Essential Oil Composition Analysis of Cymbopogon Species from Eastern Nepal by GC-MS and Chiral GC-MS, and Antimicrobial Activity of Some Major Compounds

Cymbopogon species essential oil (EO) carries significant importance in pharmaceuticals, aromatherapy, food, etc. The chemical compositions of Cymbopogon spp. Viz. Cymbopogon winterianus (citronella) Cymbopogon citratus (lemongrass), and Cymbopogon martini (palmarosa) were analyzed by gas chromatography–mass spectrometry (GC-MS), enantiomeric distribution by chiral GC-MS, and antimicrobial activities of some selected pure major compound and root and leaves EOs of citronella. The EO of leaves of Cymbopogon spp. showed comparatively higher yield than roots or other parts. Contrary to citral (neral and geranial) being a predominant compound of Cymbopogon spp., α-elemol (53.1%), α-elemol (29.5%), geraniol (37.1%), and citral (90.4%) were detected as major compounds of the root, root hair with stalk, leaf, and root stalk with shoot of citronella EO, respectively. Palmarosa leaves’ EO contains neral (36.1%) and geranial (53.1) as the major compounds. In the roots of palmarosa EO, the prime components were α-elemol (31.5%), geranial (25.0%), and neral (16.6%). Similarly, lemongrass leaves’ EO contains geraniol (76.6%) and geranyl acetate (15.2%) as major compounds, while the root EO contains a higher amount of geraniol (87.9%) and lower amount of geranyl acetate (4.4%). This study reports for the first time chiral terpenoids from Cymbopogon spp. EOs. Chiral GC-MS gave specific enantiomeric distributions of nine, six, and five chiral terpenoids in the root, root stalk with a shoot, and leaves of citronella EOs, respectively. Likewise, four and three chiral terpenoids in the root and leaves of lemongrass oil followed by two chiral terpenoids in the leaves and root of palmarosa EOs each. Additionally, the root and leaves’ EOs of citronella exhibit noticeable activity on bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes and fungus such as Candida albicans, Microsporum canis, and Trichophyton mentagrophytes. So, geranial-, neral-, geraniol-, and citronellal-rich EOs can be used as an alternative antimicrobial agent.


Introduction
The genus Cymbopogon (Poaceae) comprises up to 144 species distributed in Asia, America, and Africa [1,2], among which Cymbopogon winterianus Jowitt ex Bor, Cymbopogon citratus Stapf, and Cymbopogon martini (Roxb.) Will. Watson are the economically important species of the genus [3]. The species C. winterianus is popularly known as citronella grass, citronella, or Java citronella [4]. Citronella is one of the industrially important essential oils (EO), which is produced by steam or hydro-distillation of the whole plant [5] and has a characteristic lemon odor [6]. The world consumption of citronella EO has amounted to several thousand tons annually due to being the most important source of geraniol and citronellal [7]. It is also known for its natural insect-repellent property and is of great

Essential Oil Yields
The EO yields of three Cymbopogon species, namely C. winterianus, C. citratus, and C. martini are presented in Table 1. The hydro-distillation of leaves of all three Cymbopogon species yielded 1.3-2.2% essential oil, which is remarkably high compared with other parts of the plant. The previously reported hydro-distillation yield of C. winterianus was 1.50 ± 0.15% (w/w) and with better extraction yield in comparison with steam distillation [39]. Similarly, the hydro-distillation extraction yield of C. citratus was 0.98%, which was quite lower as compared with microwave-assisted hydro-distillation. Our extraction yield was closely in agreement with previously reported microwave-assisted hydro-distillation yields [40]. In the case of C. martini leaves, the yield of extraction was (1.4756 w/w %), which is in close agreement with our hydro-distillation yield [41].

Chemical Composition of Cymbopogon Species
The major compounds present in the different tissues of cymbopogon species are presented in Figure 1. Several studies have been reported on the EO composition of C. winterianus, which reveals high variability in its chemical fingerprint. So, to reveal the mystery behind this variability for the first time, we studied the composition of EOs extracted from different parts of C. winterianus. The results of GC-MS analysis of C. winterianus EOs from different parts are presented in Supplementary File (Table S1), and Table 2 shows only the major selected constituents.

