Chemical composition, antimicrobial and antioxidant activities of Algerian Citrus sinensis essential oil extracted by hydrodistillation assisted by electromagnetic induction heating

Abstract

Hydro-distillation assisted by electromagnetic induction heating (H-EMIH) was employed to extract essential oil (EO) from Algerian fresh orange peels (Citrus sinensis). H-EMIH was compared with conventional hydro-distillation (C–H) in terms of hydro-distillation time, yield, chemical composition and, antibacterial and antioxidant activities. It was found that extraction of EO with H-EMIH gave a maximal yield of 3.77% in 35 min whereas C–H gave 2.72% in 41 min. The extracts obtained by both techniques were analyzed by Gas Chromatography-Mass Spectrometry. Their chemical compositions are relatively similar; limonene and β-myrcene were found as the principal compounds. The antioxidant activity results demonstrated that EO extracted by H-EMIH showed the highest capacity of radical scavenging than EO isolated by C–H process. Otherwise, it was found that EO extracted by H-EMIH exhibited an antimicrobial potential slightly higher than that extracted by C–H.

Introduction

The Citrus variety has approximately 16 species in Rutaceae family, largely cultivated in subtropical regions (Fisher and Phillips 2008). During 2015 and 2016, about 1289.9 and 1372.4 thousand of tons of citrus production were in Algeria which is the 5th production in the Mediterranean region (Statistical Bulletin 2017). The food and agro-food processing industries using citrus fruits yield considerable amounts of wastes or by-products in form of pulps, seeds and peels which represent 50% of raw fruits (Anwar et al. 2008). These by-products constituted an important source of different bioactive products like essential oils (EOs) (Senevirathne et al. 2009).

EOs are important natural chemical derivatives that can be employed in the preparation of different products (food and pharmaceutical). In addition, substantial amounts of EOs are also employed in the formulation of cosmetic products (Sahraoui et al. 2011). The qualitative and quantitative contents of EOs determine the characteristics of oils; this can be due to several factors such as fruit quality, stage of growth when extracted, ecological conditions and extraction methods (Rivera and Vilarem 2007).

In distilled oils, differences in quality are likely to arise from variations in distillation time, distillation rate, efficiency of steam condensation and separation methods (Babu and Kaul 2005). There are many practical technologies denoted to produce EOs at an optimum output to achieve acceptable production rate, energy consumption and process minimization (Muhammad Hazwan et al. 2012). Recently, interest was devoted to electromagnetic induction heating (EMIH) as an emerging novel technology. It was used by Rivera and Vilarem (2007) to extract EOs from caraway (Carum Carvi L.). They found that the time of extraction is much shorter than that of conventional hydro-distillation (C–H). Zouambia et al. (2017) also studied the impact of EMIH on the characteristics of pectic substances extracted from citrange albedos; they reported that EMIH preserves the physicochemical characteristics of the extracted substances and minimizes the extraction time compared with C–H. Similar effects of extraction time and yield, using EMIH process, were reported by Terkmane et al. (2016) to extract inulin from globe artichoke, and by Megateli et al. (2018) to extract flavonoid and phenolic components from Rosmarinus officinalis L.

The principle basic application of EMIH in a reservoir is to transform the electromagnetic energy to a heat one (Greff and Babadagli 2013). The produced current dissipates heat in the material in which it is placed. As reported by Bera et al. (2015), EMIH is more efficient in energy terms than other aqueous thermal heating methods. It is a time-saving process (within shorter time than other heating processes) wherein it can increase the temperature and therefore enhances the production rate.

The aims of our work were to explore the beneficial use of H-EMIH as an alternative process to extract EOs from peels of Citrus sinensis, and compare the results with those acquired by C–H, in terms of some important parameters like the yield, production quality and time of extraction. In addition, biological activities (antioxidant and antimicrobial) of EOs extracted by both techniques were evaluated.

Materials and methods

Raw materials

Sweet orange fruits (C. sinensis) used as raw materials were collected in the region of Mitidja (North of Algeria). The fruits were washed by water and peeled. Fresh peels were used in essential oil extraction.

Extraction method

EO was extracted from Citrus sinensis peels by hydro-distillation using two modes of heating: conventional method by heating mantle (C–H) and electromagnetic induction heating method (EMIH). The fresh plant material was submerged in distilled water with a solid/liquid ratio 1:2 (w/v).The extraction was continued until no supplemental EO was obtained.

