Studies on Antioxidant and Cryoprotective Flavonoids from the Stem bark of Semecarpus anacardium

CHAPTER-I INTRODUCTION AND REVIEW OF LITERATURE

1.1. INTRODUCTION

Oxidative stress, excess generation of Reactive Oxygen Species (ROS), is a common event in many pathological conditions including cancer. The generation of reactive oxygen species (ROS) is an inevitable aspect of life under aerobic conditions. ROS are continuously produced as byproducts of certain metabolic pathways and also by some specific systems under fine cellular control. At the same time, ROS are degraded via several specific and nonspecific mechanisms. These two processes are usually under tight cellular control and very low (<10-8 M) steady-state levels are maintained (Halliwell and Gutteridge, 1989). However, under some special conditions, the balance between ROS production and elimination is disturbed and leading to generation of super oxides (O2-), hydroxyl radical (OH-) and hydrogen peroxide (H2O2).

H2O2, a common intermediate of various enzymatic and non enzymatic reactions including electron transport chain, peroxisomal oxidases, flavoproteins as well as D-amino acid oxidases, L-hydroxy acid oxidases and fatty acyl oxidases. In endoplasmic reticulum, cytochrome P-450, P-450 reductase and cytochrome b -5 reductase generate superoxide anion and hydrogen peroxide during their catalytic cycles. The catalytic cycle of xanthine oxidase has emerged as an important source of O2 – and H2O2. During phagocytosis, the phagocytic cells such as neutrophils generate O2– and H2O2 by NADPH oxidase. Inflammation is also one of the major source of ROS. Spontaneous dismutation of O2– at neutral pH or dismutation by superoxide dismutase also results in H2O2 production (Bandyopadhyay et al., 1999). Substantial evidence has indicated that H2O2 play a key role in wide range of diseases including CNS , autoimmune, cardiac, alveolar and hepatic disorders (Rahal et al., 2014). The pathological role of H2O2 is well established in many acute and chronic disorders such as ischemia, atherosclerosis, diabetes, aging, immuno suppression and neuro degeneration (Saeed et al., 2012).

H2O2 is an important metabolite produced in cells during metabolic reactions, plays an important role in cell death (or) apoptosis. Accumulating data suggest that an increase in the cellular concentrations of H2O2 associated with DNA damage and mutations which leads to the prevalence of various disorders. H2O2 induced DNAf damage seems to be mediated by OH.- generated from H2O2. Several studies have also demonstrated that increased levels of H2O2 can induce cell death (Lazaro et al., 2006).

Studies over the years have demonstrated the importance of H2O2 in intracellular signalling mechanisms of apoptosis and proliferation. H2O2 also mediates the activation of important protein kinases including Mitogen activated protein kinase (MAPK), tyrosine kinase and protein kinase-c (Aslan et al., 2003, Frank et al., 2003). It was previously reported that p38 MAPK up-regulate the antioxidant enzymes such as catalase and superoxide dismutase-2 mRNAs in response to H2O2 (Alvaro et al., 2011). Studies also focused on activation of Mitogen-activated protein kinases (MAPKs) related to cell growth and stress response. In eukaryotes, three major MAPK pathways are represented by kinase cascades leading to activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPKs. Generally, ERK modulates the responses of cell differentiation, whereas JNK and p38 MAPK are activated by stress-associated stimuli, such as heat shock, inflammation, ultraviolet light and irradiation (Yeh and Yen, 2006). Coyle, (2004) reported the role of intracellular redox state which is controlled by the ratio of oxidant to antioxidant, in regulating gene expression of the various proteins which confers protection from the ROS mediated damage.

Lung and Liver are most important organs exposed to toxic chemicals. The primary vital organ, lung has antioxidant defence systems including antioxidant enzymes such as superoxide dismutases (SOD), catalases, glutathione peroxidases (GPxs), which protects alveolar tissues (MacNee, 2005). Many of these antioxidant enzymes are upregulated during lung disorders such as lung fibrosis via the Nrf2 redox-sensitive transcription factor (Messier et al., 2013). Nrf2 is also critical for cytoprotection by co-ordinated activation of detoxification genes and preventing the pathogenesis of liver (Niture et al., 2010).

The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a leucine zipper (bZip) transcription factor plays a vital role in protecting wide range of tissues such as lung, liver, kidney, small intestine, stomach, central nervous system, splenocytes and macrophages (Lee et al., 2005). Nrf2 is a master regulator of cytoprotective genes including antioxidant enzymes, anti-inflammatory mediators and detoxification enzymes. It plays an important role in the maintenance of cellular homeostasis (Qiang Ma, 2013). Yu et al., (2000) reported that, Nrf2 knockout mice (Nrf2-/-) exhibited lower levels of Phase II antioxidant enzymes and are more susceptible to oxidative stress and carcinogen induced tumorogenesis.

1.2. REVIEW OF LITERATURE

Antioxidants are scavengers of free radicals and modifiers of various enzymatic functions. These antioxidant enzymes inhibit the generation of ROS either by removing potential oxidants or by transforming ROS into relatively stable compounds (Evans, 2001). Superoxide dismutase and catalase are are mutally supportive team of defense enzymes against ROS. Superoxide dismutases (SODs) are classified by their metal cofactors into three known types, the copper/zinc (Cu/Zn-SOD), the manganese (Mn-SOD) and the iron (Fe-SOD). The Mn-SOD is found in the mitochondria of eukaryotic cells and in peroxisomes (del Rio et al., 2003); whereas Cu/Zn-SOD isozymes are found in the cytosolic fractions and also in chloroplasts of higher plants (del Rio et al., 2002). Superoxide dismutase (SOD) is the most effective intracellular antioxidant enzyme, provide the first line of defense against the toxic levels of ROS. The SOD2 removes superoxide (O2-) by catalyzing its dismutation, one O2- being reduced to H2O2 and another one oxidized to O2. This reaction has a 10,000 fold faster rate than spontaneous dismutation. Catalase (CAT) is a tetrameric heme containing enzyme directly convert H2O2 into H2O and O2. It is also indispensable for detoxification of aerobic oxidative stress (AOS) during stress conditions (Garg and Manchanda, 2009).

