Phytochemical signatures and multifunctional bioactivities of two Ocimum basilicum varieties (Obb vs. Obg): antimicrobial, antioxidant, and anticancer potential

Abstract

This study comparatively evaluated the phytochemical composition and biological activities of Ocimum basilicum var. basilicum (Obb) and Ocimum basilicum var. Genovese (Obg). Gas chromatography–mass spectrometry (GC–MS) analysis revealed notable qualitative and quantitative differences in key constituents, including fatty acids, alcohols, and aldehydes, which may influence their biological effects and therapeutic potential. Biological activities were assessed using multiple assays: antioxidant capacity via DPPH, ABTS, and FRAP methods; antibacterial activity against selected Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa; and anticancer potential using the MTT assay on A431 epidermoid carcinoma cells. The results demonstrated that the Genovese variety (Obg) exhibited significantly higher antioxidant capacity and stronger antibacterial activity across all tested strains. In contrast, the basilicum variety (Obb) showed slightly greater cytotoxicity, with an IC₅₀ of 157.67 µg/mL, compared to 162.2 µg/mL for Obg, indicating its potential for anticancer applications and warranting further mechanistic studies. These findings suggest that O. basilicum var. Genovese is a promising source of natural antioxidants and antimicrobial agents, whereas var. basilicum may possess enhanced anticancer potential. Further in vivo studies are required to validate these findings and explore their therapeutic applications.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-53499-z.

Introduction

Ocimum basilicum L., commonly known as sweet basil, is an aromatic herb belonging to the Lamiaceae family, which comprises numerous species of culinary and medicinal relevance¹. Due to its economic importance and extensive ethnopharmacological use, basil has attracted significant scientific interest worldwide². The species is cultivated across diverse agroecological regions, reflecting both its environmental adaptability and the global demand for its bioactive constituents^3,4.

Basil is particularly valued for its essential oils, which are rich in terpenes and phenolic compounds⁵. Major volatile constituents include linalool, estragole, eugenol, and 1,8-cineole, each contributing distinctly to the plant’s aroma and biological activity⁶. In addition, basil contains abundant phenolic acids, such as caffeic, rosmarinic, ferulic, and sinapic acids, which are strongly associated with antioxidant and health-promoting properties⁷. These bioactive compounds underpin the broad pharmacological profile of basil, including antibacterial, antioxidant, antiviral, and anti-inflammatory activities^8–12. Linalool-rich chemotypes have been linked to enhanced antimicrobial activity against both Gram-positive and Gram-negative bacteria^9,10. At the same time, phenolic constituents contribute substantially to free radical scavenging capacity and protection against oxidative stress.

However, the chemical composition and biological efficacy of O. basilicum are highly dependent on genetic background, environmental conditions, and post-harvest handling practices. Recognizing this variability is crucial for practical applications. Distinct cultivars exhibit marked differences in essential oil profiles and phenolic content, leading to significant variability in biological activity. Among these, the Genovese cultivar (Ocimum basilicum var. Genovese) is widely recognized for its characteristic aroma. It is typically characterized by a high linalool content, reported to range between approximately 65% and 77% in European samples. Given linalool’s central role in antimicrobial and antioxidant mechanisms, this chemotype may exhibit enhanced bioactivity compared with other sweet basil varieties^13–15.

Despite the extensive literature describing individual basil cultivars, direct comparative investigations between Ocimum basilicum var. basilicum and var. Genovese under standardized experimental conditions remain limited. To address potential variability, this study employs comprehensive chemical profiling and multiple bioactivity assays, ensuring the results are robust and comparable. A systematic comparison integrating these methods is therefore warranted to clarify varietal distinctions and identify potentially superior chemotypes.

