Medicinal plants constitute an effective source of both orthodox and traditional
medicine, herbal medicine has been shown to have genuine utility with about
80% of rural dwellers depending solely on it for primary health care (Akinyemi
et al., 2005).
In Nigeria, over 300 plants are used for treating various diseases including
HIV/AIDS opportunistic infections such as pneumonia, diarrhea, typhoid fever,
candidiasis, tuberculosis and other ailments (Sofowora, 1986;
Medicinal plants are known to owe their curative potentials to a certain biological
active substances which are refered to as active principles or phytochemical
substances and these include terpenes, flavonoids, saponins, anthraquinones,
glycosides etc (Iwu et al.,1993).
Essential oils are important constituents of some higher plants comprising
monoterpenes, sesquiterpene, arylpropanoids and fatty acid derivatives. They
have been recognized long ago to possess antimicrobial activities (Del-Vechio
et al., 2009).
Gongronema latifolium (Endl.) Decne commonly called Utazi and Arokeke
or Madumaro in the South eastern and south western part of Nigeria respectively
belongs to the family Asclepiadaceae. It is a tropical rain forest plant primarily
used as spice and vegetable in traditional folk medicine (Ugochukwu
and Babady, 2003; Ugochukwu et al., 2003).
Few studies have been carried out on the phytochemical composition, antimicrobial
properties and essential oil constituents of the plant. Aqueous and Ethanolic
extracts of the plant have been reported to have inhibitory effect on pathogenic
microoganisms (Eleyinmi, 2007). Its hypoglycemic,
hypolipidemic and antioxidative properties had also been reported (Ugochukwu
and Babasdy, 2003). Due to wide spread use of this plant by traditional
healers detail scientific investigation is needed.
This present study was therefore embarked upon to achieve the following objectives; (1) To identify the essential oil constituents (2) determine the antibacterial and antifungal effects of the essential oil and other extracts of the plant on bacteria and Yeast causing blood stream infection in HIV patients in Lagos with a view to determine whether this plant can serve as an alternative medical therapy for managing HIV blood stream infections in this resource limited environment.
MATERIALS AND METHODS
Sources and collection of plants material: Fresh plant samples of
Gongronema latifolium were collected from Ikorodu market and Mile 12
Market in Kosofe Local Government Area, Lagos State. The fresh leaves samples
were authenticated at the Department of Botany and Microbiology, Faculty of
science, University of Lagos. This study commenced in November 2008 and was
concluded in September 2009.
Phytochemical screening: Preliminary phytochemical tests as described
by Harbone (1984), Sofowora (1986)
were carried out on the aqueous extract of Gongronema latifolium.
These tests involve the addition of appropriate chemical agents to the aqueous extracts of the plant in a test tube.
Alkaloids, Saponins, Tannins, flavonoids and other compounds were tested for using these methods.
Preparation of extracts: The leaves sample were rinsed and air-dried. They were further dried in vacuum oven at 50°C for 10-15 h. The leaves were milled completely into coarse powder by grinding. The powdery form of the leaves was further treated to extract active ingredient.
Aqueous extract: Aqueous extract was carried out as described by Eleyinmi
(2007) and Adeleye et al. (2008a). One hundred
and eighty gram of dried milled leaves powder was soaked in 300 mL of sterile
distilled water for 5 days at 4°C. The solution (i.e., powdered leaves and
water) was centrifuged at 10,000 rev min-1 for 5 min and was filtered
with Whatmann No. 1 filter paper.
The filtrate was poured into a 250 mL beaker and labeled appropriately. The filtrate was dried at 50°C for 2 weeks until a constant dry weight of the extract was obtained in a Vacuum oven.
Ethanol extract: Method of ethanol extraction was similar to the aqueous
extraction as described by Eleyinmi (2007) and Adeleye
(2008a). One hundred and eighty gram powder leaves material was soaked in
300 mL of 70% ethanol for 5 days at room temperature.
The solution was also centrifuged at 10,000 rev min-1 for 5 min and this was filtered with whatman No. 1 filter paper into 250 mL conical bottle flask. The ethanol filtrate was placed in a vacuum oven at 50°C and dried for 2 weeks to evaporate the alcohol. This was also labeled accordingly.
