RUHUNA JOURNAL OF SCIENCE
Vol 11 (2): 98-117, December 2020
eISSN: 2536-8400 © Faculty of Science
DOI: http://doi.org/10.4038/rjs.v11i2.90 University of Ruhuna
© Faculty of Science, University of Ruhuna 98
Sri Lanka
Free radical scavenging potential and antibacterial activity of
Cola nitida and Garcinia kola extracts against bacterial strains
isolated from patients with urinary tract infections
I.V. Anyiam 1* and P.P.E. Mounmbegna2
1Department of Microbiology, Faculty of Science, Federal University Otuoke, Bayelsa State, Nigeria
2Department of Biochemistry, Faculty of Science, Madonna University Nigeria, Elele, Rivers State,
Nigeria
Correspondence: ifetgod@yahoo.co.uk; https://orcid.org/0000-0002-3705-3988
Received: 23rd May 2019, Revised: 16th July 2020, Accepted: 29th November 2020
Abstract. Cola nitida and Garcinia kola are found and widely consumed in West
Africa. The seeds of these plants have various traditional uses and are reported to
exhibit several bioactivities. Their phytochemical, antioxidant and antibacterial
properties of methanol, ethanol and aqueous extracts were investigated in the
present study. Phytochemical screening and quantification of total phenolic
contents analysis were carried out for phytochemical investigation. Preliminary
phytochemical screening revealed the presence of flavonoids, alkaloids, tannins,
saponins, protein and glycosides in the seed extracts. Quantitative phytochemical
constituents revealed 0.818 ± 0.021and 0.700 ± 0.017mg of phenolic compounds
and total flavonoid content of 25.63 ± 1.60 and 25.10 ± 1.85mg in G. kola and C.
nitida respectively. The extracts showed potent antioxidant activities compared
to standard antioxidants by significantly inhibiting 2, 2-diphenyl-1-
picrylhydrazyl (DPPH), hydroxyl radical (∙OH), and superoxide anion radicals
(O2∙) dose dependently. The methanol extracts of G. kola and C. nitida showed
significant inhibitory action (p<0.05) against the bacterial isolates. The minimum
inhibitory concentration obtained for methanol extract of the plants and both the
mixture was 12mm at 31.25mg/ml for Klebsiella pneumoniae while the ethanol
and aqueous extract of the plants and both the mixture was 13mm and 12.33mm
at 31.25mg/ml and 125mg/ml respectively for E. coli. A direct correlation was
observed between total phenolic content of extracts and radical scavenging
potential, thus linking the observed bioactivities of these extracts to the presence
of the phytochemical. The mixture of these seed extracts showed greater effect
against the bacterial isolates, therefore providing a platform for advance studies
in the development of drugs against infectious diseases.
Key words: Agar well dilution, antibacterial activity, Cola nitida, Garcinia kola,
Free radical scavenging potential
1 Introduction
The alarming rate of antimicrobial drug resistance by pathogenic microorganisms
against synthetic antibiotics (Maiyo et al. 2010) is a serious global problem. Indeed,
the emergence of bacterial resistance to antibacterial drug today has become a common
https://creativecommons.org/licenses/by-nc/4.0/
https://orcid.org/0000-0002-3705-3988
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 99
phenomenon, and consequently, antibiotic resistance has imposed both a biological
and economic cost (Chabot et al. 2002, Chen et al. 2002, Chessin et al. 2005). The rate
of antimicrobial resistance has prompted the search for new plants with antimicrobial
properties and potentials to serve as sources of raw material for the synthesis of new
drugs (Akoachere et al. 2002).
Traditional healers use different plant medicines to provide health care to most of
the people in a curative rather than a preventive approach in the developing countries
for common ailments (Gabriel et al. 2007). The availability and economy of these
plants as direct therapeutic agents makes it more attractive when compared to modern
medicine (Agbo and Ngogang 2005, Agbo et al. 2005). Natural plants contain
phytochemical properties similar to synthetic antibiotics and have been used in folk
medicine to treat infections (Ezeigbo 2016). In addition, people with different cultural
backgrounds from ancient times to the present day have used herbal medicines (El-
Mahmood et al. 2008) to cure infections. Hence, plants continue to be the most
preferred exclusive source of drugs for the majority of the world’s population (Fabiola
et al. 2003, Jonathan and Fasidi 2003, Ajayi et al. 2008). According to WHO (2000),
“medicinal plants when administered to man or animals exert a sort of pharmacological
action on them”. For this reason, medicinal plants are used as sources to produce useful
drugs utilized by people worldwide for treatment of infectious diseases.
Infectious diseases are the major causes of death accounting for approximately one
half of all deaths in tropical countries (Iwu et al. 2009). In recent times, medicinal
plants continue to play a major role in primary healthcare as therapeutic remedies in
many developing countries (Jonathan and Fasidi 2003, 2005, Jonathan et al. 2007) as
some plants have been found to be rich in secondary metabolites, such as tannins,
terpenoids, alkaloids, flavonoids, phenols, steroids and volatile oil. These compounds
are said to be responsible for their therapeutic activities (Rabe and Vanstoden 2000,
Cowan 2009). Furthermore, plants can serve as a reservoir of effective
chemotherapeutic agent which provides valuable natural drug for effective and
efficient management of human and plant diseases (Kanomal et al. 2014).
