Evaluation of the Chemical Composition and Antioxidant Activity of the Peel Oil of Citrus nobilis

Abstract

Peel oil of Citrus nobilis (Lour) was analyzed for determining its chemical composition. Fourteen identified components accounted for 99.1% (GC) and 100.0% (FID) of the total oil. Major component of the oil was limonene (76.8%-GC and 86.2%-FID). Essential oil was also evaluated for its antioxidant activity in four complementary test systems namely; β-carotene/linoleic acid, DPPH radical scavenging, reducing power and metal chelating activities. In the first system, antioxidant activity increased with the increasing concentration. At 20.0 mg.ml⁻¹ concentration, antioxidant property of the oil was 96.8% ± 0.2 and as strong as the positive controls BHT and α-tocopherol. Scavenging effect of the oil was superior to the positive controls BHT and α-tocopherol at 1.5 mg.ml⁻¹ concentration (96.4% ± 0.1). Reducing power and chelating effect of the essential oil increased with the increasing concentration.

Keywords:

• Citrus nobilis
• Peel oil
• Chemical composition
• Antioxidant activity

INTRODUCTION

It is well known that oxidative damage of biological molecules in human body is involved in degenerative or pathological processes such as aging, coronary heart disease (CHD) and cancer. These oxidative damages could be retarded by endogenous defense systems such as catalase, superoxide dismutase, and the glutathione peroxidase system, but these systems are not completely efficient.[1,2] In the past decade, lots of epidemiological studies have conformed that intake of exogenous antioxidants is effective in preventing or suppressing such diseases.[3,4] Several synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene (BHT), and tetr-butylhydroquinone (TBHQ) are commercially available and currently used. However, these substances may be inappropriate for chronic human consumption, because recent publications have mentioned their possible toxic properties for human health and environment.[5,6] Hence, the development of alternative antioxidants from natural origin has attracted considerable attention and is thought to be a desirable development.

The production of citrus fruits across the world continues to show a tremendous growth. Its global production reached 110 million metric tones in 2004 and has increased about 26% over the past 10 years.[7] Citrus fruits are largely processed for their juice, one of the most important commodities, as well for their essential oil. Despite the fact that USA and Brazil are the main producers of citrus fruits, south-east Asia is believed to be the place of origin of citrus fruits. Ample studies have been carried out in order to investigate the volatile compounds present in many citrus fruits. The scope of the research ranges from the famous cultivars, such as Valencia and Navel oranges, blood and blond oranges, mandarins and Dancy tangerines and ponkans, the local cultivars such as Italian blood and blond oranges, Japanese yuzu and Turkish kozan.[8–13] Research was also extended from the citrus essential oils to the juices, as they have different aroma profiles.[14–19]

The reason for this study is that antioxidant activity of the peel oil of Citrus nobilis (Lour) has not previously been reported in the literature although the people have been using the peel for its aroma for the several purposes. Therefore, the aim of present study was to is identifying the chemical composition of the peel oil of C. nobilis and evaluating its antioxidant potential by four different antioxidant test systems namely β-carotene/linoleic acid, DPPH, reducing power and chelating effect.

MATERIALS AND METHODS

Chemicals

Potassium ferricyanide, ferrous chloride, ferric chloride, Folin-Ciocalteu's reagent (FCR), methanol, and trichloroacetic acid (TCA) were obtained from E. Merck (Darmstadt, Germany). 1.1-Diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), and α-tocopherol (TOC) were obtained from Sigma Chemical Co. (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals and solvents are of analytical grade.

Plant Material and Extraction of the Essential Oil

Citrus nobilis fruits were collected in 2006 from Duzici, Osmaniye, Turkey. Peels (100 g) of the fresh fruits were submitted for 3 h to water-distillation (at 100°C) using a Clevenger-type apparatus (yield 3.15%, v/w). Essential oil was condensed by external cooling water at 15°C and collected. Obtained essential oil was dried over anhydrous sodium sulphate and after filtration, stored in a sealed bottle at +4°C until tested and analysed.