Flavonoids are the one the most important antioxidants reported to alleviate a wide array of ROS mediated injuries in humans (Agati et al., 2001; Tapas et al., 2008). Flavonoids reported to inhibit the generation of reactive oxygen species (ROS) by quenching and by donating hydrogen. Indeed, the phenolic groups of flavonoids serve as a source of a readily available hydrogen atoms such that the subsequent radicals produced are delocalized over the flavonoid structure (Sandhar et al., 2011). Antioxidative action of flavonoids have been attracted attention of many investigators and ample number of reviews were published. These studies, mostly centered to the direct scavenging action of flavonoids against free radicals and active oxygen species, via superoxide dismutase, catalase, glutathione peroxidase (Cheng-Peng et al., 2014).

Nagata et al., (1999) reported that some flavonoids display their antioxidative functions through the interaction with SOD2 and CAT. For a long time flavonoids have been part of the herbal treatment by local practitioners. Recently flavonoids were recognized as effective drugs for the treatment of atherosclerosis, cardiovascular and inflammatory diseases and cancer (Havsteen, 2002). In addition, flavonoids are reported to act as developmental regulators/signaling molecules. Flavonoids mediates intracellular signaling cascades by interacting with a variety of protein kinases, critical for cell growth and differentiation. The beneficial health effects of flavonoids are also thought to control the activity of several protein kinases, including the MAPK (Brunetti et al., 2013).

The medicinal plants are extensively utilized throughout the world in two distinct areas of health management, traditional and modern systems of medicine. The traditional system of medicine mainly functions through local or folk or tribal medicine. It is estimated that 40% of the world populations depends directly on plant based medicine for their health care due to low cost and safety (World Health Organization, 2003). Over the centuries, the use of medicinal plants has become an important part of daily life despite the progress in modern medical and pharmaceuticals research. Approximately, 3000 plants species are known to have medicinal properties in India (Prakasha et al., 2010).

Medicinal plants contain an innumerable number of bioactive constituents with antioxidant activity including flavonoids and phenols (Latha and Daniel, 2001; Packer et al., 1999). The antioxidant potentials of natural flavonoid is similar to synthetic antioxidants of related structures (Yu-Ling et al., 2012).

Since several decades flavonoids have been reported in several medicinal plants (Osawa et al., 1981; Mackeen et al., 1997). Sabu et al., (2002) demonstrated the antioxidant activity of flavonoids in Terminalia chebula, Terminalia arjuna and Emblica officinalis. Reddy et al., (2005) reported the antidiabetic and antioxidant activity of Hydnocarpus wightiana seeds. Menon and Sudheer (2007) reported the antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal and anticancer flavonoids of Azadirachta indica and Curcuma longa. Sahreen et al., (2013) reported the antimicrobial, cytotoxic and antitumor flavonoids in Carissa opaca fruits. Eghdami et al., (2013) reported the antiproliferative and antioxidant flavonoids in the aerial parts of Hypericum perforatum. Paredes et al., (2013) reported the Antibacterial, antiparasitic, anti-flammatory, and cytotoxic flavonoids in leaves and fruits of Lopezia racemosa.

Recently, phenolic acid content, antioxidant and antimicrobial activity of Lingusticum mutellina was reported (Sieniawska et al., 2012). Hoessini et al., (2014) reported the anti diabetic activity of flavonoids in the leaves of Juglans regia leaves. Recently, Le Son and Thao (2014) reported the antioxidant and anticancer flavonoids in the leaves of Pteris multifida Poir. Ghasemzadeh et al., (2014) reported the anticancer flavonoids of Murraya koenigii.

Semecarpus anacardium : It is a deciduous tree belongs to the family Anacardiaceae, distributed in sub-Himalayan tract and hot parts of India (Kirtikar and Basu, 1975). S.anacardium is a deciduous tree having a height of about 10 m and growing naturally in tropical and dry climate. It has lots of applications in indigenous system of medicine (Khare, 1982). It is used by Ayurvedic practitioners/traditional healers across the country albeit with caution. Various parts of S.anacardium are traditionally used for the treatment of rheumatism, asthma, neuralgia, helminthic infection, cancer and psoriasis. This plant has potential to produce allergic manifestations through contact dermatitis. The tree has several medicinal applications.

Figure 1.1 Monograph of Semecarpus anacardium

illustration not visible in this excerpt

Various parts of S.anacardium have been widely used in traditional and folk medicine. The fruits are astringent, digestive, carminative, appetiser, anthelmintic, purgative, liver tonic, respiratory stimulant, expectorant, alterant, aphrodisiac, antiarthritic, stimulant, antiseptic, anti-inflammatory, cardiotonic, rejuvenating and tonic. They are useful in beriberi, cancer, sciatica, rheumatism, palsy, epilepsy, nervous debility, neurotic cardiac disorders, neuritis, cough, asthma, dyspepsia, constipation, flatulence, colic, haemorrhoids, helminthiasis especially hookworms, liver disorders, splenopathy, leprosy, leucoderma, scaly skin eruptions, inflammations, fever, diabetes, dysmenorrhoea, amenorrhoea, syphilis, scrofula, tumors, ulcers and general debility . The gum exuding from the bark is useful in scrofula, venereal diseases and leprous affections. Oil is powerful antiseptic and cholagogue. It is used in scaly skin eruptions like psoriasis and leucoderma (Chattopadhyaya and Khare, 1969).

