Photochemistry and Photobiology, 2014, 90: 702–708
Biochemical Composition and Antioxidant Properties of Lavandula angustifolia Miller Essential Oil are Shielded by Propolis Against UV Radiations Gismondi Angelo, Canuti Lorena, Grispo Marta and Canini Antonella* Department of Biology, University of Rome “Tor Vergata”, Rome, Italy Received 27 November 2013, accepted 17 December 2013, DOI: 10.1111/php.12229
evidences reported that UVA also may contribute to the development of melanomas and other skin cancers (5). During the evolution, animals developed the melanin production as defense mechanism against UV radiation damaging effects. Sometimes, this feature was revealed insufﬁcient and even able to contribute to melanoma initiation (6). In fact, over the last few decades, innumerable premature deaths and cases of skin pathologies have been associated with excessive UV exposure (7). Unfortunately, this alarming condition is continuously worsened by anthropogenic air pollution that, causing ozone layer depletion, facilitates UV penetration in the atmosphere (8). Sunscreens application efﬁciently reduces the risk of UV-induced skin damages. However, it is not commonly known that several sun cream components (i.e. petroleum derivatives, silicones) can be cytotoxic or able to generate harmful reactive species (9). For these reasons and to increase their protective effects, sunscreens are usually supplemented with plant extracts rich in antioxidant and UV light–capturing molecules (10). The synthesis of these compounds, typical of vegetal cell, was evolved for different functions (i.e. propagation, defense against biotic agents) but, in particular, to shield plants from UV radiations (11). Secondary metabolite light-capturing properties and correlated antiradical activity essentially derive from their characteristic chemical structures: aromatic rings, double bonds and spatial arrangement and typology of substituents (12). Essential oils are examples of plant products full of vegetal secondary compounds; in fact, secreted by glandular trichomes, they contain the lipophilic fraction of plant pythocomplex and are basically made up of terpenic and phenolic molecules (13,14). On the basis of these considerations, aim of this research was the analysis of biochemical proﬁle alteration of a vegetal product, the Lavandula angustifolia Miller essential oil that is a frequent ingredient of moisturizers, face and hand creams, barrier lotions, cosmetics and suntan creams, after exposure to UV radiations. This work also evaluated the change in lavender extract radical scavenging function, subjected to UV, both in vitro and directly on murine B16-F10 melanoma cell line. Moreover, another important goal of this study was the investigation of propolis (bee glue) addition to the essential oil as protective agent against UV photodegradation. Propolis is a resinous material derived from plant exudates that honeybees collect and reelaborate with waxes, pollens and proteins, to obtain a multifunctional substance indispensable for the construction, protection and maintenance of the hives. The choice of this natural additive was determined by its great antioxidant and preservative power; in fact, bee glue is rich in amino acids and secondary
UV radiations are principal causes of skin cancer and aging. Suntan creams were developed to protect epidermis and derma layers against photodegradation and photooxidation. The addition of antioxidant plant extracts (i.e. essential oil) to sunscreens is habitually performed, to increase their UV protective effects and to contrast pro-radical and cytotoxic compounds present in these solutions. According to these observations, in the present work, the alteration of chemical composition and bioactive properties of Lavandula angustifolia Miller essential oil, exposed to UV light, was investigated. UV induced a signiﬁcant deterioration of lavender oil biochemical proﬁle. Moreover, the antioxidant activity of this solution, in in vitro tests and directly on B16-F10 melanoma cells, greatly decreased after UV treatment. Our results also showed that essential oil was shielded from UV stress by propolis addition. Even after UV treatment, bee glue highly protected lavender oil secondary metabolites from degradation and also preserved their antiradical properties, both in in vitro antioxidant assays and in cell oxidative damage evaluations. This research proposed propolis as highly efﬁcient UV protective and antiradical additive for sunscreens, cosmetics and alimentary or pharmaceutical products containing plant extracts.
