1. Introduction
Free radicals generated by increasing the cell oxidation contribute to many diseases like cardiovascular disease, tumor growth, wrinkled skin, cancer, Alzheimer’s disease and even a decline in energy and endurance [1] -[4] . Several free radicals such as superoxide anions, hydroxyl radicals and hydrogen peroxide are toxic to cell membrane present in the body organs [5] . Natural antioxidants provide a protection to living organisms from damage caused by reactive oxygen species and concomitant lipid peroxidation, protein damage and DNA strand breaking [6] . At the present time, the most commonly used antioxidants butylated hydroxyanisole (BHA) and butylated hydroxy toluene (BHT) which are suspected of being carcinogenic and causing liver damage. Therefore, there is a growing interest on natural and safer antioxidants [7] . Naturally occurring antioxidants such as ascorbic acid, vitamin E, phenols and flavonols possess the ability to inhibit the free radical generation associated with many diseases, including cardiovascular disease, cancer, diabetes, arthritis, immune deficiency diseases and aging [8] .
Crossandra infundibuliformis L. Leaf belongs to the family Acanthaceae, traditionally used in the treatment of migraine in Belgaum district, Karnataka, India. It is distributed in India, Bengal, Malaya, Sri Lanka, and Nepal. The plant has aphrodisiacal properties [9] and crushed plant material applied to a cattle is bitten by a dog [10] . The leaves of C. infundibuliformis reported for several activities including hepatoprotective [11] and antimicrobial properties were studied [12] . So far, no in vitro antioxidant study has been reported on C. infundibuliformis leave extracts. Hence, the present study is designed to give information and application for traditional use of C. infundibuliformis by conducting in vitro antioxidant assays of leave extracts.
2. Experimental
2.1. Plant Material
The leaves of C. infundibuliformis were collected from Raibag Taluk, Belgaum District, Karnataka, India. This plant was authenticated by Dr. Shiddamallayya N, Regional Research Institute Bangalore, where a voucher specimen has been preserved for future identification. Collected leaves were then washed with running water to remove the dirt, shade dried and ground to obtain course powder.
2.2. Chemicals
Folin-Ciocalteau reagent, ascorbic acid, gallic acid and DPPH were purchased from Sigma Chemicals, Germany. Potassium dihydrogen phosphate, potassium hydroxide, ferric chloride, ferrous sulphate, potassium ferricyanide, butylated hydroxy anisole (BHA) and thiobarbituric acid (TBA), trichloro acetic acid (TCA) were purchased from Merck (Mumbai, India). All the other solvents used in this study were of analytical grade and purchased from Sigma Aldrich, Bangalore.
2.3. Extraction
The powdered plant material (250 g) was extracted successively with 1500 ml each of petroleum ether (60˚C - 80˚C), chloroform, ethyl acetate and methanol in Soxhlet extractor for 18 - 20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature (40˚C - 50˚C) in rotary evaporator. The percentage yield of all the extracts was given in Table 1. The extracts were then stored at 4˚C in refrigerator for further analysis.
2.4. Determination of Total Phenol Content
The total phenol content of the extracts was measured by using Folin-Ciocalteau method with minor modifications [13] . Aliquots of the extracts 0.1 ml was mixed with 2.5 ml of Folin-Ciocalteau reagent (previously diluted with distilled water 1:10 v/v) and 2 ml (7.5%) of sodium carbonate. After incubation at 40˚C for 30 min, the absorbance of the reaction mixture was measured at 760 nm by using a spectrophotometer (Shimadzu UV-160 spectrophotometer). Gallic acid was used as a standard and total phenol content of the extracts was expressed in milligram gallic acid equivalents (mg GAE) per gram of extract. All determinations were carried out in triplicates.
Table 1. Percentage yield, total phenolic, flavonol and total antioxidant capacity of successive extracts of Crossandra infundibuliformis leaves.
aMean of three replicate determination ± SEM, standard error mean, bgallic acid, crutin, equivalent in mg/g of extract. dThe total antioxidant capacity expressed as millimolar equivalent to ascorbic acid. Values with different symbols in the same column were significantly different (p < 0.05) from each other.
2.5. Determination of Total Flavonol Content
Total flavonol content of extracts was determined by using the aluminium chloride method with some modifications [14] . Aliquots of the extracts 0.5 ml was mixed with 1.5 ml aluminium chloride (10%) and 0.1 ml potassium acetate (1 M). After incubation at room temperature for 30 min, 2.8 ml of distilled water was added. The absorbance of the reaction mixture was measured at 415 nm. Total flavonol content was expressed as milligram rutin equivalents per gram of extract. All determinations were carried out in triplicates.
