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Original Article
Chemical Analyses, Antimicrobial and Antioxidant Activities of
Extracts from Cola nitida Seed
Julius K. Adesanwo1*, Seun B. Ogundele1, David A. Akinpelu2 and Armando G. McDonald3
1Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria; 2Department of Microbiology, Obafemi Awolowo University,
Ile-Ife, Nigeria; 3Department of Forestry, Rangeland and Fire Sciences, University of Idaho, Moscow, ID 83843, USA
Abstract
Background and Objecves: Medicinal plants are the richest, cheapest and most readily available source of drugs,
nutraceucals and food supplements. Pharmaceucal industries sll rely largely on medicinal plants for interme-
diates due to their chemical diversies. This study, therefore, invesgated the chemical constuents, thermal
decomposion products and biological acvies of extract from seeds of Cola nida (the ‘kola nut’).
Methods: The pulverized seed was sequenally extracted with dichloromethane and methanol CH3OH. The ex-
tracts were analysed directly by Fourier Transform Infra-Red, electrospray ionizaon mass spectrometer and as
fay acid methyl ester and trimethylsilyl derivaves by gas chromatography-mass spectrometry (GC-MS). The
CH3OH extract was analysed by high-performance liquid chromatography for sugars. The intact and extracted
seed biomasses were analysed directly by pyrolysis GC-MS. For isolaon of chemicals and assessment of biologi-
cal acvity, a large scale CH3OH extracon was performed and the extract paroned with n-hexane, ethyl ac-
etate (EtOAc) and butanol. Fraconaon was done using various chromatographic techniques. Anmicrobial and
anoxidant acvies of the extract, fracons and isolated caeine were respecvely determined by the methods
of agar-well diusion and 2,2-diphenyl-1-picrylhydrazyl radical scavenging.
Results: Caeine and hexadecanoic acid were isolated from the EtOAc fracon while theobromine, caeine, cat-
echins, procyanidins, proanthocyanidins, sugars, fay acids, alcohols and sterols were idened in the extracts.
Multude (62) biomass degradaon products were idened in pyrolysed seed samples. The extract and frac-
ons showed varying acvies against most of the tested microbes, except against Shigella species, for which
neither the extract nor fracons elicited any response. The butanol fracon exhibited the highest anoxidant
acvity.
Conclusions: This report gives insight into the chemi-
cal constuents in Cola nida seed, details the ther-
mal decomposion constuents and establishes the
anmicrobial and anoxidant acvies of the seed
extract and fracons, thereby contribung to the
knowledge on the chemistry and pharmacology of
the genus.
Keywords: Cola nitida; Caffeine; n-Hexadecanoic acid; Catechin; Antimicrobial;
Antioxidant; GC-MS; ESI-MS.
Abbreviations: AGC, Acelerated Gradient Chromatography; 13CNMR, Carbon
13 Nuclear magnetic resonance spectroscopy; CND, Cola nitida dichloromethane
extract; CNM, Cola nitida methanol extract; IC50 value, concentration of samples
leading to 50% reduction of initial DPPH radical concentration; CDCl3, deuterated
chloroform; DCM or CH2Cl2, dichloromethane; DPPH, 2,2-diphenyl-1-picrylhydra-
zyl; ESI-MS, electrospray ionization mass spectrometer; EFAs, essential fatty acids;
EtOAc, ethyl acetate; FAME, fatty acid methyl ester; FAs, fatty acids; FTIR, fourier
transform infrared; GC-MS, gas chromatography-mass spectrometry; GRAM +ve,
Gram positive; GRAM −ve, Gram negative; HPLC, high-performance liquid chroma-
tography; IS, internal standard; CH3OH, methanol; 1HNMR, proton nuclear magnetic
resonance spectroscopy; Pyro GC-MS or Py GC-MS, pyrolysis gas chromatography-
mass spectrometry; Ag, silver; TLC, thin-layer chromatography; TMS, trimethylsilyl;
UV, Ultra- violet; ZnSe, Zinc Selenium.
Received: April 28, 2017; Revised: June 23, 2017; Accepted: June 28, 2017
*Correspondence to: Julius K. Adesanwo, Department of Chemistry, Obafemi
Awolowo University, Ile-Ife, Nigeria. Tel: +234-8030821561, E-mail: adesanwojk@
yahoo.com or julius08@oauife.edu.ng
How to cite this article: Adesanwo JK, Ogundele SB, Akinpelu DA, McDon-
ald AG. Chemical Analyses, Antimicrobial and Antioxidant Activities of Extracts
from Cola nitida Seed. J Explor Res Pharmacol 2017;2(3):67–77. doi: 10.14218/
JERP.2017.00015.
