Enhancing antioxidant and antibacterial activities of Cuminum cyminum, Origanum vulgare, and Salvia officinalis essential oils through a synergistic perspective

Abstract

Essential oils (EOs) from aromatic and medicinal plants have antioxidant and antibacterial properties and are commonly used as preservatives in the food industry. Combining EOs can produce a synergistic effect, enabling their application as condiments at reduced dosages. Therefore, this study aimed to evaluate the antioxidant and antibacterial activities of the combination of three EOs extracted from Sage (Salvia officinalis), Oregano (Origanum vulgare), and Cumin (Cuminum cyminum). This experiment involved a three-factor simplex lattice design comprising 14 experimental runs with three replications. Gas chromatography–mass spectrometry (GC–MS) analysis identified carvacrol (39.54%) and γ-terpinene (23.22%) as the principal components of the EO from O. vulgare, while cumin aldehyde (24.24%) and lavandulyl acetate (19.56%) and γ-terpinene (18.78%) were dominant in the EO of C. cyminum; additionally, S. officinalis exhibited α-thujene (34.85%) and pinocarvone (18.44%) as its main constituents. The optimal conditions for the independent variables were established at 15.0553% for C. cyminum, 74.0453% for O. vulgare, and 10.90% for S. officinalis. These conditions resulted in notable increases in antioxidant activity, as demonstrated by a DPPH scavenging activity of 88.1% and a FRAP assay value of 11.3 μmol Fe(II)/g dry weight (DW). Additionally, the optimal conditions yielded significant inhibition zones against various bacterial strains, measuring 18.59 mm for Salmonella enterica, 22.97 mm for Escherichia coli, and 19.26 mm for Staphylococcus aureus. The predictive models showed high and statistically significant regression coefficients for all the parameters (p < 0.05). The measured parameters were in close agreement with the predicted values from the quadratic polynomial equation. Correlation analysis tools can effectively identify EOs’ synergistic properties and predict active compounds’ optimal characteristics. These findings confirm that the antioxidant and antibacterial activities are enhanced through the synergistic combination of EOs, underscoring their potential as effective natural preservatives in industrial applications.

Introduction

In recent years, synthetic preservatives have been widely used to prevent microbial contamination and oxidation in various food products, but concerns have arisen regarding their safety due to health risks and effects associated^1,2. Phytochemicals, due to their antioxidant and antibacterial properties, represent a promising natural alternative for the food industry to avoid the adverse effects of synthetic additives³. Natural food preservatives, such as essential oils (EOs), have proven effective in suppressing the growth of pathogenic microorganisms and offer promising alternatives to chemical antibiotics^2,4. EOs are considered vital compounds for pharmaceutical and biomedical applications due to their properties, which include anti-inflammatory, analgesic, sedative, antibacterial, and antifungal activities ⁵. Due to their high antioxidant properties, antimicrobial effects, and strong medicinal qualities, EOs have been used as flavoring agents and preservatives in food⁶. High doses of EOs in food storage can negatively impact taste. However, combining different EOs can help preserve the flavor and aroma⁷. This combination may result in antagonism, synergistic effects, or no effect⁸. Minor components’ activity can enhance the effectiveness of major constituents in EOs⁹. Moreover, EOs’ strong insect-repellent activity is attributed to their complex mixture of hydrocarbons ¹⁰. By optimizing the efficiency of these combined agents, it is possible to reduce the required dosage while simultaneously increasing their antioxidant and antimicrobial activities ^11,12. Components with distinct structures in EOs compositions synergistically enhance each other’s effects and influence bacterial biochemical processes. The combination of these agents contributes to the complex nature of the compound, amplifying its antibacterial properties ^11,13. Recently, many studies have reported a synergistic impact to enhance the antioxidant and antibacterial activities of EOs ^12,14,15. This study investigates the antimicrobial and antioxidant properties of EOs extracted from three medicinal plants, including sage (Salvia officinalis), oregano (Origanum vulgare), and cumin (Cuminum cyminum).

Salvia officinalis, belongs to the Lamiaceae family and is recognized as one of the most significant sub-shrub perennial medicinal plants¹⁶. Numerous studies have highlighted the properties of S. officinalis, including its anticancer, antimicrobial, antioxidant, antispasmodic, antiseptic, and skin-related benefits. Additionally, it is a valuable natural source for cosmetics and food products^16,17. The EOs extracted from the leaves of S. officinalis contain notable monoterpenoids such as thujone, camphor, 1,8-cineole, and limonene, as well as diterpenes like carnosic acid, which are renowned for their ability to scavenge free radicals and promote human health¹⁸.

Origanum vulgare is another valuable medicinal plant from the Lamiaceae family, rich in phenolic compounds, sterols, and triterpenoids. Its leaves and flowers are used as spices and herbal remedies¹⁹. The EOs of O. vulgare, including carvacrol, thymol, linalool, and p-cymene, exhibit significant antioxidant activity and notable pharmacological properties, such as antibacterial, antimalarial, and antitumor effects^20–22. Furthermore, C. cyminum is an herbaceous bushy annual plant belonging to the Apiaceae family²³. The extracts and EOs of C. cyminum contain key compounds like sabinene, flavonoids, coumarin, and cuminaldehyde, which possess antibacterial and antioxidant properties, making them valuable in the food, cosmetic, and hygiene industries. C. cyminum is widely used as a flavoring agent in culinary applications^24,25.

