Influence of phenological stage on chemical composition and antioxidant activity of Salvia lavandulifolia Vahl. essential oils

Industrial Crops and Products 53 (2014) 71–77

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Influence of phenological stage on chemical composition and antioxidant activity of Salvia lavandulifolia Vahl. essential oils María Porres-Martínez, Elena González-Burgos, M. Emilia Carretero, M. Pilar Gómez-Serranillos ∗ Department of Pharmacology, Faculty of Pharmacy, University Complutense of Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 27 July 2013 Received in revised form 22 October 2013 Accepted 14 December 2013 Keywords: Essential oils Salvia lavandulifolia Chemical composition Antioxidant activity Cytoprotection Astrocytes

a b s t r a c t The influence of phenological stages (vegetative and full flowering stages) on chemical composition and antioxidant activity of essential oils of Salvia lavandulifolia were investigated. GC analysis of the essential oil samples pointed to a quantitative variability of components; terpene hydrocarbons derivatives predominate at the vegetative stage whereas oxygenated derivatives are the main components in essential oil samples from plants at the full flowering stage. Moreover, ORAC assay, used for measuring the peroxyl-radical scavenging capacity, revealed that the essential oil samples from plants collected at the full flowering stage possessed the most potent antioxidant potential. Furthermore, this study assessed whether the investigated essential oil samples protect the human astrocytoma cell line from H2 O2 -induced oxidative stress. This paper reports that pretreatments with essential oils from both phenological stages exerted a cytoprotective effect by increasing cell viability, recovering changes in cell morphology and in GSSG/GSSG + GSH ratio, inhibiting lipid peroxidation and caspase-3 activation and inducing antioxidant enzymes expression and activity. Essential oils samples from full flowering state versus flowering stage at the concentration of 50 ␮g/mL were the most effective in conferring protection. On the basis of these results, S. lavandulifolia essential oils as antioxidants are of interest for its use for industry and human health purposes. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Under physiological conditions, there exists equilibrium between the levels of free radicals production and the antioxidant defenses capacity in the body. However, when this redox equilibrium is disturbed in favor of increasing of free radicals formation, these oxidizing agents can indiscriminately damage cellular structures (lipids, DNA and proteins), leading even to cell death (Jones, 2008). The accumulation of oxidative damage products are major pathophysiological mechanisms in several neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases (Gandhi and Abramov, 2012). It has been recognized that the use of natural antioxidants may chemoprevent and protect the harmful and degradative effects free radical-induced in human body (Niki, 2012). In this context,

Abbreviations: CAT, catalase; DCFH-DA, 2 ,7 -dichlorofluorescin diacetate; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; HO-1, hemeoxygenase-1; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide; Nrf2, nuclear factor-erythroid 2; ORAC, oxygen radical absorbance capacity; ROS, reactive oxygen species; SOD, superoxide dismutase. ∗ Corresponding author. Tel.: +34 913941767; fax: +34 913942276. E-mail address: [email protected] (M.P. Gómez-Serranillos). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.12.024

essential oils from plants contain a large and complex variety of compounds, mostly hydrocarbons and oxygenated-types, with potential antioxidant properties utilizing in pharmaceutical, cosmetic, and other industries (Miguel, 2010). To name a few examples, several members of Lamiaceae family such as thyme, sage and rosemary are valuable in preventing oxidation of refined oils during storage (M’Hir et al., 2012). Moreover, essential oils obtained from some Lamiaceae plants are used in cosmetic preparations as aromatic ingredients, but they are also employed for its antioxidant properties to prevent or reduce ROS-induced photoaging of the skin (Ziosi et al., 2010). Previous works have demonstrated that the composition and activity of essential oils depend on external factors (i.e. vegetative cycle, environmental factors and cultivation practices) and internal factors (i.e. chemotype and biotype of the plant) (Duarte et al., 2010; Tundis et al., 2005). Spanish sage (Salvia lavandulifolia Vahl.) of Lamiaceae family is an annual herb originated in Iberian Peninsula, and it is widely distributed within East Spain, South France and North Africa. This aromatic shrub of about 17–100 cm high has opposite, simple green or light gray leaves and flowers with a bright blue-purple color (Sáez, 2010). In the Mediterranean area, the aerial parts of S. lavandulifolia, which are rich in essential oils, are used as herbal remedy regarding sedative, analgesic, antioxidant and antiseptic properties (Perry et al., 2003).

