxidant activity while reducing extraction time and solvent consumption. RP‐HPLC‐PDA analysis identified nine phenolic compounds, with gallic acid being the most abundant constituent, which highlights the distinctive phytochemical profile of the studied extract.

Molecular docking analysis indicated that gallic acid formed favourable interactions with the aromatase active site, providing preliminary insight into its potential biological activity. However, these computational predictions require further validation through docking protocol validation and experimental biological assays.

Overall, the integration of extraction optimisation, phytochemical profiling, antioxidant evaluation, and computational analysis provides a comprehensive assessment of E. alata and supports its potential as a natural source of bioactive compounds for nutraceutical and pharmaceutical applications. Future studies should focus on LC–MS/MS confirmation of the identified phenolic compounds, isolation of the major bioactive constituents, experimental validation of their biological activities, and toxicity and in vivo investigations to further establish their therapeutic potential.

4. Materials and Methods

4.1. Chemical Products

All solvents and analytical‐grade reagents were purchased from Sigma‐Aldrich (Taufkirchen, Germany), including dichloromethane, chloroform, acetone, ethyl acetate, methanol, Folin‐Ciocalteu reagent, sodium carbonate, sodium acetate, sodium phosphate, gallic acid, ascorbic acid, catechin, quercetin, vanillin, sodium nitrite (NaNO₂), aluminum chloride (AlCl₃), sodium hydroxide (NaOH), hydrochloric acid (HCl), sulfuric acid (H₂SO₄), ammonium molybdate, and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH).

4.2. Plant Collection

E. alata was collected in May 2022 from Biskra, 50 km north of Oran city (Northwest Algeria) (0° 41’ West longitude, 35° 37’ North latitude), in Algeria. This plant was identified by Dr. BABA ALI B., Department of Biology and Environment, Tlemcen University, Algeria. Voucher specimen number (E. alata Poir.: No. 59) was deposited in the laboratory of Natural Products, Tlemcen.

4.3. Preparation of Plant Samples

The dried stems of E. alata were ground into a fine powder prior to extraction. Three extraction techniques, namely maceration, sonification, and ultrasound‐assisted extraction (UAE), were employed using five solvents of increasing polarity: dichloromethane (DCM), acetone (Ace), ethyl acetate (EtOAc), methanol (MeOH), and a methanol/water mixture (70:30, v/v). For each extraction, 6 g of the powdered plant material were mixed with 120 mL of the selected solvent. Sonification was performed using an UP200S ultrasonic homogenizer (Hielscher Ultrasonics GmbH, Teltow, Germany; 24 kHz, 200 W), whereas ultrasound‐assisted extraction was carried out using a Sonorex RK 100 H ultrasonic bath (BANDELIN electronic GmbH & Co. KG, Berlin, Germany; 35 kHz, 160 W). All extractions were conducted at room temperature for 24 h. The resulting extracts were filtered through Whatman No. 1 filter paper, and the filtrates were concentrated under reduced pressure using a Rotavapor R‐300 (BÜCHI Labortechnik AG, Flawil, Switzerland) at 40°C until complete solvent removal. The dried extracts were subsequently weighed, and the extraction yield (%) was calculated on a dry weight basis.

4.4. Phytochemical Analysis

Total polyphenols were assayed by the Folin‐Ciocalteu reagent and gallic acid as standard according to Singleton and Rossi [53]. Absorbance was checked at 765 nm. Contents were expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW) through the calibration curve with gallic acid, ranging from 0 to 500 mg/mL. All samples were analyzed in triplicate.

TFC was assayed by the aluminum chloride reagent and catechin as a standard according to Zhishen et al. [54]. Absorbance was checked at 510 nm. Contents were expressed as mg catechin equivalent per gram of dry weight (mg CE/g DW) through the calibration curve with catechin. All analyses were performed in triplicate.

The proanthocyanidin content was evaluated by vanillin assay and catechin as a standard according to Julkunen‐Titto [55]. Absorbance was checked at 550 nm. Contents were expressed as mg catechin equivalent per gram of dry weight (mg CE/g DW) through the calibration curve with catechin. Triplicate measurements were carried out for all samples.

