This article was downloaded by: [Selcuk Universitesi] On: 25 December 2014, At: 02:48 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam. a
a
Diogo Alexandre Siebert , Adrielli Tenfen , Celina Noriko a
a
Yamanaka , Caio Maurício Mendes de Cordova , Dilamara Riva b
b
a
Scharf , Edésio Luiz Simionatto & Michele Debiasi Alberton
Click for updates
a
Departamento de Ciências Farmacêuticas, Universidade Regional de Blumenau, Rua São Paulo, 2171, CEP 89030-000, Blumenau, SC, Brazil b
Laboratório de Cromatografia, Universidade Regional de Blumenau, Rua São Paulo, 3250, CEP 89030-000, Blumenau, SC, Brazil Published online: 15 Sep 2014.
To cite this article: Diogo Alexandre Siebert, Adrielli Tenfen, Celina Noriko Yamanaka, Caio Maurício Mendes de Cordova, Dilamara Riva Scharf, Edésio Luiz Simionatto & Michele Debiasi Alberton (2015) Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam., Natural Product Research: Formerly Natural Product Letters, 29:3, 289-292, DOI: 10.1080/14786419.2014.958736 To link to this article: http://dx.doi.org/10.1080/14786419.2014.958736
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 3, 289–292, http://dx.doi.org/10.1080/14786419.2014.958736
SHORT COMMUNICATION Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam. Diogo Alexandre Sieberta, Adrielli Tenfena, Celina Noriko Yamanakaa, Caio Maurı´cio Mendes de Cordovaa, Dilamara Riva Scharfb, Ede´sio Luiz Simionattob and Michele Debiasi Albertona*
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
a
Departamento de Cieˆncias Farmaceˆuticas, Universidade Regional de Blumenau, Rua Sa˜o Paulo, 2171, CEP 89030-000, Blumenau, SC, Brazil; bLaborato´rio de Cromatografia, Universidade Regional de Blumenau, Rua Sa˜o Paulo, 3250, CEP 89030-000, Blumenau, SC, Brazil (Received 18 June 2014; final version received 21 August 2014) This study describes the seasonal composition and the antibacterial, antioxidant and anticholinesterase activity of the essential oil from Eugenia brasiliensis leaves. Analysis by using GC allowed the identification of 40 compounds. It was observed that the monoterpenes varied more (42%) than the sesquiterpenes (14%), and that the monoterpene hydrocarbons suffered the greatest variation throughout the year (64%). Major compounds were spathulenol in the spring (16.02 ^ 0.44%) and summer (18.17 ^ 0.41%), t-cadinol in the autumn (12.83 ^ 0.03%) and a-pinene (15.94 ^ 0.58%) in the winter. Essential oils were tested for their antibacterial activity, and the best result was obtained from the autumn oil, with MIC ¼ 500 mg mL21 against Staphylococcus saprophyticus and Pseudomonas aeruginosa. Antioxidant activity was evaluated using DPPH, lipid peroxidation and iron-reducing power assays, as well as the anticholinesterase activity. Both tests showed a weak performance of the essential oils. Keywords: Eugenia brasiliensis; essential oil; antibacterial; antioxidant; anticholinesterase
1. Introduction Essential oils are complex natural products produced by the secondary metabolism of aromatic plants, with a mixture of compounds at different concentrations, consisting primarily of low molecular weight molecules such as terpenes. Due to the fact that they are responsible for a wide range of biological activities, such as antimicrobial, antioxidant, anti-inflammatory, antiviral, among others, they have been extensively studied by the scientific community (Bakkali et al. 2008). However, essential oils can vary in quality, quantity and in composition according to climate, soil composition, plant organ, age and vegetative cycle stage (Masotti et al. 2003; Angioni et al. 2006). Eugenia brasiliensis Lam. (Myrtaceae) popularly known in Brazil as ‘grumixama’, ‘grumixameira’ and ‘cereja-brasileira’, occurs in the Atlantic rain forest of southern Brazil, and in Santa Catarina state in the regions of Floriano´polis, Brusque, Ibirama, Itajaı´ and Joinville (Reitz & Klein 1969). This species is used in folk medicine in the form of infusion of the leaves, for the treatment of rheumatism and arthritis, diarrhoea and as a diuretic (Revilla 2002). Previous reports regarding the biological activities of the essential oil from the leaves of E. brasiliensis
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
290
D.A. Siebert et al.
