Antioxidant Activity of Nepeta nuda L. ssp. nuda Essential Oil Rich in Nepetalactones from Greece

Abstract

Essential oils from air-dried leaves and verticillasters of Nepeta nuda ssp. nuda from Greece were analyzed by means of gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. The dominant constituent in the verticillaster oils was 4aα,7α,7aβ-nepetalactone (75.7%). The main metabolites of the leaf oil were 1,8-cineole (16.7%), 4aα,7α,7aβ-nepetalactone (24.7%), and caryophyllene oxide (16.3%). The oils were examined for their antioxidant activity. Neutralization of stable 2,2-diphenyl-1-picrylhydrazyl radical ranged from 10.83% (2.50 μg/mL) to 58.64% (50.00 μg/mL) for verticillaster oil and from 6.25% (2.50 μg/mL) to 57.79% (50.00 μg/mL) for leaf oil. The essential oil from verticillasters had significant effects on lipid peroxidation (in the range of 41.18–59.23%), compared to tert-butylated hydroxytoluene (37.04%). In contrast, the essential oil from leaves exhibited pro-oxidant activity at the highest concentration applied.

Introduction

N epeta L. is a genus of annual or perennial herbs found in temperate Europe, Asia, and North Africa and in mountains of tropical Africa and comprises approximately 250 species.¹ In the literature, the Nepeta genus is characterized by the presence of monoterpene nepetalactones, other terpenoids, iridoids, and flavonoids.^2

–7

Nepeta species are known for their utilization in traditional folk medicine and are reported to have diuretic, antitussive, expectorant, antispasmodic, anti-asthmatic, and antiseptic activities.⁸ Their extracts and essential oils have exhibited anti-inflammatory, analgesic, antioxidant, antiviral, and antimicrobial activities.^9

–15

The leaves and the flowering tops of Nepeta cataria L. (catnip) are recommended for use as a flavoring in sauces and cooked foods and dried in mixtures for soups and stews. Moreover, the tops are currently used in teas.¹⁶ Nepeta nuda L. ssp. nuda is a common species of this genus found throughout Europe.¹⁷ The leaves and the flowering tops of this taxon are used locally in Greece by shepherds to make tea. In our continuing research on Greek aromatic plants, we have analyzed the essential oils from leaves and verticillasters of N. nuda ssp. nuda and investigated their potential antioxidant activity.

Materials and Methods

Plant material

Aerial parts of N. nuda ssp. nuda were collected during the flowering stage from natural populations in Greece from Mount Parnassos, in the prefecture of Viotia, at an altitude of 1,940 m, in August 2003. A voucher specimen has been deposited in the Herbarium of the University of Athens, Athens, Greece.

Essential oil

The plant material was dried in the shade to constant weight. Dried leaves and verticillasters were ground into small pieces and subjected separately (50 g) to hydrodistillation in a modified Clevenger apparatus for 3 hours with 300 mL of deionized water. The essential oils obtained were dried over anhydrous sodium sulfate and, after filtration, stored at 4°C. The resulting essential oils had a characteristic odor and were diluted in n-hexane and subjected to chemical analyses, as well as for antioxidant activity assays. The yields of the essential oils obtained from the leaves and verticillasters were 0.97% and 3.80% (wt/wt), respectively.

Chromatographic analysis

Gas chromatography (GC) analysis of the oils was carried out using an SRI (Brooks, Hatfield, PA, USA) model 8610C GC-flame ionization detector (FID) system, equipped with a DB-5 capillary column (30 m × 0.32 mm; film thickness, 0.25 μm) and connected to an FID detector. The injector and detector temperature was 280°C. The carrier gas was He, at a flow rate of 1.2 mL/minute. The thermal program was 60°C to 280°C at a rate of 3°C/minute.

GC-mas spectrometry (MS) analyses were carried out using a Hewlett Packard GmbH (Waldbronn, Germany) model 5973-6890 GC-MS system operating in electron ionization mode at 70 eV, equipped with a split-splitless injector (200°C). Helium was used as the carrier gas (1 mL/minute). The capillary columns used were an HP-5MS (30 m × 0.25 mm; film thickness, 0.25 μm; Agilent, Palo Alto, CA, USA) and an HP-INNOWax (30 m × 0.25 mm; film thickness, 0.50 μm; Hewlett Packard, Palo Alto). The temperature programs were 60°C to 280°C at a rate of 3°C/minute and 60°C to 260°C at a rate of 3°C/minute, respectively, with a split ratio of 1:10. The essential oils obtained by hydrodistillation were dissolved in n-hexane (100 μL/mL). The injected volume was 1 μL.

