In vitro Antibacterial Activity of crude extracts of leaf, bark and root of Azadirachta indica on some selected medically important Bacteria

1. INTRODUCTION

Azadirachta indica (Meliaceae) commonly known as neem is native of India and naturalized in most tropical and subtropical countries and is of great medicinal value and distributed wide spread in the world. The Neem tree (known as DogonYaro in Hausa) is a member of the Kingdom Plantae; Division Magnoliophyta; Order Sapindales and Family Meliaceae. It is native to Bangladesh, India, Myanmar, and Pakistan. It is found growing in tropical and semi-tropical regions (Wikipedia, 2008). It is now abundant in the tropical belt from Somalia in the east to Mauritiania in the west and it spreads in the South Pacific (Mahmood, 2004; Mohan and Nair, 2007). The tree is also commonly found in Nigeria where it is used for aforestation. The major active constituents in neem are terpenoids such as azadirachtin, which are considered to have antimicrobial and insecticidal properties among many other actions.

Historically, bacteria have been the cause of some of the most deadly diseases and widespread epidemics of human civilization Bacteria of medical importance are those group of organisms that are recognized as the most difficult healthcare-associated infections to control and treat and among the most important medically bacteria salmonella typhi, staphylococcus aureus and pseudomonas aeuroginosa are considered serious threats to public health. Microorganisms have developed resistance against many antibiotics due to the indiscriminate use of antimicrobial drugs (Ahmad et al., 1998) Evolution of highly resistant bacteria strain has compromised the use of new generations of antibiotics (Levy, 2008). Furthermore, antibiotics are sometimes associated with side effects (Cunha, 2001).

It is known that more than 400, 000 spp. of tropical flowering plants have medicinal properties and this has made traditional medicine cheaper than modern medicine (Odugbemi, 2006). Every part of the neem tree has been used as traditional medicine for house-hold remedy against various human ailments from antiquity. The tree is still regarded as village dispensery because of its wide spectrum of activity and known for over 2000 years as one of the most versatile medicinal plants (Larkshamanan and Subramanian, 1996). Extracts from the bark, leaves, fruits and roots have been used to control leprosy, intestinal helminthosis and respiratory disorders (Ketkar and Ketkar, 1995). However, there has been seldom effective collaboration between the traditional and western medical therapeutics, largely due to the perception that the use of traditional and herbal medicines has no scientific basis. According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy. The objectives of this research work is to determine the phytochemical component of leaf and root extract of Azadirachta indica, to test for its in vitro antibacterial activity on some selected medically important bacteria at various concentration on Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi.

2. MATERIALS AND METHOD

2.1 Collection and processing of plant material: leave, root and bark of Azadirachta indica were collected and then washed under running tap water to eliminate dust and any other foreign particles and then dried under shade for six weeks in post graduates laboratory of Abubakar Tafawa Balewa University. The dried materials were pulverized in mortar, packaged and labeled in cellophane bags until it was required for use (Abalaka et al., 2012).

2.2 Preparation of plant extract: Four hundred grams of dried ground sample of Azadirachta indica leaves, bark and root were macerated in 2.5 liters of acetone followed by ethanol and then methanol for 72 hours, shaking was done continuously using an orbital shaker. The extract was filtered twice through cotton wool and then through whattman No. 1 filter paper and then concentrated using a rotary evaporator at a temperature not exceeding 40^oC. The concentrated extract was dried to complete dryness in an aerated oven at 40^oC. This was then kept in refrigerator at 4^oC until required for use. As described by Supelco Bulletin (1998), Samuelsson (2004).

2.3 Phytochemical Analysis: The extracts obtained were analyzed by following the procedures of Talukdar and Choudhary, (2010). To test for the presence of the alkaloids, saponins, tannins, Terpenoids, flavonoids, glycosides, volatile oils and reducing sugars

2.3.1 Saponins: Saponins were detected using the froth test. 1g of the sample was weighed into a conical flask in which 10ml of sterile distilled water was added and boiled for 5 minutes. The mixture was filtered and 2.5 ml of the filtrate was added to 10ml of sterile distilled water in a test tube. The test tube was tapered and shaken vigorously for about 30 seconds. It was then allowed to stand for half an hour. Honeycomb froth indicated the presence of saponins.