INTRODUCTION Solar radiations daily reach the earth’s surface and regulate life processes. They consist of different types of emissions that are clustered in visible light (~45%), ultraviolet one (UV, ~5%) and infrared rays (~50%). UV radiations, in turn, can be divided, according to their wavelength, in three main groups: UVA (400– 315 nm), UVB (315–280 nm) and UVC (280–100 nm) (1). Stratospheric ozone entirely (~100%) ﬁlters UVC light and partially (~95%) screens from UVB rays. Therefore, only UVA and a little percentage of UVB go right through the atmosphere (2). UVA rays deeply penetrate in animal skin up to the derma where they deteriorate connective and vascular tissues causing erythemas, inﬂammation and photoaging. UVB radiations are more aggressive than UVA, although they restrict their action to the epidermis layer; they are able to penetrate into cell nucleus producing DNA alterations and inducing tumors (3,4). Scientiﬁc *Corresponding author e-mail: [email protected]
(Canini Antonella). © 2013 The American Society of Photobiology
Photochemistry and Photobiology, 2014, 90 metabolites, such as phenolic acids, ﬂavonoids and terpens (15,16).
MATERIALS AND METHODS Sample preparation. L. angustifolia Miller essential oil and propolis were produced and kindly provided by the herbalist’s laboratory of Sarandrea Marco & C. s.r.l. (Collepardo, FR, Italy). Propolis solution was prepared dissolving 30% (w/v) of powdered propolis in ethanol, for 24 h in agitation at room temperature, and then sieving with 0.22 lm ﬁlters. For this study, lavender essential oil was used pure (EO sample) or supplemented with 1% of propolis solution (EO + P sample). These preparations were irradiated for 6 h every day per 7 days with UV light (Spectroline EN16/F UV lamp 230 V; 50 Hz; 0.15 Amp with an emission peak of 365 nm), respectively, EOuv and EO + Puv samples. P sample consisted in a dilution of propolis solution to 1%. All samples were stored in the dark at 4°C. Gas chromatography–mass spectrometry (GC-MS). Oil sample composition was studied using gas chromatography coupled to a mass spectrometer (GC-MS). The GC-MS QP2010 (Shimadzu, Japan) was equipped with a Restek Rtx-5MS column (30 m 9 250 lm i.d. 9 0.25 lm ﬁlm thickness). A volume of 2 lL of the sample diluted with n-hexane (1:20) was injected (using a split ratio of 20:1) at an inlet temperature of 280°C. The GC oven temperature was set at 60°C for 5 min, then 270°C at a rate of 4°C/min for 10 min and followed by a temperature of 240°C at a rate of 1°C/min. Helium was used as a carrier gas at a constant ﬂow of 1.0 mL/min. Spectra of singles molecules was obtained on electron impact (EI) at 70 eV, scanning from 100 to 600 m/ z. The individual compounds were identiﬁed by comparing retention times of standards of principal molecules and according to their mass spectra to those of NIST library of mass spectral data associated with the software. In vitro antioxidant assays. FRAP (2,4,6-tris(2-pyridyl)-s-triazine; Sigma Aldrich) and DPPH (stable free radical 2,2-diphenyl-1-picrylhydrazyl; Merck) antiradical tests were performed as reported in Gismondi et al. (17). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging assay was carried out according to Re et al. (18) method opportunely modiﬁed. Ten microliters of sample was diluted with 180 lL of ethanol, mixed with 2 mL of working solution (7.4 mM ABTS in H2Odd, 2.6 mM K2S2O8 in H2Odd, methanol 100%; 1:1:30 v/v/ v) and 200 lL of H2Odd and then stored at 37°C for 10 min in the dark. The test measured the capacity of the antioxidant samples to reduce the dark green radical ABTS compound to its transparent oxidized form, analyzing the absorbance change in the solution at 734 nm (UV–visible spectrophotometer Cary 50; Bio Varian). In FRAP and ABTS assays, both ascorbic acid and FeSO4 were used to obtain standard solutions (50–500 lM). Relative results were expressed as micromoles of each standard equivalent per liter of solution. Cell culture and treatments. Highly metastatic murine B16-F10 melanoma cells were maintained and propagated as described in Gismondi et al. (19). Cell treatments were performed by adding, for 6 h, 1 lL of sample solutions, diluted 1:10 (v/v) in DMSO, per mL of culture media. Control cells (CNT) were treated, for the same time, with 1 lL of DMSO. 2′-7′-dichlorodihydroﬂuorescein diacetate (DCFH-DA) test. DCFHDA assay was performed according to Barzegar and Moosavi-Movahedi (20) method with some modiﬁcations. DCFH-DA molecule is able to penetrate the cell membrane and to be metabolized to DCFH. Intracellular reactive oxygen species (ROS) oxidize this compound generating the ﬂuorescent DCF. Therefore, DCF can be directly used to detect the ROS generation in cells and to quantify intracellular oxidative stress levels. Cells were incubated, for 15 min at 37°C, in presence of DCFH-DA 10 lM. Then, they were washed three times with PBS and collected in cytoﬂuorimetric tubes for the analysis. The amount of ﬂuorescence inside cells was revealed and measured, respectively, by FACSCalibur instrument (Becton Dickinson, Milan, Italy), using an appropriate ﬁlter for ﬂuorescein (FITC, FL1-H), and CellQuest software. Western blotting analysis. Protein extraction method, western blotting protocol and detection and quantization of the antibody signals were exactly carried out as reported in Gismondi et al. (21). Rabbit polyclonal anti-b-actin, goat polyclonal antisuperoxide dismutase 1 (SOD), mouse monoclonal anticatalase (CAT) and the respective horseradish peroxidase-conjugated secondary antibodies were purchased by Sigma-Aldrich
(Milan, Italy). Mouse monoclonal antiglutathione peroxidase (GPx) was acquired by Santa Cruz Biotechnology (Dallas, TX). Detection of carbonylated proteins. Carbonylated proteins were detected using the OxyBlot Protein Oxidation Detection Kit (Millipore, Vimodrone, Italy). Brieﬂy, 25 lg of cell proteins was labeled with 2,4-dinitrophenylhydrazine (DNP) during an in vitro reaction, at room temperature, for 10 min, as reported on the manufacturer’s instructions. Then, proteins were resolved on 12% denaturing polyacrylamide gel and blotted onto a nitrocellulose ﬁlter. DNP-derivatized proteins were identiﬁed by immune reaction using a speciﬁc anti-DNP antibody. For carbonyl quantization, the signal of all labeled proteins was measured and then normalized for b-actin, used as loading control. Data analysis. Each experiment was performed at least three times and all data were expressed as means standard deviation (SD) of the three independent measurements. Analysis of the signiﬁcance was assessed by Student’s t-test (P values < 0.05 were considered signiﬁcant).
