The effect of soursop‐flower‐enriched fried palm olein on some biochemical and hematological parameters of rats

Abstract This work set out to, first, assess the role of soursop flower extracts (SFE) in limiting palm olein oxidation during the production of plantain chips, before ascertaining the effect of these soursop‐flower‐enriched fried palm olein on some biochemical and hematological parameters of rats. The extracts were added to 1.5 kg of oil at 1000, 1400, and 1800 ppm, while BHT at 200 ppm served as a positive control (PO+BHT), and the oil without additives was the negative control (PO). The samples were subjected to 15 frying cycles. Total oxidation values varied between 5.94 ± 0.0 and 31.58 ± 0.37; 8.08 ± 0.25 and 28.24 ± 0.00 and 13.71 ± 0.24 and 42.71 ± 0.40 respectively for palm olein enriched with SFE, for PO+BHT and for PO. Twenty‐one groups each comprising five rats received, through dietary supplementation, oils subjected to 0, and 5, 10 and 15 frying cycles for a duration of 30 days. The alanine transaminase and aspartate transaminase of rats fed with oils enriched with SFE at fresh states and at 5 frying cycles was comparable to that of the neutral control group (23.45 ± 2.65 and 93.10 ± 3.53 U/L) and lower than that of the negative control group (52.15 ± 2.01 and 124.07 ± 1.89 U/L). The HDL cholesterol of these animals was also comparable to that of the neutral control group (67.82 ± 4.06 mg/dl) and higher than that of the negative control group (50.25 ± 5.20 mg/dl). White blood cells and mean corpuscular volume of rats fed with fried olein previously enriched with SFE were lower than those fed with fried olein without additives. These extracts are recommended as natural antioxidants for the stabilization of palm olein.

. Unfortunately, inappropriate culinary treatments such as prolonged frying to which vegetable oils are generally exposed lead to the oxidation of these unsaturated fatty acids, resulting in the formation of oxidation products including hydroxyl radicals, hydroperoxides, alkyls, alkoxyls, aldehydes, alcohols, and ketones (Duguma & Abebaw, 2020;Fadda et al., 2022).
All these compounds are very toxic because once ingested through the fried food, they can cause various types of pathologies such as inflammations, arteriosclerosis, cancer, and other diseases related to oxidative stress (Domínguez et al., 2019). In fact, aldehydes such as formaldehyde, acetaldehyde, 4-hydroxy-trans-2-nonenal and hexenal have neurotoxic and pro-inflammatory properties (Grootveld, 2022).
In addition, animal experiments have shown that the consumption of deep-fried vegetable oils has hepatotoxic and nephrotoxic effects (Ambreen et al., 2020;Amsalu et al., 2020). The investigations of Syamsunarno et al. (2020) and Islam et al. (2019) also show that the ingestion of thermooxidized dietary oils by rats compromises their health status, resulting in leukocytosis and an increase in serum concentrations of total cholesterol, LDL-cholesterol, and triglycerides followed by a decrease in HDL-cholesterol.
The use of antioxidants remains an effective means in the fight against oxidation because they have the ability to donate an electron or a hydrogen atom to free radicals in order to stabilize them, and this leads to an inhibition of the reaction (Al-Mamary & Moussa, 2021).
Antioxidants are naturally present in virgin oils in the form of tocopherols, carotenoids or polyphenols (Fadda et al., 2022), but, unfortunately, they are destroyed during the refining of these oils. In order to overcome this problem, the food industry supplements refined oils with synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and therbuthyl hydroquinone (THQ), and propyl gallate (PG) (Fatourehchi, 2020;Lourenço et al., 2019), which unfortunately are very toxic compounds. Indeed, studies have shown that prolonged consumption of BHA and BHT causes hepatotoxicity, DNA damage, gastrointestinal tract damage and tumor cell formation (Fatourehchi, 2020;Lourenço et al., 2019;Xu et al., 2021).
These setbacks have, at least, in part, led to growing interest in using natural antioxidants instead of these chemical additives.
So far, several research works (Djikeng, Womeni, Enti et al., 2017;Djikeng et al., 2022;Li et al., 2020;Mahmud & Muhammad, 2021;Şahin et al., 2020) have concluded that natural antioxidants from various plant matrices such as extracts of ginger rhizomes, rosemary leaves, olive leaves, soursop flowers, cinnamon, mangosteen, turmeric, and cassumunar ginger powders have the potential to limit the oxidation of vegetable oils during storage and frying. The findings from these research works also converge on the premise that the stabilizing activity of these natural antioxidants is closely related to the amount and nature of the phenolic compounds they contain. Studies conducted on Soursop Flower Extracts (SFEs) have revealed that this matrix has a total phenolic compound content of 51.33 mg/g GAE (Womeni et al., 2016), while phytochemical analysis detected the presence of vanillic acid, caffeic acid, gallic acid, ferulic acid, and quercetin which are phenolic compounds with good antioxidant activities. Furthermore, Djikeng, Womeni, Enti et al. (2017) showed that these extracts can limit the oxidation of palm olein during storage at 180°C after 24 h of heating.
Based on our search, no work has explored neither the stabilizing effect of soursop flower extracts in palm olein during deep frying, nor the harmful effects of fried oleins on some biochemical and hematological parameters of rats, a task which this paper seeks to accomplish.

