Antioxidant Activity of the Essential Oils of Different Parts of Juniperus communis. subsp. hemisphaerica. and Juniperus oblonga.

Abstract

The essential oils of different parts of Juniperus communis. subsp. hemisphaerica. (Presl) Nyman (Cupressaceae) and Juniperus oblonga. M. B. were examined for their potential radical scavenging activity. The compositions of the essential oils of these plants were studied qualitatively and quantitatively by GC and GC-MS. The main components of the essential oils as well as positive controls were subjected to antioxidant testing. A rapid evaluation for antioxidants, using two TLC screening methods, showed that all tested oils and their main components have antioxidant activity. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms for electron were checked in the diphenylpicrylhydrazyl (DPPH) assay. In the DPPH assay, the strongest effect among the essential oils was measured for the oil of leaves of male J. communis. subsp. hemisphaerica. at a concentration of 4 µL/mL (24.0%) In the deoxyribose degradation assay, the essential oils, pure components, and positive controls were tested at different concentrations. Most of the tested compounds showed some antioxidant effects. The fruit oil of J. oblonga. has the strongest effect among the tested volatile oils.

The deoxyribose assay was modified in three different ways to assess whether the oils exhibited site-specific effects. The results of the current study, which demonstrate the DPPH scavenging activity of the essential oils of the of leaves of male J. communis. subsp. hemisphaerica. and the OH radical scavenging effects of the fruit oil of J. oblong. suggest the use of these two essential oils in very low concentrations for preserving food materials.

Keywords:

• Antioxidant activity
• deoxyribose assay
• DPPH assay
• essential oil
• J. communis. subsp. hemisphaerica
• J. oblonga
• TLC screening

Introduction

Conifers are a small group of the flora of Iran (eight species of 8000 species). All aromatic Iranian conifers belong to the Cupressaceae family. In Iran, this family consists of one species of Platycladus., one species of Cupressus., and five species of Juniperus.. The genus Juniperus. has three subgenera: Caryocedrus., Sabina., and Oxycedrus.. Two species of Iranian Juniperus. belong to the later subgenus. They are Juniperus communis. subsp. hemisphaerica. and Juniperus oblonga. (Riedl, 1968; Assadi, 1998). This study is a part of a systematic investigation on the various aromatic Iranian conifers.

Juniperus communis. L. subsp. hemisphaerica. (Presl) Nyman (syn.: J. hemisphaerica. Presl), J. communis. L. subsp. communis. auct. Pl. fl. Iran, J. communis. L. var. rectifolia. parsa) (Cupressaceae) is a dioecious and evergreen prostrate shrub with a height of 20 cm to 1.5 m. This shrub is native to Europe, Turkey, and Iran as well as the Caucasus. Its Persian names are “piru” or “lambir” (Sabeti, 1976; Assadi, 1998).

J. communis. subsp. hemisphaerica. is a medicinal plant. This plant is used to lower blood pressure and as an antidiabetic and diuretic (De Medina et al., 1994; Anonymous, 2004). This plant is used externally for rheumatic symptoms and internally to regulate menstruation, to relive menstrual pain, and as a contraceptive (Agrawal et al., 1980; Anonymous, 2004). Few papers have been published on phytochemical studies of J. communis. subsp. hemisphaerica. (San Feliciano et al., 19911995).

Juniperus oblonga. M. B. (syn.: J. communis. var. oblonga. Medw., J. communis. var. caucasica. Endl. ex Medw.) is a dioecious evergreen and a rather low tree. This tree is native to Iran, Turkey, and the Caucasus. Its Persian name is “chatanah” (Sabeti, 1976).

Juniperus oblonga. is also a medicinal plant. Ripe berries of this plant have diuretic and antiscorbutic effects (Komarov, 1968).