Enantiomeric Distributions Analysis of Cymbopogon Species Essential Oils
The enantiomeric distributions of chiral terpenoids present in Cymbopogon specie EOs are presented in Table 5. In previous studies, the enantiomeric distributions of chir   [42,43]. "t" indicates trace (≤0.05%) and "-" indicates not detected.
The results of GC-MS analysis of Cymbopogon citratus EOs extracted from roots and leaves are tabulated in Supplementary File (Table S2), and Table 3 shows only the selected major constituents.
The results of GC-MS analysis of Cymbopogon martini EOs extracted from leaves and roots are presented in Supplementary File (Table S3), and Table 4 shows only the selected constituents. The C. martini leaves' EO contains geraniol (76.6%), and geranyl acetate (15.2%) as major compounds, which is comparable to the previous study conducted by Jnanesha et al. [27]. Geraniol content in the C. martini leaves' EO up to 80 days was noticeably increased, whereas geranyl acetate decreased significantly at that time and positively correlated [3]. Interestingly, one of the C. martini cultivars from India showed limonene as a major compound [47], while, the C. martini root EO contains a higher amount of geraniol (87.9%) compared with the leaves' oil, which ultimately leads to a decrease in the content of geranyl acetate (4.4%). The underlying reasons for the differences in EOs composition could be attributed to genotype, edaphic variables, geographical location, pedo-climatic conditions, harvest time, extraction procedure, maturity of plant, different part of plant material, and analytical procedures.  [42,43]. "t" indicates trace (≤0.05%).

Enantiomeric Distributions Analysis of Cymbopogon Species Essential Oils
The enantiomeric distributions of chiral terpenoids present in Cymbopogon species EOs are presented in Table 5. In previous studies, the enantiomeric distributions of chiral terpenoids have been successfully used for species identification and adulteration detection of different EOs [38,48]. To the best of our knowledge, this is the first report on enantiomeric distributions of chiral terpenoids from Cymbopogon species Viz C. winterianus, C. citratus, and C. martini. There were, altogether, nine chiral terpenoids detected in various parts of Molecules 2023, 28, 543 6 of 12 citronella Eos, among which linalool, terpinen-4-ol, bornyl acetate, borneol, α-terpineol, and (E)-β-caryophyllene were levorotatory. However, citronellal and citronellol were detected as dextrorotatory compounds. On the contrary, germacrene D was levorotatory in the root and dextrorotatory in leaves, as well as root stalk and shoots.  There were, altogether, five chiral terpenoids detected in lemongrass root and leaves EOs. Among these, linalool and (E)-β-caryophyllene were levorotatory and detected in both root and leaf oil. However, citronellol and citronellal were dextrorotatory and detected only in root EO. Germacrene D, on the other hand, was dextrorotatory but detected only in the lemongrass leaves' oil. Lastly, only two chiral terpenoids were detected in palmarosa root and leaves' EOs, namely linalool and (E)-β-caryophyllene; linalool was dextrorotatory and (E)-β-caryophyllene was 100% levorotatory.
Thus, the enantiomeric distributions of chiral terpenoids present in Cymbopogon species Viz C. winterianus, C. citratus, and C. martini will be helpful to establish the chemical fingerprint of these species and also in the adulteration detection of EOs of these species.

Antimicrobial Activity of Cymbopogon Winterianus Essential Oil and Some Major Compounds
The MIC values of Cymbopogon winterianus EOs and some pure compounds such as geraniol, (±) citronellol, citral, and (−) citronellal, and that of the positive control gentamicin against a panel of bacterial and fungal strains, were determined through a two-fold broth microdilution method. This study showed that the assayed root and leaves of C. winterianus EOs have variable microbial inhibitory activities, as presented in Table 6. Plants having secondary metabolites and the EOs of C. winterianus have demonstrated a broad range of antimicrobial activities against different pathogens [11,12,49,50]. The leaf part of C. winterianus EO showed effectiveness against Pseudomonas aeruginosa, with an MIC of 78.1 µg/mL and noticeable activity against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes, with an MIC of 156.3 µg/mL, while other panels of bacterial strains had no surprising results. The Eos of C. winterianus demonstrated weaker antibacterial activities than those of the positive control, gentamicin (MIC < 19.5 µg/mL). It is difficult to speculate as to which components in the root and leaves of C. winterianus Eos may be responsible for the antibacterial activity. In the case of Staphylococcus aureus, pure component citral (MIC = 78.1 µg/mL) is more active than EO. It might be due to the antagonistic effect of individual components present in EO. In the case of Pseudomonas aeruginosa, the leaf EO is more potent than either of the tested pure components, which might be due to the synergetic mechanism among components of EO.
The EO from the leaves of C. winterianus displayed potent antifungal activity against Aspergillus niger, Aspergillus fumigatus, and Trichophyton mentagrophytes (MIC = 78.1 µg/mL). Both the leaves and root parts of C. winterianus EOs showed good activity against Candida albicans, Microsporum canis, and Trichophyton mentagrophytes, with MIC values of 156.3µg/mL. The EOs of C. winterianus demonstrated weaker antifungal activities than those of the positive control, amphotericin B (MIC < 19.5 µg/mL). In the cases of Trichophyton mentagrophytes, Aspergillus fumigates, and Aspergillus niger, the synergetic effect is more pronounced in the leaves as compared with root EO. In the case of, Aspergillus niger, pure component (−) citronellal (MIC = 78.1 µg/mL) is more active than root EO. On the other hand, despite the absence of (−) citronellal, as indicated by chiral GC-MS analysis in leaves' oil, it shows effectiveness against Aspergillus niger, which might be due to the synergistic effect of individual components of leaves EO. However, in the cases of Candida albicans and Trichophyton rubrum, there was no antagonistic and synergetic effect pronounced. C. winterianus leaves EO showed promising antimicrobial properties and can be used in lieu of synthetic chemicals to counter microbial attacks. Additionally, the leaves of C. winterianus EO were more potent than the root oil. This may be due to differences in chemical compositions of EOs in roots and leaves. Alternatively, the antimicrobial properties of C. winterianus EO may be the presence of secondary metabolites such as citronellal, citronellol, geraniol, neral, geranial, and other components by synergistic and antagonistic mechanisms. The antifungal and antibacterial mechanisms of action of EOs are not clearly understood yet. However, it has been postulated that the hydrophobic constituents either disrupt cytoplasmic membranes via a cascade of different reactions leading to cytoplasmic leakage, cell lysis, and ultimate death, or via the inhibition of sporulation [51,52].

