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INTRODUCTION

Tanacetum vulgare L. (Asteraceae), known as “tansy” in Europe and “Palma real” in Peru, is an aromatic plant widely used in folk medicine as a vermifuge and anti-inflammatory.^1,2 Also, the aerial parts of this plant are popularly used to treat migraine, neuralgia, and rheumatism, and as an anthelmintic and insect repellent.³ Tansy extract has been reported to exhibit antitumor,⁴ anti-inflammatory,⁵ antioxidant,⁶ and antimicrobial activity.⁷ Papers on the composition of the oils of this species published up to 1996 have been reviewed.⁸ Twenty-three chemotypes of the oils of tansy plants have been determined according to the most dominant constituent in the essential oils.⁸ The most common, a β-thujone chemotype was identified in nine countries: Argentina, Belgium, Canada, Holland, Poland, Hungary, Finland, India and Italy, while an α-thujone chemotype occurred in three countries: France, Germany and Italy. Camphor type oils were collected in plants from seven countries (Belgium, Germany, Holland, Hungary, Finland, Kazakhstan and USA) and of trans-chrysanthenyl acetate chemotype – in five ones (Belgium, Finland, Holland, Hungary and Italy). The 1,8-cineole and artemisia ketone chemotypes were present in Holland, Hungary and Finland. Other 16 chemotypes in which the dominant constituent is one of the following α-pinene, sabinene, g- terpinene, thujyl alcohol, thujyl acetate, umbellulone, chrysanthenone, chrysanthenone oxide, isopinocamphone, borneol, piperitone, trans-dihydrocarvone, lyratol, lyratyl acetate, davanone or germacrene D occurred in few countries.⁸ Papers on the composition of tansy oils published later,^9-13 only found one new chemotype in which dominant components were β-thujone-trans-chrysanthenyl acetate.¹⁴,¹⁵

The well-known and widely used peppermint (Mentha . piperita L.) (Lamiaceae) is a cultivated natural hybrid of Mentha aquatica L. (water mint) and Mentha spicata L. (spearmint). Although is native to the Mediterranean region, it is cultivated all over the world for its fragrant oil. Peppermint essential oil is used in flavor, fragrance, medicinal, and pharmaceutical applications.^16-19 More than 300 constituents have been identified in the essential oil, where the major constituents are menthol (35-60 %), menthone (2-44 %), menthyl acetate (0.7-23 %), 1,8-cineole (1-13 %), menthofuran (0.3-14 %), isomenthone (2-5 %), neomenthol (3-4 %) and limonene (0.1-6 %).^20,21

Despite the medicinal potential of plants in Peru being considerable, knowledge of this area and studies on the biological activities of these plants remained scarce. Furthermore, as far as our literature survey could as certain, antioxidant activities of the Peruvian aromatic plants Tanacetum vulgare L. and Mentha . piperita L. var. vulgaris have not previously been published, although, there are many reports concerning essential oils from these species in other countries. Moreover, knowing that the chemical composition and activity of essential oils from aromatic plants depends on several factors such as the geographical origin, the aims of this study were (i) to determine the composition of the essential oils isolated from the leaves of Tanacetum vulgare L. and Mentha . piperita L. var. vulgarisgrown wild at high height above sea level and (ii) to assess the antioxidant activities of these essential oils.

MATERIALS AND METHODS

Plant material and isolation of essential oil

Leaves of T. vulgare and Mentha . piperita var. vulgariswere collected in summer (February 2015) in the Cuzco region, in southeast Peru (3,618 m height above sea level). The species were identified, and the voucher specimens (accession numbers 24273 CUZ and 24274B CUZ, for T. vulgare and M. . piperita var. vulgaris, respectively) were deposited at the herbarium of the National University of San Antonio Abad del Cusco. Three samples of fresh chopped plants (ca. 200 g) were extracted using hydrodistillation for 3 h in a Clevenger-type apparatus.

GC-FID and GC-MS

Analyses of the essential oils were performed by gas chromatography with a flame ionization detector (GC-FID) on a Konik 4000A (Konik, Barcelona) equipped with a 30 m x 0.25 mm i.d. x 0.25 mm DB-Wax (Agilent Technologies, Santa Clara, CA, USA) or 30 m x 0.25 mm i.d. x 0.25 mm DB-5MS fused-silica capillary columns (J & W Scientific, Folsom, CA, USA). The analyses were conducted under the following conditions for both columns: oven temperature program, 60 oC (2 min), 60-220 oC (4 oC/min) and 220 oC (5 min); carrier gas Helium flow rate 1 mL/min; injector and detector temperatures 250 oC, injection volume 0.2 μL and split ratio 20:1.

Essential oils were also analyzed by gas chromatography-mass spectrometry (GC–MS) using a Hewlett Packard 6890, fitted with the same columns interfaced with an Hewlett Packard mass-selective detector 5973 (Agilent Technologies, Palo Alto, CA, USA). GC parameters were similar to GC-FID and interface temperature: 250 oC; MS source temperature: 230 oC; MS quadrupole temperature: 150 oC; ionization energy: 70 eV; mass range: 35-400 m/z.

