Antioxidant Activity and Total Phenolic Content of Moringa oleifera Leaves in Two Stages of Maturity
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- Sreelatha, S. & Padma, P.R. Plant Foods Hum Nutr (2009) 64: 303. doi:10.1007/s11130-009-0141-0
Antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to human against infections and degenerative diseases. Current research is now directed towards natural antioxidants originated from plants due to safe therapeutics. Moringa oleifera is used in Indian traditional medicine for a wide range of various ailments. To understand the mechanism of pharmacological actions, antioxidant properties of the Moringa oleifera leaf extracts were tested in two stages of maturity using standard in vitro models. The successive aqueous extract of Moringa oleifera exhibited strong scavenging effect on 2, 2-diphenyl-2-picryl hydrazyl (DPPH) free radical, superoxide, nitric oxide radical and inhibition of lipid per oxidation. The free radical scavenging effect of Moringa oleifera leaf extract was comparable with that of the reference antioxidants. The data obtained in the present study suggests that the extracts of Moringa oleifera both mature and tender leaves have potent antioxidant activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage.
KeywordsMoringa oleiferaAntioxidantsFree radicalsScavenging activity
The potentially reactive derivatives of oxygen, ascribed as ROS (reactive oxygen molecules) such as O2−, H2O2 and OH, are continuously generated inside the human body as a consequence of exposure to a plethora of exogenous chemicals in our ambient environment and/or a number of endogenous metabolic processes involving redox enzymes and bioenergetic electron transfer . Under normal circumstances, the ROS generated are detoxified by the antioxidants present in the body and there is an equilibrium between the ROS generated and the antioxidants present. However, owing to ROS overproduction and/or inadequate antioxidant defense, this equilibrium is hampered favouring the ROS upsurge that culminates in oxidative stress . The ROS readily attack and induce oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA . This oxidative damage is a crucial etiological factor implicated in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, neurodegenerative diseases and also in the ageing process [4, 5].
Based on growing interest in free radical biology and the lack of effective therapies for most chronic diseases, the usefulness of antioxidants in protection against these diseases is warranted. Epidemiological studies have found that the intake of antioxidants such as vitamin C reduces the risk of coronary heart disease and cancer . The antioxidants may mediate their effect by directly reacting with ROS, quenching them and/or chelating the catalytic metal ions . Several synthetic antioxidants, e.g., BHA (butylated hydroxy anisole) and BHT (butylated hydroxy toluene) are commercially available but are quite unsafe and their toxicity is a problem of concern . Natural antioxidants, especially phenolics and flavonoids, are safe and also bioactive.
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 novel drugs . 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, etc. . A native of the sub-Himalayan regions of North West India Moringa oleifera is indigenous to many countries in Africa, Arabia, South East Asia, the Pacific, Caribbean Islands and South America. Although there are 12 varieties of Moringa species Moringa oleifera is the best known of all species of the genus Moringaceae . 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 disorders . The flowers and roots are used in folk remedies, for tumors, the seeds for abdominal tumors, leaves applied as poultice to sores, rubbed on temples for headaches and are said to have purgative properties . Moringa oleifera is called “Miracle Vegetable” because it is both a medicinal and a functional food . Administration of Moringa oleifera leaf extract inhibited the growth of pathogenic gram positive and gram negative bacteria  and exerted chemo-modulatory effect against skin papillomagnesis in mice . Moringa oleifera has the highest proportion of essential amino acids and significant quantities of minerals  when analyzed. Moringa oleifera is rich in compounds like glucosinolates and isothiocyanates  and the stem bark has been reported to contain alkaloids namely Moringinine and Moringine . Flowers contain pigments such as alkaloids, kaempferol, rhamnetin, isoquercitrin and kaempferritin . 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. In view of the several ethno botanical uses of Moringa oleifera described above, it was proposed to screen its successive extracts for the in vitro antioxidant activity using standard procedures at two different stages of maturity.
Materials and Methods
2, 2-Diphenyl-2-picryl hydrazyl (DPPH) was obtained from Sigma–Aldrich Co., St. Louis, USA. Naphthyl ethylene diamine dihydrochloride (NEDD) was from Roch-Light Ltd., Suffolk, UK, ascorbic acid, nitro blue tetrazolium (NBT) and butylated hydroxyl anisole (BHA) were from SD Fine Chemicals Ltd., Mumbai, India. Sodium nitroprusside and Silymarin were from Ranbaxy Laboratories Ltd., Mohali, India. Sulphanilic acid used was from E-Merck (India) Ltd., Mumbai, India. All chemicals used were of analytical grade.
