Phytochemical Screening And Antibacterial Activity Of Polyalthia

Longifolia Crude Extracts

* D. Uzama, M.B. David, R. Ahmadu and S.A. Thomas

Sheda Science and Technology Complex (SHESTCO), P.M.B.186,

Garki, Abuja.

*Corresponding author: [email protected]

+2348069612751

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Abstract

Crude extracts of the leaves of Polyalthia Longifolia were subjected to

phytochemical investigations using standard methods. The tests conducted
indicated the presence of alkaloids, tannins, saponins and glycosides. The crude
methanolic, ethanolic, ethyl acetate and petroleum ether extracts of the leaves of
Polyalthia Longifolia inhibited Bacillus subtilis and staphylococcus aureus at
25mg/ml.The minimum inhibitory concentrations of the crude extracts were
determined for the various organisms which ranged between 0.01 and
0.5mg/ml,while the minimum bactericidal concentration ranged between 0.01 and
1.3mg/ml.Polyalthia longifolia could be a potential source of antimicrobial agents.

Key words: Phytochemical screening, antibacterial activity, plant extracts,

polyalthia longifolia, minimum inhibitory concentration.

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INTRODUCTION

Medicinal plants are of great value to mankind. They are nature's gift to human beings

to lead a disease – free, healthy life. They play a vital role in preserving our health

(Bhagwati, 2003). The medicinal value of plants can be observed from the chemical

agents they possess which may alter certain physiological actions in the human body

(Raghunathan and Mitra,1982).The most important of these bioactive constituents of

plants are alkaloids,tannins,flavonoids and phenolic compounds. Many of these

medicinal plants are used as spices and food plants. The plant medicines are

considered as alternatives to synthetic drugs because of their relatively lower

incidence of adverse reactions and reduced cost (Gislence et al., 2000).

Herbal molecules are safe and will overcome the resistance produced by the

pathogens as they exist in a combined form or in a pooled form of more than one

molecule in the protoplasm of the plant cell (Motamedi et al., 2009).

The genus polyalthia belongs to the family Annonaceae. Polyalthia is a Greek word,

with poly meaning much or many and althia meaning to cure (Wu et al, 1990). The

plant is a lofty ever green tree, which exhibits symmetrical pyramidal growth with

willowy weeping pendulous branches and long narrow lanceolate leaves with

undulating margins (Krishnamurth, 1987) .The plant, occurs mainly in Africa, Asia,

Australia, India and New Zealand.

Nowadays the use of antibiotics to control diseases is producing adverse toxicity to

the host organs, tissues and cells. The toxicity produced by the antimicrobial agents

can be prevented by using herbs.

The bark of polyalthia longifolia is traditionally reputed to lower blood pressure,

stimulate respiration and help in fever, skin diseases, diabetes and hypertension (Nair

et al., 2004, Saleem et al., 2005).The methanolic extract of the roots of polyalthia

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longifolia showed a maximum inhibition of oedema (Ramakrishna et al., 2000).The

plant contains antitumor and anticancer active principles (Chen et al., 2000).

In the present investigation, different organic solvent extracts of polyalthia longifolia

leaves have been evaluated for their antibacterial potential.

MATERIALS AND METHODS

Collection of plant material

Mature leaves of polyalthia longifolia were collected from the staff quarters of Sheda

Science and Technology Complex (SHESTCO), Abuja, during the rainy season. The

leaves were rinsed with water and air dried in the laboratory for two weeks. They

were ground with Excella mixer grinder and sieved with a mesh of size 0.5mm.The

powdered samples obtained were stored in clean air tight containers at ambient

temperature until when needed for use.

Preparation of Extracts

Aqueous extract

The leaves powder (20g) was placed in a 500ml conical flask. To this was added

200ml of distilled water and boiled until the volume of the water reduced to 50ml.The

water extract was filtered through a 420µm stainless steel filter. The filtrate was

further concentrated to dryness using water bath.

