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Home , Cucumis, Melon, Watermelon, Watermelon seed oil

SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

ANALYSIS OF ESSENTIAL FROM

Mansur Ahmad, Riskuwa Faruk, Kasimu Abubakar Shagari and Shehu Umar Shehu Shagari College Of Education, Sokoto- [email protected] ABSTRACT Watermelon belongs to the family of and it well known in Nigerian modern and traditional system for its modern and traditional uses. The present investigation was carried out to determine the chemical properties, proximate composition as well as the possible bioactive components of watermelon seeds using GCMS analysis. The oil from the seeds was extracted using soxhlet extraction procedure. Moisture was determined directly on the seeds by oven drying at 105oC for 6 hours. The yield of the dry seeds from the sample was determined. The ripened seeds and dry seeds were then ground in some blender, separately, and placed in a vacuum oven at 60oC for 6 hours and finally stored in a desiccator until analyzed. Proximate analyses were performed in triplicate in accordance with the AOAC procedures (AOAC, 1990). The ash was determined by heating overnight at 500oC and the content of the seeds by standard Kjeldahl (total %N) procedure. Wiss and Devine (1961) method was adopted for the chemical analysis. Free Fatty Acid of the oil (2.46±0.01%) which shows the better quality of the oil. The acid value (7.60mg/NaOH/g) fall within acceptable limits for edible (≤10mg/NaOH/g). The GC-MS analysis was carried out on a GC-MS-QP 2010 Shimadzu system using NIST database. Seven (7) components from watermelon seeds were identified. The prevailing elements in the watermelon seeds were , carbonic acid, , Dioctylester, propanedioic acid, chloride and delta-tocopherol. The presence of various bioactive compounds confirms the applications of watermelon for multiple ailments by traditional and modern practitioners. However, isolation of individual phytochemical constitutes may proceed to find a novel drug or lead compound.

Keywords: Watermelon, Seeds, GC-Ms, Proximate Analysis, Essential Oil

INTRODUCTION The use and dependence on as medicines by man has been in existence since time immemorial, and man continues to search for plants as drugs for a particular disease within his reach. Synthetic medicines are safe than herbal medicines because the phytochemical in the extract target the biochemical pathway (Zaidan and Badrul, 2005). Medicinal plants are an expensive gift from nature to human. The approval of traditional medicines as an alternative form of healthcare and the improvement of microbial resistance to the existing antibiotics has lead researchers to scrutinize the antimicrobial compounds (Parvathi, S., 2010). Medicinal plants have been used all over the world for the treatment and prevention of various ailments especially in developing countries where infectious diseases are endemic and modern health facilities, and services are inadequate (Zaida and Badrul, 2005). The medicinal actions of plants unique to particular plant species or groups are consistent with the concept that the combination of secondary products for a specific plant taxonomically distinct (Wink et at., 1999). 1

Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

Screening active compounds from the plant have led to the invention of new medicinal drugs which have protection and treatment roles against various diseases including cancer, Alzheimer‟s diseases (Sheeja and Kuttan, 2007). Plant remains a vital source of drugs and nowadays much emphasis has been given to nutraceuticals. Watermelon ( lanatus), family Cucurbitaceae is a -like originally from southern . Its , which is also called watermelon is a referred to by botanists as a pepo, a which has a thick rind (exocarp) and fleshy center (mesocarp and endocarp) pepos are derived from an inferior and are characteristic of the Cucurbitaceae. The watermelon , loosely considered a type of -although not in the genus has a smooth exterior rind (green, yellow and sometimes white) and a sweet interior flesh (usually pink, but sometimes orange, yellow, red and sometimes green if not ripe). It is also commonly used to make a variety of salads, most notably fruit salad (Pomeranz and Clifton, 1981).

