Keywords:
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| Butea monosperma, Antioxidant, DPPH, Reducing power, Oxidative stress |
INTRODUCTION
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| Oxidative stress is involved in the pathogenesis of various chronic diseases.[1] Antioxidants have ability to neutralize excess oxidant production including enzymes that convert oxidants into less harmful or harmless species and small molecules that serve as oxidant sinks or scavengers.[2] The carcinogenic adverse effects of synthetic antioxidants such as butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) has led to a growing interest towards natural antioxidants of plant origin in recent years.[3,4,5,6] |
| Plants are rich in photochemical. Common dietary antioxidants primarily include ascorbate, tocopherols, carotenoids and bioactive plant phenols. The health benefits of fruits and vegetables are principally due to the antioxidant vitamins and large number of phytochemicals, some with greater antioxidant properties.[7] These phytochemicals mainly includes secondary constituents or metabolites like carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols and tocotrienols and are believed to have an crucial role in the maintenance of human health because endogenous antioxidants provide insufficient protection against the constant and unavoidable challenge of reactive oxygen species in certain conditions like cancer, cardiovascular disease, atherosclerosis, diabetes, cataracts, arthritis, immune deficiency diseases, aging and central nervous system disorders.[8,9] |
| Butea monosperma is commonly known as ‘Flame of forest’, belongs to the family Fabaceae.10 It is locally called as Palash, Palas, Mutthuga, Bijasneha, , Khakara, Dhak Chichra, Bastard Teak, Bengal Kino, Nourouc and is common throughout India, Burma and Ceylon except in very acrid parts.[10,11,12] Almost all the parts of plant including flowers, seeds, leaves and barks possess medicinal property.[13] The plant holds a significant place because of its medicinal and other miscellaneous uses of economic value.[10] Flowers are large, in a rigid racemes 15 cm long, 3 flowers together form the tumid nodes of the dark olive-green velvety rhachis.[10,11] Flowers used as vegetable by tribals and used for the preparation of dye for colouring garments and for making skin antiseptic ointments.[10] Flowers are rich in triterpene[14], butein, butin, isobutrin[13,15], coreopsin, isocoreopsin (butin 7- glucoside)[16], sulphurein, monospermoside (butein 3-β-D-glucoside) and isomonospermoside, chalcones, aurones, flavonoids (palasitrin, prunetin)[17,16,18], steroids, sugar and amino acids[19,20], myricyl alcohol, stearic, palmitic, arachidic and lignoceric acids[21]. Traditionally flowers are used in various conditions like astringent to bowel, increase “Vata” cure “Kapha”, leprosy, strangury, thirst, gout, skin diseases, burning sensation, enlargement of spleen, diarrhea, to reduce body heat and chronic fever, to prevent pus from urinogenital tracts of males, leucorrhoea, flower juice is useful in eye diseases.[10,11,12] Flower is bitter, aphrodisiac, expectorant, tonic, emmenagogue, diuretic, good in biliousness, inflammation and gonorrhea.[10] Plant parts has been evaluated for various activities such as antistress activity[22,23], anticonvulsive activity[24], nootropic activity, antiestrogenic activity25, hypoglycemic and antidiabetic activity[26,27,28], osteogenic activity[16,29] and postcoital anticonceptive activity[30], antiovulatory and anti-implantation activities[31], leprosy, leucorrhoea and gout[32], chemopreventive and anti-cancer properties[32,33], anti-inflammatory[34,35,36], anthelmintic activity[37,38], antioxidant[39], aphrodisiac activity[40], antimicrobial[41], wound healing activity[42,43], thyroid inhibitory and antiperoxidative[44] and antidiarrhoeal activity.[45] |
| Hence, the present study aimed to assess antioxidant potential and free radical scavenging activities of various fractions of Butea monosperma flowers. |
MATERIALS & METHODS
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| Chemicals |
| 1- diphenyl-2-picryl hydrazly (DPPH) was purchased from Sigma Aldrich, India. Trichloroacetic acid (TCA), N-(1- Naphthyl)ethylenediamine dihydrochloride were obtained from S.D. Fine chemicals, India. Ascorbic acid, Butylated hydroxyl anisole (BHA), Potassium ferricyanide, Ferric chloride (FeCl3), Sodium nitroprusside, Disodium hydrogen phosphate, Potassium dihydrogen phosphate, Sodium chloride, Ammonium molybdate, Sodium phosphate, Sulphuric acid were purchased from Loba chieme, India. Methanol, Sulphanilamide , Phosphoric acid were obtained from Qualigens Fine Chemicals, India. |
| Plant material |
| Flowers of Butea monosperma were collected from Toranmal, Maharashtra, India. The plant was botanically identified and authenticated by Dr. D. A. Patil botanist SSVPS college, Dhule, Maharashtra, India and a voucher specimen was deposited at the departmental herbarium. |
