Abstract: Ethanolic leaf extract from Aframomum melegueta was accessed for in vitro antimicrobial activity as well as phytochemical constituents. Refuse dump materials were screened for resident micro floral with antagonistic activities against the pathogens. Thirteen pure isolates were obtained in all including nine bacteria and four fungi. Five pathogenic bacteria; Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Klebsiella pneumoniae were tested for their susceptibilities to the antimicrobial activity of the leaf extract as well as the antagonistic potential of the isolates refuse Standard antibiotics were also tested on the pathogens and their susceptibilities investigated. The effectiveness of the antimicrobial phytoconstituents from Aframomum melegueta and microbial antagonisms was compared with that of the standard antibiotics in which the former fairly compete with the latter. The plant extract of Aframomum melegueta was found inhibitory to the growth of Salmonella typhi and Klebsiella pneumoniae while the refuse isolates were more effective than the bacteria as shown growth inhibitory indices. Seven of the nine isolates inhibited the growth of at least one of the pathogenic organisms; Klebsiella pneumoniae and Staphylococcus aureus being the most susceptible. The findings revealed that the extract from the plant and the refuse isolates offer an enormous potential as biocontrol of these pathogens and source of antimicrobial agent of therapeutic importance.
INTRODUCTION
In recent years, acquired resistance to most commonly available standard antibiotics like chloramphenicol and ampicillin by most commonly encountered pathogenic microbes like Shigella dysenteriae and Haemophilus influennzae type b is a growing world wide problem (Monica, 2000). It has gotten to the extent that one could correctly predict the resistance of certain organisms to specific antimicrobials. Monica, 2000 has stated that Proteus species are generally resistant to nitrofurantoin and tetracycline, Streptococcus pyogenes to penicillin, Klebsiella pneumoniae to ampicillin and anaerobes to metronidazole. There have also been reported cases of drug hypersensitivity and many third generation antibiotics have prolific side effects on patients’ normal flora. Protracted use of these refined drugs also results in immunosuppression.
It has therefore become expedient that an alternative therapeutic measure be adopted. This may include the use of medicinal plant extract and general traditional medicine. There is also a glimpse into biocontrol, which is the use of antagonistic microorganism to control and curtail these deleterious creatures.
Traditional medicine is wide spread throughout the world. It is the total combination of knowledge and practices, whether explicable or not, used in diagnosing, preventing, or eliminating a physical, mental, or social disease. This may rely exclusively on past experience and observation handed down from generation to generation, verbally or in writing; while bearing in mind the original concept of nature which includes the material world, the sociological environment, whether living or dead and the metaphysical forces of the universe (Sofowora, 1993).
The term, traditional medicine is interchangeably used with herbal medicine and natural medicine (Hazan and Atta, 2005). Since antiquity, man has used plants to treat common infectious disease and even long before mankind discovered the existence of microbes, the idea that certain plants had healing potential was well accepted (Rios and Recio, 2005).
A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purpose or which are precursors for the synthesis of useful drugs. A number of plants have been used in Traditional medicine for many years (Sofowora, 1993).
The fact that plants have anti-microbial properties has been proven by many scholars. Specifically, the medical value of these plants lies in some chemical substances that produce a definite physiological action on the human or animal body (Edeoga et al., 2005). The most important of these bioactive constituents which, are majorly secondary metabolites are alkaloids flavonoids tannins and phenolic compounds These phytochemicals are toxic to microbial cells.
Aframomum melegueta, alligator pepper is a Tropical plant of the genus Aframomum, belonging to the family Zingiberaeae of the angiosperms in the kingdom planta. It is a perennial herb of about 3 ft, with narrow leaves. The fruits are ovate with reddish-brown irregular seed that is strongly aromatic and of pungent taste (Gordian, 2005). It is widely spread across west tropical Africa including Cameroon, Cote D’ Ivoire, Ghana, Liberia, Nigeria, Sierra Leone and Togo; often used as food additives and flavouring agents apart from serving medicinal purposes.
This plant has been found to exert some physiological affect on human body. Gordian (2005) reported that it is being used in Nigeria as stimulant. Antimicrobial properties investigation carried out by Razaq et al. (2003) revealed that its extract is a broad spectrum antimicrobial agent.
