Assessment of Nutrient Composition and Antioxidant Activity of Some Popular Underutilized Edible Crops of Nagaland, India

In Nagaland ~70% of population lives in rural areas and depends on forest products for livelihood. Being part of the biodiversity hotspot, state is rich in biodiversity. The present study was an attempt made to understand the nutritional properties of 22 popular underutilized edible plants (UEP) Kohima, Phek, Tuensang districts. Results revealed moisture content of 22 studied plants ranged between 4.8 to 88.15 g/100g, while protein content varied between 0.00269 0.773 g/100g with highest in Terminalia chebula (0.773 g/100g) fruit while lowest protein content was in Setaria italica (0.00269 g/100g). Total carbohydrate content was between 0.198 5.212 g/100g with highest in Setaria italica (5.212 g/100g) and lowest in Juglans regia (0.198 g/100g). Of the 22 samples, maximum antioxidant activity was in Terminalia chebula fruits (37.49 μg/ml) followed by Clerodendrum glandulosum (65.29 μg/ml) leaves, Phyllanthus emblica (79.08 μg/ml) fruits against Trolox (96.89 μg/ml). Highest total phenol content (TPC) was recorded in Terminalia chebula (53.11 mg GAE/g) and Rhus chinensis (43.99 mg GAE/g) while in other 20 crops the values varied from 0.09 8.44 mg GAE/g. Total flavonoid content (TFC) varied between 0.004 43.67 mg QE/g with clerodendrum glandulosum (43.67 mg QE/g) and Terminalia chebula (27.78 mg QE/g) were found to be highest among the 22 plant samples. Findings suggest that these underutilized edible plants should be popularized as they can contribute to nutritional support to different region of the state for health improvement and cultivated them commercially to help and develop various value added local product to improve the livelihood status of the rural population and also add to the economy of the state and region.


Introduction
Underutilized edible plants are under exploited of its potential for contributing to food security (nutritional/medicinal), income generation and environmental services [1]; they are locally abundant instead of globally, lack scientific knowledge concerning their physiological, agronomic and ecological properties and have a limited current use relative to their economic potential. They have the potential to fight against poverty and starvation faced by many developing countries and can help promote crop diversification to encounter such problems and also improve the rural economy [2] [3]. Though they are not significant in terms of global production and consumption systems but are rich in macronutrients and micronutrients and comparatively they require relatively low inputs thereby contributing to rural sustainable agricultural production and combating the "hidden hunger" caused by micronutrients deficiencies [2] [3] [4] [5]. They have the capability to deliver many health benefits apart from fulfilling physiological needs and had been conferred the status of functional foods [6].
A diet rich in higher antioxidant activities with free radical scavenging molecules (flavonoids, anthocyanins, cartenoids, and dietary glutathionine), vitamins and endogenous metabolites can protect the human body against cellular oxidation reactions, cardiovascular events, cancer, and other age-related degenerative diseases [7] [8] [9]. Fruits, nuts and vegetables play a significant role in human nutrition especially as source of vitamins, minerals and dietary fiber [10]. They offer advantages over dietary supplements because of low cost and wide availability; as such they remain an important source of nutrients in many parts of the world. Nuts are good source of essential fatty acid, fiber, vitamin E and mineral [11]. Some components of fruits and vegetables are strong antioxidants and function to modify the metabolic activation and detoxification/disposition of carcinogens or even influence processes that alter the course of the tumor cell [12]. A high dietary intake of fruits and vegetables is strongly associated with a reduced risk of developing some chronic diseases, such as various types of cancer, cardiovascular disease, type II diabetes and other degenerative or age-related diseases, which causes death in many developed countries was established by clinical trial and epidemiological studies [8]. With the advantages of medical science, people live longer and are faced with diseases that come along with age and illnesses such as cancer and diabetes mellitus [13]. The antioxidants from plant origin are beneficial compared to synthetic ones as they do not have any side effect/genotoxic effect [14] [15], further natural antioxidants are advantageous due to cost effectiveness and easy accessibility in the form of vegetables and fruits [16]. Data on epidemiological and in vitro studies strongly suggest that foods containing phytochemicals with anti-oxidation potential have strong protective effects against gained increasing interest among consumers and the scientific community because epidemiological studies have indicated that frequent consumption of natural antioxidants is associated with a lower risk of cardiovascular disease and cancer [18]. Potential sources of antioxidant compounds have been searched in several types of plant materials such as vegetables, fruits, leaves, oilseeds, cereal crops, barks and roots, spices and herbs, and crude plant drugs [19]. Phenolic substances such as flavonoids are the most common compounds in fruits and vegetables and have strong antioxidant capacity [20] [21]. Crop improvement has played important role in sustaining and strengthening food and nutrition, health and livelihood in the world from the very beginning of domestication yet millions of people are still under-nourished [22] [23].
Nagaland, a small state of North-Eastern region of India very rich in its flora and fauna. Being a part of Indo-Burma biodiversity hotspot, the region is a home for unique biodiversity and the indigenous Naga tribes depends greatly on forest products as food source and is an integral part of their daily lives [2] [3]. Nagaland is basically an agricultural based state and cultivation of cereal crops plays a big part in agricultural system of the state. Millets and jobs tear are important traditional Naga cereal crops which have been passed down from generation through traditional preservation method. Forest has a large and indispensable role in improving the food security and livelihood of the tribal society [24]. About 70% of ethnic tribes are living in rural hilly areas and depends on minor forest products for livelihood including food and medicines. During 2012-16 three hilly districts (Kohima, Phek and Tuensang) of Nagaland, India was surveyed for underutilized wild edible plant species and 142 species were collected, documented [3]. Of these 142 species, 22 species were found to be popular among the tribal population. Following documentation, an attempt was made to understand the nutritional properties of these 22 popular underutilized edible plants (UEP).

