Introduction
Moringa (Moringa oleifera Lam) is one of the thirteen species belonging to the Moringaceae family. It is a fast-growing perennial tree, native to northwestern India, distributed throughout the Middle East and in African and Asian countries and widely cultivated in tropical and subtropical areas due to its adaptability to climatic conditions (Leone et al., 2016). All plant parts of the moringa tree (leaves, flowers, bark, roots, and seeds) are edible and are also used as tea or infusions in traditional medicine to treat several ailments, including malaria, typhoid fever, parasitic diseases, arthritis, swelling, cuts, diseases of the skin, and others (Al-Asmari et al., 2015; Bakre, Aderibigbe & Ademowo, 2013; Ouédraogo et al., 2011; Vasanth et al., 2015; Yassa & Tohamy, 2014). Moringa seeds represent a source of protein and oil. Interest in the oil extracted from moringa seeds has grown significantly over the years, being identified among the most promising emergent seeds for biodiesel production (Martín et al., 2010). A single moringa tree can produce 15 000 to 25 000 seeds per year (Foidl et al., 2001). The seeds are round-shaped, containing a kernel protected by a brownish semi-permeable hull (shell) and have three papery wings (Figure 1) (Leone et al., 2016). Besides conventional oilseeds, moringa seeds are dehulled before oil extraction to reduce oil absorption in the cake, avoid undesirable characteristics of the extracted oils such as increased pigments and waxes contents and increased oil extraction yields (Boukandoul, Casal, Cruz, Pinho & Zaidi, 2017). On the other hand, the shells obtained are discarded or burnt due to a lack of economic value (Martín et al., 2010).
For a rational utilization of moringa seeds, it is necessary to investigate the nutritional and antinutritional chemical composition and the nutraceutical properties of each anatomical part. Moringa whole seed and its kernel fraction have been widely studied regarding its nutritional content and antioxidant capacities (Ferreira, Farias, Oliveira & Carvalho, 2008; Singh, Negi & Radha, 2013). However, there is no information available on the shells and wings fractions. In order to contribute to the understanding of their composition and elucidate potential uses as ingredients or nutraceutical applications, in this work, partial characterization of moringa seeds and its anatomical fractions (kernel, shells, and wings) was performed, including proximate chemical composition, antinutritional factors, and antioxidant capacity.
Materials and methods
Plant material
Moringa seeds used in this study were obtained from mature pods of trees harvested in Culiacan, Sinaloa, Mexico (24° 59' 16.15" N; 107° 34' 25.64" W). The seeds were collected, cleaned, and stored at 4 °C until analysis.
Seed physical characteristics
Physical dimensions, namely seed length and width, of 25 seeds randomly selected were determined using a digital micrometer with 0.01 mm accuracy (Milán-Carrillo, Reyes-Moreno, Armienta-Robledo, Cárabez-Trejo & Mora-Escobedo, 2000). For 1000-seed weight determination, 100 seeds were randomly selected and weighted. The weight obtained was then multiplied by ten (Bortey, Sadia & Asibuo, 2016). Hectoliter weight (Kg/hL) was estimated in triplicate using a 1-liter graduated cylinder filled with seeds consistently and uniformly before weighting. The mass obtained was then multiplied by 100.
Seed fractionation and sample preparation
Seeds were manually fractionated into kernel, shells, and wings. Whole seeds and seed fractions were then ground into powder, passed through a 60-mesh screen sieve, packed, and stored at -20 °C until analysis.
Proximate composition
Proximate chemical composition analyses were conducted according to the standard methods (Association of Official Analytical Chemists [AOAC], 1999). The samples were dried at 130 °C to determine moisture (method 925.09 B), defatting in a Soxhlet apparatus with petroleum ether for lipid analyses (method 920.39 C), and a micro-Kjeldahl was used for protein quantification (N x 6.25) (method 960.52). Soluble and insoluble dietary fiber (SDF/IDF) were both determined using the total dietary fiber assay kit (Sigma-Aldrich [TDF 100 A]) according to the manufacturer instructions.
Phytate
The phytate content of the samples was determined by the colorimetric procedure described by Vaintraub & Lapteva (1988) with minor modifications. Phytate was extracted using 3.5% HCl (w/v). Suitable aliquots were diluted with distilled water to make 3 mL. They were treated with 1 mL Wade reagent (0.03% FeCl3∙6H2O + 0.3% sulfosalicylic acid) and determined spectrophotometrically at 500 nm using phytic acid as a standard. The phytate content was expressed as mg Phytic Acid Equivalent (PAE)/100 g (dw) (León-López, Dávila-Ortiz, Jiménez-Martínez & Hernández-Sánchez, 2013).
