GC-MS Profile, α-glucosidase Inhibition Potential, Antibacterial and Antioxidant Evaluation of Peels Citrus aurantium (L), Essential Oil

a Research Unit Valorization and Optimization of Resource Exploitation (UR16ES04), Faculty of Science and Technology of Sidi Bouzid, University of Kairouan, Campus University Agricultural City Sidi Bouzid 9100 Tunisia. b Regional Center of Agricultural Research (CRRA) Sidi Bouzid, Gafsa Road Km 6, PB 357, Sidi Bouzid 9100, Tunisia. c Laboratory of Valorization of Unconventional Waters, INRGREF, University of Carthage, Tunisia. d Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia. e University of Monastir, Faculty of Sciences of Monastir, Avenue of the Environment, Monastir 5019, Tunisia. f Department of Biology, Hail University, College of Science, P.O. Box 2440, 81451 Ha’il, Kingdom of Saudi Arabia. g Laboratory of Genetic, Biodiversity and Valorization of Bioressources, Higher Institute of Biotechnology of Monastir, University of Monastir, Avenue Taher Hadded BP 74, 5000 Monastir, Tunisia. h Laboratory of Bioressources: Integrative Biology and Recovery, High Institute of BiotechnologyUniversity of Monastir, Monastir 5000, Tunisia. i Faculty of Science and Arts in Baljurashi, Albaha University, P.O. Box (1988), Albaha, Saudi Arabia. j Faculty of Science of Sfax, Department of Chemistry, University of Sfax, B.P. 1171, 3000, Sfax, Tunisia.


INTRODUCTION
The genus Citrus belongs to the family Rutaceae, with important crops like orange, lemons, pummelos, grapefruits, limes, and so on [1]. Citrus fruits with high nutritional value, along with potential several secondary metabolites, including flavones, flavanones, flavonols, flavans, and anthocyanins are recognized to have beneficial and healthy effects for human. Among the most common Citrus species, Citrus aurantium L., also known as Seville orange, sour orange, or bitter orange, originating in Eastern Africa, and Syria, and was cultivated in Spain, Italy, and North America [2]. In addition to the richness in bioactive molecules, they have demonstrated several health effects such as antioxidant, antimicrobial, anti-inflammatory, antihypertensive, neuroprotective, antimutagenic, and antiallergic properties [3,4]. Citrus are sources of essential oils due to their aromatic compounds which are used in drinks, confectionery, cookies, desserts, cakes, and ice cream [5,6].
Citrus essential oil is largely present in the peels compared to other parts. It is represent an abundant and inexpensive source of terpenes and oxygenated terpenes which are of interest to many sectors, in particular; food industry, pharmaceuticals, cosmetics, the aroma and perfume industry; molecules, such as myrcene and linalool, are contained in small quantities in essential oils and which have high added value due to their particularly desirable sensory profile; although the non-oxygenated terpene, limonene is a major component of all essential oils in citrus fruits [11,12].
Citrus aurantium (L), has been used in herbal medicine as a stimulant and appetite suppressant; it has also been used in traditional Chinese medicine to treat nausea, indigestion, and constipation as well as cancer and cardiovascular diseases [13]. Furthermore, recent studies have been improved the efficiency of EOs and extracts as well as their secondary metabolites from as antimicrobials and antidiabetics gent [14][15][16][17][18][19]. Also, immature peels and EOs are used to treat intestinal diseases and antidiabetic effect [8,20,21]. These studies are focused on the search for potential inhibitors of the two enzymes α-glucosidase and α-amylase, in order to treat type 2-diabetes [22]. Furthermore, recent research has emphasized the importance of promoting safer and tolerable inhibitors for the two enzymes α-glucosidase and α-amylase that are naturally extracted from medicinal plants, fruits, and vegetables at a lower cost, particularly Citrus fruits.
In this optic, the present study was conducted to explored CAEO chemical composition and its anti-α-glucosidase, antioxidant and antibacterial activities.

Plant Material and Essential Oil Isolation
Citrus aurantium L. fruits were harvested from a garden of Faculty of Sciences and Technology of Sidi Bouzid (Centre of Tunisia) and identified according to the flora of Tunisia. The essential oil extraction was carried out from the fresh peel of bigarade. The freshly harvested fruits were carefully washed to remove dust then peeled and cut into small pieces. An amount of 100 g of fresh peels was transferred to hydro-distillation for 3 hours with 500 mL distilled water using a Clevenger-type apparatus. The distilled EO was dried over anhydrous sodium sulfate, filtered, and stored at 4°C. The yield was calculated based on the dried weight of the sample.

