Elevational gradients shape total phenolic content and bioactive potential of sweet marjoram (Origanum majorana L.): A comparative study across altitudinal zones

Emel Karaca Öner; Meryem Yeşil; Sinem Aydın; Fatih Öner

doi:10.1515/chem-2025-0139

Article Open Access

Elevational gradients shape total phenolic content and bioactive potential of sweet marjoram (Origanum majorana L.): A comparative study across altitudinal zones

Emel Karaca Öner , Meryem Yeşil , Sinem Aydın and Fatih Öner

Published/Copyright: April 29, 2025

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From the journal Open Chemistry Volume 23 Issue 1

Abstract

Sweet marjoram (Origanum majorana L.) is a species of economic and industrial importance from the Lamiaceae family, whose leaves are used as spices and condiments, fresh or dried. Additionally, it comprises bioactive substances that have applications in the nutraceutical and pharmaceutical industries. This study aims to evaluate the effects of different altitudes on some biological activities in O. majorana. The total phenolic content (TPC) was identified by the Folin–Ciocalteu assay, and antioxidant capacities were evaluated by metal chelating activity, cupric reducing antioxidant, and 2,2-diphenyl-1-picrylhydrazyl assays. TPC was the highest in the altitudes 1,300–1,400 m (188.67 mg QE/g) and 1,100–1,200 m (185.03 mg QE/g), respectively. The maximum antioxidant activities were found to be 241.34 µg AAE/mL (1,300–1,400 m) and 210.63 µg AAE/mL (800–900 m), respectively. EOs exhibit strong antimicrobial activity, particularly against Salmonella typhi, Enterococcus facelis, and Staphylococcus aureus. The findings indicate that the biological activity of O. majorana may vary depending on different altitudes.

Keywords: altitude; anti-radicals; bioactivity; sweet marjoram

1 Introduction

Medicinal and aromatic plants are recognized for their capacity to generate aromatic chemicals from a variety of organs, including leaves and flowers, which are used in the cosmetics and culinary sectors. In contrast, the World Health Organization categorizes medicinal plants as those containing active ingredients that can prevent, alleviate, or heal illnesses or act as precursors for new pharmaceuticals. These bioactive compounds, often referred to as secondary metabolites, are products of metabolic pathways that intersect primary and secondary metabolism [1,2]. The diversity of secondary metabolites in plants contributes to their significant biological effects. The Lamiaceae family is extensively researched for its secondary metabolites, encompassing both volatile and non-volatile substances that form intricate mixtures. These mixtures are known for their potent biological activities, rendering Lamiaceae plants valuable in the food, which makes the plants of the Lamiaceae family valuable in food, cosmetic, and pharmaceutical sectors [3,4]. Origanum majorana L., a member of the expansive Lamiaceae family, which includes around 230 genera and nearly 7,000 species [5], is a perennial aromatic herb that is also cultivated annually. It is synonymous with Majorana hortensis Moench., Origanum dubium Boiss., Origanum confertum Savi and is commonly referred to as sweet marjoram. Indigenous to regions like Cyprus, Greece, and Turkey, sweet marjoram has also been cultivated in North African countries such as Morocco, Egypt, Tunisia, and Algeria [6]. O. majorana, a species of the Lamiaceae family, has been extensively researched for its complex chemical composition, which encompasses both its essential oil (EO). This composition has led to O. majorana being recognized for its significant pharmacological potential [7,8]. The EO, in particular, extracted from the plant's aerial parts, has been the subject of numerous studies. This EO, a complex mix of secondary metabolites, has been credited with a range of properties, including but not limited to antioxidant, antimicrobial, anti-inflammatory, anti-acetylcholinesterase, anti-cancer, anti-depressant, and analgesic effects [9,10]. Beyond these, the repellent and insecticidal properties of EOs are also highly regarded [11,12,13].

