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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 11, Num. 2, 2008, pp. 215-219
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TOXICOSIS OF NON-STEROIDAL ANTI-INFLAMMATORY AGENTS IN RATS
African Journal of Biomedical Research, Vol. 11, No. 2, May, 2008, pp. 215-219
Full Length Research Article
Antimicrobial
Effect of Phyllanthus amarus and Parquetina nigrescens on Salmonella
typhi
Flora Oluwafemi* and
Folasade Debiri.
Department of
Microbiology, University of Agriculture,
P.M.B. 2240, Abeokuta, Ogun State, Nigeria.
*Corresponding
Author: foluwafemi2000@yahoo.co.uk
Received: July
2007
Accepted
(Revised): January
2008
Published: May
2008
Code Number: md08029
ABSTRACT
Phyllanthus
amarus and Paraquetina nigrescens
are economic plants grown in West Africa for antimicrobial properties. Crude
aqueous (hot and cold water) and ethanolic extracts of the plants were investigated
for antimicrobial activity against Salmonella typhi. The organism
was collected from the University College Hospital, lbadan, Nigeria and was
exposed to ten standard different antibiotics and also to crude extract of P.
amarus and P. nigrescens. Agar cup diffusion method was employed fro
the plants extracts while disk diffusion method for the standard antibiotics.
Ethanolic extracts of P. amarus had the strongest activity against Salmonella
typhi with 8.0mm zone of growth inbibition followed by hot water (4.7mm)
and cold water (3.8mm). This was statistically significant at P= 0.05 when
compared with hot and cold water extracts. Amongst the commercial antibioticis
examined, ciprofloxacin had the highest zone of growth inhibition of 9.0mm;
Oflxation (6.0mm) Amoxycillin, (4.0mm) while other antibiotics had on effect on
test organism. Screening carried out on P. amarus and P. nigrescens using
standard methods revealed the presence of saponins, alkaloids, tannins and
cardiac glycosides. P. amarus possesses significant antimicrobial activity and
confirms the justification by herbalists as extract used for treatment of
typhoid fever.
Key Words: P. amarus, P. nigrescens, antimicrobial activity,
standard antibiotics, typhoid fever
INTRODUCTION
There
has been an increasing interest world wide on therapeutic values of natural
products. It is believed that the cure to any debilitating human ailments and
diseases may be found among the worlds flora in natures pharmacy (Olowosulu
and Ibrahim, 2006). In addition, nature has presented to humanity the gift of
vast therapeutic workshop with a wide variety of medicinal plants. There are
multitudes of potential useful bioactive substances to be derived from these
plants. These phytochemicals have made significant contribution in maintaining
human health. There are multitudes of potential useful bioactive substances to
derive from these plants. Theses phytochemical have made significant
contribution in maintaining human health. The significant of drugs derived from
plants cannot be over emphasized with the recent trend of high percentage of
resistance of microorganisms to the present day antibiotics (Ibekwe et al, 2000).
Effort has been intensified by researcher towards a search for more source of
antimicrobial agents. Phyllanthus amarus is a herb common to central and
southern lndia and can grow to 30-60cm in height. Al parts of the plant are
employed therapeutically. Phyllanthus species can also be found in other
countries including China (e.g. P. uminaria), The Phillipines, Cuba, Nigeria
and Guam (Bharatiya, 1992). It blocks DNA polymerase in the case of hepatitis B
virus during reproduction. In one study, 50% of those infected with chronic
viral hepatitis B lost one of the major blood markers of hepatic B virus
infection (e.g. hepatitis B surface antigen) after using Phyllanthus for 30
days.
Parquetina
nigrescens is commonly found in secondary forest and around villages in Senegal
and Nigeria. It is a perennial plant with twining stems and a woody base,
shortly tapering 10-15cm long, 6-8cm broad, smooth, long stem. The leaves and
whole plant are usually used for the treatment of gonorrhoea, jaundiced,
rickets and asthma (Schlage et al, 1992). It is mostly used by traditional
healers (Leaman et al, 1995). This paper, therefore, reports investigation into
the antibacterial activities of extracts of P. amarus and P. nigrescens against
clinical isolate of S. typhi and the results of preliminary phytochemical
screening of the aforementioned plants.
MATERIALS
AND METHOD
Plant
Collection and Identification: The
two plant used in this study, Phyllanthus amarus and Parguetina nigrescens were
both collected from the Department of Forestry, University of Agricultural,
Abeokuta. The plants were identified by carrying out macroscopical examination
on plant samples as stipulated by Dalziel (1968) and confirmed at Department of
Forestry, University of Agriculture, Abeokuta, Nigeria. The leaves of both
plants were chopped into small pieces and thereafter, pulverized in a domestic
mill. The powdered mass was later used for extraction.