Saponins
Total saponin content was determined following the method described by Hiai, Oura & Nakajima (1976). The extract was obtained with 80% aqueous methanol. The quantification was done using a standard curve of diosgenin, and the content of saponins was expressed as mg equivalents of diosgenin (DE)/100 g (dw) (León-López et al., 2013).
Total phenolic content
Free and bound phenolics were extracted using 80% aqueous methanol according to the procedure described by Mora-Rochín et al. (2010). Free phenolics were obtained by mixing 0.5 g of sample with a 10 mL extraction solvent for 10 min. The blends were centrifuged (3000 g, 10 min), and the supernatant was recovered and concentrated to 2 mL at 45 °C using a vacuum evaporator and stored at −20 °C. Bound phenolic compounds were released and retrieved from the leftover pellet. The content of free and bound phenolic compounds was determined using the colorimetric method described by Singleton, Orthofer & Lamuela-Raventós (1999). A calibration curve was prepared using gallic acid as standard, and total phenolic content (TPC) was expressed as mg gallic acid equivalents (GAE)/100 g dry weight (dw). TPC was calculated as the sum of free and bound phenolic compounds.
Tannins
Tannins were extracted with 80% methanol (Xu & Chang, 2007), assayed colorimetrically according to the modified vanillin method of Price, van Scoyoc & Butler (1978) and expressed as mg catechin equivalents (CE)/100 g (dw).
Trypsin inhibitor activity
The trypsin inhibitor activity was tested according to the enzymatic assay described by Welham & Domoney (2000) using a-N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) as trypsin substrate. Trypsin inhibitor activity expressed as trypsin inhibitor units TIU/mg sample (dw) was calculated from the absorbance read at 410 nm against a reagent blank.
Antioxidant capacity (AoxC)
The antioxidant capacity (AoxC) of aqueous methanol extracts assayed for free and bound phenolic was determined by the oxygen radical absorbance capacity (ORAC) assay using fluorescein as a probe, as described by Ou & Hampsch-Woodill & Prior (2001). Trolox was used as a standard reference, and results were expressed as µmol Trolox equivalents (TE)/ 100g (dw). Total AoxC was calculated as the sum of AoxC from free and bound 80% methanolic extracts. All measurements were made in triplicate.
Statistical analysis
Results obtained were expressed as mean ± standard deviations. One-way ANOVA and Duncan’s multiple range test for means comparisons were applied to establish significant differences among the anatomical seed fractions of moringa, with a significance level of p < 0.05 using the Statgraphics Centurion XVII statistical package.
Results
Seeds physical Properties
The physical properties of moringa seeds are presented in Table 1. The average length and width of the whole moringa seed (MWS) were 1.05 cm and 0.86 cm, respectively. MWS show an average weight of 0.22 g, a 1000-seed weight of 212.82 g, and a hectoliter weight of 18.10 Kg/hL. Table 1 also shows the yield of seed anatomical fractions based on 100g of MWS seeds. Seed kernel fraction (SK) represents more than 70% of the whole seed weight, while the shells (SS) and wings (SW) correspond to 23.37% and 3.51%, respectively.
Parameter | Value |
Length * (cm) | 1.05 ± 0.07 |
Width * (cm) | 0.86 ± 0.08 |
Seed weight *(g) | 0.22 ± 0.06 |
1000-seed weight ** (g) | 212.82 ± 6.71 |
Hectolitre weight ** (kg/hL) | 18.10 ± 4.12 |
Wings, % of whole seed weight | 3.51 ± 0.09 |
Shell, % of whole seed weight | 23.37 ± 0.05 |
Kernel, % of whole seed weight | 71.78 ± 0.05 |
*Mean of 25 seeds; ** Mean of triplicate determinations.
Source: Authors’ own elaboration.
Proximate composition of whole seeds and seed fractions
Chemical proximate composition of moringa seeds and its fractions are given in Table 2. MWS contained significant amounts of crude oil (29.40%), crude protein (25.47%), and dietary fiber (28.12%). Significant differences in composition were observed among the seed anatomical fractions, showing the macronutrient distribution over the seed (p < 0.05). SW and SS fractions are mainly composed of carbohydrates (26.38% and 19.73%, respectively) and insoluble fiber (51.06% and 70.94%, respectively). As is well known, the SK fraction is rich in protein (32.19%) and oil (38.72%).