As mentioned, the constituents of the EOs of S. officinalis, O. vulgare, and C. cyminum are different in chemical composition and bioactivity. It is hypothesized that the synergistic effects of these plants’ EOs enhance pharmacological activities, particularly antioxidant and antibacterial properties. Therefore, the present study evaluated the synergistic effects of EOs from S. officinalis, O. vulgare, and C. cyminum on DPPH scavenging activity, the ferric reducing antioxidant power (FRAP) assay, and antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Salmonella enterica.

Results and discussion

Statistical validation of the model

This experiment used a simplex lattice design to combine the EOs of plants, C. cyminum (X₁), O. vulgare (X₂), and S. officinalis (X₃) (Table 1). The mixture of EOs, the response, and the prediction of each experiment are presented in Table 2. The analysis of variance for various variables is shown in Table 3. According to the ANOVA, the effect of the main regression was statistically significant in all five cases. Statistical significance was confirmed based on p values (p ≤ 0.05). The lack of fit for the variables DPPH scavenging activity and FRAP assay, along with the bacterial strains S. enterica, E. coli, and S. aureus, were found to be 0.076, 0.33, 0.86, 0.26, and 0.10, respectively, indicating the adequacy of the model. The R-squared (R²) values further suggest the significance of the models obtained; R² demonstrates satisfactory agreement between the predicted and actual values. Specifically, the R² values for DPPH scavenging activity (0.92), FRAP assay (0.83), S. enterica (0.87), E. coli (0.87), and S. aureus (0.88) demonstrate a strong agreement between the observed and predicted data (Table 2). The R² and adjusted R² values differed by less than 0.2, suggesting that the model demonstrates a reasonable level of consistency and is not overfitting the data¹². Therefore, the quadratic models fitted to the experimental data, as presented in Eqs. (1)–(5), effectively explain the observed variations in the results.

1

2

3

4

5

Table 1.

Identification of independent variables for the augmented simplex-lattice design.

Components	Composition (µg/ml)
	Coded variables	Level−	Level +
Cuminum cyminum	X1	0	1
Origanum vulgare	X2	0	1
Salvia officinalis	X3	0	1
Sum of proportions		0	1

Table 2.

Matrix design, results, and predicted value of experiment for antioxidant, and antibacterial activity (inhibition zones).

Run	Cuminum cyminum (X1)	Origanum vulgare (X2)	Salvia officinalis (X3)	DPPH(%)		FRAP(μmol Fe(II)/g DW)		Salmonella enterica (mm)		Escherichia coli(mm)		Staphylococcus aureus (mm)
				Measured	Predicted	Measured	Predicted	Measured	Predicted	Measured	Predicted	Measured	Predicted
1	1	0	0	72	73.60	10.21	10.38	13.54	14.20	15.8	17.85	18.8	19.38
2	0.333	0.333	0.333	69	75.04	10.52	10.28	18.23	16.28	24.2	24.13	20.43	19.07
3	0.166	0.666	0.166	92	85.84	12.61	11.48	20.42	19.24	19.45	23.16	20.98	18.86
4	1	0	0	75	73.60	10.75	10.38	14.87	14.20	19.11	17.85	20.43	19.38
5	0.5	0.5	0	90.32	90.14	9.21	10.15	17.12	14.37	23.72	22.26	22.72	23.02
6	0	1	0	91	90.65	11.65	11.22	17.97	17.31	22.09	21.94	18.25	18.23
7	0	0.5	0.5	76.43	78.45	12.19	12.99	23.12	24.17	21.72	20.22	12.65	14.18
8	0	0	1	65	67.74	9.32	8.96	12.3	9.86	11.1	12.31	15.76	14.93
9	0	1	0	89	90.65	10.62	11.22	16.61	17.31	22.81	21.94	17.87	18.23
10	0.666	0.166	0.166	76.32	73.43	9.43	9.41	14.04	12.96	25.04	23.34	20.54	20.80
11	0.5	0	0.5	57.32	58.25	7.2	7.63	7.43	8.44	24.55	24.85	18.1	18.83
12	0	0	1	70	67.74	8.66	8.96	8.123	9.86	13.34	12.31	14.45	14.93
13	0.5	0.5	0	88.22	90.14	10.43	10.15	9.87	14.37	21.7	22.26	21.91	23.02
14	0.166	0.166	0.666	71.32	67.58	10.33	9.88	13.76	14.78	20.69	20.82	16.42	16.37

Table 3.

Variance analysis (ANOVA) for the quadratic models.

