Evaluation of antioxidant potential and antimicrobial studies of bark of medicinal plant , Mallotus tetracoccus ( Roxb . )

The present study was aimed to investigate the total phenols, flavonoids, carotenoids, antioxidant activity and antimicrobial activity of the bark extract of Mallotus tetracoccus (Roxb.) Kurz. Total phenols, flavonoids and carotenoids in the extract were found to be 55.35 ± 0.13 mg GAE, 260.3053 ± 1.413 mg QE and 0.182 ± 0.005 mg/g, respectively. The reducing power and metal chelating activity of the bark extract was said to be increasing with increasing concentration. The metal chelating activity (IC50) of the M. tetracoccus bark extract was found to be 647.4 ± 0.321 μg/ml, which was higher compared to the standard, ethylenediaminetetraacetic acid (EDTA; 1.89 ± 0.03 μg/ml). Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods. The antioxidant activity (IC50) of the bark extract was said to be 0.504 ± 0.002 μg/ml by (DPPH) method. The total antioxidant activity of M. tetracoccus ethanolic bark extract was found to be 275.21 ± 0.21 mg equivalent of ascorbic acid/100 g of plant extract as determined by phosphomolybdenum method. The antibacterial studies of the ethanolic bark extract was also tested at different concentration of extracts, where 250 mg/ml concentration of extract showed good inhibitory activity against all the clinically important disease pathogens compared with standard antibiotics like streptomycin and penicillin. The highest inhibition was noted in order of Klebsiella pneumonia (25.33 ± 0.37 mm), Staphylococcus aureus (22.83 ± 0.31 mm), Vibrio cholerae (20.00 ± 0.31 mm) and Escherichia coli (20.22 ± 0.26 mm). Thus, the bark extract of M. tetracoccus was said to possess significant antioxidant and antimicrobial activity, which suggests its use in traditional medicine.

Although researches have reported on M. tetracoccus (Roxb.) Kurz. leaves, a literature search revealed no references to investigation on its bark. Thus, the objective of the study was to study the antioxidant and antimicrobial property of M. tetracoccus bark extract.

Collection of plant material
The fresh bark of M. tetracoccus (Roxb.) Kurz. were collected from the Agasthiar Malai Reserved Forest, Western Ghats, South India and authenticated by the Director, Centre for Biodiversity and Forest Studies, Madurai Kamaraj University. The voucher specimens were deposited in the herbarium of Centre for Biodiversity and Forest Studies of our university (No.AM-07).

Preparation of extract
Fresh barks were shade dried, powdered and extracted with ethanol for 6 to 8 h using soxhlet apparatus, at a temperature of 80°C. The extract was then filtered through muslin, evaporated under reduced pressure and vacuum dried to get the viscous residue. The ethanolic extract of the bark was used for antioxidants and antimicrobial studies.

Determination of total phenols and flavonoids
The total phenolic content was determined according to the method described by Siddhuraju and Becker (2003). Aliquots of each extracts were taken in test tubes and made up to the volume of 1 ml with distilled water. Then 0.5 ml of Folin-ciocalteu phenol reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube. Soon after vortexing the reaction mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725 nm against the reagent blank. Total phenol contents were expressed in terms of gallic acid equivalent (mg/g of dry mass), which is used as a reference compound.
Flavonoid content was determined according to the aluminium chloride colorimetric method by Chang et al. (2002) with some modifications. Quercetin was used as a standard to make the calibration curve. The sample solution (0.5 ml) was mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminium chloride hexahydrate, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 40 min, the absorbance of the reaction mixture was measured at 415 nm. The same amount (0.1 ml) of distilled water substituted for the amount of 10% aluminium chloride as the blank and a seven point standard curve (0 to 500 µg/ml) was obtained.

Determination of carotenoids
Total carotenoids were determined by the method of Jensen (1978). One gram sample was extracted with 100 ml of 80% methanol solution and centrifuged at 4000 rpm for 30 min. The supernatant was concentrated to dryness. The residue was dissolved in 15 ml of diethyl ether and after addition of 15 ml of 10% methanolic KOH, the mixture was washed with 5% ice-cold saline water to remove alkali. The free ether extract was dried over anhydrous sodium sulphate for 2 h. The ether extracts were filtered and its absorbance was measured at 450 nm using ether as blank.