Solvent extracts

The leaves powder (10g) was extracted with solvents of different polarities (methanol,

ethanol, ethyl acetate, petroleum ether and acetone) by cold maceration for 24h.The

extracts were filtered through Whatman No.1 filter paper. The extracts were

concentrated to dryness using rotary flask evaporator under reduced pressure.

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Phytochemical screening of crude extracts

The phytochemical components of the leaves of polyalthia longifolia were screened

using the standard methods of Hassan et al.,2005, and Odebiyi and

Sofowora,1978.The components analysed for were saponins, carbohydrates, alkaloids,

tannins, resins, steroids, glycosides and flavonoids.

Collection and maintenance of microbial culture

The bacterial strains such as,Bacillus subtilis, Klebsiella pneumoniae, Staphylococcus

aureus and Escherichia coli used during the present study were obtained from the

Medical Laboratory of the University of Abuja Teaching Hospital. The bacteria were

grown in nutrient broth at 37oC and maintained on nutrient agar slants at 4oC.

Antibacterial sensitivity

Invitro antimicrobial screening of the crude extracts of polyalthia longifolia leaves

was carried out by well diffusion method according to Perez et al (1990). The plates

were prepared by pouring sterile Muller Hinton agar (oxoid, England) into sterile

Petri dishes that were previously autoclaved. Sterilized cotton swabs were dipped in

the bacterial culture in nutrient broth and then swabbed on the agar plates. Wells of

equal size were cut with proper gaps in the medium and the extracts were added into

it. The plates were allowed to stand for one hour, to allow pre-diffusion of the extract

into the medium (Esimone et al, 1998). The plates were incubated at 37oC for 24

hours.

The standard drugs used were Ciprofloxacin and Chloramphenicol. At the end of the

incubation period, inhibition zones were measured in millimetre. This study was

carried out in triplicate.

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Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentration is the least extract concentration which

inhibited the growth of the test organisms. The minimum inhibitory concentration was

determined by incorporating various concentrations of solutions of extract in Muller

Hinton agar(200 to 0.4mg).A loop full of standard test bacterial broth culture was

used to streak plates. The plates of bacteria were incubated at 37oC for 24h. A positive

control containing only the growth medium and extract was also set up. The MIC was

regarded as the lowest concentration of the extract that did not permit any visible

growth of the organism.

Determination of minimum Bactericidal Concentration (MBC)

The MBC was determined by sub culturing the test dilution on Muller Hinton agar

without any growth on Muller Hinton broth and further incubated for 24h at 36oC.

The lowest concentration that yielded no bacterial growth was taken as the minimum

bactericidal concentration.

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RESULTS AND DISCUSSION

Phytochemical analysis

The results of the phytochemical screening (Table1) revealed the absence of

carbohydrates, resins, steroids and flavonoids in all the extracts. Alkaloids were

present in methanol and petroleum ether extracts. Tannins was present in methanol,

ethanol and water extracts.Saponins was present in methanol, petroleum ether and

water extracts. Glycosides were present only in methanol extract.

These chemical constituents are responsible for the medicinal and physiological

activities of the leaves of the plant. The active compounds detected may be

responsible for the antimicrobial activity of tested organisms.

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Table1: Phytochemical analysis of the leaves of polyalthia longifolia in different
solvent extracts

S/N Compound Extracts

-------------------------------------------------------------------------------
BZ MT PE ET AT EA AQ

1 Carbohydrates - - - - - - -

2. Alkaloids - + + - - - -

3. Tannins - + - + - - -

4. Resins - - - - - - -

5. Steroids - - - - - - -

6. Saponins - + + - - - +

7. Glycosides - + - - - - -

8 Flavonoids - - - - - - -

BZ - Benzene AT - Acetone
MT - Methanol EA - Ethyl acetate
PE - Petroleum ether AQ - Aqueous
ET - Ethanol + = Present
- = Absent

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Antibacterial sensitivity

The results of antibacterial screening of the plant extracts by well diffusion method

are given in Table 2. The results indicated that the extracts are potent antimicrobials

against Bacillus subtilis and staphylococcus aureus. The antibacterial activity was

screened from the zone of inhibition. The inhibitory effects of the extracts were

compared with standard antibiotics like chloramphenicol and ciprofloxacin.