The objectives of the present investigation are to isolate, characterize and identify microorganisms associated with refuse dump site and assay their antagonistic effect on some pathogens. Comparative evaluation of the antimicrobial activities of the crude extracts from Aframomum melegueta on the pathogenic organisms as well as screening for the phytochemicals of pharmacological importance was also determined.
MATERIALS AND METHODS
Test Organisms
The pure culture of Escherichia coli, Samonella typhi,
Klebsiella pneumoniae, Staphylococcus aurerus and Bacillus ceraus.
were collected from the Medical Microbiology Laboratory of the University College
Hospital (UCH), Ibadan, Oyo State, Nigeria.
Plant Sample: Source, Extraction and Fractionation
The leaves of plant, Aframomum melegueta were bought from central market,
Akure, Ondo State. The leaves were air dried and grinded into fine powder. About
500 g of the pulverized plant sample was extracted with 60% ethanol. The sample
was soaked for 72 h after which the resultant mixture sieved with muslin cloth.
The sieved extract was concentrated to dryness using a rotary evaporator The
extract was dissolved in 0.1M Tris-HCL buffer (pH 7.0, 5 mL) and applied to
a column (5x85 cm) of Sephacryl S-300 HR, pre-equilibrated and developed with
the same buffer. Fractions corresponding to the peak were pooled together concentrated
and freeze dried. The powder was dissolved in water and applied to a Sephadex
G-25, column (1.5x50 cm), then eluted with water and fractions were collected.
The eluate obtained was concentrated and lyophilized.
Refuse Dump Sample Collection and Isolation
Both decomposing and decomposed refuse materials were separately collected
in sterile MacCarney bottles. The samples were collected from three different
dump sites in Akure, Ondo State, Nigeria. Microorganisms associated with the
refuse was isolated and identified using standard methods (Prescott et al.,
2002).
Detection of Antagonistic Activity
The antagonistic activities of the refuse isolates against the pathogenic
organisms were determined using the modified agar diffusions assay described
by Schillinger and Lucke (1989). Old culture of the refuse bacterial (isolates
on slant) was inoculated asceptically into the tubes of fresh nutrient broth
and incubated at 37°C for 24 h. After 24 h incubation, the culture supernatant
of broth was obtained by configuration.
Wells were made with an 8 mm cork borer in plates containing solidified nutrients agar already seeded with 0.1 mL of the pathogenic isolates from 24-h-old culture. Then, few drops of culture supernatant of actively growing refuse isolates were introduced into the wells with the aid of sterile syringes. The plates were incubated at 37°C for 24 h after which zones of inhibition around each well were examined.
Antimicrobial Screening of the Plant Extract
The antimicrobial study of the plant extract was carried using the agar
well diffusion method described by Cowan (1999).
Phytochemical Screening
The bioactive molecules were screened for using the methods of Harbone (1984).
Antibiotic Sensitivity Tests
To determine the antimicrobial activities of the standard antibiotics, the
disc diffusion method described by Brock and Madigan (1988) was employed.
RESULTS AND DISCUSSION
The total number of distinct bacterial isolates obtained from the refuse samples based on their colonial, morphological and biochemical characteristics were nine (Table 1) and four fungi species (Table 2). It shows that refuse dump is inhabited by myriad of biota as reported by Koestler (2006).
The results of the antimicrobial activities of the extract are presented on Table 3. It was unable to inhibit the growth of all the organisms at lower concentration of 100 and 200 mg mL-1. This suggests that the inhibitory potential of the extract is concentration dependant. Similar findings had been reported by Cowan (1999). However, at 300 mg mL-1 zones of inhibition of 5.0 mm and 6.5 mm were observed with Salmonella typhi and Kebsiella pneumoniae conforming to the result obtained by Razaq et al. (2003).
