Journal of Pharmaceutical and Allied Sciences

JOPHAS
ANTIBACTERIAL ACTIVITY OF GLOBIMETULLA BROWNI (MISTLETOE) LEAF EXTRACT AGAINST FOUR CLINICAL BACTERIAL ISOLATES ADEWUMI, A.A.J., *AINA, V.O., ALHASSAN SALMANU AND ADEBAYO, F.I. DEPARTMENT OF APPLIED SCIENCE, COLLEGE OF SCIENCE AND TECHNOLOGY, KADUNA POLYTECHNIC, KADUNA NIGERIA. Corresponding Author E-mail: vocwummi2006@yahoo.com
ABSTRACT The antibacterial properties of ethanolic extracts of Globimetulla browni (Mistletoe) against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae were investigated using agar well diffusion method. The ethanolic extract of Globimetulla browni contained appreciable amount of tannins, moderate amount of alkaloids, cardiac glycosides, and saponins, trace amounts of steroids and total absence of flavonoids. At both 1000 mg/ml and 100 mg/ml concentrations, the antibacterial activity of the extract revealed high zones of inhibition against Staphylococcus aureus while the least zone of inhibition was observed against Klebsiella pneumoniae. The minimum inhibitory concentration (MIC) was determined using spread plate method. The MIC was observed at the concentration of 500 mg/ml against Staphylococcus aureus and Escherichia coli. At 1000 mg/ml, the MIC was observed against Pseudomonas aeruginosa, but the extract showed no effects against Klebsiella pneumoniae, while the minimum bactericidal concentration (MBC) was observed at the concentration of 500 g/ml against Staphylococus aureus. Keywords: Globimetulla browni, Staphylococcus aureus, Phytochemical, Agar-Well Diffusion method, McFarland Standard, Steroids.

INTRODUCTION Plant extracts continue to provide health coverage for over 80% of the world population, especially in the developing world (1). The medicinal values of these plants lie in their component phytochemicals, which produce definite physiological actions on human body. Some of these phytochemicals are alkaloids, tannins, flavonoids, cardiac glycosides and saponins (2). Different antibacterial phytochemical constituents of medicinal plants have been used for the treatment of bacterial infections as possible alternative to chemically synthetic

drugs to which many infectious microorganisms have become resistant (3). Medicinal plants such as mistletoe have been used traditionally and possess the following ascribed properties: antidiabetic, antihypertensive, anticancer, immunostimulator amongst others. There are advantages and disadvantages in the use of medicinal plants just like modern drugs.
Journal of Pharmaceutical and Allied Sciences Vol. 8 No. 3 (2011) ISSN: 1596-8499 Website: http://ajol.info/index.php/jophas

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Among the advantages are cost, accessibility, potential source of new drugs, use of natural products and its multi-component preparation aimed at healing several ailments simultaneously. Modern medicine is expensive and not within the reach of the majority of people in developing countries due to shortage of hospitals, health centres and the bureaucracy involved before the patient can get to the modern doctors (4). Mistletoe plant, called Kauche in Hausa, Afomo in Yoruba and Awushie in Igbo was originally applied to Viscum album (European mistletoe) and later the name was further extended to other related species such as Pharadendron flavescence (American mistletoe). Being a parasite, mistletoe grows as a hanging bush on the branches of trees with its cylindrical stem, ramified in pairs and slightly thickened at the nodes (5). Mistletoe belongs to the family, Lorantheceae. It grows on a tree and uses its root to invade the trees bark which allows mistletoe to absorb nutrients and water from the trees branches. Mistletoe extracts are prepared as water based solutions of water and alcohol. Preliminary trials carried out using oral mistletoe have indicated that mistletoe reduces the symptoms of high blood pressure, particularly headaches and dizziness. Mistletoe in low doses can relieve panic attacks and improve the ability to concentrate. The leaves and young twinges are used by herbalists in Europe for the treatment of circulatory and respiratory system problems (6). The use of medicinal plants is the most ancient approach to healing practiced by traditional medicine practitioners. There are various theories about the origin of the use of medicinal plants, depending on the various communities (7).

The aim of this present study is to determine the active components of the mistletoe leaf through phytochemical screening and evaluate the antibacterial activities of the active components on four bacterial species namely: Staphylocosus aureus, Eshcerichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. MATERIALS AND METHODS Collection of Plant Material (Globimetulla browni) The fresh leaves of Globimetulla browni (Mistletoe) were collected from the host plant Cordiea platyphlla (Crofan plant) in front of the Department of Applied Sciences, Kaduna Polytechnic in the month of April, 2010. The fresh leaves were washed and air-dried for three weeks and then pulverized (8). This research work was carried out between April-November, 2010 in the Microbiology Laboratories of the Department of Applied Sciences, Kaduna Polytechnic, Kaduna -Nigeria. Authentication and confirmation of the taxonomic identity of the plant material was carried out using voucher specimen in the herbarium of Biology/Microbiology Unit of Kaduna Polytechnic. Extraction Procedure Powdered sample (50 g) of the extract was weighed using a weighing balance (model number 1401 Binatone). The weighed sample was properly tied in a clean white cloth and then placed into an extraction thimble of a soxhlet apparatus. The boiling flask of the soxhlet apparatus was filled with 250 cm3 of 90% ethanol solvent, placed on a heating mantle and allowed to reflux for 2 hours. Thereafter, the thimble was carefully removed, when the boiling flask was almost free of ethanol which implies 1356

