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Antibacterial potency and differential synergistic effects of Zingiber officinale and Capsicum annum extracts against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa

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Antibacterial potency and differential synergistic effects of Zingiber officinale and Capsicum annum extracts against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa

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ABSTRCT There is need to look for nontoxic cheap alternative ways of fighting mutating and resistant disease causing microbes especially in sub-Saharan region. This is because of the lower average immune system strength due to bad feeding habits, high population and increase of disease causing organisms which are exacerbating the HIV and AIDS menace. The use of species to preserve, give flavour to food and to fight illnesses has been practiced in Africa since the invasion of Asian populations in the coastal regions. In this study, we investigated the potential of using common spices like Ginger (Zingiber officinale) and Chilli pepper (Capsicum annuum) against Escherichia coli, Pseudomonas aeruginosa and staphylococcus aureus which are common bacteria mostly found in contaminated food. This was done using the Kirby Bauer diffusion method. Ginger and chili pepper were found to inhibit the growth of the tested bacteria especially staphylococcus aureus through comparing the diameter of inhibition zones. The results indicated that extracts of ginger and chilli pepper had antibacterial activity in the range of 15-33mm. E. coli and Staphylococcus aureus were more affected by the extracts than Pseudomonas aeruginosa. However, a mixture of the two extracts produced a greater antibacterial activity than the individual extracts with p values nearing 0.05. Therefore, the extracts displayed an antimicrobial activity with greater efficacy when acting synergistically on the test organisms. Therefore, the potential of using the combination as a naturopathy is still high despite the perceived microbial resistance after a long time of usage in human populations.
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American Journal of Research Communication www.usa-journals.com
Masila, et al., 2017: Vol 5(2) 66
Antibacterial potency and differential synergistic effects of Zingiber officinale
and Capsicum annum extracts against E. coli, Staphylococcus aureus and
Pseudomonas aeruginosa
Ednah Mutindi Masila2 and Josphert N. Kimatu1*
2Department of Biochemistry and Molecular Biology, South Eastern Kenya University, P.O. Box
170-90200, Kitui, Kenya
1Directorate of Research, Innovation and Technology, Department of Biology, South Eastern
Kenya University, P.O. Box 170- Kitui, Kenya
*Corresponding author: Email: jkimatu@selu.ac.ke
ABSTRCT
There is need to look for nontoxic cheap alternative ways of fighting mutating and resistant
disease causing microbes especially in sub-Saharan region. This is because of the lower
average immune system strength due to bad feeding habits, high population and increase of
disease causing organisms which are exacerbating the HIV and AIDS menace. The use of
species to preserve, give flavour to food and to fight illnesses has been practiced in Africa
since the invasion of Asian populations in the coastal regions. In this study, we investigated
the potential of using common spices like Ginger (Zingiber officinale) and Chilli pepper
(Capsicum annuum) against Escherichia coli, Pseudomonas aeruginosa and staphylococcus
aureus which are common bacteria mostly found in contaminated food. This was done using the
Kirby Bauer diffusion method. Ginger and chili pepper were found to inhibit the growth of the
tested bacteria especially staphylococcus aureus through comparing the diameter of inhibition
zones. The results indicated that extracts of ginger and chilli pepper had antibacterial activity in the
range of 15-33mm. E. coli and Staphylococcus aureus were more affected by the extracts than
Pseudomonas aeruginosa. However, a mixture of the two extracts produced a greater antibacterial
activity than the individual extracts with p values nearing 0.05. Therefore, the extracts displayed an
antimicrobial activity with greater efficacy when acting synergistically on the test organisms.
Therefore, the potential of using the combination as a naturopathy is still high despite the perceived
microbial resistance after a long time of usage in human populations.
