Nutritional, phytoconstituents, and free radical scavenging potentials of Cola lepidota K. Schum. and Irvingia gabonensis (Aubry-Lecomte ex O’Rorke) Baill. leaves

This study was carried out to determine the essential oil components, protein qualities, fatty acid composition, and free radical scavenging potentials of leaves of Cola lepidota K. Schum. (Malvaceae) and Irvingia gabonensis (Aubry-Lecomte ex O’Rorke) Baill. (Irvingiaceae) using chromatographic and spectrophotometric methods. Thirty five bioactive components were isolated from C. lepidota leaves with myrcene, phytol, ephedrine, hexadecanoic acid, and 1,14-tetradecanediol as the main compounds while phytol, 2-furancarboxaldehyde, 5-(hydroxymethyl)-, 1-hexadecyne, carotene, and humulene were the predominant components of the I. gabonensis leaves. Leucine and arginine were the predominant essential amino acids, whereas glutamic acid and serine were the main nonessential amino acids in both leaves. The total amino acid (TAA) (70.92 g/100g), total non-essential amino acid (TNEAA) (45.87 g/100 g), and total acidic amino acid (TAAA) (23.01 g/100 g) of C. lepidota were high whereas I. gabonensis recorded higher Total essential amino acid (TEAA) (28.98 g/100 g), total aromatic amino acid (TArAA) (7.21 g/100 g), total branched chain amino acid (TBCAA) (14.28 g/100g), predicted protein efficiency ratios (P-PERs), and essential amino acid index (EAAI). C. lepidota contained 55.72% of unsaturated fatty acids, with predominance of linolenic and linoleic acids, while I. gabonensis produced 74.46% of saturated fatty acids, having myristic, lauric, and palmitic acid as the main compounds. All the radical scavenging potentials of both leaves were concentration dependent and produced higher DPPH, hydrogen peroxide, and ABTS radical scavenging potentials than the standards. This study has thus provided the scientific backing for the inclusion of both leaves for dietary and therapeutic purposes.


Introduction
The paucity of information, and non existence of a database for properties of local foods and fruits, are major contributors to the under-exploitation of some of the available medicinal plants in nature (Grivetti and Ogle, 2000).
Evidently, in Nigeria, many local fruits and leaves are underutilized and could face extinction. Erosion of practices encouraging the utilization of local medicinal plants could be drawn to the behavioural or lifestyle changes, massive migration to urban areas, and overdependence on popularly positioned resources (Joshi and Joshi, 2005), therefore accounting for the high rate of diet related diseases and malnutrition. In other words, proposing a return to the investigation of local plants holds the key to the adequate provision of nutrients, and other bioactive components for the populace.
Of such underexploited plants, are the leaves of Cola lepidota K. Schum. (Malvaceae) and Irvingia gabonensis (Aubry-Lecomte ex O'Rorke) Baill. (Irvingiaceae). C. lepidota also known as the monkey kola, is one of the members of the Cola species, and the Family Malvaceae that bear sweet edible fruits. In Nigeria, they are mostly consumed by the southerners and some Eastern part of Nigeria. The fruits of C. lepidota have been widely evaluated for its nutritional and biochemical properties (Pamplona-Roger, 2008;Singh et al., 2010). However, the leaves of this plant have been surprisingly overlooked in terms of nutritional and ethnomedicinal applications.
I. gabonensis is known as bush mango by local inhabitants, and belongs to the Family Irvingaceae which are green tall trees with perennial life cycle. The bush mango boasts of rich ethnobotanical profile due to extensive tradomedical applications for several disease conditions (Lowe et al., 2000;Ezeasor et al., 2017). The seeds also called Ogbono or Ugiri seeds in Igbo, is a known soup thickener, and have been shown to posses weight loss and hypoglycaemic effects coupled with other therapeutic potentials (Rosen and Spiegelman, 2000;Ngondi et al., 2009). However, the leaves of this popular culinary plant have seen little utilization regarding its ethnomedicinal potentials.
We understand that the under-exploitation of the leaves of both C. lepidota and I. gabonensis have occurred as a consequence of paucity of information about their nutrient composition. Hence, this study was carried out to determine the essential oil, fatty acid, protein qualities, and in vitro antioxidant potentials of C. lepidota and I. gabonensis leaves.

