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Free radical scavenging activity, phenolic contents andphytochemical evaluation of different extracts of stembark of Butea monosperma (Lam.) KuntzeRaj Kumar Salar a & Leena Seasotiya aa Department of Biotechnology, Chaudhary Devi Lal University, Sirsa, 125 055, India

Version of record first published: 27 Mar 2012.

To cite this article: Raj Kumar Salar & Leena Seasotiya (2011): Free radical scavenging activity, phenolic contents andphytochemical evaluation of different extracts of stem bark of Butea monosperma (Lam.) Kuntze, Frontiers in Life Science,5:3-4, 107-116

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Frontiers in Life ScienceVol. 5, Nos. 3–4, September–December 2011, 107–116

Free radical scavenging activity, phenolic contents and phytochemical evaluation of differentextracts of stem bark of Butea monosperma (Lam.) Kuntze

Raj Kumar Salar∗ and Leena Seasotiya

Department of Biotechnology, Chaudhary Devi Lal University, Sirsa - 125 055, India

(Received 24 September 2011; final version received 24 October 2011 )

The antioxidant activity, phenolic contents and phytochemical profile of acetone, methanol, water and ethyl acetate extractsof the stem bark of B. monosperma were investigated. Extracts showed highest antioxidant activity at 100 μg ml−1 withapplication of different assays: 2′-2′ diphenyl–2′ picrylhydrazyl (DPPH) scavenging (83.5% and 78.1%), reducing power(1.85 and 1.78), deoxyribose degradation site-specific (65.5% and 61.5%) and non-site-specific (67.1% and 66.5%) andchelating power (63.4% and 58.9%) in decreasing and increasing polarity of acetone extracts, respectively. However, nitrobluetetrazolium (NBT) showed scavenging of O2• radicals by 56.4% and 53.1% in decreasing and increasing polarity methanolextracts, respectively. Similarly, electron paramagnetic resonance (EPR)/spin-trapping exhibited highest radical scavengingactivity with acetone extracts (12.6 mg g−1 Trolox). The results pointed to the significant antioxidant activities in acetoneand methanol extracts. In most cases, the extracts obtained through decreasing polarity showed higher antioxidant activity.The phenolic content exhibited a strong association (r2 > 0.9) with antioxidant activity. These results suggest that bark ofB. monosperma can be a potential source of natural antioxidants.

Keywords: Butea monosperma; antioxidant activity; phytochemical profile; EPR spectroscopy; free radical; spin trap

IntroductionDrugs from plant origin are relied upon by 80% of theworld’s population (World Health Organization 2008). InIndia, the use of herbal drugs is an important componentof the traditional system of medicine. In the past decade,considerable attention has been paid to the improvement ofhuman health by consumption of functional food and herbalproducts. Many plants synthesize substances including phe-nolic compounds that are useful in the maintenance of goodhealth in humans and animals. There is much interest in thestudy of phenolic compounds because of their antioxidant,anti-inflammatory, antimicrobial and anti cancerous activity(Jiménez et al. 2010; Pandey and Mishra 2010).

Free radicals are known be the product of normalmetabolism. However, when oxygen is supplied in excess orits reduction is insufficient, reactive oxygen species (ROS)such as superoxide anions, hydroxyl radicals, singlet oxy-gen and hydrogen peroxide are generated (Aruoma 1999)that can cause cellular injuries and initiate peroxidation ofpolyunsaturated fatty acids in biological membranes. ROSmay also cause tissue injury leading to DNA strand break-age, protein damage and oxidation of important enzymesin the human body. These events could consequently leadto free-radical related several chronic and degenerativediseases such as carcinogenesis, malaria, heart diseases,

∗Corresponding author. Email: rajsalar@rediffmail.com

arteriosclerosis, diabetes and many other ailments related toageing (Halliwell and Gutteridge 1984; Honda et al. 2004).

It is well known that the majority of plants are anexcellent source of phytochemicals such as phenolic andpolyphenolic compounds (e.g. phenolic acids, flavonoids,tannins, etc.), many of which have potent antioxidant activ-ities which can be exploited in the preparation of food andpharmaceutical products (Liu et al. 2008; Li et al. 2009).Phenolic compounds exert their protective effect mainlybecause of their redox properties, which allow them to actas reducing agents, hydrogen donators and singlet oxygenquenchers. Earlier studies have shown that phenolic com-pounds can be beneficial as prophylactic agents owing totheir antibacterial, anticancer, anti-inflammatory, anti-viral,anti-allergic and immune-stimulating activities (Liu et al.2008; Viuda-Martos et al. 2008). Nevertheless, all aerobicorganisms, including humans, have antioxidant defencesthat protect against oxidative harm and repair damagedmolecules. However, as natural antioxidant mechanismscan be inadequate, the supply of antioxidants throughdietary ingredients is of great interest for a healthy life.A number of plants have been documented for their healthpromoting benefits (Scalbert and Williamson 2000). Theuse of these plant materials as a source of natural antiox-idants and for other applications is important not only for

ISSN 2155-3769 print/ISSN 2155-3777 online© 2012 Taylor & Francishttp://dx.doi.org/10.1080/21553769.2011.635813http://www.tandfonline.com

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food safety reasons, but also because they are natural andare more readily acceptable to consumers.