Preparation of the Extract
The leaves were chopped to small pieces and dried in shade. The dried leaves were powdered and passed through sieve no. 20 and extracted (100 g) successively with 600 ml of water in a soxhlet extractor for 18–20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature (40–50 °C). The yield (w/w) of the extract from fresh leaves was about 10%. The extracts were prepared in duplicate and all analysis was carried out in triplicates.
Determination of Enzymatic Antioxidants
Advances in nutrition research during the past few decades have changed scientists’ understanding of the contribution of vegetarian diets to human health and disease. Phytochemicals, the bioactive non-nutrient plant compounds in fruits, vegetables, grains and other plant foods have been linked to reductions in the risk of major chronic diseases. Fruits and vegetables contain a wide variety of antioxidant phytochemicals such as phenolics and carotenoids that may help to protect the cellular systems from oxidative damage and lower the risk of chronic diseases. Many medicinal plants that were discovered by early people are still in use today and medicines are still being discovered in plants .
Catalase activity was measured according to the method where  one unit of catalase was defined as the amount of enzyme required to decompose 1 μM of H2O2 in 1 min. The reaction was initiated by the addition of 1.0 ml of freshly prepared 20 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm for 1 min. The enzyme activity was expressed as units/g. One unit is the amount of enzyme activity required to decrease the absorbance at 240 nm by 0.05 units. The activity of SOD was measured according to the principle where  superoxide dismutase (SOD) was assayed based on the inhibition of production of nitro blue tetrazolium formazone. SOD activity was then measured at 560 nm. The enzyme activity was expressed as units/g. Where one unit is the amount of enzyme that gives 50% inhibition of the extent of NBT reduction in 1 min.
GPX activity was measured according to procedure where the  reaction mixture consisted of sodium phosphate buffer, pH 7.0, 1 mM sodium azide, 1 U/ml of reduced glutathione, extract, 0.25 mM H2O2 in a total volume of 1 ml. The tubes were incubated at 37 °C for 3 min. The reaction was terminated by TCA, and to the residual glutathione content, disodium hydrogen phosphate and DTNB solution were added. Yellow colour developed at 412 nm was recorded at 25 °C. One unit of GPx activity was expressed as μg of glutathione consumed/min.
GST activity was assayed accordingly  with some modifications. The reaction was carried out in 0.1 M potassium phosphate buffer (pH 6.5), 1 mM GSH, and 1 mM 1-chloro-2, 4-dinitrobenzene in a 50 μl sample. An increase in absorbance was monitored with a wavelength of 340 nm at 25 °C for a 4 min time period, and the activity of enzyme was expressed as μmol/min/g.
Determination of Non-enzymatic Antioxidants
The method described by Zakaria et al.  was followed for the estimation of total carotenoids. The total carotenoids in the sample can be extracted in petroleum ether and estimated in UV/visible spectrophotometer at 450 nm.
Ascorbic acid, a scavenger of oxy radicals was assayed by the method where  ascorbate is converted to dehydroascorbate by the treatment with activated charcoal. Dehydro ascorbic acid then reacts with 2,4-dinitrophenyl hydrazine to form osazones, which dissolves in sulphuric acid to give an orange colored solution, whose absorbance can be measured spectrophotometrically at 540 nm.
The method described by Rosenberg  was followed for the estimation of tocopherol. Emmerie-Engel reaction is based on the reduction of ferric to ferrous ions by tocopherols, which then forms a red color with 2, 2′-dipyridyl. Tocopherols and carotenes are first extracted with xylene and the extinction read at 460 nm to measure carotenes. A correction is made for this after adding ferric chloride and read at 520 nm.
DPPH (2, 2-Diphenyl-1-Picrylhydrazyl)—Scavenging Activity
Scavenging of Superoxide Radical
To the reaction mixture containing 0.1 ml of NBT (1 mg/mL solution in DMSO) and 0.3 ml of the extracts, the compound and standard in dimethyl sulphoxide (DMSO), 1 ml of alkaline DMSO (1 ml DMSO containing, 5 mM NaOH in 0.1 ml water) was added to give a final volume of 1.4 ml and the absorbance was measured at 560 nm .
Scavenging of Nitric Oxide Radical
Scavenging of nitric oxide (NO) radical was determined by incubating sodium nitroprusside (SNP) (5 mM, in PBS) with different concentrations of Moringa oleifera leaf extract at 25 °C. After 120 min, 0.5 ml of the incubation solution was withdrawn and mixed with 0.5 ml of griess reagent . The absorbance was measured at 550 nm. Percent inhibition of the nitric oxide generated was measured by comparing the absorbance values of control and test preparations. Cur cumin was used as a reference standard.
