Comprehensive phytochemical profiling of bioactive compounds from Barleria prattensis for their antioxidant and cytotoxic capacity and its characterization using GC-MS

Abstract

Barleria prattensis Santapau has been traditionally used for its medicinal properties, particularly for its antimicrobial and antioxidant benefits. This study evaluates its phytochemical composition, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and anticancer potential. Phytochemical screening was conducted using standard qualitative assays, while TPC and TFC were quantified using the Folin–Ciocalteu and aluminum chloride methods, respectively. The TPC values for methanol (MeOH), chloroform (CHCl₃), and petroleum ether (PE) extracts were 72.9, 37.3, and 16.6 mg GAE/g, whereas TFC values were 43.4, 28.1, and 18.3 mg QE/g, respectively. Antioxidant activity was assessed using the DPPH radical scavenging assay, with IC₅₀ values of 7.46, 16.13, 66.95, and 112.97 μg/mL for ascorbic acid (AA), MeOH, CHCl₃, and PE, respectively. Cytotoxicity against the MCF-7 breast cancer cell line was evaluated using the MTT assay, with IC₅₀ values of 293.6 μg/mL for MeOH, 260.0 μg/mL for CHCl₃, and 60.1 μg/mL for PE. Morphological analysis confirmed apoptotic changes in treated MCF-7 cells compared to untreated controls. GC-MS analysis identified key bioactive compounds, including phytol, squalene, neophytadiene and β-sitosterol, which are known for their antioxidant and anticancer properties. The methanolic extract exhibited the highest antioxidant activity, comparable to ascorbic acid, while the PE extract showed the strongest cytotoxic effect. This study provides novel insights into the dual therapeutic potential of B. prattensis, underscoring its role as a rich source of antioxidant and anticancer phytochemicals. The superior cytotoxicity of the PE extract and the methanolic extract's radical scavenging efficacy highlight its viability for developing plant-based therapies for oxidative stress-related disorders and breast cancer. Further isolation and characterization of bioactive compounds, including squalene, phytol, and neophytadiene, which are the most abundant in this plant, could support drug discovery and the development of new therapeutic agents.

Graphical abstract

Successive Soxhlet extraction of Barleria prattensis leaves (PE, CHCl₃, MeOH) followed by phytochemical profiling (TPC, TFC, GC-MS) and bioactivity assessment: DPPH antioxidant assay and MTT cytotoxicity testing on MCF-7 breast cancer cells.

Highlights

• •Comprehensive phytochemical profiling of Barleria prattensis extracts via GC-MS.
• •Methanol extract exhibits antioxidant activity comparable to ascorbic acid.
• •Petroleum ether extract shows potent cytotoxicity against MCF-7 breast cancer cells.
• •Squalene, phytol, and neophytadiene were identified as key bioactive compounds.
• •High phenolic and flavonoid content linked to observed bioactivities and antioxidant capacity.

1. Introduction

Plants of the genus Barleria (Acanthaceae) have long been valued in traditional medicine for their diverse therapeutic properties, including wound healing, anti-inflammatory, antibacterial, antioxidant, and cytotoxic effects [1]. Despite its ethnomedicinal significance, B. prattensis, a species endemic to Gujarat, where it holds cultural and therapeutic importance, remains severely understudied compared to well-characterized relatives such as B. cristata and B. lupulina. The increasing demand for plant-derived bioactive compounds as sustainable alternatives to synthetic drugs highlights the need for a systematic investigation of neglected species like B. prattensis to elucidate their phytochemical diversity and pharmacological potential [2].

Phytochemical screening, a fundamental step in natural product research, aids in the identification of secondary metabolites such as phenolic acids, flavonoids, and alkaloids, which play key roles in antioxidant and cytotoxic mechanisms. Quantification of total phenolic content (TPC) and total flavonoid content (TFC) provides critical insights into antioxidant capacity, as these compounds neutralize free radicals and mitigate oxidative stress, a major factor in chronic diseases, including cancer, neurodegenerative disorders, and cardiovascular ailments [3]. However, these phytochemical parameters remain unreported for B. prattensis, limiting its integration into evidence-based therapeutic applications [4,5].

Gas Chromatography-Mass Spectrometry (GC-MS) has revolutionized the identification of volatile and semi-volatile bioactive compounds in medicinal plants. While GC-MS profiling has been used to characterize other Barleria species, its application to B. prattensis remains scarce, and the molecular basis of its purported bioactivity is unknown. This knowledge gap hinders both the validation of its traditional medicinal uses and the exploration of its potential as a source of targeted therapeutics [6].

Similarly, cytotoxic screening of plant extracts against cancer cell lines is essential for anticancer drug discovery. Natural products with selective cytotoxicity are particularly valuable for overcoming the limitations of conventional chemotherapeutics, such as toxicity and resistance [8]. Despite the global burden of breast cancer and the urgent need for novel therapeutic agents, no studies have investigated the cytotoxic potential of B. prattensis extracts against breast cancer models [7].

This study addresses critical research gaps by conducting the first comprehensive phytochemical and pharmacological investigation of Barleria prattensis, a species with limited prior scientific evaluation despite its ethnomedicinal relevance. It uniquely integrates qualitative phytochemical screening with GC-MS-based identification of polar and non-polar metabolites and provides comparative quantification of total phenolic and flavonoid content across solvent systems. Unlike previous fragmented studies on related species, this work systematically evaluates antioxidant activity using DPPH and total antioxidant capacity (TAC) assays. It is also the first to investigate the cytotoxic effects of B. prattensis extracts against hormone-responsive and triple-negative breast cancer cell lines. By correlating chemical profiles with biological activity, this study bridges traditional medicinal knowledge with modern pharmacological evidence and highlights B. prattensis as a novel source of antioxidant and anticancer agents for potential application in oxidative stress-related disorders and oncology drug development.

2. Methods and materials

2.1. Materials and reagents

Analytical reagent (AR) grade ascorbic acid was purchased from Sigma-Aldrich (CAS No: 50-81-7, Product Code: A2218). Trypsin, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and penicillin/streptomycin were sourced from HiMedia, India. Extra-purity (95 %) 2,2-diphenyl-1-picrylhydrazyl (DPPH), phosphate-buffered saline (PBS), Folin–Ciocalteu reagent, dimethyl sulfoxide (DMSO), and AR-grade petroleum ether, chloroform, and methanol were obtained from SRL, India.