Butea monosperma (Lam.) Kuntze, also known as flameof the forest, is found in greater parts of India, Burmaand Sri Lanka and belongs to the family Fabaceae. Itis a sacred deciduous tree, attaining a height of 12–15 m. It is reported to have excellent medicinal properties.B. monosperma flowers are reported to contain butrin,isobutrin, butein, butin, flavonoids, glycosides and steroids.It has been dubbed a wonder drug and is reported to pos-sess chemopreventive and anticancer (Choedon et al. 2010),anti-hepatotoxic (Sharma and Shukla 2011), and antidia-betic (Somani et al. 2006) properties. The bark exudes a redjuice that dries to form Butea gum. Roots of B. monospermaare useful in the treatment of filariasis, night blindness,helminthiasis, piles, ulcers and tumours. Pippali rasayana,an Indian ayurvedic drug, employs B. monosperma andis used in the management of giardiasis (Aggarwal et al.1997). Its gum contains leucocyanidin tetramers, pro-cyanidin and gallic acid which are reported to possessanti-implantation, anti-ovulatory activity and is useful inelephantiasis and in curing night blindness and other eye-sight defects. The bark is reported to possess astringent,pungent, alliterative, aphrodisiac and antihelminthic prop-erties. It is also useful in tumours, bleeding piles, gonor-rhoea and ulcers (Burli and Khade 2007). Thus, most ofthese studies reported a good pharmaceutical potential ofB. monosperma. However, to the best of our knowledge,antioxidant studies on the bark of this plant and their corre-lation with phenolics are scarce. Therefore, the objectivesof the present investigation were to evaluate the antioxi-dant activity of various extracts/fractions of the bark of B.monosperma by employing a number of antioxidant assaysand solvent systems in addition to provide data on totalphenolics and phytochemical profile of the extracts.

Materials and methodsChemicals and plant material2′-2′ Diphenyl–2′ picrylhydrazyl (DPPH), 2–thiobarbituricacid (2–TBA), deoxyribose, ferrozine, potassium ferri-cyanide, ferric chloride, EDTA, hydrogen peroxide, L-ascorbic acid, sodium hydroxide, trichloroacetic acid, gallicacid, sodium carbonate, butylated hydroxyanisole (BHA)butylated hydroxytoluene (BHT) and chemicals for phyto-chemical screening were of analytical grade and obtainedfrom Hi-media Pvt. Ltd., Mumbai, India. Other chemicals,namely Folin Ciocalteu reagent and organic solvents wereobtained from Fischer Scientific, Mumbai.

Stem bark of Butea monosperma (Lam.) Kuntze. wasobtained from a tree growing in a locality in Sirsa town,Haryana, India in the month of February. Its bark is hard, fis-sured and ash coloured. It was washed thoroughly initiallywith tap water and then with distilled water to remove anydebris or dust particles and allowed to dry in an oven below

40◦C for 24 h. The dried bark was ground to powder andstored in airtight containers until used for further studies.

Preparation of extractsFor preparation of extracts two methods were followedbased on solvent polarity. In increasing order of solventpolarity, extraction was initiated with a least polar solvent,i.e. hexane, which was followed by chloroform → ethylacetate → acetone → methanol → water. In decreasingorder of solvent polarity, the extraction was started withmost polar solvent (water), followed by methanol → ace-tone → ethyl acetate → chloroform → hexane. In bothmethods, 500 g of bark powder was soaked with 1500 ml ofeach solvent for 24 h at room temperature. After recoveringthe supernatant, the respective solvents were added twice tothe residue. All the three supernatants recovered from eachsolvent were combined and the solvents were eliminatedusing a rotary vacuum evaporator (Buchi type) to obtain adry extract. The ethyl acetate extract was further partitionedinto water fraction (WF) and ethyl acetate fraction (EAF).All extracts were stored at 4◦C until use. Extractive valuewas calculated as follows:

Extractive value = mass obtained after extractiontotal mass taken for extraction

× 100.(1)

Determination of total phenolic contentThe total phenolic content (TPC) of the extracts was deter-mined using Folin–Ciocalteu reagent following Kujala et al.(2000). In this method, briefly, to 100 μl of extract (100 μgdry extract per ml solvent) were added 500 μl of appropri-ately diluted (50%) Folin–Ciocalteu reagent followed by1 ml of 20% Na2CO3 aqueous solution. The mixture wasincubated at room temperature for 20 minutes before record-ing the absorbance at 730 nm. The results were expressedas gallic acid equivalents (GAE) in mg g−1 of dry plantmaterial and correlated with different antioxidant assays.

