Journal of Biosciences and Medicines, 2014, 2, 37-42
Published Online May 2014 in SciRes. http://www.scirp.org/journal/jbm
http://dx.doi.org/10.4236/jbm.2014.23006
How to cite this paper: Muhammad, S. and Fatima, A. (2014) Studies on Phytochemical Evaluation and Antibacterial Prop-
erties of Two Varieties of Kolanut (Cola nitida ) in Nigeria. Journal of Biosciences and Medicines, 2, 37-42.
http://dx.doi.org/10.4236/jbm.2014.23006
Studies on Phytochemical Evaluation and
Antibacterial Properties of Two Varieties of
Kolanut (Cola nitida) in Nigeria
S. Muhammad*, A. Fatima
Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
Received March 2014
Abstract
Kola nut is a native stimulant which is commonly chewed in many West African cultures, indivi-
dually or in a group setting. It is often used ceremonially and to honour guest. Phytochemical
analysis and antibacterial screening of red and white Cola nitida (kola nut) extracts were eva-
luated using qualitative and quantitative method. Thin layer chromatography (TLC) was used to
evaluate the phenolic content. Antibacterial screening was done using agar well diffusion method
against odontopathogens; (Lactobacillus sp., Corynebacterium haemolyticum, Proteus vulgans, Ba-
cillus lentus, Streptococcus anginosus and Pseudomonas aeruginosa) obtained from stock cultures.
Phytochemical screening revealed the presence of alkaloids, flavonoids, saponin, tannins, glyco-
side, steroids, volatile oil and balsams. Higher content of alkaloids, saponin and glycoside was ob-
tained in the aqueous extract than the methanol for both red and white kola nut. The results of
TLC analysis revealed the presence of quercetin and coumarin in red kola and keamferol and
coumarin in white kola respectively. The result of antibacterial activity of red C. nitida showed a
zone of inhibition of 18 mm and 23 mm at 60 mg/ml for aqueous extract on Proteus vulgans and
Streptococcus anginosus. Similarly white kola inhibited the growth of S. ang ino su s at 90 mg/ml
with a zone of 18 mm. Methanol extract of red kola inhibited the growth of P. vulgans and S. angi-
nosus at 60 mg/ml with a zone of 16 mm and 20 mm respectively. Acetone and ethyl acetate ex-
tracts of both red and white kola nut did not show any activity against the tested organism at all
concentrations. The results obtained indicate that C. nitida which posses promising chemothera-
peutic and antibacterial potentials respectively that could be useful against odontopathogens. The
study may serve as baseline for further studies.
Keywords
Kola Nut. Phenol Zone of Inhibition and Odontop ath ogen s
1. Introduction
Treatment of anaerobic infections is usually empirical but the universality of susceptibility of anaerobes to the
*Corresponding author.

S. Muhammad, A. Fatima
38
usual drugs cannot always be assumed as reports of resistant strains have been growing in the literature from all
over the world; [1]-[4]. There is therefore a need for continuous search for an urgent alternative to synthetic
drugs. In Africa, Asia, North and South America, medicinal plants were used for the treatment of infections be-
fore the introduction of antibiotics and other modern drugs [5]-[7].
Traditional chewing sticks are commonly used especially by rural dwellers in maintaining oral hygiene [8]
with about 80% - 90% of Nigerians using chewing sticks from a variety of plants [9]. The reasons can be attri-
buted to culture, affordability, accessibility as well as the popular thought misguided belief that natural medi-
cines have no side effects [10]. Studies of many plant materials and standardization of the dosages have being
reported [11]-[13]. Medicinal plants such as Vernonia amygdalina, Terminalia glaucesens, Nauclea latifolia,
Serindeia warneckei, and kola nut are known to inhibit the activities of some anaerobic pathogens [14]. Gener-
ally, the mechanical cleansing effect and antimicrobial substances in the seed are seen as major beneficial effects
of chewing this nut. The chemical composition of kola nut includes: caffeine (2% - 3.5%), theobromine (1.0% -
2.5%), theophylline, phenolics (phlobaphens, epicatechin, D-catechin, tannic acid), sugar (cellulose), water [15].
Worldwide, approximately 2.43 billion people (36% of population) have dental carries in their permanent
teeth. In baby teeth it affects about 620 million (9% of world population) [16]. It is the primary pathological
cause of tooth loss in children and about 29% - 59% of adult over the age of 50 experience dental carries [17].
Bacterial dental plaque is involved in the initiation of gingivitis and the progression of periodontitis and pla-
que control is of great importance in the prevention of these diseases [18]. Adequate plaque control using me-
chanical methods is difficult to achieve for most individuals. Therefore, a number of chemical dental pla-
que-control agents have been used to supplement routine oral procedure such as mouth washing or oral irriga-
tion [19]. Antimicrobial agents such as antibiotics, enzymes, chlorhexidine and phenol have shown anti-plaque
effect both in vitro and in vivo [20].
