Introduction
Today the world appears to be increasingly interested in the health benefits of food and has begun to look beyond the basic nutrional benefits of foodstuffs to disease prevention. It is generally accepted that the beneficial effects of herbal remedies can be obtain from active constituent present in whole plant, part of the plant (e.g. flowers, fruits, roots, stem, leaves, or herbs), or plant materials or combinations thereof, whether in crude or processed state.1 Free radicals’ which are delivered as a consequence of typical biochemical responses in the body are involved in cancer, ischemic heart disease, inflammation, diabetes, aging, arthrosclerosis, immunosuppressant and neurodegenerative disorders.2 The human body has complex antioxidant defense systems that use antioxidant enzymes (eg.-catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. glutathion, melatonin carotenoids thiol antioxidants and vitamins E & C). Almost all organisms’ posses’ antioxidants and repair systems to protect themselves against oxidative damage, but these systems cannot completely prevent damage.2 This deficiency may result in oxidative stress.3 Antioxidants can be broadly defined as any substance that delays or inhibits oxidative damage to target molecules. The main characteristic of antioxidants is its ability to trap free radicals.4
Opuntia ficus-indica was screened for the phytochemical and antioxidant evaluation. The fruit part of the plant was selected for the present study.
Opuntia ficus-indica is a species of cactus that has long been a domesticated crop plant important in agricultural economies throughout arid and semiarid parts of the world. It is thought to possibly be native to Mexico. Some of the common English names for the plant and its fruit are Indian fig opuntia, barbary fig, and prickly pear, although this last name has also been applied to other less common Opuntia species.5 The fruit ripen from August through October. The fruit are typically eaten, minus the thick outer skin, after chilling in a refrigerator for a few hours. They have a taste similar to a juicy, extra sweet watermelon.Table 1 The bright red/purple or white/yellowish flesh contains many tiny hard seeds that are usually swallowed, but should be avoided by those who have problems digesting seeds.6
Material and Methods
Table 1
Detail of plant extract
|
Plant Name |
Family |
Species |
Drug |
|
Opuntia Ficus Indica |
Cactaceae |
Opuntia Ficus Indica |
Fruits |
Qualitative phytochemicals analysis
Qualitative analysis for various phytochemicals viz. alkaloids, carbohydrates, flavonoids, glycosides and saponin glycoside, tannins, phenol, steroids, amino acid, proteins was carried out for opuntia ficus indica. Test method & result shown in Table 2 .
In vitro anti-oxidants activities
DPPH Assay
DPPH (1, 1-Diphenyl-2-picrylhydrazyl) is a stable free radial with red colour (absorbed at 517nm). If free radicals have been scavenged, DPPH will generate its colour to yellow. This assay uses this character to show herbs free radical scavenging activity.7
Procedure
Scavenging activity on DPPH free radicals by the extract was assessed according to the method Reported by Gyamfi et al.7 Briefly, 50 íL of the ethanol extract Containing varied amounts of powdered ethanol extract (1, 5, 10, and 50 µg/mL distilled water, respectively, in each reaction) was mixed With 1 mL of 0.1 mM DPPH-ethanol solution and 450 íL of 50 mM Tris-HCl buffer (pH 7.4). After the solution incubated for 30 min incubation at room temperature, reduction of DPPH free radicals was measured by reading the absorbance at 517 nm. The assays were carried out in triplicate and the results were expressed as mean values ± standard deviations.8
In the experiment, prepare control using solvent of dilution & 0.5ml of DPPH.
Percentage inhibition (I %) was calculated
Using the formula,
Where Ac is the absorbance of the control and as is the absorbance of the sample
Reducing power assay
In this assay, the yellow color of the test Solution changes to various shades of green and blue, Depending on the reducing power of each compound. Presence of reducers causes the conversion of the Fe3+/ferricyanide complex used in this method to the ferrous form.9
Procedure
Reducing power ability the reducing power was assayed as described in kuda et al.2005 with some modifications. Different Deore S. L...et al /Int.J. ChemTech Res.2009, 1(2) 175 Concentrations of ethanolic extracts (1.0 ml) were mixed With 2.5 ml of phosphate buffer (0.2 molar, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was then incubated at 50 oC for 20 min. After, 2.5 ml of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 1.25 ml from the supernatant was mixed with 1.25 ml of distilled water and 0.25 ml FeCl3 solution (0.1%, w/v). The absorbance was measured at 700 nm. The assays were carried out in triplicate and the results were expressed as mean values ± standard deviations. Increased absorbance values indicate a higher reducing power.10
Scavenging of hydrogen peroxide
The ability of the extracts to scavenge hydrogen peroxide was determined.