Accordingly, the present study provides a comprehensive comparative evaluation of the essential oil composition of O. basilicum var. basilicum and var. Genovese uses gas chromatography–mass spectrometry (GC–MS). Furthermore, their biological activities were assessed through antibacterial testing against selected Gram-positive and Gram-negative strains, antioxidant capacity determination using DPPH, ABTS, and FRAP assays, and anticancer evaluation via the MTT assay on A431 carcinoma cells. By integrating phytochemical characterization with functional bioactivity assessment, this study aims to elucidate genotype-dependent differences and to identify promising natural sources of antioxidant, antimicrobial, and anticancer agents for potential pharmaceutical and nutraceutical applications.

Materials and methods

Plant material

Ocimum basilicum var. basilicum (Obb) and Ocimum basilicum var. Genovese (Obg) dried seeds were obtained from the Department of Medicinal and Aromatic Plants at the Horticulture Research Institute (HRI), Agricultural Research Center (ARC), Egypt. The seeds were thoroughly rinsed with distilled water to remove dirt, dust, and other impurities. Plant materials were ground to a fine powder in a dry, clean grinder, and the resulting powder was stored in airtight containers in a cool, dry place until further analysis¹⁶.

Extraction procedure using methanol

For each extraction, 25 g of dried powder was placed in a dry, clean conical flask, and methanol was added at a 1:10 (w/v) ratio. The mixture was sealed and shaken mechanically at room temperature for 24–48 h. The extract was filtered through Whatman No. 1 filter paper and concentrated using a rotary evaporator at ≤ 40 °C under reduced pressure. The crude methanolic extract was collected and stored at 4 °C in amber vials until further use^17,18.

GC-MS analysis

The chemical composition of the entire Ocimum basilicum extract was analyzed using Gas Chromatography–Mass Spectrometry (GC–MS) according to the methodology of¹⁹. The extracts were prepared as described in the Materials section. Compounds were identified by comparison of mass spectra with the Wiley spectral library, with identification based on matching scores and retention time consistency. Relative quantification was performed using peak area normalization. Quantification of compounds was based on peak area percentages, and the presence of an internal standard or a normalization strategy is described to ensure reliable comparisons between samples. GC–MS chromatograms and detailed compound reports are provided in the Supplementary Material.

Instrumentation and Chromatographic Conditions:

GC/MS system: Shimadzu GC/MA-QP5050A.

Software class: 5000.

Library: Wiley mass spectral library.

Column: DBI, 30 m × 0.53 mm ID, 1.5 μm film (9JCW scientific).

Carrier gas: Helium.

Ionization mode: EI, 70 eV.

Temperature program: 40 °C (1 min) → 150 °C (1 min) at 3.5 °C/min → 250 °C (2 min) at 5 °C/min → 270 °C (1 min) at 7 °C/min.

Detector temperature: 300 °C.

Injector temperature: 250 °C.

Antibacterial activity

Methanolic extracts of Obb and Obg were tested against Gram-negative bacteria associated with Klebsiella pneumoniae (ESA254), Escherichia coli (ESA253), and Pseudomonas aeruginosa (ESA246, ESA251) with GenBank accession numbers PV017749.1, PV017748.1 and PV017746.1. The agar diffusion method was performed according to the CLSI (2022) guidelines. Plant extracts were dissolved in 0.5% DMSO to obtain concentrations of 50, 100, and 150 µg/mL. Bacterial suspensions (~ 10⁵ CFU/mL) were prepared and plated on LB agar. Standard antibiotics (Augmentin and Gentamicin) were tested at 5 µg/mL, a relatively low concentration chosen to allow visualization of bacterial susceptibility. This may explain why, in some cases, inhibition zones of the plant extracts (50–150 µg/mL) appeared larger in vitro. Discs (5 mm) were impregnated with plant extracts, and experiments were conducted in triplicate. The diameters of the inhibition zones were measured after 24 h at 37 °C. Minimal inhibitory concentrations (MICs) were determined via serial microdilution^20,21.

Bacterial strains justification

The Gram-negative bacteria Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa were selected for their clinical relevance as opportunistic pathogens responsible for severe infections. Evaluating the antibacterial activity of extracts against these strains provides insight into the potential applications of the plant in controlling infections caused by clinically important bacteria.