Component (g/100g sample) |
Whole seeds | Wings | Seed fractions Shell |
Kernel |
Crude Protein | 25.47 ± 0.43 | 12.67 ± 0.47b | 5.20 ± 0.50c | 32.19 ± 0.75a |
Lipids | 29.40 ± 0.14 | 2.56 ± 0.48b | 0.82 ± 0.16c | 38.72 ± 0.09a |
Ash | 2.97 ± 0.10 | 3.57 ± 0.02a | 3.32 ± 0.19a | 3.39 ± 0.34a |
Carbohydrates | 14.04 ± 4.03 | 26.38 ± 1.68a | 19.73 ± 2.41b | 12.58 ± 5.03b |
Dietary fiber | 28.12 ± 3.36 | 54.82 ± 0.98b | 70.94 ± 1.56a | 13.31 ± 3.85c |
Soluble | 2.88 ± 0.06 | 3.77 ± 0.43a | 2.82 ± 0.30b | 1.52 ± 0.10c |
Insoluble | 25.24 ± 4.06 | 51.06 ± 1.08b | 68.12 ± 0.51a | 11.79 ± 2.66c |
All values are means of triplicate determinations ± standard deviation. Means within rows with different letters are significantly different (p < 0.05). All data are expressed on dry weight basis (dw).
Source: Authors’ own elaboration.
Antinutritional factors content of whole seeds and seed fractions
The antinutritional components (phytate, saponins, phenolics, tannins, and trypsin inhibitors) in MWS and seed fractions are shown in Table 3. Phytate was the most abundant antinutritional factor found in moringa seeds. The phytate content in MWS was 1380.6 mg PAE/100 g, and these compounds were detected in all anatomical fractions assayed; SW (604.0 mg PAE/100 g), SS (1453.0 mg PAE/100 g), and SK (1674.9 mg PAE/100 g). Saponins were also detected in MWS (154.95 mg DE/100 g) and all anatomical fractions assayed; SW (168.7 mg DE/100 g), SS (197.4 mg DE/100 g) SK (130.4 mg DE/100 g). MWS shows a good TPC (212.8 mg GAE /100 g). The highest-level TPC was detected in SS (386.9 mg GAE /100 g), followed by SK (236.0 mg GAE /100 g) and SW (218.5 mg GAE /100 g). In MWS and its fractions, phenolic in the bound form represents the bulk of the TPC; 92.5% in MWS, 73.5% in SW, 62.5% in SS, and 92.9% in SK. Meanwhile, tannins quantified by the vanillin test were present in an amount of 62.6 mg CE/ 100g in MWS. The highest values were found in SW and SS fractions (189.7 mg CE/ 100g and 196.0 mg CE/ 100g, respectively), and no significant differences were found between them. The lowest content of tannins corresponded to the SK fraction (34.3 mg CE/ 100g). On the other hand, trypsin inhibitor activity detected in MWS was 5.8 TIU/ mg sample. SW fraction presented the lowest trypsin inhibitor activity (1.1 TIU/ mg sample), while SS and SK fractions show the highest activity (5.9 TIU/ mg sample and 5.7 TIU/ mg sample, respectively) without significant difference (p < 0.05) between them.
Component | Whole seeds | Seed fractions | ||
Wings | Shell | Kernel | ||
Phytic acid1 | 1380.6 ± 33.8 | 604.0 ± 38.0c | 1453.0 ± 11.2b | 1674.9 ± 11.6a |
Saponins2 | 154.95 ± 6.6 | 168.7 ± 7.6b | 197.4 ± 5.14a | 130.4 ± 8.8c |
Total phenolics3 | 212.8 ± 10.5 | 218.5 ±18.1b | 386.9 ± 15.2a | 236.0 ± 5.8b |
Free3 | 15.8 ± 1.2 | 57.9 ± 4.1b | 144.9 ± 7.8a | 16.7 ± 2.9c |
Bound3 | 196.9 ± 9.3 | 160.6 ± 13.9b | 242.0 ± 13.9a | 219.2 ± 2.9a |
Tannins4 | 62.6 ± 5.40 | 189.7 ± 10.5a | 196 ± 2.7a | 34.3 ± 2.0b |
Trypsin inhibitors5 | 5.8 ± 0.0 | 1.1 ± 0.0b | 5.9 ± 0.3a | 5.7 ± 0.1a |
All values are means of triplicate determinations ± standard deviation. Means within rows with different letters are significantly different (p < 0.05). 1mg Phytic Acid Equivalent (PAE) /100 g (dw); 2mg Diosgenin Equivalent (DE)/100 g (dw); 3mg Gallic Acid Equivalent (GAE)/100 g (dw); 4mg Chatequin Equivalent (CE)/ 100 g (dw); 5Trypsin Inhibitor Units (TIU)/mg sample.
