Proximate, mineral, vitamin and anti-nutrient content of Celosia argentea at three stages of maturity

South African Journal of Botany 124 (2019) 372–379

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Proximate, mineral, vitamin and anti-nutrient content of Celosia argentea at three stages of maturity O.D. Adegbaju, G.A. Otunola ⁎, A.J. Afolayan Medicinal Plants and Economic Development Research Centre, Department of Botany, University of Fort Hare, Alice 5700, South Africa

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Article history: Received 3 January 2019 Received in revised form 3 May 2019 Accepted 26 May 2019 Available online 13 June 2019 Edited by L Sebastiani Keywords: Proximate Celosia argentea Mineral Growth stages Anti-nutrients

a b s t r a c t The distribution of nutrients and metabolites in different organs and tissues is in a constant state of flux throughout the growth and development of a plant. This study was designed to evaluate the nutritional composition of Celosia argentea at three different stages of maturity to establish the best time of harvest for optimal nutritional benefits. Three growth stages: Pre-flowering (PRF), flowering (FLW) and post-flowering (PST) stages of two trials were investigated. Proximate, vitamins and anti-nutrients were performed using AOAC methods while minerals were determined using inductively coupled plasma-optical emission spectrometer (AOAC). For both trials, the PRF had the highest ash (28.15 ± 0.10% and 26.22 ± 0.10%) and crude protein (19.50 ± 0.17% and 25.80 ± 0.20%) contents. However, the PST had the highest carbohydrate (28.51 ± 0.20% and 36.16 ± 0.22%), crude fiber (33.41 ± 0.87% and 25.20 ± 0.20%) and energy (435.28 ± 27.6% and 426.08 ± 1.08%) but was the lowest in fat and moisture (8.43 ± 0.15% and 6.35 ± 0.09%) contents respectively. In the mineral composition, there was no marked demarcation in the amount of Mg, Na and Fe. However, the PRF, had the highest amount of Ca, K, P and Cu. Zinc was highest at the flowering stage of growth, while Vitamins A, C and E concentration decreased as the plant approached maturation. PRF stage had the highest vitamin concentrations; alkaloid and saponin contents were highest at the PRF stage while oxalate and phytate content was not dependent on growth stages. The study revealed that although, the nutrient composition of Celosia argentea is strongly influenced by maturity stages, the best age of harvest, would be determined by the deficiency to be corrected. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Increase in agricultural productivity towards reduction of hunger has been recorded worldwide over the past two decades. However, over 1 billion adults are overweight (Remans et al., 2011), more than 900 million people are undernourished and over 2 billion people are afflicted by one or more micronutrient deficiencies (WHO, 2007; FAO, 2010). In a bid to produce enough calories necessary for a healthy living, provision of adequate diversity of nutrients necessary for a healthy life is often overlooked by agricultural and food systems. Arising from the afore-mentioned, nutritional scientists have developed different methods of introducing nutrients or food items to accommodate the bigger picture of diet diversity by encouraging the consumption of fruits and vegetables; at least one serving per day for essential nutrients and energy (Barnes et al., 2013; Weaver and Marr, 2013). Food plants such as leafy vegetables have played an important role in human nutrition especially in the aspect of food security and Abbreviations: PRF, Pre-flowering; FLW, Flowering; PST, Post-flowering. ⁎ Corresponding author. E-mail address: [email protected] (G.A. Otunola).

https://doi.org/10.1016/j.sajb.2019.05.036 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

micronutrient deficiencies (Borokini et al., 2017). However, antinutrients substances such as phytic acid, flavonoids, tannins, saponins, oxalates and alkaloids are present in most vegetables (Aregheore, 2012). Although most leafy vegetables contain anti-nutrients, most of these acts as antioxidants and are responsible for their therapeutic properties (Jimoh and Afolayan, 2009). Celosia argentea Linn. (Amaranthaceae) is a widely cultivated vegetable in tropical and sub-tropical Africa, North America and Asia (Yarger, 2007). It is a leading vegetable of high economic value, which serves as a source of living for most rural vegetable farmers during the dry season, especially in the south western part of Nigeria where it is known as Sokoyokoto (Akinfasoye et al., 2008). This plant has high protein and vitamin contents and is also a good source of calcium, iron, carbohydrates and phosphorus (Ayodele and Olajide, 2011). Apart from its nutritional properties, various ethnopharmacological studies have reported its medicinal properties which include anti-inflammatory, antidiarrhea, anti-urolithiatic and wound healing properties (Nidavani et al., 2014; Varadharaj and Muniyappan, 2017). Despite its nutritional popularity in the tropical regions of Africa and Asia, it is considered as weed and not consumed in most parts of Southern Africa and other parts of the globe (Council, 2006).

O.D. Adegbaju et al. / South African Journal of Botany 124 (2019) 372–379

Plant growth and development is highly dependent on mineral nutrient uptake and the concentrations of these nutrients fluctuate greatly in space and time. This fluctuation is influenced by various factors such as; cultural practices, stage of maturity, harvesting methods and postharvest handling procedures as well as the interactions among these factors (Flyman and Afolayan, 2008; Maathus and Diatloff, 2013). Mineral composition of each plant organ is determined by a sequence of events that begins with bioavailability of mineral nutrients in soils and membrane transport in the roots to the final deposition of these nutrients in to one or more cellular compartments (Grusak, 2002). Plants accumulate and redistribute macro- and micronutrients throughout their life cycle. While some nutrients are relatively immobile in plants, some are easily redistributed to other parts, especially during abiotic stress conditions (e.g., nutrition depletion in the soil). Some nutrients may be translocated from mature leaves and fruits to the younger leaves. Also, in order to optimize reproductive potential, most plants need to remobilize nutrients from sources in a highly orchestrated way towards flowering. This process is spatially and temporally dependent on the stage of the plant's life cycle. Furthermore, changes in mineral nutrient homeostasis have been reported to influence multivariate changes in maturity of plants (Bennett et al., 2012). Most vegetables like C. argentea are usually harvested by uprooting or repeated cutting, which invariably results to an uneven distribution of nutrients in the harvested vegetables. The nutritional values of C. argentea have been comprehensively explored by different researchers; however, none reported the nutrient and antinutrients compositions at different stages of maturity, except for the reports of Adediran et al. (2015) on the influence of leaf position on nutritional composition of C. argentea. This study was therefore designed to evaluate the impact of maturity on the nutritional composition of C. argentea at three different stages to establish the best time of harvest for optimal nutritional benefits.