2.2. Collection of plant material

Barleria prattensis Santapau (Acanthaceae) was collected in November 2022 from the Dang district of Gujarat, India (20°56′44″N, 73°39′05″E) at an elevation of 824 m. The region has a predominantly dry climate, with the southwest monsoon occurring from June to September. The collection site lies within a dry deciduous forest zone with lateritic soils, which may influence the plant's phytochemical profile [8]. The specimen was identified by Dr. Padmanabha S. Nagar, Associate Professor, Department of Botany, The Maharaja Sayajirao University of Baroda, and is preserved in the herbarium under accession number AN30784.

2.3. Extraction of plant material

The plant material was thoroughly washed with tap water to remove surface contaminants, followed by distilled water for further purification. It was then dried in a shaded area for two weeks to prevent the degradation of thermolabile bioactive compounds. Once completely dried, the material was ground into a fine powder using a high-speed blender to ensure uniform particle size, enhancing extraction efficiency.

The finely powdered plant material was subjected to Soxhlet extraction using solvents of increasing polarity: petroleum ether (PE), chloroform (CHCl₃), and methanol (MeOH) (Fig. 1). Each extraction continued until the solvent became colorless, indicating the complete extraction of phytochemicals. The collected extracts were filtered to remove particulate matter, concentrated under reduced pressure using a rotary evaporator to eliminate excess solvent, and stored at 4 °C for further phytochemical and biological analyses [9].

Fig. 1.

Schematic workflow of the extraction procedure for Barleria prattensis leaves. The process includes leaf collection, botanical authentication, washing with tap and distilled water, shade drying, and grinding. The powdered material undergoes sequential extraction with petroleum ether (PE), chloroform (CHCl₃), and methanol (MeOH), yielding solvent-specific extracts for subsequent phytochemical and biological evaluation.

2.4. Phytochemical screening

2.4.1. Qualitative phytochemical analysis

Preliminary phytochemical screening of B. prattensis extracts was carried out using standard qualitative procedures described by Shaikh et al. (2020) to identify major bioactive constituents, including alkaloids, phenols, tannins, flavonoids, terpenoids, steroids, saponins, and reducing sugars [10]. In brief, flavonoids were confirmed using three tests. In the lead acetate test, 1 mL of plant extract was treated with a few drops of 10 % lead acetate, yielding a yellow precipitate. In the ferric chloride test, mixing the extract with 10 % ferric chloride resulted in a greenish-black color. In the concentrated sulfuric acid test, the appearance of an orange color upon treatment with H₂SO₄ indicated flavonoids. Alkaloids were detected by Mayer's Test and the iodine test. The addition of Mayer's reagent produced a creamy white or yellow precipitate, while iodine solution gave a blue color that disappeared on boiling and reappeared on cooling. Phenolic compounds were confirmed through the ferric chloride test, which produced a dark green or bluish-black coloration, and the lead acetate test, which resulted in a white precipitate. Tannins were identified using Braymer's test, where ferric chloride yielded a blue-green color, and the lead acetate test, which produced a creamy gelatinous precipitate. Terpenoids were detected using the Salkowski test and copper acetate test. The Salkowski test involved layering the extract with concentrated sulfuric acid and chloroform, resulting in a reddish-brown interface. In the copper acetate test, the formation of an emerald green color indicated terpenoids. Steroids were confirmed through Liebermann–Burchard's test, where treatment with acetic anhydride and concentrated sulfuric acid produced a green to blue coloration. Saponins were identified using the Foam test. The formation of a stable froth after vigorous shaking with distilled water, which persisted for at least 10 min, indicated the presence of saponins. Reducing sugars were detected using Fehling's test and Borntrager's test. In Fehling's test, a red precipitate formed upon heating the extract with Fehling's solutions A and B. In Borntrager's test, a pink-colored solution appeared after treating the extract with benzene and ammonia.

2.4.2. Quantitative phytochemical analysis

2.4.2.1. Total phenolic content

Phenol estimation in plant extracts was conducted using the Folin–Ciocalteu method, with gallic acid serving as the standard. Plant extracts (1 mg/mL) and gallic acid solutions at varying concentrations (10, 20, 30, 40, 50, and 60 μg/mL) were prepared. In a 96-well plate, 100 μL of the sample or standard solution was added in triplicate, followed by 100 μL of 20 % Na₂CO₃ solution. The mixture was thoroughly homogenized, and 20 μL of Folin–Ciocalteu reagent was subsequently added, followed by another round of mixing. The plate was incubated at room temperature for 30 min in the dark, after which the absorbance was measured at 760 nm using a microplate reader (Synergy HTX multi-mode reader) [11].

The concentration of phenolic compounds in each extract was determined from the regression equation based on their absorbance measurements [12]. The results were ultimately expressed as TPC in milligrams of Gallic acid equivalent per gram of dry extract (mg GAE/g) via Equation (1):

$$T=C+\frac{V}{m}$$ 1

Where T represents the total phenolic content (TPC) in mg/g, expressed in Gallic acid equivalent (GAE), C denotes the concentration of Gallic acid derived from the calibration curve in mg/mL, V signifies the volume of the extract in mL, and m indicates the weight of the dried plant extract in grams [13].

2.4.2.2. Total flavonoid content

The total flavonoid content (TFC) was determined using the AlCl₃ colorimetric assay (Sari, K. R. P 2023) with slight modifications. A quercetin calibration curve was employed for TFC calculation. Plant extracts (1 mg/mL) and quercetin solutions at concentrations of 10, 20, 30, 40, 50, and 60 μg/mL were prepared. In a 96-well plate, 100 μL of each sample or standard solution was added in triplicate, followed by 50 μL of 7 % AlCl₃ solution. The mixture was well-mixed, and 50 μL of sodium acetate was added, followed by another round of homogenization. The plate was incubated at room temperature in the dark for 45 min. Absorbance was then measured at 415 nm using a Synergy HTX multi-mode microplate reader. The flavonoid concentration in each extract was determined using the regression equation derived from the absorbance readings [14]. The results were expressed as TFC in milligrams of quercetin equivalent per gram of dry extract (mg Q/g), according to Equation (2):

$$T=C+\frac{V}{m}$$ 2

where T represents the TFC, C expressed as milligrams of quercetin equivalent (QE) per gram of dry extract (mg QE/g). C denotes the concentration of gallic acid obtained from the calibration curve (mg/mL), V is the volume of the plant extract used in the assay (mL), and m is the weight of the dried plant extract (g) [14].

