INTRODUCTIONCereals can be defined as classes of grass plantedand harvested for food purposes [1]. Different typesof cereals are cultivated all over the world and occupyan area of about 60%. They have notable benefitscontributing to human health due to nutrients andbiologically active substances in their composition [2–4].In the present study, sorghum was of our interest.Sorghum is also called Jowar in India, Guinea cornin West Africa, and Kaoliang in China. In Nigeria, itis called Oka by the Yorubas, Dawa by the Hausas,and Sorghum by the Igbos. There are also othernomenclatures for sorghum and its role in the food chainis well documented by Sarwar et al. [5].Varoquax et al. reported that sorghum is highlyresistant towards drought and heat, which allowsit to flourish and thrive even under hot and aridenvironmental conditions [6]. Sorghum is known tobe able to boost blood level. This is of tremendousimportance to human health. For instance, women whosuffer from myoma have anemia due to the excessiveblood losses, especially during menstrual period.372Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378Sorghum, with its high nutraceutical value, could helpthese women [7].In Africa, where diverse species or cultivars ofsorghum are cultivated, this cereal is served as animportant food crop. In Nigeria, sorghum is classifiedinto three cultivars depending on the nature and colorof their seminal glands and endosperm, namely Guines,Kaura, and Farafara [8]. Nonetheless, small-scalefarmers prefer Farafara to Kaura due to the fact that theformer is known to have a better storage behaviour andattributes.Sorghum is classified as a tall grass that often growsto as high as two to eight feet, occasionally being as highas fifteen feet. Generally, a whitish wax coating coversthe stalks and leaves of sorghum; while specifically,stalk’ piths of some species are juicy and sweet [9, 10].A well manured sorghum leaf is around 76 cm long and5 cm wide. Panicle portion of sorghum is responsible forthe production of tiny flowers which can be from looseto dense, with clusters containing 800–3000 kernels.The diversity of species is identifiable by the coloration,shape, and size of the seeds, which are smaller thanwheat seeds [10].Statistics in 2016 showed that Nigeria provided 23%of the total sorghum production in African, which madeNigeria the largest producer of sorghum in Africa [11].Mathur et al. documented the emergence of Sorghumbicolor as a viable option for producing lignocellulosicbiofuel [12]. Vanamala et al. reported that sorghumcontains bioactive compounds that play a crucial role inits pharmacological potential and immune modulatoryproperties [13]. Hassan et al. studied the effect ofultrasonic waves and microwaves on extraction of thelipid fraction from sorghum. They revealed that thesetechniques increased the oil yield [14].Since sorghum seed oil and its defatted extractsare widely used in Africa, the aim of this study was toevaluate the nutraceutical potential, phytochemicalcomponents, and secondary metabolites of sorghumfrom Ota (Nigeria).STUDY OBJECTS AND METHODSSample collection and preparation. Sorghum seedswere purchased from a local market in Ota, Nigeria.The seeds were washed, air-dried, and finally dried ina Thermofisher vacuum oven until constant weight wasachieved. The seeds then were finely powdered with amechanical blender. Prior to extraction, the powderedseeds were protected from sunlight, dust, as well asother particulate matter to avoid oxidation and microbialcontamination.Oil extraction. 200 g of the powdered seeds ofsorghum was weighed, carefully wrapped in Whatmanfilter paper, and mounted up on a Soxhlet extractor. Oneliter of petroleum ether was transferred into a roundbottom flask connected with a thimble with the samplein. When the extraction process completed, the petethersolvent was removed with an IKA® RV 10 rotaryevaporator, and the sample was stored in a refrigerator.GC-MS analysis condition. Agilent 7890BGC/5977 MS was utilized for the GC-MS analysis ofthe extract using the given conditions: column – HP 5capillary (60 m×0.25 mm×0.25 μm); oven temperatureprogram – the column was held initially at 50°C for1 min after injection, then ramped to 300°C at 7°Cper minute and held for 14 min; injector temperature –250°C; detector (MS) temperature – 275°C; carrier gas –helium; inlet pressure – 40.65 psi; linear gas velocity –39 cm/s; column flow rate – 2.7 mL/min; split ratio –10:1; injection volume – 1 μL. The components wereidentified by retention time determination on thecapillary column as well as by matching mass spectrawith the data of the NIST mass spectral library.Phytochemical tests. Terpenoids. 