2.3.2 Tannins: To a portion of the extract diluted with water, 3-4 drops of 10% ferric chloride solution was added. A blue color is observed for gallic tannins and green color indicates catecholic tannins.

2.3.3 Reducing Sugars: To 0.5ml of plant extracts, 1ml of water and 5-8 drops of Fehling’s solution was added and heated over water bath. Brick red precipitate indicates the presence of reducing sugars.

2.3.4 Glycosides: 25ml of dilute sulphuric acid was added to 5ml extract in a test tube and boiled for 15 minutes, cooled and neutralized with 10%NaOH, then 5ml of Fehling solution added. Glycosides are indicated by a brick red precipitate.

2.3.5 Alkaloids: 2ml of extract was measured in a test tube to which picric acid solution was added. An orange coloration indicated the presence of alkaloids.

2.3.6 Flavonoids: 4ml of extract solution was treated with 1.5 ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was added and red color was observed for flavonoids and orange color for flavones.

2.3.7 Volatile oils: 2ml of extract was shaken with 0.1ml dilute NaOH and a small quantity of dilute HCl. A white precipitate is formed if volatile oils are present.

2.3.8 Terpenoids: Four milligrams of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and red violet color was observed for terpenoid.

2.4 Collection and Cultivation of Test Organisms

Isolates of Pseudomonas aeroginosa, Staphylococcus aureus and Salmonella typhii. were obtained from Abubakar Tafawa Balewa university teaching hospital Bauchi. These clinical isolates were further characterized using both morphological (Gram staining) and biochemical test (urease, catalase, coagulase motility test, etc) to confirm the exact species of isolates. The clinical isolates were inoculated into nutrient agar slants and then stored in the refrigerator at 4^oC until required.

2.5 Growth Media

Muller Hinton agar and Nutrient broth were use in this study. The media were prepared according to manufacturers’ guide (Cheesbrough, 2005).

2.6 Preparation of turbidity standard

Barium sulphate (BaSo₄) standard suspension was used as turbidity standard. This was prepared as follows: one percent (1% v/v) solution of H₂SO₄ was prepared by adding 1ml of conc. H₂SO₄ into 99ml of water. 1% w/v solution of barium chloride (Bacl) was prepared by dissociating of 0.5g of dehydrated Bacl in 50ml distilled water, then 0.6ml of the Bacl solution was combined with 99.4ml of H₂SO₄ solution to yield 1% w/v barium sulphate (Baso₄) suspension. The turbidity solution formed was transferred into the test tube as the standard for comparison (Cheesbrough, 2005).

2.7 Standardization of the Inoculum

Using inoculums loop, enough material from the overnight culture of test organisms was transferred into a tube containing about 2.0ml normal saline until the turbidity matched the turbidity standard of 1% Baso_4, (Cheesbrough, 2005).

2.8 Preparation of Sensitivity Discs

Whattman No. 1 filter paper was punched using paper punch to produce discs of 6mm in diameter. The disc was then put in 50ml conical flask with the mouth plugged with cotton wool for sterilization in dry heat at 140⁰c for 1 hour. The discs were then allowed to cool and stored in the refrigerator at 4⁰C until required for use (Okoro et al., 2012).

2.9 Preparation of Stock Solution

Using screw capped bottle, different stock solutions of the plant extracts were prepared using Dimethyl sulphoxide (DMSO). Varying the concentration of the crude extracts (1000mg/ml, 500mg/ml, 200mg/ml and 100mg/ml) were prepared by weighing 1.0,0.50, 0.20, 0.10g respectively using an electric meer balance with model No. H30 and then each were dissolved in 1ml of diluents called dimethyl sulphoxide (DMSO) under aseptic condition. The sterilized discs were then put into the bottles each containing discs so that each will absorbequal volume(Baker et al, 2003).

