Research J. Pharm. and Tech.

5(12): December 2012


In vitro Antioxidant Activities of Different parts of the Plant Moringa oleifera Lam.
Nurul Huda Md. Masum1, Kaiser Hamid2*, Abu Hasanat Md. Zulfiker3, Md. Kamal Hossain4, Kaniz Fatima Urmi5
Department of Pharmacy, Southeast University, Bangladesh Lecturer, Department of Pharmacy, East West University, Bangladesh 3 Department of Pharmacy, Southeast University, Bangladesh 4 Vetafarm Manufacturing Pty. Ltd, Wagga Wagga, NSW, Australia 5 Department of Pharmacy, Jahangirnagar University, Bangladesh *Corresponding Author E-mail:,
2* 1

The aim of the present study was to evaluate and compare the in vitro antioxidant activity of different parts of the plant Moringa oleifera lam. Antioxidant activity was measured based on the DPPH radical scavenging assay, Nitric oxide scavenging assay, determination of total phenol, total flavonoids, total antioxidant content and reducing power assay. Ethyl acetate fraction of both bark and leaves showed potent free radical scavenging activity with IC50 value of 14.47 and 29.91 (µg/ml) respectively. The IC50 value of standard (Ascorbic acid) was 33.77 (µg/ml). It was observed that ethyl acetate fraction of fruit contain highest amount of phenolics (613.023±4.11108) expressed as mg Gallic acid equivalents (GAE)/gm of plant extract. In case of total flavonoid content, pet ether fraction of bark contains 2453.1±16.4443 (expressed as mg of quercetin equivalents/gm of plant extract). All fractions contains significant amount of ascorbic acid equivalents ranging from 445.581±0.8222-1162.44±82.222 as mg of ascorbic acid equivalents per gram of plant extract. In case of nitric oxide scavenging assay, ethyl acetate and chloroform fractions of bark and fruit of Moringa oleifera showed potential antioxidant effect having IC50 values ranging from 2.9-87.83 µg/ml. For reducing power assay, all extracts showed increase in the absorbance with increase in concentration. The present study supplements and at the same time compared the presence of antioxidant compounds in different parts of the plant M. oleifera. The next step is to identify the individual compound and their role in different chronic diseases.

KEYWORDS: Antioxidant, Moringa oleifera Lam and different parts. INTRODUCTION:
The most widely used synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are suspected to cause some safety concerns. For this reasons in spite of having marked antioxidant activity, these antioxidants have been restricted recently. Because such materials may cause liver swelling and influence liver enzyme activities [6, 7]. α-Tocopherol, a natural antioxidant, is an effective antioxidant for lipid-containing foods but has limited usage [8]. The fact that various antioxidants occur [9-11] . Therefore, Antioxidants are substances that could attenuate this naturally in plants has been proven oxidative damage of a tissue indirectly by enhancing natural identification and development of safer, natural antioxidants defenses of cell and/or directly by scavenging the free is more beneficial. Moreover, there is also a considerable amount of evidence revealing an association between radical species[4-5]. individuals who have a diet rich in fresh fruits and vegetables and the decreased risk of cardiovascular diseases and certain forms of cancer [12,13], and it is generally Received on 01.10.2012 Modified on 18.10.2012 assumed that these dietary elements, responsible for the Accepted on 25.10.2012 © RJPT All right reserved protective effects, are antioxidant nutrients. Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1532-1537 Excessive free radicals (commonly designated as reactive oxygen species, ROS) production has been ascertained to play multiple roles in tissue damage and loss of function in a number of tissues and organs [1]. Subsequently, these free radicals contribute to more than one hundred disorders in humans including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer and AIDS [2, 3].


