Antimicrobial potential of ethanolic extract of Psidium guajava leaf and its isolated fraction against some pathogenic microorganisms

¹M. Shakeera Banu and ²K. Sujatha

¹P.G. Department of Biotechnology, Sree Narayana Guru College, Coimbatore, Tamil Nadu, India.

²P.G. and Research Department of Zoology, Government Arts College, Tamil Nadu, India.

ABSTRACT:

The present investigation focuses on the antimicrobial potential of leaf extract of Psidium guajava and its isolated fraction against the selected bacterial strains. Ethnolic leaf extract of Psidium guajava and its isolated fraction was more potent in inhibiting the growth of pathogenic Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis with different degree of inhibition. The antibacterial activity was more effect in isolated fraction compound than the plant extract. The results support the notion that plant extract containing compound may have many roles in pharmaceuticals as antimicrobial formulations.

KEYWORDS: Psidium guajava, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis

INTRODUCTION:

Although this era witness amazing success in development of technology, science and medicine, but we failed to control the dramatic spread of infectious diseases. Microbes are existing in earth since million years ago, being one of the oldest creatures in this planet. These tiny microorganisms are adapted, developed and survived in this changing nature throughout the eras, while other advanced huge ancient animals and plants perished. Though, microbes considered as one of the most adaptive and successive creatures in nature ¹.

Interestingly, traditional medicine (including herbal medicine) is currently considered as a rapidly growing health system worldwide, it remains widespread in developing countries and it is increasing greatly in developed countries ². The applications of traditional medicinal plants have been employed as remedy long before the development of western medicine with the advent of science and technology. Though, it could be effective alternative source for many therapeutics, particularly after the recent dramatic failures of antibiotics against multi-drug resistant microorganisms.

Psidium guajava Linn. is one of such medicinal plants belonging to the family Myrtaceae that is also used as a source of food. It is a native of Central America but is now widely cultivated throughout the tropics. Psidium guajava fruit is still enjoyed as a sweet treat by indigenous peoples throughout the rainforest region. The leaves and bark of Psidium guajava tree have a long history of medicinal uses that are still employed today ³. The present study is aimed at evaluating the antimicrobial potential of leaf extracts of Psidium guajava and its isolated fraction against some pathogenic microorganisms.

MATERIALS AND METHODS:

Collection and preparation of plant material

Fresh leaves of Psidium guajava were collected in Coimbatore, during the months of April-May. The plant was authenticated by Botanist at the Government Arts College, Coimbatore. Plant material was dried under shade at room temperature, pulverized by a mechanical grinder and sieved through 40 meshes, then stored in airtight closed bottles until required.

Extraction and isolation

The coarse powder (100 g) was extracted successively with ethanol (250 ml) by hot continuous percolation method in a Soxhlet apparatus for 24 hrs. The extracts was concentrated and re-concentrated in petroleum ether (40°-60°C) (fraction-I), diethyl ether (fraction-II) and ethyl acetate (fraction-III) in succession in the ratio of 1:2:1. Each of the steps was repeated three times to ensure complete extraction in each case. Fraction I was rejected since it was rich in fatty substances whereas fraction II was analysed for the free flavonoids in each of the samples. Fraction III of each of the test samples was hydrolysed by refluxing with 7% H₂SO₄ (10 ml/gm residue) for 5 hrs. The mixture was filtered and the filtrate extracted with ethyl acetate in a separating funnel. The ethyl acetate layer was washed with distilled water till neutrality and dried in vacuo. The residues were taken up in small volumes of ethanol separately and then the fraction was subjected to TLC to confirm the isolated fraction (quercetin).

Test Microorganism and culture media

The bacterial strains used for the test were both pathogenic and non pathogenic such as, Escherichia coli (cause serious food poisoning in humans), Staphylococcus aureus and (cause a range of illnesses, from minor skin infections such as pimples, impetigo, boils and cellulitis folliculitis, to life-threatening diseases such as pneumonia, meningitis, bacteremia and sepsis) and Staphylococcus epidermidis (is not usually pathogenic). All stock cultures obtained from Department of Microbiology, from Karpagam University, Coimbatore. Nutrient Agar (Himedia, India) was used as the media for the culturing of bacterial strains.

Screening antimicrobial activity

Anti-microbial activity was performed by the direct plate method. From the nutrient broth of about 50 μl of each of the test organisms were transferred in to different nutrient agar plate containing (20ml NA) and organism the organism was spread throughout the medium by sterile L rod. After spread plate is done, a 5mm diameter cork borer was used to make four wells in to each plate and in different concentrations (10 μl, 50 μl, and 100 μl) of ethanolic leaf extracts of Psidium guajava and its isolated fraction was introduced in to each plate. Control plates were also maintained. This assay was conducted at regular intervals of 24 hrs. The plant extract and its isolated fraction used at a concentration of 10 μl (a – plant extract and e – isolated fraction), 50 μl (b – plant extract and f – isolated fraction), 100 μl (c – plant extract and g – isolated fraction) and d – control.

