Abstract: Peltophorum pterocarpum, a large tree of wide occurrence, posses varied medicinal properties. In the present study, its flowers were successively extracted with pet. ether, dichloromethane, ethyl acetate and methanol and screened for their potential antimicrobial and antioxidant efficacies. In antibacterial screening, ethyl acetate fraction exhibited maximum activity against S. aureus and E. aerogenes (IZ 16.00±0.57 and 15.33±0.32 mm, respectively), while dichloromethane fraction was found active against R. planticola and E. aerogenes (IZ 15.66±1.19, 14.66±0.66 mm, respectively). In antifungal screening, ethyl acetate fraction showed appreciable activity against T. rubrum and P. crysogenum (IZ 15.00±0.00 mm). The 6.5 μg mL-1) as compared to the standard (quercetin). From the results it is evident that the flowers of P. pterocarpum. However, in antioxidant screening, methanol fraction showed highest activity (RC50 possess very potent antimicrobial and antioxidant efficacies.
INTRODUCTION
Peltophorum pterocarpum (DC.) Baker ex. K. Heyne (Vern. name: Peela Gulmohar; Fam. Caesalpiniaceae) is a deciduous tree growing up to 25-25 m. The bark of tree is used in dysentery, for gargles, tooth powders and externally in eye diseases, muscular pains and sores as lotions (Deshaprabhu, 1966). The bark also gives a dye of a yellow color. From its flowers crystalline compound, peltophorin which was later called as berginin, β-sitosterol, lupeol and a flavonoid glycoside naringenin-7-glucoside (Rao, 1965; Joshi and Kamat, 1969; Rahman et al., 1969; Varshney and Dubey, 1969) have been isolated. Antimicrobial activity of benzene and methanol fractions of flowers has been conducted against several Gram-ve and Gram+ve bacteria, where significant results have been demonstrated (Sethuraman et al., 1984; Duraipandiyan et al., 2006). Likewise, whole plant extracts have been tested against other microbes (Ali et al., 2001; Rahman et al., 2007; Satish et al., 2007, 2008; Voravuthikunchai and Mitchell, 2008). However, in the present study antimicrobial activity against some select bacteria and fungi including its antioxidant activity has been carried out.
MATERIALS AND METHODS
Plant material: During the course of studies, authenticated sample of Peltophorum pterocarpum (DC) Baker ex K. Heyne (Caesalpineaceae) were procured from Campus of University of Rajasthan, Jaipur and others in the month of July-September, 2008-09. Their voucher specimens were deposited in the Herbarium, Botany Department, University of Rajasthan, Jaipur.
Preparation of test extracts: Flowers were shade-dried, crushed and Soxhlet extracted in succsession with pet. ether (A), dichloromethane (B), ethyl acetate (C) and methanol (D) for 72 h. These extracts were filtered, concentrated to dryness in vacuo and stored at appropriate temperature for further studies. The yield (%) of each fraction (A-D) was found to 2.45, 0.80, 0.78 and 6.50, respectively.
Antimicrobial efficacy
Bioassays: Pure cultures of test bacteria, Bacillus subtilis (MTCC
441), Enterobacter aerogenes (MTCC 111), Escherichia coli (ATCC
443), Pseudomonas aeruginosa (ATCC 741), Raoultella planticola (MTCC
530) and Staphylococcus aureus (ATCC 740) were obtained from the IMTECH,
Chandigarh, India. These cultures were grown and maintained on Nutrient broth
medium (NB) at 27°C for 48 h. In fungus, Aspergillus flavus (16870)
A. niger (ATCC 322), Candida albicans (ATCC 4718), Penicillium
crysogenum (ATCC 5476) and Tricophyton rubrum (ATCC 2327)
obtained from IARI, New Delhi, India, were cultured on Sabouraud’s dextrose
agar (SDA) at 37°C for 48 h. Antimicrobial tests were performed by agar
well diffusion method (Boyanava et al., 2005).
Inoculum of each bacterial in nutrient broth and fungi on SDA broth were prepared
at 25°C for the period of 36 h for the adjustment of appropriate concentration
(106-107 cfu mL-1). Twenty microlitter of bacterial
inoculum and 80 μL of fungal inoculum are inoculated in Müller-Hinton
and SDA, respectively. Four milligram extract was delivered to each well. To
ensure diffusion of sample into agar, plates were incubated at 4°C for 1
h, which were then incubated at 37°C for bacteria and 25°C in fungi
for appropriate time periods under aerobic conditions. The diameter of the inhibition
zone around each hole was measured and recorded (Inhibition zone recorder, HiMedia,
India). Three replicates were used and the average value was statistically analyzed.
Gentamycin (10 mcg disc-1) and ketonocozole (10 mcg disc-1)
were used as positive controls.
Total phenolic contents: The total phenolic contents were determined using Folin-Ciocalteau reagent (Maehly and Chance, 1954). Optical Density (OD) was measured at 750 nm (Pharmaspec UV- Vis spectrophotometer by Shimadzu). A standard calibration curve of gallic acid (10-500 mg L-1) was prepared and total phenolics in the extract were expressed in mg of gallic acid equivalents (mg GAE/100 g of extract). All determinations were carried out in triplicate and statistically analyzed.
Total flavonoids contents: Total flavonoids were estimated by AlCl3 spectrophotometric method (Zhishen et al., 1999). Standard curve of quercetin (10-100 mg mL-1) was prepared. The total flavonoids were expressed as mg of quercetin equivalents (mg QE/g) of extract and statistically analyzed.
