Yield
As presented in Table 1, the yield of the essential oil obtained from Citrus sinensis (L.) peel was 2.05% (v/p).
Table 1. Yield of Citrus sinensis (L.) peel oil.
Plant Sample
|
Month/Year
|
Temperature
|
Sample Label
|
Plant Mass
|
Yield
|
Citrus sinensis (L.) peel oil
|
Nov./2019
|
27 °C
|
CsPO*
|
400 g
|
2.05%
|
Santos and colleagues [36] conducted research on the extraction of essential oil from Citrus sinensis (L.) peel and obtained a yield of 0.4% (v/p) from segregated whole peels. In a separate study, Fernandes and colleagues [37] obtained an essential oil yield of 2.93% (v/p) for segregated and crushed peels, and 0.53% (v/p) for segregated whole peels in distillations lasting three hours. Due to the variations in extraction methods based on plant parts and intended applications [38], the yield of a specific oil can vary significantly depending on the chosen extraction method.
Chemical Composition
The chemical composition of the CsPO under consideration, along with the area, retention time, calculated Kovats index, and tabulated Kovats index for each compound, are detailed in Table S1 (Supplementary material).
A total of thirty-three compounds, including both monoterpenes and sesquiterpenes, were detected and observed on the chromatogram through GC-MS analysis. Among these, the predominant compounds identified encompass the hydrogen monoterpenes β-citronelene (7.33%) and β-pinene (9.63%), the oxygenated monoterpenes car-3-en-2-one (8.43%), eugenol (4.24%), and limonene-10-ol (8.26%), as well as the hydrogen sesquiterpenes α-humulene (10.52%), α-neo-Clovene (4.83%), and β-acoradiene (12.47%).
In Ganzhou, China, the essential oil obtained from Citrus essensis peel was found to comprise 37 components, constituting 98.05% of the total essential oil [39]. The primary constituents identified were limonene (88.25%), terpinen-4-ol (1.98%), β-myrcene (1.90%), and linalool (1.50%). Similarly, in Guangdong, China, Li and colleagues [40] conducted a characterization of the Citrus sinensis peel oil, revealing the presence of 28 components accounting for 85% of the total essential oil. Notably, the key components in this case were d-limonene (49.14%), neral (9.54%), and eucalyptol (3.55%).
Conversely, in Brazil, Dias and colleagues [41] extracted essential oil from Citrus sinensis peel collected in Rio Verde (GO). Their analysis identified four components, primarily limonene (98.54%), δ-2-carene (0.27%), δ-2-carene (0.74%), and tricyclene (0.25%), collectively amounting to 99.80% of the oil’s content.
In this investigation, the CsPO sourced from Rio Preto (AM), Brazil, exhibited a profile of 33 compounds. Surprisingly, limonene did not dominate the composition, marking a departure from findings in other studies. Instead, the principal constituents in this instance were β-acoradiene (12.47%) and α-humulene (10.52%), two components not previously reported by the mentioned researchers. Moreover, it should be noted that the main components demonstrated a more balanced distribution within this study.
Biological Assays
Determination of Antimicrobial Activity
The antimicrobial activity of CsPO, evaluated by means of the agar-diffusion technique, is depicted in Table 2 and Fig. S2 (supplementary material).
Table 2. CsPO antimicrobial activity against Enterococcus faecalis, Escherichia coli, Candida albicans, and Mycobacterium smegmatis.
Strains
|
Sample
|
E. faecalis
|
E. coli
|
C. albicans
|
M. smegmatis
|
CsPO
|
++
|
+
|
++
|
++
|
Low (+): diameter of inhibition halo between 7 and 12 mm; Moderate (++): diameter of inhibition halo between 13 and 16 mm; High (+++): diameter of inhibition halo greater than 17 mm; Negative (-): absence of halo.
The findings indicate notable antimicrobial efficacy against Enterococcus faecalis, Escherichia coli, Candida albicans, and Mycobacterium smegmatis, with inhibition halos measuring 13 mm (++), 12 mm (+), 13 mm (++), and 16 mm (++), respectively.
According to Bajpai and colleagues [42], the antimicrobial efficacy of essential oils may be attributed to their ability to permeate the bacterial membrane and access the cell interior. This process inhibits the functional attributes of the cell. The presence of porin proteins in the bacterial membrane, the existence of these proteins in gram-negative bacteria, and the intracellular distribution of essential oil constituents are pivotal factors influencing the diffusion and action of essential oils within the cells. These elements contribute to the variation in essential oil activity against different bacterial groups [43, 44]. This alteration in permeability is also reported by Lambert and colleagues [43], supporting the notion that hydrophobic compounds undermine the integrity of bacterial cell membranes.