| Materials
Soursop flowers were collected from a multipurpose research station of the Institute of Agricultural Research for Development (IARD) in Foumbot-Cameroon, in September 2019. Palm olein without additives was purchased from SCS/RAFCA in Bafoussam-Cameroon, in November 2019.

| Preparation of extracts
The protocol of Djikeng, Womeni, Marrapu et al. (2017)  and 1800 ppm respectively. In the fourth bottle, BHT (synthetic antioxidant) was introduced at its legal limit concentration of 200 ppm (Metzner et al., 2020) and represented the positive control. The fifth bottle did not receive any additive except 5 ml of methanol. Once the different oil samples were made up, they were shaked regularly for 3 h before being placed uncovered in an oven at 45°C for 24 h for maximum evaporation of the solvent. All oil samples (stabilized and non-stabilized) were prepared under the same conditions.

| Frying of plantain chips
The frying process was done according to the protocol of Leong et al. (2010) with some modifications. One hundred gram of fresh oil was collected first before the frying process. Palm olein sample was heated at 180°C in an electric fryer, and then 50 g of unripe plantain previously cleaned and cut into strips was introduced. After 3 min, the plantain chips were removed from the oil. Then, the hot oil left to cool at room temperature for 5 h before the collection of 100 g of the sample for analysis. The pre-cooled oil was used to frying another batch of plantains without adding new oil. The operation was repeated 4, 7, 9, and 14 times in order to obtain oil frying at 5, 8, 10, and 15 times. All oil samples (stabilized and non-stabilized) were fried under the same conditions.

| Measurement of oxidation parameters of the oil samples
The measurement of oxidation parameters was done according to standard methods as follow:

| Treatment of animals
Animals were treated according to the protocol of Zeb and Khan (2019) with some modifications. One hundred five albino Wistar rats were individually divided into 21 groups of five rats each. They weighed between 150 and 168 g. They were acclimatized for 1 week according to the rearing conditions prescribed by OECD (2008). The ambient temperature of the animal house was 25°C with a 12-h light/dark cycle.
They had free access to water and food. The staple food (SF) for the animals was composed according to the food composition for animals of Doungue et al. (2020) as follows: maize meal (68%), soybean meal (20%), fish meal (10%), bone meal (1%), cooking salt (0.8%), and vitamin complex (0.1%). The neutral control group received the staple food.
The other groups received the corresponding oil sample by dietary supplementation of 2% (2 ml oil in 100 g food). Table 1 shows the distribution of the groups of animals and the composition of their diets.