In the current agricultural landscape, there is an ongoing debate regarding the adverse effects of synthetic pesticides on both human health and the environment [14]. As a result, there is a growing interest in biological alternatives, such as EOs, which could potentially replace synthetic pesticides [15]. A substantial body of research is dedicated to exploring the biological activities of EOs, particularly their effectiveness as pesticides against various insect species [16,17]. These natural products are sought after for their ability to safeguard crops in an environmentally friendly way without compromising human health. Specifically, the EO of O. majorana has been studied for its insecticidal, larvicidal, repellent, and fumigant properties [18,19].

This research, as emphasized in previous studies, was carried out to investigate the phytochemical changes of O. majorana species depending on different altitudes and the effects of this change on biological activity. The objectives are two-fold: (a) to compile and analyze data on O. majorana phytochemical composition as documented by researchers worldwide, and (b) to examine changes in antibacterial and antioxidant activities depending on different altitudes.

2 Materials and methods

2.1 Isolation of EO

Specimens for this study were procured from the southwest of Turkey, specifically between latitudes 36°07′ and 37°20′N and longitudes 29°20′ and 32°35′E. The O. majorana samples were harvested from Akseki natural habitats at seven distinct elevations, each separated by 100 m, ranging from 766 to 1,387 m (Figure 1).

Figure 1

The location map of the collection area [20].

Harvesting involved plucking the upper two-thirds of the plants at both 9:00 am and 11:00 pm for EO; the collected samples underwent leaf-stem separation at each altitude level. Plants were collected if more than 50% of the samples were in the flowering period of collection, depending on the altitude, and the flowering period varied at each altitude. The leaves were then air-dried, pulverized, and 100 g from each sample was distilled for 3 h using a Clevenger apparatus. The resulting EO was measured with a graduated cylinder, transferred into vials, and stored at 4°C for subsequent analysis.

2.2 Microorganisms

The antimicrobial activity was performed using three Gram-positive bacteria: Bacillus subtilis ATCC 6051, Staphylococcus aureus ATCC 25923, and Enterococcus facelis ATCC 29212, and two Gram-negative bacteria: Salmonella typhi ATCC 14028 and Escherichia coli ATCC 25922.

2.3 Disc diffusion method

Dimethyl sulfoxide (DMSO) containing 20 mg/ml EOs was used to dissolve them and then filtered through 0.45 μm millipore filters for sterilization [21]. Gentamicin was employed as a common antimicrobial agent. A negative control was DMSO. Mueller-Hinton agar (MHA) plates were inoculated with bacterial suspensions that had been turbidity adjusted to 0.5 McFarland standard (10⁸ CFU/mL) and left to dry. Discs with a diameter of 5 mm were placed on infected agar. Discs were each filled with 20 mg/mL of Origanum species EO [22]. The infected plates were kept in the fridge for 1 h before being incubated for the entire night at 37°C. Zone diameters were measured.

2.4 Determination of minimum inhibitory concentration (MIC)

The MIC of EOs of six Origanum species obtained from different altitudes was determined [23]. EOs exhibiting inhibitory zones ≥10 mm were examined in the MIC assay [24]. A micro-dilution broth susceptibility assay was employed to determine MIC. EOs from six Origanum species were produced as two-fold serial dilutions (in DMSO) ranging from 0.009765 to 20 mg/mL in a 96-well microplate. To prepare the 96-well plates, 5 µL of the inoculum and 95 µL of Mueller Hinton Broth were dispensed into each well. The first wells received 100 µL of Origanum EO applied to them. Following that, 100 µL of each of their serial dilutions were added to a new well. The final well served as the negative control and held 195 µL of nutritional broth devoid of compound and inoculum on each strip. Six different Origanum species’ EOs underwent this process separately. For 24 h, plates were incubated at 37°C [25].

2.5 Total phenolic content (TPC)

The test tube was filled with 0.1 mL of EO, 4.5 mL of distilled water, 0.1 mL of the Folin reagent, and 0.3 mL of Na₂CO₃ (2%) in that order. The mixture was incubated for 90 min, and the absorbance measurements at 760 nm were obtained [26]. The oils’ TPC was measured in µg gallic acid equivalents (GAE)/mL.

