2.2. 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.
In the root oil of C. winterianus, sesquiterpenoids were predominant, representing 84.5% of the total oil in which monoterpenoids were 9.8% and sesquiterpenes 4.7%. The result shows that the roots were rich in α-elemol (53.1%), α-eudesmol (18.9%), γ-eudesmol (7.5%), whereas the root hair and stalk oils are dominated by more than half by the monoterpenoids (52.1%), sesquiterpenoids (41.8%), and sesquiterpenes (3.1%) of the total oil. The principal compound of root hair and stalk was α-elemol (29.5%), geranial (10.7%), citronellal (8.6%), geraniol (8.6%), citronellol (7.5%), neral (6.5%), and geranyl acetate (5.3%). The composition of root and root hair and stalk oils are almost similar, presenting α-elemol as the major compound. The percentage of some compounds such as citronellol, citronellal, neral, geraniol, geranial, and geranyl acetate are higher in root hair and stalk EO, while, other compounds such as α-elemol, α-Eudesmol, and γ-Eudesmol are higher in the root oil of C. winterianus.
The leaves and root stalk and shoot oils largely contained monoterpenoids as their major components. The leaves’ EO contains (90.4%) monoterpenoids, (3.9%) sesquiterpenes, and (2.6%) sesquiterpenoids. The major compounds of leaves were geraniol (37.1%), followed by citronellal (13.7%), geranyl acetate (11.3%), geranial (11.0%), neral (8.3%), and citronellol (5.1%); this was in a close agreement with studies conducted by Rodrigues et al. [
4], except the presence of α-elemol in high amounts, which might be due to the addition of roots in their studies during EO extraction. In another study, it is unclear whether the part used in their extracted EO is either the leaves, stems or both of
C. winterianus; anyways, the principle components are citronellal, geraniol, and citronellol [
44]. Similarly, contrasting the type of
C. winterianus leaves EO composition and major component was geraniol (18.88%), citronellal (16.95%), elemenol (14.08%), and citronellol (12.57%), and we confirm that was not pure single-tissues EO, and there might be addition of other tissues [
45]. Meanwhile, 94.2% monoterpenoids, 1.8% sesquiterpenoids, and 0.5% sesquiterpenes were found in root stalk and shoot EO, where citral was the predominant constituent, consisting of geranial (59.0%) and neral (31.5%).
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.
C. citratus leaves’ EO contains neral (36.1%) and geranial (53.1%) monoterpene aldehydes as the major compounds, whereas (
E)-β-caryophyllene (1.0%) and caryophyllene oxide (1.3%) contain γ-cadinene (0.81%) in lower concentrations, which is similar to the study previously conducted by Boukhatem et al. [
18], except for the presence of β-myrcene in a significant amount.
C. citratus leaves’ EO from Vietnam also showed a comparable content of citral (neral and geranial), and the content of β-myrcene can be used to distinguish the origin of the EO [
46]. In the roots of
C. citratus EO, a sesquiterpene alcohol and monoterpene aldehydes were predominant compounds. The prime components were α-elemol (31.5%), geranial (25.0%), neral (16.6%), α-eudesmol (11.3%), and γ-eudesmol (5.2%);
trans-β-elemene (1.4%) was present in a lesser quantity. Thus, compounds such as α-elemol, α-eudesmol, and γ-eudesmol can be used to identify the source of
C. citratus EO.
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.
2.3. 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 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.
2.4. 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].