2.10 Bioassay

Sensitivity tests were carried out using Agar disc diffusion method (Baker and Silverton 1993 and Mukhtar and Tukur 2000). The organisms were inoculated by using streak plating method in which the surface of Huller Hinton agar plates were streaked with sterile swabs containing each of the standard inoculums. The filter paper discs impregnated with the above concentrations of extracts were placed on the surface of the inoculated Muller Hinton agar plates with the aid of sterilized pair of forceps.

Discs impregnated with DMSO only was placed at the centre of some plates to serve as negative controls while disc impregnated with perfloxacin was placed at the centre of some plates to serve as positive controls. A pre – diffusion time of 30 seconds was allowed for the extracts to diffuse from the discs into the agar medium before incubation. The plates were inverted and incubated at 37°C for 24 hours.

The degree of sensitivity of the organisms to the extracts was determined by measuring diameter of visible zones of inhibition to the nearest millimeter with respect to each isolate and extract concentration (Mukhtar and Okafor, 2002)

3. RESULTS

3.1 Qualitative Phytochemical Analysis

The preliminary phytochemical analysis performed was of qualitative type. Table 1 shows the phytochemical component present in the leaf extract of Azadirachta indica saponins, volatile oil and terpenoids were found to be present in both the methanol, ethanol and acetone extract of the leaf. Reducing sugar was present in the methanol and ethanol leaf extract while tannin and glycosides were only present in the methanol fraction of the leaves.

The phytochemical analysis of stem/bark extract using acetone, ethanol and methanol was shown in table 2. From the phytochemical analysis saponins volatile oil and flavonoids were found to be absent in the stem/ bark of Azadirachta indica extracted using the solvents acetone, ethanol and methanol. Tannins, reducing sugars and terpenoids were found to be present in the stem/ bark of Azadirachta indica using the solvent acetone ethanol and methanol. The methanol and ethanol extract only showed the presence of alkaloids and glycosides.

The phytochemical constituents of the root of Azadirachta indica using acetone, ethanol and methanol was showed in table 3 saponins and volatile oil were found to be absent in the root extract of Azadirachta indica using both the three solvents while tannins, alkaloids and reducing sugars were observed in the root of Azadirachta indica using the three solvents, however glycosides and alkaloids were only observed in the methanol and ethanol extract of the root of Azadirachta indica

3.2 In vitro antibacterial activity of the extract Azadirachta indica

Antibacterial activity of the leaves extract of Azadirachta indica at concentration of 1000mg, 500mg, 200mg and 100mg is shown in table 4. Methanol extracts of the leaves of A. indica showed the maximum zone of inhibition (15mm) against salmonella typhi at a concentration of 1000mg/disc, this is followed by staphylococcus aureus (12mm), pseudomonas aeruginosa (11mm). At concentration of 500mg/disc 9mm zone of inhibition was recorded on staphylococcus aureus and pseudomonas aeruginosa while 11mm zone of inhibition was recorded on salmonella typhi.8mm,7mm and 9mm zone of inhibition was recorded on staphylococcus aureus pseudomonas aeruginosa and salmonella tyhi respectively at concentration of 200mg/disc. At concentration of 100mg/disc 6mm, 7mm and 8mm zone of inhibition was recorded on staphylococcus aureus, pseudomonas aeruginosa and salmonella typhi respectively.

Ethanol extract of the leaves of A. indica showed the highest zone of inhibition of (13mm) on salmonella typhi, this is followed by staphylococcus aureus (11mm) and then pseudomonas aeruginosa (10mm) at concentration of 1000mg/disc. 10mm, 9mm and 8mm zone of inhibition was recorded on salmonella tyhi, staphylococcus aureus, and pseudomonas aeruginosa respectively at concentration of 500mg/disc. At concentration of 200mg/disc, 8mm zone of inhibition was recorded on salmonella typhi and staphylococcus aureus while 7mm was recorded on pseudomonas aeruginosa. At 100mg/disc, 6mm zone of inhibition was recorded on staphylococcus aureus, and pseudomonas aeruginosa and 7mm on salmonella typhi.