2.6 Total antioxidant activity

Using the phosphomolybdenum technique, the overall antioxidant capacity of EOs was assessed in accordance with the protocol outlined by Prieto et al. [27]. The reference was ascorbic acid. The unit of measurement for the total antioxidant capacity was µg ascorbic acid equivalent (AAE)/mL.

2.7 Metal-chelating activity assay

A study of the chelation of ferrous ions by EOs and EDTA was conducted using the method outlined in the study of Dinis et al. [28]. To a solution of 2 mM FeCl₂ (0.1 mL), 5 mL of oils were added at varying concentrations (250–1,000 µg/ml). The process was started by the addition of 5 mM ferrozine (0.2 mL). After allowing the mixture to sit at room temperature for 10 min, the absorbance was measured at 562 nm. The following formula was used to determine the metal chelating activity: [(A0 − A1)/A0] × 100 is the metal chelating activity (%) (A0: control absorbance; A1: standard or EO’s absorbance).

2.8 CUPRAC assay

A test tube was filled with 0.5 mL of EO, 1.0 mL of CuCl₂ solution, 1.0 mL of neocuproine solution, and 1.0 mL of ammonium acetate buffer. The absorbance at 450 nm was measured 30 min after the tube was placed in a dark area [29].

2.9 Metal chelating and DPPH assay

We combined oils (250, 500, 750, and 1,000 mg/mL) with 0.1 mL of FeCl₂. To the mixture, 0.2 mL of 5 mM ferrozine was added. The mixture was measured using a spectrophotometer at 562 nm after incubating for 10 min [28]. We evaluated the capacity of the oils to scavenge DPPH free radicals using the standard method. A 6 × 10⁻⁵ M methanolic solution of DPPH was combined with 1.5 mL of diluted solutions (0.75 mL each). After incubating at room temperature for 30 min in the dark, the absorbance was measured at 517 nm.

The following formula was used to determine the metal-chelating activity and DPPH radical scavenging activity: metal chelating activity (%) or DPPH radical scavenging activity (%) = [(A0 − A1)/A0] × 100 (A0: control absorbance; A1: standard or EO’s absorbance).

3 Results and discussion

3.1 Antibacterial activity

Table 1 shows the inhibition zones of EOs. The EOs of O. majorana, at altitudes of 900–1,000 and 1,000–1,100 m, did not show antibacterial activity against the bacteria tested. The EOs derived from plants collected from altitudes other than 800–900 m exhibited the highest antibacterial activity against E. coli (20 mm zone). The EOs collected from altitudes of 800–900 m exhibited the highest antibacterial activity against E. coli (20 mm zone), with the exception of the EOs of O. majorana at 1,300–1,400 and 1,200–1,300 m. The next highest value was observed at an altitude of 800–900 m, with a zone diameter of 17 mm against S. typhi, S. aureus, E. coli, and E. faecalis. The lowest activity detected was 7 mm against S. aureus in the EO of O. majorana at an altitude of 1,200–1,300 m. Gentamycin demonstrated a higher level of activity when compared with the EOs of O. majorana and DMSO, which was used as a negative control and had no effect against bacteria. In this study, it was determined that altitude was effective in biological activities as an antibacterial in O. majorana.

Table 1

Inhibition zones of the O. majorana EOs, gentamicin, and DMSO (mm)

Bacteria	1,300–1,400 m	1,200–1,300 m	1,100–1,200 m	1,000–1,100 m	900–1,000 m	800–900 m	Gentamicin	DMSO
S. typhi	17	11	15	NA	NA	11	12	NA
B. subtilis	NA	NA	13	NA	NA	10	25	NA
S. aureus	16	7	17	NA	NA	9	21	NA
E. coli	14	15	17	NA	NA	20	20	NA
E. faecalis	16	12	15	NA	NA	17	19	NA

NA: no activity.

