|Year : 2011 | Volume
| Issue : 2 | Page : 226-229
In vitro antimicrobial activity of methanolic leaf extract of Psidium guajava L.
Anju Dhiman1, Arun Nanda1, Sayeed Ahmad2, B Narasimhan1
1 Department of Pharmaceutical Sciences, M.D. University, Rohtak, Haryana, India
2 Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
|Date of Submission||14-Oct-2010|
|Date of Decision||12-Feb-2011|
|Date of Acceptance||10-Mar-2011|
|Date of Web Publication||12-May-2011|
Department of Pharmaceutical Sciences, M.D. University, Rohtak, Haryana
| Abstract|| |
Aim: This study was designed to examine the chemical composition and in vitro antimicrobial potential of methanolic extract of Psidium guajava Linn (Myrtaceae). Materials and Methods: The inhibitory effect of methanolic extract of P. guajava was tested against three bacterial and two fungal strains by using the paper disc diffusion method. Results: The methanolic extract exhibited antibacterial activity against E. coli with minimum inhibitory concentration, 0.78 μg/ml, minimum bactericidal concentration of 50 μg/ml, and appreciable antifungal activity with minimum inhibitory concentration of 12.5 μg/ml. Preliminary phytochemical analysis of methanolic extract revealed the presence of antimicrobial compounds such as flavonoids, steroids, and tannins, which may contribute for the antimicrobial action of P. guajava. Conclusion: The extract was found to be bacteriostatic and fungistatic in action.
Keywords: Antimicrobial activity, paper disc diffusion method, Psidium guajava
|How to cite this article:|
Dhiman A, Nanda A, Ahmad S, Narasimhan B. In vitro antimicrobial activity of methanolic leaf extract of Psidium guajava L. J Pharm Bioall Sci 2011;3:226-9
|How to cite this URL:|
Dhiman A, Nanda A, Ahmad S, Narasimhan B. In vitro antimicrobial activity of methanolic leaf extract of Psidium guajava L. J Pharm Bioall Sci [serial online] 2011 [cited 2014 Apr 23];3:226-9. Available from: http://www.jpbsonline.org/text.asp?2011/3/2/226/80776
Finding healing powers in plants is an ancient idea. People in all continents have long applied poultices and imbibed infusions of hundreds, if not thousands, of indigenous plants, dating back to prehistory.  Since times immemorial, different parts of medicinal plants have been used to cure specific ailments in India. Now-a-days there is widespread interest in evaluating drugs derived from plant sources.  It is generally recognized that an increased consumption of vegetable and fruits protects against cancer and cardiovascular diseases. An attractive hypothesis is that vegetable and fruits contain bioactive compounds that have a protective effect.  One of the plants used traditionally is guava, Psidium guajava Linn (Myrtaceae). It is commonly called guave, goyave, or goyavier in French; guave, Guayebaum, and guayave in German; banjiro in Japanese; goiaba, goiabeiro in Portugal; araca-goiaba, araca-guacu, and guaiaba in Brazil; guayaba, guayabo in Espanol, and guava in English.  P. guajava contains a number of chemical constituents, which are reported to possess antibacterial, antidiarrheal, antimycobacterial, antihyperglycemic, antimalarial, cytotoxic, and antioxidant activities.  In a recent study, it was shown that the P. guajava aqueous extract possessed antibacterial activity against Salmonella More Details typhi and Klebsiella pneumoniae, but no effect on the growth of Escherichia More Details coli, Staphylococcus aureus, and Streptococcus fecalis organisms. 
The inhibitory effects of aqueous and alcoholic extracts of the P. guajava (root as well as leaves) on the growth of Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella enteritidis, Bacillus cereus, Proteus spp., Shigella spp., and Escherichia coli, causal agents of intestinal infections in humans were examined using the in vitro agar well diffusion method.  Results supported the utilization of P. guajava in traditional medicines for intestinal diseases produced by microorganisms.
In another study, antibacterial activity of P. guajava Linn., leaf extracts on diarrhea-causing enteric bacteria isolated from seabob shrimp, Xiphopenaeus kroyeri (Heller) were evaluated and the results supported the use of guava leaf-made medicines in diarrhea cases where access to commercial antibiotics is restricted. 