Antioxidant activityDPPH (2′-2′ diphenyl–2′ picrylhydrazyl) radicalscavenging assayThe free radical scavenging capacity of different sampleswas measured by the DPPH radical scavenging methodof Yen and Chen (1995) with slight modifications. Themethod involves the reaction of the antioxidants presentin the extracts with the stable DPPH in 0.1 mM methanolsolution. Briefly, the reaction mixture contained 300 μl ofextract of varying concentrations (10–100 μg ml−1) and2 ml of DPPH solution. After 10 minutes, the change inabsorbance was recorded at 517 nm in a spectrophotome-ter against a blank, which did not contain the extract.L-ascorbic acid was used as a positive control. The DPPHradical scavenging capacities were expressed as Vitamin-C

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Equivalent Antioxidant Capacity (VCEAC) in mmol g−1 ofextract.

The % DPPH• scavenging activity was calculated by theequation:

% DPPH• scavenging activity = Acontrol − Asample

Acontrol× 100.

(2)In order to calculate the IC50 value, which is the amountof sample necessary to decrease the absorbance of DPPHradical by 50%, the decolourization was plotted against theconcentration of sample extract.

Reducing power assayReducing power of the plant extracts was determined fol-lowing the method of Oyaizu (1986). Briefly, to differentconcentrations of extracts (1 ml) were added 2.5 ml ofphosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassiumferricyanide (1%). The reaction mixture was allowed toincubate at 50◦C for 20 min. Then 2.5 ml of trichloroaceticacid (10%) was added to the reaction mixture, which wasthen centrifuged at 9500 rpm for 10 min. The upper layer ofsolution (2.5 ml) was recovered and mixed with 2.5 ml dis-tilled water and 2.5 ml FeCl3 (0.1%). The absorbance wasrecorded at 700 nm in a spectrophotometer. An increase inthe absorbance of reaction mixture indicated the increasedreducing power.

Deoxyribose degradation assayThe non-site and site-specific deoxyribose assays were per-formed respectively following the method of Halliwell et al.(1987) and Arouma et al. (1987) with some modifica-tions. In non-site specific deoxyribose assay, the extracts(10–100 μg ml−1) were mixed with a Haber–Weiss reac-tion buffer [10 mM FeCl3, 1 mM EDTA (pH 7.4), 10 mMH2O2, 10 mM deoxyribose, and 1 mM L-ascorbic acid]and the final volume of all mixtures was made to 1.0 ml.The mixture was then incubated at 37◦C for 1 h follow-ing heating at 80◦C for 30 min with 1 ml of 2-TBA (0.5%2-TBA in 0.025 M NaOH, 0.02% BHA) and 1 ml of 10%trichloroacetic acid (TCA) in water bath for 45 min. Aftercooling, the absorbance of mixture was measured at 532 nm.A site-specific assay was performed following slight modi-fications where the EDTA was replaced with a same volumeof phosphate buffer. The percentage inhibition was calcu-lated employing the formula as given for DPPH scavengingassay and was correlated with total phenolic content. TheIC50 value was also calculated.

Chelating effects on ferrous ionsThe chelating effect on ferrous ions was determined asper Dinis et al. (1994) with certain modifications. Briefly,the extracts (250 μl) were mixed with 1.75 ml of methanoland 250 μl of FeCl2 (250 mM) followed by the addition of

250 μl of 2 mM ferrozine, and allowed to react for 10 minat room temperature before measuring the absorbance ofmixture at 562 nm in a spectrophotometer. The chelatingeffect (%) was also calculated from the formula as given forDPPH radical scavenging assay.

NBT superoxide radical scavenging assayThe scavenging activity of the plant extracts towards super-oxide anion radicals was measured by following the methodof Liu et al. (1997). The superoxide anion was generatedin 3 ml of Tris-HCL buffer (100 mM, pH 7.4) containing750 μl of NADH (936 μM) solution and 300 μl of differentconcentrations (10–100 μg ml−1) of extracts. L-ascorbicacid was used as positive control. The reaction was initiatedby adding 750 μl of PMS (120 μM) to the mixture. After5 min of incubation at the room temperature, the absorbancewas measured at 560 nm. The per cent NBT decolourizationof the sample was calculated by the equation:

Per cent inhibition = (A0 − A1/A0) × 100, (3)

where A0 is the absorbance of the negative control and A1 isthe absorbance of the reaction mixture. The IC50 values (theamount required to inhibit superoxide radical formation by50%) of plant extracts were calculated and compared withthat of L-ascorbic acid used as a positive control.