In a study conducted by Ezzeddine et al. [30], the most susceptible bacterial strains of O. majorana EO were E. coli, S. aureus, S. dysenteria, and S. enteritidis, while the least susceptible bacterial strain was Pseudomonas aeruginosa. The EO of O. majorana showed strong antimicrobial activities owing to the oxygenated monoterpenes in its structure [31]. All results are expressed as mean ± standard deviation (SD) of each triplicate test.

Table 2 shows MIC values of EOs. The highest and the lowest values were found to be 10 mg/mL against E. coli at 800–900 m and 0.01953 mg/mL against S. typhi in EOs of O. majorana at 1,300–1,400 m, respectively. MIC values could not be determined at some altitudes.

Table 2

MIC values of the EOs of O. majorana (mg/mL)

Bacteria	Mic (mg/mL)	800–900 m	900–1,000 m	1,000–1,100 m	1,100–1,200 m	1,200–1,300 m	1,300–1,400 m
S. typhi	0.128	0.625	0.15625	0.078125	0.078125	0.0390625	0.01953
B. subtilis	0.008	1.25	0.625	0.625	0.3125	0.3125	0.078125
S. aureus	0.016	—	—	—	0.15625	0.15625	0.078125
E. coli	0.016	10	5	5	2.5	2.5	1.25
E. facelis	0.032	—	—	5	1.25	0.3125	0.15625

3.2 TPC

Table 3 summarizes the TPC of EOs of O. majorana. While the highest TPC was found in the EO to be 188.67 ± 0.010 µg GAE/mL at 1,300–1,400 m, the lowest content was found in the EO to be 11.60 ± 0.011 µg GAE/mL at 800–900 m. There is an effect on the TPCs of the EOs collected from different altitudes.

Table 3

TPCs of O. majorana EOs

Altitude (m)	TPC (µg GAE/mL)
800–900	11.60 ± 0.011
900–1,000	37.13 ± 0.002
1,000–1,100	36.67 ± 0.011
1,100–1,200	57.80 ± 0.006
1,200–1,300	185.03 ± 0.007
1,300–1,400	188.67 ± 0.010

3.3 Antioxidant activity

Table 4 summarizes the total antioxidant capacity of EOs of O. majorana. While the highest total antioxidant capacity was found at 1,300–1,400 m (241.34 ± 0.081 µg AAE/mL), the lowest total antioxidant capacity was found at 1,000–1,100 m (25.65 ± 0.011 µg AAE/mL). The total antioxidant capacity of EOs is affected by altitude. Several studies have indicated that sweet marjoram exhibits a high antioxidant capacity [31,32,34].

Table 4

Total antioxidant capacity of O. majorana EOs

Altitude (m)	Total antioxidant capacity (µg AAE/mL)
800–900	25.65 ± 0.011
900–1,000	105.45 ± 0.041
1,000–1,100	144.49 ± 0.057
1,100–1,200	198.77 ± 0.080
1,200–1,300	210.63 ± 0.028
1,300–1,400	241.34 ± 0.081

3.4 Metal-chelating activity

Table 5 shows the metal-chelating activity of the EOs. The activity increased (from smallest to highest) in the following order at 1,000 µg/mL concentration: 800–900 m, 1,000–1,100 m, 1,300–1,400 m, 1,200–1,300 m, 1,100–1,200 m, 900–1,000 m and EDTA. Moreover, the activity increased in increasing concentrations. Altitudes of EOs had no effect on the metal-chelating activity.

