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             \author[1]{Rufai  Y}

             \affil[1]{  Chemistry Department, Federal College Of Education Okene, P.M.B 1026 Kogi State, Nigeria}

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\date{\small \em Received: 11 April 2015 Accepted: 3 May 2015 Published: 15 May 2015}

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\begin{abstract}
        


Day by day, faith of people on herbal medicine increases due to the side effect of synthetic drugs; this has resulted into people falling back to the traditional knowledge of plant for their health care. Certain local practitioner and traditional healers use the fruits of Citrus aurantium var. Dulcis L pulps in various disease management and so, they advise to eat the pulps along with the drinking of the juice. The present study deals with preliminary phytochemical analysis of the fruit of Citrus aurantium var. Dulcis L pulp using 95% ethanol for its extraction. The fruits of Citrus aurantium var. Dulcis L pulp ethanolic extract revealed the presence of all tested phytochemical compounds except protein and glycoside. These include Alkaloids, Tannins, Phenolic, Quinine, Reducing Sugar, Coumarins, Flavonoids, Saponins, and Steroids. During the analysis, the quantitative fractionation of the ethanolic extract showed a reasonable amount of saturated hexane fraction (40g), unsaturated hexane fraction (2.0g), methanolic fraction (1.3g), acidic fraction (1.2g) and basic fraction (0.3g).

\end{abstract}


\keywords{citrus aurantium var. dulcis L pulp, preliminary phytochemical analysis, quantitative fractionation.}

