Ethnobotanical Leaflets 13: 254-58. 2009.

In Vitro Antibacterial Prospective of Crude Leaf Extracts of Melia

azedarach Linn. against Selected Bacterial Strains
S Ramya1, PJ Jepachanderamohan1, N Alaguchamy1, M Kalayanasundaram1 and R
Jayakumararaj2

1
Department of Zoology, Raja Doraisingam Government Arts College, Sivagangai – 630561
2
Department of Botany, RD Government Arts College, Sivagangai – 630561

Issued 10 January 2009

ABSTRACT
The present study was carried out to evaluate the antibacterial activity of the crude leaf extracts
of Melia azedarach against selected Gram positive and Gram negative bacterial strains. Five plant
extracts (Methanol, Ethanol, Dichloromethane, Ethyl acetate and Aqueous) under five different
concentrations (1mg/ml and 5mg/ml) were tested by Disk diffusion method. Methanol, Ethyl acetate
and Aqueous extracts of M. azedarach showed significant inhibition against bacteria tested.

KEY WORDS
Melia azedarach; Antibacterial activity; growth inhibition; leaf extracts; Disc Diffusion Assay (DDA).

INTRODUCTION
Plants have been exploited for treatment of human diseases by different ethnic groups in
different parts of the world since the dawn of civilization. But the traditional cultures without proper
scientific evidence are not able to ponder the importance of plant species for treatment of human
diseases. Therefore, to ascertain the medicinal value of the phytochemicals pharmacological studies
have been carried out by different groups world over (Prusti et al., 2008). Also because the
phytochemicals from medicinal plants serve as lead compounds in drug discovery and design (Ebi and
Ofoefule, 2000).
According to World Health Organization (WHO), infectious diseases are the number one cause
of deaths world wide. Infectious diseases alone account for more than 50 % of the deaths in tropical
countries. To combat these diseases in the last few decades, pharmacological industries have produced
a number of antibiotics, but the resistance of microbes has also increased parallely. Further, it has been
reported that bacterial strains have developed resistance to almost all the antibiotics that are available
in the market. This has resulted multiple drug resistance in both human and plant pathogens due to
indiscriminate use of synthetic drugs especially in the developing countries (Hart and Karriuri, 1998).
Further more, some antibiotics have serious undesirable side effects which limit their applications,
hence, the ultimate goal of the leading drug companies and the academia is to hunt for novel
therapeutic/ antimicrobial agents that are effective with minimal side effects.
According to WHO, about 80% of the population in the developing countries use traditional
medicine in the treatment of various aliments. Nearly, 25 to 45% of modern prescriptions contain plant
derived lead molecules as a basic source in drug formulations. Nevertheless, ruthless hunting has
resulted in inclusion of their name in the red data book (Jain and Sastry, 1979; Ahmedullah and Nayar
1999). Therefore, plant species used by different ethnic groups should be investigated in order to tap
the incredible bioresources for sustainable harvest of novel bioactive phyto pharmaceuticals (Cox,
1994). In-depth investigation is expected to provide better understanding of pharmacological
properties, safety and efficacy (Chopra et al., 1997). In the last few years, a number of studies have
been conducted worldwide to prove such efficacy (Khan et al., 2002). In view of enormous
ethnomedicinal uses and medicinal applications, antimicrobial properties of Melia azedarach L.
(Meliaceae), was investigated against selected bacterial strains.

Description of the plant:

Azadirachta indica and Melia azedarach are two closely related species of Meliaceae. The
former is popularly known as Indian Neem (margosa tree) or Indian lilac, and the latter as the Persian
lilac (Biswas et al., 2002). The plant is a small-to medium-sized deciduous tree. It grows to a height of
5 to 15 m tall and 30 to 60 cm in diameter. The plant is characterized by the presence of a spreading,
dense and dark green crown. Its bark is dark brown in color, relatively smooth, and fissured. The
leaves are alternate, leaflets are short stalked and thin, hairless, dark green (ventral) and relatively pale
(dorsal). Flowers are white with purple stripes and are charcteised by the presence of a typical
fragrance (odor). Fruits or berries are yellow, round, smooth, and fleshy. Dried fruits are hard with 4 to
5 seeds. M. azedarach Linn. is native to tropical Asia. It is widespread and naturalized in most of the
tropics and subtropical countries (Asolkar et al., 1992).

Medicinal uses and Pharmacology:

Leaves: leprosy, scrofula, anthelmintic, antilithic, diuretic, deobstruent, resolvent. Root:
resolvent, deobstruent. Seeds: rheumatism. Leaves: Leaf extract has insecticidal property (azadirachtin)
that repels insects in clothing. The leaves can also serve as feed for goats. Seed oil: The oil is the most
active medicinal product of the plant. It is used as antiseptic for sores and ulcers that show no tendency
to heal. It is also used for rheumatism and skin diseases such as ringworm and scabies. Internally, the
oil is useful in malaria fever and leprosy. Powdered dust of fruit insecticidal, crude extract from wood
and bark insecticidal, oil antibacterial. Alcoholic extract (50% EtOH) of leaf anthelmentic, oil with
unspecified extract central nervous system depressant, mild analgesic, depression followed by
stimulation in animals. Alcoholic extract (50% EtOH) of stem bark anticancerous, antispasmodic,
antiviral (Rastogi and Mehrotra, 1991, 1993, Rastogi, 1998).

Table 1. Ethnomedicinal uses of Melia azedarach.

