Physicochemical Indexes and Evaluation of Antioxidant, Antihemolytic and Antibacterial Activities of Citrus sinensis and Citrus limon Peel Essential Oils

. In this study, the physicochemical indexes, phytochemical screening by thin layer chromatography of essential oils (EOs) obtained from C. limon L. and C. sinensis peels and their antioxidant, antihemolytic and antibacterial activities were investigated. The highest yield (0.71%), density (0.918 ±0.05), refractive index (1.974±0.02), saponification value (186.11±1.13 mg KOH/g) where obtained in C. limon, where the highest acid value ( 0.45±0.2mg KOH/g) was for C sinensis . Eight and six spots were identified in C. sisensis and C. limon respectively using thin-layer chromatography (TLC). IC50 for DPPH radical-scavenging activity were 7.04 ±1.03 and 8.39 ± 0.9 µg/mL for C. limon and C. sinensis essential oils, respectively. C. limon EO was also demonstrated better antihemolytic activity by peroxide scavenging, IC50 were 12.76± 0.6 for C. limon and 14.26 ±0.3 µg/mL for C. sinensis essential oils. The C. limon peel EOs showed, also, a higher antibacterial effect on S. aureus and E. coli with 20.5±0.2 and 11.5 ±0.01 mm, respectively. Our data showed that both C. limon and C. sinensis EO were inactive against P. aeruginosa.


pH Determination
The pH was determined using pH paper and an approximate value was obtained.

Specific Gravity (Density)
The relative density of EO was defined as the ratio of the mass of a certain volume of oil at 20°C to the mass of equal volume of distilled water at 20°C. The measurement was carried out using an electronic densimeter. The relative density at 20°C was calculated using the following formula: d 20 = dt + (t-20) x 0.00068 Where: dt: density at room temperature / t: ambient temperature / d20: density at 20°C.

Acid Value
The acid index was determined by mixing of 0.125g of EO with 2.5mL of ethanol, then 2 drops of phenolphthalein were added. The whole was titrated with ethanolic solution of KOH 0.1N until the color changes to pink. The acid index (AI) was determined by the formula:

AI ꞊
Where: V.KOH: Volume of KOH / W.H.E: Weight of essential oil.

Saponification Index
The saponification index was carried out by mixing of 0.25g of EO and 5 mL of ethanolic solution of KOH 0.5 N. The mixture was heated for 30 minutes. After cooling, 10 mL of distilled water and 5 mL of ethanolic solution of KOH 0.5 N were added then the mixture was returned to the water bath for 30 min. After cooling, three drops of phenolphthalein were added and the soapy solution was titrated with HCl (0.5N). The control test was prepared without essential oil. The saponification index (SI) was determined by the formula: SI = ((V0-V1)×M×N×F)/m Where : V0: volume (mL) of the HCl solution used for the control / V1: volume (mL) of the HCl solution used for the test sample / M: molar mass of KOH /N: normality of KOH solution/ F: correction factor for the normality of KOH solution /W: Weight of the test sample.

Phytochemical Screening of Essential Oils by Thin Layer Chromatography
Thin layer chromatography (TLC) is a fast, simple analytical technique. The stationary phase used is 60F 254 silica gel plates on aluminum foil. Two mobile phases were tested: the first consists of butanol/ethyl acetate (8/1.9: V/V) and the second is a mixture of dichloromethane/Hexane (9/1: V/V). Approximately one microliter (μL) of each essential oil is deposited on the plate at a reference point located above the surface of the mobile phase. The plates are revealed chemically using (Godin reagent), composed of sulfuric vanillin (1%) in a concentrated sulfuric acid and ethanol 95% (2/98 :v/v). After spraying, the plate was heated in an oven for a few minutes at 110°C. Each spot is characterized by its coloration after revelation and its retention factor (Rf), this is calculated using the equation: Where d: Distance traveled by the component/D: Distance traveled by the eluent front [14,15,16].