2. Materials and methods 2.1. Procurement and preparation of plant materials Mature seeds of C. argentea were obtained from an Agro shop in Nigeria. Seeds were raised in rectangular seedling trays measuring 65 × 100 cm2 with 200 bottom holes. They were transplanted into 72 plastic pots filled with compost soil (Khanya Nursery, Alice Eastern Cape, South Africa) at the green house of the University of Fort Hare, Alice, 5700 Eastern Cape, South Africa. The geographical location of the study site lies at latitude 32° 47′–19° 26′ S; longitude 26° 50′–42° 306′ E and altitude of 514.70 m above sea level. All plants were sufficiently watered every 2 days until the experiment was terminated at 12 weeks after transplanting and three randomly selected plants were harvested per row for each harvest period. Three harvest periods of two trials: Pre-flowering (PRF, leaves and stem were harvested when the first flower was sighted), flowering (FLW, harvest was done when 50% of all the plants had flowered) and post flowering (PST, harvest was done when the flowers were few and dropping) were investigated. The first trial was conducted from October 2017 to January 2018 while the second trial took place from March to May 2018. A voucher specimen (Ade/med/2017/01) at maturity stage was deposited at the Giffen Herbarium, University of Fort Hare. The leaves and stem were rinsed with deionized water and gently blotted with paper towel, sliced into small bits and oven-dried (LABOTEC, South Africa) at 55 °C for 72 h until constant weight was achieved and then ground into powder by an automated motor blender (Polymix® PX-MFC 90D Switzerland). Physiochemical parameters of the soil before planting were determined for both trials using the Inductively Coupled Plasma – Optical Emission Spectrometer (ICP-OES) as outline by Agrilasa (2008).

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2.2. Proximate 2.2.1. Moisture content Moisture content was measured using air-oven following methods of Association of Official Analytical Chemists (AOAC, 2000). A material test chamber M720 (LABOTEC, South Africa) was used to dry an empty weighing vessel at 105 °C for 1 h (W1) and weighed (W2). The dry sample (5 g) was then poured in to the vessel, oven dried at 105 ± 1 °C until constant weight was attained. This was then cooled in a desiccator, after which it was weighed (W3). The percentage moisture was calculated as: %Moisture content ¼ W2−W3 W2−W1  100 where W1 = weight of the empty vessel. W2 = weight of the vessel + sample. W3 = weight of vessel + dried sample. 2.2.2. Ash content The ash content was determined using a dry ashing method (Agrilasa, 2007). A porcelain crucible was dried at 105 °C for 1 h, after cooling in a desiccator, and then weighed (W1). The samples (2 g) were placed in the previously weighed crucible and reweighed (W2). The crucible with its content was then ashed first at 250 °C for 1 h at 550 °C for 5 h. (Furnace E-Range, E300-P4, MET-U-ED South Africa) and allowed to cool and the weight was taken (W3). The percentage ash was calculated as: %Ash content ¼ W2W3 W2W1  100 where W1 = weight of a dried porcelain crucible. W2 = weight of the crucible + sample. W3 = weight of the crucible + ashed sample. 2.2.3. Crude lipid Crude lipid was determined using the Soxhlet extraction technique (AOAC, 2005). The lipid content of the sample (5 g) was extracted using 100 mL of petroleum ether. The mixture was filtered, and its lipid content was collected in a pre-weighed (W1) clean beaker. Thereafter exhaustive lipid extraction was done on the same sample with 100 mL of petroleum ether for 24 h. It was then filtered and decanted into (W1) beaker. The lipid content was concentrated to dryness in a steam bath and oven dried at 40–60 °C and the beaker was reweighed (W2). Percentage of lipid was calculated as; ð%Þ Crude lipid ¼ Weight ofW2−W1  100 original sample 2.2.4. Crude fiber A modification of the acid/base digestion method described by Aina et al. (2012) was used to determine the dietary fiber. A 5 g of sample was digested with 100 mL of 0.25 M sulfuric acid solution by boiling under reflux for 30 min and quickly filtered. The insoluble matter was rinsed four times with boiling water to remove the remaining acid. This process was repeated on the residue using 100 mL of 0.31 M sodium hydroxide solution. The final residue was washed with water until it was free of base. It was then oven-dried at 100 °C, cooled in a desiccator and weighed (C1). The weighed sample was incinerated in a muffle furnace at 550 °C for 5 h, transferred to cool in a desiccator and weighed (C2). The percentage crude fiber was calculated as: C2−C1 ð%Þ Crude fiber ¼ Weight  100 of sample

2.2.5. Determination of crude protein The total nitrogen amount in the sample was determined following the micro Kjeldahl method (AOAC, 2005). Digestion of the sample (2 g) was done in a Kjeldahl flask by boiling 20 mL of concentrated H2SO4 and a Kjeldahl digestion tablet until a clear mixture was obtained. The digest

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was filtered into 250 mL volumetric flask, made up to mark with distilled water and set up for distillation. Ammonia was steam-distilled from the digest to which 50 mL of 45% NaOH solution has been added. The distillate (150 mL) was collected into a conical flask containing 100 mL 0.1 N HCl and methyl orange was used as an indicator. The ammonia reacted with the acid in the receiving flask and percentage nitrogen (N) was estimated by back titration against 2 M NaOH. Nitrogen calculated using the following equation. ½ðmL standard acidN of acidÞ–ðml blankN of baseÞ–ðml std:baseN of baseÞ Weight of sample in grams