Antioxidant assay
The antioxidant activity of the plant extracts was tested using two methods: ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods.

Ferric thiocyanate (FTC) method
The standard method described by Kikuzaki and Nakatani (1993) was used. A mixture of 4.0 mg plant extract in 4 ml absolute ethanol, 4.1 ml of 2.5% linolenic acid in absolute ethanol, 8.0 ml of 0.05 M phosphate buffer (pH 7.0) and 3.9 ml of water was placed in a vial with a screw cap and then placed in an oven at 40°C in the dark. To 0.1 ml of this solution was added 9.7 ml of 75% ethanol and 0.1 ml of 30% ammonium thiocyanate. Precisely 3 min after addition of 0.1 ml of 0.02 M ferrous chloride in 3.5% HCl to the reaction mixture, the absorbance of red colour was measured at 500 nm each 24 h until the day after absorbance of control reached maximum. Beta hydroxyl toluene (BHT) and α-tocopherol were used as positive controls, while the mixture without plant sample was used as the negative control.

Thiobarbituric acid (TBA) method
The method of Ottolenghi (1959) was referred. Briefly, Two ml of 20% trichloroacetic acid and 2 ml of 0.67% 2-thiobarbituric acid was added to 1 ml of sample solution, as prepared in FTC method. The

Determination of Phyto-constituent
Mallotus tetracoccus bark extract Total phenols 55.35 ± 0.13 mg GAE Flavonoids 260.3053 ± 1.413 mg QE Carotenoids 0.182 ± 0.005 mg/g mixture was placed in a boiling water bath and after cooling was centrifuged at 3000 rpm for 20 min. Absorbance of supernatant was measured at 552 nm. Antioxidant activity was based on the absorbance on the final day of FTC method.

DPPH free radical scavenging activity
The free radical scavenging activity of the extracts, based on the scavenging activity of the stable 2,2-diphenyl-2-picrylhydrazyl (DPPH) free radical was determined by the method described by Braca et al. (2002). Plant extract (0.1 ml) was added to 3 ml of a 0.004% methanol solution of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage inhibition activity was calculated as [(A0-A1)/A0] × 100, Where A0 is the absorbance of the control and A1 is the absorbance of the extract/ standard. A blank is the absorbance of the control reaction (containing all reagents except the test compound). A percent inhibition versus concentration curve was plotted and the concentration of sample required for 50% inhibition was determined and represented as IC50 value for each of the test solutions.

Reducing power assay
Aliquots of each extracts were taken in test tubes and dissolved in 1 ml of 0.2 M phosphate buffer in a test tube to which was added 5 ml of 0.1% solution of potassium ferric cyanide (Siddhuraju et al., 2002). The mixture was incubated 50°C for 20 min. Subsequently, 5 ml of trichloroacetic acid (10%) (w/v) solution was added and the mixture was then centrifuged at 7000 rpm for 10 min. A 5 ml of aliquot of the upper layer was combined with 5 ml of distilled water and 1 ml of ferric chloride solution (0.1%) and absorbance was recorded at 700 nm against reagent blank. A higher absorbance of the reaction mixture indicates greater reducing power of the sample.

Metal chelating activity
The chelating of ferrous ions by M. tetracoccus (Roxb) Kurz. ethanolic bark extract was estimated by the method of Dinis et al. (1994). Briefly, the extract samples (250 µL) were added to a solution of 2 mmol/L FeCl2 (0.05 ml). The reaction was initiated by the addition of 5 mmol/L ferrozine (0.2 ml) and the mixture was shaken vigorously and left standing at room temperature for 10 min, after which the absorbance was measured spectrophotometrically at 562 nm. The chelating activity of the extracts was evaluated using EDTA as standard. The metal chelating activity of the extract is expressed as mg EDTA equivalent/g extract.

Phosphomolybdenum activity
The antioxidant activity of samples was evaluated by the green phosphomolybdenum complex formation according to the method of Prieto et al., 1999. An aliquot of 100 µl of sample solution was combined with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in a 4 ml vial. The vials were canned and incubated in a water bath at 95°C for 90 min. After the samples have cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank.
The results were reported (ascorbic acid equivalent antioxidant activity) as mean values expressed in g of ascorbic acid equivalents/100g extract.