have demonstrated promising results for antibacterial activity against Staphylococcus aureus (Magina et al. 2009). Some studies have investigated the chemical composition of the essential oil from the leaves of this species collected in the south and southeast of Brazil, demonstrating different results. Basically, monoterpenes such as a-pinene and b-pinene, oxygenated monoterpenes such as a-terpineol, sesquiterpenes as caryophyllene, and oxygenated sesquiterpenes such as spathulenol, a-cadinol and t-cadinol comprise the E. brasiliensis essential oil. However, it is observed that the quantitative and qualitative compositions vary according to the period and location of collection (Fischer et al. 2005; Ramos et al. 2006; Moreno et al. 2007; Lima et al. 2008; Magina et al. 2009). Therefore, the aim of this work was to evaluate the seasonal chemical composition of the essential oil from E. brasiliensis fresh leaves collected in Floriano´polis-SC in all seasons of the year. Furthermore, antibacterial, antioxidant and anticholinesterase activity of these essential oils were also evaluated. 2. Results and discussion 2.1. Seasonal chemical composition The yield obtained from the extraction of essential oils was different in each season. The yield was 0.14% (w w21) in the summer, 0.14% (w w21) in the winter, 0.10% (w w21) in the autumn and 0.08% (w w21) in the spring. A total of 40 compounds were identified accounting 97.29 ^ 0.11, 98.53 ^ 0.13, 92.24 ^ 0.04 and 95.36 ^ 0.06% from the essential oils of spring, summer, autumn and winter, respectively. Supplementary Table S1 shows the qualitative and quantitative chemical compositions of the samples studied. In all analysed oils, the sesquiterpene fractions (spring: 81.96 ^ 0.11%; summer: 70.90 ^ 0.15%; autumn: 74.44 ^ 0.03%; winter: 57.55 ^ 0.04%) were higher than the monoterpene fractions, with the oxygenated compounds being most prevalent among the sesquiterpenes. All samples, except the essential oil from winter, also showed sesquiterpene as their major compound. In these samples, the major compounds were spathulenol in the essential oils from spring (16.02 ^ 0.44%) and summer (18.17 ^ 0.41%) and t-cadinol in autumn (12.83 ^ 0.03%). The most prevalent monoterpenes in these oils were a-terpineol in the spring (3.48 ^ 0.14%) and a-pinene in the essential oils of summer (9.05 ^ 0.37%) and autumn (5.14 ^ 0.15%). As regards the essential oil extracted in the winter, the higher concentration was of monoterpenes (37.81 ^ 0.08%), with the major compound of this season being a-pinene (15.94 ^ 0.58%). In this essential oil, the most prevalent sesquiterpene was t-cadinol (10.38 ^ 0.17%). The chemical composition of the essential oils is in agreement with the reports of Ramos et al. (2006) and Magina et al. (2009), for which the leaves were collected in Campinas (SP, Brazil) and Santo Amaro da Imperatriz (SC, Brazil), respectively, and indicate spathulenol as the major compound, in agreement with the essential oils from the spring and summer. However, a study from Moreno et al. (2007), with leaves collected in Moji-Guac u (SP, Brazil) points out the monoterpenes a-pinene and b-pinene as the major compounds, in agreement with the essential oil extracted in the winter. Among the quantified compounds, 16 showed high variation, 18 varied moderately and only 3 had shown low variation. Among the compounds that showed high variation, we highlight a-pinene (76% variation) and spathulenol (41%). Among those with moderate variation, the t-cadinol, with 16% of variation, is noticeable, and regarding the compounds with low variation, the most relevant is the a-cadinol, with 14% of variation. Other compounds, besides those mentioned, also showed high levels of variation, but they are minor compounds, and therefore less relevant to the total oil composition. Analysing the results, it is observed that the
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
Natural Product Research
291
