Research J. Pharm. and Tech.

5(12): December 2012

ISSN 0974-3618 RESEARCH ARTICLE

www.rjptonline.org

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: kaiserpharm_1134@yahoo.com, kaiserpharm@gmail.com
2* 1

ABSTRACT:
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 (g/ml) respectively. The IC50 value of standard (Ascorbic acid) was 33.77 (g/ml). It was observed that ethyl acetate fraction of fruit contain highest amount of phenolics (613.0234.11108) expressed as mg Gallic acid equivalents (GAE)/gm of plant extract. In case of total flavonoid content, pet ether fraction of bark contains 2453.116.4443 (expressed as mg of quercetin equivalents/gm of plant extract). All fractions contains significant amount of ascorbic acid equivalents ranging from 445.5810.8222-1162.4482.222 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 g/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]. -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].

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method described by Braca et al., 200127. Plant extract (0.1 ml) was added to 3 ml of a 0.004% methanol solution of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage inhibition activity was calculated from [(A0A1)/A0] x100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/ standard. The inhibition curves were prepared and IC50 values were Almost all the parts of this plants have been used for calculated. various ailments in the indigenous medicine of South Asia, including the treatment of inflammation and infectious Nitric oxide scavenging assay diseases along with cardiovascular, gastrointestinal, Nitric oxide radical scavenging was estimated on the basis hematological and hepatorenal disorders [14, 15]. Leaves of of Griess Illosvoy reaction using method followed by M. oleifera have been reported to regulate thyroid status Govindarajan et al., 200328. In this investigation, Griessand possess radioprotective [16] and antitumor activities [17]. Illosvoy reagent was modified by using naphthyl ethylene Fruits are found to have hypocholesterolaemic activity in diamine dihydrochloride (0.1% w/v) instead of 1Wistar rats and rabbits, respectively [18, 19]. Pod has been napthylamine (5 %). The reaction mixture (3 ml) containing reported to shown hypotensive effect [20]. Seeds have been sodium nitroprusside (10 mM, 2 ml), phosphate buffer reported for coagulative, antimicrobial and antitumor saline (0.5 ml) and plant extract (5 to 250 g/ml) or activity [21]. Roots possess antimicrobial and anti- standard solution (ascorbic acid, 0.5 ml) was incubated at inflammatory activity [22]. 25 C for 150 min. After incubation, 0.5 ml of the reaction mixture mixed with 1 ml of sulfanilic acid reagent (0.33% M. oleifera Lam. has been reported to contain various in 20% glacial acetic acid) and allowed to stand for 5 min phytochemicals which includes carotenoids, vitamins, for completing diazotization. Then 1 ml of naphthyl minerals, amino acids, sterols, glycosides, alkaloids, ethylene diamine dihydrochloride was added, mixed and flavonoids and phenolics [20, 23]. We are investigating the allowed to stand for 30 min at 25C. A pink coloured medicinal plants of Bangladesh for their antioxidant, chromophore formed in diffused light. The absorbance of cytotoxic, antimicrobial and antidiabetic activity. these solutions was measured at 540 nm against the Previously we reported the antioxidant activity of Ipomoea corresponding blank solutions. aquatica, Pandanus odorus, Ficus rasemosa seeds [24-26]. To the best of our knowledge, antioxidant activity of the leaves Determination of total antioxidant capacity of M. oleifera Lam. has been reported earlier. But, 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, bark and fruits of this plant and at the described by Prieto et al., 199929. 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. formation of a green phosphate/Mo (V) complex at acidic pH. A 0.3 ml extract was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and EXPERIMENTAL: 4 mM ammonium molybdate). The tubes containing the Plant materials Different parts of the test plant was collected during the reaction solution were incubated at 95C for 90 min. Then month of January, 2010 from Ramnagar, Comilla, 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, Dhaka where a voucher specimen was room temperature. Methanol (0.3 ml) in the place of extract is used as the blank. The antioxidant activity is expressed as deposited having the accession no. 35199 the number of equivalents of ascorbic acid. Preparation of Crude Plant Extract About 200 g of dried, ground separate parts of the plant Total phenols determination phenols were determined by Folin Ciocalteu were soaked in 1.5 L of 98% methanol for 5-7 days, stirring Total 30 every 18 h using a sterilized glass rod, separately. The final reagent . A dilute extract of each plant extract (0.5 ml of extracts were passed through No. 1 Whatman filter paper 1:10 g ml-1) or gallic acid (standard phenolic compound) (Whatman Ltd., UK) that is followed by solvent-solvent was mixed with Folin Ciocalteu reagent (5 ml, 1:10 diluted partitioning with petroleum ether, chloroform and ethyl with distilled water) and aqueous Na2CO3 (4 ml, 1 M). The acetate. 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 4C phenols were determined by colorimetry at 765 nm. The standard curve was prepared using 0, 50, 100, 150, 200, 250 for further use. mg/L solutions of gallic acid in methanol: water (50:50, v/v). Total phenol values are expressed in terms of gallic Tests For Antioxidant Activity acid equivalent (mg/g of dry mass), which is a common DPPH radical scavenging activity The free radical scavenging activity of the extract, based on reference compound. the scavenging activity of the stable 1, 1-diphenyl-2picrylhydrazyl (DPPH) free radical was determined by the Moringa oleifera Lam. commonly known as sajna in Bangladesh, a member of the family Moringaceae, is a small-medium sized tree, 1015m high, widely cultivated in East and Southeast Asia, Polynesia and the West Indies. Different parts of the M. oleifera tree are reported to possess various pharmacological actions.