DPPH Radical Scavenging Assay
The method described by Sanchez-Moreno (1998) [17] was used to evaluate antioxidant activity of essential oils. A volume of 50 μL of each essential oils at different concentrations (diluted in methanol) was added to 1950 μL of freshly prepared methanolic solution at 0.024 g/L of DPPH (1,1diphenyl-2-pycrylhydrazile). Ascorbic acid was used as a standard antioxidant while a negative control was prepared by the addition of 50 μL of methanol to 1950 μL of DPPH solution. The absorbance was measured at 517 nm after incubation for 30 min in the dark at room temperature, Radical-scavenging activity was calculated as a percentage of DPPH discoloration using the following equation: Where, A control: absorbance of the DPPH solution without essential oil, A sample: absorbance of the solution containing the essential oil. The antioxidant activity of the essential oils was expressed as IC50 (half-maximal inhibitory concentration) values (μg/ mL) which were calculated from the graph representing the percentage of inhibition according to different concentrations of extracts.

Antihemolytic Activity
Blood from a healthy donor, collected in EDTA, was used to evaluate the antihemolytic activity of essential oils. after centrifugation for 5 min at 1000x g and elimination of supernatant, the pellet was washed three times with PBS (0.2 M, pH 7.4) and resuspended in a saline solution (4%). then 0.5 mL of essential oils at different concentrations in DMSO were added to 2 ml of suspension of erythrocytes and incubated at ambient temperature for 20 min. to cause the oxidative degradation of the membrane lipids, 0.5 ml of H 2 O 2 solution was added to the reaction media. after centrifugation for 10 min at 1000xg, the absorbance of the supernatant is read at 540 nm. the positive control was ascorbic acid while DMSO was taken as a negative control. The percentage of hemolysis was calculated using the formula: Ac: absorbance of the negative control/At: absorbance of the essential oil tested [18].

Statistical Analysis
Data were analyzed using Excel (Microsoft Inc.). The different tests were carried out in triplicate and the results were illustrated as the means standard deviation of three independent measurements.

Extraction Yield
As it can be seen in Figure 1, the yield of the hydrodistillation essential oil of C. limon peel was 0.71% and it was characterized by a pale yellowish color and pleasant aromatic fragrance. Whereas, orange peels seem less rich in essential oils with a yield of 0.55 %, these oils was characterized by pleasant aromatic fragrance, a pale yellowish color for lemon and darker yellow color for C. sinensis.

Physicochemical Indexes of Essential Oils
Examination of Table 1 reveals that the measured physicochemical indexes conform to international standards described by AFNOR (According to the French Association for Standardization) [13]. The essential oil from the peels of C. sinensis has a relatively lower density and refractive index, compared with C. limon. The acid index of C. sinensis evaluated at 0.45, is slightly higher than that of C. limon (0.39±0.01) While saponification index showed the opposite.

Phytochemical Screening of Essential Oils by Thin Layer Chromatography
The best chromatographic separation was obtained with dichloromethane/Hexane (9/1: V/V) mobile phase. These chromatograms were shown in Figure

DPPH Radical Scavenging Assay
The results of DPPH Radical Scavenging power of EOs are illustrated in Figure 3. Essential oil obtained from C. limon showed highest radical-scavenging activity than that of C. sinensis such the IC 50 values were 7.04±1.03 and 8.39±0.9μg/mL respectively. These two oils remain less important than that of ascorbic acid giving an IC 50 equal to 6.46±0.05μg/mL.

Antihemolytic Activity
Hemolysis inhibition activity caused by H 2 O 2 shows that C. limon EO is more inhibiting than C. sinensis EO (Figure 4), this is evidenced by their IC50 evaluated at 12.76 ± 0.6 and 14.26±0.3μg/mL respectively. But without as much reaching the activity of ascorbic acid proved to be the most effective, its IC50 was 6.77 ±0.01 μg/mL.