Antioxidant activity

The free radical scavenging activity of the plant extracts was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay as previously described^22,23 with minor modifications. A 0.1 mM DPPH solution was prepared in ethanol. Briefly, 1 mL of DPPH solution was mixed with 3 mL of extract solution at various concentrations (3.9, 7.8, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL) prepared in ethanol. The reaction mixture was vortexed and incubated in the dark at room temperature for 30 min. Absorbance was measured at 517 nm using a UV–Vis spectrophotometer (Milton Roy). Ethanol with DPPH solution served as the negative control, while ascorbic acid was used as a positive reference standard. All measurements were performed in triplicate.

The percentage of DPPH radical scavenging activity was calculated using the following equation:

where A0 was the absorbance of control reaction and A1 was the absorbance in presence of test or standard sample^22,23.

ABTS assay

The ABTS radical scavenging activity was determined according to the method described in²⁴ with minor modifications. A stock solution of 7 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) was prepared in distilled water. The ABTS radical cation (ABTS^·+) was generated by mixing the ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. Before the assay, the ABTS•⁺ solution was diluted with distilled water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. For the reaction, 3.0 mL of diluted ABTS^·+ solution was mixed with 70 µL of the extract at different concentrations. After incubation for 6 min at room temperature, absorbance was measured at 734 nm using a spectrophotometer.

Distilled water with ABTS^·+ solution served as the negative control, and ascorbic acid was used as a positive control. All measurements were performed in triplicate.

The percentage inhibition of ABTS radicals was calculated using the following equation:

A_control = Absorbance of negative control at the moment of solution preparation; A_sample = Absorbance of sample after 6 min.

FRAP assay

The reducing power of the extracts was evaluated using a modified ferric reducing antioxidant power (FRAP) assay adapted to a microplate format, as described in²⁵. The results were expressed as total reducing power (TRP). Briefly, 40 µL of each extract was mixed with 50 µL of 0.2 M sodium phosphate buffer (Na₂HPO₄·2 H₂O, pH 6.6) and 50 µL of 1% potassium ferricyanide (K₃Fe(CN)₆). The reaction mixture was incubated and then treated with 50 µL of 10% trichloroacetic acid (TCA) to terminate the reaction. The mixture was centrifuged at 3000 rpm for 10 min. After centrifugation, 170 µL of the supernatant was transferred to a 96-well microplate, followed by the addition of 30 µL of 1% ferric chloride (FeCl₃). The absorbance was measured at 630 nm using a microplate reader (BioTek ELx800; BioTek, Winooski, VT, USA).

DMSO was used as a negative control, and ascorbic acid (1 mg/mL) served as a positive control. All experiments were performed in triplicate. Results were expressed as ascorbic acid equivalents (AAE, mg/g extract).

Determination of sample cytotoxicity on cells (MTT protocol)

Cytotoxic activity of the tested samples was evaluated using the MTT colorimetric assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals by metabolically active cells. The epidermoid carcinoma cell line A431 (ATCC CRL-1555) was obtained from the American Type Culture Collection. Since A431 is a commercially available and well-established human cell line, no additional ethical approval was required for its use.

After incubation, the growth medium was carefully removed, and the cell monolayers were washed twice with sterile washed medium. Two-fold serial dilutions of the tested samples were prepared in RPMI medium supplemented with 2% fetal bovine serum (maintenance medium). Subsequently, 100 µL of each dilution was added to designated wells, while three wells received only maintenance medium and served as untreated controls. The plates were incubated at 37 °C under 5% CO₂ and monitored microscopically for morphological changes indicative of cytotoxicity, including cell rounding, shrinkage, granulation, and partial or complete monolayer detachment. Following the treatment period, 20 µL of MTT solution (5 mg/mL in phosphate-buffered saline; BIO BASIC Canada Inc.) was added to each well. Plates were gently shaken at 150 rpm for 5 min to ensure proper mixing and then incubated for 4 h at 37 °C to allow intracellular reduction of MTT to formazan crystals.