Keywords: Ginger; Chili Pepper; Resistance; Immune systems; Inhibition zone; Sub-Saharan
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Masila, et al., 2017: Vol 5(2) 67
{Citation: Ednah Mutindi Masila, Josphert N. Kimatu. Antibacterial potency and differential
synergistic effects of Zingiber officinale and Capsicum annum extracts against E. coli,
Staphylococcus aureus and Pseudomonas aeruginosa. American Journal of Research
Communication, 2017, 5(2): 66-76} www.usa-journals.cmom, ISSN: 2325-4076.
INTRODUCTION
The use of medicinal plant to treat ailment associated with pains is well known through history.
Such plants can play important role in drug discovery and such practice has been regarded as
logical research strategy for searching new drugs (Auta et al., 2011). The acquaintance with
different ethnic groups has contributed to the developed of research on natural products. This has
been used to decipher many phenomenon like the close relationship between the chemical structure
of a compound and its biological property in many animal-plant interrelations. Spices and herbs
have been used for thousand of centuries by many cultures to enhance the favour and aroma of
food. Microbial inhibitory activities of spices and their derivatives have also been identified.
(Shelef et al, 1983). A number of spices have been screened for antimicrobial activity (Premanth et
al, 2011) and efforts have been done to identify their active constituents. Studies have shown that
both gram negative and positive food-borne and water–borne bacteria are inhibited by garlic,
onion, cinnamon, cloves, thyme and sage (Auta et al, 2011).
Ginger is a member of the family Zingiberaceous, a small family with more than 45 genera and
spices. Ginger is the underground rhizome of the ginger plant with a firm striated texture (Hoffman
et al, 2012). On the other hand, chili pepper is a member of Solanaceae family and the capsicum
species. Research has shown that it has activity against food borne pathogenic bacteria (Dasgupta
et al, 2011). Ginger and chilli pepper are reputed to be effective against E. coli, Staphylococcus
aureus, Bacillus subtilis and Pseudomonas aeruginosa which cause significant illness in humans
(Hara et al, 2006). Zingiber officinale has antimicrobial activity and thus can be used in treatment
of bacterial infections (Tan and Vanitha et al,. 2004). This this because of plants have almost
limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen
substituted derivatives most of which are secondary metabolites (Geissman et al,.1963). The
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Masila, et al., 2017: Vol 5(2) 68
capsaicinoids of chili pepper have demonstrated a degree of biological activity affecting the
cardiovascular and digestive system (Virus and Gebhart et al, 1979). Antimicrobial activity of chili
pepper has been shown.
The use of spices and other plants as antimicrobial agents developed due to emergence of multiple
antibiotic resistances among such pathogen as Staphylococcus aureus, E. coli and Pseudomonas
aeruginosa (Wald et al, 2000). This is because of intrinsic virulence, its ability to cause a diverse
array of life threatening infection and the capacity to adapt to different environmental infections
(Lowry et al, 1998). Pseudomonas aeruginosa is not easily controlled by antibiotics because it is
gram negative and has fairly impermeable outer and can also turn on pumps that remove antibiotics
from the cell. Microbes attain resistance because of adaptability feature conferred by plasmids
which carry integrin and gene cassettes (Kumarasamy et al, 2010).
Research by BMJ in 2015 showed that people who eat spicy foods nearly every day have 14%
chance of living longer than those who consume spicy foods less than once a week .Regular spicy
food eaters also are less likely to die from cancer, heart and respiratory diseases than those who eat
spicy foods infrequently. Bioactive ingredients in spices have been found to have anti- obesity,
antioxidant, anti-inflammation and anti-cancer properties. This research was set out to investigate
the differences in microbial potency of the ginger and capsicum spices when used individually and
when used in combinations, the research also examined the magnitude of the effect of the two
spices on Staphylococcus aureus and E. coli in relation to Pseudomonas aeruginosa.
Methodology
The study was carried out under controlled environmental conditions at both the South-Eastern
Kenya University laboratory and the institute of Biotechnology Research laboratory at Jomo
Kenyatta University of Agriculture and Technology in Kenya. The plant material of Zingiber
officinale rhizomes and Capsicum annuum fruits were obtained at a garden in Institute of
Biotechnology Research in Nairobi.
