Biological Research

Irvingia gabonensis seeds yield a large amount of oil which can be evaluated for its fuel potential. In this study, Irvingia gabonensis seeds were analysed for their chemical composition. The analysis was carried out on the seeds using standard methods. Selected metals were determined in the seeds by dry ashing method using atomic absorption spectroscopy. Oil was extracted with n-hexane by sonication. Biodiesel (methyl esters) was produced from the oil by transesterification. The relative abundance of some fatty acid methyl esters were determined by gas chromatography-mass spectroscopy. The fuel properties of the oil methyl ester (biodiesel) were tested using standard methods. The results showed that the average moisture content was 3.21%, ash level was 1.73%, crude fat was 52.67%, while crude fibre, crude protein and carbohydrate contents were 2.63, 7.31 and 10.15% respectively. The seeds contained Na, K, Ca, Mg, Pb, Zn, Cu, and Cd which were in various amounts. Linoleic acid and methyl ester had the highest abundance at 48% respectively while oleic acid was of least abundance at 1.2%. The fuel properties showed that the properties of biodiesel from Irvingia gabonensis were within the acceptable quality standards for application in diesel engines.


Irvingia
gabonensis commonly called Af rican/bush/wild mango is a deciduous tree, grown in West and Central Af rican countries (Mgbemena et al., 2019). The f ruits have greenish mesocarp when unripe, and yellowish or light orange when ripe (Etebu and Tungbulu, 2015). The seeds have been the subject of research due to claims of their health benef its and nutritive content. Ngondi et al. (2005) opined that the seed is capable of reducing f asting blood glucose levels. Oben (2011) suggested that the seed extracts of Irvingia gabonnsis can be applied to manage metabolic syndrome with oxidative stress of dif ferent components and to signif icantly reduce the plasma levels of the inf lammatory marker in overweight and obese patients. A research result revealed that ethanolic extract of Irvingia gabonensis at doses studied caused a reduction in the body weight in mice (Adesanya et al., 2019).
Due to various claims on the health benef its, the trees are being exploited f or economic reasons. Consequently, Irvingia gabonensis is in the list of endangered plant species (Kof f i et al., 2018). Apart f rom the medicinal properties associated with Irvingia gabonensis, the oil content of the seed is known to be high, thereby making it usef ul f or making various products like cosmetics and soap. It became imperative to produce biodiesel f rom seed oil and check its properties.
Because of these applications, searches were made on basis of the various assertions on Irvingia gabonensis . Literature search revealed scanty inf ormation on the analysis of Irvingia gabonensis seed f or chemical composition. For example, Giami et al (1994) studied the chemical composition and f unctional properties of raw, heat-treated and partially proteolysed wild mango seed f lour. Also, Ekpe et al (2007) studied the proximate composition and amino acid prof ile of bush mango seeds, while Ogunsina et al (2012) investigated the proximate composition of Af rican bush mango kernels (Irvingia gabonensis ) and characteristics of its oil. Theref ore, this study investigated the chemical composition of Irvingia gabonensis The seed oil is assessed f or its f uel potential f or application in diesel engines.

Sample collection and preparation of Irvingia gabonensis seed
Irvingia gabonensis f ruits were picked up under the Irvingia gabonensis trees that were f ound at several locations in Nsukka town, Enugu State, Nigeria in 2020. The f ruits were washed and identif ied by a botanist at the University of Nigeria, Nsukka, Nigeria. The mesocarp of the f ruits and the seed coats were peeled of f with a knif e. The seeds were lef t to dry in the solar dryers at the National Center f or Energy Research and Development, University of Nigeria, Nsukka, f or two months. This was due to f luctuating temperatures at the time. The dried seeds f rom dif f erent f ruits were ground and screened through a sieve of 250-micron pore size. The samples that passed through the sieve pores were mixed thoroughly and f ormed a composite mixture which was packaged in plastic bags. The labelled bags containing the samples were lef t in a cupboard which was dry and dark, to prevent degradation of the samples, bef ore they will be used f or pending analysis.
This study was carried out at the Biomass Laboratory, National Center f or Energy Research and Development, University of Nigeria, Nsukka, Nigeria, between 2020 and 2021.