HISTORY Watermelon is thought to have originated in southern Africa, where it is growing wild. Alphonse de Candolle, in 1882 already considered the evidence sufficient to prove that watermelon was indigenous to tropical Africa (North Carolina State University). Though Citrullus cololyn this often supposed to be a wild ancestor of watermelon and is now found a native in North and West Africa. (Dane and Liu, 2007) suggest by chloroplast DNA investigations that the cultivated and wild watermelon appears to have diverged independently from a common ancestor, possibly Citrullus ecirrhosus from Namibia. It is not known when the plant was first cultivated, but Zohary and Hopf note evidence of its cultivation in the valley from at least as early as the second millennium BC. Although watermelon is not depicted in any Egyptian hieroglyphic text nor makes any ancient writer mention, it finds of the characteristically large seeds is reported in Twelfth dynasty sites numerous watermelon seeds were recovered from the tomb of Pharaoh Tutankhamen (Daniel and Maria, 2000). By the 10th century AD, were cultivated in , which is today the world‟s single largest watermelon producer. By the 13th century, Moorish invaders had introduced the fruit to according to John Mariana the Dictionary of American food and Drink. Charles Fredric Andrus, a horticulturist at the USDA Breeding laboratory in Charleston South Carolina, set out to produce a disease-resistant and wilt-resistant watermelon. The result was that gray melon from Charleston. Its oblong shape and hard rind made it easy to stack and ship. Its adaptability meant it could be grown over a wide geographical area. It produced high yields and was resistant to the most severe watermelon disease Anthracnose and Fusarium wilt. In , farmers of the Zentsuji region found a way to grow cubic watermelons by planting the fruits in glass boxes and letting them naturally assume the shape of the receptacle. The square is designed to make the easier to stack and store, but the square watermelons are often more than double the price of normal ones. Pyramid shaped watermelons have also been developed, and any polyhedral shape may potentially also be used.

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

VARIETIES There are more than 1200 varieties of watermelons ranging from less than a pound to more than two hundred pounds, with flesh that is red, orange, yellow or white (Baker Creek, 2008). Several notable varieties are as follows:

Carolina cross: - This variety of watermelons produced the current world record melons weighing 262 pounds (119kg). It has green skin, red flesh and commonly produces fruits between 65 and 150 pounds (29 and 68 kg). It takes about 90 days from planting to harvest, (Baker Creek, 2008).

Yellow Crimson: - Variety of watermelon that has a yellow colored flesh. This particular type of watermelon is “sweeter” and more “honey” flavored than another red flesh watermelon (Anioleka et al., 2007).

Orangeglo: - This variety has a very sweet orange pulp, and is a significant oblong fruit weighing 9kg (20-30 pounds). It has a light green rind with Jaggiest dark green stripes. It takes about 90-100 days from planting to harvest. The Moon and Stars variety of watermelon has been around since 1926 (Moons and star, 2008). It has many small yellow circles with purple-black rind, (Stars) and one or two large yellow circles (Moon). The watermelon weights 9-23kg (20-50 pounds) (Johnson and Sahaya, 2012). The flesh is pink or red and has brown seeds. The foliage is also spotted. The time from planting to harvest is about 90 days.

Cream of Saskatchewan: - This variety consists of small round fruits around 25cm (10 inches) in diameter. It has a quite thin, light green with dark green striped rind, with sweet white flesh and black seeds. It can grow well in cold climates. This variety was brought to Saskatchewan, by Russian immigrants. These watermelons take 80-85 days from planting to harvest.

Melitopolski: - This variety has small round fruits roughly 28-30cm (11-12 inches) in diameter. It is an early variety that originated from the Volga River region of are known for the cultivation of watermelons. This watermelon is seen piled high by vendors in Moscow in summer. This variety takes around 95 days from planting o harvest.

