Balanites Aegyptiaca) ESSENTIAL OIL Yasir W.A.Mustafa1* and Hassan

Volume 2 Issue 2, May 1 2021

SUDANESE ONLINE RESEARCH JOURNAL

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V olume (2) Issue (2) MAY 1st, 2021

APPLIED SCIENCE GC-MS ANALYSIS AND ANTIMICROBIAL ACTIVITY (VITRO TEST) OF (Balanites aegyptiaca) ESSENTIAL OIL Yasir W.A.Mustafa1* and Hassan. N. Ali2 1Faculty of science, Sudan University of Science and Technology 2Faculty of applied science, International University of Africa *Corresponding Author: [email protected]

Received 10th NOV 2020 Accepted 20th APR 2021 Published 1st MAY 2021

ABSTRACT The aim of the present study was to extract oil from Heglig MATERIALS AND METHODS seeds (Balanites aegyptiaca) to indicate phytochemical Plant material: properties, GC/MS analysis and anti- biological activity The Heglig ( Balanites aegyptiaca ) fruits were collected (Vitro test). The kernel seeds were ground, and 250 g were from Alrahed North Kordofan , Sudan in 2017 . Then was transferred into a soxhelt unit and extracted by using normal treated to obtain the kernel seeds which contain the oil as a hexane. major component. The chemical extraction followed by GC-MS analysis result Instruments in 25 compounds, the compound which had higher A shimadzo GC-MS – Q P 2010 Ultra instrument with a percentage are: RTx-5MS column ( 30m, length ; 0.25um, thickness) with 9-Octadecanoic acid (Z)-methyl ester 29.46%, 9, 12- Helium as a carrier gas . Octadecadienoic acid (Z, Z)-, methyl ester 29.18%, Methyl Test organisms stearate 18.71%, Hexadecanoic acid, methyl ester 16.70%. Balanitis aegypetiaca oil was screened for anti-bacterial and and Eicosanoic acid, methyl ester 1.81%. anti fungal activity using standard micro organisms showed The oil showed activity only against Candida Albicans. in table (1) & (2).

KEYWORDS: Antimicrobial activity, Balanites aegyptiaca Table (1): test organisms , Essential oil , GC-MS. Ser. Microorganism Type No INTRODUCTION 1 Bacillus subtilis G +ve Balanites aegyptiaca is a species of tree, classified either as 2 Staphylococcus aureus G +ve a member of the Zygophyllaceae or the Balanitaceae.[1] This tree is native too much of Africa and parts of the Middle 3 Escherichia coli G -ve East[2]. 4 Aspergillus Niger Fungus The Balanites aegyptiaca tree reaches 10 m (33 ft) in height 5 Candida albicans Fungus with a generally narrow form[5]. Many parts of the plant are used as famine foods in Africa; the leaves are eaten raw or cooked, the oily seed is boiled to make it less bitter and eaten mixed with sorghum, and the METHODS flowers can be eaten.[3]. Phytochemical’s screening Desert date fruit is mixed into porridge and eaten by nursing 250g) from Heglig kernel seeds powder loaded into the mothers, and the oil is consumed for headache and to thimble, which was placed inside the soxhelt and extracted improve lactation.[3] Bark extracts and the fruit repel4] or with 500ml of hexane 95% until complete the extraction destroy[5] freshwater snails and copepods, organisms that act after 24 hours .this prepared extract (PE) was used for as intermediary hosts host the parasites Schistosoma, phytochemical screening according to the methods describe including Bilharzia, and guinea worm, respectively. Existing by Harborne[6]. worm infections are likewise treated with desert date, as are Determination peroxide value liver and spleen disorders. A decoction of the bark are also One gram of oil was weighted into a clean dry boiling tube used as an Abortifacient and an antidote for arrow-poison in and one gram of powdered potassium iodine and 20 ml of West African traditional medicine.[5] The seed contains 30- solvent mixture (glacial acetic acid + chloroform) were 48% fixed (non-volatile) oil, like the leaves, fruit pulp, bark added [8]. and roots, and contains the sapogeninsdiosgenin and The tube was placed in boiling water, and then was yamogenin.[4][5]Saponins likewise occur in the roots, bark transferred to conical flask containing 20 ml of potassium wood and fruit.[5] iodine solution, the tube was washed twice with 25 ml of water and collected in the conical flask. The mixture was titrated against sodium thiosulphate

