A Comparative Study on the Sustainability of Groundnut Oil Storage Stability using Ethylacetate Extract from Piliostigma reticulatum and Butylated Hydroxyanisole

Lipid oxidation poses a significant challenge, adversely affecting the long-term stability of vegetable oils. This research aimed to evaluate the effectiveness of ethyl acetate extract from Piliostigma reticulatum leaves and butylated hydroxyanisole (BHA) in preserving freshly extracted groundnut oil during a four-month storage study. The groundnut oil was divided into five treatment groups and the progression of oxidative changes in each of these groups was diligently monitored every two weeks over the course of four months. Key parameters, including free fatty acid (FFA), peroxide value (PV), iodine value (IV), total phenolic content (TPC), and total carotenoid, were analyzed. The results of the stability study revealed that FFA and PV increased in all treatment groups over time, but the oil protected with P. reticulatum plant extract exhibited the lowest values. IV, TP, and total carotenoid were also observed to decrease in all groups, with the plant extract-protected oil sample showing the least reduction. This study concludes that the ethyl acetate fraction of P. reticulatum demonstrates superior antioxidant properties compared to BHA and can be considered a promising natural alternative for safeguarding vegetable oil against lipid oxidation.


Introduction
Vegetable oils serve as vital culinary and industrial ingredients, contributing to the rich flavours and textures of a myriad of dishes and serving as essential components in various industrial applications.However, these oils are inherently susceptible to a chemical process known as lipid oxidation, which can jeopardize their quality and shelf life [1].Lipid oxidation is a complex phenomenon involving the reaction of unsaturated fatty acids in the oil with oxygen, IOP Publishing doi:10.1088/1755-1315/1342/1/012012 2 leading to the formation of detrimental compounds such as free radicals, peroxides, and aldehydes [2].
The challenge posed by lipid oxidation in vegetable oils is multifaceted, given its far-reaching consequences.Beyond the degradation of sensory properties and nutritional quality, oxidized oils may exhibit reduced shelf life, which is a pressing concern for both manufacturers and consumers alike [2].As such, addressing lipid oxidation is essential to maintaining the quality and safety of vegetable oils.The management of oxidation processes has been accomplished through the use of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG).These synthetic antioxidants have the capacity to prolong the shelf life of vegetable oils by retarding or impeding the breakdown of lipids.Nevertheless, apprehensions regarding the safety of synthetic antioxidants, coupled with consumers' increasing preference for natural food items, have catalyzed the development of methods centered on using natural compounds to impede oil oxidation [3,4].
Piliostigma reticulatum is a tree that is native to Africa.This tree is well-known for its bioactive compounds, such as flavonoids, tannins, and alkaloids, which may be found in its bark, leaves, and roots.It is a useful resource in traditional medicine, with a history of usage in treating a variety of diseases because of these bioactive chemicals [5,6].Previous research conducted within our research team has confirmed that Piliostigma reticulatum (DC) Hochst is abundant in a diverse array of phenolic compounds, all possessing robust antioxidant properties [7,8].
In our earlier investigation [9], it was found that the ethyl acetate fraction of Piliostigma reticulatum exhibited the most potent antioxidant activity (EC50 value of 8.25 ± 0.89 μg/mL) compared to other fractions tested.This present study aims to conduct a comparative analysis of total carotenoid content, total phenolic content, and some lipid oxidation parameters in

Materials and Methods
The plant sample (Piliostigma reticulatum), collected in Ikire, Osun State, Nigeria, was subjected to authentication procedures at the Department of Botany, Obafemi Awolowo University, Ile-Ife.Subsequently, the plant material was air-dried for a duration of three weeks and then finely ground using a Norris grinding machine (Model no.7445, Christ and Norris Ltd., Germany).Freshly harvested groundnut was procured from a farm located in Ogbomoso, Oyo State, Nigeria.Essential chemicals, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), gallic acid, and Folin-Ciocalteu reagent, were obtained from Sigma-Aldrich in Johannesburg, South Africa.All other chemicals and reagents employed in this study met analytical-grade standards.

The process of preparing the crude extract
A crude extract was prepared from 350 g of pulverized leaves, employing the procedure outlined by Falade et al. [9].The resultant extract was concentrated through vacuum evaporation at 40 o C, utilizing a rotary evaporator (Heidolph Laborota 4001, Schwabach, Germany), ultimately yielding the crude extract.

Solvent partitioning of the crude extract
The crude extract, once obtained, was mixed with 250 mL of distilled water and subjected to sequential extractions with hexane (500 mL × 2), dichloromethane (500 mL × 2), and ethyl acetate (750 mL × 2) in that order, using a separating funnel.The resulting fractions, except for the ethyl acetate portion, were discarded.The ethyl acetate fractions were pooled together and then subjected to concentration under vacuum at 40 o C using a rotary evaporator, resulting in the formation of the ethyl acetate (EtOAc) solvent fraction.The selection of the ethyl acetate extract for this stabilization study was based on previous research conducted in our laboratory, 4 which involved the screening of various Nigerian medicinal plants, including Bauhinia purpurea, Bauhinia monandra, Bauhinia tomentosa, Bauhinia rufescens, and Piliostigma reticulatum, for antioxidant activity.Among all the plants tested, the ethyl acetate extract from Piliostigma reticulatum exhibited the highest level of antioxidant activity, as reported in the study by Falade et al. [9].