Fruit extract exhibited hypocholesterolemic action and prevented cholesterol induced atheroma in hypercholesterolaemic rabbit (Sharma et al., 1995). Fruit extract exhibit anti-atherogenic, antiinflammatory, antioxidant, antimicrobial, anti-reproductive, CNS stimulant, hypoglycemic, anticarcinogenic and hair growth promoter activities. Methanolic, ethanolic, chloroform, ethyl acetate and petroleum ether extracts of fruits of S. anacardium exhibited antiinflammatory activity against carrageenan-induced paw edema albino rats (Sugapriya et al. 2008 and Bhitre et al., 2008).

Nair and Bhide, (1996) have shown anti-bacterial properties of alcoholic extracts of leaves, twigs and green frui.. Bondre and Nathar (2011) reported a large number of metabolites such as alkaloids, flavonoids and phenols and also the minerls like Sulphur (S), Calcium (Ca), Magnesium (Mg), Phosphorus (P) and Iron (Fe) in S.anacardium leaves. Barman et al., (2013) reported the antioxidant potential of ethanolic, acetone and aqueous extracts of leaves.

The nut is commonly known as ‘marking nut’ and in the vernacular as ‘Ballataka’ or ‘Bhelwa’. Phytochemical studies on nut extracts of S.anacardium revealed the presence of phenolic compounds, bhilawanols , biflavonoids, anacardic acid, alkenyl catechols and have been reported in nuts of S.anacardium. Nut extract reported to possess several biological activities such as anti-arthritis, antispermatogenic, antimicrobial and mutagenic properties (Ashwinikumar et al., 2007). Mohanta et al., (2007) reported that aqueous and petroleum ether extracts of nuts showed inhibitory activity against Staphylococcus aureus and Shigella flexneri, chloroform extract showed against Bacillus licheniformis, Vibrio cholerae and Pseudomonas aeruginosa, the ethanol extract showed against Pseudomonas aeruginosa and S. aureus. Pal et al., (2008) demonstrated that petroleum ether and ethanol extracts of nuts showed higher DPPH scavenging activity.

Semecarpus anacardium nuts has been used in clinical practice and encouraging results have been reported, particularly for cancer of the oesophagus, liver, urinary bladder and leukaemia. Premalatha et al., (1999) revealed that nut extract shown potent anticarcinogenic activity against AFB1 mediated hepatocellular carcinoma. Mathivadhani et al., (2007) reported that nut extract triggers apoptosis in human breast cancer cells (T47D) by rapid mobilization of calcium from intracellular stores and changing mitochondrial transmembrane potential by decreaseing Bcl(2) and increasing Bax, cytochrome c, caspases and cleaved PARP, and DNA fragmentation. Patel et al., (2009) reported significant antiproliferative activity of methanolic nut extract against hep 2 and vero cell lines. The flavanoids from nut have the ability to prevent various cancers (Jain and Sharma, 2013 ).

Premlatha and Sachdanandam (2000) reported the immunomodulatory potency, antioxidant, membrane stabilizing and tumors marker regulation properties of nut extract of S. anacardium in hepatocellular carcinoma. Tripathy and Pandey (2004) reported the hypolipidemic and hypocholesterolemic activity of nut oil fraction in rats fed with atherogenic diet. Ramprasath et al., (2005) reported that ethanolic nut extract modulates reactive oxygen/nitrogen species and antioxidative system in adjuvant arthritic rats. Antiinflammatory activities of crude ethanolic extract of nuts in was reported in healthy individuals as well as rheumatoid arthritis (RA) patients. It is significantly decreased the carrageenan-induced paw edema and cotton pellet granuloma. Inhibition of pro-inflammatory cytokine production by nut extracts was reported ( Singh et al., 2006 ) and (Ramprasath et al., 2006). Mythilypriya et al., (2008) reported the antiinflammatory activity of nut extract in adjuvant-induced arthritic rat (AIA) model, with reference to mediators of inflammation (lysosomal enzymes) and its effect on proteoglycans.

Antispermatogenic effect of nut extract evidenced by reduction in numbers of spermatogenic cells and spermatozoa was reported in male albino rats (Sharma et al., 2003). Semecarpus anacardium nut extract feeding caused antispermatogenic effect evidenced by reduction in numbers of spermatogenic cells and spermatozoa in male albino rats (Sharma et al., 2003). Extraction of nuts of S. anacardium with milk showed effect on locomotor and nuerotropic activities of central nervous system (CNS) in different experimental animal models (Farooq et al., 2007).

Arulkumaran et al., (2007) showed the protective efficacy of Kalpaamruthaa (KA) against peroxidative damage in 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary carcinoma rats. Kalpaamruthaa (KA), an indigenous-modified Siddha formulati on of S.anacardium nut extract, dried powder of Emblica officinalis (EO) fruit along and honey has been reported as potent antioxidant analgesic, antipyretic and anti-ulcerogenic agent (Mythilpriya et al., 2008). Pal et al., (2008) demonstrated that petroleum ether and ethanol nut extracts showed remarkable antioxidant activity due to the presence of flavonoids, ascorbic acid and tannins. Verma and Vinayak, (2008) reported that aquoeus extracts of nuts had significantly increased the antioxidant enzyme activities in lymphoma-transplanted mouse . Barman et al., (2013) reported the radical scavenging ability of acetone and aqueous extracts of nuts .

