INTRODUCTION
Plants produce many bioactive principles that have found use in the pharmaceutical industry, thus impacting the healthcare system in positive ways. These compounds protect against free radical damage by acting as natural antioxidants (Altemimi, Lakhssassi, Baharlouei, Watson, & Lightfoot, 2017; Du, Ahn, & Du, 2002; Ogunwusi & Ibrahim, 2016). The potentials of edible seed oils have been exploited in the management of diseases. Diets rich in seed oils have provided micronutrients that helped the body defense mechanisms against reactive oxygen species; and generated biologically active metabolites that have played essential roles in the physiological resolution of inflammatory processes (Mazzocchi et al., 2021).
Depending on plant species, seed oils have been demonstrated as a viable and renewable source of bioactive compounds with great health-promoting activities in human nutrition and cosmetic applications (Quilez et al. 2020). Several seed oils, including Satureja hortensis (Quílez, Ferreres, Lopez-Miranda, Salazar, & Jordan, 2020) ; Punica granatum L and Momordica charantia L. (Yoshime, Melo, Sattler, Torres, & Manchini-Filho, 2019); Cucurbitaceae (Rezig, Chouaibi, Meddeb, Msaada, & Hamdi, 2019) and tomatoes (Szabo et al., 2019) , have been studied and presented as alternative plant oils that can serve as raw materials for food applications.
The African oil bean tree (Pentaclethra macrophylla Benth) is an indigenous forest fruit tree in Nigeria belonging to the family Leguminosae cultivated in homestead (Omokhua, 2009). The tree can reach a height and width of approximately 21 and 6 m, respectively. Its bark is greyish to dark reddish-brown and used for treating leprosy sores, while the seed helps prepare delicacies when cooked and fermented (Aseogwu, Ohanyere, Kanu, & Iwueke, 2006). The extract of stem bark, leaf, root bark and seed pulp of P. macrophylla oil has anti-inflammatory, and anthelmintic activities, hence used to treat gonorrhoea, convulsions, dysentery, taken as an analgesic, laxative, emollient against itch and is used to induce abortion (Oboh, 2007). It is an exceptionally highly sought-after food supplement for both local consumption and export due to its richness in vitamins and minerals make (Aseogwu et al., 2006; Enujiugha & Agbede, 2000). The seed with 58.15% oil content serves as a source of oils for candle making, cooking and soap) (Ogunwusi et al., 2016; Tico, 2005).
Africa seed oil is equally rich in phytochemicals, including alkaloids, tannins, saponin, oxalate, phytate and ascorbic, much as it contains minerals such as potassium, magnesium, sodium, iron, calcium, manganese and zinc (Aladekoyi, Orungbemi, Karimu, & Aladejimokun, 2017). The oil of P. macrophylla seed had been reported to inhibit the growth of Bacillus licheniformis, B. cereus, Lactobacillus species, Escherichia coli and Candida albicans, thus confirming that it is possible its antimicrobial effects and medicinal values (Okoye et al., 2014). The seed oil was further shown to protect against atherosclerosis due to its anti-atherogenic property and is edible because of its low free fatty acid value (Anioke, 2019; Ikhuoria, Aiwonegbe, Okoli, & Idu, 2008). The present study aimed to conduct a detailed characterization of the bioactive compounds in the seed oil of P. macrophylla to further its exploitation for industrial uses in cosmetics and sustain pharmaceutical uses.
MATERIALS AND METHODS
Plant materials
A botanical garden at Ilaro Federal Polytechnic's Department of Science Laboratory Technology provided the seeds, which were harvested between July and October of last year. At the Forestry Research Institute of Nigeria (FRIN), voucher number FHI 111205 was used to identify seed samples.
Chemicals and reagents
Standard tocopherols, cholesterol, stigmasterol, and sitosterol were purchased from Sigma Chemical Co. n-Hexane (HPLC grade) and pyridine were procured from (Merck, Vikroli Mumbai, India) (St. Louis, Mo., USA). It was acquired from Supelco as Sylon BFT (Bis-(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane (Bellefonte, Pa., USA).
Sample preparation
Following a previously published process, the Soxhlet extractor was used to extract n-Hexan (Oyedeji, Azeez, & Osifade, 2017). Solvent removal was completed in a rotary evaporator (Model RE300, Bibby Scientific Ltd., United Kingdom) at reduced pressure while traces of n-hexane was expelled under nitrogen gas.
Analytical method
Carotenoids, terpenoids, and sterols in the seed oils were determined using Gas Chromatography-Flame iIonization Detector as (Du et al., 2002) described.
