1. Introduction

Plant-derived essential oils are known and primarily used for their biological properties (Mesquita et al., 2005). Combined with this, the major interest of the pharmaceutical, food and cosmetics industries in the use of new oils as well as the consumer receptivity to new products of natural origin, transformed the evaluation methods of these plants into widely used tools in the search for new products (Aćimović et al., 2022).

The proportion of individual compounds in the oil composition differs from trace levels to over 90% (Bassolé and Juliani, 2012). Then, the complete separation and the correct identification of the essential oil compounds appear to be very important to a better understanding of the mechanisms involved in those biological activities and the prospection of new active compounds (Cagliero et al., 2022).

Mangifera indica L., belonging to the Anarcadiaceae family, is one of the 40 species of the Mangifera genus that can be found in tropical and subtropical regions of Southeastern Asia, Africa, and Latin America (Nikhal and Mahajan, 2010). Its fruits are considered multifunctional foods. However, other parts of this plant, such as bark, flowers, branches, and leaves, have also bioactive compounds (Gupta et al., 2022).

The leaves of M. indica have been used in the medical system of India to treat diseases such as asthma, dysentery, cough, leucorrhea, jaundice, pain, and malaria (Basha et al., 2011). In Brazil, the leaves are used as analgesic, anti-inflammatory and the treat hepatitis (Oliveira et al., 2022). The study of the aqueous extracts of the bark of a selected variety of M. indica resulted in a pharmaceutical formula, commercially named Vimang. The volatile constituents of M. indica fruits present a considerable variation in their chemical composition, which has been extensively investigated (Dzamić et al., 2010). The variability of the volatile constituents can be influenced by factors such as the stage of development, variety, and extraction method (Pino et al., 2005).

The essential oils of M. indica leaves from Egyptian varieties have antimicrobial activity (Ouf et al., 2021). The latex essential oil of M. indica from the ‘rosa’ and ‘espada’ varieties showed cytotoxic activity against HL-60 human tumor cells (Ramos et al., 2014). The volatile compounds in M. indica were usually obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry (GC/MS) (Ansari et al., 2000; Berenbaum et al., 1985; Dzamić et al., 2010; Moreno et al., 2010; Oliveira et al., 2017; Pino et al., 2005).

Due to the volatility and polarity of essential oils components, capillary gas chromatography is the preferable technique for their analysis because essential oils are generally complex mixtures of components with similar physicochemical characteristics (Aspromonte et al., 2019; Rubiolo et al., 2010). However, the satisfactory separation of a complex sample requires a higher peak capacity. In this case, comprehensive two-dimensional gas chromatography (GC×GC), a relatively new technique, can be the best alternative (Keppler et al., 2018).

Comprehensive GC×GC, idealized by Liu and Phillips (1991), has since emerged as the most powerful separation technique for analyzing volatile compounds. The satisfactory separation in complex samples, such as some essential oil, requires a higher peak capacity, achieved using GC×GC. In this technique, two independent separation mechanisms are used to resolve the compounds of complex samples within a single analysis, based on applying two GC columns with different stationary phases connected in series, with a transfer device defined as a modulator. The modulator’s function is continuously isolating, reconcentrating, and introducing small portions of the first (1D) effluent onto a second column (2D). The time required to complete this process is defined as the modulation period. Each 1D peak is modulated several times, preserving the 1D separation (Adahchour et al., 2008; Stefanuto et al., 2021).

The GC×GC has the advantage of increasing the resolution and sensitivity of the analysis due to the concentration of the sample fraction through the modulation process allowing the detection of compounds in trace levels as well as the separation of related compounds in the second dimension (Baharum et al., 2010). GC×GC is the most powerful separation system now available when combined with mass spectrometry (MS).

The time-of-flight mass spectrometer (TOFMS) can obtain high spectra acquisition rates for the correct peak assignment and quantification in GC×GC. However, its high cost limits its laboratory utilization. Some studies used GC×GC with a time-of-flight mass spectrometry detector (GC×GC/TOFMS) to analyze essential oils (Eyres et al., 2007; Ieri et al., 2019; Jalali et al., 2012; Rubiolo et al., 2010; Wang et al., 2012). The results obtained by these studies showed an important improvement in the characterization of these samples by GC×GC.

In the present study, the volatile compounds of two M. indica were analyzed by GC×GC/TOFMS to evaluate the difference between the compounds in the varieties, allowing an adequate selection for medicinal and industrial purposes.

2. Experimental

2.1 Samples

The leaves of the M. indica variety were collected in Campo Grande/MS, Brazil. The ‘espada’ variety (collected at 20°30’7” S and 54°37’17” W) and the ‘coração de boi’ variety (20°30’13” S and 54°37’14” W) were identified by Dr. Ronaldo Posella Zaccaro (Centro Universitário Moura Lacerda, Ribeirão Preto/SP, Brazil) and deposited with voucher specimens’ numbers CM105 and CM 107, respectively. All the used solvents and reference standards (linear alkanes) were HPLC grade (JT Baker and Sigma Aldrich). The collection was recorded in the SisGen, number AF9B3C3.