RESULTS GC-MS analysis L. angustifolia essential oil samples, supplemented or not with propolis (EO + P, EO) and subjected to UV stress (EO + Puv, EOuv), were analyzed by GC-MS, to determine their biochemical composition. GC proﬁles were shown in Data S1. In total, 58 molecules were detected and their relative abundance for each specimen was reported in Table 1. EOuv, with respect to EO, presented the reduction in the levels of some molecules, as described: 24 compounds decreased their amount between 10% and 49%, 8 substances between 50% and 89% and 2 between 90% and 99%. Moreover, 16 compounds preserved their quantity approximately unchanged (0–9% of decline) while 8 secondary metabolites were completely degraded by UV action. On the other hand, EO + Puv, compared to EO + P, did not show great changes in the abundance of its components: 53 molecules minimally presented alterations in their levels (between 0% and 9%) and 5 compounds only reduced their quantity between 10% and 49%. In this sample, no metabolite was deteriorated more than 50% by UV stress. In vitro antioxidant activity The antiradical properties of the samples were evaluated with three different in vitro assays. FRAP test indicated an antioxidant power of the EO, EOuv, EO + P, EO + Puv and P preparations, respectively, of 752.1, 434.6, 1086.2, 1048.2 and 65.6 lmol/L of ascorbic acid equivalent (Fig. 1a). Therefore, EOuv showed an antioxidant activity reduced of 43% with respect to EO, whereas EO + Puv and EO + P had a difference of only 4%. Free radical scavenging properties of EO, EOuv, EO + P, EO + Puv and P samples, according to ABTS assay, were estimated, in that order, 803, 395.7, 7236.4, 7125.6 and 89.7 lmol/L of ascorbic acid equivalent (Fig. 1b). In this case, UV radiations decreased essential oil antioxidant properties of 51%, compared to its standard condition (EOuv vs EO). Otherwise, EO + Puv, with respect to EO + P, reduced its antiradical effect of 2%. Obtained results in FRAP and ABTS tests were also expressed as lmol/L FeSO4 equivalent, conﬁrming the trend observed with ascorbic acid standard curve (Data S2). DPPH analysis (Fig. 1c) showed an IC50 value of 0.242, 1.56, 0.03, 0.041 and 0.42 lL, respectively, for EO, EOuv, EO + P, EO + Puv and P solutions: EOuv decreased its antiradical effect of 644%, with respect to EO sample, and EO + Puv of 136%, compared to EO + P.
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Table 1. Essential oil biochemical compounds. The molecules detected by GC-MS analysis were reported with their relative abundance (in arbitrary units) for each sample (EO, EOuv, EO + P and EO + Puv). In brackets, it was also indicated the quantity in percentage of metabolite levels in EOuv and EO + Puv samples, respectively, compared with EO and EO + P specimens, considered as unit (100%). Signiﬁcant changes were shown in bold. Sample Molecule 1-hexanol Alpha-pinene Camphene Beta-pinene 1-hepten-3-ol 3-octanone Beta-geraniolene 3-octanol Acetic acid, hexyl ester 1,3,8-p-menthatriene D-limonene Eucalyptol Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl () Beta-ocimene 2-furanmethanol, 5-ethenyltetrahydro-alpha 1-octanol Terpinolene 2-furanmethanol, 5-ethenyltetrahydro-alpha Octen-1-ol, acetate (-)-alcanfor 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)Borneol Terpinen-4-ol Furan,2,4-dimethyl Butyric acid, hexyl ester Alpha-terpineol Beta-myrcene 9-decen-2-one, 5-methylene Butanoic acid, 2-methyl-, hexyl ester Hexyl n-valerate Linalyl acetate 1,4-hexadiene, 3-ethyl-4, 5-dimethyl 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl) Cyclobutanecarboxylic acid, hexyl ester 2,7-octadiene-1,6-diol,2,6-dimethyl-,(Z) Neryl acetate Geraniol acetate ( )-zingiberene Aromadendrene Alpha-bergamotene Coumarin Beta-farnesene () farnesene Germacrene D Alfa-farnesene Trans-alpha-bergamotene Caryophyllene oxide Tau-cadinol Alpha-bisabolol 1,2-benzenediol, O-(5-chlorovaleryl)-O-(2-methylbenzoyl Linalyl iso-valerate Limonene oxide, transAlpha-bourbonene 4(10)-thujene Geraniol butyrate Ocimene Beta-linalool
