Antioxidant and Antimycotic Activities of Two Native Lavandula Species from Portugal

The antioxidant and antimycotic activities of the essential oils and extracts of two native Portuguese Lavandula species, L. stoechas subsp. luisieri and L. pedunculata, were evaluated by in vitro assays. The total phenolics and flavonoids content were also determined. The antioxidant potential was assessed through DPPH radical scavenging, inhibition of lipid peroxidation (ILP), and DNA protection assays. All samples displayed a high DPPH scavenging activity, some of them showing concentration dependence. The majority of the samples were also able to inhibit lipid peroxidation. A strong correlation was observed between the results of DPPH and ILP assays and the flavonoids content of the samples. In the DNA protection assay, all the extracts were able to preserve DNA integrity. The antimycotic activity was performed against twelve fungi belonging to Basidiomycota and Ascomycota Divisions. L. stoechas subsp. luisieri exhibited the broadest activity spectra. L. pedunculata extracts were active against five fungi. Cryptococcus neoformans was the most sensitive, being inhibited by all the extracts. Our results led to the conclusion that L. stoechas subsp. luisieri and L. pedunculata can be useful as new sources of natural antioxidants and antimycotic agents, providing a possible valorization of the existing biodiversity and resources of Portuguese flora.


Introduction
Southwestern Iberia includes a peculiar and unique ecosystem, the montado, which combines agrosilvopastoral system with crop cultivation, maintaining high levels of biodiversity. It is protected under the Pan-European-Natura 2000 network [1,2]. Many native species are found at the montado, including some endemics. All of them are adapted to very difficult edaphoclimatic conditions thus developing several functional and chemical features that allow their survival. Some of these plants have been used in traditional medicine and might provide bioactive natural compounds or extracts that may enable different approaches in the pharmaceutical area. This is the case of Lavandula genus (Lamiaceae) that comprises 39 species, some of them being used for centuries either dried or as essential oil for a variety of therapeutic, cosmetic, fragrance and condiment purposes. In addition to the essential oils with great economic value, several Lavandula species are also distinguished for their antioxidant potential and biological activities against the growth of a wide range of microorganisms [3][4][5][6][7][8].
L. stoechas subsp. luisieri is considered endemic in Iberia [9]. It is a perennial shrub that grows up to 20-60 cm, characterized by the opposite, narrow oblong-lanceolate gray 2 Evidence-Based Complementary and Alternative Medicine leaves of different sizes and small lilac flowers arranged in dense short-stalked spikes (0-30 mm), all of them densely covered by an indumentum that includes several types of secretory hairs [10,11]. L. pedunculata is more commonly found throughout the Iberic Peninsula and can reach up to 70 cm tall. Its long-stalked spikes, full of lilac flowers, can reach 24 cm and are covered by different kinds of glandular trichomes [10,11].
In order to make a step change on the conservation and valorization of the Portuguese montado flora, our goal is to study its potential through the botanical, phytochemical, and biological characterization of some selected plants. In previous work, we evaluated several montado plant species for their antimicrobial activity [6,12]. In those studies, a total of forty extracts with different polarities and chemical compositions, were assessed against a panel of sensitive and resistant Gram positive and Gram negative bacteria, as well as Mycobacterium smegmatis and Candida albicans. Very interesting results were obtained with the most polar extracts of P. broteroi, E. lusitanica, and Q. faginea, when tested against sensitive and resistant Staphylococcus aureus strains [6]. In the present work, the essential oils and ten extracts of Lavandula stoechas subsp. luisieri and Lavandula pedunculata, were evaluated and compared for their in vitro antioxidant and antimycotic activities. The evaluation of the antioxidant activity was performed using chemical and biochemical experiments, namely, DPPH radical scavenging, inhibition of lipid peroxidation, and DNA protection assays. The antimycotic activity was performed by the broth microdilution method against twelve fungi species, chosen primarily on the basis of their importance as opportunistic pathogens of humans. Furthermore, the total phenolics and flavonoids content of essential oils and extracts were also determined in order to try to establish a correlation between the chemical composition and the biological activities.

