Chemical profiling of essential oil from Piper betle L. cv. Mysore local by GC–MS/MS

Introduction

Piper betle L., commonly known as betelvine, is an important perennial climber belonging to the family Piperaceae and is widely cultivated in tropical and subtropical regions. The leaves are traditionally consumed as a masticatory and are also valued for their culinary, cultural and medicinal significance. Betelvine germplasm comprises numerous cultivars and locally recognized types that differ in morphological, sensory and biochemical characteristics (Chattopadhyay and Maity, 1967) [4] . The characteristic aroma of betel leaves is primarily attributed to their essential oils, which contain diverse volatile constituents, including phenolic and terpene-like compounds (Bajpai et al., 2010a) [2] . Chemical profiling of betelvine leaf essential oils has demonstrated variation in their chemical composition among different landraces, with distinct chemical profiles reported for major betelvine groups (Rawat et al., 1989) [13] . Such variation in volatile composition may contribute to differences in the characteristic aroma and other phytochemical properties of betelvine types. Chemical characterization of essential oils provides a useful approach for documenting variation in volatile composition among plant materials. Gas chromatography coupled with mass spectrometry (GC-MS) is a widely used analytical approach for separation and characterization of volatile constituents based on their chromatographic and mass-spectral properties. Previous studies have reported considerable variation in the essential-oil composition of P. betle and have identified diverse volatile constituents associated with the characteristic chemical profile of betel leaves (Bajpai et al., 2010a; Rawat et al., 1989) [2,13] . The reported biological properties of betelvine essential oils and their constituents further emphasize the importance of characterizing the volatile fraction (Kumar et al., 2010; Rai et al., 2011) [8,12] .

Although several betelvine types have been characterized for their chemical constituents, cultivar-specific information on essentialoil composition remains valuable for documenting the diversity of volatile compounds. Mysore Local is one of the betelvine types maintained among the germplasm evaluated in the present study. Therefore, the present investigation was undertaken to characterize the essential-oil profile of Piper betle L. cv. Mysore Local using GC-MS/MS and to identify its major volatile constituents based on retention-index and mass-spectral information.

Materials and Methods

Plant material

Fresh leaves of Piper betle L. cv. Mysore Local were used for essential-oil analysis. The cultivar was maintained as part of the betelvine germplasm evaluated in the study.

Essential oil extraction and sample preparation

Essential oil from P. betle cv. Mysore Local was obtained by hydrodistillation using a standardized procedure developed during the study. The resulting essential oil was subjected to GC-MS/MS analysis for characterization of its volatile constituents.

GC-MS/MS analysis

The essential oil was analyzed using a Shimadzu GC-MS/MS-TQ8040 system equipped with a benchtop triple-quadrupole mass spectrometer operated in electron ionization (EI) mode. An AOC-20i autosampler was used for sample injection. Chromatographic separation was performed using an SP™ 2560 capillary column (100.0 m length × 0.25 mm internal diameter × 0.2 µm film thickness). The oven temperature was initially maintained at 100 °C for 5 min, followed by an increase at 4 °C/min to 220 °C, where it was maintained for 30 min, resulting in a total run time of 65 min. Helium was used as the carrier gas at a constant flow rate of 1.00 mL/min. An injection volume of 1 µL was used, with the injector temperature maintained at 220 °C and a split ratio of 40:1. The MS interface and ion-source temperatures were maintained at 220 °C, and the solvent cut time was 13.2 min. The mass scan range was 45-500 m/z, with a sampling speed of 1666. The relative abundance of individual volatile constituents was expressed as percentage peak area based on the total chromatographic peak area. Compound identification was carried out by comparing retention indices calculated using a homologous series of n-alkanes (C5-C32) with reference information and by matching the acquired mass spectra against the NIST 2015 spectral library.

Results

Essential-oil composition of P. betle cv. Mysore Local

GC-MS/MS analysis of the essential oil of P. betle cv. Mysore Local revealed 25 identifiable volatile constituents, accounting for approximately 100% of the total chromatographic peak area (Table 1). Considerable variation was observed in the relative abundance of individual compounds (Figure 1). Caryophyllene was the predominant constituent, accounting for 13.08% of the total peak area, followed by Germacrene D (10.52%) and Cubenene (8.95%). Other major constituents included β-elemene isomer (6.38%), γ-muurolene (5.77%), Bicyclo [5.2.0] nonane, 2-methylene-4,8,8-trimethyl-4-vinyl-(5.37%), and Spathulenol (5.32%). Eucalyptol (4.50%), α-Cadinol (4.29%), (-)-Globulol (4.21%), β-Ocimene (3.20%), Viridiflorol (3.33%), Epicubenol (2.95%) and Neointermedeol (2.70%) were also detected. Other constituents included Virdiflorene (3.46%), δ-Elemene (1.42%), α-Copaene (2.35%), Linalool (2.22%), Eremophylene (2.27%), Bicyclogermacrene (2.09%), Safrole (0.81%), Junenol (1.24%), Rosifoliol (1.50%), Methyleugenol (0.69%) and τ-Cadinol (1.36%).