Studies on stem bark extract revealed that the ethyl acetate extract of showed strong inhibitory activity on pro-inflammatory enzymes such as cyclooxygenase and acetyl cholinesterase (Selvam et al., 2004 and Vinutha et al., 2007). Sahoo et al., (2008) reported significant antioxidant activity of ethyl acetate extract of stem bark and presence of phenolic compounds.

Singh et al., (2010) reported that the aqueous ethanolic (50%) extracts of S.anacardium stem bark exhibited potent antibacterial and antifungal activities against the microorganisms Micrococcus luteus, Enterobacter aerogenes, Klebsiella pneumonia, Salmonella typhimurium, Streptococcus pneumonia, Candida albicans, Trichophyton rubrum, Aspergillus niger and A. flavus. Recently, Ali et al., (2012) reported that ethanolic extract of stem bark exhibited anti diabetic activity in normal and alloxan induced diabetic rats.

A good number of secondary metabolites bhilwanols, phenolic compounds, biflavonoids (Govindachary, 1971 and Rao et al., 1973), sterols and glycosides (Ishatulla, 1977) have been reported in nuts. Bhilwanol, a mixture of cis- and transisomers of ursuhenol also reported in fruits (Indap et al., 1983). Other components reported including semecarpuflavanone (Murthy, 1983), jeediflavanone (Murthy, 1984; and Murthy, 1985), nallaflavanone (Murthy, 1987), semecarpetin (Murthy, 1988), anacardoside (Farnsworth, 1991), galluflavanone, anacarduflavone (Murthy, 1992), mono-olefin I, diolefin II, bhilawanol-A, bhilawanol-B, amento flavone, tetrahydro amentoflavone semicarpol, anacardic acid. Selvam et al., (2004) reported two chalcone derivatives (3,2,2,4-terta hydroxy chalcone and 7,3,4-trihroxy flavone) with cyclooxygenase inhibitory activity in ethylacetate extracts of stembark.

Earlier, the aqueous stem bark extract was reported to have antimicrobial, CNS stimulant, hypoglycemic, anti-atherogenic and anti-carcinogenic activities (Kirtikar and Basu; 1975). The stem bark of Semecarpus anacardium was used for the treatment of various ailments especially radical related damages, inflammation and cancer in folk medicine. Extensive literature survey revealed that very limited studies have been reported on stem bark of S.anacardium. Hence, the current study was aimed to evaluate the antioxidant and cytoprotective activities of stem bark of S.anacardium to confirm its claims in traditional medicine.

CHAPTER-II PHYTOCHEMICAL ANALYSIS OF Semecarpus anacardium STEM BARK FOR FREE RADICAL SCAVENGING, ANTIOXIDANT AND CYTOPROTECTIVE ACTIVITIES

2.1 INTRODUCTION

Reactive oxygen species (ROS) play a key role in the prevalence of diseases at both cellular and molecular levels (Nordberg and Arner, 2001 ; Stamler and Hauslden, 1998). It is increasingly realized that many diseases are due to the ROS mediated damage of biological macromolecules such as proteins, lipids and DNA in cells. These changes can contribute to atherosclerosis, cardiovascular and inflammatory diseases and cancer (Braca et al., 2002). Oxidative stress can be initiated either by injury or infection. Activation of phagocytes leads to the production of radical and non radical species of both oxygen and nitrogen, which affects surrounding tissue either directly or indirectly. This initiates lipid per oxidation resulting in membrane destruction of cells and cell organelles. Lysosomal enzymes also released during inflammation produces a variety of disorders which leads to the tissue injury by damaging the macromolecules and lipid peroxidation of membranes. Erythrocyte membrane is structurally analogous to the lysosomal membrane. Therefore, the use of erythrocyte membrane is good model to study the protective effect of medicinal plant extracts (Chou, 1997). Previously, stabilization of hypo tonicity induced human red blood cell membrane (HRBC) was used as an in vitro model to study the cytoprotective activity of medicinal plant extracts (Murugasan et al., 1981). However, H2O2 induced damage in RBC membrane appears to mimic natural oxidative damage (Winrow et al., 1993).

The uses of medicinal plants as traditional medicine is wide spread and represent a large source of natural anti-oxidants that might serve as leads for the development of the novel drugs (NaveenKumar et al., 2013). Recently, much attention has been directed towards the development of ethno-medicines with strong antioxidant properties (Maxwell, 1995). Semecarpus anacardium (Anacardiaceae) is well-known for medicinal value in ayurvedic and siddha system of medicine (Khare, 1982). Different parts of this plant have been traditionally used to treat rheumatism, asthma, neuralgia, helminthic infection, psoriasis and cancer (Chadha, 1989). The present study was focused to evaluate the antiradical and antioxidant activities of methanolic extract of S.anacardium stem bark using widely accepted methods.

2.2 OBJECTIVES

- To determine the total antioxidant ability of stem bark extracts of S.anacardium by using FRAP methods.
- To evaluate the radical scavenging activity of stem bark extract of S.anacardium by using hydroxyl, superoxide radical and hydrogen peroxide scavenging methods.
- To determine the cytoprotective activity of stem bark extract using in vitro inhibition of lipid peroxidation and HRB membrane stabilization assays.
- To screen the stem bark extract of S.anacardium for phytochemicals by qualitative and quantitative methods.

2.3 MATERIALS AND METHODS

Plant material

The stem bark of S.anacardium was collected from the Eastern Ghats of Vizianagaram region and authenticated by the faculty in the department of Botany, Andhra University, Visakhapatnam.