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solvent recovery. The extract was transferred into a conical flask and placed on a water bath to concentrate. The crude extract was allowed to cool after which it was weighed and recorded. Phytochemical Screening of the Extract The crude extracts were subjected to phytochemical screening for the following components: alkaloid, saponins, tannins, steroids, flavonoids and cardiac glycosides using standard methods (8). Bacterial Isolates The test microorganisms used in this study, Stephylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae were collected from the Microbiology section of the pathology laboratory at the Nigerian Army Reference Hospital (44), Kaduna. The bacterial isolates obtained were subcultured and identified by Gram staining. Preparation of Culture Media a) The Nutrient Agar: Nutrient agar powder (7.0 g) was carefully weighed into a clean conical flask and properly mixed with 250 ml of sterile distilled water. The mixture was thoroughly shaken and autoclaved at 1210C for 15 minutes. The slant cultures of the organisms were prepared aseptically using the sterile nutrient agar and incubated at 370C for 24 hours to obtain pure cultures of Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae used for the bioassay. b) The Nutrient Broth: Nutrient broth powder (6.3 g) was weighed into a clean conical flask, then dissolved with 250 ml of distilled water and mixed homogenously. A 10-ml volume of the homogenous mixture was transferred into 4 different test tubes, plugged with cotton wool and aluminum foil and then autoclaved at 1210C for 15 minutes. It was allowed to cool and a loopful of the test

organisms from the stored slant cultures were separately and aseptically introduced into the sterile nutrient broth in the test tubes which then served as the overnight broth (8). Preparation of McFarland Standard McFarland equivalent turbidity standard (0.5) was prepared by adding 0.6 ml 1% barium chloride solution of 99.4 ml H2SO4 and mixed thoroughly. A small volume of the turbid solution was transferred to a capped tube of the same type that was used to prepare the test and control inocula. Exactly 0.5 McFarland gives equivalent approximate density of bacteria 1 x 108 cfu (9). Antibacterial Activity Using Agar-Well Diffusion Method The ethanolic extract was used to prepare two different concentrations (1000 g/ml and 100 g/ml). For the concentration at 1000 g/ml, 0.02 g of the extract was accurately weighed, dissolved in 2 ml of sterile distilled water, and this then served as stock solution for testing at 1000 g/ml. For the concentration at 100 g/ml, 0.1 ml from the stock solution for testing at 1000 g/ml was measured and dispensed into a separate test tube containing 0.9 ml sterile distilled water which served as stock solution for testing at 100 g/ml. The test organisms were inoculated on solidified nutrient agar plates and then spread evenly with a sterile swab stick. Walls were then bored into the agar using a sterile 6 mm diameter cork borer. A 0.1 ml quantity of the various concentrations was introduced into the walls, allowed to stand at room temperature for 1 hour and then incubated at 370C. Another solidified nutrient agar plate was inoculated with the test organisms and wells were then bored into the agar. A 0.1 ml volume of distilled water was introduced into the wells, allowed to stand at room temperature for 1 hour and then incubated at 370C for 24 hours. The mean zones were measured in

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millimeters and recorded against the corresponding concentration. Determination of Minimum Inhibitory Concentration (MIC) Three different concentrations (1000 g/ml, 500 g/ml and 250 g/ml) were prepared for the extract. For the concentration at 1000 g/ml, 0.1 g of the extract was dissolved in 5 ml of sterile distilled water; for the concentration at 500 g/ml, 0.05 g of the extract was dissolved in 5 ml of sterile distilled water and for the concentration at 250 g/ml, 0.025 g of the extract was dissolved in 5 ml of distilled water. These three served as stock solutions. From each of the extract solution, 1 ml was pipetted into sterile labeled Petri plates containing molten nutrient agar and then mixed thoroughly to make final concentrations of 1000 g/ml, 500 g/ml and 250 g/ml respectively and allowed to solidify. Each of the plates was aseptically inoculated with a loopful of the standardized bacterial culture using the spread plate method and incubated at 370C for 24 hours after which they were examined for the presence or absence of growth. Determination of Minimum Bactericidal Concentration (MBC) The minimum inhibitory concentration (MIC) of each organism that does not show visible growth on the plates after 24 hours was sub-cultured into freshly prepared sterile nutrient agar plates labeled 1000 g/ml, 500 g/ml and 250 g/ml and then incubated at 370C for 24 hours. The lowest concentration of the extracts that

does not produce growth after 24 hours was regarded as the minimum bacterial concentration (MBC) (10).