Plant Material and Preparation of Samples.
Aerial parts of L. stoechas subsp. luisieri and L. pedunculata were collected in 2010 during the flowering phase, from two study locations, at Southwestern and Center regions of Portugal, respectively. Plants were identified at the Lisbon Botanical Garden Herbarium (LISU) and Lisbon Agronomic Institute Herbarium (LISI), where vouchers were deposited. The plant material was dried in the shadow at room temperature and powdered before use. The crude extracts were prepared as previously reported [6]. Briefly, 100 g of powdered plant material were sequentially extracted with 300 mL of n-hexane (n-Hex), dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), methanol (MeOH) and water (H 2 O), during 24 h, at room temperature with occasional shaking. After filtration, the extracts were concentrated under reduced pressure at 40-45 ∘ C, and stored at −20 ∘ C until further use. Essential oils of the powdered air-dried aerial parts (150 g of each species) were obtained by hydrodistillation during 3 h, using a Clevenger apparatus, according to European Pharmacopoeia (Council of Europe, 2007). The essential oils were stored at −20 ∘ C until further use.

Evaluation of Total Phenolics Content.
The concentrations of phenolic compounds were assessed using the Folin-Ciocalteu reagent according to the method described by Singleton and Rossi [13], with minor modifications. 100 L of extracts (2 mg/mL) were added to 0.2 mL of Folin-Ciocalteu reagent. After 3 min, 1 mL of Na 2 CO 3 (15%) and 2 mL of water were added, and the mixture was kept in the dark at room temperature for 2 h. The absorbance was measured at 765 nm (Shimadzu UV-160 spectrophotometer). A calibration curve of gallic acid was prepared and the results were expressed as gallic acid equivalents (mg GAE/g extract) based on five points regression curve, 0.00-0.20 mg/mL, R 2 = 0.9983. Results are expressed as the mean ± SD from experiments performed in triplicate.

Evaluation of Total Flavonoids
Content. The flavonoids quantification was performed according to the technique presented by Zhishen et al. [14]. Briefly, 500 L of extracts (2 mg/mL), 2 mL of water and 150 L de NaNO 2 5% were mixed. After 5 min incubation at room temperature, in the dark, each sample was stirred with 150 L of AlCl 3 10%, 1 mL of NaOH 1 M e 2 mL of water. After 10 min incubation in the dark at room temperature the absorbance was measured at 510 nm (Shimadzu UV-160 spectrophotometer). Rutin was used as the standard for a calibration curve. The total flavonoids content was expressed in rutin equivalents (mg RE/g extract) based on four points regression curve, 0.005-0.080 mg/mL, R 2 = 0.9923. Results are expressed as the mean ± SD from experiments performed in triplicate.

Chemical and Biochemical Assays to Evaluate
Antioxidant Potential 2.3.1. DPPH Radical-Scavenging Activity. The assay was performed according to the method described by Sarikurkcu et al. [15]. A methanol solution (1.5 mL) of each sample at three different concentrations (50, 200, and 400 g/mL) was added to 9 mL of DPPH (in methanol, 60 mM). After vigorous shaking, the mixtures were incubated for 30 min in the dark. The absorbance was measured at 517 nm. For each dilution of the extracts, the DPPH scavenging activity (I, %) was determined according to the following expression:

DNA Protection
Assay. Antioxidant potential of plant extracts was evaluated as described by Inayatullah et al. [17]. Total DNA from Escherichia coli ATCC 25922 was isolated with the JetFlex Genomic DNA Purification Kit (Genomed) according to manufacturer's protocol. 5 L of DNA dissolving in buffer TE (10 mM Tris, 1 mM EDTA) was mixed with three different concentrations of extracts (10, 100 and 1000 ppm). Fenton reaction was induced by addition of H 2 O 2 (45 L, 30%) and FeSO 4 (3 L, 2 mM). Four controls (untreated DNA, DNA treated with FeSO 4 , DNA treated with H 2 O 2 , DNA treated with both FeSO 4 and H 2 O 2 ) were run simultaneously. Samples were incubated at 37 ∘ C in the dark for one hour. The reaction was stopped by addition of 2 L of 6x bromophenol blue. The samples were electrophoresed on a 1% agarose gel at 100 V and then were stained with the ethidium bromide solution and finally visualized in a gel documentation system (UVtec). Untreated DNA was used as positive control and revealed a band corresponding to the intact supercoiled DNA.

MIC Determination.
The antifungal in vitro evaluation of microorganisms susceptibility was performed by the broth microdilution standardized method described by CLSI [18]. Briefly, a stock solution of the extracts in dimethyl sulfoxide was diluted with culture medium to a final concentration of 1 mg/mL. 98-wells microplates were filled with 100 L of RPMI medium. 100 L of the extract solutions were placed in the first well and serial two fold dilutions was performed. 10 L of a suspension of microorganisms (1.0 × 10 3 -1.5 × 10 3 cells/mL) were added to each well and the plates were incubated for 24 h at 30 ∘ C. Ketoconazole and amphotericin B were used as positive controls. The minimal inhibitory concentration (MIC) was defined as the lowest concentration where no visible growth was observed.

