Original 1 Research Article
PHYTOCHEMICAL AND ANTIOXIDANT PROPERTIES OF STEM BARK EXTRACTS OF ANTHOCLEISTA
Aim: This study was carried out to determine the phytochemical and antioxidant properties of extracts of Anthocleista nobilis.
Methodology: Acetone and methanol extracts of A. nobilis were investigated for their free radical scavenging activities in the presence of diphenyl picrylhydrazyl (DPPH) using ascorbic acid as positive control.
Results: In the phytochemical screening of the plant extracts, results showed that both extracts recorded the presence of alkaloids, tannins, flavonoids, and glycosides. The acetone and methanol extracts of A. nobilis exhibited significant free radical scavenging activities in the DPPH assay with the acetone extract recording better activity. The antioxidant activity of the two extracts was however, lower than that recorded by the positive control-ascorbic acid.
Conclusion: The result suggests that the extracts have potential antioxidant properties which could be exploited in medicine and food industry.
Keyword: Anthocleista nobilis, antioxidant, DPPH.
Plants have been utilized by humans to treat various infectious and non-infectious diseases since the beginning of time as they constitute a reliable source of therapy. In addition, they are also the primary source for many of today’s medicines [1,2,3]. For instance, purified secondary metabolites such as vinca alkaloids are used widely in cancer chemotherapy .Quinine and artemisinin, derived from the bark of the cinchona and qinghaosu trees respectively and their derivatives have been widely used for the treatment of malaria [5,6,7].
Some of these secondary metabolites are synthesized for specific purposes by plants. Others may be by-products of plant metabolism which currently have no known biological function. Hydroxylated coumarins have been reported to accumulate in carrots in response to fungal invasion , glucosinolates, recognized for their antimicrobial properties, have also been reported in Brassia rapa in response to fungal infection/attack .
These secondary metabolites fall under one of the major phytochemicals such as flavonoids, tannins, glycosides, steroids, terpenes, etc. Although these compounds are known to be bioactive, a lot is yet to known about their mechanisms of action. Some of these groups of compounds such as flavonoids, proanthocyanidins and tannins are polyphenols or phenolic . Phenolics are known for their antioxidant properties .
Antioxidants are molecules that halt oxidation processes while the molecules get oxidized in the process. The antioxidant activity of phenolics is derived from their ability to act as reducing agents, donating hydrogen, electrons and stabilizing reactive oxygen species (ROS) . ROS such as hydroxyl radicals (OH.), superoxide anion radicals (O2 .) and singlet oxygen (145 O2) have been implicated in many disease processes such cancer, diabetes, ageing, atherosclerosis and neurodegeneration. Consumption of fresh fruits and vegetables rich in plant polyphenols(antioxidants) as food has been reported as a protection against several diseases which include cancer, cardiovascular diseases, diabetes, asthma etc.  suggesting that the mechanism of action of the secondary metabolites can be traced to their antioxidant properties. ROS can cause the deterioration of food by causing lipid peroxidation. The rancid odor and taste of lipid containing food such as palm oil occur as result of lipid peroxidation which in turn affects the nutritional value and safety 52 of such food items .
Recently, the use of synthetic preservatives and other additives has been linked to increased prevalence of cancer. For instance, sodium nitrate and sodium nitrite used to preserve processed meat has been implicated in bowel cancer . As a result, the demand for natural food preservatives has been rising steadily  and that prompted the European Union funded AGROCOS to include the replacement of synthetic preservatives and other ingredients as part of the FP7 research topics which has yielded about 30 natural compounds for the food and cosmetic industries presently being further tested by Greek and German companies .
Anthocleista nobilis which is commonly called the candelabrum or cabbage tree in English language, Duwa Kuchi in Nupe language, Kwari in Hausa language and Apa Ora in Yoruba language belongs to the family Loganiaceae . Anthocleista nobilisis used in local medicine in parts of West Africa for curing fever, stomach ache, diarrhoea, and gonorrhoea, and are also aspoultice for sores [20,21]. The present study was designed to investigate the antioxidant properties of extracts of Anthocleista nobilis by measuring their free radical scavenging properties with aim of confirming the ethnobotanical use and assessing their suitability as preservatives in the food industry.