EO 281689 386693 329143 317933 488722 585234 1160605 190831 579748 264333 2148141 6967646 2146078 3779091 295275 198882 287655 176717 552141 18295949 761960 4320213 3153247 116991 1633947 1916258 1339525 185783 233306 361673 892530320 254996 3410553 403452 133683 882814 1732791 121464 3310829 159025 182858 1642439 144000 469963 579915 100044 461268 163644 433967 113000 220678 150234 43234 80234 60789 70567 74294396
Cell oxidative stress evaluation Culture media were, respectively, supplemented with EO, EOuv, EO + P, EO + Puv and P solutions, as reported in the Materials and Methods section, and the amount of reactive species inside B16-F10 cells was measured by DCF ﬂuorescent marker
EO + P
EOuv 235886 216340 261663.5 250577 231792.5 533245 1064579 122831 487733 188053 1222205 4573768 2060185 2546983 185153 146816 177258 174136.5 505089 8524358 738260 4246037 3120303 0 1621000 1895356 1300038 90694 227775 301682.5 870974350 246567 3405519 402689 110456 865260 1698160 70977 3210077 59678 0 1376009 0 287819 0 0 380499 29789 289241 0 215593 62025 4321 0 4567 0 70527835
(83.7) (55.9) (79.5) (78.8) (47.4) (91.1) (91.7) (64.4) (84.1) (71.1) (56.9) (65.6) (95.9) (67.4) (62.7) (73.8) (61.6) (98.5) (91.5) (46.6) (96.9) (98.3) (98.9) (0) (99.2) (98.9) (97.1) (48.8) (97.6) (83.4) (97.6) (96.7) (99.9) (99.8) (82.6) (98.0) (98.0) (58.4) (96.9) (37.5) (0) (83.8) (0) (61.2) (0) (0) (82.5) (18.2) (66.7) (0) (97.7) (41.3) (9.9) (0) (7.5) (0) (94.9)
225860 235315.5 192929 197502 216962 509420 1098747.5 185034 459209 233694 1136485 6231713 2144045.5 2965759.5 285132 204098 189745.5 204957 54678 17569760 756346 4713872 4027161 198412 163984 1542303 1320234 188655 236181 335537 880765567 263456 3180430 407056 134560 880234 1728104 120879 3631015 158675 184234 1654350 142345 450345 439192 99345 417860 162345 430123 113039 215678 149282 42356 81234 61230 70234 71429817
EO + Puv 220440 239546 182234 187549 198670 498760 1048560 176540 429760 219650 1100345 5229738 2107680 2888286 265420 200567 188240 192345 53467 16547346 740234 4563246 3747066 188871 156435 1498641 1298560 178549 233212 332456 878679789 260567 3000546 402574 132456 876078 1658432 119456 3455673 150234 180456 1605677 140001 445678 322087 98657 410789 154326 399678 100345 210899 140345 39786 79456 59678 56789 69606106
(97.6) (101.8) (94.5) (94.9) (91.6) (97.9) (95.4) (95.4) (93.6) (94.0) (96.8) (83.9) (98.3) (97.4) (93.1) (98.3) (99.2) (93.8) (97.8) (94.2) (97.9) (96.8) (93.0) (95.2) (95.4) (97.2) (98.4) (94.6) (98.7) (99.1) (99.8) (98.9) (94.3) (98.9) (98.4) (99.5) (95.9) (98.8) (95.2) (94.7) (97.9) (97.1) (98.3) (99.0) (73.3) (99.3) (98.3) (95.1) (92.9) (88.8) (97.8) (94.0) (93.9) (97.8) (97.5) (80.9) (97.4)
(Fig. 2a). With respect to control cells (CNT), in that order, all samples showed a decrease in radical ions of 42.8%, 12.2%, 47.1%, 36.3% and 14.2%. In particular, EOuv and EO + Puv increased ROS levels of 53.5% and 20%, respectively, compared with EO and EO + P solutions (Fig. 2b). Moreover, the variation in the oxidative status in murine melanoma cells, after treatment
Photochemistry and Photobiology, 2014, 90 (a)
Figure 1. In vitro antioxidant assays. Results obtained for each sample in FRAP (a) and ABTS (b) tests were reported as micromoles of ascorbic acid equivalent per liter of solution. The outcomes of DPPH assay (c) were measured as IC50 (lL of sample solution). Data were reported as the mean of three different experiments SD (P < 0.001, vs EO).