monoterpenes vary more (42%) than the sesquiterpenes (14%). Within the class of monoterpenes, it was observed that the monoterpene hydrocarbons are the ones which suffer most variation throughout the year, reaching 64% of variation, whereas these compounds when oxygenated show a much smaller variation of 18%. Correlating the chemical composition data with the climatic variations of the region and time of collection (Supplementary Table S2), it is noted that the content of monoterpenes and sesquiterpenes is apparently influenced by these conditions. It is also observed that the increase in the content of monoterpenes is apparently favoured at lower average temperature and higher average relative humidity. In contrast, the content of sesquiterpenes becomes lesser in these conditions, probably due to the increase in the proportion of monoterpenes. These results can be explained by the fact that monoterpenes are smaller molecules and therefore lighter, getting fixed in the plant in more humid and less hot climates, which would prevent their volatilisation. A similar phenomenon has been noticed in the study of the mechanism of volatilisation of monoterpenes from Salvia mellifera, when a strong correlation between the relative humidity and temperature with the content of monoterpenes in the plant was observed (Dement et al. 1975). All data presented indicate that factors related to the season and microclimate can influence the variation of the chemical composition of the essential oil of plant species, but a further quantitative study in the subject is needed to draw better conclusions. 2.2. Antibacterial, antioxidant and anticholinesterase activity of essential oil For the evaluation of the antibacterial activity, a criterion established by Machado et al. (2005) was used. Samples with MIC values lower than 10 mg mL21 were considered to have excellent antibacterial activity; values between 10 and 100 mg mL21 were considered good; values between 100 and 500 mg mL21 were considered moderate; values between 500 and 1000 mg mL21 were considered low, and for MIC values above 1000 mg mL21, samples were considered inactive. Results for MIC of all essential oil samples are shown in Supplementary Table S3. For cell-wall bacteria, some tested oils were active. Against Staphylococcus saprophyticus, all samples were active, with moderate activity for oils from autumn and winter (MIC 500 mg mL21) and weak activity for oils from summer and spring (MIC ¼ 1000 mg mL21). None of the essential oils was able to inhibit S. aureus growth, having an MIC higher than 1000 mg mL21. Against Escherichia coli, only the autumn oil showed weak activity (1000 mg mL21). Finally, against Pseudomonas aeruginosa, the essential oil obtained from the autumn showed moderate activity (500 mg mL21) and the winter oil presented weak activity (1000 mg mL21). As it can be noticed, the antibacterial activity of essential oils against some of the tested microorganisms seems to vary according to the extraction season. As for mollicutes, the essential oils were not active, suggesting that the mechanism of action of these oils is at the cell wall level, since they do not have cell walls but plasma membranes rich in cholesterol. An important characteristic of essential oils and their components, such as terpenes, is their hydrophobicity, which enables them to partition in the lipids of the bacterial cell membrane and mitochondria, and so the mechanism of action of terpenes might be associated with a damage of the plasma membrane stability (Trombetta et al. 2005). The antioxidant activity of the essential oils from each season as evaluated by the determination of free radical scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Cavin et al. 1998), inhibition of lipid peroxidation (ILP) (Mokbel & Hashinaga 2006) and iron-reducing power (Waterman & Mole 1994), as well as the determination of the IC50 for the anticholinesterase activity are reported in Supplementary Table S4. As it can be observed, all oil samples showed weak activity. Regarding the DPPH assay, all samples were unable to reduce its DPPH radical to levels below 50% at a maximum concentration of 500 mg mL21. ILP showed
292
D.A. Siebert et al.
poor results, with the best percentage of inhibition obtained with the essential oil from the spring (14.05%). The reduction potential showed similar results in all samples, and the best registered result was from the autumn (94.32 mg AA g21). At last, for the anticholinesterase activity, all samples had IC50 of more than 1000 mg mL21.