Phytochemical studies of Juniperus oblonga. indicated monoterpenes like α.-pinene, sabinene, limonene, terpinene-4-ol, and thujyl alcohol are the main composition of volatile oils of this plant (Goryaev et al., 1964; Adams, 2000; Pisarev et al., 2005).

There are no published reports on antioxidant activity of the essential oils obtained from different parts of these plants. Also, no previous reports have been published concerning chemical composition and/or antioxidant activity of essential oil of these plants growing in Iran.

Essential oils are known to possess potential as natural agents for food preservation. Many of them recently have been qualified as natural antioxidants and proposed as potential substitutes for synthetic antioxidants in specific sectors of food preservation where their use is not in contrast with their aroma (Ruberto & Baratta, 2000).

A plethora of different antioxidant assays is available and, because results rely on different mechanisms, they strictly depend on the oxidant/antioxidant models employed and on lipophilic/hydrophilic balance. A single-substance/single-assay produces relative results, and it is perceived as a reductive approach whenever a phytocomplex is involved. Therefore, a multiple-test and a simultaneous chemical characterization must be taken into account whenever assays of essential oils are performed to allow a balance between the sensory acceptability and functional properties (Saccheti et al., 2005).

In this article, we report the results of a study aimed to define DPPH radical scavenging and anti-OH radical activities of the essential oils obtained from different parts of J. communis. subsp. hemisphaerica. and J. oblonga.. Also, we analyzed the essential oils by GC and GC-MS in order to identify the constituents of the oils and to attempt to determine which components contribute to the antioxidant effect.

Materials and Methods

Chemicals

Chemicals were obtained from Sigma (Sigma Aldrich GmbH, Sternheim, Germany). α.-Thujene, α.-pinene, sabinene, β.-pinene, limonene, and γ.-terpinene were purchased from Roth (Karlsruhe, Germany). TLC was carried out on silica gel F₂₅₄ aluminum sheets (Merck, Darmstadt, Germany).

Plant material

J. communis. subsp. hemisphaerica. fruits and leaves of male and female were collected from the Damlou and Sefali regions, Golestan province, at the height of 2063 m from the sea, located in northern Iran.

The fruits and leaves of male and female J. oblonga. were collected from Makidi and Winagh, 1400 m, Arasbaran, Eastern Azarbaijan province, northwestern Iran.

These plants were identified by Mr. Zaree from the Noushahr Ecological Garden, where voucher specimens of both J. communis. subsp. hemisphaerica. and J. oblonga. were deposited. The herbarium numbers are 97-1015-11 and 97-1003-08, respectively. The collected materials were stored at −20°C in order to avoid unfavorable changes in the chemical components (Adams et al., 1984).

Isolation of the essential oil

Fresh leaves of male and female plants (800 g fresh weight) as well as fruits (400 g fresh weight) were cut to small pieces and then ground by a blender. The volatile oils were isolated by steam distilled using a manufactured apparatus with a condenser. Distillation continued for about 3 h and the volatile compounds containing the water-soluble fraction were allowed to settle for 30 min (Kirk-Othmer, 1978). The essential oils were separated from the aqueous layer and dried over anhydrous sodium sulfate. The dried filtered oils were stored under nitrogen gas in a sealed vial at −20°C until analyzed. The yield percentage and composition of the essential oils were expressed in mL/100 g of fresh plant materials.

GC and GC-MS analysis

The component of volatile oil samples from the leaves of male and female plants and fruits were identified using gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) analysis.

The GC-MS apparatus was a Varian GC-MS spectrometer consisting of a Varian star 3400 gas chromatograph equipped with a fused-silica column (DB-5, 30 m × 0.32 mm i.d., film thickness 0.25 µm; J&W Scientific Inc), interfaced with a mass spectrometric detector (Varian Saturn 3). The operating conditions were oven temperature, 60–280°C with the rate of 3°C/min; injector temperature, 280°C; injector mode, split injection; with the carrier gas, He; flow rate, 2 mL/min; ionization mode, electron impact (EI) at 70 eV; interface temperature, 300°C; scan range, 40–300 u.

