Research Article
Antimicrobial Evaluation of Kigelia africana (Lam)
How to cite this article:
Jeyachandran, R. and A. Mahesh, 2007. Antimicrobial evaluation of Kigelia africana (Lam). Res. J. Microbiol., 2: 645-649.
ABSTRACT
The evaluation of the activity of the aqueous, methanol and chloroform extracts of the bark of the Kigelia africana (Lam) was tested against E. coli, Enterobacter aerogens, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Pseudomonas aeruginosa (Gram-negative), Staphylococcus aureus and Bacillus cereus (Gram-positive) by disc diffusion method. The methanol extracts presented a higher activity than the aqueous and chloroform extracts. It exhibits the greatest activity against Salmonella typhi and Proteus vulgaris moderate activity against E. coli, Staphylococcus aureus and Bacillus cereus. The remaining strains viz., Enterobacter aerogens, Klebsiella pneumoniae and Pseudomonas aeruginosa were presented less activity. The inhibition zone was recorded and compared with standard antibiotic drug streptomycin. Results support the traditional use of Kigelia africana (Lam) bark as a good source of antimicrobial agent.
INTRODUCTION
Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world (Saxena and Sharma, 1999; Ahmad and Beg, 2001). Various medicinal plants have been used for years in daily life to treat diseases all over the world. It has been used as a source of medicine. Higher plants, as source of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient time (Farombi, 2003). Over 50% of all modern clinical drugs are natural products origin and natural products play an important role in drug development programmes in the pharmaceutical industry (Baker et al., 1995).
The success story of chemotheraphy lies in the continuous search for new drugs to counter the challenge posed by resistant strains of microorganisms (Khan et al., 2003). Antibiotics are sometimes associated with adverse effects on hosts which include hypersensitivity, depletion of beneficial gut, mucosal microorganisms, immuno suppression and allergic reactions (Idose et al., 1968; Ahmed et al., 1998). The investigation of certain indigenous plants for their antimicrobial properties may yield useful (Khan et al., 2003) and there is increasing interest in plants as source of agent to fight microbial diseases and treatment of several infections (Chariandy et al., 1999; Aburjai et al., 2001).
Kigelia africana is one of highly valuable ethnomedicinal plants belonging to the family Bignoniaceae and vernacular name is marachurai. The plant bark is used for rheumatism, dysentery and veneral diseases and also used for ring worm, tape worm, haemorrhaging, malaria, diabetes, pneumonia and tooth ache (Akunyili and Houghton, 1993; Kolodziej, 1997). However to the best of our knowledge, there is no previous study on this particular plant. Therefore, the lack of the information in the literature prompted this investigation in order to evaluate the antimicrobial activity of Kigelia africana.
MATERIALS AND METHODS
The medicinal plant Kigelia africana (Lam) bark, used in this study, were collected around Tiruchirappalli district, South India. The collected plant materials were identified at Rapinat Herbarium, St. Joseph's College, Tiruchirappalli, South India (Mathew, 1983). The bark were shade-dried at room temperature for 10 days.
Extraction Procedure
The dried and powdered plant materials (100 g) were extracted successively with 600 mL of aqueous, methanol and chloroform (1:6 w/v) by using soxhlet extractor for 48 h at a temperature not exceeding the boiling point of the solvent (Lin et al., 1999). The extracts were filtered using Whatman No.1 filter paper and then concentrated in vacuum at 40°C using a Rotary evaporator. Each extracts transferred to glass vials and kept at 4°C before use.
Bacterial Strains
Eight different laboratory bacterial strains were used namely, Escherichia coli, Enterobacter aerogens, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris and Pseudomonas aeruginosa (gram-negative), Staphylococcus aureus and Bacillus cereus (gram-positive). The bacterial strains were supplied by the Department of Microbiology and institute of Basic Medical Science, Chennai, India.
Preparation of Inoculum
The bacterial strains preserved in the nutrient agar at 4°C were revived in nutrient broth (liquid medium) and incubated at 37±1°C for overnight and the suspensions were checked to provide approximately 105 cfu/mL.
Microbiological Tests of Plant Extracts
The disc diffusion assay methods of Iennette (1985) as described by Rosoanaivo and Ratsimanaga-Urverg (1993), Rabe and Van Staden (1997) were used with modification to determine the growth inhibition of bacteria by plant extracts. The diluted bacterial culture (200 μL) was spread over nutrient agar plates using sterile glass L-rod. One hundred microliter of the each extracts was applied per filter paper disc (Whatman No. 1, 6 mm dia) and was allowed to dry before being placed on the layer of the agar plate. Each extracts was tested in triplicate (3 discs/plate) and the plates were inoculated at 37±1°C for 24 h. After incubation, the diameter of inhibition zones and the sensitivity were measured with a caliper. Standard antibiotic of streptomycin (10 mg/disc) was used as reference of positive control.