 1 : 4007

where, N = normality, percentage crude protein was obtained by multiplying the nitrogen value by a factor of 6.25.% crude protein = Nitrogen in sample × 6.25. 2.2.6. Total carbohydrate content The carbohydrate content was estimated by deducting the total crude protein, crude fiber, ash and lipid from the total dry matter as: % Total carbohydrate = 100 – (% Moisture content + % Total Ash + % crude fat + % crude fiber +% crude protein). 2.2.7. Energy content Total energy of the samples was calculated by the difference method. The atwater factors: 4, 9 and 4 kcal were employed to calculate the caloric value by summing the multiplied values for crude protein, crude lipid and carbohydrate respectively as: Energy value(kcal/100 g) = (crude protein × 4) + (crude lipid × 9) + (total carbohydrate × 4). 2.3. Elemental analysis All elemental analyses were carried out in three replications as described by Bvenura and Afolayan, (2012) using Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES; Varian 710–ES series, SMM Instruments, Cape Town, South Africa). Wavelengths, slits and lamp current used for the determination of nine elements were 213.9 nm, 0.5 nm, 4.0 mA (zinc); 422.7 nm, 1.2 nm, 4.0 mA (calcium); 422.7 nm, 1.2 nm, 3.0 mA (copper); 589.0 nm, 0.8 nm, 3.0 mA (sodium); 248.3 m, 0.2 nm, 6.0 mA (iron); 285.2 nm, 0.1 nm, 4.0 mA (magnesium); 279.5 nm, 0.2 nm, 5 mA (manganese); 213.6 nm, 1.0 nm, 10 mA (phosphorus) and 766.5 nm, 0.8 nm, 4.0 mA (potassium),respectively. The results for mineral contents were expressed as mg/100 g dry weight (DW). 2.4. Vitamins 2.4.1. Vitamin A The retinol content in the samples was estimated using the method described by Onyesife et al. (2014). Briefly, 1 g of sample was macerated with 20 mL of petroleum ether. The solution was incubated for 2 h and filtered, evaporated to dryness and 0.2 mL of chloroform-acetic anhydride (1:1 v/v) added to the residue. Thereafter, 2 mL of 30% TCAchloroform was added to the mixture and absorbance was measured at 620 nm using a UV-3000PC spectrophotometer. Retinol standard was prepared in similar fashion. The concentration of vitamin A in the sample was extrapolated from the standard curve using the equation: Y = 0.001x + 0.0008, R2 = 0.9969. 2.4.2. Vitamin C Ascorbic acid (Vitamin C) was quantified using the 2,6-Dichlorophenol-indo-phenol, sodium salt (DPIP) titration method as described by Adebooye (2008). The samples (1 g) were separately homogenized with 40 mL of a buffer solution made up of 1 g/L oxalic acid and 4 g/L sodium acetate anhydrous. The mixture was titrated against a solution

containing 295 mg/L DPIP and 100 mg/L sodium bicarbonate. The endpoint of the titration was identified by the disappearance of the initial blue color and results were expressed as mg 100/g dry weight. 2.4.3. Vitamin E The vitamin E (α-tocopherol) content of the samples was determined using the method of Njoku et al. (2015). A known weight, 1 g of the sample was macerated with 20 mL of ethanol. The solution was filtered with Whatman No. 1 filter paper, then 1 mL of the filtrate was added to 100 μL of 0.2% ferric chloride in ethanol and 1.0 mL of 0.5% α,α′-dipyridyl solution. The solution was diluted with distilled water to make the solution up to 5 mL. Absorbance was measured at 520 nm using UV-3000PC spectrophotometer. The concentrations of the standard solutions ranged from 10 to 100 μg/mL. The concentration of vitamin E in the samples was extrapolated from the standard curve using the equation: Y = 0.0086x − 0.0216, R2 = 0.9985. The vitamin E content in the samples was expressed as mg/100 g DW. 2.5. Anti-nutrient composition 2.5.1. Alkaloid content The alkaloid content was determined according to the method described by Omoruyi et al. (2012). Five grams of sample was macerated in 200 mL of 10% acetic acid in ethanol. The mixture was covered and allowed to stand for 4 h. After which the mixture was filtered, and the filtrate concentrated in a water bath to one-fourth of its original volume. Concentrated ammonium hydroxide was added drop wise to the concentrated solution until precipitation (cloudy fume) was completed. The solution was allowed to settle, washed with dilute ammonium hydroxide and then filtered. The residue collected with Whatman No. 1 filter paper, was dried, weighed and the alkaloid content calculated as: Weight of precipitate Alkaloidð%Þ ¼ Weight  100 of original sample

2.5.2. Oxalate content The titration method described by Day and Underwood (1986) was used to determine the oxalate content of the sample. One gram of sample was macerated with 75 mL of 3 mol/L H2SO4 in a conical flask. The mixture was stirred with a magnetic stirrer for 1 h and filtered. The filtrate (25 mL) was collected and heated to 80–90 °C then maintained at 70 °C. The hot aliquot was titrated continuously with 0.05 mol/L of KMnO4 until the end point of a light pink color which persists for 15 s. The oxalate content was calculated by taking 1 mL of 0.05 mol/L of KMnO4 as equivalent to 2.2 mg oxalate. 2.5.3. Phytic acid Phytic acid was determined as described by Damilola et al. (2013). The sample (2 g) was weighed into a flask, macerated with 100 mL of 2% HCl and allowed to stand for 3 h. The mixture was centrifuged for 10 min at 13,000 rpm, then 25 mL of the supernatant was placed in a separate 250 mL conical flask with 5 mL of 0.3% ammonium thiocyanate solution as indicator and 53.5 mL of distilled water was added to the mixture. The mixture was titrated against standard iron III chloride solution (0.001 95 g of iron per mL) until an end- point of slightly brownish yellow color persisted for 5 min. Phytic acid was calculated as: Phytic acid(%) = Titer value × 0.00195 × 1.19 × 100. 2.5.4. Saponin content Saponin content was estimated using the method previously described by Otang et al. (2012). One gram of sample was mixed with 40 mL of 20% ethanol, homogenized and allowed to stand in a water bath for 4 h at 55 °C. The resulting mixture was filtered using a vacuum pump. Residue was collected and re-extracted with 20 mL of 20% ethanol. The filtrates form the residue were combined and reduced to 40 mL