| Extraction and fractionation |
| The Flowers were shade dried at room temperature and the dry material was ground to a fine powder using pulverizer. The powdered plant material was extracted using methanol by soxhlet extraction. Solvent was removed with a rotary evaporator (Equitron Rotaeva-8703). The extract was filtered and concentrated. The dried extract was suspended in water and fractionated with pet ether, n-butanol and ethyl acetate. The n-butanolic and ethyl acetate fractions and the remaining aqueous phase were concentrated. All the solvent were removed with a rotary evaporator to obtain the fractions in the yield of 5.5 % gm for n-butanolic and 1.1% for ethyl acetate. |
| Photochemical prospective |
| The phytochemical tests of fractions were performed to detect the presence of saponins, tannins, flavonoids, steroids, triterpenes, glycosides, proteins, carbohydrates and alkaloids. The tests were based on the visual observation of a change in color or formation of precipitate after the addition of specific reagents. |
| DPPH radical scavenging activity[46,47,48] |
| The capacity to scavenge the ‘‘stable’’ free radical DPPH by n-butanolic and ethyl acetate fraction was measured according to Hanato et al (1998) which is based on the reduction of methanolic solution of the coloured free radical of 1, 1- diphenyl-2-picryl hydrazyl (DPPH). A methanol DPPH solution (0.1 mM, 1 ml) was mixed with serial dilutions (10, 20, 40, 60, 80μg/ml) of the n-butanolic fraction and ethyl acetate fraction and incubated for 30 min at room temperature (250C). For each concentration the assay was run in triplicate and the absorbance was read at 517 nm using microplate reader (Powerwave XS, Biotek, USA). Ascorbic acid (Loba chiemie, India) was used as standard to compare with fractions. IC50 (the antiradical dose required to cause a 50% inhibition) for ascorbic acid, n-butanolic and ethyl acetate fraction was determined. The ability to scavenge the DPPH radical was calculated using the following equation: |
| DPPH scavenging effect (%) = (ADPPH - Atest) / ADPPH X 100 |
| Where ADPPH is absorbance of 0.1mM DPPH solution and Atest is absorbance of fractions. |
| Reducing power assay[3,49,50,51] |
| The reducing power was determined according to the method of Oyaizu (1986). Various concentration of the n-butanolic and ethyl acetate fraction (10, 20, 40, 60, 80μg/ml) were mixed with 2.5ml of 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 500C for 2 min. Then 2.5 mL of 10% tricloroacetic acid (w/v) were added, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant (2.5mL) was mixed with 2.5mL of demonized water and 0.5 mL of 0.1% of ferric chloride and the absorbance was measured spectrophotometrically at 700 nm using microplate reader (Powerwave XS, Biotek, USA). Increase in the absorbance of the reaction mixture indicates reducing ability of fractions and which is compared with ascorbic acid as a standard. |
| Nitric oxide scavenging activity[49,51,52,53] |
| The nitric oxide scavenging activity was determined according to method reported by Sreejayan (1997). The method is based on inhibition of nitric oxide (NO) radicals generated from sodium nitroprusside solution at physiological pH. Sodium nitroprosside (1ml of 10mM) was mixed with 1ml fractions of different concentrations (150- 300μg/ml) in phosphate buffer (pH 7.4). The mixture was incubated at 250C for 150 min. To 1 ml of incubated solution, 1ml of Griess reagent (- naphthyl-ethylenediamine dihydrochloride 0.1% in water and sulfanilamide 5% in H3PO4) was added. The same reaction mixture without fractions but equivalent amount of distilled water was served as control. Absorbance was measured at 546 nm using microplate reader (Powerwave XS, Biotek, USA) and percentage inhibition was calculated using following formula: |
| % Inhibition = (AControl – Afraction) / AControl X 100 |
| Where AControl is absorbance of control and Afraction is absorbance of fractions. The antioxidant activity of the factions was expressed as IC50 value was defined as concentration (in μg/ml) of fractions that inhibits the formation of nitric oxide radicals by 50%. |
| Evaluation of total antioxidant capacity by phosphomolybdenum method[54,55,56,57,58] |
| The total antioxidant capacity of fractions was evaluated by the method of Prieto et al (1999). The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH (9). 0.1 ml of fraction (100-800 μg/ml) was mixed with 1 ml of the reagent solution (28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid) and sample was incubated at 950C for 90 min. After the mixture had cooled to room temperature, the absorbance of each solution was measured at 695 nm using an UV/Vis spectrophotometer (Beckman DU-530). The values are presented as the means of triplicate analysis. The antioxidant capacity was expressed as ascorbic acid equivalent by using the standard ascorbic acid graph. |