Extraction of essential oil: Extraction of essential oil from vegetable
material was carried out by hydro-distillation process as described by Nenad
et al. (2007).
Sources of microoganisms: The test organisms employed for screening
plant for antimicrobial activity of essential oil and Gongronema latifolium
extracts were isolates from the blood stream infections of HIV infected patients
obtained from the Lagos University Teaching Hospital Complex (LUTH) and Nigeria
Institute of Medical Research (NIMR) Yaba, Lagos (Adeleye,
The organisms were Shigella dysentriae, S. flexneri, Staphylococcus aureus, S. chromogenes, S.c. cohnii, S.c. urealyticum, S. warnei, S. sciuri, S. epidermdis, Escherichia coli, Salmonella typhi, S. typhimunium, Klebsiella pneumonia, Pseudomonas aeruginosa, P. flourescence, Onchrobactrum anthropi and Candida albicans.
They were all sub-cultured unto fresh nutrient agar plates and incubated 24 h before use.
Preparation of inoculum: Active cultures for screening were prepared by transferring a loopful of cells from the stock cultures to test tube of Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi and were incubated without agitation for 24 h at 37°C and 72 h at 25°C, respectively. The cultures were serially diluted with fresh Mueller-Hinton broth and Sabouraud dextrose broth to achieve a McFarland standard of 0.5 corresponding to a cell density of 1.5x108 cfu mL-1 for bacteria and 1.5x105 spore mL-1 for fungal.
These were use to inoculate the Mueller-Hinton plates by using 0.1% inoculum suspension to swab uniformly using sterile cotton wool.
Antimicrobial screening: The agar diffusion method of NCCLS in 2003 was employed for the screening of antimicrobial activities of extracts. Sterile cork borer of 6.0 mm diameter were used to bore holes into the organisms seeded plates and three drops of the reconstituted water and ethanol extract were dropped, into the holes.
Three drops of essential oil was also dropped in other holes. Sterile distilled water was used as positive control, while Ampicillin, Ciprofloxacin and Chloramphenicol were used as negative control. These were done in triplicates under aseptic condition.
All the plates containing the test organisms and extracts (Ethanol, Water and Essential oil) were incubated at 37°C for 24 h for bacteria and at 25°C for 48 h for yeast, respectively. Staphylococcus aureus ATCC 25923 and Escherichia coli NCTC 10148 were used as standard test organisms. Zones of inhibition were measured in millimeters (mm).
Minimal Inhibitory Concentration (MIC): The Minimum inhibitory concentration of the extract (aqueous and ethanol extracts were determined for each of the isolate using dilution susceptibility test. Two fold serial dilutions of each extract i.e., 100 mg mL-1 was carried out in seven tubes containing sterile 2 mL Mueller Hinton broth to give the following extract concentration of 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mg mL-1.
A loopful of the standardized test organisms was inoculated aseptically into the tubes containing the serially diluted extracts and incubated at 37°C for 24 h.
Minimum Bactericidal Concentration (MBC): This is the lowest extract concentration at which the organisms did not recover and grow when transferred into a fresh medium. This was determined by subculturing from the tubes not showing visible growth after 24 h on MacConkey agar plates for bacteria and incubated at 37°C for 24 h and Sabouraud dextrose agar plate for yeast at 25°C after 48 h.
Essential oil-MIC and MBC Determination: Serial dilutions of the essential oil was done using 10% Tween 80 in sterile nutrient broth and Sabouraud dextrose broth for bacteria and yeast (Candida albicans), respectively.
This was to facilitate essential oil dispersion. The 0.5 mL of the essential
oil was added to 2 mL of the media and double fold serial dilution was carried
out to give a concentration of 1, 2, 5, 10 and 40 μg mL-1. Each
strain of test organism was tested with essential oil by inoculating with 50
mL physiological saline containing 5x106 cells for bacteria and 5x105
for Yeast, respectively. They were incubated at 35°C for 18-24 h (bacteria)
and at 22°C for 48-72 h (yeast) (Panizzi et al.,
1993; Del-Vechio et al., 2009). MIC and MBC
were determined as described above by sub-culturing from the tubes not showing
visible growth after 24 h on MaConkey agar and sabouraud dextroxe agar and incubated
at 37°C for 24 h (Bacteria) and 25°C for 48 h (yeasts). Results were
recorded in millimeter after measuring the diameter of zone of inhibition.
