SCREENING OF SECONDARY METABOLITES, ANTIOXIDANT AND ...

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Sumathy et al. World Journal of Pharmacy and Pharmaceutical Sciences

SCREENING OF SECONDARY METABOLITES, ANTIOXIDANT AND

ANTIMICROBIAL ACTIVITY FROM THE PETALS OF MORINGA

OLEIFERA

*Sumathy R1, Vijayalakshmi. M1, Deecaraman. M1, Sankaranarayanan S2 , Bama P3.,

Ramachandran J4.,

1Department of Biotechnology, Dr. M.G.R. Educational and Research Institute University, Maduravoyal,

2Sairam Centre for Advanced Research, Sai Leo Nagar, West Tambaram, 3Department of Biochemistry, Sri Sairam Siddha Medical College and Research Centre, Sai

Leo Nagar, West Tambaram, 4Gloris BioMed Research Centre (P) Ltd, Vijayaragavapuram, Saligramam, Chennai, Tamil

Nadu, India

ABSTRACT

Moringa oleifera is a small, fast-growing evergreen or deciduous tree

and known for its various medicinal properties. The in vitro

antimicrobial activity of aqueous methanol extract of Moringa oleifera

petals was investigated against Staphylococcus aureus, Escherichia

coli, Bacillus subtilis, Pseudomonas aeruginosa and Proteus vulgaris

using the disc diffusion method. The methanol extract inhibited the

growth of all the presently investigated bacteria with zone of inhibition

between 12.4 – 23.4 mm at 20 µl/ml whereas the minimum inhibitory

effect of the methanol extract of Moringa oleifera petals was effective

at the highest concentration (20 µl/ml) against pathogenic

microorganisms. Preliminary phytochemical analysis showed that the methanol extracts of

Moringa oleifera petals contain flavonoids, tannins, saponins, alkaloids and glycosides.

Studies have shown that the petals of Moringa oleifera may be a great natural source for the

development of new drugs and might be useful for treating the bacterial diseases.

Keywords- Moringa oleifera, Antimicrobial activity, Phytochemical screening, Minimum

inhibitory concentration (MIC),

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Article Received on 16 April 2014, Revised on 08 May 2014, Accepted on 26 May 2014

*Correspondence for Author

Sumathy R

Department of Biotechnology,

Dr. M.G.R. Educational and

Research Institute University,

Maduravoyal


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INTRODUCTION

Medicinal plants have been used for centuries before the advent of orthodox medicine.

Secondary metabolites produced by plants constitute a major source of bioactive substances.

These secondary metabolites have recently been referred to as phytochemicals. The medicinal

values of the plants lie in their component phytochemicals, which produce definite

physiological actions and pathological behavior infective disease on the human body. The

most important of these phytochemicals are alkaloids, tannins, flavonoids, phenolic

compounds and around 40% of modern medicine are derived from medicinal plants [1] which

possess antibacterial properties [2]. It is reported that phenolic compounds in plants possess

strong antioxidant activity and may help to protect cells against the oxidative damage caused

by free radicals [3].

Traditional medicine using plant methanol extracts continues to provide health coverage for

over 80% of the world’s population, especially in the developing world (WHO, 2002).

Infectious diseases continue to be the major concern in all over the world (accounting for

over 50, 000 deaths every day), especially with the current increasing trends of multidrug

resistance among emerging and re-emerging bacterial pathogens to the available modern

drugs or antibiotics [4, 5]. The search for newer sources of antibiotics is a global challenge,

since many infectious agents are becoming resistant to synthetic drugs [6]. It is therefore very

necessary that the search for newer antibiotic sources be a continued process. Plants are

inexpensive and safer alternative sources of antimicrobials [7, 8, and 9]

Moringa oleifera is one of the 14 species of family Moringaceae and is commonly known as

“Drumstick”. Moringa oleifera is used as a highly nutritive vegetable in many countries. Its

young leaves, flowers, seeds and tender pods are commonly consumed and they are having

some medicinal properties. Traditionally its roots are applied as plaster to reduce the swelling

and rheumatism. The root, flower, fruit and leaf have analgesic and anti inflammatory

activity. Moringa leaves contains phytochemical having potent anticancer and hypotensive

activity and are considered full of medicinal properties and used in siddha medicine [10]. The

whole Moringa oleifera plant is used in the treatment of psychosis, eye diseases and fever.