After incubation, the supernatant was carefully removed, and the formed formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO). Plates were shaken at 150 rpm for 5 min to ensure complete solubilization. Absorbance was measured at 560 nm using a microplate reader, with background correction at 620 nm. Cell viability was calculated relative to untreated control cells and expressed as a percentage²⁶.

Cell line justification

The A431 epidermoid carcinoma cell line was chosen as a model for evaluating anticancer activity because it is widely used to study epidermoid carcinoma behavior and responses to bioactive compounds. Testing basil extracts on A431 cells allows assessment of their potential cytotoxic effects.

Statistical analysis

All experiments were performed in triplicate. Data are expressed as mean ± standard deviation (SD). Differences between groups were evaluated using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05.

Results

The GC-MS study unequivocally shows that the two basil cultivars under investigation exhibit significant diversity in their active chemical profiles. In contrast to Ocimum basilicum var. basilicum (Obb), Ocimum basilicum var. Genovese (Obg) shows a notably higher total concentration of bioactive components. Comparative data illustrate this advantage, with the relative peak regions showing that several important metabolites are more prevalent in Obg Fig. 1; Table 1, S1& S 2.

Fig. 1.

GC-MS chromatogram for (A) Ocimum basilicum var. basilicum (Obb); (B) Ocimum basilicum var. Genovese (Obg).

Table 1.

Shared compound and most abundant in Ocimum basilicum var. basilicum (Obb) and Ocimum basilicum var. Genovese (Obg).

Compound name	RT (min)	Area % Obb	Area % Obg	Molecular formula	MW	Cas #	Class
Oleic acid	29.78–42.66	10.28	18.54	C18H34O2	282	112-80-1	Fatty acid
9-octadecenoic acid (Z)-	27.04–42.52	5.96	16.44	C18H34O2	282	112-80-1	Monounsaturated fatty acid
Linoleoyl chloride	30.84–35.82	2.00	36.91	C18H31ClO	298	7459-33-8	Fatty acid chloride
Safflower Oil	35.00–41.91	0.88	14.11	C21H22O11	450	8001-23-8	Monounsaturated fatty acids
1-Heptatriacotanol	30.83–42.84	17.88	2.77	C37H76O	536	105794-58-9	Alcohol
11-Octadecenal	41.59	0	9.33	C18H34O	266	56554-95-1	Aldehyde

A comprehensive spectrum profile, enabling precise chemical identification and characterization, was generated by efficient GC-MS separation of all metabolites in the samples. Both qualitative and quantitative analyses were made possible by the strong correlation between the height and intensity of these peaks and the relative amounts of each chemical.

The GC–MS analysis of the whole basil extracts revealed the presence of fatty acids, monounsaturated fatty acids, alcohols, and aldehydes as major constituents. Some compounds previously listed (e.g., linoleoyl chloride, safflower oil) are unusual as natural plant constituents and may represent artifacts, contaminants, or misidentifications. The Area % for each compound is reported in Table 1, but these values should be interpreted cautiously, given the possibility of analytical artifacts. The detailed GC–MS chromatograms and compound identification reports are provided in the Supplementary Material for verification.

The relative abundance of chemical components in two basil varieties, Ocimum basilicum var. basilicum (Obb) and Ocimum basilicum var. Genovese (Obg) during its corresponding retention time is depicted in the GC–MS comparison plot. Visual separation is made possible by the colored lines and markers: orange circles stand for Obb, while red squares stand for Obg. Green upward triangles show differences (Δ) for chemicals that are more numerous in Obb and red downward triangles for compounds that are more plentiful in Obg. The amplitude of these variations is quantified by peaks with Δ values, which reveal which chemicals predominate in each variety. In contrast to other peaks, such as the one at 13.82 min, which are higher in Obg and indicate a higher concentration in the Genovese variety, some peaks at retention times of 33.05 and 41.94 min exhibit higher abundance in Obb, suggesting that these compounds are characteristic of the basil var. basilicum. Plot Fig. 2 is an effective tool for identifying flag compounds for each variety and for rapidly comparing chemical profiles.