Densuke: - This variety has round fruit up to 25 16 (11 kg). The rind is black with no stripes or spots. It can only grow on the Island of Hokkaido, Japan, where up to 10,000 watermelons are produced every year. In June 2008, one of the first harvested melons is being sold at an auction for 650,000 yen (6300 USD) making the most expensive watermelon ever sold. The average selling price is generally around 25,000 yen (250 USD) (Vohra and Kaur, 2011). LITERATURE REVIEW

Definition of and Oils Fats and oils can be defined by layman as the substances that are clearly fatty in nature, greasy in texture, and are immiscible with biochemically , fats and oils are defined as substances that are insoluble in water and can be extracted by organic solvents of low polarity 3

Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161 such as petroleum ether chloroform, methanol, n-hexane, etc. Chemists defined fats and oils as the triglycerides of fatty acids in which one molecule of glycerol combined with three molecules of long chain fatty acid with the elimination of water molecule.

Sources of Fats and Oils Fats and oils from plants are found mostly in fruits and seeds while in animals it‟s stored as fatty tissues which protect certain parts of the body and as insulation against cold. Synthetic fats and oils are produced by oxidation of hydrocarbons to fatty acids, which are then esterified with glycerol, or synthesis of fatty acids from by microorganisms.

Essential Oils Essential oils are highly volatile substances isolated by physical process from an odoriferous plant of a single botanical species. Therefore the fragrance of buds flowers leaves or bark, and terminal branches of individual plants is due to the presence of these volatile oils. They bear the name of the plant from which it is derived from example peppermint oil or rose oil. Such essentials oil was thought to represent their very essence of odor and flavor. After many years of research, it is now known that these essential oils are a mixture of up to twenty or more fragrance chemical compounds of highly aromatic substances mostly benzene straight chain hydrocarbons compounds of intermediate molecular lengths and terpene derivatives.

ECONOMIC IMPORTANCE OF WATERMELON

Nutritive Values Watermelon raw (edible part) has the total energy of about 127kj (30kcal), Carbohydrates content 7.55g, (6.2g), (0.4g) (0.15g) Protein (0.61g) Water (91.45g),

Vitamin A equivalent to 28µg (3%), Thiamin (Vit.B1) 0.033mg (3%), Riboflavin (vit.B2)0.021mg (1%), Niacin (Vit.B3) 0.178mg (1%), Pantothenic acid (B5) 0.221mg (4%), Vit.B6 0.045mg (3%), Folate (Vit. B9)3.4g (1%), Vit.C 8.1mg (14%), Calcium 7mg (1%) Iron 0.24mg (2%), Magnesium 10mg (3%), 11mg, Potassium 112mg (2%), 0.10mg (1%), (Iakshmi and Kaul, 2011).

Sources of Other Compounds/Substances Watermelons contain a significant amount of citrulline, and after consumption of several kilograms of watermelon, an elevated concentration is detected in the blood plasma, this could be mistaken for citrullinaemia or other urea cycle disorders (Martin and Negro, 2007). Watermelon rinds usually a light green or white color is also edible and contains many hidden that most people avoid eating due to its unappealing flavor. They are sometimes used as a vegetable (Martin and Negro, 2007) In China, watermelons are stir-fried, stewed or more often pickled. When stir-fried the skinned and fruited rind is cooked with , garlic, chili peppers, scallions, sugars, and rum. Pickled watermelon rind is also commonly consumed in the southern us. Watermelon juice can also be made into . Watermelon is also mildly diuretic and contains significant amounts of beta-carotene. Watermelon with red flesh is a significant source of , (Hdider and Lenucci, 2011). 4

Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

Health Benefits of Watermelons Watermelon has plenty of potassium that is useful in cleansing the toxic depositions of kidneys. Additionally, it‟s helpful in lowering amount of uric acid in the body, thus reducing the risk of kidney problems and development of renal calculi. Besides these, being loaded with water contents, it encourages frequent urinating; again it‟s useful for kidney cleansing. Furthermore, the antioxidants in the watermelons ensure the well-being of kidneys. A great deal of magnesium and potassium found in the watermelons are incredibly effective in reducing the blood pressure level. The contained in the fruit protect walls of arteries and veins from hardening, thus helping to lower blood pressure level, (Poduri et al., 2012). Alongside tomatoes, watermelon has moved up to the front of the line in recent research studies on high-lycopene foods. Lycopene is a phytonutrient that is especially important for our cardiovascular health, and an increasing number of scientists now believe that lycopene is an important for bone health as well. Among whole, fresh fruits that are commonly eaten in the U.S., watermelon now accounts for more U.S. intake of lycopene than any other fruit, (Saha and Poduri, 2012). Health scientists are becoming more and more interested in the citrulline content of watermelon. Citrulline is an that is commonly converted by our kidneys and other organ systems into arginine (another amino acid). The flesh of a watermelon contains about 250mg of citrulline into arginine. In particular, if a person‟s body is not making enough arginine, higher levels of arginine can help improve blood flow and another aspect of our cardiovascular health, (Azevedo et al., 2007). There is also some preliminary evidence from animal studies that greater conversion of citrulline into arginine may help prevent excess accumulation of fat in fat cells due to a blocked activity of an enzyme called alkaline phosphatase, or TNAP, (Saha et al., 2012).

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

MATERIALS AND METHODS

MATERIALS: The material used includes the fruits of fresh watermelons.

CHEMICALS REAGENTS MANUFACTURER %PURITY

Petroleum ether B.O.H Chemical Ltd. England 99.8% Sulphuric acid B.O.H Chemical Ltd. England 97% Sodium hydroxide B.O.H Chemical Ltd. England Conc. HCl B.O.H Chemical Ltd. England 99% Phenolphthalein B.O.H Chemical Ltd. England Pyridine B.O.H Chemical Ltd. England 96% Ethanol B.O.H Chemical Ltd. England 99.9% Starch C. Gerhard Gmbu Company Potassium iodide B.O.H Chemical Ltd. England 97% Iodine James Borough Puy Ltd. London 99% Chloroform B.O.H Chemical Ltd. England 99.4% Sodium thiosulphate B.O.H Chemical Ltd. England 98% Glacial acetic acid B.O.H Chemical Ltd. England 99.8% Potassium chloride B.O.H Chemical Ltd. England 97% Ammonium sulfate B.O.H Chemical Ltd. England 98% Boric acid indicator B.O.H Chemical Ltd. England Sodium sulfate B.O.H Chemical Ltd. England 96% Copper sulfate B.O.H Chemical Ltd. England 95%

APPARATUS APPARATUS TYPE/MODEL COMPANY Spatula Glass Pyrex glass England Volumetric flask Glass Pyrex glass England Measuring cylinder Glass Pyrex glass England Burette Glass Pyrex glass England Conical flask Glass Pyrex glass England Tripod Metallic Pyrex glass England Soxhlet set Hot air oven Metallic, HQ4400 Muttle, Switzerland GC-MS machine QP 2010 Shimadzu, Japan Crucibles Ceramic Muttle, Switzerland

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

Sampling Watermelon fruits were purchased from Ramen-Kura, a local market for in Sokoto state, in the northern part of Nigeria. The flesh is removed and the seeds collected were washed and dried for easy removal of the epicarp.

Sample Preparation The melon seeds were then subjected to sun-drying the melon seeds of 100g were dry-milled, and the oil content was extracted by Soxhlet extraction method before being subjected to chemical, proximate and GC- MS analyses respectively.

Extraction of Oil from Watermelon Seeds From the grounded sample of watermelon seeds, 50g was weighed into an empty thimble which was placed in a Soxhlet extraction set. 250ml of n-hexane solvent was then poured into the extraction flask. The bottom of the extractor containing the sample was connected to the extraction flask and placed on the heating mantle. As temperature increased steadily n-hexane boiled and formed a vapor which was condensed by the condenser formerly attached to the extractor and then dropped into the thimble dissolving and extracting oil present in the powdered seeds. The extraction was carried out continuously for 6 hours. After the extraction procedure, the thimble was removed then the n-hexane formed a vapor which condensed and was collected in the receiver of the Soxhlet extractor while the oil remained in the flask allowed to cooled and weighed. The procedure was repeated for the remaining grounded sample of the watermelon seeds, and the oil was obtained.

