208 J.F.Martucci et aL Industrial Crops and Products 71(2015)205-213 Table 1 (Inouye et al.,2001).Gram-positive bacteria,in turn,lack the rigid Main compounds,expressed as percentage of chromatographic area of L officinalis and O.vulgare essential oils. outer membrane being more susceptible to the action of EOs. The effectiveness of pure constituents of OEO and LEO against Components() the studied bacteria was statistically lower than that of EOs Lavander (Lavandula officinalis) Oregano (Origanum vulgare) (Table 2)indicating that minor ingredients are critical to the activ- Linalool 53.50 ity,as previously observed by others (Kulevanova and Panovska Carvacrol 26.70 Camphor 8.40 p-Cimene 1520 2001:Canillac and Mourey,2001:Gende et al.,2010:Danh et al. Terpinen-4-ol 7.60 y-Terpinene 15.10 2012:Teixeira et al.,2013a).Furthermore,both pathogens were 18-cineol 6.80 Terpinene 7.50 less susceptible to the action of lavender oil and to its major compo- Borneol 4.70 o-Pinene 5.60 nent,linalool (Table 2),suggesting that phenolic compounds seem Linalyl acetate 420 Iso borneol 3.80 080 3.40 to govern the antibacterial ability of Eos(Burt,2004:Tassou et al. Lavandulvl acetate Terpinolene Hexilacetate 0.60 B-Myrcene 3.40 2000).Similar trend was previously observed for thyme and basil 1-0cten-3-0 055 c-Thujene 3.40 essential oils(and their major compounds:thymol,estragol,car- 3-octanone 0.40 o-Terpineol 2.30 vacrol,linalool,and p-cymene)being carvacrol the one exerting Myrcene 030 Methyl carvacrol 220 the strongest antibacterial activity against E.coli followed by thy- Caryophyllene 1.40 Sabinene 1.40 mol.Estragol and linalool,in turn,exhibited limited antibacterial Endo borneol 120 activity (Bagamboula et al.,2004). Thymol 1.10 The potential antagonistic or synergic effect among components Terpinen-4-ol 1.10 of both essentials oils was experimentally analyzed on mixtures B-Phellandrene 0.60 Camphene 0.40 of LEO:OEO 50:50 The obtained results (Table 2)reflected that 1.8-cineol 0.30 the tested microorganisms were less susceptible to the action of Total 87.85 Total 96.1 the mixture,resulting in a lower antimicrobial effect than the sum of the single effects produced by each essential oil individu- ally,thereby indicating a possible antagonistic effect between their 3.Results and discussion components(Canillac and Mourey,2001:Sadaka et al.,2013). The antioxidant capacity of EOs was revealed through their 3.1.Characterization of oregano and lavender essential oils reducing power and DPPH radical scavenging efficiency,respec- tively.In Fig.1 a,the FRAP value points reflect a ten-fold higher Active components in oregano and lavender essential oils iden- reducing power of OEO than that of lavender oil,in accordance with tified by GC/MS are summarized in Table 1.The quantitative the presence of electron donor chemicals such as carvacrol and thy- analysis of lavender essential oil revealed a prevalence of linalool mol,which can react with free radicals and turn them into more (53.50%),followed by camphor (8.40%).terpinen-4-ol (7.60%)and stable products,and so terminate radical chain reactions (Burt 1.8-cineol(6.80%).The results of the chemical profile of LEO com- 2004).Moreover,OEO exhibited a strong dose-dependent reducing pare favorably with those of earlier studies (Inouye et al.,2001 power rising from 11 ppm of AA for 200 ppm up to 114 ppm of AA Dahn et al.,2012 Teixeira et al.,2013a).The prevailing compounds for 3000 ppm.Higher OEO concentrations did not induce signifi- of oregano essential oil were aromatic monoterpenes:carvacrol cant changes in the reducing power,indicating a saturation level (26.70%)and thymol (1.10%)and aliphatic monoterpenes being p- of around 3000ppm.By contrast,no significant variations were cymene(15.20%).y-terpinene(15.10%)and terpinene(7.50%)the evidenced for LEO activity which exhibited a lag phase of up to major constituents of this group.Plants that produce carvacrol and 2000 ppm followed by a minor increment of up to 17 ppm for a thymol almost always have relatively high amounts of p-cymene concentration of 6000 ppm.These results are in agreement with and y-terpinene,which are biosynthetic precursors for both phe- OEO composition constituted by phenolic compounds(Table 1). nolic monoterpenes (Poulose and Croteau,1978).Differences in The phenolic compounds are free radical acceptors that delay or essential oil compounds may be influenced by geographical vari- inhibit the autoxidation initiation step or interrupt the autoxida- ables,time of plant harvesting.preparation process extraction tion propagation step (Kacaniova et al..2012:Eca et al..2014) method and quantification conditions (Burt,2004:Gende et al. Fig.1 b clearly shows that the radical