Test organisms
The ethanolic bark extract of M. tetracoccus (Roxb.) Kurz. was screened against four bacterial pathogens. The clinical isolates used for study are Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Vibrio cholerae.

Antibacterial Screening
The different concentrations of the bark extracts (50, 100 and 250 mg/L) were tested for antibacterial activity using agar disc diffusion assay according to the method of Bauer et al. (1966). The strains of microorganisms obtained were inoculated in conical flask containing 100 ml of nutrient broth. These conical flasks were incubated at 37°C for 24 h and were referred to as seeded broth. Media were prepared using Muller Hinton Agar (Himedia, Mumbai, India), poured on Petri dishes and inoculated with the test organisms from the seeded broth using cotton swabs. Sterile discs of 6 ml width had been impregnated with 20 µL of test extract and introduced onto the upper layer of the seeded agar plate. The plates were incubated overnight at 37°C. Antibacterial activity was assigned by measuring the inhibition zone formed around the discs. The experiment was done three times and the mean values were presented. Streptomycin (10 µg/disc) and penicillin (10 µg/disc) were used as standards.

Determination of total phenols and flavonoids
The percentage yields of the ethanolic bark extract of M. tetracoccus (Roxb.) Kurz. were found to be 14.89%. Total phenolic content of the ethanolic extract of M. tetracoccus (Roxb.) Kurz. bark extract was 55.35 ± 0.13 mg gallic acid equivalent per gram of plant extract (Table 1). The flavonoid contents of the M. tetracoccus ethanolic bark extract was 260.305 ± 1.413 mg Quercetin equivalent per gram of plant extract (Table 1).
While the carotenoids contents of the M. tetracoccus ethanolic bark extract were found to be 0.182 ± 0.005 mg/g (Table 1).

Ferric thiocyanate (FTC) method
As shown in Figure 1, the absorbance of the M. tetracoccus bark extract and beta hydroxyl toluene (BHT) at day 6 and 7 was significantly (P < 0.05) decreased when compared with the absorbance of standard (α-tocopherol) at day 6 and 7. The control had the highest value throughout the study. standard BHT, where significantly higher than the αtocopherol.

DPPH free radical scavenging activity
The radical scavenging activity of the M. tetracoccus ethanolic bark extract was tested using stable free radical DPPH (deep purple colour), as DPPH has the advantage of being unaffected by certain side reactions. Figure 3 shows the DPPH radical scavenging activity of M. tetracoccus with ascorbic acid as reference, where the IC 50 values for the M. tetracoccus ethanolic bark extract (0.504 ± 0.002 µg/ml) which was said to be significant than the standard ascorbic acid (IC 50 = 30.31 ± 0.01 µg/ml). Figure 4 shows the reducing power of the M. tetracoccus ethanolic bark extract using potassium ferricyanide reduction method. The absorbance value of the extract shows higher increase with increase in concentration, when compared to standard ascorbic acid. Figure 5 shows the metal chelating activity of the M. tetracoccus bark extract, where IC 50 of plant extract was said to be 647.4 ± 0.321 µg/ml, which was higher compared to the standard, EDTA (1.89 ± 0.03 µg/ml).

Phosphomolybdenum activity
The total antioxidant activity of M. tetracoccus (Roxb.) Kurz. ethanolic bark extract was found to be 275.21 ± 0.21 mg equivalent of ascorbic acid/100g of plant extract as determined by phosphomolybdenum method.

DISCUSSION
The phenolic compounds present in natural products have higher antioxidant activity than synthetic antioxidants, also by acting as free radical terminators (Lu and Foo, 2000). The total phenolic content of the stem bark of Gmelina arborea was 356 ± 1.4 mg g -1 (Patil et al., 2009). The bark extract of Acacia confusa showed total phenolic content of 470.6 mg/g, where the antioxidant activity by DPPH possessed IC 50 values ranging from 1 to 5 µg/ml (Chang et al., 2001). Chamaecyparis obtusa various fractions of bark extract was also evaluated for antioxidant activity, and the total phenolic content varied from 27.71 to 102.86 mg GAE g −1 dry weight for fractions (Marimuthu et al., 2008). The flavonoids possess antioxidant activity acting through scavenging or chelating process, thereby having considerable effect on human health and nutrition (Kessler et al., 2003). Since the bark extract of M. tetracoccus showed higher amount of phenolic compounds, this suggests their usage as a good source of natural antioxidant, preventing free radical-mediated oxidative damage.