4.5. Antioxidant Activity

The TAC of the plant extracts was evaluated by the phosphomolybdenum method according to Prieto et al. [56]. The absorbance was checked at 695 nm. The results were expressed as milligrams of ascorbic acid equivalents per gram of dry weight (mg AAE/g DW) through the calibration curve with ascorbic acid (y = 2.15 + 0.44, R² = 0.99). Every sample was subjected to three independent analyses.

DPPH scavenging activity was determined according to Sanchez‐Moreno et al. [57]. Briefly, methanolic solution at different concentrations were added to 0.0025 mg/mL of 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) methanolic solution, and then the absorbance was measured at 515 nm. The ascorbic acid was used as a positive reference. DPPH radical scavenging activity was expressed as IC₅₀, defined as the concentration of the extract generating 50% inhibition. All analyses were performed in triplicate.

4.6. RP‐HPLC‐PDA Analysis

Only the active extract was selected for the characterization of phenolic compounds, which was performed using reverse‐phase high‐performance liquid chromatography coupled with a photodiode array detector (RP‐HPLC‐PDA). The methanolic extract obtained by sonication was analyzed on a PerkinElmer Flexar system equipped with a binary pump delivery system and a C18 column (150 mm × 4.6 mm, 5 µm). The mobile phase consisted of solvent A (2% acetic acid in water) and solvent B (acetonitrile). The gradient elution system was started with 90% A, followed by a decrease to 10% A over 25 min, then a linear increase to 100% solvent B over 15 min, and finally a 20 min re‐equilibration step. The flow rate was set at 1 mL/min. The chromatograms were monitored at 280 nm. All chromatographic conditions were performed according to the method described previously by El Haci et al. [58]. Identification of phenolic compounds was carried out qualitatively by comparison of their retention times and UV‐Vis spectra with those of pure standards. Since the RP‐HPLC–PDA analysis was performed exclusively for qualitative phytochemical profiling, calibration curves, limits of detection (LOD), limits of quantification (LOQ), and other analytical validation parameters were not determined. Although RP‐HPLC–PDA provides reliable preliminary identification of phenolic constituents, unequivocal structural confirmation would require complementary techniques such as LC–MS/MS, which was beyond the scope of the present study.

4.7. Molecular Docking Study

A molecular docking study was performed to investigate the interaction between gallic acid, the predominant phenolic compound identified by RP‐HPLC–PDA analysis, and the aromatase enzyme, a validated therapeutic target in hormone‐dependent breast cancer. The crystal structure of human aromatase (PDB ID: 5JKV; resolution: 2.75 Å) was retrieved from the Protein Data Bank (https://www.rcsb.org/). The three‐dimensional (3D) structure of gallic acid (PubChem CID: 370) was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The receptor and ligand structures used for molecular docking are presented in Table 5.

TABLE 5.

The structure of gallic acid with its CID (PUBCHEM) and the aromatase receptor.

Gallic acid	CID	Aromatase
	370

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The ligand geometry was energy‐minimized using Chem3D Ultra 8.0 with the MM2 force field. Receptor and ligand preparation, including the addition of polar hydrogen atoms, assignment of Kollman charges, and conversion of PDB files to PDBQT format, was carried out using AutoDock Tools 1.5.6. The docking calculations were performed using AutoDock 4.2 [59]. The grid box was centered at x = 89.072 Å, y = 52.260 Å, and z = 41.053 Å, with dimensions of 40 × 40 × 40 Å and a grid spacing of 0.375 Å. Binding affinity was evaluated based on the predicted binding energy and ligand–protein interactions.

Docking poses and intermolecular interactions were visualized and analyzed using Discovery Studio Visualizer, which was employed to identify interacting amino acid residues and measure hydrogen‐bond distances [60].