Costa and colleagues [45] demonstrated that the antimicrobial efficacy of essential oils derived from Citrus fruit peel is closely tied to their chemical composition, which can be shaped by the extraction technique and the plant’s growth environment. In the context of this research, CsPO displayed a moderate level of antimicrobial activity against E. faecalis. This suggests that the activity could stem from major components like limonen-10-ol and β-pinene, or possibly from a synergic effect among the various constituents.
Ambrósio and colleagues [46] propose that gram-negative bacteria tend to display higher tolerance to essential oil treatments due to their comparatively impermeable outer membranes. This outer membrane structure obstructs the penetration of hydrophobic essential oils. This phenomenon was observed in the case of E. coli (a gram-negative bacterium), which exhibited greater resilience and consequently a smaller inhibition halo (+) when exposed to 30 µL of the essential oil in the agar-diffusion technique. In the study conducted by Atolani and colleagues [47], the inhibition halo sizes for E. coli ranged from 10 to 18 mm, and for the fungus C. albicans, they varied between 10 and 16 mm. Conversely, with the gram-positive bacterium E. faecalis, CsPO demonstrated significant inhibition. This finding suggests that E. faecalis poses a more fragile barrier against external agents compared to other bacterial strains.
The research conducted by Abers and coauthors [48] investigated the antimicrobial potential of the essential oil from C. sinensis peel against the bacterium M. smegmatis. They experimented with varying volumes of oil, from 20 to 40 µL, and achieved inhibition halos ranging from 19 to 22 mm, thus confirming its efficacy. The inhibition halos of 13-16 mm observed at 30 µL underscore the efficiency and applicability of the antimicrobial attribute of this essential oil.
Assessment of Allelopathic Effect
Aromatic fruit essential oils play several roles in plant interactions, apart from being crucial allelochemical sources [49] that influence the germination of different species [50]. It is important to recognize that allelochemicals, contingent on their concentration and the species under examination, can either promote or hinder seed development by modifying numerous metabolic processes linked to germination [51, 52].
CsPO was subjected to an allelopathic investigation by employing various oil concentrations and assessing their impact on seed germination. Table 3 highlights that the rate index, mean germination time, and rate coefficient remained consistent, indicating that CsPO did not influence these parameters. However, a notable contrast in root growth and germination rate of butter bean seeds was evident in Treatment 4 (T4), in which 500 µL of CsPO was employed. CsPO fostered significant root growth, surpassing the outcomes observed in the treatment utilizing soybean oil. This suggests a favorable allelopathic effect of CsPO, stimulating seed development.
Table 3. Summary of butter bean seed germination means under the effect of CsPO at different concentrations.
Treatment
|
Germination
|
AGTa
|
GRb
|
GRIc
|
RLd
|
T0
|
56 Af
|
2.15 A
|
0.46 A Bg
|
16.74 A
|
22.96 A B
|
T1
|
43 A
|
2.72 A
|
0.37 A
|
10.77 A
|
15.46 A
|
T2
|
59.5 A
|
2.12 A
|
0.48 A B
|
19.32 A
|
23.22 A B
|
T3
|
55.5 A
|
2.00 A
|
0.50 A B
|
16.95 A
|
26.63 A B
|
T4
|
55 A
|
1.75 A
|
0.59 B
|
17.75 A
|
36.09 B
|
T5
|
42 A
|
2.60 A
|
0.41 A B
|
10.73 A
|
21.55 A B
|
RCe
|
32.04
|
22.23
|
19.14
|
40.17
|
36.00
|
Average
|
51.83
|
2.22
|
0.47
|
15.38
|
24.32
|
aAGT: average germination time; bGR: germination rate; cGRI: germination rate index; dRL: Root length; eRC: rate coefficient; fA: highest average (highest activity); gB: lowest average (lowest activity).
The rate coefficient values exhibited considerable variability, suggesting the possibility of test-related issues. Seed storage methods stand out among the analysis criteria, as seeds are often treated with plant-derived substances (powders, oils, extracts) before storage. These substances can both eliminate and deter insects, thereby hindering their growth and development. These treatments offer the advantage of uniform seed coverage. To prevent excessive moisture absorption and interference with germination, these treatments should be of short duration [53].