Sample collection, identification and preparation
Freshly harvested leaves of C. lepidota, and I. gabonensis were obtained from farmlands at Mbaise in Imo State Nigeria. The leaves were identified by the botanist at Department of Plant Science and Biotechnology (DPSB), Imo State University Owerri and the voucher specimen deposited in the DPSB herbarium.
After cleaning the samples of debris using distilled water, the leaves were dried under open air, and ground continuously to powder size.

Extraction and analysis
Using a Clevenger type apparatus, the hydrodistilled extracts of the samples (100 g) were obtained and analyzed in a HP Chem Station Rev. A 09.01 machine, under the following conditions; Detector; FID, Injection type and ratio; split and 20:1, inlet temp; 150 °C, Column; HP5MS (of dimension 30 m x 0.25 mm x 0.25 µm, carrier gas; hydrogen, flow rate; 1.0 mL/min, carrier gas pressure; 22 psi, compressed air pressure; 28 psi, oven parameters; initial temp of 40 °C, run rate at for 2 min at 200 °C, ramping (5 °C/min first and 200 °C/min final).

Identification of the components of essential oils
The chromatogram was interpreted using NIST database from the comparison of the spectrum of the unknown components with the spectrum of the known components stored in the NIST library. The name, percentage areas, and percentage concentrations of the components of the test materials were thus ascertained.

Amino acid analysis of the leaves
The method of Benitez, (1989) was applied for the amino acid content determination of the leaves. Briefly, 100g of the samples were immersed in 500 mL of mixture of chloroform and methanol (2:1) and sieved after 48 h. The extracts were concentrated in a rotary evaporator and were transferred the Amino Acid Autoanalyzer (Technicon Sequential Multi-Sample). Tryptophan was measured colorimetrically following the procedure of Rama Rao et al. (1974).

Determination of protein quality parameters
The groups of amino acids were arithmetically determined following the procedure of Amadi et al. (2017). The estimation of the indicators of protein qualities; Predicted protein efficiency ratios (P-PERs I, II, and III) were carried out using standard equations of Chavan et al. (2001)  The method of Steinke et al. (1980) as modified, was adopted for the determination of essential amino acid index (EAAI) shown below; EAAI= 9� mg of Lys in 1g of test protein × essential amino acids +His mg of Lys in 1g of Reference protein

Determination of fatty acid methyl esters
The procedure of Agomuo et al. (2017) was adopted for the extraction of the oils from the leaves using a mixture of n-hexane and isopropyl alcohol in a ration of 3:2. The Fatty acid methyl esters (FAMES) were generated by saponifying the extracted oils in a mixture of 3 mL 0.5 M KOH and 2 mL methanol and neutralized in 3 mL of 14% BF3 in 100 mL hydrochloric acid. Methylation was completed after heating at 90 °C for 10 min. Precisely 1 µL of the FAMES was drawn out and transferred to the chromatography machine for analysis. The chromatography conditions were as follows: Carrier gas and compressed air pressure: 22psi and 35psi Oven conditions: Initial temp (60 0 C), ramping (first, 12 0 C/min for 20 min and second, 15 0 C/min for 3min). The identification of components and peak areas and percentages were obtained using standard chemstation system after matching with standard methyl esters.

