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Cyclodextrins enhance the antioxidant activity of essential oils from three Lamiaceae species
Industrial Crops and Products 70 (2015) 341–346
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Cyclodextrins enhance the antioxidant activity of essential oils from three Lamiaceae species Patrícia Costa 1 , Bruno Medronho, Sandra Gonc¸alves, Anabela Romano ∗ Faculty of Sciences and Technology (MEDITBIO), University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 16 March 2015 Accepted 18 March 2015 Available online 29 March 2015 Keywords: Essential oils Lavandula viridis Lavandula pedunculata Thymus lotocephalus Cyclodextrins Antioxidant activity
a b s t r a c t In the present study, the effect of -cyclodextrin (-CD) and 2-hydroxypropyl--cyclodextrin (HP--CD) on the antioxidant activity and storage stability of the essential oils (EOs) from Lavandula viridis, Lavandula pedunculata subsp. lusitanica and Thymus lotocephalus (Lamiaceae) was investigated. The presence of both cyclodextrins (CDs) was found to produce a remarkable enhancement on the antioxidant activity as assessed by the oxygen radical absorbance capacity (ORAC) assay. The highest free radical-scavenging effect was observed in the EO from T. lotocephalus, particularly at the highest concentration of HP--CD (i.e., 1900 molTE /gEO ). Moreover, the antioxidant activity of the EO/HP--CD complexes was found to be considerably stable over 30 days, at 4 ◦ C, possibly due to the higher solubility of the modified -CD. The results obtained suggest that these systems can be a very interesting alternative as a natural source of antioxidants for different applications such as in the food preservation field. © 2015 Elsevier B.V. All rights reserved.
Introduction Essential oils (EOs) are natural complex mixtures of volatile components with different functional group classes that are produced as secondary metabolites by aromatic plants. Because EOs have been granted GRAS (Generally Recognized as Safe) by the Food and Drug Administration (CFR 2009) these natural compounds are very sought by the pharmaceutical, cosmetic and food industries (Bakkali et al., 2008). Due to their antioxidant and antimicrobial effects, EOs have a large potential as food preservatives reducing the oxidative reactions and microbial contaminations during food handling, processing and storage (Hyldgaard et al., 2012). However, their limited water solubility, volatility and sensitivity to oxygen, light and heat can considerably decrease their bioavailability and, consequently, restrict its application. For instance, it has been reported that linalool, frequently incorporated in fragranced products, oxidizes on air exposure (autoxidation) generating allergenic oxidation products (Skold et al., 2002). In addition, chemical reactions between EOs and the food ingredients should not be neglected (Hyldgaard et al., 2012).
∗ Corresponding author. Tel.: +351 289800910; fax: +351 289818419. E-mail address: [email protected] (A. Romano). 1 Present address: LSRE-Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. http://dx.doi.org/10.1016/j.indcrop.2015.03.065 0926-6690/© 2015 Elsevier B.V. All rights reserved.
The association of EOs with different systems has been indicated as an efficient strategy to limit the above-mentioned constraints (Pinho et al., 2014). For this purpose, a vast role of molecules has been extensively used as association agents, such as chitosan (Zivanovic et al., 2005), proteins (Baranauskiene et al., 2006), gums (Chang et al., 2006) and starch-based compounds (Hadaruga et al., 2007; Ayala-Zavala et al., 2008; Petrovic et al., 2010; Ciobanu et al., 2012). Among them, cyclodextrins (CDs) are of particular interest due to their unique structural features and amphiphilic properties. CDs are natural cyclic oligosaccharides with a truncated cone shape containing 5 or more d-glucopyranose units netted through covalent bonds by C1 and C4 carbons (Fig. 1A). The -CD, with 7 sugar units, has been the most commercially attractive (more than 95% of CDs produced and consumed) because of its simple synthesis, availability and price. Nevertheless, at room temperature, -CD has a relatively low solubility in water (1.8 wt%) when compared to other CDs (Szente and Szejtli, 1999). This low solubility has triggered the synthesis of new CD derivatives. While possessing a safe biological profile, 2-hydroxypropyl--cyclodextrin (HP--CD) is a -CD derivative with improved water solubility (ca. >70 wt%) despite being structurally less hydrophilic. Thus, the verified increase in water solubility due to the hydrophobic substitution may come as unexpected at a first glance. However, this effect comes from the bulky hydroxypropyl substitution which presumably destabilizes the crystalline state (Szente and Szejtli, 1999). In fact, this effect is rather general and a similar behavior can be identified, for example,
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Fig. 1. Chemical structure (A) and schematic representation of the truncated conical shape (B) of cyclodextrins (adapted from Pazos et al., 2010). The “n” represents the number of repeating sugar units. In the modified HP--CD, the OH groups at the outer hydrophilic surface are substituted by hydroxypropyl moieties.
in the case of cellulose and its derivatives (Medronho and Lindman, 2014). The CD cavity is lined by the hydrogen atoms and the glycosidic oxygen bridges. The nonbonding electron pairs of the glycosidic oxygen bridges are directed towards the inside of the cavity, which results in a high electron density, lending it some Lewis base character. As a result of this spatial arrangement of the functional groups in the CDs molecules, the cavity is relatively hydrophobic while the external surfaces are hydrophilic (Fig. 1B). Therefore, CDs are expected to considerably change the physicochemical properties of the solutions, forming for instance, inclusion complexes with a wide range of molecules (i.e., flavoring agents, fragrances, EOs, metallic cations, pesticides) (Marques, 2010). Lamiaceae is a diverse and widespread family of plants, where many of which are of great economic importance, particularly due to their EOs production. Among others, Lavandula and Thymus genera comprise popular aromatic plants extensively used as flavoring agents, pleasant fragrances, or in culinary and medical applications (Boelens, 1995; Sáez and Stahl-Biskup, 2002). Their EOs are of great economic value and, therefore, strictly regulated by international ISO standards (ISO14715, 2010; ISOTC54-ISO/CD8902, 2007; ISOTC54N-ISO/WD4719, 2009). Though, there are species from these genera with a more restrict distribution and less explored, such is the case of Lavandula viridis L’Hér, Lavandula pedunculata subsp. lusitanica (Chaytor) Franco and Thymus lotocephalus G. López & R. Morales. Previous phytochemical and biological studies of their EOs highlighted the abundance of oxygen-containing monoterpenes and revealed the presence of antioxidants and cholinesterase
inhibitors (Costa et al., 2012a,b, 2013). In addition, EOs from L. viridis, T. lotocephalus and other subspecies of L. pedunculata have been shown to possess remarkable antimicrobial properties (Faleiro et al., 2003; Zuzarte et al., 2009, 2011). This is particularly important for applications in the food preservation domain (Singh et al., 2011; Hyldgaard et al., 2012). Therefore, and considering the impressive biological potential of EOs from the cited species, this work intends to investigate the effect of -CD and HP--CD on their antioxidant activity and storage stability. 2. Materials and methods 2.1. Chemicals Fluorescein was obtained from Panreac (Barcelona, Spain). 2,2 Azobis(2-methylpropionamidine) dihydrochloride (AAPH) and 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Acros Organics (Geel, Germany). Cyclodextrin and 2-hydroxypropyl--cyclodextrin were purchased from Sigma–Aldrich (Steinheim, Germany). All chemical were used as received. 2.2. Plant material and preparation of the EOs The aerial parts of L. viridis plants were collected during the flowering period in the area of São Bartolomeu de Messines (Algarve, Portugal). A voucher specimen was deposited in the herbarium of the Botanical Garden of the University of Lisbon under the
Fig. 2. (A) Fluorescence decay curves induced by AAPH of blank (black circles) and Trolox (50 M) (white circles). (B) Linear regression of Net AUC of Trolox at different concentrations tested.
