Introduction
Essential oils and their components for a long time have been used in health, food, and aromatherapy. Essential oils are composed of many components, of varying concentrations and chemical structures [
1,
2]. The growing interest in food safety is today gaining momentum for both the food industry and consumers as all are becoming more careful about the several health and environmental effects of foods. Natural products used in cosmetics and pharmaceutics processing today play a great role; their positive benefits on both health and the environment have made them the main principles of what formed a healthy diet today and, in the future [
1,
2].
Citrus plant production has reached more than 126 million tons per year [
3], where the Mediterranean region obsesses about one-fifth. This makes
Citrus an economically important tree crop.
Citrus is known to be rich in vitamins as B
9, C and E, coumarins, dietary fiber and antioxidants [
3‐
6].
The chemical composition of
Citrus essential oil has been extensively studied and several compositional patterns owing to the species/cultivars, origin, climate, season, ripening stage, extraction, and analytical methods have been published. (
Citrus aurantium L.) is a member of the family Rutaceae. It is famous in the Mediterranean region for many ornamental and agronomic uses. Peels and flowers represent the most-used parts of
C. aurantium L. thus forming the base for many promising research groups due to their many medicinal properties [
3].
Citrus aurantium L. var
amara commonly named bitter or sour orange “Narenja Agria” [
4], is widespread in Central and South America but mainly in the Mediterranean countries [
5], where it is used in a large area from the industry as a flavoring agent or a fragrance to medicine as a nasal decongestant and a dieting agent [
6]. The chemical compositions of leaves essential oils (LEO) and peel essential oils (PEO) from bitter oranges have been reported in the literature, where the main compounds in the LEO were linalool and linalyl acetate [
7] whereas the PEO was dominated by limonene [
7].
The chemical composition of the essential oil of sour orange (
Citrus aurantium) was assessed in different plant parts during different seasons. Many studies were focused on the essential oil extracted from
C. aurantium peels and limonene was found to be the major component. In contrast, other studies carried out on essential oil extracted from
C. aurantium flowers showed that linalool and linalyl acetate are the main components. However, the volatile oil constituents from leaves have not received much attention in the literature. Indeed, the few reported studies that focused on
C. aurantium leaves showed that linalool is the main component of essential oil. In addition, a large number of studies on
C. aurantium were performed in Tunisia and Greece [
3].
Sweet and bitter oranges are two of the most commercially important fruits with a huge world production due to their vast array of nutritive and therapeutic potentials. Sugar/acid ratios are regarded as the main index of citrus fruit maturity and one of the major analytical measures of its flavor quality [
8]. Sweet orange plays an important therapeutic role in being active as an anti-diabetic, anti-obesity, and hypocholesterolemic agent. The peels and other waste by-products of the two kinds of citrus have a lot of therapeutic applications as nutraceuticals and functional foods. Peels and flowers, the most-used parts of
Citrus aurantium, have constituted a largely promising area of research for their many medicinal properties. However, the leaves of sour orange have not yet been studied extensively. The essential oil of the bitter orange leaves from Algeria was analyzed using GC/MS. Forty-three volatile compounds were detected in essential oil and the majority of them were linalool, linalyl acetate, and
a-Terpineol. The essential oil had an interesting level of elastase and collagenase inhibition and thus can be applied as an anti-aging agent [
3,
9,
10]. This research article targeted the study of the effect of changing the essential oil extraction method on the composition of the essential oil of the leaves of
Citrus aurantium together with the evaluation of its antioxidant, antidiabetic, and neuroprotective activities.
Discussion
Plant extracts and essential oils have gained much interest recently due to their interesting chemical composition together with potent biological activities which positioned them on top of research interests worldwide [
28‐
30]. Genus
citrus is famous for its well-known species viz. orange, lemon, lime, grapefruit, and many others.