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solution until yellow colour was almost disappears. A 0.5 ml Anti-microbial assay: of starch was added, shaked vigorously and was titrated Preparation of bacterial suspensions: carefully till the blue colour just disappeared [8]. One ml aliquots of a 24 hour broth culture of the test Determination acidity number: organisms were aseptically distributed onto nutrient agar (2g) of oil were dissolved in 25 ml of ethanol then titrated slopes and incubated at 37 ºC for 24 hours. The bacterial against alcoholic potassium hydroxide (0.1 ml) in present of growth was harvested and washed off with 100 ml sterile phenolphthalein as indicator until the end point[8]. normal saline, to produce a suspension containing about 108- Determination saponification value: 109 C.F.U/ ml. The suspension was stored in the refrigerator (2g) of oil were weighted in round flask and 20 ml of at 4° C till used. alcoholic potassium hydroxide were added, then placed in The average number of viable organisms per ml of the stock soxhelt for 1 hour, after that the mixture was titrated against suspension was determined by means of the surface viable hydrochloric acid (0.5 M) in presence of phenolphthalein counting technique. Serial dilutions of the stock suspension indicator until the end point[8]. were made in sterile normal saline solution and 0.02 ml Determination iodine value: volumes of the appropriate dilution were transferred by (2g) of oil were weighted and 25 ml of wajs solution were micro pipette onto the surface of dried nutrient agar plates. added, the mixture was putted in dark place for 15 minutes . The plates were allowed to stand for two hours at room 10 ml of potassium iodine (15%) were added. The excess of temperature for the drops to dry and then incubated at 37 ºC potassium iodine was titrated against sodium thiosulfate for 24 hours. After incubation, the number of developed pentahydrate in present of starch indicator until the end colonies in each drop was counted. The average number of point, the blank solution also was prepared [8]. colonies per drop (0.02 ml) was multiplied by 50 and by the dilution factor to give the viable count of the stock GC-MC Analysis: suspension, expressed as the number of colony forming The oil from seeds of balanitis aegypetiaca was analyzed by units per ml suspension. gas chromatography mass spectrometry . A shimadzo MC- Preparation of fungal suspension: MS - QP2010 ultra instrument with Rtn-5MS column ( 30 m The fungal cultures were maintained on Sabouraud dextrose length,0.25 mm diameter , 0.25 µm, thickness ) was used . agar, incubated at 25 ºC for 4 days. The fungal growth was Helium (purity 99, 99%) was used as carrier gas . oven harvested and washed with sterile normal saline and finally temperature program is given in table (2), which the suspension in 100 ml of sterile normal saline, and the chromatographic conditions are depicated in table (3) suspension were stored in the refrigerator until used. Table (2): oven temperature program Testing of antibacterial susceptibility Rate Temperature ( co) Hold time (min-1 ) The paper disc diffusion method was used to screen the - 150.0 1.00 antibacterial activity of plant extracts and performed by 4.00 300.0 0.00 using Mueller Hinton agar (MHA). The experiment was carried out according to the National Committee for Clinical Table (3): chromatographic conditions Laboratory Standards Guidelines (NCCLS, 1999). Bacterial o suspension was diluted with sterile physiological solution to Column oven temperature 150.0 c 8 Injection temperature 300.0 co 10 cfu/ ml (turbidity = McFarland standard 0.5). One hundred micro liters of bacterial suspension were swabbed Injection mode Split uniformly on surface of MHA and the inoculums was Flow control mode Linear velocity allowed to dry for 5 minutes. Sterilized filter paper discs Total flow 139.3 kpa (Whatman No.1, 6 mm in diameter) were placed on the Column flow 1.54 ml/min surface of the MHA and soaked with 20 µl of a solution of Linear velocity 47.2 cm/sec each plant extracts. The inoculated plates were incubated at Purge flow 30ml/min 37 ºC for 24 h in the inverted position. The diameters (mm) Split ratio -1.0 of the inhibition zones were measured.

RESULTS AND DISCUSSION Phytochemical screening: Phytochmical screening of Balanitis aegypetiaca fruits gave positive for : Alkoloids , flavonoids , tannins , saponins and carbohydrates (table 4). Species flavonoids tannins Alkoloids saponins carbohydrates

Balanitis + ve + ve + ve + ve + ve aegypetiaca

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Chemicals properties of Heglig oil : identification of constituents was initially accomplished by Table 5: Chemical properties of Heglig oil comparison with the MS library (NIST) and further No. Chemicals propertis Results confirmed by interpreting the observed fragmentation 1 Acidity number 0.42 pattern . comparison of the mass spectra with the data base 2 Peroxide value 2.86 on MS library revealed about 90% - 95% match . 3 Saponnification value 189.71 Constituents of oil 4 Iodine value 98.76 The GC-MS chromatography analysis of the studied oil revealed the presence of (25) components (table 6). GC-MC analysis of Heglig oil The typical total ion chromatograms (TIC) are show in GC-MS analysis of Heglig oil was conducted and the figure (1)

Figure (1) GC-MS chromatogram of Higlig oil

Major constituent were detected in the chromatograms 4. The peak at m/z 74, which appeared at R.T 17.643 in total 9-Octadecanoic acid (Z)-methyl ester (29.46%) ion chromatogram, corresponds to M+[C19H38O2]+ . The EI mass spectrum of 9-Octadecanoic acid (Z)-methyl Hexadecanoic acid, methyl ester (16.70%) ester is shown in figure 2. The peak at m/z 55, which The EI mass spectrum of Hexadecanoic acid, methyl ester is appeared at R.T 17.497 in total ion chromatogram, shown in figure 5. The peak at m/z 74.05, which appeared at corresponds to M+[C19H36O2]+ . R.T 15.693 in total ion chromatogram, corresponds to 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (29.18%) M+[C17H34O2]+ . The EI mass spectrum of 9,12-Octadecadienoic acid (Z,Z)-, Eicosanoic acid, methyl ester (1.81%) methyl ester is shown in figure 3. The peak at m/z 67, which The EI mass spectrum of Eicosanoic acid, methyl ester is appeared at R.T 17.410 in total ion chromatogram, shown in figure 6. The peak at m/z 74.05, which appeared at corresponds to M+[C19H34O2]+. R.T 19.303 in total ion chromatogram, corresponds to Methyl stearate (18.71%): M+[C21H42O2]+ . The EI mass spectrum of Methyl stearate is shown in figure