Various parts of the plant such as the leaves, roots, seed, bark, fruit, flowers and immature

pods act as cardiac and circulatory stimulants, possess antitumour, antipyretic, antiepileptic,

anti- inflammatory, antiulcer activity [11]. The flowers and roots contain an antibiotic that is

highly effective in the treatment of cholera .The juice of the leaves is believed to stabilize


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blood pressure, flowers are used to cure inflammations, pods are used for joint pain, and the

roots are used to treat rheumatism [12]

In view of the importance of Moringa oleifera in ethanobotany as health remedy and the

antimicrobial property of crude extracts of the petals of Moringa oleifera has been studied as

part of the exploration for new and novel bio-active compounds

MATERIALS AND METHODS

Collection of plant material

The petals of Moringa oleifera were collected from the Sri Sairam Siddha Medical College

and Research Centre, Herbal garden, Tamilnadu, India. It was ensured that the plant was

healthy and uninfected. The plant was identified with the help of available literature and

authenticated by Dr. S. Sankaranarayanan, Head of the Department, Department of Medicinal

Botany, Sri Sairam Siddha Medical College, West Tambaram, Chennai. The petals were

washed under running tap water followed by distilled water to eliminate dust and other

foreign particles and shade dried for 5 days.

Preparation of extract

The dried petals were then blended using a household electrical blender. 20g of petal powder

was extracted in 70% methanol. The extracts was then filtered using Whatman No. 1 filter

paper. The methanol filtrate was concentrated to dryness in using a rotary evaporator at 40º

C. The residue was partitioned with Petroleum ether, chloroform and ethyl acetate. The ethyl

acetate extract was used for further studies.

Determination of phytochemical constituents

The freshly prepared extracts were subjected to standard phytochemical analyses for different

constituents such as tannins, alkaloids, flavonoids, glycosides, saponins and terpenoids as

described by Jigna et al & Harborne et al [13,14].

Determination of total phenol content

The amount of total phenol content was determined by Folin-Ciocalteu’s reagent method

[15]. 0.5 ml of extract (1 mg/ml) and 0.1 ml Folin-Ciocalteu’s reagent (0.5 N) was mixed and

the mixture was incubated at room temperature for 15 min. Then 2.5 ml saturated sodium

carbonate solution was added and further incubated for 30 min at room temperature and the


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absorbance was measured at 760 nm. Gallic acid was used as a positive control. Total phenol

values are expressed in terms of gallic acid equivalent (mg/g of extracted compound).

Reducing power assay

The reducing power was determined by the Fe3+ - Fe2+transformation in the presence of the

extracts as described in the literature [16]. The Fe2+ can be monitored by measuring

theformation of Perl’s Prussian blue at 700 nm. The different concentration of methanol

extract (5-20µl) was mixed with 2.5 ml of phosphate buffer (pH 6.6) and 2.5 ml of1%

potassium ferricyanide were incubated at 50°C for 30 minand 2.5 ml of 10% trichloroacetic

acid was added to the mixture and centrifuged at 3000 rpm for 10 min. Supernatant (2.5 ml)

was diluted with 2.5 ml of water and shaken with 0.5 ml of freshly prepared 0.1% ferric

chloride. The absorbance was measured at 700 nm. Increased absorbance of the reaction

mixture indicated greater reducing power. Vitamin C was used as a positive control.

Metal chelating assay

To determine metal chelating ability the protocol according to Eric Decker and Welch was

used [17]. The different concentration of extract (5-20 µl) was added to a solution of 0.1 ml

of 2 mM FeCl2. This was followed by the addition of 0.2 ml of 5 mM ferrozine solution,

which was left to react at room temperature for 10 min under shaking conditions before

determining the absorbance of the solution at 562 nm. The percentage inhibition of

Ferrozine– Fe2+ complex formation was calculated using the formula

I= (Abs control – Abs sample) / Abs control X 100

EDTA was used as positive control

Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging assay was carried out following the procedure of Ruch et

al. (1989)[18]. A solution of H2O2 (40 mM) was prepared in phosphate buffer (0.1 M, pH

7.4). The different concentration of Crude extract (5- 20 µl) was added to 0.6 ml of H2O2

solution (0.6 ml, 43 mM). The absorbance value of the reaction mixture was recorded at 230

nm. Blank solution contains sodium phosphate buffer without H2O2. The percentage of H2O2

scavenging of crude extract and standard compounds were calculated using the following

equation

H2O2 scavenging effect (%) = (1 – As/Ac) × 100


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where AC is the absorbance of the control and AS is the absorbance in the presence of the

sample extract or standards.