Citrus essential oils were documented to contain many oxygenated and non-oxygenated terpenes. The volatile components were usually isolated from the leaves [
3,
31], flowers [
32], fruits [
33], fruit rind [
34], bark [
35], and even the seeds [
36]. Many biological activities were reported for the
Citrus essential oils including; antiviral [
29,
37], antibacterial [
28,
38,
39], anti-inflammatory [
40,
41], antioxidant [
38,
42], cytotoxic [
43‐
46], etc.
Sour orange also known as Citrus aurantium is one of the famous and well-studied Citrus species, especially its essential oil content. Herein, one hundred and two volatile components were identified and quantified from three essential oil samples of C. aurantium and they were different in the oil preparation method. As discussed earlier in the results, the hydrodistilled and microwave-assisted extraction of the sour orange essential oils showed nearly similar volatile oil contents with the same components in both of them with differences in the compositions of these components. Moreover, the hydrodistilled essential oil (HD) showed the unique presence of camphene while the microwave-assisted (MV) showed the presence of β-ocimene which was absent in the HD essential oil. On the other hand, the steam-distilled (SD) essential oil presented a unique chemical composition when compared to the two other oil samples. β-Pinene, β-myrcene, linalool, and α-terpineol were found to be common components for the three essential oil samples while the latter two components were major in the HD and MV essential oils they represented minor or even trace components of the SD essential oil.
As reported in the literature, steam distillation marks the most recommended method for extraction of
Citrus essential oils compared to other conventional methods [
47] due to its ability to allow the liberation of the volatile components under controlled conditions of pressure and temperature compared to the atmospheric pressure in case of hydrodistillation for example [
48,
49]. Other studies on
C. aurantium essential oil addressed the effect of the isolation technique on both the quality and the essential oil yield. One study compared the essential oil components from
C. aurantium blossoms obtained through seven isolation methodologies namely; commercial hydrodistillation, hydrodistillation, steam distillation, ohmic-assisted hydro distillation, solvent-less microwave extraction, solvent-free microwave extraction and microwave-assisted hydrodistillation where hydrodistillation showed the highest essential oil yield compared to the other methods [
50].
Another study compared four isolation techniques namely; hydrodistillation, solvent extraction, microwave-assisted extraction, and ultrasound-assisted extraction regarding the essential oil composition of the peels of
C. aurantifolia, C. limon and
C. sinensis. This study concluded that both hydrodistillation and solvent extraction were superior in the isolation of mono- and sesquiterpenes while the two other methods led to the isolation of essential oils with higher percentages of hydrocarbons, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes and fatty acids [
51]. Thus, upon the results of such studies and when comparing them to our current study. The essential oil composition can vary according to many factors mainly centered on the isolation technique, the part used in the extraction process, the different species also the seasonal variation. All or part of these factors eventually led to new essential oil components even from the same plant species thus proper selection and optimization of the distillation technique plays crucial role in the quality and quantity of the resulting essential oil.
Many earlier studies had highlighted the chemical components and medical importance of sour orange essential oils. The essential oil from
C. aurantium peels was rich with limonene as the main component with about 90% of the total oil content and it exhibited potent antioxidant activities with ABTS
•+ (44.93 ± 1.45%) and DPPH
• (11.03 ± 1.08%) inhibition [
52]. The essential oil of
C. aurantium leaves was evaluated for its composition together with its potential antioxidant and anti-inflammatory activities. The essential oil contained forty-three components (yield = 0.57%). Linalool, linalyl acetate, and α-terpineol composed the bulk of the essential oil composition. The antioxidant activity was evaluated through a DPPH assay (IC
50 > 10,000 mg/L) [
3].
The hydrodistilled essential oil contents of the leaves and peel of
C. aurantium were compared where the leaves essential oil contained linalool (18.6%), γ-terpinene (6.9%) and α-terpineol (15.1%) as its main compounds while the peel essential oil was rich in linalool (12%),
cis-linalool oxide (8.1%),
trans-carveol (11.9%),
endo-fenchyl acetate (5.5%) and carvone (5.8%) [
7].