Figure 2. Intracellular reactive oxygen species (ROS). After treatment for 6 h with each sample solution, cells were treated with DCFH-DA and then analyzed by ﬂow cytometry to monitor the increase in intracellular ﬂuorescence (a). Dotted line was added to facilitate the evaluation of ﬂuorescence shift in the different samples, compared to untreated cells (CNT). Relative results were expressed as% of cell ﬂuorescence variation with respect to the control (100%) (b). Results were reported as the mean of four different experiments SD (P < 0.01, vs control).
with the different preparations, was analyzed by calculating the quantity of protein carbonylation (Fig. 3). Compared to the control (CNT), EO, EO + P, EO + Puv and P samples decreased the amount of oxidative modiﬁcations of total proteins of 44.2%, 29.2%, 33.2% and 19.8%, whereas EOuv increased it of 54.8%. According to these results, UV radiations induced the increase in carbonyl groups in cell proteins of 168.5% and 0.7% in EOuv and EO + Puv samples, correspondingly, compared to their respective unstressed specimens (EO and EO + P).
tial oil and propolis (Fig. 4). CAT levels increased of 351.7%, 35%, 273.3%, 497.2% and 50.1%, respectively, in EO, EOuv, EO + P, EO + Puv and P samples, compared to control cells (CNT). In the same order, with respect to the control, SOD was modulated of 76%, 2.8%, 70.7%, 83% and 21.9%. Finally, GPx amount was enhanced of 254.2%, 121.7%, 298.9%, 379.1% and 129.9%, correspondingly, in EO, EOuv, EO + P, EO + Puv and P treated cells, compared to CNT ones.
DISCUSSION Enzymatic antioxidant levels The amount of endogenous antioxidant enzymes was analyzed in B16-F10 cells after treatment with the different samples of essen-
The application of L. angustifolia essential oil dates back to Ancient Egypt when priests used it to embalm and perfume mummies. Thenceforth, the economic and cultural value of this
Gismondi Angelo et al.
β-Actin Figure 3. Protein oxidative damage evaluation. Carbonylated proteins were detected by a speciﬁc Western blotting analysis, as reported in the Materials and Methods section. A representative immunoblot of three independent experiments, with similar results, and protein size ladder, acquired with white light scanning, were shown (a). b-actin was employed as loading control. Results were expressed as percentage variation with respect to the control (100%) (b). Data were reported as the mean of three different experiments SD (P < 0.01, vs control).
β-Actin CAT SOD GPx Figure 4. Endogenous enzymatic antioxidants. Detection of CAT, SOD and GPx protein levels by immunoblot analysis was carried out. Three independent experiments, exhibiting similar results, were performed and an example of them was shown (a). b-actin was used as loading control. Data were expressed as percentage with respect to the control (100%) (b). Each value represented the mean of the three different determinations SD (P < 0.05, vs control).