SOV	DPPH scavenging activity					FRAP assay
	SS	df	MS	F-value	p value	SS	df	MS	F-value	p value
Quadratic model	1447.44	5	289.49	18.46	0.0003***	21.97	5	4.39	8.26	0.0051***
Residual	125.48	8	15.68			4.26	8	0.532
Lack of fit	104.27	4	26.06	4.9	0.076	2.62	4	0.655	1.60	0.3301
Pure error	21.205	4	5.30			1.64	4
Cor total	1572.9	13				26.23	13

SOV	S. enterica					E. coli
	SS	df	MS	F-value	p value	SS	df	MS	F-value	p value
Quadratic model	215.88	5	43.18	7.18	0.0079***	205.87	5	41.17	10.77	0.0021***
Residual	48.1	8	6.01			30.58	8	3.82
Lack of fit	11.29	4	2.82	0.3067	0.8606ns	20.29	4	5.07	1.97	0.2634ns
Pure error	36.81	4	9.20			10.29	4	2.57
Cor total	263.98	13				236.45	13

SOV	S. aureus						R2	A. R2	P. R2	A. P
	SS	df	MS	F-value	p value	DPPH	0.92	0.87	0.80	12.49
Quadratic model	19.45	5	19.45	11.89	0.0015**	FRAP	0.83	0.73	0.27	11.22
Residual	13.09	8	1.64			S. enterica	0.81	0.70	0.38	9.78
Lack of fit	10.51	4	2.63	4.06	0.1017ns	E. coli	0.87	0.78	0.59	9.79
Pure error	2.59	4	0.46			S. aureus	0.88	0.80	0.46	10.56
Cor total	110.36	13

NS, and *** are non-significant, significant at p ≤ 0.001 respectively; SOV source of variation; SS sum of squares; MS mean square; S. aureus Staphylococcus aureus; E. coli Escherichia coli; S. enterica Salmonella enterica; Df degree of freedom; R² Coefficient of determination; A R² adjusted R²; P R² predicted R² and A. P adeq precision.

Chemical composition of essential oils

The extracted EOs products differ in terms of stereochemical types and the number of constituent molecules²⁶. Various factors, such as genetic, climatic variations, harvest time, environmental conditions, and the specific part of the plant utilized, influence the chemical constituents of EOs^27,28. Furthermore, specific temperature ranges and altitudes may lead to the production of distinct secondary metabolites²⁹. EOs must be extracted under uniform conditions to obtain a consistent chemical composition, after which they are chemo-typed based on analytical results^5,30.

The EOs from O. vulgare, C. cyminum, and S. officinalis, were analyzed through GC/MS and are presented in Table 4. Due to the morphological and chemical diversity within the Origanum genus, a wide variation in the EOs composition has been reported^31,32.

Table 4.

Percentage composition of EOs obtained from Origanum vulgare, Cuminum cyminum, and Salvia officinalis.

RI	Compounds	Formula	O. vulgare	C. cyminum	S. officinalis
938	α-pinene	C10H16	0.61	0.72	4.77
959	Camphene	C10H16	–	–	4.13
974	Sabinene	C10H16	0.83	0.73	0.17
980	β-pinene	C10H16	–	11.34	1.76
978	Octen-3-ol	C8H16O	2.68	–	–
990	Myrcene	C10H16	0.23	0.6	0.93
1006	α-phellandrene	C10H16	0.16	0.44	–
1016	α-terpinene	C10H16	2.44	0.18	–
1024	p-cymene	C10H14	9.83	7.23	0.32
1029	Limonene	C10H16	0.31	0.62	1.29
1031	1,8-cineole	C10H18O	0.67	0.26	12.61
1060	γ-terpinene	C10H16	23.22	18.78	0.26
1080	Terpinolene	C10H16	0.34	–	–
1098	trans sabinene hydrate	C10H18O	–	0.1	–
1140	trans-pinocarveol	C10H16O	–	0.07	–
1144	Pinocarvone	C10H14O	–	–	18.44
1161	Terpinene-4-ol	C10H18O	1.02	0.17	–
1112	α-thujene	C10H16	1.42	0.29	34.85
1120	β-thujene	C10H16	–	–	9.96
1172	α-terpineol	C10H18O	1.34	1.19	0.12
1191	Hexyl butanoate	C10H20O2	–	0.08	–
1236	Methyl ether carvacrol	C11H16O	6.43	–	–
1239	Cuminaldehyde	C10H12O	–	24.24	–
1271	Geranial	C10H16O	–	0.1	–
1281	Bornyl acetate	C12H20O2	–	12.19	0.22
1288	Lavandulyl acetate	C12H20O2	–	19.56	–
1161	Borneol	C10H18O	–	–	1.08
1166	Terpinene-4-ol	C10H18O	–	–	0.40
12.92	Thymol	C10H14O	3.33	–	–
1303	Carvacrol	C10H14O	39.54	–	–
1410	E-caryophyllene	C15H24	1.22	–	2.03
1418	Aromadendrene	C15H12O6	–	–	0.25
1420	trans-caryophyllene	C15H24	–	0.09	–
1445	cis-β-farnesene	C15H24	–	0.15	–
1455	α-humulene	C15H23	–	–	1.97
1465	β-acoradiene	C15H24	–	0.09	–
1497	Germacrene D	C15H24	0.51	0.17	–
1505	Germacrene A	C15H24	2.01	–	–
1576	Caryophyllene oxide	C15H24O	–	–	0.91
1594	Carotol	C15H26O	–	0.12	–
–	Monoterpene hydrocarbons	–	39.39	40.93	58.44
–	Oxygenated monoterpene	–	45.9	26.21	32.55
–	Sesquiterpene hydrocarbons	–	3.74	0.62	4
–	Oxygenated sesquiterpenes	–	0	0	1.16
–	Other	–	9.11	31.75	0.22
–	Total	–	98.14	99.51	96.3

The EOs of O. vulgare were identified to contain 20 compounds, collectively representing 98.14% of the total oil. In O. vulgare EOs, monoterpene hydrocarbons (39.399%), oxygenated monoterpenes (45.9%), and sesquiterpene hydrocarbons (3.74%) were identified. The main constituents include carvacrol (39.54%), γ-terpinene (23.22%), p-cymene (9.83%), methyl ether carvacrol (6.43%), and thymol (3.33%). The health benefits, pharmacological effects, and phytochemical content of O. vulgare EOs have garnered significant attention. Azimzadeh et al.³³ identified 27 compounds in the EOs of O. vulgare, with carvacrol (40.93%) and γ-terpinene (17.73%) as the main constituents.