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The current work was conducted to evaluate the chemical composition of S. lavandulifolia essential oil and its antioxidant activity employing non cell-based and cell-based in vitro assays and investigate for the first time how both (composition and activity) may be affected by phenological stages (vegetative and full flowering stages). The findings of this study contributes to the best knowledge of the Spanish sage as antioxidant medicinal plant for its use in human health.

reader (BMG LABTECH GmbH, Offenburg, Germany). Inhibition of peroxyl radical induced oxidation was quantified by comparing the area under the curve to that of Trolox. Results were expressed as micromoles of Trolox equivalents (TE) per gram. 2.4. Cell culture

2. Material and methods

The human astrocytoma U373-MG cell line was cultured in DMEM with 10% FBS and 0.5% gentamicin (50 mg/mL) at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air.

2.1. Chemicals and plant material

2.5. MTT assay for cell viability

Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). 2,2 -Azobis (2-methylpropionamidine) dihydrochloride (AAPH), Trolox, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), 2 ,7 -dichlorofluorescein diacetate (DCFH-DA), oxidized glutathione (GSSG), reduced glutathione (GSH), hydrogen peroxide (H2 O2 ), and the antibodies for catalase (CAT), superoxide dismutase (SOD) and ␤-actin were obtained from Sigma–Aldrich (St Louis, MO, USA). The antibodies for glutathione peroxidase (GPx) and glutathione reductase (GR) were supplied by Abcam (Cambridge, UK) and for heme oxygenase1 (HO-1) and Nrf-2 factor by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Aerial parts of S. lavandulifolia Vahl. were collected at vegetative stage (VS) and at full flowering stage (FF) from the experimental field located in Aranjuez, Madrid (Spain) in March 2010 and June 2010, respectively. The specimens were identified and authenticated by Dr. Ma Ángeles Cases Capdevila from the National Institute of Agricultural and Food Research and Technology (INIA, Ministerio de Agricultura, Alimentación y Medio Ambiente, Spain).

Cell viability was measured using MTT assay as described previously (Mosmann, 1983). Briefly, cells were incubated with the MTT solution (final concentration 2 mg/mL in PBS) for 1 h at 37 ◦ C. After incubation, the dark blue formazan crystals formed in viable cells were dissolved with DMSO and their amount was determined by measuring absorbance at 550 nm using a microplate reader (Digiscan 340, Asys Hitech GmbH, Eugendorf, Austria). The results were expressed as percentage of cell viability compared to control cells (untreated cells was 100% of cell viability).

2.2. Essential oil extraction and analysis The essential oil of S. lavandulifolia was obtained using the hydrodistillation method described in The Royal Spanish Pharmacopeia (2002). Aerial parts of S. lavandulifolia were hydrodistilled for 2 h employing a Clevenger type apparatus. Oil samples were dried with anhydrous Na2 SO4 and stored in dark glass vials at 4 ◦ C until use. The analysis of the volatile components of the essential oil of S. lavandulifolia was performed with a Hewlett Packard 6890 gas chromatograph equipped with a flame ionization detector (FID) and a 30 m × 0.25 mm of 5% phenyl-methyl-silicone column. The injection volume was 2 ␮l of 1% solution of essential oil in diethyl ether, in the split mode (1:100) and the injector temperature was kept at 250 ◦ C. The oven temperature was programmed at 3 ◦ C/min from 70 ◦ C to 240 ◦ C and then held it for 2 min. Carrier gas flow is helium at 100 mL/min with 5 atm of pressure. Most constituents were identified by comparing their retention indices with those of authentic standards available in the Spanish National Institute for Agricultural and Food Research and Technology (INIA) or with those based on the references presented in the literature. 2.3. ORAC method for antioxidant activity The antioxidant activity was evaluated by measuring the peroxyl-radical scavenging capacity using ORAC method (Dávalos et al., 2004). The essential oil samples and Trolox (antioxidant reference compound) (20 ␮L) were incubated with fluorescein (120 ␮L, 70 nM) for 10 min at 37 ◦ C. Then, 60 ␮L of the radical initiator AAPH were added to the mixture. The fluorescence was read at an excitation wavelength of 485 nm and at an emission wavelength of 520 nm for 2.5 h using FLUOstar OPTIMA fluorimeter microplate