The gas chromatograph (GC) was a Shimadzu GC-17 equipped with a FID detector, fused-silica column (BP-5, 25 m × 0.22 mm i.d., film thickness 0.25 µm). The operating conditions were oven temperature, 60–280°C with the rate of 8°C/min; injector temperature, 280°C, split ratio 1:10, with the carrier gas, N₂; detector temperature, 300°C.

The oil components were identified from their retention indices (RI) obtained with reference to n.-alkane series (Sigma, UK) on DB-5 column, mass spectra with those of authentic samples (28), composition of their mass spectra and fragmentation patters reported in literature, computer matching with MS-data bank (Saturn version 4). Quantification of the relative amount of the individual components was performed according to the Area Percentage Method without consideration of calibration factor (Adams, 2001).

Antioxidative assay

Rapid TLC screening for antioxidant

Each essential oil sample was spotted on two different silica gel sheets and developed in toluene-ethyl acetate (97:3 v/v). One of the plates was sprayed with β.-carotene–linoleic acid reagent (Pratt & Miller, 1984). Active compounds were detected as yellow spots on a white background zones, where the colors changed within 30–60 min (after spraying) were taken as positive results. The other developed TLC sheet was sprayed with a 0.2% solution of the stable radical diphenylpicrylhydrazyl (DPPH) (Cuendet et al., 1997; Kirby & Schmidt, 1997). In these plates, active compounds were detected as yellow spots on a purple background. Zones where the color changed within 30 min (after spraying) were taken as positive results. In both cases, some potential radical scavenging compounds (vitamin C, quercetin) were used as positive controls. The following seven pure component, α.-thujene, α.-pinene, sabinene, β.-pinene, limonene and γ.-terpinene, were also spotted as pure compounds.

DPPH free radical scavenging activity

In this spectrophotometric method, DPPH was used as a reagent in order to measure DPPH free radical scavenging activity (Cuendet et al., 1997; Kirby & Schmidt, 1997). Various concentrations (50 µL) of the volatile oils and some of the main components of the essential oils as well as butylhydroxytoluene (BHT) and vitamin E were added to 2.5 mL of an 0.004% solution of DPPH in methanol. The reaction mixture was shaken and then incubated for 30 min at room temperature. The amount of DPPH remaining was determined at 517 nm against a blank using a spectrophotometer (Milton Roy Company Spectronic 2OD). Quercetin and ascorbic acid were used as positive controls. All tests were carried out five-times.

Deoxyribose degradation assay

OH free radical scavenging activity of samples was measured with the procedure described by Burits et al. (2001). All solutions were prepared freshly. In this method, 100 µL of 28 mM, 2-deoxy-d-ribose in phosphate buffer (pH 7.4), 500 µL of solutions of various concentrations of the tested oils or the other pure compounds as well as quercetin and dimethylsulfoxide, DMSO (in phosphate buffer containing 1.5% of Tween 80), 200 µL of mixture of 1.04 mM EDTA and 200 µM FeCl₃ (1:1 v/v), 100 µL of 1.0 mM H₂O₂, and 100 µL of 1.0 mM ascorbic acid were mixed. The reaction mixtures (final volume 1.0 mL) were incubated for 1 h at 37°C. To each of these solutions, 1 mL of thiobarbituric acid (TBA) (1%), and 1.0 mL of trichloroacetic acid (TCA) (2.8%), were added, and again incubated for 20 min at 100°C. After cooling, absorbance was measured at 532 nm against a blank containing dexoyribose and buffer. Reactions were carried out in triplicate. Inhibition (I.) of deoxyribose degradation in percent was calculated in the following way: where A.₀ is the absorbance of the control reaction (reaction, containing no test compound) and A.₁ is the absorbance of the test compound.