Cuminaldehyde was identified as the predominant component (24.24%) in the EOs of C. cyminum, followed by lavandulyl acetate (19.56%), γ-terpinene (18.78%), bornyl acetate (12.19%), β-pinene (11.34%), and p-cymene (7.23%) (Table 4). Sharifi et al.³⁴ identified 27 compounds in the EOs extracted from C. cyminum, with cuminaldehyde as the predominant component, accounting for 38.26%. Furthermore, in the EOs of S. officinalis, 20 compounds were identified, representing 96.37% of the overall oil composition (Table 4).

In EOs of S. officinalis, 58.44% of the compounds were composed of monoterpene hydrocarbons, 32.55% oxygenated monoterpenes, 4% sesquiterpene hydrocarbons, and 1.16% oxygenated sesquiterpenes. The main component was α-thujene, which constituted 34.85% of the total EOs, followed by β-thujene (9.96%), α-pinene (4.77%), camphene (4.13%), 1,8-cineole (12.61%), and pinocarvone (18.44%). Jažo et al.¹⁸ reported that 25 compounds were identified, and α- and β-thujone, camphor, and 1,8-cineol were the primary constituents of the EOs of S. officinalis.

Antioxidant activity

The influence of three independent variables on the dependent variable is illustrated using 2-D response surface plots (Fig. 1a–e). In these triangle graphs, red indicates a high degree of interaction between the parameters, while blue represents the lowest level of interaction.

Fig. 1.

2D Mixture plot showing the antioxidant and antibacterial activity responses of the mixture of the three plants’ EOs extracts. X₁: C. cyminum, X₂: O. vulgare, X₃: S. officinalis. (a) DPPH scavenging activity, (b) FRAP assay, (c) S. aureus, (d) E. coli, and (e) S. enterica.

The antioxidant activity of EOs was evaluated using DPPH scavenging activity and FRAP assays. The results demonstrated significant DPPH scavenging activity differences among the various EOs, ranging from 57.32 to 92% (Fig. 1a). The arrangement of DPPH scavenging activity among the EOs extracted from the three plants was as follows: O. vulgare > C. cyminum > S. officinalis. The desirability test identified the optimal combination of 29.5455% C. cyminum EOs and 70.4554% O. vulgare EOs, resulting in the highest DPPH scavenging activity value of 92.28% (Fig. 2a). This value was higher than that of each EOs. Likewise, when 44.1178% C. cyminum and 55.8822% S. officinalis EOs were combined, they showed antagonistic effects, resulting in a DPPH scavenging activity value of 58.08%, the lowest recorded (Fig. 1a).

Fig. 2.

The optimal proportions of C. cyminum, O. vulgare, and S. officinalis EOs in the maximum extraction of (a) DPPH scavenging activity, (b) FRAP assay, (c) S. aureus, (d) E. coli, and (e) S. enterica.

Figure 1b displays the FRAP responses for the EOs of C. cyminum, O. vulgare, and S. officinalis. This study found FRAP values ranging from 7.2 to 12.61 μmol Fe(II)/g DW to assess the influence of the EOs. Experimental runs 3 and 7 exhibited synergistic interactions, resulting in higher FRAP assay values than the individual EOs of C. cyminum, O. vulgare, and S. officinalis. The combination of EOs of 0.66% O. vulgare, 0.16% C. cyminum, and 0.16% S. officinalis demonstrated synergistic effects, yielding the highest FRAP assay value of 12.61 μmol Fe(II)/g DW. Also, the desirability test revealed synergistic interactions between 0.7% O. vulgare and 0.3% S. officinalis EOs, resulting in a maximum FRAP (12.94 μmol Fe(II)/g DW) (Fig. 2b). In contrast, 41.3138% C. cyminum and 58.6852% S. officinalis exhibited antagonistic interactions, yielding a minimum FRAP assay (7.57 94 μmol Fe(II)/g DW).

The antioxidant activity appears to depend on the concentration of O. vulgare in many mixtures. The various components of EOs play a role in determining antioxidant properties, color, and aroma^11,35. Specifically, the interactions between EOs derived from various medicinal plants can significantly enhance their biological activities compared to when the EOs are used individually^14,36. The appropriate composition and concentration of EOs can enhance their antioxidant activity through synergistic effects ¹¹. Yeddes et al.¹⁵ demonstrated that the antioxidant activity of combined EOs from cloves (Syzygium aromaticum), thyme (Thymus vulgaris), and lemon (Citrus limon), as assessed by FRAP assay and ABTS scavenging activity, was greater than that of each EOs when tested individually. Various chemical classes within the EOs significantly influence their antioxidant properties³⁷. Notably, sesquiterpenes and monoterpenes have shown considerable efficacy in neutralizing and mitigating the effects of free radicals³⁸. Walasek-Janusz et al.³⁹ reported that the EOs of O. vulgaris are rich in carvacrol and thymol, exhibiting strong antioxidant and antimicrobial activity.

