Composition and Antioxidant Effect of the Essential Oils of the Flowers and Fruits of Ducrosia assadii Alava., a Unique Endemic Plant from Iran

INTRODUCTION

Ducrosia belongs to the family of Apiaceae (Umbelliferae) and comprises about only 4 known species which occur largely in Africa and Asia especially in Iran, Afghanistan, Pakistan, Syria, Lebanon and Iraq (Aynehchi, 1990; Zargari, 1996). This genus comprises about 3 habitat species: Ducrosia anethifolia Boiss., Ducrosia flabellifolia Boiss. and Ducrosia assadii Alava in Iran (Rechinger, 1987). The plant of D. assadii is described by Mozaffarian for a vast region in Kerman province between Dehbakri and Jiroft, Lalehzar and Baghin regions and known in Persian as “Moshgake bakraei” (Mozaffarian, 1998). Phytochemical studies have demonstrated the presence of some coumarins such as pangelin in D. anethifolia (Starvi et al., 2003). The flowers and fruits of D. assadii Alava have been used as a flavoring agent, antispasmodic and carminative in folk medicine. Janssen et al. (1984) reported α-pinene (59.2%), myrcene (11.6%) and limonene (7.5%) as the major compounds of the essential oil of D. anethifolia (Janssen et al., 1984). In another report, the major compounds of the oil of D. anethifolia were reported to be α-pinene, terpineolene and ocimene (Mostafavi et al., 2008). Haghi et al. (2004) reported the presence of α-pinene, citronellal, limonene, linalool and myrcene in this oil (Haghi et al., 2004) while in the another report, n-decanal constitutes more than 70% of the essential oil which has exhibited antianxiety effect in mice (HajhashemiI et al., 2010). There are lesser studies for D. assadii in the literature. A report shows n-decanal (36.4%) as the main component among the 29 constituents characterized in the oil of D. assadii (Rustaiyan et al., 2006), meanwhile Mostafavi et al. (2008) reported that the essential oil of the aerial parts of the plant is composed of n-decanal (74%), dodecanal (7.2%) and α-pinene (4.0%) (Mostafavi et al., 2010).

Several studies suggest that free radicals can damage cellular macromolecules (Bergendi et al., 1999). Some of plant secondary metabolites especially essential oils exhibit antioxidant effect (Bensky et al., 2004; Sharififar et al., 2003, 2007a, b, 2009, 2011; Yassa et al., 2005). As a part of a project on the volatile composition of Ducrosia species endemic to Iran, we have studied the oil composition of the aerial parts of D. assadii in two separate stages of flowering and fruiting times by GC and GC/MS. We think that the knowledge of the essential oils may be useful to clarify the systematic status of D. assadii. On the other hand the antioxidant effect of these essential oils has been studied using DPPH free radical scavenging assay.

METHODS AND MATERIALS

Plant materials: The air-dried tops of D. assadii were gathered at flowering and fruiting stages (late June and August, 2011, respectively) from Dehbakri, Kerman, Iran (at altitude of 1900 m above sea level). Voucher specimen was deposited at the Herbarium of department of Pharmacognosy of Faculty of Pharmacy in Kerman University of Medical Sciences, Kerman, Iran (KF1387).

Isolation of essential oil: The gathered parts of the plant were left to dry at room temperature and 100 g of flowering and fruiting tops of the plant were coarsely minced and placed in a flask containing 500 mL of water and steam distilled in a Clevenger apparatus for 4 h. The obtained essential oils were dried over anhydrous sodium sulfate and stored at 4°C in the darkness. Essential oil yield was 0.71% and 0.49% based on dried weight of sample for flower and fruit tops, respectively.

Gas chromatography: The essential oils were analyzed using a Shimadzu QP 5000 gas chromatograph equipped with a FID detector and HP-5 MS capillary column (30 m*, 0.25 mm, film thickness 0.25 m). Injector and detector temperatures were set at 220 and 290°C, respectively. Oven temperature was kept at 50°C for 3 min, then gradually raised to 160 at 3°C min^-1, held for 10 min and finally raised to 240 at 3°C min^-1. Helium was the carrier gas, at a flow rate of 1 mL min^-1. Diluted samples (1/100 in acetone, v/v) of 1,0 L were injected manually in the splitless mode. Quantitative data were obtained electronically from area percent data without the use of correction factors.

Gas chromatography/mass spectrometry: GC-MS analysis of the essential oils was performed under the same conditions with GC (column, oven temperature, flow rate of the carrier gas) using a Shimadzu QP 5000 gas chromatograph equipped with a Shimadzu QP 5050 mass selective detector in the electron impact mode (70 ev). Injector and MS transfer line temperatures were set at 220 and 290°C, respectively.

Qualitative and quantitative analysis: The components of the oils were identified based on the comparison of their relative retention times and mass spectra with those of standards. The individual compounds were identified by MS and their identity was confirmed by comparing their retention indices relatives to n-alkanes and by comparing their mass spectra and retention times with those of authentic samples or with data already available in the NIST and Wiley library and literature (Adams, 1995). GC and GC/MS analysis results are given in Table 1.

Table 1:	GC/MS analysis of the essential oil of Ducrosia assadii in two stage of flowering (FLD) and fruiting (FRD)

DPPH free radical scavenging assay: The antioxidant activity of FLD and FRD was determined in terms of hydrogen donating ability using the DPPH free radical scavenging assay as modified by Sanchez-Moreno et al. (1998). At first a serial dilution of the essential oils was prepared . A volume of 0.1 mL of each sample was added to 2.9 mL of DPPH solution (60 μM). The absorbance was read for 30 min at 5 min intervals using a spectrophotometer. The remaining concentration of DPPH in the reaction medium was calculated through the linear regression. The percentage of remaining DPPH was expressed as following formula:

where, DPPH₃₀ is DPPH concentration after 30 min and DPPH₀ is the initial concentration. The percentage of DPPH inhibition of different concentration of essential oils was determined as follow equation:

where, A_blank is the absorbance of the control reaction (containing all reagents except the test compound) and A_sample is the absorbance of the test compound. Each experiment was performed in triplicate and data was reported as Mean±SD.