Original Article

Qualitative and Quantitative Analysis of Nepeta leucostegia Essential Oil Components by GC–MS/FID Along With Antioxidant Activity

Please cite this article as follows: Hamah Ameen B, Ghaderi H, Dastan D. Qualitative and quantitative analysis of nepeta leucostegia essential oil components by GC–MS/FID along with antioxidant activity. Avicenna J Med Biochem. 2023; 11(1):55-59. doi:10.34172/ajmb.2023.2433

Background

Medicinal plants and natural compounds are among the main sources for discovering new drugs. Recently, due to their availability and fewer side effects, natural products have gained significant popularity among people. Plants, especially edible ones, are more important in terms of daily use. In addition to nutrition, plants are suitable sources of useful compounds such as antioxidants, antibacterials, and anti-inflammatory agents (1,2).

Free radicals produced by various factors in the body can cause various problems such as inflammation and cancer. For this reason, preventing the formation of free radicals in the body or removing them is one of the most important ways to maintain health and prevent diseases. Natural compounds found in plants such as phenols, flavonoids, and terpenes have shown significant antioxidant effects due to their structure and functional groups. Therefore, the oral use of plants as well as natural products such as medicine and nutritional supplements containing antioxidant compounds plays an important role in controlling and eliminating free radicals (3,4).

Nepeta is a large genus belonging to the Lamiaceae family and comprises 280 species. In previous studies, different medicinal properties of the species of this genus, such as sedative, diuretic, anti-spasm, anti-asthma, anti-allergic, diaphoretic, and febrifugal effects, were reported (5-8). Roughly 67 species of the genus Nepeta are found in Iran. Nepeta leucostegia is indigenous to Kurdistan province (Sanandaj) in western Iran. The phytochemical and biological evaluation of N. leucostegia essential oil was done for the first time in this study.

Materials and Methods

Preparation of Plant Material

The aerial parts of N. leucostegia were gathered in June 2021 from Kurdistan province (Hawraman mountains), Iran, and identified by Hiva Ghaderi according to voucher number 3104 in Herbarium of the Research Institute of Forests and Rangeland, Sanandaj, Iran.

Preparation of Essential Oil

The extraction of the essential oil was done by distillation using a Clevenger apparatus. For this purpose, 100 g of the aerial parts of the plant was transferred into the flask containing water, and the distillation process was carried out for 3 hours. In the end, the resulting essential oil was collected and its water was removed using sodium sulfate (9).

Analysis of the Essential Oil

Briefly, 1 mL of essential oil extracted by water distillation was injected into a gas chromatography device coupled with a mass spectrometer (GC/MS) to identify compounds. The column of this device was 60 m in length, 0.25 mm in diameter, and 0.25 µm in thickness in the inner layer. The carrier gas of this device was helium with a flow of 1.1 mL/min. The temperature program of the column was set from 60 to 250°C at a rate of 5°C/min and was kept at 250°C for 2 minutes. The detector of the device had a power of 70 eV. The temperature of the injection site and detector was set at 250 and 300°C. The separated compounds were identified using reference libraries (the Adams Library and Wiley Library) as well as the calculation of the Kovats index and comparison with the sources. The Kovats index was obtained based on the retention index of normal alkanes (C6-C30) under the same temperature and injection conditions as the sample (5,10). GC-FID was used to determine the amount of essential oil compounds. The temperature program, injection amount, column, and other parameters were similar to GC-MS device. The peak area percentages of GC-FID were calculated using DB‐5 column without using correction factors (11).

Antioxidant Properties

The antioxidant activity of the essential oil was evaluated by measuring free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The samples with different concentrations were mixed with 1 mL of 90 μM DPPH° solution, made up to 4 mL with 95% methanol, and stirred for one hour in the dark. The absorbance of the samples and control solutions was read after one hour at a wavelength of 517 nm using a spectrophotometer. Tests were carried out in triplicate and butylated hydroxytoluene was used as positive control (12).

Results

The yield of the essential oil of N. leucostegia aerial parts was reported to be 0.4% v/w of the plant. According to the result of gas chromatography–mass spectrometry (GC-MS) and GC with flame ionization detection (GC-FID) analysis of N. leucostegia essential oil, 36 compounds were identified which comprise 96.63% of total essential oil (Figure 1). The essential oil of the aerial parts of N. leucostegia was rich in oxygenated monoterpenes. The major component was 1,8-Cineole (39.1%) and the other compounds with almost high percentages were epi-α-cadinol (6.9%), α-terpineol (6.0%), α-fenchene (5.8%), and camphene (5.5%), respectively (Table 1, Figure 2). Essential oil compounds mostly included oxygenated monoterpenes, which comprise 57.3% of the total compounds. The most abundant constituents of essential oil were oxygenated sesquiterpenes, followed by monoterpene hydrocarbons The sesquiterpene hydrocarbons and other compounds formed the smallest group of essential oil compounds. According to DPPH assay, the essential oil showed a significant antioxidant effect (IC₅₀ = 75 μg/mL).