Lipid Peroxidation (LPO)
LPO was induced and assayed in goat liver homogenates according to the method where  the reaction mixture, in a total volume of 1.0 ml, contained 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml of liver homogenate (10%, w/v), 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric chloride (100 mM) was incubated at 37 °C in a shaking water bath for 1 h. The reaction was stopped by the addition of 1.0 ml TCA (10%, w/v). Then, 1.0 ml of TBA (0.67%, w/v) was added and all the tubes were placed in a boiling water bath for 20 min. At the end, the tubes were shifted to an ice-bath and centrifuged at 2,500 × g for 10 min. The amount of malondialdehyde formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm against a reagent blank. The molar extinction coefficient for MDA was taken to be 1.56 × 105 M−1 cm−1.
DNA Damage Using λ DNA
The extent of DNA damage induced in λ DNA was followed by the difference in migration pattern on agarose gel . The reaction was conducted in a total volume of 30 μl containing 5 μl of tris buffer, 5 μl of λ phage DNA and 5 μl of plant extract prepared in tris buffer. Then, 10 μl of H2O2 and 5 μl of FeCl3 were added and incubated at 37 °C for 30 min. The reaction mixture was then mixed with 6 μl of gel loading dye, loaded into 1% agarose gel and run at 100 V for 15 min in a submarine gel electrophoretic apparatus. The DNA was visualized and photographed using an Alpha Digidoc digital gel documentation system.
Total Phenolics and Flavonoids
Antioxidant compounds generally contain phenolic group(s) and hence, the amounts of phenolic compounds in the extracts of the leaves were estimated by using Folin–Ciocalteau reagent . In a series of test tubes, 0.4 ml of the extract in methanol was taken, mixed with 2 ml of Folin–Ciocalteau reagent and 1.6 ml of sodium carbonate. After shaking, it was kept for 2 h and the absorbance was measured at 750 nm using a Shimadzu-UV-160 spectrophotometer. Using gallic acid monohydrate, a standard curve was prepared. The linearity obtained was in the range of 1–10 μg/ml. Using the standard curve, the total phenolic compounds content was calculated and expressed as gallic acid equivalent in mg/g of extracts.
Flavonoids were extracted and estimated by the method where  an aliquot of the extract was pipetted out and evaporated to dryness. 4.0 ml of vanillin reagent was added and heated for 15 min in a boiling water bath. The standard was also treated in the same manner. The optical density was read at 340 nm. The values are expressed as mg flavonoids/g leaf.
TLC of Alkaloids, Phenolics and Flavonoids
The extracted fractions of Moringa oleifera leaves of both mature and tender are subjected to TLC on silica gel G60 F254 plates (Merck) as described . The alkaloid fraction was developed with CH2Cl2: ethanol: 28% NH4OH (85:14:1) and sprayed with Dragendroff’s reagent. Phenolics were separated with acetic acid: chloroform (45:55) and flavonoids with n-butanol:acetic acid:water (4:1:5) and both were detected with vanillin-H2SO4 (10% vanillin in ethanol: concentrated sulphuric acid in 2:1 ratio) spray reagent. The Rf values of the spots were calculated as the ratio of the distance traveled by the solute to that by the solvent front.
The data were subjected to statistical analysis to verify and evaluate the difference between the antioxidant activities of the two leaf extracts. The data were expressed as mean ±S.D (n = 6) where ‘n’ represents the no. of samples. Results were analyzed statistically by one-way ANOVA, followed by post hoc analysis using Fischer’s LSD, Sigma Stat statistical package (Version 3.1). The difference was considered significant if P < 0.05.