Then 1 ml of naphthyl minerals. which is a common DPPH radical scavenging activity The free radical scavenging activity of the extract. 1 Whatman filter paper 1:10 g ml-1) or gallic acid (standard phenolic compound) (Whatman Ltd. A 0. glycosides. and A1 is the absorbance of the extract/ standard. Plant extract (0. Total phenol values are expressed in terms of gallic Tests For Antioxidant Activity acid equivalent (mg/g of dry mass). where A0 is the absorbance of the control. antimicrobial and antidiabetic activity.3 ml) in the place of extract is used as the blank. 25 °C for 150 min. Absorbance at 517 nm was determined after 30 min. The antioxidant activity is expressed as deposited having the accession no. Preparation of Crude Plant Extract About 200 g of dried.6 M sulfuric acid.5 ml of the reaction mixture mixed with 1 ml of sulfanilic acid reagent (0.5 ml) was incubated at inflammatory activity [22]. 1:10 diluted partitioning with petroleum ether.. these solutions was measured at 540 nm against the Previously we reported the antioxidant activity of Ipomoea corresponding blank solutions. ground separate parts of the plant Total phenols determination phenols were determined by Folin Ciocalteu were soaked in 1. 199929. respectively [18.5 L of 98% methanol for 5-7 days. oleifera Lam. 0. The absorbance of cytotoxic. vitamins. a member of the family Moringaceae. 15]. chromophore formed in diffused light. 28 mM sodium phosphate and EXPERIMENTAL: 4 mM ammonium molybdate). The reaction mixture (3 ml) containing reported to shown hypotensive effect [20]. is a small-medium sized tree. 1-diphenyl-2picrylhydrazyl (DPPH) free radical was determined by the Moringa oleifera Lam. The tubes containing the Plant materials Different parts of the test plant was collected during the reaction solution were incubated at 95°C for 90 min.004% methanol solution of DPPH. Pandanus odorus. based on reference compound. Griessand possess radioprotective [16] and antitumor activities [17].33% M. 50. phosphate buffer reported for coagulative. A dilute extract of each plant extract (0. 0. various ailments in the indigenous medicine of South Asia. Then month of January. oleifera have been reported to regulate thyroid status Govindarajan et al. oleifera Lam. 2010 from Ramnagar.. Methanol (0. Nitric oxide radical scavenging was estimated on the basis hematological and hepatorenal disorders [14. gastrointestinal.5 ml of extracts were passed through No. amino acids.5 ml) and plant extract (5 to 250 µg/ml) or activity [21]. Polynesia and the West Indies. 19]. The standard curve was prepared using 0. 200. including the treatment of inflammation and infectious Nitric oxide scavenging assay diseases along with cardiovascular.1% w/v) instead of 1Wistar rats and rabbits. commonly known as “sajna” in Bangladesh. To the best of our knowledge. Seeds have been sodium nitroprusside (10 mM.standard solution (ascorbic acid. 250 for further use. The inhibition curves were prepared and IC50 values were Almost all the parts of this plants have been used for calculated. formation of a green phosphate/Mo (V) complex at acidic pH. separately. Comilla. Dhaka where a voucher specimen was room temperature. The final reagent . v/v)..1 ml) was added to 3 ml of a 0. We are investigating the allowed to stand for 30 min at 25°C. UK) that is followed by solvent-solvent was mixed with Folin Ciocalteu reagent (5 ml. the scavenging activity of the stable 1. and the percentage inhibition activity was calculated from [(A0–A1)/A0] x100.Research J. this The antioxidant activity of the extract was evaluated by the article is the first of its kind reporting the antioxidant phosphomolybdenum method according to the procedure activity of the leaves. The assay is based on the same time comparing their potentiality of the mentioned reduction of Mo (VI)–Mo (V) by the extract and subsequent effect. Different parts of the M. 200127. 