Seeds, which are valuable sources of fats and oils, comprise an essential component of the human diet (Hao et al., 2020). Seed oils are finding more and more uses in various industries, e.g., as flavors and textures in food industries and as oleo-chemicals for petrochemical industries (Anyasor et al., 2009). Africa, like most other (sub-)tropical continents, has these seeds and nuts in abundance but is yet to wholly utilize them, owing to the scarcity in data concerning their physical, chemical, biochemical, and industrial properties (Abdulkadir et al., 2013). Nevertheless, some seed oils, like those from Arachis hypogea (groundnut) and Glycine max (soya beans), have received considerable attention and already play important roles in countries such as Nigeria (Esonu et al., 2014). However, most seeds remain underutilized, one of which is Hura crepitans (Abdulkadir et al., 2013; Hao et al., 2020).

H. crepitans L. (common name: Sandbox tree) is an evergreen, perennial and dicotyledonous plant of the spurge family (Euphorbiaceae). It has short, dark, thickly parked, and pointed spines on the trunk and branches and is often planted as shade trees in towns and villages in Nigeria (Ezeh et al., 2012). H. crepitans seeds embody a very vital source of oil, with diverse potentials. The seeds contain amino acids at levels comparable to the other utilized seeds, with even higher lysine, cysteine methionine, threonine, and histidine (Ezeh et al., 2012; Esonu et al., 2014). Moreover, the bark has been used as a traditional medicine to treat constipation, skin irritations, microbial and fungal diseases in humans and in veterinary practices (Adindu et al., 2015). In Nigeria, however, the seeds are discarded as waste since there is no definite use for the H. crepitans seed oil (Adewuyi et al., 2014). Oil from Arachis hypogea (AHO), known as groundnut oil or peanut oil, is one of the major vegetable oils, with as much as 6.05 million metric tons of AHO produced globally in 2019/20 (Akhtar et al., 2014; Arya et al., 2016). AHO is rich in essential vitamins and unsaturated fats but contains a low proportion of saturated fats; it also has good antioxidant properties (Arya et al., 2016). It is therefore not surprising that it is one of the most utilized seed oils, whereas that from H. crepitans remain under-utilized, despite boasting comparable physical and chemical properties. The physicochemical properties and fatty acid composition of the seed and seed oil of H. crepitans have been reported (Oyeleke et al., 2012; Oyekunle and Omode, 2008). Besides, previous studies demonstrated the antimicrobial potentials of essential oil from H. crepitans (Abdulkadir et al., 2013; David et al., 2014) but left issues regarding its safety and other biochemical outcomes unexplored.

This study reports, for the first time, the functional and inflammatory responses to H. crepitans seed oil, in comparison with that of A. hypogea oil (a well-utilized oil), in a bid to fill the dearth of information regarding the biochemical characteristics of this under-utilized seed oil.

The unprecedented surge in the world’s population, along with the resultant increased consumption of food and fuel, has made the search for alternative food and fuel sources a priority in the area of study. Seed oils now occupy influential positions in human diets and as alternative biofuels, owing mostly to their tolerability, availability, inexpensiveness, and a plethora of applications (Anyasor et al., 2009; Lei et al., 2018; Pachuau et al., 2019). Despite the intensifying enthusiasm to elucidate the diverse health relevance and bioactivities of these seed oils, many are still under-studied, and one of such is H. crepitans (Abdulkadir et al., 2013), partly due to lack of information regarding its biochemical properties. This study thus investigates the functional and pro-inflammatory responses to H. crepitans seed oil (HCSO), in comparison with A. hypogea oil (AHO), in a bid to fill that dearth of information.

Saponification value correlates inversely with average molecular weight (or, by extension, chain-length) of fatty acids in oil, implying that the lower the saponification value the higher the average molecular weight (or longer the fatty-acid chain) and vice-versa (Gunstone, 2009). Peroxide value is used to monitor the oxidative deterioration (rancidity) of oils. Thus, a high peroxide value may indicate increased oxidation and formation of hydroperoxides (Gordon, 2004). The acid number is a measure of the number of carboxylic acid groups, i.e., the acidity of the oil sample, and a high acid value indicates oil with a reduced quality (Kardash and Tur’yan, 2005). In-vitro evaluations revealed similar physical properties (color, odor, and yield) between HCSO and AHO. However, the saponification, peroxide, and acid values were lower in the HCSO, indicating that HCSO may be of even better quality than AHO. Moreover, the values of these chemical properties obtained for HCSO compared favorably with previous values obtained for more utilized seed oils like those of soy bean, sunflower, olive, linseed, etc. (Gunstone, 2009), which is suggestive of the quality of HCSO.