2.6. Preparation of cellular lysates (total, cytosolic and nuclear) Cells were incubated with essential oils (50 and 15 ␮g/mL) for 24 h. Then, cells were exposed to oxidative stress inductor (1 mM H2 O2 ) for 30 min. After treatments, cells were washed with PBS and scrapped off from the dish. Samples were centrifuged at 800 rpm for 5 min, obtained the pellet. Then, for total cellular extracts, pellets were resuspended in 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100 and a protease inhibitor cocktail [leupeptin (1 mg/mL), PMSF (0.5 mg/mL) and pepstatin (1 mg/mL)] for 20 min on ice. The samples were then centrifuged at 2500 rpm for 10 min at 4 ◦ C, and the supernatant was employed for subsequent experiments. For cytosolic cellular extracts, pellets were resuspended in a solution containing 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 10 mM Hepes (pH 7.9), 10 mM KCl, 5 mM NaF, 10 mM Na2 MoO4 , 1 mM NaVO4 , 10 ␮g/mL leupeptin, 1 ␮g/mL pepstatin and 0.5 mM PMSF for 15 min on ice. Following incubation time, a volume of 10 ␮L of 0.5% Nonidet P-40 was added to samples, and they were then centrifuged at 13,000 rpm for 30 s at 4 ◦ C. The supernatant was used for subsequent experiments and the pellet was employed for preparation of nuclear extracts. For nuclear extracts, pellets obtained from the preparation of cytosolic extracts were resuspended in a solution containing 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 20 mM Hepes (pH 7.9), 0.4 mM NaCl, 5 mM NaF, 10 mM Na2 MoO4 , 1 mM NaVO4 , 10 ␮g/mL leupeptin, 1 ␮g/mL pepstatin and 0.5 mM PMSF and the mixture was shaken on vortex for 30 min at 4 ◦ C. Finally, samples were centrifuged at 15,000 rpm for 5 min at 4 ◦ C, and the supernatant was collected for subsequent experiments. 2.7. Protein concentration determination The determination of protein content was done using the bicinchoninic acid method with bovine serum albumin (BSA) as standard (Smith et al., 1985). 2.8. Thiobarbituric acid reactive substances (TBARS) for lipid peroxidation Mihara and Uchiyama (1978) method was used to determinate lipid peroxidation, by measuring the formation of thiobarbituric acid reactive substances (TBARS) in cells. Total extracts were three times frozen at −80 ◦ C and defrost at room temperature. Then,