Introduction
Cola nitida (Vent) Schott et Endl, family Malyaceae, is a tree na-
tive to the rainforest of tropical West Africa. The seed or nut com-
monly called ‘kola nut’ is a popular stimulant in West Africa. It
is called “Obi gbanja” by Yorubas in western Nigeria, “Goro” by
Hausas in the north, and “Oji” by Ibos in the east.1 It is mainly used
for nutritional purpose, eaten across cultures in West Africa, and
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Adesanwo JK.. et al: Medicinal activities of Cola nitida seed
J Explor Res Pharmacol
has great social, religious and medicinal importance as well.2 Kola
nut extracts are used in the manufacture of various non-alcoholic
beverages, soft drinks, wines, chocolate and sweets.3–5
Previous researchers have reported a number of secondary me-
tabolites from the kola nut, including caffeine, theobromin, cate-
chin, epicatechin, procyanidins and proanthocyanidins.6–8 Aliphat-
ic and heterocyclic amines in different species of cola have been
reported as well,9 and quinic, tannic and chlorogenic acids have
been found as present in the kola nut.10 Caffeine was the major
alkaloid identied in Cola seeds and was considered as one of the
signature compounds due to its concentration range.6
Knebel and Higler showed that fresh kola nut contained a glu-
coside named “kolanin”, reporting that it is readily hydrolysed or
split into glucose and caffeine in ripe or dried fruit.7 Kola nuts are
rich in xanthine alkaloids, such as theobromine, caffeine, kolatin
and kolanin. While caffeine stimulates the body, kolatin stimu-
lates the heart.11,12 These constituents (caffeine, theobromine, and
theophylline) in kola nut extract have been shown to contribute
to the anti-photodamage effect, including wrinkles, and to elicit
antioxidant and anti-aging activities, including suppression of UV-
induced erythema, and to decrease skin roughness and scaling by
topical or oral application.13
Nowadays, pathogenic microorganisms are developing resist-
ance to existing antibiotics at an alarming rate. Extract of the
leaves, root and stem bark of Cola nitida are extensively used in
folk medicine.6 Different parts of Cola nitida have been exploit-
ed for different biotechnological applications. For instance, cola
pods have been used for production of fructosyltransferase, an
important enzyme for the synthesis of fructooligosaccharides,14
and most recently for the biosynthesis of silver (Ag) nanoparticles
and silver-gold alloy nanoparticles for diverse biomedical appli-
cations,15–19 such as antimicrobial, antioxidant, anticoagulant and
thrombolytic activities.
Similarly, the seed and seed shell extracts of Cola nitida have
been used for green synthesis of Ag nanoparticles, showing pro-
found antibacterial activities.20 In Uganda, extracts of the seed and
leaves are used in treatment of sexual and erectile dysfunctions.21
In Nigeria, methanol extract of the seed is used against emesis and
migraine.22 The aqueous extract of the seed has also been used as
avouring in carbonated drinks.23 Kola nut extract also demon-
strates good protective property against red cell degradation.9
The aims of this study were to: (i) determine the composition of
kola nut dichloromethane (CH2Cl2) and CH3OH extracts by a com-
bination of electrospray ionization-mass spectrometry (ESI-MS)
and gas chromatography-mass spectrometry (GC-MS) analyses;
(ii) determine the antimicrobial and antioxidant activity of the ex-
tracts; and (iii) isolate the active principle component(s).
Experimental
General
All thin-layer chromatography (TLC) analyses were performed at
room temperature using pre-coated plates (silica gel 60 F254 0.2
mm; Merck). Detection of spots was achieved by staining with
iodine crystals and exposure to ultraviolet light (254 and 366
nm). Melting point determination was carried out using a Gallen-
kamp apparatus. Accelerated gradient chromatography (Baeck-
strom Separo AB) was carried out using silica gel (Kieselgel 60G
0.040–0.063 mm) and column chromatography using silica gel,
with a 60–200 mesh for fractionation. Proton Nuclear magnetic
resonance (1HNMR) spectroscopic data were recorded on a NMR
machine (Agilent Technologies) at 400 MHz and at 100 MHz for
13CNMR. Chemical shifts of signals were reported in parts per mil-
lion (ppm).
Collection of plant material
The plant material (kola nut) was collected at Alaro Farm Settle-
ment, Ile-Ife, identied and authenticated at the Department of
Pharmacognosy, Obafemi Awolowo University (Voucher Number
FPI 2052).
Sample preparation and extraction
The seeds were dried at 60 °C for 48 h, and milled to particle size
of 1 mm. Moisture content was determined in duplicate before
extraction. The plant material was Soxhlet extracted (40 g, in du-
plicate) rst with dichloromethane (DCM) (150 mL) for 24 h and
then with CH3OH (150 mL) for 48 h. The extracts were concen-
trated in vacuo at 40 °C to give yield of 0.77% and 17.62% for the
DCM and CH3OH extracts, respectively.
Fourier transform infrared (FTIR) spectroscopic analysis
The functional groups in the milled samples and extracts, and the
extracted biomasses were determined by FTIR spectroscopy using
a Nicolet iS5 spectrometer (ThermoScientic) using a ZnSe at-
tenuated total reection probe. Spectra were collected in duplicate.
The absorbance spectra were baseline-corrected and averaged us-
ing Omnic v9.0 software (ThermoScientic).24,25
ESI-MS experiment
The samples (about 1.0 mg each of DCM and CH3OH extracts, in
duplicate) were added to CH3OH (1 m:) and acetic acid (10 µL).