There is growing interest in correlating phytochemical constituents of plants with their pharmacological activity.  Since, the earlier reports on the plant leaf for antimicrobial activity are on the preliminary level even without MIC (minimum inhibitory concentration) values and bactericidal values as well as no report on the disc diffusion method. Hence, the objective of this study was to determine the antimicrobial activity of the methanolic extract of P. guajava Linn leaf with reference to gram positive, gram negative bacterial strains, and pathogenic fungal strains using the disc diffusion method and to correlate the activity with the plant constituents present.
| Materials and Methods|| |
Plant collection and identification
The fresh leaves of P. guajava were collected from Bahadurgarh, India in December 2009. The plant was identified and authenticated for taxonomic identity in Department of Biosciences, M.D. University, Rohtak, India, by Dr. J.P. Yadav, and a voucher specimen was deposited in Herbarium of Department of Pharmaceutical Sciences, M.D. University (voucher number DPS 0015).
Processing and extraction of plant material
The leaves were chopped and shade dried at room temperature for 2 weeks and then grounded to coarse powder using a homogenizer for ease of extraction of active compounds. The powdered plant material (100 g) was packed into a soxhlet apparatus (1 l) and extracted up to 4 h with petroleum ether (60-80 °C) for defatting. It was then extracted with methanol for further 4 h. The extract was filtered, and the solvent was evaporated under reduced pressure using a rotary vacuum evaporator, which was kept in a desiccator for further use.
Concentration of extract
The stock solution of the extract was prepared by dissolving 10 mg of plant extract in 10 ml DMSO. The following concentrations were prepared, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μg/ml of the crude extract for determination of minimum inhibitory concentration, minimum bactericidal concentration (MBC), and minimum fungicidal concentration. For antimicrobial assay, concentrations taken were from the range 1-20 μg/ml. A standard antibacterial agent, norfloxacin (Belco Pharmaceuticals, Bahadurgarh) and an antifungal agent, fluconazole served as a positive control. Dimethylsulfoxide (HPLC grade) was used as a negative control.
The crude methanolic extract of P. guajava was subjected to qualitative chemical screening for identification of various classes of active chemical constituents such as carbohydrates, tannins, saponins, cardiac glycosides, steroids/triterpenoids, flavonoids, anthraquinones, and alkaloids. The phytochemical analysis was done according to standard methods. ,
Tested bacterial strains (Bacillus subtilis, Staphylococcus aureus and Escherichia coli), and fungal strains (Candida albicans and Aspergillus niger) were obtained from the Department of Microbiology, Guru Jambeshwar University, Hissar, India.
Double strength nutrient media used for antibacterial studies were prepared using peptone (1 g), yeast (0.3 g), sodium chloride (0.5 g) and dissolving these ingredients in water q.s. to make 50 ml. The media were then sterilized by autoclaving at 15 lb/psi pressure for 15 min. Double strength Sabourauds glucose broth used for antifungal studies was prepared by dissolving glucose (8 g), peptone (2 g) in distilled water q.s. to make 100 ml with aid of heating. Then, the medium was cooled and filtered, pH was adjusted to 5.4 with 10% lactic acid. The media were sterilized by autoclaving at 15 lb/psi pressure for 15 min. 
Cultures were maintained at refrigerated conditions. For culture studies, the fresh 24-h-old cultures were prepared in the case of E. coli, S. aureus, B. subtilis, 48 h culture for C. albicans, and 7 days culture for A. niger. The bacterial/ fungal suspension was prepared in normal saline by transferring the organism from fresh cultures (1 × 10 8 cells/ml) [Table 1].
|Table 1: Classification and optimal growth parameters for microbes used in this study|
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Paper disc diffusion method
To determine the antibacterial activity of the crude extract, 0.2 μl individual bacterial and fungal cultures were poured with a nutrient agar medium (30 ml) in petri plates (90 mm). Sterilized filter paper discs (Whatman No. 1; 6 mm in diameter) soaked in different beakers containing the dissolved extracts of different concentrations were taken out with sterilized forceps and air-dried and placed on plates with the different organisms. The plates were incubated at 37°C for 24 h for bacterial strains, for 2 days at 37°C for C. albicans and for 7 days at 25°C for A. niger. After incubation, the inoculated plates were observed for zones of inhibition in millimeter diameter using a transparent ruler.
The sensitivity or susceptibility of the test bacteria and fungi to the standard drug was tested using an inoculated agar plate and norfloxacin and fluconazole at different concentrations. The zones of inhibition were measured and compared with those of the plant extract.  The experiment was performed in triplicate, and the results are reported as mean ± SD.
Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC)
The turbidimetric method or tube dilution method was used for determination of minimum inhibitory concentration, minimum bactericidal concentration, and minimum fungicidal concentration. The extract was serially diluted to give a concentration of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μg/ml in test tubes containing 1 ml sterile nutrient broth. Then, the tubes were inoculated with 100 μl of bacterial suspension in the saline and incubated at 37 °C for 24 h (for plates containing bacterial cultures), 37 °C for 2 days (for plates containing C. albicans culture), and 25 °C for 7 days (for plates containing A. niger culture). A tube containing nutrient broth only was seeded with the test organism to serve as control. All the tubes were then incubated at 37 °C for 24 h and then examined for growth by observing turbidity. The MBC of the plant extract on the clinical bacterial isolates was carried by pipetting out 0.1 ml bacterial culture from the mixture obtained in the determination of MIC tubes which did not show any growth and subcultured on to nutrient media and incubated at 37 °C for 24 h. After incubation, the concentration at which there was no single colony of bacteria was taken as MBC. MFC was also determined similar to MBC. 
| Results and Discussion|| |
Investigating the antimicrobial effect of guava leaves involved a comparison of its methanolic extract [Table 2] and [Table 3] with commercially available antibiotics by comparing the inhibition zones. We have noted that the commercial antibiotics showed a larger inhibitory effect than the guava leaf methanolic extract. This is not surprising and reinforces the position that commercially perfected and tested antibiotics should be used in treatments whenever available. The demonstration of activity of the extract against both gram positive and gram negative bacteria is an indication of the broad spectrum of activity and thus can be used to source antibiotic substances for drug development that can be used in the control of these bacterial infections. 
|Table 2: Zone of inhibition (mm) of different bacterial agents at various concentrations of the test extract of P. guajava and the standard norfloxacin|
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|Table 3: Zone of inhibition (mm) of different fungal agents at various concentrations of the test extract of P. guajava and the standard fluconazole|
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In the previous investigations, some results were reported, in which P. guajava leaves were subjected to ethanolic extraction, fractionation, and isolation of the flavonoidal compounds. Fractions together with the isolates were tested for their antimicrobial activity using the agar diffusion technique, against bacteria such as S. aureus, E. coli, P. aeruginosa, and the fungus C. albicans;  while our results showed that the methanolic extract of P. guajava can inhibit the growth of S. aureus, E. coli, P. aeruginosa, and the fungus C. albicans and A. niger by the paper disc diffusion method supported by the tubidimetric method.
Noscomial aspergillosis is a life threatening infection in immunocompromised patients caused by A. niger and studies are being conducting to explore genetics of A. niger and therefore, to unravel resistance development. 
It was found that the methanolic extract exhibited antibacterial activity against E. coli with MIC, 0.78 μg/ml, and MBC of 50 μg/ml, and antifungal activity with the MIC of 12.5 μg/ml [Table 4]. The extract was found to be bacteriostatic and fungistatic in action. The phytochemical screening results indicated the presence of carbohydrates, flavonoidal glycosides, steroids, and tannins as the main constituents that might be responsible for antimicrobial activity of the test extract.
|Table 4: MIC and MBC/MFC of P. guajava methanolic leaf extract against microbial strains|
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Flavonoids are hydroxylated phenolic substances; occur as a C 3 -C 6 unit linked to an aromatic ring. They are known to be synthesized by plants in response to microbial infection. Their activity is probably due to their ability to complex with extracellular and soluble proteins, and to complex with bacterial cell walls. More lipophilic flavonoids may disrupt the microbial membranes.  The flavonoids extracted from guava leaves were believed to be responsible for the antimicrobial activity. 
Tannins have received a great deal of attention in recent years, since it was suggested that the consumption of tannin-containing beverages, especially green teas and red wines, can cure and prevent a variety of ills. Their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cells envelop transport proteins, etc. They also combine with polysaccharides. 
The mode of action of antibacterial effects of saponins seems to involve membranolytic properties, rather than simply altering the surface tension of the extracellular medium. 
| Conclusion|| |
As the rapid emergence of drug-resistant organisms necessitates the continuous search of new antimicrobial substances, natural products may act as alternative for antibiotics and chemotherapeutic agents in certain circumstances. The results showed that the methanolic extract of P. guajava leaf was able to inhibit all of the bacteria and fungi used in this study with different degree of inhibition. The information obtained may provide validation for its reported medicinal uses. In conclusion, the guava leaf methanolic extract is most effective against the tested bacterial strains than the fungal strains.
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[Table 1], [Table 2], [Table 3], [Table 4]
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