Chemical variability in the essential oils from leaves of Syzygium jambos

Chemical variability in the essential oils from leaves of Syzygium jambos

Wilma P. Rezende^I; Leonardo L. Borges^I; Nilda M. Alves^II; Pedro H. Ferri^III; José R. Paula^I

^ILaboratório de Pesquisa de Produtos Naturais, Faculdade de Farmácia, Universidade Federal de Goiás, Brazil

^IIEscola Superior de Ciências da Saúde, Departamento de Farmácia e Bioquímica, Universidade de Rio Verde, Brazil

^IIILaboratório de Bioatividade Molecular, Instituto de Química, Universidade Federal de Goiás, Brazil

^{Correspondence}

ABSTRACT

Syzygium jambos (L.) Alston, Myrtaceae, occurs in tropical regions and is a widespread medicinal plant used to treat several diseases, such as hemorrhage, dysentery, diabetes, inflammation, diabetes and gastrointestinal disorders. Leaf essential oils of ten specimens of S. jambos collected from two localities of Central Brazilian Cerrado were investigated by GC-MS. Soil and foliar nutrients were analyzed to determine the mineral compositions. The aims of this study was to evaluate the influence of environmental factors on chemical composition of leaf essential oils of S. jambos. Studies on the influence of environmental factors over composition of essential oils are important because they contribute data for its cultivation, harvest and establish parameters to essential oil components. The data were analyzed using stepwise Multiple Regression and Cluster Analysis, and the results suggest that the main factor capable to influence the chemical composition of leaf essential oils was the collection period and the collection site had a minor effect. The results also suggest that the leaf essential oils composition was influenced mainly by foliar nutrients (N, Mn, Co, Fe, S and Mg) and soil nutrients (Na, Al, S and H+Al). The compound with the best model obtained was the (E)-caryophyllene, with a coefficient of determination equal 0.8113.

Keywords: medicinal plants; Myrtaceae; seasonality; mineral nutrients; statistical analysis

Introduction

The Brazilian Cerrado stands out as the richest source of biodiversity, with about 6500 plant species cataloged and approximately 220 of which have medicinal uses (MMA, 2009). Several studies by authors suggest that, the percentage of species present in this biome may represent 20 to 50% of the total found in the country (Machado et al., 2004). The Myrtaceae family is one of the principal floras, with 23 genera and about 130 species, and many species are used popularly against gastrointestinal disorders, infectious diseases and hemorrhagic conditions. This family also includes many species that are characterized by the presence of essential oils (Rodrigues & Carvalho, 2001; Holetz et al., 2002; Amaral et al., 2006; Gondim et al., 2006; Sá et al., 2012). Among the species of this family, Syzygium jambos (L.) Alston (syn Eugenia jambos) stands out due its medicinal properties.

This genus has been reported for their different medicinal uses. Syzygium cumini, S. aromaticum, Syzygium jambolanum and Syzygium jambos are the most pharmacologically studied species, and have been recommended to treat haemorrhage, dysentery and gastrointestinal disorders (Moreira, 1978; Cruz, 1979), diabetes (Kelkar & Kaklij, 1996; Stanely Mainzen et al., 1998) and inflammation (Kim et al., 1998; Muruganandan et al., 2001). They have also been employed as sedative and anticonvulsivant (De Lima et al., 1998), as antihypertensive (Bhargava et al., 1968), against herpes virus (Kurokawa et al., 1998) and as inhibitor of histamine release ((Kim et al., 1998). Djipa et al., (2000) tested acetone and aqueous extracts from the barks of S. jambos for antimicrobial activity in vitro by the agar dilution method in petri dishes, and both extracts showed activity against Staphylococcus aureus, Yersinia enterocolitica and coagulase negative staphylococci among which Staphylococcus hominis, Staphylococcus cohnii and Staphylococcus warneri. The leaves of S. jambos are widely used in folk medicine to inflammation, digestive ulcers and high fever (Kan, 1987; Slowing et al., 1994a; 1994b; 1996; Rodrigues & Carvalho, 2001; Souza et al., 2002; Di Stasi et al., 2002; Pessini et al., 2003; Fiuza et al., 2008).

One property known of essential oils is their antimicrobial activity, particularly antibacterial and antifungal activities. Some of these species include the following: Syzygium aromaticum (L.) Merrirl et L. M. Perry, Thymus sp., Lavandula sp., Origanum vulgare L., Rosmarinus officinalis L. and Eucalyptus globulus Labill (Simões & Spitzer, 2004; Cunha, 2005).

It is important consider the chemical variations in essential oils caused by genetic, physiological or environmental variables when domesticating and improving plants of medicinal interest. Therefore, it is necessary studies that demonstrate that the chemical composition of the essential oils, although genetically determined, can suffer influence as a result of several environmental factors especially when referring to vegetal material used in chemical, pharmacological and agronomic studies that aim to obtain herbal medicines, because pharmacological properties can differ due to differences in essential oil composition (Lorenzi & Matos, 2002; Bergo et al., 2005; Lima et al., 2006; Potzernheim et al., 2006; Paula et al., 2011). The aim of our investigation was to study the chemical variability and environmental influence of the essential oil of S. jambos leaves in two samples collected from two different sites.