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
3. Conclusions The results presented in this work allow inferring that, when correlating the chemical composition with the season of collection, factors such as the microclimate can influence the chemical composition of the essential oil of plant species as well as the biological activities. Therefore, further quantitative studies, increasing the follow-up samples, broadening listed individuals and within the possibilities controlling the growing environment of the plant are necessary. Supplementary material Experimental details are available online, alongside with Tables S1 –S4. Acknowledgements The authors are grateful to CAPES and FURB for the financial support.
References Angioni A, Barra A, Coroneo V, Dessi S, Cabras P. 2006. Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp. stoechas essential oils from stem/leaves and flowers. J Agric Food Chem. 54:4364–4370. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils: a review. Food Chem Toxicol. 46:446–475. Cavin A, Hostettmann K, Dyatmyko W, Potterat O. 1998. Antioxidant and lipophilic constituents of Tinospora crispa. Planta Med. 64:393–396. Dement WA, Tyson BJ, Mooney HA. 1975. Mechanism of monoterpene volatilization in Salvia mellifera. Phytochemistry. 14:2555–2557. Fischer DCH, Limberger RP, Henriques AT, Moreno PRH. 2005. Essential oils from leaves of two Eugenia brasiliensis specimens from Southeastern Brazil. J Essent Oil Res. 17:499–500. Lima NP, Cerqueira SHF, Fa´vero OA, Romoff P, Lago HG. 2008. Composition and chemical variation of the essential oil from leaves of Eugenia brasiliensis Lam. and Eugenia sp. (Myrtaceae). J Essent Oil Res. 20:223–225. Machado KE, Cechinel Filho V, Tessarolo R, Mallmann C, Meyre-Silva C, Bella Cruz A. 2005. Potent antibacterial activity of Eugenia umbelliflora. Pharm Biol. 43:636–639. Magina MDA, Dalmarco EM, Wisniewski A, Simionatto EL, Dalmarco JB, Pizzolatti MG, Brighente IMC. 2009. Chemical composition and antibacterial activity of essential oils of Eugenia species. J Nat Med. 63:345–350. Masotti V, Juteau F, Bessie`re JM, Viano J. 2003. Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J Agric Food Chem. 51:7115–7121. Mokbel MS, Hashinaga F. 2006. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 94:529–534. Moreno PRH, Lima MEL, Sobral M, Young MCM, Cordeiro I, Apel MA, Limberger RP, Henriques AT. 2007. Essential oil composition of fruit colour varieties of Eugenia brasiliensis Lam. Sci Agric. 64:428–432. Ramos MFS, Siani AC, Souza MC, Rosas EC, Henriques MGMO. 2006. Avaliac a˜o da atividade anti-inflamato´ria dos o´leos essenciais de cinco espe´cies de Myrtaceae [Evaluation of anti-inflammatory activity of the essential oils from five species of Myrtaceae]. Fitos. 2:58–66. Portuguese. Reitz RD, Klein RM. 1969. Mirta´ceas: Flora Ilustrada Catarinense [Myrtaceae: illustrated catarinense’s flora]. Itajaı´: Herba´rio Barbosa Rodrigues. Revilla J. 2002. Plantas u´teis da bacia amazoˆnica [Useful plats from the Amazon basin]. Rio de Janeiro: INPA. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, Saija A, Mazzanti G, Bisignano G. 2005. Mechanism of antibacterial action of monoterpenes. Antimicrob Agents Chemother. 49:2474–2478. Waterman PG, Mole S. 1994. Analysis of phenolic plant metabolites. Oxford: Blackwell Scientific.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam. a
a
Diogo Alexandre Siebert , Adrielli Tenfen , Celina Noriko a
a
Yamanaka , Caio Maurício Mendes de Cordova , Dilamara Riva b
b
a
Scharf , Edésio Luiz Simionatto & Michele Debiasi Alberton
Click for updates
a
Departamento de Ciências Farmacêuticas, Universidade Regional de Blumenau, Rua São Paulo, 2171, CEP 89030-000, Blumenau, SC, Brazil b
Laboratório de Cromatografia, Universidade Regional de Blumenau, Rua São Paulo, 3250, CEP 89030-000, Blumenau, SC, Brazil Published online: 15 Sep 2014.
