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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 404-410
ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 404-410
http://www.ijcmas.com
Original Research Article
Effect of Ethanol and Aqueous Solutions as Extraction Solvents on Phytochemical
Screening and Antibacterial Activity of Fruit and Stem Bark Extracts of
Tetrapleura tetrapteraon Streptococcus salivarus and Streptococcus mutans
Gberikon, G.M*, Adeoti, I.I and Aondoackaa, A.D.
University of Agriculture Makurdi, Benue state
*Corresponding author
A B S T R A C T
Phytochemical screening and antibacterial activity of fruit and stem bark of
Keywords Tetrapleura tetraptera using ethanol and aqueous solvents as means of extraction
against S. mutans and S. salivarius was investigated. Samples of T. tetraptera fruits
Streptococcus were purchased in Wukari market, Taraba State. Stem bark samples were collected
mutans, from Vandeikya, Benue State of Nigeria. Phytochemical analyses were carried out on
Streptococcus both samples in the laboratory, Department of Biological Sciences, University of
salivarius, Agriculture Makurdi. Phytochemical screening showed the presence of flavonoids,
Tetrapleura saponins, tannins, steroids, alkaloids, phlobatannins, anthraquinones, glycosides and
tetraptera reducing sugars in varying concentrations in fruit and stem bark samples of the test
flavonoids, plants using ethanol and aqueous solutions. Antibacterial activity of ethanolic and
saponins, aqueous extracts of Tetrapleura tetraptera was also studied against Streptococcus
mutans and Streptococcus salivarius. Ethanol extracts showed strong antibacterial
tannins, steroids, activity, aqueous extracts did not show any antibacterial activity on the test organisms,
alkaloids, hence, no antibacterial activity with extracts using aqueous solution. Ethanol extract of
Phlobatannins, fruit gave an inhibition zone of 08.33mm against S. mutans and 16.33mm against S.
Anthraquinones, salivarius. Also ethanol extract of stem bark gave an inhibition zone of 12.00mm
Glycosides against S. mutans and no inhibition zone against S. salivarius. There was significant
reducing sugar difference (p 0.025) between antibacterial effect of ethanolic and aqueous extracts of
fruit and stem bark as shown in this study. Ethanolic extracts of fruit and stem bark of
Tetrapleura tetraptera was more potent against the test organisms than the aqueous
extracts.
Introduction
Plants have being the basis for medical known as herbal or botanical medicine refers
treatments through much of human history to the use of plant seeds, fruits, roots, leaves
(Nunn, 2002). Traditional medicine is and stem bark for treatment of aliments
valued in most part of the world and it is still (Robertson and Baek, 2009).
widely practiced today (Hong, 2004). Tetrapleura tetraptera, belongs to the family
Medicines from plant source otherwise Fabaceae, it is highly valued in Nigeria and
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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 404-410
beyond for its medicinal properties (Essien Sciences Department, University of
et al., 1994). The aqueous fruit extract has Agriculture, Makurdi for extraction process.
also been shown to possess hypoglycaemic
properties (Ojewole and Adewunmi, 2004). Preparation of plant for extraction using
Phytochemical screening revealed the aqueous and ethanol as solvents
presence of tannins, phenolic compounds,
saponins, alkaloids, steroids and flavonoids The extracts of the plant materials were
which could be assumed to be responsible obtained using the cold maceration method
for its varied biological and pharmacological described by Umeh et al. (2005). Fifty gram
properties. (50g) of powdered plant materials (fruit and
stembark) was weighed into clean sterile
Streptococcus mutans is commonly found in bottles. Each weighed-out plant parts was
the human oral cavity and a significant extracted using 250ml aqueous and ethanol
contributor to tooth decay (Ryan and Ray, separately in tightly covered bottles and left
2004). Streptococcus salivariusis a for 48hours at room temperature. The
prominent member of the oral microbiota resultant suspensions were filtered into
(Ryan and Ray, 2004). Therefore subjecting sterile beakers, and filtrates collected was
these organisms to antibacterial activity of re-filtered using Whatman No. 1 filter paper
Tetrapleura tetraptera of aqueous and into sterile sample bottles. They were
ethanolic extract of fruit and stem bark will labelled appropriately and stored in plastic
be a head way for caring about the hygienic bags at -20 °C for further analyses.
situation of the oral cavity.