natural product was greatly ampliﬁed, especially after the ﬁrst scientiﬁc evidences about its antioxidant, anxiolytic, antifungal, sedative, spasmolytic, antihypertensive, antimicrobial, analgesic and aromatherapic properties (22,23). Recently, plant extracts (including essential oils) and speciﬁc secondary metabolites were largely employed, with great success, as additives for sunscreens, because of their UV screening property, their preservative activity against natural or induced oxidative processes and to rescue the adverse effects that some pharmaceutical products usually cause in consumers (24–28). By literature, no scientiﬁc paper reported UV-induced damages directly on vegetal preparations that should protect skin against UV stress. For these reasons, object of this research was the study of the alteration of L. angustifolia essential oil composition and antioxidant activity after exposure to UV radiations. In particular, natural solutions were subjected to UVA light because, as reported also in introduction, this is the principal component of UV radiations that go right through the atmosphere and reach earth’s surface, plants and animals (2). Moreover, UVA rays were recently associated with skin cancer development (5). Lavender essential oil, subjected (EOuv) or not (EO) to UV light (as indicated in the Materials and Methods section), was typed by GC-MS. The
overlapping of the GC molecular proﬁles (Data S1a) clearly evidenced that UV treatment (EOuv) caused a huge alteration of oil biochemical content, with respect to EO sample. Based on these preliminary observations, to identify a natural product that could be able to preserve and stabilize essential oil features from UV damages, propolis (Apis mellifera glue) was added to the samples both exposed to UV light (EO + Puv) and not (EO + P). In these two last cases, GC chromatograms did not show excessive alterations (Data S1b), suggesting a protective function of the propolis on the essential oil in presence of UV. On the other hand, propolis addition to the samples did not alter GC analysis because it was diluted, into the oil, to a ﬁnal concentration that was too much low to be detected. Lavender oil generally contains about 60–70 low-molecular-weight compounds but only few molecules are detectable in high concentrations (29). Levels and identity of each molecule, detected and quantiﬁed by GCMS in all specimens, were reported, in detail, in Table 1. These results demonstrated that UV caused a strong degradation of oil constituents. About 2/3 of secondary metabolites originally present in EO highly reduced their amount in EOuv sample; moreover, UV caused the complete deterioration of eight compounds: coumarin; () farnesene; alfa-farnesene; trans-alpha-bergamoten-
Photochemistry and Photobiology, 2014, 90 e; 1,2-benzenediol,O-(5-chlorovaleryl)-O-(2-methylbenzoyl); 4 (10)-thujene; ocimene and furan,2,4-dimethyl. UV adverse effects on essential oil biochemical composition were extraordinarily decreased or abolished in presence of propolis. In fact, EO + Puv, with respect to EO + P sample, presented only 1/12 of its metabolites that minimally reduced their concentration. The second part of the work was focused on the analysis of lavender essential oil (propolis) antioxidant property modulation after UV exposure. In accord, FRAP, ABTS and DPPH in vitro assays (Fig. 1 and Data S2) showed that essential oil greatly decreased its antiradical power when subjected to UV radiations (EOuv), with respect to EO sample. This discrepancy remarkably disappeared, or was reduced at the minimum, when the oil was enriched with propolis (EO + P vs EO + Puv). Moreover, the antioxidant activity of the propolis alone (P sample) was evaluated to demonstrate that this additive could not be directly responsible for the signiﬁcant increment of the free radical scavenging effect of the essential oil in EO + P samples. In fact, the low concentration (1%) of propolis justiﬁed the little antioxidant activity of the P sample. According to these data, propolis resulted able to extraordinarily amplify lavender oil antiradical properties (see EO + P sample) and to prevent its degradation by UV (see EO + Puv sample), probably because of the synergistic effect between bee glue polyphenolic constituents and oil terpene components (30,31). In all antioxidant assays, we also investigated the protective effects of the propolis at 2% and 3% on lavender essential oil (data not shown). These preliminary experiments produced very similar results compared to samples supplemented with propolis 1%. Therefore, since additives should not generally exceed in quantity and high concentration of propolis could affect and alter essential oil sensorial properties, integrity and function, we decided to continue our research using the minimal concentration of propolis that anyway showed a protective function. Redox reactions are essential processes in cell metabolism and their equilibrium must be always maintained constant. In