Figure S3 (Supplementary material) illustrates the root growth of butter bean seeds, where genetic factors could have impacted their germination rate. Some scholars have linked the allelopathic attributes of essential oils, alongside other characteristics, to the presence of terpenoids. These compounds can modify cell membrane permeability, diminish enzymatic activity, and disrupt DNA transcription and RNA translation, consequently impeding both seed germination and seedling growth [54].
The substitution of agrochemicals with plant extracts possessing fungicidal properties has undergone extensive investigation in the agricultural domain. Numerous studies have showcased the effectiveness of essential oils and various other plant extracts in suppressing phytopathogenic fungi [55, 56].
In-Vitro Assessment of Acetylcholinesterase Inhibition
The CsPO under investigation displayed significant activity in the acetylcholinesterase inhibition assay, with an IC50 value of 72.0 µg/mL. This could be attributed to the presence of acyclic and monocyclic monoterpenes in the oil, e.g., linalool and γ-terpinene, respectively. Certain characteristics, such as the presence of a hydrophobic ligand, might contribute to heightened inhibitory effectiveness, given the known susceptibility of the AChE active site to hydrophobic interactions. These characteristics inherent to cyclic and acyclic monoterpenes could influence AChE-inhibiting activity. In the case of bicyclic monoterpenes possessing the carene or pinnae skeleton, the potential for AChE inhibition has been linked to the position of the double bond [8].
Research into the structure-activity relationship between AChE and monoterpenoids has demonstrated that hydrocarbons exhibit more robust inhibition compared to alcohols and ketones. The presence of an oxygenated functional group diminishes the potency of AChE inhibition [7]. More recent investigations on the structure-activity relationship with bisabolane-type sesquiterpenoids, such as that conducted by Fujiwara and colleagues [57], suggest that AChE inhibition potency follows this order: ketones < alcohols < hydrocarbons.
Additionally, certain compounds like coumarins have demonstrated inhibitory effects on AChE. For instance, the compound citroptene (5,7-dimethoxycoumarin), found in citron peel, and aurapten (7-27 geranyloxycoumarin), present in C. paradisi Macfad., have been proposed as potential erythrocyte AChE inhibitors [8].
In Silico Molecular Docking Determination of Acetylcholinesterase Inhibition
Molecular docking investigations were performed, involving the three-dimensional structure of AChE (1QTI) and the key CsPO components currently under scrutiny. These investigations were targeted at the active site region, previously occupied by galantamine. The active site of this enzyme is situated at the base of its cavity. The so-called ‘catalytic triad’ — consisting of three amino acid residues, namely, Ser200, His440, and Glu327 — plays a direct role in the hydrolysis of acetylcholine [58]. Moreover, amino acid residues from the anionic subsites (Trp86 and Phe330) and the peripheral subsite (Trp279) are also significant for ligand recognition [59, 60].
Table S2 displays the outcomes of molecular docking investigations involving the primary components of CsPO and AChE. The binding energy values ranged from -6.2 to -9.6 kcal/mol, which closely aligns with the values observed for the acetylcholinesterase inhibitor galantamine (redocking binding energy = -9.8 kcal/mol; RMSD = 0.3678 Å).
The interactions observed for galantamine (Fig. S4 - Supplementary material)) concur with those detailed by Bartolucci and colleagues [32]. These interactions highlight the hydrogen bond formed between the Ser200 residue and the oxygen atom of the O-methyl group, alongside π-alkyl interactions in the anionic subsite residues (Trp84 and Phe330). As outlined by the aforementioned researchers [32], the hydroxyl group of the galantamine molecule engages with two water molecules, both of which are firmly connected to the catalytic pocket via Ser200. Meanwhile, the cyclohexene ring and two methylene groups of the tetrahydroazepine ring of the galantamine molecule align with the Trp84 indole ring. The double bonds of the cyclohexene ring converge towards the π system of the indole ring, facilitating π-π interactions.
Among the compounds subjected to testing, α-humulene displayed the most favorable binding energy value (-9.6 kcal/mol). The primary interactions identified involved anionic residues: (1) Try121, Try334, and Phe330, participating in π-alkyl interactions, and (2) Trp84, contributing to π-sigma interactions. These specific residues hold significance as they are linked to the presumed ‘anionic’ bond with choline in the AChE cavity [60]. The remaining compounds also engaged in π-sigma and π-alkyl interactions with these residues (Figs. S5 and S6 - supplementary material). This implies that these substances establish interactions capable of inhibiting AChE, a conclusion supported by the biological inhibition data.