Phytochemical screening
Materials and Methods
Plants material extracted using ethanol and
Collection of plant materials aqueous solutions were subjected to
phytochemical screening according to the
The stem bark of the plant (Tetrapleura method described by Odebiyi and Sofowora
tetraptera) for this study was collected in (1978); Okerulu and Ani (2001) to ascertain
Vandeikya local government area of Benue the presence or absence of some specific
State. Fruit samples were bought from active metabolites such as tannins, saponins,
railway market at Makurdi and from new flavonoids, reducing sugars, alkaloids,
market, Wukari local government area in steroids, anthraquinones, glycosides and
Taraba state. The plant parts were package phlobatannins
in sterile polythene bags and transported to
the laboratory, Department of Biological Identification and confirmatory test on
sciences, University of Agriculture, Makurdi test organisms
for identification and analyses.
Test organisms used for this study were
Preparation of plant material Streptococcus mutans and Streptococcus
salivarius the stock isolates of these
The collected plant parts were shade dried at organisms were obtained from Tosema
27 °C for a period of one week and crushed specialist diagnostic laboratory, Makurdi. A
into small pieces using a clean mortar and well isolated colony of the bacteria was
pestle, crushing was done separately. They picked using sterile inoculating wire-loop
were later taken to the laboratory, Biological and transferred into nutrient agar and blood
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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 404-410
agar slant, and incubated at 37oC for (quantitative) tests to determine their
24hours before susceptibility test. The agar minimum inhibitory concentrations (MICs).
o
slants were stored at 4 C.Identification and
confirmatory test were carried out on the Determination of minimum inhibitory
organisms using appropriate biochemical concentration (MIC)
tests like catalase, coagulase
Pyrrolidonylarylamidase (PYR) test, Extracts that showed potent antibacterial
oxidase, bile solubility test, Optochin activity was further tested to determine the
susceptibility test and Glucose fermenting minimum inhibitory concentration (MIC) for
test. the bacterial samples. The MICs of these
extracts was determined by broth micro
Determination of antibacterial activity dilution method.
The disc diffusion method was used (Salie et Dilution of extracts
al., 1996; Nostro et al., 2000). Stock
solutions used contain 200mg/ml of each The stock solution was serially diluted with
extract for both fruit and stembark. Blood the extraction solvent (ethanol and aqueous),
agar plates were inoculated with the in sterile test tubes labelled and arranged
organisms, within 15min of inoculation of from the highest to lowest concentration of
the plates, the drug/extract-impregnated disc extract desired (400mg, 200mg,150mg,
was placed on the agar surface, with at least 100mg,50mg and 25mg). Using a sterile
24mm (centre to centre) (Jorgensen and pipette (or 2ml needle and syringe), 1 ml of
Turnidge, 2003). The disc was placed with a solvent was added to each of the 6 tubes,
sterile forceps and then gently pressed down except the first and second tubes. 2ml of
onto the agar surface to provide uniform extract was added to the first tube (400mg),
contact. The plates were allowed to stand for 1ml of the extract (200mg/ml) was added to
few minutes to enable the extracts diffuse the second and third tubes, and the contents
into the agar. Standard ofloxacin antibiotic of the third tube agitated on a Vortex mixer.
