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Effects of Different Altitudes on Essential Oil Composition of Origanum majorana Species

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06 March 2023

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07 March 2023

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Abstract
Altitude is an important ecological factor that significantly affects essential oil content, yield and composition. The Origanum majorana, belonging to Origanum genus of Lamiaceae family, is a species with antibacterial and antiviral effects, widely used as a spice and also used in folk medicine for treatment of diseases such as asthma, indigestion, headache and rheumatism. In this study, conducted to examine the effects of altitude on essential oil content and composition of origanum majorana, plant samples were collected from the southern region of Turkey at the beginning of flowering period from seven different altitudes (700-800 m, 800-900 m, 900-1000 m, 1000-1100 m, 1100-1200 m, 1200-1300 m and 1300-1400 m) at 100 m intervals. The highest percentage of essential oil (6.50%) obtained by hydro-distillation was determined at 700-800 m altitudes. The GC-MS analyses revealed that low altitude affected some essential oil components positively. The linalool ratio, which is the major component of the essential oil of Origanum majorana species, was the highest at 700-800 m (79.84%) altitudes. Borneol (0.97%), linalool oxide (1.28%), trans-linalool oxide (1.47%), caryophyllene (2.78%), a- humulene (0.21%), germacrene-D (0.31%) and bicyclogermacrene (2.64%) components yielded high values at 800-900 m altitudes. Thymol (6.28%) and a-terpineol (1.04%), which have an important place in the essential oil composition, increased at 1100-1200 m altitudes; a- terpinene (0.17%), cis-sabinene hydrate (0.82%), terpinene-4-ol (0.55%) and carvacrol (21.50%) increased at 1300-1400 m altitudes.
Keywords: 
Subject: Biology and Life Sciences  -   Agricultural Science and Agronomy

1. Introduction

Thyme is a pungent herb of Lamiaceae family and has several species used for medicinal and aromatic purposes. In Turkish flora, several species of Origanum, Thymus, Satureja, Thymbra and Coridothymus genera are known as thyme [1,2]. It has a great morphological and chemical diversity and has a natural widespread with 42 species in Eurasia and North Africa [3,4]. It is represented by 22 species in Turkish flora and endemism rate is around 63% [5].
Thyme has a great economic importance. It is generally consumed as a spice and in small amounts in the form of thyme tea, thyme oil or thyme juice. Since thyme contains essential oils with highly strong antimicrobial and antioxidant effects, it is of great importance as an additive in foods, perfumes, cosmetics, medicines, lotions, soaps and toothpastes [6,7,8]. Thyme essential oil is composed of terpineol, linalool, carvacrol, cymol, thymol, p-cymene and borneol. Thymol and carvacrol are responsible for the scent of the plant. These substances constitute the main components of thyme essential oil. Carvacrol is mostly found in the essential oil of Thymbra, Origanum and Satureja species and thymol is found in higher proportions in the essential oil of Thymus species [9].
Origanum majorana species belonging to the Origanum genus, also known as Marjoram, is widely used as a spice, also used in folk medicine for the treatment of diseases such as asthma, indigestion, headache and rheumatism [10]; antibacterial [11] and antiviral [12] effects have also been reported. Its main components were reported as sabinene linalyl acetate and cis-sabinene by [13]; as carvacrol (65%), thymol (4%), terpinene-4-ol (31.15%), cis-sabinene hydrate (15.76%), p-cymene (6.83%), sabinene (6.91%), trans-sabinene hydrate (3.86%) and a-terpineol (3.71%) by [14] as terpinene-4-ol (38.40%), cis-sabinene hydrate (14.95%), p-cymene (7.01%) and a-terpineol (4.88%) by [15].
Chemical composition of Origanum species is largely designated by different chemotypes, geographical origins and harvest times [16,17]. Although the effect of altitude on essential oil components of Origanum species has been investigated by some researchers [4,18,19,20], no comprehensive study has been found in Origanum majarona species.
Secondary metabolites are not solely under the control of genetic factors. Biotic and abiotic factors that alter essential oil composition and rates include altitude, temperature, precipitation, humidity, wind exposure, light intensity, plant vegetative composition and plant growth periods [4,19,21,22,23,24,25,26].
This study was conducted to investigate the effects of natural altitudes on essential oil content and composition of Origanum majorana species.