Chromatographic conditions
HP Gas chromatograph 5890 series II, software - HP ChemStation Rev.A 09.01[1206], Column type – HP -5MS and HP INNOWax, Agilent Technologies India Pvt. Ltd, Hyderabad, India. Injection temperature - split injection; split ratio - 20:1; carrier gas – nitrogen, inlet temperature – 250oC; column dimension (30 m × 0.25 mm × 0.25 µm); detector and detector temperature – fid (320oC); flow rate – 1.0 ml/min; hydrogen gas pressure – 30 psi; compressed air – 35 psi; oven programs (Carotenoids and sterols - initial temperature @ 60oC. first ramping @ 10 oC/min for 20 min, maintained for 2 min second ramping @ 15oC/min for 4 min.).
RESULTS AND DISCUSSION
Carotene, xanthophyll, lutein and neo-xanthin, were the major carotenoids determined in the seed oil of P. macrophylla (Table 1). Their respective concentrations were 15.89, 10.32, 9.22 and 5.46 mg/100 g. Carotene was the major carotenoid content with a 15.89 mg/100g (32%) concentration, followed by xanthophylls 10.32 mg/100 g (21%). Other carotenoids include lutein, neo-xanthin, viola-xanthin, and authera-xanthin with 5.46, 3.91, 3.57 and 0.51 mg/100 g, respectively. Others, which occurred in trace amount were β-cryptoxanthin (8.27 x 0-3 mg/100 g), malvidin (1.08 x 10-4 mg/100 g) and lycopene (3.91 x 10-6 mg/100 g).
Table 1
Carotenoids and sterols of P. macrophylla seed oil
P. macrophylla seed oil contained nine different antioxidants, with carotene been the most abundant at about 35% and lower to 94.8% in palm oil (Zeb & Mehmood, 2004) .
The total concentration of carotenoids in this study, as shown in Table 1, was (48.89 mg/100 g) and is higher than 0.036 and 0.051 mg/100 g in Gordial olive fruit oil and Hojiblanca oil, respectively (Gandul-Rojas, Cepero, & Minguez-Mosquera, 1999). The carotenoid content of sea buckthorn (1167 mg/100 g) is higher than that obtained in this study (48.89 mg/100 g) (Zeb et al., 2004) .
In addition to vitamin A activity, carotenoids, particularly -carotene, operate as a scavenger of reactive free radicals and a quencher of chlorophyll and singlet oxygen excited states. Carotenoids can operate as potent peroxidation inhibitors when exposed to light. This function is, however, reduced due to heat-induced degradation with increasing temperature. The antioxidant activities of β -carotene are enhanced in the presence of α-tocopherol (Ghazani & Marangoni, 2013).
Sitosterol, campesterol, stigmasterol and Δ5-avenasterol were the major sterols determined in the seed oil of P. macropylla. Their respective concentrations were 457.58, 34.54, 18.52, and 3.32 mg/100 g (Table 1). Cholesterol, ergosterol and cholestanol, were also determined in trace amounts of 2.20 x 10-4, 5.56 x 10-6 and 7.01 x 10-6 mg/100 g, respectively. The total sterols determined in this study were 513.97 mg/100 g and are much higher than the total sterol content of 295 mg/100 g measured in the seed oil of Cucurbita maxima (Montesano, Blasi, Simonetti, Santini, & Cossignani, 2018). Sitosterol accounted for 89% of the total sterols and was distantly followed by campesterol (7%). Sitosterol percentage was higher in P. macrophylla than olive, peanut, sunflower, soya bean, and rapeseed oils with 84, 68, 62, 55 and 52%, respectively (Philips, Ruggio, Toivo, Swank, & Simpkins, 2002).
The amount of stigmasterol (3%) to other sterols in this study was found to be higher than that reported for different species of berry and kiwi seed oils that ranged between 0.3 - 2.4%. There is evidence that stigmasterol serves as a precursor of progesterone, an important human hormone that plays a critical role in regulation and tissue repair mechanisms (Hoed, Clereq, Echim, & Andgelkovic, 2009) . The 7% campesterol of the sterols in this study was lower compared to other vegetable oils such as soya bean (19.8%), rapeseed (31.9%), sunflower (10.5%), sesame seed (18.25%), and Arachis seed oils (17.3%) but higher than other oils that include apricot (3.8%), olive (2.85), and hazelnut seed oils (4.31%). There is evidence that stigmasterol serves as a precursor of progesterone, an important human hormone that plays a critical role in regulation and tissue repair mechanisms (Hilali & Charreuf, 2007).
Vegetable oil sterols have been shown to reduce total and low density lipoprotein (LDL) cholesterol levels in people by inhibiting gastrointestinal absorption of cholesterol (Lim, Tan, Karim, Ariffin, & Bakar, 2010). It has been found that a number of plant steroid compounds have anti-tumor properties, mainly in breast, colon, and prostate cancers. Cancer cell tumour inhibition and an increase in cancer cell apoptosis are linked to their mode of action (Fabrikov, Guil-Guerrero, Gonzalez-Fernandez, Rodríguez-Garcia, & Gomez-Mercado, 2019). From the preceding, the oil of P. macropylla could be deployed to deliver synthesized anti-cancer nanoparticles and drugs to enhance their potency. African oil bean seed in a previous study was demonstrated to have a positive effect on lipid metabolism in rats and showed an anti-atherogenic property indicating that it is protective against atherosclerosis, thus establishing its richness in sterols (Anioke, 2019).