Chemicals and Other Reagents

Thiobarbituric acid (TBA), 1,2-dipheny1-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-striazine (TPTZ), nitroblue tetrazolium (NBT) , were purchased from Sigma, USA. Quercetin, ascorbic acid, butylated hydroxyl toluene (BHT), sodium acetate, ferric chloride, tris-HCl, ferrous sulphate, sodium dodecyl sulphate (SDS), acetic acid, butanol, pyridine, deoxyribose, EDTA, H2O2, tricholoro acetic acid (TCA), potassium ferro cyanate , sodium carbonate, hydroxylamine hydrochloride and aspirin were purchased from Merck, Germany. The other chemicals and solvents used in the present study were of analytical grade obtained from local suppliers.

2.3.1 Preparation of Plant Extract

The stem bark was thoroughly cleaned, shade dried and powdered using mechanical grinder. The fine powder was successively extracted with n- hexane, ethyl acetate, methanol and water for 48 hrs using soxhlet extractor. The extracts were concentrated and dried using rota evaporator. The dried extracts were suspended in aforesaid solvents (1mg/ml) and DPPH radical scavenging ability and total antioxidant activity was determined. Further, methanolic extract at different concentrations (25, 50, 100, 250, 500, 750 and 1000µg) was used to determine antioxidant and cytoprotective activities.

2.3.2 DPPH Radical Scavenging Activity

DPPH radical scavenging activity was measured by the method of Cuedent et al., (1997). To 3.0 ml of methanolic solution of DPPH (0.1mM), 1.0ml of extract was added. In control, the extract was replaced by methanol. The reaction mixture was incubated for 30 min at 37oC and absorbance was measured at 517nm using UV-visible spectrophotometer. The percent of inhibition was calculated from the following equation: A0-Ax100/A0, where A0 and A are the absorbance of control and test sample, respectively. Quercetin was used as standard.

2.3.3 Total Antioxidant Activity by FRAP Method

The total antioxidant status of stem bark extract of S.anacardium was assayed by using FRAP method with some modifications (Wong et al., 2006). Briefly, 0.2ml of methanolic extracts (25, 50, 100, 250, 500 ,750 and 1000µg/ml) was added to 3.0ml of FRAP reagent (mixture of 300mM sodium acetate buffer pH 3.6, 10mM TPTZ solution and 20 mM FeCl3 in a ratio of 10:1:1. The reaction mixture was incubated in a water bath at 37oC for 30min. The increase in the absorbance was measured using spectrophotometer at 593 nm. The percent of total antioxidant activity was calculated using a formula, = [(A593 of sample – A593 of control)/ A593 of sample] x 100.

2.3.4 Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity was carried out by measuring the competition between deoxyribose and the extract for hydroxyl radical’s generated by Fenton reaction, a method originally described by Gulhan et al., (2003). 0.1ml of extract or quercetin was added to the reaction mixture containing 0.1ml of 3.0mM deoxyribose, 0.5ml of 0.lmM FeCl3, 0.5ml of 1mM H2O2 and 0.8ml of 20mM phosphate buffer, pH 7.4 in a final volume of 3.0ml and incubated at 37°C for 1hr. The reaction mixture without plant extract was served as control. The thiobarbituric acid reactive substances (TBARS) formed were measured by treating with 1.0 ml of TBA (1.0%) and 1.0ml of TCA (2.8%) at 100°C for 20min. After the mixture was cooled, absorbance was measured at 532 nm against control, which is devoid of plant extract. Percent of inhibition was calculated as

(I) = [(A532 of control- A532 of test / A532 of control)] x 100.

2.3.5 Superoxide Radical Scavenging Activity

The superoxide radical scavenging ability of stem bark extract of S.anacardium was determined by the method of Beauchamp and Fridovich (1976). To 0.5ml of extract, 1.0ml of 0.12M sodium carbonate, 0.4ml of 25µM NBT and 0.2ml of 0.1mM EDTA were added. The reaction was initiated by adding 0.4ml of 1.0mM hydroxylamine hydrochloride and incubated for 20min (25, 50, 100, 250, 500, 750 and 1000µg/ml). The absorbance was measured at 560nm using spectrophotometer. The super oxide anion scavenging activity was calculated as percent inhibition of absorbance compared to the control.

2.3.6 Hydrogen Peroxide Scavenging Assay

Hydrogen peroxide scavenging activity of the extract was estimated by the method of Zhang (2000). 1.0ml of 0.1mM H2O2, 1.0 ml of methanolic extract or quercetin, 2 drops of 3% ammonium molybdate, 10 ml of 2M H2SO4 and 0.7ml of 1.8M KI solutions were mixed well. The solution was titrated with 5.09mM Na2S2O3 until yellow colour was disappeared. All reagents were added except plant extract for control. Percent of hydrogen peroxide scavenging was calculated as: Inhibition = (V0- V1) /V0 × 100, where V0 was volume of Na2S2O3 solution used to titrate the control sample, V1 was the volume of Na2S2O3 solution used in the presence of the extract.

