Nutritional composition, vitamins, minerals and toxic heavy metals analysis of Trianthema portulacastrum L., a wild edible plant from Peshawar, Khyber Pakhtunkhwa, Pakistan

This study aimed at analyzing the nutritional potential of a wild edible plant, Trianthema portulacastrum L. widely used in Peshawar, Khyber Pakhtunkhwa, Pakistan. The nutritional composition, vitamins, minerals and heavy elements were analyzed following the standard methods of Association of Official Analytical Chemists (AOAC). The nutritional assessment included determination of moisture, ash, lipid, fiber, protein, carbohydrate and energy. Among the nutrient values, fiber was found to be the highest (430.0 mg/g), followed by ash (348.0 mg/g), total protein (91.9 mg/g), moisture (80.0 mg/g), carbohydrate (30.2 mg/g) and total lipid (20.0 mg/g). The vitamins analyzed were found to have greater value for riboflavin (2.02 mg/g), than retinol (0.81 mg/g). Among the macro minerals, potassium was present in high concentration (51.6 mg/g) than sodium (44.0 mg/g). The trace elements were assessed using atomic absorption spectrophotometer (AAS) and their decreasing order was Fe>Zn>Mn>Ni>Cu. Two toxic metals, Pb and Cd were present in very minute quantities of 0.08 and 0.0006 mg/g, respectively. The results suggest that T. portulacastrum L. is a good source of fiber, proteins, riboflavin, potassium, sodium and iron.


INTRODUCTION
Nutrition is the most important fundamental needs of humans, for development, productivity and mental health. In developing world, the high rate of population growth, cause shortage of fertile land, leading to hunger and malnutrition problems (Pelletier et al., 1995). Plants have been handled by human societies for food purposes, since time immemorial. It is true that today, human plant food is more than 85 to 90% based on mainly twelve crops, but it is also fact that in many parts of the world, wild plants have major contribution in daily intake of food (Prescott-Allen and Prescott-Allen, 1990;Scherrer et al., 2005;Bussmann et al., 2006;Bussmann and Sharon, 2006;Kunwar et al., 2006;Cavender, 2006;Pieroni et al., 2007). Green leafy vegetables are well known for their *Corresponding author. Email: nkhan812@gmail.com. Tel: +92-333-9302834. nutritional importance as rich sources of proteins, vitamins like ascorbic acid, carotene, folic acid, riboflavin, and minerals like calcium, iron and phosphorus (Kris-Etherton et al., 1988;NRC, 1996;Osler et al., 2001;Sheela et al., 2004). Green wild edible plants are commonly found in countries with varied climates. Many research studies have shown several wild species fit for human consumption due to good proximate and nutritional values. Even in some modern cultures today, people consume wild plants as a normal food source, for obtaining good amounts of several nutrients and it is widely accepted that leafy green vegetables are significant nutritional sources of minerals (Grau et al., 1989;Kuhnlein, 1990;Khan et al., 2013a). Around the world, many workers have reported the nutritional compositional evaluation and functional properties of various types of edible wild plants used in the developing countries (Lockeett et al., 2000;Akindahunsi and Salawu, 2005;Edeoga, 2006;Hassan and Umar, 2006;Ekop, 2007;Adnan et al., 2010;Mohan and Kalidass, 2010;Hussain et al., 2010;Gafar and Itodo, 2011;Vishwakarma et al., 2011;Valvi and Rathod, 2011;Naryan et al., 2011;Seal, 2011). Since Pakistan came into being, numerous wild vegetables are used as food in both urban and rural areas. The researchers have investigated several wild plants of Pakistan, for nutritional composition (Khattak et al., 2006;Imran et al., 2007;Qureshi and Bhatti, 2009;Hussain et al., 2009;Marwat et al., 2010;Jan et al., 2011;Khan et al., 2011Khan et al., , 2013b. The database of the nutrient and chemical compositions of these plant foods is still incomplete as many plants are still not investigated properly and thus much work is still needed to be done. In Peshawar Pakistan, many of the local wild vegetable materials are used as food without any nutritional studies.
In order to contribute to the growing body of knowledge and to narrow the growing gap of this subject, the present study was designed on wild edible plant, Trianthema portulacastrum L. commonly used as food by the people in Peshawar, Khyber Pakhtunkhwa, Pakistan, to analyze for proximate and nutrient analysis, vitamins, minerals, trace and toxic trace metals, using standard AOAC methods.