Survey Areas
Kohima District: Kohima is the capital city of Nagaland state, India, located between the geographical coordinates of 25.6701˚N and 94.1077˚E surrounded by the Dimapur district to the west, Phek and Zunheboto district to the east, Wokha district to the north and Manipur state to the south with a total geographical area of 1463 sq. km and an average elevation of 1444 m (4738 ft) above sea level. It is the home land of the Angami Naga tribe and agriculture is the main occupation.
Phek district: Phek district lies in the South-East of Nagaland with geographical coordinate of 25.

Sample Preparation
The 22 selected UEP considered for the assessment consists of 2 cereal crops (Coix-lcryma jobi and Setaria italica), 9 leafy vegetables (Centalla asiatica, Clerodendrum glandulosum, Elatostema platyphyllum, Lasia spinosa, Polygonum chinense, Polygonum molle, Trichodesma khasianum, Plantago erosa, Zanthoxylum oxyphyllum) and 11 fruits and nuts (Actinidia callosa, Hodgsonia macrocarpa, Ficus auriculata, Ficus semicordata, Juglans regia, Olax imbricata, Phyllanthus emblica, Rhus chinensis, Spondias pinnata, Stixis suaveolens, Terminalia chebula). Fresh plant samples were harvested during right harvesting period in different seasons from the districts of Kohima, Phek and Tuensang and brought to the laboratory. The plant samples were rinsed with water, air dried and grounded into powder for the assessment. Thereafter, the prepared samples were subjected to analysis for nutritional and phytochemical content and antioxidant properties using different analytical methods. Details of the 22 selected UEP are listed in Table 1 along with the botanical name, common name and the parts used of the plants.