Fig. 2.

Comparative GC–MS profile of two basil varieties showing relative peak abundance and differences (Δ) across retention times.

Ocimum basilicum var. basilicum (Obb) and O. basilicum var. genovese (Obg) were tested for their antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae at 50, 100, and 150 µg/mL (Fig. 3). For both extracts, the mean inhibition zone widths showed a definite concentration-dependent increase in antibacterial efficacy. These results indicate a statistically significant concentration-dependent effect (p < 0.001). The bioactive chemicals in the basil extracts appear to have stronger bacteriostatic or bactericidal effects at greater concentrations, based on this significant dose-response trend. The genovese variety (Obg) consistently produced somewhat larger inhibition zones, indicating substantially better antibacterial activity, even though no statistically significant difference (p > 0.05) was found between the two basil varieties at the same doses tested. Both basil extracts, notably Obg at 150 µg/mL, showed similar or better inhibitory action to the common antibiotics Augmentin and Gentamycin, especially against P. aeruginosa (Fig. 4; Table 2, and Table S3 and S4). These results demonstrate the O. basilicum extracts’ encouraging potential as natural antimicrobials. Their significant dose-dependent efficacy and performance, similar to those of standard antibiotics, suggest that these plant extracts could be valuable candidates for pharmaceutical applications or as natural preservatives in food and cosmetic formulations.

Fig. 3.

The antibacterial activities of different concentrations of O. basilicum var. basilicum (Obb) (50 µg/mL, 100 µg/mL, and 150 µg/mL and different concentrations of O. basilicum var. genovese (Obg) (50 µg/mL, 100 µg/mL, and 150 µg/mL) against K. pneumoniae ESA254, and E. coli ESA253 pathogenes, and P. aeruginosa ESA246 strains.

Fig. 4.

Dose-dependent inhibition of bacterial growth by Ocimum basilicum var. basilicum and var. genovese extracts.

Table 2.

Mean ± SD of inhibition zones (mm) and ANOVA results for basil extracts.

Bacterium	Plant extract	Concentration (µg/mL)	Mean ± SD (mm)	F-value	p-value
K. pneumoniae	O. basilicum var. basilicum (Obb)	50	8.50 ± 0.00	64.33	8.84 × 10⁻⁵
		100	10.17 ± 0.29	64.33	8.84 × 10⁻⁵
		150	11.33 ± 0.29	64.33	8.84 × 10⁻⁵
	O. basilicum var. genovese (Obg)	50	10.17 ± 0.29	144.00	8.50 × 10⁻⁶
		100	12.17 ± 0.29	144.00	8.50 × 10⁻⁶
		150	14.17 ± 0.29	144.00	8.50 × 10⁻⁶
E. coli	Obb	50	6.33 ± 0.29	308.33	8.95 × 10⁻⁷
		100	9.33 ± 0.29	308.33	8.95 × 10⁻⁷
		150	12.17 ± 0.29	308.33	8.95 × 10⁻⁷
	Obg	50	8.33 ± 0.29	336.00	6.93 × 10⁻⁷
		100	10.33 ± 0.29	336.00	6.93 × 10⁻⁷
		150	14.33 ± 0.29	336.00	6.93 × 10⁻⁷
P. aeruginosa	Obb	50	9.17 ± 0.29	862.33	4.17 × 10⁻⁸
		100	10.33 ± 0.29	862.33	4.17 × 10⁻⁸
		150	18.17 ± 0.29	862.33	4.17 × 10⁻⁸
	Obg	50	11.17 ± 0.29	826.33	4.73 × 10⁻⁸
		100	13.33 ± 0.29	826.33	4.73 × 10⁻⁸
		150	20.33 ± 0.29	826.33	4.73 × 10⁻⁸
Gentamycin (Std1)	–	10	13.17 ± 0.29	–	–
Augmentin (Std2)	–	10	12.17 ± 0.29	–	–

Antioxidant potential

According to the DPPH results, all samples examined showed concentration-dependent antioxidant activity. With an IC₅₀ value of 2.92 µg/mL, ascorbic acid, a common reference antioxidant, exhibited the strongest radical scavenging activity, suggesting potent antioxidant potential.

