Chemical composition and antioxidant activity of essential oil , various organic extracts of Cistus ladanifer and Cistus libanotis growing in Eastern Morocco

In the present work, we studied the chemical composition of the essential oil of Cistus ladanifer and Cistus libanotis growing in Eastern Morocco. The essential oils were obtained by hydrodistillation and their chemical composition was analysed using gas chromatographymass spectrometry (GC–MS). Camphene, borneol, cyclohexanol-2, 2, 6 tremethyl, terpineol-4 and α-pinene were the main constituents of the essential oil of C. ladanifer, while in the essential oil of C. libanotis we obtained terpineol-4, γterpinene, camphene, sabinene, α-terpinene and α-pinene. The antioxidant potential of various extracts (water, ethanol, ethanol: water (50:50), methanol, methanol: water (50:50), acetonitrile) and essential oils of C. ladanifer and C. libanotis were carried out by the method of 1,1-diphenyl-1-picrylhydrazylhydrate (DPPH) free radical scavenging. Total phenolic and flavonoid contents were determined. The result show that C. ladanifer of the leaves of methanol: water (50:50) extract had the highest value of total phenolic content and the lowest was present in ethanol: water (50:50) extract of the stem and acetonitrile extract of the flowers of C. libanotis. From our experimental results, the extract of flowers, fruit, stem and leaves of those plants showed highest potential as free radical scavengers.


INTRODUCTION
Cistaceae comprises eight genera and about 180 species.They are a medium-sized family typically consisting of heliophyte shrubs, subshrubs and herbs occurring in open areas on poor soils (Guzma´n and Vargas, 2009).They are pyrophylic (Alonso et al., 1992) and they are adapted to fires in Mediterranean forests.
In the current study, our objective was to evaluate the chemical composition of the essential oil of C. ladanifer and C. libanotis and to examine the antioxidant potential of oils and various solvent extracts of plant parts (fruits, flowers, stems and leaves) by 1,1-diphenyl-2picrylhydrazyl-hydrate (DPPH) free radical scavenging assay.Additionally, total flavonoid and phenolic contents were also determined.

Plant material
C. ladanifer and C. libanotis were collected from Tafoughalt (Eastern of Morocco) and they were identified by Dr Haloui of the Department of Biology (University Mohamed Premier Oujda).Collected plants (5 May, 2012) were dried in the laboratory under normal air at room temperature.

Preparation of extract and isolation of essential oil
A weighed portion (2 g) of each dried and powdered sample (fruits, stems, flowers and leaves) was extracted with 100 ml of different solvent (water, ethanol, ethanol: water (50:50), methanol, methanol: water (50:50), acetonitrile) for 8 days at room temperature.The extracts were filtered and evaporated to dryness in a rotary evaporator.The extracts were dissolved in ethanol at 25 mg ml -1 concentration and stored until used.The essential oil from leaves of C. ladanifer and C. libanotis was obtained by hydrodistillation in a Clevenger-type apparatus during 5 and 4 h, respectively.Both oils were dried under anhydrous sulphate and stored at 4°C until used.

Gas chromatography-mass spectrometry (GC-MS) analysis
Analysis of essential oils was carried out by GC-MS using a Hewlett Packard 6890 gas chromatography equipped with a Hewlett Packard 6890 mass selective detector and a HP-5 MS capillary column 30 m x 0.25 mm (cross-linked Phynel-Methyl Siloxane) and film thickness of 0.25 µm.The column was temperature programmed as follows: 40°C for 5 min, and then the temperature was increased to 280°C at a rate of 10°C/min.Helium was used as a carrier gas and the oils (0.1 µl) were injected without any dilution in the splitless mode.
Mass analyzed conditions were ion source of 230°C, ionization energy of 70 eV, electron current of 2 A, and resolution of 1000.Mass unit was monitored from 30 to 450 m/z.Identification of components in the oil was based on retention indices relatives with n-alkanes and computer matching with the WILLEY 275.L library, as well as by comparison of the fragmentation patterns of mass spectra with those reported in the literature (Adams, 1995).The relative proportions (%) of each volatile compound as the percentage ration of the peak area of each compound to the total peak area of all identified compounds were calculated.

DPPH radical scavenging effect
Antioxidant activity was evaluated according to the method cited by Xiao-Juan et al. (2012).100 µl of different concentration of extract or essential oils or standard (1 to 15 mg ml -1 ) was added in 1, 4 ml of ethanol and 1 ml of solution of DPPH (0.004 %).The absorbance of the discoloration of the solution was measured at 517 nm.The percentage of inhibition was calculated by: A DPPH is the absorbance of DPPH solution and A sample is the absorbance of the test sample.All tests were performed in triplicate.The lower absorbance of the reaction mixture indicates higher free radical scavenging activity.