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number LISU 173992. The aerial parts of L. pedunculata and T. lotocephalus plants were collected during the flowering period in Campus de Gambelas (Algarve, Portugal) and vouchers specimens were deposited in the herbarium of the University of Algarve under the number ALGU 8080 and 8081, respectively. The plant material was dried at room temperature until constant weight, then blended to powder to achieve a mean particle size lower than 2 mm using a sieve (Retsch, Germany) and finally stored at −20 ◦ C until further use. The EOs from air-dried plant material (50 g) were isolated by hydrodistillation for 3 h in a Clevenger-type apparatus, according to the method recommended by the European Pharmacopoeia (Council of Europe, 2007). Since the extracted EOs were virtually water-free it was not necessary the use of anhydrous sodium sulfate or any other “drying” procedure.
The inclusion complexes were prepared by mixing a fixed amount of EOs from L. viridis, L. pedunculata or T. lotocephalus (i.e., 31 g/ml) with different concentrations of -CD (i.e., 0, 2.25, 4.5 and 9 mg/ml) and HP--CD (i.e., 0, 2.25, 4.5, 9 and 18 mg/ml). The EOs and CDs solutions were prepared in ethanol and water, respectively. The mixtures were sealed to avoid loss of volatiles and shaken for 2 h at room temperature on an orbital shake to allow for complex formation (VWR Collection Tube Rotator, EU plug) and, afterwards, stored at 4 ◦ C and protected from light during 30 days.
μmol TE / gEO
2.3. Association of EOs and CDs
2.4. Evaluation of antioxidant activity by oxygen radical absorbance capacity (ORAC) assay The antioxidant capacity was evaluated as described by Gillespie et al. (2007) using fluorescein as the fluorescent probe and AAPH as a peroxyl radical generator. In ORAC assay, a fluorescent probe is used to compete with antioxidants for peroxyl radicals generated by thermal decomposition of AAPH (Prior et al., 2005). A black microplate (NUNC, Rochester, New York, USA) was loaded with fluorescein (0.08 M) and EO/CD complex, Trolox standard (6.25–50 M), phosphate buffer or empty CDs (blanks). After a 10 min incubation period at 37 ◦ C, the reaction was initiated by adding AAPH (150 mM) to each well. The reduction in fluorescence was determined by reading fluorescein excitation at 485 nm and emission at 530 nm, every minute, for 1 h. The EO/CD complex stability was evaluated over a 30 day period following the alluded procedure. All experiments were carried out in triplicate. The ORAC value for each sample was calculated by subtracting the area under the blank curve from the area under the sample curve to obtain the net area (Net AUC). Such procedure is illustrated in Fig. 2 where one can see (a) typical fluorescence decay curves, induced by AAPH in the absence (blank) and presence of Trolox (e.g., 50 M) and (b) the linear regression of Net AUC of Trolox at the different concentrations tested. The results are expressed as Trolox equivalents per gram of EO (TE/gEO ).
2.5. Statistical analysis The data is expressed as the mean ± standard error and was subjected to one-way analysis of variance (ANOVA). To analyze the effect of CD concentration and storage period on the antioxidant activity, means were compared by Duncan’s New Multiple Range Test. All statistical analysis was carried out using the SPSS statistical package for Windows (release 18.0; SPSS Inc., Chicago, IL, USA).
β Fig. 3. Effect of the concentration of -CD on the antioxidant activity of the Lamiaceae essential oils. No antioxidant activity was observed for free EOs and CDs alone at the tested concentrations.
3. Results and discussion 3.1. Antioxidant activity of EOs complexed with CDs The effect of -CD on the antioxidant capacity of the EOs is showed in Fig. 3. It is important to highlight that neither the EOs nor the CDs alone show free radical-scavenging ability at the tested concentrations. In fact, to detect any antioxidant activity in its free state, a much higher concentration of EOs would be needed as proved in previous studies (i.e., up to sixteen times higher than the one used in this work) (Costa et al., 2012a,b). Aiming a potential application in the food domain, it was intended to reduce the concentration of the active ingredient (EOs) as much as possible while achieving a remarkable antioxidant activity via EOs/CDs complexation. From Fig. 3, it is clear that when the EOs are in the presence of -CD, a remarkable antioxidant activity is observed despite the use of a much smaller quantity of EO. The EOs from L. viridis and T. lotocephalus were found to be the most active radical scavengers in the presence of -CD. Moreover, the highest antioxidant activity of L. pedunculata and T. lotocephalus was observed in a higher concentration of -CD used (i.e., 9 mg/ml) whereas no significant effect of the -CD concentration was observed in the L. viridis EO. Likewise, the activity of all the studied EOs was enhanced by the presence of HP--CD and, in this case, as can be seen in
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μmol TE / gEO
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β Fig. 4. Effect of the concentration of HP--CD on the antioxidant activity of the Lamiaceae essential oils. No antioxidant activity was observed for free EOs and CDs alone at the tested concentrations.