The mixture was subjected to sonication to ensure total dissolu-
tion. Electrospray mass spectrometric analyses were performed on
a 5989A device (Hewlett-Packard) equipped with an electrospray
interface 59987A. Nitrogen was used as nebulizing gas, at a pres-
sure of 50 psi and a temperature of 300 °C. Sample analysis was
performed by direct infusion in ESI-MS using a syringe pump
(Harvard Apparatus) at a ow-rate of 10 mL/min. Mass spectra
were acquired in scan mode detection, and ESI-MS conditions
were optimized using available standards.
GC-MS of fatty acid methyl ester (FAME) derivatives
Extracts (about 2.0 mg, in duplicate) were prepared by adding
a solution (2 mL) of CH3OH/H2SO4/CHCl3 (1.7:0.3:2.0 v/v) in
which the CHCl3 contained 1-naphthaleneacetic acid (100 µg/
mL) as an internal standard. The mixture was heated for 90 min
at 90 °C in a sealed vial. Water was added to the mixture and the
CHCl3 layer was removed, dried and transferred to a GC vial.
The prepared FAME derivatives were analysed by electron impact
ionization GC-MS on a Focus ISQ (ThermoScientic) with a ZB5
column (30 m × 0.25 mm; Phenomenex) and a temperature pro-
le of 40 °C (1 min) to 320 °C (10 min) at 5 °C/min. The eluted
compounds were identied with authentic standards (C12 to C20
fatty acids) and by spectral matching with the NIST 2008 spectral
library.
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GC-MS of extracts for trimethylsilyl (TMS) derivatives
Extracts (about 1.0 mg, in duplicate) were weighed in GC vials, to
which CH2Cl2 (1 mL) containing anthracene as an internal stand-
ard (IS; 50 µg/mL) was added. The samples were silylated with
addition of N,O-bis(trimethylsilyl)-triuoro-acetamide (BSTFA)
containing 1% trimethylchlorosilane (TMCS; 50 µL) and pyridine
(50 µL) and heated for 30 min at 70 °C or longer until the solu-
tion became clear. The prepared TMS derivatives were analysed by
GC-MS (as described above).
Analytical pyro (Py)-GC-MS of samples
Analytical Py-GC-MS was carried out on the milled sample before
and after extraction using a Pyrojector II (SGE Analytical Science)
at 500 °C in He coupled to a GC-MS (FOCUS-ISQ; ThermoScien-
tic) instrument operating in the electron impact ionization mode.
The compounds were separated on the ZB5-MS capillary column
(30 mm × 0.25 mm; Phenomenex) with temperature programmed
to be 50 °C to 3000 °C, at 5 °C min−1. The eluted compounds
were identied by their mass spectra, authentic standards, and with
NIST 2008 library matching.
High-performance liquid chromatography (HPLC) analysis
Sugars were quantied by HPLC using a Rezex ROA column (7.8
mm × 30 cm; Phenomenex) and a Waters HPLC (Waters Corp.)
equipped with differential refractive index detector (Shodex SE61;
Showa Denko America, Inc.), on elution with 0.01 M H2SO4 (0.5
mL/min) at 65 °C. The CH3OH extract (10 mg) was dissolved in
0.01 M H2SO4 (5 mL), centrifuged and the supernatant ltered
(0.45 µm). Data was acquired and analysed using the N2000 chro-
matography software (Science Technology Inc.). The sugar con-
tents were determined from peak area using the external standard
method with standard sugars (glucose, fructose, xylose, myo-ino-
sitol, sucrose, maltose and turanose).
Isolation of chemical compounds
The seeds (2 kg) were extracted with CH3OH and concentrated
in vacuo to obtain CH3OH extract (80 g, 4% yield). The crude
extract was suspended in water and partitioned with n-hexane,
ethyl acetate (EtOAc) and n-butanol to give respective fractions.
The EtOAc fraction was subjected to Acelerated Gradient Chro-
matography (AGC) fractionation. Fractions 24 through 76 were
bulked together and separated by column chromatography (silica
gel 60–200 mesh) and elution was monitored by TLC. Fractions 36
through 67 were evaporated to yield a white crystalline solid (caf-
feine) with melting point of 230–233 °C. Pooled AGC fractions
8 through 23 were concentrated and a yellow viscous solid was
obtained, which was further puried by column chromatography,
yielding a colourless solid (hexadecanoic acid) with minor impuri-
ties as accessed by TLC. Both compounds were characterized by
NMR spectroscopy (400 MHz; Agilent).
Antimicrobial assay
The antimicrobial activity of CH3OH extract, solvent fractions and
isolated caffeine were determined using the agar-well diffusion
method.26,27 The bacteria were grown in a nutrient broth before
use, while the fungi were grown on potato dextrose agar medi-
um until they sporulated, at which time they were harvested. The
standardized bacteria suspension was spread on Muller-Hinton
agar and allowed to set. Wells were then bored with a 6-mm borer
and lled with the respective sample solutions at 10 mg/mL, with
ampicillin and nystatin added at 1 mg/mL, which was followed by
incubation at 37 °C for 24 hrs. The fungal plates were incubated at
25 °C for 96 hrs.