Proximate Composition
Moisture content: Moisture content was estimated following Association of Official Agricultural Chemists [25] method with slight modification. Two g of the collected each sample was taken in a separate pre-weighed dish and oven dried at 70˚C for ~4 h for leafy vegetables and ~18 h for fresh fruits, nuts and cereals. The moisture content was calculated by using the formula.  Protein content: Protein estimation was determined by using the Colorimetric method described by Lowry [26]. About 500 mg of the oven dried sample was grounded and homogenized with 10 ml of 0.1M Phosphate buffer (pH 7.4) and centrifuged the solution at 1000 rpm for 10 min. The supernatant was filtered with Whatman filter paper and the filtrate was used for protein analysis.
Pipette out 1 ml of the extract to which 5 ml of the Lowry's regent was added.
Mixed the solution well and allowed the mixture to stand for 10 min. Added 1N of Folin-ciocalteau reagent to the solution followed by incubation at room temperature in the dark for 30 min. Absorbance was taken at 660 nm and the standard curve was prepared with Bovine Serum Albumin.
Total carbohydrate: Total carbohydrate was estimated using the Phenol-Sulphuric Acid Method [27]. About 300 mg of sample was weighed and hydrolyzed by keeping it in a boiling water bath for 3 h with 5 ml of 2.5N HCl and cool to room temperature. The volume was made to 50 ml with distilled water and cen- trifuged at 1000 rpm for 10 min and the supernatant was filtered and the filtrate to be used for the estimation of total carbohydrate. To 1 ml of the extract, added 1 ml of 5% Phenol solution followed by 5 ml sulphuric acid (96%) and mixed well by shaking for 10 min. The content of the tube was placed on a water bath 25˚C to 30˚C for 20 min and the absorbance was read at 490 nm and the standard curve was prepared using glucose.
Reducing sugar estimation: Reducing sugar was estimated using the method of 3,5-dinitrosalicylic acid reagent [28]. One g sample was weighed and grounded with 10 ml of ethanol (80%, v/v) and the solution was centrifuged at 1000 rpm for 10 min, supernatant was then filtered and the filtrate was used for estimation of reducing sugar. To 1 ml of extract 3 ml of DNS reagent was added followed by heating the solution in a boiling water bath for 5 min. Added 1 ml of 40% (w/v) Rochelle salt solution (Potassium Sodium Tartrate) in the cooled reaction mixture. The absorbance was read at 510 nm and compared with the standard curve prepared using glucose.
Preparation of Sample extracts for Antioxidant activity, Total phenol content and Total flavonoid content: Methanolic extraction was prepared for estimation of antioxidant activity, total phenolic content, total flavonoid content. Ten g of dried sample was grinded in liquid nitrogen using mortar and pestle, powder was mixed with 100 ml of methanol and incubated for 24 h at 25˚C under continuous stirring at 150 rpm. The process repeated until the extraction solvent became colorless. The extract was then filtered on filter paper Whatman No. 4 and the filtrate was used for analysis.

Antioxidant Activity
DPPH Radical Scavenging Assay: 2, 2-Diphenyl-1-picrylhydazyl (DPPH) stable free radical method was used to determine the antioxidant activity of the plant extract following Aoshima et al. [29]. To 0.1 ml of sample extract with different extract concentrations (10 µg/ml, 50 µg/ml, 100 µg/ml, 150 µg/ml, 200 µg/ml), 2.9 ml of DPPH (0.1 mM DPPH prepared in methanol) was added and vortexed vigorously. Incubation of the reaction mixture for 30 min was done in the dark at 30˚C and the absorbance of the mixture was measured at 517 nm. Trolox was used as the standard for the evaluation of antioxidant activity. Inhibition of free radical by DPPH was calculated by the following equation: Total phenolic content: The presence of polyphenols was determined by Folin-Ciocalteu method [30]. The resulting absorbance of the blue colored solution was measured at 765 nm and the total phenolic content of the mushroom samples were expressed as g of gallic acid equivalents (GAE) per g of the extract.
Total flavonoid content: The flavonoids content was estimated by spectrophotometric method following Sahreen et al. [31]. The absorbance of the solu-tion was measured at 510 nm. Quercetin was used as the standard and the results expressed as g of quercetin equivalents (QE) per g of the extract.

Statistical Analysis
All the experiments were carried out in triplicate (n = 3) and data expressed as Mean ± Standard deviation.