GC-MS Analysis Conditions

GC-MS analyses were performed by using an Agilent-5973 Network System. A mass spectrometer with an ion trap detector in full scan mode under electron impact ionization (70 eV) was used. The chromatographic column used for the analysis was HP-5 capillary column (30 m 0.32-mm i.d., film thickness 0.25 lm). The carrier gas used was helium, at a flow rate of 1 mL/min. The injections were performed in splitless mode at 230°C. One microliter essential oil solution in hexane (HPLC grade) was injected and analyzed with the column held initially at 60°C for 2 min and then increased to 260°C with a 5°C/min heating ramp and subsequently kept at 260°C for 13 min. The relative percentage amounts of the separated compounds were calculated from total ion chromatograms by a computerized integrator.

Total Antioxidant Activity by β-Carotene--Linoleic Acid Method

In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation.[20] A stock solution of (-carotene--linoleic acid mixture was prepared as following: 0.5 mg β-carotene was dissolved in 1 ml of chloroform (HPLC grade). 25 μl linoleic acid and 200 mg Tween 40 was added. Chloroform was completely evaporated using a vacuum evaporator. Then 100 ml of oxygenated distilled water was added with vigorous shaking; 2.5 ml of this reaction mixture was dispersed to test tubes and 0.5 ml of various concentrations (0.5–20 mg.ml⁻¹) of the oils in methanol were added and the emulsion system was incubated for up to 2 h at 50°C. The same procedure was repeated with the positive control BHT, (-tocopherol. and a blank. After this incubation period, absorbance of the mixtures was measured at 490 nm. Measurement of absorbance was continued until the color of β-carotene disappeared. The bleaching rate (R) of β-carotene was calculated according to Eq. (1).

(1)

where ln: natural log; a: absorbance at time 0; and b: absorbance at time t (120 min).[21] The antioxidant activity (AA) was calculated in terms of percent inhibition relative to the control using Eq. (2).

(2)

Antioxidative activities of the oil was compared with those of BHT and α-tocopherol at 0.5 mg ml⁻¹ and blank consisting of only 0.5 ml methanol.

Scavenging Effect on 1,1-Diphenyl-2-Picrylhydrazyl

The hydrogen atoms or electrons donation ability of the corresponding samples and some pure compounds were measured from the bleaching of purple colored methanol solution of DPPH. The effect of the oil on DPPH radical was estimated according to Hatano et al.[22] Four ml of various concentrations (0.25–1.50 mg.ml⁻¹) of the oils in methanol was added to a 1 ml of DPPH radical solution in methanol (final concentration of DPPH was 0.2 mM). The mixture was shaken vigorously and allowed standing for 30 min; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). Inhibition of free radical DPPH in percent (I%) was calculated as:

where A_Control is the absorbance of the control reaction (containing all reagents except the test compound); and A_Sample is the absorbance of the test compound. BHT and α-tocopherol were used as a control.

Reducing Power

The reducing power was determined according to the method of Oyaizu.[23] The essential oil (1–6 mg.ml⁻¹) in methanol (2.5 ml) was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricynide and the mixture was incubated at 50 (C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid were added, and the mixture was centrifuged at 200 g (MSE Mistral 2000, London, UK) for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml of deionized water and 0.5 ml of 0.1% ferric chloride. Finally the absorbance was measured at 700 nm against a blank. BHT and (-tocopherol were used as a control.

Chelating Effects on Ferrous Ions

The chelating effect was determined according to the method of Dinis et al.[24] Briefly, 2 ml of various concentrations (0.0625–0.50 mg.ml⁻¹) of the oils in methanol was added to a solution of 2 mM FeCl₂ (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml). Total volume was adjusted to 5 ml with methanol and then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was measured spectrophotometricaly at 562 nm. The inhibition percentage of ferrozine--Fe²⁺ complex formation was calculated by using the formula given below:

where A_Control is the absorbance of control (the control contains FeCl₂ and ferrozine, complex formation molecules); and A_Sample is the absorbance of the test compound. BHT and α-tocopherol were used as a control.

RESULTS AND DISCUSSION

Chemical Composition of the Essential Oil

GC analysis of the peel oil of C. nobilis was confirmed by FID and the results are presented in Table 1. The total of the 14 identified components accounted for 99.11% (GC) and 100.00% (FID) of the total oil. It can be seen from these results that FID analysis gave the more consistent outputs. Major components of the oil were limonene (76.77%-GC and 86.17%-FID). This component was followed by γ-terpinene (8.24%-GC and 4.02%-FID), linalool (3.01%-GC and 1.46%-FID), and myrcene (2.38%-GC and 2.22%-FID), respectively. According to the results obtained from both systems, monoterpenes consist of 90% of the total oil approximately. The second large compound class was monoterpeniods. Sesquiterpenes were represented in a small quantity in the oil.

