Phytochemical Screening and Antibacterial Properties of Organic Solvent Fractions of Psidium guajava Aqueous Leaf Extracts
Resistance of some bacteria, especially some stains of E. coli to common antimicrobial agents has created an urgent need to develop alternative antimicrobial drugs from herbs that are safe, cheap and may overcome the resistance of the pathogens. The crude aqueous extract of Psidium guajava leaf which is known to possess some antibacterial properties was further subjected to sequential fractionation with organic solvents (chloroform, ethyl acetate, normal butanol) of different polarity. This was done until the organic layer was visibly clear to obtain chloroform, ethyl acetate and n-butanol soluble fractions and residual aqueous fraction. Phytochemical screening and antibacterial activity of organic solvents soluble fractions and residual fraction of the extract on some gram positive and gram negative microbes were carried out. The different fractions showed variation in phytochemical constituency and thus in their antibacterial properties. The ethyl acetate soluble fraction of the extract showed broad spectrum antibacterial properties against all the organisms tested. The fraction also showed a good activity against E. coli at a relatively lower concentration and hence could possibly be use against E. coli infections.
Infections with Avian Pathogenic Escherichia coli (APEC) cause colibacillosis, an acute and mostly systemic disease resulting in significant economic losses in poultry industry worldwide (Ewers et al., 2003). Antimicrobials are valuable tools in colibacillosis control, however, a substantial proportion of avian pathogenic E. coli strains have developed resistance to antimicrobial drugs commonly used in poultry production (Yang et al., 2004). Of particular concern is the emergence of resistance to frontline antimicrobials, such as the fluoroquinolones, which because of their low toxicity and relatively broad-spectrum coverage are extremely valuable for treating infections (Angulo et al., 2000; Livermore et al., 2002). The emergence of these resistant bacteria has created a major concern and an urgent need for new antibacterial agents (Emori and Gaynes, 1993; Davis, 1994; American Society of Microbiology, 1995). Furthermore, using antibiotics to subside infection produces adverse toxicity to host organs, tissues and cells (Prakash, 2006). Herbal molecules are safe, will overcome the resistance produced by the pathogens since they are in combined form or in pooled form of more than one molecule in the protoplasm of the plant cell (Prakash, 2006).
Psidium guajava leaves are employed in the treatments of a cascade of
diseases including diarrhea, dysentery and vertigo. It is also used in regulating
menstrual periods and dressing wounds in many parts of Africa (Iwu, 1993). The
crude extract of Psidium guajava has been reported to possess some antibacterial
properties (Guan and Damello, 1999; Geidam et al., 2007). Majority of
medicinal plants used for herbal treatments are flowering plants (angiosperms)
and are readily available in the rural areas (Farnsworth and Morries, 1976)
and this has made traditional medicine relatively cheaper than orthodox medicine
in the third world countries. Since over 80% of the worlds population
use plant as their primary source of medication (Farnsworth et al., 1985;
Cordell, 2000) and antibiotics are sometimes associated with adverse side effects
to the host including hypersensitivity, immuno-suppressive and allergic reactions
(Ahmed et al., 1998; Prakash, 2006), it is important to develop alternative
antimicrobial drugs that are herbal based, for the treatment of infectious diseases
(Clark, 1996; Cordell, 2000). Some herbs have been recommended for the treatment
of some diseases like tuberculosis (Mata et al., 2004) and ischemic heart
disease (Gauthaman et al., 2005). McLaughlin (1991) reported that using
bioassay guided screening and fractionation of plant extracts ensures that the
compounds will have better biological activity and therefore, stands a good
chance for drug discovery through subsequent structure-activity relationship
studies to obtain a compound with improved activity and least toxicity.
The present study is therefore to further determine the phytochemical constituents and the antibacterial activity of the organic solvent fractions of Psidium guajava on some bacterial organisms. The result of the antimicrobial study would identify the most active organic solvent fraction of Psidium guajava leaf and give validity to its possible use as an antibacterial agent against E. coli infections.
MATERIALS AND METHODS
Sample collection, identification and preparation of extract: Fresh samples of the leaves without stalk were collected in February, 2006 from the University of Maiduguri campus, Maiduguri, Nigeria. The plant was identified and authenticated by Dr. S.S. Sanusi of the Department of Biological Sciences, University of Maiduguri, Nigeria. A voucher specimen (Chemistry 242 B) was deposited in the Department of Chemistry, University of Maiduguri, Maiduguri.
The fresh leaves of P. guajava collected were air-dried in the laboratory, ground into fine powder and stored in a glass container at 4°C. Eight hundred grams of the powdered sample was exhaustively extracted with distilled water using a reflux method. The crude aqueous extract obtained was concentrated in vacuo, brown in colour and yielded 33.75% (w/w). It was properly labeled and stored in the refrigerator at 4°C until used (Trease and Evans, 1989). All work was carried out in accordance with the general guidelines for methodologies on research and evaluation of traditional medicine (WHO, 2000).