Ferric thiocyanate (FTC) method
The ferric thiocyanate (FTC) results showed that M. tetracoccus bark extract had greater antioxidant activity when compared with α-tocopherol. The absorbance values for all the samples were maximal on the 6th day and then dropped at the 7th day due to malondialdehyde (MDA) compounds from linoleic acid oxidation, in which peroxide reacts with ferrous chloride to form a reddish ferric chloride pigment. The control had increasing absorbance values from 0 day until the absorbance reached the maximal level at 6 day and the absorbance value dropped at 7 day.
The reaction in the FTC method is due to the MDA compounds from the linoleic acid oxidation in which peroxide reacts with ferrous chloride to form a reddish ferric chloride pigment (Al-Naqeeb et al., 2009). Peroxides are slowly decayed to lower molecular compounds during the oxidation course (Behbahani et al., 2007). The bark and leaf extracts of M. germanica L. also exhibited higher antioxidant activity in the ferric thiocyanate (FTC) method showing higher peroxidation inhibition than vitamin C and BHA at the 96th hour (p < 0.05) (Nabavi et al., 2011).

Thiobarbituric acid (TBA) method
The TBA results were in agreement with the FTC results. After sometime, malonaldehyde a secondary product of lipid peroxidation changes into alcohol and acid. The alcohol and acid cannot be detected with a spectrophotometer (Rahmat et al., 2003). The FTC method indicates the amount of peroxide in the initial stages of lipid peroxidation (Saha et al., 2004;Rahmat et al., 2003), whereas the thiobarbituric acid method shows the amount of peroxide in the secondary stage of lipid peroxidation (Rahmat et al., 2003). Therefore, the higher antioxidant activity found from the ferric thiocyanate method indicated that the amount of peroxide in the initial stage of lipid peroxidation was greater than the amount of peroxide in the secondary stage (Rezaeizadeh et al., 2011).

DPPH free radical scavenging activity
The antioxidants present in the extract quenches the DPPH free radicals (by providing hydrogen atom or by electron transfer, conceivably via a free radical attack on the DPPH molecule) and convert them to a colourless product (2, 2-diphenyl-1-picrylhydrazyl, or a substituted analogous hydrazine), resulting in a decreasing absorbance at the 517 nm (Yamaguchi et al., 2002). The antioxidative effect of the bark extract in these studies may be due to the presence of thiocyanic acid, furfural and 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl compounds (unpublished results). In addition, antioxidative activities observed in the extracts could be the synergistic effect of more than two compounds that may be present in the plant. It has been reported that most natural antioxidative compounds often work synergistically with each other to produce a broad spectrum of antioxidative activities that creates an effective defence system against free radical attack (Lu and Foo, 1995). Stem bark extract of M. germanica L. (aqueous and methanol) showed best activity in 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity with IC 50 = 10.7 ± 0.6 and 11.4 ± 0.8 µgml -1 , respectively (Nabavi et al., 2011). The alcoholic extract of stem bark of Terminalia arjuna showed potent antioxidant activity with EC 50 value of 2.491 ± 0.160 (Viswanatha et al., 2010). Stem bark of Gmelina arborea studied for antioxidant activity also possessed IC50 values of extract and standard were 46.2 ± 1.2 and 28.80 ± 0.50, respectively (Patil et al., 2009). Ethyl acetate extract of Tectona grandis bark showed very high activity with 98.6% inhibition against DPPH and ABTS+ free radicals (Krishna et al., 2010). Moreover, the Ethyl acetate soluble fraction of Chamaecyparis obtusa bark was found to be the best antioxidant-rich fraction in terms of DPPH (0.15 to 0.26 mmol L −1 Trolox equivalents) and reducing power assays (Marimuthu et al., 2008).

Reducing power assay
The yellow colour of test solution changes to various shades of green and blue due to the reduction of Fe 3+ / Ferric cyanide complex to ferrous form by the antioxidants present in the extract. Thus, the reducing power of medicinal plants and vegetables are said to be well associated with the antioxidant activity and its phenolic constituents (Siddhuraju and Becker, 2003). The leaves and bark extracts of M. germanica L. also showed good reducing power than fruit extract (Nabavi et al., 2011(Nabavi et al., , 2003.