Table 1 Chemical composition of the peel oil of C. nobilis

		GC			FID
No	Compounds	% Area	Exp. RT ^a	Exp. RI ^b	% Area	RI ^c	Ident. RI ^d
1	α-Thujene	0.18	4.96	929	0.73	931	930
2	α-Pinene	1.08	5.14	936	0.30	940	939
3	β-Pinene	0.77	6.32	954	0.21	960	956
4	Myrcene	2.38	6.79	995	2.22	993	991
5	Limonene	76.77	8.26	1032	86.17	1031	1029
6	γ-Terpinene	8.24	9.18	1067	4.02	1063	1060
7	Terpinolene	0.71	10.25	1091	0.27	1090	1089
8	Linalool	3.01	11.02	1102	1.46	1099	1097
9	Terpinen-4-ol	0.73	14.17	1189	0.29	1175	1177
10	Methyl chavicol	3.65	15.01	1201	1.62	1192	1196
11	Exo-Fenchyl Acetate	0.33	16.23	1236	1.44	1228	1233
12	Perilla aldehyde	0.39	18.19	1274	0.47	1269	1272
13	β-Elemene	0.36	23.01	1396	0.59	1395	1391
14	E,E-α-Farnesene	0.51	27.83	1511	0.21	1508	1506
	Total Identification	99.11			100.00
	Compound Classes	% Area			% Area
	Monoterpenes	90.13			90.23
	Monoterpenoids	8.11			5.28
	Sesquiterpenes	0.87			0.80
^aExp. RT: experimental retention time (as minutes); ^bExp. RI: experimental retention indices; ^cRI: retention indices; and ^dIdent. LRI: identification library retention indices.

For most of the Citrus, mandarin peel oil and leof or “petit grain” oil can be obtained, respectively, by cold pressing and hydrodistillation of the fresh material. Studies concerning the chemical composition of peel and leaf oils of mandarins have been reviewed.[25–28] Nevertheless, in the most cases, species and/or the varieties remained unspecified and the results are not useful from the taxonomic point of view.[16]

Lota et al.[16] studied the chemical composition of the peel oils of more than 58 mandarin cultivars, belonging to 15 different species. They have divided these cultivars into several chemotypes according to the compositions of their peel oils such as; limonene chemotype, limonene/γ-terpinene chemotype, linalyl acetate/limonene chemotype, sabinene/linalool chemotype, γ-terpinene/linalool chemotype, and methyl N-methylanthranilate chemotype. According to this report, chemical composition of the cultivars included in this chemotype was dominated by limonene (55.8%−79.0%) associated with γ-terpinene (0.1–19.9%) and p-cymene (0–12.0%). This chemotype has also been described in the oils of sanderson cultivar from USA,[29] “comun” and “malvasio” cultivars from Uruguay,[30] unspecified cultivars from Italy, Argentina,[31,32] and from France.[33]A great number of commercial oils (266 samples) of unspecified cultivar from Italy and belonging to this chemotype were investigated by Verzera et al.[34] Lota et al.[16] were characterized the cultivar C. nobilis as chemotype dominated by the limonene associated with γ-terpinene.

Antioxidant Activity

Antioxidant activity of the peel oil of C. nobilis has been tested by four complementary test systems. Results from these experiments are shown is Table 2. In β-carotene/linoleic acid system, antioxidant activity of the oil increased with the increasing concentration. At 20.0 mg.ml⁻¹ concentration, antioxidant property of the oil was 96.8% ± 0.2 and as strong as the positive controls BHT and α-tocopherol (98.1% ± 0.8 and 97.7% ± 0.5, respectively).