RESULTS AND DISCUSSION:

Psidium guajava plant has wide uses and it has some activities against the bacteria, fungus etc. It has anti-bacterial, anti-fungal, treatment of cough, anti-diarrhoeal, anti-malarial. The antimicrobial activity of dried leaf extract of Psidium guajava and its isolated fraction is presented in Fig. 1-3. The results of the present study showed that the ethanolic leaf extract of Psidium guajava able to inhibit Escherichia coli (Fig. 1 a, b and c), Staphylococcus aureus (Fig. 2 a, b and c) and Staphylococcus epidermidis (Fig. 3 a, b and c) with different degree of inhibition. The maximum inhibiting activity showed in 50 μl and 100 μl volume containing plate.

In view of the attributed medicinal properties, the present study was undertaken fractions of Psidium guajava leaves were also tested for its antimicrobial activity. The isolated fraction compound showed inhibition activity against all tested organisms Escherichia coli (Fig. 1 e, f and g), Staphylococcus aureus (Fig. 2 e, f and g) and Staphylococcus epidermidis (Fig. 3 e, f and g). Staphylococcus epidermidis was the most sensitive strains to isolated fraction. The antibacterial activity of isolated fraction compound showed highest activity against the all bacterial pathogen compared with leaf extract. These results were compared with positive control (without tested compounds).

Iwu, ⁴ tested the extract of Psidium guajava showed in vitro antimicrobial activity against Escherichia coli, Salmonella typhi, Staphylococcus aureus, Proteus mirabilis, and Shigella dysenteria. Cáceres et al. ⁵ tested Psidium guajava leaf extracts with three solvents of different polarities (n-hexane, acetone and ethanol) and discovered that the ethanol extract was the most efficient against the pathogenic enterobacteria tested.

Psidium guajava shown to antibacterial against Staphylococcus aureus and it was also useful against Streptococcus species⁶. Gnan and Demello⁷ also reported a complete inhibition of a hot water (80ºC) extract of Psidium guajava leaves against Staphylococcus epidermidis and Salmonella typhimurium. In addition, Coutino-Rodríguez et al.⁸ confirming its growth inhibition effect particularly on Staphylococcus aureus, Escherichia coli, and other common entero-pathogenic cultures.

Vieira et al.⁹ have also reported Psidium guajava leaves extracts found to inhibited the growth of the Staphylococcus aureus. The methanolic plant leaf extracts of Psidium guajava and barks of this plant have anti-microbial activity against Salmonella species, Bacillus species, and the concentrations vary according to the organisms¹⁰. Psidium guajava have shown potent antimicrobial activities against Propionibacterium acnes and may be beneficial in treating acne ¹¹.

Although, plants have many other defense mechanisms against micro and macro organisms. Medicinal plants are rich in a numerous variety of secondary metabolites of antimicrobial properties such as saponines, tannins, alkaloids, alkenyl phenols, glycoalkaloids, flavonoids, sesquiterpenes lactones, terpenoids and phorbol esters ^{12, 13}. Accordingly, since times immemorial, plants have been resisting the continuous attacks of microorganisms by producing endless secondary metabolites. However, plants are able to develop new, faster and natural antimicrobials and then man-made remedies ¹⁴, and that is explaining why plants succeed in its fighting against microbes since millions of years while human failed.

In the present study we have isolated quercetin from the ethnolic extract of Psidium guajava leaf and the isolated fraction compound quercetin was confirmed by TLC. Interestingly the isolated fraction showed more potent antimicrobial activity than the plant extract.

Four antibacterial compounds were isolated from the leaves of Psidium guajava and the flavonoids extracted from Psidium guajava leaves were found to be effective against the several strains of food borne pathogenic bacteria ¹⁵. The leaves are rich in tannin, and have antiseptic properties ¹⁶. The microbicidial activity of Psidium guajava was attributable to guajaverine and to psydiolic acid. The active flavonoid compound guaijaverin extracted from leaves of same plant was reported to have high potential antiplaque activity ^{17, 18}. Furthermore, the leaves contain large amounts of tannin, riterpernoids (crateogolics, guaijavolic, oleanolics and ursolic acid) and essential oils containing β-sitosterol, β-bisabolene, β-cariophyllene, aromadendrene, β-salinene, guaijaverine, nerolidiol and sel-11-en-4α-ol ¹⁹.

CONCLUSION:

The antimicrobial studies showed good activities for the extracts of Psidium guajava leaf and its isolated compounds. This study has provided a scientific basis of the used of extracts of Psidium guajava and its isolated fraction making up important potential sources of new antimicrobial compounds.

ACKNOWLEDGEMENT:

I gratefully acknowledge Dr. V. K. Gopalakrishnan, Head, Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore, for providing me the facility to do the experiments and his useful guidance.

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Received on 22.03.2012

Modified on 01.04.2012

Accepted on 06.04.2012

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Research J. Pharmacology and Pharmacodynamics. 4(3): May-June, 2012, 169-171