Antioxidant activity
Free radical scavenging activity by 2, 2-diphenyl-1-picryl-hydrazyl:
The effect on DPPH radical was determined using the method by Fogliano
et al. (1999). Absorbance was measured at 517 nm using a UV-Vis spectrophotometer.
The capability to scavenge the DPPH radical was calculated using following equation:
where, A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of given extract.
Total reduction capability by FRAP method: Ferric ion Reducing Antioxidant Potential (FRAP) of extracts was determined according to Yen and Chen (1995). The absorbance was measured at 700 nm using a UV-Vis spectrophotometer. Higher absorbance of the reaction mixture indicated greater reducing power. Standard calibration curve of ascorbic acid was prepared using 10-500 mg L-1 and total antioxidant potentials were calculated in mg of ascorbic acid equivalents (mg AAE/g of extract). All determinations were carried out in triplicate and statistically analyzed.
Metal chelating activity on ferrous ions: Ferrous ion (Fe+2) chelation was estimated by the ferrozine assay (Dinis et al., 1994). Optical density was measured at 562 nm. All tests were run in triplicate and averaged. The % inhibition of ferrozine Fe+2 complex formations was calculated as follows:
where, A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of given extract. The control contained FeCl2 and ferrozine complex molecules.
RESULTS
Total yield of crude extracts was found to be variable. The extracts were prepared on the basis of polarity of different secondary metabolites. All the four extracts (pet. ether, dichloromethane, ethyl acetate and methanol) demonstrated appreciable activity against most of the test bacteria and fungi (Table 1). In antibacterial screening, the potential activity 16.00±0.57 and 15.33±0.32 mm (with MIC 125 μg mL-1 in both cases) was demonstrated by ethyl acetate extract at 4 mg/well concentration against R. planticola and S. aureus, respectively. Likewise, against fungi, pronounced activity 14.33±0.33, 15.00±0.00 and 15.00±0.00 mm was recorded in same extract against C. albicans, P. crysogenum and T. rubrum (MIC and 62.5 μg mL-1 in P. crysogenum and T. rubrum, respectively).
On investigation of total phenolics and flavonoids, methanol extract showed maximum levels of phenolics (74.66±0.32 mg GAE/100 g) while flavonoids were higher in ethyl acetate extract (29.61±0.43 mg QE/g) (Table 2).
It is evident that methanol extract was the most active among all the four extract. In DPPH method, % inhibition of methanol and ethyl acetate extracts were 4 and 6.5 μg mL-1, respectively (Table 3). Methanol extracts exhibited antioxidant activity in concentration dependent manner viz., 80 μg mL-1 showed 84.43% inhibition as compared to 94.71% as compared to quercetin. In FRAP method, maximum absorbance was demonstrated by methanol extract where 1000 μg concentration demonstrated 625.00±0.64 mg AAE (Table 2).
| Table 1: | Antibacterial activity of P. pterocarpum flowers |
| aTest samples 4 mg/well. Standard test drugs: Gentamycin for bacteria, ketonocozole for fungi (10 mcg disc-1).b IZ: Inhibition zone (in mm) including the diameter of well (6 mm). MIC: Minimum inhibitory concentration in μg mL-1. AId: Activity index = Inhibition zone of the sample/Inhibition zone of the standard. | |
In ferric thiocynate method, methanol extract exhibited maximum activity, i.e., % inhibition 90.74 (at 80 μg mL-1 concentration).
| Table 2: | Total phenolics, flavonoids and antioxidant activity by FRAP method |
| aGAE: Gallic acid equivalents, bQE: Quercetin equivalents, cAAE: Ascorbic acid equivalents | |
| Table 3: | Antioxidant activity by DPPH and ferrous ion chelating method |
| % Inhibition = 1-(Absorbance of the sample/Absorbance of the control)x100 | |
DISCUSSION
Most active antimicrobial activity was demonstrated by ethyl acetate extract having higher flavonoids content. Antibacterial activity of flavonoids has earlier been documented (Bylka et al., 2004; Cushnie and Lamb, 2005; Kosalec et al., 2008). In the present study antioxidant activity were better in methanol extract and demonstrated using DPPH method were DPPH accept free radical and become stable diamagnetic molecule that absorb maximum at 517 nm. Antioxidant molecule absorbs free radical and cause reduction in absorbance color changes purple to yellow (Chang et al., 2002), whereas, in ferrous ion chelating activity ferrozine quantitatively form complex with Fe+2. In the presence of antioxidant compound this complex gets disrupted and formation of red color is imparted (Yamaguchi et al., 2000). Several diseases such as coronary heart disease, atherosclerosis, cancer, AIDS and ageing are caused due to oxidative damage (Finkel and Holbrook, 2000). Antioxidants are compounds that slow or prevent oxidation of biomolecules they prevent the formation of free radical as well as react with active electrons by their scavenging effects (Buyukokuroglu et al., 2001). Phenolics and flavonoids are the compounds which are widely distributed throughout the plant system demonstrated variable biological activities including antioxidant antiinflammatory; anticarcinogenic and antibacterial activities (Gryglewski et al., 1987).
CONCLUSION
The results further established the appreciable antioxidant and antibacterial activities which are attributed to the higher level of total phenolics, as present in methanolic extract. Similar observations have also been recorded by earlier workers (King et al., 1972; Dutra et al., 2008; Shukla et al., 2009).
ACKNOWLEDGMENTS
Authors are thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for partial financial support.