Kola nut contain caffeine, which may explain their popular use in energy tonics. Native of Africa, the nuts of
this towering tree are also a traditional medicine thought to enhance male potency. Kola is listed as having aph-
rodisiac stimulant, cardiotonic properties in Duke phytochemical database and to maintain alertness. Kolanut is
used in peace mission or to invite some highly placed personelities to an occasion. There is a popular saying in
Nigeria about kolanut. “Yoruba’s produce it, Hausa’s chew it the most and Igbo ritualize it the most”.
Considering the use of kola nut by people in Nigeria, who believe that kola nut may prevent dental plaque if
proper hygiene is maintained. Also, the antimicrobial activity of phenolic compound, there is the need for tho-
rough biochemical evaluation of kola nut with the view to obtaining the potential which could be vital in pro-
viding dental care.
The aim of the study is to evaluate the phytochemical content of kola nut and its antibacterial activity against
odontopathogens.
2. Methodology
Two varieties of kola nut (Red and White) Cola nitida were collected from major cola nuts market across Nige-
ria. The plant material was authenticated at the herbarium, Department of Biological Sciences, Usmanu Danfo-
diyo University Sokoto. A portion of it was shaded dried and pulverized into powder using a mortar and pestle.
2.1. Extraction of Red and White Variety of Kola Nut
A portion of about 250 g of each pulverized kola nut was socked in 1liter of water, methanol, acetone and ethyl
acetate each respectively in separate conical flask and corked. These were allowed to stand for 24 hrs, filtered
and the powered extract was collected separately for red and white variety.
Prepared extract of Cola nitida were subjected to standard phytochemical analysis for different constituents
such as tannins, alkaloids, flavonoids, anthraquinones, glycocides, Saponins and phenols as described by [21]
Jigna et al. (2006). The following test were conducted Test for flavonoid Test for tannins, Test for saponin [22].
Test for glycoside and Test for alkaloids [23].
2.2. Antibacterial Assay
Nutrient agar was used for testing of these extracts. Three wells were made on each plate agar using sterilized 14
mm cork borer. Each plate agar was inoculated with different isolates separately. This was done with the use of
sterilized wire loop after standardization of each inoculum.

S. Muhammad, A. Fatima
39
After inoculation, different concentration of the plant extract was mixed with little plain agar and pour into
designated well which carry the specific concentration of the extract. The inoculated plates were allowed to
stand for 15 minute before incubating then for 24 hours at 37˚C. In a good light and colour background the tur-
bidity of the suspension was matched with that of the turbidity standard. The turbidity of the test inoculum was
adjusted by either adding more Candida albican or more physiological saline until a good match is obtained.
Minimum Inhibitory Concentration (MIC) was then conducted on positive inhibited test organisms.
3. Results and Discussion
The results of qualitative phytochemical screening of two varieties of kola nut are represented in Table 1. The
results revealed the presence of alkaloid, saponin, and glycosides were detected in the aqueous extract of red and
white C. n itida. However, saponin and steroids were detected in methanol extract while flavonoids, tannins, sa-
ponin glycoside, cardiac glycoside, anthraquinone, volatile oil and balsam were little or not detected in both
aqueous and methanol extract (Table 1).
The results of quantitative phytochemical analysis of both red and white kola nut are presented in Table 2.
Result shows that there was a significant difference (p < 0.05) in the contents of tannins, saponins and phenolic
in red variety compared to the white variety. From the result, it was observed that the white variety had the
highest percentage of saponin with 1.10% while red had 0.90%. Similarly, white variety had 0.25% phenol
while red variety had 0.21%. Moreover, tannins content of white variety was also high 0.12% as compared to
red kola with 0.11%.
The involvement of anaerobes in human infections has generated global interest in recent time. Oral environ-
ment tends to favour the growth of both aerobic and facultative anaerobic bacteria. Bacteria accumulate on both
the hard and soft oral tissue in biofilms [24]. Dental plaque is a biofilm that build up on the teeth and consist of
bacteria and their products all in close proximity to the surface of the teeth. The accumulation of food debris on
the teeth, might give room for pathogenic bacteria to grow thereby leading to oral infections like dental carries,
peridontal infections.
Kola nut is regularly chewed and by Nigerian, posses polyphenols which have been reported to exhibit anti-
bacterial activities with distinguished characteristics in their reactivity with protein related polyamide polymer
[25]. In this study, red and white variety of kola nut aqueous and methanol extracts showed antibacterial
Table 1. Results of qualitative phytochemical content of aqueous and methanol extract of red and white C. nitida.