2. Materials and Methods

2.1. Plant Material

Samples were collected from Antalya province, located in southwest of Turkey between the 36o 07' - 37o 20' north latitudes and 29o 20' - 32o 35' east longitudes [27]. The Origanum majorana samples were collected from natural flora of Akseki district at seven different altitudes in 100 m intervals (700-800 m, 800-900 m, 900-1000 m, 1000-1100 m, 1100-1200 m, 1200-1300 m, 1300-1400 m). Samples were collected at the beginning of 2021 vegetation period between the hours 9:00-11:00 a.m. (Fig 1). Collected species were identified at Akdeniz University Manavgat Vocational School and their herbariums were taken under protection at Plant and Animal Production Department of Ordu University Technical Sciences Vocational School.

2.2. Essential oil extraction

Leaf-stem separation was made on samples collected from each altitude. Leaves were dried in shade and ground. Ground leaves (100 g in each replicates) were subjected to distillation process for three hours in a Clevenger device. Essential oil quantities were determined with the aid of a graduated tube and transferred to vials and stored at +4 0C until the relevant analyses.

2.3. Analysis of the essential oil

Essential oil components were determined with the use of a GC-MS (Agilent Technologies) device. Helium was used as carrier gas and GC-MS parameters are provided below:

2.3.1. GC-MS analysis

Essential oil components were determined by GC-MS (Agilent Technologies) instrument. Helium was used as carrier gas, GC-MS parameters are presented below.
Column: DB-WAX (60 m x 0.25 mm i.d., x film thickness 0.25 pm)
Dedektör: Agilent Technologies 5977A MS
Injection Temp: 250 °C
Injection Mode: Split
Split ratio: 150
Flow Control Mode: Pressure
Pressure: 80 kPa
Split Ratio: 25
Column Temprerature programme: 40 0C 2 min, 4 0C /min 240 0C 3 min
Ion source Temp: 200 0C
Interface Temp: 250 0C
Start Time: 0 mim End Time: 55 min
Acquisition Mode: Scan
Start m/z: 40 End m/z: 4000
W9N11.L and MPW2011.L libraries were used for identification of essential oil components.

2.4. Statistical analysis

Experiments were conducted in randomized plots design with three replications. Experimental data were subjected to analysis of variance with the use of SAS-JMP 13.0 statistical software and significant means were compared with the use of LSD multiple comparison test.

3. Results and Discussion

3.1. Essentional oil ratio

The essential oil content and composition of the plant part to be used as a drug in medicinal and aromatic plants are the most important criteria that designate the quality of the product [28,29]. Previous studies have revealed that genetic structure of the plant material used in production and ecological conditions of the region where the production is made had significant effects on essential oil ratios of aromatic plants [30,31,32,33,34].
Effects of altitude on essential oil ratios are provided in Table 1 and altitude differences were found to be significant (p<0.001). Essential oil ratios of the samples varied between 4.67 - 6.50%, with the highest value (6.50%) from 700-800 m altitudes and the lowest value (4.67%) from 1000-1100 m altitudes. Essential oil ratios decreased with increasing altitudes, but such a decrease was placed into the same statistical group, except for the altitudes of 800-900 m (5.43%) and 1000-1100 m (4.67%). Altitude-dependent differences in essential oil ratios were mainly attributed to ecological factors that change with altitudes [35]. Essential oil ratios of medicinal and aromatic plants usually vary with the climate components such as temperature, precipitation, relative humidity, day light hours, light intensity, soil conditions and development period [31,36,37]. The highest essential oil ratio of 700-800 m altitudes could be attributed to greater air temperature and relative humidity than the other altitudes (Table 1).
The factors that alter essential oil ratios of aromatic plants should be well-comprehended [34,38], since pharmacopoeias specify the minimum essential oil ratios that aromatic plants must contain in order to be accepted as drugs and used as medicine and drugs containing essential oils below these ratios are not allowed to be used as medicine [39].