In the first method, Citrus sinensis peels was subjected to conventional hydro-distillation using a classical heating process, with an optimum heat setting to ensure stability of extraction at a well-controlled constant speed. In the second method, the apparatus of hydro-distillation is assisted by EMI heating (H-EMIH). The extraction was carried out in a magnetizable container (1500 mL) placed on an induction ceramic plate assisted by magnetic field with 9 power levels. For an accurate comparison, between these two procedures, the EMI power was fixed correspondingly to that of C–H. The vapor produced was condensed in the refrigeration part. In both procedures, The EO was collected after hydro-distillation, dried over anhydrous Na₂SO₄ and stored away from light at 4 °C until used.

GC–MS analysis

The EO was analyzed by gas chromatography coupled with mass spectrometry (HP 6890 (II) interfaced with a HP 5973 mass spectrometer) using a capillary column RTX-5MS (30 m, ID 0.25 mm, film thickness 0.25 μm) with Helium (1 mL/min) as a carrier gas. The temperature program of column was fixed at 40 °C during 8 min, and was increased to 180 °C at a rate of 3 °C/min, then with 20 °C/min increased to 230 °C. The identification of EOs composition was performed based on pure compounds GC–MS spectra registered in MS libraries of NIST-MS (National Institute of Standards and Technology-Mass Spectra) and standard Wiley register of mass spectra.

Physicochemical properties

The usual chemical and physical constants defining the characteristics of EOs, such as refractive index, specific gravity, optical rotation and solubility in 95% ethanol, were determined at 20 °C.

Antioxidant activity assays

The percentage of antioxidant capacity of orange EO was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay as reported earlier by Benhammou et al. (2007). 1.5 mL of each dilution of EO in ethanol (ranging from 1.74 to 8.74 mg/mL) was mixed with 1.5 mL of ethanolic solution of DPPH (0.004%). When DPPH radical reacts with an antioxidant compound, which can give a hydrogen atom, it is reduced. The absorbance of samples was measured at 517 nm after an incubation of 30 min in darkness and at ambient temperature. The percentage of DPPH reduction (AA, %) was calculated as:

$$AA\left(\%\right)=\left[\frac{A0-A1}{A0}\right]\times 100$$ 1

where A0 is the absorbance of the blank (with only DPPH without the test compound), and A1 is the absorbance of the sample. The positive control used was ascorbic acid.

Tests of bioactivity

Growth inhibition activities of EO were evaluated by the agar diffusion and microdilution methods (NCCLS 1999). Four microbial strains were used; they include one gram-positive (Staphylococcus aureus ATCC 29213), two gram-negative (Escherichia coli ATCC 11105, Pseudomonas aeruginosa ATCC 9027) and, Condidas albicans (ATCC 10231) as a yeast strain. The active cultures were diluted with sterile physiological solution in order to get inoculums of about 10⁸ CFU/mL. Initially, Petri plates containing 10 mL of Mueller–Hinton (MH) agar medium were inoculated by spreading uniformly 1 mL of standardized inoculum containing 10⁸ CFU/mL of bacterial suspension, then dried for about five minutes. Thereafter, impregnated discs with pure EO were subsequently applied to the inoculated agar plates and then incubated for 24 h at 37 °C. Antibacterial effect was assessed by measuring of the inhibition zones diameters against the tested bacteria. The minimum inhibitory concentration (MIC) of the tested EO was determined using broth microdilution method as described by Smith-Palmer et al. (1998) with some modifications. Briefly, EO was incorporated into MH broth medium tubes containing Tween 80 (0.01%, v/v) to attain a final concentration ranging from 34.96 to 1.09 mg/mL. Then, 0.5 mL of standardized suspension of each fresh tested organism (10⁸ CFU/mL) was added to each tube. After agitation in vortex, the tubes were incubated at 37 °C during 24 h for bacteria and at 30 °C during 48 h for yeast.

