Phytochemical composition of desert date kernel (Balanites aegyptiaca) and the physical and chemical characteristics of its oil

The desert date (Balanites aegyptiaca) is an important tree found in some African countries. In this study the phytochemical composition of the desert date kernel and some physical and chemical properties of its oil were analyzed using standard procedures. The results of the phytochemical screening revealed the presence of alkaloids, saponins, flavonoids, tannins, steroids and glycosides. While the results for the physical analysis of the kernel oil revealed the following: Color, pale yellow; density, 0.910 g/cm3; specific gravity, 0.907; refractive index, 1.458; and viscosity, 19.68. And on the other hand, the results for the chemical analysis of the kernel oil revealed the following: Acid value, 3.06 mgKOH/g; free fatty acid, 1.27; peroxide value, 3.71 mEq/Kg; saponification value, 198 mg/KOH/g; and iodine value, 98.73 100/g. All the results were compared and found to be within the FAO/WHO standards.


Introduction
Desert date (Balanite aegyptiaca Linn.) is an important multipurpose tree found in most African countries (Clement et al., 2011). Balanites Aegyptica has multiple applications with almost every part of the plant being useful. The tree is actually valued more for its fruits and seeds: The fruits and kernel are widely used in many ways in different countries including Nigeria especially during the dry season and drought periods (Lockett et al., 2000). The kernel of this plant is traditionally used in the treatment of various ailments such as jaundice, intestinal worm infection, malaria, syphilis, epilepsy, dysentery, constipation, haemorroid among others (Daya and Vaghasiya, 2011). The stem of the plant is used as timber, poles, charcoal, firewood, fodder, food (Elseed et al., 2002), gum, shade, gum and windbreak (Guinand and Lemessa 2001). The plant may be grown for its fiber, oil and/or for medicinal values. It is also used in treatment of several diseases and disorders since ages.
Balanites Aegyptica fruit is readily available in northern Nigeria and the seed which is obtained after it is being sucked is seen as a nuisance along markets and settlements in communities (Jock 2017). This is because no suitable application for the seed kernel has been found in most developing countries. However, the seed kernel oil of Balanites aeyptiaca is a good source of raw material for food, cosmetic and pharmaceutical industries (Jock 2017).
This multipurpose tree is used for food and fodder in almost all parts of Africa and South Asia (Billore 1988; Elseed et al., 2002). Among the various useful parts of the plant, the fleshy pulp of the fruit is eaten fresh or dried; it also used as a food, beverage and medicines. The protein content of the fruit of this plant is believed to be superior to that in guava, mango, banana and papaya. The fleshy fruit contains high carbohydrates and steroidal saponins (Al-Thobaiti and Abu-Zeid 2018), vitamin A, vitamin C and other essential minerals for human. Seed kernel also widely used for oil. The kernel produces high quality edible oil (Obidah et al., 2009) with large number of medicinal properties (Hanan et al., 2009), and is also rich in protein and minerals contents (Elfeel and Warrag, 2011). In quality aspect, it is similar to sesame and groundnuts oils (Abu Al-Futuh, 1983; Obidah et al., 2009), with the oil used as a biodiesel (Chapagain et al., 2009;Gutti et al., 2012;Kumawat et al., 2012).
The aim of this study is to determine the phytochemical constituents of desert date kernel Balanites aegyptiaca, and also to investigate the physical and chemical characteristics of the desert date kernel oil.

Sample Collection and Preparation
The fruits of Balanites aegyptiaca (desert date) were purchased from Rimi market in Kano State, Nigeria and brought to the department of Biological Sciences, Yusuf Maitama Sule University, Kano, for identification by a plant taxonomist. The plant was identified and authenticated and issued a voucher specimen number YUHAN 0058. The fruits were crushed using a steel hammer to obtain the kernels, which were then air-dried and then ground using mortar and pestle (Bayero et al., 2019). The ground kernel was then packed in an air tight container and then stored in a desiccator (containing silica gel) ready for further analysis.
All the chemicals and reagent used were of analytical grade.

Extraction of the Sample
The powdered sample (50 g) was soaked in 300 ml of absolute ethanol for 72 hrs, and stored away from direct light. The supernatant was decanted and filtered using filter paper and the filtrate was evaporated to dryness, and then stored in sample bottles at room temperature to avoid any biological degradation (Bayero et al., 2019).

