Quercus infectoria galls possess antioxidant activity and abrogates oxidative stress-induced functional alterations in murine macrophages

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Abstract

The present study reports the antioxidant activity of ethanolic extract of Quercus infectoria galls. The antioxidant potency of galls was investigated employing several established in vitro model systems. Their protective efficacy on oxidative modulation of murine macrophages was also explored. Gall extract was found to contain a large amount of polyphenols and possess a potent reducing power. HPTLC analysis of the extract suggested it to contain 19.925% tannic acid (TA) and 8.75% gallic acid (GA). The extract potently scavenged free radicals including DPPH (IC50∼0.5 μg/ml), ABTS (IC50∼1 μg/ml), hydrogen peroxide (H2O2) (IC50∼2.6 μg/ml) and hydroxyl (radical dotOH) radicals (IC50∼6 μg/ml). Gall extract also chelated metal ions and inhibited Fe3+–ascorbate-induced oxidation of protein and peroxidation of lipids. Exposure of rat peritoneal macrophages to tertiary butyl hydroperoxide (tBOOH) induced oxidative stress in them and altered their phagocytic functions. These macrophages showed elevated secretion of lysosomal hydrolases, and attenuated phagocytosis and respiratory burst. Activity of macrophage mannose receptor (MR) also diminished following oxidant exposure. Pretreatment of macrophages with gall extract preserved antioxidant armory near to control values and significantly protected against all the investigated functional mutilations. MTT assay revealed gall extract to enhance percent survival of tBOOH exposed macrophages. These results indicate that Q. infectoria galls possess potent antioxidant activity, when tested both in chemical as well as biological models.

Introduction

Oxidative damage to cells is believed to be a crucial factor implicated in the pathogenesis of several clinical disorders and in the normal process of ageing [1], [2]. Potently reactive derivatives of oxygen, known as reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2) and hydroxyl radical (radical dotOH) are continuously generated by normal metabolic process or from exogenous chemicals in our ambient environment. Normally, ROS are efficiently neutralized by potent antioxidant armory of the cells without any untoward effect. However, hampering the balance between ROS generation and antioxidant defense leads to the onset of oxidative stress, which following a series of events, dysregulates cellular physiology leading to several chronic ailments such as cancer, atherosclerosis, arthritis, diabetes, etc. Intake of antioxidants can avert or impede the oxidative damage and therefore, thwart the disorders implicating oxidative stress in their pathophysiology. Antioxidants mediate their effect by directly reacting with ROS, quenching them and/or chelating the catalytic metal ion [3]. Synthetic antioxidants such as BHA and BHT are available; however, their toxicity is a problem of concern [4]. This has led to a growing interest in exploring natural antioxidants.

Macrophages, the major phagocytic cells, are the chief constituents of body's cell mediated immune system. These cells possess a high proportion of polyunsaturated fatty acids in their plasmamembrane and are exposed to a huge amount of oxidants during respiratory burst, a key step involved in the destruction of engulfed microbes. These two factors together make macrophages remarkably perceptive to alterations in oxidant–antioxidant equilibrium [5]. Macrophages, therefore, provide an excellent model system to explore the efficacy of antioxidant compounds against oxidative injury induced to cellular systems. In the present study, we investigated the antioxidant activity of ethanolic extract of Quercus infectoria galls employing several in vitro assays and also investigated the protective effect of extract against tertiary butyl hydroperoxide (tBOOH) induced oxidative stress and physiological alterations produced by it in murine macrophages.

Q. infectoria Olivier (Fagaceae) is a small tree widely distributed in Greece, Asia Minor and Iran. The tree bears galls that emerge on its shoots as a consequence of attack of gall wasp, Cypnis gallae-tincotoriae. Galls are of great medicinal value and have widely been used in folklore medicines mainly as astringent and against inflammation. Pharmacological evaluation of galls has deciphered them to be astringent, antiparkinsonian, antitremorine and antidiabetic [6], [7], [8]. We have previously revealed galls to possess potent anti-inflammatory activity [9]. The present study explores the antioxidant activity of ethanolic extract of Q. infectoria galls.

Section snippets

Chemicals

Thiobarbituric acid (TBA), RPMI-1640 media, reduced nicotinamide adenine dinucleotide (NADH), 2-mercaptoethanol, ferricytochrome c, horseradish peroxidase, mannose-BSA, mannan, chloramine T, β-glucuronidase, phenolphthalein glucuronic acid, phenol red, dithionitrobenzene (DTNB) and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Na125I was purchased from Board of Radiation and Isotope Technology, Bhabha Atomic Research Center, Mumbai. All other chemicals and reagents used were of

Total phenolics and reducing power

The yield of extract obtained from Q. infectoria galls using ethanol was 23.5% (w/w). The extract was found to contain 416 ± 10.6 mg/g of total polyphenolics expressed as gallic acid equivalent (GAE, mg/g of extract). Since antioxidant activity may be directly correlated to the polyphenolic content, the obtained amount of total polyphenolics in gall extract indicated it to possess a high antioxidant activity. The reducing capacity, another vital indicator of antioxidant activity, was also found to

Discussion

Various degenerative disorders implicate a deficient natural antioxidant defense as their etiological or pathological factor. The onset/progression of these disorders can be held or delayed by supplementing with antioxidant defense. Plant-based antioxidants such as resveratrol and quercetin potentiate body's antioxidant defense and are relatively safe. Thus, a huge body of scientific research is focused on exploring plants with antioxidant potential. In the present study, we have revealed Q.

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