Determination of in vitro antioxidant properties: DPPH radical scavenging potential
The spectrophotometric method of Shimada et al. (1992) was adopted for the determination of DPPH radical scavenging potentials of the leaves.
The hydrogen peroxide radical % inhibition of the leaves was estimated following the procedure of Ruch et al. (1989) by spectrophotometry.
Nitric oxide radical scavenging potentials of the leaves were spectrophotometrically determined by the procedures of Alam et al. (2013).
Valine and methionine found in this study for both leaves were high than the medicinal plants; C. esculenta and I. batatas reported by (Inyang, 2016). Arginine, histidine, of both plants, and threonine levels of I. gabonensis surpassed their respective recommended daily allowances (Ijarotimi and Olopade, 2009). Both plants similarly produced appreciable amounts of non-essential amino acids. The total amino acids for both plants were comparable whereas I. gabonensis produced slightly greater total non essential amino acids with and without histidine, total aromatic, basic, sulphur, and branched chain amino acids, while the amount of acidic amino acids were greater in C. lepidota. Only I. gabonensis produced aromatic amino acids equivalent to the recommended levels for infants . Further, the protein quality indices showed the range of P-PERs as 1.03-2.02 for I. gabonensis, and 0.81-1.66 for C. lepidota. Both values were equivalent to those reported for millet and cowpea  while the EAAI values of both leaves compared with soy (Nielson, 2002) and peanut (Adeyeye, 2010) seeds, thus implying that both leaves are suitable protein sources for diet formulations. The fatty acid compositions of leaves of C. lepidota and I. gabonensis were presented in Table 4. C. lepidota contained a greater proportion of unsaturated fatty acids, with predominance of linolenic and linoleic acids, while I. gabonensis produced 74.46% of saturated fatty acids, having myristic, lauric, and palmitic acid as the main compounds. The result further indicated that both leaves are not richly supplied with short and very long chain fatty acids. For the unsaturated fatty acids, the linoleic acid contents of both plants were similar to those of broccoli, red cabbage and radish, whereas the linolenic acid contents of C. lepidota leaves in this study were higher than those reported for artichoke, asparagus, green pepper and egg plant and that of I. gabonensis compared with those for chicken pea and carrots (Virdih et al., 2009). Both leaves in this study were similar to wheat germ and olive oil, in terms of their palmitic acid contents, while out of the edible oils (Orsavova et al., 2015) like groundnut oil, sesame oil, rapeseed oil and almond oils, only coconut oil contained higher lauric acids than I. gabonensis leaves, but had lower myristic acid level. With higher unsaturated fatty acids, the consumption of C. lepidota leaves could improve heart health whereas the higher percentage of unsaturated fatty acids in I. gabonensis would require supplementation with richer sources of unsaturated fatty acids.
Braz. J. Biol. Sci., 2020, Vol. 7, No. 15, p. 43-57. Results were mean ± S.D of duplicate determination. Figure 1 to 5 contains the free radical scavenging properties of C. lepidota and I. gabonensis leaves. All the radical scavenging potentials of both leaves illustrated in this study were concentration dependent.
The DPPH and hydrogen peroxide radical scavenging potentials of both leaves, presented in Figure 1 and 2, respectively, showed that both leaves produced higher antioxidant effects than those for standard ascorbic acid.  Both leaves produced similar potentials to scavenge nitric oxide and ABTS radical scavenging potentials (Figure 3). The nitric oxide radical scavenging potentials of both leaves were similar to the standard at 30 and 40 ppm and at 10 ppm for ABTS. At 10 ppm, I. gabonensis produced lower FRAP than the standard trolox, but produced comparable effects with C. lepidota and trolox at 20 ppm, and higher effects than the trolox at 30 and 40 ppm. The high DPPH and ABTS radical scavenging effects of both leaves imply both leaves are richly supplied with phytoconstituents with hydrogen donating capacities, whereas their rich phenolic acids compositions were implicated by their potent nitric oxide scavenging (Kedare and Singh, 2011;Aluko et al., 2013). Hence, both leaves are dependable sources for the relief of oxidative stress in the system.

Conclusion
This study has shown that the leaf of C. lepidota is richly supplied with components with potent antioxidant and immune boosting potentials whereas I. gabonensis leaves were found to contain high quantities of anti-inflammatory and anti-sickling agents. Also both leaves produced high amounts of essential amino acids, and unsaturated fatty acids on par with popularly consumed vegetables as well as demonstrated strong free radical scavenging potentials which in most of the assays were greater than standard antioxidants.