The methanolic extract showed a significant inhibition against Bacillus subtilis

(24mm), which was comparable with that of the standards used, chloramphenicol

(22.3mm) and ciprofloxacin (24mm). Bacillus subtilus was also found to be

significantly inhibited by ethanolic extract. Acetone(22.3mm) and petroleum ether

(21.7mm) extracts significantly inhibited staphylococcus aureus,but methanolic

extract (22.6mm) inhibited staphylococcus aureus most effectively.

Among various solvent extracts studied, methanol extract showed higher degree of

inhibition followed by ethanol, ethyl acetate, acetone and petroleum ether. The

diameter of inhibition zones for each of the samples were compared with

standard antibiotics chloramphenicol and ciprofloxacin. Highest antibacterial

activity was against Bacillus subtilis in methanol extract, followed by staphylococcus

aureus. No inhibition was recorded with Klebsiella pneumonia and Escherichia

coli for all the extracts.

The results in Table 3 indicate that the minimum inhibitory concentration (MIC) of

the leaves extracts of polyalthia longifolia ranged between 0.01 and 0.5 mg/ml. An

antimicrobial agent with high activities against an organism yields a low MIC

while an antimicrobial agent with low activity has a high MIC.The minimum

inhibitory concentration values indicated that the petroleum ether extract was

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highly active against staphylococcus aureus (0.01mg/ml) and Bacillus subtilis

(0.01mg/ml). The MIC also indicated that the ethanolic, ethyl acetate and acetone

extracts were also highly active against Bacillus subtilis.The control has not

shown any inhibitory effect.

The minimum bactericidal concentration (MBC) of the leaves extracts of

polyalthia longifolia ranged between 0.01 and 1.3mg/ml (Table 4 ). The MIC and

MBC are normally used to evaluate the efficacy of the agents.

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Table 2: Antimicrobial activity of polyalthia longifolia leaves extracts

S/N Test Organism Zone of inhibition (mm)

-------------------------------------------------------------------------------
Antibiotics Control
-------------------------------
MT ET EA PE AT CP CF DMSO

1. Bacillus subtilis 24±0.11 20±0.13 19±0.24 17±0.13 19±0.15 22±0.12 24±0.01 10±0.12

2. Klebsiella
pneumonia 10±0.13 10±0.20 10±0.21 10±0.12 10±0.23 19±0.13 10±0.05 10±0.11

3. Staphylococcus 23±0.21 20±0.31 20±0.15 22±0.19 22±0.25 24±0.15 31±0.04 10±0.13

Aureus

4. Escherichia 10±0.15 10±0.23 10±0.25 10±0.21 10±0.21 31±0.16 10±0.08 10±0.15

coli

MT - Methanol ET - Ethanol EA - Ethyl acetate

PE - Petroleum ether AT - Acetone CP - Chloramphenicol
CF - Ciprofloxacin

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Table 3: Minimum inhibitory concentration (mg/ml) of the polyalthia longifolia
leaves extracts against Test organisms.

S/N Extracts Staphylococcus aureus Bacillus subtilis

1. Methanol 0.4 0.5

2. Ethanol 0.1 0.01

3. Ethyl acetate 0.1 0.01

4. Acetone 0.1 0.01

5. Petroleum ether 0.01 0.01

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Table 4: Minimum Bactericidal Concentration (mg/ml) of Polyalthia longifolia
leaves extracts against Test organisms

S/N Extracts Staphylococcus aureus Bacillus subtilis

1. Methanol 1.3 1.3

2. Ethanol 0.1 0.02

3. Ethyl acetate 0.1 0.01

4. Acetone 0.1 0.01

5. Petroleum ether 0.2 0.01

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CONCLUSION

The study has confirmed that the crude extracts of the leaves of polyalthia

longifolia contain alkaloids,tannins,saponins and glycosides. The leaves extracts of

the plant studied can be seen as a potential source of antibacterial. There is the need

for further studies on other parts of the plant in order to isolate, identify, characterize

and elucidate the structure of antimicrobial compounds.

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