Assay for site-specific actions

This assay presented only another feature of the deoxyribose method, in order to assess possible site-specific action (Halliwell et al., 1987; Aruoma & Guppett, 1997).

Performing these tests indicates whether the oils possess pro-oxidative activities, if they can stimulate an oxidative process or if they generate fragments, which react with thiobarbituric acid. The described deoxyribose method was adapted in three ways:

1. FeCl₃ was used instead of a solution of Fe³⁺-EDTA.

2. Ascorbic acid was omitted from the test system.

3. The reaction was performed without deoxyribose. The required volume was made up with phosphate buffer pH 7.4. All experiments were carried out in triplicate (Burits et al., 2001).

Statistical analysis

Values expressed are means±standard deviation. All statistical analyses were carried out using SPSS 11.05 for Windows.

Results and Discussion

GC-MS analysis

The essential oils isolated separately from the leaves of male and female plants as well as the fruits of J. communis. subsp. hemisphaerica. were clear and light-yellow and possessed a strong odor. The various parts of the plant yielded 0.63%, 0.60%, and 1% (v/w) of volatile oil, respectively. J. oblonga. leaves of male and female yielded 0.6%, 0.30% (v/w) pale-yellow essential oils, respectively, and the fruits of this plant yielded 0.70% (v/w) colorless oil. The essential oils were analyzed by GC and GC-MS.

The constituents of each essential oil are listed in Tables 1 and 2 in order of elution from the DB5 column. In the oils of leaves of both male and female J. communis. subsp. hemisphaerica., 21 compounds (92.7% and 91.5% of the essential oils compounds, respectively), and in the oil of fruits of this plant, 20 compounds (95.1% of the essential oil compounds), were identified (Table 1).

Table 1.. Chemical composition of the volatile oil of fruits and male and female leaves of Juniperus communis. subsp. hemisphaerica..

Components	Retention index^a.	Male leaves %	Female leaves %	Fruits %
α.-Thujene	935	1.7	1.6	0.9
α.-Pinene	940	12.1	15.8	13.6
Sabinene	977	21.9	20.3	25.1
Myrcene	997	2.2	3.2	3.6
α.-Terpinene	1016	0.7	0.8	1.1
Cymene (para.)	1028	1.2	2.7	2.3
Limonene	1035	7.1	9.2	9.1
γ.-Terpinene	1065	7.2	3.9	5.2
Terpinolene	1090	6.1	3.1	3.0
4-Terpineol	1180	8.5	5.3	8.7
Citronellol	1231	0.4	0.8	0.9
Bornyl acetate	1288	0.9	1.1	—
α.-Terpinyl acetate	1348	2.1	2.9	2.8
β.-Elemene	1385	1.4	1.8	2.5
β.-Caryophyllene	1420	3.1	2.2	2.9
α.-Humulene	1457	1.6	1.3	0.7
Germacrene D	1479	4.9	6.1	5.3
α.-Bisabolen	1506	1.2	1.3	t
Cadinene-delta	1528	t	1.1	0.5
Caryophyllene oxide	1583	5.7	4.2	6.5
α.-Bisabolol	1683	2.7	2.9	0.4
Grouped compounds:
Monoterpene hydrocarbons		60.2	60.6	63.9
Oxygen-containing monoterpenes		11.9	10.1	12.4
Sesquiterpene hydrocarbons		12.2	13.7	11.9
Oxygen-containing sesquiterpenes		8.4	7.1	6.9

t = trace (< 0.1%).

^a.The retention Kovats indices were determined on DB-5 capillary column.

Table 2.. Chemical composition of the volatile oil of fruits and leaves of male and female Juniperus oblonga..