Table 5

Metal-chelating activity of the O. majorana EOs and EDTA

Altitude (m)	Concentration (µg/mL)	Metal-chelating activity (%)
800–900	250	NA
	500	2.34 ± 0.01
	750	8.1 ± 0.01
	1,000	15.22 ± 0.00
900–1,000	250	NA
	500	2.4 ± 0.03
	750	12.36 ± 0.01
	1,000	23.91 ± 0.02
1,000–1,100	250	21.13 ± 0.03
	500	23.12 ± 0.02
	750	24.13 ± 0.03
	1,000	29.57 ± 0.02
1,100–1,200	250	24.62 ± 0.03
	500	25.41 ± 0.02
	750	25.92 ± 0.02
	1,000	30.8 ± 0.03
1,200–1,300	250	25.34 ± 0.02
	500	26.01 ± 0.02
	750	27.12 ± 0.01
	1,000	31.31 ± 0.02
1,300–1,400	250	24.24 ± 0.03
	500	30.96 ± 0.01
	750	32.57 ± 0.03
	1,000	33.98 ± 0.02
EDTA	250	82.69 ± 0.09
	500	98.84 ± 0.01
	750	98.95 ± 0.01
	1,000	99.11 ± 0.00

3.5 CUPRAC activity

Table 6 shows the CUPRAC activity of the EOs. The EOs of O. majorana at 1300–1,400 and 1,300–1,200 m showed higher activity than BHT, which is a standard antioxidant agent. The CUPRAC activity increased (from smallest to largest) in the following order at 1,000 µg/mL concentration: 1,000–1,100 m, 900–1,000 m, 800–900 m, 1,200–1,300 m, 1,300–1,400 m, and 1,100–1,200 m. Moreover, the activity increased in increasing concentrations. The altitudes of EOs had no effect on the CUPRAC activity of EOs.

Table 6

Cuprac activity of the O. majorana EOs and standard (nm)

Altitude (m)	Concentration (µg/mL)	Cuprac activity (nm)
800–900	250	0.029 ± 0.01
	500	0.051 ± 0.00
	750	0.053 ± 0.01
	1,000	0.068 ± 0.01
900–1,000	250	0.013 ± 0.01
	500	0.086 ± 0.02
	750	0.171 ± 0.02
	1,000	0.228 ± 0.02
1,000–1,100	250	0.135 ± 0.02
	500	0.276 ± 0.01
	750	0.418 ± 0.02
	1,000	0.536 ± 0.09
1,100–1,200	250	0.378 ± 0.06
	500	0.606 ± 0.00
	750	0.774 ± 0.02
	1,000	1.026 ± 0.04
1,200–1,300	250	1.199 ± 0.02
	500	1.855 ± 0.04
	750	1.944 ± 0.04
	1,000	1.953 ± 0.03
1,300–1,400	250	1.815 ± 0.04
	500	1.905 ± 0.04
	750	1.906 ± 0.03
	1,000	1.956 ± 0.07
BHT	250	0.795 ± 0.07
	500	0.834 ± 0.03
	750	0.951 ± 0.03
	1,000	1.273 ± 0.04

3.6 DPPH radical scavenging activity

Table 7 shows the DPPH radical scavenging activity of the EOs. BHT and rutin had higher activities than those of EOs of O. majorana. The DPPH radical scavenging activity increased (from smallest to largest) in the following order at 1,000 µg/mL concentration: 800–900 m, 900–1,000 m, 1,000–1,100 m, 1,100–1,200 m, 1,200–1,300 m, 1,300–1,400 m, BHT and rutin. Moreover, the activity increased in increasing concentrations. The altitudes of EOs had no effect on the DPPH radical scavenging activity.