Plant Material and Isolation of Essential Oils
Three cultivated species of Cymbopogon, namely C. winterianus, C. citratus, and C. martini were collected in March, 2021 from Sunsari (26 • 42 19.6 N 87 • 15 29.7 E), Nepal, presented in Figure 2. Different parts of plants were separated, washed, and then hydrodistilled for 6 h using a Clevenger apparatus, as previously described [48]. The obtained EOs were dried with anhydrous sodium sulfate and stored in bottles at 4 • C until further research was conducted. The Cymbopogon species EOs yields are summarized in Table 1.

Plant Material and Isolation of Essential Oils
Three cultivated species of Cymbopogon, namely C. winterianus, C. citratus, and C. martini were collected in March, 2021 from Sunsari (26°42′19.6″ N 87°15′29.7″ E), Nepal, presented in Figure 2. Different parts of plants were separated, washed, and then hydrodistilled for 6 h using a Clevenger apparatus, as previously described [48]. The obtained EOs were dried with anhydrous sodium sulfate and stored in bottles at 4 °C until further research was conducted. The Cymbopogon species EOs yields are summarized in Table 1.

Chemical Composition Analysis by Gas Chromatography/Mass Spectrometry (GC-MS)
Analysis of the chemical constituents in the Cymbopogen species (C. winterianus, C. citratus, and C. martini) EOs was carried out using Shimadzu GCMS-QP2010(Shimadzu Corp, Columbia, MD, USA) Ultra under the following condition: mass selective detector (MSD), operated in the EI mode (electron energy = 70 eV), with scan range = 40-400 m/z and scan rate of 3.0 scans/s, as previously described [53,54]. Identification of the individual components of the EOs was determined by comparison of the retention indices determined by reference to a homologous series of n-alkanes and comparison of the mass spectral fragmentation patterns (over 80% similarity match) with those reported in the literature [43] and our own in-house library [42] using the LabSolutions GC-MS solution software version 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA).

Enantiomeric Analysis by Chiral Gas Chromatography-Mass Spectrometry (CGC-MS)
A Shimadzu GC-MS-QP2010S with EI mode (70 eV) and B-Dex 325 chiral capillary GC column was used to perform the enantiomeric analysis of Cymbopogen species (C. winterianus, C. citratus, and C. martini) Eos, as previously described [55]. A comparison of retention times and mass spectral fragmentation patterns with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA) was used to identify the enantiomers. Table 5 shows the enantiomeric distribution of chiral terpenoids from Cymbopogen species (C. winterianus, C. citratus, and C. martini) EOs

Conclusions
As far as we are aware, this is the first report on the EOs of three Cymbopogon species (C. winterianus, C. citratus, and C. martini) from Sunsari, eastern Nepal that includes not only chemical composition analysis by GC-MS but also enantiomeric composition by chiral GC-MS. The results show variations in volatiles' compositions and enantiomeric distributions of chiral terpenoids. The yield of extraction varies depending upon the part used. The study can be used to create a benchmark for future Cymbopogon species' EOs assessments, as well as authentication for adulteration or consumer safety. In addition, the antibacterial and antifungal activity of some selected pure compounds and leaves of Cymbopogon winterianus EO (rich in citral, citronellal, citronellol, and geraniol) suggests that it can be used in lieu of synthetic antimicrobial agents. It is unclear which of the individual components is responsible for the antimicrobial activity. However, it is likely that synergistic effects are more pronounced for the components' activity.