2.5. GC–MS identification of compounds

Analysis by GC-MS was performed using a PerkinElmer Clarus 680 gas chromatograph with an attached SQ8C single quadrupole mass analyzer. Separation was achieved on an HP-5MS capillary column (30 m × 0.25 mm ID, 0.25 μm film) under an inert carrier gas flow of helium kept constant at 1.2 mL/min. The injection volume was 2.0 μL in splitless auto-injection mode. The column oven temperature was initially set at 70 °C and ramped up to 200 °C with an increase rate of 10 °C/min and held for 2 min. It was then raised from 200 °C to 280 °C at a rate of 5 °C/min and held for 14 min. The total run time was 45 min for MeOH and CHCl₃ extracts and 60 min for the PE extract. The mass analyzer operated in electron ionization mode at 70 eV conditions with ion source, transfer line, and quadrupole temperatures maintained at 250 °C, 280 °C, and 150 °C, respectively. The mass spectra were recorded in scan mode for 50–600 m/z at a rate of 1.56250 pt/s. The data processing was carried out using PerkinElmer TurboMass™ software version 6.1. Compounds were identified by comparing the obtained mass spectra with the NIST spectral library, and relative abundances were calculated based on peak area percentages [15,16].

2.6. Antioxidant activity

2.6.1. DPPH free radical scavenging assay

The antioxidant capacity of B. prattensis extracts was assessed utilizing the DPPH method. Ascorbic acid (AA) functioned as a positive control to assess the free radical scavenging capacity of B. prattensis extracts. B. prattensis extracts and AA were produced in methanol at concentrations of 20, 40, 60, 80, 100, and 120 μg/mL. A 0.1 mM DPPH solution in methanol was incubated in the dark for 30 min 100 μL of B. prattensis extracts and ascorbic acid at varying doses were administered to each well of a 96-well plate, except the control wells, which were allocated 100 μL of methanol. Subsequently, 100 μL of the 0.1 mM DPPH solution was dispensed into each well. A separate well was filled with 200 μL of methanol to serve as a blank. The plate was incubated for 30 min, and absorbance was measured at 517 nm using a Synergy HTX multi-mode microplate reader [17]. The scavenging activity was determined using the subsequent formula (Equation (3)):

$$DPPHscavengingactivity(\%)=\frac{\left(ControlAbs-SampleAbs\right)}{ControlAbs}\ast 100$$ 3

Where Control Abs represents the absorbance of the control (MeOH + DPPH) and Sample Abs denotes the absorbance of the sample (plant extracts or AA + DPPH). All experiments were conducted in triplicate with minimal light exposure to ensure accurate and reliable results [17].

2.6.2. Total antioxidant capacity

The total antioxidant capacity (TAC) of the B. prattensis extracts was assessed by the phosphomolybdenum assay, which was expressed as ascorbic acid equivalents, following the protocol of Nurcholis. W. et al., 2021 with minor modifications [14]. The total antioxidant capacity (TAC) was assessed by preparing 100 μL aliquots of the sample solution and a standard at varying concentrations (100, 200, 300, 400, 500, and 600 μg/mL). Each aliquot was mixed with 1 mL of a reagent solution in an Eppendorf tube. The reagent solution contained 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The tubes were tightly sealed and incubated at 95 °C for 90 min in a thermal block. Following incubation, the samples were cooled to room temperature, and the absorbance was measured at 695 nm against a blank using a Synergy HTX multi-mode microplate reader. The blank consisted of 1 mL of the reagent solution and an equivalent volume of the solvent used for the samples (distilled water with 2 % DMSO), treated under the same conditions as the test samples. The experiments were performed independently in triplicate, and the mean and standard deviation (SD) were calculated. Antioxidant capacities for B. prattensis extracts were expressed as equivalents of ascorbic acid [[18], [19], [20]].

2.7. Cytotoxicity of B. prattensis

The cytotoxicity of the B. prattensis extracts tested on MCF7 human breast cancer cells was assessed via the MTT dye reduction assay. MCF-7 cells were acquired from India's National Centre for Cell Science in Pune. The cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin/streptomycin. In addition, MCF-7 cells were grown at 5 % CO₂ in a humidified CO₂ incubator. Cultures were examined under an inverted microscope to determine confluence and confirm the absence of bacteria and fungi [21].

With a slight modification of the methodology of Ali et al. (2024) [22], 100 μL of a suspension containing 10,000 MCF-7 cells was added to each well of a 96-well plate. After the cells were incubated for 24 h, next, 100 μL of plant extracts in DMEM were added to Row A at a 500 μg/mL concentration. A series of dilutions was then performed to achieve a final concentration of 31.25 μg/mL. In addition to the control group (PBS containing 0.5 % DMSO), the cells were incubated for 24 h after B. prattensis extract administration. After 24 h of treatment, images of the cells were taken using an Eclipse Ti2-E inverted fluorescence microscope (Nikon, Japan). Following imaging, 10 μL of a 5 mg/mL MTT solution was added to each well. The cells were incubated for 4 h before the medium was removed from each well. The formazan crystals were dissolved in 100 μL of DMSO, and the plates were incubated for 15 min. The OD was measured at 570 nm with a microplate reader (Synergy HTX multimode reader).

The OD was used to determine cell viability via Equation (4):

$$Cellviability(\%)=\frac{ODofSample}{ODofControl}\ast 100$$ 4

where the mean optical density (OD) value of the untreated experimental control is referred to as the OD of the control, and the mean OD value of the experimental sample is referred to as the OD sample [23].