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50 ␮L of total extracts were mixed with 100 ␮L of TBA-TCA-HCl reactive, and samples were then boiled at 100 ◦ C for 10 min. Finally, the reaction was stopped on ice and samples were centrifuged at 3000 rpm for 10 min at 4 ◦ C. The absorbance was read at 535 nm using a microplate reader (Digiscan 340, Asys Hitech GmbH, Eugendorf, Austria). 2.9. Measurement of glutathione levels The glutathione levels were measured using the fluorochrome O-phthalaldehyde (OPT) as substrate (Hissin and Hilf, 1976). Briefly, GSH levels were determined by incubating total extracts with 0.1 M sodium phosphate buffer (pH = 8) and O-phthalaldehyde (1 mg/mL methanol) for 15 min in the dark at room temperature. GSSG levels were determined by incubating total extracts with N-ethylmaleimide (NEM) for 15 min at room temperature, before mixing with NaOH 0.1 N and O-phthalaldehyde (1 mg/mL methanol). Fluorescence was read at an excitation wavelength of 528 nm and at an emission wavelength of 485 nm using a microplate fluorescence reader (FLx800, Bio-Tek Instrumentation). GSH and GSSH contents in cells were expressed in ng GSSG or GSH/mg protein. Final results were expressed as Redox Index (RI = GSSG/GSH + GSSG). 2.10. Enzymatic activity assays The activity of the antioxidant enzymes CAT, SOD, GR, GPx and HO-1 were determined using specific spectrophotometric methods. For CAT activity, total cell lysates were added to a solution containing 50 mM of phosphate buffer (pH = 7.4) and 15 mM H2 O2 , and the decomposition of H2 O2 was recorded by measuring absorbance at 240 nm for 1 min (Aebi, 1984). For SOD activity, total cell lysates were added to a solution containing 50 mM Tris–DTPA buffer (pH = 8.2), and the inhibition of pyrogallol auto-oxidation was recorded by measuring absorbance at 420 nm for 1 min (Marklund and Marklund, 1974). For GPx activity, total cell lysates were added to a solution containing 50 mM phosphate buffer (pH = 7.4), 4 mM sodium azide, 1 mM EDTA, 4 mM GSH, 27 U of GR and 0.2 mM NADPH. To initiate the assay, H2 O2 was added for measuring selenium-dependent glutathione peroxidase activity and cumene for seleniumindependent GPx activity, and the decrease in absorbance was then recorded at 340 nm for 3 min (Paglia and Valentine, 1967). For GR activity, total cell lysates were added to a solution containing 50 mM phosphate buffer, 6.3 mM EDTA (pH 7.4), 80 mM GSSG and 6 mM NADPH, and the decrease in absorbance was recorded at 340 nm for 4 min with a delay time of 60 s (Barja de Quiroga et al., 1990). For HO-1 activity, total cell lysates were added to a solution containing 8 mM NADPH, 2 mM glucose-6-phosphate, 0.2 U glucose-6-phosphate-1-dehydrogenase and 2-mg liver cell cytosolic proteins of rat and 10 ␮M of hemin in potassium phosphate buffer. The reaction was incubated for 1 h at 37 ◦ C in darkness and stopped on ice. The difference in absorption between the wavelength of 464 and 530 nm was used to determine bilirubin content (Motterlini et al., 1996). On the other hand, the activity of caspase-3, apoptosis-related enzyme, was determined using a specific fluorimetric method. Briefly, 20 ␮g proteins of total cellular extracts were incubated with the substrate Ac-DEVD-AMC for 1 h at 37 ◦ C in darkness, and the fluorescence of samples was recorded at an excitation wavelength of 380 nm and at an emission wavelength of 460 nm using a fluorimeter microplate reader Bio-Tek FL 800. Results were expressed as percent of control (100%).

A

73

B

C

D

O O

Fig. 1. Molecular structures of major compounds identified in S. lavandulifolia essential oils: (A) 1,8-cineole, (B) camphor, (C) ␣-pinene and (D) ␤-pinene.