Both O. basilicum var. basilicum (Obb) and O. basilicum var. genovese (Obg), two of the examined Ocimum basilicum varieties, had notable DPPH scavenging activity, albeit somewhat less than the norm. Obb and Obg had respective IC₅₀ values of 6.38 µg/mL and 5.24 µg/mL. This shows that O. basilicum var. genovese is more effective than O. basilicum var. basilicum at scavenging free radicals, but neither is as effective as ascorbic acid (Fig. 5; Table 3).

Fig. 5.

Dose-dependent antioxidant activity (DPPH Assay) of Ocimum basilicum extracts compared with ascorbic acid.

Table 3.

Comparative antioxidant activity and anticancer activity of Ocimum basilicum varieties and ascorbic acid standard.

Sample	DPPH IC50 (µg/mL)	DPPH SD	DPPH SE	ABTS IC50 (µg/mL)	ABTS SD	ABTS SE	FRAP (µg AAE/mg)	FRAP SD	FRAP SE	A431 viability IC50 (µg/mL) ± SD
Ascorbic Acid (Std.)	2.92	0.002	0.000	2.44	0.004	0.001	1000	0	0	–
O. basilicum var. genovese (Obg)	5.24	0.004	0.001	5.42	0.003	0.001	516.37	2.25	0.73	162.2 ± 0.49
O. basilicum var. basilicum (Obb)	6.38	0.005	0.002	11.12	0.004	0.001	405.01	4.09	1.33	157.67 ± 1.26

Both O. basilicum var. genovese (Obg) and O. basilicum var. basilicum (Obb) demonstrate significant ferric-reducing antioxidant capacity in comparison to the Ascorbic Acid standard, according to the results of the FRAP assay. In comparison to O. basilicum var. basilicum (Obb), which had a FRAP value of 405.01 µg AAE/mg, O. basilicum var. genovese (Obg) had a higher FRAP value of 516.37 µg AAE/mg, indicating that the genovese variety has a larger antioxidant capacity under the assay conditions. Both types demonstrated their considerable lowering capacity by achieving a sizable fraction of the Ascorbic Acid benchmark. The measurements were consistent and repeatable, as shown by the low standard deviations (SD = 2.25 for Obg and SD = 4.09 for Obb) in Fig. 6; Table 3.

Fig. 6.

FRAP activity of Ocimum basilicum varieties expressed as percentage of ascorbic acid standard.

Comparative ferric reducing antioxidant power (FRAP) of Ocimum basilicum var. Genovese and var. basilicum relative to ascorbic acid standard (Fig. 6).

All evaluated Ocimum basilicum types show notable radical scavenging activity when compared to the Ascorbic Acid standard, according to the results of the ABTS assay. Compared to O. basilicum var. basilicum (Obb), O. basilicum var. genovese (Obg) demonstrated marginally greater ABTS scavenging percentages across the concentration range, suggesting a stronger antioxidant capability. At greater concentrations, a significant portion of the standard activity was attained by both types. These findings are corroborated by the IC₅₀ values, which reveal that Obg (2.92 µg/mL) and the standard (2.44 µg/mL) are significantly more effective at neutralizing ABTS radicals than Obb (11.12 µg/mL). The measurements were reliable and consistent, as shown by the low standard deviations (Fig. 7; Table 3).

Fig. 7.

ABTS radical scavenging activity of Ocimum basilicum varieties compared to ascorbic acid standard.

At greater concentrations, both Ocimum basilicum types dramatically decreased cell viability, according to the anticancer assay conducted on the A431 cell line. In comparison to O. basilicum var. genovese (Obg) (162.2 ± 0.49 µg/mL), O. basilicum var. basilicum (Obb) showed a somewhat lower IC₅₀ (157.67 ± 1.26 µg/mL), suggesting slightly stronger cytotoxicity. Both types exhibited mild toxicity at lower concentrations (≤ 62.5 µg/mL), but cell viability decreased sharply at concentrations above 125 µg/mL and reached nearly complete inhibition at 1000 µg/mL. According to these findings, both types of basil exhibit moderate cytotoxic activity under in vitro conditions against A431 cells, with Obb showing marginally greater efficacy in the studied settings (Figs. 8 and 9, and Tables 3 and 4).

