Evaluation of vitamin C of ethno-wild edible plants in Northeast India

Ethnobotany deals with the usage of plants by different ethnic communities throughout the world for various purposes such as food, shelter, clothing, medicine, tools etc. North East India region is a part of eastern Himalayas with rich plant diversity. About 250 ethnic tribes inhabit the region with diverse cultures and traditional practices. Most of the ethnic communities in the region depend on natural resources directly for their daily needs and wild edible plants are one of them as they live in the vicinity of forests. This study deals with 20 wild edible plants, traditionally used by indigenous people. 18 genera belonging to 15 families with 3 species represented by family Lamiaceae. Different parts of plants are traditionally consumed as vegetables, of which 19 species represented with leaves as edible part. Berry, petiole and rootstalk constitute other edible parts. Most of the plant species were found to be rich source of vitamin C. Vitamin C content ranges from 6.24±0.34 mg to 79.91±1.52 mg/100 g fresh weight and maximum was recorded in Alocasia indica (leaves) and Oxalis corniculata and the least in Tamarindus indica. The findings indicated that these ethno-botanicals can be grown in homestead as sources of vitamin C in regular diet.


Introduction
Northeast India (NEI) comprises of eight states viz. Assam, Arunachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Tripura and Sikkim. NEI lies between 21°34'N to 29°50'N latitude and 87°32'E to 97°52'E longitudes covering an area of 262060 sq. km. The region is known for its richness in plant biodiversity, folk traditions and cultures. The region harbors about 50% of the flowering plants recorded in India. It is inhabited by more than 250 tribes of different ethnic groups and speaks about 200 dialects with distinct cultural entities and rich indigenous traditional knowledge; a paradise for ethnobotanists and anthropologists (1). The relationship between plants and human cultures is not only limited to the use of plants for food, clothing and shelter but include their uses for religious ceremonies, ornamentation and health care (2). The region is not only rich in cultures and traditions but abounds with different topography of varying climatic conditions suitable for fauna and flora to flourish making the whole communities directly or indirectly dependent upon its available natural resources for food, shelter, medicine etc. for their daily needs. There are hundreds of sacred groves in northeastern states viz. 101 in Arunachal Pradesh (3), 27 in Assam (4), 365 in Manipur (5), 111 in Meghalaya (6) and 19 in Sikkim (7), which are directly associated with religious practices and community sense of conservation of threatened flora and fauna insitu conditions and act as a store house of innumerable medicinal plants, fruits, fuel wood etc. (8).
Wild edible plants (WEPs) are neither cultivated nor domesticated but growing in wild and are edible (9). There are about 800 different species of wild edible plants found in India, of which 300 species are used mostly by the tribal and rural population of the Northeastern region alone (10). The northeastern region is rich in wild edible plants and different parts of the plants, such as flower, fruit, leaf, stem, tuber, root etc. are consumed by different ethnic groups. There are several reports on WEPs of this region by various workers. The highest number of wild edible plants reported so far from this region is 628 species (11) consumed by Naga tribes residing in different parts of the biodiversity rich areas, Eastern Himalayas; 51 wild edible fruits from Mizoram (12); 61 WEPs from Tripura (13); 61 condiments and spices (14) and a total of 86 wild fruits (15) from Assam (16); 33 wild edible mushrooms from Nagaland (17); 22 wild edible mushrooms of Meghalaya (18); 384 wild food plants from Arunachal Pradesh (19,20) and a total of 84 WEPs from Manipur (21,22). Though there are a total of more  (26,27).
Indigenous people are the custodian of these plant resources and the traditional knowledge associated with those plants (28). However, traditional knowledge is declining due to the influence of modern life and changes in land use patterns due to various anthropological developmental activities and encroachment of forest for agricultural practices for more food to feed the ever-increasing population.
Indigenous people have the knowledge of edible wild plants on when, where and how to gather and take either in raw form or by cooking and make various ethnic delicious dishes. WEPs are available in their natural habitats and some species within the vicinity of human inhabitants. In the past, people were heavily depended upon the wild plants to sustain themselves. However, the modern system of agricultural practices has afforded the choices to make of various food products that are commercially available thereby overlooking the necessity and importance of wild edible plants. On the other hand, commercially available crops make us depended on limited food sources thereby limit our dietary diversity which lead to nutrient deficiency and thereafter physiological problems and ailments. WEPs are lesser known to many; the reason of which could be due to non-availability or less palatability. However, these resources could be utilized for food security, either as emergency foods or as an integral part of our daily diet, as these are usually rich in vitamins and minerals than those of exotic or commercially available crops in the markets. Therefore, the present work is an attempt to substantiate the use of ethno-botanicals with scientific validation focusing on Vitamin C content on 20 species of WEPs from northeast region to sustain the confidence of the consumers and promote the acceptability of the same among others. Vitamin C is essential physiologically and as potent antioxidant, immunity booster to protect oneself from infectious diseases and healing from ageing and numerous diseases due to damage caused by free radicals and reactive oxygen species.