2.11. Western blotting Proteins from total extract (for antioxidant enzymes) and from nuclear and cytosolic extracts (for Nrf2 factor) were separated on 10–15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were then blocked with 5% milk dissolved in Tris-buffered saline for 1 h and half at room temperature. Following, they were immunoblotted at 4 ◦ C overnight with the primary antibodies CAT (1:1000), SOD (1:1000), GPx (1:2000), GR (1:3000), HO-1 (1:1000), Nrf-2 (1:500) and ␤-actin (1:5000). Next day, membranes were washed with PBS-Tween and then exposed to anti-mouse and anti-rabbit secondary antibodies for 2 h at room temperature. The blotted proteins were visualized using the ECL Advance detection kit (GE Healthcare, Chalfont St. Giles, UK) and quantified with Syngene Multigenius BioImaging System (Frederick, MD, USA) and Image Quants. 2.12. Statistical analysis The statistical analysis was performed using Statgraphics Plus Version 5.1 (Statpoint Technologies, Inc., Warrenton, VA). The statistically significant differences were determined by one-way ANOVA, followed by Fisher’ test at the 5% level of significance. All results were expressed as the mean ± standard deviation of at least three independent experiments. 3. Results and discussion 3.1. Constituents of essential oils of S. lavandulifolia of different phenological stages Many previous reports have indicated that the yield and chemical composition of essential oils may be significantly affected by various environmental and genetic factors (Tundis et al., 2005; Duarte et al., 2010). In the present work we have examined for the first time how phenological stages (vegetative and full flowering stages) may influence in S. lavandulifolia essential oil composition and therefore in the activity. The essential oil yield was 1.54% and 1.75% for the vegetative stage (VS) samples and full flowering (FF) samples, respectively. As Zrira et al. (2004) and Karamanos (2000) reported previously, the highest oil yield was obtained during the flowering period for plants growing in warmer and dried regions. As shown in Table 1, the GC analysis of the essential oil of S. lavandulifolia identified the presence of a total of 28 compounds in both investigated phenological stages, which account for 86.11% for vegetative stage samples and 92.15% for full flowering samples. 1,8-Cineole was found as major component (25.20% VS, 31.30% FF), followed by camphor (10.99% VS, 15.59% FF), ␤-pinene (9.77% VS, 11.83% FF), ␣-pinene (10.90% VS, 7.52% FF) and camphene (6.87% VS, 6.27% FF) (Fig. 1). Comparing these results with previous works, the composition of the essential oil of S. lavandulifolia of our study is very similar to that of samples collected in closer regions (Herraiz˜ et al., 2010). However, more differences in composition Penalver have been found with S. lavandulifolia samples collected from other regions of Spain, further than those of our study. As example,

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Table 1 Chemical composition of S. lavandulifolia essential oils of different phenological stages. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Compounds (%)

Phenological stages VS

Rt (min)

FF

Rt (min)

Sabinene ␣-Pinene Camphene ␤-Pinene Myrcene 3-Octanyl Limonene 1,8-Cineole ␥-Terpinene 1-Octanol Linalol Linalol oxide Camphor Iso-borneol Borneol Terpineol Lavandulol Verbenone Phenylacetate Linalyl acetate Carvone Thymol Bornyl acetate Neryl acetate Terpenyl acetate Sabinyl acetate ␣-Caryophyllene Nerolidol

0.11 10.90 6.87 9.77 7.62 0.20 3.38 25.20 0.24 0.20 0.53 0.08 10.99 0.19 3.67 0.49 0.20 0.15 0.12 0.50 0.10 0.68 0.12 0.05 1.78 1.06 0.18 0.73

3.515 4.876 5.175 5.791 6.014 6.657 6.982 7.238 8.096 8.764 9.106 9.726 10.936 11.117 11.657 12.497 12.716 13.92 14.452 15.008 15.670 16.346 19.006 20.730 22.033 23.431 24.600 29.800

0.06 7.52 6.27 11.83 3.88 0.19 0.11 31.30 1.36 0.20 0.36 0.17 15.59 0.24 3.51 0.77 0.16 0.09 0.07 0,34 0.06 1.06 0.10 0.06 3.00 2.49 0.06 1.30

3.517 4.860 5.169 5.800 5.985 6.657 6.975 7.245 7.864 8.764 9.122 9.185 1.098 11.172 11.657 12.516 12.722 13.917 14.454 14.997 15.672 16.364 18.990 20.437 22.067 23.482 24.541 29.800

Percentage of total (%)