Metal chelating activity
In this metal chelating activity, the presence of chelating agents in the extract of M. tetracoccus disrupts the ferrozine-Fe 2+ complex formation, thus decreasing the red colour. It is reported that chelating agents are effective as secondary antioxidants as they stabilise the oxidised form of the metal ion by reducing the redox potential (Gardner et al., 2000). Iron can generate free radicals from peroxides and may be implicated in human cardiovascular disease (Halliwell and Gutteridge, 1990). Bivalent transition metal ions play an important role as catalysts of oxidative processes, leading to the formation of hydroxyl radicals and hydroperoxide decomposition reactions via Fenton chemistry (Halliwell, 1997). These processes are delayed by iron chelators, which mobilize tissue iron by forming soluble and stable complexes (Nabavi et al., 2011). Ferrozine can quantitatively form complexes with Fe 2+ . In the presence of other chelating agents, the ferrozine complex formation is disrupted, resulting in a decrease in the red color of the complexes (Ebrahimzadeh et al., 2008).

Phosphomolybdenum activity
This method is based on the formation of green phosphomolybdenum complex at 95°C measured at an intensity of absorbance at 695 nm. In this method, reduction of Mo (VI) to Mo (V) by the antioxidant compounds present in the plant extract, forming green phosphate/Mo (V) complex takes place (Sowndhararajan et al., 2010).

Antibacterial screening
The different fractions of methanolic, ethanolic and aqueous extracts of stem bark of Acacia leucophloea was studied for their antimicrobial activity against different bacterial strains and fungal strains. Methanol extract exhibited a broad spectrum activity on all strains also high antioxidant activity due to the presence of various phytochemicals (Anjaneyalu et al., 2010). The isolated compounds of stem bark of Garcinia malaccensis, α-and β-mangostin and cycloart-24-en-3β-ol showed high cytotoxic, antioxidant and antibacterial activity (Taher et al., 2012). In addition, the aqueous and alcoholic extract of stem bark of Cassia fistula was tested for their antibacterial activity. Alcoholic extract (7.0 to 12.0 mm) showed greater inhibition against S. aureus compared to aqueous extract (7.0 to 11.6 mm) (Vimalraj et al., 2009). The stem bark Manilkara zapota was also evaluated for in vivo antitumor activity against EAC in Swiss albino mice, and the ethyl acetate fraction was said to possess significant antitumor activity (Osman et al., 2011).
Furthermore, the bark extracts of Bauhinia purpurea were phytochemically analyzed and evaluated for antimicrobial and antioxidant activities. Among the different solvent extracts, aqueous and methanolic extract exhibited a broad spectrum of antimicrobial (antibacterial and antifungal) activity and 80 to 90% antioxidant activity in terms of DPPH radical scavenging activity (Avinash et al., 2011). The antimicrobial and phytotoxic activities of the crude methanolic extract and its subsequent solvent fractions of Pistacia integerrima bark were investigated. The outstanding activity was shown by the ethyl acetate fraction followed by aqueous fraction against S. aureus having zone of inhibition of 19 and 15 mm, respectively (Rahman et al., 2011). The antibacterial activity of all extracts from Tectona grandis (Teak) were also checked against important hospital strains by disc diffusion assay, where chloroform extract showed inhibition to growth of S. aureus (14 mm) and K. pneumoniae (8 mm), and also a high cytotoxicity against chick embryo fibroblast (CEF) and human embryonic kidney (HEK 293) cells with 87 and 95.3% inhibition, respectively (Krishna et al., 2010).

Conclusion
The present study indicates that the ethanolic bark of M. tetracoccus (Roxb.) Kurz. is high in phenolic and flavonoid content. Also, the results of scavenging activities observed against DPPH, reducing power, phosphomolybdenum activities, metal chelating shows that M. tetracoccus (Roxb.) Kurz. provide promising natural sources of antioxidants suitable for preventing free radical-mediated diseases. The extract also showed high antibacterial against important clinical bacterial isolates. Further studies are needed to explore the potential phenolics and flavonoid compounds from M. tetracoccus bark for application in drug delivery, nutritional or pharmaceutical fields.

ACKNOWLEDGEMENT
The author Ramalakshmi S is thankful to UGC for providing meritorious fellowship (UGC-BSR).