PRODUCTOS NATURALES

 

Chemical composition of the Schinus molle L. essential oil and their biological activities

 

Composición química y actividad biológica del aceite esencial de Schinus molle L

 

Lic. Paulo Steider Doleski Muhd^I, Lic. Camila Helena Ferreira Cuelho^I, Lic. Juliana Calil Brondani^I, Dr. C. Melânia Palermo Manfron^II

^I Phyitochemical Research Laboratory, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
^II Associate Professor, Industrial Pharmacy Department, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.

 

 

---

ABSTRACT

Introduction: essential oils and aromatic plant extracts have been recognized for many years as a great source of pharmaceutical agents. It is important to research on the chemical composition of the essential oils of plants widely used by the population because its usability becomes safer.
Objective: to identify the chemical characterization of Schinus molle L. essential oils and their biological activities and to compare them with the biological activities of the main compounds found in literature.
Methods: fifty grams of leaves were used to extract the oils through distillation in a modified Clevenger apparatus. The chemical analysis of volatile oils was carried out with capillary gas chromatography using flame ionization detector for quantitative analysis of its elements and, subsequently, a mass detector for qualitative analysis.
Results: nineteen substances were separated and the major compounds were bicyclogermacrene (20.5 %), betacaryophyllene (19.7 %) and spathulenol (19.2 %).
Conclusions: given the extensive distribution of the raw material in Rio Grande do Sul state, Brazil, more studies on the chemical properties and biological activities of Schinus molle essential oil are needed.

Keywords: essential oil, biological activities, Schinus molle.

---

RESUMEN

Introducción: los aceites esenciales y extractos de plantas aromáticas han sido reconocidos desde hace muchos años como una gran fuente de agentes farmacéuticos. Es importante la investigación de la composición química de los.aceites esenciales de las plantas utilizadas ampliamente por la población porque su aplicabilidad se torna más segura.
Objetivo: identificar la composición química del aceite esencial de las hojas de Schinus molle L. y correlacionarlas con las actividades biológicas de los principales compuestos localizadas en la literatura.
Métodos: cincuenta gramos de hojas se sometieron a extracción a través de destilación usando un equipo Clevenger modificado. Para el análisis químico, los aceites volátiles se sometieron a cromatografía capilar de gases usando un detector de ionización de llama para el análisis cuantitativo de sus elementos constituyentes y, posteriormente, un detector de masas para el análisis cualitativo.
Resultados: diecinueve compuestos fueron separados y los principales eran biciclogermacreno (20,5 %), betacariofileno (19,7 %) y espatulenol (19,2 %).
Conclusiones: dada la extensa distribución del material crudo en el estado de Río Grande do Sul, Brasil, se debe incrementar los estudios sobre las características químicas y las actividades biológicas del aceite esencial de Schinus molle.

Palabras clave: aceite esencial, actividades biológicas, Schinus molle.

---

 

 

INTRODUCTION

Schinus molle L. commonly called "peppertree, molle and aguaribay" is tree native to South America that belongs to the Anacardiaceae family. Nowadays it is distributed through Argentina, south-eastern Brazil, Peru, Colombia, Ecuador, Uruguay, Mexico, Central and Southern of California and West Texas, United States.¹

In folk medicine, S. molle has been used as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural, anti-spasmodic, astringent, digestive stimulant, tonic, diuretic, wound healing, an analgesic agents, as well as a stimulant and an antidepressant.^2-9 It has also been used in the treatment of toothache, rheumatism, menstrual disorders, and respiratory and urinary tract infection.^10,11

The essential oil (EO) in their chemical composition might justify the therapeutic use of this plant since the essential oils (EOs) and extracts of aromatic plants have been recognized for many years as a great source of pharmaceutical agents and food additives.¹² Their antioxidant capacity for acting in metabolic response to the endogenous production of free radicals and other oxidant species has been demonstrated.¹³ Furthermore, EOs have shown important in vitro antimicrobial properties against pathogens and foodborne agents causing diseases.¹⁴ The anti-in-ammatory activity of EOs has been investigated in in-ammatory diseases such as allergy, rheumatism, arthritis and bronchitis.¹⁵ They tend to have low mammalian toxicity, less environmental effects and wide public acceptance.¹⁶

In the present study, was analyzed the chemical characterization of essential oil extracted from the leaves of S. molle L.

