INTRODUCTION:

In recent years much attention has been devoted to natural antioxidant and their association with the health benefits¹. Plants are potential sources of natural antioxidants. It produces various antioxidative compounds to counteract reactive oxygen species (ROS) in order to survive². The most frequently encountered free radicals are the hydroxyl radical (HO^.), the superoxide radical (O₂^.), the nitric oxide radical (NO^.) and the lipid peroxyl radical (LOO^.) while non-free radical species principally being H₂O₂ and singled oxygen (O₂^.)³. These molecules are exacerbating factors in cellular injury and ageing process⁴. Free radicals play an important role in the pathogenesis of several human diseases, such as cancer, rheumatoid arthritis, and cardiovascular diseases⁵. Strong epidemiological evidence suggests that regular consumption of fruits and vegetables, which are a rich source of antioxidants, can reduce cancer and coronary heart diseases^6,7.

Antioxidants thus play an important role of protecting the human body against damage by reactive oxygen species^8,9. Several synthetic antioxidants, e.g., BHA (butylated hydroxyl anisole) and BHT (butylated hydroxyl toluene) are commercially available but are quite unsafe and their toxicity is a problem of concern¹⁰. Natural antioxidants, especially phenolic and flavonoids are safe and also bioactive. They have been reported to exert multiple biological effect including antioxidant, free radical scavenging abilities, anti-inflammatory, anticarcinogenic etc. Therefore, in recent years, considerable attention has been directed towards identification of plants with antioxidant ability that may be used for human consumption¹¹. The use of traditional medicine is widespread, and plants still present a large source of natural antioxidants that might serve as leads for the development of novel¹².

Moringa oleifera commonly known as (family: Moringaceae) Horse radish tree or Drumstick tree is both nutritional and medicinal with some useful minerals, vitamins, amino acids, etc¹³.

Almost all the parts of this plant: root, bark, gum, leaf, fruit (pods), flowers, seed and seed oil have been used for various ailments in the indigenous medicine of South Asia, including the treatment of inflammation and infectious diseases along with cardiovascular, gastrointestinal, hematological and hepatorenal disorders¹⁴.

Although much has been learned about the nutritional value of Moringa oleifera additional knowledge remains to be secured. Therefore, in recent years; considerable attention has been directed towards identification of plants with antioxidant ability that may be used for human consumption. Thus, the aims of present study are to investigate the phytochemical profile and antioxidant activity of pods of Moringa oleifera against free radicals using specific in vitro standard procedures so as to assess the medicinal potential of the plant and justify its folklore use.

MATERIALS AND METHODS:

Collection of Sample:

The pods of Moringa oleifera (Gaertn) were collected from Krishi Vigyan Kendra, Banasthali University, Banasthali, Tonk district, Rajasthan, India. The plant material was taxonomically identified by Botanist of Krishi Vigyan Kendra, Banasthali, Tonk district. The collected pods were shade dried and milled into coarse powder with an electrical grinder and further passed through sieve-mesh 40 and stored in an air-tight container at 25⁰ C.

Chemicals and reagents:

DPPH (1,1-diphenyl-1,2-picryl hydrazyl), TPTZ (2,4,6,-tripyridyl-s-triazine), Ferrozine, Deoxyribose were purchased from Sigma Chemical Co. Ltd USA. Trichloroacetic acid (TCA), thiobarbituric acid (TBA), butylated hydroxyl toluene (BHT), butylated hydroxyanisole (BHA), L-Ascorbic acid, ammonium molybdate, nitro blue tetrazolium (NBT), PMS (phenazine methosulfate), reduced NADH (nicotinamide adenine dinucleotide), quercetin were purchased from HI Media, Mumbai. DMSO (Dimethyl sulfoxide) was purchased from Merck Co. (Germany), Mumbai. All other unlabelled chemicals and reagents were of analytical grade and used without further purification.

Extraction:

Dried powdered material was placed in the Soxhlet thimble with 80% ethanol in 250 ml flat bottom flask. Further refluxed for 18 h at 80⁰ C for two days. Collected solvent were cooled and poured in a glass plate. The marc was dried in hot air oven below 50⁰ C for 48 h Collected the dried extract and stored at 25⁰ C in air-tight containers.

Preliminary Phytochemical Screening:

Qualitative screening:

The freshly prepared hydro-ethanolic extract of plant (MOEE) and aqueous extract (MOAE) was qualitatively tested for the presence of chemical constituents. Phytochemical screening of extracts was carried out using the following reagents and chemicals according to the methods described by Parekh and Chanda¹⁵: Test for alkaloids performed with Dragendorffs reagent, flavonoids with the use of ammonia and concentrated H₂SO₄, tannins with ferric chloride and potassium dichromate solutions, phenolics with FeCl₃, Saponins (frothing test), Steroids (Liebermann-Burchard test), terpenoids with Fehling’s solution, Cardiac glycosides (Keller-Kinliani test).