Phytochemical Analysis
The crude ethanol extract was phytochemically screened for the presence of secondary metabolites such as alkaloids, saponins, phenols, flavonoids, tannins, steroids, reducing sugars, terpenoids, glycosides, volatile oils and anthraquinones in accordance with standard methods by Harborne (1998)

Test for Alkaloids
The ethanol extract (2 g) was dissolved in dilute 1% hydrocloric acid and filtered.
Mayer's Test: A portion of the filtrate (1 ml) was treated with 4 drops of Mayer's reagent (potassium iodide). Formation of white or pale yellow precipitate indicates the presence of alkaloids.

Test of Saponin
Froth Test: A portion of the ethanol extract (2 ml) was diluted with distilled water to 5 ml and then shaken in a graduated cylinder for 5 minutes. Formation of honeycomb froth confirms the presence of saponin.

Test for Phenols
A portion of ethanol extract (2 ml) was treated with 4 drops of ferric chloride (FeCl3) solution. Formation of a bluegreen colour confirms the presence of phenols.

Test for Flavonoids
Alkaline Reagent Test: A fraction of the extract (2 ml) was treated with 4 drops of 1% sodium hydroxide. Formation of yellow colour indicates the presence of flavonoids.

Test for Tannins
Lead Acetate Test: A portion of the filtrate (1 ml) was treated with 3 drops of 1% lead acetate in a test tube. Formation of a blue-black colour indicates the presence of tannins.

Test for Steroids
Salkowski Tests: Chloroform (5 ml) was added to 0.5 ml of the filtrate in a test tube, and then an equal volume of concentrated sulphuric acid (5 ml) was added by the sides of the test tube. Formation of a red colour on standing indicates the presence of steroids.

Test for Reducing Sugars
Benedict's Test: Benedict's solution (2.5 ml) was added to the extract (1 ml) in a test tube, and then warmed over water bath for about 5 minutes. Formation of a green, red or yellow coloration indicates the presence of reducing sugar.

Test for Terpenoids
Chloroform (2 ml) was added to 5 ml of the ethanol extract (5 ml), and carefully followed by 3 ml of Conc H2SO4 to form a layer. Appearance of a reddish brown coloration indicates the presence of terpenoids.

Test for Glycosides
The sample extract (5 ml) was mixed with 2 ml of glacial acetic acid containing 1 drop of FeCl3 solution, and then 1 ml of Conc. H2SO4 was further added. Formation of a brown ring of interface indicates the presence of glycosides.

Test for Volatile Oils
Five (5) grams of the ethanol extract was dissolved in 90% alcohol and 4 drops of ferric chloride were added. Formation of green coloration indicates the presence of volatile oils.

Test for Anthraquinones
Sulphuric acid (10 ml) was added to 0.5 g of the extract and then boiled and filtered while hot. The filtrate was further shaken with 5 ml of chloroform, and the chloroform layer transferred to another test tube and 1 ml of dilute ammonia solution was added. Formation of a pink, red or violet colour indicates the presence of anthraquinones.

Extraction and Purification of the Oil
The oil was extracted from the kernels of the desert date using the solvent extraction process, using petroleum ether as a solvent by soxhlet apparatus (Karamat et al., 2003;Adejumo et al., 2013;Ratna et al., 2014). For purification, the oil was taken in a separating funnel along with water (100 ml), ether (200 ml) and saturated sodium chloride the content was well shaken and then allowed to stand. The aqueous layer was then discarded and the process was repeated three times with organic layer. Finally the ethereal extract was taken in a conical flask and then dried over 20 g anhydrous sodium sulfate and was evaporated at a temperature of 40°C to get the purified oil (Karamat et al., 2003;Ratna et al., 2014).

Color of Oil
The color of the extracted oil from the desert date kernel was observed visually as reported by Ogala et al., (2018).

Determination of Density and Specific Gravity of the Oil
The density of the desert date oil was determined using pre-washed, dried and labeled density bottles. The density bottled was then filled up to the volume mark with the oil sample, and the weight of the density bottle with the oil was determined. The density bottled was also filled up to the volume mark with the water, and the weight of the density bottle with the water was determined (Myles 2001;Brosk 2014). The density and the specific gravity of the oil were calculated using the formulae: Where; W1 = Weight of Empty Density Bottle W2 = Weight of Density Bottle + Oil W3 = Weight of Density Bottle + Water V = Volume of Oil.

Refractive Index of the Oil
The refractive index of the oil was determined using Abbe-60 refractometer (NYRL 3-Leica Mark, Leica Inc., Buffalo, New York) as described by AOAC (2008).