Statistical Analysis
Random sampling was used for the entire test in triplicates. Calculations were carried out in triplicate with their mean values and standard deviation by using the formula given by Gupta (1977). Positivity index was calculated by comparing the zone of inhibition of bark extracts with standard antibiotics.
RESULTS AND DISCUSSION
The antibacterial activity of Kigelia africana bark extract (aqueous, methanol and chloroform) against Escherichia coli, Enterobacter aerogens, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus by disc diffusion method showed that the methanolic bark extracts highly affected the activity of Salmonella typhi and Proteus vulgaris
The inhibition against Escherichia coli, Staphylococcus aureus and Bacillus cereus was moderate and less inhibition was associated with Enterobacter aerogens, Klebsiella pneumoniae and Pseudomonas aeruginosa. Chloroform extracts inhibited moderate activity against Proteus vulgaris and Staphylococcus aureus and the other strains exhibited less activity. The aqueous extracts were exhibited less activity of Staphylococcus aureus and all the remaining strains showed very poor activity. These results were compared with standard antibiotic, streptomycin as a standard
The aqueous and organic extracts exhibited different activities. Organic extracts showed greater activity than aqueous extract. Because most of the antibacterial principles were either polar or non-polar and were extracted only through the organic solvent medium (John Britto, 2001). It was reported that methanol was a better solvent for the consistent extraction of antimicrobial substances from medicinal plants when compared to other solvents such as aqueous, ethanol, chloroform and hexane (Lin et al., 1999; Ahmad et al., 1998; Eloff, 1998). Present observation suggested that the organic solvent extraction method was suitable to verify antibacterial activity. Similar conclusions were drawn by Krishna et al. (1997) and Singh and Singh (2000) in their studies.
The antibacterial activity of plant extracts can be attributed to not only a single bioactive principle but also due to the combined action of other compounds (Sunayana et al., 2003). A number of phytochemicals have been studied for their antibacterial activity which are potentially useful against infectious diseases. It is clear that the chemical structure of the antimicrobial agents found in higher plants belong to most commonly encountered classes of higher plant secondary metabolites such as flavonoids (Watchter et al., 1999), terpenes (Conveney et al., 1985), terpenoids (Osawa et al., 1990; Habibi et al., 2000) and phenolic acids (Fernandez et al., 1996).
From the results, it can be concluded that plant extracts have great potential as antimicrobial principles against microorganisms and that they can be used in the treatment of infectious disease caused by resistant microorganisms. Kigelia africana showed maximum antibacterial activity and hence this plant can be used to discover bioactive natural products that may serve as leads for the development of new pharmaceuticals. This will also offer a great help in facing the emergence for spread of antimicrobial resistance.
ACKNOWLEDGMENT
We are grateful to the Management, St. Joseph's College, Tiruchirappalli for providing necessary facilities.
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Ethnobotanical Leaflets 14:797-806. 2010.
Epicuticular Wax and Volatiles of Kigelia pinnata Leaf Extract
Atolani Olubunmi1* and Olatunji A. Gabriel2
1Department of Chemical Sciences, Redeemer’s University, PMB 2011, Ogun State, Nigeria
2Department of Chemistry, University of Ilorin, P. M. B 1515, Ilorin, Nigeria
Issued: July 1 2010
Abstract
The fractions of volatile compounds in plants are essential in defining the sensory impact of an aromatic smell, as well as being of some medicinal importance. Epicuticular wax in plants also determine its susceptibility to disease, and herbivore, desiccation and ultraviolet radiation damage. The epicuticular wax consisting of hydrocarbons and some volatile compounds of the leaves of Kigelia pinnata (Kigelia africana; family Bignoniaceae), a multimedicinal plant, was extracted, isolated and analyzed qualitatively and quantitatively for the various chemical compositions using GC (equipped with a flame thermionic detector -GC/FTD) and GC-MS (“GCMS-QP2010 Plus, which employs a dual inlet Turbo Molecular Pump (TMP) that provides increased sensitivity and column flow capacity for improved performance and confident results. The evaluation is useful for the future comprehensive analysis of the constituents responsible for the various documented medicinal uses of the leaf. The extracted constituent revealed twelve compounds with the major ones identified as n-hentriacontane (55.40%): a probable anti-tumour compound; 1-tricosene, (18.45%); 11- (2,2-dimethylpropyl) heneicosane, (9.66%); 2,6,10-trimethyldodecane, (4.43%); pentafluoroheptadecyl ester, (4.40%); 2-ethylhexyloctadecyl sulfurous acid ester, (3.05%); heneicosane, (1.61%); and hexyloctyl sulphurous acid ester, (1.42%). Other compounds are recorded in minute quantities. The major component, identified as hentriacontane, a C-31 saturated hydrocarbon apparently is responsible for the plant susceptibility to diseases, protection against UV radiation and for the antitumour and antioxidative potential of the plant. This will give credence to the traditional use of the plant as an anti-inflammatory and anticancer. The ability of the plant to act as anti-sun burn may be due to the presence of the wax.