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Research J. Pharm. and Tech. 5(12): December 2012 Table 1: DPPH free radical scavenging by Moringa oleifera lam Sample IC50 values(g\ml) Petroleum ether fraction 74.78425197 Ethyl acetate fraction 29.91105464 Leaf Chloroform fraction 47.59137344 Petroleum ether fraction 105.2477876 Ethyl acetate fraction 14.47263017 Bark Chloroform fraction 28.02124834 Petroleum ether fraction 166.0771704 Fruit Ethyl acetate fraction 58.15717822 Chloroform fraction 45.80625 Standard Ascorbic acid 33.7712895 R2 R = 0.927 R = 0.740 R = 0.948 R = 0.989 R = 0.537 R = 0.647 R = 0.858 R = 0.984 R = 0.848 R = 0.819

Regression equation y = 0.635x + 2.512 y = 0.787x + 26.46 y = 0.881x + 8.072 y = 0.452x + 2.428 y = 0.749x + 39.16 y = 0.753x + 28.90 y = 0.311x - 1.650 y = 0.808x + 3.009 y = 0.960x + 6.026 y = 0.822x + 22.24

Table 2: Determination of Total Phenol Content, Total Flavonoid content and Total Antioxidant content of Moringa oleifera lam. 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 107.2090.8221 359.530.82221 604.3028.2222 Chloroform Fraction 7.791.6443 3.7212.46665 631.6287.4 Pet Ether Fraction 7.2090.82221 47.3261.64443 560.6984.1111 Fruit Ethyl Acetate Fraction 613.0234.11108 6.0470.82221 983.9530.8222 Chloroform Fraction 27.5586.57773 208.370.82221 445.5810.8222 Pet Ether Fraction 9.5350.82221 8.3920.82221 440.34914.8 Bark Ethyl Acetate Fraction 387.4422.46665 16.5120.82221 988.023164.44 Chloroform Fraction 157.7911.64443 202.46665 1162.4482.222 Pet Ether Fraction 14.1860.82221 2453.116.4443 986.2790.8222 Table 3: Reducing Power capacity assessment of Moringa oleifera lam Leaf Bark Concen tration (g\ml) Absorban ce of Pet Ether fraction 0.163 0.229 0.401 0.568 1.156 Absorban ce of Ethyl Acetate Fraction 0.158 0.326 0.657 0.75 1.36 Absorban ce of Chlorofor m Fraction 0.17 0.372 0.545 1.19 1.992 Absorban ce of Pet Ether fraction 0.47 0.568 0.594 0.805 0.924 Absorban ce of Ethyl Acetate Fraction 0.519 0.523 0.67 0.849 1.183 Absorban ce of Chlorofor m Fraction 0.543 0.803 1.009 1.627 2.487

Fruit Absorban ce of Pet Ether fraction 0.579 0.584 0.529 0.65 0.79 Absorban ce of Ethyl Acetate Fraction 0.536 0.589 0.729 1.148 1.474 Absorbanc e of Chlorofor m Fraction 0.147 0.295 0.388 0.716 1.172

Ascorb ic Acid A(STD)

5 25 50 100 200

0.2895 0.394 0.538 0.7755 1.0155

Total flavonoids determination Aluminum chloride colorimetric method was used for flavonoids determination31. Each plant extracts (0.5 ml of 1:10 g ml-1) in methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solutions at concentrations 12.5 to 100 g/ ml in methanol. Reducing power The reducing power of the extracts was determined according to the method of Oyaizu, 198632. Different amounts of extracts (50250 mg) in 1ml of methanol were mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50 1C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged (650 g at room temperature) for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and

the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

RESULTS:
DPPH Free Radical scavenging activity: Leaf and bark of Moringa oleifera lam possess greater DPPH free radical scavenging capacity than fruit. Ethyl acetate fraction of both bark and leaves showed potent free radical scavenging activity with IC50 value of 14.47 and 29.91 (g/ml) respectively. In case of fruit it was 58.15 (g/ml) (Table 1). The IC50 value of standard (Ascorbic acid) was 33.77 (g/ml) Total Phenol, Total Flavonoid and Total Antioxidant Content Ethyl acetate fraction of leaf, bark and fruit part of Moringa oleifera lam contains 107.2090.8221, 387.4422.46665 and 613.0234.11108 mg Gallic acid equivalents (GAE)\gm of plant extract respectively.