Statistical
Analysis. The data are expressed as means ± standard deviation (SD) and individual experiments were performed in triplicate. The statistical analysis was kindly performed by Professor Humberto Ferreira (DQFT, Faculty of Pharmacy, University of Lisbon). The analyses were done using SPSS version 21.0 software. One Way Analysis of Variance (ANOVA) coupled with the Tukey's post-hoc tests were used to compare the data and to identify means with significant differences. When non statistical differences were detected the value was presented. Two-sided values of < 0.001 and < 0.05 were considered significant.

Total Phenolics and Flavonoids Content.
Phenolic compounds and flavonoids are important secondary metabolites that are synthesized by plants during development. They possess an array of health-promoting benefits and have engaged a great deal of scientific interest due to their health promoting effects as antioxidants [19]. Phenolic compounds are ubiquitous in plants and they can act as chelators and free radical scavengers especially over hydroxyl, peroxyl, superanions, and peroxynitriles. Flavonoids are highly effective scavengers of most oxidizing molecules and of several free radicals related to various pathologies. They are also able to chelate metals and activate some antioxidant enzymes [20]. Therefore, it is important to correlate the total phenolics and flavonoids content with the antioxidant potential of each plant extract. The total phenolics content expressed as mg GAE/g extract and the total flavonoids content expressed as mg RE/g extract are shown in Figures 1 and 2, respectively. As can be observed, for both Lavandula species, the concentration of total phenolics has a strong dependence on the solvent used on extraction. As expected, the amount of total phenolics was higher in polar fractions, namely in methanol and water extracts ( Figure 1). L. stoechas subsp. luisieri extracts displayed a total phenolics content ranging from 67.4 ± 0.7 (CH 2 Cl 2 extract) to 1689.3 ± 41.9 mg GAE/g (H 2 O extract). Comparatively, L. pedunculata extracts had lower content of total phenolics, which ranged from 50.0 ± 0.2 (essential oil) to 1040.3 ± 17.8 GAE mg/g (MeOH extract).
Concerning the total flavonoids content, it can be observed in Figure 2 that, excepting the essential oil, their distribution is more homogeneous among all the extracts of both species, probably due to the polarity of these compounds family. Comparing both plant extracts, L. stoechas subsp. luisieri had the highest content of flavonoids in the MeOH (506.2 ± 45.7 mg rutin/g) and H2O (458.6 ± 11.8 mg rutin/g) extracts, followed by the CH 2 Cl 2 extract (392.3 ± 7.8 mg rutin/g). Regarding L. pedunculata, n-Hex, CH 2 Cl 2 and MeOH (310.7 ± 10.6, 344.7 ± 8.8 and 371.9 ± 29.8 mg rutin/g, resp.) were the extracts that displayed high levels of flavonoids.