2.7.1. Statistical analysis

Data analysis was conducted using Microsoft Excel, GraphPad Prism 8, and Origin Pro 2018. All results are expressed as the mean ± standard deviation (SD) from three independent experiments. Statistical comparisons between groups were performed using t-tests and one-way ANOVA, with a significance threshold set at p < 0.05.

3. Results and discussion

3.1. Extract yield

The sequential Soxhlet extraction of B. prattensis using solvents of increasing polarity resulted in different yields, as shown in Table 1. The methanol extract had the highest yield at 3.36 %, followed by chloroform at 1.92 %, and petroleum ether at 0.94 %. The larger yield from methanol suggests that B. prattensis contains more polar compounds. This pattern, where the yield increases from non-polar (petroleum ether) to polar (methanol) solvents, shows that B. prattensis is mainly made up of polar constituents. While the pattern of increasing yield suggests the presence of polar compounds in B. prattensis, the extraction process is more complex. Solvent-solute interactions are influenced by factors such as hydrogen bonding and the ability of solvents to disrupt the plant matrix, which contains compounds with varying solubility profiles [24]. The variation in extraction yields observed between methanol, chloroform, and petroleum ether is likely influenced by the polarity and dielectric constant of the solvents. Methanol, being a polar solvent, is more effective in extracting polar compounds, leading to a higher yield. In contrast, chloroform and petroleum ether, being non-polar, are more selective for non-polar compounds, resulting in lower yields [25].

Table 1.

Percentage yields of different solvent extracts of B. prattensis.

S·NO	Extract	Yield %
1	Petroleum Ether	0.94
2	Chloroform	1.92
3	Methanol	3.36

3.2. Phytochemical screening

3.2.1. Qualitative phytochemical analysis

The qualitative phytochemical analysis verified the existence of flavonoids, phenols, and tannins in all three extracts (petroleum ether, chloroform, and methanol) of B. prattensis (Table 2). Alkaloids, terpenoids, and steroids were identified in both chloroform and methanol extracts but were not present in the petroleum ether extract. This extraction pattern indicates the comparatively polar characteristics of these chemicals in B. prattensis. However, reducing sugars were absent in all extracts, indicating their absence in the plant material. Similar phytochemical profiles have been reported in other studies. Barlaria species, such as B. cristata and B. lupulina, also show flavonoids, alkaloids, and phenolic compounds [1]. The presence of bioactive chemicals, including flavonoids and phenols, in all extracts indicates potential therapeutic uses of B. prattensis, necessitating further exploration of its biological activity.

Table 2.

Qualitative phytochemical profile of B. prattensis extracts.

Phytochemical	Reagent test	Result observed	Solvent
			Petroleum ether	Chloroform	Methanol
Flavonoid	Lead acetate test Ferric chloride test Conc. H2SO4 test	A yellow precipitate A green precipitate An orange colors	+	++	+++
Alkaloid	Mayer's Iodine Test	A creamy white/yellow precipitate A blue color, which disappears on boiling and reappears on cooling	_	+++	++
Phenol	Ferric chloride test Lead acetate test	Dark green/bluish black color A white precipitate	+	++	+++
Tannin	Braymer's test Lead acetate test	Blue-green color A creamy gelatinous precipitate	++	++	+
Terpenoids	Salkowski test Copper acetate test	Reddish-brown Emerald green color	_	+	++
Steroids	Lieberman Burchard'stest	A green, blue color appears	++	++	++
Saponin	Foam test	Stable froth lasts 10 min.	+	++	+++
Reducing sugar	Fehling's test Borntrager's test	A red precipitate A pink colored solution	_	_	_

Note: A negative test is represented as '_', while a positive test is indicated as '+' for low presence, '++' for moderate presence, and '+++' for high presence.

The qualitative phytochemical analysis of B. prattensis extracts revealed the presence of flavonoids, phenols, and tannins in all three solvent systems. Flavonoids are a diverse group of polyphenolic compounds commonly classified into flavonols, flavones, flavanones, and others. Although individual subclasses were not identified in this preliminary study, previous reports on Barleria species suggest the likely presence of flavonols (e.g., quercetin-type compounds) and flavones (e.g., luteolin derivatives), both of which are known for their potent antioxidant and cytotoxic properties [1]. These compounds differ in hydroxylation and saturation patterns, which influence their free radical scavenging capacity and interactions with cellular signaling pathways [26]. Phenols and tannins, also detected in the extracts, are known to act through mechanisms such as metal ion chelation, inhibition of oxidative enzymes, and disruption of cellular membranes [27]. The combined presence of these phytochemicals may explain the observed antioxidant and cytotoxic activities and suggests a synergistic effect among these bioactive classes [28]. Further studies involving chromatographic separation and structural characterization are needed to confirm the specific flavonoid subclasses and their functional contributions.

3.2.2. Quantitative phytochemical analysis

3.2.2.1. Total phenolic content

The total phenolic content (TPC) of the plant extracts, quantified by the Folin-Ciocalteu technique and represented as gallic acid equivalents (mg GAE/g of dry extract), exhibited substantial variation based on the extraction solvent (Fig. 2c). The methanol (MeOH) extract exhibited the greatest total phenolic content (TPC) at 72.92 ± 2.2 mg GAE/g, followed by chloroform (CHCl₃) at 37.30 ± 3.2 mg GAE/g, and petroleum ether (PE) at 16.6 ± 0.84 mg GAE/g. A robust linear correlation (R² = 0.999) in the gallic acid standard curve (Fig. 2a; equation y = 0.0219x + 0.043) validates the accuracy of the test.

Fig. 2.

(a) Standard curve of gallic acid for total phenolic content (TPC) estimation (R² = 0.999). (b) Standard curve of quercitrin for total flavonoid content (TFC) estimation (R² = 0.9934). (c) TPC of methanol, chloroform, and petroleum ether extracts expressed as mg GAE/g. (d) TFC of the same extracts expressed as mg QE/g, with the methanol extract showing the highest TPC (72.9 mg GAE/g) and TFC (43.4 mg QE/g). All assays were performed in triplicate (n = 3).

