Antioxidant Activity of Leaf Extract of Aegle marmelos

Introduction: Leaf extracts of Aegle marmelos are reported to have hypoglycemic and anticancer effects. While a lot of information is available about the antioxidant activity of fruit of A. marmelos not much information is available about the antioxidant activity of the leaf of A. marmelos. Objective: The primary objective if this study was to ascertain the antioxidant activity of leaf extract of A. marmelos (AME). Methods: AME was evaluated for total phenolic content (TPC) and total flavonoid content (TFC) by Folin-Ciocalteau reagent method and by aluminium chloride method, respectively. Antioxidant activity of AME was assessed by FRAP assay, DPPH assay, ABTS cation scavenging activity and by reducing power determination. Results: High levels of TPC and TFC were found in AME which showed antioxidant activity comparable to vitamin C. Significant correlation between TPC, TFC and antioxidant activity of AME was found when Pearson’s correlation is applied. Conclusion: This study proved that the leaves of A. marmelos have high antioxidant component.


INTRODUCTION
Aegle marmelos is a medium size perennial tree of Rutaceae family commonly known as Bael or Bilwa, grows in tropical and subtropical region of South East Asia and India.All parts of the tree are used as herbal medicine in Ayurved, Unani and Siddha systems of medicine for the treatment of various ailments like dysentery, dyspepsia, chronic diarrhea. 1,2Soft drinks prepared from the fruit pulp are used in India to keep the body cool during summers.Aqueous extract of bael fruit has shown hypoglycemic effects in streptozotocin induced diabetes in rats when administered orally and intra peritoneally. 3Leaf extracts of A. marmelos are reported to have cardiotonic, hypoglycemic, antidyslipidemic, anticancer effects and also used to cure opthalmia, ulcers, dropsy, cholera and beri beri.][6] Free radicals are generated in the body due to oxidative stress developed during normal metabolic processes or upon exposure to various environmental or chemical factors which subsequently damage the macromolecules like DNA, proteins and lipids.Oxidative stress is one of the contributing factors of various diseases like cancer and diabetes. 7atural products have been reported to have stores of large amount of antioxidants other than Vitamin C, E and carotenoids.Antioxidants delay or prevent free radical catalyzed reactions.Various phenolic compounds and flavonoids are responsible for the antioxidant activity of medicinal plants. 8,9Therefore, it was thought worthwhile to evaluate antioxidant activity of A. marmelos leaf extract.In the present investigation, antioxidant activity of A. marmelos leaf extract was assessed using various methods.

Plant material
The leaves of A. marmelos were collected from New Delhi India during the month of December.The leaves were shade dried for 20-25 days.The plant material was authenticated by Dr. Sunita Garg, Chief Scientist, Raw Materials Herbarium & Museum, CSIR-NISCAIR, New Delhi, India and specimen was deposited in Raw Materials Herbarium & Museum, CSIR-NISCAIR, New Delhi, India.

Preparation of Extracts
The air dried leaves were powdered (about 350 gm) and extracted with 50% ethanol using Soxhlet apparatus.The extract was filtered through Whatmann filter paper and the filtrate was concentrated in Rotary evaporator.Dried extract was stored at 4°C and methanol soluble portion (AME) is used for further studies.The percentage yield of the extract was 20%.

Quantitative phytochemical analysis Total phenolic content (TPC)
Total phenols were determined by Folin Ciocalteu reagent. 102 mL of the 1 mg/mL of AME was mixed with 2 ml of Folin-Ciocalteu reagent, followed by addition of 2 mL of 7.5%, w/v solution of sodium carbonate.The mixture was stirred and measured at 765 nm after keeping in the dark for 30 min.A blank sample consisting of methanol and reagents was used as a reference.A sample color blank was examined using the AME and water to exclude the color interference of the AME.The results were expressed as mg of gallic acid equivalents per 100 g of dry weight (mg GAE/100 g DW), utilizing a calibration curve of gallic acid in a concentration range of 10-60 µg/mL.

Total flavonoid content (TFC)
Aluminium chloride colorimetric method was used for flavonoids determination. 111 mL of 1mg/mL AME was mixed with 4 mL of distilled water and 0.3 mL of 5% solution of sodium nitrite.After 5 min, 0.3 mL of 10% solution of aluminium chloride was added and followed by addition of 2 mL of 1 M sodium hydroxide.The entire mixture was immediately diluted to 10 mL with distilled water.The mixture was mixed thoroughly and read at 510 nm against a blank.A calibration curve was plotted by using serial dilutions of quercetin solution at concentrations 20 to 100 mg/mL in methanol.The results were ex-Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 pressed as mg of quercetin equivalent per 100 g of dry weight (mg QE/100 g DW).