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in a water bath at 90 °C; the concentrate was transferred into a 200 mL separating funnel, 20 mL of diethyl ether was added and mixed vigorously. The lower fraction was collected while the upper layer (Ether) was discarded. The lower fraction was re- introduced into the separating funnel and 20 mL of butan-1-ol was added, mixed vigorously, followed by 5 mL of 5% aqueous sodium chloride. The upper fraction (butan-1-ol) was collected and evaporated to constant weight in the oven. The saponin content in the sample was calculated using the equation: %saponin content ¼

Weight of fraction  Weight of sample

100:

2.6. Statistical analysis All data were expressed as mean ± standard deviation and were subjected to one-way analysis of variance (ANOVA). Where data showed significance (p b .05), Fischer's least significance difference (LSD) test was applied using MINITAB 17 statistical package. 3. Results 3.1. Soil composition Table 1 revealed that the soil used for planting C. argentea at the two trials had all the elements necessary for normal growth and development of the plant. 3.2. Proximate composition The proximate analysis of C. argentea at different growth stages of the two trials is represented in Table 2. The moisture content ranged from 8.43 ± 0.15 to 10.42 ± 1.33% in the first trial and from 6.35 ± 0.09 to 9.42 ± 0.30% in the second trial. There was no significant difference in moisture content at all stages of growth in the first trial. The ash content was highest at the PRF stage (28.20 ± 0.10%) of the first trial and lowest at the PST (15.51 ± 0.02%) of the second trial. The fat content ranged from 2.01 ± 0.30 to 2.50 ± 0.03% in the first trial and from 3.07 ± 0.14 to 5.70 ± 0.00% in the second trial. There was no significant difference in the fat content at all the growth stages of the first trial. Crude fiber content ranged from 22.40 ± 0.54 to 33.41 ± 0.87% in the first trail and from 15.50 ± 0.15 to 25.20 ± 0.20% in the second trial. The percentage crude protein ranged from 10.61 ± 0.34 to 19.50 ± 017% in the first trial and from 13.80 ± 0.13 to 25.80 ± 0.30% in the second trial. The carbohydrate content ranged from 17.85 ± 0.44 to 28.50 ± 0.20 in the first trial and from 18.25 ± 0.50 to 36.20 ± 0.22% in the second trial. The overall estimated energy value for both trials ranged from 315.36 ± 0.40% in the PRF stage of the second trial to 435.30 ± 1.06% of the PST stage of the first trial. There was no significant difference in the percentage energy composition at all growth stages of the first trial. For both trials, the PRF stage of growth had the highest ash and crude protein content, while the PST stage had the highest carbohydrate, energy, fiber but the lowest fat and moisture contents. 3.3. Mineral composition Table 3 shows the result of the mineral composition of C. argentea at different growth stage of two trials. Calcium concentration was highest during the PRF stage and lowest at the PST stage in both trials. The FLW stage of the first trial had the highest magnesium content of

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765 mg/100 g while the PRF stage of the second trial had the highest magnesium composition of 835 mg/100 g. However, there was no significant difference in the magnesium concentration at the three stages of growth in both trials. Similarly, no significant difference was observed in all the growth phases of both trials for sodium. However, the PST stage of the first (65 mg/100 g) and second trial (87 mg/100 g) had the highest sodium content. Phosphorus content was highest at the PRE stage (845 mg/100 g) of the first trial. It however decreased as it tends towards maturity in the first trial. In the second trial, Table 3 shows the result of the mineral composition of C. argentea at different growth stage of two trials. Calcium concentration was highest in the PRE stage and lowest in the PST stage in both trials. The FLW stage of the first trial had the highest magnesium content of 765 mg/100 g while the PRE stage of the second trial had the highest magnesium content of 835 mg/100 g. The phosphorus content of the plant was not affected by age as the composition (505 mg/100 g) was the same in all the growth stages. The zinc content was highest in the FLW stage of the first (12.30 mg/100 g) and second trial (8.7 mg/100 g). Statistically, no significant difference was recorded in the zinc composition of C. argentea at all growth stages of the second trial (Table 3). The highest manganese composition of C. argentea in the first trial was recorded in the PST stage (7.65 mg/100 g). While the highest composition for the second trial was recorded at the PRE stage (6.15 mg/100 g). Also, there was no statistical difference in the manganese content of the second trial. At all growth stages, copper content was highest at the PRF stage of growth for both trials (1.74 mg/100 g for first trial and 1.45 g/100 g for the second trial); and was not significantly different at all growth stages of the second trial. The amount of Iron in the plant ranged from 15.8 to 21.3 mg/100 g for the first trial and 12.5 to 13.1 mg/100 g in the second trial. Although, no significant difference was observed in all the three growth stages in both trials, PST had the lowest iron content, while potassium content ranged from 5995 to 10,340 mg/100 g in the first trial and from 5740 to 10,160 mg/100 g in the second trial and the PRE stage had the highest potassium content in both trials. 3.4. Vitamin content Table 4 shows the vitamin contents of C. argentea at three different growth stages of two trials. Vitamin A content of the plant samples ranged from 4.13 mg/g DW (PST first trial) to 9.73 mg/g DW (PRE second trial), while vitamin C ranged from 2.31 mg/g DW (PST stage of the first and second trial) to 4.10 mg/g DW (PRE stage second trial). There was no significant difference in the vitamin C content of the PRE and FLW stage in both trials. Vitamin E was highest (8.68 mg/g) at the PRE stage of growth of the second trial and lowest at the PST stage of growth of the same trial. Overall, the PRE stage of growth had the highest vitamin A, C and E content at both trials. NOTE: in Table 4 vitamin A content is in (μg retinol/100 g DW), vitamin C in (mg ascorbic acid/100 g DW) and vitamin E in (mg α-tocopherol/100 g DW), units of these three vitamins are not in mg/g DW, therefore they cannot be compared. Anti-nutrient composition Table 5 shows that phytate was the highest with a percentage value of 8.57 ± 0.06% at the PRE stage of the first trial, followed by saponin (4.85 ± 0.21%) at the FLW stage of the first trial and oxalate (3.70 ± 0.21%) at the PRE stage of the second trial. Alkaloid content was lowest (0.26 ± 0.04%) at the PST stage of the second trial. For both trials, the