Table 7

DPPH radical scavenging activity of O. majorana EOs and standard (%)

Altitude (m)	Concentration (µg/mL)	DPPH radical scavenging activity (%)
800–900	250	6.50 ± 0.02
	500	7.29 ± 0.01
	750	8.45 ± 0.00
	1,000	10.05 ± 0.00
900–1,000	250	11.75 ± 0.03
	500	13.46 ± 0.02
	750	13.73 ± 0.01
	1,000	15.81 ± 0.01
1,000–1,100	250	8.47 ± 0.06
	500	15.09 ± 0.02
	750	15.81 ± 0.01
	1,000	18.03 ± 0.01
1,100–1,200	250	17.19 ± 0.01
	500	19.70 ± 0.03
	750	22.54 ± 0.02
	1,000	24.85 ± 0.02
1,200–1,300	250	30.16 ± 0.02
	500	40.80 ± 0.01
	750	49.53 ± 0.02
	1,000	54.34 ± 0.01
1,300–1,400	250	34.12 ± 0.03
	500	41.79 ± 0.01
	750	50.34 ± 0.00
	1,000	56.06 ± 0.01
BHT	250	89.85 ± 0.02
	500	90.47 ± 0.01
	750	92.95 ± 0.01
	1,000	95.17 ± 0.01
Rutin	250	87.89 ± 0.01
	500	89.90 ± 0.01
	750	91.63 ± 0.00
	1,000	93.17 ± 0.01

In a study conducted by Hossain et al. [33], DPPH radical scavenging activity of O. majorana solutions prepared with ethanol, methanol, and water were investigated and measured as 24.7, 77.2, and 78.4%, respectively.

4 Conclusion

Turkey has emerged as a significant exporter of Origanum herb, and its EO to global markets is attributed to the quality of its products. The fresh or dried leaves and flowering tops of sweet marjoram find widespread use in culinary endeavors, enhancing the flavor profiles of various dishes. Furthermore, its EO boasts numerous applications in pharmaceuticals, perfumery, and cosmetics. In this study, we investigated the phytochemical changes in O. majorana as a function of altitude and determined the effects of these changes on biological activity.

The highest antibacterial activity was measured as a 20 inhibition zone against E. coli from sweet marjoram EOs collected at an altitude of 800–900 m. The highest MIC value of the EOs was found to be 5 mg/mL against E. coli at 1,300–1,400 m. The highest TPC and the highest total antioxidant capacity of EOs were identified at an altitude of 1,300–1,400 m, with 188.67 µg GAE/mL and 241.34 µg AAE/mL, respectively.

The highest antioxidant capacities were measured at an altitude of 900–1,000 m as 33.98% for metal chelating activity, 1.956 nm for CUPRAC activity at an altitude of 1,100–1,200 m, and 56.06% for DPPH radical scavenging activity at an altitude of 1,100–1,200 m.

The study reveals that the EOs of sweet marjoram (O. majorana) possess strong antibacterial and antioxidant properties, along with a high TPC. This makes sweet marjoram a valuable source of biologically important compounds. As a result, it can be effectively used as a natural preservative in food products, ensuring longer shelf life and safety. Additionally, its potent antioxidant properties make it an excellent ingredient in cosmetics, helping to protect the skin from damage and aging. In the pharmaceutical industry, sweet marjoram can be utilized for its health benefits. Interestingly, the biological activities of its EOs vary depending on the altitude at which the plant is grown, highlighting the importance of sourcing.

Acknowledgments

The author expresses his gratitude to the professors of the Medical Aromatic Plants Program at Ordu University’s Vocational School of Technical Sciences, Prof. Dr. Emel Karaca Öner and Prof. Dr. Meryem Yeşil for their invaluable assistance in gathering information and procuring essential oils. Prof. Dr. Sinem Aydın contributed to this study with her scientific opinions.

Funding information: The authors state no funding involved.
Author contributions: Emel Karaca Öner: planning of the experiment, collection of material, and oil extraction. Meryem Yeşil: planning of the experiment, collection of material and oil extraction. Sinem Çoban: determination of antimicrobial activity in the laboratory and article writing. Fatih Öner: investigation, funding acquisition, data curation, writing – review editing, original draft, and visualization.
Conflict of 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 manuscript.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2024-10-29

Revised: 2025-02-06

Accepted: 2025-02-06

Published Online: 2025-04-29

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/chem-2025-0139

Keywords for this article

altitude; anti-radicals; bioactivity; sweet marjoram

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