Table 2 Antioxidant activity of the peel oil of C. nobili s. ^a

	Sample concentration (mg.ml^−1)	Essential Oil	BHT ^b	TOC ^c
β-Carotene Linoleic Acid (%)	0.50	71.21 ± 1.29	98.13 ± 0.76	97.71 ± 0.52
	2.00	86.78 ± 0.53	–	–
	8.00	94.62 ± 0.31	–	–
	20.00	96.80 ± 0.16	–	–
DPPH (%)	0.025	–	92.26 ± 0.87	76.75 ± 0.48
	0.25	18.95 ± 0.46	–	–
	0.50	48.46 ± 1.23	–	–
	1.00	82.51 ± 0.59	–	–
	1.50	96.44 ± 0.10	–	–
Reducing power (%)	1.00	0.29 ± 0.07	3.379 ± 0.043	2.181 ± 0.033
	2.00	0.36 ± 0.05	–	–
	4.00	0.79 ± 0.03	–	–
	6.00	1.26 ± 0.05	–	–
Chelating effect (%)	0.0625	48.13 ± 0.52	19.01 ± 0.52	42.40 ± 0.74
	0.125	62.21 ± 1.14	30.19 ± 1.01	44.72 ± 0.39
	0.25	89.44 ± 0.88	41.39 ± 0,64	50.70 ± 1.18
	0.50	92.26 ± 0.13	68.20 ± 1.25	56.55 ± 0.66
^aValues expressed are means ± S.D. of three parallel measurements; ^bBHT: butylated hydroxy toluene; and ^cTOC: α-Tocopherol.

The radical scavenging activity of the oil was tested using a methanolic solution of the “stable” free radical 1, 1-Diphenyl-2-picrylhydrazyl (DPPH). The oil did not show any radical scavenging effect at 0.025 mg.ml⁻¹ concentration. On the other hand, scavenging effect of the oil was superior to the positive controls BHT and α-tocopherol at 1.50 mg.ml⁻¹ concentration (96.44% ± 0.10).

In the reducing power assay, the yellow color of the test solution changes to various shades of green and blue, depending on the reducing power of each compound. The presence of reducers (i.e., antioxidants) causes the reduction of the Fe³⁺/ferricyanide complex to the ferrous form. The reducing power of the essential oil increased with concentration. The reducing power was measured as 1.26% ± 0.05 at 6.00 mg.ml⁻¹ concentration.

Ferrous ions could stimulate lipid peroxidation by Fenton reaction, and could also accelerate peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen and perpetuate the chain reaction of lipid peroxidation.[35] As it can be seen from the Table 2 that chelating capacity of the essential oil was increased with the increasing concentration. Chelating effect was measured as 92.26% ± 0.13 at 0.5 mg ml⁻¹ concentration. It is extremely important to point out that, the oil showed better chelating effect than those of the standards at the all concentration values.

As it can be seen from the Table 1, limonene is the major compound for the peel oil of C. nobilis. Limonene, a monocyclic monoterpene, is a natural component of a variety of foods and beverages. It was found in many fruits (especially in Citrus fruits), vegetables, and spices.[36] As the main odor constituent of citrus, limonene is used in food manufacturing as a flavoring and added to cleaning products to give a lemon-orange fragrance.[37] Large dose of limonene has been proved to have anticancer activity and minimal toxicity in preclinical and clinical studies.[38–45] Low dose of limonene showed carminative and cholagogue effects and was used in the treatment of gallstone, cholecystitis and angiocholitis.[46] As a result, considerable research interest has been focused on this compound.

As far as our literature survey could ascertain, no information available in the literature directly concerning the antioxidant action of limonene. On the other hand, some reports confirmed that the antioxidant activities of the plants rich in limonene.[47–48] In the light of data given in Table 1, and the supporting results presented in Table 2 show the antioxidant potential of limonene as a strong radical scavenger. These findings have also been supported the earlier results of by Ruberto and Baratta.[49]

CONCLUSIONS

In general, antioxidant properties of the essential oil were found promising. It was significantly increased with increasing concentration and exhibited highly comparable activity with BHT and α-tocopherol. Especially free radical scavenging action of the oil was superior to the positive controls BHT and α-tocopherol at 1.5 mg.ml⁻¹ concentration (96.4% ± 0.1). Although the people have been using the peel of the Citrus fruits for their aroma for the several purposes, there is no detailed report in the literature concerning its antioxidant capacity. Therefore, identifying the biological properties of these oils, as natural sources, could be useful for the food, cosmetic and health care industries. From this point of view, this study could be assumed as the first report on this species.