Antioxidant activity in a hemoglobin induced linoleic acid

The antioxidant activity of Methanol extracts was determined by the method of Kuo et al

(1999) [19]. Methanol extracts (5-20 µl/ml) was mixed with 1 ml of 1 mmol/l of Potassium

phosphate buffer (pH-6.5) followed by the addition of 20 µl of 0.0016% hemoglobin was

shaken vigorously. The mixed solution was incubated at 37°C for 45 minutes. After

incubation, 2.5 ml of 0.6% Hcl in ethanol was added and mixed thoroughly to stop the lipid

peroxidation. Then, 100 µl of 0.02 mol/l FeCl3 and 100 µl of ammonium thiocyanate

(15g/50ml) was added and vortexed thoroughly. The total antioxidant activity determination

was performed in triplicate using the thiocyanate method by reading the absorbance at 480

nm.

Test organisms

The bacterial cultures were Staphylococcus aureus MTCC 29213, Bacillus subtilis

MTCC441, Gram negative; Escherichia coli MTCC 25922, Pseudomonas aeruginosa MTCC

2488, Proteus vulgaris MTCC 1771 used for the antibacterial activity .They were obtained

from the Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacterial

cultures were maintained at 4°C on nutrient agar.

Antimicrobial assay

Antibacterial activity was carried out using disc diffusion method of Sathyabama et al [20].

Petriplates were prepared with 20 ml of sterile nutrient agar (HIMEDIA). The tested cultures

were swabbed on top of the solidified media and allowed to dry for 10 minutes. The crude

extract impregnated discs (Whatman No.1 filter paper was used to prepare discs) were

prepared and air dried well. The test was conducted at four different concentrations of the

crude extract (5, 10, 15 & 20 µl/ml) with 3 replicates. The loaded discs were placed on the

surface of the medium and incubated at room temperature for 24 hrs. The relative

susceptibility of the organisms to the crude extract indicated by the clear zone of inhibition

around the discs, were observed, measured and recorded in millimeters.

Determination of Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration was determined according to method of Velickovic

Ana et al [21]. The different concentration of methanol extracts (5-20µl/ml) was mixed with


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0.5 ml bacterial cultures were incubated at 37°C for 18 hrs and OD was measured

spectrophotometrically at 580 nm.

RESULTS AND DISCUSSION

Phytochemical Screening of Moringa oleifera Petals

Investigations on the phytochemical screening of Moringa oleifera aqueous methanol extracts

revealed the presence of saponins, tannins, glycosides, alkaloids and flavonoids (Table

1).These compounds are known to be biologically active and therefore aid the antimicrobial

activities of Moringa oleifera. These secondary metabolites exert antimicrobial activity

through different mechanisms.

Tannins have been found to form irreversible complexes with proline rich protein [22]

resulting in the inhibition of cell protein synthesis. Tannins have stringent properties, hasten

the healing of wounds and inflamed mucous membranes [23].Herbs that have tannins as their

main components are astringent in nature and are used for treating intestinal disorders such as

diarrhea and dysentery [24]. These observations therefore support the use of Moringa oleifera

in herbal cure remedies.

Another secondary metabolite compound observed in the petals of Moringa oleifera was

alkaloid. One of the most common biological properties of alkaloids is their toxicity against

cells of foreign organisms. These activities have been widely studied for their potential use in

the elimination and reduction of human cancer cell lines [25]. Alkaloids which are one of the

largest groups of phytochemicals in plants have amazing effects on humans and this has led

to the development of powerful pain killer drugs [26].Pure isolated alkaloids and their

synthetic derivatives are used as basic medicinal agents for their analgesic, antispasmodic and

bactericidal effects [27] .