Fig. 4, the effect was significantly (p < 0.05) more pronounced in the T. lotocephalus EO, particularly at the highest concentration of HP--CD (i.e., 18 mg/ml). In this case, when the concentration of HP--CD increased from 2.25 mg/ml to 18 mg/ml, the free radicalscavenging potential almost doubled, reaching the highest ORAC value observed (i.e., 1900 molTE /gEO ). On the other hand, no significant effect of the HP--CD concentration was detected in the activity of the Lavandula species. As mentioned above, the activity of CDs alone was evaluated and no effect was observed for all the studied concentrations. This lack of antioxidant activity of CDs is expected and has also been reported in previous studies (Lucas-Abellan et al., 2008). These results suggest that the increase on the antioxidant activity is linked to the enhancement of solubility of the EOs since, in its free state and at the same low concentration of 31 g/ml, no measurable activity was detected. Furthermore, due to the higher solubility in water, the process of complexation with HP--CD is much facilitated compared with the native -CD. Different studies have reported the association of some volatiles found in the studied species and CDs complexes. For instance, Astray et al. (2010) investigated the binding constants of different flavors, including cineole and camphor, to ␣- and -CDs and concluded that the main driving force for CDsflavor complex formation is related to the amphiphilic features of the flavors. Additionally, it has been reported that the majority of
volatiles have a 1:1 (volatile:CD) stoichiometry (Szente and Szejtli, 2004; Ciobanu et al., 2013). The inclusion complex ability of CDs depends on the geometric accommodation in the internal cavity and thermodynamic interactions with the guest (Del Valle, 2004). From this point of view, no significant differences are expected to be found between the CDs used in this work, since both possess the same number of sugar units. On the other hand, and additionally to the entrapment in the internal cavity of CD (can be totally or partially), the hydroxyl groups located on the outer surface of the CDs can also interact with guest molecules (Loftsson and Duchene, 2007). Both CDs have this capacity, however the chemical structure of the hydroxypropyl moieties in HP--CD not only makes this derivative more soluble than the native -CD but also changes the surface’s chemistry. Therefore, less polar compounds, which have not been trapped in the HP--CD cavity (and do not strongly interact with simple hydroxyl groups), are expected to be absorbed at the HP--CD external surface. This will in turn increase the EOs solubility and consequently enhance the antioxidant activity. The EOs of L. viridis, L. pedunculata and T. lotocephalus were previously characterized and the main compounds identified are showed in Table 1. Camphor was the main constituent identified in the EOs of L. viridis (Costa et al., 2012a) and L. pedunculata (Costa et al., 2013), whereas linalool was the major compound in the EO of T. lotocephalus (Costa et al., 2012b). Camphor and linalool are high-value aromatic compounds for many industries and both have previously demonstrated radical scavenging abilities (Celik and Ozkaya, 2002; Zaouali et al., 2010). Recently, their interactions with CDs and -CD polymers in Lavandula angustifolia EO were investigated (Ciobanu et al., 2012). Authors reported that -CD has more affinity for linalool and camphor than for ␣- or ␥-CD. Moreover, authors found that camphor has apparently a better geometric accommodation inside the CD cavity and, consequently, a greater stability than that of linalool. Nevertheless, there are several examples (Costa et al., 2012a; Nakatsu et al., 2000) indicating that the minor compounds cannot be neglected as it may positively contribute to the activity of the whole EO. Since the overall effect of the EOs results from the synergy of all compounds it is not trivial to rationalize the higher antioxidant activity generally observed in this work for the EO obtained from the T. lotocephalus. Therefore, and due to the fact that the EOs are multicomponent systems, the determination of their mechanism of association with CDs, association constants and association stoichiometries are quite laborious. Although, these are relevant questions for a complete understating of the system, such is outside the scope of the present paper. These pertinent issues are expected to be addressed and clarified in other publication where sound nuclear magnetic resonance methodologies will be explored (Medronho et al., 2014). 3.2. Storage stability of EO–CD complexes In food quality, time and temperature are important factors. Fresh food products, e.g., dairy products, fruits and vegetables, are known to have a short shelf life (typically less than 30 days) requiring some kind of protection to avoid their fast deterioration during storage which is typically caused by different events such as proliferation of microorganisms, enzymes and oxidation (Del Nobile et al., 2012). Natural antimicrobial and antioxidant compounds have been added to food to prevent, to some extent, degradation (Ayala-Zavala and Gonzalez-Aguilar, 2010) but their activity can also be susceptible to chemical and biological modifications during storage process (Marques, 2010). Even at refrigerator temperatures (ca. 4–5 ◦ C), perishable food will deteriorate, but this process is considerably delayed due to the slow growth rate of the microorganisms already present in the product. It was seen in the previous section that, given the proper conditions, the association between CDs and EOs is beneficial when a remarkable enhancement of the
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Table 1 Main compounds (relative %) identified in L. viridis, L. pedunculata and T. lotocephalus essential oils. L. viridisa
L. pedunculatac
T. lotocephalusb
Camphor (31.59%) 1,8-Cineole (21.31%) Verbenone (3.54%) Norinone (2.12%) trans-Linalool oxide (1.78%) Linalool (1.75%) Myrtenol (1.77%) Myrtenal (1.22%) Caryophyllene oxide (1.21%)
Camphor (40.8%) Fenchone (38.2%) ␣-Fenchol (2.6%) Linalool (2.0%) Verbenone (1.5%) Eremophyllene (1.4%) ␣-Cadinol (1.3%) p-Cymen-8-ol (1.3%) 1,8-Cineole (0.9%)
Linalool (10.43%) Caryophyllene oxide (8.71%) Camphor (7.97%) Borneol (5.62%) ␣-Terpineol (4.46%) 1,8-Cineole (3.82%) Globulol (3.70%) Viridiflorol (2.69%) Geranyl acetate (2.20%)
a b c
Costa et al. (2012b) Costa et al. (2012a) Costa et al. (2013)
2500
L. viridis
2000 1500 1000 500 0 2500
L. pedunculata
2000
μmol TE / gEO
μmol TE / gEO
1500 1000 500 0 2500
T. lotocephalus
2000 1500 1000 500 0 0
6
12
18
24
30
Time (days) Fig. 5. Storage stability of the Lamiaceae essential oils for a -CD concentration of 2.25 mg/ml (black circles) and 9 mg/ml (white circles). Samples were stored at 4 ◦ C.