Discussion

The essential oil of Piper betle L. cv. Mysore Local exhibited a chemically diverse profile comprising 25 tentatively identified volatile constituents, representing different classes of terpenoid and related compounds. Considerable variation in the relative abundance of individual constituents was observed, with caryophyllene (13.08%), germacrene D (10.52%) and cubenene (8.95%) representing the three major components. Together, these compounds accounted for approximately 32.55% of the total chromatographic peak area, indicating their substantial contribution to the volatile profile of cv. Mysore Local. The occurrence of diverse sesquiterpenes, monoterpenes and phenylpropanoid-related compounds is consistent with the reported chemical complexity of P. betle essential oils (Bajpai et al., 2010a; Rawat et al., 1989) [2,13] . Caryophyllene was the predominant constituent in the essential oil of Mysore Local, accounting for 13.08% of the total peak area. Caryophyllene has previously been reported as an important constituent of P. betle essential oils, although its relative abundance varies considerably among genotypes and cultivars. Earlier studies have reported caryophyllene in different P. betle materials, including Bangla betel leaves and cured betel leaves (Joseph and Kalesh, 2015; Saini et al., 2020; Madhumita et al., 2019) [7,14,10] . The relatively high abundance of caryophyllene observed in Mysore Local therefore contributes substantially to the sesquiterpene-rich character of its essential-oil profile. Germacrene D was the second major constituent, accounting for 10.52% of the total peak area. Germacrene D has been reported as a prominent sesquiterpene in certain P. betle varieties, including Sirugamani-1 and Kapoori (Sugumaran et al., 2011; Chakravarthi et al., 2018) [16,3] . The relatively high level of germacrene D in Mysore Local is therefore consistent with previous observations demonstrating the contribution of germacrene-type sesquiterpenes to the volatile composition of betelvine. Cubenene was the third major constituent, representing 8.95% of the total chromatographic area. Other relatively abundant constituents included β-elemene isomer (6.38%), γ-muurolene (5.77%) and spathulenol (5.32%). The occurrence of these compounds indicates that sesquiterpenes and oxygenated sesquiterpenes constitute an important component of the essential-oil profile of Mysore Local. Considerable variation in germacrene-related compounds, muurolenes and oxygenated sesquiterpenes has also been reported among P. betle accessions from different geographical regions (Gocchi et al., 2023; Saini et al., 2020) [6,14] .

The presence of γ-muurolene (5.77%) further supports the importance of sesquiterpenes in the essential oil of Mysore Local. Previous studies have reported γ-muurolene in P. betle essential oils at variable concentrations, demonstrating differences in its accumulation among betelvine genetic materials (Gocchi et al., 2023; Saini et al., 2020) [6,14] . Similarly, the detection of oxygenated sesquiterpenes such as spathulenol, α-cadinol, viridiflorol and globulol indicates the chemical complexity of the oil. Comparable oxygenated sesquiterpenes have been reported in P. betle accessions from different geographical regions, including δ-cadinol, caryophyllene oxide, spathulenol and α-cadinol. Monoterpene-related constituents were also detected in the essential oil of Mysore Local. Eucalyptol (4.50%), β-ocimene (3.20%) and linalool (2.22%) were present at lower relative abundances than the major sesquiterpene constituents. Linalool has previously been reported in P. betle essential oils, with considerable variation among genotypes. Saini et al. (2020) [14] reported relatively high linalool content in Bangla betel, while its occurrence in Sri Lankan accessions has also been documented (Kumaratunge et al., 1999; Saini et al., 2020) [9,14] . The moderate level of linalool observed in Mysore Local indicates that it contributes to the volatile profile without being a predominant constituent of this cultivar. Phenylpropanoid-related constituents were detected at comparatively low levels in Mysore Local. Safrole (0.81%) and methyleugenol (0.69%) were present in the analyzed oil. The relatively low abundance of these compounds, compared with the major sesquiterpenes, suggests that the essential-oil profile of Mysore Local is predominantly characterized by terpenoid constituents rather than being strongly phenylpropanoiddominant. Safrole-rich chemotypes have been reported in several P. betle cultivars and geographical types, demonstrating substantial variation in phenylpropanoid composition among betelvine genetic materials (Arambewela et al., 2005; Saxena et al., 2014; Dwivedi and Tripathi, 2014) [1,15,5] . The identification of 25 volatile constituents using retentionindex information together with mass-spectral library matching provides a useful chemical profile of P. betle cv. Mysore Local. The combined use of retention indices and mass-spectral comparison provides greater confidence in compound annotation than reliance on mass-spectral matching alone. However, since authentic reference standards were not used for confirmation of each individual compound, the detected compounds are appropriately described as tentatively identified rather than definitively identified. [1, 15, 16, 6] . The variation in major volatile constituents reported among P. betle genotypes supports the usefulness of essential-oil profiling for documenting chemical diversity within the species. In the present study, the predominance of caryophyllene, germacrene D and cubenene distinguishes the volatile profile observed in Mysore Local from profiles dominated by other compounds in different betelvine genetic materials. Such cultivarassociated differences may contribute to variation in the characteristic aroma and other phytochemical attributes of betelvine. However, direct relationships between individual volatile constituents and sensory characteristics would require dedicated sensory and aroma studies. Overall, the essential oil of P. betle cv. Mysore Local was characterized predominantly by sesquiterpenoid constituents, with caryophyllene, germacrene D and cubenene as the major components. The presence of multiple sesquiterpenes, oxygenated sesquiterpenes, monoterpenes and phenylpropanoidrelated compounds highlights the chemical complexity of this cultivar. The profile obtained in the present study provides useful baseline information for comparison with other P. betle cultivars and for future investigations into the relationships between essential-oil composition, aroma characteristics and biological properties.

Conclusion

The essential oil of Piper betle L. cv. Mysore Local exhibited a diverse volatile profile comprising 25 tentatively identified constituents. Caryophyllene (13.08%), Germacrene D (10.52%) and Cubenene (8.95%) were the predominant constituents, followed by β-elemene isomer, γ-muurolene, a bicyclo[5.2.0]nonane derivative and spathulenol. The GC-MS/MS profile demonstrated considerable chemical diversity within the essential oil and provided useful information for the chemical characterization of the Mysore Local cultivar. The volatile profile obtained in the present study provides a useful basis for future comparative studies of essential-oil composition among betelvine cultivars.

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