Identification of the constituents was based on comparison of the retention times with those of authentic compounds either purchased or isolated and identified,³ comparing their linear retention indices relative to the series of n-hydrocarbons, on computer matching against commercial (Wiley) (available through Hewlett Packard) and MS literature data^18
–20 and with our own data (MS of pure compounds). Authentic reference chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The percentage composition of the essential oils is relative, computed from peak areas without correction factors.

Antioxidant activity

2,2-Diphenyl-1-picrylhydrazyl assay

Free radical scavenging capacity (RSC) was evaluated measuring the scavenging activity of examined essential oils against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The DPPH assay was performed as described before,²¹ with small modifications. The samples (from 2.50 to 50 (μg/mL) were mixed with 1 mL of 90 (μM DPPH^· solution (Sigma) and filled up with 95% methanol to a final volume of 4 mL. The absorbance (A) of the resulting solutions and the blank (with same chemicals, except for the sample) was recorded after 1 hour at room temperature, against tert-butylated hydroxytoluene (BHT) (Fluka AG, Buchs, Switzerland) as a positive control. For each sample four replicates were recorded. The disappearance of DPPH^® was measured spectrophotometrically at 515 nm using a Beckman (Palo Alto) DU^®-65 spectrophotometer. Percentage of RSC was calculated using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} RSC \ (\%) = 100 \times ([A_{\rm blank} - A_{\rm sample}]/A_{\rm blank}) \end{align*} \end{document}

The IC₅₀ value, which represented the concentration of the essential oil that caused 50% neutralization, was determined by linear regression analysis from the RSC values obtained.

Rapid screening for scavenging compounds of essential oils

For fast screening of essential oil compounds on RSC, the dot-blot test on thin-layer chromatography silica gel F254 aluminum plates (Merck, Darmstadt, Germany) stained with the free DPPH^® was used.²¹ An appropriate amount of pure essential oil (5 (L) was placed on a silica gel plate and developed in benzene:ethyl acetate (95:5 vol/vol). After drying, the plates were sprayed with a 0.4 mM solution of DPPH^® in methanol, using a DESAGA GmbH (Wiesloch, Germany) spray gun. Sprayed plates gave a purple background with yellow spots at the location of those compounds that possess high RSC. Essential oil compounds responsible for scavenging activity were identified comparing the DPPH-thin-layer chromatogram with the control, treated with vanillin-sulfuric acid spray reagent.

Lipid peroxidation assay

The extent of lipid peroxidation (LP) was determined by measuring the color of the adduct produced in the reaction between thiobarbituric acid (TBA) and malondialdehyde (MDA), as an oxidation product in the peroxidation of membrane lipids, by the TBA assay.²¹

A commercial preparation of liposomes (PRO-LIPO S, Lucas-Meyer, Hamburg, Germany), pH 5–7, was used as a model system of biological membranes. The liposomes, 225–250 nm in diameter, were obtained by dissolving the commercial preparation in demineralized water (1:10), in an ultrasonic bath.

For the experiment three concentrations of essential oil of leaves (0.425, 1.065, and 2.13 (μg/mL) and six for essential oil of verticillasters (ranging from 0.425 to 10 (μg/mL) were prepared. Essential oils and BHT were diluted in n-hexane, and the control with n-hexane instead of sample was also analyzed.

Sixty microliters of suspension of liposomes was incubated with 20 (μL of 0.01 M FeSO₄, 20 (L of 0.01 M ascorbic acid, and 10 (μL of essential oil samples in 2.89 mL of 0.05 M KH₂PO₄-K₂HPO₄ buffer, pH 7.4 (3 mL final solution). Samples were incubated at 37°C for 1 hour. LP was terminated using the reaction with 1.5 mL of TBA reagent and 0.2 mL of 0.1 M EDTA, heating at 100°C for 20 minutes. After cooling and centrifugation of precipitated proteins (4,000 rpm with a Sigma GmbH [Osterode am Harz, Germany] centrifuge for 10 minutes), the content of MDA (TBA-reactive substances) was determined by measuring the absorbance of the adduct at 532 nm.