86.11

92.15

Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others

38.89 45.91 0.18 0.73 0.40

31.03 59.37 0.06 1.30 0.39

␣-thujone, which has been not detected in our plant samples, has been identified as major component in the essential oil of Spanish sage from northeast Spain (Guillén et al., 1996). The chemical composition analysis pointed to a quantitative variability of components between essential oil samples obtained from plants collected at both phenological stages. Concerning the concentrations of the different plant secondary metabolites identified, the hydrocarbons derivatives (monoterpenes and sesquiterpenes) predominate at the vegetative stage versus flowering stage whereas oxygenated derivatives (monoterpenes and sesquiterpenes) are the main components in essential oil samples from plants at the full flowering stage. Among particular components, the major quantitative differences between the essential oils obtained from both phenological stages were found for camphor (10.99% VS, 15.59% FF), 1,8-cineole (25.20% VS, 31.30% FF), myrcene (7.62% VS, 3.88% FF), limonene (3.38% VS, 0.11% FF), thymol (0.68% VS, 1.06% FF), terpenyl acetate (1.78% VS, 3.00% FF), sabinyl acetate (1.06% VS, 2.49% FF) and nerolidol (0.73% VS, 1.30% FF). 3.2. Effects of essential oils of S. lavandulifolia of different phenological stages on antioxidant activity Peroxyl radicals are major oxidative products occurring during lipid peroxidation in biological models. The antioxidant activity was evaluated by measuring the peroxyl-radical scavenging capacity using ORAC method with Trolox as positive standard. ORAC method is a very common, easy, useful and fast chemical assay to determine the antioxidant properties of extracts, essential oils and pure compounds from plants by quenching peroxyl free radicals via hydrogen donation (Prior et al., 2005). Essential

oil from plants samples from full flowering stage (ORAC value 0.443 ␮mol Trolox/mg) possessed a statistically significant higher antioxidant activity than those from the vegetative stage (ORAC value 0.339 ␮mol Trolox/mg). 3.3. Effects of essential oils of S. lavandulifolia of different phenological stages on cell viability in cultured human astrocytoma Because the pathophysiology of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases are related to ROS-induced oxidative cell damage, the use of essential oils with antioxidant activities may have beneficial effects on human health in the prevention and treatment of these common age-related diseases (Gandhi and Abramov, 2012). Astrocytes are the major cell type in the central nervous system and they exert multiple key biological functions including physical, metabolic and nutritional support to neurons, regulation of microenvironment for maintaining neuronal function and guide neural migration. Because of the strong relationship between neurons and astrocytes, considerable attention has been paid to astrocytes as potential therapeutic targets in neurodegenerative diseases (Maragakis and Rothstein, 2006). Therefore, in the current work we have evaluated for the first time the antioxidant properties of essential oils of S. lavandulifolia of different phenological stages in an oxidative stress model using culture human astrocytes as cellular model (Porres-Martínez et al., 2013). First of all, the effects of different concentrations of essential oils of S. lavandulifolia on cell viability were investigated. As shown in Fig. 2A, the concentrations ranging from 50 ␮g/mL to 5 ␮g/mL did not result in cytotoxic for the cultured human astrocytoma U373-MG cells. Concentrations higher than 75 ␮g/mL caused a significant loss in viability, being these concentrations discard for further experiments. 3.4. Protective effects of essential oils of S. lavandulifolia of different phenological stages on cell viability and morphology damage H2 O2 -induced in cultured human astrocytoma Fig. 2B shows the protective activity of essential oils of S. lavandulifolia of different phenological stages on cell viability assessed by MTT assay. As shown, all non-cytotoxic concentrations tested (from 50 ␮g/mL to 5 ␮g/mL) significantly increased cell viability as compared to H2 O2 -treated cells. Hydrogen peroxide caused a cell death over 40%. However, both pretreatments essential oils protect astrocytes from H2 O2 -induced cell death. The major protective effect was observed for plant samples from full flowering stage. Further, the effect of essential oils on cell morphology was investigated using the concentrations of 15 and 50 ␮g/mL. Fig. 2C shows a loss of the typical star-shaped appearance of astrocytes after H2 O2 -treatment. Both essential oil samples and both assayed concentrations recovered cellular morphology, supporting the potential protective role of essential oils of S. lavandulifolia against the oxidative damage H2 O2 -induced. 3.5. Effects of essential oils of S. lavandulifolia of different phenological stages on oxidative stress parameters in cultured human astrocytoma Malondialdehyde (MDA), end-products of lipid peroxidation and a common oxidative stress marker, has been detected in increased levels in brains of patients suffered from free radicalrelated neurodegenerative diseases (Molina et al., 1992; Dib et al., 2002). The MDA levels were quantified using the well-establish method called as the TBARS assay. The thiobarbituric acid reacts