Enzymatic Antioxidants and Non-enzymatic Antioxidants
Activities of enzymatic antioxidants in Moringa oleifera leaves
14.64 ± 0.01d
13.64 ± 0.02
106.84 ± 0.07d
71.06 ± 0.02
163.68 ± 2.36d
142.22 ± 0.37
0.30 ± 0.008d
0.2 ± 0.008d
Levels of non-enzymatic antioxidants in Moringa oleifera leaves
Ascorbic acid (mg/g)
6.60 ± 0.01a
5.81 ± 0.01
6.53 ± 0.01a
5.63 ± 0.008
Total carotenoids (mg/g)
92.38 ± 0.11a
85.20 ± 0.14
DPPH Free Radical Scavenging Activity
Antioxidant profiles of Moringa oleifera (Concentration in micrograms (μg /ml) needed for 50% inhibition)
Antioxidant and radical scavenging activities of Moringa oleifera extract
IC 50 (µg/ml) extract/standard
Mature leaf extract
Tender leaf extract
λDNA damage assay
72.45 ± 0.23a
82.63 ± 0.51
DPPH scavenging activity
18.15 ± 0.92a
19.12 ± 0.75
Superoxide scavenging activity
12.71 ± 0.15a
15.51 ± 0.25
Nitric oxide scavenging activity
56.77 ± 0.45a
65.88 ± 0.65
25.32 ± 0.54a
30.15 ± 0.23
Superoxide Anion and Nitric Oxide Radical Scavenging Activity
Moringa oleifera leaf extract scavenged O2− significantly as shown in Table 3. The mature leaf extract (12 µg) was found to be better scavenger than the tender leaf extract. IC50 value of the standard ascorbic acid was achieved at 9 µg concentration. Moringa oleifera leaf extract also quenched NO released by a NO donor, SNP (sodium nitro prusside). The extract effectively decreased the release of NO. In comparison the IC50 value of the standard curcumin was achieved at 51 µg concentration. It is evident from this observation that Moringa oleifera leaves possess active components that seem to contribute to radical scavenging activity. However the activity remains to be below the standards as observed.
The lipids in membrane are continuously subjected to oxidant challenges. Oxidant induced abstraction of a hydrogen atom from an unsaturated fatty acyl chain of membrane lipids initiates the process of LPO, which propagates as a chain reaction. In the process, cyclic peroxides, lipid peroxides and cyclic end peroxides are generated, which ultimately are fragmented into aldehydes like MDA. MDA forms a pink chromogen with TBA that absorbs at 535 nm. Moringa oleifera leaf extract inhibited the amount of MDA generated (and thus lipid per oxidation) in liver homogenate which is presented in Table 3. Thus, the decrease in the MDA level in the leaf extracts indicates the role of the extracts as an antioxidant where mature leaf extract showed higher inhibition than the tender leaf extract.
Extent of DNA Damage
Amount of Total Phenolic Compounds
Total phenolic & flavonoid contents in Moringa oleifera leaf extract
Extract/standard contents (mg/g)
Total phenolic contents
Mature leaf extract
45.81 ± 0.02
27 ± 0.03
Tender leaf extract
36.02 ± 0.01
15 ± 0.02
Phytochemical screening was conducted in order to identify the chemical nature of active principles possibly rendering antioxidant protection. Qualitative analysis of the extracts revealed the presence of phenolics, flavonoids and trace amounts of alkaloids, in both mature and tender leaves. Since phenolics and flavonoids were found to be maximum, the phenolics and flavonoid fractions were subjected to TLC in different solvent mixtures, acetic acid : chloroform (1:9) for phenolics, and n-butanol : acetic acid : water (4:1:5) for flavonoids and developed with the respective spraying reagent as described in methodology. Mature leaves showed two spots (Rf values 0.68 and 0.85) for phenolics and two spots (Rf values of 0.75, 0.80) for flavonoids and a single spot for alkaloids (Rf value of 0.72). Tender leaves also showed two spots (Rf values of 0.68, 0.84) for phenolics and two spots (Rf values of 0.76 and 0.85) for flavonoids and a faint spot for alkaloids. The phenolic compounds may contribute directly to the antioxidative action . Thus, the antioxidant properties of Moringa oleifera may possibly be attributed to the phenolic compounds present.
Plant extracts and plant-derived antioxidants can elicit a number of in vivo effects such as promotion of increased synthesis of endogenous antioxidant defenses or themselves acting directly as antioxidants . It is also reported that the composition of antioxidants varies widely with several factors like the stage of maturity, variety, climatic conditions, part of the plant analyzed, post-harvest handling, processing, and storage . The results in the present study also show that the antioxidants vary with the stage of maturity in Moringa oleifera leaves. Zimmermann and Zentgraf  have reported that the components of both the enzymatic and the non-enzymatic antioxidant system correlate well with oxidative stress during senescence and plant development. SOD and CAT activities declined in Triticum aestivum leaves upon senescence  and with maturity of blackberry . The effects of senescence and aging on the expression of antioxidant gene products have been studied in many plants which report the variation in levels of antioxidants [43, 44]. In the present study, the activities of all the enzymatic antioxidants studied showed higher values at the mature stage than the tender stage. Thus, in light of the reports, it is clear that the response of enzymatic antioxidant components to maturity process is not uniform in all the plants.
