The fruits of B. aegyptiaca were harvested from Central of Sudan-Eldamazen. The kernels of the fruits were separated and powdered using a mortar and pestle. 200 gm of B. aegyptiaca seeds weighed and packed in soxhlet apparatus. The powdered seeds were extracted with petroleum ether 40˚C - 60˚C. The solvent was then collected and evaporated under reduced pressure using rotary evaporator apparatus. The petroleum ether extract yielded pale yellow colour oil which was then stored in dark bottles at room temperature till use [16] .

The triglycerides of the B. aegyptiaca oil trans-esterified using the following procedures [17] [18] . Typically, the oil (1 ml) was diluted by 1 ml of hexane. Then 6 ml of NaOH (0.5 M) in methanol were added. The mixture was refluxed for 2 - 3 min. Next, 6 ml of H₂SO₄ 1% were added and shook well-over night, followed by the addition of 2 ml of hexane and shook after that saturated NaCl was added. Then to the upper layer (hexane) a few amount of Na₂SO₄ was added. After decantation the fatty acids composition was determined using gas chromatography (GC-2010, SHIMADZU-Japan).

Iron oxide nanoparticles were prepared by a co-precipitation method with the addition of B. aegyptiaca oil as a surfactant. Typically, 50 ml of 1 M solution FeCl₃・6H₂O and 50 ml of 2 M FeSO₄・7H₂O were mixed and dissolved in deionized water. Then 3 M of Sodium hydroxide were added into the above solution and the pH value was maintained between 10 - 11 with continuous stirring for 30 minutes and a dark precipitate was formed. 2 ml of B. aegyptiaca oil were heated to 70˚C under nitrogen atmosphere and added in the above solution with continuous stirring for 3 h at 80˚C. After that, the magnetic precipitate was isolated from the solvent by a permanent magnet, which then washed several times with distilled water and ethanol in sequence. Finally it was dried at 50˚C for 24 h and grinded to fine powder.

In antibacterial studies, agar well disk diffusion susceptibility test was used to measure efficacy of iron oxide nanoparticles against Gram-positive bacteria Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis) and Gram-negative bacteria Escherichia coli (E. coli) in bacterial sensitivity to antibiotics is commonly tested using a disk diffusion test, employing antibiotic impregnated disks [19] . A similar test with nanoparticles laden disks was used in this study. Briefly, A 5 ml suspension of nanoparticles (5 mg ml⁻¹) was sonicated and subsequently filtered through a membrane filter (0.2 µm, 47 mm diameter). The nanoparticles laden filter paper was dried in an oven for 1 h and small disks of uniform size (6 mm diameter) containing 100 ± 15 µg nanoparticles were punched out and stored in a desiccators at room temperature. The bacterial suspension (100 µl of 104 - 105 CFU ml⁻¹) was applied uniformly on the surface of a beef agar plate before placing the disks on the plate (3 per plate). The plates were incubated at 35˚C for 24 h, after which the average diameter of the inhibition zone surrounding the disk was measured with a ruler with up to 1 mm resolution terms of zone inhibition [20] .

In order to determine the fatty acids composition of B. aegyptiaca seed oil gas chromatography analysis of the petroleum ether extract of the seed oil revealed the presence of stearic acid M. (66.9%%), Cis-10-Heptadecenoic acid (22.5%), oleic acid (8.3%) and palmitic acid (2.1%). The fatty acids composition of B. aegyptiaca seed oil is presented in Figure 1. The chromatograms of B. aegyptiaca oil are composed of essentially four carboxylates, corresponding to four fatty acid. Cis-10-Heptadecenoic acid (17:1) has long been recognized as minor constituent of ruminant fats and its isomeric definition remains undefined in most reports on ruminant milk and intramuscular fat [21] [22] . Recently it was proposed, along with other odd-chain fatty acids, as a potential marker of microbial biomass [23] .

Transmission electron microscopy allows obtaining information about the shapes and sizes of the particles. Figure 2 shows the TEM images and histogram of the size distribution of the magnetite particles with and without B. aegyptiaca oil coating. Figure 2(a) denotes presence of agglomeration of the particles in pure Fe₃O₄, while Figure 2(b) shows each particle separated from the neighbors because of the organic chain absorbed on its surface of iron oxide nanoparticles. Surfactant molecules (also called stabilizing agents) are attached to the nanoparticle surface, preventing clotting and providing the desired dispersion and surface properties. It is well known that magnetic Fe₃O₄ prepared by the co-precipitation method has a large number of hydroxyl groups on its surface in contact with the aqueous phase. The OH groups on the surface of Fe₃O₄ particles react readily with carboxylic acid head groups of oil fatty acids molecules. Excess fatty acids on oil of B. aegyptiaca and then adsorbed the fatty acids to form as hydrophobic shell [24] . The size and shape of magnetite nanoparticles may vary depending on the nature and concentration of the stabilizing agent [25] . The size of bare Fe₃O₄ nanoparticles is 29 nm with a dominant population at 5 - 10 nm and the size of nanoparticles coated B. aegyptiaca oil is 35 nm with a dominant population at 11 - 12 nm. The nanoparticles coated B. aegyptiaca oil has a size slightly higher than the nanoparticles without B. aegyptiaca. A mean size of samples was calculated by programme nano measurer 1.2.