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Figure 1.

GC-MS Chromatogram of the Essential Oil of Nepeta leucostegia

Figure 1.

GC-MS Chromatogram of the Essential Oil of Nepeta leucostegia

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Figure 2.

The Major Compounds of the Essential Oil of Nepeta leucostegia

Figure 2.

The Major Compounds of the Essential Oil of Nepeta leucostegia

Discussion

In terms of efficiency, N. leucostegia essential oil is in the range of ordinary plants, and its density is lower than water. 1,8-cineole is the main compound of essential oil, which is one of oxygenated monoterpenes according to its structure. 1,8-cineole has a special structure that, due to its oxygen bridge and stereochemistry, has the ability to interact with different receptors, and as a result, it is effective. Due to the high content of 1,8-cineole (40%) in the essential oil of the plant as well as its special structure, N. leucostegia is suggested as a potential source of 1,8-cineole.

According to a review on Nepeta genus, there are two main essential oil chemotypes in Nepeta species including nepetalactone and 1,8-cineole and/or linalool chemotype. In other words, N. leucostegia essential oil belongs to 1,8-cineole and/or linalool chemotype, indicating that it can be used in the chemotaxonomic study of Nepeta genus (13,14). The essential oils of herbal medicine, which showed extensive pharmacological properties including anti-inflammatory and antioxidant and were used for the treatment of respiratory diseases, cardiovascular diseases, and so on, are rich in 1,8-cineole. Therefore, N. leucostegia can be used in medicine and food industry as an antioxidant and anti-inflammatory agent (15).

The monoterpene compounds make up the largest percentage of essential oils and consist of two isoprene units. Due to their lower molecular weight compared to other terpenoids, monoterpenes comprise a higher percentage of plant essential oils. For this reason, perfume and cosmetic products are widely used in aromatherapy. Additionally, various biological effects such as antioxidant, antimicrobial, and anti-inflammatory activities have been reported for them (16,17).

In traditional medicine of different countries including Iran, India, Pakistan, China, Nepal, and Turkey, different species of Nepeta genus are used for treating various diseases, including Asthma, respiratory disorders, influenza, pneumonia, tuberculosis, chicken pox, and eye irritation (18). N. leucostegia is used in traditional and ethnobotanical medicine to treat stomach problems, colds, and coughs. According to DPPH assay, the essential oil showed a significant antioxidant effect (IC₅₀ = 75 μg/mL). It should be noted that the IC₅₀ value for the BHT standard was reported to be 26 (Table 2). Due to the side effects of synthetic antioxidant compounds, the use of natural compounds with antioxidant properties in the pharmaceutical, cosmetic, and food industries is widely accepted (19). Compared to the essential oil of other Nepeta species, it has significant antioxidant properties. In previous studies, essential oils of N. cataria, N. sintenisii, and N. curviflora species showed antioxidant effects with IC₅₀ of 80.62, 716.0, and 180.0 μg/mL,respectively (20-22). Recent studies on the antioxidant activity of essential oils reported radical scavenging activity that occurs through their constituents (23). The antioxidant effect is caused by the components of the essential oil, especially 1,8-cineole and epi-α-cadinol. In the study conducted by Ciftci et al, 1,8-cineole showed antioxidant activity in rats and eliminated TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced oxidative stress (24).

Conclusion

The phytochemical and biological evaluation of N. leucostegia essential oil was done for the first time in this study. The yield of plant essential oil was 0.4% v/w and 96.63% of the total essential oil compounds were identified. The main group of the essential oil belonged to oxygenated monoterpenes and the main composition of the essential oil was 1,8-Cineole. The results of this study complete the information on the Nepeta genus and can be used in the chemotaxonomic study of the Nepeta genus. Due to its antioxidant effect (IC₅₀ = 75 μg/mL), N. leucostegia has the potential for use in pharmaceutical and food industries. Therefore, other properties of this plant include its nutritional values and it is suggested that its interaction with other foods should be studied in the future.

Acknowledgements

We are grateful to the Vice-chancellor for Research and Technology, Hamadan University of Medical Sciences (No. 9806264980) for the financial support of this work.

Authors’ Contribution

Conceptualization: Dara Dastan.

Data curation: Dara Dastan.

Formal analysis: Dara Dastan, Baram Hamah Ameen.

Funding acquisition: Dara Dastan.

Investigation: Dara Dastan.

Methodology: Dara Dastan.

Project administration: Dara Dastan.

Resources: Hiva Ghaderi.

Supervision: Dara Dastan.

Validation: Dara Dastan.

Visualization: Dara Dastan.

Writing–original draft: Dara Dastan.

Writing–review & editing: Baram Hamah Ameen.

Competing Interests

The authors declare no conflict of interests regarding the publication of this paper.

Ethical Approval

Not applicable.

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