2.2 Essential oil

Each essential oil was isolated from a 400 g sample of fresh leaves of M. indica by hydrodistillation using a Clevenger-type apparatus. The essential oils were recovered, dried with anhydrous sodium sulfate, transferred to dark vials, and finally stored at –4 °C for further analysis. Before gas chromatographic analysis, the essential oils (1 mg) were diluted in 1 mL n-hexane. The essential oil yield calculated based on fresh leaves was 0.2% for ‘coração de boi’ and 0.3% for ‘espada’.

2.3 Chromatographic analysis

A GC×GC/TOFMS Pegasus-IV system (LECO, St. Joseph, USA) was equipped with a liquid nitrogen quad-jet modulator and CTC Combi Pal autosampler (CTC Analytics, Carrboro, NC, USA). Electron ionization was 70 eV, the mass acquisition was performed in the range of 50 to 550 amu at 100 Hz, and the detector voltage was –1,706 V. The injector, transfer line and detector temperature were maintained at 250 °C. A conventional column set was employed: DB-5 (5% phenyl–95% dimethylpolysiloxane) with 60 m length, an internal diameter of 0.25 mm, and 0.10 μm of film thickness in the first dimension and a DB-17ms (50% phenyl–50% dimethylpolysiloxane) with 2.15 m length, the internal diameter of 0.18 mm and 0.18 μm of film thickness. Both columns were acquired from Agilent Technologies – J&W Scientific (Palo Alto, CA, USA). The temperature program of the first column started at 50 °C for 5 min, heating at 3 °C min^–1 till 250 °C. The second column temperature was maintained 10 °C above the temperature of the first column. The modulation period was 10 s, and the Hot pulse was 40% of the modulation period. ChromaTOF software version 3.32 was employed for data processing the total ion current chromatogram, including tools such as peak finder and mass spectra deconvolution. Data processing was performed using a signal-to-noise ratio equal to three. The criterium for accepting a detected compound was a minimum of 80% similarity with the library.

Temperature-programmed retention indices (Mota et al., 2013) were calculated using a mixture of linear alkane (C6-C30), which was analyzed under the same conditions as the chromatographic analysis of the samples. The volatile components’ identification was based on comparing their mass spectra with those of the database NIST 2.0, the comparison of their retention index and mass spectrum (Adams, 2007) and the interpretation of the mass spectrum.

The results obtained in this study were compared with the data obtained using GC/MS by Oliveira et al. (2017).

3. Results and discussion

Some studies show that the main compounds of the essential oils obtained from mango leaves are sesquiterpenes and monoterpenes (Dzamić et al., 2010; Gerbara et al., 2011; Moreno et al., 2010; Pino et al., 2005). These results of this study corroborate the data on the composition of M. indica leaves in the ‘espada’ and ‘coração de boi’ varieties.

The essential oil from mango contains constituents such as α-gurjunene, trans-caryophyllene, α-humulene, α-selinene, and camphor (Kumar et al., 2021).

Ramos et al. (2014) identified 25 compounds in the essential oil of M. indica leaves using gas chromatography with flame ionization detection (GC-FID) and GC/MS. Fontenelle et al. (2017) studied the essential oil from different M. indica leaves by GC/MS, obtaining 20 compounds for the ‘Tommy Atkins’ variety, 13 for the ‘rosa’ variety, 6 for the ‘muscat’ variety and 15 for the ‘jasmine’ variety. Ouf et al. (2021) identified 31 compounds in the ‘Alphonso’ variety, 33 compounds in the ‘Sidik’ variety, 29 compounds in the ‘waste’ variety, 26 compounds in the ‘zebda’ variety and 31 compounds in the ‘fagri-kalan’ variety and trans-caryophyllene (8.06–18.88%), α-selinene (4.33–16.92%), and α-humulene (8.48–25.98%) were found in the higher concentrations. For the ‘coração de boi’ variety, cyperene, (E)-caryophyllene and α-humulene are the predominant compounds. Those compounds were also reported to be the most important in the leaves of the ‘coquinho’ variety by GC/MS analysis (Gerbara et al., 2011).

The study performed by Oliveira et al. (2017) identified 23 volatile compounds such as monoterpenes and sesquiterpenes from the leaves of M. indica ‘espada’ and ‘coração de boi’ varieties extracted by hydrodistillation and analyzed by GC/MS. In the essential oil of leaves obtained from the ‘espada’ variety, the major compounds were β-selinene (34.90%), cyperene (22.40%), (E)-caryophyllene (16.39%), α-humulene (10.84%), terpinolene (2.31%), and α-selinene (2.31%), while in ‘coração de boi’ variety, the major compounds were cyperene (32.62%), (E)-caryophyllene (26.91%), α-humulene (17.12%), β-selinene (5.70%), myrcene (2.80%), and b-phellandrene (2.70%) Oliveira et al. (2017).