Quantitative screening:

Determination of total Phenolics:

The content of the total phenolic in plant extract is determined by Folin Ciocalteu method ¹⁶ spectrometrically. To 1 ml of Folin-Ciocalteu’s reagent, previously diluted (1:20), was added to 1 ml of samples (250 µg/ml) and mixed thoroughly. To the mixture, 4 ml of sodium carbonate (75 g/L) and 10 ml of distilled water were added and mixed well. The mixture was allowed to stand for 2 h at room temperature. Contents were then centrifuged at 2000 g for 5 min and the absorbance of the supernatant was taken at 760 nm. A standard curve was obtained using various concentrations of gallic acid. Results were expressed as percentage of gallic acid equivalents (GAE).

Determination of Total Flavonoids:

Total flavonoid contents were determined using the method of Ordon¹⁷. A volume of 0.5 ml of 2% AlCl₃ ethanol solution was added to 0.5 ml of sample solution. After one hour at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of Flavonoids. Extract samples were evaluated at a final concentration of 1mg/ml. Total Flavonoids content were calculated as rutin equivalent (mg/g). The amount of flavonoids in plant extract in rutin equivalent (RE) was calculated by the following formula:

X= (A. m₀)/ (A₀. m)

Where,

X- The total flavonoid content, mg/mg plant extract in RE,

A- The absorption of plant extract solution,

A0- The absorption of standard rutin solution,

m- The weight of plant extract (mg)

m0- The weight of rutin in the solution, (mg)

Determination of total Flavonols:

Total flavonols in the plant extracts were estimated using the earlier described method¹⁸. To 2.0 ml of 2% AlCl₃ ethanol and 3.0 ml (50 g/l) sodium acetate solutions were added in 2.0 ml of extract solution. The absorption at 440 nm was read after 2.5 h at 20°C. Sample extract were evaluated at a final concentration of 1mg/ml. Total flavonoid content was calculated as rutin equivalent (mg/g). Total flavonol content was calculated as rutin (mg/g) using the following equation based on calibration curve: y = ax + b, where x was the absorbance and y was the rutin equivalent (mg/g).

Determination of Antioxidant activity:

DPPH free radical scavenging activity:

The free-radical scavenging activity of MO pod extract was measured by decrease in the absorbance of methanol solution of DPPH. A stock solution of DPPH (33 mg in 1 L) was prepared in methanol, which gave initial absorbance of 0.493, and 5ml of this stock solution was added to 1 ml of MO pod extract solution at different concentrations (100-1000 µg/ml). After 30 min, absorbance was measured at 517 nm and compared with standards (100-1000 µg/ml). Scavenging activity was expressed as the percentage inhibition calculated using the following formula:

% Anti-radical activity = Control Abs – Sample Abs × 100

Control absorbance

Scavenging of Hydrogen Peroxide:

A solution of hydrogen peroxide (40mM) was prepared in phosphate buffer (pH 7.4). Different concentrations (100-1000 µg/ml)) of MO pod extract were added to a hydrogen peroxide solution (0.6 ml, 40mM). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min. against a blank solution containing phosphate buffer without hydrogen¹⁹. The percentage scavenging of hydrogen peroxide of MO and standard compounds was calculated using the following formula:

% Scavenged [H₂O₂] = [(A₀ – A₁)/ A₀] × 100

Where A₀ was the absorbance of the control, A₁ was the absorbance in the presence of the sample of Moringa oleifera and standards.

Super oxide anion scavenging activity:

To 1ml of NBT solution (144µM in 100mM phosphate buffer, pH 7.4), 1ml of reduced NADH (677µM in100mM phosphate buffer, pH 7.4) and 0.5 ml of sample extract was mixed and the reaction was started with adding 100µl of PMS solution (60µM PMS in100mM phosphate buffer, pH 7.4). The reaction mixture was incubated at 25°C for 5 min, and the absorbance 560 was measured against blank²⁰ and compared with standards. Decreased absorbance of reaction mixture indicated increased super oxide anion scavenging activity. The percentage inhibition of super oxide anion generation was calculated using the following formula:

% Inhibition = [A_° - A₁/A_°] × 100

Where, A_° = Absorbance of the control.

A₁ = Absorbance of test sample.

Non Specific Assay:

Different concentrations of extract were mixed with 1 ml of reaction buffer (100 μM FeCl₃, 10 μM EDTA, 1.5 mM H₂O₂, 2.5 mM Deoxyribose and 100 μM L- ascorbic acid, pH 7.4) and incubated for 1 h at 37º C. 1 ml of 0.5 % 2- thiobarbituric acid in 0.025 M sodium hydroxide and 1 ml of 2.8 % trichloroacetic acid was added to the mixture and heated for 30 min at 80º C. Finally the mixture was cool and absorbance was measured at 532 nm using spectrophotometer.