Components	Retention index^a.	Male leaves %	Female leaves %	Fruits %
α.-Thujene	935	3.4	5.1	2.6
α.-Pinene	940	15.6	13.6	33.3
Sabinene	977	16.4	19.5	23.8
β.-Pinene	990	3.7	4.2	20.8
Myrcene	997	4.2	5.2	—
δ.-2-Carene	1011	2.6	1.2	—
α.-Terpinene	1020	—	0.9	0.3
Limonene	1034	5.5	5.2	4.4
γ.-Terpinene	1064	3.3	5.3	0.9
Terpinolene	1092	2.7	3.4	1.9
4-Terpineol	1182	3.7	4.8	0.1
α.-Terpinyl acetate	1348	3.4	1.5	t
β.-Elemene	1383	1.5	1.6	0.3
β.-Caryophyllene	1422	4.0	6.4	0.2
γ.-Elemene	1435	6.7	5.4	4.5
α.-Humulene	1452	1.3	t	0.1
Germacrene D	1482	8.2	5.7	1.5
β.-Bisabolen	1511	4.0	3.2	—
δ.-Cadinene	1522	2.7	2.3	—
Germacrene B	1565	—	—	1.7
α.-Bisabolol	1688	2.7	1.6	—
Grouped compounds:
Monoterpene hydrocarbons		57.4	63.6	88.0
Oxygen-containing monoterpenes		7.1	6.3	0.1
Sesquiterpene hydrocarbons		28.4	24.6	8.3
Oxygen-containing sesquiterpenes		2.7	1.6

t = trace (< 0.1%).

^a.The retention Kovats indices were determined on DB-5 capillary column.

The major components in essential oils were monoterpene hydrocarbons. The main monoterpenes of the oils of the fruits and both male and female leaves were α.-pinene (13.6%, 12.1%, and 15.8%), sabinene (25.1%, 21.9%, and 20.3%), limonene (9.1%, 7.1%, and 9.2%), and 4-terpineol (8.7%, 8.5%, and 5.3%), respectively, for J. communis. subsp. hemisphaerica..

Analysis of J. oblonga. essential oils leads to identifying 19 and 20 compounds in the oil of leaves of both male and female, respectively (95.6% and 96.1% of the essential oil compounds, respectively) and 14 compounds in the oils of fruits (96.4% of the essential oil compounds (Table 2). α.-Pinene (33.3%, 15.6%, and 13.6%), sabinene (23.8%, 16.4%, and 19.5%), and limonene (4.4%, 5.5%, and 5.2%), respectively, were identified as the major components of fruits and leaves of male and female essential oils of J. oblonga.. β.-Pinene (20.8%) was also one of the major components of J. oblonga. fruits oils. The amounts of these components are different from other published data (Adams, 2000; Mastelic & Milos, 2000). These differences are very common as studies have shown that the oil composition of individual plants may vary widely (Hoerster, 1974).

Antioxidative assay

Rapid TLC screening

Essential oils of different parts of the plant, their pure components, and positive controls were tested for their antioxidant activity using a rapid TLC screen. First, the TLC sheet with the tested compounds was sprayed with β.-carotene–linoleic acid reagent. In the case of essential oils, only one yellow zone related to each oil was detectable. Also, sabinene produced one active spot. In plate sprayed with DPPH reagent, also, one spot appeared for each essential oil (like the plate sprayed with β.-carotene–linoleic acid reagent). All the pure compounds produced yellow spots. The fact that the polarity of monoterpene hydrocarbons of the essential oils are very similar (therefore they generally should accumulate on the same area) may explain why only one yellow zone appeared for each essential oil. Therefore, the yellow zones in these TLC plates cannot be attributed to a specific compound. Because each essential oil contains several different components, it is more appropriate if each zone relates to a group of compounds. The results of the TLC method can be used as a rapid test to detect antioxidant effects of samples, but it is not appropriate to identify which compounds in an essential oil correspond to the antioxidant effect. Therefore, the essential oils and other pure components that possessed antioxidant activity were subjected to further testing.