2.6. In Vitro Antioxidant Activity
2.6.1. Total Antioxidant Capacity Assay
The total antioxidant activity of successive extracts was determined by phosphomolybdenum method [15] . An aliquot of 0.1 ml of extract or standard in DMSO was combined with 1 ml of reagent solution (sulphuric acid 0.6 M, sodium phosphate 28 mM and ammonium molybdate 4 mM) in an Eppendroff tube. The tubes were caped and incubated in water bath at 95˚C for 90 min. The blank solution contained 1 ml of reagent solution and 0.1 ml of methanol. After cooling to room temperature, absorbance was measured at 695 nm against a blank. The results were expressed as millimolar equivalents of ascorbic acid.
2.6.2. DPPH Radical Scavenging Assay
DPPH radical scavenging activity of the extracts was determined by minor modifications [16] . Different concentrations of extracts 1 ml was added to 1
ml of DPPH (0.2
mM in methanol). The reaction mixture incubated at 30˚C for 30
min. The control was prepared as above without any extract and methanol was used for the baseline correction. The changes in absorbance were measured at 517
nm using a spectrophotometer. All the measurements were done in triplicate.
2.6.3. Hydrogen Peroxide Scavenging Assay
The hydrogen peroxide scavenging assay was determined by the method prescribed Jaishree et al., (2008) [17] . A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer saline (PBS, pH 7.4). Various concentration of the extract or standard in methanol (1 ml) was added to 2 ml of hydrogen peroxide solution in PBS. After 10 min, the absorbance was measured at 230 nm.
2.6.4. Lipid Peroxidation Inhibitory Assay
Lipid peroxidation inhibitory activity was measured according to the modified method described by Jaishree et al., (2008) [17] . Lipid peroxidation was induced by adding ferric chloride 10 µl (400 mM) and Lascorbic acid 10 µl (400 mM) to a mixture containing egg lecithin (3 mg/ml) in phosphate buffer solution and different concentration of extracts (100 µl). After incubation for 1 h at 37˚C, the reaction was stopped by adding 2 ml of 0.25 N hydrochloric acid containing 15% w/v trichloroacetic acid and 0.375% w/v thiobarbituric acid, boiled for 15 min, cooled, centrifuged and absorbance of supernatant was measured at 532 nm.
2.6.5. Nitric Oxide Scavenging Assay
Nitric oxide scavenging assay was determined with minor modifications [17] . The reaction mixture (6 ml) containing sodium nitroprusside (10 mM, 4 ml), PBS (1 ml) was incubated at 25˚C for 150 min. After incubation, 0.5 ml of the reaction mixture was removed and 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) was mixed and allowed to stand for 5 min for completion of diazotization reaction. Naphthyl ethylene diamine dihydrochloride (1 ml) was added to the mixture and allowed to stand for 30 min in diffused light. The absorbance was measured at 540 nm.
2.6.6. Reducing Power Assay
Reducing capacity of extract was dmeasured with minor modifications [18] . 1.0 ml of extract was mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of potassium ferricyanide (30 mM). The mixture was incubated at 50˚C for 20 min. Thereafter, 2.5 ml of trichloroacetic acid (600 mM) was added to the reaction mixture and centrifuged for 10 min at 3000 rpm. The upper layer of solution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride (6 mM) and absorbance was measured at 700 nm.
3. Statistical Analyses
All assays were carried out in triplicates and results are presented as Mean ± SD (n = 3). One way analysis of variance was performed (one way ANOVA) and the significant values were determined by Tukey’s test level of significance of p < 0.05. The statistical analyses were carried out by using PRISM 5.
4. Results and Discussion
The total phenol and flavonol content of successive extracts of C. infundibuliformis were shown in Table 1. The methanol extract was found to be the highest amount of total phenols (98.52 ± 0.61 mg GAE/g of extract) and flavonols (84.59 ± 0.29 mg rutin equivalent to/g of extract) contents than other extracts.