We also examined the effects of HCSO on the function of some vital organs. The liver, the largest internal organ, performs various metabolic functions that are essential for the continuity of life. Due to its strategic position and diverse roles, it is particularly susceptible to diseases (Owojuyigbe et al., 2020). Liver disease, currently among global health issues (Byass, 2014), can be detected by carrying out liver function tests in the blood, such as levels of bilirubin and albumin, as well as the activities of some liver enzymes (Cheesbrough, 2006; Owojuyigbe et al., 2020). Bilirubin is formed from the breakdown of heme. This water-insoluble bilirubin (unconjugated or indirect bilirubin) is transported to the liver, via the blood, where it is conjugated with glucuronic acid (by glucuronosyltransferase) to form water-soluble bilirubin glucuronides (conjugated or direct bilirubin), which are then excreted via biliary excretion. Total bilirubin refers to both direct and indirect bilirubin (Cheesbrough, 2006). Liver enzymes (such as AST, ALT and GGT) are generally useful and rather sensitive markers of liver disease. Localized within the hepatocytes, these enzymes are released into circulation, following the compromise of the cell membrane (resulting from liver injury or disease) (Niemelä and Alatalo, 2010). Increased levels or activities in these biomarkers typically characterize liver damage or disease (Owojuyigbe et al., 2020). Indeed, the hepatoprotective properties of many treatments are assessed based on the reductions in the blood levels of these liver biomarkers. In this study, the activities and levels of these biomarkers in rats fed with HCSO-supplemented diets were not significantly different from that in rats fed with 15% AHO, indicating that HCSO did not damage the liver or impair its function. These findings are corroborated by Igwenyi et al. (2017), who reported decreased liver biomarkers (AST, ALT, total and direct bilirubin) following treatment of diabetic rats with H. crepitans seed extract (HCSE). They attributed these effects to its rich phytochemical constituents, such as alkaloids, carotenoids, flavonoids, etc., previously characterized by Adindu et al. (2015) .

The kidney, another major organ, primarily excretes wastes from the blood and are involved in other regulatory processes. However, because they are metabolically active and receive a quarter of cardiac output (despite weighing below 1% of total body weight); while also filtering out water from the filtrate (and may thus concentrate and accumulate toxic substances), the kidneys are particularly vulnerable to injury (Gheshlaghi, 2012; Shaterzadeh-Yazdi et al., 2018). Clinically relevant biomarkers of renal damage include urea, uric acid, and creatinine. Creatinine is produced by the muscle during unaltered catabolism excreted by the kidney, and may thus serve as an important criterion for kidney function. Urea, a waste product from dietary protein, is also filtered into the urine by the kidneys (Burns and Wortmann, 2011). Uric acid, a product of purine metabolism, is a normal component of urine produced in conditions where there is cellular destruction and thus, degradation of the nuclear material and release of purine bases. These purines are, via a series of reactions, converted into either hypoxanthine or xanthine, which are then broken down by xanthine oxidase into uric acid. The excessive build-up of uric acid results in gout (Kanwal et al., 2018). However, these biomarkers (creatinine, urea, and uric acid) are liberally filtered by the glomerulus and efficiently excreted via the urine with negligible metabolism by the kidney. Thus, increased levels in the blood, in most cases, may evince the onset of kidney failure (Burns and Wortmann, 2011). While this current study did not carry out urinalysis, in light of the observed significantly (p < 0.05) reduced plasma levels of uric acid and creatinine in all HCSO groups compared to the AHO group, we hypothesize that HCSO promotes renal clearance of these excretory products, suggestive of its reno-beneficial potentials. Interestingly, the levels of urea were significantly (p < 0.05) higher in rats fed with the HCSO-supplemented diet compared to those fed the AHO supplemented diet. We attribute this elevation in urea level to the high amino acid content of the H. crepitans seed oil (Esonu et al., 2014), especially glutamate (about 14.41 g/100 g protein), as per the reports of Fowomola and Akindahunsi (2007). Thus, the increased urea level may be a consequence of increased amino acid catabolism, which obligates increased urea production (Higgins, 2016).