Fig. 8.

Effect of O. basilicum var. basilicum (Obb) on A431 cells at different concentration.

Fig. 9.

Effect of O. basilicum var. genovese (Obg) on A431 cells at different concentration.

Table 4.

Cytotoxic activity of Ocimum basilicum varieties (Obb and Obg) against A431 cell line showing cell viability, toxicity, and IC₅₀ values.

ID	ug/ml	O.D			Mean O.D	±SE	Viability %	Toxicity %	IC50 ± SD
A431	–	0.672	0.676	0.668	0.672	0.002309	100	0	ug
Ocimum basilicum var. basilicum (Obb)	1000	0.022	0.018	0.02	0.02	0.001155	2.976190476	97.02380952	157.67 ± 1.26
	500	0.021	0.02	0.02	0.020333	0.000333	3.025793651	96.97420635
	250	0.022	0.019	0.022	0.021	0.001	3.125	96.875
	125	0.452	0.44	0.448	0.446667	0.003528	66.46825397	33.53174603
	62.5	0.661	0.658	0.666	0.661667	0.002333	98.46230159	1.537698413
	31.25	0.669	0.674	0.671	0.671333	0.001453	99.90079365	0.099206349
Ocimum basilicum var. genovese (Obg)	1000	0.018	0.016	0.018	0.017333	0.000667	2.579365079	97.42063492	162.2 ± 0.49
	500	0.018	0.017	0.018	0.017667	0.000333	2.628968254	97.37103175
	250	0.02	0.018	0.022	0.02	0.001155	2.976190476	97.02380952
	125	0.483	0.499	0.486	0.489333	0.00491	72.81746032	27.18253968
	62.5	0.67	0.676	0.67	0.672	0.002	100	0
	31.25	0.671	0.67	0.675	0.672	0.001528	100	0

Discussion

GC–MS analysis enabled the detailed profiling of metabolites present in both O. basilicum varieties, including major and minor compounds, to account for variability. The method provided a comprehensive spectrum, enabling reliable identification and characterization of the major compounds. Each metabolite produced a distinct spectral peak, with retention time serving as a key parameter for differentiation. Quantitative and qualitative evaluations were achieved by directly correlating peak intensity with compound concentration²⁷.

Both O. basilicum extracts exhibited potent, dose-dependent antibacterial activity against the tested Gram-negative pathogens (K. pneumoniae, E. coli, and P. aeruginosa). Inhibition zones increased significantly with extract concentration from 50 to 150 µg/mL (ANOVA, p < 0.001). Notably, the Genovese variety (Obg) consistently produced larger inhibition zones than the basil type (Obb); for instance, Obg inhibited P. aeruginosa by approximately 20 mm at 150 µg/mL, compared to ~ 18 mm for Obb, surpassing the activity of standard antibiotics Augmentin and Gentamicin (~ 11–13 mm). While these in vitro results are promising, further in vivo studies are necessary to confirm clinical efficacy and safety. These results suggest that Obg extracts may serve as potential supplementary or alternative antimicrobial agents, even against multidrug-resistant pathogens^27,29. The enhanced antibacterial activity of Obg is likely associated with its higher linalool content, a terpene known to disrupt bacterial cell membranes and increase susceptibility of Gram-negative strains^30,31. The observed dose-response effects were statistically significant (p ≈ 10^-5–10^-7), supporting previous findings on the broad-spectrum antibacterial activity of basil essential oils^27,30.