antioxidant activity is achieved. However, if EOs want to be recognized as valuable alternatives to enhance the life of a certain fresh product, it is thus relevant to know if the EO–CD complex formed is stable over time and, more importantly, if it maintains its high antioxidant activity when stored at lower temperatures. In this context, the effect of -CD at different concentrations (2.25 and 9 mg/ml) on the activity of the EOs was assessed and the results are displayed in Fig. 5. After 30 days, regardless of the concentration of -CD, the Lavandula spp. showed a moderate decrease on the antioxidant activity. Conversely, the EO of T. lotocephalus
Fig. 6. Storage stability of the Lamiaceae essential oils for a HP--CD concentration of 2.25 mg/ml (black circles) and 18 mg/ml (white circles). Samples were stored at 4 ◦ C.
revealed high storage stability with no statistical significant change in activity after 30 days, particularly when associated with -CD at the highest studied concentration. When HP--CD was used, all species showed a remarkable stable activity profile over 30 days (Fig. 6). Overall, the activity was observed to be more stable over time in the presence of HP--CD than with -CD. It is expectable that these results are related not only to the general higher solubility of HP--CD but also with predictable favored interactions between the hydroxypropyl moieties and the EOs constituents.
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4. Conclusions In this study, it is reported for the first time the effect of two CDs, -CD and HP--CD, in the antioxidant activity of the EOs from L. viridis, L. pedunculata and T. lotocephalus. Alone, both EOs and CDs did not show any measurable antioxidant capacity for the investigated concentrations. However, when the Lamiaceae EOs are associated with -CD or HP--CD, an impressive antioxidant activity was observed. Most likely, this behavior is due to an enhancement in solubility of some of the less polar compounds of the EOs but the mechanism is not clear yet. For the lower concentrations of CDs, little effect on the antioxidant activity of Lavandula EOs was detected. On the other hand, the activity increased for the highest CD concentration tested (i.e., 9 mg/ml for -CD and 18 mg/ml for HP--CD). The modified CD, HP--CD, was shown to have a more pronounced effect in EOs activity, in particular for ;1;T. lotocephalus. All formulations with HP--CD are considerably stable over time showing a remarkable high activity after 30 days of storage. In the case of -CD, the complexes seem less stable and the activity slightly decreased during the same period. Nevertheless, and in the worst case, the activity measured at the end of the storage period is still quite appreciable. It was clearly demonstrated that CDs (particularly HP--CD) have a significant positive impact on the antioxidant activity of EOs from three Lamiaceae species in aqueous systems. Since these EOs can be seen as a natural source of antioxidants and antimicrobial compounds, it is believed that these systems are rather good candidates to be used particularly in food preservation applications. Acknowledgements The authors acknowledge support from the Portuguese Foundation for Science and Technology (FCT, project PTDC/AGRTEC/4049/2012, post-doc grants assigned to Patrícia Costa: SFRH/BPD/93108/2013, Bruno Medronho: SFRH/BPD/74540/2010 and Sandra Gonc¸alves: SFRH/BPD/84112/2012). References Astray, G., Mejuto, J.C., Morales, J., Rial-Otero, R., Simal-Gandara, J., 2010. Factors controlling flavors binding constants to cyclodextrins and their applications in foods. Food Res. Int. 43, 1212–1218. Ayala-Zavala, J.F., Gonzalez-Aguilar, G.A., 2010. Optimizing the use of garlic oil as antimicrobial agent on fresh-cut tomato through a controlled release system. J. Food Sci. 75, M398–M405. Ayala-Zavala, J.F., Soto-Valdez, H., Gonzalaz-Leon, A., Alvarez-Parrilla, E., Martin-Belloso, O., Gonzalez-Aguilar, G.A., 2008. Microencapsulation of cinnamon leaf (Cinnamomum zeylanicum) and garlic (Allium sativum) oils in beta-cyclodextrin. J. Inclusion Phenom. Macrocyclic 60, 359–368. Bakkali, F., Averbeck, S., Averbeck, D., Waomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. Baranauskiene, R., Venskutonis, P.R., Dewettinck, K., Verhe, R., 2006. Properties of oregano (Origanum vulgare L.), citronella (Cymbopogon nardus G.) and marjoram (Majorana hortensis L.) flavors encapsulated into milk protein-based matrices. Food Res. Int. 39, 413–425. Boelens, M.H., 1995. Chemical and sensorial evaluation of Lavandula oils. Perfumer Flavorist 3, 23–51. Celik, S., Ozkaya, A., 2002. Effects of intraperitoneally administered lipoic acid, vitamin E, and linalool on the level of total lipid and fatty acids in guinea pig brain with oxidative stress induced by H2 O2 . J. Biochem. Mol. Biol. 35, 547–552. Chang, C.P., Leung, T.K., Lin, S.M., Hsu, C.C., 2006. Release properties on gelatin-gum arabic microcapsules containing camphor oil with added polystyrene. Colloid Surf. B 50, 136–140. Ciobanu, A., Landy, D., Fourmentin, S., 2013. Complexation efficiency of cyclodextrins for volatile flavor compounds. Food Res. Int. 53, 110–114. Ciobanu, A., Mallard, I., Landy, D., Brabie, G., Nistor, D., Fourmentin, S., 2012. Inclusion interactions of cyclodextrins and crosslinked cyclodextrin polymers with linalool and camphor in Lavandula angustifolia essential oil. Carbohydr. Polym. 87, 1963–1970. Council of Europe (COE) – European Directorate for the Quality of Medicines (EDQM), E.P. 2007. Costa, P., Goncalves, S., Grosso, C., Andrade, P.B., Valentao, P., Bernardo-Gil, M.G., Romano, A., 2012a. Chemical profiling and biological screening of Thymus