Sample collection
Ethnobotanical information and uses of the WEPs were gathered from the local people through personal interviews for the present study. 20 edible wild species were collected in April and May, 2020  Table 1). Plant species were identified with the help of experts, local flora and literatures. Freshly collected vegetables were cleaned by soaking in water for about 30 min. followed by rinsing in distilled water. External moistures were blotted dry with tissue paper followed by air drying briefly and non-edible portions were sorted and discarded. Edible portions of WEPs were chopped into small pieces and used for ascorbic acid analysis. Leaf and petiole of Alocasia indica were processed in the same manner, but separately and so also the leaf and rhizome of Houttuynia cordata.

Vitamin C determination
Ascorbic acid content was determined according to standard method (29). Ten (10) g sample in 3% metaphosphoric acid was blended and final volume made upto 100 ml. Mixture was filtered using muslin cloth. 5 ml of the extract/filtrate was titrated against 2, 6 dichlorophenol indophenol to light pink endpoint which persist for about 15 sec. Experiments were conducted in triplicates. Vitamin C content was calculated by the formula: Data were analyzed statistically using analysis of variance (ANOVA) and differences among the means were determined for significance using LSD (Least Significant Difference) test.

Results and Discussion
Selected 20 species of WEPs in the present investigation falls under 15 families and 18 genera, among which family Lamiaceae has maximum representative with three species viz., Mentha spicata, Clerondendrum colebrookianum and Pogostemon benganlensis while others are represented by either two or one species each ( Fig. 1 & Table 1). The detail information on recipes, part(s) used and modes of consumption by the ethnic communities in the region is presented in Table  1. Edible parts of WEPs constitute mostly leaf, stem, flower, fruit, rootstalk ( Table 1). The present study indicated that ethnic communities use more of leafy portion as vegetables besides petiole (Alocasia indica), rhizome/rootstalk (Houttuynia cordata) and berry fruit (Solanum torvum) as food. These WEPs plants are traditionally preferred as vegetables by the ethnic groups in the region. These plants can be grown hygienically in home/kitchen garden, harvested and cleaned to be consumed raw, except Alocasia indica, as salads and for garnishing curry or ethnic dish such as eromba or shingju to reap maximum health benefits. For example, Brassica juncea tender leaves are sorted into pieces and relished raw with chutney or eromba by some communities in Manipur. Food and health are strongly inter-related which can be observed in the usage of the same plant as food and remedy for ailments by indigenous folks (30).  This is reflected in the present study where almost all the plants investigated are used as food and medicine at the same time (Table 1). Vitamin C is one of the essential nutritional components as it acts as natural antioxidant and need to be taken through diet or supplement. It cannot be synthesized in human body (31). Keeping in mind the roles played by vitamin C in health and diseases in human body, this study was undertaken to determine vitamin C content in commonly use wild edible plants. These plants can be broadly categorized into three groups based on Vitamin C content (Table 2) viz., high, moderate and low.

