Comparative Study of the Phytochemical Profile, Proximate Content and Antioxidant Properties of Leaves, Seeds and Pods of Moringa Oleifera

Moringa oleifera (Moringa) is a highly valued plant and various parts of this plant are employed for the therapeutic purposes in the indigenous system of medicine. This study was undertaken to compare the phytochemical, mineral, proximate and antioxidant constituents of the aqueous, ethanol and coconut oil extracts of M. oleifera leaf, seed and pod. Pulverized samples of the leaves, seeds and pods were extracted separately with distilled water, ethanol and coconut oil. The proximate analysis was carried out using standard AOAC protocols while the mineral contents were analyzed through atomic absorption spectrometry. The phytochemicals and antioxidants constituents of the extracts were analysed using standard protocols. The phytochemical screening of M. oleifera revealed the presence of alkaloids in each extract of the leaf, seed and pod in varying concentrations with the absence of phlobatannins in all sampled parts. Proximate composition of M. oleifera revealed higher percentage of crude protein (26.05±0.01 %) and total fatty acid (5.42±0.01 %) in the seed, while the leaf had higher percentage of fibre (8.12±0.02 %) and ash (7.82±0.02 %). The pod showed higher percentage of carbohydrate (72.05±0.2 %) and the seed revealed lowest moisture content (3.12±0.01 %) compared to other sampled parts. Sodium was revealed as the highest composition of mineral in each part of the plant. The result for antioxidant activity revealed that the leaf, seed and pod extracts have stronger scavenging effect on nitric oxide radical compared to the standards (ascorbic acid 8467.15μg/ml) and the least scavenging effect on FRAP. The results obtained from this study validate the pharmacological and nutritional potentials of M. oleifera and its use in treating different ailments as used in traditional medicine.


Introduction
Medicinal plants are known to be rich sources of bio-active phytocomponents or bio-nutrients (Nandi et al., 2006).They are described as enriched resources that can be used in drug development and as vital deposit of nutrients and therefore, recommended for their therapeutic values (Khan, 2018).Phytochemicals are defined as substances produced by plants, which exhibit biological activity; they include alkaloids, terpenes, steroids, stilbenes.saponins, xanthones, carotenoids, flavonoids, glycosides, phenolic acids.The study of these chemicals includes the extraction, the biosynthesis of the chemicals, and the functions of the chemicals in plants, animals and humans (Mendoza and Silva, 2018).Moringa oleifera Lam.(drum stick tree/ miracle tree/ ben oil tree/ horseradish tree) is a useful plant which has benefited humanities for centuries because of its medicinal properties and health benefits.Its antifungal, antiviral, antidepressant and anti-inflammatory properties have been reported in literatures such as Abdull Razis et al. (2014), Biswas et al. (2020) and Ahmadua et al. (2021).According to Palafox et al. (2012), oil extracted from Moringa seeds (Ben oil) contains 70% oleic acid and showed stronger antifungal activity against a zoophilic dermatophyte which causes marked inflammatory reactions in humans.The leaves of M. oleifera could serve as a great benefit to people who cannot obtain proteins from meat.Many companies across the world manufacture various products from Moringa leaf such as tea, tablets, capsules, leaf powder, soaps and facewash (Mishra et al., 2012).Dried ground powder made from young root of Moringa plant gives a flavour similar to that of horseradish and can be used as a hot seasoning base (Builders et al., 2014).This study was designed to compare the phytochemical components, mineral content and antioxidant properties of the leaves, seed and pod of Moringa oleifera using different media of extraction.

Sample Collection
The leaves, pods and seeds of Moringa oleifera were obtained from trees in various locations in the eastern part of Nigeria.They were authenticated and deposited at the Lagos University Herbarium (University of Lagos, Nigeria) with voucher identification number LUH 8922 for the seeds and pods and LUH 8978 for the leaves.

Sample Treatment and Preparation
The fresh leaves and pods were separated manually, air dried for seven days and pulverized into fine particles.The seeds were deshelled, air dried for two weeks and pulverized using an electric grinder.An amount of 25 g of each pulverized sample was macerated separately with ethanol for 72 h, coconut oil for 168 h and decocted with distilled water for ½ h.The ethanol extract was concentrated to dryness using rotator evaporator at 40℃, while the aqueous extract was concentrated to dryness using water bath.The dried powdered sample was used for the proximate analysis and mineral content determination.The extracts of each of the sampled parts of M. oleifera was used for the phytochemical screening and determination of the antioxidant activity.

Mineral Analysis
Digestion method was according the protocols of Hernández et al.