Key words; Kigelia pinnata, Kigelia africana, Epicuticular wax, Bignoniaceae, volatiles, GC-MS, hentriacotane.
Introduction
The pharmaceutical, cosmetic and food industries are constantly being faced with the challenge of identification, isolation and characterization of volatile compounds of medicinal importance in plant materials. Kigelia pinnnata (Jacq) DC, also known as Kigelia africana (Lam) Benth and sausage tree on account of its large fruits, is a member of the family Bignoniaceae. It is a multipurpose medicinal plant with great potentials to be developed as drug by pharmaceutical industries (Sangita et al., 2009, Olatunji and Atolani, 2009). The traditional uses of the stem, bark, root, leaves and fruit are enormous. Studies that have been reported on the leaf of this plant are rather limited. The plant’s crude extract is used as antiageing and antiburn in cosmetic such as cream. The plant is rich in volatile constituents. Phytochemical studies on the stem bark revealed the presence of Kigelinone and isopinnatal (Akunyili and Houghton, 1993), specioside, verminoside and minecoside (Picerno et al., 2005; Neelam et al., 2006) and phenylpropanoid (Gauda et al., 2006). The root has been reported to possess isopinnatal, kigelinol and isokigelinol (Moideen et al., 1999) and p-coumaric acid (Binutu et al., 1997). Flavonoids and irridoids have been isolated from its leaves (Gouda et al., 2003).
The anti-diarrhoea activity of the aqueous leaves extract has been confirmed (Akah, 1996). The ethanolic extract of the stem bark was examined to show strong analgesic and anti-inflammatory activities by inhibiting the synthesis of prostaglandins and other inflammatory mediators, (Owolabi and Omogbai, 2007). The stem bark extract have been reported for their cytotoxicity activities (Houghton et al., 1994), Central Nervous System Stimulation (Owolabi et al, 2008), antifungal activity (Jain and Belsare, 2009) and anti-trypanosomal activity against trypanosome brucel brucei in vitro (Moideen et al., 1999), while the root extract have been reported for its positive anti- uterus cancer against malignant melanoma (Skin cancer) (Houghton et al., 1994 and Msouthi and Mangombo, 1983).
Epicuticular waxes are partially saturated lipid compounds located on the above-ground portions of land plant (Post-Beittenmiller, 1996). The chemical composition of waxes varies by species: in maize, the epicuticular wax is composed of fatty acyl chains that are later converted to alcohols, esters and aldehydes and a small proportion of alkanes (Bianchi et al., 1985 and 1989) Flavonoids, fatty acid esters could also be found in epicuticular waxes (Schmutz et al., 1994; Whitaker et al., 2001). Epicuticular wax have been extracted, purified by column and thin layer chromatography (Kolattukudy, 1965 and 1966) and reported to determine plant susceptibility to diseases, herbivore, desiccation (Juliana and Silvana, 2006) and ultraviolent radiation protectant (Lacy et al., 2003)
To the best of our knowledge, very little has been reported on the phytochemistry and medicinal use of the leaves of K. pinnata, despite its many traditional applications. Hence it is important to demystify the compositions of the components of the leave. This constitutes the main objective of this study.
Materials and Method
All the solvents used were obtained from the Department of Chemistry, University of Ilorin, Ilorin, Nigeria and were pre-distilled before use. Leaves of Kigelia pinnata were collected from a fruiting tree in Ado Ekiti, Ekiti State, Nigeria during the summer time. Prof. Oladele in the Department of Plant Biology, Faculty of Science of the University of Ilorin, Ilorin Nigeria, carried out taxonomic authentication of the plant.
Extraction
Kigelia pinnata leaves were dried at room temperature. The air-dried plant material was blended into powder at a low temperature. 240g of the powdered plant material was extracted exhaustively with cold hexane for six days. The extract was combined, concentrated in vacuum and 3.06g of brownish-green syrup was obtained.
Isolation of volatiles
The syrup was applied to a column chromatography (CC) packed with Silica gel and eluted with hexane. Eleven fractions were collected, examined (talc) and combined as appropriate. The major combined fraction was subjected to a silica gel flash chromatography. Six yellow fractions with same Rf (silica gel) were obtained, combined and evaporated to dryness. This was coded KPLH/CC/FC/0.5. The chromatoplate was sprayed with vanillin spray reagent for detection of other spots.