The higher TPC in the methanol extract reflects not only its polarity but also its ability to disrupt cell walls and release bound phenolics. Methanol penetrates plant tissues effectively, extracting both free and glycosylated phenolics. In contrast, non-polar solvents like chloroform and petroleum ether primarily extract non-phenolic compounds. These differences highlight the complex influence of solvent–matrix interactions on phenolic yield [27].

The elevated TPC in the methanol extract corresponds with the polar characteristics of phenolic chemicals, which solubilize more effectively in polar solvents. This aligns with the Folin-Ciocalteu technique, which depends on the reduction of phosphomolybdic acid by phenolic hydroxyl groups an interaction enhanced in polar conditions. The robust linearity of the standard curve (R² = 0.999) further corroborates the precision of the approach for assessing phenolics in plant extracts. The elevated TPC in the MeOH extract (72.92 mg GAE/g) indicates that the plant is a substantial source of antioxidants, given that phenolic compounds are recognized for their capacity to neutralize free radicals and chelate metal ions [13].

3.2.2.2. Total flavonoid content

The total flavonoid content (TFC) varied across the plant extracts, with the methanol (MeOH) extract having the highest TFC at 43.4 mg QE/g, followed by chloroform (CHCl₃) at 28.1 mg QE/g, and petroleum ether (PE) at 18.3 mg QE/g (Fig. 2d). The strong correlation in the quercetin standard curve (Fig. 2b; y = 0.0191x-0.0047; R² = 0.9934) confirms the method's accuracy.

Flavonoids are well known for their antioxidant effects. The higher TFC in the MeOH extract indicates it may have strong antioxidant properties, helping to neutralize free radicals and reduce oxidative stress, a key factor in many diseases. This makes the MeOH extract a valuable source of bioactive flavonoids with potential therapeutic benefits.

3.3. GC–MS identification of compounds

Petroleum ether, chloroform, and methanol extracts of B. prattensis were subjected to GC-MS analysis (Fig. 3), identifying several bioactive chemicals with strong cytotoxic and antioxidant properties. Every extract had a distinct phytochemical makeup that added to the plant's overall pharmacological profile. Ten primary components were found in the petroleum ether (PE) extract (Fig. 3a), with the most common ingredients being β-sitosterol (12.39 %), squalene (17.58 %), and stigmasterol (13.46 %) (Table 3). Strong antioxidants like squalene and tocopherols shield cells from oxidative stress, whereas β-sitosterol and stigmasterol have been shown to have cytotoxic effects that stop the growth of cancer cells [29]. In line with the plant's historical medicinal uses, the presence of fatty alcohols and hydrocarbons, such as octadecanal and nonadecane, suggests additional antibacterial and etabolic benefits [30]. Nine significant phytochemicals were found in the chloroform (CHCl₃) extract (Fig. 3b), including n-hexadecanoic acid (6.58 %), 13-docosenamide, (Z)- (8.96 %), and n-neophytadiene (11.31 %) (Table 4). The pharmacological relevance of the extract is attributed to neophytadiene's well-known anti-inflammatory, antibacterial, and antioxidant properties [31]. By altering cellular pathways and halting lipid peroxidation, fatty acids like n-hexadecanoic acid and 17-octadecenoic acid have anticancer and antioxidant properties [32]. Furthermore, 1,6,10,14,18,22- tetracosahexaen-3-ol (4.18 %) and phytol (6.27 %) strengthen the extract's cytotoxic and antioxidant qualities, hence enhancing its potential for therapeutic use [33].

Fig. 3.

GC-MS chromatographic profiles of B. prattensis extracts using different solvents: (a) petroleum ether (PE), (b) chloroform (CHCl₃), and (c) methanol (MeOH). The x-axis shows retention time (min), and the y-axis shows relative abundance (%). Each chromatogram reveals distinct phytochemical compositions, emphasizing solvent-dependent variation in volatile and semi-volatile constituents.

Table 3.

GC-MS of PE extract of B. prattensis.

S.N	RT	Area %	Compound Name	Chemical Formula	MW(g/mol)	Reported activity
1	18.49	5.73	Phytol	C20H40O	296.53	Anxiolytic, cytotoxic, antioxidant, apoptotic, anti-inflammatory, antimicrobial [33,40].
2	28.82	2.79	9-Octadecenamide	C18H35NO	281.5	Anti-inflammatory activity and antibacterial activity [41].
3	29.25	17.58	Squalene	C30H50	410.7	Antioxidant, antitumor, antibacterial, and detoxification effects [42].
4	30.43	6.05	Nonadecane, 2-methyl-	C20H42	282.5	Antioxidant [43], antibacterial [44].
5	32.40	4.22	Octadecanal	C18H36O	268.5	Antibacterial activities [45].
6	33.36	3.40	Eicosane, 7-hexyl-	C26H54	366.7	Antibacterial and antifungal activities [46].
7	33.45	2.63	1-Hexadecanol	C16H34O	242.44	Antimicrobial, Anti-inflammatory [47].
8	33.79	3.94	dl-à-Tocopherol	C29H50O2	430.7	Antioxidant [48].
9	36.24	13.46	Stigmasterol	C29H48O	412.7	Anticancer, anti-inflammatory, antimicrobial, and antioxidant properties [49].
10	37.66	12.39	β- -Sitosterol	C29H50O	414.72	Anxiolytic & sedative, anti-inflammatory, antibacterial anticancer [50].

Table 4.

GC-MS of CHCl₃ extract of B. prattensis.