Extraction of groundnut oil
Groundnuts and their pods were thoroughly cleaned to remove sand particles, and any rotten nuts were removed.The high-quality nuts were air-dried in the lab for 7 days, then manually dehulled.The dehulled seeds were finely ground using a blender (MX-795N, Metsushita Electric Industries Co. Ltd., Malaysia) and stored in a refrigerator for up to two days.Oil was extracted from the ground seeds using the Soxhlet extraction method [10].The extracted oil was stored in amber-colored bottles in a dark place until needed.

Sample treatment
The study involved five treatment groups (A, B, C, D, and E) with 500 mL of groundnut oil each, stored in different conditions.Groups A, B, and E were kept in dark bottles, while C and D were in transparent bottles under light.Oil from groups B, C, and E was mixed with antioxidants (P.reticulatum extract in B and C, BHA in E) at a concentration of 600 mg/L and homogenized (Kika Labortechnik T25 basic homogenizer, Germany) for 2 minutes at 9500 rpm.All groups were heated in a water bath at 50°C.Groups C and D were exposed to oxygen bubbling and treated with iron II sulphate.Over the course of four months, the progression of lipid oxidation was tracked by measuring free fatty acids, peroxide value, iodine value, phenol content, and total carotenoid content every two weeks.

Determination of peroxide value (PV)
The peroxide value of the oil samples was assessed following the methodology described by Romano et al. [11].In this process, a standardized sodium thiosulfate solution was employed to titrate the mixture of the oil sample, potassium iodide, and acetic acid.The resulting peroxide value was expressed in milliequivalents of oxygen per kilogram (meq O2 / kg).

Determination of iodine value (IV)
The iodine value of the oil samples was determined using the titrimetric method outlined in the study by Okidhika and Ekpete [12].

Determination of free fatty acid
The analysis of free fatty acid (%) followed the method described by Chinedu et al. [13].
Initially, 5ml of diethyl ether and 5ml of ethanol were measured and added to a 250ml conical flask.Then, 0.5ml of a 1% phenolphthalein solution was introduced to this mixture.Subsequently, 2 g of the oil sample was accurately measured and introduced into the conical flask, followed by titration with a 0.1M solution of potassium hydroxide (KOH).The process included consistent agitation of the conical flask while the titration progressed until a persistent pink coloration appeared for approximately 15 seconds.The amount of KOH consumed to reach this endpoint was recorded.

Determination of total phenolic content
The spectrophotometric method detailed by Falade et al. [14] was employed to determine the total phenolic content of the oil samples.The quantified total phenolic content in the oil samples was standardized against gallic acid, and the result was expressed in micrograms of gallic acid equivalent per gram of oil (µg GAE/g).

Determination of total carotenoid content of the oil samples
The quantification of total carotenoid content was carried out through the application of the spectrophotometric method as described in the AOAC [15] standard.The concentration of total carotenoids was subsequently computed using the following formula, leveraging the extinction coefficient of β-carotene (2480) as proposed by Klein and Perry [16].Where A is the absorbance, V is volume of the ether extract (10mL) and E 1% 1cm is the extinction coefficient (2480)

Statistical analysis
The collected data underwent analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (IBM SPSS; version 22, Armonk, USA).Significant differences between specific means at a confidence level of 95% (p < 0.05) were determined through the application of Tukey's HSD post-hoc test.

Free fatty acid content of the oil samples
The study began with a 0.50% free fatty acid (FFA) level (Table 1), higher than a previous report of 2.8% (18), possibly due to differences in kernel handling and extraction methods (17,19).After 14 days, untreated oil (A) had a 10% FFA increase, while plant extract-protected oil (B) showed only a 2% increase.Oil treated with pro-oxidants (D) had the highest FFA level, a 28% increase compared to sample B. Sample C, treated with both plant extract and prooxidants, had a 16% FFA increase.Over 112 days, the FFA increase varied significantly, with unprotected oil, which also contains pro-oxidants (D) showing the highest increase (272%) and plant extract-protected oil (B) having the smallest increase (56%).This suggests that antioxidants, possibly through steric hindrance, may reduce triacylglycerol hydrolysis into fatty acids, making them less susceptible to oxidation than free fatty acids.

Peroxide value of the oil samples
Peroxide formation serves as an indicator for the initial phase of lipid oxidation [20].Table 2 reveals that the initial peroxide value (PV) of groundnut oil was 0.75 meq O2/kg oil, significantly lower than the 10.6 meq O2/kg reported in a prior study by Falade et al. [14] but within the range reported by Ozcan and Seven [21].This discrepancy could be attributed to the use of freshly harvested groundnuts in this study compared to those previously stored on the market, highlighting the role of endogenous lipases in releasing free fatty acids that can undergo oxidation.