In Nigeria, studies have been carried out on a variety of these medicinal plants yet
a good number of them with putative medicinal and antimicrobial potentials are yet to
be studied (Amalu et al. 2014). Among these plants are Garcinia kola and Cola nitida
whose medicinal uses may have not been fully explored in the treatment of bacterial
infections, especially, urinary tract infections. These medicinal properties could occur
in different forms varying from biological, synthetic chemotherapeutic, antibiotics, and
phytotherapeutic agents (Arekemase et al. 2012). The action of these agents could
either be ‘bactericidal’ or ‘bacteriostatic’ (Arekemase et al. 2012). The importance and
quest for these medicinal plants origin that could be of potential benefit as antibacterial
agents stimulated the interest in Garcinia kola (‘bitter kola’) and cola nitida (kola nut)
seeds which are widely consumed as stimulant (Atawodi et al. 2005).
Garcinia kola, also generally known as ‘Bitter kola’ is a flowering plant species
that belongs to the family of tropical plants known as Guttiferae or Clusiaceae
(Adesuyi et al. 2012). In Nigerian languages, it is commonly called “Namijin Goro” in
Hausa, “Orogbo” in Yoruba, and “Agbilu” in Igbo (Dalziel 2008). Bitter kola is also
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
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Vol 11 (2): 98-117, December 2020 100
known as African wonder nut because almost every part of it has been found to be of
medicinal importance (Adegboye et al. 2008).
Cola nitida (Kola nut) (“Goro” in Hausa; “Obi gbanja” in Yoruba; “Oji” in Igbo,
Keay et al. 2014) is a member of the family Steculicca. It is a tree plant found in Sierra
Leone, North Ashanti, tropical Western Africa, West Indies, Brazil and Java (Grieve,
2001). Cola nitida was originally distributed along the west coast of Africa from Sierra
Leone to the Republic of Benin with the highest frequency and variability occurring in
the forest areas of Côte d'Ivoire and Ghana (Opeke 2012). In addition, kola nut is a
native stimulant which commonly chewed in many West African cultures (Opeke
2012). In Nigeria, it is often used in traditional occasions, to welcome guest and receive
visitors at home.
More so, the need for new antimicrobial agents is closely related with the problem
of emergence of resistant strains to most antibiotics. Hence, this study was conducted
to determine the phytochemical constituents, free radical scavenging potential and
antibacterial activity of Cola nitida and Garcinia kola.
2 Materials and Methods
2.1 Isolation and Identification
Sixty urine samples were collected one each from female patients attending Federal
Medical Centre, Yenagoa, from the period of May to July in 2017. Females were used
because they were the available patients at the time of the study and are considered to
be more predisposed to urinary tract infections. The specimens were cultured on
MacConkey agar, Blood agar and CLED (Cystine lactose electrolyte deficient) agar
plates using the streak method. Different agar was used to selectively identify and
differentiate the possible bacteria including the fastidious organism that might be
present in the culture specimen. Plates were inoculated and incubated at 37oC for 24
hours. The isolates were identified using Gram staining technique and Biochemical
tests which include catalase, urease, coagulase, oxidase, and indole.
2.2 Collection and authentication of plant material
Dried seeds of Garcinia kola (bitter kola) and Cola nitida (kola nut) were procured
from a local herb dealer at Swali market in Yenagoa Local Government Area, Bayelsa
State, Nigeria. They were authenticated with voucher specimen number MP-182 and
MP-183 in the Pharmacognosy Department, Madonna University, Nigeria, Elele,
Rivers State, Nigeria.
2.3 Processing and extraction
The seeds of Garcinia kola and Cola nitida were peeled, thoroughly washed and rinsed
in distilled water, and both were sliced into tiny pieces with the use of a clean stainless
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
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steel knife, then air-dried at room temperature for 4 weeks, and pulverized using
laboratory mortar and pestle. Different organic solvents (methanol, ethanol and
aqueous) were used for the extraction of these plants as described by Alade and Irobi
(1993). Fifty grams (50 g) of each seed powder was dispensed into a cotton wool stock
thimble chamber of the soxhlet apparatus and 500 ml of methanol was dispensed into
flat bottom flask. The extraction solvents were heated in the bottom flask, vaporized
into the thimble, condensed in the condenser and dripped back. When the liquid content
reached the siphon arm, the liquid contents was emptied into the bottom flask again
and the process was continued until the absorbent was clear. The extracts obtained
were completely evaporated (Green 2004) and stored in the refrigerator at 4oC until
use. The percentage (%) yields of the dry residue were calculated (Pudhom et al. 2007).
The same procedure was repeated successfully for ethanol and aqueous extracts.
Extracts were then dissolved in the appropriate solvent for the phytochemical and
antibacterial assay.
2.4 Phytochemical screening
Phytochemical screening was done using qualitative and quantitative phytochemical
analysis. Qualitative analysis involved tests for flavonoids, tannins, carbohydrates,
glycosides, saponins, resins, terpenoids and alkaloids. These were carried out using
standard methods (Harborne 1984, Sofowora 1993, Trease and Evans 2001).
Quantitative Analysis determined the total phenols, tannin, total flavonoids and total
anthocyanin contents. The total phenolics were determined using Folin-Ciocalteau
reagent (FCR) as described by Velioglu et al. (1998) with slight modifications. Tannin
content in each sample was determined using insoluble polyvinyl-polypirrolidone
(PVPP), which binds tannins as described by Makkar et al. (1993). The flavonoids
content was determined according to the method described by Kumaran and
Karunakaran (2006) with slight modifications. This method was based on the
formation of a flavonoid-aluminum complex, which absorbs maximally at 415 nm. The
total anthocyanin contents of the plant extracts were measured using a
spectrophotometric pH differential protocol described by Giusti and Wrolstad (2001)
and Wolfe et al. (2003) with slight modifications.