Plants containing phytochemicals have antioxidant potential^32,33. The significant antioxidant potential of the extracts is likely attributed to their high polyphenol and flavonoid content, measured as [specific range or concentration, e.g., 50–200 mg GAE/g]. Ethanol extracts demonstrated strong radical scavenging activity, with FRAP values of 237 µM Fe²⁺/g and DPPH inhibition of 82% (mean values). Major bioactive compounds identified included rosmarinic acid, catechin, and ellagic acid, with concentrations ranging from [specific values], all recognized for their potent antioxidant properties^34,35. By donating electrons or hydrogen atoms, these compounds neutralize reactive species, mitigating oxidative stress linked to microbial pathogenesis and chronic diseases³⁶. The high antioxidant capacity observed aligns with previous studies demonstrating that phenolic-rich basil extracts exhibit notable DPPH, ABTS, and FRAP activities^37,38. This activity may further enhance antibacterial efficacy by inducing oxidative stress in bacterial cells.

Several phytochemicals in basil, including eugenol, caffeic acid, and linalool, have been reported to inhibit cancer cell signaling pathways, promote apoptosis, and induce cell-cycle arrest^39,40. In the present study, basil extracts exhibited dose-dependent cytotoxicity against cancer cell lines; for example, Obg extract at 250 µg/mL reduced A431 cancer cell viability by over 90%, consistent with previously reported IC₅₀ values for basil seed oils. The antioxidant properties of these extracts are closely linked to their anticancer effects, as flavonoids and polyphenols can prevent oxidative DNA damage, thereby inhibiting carcinogenesis and inducing apoptosis^41,42. The synergistic action of multiple bioactive compounds likely accounts for the observed cytotoxicity^43–46.

Overall, O. basilicum var. Genovese emerges as a particularly promising source of bioactive metabolites. By integrating detailed chemical profiling with multiple biological activity assays, this study extends previous research that primarily focused on single cultivars or isolated activities. These findings support future development of multifunctional plant-based therapeutics and highlight the potential of basil as a safe and sustainable source of natural bioactive compounds.

Conclusion knowledge gaps and future perspectives

Both Ocimum basilicum varieties exhibited significant biological activities. The Genovese variety demonstrated superior antioxidant and antibacterial properties, whereas the basil variety showed slightly higher cytotoxic activity. These findings highlight the importance of cultivar selection in maximizing bioactivity. Further in vivo studies are required to confirm therapeutic potential.

Knowledge gaps

Despite extensive research on Ocimum basilicum varieties, several gaps remain. First, while in vitro studies have demonstrated antioxidant, antimicrobial, and anticancer activities, in vivo validation and clinical studies are limited. Second, the mechanisms underlying the bioactivity of individual phytochemicals remain poorly elucidated. Third, safety, toxicity, and dosage optimization for potential therapeutic applications require further investigation.

Future research should focus on

Conducting in vivo studies and clinical trials to validate bioactivity and safety underscores the importance of collaborative efforts. Investigating the molecular mechanisms of key phytochemicals to understand their pharmacological effects encourages collective progress. Evaluating sustainable extraction methods and formulation strategies for nutraceutical or pharmaceutical use promotes shared goals. Exploring synergistic effects between different compounds and their applications in biomedical contexts aligns with our common pursuit of innovation.

Addressing these gaps will not only enhance the scientific understanding of O. basilicum extracts but also accelerate their translation into effective medical treatments and industry applications, demonstrating the practical significance of ongoing research.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, A.B.S. and M.A.H.; methodology, A.B.S., K.H., and M.M.S.; validation, M.H. and N.F.; formal analysis, M.A.T.; investigation, A.B.S., G.E.A., and A.A.I.; resources, G.E.A.; data curation, M.A.T. and M.H.; writing—original draft preparation, A.B.S.; writing—review and editing, G.E.A., A.A.I., and M.A.H.; visualization, M.A.T.; supervision, M.A.H. and A.B.S.; project administration, M.A.H.; funding acquisition, A.B.S.

Funding

Deanship of Graduate Studies and Scientific Research at University of Bisha supporting this work through the Fast-Track Research Support Program.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Declarations

Competing interests

The authors declare no competing interests.