Collection of samples
The plant materials were collected from the provincial capital city, Peshawar, as wild grown plants in agriculture fields. Ten samples were collected, at three times from different fields, during July to November 2011, and each sample was studied in triplicate, thus making a total of 90 samples analyzed. Standard methodology was used for collection of plant samples (Humphry, 1993). Specific samples were obtained with the aid of interpreters and field guides. Genus and species was identified by plant taxonomist, by comparison with herbarium reference materials. The voucher specimen was preserved in Department of Plant Sciences, KUST, Kohat, for future references. The samples were shed dried, pulverized and stored in an airtight container. Details of each plant species, in respect of their scientific, family and local names, part used and status are elaborated in Table 1 (Shah et al., 2006;ENVIS, 2012).

Nutritional composition
The content of moisture was measured by keeping the sample in oven (HB-502M Hanbaek Co, Korea) at 100 to 110°C for overnight and then cooling in desiccator to constant weight. The loss in weight was regarded as a measure of moisture. For determination of ash, the sample was heated in furnace (F6010, Branstead Thermolyne Co., Dubuque, lA, USA), at 550°C, until white or grayish white ash was obtained. Weight of the ash was noted directly. Crude fiber was determined by treating the sample with 1.25% H 2 SO 4 , 1.25% NaOH and then 1% HNO 3 , filtered and washed with hot water after each step. The residue obtained was dried in oven at 130°C and ashed at 550°C in furnace. The loss in weight on ignition was expressed as content of crude fiber (AOAC, 2000). Total lipid was extracted from the sample with petroleum ether (60 to 80°C) in a Soxhelt apparatus for about 6 to 8 h. The

Vitamin Quantity (mg/g ± SD)
Values are mean ± standard deviations of three (n = 3) measurements. Different superscript letters within columns are significantly different (p < 0.05). residual solvent was evaporated in a pre-weighed beaker and increase in weight of beaker gave total lipid (AOAC, 2000). Total lipid content was fractionated into saponfiable and non-saponfiable lipids by the saponification of total lipid followed by extraction of non-saponifiable fraction with petroleum ether, 40 to 60°C (AOCS, 1993). Nitrogen content in the sample was estimated by using micro Kjeldahl method and crude protein was calculated by multiplying the evaluated nitrogen by 6.25. The value of total carbohydrate was given by: 100-(percentage of ash + percentage of total lipid + percentage of protein + percentage of crude fibre) (AOAC, 2000). The calorific value was calculated by multiplying the values of total carbohydrate, lipid and protein by the factors 4, 9 and 4 respectively, taking the sum of the products and expressing the result in kilocalories (Guil-Guerrero et al., 1998).

Vitamins analysis
Vitamin A was estimated by extraction with ethanol and then mixing with petroleum ether. The amount in extract was determined by UV-Visible Spectrophotometer (Hitachi U-2000, Japan) at 450 nm using Fikselova (2008). For riboflavin (vitamin B 2 ), the ethanol extract was added to potassium permanganate and H 2 O 2 and allowed to stand over hot water. Then 40% sodium sulphate was added and the absorbance was measured at 510 nm by spectrophotometer (James, 1995).

Minerals and toxic heavy metals analysis
Two macro minerals, sodium and potassium were estimated by using flame photometer (Model 410 Corning, Germany). Standard solution of each was used for calibration of the instrument before analysis (AOAC, 2000). The micro minerals including Zn, Cu, Ni, Mn, and Fe along with toxic heavy metals Pb and Cd were determined by wet digestion of the sample followed by analysis using atomic absorption spectrophotometer (A-Analyst 700 Perkin Elmer/ USA) equipped with standard burner, air acetylene flame and hollow cathode lamps as radiation source (Indrayan et al., 2005).

Statistical analysis
Data were reported as mean ± standard deviation of triplicate measurements. Significant differences (p < 0.05) within means were analyzed by analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) test in the SPSS Statistics Software Version 20 (IBM, New York, USA).

RESULTS AND DISCUSSION
The proximate and nutritional contents are mentioned in Table 2, vitamins constituents in Table 3, while minerals and toxic heavy metals are given in Table 4. The results are discussed in comparison with published literature and biological applications under the following four headings.