Materials and Methods

Plant material

Leaves of five specimens of Syzygium jambos (L.) Alston, Myrtaceae, were collected in the municipality of Rio Verde, Goiás state, Brazil (17º 48' 33,9" S; 50º 56' 39,1" W; 710 m), (17º 46' 33,6" S; 50º 54' 13,2" W; 688 m), (17º 46' 27,1" S; 50º 54' 52,2" W; 750 m), (17º 46' 09,6" S; 50º 54' 52,6" W; 781 m), (17º 46' 41,2" S; 50º 56' 43,1" W; 758 m) and five specimens in the municipality of Nova América, Goiás state, Brazil (15º 01' 12" S; 49º 52' 33,4" W; 782 m), (15º 01' 48,7" S; 49º 51' 27,9" W; 657 m), (15º 01' 48,6" S; 49º 51' 29,9" W; 652 m), (15º 02' 58,5" S; 49º 51' 53,4" W; 614 m). All samples were collected twice, in January and July 2011. The plants were identified by Prof. José Realino de Paula and a voucher was deposited at the Herbarium of the Federal University of Goiás under code number 47579. The leaf samples air-were dried in a chamber at 40 ºC and ground into a powder.

Essential oil extraction

The essential oils from leaves of S. jambos were submitted to hydrodistillation in a modified Clevenger-type apparatus (2 h). Each essential oil was dried over anhydrous sodium sulfate and stored at - 20 ºC for further analysis.

Essential oil analyses

Leaf essential oils obtained were analyzed using a gas chromatograph coupled to a mass selective detector (GC-MS), Shimadzu QP5050A, using an ionization voltage of 70 eV. A fused silica capillary column was utilized (CBP - 5; 30 m x 0.25 mm x 0.25 µm) and helium was used as the carrier gas at a flow rate of 1 mL min^-1. The temperature program used was as follows: ramp up from 60 to 240 ºC at 3 ºC min^-1, increase to 280 ºC at 10 ºC min^-1, and complete with 10 min at 280 ºC. The injection volume was 1 µL diluted with CH₂Cl₂ at a ratio 1:5. The essential oil constituents were identified by comparing their mass spectra with those from the National Institute of Standards and Technology (NIST, 1998), as well as by comparing the mass spectra and calculated linear retention indices (RI) with values in the literature (Adams, 2007). Retention indices were obtained by co-injection with a mixture of linear hydrocarbons, C9-C22 (Sigma, USA) and calculated using the equation of Van Den Dool & Kratz (1963). The percentage of each component was calculated to normalize for the area in the chromatogram obtained using a Varian gas chromatograph (FID) equipped with a ZB-5 fused silica capillary column that was 30 m X 0.25 nm with 0.25 µm film thickness (5% phenylmethylpolysiloxane). The following temperature program was used: increase from 60 to 240 ºC at 3 ºC min^-1, followed by an increase to 280 ºC at 10 ºC min^-1, and complete with 10 min at 280 ºC. The carrier gas was N₂, at a flow rate of 1.0 mL/min; the injector port and detector temperatures were 220 and 240 ºC, respectively. Samples were injected by splitting, and the split ratio was 1:20.

Chemical analysis of leaves and soil

Chemical analysis of soil and leaf samples were performed at the Solocria Agricultural Laboratory, School of Agronomy, Federal University of Goiás, following standard procedures (Silva, 2009). For the analysis of foliar nutrients, the nitrogen (N) was extracted by digestion with H₂SO₄ and catalysts. The minerals phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) were extracted by digestion with HClO₄ and HNO₃. For the analysis of nutrients in the soil samples were collected at a depth of 0-20 cm in four locations around each specimen of S. jambos, subsequently homogenized and then air dried. A mass of 500 g was packed in plastic bags. The pH was determined in a volume of water-soil at 1:1. Ca, Mg and Al were extracted with KCl 1M, and P, K, Zn, Cu, Fe and Mn were extracted with Mehlich's solution. Organic matter (OM), cation exchange capacity (CEC), potential acidity (H+Al), base saturation (V) and aluminum saturation (m) were determined by standard methods (Silva, 2009). The quantitative determination of minerals in leaves and soil was performed according to the methodology described by (Silva, 2009). Nitrogen was determined by distillation (semi-micro Kjeldahl method), phosphorus by colorimetry, potassium by flame photometry and sulfur by turbidimetry. Calcium, magnesium, copper, iron, manganese and zinc were determined by atomic absorption.

Climate data

The monthly climate data (average temperature and average daily precipitation) for the collection months were collected from the official site of the National Institute for Space Research (Instituto Nacional de Pesquisas Espaciais).

Statistical analysis

The relationship between the components found in leaf essential oils (dependent variables) from S. jambos and the environmental factors (independent variables) were investigated by stepwise Multiple Regression and Pearson's Correlation Analysis implemented using SAS GLM and SAS CORR procedure, respectively (Draper & Smith, 1981). Cluster Analysis was also applied to the study of similarity of samples on the basis of constituent distribution and hierarchical clustering was performed according to Ward's variance minimizing method (Ward, 1963). Prior to the Cluster Analysis, the data were preprocessed by auto-scaling and mean centering. All procedures were performed using the software SAS (Statystical Analysis System) and Statistica 7 (Inc, 2002; Stat Soft, 2004).