Proximate Analysis
The protocols described by the Association of Official Analytical Chemists (AOAC, 2012) was used for moisture, ash, crude fiber and crude protein content.The carbohydrate content was calculated by subtracting the sum of the percentage ash, crude lipid, moisture, crude protein and crude fibre from 100 % according to the methods described by Thiex (2009) and Maisarah et al. (2014)

Determination of Antioxidant Activities Ferric Reducing Power Assay
The method of Rajurkar and Hande, (2011) was employed in determining the reducing power capacity of the extracts/fraction.The protocol involves a mixture containing 1 ml of sample (25-100 µg/ml), 2.5 ml of 0.1 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1%, w/v potassium ferrocyanate [K3Fe(CN)6] and incubated at 50 °C for 20 min.Trichloroacetic acid (2.5 ml, 10%, w/v), was added to the mixture and centrifuged at 5000 rpm for 10 min.Equal volume of Fresh FeCl3 ( 5ml 0.1%, w/v) was mixed, the upper layer (5 ml) of the centrifuged mixture and the absorbance was taken at 700 nm against a control (blank).

Free Radical Scavenging using DPPH Assay
The method of Kordali et al. (2005) was followed in accessing the free radical scavenging potential of the extracts/fraction.Precisely, 1 ml of 0.1 mM of DPPH in ethanol solution was added to 1 ml of extracts/fraction in water at different concentrations (25-100 μg/ml).The mixture was briskly agitated and allowed to stand at 25 ℃ for 30 min and the absorbance taken using a UV-Visible Spectrophotometer at 517 nm.The percent DPPH scavenging effect was calculated using the following equation: Where A0 was the absorbance of the control and A1 was the absorbance in the presence of the standard sample or extract.The IC50 value represented the concentration of the compounds that caused 50 % inhibition of DPPH radical formation.

Nitric Oxide Assay
The antioxidant capacity of the extract using Nitric oxide assay was investigated using the methods of Lee et al. (2011) and Alam et al. (2013).In a beaker was added 2 ml of 10 mM sodium nitroprusside in 0.5 ml phosphate-buffered saline (pH 7.4), and 0.5 ml of extracts/ fraction was added at varying concentrations.The mixture was incubated for 150 min at room temperature.In a test tube, 0.5 ml of the incubated mixture was added into 1.0 ml sulphanilamide solution (0.3 % in 20 % glacial acetic acid) and the mixture reincubated for 5 mins at 25 o C. Lastly, 1.0 ml naphthylethylenediamine dihydrochloride (0.1 % w/v) was added and the resultant mixture incubated and the room temperature maintained for 30 min.The absorbance was taken at 546 nm for the sample and the standard.The percentage inhibition was calculated according to the following equation.
Where Ai = Absorbance of the extract; A0 = Absorbance of the control/blank.

Lipid Perioxidation
The thiobarbituric acid assay protocol used by Ganhão, et al. (2011) was employed in determining the lipid peroxidation activity of the extracts/fraction.10 μL of samples at different concentrations (25, 50, 75 and 100 μg/mL)/ standard solution (1,1,3,3tetramethoxypropane, TEP) and 40 μL of 20 mM phosphate buffer (pH 7.0) were added to a test tube on ice bath.In each tube, 50 μL of 3% sodium dodecyl sulfate (SDS), 200 μL of 0.1 N HCl, 30 μL of 10% Phosphotungstic acid, and 100 μL of 0.7% of 2-thiobarbituric acid (TBA) were added.The tubes were firmly closed and boiled at 100 °C for 30 min in water bath.The reaction mixture was mixed with 400 μL of n-butanol and then centrifuged at 3000 rpm for 10 min.Supernatants were collected and pass through a UV/VIS spectrophotometer at a wavelength of 515 nm/555 nm.
Lipid Perioxidation (%) = (control − test) x 100 control where control = absorbance of control sample and test = absorbance in the presence of the samples of extracts or standards.

Statistical Analysis
All experiments were carried out in triplicates.Data were presented as mean and standard deviation.Statistical Package for Social Science (SPSS) version 22 was used for the data analysis.

