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\title{Phytochemical Screening and in Vitro Antioxidant Activity of Aqueous Extract of Blighia Sapida Stem Bark}
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             \author[1]{Amira Philip  Olaniyi}

             \affil[1]{  Federal Polytechnic,}

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\date{\small \em Received: 9 December 2016 Accepted: 2 January 2017 Published: 15 January 2017}

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\begin{abstract}
        


Blighia sapida is a plant belonging to the family of sapindaceae. In this study we aimed to carry the phytochemical screening of aqueous extract of Blighia sapida stem bark and evaluate its in vitro antioxidant activity. It was found that the aqueous extract of Blighia sapida stem bark showed the presence of tannin, saponin, flavonoid, alkaloid, phenols and ascorbic acid. It also contains trace elements zinc and selenium. Furthermore it showed some scavenging activity but not as such could be compared with the various standards used except for nitric oxide scavenging activity. These are indications that the aqueous extract ofBlighia sapida contains some physiologically significant secondary metabolites and also possesses appreciable in vitro antioxidant activity.

\end{abstract}


\keywords{blighia sapida, phytochemicals, antioxidant, metabolites.}

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\let\tabcellsep& 	 	 		 
\section[{I. Introduction}]{I. Introduction}\par
hytochemicals are naturally occurring in the medicinal plants, leaves, vegetables and roots that have defense mechanism and protect from various diseases. In fact, phytochemicals (or antioxidants) such as phenolic compounds (tocopherols, flavonoids and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids and amines), carotenoids and ascorbic acid  {\ref (Hall and Cuppett, 1997;}\hyperref[b13]{Larson, 1988)} compounds inhibit, or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reaction.\par
Blighia sapida is a plant belonging to the family of Sapindaceae. It is commonly known as ackee. In Nigeria, it is called Gwanja Kusa (Hausa), Isin (Yoruba) and Okpu (Igbo). It is an evergreen tree of about 33 to 40ft (10-12m) with a dense crown of spreading branches. It is rather hand usually with a short trunk to 6ft (1.8m) in circumference. Its bark is grey and nearly smooth. The leaves are compound with three to five pairs of oblong, ovate-oblong, or elliptical leaflets 1.5-3.0cm long. The seed of the fruit is not edible, whereas the fleshy aril is edible. The fruit is known to contain Most of the earlier studies on Blighia sapida have been on the nutritional qualities of the root (Abolaji et al, 2007) and the leaves as a dry season feed resource for West African dwarf goats in the Northern savanna zone of Nigeria \hyperref[b1]{(Aderinola et al, 2007)}. The repellant potential of the fruit part components against stored-product insect pests \hyperref[b12]{(Khan and Gumbs, 2003)} as well as neutropenia and thrombocytopenia effects of the aqueous and lipid extracts of the unripe fruit have been investigated in mice \hyperref[b5]{(Gardiner et al, 1996)}. More recently, the physicochemical properties of the oil from the fruit of the species and toxicological evaluation of the oil -based diet in Wister rats have been investigated \hyperref[b18]{(Oladiji et al, 2009)}.\par
Tree bark is an important component of African traditional medicine as herbal medicine is still the main source of health care for the majority of Africans and in particular, Nigerians.There has been increasing demand for the use of plant products with therapeutic activity. The high cost, availability, uncertainty of use during pregnancy and undesirable side effects of synthetic drugs or drugs from other animal sources are some of the factors leading to a strong preference for drugs of plants origin.\par
This study is thus aimed at investigating the phytochemicals and in vitro antioxidant activity of the readily available Blighia sapida stem bark. 
\section[{II. Materials and Methods}]{II. Materials and Methods}\par
Chemicals: All chemicals used were of analytical grade and items are products of BDH and Sigma Chemical Ltd., UK and Roche Diagnostic, Germany.\par
Sourcing for the Tree Bark of Blighia sapida: A sizeable quantity of the tree bark of Blighia sapida was obtained from the compound of the Federal Polytechnic, Ado Ekiti, Nigeria.\par
Identification of Plant: The fruits and leaves of Blighia sapida plant were obtained from the compound of the The air-dried tree bark of Blighia sapida was pulverized.\par