| Statistical analysis |
| All determinations were run in triplicate and the results were reported as the mean and standard deviation. Statistical analysis was carried out with one way ANOVA followed by Dunnet using graph pad prism software. |
RESULT
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| Photochemical Prospective |
| Table 1 shows presence of various metabolites such as flavonoids, glycosides, tannins, steroids, alkaloids, proteins, carbohydrate and phenol in extract and further fractions contains only flavonoids, steroids and phenols. |
| Inhibition of DPPH radical |
| The scavenging activity of n-butanolic fraction and ethyl acetate fraction of Butea monosperma flowers is shown in figure 1 and compared with that of ascorbic acid. The scavenging effect of extract, fractions and standard on the DPPH radical was expressed as percentage inhibition. Fractions show concentration dependant reduction in absorbance and exhibits effective antioxidant activity. |
| Reducing ability |
| Figure 2 shows the reducing capabilities of nbutanolic and ethyl acetate fractions of Butea monosperma flowers compared with ascorbic acid. The reducing power of fractions increased with increase in concentration. |
| Inhibition of nitric oxide radical |
| Nitric oxide radical generated from sodium nitroprusside at physiological pH was found to be inhibited by fractions of Butea monosperma flowers when compared with ascorbic acid. Figure 3 shows significant reducing capabilities of n-butanolic fraction. |
| Total antioxidant capacity |
| Figure 4 illustrates the total antioxidative capacities of various concentrations of nbutanolic and ethyl acetate fractions of Butea monosperma flowers. |
DISCUSSION
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| Oxidative stress is classically defined as a redox unbalance with an excess of oxidants or a fault in antioxidants.[59] The sources of oxidants are abundant and are generally extremely reactive and unstable.[60] Most oxidants are derived from enzymatic or chemical reactions that produce superoxide anion, hydroxyl, peroxyl (RO2*), alkoxyl (RO*), and hydroperoxyl (HO2*) or nitric oxide (NO). Further these are converted to secondary very reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydrogen peroxide, hypochlorous acid (HOCl), hypobromous acid (HOBr) and per-oxynitrite (ONOO-).[2,60,61] In normal physiology ROS and RNS serve as important regulators of signal transduction and protein function and both produced in a well regulated manner to maintain homeostasis at the cellular level in the normal healthy tissues.[62] However elevated levels of ROS or RNS can damage vital cellular components such as structural and regulatory proteins, membrane lipids, DNA.[2,63] Cellular antioxidants include non-enzymatic endogenous antioxidants like vitamins C and E and coenzyme Q, bcarotene, and glutathione and enzymatic antioxidant defenses like superoxide dismutase, glutathione peroxidase and catalase that convert free radicals to more benign molecules.[64,65] These molecules are capable to donate an electron and neutralize free radicals but are destroyed upon oxidation.[2] Different mechanisms such as neutralizing reactive species (scavenging activity), sequestering transition metal ions (chelation activity), inhibiting enzymes involved in the over production of reactive species and modulating gene expression (e.g. ARE/Nrf-2 pathway) are responsible for its protective effect as antioxidant.[65] In recent years herbal drugs were found to be effective in combating free radical induced physiologic and pathologic conditions. |
| Previous animal study has shown that dietary phytochemical antioxidants like phenolic and polyphenolic compounds such as flavonoids and catechin from edible plant exhibit potent antioxidant activities and capable of removing free radicals. Phytochemical are effective in the management of conditions associated with ROS formation like β-carotene reacts with a peroxyl radical to form a resonance-stabilized carboncentered radical within its conjugated alkyl structure thereby arresting the chain propagation effect of ROS. Brussels sprouts (300 g/d) markedly minimizes the urinary excretion of 8- hydroxydeoxyguanosine in humans indicating a reduction of DNA oxidation.[66,67] Phytochemical investigation of extract and fractions shows that both are rich in the phenolic and polyphenolic compounds. |