Antioxidant Activity.
Oxidants or reactive oxygen species (ROS) includes radicals and chemical species that are produced by living organisms during normal oxidative processes (e.g., production of energy, metabolic regulation and signal transduction) or as an answer to stress situations like temperature or light radiation. If there is an imbalance between ROS and antioxidant defense molecules, several diseases might succeed including atherosclerosis, carcinogenesis, neurodegenerative and chronic inflammatory pathologies [21]. Antioxidants are molecules that play an important role on scavenging the ROS species, preventing the initiation chain, acting on the catalytic systems that neutralized ROS (enzymes) or inactivating metal ions [22]. Based on ROS inactivation mechanism, most of the in vitro antioxidant assays have been divided in two major groups: (1) hydrogen atoms transfer (HAT) and single electron transfer (ET)      reactions based assays. In this work and according to these data, two methods for evaluating the antioxidant activity of Lavandula species were selected: the DPPH assay that is included in the ET techniques and the inhibition of lipid peroxidation assay, a HAT method [22]. According to Ou et al. [23], the HAT plays a dominant role in biological redox mechanisms, competing in vivo with the ET reactions.
The DPPH radical-scavenging activity of the ten extracts and essential oils of L. stoechas subsp. luisieri and L. pedunculata were determined and the results are shown in Figures 3 and 4. BHT and ascorbic acid were used as positive controls (20, 40 and 80 M). Three concentrations were assayed for each extract (50, 200 and 400 g/mL). All samples showed radical scavenging activity. Some of them  The results of DPPH scavenging assay revealed, for almost all the data, a very good correlation with the flavonoids content, displaying the L. pedunculata extracts the highest correlation. Nevertheless, it must be noticed that other compounds like terpenes, alkenes, carbonyl compounds (constituents for instance of the essential oil), lignans, and anthocyanines, among other compounds, may be related to the scavenging activity and their presence was not evaluated in this work.
The antioxidant activity of the extracts and essential oils of L. stoechas subsp. luisieri and L. pedunculata was also evaluated measuring the inhibition of oxidation of the exogenous linoleic acid by a thermal free radical producer (AAPH). All samples were tested at 0.2 mg/mL. BHT was used as positive control (0.2 mg/mL). From Figure 5, it was evident that, excepting the L. stoechas subsp. luisieri and L. pedunculata essential oils (14.8 ± 3.1% and 17.2 ± 1.3%, resp.) and the EtOAc extract from L. stoechas subsp. luisieri (18.1±0.8%), all the tested extracts displayed similar or higher lipid peroxidation inhibition activity than BHT (22.6 ± 2.5%). The MeOH and H 2 O extracts of both species exhibited the highest % inhibition capacity with values that range between 34.6 ± 1.5% and 49.1 ± 0.7%. It is interesting to note that the highest lipid peroxidation inhibition activity observed in the most polar extracts were correlated with those that displayed higher phenolics and flavonoids content (Figure 2).
The antioxidant potential of essential oils and extracts was further investigated by the protection of DNA against damage induced by Fenton reaction (Figure 6). This assay aims to evaluate the ability of the extracts to prevent the DNA degradation induced by the hydroxyl radicals formed after the addition of FeSO 4 and H 2 O 2 . Hydroxyl radicals from the Fenton reagent are able to react with the nucleotides in DNA and cause strand breakage, which can have an important role in many biological processes including aging, and carcinogenesis [24]. Although all the ten extracts and the essential oils were assayed at three different concentrations (10, 100 and 1000 ppm), the results obtained were rather similar to those displayed in Figure 6, which exhibit the effects on DNA protection obtained with the MeOH extracts of L. stoechas subsp. luisieri (lanes 5-7) and L. pedunculata (lanes 8-10). As can be observed, the supercoiled DNA appears as a predominant and well defined band (lane 1) and no alteration were induced by the addition of FeSO 4 (lane 2) or H 2 O 2 (lane 3). When both reagents are present along with the DNA, a random cleavage is induced by the hydroxyl radicals being that phenomenon evident by the smear observed in lane 4.
No apparent DNA damage was observed in any of the tested samples (lanes 5 to 10). In conclusion, there is evidence that all samples exhibited significant DNA protection activity and were able to preserve DNA integrity. It is noteworthy that L. angustifolia is the only Lavandula specie included in pharmaceutical formulations [25]. The antioxidant studies on the essential oils and extracts of this specie have shown discordant results. Some authors reported no activity [26,27] while others stated the existence of antioxidant activity [28]. However, all of them sustained a modest role as antioxidants, less potent than other members of the Lamiaceae family. The inconsistencies between those observations and the results presented in this work might be related to differences observed on the chemical composition of Lavandula species chemotypes. Several authors have already pointed out the fact that differences in the yield and in the chemical composition of the essential oils and extracts of aromatic species are common. These differences should be probably due to several conditions, particularly the genotype, the environment, the geographic origin, the period of harvest, the conditions of drying and the method of extraction including the scientific equipment used [5,[29][30][31].