Extraction of plant material
The ginger rhizomes were washed with distilled water and allowed to air dry for 2 days.
Extraction was done using the crude extraction method using methanol as described by Fatope et
al,.1993. About 40g of ginger was blended into powder and soaked into 100 ml of methanol
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for 24 hours with intermediate shaking. The extract was stored in refrigerator until use.
Chilli pepper fruits extracts were prepared by macerating pepper powder in methanol for 24
hours then filtered. The residue was washed twice with fresh methanol and the filtrates
combined with the first filtrate. Combined filtrate was evaporated to dryness to produce sticky
materials from the chili pepper extract
Culturing the test micro-organisms and their inoculation
Pure isolates of E. coli, Pseudomonas aeruginosa and staphylococcus aureus were provided by the
Institute of Biotechnology Research (IBR) then maintained in nutrient agar slants and incubated
to match turbidity of 0.5 according to McFarland standards. Mueller Hinton agar was prepared
according to the manufacturer’s instruction then dispensed into petri dishes. The agar plates
containing the Mueller Hinton Agar medium was inoculated with 0.01ml of the inoculum using an
inoculating loop and spread using a spreader. Assay discs around 6mm diameter were soaked on
the extracts then transferred on the media surface to determine the antibacterial activity according
to Bauer AW et al, 1966. All plates were incubated at 370C for 15 hours and the zones of inhibition
measured using a ruler after 15 hours and 30 hours. The experiment was carried out in triplicates
and for the control the disks, pure methanol was used since the extraction was done using
methanol.
Statistical analysis
The results were expressed as means of diameter of zones of inhibition in millimeters at 15 hours
and 30 hours and calculation done using Microsoft excel and R software where Welch Two
Sample t-test was done to determine the significant time. Graphs were included to illustrate and
compare the inhibitory and synergetic effects of the spice extracts.
RESULTS
Comparing the inhibition zones
The methanolic extracts of ginger, chili pepper and combined ginger and chili pepper plants
material showed antimicrobial activity on the tested organisms. The highest susceptibility was
recorded with the combined ginger + chili pepper extract followed by extracts of chili pepper then
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ginger extracts. (Figure 1) The antibacterial efficacy of the mixture was highest compared to the
individual extracts. The control didn’t have zones of inhibition hence did not show antibacterial
activity. The p values for the comparisons was > 0.05 hence not significant with the ginger + chili
pepper mixture which had a p value close to 0.05.
Figure 1. A clustered column of the inhibition zones(mm) of various extracts against the
three microbes. The Line in the chart show the level of inhibition exhibited by the P.
aeruginosa. The combination of the two extracts show antimicrobial synergy.
Differential Comparative Antibacterial Potency
An analysis of the inhibition zones showed that the mixture of the ginger and pepper had the
highest zone of inhibition. The analysis of the three microorganisms showed that the
Staphylococcus aureus was the most inhibited by the three combinations of the extract. The
Pseudomonas aeruginosa was the least inhibited by the extracts. The ginger extract showed the
least effect of antibacterial effect on the P. aeruginosa but it was surprising that its effect on E.
0
5
10
15
20
25
30
35
40
45
Pepper (15
hours)
Pepper (30
hours)
Ginger + Pepper
extract (15
hours)
Ginger + Pepper
extract (30
hours)
Ginger (15 hours) Ginger (30 hours)
Inhibition Zone (mm)
Different Extracts
Staphylococcus aureus E.coli Pseudomonas aeruginosa
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coli was reduced in the combined extract with pepper (Figure 2).
Figure 2. A clustered column chart showing different synergetic effect of spice extracts as
differential antibacterial agents. The E. coli had a negative effect on the combined ginger
and pepper extracts.