Table 2
Terpenes, terpenoids, tocoperols and other components of P. macrophylla seed oil
Beta-caryophyllene, ϒ-elemene, α-caryophyllene, β-elemene, D-limonene and linalool were the major terpenes determined in the seed oil of P. macrophylla (Table 2). Their respective concentrations per 100 g oil were 45.67 mg for β-caryophyllene being the major terpene, ϒ-elemene (17.28 mg), α-caryophyllene (13.06 mg), β-elemene (12.43 mg), D-limonene (6.11 mg) and linalool (2.90 mg). Other terpenes were also determined in trace amounts at concentrations ranging between 0.02 and 0.59 mg/100 g (Table 2). The terpene content in cannabis seed oil mainly includes caryophyllene and myrcene (740 and 160 mg/L, respectively), which has a higher concentration than the terpene contents in P. macrophylla (58.73 and 0.52 mg/100 g, respectively). Terpenes give oils their characteristic smell and are primarily found in the essential oil of cannabis rather than in the seed oil (Adumanya, Uwakwe, & Essien, 2014; Bourgou, Pichette, Marzouk, & Legault, 2010).
Tocopherols are valuable antioxidants for cellular buffer and protection against diseases in humans. The major tocopherols obtained in the seed oil of P. macrophylla were ϒ-tocopherols, α-tocopherols, δ-tocopherols, β- tocopherols with concentrations of 43.83, 3.35, 0.40 and 2.42 x 10-2 mg/100 g, respectively (Table 2). The total tocopherol determined was 47.61 mg/100 g. The tocopherols are isomers of vitamin E with γ-tocopherol been the most abundant at approximately 92%, similar to that of soya bean oil (88%) and higher than 10.5, 58, and 46% for Brazilian nut (Bertholletia excelsa) oil, olive oil and walnut oil, respectively (Chunhieng, Haidi, Pioch, Brohier, & Montet, 2008; Nehdi et al., 2012). The total vitamin E in the study is greater than 33, and 42.54 mg/100 g for palm kernel and cold-pressed olive oils (Ellwood et al., 2014). Tocopherols are noted for their antioxidant activities; α-tocopherols has been the most active in capacity in vivo compared with other homologues. The α-tocopherols level in this study is suitable for the daily recommended level of 5–9 mg when P. macrophylla oil is used in food systems (Ghazani et al., 2013). Due to its high levels of γ-tocopherol, P. macrophylla oil can find use in cosmetic and pharmaceutical formulations by adding value and stability to those products because of their inherent antioxidant capacity and improves their emollient effects.
Total terpenoids content in the seed oil was 1.03 mg/100 g, as shown in Table 2. Terpenoids such as α- and β-amyrin have been reported to have anti-inflammatory effects in rodents, while some authors have also reported their usefulness as painkillers and for treating inflammation (Ebajo, Shen, Consolacion, & Ragasa, 2015; Okoye et al., 2014). These compounds in the seed oil of P. macrophylla suggest their usefulness as a painkiller and an anti-inflammatory agent.
The oil of P. macrophylla has chlorophyll at a concentration of 39.4 mg/100 g and above the 30 mg/kg specification for crude vegetable oils in Canada. Chlorophyll, a prooxidant, is involved in autoxidation, and photooxidation mechanisms reduce the shelf-life of oils as it contains pheophytin and pheophorbide as derivatives are therefore undesirable. The level of chlorophyll in the oil of P. macrophylla can be reduced to an acceptable level by bleaching it with activated charcoal (Ghazani et al., 2013; Mraihi, Journi, Cherif, & Trabelsi-Ayadi, 2013). The refractive index (RI) is a physical property that can be related to the structure that includes double bonds in fatty acids, and it increases as a result of an increase in conjugation, the formation of hydroperoxide and polymerization (Arya, Ramanujam, & Vijayaraghavan, 1969; Ospina, Tovar, Flores, & Orozco, 2016). The RI of P. macrophylla seed oil is 1.46 at 40oC, and it is consistent with the literature values of RI for most vegetable oils.
CONCLUSION
This study revealed the abundance of tocopherols, tocotrienols, terpenes and terpenoids and sterol in the seed oil of P. macrophylla. This study further showed that P. macrophylla oil has excellent nutritional potentials that are underexploited. Disease, including coronary disease, could be prevented with oil either directly or through many food products, serving as an antioxidant and as an anti-inflammatory agent. Related to the relative abundance and low cost of P. macrophylla, the tree holds good promise concerning the commercial production of oils.