The antagonistic activities of the refuse isolates on the pathogens are presented on Table 4. All the isolates were found to posses’ antagonistic activity. Microorganisms associated with biodegradation had been reported to produce toxic metabolite to other competing micro floral (Koestler, 2006). Staphylococcus aureus was the most susceptible of the pathogens. Escherichia coli were resistant to all the isolates. The variation in the susceptibility and resistivity may be due to some inherent genetic factors in the organisms which may be chromosomal or plasmid coded. It may also be as a result of their gram reaction owing to difference in chemical composition that determines the structural integrity of each organism. All the fungi inhibited the growth of all the selected pathogens (Table 5). Aspergillus repens was the most effective having zones of inhibition ranging from 11 mm to 13 mm. Fungi had been shown to be an excellent reservoir for antibiotics (Prescott et al., 2002)
The sensitivity of the organisms to the standard antibiotics varies. Susceptibility of the gram negative ones was higher (Table 6). None of the organisms however, is susceptible to Cefunxine. Only Salmonella was susceptible to Ofloxacin while Ciprofloxacin was effective against all the organisms. The reason for the observed trend may be similar to those ascribed for the refuse isolates. The potency of the antibiotics was found to be higher than that of the extract and the antagonist in some cases. This can be due to high purification and concentration of active antimicrobial ingredients in the antibiotics.
| Table 1: | Characterization and identifinat of refuse bacterial isolates sugar fermentation |
| Table 2: | Cultural and microscopic characterization of fungi isolates |
| Table 3: | Antimicrobial activties of leaf extract of Aframomum melegurta |
| NI = Inhibition | |
| Table 4: | Antagonistic effects of refuse bacterial isolates on pathogenic isolates |
| NI = No Inhibition | |
| Table 5: | Antagonistic effects of refuse fungi on pathogenic bacteria |
Phytochemical screening revealed that Aframomum melegueta contains antinutritional constituents like Saponins and Tannins, Cardiac glycosides, flavonoids and terpenoids. However, alkaloids, anthraquinonones and Phlobatannins were absent (Table 7). The presence of these bio-molecules may be responsible for the antimicrobial activities of the extract while the absence of some may be responsible for its inactivity at low concentration.
| Table 6: | Antibiotic sensitivity of pathogenic bacteria isolates |
| OF = Ofloxacin, C = Chloranphenicol, CF = Cefunoxine, AM = Ampicillin, N = Nitrofurantione, CIP = Ciprofloxacin, TE = Tetracycline, NF = Narfloxacin, TE = Tetracyclin, AX = Amoxycillin, FX = Floxapen, AU = Augumentin, E = Erythromycin, CD = Clidamycin, GN = Gentamycin, Cx = Cephaplexin, CO = Cotrimoazole, AP = Cloxacilin NI = No Inhibition | |
| Table 7: | Phytochemical constituents of Aframomum melegueta |
| + = Present, - = Not present | |
Although the absence of Alkaloids, Anthraquinones and Phlobatanniins lowered its microbial activity, other Phytochemical Constituents preset justified their medicinal used as stated by Gordian, (2005). Alkaloids were mentioned alongside with other phytochemicals like tannins, flavonoids and phenolic compounds as most important bioactive constituents of leaf extracts (Edeoga, et al. (2005) and these compounds have in vitro antimicrobial properties according to Cowan (1999).
Almost all the refuse isolates were active against at least one of the pathogens.It therefore mean that they possess broad spectrum of activity more than the antibiotics. Krasil (1997) stated that the properties of antagonism is characteristic of all species of microorganisms. The results obtained here were in conformity with the report of Brock and Madigan, (1988) that antibiotics-producing microorganism occur very frequently in natural habitats However, better consistency was observed with the use of standard antibiotic discs.
CONCLUSIONS
In general, microbe-microbe interactions have not yet been extensively explored. The present study offers further knowledge on the subject matter of microbe interaction and specifically, antagonism. With further improvements, there is possibility of refuse isolates being used as biocontrol for pathogenic organisms.
The need to study medicinal plants cannot be overemphasized for a vista of reasons including inter-alia widespread use of plants in folk’s medicine, rescuing traditional plants and knowledge about them from imminent loss as well as the need for health for all. The plant Aframomum melegueta can be of immense use in phytomedicine and can then be included in health care delivery system particularly in the developing economy. Further studies on more effective method of extracting only the necessary constituents and standard reconstitution means as well as other better processing, refining and purification measures using HPLC, GC, NMR and mass spectroscopy for structural elucidation of the biologically active molecules would be necessary.