2. MATERIALS AND METHODS
2.1 Sample collection
The stem barks of A. nobilis were collected in March, 2014 from Ezza Community in Ebonyi state, Nigeria. They were identified by a plant taxonomist in the Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria. The plant material was pulverized into fine powder using a mechanical grinding machine.
500 g of thepulverized plant was macerated in 1 Liter of acetone and methanol respectively for 48 hrs.The mixture was sieved using porcelain cloth and was further filtered using No. 1 Whatman filter paper. The filtrate was concentrated using rotary evaporator and the crude concentrate was then stored at 4o80 C until required for further experiment.
2.3 Phytochemical Screening
In the phytochemical analysis of the extracts of A. nobilis stem barks, tests for alkaloids, tannins, saponins, flavonoids, steroids, cardiac glycosides, and terpenoids were carried out using standard methods reported by Trease and Evans .
2.4 In vitro free radical scavenging activity of the extract (Diphenyl dipicrylhydrazyl (DPPH) free radical scavenging activity)
The free radical scavenging activity of the various extracts and ascorbic acid was measured with DPPH. Three (3) mL of DPPH in methanol solution was added 100 μL of different concentrations of extracts (10-1000 μg/mL). After 30 minutes, the absorbances of the extract solutions ion methanol were measured at 517 nm after calibration with methanol. Lower absorbance indicated higher free radical scavenging activity. The antioxidant activity of the extracts was expressed as IC50 which was defined as the concentration of the extracts that inhibited the formation of DPPH radical by 50 %. Each experiment was carried out in triplicate. The average absorbance for each triplicate was calculated and the percentage inhibition of the extracts at different doses calculated using the formula:
Where Ao is absorbance of control and As the absorbance of tested extracts.
3. RESULTS AND DISCUSSION
The antioxidant activity of the extracts of A. nobilis was investigated and the results were compared to that of ascorbic acid which was used as the positive control. The results are shown in Figures 1. Also, the result of the phytochemical analysis links the presence of flavonoids and other polyphenols to the antioxidant activities observed. Different levels of the secondary metabolites based on the tests carried out were observed as displayed in Table 1.
Table 1. Result of the Phytochemical Analysis of A. nobilis Extracts
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Alkaloids Tannins Saponins Flavonoids Glycosides Terpenoids Steroids
++ +++ ++ + ++ – –
+ ++ – ++ + ++ –
+++ = Abundantly present; ++ = Moderately present; += Mildly 108 present; – =Absent
Figure 1. Comparison between free radical scavenging activities of acetone and methanol extracts of A. nobilis and ascorbic acid determined using DPPH. IC50 for Acetone extract= 400 μg/mL; Methanol extract= 800 μg/mL; Ascorbic acid= 30 μg/mL.
The bark of A. nobilis is used as warm expellant and as antimalarial remedy amongst other ethnobotanical uses by the Ezza people in Nigeria. The result of this study showed that the crude extracts showed significant antioxidant properties with the acetone extract of A. nobilis recording an IC50 of 400 μg/ml. The methanol extract of A. nobilis had the lowest antioxidant activity with an IC50 of 800 μg/mL.
However, the acetone extract of A. nobilis which had the best antioxidant activity compared to the methanol extract had moderate presence of terpenoids, flavonoids and tannins. Flavonoids and tannins are polyphenols which are known to have potent antioxidant properties due to there reducing ability . The lower activity observed can be explained by the fact that flavonoids only exihibit antioxidant properties if features such ortho-dihydroxy substitution in the B-ring, C2-C3 double bond and a carbonyl group in C-4 of the C-ring are present . Quercetin is a good example of a flavonoid with such structural features and it has a high antioxidant property .
Although the free radical scavenging activities observed for the extracts were not as much as that observed for ascorbic acid which was the positive control, the antioxidant activity of the extracts, can be said to be significant considering that the extracts were in the crude form. Further purification of the aqueous extract is expected to produce pure compounds with improved antioxidant property.
The findings of this study reveal that Anthocleista nobilis possess antioxidant property. This provides a scientific basis for the ethnomedicinal utilization of this plant. The antioxidant property of this plant may qualify it for use as preservatives of natural origin in the food industry. Further tests are needed to explore the exact mechanism of action at the molecular level and to know the actual 138 constituents responsible for these activities.
% Inhibition Versus Concentration
UNDER PEER REVIEW