Phytochemical Screening
The phytochemical assay revealed that Moringa oleifera seed, leaf and pod contain various components such as alkaloids, anthraquinone, flavonoids, phenols, reducing sugars, tannins, saponins, steroids, in varying concentrations with the absence of phlobatannins as shown in Table 1.This is similar to the results reported by Nepolean et al., (2009); Fowoyo and Oladoja, (2015), and Nkot et al., (2018) on M. oleifera.The coconut oil extract contains higher concentration of saponins, steroids and reducing sugars while aqueous extract contains high concentration of saponins.Ethanolic extract contains all tested phytochemicals except phlobatannins with high concentration of steroids and reducing sugars.Ethanol was observed to be the best solvent of extraction for the seed.The aqueous extract of the pod showed higher concentration of the phytochemicals presents than other extracts.Alkaloids were found to be the only phytochemical present in all extracts of the leaf, seed and pod.Literatures have it that medicinal plants containing alkaloids, flavonoids and phenols as bioactive metabolites have good antibacterial properties and the presence of these metabolites in Moringa oleifera could make it a potent antibacterial agent (Akintelu et al., 2021).Flavonoids are responsible for the medicinal qualities accorded in the leaf.Foye et al. (2008) reported that they also induce mechanisms that may kill cancer cells and inhibit tumor invasion.Saponins were observed in the aqueous and ethanolic extract of the leaf, seed and pod samples as well as in the coconut oil extract of the seed and pod of Moringa oleifera.Due to its ability to form froth, soap can be produced locally for bathing from any plant containing Saponins (Oman, 2013).Saponins are also used as an adjuvant in the production of vaccines and also possess antioxidant, anti-inflammatory, anti-apoptosis and immune-stimulant as reported by Woods et al. (2017).Alkaloids are nitrogen-containing naturally occurring compounds, commonly found to have antimicrobial properties and are used as antimalarial, pesticides, tranquilisers and stimulants (Galeotti et al., 2008).

Proximate Analysis
The results obtained from the proximate analysis of M. oleifera revealed high content of carbohydrates (72.05±0.2%) in the pod and protein (26.05±0.01%) in the seed compared to other compositions as confirmed with the result obtained by Sodamade et al. (2013).
Carbohydrates was also found to be in high concentration (68.23±0.3%) in the leaf.Carbohydrate is suitable for optimal functioning of the brain, heart, nervous, digestive and immune system while depletion of body tissue is the consequence of its deficiency (Offor et al., 2014).Proteins are known to be body building blocks; thus, protein molecules carry out all the major structural and functional aspects of the body.According to Vasudevan et al. (2019) an abnormality in protein structure could lead to molecular diseases with severe alterations in metabolic functions.This supports the report that M. oleifera plant could serve as food supplement to increase the nutritional values of foods that lack protein, carbohydrate and lipid as asserted by Fowoyo and Oladoja, (2015).This would be a great boom to people who do not get protein from meat and who need more energy for metabolism.The seed showed low moisture content (3.12±0.01%)than other parts of the plant, as a result, slows down the growth of microorganisms hence increasing the storage life capacity.The result on the proximate content is as shown in Table 2.The pod showed the lowest amount of total fatty acid (1.15±0.01%).

Mineral Content
The mineral content of Moringa oleifera plant samples revealed the presence of varying amounts of minerals with sodium having the highest percentage as displayed in Table 3. Cobalt was not detected in any of the sample.The mineral content of the seed indicated that sodium (5.550±0.01mg/L), potassium (1.511±0.00mg/L), magnesium (0.872±0.00 mg/L) and zinc (0.361±0.00 mg/L) are the most abundant as justified by Kawo et al., (2009) and Sodamade et al., (2013) with little difference in their values.Sodium is present in higher concentration in the leaf and pod with a large significant difference.Sodium is both an electrolyte and mineral; it helps regulate blood fluids, balance the body and prevents low blood pressure, it is necessary for the health of the heart, liver and kidneys.Potassium plays a role in regulating the movement of nutrients into   et al., 2016).Magnesium helps in promoting skin cells from damage.Majekodunmi (2015) reported that zinc is useful for protein synthesis, normal body development and recovery from illness.

Antioxidant Activity
The antioxidant potential of Moringa oleifera extracts revealed increase in reductive potential with increasing concentration of the extracts while the aqueous extract was observed to have the least scavenging activity as shown in

Conclusion
Moringa oleifera is one of the medicinal plants reported in folklore to have been explored for its beneficial purposes.The presences of various phytochemicals, nutritional and high antioxidant activities as asserted in this study lay credence to the fact the leaf, seed and pod of M. oleifera may be a good source of nutrient and therapeutic material to treat a number of diseases.

Table 4 .
Antioxidants are inhibitors that the DPPH radical scavenging activities of M. oleifera leaf which tallies with the work carried out byIram et al. (2016)and can therefore be safely ingested.The result obtained also supports the use of M. oleifera seed for natural antioxidant, anti-inflammatory, anti-diabetic, anti-hypertensive and anti-ulcer activities.

Table 1 : Presence of Phytochemicals Assayed in Sampled Parts of Moringa oleifera Asekunowo
et al., 2022 Comparative Study of the Phytochemical Profile 65| This journal is © The Nigerian Young Academy 2022 Annals of Science and Technology 2022 Vol.7 (2) 62-68