100g of the pulverized sample was extracted with 800ml of distilled water for seventy-two (72) hours in an extractor. The aqueous extract was obtained by filtering with Whatman filter paper and subsequently freeze-dried in Armfield freeze-drier for ten \hyperref[b8]{(10)} days.\par
Qualitative Phytochemical Analysis: Chemical tests were carried out on the aqueous extract using modified standard procedures to identify the constituents as described by  {\ref Sofowora(1993)},  {\ref Trease and Evans(1989)} and Harborne  {\ref (1973)}.\par
Quantitative Determination of Phytochemicals Determination of Tannin: The tannin content of the extract was determined by using the modified procedure of \hyperref[b15]{Makkar (1994)}.\par
Determination of Saponin: The method used was that described by \hyperref[b16]{Obadoni and Ochuko (2001)}.\par
Determination of Flavonoid: The method of Boham and Kocipal-Abyazam (1994) was used.\par
Determination of Alkaloid: The total alkaloid content of the extract was determined using the method described by Harborne (1973).\par
Determination of Total Phenols: The total phenolic content was determined using the method described by Singleton and Rossi(1965) using Folin-Ciocalteu's phenol reagent.\par
Determination of Zinc and Selenium: The level of zinc and selenium were determined by the method described by AOAC (2006).\par
In vitro Antioxidant Assay DPPH free radical scavenging assay: The hydrogen or radical scavenging properties of the extract was determined using the stable radical DPPH (2, 2-Diphenyl-1-picrylhydrazyl hydrate) according to the method proposed and described by \hyperref[b3]{Blois (1958)}.\par
Hydroxyl radical scavenging assay: Deoxyribose assay was used to determine the hydroxyl radical scavenging activity in an aqueous medium .\par
Hydrogen peroxide decomposition assay: This activity was determined according to a method described by Long et al., (1999).\par
Nitric oxide (NO) scavenging assay: At physiological pH, nitric oxide generated from aqueous sodium nitroprusside (SNP) solution interacts with oxygen to produce nitrite ions, which may be quantified by the Griess Illosvoy reaction  {\ref (Garratt, 1964)}.\par
Ferric reducing antioxidant assay (FRAP): The FRAP assay uses antioxidants as reductants in a redox-linked colorimetric method with absorbance measured with a spectrophotometer \hyperref[b2]{(Benzie and Strain, 1999)}. The principle of this method was based on the reduction of a colourless ferric-tripyridyltriazine complex to its blue ferrous colour formed owing to the action of electron donation in the presence of antioxidants.\par
Determination of total antioxidant capacity: The total antioxidant capacity was determined in accordance with the method described by Prieto et al.,  {\ref (1999)}. Statistical Analysis: Data were expressed as mean + S.E.M. of five replicates. 
\section[{III. Results}]{III. Results}\par
Phytochemical screening of extract: The result of phytochemical screening of aqueous extract of Blighia sapida stem bark shows the presence of tannin, saponin, flavonoid, alkaloid, phenols and ascorbic acid.\par
It also shows the presence of the trace elements zinc and selenium (Table \hyperref[tab_0]{1 and 2}).\par
In vitro antioxidant activity: In vitro antioxidant studies revealed that aqueous extract of Blighia sapida stem bark showed some scavenging activity by total antioxidant capacity (TAC) with the IC 50 value of 16.63± 0.51µg/ml, DPPH radial scavenging activity with the IC 50 value of 23,938± 169.28 µg/ml, total phenol of 5.47± 0.93 µg/ml GAE, hydrogen peroxide radical decomposition activity with the IC 50 value of 5,133.50± 191.70 µg/ml, hydroxyl radical scavenging activity with IC 50 value of 6904.00± 751.90 µglml and nitric oxide scavenging activity with IC 50 value of 447.57± 153.28 µg/ml. However, apart from the nitric oxide scavenging activity value that compares with that of the standard (i.e. ascorbic acid), all other values are a lot higher than those of the standards used (Table \hyperref[tab_1]{3}).   
\section[{IV. Discussion}]{IV. Discussion}\par
Antioxidants such as phenolic compounds (tocopherols flavonoids, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids and amines), carotenoids and ascorbic acid  {\ref (Hall and Cuppett, 1997;}\hyperref[b13]{Larson, 1988)} compounds inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions.\par
