Antioxidant and free radical-scavenging properties of seeds flavonoids extract of Cedrus atlantica Manetti , Linum usitatissimum L . and Ocimum basilicum L . species

1 Plant Biotechnology Unit, National Institute of Medicinal and Aromatic Plants-Taounate, Sidi Mohamed Ben Abdellah University -Fez, Morocco. 2 Pharmacology and Toxicology Laboratory, National Institute of Medicinal and Aromatic Plants-Taounate, Sidi Mohamed Ben Abdellah University –Fez, Morocco. 3 BiochemistryLaboratory, National Institute of Medicinal and Aromatic Plants-Taounate, Sidi Mohamed Ben Abdellah University –Fez, Morocco.


INTRODUCTION
Flavonoids are considered as mainly the most important phenolics, due to its wide range of chemical and biological activities, including antioxidant and free radical scavenging properties (Kahkonen et al., 1999).In fact, many reports reflect, the antioxidant, scavengers of a broad spectrum of reactive oxygen species and inhibitors of lipid peroxidation effect of flavonoids (Williams et al., 2004).Many of These compounds, which are widely distributed across the plant kingdom and currently used in large amounts in our daily diet, represent the most abundant antioxidants in the diet and they have gained tremendous interest as potential therapeutic agents against a wide variety of diseases, most of which involve oxidant damage (Ross and Kasum, 2002).
Reactive oxygen species (ROS) leads to considerable oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA.The oxidative damage has been implicated in several chronic human diseases, namely cardiovascular diseases, rheumatism, diabetes mellitus and cancer (Pong 2003).Founded on development in free radical biology and thedeficiency of a specific function in fact, of effectual therapies for most chronic diseases, the utility of antioxidants in protection against these diseases is warranted.Antioxidants are chemical substances that reduce or prevent oxidation.They have the ability to counteract the damaging effects of free radicals in tissues and thus are believed to protect against cancer, arteriosclerosis, heart disease, and several other diseases (Bandyopadhyay, 2007).Many studies have shown that phenolic compounds display antioxidant activity as a result of their capacity to scavenge free radicals (Seyoum et al., 2006).Phenolic compounds can also act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system (Al-Azzawie and Alhamdani, 2006).
Cedrus atlantica Manetti, Members of the pine family (Pinaceae), especially species of Cedrus dominates many of Morocco forests; represent a potential antioxidant effect reported by many studies.Ocimum basilicum L. is a member of Lamiaceae, Basil extract has antimicrobial and antioxidant activities due to its phenolic and aromatic compounds (Jayasinghe et al., 2003).The phenolics reported in basil are phenolic acids and flavonol glycosides (Kashyap et al., 2011).The seeds of Linum usitatissimum L. are known to be a rich source of the polar glycosidic-dibenzyl-butanediol-type lignin secoisolariciresinoldiglucoside (SDG) which is considered avaluable food constituent due to its function as precursor for enterolignans (Westcott and Muir, 2003).These métabolites formed by the human enteral microflora have been demonstrated to possess chemopreventive activity against various tumors and cardiovascular disorders (Rickards-Bon and Thompson, 2003).

Plant material
Seeds of Atlas cedar are collected from Forestry center « Azrou » as native coniferal species of Morocco (in November 2013), Lin seeds from cork oak Maâmora forest region and Basilic seeds from Taounate region.Then they are air-dried at 40°C with forced ventilation for two days.The Botanical identification and the Authenticated voucher specimens deposited in the Herbarium of The National Institute of Medicinal and Aromatic Plants.

Phytochemical screening
The identification of flavonoids in our extracts (using the samples of seeds ethanolic extract of each species) was done using the reaction Cyanidin test would detect compounds having the γbenzopyrone nucleus (Farnsworth, 1966).In the presence of alcohol and hydrochloric magnesium appears coloration ranging from orange to red characteristic flavonoids.The principle is to react at equal volume to be extracted with a hydrochloric solution of alcohol.
A mixture to which is added some magnesium swarf then isoamyl alcohol, 1/5 th of the volume is compared to the hydrochloric alcohol.

Flavonoids extraction
For each accession, Cedrus, Linum and Basilicum dried seeds.One hundred grams of dried seeds were ground in fine powder and soaked in 50% aqueous ethanol for 4 h is heated to 90°C under reflux.The extract is filtered and the aqueous phase is then extracted with n-butanol and then acidified with 10% HCl to pH 3.The butanol layer was concentrated in vacuum by means of rotavaporand then evaporated to dryness at room temperature.The concentrated extract was then dissolved as extract three times with 200 ml of distilled water/ethyl acetate mixture (1v:1v) for one hour.The organic layer was basified with NaHCO3 to pH 9.After 15 min the residual organic phase (flavonoids) was concentrated in vacuum by means of rotavapor at 40°C (Lee et al, 1995)with slight modifications.

DPPH free radical-scavenging activity
The DPPH free radical-scavenging assay was carried out, as previously reported by Cheel et al. (2007) with some modifications.The flavonoids extract separated from Cedrus, Linum & Basilicum seeds at various concentrations (1, 0.5, 0.025 mg/ml), (2, 1, 0.5, 0.25 mg/ml) & (1, 0.0625, 0.03125mg/ml) respectively were added to a 4% DPPH* mixture 1v:1v solution in ethanol and the reaction mixture was shaken vigorously.After incubation for 30 min at room temperature in obscure, the absorbance at 517 nm was recorded spectrophotometrically.Extracts which displayed promising activity (≥50% decolorisation at 1mg/ml) were retested at lower concentrations using serial dilutions.BHT was used as a reference compound.
A control solution, without the tested compound, was prepared in the same manner as the assay mixture.All the analyses were done in triplicate.The degree of discolorisation indicates the free-radical scavenging (FRS), efficiency of the substances.The antioxidant activity of Cedrus, Linum and Basilicum extracts was calculated as an inhibitory effect (IE %) of the DPPH radical formation as follows: A517 (control): Absorbance of the control (DPPH) at 517nm A517 (sample): Absorbance of the Sample at 517nm and expressed as IC50.
The IC50 value was defined as the concentration (in mg/ml) of the flavonoid extract required to scavenge the DPPH radical by 50%.