Similarly, higher levels of total phenolics, total flavonoids and antioxidant capacity were observed in spinach leaves at the mid-maturity stage, compared to the immature stage . The levels of chlorophyll and carotenoids in the leaves of Zea mays have been shown to be dependent on age . Total carotenoids increased with maturity in pineapple  and in guava . Chlorophyll content increased with maturity in Cistus chesli plants . In a sweet pepper variety; it was shown that red ripe fruits had the highest content of vitamin C and provitamin A compared to the immature green peppers . β-carotene and α-tocopherol increased with maturity in the leaves of Pistacia lentiscus, while ascorbate levels showed no difference . Our results show that the non-enzymatic antioxidants increase with maturity in Moringa oleifera leaves. From our results, it can be deduced that the best stage of Moringa oleifera suited for consumption is the mature stage, when the maximum benefit of the antioxidant content can be derived.
The antioxidants react with DPPH, a purple colored stable free radical and convert it into a colorless α-α-diphenyl-β-picryl hydrazine. Antioxidants, on interaction with DPPH, either transfer an electron or hydrogen atom to DPPH, thus neutralizing its free radical character . Moringa oleifera leaf extract significantly reduced DPPH radicals. The degree of discoloration indicates the scavenging potential of the antioxidant extract, which is due to the radical scavenging ability. Superoxide anion radical (O2−) is a precursor to active free radicals that have the potential of reacting with biological macromolecules and there by inducing tissue damage . NO, is a key signaling molecule in physiological and pathological conditions and when commonly consumed vegetables were screened for their inhibitory effect on NO production, the aqueous extracts of several of them exhibited over 80% inhibition . Therefore, Moringa oleifera leaf extracts can significantly scavenge free radicals and hence inhibit cellular damage.
LPO has been used as a reliable marker of oxidative stress, both in vitro and in vivo. Several plant extracts have been shown to inhibit LPO as measured by the levels of TBARS. Polyphenols other than vitamin E have been known to exert powerful antioxidant effect in vitro. They inhibit lipid per oxidation by acting as chain-breaking peroxyl-radical scavengers, and can protect LDL from oxidation . It has been found in the present study that the Moringa oleifera leaf extract contains polyphenols, therefore the antioxidant effects of the leaf extract may depend on its phenolic components.
DNA contains reactive group in its bases that are highly susceptible to free radical attack . H2O2 plays an important role in the generation of free radical induced DNA damage, and mutations . Many studies have reported the protection against oxidative DNA damage by herbal extracts and formulations. Oxidative damage mediated as single strand breaks in super coiled PTZ18U plasmid DNA has been reported to be suppressed by 6-gingerol (a phenolic compound in ginger) . Methanolic extracts of Nelumbo nucifera inhibited H2O2-induced damage to pUC18 DNA . Oxidative DNA damage has been shown to be reverted upon treatment with many antioxidants. A reduction in the basal levels of oxidative DNA damage upon treatment with 4-coumaric and protocatechuic acids were reported . The results of the present study are also in agreement with the above reports. These findings support the use of the leaves of Moringa oleifera to protect against oxidative DNA damage.
Natural antioxidants that are present in herbs are responsible for inhibiting or preventing the deleterious consequences of oxidative stress. Herbs contain free radical scavengers like polyphenols, flavonoids and phenolic compounds. A number of scientific reports indicate certain terpenoids, steroids and phenolic compounds such as tannins, coumarins and flavonoids have protective effects due to its antioxidant properties . Phenolics are the most wide spread secondary metabolite in plant kingdom. These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelator. It has been reported that the antioxidant activity of phenol is mainly due to their redox properties, hydrogen donors and singlet oxygen quenchers . Several studies have shown that the higher antioxidant activity associated with medicinal plants is attributed to the total phenolic compounds . Thus, the outcome of the present study highlights the antioxidant effects rendered by mature and tender leaf extracts of Moringa oleifera leaves under oxidative stress conditions.
Overall, it could be concluded that Moringa oleifera leaves bear a potent antioxidant activity. The analysis revealed only minor differences in the antioxidant activity in the two maturity stages, mature and tender leaves. Their constituents scavenge free radicals and exert a protective effect against oxidative damage induced to cellular macromolecules. The antioxidant potential may be attributed to the presence of polyphenolic compounds and consumption of both mature and tender leaves, and might be equally beneficial to human antioxidant protection system against oxidative damage. These results are encouraging enough to pursue characterization of these fractions.