EPR (electron paramagnetic resonance) spin trappingtechniqueThe thermal decomposition of potassium persulfate(K2S2O8) in dimethyl sulphoxide (DMSO) at 333 K wasused as a source of reactive radicals. To measure the radicalscavenging capacity of acetone and methanol extracts of B.monosperma, the EPR spin trapping technique (Rapta et al.2005) was used, employing 5,5-dimethylpyrroline N -oxide(DMPO, Sigma-Aldrich, St. Louis, MO, USA) as a spintrap. Sulfate radical anions (SO•−

4 ) generated upon thermaldecomposition of K2S2O8 represent reactive species withhigh reduction potential, capable of reacting with a varietyof organic compounds. In DMSO solvent these paramag-netic species are added to the double bond of DMPO spintrapping agent producing the corresponding spin adducts(Zalibera et al. 2009). All EPR measurements were car-ried out in a single 4 mm flat quartz cell in a Bruker TE102(ER 4102 ST) cavity using the EMX EPR spectrometer(Bruker, Rheinstetten, Germany) working in the X-band.The ER 4111 VT temperature unit (Bruker, Germany) wasused for temperature regulation. The reaction mixture con-sisted of 25 μl of respective extracts dissolved in DMSO(pure DMSO in reference), 175 μl DMSO, 25 μl of 0.2 MDMPO dissolved in DMSO and 25 μl of 0.01 M K2S2O8(DMSO). A time course of EPR spectra of the DMPOspin adducts was recorded in 66 s intervals for 22 minutesat 333 K (each spectrum was an accumulation of threescans). The integral EPR intensity (double integral) found

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after 22 minutes of thermal treatment for the sample solu-tion was compared with the reference measurement. Thedifference between the integral EPR intensities of the ref-erence and the samples in the 22nd minute characterizesthe amount of radicals scavenged by the various compo-nents present in the sample acting as radical scavengers.The radical scavenging capacity (RSC) values were calcu-lated as a percentage of scavenged radicals relative to thereference sample (DMSO). These values were recalculatedto Trolox equivalent antioxidant capacity (TEAC) using acalibration curve measured analogously for Trolox solu-tions in K2S2O8/DMPO/DMSO systems, and so obtainedradical scavenging characteristics of investigated sampleswere evaluated in mmol of Trolox per 1 g of extract.

Phytochemical screeningPhytochemical screening of various extracts/fractions wascarried out according to the standard methods as describedby Trease and Evans (1996).

Statistical analysis and correlationsThe mean values and the standard deviations were cal-culated from the data obtained from three independentexperiments. Statistical differences at p < 0.05 were con-sidered to be significant coefficient of determination (r2)

to determine the relationship between two variables werecalculated using Microcal Origin 5.0 and Microsoft Excel.

Results and discussionPhytochemicals and total phenolic contentsResults of phytochemical analysis showed that bothincreasing and decreasing solvent polarity extracts exhib-ited the presence of various phytochemicals (Table 1).Qualitatively, acetone and methanol extracts of decreasing

solvent polarity exhibited the presence of most phytochemi-cals investigated. The highest extractive value in decreasingsolvent polarity was obtained in water extract (4.03%)indicating the presence of a high amount of water solublephytoconstituents. Conversely, increasing polarity extractsexhibited maximum extractive value (2.09%) in acetoneextracts.

Phenolic compounds have been widely investigated inmany medicinal plants, fruits and vegetables (Djeridaneet al. 2006). Phenolic compounds or polyphenols are theprincipal antioxidant constituents with more than 8000 phe-nolic structures currently known. The total phenolic contentof different extracts of stem bark of B. monosperma wasestimated by the Folin–Ciocalteu method. From the results,it was observed that total phenolics ranged from 45 to141 mg GAE/g of extract (Table 2). Among the extractsinvestigated, the amount of phenolic compounds in ace-tone extracts of both solvent polarities were highest, andlowest in ethyl acetate extract (p < 0.05). Tawaha et al.(2007) estimated that a total phenolic content higher than20 mg GAE/g dry weight could be considered as very high.This suggests that, the methanol, acetone, ethyl acetate andwater extracts of B. monosperma bark must be considered asgood source of phenolic compounds. However, a very highamount of phenolic content (>775 mg GAE/g extracts) wasreported in Acacia auriculiformis (Singh et al. 2007). Thistremendous increase in phenolic content could be explaineddue to further fractionation of the crude extracts used in theirstudy.