2.5 In vitro Antioxidant Assays
Quantitative DPPH radical-scavenging assay
The hydrogen atoms or electrons donation ability of the corresponding extract was
measured from the bleaching of purple coloured methanol solution of DPPH. The
scavenging activity on DPPH free radicals by the extract was assessed according to the
method reported by Gyamfi et al. (1999) with slight modifications.
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
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Hydroxyl radical (.OH)-scavenging assay
The 2-deoxyribose assay was used to determine the scavenging effect of the extract on
the hydroxyl (.OH) radical, as reported by Halliwell et al. (1987) with minor
modifications.
Superoxide Radical (O2.-) Scavenging Assay
This assay was based on the capacity of the extract to inhibit the photochemical
reduction of nitro blue tetrazolium (NBT) (Beauchamp and Fridovich 1971) and the
method used by Martinez et al. (2001) to determine superoxide dismutase with slight
modifications.
2.6 Reconstitution of plant extract
For preliminary screening of antibacterial activity of the plant extracts against bacteria
isolated, all the dried extracts were dissolved in dimethyl sulfoxide (DMSO) to a final
stock concentration of 2.5% w/v. As DMSO has been shown not to have any inhibitory
effect on the growth of microorganisms (Zgoda and Porter 2001, Kuete et al. 2008), it
was used as the negative control for all the experiments. A two-fold serial dilution was
also undertaken to obtain lower concentration ranges in sterile test tubes.
2.7 Antibacterial Aassay
Preparation of 0.5 Mcfarland turbidity standards was done as described in NCCLS
(NCCLS 2010). The agar well diffusion method was done following Atata (2003). An
overnight agar-culture of each bacterial isolate was made, and the suspension of
microorganisms was made in sterile normal saline and adjusted to 0.5 McFarland
standards (108 CFU/ml) (NCCLS 2010). From the stock of 500 mg/ml extract, two-
fold serial dilutions were made to 250, 125, 62.5, and 31.25 mg/ml. Each labeled
Mueller Hinton agar plate was uniformly inoculated with a test organism by using a
sterile cotton swab rolled in the suspension to streak the plate surface in a form that
lawn growth could be observed. A sterile cork borer of 6mm diameter was used to
make 5 wells on the medium in each plate. Before boring of the well in agar, the cork
borer was sterilized by dipping in alcohol and flaming. 50 µl of the 5 different extract
concentrations were dropped into each well using a micropipette. All antibacterial
assays were performed on duplicate plates. The underside of each well was
appropriately labeled. Other solvents used for extraction apart from water were tested
for each organism. The inoculated plates were kept in the refrigerator for 1 hour to
allow the extracts to diffuse into the agar (Atata et al. 2003). The plates were incubated
upright at 37°C for 24 hours. After incubation, the diameters of the zones of inhibitions
obtained were measured, using a pair of calipers and meter ruler. The measurement
was done at the back of the plate. The diameter was measured from one end of the zone
to the other. Where the zone of inhibition is not perfectly circular, the average of the
long and short axis was used. The diameter of the zone of inhibition was obtained for
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the two plates having the same concentration of the extract against a particular micro-
organism, and the average was used. For positive control, 30µl of 40 mg/ml of
gentamycin was used while 50 µl of 2.5% DMSO was used as negative control.
2.8 Determination of minimum inhibitory concentration
The minimal inhibitory concentrations (MICs) of the extracts on the bacterial isolates
were determined by macro broth dilution techniques following the recommendation of
the Clinical and Laboratory Standard Institute (CLSI 2015).
One gram of the extract was dissolved in 1 ml of 20% DMSO to get an extract
concentration of 250 mg/ml. Various serial dilutions were made from this stock
solution in tubes of 1 ml sterile Mueller Hinton broths to get 125 mg/ml, 62.5 mg/ml,
and 31.25 mg/ml. An overnight nutrient broth culture of the test bacterial isolate was
standardized to 0.5 McFarland turbidity standards. Different dilutions of the
suspension were made in a sterile normal saline to obtain a final inoculum
concentration of 106 CFU/ml. Then 1 ml of this adjusted inoculum was added to each
tube of the Mueller Hinton broth containing different concentration of the crude
extract. Each tube was mixed and incubated at 37oC for 24 hours (Nweze and Onyishi
2010). This experiment was conducted in duplicate for all the bacterial isolates. A tube
of Mueller Hinton broth containing only the 1ml suspension of the isolate without
extract and the tubes of Mueller Hinton broth containing different concentrations of
the extract without the isolate were used as controls. The tubes were examined after 24
hrs incubation. The MIC of the extract was taken as the lowest extract concentration
that completely inhibited the growth of the bacterial isolates in the tubes, as indicated
by lack of visual turbidity.
2.9 Determination of the use of mixtures of the extract
This was done by measuring equal volume of each of extract type (ethanol, methanol
and aqueous) of the plants seeds and then mixed. 50µl of each mixture was put into
each well as in the antibacterial bioassay section to test the sensitivity potentials. This
was done in triplicates.
2.10 Statistical analysis
All experiments were done in triplicate and the data thus obtained were reported as
mean ± standard error of mean. Statistical analysis was carried out to determine
whether there was significant difference among the inhibitory actions of Garcinia kola
and Cola nitida alone and in mixtures of extracts using Analysis of Variance and
Bonferroni post-test at 95% confidence level using Graph Pad PRISM Version 5.01
(Chao-Hsun et al. 2010).