0.30 g of the seedpowder was carefully transferred into a 250 mL beakerand extracted with 30 mL of chloroform for 2 h. 2 mLof trichloromethane and 3 mL of concentrated sulphuricacid were added to 5 mL of the extract, thereby forminga layer. Reddish brown color at the interface confirmedthe presence of terpenoids [15].Cardiac glycosides were determined by twomethods. According to the Raymond method, 50%C2H5OH was gradually added to the extract in atest-tube, followed by 0.10 mL of 1% ethanolicm-dinitrobenzene. The resulting mixture then wastitrated with 20% NaOH. Violet coloration confirmedthe presence of active methylene group. According tothe Killer Killiani method, the extract was solubilizedin 1% FeSO4 in 5% glacial acetic acid, followed by theaddition of concentrated H2SO4. The development ofblue coloration indicated the presence of deoxy sugar.Quinones. Diluted NaOH was added to 1 mL of thesorghum extracted. Blue green or red coloration impliedthe presence of quinones [16].Saponins. The extract sample was vigorously mixedwith 5 mL of distilled H2O. The frothing was mixedwith few drops of olive oil and shaken vigorously. Theappearance of foam demonstrated the presence ofsaponins.Steroids. 2 mL of acetic anhydride was introducedinto 0.5 mL of the extract, followed by the addition of2 mL of sulphuric acid. Change in the extract colorationfrom violet to blue or green indicated the presence ofsteroids [16].Tannins. 10 mL of bromine water was introducedinto 0.5 g of the extract sample. Decolorization ofBr2/H2O showed the presence of tannins.Proximate determination. Proximate analysiswas carried out using combination of techniques andmethodologies earlier reported. For instance, crudeprotein and moisture contents were determined using themethod by Ajani et al., carbohydrate by Owoeye et al.,Molisch’s test by Gangwal et al., Biuret test by Suneethaet al., and total ash by Abdulkadir et al. [2, 3, 17–19].RESULTS AND DISCUSSIONSorghum has been identified and rated as the fifthcereal crop of greatest significance globally [20, 21].373Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378Sorghum is used in food and feed production, inwallboards, fences, biodegradable packing material,as well as for ethanol production [22, 23]. According tothe data in [24], before the outbreak of COVID-19, thetrend in the world sorghum production from 2012 to2019 fluctuated between 57 to 59 million tons (Fig. 1).Based on the world sorghum production data, there wasa drastic increase in 2015 which was as a result of theincreaseв sorghum usage by the Chinese in livestockfeed meal. This made them to purchase large volumes ofsorghum from the USA.In our previous works we studied seed oils and theirextracts, namely from Caryota mitis L., Adenantherapavoninalinn L., and sandbox tree (Hura crepitans L.)[2, 3, 25]. Continuing our research, this work featuredthe phytochemical screening, proximate determination,and GC-MS analysis of extract from sorghum fromOta (Nigeria) in order to investigate its nutraceuticalpotential and add more secondary metabolites to theorganic structure database.Seeds of sorghum were harvested from the plant. Itwas crushed and mounted on Soxhlet extractor to obtainthe oil while the remaining defatted component washerein referred to as the crude extract. The processingstages of the sorghum to identify the secondarymetabolites is demonstrated in Figure 2.Phytochemical screening. The phytochemicalscreening of the sorghum extract under study wasperformed by using standard methods as reported inour previous work [25]. The qualitative