Proximate analysis
The ground seed samples were analysed for moisture, ash, carbohydrate, protein and f at contents f ollowing AOAC methods (Mansouri et al., 2018). For the moisture determination, concisely, the ground and sieved samples (0.500 g) were weighed out and placed in the sample compartment of a moisture analyser, MB 35 Halogen moisture analyser. The equipment was operated according to the manuf acturer's instructions and set at 110 °C. The results were recorded.Ash content determination involved using a muf f le f urnace (Vecstar, model LF3, Chesterf ield, United Kingdom) in which the crucibles containing the sieved samples were af ter weighing. The f urnace was set at 400 °C f or 30 minutes, then at 850 °C f or 1 h 45 minutes. Af ter, the covered crucibles with the ash when cooled were brought out f rom the f urnace and reweighed and then gravimetrically estimated. Crude protein was determined using the Kjeldahl method (Maehre et al., 2018). Crude f at was determined by extraction method using a sonicator (Model SALD-BS2 Beaker type, Shimadzu Corporation). In this process, two grams of the dried sample were placed in the sonicator bath of the sonicator. The stirrer and the ultrasonic pulse were activated and the extraction was allowed to proceed f or 25 minutes. Then the extracts were decanted. Af ter the extraction by sonication, the extracts were placed in tubes at the same level and placed in dif f erent compartments in the centrif uge.The centrif uge was set at 3500 rpm f or 30 minutes. The extract was thereaf ter decanted and f iltered.

Determination of Selected Metals in Irvingia gabonensis Seed
The ash f rom the ash content determination was dissolved in concentrated HCL. The solution was decanted into the standard f lask and made up to mark with distilled water.
The concentrations of Fe, Zn, Cd, Cu, Fe, and Pb in the sample solution were analysed with a Flame atomic absorption spectrophotometer (FAAS) (Model AA-6800, Shimadzu Corporation, Japan) using an air-acetylene f lame which has a digital read-out system. The equipment was calibrated with standard solutions f or the metals to be analysed. Each metal was determined using its lamp. The concentrations of the metals were obtained af ter subtracting f rom the blank solution. The analysis were done in duplicate as a quality assurance measure. A recovery experiment was carried out as outlined by Ugwu and Of omatah (2021).

Determination of Sodium and Potassium by Flame Photometry
A f lame photometer, the Gallenkamp f lame photometer, was used f or the determination of Na and K in the sample. The sample solution was prepared f rom the sample ash. The photometer was operated f ollowing the instructions of the equipment manuf acturer. Calibration of the equipment was done with the standard solutions of the metals being determined. The meter of the instrument was set at 100% E (Emission) while the concentration of the standards is aspirated. The %T of all the intermediate standard solutions was recorded. The blank was used to reset the equipment. Each of the sample solutions was aspirated and the readings (T) recorded. The plot of the standard curve was made on linear graph paper. The concentration of each element in the sample solution is read f rom the standard curve which was obtained f rom the standard solution.

Determination of Calcium and Magnesium by Titrimetric Titration
Exactly 10 mL of sample solution obtained f rom the dry ashing digestion were pipetted into a separate 350 mL conical f lask. About 25 mL of NH3-NH4Cl buf f er solution was added. 25 mL of water and 2 drops of Erichrome Black-T indicator were added. The solution was titrated against 0.01 M EDTA solution until a very light blue colour was obtained as the endpoint. The volume of EDTA used is the volume equivalent of calcium and magnesium in the admixture. All the determinations were done in triplicate.

Transesterification of the seed oil of Irvingia gabonensis
The f ree f atty acid content in the oil was determined by the titrimetric method. The seed oil of Irvingia gabonensis was extracted by sonication using a sonicator. The transesterif ication process was used to produce the biodiesel f ollowing the method of Sokoto et al. (2018). The seed oil was pretreated with methanol using an H2S04 acid catalyst to convert f ree f atty acid to ester. The reaction was conducted at a temperature of 65 °C f or 60 minutes. Af ter esterif ication, the alcohol layer was removed f rom the preheated oil bef ore transesterif ication, in which methanolic sodium hydroxide was poured into a f lat bottom f lask containing the esterif ied oil. The mixture was ref luxed at a constant stirring speed and was transf erred into a separating f unnel. The mixture was allowed to separate overnight due to the inf luence of gravity. The dark bottom layer (glycerol) was drained out and the light upper layer (biodiesel) was recovered and washed with a 20% volume of warm distilled water. The mixture was gently agitated f or 5 min and allowed to settle such that two layers were f ormed, and the biodiesel was separated into another dry container (Meher et al., 2010).