S.N	RT	Area %	Compound Name	Chemical Formula	MW(g/mol)	Reported activity
1	14.15	11.307	Neophytadiene	C20H38	278.5	Anti-inflammatory, antimicrobial, and antioxidant properties [31]
2	16.17	6.584	n-Hexadecanoic acid	C16H32O2	256.4	Anti-inflammatory antioxidant and antibacterial activities [35,51]
3	16.59	4.181	Cycloeicosane	C20H40	280.5	Cytotoxic, anticancer, and bio-pesticide properties [52]
4	18.56	6.272	Phytol	C20H40O	296.5	Anxiolytic, cytotoxic, antioxidant, apoptotic, anti-inflammatory, antimicrobial [33,40]
5	19.12	5.84	17-Octadecynoic acid	C18H32O2	280.4	Cytochrome P450 inhibitors [53]
6	19.61	5.561	Dodecanoic acid, n.-octyl ester	C20H40O2	312.5	Antioxidant, antibacterial, cox-1 & cox-2 inhibitor, antiviral [54]
7	19.96	5.76	Nonacos-1-ene	C29H58	406.8	–
8	30.55	8.955	13-Docosenamide, (Z)-	C22H43NO	337.6	Antimicrobial, Anti-nociceptive [55]
9	31.14	4.178	1,6,10,14,18,22-Tetracosahexaen-3-ol,2,6,10,15, 19,23-hexamethyl-, (all-E)-(ñ)-	C30H50O	426.7	–

Eight primary chemicals were identified in the methanol (MeOH) extract (Fig. 3c), with the most prevalent ingredients being 1-Propanol, 3-(dimethylamino) (29.09 %), 9,12,15-Octadecatrienoic acid, methyl ester (10.74 %), and phytol (7.07 %) (Table 5). Fatty acid derivatives, including hexadecanoic acid, methyl ester (8.11 %), and n-hexadecanoic acid (3.66 %), contribute to antioxidant and anti-inflammatory actions, while the presence of 1-propanol and 3-(dimethylamino) implies pharmacological importance [34,35]. The discovery of 9,12,15-octadecatrienoic acid, methyl ester, and 9,12-octadecadienoic acid, methyl ester supports the extract's traditional therapeutic usage by highlighting its capacity to regulate inflammation and oxidative stress [36]. Additionally, the existence of bioactive lipids is confirmed by the discovery of methyl stearate (1.86 %), which enhances the plant's medicinal effectiveness.

Table 5.

GC-MS of MeOH extract of B. prattensis.

S.N	RT	Area %	Compound Name	Chemical Formula	MW(g/mol)	Reported activity
1	6.71	29.09	1-Propanol, 3-(dimethylamino)-	C5H13NO	103.16	–
2	13.55	3.50	Ethyl à-d-glucopyranoside	C8H16O6	208.21	Preservative [56]
3	16.67	8.11	Hexadecanoic acid, methyl ester	C17H34O2	270.45	Antibacterial [57]
4	17.47	3.66	n-Hexadecanoic acid	C16H32O2	256.42	Antioxidant and antibacterial [35]
5	19.54	2.28	9,12-Octadecadienoic acid, methyl ester	C19H34O2	294.47	Anticancer activity [58]
6	19.67	10.74	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	C19H32O2	292.50	Hepatoprotective, antihistaminic, hypocholesterolemic, anti-eczemic [59]
7	19.87	7.07	Phytol	C20H40O	296.53	Anxiolytic, metabolic, cytotoxic, antioxidant, apoptotic, anti-inflammatory, antimicrobial [33,40]
8	20.11	1.86	Methyl stearate	C19H36O2	298.50	Antidiarrheal, cytotoxic, anti-proliferative [60]
9	41.38	2.77	β -Sitosterol	C29H50O	414.72	Antioxidant, anticancer, anti-diabetic, antimicrobial and immunomodulatory activities [50]

Overall, the wide range of bioactive substances found in the three extracts validates the findings of the phytochemical screening and is consistent with the cytotoxic and antioxidant properties of B. prattensis that have been reported. Fig. 4 illustrates the structural diversity of the most abundant compounds identified across the petroleum ether, chloroform, and methanol extracts, including phytol, neophytadiene, squalene, and fatty acid derivatives, further emphasizing their pharmacological relevance. Its promise as a natural source of therapeutic agents is further supported by sterols, fatty acids, tocopherols, and other pharmacologically significant metabolites, which call for further research into its precise biological processes and therapeutic uses. These findings highlight B. pratensis as a promising source of bioactive compounds with antioxidant and anticancer activities. The sterols, fatty acids, and lipids identified in B. prattensis extracts play significant roles in human health and disease. Sterols, such as β-sitosterol, exhibit anti-inflammatory, anticancer, and cholesterol-lowering properties. Fatty acids, particularly unsaturated types, have documented anti-inflammatory and anticancer effects [37]. These compounds can interact with other phytochemicals, such as flavonoids and phenols, to enhance their biological activities through synergistic effects [38]. Furthermore, lipids contribute to the bioavailability and stability of fat-soluble compounds, improving their absorption and therapeutic potential [39]. These interactions highlight the importance of considering the combined effects of the diverse phytochemicals in B. prattensis when evaluating their therapeutic potential.

Fig. 4.

Three-dimensional chemical structures of major bioactive compounds identified in B. prattensis extracts: neophytadiene, 9,12,15-octadecatrienoic acid methyl ester (Z,Z,Z), phytol, 3-(dimethylamino)-1-propanol, and squalene. These compounds have been reported to exhibit antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, supporting the pharmacological potential of B. prattensis.

3.4. Comparative analysis of major phytoconstituents across Barleria species

To underscore the phytochemical significance of B. prattensis, Table 6 presents a comparative analysis of its major bioactive constituents, quantified as area percentages (% area) based on GC-MS analysis, alongside those reported in other Barleria species and Ocimum tenuiflorum, a well-known member of the Lamiaceae family recognized for its potent bioactivities. Notably, B. prattensis exhibits comparatively high area% % values of neophytadiene (11.31 %), squalene (17.58 %), stigmasterol (13.49 %), and β-sitosterol (12.39 %), while these compounds are either absent or detected at lower levels in other Barleria species. This distinctive chemical profile suggests that B. prattensis may serve as a valuable natural source of therapeutically relevant metabolites. The elevated area% of squalene and phytosterols, in particular, corresponds well with the observed antioxidant and cytotoxic properties, reinforcing its pharmacological potential. Moreover, this comparative analysis indicates that B. prattensis may possess unique or enriched bioactive compounds not commonly found in related species. Therefore, this plant represents a promising source for the isolation of these compounds due to their high yield, supporting further research into compound isolation and bioassay-guided evaluation.

Table 6.

Major phytoconstituents (area %) identified in B. prattensis, other Barleria species, and Ocimum tenuiflorum based on GC-MS analysis. Values represent the percentage of each compound based on total peak area.