2 ml). has been reported to contain various in 20% glacial acetic acid) and allowed to stand for 5 min phytochemicals which includes carotenoids. 1533 . After incubation. 1 M). for completing diazotization. aquatica.. 35199 the number of equivalents of ascorbic acid. The filtrates obtained were concentrated under mixtures were allowed to stand for 15 min and the total vacuum in a rotary evaporator at 40 °C and stored at 4°C phenols were determined by colorimetry at 765 nm. But. widely cultivated in East and Southeast Asia. antimicrobial and antitumor saline (0. the absorbance of the solution was measured at 695 nm Bangladesh and identified from the Bangladesh National using a spectrophotometer against blank after cooling to Herbarium. 200328. chloroform and ethyl with distilled water) and aqueous Na2CO3 (4 ml. antioxidant activity of the leaves Determination of total antioxidant capacity of M. 150. Illosvoy reagent was modified by using naphthyl ethylene Fruits are found to have hypocholesterolaemic activity in diamine dihydrochloride (0. Leaves of of Griess Illosvoy reaction using method followed by M. ethylene diamine dihydrochloride was added. has been reported earlier. In this investigation. sterols. and Tech. 100. Pharm. alkaloids. 23]. mg/L solutions of gallic acid in methanol: water (50:50. 10–15m high. Ficus rasemosa seeds [24-26]. oleifera tree are reported to possess various pharmacological actions. 5(12): December 2012 method described by Braca et al.3 ml extract was combined with 3 ml of reagent solution (0. Roots possess antimicrobial and anti. mixed and flavonoids and phenolics [20. The acetate. A pink coloured medicinal plants of Bangladesh for their antioxidant. bark and fruits of this plant and at the described by Prieto et al. stirring Total 30 every 18 h using a sterilized glass rod. Pod has been napthylamine (5 %).

6443 3.302±8.5 ml) of trichloroacetic acid (10%) was added to the mixture.388 0.805 0.91105464 Leaf Chloroform fraction 47.452x + 2. The mixture was incubated at 50 1C for 20 min.657 0.Research J. A portion (2.2895 0.59137344 Petroleum ether fraction 105. 0.24 Table 2: Determination of Total Phenol Content. Pharm.172 Ascorb ic Acid A(STD) 5 25 50 100 200 0. Each plant extracts (0.82221 604. and the absorbance was measured at 700 nm.64443 20±2.487 Fruit Absorban ce of Pet Ether fraction 0.82221 440.8221.023±164.372 0.401 0.394 0.753x + 28.628±7.82221 988.11108 6.222 Pet Ether Fraction 14.349±14.519 0.535±0. The IC50 value of standard (Ascorbic acid) was 33. Ethyl acetate fraction of both bark and leaves showed potent free radical scavenging activity with IC50 value of 14. pH 6.927 R² = 0.1%).579 0.44 Chloroform Fraction 157.819 Regression equation y = 0.808x + 3. 