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\let\tabcellsep& 	 	 		 
\section[{I. Introduction}]{I. Introduction}\par
edicinal plants are of great importance to the health of individuals and communities. The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body \hyperref[b0]{[1]}. The most important of these bioactive constituents of plants are Alkaloids, Tannins, Flavonoids, and Phenolic compounds \hyperref[b1]{[2]}. Many of these indigenous medicinal plants are used as spices and food plants. They are also sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes  {\ref [3;4]}. This field of natural products research is currently being carried out intensively though it remains far from exhaustion. An attempt to obtain bioactive agents from plants is a worthwhile exercise since only 10\% of all plants have been investigated in detail \hyperref[b4]{[5]}. However, as at the time of this study, a higher percentage of bioactive compounds could have been discovered. The majority of these bioactive compounds are Sesquiterpenes, Diterpenes, Triterpene Saponins, Triterpene Aglycones, and Monoterpenes. It is imperative that ethnobotanical researches and phytochemical tests have led to some patent-able and industrially exploitable compounds for drug development. Plants fulfill the needs of not only human being but also entire animal kingdom.\par
Man as a unique creation of God \hyperref[b5]{[6]} is but a part of the universe that relates domestically with other living creatures, man has been provided with food, water, shelter and herbal medicine around his habitat. However, the orange pulps of the fruits of Citrus aurantium var. Dulcis L. which is part of man's food are more beloved to domestic animals like goat and sheep for reason not yet proven scientifically. Domestic animal kept looking at human when eating or drinking such food and most times compete with them self in eating the thrown away part by human.\par
The popular orange tree (Citrus aurantium var. Dulcis L.) belongs to the plant family Rutaceae. It is a small tree with grayish-brown branches that are widely spread. The petioles of the leaves are winged and the leaves are ova, alternate, and have a deep green colour. The calyx is bell-shaped and bisexual flowers are pure white. The fruit is round and green and yellow when ripe. It is widely used for it juice which is sweet \hyperref[b6]{[7]}.  
\section[{Part}]{Part}\par
Medicinal Uses 
\section[{Leave}]{Leave}\par
The infusion of the leaves, mixed with a little honey, is used for controlling cough. 
\section[{Pulp}]{Pulp}\par
The pulp should be eaten instead of drinking only the juice as it ease bowel movement. 
\section[{Fruit}]{Fruit}\par
The fruit in general is good for cases of arthritis, asthma, respiratory problems, pneumonia, hysteria, neurasthenia, neuralgia, headache, colds, cough, fevers and influenza. It is highly recommended for scurvy. 
\section[{Rind}]{Rind}\par
Fresh rind rubbed on the face is a good remedy for acne. 
\section[{Juice}]{Juice}\par
The consumption of orange juice strengthens the stomach, increases Musa, The back of the oranges were pealed, the pulps were collected after juice extraction and washed with pure water, air dried as shown below and pulverized into a fine powder using a commercial blender. 
\section[{III. Extraction and Fractionation Procedure}]{III. Extraction and Fractionation Procedure}\par
Extraction and fractionation of the pulp ethanolic extract was carried out by bioassay guided fractionation protocol \hyperref[b7]{[8]}. The procedure was carried out using ethanol-water (95:5v/v) and different organic solvent in order of polarity (Hexane, chloroform and Methanol) using separatory funnel to fractionate them into different fractions. One thousand grams of the powdered fruits of Citrus aurantium var. Dulcis L pulp materials (20 mesh\textasciitilde 1g) were extracted using percolation process in a mixture of 95ml of distilled ethanol and 5 ml of distilled water at ambient temperature overnight. The extractives was filtered and re-extracted three times. The combined extract were filtered through a Whatman No. 1 paper and then concentrated invacuo at 40 0 C using a rotary evaporator, model W2-100 SENCO® @ rpm of 100; Shanghai SENCO technology Co, Ltd Japan. The various extractive concentrates were evaporated to dryness using water bath for some days and residues were obtained in gram for basic, acidic, polar and non-polar fraction as 0.3g, 1.2g, 1.3g, and 40g.\par
Preliminary Phytochemical screening was done using standard procedures to identify constituents, as described  {\ref [9;10]}   
\section[{Figure (b) c) Test for Proteins (Biurret Test)}]{Figure (b) c) Test for Proteins (Biurret Test)}\par
To the small quantity of extract 1-2 drops of Biurret reagent was added. Formation of violet colourprecipitate showed presence of proteins. 
\section[{d) Million's Test}]{d) Million's Test}\par
To the small quantity of extract 1-2 drops of Million's reagent was added. Formation of white colour precipitate showed presence of proteins. 
\section[{e) Test for Anthraquinone glycosides f) Borntrager's Test}]{e) Test for Anthraquinone glycosides f) Borntrager's Test}\par
To the 3ml of extract, dil. H 2 SO 4 was added. The solution was then boiled and filtered. The filtrate was cooled and to it equal volume of benzene was added. The solution was shaken well and the organic layer was separated. Equal volume of dilute ammonia solution was added to the organic layer. The ammonia layer turned pink showing the presence of glycosides. 