Fuel properties of the biodiesel from Irvingia gabonensis seed oil
The f uel properties of the biodiesel produced f rom Irvingia gabonensis seed oil were determined using standard methods. Thes e include specif ic gravity, viscosity and f lash point (Ibeto et al., 2011). Viscosity was determined with an Oswald portable capillary viscometer. This investigation was done at the temperature of the laboratory (31ºC) by recording the time required f or the biodiesel to pass between two marks in the viscometer (Ugwu and Eze, 2014).
Flashpoint was also determined f or the biodiesel. It is used to assess the overall f lammability hazard of a material. The f lash point f or the biodiesel produced in this study was measured by using a Pensky Martens semiautomatic multi-f lash closed cup f lash point tester (Made in Japan) ((Ugwu and Eze, 2014). The diesel index, cloud point, pour point, and cetane number were determined as described by Enweremadu et al. (2011).

GC-MS analysis of the biodiesel from Irvingia gabonensis seed oil
A gas chromatography/mass selective detector (GC/MSD) (GC model: 7890A; MSD model: 5975, Agilent Technologies, USA) in Selected Ion Monitoring (SIM) mode was used f or the characterization of the f atty acid methyl esters in the biodiesel based on their boiling points and polarity. The analytes were separated in the capillary column of the machines. This was done af ter the injection of the derivatized sample into the equipment. A helium carrier gas was used f or the analysis, and the oven program was initially at 65 °C f or 1 min, up to 290 °C, f or 11 min. The run time was 30 min, and at splitless injection, mode using an auto-sampler. The mass spectrometer quadrupole analyzer was used in electron ionization mode at 70 eV. Bef ore the sample analysis, standards of f atty acids were analyzed with the equipment in SIM mode f irst to ascertain the f ragmentation pattern. The target compounds and qualif ier ions were determined af ter scanning the standard. The target compounds were identif ied by comparing the retention time of the compounds in the samples with the time of the standards used in the calibration of the equipment. The mass spectra of the target ions ratios were compared with the library database spectra of the National Institute of Standards and Technology (NIST). The concentrations were automatically read out f rom the instrument.
This study was conducted at the National Centre f or Energy Research and Development, University of Nigeria, Nsukka.