DISCUSSIONS
The study shows that the spices studied possess significant antimicrobial properties, with a greater
antimicrobial efficacy when used synergistically. This might be due to resultant effect of the active
agents in the spices. The Capsicum annum extract tested showed antibacterial properties on both
gram positive and gram negative bacteria used in the investigation. This is in conformity with
previous studies carried out by Soetarno et al,.1997. This means tropical capsicum annum is a
potential antibacterial agent. Furthermore, these antimicrobial activities shown by ginger and chilli
pepper rhymes with later findings of others (Hara et al, 1998). The antibacterial potential of ginger
and chili pepper is perhaps due to compounds like flavonoids, gingerols, volatile oils, terpenoid and
volatile oils which are secondary metabolites produced by the plants in response to stress and have
protective properties were dissolved in organic compounds according to Jones, et al, 1997 and
Avatope et al., 2000.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Pepper Ginger + Pepper Ginger
Inhibition Zone gap
Spice Extract
Staphylococcus aureus E.coli Pseudomonas aeruginosa
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Pepper extracts
The sharp taste of capsicum peppers is due to the mixture of seven related alkaloids of which
capsaicin is the most prevalent. Capsaicinoids are mainly found in the seed and placental area of
the chilli pepper (Dong et al,. 2000). The substances’ responsible for the pungency are the
capsaicinoids alkaloids. They are characterized by high biological activity and their
pharmacological activities. The use of the whole fruit in these studies could have contributed more
to the antibacterial potency demonstrated both when used independently and when used in
combination with ginger.
Ginger extracts
Comparatively, the ginger extracts were less active against Pseudomonas aeruginosa which is
naturally resistant to antibacterial agents (Walker and Edward et al, 1999). However, this results
are in accordance with the results obtained in this study since in the replicates most of zones of
inhibition remained constant after 15 hours while zones of inhibition for the other bacteria were
increasing. This is probably due to high calorific value per 100g of ginger compared to pepper
which seem to feed the microbes rather than kill them. (Figure 3). In similar study by Bonjar et al.,
2004, the methanol extracts of ginger rhizomes were active against all gram-positive bacteria and
gram-negative bacteria in accordance to the results obtained in this study. The active compounds
in chilli pepper and ginger, which have antibacterial activity dissolved in methanol during
extraction were actually demonstrated to not only inhibit growth of microbes but also kill the tested
bacteria species (Jones, et al, 1997). Greater antibacterial efficacy is seen when the extracts are
used synergistically due to probably non-neutralizing resultant effect of the active agents in the
plant material. Gram negative bacteria are more resistant to antibiotics than gram positive. The
resistance is due to their differences in their cell wall composition. In gram negative the outer
membrane acts as great barrier to many environmental substances including antibiotics (Tortora et
al, 2001).
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Figure 3. The differences in calorific values of the two spices in per 100 g. (Source, Periodic
Table of vegetables, Goodfellow. Cambridge Science Park, USA).
CONCLUSION
In conclusion, the results have provided the justification for therapeutic potential of spices and also
opened a new field of studying positive synergetic influences of spices. There might be some cases
of negative synergetic effects of some spices. The use of the combined mixtures of spices as
supplementary or alternative medicine can be an avenue to reduce the problem of drug resistance,
side effects of medicine and reduce cost of treatment. This is feasible even in developing countries
who are experiencing high degree of land fragmentation, high population, low immunity levels due
to the HIV and AIDS epidemic. This furthermore more welcome in the growing urbanization of
agricultural lands since ginger and chili pepper are natural spices which are readily available all
year round in markets. They are also comparatively cheap and fairy nonperishable, hence requiring
non-sophisticated post-harvest management procedures.
0
10
20
30
40
50
Amonut of Calories /100g
Spice
Ginger Pepper
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... Result of analysis showed that ginger had the zone of inhibition about 24 mm [62]. In a survey conducted on the antimicrobial activity of ginger extract, MIC value was determined 40 mg/ml for E. coli [63]. ...
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