Thin layer chromatography
The three extracts, were examined with thin layer chromatography about 5 μl of each extract (100 mg/ml) were deposited on fluorescence silica gel plates (0.2 cm thickness 60 F254) and migration was conducted with mixture of ethyl acetate/methanol/H2O (100.0/13.5/10.0).After drying plate, revelation was conducted using a UV lamp at 254 and 366 nm.

Statistical analysis
Data were collected and expressed as the mean ± standard deviation of three independent experiments and analysed for statistical significance from control, using the Dunnett test (SPSS 11.5 Statistics Software; SPSS, Chicago, IL).The criterion for significance was set at p < 0.05.IC50 values, from the data, were calculated by regression analysis.

Antioxidant tests
Although there is no standardized method to evaluate the antioxidant potential of foods and biological systems, it is recommended to evaluate the antioxidant activity by different methods FRAP, ABTS etc. (Frankel and Meyer, 2000), in this study we use a DPPH method for the aim to evaluate in general; the antioxidant activity.

RESULTS AND DISCUSSION Determination of free radical scavenging activity by DPPH method
The radical scavenging assay was carried out according to (Mighri et al., 2010), with slight modifications.Briefly, 100 μl of various concentrations of the extract was added to 10 ml of an ethanol/DPPH solution.The mixture was vigorously shaken and then allowed to stand at a room temperature for 30 min in the dark.The absorbance of the mixture measured at 517 nm by using a double-beam UV-vis Camspec M550 spectrophotometer.A mixture of 100 μl of ethanol and 10 ml of DPPH solution is used as control.The scavenging activity was expressed as percentage of inhibition using the following equation: Where AB is the absorbance of the control reaction (containing all reagents except the extract), and ABS is the absorbance of the extract.BHT (butylated hydroxytolune) used as positive control.

DPPH radical-scavenging activity
The DPPH* test is largely used in plant or food biochemistry to evaluate the free radical-scavenging effect of specific compounds or extracts.This stable free radical accepts an electron or hydrogen radical to become a stable diamagnetic molecule.In its radical form, DPPH* has a broad absorption band with a maximum at 517 nm, while if it is protonated by an antiradical compound, it loses this property (Lo Scalzo 2008).The extract concentration providing 50% inhibition (EC50) was calculated from the curve of FRS inhibition percentage against extract concentration.
In this assay, all test extracts effectively reduced the stable radical DPPH* to the yellow color and their scavenging effect was dose-dependent (Fig. 1).
It was also found that Cedrus extract possesses the most potent DPPH radical-scavenging activity, with an IC50 value of 0.40 mg/ml, followed by a Basilicum extract with 0.41 mg/ml, and Linum extract with 1.21 mg/ml, while to BHT (0.003 mg/ml) used as positive control.Based on the IC50 values, the potency of DPPH free radical-scavenging activity of the tested flavonoids extract was in the order of Cedrus (0.40 mg/ml) > Basilicum (0.41 mg/ml) > Linum (1.21 mg/ml).The effect is more significant at higher doses with 2 and 1 mg/ml which show maximum inhibition effects of 86 ± 0.09, 75 ± 3.13, 46 ± 1.40% FRS (= %IE) at a concentration of1 mg/ml, respectively.The thin layer chromatography with ethyl acetate/methanol/H 2 O revealed in the Basilic seeds extract (BE), and the Cedar seeds extract (CE) the same spot with R f 0.43.
Four spot were obtained with ethyl acetate/Methanol/ H2O, and two spots were obtained with the same mixture with DPPH spray with BE.One spot were obtained with Linum extract (LE).Four spots with the mixture, no spots with DPPH spray were obtained with CE.These preliminary finding indicate the presence of flavonols subclass of flavonoids, the flavonol substrate, may be present in the Basilic and Cedar extract.
The mechanism of reaction between antioxidant and DPPH* depends on the structural conformation of the antioxidant.Some compounds react very quickly with DPPH, reducing a number of DPPH molecules equal to the number of the hydroxyl groups (Bondet et al., 1997).
Our results on the efficiency of flavonoids extract in inhibiting DPPH free radical are generally consistent with these criteria.Hence, Linum flavonoids extract, showing the lowest potency.
Finally, although a large number of antioxidant assays are available, the DPPH free radical is very stable and thus allows for easy handling and manipulation.Furthermore, its stability implies that a potential antioxidant will react with other wellknown free radical entities, which are more unstable, and therefore more reactive (Frum et al., 2007).Thus, an antioxidant candidate which proves promising in the DPPH antioxidant assay would provide an optimistic scaffold for prospective studies.

CONCLUSION
In this study, the antioxidant potential of flavonoids extracted from Cedrus, Basilicum and Linum species seeds was evaluated by using DPPH assay.Our study provides evidence that the tested flavonoids extract exhibit interesting antioxidant properties, expressed either by their capacity to scavenge free radicals.Briefly, the information presented here could be used as preliminary data and biologically more relevant experiments that will examine the therapeutic potential of the Cedrus, Basilicum, and Linum seeds flavonoids extract will be designed.This study submit that Cedrus atlantica Manetti ,Ocimum basilicum and Linum usitatissimum; providing antioxidant properties and suggest potential functional protection from free radicalsmay act as a chemopreventive agent.Then, it's necessary to identify and isolate the compounds that are responsible to the antioxidant effects.