Reagents Preparation

Na2S203.5H20 (0.1M) Six (6.0g) of sodium thiosulphate was dissolved in a volumetric flask and made up to mark 250cm3 with distilled water. Starch Indicator (1%) One (1.0g) of starch was dissolved in 5sm3 distilled water and then made up to 100cm3 volumetric flask with hot distilled water refluxed for 5minutes and then allowed to cool. KI (10%) Ten (10g) of KI was dissolved in a beaker containing 90ml distilled water and then made up to 100cm3 mark volumetric flask with distilled water. KOH (0.1M) One and a half i.e. (1.5g) of KOH was dissolved in distilled water and made up to mark 250cm3 distilled water. Alcoholic KOH (0.1M) A pellet of (1.4g) of KOH was dissolved in 50cm3 of ethanol and made up to mark 250cm3 with distilled water. HCl (0.5M) About 10.6cml of concentrated HCl was poured into a beaker containing distilled water and made up to 250cm3 with distilled water. Phenolphthalein (1%) 7

Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

One gram (1.0g) of phenolphthalein was dissolved in 100cm3 of ethanol. Solvent Mixture The solvent mixtures were prepared by mixing 125cm3 of each of toluene and isopropyl alcohol and 4cm3 of 1% phenolphthalein in isopropyl alcohol. Na0H (0.25M) One gram, i.e. (1.0g) of Na0H was dissolved in a 1dm3 volumetric flask and made up to mark with distilled water. ICI (Iodine Monochloride)

Pyridine 8.2ml by volume and 6ml of conc. H2S04 was mixed in 20ml cooled glacial acetic acid, and 26ml of bromine was added to this solution. The mixture was then diluted to Idm3 with glacial acetic acid and was kept in the dark.

ANALYSIS AND DETERMINATIONS: PROXIMATE ANALYSIS

Determination of Moisture The moisture content of melon seeds was determined by putting freshly obtained seeds in an oven at 1050C. The moisture content was also determined by drying the rind and finding the loss in weight.

Procedure An empty crucible was weighed; 2g of fresh seeds were considered into it and placed in an oven at 1050C for 24 hours. After 24 hours they were removed allowed to cool and weighed. 2g of the fresh seeds were weighed and shed dried and reweighed.

Determination of Ash Contents Ash is the non-volatile inorganic matter of the sample that remains after subjecting it to high temperature. Procedure: - An empty crucible was weighed. 2g of the fresh sample was weighed and transferred into the pre-weighed crucible. It was then heated in a muffle furnace at a temperature of 6000C for s2 hours. It was allowed to cooled and weighed again. Determination of Crude Protein The Kjeldahl method was used for crude protein determination.

Principle The method is based on the transformation of protein nitrogen and other nitrogenous compounds other than nitrate and nitrite into ammonium sulfate by acid digestion with strong 4 acid usually concentrated H2S0 i.e. Sample nitrogen + H2S04 (NH4)2 SO4 The ammonia present is digested with the help of sodium hydroxide and distilled out and collected into receiving flask containing a boric acid indicator which changed color from pink to green.

(NH4)2S04 + Na0H 2NH3 + H20 + NaS04 + NH3 + H3BO NH 4 + H2B03

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

The nitrogen content was thus estimated by titration the content in the receiving flask with standard acid (H2 S04). + H H2B03 H3B03 The amount of H+ consumed in the reaction is equivalent to the amount of nitrogen present in the sample. The amounts of protein were then estimated by multiplying the concentration of nitrogen in the sample by a conversion factor of 6.6 which is equivalent to 16gN/100g protein.

Procedure From the dried grounded sample, 2grams was weighed into Kjeldahl digestion flask, and a catalyst mixture was added (NaS04, CuS04 and Selenium oxide in the ratio 10:5:1). The content was heated in the sulfuric acid (H2S04). The content was heated in the Kjeldahl digestion unit until the digestion was completed (approximately 30minutes). The flask was cooled, diluted volumetric flask and made up to the mark with distilled water. 10ml of the aliquot was then taken into the digestion flask, and 20mls of 45% Na0H solution was added. The content was diluted to about 200ml with distilled water and distilled using Kjeldahl distillation apparatus. The distillates were received into receiving flask containing 10mls boric acid solution indicator. After distillation, the distillate was titrated with standardized 0.1M HCl to the endpoint. The blank was determined in the same way without the sample.