The ‘espada’ and ‘coração de boi’ varieties generated essential oils of leaves with 125 and 95 tentatively identified compounds using the GC×GC/TOFMS technique. Due to its superior performance over the GC/MS, the GC×GC/TOFMS increased the number of identified peaks in M. indica essential oils.

In the column setup used, nonpolar in the 1D and medium polar in the 2D, the compounds are separated in the first dimension based on their different volatilities. In the second dimension, the separation is governed by polarity. Consequently, compounds with similar volatility had similar or even exact retention times in the 1D and will be resolved in the 2D. The GC×GC/TOFMS analyses revealed a complex organic compound mixture (Figs. 1 and 2). Combining a low polar 5% phenyl phase in the first dimension with a medium polar 50% phenyl phase in the second dimension allowed efficient use of the available chromatographic space. Figures 1 and 2 highlight the complexity of the M. indica essential oil and the efficiency of GC×GC to reduce the peak coelution, obtaining pure MS spectra and increasing peak detectability.

The experimental linear retention indices show a good agreement between the identified compounds and the linear retention indices reported by literature for 1D-GC (Bogusz Junior et al., 2011). The list of identified compounds is shown in Table 1.

Furthermore, the GC×GC increased the detectability of the compounds due to using the modulator (Baharum et al., 2010), as can be observed in the increase in the number of compounds. These peaks were present at low concentrations, but the improvement of their signal by GC×GC achieved better mass spectra and separation than in the 1D-GC. In several cases, it was found that, despite using two chromatographic separation columns, some compounds were still coeluting. The essential oils have many isomers with similar retention times and mass spectra, especially sesquiterpenes and oxygenated sesquiterpenes. However, peak deconvolution algorithms allowed for resolving chromatographic solutions and extracting the mass spectrum of each compound, even in such situations. GC×GC promoted the identification of fivefold more compounds in the two essential oils than GC/MS.

The major constituents of ‘espada’ variety essential oil identified by GC×GC/TOFMS were β-selinene (10.2%), α-humulene (6.9%), α-selinene (6.7%), (E)-caryophyllene (5.2%), ciperene (5.0%), Drima-7.9(11)-diene (4.8%), γ-carene (4.2%), caryophyllene oxide (3.95%), italicene (3.5%) and β-biotol (3.4%). Ninety-five compounds were identified in essential oil of the ‘coração de boi’ variety by GC×GC/TOFMS and the major constituents were β-selinene (10.4%), α-humulene (7.2%), ciperene (7.0%), (E)-caryophyllene (6.5%), α-selinene (5.0%), Drima-7.9(11)-diene (4.9%, β-biotol (4.0%) and β-elemene (3.1%). A total of 31 compounds were identified exclusively in the ‘espada’ variety. The two essential oils were characterized by the predominance of β-selinene, (E)-caryophyllene and α-humulene. Also, monoterpenes were found in small concentrations in the two oils.

The use of GC×GC provided enhanced efficiency, mainly for minor compounds. The results showed a considerable increase in the number of separated compounds. In addition, the analysis of mass spectra data together with the retention index allowed the identification of three times more compounds which reflected a differentiation between the essential oils studied.

4. Conclusions

This study demonstrates the applicability of the GC×GC/TOFMS for the comprehensive profiling of M. indica essential oils. It also indicated that two-dimensional gas chromatography had a superior resolution, making it possible to identify more compounds. One hundred and twenty-five and ninety-five compounds were tentatively identified in the two studied essential oils of the ‘espada’ and ‘coração de boi’ varieties, respectively. These results showed that the compositions of the two analyzed essential oils showed differences in relation to the GC×GC/TOFMS and conventional chromatography technique, the GC/MS.

Authors’ contribution

Conceptualization: Cardoso, C. A. L.;

Data curation: Cardoso, C. A. L.;

Formal Analysis: Cardoso, C. A. L.; Castro, T. L. A.;

Funding acquisition: Cardoso, C. A. L.;

Investigation: Cardoso, C. A. L.; Simionatto, E.;

Methodology: Cardoso, C. A. L.; Caramão, E.;

Project administration: Cardoso, C. A. L.;

Resources: Cardoso, C. A. L.;

Software: Cardoso, C. A. L.;

Supervision: Cardoso, C. A. L.; Caramão, E.;

Validation: Cardoso, C. A. L., Simionatto, E.;

Visualization: Cardoso, C. A. L.;

Writing – original draft: Cardoso, C. A. L.; Simionatto, E.; Caramão, E.;

Writing – review & editing: Cardoso, C. A. L.; Castro, T. L. A.

Data availability statement

All data sets were generated or analyzed in the current study.

Funding

Not applicable.

Acknowledgments

Not applicable.

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Notas de autor

claudia@uems.br