2.3.7 Inhibition of Lipid Peroxide Formation

Lipid peroxidation induced by FeSO4-ascorbate system in sheep liver homogenate is estimated as thiobarbituric acid reacting substances (TBARS) by the method of Ohkawa, Ohishi and Yagi (1979). The reaction mixture containing 0.1ml of sheep liver homogenate (25%) in 20mM Tris-HCl buffer (pH 7.0; KCl (30mM); FeSO4 (NH4) SO4.7H2O (0.06 mM) and plant extract or quercetin in a final volume of 0.5ml and incubated at 37oC for 1h. After incubation, 0.4ml of reaction mixture was taken and treated with 0.2ml sodium dodecyl sulphate (8.1%), 1.5ml thiobarbituric acid (TBA) (0.8%) and 1.5ml of trichloroacetic acid (20%). The total volume was made up to 4.0ml with distilled water and then kept in a water bath at 95oC for 1h. After cooling, 1.0ml of distilled water and 5.0ml of n-butanol and pyridine mixture (15:1) are added to the reaction mixture, shaken vigorously and centrifuged at 4000g for 10 min. The butanol pyridine layer was removed and its absorbance was measured at 532 nm. Control is also run in the same manner but plant extract is replaced with methanol. Inhibition of lipid peroxidation was determined by comparing the optical density of the test sample with that of the control.

Percent of inhibition of lipid peroxidation (I) = [(A532 of control- A532 of sample / A532 of control)] x 100.

2.3.8 HRB Membrane Stabilization Test

Protection of human RBC membrane from heat induced haemolysis was carried out as described by Sadique et al., (1989). Fresh human whole blood (10ml) was collected from volunteers and centrifuged at 3000 rpm for 10min and were washed three times with equal volume of normal saline. The volume of blood sample was measured and reconstituted as 10% v/v suspension with normal saline. The reaction mixture (2.0ml) consists of 1.0ml of extract or standard diclofenac sodium and 1.0ml of 10% RBC suspension was incubated in water bath at 56⁰C for 30min. After incubation the reaction mixture was cooled to room temperature under running tap water, centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was measured at 560 nm. The reaction mixture with saline was served as control. The experiment was performed in triplicates for all the test samples. Percent of RBC membrane protection was calculated by the formula :

Percent of membrane protection = (A560 of Control – A560 of Sample)/A560 of Control x100.

2.3.9 Preliminary Phytochemical Analysis of Methanolic Extract of S.Anacardium Stem Bark.

Preliminary phytochemical analysis of methanolic extract of S.anacardium stem bark was carried out using most widely accepted qualitative tests (De et al., 2010 and Manjamalai et al., 2010).

2.3.9.1 Tests for Polyphenolics

Ferric chloride test: To 1.0ml of plant extract added 3.0ml of alcoholic ferric chloride. Brown colour precipitate indicate positive test for the presence of polyphenol compounds.

2.3.9.2 Tests for Flavonoids

Zinc Hydrochloride reduction test:

To 1.0ml of plant extract, a mixture of Zinc dust and conc. HCl was added and mixed well. Formation of red colour after few min indicates the presence of flavonoids.

Shinoda’s test:

1.0ml of plant extract was treated with 1.0ml of conc. HCl and few pieces of metal magnesium and boiled for few minutes. If test is positive, it gives red colour.

Lead acetate test:

1.0ml of plant extract was treated with basic lead acetate. Formation of yellow colour precipitate indicates positive test.

2.3.9.3 Tests for Alkaloids

Mayer’s test:

To 1.0ml of plant extract, two drops of Mayer’s reagent was added through the walls of the test tube. The test is positive, given creamy coloured precipitate.

Dragendroff’s test:

To 1.0ml of plant extract, two drops of Dragendroff’s reagent was added along the walls of the test tube. Positive test given orange brown coloured precipitate.

Wagner’s test:

To 1.0ml of plant extract, two drops of Wagner’s reagent was added through the walls of the test tube. Reddish brown coloured precipitate indicates presence of alkaloids.

2.3.9.4 Test for Saponins

Foam and froth test:

To 1.0ml of extract 9.0ml of distilled water was added. The suspension was shaken for 15min. Formation of two cm layer of foam which is stable for 1min indicates the presence of saponins and absence of foam indicates negative for saponins..

2.3.9.5 Test for Tannins

Potassium dichromate test:

To 1.0ml of plant extract few drops of water was added and filtered. The filtrate was treated with 10% aqueous potassium dichromate. Formation of yellow brown precipitate indicates the presence of tannins.

2.3.9.6 Quantification of Total Phenolics

Total phenolic content of butanol fraction was determined using the method of Mc Donald et al. (2001) with slight modification. A diluted fraction (1.0ml) of the sample was mixed with 1.0ml of the 50% Folin Cio-calteu reagent, 1.0ml of 2% sodium carbonate centrifuged at 10,000g for 15min and the absorbance was measured with spectrophotometer (Hitachi U2000) at 750nm after 30min of incubation at room temperature. The calibration curve was prepared using gallic acid at a concentration ranging from 20 to 100µg. Total phenolic content was expressed as gallic acid equivalents.

2.3.9.7 Quantification of Total Flavonoids

Aluminum chloride colorimetric method was used for the determination of total flavonoids (Chang et al., 2002). Each fraction (0.5ml) was mixed with 1.5ml of methanol, 0.1ml of 10% aluminum chloride, 0.1ml of 1M potassium acetate and 2.8ml distilled water. The absorbance of the reaction mixture was measured at 415nm after 30min at room temperature. The calibration curve was prepared by using at a concentration ranging from 40 to 200µg. Total flavonoid content is expressed as quercetin equivalents.

2.4 STATISTICAL ANALYSIS

Each experiment was carried out three times separately. Data was expressed as mean ±SE of minimum of six independent experiments. Statistical differences between control and target groups for all experiments were determined using Student’s t -test. The statistical significance was determined at 5% (p < 0.05) level. IC50 values are calculated at 90 % confidence interval using regression analysis.