Fractionation of the aqueous extract: The crude aqueous extract obtained was suspended in cool distilled water and then filtered using Whatman No. 1 filter paper. The filtrate was thereafter fractioned successively with chloroform, ethyl acetate and normal butanol (Fig. 1). The fractionation with the organic solvents which are of different polarity was done until the organic layer were visibly clear to get chloroform, ethyl acetate and n-butanol soluble fractions and residual aqueous fraction, in sequence as described by Cho et al. (2003) and Motohashi et al. (2004).
Phytochemical analysis: The P. guajava organic solvent fractions
were subjected to qualitative chemical screening for identification of the various
classes of active chemical constituents such as carbohydrates, tannins, phlabotannins,
saponins, glycosides, steroids, triperpinnoids, flavanoids, anthraquinones and
||Schematic diagram of extraction and fractionation of P.
guajava leaf extract into organic solvent fractions
The phytochemical analysis was done by standard methods (Trease and Evans,
1989; Trease and Evans, 1997).
Microbial cultures: Laboratory isolates of the pure culture of gram-positive (Staphylococcus aureus and Streptococcus fecalis) and gram-negative (Salmonella typhi, Klebsiella pneumoniae and Escherichia coli) bacteria were obtained from the Veterinary Medicine Research Laboratory, University of Maiduguri, Nigeria.
The isolates were propagated and stored on nutrient agar plate. The nutrient agar medium was obtained in dehydrated powdered form (Oxoid Ltd., England) and was prepared according to the manufacturers specification. All stock cultures were maintained in nutrient agar plate at 4°C and sub-cultured in nutrient broths (Oxoid Ltd., England) at 37°C for 8 h prior to antimicrobial testing. One milliliter of the broth culture was then used to flood the agar plates.
Extracts concentration: Stock solutions of the different organic solvents soluble fractions were prepared by dissolving 100, 200 and 400 mg of the extract in 1 mL of distilled water. The following concentrations of each fraction were prepared, 100, 200 and 400 mg mL-1. Standard antibacterial agent (oxytetracycline, Cipla Ltd., Mumbai, India) at a concentration of 10 mg mL-1 was also used on all the bacteria organisms and their zones of inhibition were compared with those of the extract.
Antimicrobial sensitivity testing: Disc diffusion method as described by the National Committee of Clinical Laboratory Standards (1993) was used to determine the antibacterial activity of the various organic solvent fractions of Psidium guajava leaf. Discs containing different concentrations of dissolved extracts was prepared with sterilized filter papers (Whatman No. 1; 6 mm in diameter) soaked in different beakers containing different concentrations (100, 200 and 400 mg mL-1) of the extracts. The discs were dried at 50°C.
Overnight cultures of each bacterial isolate were diluted using sterile normal saline to give an inoculums size of about 106 Cfu mL-1. The inocula were spread on the surface of dried nutrient agar plates with cotton wool swabs, which have been dipped in the diluted suspension of the organisms. The plates were incubated at 37°C for 30 min before the discs were applied aseptically. The treated plates were incubated at 37°C for 48 h. The same procedure was carried out using oxytetracyclin (10 mg mL-1) as the positive control. Plates without the antibiotic or extract discs were set up as the negative control experiment. The zone of inhibition above 6 mm diameter of each isolate was used as a measure of susceptibility to the extracts and this was compared to that of the standard antibiotic.
Minimal inhibitory concentration: The Minimum Inhibitory Concentrations
(MIC) of the ethyl acetate soluble fraction of aqueous extract of Psidium
guajava leaf were determined using the method described by Greenwood (1989).
Six sterile test tubes were arranged in five rows in a test tube rack, each
row for one of the five microorganisms used for the test. Half a milliliter
of sterile nutrient broth was pipetted into all the tubes. In addition, 0.5
mL of the ethyl acetate soluble fraction of the extract containing 200 mg mL-1
was pipette into the first tubes of the 5 rows to obtain a concentration of
100 mg mL-1. Thereafter there was a serial dilution of the extract
in each row to obtain concentrations of 50, 25, 12.5, 6.25 and 3.13 mg mL-1,
respectively. The test organisms (0.5 mL) were pipetted into each of the test
tubes and incubated at 37°C for 24 h. The MIC was recorded as the least
concentration of the extract that completely inhibited the growth of the test
organisms. The content of the tubes were further sub-cultured for 24 h to determine
bactericidal or bacteriostatic activity. Bactericidal effect was demonstrated
when no growth occurred on the sub-cultured medium after MIC determination.