According to the literature, thymol and carvacrol, as phenolic monoterpenoids, are reduce DPPH radicals by converting them into their reduced (non-radical) form⁴⁰. The synergistic role of thymol and carvacrol is further supported by their contribution to increasing the antioxidant activity of EOs compared to the individual⁴¹. In this study, runs 3 and 7 demonstrated a greater antioxidant response in DPPH scavenging activity and FRAP assays compared to C. cyminum and S. officinalis when tested individually. The interactions between carvacrol and other monoterpenes, such as cuminaldehyde, α-thujene, γ-terpinene, 1,8-cineole, thymol, and p-cymene, result in synergistic effects, which likely explain the observed increase in antioxidant activity in this study. Also, C. cyminum, as previously mentioned, possesses significant antioxidant activity, likely due to its major constituent, cuminaldehyde⁴². As an aromatic and unique flavor monoterpenoid, the bioactive compound, cuminaldehyde, has demonstrated high antioxidant activity and can also decrease reactive oxygen species⁴³. The antioxidant potential observed in the EOs of S. officinalis can be ascribed to the activity of α-thujene⁴⁴. Similar to our data, Baja et al.⁴⁵ have reported the highest antioxidant potency 90% (DPPH) obtained for the composition mixture EOs of Origanum majorana (75%), Ocimum basilicum (8%), and Rosmarinus officinalis (17%), compared to the activity of the individual component O. majorana (88%). Assaggaf et al.⁴⁶ reported that a mixture of EOs of 20% Cymbopogon flexuosus, 53% Carum carvi, and 27% Acorus calamus exhibited the best DPPH IC50 value of 185.34 μg/mL.

Antibacterial activity

The results demonstrate the significant effects of the EOs from C. cyminum, O. vulgare, and S. officinalis against S. enterica, E. coli, and S. aureus (p < 0.01) (Figs. 1 and 3). Findings from the antimicrobial studies demonstrated synergistic effects in combinations of EOs against S. enterica, with inhibition zones measuring between 7.43 and 23.12 mm (Table 2).

Fig. 3.

Antimicrobial activity of various experimental runs of EOs against (a) S. aureus, (b) E. coli, and (c) S. enterica.

Figure 2c illustrates that the optimal mixture of 0.59% O. vulgare and 0.40% S. officinalis was required to achieve maximum inhibition of S. enterica (24.49 mm), showing greater inhibition than the individual EOs. Conversely, the combination of 0.34% C. cyminum and 0.65% S. officinalis exhibited antagonistic interactions, resulting in the minimum inhibition of S. enterica (8.11 mm) (Fig. 1c). Additionally, the combination of EOs from Cuminum cyminum at 0.16%, Origanum vulgare at 0.66%, and Salvia officinalis at 0.16% exhibited synergistic interactions, leading to enhanced inhibition zones against S. enterica.

The combined EOs of C. cyminum, O. vulgare, and S. officinalis were more successful in inhibiting E. coli growth than each EO used separately (Fig. 1d). Synergistic effects were observed against E. coli when EOs were combined at 0.43% C. cyminum, and 0.56% S. officinalis, showing the highest inhibition zone of 25.04 mm (Fig. 2d). In contrast, EOs from 0.5% C. cyminum exhibited antagonistic interactions when combined with 0.5% O. vulgare, resulting in average inhibition zones of 10.73 mm.

The interactions between 0.56% C. cyminum and 0.43% O. vulgare EOs demonstrated synergistic effects against S. aureus, resulting in the maximum inhibition zone of 23.03 mm (Fig. 2e). In contrast, the combination of 0.32% O. vulgare and 0.67% S. officinalis exhibited antagonistic effects, producing a minimum inhibition zone of 13.89 mm. S. aureus treated with a mixture of EOs in runs 2, 3, 5, and 10 exhibited significantly higher inhibition than the individual EOs, particularly with the combination of C. cyminum and O. vulgare.