DETERMINATION OF CRUDE FIBRE (AOAC 1990)

Procedure From the grounded sample, 2grams was weighed and placed in a conical flask containing

200ml of 1.15% H2S04 sulphuric acid and boiled gently for 39 minutes. The content was filtered, and the residues were scraped into the container with a spatula. The oxidant 1.25% sodium hydroxide Na0H was added and allowed to boil gently for 30minutes. The content was then filtered and washed thoroughly with hot distilled water. The precipitate was allowed to dry, and the residue was scraped into a crucible and dried overnight at 105OC in a hot air oven. It was then removed and cooled in a desiccator. The sample was then weighed and ashed at 6000C for ninety minutes in a furnace. This was finally cooled in a desiccator and weighed.

DETERMINATION OF CRUDE LIPID Soxhlet extractor used for crude lipid determination. 2grams each of the dried grounded samples were weighed into a porous thimble and its mouth covered with cotton. The thimble was then placed in an extractor chamber, which was suspended above a receiving flask containing petroleum ether (boiling point 40-600C). The flask was heated on a hot mantle and the oil extracted.

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

The extraction continues for about eight hours (8) after which the thimble was removed from the soxhlet, and the apparatus was reassembled and heated on water-bath apparatus for solvent recovery. The flask containing the crude oil was then disconnected, cleaned up and placed in an oven at 1000C for thirty minutes (30). The flask was then cooled in a desiccator and weighed.

DETERMINATION OF CARBOHYDRATES To obtain the % content, all the above proximate analysis parameters have to be summed and subtracted the value obtained out of 100. The remaining value obtained after substitution is our percentage carbohydrate content in the sample. %CH0= 100- (% Ash +% crude protein +% crude fiber + % crude lipid).

ANALYSIS AND DETERMINATION PHYSICO-CHEMICAL ANALYSIS

DETERMINATION OF PEROXIDE VALUE Peroxide value is an index of lipid deterioration expressed as peroxide per kg of fat or mili- equivalent. It is the first products of unsaturated oil when peroxide value reaches a certain level; complex change occurs with formation of ketone, aldehyde and hydroxyl group all these are volatile and responsible for odor and flavor.

Procedure From the extracted oil, 2g was weighed into a conical flask, and 25ml of glacial acetic acid and chloroform was added. 1ml of KI solution was added and allowed to stand in the dark for 1 minute so that the solution becomes straw yellow. 35 ml of distilled was also added and then followed with ten drops of 1% starch indicator. This was titrated against 0.004M

Na2S203.5H20 till blue color disappeared. The procedure was done in triplicate and blank was estimated without adding oil.

DETERMINATION OF ACID VALUE It is the percentage of free fatty acid expressed as oleic acid.

Principle Acid value of oil is determined by titration of known weight of oil against 0.25N Na0H using phenolphthalein as indicator

C17H33COOH + Na0H C17H33C00Na +H20

Procedure From the extracted watermelon oil, 1g was weighed in a conical flask 50ml of denatured alcohol was then added and shaken and two drops of phenolphthalein indicator were added to the mixture then titrated against 0.25M Na0H with vigorous shake until the permanent light pink color was observed.

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

SAPONIFICATION VALUE William and Devine's method was used (1961). Saponification number is the amount of milligram of potassium hydroxide (KOH) required to completely saponify (I.0g) of oil.

Principle When oils are treated with excess alcoholic (KOH), the oil gets saponified, and a definite amount of (KOH) is used up. The excess KOH left unused may then be found by titrating against 0.5M HCl.