Viscosity of the Oil
The viscosity of the oil sample was investigation using the Ostwald-U-tube viscometer according to AOAC (2008). The viscometer was first suspended in a constant temperature water bath so that the capillary was vertical. The instrument was filled with the oil to the mark at the top of the lower reservoir with the aid of a pipette inserted in the side arm in such a way that the tube wall above the mark was not wetted. The instrument was then left to stand for 3 minutes before reading in order to equilibrate the sample temperature with that of the instrument (35°C). By means of pressure on the respective arm of the tube, the oil moved into the other arm so that the meniscus was above the mark at the top of upper reservoir. Finally, the liquid was allowed to flow freely through the tube and the time required for the meniscus to pass from the mark above the upper reservoir to that at the bottom of the upper reserve was recorded.

Acid Value of the Oil
Here 2 g of the test sample was transferred into a conical flask, and then 50 cm 3 petroleum ether added and gently mixed. Ethanol (50 cm 3 ) was then added into the mixture and titrated with 0.1 M KOH to pink colour (AOAC, 2008). The Acid value was calculated using the formula:

Peroxide Value of the Oil
The peroxide value (PV) of desert date kernel oil was determined according to the procedure reported by Wail et al., (1995) and adopted by Mohamed and Mohammed (2018), where 1 g of the test sample was transferred into 250 ml conical flask, then 30 ml of a glacial acetic acid/chloroform solution (ratio 3:2) was added, and the flask was gently shaken until the sample was dissolved, and then 0.5 ml of saturated potassium iodide was added. The solution was one again gently shaken for 1 minute, and 30 ml of distilled water was added followed by 0.5 ml of 1% starch solution. The content of the flask was later titrated with 0.1 N sodium thiosulphate with constant and vigorous shaking until the blue colour just disappeared. A blank test was also carried out in similar manner. The volume of the 0.1 N sodium thiosulphate required was recorded.

= ( − ) 100
Where; Va = Volume of sodium thiosulphate solution used in the titration Vb = Volume of sodium thiosulphate solution used in the blank test W = Weight of the sample in grams N = Normality of sodium thiosulphate.

Saponification Value of the Oil
Determination of the saponification value of the desert date kernel oil was carried out according to the AOAC (2008) method, where 1 g of the oil sample was transferred into 200 ml conical flask, and the 25 ml of 0.1N alcoholic KOH solution was added. The flask and its content were boiled under reflux for one hour with frequent rotation, then 1 ml of phenolphthalein indicator was added while the solution was still hot, and the excess alkali was titrated with 0.5N HCl, with the volume of HCl required to complete the titration recorded. The same procedure was repeated for the blank and the volume of HCl required to complete the titration also recorded.

= ( − ) x 0.02805 x 1000
Where; a = Volume of HCl used to titrate the sample b = Volume of HCl used to titrate the blank S = Weight of oil in gram.

Iodine Value of Oil
The iodine value (IV) of the oil sample was determined according to the BSI (1985) method as adopted by Mohamed and Mohammed (2018), where 0.26 g of the sample was weighed into a glass stoppered flask and then dissolved with 10 ml cyclohexane. Then 20 ml of Wijs solution (iodine monochloride dissolved in acetic acid) was added and the flask was stoppered and then allowed to stand in the dark for 30 minutes at a temperature of 25 O C after which 20 ml of 10% potassium iodide solution was added. The mixture was then titrated against 0.1 M Na2S2O3 using starch as the indicator. The analysis was then carried out using a blank, and the iodine value was calculated using the following formula (AOAC, 2008).

= 12.69 ( 1 − 2) Weight of
Where; C = Concentration of Na2S2O3 solution, V1 = Volume of Na2S2O3 used for the blank, V2 = Volume of Na2S2O3 used for the sample

Determination of Fat Content
The Gerber method for the determination of the fat content of the desert date kernel oil was employed in accordance with the procedure reported by Richardson, (1985) and adopted by Gemechu et al., (2015). Here 5 ml of each of the oil sample was mixed with 10 ml of sulphuric acid (specific gravity 1.82) into butyrometer and 1 ml of amyl alcohol was then added. The butyrometer was then closed with rubber cork, and the content was vigorously shaken until all the oil was digested by the acid. The butyrometer was then placed in a water bath at 65 O C for 5 minutes. The sample was centrifuged for 5 minutes, and then transferred back to the water bath at 65 O C for 5 minutes, and the percentage fat was recorded from the butyrometer.