Source: Authors’ own elaboration.
Antioxidant capacity
Antioxidant capacity of aqueous methanolic extract from MWS and moringa seed fractions are shown in Table 4. The SK fraction's total antioxidant capacity was 2.6 and 1.8 times higher than the SS and SW fractions, respectively. Most of the antioxidant capacity was found in the free aqueous methanolic extracts from all samples tested; SK presents the higher free antioxidant capacity followed by the SS fraction and the SW fraction. The antioxidant capacity of bound aqueous methanolic extracts showed a similar behavior; the highest bound antioxidant capacity was found in SK fraction followed by the SS fraction and the SW fraction.
Aqueous Methanolic Extract |
Whole seeds | Wings | Seed fractions Shell |
Kernel |
Free | 7818.2 ± 720.8 | 3562.1 ± 60.2c | 5193.4 ± 448.8b | 8703.0 ± 343.8a |
Bound | 2262.3 ± 197.9 | 2316.7 ± 316.7c | 3306.1 ± 297.7b | 6548.9 ± 252.5a |
Total | 10 080.6 ± 918.7 | 5878.9 ± 376.9c | 8499.5 ± 746.5b | 15 251.9 ± 596.4a |
Data are expressed as means ± standard deviation of triplicate samples. Means with different superscripts in the same row show a significant difference (p < 0.05). 1μmol Trolox equivalents (TE)/100 g (dw).
Source: Authors’ own elaboration.
Discussion
Moringa seeds are used to obtain oil and proteins. During these processes, the seeds are dehulled, and the obtained fraction is discarded and subtilized. On the other hand, the population in developing countries consumes the whole seeds without any recommendation or prescription by health promoters. In the current study, moringa seeds cultivated in Sinaloa, México, show different dimensions (length and width) compared to other authors who found values from 2 cm to 2.5 cm for height and 0.4 cm-0.7 cm for width (Leone at al., 2016; Nouhi, Kwaambwa, Gutfreund & Rennie, 2019). Average weight and 1000-seed weight were similar to previous results reported by Olson & Fahey (2011), while hectoliter weight (18.10 kg/hL) is published for the first time, as far as it is known. This parameter describes a low-density seed.
Moringa SK fraction represents more than 50% of the seed's total weight, being similar to other nonconventional oilseeds such as Jatropha curcas and Azadirachta indica (Neem) (Martín et al., 2010). Besides, moringa SK presents high crude oil content very similar to neem seeds (39.7%). In this study, SS and SW fractions yield and composition are reported separately for the first time. Moringa SS fraction is composed mainly of dietary fiber with low protein content, indicating a poor nutritional value, whereas SW fraction, also rich in dietary fiber, showed considerable protein content.
Conventional antinutritional factors such as trypsin inhibitors, saponins, and tannins have been little studied in moringa seeds. However, in the seeds used in this research, tannins, phytates, and saponins were detected at considerable levels. Phytic acid and its salts, collectively referred to as phytate, are potent inhibitors of iron and zinc absorption (Gibson, Raboy & King, 2018). In legumes and most oilseeds, phytate is uniformly distributed within the protein bodies of the endosperm or kernel (Gibson et al., 2018; Gupta, Gangoliya & Singh, 2015). Despite being present in all anatomical fractions, the highest proportion of phytate in moringa was found in the SK fraction (1674.9 mg PAE/100 g). This value, apparently high, still falls within the range reported for other oilseeds as soybeans, sesame seeds, sunflower kernels, linseeds, and rape seeds (1000-5400 mg/100 g) (Gupta et al., 2015). Although most reports underline the phytate action as an antinutritional agent, in recent studies, dietary phytate has been associated with specific therapeutic effects, including anti-diabetic, anti-cancer, and anti-inflammatory properties, among others (Abdulwaliyu et al., 2019).
The saponin content in moringa whole seed (MWS) found in this study differs from a previous study by Ijarotimi, Adeoti & Ariyo (2013), which reported a substantially lower saponin amount (9.83 mg/100 g). However, the saponin content found in MWS (154.95 mg DE/100 g), or in any of the anatomical seed fractions, is lower when compared with those values reported for quinoa seeds (1260 mg/100 g) (Lim, Park & Yoon, 2020) and other edible legumes such as soybean (800 mg/100g) (Mohan, Tresina & Daffodil, 2016).