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Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53, 4290–4302. Sáez, F., Stahl-Biskup, E., 2002. Essential oil polymorphism in the genus Thymus. In: Stahl-Biskup, E., Sáez, F. (Eds.), Thyme: The Genus Thymus. Taylor and Francis, London and New York, pp. 124–143. Singh, G., Kapoor, I.P.S., Singh, P., 2011. Effect of volatile oil and oleoresin of anise on the shelf life of yogurt*. J. Food Process. Preserv. 35, 778–783. Skold, M., Borje, A., Matura, M., Karlberg, A.T., 2002. Studies on the autoxidation and sensitizing capacity of the fragrance chemical linalool, identifying a linalool hydroperoxide. Contact Dermatitis 46, 267–272. Szente, L., Szejtli, J., 1999. Highly soluble cyclodextrin derivatives: chemistry, properties, and trends in development. Adv. Drug Deliver Rev. 36, 17–28. Szente, L., Szejtli, J., 2004. Cyclodextrins as food ingredients. Trends Food Sci. Tech. 15, 137–142. Zaouali, Y., Bouzaine, T., Boussaid, M., 2010. Essential oils composition in two Rosmarinus officinalis L. varieties and incidence for antimicrobial and antioxidant activities. Food Chem. Toxicol. 48, 3144–3152. Zivanovic, S., Chi, S., Draughon, A.F., 2005. Antimicrobial activity of chitosan films enriched with essential oils. J. Food Sci. 70, M45–M51. Zuzarte, M., Goncalves, M.J., Cavaleiro, C., Canhoto, J., Vale-Silva, L., Silva, M.J., Pinto, E., Salgueiro, L., 2011. Chemical composition and antifungal activity of the essential oils of Lavandula viridis L’Her. J. Med. Microbiol. 60, 612–618. Zuzarte, M., Goncalves, M.J., Cavaleiro, C., Dinis, A.M., Canhoto, J.M., Salgueiro, L.R., 2009. Chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav. Chem. Biodivers. 6, 1283–1292.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Cyclodextrins enhance the antioxidant activity of essential oils from three Lamiaceae species Patrícia Costa 1 , Bruno Medronho, Sandra Gonc¸alves, Anabela Romano ∗ Faculty of Sciences and Technology (MEDITBIO), University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 16 March 2015 Accepted 18 March 2015 Available online 29 March 2015 Keywords: Essential oils Lavandula viridis Lavandula pedunculata Thymus lotocephalus Cyclodextrins Antioxidant activity
a b s t r a c t In the present study, the effect of -cyclodextrin (-CD) and 2-hydroxypropyl--cyclodextrin (HP--CD) on the antioxidant activity and storage stability of the essential oils (EOs) from Lavandula viridis, Lavandula pedunculata subsp. lusitanica and Thymus lotocephalus (Lamiaceae) was investigated. The presence of both cyclodextrins (CDs) was found to produce a remarkable enhancement on the antioxidant activity as assessed by the oxygen radical absorbance capacity (ORAC) assay. The highest free radical-scavenging effect was observed in the EO from T. lotocephalus, particularly at the highest concentration of HP--CD (i.e., 1900 molTE /gEO ). Moreover, the antioxidant activity of the EO/HP--CD complexes was found to be considerably stable over 30 days, at 4 ◦ C, possibly due to the higher solubility of the modified -CD. The results obtained suggest that these systems can be a very interesting alternative as a natural source of antioxidants for different applications such as in the food preservation field. © 2015 Elsevier B.V. All rights reserved.
Introduction Essential oils (EOs) are natural complex mixtures of volatile components with different functional group classes that are produced as secondary metabolites by aromatic plants. Because EOs have been granted GRAS (Generally Recognized as Safe) by the Food and Drug Administration (CFR 2009) these natural compounds are very sought by the pharmaceutical, cosmetic and food industries (Bakkali et al., 2008). Due to their antioxidant and antimicrobial effects, EOs have a large potential as food preservatives reducing the oxidative reactions and microbial contaminations during food handling, processing and storage (Hyldgaard et al., 2012). However, their limited water solubility, volatility and sensitivity to oxygen, light and heat can considerably decrease their bioavailability and, consequently, restrict its application. For instance, it has been reported that linalool, frequently incorporated in fragranced products, oxidizes on air exposure (autoxidation) generating allergenic oxidation products (Skold et al., 2002). In addition, chemical reactions between EOs and the food ingredients should not be neglected (Hyldgaard et al., 2012).
∗ Corresponding author. Tel.: +351 289800910; fax: +351 289818419. E-mail address: [email protected] (A. Romano). 1 Present address: LSRE-Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. http://dx.doi.org/10.1016/j.indcrop.2015.03.065 0926-6690/© 2015 Elsevier B.V. All rights reserved.
The association of EOs with different systems has been indicated as an efficient strategy to limit the above-mentioned constraints (Pinho et al., 2014). For this purpose, a vast role of molecules has been extensively used as association agents, such as chitosan (Zivanovic et al., 2005), proteins (Baranauskiene et al., 2006), gums (Chang et al., 2006) and starch-based compounds (Hadaruga et al., 2007; Ayala-Zavala et al., 2008; Petrovic et al., 2010; Ciobanu et al., 2012). Among them, cyclodextrins (CDs) are of particular interest due to their unique structural features and amphiphilic properties. CDs are natural cyclic oligosaccharides with a truncated cone shape containing 5 or more d-glucopyranose units netted through covalent bonds by C1 and C4 carbons (Fig. 1A). The -CD, with 7 sugar units, has been the most commercially attractive (more than 95% of CDs produced and consumed) because of its simple synthesis, availability and price. Nevertheless, at room temperature, -CD has a relatively low solubility in water (1.8 wt%) when compared to other CDs (Szente and Szejtli, 1999). This low solubility has triggered the synthesis of new CD derivatives. While possessing a safe biological profile, 2-hydroxypropyl--cyclodextrin (HP--CD) is a -CD derivative with improved water solubility (ca. >70 wt%) despite being structurally less hydrophilic. Thus, the verified increase in water solubility due to the hydrophobic substitution may come as unexpected at a first glance. However, this effect comes from the bulky hydroxypropyl substitution which presumably destabilizes the crystalline state (Szente and Szejtli, 1999). In fact, this effect is rather general and a similar behavior can be identified, for example,
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Fig. 1. Chemical structure (A) and schematic representation of the truncated conical shape (B) of cyclodextrins (adapted from Pazos et al., 2010). The “n” represents the number of repeating sugar units. In the modified HP--CD, the OH groups at the outer hydrophilic surface are substituted by hydroxypropyl moieties.