Results and discussion

Influence of the heating mode on the extraction efficiency

The experimental yield values of C. sinensis EO extracted by H-EMIH and C–H processes are presented in Fig. 1. It was observed that the extraction method had a significant effect on the oil yield where H-EMIH gave a higher yield (3.77%) than that found when C–H was employed (2.86%). The evolution of EOs yield versus extraction time obtained for both heating methods presents similar appearance characterized by three distinct extraction periods. The first fraction of extracted EO, accounting for approximately 85% yield can be reached during the earlier period rising 7 min in which a rapid increase in the yield can be observed. Boukroufa et al. (2015) indicated that this fraction of EOs is found at the surface of vegetable particles. A second increasing line (step 2) is displayed, due to the internal diffusion of EO from the medium of the particles to the external medium involved by the intern warming of the water situated in the plant cells (Boukroufa et al. 2015). During this stage, the proportion of oil distillation decelerated because the EO diffusion is slow (Milojevic et al. 2008). So, approximately 9.81% of the global yield was obtained during 28 min for H-EMIH while it reached 16% after 34 min of C–H extraction method. A horizontal line marks the final part (step 3) of the extraction process when all volatile aromatic oils have already been exhausted from the plant material, which is achieved after 35 min by H-EMIH and after 41 min by C–H process.

Fig. 1.

Yield profiles as a function of time for H-EMIH and C–H assisted extraction methods of essential oils from orange peels

Hence, H-EMIH is significantly more efficient on EO yield than C–H. Under the selected operating conditions H-EMIH process is clearly faster than C–H with an obvious gain of time. As previously described by Rivera et al. (2007), this effect can be referred to the ability of the magnetic field to increase the hydro-distillation rate, by increasing hydro-diffusion from either the ionization of the extraction medium or reduction of the size of water aggregates. Zouambia et al. (2017) reported also the same influence of extraction time in pectin extraction from citrange albedos using EMIH. These authors suggested that the use of an electromagnetic field generates an agitation of ions in the electrolyte solution which leads to increase the temperature. After a few minutes, the aqueous solution initiates a motion with the solids and then a homogeneous boiling starts, allowing greater heat transfer, which increases the extraction rate. The same observations were also reported by Ferhat et al. (2006) about microwave heating; they found that this technique of heating caused the rupture of glands more rapidly than in conventional extraction.

Chemical composition

The retention times and area percentages revealing concentrations and content of C. sinensis EOs obtained by GC–MS are indicated in Table 1. Comparing both of extraction processes, a clear similarity in the fraction of the most representative compounds is observed (Fig. 2a, b). The monoterpene hydrocarbons (limonene with 94.42% for H-EMIH process and 93.25% for C–H process, and β-myrcene, which presented 2.15 and 2.27%, for H-EMIH and C-H processes respectively) are the main components. The oxygenated monoterpene was mainly represented by linalool, with 0.29 and 0.62%, respectively for H-EMIH and C–H, and by Neral whose content does not exceed 0.11%. Sesquiterpene hydrocarbons are mostly exhibited by valencene, 0.16% for H-EMIH and 0.04% for C–H. The fractions of non terpenic compounds occur as alcohols, and aldehydes.

Table 1.

Chemical composition of orange EOs obtained by H-EMIH and C–H methods

N°	Compound	R.Time	H-EMIH(%)	C–H(%)
Monoterpenes hydrocarbons			98.087	97.079
1	α-Pinene	10.95	0.537	0.592
2	Sabinene	13.54	0.463	0.767
3	β-Myrcene	14.94	2.158	2.278
4	α-Phellandrene	15.46	0.072	0.042
5	γ-terpinene	15.79	0.328	0.120
6	Limonene	17.28	94.427	93.258
7	Ocimene	18.39	0.030	0.026
8	α- terpinene	20.46	0.072	0.022
Oxygenated monoterpenes			0.68	1.01
9	Linalool	21.33	0.293	0.626
10	Citronellal	24.18	0.095	0.041
11	γ-Terpineol	26.03	0.068	0.085
12	Neral	28.63	0.102	0.098
13	β-Citral	30.11	0.122	0.151
Sesquiterpene hydrocarbons			0.245	0.107
14	β-Cubebene	37.28	0.026	0.018
15	Germacrene-D	39.51	0.026	0.025
16	Valencene	40.04	0.165	0.044
17	δ-Cadinene	41.37	0.028	0.020
Others			0.977	1.014
18	Octanal	15.63	0.435	0.780
19	Nonanal	21.56	0.102	0.125
20	Decanal	26.94	0.377	–
21	n-Dodecanal	36.55	0.063	0.064
22	Hexadecanol	26.94	–	0.0454

The amounts of the most representative compounds are indicated in bold

Fig. 2.