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Fig. 2. Effect of S. lavandulifolia essential oils of different phenological stages (A) on U373-MG cell viability, (B) against cell death H2 O2 -induced and (C) against morphological changes H2 O2 -induced. Values were expressed as mean ± SD. *p < 0.5 versus control; # p < 0.5 versus H2 O2 ; and ## p < 0.5 VS (vegetative stage) versus FF (full flowering).

Table 2 Effect of S. lavandulifolia essential oils of different phenological stages on oxidative stress biomarkers. VS (vegetative stage), FF (full flowering), TBARS (thiobarbituric acid reactive substances) and RI (redox index). Compounds

TBARS (pmol/mg protein)a

Control H2 O2 VS 50 ␮g/mL + H2 O2 VS 15 ␮g/mL + H2 O2 FF 50 ␮g/mL + H2 O2 FF 15 ␮g/mL + H2 O2

1.20 3.89 2.87 2.96 2.17 3.42

a * # ##

± ± ± ± ± ±

0.20 0.05* 0.06# 0.20# 0.70#,## 0.10

RI = GSSG/ (GSSG + GSH)a 0.32 0.37 0.34 0.34 0.27 0.31

± ± ± ± ± ±

0.03 0.05 0.05 0.06 0.03 0.08

Values were expressed as mean ± SD. p < 0.5 versus control. p < 0.5 versus H2 O2 . p < 0.5 VS (vegetative stage) versus FF (full flowering).

with MDA (2:1) resulting in a colored derivative compound detected spectrophotometrically (Mihara and Uchiyama, 1978). As shown in Table 2, exposure of U373-MG cells to H2 O2 increased significantly MDA levels over 3 fold as compared to control cells. Pretreatments with essential oils of S. lavandulifolia of different phenological stages inhibited the accumulation of lipid peroxidation, being the most active samples those corresponding to full flowering stage at the concentration of 50 ␮g/mL. Our data support the reported previous findings on the antiperoxidative potential protective role of Salvia spp. Previous works have demonstrated that Salvia officinalis, Salvia miltiorrhiza, Salvia candelabrum and Salvia ringens, among others, act as active medicinal plants in

inhibiting lipid peroxidation (Hohmann et al., 1999; Zhang and Chen, 1994; Zupkó et al., 2001). Next, we examined the effect of essential oils of S. lavandulifolia on glutathione levels. The reduced-type glutathione (GSH), through its cysteine moiety, act as a potent endogenous antioxidant compound. The ratio between the oxidized-type and reduced-type glutathione levels is often used as indicative of the cellular reducing power (Schulz et al., 2000). As shown in Table 2, GSH/(GSSG + GSH) ratio was increased in H2 O2 -treated cells than in control cells. Pretreatments with essential oils of S. lavandulifolia, at both phenological stages investigated and at both concentrations assayed, reduced the H2 O2 -induced increase in the glutathione ratio. The most active sample was the essential oil obtained from full flowering plants at the concentration of 50 ␮g/mL. 3.6. Effects of essential oils of S. lavandulifolia of different phenological stages on enzymatic activity in cultured human astrocytoma The induction of antioxidant enzymes such as CAT, SOD, GR, GPx and HO-1 are among the described mechanisms through which essential oils may prevent and protect cells from ROSinduced oxidative stress damage. CAT catalyzes the decomposition of hydrogen peroxide, SOD catalyzes the dismutation of the superoxide anion radical, HO-1 catalyzes the oxidative heme degradation and GPx catalyzes the reduction of hydroperoxides in a reaction coupled with GR (Krishnamurthy and Wadhwani, 2012). Therefore, we further investigate the effect of essential oils of S. lavandulifolia on activity and protein expression of these enzymes. As shown