Antioxidant activity FRAP assay
The FRAP assay was determined by modified method of Müller et al.  (2011). 12The working FRAP solution was freshly prepared by mixing 300 mM acetate buffer of pH 3.6,10 mM TPTZ (2,4,6-Tris(2-pyridyl)-striazine ) solution in 40 mM HCl and 20 mM ferric chloride solution in a ratio of 10:1:1.The working solution was warmed at 37°C before use.An aliquot of 100 µL of 1mg/mL of AME was mixed with 3 mL FRAP solution.After 4 min of incubation at 37°C, the absorbance was read at 593 nm.A calibration curve was made by ferrous sulphate and results were expressed as mM Fe 2+ per g dried weight (mM Fe 2+ /g DW) from four determinations.
DPPH radical scavenging activity DPPH radical scavenging activity was conducted according to the method described by Gülçin (2006), with some modifications. 13An aliquot of 4 mL 0.1 mM DPPH radical in buffered methanol was mixed with 2 mL AME at different concentrations or methanol as negative control.The mixture was kept in dark for 30 min and then read at 517 nm with a blank contain only DPPH solution and methanol.The sample colour blank was examined containing AME and methanol to exclude the colour interference of sample extract.The DPPH radical-scavenging activity (%) was calculated using Eq.( 1). ( Where, A sample is the absorbance in the presence of extracts, and A control is the absorbance of the control. 13TS radical cation scavenging activity ABTS radical cation scavenging activity was assayed according to method of Re et al. (1999), with minor variation.14 The ABTS + solution was prepared by reacting 7 mM ABTS solution and 2.45 mM potassium persulfate (final concentration) in the dark for at least 16 h at room temperature.The solution was then diluted 65 times with phosphate buffer of pH 7.4 to an absorbance of 0.759 at 728 nm as the working solution.An aliquot of 5 mL working solution was mixed with 50 µL of AME at different concentrations and allowed to react at 30°C for 20 min.A blank containing ABTS + working solution and ethanol was detected.The absorbance was measured at 734 nm.The radical scavenging activity of the samples was calculated using Eq. ( 1).

Reducing power
The reducing power of AME was determined by the method of Zhang et al. (2009), with minor modifications. 15Briefly, an aliquot of 1 mL AME at various concentrations was mixed with 1 mL of 1% w/v potassium ferricyanide in 0.2M PBS of pH 6.6.The mixture was incubated at 50°C for 20 min and then added to 1 mL of 10% w/v trichloroacetic acid.This 3 mL mixture is diluted with equal amount of distilled water and the absorbance was measured at 710 nm after 30 min. of adding 200 µL of 0.1% w/v ferric chloride.The blank contained all reagents except the sample extract.

Statistical analysis
All data were expressed as Mean±SD.The correlations between phytochemical contents and antioxidant activities were statistically evalu-  Scavenging activity (%) = (A control -A sample ) × 100 (A control ) ated by two tailed bivariate correlate analysis, and were indicated by Pearson's coefficient indexes and p<0.05 was considered as statistically significant.

RESULTS AND DISCUSSION
TPC and TFC are used as indicators of overall antioxidant activity of the herbal drugs. 16Poly phenols have been recognized to show medicinal properties and exhibit physiological activity. 7The TPC of AME was tested by Folin-Ciocalteu method with gallic acid as standard.The TPC of the AME was found to be 1118.12±79.19mg GAE/100 g of dried material.The TPC over 500 mg GAE/100 g is considered as a high category antioxidant activity.Flavonoids are hydroxyls containing secondary metabolites present in plants and exhibit free radical scavenging activities. 17In this study, TFC of AME was found to be 914.67±118.51mg of QE/100 g of dried material.The results were comparable with the reported results of TPC of the fruit pulp of A. marmelos. 3ntioxidant activity of AME was evaluated by different detection methods.Primary antioxidants have been shown to react against the oxidative stress by breaking chain reaction or by scavenging free radicals.While, secondary antioxidants deactivate metals, inhibit the breakdown of lipid peroxides, regenerate primary antioxidants, and quench singlet oxygen. 18herefore, various chemical based assay methods have been developed and attuned for the detection of antioxidant activities. 19,14RAP assay quantitate the ability of an antioxidant to reduce the Fe 3+ / tripyridyl-s-triazine complex. 12As shown in Table 1, AME had FRAP values of over 147 mM Fe 2+ /g DW.The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity.The DPPH and ABTS method focus on the non-specific radicals existed in the reaction system.The DPPH method is based on the reduction of DPPH• into diphenyl picryl hydrazine in the presence of a hydrogen donating antioxidant.AME reduces the colour of DPPH due to its hydrogen donating ability. 7The IC 50 values of DPPH and ABTS radical scavenging activities were 160.47 ± 8.51µg/mL and 282.46 ± 44.11µg/mL, respectively as shown in Table 1.The total reducing power serves as a significant indicator of the potential antioxidant capacity, and is expressed as the increased absorbance of the reaction mixture at 710 nm. 15 Figure 1 shows the reduction of Fe 3+ to Fe 2+ in the presence of AME.The IC 50 value is defined as the effective concentration at which the absorbance was 0.5.As displayed in the Figure 1, the absorbance of the Vitamin C dramatically increased in a dose dependent-manner while AME also reduce Fe 3+ in a dose dependent manner.The TPC and TFC of herbal drugs have been proved to be positively correlated with their antioxidant activities. 16,17Table 2 lists the Pearson's coefficients between TPC, TFC and various antioxidant capacities.The TPC showed a high positive correlation with TFC, FRAP, DPPH, ABTS cation scavenging and reducing power (r 2 =0.9808 ** , 0.9556 * , 0.9365 * , 0.9207 * and 0.9273 * , respectively).The correlation coefficients of the TFC to the FRAP, DPPH, ABTS cation scavenging and reducing power were 0.9945 ** , 0.9286 * , 0.9376 * and 0.9801 * , respectively.The antioxidant constituents provide a fillip to the antioxidant defense mechanism in cancer and increase the uptake of glucose by muscles by modulating lipid peroxidation, scavenging free radicals and reducing the generation of reactive oxygen species (ROS). 3,6

CONCLUSION
The antioxidative effect of the AME is ascribed to the free radical scavenging property due to the presence of phenolics and flavonoids.Hence, the hypoglycemic and anticancer effect of leaves of A. marmelos could be credited to its antioxidant property.Further studies are required to identify the specific active principles of the plant for this significant antioxidant effect.

Figure 1 :
Figure 1: Reducing power of AME and vitamin C.