4.8. Experimental Design and Modeling

To evaluate the individual and combined effect of categorical factors on the phytochemical and antioxidant responses, a full factorial design was implemented. The factors investigated were the extraction solvent (five levels: DCM, AC, AC_ET, MeOH, MeOH_H₂O) and the extraction method (three levels: maceration, sonification, and ultrasound). A total of 30 experiments were carried out using n = 2 replicates for each combination of the Solvent × Method. The statistical analysis was conducted using R software for the two‐way ANOVA (screening phase) and MATLAB for the subsequent Response Surface Methodology (RSM) based on the BBD optimization and graphical representation of contour plots. The screening data were specifically analyzed using a two‐way ANOVA with interaction in R. A probability value of p < 0.05 was considered to indicate statistical significance for all tests. The common significance codes were used: ***p < 0.001, **p < 0.01, and *p < 0.05.

4.9. Correlation Analysis Between Phytochemical Composition and Antioxidant Activity

The association between phytochemical composition and antioxidant activity was evaluated using Spearman's rank correlation analysis. Correlations were calculated among TPC, TFC, tannin content, TAC, and DPPH IC₅₀ values obtained from the fifteen solvent–extraction method combinations (n = 15; n = 14 for analyses involving DPPH IC₅₀ due to one missing value). A nonparametric approach was adopted because all variables deviated significantly from a normal distribution according to the Shapiro–Wilk test (p < 0.05; Table S3), and isolated outliers were observed in the TFC and DPPH IC₅₀ datasets.

Author Contributions

Nadjat Chekroun‐Bechlaghem: writing – original draft, experience, review & editing, validation, formal analysis, data curation, supervision, resources, funding acquisition. Nabila Belyagoubi‐Benhammou: validation, software, data curation, review & editing. Zineb kaid: software, data curation, review & editing. Ahmed Djafri and Amel Zitouni: review & editing. Imad Abdelhamid El Haci: investigation, experience, writing. Abdelghani Bouchama: investigation, funding acquisition, data curation.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71618‐sup‐0001‐SuppMat.docx.

CBDV-23-e71618-s001.docx^{(28.5KB, docx)}

Acknowledgments

Nadjat Chekroun‐Bechlaghem sincerely thanks Dr. Imad El Haci, Abdelghani Bouchama, and Ahmed Djafri from ‘Centre de Recherche Scientifique et Technique en Analyses Physico‐chimiques (CRAPC)’, Tipaza, Algeria, for their help in the HPLC analysis and for their docking study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File 1: cbdv71618‐sup‐0001‐SuppMat.docx.

CBDV-23-e71618-s001.docx^{(28.5KB, docx)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Gallic Acid–Rich Ephedra alata: Integrated Phytochemical Profiling, Aromatase Docking, and Optimized Extraction via Box–Behnken Design

Nadjat Chekroun‐Bechlaghem

Nabila Belyagoubi‐Benhammou

Zineb Kaid

Amel Zitouni

Imad Abdelhamid El Haci

Abdelghani Bouchama

Ahmed Djafri

ABSTRACT

Abbreviations

1. Introduction

2. Results and Discussion

2.1. Yields and Quantification of Polyphenolic Compounds

FIGURE 1.

2.2. Evaluation of Antioxidant Activity

FIGURE 2.

2.3. RP‐HPLC‐PDA Analysis

FIGURE 3.

2.4. Molecular Docking Results

TABLE 1.

FIGURE 4.

2.4.1. Factor Significance

TABLE 2.

2.5. Effect of Extraction Parameters on Polyphenol Content and Antioxidant Activity

TABLE 3.

2.6. Effects of Extraction Parameters

TABLE 4.

FIGURE 5.

FIGURE 6.

2.7. Correlation Analysis Between Phytochemical Content and Antioxidant Activity

FIGURE 7.

3. Conclusions

4. Materials and Methods

4.1. Chemical Products

4.2. Plant Collection

4.3. Preparation of Plant Samples

4.4. Phytochemical Analysis

4.5. Antioxidant Activity

4.6. RP‐HPLC‐PDA Analysis

4.7. Molecular Docking Study

TABLE 5.

4.8. Experimental Design and Modeling

4.9. Correlation Analysis Between Phytochemical Composition and Antioxidant Activity

Author Contributions

Conflicts of Interest

Supporting information

Acknowledgments

Data Availability Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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