phytochemicalconstituent composition was determined in the sorghumseed oil obtained with the help of two different solvents:petroleum ether and ethanol (Table 1). Saponinavailability in the sorghum extracts was established withfoam test and Froth test. In the extract obtained withpetroleum ether saponins were not detected, while theextract obtained with ethanol contained saponins.Saponins contain an agent with surface activitydue to the sugar units which are very soluble in water.Although, the sapogenin units in saponins have highFigure 1 Statistics of world sorghum production from 2012 to 2019 [24]Figure 2 Stages of sorghum processing to identify secondary metabolites505356596265682012-13 2013-14 2014-15 2015-16 2016-17 2017-18 2018-19Quantity Produced (MMT)Year of ProductionWorld Sorghum Production in MMT5.00 10.00 15.00 20000004000000600000080000001e+071.2e+071.4e+071.6e+071.8e+072e+072.2e+072.4e+072.6e+072.8e+07Time-->Table 1 Phytochemical screening of sorghum extractPhytochemicals Type of test Petroleumether extractEthanolextractSteroids Salkowski – +Saponins Foam test/Froth – +Tannins Ferric chloride – –Terpenoids Acidifiedchloroform+ +Alkaloids Dragendroff + –CardiacglycosidesKiller Killiani + +Phenol Ferric chloride – –Oxalates Acid digestion – –Quinone Dilute NaOH + +The symbol (–) represents absence, while symbol (+) representspresence374Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378lipophilicity, which make them soluble in fat [26]. DEBruijn found that a wide variety of leguminous plantscontains diverse saponins; for instance, five classesof saponins were reported in soya beans [27]. Thisexplained why the extracts obtained with ethanol hadpositive saponin test. Saponins play a significant rolein the reduction of plasma cholesterol as a result of theeffective inhibition of cholesterol absorbing capacityin the intestinal tract of experimentally investigatedanimals [28].Cardiac glycosides are present in the bothpetroleum ether and ethanol extracts. These valuablesecondary metabolites are able to enhance myocardialcontraction, treating thereby congestive heartfailure [29]. Cardiac glycosides also indirectly effecton vascular resistance [30]. Thus, the presence ofcardiac glycosides in sorghum could be exploited fortheir medicinal potential. The presence of cardiacglycosides in the sorghum extracts was detectedby Raymond and Killer Killiani tests; the lattereffectively transformed 2-deoxy-sugars of cardiacglycoses by distinctive coloration, which made thequalitative and quantitative monitoring easier [29].Molecules of cardiac glycosides are capable of inhibitingNa+/K+- ATPase [31].Both petroleum ether and ethanol extracts ofsorghum contained terpenoids. Terpenoids form a groupof compounds, the majority of which occur in the plantkingdom. Simpler mono- and sesquiterpenes are themain constituents of essential oils [32]. Because of theirsweet smell, these essential oils are used in perfumeryin cosmetic chemistry [33]. Quinone was also presentin the tested sorghum extracts. Naturally, quinone playsan important role in transduction and accumulationof energy, which is necessary in such processes asrespiration and photosynthesis [34]. Alkaloids werefound in the sorghum extracts, while tannins, phenol,and oxalates were not detected in the both ethanol andpetroleum ether samples.Proximate and physico-chemical analyses. Theproximate analysis results are presented in Table 2. Theimportance of oil in human dietary intake cannot beoverestimated. Its biological availability and fatty acidprofile depends in most cases on environmental, crop,and genetic conditions [35]. The Soxhlet extractionwith n-hexane as a solvent revealed the crude fat of theextract of 11.00 ± 0.34%. This was a higher yield thanthe fat content (9.32%) reported from the distiller driedgrain (DDG) sorghum [36].The ash content in the sorghum extracts understudy was 3.05 ± 0.11%, which is in accordance withthe results obtained by Mohammed et al. who studiednutritional composition of three commonly consumedvarieties of sorghum [8]. The ash content may beaffected by the nature and amount of ions in the