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Table 3 Effect of S. lavandulifolia essential oils of different phenological stages on enzymatic activity. Compounds

CATa (UI/mg protein)

Control H2 O2 VS 50 ␮g/mL + H2 O2 VS 15 ␮g/mL + H2 O2 FF 50 ␮g/mL + H2 O2 FF 15 ␮g/mL + H2 O2

42.9 34.1 38.8 38.6 43.3 38.2

a * # ##

± ± ± ± ± ±

2.8 1.0 6.4 3.3 3.6# 1.8

SODa (UI/mg protein) 7.8 3.9 5.3 4.0 5.2 5.0

± ± ± ± ± ±

1.5 0.5* 1.4 0.5 0.1 0.7

Total GPxa (nmol NADPH/min mg protein) 86.0 75.0 75.4 82.3 82.0 94.8

± ± ± ± ± ±

1.6 0.3 0.5 2.3 9.4 4.7#

GRa (nmol NADPH/min mg protein) 21.4 19.4 21.6 24.9 31.5 32.5

± ± ± ± ± ±

1.1 1.3 2.6 3.2 6.5#,## 3.3#,##

HO-1a (pmol bilirubin/mg protein/h) 25.8 13.1 21.1 20.6 29.7 21.4

± ± ± ± ± ±

2.9 2.5* 6.7 6.4 7.3# 3.1

Caspase-3a (%) 100 201.4 91.6 66.7 77.5 70.6

± ± ± ± ± ±

4.4 2.8* 1.1# 1.1# 2.8#,## 4.1#,##

Values were expressed as mean ± SD. p < 0.5 versus control. p < 0.5 versus H2 O2 . p < 0.5 VS (vegetative stage) versus FF (full flowering).

Fig. 3. (A) Effect of S. lavandulifolia essential oils of different phenological stages on antioxidant enzymatic expression. (B) Effect on Nrf2 transcription factor. U373-MG were pretreated with essential oils (15 and 50 ␮g/mL) for 24 h, prior to H2 O2 exposure (1 mM, 30 min). Values were expressed as mean ± SD. *p < 0.5 versus control; # p < 0.5 versus H2 O2 ; and ## p < 0.5 VS (vegetative stage) versus FF (full flowering).

in Table 3 and Fig. 3A, both activity and protein expression of the antioxidant enzymes studied were decreased in H2 O2 -treated cells as compared to control cells. Pretreatments with essential oils of S. lavandulifolia induced the activation of antioxidant enzymes, protecting against the adverse effects of H2 O2 . Essential oils samples from full flowering state at the concentration of 50 ␮g/mL were the most effective in conferring protection. The induction of the expression of the genes encoding for antioxidant and detoxifying enzymes is regulated by the transcription factor Nrf2 (Ma and He, 2012). The effect of essential oils on Nrf2 nuclear ratio is shown in Fig. 3B. Data indicated that essential oils samples from full flowering state were the most effective inducing Nrf2 expression. Accumulation of H2 O2 -induced oxidative injury to lipids, nucleic acids and proteins can trigger apoptosis as common cell

death mechanism. Caspase-3 is a key enzymatic mediator in external and internal apoptosis pathways. The direct suppression of active caspase-3 contributes to the cellular protection against oxidative stress (Ozben, 2007). Table 3 shows that caspase-3 activity is increased two fold in H2 O2 -treated cells as compared to control cells. Pretreatment with both essential oil samples inhibited caspase-3 activation; plants samples from full flowering stage at 15 and 50 ␮g/mL and from vegetative stage at 15 ␮g/mL exhibited the highest inhibitory effect. 4. Conclusions As a global conclusion of this work, these findings revealed that the phenological stages (vegetative and full flowering stages) influence in both chemical composition and antioxidant activity

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