Groups
Codes Diet The experiment lasted 30 days after which the animals were anesthetized with chloroform vapor. The blood of each animal was collected by heart puncture, and each blood sample was divided into two parts. The first was placed in EDTA tubes for hematological evaluations, and the second was placed in dried tubes and the serum was obtained by centrifugation (1008 g for 15 min).

| Determination of biochemical parameters
The parameters assessed were transaminases (ALAT/ASAT), total protein, creatinine, triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol. With the exception of the total protein level which was determined with the BIOLABO kit, all other parameters were obtained from the SPINREACT kits.

| Determination of hematological parameters
Hematological analyses were performed on blood samples taken in Ethylene Diamine Tetraacetic Acid (EDTA) tubes by a blood count using an automatic hematological analyzer (SFRI H18 LIGHT auto hematology analyzer).

| Statistical analysis
The raw data were analyzed using the SPSS for Microsoft version 26.0. The results were presented as means ± standard deviation.
Multiple comparisons were performed using the Waller-Duncan test and the results were considered significant at p ˂ .05.

| Peroxide value
This parameter provides information on the primary oxidative state of fat. The variation of peroxide value (PV) of all the sample is presented in Figure 1A. In general, the peroxide value of all oil samples increases with the number of frying times, and the sample supplemented with BHT showed the highest peroxide value after 15 frying times (7.3 meq O2/kg). This increase can be linked to the production of hydroperoxides. As has been previously reported, under the effect of heat, unsaturated fatty acids easily lose a hydrogen atom in the α position on their side chains with the formation of alkyl radicals which react with triplet oxygen to produce peroxyl radicals, which will in turn abstract a hydrogen atom from another fatty acid in the medium and then form hydroperoxides

| p-anisidine value and thiobarbituric acid value
The anisidine value and thiobarbituric acid value help to measure the secondary oxidation products, which are 2, 4-dienals, 2-alkenals, and malondialdehydes, respectively. As seen in

| Total proteins
Proteins are biochemical macromolecules involved in both structural and biological functions of organisms and are also among the preferred targets of oxidation products. The details presented in Table 2 show that the consumption of the different oil samples enriched with plant extracts in the fresh state, as well as after 5 and 10 frying cycles led to a non-significant (p ˃ .05) variation in total protein concentration in the different test groups compared with the neutral control group. Nevertheless, a significant (p ˂ .05) decrease in this parameter is observed in all the groups that received the different oil samples that underwent 15 frying cycles. In addition to this, the groups of rats that consumed the non-enriched oil samples presented the lowest (p ˂ .05) serum protein concentrations compared with all the other groups. The decrease in protein levels in the different groups of animals can be due to the decrease in the digestibility of these molecules following their oxidation by free radicals. Aldehydes, which are among the major degradation products of fat oxidation, can attack the various amino acids (histidine, proline, tryptophan, cysteine, and tyrosine) contained in proteins, causing their oxidation (Hawkins & Davies, 2019;Kehm et al., 2021). This phenomenon generally leads to the formation of protein aggregates, making them unusable by the organism.
These protein aggregates are noticeably involved in the development of proteinopathies, such as Alzheimer's disease, Parkinson's disease, and prion disease (Lévy et al., 2019). The high total protein concentrations recorded in the groups fed with samples of oil enriched with plant extracts would be due to the action of natural antioxidants. The interactions between proteins and polyphenols have been found to improve oxidation resistance capacities (Li et al., 2021). Furthermore, the low concentrations observed in the groups fed with oil samples without additive would be related to the high rancidity of these oils, as previously observed in the chemical characterization tests. These results are consistent with those of Ambreen et al. (2020), who found that the consumption of repeatedly heated mixed vegetable oils results in a decrease in serum total protein concentration in rabbits.

| Creatinine
The change in serum creatinine concentration is an indicator of the pathological state of the kidneys. The results of this parameter are presented in Table 2   the phenomena of glomerular filtration, reabsorption, and tubular excretion during the production of urine, with the consequence of renal insufficiency which can be reflected in an increase in serum creatinine (Guerreiro et al., 2022). The low serum creatinine concentration observed in groups of animals that consumed palm olein enriched with soursop flower extracts is relatable to the low oxidative status of these oils This low serum creatinine concentration can effect the phenolic acids and flavonoids contained in the extracts (Womeni et al., 2016). According to Ashkar et al. (2022), caffeic acid possess anti-inflammatory, antioxidant, and immunomodulatory effects and can inhibits lipid peroxidation in renal tissues.