The acetone fraction of the leaves of A. indica highest zone of inhibition was seen on salmonella typhi and staphylococcus aureus (9mm) at concentration of 1000mg/disc and 8mm on pseudomonas aeruginosa (6mm). At concentration of 500mg/disc (7mm) zone of inhibition was recorded on staphylococcus aureus, (6mm) on pseudomonas aeruginosa and 8mm on salmonella typhi. (6mm) zone of inhibition was recorded on pseudomonas aeruginosa and staphylococcus aureus and (7mm) on salmonella typhi at concentration of 200mg/disc. 6mm zone of inhibition was recorded on all the bacterial isolates tested at 100mg/disc.

Table 5 shows the inhibitory activity of the stem/bark of A. indica, here methanol extract showed the highest zone of inhibition (17mm) on salmonella typhi at concentration of 1000mg, this was followed by staphylococcus aureus (15mm), pseudomonas aeruginosa (13mm). At concentration of 5000mgdisc 15mm, 9mm and 11mm zone of inhibition was observed on salmonella typhi, staphylococcus aureus and pseudomonas aeruginosa respectively. At concentration of 200mg/disc, 12mm zone of inhibition was recorded on salmonella typhi, (7mm) on pseudomonas aeruginosa and (8mm) on staphylococcus aureus . Least zone of inhibition (7mm) was observed on staphylococcus aureus and pseudomonas aeruginosa while (10mm) zone of inhibition was recorded on salmonella typhi at concentration of 100mg/disc

Ethanol extract of the stem/bark of A.indica produced the highest zone of inhibition (14mm) on salmonella typhi this is followed by (13mm) on pseudomonas aeruginosa and then (11mm) on staphylococcus aureus at the concentration of 1000mg/disc. At 500mg/disc concentration, 11mm zone of inhibition was recorded on staphylococcus aureus,(9mm) on pseudomonas aeruginosa and (12mm) on salmonella typhi. 9mm, 8mm and 10mm zone of inhibition was recorded on staphylococcus aureus, pseudomonas aeruginosa and salmonella typhi respectively at concentration of 200mg/disc. Least zone of inhibition (6mm) was observed on staphylococcus aureus and pseudomonas aeruginosa while (7mm) zone of inhibition was recorded on salmonella typhi at concentration of 100mg/disc.

Acetone fraction of the stem/bark of A. indica shows the highest zone of inhibition of (12mm) on pseudomonas aeruginosa, this is followed by staphylococcus aureus (10mm) and then salmonella typhi (9mm) at the concentration of 1000mg/disc. staphylococcus aureus and pseudomonas aeruginosa recorded the lowest zone of inhibition (6mm) at concentration of 100mg/disc.

Antibacterial activity of the root extract of Azadirachta indica is presented in table 6. For the methanol fraction highest zone of inhibition (23mm) was recorded on salmonella typhi this is followed by (19mm) on staphylococcus aureus, (17mm) on pseudomonas aeruginosa at concentration of 1000mg/disc. At concentration of 500mg/disc15mm,13mm and 19 mm zone of inhibition was recorded on staphylococcus aureus, pseudomonas aeruginosa and salmonella typhi respectively. 10mm, 8mm and 16mm zone of inhibition was recorded on staphylococcus aureus, pseudomonas aeruginosa and salmonella typhi respectively, at concentration of 200mg/disc and at 100mg/disc, 10mm was recorded on salmonella typhi, 6mm on pseudomonas aeruginosa and 7mm on staphylococcus aureus.

Ethanol extract of the root of A. indica shows the highest zone of inhibition (17mm) on salmonella typhi, this is followed by staphylococcus aureus (14mm), pseudomonas aeruginosa (12mm) at concentration of 1000mg/disc. salmonella typhi (15mm), staphylococcus aureus (11mm) and pseudomonas aeruginosa (9mm) at concentration of 500mg/disc, at concentration of 200mg/disc 8mm, 6mm and 12mm zone of inhibition was recorded on staphylococcus aureus, pseudomonas aeruginosa and salmonella typhi respectively. 6mm zone of inhibition was recorded on staphylococcus aureus, 7mm on pseudomonas aeruginosa and 9mm on salmonella typhi.