Disease Plant part used and mode of application

 Burns Fresh leaf extract is applied externally.
Gingivitis Fresh leaf extract is used as mouth wash
Gonorrhea Stem bark infusion 30-50 ml is administered orally twice a day
Piles Leaf extract 5 ml is administered orally thrice a day.
Pyrexia Leaf extract 5-10 ml is administered orally twice a day for 7 days
Source (Khan et al., 2002).

MATERIALS AND METHODS

Collection of Plant Material
Mature leaves of M. azedarach were collected from the wild in Vellore District, Tamilnadu,
India during Apr – Jun 2008. The Flora of Presidency of Madras (Gamble, 1935) and The Flora of
Tamil Nadu Carnatic (Matthew, 1983) were used for identification and authentication of the plants.
Collected material was washed thoroughly in running tap water, rinsed in distilled water and shade
dried in open air and ground to powder.

Preparation of Phytochemical Extracts

The powder was extracted by maceration in double distilled water. The plant extracts were
concentrated using rotary evaporator (Buchi, Switzerland) and stored at 4℃ until used in the assay.

Test Organisms
Eight strains of Gram-positive bacteria - Micrococcus glutamicus, Lactobacillus bulgaris,
Streptococcus faecalis, Staphylococcus aureus, Bacillus stearothermophilus, Staphylococcus pyogenes,
Micrococcus luteus, Bacillus cereus and two strains of Gram negative bacteria - Escherichia coli and
Pseudomonas aeruginosa were used to evaluate the antibacterial activity (Table 2). All bacterial
cultures were maintained in NA slants/ plates; stored at 4℃ and periodically sub-cultured.

Antimicrobial Activity Test

Antimicrobial activity was tested using a modified disc diffusion assay (DDA) method
originally described by Bauer (1966) and Ncube et al (2008). Plant extracts were dissolved in 20%
DMSO treated water. The inoculums for each microorganism were prepared from broth cultures (105
CFU/ml). A loop of culture from the NA slant stock was cultured in LB medium overnight and spread
with a sterile swab into Petri-plates. Sterile disc (6 mm, Hi-media, Mumbai, India) impregnated with
the plant extracts (1.0 mg/disc and 5.0 mg/disc) were placed on the cultured plates and incubated for 24
h at 37℃. The solvent loaded disc without extracts in it served as control in the study. The results were
recorded by measuring the zones of growth inhibition. Clear inhibition zones around discs indicated
the presence of antimicrobial activity. All data on antimicrobial activity were average of triplicate.

RESULTS AND DISCUSSION

Dichloromethane leaf extract of M. azedarach was found to be more effective against gram
positive than gram negative bacterial strains used in the present study. While ethanol extract inhibited
the growth of three gram positive and two gram negative bacterial strains, ethyl acetate, methanolic
fraction and aqueous extract of M. azedarach were found to be effective against all tested bacterial
strains (Table 2).
Results of antibacterial activity of M. azedarach depict that all the extracts effectively inhibited
the growth of B. subtilis with maximum zone of inhibition on the disc loaded with phytochemical
extract (1 and 5 mg/ml/disk). Ethanol extract inhibited the growth of all the ten tested bacterial strains;
however, maximum inhibition zone was recorded against the gram negative strains namely E. coli and
P. aeruginosa. All the tested bacterial strains showed sensitivity against the ethyl acetate fraction S.
aureus and B. subtilis. While methanolic fraction was also found to be effective against all the strains
and maximum inhibition was recorded against B. subtilis. Aqueous leaf extract showed moderate
degree of sensitivity against all tested bacterial strains.
From the results it is clear that leaves of M. azedarach are effective against both gram positive
and gram negative strains. Ethyl acetate fractions exhibited maximum inhibition followed by
Methanolic fractions. The in vitro screening studies confirm medicinal uses reported earlier (Kirtikar
and Basu, 1935; Asolker et al., 1992; Khan et al., 2002; Jain 1991; Fransworth, 1988; Khan et al.,
2002; Valsaraj et al., 1997).

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Table 2. Bacterial strains used in the present study.

S. No Bacterial strain Gram (+/-)
1 Escherichia coli −
2 Pseudomonas aeruginosa −
3 Bacillus cereus +
4 Bacillus subtilis +
5 Lactobacillus bulgaris +
6 Micrococcus glutamicus +
7 Micrococcus luteus +
8 Staphylococcus aureus +
9 Staphylococcus pyogenes +
 10 Streptococcus faecalis +

Table 3. In vitro antibacterial potential of Melia azedarach crude leaf extracts.

Organism E M DCM EA A
C 1 5 C 1 5 C 1 5 C 1 5 C 1 5
Escherichia coli − ++ ++ − + + − ++ + − + + − + +
+
Pseudomonas − ++ ++ − + + − ++ + − + + − + +
aeruginosa +
Bacillus cereus − + ++ − + + − ++ + − + + − + +
Bacillus subtilis − + ++ − + + − ++ + − + ++ − + +
Lactobacillus − + ++ − + + − ++ + − + + − + +
bulgaris
Micrococcus − − − − − − − − − − − − − − −
glutamicus
Micrococcus − − + − − − − + − − − + − − −
luteus
Staphylococcus − − + − − − − + − − + ++ − − −
aureus
Staphylococcus − − + − − − − + − − − + − − −
pyogenes
Streptococcus − + ++ − + + − ++ + − + + − + +
faecalis

(Growth analysis: +++ = abundant; ++ = normal; + = less; − = no; C = control; 1 = 1 mg/ml; 5 = 5 mg/ml)
E = Ethanol M = Methanol DCM = Dichloromethane
EA = Ethyl acetate A = Aqueous