Nutritional composition
Moisture is considered as a good source of water and it is necessary that 20% of the total water consumption must come from food moisture (FNB, 2005). The average moisture content of T. portulacastrum L. studied was 8.0% (Table 2), very low compared to other wild edible plants such as Amanthus viridus, Chenopodium murale, Nastrium officinale and Scandex pectin-veneris with 88.90, 89.50, 90.54 and 81.31% respectively (Imran et al., 2007). However, it was very close to some common leafy vegetables such as Xanthosem sagittifolum (13.17%) and Adensonia digitata (9.5%) (Ladan et al., 1996). Ash content is the index of mineral contents in plants such as calcium, sodium, potassium, nickel and zinc. The ash content of the sample studied was 34.8% (Table 2), which showed that the leaves were rich in minerals. The value obtained was higher compared to 1.8% reported in sweet potato leaves, and 5% in Tribulus terristris leaves, 1.85% in A. viridus leaves, 2.70% in C. murale leaves, 1.77 and 3.10%, in N. officinale and S. pectenveneris leaves respectively (Imran et al., 2007). The crude protein content of the sample was found to be 9.19% (Table 2). This value was higher compared to 2.11% in A. viridus, 2.98% in C. murale leaves, 2.76% in N. officinale, 6.30% in water spinach and 6.40% in Momordica foecide leaves (Imran et al., 2007). But lower than 11.29% in balsam apple leaves, 24.85% in sweet potato leaves, Piper guineeses and Talinum triangulare with values of 29.78 and 31.00%, respectively (Akindahunsi and Salawu, 2005).
Among the reported wild edible plants of the same family, sample leaves contain higher crude protein value. According to the WHO recommended dietary allowance (RDA) of protein, for children, adult male and adult female is 28, 63 and 50 g respectively (Akindahunsi and Salawu, 2005), while in Pakistan the average protein intake is 43.4g/day (Figure 1) (NNSP, 2011). As the plant protein is also considered as biological value, so for 100 g of T. portulacastrum L. provide 9.19 g of proteins indicate that the sample plant is a good source of daily proteins. Lipid in food is considered as a chief source of storage form of energy, essential fatty acids and fat soluble vitamins and precursors of vitamins. The sample plant contained 2.0% crude lipid (Table 2). It is lower than 11% in water spinach leaves, 12% in Senna obtusfolia, 11% in Amaranthus caudatus leaves, 28.2% in Centilla asiatica leaves, 29% in Bahunian purpurea leaves and 60% in Amaranthus hybridus but higher than 0.47% in Amaranthus viridus, 0.54% in C. murale and 0.63% in S. pectenveneris leaves (Imran et al., 2007). The crude fiber content of sample leaves was 43% which is higher compared to 7.20% in sweet potato leaves, 13% in Tribubus terristris leaves, 29.0% in balsam apple leaves, 1.93% in A. viridus, 3.82% in S. pectenveneris leaves (Akindahunsi and Salawu, 2005).
Dietary fiber helps to reduce serum cholesterol level, risk of coronary heart diseases, colon and breast cancer and hypertension. The high level of fiber in diet can cause intestinal irritation, lower digestibility, difficult absorption of minerals found in plant and overall decrease nutrient utilization (Imran et al., 2007). The carbohydrate content of sample leaves was 3.02%, considerably low when compared to other wild edible plants such as A. caudatus leaves (61.03%), 55.67% in T. terristris, 54.2% in water spinach leaves, 75% in sweet potato leaves, 82.8% in Corchorus triden leaves. But closely similar when compared to A. viridus, C. murale, Nastritium officinale and S. pectenveneris leaves that is 4.74, 3.41, 3.38 and 7.32% respectively (Imran et al., 2007). Carbohydrates are principal and indispensible source of energy. The RDA for carbohydrates is 130 g (FAO, 1998), while in Pakistan 349 g of carbohydrate intake is reported (Figure 1) (Ministry of Health and Nutrition, 1994). Due to carbohydrates content sample, plant can be a good food source. The 100 g of T. portulacastrum L. provide 76.01 kcal of energy. This reveals that the sample plant can contribute meaningfully to the daily energy requirement of a person.

Vitamin analysis
Vitamin A is necessary for vision process and also plays a role in skin mucosa, normal reproductive capabilities and is an important anti oxidant (FAO, 2001). The sample contained 0.081 mg/100 g of vitamin A (Table 3). The RDA for vitamin A is 1.5 mg/100 g (Figure 2). The results show that the sample is not a good source of vitamin A. The sample contained 0.202 mg/100 g of vitamin B 2 . The RDA for riboflavin is 1.7 mg. The results show that it is a rich source of riboflavin compared to vitamin A. Riboflavin is present in body as co-enzyme which acts as hydrogen acceptor in amino acid metabolism (FAO, 2001).