To cite this article: Diogo Alexandre Siebert, Adrielli Tenfen, Celina Noriko Yamanaka, Caio Maurício Mendes de Cordova, Dilamara Riva Scharf, Edésio Luiz Simionatto & Michele Debiasi Alberton (2015) Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam., Natural Product Research: Formerly Natural Product Letters, 29:3, 289-292, DOI: 10.1080/14786419.2014.958736 To link to this article: http://dx.doi.org/10.1080/14786419.2014.958736
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 3, 289–292, http://dx.doi.org/10.1080/14786419.2014.958736
SHORT COMMUNICATION Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam. Diogo Alexandre Sieberta, Adrielli Tenfena, Celina Noriko Yamanakaa, Caio Maurı´cio Mendes de Cordovaa, Dilamara Riva Scharfb, Ede´sio Luiz Simionattob and Michele Debiasi Albertona*
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
a
Departamento de Cieˆncias Farmaceˆuticas, Universidade Regional de Blumenau, Rua Sa˜o Paulo, 2171, CEP 89030-000, Blumenau, SC, Brazil; bLaborato´rio de Cromatografia, Universidade Regional de Blumenau, Rua Sa˜o Paulo, 3250, CEP 89030-000, Blumenau, SC, Brazil (Received 18 June 2014; final version received 21 August 2014) This study describes the seasonal composition and the antibacterial, antioxidant and anticholinesterase activity of the essential oil from Eugenia brasiliensis leaves. Analysis by using GC allowed the identification of 40 compounds. It was observed that the monoterpenes varied more (42%) than the sesquiterpenes (14%), and that the monoterpene hydrocarbons suffered the greatest variation throughout the year (64%). Major compounds were spathulenol in the spring (16.02 ^ 0.44%) and summer (18.17 ^ 0.41%), t-cadinol in the autumn (12.83 ^ 0.03%) and a-pinene (15.94 ^ 0.58%) in the winter. Essential oils were tested for their antibacterial activity, and the best result was obtained from the autumn oil, with MIC ¼ 500 mg mL21 against Staphylococcus saprophyticus and Pseudomonas aeruginosa. Antioxidant activity was evaluated using DPPH, lipid peroxidation and iron-reducing power assays, as well as the anticholinesterase activity. Both tests showed a weak performance of the essential oils. Keywords: Eugenia brasiliensis; essential oil; antibacterial; antioxidant; anticholinesterase
1. Introduction Essential oils are complex natural products produced by the secondary metabolism of aromatic plants, with a mixture of compounds at different concentrations, consisting primarily of low molecular weight molecules such as terpenes. Due to the fact that they are responsible for a wide range of biological activities, such as antimicrobial, antioxidant, anti-inflammatory, antiviral, among others, they have been extensively studied by the scientific community (Bakkali et al. 2008). However, essential oils can vary in quality, quantity and in composition according to climate, soil composition, plant organ, age and vegetative cycle stage (Masotti et al. 2003; Angioni et al. 2006). Eugenia brasiliensis Lam. (Myrtaceae) popularly known in Brazil as ‘grumixama’, ‘grumixameira’ and ‘cereja-brasileira’, occurs in the Atlantic rain forest of southern Brazil, and in Santa Catarina state in the regions of Floriano´polis, Brusque, Ibirama, Itajaı´ and Joinville (Reitz & Klein 1969). This species is used in folk medicine in the form of infusion of the leaves, for the treatment of rheumatism and arthritis, diarrhoea and as a diuretic (Revilla 2002). Previous reports regarding the biological activities of the essential oil from the leaves of E. brasiliensis
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
290
D.A. Siebert et al.