Separation of Component
The volatile fraction (KPLH/CC/FC/0.5) was analysed by gas chromatography (GC) using a GC 2010 gas chromatography (Shimadzu, Japan) equipped with a flame thermionic detector -GC/FTD and an electronic high pressure control injector. The flow of the carrier gas (He at 100.2 kpa) 1.61mL/min; linear velocity: 46.3cm/sec; total flow: 6.2mL/min; purge flow: 3.0mL/min. The split ratio is 1:0. The analysis was performed using the following program; oven kept isothermally at 60oC for 6 min, increased from 60oC to 220c at a rate of 25oC/min and kept isothermally at 220oc during 5min. It was increased to 280oC at a rate of 5oC/min for 20min; Equilibrium time, 3min. Total ion chromatogram (TIC), 1.0
GC-MS Analysis
Gas Chromatography-Mass Spectroscopy, GC-MS System; GCMS - QP2010 PLUS (SHIMADZU JAPAN) interfaced with a finigan MAT ion trap detector ion source Temp., 200oC; interface Temp, 250oC; solvent cut time, 2.50min; relative detector gain mode, ACQ mode; Scan: start time - end time; 3.00min - 46.00min; event time, 0.50 sec; Scan Speed, 1428. Identification of the volatile components was carried out using the peak enrichment technique of reference compounds and as final confirmation of the peak identification by GC-MS, comparing their spectra with those of the NIST commercial library mass spectra.
FT-IR analysis
The infrared spectrum of the volatile was recorded on a Shimadzu (8400S) Fourier Transform-Infrared Spectroscopy (FTIR) Spectrum Spectrophotometer using KBr pellet.
Results and Discussion
A total of twelve compounds were detected in the fraction (KPLH/CC/FC/0.5) of Kigelia pinnata examined. The compounds identified, the retention times, peak areas, percentage yields and base peaks are shown in Table 1. The IR spectrum of the isolated epicuticular wax showed υmax (cm-1, KBr) 3479 – 3282, (OH); 2955 – 2850, (CH2, CH3); 1650, (C=O); 1462 and 1377, (CH2, CH3). About 91% of the total isolate is hydrocarbon.
Hentriacontane (55%), a saturated hydrocarbon, the major compound identified has been isolated from Scabiosa comosa (Dargaeva and Brutko, 1976). It is reported to be responsible for its uptake in the soil by plant (Éric, 1995) and shown to be involved with stimulation of fungal spore germination (Dauri and José, 1995). Hentriacontane has also been isolated from spinach leaves, and discovered to be unsaponifiable (Edith and Ida 1929) and shown to have possible anti-tumour activity (Takahashi et al., 1995). Methyl 12- Methyl tetradecanoate, a lipid of biological significant was also identified in a microscale. It has been identified in trace amount in Cryobacterium psychrophilum (Ken-Ichiro et al. 1997), Xylella fastidiosa plant (Ana Valéria et al., 2007), and Corynebacterium sepedonicum plant (Paul et al., 1988). It was reported for its inhibition capacity on the development of corneal angiogenesis, which is responsible for blindness and other infections (Cole et al., 2007). The three main non-hydrocarbons obtained are 2-ethylhexyl-octadecyl ester (3.05%), heptadecyl ester (pentafluoropropionic) (4.40%) and hexyloctyl ester (1.42%). Tetradecanoic acid (12-methyltetradecanoate) was obtained in a low quantity (0.29%). Hexadecanoic acid has earlier been reported as a component in alcohol extract of the leave of Kigelia pinnata (Grace et al., 2002). The components reported in this work have not been reported in literature before.
In conclusion, twelve volatiles were identified and characterised by the GC/GCMS. The major component, identified was hentriacontane, a C-31 saturated hydrocarbon which might be apparently responsible for the plant susceptibility to diseases, herbivore, desiccation, protection against UV radiation and for the antitumour and antioxidative potentials of the plant. This will give credence to the traditional use of the plant as an anti-inflammatory and anticancer. The ability of the plant to act as anti-sun burn when used in cream may be partly due to the presence of the wax. We hereby report that the major paraffin in the epicuticular wax of Kigelia pinnata leave is n-hentriacontane, an n-C31 hydrocarbon (Kolattukudy, 1965 and 1966).
ACKNOWLEDGEMENTS
We thank the authority of Redeemer’s University, Nigeria for the use of their Infrared Spectrometer and also acknowledge National Research Institute (NARICT), Zaria, Nigeria for the use of their GC/GCMS instrument.
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