The different components were identified using their retention indices and mass spectra. Retention indices, calculated using linear interpolation relative to retention times of C8-C24 of .-alkanes, were compared with those standards and data from the literature.22 Mass spectra were compared with corresponding reference standard data reported in the literature22 and mass spectra from NIST 05, Wiley 6, NBS 75 k, and in-house Flavorlib libraries. Moreover, the retention indices of authentic compounds and marker constituents of known essential oils were also used to authenticate the constituent’s identification.

The quantification of compounds was performed using relative percentage abundance and normalization method with correction response factors based on grouping the essential oil components by their functional groups.²³ Percentage data are the mean values of three injections per sample.

Assay of 2,2-diphenyl-2-picrylhydrazyl (DPPH•) scavenging activity

The antioxidant capacity of the essential oils was measured determining by DPPH• scavenging ability according to the method previously described24 with minor modifications. In the test tubes, 1 500 μL of DPPH (0.075 mg/mL) in ethanol was mixed with 750 μL of five concentrations of the oil sample to evaluate in a range between 100-5 000 μg/mL. A control sample (absolute ethanol) and a reference sample (750 μL absolute ethanol plus 1 500 μg/mL of DPPH solution) were also used. The decrease in the absorbance (Abs) at 515 nm was determined until the reaction plateau step was reached. Triplicate measurements were carried out. Then, the IC50 values were determined by using GraphPad Prism program (ver. 5) and it was defined as the total antioxidant compound necessary to decrease the initial DPPH radical concentration by 50 %. Their scavenging effect of the product was calculated based on the percentage of DPPH scavenged using the followed formula:

% DPPH inhibition = (control Abs – sample Abs)/(control Abs) x 100, where control Abs represent: ethanol Abs + DPPH and sample Abs: sample Abs + DPPH

RESULTS AND DISCUSSION

The extraction yield of T. vulgare and M. . piperita var. vulgaris essential oils were 0.4 % and 0.8 % (v/m), respectively.

Quantitative data expressed as average percentage values and standard deviations, of single components in the essential oils are listed in Table 1. Fifty components were identified in the essential oil from T. vulgare leaves. As can be seen, oxygenated monoterpenes were the most represented class of volatile with 92.6 %. Amongst their derivative, trans-thujone or β-thujone was the major compound. This chemical composition suggests that the analyzed tansy plant belong to the most common trans-thujone chemotype. This monoterpenic ketone is a bioactive compound with medicinal properties, but at high concentrations it exhibits toxicity.^25-28

Seventy constituents were identified in the essential oil from Mentha . piperita leaves (Table 1). The essential oil contains basically oxygenated monoterpenes (67.7 %) being the major constituents menthol, menthyl acetate, and menthone. Menthofurane was not detected in the present study, but developmental and environmental factors are known to influence greatly the yield and composition of peppermint essential oils.¹⁸ The composition found in the present study is according to the general criteria that high quality peppermint essential oils have higher quantities of menthol and menthone, and lower amounts of pulegone and menthofuran.¹⁸

DPPH is a free radical compound which is usually used as in vitro test to evaluate the ability of the several compounds to free radical scavenging. At this work, the antioxidant effect of the essential oils from T. vulgare and M. x piperita var. vulgaris was tested by using this assay.

Tansy antioxidants have been studied rather scarcely and mainly as extracts.^6,29,30 The essential oils from T. vulgare had the capacity to scavenge the DPPH radical, showing a maximal effect of 58.26 ± 0.9 % at the concentration of the 4 500 μg/mL. On the other way, usually DPPH scavenging is presented by the IC50 value, concentration of antioxidant needed to scavenged 50 % of the DPPH radical present into the test solution. For this tansy essential oil the IC50 value was 3 525 ± 123 μg/mL. But, as can be seen, this effect was very lower than the observed for M. x piperita var. vulgaris essential oil and the ascorbic acid, the positive control used in the assay, which showed a percentage of inhibition of 89.0 ± 4.3 % at 100 μg/mL (Table 2).

Antioxidant effectiveness expressed as IC50, the total antioxidant capacity necessary to decrease the initial DPPH radical concentration by 50 %. Values represent average of three determinations with ± standard deviation. Ascorbic acid was used as standard.

In addition, M. x piperita var. vulgaris essential oil exhibited concentration dependent increase to DPPH radical scavenging effect, the maximum effect observed was 69.1 ± 3.4 % at 500 μg/mL. On the other hand, the IC50 value for this tested product was 136.4 ± 35 μg/mL. Studies performed for evaluating the free radical scavenging capacity of essential oils from other species of Mentha showed the antioxidant properties of this species, where one variety of M. x piperita var. vulgaris expressed IC50 values of 2.53 µg/mL.³¹ At this study the most powerful scavenging compounds associated to antioxidant activity of M. x piperita were monoterpene ketones, particularly the menthone and iso-menthone. Comparison of the DPPH scavenging property from oils present in the M. x piperita var. vulgaris evaluated by us and previously reported31 for M. x piperita and the found for ascorbic acid (51.2 ± 2.9 μg/mL at 100 μg/mL) in our study, the essential oil evaluated at the present study exhibited weakest antioxidant effects than the previously report for the specie and the standard antioxidant.

CONCLUSIONS

The essential oil from Tanacetum vulgare has lower antioxidant activity than Mentha . piperita var. vulgaris, but both have potential use in food industry, cosmetic or pharmaceutical industries.

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