Antioxidant activityAntioxidant activity may possibly result by the synergybetween a number of mechanisms. Owing to complexnature of the phytochemicals present (Hall and Cuppett1997), the antioxidant activity of plant extracts cannotbe evaluated by a single method. In order to explore

Table 1. Phytochemical analysis and extractive value of different solvent extracts of B. monosperma bark in increasing and decreasingorder of solvent polarity.

Extractsa

Increasing polarity Decreasing polarity

S. No Phytochemicals 1 2 3 4 5 6 7 1 2 3 4 5 6 7

1 Alkaloids − − − + + + − − − − − + + +2 Carbohydrates − − − − − − − − − − − + + +3 Sterols − − − − + − − − + − + + + +4 Saponins − − − − − − − − − − − + + +5 Tannins − − − − + + − − − − − + + +6 Flavonoids − + − − − − − − + − − − + −7 Amino acids − − − − + − − − − − − + + +8 Proteins − − − − + − − − − − − + + +9 Resins + − − − − + − + + − − − + +Extractive value (%) 0.36 0.41 0.18 0.73 1.14 2.09 ND 0.11 0.22 0.26 0.10 0.51 1.98 4.03

Notes: aExtracts: 1, hexane; 2, chloroform; 3, ethyl acetate fraction; 4, water fraction; 5, acetone; 6, methanol; 7, water; ND, not determined.“+” Indicates presence of the phytochemical, “−” indicates absence of the phytochemical.

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Table 2. Total phenolic content (as mg gallic acid equivalents(GAE) per g of extract) in different extracts of B. monosperma.

Total phenolic content (mg GAE/g of extract)

Plant extracts Increasing polarity Decreasing polarity

Acetone 141ax 128ay

Methanol 117bx 121ax

Ethyl acetate 45cx 47cx

Water 45cx 93by

Notes: a–cwithin a column different letters are significantly dif-ferent (p < 0.05).x−ywithin a row different letters are significantly different(p < 0.05).

these additional mechanisms, several antioxidant assays,namely DPPH, reducing power, deoxyribose degradation,chelating power, NBT and EPR, were employed in thepresent investigation to evaluate antioxidant activity of B.monosperma bark extracts and were correlated with theirtotal phenolic contents.

DPPH radical scavenging activityThe stable DPPH free radical has been widely used to testthe antiradical power of various natural products (Brand-Williams et al. 1995) and has been accepted as a modelcompound for free radicals originating in lipids (Yasudaet al. 2000). In the present study, various extracts of B.monosperma were effective in reducing the stable DPPHradical to yellow coloured diphenylpicrylhydrazine, indi-cating that these extracts are active in DPPH radical scav-enging (Table 3). Strikingly, acetone and methanol extractsof B. monosperma tested had exceptionally high scaveng-ing activity when compared with the water and ethyl acetateextracts. In this study, we expressed DPPH antioxidantcapacity results by considering kinetic parameters by testingdifferent initial concentrations (20–100 μg ml−1) of the testsamples. In the present investigation, we also established theIC50, which was the amount of sample needed to scavenge50% of the initial concentration of the free radical (DPPH).Results of the IC50 values revealed that acetone extracts of

increasing polarity were more powerful and depleted theinitial concentration of DPPH by 50% with 44.77 μ g ml−1

extract concentration in 10 minutes. Meanwhile this con-centration was highest (139.88 μ g ml−1) in water extract.Similarly, in case of decreasing polarity extracts, the IC50value was also found to be minimum (34.96 μg ml−1) inacetone extract and maximum (71.37 μg ml−1) in ethylacetate fraction. The lower the IC50 value the higher thefree radical scavenging activity of a sample.

The DPPH scavenging activity of B. monospermaextracts in increasing order of solvent polarity rangedbetween 36.68% and 78.10% at 100 μ g ml−1 extractconcentration. For decreasing polarity extracts it rangedbetween 59.26% and 83.02% at 100 μ g ml−1 extract con-centration (Table 3). A linear correlation between DPPHscavenging activity and total phenolic content showed astatistically significant correlation with r2 of 0.809 to 0.980(not shown) with different extracts. These data are in accor-dance with earlier research (Liu et al. 2008; Amensouret al. 2010; Kuate et al. 2010), suggesting that a high totalphenolic content increases antioxidant activity.