 

METHODS

PLANT MATERIAL

The leaves of S. molle L. were collected on Morro do Cechella in Santa Maria (Rio Grande do Sul, State of Brazil), coordinates 29°40’35’’S and 53°47’06”W. Exsiccate was identified by Pharmacobotanic Laboratory (Industrial Pharmacy Department/UFSM).

ESSENTIAL OILS EXTRACTION, ISOLATION AND IDENTIFICATION

Fifty grams of leaves were subjected to extraction through distillation in a modified Clevenger¹⁷ apparatus. After 2 hours, the fraction of oil was collected in ethyl ether, being dehydrated with sodium sulfate and concentrated on a water bath.

For chemical analysis, the volatile oils were diluted in ethyl ether in the ratio 2:100 (v / v). Subjected to capillary gas chromatography using flame ionization detector (GC/FID) for quantitative analysis of its constituents and, subsequently, to the mass detector (GC/MS) for qualitative analysis.

The separation and quantification of volatile oils constituents were performed on a gas chromatograph equipped with flow divider (spliter) with partition of 1:50. Helium was used as carrier gas at a pressure of 80 kPa and linear speed of 1 mL/min. Nitrogen, synthetic air and hydrogen were used as auxiliary gases to flame detector in 1:1:10 ratio, respectively. The quantification was obtained by electronic integration (normalization technique). For the separation of the constituents was used Durabond-DB5 column, with 30 m long and 0.25 mm internal diameter, filled with dimethyldiphenylsiloxane containing 5 % phenyl groups on a 0.25 mm thick film.

Qualitative analysis was performed using the same equipment, however coupled to a mass detector GC/MS-QP5000, equipped with cylindrical quadrupole, operated with ionization energy of 70 eV and sample partition of 1:20. The ionization was obtained by the technique of electron impact.

First of all, the chromatograms obtained by GC/FID and GC/MS of volatile crude oil were compared and the linear retention indices corresponding to each peak were calculated. A comparison was made with the retention times of the sample and a mixture of linear alkanes. The characterization of the constituents was based on Kovats indices (KI)¹⁸ and their mass spectra, by comparison of these with authentic samples and literature data.¹⁹

STATISTICAL ANALYSIS

All the constituents identified in the essential oils were subjected to analysis of variance (ANOVA) and comparison of test averages (Tukey 5 %). Statistical analyses were performed by SPSS (version 10.1) and differences were considered statistically significant when p<0.05, p<0.01 and p<0.001.

 

RESULTS

The chromatogram obtained by GC/FID and GC/MS of volatile crude oil were compared and the linear retention indices corresponding to each peak were calculated. The chromatogram developed (figure) resulted in the separation of nineteen substances.

Each peak in the chromatogram was identified by its mass spectrum, by comparison with library equipment, by consulting the literature^{20, 21} and by injection of standards. Have the quantification of the relative percentage of identified constituents was obtained based on the areas of the chromatographic peaks for the method of normalization.

Statistical analysis no showed variation on the chemical composition of the EO, for the same sample.

Results of gas chromatographic analysis of S. molle EO are summarised in Annex, Nineteen components were identified in leaf EO, representing 2.8 % of monoterpenes and 92.2 % of sesquiterpenes. Leaf EO was characterized mainly by bicyclogermacrene (20.5 %), beta-caryophyllene (19.7 %), spathulenol (19.2 %), globulol (9.5 %), germacrene-D (7.4 %), caryophyllene oxide (5.3 %) and terpinen-4-ol (1.2 %).

Annex. EOs isolated from the leaves of Schinus molle L. by gas chromatography.

 

 

Results of this work showed that major compounds were bicyclogermacrene (20.5 %), beta-caryophyllene (19.7 %) and spathulenol (19.2 %). Furthermore, sesquiterpenes hydrocarbons were the dominant components in studied EO.