The commercial synthetic antioxidant BHT (0.5 M stock solution, concentration 220.4 (μg/mL) was used as a positive control. All the reactions were carried out in triplicate.

The percentage of LP inhibition (I) was calculated by the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} I (\%) = ([A_0 - A_1]/ A_0) \times 100 \end{align*} \end{document}

where A ₀ was the absorbance of the control reaction (full reaction, without the test compound) and A ₁ was the absorbance in the presence of the inhibitor.

Statistics

All data except GC-MS analysis were reported as mean ± SD (SEM) values calculated from replicates (n ≥ 3). From the obtained RSC and values of the inhibition of LP, the IC₅₀ values representing the concentrations of the investigated essential oils that caused 50% neutralization or inhibition were determined by the linear regression analysis in the Microsoft (Redmond, WA, USA) Excel program for Windows, version 2000.

Results

Chemical composition of the essential oils

The leaves and verticillasters of N. nuda subsp. nuda from Greece were distilled, and the resulting oils were analyzed by GC-FID and GC-MS. Table 1 lists components identified, together with their linear retention indices on both the HP5-MS and HP-INNOWax columns and their percentage abundances. Fifty-five components were detected and identified, representing 93.3% and 99.1%, respectively of the total oils. The chemical composition of the analyzed samples was qualitatively similar, with nepetalactones along with 1,8-cineole making up more than 50% of the oils investigated. Oxygenated monoterpenes were the main components in the oils studied, accounted for 49.2% in leaf oil and 82.5% in verticillaster oil. The verticillaster oil was richer in the three nepetalactones detected (4aα,7α,7aα-nepetalactone, 4aα,7α,7aβ-nepetalactone, and 4aβ,7α,7aβ-nepetalactone), with 4aα,7α,7aβ-nepetalactone being the dominant constituent (75.7%). In the leaf oil the main metabolites were 1,8-cineole (16.8%), 4aα,7α,7aβ-nepetalactone (24.7%), and caryophyllene oxide (16.3%).

Table 1.

Chemical Composition of N. nuda ssp. nuda Essential Oils

	RI ^b		%
Component ^a	HP5-MS	HP-INNOWax	L	V
α-Thujene	925		Tr	—
α-Pinene	934	1,030	1.4	0.2
Benzaldehyde	956		Tr	—
Sabinene	970	1,125	1.9	0.4
α-Pinene	974	1,101	Tr	Tr
1-Octen-3-ol	975	1,436	—	0.2
3-Octanone	979		—	Tr
Myrcene	986	1,156	—	Tr
p-Cymene	1,020	1,268	Tr	Tr
1,8-Cineole	1,026	1,202	16.7	3.3
(Z)-β-Ocimene	1,037		Tr	Tr
γ-Terpinene	1,048	1,247	Tr	Tr
cis-Sabinene hydrate	1,057		Tr	Tr
Terpinolene	1,079	1,278	Tr	Tr
Linalool	1,092	1,524	Tr	Tr
Nonanal	1,096		0.8	Tr
p-Menth-2-en-1-ol	1,112		—	Tr
α-Campholenal	1,116		Tr	Tr
trans-Pinocarveol	1,126	1,638	Tr	Tr
cis-Verbenol	1,129		Tr	—
trans-Verbenol	1,132		Tr	—
Pinocarvone	1,147	1,554	Tr	Tr
δ-Terpineol	1,152	1,656	Tr	Tr
Terpinen-4-ol	1,161	1,579	Tr	Tr
p-Cymen-8-ol	1,171		Tr	—
α-Terpineol	1,175	1,673	1.0	0.6
Myrtenal	1,179		Tr	Tr
Verbenone	1,190		Tr	—
(Z)-3-Hexenyl pentanoate	1,216		Tr	0.2
Carvone	1,227		Tr	—
Dihydroedulan II	1,264		Tr	—
Dihydroedulan I	1,303		Tr	—
4aα,7α,7aα-Nepetalactone	1,337	1,975	3.5	2.3
α-Copaene	1,350	1,471	4.4	2.6
β-Bourbonene	1,363	1,500	4.7	3.1
4aα,7α,7aβ-Nepetalactone	1,367	2,024	24.7	75.7
4aβ,7α,7aβ-Nepetalactone	1375	2040	—	Tr
β-Caryophyllene	1,394	1,610	3.6	—
β-Gurjunene	1,409		Tr	—
Aromadendrene	1,414		—	—
α-Humulene	1,424		0.6	1.5
Geranyl acetone	1,427		Tr	—
(E)-β-Farnesene	1,430	1,648	Tr	1.2
allo-Aromadendrene	1,432		Tr	—
α-Amorphene	1,456	1,743	Tr	—
Germacrene-D	1,458	1,687	1.8	1.9
α-Zingiberene	1,468		—	Tr
β-Bisabolene	1,480	1,698	Tr	1.4
δ-Cadinene	1,494	1,733	2.0	—
β-Sesquiphellandrene	1,504		—	1.4
α-Calacorene	1,521	1,905	1.5	Tr
Elemol	1,525		1.2	0.6
Caryophyllene oxide	1,557	1,983	16.3	2.0
Humulene epoxide II	1,581		2.3	0.3
cis-Calamenen-10-ol	1,632		1.6	—
14-Hydroxy-9-epi-(E)-caryophyllene	1,648		3.3	0.2
Total			93.3	99.1

