Evaluation of the Nutritional Value of Selected Leafy Vegetables Grown in Sri Lanka

Sri Lanka is blessed with a wide variety of leafy vegetables. However, the information on their nutritional value is scanty. In the present study, ten leafy vegetables commonly grown in Sri Lanka, namely Centella asiatica (Gotukola), Alternanthera sessilis (Weda Mukunuwenna), Alternanther asessilis (Common Mukunuwenna), Sesbania grandiflora (Kathurumurunga), Amaranthus viridis (Red thampala), Amaranthus viridis (Green Thampala), Dregea volubilis (Aguna kola), Coccinia grandis (Kowakka), Costus speciosus (Thebu kola) and Trianthema portulacastrum (Sarana) were evaluated for their nutritional composition. Samples of each vegetable species were collected from different geographical locations of the country representing all agro-climatic zones. Healthy, undamaged plant parts were collected, cleaned and air dried at 45 °C, pooled in similar quantities and stored at 4 °C for further analysis. The proximate and mineral composition of each sample were determined and presented on fresh weight basis. Moisture, ash, crude fat, crude protein and total carbohydrate contents were determined using AOAC standard methods. The moisture content ranged between 76.99 and 89.55% while the crude protein content ranged between 1.73 and 7.17%. The corresponding ranges for ash, total carbohydrates and crude fat were 1.28 – 3.86%, 4.41 – 10.76% and 0.29 – 1.81%, respectively. Comparatively, A. viridis was found to be the richest (P<0.05) source of essential mineral elements.


INTRODUCTION
An increasing interest has been developed among Sri Lankans towards the consumption of leafy vegetables as means of obtaining nutrients especially micronutrients.Furthermore, many leafy vegetables have been used in traditional medicine for therapeutic and curative purposes.Moreover, many nutritional studies have given an important consideration for dark green leafy vegetables in dietary guidelines as rich sources of vitamins, especially vitamins C and A; minerals, including calcium, iron and phosphorus; and phytochemicals, particularly antioxidants.
The dietary intake of leafy vegetables is negatively associated with many chronic disease conditions such as cardiovascular diseases, cancer, diabetes mellitus, Alzheimer disease, cataract, and age-related functional decline (Willett, 1995;Temple, 2000).The study of Jayawardena et al. (2012) provides evidence that a substantial proportion of the Sri Lankan population acquires meals that include no servings or very few servings of vegetables, though Sri Lanka produces a significant quantity.The daily intake of vegetable portions (1.73) is notably below the national and international recommendations (5 portions).Some part of the population still suffers from one or more micronutrient deficiencies as a result of diets that are deficient in micronutrient-rich foods.The knowledge of nutritional significance can be used for recommendation of leafy vegetables in the diet, mediating nutrient deficiency, nutrient adequacy and nutrient toxicity of a population.Furthermore, compositional data can be incorporated into food consumption surveys, nutrition, agriculture and health policies and programmes.However, nutritional data of green leafy vegetables commonly grown in the country is quite scanty.Therefore, this study was carried out to determine proximate and mineral composition of selected ten leafy vegetables commonly grown and used in food preparations in Sri Lanka namely, Centella asiatica (Gotukola), Alternanthera sessilis 1 (Weda Mukunuwenna), Alternanthera sessilis 2 (common Mukunuwenna), Sesbania grandiflora (Kathurumurunga), Amaranthus viridis 1 (Red Thampala), Amaranthus viridis 2 (Green Thampala), Dregea volubilis (Aguna kola-Thiththaaguna), Coccinia grandis (Kowakka), Costus speciosus (Thebu kola) and Trianthema portulacastrum ( Sarana) (Figures 1-3).

Materials
Chemicals: petroleum ether, potassium sulfate, copper sulfate, boric acid, sulphuric acid, sodium hydroxide, bromocresol green and methyl red mixed indicator, hydrochloric acid, sodium carbonate, Devarda's alloy, Phenol, glucose, nitric acid, Rh and Re standards and multi-elemental standards for ICP-MS analysis were purchased from Sigma-Aldrich ™ , USA.