Results and Discussion

The environmental variables are presented in Tables ¹, ², ³ and ⁴. The compounds of leaf essential oils analyzed are shown in Table ⁵.

A total of 62 compounds were identified, however just the components that appear in most amounts or with more frequency were chosen for statistical analysis. The following compounds were selected: (E)-caryophyllene, α-humulene, α-zingibirene, hydroxytoluene butylated, caryophyllene alcohol, caryolan-8-ol, caryophyllene oxide, thujopsan-2-α-ol and n-heneicosane.

From the stepwise Multiple Regression, were obtained the following equations with the significant (p-value less than 0.05) variables (l=leaf and s=soil):

butylated hydrixytoluene = Significant coefficients were not found for this model

caryolan-8-ol= Significant coefficients were not found for this model

Multiple Regression Analysis suggest that there the main factors capable to influence the levels of the compounds analyzed were: Als, Mgs, H+Al, Nas, Ss, Nl, Mnf, Col and Fel.

Multiple coefficient of determination (R²) means the total proportion of the total variation that is explained by the overall regression model (Bowerman et al., 2005), when the R² is higher, better the model fits the data. Multiple correlation coefficient (R) is the positive squared root of R², and is the method in regression that is employed to look how far the relationship between two variables (Levinm & Rubin, 1994).

Equations 1, 2 and 3 showed a high explicability as can be seen by the higher R² values, which the main environmental factors that could predict the concentration of these compounds were foliar nutrients. The Equation 1 presents a very strong correlation (R>0.90) between (E)-caryophyllene and the set of variables (Nas, Nl, Mnl, Col), mainly the foliar nutrients were the factors capable to influenced the levels of (E)-caryophyllene (Piaw, 2006). These results suggests a strong association between (E)-caryophyllene levels and foliar nutrients. The compound (E)-caryophyllene stands out, due its many known pharmacological properties, such as: anti-inflammatory (Tambe et al., 1996), anticarcinogenic (Zheng et al., 1992), cytotoxic (Kubo et al., 1996), spasmolytic and local anesthetic (Cabo et al., 1986). Besides, this component may suffer oxidation when air exposure, resulting (E)-caryophyllene oxide, which present moderate allergenic activity (Skold et al., 2006).

The observed positive relationship between Mnl and (E)-caryophyllene, α-humulene and α-zingiberene is in agreement with the requirement of sesquiterpenes synthases for a divalent metal ion as cofactor. The observed positive relationship between Mnl and (E)-caryophyllene, α-humulene and zingiberene is in agreement with the requirement of sesquiterpenes synthases for a divalent metal ion as cofactor. The nutrient Mg also showed positive influence over caryophyllenyl alcohol and caryophyllene oxide, because Mg also is a divalent cation (Picaud et al., 2005; Duarte et al., 2010; 2012). The formation of sesquiterpenes in ginger (Zingiber officinale Roscoe; Zingiberaceae) is favored with Mg²⁺ as cofactor, which corroborates the results found in this paper (Picaud et al., 2006).

Cluster analysis employing Ward's method was also applied to the study of similarity of samples on the basis of constituent distribution and the samples showed a highly chemical variability within the essential oil from leaves of S. jambos. The dendrogram represented in Figure 1 suggests that the main factor that seems to influence the composition is the collection time, due of the formation of two clusters, one of them with samples of January (Cluster I) and other group with samples of July (Cluster II). The Cerrado is characterized by two seasons: dry (April to September) and wet (October to March), which may have influenced the similarity profile of the samples in leaf essential oils from S. jambos, found in Cluster analysis (Santos et al., 2006).

The chemical variability in leaf essential oils from S. jambos determined by statistical analysis may reflect environmental influence on oil composition, although it may also have been promoted by genetic factors in cultivated samples (Duarte et al., 2010). This work suggests that there is an influence of environmental factors on the chemical composition of leaf essential oils of S. jambos, mainly foliar nutrients (N, Mn, Co, Fe, S and Mg) and soil nutrients (Na, Al, S and H+Al). The climate data seems had exerted a low influence due no significant correlation found in Multiple Regression Analysis, which is in agreement with Figure 1, where the dendrogram represents the localities as the main factor in the differences in samples.

Acknowledgment

We would like to thank the Brazilian government funding agencies CAPES and CNPq for the financial support.

Author's contributions

WPR contributed on interpretation of chromatograms and identification of structures; LLB contributed on statistical analysis, interpretation of results and drafting of the article; NMA in the extraction of essential oils; PHF contributed to essential oils analysis by GC-MS and JRP designed the study and drafting the manuscript.

Received 20 Dec 2012

Accepted 15 Apr 2013

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Publication Dates

• Publication in this collection
  14 May 2013
• Date of issue
  June 2013

History

• Received
  20 Dec 2012
• Accepted
  15 Apr 2013