fact, the unbalance of these events is correlated with the development of pathologies and even with cell senescence and death (32). Different works, in literature, reported that plant molecules were able to rescue the oxidative stress directly generated in cell cultures or artiﬁcially in industrial matrixes. For these reasons, an adequate addition of vegetal substances is more and more recommended in alimentary, cosmetic and pharmaceutical products (10,33). Therefore, the antioxidant characteristics of the various essential oil solutions were also directly investigated on B16-F10 cells, by adding sample solutions to the culture medium and measuring their ability in reducing cell oxidative stress; in particular, the amount of intracellular reactive species (Fig. 2) and the oxidative damage on proteins (Fig. 3) were evaluated. These experiments were performed on melanoma cell line because melanocytes are one of the principal targets of UV radiations and in some way in direct contact with sunscreens (34). Compared to the control (CNT), ROS concentration and carbonylated protein amount decreased in EO, EO + P and EO + Puv treated cells, while returned to high levels in EOuv cells. These results were in accordance with in vitro antioxidant assays. In fact, EO, EO + P and EO + Puv preparations showed great antiradical properties that could justify the relative decrease in reactive species and protein oxidative posttranslational modiﬁcations in cells. Moreover, the low antiradical effect of EOuv sample, evidenced by FRAP, ABTS and DPPH tests, clearly reﬂected the relative increase or stabilization of cell
oxidative stress. All these data conﬁrmed that propolis was able to shield oil molecular components from UV degradation and to preserve their integrity and antioxidant property, both in vitro than directly in cells. Finally, the levels of principal endogenous enzymatic antioxidants in B16-F10 cells, treated or not with essential oil samples, were analyzed. Our results were quite in accordance with previous ones; SOD, CAT and GPx levels increased in all samples, with respect to the control, except that in cell grown in EOuv supplemented medium, where the increase in these proteins was negligible. P treatment on B16F10 did not produce any particular effect, with respect to the control, excluding the possibility that propolis could alter the essential oil properties. According to all previous results on B16-F10, we concluded that, in EOuv sample, UV radiations maybe decomposed speciﬁc essential oil molecules that were able to penetrate into cells and operate as direct antioxidants (on cytosolic ROS and other radical compounds) or indirect ones (stimulating signal transduction pathways that activate endogenous antioxidant enzymes, such as SOD, CAT and GPx, or transcription factors regulating cell redox balance) (35–38). On the other hand, data described in this study suggested that these mechanisms could be induced in melanoma cells treated with essential oil (propolis), not subjected to UV light, or with UVstressed samples previously supplemented with bee glue. In conclusion, this study reported scientiﬁc evidences about: (1) UV photodegradation of L. angustifolia essential oil secondary metabolites; (2) reduction in lavender oil antioxidant power, in in vitro tests and directly on B16-F10 cells, after UV stress; (3) identiﬁcation of a natural matrix, the propolis, able to preserve plant extracts (i.e. essential oils), as much as sunscreens containing vegetal molecules, from UV decomposition and oxidation; (4) proposal of the propolis as highly efﬁcient UV protective and antiradical additive for cosmetics and alimentary and pharmaceutical products. Acknowledgement—We thank Mr. Roberto Targa for his technical support.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Data S1. GC proﬁles. GC biochemical proﬁles of the essential oil samples, subjected (EOuv, black line) or not (EO, grey line) to UV radiations, were reported overlaid (a). GC biochemical proﬁles of the essential oil samples, supplemented with propolis and treated (EO + Puv, black line) or not (EO + P, grey line) with UV radiations, were showed overlaid (b). Each reported proﬁle represented only one of the three independent GC analysis performed. A little slipping of the overlapped proﬁles was executed during image preparation to better show the observed differences. In the graphs, Y-axis indicated the relative percentage abundance (in arbitrary units) of the molecules, whereas Yaxis showed the chromatographic retention time of the compounds in the column. The magniﬁcations were also reported on the left of the tables (10.000.000X). Data S2. In vitro antioxidant assays. FRAP (a) and ABTS (b) results about the antiradical power of each sample were reported as lmol of FeSO4 equivalent per L of solution. Outcomes were expressed as the mean of three independent experiments SD (P < 0.001, vs EO).
Gismondi Angelo et al.
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