EOs are commonly utilized in food preservation due to their notable antibacterial and antioxidant activities⁴⁷. Moreover, combining various EOs can enhance their synergistic effects, leading to more effective control of pathogenic bacteria growth⁴⁸. The combination of EOs can enhance food preservation, even at lower doses¹³. This study showed that the synergistic effect of EOs contributed to a significant reduction in bacterial growth. These findings align with Benkhaira et al.⁴⁹, who demonstrated that the combined EOs of Clinopodium nepeta, Ruta montana, and Dittrichia viscosa significantly enhance their inhibitory effects against S. aureus and Pseudomonas aeruginosa. Combining different EOs enhances their ability to disrupt and destroy bacterial cell membranes, inhibiting cell respiration, and resulting in the leakage of cellular components⁴⁸. The chemical composition of EOs directly influences their antibacterial activity. Thus, the interaction between minor and major components of EOs enhances their antimicrobial activity. Studies have indicated that while the main constituents play a significant role, the complete EOs exhibit greater antimicrobial activity than the major components alone³. Gallucci et al.⁵⁰ reported that the synergistic interaction between eugenol, the major component of Ocimum gratissimum EOs, and linalool, a component of Cymbopogon flexuosus, may contribute to the enhanced antibacterial activity. In this study, the increased antibacterial effect of O. vulgare against S. enterica and E. coli may be due to carvacrol, a compound known for its strong antibacterial properties. Studies have shown that carvacrol, a natural monoterpene phenol, is effective against various types of bacteria⁵¹. The synergistic effect of carvacrol in combination with other compounds may enhance its bacterial inhibitory activity⁵². Monoterpene hydrocarbons, such as limonene, terpinene, and myrcene, exhibited marked enhancement in their antimicrobial properties when used with carvacrol instead of their application alone⁴⁸. Monoterpene compounds induce swelling in the bacterial cell membrane, thereby increasing the membrane’s permeability to carvacrol, resulting in a synergistic antimicrobial effect^51,52. Similarly, cuminaldehyde and α-thujene, the major components of the EOs from C. cyminum and S. officinalis, respectively, may play a significant role in their antibacterial activities and have been previously reported to exhibit strong antibacterial properties. Reyes-Jurado et al.⁵³ reported that p-cymene and carvacrol in Mexican oregano (Lippia berlandieri Schauer) EOs, p-cymene, linalool, and thymol in thyme (Thymus vulgaris) EOs, as well as allyl isothiocyanate in mustard (Brassica nigra) EOs, are likely major contributors to the antibacterial activity against Listeria monocytogenes, S. aureus, and S. enterica. It has been reported that sesquiterpene compounds, such as β-caryophyllene and Germacrene A, D, found in EOs, possess antibacterial properties effective against gram-negative and gram-positive bacterial strains^54,55. Santos et al.⁵⁶ demonstrated that β-caryophyllene is the major component in Aloysia gratissima (Gillies & Hook) EOs had antibacterial action against S. aureus, E. coli, and Pseudomonas aeruginosa. Antibacterial activity, with antioxidant mechanisms, can disrupt bacterial cell membranes, resulting in the loss of cellular components and an imbalance of electrolytes⁵⁷. Alizadeh Behbahani et al.⁵⁸ highlighted the synergistic antimicrobial and antioxidant effects of Satureja intermedia and Ducrosia anethifolia EOs in inhibiting Candida albicans, Klebsiella aerogenes, and Listeria monocytogenes. Furthermore, Sharma et al.⁵⁹ reported that a synergistic combination of EOs derived from six medicinal plants (Callistemon lanceolatus, Ocimum gratissimum, Cymbopogon winterianus Jowitt, Cymbopogon flexuosus, Mentha longifolia, and Vitex negundo) enhanced antibacterial activity (against S. aureus, Micrococcus luteus, Bacillus subtilis, E. coli, and Klebsiella pneumoniae).

Desirability of the assumed model

This research aimed to determine the optimal formulation of a combination of three EOs to achieve maximum independent variable, compared to the effects of each EOs used individually. This illustration demonstrates that the optimal mixture consists of 15.0553% C. cyminum, 74.0453% O. vulgare, and 10.90% S. officinalis, leading to maximum DPPH scavenging activity (88.1%), FRAP assay result (11.36 μmol Fe(II)/g DW), and inhibition zones against S. enterica (18.59 mm), E. coli (22.97 mm), and S. aureus (19.26 mm), with overall desirability of 0.77 (Fig. 4). This desirability outcome demonstrates the strong alignment between the prediction model and the experimental results. Therefore, using a simplex lattice design, the optimal mixture ratio of the three EOs was determined to achieve the most effective antioxidant and antibacterial activity formulation. In this study, three bacterial strains and two antioxidant methods were utilized. Identifying a blend of EOs that can maximize antioxidant and antibacterial effects could be significant for the food and cosmetic industries. Such compounds are often incorporated into food products or pharmaceutical formulations for their antimicrobial and antioxidant properties. In other words, the multifunctional nature of formulated EOs could hold a unique and promising position in these industries.

Fig. 4.

The desirability plot illustrates the optimal proportions of C. cyminum, O. vulgare, and S. officinalis EOs in the optimizing extraction of DPPH scavenging activity, FRAP assay results, and antimicrobial activity against S. enterica, E. coli, and S. aureus.

Mantel test analysis, correlations, and multivariate analysis of combined data

This research aims to enhance the antioxidant and antibacterial properties of EOs formulations by introducing innovative and efficient techniques for their utilization at lower dosages. Recent studies have demonstrated notable improvements in the combined application of EOs, leading to reduced dosages and more reliable outcomes. Correlation analysis revealed the relationship between EOs components and the factors under investigation (Fig. 5a). The results indicated that O. vulgare EOs significantly affected all factors, with the greatest impact (highlighted in red) on antioxidant activity as measured by the DPPH scavenging activity method. Additionally, S. officinalis significantly influenced the analyzed factors (p < 5%). A positive and significant correlation was observed between the FRAP assay and DPPH scavenging activity (p < 0.05), and between the FRAP assay and S. enterica (p < 0.001).

Fig. 5.

(a) A Mantel analysis test and Spearman’s correlation, a correlation analysis was conducted between independent (C. cyminum, O. vulgare, and S. officinalis) and dependent variables (DPPH scavenging activity, FRAP assay, S. enterica, E. coli, and S. aureus), (b) Correlation analysis, (c) Principal component analysis (PCA) based on all studied traits, (d) Two-way hierarchical cluster analysis (HCA), (e) Circular clustering of all combined attributes.