soilfrom which plants draw nutrients. In our work, theash content is within acceptable limit (< 5%) [37]. Soilcomposition has a partial but direct effect on ashcontent [38], as the ash content of seeds may partiallybe a function of the soil composition on which the plantsgrow [39].The protein content was determined to be 5.54 ±0.15%, which was within the range of earlier reportedvalues, namely 4.82 ± 2.39% for white sorghum and6.06 ± 0.40% for red sorghum. The moisture content ofthe sorghum tested was 5.94 ± 0.18%, which indicatedmoderate shelf-life of sorghum [19].In addition, the investigated sorghum herein had thefiber content of 0.20 ± 0.07%. Dietary fiber is valuablein digestion, hormone production, and cardiovascularhealth. This also assists in the reduction of low-densitylipoprotein cholesterol due to its bile reabsorbedreduction capability in the intestinal tract. Fibers in foodprevent excess starch in the body and regulate metabolicconditions such as diabetes and hypercholesterinemia[40].The carbohydrate content, which generally referredto the readily digested carbohydrates like sugar, starch,as well as organic acids, amounted for 74.27 ± 0.85%.High carbohydrate content supplies energy for themetabolic process, thus stabilizing health status of theconsumers [41]. The proximate analysis showed that thestudied sorghum extract contained 91.01 ± 0.93% of totalorganic matter.The physicochemical parameters of the sorghumextract included its refractive index, density, and cloudpoint (Table 2). According to the refractive indexvalue, the oil was of a good quality, so it can be usedfor homogeneous binary mixture formation. In theirresearch, Ospina et al. reported that the above mentionedparameters are characteristics for fast and cheap testingof the purity of essential oils [42]. The density of theseed oil of sorghum was 0.81 g/cm3, which was lowerthan that of Caryota mitis (0.93 g/cm3) and Adenantherapavonina (0.85 g/cm3) in our previous works [2, 3]. Thisalso implies that the oil of sorghum was less viscousthan that of Adenanthera pavonina and Caryota mitis.Table 2 Physicochemical and proximate determinationof sorghum extractParameters Obtained valuesProximate determination parametersMoisture content 5.94 ± 0.18%Ash content 3.05 ± 0.11%Crude fiber 0.20 ± 0.07%Protein 5.54 ± 0.15%Crude fat 11.00 ± 0.34%Carbohydrates 74.27 ± 0.85%Organic matter 91.01 ± 0.93%Selected physicochemical parametersRefractive index 1.46Density 0.81Cloudy 0.40°CValues are mean ± SD for triplicate measurement375Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378GC-MS analysis. Based on the GC-MS analysis,Figure 3 demonstrates the chromatogram of thesorghum extracts under study. The chromatogramallows us to compare spectra of each composition andthe NIST library data. The molecular structures of theidentified constituents are shown in Figure 4, with9,12-Octadecadienoic acid, methyl ester bring being thepredominant fatty acid.The mass spectrum chromatography assay showedthat the major constituents of the sorghum includedorganic acids, esters, sterols, tocopherols, and fattyaldehyde. Organic acids alone accounted for about 72%of the sorghum composition. Overall, the most abundantcompounds are 9,12-Octadecadienoic acid (Z,Z)-,Stigmasterol, 8-Dodecen-1-ol, acetate, (Z)-, vitamin E,Linoleic acid ethyl ester and 9,12-Octadecadienoic acid,and methyl ester, which accounted for about 85% of thesorghum composition.Among the organic acids, 9,12-Octadecadienoic acid(Z,Z)-, Linoleic acid ethyl ester and hexadecenoic acidswere contained in high concentrations. Hexadecanoicacids had three isomers, namely hexadecanoic acid,methyl ester; hexadecanoic acid, ethyl ester; andn-Hexadecanoic acid, making up about 5% of thecomposition.9,12-Octadecadienoic acid (Z,Z)-, was thepredominant fatty acid. It had two double bonds(C=C), which qualified it as an unsaturated fatty acid.9,12-Octadecadienoic acid (Z,Z)-, occurs as glycosidesin plants, which corroborates the presence of cardiacglycosides in their phyto-constituents. This essentialfatty acid is a functional component of human foodFigure 3 GC-MS chromatogram of