High Vitamin C
Leaves and petiole of A. indica are widely used as vegetable either fresh or in dried form by different communities in Manipur. The petiole is consumed as a blood purifier and the leaf is used for treating boils. It is hepatoprotective, antioxidant, analgesic, antiarthritic, anti-inflammatory, anti-tumour and antipyretic (32). A. indica corm/tuber possessed both ascorbic acid (76.65 mg/100 g) and alpha-tocopherol (69.54 mg/100 g) on fresh weight (33). These vitamins are most commonly found in vegetables and act synergistically as a potent antioxidant, thus, preventing cellular damage caused by free radicals and reactive oxygen species (33). Present study revealed that A. Indica leaf contain higher vitamin C (79.91 ± 1.52 mg/100 g) than tuber (76.65 mg/100 g) (33) and has the highest content of Vitamin C among the undertaken WEPs. A. indica petiole has 11.07 ± 0.39 mg/100 g fresh weight. Oxalis species viz., O. corniculata (77.69 ± 1.05 mg/100 g) and O. debilis (56.60 ± 0.39 mg/100 g) showed the second highest content of vitamin C. These species are used by indigenous people in NEI for treating different ailments, for instance, O. corniculata is used for treating diarrhea and dysentery by Poumai-Naga and O. debilis is used against gastritis by people of Assam.
B. juncea is a semi-cultivated species and one of the most popular leafy vegetables consumed by every house-hold in its season in Manipur. B. juncea leaves are one of the highest sources of vitamin K among the leafy vegetables and low in calories and fats with a rich source of phytochemicals. Moreover, mustard leaves are source of vitamin C, several essential minerals such as calcium, iron, magnesium, potassium, zinc, selenium and manganese (34). Leaf of B. juncea was found to contain high amount of vitamin C (54.58 ± 0.36 mg/100 g).

Moderate Vitamin C
The young and tender leaves of mulberry (M. nigra) are used as vegetable by two ethnic communities (Naga and Kuki) in Manipur. It is also used as feeds for silk worm culture in sericulture. Ripened fruits are relished fresh and used for making jam. In the present investigation, M. nigra leaves were found to contain sufficient amount of vitamin C (34.16 ± 0.39 mg/100 g). Turkish mulberry fruits (M. alba, M. nigra, and M. rubra) ascorbic acid content ranges from 19.4 to 22.4 mg/100 ml (35). H. cordata whole plant is consumed as vegetables by different ethnic communities. The leaves and rootstalks are consumed either raw or cooked. Rhizomes/rootstalks are preferred over its leaves for making ethnic special spicy chutney with other ingredients among the folks in Manipur, whereas leaves are more popular among ethnic groups in Assam. The plant is anti-infectious and anti-inflammatory (36,37) and also used for treating measles, gonorrhoea, skin ailments, anti-tumour, pneumonia, bronchitis, stomach ulcer (38,39). While analyzing the vitamin C content on leaves and rhizomes, leaves showed higher value (24.36 ± 0.34 mg/100 g) than the rhizomatous parts (10.09 ± 0.39 mg/ 100 g). Dried leaves were reported with 15.23 mg/100 g (40). Many communities in Manipur prefer rhizome over leaves and leaves are usually ignored. However, from the present findings the leaves should be preferred over rhizomatous roots and can also be harvested frequently. By doing so it can be protected from uprooting from its natural habitats which is not the sustainable way of harvesting of WEPs. Also, it justifies its uses against infections, as the vitamin C is known to be a potent non-enzymatic antioxidant and known to enhance immunity. A. philoxeroides is a common WEP and often sighted at the local markets consumed as vegetable by the people of Assam, though, it is not popular among other communities of NE states. A. philoxeroides possesses high nutritional and low anti-nutritional factors and therefore recommended to be included in the diet (41). However, it is reported to accumulate heavy metals (42) by other researchers and therefore it could pose health hazards. It was observed to have high content of vitamin C (34.52 ±0.39 mg/100 g) as compared to findings (41). Leaf and corm of A. galanga are commonly used as spices or condiments in nonvegetable food items by Naga communities. The plant is used for treating dyspepsia, fevers, incontinence of urine, halitosis and voice hoarseness in throat infections (43). A. galanga is available in the forests of NE region and the leaves are found to contain a moderate amount of vitamin C (20.81 ± 0.39 mg/100 g). A. viridis is a very popular leafy vegetable commonly available in the paddy field or furrow land and the tender shoots are harvested and sold at local markets. It tastes at its best in March-April, though it is available almost throughout the year. A. viridis is an excellent source of protein. The amino acid composition of the plant is comparable to that of a World Health Organization (WHO) protein standard. Two fatty acids (linoleic and α-linolenic) essential for humans and number of minerals including iron, magnesium, calcium and zinc were also reported (44). Leaves of A. viridis showed low vitamin C (23.58 ± 0.79 mg/100 g) as compared to (106.64 mg/100 g fresh weight) (45). S. nigrum leaves are cooked as vegetable and the ripened berries are eaten raw. Content of vitamin C in leaves was estimated to be 30.43 ± 0.39 mg/100 g on fresh weight which is slightly lower than value reported earlier (35.18 mg/100 g) (46). C. colebrookianum is widely used as vegetable by different ethnic groups in the northeastern region. Leaves are either boiled/cooked with dry fish or meat and consumed. It is widely used as a home remedy for hypertension by North East people. It is found to be practically non-toxic (47). Vitamin C content in the leaves of C. colebrookianum was 14.05 ± 0.36 mg/100 g. M. koenigii is widely used as spice or flavor in curry, salad, chutney, pickle etc. It is used for treating nausea, vomiting, diarrhea, dysentery, blood purification, tonic and stomachic (48). Leaves were found to contain moderate vitamin C (25.69 ± 0.39 mg/100 g fresh weight) as compared to the reported (815 ± 0.80 mg / 100 g dry weight) (49). P. microcephalum leaves are used as souring agent in the curry by the ethnic people. It was found to have 29.38 ± 0.39 mg/100 g in fresh weight vitamin C. P. benghalensis leaves are used as vegetable, also in preparing side-dish like "pakora" by the ethnic communities. Leaves are cooked and given for clearing uterus to women post-delivery by ethnic groups in Assam and root for treating fever (50). Vitamin C content in leaf of P. benghalensis was 14.09 ± 0.3 mg /100 g. P. major is used throughout the world for treating several diseases such as skin diseases, infectious problems related to digestive organs, respiratory organs, reproduction, blood circulation, anti-tumour, pain relief and reducing fever (51). In India, different ethnic groups use it for treating diarrhoea, dysentery, ulcer, urinary tract infections, bee stings, pus-formation in impetigo, diurectic, stimulant (52); anti-inflamatory (53); cuts and wounds (54); menstrual disorders (55); astringent effects (56). Ethnic communities from the north east region use it for various purposes. Its leaves are used as vegetable and ethno-medicine by indigenous people. It is commonly available in the fields. Naga tribes use it as a haemostatic on wounds and cuts (57); Khasi tribe uses it for treating burns (58); People of Assam and Arunachal use it for treating any inflammatory ailment (59). P. major leaf was found to contain moderate amount of vitamin C (11.9 ± 0.39 mg/100 g) as compared to the other wild edible plants undertaken for the study.

