Nutritive scenario of wild consumable plants of Meghalaya state in India together with heavy metals, anti-nutritional, vitamin and toxicity studies

Tapan Seal

Plant Chemistry Department, Botanical Survey of India, AJC Bose Indian Botanic Garden,

Shibpur, Howrah 711103  India

*Corresponding Author E-mail: kaktapan65@yahoo.co.in

ABSTRACT:

The present exploration was projected to assess the nutritive potential, minerals content, vitamin quantity, antinutritional properties and toxicity investigations in five wild edible plants for example Agapetes variegata, Ficus virens, Begonia  roxburghii, Clerodendrum serratum and Rhododendron  arboreum, devoured by the diverse ancestral individuals of Meghalaya State in India as their sustenance.The proximate parameters,minerals,toxic heavy metals and antinutritional components were evaluated using standard food analysis techniques. The water soluble vitamins were carried out by HPLC.  The in vitro haemolytic assays of plants were carried out on rat erythrocytes. Assessments of cytotoxicity of edible plants were evaluated by MTT assay. The genotoxicity were tested by the single-cell gel electrophoresis comet assay. The outcomes of examination demonstrated that these plants are rich wellsprings of protein, minerals and vitamins. The estimations of antinutrients and substantial metals in the plants are beneath the lethal levels and furthermore safe to consume.

KEYWORDS: Wild edible plants, nutrients, antinutrients,minerals,vitamin,toxicity, India.

1. INTRODUCTION:

As of late, a ton of intrigue has been engaged to assess different wild consumable plants since they fill in as a key constituent of human eating regimen renewing the body with minerals, nutrients and certain hormone antecedents, notwithstanding protein and vitality. Because of the nearness of protein, carbohydrate and different full scale supplements, the wild edible plants assume a significant job to decrease the danger of different maladies like disease, coronary heart attack, diabetes, and so on. The wild vegetables likewise these days have turned into a business crop with expanding market potential because of the nonappearance of deposits from pesticides or manures¹. The wild palatable plants additionally contribute some valuable items like drug, fiber, grub, colors, and so forth. These plants which are generally devoured by the ancestral and provincial individuals as sustenance and prescriptions, yet adequate logical information on the proximate and substance arrangement of those wild vegetables still obscure to them, and individuals don't have satisfactory learning about their advantageous and dangerous properties. In the greater part of distributed writing, it was advised that the nutraceutical estimation of capricious plants sustenances could be practically identical to or even in some cases better than the common vegetables ². In this background the examination of wild edible plants is essential to recognize the potential sources which could be abused as elective sustenance. In spite of the fact that the wild palatable plants are delectable and nutritious, however overabundance utilization of such plants might be hurtful to our body due to having some antinutritional mixes in the plants. The antinutritional factors, for example, phyticacid, tannin, saponin, oxalic acid, cyanogen glycoside have antagonistic impact on wellbeing through restraint of protein assimilation, development, iron and zinc retention ^3,4. Phytic acid brings down the bioavailability of minerals ⁵,  tannins bond to proteins through hydrogen bonding and hydrophobic cooperations, in this way decreasing their dietary quality ⁶. Since antiquated occasions plants have been utilized as sustenance and medications and it is likewise realized that, by and large, green plants are an essential wellspring of antimutagens just as normal harmful specialists⁷. So, it is basic to decide if the wild plants can create unfriendly impacts on living being before utilization.

Meghalaya is a little state in north-eastern India. An enormous part to the locale is naturally under-investigated or even unfamiliar The timberlands of Meghalaya give countless plants whose organic products, seeds, tubers, shoots, and so on make a significant commitment to the eating regimen of the innate individuals. Along these lines, this examination was directed with the expect to assess the healthy benefit, nutrient substance, antinutritional properties and poisonous quality investigations of five wild consumable plants viz. Agapetes variegata, Ficus virens, Begonia  roxburghii, Clerodendrum serratum and Rhododendron  arboreum, allegedly consumed by the ancestral individuals of Meghalaya.

2. MATERIALS AND METHODS:

2.1 Collection of plant materials

The edible part of five plant materials, for example Agapetes variegata (Roxb.) D.Don ex G.Don (Ericaceae), Ficus virens Aiton (Moraceae), Begonia roxburghii A.DC. (Begoniaceae), Clerodendrum serratum L. Moon (Lamiaceae) and Rhododendron  arboreum Sm. (Ericaceae) were collected from different places of Meghalaya State, India and idenfications were authenticated in our office. The voucher examples were preserved at the Plant Chemistry department of our office under registry no. BSITS 96, BSITS 97, BSITS 98, BSITS 99 and BSITS 100 separately. The plant parts were shed-dried, pounded and put away in an impenetrable compartment and proximate composition, and mineral substance, vitamin content and toxicity studies were completed in our research facility.

2.2 Estimation of proximate composition

The nutritional composition of the powdered edible plants were analyzed in our laboratory following the standard food analysis methods ⁸.

Ash content was estimated by heating plant sample  in a muffle furnace for about 5-6 h at 500 °C  whereas moisture content were determined by heating plant sample in an air oven at 100–110°C. The crude fat was extracted from moisture free sample with petroleum ether (60-80°C) in a Soxhlet apparatus for about 6-8 h.  Estimation of crude fibre content in the plant materials were carried out by treating the fat and moisture free materials with 1.25% dilute acid and 1.25% alkali followed by washing with water and ignition of the residue. The crude protein was determined using micro Kjeldahl method as described in Datta et al. 2019. The  total  carbohydrate  content  was  estimated  as described in  the  method  of  Hedge and Hofreiter ⁹.  The energy content of each plant samples were determined by multiplying the values obtained for protein, fat and available carbohydrate by 4.00, 9.00 and 4.00, respectively and adding up the values ⁸.