387.568 1.7755 1.186±0.568 0.53±0.523 0.009 1.627 2.79 Absorban ce of Ethyl Acetate Fraction 0.716 1.6) and potassium ferricyanide [K3Fe(CN)6] (2.0155 Total flavonoids determination Aluminum chloride colorimetric method was used for flavonoids determination31.5 to 100 g/ ml in methanol.67 0.82221 445.147 0.581±0.326±1. Total Flavonoid content and Total Antioxidant content of Moringa oleifera lam.948 R² = 0.183 Absorban ce of Chlorofor m Fraction 0. 0.229 0.5 ml. 5(12): December 2012 Table 1: DPPH free radical scavenging by Moringa oleifera lam Sample IC50 values(µg\ml) Petroleum ether fraction 74.36 Absorban ce of Chlorofor m Fraction 0.849 1.64443 560.442±2.2 mol/l.46 y = 0.1111 Fruit Ethyl Acetate Fraction 613.858 R² = 0.8 ml of distilled water. 0. Total Flavonoid and Total Antioxidant Content Ethyl acetate fraction of leaf.047±0.026 y = 0.17 0.749x + 39.16 y = 0.295 0.47 0.79±1.82221 983.729 1. Reducing power The reducing power of the extracts was determined according to the method of Oyaizu.072 y = 0.647 R² = 0.156 Absorban ce of Ethyl Acetate Fraction 0. Sample Total Phenol Total Flavonoid Total Antioxidant Content (expressed as mg of ascorbic Content(expressed as mg Content(expressed as mg of Gallic acid equivalents Quercetin equivalents\gm of acid equivalents per gram of (GAE)\gm of plant extract) plant extract) plant extract) Leaf Ethyl Acetate Fraction 107.650 y = 0.209±0. 198632.7712895 R2 R² = 0.442±2.44±82.787x + 26.279±0.148 1.65 0.8221 359.924 Absorban ce of Ethyl Acetate Fraction 0.2477876 Ethyl acetate fraction 14.5 ml.881x + 8.75 1.023±4.529 0.82221 2453.91 (µg/ml) respectively. In case of fruit it was 58.15717822 Chloroform fraction 45.163 0. RESULTS: DPPH Free Radical scavenging activity: Leaf and bark of Moringa oleifera lam possess greater DPPH free radical scavenging capacity than fruit.8222 Table 3: Reducing Power capacity assessment of Moringa oleifera lam Leaf Bark Concen tration (µg\ml) Absorban ce of Pet Ether fraction 0. and Tech.698±4.209±0.5 ml of 1:10 g ml-1) in methanol were separately mixed with 1.02124834 Petroleum ether fraction 166.11108 mg Gallic acid equivalents (GAE)\gm of plant extract respectively.4 Pet Ether Fraction 7.46665 and 613.80625 Standard Ascorbic acid 33. which was then centrifuged (650 g at room temperature) for 10 min. 0.2222 Chloroform Fraction 7.023±4.512 y = 0.848 R² = 0. The calibration curve was prepared by preparing quercetin solutions at concentrations 12.209±0.984 R² = 0.538 0.46665 1162.5 ml) and FeCl3 (0.57773 208.311x .1.635x + 2.15 (µg/ml) (Table 1).009 y = 0.960x + 6. It remained at room temperature for 30 min.47263017 Bark Chloroform fraction 28.82221 8.953±0.536 0.78425197 Ethyl acetate fraction 29.584 0.0771704 Fruit Ethyl acetate fraction 58.594 0.46665 16. bark and fruit part of Moringa oleifera lam contains 107.1 ml of 1 M potassium acetate and 2.512±0.392±0.8222 Chloroform Fraction 27.545 1.428 y = 0.989 R² = 0.543 0.537 R² = 0.326 0. Different amounts of extracts (50–250 mg) in 1ml of methanol were mixed with phosphate buffer (2.589 0.47 and 29.5 ml.822x + 22.4443 986. 1534 .1±16.992 Absorban ce of Pet Ether fraction 0.19 1.46665 631.37±0. The upper layer of solution (2.740 R² = 0.8222 Pet Ether Fraction 9. Increased absorbance of the reaction mixture indicated increased reducing power.791±1. 1%).5 ml of methanol. the absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible spectrophotometer (USA).5 ml) was mixed with distilled water (2.558±6.82221 47.90 y = 0.77 (µg/ml) Total Phenol.1 ml of 10% aluminum chloride.8 Bark Ethyl Acetate Fraction 387.474 Absorbanc e of Chlorofor m Fraction 0.721±2.803 1.158 0.