Table 1 Mineral and physiochemical components of soil before planting C. argentea for both trials. Samples

Sample density g/mL

P mg/L

K mg/L

Ca mg/L

Mg mg/L

pH (KCl)

Zn mg/L

Mn mg/L

Cu mg/L

First trial pre-planting soil Second trial pre-planting soil

0.71 0.80

90 140

736 1626

3057 4272

783 976

4.92 6.61

25.7 27.5

77 3

5.2 6.2

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Table 2 Proximate composition of Celosia argentea dry weight (DW) at different growth stages of two trials.

First trial

Second trial

Growth stages

Carbohydrate (%)

Moisture (%)

Ash (%)

Crude protein (%)

Crude fiber (%)

Crude fat (%)

Energy (%)

Pre-flowering Flowering Post-flowering Pre-flowering Flowering Post-flowering

17.85 ± 0.44b 19.80 ± 2.70b 28.51 ± 0.20a 18.30 ± 0.50c 27.01 ± 0.30b 36.20 ± 0.22a

9.70 ± 0.37a 10.42 ± 1.33a 8.43 ± 0.15a 8.60 ± 0.00b 9.42 ± 0.30a 6.35 ± 0.09c

28.20 ± 0.10a 26.25 ± 1.00b 17.03 ± 0.23c 26.22 ± 0.10a 20.27 ± 0.10b 15.51 ± 0.02c

19.50 ± 0.17a 17.24 ± 0.31b 10.61 ± 0.34c 25.80 ± 0.30a 18.50 ± 0.10b 13.80 ± 0.13c

22.40 ± 0.54c 23.83 ± 3.92b 33.41 ± 0.87a 15.50 ± 0.15c 21.18 ± 0.90b 25.20 ± 0.20a

2.50 ± 0.13a 2.50 ± 0.03a 2.01 ± 0.30a 5.70 ± 0.00a 3.65 ± 0.30b 3.07 ± 0.14b

350.5 ± 0.80a 362.68 ± 2.60a 435.30 ± 1.06a 315.36 ± 0.40c 372.58 ± 0.90b 426.08 ± 1.10a

Values shown are mean ± SD; different letters along a column represent significant differences at p b .05 among the growth stages.

FLW stage had the lowest oxalate content, while the PST had the lowest alkaloid, phytate and saponins content. 4. Discussion The distribution of nutrients and metabolites in different organs and tissues is in a constant state of flux throughout the growth and development of a plant, leading to profound changes at crucial stages during their life cycle such as vegetative, flowering and senescence and nutritional content (Bennett et al., 2012). This study revealed that the nutrient content of C. argentea varied across the three growth stages investigated. Moisture content is an index of water activity of many foods which helps in maintaining protoplasmic content of the cell and leaf texture. It also promotes the activity of water-soluble enzymes and coenzymes involved in metabolic activities of plants (Arasaretnam et al., 2018). However, microbial contamination and chemical degradation is associated with high moisture content in foods (Ooi et al., 2012). In all the growth stages of both trials the moisture content of pulverized samples of C. argentea was within the acceptable range for food preservation. The relatively low moisture content observed at the PST stage of growth of the first and second trials indicates that the plant could possess a longer shelf life at that stage of growth. This result is comparable to the findings of Ayodele and Olajide (2011) who reported the value of 8.84% for the moisture content of the same species. The ash content of any food sample depicts the quality of its elemental composition (Hofman et al., 2002). At the pre-flowering stage, high levels of total ash were recorded for both trials. The ash content in this study was higher than those reported by Ilodibia et al. (2016) (23.91%) and Ayodele and Olajide (2011) (22.43%) for the same species. It could be an indication that C. argentea possess more minerals at the pre-flowering stage of growth and should be maximized for enriching the diet with micronutrients. Fat in food is one of the major sources of energy in human diet. It plays a major role in the tastes perception of foods and helps the body in the absorption of some fat-soluble vitamins. However excess fat (saturated) can lead to increased cholesterol level which is a major cause of cardiovascular disorders (Singh et al., 2009). Generally, green leafy vegetables are regarded as a healthy source of dietary fat because of their low to no fat content. The very low crude fat content recorded at PST when compared to other growth stages suggests that C. argentea would be more suitable for the management of weight loss and some chronic diseases associated with excess fat.