 

DISCUSSION

Regarding the chemical composition of EOs, a study analyzed the EO composition of leaves and its relationships within and among 11 populations of S. molle, collected at different locations over the distribution area of this species in State of Rio Grande do Sul (Brazil) and Uruguay. The populations analyzed were: Alto Alegre; Bagé; Caçapava do Sul; Caxias do Sul; Dom Pedrito; Erechim; Pinheiro Machado; Quaraí; Sant’Ana do Livramento; São Borja (Brazil) and Rivera (Uruguay), each population composed by 5–8 individuals. The EO composition of the 11 populations (79 samples), extracted by steam distillation and analyzed by GC–MS, identified 22 compounds, including oxygenated and non-oxygenated forms of monoterpenes and sesquiterpenes, that represented between 73.5 % and 96.9 % of the total EO extracted. Four groups were formed by the Average Linkage cluster analysis. The first group was characterized by the compound sabinene; the second, which was the largest group, was characterized by the presence of alfa and betapinene; the third group comprised the samples corresponding to the São Borja population and was characterized by the high contents of alfacadinol; and the fourth group was characterized by the high concentrations of myrcene.22

Another study, was performed to investigate the allelopathic potential of Schinus molle and Schinus terebinthifolius EOs on onion and lettuce germination and initial growth, given the well-known biological activities of several of the terpenoid compounds found in EOs. Leaves were collected in Porto Alegre City (30° 1′ 39.73″ S51°13′ 43.45″ W), State of Rio Grande do Sul, Brazil. At least six plants of each species were sampled. S. molle EO contained betapinene as a major compound, also containing limonene and betapinene.²³

Twenty-two components were identified in leaf EO of S. molle, collected in autumn in the Évora region, in southeast Portugal; representing 69.3 % of monoterpenes and 17.0 % of sesquiterpenes. Leaf EO was characterized mainly by alfaphellandrene (25.9 %), limonene (11.7 %), betamyrcene (11.1 %), betaphellandrene (10.5 %) and elemol (9.0 %).²⁴

Was observed in this study, compared with previously published studies,^22-24 there was a difference in the chemical composition of the EOs from samples collected in the same region. Differences on chemical composition suggest the presence of different chemotypes of S. molle.²⁵ In EOs, constituents and concentrations depend not only on the plant species. Among the various factors that influence the chemical composition, the most important are the origin of the plant, plant part used, the stage of plant development, climate and growing conditions such as temperature, soil and fertilizer and distillation conditions and storage.²⁶

The identification of the presence of bicyclogermacrene (20.5 %), betacaryophyllene (19.7 %) and spathulenol (19.2 %) in the present leaf EO can justify the use of S. molle in traditional medicine.

The bicyclogermacrene is not cited as the active bactericidal, but it has demonstrated larvicidal potential through testing with the larvae of the mosquito Aedes aegypti. The results showed that the oil of Cordia leucomalloides was able to kill 98.7 % of the larvae in the concentration of 100 ppm.²⁷ Anti-inflammatory and anti-ulcer activity were also tested and its inhibited 90 % of stress-induced gastric ulcers while cimetidine inhibited 70 %.²⁸

Betacaryophyllene is the main volatile constituent found in large amounts in the essential oils of different plants and spices, such as oregano ( Origanum vulgare L.), cinnamon (Cinnamomum spp.) and black pepper (Piper nigrum L.).^29,30 It is known as a potential agent anti-inflammatory, antioxidant, protector of gastric mucosal, local anesthetic, anti-acne and anticarcinogenic, due to its capacity to detoxify xenobiotics or to attack cancerous cell lines.^31,32

Cunico et al. extracted three essential oils from leaves, fruits and roots of Ottonia martiana Miq. (Piperaceae), common species in Brazilian Rain Forest, and analyzed by GC-MS and tested in an antibacterial assay against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Pseudomonas aerogenes (ATCC 27853) and Escherichia coli (ATCC 25922). Spathulenol was the major component of the oil extracted from the roots (17.83 %) and fruits (17:37 %). The oils showed an activity against S. aureus with the MIC of 5 mg mL^-1 (fruit oil) and 5 µg mL^-1 (root oil). This oils also showed an activity against S. epidermidis with the MIC of 5 µg mL^-1 (fruit oil) and 5 µg mL^-1 (root oil).³³

The methanol extract of Salvia mirzayanii has shown an immunomodulatory effect on peripheral blood lymphocytes in study developed by Ziaei et al. Fractionation of the methanol extract and purification of the components using normal column chromatography and preparative thin layer chromatography resulted in identification of the bioactive compound, spathulenol, with an immunoinhibitory effect. Treatment of activated lymphocytes with a concentrated fraction containing 62 % of spathulenol showed a decrease in the proliferation of lymphocytes with an IC₅₀ of 85.4 ± 11.08 µg/mL. Spathulenol showed the capacity to inhibit proliferation in the lymphocytes and to induce apoptosis in these cells possibly through a caspase-3 independent pathway.³⁴

It is demonstrated that the chemical characteristics of the S. molle essential oil encourages more studies regarding biological activities, becoming economically viable, because, the raw material is widely distributed in the state of Rio Grande do Sul, Brazil

 

REFERENCES BIBLIOGRAPHICAL

1. Zunino MP, López ML, Zygadlo JA. Medicinal plants of Argentina. Pharmacological properties and phytochemistry. In: Imperato F. Advances in phytochemisty. Trivandrum: Research Singpost; 2003. p.209-45.