Iodine value of the oil samples
The initial iodine value of freshly extracted groundnut oil, as shown in Table 3

Total phenolic content of the oil samples
In the first 14 days of storage, oil samples A, B, C, D, and E experienced reductions in total phenol content (Table 4).After 112 days, samples A, B, C, D, and E registered further reductions.The addition of the plant extract to sample B slowed down the deterioration of phenolic compounds, used in sample A as defense against lipid oxidation.Strong correlations were found between total phenol in unprotected oil (sample A) and peroxide value (r = -0.907),free fatty acid (r = -0.916),and iodine value (r = 0.908).Samples C and D, both with 96% and 98% reductions in total phenol, had almost entirely depleted their phenol contents, suggesting active participation of phenolic compounds in shielding against lipid oxidation.The plant extract outperformed BHA in protecting oil and its phenolic compounds, with sample B showing a 66% reduction compared to 83% recorded in E.

Total carotenoid content
The carotenoid levels in the oil samples are detailed in

Conclusion
In this study, the ability of an ethyl acetate extract from Piliostigma reticulatum to protect groundnut oil from oxidative degradation was tested.The findings indicated that, when compared to synthetic antioxidants, the plant extract proved more proficient in retarding the oxidation process in groundnut oil.The incorporation of this plant extract notably enhanced the chemical stability of the oil, resulting in a slight reduction in total phenolic content and total carotenoids, a lower peroxide value, and free fatty acid formation, along with a minor decrease in iodine value.Consequently, the utilization of ethyl acetate extract from Piliostigma (µ/) =  × Volume extract (mL) × 104 E1% 1cm × sample weight IOP Publishing doi:10.1088/1755-1315/1342/1/0120126

Table 1 :
Percentage Free Fatty Acid of the Oil Samples Results are means of triplicate determination ± standard deviation.Mean values in the same row with different superscript letters are significantly different (p < 0.05).Sample A: groundnut oil without plant extract (control); Sample B: groundnut oil with plant extract (to compare A); Sample C: groundnut oil with plant extract and prooxidants (light, Fe 2+ and Oxygen); Sample D: groundnut oil without plant extract but with pro-oxidants (to compare C); Sample E Groundnut oil with BHA (to compare B).

Table 2 :Table 2 :
Peroxide Value of the Oil Samples (meq O2 / kg oil) Results are means of triplicate determination ± standard deviation.Mean values in the same row with different superscript letters are significantly different (p < 0.05).Sample A: groundnut oil without plant extract (control); Sample B: groundnut oil with plant extract (to compare A); Sample C: groundnut oil with plant extract and prooxidants (light, Fe 2+ and Oxygen); Sample D: groundnut oil without plant extract but with pro-oxidants (to compare C); Sample E Groundnut oil with BHA (to compare B).
[26]s 265.83 g I2 per 100 g of oil, a significant reduction from the previously reported range of 76.36 to 97.13 g I2 per 100 g of oil for groundnut oil by Nkafamiya et al.[26].This discrepancy can be attributed to the various treatments applied to the groundnut seeds before oil extraction, such as sun drying and roasting, which are known to intensify lipid oxidation and polymerization.
significant protective effect.Comparing the oil sample protected with the plant extract (Sample B) to the one protected with BHA (Sample D), the plant extract proves superior.After 112

Table 3 :
Iodine Value of the Oil Samples (g I2 /100g oil) Results are means of triplicate determination ± standard deviation.Mean values in the same row with different superscript letters are significantly different (p < 0.05).Sample A: groundnut oil without plant extract (control); Sample B: groundnut oil with plant extract (to compare A); Sample C: groundnut oil with plant extract and prooxidants (light, Fe 2+ and Oxygen); Sample D: groundnut oil without plant extract but with pro-oxidants (to compare C); Sample E Groundnut oil with BHA (to compare B).

Table 4 :
Total Phenol Content of the Oil Samples (μg GAE / g oil) Results are means of triplicate determination ± standard deviation.Mean values in the same row with different superscript letters are significantly different (p < 0.05).Sample A: groundnut oil without plant extract (control); Sample B: groundnut oil with plant extract (to compare A); Sample C: groundnut oil with plant extract and prooxidants (light, Fe 2+ and Oxygen); Sample D: groundnut oil without plant extract but with pro-oxidants (to compare C); Sample E Groundnut oil with BHA (to compare B)

Table 5 .
[29]value of 2.00 ± 0.06 µg/g oil obtained for freshly extracted groundnut oil in this study exceeded the previously reported value of 0.18 -0.51 mg/kg for groundnut oil by Idrissi et al.[28].However, it fell below the range of 5-15 µg/g reported for rape seed oil by Saskia et al.[29].

Table 5 :
Total Carotenoid Content of the Oil Samples (µg / g oil)Results are means of triplicate determination ± standard deviation.Mean values in the same row with different superscript letters are significantly different (p < 0.05).Sample A: groundnut oil without plant extract (control); Sample B: groundnut oil with plant extract (to compare A); Sample C: groundnut oil with plant extract and prooxidants (light, Fe 2+ and Oxygen); Sample D: groundnut oil without plant extract but with pro-oxidants (to compare C); Sample E Groundnut oil with BHA (to compare B).