Determination of total phenolic content
The concentration of total phenolics in different organic extracts was determined by using Folin Ciocalteu reagent (Singleton and Rossi, 1965); briefly, 20 µl of sample extract (1 mg ml -1 ) was added to 100 µl of Folin Ciocalteu reagent and 300 µl of Na 2 CO 3 .2.5 of distilled water was added after 5 min.The mixture was left 2 h and the supernatant was measured at 576 nm against a blank.The calibration curve was prepared by Ferulic acid at different concentration (0.2 to 2 mg ml -1 ; Y= 0.0042X + 0.0014; R 2 = 0.9991).A test was carried out in triplicate.

Determination of total flavonoid content
Total flavonoid was determined using the method described by Pourmorad et al. (2006); briefly 0.5 ml of each extract (1 mg ml -1 ) was added to 1.5 ml of ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water.The resulting solution was left at room temperature for 30 min.The absorbance was measured at 415 nm.The calibration curve was prepared by different concentration of quercetin solutions in ethanol (0.01 to 0.08 mg ml -1 ; Y= 0.0048X -0.0128; R 2 = 0.9978).A test was carried out in triplicate.

Statistical data
All extractions were conducted in triplicates.Phenolic and flavonoid data are expressed as mean ± standard deviation.The means were compared by using the one-way and multivariate analysis of variance (ANOVA) followed by TuKey's using the SPSS 11.5 for Windows.The differences between individual means were deemed to be significant at p < 0.05.

Chemical composition of the essential oils
The essential oil yields in leaf of C. ladanifer and C. libanotis were respectively 0.14 and 0.13% on dry weight basis (w/w).The essential oil volatile compounds of plants, their kovalts indices and their percentage are presented in Table 1.
As can be seen from Table 1, camphene is the major compounds of the C. ladanifer oil together with borneol and cyclohexanol-2, 2, 6 tremethyl in the present study.When compared with these compounds, bis (2-ethylhexyl) phthalate and cyercene were detected in a small quantity in the oil.Another study from Portugal has investigated the main constituents in the oil of C. Values are mean ± SD (standard mean deviation) (n=3).In each column, different letters mean significant differences between plant parts (P < 0.001).Values followed by the same letter under the same column are not significant different (P ≥ 0.05).FA, Ferulic acid equivalents; QE, quercetin equivalents; Fl, flowers; F, fruit; S, stem; L, leaves.

Contents of total phenols and flavonoids
Phenolic compounds are the main class of natural antioxidants and there is a close relationship and positive correlation between the phenolic content and antioxidant activity of plant tissues.Table 2 summarises the results from the quantitative determination of the phenol and flavonoid content of the different extracts.Total phenol content was determined as ferulic acid equivalents in milligrams per gram (FA mg/g dry weight) while total flavonoid contents were calculated as quercetin equivalents in milligrams per gram (mg QE mg/g dry weight).The total phenol contents varied between different plant parts: aqueous methanolic extract from C. ladanifer flowers had higher total phenol content (182.41 mg FA/g) than other extract.Significant differences were also found among different plant parts.
As for total flavonoid content, the ethanolic extract of C.  -catalán et al., 2011).

DPPH radical scavenging activities
Free radical scavenging properties of different extracts from different C. ladanifer and C. libanotis parts and For C. ladanifer, methanolic extract were observed in this order: 97.8% (leaf) > 97.3% (stem) > 96.1% (flower) > 95.9% (fruit) whereas for C. libanotis, it was found as 96.9% (flower and stem) > 95.9% (fruit) > 93.4% (leaf).In addition, DPPH scavenging ability of the extracts of plant parts was similar to ascorbic acid, suggesting that plant extracts have a potent antioxidant activity mainly due to their richness on phenolic compounds.Oils was found less effective than the control.The use of water with alcoholic solvent presents a good advantage to extract polyphenols and can also modulate the polarity of alcohol solvent used (Mohammedi and Atik, 2011).These extract possess a remarkable potential in scavenging of free radicals since they present a number of hydroxyls acting as hydrogen donators.

Conclusion
There is a growing interest of industry to replace synthetic chemicals from natural products with bioactive properties from plant origin.The results indicate a significant qualitative difference found in terms of chemical composition in the oils.The most abundant compounds in C. ladanifer were camphene, borneol, cyclohexanol-2, 2, 6 tremethyl, terpineol-4 and α-pinene, while the essential oil of C. libanotis were terpineol-4, γterpinene, camphene, sabinene, α-terpinene and αpinene.The present data suggest the importance of comparing and exploring the variety of essential oil and phenolic content from different C. ladanifer and C. libanotis parts, since this variability of repartition of bioactive substances between leaf, stem, flower and fruit entrained the variability of their potential antioxidant activities.

Figure 3 .
Figure 3. DPPH radical scavenging activities of essential oil of C. ladanifer and C. libanotis.

Table 1 .
Chemical composition of the essential oil of C. ladanifer and C. libanotis .
%, Relative percentage area; ND, compound not identified in this sample; KI, Kovalts indices calculated using an apolar column (HP-5).

Table 2 .
Comparison of total phenolic and flavonoid contents of C. ladanifer and C. libanotis.