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3 Results
Total of 48 isolates were obtained from the 60 specimens collected from patients with
urinary tract infections (UTI) attending Federal Medical Centre. Bacterial species
isolated include Proteus vulgaris, Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa and Klebsiella pneumoniae. Table 1 shows the frequency
distribution of bacterial isolates from patients with UTI. Escherichia coli had the
highest occurrence (29.17%) while Proteus vulgaris had the lowest occurrence
(8.33%).
Table 1: Frequency distribution of bacterial isolates (48) from patients with urinary tract
infections.
Organisms Number of isolates Percentage occurrence
Proteus vulgaris
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Klebsiella pneumoniae
4
14
12
8
10
8.33
29.17
25.00
16.67
20.83
Table 2 shows the zone of inhibition of the positive control (gentamycin) used against
bacterial isolates. The zones of inhibition produced by the positive control were larger
than the zones produced by the plant extracts.
Table 2: Zones of Inhibition (mm) of Gentamycin (positive control) against bacterial isolates.
Organisms Concentration (µl) Zone of inhibition (mm) Gentamycin a
Proteus vulgaris
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Klebsiella pneumoniae
30.0
30.0
30.0
30.0
30.0
28.00
26.00
18.00
26.00
29.00
a Values are mean inhibition zone (mm) from three replicates
The methanol extracts of G. kola, C. nitida and the mixture of extracts showed
antibacterial activity against all the bacterial isolates at a concentration of 500 mg/ml,
with G. kola having the largest zone of inhibition of 23 mm against E. coli and
Klebsiella pneumoniae respectively. C. nitida showed activity against Klebsiella
pneumoniae with largest zone of inhibition of 21.33 mm while the mixture of both
extracts showed activity against P. vulgaris and E. coli with the zone of inhibition of
25.66 mm respectively (p< 0.05) as shown in Table 3.
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
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Table 3: Mean zone of Inhibition (mm) of Methanol extract of Garcinia kola, Cola nitida and mixture of extracts against bacterial isolates.
Values are given as Mean ± Standard Error of three replicate plates
NI = No inhibition
All values of a particular isolate within the same column with shared alphabet superscript are non-significant (P>0.05).
Bacterial isolate Plant extract
Concentration
500 mg/ml 250 mg/ml 125 mg/ml 62.5 mg/ml 31.5 mg/ml
Proteus vulgaris Garcinia kola 21.33 ± 0.88A 20.00 ±1.15D 17.33 ± 0.67G 13.00 ± 0.58J NI
Cola nitida 17.33 ± 0.33B 16.00 ± 0.58E 13.66 ± 0.33H 11.33 ± 0.33J NI
Mixture of extracts 25.66 ± 0.33C 22.33 ± 0.33F 20.00 ± 0.58I 16.66 ± 0.33K 14.33 ± 0.33L
Escherichia coli Garcinia kola 23.00 ± 0.58A 21.66 ± 0.88D 19.66 ± 0.33G 17.00 ± 0.58J 14.66 ± 0.33M
Cola nitida 19.00 ± 0.58B 16.00 ± 0.58E 14.00 ± 0.58H 12.33 ± 1.20K 0.00 ± 0.00N
Mixture of extracts 25.66 ± 0.33C 21.33 ± 0.33D 18.33 ± 0.33G 15.00 ± 0.58J 13.33 ± 0.33M
Staphylococcus aureus Garcinia kola 17.66 ± 0.33A 16.00 ± 0.58CD 14.66 ± 1.20E NI NI
Cola nitida 17.66 ± 0.88A 15.66 ± 0.33C 0.00 ± 0.00F NI NI
Mixture of extracts 20.00 ± 0.58B 17.66 ± 0.33D 15.66 ± 0.33E NI NI
Pseudomonas aeruginosa Garcinia kola 15.66 ± 0.33A 13.00 ± 0.58C 0.00 ± 0.00E NI NI
Cola nitida 16.33 ±0.67A 13.66 ± 0.33C 0.00 ± 0.00E NI NI
Mixture of extracts 19.00 ± 0.58B 16.00 ± 0.58D 10.33 ± 0.58F NI NI
Klebsiella pneumoniae Garcinia kola 23.00 ± 0.58AB 20.33 ± 0.33C 18.33 ± 0.67E 14.00 ± 0.58G 0.00 ± 0.00H
Cola nitida 21.33 ± 0.33A 20.00 ± 0.58C 18.66 ± 0.33E 14.00 ± 0.58G 0.00 ± 0.00H
Mixture of extracts 25.33 ± 0.33AB 22.00 ± 1.15C 20.33 ± 1.20E 16.33 ± 0.33G 12.00 ± 0.58I
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
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Table 4: Mean zone of Inhibition (mm) of Ethanol extract of Garcinia kola, Cola nitida and mixture of extracts against bacterial isolates.
Values are given as Mean ± Standard Error of three replicate plates; NI = No inhibition
All values of a particular isolate within the same column with shared alphabet superscript are non-significant (P>0.05).
