Antibacterial Activity of Marula [Sclerocarya Birrea] and Brominated Marula Seed Oil

Over the last decade, the use of hand sanitizers has increased substantially. Recently, with this outbreak of COVID 19 virus, all natural hand sanitizers have taken the spotlight for their simplicity, powerful cleaning abilities, natural moisturizing formulas and the ability to never leave the skin feeling dry. Marula [Sclerocarya birrea] seed oil was soxhlet extracted with n-hexane and then brominated. The oil quality parameters of marula oil (MO) and brominated Marula oil (BMO) were found to be; for MO: oil content (58.5 ± 0.435 %), moisture content (4.3 ± 0.158 %), saponification value (180 ± 0.7150 mgKOH/g ), peroxide value (10.52 ± 0.006 meq/kg), acid value (3.48 ±0.002) meq/kg ), FFA (1.73 ± 0.000 % as oleic),iodine value (61.02 ± 0.477 gI2/100g ). For brominated marula oil (BMO) ; saponification value ( 168.7 ± 0.930 mgKOH/g ), iodine value ( 8.6 ± 0.490 gI2/100g ), peroxide value (10.63 ± 0.505 meq/kg ), acid value ( 3.42 ± 0.000 meq/kg ), FFA (1.61 ± 0.000 % as oleic). Fourier Transform Infrared (FTIR) spectroscopy was used to confirm the bromination process. The following peaks of absorptions were observed for BMO: C–Br at 600 cm -1 , C–H stretch, (2950 to 2850 cm -1 for alkane), 1750cm -1 for carbonyl stretch, 1450 cm -1 for ester, 1680 cm -1 for alkene. The BMO showed a broad spectrum activity against E. coli, Salmonella sp, Staphylococcus aureus and Streptococcus epidermidis microorganism which gave the inhibitions zones of 19.50 mm, 20.50 mm, 19.00 mm, 18.60 mm respectively. DMSO was used as the negative control while streptomycin as the positive control at 400 mg/ml concentration. Based on these findings, BMO may be use as disinfecting oils or hand sanitizers and will discourage its use as food additive.


INTRODUCTION
Brominated Vegetable Oils (BVO) are complex mixtures of plant derived triglycerides that have been reacted to contain atoms of the element bromine bonded to the molecules [12]. Brominated vegetable oil has extensively been used as a food additive in soft drink manufacture, where it has been used as an emulsifying agent. However, in other applications, it has been utilized for its fire retardant properties a practice that have brought to question in its continued use as a food additive [10]. This is arising from reported cases of Bromism [5] and the observed increase in the level of brominated fatty acids that have been noted to be stored away in the body's lipid [10,11]. To support this assertion is the reported effort of BVO on yeast population [7] and thus its possible effect on human body cells, a fact that have been pointed out in studies with experimental animals. Marula oil is extracted from the kernels (nuts) of the marula tree (Sclerocarya birrea), from the Anacardiaceous family. Marula oil is traditionally used in cosmetics, in food as cooking oil and as a meat preservative and to treat leather [4].

II. MATERIALS AND METHODS
The kernel of plant Sclerocarya birrea was obtained within A T B U Bauchi metropolis. The sample was authenticated by the biological science department A.T.B.U Bauchi Nigeria.
The marula oil was soxhlet extracted using n-hexane and was brominated.The oil quality parameters of marula oil (MO) and brominated marula oil (BMO) was determined using a standard method by [1]. Infrared (Ir) spectroscopy was use to confirm the bromination process.

 Bromination process
This involves direct bromine adding with a dropping funnel into the oil sample in a Erlenmeyer flask at a maintained temperature of not more than 50 0 C. The reaction took up to 8 hours under vigorous strirring with magnetic stirrer with a condenser connected under a fume curpboad.

 Antibacterial Evaluation of MO and BMO
The Disc Diffusion Method for antimicrobial susceptibility testing was adopted [6,9] to access the presence of the antibacterial activity of MO and BMO. Bacteria culture of E Coli, Salmonella species, Staphylococcus aureus and Streptococcus epidermidis were used. In this method discs which have been impregnated with MO and BMO was placed on the Muller-Hinton Agar (MHA) plate surface, with each test plate comprising of 6 discs. The plates were incubated at 37 0 C for 18 to 24 hours, after which the plates was examined for inhibition zones diameter was measured using calipers and were recorded. Controls were conducted in line with the above procedure, using streptomycin antibiotic for positive control while dimethylsulphoxide (DMSO) for negative control.

 Extract Preparation
About 0.4ml of the extract (each of MO and BMO) were dissolved and made up to 1ml with DMSO to give a concentration of 400mg/ml each.

III. RESULTS AND DISCUSSION
The moisture content of the Marula oil (4.3%) was moderately high which could be responsible for the hydrolysis of triglycerides to yield high level of free fatty acids and will lead to high refining losses of oil yield (58.5%) as consequences as shown in table 1. Other parameters which include; acid value (AV), perioxide value (PV), saponification value (SV), free fatty acid (FFA), and iodine value (IV) were also presented at  Table 1:-Oil quality parameters of /MO and BMO The results are express in terms of mean ± standard deviation) Iodine value (IV) is the measure of the overall unsaturation in the structural matrix of the oil and it is usually used to characterize oils and fats as nondrying, semi drying and drying. However, the IV of MO and BMO falls within the nondrying class. The reduction of IV in BMO (8.6 gI 2 /100g) from (61.02 gI 2 /100g) for MO (table 1.) indicate that bromination has occurred but not 100% completion.
Evidence that bromination has occurred, is the sharp weak absorption of carbon-bromine bond at 600cm -1 at table 3. And its absence in that of MO at table 2. Presence of some targeted functional groups like the carbonyl stretch 1750cm -1 ,1400cm -1 for C-O ester and 1660 to 1680cm -1 for C=C alkene were observed in both IR spectra of MO and BMO (table 2&3).   BMO showed a broad activity against E coli, salmonella sp, staphylococcus aureus and streptococcus epidermidis microorganism which gave the inhibition zones of 17 mm, 16.5 mm, 15 mm and 16.0 mm respectively. Dimethylsulphoxide (DMSO) was used as the negative control while Streptomycin as the positive control at 400mg/ml sample concentration.

IV. CONCLUSION
This research work was successful since marula oil was extracted and brominated. The oil quality parameters of the marula oil and its brominated derivative was determined. The IR spectroscopy of MO and BMO were conducted to verify the presence of some targeted functional groups and to confirm the bromination process.
Antibacterial activity of the two (MO and BMO) shows activity with microbes that was used.
Focusing on the activity BMO has on some bacteria, a suggestion of it as an antimicrobial agent, can be explored and exploited for it application as sanitary products such as hand sanitizers or as disinfecting oils.