scavenging activity of OEO is 2010).Several studies on O.vulgare ssp.cultivated in different significantly higher as compared to that of LEO for the same concen- regions worldwide have centered their attention on the variabil- trations.As it can be observed in Fig.1 b,OEO free radical scavenging ity of chemical composition.Different percentages of thymol and effect also exhibited a dose-dependent increase having a 74%RSA carvacrol have been reported as majority compounds in this vegetal for 6000 ppm,in line with the results reported by Kacaniova et al. specie(D'Antuono et al.,2000;Oussalah et al.,2004:Gomez-Estaca (2012)for the same concentration of oregano essential oil.LEO did etal,2009). not show significant activity for all concentrations tested,i.e.,from EOs antibacterial efficiency was quantified by determining the 2000 to 6000 ppm.The weakest activity of LEO could be associated minimum inhibitory concentration (MIC).Oregano and lavender with the absence of phenolic compounds in its composition. essential oils,together with their major components.c.a.carvacrol and linalool,were evaluated against E.coli and S.aureus.MIC values Table 2 are summarized in Table 2.It is noteworthy that greater inhibition Antimicrobial activity of essential oils against E coli and S.aureus strains. of oils and pure compounds was observed against S.aureus.This finding is in line with early studies focused on the action of whole Minimum inhibitory concentration(MIC) E.coli S.aureus EOs against food spoilage organisms and food-borne pathogens Lavender EO (LEO) 2000 1000-1200 (Inouye et al.,2001:Burt,2004)and it is thought to arise as a Oregano Eo(oEO 1600-1800 800-900 LE0:0E050-50 >2000 1600-1800 result of the differences in their cell membrane structure and Thymol 1000 800-900 the hydrophobic character of essential oils and their components Carvacrol 600 500-600 (Vaara,1992:Inouye et al.,2001 Inouye et al.,2001).Gram nega- Linalool 2000 2000 tive bacteria possess an outer membrane surrounding the cell wall Chloramphenicol 8 2 composed by hydrophilic polysaccharides which restricts diffusion Notes:Data are MIC (ug/mL)range values.The antimicrobial activity was deter- of hydrophobic compounds such as OEs and their main constituents mined by triplicate analyses for oil and strains.208 J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 Table 1 Main compounds, expressed as percentage of chromatographic area of L. officinalis and O. vulgare essential oils. Components(%) Lavander (Lavandula officinalis) Oregano (Origanum vulgare) Linalool 53.50 Carvacrol 26.70 Camphor 8.40 p-Cimene 15.20 Terpinen-4-ol 7.60 −Terpinene 15.10 1,8- cineol 6.80 Terpinene 7.50 Borneol 4.70 − Pinene 5.60 Linalyl acetate 4.20 Iso borneol 3.80 Lavandulyl acetate 0.80 Terpinolene 3.40 Hexilacetate 0.60 - Myrcene 3.40 1-octen-3-ol 0.55 − Thujene 3.40 3- octanone 0.40 - Terpineol 2.30 Myrcene 0.30 Methyl carvacrol 2.20 Caryophyllene 1.40 Sabinene 1.40 Endo borneol 1.20 Thymol 1.10 Terpinen-4-ol 1.10 - Phellandrene 0.60 Camphene 0.40 1,8- cineol 0.30 Total 87.85 Total 96.1 3. Results and discussion 3.1. Characterization of oregano and lavender essential oils Active components in oregano and lavender essential oils iden￾tified by GC/MS are summarized in Table 1. The quantitative analysis of lavender essential oil revealed a prevalence of linalool (53.50%), followed by camphor (8.40%), terpinen-4-ol (7.60%) and 1.8-cineol (6.80%). The results of the chemical profile of LEO com￾pare favorably with those of earlier studies (Inouye et al., 2001; Dahn et al., 2012 Teixeira et al., 2013a). The prevailing compounds of oregano essential oil were aromatic monoterpenes: carvacrol (26.70%) and thymol (1.10%) and aliphatic monoterpenes being p￾cymene (15.20%), - terpinene (15.10%) and terpinene (7.50%) the major constituents of this group. Plants that produce carvacrol and thymol almost always have relatively high amounts of p-cymene and -terpinene, which are biosynthetic precursors for both phe￾nolic monoterpenes (Poulose and Croteau, 1978). Differences in essential oil compounds may be influenced by geographical vari￾ables, time of plant harvesting, preparation process extraction method and quantification conditions (Burt, 2004; Gende et al., 2010). Several studies on O. vulgare ssp. cultivated in different regions worldwide have centered their attention on the variabil￾ity of chemical composition. Different percentages of thymol and carvacrol have been reported as majority compounds in this vegetal specie (D’Antuono et al., 2000; Oussalah et al., 2004; Gómez-Estaca et al., 2009). EOs antibacterial efficiency was quantified by determining the minimum inhibitory concentration (MIC). Oregano and lavender essential