2. Ruffa MJ, Ferraro G, Wagner ML, Calcagno ML, Campos RH, Cavallaro L. Cytotoxic effect of argentine medicinal plant extracts on human hepatocellular carcinoma cell line. J Ethnopharmacol. 2002;79(3):335–9.

3. Yueqin Z, Recio MC, Máñez S, Giner RM, Cerdá-Nicolás M, Ríos JL. Isolation of two triterpenoids and a biflavanone with anti-inflammatory activity from Schinus molle fruits. Planta Med. 2003; 69(10):893–8.

4. Murray AP, Frontera MA, Tomas MA, Mulet MC. Gas chromatography–mass spectrometry study of the essential oil of S. longifolia (Lindl.) speg., Schinus fasciculata (Griseb.) I.M. Johnst., and Schinus areira L. Z Naturforsch C. 2005;60(1-2):25–9.

5. Taylor L. The healing power of rainforest herbs: a guide to understanding and using herbal medicinals. New York: Square One Publishers; 2005.

6. Alanís-Garza BA, González-González GM, Salazar-Aranda R, Waksman de Torres N, Rivas-Galindo VM. Screening of antifungal activity of plants from the northeast of Mexico. J Ethnopharmacol. 2007;114(3):468–71.

7. Machado DG, Bettio LEB, Cunha MP, Santos ARS, Pizzolatti MG, Brighente IMC, et al. Antidepressant-like effect of rutin isolated from the ethanolic extract from Schinus molle L. in mice: evidence for the involvement of the serotonergic and noradrenergic systems. Eur J Pharmacol. 2008;587(1-3):163–8.

8. Molina-Salinas GM, Pérez-López A, Becerril-Montes P, Salazar-Aranda R, Said-Fernández S, de Torres NW. Evaluation of the flora of Northern Mexico for in vitro antimicrobial and antituberculosis activity. J Ethnopharmacol. 2007;109(3):435–41.

9. Guala MS, Elder HV, Perez G, Chiesa A. Evaluación del poder antioxidante de fracciones de aceite esencial crudo de Schinus molle L. obtenidas por destilación al vacío. Inf Tecnol. 2009;20(2):83–8.

10. Barrachina MD, Bello R, Martínez-Cuesta MA, Primo-Yúfera E, Esplugues J. Analgesic and central depressor effects of the dichloromethanol extract from Schinus molle L. Phytother Res. 1997;11(4):317–19.

11. Bello R, Beltrán B, Moreno L, Calatayud S, Primo-Yúfera E, Esplugues J. In vitro pharmacological evaluation of the dichloromethanol extract from Schinus molle L. Phytother Res. 1998;12(7):523–5.

12. Joy B, Rajan A, Abraham E. Antimicrobial activity and chemical composition of essential oil from Hedychium coronarium. Phytother Res. 2007;21(5):439–43.

13. Wei A, Shibamoto T. Antioxidant/lipoxygenase inhibitory activities and chemical compositions of selected essential oils. J Agric Food Chem. 2010;58(12):7218–25.

14. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol. 1999;86(6):985–90.

15. Süntar I, Tumen I, Ustün O, Keles H, Akkol EK. Appraisal on the wound healing and anti-inflammatory activities of the essential oils obtained from the cones and needles of Pinus species by in vivo and in vitro experimental models. J Ethnopharmacol. 2012; 139(2):533–40.

16. Koroch A, Juliani HR, Zygadlo JA. Bioactivity of essential oils and their components. In: Berger RG. Flavours and fragrances chemistry, bioprocessing and sustainability. Berlin: Springer Verlag. 2007;p. 87–115.

17. Wasicky R, Akisue G. Um aparelho aperfeiçoado para a extração de óleos essenciais. Rev Fac Farm Bioquim Univ São Paulo. 1969;7(2):339-405.