| Antioxidant behavior can be assessed either by activity in foods or bioactivity in humans. Antioxidant activity cannot be measured directly but rather by the effects of the antioxidant in controlling the extent of oxidation. DPPH radical bleaching is one of the methods used to evaluate the antioxidant properties of phytoconstituents and is based on the capacity of herbal extract to bleach the DPPH radical, a nitrogen-centred free radical. Figure 1 shows the results of scavenging DPPH radical ability of n-butanolic and ethyl acetate fractions of Butea monosperma flowers in comparison with same doses of ascorbic acid as a standard. The IC50 value of n-butanolic and ethyl acetate fractions was 69.09 and 95.16 showed dose-dependent DPPH radicals scavenging activity. This decrease in absorbance is due to scavenging of the formed radicals by donated hydrogen from antioxidants. Similar effects have been reported by many authors.[68] |
| NO is one of the most common signaling molecules and involved in various cellular metabolic pathways in the body. Physiologically it is essential for regulating the relaxation and proliferation of vascular smooth muscle cells, platelet aggregation, leukocyte adhesion, angiogenesis, thrombosis, vascular tone, hemodynamics, neurotransmitter synthesis and immune response. However increase level of No causes oxidation of various biomolecules (e.g., protein, amino acids, lipid, and DNA) which leads to cell injury and death. Removal of such free radicals is achieved through enzymatic (SOD, GSH, GSH peroxidases, glutathione reductase, catalase) and non-enzymatic reactions in the body.[61,66] The n-butanolic and ethyl acetate fractions of flowers of Butea monosperma significantly scavenges the NO formed from sodium nitroprusside. Figure 3 illustrates the percentage inhibition of nitric oxide generation by fractions. |
| The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant.[3,5] Fe3+/Fe2+ transformation was observed in the presence of fractions for the measurements of the reductive ability. Okuda et al reported that tannins inhibits the formation of lipid peroxides and thereby prevents liver damage.[50] Reducing power of the n-butanolic fraction was found to be significant as that of standard ascorbic acid. |
| Phosphomolybdenum assay used to determine the total antioxidant capacity of fractions which is based on formation of phosphate/Mo (V) complex because of reduction of Mo (VI) by the fraction.[54,55] Figure 4 illustrates the antioxidative capacities of various concentrations of factions. The n-butanolic fraction of Butea monosperma flowers presented a strong total antioxidant activity. The antioxidant activity of fraction might be attributed to the presence of antioxidant phytochemicals such as flavonoids and phenolic compounds. |
| Till date over eight thousand naturally occurring phenolic compounds are known. Major classes of plant phenolics with ‘the type of carbon skeleton, class name (example)’ format include: C6, simple phenols (resorcinol); C6-C1, phenolic acids (p-hydroxybenzoic acid); C6-C2, acetophenones and phenylacetic acids; C6-C3, hydroxycinnamic acids (caffeic acid); C6-C4, hydroxyanthraquinones (physcion); C6-C2- Molecules 2007, 12 1498C6, stilbenes (resveratrol); C6-C3-C6, flavonoids (quercetin); (C6-C3)2, lignans (matairesinol); (C6-C3-C6)2, biflavonoids (agathisflavone); (C6-C3)n, lignins; (C6-C3-C6)n, condensed tannins (procyanidin).[67,69] The admirable antioxidant capacity of polyphenols is due to presence and distribution of numerous hydroxyl groups in the chemical structure. They either chelate transition metal ions or inhibit the activity of many enzymes participating in the formation of free radicals.[70,71] Among polyphenols, flavonoids constitute the most important single group, including more than 5000 compounds that have been thus far identified and exhibits a broad spectrum of chemical and biological activities including radical scavenging properties.[57,67,72] The potent antioxidative activity of fractions of Butea monosperma flowers ascribed to their free-radical scavenging capacity as well as their ability to form chelation with metal ions or both. |
CONCLUSION
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| The n-butanolic fraction and ethyl acetate fractions were found to have potent free radical scavenging activity and significant in vitro antioxidant activity. The observed activity may be due to the steroids, flavonoids and phenolic content presents in the fraction. The component and exact mechanism responsible for antioxidant potential is still needs to evaluate. Determination of these natural antioxidant compounds of plant extracts will help to develop new drug candidates for antioxidant therapy. |
CONFLICT OF INTEREST STATEMENT
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| We declare that we have no conflict of interest. |
Tables at a glance
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| Table 1 |
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Figures at a glance
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| Figure 1 |
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Figure 4 |
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