Statistical analysis:

The experimental results were expressed as mean ± standard deviation (SD) of three replicates. The data were subjected to one way analysis of variance (ANOVA) and differences between samples were determined by Bonferroni’s multiple comparison test using the SPSS 16.0 (Statistical program for Social Sciences) program. Results with p<0.05 were regarded as statistically significant and considered p<0.001 as very significant.

RESULTS AND DISCUSSION:

Phytochemical screening of extracts:

Phytochemical screening of the hydro-ethanolic and aqueous extracts of Moringa oleifera pods revealed the presence of various bioactive components of which alkaloid, phenolics, flavonoids, flavonols, proanthocyanidins, terpenoids, tannin, and cardiac glycosides are the most prominent components and the result of phytochemical test is presented in Table 1. All these phytochemicals possess good antioxidant activities and has been reported to exhibit multiple biological effects including anti-inflammatory and antitumor activities. MOEE is tested negative for alkaloids but plant phenolics like flavonoids and tannins which act as primary antioxidants or free radical scavengers show their presence in the extracts of Moringa oleifera.

Table 1. Qualitative analysis of the phytochemicals of aqueous and hydro-ethanolic extracts of Moringa oleifera pods.

Phytochemicals	Aqueous extract	Hydro-ethanolic extract
Alkaloids	+	-
Saponins	+	+
Phytosterols	+	+
Phenols	+	+
Flavonoids	+	+
Terpenoids	-	+
Tannins	+	+
Phlobatannins	+	+

+ Presence of the compound; - Absence of the compound.

Total Phenolic, Flavonoids and Flavonols content:

The observations made in the present investigation strongly suggest that phenolics are important constituent of this plant and some of their pharmacological effects could be attributed to the presence of these valuable constituents. The antioxidant activity of Moringa oleifera is probably due to its phenolic content. It is well known that phenolic compounds are constituents of many plants and they have attracted a great deal of public and scientific interest because of their health promoting effects as antioxidants. Table 2 shows total phenolic content of MOEE and MOAE, determined in terms of gallic acid equivalents. Results obtained in the study revealed that the level of phenolic compound in MOAE was higher than that of MOEE when compared to that of standard compound used in the study. Total phenolic content was expressed as gallic acid equivalents (GAE) (mg/g). On the basis of the present investigation both the extract showed high significance (p<0.001) as compared to standard. Table 2 shows total flavonoids and flavonol content of MOEE and MOAE extracts which were expressed in terms of rutin equivalents. Total flavonoid content was calculated as rutin (mg/g). Total flavonols content was calculated as rutin (mg/g).

Table 2: Polyphenol contents of the hydro-ethanolic and aqueous extracts of the pods of Moringa oleifera.

Phenolics	MOEE	MOAE
Total phenolics^a	0.612 ± 0.244	2.360 ± 0.096
Flavonoids^b	0.803 ± 0.072	0.678 ± 0.061
Total Flavonols^c	1.515 ± 0.013	2.738 ± 0.086

^aExpressed as mg gallic acid/g of dry plant material; ^bExpressed as mg rutin/g of dry plant material; ^cExpressed as mg rutin/g of dry plant material; Data are presented as the mean ± SD of each triplicate test.

From the aforementioned results it can be concluded that polyphenols are the major plant compounds with antioxidant activity. This activity is believed to be mainly due to their redox properties²¹, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides.

DPPH Radical Scavenging Activity:

DPPH is known to abstract labile hydrogen and the ability to scavenge the DPPH radical is related to the inhibition of lipid per oxidation.²² DPPH radical was used as a substrate to evaluate free radical scavenging activity of MOEE and MOAE. BHT, ascorbic acid and BHA were used as standards. All the concentrations of both the extracts (MOEE and MOAE) demonstrated H-donor activity. Fig. 1 shows comparisons of MOEE and MOAE with ascorbic acid, BHA, and BHT. It was observed that scavenging effect of hydro-ethanolic extract of Moringa oleifera had higher activity than that of aqueous extract. At concentration of 1mg/ml the scavenging activity of ethanolic extract was reached to 50.6% while that of aqueous extract was only 27.27%. On the other hand the ascorbic acid, BHA and BHT at 1mg/ml concentration had 62.6%, 44.2% and 48.4% inhibition of free radical, respectively. Our finding reveals that there is a correlation between the free radical scavenging activity and MOEE of proanthocyanidin content (r = 0.977) and MOAE of phenolic content (r = 0.941).

Figure1: DPPH activity at different concentrations of MOEE, BHT, BHA and Ascorbic acid.