Figure 3 shown the composition of the Fe₃O₄ with B. aegyptiaca was confirmed by X-ray EDS microanalysis of the analyzed point. The presence of Fe and O can be observed, with iron abundance higher than oxygen signals that indicate oil coating and surrounding iron oxide nanoparticles. Moreover, together with Fe and O ones, the copper signal is present because of the copper grids used for sample preparation, in which traces of Cu are present, as reported by the manufacturer. The C signals are attributed mainly to organic molecules (fatty acid) in B. aegyptiaca oil.

The magnetic properties of the B. aegyptiaca coated Fe₃O₄ nanoparticles were investigated with a vibrating sample magnetometer at 300 k. Figure 4 is shown the magnetic hysteresis loop at 300 k. The saturation magnetization (Ms), remanence (Mr), coercivity (Hc) and loop squareness ratio (Mr/Ms) of B. aegyptiaca coated iron oxide nanoparticles is shown in Table 2. The coated sample had a higher Ms but a lower Mr, Hc and Mr/Ms. The increase in magnetization of the coated sample is probably due to the nanoparticles, spinal structure, confirmed that the good paramagnetic property of the resulting sample at room temperature [26] .

To understand the adsorption spectrum of the B. aegyptiaca oil on the surface of Fe₃O₄ nanoparticles, infrared measurements were carried out on the B. aegyptiaca oil, pure Fe₃O₄ and the composite Fe₃O₄ nanoparticles coated with B. aegyptiaca oil. Figure 5 shows the typical IR spectrum of the B. aegyptiaca oil. The functional groups present in the ester B. aegyptiaca oil are explained as follows: The vibration caused by C-H stretching could be found at wavelength around 3000 cm⁻¹ (Figure 5(c)). This indicates the presence of aliphatic hydrocarbons. Between 1600 and 1800 cm⁻¹ another distinctive peak could be noticed. This is C=O, i.e. carbonyl group, which could account for the carboxylic acid of B. aegyptiaca oil. Lastly, C-O ether groups can be seen between 1000 and 1100 cm⁻¹.

In Figure 5(c) two sharp bands at 2955 and 2852 cm⁻¹ were attributed to the asymmetric CH₂ stretch and the symmetric CH₂ stretch, respectively which were not found in the pure Fe₃O₄. The intense peak at 1745 cm⁻¹ was

derived from the existence of the C=O stretch and the band at 1346 cm⁻¹ exhibited the presence of the C-O stretch. The O-H in-plane and out-of-plane bands appeared at 1462 and 955 cm⁻¹, respectively. In the Figure 5(b), the asymmetric CH₂ stretch and the symmetric CH₂ shifted to 2916 and 2850 cm⁻¹, respectively. The surfactant molecules in the adsorbed state were subjected to the field of the solid surface. As a result, the characteristic bands shifted to a lower frequency region which indicated that the hydrocarbon chains in the monolayer surrounding the nanoparticles were in a closed-packed, crystalline state [27] . Which were characteristic of the asymmetric in as (COO^?) and the symmetric in s (COO^?) stretch, instead. This result can be explained that the bonding pattern of the carboxylic acids on the surface of the nanoparticles was a combination of molecules bonded symmetrically and molecules bonded at an angle to the surface [28] . The characteristic band for pure Fe₃O₄ usually appears at 570 cm⁻¹, whereas the present sample was shifted to 579 cm⁻¹. This can be explained as the carboxyl groups of B. aegyptiaca oil combined with the Fe atoms on the surface of the Fe₃O₄ nanoparticles and render a partial single bond character of the C=O bond to weaken the bond, and then shift the stretching frequency to a lower value [29] .

The crystallinity of the magnetic nanoparticles was investigated by powder XRD (Figure 6). Both Fe₃O₄ and Fe₃O₄ B. aegyptiaca oil nanoparticles exhibit six characteristic peaks at 2θ = 30.1, 35.3, 43.6, 53.6, 57.3 and 62.8, indexed as (220), (311), (400), (422), (511) and (440), respectively. The peak positions and relative intensities of the nanoparticles match well with those from JCPDS card 19-0629 for magnetite [30] , All the reflections in the pattern can be indexed from those of a standard sample of the Fe₃O₄ spinel structure [JC-PDS card No: 26-1136], indicating that the pure Fe₃O₄ has been obtained [31] .

Antimicrobial activity of B. aegyptiaca synthesized iron oxide nanoparticles suspension was tested against different gram positive (Staphylococcus aureus, Bacillus subtilis) and gram negative Escherichia coli (E. coli). It was shown in Figure 7 moderate activities against most of the tested microorganisms. The antimicrobial activity test of the fixed oil against selected strains of Gram-positive and negative was shown the highest activity on the gram positive bacteria. While B. aegyptiaca oil didn’t shown any result for gram negative bacteria, in contrasted it was shown highest antimicrobial activity against gram negative bacteria when used Fe₃O₄ with B. aegyptiaca oil and no result in gram positive bacteria these results are consistent with recent reports the bacitracin-conjug- ated Fe₃O₄@PAA nanoparticles also shown antibacterial ability towards Gram-negative E. coli bacteria [30] . The antimicrobial activity of this nanoparticles synthisezed by oil Fe₃O₄ is less than standard B. aegyptiaca oil that as reflected by lower size of the inhibition zone. The inhibition zones were calculated and taken as the measure of the inhibitory power of the oil [32] . The picture was shown that the oil at the tested dose of 10 μl/disk had antimicrobial activity with maximum inhibition zone against Staphyloccous aureus (S. aureus) compared with Fe₃O₄-B. aegyptiaca with maximum inhibition against Escherichia coli (E. coli).