in the case of cellulose and its derivatives (Medronho and Lindman, 2014). The CD cavity is lined by the hydrogen atoms and the glycosidic oxygen bridges. The nonbonding electron pairs of the glycosidic oxygen bridges are directed towards the inside of the cavity, which results in a high electron density, lending it some Lewis base character. As a result of this spatial arrangement of the functional groups in the CDs molecules, the cavity is relatively hydrophobic while the external surfaces are hydrophilic (Fig. 1B). Therefore, CDs are expected to considerably change the physicochemical properties of the solutions, forming for instance, inclusion complexes with a wide range of molecules (i.e., flavoring agents, fragrances, EOs, metallic cations, pesticides) (Marques, 2010). Lamiaceae is a diverse and widespread family of plants, where many of which are of great economic importance, particularly due to their EOs production. Among others, Lavandula and Thymus genera comprise popular aromatic plants extensively used as flavoring agents, pleasant fragrances, or in culinary and medical applications (Boelens, 1995; Sáez and Stahl-Biskup, 2002). Their EOs are of great economic value and, therefore, strictly regulated by international ISO standards (ISO14715, 2010; ISOTC54-ISO/CD8902, 2007; ISOTC54N-ISO/WD4719, 2009). Though, there are species from these genera with a more restrict distribution and less explored, such is the case of Lavandula viridis L’Hér, Lavandula pedunculata subsp. lusitanica (Chaytor) Franco and Thymus lotocephalus G. López & R. Morales. Previous phytochemical and biological studies of their EOs highlighted the abundance of oxygen-containing monoterpenes and revealed the presence of antioxidants and cholinesterase
inhibitors (Costa et al., 2012a,b, 2013). In addition, EOs from L. viridis, T. lotocephalus and other subspecies of L. pedunculata have been shown to possess remarkable antimicrobial properties (Faleiro et al., 2003; Zuzarte et al., 2009, 2011). This is particularly important for applications in the food preservation domain (Singh et al., 2011; Hyldgaard et al., 2012). Therefore, and considering the impressive biological potential of EOs from the cited species, this work intends to investigate the effect of -CD and HP--CD on their antioxidant activity and storage stability. 2. Materials and methods 2.1. Chemicals Fluorescein was obtained from Panreac (Barcelona, Spain). 2,2 Azobis(2-methylpropionamidine) dihydrochloride (AAPH) and 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Acros Organics (Geel, Germany). Cyclodextrin and 2-hydroxypropyl--cyclodextrin were purchased from Sigma–Aldrich (Steinheim, Germany). All chemical were used as received. 2.2. Plant material and preparation of the EOs The aerial parts of L. viridis plants were collected during the flowering period in the area of São Bartolomeu de Messines (Algarve, Portugal). A voucher specimen was deposited in the herbarium of the Botanical Garden of the University of Lisbon under the
Fig. 2. (A) Fluorescence decay curves induced by AAPH of blank (black circles) and Trolox (50 M) (white circles). (B) Linear regression of Net AUC of Trolox at different concentrations tested.
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number LISU 173992. The aerial parts of L. pedunculata and T. lotocephalus plants were collected during the flowering period in Campus de Gambelas (Algarve, Portugal) and vouchers specimens were deposited in the herbarium of the University of Algarve under the number ALGU 8080 and 8081, respectively. The plant material was dried at room temperature until constant weight, then blended to powder to achieve a mean particle size lower than 2 mm using a sieve (Retsch, Germany) and finally stored at −20 ◦ C until further use. The EOs from air-dried plant material (50 g) were isolated by hydrodistillation for 3 h in a Clevenger-type apparatus, according to the method recommended by the European Pharmacopoeia (Council of Europe, 2007). Since the extracted EOs were virtually water-free it was not necessary the use of anhydrous sodium sulfate or any other “drying” procedure.
The inclusion complexes were prepared by mixing a fixed amount of EOs from L. viridis, L. pedunculata or T. lotocephalus (i.e., 31 g/ml) with different concentrations of -CD (i.e., 0, 2.25, 4.5 and 9 mg/ml) and HP--CD (i.e., 0, 2.25, 4.5, 9 and 18 mg/ml). The EOs and CDs solutions were prepared in ethanol and water, respectively. The mixtures were sealed to avoid loss of volatiles and shaken for 2 h at room temperature on an orbital shake to allow for complex formation (VWR Collection Tube Rotator, EU plug) and, afterwards, stored at 4 ◦ C and protected from light during 30 days.
μmol TE / gEO
2.3. Association of EOs and CDs
2.4. Evaluation of antioxidant activity by oxygen radical absorbance capacity (ORAC) assay The antioxidant capacity was evaluated as described by Gillespie et al. (2007) using fluorescein as the fluorescent probe and AAPH as a peroxyl radical generator. In ORAC assay, a fluorescent probe is used to compete with antioxidants for peroxyl radicals generated by thermal decomposition of AAPH (Prior et al., 2005). A black microplate (NUNC, Rochester, New York, USA) was loaded with fluorescein (0.08 M) and EO/CD complex, Trolox standard (6.25–50 M), phosphate buffer or empty CDs (blanks). After a 10 min incubation period at 37 ◦ C, the reaction was initiated by adding AAPH (150 mM) to each well. The reduction in fluorescence was determined by reading fluorescein excitation at 485 nm and emission at 530 nm, every minute, for 1 h. The EO/CD complex stability was evaluated over a 30 day period following the alluded procedure. All experiments were carried out in triplicate. The ORAC value for each sample was calculated by subtracting the area under the blank curve from the area under the sample curve to obtain the net area (Net AUC). Such procedure is illustrated in Fig. 2 where one can see (a) typical fluorescence decay curves, induced by AAPH in the absence (blank) and presence of Trolox (e.g., 50 M) and (b) the linear regression of Net AUC of Trolox at the different concentrations tested. The results are expressed as Trolox equivalents per gram of EO (TE/gEO ).
2.5. Statistical analysis The data is expressed as the mean ± standard error and was subjected to one-way analysis of variance (ANOVA). To analyze the effect of CD concentration and storage period on the antioxidant activity, means were compared by Duncan’s New Multiple Range Test. All statistical analysis was carried out using the SPSS statistical package for Windows (release 18.0; SPSS Inc., Chicago, IL, USA).
β Fig. 3. Effect of the concentration of -CD on the antioxidant activity of the Lamiaceae essential oils. No antioxidant activity was observed for free EOs and CDs alone at the tested concentrations.