Dietary fiber plays a vital role in the regulation of bowel movement, prevents coronary disease and slow down cholesterol absorption (Viuda-Martos et al., 2010). The fiber content of C. argentea at all growth stages was higher than the values reported for Amaranthus cruentus (8.45%) and Solanum nigrum (9.56%) (Ajayi et al., 2018). The high fiber content recorded at the PST of C. argentea is an indication that as plant grow older, their leaves and stem becomes woody and more fibrous which could help in the regulation of intestinal transit, improve dietary bulk and also decrease the risk of several metabolic disorders caused by inadequate crude fiber intake such as colon cancer, obesity and diabetes in the human body (Unuofin et al., 2017 not included in references). The protein content of most leafy vegetables ranged from 1 to 7% of fresh weight or 8 to 30% of dry weight (Uusiku et al., 2010). From the results of this study, the protein content of C. argentea varies with the age of the plant. The highest protein content recorded of the pre-flowering stage of the second trial (25.80 ± 0.30%) is higher than the value reported for Brassica oleracea (24.32%), A. cruentus (11.32%), S. nigrum (15.06%) (Ajayi et al., 2018) and Moringa oleifera leaf (17.09%) (Ogbe and Affiku, 2011). This implies that C. argentea at the pre-flowering stage can provide more than 12% of the recommended dietary allowance (RDA) of protein for children, men and women (Otten et al., 2006). Furthermore, C. argentea protein has been reported to contain all the major essential amino acids (Ayodele and Olajide, 2011). Therefore, C. argentea could serve as a very good source of protein for the alleviation of protein malnutrition (Bauer et al., 2013). The significant reduction of protein as the plant approaches maturation is in line with the findings of Adediran et al. (2015). The low protein content at the PST stage in both trials could be as a result of leaf senescence (Table 2). A study performed on plant life cycle showed that as plant approaches senescence, photosynthesis decreases, this triggers events of chain reactions which result in a decrease in the nitrogen contents which is responsible for the amino acids/proteins of the plant (AvilaOspina et al., 2014). Carbohydrate is the primary source of energy for bodily functions. The highest level of total carbohydrates was recorded at PST (Table 2). This suggests that consumption of this vegetable at the PST stage of growth could help in providing optimal nutrition by increasing the energy content of the diet. The levels of carbohydrates in C. argentea is higher than the value reported for A. viridis (Nisha et al., 2012) but lower than that of Ocimum grattisimum (55.42%) (Ajayi et al., 2018). Similarly, the overall estimated energy content of C. argentea was undoubtedly highest at PST because of its higher carbohydrate content.

Table 3 Elemental composition (mg/100 g) of C. argentea at three different growth stages (PRF, FLW and PST) of two trials. Composition (mg/100 g) Trials First trial

Growth stages Calcium

Pre-flowering Flowering Post-flowering Second trial Pre-flowering Flowering Post-flowering

1540 ± 0.02a 1445 ± 0.02b 1365 ± 0.02c 1315 ± 0.01a 1160 ± 0.16a 985 ± 0.01a

Magnesium Sodium

Phosphorous Potassium

Zinc

Manganese

Copper

Iron

Na+/K+

755 ± 0.01a 765 ± 0.04a 760 ± 0.00a 835 ± 0.01a 785 ± 0.01a 685 ± 0.01a

845 ± 0.02a 770 ± 0.03a 645 ± 0.02b 505 ± 0.01a 505 ± 0.10a 505 ± 0.00a

11.80 ± 0.1ab 12.30 ± 0.04a 10.00 ± 0.70b 7.05 ± 0.50a 8.70 ± 3.60a 4.95 ± 0.10a

6.30 ± 0.00b 6.10 ± 0.50b 7.65 ± 0.50c 6.15 ± 0.05a 6.20 ± 0.80a 4.80 ± 0.00a

1.70 ± 0.00a 1.55 ± 0.05b 1.2 ± 0.10c 1.45 ± 0.05a 1.25 ± 0.30a 0.95 ± 0.10a

16.0 ± 0.80a 21.3 ± 1.80a 15.8 ± 0.00a 23.1 ± 4.20a 14.8 ± 3.00a 12.35 ± 0.15a

0.00 ± 0.00 0.01 ± 0.10 0.01 ± 0.11 0.01 ± 0.00 0.01 ± 0.01 0.02 ± 0.10

45 ± 0.01a 55 ± 0.01a 65 ± 0.02a 85 ± 0.01a 85 ± 0.03a 87 ± 0.01a

10,340 ± 0.10a 8965 ± 0.09b 5995 ± 0.05c 10,160 ± 0.11a 9575 ± 1.43a 5740 ± 0.01b

Values shown are mean ± SD; different letters along a column represent significant differences at p b .05 among the growth stages for each trial.

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Table 4 Vitamins A, C and E contents of Celosia argentea at different growth stages of two trials.

First trial

Second trial

Growth stages

Vitamin A (μg retinol/100 g DW)

Vitamin C (mg ascorbic acid/100 g DW)

Vitamin E (mg α-tocopherol/100 g DW)

Pre-flowering Flowering Post-flowering Pre-flowering Flowering Post-flowering

28.64 ± 0.20b 20.7 ± 0.10d 13.72 ± 0.31e 32.42 ± 0.99a 23.03 ± 0.40c 21.05 ± 0.33d

7.70 ± 0.00a 6.70 ± 0.90a 4.62 ± 0.13b 8.21 ± 0.90a 7.69 ± 1.53a 4.62 ± 1.53b

8.06 ± 0.18c 7.26 ± 0.15d 5.90 ± 0.05e 12.96 ± 0.72a 11.76 ± 0.04b 4.60 ± 0.05f

Values shown are mean ± SD; different letters along a column represent significant differences at p b .05 among all the growth stages of both trials.