Different herbal extracts and essential oils represent promising reservoirs of naturally occurring compounds that exhibit antioxidant properties. A prior investigation has documented the most robust DPPH scavenging activity within the
C.
aurantium extract, demonstrating IC
50 values of 96.07 µg/ml, and its corresponding essential oil, with IC
50 values of 393.71 µg/ml [
53]. The essential oil extracted through SD exhibited elevated concentrations of both monoterpene and sesquiterpene components. A prior investigation showed their antioxidant effectiveness, which closely paralleled that of the phenolic constituents found in
C.
aurantium [
53]. These molecules effectively disrupted free-radical chain reactions and induced their transformation into inert compounds, as supported by additional studies [
54,
55]. The findings of our study align with those of several other researchers who have affirmed the antioxidant capabilities of
Citrus essential oils [
3,
53,
54,
56]. Nonetheless, presenting their antioxidant efficacy in IC
50 values makes it challenging to make a direct comparison with the current study, given the difference in units of measurement.
The current study assessed the therapeutic potential of essential oil extracted from
C. aurantium using three distinct extraction methods in the context of AD treatment. This evaluation involves the examination of their effects on AChE and BChE inhibition assays. In AD pharmacotherapy, AChE and BChE inhibitors are frequently used to target essential enzymes, easing cognitive symptoms and slowing disease progression [
25]. The elevated monoterpenes content in Citrus essential oils provides a plausible rationale for their bioactivity. Numerous studies have underscored the AChE inhibitory properties associated with this category of secondary metabolites. Previous research has explored the potential of various monoterpenoids from
Citrus essential oils, including monoterpene, oxygenated monoterpene, and sesquiterpene compounds, for inhibiting cholinesterase enzymes, with a significant focus on AChE inhibition [
57,
58]. There are reports of certain citrus essential oils displaying radical scavenging capabilities [
59]. For instance, Eureka lemon essential oil, known for its potent tyrosinase inhibitory properties, has also been observed to exhibit DPPH radical scavenging activity [
60]. Previous study and the present results suggests that citrus essential oils, composed of a diverse array of compounds, may effectively inhibit melanogenesis through a multifaceted range of mechanisms and activities.
To manage diabetes, reducing post-meal high blood sugar levels involves inhibiting two crucial digestive enzymes: α-amylase and α-glucosidase, responsible for breaking down complex carbohydrates into simpler sugars. This study unveiled the potential antidiabetic properties of
C. aurantium essential oil by demonstrating their inhibition of α-amylase and α-glucosidase in vitro, corroborating prior citrus extract research [
61,
62]. These findings, along with previous studies, suggest that the presence of monoterpenes and sesquiterpenes, along with their synergistic interactions, likely underlie these enzymes’ suppressive effects of the essential oils.
The observed enzyme inhibitory effects can be explained by the presence of some volatile components in the tested essential oils. For example, the SD essential oil was rich in eucalyptol and the compound had a potent cholinesterase inhibition [
63‐
65], amylase inhibition [
66] and glucosidase inhibition [
67]. Regarding the HD and MW essential oils contained higher concentration of linalool and linalool acetate. Similarly, some studies have been found significant enzyme inhibitory properties of the compounds; Acetylcholinesterase; [
68,
69], tyrosinase [
70], glucosidase inhibition [
71]. However, as can be seen in Table
1, the essential oils contained several compounds, so their synergistic and antagonistic effects are possible. In this sense, we proposed further studies to isolate the components and test their enzyme inhibitory potential as individuals.
In the current study, components like linalool acetate, α-terpinyl acetate, linalool, and eucalyptol represented the main essential oil components obtained through the three studied distillation techniques. The aforementioned components belong to the oxygenated monoterpene class of volatile constituents which is well-known for its potent antioxidant and neuroprotective effects through their ability to fight against oxidative stress and support the natural oxidant-antioxidant balance [
72‐
74] as well as their potential antidiabetic activity through inhibition of α-amylase and α-glucosidase as the main enzymes involved in the process [
75]. Moreover, they can modulate enzymes and proteins that contribute to insulin resistance and other pathological events caused by
Diabetes milletus [
76].
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