The plausible effects of the EOs mixture can be systematically investigated through the robust analytical correlation analysis method. There was a positive correlation between DPPH scavenging activity and FRAP assay and S. enterica (Fig. 5b). DPPH scavenging activity also positively correlated with E. coli (0.66) and S. aureus (0.45). In contrast, FRAP activity did not significantly correlate with E. coli (0.09) but was negatively associated with S. aureus (−0.06). One of the most widely used techniques in statistical analyses is the application of principal component analysis (PCA). PCA revealed significant variations in the evaluated traits, with the first and second principal components accounting for Dim1 (52.2%) and Dim2 (27.7%) of the total variance, respectively (Fig. 5c). These results suggest that the synergistic interactions among EOs are positively correlated with enhanced antioxidant and antibacterial activities. DPPH scavenging activity, E. coli, and S. aureus were in the positive section. Additionally, FRAP and S. enterica were positioned in the upper right section. Experimental runs 8 and 12, along with the minimum desirability, were situated opposite to the location of maximum desirability. According to this analysis, experimental run 9 was closer to the maximum desirability, indicating that this combination represents a more suitable mixture for achieving synergistic effects of EOs than other mixtures. Other correlations related to EOs mixtures are illustrated in Fig. 5c.

Bidirectional hierarchical clustering was employed to cluster and classify the EOs mixtures and desirability data (Fig. 5d). Based on this analysis, the combinations of 0.16% C. cyminum, 0.66% O. vulgare, and 0.16% S. Officinalis were closer to the optimal desirability values. Moreover, the minimum desirability and experimental runs 8, 11, and 12 were grouped in the same cluster. The most effective and optimized combinations closer to the highest desirability can be utilized for EOs formulation. Circular clustering analysis was employed to investigate the synergistic effects of EOs further. Based on this analysis, the evaluated traits were classified into three main groups. Compounds exhibiting more potent synergistic antibacterial and antioxidant properties and those closer to optimal desirability values were clustered within the same category (Fig. 5e). Based on these findings, EOs combinations can significantly reduce bacterial growth. However, the effectiveness varies among different formulations. Variations in the chemical composition of EOs may contribute to better optimization in antibacterial applications. The relationships between EOs mixtures, their chemical profiles, and the evaluated traits further strengthen the predictive models and enhance their applicability in food industry programs.

Conclusion

Combining EOs resulted in synergistic interactions, enhancing antimicrobial and antioxidant activities. Combining essential oils can reduce high costs, minimize side effects, and lower the toxicity of higher doses. Therefore, employing mixture design experiments serves as a valuable approach for developing effective formulations to improve the shelf life of food and pharmaceutical products. This study found that a combination of 15.0553% C. cyminum, 74.0453% O. vulgare, and 10.90% S. officinalis demonstrated the most effective DPPH scavenging activity (88.1%), FRAP assay result (11.36 μmol Fe(II)/g DW), and inhibition zones against S. enterica (18.59 mm), E. coli (22.97 mm), and S. aureus (19.26 mm). Correlation and multivariate analysis of trait and EOs mixtures can help make their use more targeted and efficient. Further studies on the individual performance of essential oil components, the mechanisms underlying synergistic or antagonistic interactions, and the diverse effects resulting from component interactions are necessary to formulate novel and effective combinations. These findings could support and contribute to efforts to improve the optimal use of natural compounds within the food and pharmaceutical industries.

Materials and methods

Chemicals and reagents

All chemical reagents, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride hexahydrate (FeCl₃·6H₂O), and 2,4,6-tripyridyl-s-triazine (TPTZ), were purchased from Sigma-EOs. Constituents were identified by comparing their mass (Canada). Dimethyl sulfoxide (DMSO), agar, methanol, and ethanol were obtained from Merck (Rahway, New Jersey, USA). Gas chromatography–mass spectrometry (GC–MS) analysis was conducted using an Agilent system (Agilent Technologies, Wilmington, USA). A spectrophotometer (Dynamic HALO DB-20, UK) was also used in this study.

Plant material

In this study, the EOs of three medicinal plants were used. Sage (S. officinalis L.) and Oregano (O. vulgare L.) aerial parts were harvested from the Horticultural Sciences Department at Urmia University, West Azerbaijan Province, Iran. Furthermore, Cumin seeds (C. cyminum L.) were purchased from Pakan Bazr Company, Isfahan. The plant samples were stored in a dark, dry place with proper ventilation. The schematic design outlining the study’s stages on the synergistic antioxidant and antibacterial activity of a three-component EOs mixture comprising C. cyminum, O. vulgare, and S. officinalis is illustrated in Fig. 6.

Fig. 6.

Schematic representation illustrates the synergistic antioxidant and antibacterial activity of a three-component EOs mixture: C. cyminum, O. vulgare, and S. officinalis. The main compounds of the EOs mixture were analyzed (GC–MS), and the structure of the mixture points was determined using the simplex lattice design method.