discs (10microgram/disc) were used as 1 ml of the solution in the third tube was
control and were similarly applied on plates transferred to the fourth tube, and the
seeded with the organism. Sterile disc process continued through the next to the
loaded with 0.1ml of sterile distilled water last tube from which 1 ml was removed and
was used as negative control. Within discarded. 0.25ml of extract was later added
15minutes of applying the disc, the plates to the third tube to make the concentration
were inverted and incubated at 37ºC for 150mg. No extract was added to the 7th tube
24hrs (Salie et al., 1996). All tests were which served as a negative growth control,
performed in triplicate and the antibacterial 10microgram of ofloxacin was used as
activity was expressed as the mean diameter positive control (that prevented bacterial
of inhibition zones (mm) produced by the growth). An equal volume of a fixed
plant extracts. The diameters of the zones of bacterial culture was added to the tubes and
inhibition produced around the disc were incubated at 37 °C for 24 hrs. After which
measured with a transparent ruler to the tubes were examined for turbidity. The
nearest millimetre (Salie et al., 1996). The lowest concentration that shows no visible
measurements taken were recorded. Extracts growth (turbidity) was noted and recorded as
of the fruit, and stembark that inhibited the MIC values (Salie et al., 1996).
bacterial growth were subjected to further
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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 404-410
Statistical analysis and as such they are of tremendous
importance and interest in pharmaceutical
Statistical package for the social sciences research. Aqueous extracts of T. tetraptera
(SPSS) version 20 was used to analyse the did not show any antibacterial activity
data obtained. against the tested organisms. This is because
water is not a good solvent for extraction.
Results and Discussion This contrast the work of Uchechi and
Chigozie (2010), where aqueous extract of
Phytochemical screening using ethanol on this same Tetrapleura tetraptera exhibited
stem bark of Tetrapleura tetraptera showed activity against some bacteria namely
appreciable amounts of phytochemicals than Escherichia coli, Salmonella typhi and
its aqueous counterpart. These result Pseudomonas aeruginosa.
confirmed the evidence in previous studies
that alcoholic solvents like ethanol and Tetrapleura tetraptera possess antibacterial
methanol are more suitable than other activity as shown in this study. The ethanol
solvents such as water in extracting extract of the fruit showed the highest
components of medicinal plants (Ahmad et inhibitory effect on the test organisms while
al., 1998; Cowan, 1999;Emadet al., 2009). aqueous extract did not inhibit any of the
tested organisms. This is because ethanol
The ethanolic extract of the fruit of extracted more phytochemicals that will
Tetrapleura tetraptera exhibited activity inhibit growth of bacteria as opposed to
against S. mutans and S. salivarius showing aqueous solution.
maximum zone of inhibition of 08.33 and
16.33 respectively. Ethanolic extract of stem Sensitivity pattern of test organisms to the
bark of Tetrapleura tetraptera exhibited fruit extracts (aqueous and ethanol) of
activity against S. mutansonly showing Tetrapleura tetraptera was higher than the
maximum zone of inhibition of 12.00. There stem bark extract. Results showed that
was no activity on S. salivarius.These also ethanol extract of fruit of Tetrapleura
showed that the antibacterial activity and tetraptera produced clear zones of inhibition
susceptibility test obtained in this study on the tested organisms (Streptococcus
varied according to the extraction solvent mutans and Streptococcus salivarius).The
and parts of the plant used. The variation mean zones of inhibition were 8.33mm and
may probably be due to the type of bioactive 16.33mm respectively.
compounds present in the different
extraction solvents as suggested by Abiodun The aqueous extract of the plant produced
et al. (2007). In the phytochemical analyses, no inhibitory effect on the tested organisms,
saponins, tannins, steroids, phlobatannins, hence no visible zone of inhibition.
alkaloids, anthraquinones, and flavonoids Sensitivity of test organisms to the stembark
were present in highest concentration in one extracts of Tetrapleura tetraptera was not as
extract than the other. These groups of high as fruit extract. It was clear from the
compounds form the active principles that data shown that there was no zones of
confer antibacterial activity on the plant. inhibition on Streptococcus salivarius
except against Streptococcus mutans and it
Steroids and phlobatannins which were gave a mean clear zone of inhibition of
found to be present in all the extracts of the 12.00mm.
plant parts tested are steroidal compounds
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