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Table 6: GC-MS chromatography analysis of Higlig oil No. R.Time Area% Name 1 13.529 0.18 Methyl tetradecanoate 2 14.338 0.00 Cis-5-Dodecenoic acid 3 14.443 0.00 5-Octadecadienoic acid, methyl ester

4 14.603 0.01 Pentadecanoic acid, methyl ester 5 15.332 0.00 7 10-hexadecadienoic acid, methyl ester 6 15.393 0.08 7-Hexadecenoic acid, methyl ester, (Z)- 7 15.438 0.65 9-Hexadecenoic acid, methyl ester, (Z)- 8 15.533 0.02 trans-13-Octadecenoic acid, methyl ester 9 15.693 16.70 Hexadecanoic acid, methyl ester 10 16.402 0.25 cis-10-Heptadecenoic acid, methyl ester 11 16.612 0.50 Heptadecanoic acid, methyl ester 12 17.410 29.18 9,12-Octadecadienoic acid (Z,Z)-, 13 17.497 29.46 9-Octadecenoic acid (Z)-, methyl ester 14 17.643 18.71 Methyl stearate 15 18.229 0.07 cis-10-Nonadecenoic acid, methyl ester 16 18.445 0.05 Nonadecanoic acid, methyl ester 17 18.906 0.52 6,9-Octadecadienoic acid methyl ester 18 19.103 0.65 cis-11-Eicosenoic acid, methyl ester 19 19.303 1.81 Eicosanoic acid, methyl ester 20 19.470 0.05 9,12,15-Octadecatrienoic acid, methyl ester 21 20.919 0.27 Docosanoic acid, methyl ester 22 22.421 0.40 Tetracosanoic acid, methyl ester 23 23.133 0.13 Pentacosanoic acid, methyl ester 24 23.821 0.26 Hexacosanoic acid, methyl ester 25 25.529 0.04 Vitamin E

Figure 2: Mass spectrum of 9-Octadecenoic acid (Z)-, methyl ester

Figure 3: Mass spectrum of 9,12-Octadecadienoic acid (Z,Z)-, methyl ester

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Figure 4: Mass spectrum of Methyl Stearate

Figure 5: Mass spectrum of Hexadecanoic acid, methyl ester

Figure 6: Mass spectrum of Eicosanoic acid, methyl ester

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Anti-microbial activity The oil was screened for anti-microbial activity against standard microorganisms. The average of diameters of growth inhibition zone is shown in table (6). The results were interpreted in terms of the commonly used ( 18mm: very active).

Table 7: Anti-bacterial activity of Higlig oil M.D.I.Z (mm) Drug Conc Ec Ps Sa Bs Ca An mg/ml Higlig oil 100 _ _ _ _ 10 _

Sa: Staphylococcus aureus. Ec: Escherichia coli. Pa: Pseudomonas aeruginosa. An: Asperyillus niger. Ca: candida albicans. Bs: Bacillus subtilis. M.D.I.Z: growth inhibition zone (mm). Average or two replicates, inhibition zone >= 15: sensitive, < 15: resistant. The oil showed activity only against candida albicans (table 6). CONCLUSION In conclusion, this study reports analysis of oil from Hegilig seeds (Balanites aegyptiaca) after extraction, the oil was characterized using manual techniques and spectroscopic analytical techniques such as GC-MS and IR. Antimicrobial activity of the oil was also investigated. Good antibacterial activity was observed. The oil of (Balanites aegyptiaca) may be developed to drug in the future for treatment of diseases caused by the tested pathogenic bacterial strain.

ACKNOWLEDGEMENTS Authors Extend their thanks to Dr. Mahmoud Ali, Faculty of applied science, International University of Africa for his support.

REFERENCES 1- Delile , ( 2016) . "Balanites aegyptiaca (L.) ". The Plant List p 35. 2- "Zygophyllaceae". (2016),The Plant List. 3- "Genus, (2016), Balanites Delile". U.S. National Plant Germplasm System, Retrieved, p 12. 4- Kinloch, Bruce (1972). The shamba raiders: memories of a game 5- Robert Freedman, (2016) , "BALANITACEAE". Famine Foods p 132. warden (3rd ed.). Hampshire: Ashford. p. 217. ISBN 1852530359. 6- Harborne ,J.B," phytochemical methods ", chapman and Hall, London, 1979. 7- Firestone D (May. June 1994) ." determination of iodine value of oils and fats: summary of collaborative study " . 8- BSI (1976). Methods of test for Essential oils. British standard institution (BSI), 2park street London W1A2BS, UK. 28A[35-46].