Antimicrobial Activity
The antibactrial activity of the C. limon and C. sinensis EOs was tested against S.aureus, E. coli and P. aeruginosa. The effectiveness of antibacterial activities was evaluated by measuring the zone of inhibition and summarized in Figure 5 (A and B). The result indicates that the C. limon and C. sisensis EO showed very active against S. aureus forming zones of inhibition of 20.5±0.2 and 17± 0.05 mm respectively with a MIC was less than 14 μg/ml for C. limon and between 26.4 and 12.89 μg/ml for C. sinensis . The essential oils of C. limon and C. sinensis seem to have very few activity against E. coli, the measured zones of inhibition were 11.5± 0.01 and 10±0.5 mm respectively with MIC between 26.4 and 12.89 μg/ml. whereas, P. aeruginosa.

Discussion
Extraction yield of essential oil from dry C. limon and C. sinensis peels is within the range given by AFNOR (2000) ranging from 0.5 to 2%. This compares favorably with the findings of other researchers. The percentage yield of volatile oil from C. sinensis and C. limon has been reported by hydrodistillation as 2 and 1.5 %, respectively by Sharma and Vashist (2015) [22], as 0.81 ± 0.092% for C. limon [23], as 0.57 to 3.24 for C. sinensis by Soxhlet apparatus [24]. Golmakani and Moayyedi (2015) [25] mentioned 1.36 ± 0.06 %, 1.18± 0.08 and 1.22±0.14 % in C. limon peel oil in different method of oil extractions viz., solvent-free microwave extraction, microwave assisted hydrodistillation and hydrodistillation respectively. It was articulated that the quality, composition, quantity and aroma of pure essential oils may vary depending on growing habitat, environmental factors, the extraction technique, the harvest, drying period, the degree of freshness and the variety [26,27]. In addition, the flavedo ratio (superficial layer rich in HEs / albedo (internal white layer with a spongy quality) in the peels influences the yield of the extraction [28]. Physicochemical indexes of an essential oil is a very important criterion for evaluating its quality as well as for identification. The measured lemon's essential oils density and refractive index shows slightly important than those obtained by Boughendjioua et Djeddi (2017) [29] and Benoudjit et al. (2020) [30] with the respectively values of 0,855±0,005 and 0.894 for density and 1,4700±0,005 and 1.475 for Refractive index. The refractive index increases with the introduction or presence of secondary products in the oil and extraction temperature [31]. It essentially varies with the content of monoterpenes and oxygenated derivatives. A high content of monoterpenes will give a high index [32]. Kanko et al. (2004) [33] showed that a low refractive index of the essential oil indicates its low refraction of light, which could favor its use in cosmetic products. An approximate pH studied essential oils is slightly acidic which supposed the richness of oil with acidic character compounds. The low acidity of oils is considered as neutralized and safe for making skin care products [34]. The present study showed, comparatively, lower acid value than that of finding of Ali (2015) [35] with 1.79 and 1.99 mg/KOH/g oil in C. limon and C. sisensis respectively. However, this same study shows very low saponification values (13.5 and 13.7 mg/KOH/g for C. limon and C. sisensis respectively). On the other, Khan (2013) [36] was mentioned a saponification index of C. sisensis essential oil very close to our results (183 mg/KOH/g). The acid and saponification indexes are quality criterions indicating the quantity of free fatty acids present in our essential oil and the susceptibility of the essential oil to undergo alterations, in particular oxidation. The lower acid value indicated the high quality of product, but the relatively high saponification value recorded is indicative that it has potential for use in the industry [34,30]. Several spots were revealed by Godin reagent, it detects terpene compounds by coloring them in the visible in purple, blue, green, orange, pink and yellow [37]. These results are confirmed by the GC-MS profiles described in the literature. Indeed, Ben Miri (2018) [38] identified the presence of different group of terpenoid compounds. The monoterpene hydrocarbons are mostly represented by limonene with 54.95 % in C. limon EO and 82.6 % in C. sinensis EO. The oxygenated monoterpenes