Reducing power assayReducing power is based on the reduction of Fe3+ to Fe2+in the presence of reductants present in the extracts. Table 4shows the relative reducing power of various extracts ofB. monosperma prepared by increasing and decreasing sol-vent polarities. It is clear from the results that regardlessof extraction procedure, acetone extracts exhibited max-imum (1.779, 1.852) reducing potential, whereas waterextracts depicted minimum (0.985, 0.838) reducing poten-tial in increasing and decreasing order, respectively, ofsolvent polarities (p < 0.05), at 100 μg ml−1 extract con-centration. There was no significant difference of activitiesas shown by increasing and decreasing solvent polarityextracts. Reducing power assay is an important parameterused in evaluating antioxidant activities of natural extracts.It inhibits lipid peroxidation (LPO) by donating a hydrogenatom, resulting in termination of free radical chain reaction(Yen and Chen 1995). In this study, reducing potential

Table 3. Percentage inhibition of DPPH radical in the presence of different concentrations of increasing and decreasing polarity extractsof B. monosperma.

DPPH scavenging activity (%)

Increasing polarity Decreasing polarity

Plant extracts IC50 IC50(μg ml−1) 20 40 60 80 100 (μg ml−1) 20 40 60 80 100 (μg ml−1)

Acetone 38.26a 45.83a 55.12a 74.75a 78.10a 44.77 42.94a 50.84a 58.96a 76.06a 83.02a 34.96Methanol 37.28a 46.97a 56.02a 67.65a 73.75a 45.69 27.07b 34.96b 55.23a 79.57a 83.41a 51.77Ethyl acetate 24.59b 39.24b 44.19b 51.50b 58.85b 74.77 23.66b 35.75b 46.65b 55.77b 59.26b 71.37Water 19.44c 24.42c 26.71c 35.59c 36.68c 139.8 23.69b 42.46b 56.56a 60.16b 65.44b NDAscorbic acid 22.14b 43.19a 63.97a 72.19a 80.75a 62.87 22.14b 43.19b 63.97a 72.19a 80.75a 62.87

Notes: a–cwithin a column different letters are significantly different (p < 0.05); ND: not determined.

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Table 4. Relative reducing power of different concentrations of increasing and decreasing polarity extracts of B. monosperma.

Relative reducing power

Increasing polarity Decreasing polarity

Plant extracts (μg ml−1) 20 40 60 80 100 20 40 60 80 100

Acetone 0.926a 1.250a 1.382a 1.558a 1.779a 0.455b 0.705b 0.970b 1.294b 1.852a

Methanol 0.529b 0.838b 1.073b 1.117b 1.235b 0.367b 0.661b 0.985b 1.264b 1.588b

Ethyl acetate 0.366c 0.605c 0.843c 0.923c 1.098b 0.220c 0.426c 0.676c 0.955c 1.147c

Water 0.205d 0.500c 0.764c 0.897c 0.985c 0.235c 0.500c 0.661c 0.750d 0.838d

Butylated hydroxytoluene (BHT) 0.779a 1.235a 1.382a 1.735a 1.823a 0.779a 1.235a 1.382a 1.735a 1.823a

Notes: a–d within a column different letters are significantly different (p < 0.05).

Figure 1. Scavenging of hydroxyl radicals by various extracts of B. monosperma in (a) site specific and (b) non-site specific deoxyribosedegradation assay.

increased in a dose dependent manner which is concomitantwith the study of Yen and Duh (1993), who reported thatthe reducing power of peanut hull extract increased withincrease in concentration and correlated (r2 = 0.979) wellwith the extent of antioxidant activity.

Deoxyribose degradation assayHydroxyl radical is the most reactive ROS and attacksalmost every molecule in the body resulting in peroxidationof cell membrane lipids and in formation of malondialde-hyde, which is mutagenic and carcinogenic. The resultsobtained in deoxyribose degradation assay (site-specific andnon-site-specific) to prevent 2-deoxy-D-ribose oxidationmediated by OH• radicals are presented in Figures 1(a)

and 1(b), respectively. This assay was carried out undertwo different experimental conditions: in the presence ofEDTA (non-site-specific) which allows the study of OHradical scavenging activity; and in the absence of EDTA(site-specific) which allows evaluation of the extracts’ abil-ity to bind to Fe. It was observed that in general, all theextracts of B. monosperma displayed a protective effectdepending on the concentration of the extracts. However,the effect was slightly more in the non-site-specific assay,which indicated the suppression of OH• radicals; this hasbeen implicated as a highly damaging species in free radi-cal pathology, capable of damaging almost every moleculefound in living cells (Yasuda et al. 2000).