Dietary mineral elements are crucial for good and balanced human nutrition. They support a wide variety of bodily functions, such as; building and maintaining healthy bones and teeth, keeping the muscles in shape and improving the functions of the heart and brain (Jéquier and Constant, 2010). This study revealed that C. argentea at pre-flowering stage of both trials exhibited the highest content of most minerals evaluated except zinc. Potassium which was the highest mineral recorded at the pre-flowering stage, is an important mineral required in the body for blood pressure regulation, nerve transmission, contraction, regulates waste elimination and responsible for proper fluid balance (Gharibzahedi and Jafari, 2017). The high potassium content at the PRF stage of growth could have been influenced by the concentration in the soil (Table 1), since plant growth and mineral uptake is highly dependent on soil content (Aibara and Miwa, 2014). The recommended daily allowance (RDA) of potassium for adults is 4700 mg. C. argentea at the PRF stage is capable of contributing more than two times the RDA for potassium This result is similar to the findings of Makobo et al. (2010) who recorded the highest potassium content of A. cruentus at six weeks after sowing; an age similar to the PRF stage of growth in this present study. The calcium content at the PRF stage of growth was also highest and could contribute almost two times the RDA of 1000 mg/day required for humans for calcium (Khan et al., 2011). The calcium content at all the growth stages could also reflect the pre-planting soil which was rich in calcium (Table 1). Calcium plays key roles in cell signaling, muscle contraction and nerve function. It also supports the skeletal structure by maintaining strong bones and teeth. Cells in the body use calcium to activate certain enzymes, transport ions across the cellular membranes, send and receive neurotransmitters during communication with other cells (Sadler, 2011). Adequate intake of C. argentea could reduce the risk of fractures and osteoporosis in human. Phosphorus is needed for cell growth and maintenance, helps with kidney performance and repair. It is responsible for the formation of adenosine triphosphate ATP and synthesis of DNA and RNA. It aids the absorption of calcium and maintains acid–base balance (Gharibzahedi and Jafari, 2017). In the study, the phosphorus content decreased as the plant approached maturation; this is like the report of Makobo et al. (2010) who recorded highest phosphorus content for A. cruentus at four weeks old and a decrease as the plant aged. Phosphorus concentration at the PRF (845 mg/100 g) endorse C. argentea as a good supplement for phosphorus in the diet as 118.3 mg of C. argentea will provide the daily 1000 mg/day amount required by adults (NHMRC, 2006). Magnesium is required for various biochemical reactions such as the

regulation of protein synthesis, muscle and nerve functions, oxidative phosphorylation and glycolysis. It is also responsible for the formation of protein and nerve transmission in the body (Gröber et al., 2015). From the results obtained in this study, there was no significant difference in the magnesium content of C. argentea at all growth stages, as such; only 53.9 mg of C. argentea will be required to meet the 450 mg/day of magnesium required for healthy living. This result corroborates the findings of Adediran et al. (2015) who also reported no significant difference in the magnesium content of C. argentea at different age of harvest. The sodium content of the plant across developmental stages is low when compared to the 1500 mg RDA of sodium for an adult (McCarron et al., 2013). Sodium is responsible for the maintenance of electrolyte in the body. The high content of potassium in relation to the low sodium observed in this study led to a very low Na+/K+ ratio (Table 1). The ratio of Na+/K+ in any food item is a crucial factor in nutrition, diet with Na+/K+ ratio that is less than one (Na+/K+ b 1) reduces the risk of hypertension (Yang et al., 2011). Iron is one of the most common deficient micronutrients among school children. Iron deficiency has been implicated for anemia, fatigue and other blood related diseases (Haimi and Lerner, 2014). The iron content of 100 g of C. argentea at the PRF stage was higher than the RDA of 9 and 15 mg/day respectively for children and adults (JahnenDechent and Ketteler, 2012). Iron is a vital element needed for the formation of hemoglobin in red blood cells. It serves as a carrier of oxygen to the tissues from the lungs. Iron is an integrated part of some enzyme systems in various tissues and crucial for energy production (Gharibzahedi and Jafari, 2017). The FLW stage of growth had the highest zinc content at both trials. This agrees with the reports of Adediran et al. (2015) who reported zinc content of C. argentea to be at its peak at seven weeks after sowing (like FLW stage of growth in this study). Zinc is another important micronutrient playing important roles in fetal development, normal growth and sexual maturation of human beings. Deficiency in zinc could lead to low immune system (Roohani et al., 2013). In this study, zinc content progressively declined with advancing maturity: a trend also reported by Flyman and Afolayan (2008) for Mormodica. balsamina and Vigna unguiculata. With the zinc content recorded at the FLW stage, 32.52 mg and 113.82 mg of C. argentea respectively can provide the 4 and 14 mg/day requirement of zinc for children and adult (NHMRC, 2006). Copper and manganese are essential trace elements that are needed only in minute amounts by the human body for biochemical functions. Manganese acts as a co-factor to many enzymes, it is important for the normal functioning of the brain and proper activity of

Table 5 Anti-nutrient content of Celosia argentea at different growth stages of two trials.

First trial

Second trial

Growth stages

Oxalate (%)

Phytate (%)

Alkaloid (%)

Saponins (%)

Pre-flowering Flowering Post-flowering Pre-flowering Flowering Post-flowering

1.54 ± 0.18b 1.25 ± 0.21d 2.64 ± 0.18c 3.70 ± 0.21a 2.42 ± 0.18c 3.08 ± 0.18d

8.57 ± 0.06a 5.71 ± 0.50c 5.69 ± 0.41c 4.04 ± 0.14d 6.99 ± 0.8b 2.73 ± 0.30e

3.18 ± 0.21b 2.67 ± 0.24d 1.98 ± 0.10e 3.34 ± 0.37a 2.85 ± 0.21c 0.26 ± 0.04f

3.10 ± 0.10c 4.85 ± 0.21a 2.82 ± 0.15c 1.82 ± 0.15d 3.91 ± 0.13b 1.00 ± 0.00e

Values shown are mean ± SD; different letters along a column represent significant differences at p b .05 among all the growth stages of both trials.