and Tech. which is due to the hydrogen donating or radical scavenging ability33.82221. 201034. This observed result is similar to the findings by Atawodi et al. Nitric Oxide Scavenging Capacity Ethyl acetate and Chloroform fractions of bark and fruit of Moringa oleifera showed potential antioxidant effect having IC50 values ranging from 2.4443 mg of quercetin equivalents/gm of plant extract.328 R² = 0.282x + 25.296x + 44.341x + 12.1333 Ethyl acetate fraction 27. Thus. it would be valuable to determine the total phenolics content (TPC) and total flavonoids content (TFC) of extracts.269x + 42.75 Ethyl acetate fraction 8.83688 Standard Ascorbic acid 71.251x + 47. it may due to the presence of highest total antioxidant content of this fraction that is mg of ascorbic acid equivalents per gram of plant extract which is a potent reducing agent.9-87. scavenge oxygen free radicals. The existence of reductones are the key of the reducing power which exhibit their antioxidant activities through the action of breaking the free radical chain by donating hydrogen atom41.44±82.326693 Chloroform fraction 9.443 R² = 0. Several studies have amply demonstrated that quercetin and kaempferol43. oleifera lam showed highest radical scavenging activity with IC50 value of 14.33 Y = 0.45 Y = 0. In case of total flavonoids.306 R² = 0.47 µg/ml which is followed by the chloroform fraction of bark IC50 value of 28.37±0. oleifera showed to contain highest total phenol with the value of 613.1±16.739 Regression equation Y = 0. The above findings do not correlate the amount of phenolics with the reducing power of the extracts.05505 R2 R² = 0.02 µg/ml.231x + 49.1±16. Phenolic compounds undergo a36 complex redox reaction with phosphotungstic and phosphomolybdic acids present in the Folin. and prevent the oxidation of low-density lipoprotein52. M. the ethylacetate fractions of the leaves of M.150x + 27.18 Y = 0. The reducing power of the extracts increased with an increase in concentration.11108 which expressed as mg Gallic acid equivalents (GAE)/gm of plant extract. DPPH is a stable free radical with characteristic absorption at 517 nm and antioxidants react with DPPH and convert it to 2.669 R² = 0.83951 Bark Petroleum ether fraction 18. The ethylacetate fraction of the fruit of 1535 . 208. oleifera. Flavonoids are one of the most diverse and widespread group of natural compounds. 2453.4443 expressed as mg of Quercetin equivalents/gm of plant extract). Furthermore. oleifera lam. The chloroform fraction of the bark showed to contain highest amount of total amount of total antioxidant (1162.30 Y = 0.222 as mg of ascorbic acid equivalents per gram of plant extract (Table 2).243x + 32. the phenolic and flavonoid compounds may contribute directly to antioxidant potential38.8 Y = 0.322034 Fruit Petroleum ether fraction 110. are probably the most important natural phenolics.900433 Chloroform fraction 87.236x + 47. possess remarkable antioxidant activities. However other workers have also reported the presence of other antioxidants like b-carotene.368 R² = 0.23 Y = 0. 53.82221. oleifera may be connected with the polyphenol profile42-44. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging properties35. chloroform fraction of fruit and pet ether fraction of bark contains 359. and vitamin E in different parts of the plant45-51. Reducing Power Capacity Assessment All extracts showed increase in the absorbance with increase in concentration (Table 3). However.Research J. vitamin A.31 Y = 0. The degree of discoloration indicates the scavenging potential of the antioxidant extract. 52-54 as well as chlorogenic acid55 and their derivatives.53±0.91 Y = 0.504 R² = 0.023±4.5279 Ethyl acetate fraction 2.348x + 25. All fractions contains significant amount of ascorbic acid equivalents ranging from 445.44±82.721 R² = 0. Pharm.357 R² = 0. However.8222-1162. This observed antioxidant properties of extracts of different parts of M. Because the highest reducing power was observed with the chloroform fractions of the bark. the presence of a negligible concentration of ascorbic acid in the respective extracts contributed to the effectiveness of phenolics-induced reducing power.222 expressed as mg of ascorbic acid equivalents per gram of plant extract) The reducing capacity of a compound Fe3+/ferricyanide to the ferrous form may serve as a significant indicator of its antioxidant capacity39. therefore. 40.437 R² = 0. the Pet ether fraction of bark contain highest amount (2453. 5(12): December 2012 Table 4: Nitric Oxide Free Radical Scavenging by Moringa oleifera lam Sample IC50 values(µg\ml) Leaf Petroleum ether fraction 152. Ethyl acetate and chloroform fractions of leaf part also possess small IC50 values (Table 4).2-diphenyl-1picrylhydrazine..73234 Chloroform fraction 72. Among all the fractions assayed.21 Ethyl acetate fraction of leaf.54 Y = 0. it is necessary to determine the reducing power of phenolic constituents to elucidate the relationship between their antioxidant effect and their reducing power.581±0. which were also detected in the leaf extract of M. DISCUSSION: In the present study we investigated and compared the invitro antioxidant activity of different parts and fractions of M.Ciocalteu reagent37.83 µg/ml. Quercetin is a strong antioxidant because it can chelate metals.

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