On the other hand, tannin content in MWS found in this study (62.6 mg CE/100 g) was higher than that reported by Igwilo, Okonkwo, Ugochukwu, Ezekwesili & Nwenyi (2017), who described a 40 mg/100 g value. The SK fraction showed a lower tannin content than those reported in commercially important edible nut seeds (100 CE/100 g-800 CE/100 g) (Venkatachalam & Sathe, 2006). Tannin content found in moringa SS and SW fractions were almost 6-fold higher than in the SK fraction. Similar behavior was observed on tannin distribution in Mexican pecan nut. Flores-Cordova et al. (2017) found a tannin content of 7-fold to 8-fold higher in pecan shell than it was found in the edible part.
Trypsin inhibitor activity detected in MWS (5.8 TIU/ mg) was similar to the one reported for different varieties of lentils (4.98 TIU/ mg-6.29 TIU/ mg), and it was lower compared to chickpeas (14.22 TIU/mg- 15.96 TIU/mg) and common beans (15.18 TIU/mg-20.83 TIU/mg) (Shi, Mu, Arntfield & Nickerson, 2017). Generally, trypsin inhibitor activity tends to be higher in the cotyledon fractions than in whole seeds. In this study, no significant difference was observed among the trypsin activity found in MWS and moringa SK fraction.
Despite the appreciably high protein contents in MWS and moringa SK fraction, the presence of antinutritional components could be a restriction to their incorporation into food matrixes. Moringa SK fraction, which is particularly rich in protein and fat, once defatted, results in a protein-rich meal. Based on its amino acid composition, it is expected that this meal support growth when tested as feed-in animal models. However, Igwilo et al. (2013) determined the effect of defatted Moringa seed powder on growth rate and some biochemical parameters in albino rats for 21 days. They observed a reduction in growth accompanied by body weight loss. The results obtained by Igwilo et al. (2013) suggested that moringa seed proteins have a low digestibility due to the presence of antinutritional factors. On the other hand, the whole Moringa seeds are consumed by the population as traditional medicine; however, it is crucial to know the chemical composition of each anatomical part in order to recommend people how to use this type of seeds either as a source of protein or as a source of phytochemicals.
Likewise, the results clearly show MWS and its fractions contained substantial amounts of TPC. Some studies have demonstrated a linear relationship between TPC and AoxC. However, in this work, AoxC evaluated by the ORAC assay was not correlated with TPC in moringa samples (r 2 = 0.1387). Moringa SS, particularly rich in phenolics, including tannins, did not show the higher values of AoxC. This behavior could be attributed to other compounds that also exert antioxidant capacities in the extracts assayed for AxoC, such as saponins.
Antioxidant capacity of aqueous methanolic extracts from MWS and moringa seed fractions showed a considerable antioxidant capacity. The antioxidant capacity values found in all moringa samples were higher than those present in 100 g of almonds (4282 µmol TE) (US Department of Agriculture, Agricultural Research Service, 2010). The SK fraction exerts the highest activity among the samples assayed (15 252 µmol TE / 100 g, dw), this value is similar to those found in nuts like walnuts (13 057 µmol TE / 100 g, dw) or pecans (17 524 µmol TE/ 100 g, dw) (US Department of Agriculture, Agricultural Research Service, 2010).
Assuming that µmoles Trolox are equivalent to µmoles tocopherol, and 0.67 mg α-tocopherol represent 1 IU vitamin E (Apak et al., 2013), in 100 g dry weight of whole moringa seeds an antioxidant activity equivalent to 6479 IU vitamin E is found. The SK fraction exerts the highest antioxidant capacity, providing 9802 IU vitamin E/100g sample, followed by SS and SW fractions that exert an antioxidant capacity equivalent to 5463 vitamin E/100g and 3778 vitamin E/100g, respectively.
Conclusions
Moringa seed kernel fraction seems to be a promising source of protein. However, due to its considerable antinutritional factors content, more in-depth research is needed regarding its suitability and utilization as food or food ingredients in the human diet. All anatomical seed fractions of moringa are a reservoir of dietary fiber and antioxidants. Seed shells and wings fractions, generally removed before moringa processing for oil production, represent a good alternative for the extraction of natural antioxidant phenolic compounds, including tannins, which could be used for different purposes an ingredient in both pharmaceutical and food industries.