\section[{g) Test for Cardiac glycosides (Keller-Killiani Test)}]{g) Test for Cardiac glycosides (Keller-Killiani Test)}\par
To the 5ml of extract, 1ml of conc. H 2 SO 4, 2ml of Glacial acetic acid and 1 drop of FeCI 3 solutions was added. Appearance of Brown ring shows the presence of cardiac glycosides. 
\section[{h) Test for Coumarins}]{h) Test for Coumarins}\par
To the 2ml of extract 10\% NaOH was added and shake well for 5mm shows the yellow colour. 
\section[{i) Tests for Quinone}]{i) Tests for Quinone}\par
To the 2ml of extract conc. H 2 SO 4 added and shake well for 5 mm shows the Red colour. 
\section[{j) Test for steroids (Salkowski Test)}]{j) Test for steroids (Salkowski Test)}\par
To 2 ml of extract, 2 ml of chloroform and 2 ml of conc. H 2 SO 4 was added. The solution was shaken well. As a result chloroform layer turned red and acid layer showed greenish yellow fluorescence. 
\section[{Figure (c) k) Test for alkaloids(Hager's Test)}]{Figure (c) k) Test for alkaloids(Hager's Test)}\par
To the 2-3 ml of filtrate, 1ml of dil. HCI and Hager's reagent was added and shake well. Yellow precipitate was formed showing the presence of alkaloids. 
\section[{l) Mayer's Test}]{l) Mayer's Test}\par
To the 2-3 ml of filtrate, 1 ml of dil. HCI and Mayer's reagent was added and shake well. Formation of yellow precipitate showed the presence of alkaloids. 
\section[{m) Dragendroff's Test}]{m) Dragendroff's Test}\par
To the 2-3ml of filtrate, 1ml of dil. HCI and Dragendroff's reagent was added and shake well. Formation or orange-brown precipitate showed the presence of alkaloids.  
\section[{n) Wagner's reagent test}]{n) Wagner's reagent test}\par
To the 2-3ml of filtrate, 1ml of dil. HCI and Wagner's reagent was added and shake well. Formation of reddish-brown precipitate showed the presence of alkaloids. 
\section[{o) Test for Flavonoids (With Lead Acetate )}]{o) Test for Flavonoids (With Lead Acetate )}\par
To the small quantity of extract lead acetate solution was added. Formation of yellow precipitate showed the presence of flavonoids. 
\section[{p) Test for Tannins and Phenolic compounds (FeCI 3 Solution Test)}]{p) Test for Tannins and Phenolic compounds (FeCI 3 Solution Test)}\par
On addition of 5\% FeCI 3 solution to the extract, deep blue black colour appeared. 
\section[{q) Lead Acetate Test}]{q) Lead Acetate Test}\par
On addition of lead acetate solution to the extract white precipitate appeared. 
\section[{r) Test for Saponins (Foam Test)}]{r) Test for Saponins (Foam Test)}\par
To 1mol extract 20ml distilled water was added and shakes well in measuring cylinder for 15min. Then 1cm layer of loam was formed. Above phytochemical analysis will be carried out using standard procedure  {\ref [11;12]}.  
\section[{Key: + = Present -= Absent}]{Key: + = Present -= Absent}\par
These include Alkaloids, Saponins, Steroid, Carbohydrate, Tannins, Quinone, Coumarins, Phenolics, Terpenoids Fixed Oil, Fat and Flavonoids as shown in Table \hyperref[tab_2]{2}. As it is expected for ethanolic solvent used being an active component extractor \hyperref[b12]{[13]}. Therefore, the presence of these secondary compounds validates the use of oranges pulps as herbal drugs anywhere they are found. On carrying out phytochemical analysis, crude extracts were fractionated into acidic, basic, polar and nonpolar fractions as shown in Table \hyperref[tab_3]{3}. The highest quantity of phytochemical was found to be oil from hexane fraction thereby indicating steroidal properties responsible in the hormonal production and enhancement. Most Alkaloid fraction is known to be poisonous. Thus, it was the least fraction obtained from the fruitof Citrus aurantium var. Dulcis L. pulp showing their friendly and less harmful as to be used in medicine.\par
Each fraction obtained through the bioassay fractionation protocol showed fluorescence under the UV observation. Thus, wavelength between 254-365nm has indicated the presence of secondary metabolites in the fractions. The ultraviolet region extends from about 10 to 380nm, but the most useful region in analysis is from 200 to 380nm, called the near-ultraviolet or quartz UV region. This is as a result of chromophores acting as chromatogram and conjugation (where multiple e.g., double and triple bonds are separated by just one single bond each) between the double bonds from oxygen atoms with the single bonds present in the structure. The different colours of the fluorescence rings are due to different atoms present in the compound having different wavelengths. When atoms are excited to a higher energy level, they may fall back to their original position using the same or a different wavelength resulting to emission of different colours \hyperref[b14]{[14]}. At still higher energies (visible and ultraviolet wavelengths) different levels of electronic transition take place, and rotational and vibrational transitions are superimposed. Thus, indicating that important medicinal compound could be present in the fruit of Citrus aurantium var. Dulcis L. pulp fractions. Phytochemicals are known to possess antimicrobial properties as reported \hyperref[b12]{[13]}. This showed that the orange pulps were rich in chemical constituents. These principles have been known for many years to exhibit biological activity, such as effects on the central nervous system, and antibacterial, antiturmour, and anthehelmintic activity \hyperref[b16]{[16]}. Many