DPPH free radical scavenging activity

In this assay, the abilities of the test compounds (both the essential oils and their main components) to donate hydrogen atoms or electrons were measured spectrophotometrically. Each of the tested materials that reduced DPPH to the yellow-colored product, diphenylpicrylhydrazine, and decrease the absorbance at 517 nm, possess antioxidant activity.

Pure compounds use as standards showed very different antioxidant activity, ranging from 17.74 (in concentration of 4 µL/mL) for γ.-terpinene, no activity for α.-pinene, and extremely low activity for β.-pinene and limonene. Positive controls, quercetin and ascorbic acid, possessed relatively high antioxidant effect (77.74% and 38.06%, and 23.5%, respectively). The strongest effect among the essential oils was measured for the oil of leaves ofmale J. communis. subsp. hemisphaerica. in a concentration of 4 µL/mL (24.0%) and the weakest effect was related to oil of J. oblonga. fruits (1.74%).

The strong antioxidant activity of the leaves of male J. communis. subsp. hemisphaerica. may be partially due to the high amounts of γ.-terpinene (7.2%) in the leaves, which is a strong antioxidant compound (Ruberto & Baratta, 2000). However, the low antioxidant activity of J. oblonga. fruits may be partially due to the low amounts of γ.-terpinene (0.9%) as well as high amounts of α.-pinene (33.3%) and β.-pinene (20.8%), both of which are inactive in DPPH test.

This can be generally observed when the higher antioxidant activity of the leaves and fruits of J. communis. subsp. hemisphaerica. (contain no detectable amount of β.-pinene and low amount of α.-pinene, higher amount of γ.-terpinene) compares with lower antioxidant activity of the leaves and fruits of J. oblonga. (contain high amount of α.-pinene and β.-pinene and low amount of γ.-terpinene) in the DPPH test (Table 3).

Table 3.. Antioxidant activity of the test compounds in DPPH assay.

	Concentration (µL/mL)
	0.1	0.5	1	2	4
Quercetin	3.26 (0.44)^a.	10.59 (0.82)	21.84 (0.54)	37.92 (1.63)	77.74 (3.21)
Vitamin C	1.3	5.28 (0.97)	11.06 (1.75)	19.26 (0.32)	38.06 (1.03)
BHT	0.5	1.94 (0.32)	2.58 (0.39)	4.76 (0.6)	13.5 (3.62)
DMSO	0.56 (0.42)	0.3 (0.24)	0.98 (0.39)	0.66 (0.32)	2.44 (0.85)
Vitamin E	NA	NA	NA	1.10 (1.1)	5.26 (0.32)
γ.-Terpinene	0.82 (0.39)	3.26 (0.44)	5.58 (0.36)	8.48 (0.64)	17.74 (0.32)
δ.-2-Carene	0.5	1.94 (0.32)	2.58 (0.39)	4.76 (0.60)	13.5 (3.62)
Limonene	0.12 (0.24)	NA	2.14 (0.32)	0.36 (0.29)	0.96 (0.44)
β.-Pinene	0.36 (0.29)	NA	NA	0.12 (0.24)	0.96 (0.44)
α.-Pinene	NA	NA	NA	NA	NA
α.-Thujene	NA	0.54 (0.44)	0.9	1.62 (0.36)	5.38 (0.74)
Sabinene	NA	1.26 (0.44)	1.08 (0.36)	1.82 (0.04)	4.82 (0.45)
J. communis. M	1.4	2.8	6.3 (0.89)	12.6	24.4 (1.03)
J. communis. F	1.4	1.4	3.5 (1.08)	5.18 (0.84)	9.01 (0.63)
J. communis. FT	NA	0.56 (0.69)	0.84 (0.69)	1.68 (0.56)	5.6
J. oblonga. M	0.3	1.26 (0.32)	1.26 (0.32)	1.9	7.5 (0.88)
J. oblonga. F	NA	0.12 (0.15)	0.34 (0.4)	0.78 (0.39)	5.74 (0.32)
J. oblonga. FT	NA	NA	0.06 (0.12)	NA	1.74 (0.32)

NA, not active; M, male; F, female; FT, fruit.