Sample collection and preparation
A total of 500 g of healthy, undamaged, edible leaves and stems of Centella asiatica, Alternanthera sessilis, Amaranthus viridis and The plant samples were photographed, identified and authenticated by taxonomists.They were sorted and cleaned appropriately and oven dried at 45 °C (Phoenix ™ , Germany).The oven dried samples from each agro ecological zone were disintegrated in a stainless steel grinder (Apex ™ , India), pooled separately in similar quantities and homogenized assuring uniform distribution to prepare the working samples.Oven-dried samples were stored at 4°C (LG ™ , Japan) for further analyses.

Proximate analysis
Methods approved by AOAC (2009) were employed to determine the moisture content of fresh samples and crude protein, fat and ash contents of oven dried samples.The crude protein and crude fat contents were determined using Kjeldahl method (Digester -VELP Scientifica ™ , DK 6, Italy and Distillation unit -VELP Scientifica ™ , UDK 129, Italy) and Soxhlet extraction methods respectively.The moisture content was determined by drying the sample to a constant weight at 105 °C in a forced air-oven (SIBATA, SPF-600, Japan).The ash content was determined using gravimetric method by muffle furnace (HOBERSAL ™ , HP-150, Spain).

Determination of total carbohydrate content
The total carbohydrate content was determined according to the method described in AACC 2003 (American Association of Cereal Chemists).A sample (100 mg) was acid-hydrolyzed in a boiling water bath for 3 h with 5 mL of 2.5 N HCl.Then the sample was cooled to room temperature, neutralized with sodium carbonate until the effervescence ceased, made up the volume to 100 mL and centrifuged at 4000 rpm for 10 min.A sample (0.1 mL) of the supernatant collected was pipetted out and volume was made up to 1 mL with distilled water.Blank was set with 1 mL of distilled water.Five percent of Phenol solution (1 mL) and 5 mL of 96% sulphuric acid were added into each tube, the contents were shaken for 10 minutes and placed in a water bath at 25-30 °C for 20 min.
All analyses were carried out in triplicate and absorbance was taken using UV/Visible microplate spectrophotometer (Thermo Scientific ™ , Genesys 10S, UK) at 490 nm.Total carbohydrate contents were calculated as percentages using the glucose standard calibration curve (0.0028-0.0143 ppm).The absorbance of the blank was read and necessary corrections were made.

Quantification of the mineral content
Mineral analysis by inductively coupled plasma mass spectrometer (ICP-MS, iCAPQ, Thermo Scientific, UK) was performed according to the method described by De la Rocha (2009) with some minor modifications.Acid digestion of the oven dried samples (0.2 g) was performed with 10 mL of nitric acid (65%, w/v) using a commercial high-pressure laboratory microwave oven (CEM ® Corporation, BR601050, USA), operating by setting the program for 15 min ramp time, 30 min holding time during digestion at 180 °C and 15 min cooling time.Digested samples were transferred into a 50 mL volumetric flask, made up to volume with de-ionized water, filtered and stored at 4 °C.Test solutions contained 100 µg/L of Rh and Re, which were used as internal standards to correct instrumental drift and possible matrix effects.For calibration, multi-elemental standard solutions were prepared with the following concentrations: 0, 5, 10, 30 and 50 mg/L for Na, Mg, K, Al, Mn, Zn, Fe, Rb, Sr and Ba; and 0, 0.8, 3.0 and 10.0, 20.0 µg/L for Cr, Co, Ni, Cu, As, Se, Li, Be, V, Ga, Mo, Cd and Bi.The operating conditions of the ICP-MS for the analysis of the leafy vegetable samples are shown in Table 1.Elements with the highest isotopic abundance were selected as analytical mass.For the within-run assessment, the accuracy and precision of the assays were examined in a single run, with six replicates.For the between-run assessment, TM 25.4 (Environment Canada) for water was analyzed as certified reference material which covered the range of the elements studied such as Al, Cr, Mn, Co, Ni, Cu, Zn, As, and Se in six separate runs.Accuracy was defined as the percentage difference between the monitored concentration and the expected value (%D).Precision was expressed as the percentage of the variation coefficient (%CV).
Acceptance criteria for accuracy and precision, derived from International Conference on Harmonization (ICH) (1996) rules, were as follows: the mean %D and %CV should not exceed ±15%; 75% of all samples had to be within ±15% of expected concentrations.In the experiment, no more than one of the six samples within a given concentration could exceed ±15% of their expected values.