Comp onent Aqueous extract Methanol extract
Red White Red White
Alka lo ids +++ +++ ND ND
Flavonoids + Trace ND ND
Saponins ++ ++ +++ +
Tannins + + ND ND
Glycosides ++ +++ + +
Saponin glycoside Trace Trace ND ND
Cardiac glycoside ND ND ND ND
Steriods + + +++ +
Anthraquinone ND ND ND ND
Volatile oil Trace Trace + +
Balsams + + + +
Key: +++ = Present in high concentration; ++ = Moderately present; + = Slightly present; ND = not detected.
Table 2. Results of quantitative analysis of red and white Cola nitida Kola nut variety % constituent (mg/ml).
Tannins Saponi ns Phenols
White variety 0.12 1.10 0.21
Red variety 0.11 0.90 0.25

S. Muhammad, A. Fatima
40
activity against Streptococcus anginosus, gram positive bacteria which, is a member of the viridian Streptococci.
These are heterogenic bacteria with unique pathogenicity than other Streptococci [26]. However, red variety also
showed activity against Proteus vulgans, gram negative bacteria at 60 mg/ml, which gave a zone of inhibition of
18 mm and 16 mm at 60 mg/ml for methanol and aqueous extracts. Similar report was seen in the work of [27]
on kola extract against Proteus mirabilis, which gave an inhibition zone of 16 mm at 1000 µg/ml.
The inhibition of micro-organisms by kola nut which contain phenolic compounds may be due to iron depri-
vation or hydrogen bonding with vital proteins such as micro- enzyme. Coumarine, quercetin and kaeferol which
might be present in kola nut as detected from the TLC ran on both red and white varieties, are belief to posses
inhibitory characteristics against gram positive and gram negative bacteria [28]. Though inhibition was not ob-
served on other odontopathogens used in this study that is (Lactobacillus sp , Corynebacterium haemolyticum,
Bacillus lentus and Pseudomonas aeruginosa). C. nitida inactivity exhibited against these organisms and also
that of acetone and ethyl acetate extract of both red and white variety, may be due to the absence of inhibitory
alkaloids against the tested organisms. This is because alkaloids have been chained to be responsible for antimi-
crobial effect [29] [30]. However, the inactivity may not be justified that no other bioactive agent present in the
extract. This is similar to the report of [31], who showed that no active substance exhibited its maximum activity
under laboratory experimental condition. Therefore, if there is increase in concentration these may enhance ac-
tivity.
Preliminary qualitative test is useful in the detection of bioactive principles and subsequently may lead to
drug discoveries and development [32] [33] conducted phytochemical screening of leaves of twenty eight woo-
dy species from different plants families in Nigeria and discovered the presence of tannins, alkaloids and flavo-
noids in all samples, thus these secondary metabolites seem to be cosmopolitan in plants but, in varying degrees
and type.
In this study, the presence of alkaloids, saponins, and glycoside were present in aqueous extract of both red
and white kola nut. However, saponin and steroid were denser in methanol extract of red kola nut. Anthraqui-
none and cardiac glycoside were not detected in both extracts of red and white C. nitida. This is similar to the
finding of [34] on some Niger Delta plants who observed the presence of flavonoid, tannins, alkaloids and sapo-
nin in large quantity. The presence of alkaloids and saponins might also explain the therapeutic properties of this
plant, since alkaloid-containing plant has been used by humans since ancient times for therapeutic and recrea-
tional pursoses [35].
4. Conclusions
Phytochemical investigation of Cola nitida traditionally used in Nigeria revealed the presence of medicinal na-
turals. kaemferol, quercetin and coumarin were identified by using simple TLC method. The qualitalitative
analysis also revealed the presence of alkaloids, flavonoids, saponin, tannins, steroids, volatile and balsam.
These result showed varying degrees of antibacterial activities on some odontopathogens.
The red kola aqueous and methanol extract impaired the growth of Proteus vulgans and Streptococcus angi-
nosus. These organisms help in plague formation therein, initiates different dental problems.
The C. nitida extract showed its potential in the impairment of growth of test isolates and can serve as an al-
ternative remedy for oral infections. This is a step forward for further evaluation of the plant for bio-remedies.
Recommendation
Based on the result of the study, the following recommendations are provided:
1) Considering the antibacterial activity of red Cola nitida further studies should be carried out on odontopa-
thogens and how best it can be recommended for use against dental infections.
2) The phenolic compounds present in C. nitida should be isolated, purified and tested to obtain their maxi-
mum therapeutic potentials.
3) Vigorous research work should be geared towards the investigating the antimicrobial potential of Cola ni-
tida, for harnessing the numerous volatile compounds from the plant for industrial uses to better the lots of hu-
man ity.
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