α-Glucosidase Inhibitory Assay
The α-glucosidase assay of the tested EO was conducted according to the standard method with slight modification [24].

Scavenging ability on 2,2-diphenyl-1picrylhydrazyl (DPPH) Radical
The DPPH quenching ability of the EO was measured according to the method cited by Felhi et al. [25].

Reducing power
The ability of the EO to reduce Fe 3+ was assayed using the method cited by Hajlaoui et al. [26] and Bakari et al., [27]. Butylated hydroxytoluene (BHT) was used as positive control.

Disc-diffusion assay
The bacterial strains tested in this study belonged to 8 references, which are presented in Table 3. The bacterial species consisted of 5 Gram-positive and 3 Gram-negative bacterial strains. The disc-diffusion assay was performed according to the protocol cited by Hajlaoui et al. [20].

Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values
were determined for all bacterial strains used in this study as described by Hajlaoui et al. [20].

Essential Oil Composition of Citrus aurantium Essential Oil
In this part, chemical composition identification of CAEO was carried out by calculating the retention index (IR) for each compound and their percentage. The constituents of this EO are listed in Table 1.
GC-MS analysis of CAEO showed the presence of 37 compounds accounting for 99.3% of the EO. The major compounds are: limonene The classification of these compounds shows that CAEO peels is particularly rich in hydrocarbon monoterpenes (75.7%), followed by oxygenated monoterpenes (19.16%). While the percentage of hydrocarbon and oxygenated sesquiterpenes does not exceed 5%. This chemical composition remains specific and characteristic of bitter orange plants in the garden of the FST of Sidi Bouzid. This specificity was related to bioclimatic stage. In fact, each time the place of harvest changes, the chemical composition changes also [28]. In addition, the chemical composition of EO changes also according to the plant organs. Indeed, Bnina et al. [29] reported that EOs isolated from flowers and leaves of C. aurantium were particularly rich in oxygenated monoterpenes (59.02-69.21%) represented by linalool (41.82-37.24%) and linalyl acetate (13.75-7.87%), followed by hydrocarbon monoterpenes (24.61-32.28%), with the most important hydrocarbon monoterpenes were αthujene (6.15-10.65%) and β-pinene (9.21-9.68%). In contrast, the EO isolated from the peels was dominated by limonene (monoterpene hydrocarbon) (73.60%), with oxygenated

α-Glucosidase Inhibitory Assay
In this part, Fig. 1 showed the inhibitory effect of different concentrations of CAEO peels on αglycosidase activity compared to Acarbose.
Based on these results, EO and Acarbose exert an inhibitory effect on α-glycosidase. This inhibition increases in proportion with the concentration of the samples. The inhibition of Acarbose is found to be greater than EO. Indeed, a low concentration of Acarbose can cause maximum inhibition. The IC 50 obtained ( Fig. 2) with Acarbose (0.7 ± 0.1 mg/ml) is almost 14 times lower than that obtained with EO (10 ± 1 mg / ml). These results are in agreement with other studies showing an efficacy of EOs in inhibiting the enzymatic activity of α-glycosidase, which remains lower than that of Acarbose. Several antidiabetic trials, with a wide range of extracts and EOs from plants, inhibit the enzymatic activity of α-glucosidase and αamylase. But the effectiveness of this inhibition depends on several parameters, including the composition of the bioactive mixture, the structure-function relationship, and type and stability degree of established links between enzyme and inhibitor molecule. Moreover, it has been shown that terpenes represent a good antidiabetic potential [36]. Among the active monoterpenes, p-cymene and -terpinene have revealed a powerful inhibitory effect [36,37]. The strongest α-glucosidase inhibitory effect was also displayed by EO Sideritis galactic containing a high level of α-pinene (32.2%) and all the activity was attributed to the high level of monoterpene hydrocarbons. In our study, this fraction is of 75.7% of total CAEO.

Scavenging Ability on DPPH Radical
The antiradical activity profile of CAEO compared to the synthetic antioxidant BHT is shown in Fig.  3. This result revealed that EO has a significant antiradical activity but it is lower than that obtained by BHT. In fact, 100% inhibition is achieved for a 100 µg/ml of BHT concentration. This percentage was not reached even 200µg/ml concentrations for EO.
The (IC 50 ) values (Fig. 4) shows that EO has a significant capacity for scavenging free radicals with an IC 50 = 33.66 µg/ml. This activity is 3 times less than BHT (10.33% g / ml).