Antimycotic Activity.
To perform the antimycotic assays twelve species were selected. Fungi used in this study were chosen primarily on the basis of their importance as opportunistic pathogens of humans. Candida spp. and Cryptococcus spp. are the major responsible for the nosocomial fungal infections according to National Nosocomial Infection Surveillance [32]. Aspergillus niger is a cosmopolitan fungus responsible for the production of mycotoxins that are hepatogenic and nephrogenic [33]. Rhodotorula and Trichosporon spp. are emerging opportunistic pathogens, particularly in haematological, immunosuppressed and/or cancer patients [34,35]. Saccharomyces cerevisiae is the most well characterized eukaryote and is unique in its broad applications [36].
The results are summarized in Table 1. It should be pointed out that a quite narrow endpoint criterion for MIC was considered and only crude extracts with MIC values lower than 100 g/mL were considered to be active [37,38]. The MIC endpoint criterion for in vitro testing of plant extracts is not consensual and several authors assume a much higher cut-off value [32,39,40]. As displayed in Table 1, all the extracts were active on one (L. pedunculata MeOH and EtOAc extracts) or more fungi species. L. stoechas subsp. luisieri exhibited the broadest activity spectra presenting MIC values ranging from 7.5 to 62.5 g/mL against seven out of twelve fungi species (A. niger, C. albicans, C. guilliermondii, S. cerevisiae, C. neoformans, R. rubra, and T. cutaneum). All the L. stoechas subsp. luisieri extracts were active on C. neoformans and S. cerevisiae, presenting MIC values between 15.5 and 31. It is noticeable the activity of essential oil and CH 2 Cl 2 extract of L. stoechas subsp. luisieri, which inhibited the growth of four fungi including C. albicans, one of the most common human pathogens.
L. pedunculata extracts were active on five species, namely C. guilliermondii, C. neoformans, R. rubra, S. cerevisiae, and T. cutaneum (MIC 15.5-62.5 g/mL). The most active against 8 Evidence-Based Complementary and Alternative Medicine these fungi was the n-Hex extract (MIC 31-62 g/mL). C. neoformans was the most sensitive, being inhibited by all the extracts. None of the tested extracts were able to inhibit the growth of C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis.
In general, it can be pointed out that the nonpolar extracts (essential oils, n-Hex, CH 2 Cl 2 ) were more active than the polar ones (AcOEt, MeOH and H 2 O). From our results it can be observed that fungi belonging to Basidiomycota Division are much more sensitive to the tested extracts than the species included in the Ascomycota Division, especially against C. neoformans. Cryptococcosis is until now a worldwide mycose that has been characterized by the development of resistance among many strains after antifungal therapies. Therefore, alternatives should be considered. This different activity observed against the species tested could be explained because these species are phylogenetically different, and the difference of fungi nature, the variation of metabolism trend, the wall organization, and the dimorphism are other factors that can affect the resistance of some species against antifungal agents comparatively to others.
Zuzarte et al. [41,42] reported the antifungal activity of the essential oils of both Lavandula species presented in this work, but not from our places of collection. Nevertheless, the results are not superimposed and this is related with the different composition of the essential oils. Often the species show different qualitative and quantitative composition, being the relative amounts of their main constituents altered, and so, changing their physical and biological characteristics and properties, which correspond to different chemotypes [43]. Feijão et al. [44,45] identified the essential oils components of the same species and populations used in the present study, through GCMS technique. L. pedunculata chemotype had fenchone as major component (almost 80%) [44], whereas Zuzarte et al. 's [41] chemotype presented only at maximum 45% of this compound. Similar conclusions were observed on chemical composition of L. stoechas subsp. luisieri essential oil. The chemotype studied in this work displayed not only different percentages of some constituents (limonene, camphor, -pinene, 1,8-cineole and fenchone) but also exhibited some components like pulegone or 5-methylene-2,3,4,4-tetramethyl cyclopentenone that were absent in the Zuzarte et al. 's essential oils [42,45].

Conclusions
Although in the past decades the chemical and bioactivity of Lavandula essential oils have been the midpoint of several published studies, minor attention has been handed out to the extracts of these plants. As part of our ongoing research on the properties and possible applications of the Portuguese montado flora, we investigated the antioxidant and antimycotic activities of two Lavandula species, namely L. stoechas subsp. luisieri and L. pedunculata. The results demonstrated that both species displayed a high content of phenolic and flavonoid compounds. Particularly, flavonoids had a great contribution to the antioxidant activity, showing a high correlation with the results obtained in the DPPH scavenging and inhibition of lipid peroxidation assays. However, the existence of other compounds with antioxidant properties cannot be disregarded. From the best of our knowledge the DNA protection assay was never performed in these taxa, neither with the extracts nor the essentials oils. In this work, it was established that the samples exhibited a very important effect of protecting the in vitro DNA damage induced by hydroxyl radicals, therefore being able to preserve DNA activity. Concerning the antimycotic assays, promising results were also obtained with the essential oils and the nonpolar extracts of both plants, when tested against several selected species belonging to Ascomycota and Basidiomycota Divisions.
In conclusion, L. stoechas subsp. luisieri and L. pedunculata can be useful as a new potential source of natural antioxidants and antimycotic agents and can be directly applied on the composition of dermocosmetic formulations as antiaging products or, as raw material for the isolation of bioactive chemical compounds. Ultimately, this could contribute to the valorization of the existing biodiversity and resources of Portuguese native species of Lavandula, promoting its sustainable use and producing novel economic revenues.