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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
 Author information
Journal of Exploratory Research in Pharmacology   2017;2(3):67-77

doi: 10.14218/JERP.2017.00015


Background and Objectives

Medicinal plants are the richest, cheapest and most readily available source of drugs, nutraceuticals and food supplements. Pharmaceutical industries still rely largely on medicinal plants for intermediates due to their chemical diversities. This study, therefore, investigated the chemical constituents, thermal decomposition products and biological activities of extract from seeds of Cola nitida (the ‘kola nut’).


The pulverized seed was sequentially extracted with dichloromethane and methanol CH3OH. The extracts were analysed directly by Fourier Transform Infra-Red, electrospray ionization mass spectrometer and as fatty acid methyl ester and trimethylsilyl derivatives 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 isolation of chemicals and assessment of biological activity, a large scale CH3OH extraction was performed and the extract partitioned with n-hexane, ethyl acetate (EtOAc) and butanol. Fractionation was done using various chromatographic techniques. Antimicrobial and antioxidant activities of the extract, fractions and isolated caffeine were respectively determined by the methods of agar-well diffusion and 2,2-diphenyl-1-picrylhydrazyl radical scavenging.


Caffeine and hexadecanoic acid were isolated from the EtOAc fraction while theobromine, caffeine, catechins, procyanidins, proanthocyanidins, sugars, fatty acids, alcohols and sterols were identified in the extracts. Multitude (62) biomass degradation products were identified in pyrolysed seed samples. The extract and fractions showed varying activities against most of the tested microbes, except against Shigella species, for which neither the extract nor fractions elicited any response. The butanol fraction exhibited the highest antioxidant activity.


This report gives insight into the chemical constituents in Cola nitida seed, details the thermal decomposition constituents and establishes the antimicrobial and antioxidant activities of the seed extract and fractions, thereby contributing to the knowledge on the chemistry and pharmacology of the genus.


Cola nitida, Caffeine, n-Hexadecanoic acid, Catechin, Antimicrobial, Antioxidant, GC-MS, ESI-MS


Cola nitida (Vent) Schott et Endl, family Malyaceae, is a tree native to the rainforest of tropical West Africa. The seed or nut commonly 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 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 metabolites from the kola nut, including caffeine, theobromin, catechin, epicatechin, procyanidins and proanthocyanidins.6–8 Aliphatic 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 identified 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 glucoside 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 stimulates 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 resistance 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 exploited 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 applications,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 profound 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 flavouring in carbonated drinks.23 Kola nut extract also demonstrates 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 combination of electrospray ionization-mass spectrometry (ESI-MS) and gas chromatography-mass spectrometry (GC-MS) analyses; (ii) determine the antimicrobial and antioxidant activity of the extracts; and (iii) isolate the active principle component(s).



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 Gallenkamp apparatus. Accelerated gradient chromatography (Baeckstrom 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 million (ppm).

Collection of plant material

The plant material (kola nut) was collected at Alaro Farm Settlement, Ile-Ife, identified 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 duplicate) first with dichloromethane (DCM) (150 mL) for 24 h and then with CH3OH (150 mL) for 48 h. The extracts were concentrated 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 (ThermoScientific) using a ZnSe attenuated total reflection probe. Spectra were collected in duplicate. The absorbance spectra were baseline-corrected and averaged using Omnic v9.0 software (ThermoScientific).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 dissolution. 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 pressure 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 flow-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 (ThermoScientific) with a ZB5 column (30 m × 0.25 mm; Phenomenex) and a temperature profile of 40 °C (1 min) to 320 °C (10 min) at 5 °C/min. The eluted compounds were identified with authentic standards (C12 to C20 fatty acids) and by spectral matching with the NIST 2008 spectral library.

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 standard (IS; 50 µg/mL) was added. The samples were silylated with addition of N,O-bis(trimethylsilyl)-trifluoro-acetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS; 50 µL) and pyridine (50 µL) and heated for 30 min at 70 °C or longer until the solution 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; ThermoScientific) 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 identified by their mass spectra, authentic standards, and with NIST 2008 library matching.

High-performance liquid chromatography (HPLC) analysis

Sugars were quantified 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 filtered (0.45 µm). Data was acquired and analysed using the N2000 chromatography software (Science Technology Inc.). The sugar contents were determined from peak area using the external standard method with standard sugars (glucose, fructose, xylose, myo-inositol, 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 Chromatography (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 (caffeine) 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 purified by column chromatography, yielding a colourless solid (hexadecanoic acid) with minor impurities 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 medium 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 filled 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 antioxidant assay

Quantitative antioxidant activity was determined spectrophotometrically 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 final concentrations were prepared based on the preliminary 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 antioxidant activity (%AA) values of test samples were calculated from the absorbance using the formula:

% AA={XcontrolXtestXcontrol}×100
where 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 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 afforded 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 assessment.

FTIR spectroscopic analysis

FTIR spectroscopy was used to investigate the functional groups in the sample and extracts. The spectra (Fig. 1) showed the presence of strong O–H stretching vibration at 3340 cm−1 corresponding 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 virtually absent in Figure 1b but noticeable in Figure 1C and D. Similarly, 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.

Fourier transform infrared spectra of <italic>Cola nitida</italic> seed.
Fig. 1  Fourier transform infrared spectra of Cola nitida seed.

(A) Powdered unextracted material, (B) Cola nitida dichloromethane (CND) extract, (C) Cola nitida methanol (CNM) extract, (D) Extracted biomass.

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 carbonyl 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 evaluations.

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 various flavonoid 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.

Electrospray ionization mass spectra of <italic>Cola nitida</italic> extracts positive mode.
Fig. 2  Electrospray ionization mass spectra of Cola nitida extracts positive mode.

(A) Cola nitida methanol extract, (B) Cola nitida dichloromethane extract.

Electrospray ionization mass spectra of <italic>Cola nitida</italic> extracts negative mode.
Fig. 3  Electrospray ionization mass spectra of Cola nitida extracts negative mode.

(a) Cola nitida methanol extract, (b) Cola nitida dichloromethane extract.

The positive ion ESI-MS for both extracts showed the largest peak at m/z 195 (100% intensity), which was due to caffeine ([M+H]+). In the MeOH extract (Fig. 2A), the tentative peak assignments 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 flavonoids); and m/z 1153 and 1154 (proanthocyanidins). There were many unidentified peaks. Niemenak et al.6 also reported that HPLC of Cola nitida extract had 11 unidentified compounds.

GC-MS of extracts of FAME derivatives

It is well known that fatty acids (FAs), especially the essential fatty 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 system inflammation 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 converted to FAME derivatives for identification and quantification.

The CH2Cl2 and CH3OH extracts were derivatised into FAME and analysed by GC-MS. The CH2Cl2 extract contained FAs ranging 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-oxohexadecanoic 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.

Table 1

GC–MS FAME derivatives of CND extract

S/NoCompoundMolecular formulaClassMolecular weightRetention time (min)% Extract
1Naphthalene acetic acid (IS)C13H12O220027.34
3Palmitic acidC17H34O2FA27031.8319.4
4Linoleic acidC19H34O2FA294358.6
5Oleic acidC19H36O2FA29635.1124.0
6Stearic acidC19H38O2FA29835.61.5
78,9-Methylene-8-heptadecenoic acidC19H34O2FA29436.244.9
810,12-Octadecadienoic acidC19H34O2FA29436.391.7
118-Oxohexadecanoic acidC17H32O3FA28437.2512.0
12Tetrahydropyran-2-yl ether of 7-dodecynolC17H30O2Alcohol26637.461.2
139,10-Methylene-9-octadecenoic acidC20H36O2FA30837.991.4
16Sitosterol acetateC31H52O2Sterol45652.160.7

GC-MS of extracts of TMS derivatives

Trimethylsilylating reagent is routinely used to derivatise rather non-volatile compounds, such as certain alcohols, phenols, or carboxylic acids, by substituting a TMS group for a hydrogen in the hydroxyl groups on the compounds. Both extracts were derivatised 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.

Table 2

GC-MS of Cola nitida CH3OH extracts of TMS derivatives

CompoundMolecular formulaClassMolecular weightRetention time (min)% Extract
Glycerol TMSC12H32O3Si3Alcohol30818.110.2
Malic acid TMSC13H30O5Si3Acid35023.651.5
Anthracene (IS)C14H1017830.14
Fructose TMS5C19H46O6Si4Sugar54030.702.2
Table 3

GC-MS of Cola nitida CH2Cl2 extracts of TMS derivatives

CompoundMolecular formulaClassMolecular weightRetention time (min)% Extract
Nonanoic acid TMSC12H26O2SiFA23020.140.82
Octanedioic acid TMSC14H30O4Si2FA31828.270.26
Palmitic acid TMSC19H40O2SiFA32835.071.64
Linoleic acid TMSC21H40O2SiFA35237.945.95
Oleic acid TMSC21H42O2SiFA35438.111.89
Stearic acid TMSC21H44O2SiFA35638.580.14
Linolenic acid TMSC21H38O2SiFA35039.571.08
Stigmasterol TMSC32H56OSiSterol48453.460.20
Sitosterol TMSC32H58OSiSterol48654.132.05

Caffeine (17.9% and 3.85% in CH3OH and CH2Cl2 extracts, respectively) was predominant, as observed by Niemenak et al.6 Catechin was the dominant flavonoid in the kola seed. Other identified 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.

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 anhydride (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 identified. This finding 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 identified were: CO2 (17%), acetol (11%), 1,2-benzenediol (11%), 1,2-cyclopentanedione (10%), acetic acid (7%), butylamine (5%) and caffeine (5%).

Table 4

Py-GC-MS of unextracted and extracted powdered Cola nitida seed

CompoundMolecular formulaMolecular weightRetention time (min)Kola nut, %Extracted nut, %
N-Methyl ethylamineC3H9N591.304.9
Acetic acidC3H4O3601.645.96.5
Methyl pyruvateC4H6O31023.491.71.1
2-Furfuryl alcoholC5H6O2984.902.24.4
4-Methyl-5H-furan-2-one + UnknownC5H6O298 + 1108.090.6
2 Hydroxy-3-methyl-2-cyclopenten-1-oneC6H8O21129.541.45.4
2,3 Dimethyl-2-cyclopenten-1-oneC7H10O1109.850.3
2-Methyl phenolC7H8O210810.400.7
3 Hydroxy-2-methyl-4H-pyran-4-one (Maltol)C6H6O312612.030.50.7
4-Hydroxy-3-methyl-(5H)-furanone or 3-methyl-2,4(3H,5H)-furandioneC5H6O311412.330.5
Dihydro-2H-pyran-3(4H)-one + unknownC5H8O2100+12813.160.8
Ethyl/dimethyl phenol + 3,5-dihydroxy-2-methyl-(4H)-pyran-4-oneC11H18O7122+14213.660.7
Benzene diol + 3,5 dihydroxy-2 methyl-4-pyroneC6H6O2 + C6H6O4110 + 14214.130.7
5 Hydroxymethyl-2-furaldehydeC6H6O312615.470.6
4-Methyl catecholC7H8O212417.211.52.0
Palmitic acidC16H32O225632.361.50.8
Linoleic acidC18H32O228035.441.0
Oleic acidC18H34O228235.620.90.5
Stearic acidC18H36O228436.010.3
Methyl-2,3-dicyano-3- [4-dimethylamino)phenyl]-2-propenoateC14H13N3O225543.570.6
Unknown steroid34447.500.1
6,9,10-Trimethoxy-12H-[1]benzopino[2,3,4-ij]isoquinoline (oxocularine)C19H17NO432351.170.4
6,16 Dimethylpregna-1,4,6-triene-3,20-dioneC23H30O233851.580.3
3,4,5,6-Tetrahydro alstonineC21H24N2O335252.260.4

It is noteworthy that alkanes, alkenes ethers and steroids/sterols were not identified by the Py-GC-MS of the extracted biomass; this may be explained as due to exhaustive removal of the relatively low polar compounds during the process of extraction. Analytical pyrolysis experiment showed caffeine as the major constituent of Cola nitida. Maltol, a naturally occurring organic compound, is primarily used as a flavour 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 respectively), 148.6, 141.5, 107.5 (olefinic C8, C4 and C5), 33.5, 29.7 and 27.9 (methyl groups). These correlate with the literature data for caffeine.

1,3,7-trimethyl-1 <italic>H</italic>-purine-2,6(3<italic>H</italic>,7<italic>H</italic>)-dione (Caffeine).
Compound 1  1,3,7-trimethyl-1 H-purine-2,6(3H,7H)-dione (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.

n-Hexadecanoic acid.
Compound 2  n-Hexadecanoic acid.

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 bacteria than Gram-negative bacteria. This could be a result of the morphological 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.

Antimicrobial experiment against selected fungi, Gram-positive and Gram-negative bacteria.
Fig. 4  Antimicrobial experiment against selected fungi, Gram-positive and Gram-negative bacteria.

These microorganisms are implicated in the pathogenesis of human 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 fraction was not active against Pseudomonas Spp., Clostridium sporogenes, 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 demonstrated appreciable antimicrobial activity against Bacillus cereus, 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 significantly with that of ascorbic acid. However, the EtOAc and butanol 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 seed of Cola nitida; however, current findings revealed there are yet more compounds that remain to be identified. The present work has identified 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 applications of products of Cola nitida have opened the door to a new dimension 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 quantitative HPLC analysis of the methanol extract of Cola nitida, some of the unknown compounds can be isolated and structural elucidation carried out. The chemicals identified in the kola nut can be derivatised by functional group inter-conversion to more potent and less harmful compounds for both industrial and medicinal applications.


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 identified and structurally elucidated. In this report, the FAs and sugars present in Cola nitida have been identified using the GC-MS (FAME and TMS derivatives of the extracts). With analytical Py-GC-MS experiment, the constituents of thermal decomposition of Cola nitida were also identified 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 assays. Caffeine was ineffective in antioxidant assay, but exhibited appreciable activity against Bacillus cereus, Escherichia coli and Aspergillus niger. The butanol fraction displayed higher antioxidant activity than the CH3OH extract, which might be due to the higher concentration of phenolic compounds in the butanol fraction.

Supporting Information

Table S1

In vitro antimicrobial activity of the extracts and compound 1 (caffeine) against selected microbes




Acelerated Gradient Chromatography


Carbon 13 Nuclear magnetic resonance spectroscopy


Cola nitida dichloromethane extract


Cola nitida methanol extract

IC50 value: 

concentration of samples leading to 50% reduction of initial DPPH radical concentration


deuterated chloroform

DCM or CH2Cl2





electrospray ionization mass spectrometer


essential fatty acids


ethyl acetate


fatty acid methyl ester


fatty acids


fourier transform infrared


gas chromatography-mass spectrometry

GRAM +ve: 

Gram positive

GRAM −ve: 

Gram negative


high-performance liquid chromatography


internal standard




proton nuclear magnetic resonance spectroscopy

Pyro GC-MS or Py GC-MS: 

pyrolysis gas chromatography-mass spectrometry




thin-layer chromatography




Ultra- violet


Zinc Selenium



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 International Pharma & Clinical Pharmacy Congress, November 07–09, 2016, Las Vegas, Nevada, USA.

Conflict of interest

The authors have no conflict of interests related to this publication.

Authors’ contributions

Originator of the research idea and supervising the work (JKA), involving in isolation and antimicrobial experiments (SBO), assisting in the antimicrobial study (DAA), involving in the chemical/instrumental analyses (AGM).


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Chemical Analyses, Antimicrobial and Antioxidant Activities of Extracts from Cola nitida Seed

Julius K. Adesanwo, Seun B. Ogundele, David A. Akinpelu, Armando G. McDonald
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