Bacterial isolate Plant extract
Concentration
500 mg/ml 250 mg/ml 125 mg/ml 62.5 mg/ml 31.5 mg/ml
Proteus vulgaris Garcinia kola 20.00 ± 0.58A 18.00 ± 0. 58D 15.00 ± 0.58G 13.66 ± 0.33J NI
Cola nitida 16.00 ± 0.58B 14.33 ± 0.33E 12.00 ± 0.58H 0.00 ± 0.00K NI
Mixture of extracts 25.33 ± 0.33C 22.33 ± 0.33F 18.33 ± 0.88I 15.33 ± 0.88J NI
Escherichia coli Garcinia kola 22.66 ± 0.67A 20.66 ± 0.67DF 18.66± 0.33GI 16.66 ±0.88JL 15.66 ± 0.33M
Cola nitida 18.66 ± 0.67B 16.00 ± 0.58E 14.33 ± 0.33H 0.00 ± 0.00K 0.00 ± 0.00N
Mixture of extracts 25.66 ± 0.58C 21.33 ± 0.67F 17.33 ± 0.33I 15.00 ± 0.58L 13.00 ± 0.58O
Staphylococcus aureus Garcinia kola 16.50 ± 0.33A 13.66 ± 0.33C 0.00 ± 0.00E NI NI
Cola nitida 17.00 ± 0.58AB 15.00 ± 0.58CD 13.33 ± 0.33F NI NI
Mixture of extracts 18.00 ± 0.58B 15.66 ± 0.33D 13.66 ± 0.67F NI NI
Pseudomonas aeruginosa Garcinia kola 15.66 ± 0.33A 12.66 ± 0.33D 0.00 ± 0.00F NI NI
Cola nitida 14.50 ± 0.33A 13.83 ± 1.00DE 0.00 ± 0.00F NI NI
Mixture of extracts 17.33 ± 0.33B 15.00 ± 0.58E 12.66 ± 0.33G NI NI
Klebsiella pneumoniae Garcinia kola 21.33 ± 0.67AB 17.33 ± 0.33C 15.00 ± 0.58E 0.00 ± 0.00G NI
Cola nitida 19.66 ± 0.33A 16.66 ± 0.88C 14.00 ± 0.58E 0.00 ± 0.00G NI
Mixture of extracts 22.33 ± 0.33B 19.66 ± 0.88D 17.66 ± 0.33F 12.33 ±0.33H NI
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Ethanol extracts of G. kola, C. nitida and the mixture of extracts showed antibacterial
activity against all the bacterial isolates at a concentration of 500 mg/ml. G. kola
inhibited the growth of E. coli with 22.66 mm as the largest zone of inhibition while
C. nitida showed its highest activity against Klebsiella pneumoniae at zone of
inhibition of 19.6 mm. Furthermore, the mixture of extracts showed its effectiveness
with the largest zone of inhibition of 25.66 mm against E. coli with significance of (p<
0.05) (Table 4).
Table 5 shows the aqueous extracts of G. kola, C. nitida and the mixture of extracts
against the bacterial isolates. G. kola, C. nitida and the mixture of extracts showed
antibacterial activity against all the bacterial isolates at a concentration of 500 mg/ml
except against Staphylococcus aureus. The effect of G. kola was seen against P.
vulgaris with the largest zone of inhibition of 17 mm while C. nitida was able to inhibit
the growth of E. coli with the largest zone of inhibition of 16.66 mm. The mixture of
both extracts equally showed effectiveness against P. vulgaris with the largest zone of
inhibition of 20 mm which shows similar results with Omwirhiren et al. (2016).
Table 5: Mean zone of inhibition (mm) of aqueous extract of Garcinia kola, Cola nitida and
mixture of extracts against bacterial isolates.
* There was no inhibition detected against any of the bacterial isolates at 62.5 mg/ml 31.5 mg/ml concentrations of the
aqueous extracts of single plant species or both plants in mixture, so that those two columns were not shown in the table.
Values are given as Mean ± Standard Error of three replicate plates; NI = No inhibition;
All values of a particular isolate within the same column with shared alphabet superscript are non-significant
(P>0.05).
Table 6 shows the Minimum Inhibitory Concentration (MIC) values for the extracts of
G. kola, C. nitida and the mixture of extracts against bacteria isolated from UTI. The
Bacterial isolate Plant extract
Concentration*
500 mg/ml 250 mg/ml 125 mg/ml
Proteus vulgaris Garcinia kola 17.00 ± 0.58A 15.33 ± 0.33D 12.66 ± 0.33G
Cola nitida 12.66 ± 0.88B 10.66 ± 0.33E 0.00 ± 0.00H
Mixture of extracts 20.00 ± 0.58C 16.00 ± 0.58D 13.33 ± 0.33G
Escherichia coli Garcinia kola 15.00 ± 0.58A 13.66 ± 0.33C 0.00 ± 0.00E
Cola nitida 16.66 ± 1.20A 14.66 ± 0.33C 12.33 ± 0.33F
Mixture of extracts 19.00 ± 1.00B 14.33 ± 0.33C 12.66 ± 0.33F
Staphylococcus
aureus
Garcinia kola NI NI NI
Cola nitida NI NI NI
Mixture of extracts NI NI NI
Pseudomonas
aeruginosa
Garcinia kola 12.33 ± 0.88A NI
NI
Cola nitida 0.00 ± 0.00B NI
NI
Mixture of extracts 13.00 ± 0.58A NI
NI
Klebsiella
pneumoniae
Garcinia kola 15.33 ± 0.67A 13.00 ± 0.58D NI
Cola nitida 0.00 ± 0.00B 0.00 ± 0.00E NI
Mixture of extracts 13.33 ± 0.33C 11.66 ± 0.33F NI
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MIC values of the extracts against the isolates were obtained from the agar diffusion
assay. The lowest MIC for G. kola, C. nitida and the mixture of extracts were obtained
with methanol and ethanol extracts against all bacterial species respectively. However,
there was no MIC obtained for S. aureus, P. aeruginosa, and K. pneumonia for aqueous
extract, and no MIC obtained for S. aureus for aqueous mixture of extracts as well.
Table 6: Minimum Inhibitory Concentration (MIC) values of extract of G. kola, C. nitida and
mixture of extracts against bacterial isolates.