Plant Species	Phytol (%)	Neophytadiene (%)	Squalene (%)	Stigmasterol (%)	β-Sitosterol (%)	9-Octadecanoic acid (%)	References
B. prattensis	5.73	11.307	17.58	13.49	12.39	2.79	Current study
B. cristata	–	–	–	–	–	–	–
B. albostellata	4.66	–	1.06	4.01	–	–	[61]
B. lupulina	41.80	–	2.56	–	–	–	[62]
B. cuspidata	8.5	–	–	1.76	–	1.22	[63]
B. terminalis	4.4	–	–	–	–	–	[64]
Ocimum tenuiflorum	4.86	4.33	–	–	–	–	[65]

3.5. Antioxidant activity

3.5.1. DPPH free radical scavenging assay

The DPPH radical scavenging activity (RSA%) of B. pratensis extracts (MeOH, CHCl₃, PE) and ascorbic acid (AA) demonstrated a clear concentration-dependent antioxidant efficacy (Fig. 5a), with IC₅₀ values of 7.46, 16.13, 66.95, and 112.97 μg/mL for AA, MeOH, CHCl₃, and PE, respectively (Fig. 5b). Among the extracts, the methanol (MeOH) extract exhibited potent DPPH radical scavenging activity (96.4 % RSA at 120 μg/mL), comparable to ascorbic acid (AA; 95.5 %) at the same concentration. The MeOH extract's IC₅₀ of 16.13 μg/mL confirmed its strong antioxidant efficacy, outperforming chloroform (66.95 μg/mL) and petroleum ether (112.97 μg/mL) extracts. While less potent than AA (IC₅₀ = 7.46 μg/mL), the MeOH extract's high activity highlights its potential as a natural antioxidant, likely due to polar phytochemicals like phenolics and flavonoids. This positions B. pratensis as a promising candidate for nutraceutical or pharmaceutical applications targeting oxidative stress. The antioxidant efficacy demonstrated by the extracts is consistent with the presence of bioactive phytochemicals identified via GC-MS, which have documented roles in enhancing enzymatic antioxidant defenses and mitigating oxidative damage. These observed antioxidant and cytotoxic effects can be attributed to the phytochemicals found through the use of GC-MS. Phytol, hexadecanoic acid, and β-sitosterol compounds have been shown to counteract oxidative stress by increasing the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), which help prevent free radical damage [66].

Fig. 5.

DPPH radical scavenging activity of B. prattensis extracts. (a) RSA (%) at increasing concentrations (10–120 μg/mL) for petroleum ether (PE), chloroform (CHCl₃), and methanol (MeOH) extracts, compared with ascorbic acid (AA). (b) IC₅₀ values of each extract were 7.46, 16.13, 66.95, and 112.97 μg/mL for AA, MeOH, CHCl₃, and PE, respectively. MeOH showing the lowest IC₅₀, indicating the highest antioxidant efficacy. Data are presented as mean ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001 vs. AA.

The antioxidant activity of B. prattensis suggests potential in managing oxidative stress-related conditions such as neurodegenerative diseases and metabolic disorders. Bioactive compounds like stigmasterol, phytol, and squalene may contribute through mechanisms including reactive oxygen species (ROS) scavenging, activation of the Nrf2/ARE pathway, and anti-inflammatory modulation, as supported by their pharmacological profiles [67].

3.5.2. Total antioxidant capacity

The total antioxidant capacity (TAC) of the three solvent extracts petroleum ether (PE), chloroform (CHCl₃), and methanol (MeOH) was evaluated using the phosphomolybdate assay, with results expressed in ascorbic acid equivalents (μg/mL) (Table 7). All extracts exhibited a concentration-dependent increase in TAC. MeOH showed the highest TAC (78.95 ± 1.8 to 178.00 ± 2.6 μg AA eq/mL), followed by CHCl₃ (69.43 ± 1.2 to 149.43 ± 3.6 μg AA eq/mL) and PE (23.24 ± 1.5 to 130.14 ± 2.1 μg AA eq/mL) at 100–600 μg/mL. The higher TAC of MeOH suggests a greater concentration of polar bioactive compounds with strong reducing properties, such as flavonoids and phenolics. In contrast, the lower TAC of PE indicates a lesser contribution of nonpolar molecules to antioxidant activity. This trend aligns with previous studies demonstrating that polar solvents are more effective in extracting antioxidant-rich compounds from plants [68].

Table 7.

Total antioxidant capacity (TAC) using phosphomolybdate assay.

Extract	Concentration (ug/mL) (Ascorbic Acid Equivalent)
	100	200	300	400	500	600
PE	23.24 ± 1.5	59.19 ± 3.6	73.95 ± 2.7	84.67 ± 1.5	105.38 ± 2.9	130.14 ± 2.1
CHCl3	69.43 ± 1.2	80.85 ± 3.7	98.71 ± 1.2	127.29 ± 4.9	140.14 ± 3.7	149.43 ± 3.6
MeOH	78.95 ± 1.8	88.00 ± 2.1	104.67 ± 2.2	126.09 ± 6.2	165.62 ± 1.8	178.00 ± 2.6