The ability of extracts to bind to Fe in site-specific assayvaried from 29.9 to 61.1% and 25.8 to 65.5% in increasing

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and decreasing order of solvent polarities, respectively, at100 μg ml−1 concentration, with acetone extracts being themost effective. Similarly the scavenging of OH• radicals bythe extracts in non-site-specific assay varied from 27.1 to66.5% and 24.4 to 67.1% in increasing and decreasing orderof solvent polarities, respectively, at 100 μ g ml−1 concen-tration of acetone extracts. The IC50 values were found to beminimum (75.8 μg ml−1) in acetone extract and maximum(145.21 μg ml−1) in water fraction in increasing order ofsolvent polarities. In case of decreasing polarity extracts theIC50 value was minimum (71.04 μg ml−1) in the methanolextract whereas it was maximum (182.57 μg ml−1) in thewater extract. The antioxidant potential of extracts dis-played by deoxyribose degradation (site-specific and non-site-specific) assay was found to be highly correlated totheir total phenolic content, showing a positive correlationin most of the extracts. The abilities of extracts in boththe assays indicate their effectiveness as chelating agents,as well as their capacity to scavenge OH• radicals whichare produced in solution from a Fe2+-EDTA chelate (Singhet al. 2007).

Fe2+ ion chelating activityThe results of chelating ability of various extracts ofB. monosperma towards ferrous ion, the most effective

pro-oxidant commonly found in food system (Yamaguchiet al. 1998) are presented in Figure 2(a). The ferrousstate of iron can stimulate lipid peroxidation by the Fen-ton reaction and is the most powerful pro-oxidant amongvarious species of metal ions (Kuate et al. 2010). In thisassay, per cent inhibition of formation of ferrous complexby extracts of B. monosperma was estimated. The resultsrevealed that Fe2+-ion chelating activity of extracts inincreasing order of solvent polarity was minimum (21.33%)in water fraction whereas maximum (58.87%) in ace-tone extract at 100 μg ml−1 concentration. Conversely,decreasing polarity extracts showed minimum inhibition(33.69%) in ethyl acetate fraction and maximum (63.44%)in acetone extract at 100 μ g ml−1 concentration. Further,it was observed that, in general, the Fe2+-ion chelat-ing activity increased as the concentration of the extractsincreased up to 60 μg ml−1, at which the effect levelledoff even with a further increase in extract concentration(Figure 2a). Moreover, various potencies for ion-chelationactivity with an IC50, ranging from 67.4 to 227.4 μg ml−1,were noted with various extracts depending on the pro-cedure of extraction. In general, IC50 value was observedminimum (67.43 μg ml−1) in acetone extract of decreasingpolarity. Furthermore, the antioxidant potential of ace-tone extracts evaluated by chelating power assay was alsohighly correlated (r2 = 0.908 and r2 = 0.954 for increasing

Figure 2. (a) Ferrous ion chelating power and (b) scavenging of the superoxide radical (NBT) by increasing and decreasing solventpolarity extracts of B. monosperma.

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114 R.K. Salar and L. Seasotiya

and decreasing polarity extracts) to the total phenoliccontent.

NBT (superoxide scavenging) assaySuperoxide anion is a reduced form of molecular oxy-gen and is one of the most representative free radicals.In cellular oxidation reactions, superoxide radicals arenormally formed first and their effects can be magnifiedbecause they lead to a cascade formation of other ROSand oxidizing agents in the cells (Liu and Ng 2000). O2•radicals can directly mediate protein damage and stimu-late the formation of other entities involved in the damageof biomolecules such as lipids (Halliwell et al. 1995). Inthis study, the effect of B. monosperma extract to inter-act with O2• radicals was measured as a function of itsinhibitory effect on the NBT reduction caused by theseradicals. It is clear from the data that all extracts of B.monosperma react directly with O2• radicals in a dosedependent manner (10–100 μg ml−1) (Figure 2b). How-ever, the methanol extracts showed maximum inhibition(53.06% and 56.35%) in increasing and decreasing orderof solvent polarity, respectively, at 100 μg ml−1 extractconcentration (Figure 2b). The ethyl acetate extracts hadlowest scavenging activity. The IC50 value was determinedas 88.5 μg ml−1 and 85.9 μg ml−1 in methanol extractsof increasing and decreasing polarity extracts, respec-tively, which was highly significant (p < 0.05) comparedto ethyl acetate extracts. The antioxidant potential of B.monosperma extracts evaluated by NBT assay when com-pared with total phenolic content was also found to be highlycorrelated showing a positive correlation in most of theextracts.