have demonstrated promising results for antibacterial activity against Staphylococcus aureus (Magina et al. 2009). Some studies have investigated the chemical composition of the essential oil from the leaves of this species collected in the south and southeast of Brazil, demonstrating different results. Basically, monoterpenes such as a-pinene and b-pinene, oxygenated monoterpenes such as a-terpineol, sesquiterpenes as caryophyllene, and oxygenated sesquiterpenes such as spathulenol, a-cadinol and t-cadinol comprise the E. brasiliensis essential oil. However, it is observed that the quantitative and qualitative compositions vary according to the period and location of collection (Fischer et al. 2005; Ramos et al. 2006; Moreno et al. 2007; Lima et al. 2008; Magina et al. 2009). Therefore, the aim of this work was to evaluate the seasonal chemical composition of the essential oil from E. brasiliensis fresh leaves collected in Floriano´polis-SC in all seasons of the year. Furthermore, antibacterial, antioxidant and anticholinesterase activity of these essential oils were also evaluated. 2. Results and discussion 2.1. Seasonal chemical composition The yield obtained from the extraction of essential oils was different in each season. The yield was 0.14% (w w21) in the summer, 0.14% (w w21) in the winter, 0.10% (w w21) in the autumn and 0.08% (w w21) in the spring. A total of 40 compounds were identified accounting 97.29 ^ 0.11, 98.53 ^ 0.13, 92.24 ^ 0.04 and 95.36 ^ 0.06% from the essential oils of spring, summer, autumn and winter, respectively. Supplementary Table S1 shows the qualitative and quantitative chemical compositions of the samples studied. In all analysed oils, the sesquiterpene fractions (spring: 81.96 ^ 0.11%; summer: 70.90 ^ 0.15%; autumn: 74.44 ^ 0.03%; winter: 57.55 ^ 0.04%) were higher than the monoterpene fractions, with the oxygenated compounds being most prevalent among the sesquiterpenes. All samples, except the essential oil from winter, also showed sesquiterpene as their major compound. In these samples, the major compounds were spathulenol in the essential oils from spring (16.02 ^ 0.44%) and summer (18.17 ^ 0.41%) and t-cadinol in autumn (12.83 ^ 0.03%). The most prevalent monoterpenes in these oils were a-terpineol in the spring (3.48 ^ 0.14%) and a-pinene in the essential oils of summer (9.05 ^ 0.37%) and autumn (5.14 ^ 0.15%). As regards the essential oil extracted in the winter, the higher concentration was of monoterpenes (37.81 ^ 0.08%), with the major compound of this season being a-pinene (15.94 ^ 0.58%). In this essential oil, the most prevalent sesquiterpene was t-cadinol (10.38 ^ 0.17%). The chemical composition of the essential oils is in agreement with the reports of Ramos et al. (2006) and Magina et al. (2009), for which the leaves were collected in Campinas (SP, Brazil) and Santo Amaro da Imperatriz (SC, Brazil), respectively, and indicate spathulenol as the major compound, in agreement with the essential oils from the spring and summer. However, a study from Moreno et al. (2007), with leaves collected in Moji-Guac u (SP, Brazil) points out the monoterpenes a-pinene and b-pinene as the major compounds, in agreement with the essential oil extracted in the winter. Among the quantified compounds, 16 showed high variation, 18 varied moderately and only 3 had shown low variation. Among the compounds that showed high variation, we highlight a-pinene (76% variation) and spathulenol (41%). Among those with moderate variation, the t-cadinol, with 16% of variation, is noticeable, and regarding the compounds with low variation, the most relevant is the a-cadinol, with 14% of variation. Other compounds, besides those mentioned, also showed high levels of variation, but they are minor compounds, and therefore less relevant to the total oil composition. Analysing the results, it is observed that the
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
Natural Product Research
291