oils, together with their major components, c.a. carvacrol and linalool, were evaluated against E. coli and S. aureus. MIC values are summarized in Table 2. It is noteworthy that greater inhibition of oils and pure compounds was observed against S. aureus. This finding is in line with early studies focused on the action of whole EOs against food spoilage organisms and food-borne pathogens (Inouye et al., 2001; Burt, 2004) and it is thought to arise as a result of the differences in their cell membrane structure and the hydrophobic character of essential oils and their components (Vaara, 1992; Inouye et al., 2001 Inouye et al., 2001). Gram nega￾tive bacteria possess an outer membrane surrounding the cell wall composed by hydrophilic polysaccharides which restricts diffusion of hydrophobic compounds such as OEs and their main constituents (Inouye et al., 2001). Gram-positive bacteria, in turn, lack the rigid outer membrane being more susceptible to the action of EOs. The effectiveness of pure constituents of OEO and LEO against the studied bacteria was statistically lower than that of EOs (Table 2) indicating that minor ingredients are critical to the activ￾ity, as previously observed by others (Kulevanova and Panovska, 2001; Canillac and Mourey, 2001; Gende et al., 2010; Danh et al., 2012; Teixeira et al., 2013a). Furthermore, both pathogens were less susceptible to the action of lavender oil and to its major compo￾nent, linalool (Table 2), suggesting that phenolic compounds seem to govern the antibacterial ability of Eos (Burt, 2004; Tassou et al., 2000). Similar trend was previously observed for thyme and basil essential oils (and their major compounds: thymol, estragol, car￾vacrol, linalool, and p-cymene) being carvacrol the one exerting the strongest antibacterial activity against E. coli followed by thy￾mol. Estragol and linalool, in turn, exhibited limited antibacterial activity (Bagamboula et al., 2004). The potential antagonistic or synergic effect among components of both essentials oils was experimentally analyzed on mixtures of LEO:OEO 50:50 The obtained results (Table 2) reflected that the tested microorganisms were less susceptible to the action of the mixture, resulting in a lower antimicrobial effect than the sum of the single effects produced by each essential oil individu￾ally, thereby indicating a possible antagonistic effect between their components (Canillac and Mourey, 2001; Sadaka et al., 2013). The antioxidant capacity of EOs was revealed through their reducing power and DPPH radical scavenging efficiency, respec￾tively. In Fig. 1 a, the FRAP value points reflect a ten-fold higher reducing power of OEO than that of lavender oil, in accordance with the presence of electron donor chemicals such as carvacrol and thy￾mol, which can react with free radicals and turn them into more stable products, and so terminate radical chain reactions (Burt, 2004). Moreover, OEO exhibited a strong dose-dependent reducing power rising from 11 ppm of AA for 200 ppm up to 114 ppm of AA for 3000 ppm. Higher OEO concentrations did not induce signifi- cant changes in the reducing power, indicating a saturation level of around 3000 ppm. By contrast, no significant variations were evidenced for LEO activity which exhibited a lag phase of up to 2000 ppm followed by a minor increment of up to 17 ppm for a concentration of 6000 ppm. These results are in agreement with OEO composition constituted by phenolic compounds (Table 1). The phenolic compounds are free radical acceptors that delay or inhibit the autoxidation initiation step or interrupt the autoxida￾tion propagation step (Kacániová ˇ et al., 2012; Ec¸ a et al., 2014). Fig. 1 b clearly shows that the radical scavenging activity of OEO is significantly higher as compared to that of LEO for the same concen￾trations.As it canbe observed inFig. 1 b, OEO free radical scavenging effect also exhibited a dose-dependent increase having a 74% RSA for 6000 ppm, in line with the results reported by Kacániová ˇ et al. (2012) for the same concentration of oregano essential oil. LEO did not show significant activity for all concentrations tested, i.e., from 2000 to 6000 ppm. The weakest activity of LEO could be associated with the absence of phenolic compounds in its composition. Table 2 Antimicrobial activity of essential oils against E. coli and S. aureus strains. Minimum inhibitory concentration (MIC) E. coli S. aureus Lavender EO (LEO) 2000 1000–1200 Oregano EO (OEO) 1600-1800 800–900 LEO:OEO 50:50 >2000 1600–1800 Thymol 1000 800–900 Carvacrol 600 500–600 Linalool 2000 2000 Chloramphenicol 8 2 Notes: Data are MIC (g/mL) range values. The antimicrobial activity was deter￾mined by triplicate analyses for oil and strains.