Essential oil extraction and analysis

For extraction, 50 g of C. cyminum seeds and 25 g of shade-dried O. vulgare and S. officinalis were placed in 550 mL of distilled water and subjected to steam distillation in a Clevenger apparatus for 2.5 h. The obtained EOs were collected using an insulin syringe and stored in dark vials at −4 °C for analysis by GC–MS. The GC–MS system utilized a Varian 3400 ion trap mass spectrometer with a DB-5 column (30 m long, 0.25 mm internal diameter, and 0.25 µm stationary phase thickness). The thermal programming of the column ranged from an initial temperature of 50 °C to a final temperature of 250 °C, increasing at a rate of 4 °C per minute. The injection port temperature was 10 °C higher than the final column temperature. Helium (with a purity of 99.99%) served as the carrier gas, flowing at a rate of 31.5 cm per second through the column. The scan time was set to one second, with an ionization energy of 70 electron volts, and the mass range analyzed was from 40 to 340 amu⁶⁰. EOs constituents were identified by comparing their mass spectra with those of authenticated standards and established spectral libraries. Retention indices (RIs) were calculated based on previously published data and further validated using the retention times of a homologous series of n-alkanes (C6–C24) under identical chromatographic conditions^61,62.

Measurement of antioxidant activity

Two methods were used to determine the EOs’ antioxidant activity: DPPH scavenging activity and the Ferric reducing antioxidant power (FRAP) assay.

DPPH free radical scavenging activity

For the DPPH scavenging activity measurement, 30 µL of EOs was added to 2000 µL of DPPH solution. After incubating (30 min) the mixture in the dark room, the absorbance was read using a spectrophotometer at a wavelength of 517 nm, and antioxidant activity was calculated using the following formula⁶³.

6

Here, A _control represents the absorbance of the DPPH scavenging activity solution without EOs (blank), and A _sample represents the absorbance of the DPPH scavenging activity solution with EOs.

Ferric reducing antioxidant power (FRAP) assay

In the FRAP assay, a solution was prepared by combining 250 mL of 0.3 M acetate buffer (300 mM, pH 3.6), 2.5 mL of 20 mM FeCl₃·6H₂O, and 2.5 mL of 10 mM TPTZ stock solution in a ratio of 10:1:1. Subsequently, 50 µL of the sample and 3000 µL of distilled water were added to this mixture. The resulting solution was incubated in a water bath at 37 °C for 30 min, after which the absorbance was measured at 593 nm using a spectrophotometer. A standard calibration curve was established using various concentrations of Dimethyl Sulfoxide (DMSO)⁶⁴.

Determination of antibacterial activity

The antibacterial efficacy of EOs against S. aureus (Gram-positive), S. enterica (Gram-negative), and E. coli (Gram-negative) was assessed using the Kirby-Bauer disc diffusion method. In brief, bacterial cultures were inoculated onto nutrient agar plates at an optical density of 0.08–0.13 (OD600). Sterile 5 mm paper discs, impregnated with EOs at 30 µg/mL, were placed on the agar and incubated for 24 h at 37 °C. Tetracycline (20 µg/mL) was used as a positive control, whereas distilled water acted as a negative control. The inhibition zone was measured using a digital caliper (Carbon model, China)⁶⁵.

Factors and experimental matrix for mixture design

The simplex lattice design, represented as an equilateral triangle, included three components: C. cyminum (X₁) O. vulgare (X₂), and S. officinalis (X₃). A total of 6 points and 14 experiments were conducted with three replications. Runs 1, 4, 6, 8, 9, and 12 represent three pure EOs, while 5–7, 11, and 13 correspond to binary combinations. Additionally, the central points run 2, 3, 10, and 14 are associated with ternary combinations (Fig. 7). The EOs’ components ranged from 0 to 1 in the mixture, with their total equal to 1. According to Table 2, the EOs mixtures (30 μg/mL) were prepared, and their antioxidant and antibacterial activity were evaluated. A quadratic model for the independent variables and response was formulated as follows:

7

where Y represents the dependent variable and Xi denotes the independent variable; Σ₁, Σ₂, Σ₃ correspond to the coefficients of the linear terms; Σ₁₂, Σ₁₃, and Σ₂₃ represent the coefficients of the binary terms; and ε is the error term.

Fig. 7.

The structure of mixture points was determined using the simplex lattice design method. The factors X₁, X₂, and X₃ correspond to the components C. cyminum, O. vulgare, and S. officinalis, respectively.

Statistical analysis

Experimental design (14 runs with 3 replications) and statistical analyses were performed using Design Expert (Version 13.0.0, Stat-Ease, Inc., MN, USA) software. The statistical significance was assessed using the F-ratio, which is calculated as the ratio of the mean square of the regression to that of the residuals. A higher F-value indicates that the variation in the results around the mean is statistically meaningful. The lack of fit and p value (p < 0.05) were used to assess the mathematical model’s accuracy, while R² and R² adj were utilized to evaluate the quality of the hypothetical models. ANOVA (quadratic polynomial model) was used for model validation. The multivariate analysis was conducted using R Studio version 4.1.3 (TRL http://www.rstudio.com/).

Author contributions

B.G. Investigation, Methodology, Analytical software, Statistical analysis, Writing—Original Draft. S.A-S Investigation, Methodology, Analytical software, Statistical analysis, A.H. Supervision, Review, and Editing of the manuscript. M.F. Supervision, Visualization Conceptualization, Methodology, Data curation, Writing—Review and Editing, Project administration.

Data availability

The datasets analyzed in this study are available through the main text.

Declarations

Competing interests

The authors declare no competing interests.