Low Vitamin C
Tamarindus indica, also known as tamarind, is extensively used by different ethnic communities in the world as food and traditional medicines for its wide array of applications; leaves and flowers as vegetables, fruit pulp for seasoning of food, jam and sweets, refreshing drinks and also fermented into alcoholic beverage (60 (64)(65)(66). C. crepidioide leaves are taken as vegetable or consumed raw as salad by different ethnic groups in the region. It is used as remedy for gastritis, to arrest bleeding in cuts and wounds and quick healing by Poumai and Mao communities in Manipur. Vitamin C content of its leaves was 7.64 ± 0.39 mg/100 g in fresh weight which is more or less the same as reported (9.17 mg/100 g) (67). During Covid-19 pandemic lockdown, the villagers in NE India joined the fight for controlling Covid-19 by supplying fresh wild vegetables to urban dwellers (68) thereby mitigating the short supply of fresh vegetables.

Conclusion
The findings of the present investigations confirmed that the studied ethno-wild edible plants served as foods and herbal medicines for the ethnic communities in NE India. Consumption of these 20 WEPs should be encouraged among the ethnic groups, in particular and public in general, by creating awareness about the benefits of its important constituents like vitamin C, which is essential for health and prevention of diseases. Among the ethno-wild edible plants, species with high content of vitamin C (as shown in Table 1 should be focused upon to cater the daily requirement of vitamin C in the diets for the poor and needy people. On the other hand, several species (C. colebrookianum, C. crepidioides, H. cordata, L. alba, M. spicata, P. major, S. torvum and T. indica) play dual roles in providing not only the essential nutrients or minerals supplement but also as a source of medicine for the ethnic communities. WEPs are the source of food security during emergency situations for the larger populace in the region. Therefore, the WEPs should be preserved and over exploitation from its natural habitats should be discouraged in order to maintain its germplasm for future use and development as food supplements. Global agriculture focuses on a few cultivars, usually of high yielding varieties which are costly and not affordable by poor people. Therefore, WEPs meet their daily requirement of minerals and vitamins. Moreover, these plants can be easily home grown for management or prevention of many ailments and chronic diseases at an affordable cost and for commercial production as well for an additional income for the indigenous population in the region. Lastly, the findings of this study would be helpful for researchers to carry out further studies related to nutritional supplement, biological and pharmacological functions of these plants.