3.6. Cytotoxic potential of B. prattensis extracts

MCF-7 was selected as it is a standard in vitro model for hormone-responsive breast cancer, the most commonly diagnosed cancer worldwide. The cytotoxic potential of B. prattensis extracts against MCF-7 breast cancer cells was assessed using the MTT assay. The negative control (PBS with 0.5 % DMSO) showed no significant effect, maintaining nearly 100 % cell viability, which confirms that the solvent did not affect the assay. As illustrated in Fig. 6a, all extracts exhibited dose-dependent cytotoxicity. PE demonstrating the most pronounced effect (IC₅₀ = 60.1 μg/mL), followed by chloroform (IC₅₀ = 260.0 μg/mL) and methanol (IC₅₀ = 293.6 μg/mL) Fig. 6b. At 31.25 μg/mL, PE reduced viability to 72.5 %, compared to 85.4 % and 86.8 % for chloroform and methanol extracts, respectively. The petroleum ether (PE) extract exhibited significant cytotoxicity against MCF-7 breast cancer cells in the MTT assay, suggesting the presence of bioactive lipophilic compounds. GC-MS analysis identified several such compounds, including terpenoids (phytol, squalene, dl-α-tocopherol), fatty acid derivatives (9-octadecenamide, 1-hexadecanol, octadecanal), phytosterols (stigmasterol, β-sitosterol), and aliphatic hydrocarbons (nonadecane, eicosane). These metabolites have been linked to anticancer activity in previous reports—phytol through ROS-mediated apoptosis [69], squalene via chemoprevention, and β-sitosterol through cell cycle modulation [70,71]. Stigmasterol and dl-α-tocopherol also exhibit antiproliferative and antioxidant functions [72]. While these findings suggest a possible contribution of these constituents to the observed cytotoxicity, the precise mechanisms underlying this effect remain speculative. The MTT assay was selected for cytotoxicity assessment due to its widespread use and cost-effectiveness. Nevertheless, future studies should incorporate additional assays, such as LDH release, to validate and expand upon the current findings. Future studies will focus on isolating major active compounds and employing additional mechanistic assays to validate their role in cancer cell growth inhibition and delineate the pathways involved [73]. The moderate cytotoxicity of CHCl₃, a solvent of intermediate polarity, suggests the presence of both polar and non-polar cytotoxic agents, such as terpenoids or alkaloids, which may act synergistically with other bioactive compounds. In contrast, the relatively lower cytotoxicity of the MeOH extract indicates that highly polar phytochemicals may be less effective against MCF-7 cells. While methanol is efficient in extracting phenolic compounds and flavonoids, these hydrophilic constituents may exhibit reduced permeability in cancer cell membranes, limiting their bioactivity.

Fig. 6.

(a) Cell viability (%) of MCF-7 cells treated with increasing concentrations (31.25–500 μg/mL) of petroleum ether (PE), chloroform (CHCl₃), and methanol (MeOH) extracts. A dose-dependent reduction in cell viability is observed, with PE extract demonstrating the most potent cytotoxic effect. (b) IC₅₀ values, representing the concentration required to reduce cell viability by 50 %, show that PE has the lowest IC₅₀ (60.1 μg/mL), indicating superior cytotoxicity compared to CHCl₃ (260.0 μg/mL) and MeOH (293.6 μg/mL) extracts. Data are presented as mean ± SD (n = 3).

Morphological changes in MCF-7 cells treated with PE, CHCl₃, and MeOH extracts at different concentrations are shown in Fig. 7. Untreated control cells exhibit a confluent monolayer with typical epithelial morphology. PE extract treatment results in significant cytotoxicity, with cells displaying membrane shrinkage, rounding, and detachment at higher concentrations, indicating apoptosis. CHCl₃ extract induces moderate cytotoxic effects, with cell rounding and reduced density at increasing doses. MeOH extract shows the least morphological disruption, maintaining a more adherent monolayer with minimal cell detachment. These results correlate with the MTT assay findings, highlighting the potent cytotoxicity of PE extract.

Fig. 7.

Morphological alterations in MCF-7 cells treated with B. prattensis extracts: MCF-7 cells treated with PE, CHCl₃, and MeOH extracts (31.25–500 μg/mL) showed concentration-dependent morphological changes under phase-contrast microscopy. Observed features included cell shrinkage, rounding, detachment, and reduced density, indicating cytotoxic effects. Alterations were most pronounced with PE extract at higher concentrations, consistent with apoptotic morphology and MTT assay results.

The cytotoxic efficacy of B. prattensis extracts against breast cancer cell lines suggests its potential as a source of anticancer agents. Phytol and stigmasterol may induce apoptosis via mitochondrial depolarization and caspase-3/7 activation, while squalene's anti-inflammatory action could enhance chemosensitivity. This multi-target activity may lower the risk of resistance compared to single-pathway drugs [70,74].

For cytotoxic activity, some compounds identified via GC-MS may induce apoptosis in cancer cells. For example, β-sitosterol and stigmasterol can interfere with cancer cell growth by damaging mitochondria, increasing reactive oxygen species (ROS), and activating caspase enzymes, leading to cell death [71]. These compounds are also known to block survival pathways like PI3K/Akt in cancer cells [75].

These findings align with previous research on Barleria species. Future studies should focus on isolating key bioactive constituents and elucidating their mechanisms of action in vivo models.

4. Conclusions

The comprehensive evaluation of Barleria prattensis extracts revealed significant phytochemical diversity and bioactivity, underscoring its potential as a source of natural therapeutic agents. Sequential Soxhlet extraction demonstrated that polar solvents, particularly methanol, yielded the highest extractable compounds (3.36 %), reflecting the plant's richness in polar constituents such as phenolics and flavonoids. Phytochemical screening confirmed the presence of alkaloids, terpenoids, and steroids in chloroform and methanol extracts, while petroleum ether exhibited limited diversity. Quantitative analysis further validated these findings, with methanol extract displaying the highest total phenolic (72.9 mg GAE/g) and flavonoid (43.4 mg QE/g) content, consistent with its potent antioxidant activity (DPPH IC₅₀ = 16.13 μg/mL). GC-MS profiling identified key bioactive compounds, including phytol, squalene, neophytadiene and β-sitosterol, known for their antioxidant and anticancer properties. The petroleum ether extract exhibited the most potent cytotoxic effect against MCF-7 breast cancer cells (IC₅₀ = 60.1 μg/mL), followed by chloroform (IC₅₀ = 260.0 μg/mL) and methanol (IC₅₀ = 293.6 μg/mL). Morphological analysis confirmed apoptotic changes in treated MCF-7 cells, supporting the anticancer potential of these extracts. These findings position B. prattensis as a promising candidate for managing oxidative stress-related disorders and cancer. Future studies should isolate and characterize active compounds, enhance bioavailability, and validate efficacy in vivo, bridging traditional knowledge with modern pharmacology for drug discovery.

CRediT authorship contribution statement

Saif Saleh Mohsen Ali: Writing – review & editing, Writing – original draft, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation. Pushpa Robin: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

AI tools

Were used only for language enhancement and proofreading, not for data analysis, or content generation.

Data availability

Data will be made available on request.

Funding

This research received no external funding and was fully supported by the authors' resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: