Evaluation of Antioxidant Activity of Caffeine Extracted From Kolanut (Cola Nitida)

Crude caffeine was isolated from kola nut, by treating the ltrate obtained from powdered kola nut with lead (II) acetate to precipitate acids and albumins, along with HCl to precipitate lead ions, and to make some insoluble tannins to be soluble, then Na 2 CO 3 solution was added to neutralise the acidic HCl solution. Dichloromethane is added to the ltrate to extract the caffeine from the aqueous layer and then distilled to obtain the crude caffeine. The extracted crude caffeine is recrystallized with activated charcoal and tested for its antioxidant capacity, using the DPPH assay, which screens the antioxidant activity of plant extracts. The DPPH (2, 2-diphenyl-1-picrylhydrazyl) in methanol helps to facilitate the extraction of the antioxidant compounds from the sample. Ascorbic acid (Vitamin C) is used as the standard for this assay, due to its appreciable antioxidant activity. The IC 50 value was calculated from the graph of % inhibition against concentration, and the extracted caffeine from kola nut was found out to have a high antioxidant activity comparable to that of the standard, ascorbic acid.


Introduction
Kola is a common name for a genus of about 125 species of evergreen trees and their trees are best known for their seeds or nuts which are rich in caffeine and used in the manufacturing of carbonated soft drinks known as kola beverages. They can relieve migraine, and can be used in states of depression, or weakness and even in the treatment of malaria fever [1]. They can also be used to prepare tonic, as a remedy for dysentery, coughs, diarrhoea, vomiting and chest complaints (Burkill, 1995). Kola is one of the major sources of caffeine and taking kola nut by the mouth, in large doses is likely unsafe, due to its caffeine content and frequent chewing can lead to stained teeth [2][3]. Adisa, et al, (2010) also carried out a signi cant research and reported on the effects of aqueous extract of kola nut on the reproductive hormones in rat [4]. The result of this study shows that kolanut extract signi cantly reduced plasma level of luteinizing hormone but signi cantly increased plasma testosterone level. The reduced plasma luteinizing hormone level reported in this study is in agreement with ndings of Benie and Thieulant [5], where it was reported that kolanut extract exerted an inhibitory effect on luteinizing hormone release of rat's pituitary cells, however our ndings on plasma level of testosterone was in agreement with Leehayward [6], that caffeine increases plasma level of testosterone. But contrary to the ndings of Rebecca and Elizabeth [7], who reported that bioavailable testosterone was negatively and independently associated with caffeine intake, while estrone was positively associated with high levels of caffeine consumption in post-menopausal women. The active chemical constituents of all the species of kola nut include: caffeine, glucoside, theobromine and kolatin which are stimulants [8].
The symptoms of caffeine intoxication are not unlike overdoses of other stimulants. It may include restlessness, dgetiness, nervousness, excitement, euphoria, insomnia, ushing of the face, increased urination, gastrointestinal disturbance, muscle twitching, a rambling ow of thought and speech, irritability, irregular or rapid heartbeat, and psychomotor agitation [9]. In cases of much larger overdoses, mania, depression, lapses in judgment, disorientation, disinhibition, delusions, hallucinations, and psychosis may occur, and rhabdomyolysis (breakdown of skeletal muscle tissue) can be provoked [10][11]. In large amounts, and most especially, over an extended period of time, caffeine can lead to a condition known as caffeinism [12]. Caffeinism usually combines caffeine dependency with a wide range of unpleasant physical and mental conditions including irritability, anxiety, tremulousness, muscle twitching (hyperre exia), insomnia, headaches, respiratory alkalosis, and heart palpitations [13][14]. Parvathy, et al., (2014) reported the chemical analysis of caffeine content in tea and coffee samples [15]. Okolie, (2010) reported the effect of heat on caffeine isolated from kolanut [16]. Caffeine has even been suggested as possible cause of cancer, though no studies have con rmed yet [17]. Caffeine relaxes the internal anal sphincter muscles and thus should be avoided by those with fecal in continence [18].
Caffeine citrate may confer a modest protective effect against some diseases, including Parkinson's disease [19].
Antioxidants are compounds that inhibit oxidation or substances that at low concentrations prevents the oxidation of a substrate. A chemical reaction that can produce free radicals and chain reactions that may damage the cells of organisms, have antioxidants such as thiols or ascorbic acid (vitamin C) that may act to inhibit these reactions. The antioxidant capacity is a measure of the amount of a certain free radical captured by an antioxidant sample [20]. To balance oxidative stress, plants and animals maintain complex systems of overlapping antioxidants, such as glutathione. The only dietary antioxidants are vitamins A, C, and E.
The aim of this research is to investigate the antioxidant activity of caffeine isolated from kolanut grown in Nsukka, eastern part of Nigeria. The choice of this research arises from the fact that kola nut contains most essential phytochemicals which are suspected to have antioxidant behaviour and is believed to possess numerous biological properties. Also it has been found that most of the physiological actions of kola are due to caffeine [21] and caffeine content of kola nut varies with difference in climatic and adaphic factors [22].

Materials And Method Sample Preparation
The kola nuts ( g1) used in this study was obtained from Nsukka, Enugu State, Nigeria. The kola seeds were washed, reduced in size and sun dried for four days. The reduced kola seeds were further grounded using an electric grinder and sun dried again for two days, after which it was manually grinded with a clean and dried mortar and pestle to obtain a powdered form of the kolanut as seen in g 2 below

Extraction of Caffeine from Kolanut
Caffeine was isolated and puri ed following Johnson's method as reported by Okolie, (2010). 20g of kola nut was weighed into a metal pot. 150cm 3 of water was added to it and boiled for about 12 minutes. 40cm 3 of 0.3M lead (II) acetate was added while the mixture was made hot to precipitate acids and albumin. The hot mixture of kola and water plus additives was ltered using a white cloth. The solids were ltered and re-boiled with 100cm 3 of water while a stand by hot water was used to wash the system.
After ltration, 20ml of 2M hydrochloric acid was added to the ltrate to precipitate lead ions. The lead chloride was ltered off. 30ml of 1M sodium carbonate solution was added to the solution gradually until it was slightly alkaline and concentrated to four times its initial strength. The concentrate was settled for about two minutes, decanted, and the sediment was ltered with lter paper while hot. The ltration was kept hot with steam. The latter ltrate, which has been cooled was combined with the decanted portion before drip extraction. The conc. ltrate was extracted 4 times, with 15cm 3 portions of dichloromethane.
The mixture was frequently inverted in each extraction and allowed to stand for at least 9 minutes before isolation of layers. The extracts were combined, and the chloroform was distilled to ¼ of its original volume. The remaining chloroform was evaporated to dryness to obtain the crude caffeine. The crude caffeine was puri ed by dissolving in hot water, activated charcoal was added to absorb the gas and the colouring matter, and then it was ltered. The ltrate was cooled to room temperature, followed by drip extraction. The solvent was evaporated on a hot plate. The puri ed dry caffeine was recovered and stored in a desiccator as represented in g 3.

Antioxidant Activity test
Procedure: 0.1g of the extracted caffeine and 0.1g of ascorbic acid (standard control) was weighed into different beakers, treated with 20ml of methanol respectively and stirred for ve minutes. They were ltered, and the ltrates were covered with aluminium foils and left for a while. 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0ml(s) of the respective solutions (caffeine and ascorbic acid) were measured into 6 different test tubes, and diluted serially, such that the volume of solutions in the respective test tubes were 1.0ml each.
Also, 0.0039g/L of DPPH was prepared, with methanol, and kept in a closed space, due to its high sensitivity to light (i.e. it has the ability to undergo photochemical decomposition). 5ml of the DPPH solution was added to the 1.0ml solutions in the various test tubes (which were the caffeine solution and the standard solution), and they were all con ned in a closed space for about an hour. The absorbances of these solutions were determined on a UV/V Spectrophotometer, which was set at 517nm, while using 10ml of methanol as the blank solution.  In the triplicate concentrations, absorbances and percentage inhibitions of caffeine and ascorbic acid, it was observed that the absorbances decrease with increase in concentration while percentage inhibitions increase with increase in concentration for caffeine and ascorbic acid respectively as seen in table 1.

Results And Discussion
The plot of percentage inhibition against the DPPH of concentrations of caffeine and ascorbic acid were clearly illustrated in gs 4 and 5. Fig 5 showed

Conclusion
This research revealed that caffeine isolated from kola nut exhibited potential antioxidant activity.
Chewing kola nut is therefore, said to be a healthy habit, since the antioxidant property of the caffeine it possesses can help to trap reactive oxygen species, as well as free radicals that has been generated by the body during the breaking down of food, exposure of the body to tobacco smoke or radiation and the ght against oxidative stress and damage. However, it is important to note that over intake of kolanut may impose has its own adverse effects. The phenolics and anthrocyanin present in kolanut may be responsible for its antioxidant activity [23], so further study can be done to identify the active compound responsible for caffeine antioxidant activity.

Recommendation
The caffeine extract from kola nut is a promising drug candidate for use as natural products based antioxidant for human health and it can be applied in the production of pharmaceuticals, for the prevention of variety of diseases, caused by free radicals. Also, caffeine extract from other plant source, including kola nut can be investigated for other biological activities which they may exhibit.