Free Radical Scavenging Activity of Linum arboreum.

Abstract

The methanol and water extracts of Linum arboreum.. aerial parts were screened for free radical scavenging activity. The free radical scavenging activity was determined spectrophotometrically on the basis of inhibition of cytochrome c reduction and the ability to bleach the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The methanol extracts of Linum arboreum. were shown to have potent antioxidant activity.

Keywords:

• Free radical scavenging activity
• Linum
• Linum arboreum

Introduction

Plants contain a wide range of polyphenols including anthocyanin, proanthocyanin, isoflavones, stibenes, and lignans. Several plant phenols can inhibit LDL (corrected with low density lipoproteins) oxidation in vitro., for example, caffeic acid, hydroxytyrosol, and flavonoids (Halliwell, 2000). Lignans form a group of important plant metabolities. They are defined as β-β′ dimers of phenylpropanoid derivatives and are widely spread throughout the plant kingdom. Lignans show a series of interesting biological activities including anti-HIV, immunosuppressive, hypolipidemic, antifungal, antiasthmatic, and antioxidant activities (Charlton, 1998; Ward, 1999). Several lignans have antioxidant properties (e.g., sesamolin, nordihydroguaiaretic acid, sesamin, sesamolinol) (Lewis, 1999). Several studies provide support for a productive effect of consumption of foods rich in lignans against cancer and coronary heart disease. Free radicals can cause lipid peroxidation and damage to cells. This damage can lead to coronary heart disease, stroke, aging, diabetes mellitus, rheumatic disease, carcinogenesis, liver disorders, and neurodegenerative diseases (Gassen & Youdim 1997; Halliwell & Gutteridge 1999). There is evidence that antioxidants can be useful in preventing oxidative stress and thus increasing interest in natural antioxidants that are candidates for the prevention of oxidative damage. In the course of our ongoing search for antioxidant activity from Turkish plants, we have investigated Linum arboreum. L. (Linaceae).

Materials and Methods

Plant material

Linum arboreum. L. (Linaceae) aerial parts were collected near Datça (Turkey) in April 2002. Prof. Dr. Hayri Duman confirmed the identity of the plant. A voucher specimen is deposited in the herbarium of the Faculty of Pharmacy, Ankara University.

Preparation of the extracts

The sample (weighing 40 g) was extracted with 300 ml of boiling water for 1 h. The extract was filtered. The filtrate was collected, and residue was re-extracted under the same condition. The filtrates collected from two successive extractions were combined and lyophilized. Plant material 40 g was extracted with 300 ml methanol for 24 h by using a Soxhlet apparatus. The extract was evaporated to dryness.

Chemicals

Xanthine, xanthine oxidase, cytochrome c, 2,2,diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene, and α-tocopherol were purchased from Sigma Chemical Co (St. Louis, MO).

Superoxide radical scavenging activity

The scavenger capacity of crude extract on superoxide anion was determined spectrophotometrically on the basis of inhibition of cytochrome c reduction according to the modified method of McCord and Fridowich (1969). Samples were dissolved in methanol and diluted to various concentrations. Superoxide anion was generated in the xanthine/xanthine oxidase system. The absorbance were measured spectrophotometrically at 550 nm for cytochrome c reduction. IC₅₀ values were determined from a calibration curve.

DPPH radical scavenging activity

The DPPH radical scavenging activities of these compounds were tested by their ability to bleach the stable radical 2,-2-diphenyl-1-picrylhydrazl (Blois, 1958). Reaction mixtures contained 100 µM DPPH in methanol and different concentrations of crude extract. Absorbance at 517 nm was determined after 30 min at room temperature, and the scavenging activity was calculated as a percentage of the radical reduction. Each experiment was performed in triplicate. BHT was used as a reference compound.

Results

Scavenging effects of different concentrations of methanol and water extracts on superoxide anions are shown in Table 1. The methanol extract of Linum arboreum. in the concentration range of 2.5–10 mg/ml showed superoxide anion scavenging activity. The IC₅₀ value was found to be 5.7 mg/ml. Water extracts of Linum arboreum. did not show inhibitory effects on superoxide anion.

Table 1.. Scavenger effect of Linum arboreum. L. extracts on superoxide anion.^a.

Samples	Concentration in incubation medium (mg/ml)	Inhibition of superoxide anion (O2^˙−) (% Inhibition)	IC50
Control^b.		100
Control^c.		100
Methanol extract	2.5	100±1.4	5.7
	5.0	38±4.2
	10	88±1.4
Water extract	2.5
	5.0	No effect
	10
α.-Tocopherol	0.1	100±0.7	0.22
	0.2	11±2.8
	0.4	60±1.4
	0.8	85±2.8

^a.Each value represents the mean ±SD of 2–4 independent experiments.

^b.Methanol only, control for methanol extract of Linum arboreum. L.

^c.Distilled water only, control for water extract of Linum arboreum. L.

Methanol and water extracts of Linum arboreum. have been evaluated for DPPH radical scavenging activity. The scavenging effects of different concentrations of Linum arboreum. extracts on DPPH radical are shown in Table 2.All extracts, in the concentration range of 0.125–0.5 mg/ml, have shown scavenger effects on DPPH radical. IC₅₀ values of the methanol and water extracts were found to be 0.125 and 0.14 mg/ml, respectively.

Table 2.. Scavenger effect of Linum arboreum. L. extracts on DPPH radical.^a.

Samples	Concentration in incubation medium (mg/ml)	DPPH radical scavenger activity (% Inhibition)	IC50
Control^b.		100
Control^c.		100
Methanol extract	0.125	50±1.4	0.125
	0.25	79±1.4
	0.5	93±0.7
Water extract	0.125	45±2.8	0.14
	0.25	75±1.4
	0.5	81±2.8
BHT	0.0055	16.5±4.2	0.015
	0.011	36.5±0.7
	0.022	68.8±1.4

^a.Each value represents the mean ±SD of 2–4 independent experiments.

^b.Methanol only, control for methanol extract of Linum arboreum. L.

^c.Distilled water only, control for water extract of Linum arboreum. L.

Discussion

The antioxidant effect of some plant extracts has been found to be related to polyphenolic compounds by scavenging oxygen radicals (Hanasaki et al., 1994). Therefore, we have tested Linum arboreum. plant extracts for their antioxidant activities. The methanol extracts of Linum arboreum. showed strong scavenger effects on superoxide anion and DPPH radical, whereas the water extracts of Linum arboreum. did not show any scavenger effect on superoxide anion.

In our study, the scavenger effects of water extracts of Linum arboreum. on superoxide anion and DPPH radical were not similar. Because the mechanisms of these methods are different, it is possible to observe different effects of various extracts on superoxide anion and DPPH radical interaction (Litwinienko, 2003). Superoxide anion radical and DPPH radical implicated in several pathophysiological processes. It has also been reported that antioxidant properties of some lignans are effective visa scavenging of superoxide anion and DPPH radical (Hsiao, 2001). The methanol extract of Linum arboreum. had a strong scavenger effect on superoxide and DPPH radical. Therefore, the methanol extract of Linum arboreum. can be considered as a potent antioxidant. This activity can be related to the presence of lignans in Linum arboreum. (Hemmati et al., 2004). However, at this stage, we do not know the mechanism of free radical scavenging activity. There are many reports that support the potent antioxidant activity of polyphenols (Gao et al., 1999). The findings of the current study show that in Linum arboreum., there are compounds that act as antioxidants, and further study should be conducted to identify the exact compounds and mechanism involved.