2.5 RESULTS AND DISCUSSION

Reactive oxygen species (ROS) are produced continuously in most tissues and are inextricably linked to malignant diseases, diabetes, atherosclerosis, chronic inflammation and ischemia-reperfusion injury (Droge, 2005). Superoxide and hydroxyl radicals, hydrogen peroxide, singlet oxygen are known to be cytotoxic and have been implicated in the etiology of various diseases (Bergamini et al., 2004). Epidemiological and in vitro studies have revealed that medicinal plant extracts protects biological systems from oxidative stress (Chaudhary et al., 2004). S.anacardium is a well known medicinal plant and traditionally used for the treatment of stress mediated disorders. In the present study, total antioxidant and antiradical activities were used to determine antioxidant capacity of stem bark extract S.anacardium due to its differential mechanisms of antioxidant action.

Oxygen derived radicals represent the most important class of radical species generated in living systems (Miller et al., 1990). The harmful effect of free radicals causing potential biological damage is oxidative stress (Ridnour et al., 2005). DPPH, is a stable and non-physiological radical widely used for screening antioxidant activity of plant extracts. DPPH is reduced to diphenylpicryl hydrazine with plant extracts in a concentration-dependent manner (Nanjo et al., 1996). The radical scavenging ability of different solvent extracts on DPPH radical is in the following order: methanol > ethyl acetate > aqueous > hexane with 74.85, 58.45, 30.12 and 24.52%, respectively, at 1000 μg/ml concentration (Figure 2.1).

Among the four extracts, methanol extract showed DPPH radical scavenging activity (74.85%) near to natural antioxidant, quercetin (78.35%). The methanolic extract exhibited DPPH radical scavenging activity in concentration dependent manner from 25 to 1000µg/ml . Previously ethanolic extract of nut reoprted to have 88.73% DPPH radical scavenging activity where as leaf extract showed 69.48% DPPH radical scavenging acivity (Barman et al., 2013). Though DPPH radical scavenging method is routinely used to assess the radical scavenging ability of medicinal plant extracts, just one method did not evaluate all possible mechanisms of free radical scavenging activity of methanolic extract (Ganesh babu et al., 2012). Hence, free radical scavenging activity of methanolic extract towards hydroxyl and superoxide radicals as well as hydrogen peroxide was assessed.

Figure 2.1 DPPH radical scavenging acivity of different solvent extracts of

S.anacardium stem bark.

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DPPH radical scavenging activity of n-hexane, ethylacetate, methanol and aqueous extracts of stem bark of S.anacardium. Different concentrations i.e. 25, 50, 100, 250, 500, 750 and 1000µg/ml was used to determine concentration dependent DPPH radical scavenging activity. DPPH radical scavenging activity was expressed in % control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05.

The FRAP assay is a simple, convenient and reproducible method widely employed to determined the total antioxidant activity of biological samples (Pulido et al., 2000). As shown in the figure 2.2 , methanol, ethyl acetate, aqueous and n-hexane extracts of S.anacardium stem bark exhibited 72.00, 53.48, 48.62 and 32.58%, of total antioxidant activity respectively, at 1mg/ml. Barman et al., (2013) reported that ethanolic extract of nut exhibited similar activity 72.37 where as leaf extract showed 58.42 % Fe3+ reducing activity.

Figure 2.2 Total antioxidant activity of different solvent extracts of S.anacardium stem bark.

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Total antioxidant activtity of n-hexane, ethylacetate, methanol and aqueous extracts of stem bark of S.anacardium was assessed by FRAP method as described in Materials and Methods at different concentraions (25, 50, 100, 250, 500, 750 and 1000 µg/ml) and results were expressed as percent control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05.

The results suggested that the methanolic extract showed higher reducing activity than other solvent extracts. The significant antioxidant activity of methanolic extract may be due to extraction of more hydrogen donation compounds by methanol than other solvents used in the present study. FRAP is reduced by the compounds that can donate hydrogen atoms to free radicals and convert them into stable non-reactive molecules (Gordon, 1990). Ganesh babu et al., (2012) described that the reducing ability of plant extract is a good indicator of its total antioxidant ability. The results on IC50 values of different solvent extracts of S.anacardium and standard for scavenging DPPH radical and reduction of ferric ion was depicted in Figure 2.3. The results indicate that the IC50 values of n-Hexane, ethyl acetate, methanol, aqueous extracts and quercetin were 580, 395, 185, 370 and 155µg/ml, respectively. These results indicate that crude methanolic extract is more potent radical scavenger similar to natural antioxidant, quercetin. However, Pednekar et al., (2013) reported that IC50 value of leaf extract was found to be 916.50µg/ml, which is 5 times higher than the methanolic extract stem bark (185µg/ml). But the methanolic extract (7% w/w) showed IC50 value of 60.23 µg/ml towards DPPH radical scavenging (Sahoo et al., 2008). The differential activity may be due to age of the plant and geographical conditions.

Figure 2.3 IC50 values of different extracts of S.anacardium and standard

quercetin for scavenging DPPH radical and reducing ferric ion

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IC50 values of DPPH and ferric ion reducing activity of of n-hexane, ethylacetate, methanol and aqueous extracts of stem bark of S.anacardium with respect to standards. The IC50 values were obtained from the regression analysis expressed in µg/ml. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05. QT - Quercetin.

The IC50 values of ferric ion reducing ability of n-Hexane, ethyl acetate, methanol, aqueous extracts and quercetin were 580, 395, 185, 370 and 150µg/ml, respectively. Razali et al., (2008) reported that methanolic shoot extract of Anacardium occidentalis exhibited high ferric ion reducing activity (3.591mmol/g dry weight).