Procedure From the oil extracted 0.5g was weighed in a quick-fit-reflux flask, and 25ml alcoholic KOH was then added. It was refluxed for 30minte so that it gets simmered. The flak was allowed to cool, and 1ml of phenolphthalein indicator was then added and titrated against 0.5M HCl.

IODINE VALUE The method described by Devine and Williams (1961) was used. This is the percentage of iodine monochloride (ICI) regarding iodine absorbed by the oil.

Principle The method is based on the treatment of a known weight of oil or fat with a known volume of a standard solution of iodine monochloride (ICl). Estimation of ICI is by titrating iodine liberated by adding excess potassium iodide. Titration was done against sodium thiosulphate

(Na2S203.5H20) with starch as indicator. ICI + KI KCI +I

2Na2S203+ I2 Na2S406+2NaI

Procedure

From the oil extracted, 0.3g was weighed, and 10ml of carbon tetrachloride (CCl4) was then added. Also, 25ml of Wiss solution was added the flask was shaken and allowed to stand in the dark for one hour. 15ml of 10% potassium iodide and 100ml of distilled water was added to the mixture. The mixture was titrated against (0.1M) Na2S203.5H20 until the blue coloration disappeared which indicated an endpoint. The blank solution was also titrated at the same time without oil.

DETERMINATION OF PERCENT FREE FATTY ACID From the oil extracted, 10g was weighed and then boiled with 50ml ethanol, allowed to cool and two drops of phenolphthalein indicator were added. The mixture was then titrated against 0.1M Na0H until the pink color was obtained.

Gas Chromatography-Mass Spectrometry (Gc-Ms) Analysis The GC-MS analysis was carried out on a GC-MS-QP 2010 plus Shimadzu system and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161 following conditions: column Elite – 1 fused silica capillary column (30m x 0.25mm 1D x µI composed of 100% (dimethyl-polysiloxane). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate 1ml/min, and an injection volume of 2µl was employed (split ratio of 10:1) injector temperature- 2500C ion-source temperature 2800C the oven temperature was programmed from 1100C (isothermal for 2min.) to 280oC/min, ending with a 9min isothermal at 2800C. Mass spectra were taken at 70 eV; a scan interval of 0.5 sec and fragments from 40 to 550 Da. Total GC running time was 36minutes. The relative percentage amount of each component was calculated by comparing its average peak area to the entire areas; software adapted to handle mass spectra and chromatogram was a turbo mass. The detection employed the NIST Ver. 2.0 the year 2009.

Identification Of Components The interpretation of mass spectrum of GC- MS was done using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unknown components was compared with the mass of the known elements stored in the NIST library. The name, molecular weight, and structure of the parts of the test material were ascertained.

RESULTS

Proximate Analysis The moisture content of the seed is quite low (4.56+ 0.04%) and falls within the range of moisture content of similar seeds as shows from the table below. The ash content (4.20 +0.02%) obtained is higher than the established value for animal feed. The fat, crude protein, and nitrogen-free extract are also high.

TABLE: 1 PROXIMATE COMPOSITION OF WATERMELON SEEDS Parameters Composition Moisture 4.50+0.04 Crude lipid 41.46+0.03 Crude Protein 25.50+05 Crude Fiber 6.34+0.01 Ash 4.20+0.02 Free Nitrogen 14.97+0.08

CHEMICAL ANALYSIS Free Fatty acid (FFA) value for the watermelon seeds oil is (2.46 + 0.01%). The and were all slightly higher in the oil.