Procedure
Prepared a dilution series of extract (10, 20, 40, 60, 80, 100 µg/ml). Prepared hydrogen peroxide solution (2 mM/L) with standard phosphate buffer (pH 7.4). Different concentration of the extracts in distilled water was added to 0.6 ml of hydrogen peroxide solution. Absorbance was determined at 230 nm after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage inhibition of different concentrations of the extracts was determined and compared with the standard, Gallic acid.11
Determination of total phenol content
Total phenolic compound contents were determined by the Folin-Ciocalteau method. The extract samples (0.5 ml of different dilutions) were mixed with Folin Ciocalteu Reagent (5 ml, 1:10 diluted with distilled water) for 5 min and aqueous Na2CO3 (4 ml, 1 M) were then added.12 The Mixture was allowed to stand for 15 min and the phenols were determined by spectrophotometry method at 765 nm. The standard curve was prepared by 20, 40, 60, 80 and 100 µg/ml solutions of Gallic acid in methanol: water (50:50, v/v). Total phenol values are expressed in terms of Gallic acid equivalent (mg/ g of dry mass), which is a Common reference compound.13
Determination of total flavonoid content
Flavonoids have been shown to play a number of biologically important roles such as antioxidant activity. Antioxidants may serve the task of reducing oxidative damage in humans induced by free radicals and reactive oxygen species under ‘oxidative stress’ conditions.14 The aim of this work is to develop a sensitive, diversely applicable and simple indirect spectrophotometric and fluorometric method for the determination of total flavonoids content in several plants. The method is based on the oxidation of flavonoids with Ce(IV) salts at room temperature. The spectrophotometric determination of the remaining Ce(IV) or fluoro metric determination of the formed Ce(III) was performed after reaction with flavonoids. Quercetic was used as flavonoid standard. The procedure was successfully applied to the assay of total flavonoids in the area parts of Urtica Dioica L. The obtained results were statistically compared with those obtained by known methods.15
Procedure
Take 0.5 ml of sample (1mg /ml)diluted with 2.5 ml distilled water,add0.150ml of 0.5 percent sodium nitrite stand for 6 min and then add 0.150 ml of 10%aluminium chloride after 5 minute add 1 ml of sodium hydroxide and measure the absorbance 510 nm. The standard curve was prepared by 1mg/ml 100 ,80,60 40,20 µg/ml, and solutions of Rutin in water Total Flavonoid values are expressed in terms of Rutin equivalent (mg/g of dry mass), which is a common reference compound.
Result & Discussion
Phytochemical screening of fruit extract of Opuntia ficus indica
Table 2
Phytochemical screening of fruit of Opuntia ficus indica
In vitro antioxidant assay
DPPH Assay
Table 3
Absorbance of test solution at 517nm. The results are the means of three separate experiments.
|
S. no |
Concentration µg/ml |
Absorbance |
|
|
Without DPPH |
With DPPH |
||
|
1) |
10 |
0.002 |
0.039 |
|
2) |
20 |
0.004 |
0.034 |
|
3) |
40 |
0.005 |
0.030 |
|
4) |
60 |
0.007 |
0.023 |
|
5) |
80 |
0.010 |
0.020 |
|
6) |
100 |
0.12 |
0.015 |
Table 4
Concentration vs. absorbance & calculation of % inhibition in DPPH
|
S. no. |
Concentration nm |
Absorbance AT 517nm |
% Inhibition |
|
1) |
10 |
0.042 |
49.39 |
|
2) |
20 |
0.036 |
56.62 |
|
3) |
40 |
0.032 |
61.44 |
|
4) |
60 |
0.028 |
66.26 |
|
5) |
80 |
0.023 |
72.2 |
|
6) |
100 |
0.018 |
78.31 |
Reducing power assay
Absorbance of blank = 3.080. The result is the mean of three separate experiments.
Table 5
Concentration vs. absorbance & Calculation of % reducing power
Scavenging of hydrogen peroxide
Table 6
Concentration vs. absorbance without H2O2 & with H2O2
|
S.no |
Concentration (µg/ml) |
Absorbance of Test Solution At 517 nm |
|
|
Without H2O2 |
With H2O2 |
||
|
1) |
10 |
0.011 |
0.470 |
|
2) |
20 |
0.013 |
0.415 |
|
3) |
40 |
0.015 |
0.375 |
|
4) |
60 |
0.016 |
0.350 |
|
5) |
80 |
0.018 |
0.245 |
|
6) |
100 |
0.020 |
0.200 |
Table 7
Concentration vs. absorbance &calculation of % inhibition in hydrogen peroxide scavenging assay.
|
S. no. |
Concentration µg/ml |
Absorbance AT 517nm |
% Inhibition |
|
1) |
10 |
0.61 |
48 |
|
2) |
20 |
0.49 |
58.82 |
|
3) |
40 |
0.365 |
69.33 |
|
4) |
60 |
0.305 |
74.37 |
|
5) |
80 |
0.255 |
78.57 |
|
6) |
100 |
0.19 |
84.03 |
Extract showed good hydrogen peroxide scavenging activity in range of 10 to 100 µg/ml. IC50 of extract for H2O2 was also found to be 0.381µg/ml.
Determination of total phenol content
Determination of total flavanoids content
Table 9
Concentration vs. absorbance at 515 nm
|
Drug |
Concentration µg/ml |
Absorbance (at 515 nm) |
|
Rutin |
20 |
0.08 |
|
40 |
0.19 |
|
|
60 |
0.29 |
|
|
80 |
0.37 |
|
|
100 |
0.46 |
|
|
Extract |
1000 |
0.020 |
In total flavonoid content estiofItion it was observed that extract was having 6 µg/mg extract of flavonoid (Rutin equivalent).
Conclusion
The phytochemical investigation of the ethanolic extract of, Opuntia ficus-indica fruits was carried out under the standard procedure. All the extracts showed the presence of carbohydrate, flavonoid, phenol, and steroid etc. The in vitro antioxidant study of all the plant extracts were carried out by DPPH,hydrogen peroxide radical scavenging, reducing power assay, phenolic content estimation and total flavanoid content estimation showed significant free radical scavenging activity. In DPPH assay it was observed that extract showed good antioxidant activity in range of 10 to 100 µg/ml. IC50 of extract for DPPH was found to be 4.630 µg/ml. Extract showed good hydrogen peroxide scavenging activity in range of 10 to 100 µg/ml. IC50 of extract for H2O2 was also found to be 0.381µg/ml. In total flavonoid content estiOFItion it was observed that extract was having 6 µg/mg extract of flavonoid (Rutin equivalent).