Gas chromatographic plot for C. sinensis EO obtained by (a) C–H process and (b) H-EMIH process

These results agree well with those found by Tan et al. (2011) who investigated that limonene (96.46%) was the principal constituent followed by β-myrcene (2.13%). Djenane et al. (2015) reported that 20 compounds are detected in the Algerian C. sinensis EO such as monoterpene hydrocarbons, limonene 7.37%, and minor component like β-pinène (3.45%), β-phellandrène (1.9%), naphtalène (1.41%), octanal (1.24%), linalol (1.21%), decanal and α-pinene were present in traces with 0.82% and 0.76% respectively.

The obtained results are quite different from those found for C. sinensis L. variety from Nigeria by Egharevba et al. (2016). They reported the main presence of α-terpineol (35.39%), followed by D-limonene (17.74%), linalool (9.73%), citronellol (4.88%), γ-muurolene (4.44%) and isopiperitenone (3.58%).

According to Djenane et al. (2015), several studies determined the chemical profile of citrus EOs, and showed the presence of monoterpenes as the majority compounds (97%), whereas other compounds, such alcohols, aldehydes and esters, were found with low amounts ranged from 1.80 to 2.20%.

It is also obvious from Table 1 that important proportion of monoterpene hydrocarbons against low amounts of oxygenated compounds are detected in H-EMIH orange peels EO comparing to C–H one. However, higher proportions of oxygenated monoterpenes (87.32%) were found in EO obtained using C–H process compared to that obtained by H-EMIH (0.68%). This difference can be explained by the oxidation of some compounds (Fig. 2), and could be verified by the high concentration of limonene oxidation products (limonene, terpinene and phellandrene) in the hydro-distilled oil which contains linalool (0.626%) and terpineol (0.085%). It is worth noticed that all these compounds were found at low amount in the volatiles obtained by H-EMIH.

Physical constants and sensory properties

The current findings revealed that there are no significant differences between the usual chemical and physical constants of EOs obtained by H-EMIH and C–H (Table 2). Table 2 summarizes the organoleptic properties of EOs extracted by both processes. In fact, EO isolated by H-EMIH presents a slight citrus note in comparison with that isolated by C–H. As confirmed by the GC–MS data, this is certainly due to the lower proportion of oxygenated monoterpenes as highly odoriferous compounds than monoterpene hydrocarbons concerning their contribution to the fragrance of EOs.

Table 2.

Physiochemical and sensory properties of orange EOs obtained by H-EMIH and C–H

		H-EMIH	C–H
Sensory properties
Color		Colorless	Colorless
Odor		Fresh, with a slight citrus note	Fresh, with a significant citrus note
Aspect		Watery in viscosity	Watery in viscosity
Physicochemical properties
Relative density $d_{20}^{20}$		0.851 ± 0.020	0.847 ± 0.025
Refractive index $n_D^{20}$		1.4731 ± 0.012	1.4730 ± 0.015

DPPH• scavenging capacity

The DPPH• scavenging capacity results of EOs isolated using both extraction processes are revealed that all of them are able to reduce the stable, purple-colored radical DPPH to the yellow-colored DPPH-H depending on the concentration (Fig. 3). In this work, the ability of EOs to reduce DPPH radical was measured by their concentration providing 50% inhibition (IC50).

Fig. 3.

Free radical scavenging capacities of C. sinensis EOs obtained by H-EMIH and C–H methods

EO isolated by H-EMIH showed the highest radical scavenging capacity with an IC50 value of 1.89 mg/mL than EO isolated by C–H process (IC50 = 3.7 mg/mL). The fact that EO isolated by H-EMIH had the highest scavenging activity compared with that isolated by C–H, the cause might be the higher amounts of monoterpenes, particularly β-Myrcene and limonene, which are the most abundant compounds in the examined oils (Table 1) and have been demonstrated as possessing high antioxidant capacity (Wei and Shibamoto 2007). According to several research in the chemical content and bioactivity of several citrus oils (Song et al. 2001), this behavior can be explained by the presence of authentic flavor components, such as γ-terpinene, terpinolene, geraniol, β-pinene and myrcene, which have good antioxidant activities. This might be attributed also to the synergy of some compounds with each other. However, the studied EO showed relatively low antioxidant activity than those reported by Frassinetti et al. (2011), who found that EOs of orange Bitter, sweet orange, lemon and mandarin showed higher antioxidant activity (20–70%) at concentrations ranging from 50 to 1000 μg/ml. This difference in the results is probably due to the methods used in extraction, the plant variety, and the methodology for the evaluation of the antioxidant activity.