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

TABLE 2: PHYSIC-CHEMICAL PROPERTIES OF WATERMELON SEEDS OIL Iodine value %Acid value saponification value free fatty acid peroxide value 55.42+0.07 7.60+0.3 112.58+0.5 2.46+0.01 17.47+0.4

GC-MS ANALYSIS The components present in the watermelon seeds oil were identified by GC.MS analysis as shown in the table (3) below. The active compounds with their molecular formula, molecular weight, retention time (RT) and their percentage compositions are presented in table (3). Seven (7) compounds were identified in the oil and the prevailing compounds are Palmitic acid (Hexadecanoic acid) 3.80%, Octyl propyl ester (1.03% Delta-Tocopherol (5.16%), Oleic acid (9-octadecenoic acid) (17.84%), Propanediol acid (Dicyclohexyl ester) 3.74% Dioctyl ester (1, 2- Benzenedicarboxylic acid) (0.98%) and Linoleic acid chloride 13.77% the spectrum profile of seven major components with their respective retention time were shown in the table. The mass spectrum of the compound with retention time 22.50 and 13.77 gave the primary peak.

Table 3.Detected components in watermelon seeds oil using GCMS technique. SN Name of compound Mole formula Mole weight RT % composition

1 Palmitic acid C16H3202 256 18.H 3.80

2 Carbonic acid (Octadecylpropy ester C22H4403 356 6.51 1.03

3 Delta-Tocopherol C27H 4602 402 5.7 5.16

4 Oleic acid (9 octadecenoic acid ) C18H3404 282 19.8 17.84

5 Propanedioc acid C15H2404 268 6.18 3.74

6 Dioctyl ester C24H3804 390 23.10 0.98

7 Linoleic acid chloride (19,12- C18H31Cl0 298 22.50 13.77 octadecadienoyl chloride) unidentified compounds 53.68 Total % composition 100

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

The Chromatogram of the compounds detected from the watermelon

Structures of some of the identified compounds using GC-MS analysis

Oleic acid

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Umaru Ali Shinkafi Polytechnic Sokoto, Nigeria SosPoly Journal of Science & Agriculture, Vol. 2, (Dec., 2017) ISSN: 2536-7161

DISCUSSION The low moisture content of watermelon seed is advantageous regarding the shelf life of the grain, with less moisture content seeds able to be preserved for a more extended period. From literature, ash content for nuts, seeds, and tubers should fall within acceptable limits for edible oils (1.5-2.5%) to be suitable for animal feed. The high quantity of oil content is an indication that watermelon seed is another ready source of oil like the peanuts and soybean seeds. The high protein content of the oil has a pleasant implication a society with high protein deficiency. Fatty acid value of oil is highly essential in considering the quality of oil because of the lower the free fatty acid, the better the quality of the oil which is (2.46%) and acid value (7.60mg/Na0Hg) which fall within acceptable limits for edible oils <10mg/Na0Hg. Delta-Tocopherol is a phenolic compound, and it may be used as an antioxidant antimicrobial, antifungal and anti-inflammatory agents (Duke et al., 2012). Hexadecanoic acid (Palmitic acid) is fatty, and it may be an active antimicrobial and antidiarrheal agent. By interpreting the identified compounds by GC-MS analysis, it is found that watermelon possesses various therapeutic application. The chromatogram showed the relative concentrations of the components present in the fruit. In addition to this, the results of the GC-MS profits can be used as a pharmacognostical tool for the identification of the fruit. Dioctyl ester is used in many pharmaceutical industries as an antimicrobial agent, Octadecylpropyl ester as anti-inflammatory, Linoleic acid is used in most cases to reduce mastalgia (menstrual pain), Dicyclohexyl ester as an antifungal agent, Oleic acid as an anti- allergic agent, and Delta-Tocopherol as an antioxidant, (Tlili et al., 2011).

CONCLUSION The high protein content of the watermelon seed coupled with a relatively high concentration of the amino acids makes the seed suitable for fortification of food. The watermelons seed is preferred because of its low acid value, and the oil can serve as a supplement in the formulation of animal feed. In the present study, seven components from watermelon seeds oil were identified by the GC-MS analysis. The presence of various bioactive compounds justifies the use of this fruit for multiple ailments by traditional and modern practitioners. However, isolation of individual photochemical constituents and subjecting it to biological activity will give the fruitful result. It could be concluded that watermelon contains various bioactive compounds. So it is recommended a plant of phytopharmaceutical importance. However, further studies are needed to undertake its bioactivity and toxicity profile.

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