3.2. Essential oil components

Majority of the terpenes produced by plants are products of secondary metabolism and play an important role also in primary metabolism. These terpenoid structures, with their highly complex chemical structures, show great variation within and between the species and constitute the natural defense system of plants by damaging the other living things [40].
The GS-MS analysis revealed 69 - 126 terpenoid structures for essential oils of Origanum majorana samples collected from different altitudes. Terpenoid ratios varied between 0.01 - 79.84%. The differences in ratios of all components with altitude were found to be significant (p<0.001).
Analyses on Oregano, Thyme and Marjoram species revealed that mono and sesquiterpenes were the major components of essential oils of these species [41]. In this study, the ratio of oxygenated monoterpenes that form the essential oil and the terpenoid structure of Origanum majorana plants collected from different altitudes ranged between 65.69 - 84.62% and took the first place in total amount of essential oil. Oxygenated monoterpene ratios mathematically decreased with increasing altitudes, but there was no linear increase or decrease in oxygenated sesquiterpene ratios. Similar findings were also reported by [42]. Oxygenated monoterpenes were respectively followed by phenolic monoterpene (3.29 - 25.91%), sesquiterpene hydrocarbons (2.92 - 5.94%), oxygenated sesquiterpene (0.92 - 1.63%) and monoterpenes hydrocarbons (0.04 - 0.17%) (Table 2).
In studies on essential oil composition of Origanum majorana, major components of essential oils were reported as terpinene-4-ol and sabinene hydrate by [43] and as thymol and carvacrol by [14]. In this study, the major component was linalool (60.86 - 79.84%), an Oxygenated monoterpene. Linalool is an essential oil component used in soaps, cosmetics, perfumes, cleaning products, food preservatives, herbicides and insecticides. Linalool, which has strong antimicrobial and antioxidant properties, is the essential oil component of many medicinal and aromatic plants belonging to Lamiaceae, Lauraceae and Rutaceae families [44].
Both within the oxygenated monoterpene group components and among the other components that make up the essential oil, linalool ratios varied between 79.84% at 700-800 m altitudes and 60.86% at 1300-1400 m altitudes. These values were the highest values among the total essential oil components. Linalool ratios decreased significantly (p<0.001) with increasing altitudes. Similarly, greater linalool oxide (800-900m: 1.28%) and trans-linalool oxide (800-900 m: 1.47%, 900-1000 m: 1.30%, 1000-1100 m: 1.31%) ratios were seen at lower altitudes. Linalool and linalool oxide ratios generally decrease with decreasing temperatures of high altitudes. Previous researchers emphasized that increase in phenolic components was positively affected by regional high temperatures [41,45,46,47]. The cis-sabinene hydrate (0.82%) and terpinene-4-ol (0.55%) ratios were positively affected by increasing altitudes (p<0.001) and the highest values of both were seen at 1300-1400 m altitudes. The a-terpineol ratio of 0.55% at 700-800 m altitudes increased to 1.04% at 1100-1200 m altitudes and borneol ratio of 0.32% at 700-800 m increased to 0.97% at 800-900 m altitudes and the difference was found to be significant (p<0.001). The lowest 1,8-cineol ratio was determined as 0.08% at 700-800 m and the highest ratio as 0.33% at 900-1000 m altitudes. Researchers have reported that differences in temperature, relative humidity, wind speed and light intensity as you go to higher altitudes above sea level will change the physiological reactions of the plant and thus will create variations in composition of secondary metabolites [25,48,49].
Thymol content within total essential oil was measured as 0.32% at 700-800 m, 0.44% at 800-900 m, 0.49% at 900-1000 m, 0.45% at 1000-1100 m and 0.99% at 1200-1300 m altitudes. These values were statistically placed into the same group. Thymol ratio was 6.28% at 1100-1200 m altitudes and 4.41% thymol was detected at 1300-1400 m, which is the highest altitude and the the first place statistically. High levels of carvacrol are encountered in Origanum, Thymbra, Cordiothymus, Satureja and Lippia species. Increasing carvacrol ratios were seen with increasing altitudes and the greatest value (21.50%) was seen at 1300-1400 m altitudes. The increase in both thymol and carvacrol ratio with increasing altitude may be due to the prolongation of the growth period and the emergence of sufficient vegetation period for the conversion of intermediate components to these two components [41]. At high altitudes, plants are exposed to higher light intensity and lower average temperature. Therefore, plants at high altitudes have developed a protection mechanism against climate-induced damages. Plants adapted to high altitudes perform photosynthesis more effectively at low temperatures [50,51,52]. Previous studies indicated that carvacrol ratios increased with increasing altitudes [53] and carvacrol ratios increased under stress conditions arising from environmental conditions [54].