Acetone extract of the root shows 11mm, 10mm and 8mm zone of inhibition on salmonella typhi, staphylococcus aureus and pseudomonas aeruginosa respectively at concentration of 1000mg/disc. At concentration of 500mg/disc 9mm zone of inhibition was recorded on salmonella typhi and staphylococcus aureus and 7mm zone of inhibition on pseudomonas aeruginosa. 8mm zone of inhibition was recorded on salmonella typhi and staphylococcus aureus while 6mm was recorded on pseudomonas aeruginosa at concentration of 200mg/disc and at 100mg/disc, 8mm, 6mm and 7mm zones of inhibition were recorded on salmonella typhi and staphylococcus aureus and pseudomonas aeruginosa respectively.

Positive and negative control test were also shown in tables 5, 6 and 7). For the positive control test perfloxacin antibiotic disc was used on all the bacterial isolates. 30mm zone of inhibition was observed in staphyloccus aureus, 22mm zone of inhibition on pseudomonas aeruginosa and 25mm zone of inhibition on salmonella typhi. For the negative control test, the disc was impregnated with dimethyl sulphur oxide and zero zone of inhibition was recorded on all the bacterial isolates.

Table 1: Qualitative Phytochemical Analysis of acetone, ethanol and methanol extract of Azadirachta indica leaves

Phytochemical Constituents		solvents
Phytochemical Constituents		Methanol	Ethanol	Acetone
1. Saponins		+	+	+
2.Tanins		+	_	_
3.Reducing sugars		+	+	_
4. Glycosides.		+	_	_
5. Alkaloides		_	_	_
6. Flavonoids		_	_	_
7. Volatile oils		+	+	+
8. terpenoides	+		+	+

Key: + = present,

- = absent

Table 2: Qualitative Phytochemical Analysis of acetone, ethanol and methanol extract of Azadirachta indica stem/bark

Phytochemical constituents	solvents Methanol	Ethanol	Acetone

1.Saponins	-	-	-
2.Tanins	+	+	+
3.Reducing sugars	+	+	+
4. Glycosides.	+	+	-
5. Alkaloides	+	+	-
6. Flavonoids	-	-	-
7. Volatile oils	-	-	-
8. Terpenoides	+	+	+

Key: + = present

- = absent

Table 4: Antibacterial activity of the leaves extract of Azadirachta indica

Bacteria isolates Mean values for the zones of inhibition (mm) at different concentrations(mg) based on different extraction methods
Control			Methanol				Ethanol				Acetone
	PC	NC	1000	500	200	100	1000	500	200	100	1000	500	200	100
Staphylococcus aureus	30	0	12	09	08	07	12	09	08	06	09	07	06	06
Pseudomonas aeruginosa	22	0	11	09	07	06	10	08	07	06	08	06	06	06
Salmonella typhi	25	0	15	11	09	08	13	10	08	07	09	08	07	06

Key: PC= Positive control

NC= Negative control

Table 5: Antibacterial activity of the stem bark extract of Azadirachta indica

Bacterial isolates Mean values for the zones of inhibition (mm) at different concentrations(mg) based on different extraction methods
	Control		Methanol				Ethanol				Acetone
	PC	NC	1000	500	200	100	1000	500	200	100	1000	500	200	100
Staphylococcus aureus	30	0	15	11	08	07	11	11	09	06	10	08	07	06
Pseudomonas aeruginosa	22	0	13	09	07	07	13	12	08	06	09	07	06	06
Salmonella tyhi	25	0	17	15	12	10	14	09	10	07	11	10	09	07

Key: PC= Positive control

NC= Negative control

Table 6: Antibacterial activity of the Root extract of Azadirachta indica

Bacterial isolates Mean values for the zones of inhibition (mm) at different concentrations(mg) based on different extraction methods
	Control		Methanol				Ethanol				Acetone
	PC	NC	1000	500	200	100	1000	500	200	100	1000	500	200	100
Staphylococcus aureus	30	0	19	15	10	06	14	11	08	07	10	09	08	07
Pseudomonas aeruginosa	22	0	17	13	08	07	12	09	06	06	08	07	07	06
Salmonella typhi	25	0	23	19	16	10	17	15	12	09	11	09	08	08