Saponin has the property of precipitating and coagulating red blood cells. Some of the

characteristics of saponins include formation of foams in aqueous solutions, hemolytic

activity, cholesterol binding properties and bitterness [28,29]. Saponins prevent the excessive

intestinal absorption of this cholesterol and thus reduce the risk of cardiovascular diseases

such as hypertension[30]. Recent studies have shown that, many polyphenol compounds

contribute significantly to the total antioxidant activity of many plants . The total phenols

contain good antioxidant, antimutagenic and anticancer properties [31].


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Flavonoids, another constituent of Moringa oleifera methanol extracts exhibited a wide range

of biological activities like antimicrobial,anti-inflammatory, anti-angionic, analgesic, anti-

allergic, cytostatic and antioxidant properties [32].Flavonoids, are potent water-soluble

antioxidants and free radical scavengers [33], which prevent oxidative cell damage, have

strong anticancer activity. Flavonoids in intestinal tract lower the risk of heart disease. As

antioxidants, flavonoids from these plants provide anti-inflammatory activity

Table 1: Phytochemical constituents of crude extracts of Moringa oleifera petals.

S.NO Test Methanolic extract

1 Alkaloids ++

2 Flavanoids ++

3 Saponins ++

4 Tannins ++

5 Glycosides ++

6 Terpenoids --

++ Presence of constituent -- Absence of constituent

Total phenolic content

Plant phenolics and flavonoids are a major group of compounds which have the following

effects; choleretic and diuretic functions , decreasing blood pressure, reducing the viscosity

of the blood and stimulating intestinal peristalsis [34] , as well as primary antioxidation or

free radicals scavenging activities [35].

In the present study the phenolics content of petals of M.Oleifera was found to be 62.75% in

terms of gallic acid equivalents

Reducing capacity assessment

Reducing power is associated with antioxidant activity and may serve as a significant

reflection of the antio-xidant activity [36]. Compounds with reducing power indicate that

they are electron donors and can reduce the oxidized intermediates of lipid peroxi-dation

processes, so that they can act as primary and secondary antioxidants .The maximum

reducing property was found at 20µl/ml of M.oleifera petals. (Graph 1). The reducing power

of crude extract increased gradually in concentration dependent manner.


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Metal chelating activity

The transition metal, iron, is capable of generating free radicals from peroxides by Fenton

reactions and may be implicated in human cardiovascular disease [37]. Because Fe2+ also

has been shown to cause the production of oxyradicals and lipid peroxidation, minimizing

Fe2+ concentration in Fenton reactions affords protection against oxidative damage. The

absorbance of Fe2+–ferrozine complex was decreased dosedependently,i.e. the activity was

increased on increasing concentration from 5 to 20 µl/ml. It was reported that chelating

agents are effective as secondary antioxidants because they reduce the redox potential,

thereby stabilizing the oxidized

form of the metal ion (38).

Table 1: Metal chelating activity of methanol extract of M.oleifera petals

Methanol extract

(µl/ml) aFe2+ chelating activity

5 52.93 ± 12.61

10 66.22 ± 13.42

15 81.96 ± 16.12

20 102.72 ± 20.32 a Results are expressed as % inhibition of Fe2+ chelating with respect to control. Each value

represents Mean±SD

Hydrogen peroxide scavenging activity

Hydrogen peroxide has strong oxidizing properties. It can be formed in vivo by many

oxidizing enzymes, such as superoxide dismutase and can cross cellular membranes and may


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slowly oxidize a number of intracellular compounds. The methanol extract of M.oleifera

petals was capable of scavenging H2O2 in a concentration dependent manner. Hydrogen

peroxide scavenging activity of the methanol extract was found to be 53.06 ± 3.88 at 20µl/ml.

These results showed that the extract had an effective hydrogen peroxide scavenging activity.

Table 2: Scavenging of hydrogen peroxide activity of methanol extract of M.oleifera

petals

Methanol extract

(µl/ml) a Hydrogen peroxide scavenging

activity % 5 25.15 ± 5.70

10 36.62 ± 5.28 15 45.60 ± 4.64 20 53.06 ± 3.88

Antioxidant activity in a hemoglobin induced linoleic acid

The antioxidant activity of methanol extract of M.oleifera petals of medicinal plants was

determined using hemoglobin induced linoleic acid system. The extract showed high

inhibitory ability at 20µl/ml (0.178 ± 0.015). The extracts showed a rapid and concentration-

dependent increase of antioxidant activity.