In this study, the metabolites shown in Table  {\ref 1} were known to show biological activity as well as exhibiting physiological activity  {\ref (Sofowora, 1993)}. Flavonoids are potent water-soluble antioxidants and free radial scavengers which prevent oxidative cell damage and have strong anticancer activity ( Okwuet   al., 2006). Flavonoids also lower the risk of heart disease. Saponins are capable of neutralizing some enzymes in the intestine that can become harmful, building the immune system and promoting wound healing. Alkaloids have been documented to possess analgesic, antispasmodic and bactericidal effects. Tannins hasten the healing of wounds and inflamed mucuous membrane (Okwu et al., 2006). The presence of these phytochemicals support the medicinal use of Blighia sapida (Saidu, 2012).\par
Zinc and selenium are the trace elements that have been found to be cofactors of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase \hyperref[b26]{(Weydert and Cullen, 2009)}.\par
In vitro antioxidant studies revealed that aqueous extract of Blighia sapida stem bark only showed a comparative scavenging activity when compared to the standard (i.e. ascorbic acid), by nitric oxide scavenging activity. All other parameters considered in the in vitro antioxidant studies did not show promising results when compared with the standards. 
\section[{V. Conclusion}]{V. Conclusion}\par
A major finding of the study is that aqueous extract of Blighia sapida stem bark showed the presence of metabolites such as tannin, saponin, flavonoid, alkaloid, phenols and ascorbic acid. It was also found to contain trace elements zinc and selenium.\par
The results also revealed that Blighia sapida stem bark aqueous extract showed some in vitro antioxidant activity. Ofall the parameters studied, nitric oxide scavenging activity was the only one that showed a somewhat comparable IC 50 to that of the standard while all others did not compare well with the chosen standards.   \begin{figure}[htbp]
\noindent\textbf{2} \par 
\begin{longtable}{P{0.14166666666666666\textwidth}P{0.24469696969696972\textwidth}P{0.025757575757575757\textwidth}P{0.14595959595959596\textwidth}P{0.12878787878787878\textwidth}P{0.14595959595959596\textwidth}P{0.01717171717171717\textwidth}}
\multicolumn{2}{l}{Species Extract Type}\tabcellsep Tannin\tabcellsep Saponin Flavonoid Alkaloid Phenols\tabcellsep Ascorbic Add(mg/100g)\tabcellsep Zinc (ppm) Selenium (ppm)\\
Bllghla\tabcellsep \multicolumn{3}{l}{Aqueous 22.0±1.24 21.3±3.30 13.3±1.86 17.3±4.70 5.47±0.93}\tabcellsep 22.1±6.02\tabcellsep 19.6±0.03\tabcellsep <1.0\\
saplda\tabcellsep \tabcellsep \tabcellsep \tabcellsep \end{longtable} \par
  {\small\itshape [Note: © 2017 Global Journals Inc. (US)]} 
\caption{\label{tab_0}Table 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{}
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\caption{\label{tab_1}Table 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{} \par 
\begin{longtable}{P{0.04180327868852459\textwidth}P{0.11943793911007025\textwidth}P{0.0895784543325527\textwidth}P{0.09156908665105386\textwidth}P{0.17517564402810304\textwidth}P{0.07962529274004684\textwidth}P{0.04379391100702576\textwidth}P{0.03185011709601873\textwidth}P{0.09156908665105386\textwidth}P{0.05374707259953161\textwidth}P{0.03185011709601873\textwidth}}
\tabcellsep EXTRACT\tabcellsep Total Antioxidant\tabcellsep Ferric reducing\tabcellsep DPPH free radical\tabcellsep \multicolumn{2}{l}{Total Phenol}\tabcellsep Hydrogen\tabcellsep Hydroxyl\tabcellsep Nitric oxide\\
\tabcellsep TYPE\tabcellsep Capacity (TAC)\tabcellsep antioxidant (FRAP)\tabcellsep Scavenging\tabcellsep (GAE)\tabcellsep \tabcellsep Peroxide\tabcellsep radical\\
\tabcellsep \tabcellsep \tabcellsep \tabcellsep \multicolumn{2}{l}{IC 50 Values (µg/ml)}\tabcellsep \tabcellsep \tabcellsep \\
\tabcellsep Aqueous\tabcellsep 16.63 ± 0.81\tabcellsep 9.13 ± 0.51\tabcellsep 23938.00 ± 160.28\tabcellsep \multicolumn{3}{l}{5.47 ± 0.93 5133.50 ±}\tabcellsep 6904.00 ±\tabcellsep 447.57 ± 153.28\\
Year 2017\tabcellsep Ascorbic acid Butylated\tabcellsep --\tabcellsep --\tabcellsep Standards 69.19 ± 5.02 -\tabcellsep --\tabcellsep \multicolumn{2}{l}{191.70 123.41 ± 4.84 -}\tabcellsep \multicolumn{2}{l}{751.90 -139.73 ± 31.31}\tabcellsep 264.08 ± 11.07 -\\
\tabcellsep \multicolumn{2}{l}{Hydroxytoluene}\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \\
\tabcellsep (BHT)\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \\
D D D D ) B\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \\
(\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \end{longtable} \par
  {\small\itshape [Note: signaling pathways mediators of insulin resistance and ?? ?cell dysfunction?, Diabetes, 52, 1-8.Volume XVII Issue 1 Version I]} 
\caption{\label{tab_2}}\end{figure}
 			\footnote{© 2017 Global Journals Inc. (US)} 		 		\backmatter  			  				\begin{bibitemlist}{1}
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\end{document}