monoterpenes vary more (42%) than the sesquiterpenes (14%). Within the class of monoterpenes, it was observed that the monoterpene hydrocarbons are the ones which suffer most variation throughout the year, reaching 64% of variation, whereas these compounds when oxygenated show a much smaller variation of 18%. Correlating the chemical composition data with the climatic variations of the region and time of collection (Supplementary Table S2), it is noted that the content of monoterpenes and sesquiterpenes is apparently influenced by these conditions. It is also observed that the increase in the content of monoterpenes is apparently favoured at lower average temperature and higher average relative humidity. In contrast, the content of sesquiterpenes becomes lesser in these conditions, probably due to the increase in the proportion of monoterpenes. These results can be explained by the fact that monoterpenes are smaller molecules and therefore lighter, getting fixed in the plant in more humid and less hot climates, which would prevent their volatilisation. A similar phenomenon has been noticed in the study of the mechanism of volatilisation of monoterpenes from Salvia mellifera, when a strong correlation between the relative humidity and temperature with the content of monoterpenes in the plant was observed (Dement et al. 1975). All data presented indicate that factors related to the season and microclimate can influence the variation of the chemical composition of the essential oil of plant species, but a further quantitative study in the subject is needed to draw better conclusions. 2.2. Antibacterial, antioxidant and anticholinesterase activity of essential oil For the evaluation of the antibacterial activity, a criterion established by Machado et al. (2005) was used. Samples with MIC values lower than 10 mg mL21 were considered to have excellent antibacterial activity; values between 10 and 100 mg mL21 were considered good; values between 100 and 500 mg mL21 were considered moderate; values between 500 and 1000 mg mL21 were considered low, and for MIC values above 1000 mg mL21, samples were considered inactive. Results for MIC of all essential oil samples are shown in Supplementary Table S3. For cell-wall bacteria, some tested oils were active. Against Staphylococcus saprophyticus, all samples were active, with moderate activity for oils from autumn and winter (MIC 500 mg mL21) and weak activity for oils from summer and spring (MIC ¼ 1000 mg mL21). None of the essential oils was able to inhibit S. aureus growth, having an MIC higher than 1000 mg mL21. Against Escherichia coli, only the autumn oil showed weak activity (1000 mg mL21). Finally, against Pseudomonas aeruginosa, the essential oil obtained from the autumn showed moderate activity (500 mg mL21) and the winter oil presented weak activity (1000 mg mL21). As it can be noticed, the antibacterial activity of essential oils against some of the tested microorganisms seems to vary according to the extraction season. As for mollicutes, the essential oils were not active, suggesting that the mechanism of action of these oils is at the cell wall level, since they do not have cell walls but plasma membranes rich in cholesterol. An important characteristic of essential oils and their components, such as terpenes, is their hydrophobicity, which enables them to partition in the lipids of the bacterial cell membrane and mitochondria, and so the mechanism of action of terpenes might be associated with a damage of the plasma membrane stability (Trombetta et al. 2005). The antioxidant activity of the essential oils from each season as evaluated by the determination of free radical scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Cavin et al. 1998), inhibition of lipid peroxidation (ILP) (Mokbel & Hashinaga 2006) and iron-reducing power (Waterman & Mole 1994), as well as the determination of the IC50 for the anticholinesterase activity are reported in Supplementary Table S4. As it can be observed, all oil samples showed weak activity. Regarding the DPPH assay, all samples were unable to reduce its DPPH radical to levels below 50% at a maximum concentration of 500 mg mL21. ILP showed
292
D.A. Siebert et al.
poor results, with the best percentage of inhibition obtained with the essential oil from the spring (14.05%). The reduction potential showed similar results in all samples, and the best registered result was from the autumn (94.32 mg AA g21). At last, for the anticholinesterase activity, all samples had IC50 of more than 1000 mg mL21.