3. Results and discussion 3.1. Antioxidant activity of EOs complexed with CDs The effect of -CD on the antioxidant capacity of the EOs is showed in Fig. 3. It is important to highlight that neither the EOs nor the CDs alone show free radical-scavenging ability at the tested concentrations. In fact, to detect any antioxidant activity in its free state, a much higher concentration of EOs would be needed as proved in previous studies (i.e., up to sixteen times higher than the one used in this work) (Costa et al., 2012a,b). Aiming a potential application in the food domain, it was intended to reduce the concentration of the active ingredient (EOs) as much as possible while achieving a remarkable antioxidant activity via EOs/CDs complexation. From Fig. 3, it is clear that when the EOs are in the presence of -CD, a remarkable antioxidant activity is observed despite the use of a much smaller quantity of EO. The EOs from L. viridis and T. lotocephalus were found to be the most active radical scavengers in the presence of -CD. Moreover, the highest antioxidant activity of L. pedunculata and T. lotocephalus was observed in a higher concentration of -CD used (i.e., 9 mg/ml) whereas no significant effect of the -CD concentration was observed in the L. viridis EO. Likewise, the activity of all the studied EOs was enhanced by the presence of HP--CD and, in this case, as can be seen in
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μmol TE / gEO
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β Fig. 4. Effect of the concentration of HP--CD on the antioxidant activity of the Lamiaceae essential oils. No antioxidant activity was observed for free EOs and CDs alone at the tested concentrations.
Fig. 4, the effect was significantly (p < 0.05) more pronounced in the T. lotocephalus EO, particularly at the highest concentration of HP--CD (i.e., 18 mg/ml). In this case, when the concentration of HP--CD increased from 2.25 mg/ml to 18 mg/ml, the free radicalscavenging potential almost doubled, reaching the highest ORAC value observed (i.e., 1900 molTE /gEO ). On the other hand, no significant effect of the HP--CD concentration was detected in the activity of the Lavandula species. As mentioned above, the activity of CDs alone was evaluated and no effect was observed for all the studied concentrations. This lack of antioxidant activity of CDs is expected and has also been reported in previous studies (Lucas-Abellan et al., 2008). These results suggest that the increase on the antioxidant activity is linked to the enhancement of solubility of the EOs since, in its free state and at the same low concentration of 31 g/ml, no measurable activity was detected. Furthermore, due to the higher solubility in water, the process of complexation with HP--CD is much facilitated compared with the native -CD. Different studies have reported the association of some volatiles found in the studied species and CDs complexes. For instance, Astray et al. (2010) investigated the binding constants of different flavors, including cineole and camphor, to ␣- and -CDs and concluded that the main driving force for CDsflavor complex formation is related to the amphiphilic features of the flavors. Additionally, it has been reported that the majority of
volatiles have a 1:1 (volatile:CD) stoichiometry (Szente and Szejtli, 2004; Ciobanu et al., 2013). The inclusion complex ability of CDs depends on the geometric accommodation in the internal cavity and thermodynamic interactions with the guest (Del Valle, 2004). From this point of view, no significant differences are expected to be found between the CDs used in this work, since both possess the same number of sugar units. On the other hand, and additionally to the entrapment in the internal cavity of CD (can be totally or partially), the hydroxyl groups located on the outer surface of the CDs can also interact with guest molecules (Loftsson and Duchene, 2007). Both CDs have this capacity, however the chemical structure of the hydroxypropyl moieties in HP--CD not only makes this derivative more soluble than the native -CD but also changes the surface’s chemistry. Therefore, less polar compounds, which have not been trapped in the HP--CD cavity (and do not strongly interact with simple hydroxyl groups), are expected to be absorbed at the HP--CD external surface. This will in turn increase the EOs solubility and consequently enhance the antioxidant activity. The EOs of L. viridis, L. pedunculata and T. lotocephalus were previously characterized and the main compounds identified are showed in Table 1. Camphor was the main constituent identified in the EOs of L. viridis (Costa et al., 2012a) and L. pedunculata (Costa et al., 2013), whereas linalool was the major compound in the EO of T. lotocephalus (Costa et al., 2012b). Camphor and linalool are high-value aromatic compounds for many industries and both have previously demonstrated radical scavenging abilities (Celik and Ozkaya, 2002; Zaouali et al., 2010). Recently, their interactions with CDs and -CD polymers in Lavandula angustifolia EO were investigated (Ciobanu et al., 2012). Authors reported that -CD has more affinity for linalool and camphor than for ␣- or ␥-CD. Moreover, authors found that camphor has apparently a better geometric accommodation inside the CD cavity and, consequently, a greater stability than that of linalool. Nevertheless, there are several examples (Costa et al., 2012a; Nakatsu et al., 2000) indicating that the minor compounds cannot be neglected as it may positively contribute to the activity of the whole EO. Since the overall effect of the EOs results from the synergy of all compounds it is not trivial to rationalize the higher antioxidant activity generally observed in this work for the EO obtained from the T. lotocephalus. Therefore, and due to the fact that the EOs are multicomponent systems, the determination of their mechanism of association with CDs, association constants and association stoichiometries are quite laborious. Although, these are relevant questions for a complete understating of the system, such is outside the scope of the present paper. These pertinent issues are expected to be addressed and clarified in other publication where sound nuclear magnetic resonance methodologies will be explored (Medronho et al., 2014). 3.2. Storage stability of EO–CD complexes In food quality, time and temperature are important factors. Fresh food products, e.g., dairy products, fruits and vegetables, are known to have a short shelf life (typically less than 30 days) requiring some kind of protection to avoid their fast deterioration during storage which is typically caused by different events such as proliferation of microorganisms, enzymes and oxidation (Del Nobile et al., 2012). Natural antimicrobial and antioxidant compounds have been added to food to prevent, to some extent, degradation (Ayala-Zavala and Gonzalez-Aguilar, 2010) but their activity can also be susceptible to chemical and biological modifications during storage process (Marques, 2010). Even at refrigerator temperatures (ca. 4–5 ◦ C), perishable food will deteriorate, but this process is considerably delayed due to the slow growth rate of the microorganisms already present in the product. It was seen in the previous section that, given the proper conditions, the association between CDs and EOs is beneficial when a remarkable enhancement of the
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Table 1 Main compounds (relative %) identified in L. viridis, L. pedunculata and T. lotocephalus essential oils. L. viridisa
L. pedunculatac
T. lotocephalusb
Camphor (31.59%) 1,8-Cineole (21.31%) Verbenone (3.54%) Norinone (2.12%) trans-Linalool oxide (1.78%) Linalool (1.75%) Myrtenol (1.77%) Myrtenal (1.22%) Caryophyllene oxide (1.21%)
Camphor (40.8%) Fenchone (38.2%) ␣-Fenchol (2.6%) Linalool (2.0%) Verbenone (1.5%) Eremophyllene (1.4%) ␣-Cadinol (1.3%) p-Cymen-8-ol (1.3%) 1,8-Cineole (0.9%)
Linalool (10.43%) Caryophyllene oxide (8.71%) Camphor (7.97%) Borneol (5.62%) ␣-Terpineol (4.46%) 1,8-Cineole (3.82%) Globulol (3.70%) Viridiflorol (2.69%) Geranyl acetate (2.20%)
a b c
Costa et al. (2012b) Costa et al. (2012a) Costa et al. (2013)
2500
L. viridis
2000 1500 1000 500 0 2500
L. pedunculata
2000
μmol TE / gEO
μmol TE / gEO
1500 1000 500 0 2500
T. lotocephalus
2000 1500 1000 500 0 0
6
12
18
24
30
Time (days) Fig. 5. Storage stability of the Lamiaceae essential oils for a -CD concentration of 2.25 mg/ml (black circles) and 9 mg/ml (white circles). Samples were stored at 4 ◦ C.