Statistical analyses
Data for all determinations were subjected to analysis of variance (ANOVA).Complete Randomized Design (CRD) was used as the experimental model and means of experimental data were compared by Duncan's multiple range test (P<0.05).Statistical analyses were conducted using the SAS Statistical Analysis System SAS/IML 14.1 (SAS Institute Inc., Cary, NC).

Proximate composition
The proximate composition of leafy vegetables tested in this study is presented in Table 2.
The moisture content of the samples tested ranged between 69.93 -87.40%.These values fell within the range (70 -90%) reported by FAO (2006).Protein content of the samples ranged from 1.73g/100 g of fresh weight (FW) in C. speciosus to 7.17 g/100 g FW in S. grandiflora.The values obtained were rather similar to those reported by Rajapaksha (1998), Odhav et al. (2007) and Longvah et al. (2017).However, quite higher values were reported by Patricia et al. (2014) and Gogoi and Kalita (2014).
A significantly higher (P<0.05)protein content was observed in S. grandiflora compared to all other studied leafy vegetable species.As reported in some studies, leafy vegetables are claimed to be good sources of protein and they could play a considerable role in providing cheap and accessible proteins for rural communities.It was reported that, 100 g of leafy vegetables could contribute to about 18.6 -30.92% of the daily protein requirement (71 g/day) of pregnant and lactating women (FND, 2005), assuming complete absorption of protein.Three species, namely S. grandiflora (1.81 g/100 g FW), D. volubilis (1.08 g/100 g FW) and A. sessilis (0.75 g/100 g FW) stand out to be better sources (P<0.05) of fat than other species tested.Crude fat content of studied leafy vegetables ranged from 0.29±0.00g/100 g FW to 1.81 g/100 g FW.The values obtained for lipid content in these leafy vegetable species confirmed the findings of authors such as Rajapaksha (1998), Sheela et al. (2004), Odhav et al. (2007), Lukmanji et al. (2008) and Longvah et al. (2017) which showed that leafy vegetables are poor sources of lipids (Patricia et al., 2014).Furthermore, consumption of leafy vegetables in large amount cannot be detrimental to individuals who are suffering from obesity since diet providing 20 -35% of its caloric energy as fat is stated to be adequate to maintain health and wellbeing of humans and excess fat always leads to the onset non-communicable chronic disease conditions such as cardiovascular disorders, cancer and aging (Kris-Etherton et al., 2002).The ash content of leafy vegetables tested varied from 1.28±0.03g/100 g FW in D. volubilis to 3.86±0.16g/100 g FW in A. viridis 1 .The highest total carbohydrate content was observed inA.sessilis 1 (10.76±0.11g/100 g FW) while T. portulacastrum showed the lowest (4.41±0.11g/100 g FW) among studied leafy vegetables.The ash and carbohydrate contents of most of the leafy vegetables analyzed in this study tallied with the literature (Odhav et al., 2007;Longvah et al., 2017).On the contrary, the higher values for ash and total carbohydrate contents of some studied leafy vegetable species such as A. sessilis and Amaranthus spp.were reported in some published literature such as Gogoi and Kalita (2014) and Patricia et al. (2014) while lower values were reported by Sheela et al. (2004).
Heavy metal accumulation in plants is of rising concern because of the potential human health risks.Contamination of food chain is one of the most critical pathways for the ingress of these toxic heavy metals into the human body.Accumulation of heavy metals in plants is dependent on metal absorbing efficacy of various plant species (Rattan et al., 2005).The presence of heavy metals such as Pb, Cr, As, Zn, Cd, Cu, and Hg in leafy vegetables is highly concerned due to the risk of bioaccumulation and biomagnifications of them in the food chain.These concerned heavy metals contents were comparatively higher in A. sessilis 1 , A. sessilis 2 , A. viridis 1 , A. viridis 2 and C. asiatica than other examined leafy vegetables indicating a potential heavy metal contamination.These leafy vegetable species are cultivated for commercial purpose adding fertilizers and pesticides by Sri Lankan farmers, which may be the most contributing factor for high heavy metal content in them.

Table 3 . Mineral composition of leafy vegetables studied
Data represent the mean values±SD (n=3) of three independent experiments.Means followed by the same letters in a row are not significantly different at a particular probability level by Duncan's multiple range test.