Antimicrobial assay

The antimicrobial activity was determined by the absence or presence of inhibition zones. As indicated in Table 3, EOs exhibited potential activity against S. aureus and C. albicans with inhibition diameters of 22 and 19 mm, respectively, while no effect was observed against other bacteria. These results are in accordance with those of Burt et al. (2004) which showed that Gram-negative bacteria are generally more resistant to citrus EOs than Gram-positive. According to Burt et al. (2004), the resistance of Gram-negative bacteria to EOs is attributed in part to the structures complexity of these microorganisms conversely to those of Gram-positive bacteria. As found by Djenane (2015) for other EOs. Testing EOs of orange, bergamot and lemon against S. aureus, this author proved that the lemon EO presented the highest effect. It is also obvious from Table 3, that antimicrobial effects of EOs isolated by H-EMIH are slightly higher than those isolated by by C–H.

Table 3.

Antibacterial activity of Citrus sinensis essential oil

Organisms	Amoxicillin	DIZa		MICb (mg/mL)
		H-EMIH	C–H	H-EMIH	C–H
S. aureus	38	22	20	8.74	17.48
C. albicans	NA	19	17	17.48	34.96
E. coli	25	NAc	NA	NA	NA
Ps.aeruginosa	27	NA	NA	NA	NA

^aDiameter of inhibition zones in millimeter;

^bMinimum inhibitory concentration (mg/mL);

^cNo activity

However, no activity of EOs was noticed against P. aeruginosa, this could be referring to the particular outer membrane of the strain and its capacity to metabolize a variety of organic compounds (Ferhat et al. 2010). Generally, Pseudomonas bacteria use terpene as energy and carbon sources, they transform limonene (as a model molecule of terpene) into perillyl alcohol, perillic acid, terpineol, or into limonene-6,8-diol (Poudel et al. 2015).

Table 3 shows The MIC values of Citrus EO evaluated by micro-dilution assay. It was observed that the C. sinensis EOs extracted by H-EMIH showed an inhibitory effect on the dilution of 8.74 mg/mL against S. aureus and the inhibitory effect against C. albicans was observed until the dilution corresponded to 17.48 mg/mL. In this same context, Fuselli et al. (2008) found that EOs of C. sinensis and C. limonum exhibited poor antimicrobial performance against Paenibacillus larvae at high concentrations (MIC values equal to 839.9 and 763.8 mg/l, respectively).

Comparing the MIC values of C. sinensis EOs extracted by the two processes, it seems clear that EOs isolated by H-EMIH showed better antimicrobial performance than those isolated by C–H against tested strains (S. aureus and c albicans). This is probably justified by the chemical content of EOs, which changes with the experimental extraction conditions.

Conclusion

In this research, hydro-distillation assisted by EMIH as new technology was compared to conventional hydro-distillation method to extract EOs from fresh C. sinensis peels. The obtained results confirmed the effectiveness of this new technique, which accelerates the time of hydro-distillation (35 min against 41 min for C–H) with better yield (3.77% for H-EMIH against 2.86% for C–H). Compared to C–H method, the chemical content of the C. sinensis EOs obtained by H-EMIH process, determined by GC/MS, presented higher amounts of monoterpene hydrocarbons and lower amounts of oxygenated compounds. According to our results of DPPH scavenging assay, C. sinensis EO was found as an effective antioxidant in terms of free radical scavenger, and the differences observed in antioxidant activity between EOs isolated by both processes could be due to the presence of difference in amounts of monoterpenes including; β-myrcene, α-pinene, γ-terpinene and limonene as antioxidant agents. In addition, the antibacterial activity of C. sinensis EO was related with the target organism, the methods used for the evaluation of the antimicrobial capacity and their chemical composition.

In conclusion, it will be necessary to widen the area of this investigation on other plants and organs to confirm the benefits of H-EMIH.