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging an-
tioxidant assay
Quantitative antioxidant activity was determined spectropho-
tometrically as described.28 Reactions were carried out in test
tubes, and each of the solvent fractions and CH3OH extracts were
tested at varying concentrations (µg/mL). Initial stock solutions
of 1 mg/mL were prepared for the various plant extracts. The
following nal concentrations were prepared based on the pre-
liminary qualitative TLC antioxidant screening: 60, 30, 15, 7.5,
3.75 and 1.875 µg/mL for the crude extract; 50, 25, 12.5, 6.25
and 3.13 µg/mL for n-butanol fraction; 30, 15, 7.5, 3.75 and 1.88
µg/mL for the EtOAc fraction; and 2000, 1000, 500, 250, 125 and
62.5 µg/mL for the n-hexane extract and caffeine from the stock
solution. A 0.1 mM DPPH radical solution in CH3OH (1 mL) was
added to 1 mL of the concentration series for each sample tested,
in triplicate, and allowed to react at room temperature in the dark
for 30 min. The negative control was prepared by adding 0.1 mM
DPPH radical solution (1 mL) to 1 mL of methanol, in triplicate,
and absorbance was measured at 517 nm. The percentage anti-
oxidant activity (%AA) values of test samples were calculated
from the absorbance using the formula:% AA={Xcontrol−Xtest
Xcontrol}×100where Xcontrol is the absorbance of the negative
control, Xtest is the absorbance of test samples concentrations.
Ascorbic acid (vitamin C) was used as the standard antioxidant
Fig. 1. Fourier transform infrared spectra of Cola nida seed. (A) Pow-
dered unextracted material, (B) Cola nida dichloromethane (CND) ex-
tract, (C) Cola nida methanol (CNM) extract, (D) Extracted biomass.
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Adesanwo JK.. et al: Medicinal activities of Cola nitida seed
J Explor Res Pharmacol
agent.
The IC50 value (i.e. the concentration of the test samples leading
to 50% reduction of the initial DPPH radical concentration) was
calculated from the separate linear regression of plots of the mean
percentage of antioxidant activity against the concentration of the
test samples in µg/mL.
Results and discussion
Extraction techniques
Soxhlet extraction of the kola nut with CH2Cl2 and CH3OH af-
forded yields of 0.77% and 17.6%, respectively. In comparison,
large-scale cold extraction of the kola nut afforded a low yield of
4%. Therefore, only the Soxhlet extracts were analysed in detail
by a combination of GC-MS and ESI-MS. However, the batch
CH3OH extract was used for antioxidant and antimicrobial assess-
ment.
FTIR spectroscopic analysis
FTIR spectroscopy was used to investigate the functional groups
in the sample and extracts. The spectra (Fig. 1) showed the pres-
ence of strong O–H stretching vibration at 3340 cm−1 correspond-
ing to hydroxyl groups in all, but quite pronounced in Figure 1A,
C, and D. This band is less pronounced in Figure 1B, because the
extract contained a majority of low polar compounds, whereas
Figure 1C and D contained high polar phenolic compounds and
lignocellulose respectively and containing multiple H-bonding.
The absorption at just above 3000 cm−1 in Figure 1a is due to an
aromatic C–H stretch of polyphenols and aromatics; it is virtu-
ally absent in Figure 1b but noticeable in Figure 1C and D. Simi-
larly, the substituted aromatic C–H bend band at 600–900 cm−1
is almost absent in Figure 1b but very conspicuous in Figure 1A,
C and D, indicating a low proportion of aromatics in the CND
extract.
On the other hand, the C–H sp3 stretching vibration, just below
3000 cm−1 and characteristic of long chain fatty acids [–(CH2)
n–CH3], is more conspicuously prominent in Figure 1B than in
Figure 1C and D. Carbonyl stretching absorption of carboxylic
acid aldehydes and ketones is very low in Figure 1D, as most car-
bonyl compounds had already been removed through the process
of extraction. The absorption band at 1000–1200 cm−1 that was
due to C–O stretch of esters (glycosides and cellulose) was most
intense in Figure 1C and D but low in Figure 1B. These data show
that FTIR can be used to monitor and assess the effectiveness of
the extraction process, apart from facilitating quantitative evalu-
Fig. 2. Electrospray ionizaon mass spectra of Cola nida extracts posive mode. (A) Cola nida methanol extract, (B) Cola nida dichloromethane extract.
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ations.
Electrospray mass spectrometric analysis of extracts
ESI-MS is a powerful analytical tool to rapidly analyse extracts
and isolated fractions without chromatographic separation.29–31
Moreover, ESI-MS makes it possible to discriminate between var-
ious avonoid classes, provide information on the glycosylation
position and characterize saponins.32–34 Therefore, ESI-MS was
employed to directly analyse the CH2Cl2 and CH3OH extracts in
positive (Fig. 2) and negative (Fig. 3) ion modes.
The positive ion ESI-MS for both extracts showed the larg-
est peak at m/z 195 (100% intensity), which was due to caffeine
([M+H]+). In the MeOH extract (Fig. 2A), the tentative peak assign-
ments are as follows: 181 ([M+H]+, theobromine); 295 (unknown);
and 391 (unknown). For the CH2Cl2 extract (Fig. 2B), the tentative
peak assignments are: 193 ([M+H]+, quinic acid); 313 ([M+Na]+,
catechin), 355 ([M+H]+, chlorogenic acid); and m/z 281, 294, 377
and 426 (unknowns). The negative ion ESI-MS for both extracts
(Fig. 3) showed a multitude of peaks. For the CH3OH extract (Fig.
3A), the tentative assignments are: m/z 289([M-H]−, catechin);
m/z 577 and 578 (procyanidins B1, B2 [oligomeric catechins] and
naringin avonoids); and m/z 1153 and 1154 (proanthocyanidins).
There were many unidentied peaks. Niemenak et al.6 also reported
that HPLC of Cola nitida extract had 11 unidentied compounds.
GC-MS of extracts of FAME derivatives
It is well known that fatty acids (FAs), especially the essential fat-
ty acids (EFAs), are important for optimal functioning of the body.
They regulate such bodily functions as heart rate, blood pressure,
blood clotting and fertility. They also participate in immune sys-
tem inammation against harmful waste products. Balance of
EFAs is important for good health and normal development of
humans.35,36 FAs occur widely in natural fats and dietary oils and
they play important roles as nutritious substances and metabolites
in living organisms.37 With these important biological activities of
FAs, those present in the extracts as free or glycerides were con-
Fig. 3. Electrospray ionizaon mass spectra of Cola nida extracts negave mode. (a) Cola nida methanol extract, (b) Cola nida dichloromethane extract.
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Adesanwo JK.. et al: Medicinal activities of Cola nitida seed
J Explor Res Pharmacol
verted to FAME derivatives for identication and quantication.
The CH2Cl2 and CH3OH extracts were derivatised into FAME
and analysed by GC-MS. The CH2Cl2 extract contained FAs rang-
ing from C16 to C19. Palmitic acid (C16: 0.447 mg/g), linoleic
acid (C18: 0.198 mg/g), oleic acid (C18: 0.553 mg/g), stearic acid
(C18: 0.034mg/g), 8,9-methylene-heptadec-8-oic acid (C18: 0.112
mg/g), 10,12-octadecadienoic acid (C18: 0.040 mg/g), 8-oxohexa-
decanoic acid (C16: 0.275 mg/g) and 9,10-methylene-octadec-
9-oic acid (C19: 0.032 mg/g) were the major FAs detected. The
FAs constitute about 73% of the extract. Furthermore, caffeine,
fatty alcohols and sterols were also detected (Table 1). The CH3OH
extract did not contain any FAs, only caffeine.
GC-MS of extracts of TMS derivatives
Trimethylsilylating reagent is routinely used to derivatise rather
non-volatile compounds, such as certain alcohols, phenols, or car-
boxylic acids, by substituting a TMS group for a hydrogen in the
hydroxyl groups on the compounds. Both extracts were deriva-
tised to TMS ethers/esters to conduct the analyses for non-polar
and polar components using GC-MS. The results for the CH2Cl2
and CH3OH extracts as their TMS derivatives are given in Tables
2 and 3.
Caffeine (17.9% and 3.85% in CH3OH and CH2Cl2 extracts, re-
spectively) was predominant, as observed by Niemenak et al.6 Cat-
echin was the dominant avonoid in the kola seed. Other identied
constituents of CH3OH extract included: sugars (45%); catechin
(13%); malic acid and glycerol (Table 2). The presence of glucose,
fructose and sucrose in the CH3OH extract was detected by both
GC-MS and HPLC analyses. The CH2Cl2 extract consist mainly of
FFAs (Table 3), and this observation is consistent with the results
of FAME analysis. Other compounds found in this extract were
alkaloids and sterols.