RESULTS AND DISCUSSION
Proximate analysis is a determination of the moisture, ash, oil, f ibre, protein and carbohydrate contents in a material. The mean proximate composition of Irvingia gabonensis seed, presented on Table 1, showed that the mean moisture content of I. gabonensis seed is 5.21%. This is relatively low and may be attributable to the drying in a solar dryer over a relatively long period due to f luctuations in weather. A matrice loses water as it stays longer in a dryer. It is advantageous f or the moisture content to be low since it will reduce microbial growth and deterioration over a long time.
The mean results of proximate analysis of Irvingia gabonensis seeds are presented in Table 1 Ekpe et al. (2007). The mean ash content in the present study was 2.73%. Ash content is an indication of mineral content. This indicated the low level of minerals in the seed. In comparison with other studies, ash content was 2.3% (Ogunsina et al., 2012) 6.8% (Mgbemena et al., 2019) and9.50 (Ekpe et al, 2007). . At 57.97%, the oil content in the seed is very high. These implied that oil can be obtained easily f rom the seed of I.gabonensis. Other researchers reported oil content in the sample analysed as 68.39% (Ogunsina et al., 2012) and 66.60% (Ekpe et al, 2007). The oil may be tested f or application in domestic and industrial needs. With the mean % crude f ibre at 7.63%, which is relatively low, the f ibre level will barely help in bowel f unction during the consumption of the seed. The average crude protein content is 8.31% in the present study. This is close to 8.9% reported by Ogunsina et al. (2012) and 7.6% reported by Ekpe et al. (2007) and 5.6% recorded by Mgbemena et al. (2019). The protein level is reasonable f or consumption, but I.gabonensis should be consumed with other f oods f or protein. The average level of carbohydrates in I.gabonensis is 18.15%. Ogunsina et al. (2012) reported a carbohydrate content of 18.67%. Carbohydrate level is an indication of energy content in f ood. This implied that I.gabonensis has a f air level of energy content.
The concentrations of some metals in I.gabonensis seed are shown in Table 2. Fe, Cu and Zn were essential elements required in small quantities by living organisms, including humans, to ensure good health (Brif f a et al.,  2020). These were f ound in the I.gabonensis samples. Iron is required f or the normal f unctioning of the central nervous system and blood f ormation. Other elements f ound include Na, K, Ca and Mg. These were detected in various quantities as shown in Table 2. Thes e elements all play vital roles in human health. While Na is an important component of sodium chloride, Ca and Mg are necessary f or bone development. Magnesium activates many enzyme systems and maintains the electrical potential in nerves. The levels of Cd and Pb were low. However, Cd is a known heavy metal that can cause adverse ef f ects on human health if present above a certain limit. Sodium, potassium and chlorine are important in the maintenance of osmotic balance between cells and the interstitial f luid (Soetan et al., 2010). The results of the metal analysis in the present study were compared to the results f rom a similar study by Mgbemena et al. (2019) and presented in Table  2.
The relative abundance of various f atty acid methyl esters in the Irvingia gabonensi s biodiesel presented in Table 3, shows varying compositions of the methyl esters. At 48%, linoleic acid methyl ester was most abundant. Next in abundance was myristic acid and then stearic acid at 30.54 and 10% respectively. These values show that Irvingia gabonensis oil is appropriate f or use in biodiesel production.
Some of the f uel properties of biodiesel produced f rom Irvingia gabonensis seed oil were presented in Table 4. From Table 4, It is obvious that the f uel parameters were within the specif ications of ASTM. A comparison of the cetane number of the Irvingia gabonensis biodiesel with biodiesel made f rom castor seed oil (68.55-71.16) (Auwal et al., 2022). indicates that Irvingia gabonensis biodiesel has a more f avourable cetane number f or use as biodiesel. Also, Neem oil biodiesel has Flash point, Pour point and Cloud point of 150 °C, 3 °C and 6 °C respectively (Aransiola et al., 2012), while this study has 101 °C, 4 °C and 1.9 °C f or Flashpoint, Point and Cloud point respectively. This reinf orces the suitability of Irvingia gabonensis biodiesel f or f uel application in engines. The results of tests f or f uel properties of the Irvingia gabonensis biodiesel are presented on Table 4. The variations in the composition of the Irvingia gabonensis samples f rom dif ferent locations are normal because of dif ferences in varieties, types of soil where they were grown, and climatic changes (Ibeto et al., 2011). Also, the dif f erent sample preparation methods and processing techniques will result in dif f erences in the f inal results obtained by dif ferent researchers. This research is inconclusive at this stage. Theref ore, the Irvingia gabonensis biodiesel cannot be used in engines. The biodiesel requires f urther purif ication, optimization of inputs and testing f or certif ication bef ore it may be conf irmed f or engine application. The results of the GCMS analysis of one of the samples showing relevant f atty acid methyl esters are presented graphically in Figure 1.

CONCLUSION
This study determined the chemical composition of Irvingia gabonensis f rom the proximate analysis. This revealed that Irvingia gabonensis has high oil content, low ash (mineral), low moisture, and moderate f ibre and carbohydrate contents. Irvingia gabonensis seed was studied f or some metals. It was f ound that Na, K, Ca, Mg, Cu, Fe, Zn, Pb and Cd were detected at varying amounts. Some of these elements are essential to human health. The potential of Irvingia gabonensis seed oil f or producing biodiesel was investigated using the transesterif ication process. It was f ound that Irvingia gabonensis biodiesel had qualities that complied with specif ications f or biodiesel applications. Theref ore, this study conf irmed Irvingia gabonensis as a viable source of biodiesel that can complement energy supplies f rom f ossil f uels. However, biodiesel requires f urther testing and optimization of inputs. (2018).
Ef f ect of reaction variables on biodiesel production f rom canary melon seed oil.