The heart, a muscular organ, pumps blood that carries nutrients and oxygen to other parts of the body and metabolic waste away from these parts through the blood vessels of the circulatory system (Gaze, 2012). Clinically relevant cardiac tests include CK and LDH activities in the plasma, which are quantifiable markers of the health/disease condition of the heart. CK couples the phosphorylation of creatine to phosphocreatine (PCr) with the dephosphorylation of ATP to ADP. PCr serves as an in-situ energy store for the swift regeneration of ATP. Creatine kinase (CK) is primarily cytosolic and is examined as a damage biomarker of CK-rich tissues, like the heart (Moghadam-Kia et al., 2016). LDH, another cytoplasmic enzyme, converts pyruvate to lactate during anaerobic respiration and is extensively expressed in metabolically active tissues like the heart (Hu et al., 2015). Following cardiac injury/damage, these cytosolic enzymes are released into the blood. Thus, elevated activities correlate positively with various heart diseases (Hu et al., 2015; Moghadam-Kia et al., 2016). Our results showed that the CK and LDH activities in rats fed the HCSO-supplemented diet were not significantly different from those obtained from rats fed with AHO. From these results, the HCSO did not provoke any significant damage to the organs (kidney, liver, or heart), at least in comparison with AHO.

Gene analyses revealed a dose-dependent inhibition of the relative expression of genes coding for pro-inflammatory cytokines (TNF-α, IL-1α, IL-1β, and IL-6) and receptors (IL-1R and IL-6R) in the HCSO-fed groups compared to the 15%-AHO group. To the best of our knowledge, this current study is the first to provide data regarding the in-vivo anti-inflammatory effects of H. crepitans seed oil following oral supplementation. The previously available study on the anti-inflammatory effect of H. crepitans focused on its hexane and ethyl acetate extracts and used topical application on rat paws (Avoseh et al., 2018). Although inflammation is a defence mechanism in response to noxious stimuli, e.g., infectious agents, irritants, damaged tissues, etc., if left uncontrolled, it quickly becomes damaging, and this is a contributing factor to the pathogenesis of a plethora of chronic inflammatory diseases (Chen et al., 2018). During the inflammatory response, immune cells are typically activated and they, in turn, secrete cytokines that initiate inflammatory pathways. These pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) facilitate inflammation via interaction with the receptors of TNF (TNFR), IL-1 (IL-1R), IL-6 (IL-6R), as well as the Toll-like receptors (TLRs). Following activation, the receptor initiates intracellular signalling cascades, such as the nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), activator of transcription (STAT), and Janus kinase (JAK)-signal transducer cascades (Zhang et al., 2019). These cascades play major roles in the development of many leading causes of death, like cancer, cardiovascular diseases, chronic obstructive pulmonary disease, diabetes, etc. (WHO, 2020).

The suppression of inflammatory mediators by HCSO, in a dose-dependent manner, clearly evinces its anti-inflammatory properties. It remains to be seen if these properties will prove beneficial in different models of disease conditions. Previous studies have characterized different parts of H. crepitans and enumerated various inherent phytochemical constituents, such as the nitrogen-containing alkaloids, aromatic ring-containing phenolics, and isoprene-containing terpenoids (Oyekunle and Omode, 2008; Oyeleke et al., 2012; Adindu et al., 2015). These phytochemicals have been reported to possess antioxidant and anti-inflammatory properties (Chen et al., 2018; Zhu et al., 2018) and may account for the observed effects of HCSO on organ function and inflammatory markers, and even contribute to the lower saponification, peroxide, and acid values obtained for HCSO.

Our results demonstrate that HCSO did not significantly affect the function of major organs, nor did it provoke adverse inflammatory responses. Instead, it suppressed the expression of pro-inflammatory mediators. Having characterized some of the biochemical effects of HCSO, further research may investigate its potential beneficial effects on various disease states, particularly those involving inflammation.