The a-terpinene represents monoterpenes hydrocarbons and the least encountered in monoterpene structures of Origanum majarona essential oil. The greatest value (0.17%) was seen at 1300-1400 m altitudes. Previous researchers also reported that climate and environmental factors affected the essential oil composition [20,55,56].
Caryophyllene is also a constituent of sesquiterpene hydrocarbon structure. Caryophyllene ratio was determined as 2.76% at 700-800 m and 2.78% at 800-900 m altitudes and they were placed into the same statistical group. Caryophyllene ratios decreased with increasing altitudes and decreased to 1.61% at 1200-1300 m and 1.80% at 1300-1400 m altitudes. The a-humulene (0.21%) and bicyclogermacrene (2.64%) ratios had the highest values at altitude of 800-900 m altitudes. At the highest altitudes (1300 - 1400 m), a-humulene ratio decreased to 0.11% and bicyclogermacrene to 0.84%. Like the other sesquiterpene hydrocarbons, the lowest germacrene-D ratio (0.17) was seen at 1300-1400 m altitudes and germacrene-D ratios at 800-900 m (0.31%), 1000-1100 m (0.32%) and 1200-1300 m (0.29%) altitudes were placed into the same highest group. In some specific studies, it was indicated that chemical metabolic profiles of plants belonging to Lamiaceae family with pharmacological properties, were affected by abiotic and biotic factors such as ecological conditions, soil profile, weeds, diseases and pests, harvest periods, geographical region and especially altitude [20,21,57].
Caryophyllene oxide is a compound of oxygenated sesquiterpene group. The greatest caryophyllene oxide ratio (0.35%) was seen at 1000-1100 m altitudes, while the lowest values were obtained seen at 1100-1200 m (0.15%) and 1200-1300 m (0.18%) altitudes. Spathulenol ratios varied between 0.85 - 1.28% and differences were found to be significant (p>0.001). It was determined that caryophyllene oxide and spathulenol ratios of the samples collected from both low and high altitudes did not exhibit a linear increase or decrease.
Table 2. Chemical composition of Origanum majorana essential oil.
Table 2. Chemical composition of Origanum majorana essential oil.
ALTITUDE (m)
Components f 700-800 800-900 900-1000 1000-1100 1100-1200 1200-1300 1300-1400
Monoterpenes hydrocarbons 0.06 0.04 0.08 0.07 0.11 0.13 0.17
a- Terpinene 0.0001*** 0.06 ± 0.006de 0.04 ± 0.0e 0.08 ± 0.0cd 0.07± 0.01de 0.11± 0.01bc 0.13± 0.03b 0.17 ± 0.02a
Oxygenated monoterpen 83.13 82.93 84.62 84.11 71.43 75.90 65.69
Linalool 0.0001*** 79.84 ± 1.10a 77.75 ± 3.03ab 79.68 ±2.11ab 79.20 ± 0.63ab 67.62 ± 0.13cd 71.53 ± 4.21bc 60.86 ± 5.23d
a- Terpineol 0.0001*** 0.55 ± 0.02d 0.63 ± 0.05cd 0.76 ± 0.05bc 0.88 ± 0.13ab 1.04 ± 0.00a 0.90 ± 0.02ab 0.86 ± 0.09ab
Borneol 0.0001*** 0.32 ± 0.02e 0.97 ± 0.04a 0.83 ± 0.04abc 0.87 ± 0.04ab 0.65 ± 0.01d 0.77 ± 0.01bcd 0.69 ± 0.15cd
Cis-sabinene hydrate 0.0002*** 0.27 ± 0.01b 0.28 ± 0.02b 0.30 ± 0.03b 0.44 ± 0.07b 0.39 ± 0.0b 0.43 ± 0.03b 0.82 ± 0.26a
Terpinen 4-ol 0.0001*** 0.22 ± 0.01d 0.24 ± 0.03d 0.34 ± 0.04bc 0.28 ± 0.02cd 0.42 ± 0.00b 0.43 ± 0.02b 0.55 ± 0.07a
1,8-cineole 0.0010*** 0.08 ± 0.0d 0.31 ± 0.0ab 0.33 ± 0.0a 0.14 ± 0.0bcd 0.13 ± 0.0cd 0.25 ± 0.0a'd 0.28 ± 0.0abc
Linalool oxide 0.0007*** 0.75 ± 0.02bc 1.28 ± 0.05a 1.08 ± 0.04ab 0.99 ± 0.07abc 0.63 ± 0.16c 0.74 ± 0.23bc 0.82 ± 0.07bc
Trans- Linalool oxide 0.0001*** 1.10 ± 0.03ab 1.47 ± 0.01a 1.30 ± 0.01a 1.31 ± 0.31a 0.55 ± 0.01c 0.85 ± 0.26bc 0.81 ± 0.09bc
Phenolic monoterpen 3.29 4.99 5.76 3.70 19.93 14.89 25.91
Thymol 0.0001*** 0.32 ± 0.02c 0.44 ± 0.11c 0.49 ± 0.17c 0.45 ± 0.09c 6.28 ± 0.03a 0.99 ± 0.53c 4.41 ± 1.15b
Carvacrol 0.0001*** 2.97±0.13c 4.55±1.90c 5.27±1.54c 3.25±0.79c 13.65±0.05b 13.90±3.95b 21.50±5.25a
Sesquiterpene hydrocarbons 4.82 5.94 4.44 4.89 4.78 4.01 2.92
Caryophyllene 0.0001*** 2.76 ± 0.06a 2.78 ± 0.10a 2.41 ± 0.0ab 2.42 ± 0.23ab 2.29 ± 0.01b 1.61 ± 0.03c 1.80 ± 0.25c
a-Humulene 0.0006*** 0.15 ± 0.01bc 0.21 ± 0.02a 0.15 ± 0.01bc 0.16 ± 0.02abc 0.13 ± 0.00bc 0.16 ± 0.04ab 0.11 ± 0.02c
Germacrene-D 0.0001*** 0.18 ± 0.01bc 0.31 ± 0.02a 0.20 ± 0.01bc 0.32 ± 0.01a 0.22 ± 0.01b 0.29 ± 0.04a 0.17 ± 0.0c
Bicyclogermacrene 0.0001*** 1.73 ± 0.04c 2.64 ± 0.17a 1.68 ± 0.05c 1.99 ± 0.17bc 2.14 ± 0.01b 1.95 ± 0.26bc 0.84 ± 0.02d
Oxygenated sesquiterpene 1.11 1.27 1.32 1.63 0.92 1.20 1.18
Caryophyllene oxide 0.0001*** 0.26 ± 0.02b 0.33 ± 0.03ab 0.31± 0.03ab 0.35 ± 0.01a 0.15 ± 0.01c 0.18 ± 0.040c 0.30 ± 0.04ab
Sapthulenol 0.0067*** 0.85 ± 0.22b 0.94 ± 0.15ab 1.01 ± 0.14ab 1.28 ± 0.08a 0.77 ± 0.02b 1.02 ± 0.12ab 0.88 ± 0.09b
Others 7.59 4.83 3.78 5.60 2.83 3.87 4.13
Total (%) 100 100 100 100 100 100 100
In the same row, the difference between the component ratios without a common letter is significant (***p<0.001).Climate parameters vary with the altitudes. Altitude has significant effects on yield levels, essential oil ratios and compositions [58]. Temperature, precipitation, relative humidity, day light hours, light intensity and day and night temperature differences change with altitude [31]. Altitude-dependent changes in oxygenated sesquiterpene ratios such as caryophyllene oxide and spathulenol can be explained by prevailing ecological factors of altitudes.