alkaloids are known to have effect on the central nervous system and some act as antiparasitic (such as morphine, a pain killer). Quinine was widely used against Plasmodium falciparum. In this respect, it is found from the phytochemical screening that most plants traditionally used to treat malaria contain alkaloids among other things. Analgesia is another property of many alkaloids containing plants used in traditional medicine. Degenerative disorders, such as gouts and rheumatism, have also been traditionally treated with alkaloidcontaining plants. Cochicine compounds are well known in treating gouts \hyperref[b14]{[14]}. Alkaloids which have antiinflammatory activity were present in the orange pulp and Saponins which have anti-inflammatory and considered as hemotoxic. Coumarins were present which is precursor for several anticoagulants. Tannins were present which have astringent and detergent properties were also present and can be used against diarrhea \hyperref[b15]{[15]}. The presence of these compounds in Citrus aurantium var. Dulcis L pulpwill be useful in the treatment of diseases associated with the heart, antiinflammatory action, anticoagulant, diarrhea and dysentery. Steroidal compounds are known to behave like hormones \hyperref[b16]{[16]} have reported oils, alkaloids and associated with plants to have medicinal value. Others are Tritepenoids, which include: Cardiac Glycosides, Sterols, Saponins and Tritepenes. Mode of action of compounds present in the extracts indicates that the extracts from these pulps have the potential of solving the problem of multi-drug resistance.  
\section[{VI. Conclusion}]{VI. Conclusion}\par
The study is useful for the utilization of natural product waste fruit such as the fruit of Citrus aurantium var. Dulcis L. pulpas therapeutic agents especially those that are thrown away been considered not very necessary. These may be more needed for the body wellbeing as it contains very important phytochemicals. Thus, it provides an ethnobotanical data of the medicinal fruits as used by the local practitioners, traditional healers to cure different diseases, and promote a practical use validation and to bring back the extinct knowledge for medicine. Further detailed exploration and collection of ethnobotanical information, chemical studies and screening for medicinal properties which are ignored will also provide less cost effective and reliable source of medicine for the welfare of humanity. However, the observations from the present study need to be further validated with isolations of compounds and pharmaco-chemical studies, in order to confirm their efficacy of such components present in the phytochemicals as a future drug.\begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-2.png}
\caption{\label{fig_0}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-3.png}
\caption{\label{fig_1}Figure}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-4.png}
\caption{\label{fig_2}Figure}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-5.png}
\caption{\label{figure5}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-6.png}
\caption{\label{figure6}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_0}Table 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2} \par 
\begin{longtable}{P{0.02811284046692607\textwidth}P{0.25632295719844356\textwidth}P{0.4299610894941634\textwidth}P{0.061186770428015555\textwidth}P{0.07441634241245136\textwidth}}
\tabcellsep \tabcellsep \tabcellsep \tabcellsep Year 2015\\
\tabcellsep \tabcellsep \tabcellsep \tabcellsep )\\
S/NO 1\tabcellsep Constituent\tabcellsep Chemical Hager's Reagent Dragendroff's Reagent\tabcellsep Observation + +\tabcellsep Research (\\
2 3 4 5\tabcellsep Alkaloids Carbohydrate \& reducing sugar Steriods Saponins Phenolics\& Tannin\tabcellsep Mayer's Reagent Wagner's Regent Fehling's Regent Benedict's Regent Molisch's Regent Salkowski Regent Foam Lead Acetate FeCI 3 Sol.\tabcellsep + + + + + + + + +\tabcellsep Global Journal of Medical\\
6\tabcellsep Fixed oil \& fats\tabcellsep Spot test\tabcellsep +\tabcellsep \\
7\tabcellsep Proteins\tabcellsep Biurret Reagent Million's Regent\tabcellsep --\tabcellsep \\
8\tabcellsep Anthraquinone glycosides\tabcellsep Borntrager's Reagent\tabcellsep -\tabcellsep \\
9\tabcellsep Cardiac glycosides\tabcellsep Keller-Killiani Reagent\tabcellsep -\tabcellsep \end{longtable} \par
  {\small\itshape [Note: 5 Volume XV Issue V Version I © 2015 Global Journals Inc. (US) B Preliminary Phytochemical Investigations with Quantitative Fractionation of Orange Pulp (Citrus Aurantium Var. Dulcis L.): Natural Product Waste as Medicine]} 
\caption{\label{tab_2}Table 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{P{0.04180327868852459\textwidth}P{0.3158469945355191\textwidth}P{0.12540983606557377\textwidth}P{0.21830601092896174\textwidth}P{0.14863387978142076\textwidth}}
S/No\tabcellsep Extractives\tabcellsep Weight\tabcellsep Colour\tabcellsep Texture\\
1\tabcellsep Methanolic\tabcellsep 1.3g\tabcellsep Yellow\tabcellsep Viscous\\
2\tabcellsep Basic\tabcellsep 0.3g\tabcellsep Light brown\tabcellsep Solid\\
3\tabcellsep Hexane(unsaturated)\tabcellsep 2.0g\tabcellsep Orange\tabcellsep Oily\\
4\tabcellsep Acidic\tabcellsep 1.2g\tabcellsep Brick red\tabcellsep Solid\\
5\tabcellsep Hexane(saturated)\tabcellsep 40.0g\tabcellsep Red oxide\tabcellsep Oily\end{longtable} \par
 
\caption{\label{tab_3}Table 3 :}\end{figure}
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