^a.Values in parentheses represent standard deviation.

The results of this study demonstrate the DPPH scavenging activity of some of the tested essential oils of these plants. Also, it has been reported that the methanol extract of J. chinensis. heartwood showed strong antioxidant activity when the antioxidant activity was determined by measuring the radical scavenging effect on DPPH (Lim et al., 2002).

Deoxyribose degradation assay

In deoxyribose degradation assay, most of the tested compounds showed some antioxidant effects. In this test, OH radicals were generated by the reaction of ferric-EDTA together with H₂O₂ and ascorbic acid to attack the substrate deoxyribose. The resulting products of the radical attack form a pink chromogen when heated with TBA in acid solution. Antioxidant substances, when incubated with this reaction mixture, are able to interfere with the free radical reaction, and could prevent damage to the sugar.

Positive controls, quercetin and DMSO, showed the highest activity (44.07 and 55.77 µg/ml, respectively) in this test. Some pure compounds (α.-pinene) have no effect, while some others (β.-pinene and limonene in 0.05 µL/mL) have remarkable effects.

The activities of pure standard compounds were variable. β.-Pinene had the highest activity where some other compounds, like α.-thujene, α.-pinene, sabinene, and δ.-2-carene, showed very weak antioxidant effects (Table 4). The strongest effect among the essential oils tested with this assay was measured for the oil of J. oblonga. fruits. This may be due to the high amounts of β.-pinene (20.8%) in the oil (Tables 3 and 4).

Table 4.. Antioxidant activity of the test compounds in deoxyribose assay.

	Concentration (µL/mL)
	0.05	0.1	0.2	0.5	1
Quercetin	3.37 (0.35)^a.	14.57 (0.51)	25.97 (0.74)	33.43 (0.46)	44.07 (0.90)
DMSO	7.13 (0.90)	13.13 (0.40)	25.30 (1.30)	44.37 (0.96)	55.77 (0.65)
γ.-Terpinene	8.10 (0.52)	12.23 (0.45)	12.63 (0.60)	6.97 (1.50)	17.07 (1.16)
δ.-2-Carene	NA	4.87 (0.76)	2.73 (0.64)	8.87 (1.05)	NA
β.-Pinene	47.60 (1.42)	39.53 (0.99)	34.63 (1.42)	25.23 (3.41)	31.67 (1.53)
Limonene	40.20 (1.20)	16.30 (1.01)	40.60 (2.91)	26.00 (0.92)	12.60 (2.27)
α.-Pinene	NA	NA	1.20 (0.40)	5.8 (0.79)	4.10 (1.21)
α.-Thujene	10.17 (0.61)	10.50 (0.62)	8.20 (1.54)	9.93 (0.40)	2.73 (0.61)
Sabinene	NA	12.23 (0.81)	17.0 (1.40)	8.23 (1.50)	1.27 (1.42)
J. communis. M	2.68 (0.53)	9.10 (0.50)	5.55 (0.84)	5.73 (1.39)	NA
J. communis. F	NA	2.50 (1.00)	NA	NA	5.44 (0.86)
J. communis. FT	0.77 (0.86)	1.13 (1.15)	NA	4.63 (1.15)	7.10 (1.15)
J. oblonga. M	5.93 (0.71)	7.70 (0.50)	2.27 (0.71)	1.47 (0.95)	NA
J. oblonga. F	7.80 (0.69)	5.30 (0.35)	8.20 (1.73)	NA	NA
J. oblonga. FT	10.30 (0.80)	14.53 (1.17)	13.77 (0.92)	10.57 (1.22)	NA

NA, not active; M, male; F, female; FT, fruit.

^a.Values in parentheses represent standard deviation.