Preparation
of Plant Extracts: Twenty grammes
(20g) of the pulverized leaves of each plant was decocted with 100ml of cold
water left overnight, hot water (100oC) for 5 minutes), and ethanol.
Prior to decoction, the leaves were soaked in the extracting solvent for 3
days. The mixture was then filtered and the filtrate evaporated to semi solid
mass using a rotary evaporator (Brichi, Germany) (Olowosolu and Ibrahim, 2006)
and subsequently drying in a beaker on water bath to give a dark resinous mass.
The plant extracts from the various solvents were reconstituted using 10% v/v
ethanol as solubilising agent at concentration of (10 and 100)mg/ml for
antimicrobial activity evaluation.
Test
organism: Test organism used for this
study was salmonella typhi. The stock culture maintained on nutrient
agar was obtained from the Department of Medical Microbiology, University
College Hospital, Ibadan, Nigeria.
Preparation
of Inoculum: A loopful of S.typhi
was taken and sub-cultured in test tube containing 10ml of nutrient broth. The
test-tube was incubated at 37oC for 24 hours. The broth was
standardized using sterile normal saline to obtain a population of 104cful/ml.
Antimicrobial Studies. Two approaches were used for the evaluation of
antimicrobial activity of both plant extract and commercial antibiotics.
(a)
Agar Cup Diffusion Method: Agar cup
diffusion method described by Hugo and Russel (1996) was employed.
An
overnight culture of S.typhi was standardized to contain approx. 107cfu/ml
was inoculated into 20ml of molten nutrient agar. The culture medium was
allowed to set. Thereafter, a sterile cork borer N. 4 (8.0mm diameter) was used
to punch wells in the seeded nutrient agar. The agar plugs were removed with a
flamed and cooled wire loop. Into the separate well was poured different
concentrations of the various plants extracts. The plates were incubated at 37oC
for 24 hours and the zone of inhibition was measured. The experiments were
repeated in triplicates.
(b)
Disk Diffusion Method: Disk diffusion
method was employed to determine the effect of standard antibiotics against the
test microorganism. Standard antibiotic disc with ten different antibiotics
were used against S. typhi. The nutrient agar plates were seeded with S.
typhi and filter paper strips of standard antibiotics (Nitrofurantoin,
Ciprofloxacin, Gentamycin, Ampicillin, Cefuroxin, Chloramphenicol, Ofloxacin,
Amoxyllin, Norfloaxacin, Tetracycline) were laid aseptically on the plate using
a pair of forceps. The plates were incubated at 37oC for 24 hours.
After incubation, zone of growth inhibition was measured and recorded.
RESULTS
Table
1 shows the zone of inhibition of P. amarus against S. typhi. At
24 hours contact period, ethanolic extract of P. amarus exerted greater
inhibitory activity against S. typhi with a zone of 8.3mm in diameter
The
zone of inhibition of ethanolic extract of P. amarus on S. typhi decreased from
8.3 mm to 7.7mm as the contact time increases from 24 72 hour. The zone of
inhibition using cold and hot water extract of the same plants were much lower,
however, the same decreasing pattern repeated itself as the contact time
incersased.
Table
2 shows the zones of inhibition of Parquetina nigrescens on S. typhi. The
values were much lower than those obtained with P. amarus. These value range
from 3.3mm of cold water extract to 3.7mm ethanolic extracts.
As
contact time with organism increased, the zones of inhibition decreased.
Commercial antibiotics were compared with plant extract of P. amarus and their
antimicrobial activity against S. typhi was recorded. The highest microbial
activity was exhibited by Ciprofloxacins.
Table
1: Diameters of Zones of Growth
Inhibition (MM) of Phyllanthus on S. typhi. Inhibition zone (diameters
in mm) + _ SD
Contact time (Hours) |
Cold water Extract (mg/ml) |
Hot water Extract (mg/ml) |
Ethanol extract (mg/ml) |
24
48
72 |
4.0
± 0.10
3.7
± 0.08
3.2
± 0.08 |
5.2
± 0.14
4.5
± 0.09
4.3
± 0.08 |
8.3
± 0.12
8.0
± 0.10
0.77
± 0.06 |
Table
2: Diameters of Zones of Growths
Inhibition of Parquetina nigrescens on S. typhi
Contact time (Hours) |
Cold water Extract (mg/ml) |
Hot water Extract (mg/ml) |
Ethanol extract (mg/ml) |
24
48
72 |
3.3
± 0.06
3.3
± 0.06
3.3
± 0.06 |
3.2
± 0.03
3.0
± 0.05
2.7
± 0.03 |
3.7
± 0.03
3.5
± 0.00
3.5
± 0.00 |
Table
3: Zones of inhibition of standard
antimicrobial agents S. typhi.