Compounds are listed in order of elution from an HP5-MS column.

Retention indices relative to C₉–C₂₃ n-alkanes.

L, leaf oil; V, verticillaster oil; Tr, trace (<0.1%) oil; —, compound not detected.

Antioxidant assays

Antioxidant properties of the investigated essential oils were evaluated as both free RSC and protective effect against LP. The free radical scavenging activity of the essential oils was evaluated by their ability to act as donors of electrons or hydrogen atoms to the DPPH radical, transforming it into the reduced form, DPPH-H. Both essential oils assessed were able to reduce the stable, purple-colored DPPH radical into the yellow-colored DPPH, ranging from 10.83% to 58.64% for essential oil of verticillasters and from 6.25% to 57.79% for essential oil from leaves (Table 2). In both cases this activity was dose-dependent. Although essential oils did not reach the 50% of neutralization of DPPH radical, the presumed IC₅₀ values, determined by linear regression analysis, were 39.41 μg/mL and 40.61 μg/mL for verticillasters and leaves, respectively. The compounds most responsible for RSC in both essential oils were confirmed with dot-blot co-thin-layer chromatography with previously isolated compounds³ to be the nepetalactones (Table 1), and consequently stronger scavenging activity was obtained for essential oil obtained from verticillasters.

Table 2.

Neutralization of 2,2-Diphenyl-1-Picrylhydrazyl Radicals by the Essential Oils Investigated and tert-Butylated Hydroxytoluene

	Essential oil (μg/mL)
	V	L	BHT (μg/mL)
Concentration (μg/mL)
2.50	10.83 ± 0.25	6.25 ± 0.15	23.12 ± 0.08
3.00	11.20 ± 0.10	7.39 ± 0.21	30.11 ± 0.19
5.00	12.10 ± 0.27	8.29 ± 0.17	44.71 ± 0.04
6.25	12.80 ± 0.14	9.44 ± 0.06	53.20 ± 0.10
7.20	13.44 ± 0.26	10.92 ± 0.18	59.07 ± 0.18
9.50	15.86 ± 0.04	13.98 ± 0.09	67.51 ± 0.07
12.00	22.73 ± 0.37	17.69 ± 0.14	72.12 ± 0.12
15.00	25.98 ± 0.11	22.28 ± 0.11	—
20.00	32.45 ± 0.22	28.98 ± 0.03	—
30.00	41.18 ± 0.13	40.53 ± 0.12	—
40.00	51.32 ± 0.02	49.82 ± 0.07
50.00	58.64 ± 0.06	57.79 ± 0.04	—
Equation	y = 1.0698x + 7.8403	y = 1.1384x + 3.764	y = 31.934Ln(x) + 5.529
R ² value	0.9865	0.9917	0.9957
IC₅₀	39.41	40.61	5.69

BHT, tert-butylated hydroxytoluene; IC₅₀, concentration of essential oil that caused 50% neutralization.