antioxidant activity is achieved. However, if EOs want to be recognized as valuable alternatives to enhance the life of a certain fresh product, it is thus relevant to know if the EO–CD complex formed is stable over time and, more importantly, if it maintains its high antioxidant activity when stored at lower temperatures. In this context, the effect of -CD at different concentrations (2.25 and 9 mg/ml) on the activity of the EOs was assessed and the results are displayed in Fig. 5. After 30 days, regardless of the concentration of -CD, the Lavandula spp. showed a moderate decrease on the antioxidant activity. Conversely, the EO of T. lotocephalus
Fig. 6. Storage stability of the Lamiaceae essential oils for a HP--CD concentration of 2.25 mg/ml (black circles) and 18 mg/ml (white circles). Samples were stored at 4 ◦ C.
revealed high storage stability with no statistical significant change in activity after 30 days, particularly when associated with -CD at the highest studied concentration. When HP--CD was used, all species showed a remarkable stable activity profile over 30 days (Fig. 6). Overall, the activity was observed to be more stable over time in the presence of HP--CD than with -CD. It is expectable that these results are related not only to the general higher solubility of HP--CD but also with predictable favored interactions between the hydroxypropyl moieties and the EOs constituents.
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4. Conclusions In this study, it is reported for the first time the effect of two CDs, -CD and HP--CD, in the antioxidant activity of the EOs from L. viridis, L. pedunculata and T. lotocephalus. Alone, both EOs and CDs did not show any measurable antioxidant capacity for the investigated concentrations. However, when the Lamiaceae EOs are associated with -CD or HP--CD, an impressive antioxidant activity was observed. Most likely, this behavior is due to an enhancement in solubility of some of the less polar compounds of the EOs but the mechanism is not clear yet. For the lower concentrations of CDs, little effect on the antioxidant activity of Lavandula EOs was detected. On the other hand, the activity increased for the highest CD concentration tested (i.e., 9 mg/ml for -CD and 18 mg/ml for HP--CD). The modified CD, HP--CD, was shown to have a more pronounced effect in EOs activity, in particular for ;1;T. lotocephalus. All formulations with HP--CD are considerably stable over time showing a remarkable high activity after 30 days of storage. In the case of -CD, the complexes seem less stable and the activity slightly decreased during the same period. Nevertheless, and in the worst case, the activity measured at the end of the storage period is still quite appreciable. It was clearly demonstrated that CDs (particularly HP--CD) have a significant positive impact on the antioxidant activity of EOs from three Lamiaceae species in aqueous systems. Since these EOs can be seen as a natural source of antioxidants and antimicrobial compounds, it is believed that these systems are rather good candidates to be used particularly in food preservation applications. Acknowledgements The authors acknowledge support from the Portuguese Foundation for Science and Technology (FCT, project PTDC/AGRTEC/4049/2012, post-doc grants assigned to Patrícia Costa: SFRH/BPD/93108/2013, Bruno Medronho: SFRH/BPD/74540/2010 and Sandra Gonc¸alves: SFRH/BPD/84112/2012). References Astray, G., Mejuto, J.C., Morales, J., Rial-Otero, R., Simal-Gandara, J., 2010. Factors controlling flavors binding constants to cyclodextrins and their applications in foods. Food Res. Int. 43, 1212–1218. Ayala-Zavala, J.F., Gonzalez-Aguilar, G.A., 2010. Optimizing the use of garlic oil as antimicrobial agent on fresh-cut tomato through a controlled release system. J. Food Sci. 75, M398–M405. Ayala-Zavala, J.F., Soto-Valdez, H., Gonzalaz-Leon, A., Alvarez-Parrilla, E., Martin-Belloso, O., Gonzalez-Aguilar, G.A., 2008. Microencapsulation of cinnamon leaf (Cinnamomum zeylanicum) and garlic (Allium sativum) oils in beta-cyclodextrin. J. Inclusion Phenom. Macrocyclic 60, 359–368. Bakkali, F., Averbeck, S., Averbeck, D., Waomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. Baranauskiene, R., Venskutonis, P.R., Dewettinck, K., Verhe, R., 2006. Properties of oregano (Origanum vulgare L.), citronella (Cymbopogon nardus G.) and marjoram (Majorana hortensis L.) flavors encapsulated into milk protein-based matrices. Food Res. Int. 39, 413–425. Boelens, M.H., 1995. Chemical and sensorial evaluation of Lavandula oils. Perfumer Flavorist 3, 23–51. Celik, S., Ozkaya, A., 2002. Effects of intraperitoneally administered lipoic acid, vitamin E, and linalool on the level of total lipid and fatty acids in guinea pig brain with oxidative stress induced by H2 O2 . J. Biochem. Mol. Biol. 35, 547–552. Chang, C.P., Leung, T.K., Lin, S.M., Hsu, C.C., 2006. Release properties on gelatin-gum arabic microcapsules containing camphor oil with added polystyrene. Colloid Surf. B 50, 136–140. Ciobanu, A., Landy, D., Fourmentin, S., 2013. Complexation efficiency of cyclodextrins for volatile flavor compounds. Food Res. Int. 53, 110–114. Ciobanu, A., Mallard, I., Landy, D., Brabie, G., Nistor, D., Fourmentin, S., 2012. Inclusion interactions of cyclodextrins and crosslinked cyclodextrin polymers with linalool and camphor in Lavandula angustifolia essential oil. Carbohydr. Polym. 87, 1963–1970. Council of Europe (COE) – European Directorate for the Quality of Medicines (EDQM), E.P. 2007. Costa, P., Goncalves, S., Grosso, C., Andrade, P.B., Valentao, P., Bernardo-Gil, M.G., Romano, A., 2012a. Chemical profiling and biological screening of Thymus
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