sorghum extractOOH9,12-Octadecadienoic acid (Z,Z)-Linoelaidic acidOOEthyl tetracosanoateO9,17-Octadecadienal, (Z)-OOHVitamin EOO8-Dodecen-1-ol, acetate, (Z)-O9,17-Octadecadienal, (Z)- OCyclopropaneoctanal, 2-octyl-NNNH21H-Imidazole-5-ethanamine,1-methyl- CyclononeneOHH(1S,15S)-Bicyclo[13.1.0]hexadecan-2-oneOOHexadecanoic acid, methyl esterFigure 4 Structural identification of sorghum extractcomponents analyzed chromatographicallyTable 3 Identification of sorghum constituents (GC-MS)Sample № Retention time Area Pct Library (ID) Molecular formula (molecular weight)1 12.4708 0.0443 12-Heptadecyn-1-ol C17H32O (252.44)2 13.0716 0.0063 1H-Imidazole-5-ethanamine, 1-methyl- C6H11N3 (125.17)3 13.4607 0.0376 Cyclononene C9H16 (124.22)4 14.0329 0.018 (1S,15S)-Bicyclo[13.1.0]hexadecan-2-one C16H28O (236.39)5 14.1874 0.9545 Hexadecanoic acid, methyl ester C17H34O2 (270.45)6 14.7424 1.5527 Hexadecanoic acid, ethyl ester C18H36O2 (284.48)7 15.3032 2.3082 n-Hexadecanoic acid C16H32O2 (256.42)8 15.5549 4.1409 9,12-Octadecadienoic acid, methyl ester C19H34O2 (294.47)9 16.1042 5.17 Linoleic acid ethyl ester C20H36O2 (308.50)10 16.5391 – 31.8454 55.7397 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 (280.45)11 19.9093 2.4792 Tetracosanoic acid, methyl ester C25H50O2 (382.66)12 20.3042 3.3967 Ethyl tetracosanoate C26H52O2 (396.69)13 20.864926.46673.3824 9,17-Octadecadienal, (Z)- C20H38O (294.52)14 22.2496 5.6524 Vitamin E C29H50O2 (430.71)15 23.2738 8.2181 Stigmasterol C17H32O (252.44)16 24.4526 5.866 8-Dodecen-1-ol, acetate, (Z)- C14H26O2 (226.36)17 33.5963 1.0329 Cyclopropaneoctanal, 2-octyl- C19H36O (280.49)2017-18 2018-195.00 10.00 15.00 20.00 25.00 30.0020000004000000600000080000001e+071.2e+071.4e+071.6e+071.8e+072e+072.2e+072.4e+072.6e+072.8e+07Time-->AbundanceT IC : A D D M 1 .D \ d a ta .m s376Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378which takes a part in biosynthesis of prostaglandinsand cell membranes. Other polyunsaturated fatty acidshave recently been reported to have implication oninflammatory thrombotic condition like COVID-19 [43].Prominent esters included ethyl tetracosanoateand 8-Dodecen-1-ol, acetate, (Z)-, which accountedfor about 9% of the sorghum composition. Sterols(stigmasterol), tocopherols (vitamin E) and fattyaldehyde (9,17-Octadecadienal, (Z)-, linoleate group &Cyclopropaneoctanal, 2-octyl-) were contained inless quantities, accounting for about 8%, 6%, and 4%,respectively.CONCLUSIONThe sorghum oil extract was analyzed foridentification of phytoconstituents, proximate compositions,and physicochemical parameters. It was alsocharacterized spectroscopically for the nature andstructures of its secondary metabolites using GC-MS.Proximate determination showed that the sorghumsample contained beneficial amounts of nutrients, whilephytochemical screening revealed the presence ofbioactive essential phytochemicals.Thus, sorghum and sorghum-based food couldbe of high benefit to the population with nutritionaldeficiencies, for example, to developing countries. Thiswork provided a base for a comparison of nutritionalvalue and therapeutic potential of sorghum extract withother natural food cereal sources. Sorghum requiresfurther research on fortification and functionalizationof food with sorghum extract to decrease nutraceuticalshortage in the population.CONTRIBUTIONOlayinka O. Ajani designed the work and wrotethe original draft. Taiwo F. Owoeye collectedand pretreated the sample, as well as carried outphytochemical screening. Kehinde D. Akinlabu carriedout phytochemical screening. Oladotun P. Bolade ranand discussed the GC-MS analysis. OluwatimilehinE. Aribisala carried out sample pretreatment, Soxhletextraction, and formal laboratory analysis. BamideleM. Durodola carried out laboratory testing and editingof the manuscript. All authors read and approved thefinal manuscript before submission and they are equallyresponsible for plagiarism.CONFLICT OF INTERESTThe authors state that there is no conflict of interestsrelated to the publication of this article.ACKNOWLEDGEMENTThe authors gratefully acknowledge CovenantUniversity for the support for this work.