The protective effects of the examined essential oils on peroxidation of lipids have been evaluated by the TBA assay using the Fe²⁺/ascorbate system of induction. Diluted essential oils obtained from both organs (verticillasters and leaves) of N. nuda ssp. nuda exhibited inhibition of LP (Fig. 1). In the case of verticillaster essential oil, this activity was dose-dependent and ranged from 41.18% to 59.23% (Fig. 2). In contrast, in the essential oil obtained from leaves, increasing the concentration led to a reduction of the antioxidant activity, resulting from the pro-oxidant effects of the pure essential oil (–22.90%) (Fig. 1).

FIG. 1.

Inhibition of lipid peroxidation by the essential oils investigated and BHT. Essential oils and BHT were diluted in n-hexane (the solvent had no antioxidant activity).

FIG. 2.

Inhibition of lipid peroxidation by essential oil obtained from verticillasters.

Discussion

The present study demonstrated the antioxidant activity of N. nuda subsp. nuda oil, rich in nepetalactones, by inhibition of LP and RSC.

The essential oils analyzed of N. nuda ssp. nuda from Greece were characterized by the dominance of 4aα,7α,7aβ-nepetalactone. Previous investigations on the essential oils from N. nuda plants showed considerable differences in the chemical profile due to the plant origin.^22

–26 In N. nuda from Bulgaria, 62% of the composition of the total oil was 4aβ,7α,7aα-nepetalactone.²⁴ Nepetalactone mixture, 1,8-cineole, and germacrene-D in varying amounts were present in different oils from N. nuda grown from seeds obtained from various botanical gardens in Europe.²⁶ However, nepetalactones were absent in N. nuda ssp. albiflora (Boiss.) Gams from Turkey, with the latter having been found rich in nerolidol, 1,8-cineole, and spathulenol.²³ It is noteworthy, though, that the same subspecies from another locality in Turkey was characterized by the abundance of nepetalactones.²⁵ N. nuda ssp. nuda essential oil from Turkey (central Anatolia) was different from the European ones, characterized by the presence of sesquiterpenes (caryophyllene oxide, spathulenol, allo-aromadendrene, and β-caryophyllene) and the absence of nepetalactones.²²

Although there are some reports on the biological activities of Nepeta species,^9

–15 only two of them deal with their antioxidant activity,^11,12 whereas there is no report on the antioxidant activity of N. nuda essential oil.

Terpene compounds, such as 1,8-cineol, were present in considerable amount in the leaf oil studied, and essential oils rich in this oxygenated monoterpene have already been reported to exhibit notable antioxidant activity,^11,12,27,28 but for the antioxidant effects of nepetalactones or essential oils with a high content in nepetalactones there are no data.

Single methods are not recommended for the evaluation of the antioxidant activities of different plant products, because of their complex composition.^29,30 Therefore, the antioxidant effects of plant products must be evaluated by combining two or more different in vitro assays to obtain relevant data.

Comparison of the antioxidant activity (IC₅₀ values) exhibited by the essential oils investigated and BHT showed variable effects, depending on the model system used for evaluation. Generally, the stronger antioxidant activity of the verticillaster oil points towards the importance of nepetalactones present in a very high amount in this oil (Table 1).

The verticillaster essential oil expressed significant effects on LP (in the range of 41.18–59.23%), compared to BHT (37.04%). In contrast, the leaf essential oil exhibited pro-oxidant activity at the highest concentration applied.

As far as our literature search can ascertain, this is the first report on the antioxidant activity of N. nuda ssp. nuda attributed to the content of nepetalactones.

Footnotes

Acknowledgments

The authors are grateful to Prof. A. Yannitsaros (Institute of Systematic Botany, Department of Biology, University of Athens, Panepistimioupoli Zographou, Greece) for the taxonomic identification of the specimen. The authors from Serbia are also grateful to The Ministry of Sciences and Environmental Protection, Republic of Serbia, for grant 142036, which supported part of this research work.

Author Disclosure Statement

No competing financial interests exist.

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