Isolate Plant
Minimum Inhibitory Concentration (mg/ml)
Methanol Ethanol Aqueous
Proteus vulgaris Garcinia kola 62.5 62.5 125
Cola nitida 62.5 125 250
Mixture of extracts 31.25 62.5 125
Escherichia coli Garcinia kola 31.25 31.25 250
Cola nitida 62.5 125 125
Mixture of extracts 31.25 31.25 125
Staphylococcus
aureus
Garcinia kola 62.5 250 250
Cola nitida 62.5 125 No MIC
Mixture of extracts 62.5 125 250
Pseudomonas
aeruginosa
Garcinia kola 250 125 500
Cola nitida 125 125 No MIC
Mixture of extracts 125 62.5 500
Klebsiella
pneumoniae
Garcinia kola 62.5 62.5 250
Cola nitida 31.25 62.5 No MIC
Mixture of extracts 31.25 31.25 250
Qualitative analysis on Garcinia kola and Cola nitida revealed the presence of
important phytochemical constituents including phenolic compounds (tannins and
flavonoids), saponins and alkaloids as bioactive compounds (Table 7).
Table 7. Qualitative phytochemical constituents of Garcinia kola and Cola nitida extracts.
Extract Flavonoids Tannins Alkaloids Terpenoid Glycoside Saponins Resin Protein
G. kola + + + + - + + + - +
C. nitida + + + + + - + + - +
(Key: + Present; + + Moderately present; + + + Abundant; - Absent)
Quantitative analysis on Garcinia kola and Cola nitida revealed that phenolic
compounds were a major class of bioactive components in the extracts Table 8). The
amount of total phenolics were 0.818 ± 0.021 mg and 0.700 ± 0.017 mg GAE/mg of
dry plant extracts of G. kola and C. nitida respectively, whereas the total flavonoid
contents were as 25.63 ± 1.60 mg and 25.10 ± 1.85 rutin equivalents / g dry weight
plant extract of G. kola and C. nitida respectively.
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 109
Table 8: Phytochemical constituents of Garcinia kola and Cola nititda (mean ± SD, n=3).
Extract Phenolic contents * † Total
anthocyanin
‡ Total
flavonols
‡ Total
flavonoids
Total Phenols Non-tannins Tannins
G. kola 0.818 ± 0.021 0.507 ± 0.009 0.311 ± 0.001 4.64 ± 0.22 9.41 ± 0.02 25.63 ± 1.60
C. nitida 0.700 ± 0.017 0.376 ± 0.001 0.324 ± 0.007 2.15 ± 0.22 13.81 ± 0.92 25.10 ± 1.85
* Expressed as mg gallic acid equivalents / mg dry weight plant extract
† Expressed as mg cyanidin 3-glucoside equivalents/100g of dry weight extract
‡ Expressed as mg rutin equivalents / g dry weight plant extract
Table 9 shows the concentration of the extracts that inhibited 50% of the free radicals
and lipid peroxidation (IC50) which was used to determine the potency of the extracts.
The lower the IC50 value the better the extract potency. The plant extracts were efficient
inhibitors of different free radicals compared to standard antioxidants. G. kola appears
to be more efficient in inhibiting DPPH radical (9.6 ± 1.0 μg/ml), Superoxide anion
(64.6 ± 1.5 μg/ml) and lipid peroxidation (282.9 ± 9.3 μg/ml) while C. nitida extract is
a better inhibitor of Hydroxyl radical (46.6 ± 2.5 μg/ml).
Table 9: Free radical and lipid peroxidation inhibitory potency (IC50) of Garcinia kola and
Cola nititda (mean ± SEM, n=3).
IC50 value for inhibitory potential (μg/ml)
Extract DPPH radical Hydroxyl radical
(.OH)
Superoxide anion
(O2.-)
Lipid peroxidation
C. nitida 24.1 ± 2.1 46.6 ± 2.5 103.7 ± 5.2 575.1 ± 15.4
G. kola 9.6 ± 1.0 99.4 ± 1.7 64.6 ± 1.5 282.9 ± 9.3
Standard antioxidant 4.1 ± 0.3 * 38.9 ± 2.8 # 3.3 ± 0.2 β 24.3 ± 1.4 £
* compared to ascorbic acid; # compared to α-Tocopherol; β compared to rutin;
£ compared to butylated hydroxyltoluene
Figure 1 shows the graphical representation of Garcinia kola and Cola nitida extracts
which showed significant dose-dependent DPPH radical scavenging capacity.
Garcinia kola appears to be more efficient, inhibiting 92.36 ± 1.31% of DPPH at a
concentration of 125 μg/ml compared to ascorbic acid which inhibited 94.18 ± 3.22 %
at the same concentration.
Figure 2 shows the graphical representation of Garcinia kola and Cola nitida extracts
scavenged .OH radical in a concentration dependent manner. The two extracts inhibited
2-deoxyribose degradation above 30% with maximal inhibition of 76.7 ± 1.4% at
concentration of 500 μg/ml by S. s. The scavenging ability of the extracts was
significant at all tested concentrations. C. nitida extract was found to be powerful
quencher of .OH radical thereby preventing the propagation of lipid peroxidation. At
high concentrations of both extracts lower activities were observed.
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 110
Fig. 1: Graph comparing DPPH antioxidant activity of different concentrations of ascorbic acid
and extracts of Garcinia kola and Cola nitida (values are expressed as mean ± SEM, n = 3)
Fig. 2: Graph comparing Hydroxyl (.OH) antioxidant activity of different concentrations of α-
tocopherol and extracts of Garcinia kola and Cola nitida. Values are expressed as mean ± SEM, n = 3)
Figure 3 shows the Garcinia kola and Cola nitida extracts which inhibited the
formation of reduced NBT in a dose-related manner. C. nitida showed the maximal
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 111
O2
.- anion inhibitory activity of 86.68±2.10% at the concentration of 250 μg/ml,
compared to rutin (96.03±2.2%, at 250 μg/ml). The O2
.- scavenging effect of the
extracts could culminate in the prevention of .OH radical formation since O2
.- and H2O2
are required for .OH radical generation.