GC/MS analysis of essential oils components: This was carried out using Gas Chromatography/Mass Spectrophotometry (GC/MS) method to analyze and identify the essential oil constituents.
The Gas chromatographic analyses/Mass spectrophotometry was performed with Agilent system consisting of a model 7890A, with the following parameters: Column 30x0.25 mm, idx0.25 μm HP-5 m sec fused silical capillary with a (5%phenyl)-methylpolysiloxane stationary phase (Agilentpart No. 19091S-433). Oven Temperature programme: 50 to 250°C gradient of 10oC/min, up to 200°C; injector and detector temperature 250°C; Carrier gas Helium (3.325 mL min-1), sample size: 0.5 μL.
The compounds of the oil were identified using their retention time indices(determined with reference to a homologous series of normal alkanes) and by comparison of their mass spectral fragmentation patterns(NIST data base (G 1 036 A,revision D.O.I.00/chem. Station data system (G170ICA, version CO.OO.1.08))13 and with data available in common literatures.
Phytochemical screening: Phytochemical screening revealed the presence
of compounds such as Saponins, Alkaloids, Pyhlobatinnins, Glycosides and Flavonoids
and the absence of Tannin (Table 1).
The results antimicrobial activity of aqueous, ethanolic and essential oil extracts of G. latifolium are shown in Table 2.
Staphylococcus sp., Shigella sp., Salmonella sp., Klebsiella Pneumonia, Pseudomonas sp., Escherichia coli and Onchrobactrum anthropi were all inhibited at 100 mg mL-1. The essential oil showed the highest antimicrobial and fungicidal effects against all the test organisms including Candida albicans compared to aqueous and ethanolic extracts as zones of inhibition ranged between 7.5 mm for Pseudomonas aeruginosa to 11.25 mm for Shigella flexneri, respectively.
Zones of inhibition of Aqueous extract at 100 mg mL-1 ranged from 7.2 mm (Klebsiella pneumonia) to 10.00 mm for Staphylococcus urealyticus, respectively. Ethanolic extract also ranged from 7.5 mm for Klebsiella pneumonia to 11.0 mm for Escherichia coli at 100 mg mL-1, respectively.
||Bioactive compounds in Gongronema latifolium
||MIC and MBC of aqueous, ethanolic and essential oil extracts
||(a, b)Bar chart representing zones of inhibition of extract
@100 mg mL-1
However, when compared to Ampicillin, Ciprofloxacin and chloramphenicol at 250 mg, the diameter recorded were less (Ampicilline 22.0 mm), (Ciprofloxacine 28.0 mm) and (Chloramphenicol 23.0 mm) (Fig. 1a, b).
Antimicrobial screening:The MIC and MBC values obtained for the crude extracts and essential oil varied from one organism to the other, for instance the MIC values obtained for aqueous extract of G.latifolium against Staphylococcus sp. ranged from 6.25 to 12.5 mg L-1 and 25.0 mg mL-1 for Klebsiella pneumonia while the MBC ranged from 6.25 (for E. coli) to 25.0 mg mL-1 for (Staphylococcus cohnii cohnii) (Table 2). Similarly, the MIC values obtained for ethanolic extract ranged between 3.125 (for K. pneumonia) and 12.5 mg mL-1 (for S. scuri) while the MBC ranged between 3.125 and 25.0 mg mL-1 for different organism.
For the essential oil, the MIC ranged between 5 and 10 μg mL-1 while it was bacteriocidal at 10 μg mL-1 for some Staphylococcus sp. and 40 μg mL-1 for K. pneumonieae.
However, all the extracts showed activity against standard strain Escherichia coli NCTC 10148 and Staphylococcus aureus ATCC 25923 and MIC and MBC ranged between 6.25 mg mL-1 to 12.5 mg mL-1 for aqueous and ethanolic extracts, while MIC and MBC for the essential oil was at 10 μL, respectively.