Electron paramagnetic resonance (EPR) studiesKeeping in view the potencies of extracts to scavengefree radicals as depicted by the above assays, EPR stud-ies were carried out for acetone and methanol extracts ofboth increasing and decreasing solvent polarities. The char-acteristic experimental EPR spectrum of •DMPO-SO−

4 spinadduct recorded during the thermally initiated decompo-sition of K2S2O8 in DMSO at 333 K, along with its sim-ulation (aN = 1.296 mT, aβ

H = 0.938 mT, aγ

H = 0.139 mT;g = 2.0059) were studied. Figure 3 represents the orig-inal sets of 20 individual EPR spectra monitored in thepresence of DMPO during heating of K2S2O8 at 333 Kfor the reference sample DMSO (200 μ l DMSO, 25 μl0.2 M DMPO in DMSO, 25 μl 0.01 M K2S2O8 in DMSO)and for DMSO extracts of test samples (200 μl extract inDMSO, instead of DMSO in reference sample). It shouldbe noted here that the decrease in the monitored EPR sig-nal intensity of spin adducts is influenced by concentrationof individual extracts (Figure 3). Figure 4(a) shows a timedependence of EPR integral intensities (evaluated by dou-ble integration of sets of 20 individual EPR spectra for each

Methanol IP

Acetone IP

Methanol DP

Acetone DP

0 5 10 15 20

Reference (DMSO)

Time, min

Figure 3. Time course of 20 individual EPR spectra obtainedfor samples of various extracts of B. monosperma dissolved inDMSO. All sets of 20 EPR spectra of DMPO adducts mon-itored during the thermal (333 K) decomposition of K2S2O8in the presence of DMSO extracts were taken for 22 minutesunder the same experimental conditions as for reference sample(DMSO, instead of DMSO bark extracts). Extracts concentrations:methanol I.P., acetone I.P. & D.P. (100 mg ml−1) and methanolD.P. (500 mg ml−1).

measurement, e.g. Figure 3) representatively for the sam-ples of B. monosperma extracts. EPR integral intensity after22 minutes detected for the extracts was compared to thatof the reference. The difference between these EPR inten-sities is proportional to the amount of radicals terminatedby the scavengers present in the investigated extract sample(Figure 3), and is called radical scavenging capacity (RSC,expressed in %). Finally, RSC values were recalculated tothe TEAC values (molar amount of Trolox/1 g of dry extractinducing the identical changes in RSC) using calibrationcurve obtained under strictly identical conditions for Troloxsolutions in K2S2O8/DMPO/DMSO systems (Figure 4b).The radical scavenging ability from EPR spin trapping assayof extracts ranged from 3.7 to 12.6 mg Trolox g−1 with high-est RSC displayed by acetone extracts of increasing polarity(Figure 4b). In EPR experiments we generate the SO•−

4 withhigh redox potential, which are capable of reacting with avariety of other compounds present in the extracts (e.g. lig-nans, polysaccharides, anthocyanins, phytic acid, tanninsand flavonoids) other than phenolics (Martínez-Tomé et al.2004).

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Frontiers in Life Science 115

A B C D0

5

10

15A methanol increasing polarityB methanol decreasing polarityC acetone increasing polarityD acetone decreasing polarity

TE

AC

, mg

of tr

olox

per

gra

m o

f ex

trac

t

Sample

0 5 10 15 20

0.0

2.0x10 6

4.0x10 6

6.0x10 6

8.0x10 6 Reference (DMSO)(a)

(b)

methanol IP methanol DP acetone IP acetone DP

Inte

gral

EPR

inte

nsity

Time, min

100

80

60

40

20

0

RSC

, %

Figure 4. (a) Time course of EPR integral intensities of DMPOadducts recorded during the first 22 minutes of the thermal decom-position of K2S2O8 in the presence of DMSO extracts of B.monosperma bark and (b) radical scavenging capacities equatedto actual dry weight of the extracts and expressed as TEAC g−1

of extracts.

ConclusionsIn conclusion, the present investigation reported the antiox-idant properties of bark of B. monosperma extracted accord-ing to increasing and decreasing solvent polarity. This studyalso reports the phenolic content and phytochemical profileof the various extracts. Significant variations were foundin total phenolic content, antioxidant activity and releaseof phytochemicals depending on the solvent and methodof extraction. Overall, acetone extracts of decreasing sol-vent polarity were found to be richer in antioxidant activityand phytochemicals. The amount of phenolic compoundsdetected was observed to be greater in acetone as comparedto other solvents. Although other antioxidant compoundsmay be present in the extracts, radical scavenging activ-ity investigated by various assays and the total phenolicscontent are highly correlated to each other. Further stud-ies will be focused on cytotoxic effects, fractionation andpurification of active components in acetone extracts of B.monosperma. This will most likely improve the antioxidant

activity and other potential health benefits, promoting theiruse as natural antioxidant source.

AcknowledgementsThe authors thank the Chairperson, Department of Biotechnol-ogy, Chaudhary Devi Lal University, Sirsa for providing necessarylaboratory facilities. We also wish to thank Prof. Vlasta Brezova,Faculty of Chemical and Food Technology, Slovak University ofTechnology in Bratislava, Slovak Republic for her help in EPRstudies.

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