18. Kovats VE. Gas chromatographische charakterisierung organischer verbindungen. Teil 1: retentions indices aliphatischer halogenide, alkohole, aldehyde und ketone. Helv Chim Acta. 1958;41(7):1915-32.

19. Addams RP. Identification of essential oil components by gas chromatography/mass spectroscopy. Carol Stream: Allured; 1995.

20. Addams RP. Identification of essential oil components by gas chromatography/mass spectrometry. 4. ed. Carol Stream: Allured; 2007.

21. Joulain D, König WA..The atlas of spectral data of sesquiterpene hydrocarbons. Hamburg: E. B. Verlag; 1998.

22. Gomes V, Agostini G, Agostini F, Atti dos Santos AC, Rossato M. Variation in the essential oils composition in Brazilian populations of Schinus molle L. (Anacardiaceae). Biochem Syst Ecol. 2013;48:222-7.

23. Pawlowski A, Kaltchuk-Santos E, Zini CA, Caramão EB, Soares GLG. Essential oils of Schinus terebinthifolius and S. molle (Anacardiaceae): mitodepressive and aneugenic inducers in onion and lettuce root meristems. S Afr J Bot. 2012;80:96-103.

24. Martins MR, Arantes S, Candeias F, Tinoco MT, Cruz-Morais J. Antioxidant, antimicrobial and toxicological properties of Schinus molle L. essential oils. J Ethnopharmacol. 2014;151(1):485-92.

25. Rossini C, Menéndez P, Dellacassa E, Moyna P. Essential oils from leaves of Schinus molle and Schinus lentiscifolius of Uruguayan origin. J Essent Oil Res. 1996;8(1):71–3.

26. Oladimeji FA, Orafidiya OO, Okeke IN, Dagne E. Effect of autoxidation on the composition and antimicrobial activity of essential oil of Lippia multiflora. Pharm Pharmacol Lett. 2001;11(2):64-7.

27. Santos RP, Nunes EP, Nascimento RF, Santiago GMP, Menezes GHA, Silveira ER, et al. Chemical composition and larvicidal activity of the essential oils of Cordia leucomalloides and Cordia curassavica from the Northeast of Brazil. J Braz Chem Soc. 2006;17(5):1027-30.

28. Esteves I, Souza IR, Rodrigues M, Cardoso LG, Santos LS, Sertie JAA, et al. Gastric antiulcer and anti-inflammatory activities of the essential oil from Casearia sylvestris Sw. J Ethnopharmacol. 2005;101(1-3):191-6.

29. Orav A, Stulova I, Kailas T, Müürisepp M. Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states. J Agric Food Chem. 2004;52(9):2582-6.

30. Mockute D, Bernotiene G, Judzentiene A. The essential oil of Origanum vulgare L. ssp. vulgare growing wild in Vilnius district (Lithuania). Phytochemistry. 2001;57(1):65-9.

31. Legault J, Pichette A. Potentiating effect of beta-caryophyllene on anticancer activity of alpha-humulene, isocaryophyllene and paclitaxel. J Pharm Pharmacol. 2007;59(12):1643-7.

32. da Silva SL, Figueiredo PM, Yano T. Chemotherapeutic potential of the volatile oils from Zanthoxylum rhoifolium Lam leaves. Eur J Pharmacol. 2007;576(1-3):180-8.

33. Cunico MM, Lopes AR, Côcco LC, Yamamoto CI, Plocharski RCB, Miguel MD, et al. Phytochemical and antibacterial evaluation of essential oils from Ottonia martiana miq. (Piperaceae). J Braz Chem Soc. 2007;18(1):184-8.

34. Ziaei A, Ramezani M, Wright L, Paetz C, Schneider B, Amirghofran Z. Identification of spathulenol in Salvia mirzayanii and the immunomodulatory effects. Phytoter Res. 2011;25(4):557-62.

 

 

Recibido: 14 de junio de 2014
Aprobado: 15 de agosto de 2014

 

Camila Helena Ferreira Cuelho. Phyitochemical Research Laboratory, Universidade Federal de Santa Maria, Santa Maria. Avenida Roraima, 1000, Prédio 26, sala 1107, Camobi, CEP 97105-900, Santa Maria, Rio Grande do Sul, Brazil. Teléfono: + 55 55 9622 4372. Correo eléctronico: [e-mail]