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Research J. Pharm. and Tech. 5(12): December 2012 Table 4: Nitric Oxide Free Radical Scavenging by Moringa oleifera lam Sample IC50 values(g\ml) Leaf Petroleum ether fraction 152.1333 Ethyl acetate fraction 27.73234 Chloroform fraction 72.83951 Bark Petroleum ether fraction 18.75 Ethyl acetate fraction 8.326693 Chloroform fraction 9.322034 Fruit Petroleum ether fraction 110.5279 Ethyl acetate fraction 2.900433 Chloroform fraction 87.83688 Standard Ascorbic acid 71.05505 R2 R = 0.368 R = 0.437 R = 0.504 R = 0.443 R = 0.357 R = 0.328 R = 0.721 R = 0.306 R = 0.669 R = 0.739

Regression equation Y = 0.150x + 27.18 Y = 0.269x + 42.54 Y = 0.243x + 32.30 Y = 0.296x + 44.45 Y = 0.251x + 47.91 Y = 0.236x + 47.8 Y = 0.341x + 12.31 Y = 0.231x + 49.33 Y = 0.282x + 25.23 Y = 0.348x + 25.21

Ethyl acetate fraction of leaf, chloroform fraction of fruit and pet ether fraction of bark contains 359.530.82221, 208.370.82221, 2453.116.4443 mg of quercetin equivalents/gm of plant extract. All fractions contains significant amount of ascorbic acid equivalents ranging from 445.5810.8222-1162.4482.222 as mg of ascorbic acid equivalents per gram of plant extract (Table 2). Reducing Power Capacity Assessment All extracts showed increase in the absorbance with increase in concentration (Table 3). 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 2.9-87.83 g/ml. Ethyl acetate and chloroform fractions of leaf part also possess small IC50 values (Table 4).

M. oleifera showed to contain highest total phenol with the value of 613.0234.11108 which expressed as mg Gallic acid equivalents (GAE)/gm of plant extract. In case of total flavonoids, the Pet ether fraction of bark contain highest amount (2453.116.4443 expressed as mg of Quercetin equivalents/gm of plant extract). The chloroform fraction of the bark showed to contain highest amount of total amount of total antioxidant (1162.4482.222 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, 40. 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. Thus, it is necessary to determine the reducing power of phenolic constituents to elucidate the relationship between their antioxidant effect and their reducing power. The reducing power of the extracts increased with an increase in concentration. The above findings do not correlate the amount of phenolics with the reducing power of the extracts. Because the highest reducing power was observed with the chloroform fractions of the bark. However, 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. Furthermore, the presence of a negligible concentration of ascorbic acid in the respective extracts contributed to the effectiveness of phenolics-induced reducing power. This observed antioxidant properties of extracts of different parts of M. oleifera may be connected with the polyphenol profile42-44. However other workers have also reported the presence of other antioxidants like b-carotene, vitamin A, and vitamin E in different parts of the plant45-51. Several studies have amply demonstrated that quercetin and kaempferol43, 52-54 as well as chlorogenic acid55 and their derivatives, which were also detected in the leaf extract of M. oleifera, possess remarkable antioxidant activities. Quercetin is a strong antioxidant because it can chelate metals, scavenge oxygen free radicals, and prevent the oxidation of low-density lipoprotein52, 53.

DISCUSSION:
In the present study we investigated and compared the invitro antioxidant activity of different parts and fractions of M. oleifera lam. DPPH is a stable free radical with characteristic absorption at 517 nm and antioxidants react with DPPH and convert it to 2,2-diphenyl-1picrylhydrazine. The degree of discoloration indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating or radical scavenging ability33. Among all the fractions assayed, the ethylacetate fractions of the leaves of M. oleifera lam showed highest radical scavenging activity with IC50 value of 14.47 g/ml which is followed by the chloroform fraction of bark IC50 value of 28.02 g/ml. This observed result is similar to the findings by Atawodi et al., 201034. Flavonoids are one of the most diverse and widespread group of natural compounds, are probably the most important natural phenolics. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging properties35. Phenolic compounds undergo a36 complex redox reaction with phosphotungstic and phosphomolybdic acids present in the Folin- Ciocalteu reagent37. However, the phenolic and flavonoid compounds may contribute directly to antioxidant potential38; therefore, it would be valuable to determine the total phenolics content (TPC) and total flavonoids content (TFC) of extracts. The ethylacetate fraction of the fruit of

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