Standard
agents |
Zone
of inhibition (mm) + SD |
Nitrofurantion
Ciprofloxacin
Gentaniycin
Ampicillin
Cefuroxin
Chloraphenicol
Ofloxacin
Amoxycillin
Norfloxacin
Tetracycline |
0.0 ± 0.00
9.0 ± 0.01
Nil
Nil
Nil
0.0 ± 0.01
6.0 ± 0.00
4.0
Nil
Nil |
Table
4: Mean separation of both standard
and crude antimicrobials using Dwuncan multiple range test
Antimicrobial
Plant |
Zone
of inhibition (mm) |
Duncan
multiple range |
Nitrofurantion
Ciprofloxacin
Gentaniycin
Ampicillin
Cefuroxin
Chloraphenicol
Ofloxacin
Amoxicillin
Norfloxacin
Tetracycline
Phyllanthus
amarus (Ethanol)
Parquetina
nigrescens (ethanol) |
0.0
9.0
0.0
0.0
0.0
0.0
6.0
4.0
0.0
0.0
8.0
3.6 |
f
a
f
f
f
f
c
d
f
f
b
e |
Means
within the column with the same alphabet are not significantly different
Ciprofloxacin
had the highest zone of inhibition of 9.0mm against S. typhi followed by
Ofloxacin (6.0mm), Amoxylcillin (4.0mm) while the rest antibiotics showed no
effect on S. typhi.
Table
4 shows the means zones of inhibition of commercial antibiotics (standard) and
local crude extracts of P. amarus and P. nigrescens. This results revealed that
P. amarus followed closely Ciproxacin in antimicrobial activity than all other
commercial antibiotics tested.
Phytochemical
analysis of P. amarus and P. nigrescens were carried out. Preliminary screening
revealed the presence of tannins, saponins, cardiac glycosides and alkaloids.
DISCUSSION
Several
investigations had reported that plants contain antimicrobial substances
(El-Said et al, 1971, Lewis, 1980; Zaria et al 1975, Ibekwe et al,
2001, Akujobi et al, 2004). The results of the present study agree
essentially with the reports of these previous workers. The result shows that
cold and hot water extracts of Phyllanthus amarus were not as effective in
antimicrobial activity against S. typhi as ethanolic extracts. This is not
surprisingsince ethanol is generally able to dissolve multivariable types of
compounds; polar and non-polar, simple and complex chemical structures compared
with chloroform which solubilizes mainly flavenols (Phenolic compounds from
plant) (Cowan, 1999). The relative amount of phytochemical substances from
plant extraction depends on the solubility of the phytochemical in the solvent
used for extraction (Olowosulu and Ibrahim 2006).
Although
ten commercial antibiotics (standard) were tested against S. typhi, only three
(Ciplofloxacin Ofloxacylin and Amoxycillin) had antimicrobial effect in
decreasing order. Ciprofloxacin had the highest zone of growth inhibition
against S. typhi. And remarkably has become the antibiotics of choice in the
treatment of typhoid fever. Unfortunately, resistance of salmonella typhi
strains to all of these antibiotics is becoming more common globally. As such,
appropriate treatment varies with geographic distribution of resistant strains.
The
antimicrobial properties exhibited by the extracts may be associated with the
presence of tannins, sapoins, cardiac glycosides and alkaloids found in the
plant extracts. a large number of flavonoids have been reported to possess
antimicrobial properties (Bastista et al., 1994; Tsuchiya et al
1996; Boris, 1996; Olowusulu and Ibrahim, 2006; Akimnjobi et al 2006).
Tsuchiya et al (1996) attributed the antimicrobial activities of
flavonoids to their ability to complex with extracellular and soluble proteins
as well as their ability to complex with bacterial cell walls. They suggested
that more lipophylic flavonopidsexert antimicrobial activity by disruptiog
microbial cells membranes.
The
results of this study show that Phyllanthus amarus appreciable antimicrobial
properties thus justifying its use as antimicrobial agent in Nigerian
ethnomedicine. Parquetina thus justifying its use as antimicrobial agent in
Nigerian ethnomedicine. Parquetina nigrens on the other hand was not as
effective as P. amarus Howeve, caution must be exercised until pharmacists have
adequately explored this extract in pharmaceutical preparation of antimicrobial
agents. This is necessary in order to eliminate the non-useful aspect of the
extract.
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