Key: PC= Positive control

NC= Negative control

4 DISCUSSION

Phytochemical analysis performed was of qualitative type. The aim of phytochemical screening is to confirm the presence of various constituents of azadirachta indica extract so as to be able to assess their biological activity or medicinal uses (Garima et al., 2014). In this study conducted, the medicinal value of plants is due to the presence of particular chemical substances that have a definite physiological action on the living system. The most important of these are alkaloids, glycosides, saponins, steroids, phenols, flavonoids and tannins. Phytochemical screening of neem leaves showed the presence of saponins, terpenoids, alkaloids, tannins, glycosides and steroids. These class of compounds independently or in combination may be responsible for the broad range of medicinal properties of neem as reported by Abalaka et al. (2012)

The methanolic , ethanolic and acetone extract of the leaf, bark and root of Azadirachta indica gives varying degrees of phytochemicals, the methanolic extract showed the presence of most of the phytochemical components of azadirachta indica followed by ethanolic, the solvent acetone showed the least presence of the phytochemical component hence the minimal antibacterial activity of the acetone extract.

The phytochemical components of the A. indica have been established in previous studies and these include tannins, saponins, alkaloids, carbohydrates, phenols, flavonoids, anthraquinones, cardiac glycosides, sterols and resins (De and Ifeoma, 2002; Natarajan et al., 2003; Biswas et al., 2002, El-Mahmood et al,. 2010). Several studies have linked presence of these bioactive compounds in plant materials to antimicrobial activity. The presence of these secondary metabolites in plants, produce some biological activities in man and animals and it is responsible for their use as herbs. These compounds also serve to protect the plant against infection by microorganisms, predation by insects and herbivores, while some give plants their odors and or flavors and some still are responsible for their pigments (El-Mahmood et. al,. 2008). In some cases, the activity has been associated with specific compounds or classes of compounds. These active constituents can be used to search for bioactive lead compounds that could be used in the partial synthesis of more useful drugs (Ogbonna et al., 2008; El-Mahmood et al., 2010).

The methanolic and the ethanolic extract provided more consistent and prominent antimicrobial activities as compared to the acetone extract. The acetone extract exhibited the least antimicrobial activity this could be due to lower concentration of antimicrobial compound in this extract, this agrees with the findings of Praveen and Sharmishtha (2012), another reason could be due to the fact that aromatic or saturated organic compounds are most often obtained through initial methanol and ethanol extraction when the three solvents are used together as reported by (Cowann, 1999).

Statistical analysis was carried out using analysis of variance (Anova) and the antibacterial studies of Azadirachta indica leaves, bark and root shows significant inhibitory effect on the test organism. Significant difference in response was observed between root and leaves extract and the solvents used in extracting the various plant parts, this shows that root extract of Azadirachta indica has higher inhibitory effect than the leaves and bark, this could be due to the fact that the root is more bitter than the leaves meaning that it contains more of nibidin which (Biswas et. al., 2002) showed in their work to be the main active antibacterial ingredient of Azadirachta indica. Mamman et al. (2013) shows that the stem extracts of Azadirachta indica has higher antibacterial activity than the leaves extract of Azadirachta indica.

For the purpose of comparison, positive and negative controls were used. Negative control did not show inhibitory activity against any of the test organisms while positive controls showed significant inhibitory effect on the test organism, this agrees with the work of (Rahman et al., 2011)

These findings support the traditional knowledge of the local users and it is a preliminary scientific validation for the use of these plant parts for antibacterial activity against pathogenic bacteria. The plant could be a veritable and cheaper substitute for conventional drugs since the plant is easily obtainable and the extract can easily be made via a simple process of maceration or infusion

ACKNOWLEDGEMENT

We are grateful to all those that assisted during this research work. Special gratitude goes to the laboratory technologist for providing all the necessary equipment that led to the success of this work, and to all those that helped during this work.

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