Minerals analysis
Sodium maintains fluid volume outside the cell thus normalize the cell functions. The sodium content of the sample was 44 mg/g (Table 3), slightly low compared to reported (50 mg/g) for Tribus terristis leaves and very low than 450 mg/kg in S. obtusfolia but quite high in comparison to Asparagus officinalis (0.184 mg/kg) and Momordica dioica (0.151 mg/kg) (Khan et al., 2011). The RDA for sodium is 500 mg for adults ( Figure 3). The plant sample leaves can be good source of food for hypertensive patients (FAO, 2001). Potassium content of the sample was 51.60 mg/g (  (Khan et al., 2011). The RDA for potassium is 2500 mg for adults (Figure 3), and the sample contributes good percentage to RDA, meaning the good source that can contribute to the diet of hypertensive patients (FAO, 2001).

Micro minerals analysis
The copper content of plant sample is 20 mg/kg (Table  4), which is higher compared to 0.1 mg/kg in D. mespiliformis and 0.25 mg/kg in F. bengalensis, 12.8 mg/kg in T. terristis leaves and 5.0 mg/kg in Cassia siamea leaves (Khan et al., 2011;Gafar and Itodo, 2011). The RDA value for copper is 1 to 3 mg for adult ( Figure  3). Copper contributes a role in hemoglobin formation and play a role in iron and energy metabolism (FAO, 2001). The zinc content of sample leaves was found to be 200 mg/kg, higher compared to 0.200 mg/kg in Diospyros mespiliformis, 1 mg/kg in T. terristris leaves but lower when compared to 68.5 mg/kg in C. siameea (Khan et al., 2011;Gafar and Itodo, 2011). Zinc plays a vital role in gene expression, regulation of cellular growth and participates as a cofactor of many enzymes. It also plays an important role in motility of sperm during liquation and mating. The RDA of zinc is 12 to 15 mg for adults (FAO, 2001). Iron content of sample was 6.44 mg/g, higher than other vegetables like C. siamea 700 mg/kg (Khan et al., 2011). The RDA value of iron is 10 to 15 mg/100 g. This sample is a good source of iron. Iron is required for hemoglobin formation and its deficiency leads to anemia (FAO, 2001). Manganese content of plant sample was 40 mg/kg higher than 9.8 to 38 mg/kg reported in some leafy vegetables and lower than 116 mg/kg in balsam apple leaves (Khan et al., 2011). The RDA for manganese is 2 to 5 mg. The result showed that T. portulacastrum L. is a good source to provide daily manganese.
Manganese is a co-factor for many enzymes which take part in glucose and amino acid metabolism (FAO, 2001). The amount of nickel present was 30 mg/kg in the sample. This quantity was quite higher when compared to other edible wild plants like F. bengalensis 1.14 mg/kg (Gafar and Itodo, 2011). Nickel is needed in very small amount to the body. The health benefits of Ni are healthy skin and optimal growth and also take part in iron metabolism. Higher quantity leads to toxicity (Gafar and Itodo, 2011).

Toxic heavy metals analysis
The lead content of sample was 80 mg/kg (Table 4), higher compared to F. bengalensis (0.25 mg/kg). Lead is toxic and non essential element for human body as it causes rise of blood pressure, kidney damage, miscarriage, subtle abortion, brain damage, decline fertility of men through sperm damage and diminishes learning abilities due to neuron damaging actions (Gafar and Itodo, 2011). Cadmium concentration of T. portulacastrum L. was 0.6 mg/kg higher compared to F. bengalensis (0.017 mg/kg) and lower than other usual edible plants. Cadmium is highly toxic for a body and it causes several health hazards, including cell death and cell proliferation (Gafar and Itodo, 2011).

Conclusions
The present study provides an evidence of the potential nutritional value of the selected wild edible plant, T. portulacastrum L. It showed to have good proximate values of proteins, lipids, carbohydrates, fiber and moisture. The macro minerals (Na and K), micro minerals (Fe, Cu, Ni and Mn), and vitamins (A and B 2 ) were also present in appreciable quantities. The toxic heavy metals, Pb and Cd were present in very minute quantities and therefore may not pose any threat to health. Though, T. portulacastrum L., may not provide all nutrients required by man, yet it contains sufficiently good amount of some essential nutrients like vitamin B 1 , iron and fiber. These results were making T. portulacastrum L., a good source of food and therefore could be recommended for edible purposes.