Table 2. GC-MS of Cola nida CH3OH extracts of TMS derivaves
Compound Molecular formula Class Molecular weight Retenon me (min) % Extract
Glycerol TMS C12H32O3Si3Alcohol 308 18.11 0.2
Malic acid TMS C13H30O5Si3Acid 350 23.65 1.5
Anthracene (IS) C14H10 178 30.14
Fructose TMS5C19H46O6Si4Sugar 540 30.70 2.2
Caeine C8H10N4O2Alkaloid 194 31.23 17.9
Glucose-TMS-5 C21H52O6Si5Sugar 540 32.77 0.7
D-Turanose-TMS-7 C33H78O11Si7Sugar 846 45.65 2.9
Sucrose-TMS8C36H86O11Si8Sugar 918 45.75 39.5
Catechin-TMS-5 C30H54O6Si5Flavonoid 650 48.74 12.9
Table 1. GC–MS FAME derivaves of CND extract
S/No Compound Molecular formula Class Molecular
weight
Retenon
me (min) % Extract
1 Naphthalene acec acid (IS) C13H12O2200 27.34
2 Caeine C8H10N4O2Alkaloid 194 30.43 1.7
3Palmic acid C17H34O2FA 270 31.83 19.4
4Linoleic acid C19H34O2FA 294 35 8.6
5 Oleic acid C19H36O2FA 296 35.11 24.0
6 Stearic acid C19H38O2FA 298 35.6 1.5
7 8,9-Methylene-8-heptadecenoic acid C19H34O2FA 294 36.24 4.9
810,12-Octadecadienoic acid C19H34O2FA 294 36.39 1.7
9 7-(Tetrahydro-2H-pyran-2-yloxy)-2-octyn-1-ol C13H22O3Alcohol 226 36.63 1.3
10 2-Octylcyclopropene-1-heptanol C18H34O Alcohol 266 36.79 1.7
11 8-Oxohexadecanoic acid C17H32O3FA 284 37.25 12.0
12 Tetrahydropyran-2-yl ether of 7-dodecynol C17H30O2Alcohol 266 37.46 1.2
13 9,10-Methylene-9-octadecenoic acid C20H36O2FA 308 37.99 1.4
14 1-Ethyl-(1,1-dimethylethyl)-methoxycyclohexan-1-ol C13H26O2Alcohol 214 38.97 3.2
15 Sgmastan-3,5-diene C29H48 Sterol 396 50.12 0.2
16 Sitosterol acetate C31H52O2Sterol 456 52.16 0.7
17 Sitosterol C29H50OSterol 414 52.86 0.6
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Analytical Py-GC-MS of samples
Direct analysis of the kola nut and extracted nut were performed
by analytical Py-GC-MS. The identities of the products are given
in Table 4. The main compounds in the kola nut were caffeine
(22.5%), CO2 (12.8%), methyl acetate (6.3%), acetic formic anhy-
dride (5.9%), levoglucosan (5.4%), N-methyl ethylamine (4.9%),
1,2-cyclopentanedione (3.4%) and pyrocatechol (3.0%). Aliphatic
and heterocyclic (pyrolidine, alstonine and xanthenes) amines were
identied. This nding is in accordance with the report by Atawodi
et al.9 Py-GC-MS of the extracted kola nut showed the presence
of 22 compounds (Table 4). The main compounds identied were:
CO2 (17%), acetol (11%), 1,2-benzenediol (11%), 1,2-cyclopentan-
edione (10%), acetic acid (7%), butylamine (5%) and caffeine (5%).
It is noteworthy that alkanes, alkenes ethers and steroids/sterols
were not identied by the Py-GC-MS of the extracted biomass;
this may be explained as due to exhaustive removal of the relative-
ly low polar compounds during the process of extraction. Analyti-
cal pyrolysis experiment showed caffeine as the major constituent
of Cola nitida. Maltol, a naturally occurring organic compound,
is primarily used as a avour enhancer.10 Its presence in the Cola
nitida seed may be contributory to the application of the seed in the
manufacture of soft drinks.3–5
Structure elucidation
Compound 1 (caffeine) was obtained as a white crystalline solid,
with a melting point of 230–233 °C. 1HNMR (400 MHz, CDCl3,
δ ppm): 7.46 (1Hs, 8H), 3.95, 3.54, 3.36 (s, 3H at 1,3,7). 13CNMR
(100 MHz, CDCl3, δ ppm): 155.3 and 151.6 (C6 and C2 respec-
tively), 148.6, 141.5, 107.5 (olenic C8, C4 and C5), 33.5, 29.7
and 27.9 (methyl groups). These correlate with the literature data
for caffeine.
Compound 2 (n-Hexadecanoic acid): 1HNMR (400 MHz, CDCl3,
δ ppm): 2.24–1.21 (CH2 protons), 0.78 (CH3 protons). 13CNMR
(100 MHz, CDCl3, δ ppm): 179 (C=O); 34.15–22.64 (CH2 carbon
atoms); 15.02 CH3 group.
Antimicrobial activity
The kola nut CH3OH extract and fractions, and isolated caffeine
showed varying degrees of inhibitory activities against the tested
bacterial and fungal strains (Fig. 4 and Supplementary Table S1).
Their activity was more pronounced against Gram-positive bacte-
ria than Gram-negative bacteria. This could be a result of the mor-
phological differences between these microorganisms. The Gram-
positive bacteria have an outer peptidoglycan layer, which is not an
effective permeability barrier, making these microorganisms more
susceptible to the compounds under investigation; meanwhile, the
Gram-negative bacteria have an outer phospholipidic membrane
that contains LPSs, making the cell wall of these microorganisms
impermeable.38–40.
These microorganisms are implicated in the pathogenesis of hu-
man infections. The result obtained showed the EtOAc fraction
as having the highest antimicrobial activity against most of the
organisms compared to other fractions. However, the EtOAc frac-
tion was not active against Pseudomonas Spp., Clostridium sporo-
genes, Corynebacterium pyogenes, Shigella Spp. and Candida
albicans. The activity of the extract indicated CH3OH as a good
solvent for preparation of extracts for antimicrobial assay.41,42 The
antimicrobial activity of the CH3OH extract can be attributed to
synergistic effect of compounds in the extract. Isolated caffeine
(compound 1) demonstrated higher activity than n-butanol against
Bacillus Spp., Escherichia coli and Aspergillus niger. Extract and
fractions did not show activity against Shigella Spp. Caffeine dem-
onstrated appreciable antimicrobial activity against Bacillus cere-
us, Escherichia coli, Pseudomonas vulgaris and the fungi.