Hydroxyl radical is the most deleterious and reactive radical among the ROS with shortest half-life compared to other free radicals. Hydroxyl radicals derived from oxygen in presence of transition metal ion (Fe2+) and causes the degradation of deoxyribose into malondialdehyde which produces a pink chromogen with thiobarbituric acid (Halliwell, 1987). The methanolic extract of stem bark of S.anacardium exhibited hydroxyl radical scavenging activity in a dose dependent manner and significant inhibition was observed at 1000μg/ml concentration with 73.20%. The crude methanolic extract exhibited comparable hydroxyl scavenging activity with natural antioxidant, quercetin (76.85%) (Figure 2.4). The IC50 value of methanloic extract towards hydroxyl radical scavenging was 235μg/ml compared to standard antioxidant quercetin (210μg/ml). Previous studies by Sahoo et al., (2008) determined that the methanolic extract (7% w/w) showed IC50 value of 129.51 towards hyroxyl radical scavenging.

Figure 2.4 Hydroxyl radical scavenging activity of methanolic extract of

S.anacardium stem bark.

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Hydroxyl radical scavenging activity of methanolic stem bark extract of S.anacardium at 25, 50, 100, 250, 500, 750 and 1000 µg/ml and natural antioxidant, quercetin (1000 µg/ml ) was determined as described in "Materials and Methods" and the results were expressed in percent control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05. QT - Quercetin.

Super oxide is biologically important radical as it can form singlet oxygen and hydroxyl radical which can contributes to the pathogenesis of many diseases. Over production of super oxide anion radical contributes to redox imbalance and associated with harmful physiological consequences (Gulcin et al., 2005). Super oxide anions can be generated artificially from the hydroxylamine EDTA system. The scavenging ability of extracts can be assayed by NBT reduction method. The superoxide scavenging ability of the methanolic extract of stem bark of S.anacrdium was found to be 69.45% compared to control, whereas known antioxidant quercetin exhibited 72.45% at 1000 μg/ml (Figure 2.5).

Figure 2.5 Superoxide radical scavenging activity of methanolic extract of S.anacardium stem bark.

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Superoxide radical scavenging activity of methanolic stem bark extract of S.anacardium and quercetin was determined as described in "Materials and Methods" and results were expressed as percent control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05. QT - Quercetin.

The IC50 values of extract and quercetin were 240 and 215 μg/ml, respectively (Table 2.1). Earlier Sahoo et al., 2008 reported that the methanolic extract (7% w/w) showed lowest IC50 value (78.21) towards superoxide scavenging. The variation in the scavenging activites may be due to the differneces in plant climatic conditions and the occurance of plant secondary metabolities etc (Figueiredo et al., 2008).

These results indicate that the methanolic extract of stem bark IC50 exhibited nearly equal to the pure natural compound quercetin. The concentration of methanolic extract and standard to inhibit 50% of superoxide, hydroxyl radicals and hydrogen peroxide was given in the table 2.1. The results showed that IC50 value of methanolic extract for scavenging superoxide, hydroxyl radicals and hydrogen peroxides was 240, 235 and 215 μg/ml, respectively, while natural antioxidant, quercetin was 215, 210 and 260 μg/ml, respectively. These results indicate that methanolic extract was significant scavenger of hydrogen peroxide compared to quercetin.

Table 2.1 IC50 values of methanolic extract of S.anacardium stem bark and

standards for scavenging different natural radicals and RBC

membrane protectioin and lipid peroxidation.

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Hydrogen peroxide is a non radical reactive oxygen species with weak oxidizing activity. It diffuse through cell membranes rapidly and interacts with Fe2+ and possibly Cu2+ ions to form hydroxyl radicals and other free radicals (Giorgio et al., 2007). It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. The hydrogen peroxide scavenging ability of methanolic extract of stem bark of S.anacardium is shown in the figure-2.6, and the results show that methanol extract exhibited significant hydrogen peroxide scavenging activity in a concentration dependent manner with highest activity at 1000 μg/ml (85.45%) compared to Quercetin (80.32%).

Figure 2.6 Hydrogen peroxide scavenging activity of methanolic extract of S.anacardium stem bark.

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Hydrogen peroxide scavenging activity of methanolic stem bark extract of S.anacardium (25-1000 µg/ml) and quercetin(1000 µg/ml) as mentioned in "Materials and Methods and results were expressed as percent control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05.

QT - Quercetin.

These results indicate that the methanolic extract exhibited high percent of inhibition compared to quercetin, a known antioxidant.The cytoprotective activity of methanolic extract was determined in terms of inhibition of sheep liver membrane lipid peroxidation and human RBC membrane stabilization assays. Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals and is used as an indicator of oxidative stress in cells and tissues (Halliwell and Chirico, 1993). The measurement of TBARS substances such as malondialdehyde (MDA) has been employed to monitor membrane damage by various reactive oxygen species (Trevisan et al., 2001).The results showed that the methanolic extract of S.anacardium inhibited FeSO4 induced lipid peroxidation of sheep liver tissue homogenate in a dose dependent manner. Maximum inhibition was observed at 1000μg/ml (70.65%) which was higher than the standard antioxidant quercetin (68.45%) (Figure 2.7 ).

Figure 2.7 Inhibition of lipid peroxidation by methanolic extract of S.anacardium stem bark.

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Inhibition of FeSO4-ascorbate system induced by methanolic stem bark extract of S.anacardium extract 25, 50, 100, 250, 500, 750 and 1000 µg/ml and natural antioxidant, quercetin (1000µg/ml) was evaluated as described in "Materials and Methods" and results were expressed as percent control. Each value represents mean ± SE of three independent experiments. The values are significant at p <0.05. QT - Quercetin

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