The DPPH Scavenging ability of the extract may be attributed to its hydrogen donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. The extracts showed significant scavenging activity (p<0.001) as compared to standards.

Hydrogen Peroxide Scavenging Activity:

Hydrogen peroxide scavenging activity of MOEE and MOAE on hydroxyl radical is depicted in Table 3. H₂O₂ is highly important because of its ability to penetrate biological membranes. H₂O₂ itself is not very reactive, it is a weak oxidizing agent, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells. It can cross cell membranes rapidly, once inside the cell, H₂O₂ can probably react with Fe²⁺, and possibly Cu²⁺ ions to form hydroxyl radical and this may be the origin of many of its toxic effects. It can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups.

Thus, removal of H₂O₂ is very important for protection of food systems. MOEE and MOAE also demonstrated hydrogen peroxide decomposition activity in a concentration dependent manner. The decomposition of H₂O₂ by MOEE and MOAE may at least partly result from its antioxidant and free radical scavenging activity.

Table 3 shows the H₂O₂ scavenging activity by hydro-ethanolic and aqueous MO pod extract and comparison with BHT and ascorbic acid. The percentages of H₂O₂ scavenging activity of MOEE, MOAE, Ascorbic acid and BHT were found as 81.69%, 78.27%, 38.26%, and 48.93, respectively. Our results indicate that there were a good correlation between the hydrogen peroxide scavenging activity and MOAE of flavonoid content (r = 0.932). Both the extracts showed significant value (p<0.001) as compared to standards.

Super oxide anion scavenging activity:

Super oxides are produced from molecular oxygen due to oxidative enzymes²³of body as well as via non enzymatic reaction such as autoxidation by catecholamines.²⁴Super oxide has also been observed to directly initiate lipid per oxidation. In the present study the super oxide anion radical scavenging activity of both extracts was assayed by PMS-NADH system (Fig 2). Table 3 shows the super oxide scavenging effect of both the extracts and comparison with rutin, BHA and BHT. The percentage inhibition of super oxide generation by 1mg/ml concentration of MOEE and MOAE was found as 28.52% and 54.01%. On the other hand, rutin, BHA and BHT at 1 mg/ml concentration had 52%, 56%, 42%inhibition, respectively of super oxide radical. The increase of percentage scavenging activity thus indicated the consumption of super oxide anion in the reaction mixture by plant extracts. Our finding reveals that there is a good correlation between the super oxide scavenging activity and MOAE of tannin content (r = 0.883). Both the extracts showed highly significant value (p<0.001) as compared to standards.

Figure 2: Superoxide scavenging activity at different concentrations of MOEE, BHT, BHA, rutin and ascorbic acid using PMS-NADH-NBT method.

Non-Specific Assay:

It has been observed that the extract showed excellent antioxidant activities. Concentration dependent inhibition of hydroxyl radical induced deoxyribose degradation was observed in non site-specific assay. Pro oxidant effect was not observed in case of extract. The Figure 3 shows dose dependent increase in antioxidant potential in the extract when compared to standard rutin. The extract showed highly significant value (p<0.001) as compared to standard

CONCLUSION:

The analysis revealed only minor differences in the antioxidant activity of hydro-ethanolic and aqueous extracts of pods. Their constituents scavenge free radicals and exert a protective effect against free radicals and exert a protective effect against oxidative damage induced to cellular macromolecules. The present data suggest that both the extracts could be a potential source of natural antioxidant that could have great importance as therapeutic agents in preventing or slowing the progress of ageing and age associated oxidative stress related degenerative diseases such as cancer. The antioxidant potential may be attributed to the presence of polyphenolic compounds such as flavonoids, proanthocynidin and tannins. These results are encouraging enough to pursue characterization of these fractions. Further studies are in progress in our laboratory to evaluate the in vivo antioxidant potential of these extracts in various animal models and phytochemical studies are required to establish the types of compounds responsible for the bioactivity of this medicinal plant.

ACKNOWLEDGMENTS:

The authors are grateful to UGC for providing financial assistance. The authors are also thankful to the authorities of Banasthali University for providing support to the study.

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Received on 20.10.2010 Modified on 17.11.2010

Research J. Pharm. and Tech. 4(4): April 2011; Page 566-571

Samples	DPPH radical scavenging activity (% inhibition)	Percentage scavenging of hydrogen peroxide	Superoxide anion scavenging activity (% inhibition)
MOEE	50.6 ± 0.2	81.69 ± 0.04	28.52 ± 0.28
MOAE	27.27 ± 0.09	78.27± 1.6	54.01 ± 0.08
Ascorbic acid	62.6 ± 0.4	38.26 ± 0.21	―
BHA	44.2 ± 1.6	―	56± 0.04
BHT	48.4 ± 2.2	43.93 ± 0.06	42.6± 0.28
Rutin	―	―	52 ± 0.2