The result of extraction and fractionation of the crude extract of Psidium guajava leaf is presented in Fig. 1. The n-butanol soluble fraction of the extract is more in quantity followed by ethyl acetate and chloroform soluble fractions respectively. However, the quantity of the residual fraction is more than any of the organic solvent fractions. The phytochemical investigation of the various solvent fractions of P. guajava showed the chloroform fraction of the extract to contained only steroids, which occurred in high concentration. The residual aqueous and n-butanol fractions contained similar phytochemical constituents in which the n-butanol fraction appears to contain higher concentrations of carbohydrate and flavanoid. The ethyl acetate fraction contained only tannins, steroids, glycosides and high concentrations of flavanoids. The ethyl acetate fraction did not contain any carbohydrate (Table 1).
|| Phytochemistry of organic solvent fractions of aqueous extract
of P. guajava leaf
|+ = Low concentration; ++ = Moderate concentration; +++ =
High concentration; - = Absent
|| Antibacterial efficacy of organic solvent fractions of aqueous
extract of P. guajava leaf
|R = Indicates the resistance to the test organisms
||The minimum inhibitory concentrations of ethyl acetate soluble
fraction of aqueous extract of P. guajava leaf on some bacteria
|+ = Indicates bacterial growth; - = Indicates no bacterial
Inhibition of bacterial growth with the chloroform soluble fraction was observed only on Klebsiella pneumoniae, whereas the ethyl acetate fraction showed significant inhibitory effects on the growth of the entire gram-positive and gram-negative bacteria used in this study. The inhibition of the organisms by the ethyl acetate fraction appears to be concentration and organism dependent. The E. coli was inhibited more by this fraction compared to the other organisms used in this study. The n-butanol fraction inhibited the growth of Salmonella typhi and Klebsiella pneumoniae both of which are gram negative organisms. No effect on the growth of Staphylococcus aureus, Streptococcus fecalis and E. coli was observed from the n-butanol fraction. The residual extract has no effect on any of the organisms used in this study. Oxytetracycline, the standard antibacterial agent used inhibited the growth of all the organisms used in the study. The zones of inhibition produced by the oxytetracycline on the organisms were far greater than those produced by the different concentrations of Psidium guajava leaf organic solvent fractions except that of Klebsiella pneumoniae organism where the ethyl acetate fraction had more inhibitory effect (Table 2).
The MIC of ethyl acetate soluble fraction of Psidium guajava aqueous leaf extract for the different micro organisms tested is presented in Table 3. E. coli was found to be most sensitive to the fraction since growth was inhibited at a relatively low concentration. This is followed by Staphylococcus aureus, Streptococcus fecalis and Salmonella typhi. Klebsiella pneumoniae was found to be least sensitive to the inhibitory effect of ethyl acetate fraction. There was no growth of the bacteria tested following sub-culture of contents of the tubes above the MIC.
The present study further strengthens earlier reports by Guan and Damello (1999)
and Geidam et al. (2007) that Psidium guajava leaf has antibacterial
activities. This may also be in agreement with the claim of herbal healers.
They claim to use the plant to treat diarrhea, dysentery and wounds (Iwu, 1993).
The organisms tested have been implicated in diarrhea and/or dysentery (Salmonella
sp., E. coli and Klebsiella sp.) and wound infection (staphylococcus
and streptococcus). The ethyl acetate soluble fraction showed broad spectrum
of activity against all the gram positive and gram negative bacteria used in
the study. This indicates that the ethyl acetate fraction has better antibacterial
properties as compared to the narrow spectrum of activity of the crude extract
as reported by Gnan and Demello (1999) and Geidam et al. (2007). This
is in agreement with the findings of McLaughlin (1991) who reported that fractionation
ensures better biological activity. E. coli was found to be most susceptible
to ethyl acetate fraction of the extract while Staphylococcus aureus
was the least susceptible. The activity of this fraction against E. coli
is interesting since E. coli strains have developed resistance to antimicrobial
drugs commonly used in poultry production (Yang et al., 2004) and even
to frontline antimicrobials, such as the fluoroquinolones (Angulo et al.,
2000; Livermore et al., 2002).
The antibacterial properties of ethyl acetate fraction could be due to flavanoids
and tannins contents of the fraction, both of which are known to possess appreciable
antimicrobial activities (Narayana et al., 2001). The crude extract has
been reported to contain carbohydrate, saponin, steroid and cardiac glycosides
(Geidam et al., 2007). Carbohydrates could facilitate the growth of bacterial
organisms and therefore may antagonize the antibacterial activity of the active
principles. This may account for the narrow antibacterial activity of the crude
extract and other fractions of the leaf extract of Psidium guajava that
In conclusion, this study has shown that the ethyl acetate soluble fraction of Psidium guajava aqueous leaf extract possesses broad spectrum antibacterial properties. This fraction also showed a good activity against E. coli at a relatively lower concentration and thus could possibly be use against E. coli infections. However, further studies need to be carried out.
The efforts and assistance of Hamidu Usman, Isa Gulani and Mr. Fine is highly appreciated.
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