Table 3 showed the essential oil components, retention time (Rt) and percentage composition of each compound in Gongronema latifolium leaves as analyzed by GC-MS.
Up to 56 peaks were detected in the Chromatogram of the oil and this represented 98.44% of the oil components. The leaf oil was characterized by the abundance of fatty acids. Total phthalic acids present (18.61%), fumaric acids (2.22%), oleic acids (5.2%), arachidic acids (2.34%).
The linear aliphatic compounds constitute (27.06%) tricontane (6.51%), dodecane (1.36%). Alphatic alcohols were present in fairly amount; methanol 0.84%, Pentadecanol 0.44%, Hexaconsanol 1.66%, policosanol 1.04%. Monoterpenes were also present in minute quantities; p-cymene 0.41%), camphor (1.20%) and Phytol (1.66%).
||Compounds identified from essential oil of Gongronema latifolium
GC-MS analysis of Gongronema lafifolium obtained in Lagos for this
study revealed high content of aliphatic linear compounds, unsaturated fatty
acids and monoterpenes. Ogunwande et al. (2005)
had also reported the presence of, camphor, phytol and phenol compounds and
this is in line with our present findings.
Phytochemical screening of Gongronema latifolium plant extracts revealed
the presence of Saponin, Alkaloids, Glycosides, Flavonoids. This is in line
with the findings of Morebise and Fafunso, (1998). These
compounds of Gongronema latifolium leaves extracts have been reported
to have hypoglycemic, hypolipidemic ,antioxidiative and anti-inflammatory properties
(Ugochukwu et al., 2003; Morebise
et al., 2002).
The aqueous, ethanolic and essential oil extracts of Gongronema latifolium
showed varying degrees of activities against organisms causing blood stream
infections in HIV patients. Previous studies by Eleyinmi,
(2007) has revealed antimicrobial activity of methanol extracts of Gongronema
latifolium against Salmonella enteritidis, Salmonella cholerasius ser
typhimurium and Pseudomonas aeruginosa.
Nwinyi et al. (2008) had also reported the inhibitory
activities of aqueous and ethanolic extracts of Gongronema latifolium against
Staphylococcus aureus and Escherichia coli, with MIC ranging between
2.5 and 10 mg mL-1 for ethanolic and aqueous extracts, respectively.
This is similar to our findings, as ethanolic extract of Gongronema latifolium inhibited all the organisms tested with MIC and MBC ranging from 3.125 mg-12.5 mg mL-1 and 3.125 mg-25.0 mg mL-1, respectively. However, aqueous extract was less active and this may be due to the fact that the active ingredients are more soluble in ethanol than in water.
The essential oil evaluated in these studies was found to have shown the highest
antimicrobial activity against tested organisms including Candida albicans
as larger zones of inhibitions for MIC and MBC ranging between 5-40 and 5-40
μg mL-1, respectively were recorded when compared to aqueous
and ethanolic extracts. This findings agrees with Eleyinmi
(2007) who reported the antimicrobial effect of Gongronema lafifolium
fatty acids against Staphylococcus aureus, Salmonella sp and
Escherichia coli. However, it is at variance with the study conducted by
Ogunwande et al. (2005) who reported no inhibitory
activity of the volatile oil of Gongronema latifolium against Staphylococcus
aureus and Escherichia coli. However, previous studies conducted
by Agoramoorthy et al. (2007) had confirmed that
fatty acids extracts similar to the ones recorded in the present study, obtained
from Excoecaria allocha plant, possessed antibacterial and antifungal
activities, with MIC range similar to ours.
In the present study, the Gram-positive bacteria (Staphylococcus sp.)
were more susceptible than the Gram-negative bacteria (Klebsiella pneumonia,
Salmonella typhi, Salmonella typhimurium). This is because Gram-negative
bacteria are known to be more resistant to inactivation by medium and long chain
fatty acids than Gram-positive bacteria because of their impermeability to hydrophobic
compounds (Kabara, 1981).
Based on the results of this research, ethanolic and essential oil extracts of Gongronema latifolium may be useful in the practice of ethnomedicine and in the treatment of bacterial blood stream infection in HIV patients.