Antioxidant activity
The DPPH radical antioxidant activity showed the ability of the
kola nut CH3OH extracts and fractions, and isolated caffeine to
reduce DPPH radicals through the transfer of acidic labile protons
by a free radical mechanism. None of the extracts compares sig-
nicantly with that of ascorbic acid. However, the EtOAc and bu-
tanol fractions exhibited good DPPH antioxidant activity. The IC50
values decreased in this order: ascorbic acid (3.2 ± 0.05 µg/mL),
butanol extract (9.8 ± 0.5 µg/mL), EtOAc extract (15.1 ± 0.7 µg/
mL), CH3OH extract (22.7 ± 1.7 µg/mL), hexane extract (321 ± 7
µg/mL) and caffeine (1370 ± 19 µg/mL).
Future research direction
A number of chemical compounds have been reported from the
Table 3. GC-MS of Cola nida CH2Cl2 extracts of TMS derivaves
Compound Molecular formula Class Molecular weight Retenon me (min) % Extract
Nonanoic acid TMS C12H26O2Si FA 230 20.14 0.82
Octanedioic acid TMS C14H30O4Si2FA 318 28.27 0.26
Anthracene C14H10 178 30.14
Caeine C8H10N4O2Alkaloid 194 31.32 2.95
Palmic acid TMS C19H40O2Si FA 328 35.07 1.64
Linoleic acid TMS C21H40O2Si FA 352 37.94 5.95
Oleic acid TMS C21H42O2Si FA 354 38.11 1.89
Stearic acid TMS C21H44O2Si FA 356 38.58 0.14
Linolenic acid TMS C21H38O2Si FA 350 39.57 1.08
Sgmasterol TMS C32H56OSi Sterol 484 53.46 0.20
Sitosterol TMS C32H58OSi Sterol 486 54.13 2.05
DOI: 10.14218/JERP.2017.00015 | Volume 2 Issue 3, August 201774
Adesanwo JK.. et al: Medicinal activities of Cola nitida seed
J Explor Res Pharmacol
Table 4. Py-GC-MS of unextracted and extracted powdered Cola nida seed
Compound Molecular
formula
Molecular
weight
Retenon
me (min)
Kola
nut, %
Extracted
nut, %
CO2CO244 1.12 12.8 17.4
N-Methyl ethylamine C3H9N 59 1.30 4.9
Butylamine C4H11N 72 1.38 4.9
Acec acid C3H4O360 1.64 5.9 6.5
Pentanone C5H10O86 1.65 4.6
Acetol C3H6O274 1.97 6.3 11.3
Unknown 92 3.17 1.5
Butanedial C4H6O286 3.36 1.7 3.4
Methyl pyruvate C4H6O3102 3.49 1.7 1.1
Butanedial C4H6O286 3.63 2.7
Unknown C4H4O284 3.66 1.0 1.1
2-Oxo-3-cyclopentene-1-acetaldehyde? C7H8O2124 4.37 1.3 3.2
2-Furfuryl alcohol C5H6O298 4.90 2.2 4.4
Unknown C5H8O3116 5.16 1.1
2(5H)-Furanone C4H4O284 6.34 2.1 3.9
1,2-Cyclopentanedione C5H6O298 6.67 3.4 9.9
1-Methyl-1-cyclopenten-3-one C6H8O96 7.85 1.3
4-Methyl-5H-furan-2-one + Unknown C5H6O298 + 110 8.09 0.6
Phenol C6H6O94 8.28 XX
2 Hydroxy-3-methyl-2-cyclopenten-1-one C6H8O2112 9.54 1.4 5.4
2,3 Dimethyl-2-cyclopenten-1-one C7H10O110 9.85 0.3
Unknown 116 10.00 0.8
2-Methyl phenol C7H8O2108 10.40 0.7
3-Methyl-phenol C7H8O2108 10.99 0.7
Unknown 57? 11.51 1.9 0.9
3 Hydroxy-2-methyl-4H-pyran-4-one (Maltol) C6H6O3126 12.03 0.5 0.7
3-Ethyl-2-hydroxy-2-cyclopenten-1-one C7H10O2126 12.21 0.4 1.5
4-Hydroxy-3-methyl-(5H)-furanone or 3-methyl-2,4(3H,5H)-furandione C5H6O3114 12.33 0.5
Dihydro-2H-pyran-3(4H)-one + unknown C5H8O2100+128 13.16 0.8
Ethyl/dimethyl phenol + 3,5-dihydroxy-2-methyl-(4H)-pyran-4-one C11H18O7122+142 13.66 0.7
Benzene diol + 3,5 dihydroxy-2 methyl-4-pyrone C6H6O2 + C6H6O4110 + 142 14.13 0.7
5-Hydroxymethyldihydrofuran-2-one C5H8O3116 14.42 0.8
1,2-Benzenediol C6H6O2110 14.67 3.1 11.3
1,4:3,6-Dianhydro-hexose C6H8O4144 14.86 1.0 1.9
Coumaran C8H80120 15.13 0.5
5 Hydroxymethyl-2-furaldehyde C6H6O3126 15.47 0.6
4-Methyl catechol C7H8O2124 17.21 1.5 2.0
Syringol C8H10O3154 18.69 0.5
Levoglucosan C6H10O5162 22.80 5.4
Caeine C8H10N4O2194 30.36 22.3 5.2
Theobromine C7H8N4O2180 30.75 1.8 1.0
DOI: 10.14218/JERP.2017.00015 | Volume 2 Issue 3, August 2017 75
Adesanwo JK.. et al: Medicinal activities of Cola nitida seed J Explor Res Pharmacol
seed of Cola nitida; however, current ndings revealed there are
yet more compounds that remain to be identied. The present work
has identied more chemical compounds from Cola nitida. The
presence of a number of FAs revealed in this publication serves as
evidence of the potential varied biological applications of the seed.