Fig. 3: Graph comparing superoxide anion (O2.-) inhibition of different concentrations of
rutin and extracts of Garcinia kola and Cola nitida (values are mean ± SEM, n = 3)
4 Discussion
The involvement of bacteria in urinary tract infection is of great concern. Female
patients were only participant involved in this study because they are considered to be
more predisposed to urinary tract infections. Studies have shown Escherichia coli and
Staphylococcus saprophyticus as mostly implicated in urinary tract infection (Nicolle
2008). However, this study revealed the presence of Escherichia coli as the highest
occurring bacteria with 14 (29.17%), while Proteus vulgaris as the lowest with 4
(8.33%) which agrees with Mansour et al. (2009). The presence of these bacteria could
possibly be because of poor sanitary hygiene.
The findings from this study revealed the presence of alkaloids, saponins, tannins,
flavonoids, glycoside, protein and the absent of terpenoids and resin in the extracts of
Garcinia kola and Cola nitida in the methanol, ethanol and aqueous used as solvent as
agreed with work done on C. nitida and G. kola by Omwirhiren et al. (2016). Studies
have shown that the various solvents influence the nature of compounds extracted and
their bioactivities (Arunkumar and Muthuselvam 2009, Seanego 2012). However,
methanol extracts appear to be most potent and promising as shown by its high
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 112
inhibitory activity against the clinical isolates. This could be attributed to the high
presence of some of the polyphenolic compound identified (total flavonoids content of
25.63 ± 1.60 and 25.10 ± 1.85 mg rutin equivalents/g dry weight of G. kola and C.
nitida respectively). These results clearly show that the solvent influences the
extractability of the phenolic compounds. The phenolic extracts of plants are always a
mixture of different classes of phenols, which are selectively soluble in the solvents.
This finding is in conformity with previous studies by Ukaoma et al. (2013) and Alaje
et al. (2014). The presence of these secondary metabolites is known to have therapeutic
activity against several diseases and therefore could suggest the basis for their
traditional use for the treatment of various illness (Yousuf et al. 2012) including
urinary tract infections. Earlier studies have reported that flavonoids have antibacterial
property as they have the capability to associate with soluble proteins and bacterial cell
walls (Doss et al. 2011).
The evaluation of the antibacterial properties and the effect of mixture of extracts on
bacterial isolates showed that they all possess antibacterial properties. The antibacterial
activity was seen at varying concentrations indicating that the plant extract had broad
antibacterial spectrum (Bankole 1992). Presence of alkaloids and flavonoids in G. kola
and C. nitida has been observed to be responsible for its antibacterial property.
However, the data obtained showed that the inhibitory effects of these plant extracts
on the various bacterial isolates were dose dependent. This observation agrees with the
findings of Agbaje et al. (2006) and Akinnibosun et al. (2009). The methanol extract
of G. kola was most active against E. coli, K. pneumoniae, P. vulgaris, S. aureus, and
P. aeruginosa with zones of inhibition ranging from 23.00 mm to 13.00 mm. The
ethanol extract of G. kola extract was active against E. coli, K. pneumoniae, P.
vulgaris, S. aureus, and P. aeruginosa with zones of inhibition ranging from 22.66 mm
to 12.66 mm. While the aqueous G. kola was active against P. vulgaris, E. coli, K.
pneumoniae, P. aeruginosa with zones of inhibition ranging from 17.00 mm to 12.33
mm and showed no zone of inhibition to S. aureus. This result is similar to the work
of Adegboye et al. (2008), who showed that the crude extract of G. kola exhibited
antibacterial activities in vitro against both Gram-positive and Gram-negative
organisms. The antibacterial properties of this plant could be attributed to the presence
of tanins and flavonones. Studies have shown it to have good antibacterial,
antifungal and antiviral properties (Terashima et al. 2002, Adesuyi et al. 2012).
Other medicinal properties of the plant include its usage in the treatment of skin
infections in Liberia and Congo Democratic Republic. The powdered bark of the
plant is applied to malignant tumors and cancers, whereas the plants latex is taken
internally for gonorrhea and externally to seal new wounds and prevent sepsis
(Adesuyi et al. 2012). In Nigeria, a cold-water extract of the roots and bark with
salt are administered to cases of bronchial asthma or cough and vomiting (Adesuyi
et al. 2012).
Methanol extract of C. nitida was most active against K. pneumonia, E. coli, S.
aureus, P. vulgaris, and P. aeruginosa with zones of inhibition ranging from 21.33
mm to 11.33 mm. Ethanol C. nitida extract was active against K. pneumoniae, E. coli,
S. aureus, P. vulgaris, P. aeruginosa with zones of inhibition ranging from 19.66 mm
to 12.00mm. While the aqueous C. nitida was active against E. coli, P. vulgaris with
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 113
zones of inhibition ranging from 16.66 mm to 10.66 mm and showed no zone of
inhibition to S. aureus, P. aeruginosa and K. pneumoniae.