DPPH radical is stable organic free radical with absorption band at 517 nm. The antioxidants donate the electron to DPPH radical on accepting electron, the absorption range will change in a visible to nonvisible coloration from purple to yellow [19] . Methanol extract has shown highest percentage of inhibition. Successive ethyl acetate extract of C. infundibuliformis was found to be highest IC50 value (9.79 ± 0.55 µg/ml) with excellent scavenging activity. Whereas, other extracts possess lower antioxidant activity. There was significant difference in the DPPH radical scavenging activity observed among the extracts. Reactive oxygen species such as hydroxyl radical were found by different mechanism. This is considered causative factor for many diseases [20] . Hydroxyl radicals easily penetrate in cell membranes and readily react with biomolecules including proteins, carbohydrates and lipids. These cause cell death or tissue injury. Natural antioxidants plays important role in neutralizing such type of free radicals. Among all the extracts, methanol and petroleum ether extracts of C. infundibuliformis have shown the highest scavenging ability. The IC50 values of all the extracts confirming antioxidant activity, when compared with the standard ascorbic acid (Table 2).
Free radicals induce lipid peroxidation in polyunsaturated lipid rich areas like brain and liver [21] . The assay used to evaluate lipid peroxidation inhibitory activity of C. infundibuliformis leave extracts. In this study, our results demonstrate excellent antioxidant activity. The IC50 value of methanol extract was found to be 70.66 ± 0.76 µg/ml and standard BHA 75.83 ± 0.58 µg/ml. All other extracts exhibited moderate antioxidant activity. Nitric oxide radicals are well known inducer of tissue pathogenesis leading to several diseases such as cancer, diabetes mellitus and age related disorders [22] . Many plant species with antioxidants acts as protective agents with these diseases. Among the four extracts, chloroform extract was found higher percentage inhibition and all other extracts possess moderate percentage inhibition. The IC50 value of chloroform extract is 240.50 ± 0.50 µg/ml and other extracts have shown decreasing order of antioxidant activity; petroleum ether > methanol > ethyl acetate extracts with IC50 values 370.66 ± 0.58, 814.66 ± 0.29 and 970.50 ± 0.87 µg/ml, respectively. The reducing power assay was usually used to determine the reduction capacity of antioxidants from ferric (Fe3+) to ferrous (Fe2+) [23] . Natural antioxidants play a vital role in breaking the chain reaction of free radicals by donating electron. In this study, the yellow color of test solution changes to various shades from green to blue depending on the reducing power of extracts. These results may serve significant antioxidant activity. The reducing capacity of all the extracts was shown in the
Table 2 . The methanol extract showed excellent reducing capacity with the absorbance 2.03 ± 0.005. However, petroleum ether, chloroform and ethyl acetate extracts exhibited moderate reduction capacity. In phosphomolybdenum method, molybdenum reduces to form green phosphate molybdenum complex by antioxidants, which have an absorption peak at 695 nm. Total antioxidant capacity of all the successive extracts of C. infundibuliformis was measured by monitoring the absorbance of the reaction mixture Table 2. In vitro antioxidant activity of successive extracts of Crossandra infundibuliformis leaves in different methods.
Values are means of three replicate determination ± SEM, standard error mean; NAnot analysed. aValues are significant higher reducing capacity than other groups and bvalues are significantly higher antioxidant activity when compared to other groups (P < 0.05).
at 695 nm. A high absorbance value of the reaction mixture produces higher millimolar concentration, which proves a stronger antioxidant capacity as summarised in Table 1. Chloroform extract of C. infundibuliformis has shown higher antioxidant capacity (5.03 ± 0.02 mM), followed by methanol, petroleum ether and ethyl acetate extracts of C. infundibuliformis 2.12 ± 0.01, 1.05 ± 0.04 and 0.52 ± 0.00 mM, respectively.
5. Conclusion
This study was designed to evaluate the antioxidant and free radical scavenging properties of successive extracts of C. infundibuliformis by using six different in vitro antioxidant methods. The methanol extract demonstrated the highest total phenol and flavonol contents. Among all the tested methods, methanol and chloroform extracts of C. infundibuliformis have shown strong antioxidant activity in the most of the tested methods. Considering both total phenol and flavonol contents, the antioxidant activity of methanol and chloroform extracts of C. infundibuliformis was optimal. It is indicated by the results of work that, methanol and chloroform extracts of C. infundibuliformis could serve as source of natural antioxidants.
Abbreviations
1, 1’-Diphenyl-2-picrylhydrazyl (DPPH)
Lipid peroxidation (LPO)
50 Percent inhibition concentration (IC50 value)
Butylated hydroxyanisole (BHA)
Butylated hydroxy toluene (BHT)
Dimethyl sulfoxide (DMSO)
NOTES
*Corresponding author.