Recent reports on the biotechnological/nanotechnological applica-
tions of products of Cola nitida have opened the door to a new di-
mension of research activities involving the genus, especially with
respect to waste management. The kola nut pod, which hitherto was
considered a waste product, is being converted to useful products
– turning waste into wealth. With the aid of well-planned quantita-
tive HPLC analysis of the methanol extract of Cola nitida, some of
the unknown compounds can be isolated and structural elucidation
carried out. The chemicals identied in the kola nut can be deriva-
tised by functional group inter-conversion to more potent and less
harmful compounds for both industrial and medicinal applications.
Conclusions
Previous research on Cola nitida have established the presence of
xanthine alkaloids, catechins, epicatechins, anthocyanidins and
its oligomers (proanthocyanidins). However, from the ESI-MS
experiments, we discovered there are more compounds yet to be
identied and structurally elucidated. In this report, the FAs and
sugars present in Cola nitida have been identied using the GC-
MS (FAME and TMS derivatives of the extracts). With analytical
Py-GC-MS experiment, the constituents of thermal decomposi-
tion of Cola nitida were also identied and compared with those
of extracted biomass. In the antimicrobial assay, CH3OH extract
and solvent fractions showed no activity against Shigella Spp. but
were effective against most of the tested microbes. The N-hexane
fraction was ineffective in both antimicrobial and antioxidant as-
says. Caffeine was ineffective in antioxidant assay, but exhibited
appreciable activity against Bacillus cereus, Escherichia coli and
Aspergillus niger. The butanol fraction displayed higher antioxi-
dant activity than the CH3OH extract, which might be due to the
higher concentration of phenolic compounds in the butanol frac-
tion.
Acknowledgments
The corresponding author wishes to acknowledge the Tertiary
Education Trust Fund (TETFund) Conference grant from Obafemi
Awolowo University to present part of this report at the 4th Interna-
tional Pharma & Clinical Pharmacy Congress, November 07–09,
2016, Las Vegas, Nevada, USA.
Compound Molecular
formula
Molecular
weight
Retenon
me (min)
Kola
nut, %
Extracted
nut, %
Palmic acid C16H32O2256 32.36 1.5 0.8
Linoleic acid C18H32O2280 35.44 1.0
Oleic acid C18H34O2282 35.62 0.9 0.5
Stearic acid C18H36O2284 36.01 0.3
C18:2 C18H32O2280 36.67 0.3
C19:2 C19H34O2294 37.16 0.4
Methyl-2,3-dicyano-3- [4-dimethylamino)phenyl]-2-propenoate C14H13N3O2255 43.57 0.6
Squalene C30H50 410 46.34 0.4
Cholestadiene C27H44 368 47.28 0.1
Unknown steroid 344 47.50 0.1
Sgmastan-3,5-diene C29H48 396 49.89 0.3
Nortrachelogenin C20H22O7374 50.77 0.2
6,9,10-Trimethoxy-12H-[1]benzopino[2,3,4-ij]isoquinoline (oxocularine) C19H17NO4323 51.17 0.4
6,16 Dimethylpregna-1,4,6-triene-3,20-dione C23H30O2338 51.58 0.3
Sgmasterol C29H48O412 51.94 0.2
3,4,5,6-Tetrahydro alstonine C21H24N2O3352 52.26 0.4
Sitosterol C29H50O414 52.67 0.5
Table 4. Py-GC-MS of unextracted and extracted powdered Cola nida seed - (connued)
Compound 1. 1,3,7-trimethyl-1 H-purine-2,6(3H,7H)-dione (Caeine). Compound 2. n-Hexadecanoic acid.
DOI: 10.14218/JERP.2017.00015 | Volume 2 Issue 3, August 201776
Adesanwo JK.. et al: Medicinal activities of Cola nitida seed
J Explor Res Pharmacol
Conict of interest
The authors have no conict of interests related to this publication.
Author contributions
Originator of the research idea and supervising the work (JKA),
involving in isolation and antimicrobial experiments (SBO), as-
sisting in the antimicrobial study (DAA), involving in the chemi-
cal/instrumental analyses (AGM).
Supporting Information
Supplementary material for this article is available at https://doi.
org/10.14218/JERP.2017.00015.
Table S1. In vitro antimicrobial activity of the extracts and com-
pound 1 (caffeine) against selected microbes.
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