4. Conclusions

Present findings clearly revealed that altitude had significant effects on quality criteria of origanum majorana species (essential oil ratio and secondary metabolite composition), which are largely used in folk medicine and different industries. It was determined that essential oil content of Origanum majorana species with a natural spread in the vegetation, and the linalool component, which is the main active ingredient, decreased with increasing altitudes. Borneol, linalool oxide, trans-linalool oxide, caryophyllene, a-humulene, germacrene-D, bicyclogermacrene components yielded higher values at low altitudes. On the other hand, thymol, a-terpineol, a-terpinene, cis-sabinene hydrate, terpinene-4-ol and carvacrol components were positively affected by high altitudes. Abiotic factors, such as climate and soil, affect the amount and composition of essential oil of plants that are cultivated and collected from nature. Within the scope of this study, it was determined that essential oil ratios and components changed with altitudes. Considering the essential oil ratio and the linalool ratio, which is the major component, it was anticipated that medicinal and aromatic plants should be collected from low altitudes of the natural flora.

Author Contributions

All the authors included in this article contributed equally to the work and their contributions were essential for the completion and preparation of this article.

Funding

There is no funding for this research.

Competing Interest statement

Availability of data and material not applicable. There is no conflict of interest.

Data Availability Statement

The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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Preprints 69477 g001
Figure 1. Map of Akseki district of Antalya province.
Figure 1. Map of Akseki district of Antalya province.
Preprints 69477 g001
Table 1. Descriptive statistics for essential oil ratios of Origanum majarona species.
Table 1. Descriptive statistics for essential oil ratios of Origanum majarona species.
Altitude (m) Essential oil ratio (%)
700-800 6.50 ± 0.23a
800-900 5.43 ± 0.46bc
900-1000 6.15 ± 0.46ab
1000-1100 4.67 ± 0.43c
1100-1200 5.85 ± 0.08ab
1200-1300 5.68 ± 0.28ab
1300-1400 5.73 ± 0.25ab
p: 0.0004***
Difference in means indicated with different letter in the same column is significant (***p<0.001).
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Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
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