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nervous system throughout the body (Au et al., 2008). Copper act as a co-factor and constituents of several enzymes; it is essential for the proper functioning of organs and stimulates the immune system to fight infections (DiNicolantonio et al., 2018). Deficiency in copper leads to cardiac abnormalities, while manganese deficiency results in skeletal abnormalities, impaired growth, and abnormal lipid metabolism (Au et al., 2008; DiNicolantonio et al., 2018). Copper content at all growth stages of C. argentea could provide the RDA of 0.7, or 1.1 mg/day required for children and adults respectively (Johnson et al., 2009 not included in references). While manganese content at the PRF stage of growth (first trial) could contribute two times the RDA value (NHMRC, 2006). Vitamins are organic compounds that help the body to grow and function properly by boosting the immune system. In this study, the PRF of both trials had the highest amount of all the vitamins evaluated. Vitamin A is required for the normal functioning of the visual system, growth and development, maintenance of epithelial cellular integrity, immune function and reproduction, while vitamin C helps in the biosynthesis of carnitine, hormones, collagen and iron absorption. It is a water- soluble antioxidant which acts as a free radical scavenger and stimulates the immune system; thus, promoting health in human beings. Severe deficiency of vitamin A can lead to xerophthalmia while that of vitamin C can lead to scurvy (Sommer and WHO, 1995; Locato et al., 2013). The key biological function of vitamin E is the protection of the polyunsaturated fatty acids of cell membranes from free-radical damage in oxidative stress (Korchazhkina et al., 2006). The vitamin content of C. argentea in this study decreased as the plant approached maturity. A similar trend was reported by Biesiada et al. (2007) for Leek cv. Kilima, Delikates biala and Zucchini cv Astra. This implies that the best time to harvest C. argentea for optimum vitamin content is at the PRF stage of growth. Antinutrients in foods interfere with the absorption of some minerals and other micronutrients in the digestive system which may have a negative impact on the functioning of certain organs (Gemede and Ratta, 2014). In this study, wide variation was observed in the content of the four antinutritional compounds evaluated at the three growth stages. Alkaloids are naturally occurring chemical compounds with basic nitrogen atoms; they are active components of medicinal plants. However, high percentage of alkaloid is toxic to human and animals (Matsuura and Fett-Neto, 2017). The highest alkaloid content at the PRF is within the range of alkaloids content that is perceived to have more pharmacological effect rather than toxicity (Makkar et al., 2007). Oxalate is present in the cell sap of many of the green leafy vegetable. It affects the human body by forming a strong chelate with dietary calcium and other minerals thereby rendering such nutrients unavailable for absorption and assimilation (Jiru and Urga, 1995). This insoluble calcium oxalate in the crystal form is stored in the kidney causing serious health-related problems called kidney stone (Natesh et al., 2017). The oxalate content in all the growth stages did not follow a trend in the growth stages. However, the highest values for oxalate content of C. argentea at all the growth stages in both trials are lower than that of A. cruentus (5.08 mg/g) and S. nigrum (5.88 mg/g) (Ajayi et al., 2018). Phytate is usually present as salts in grains and vegetables. Negatively charged phosphate group in phytic acid is known to inhibit the action of gastrointestinal tyrosinase, trypsin, pepsin, lipase, amylase and essential minerals (Hendricks and Bailey 1989 not included in references). The result of the phytate content of C. argentea in this study did not follow a definite trend in both trials as observed with the oxalate content. The implication of this is that the phytate content of this plant is not affected by maturity. Phytic acid at a level of 0.035% is reported to have a protective measure against fatty liver resulting from elevated hepatic lipogenesis (Onomi et al., 2004). The highest phytate content (8.57 ± 0.06%) is within the safe limit, since the inhibition of mineral

absorption by phytate only occurs at levels greater than 10% in a diet (Onomi et al., 2004). Saponins at high concentrations can affect nutrient absorption by inhibition of metabolic and digestive enzymes as well as binding with nutrients such as zinc (Shahidi, 1997). The saponin content recorded at the three growth stages in this study was low compared to the value recorded for Vernonia amygdalina(5.20 mg/100 g) (Ladi et al., 2016 not included in references). When saponin in a diet is less than 10%, it is believed to be harmless to the body (Hortwitz, 2003 not included in references). The highest content of saponin recorded at the flowering stage of growth of both trials was within the safe limit. 5. Conclusion The study confirmed that maturity stages have a significant influence on the nutritional composition of C. argentea. The antinutrient contents were low when compared to some other green leafy vegetables; therefore, interference with nutrients absorption will be negligible. This study present C. argentea as a vegetable with high potential to combat the menace of nutrient deficiency, its cultivation is therefore, highly recommended to address food and nutrient insecurity. Although, the potential of this plant as an important source of nutritional and mineral compounds is possible at all the three growth stages, the best stage of harvest would be determined by the specific nutrient need. 6. Significant statement This study provides insights into the nutritional composition of C. argentea at three growth stages. The results reveal that maturity stages significantly influence the nutrient content of this plant and serves as a baseline data for the formulation of guidelines towards the best age of harvest suitable for optimum nutrition. The consumption of C. argentea as a green leafy vegetable and source of dietary vitamin and mineral at all stages of growth is greatly encouraged. The plant can also be utilized to boost the immune system. The findings of this study are relevant in situations where a food-based strategy is sought to address specific nutrient and mineral deficiencies. Author's contributions Anthony Afolayan and Gloria Otunola conceptualized the topic; Gloria Otunola and Oluwafunmilayo Adegbaju designed the format of the experiment. Oluwafunmilayo Adegbaju carried out the experiment and wrote the manuscript. Anthony Afolayan and Gloria Otunola revise the manuscript. All the authors read the final manuscript. Acknowledgement and funding Authors acknowledge the financial support of Govan Mbeki Research Development Centre, University of Fort Hare. Grant number: C127. References Adebooye, O.C., 2008. Phyto-constituents and anti-oxidant activity of the pulp of snake tomato (Trichosanthes cucumerina L.). Afr. J. Trad. Complement. Alternat. Med. 5, 173–179. Adediran, O.A., Gana, Z., Oladiran, J.A., Ibrahim, H., 2015. Effect of age at harvest and leaf position on the yield and nutritional composition of Celosia argentea L. Int. J. Plant Soil Sci. 5, 359–365. Agrilasa, 2007. Agricultural Laboratory Association of Southern African (AgiLASA). Wet ashing Plant and feed analysis handbook, 2nd ed South Africa. Agrilasa, 2008. Method 6.1.2: wet ashing. In: Palic, C. (Ed.), Plant and feed analysis handbook. Agricultural Laboratory Association of Southern Africa, Pretoria, South Africa Unpublished manuscript. Aibara, I., Miwa, K., 2014. Strategies for optimization of mineral nutrient transport in plants: multilevel regulation of nutrient-dependent dynamics of root architecture and transporter activity. Plant Cell Physiol. 55, 2027–2036.

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