Antibacterial activity and Minimum Inhibitory Concentration of Moringa oleifera

Petals


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The antibacterial activity of aqueous methanol extract of Moringa oleifera petal at different

concentration was screened by disc diffusion technique and the zone of inhibition was

measured mm diameter (Table 2). The methanol extract of Moringa oleifera was more

effective against B.subtilis and S.aureus with a zone of inhibition percentage of 26 and 19.92

and was least effective against P. aeruginosa, P.vulgaris and E.coli with zone of inhibition

percentage of 12.70, 13.77 and 17.14 respectively at the concentration of 20µl/ml (Graph-3).

The methanol extract of Moringa oleifera petal showed the maximum inhibitory activity at

the highest concentration (20 µl/ml) than the lowest concentration (5 µl/ml) against gram

negative bacteria such as E. coli, P. vulgaris, P. aeruginosa and gram positive B. subtilis and

S. aureus (Table-3). The inhibitory effect of the methanol extract of Moringa oleifera petals

against pathogenic bacterial strains can introduce the plant as a potential candidate for drug

development for the treatment of ailments caused by these pathogens. The presence of

bioactive substances have been reported to confer resistance to plants against bacteria, fungi

and pests and therefore explains the demonstration of antibacterial activity by the plant

extracts used in this study [39].

The results of this study showed that the methanol extract was more effective and may be due

to the better solubility of the active components in organic solvents [40] There are several

reports published on antibacterial activity of different herbal extracts [41].It supports the

earlier investigation that the phytoconstituents isolated from flower possess remarkable toxic

activity against bacteria and may assume pharmacological importance [42].

Table 3: Antibacterial activity of methanol extracts of Moringa oleifera petals.

Different Concentration of extract (µl/ml)

Zone of inhibition [in mm diameter] a

S.aureus

E.coli

B.subtilis

P.vulgaris

P.aeuroginosa

5 11.36±0.77 7.43±0.60 11.4±0.79 7.06±0.40 6.43±0.60 10 13.43±0.73 11.53±0.72 16.1±0.45 8.46±0.61 7.3±0.75 15 15.3±0.75 13.96±0.45 19.3±0.81 10.43±0.60 9.3±0.81 20 17.93±0.60 15.43±0.60 23.4±0.79 12.4±0.79 11.43±0.81

aThe antimicrobial activity was determined by measuring the diameter of zone of inhibition

that is the mean of triplicates ± SD of three replicates


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Graph 3: Inhibition percentage of methanol extract of Moringa oleifera petal against

pathogenic bacteria

Table 3: Minimum inhibitory concentration of methanol extracts of Moringa oleifera

petals.

Different

Concentration of Flavanoid rich fraction

(µl/ml)

Optical density at 580 nm a

S.aureus E.coli B.subtilis P.vulgaris P.aeuroginosa

Positive control 0.642±0.010 0.666±0.012 0.759±0.011 0.513±0.009 0.609±0.011

5 0.56±0.006 0.502±0.010 0.652±0.010 0.426±0.008 0.495±0.011 10 0.523±0.010 0.416±0.009 0.523±0.011 0.305±0.011 0.403±0.010 15 0.472±0.010 0.313±0.010 0.432±0.010 0.222±0.010 0.272±0.010 20 0.312±0.010 0.180±0.011 0.206±0.010 0.162±0.010 0.168±0.012

aThe minimum inhibitory concentration was determined by optical density of inhibition that

is the mean of triplicates ±SD of three replicates.

CONCLUSION

The petals of Moringa oleifera showed promising antibacterial activities and it can play a

therapeutic role against number of epidemic and pathogen born diseases. On the basis of the

results and promising activities of methanol extract of Moringa oleifera petals could be

subjected to isolate most active components for drug development program and therapy of

infection diseases.


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ACKNOWLEDGEMENT

We are thankful to Gloris Biomed Research centre,Chennai for providing all necessary

facilities to conduct the experiment.

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