Downloaded by [Selcuk Universitesi] at 02:48 25 December 2014
3. Conclusions The results presented in this work allow inferring that, when correlating the chemical composition with the season of collection, factors such as the microclimate can influence the chemical composition of the essential oil of plant species as well as the biological activities. Therefore, further quantitative studies, increasing the follow-up samples, broadening listed individuals and within the possibilities controlling the growing environment of the plant are necessary. Supplementary material Experimental details are available online, alongside with Tables S1 –S4. Acknowledgements The authors are grateful to CAPES and FURB for the financial support.
References Angioni A, Barra A, Coroneo V, Dessi S, Cabras P. 2006. Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp. stoechas essential oils from stem/leaves and flowers. J Agric Food Chem. 54:4364–4370. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils: a review. Food Chem Toxicol. 46:446–475. Cavin A, Hostettmann K, Dyatmyko W, Potterat O. 1998. Antioxidant and lipophilic constituents of Tinospora crispa. Planta Med. 64:393–396. Dement WA, Tyson BJ, Mooney HA. 1975. Mechanism of monoterpene volatilization in Salvia mellifera. Phytochemistry. 14:2555–2557. Fischer DCH, Limberger RP, Henriques AT, Moreno PRH. 2005. Essential oils from leaves of two Eugenia brasiliensis specimens from Southeastern Brazil. J Essent Oil Res. 17:499–500. Lima NP, Cerqueira SHF, Fa´vero OA, Romoff P, Lago HG. 2008. Composition and chemical variation of the essential oil from leaves of Eugenia brasiliensis Lam. and Eugenia sp. (Myrtaceae). J Essent Oil Res. 20:223–225. Machado KE, Cechinel Filho V, Tessarolo R, Mallmann C, Meyre-Silva C, Bella Cruz A. 2005. Potent antibacterial activity of Eugenia umbelliflora. Pharm Biol. 43:636–639. Magina MDA, Dalmarco EM, Wisniewski A, Simionatto EL, Dalmarco JB, Pizzolatti MG, Brighente IMC. 2009. Chemical composition and antibacterial activity of essential oils of Eugenia species. J Nat Med. 63:345–350. Masotti V, Juteau F, Bessie`re JM, Viano J. 2003. Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J Agric Food Chem. 51:7115–7121. Mokbel MS, Hashinaga F. 2006. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 94:529–534. Moreno PRH, Lima MEL, Sobral M, Young MCM, Cordeiro I, Apel MA, Limberger RP, Henriques AT. 2007. Essential oil composition of fruit colour varieties of Eugenia brasiliensis Lam. Sci Agric. 64:428–432. Ramos MFS, Siani AC, Souza MC, Rosas EC, Henriques MGMO. 2006. Avaliac a˜o da atividade anti-inflamato´ria dos o´leos essenciais de cinco espe´cies de Myrtaceae [Evaluation of anti-inflammatory activity of the essential oils from five species of Myrtaceae]. Fitos. 2:58–66. Portuguese. Reitz RD, Klein RM. 1969. Mirta´ceas: Flora Ilustrada Catarinense [Myrtaceae: illustrated catarinense’s flora]. Itajaı´: Herba´rio Barbosa Rodrigues. Revilla J. 2002. Plantas u´teis da bacia amazoˆnica [Useful plats from the Amazon basin]. Rio de Janeiro: INPA. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, Saija A, Mazzanti G, Bisignano G. 2005. Mechanism of antibacterial action of monoterpenes. Antimicrob Agents Chemother. 49:2474–2478. Waterman PG, Mole S. 1994. Analysis of phenolic plant metabolites. Oxford: Blackwell Scientific.