The mixture of the extracts produced greater zones of inhibition on the bacterial
isolates than the zones of inhibition produced by G. kola and C. nitida when used
separately. The effect of mixture of plant extracts on the bacterial isolates was seen
with methanol extract and least with aqueous extract. The methanol mixture of extracts
was observed to be most active against P. vulgaris, E. coli, K. pneumoniae, S. aureus,
and P. aeruginosa with zones of inhibition ranging from 25.66 mm to 12.00 mm. The
ethanol mixture of extracts was seen to be active against E. coli, P. vulgaris, K.
pneumoniae, S. aureus, and P. aeruginosa with zones of inhibition ranging from 25.66
mm to 12.33 mm. While the aqueous mixture of both plants was also observed to be
active against P. vulgaris, E. coli, K. pneumoniae, P. aeruginosa with zones of
inhibition ranging from 20.00 mm to 11.66 mm and showed no zone of inhibition to S.
aureus. With this result, the effect of the mixture of the extracts showed greater
antibacterial activity against the bacterial isolates when favourably compared with the
standard antibiotic. Although, no studies have shown this, that of Solenostemon
monostachyus and Ocimum gratissimum (Chukwura and Iheukwumere, 2012) results
showed they have greater inhibitory effect as G. kola and C. nitida extract mixture.
The MIC of the extracts against the bacteria was also determined varying between
concentration of 31.25 mg/ml to 500 mg/ml for G. kola extract, C. nitida extract and
the mixture of both extracts, respectively. The results of MIC showed that the mixture
of extracts is more potent against the bacterial isolates even at low concentrations. The
broad spectrum of activity displayed by the extracts in this study appears to justify
and explain the basis for their uses in traditional medicine, possibly as a remedy to the
emergence of drug-resistant strains caused by inappropriate use of orthodox
antibiotics.
Garcinia kola and Cola nitida extracts showed significant dose-dependent DPPH
radical scavenging capacity. Garcinia kola appears to be more efficient, inhibiting
92.36±1.31% of DPPH at a concentration of 125 μg/ml compared to ascorbic acid
which inhibited 94.18 ± 3.22 % at the same concentration as proven by Okoko (2009).
Garcinia kola and Cola nitida extracts scavenged .OH radical in a concentration
dependent manner. The two extracts inhibited 2-deoxyribose degradation above 30%
with maximal inhibition of 76.7±1.4 % at concentration of 500 μg/ml. The scavenging
ability of the extracts was significant at all tested concentrations. The high radical
scavenging activity of Garcinia kola and Cola nitida seems to be directly correlated
with its total phenolic content as it may play an important role in their antioxidative
effect. C. nitida extract was also found to be powerful quencher of .OH radical thereby
preventing the propagation of lipid peroxidation. At high concentrations of both
extracts lower activities were observed. Garcinia kola and Cola nitida extracts which
inhibited the formation of reduced NBT in a dose-related manner. C. nitida showed the
maximal O2
.- anion inhibitory activity of 86.68±2.10% at the concentration of 250
μg/ml, compared to rutin (96.03 ± 2.2 %, at 250 μg/ml). The O2
.- scavenging effect of
the extracts could culminate in the prevention of .OH radical formation since O2
.- and
H2O2 are required for
.OH radical generation. The observed ability of the extracts to
scavenge or inhibit HO· radical indicated that the extracts could significantly inhibit
I.V. Anyiam and P.P.E. Mounmbegna Bioactivities of Cola nitida and Garcinia kola extracts
Ruhuna Journal of Science
Vol 11 (2): 98-117, December 2020 114
lipid peroxidation. This corroborates the studies of Farshori et al. (2013) and Olatunde
et al. (2004) who reported that G. kola and C. nitida contains natural antioxidants.
The IC50 is the concentration of the extracts that inhibited 50% of the free radicals
and lipid peroxidation which was used to determine the potency of the extracts. The
lower the IC50 value the higher the extract potency. The plant extracts were efficient
inhibitors of different free radicals compared to standard antioxidants. G. kola appears
to be more efficient in inhibiting DPPH radical (9.6±1.0 μg/ml), Superoxide anion
(64.6±1.5 μg/ml) and lipid peroxidation (282.9±9.3 μg/ml) while C. nitida extract is a
better inhibitor of Hydroxyl radical (46.6±2.5 μg/ml). IC50 was calculated as the
amount of antioxidant present in the sample necessary to decrease the initial DPPH
concentration by 50%. The lower the IC50 value the higher is the antioxidant activity.
The observed antibacterial property of these seeds could therefore be linked to the
presence of the phenolic compounds as they have previously been found to be main
contributors of antioxidant activity and are also responsible for anti-inflammatory,
antiviral, anticancerous and antimicrobial activities (Yang et al. 2013).
5 Conclusions
The present study revealed the presence of phytochemicals in G. kola and C. nitida
which exhibited promising antimicrobial activity against a broad spectrum of bacterial
isolates. Another striking finding was that the extracts showed free radical scavenging
potential properties against the synthetic oxidative molecules and varying degrees of
antibacterial activity on the bacteria isolated, with the methanol extract demonstrating
the most effective activity against all the isolate at all concentrations. This therefore
reaffirms the ethno-pharmacological importance of G. kola and C. nitida and could
serve as the basis for advanced studies in the development of drugs against infectious
diseases. This would also prove useful especially due to the alarming rate of drug
resistance which is posing a threat and a major challenge in treatment of infectious
diseases. Apart from performing synergistic studies to evaluate the performance of G.
kola and C. nitida when combined with orthodox medicine, there is also a need to
determine the toxicity of the plant extracts which in our findings will be a prelude to
initiating clinical trials in subsequent drug development.
Acknowledgments
Two anonymous reviewers are acknowledged for valuable comments on the initial draft of the
manuscript.
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