Free Fatty Acid (FFA) of the Oil Sample
To determine the free fatty acid content of the desert date kernel oil, 2 g of the oil was placed in a 250 ml conical flask and warmed, and 2.5 mL of methanol was added with constant stirring, followed by 3 drops of phenolphthalein indicator, and this was then titrated against 0.14 M potassium hydroxide solution with vigorous shaking until a permanent light pink color, which persisted for 1 min, was observed (Afolabi, 2008;Ogala et al., 2018). The end point was recorded, and the free fatty acid value was calculated using equation.
Where; V = Volume of potassium hydroxide used M = Molarity of potassium hydroxide used W = Weight of the Sample.

Results
The results for the phytochemical constituent of the desert date kernel are presented in Table 1, while the physical and chemical characteristics of the desert date kernel oil are presented in Tables 2 and 3 respectively.

Discussion
The results of phytochemical screening test of the desert date kernel oil revealed the presence of alkaloids, saponins, flavonoids, tannins, steroids, glycosides and volatile oils, while phenols, reducing sugars, terpenoids and anthraquinones were absent. These findings agree with many similar findings as reported by some researchers (Hanan et al., 2009 . Tannins, the water-soluble polyphenols are present in many plant foods, have been reported to be responsible for decreases in feed intake, feed efficiency, growth rate and protein digestibility. However, reports have indicated some carcinogenic activities of tannins (Chung et al., 1998). Steroids are known to possess many interesting medicinal, pharmaceutical and agrochemical activities, ranging from antibacterial, hepatoprotective, anti-tumor, antihelminthic, immunosuppressive, plant-growth hormone regulator, sex hormone, cytotoxic and cardiotonic activity (Patel and Savjani 2015). Glycosides family has been in clinical use for many years for the treatment of heart failure and atrial arrhythmia, and they are known to have a strong direct action on the heart and support and strengthen the rate of contraction (Vaidehi and Rajesh 2016). From ancient times, humans have used glycoside-containing plants and their crude extracts as arrow coatings, homicidal or suicidal aids, rat poisons and emetics, while in the modern times they have been adapted for the treatment of congestive heart failure and cardiac arrhythmia (Yubin et al., 2014). Volatile oils are also called essential oils or ethereal oils, and they are natural metabolic secretions of plants that are considered to be true plant hormones, as well as the fluid manifestations of the immune system of the plants, since they contribute to the removal of pests, attracting, instead, pollinating agents, which are some insects and birds (Monica and Ioan 2018). The most important feature of the volatile oils which gives them special economic value is their specific smell, and this form the basis for their use in perfumery, cosmetics and the food industry. Many essential oils have special therapeutic qualities, some of which have been known and used since ancient times (Monica and Ioan 2018).
The quality assessment of Balanites aegyptica kernel oil was carried out by analyzing some physical such as the oil color, its density, specific gravity, refractive index and viscosity. The observed colour of the oil was pale yellow, and this has been the findings of Ogala et al., (2018). The density of the kernel oil studied was found to be 0.910 which agrees with that reported by Babeker (2013)  The acid value of 3.06 mgKOH/g and observed in this study is just below the FAO/WHO standard of 4 mgKOH/g, but still shows that the oil is stable (Haftu 2015). While the free fatty acid content of 1.27% is far below the FAO/WHO standard limits of 5.78-7.28%. Oils with high acid value (above 4 mgKOH/g), also implying a high percentage free fatty acid content, will undergo rancidity due to the hydrolysis of the free fatty acids on storage. The acid value and the percentage free fatty acid of Balanites aegyptica seed kernel oil are all lower than FAO/WHO standard for edible oils. The lower the percentage free fatty acid content the less the tendency of the oil to undergo hydrolytic activities (Haftu 2015 The peroxide value, used as a measure of the extent to which rancidity reactions have occurred during storage, is used as an indication of the quality and stability of fats and oils. The peroxide value determined for the kernel oil of Balanites aegyptica was found to be 3.71 mEq/g and is within the FAO/WHO standard of less than 10 mEq/g, and the lower the peroxide value the more suitable is the oil for a long storage due to low level of oxidative and lipolytic activities

Conclusion
This research analyzed the phytochemical properties of the seed kernel of Balanites aegyptiaca and the physical and chemical properties of the oil extracted from the seed. The results suggest that the seed kernel has high potentials for medicinal applications, while the kernel oil can serve as raw material for many oil related purposes. The oil can be used for human consumption, as well as in the production of industrial products like diodiesel, lubricants, soaps, shampoos and many other cosmetic products.

Acknowledgments
Our acknowledgment goes to the management of Yusuf Maitama Sule University, Kano who gave full financial, moral and technical support to this research.

Disclosure of conflict of interest
Some of the materials required for the research had to be obtained from a sister University, and this has not been as easy as it sounds, because it contradicts the policy of our University.