Table 1: Percentage yield of the ethanolic plant extracts

Plant Infinite weight material of sample (g) Weight of % yield of extract (g) extract (g)

Globimetulla browni (Mistletoe) 50

42

84

Table 2:

Phytochemical Composition of the ethanolic crude extract of Globimetulla browni leaf extract ++ ++ ++ + +++

Constituent Alkaloids Cardiac glycosides Flavonoids Saponins Steroids Tannins

Key/legend +++ = Appreciable amount of phytochemical ++ = Moderate amount of phytochemical + = Trace amount of a phytochemical = Complete absence of phytochemical

Globimetulla browni had appreciable amount of tannin, moderate amount of alkaloids, saponins and cardiac glycosides, trace amount of steroids but flavonoids were completely absent.

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Table 3:

Antibacterial activity of Globimetulla browni against Staphylococcus aureus, Esherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae Mean zone of inhibition of plant extract using 1000 g/ml and 500 g/ml 1000 g/ml 100 g/ml 18.0 mm 13.0 mm 9.0 mm 8.1 mm

Test microorganism:

Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae

20.0 mm 14.0 mm 10.0 mm 9.5 mm

From the table above, Globimetulla browni at both 1000 g/ml and 100 g/ml concentrations had the highest antibacterial activity against Staphylococcus aureus followed by Escherichia coli, Pseudomonas

aeruginosa. The lowest antibacterial activity was against Klebsiella pneumoniae. The crude extracts of Globimetulla browni was found to have more effects on Gram positive than Gram negative bacteria.

Table 4:

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts

Test Microorganism 1000 g/ml g/ml S. aureus No growth

Concentration of the extract MIC 500 g/ml 250 1000 g/ml g/ml No growth Little growth Growth ---

MBC 500 g/ml, 250

No growth

--

E. coli

No growth

No growth

Growth

--

P. aeruginosa

No growth

Little growth

Growth

Growth

--

--

K. pneumoniae
Key: Growth = No Growth

Growth

Growth

Growth

--

--

--

Ineffective action of the extracts = Effective action of the extract

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MBC not carried out

MIC At the concentration of 1000 g/ml, effective activity of the extract was observed on the plate against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. At the concentration 500 g/ml, effective activity of the extract was observed on the plate against Staphylococcus aureus, Escherichia coli, but ineffective against Pseudomonas aeruginosa and Klebsiella pneumoniae. MBC At the concentration of 100 g/ml, inactivity of the extract was observed on the plate against Pseudomonas aeruginosa. At the concentration of 500 g/ml, effectiveness of the extract was observed on the plate against Staphylococcus aureus but there was no action against Escherichia coli. DISCUSSION Table 1 shows that a percentage yield of 84% was obtained for the extract, which may be useful in pharmacological and ethnobotanical studies. Table 2 indicates the phytochemical composition of the plant which revealed the secondary metabolite of the medicinal plant. Globimetulla browni had appreciable amount of tannins which are phytochemicals that inhibit antimicrobial activity, moderate amount of cardiac glycosides, alkaloids, saponins and trace amounts of steroids. From Table 3, at the concentration of 1000 g/ml of the extract, an excellent zone of inhibition of 20.0 mm was recorded on Staphylococcus aureus, moderate zone of inhibition of 14.0 mm on Escherichia coli, small zone of inhibition of 10.0 mm

recorded on Pseudomonas aeruginosa and 9.5 mm was observed on Klebsiella pneumoniae. At the concentration of 100 g/ml, 18.0 mm zone of inhibition was observed against Staphylococcus aureus, 13.0 mm zone of inhibition against Escherichia coli, 9.5 mm zone of inhibition against Pseudomonas aeruginosa and 8.1 mm zone of inhibition against Klebsiella pneumoniae. It was observed that zone of inhibition increased as the concentration of the extract increased. The antibacterial activity of mistletoe on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae has been reported to be due to the action of lectin in mistletoe. Differences in zones of inhibition could also be due to diffusibility of the action ingredients in the plant material and temperature of incubation could also influence this (11). The Minimum Inhibitory Concentration (MIC) of the extract was 500 g/ml concentrations which was effective on the first 2 isolates (i.e Staphylococcus aureus and Escherichia coli) and had little effects on Pseudomonas aeruginosa with no activities on Klebsiella pneumoniae. The MIC that showed no visible growth of bacteria was regarded as minimum inhibitory concentration of the extract. ACKNOWLEDGEMENT The co-authors wish to thank the Head of Department of Applied Sciences Department, Kaduna Polytechnic, KadunaNigeria for allowing access to the laboratories and reagents.

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10. Oyeleke, S.B. & Manga, S.B. (2008): Essentials of Laboratory Practicals in Microbiology 11th Edition by Tobert Publishers. Pp. 96-97. 11. Srinvansan, S.C. Burstein, A.H. & Welden, N. (2001): The effects of Mistletoe supplements on Pharmacokinetics of Saquinavir. Jorunal of Clinical Infectious Diseases. 2(24): 122-126.

6. Ernest, M. (2003) Mistletoe for Treatment of cancer. A systematic review of randomized clinical trails. International Journal of Cancer. 20:127-136.

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