) of the CAEO Peels Compared with Synthetic Antioxidant BHT
The means followed by the same letters are not significantly different at the 5% level The antioxidant properties of Citrus fruits have been described by several authors. Hamdani and Allem [38] comparing antiradical activity of the CAEOs from 4 sites in Algeria showed that the strongest antioxidant activity was characterized by CAEO from Boujlida region with IC 50 of 32.9 mg/ml, while the lowest activity was expressed by CAEO from Ouzidane region with IC 50 of 59.55 mg/ml. Results obtained from the IC 50 showed that all samples of C. aurantium have a significant antioxidant power compared to limonene (IC 50 of 258.74 mg/ml). These results are different from our study. This difference could be explained by chemical composition variation which is related to several factors namely the methodology used to obtain the extracts, the region of harvest, stage of fruit ripening, climate and fruits maturity [39,40].

Reducing power
Reducing power capacity of CAEO was shown in Fig. 5. Results indicate an increase in absorbance (OD) which refers to the increase in reducing capacity. CAEO reducing activity comparison with BHT showed a significant difference (P<0.05) for different tested concentrations. These results show significant antioxidant activity of CAEO, but it is weaker than BHT. In the present study, the CAEO peels showed significant antioxidant activity which was supported by both tests; DPPH radical scavenging capacity and iron reduction (FRAP). This activity turns out to be more interesting than others in previous work. For example, the results found by Hamdani et al. [38], working on 4 samples of CAEO, showed that IC 50 values vary from 32.9 to 59.55 mg/ml and the EC 50 values ranges from 1.369 to 2.204 mg/ml. However, limonene, the major compound, showed a low antioxidant activity, probably due to the appreciable percentage of myrcene or its combination with limonene which appears to be effective. As shown, in our study, the activity of EO is closely related to its composition, and the association of α-thujene, sabinene, linalool, linalyl acetate and neral with limonene may be also responsible for this activity.

Antibacterial Activity Evaluation
Inhibition diameters values of CAEO against all studied strains presented in Table 3, were ranged from 8.66±1.15 to 12±0 mm. These values are relatively high showing the inhibitory activity of bacterial growth of this EO despite being lower than those of gentamicin (from 20.33±0.57 to 32.67±0.58mm). Statistical analysis revealed a significant difference (P<0.05) in bacterial strains sensitivity to CAEO and gentamicin. But there is unclear difference between Gram+ and Gram-strains susceptibility to EO. However, Gram+ strains appear to be more sensitive to gentamicin than Gram-strains.
The MIC and MBC values found showed that CAEO is effective against tested strains ( Table  4). The concentrations obtained were ranged from 0.097 to 0.390 mg/ml and from 0.195 to 1.562 mg/ml, respectively. However, this activity remains less effective than gentamicin which values were ranged from 0.004 to 0.019 mg/ml for MIC, and 0.019 to 0.078 mg/ml for MBC. Based on these results, Gram+ strains appear to be less sensitive than Gram-strains to the EO and Gentamicin effects, which is in accordance with other previous work [41][42][43]. Explanation for this resistance is related to Gram-bacteria structure wall, which makes unable EO hydrophobic compounds to diffuse, unlike Gram+ Bacteria [44]. Furthermore, to better underline the capability of CAEO in destroying bacterial cells (bactericidal), the MBC/MIC ratios have been determined for each strain. As shown, CAEO was found to be bactericidal towards all tested strains.
The antimicrobial activity of EOs is closely related to their chemical composition. Actually, the mechanism of terpenes action is not fully understood, but it is believed that these compounds are involved in the damage and stability of plasma and the subsequent membrane disruption by lipophilic compounds [35,39]. Limonene and linalool, which were found to be abundant in this study, were reported as compounds with significant antimicrobial property [45]. It has also been shown that limonene, the major compound of EOs of Citrus genus, has a weaker antibacterial effect than antifungal activity. But the antimicrobial activity of Citrus EO is enhanced by the presence of bioactive alcohol, linalool, a monoterpene alcohol, known to be a potent antimicrobial [45]. On the other hand, EO activity of C. aurantium peel may be the result of a synergistic effect between these different compounds, especially since the fraction of oxygenated monoterpenes is relatively high (19.16%).

CONCLUSION
In this study, CAEO peels exhibited potent antidiabetic effect explained by a good capacity of α-glucosidase inhibition. Moreover, this EO has an important antioxidant and antibacterial activities. These potentialities are related to the chemical profiling which shows a composition rich in hydrocarbon and oxygenated monoterpenes known by their capacity to treat chronic diseases such as type 2 diabetes. In addition, this EO can be used as a food additive for its antibacterial activity.

NOTE
The study highlights the efficacy of "herbal medicine" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.