Proximate Analysis
Moisture content: Table 2 shows the moisture content of the 22 selected Protein content: Table 2 shows the protein content of the 22 plant sample varied significantly which ranged between 0.00269 -0.773 g/100g of the sample (D/W basis). The maximum protein content was recorded in the fruits of Terminalia chebula (0.773 g/100g) which was followed by leaves of Polygonum chinense (0.54 g/100g) and fruits of Phyllanthus emblica (0.439 g/100g). The lowest protein content was recorded in the cereal crops Setaria italica (0.00269 g/100g), Coix-lacryma jobi (0.00412 g/100g) followed by Ficus auriculata fruits (0.011 g/100g), Elatostema platyphyllum (0.013 g/100g) and Actinidia callosa (0.026 g/100g). Over all, the 2 cereal crops showed the lowest protein content (0.00269 g/100g in Setaria italica and 0.00412 g/100g in Coix-lacryma jobi) among the three group of the plant sample studied ( Table 2).
Carbohydrate content: Table 2 shows the data of the present analysis that revealed, the carbohydrate content in the selected 22 plant samples shows significant variation ranging from 0.198 -5.212 g/100g. The maximum carbohydrate content was recorded in Setaria italica (5.212 g/100g) followed by Spondias pinnata fruits (5.135 g/100g), Actinidia callosa (5.135 g/100g) and seeds of Coix-lacryma jobi (4.379 g/100g) and lowest was recorded in the fruits of Juglans regia (0.198 g/100g) followed by nuts of Hodgsonia macrocarpa (0.321 g/100g) and leaves of Lasia spinosa (0.417 g/100g). Over all, the highest carbohydrate content was recorded in the cereal crops amongst the three groups of plants studied.
Reducing sugar: Unlike the other nutritional parameters, in Table 2 the value of reducing sugar content in the 22 plant samples studied does not vary much which ranges from 0.009 -0.139 g/100g of sample. Reducing sugar content was highest in fruits of Ficus auriculata (0.139 g/100g) which was followed by fruits of Actinidia callosa (0.097 g/100g) and leaves of Polygonum molle (0.081 g/100g). Seeds of Coix-lacryma jobi and Juglans regia (0.009 g/100g) were found to have the lowest reducing sugar content. It was established that the leafy vegetables and fruits content higher reducing sugar then the cereal crops. Table 2 shows the proximate composition of the 22 selected UEP with the parameters: moisture content, total carbohydrate, reducing sugar and protein content.
Total phenol content (TPC): The maximum TPC was recorded in the fruits of Terminalia chebula (53.11 mg GAE/g) and Rhus chinensis (43.99 mg GAE/g). The TPC value of T. chebula and R. chinensis varies significantly from the rest of the 20 plant samples as the value of TPC of the other samples which ranges from 0.09 mg -8.75 mg GAE/g. The lowest TPC was recorded in the leaves of Elatostema platyphyllum (0.09 mg GAE/g) followed by the fruit of Ficus auriculata     Table 3 showed the proximate compositions of 22 selected UEP with the parameters: antioxidant activity, total phenol content and total flavonoid content.

Discussion
With increase in human population there is a growing concern about the food security throughout the world. with the reports of Sundriyal and Sundriyal [34]. Report of moisture percent of Centella asiatica (88%) by Rosalizan et al. [35] is higher than the present study (80%). Firdusi [36] reported the moisture content of Lasia spinosa (86.30%) which comparative higher than the present study (50.50%). The reason for this difference could be due to stage and the time of collection of the sample.
Besides moisture content, other nutritional parameters were also investigated in these selected UEPs. In the present study the protein and reducing sugar content of Spondias pinnata, Phyllanthus emblica, Hodgsonia macrocarpa, Terminalia chebula and Zanthoxylum oxyphyllum were found to be much higher compared to findings of Khomdram [32] and Sundriyal and Sundriyal [34]. Protein quality of a food depends on its amino acid content and the physiological utilization of specific amino acid after digestion, absorption and minimal obligatory inorganic commercial food products. The UEP were also found selling in local market in fresh form, dried form or roasted or made into a local product which cost around Rs. 20 -50 per 400 -500 gm packet and some are in high demand because of its taste, medicinal properties etc. [3]. Though the lifestyle and food habit of modern day has changed significantly with the advancement of Science and Technology, still over 70% of the population of ethnic tribes of Nagaland are living in rural areas with limited facilities and still depends on forest and forest products for food and health supplements/medicines. They mostly collect the vegetables, cereals etc from the forest for livelihood. These peoples have selected these lesser known forest products by trial and error methods over several generations without knowing the nutritional components. Present study is the first of its kind to know the nutritional parameters of some of the popular UEP of the region.

Conclusion
In the present investigation, assessment on the nutrient composition, phytochemical and antioxidant properties were carried out in 22 selected underutilized edible plants of Nagaland, India. The present investigation throws some light on the nutritional and antioxidant content of 22 selected underutilized edible plants which will help increase our knowledge on their use. The result from the present investigation can contribute as nutritional support to different region of the state for health improvement and help in development of the various value added local product of the underutilized edible plants which can help up-lift the socio-economic status of the local inhabitants of the state. Also, from the result obtained it was observed that many underutilized edible plants have high nutritional composition and antioxidant activity, it will be of great help if more research works are carried out in the future to study the phytochemical and nutraceutical properties of these plants which will help improve the present day medicine and its marketability.