| Lipid profile
Changes in lipid profile most often lead to dyslipidemia, which is associated with various diseases such as cancer diabetes and cardiovascular diseases (CVD). Figure (Jiang et al., 2022;Poznyak et al., 2021). The accumulation of these cells in the interstitial space also contributes to the development of atheromatous plaques followed by the onset of atherosclerosis (Poznyak et al., 2021). Several studies (Ambreen et al., 2020;Feleke et al., 2022;Zeb & Khan, 2019)  Zeb and Khan (2019), who demonstrated that alpha-tocopherol improve the lipid parameters of rats fed with a diet containing thermooxidized sunflower oil. Table 3 shows some hematological parameters of the Wistar rats fed with the different diets. In general, the white blood cells level of the animals varied according to the oxidative state of the consumed oils.

| Effect of oil consumption on the hematological profile of rats
The white blood cell concentrations of the animals fed with samples of oils enriched with plant extracts were comparable (p ˃ .05) to those of the neutral control group, whereas the animals fed respectively with the non-enriched oil and the oil containing BHT heated at 15 frying cycles showed significantly high (p < .05) white blood cell levels compared to the neutral control group. This indicates that the inflammation is caused by a state of stress in one or more organs by oxidation products. The fact that the consumption of used-cooking oil resulted in an increase in white blood cell count in animals has also been demonstrated by Syamsunarno et al. (2020).
Regardless of the type of oil consumed, no significant difference TA B L E 3 Effect of different oil samples on the white blood cells, red blood cells, and some figurative elements in the blood of rats (p ˃ .05) was observed between the red blood cell (RBC), hematocrit (HTC), hemoglobin (HGB) and mean corpuscular hemoglobin concentration (MCHC) levels of all animals in the test and neutral control groups. The results related to mean corpuscular volume (MCV) reveal that all groups of animals fed with the oils containing the plant extracts showed comparable (p ˃ .05) MCV to that of the neutral control group. On the other hand, the groups of animals fed with the non-enriched oils heated to 10 and 15 frying cycles presented the significantly elevated (p < .05) MCV compared with the neutral control group. This could reflect macrocytic anemia in these groups of animals. This type of pathology could be the consequence of a vitamin B12 or folate deficiency (Kwon & Park, 2020). It is possible that oxidation products in the oils consumed by these animals caused the inflammations of the distal ileum or jejunum in the small intestine, resulting in poor absorption of vitamin B12, folic acid, and other nutrients. These findings are at variance with those of Zeb and Khan (2019) who discovered that consumption of oxidized olive oil has no significant effect on the mean corpuscular volume of rats.

| CON CLUS ION
It emerges from this work that soursop flower extracts can enhance the oxidative stability of palm olein during the frying of plantain chips. The effect of these extracts is concentrationdependent. At 1800 ppm, their efficacy is better than that of BHT. Animals fed with the different fried oils previously enriched with soursop flower extracts showed a decrease in transaminase activities and serum creatinine concentration followed by an increase in HDL cholesterol and total protein compared to animals fed with the non-enriched fried oils. With regard to hematological parameters, it was evident that the white blood cell count and mean corpuscular volume were significantly lower among rats fed with fried oleins previously enriched with soursop flower extracts, compared to those fed with fried oils without additives. These extracts are thus recommended as natural antioxidants for the stabilization of palm olein.

ACK N OWLED G M ENTS
None declared.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare they have not conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data analyzed during this study are included in this published article.

E TH I C A L A PPROVA L
The animal testing in this work was approved by the Institutional Review Board of the University of Dschang, Cameroon. Ethically, the same body reviewed and approved the protocol and procedures used in this work.