Some of the tested essential oils and pure standard compounds showed negative effects. This may be due to a pro-oxidative effect or to the ability to produce TBARS (Halliwel, 2000). This will be examined in the assay for site-specific actions.

Assay for site-specific actions

The deoxyribose assay was modified in three different ways to assess whether the oils exhibited site-specific effects. On one occasion, the EDTA was omitted from the reaction mixture. Iron was added as ferric chloride instead of the complex form Fe³⁺-EDTA. Some of the Fe³⁺ ions bind directly to the sugar and its degradation becomes site-specific. The formed hydroxyl radicals attack deoxyribose immediately. An inhibition of this degradation in the absence of EDTA depends not only on a scavenger's ability to react with OH^⋅ but also on its potential to form complex with iron ions. None of the test compounds showed remarkable differences when EDTA was omitted.

Ascorbate was omitted from the reaction mixture on another occasion to examine the ability of a substance to reduce Fe³⁺-EDTA and decrease the rate of OH radical generation. If an agent possesses pro-oxidant activity, the deoxyribose degradation is stimulated, more fragments are produced, and the absorbance at 532 nm increases significantly (Burits & Bucar, 2000). From all tested samples, the oils of male leaves and fruits of J. oblonga., as well as standard compounds sabinene, limonene, and γ.-terpinene (l µL/mL), could induce the radical generation.

Finally, deoxyribose was omitted from the reaction mixture in order to see whether the compounds under examination themselves could form degradation products that react with TBA to make chromogen. By omission of deoxyribose from the reaction mixture, the tested oils were the only substrates to react with OH radicals and form TBA reactive species, TBARS (Burits et al., 2001). The volatile oil obtained from fruits and leaves of male and female J. oblonga., as well as standard compounds, limonene, α.-pinene, and α.-thujene, were the only substrates to react with OH radicals and to form TBARS.

Therefore, the antioxidant activity strength observed for these tested oils and compounds may not be their actual antioxidant activity. The increase of absorption in their solutions may be due to production of chromogens by various compounds in the solution, therefore, causing a false decrease in antioxidant activity.

Conclusions

The essential oils, unlike plants extracts, have special homogenicity. They contain various nonpolar compounds, mainly, terpenoids. (S.l.). Because most terpenoids show antioxidant activity, therefore, the essential oils containing these terpenoids may also show some antioxidant activity.

It is obvious that many parameters must be carefully taken into consideration when antioxidant activity of special compounds is studied. One of the most important parameters is the radical species. It appears that the antioxidant effects of a compound may depend entirely on the radical species or assay method used to determine activity, which should, therefore, always be clearly defined. However, we can suggest that antioxidant capacity data for putative scavenging compound determined via a single assay method be interpreted with some caution. On this basis, the generalized term antioxidant lacks significance, and should be replaced by more precise designation specifying the nature of radical species neutralized, such as anti hydroxyl radical, anti-oxoferryl radical, anti-superoxide ions, and so forth (Mantle et al., 2000).

The other considerable parameter is the polarity of the solvent system used in the method. Essential oils and their pure compounds are not soluble in polar solvents. Therefore, they cannot show very strong antioxidant effects. Some of the tested compounds that have no antioxidant effect in the current study were previously reported to have very high activity using two different lipid systems. For example, sabinene, α.-thujene, and γ.-terpinene, which show low activities in the deoxyribose method, have high effects in assays using two lipid solvent systems (Ruberto & Baratta, 2000).

The results of the current study, which demonstrate the DPPH scavenging activity of the essential oils of the of leaves ofmale J. communis. subsp. hemisphaerica. and the OH radical scavenging effects of the fruit oil of J. oblonga., suggest the use of these two essential oils in very low concentrations for preserving food materials.

Acknowledgments

This work was supported by a grant from Mashhad University of Medical Sciences (MUMS) Research council, Mashhad, Iran. The authors would like to thank the authorities in MUMS and in the School of Pharmacy, MUMS, for their support.