2.3 Estimation of minerals

Plant material was taken in a pre-cleaned and constantly weighed silica crucible and heated in a muffle furnace at 400°C till there was no evolution of smoke. The crucible was cooled at room temperature in a desiccator and carbon-free ash was moistened with concentrated sulphuric acid and heated on a heating mantle till fumes of sulphuric acid ceased to evolve. The crucible with sulphated ash was then heated in a muffle furnace at 600°C till the weight of the content was constant (~2–3 h). One gram of sulphated ash obtained above was dissolved in 100 ml of 5 % hydrochloric acid (HCl) to obtain the solution ready for determination of mineral elements through atomic absorption spectroscopy (AAS) (AA 800, Perkin-Elmer Germany). Standard solution of each element was prepared and calibration curves were drawn for each element and minerals were determined by atomic absorption spectrophotometry ¹⁰.

2.4 Estimation of water soluble vitamins by HPLC

2.4.1 Preparation of mixture standard vitamin solutions

The stock standard solutions of vitamin C, B1, B3, B5 and B6 and were prepared by dissolving 25 mg of the each standard in 1 ml 0.1M hydrochloric acid in 25 ml standard volumetric flask. For preparation of standard stock solutions of vitamin B9 and B2, 25 mg of the each standard were dissolved in one ml 0.1 M sodium hydroxide in 25 ml standard volumetric flask. The standard solution was stored in amber-glass bottles in the refrigerator at 4⁰C. The working standards were prepared by diluting with phosphate buffer (1M, pH 5.5).

2.4.2 Preparation of sample solution

Plant materials were washed with distilled water. The washed plant materials were cut into very small pieces, frozen in liquid nitrogen and kept at -20⁰C until analysis. 1 g each of freeze-dried sample was soaked in 10 ml water and extracted with 1 ml 0.1M NaOH and 10 ml phosphate buffer (1M, pH 5.5) were added to it and kept in dark for 24 hours. The solution was first filtered through a Whatman No. 1 filter paper and the resulting filtrate was taken in a 25 ml volumetric flask and solution was topped up to the mark with HPLC grade water. The sample solution was filtered through 0.45 mm membrane filter before injection into LC system. The stock solutions of sample were kept in a refrigerator for further use.

2.4.3 Chromatographic analysis of water soluble vitamins

The chromatographic analysis was carried out following the method as described by Seal et al. ¹¹. The mobile phase contains acetonitrile (Solvent A) and aqueous trifluoro acetic acid (TFA, 0.01% v/v) (Solvent B), the column was thermostatically controlled at 22⁰C and the injection volume was kept at 20 µl. A gradient elution was performed by varying the proportion of solvent A to solvent B. Total analysis time per sample was 35 min. HPLC Chromatograms of all vitamins were detected using a photo diode array UV/detector at four different wavelengths (210, 245, 275 and 290 nm) according to absorption maxima of analysed compounds. Each compound was identified by its retention time and by spiking with standards under the same conditions. The quantification of vitamins in the extracts was carried out by the measurement of the integrated peak area and the contents were calculated using the calibration curve by plotting peak area against concentration of the respective standard sample. The data were reported as means ± standard error of means of three independent analyses.

2.5 Estimation of antinutritional composition

Oxalate contents of edible plants were determined utilizing the strategy depicted by Munro and Bassir. One gm each of ground plants (in triplicate) was extracted thrice by warming (50⁰C) and stirring with a magnetic stirrer for 1hr with 0.3M HCl. The combined extracts were diluted to 100 ml with water and used for total oxalate estimation ¹². Phytate was determined using the method Reddy and Love. One gm of the ground plants were soaked in 100 ml of 2% HCl for 5h and filtered. To 25 ml of the filtered, 5 ml 0.3% ammonium thiocyanate solution was added. The mixture was then titrated with Iron (III) chloride solution until a brownish-yellow color that persisted for 5 min was obtained ¹³.  Saponin was determined using the method of Hudson and El-Difrawi 1979 ¹⁴. Tannins were assayed in accordance with the modified vanillin-HCl method of Price et al¹⁵ , and tannic acid was used as the reference standard. Cyanogenic glycoside contents of sample were determined by alkaline titration method where the end-point was noted as a permanent turbidity against a black background ¹⁶.

2.6 Toxicity studies of wild edible plants

2.6.1 Preparation of plant extracts

Five gm of powdered plant materials were macerated in 50 ml distilled water at room temperature for 24h and afterward separated through cotton wool. The plant materials were macerated again in the same solvent for another 24h and concentrates acquired from the first and the subsequent extractions were pooled and concentrated using a rotary evaporator under reduced pressure to obtain viscous extracts which were further dried using a freeze drier. The dry extracts were stored at -20°C until use.

Ten mg of each crude extracts were dissolved in 10 ml Phosphate buffered saline (PBS, pH 7.4) to make 1000 μg/ml. The sample solutions were passed through 0.22 μm syringe-adapted filters to eliminate any particulate matter and stored at -20°C until use.

2.6.2 Haemolytic toxicity study

Haemolytic toxicity studies of aqueous extracts of five wild edible plants were carried out following the method of Malagoli ¹⁶. The blood samples were collected from healthy rat, mixed with EDTA and centrifuged at 5,000 rpm for five minutes. The 10 % erythrocyte suspension was prepared in sterile Phosphate buffered saline (PBS, pH 7.4) for haemolytic study.

The various concentrations (100,200,300,500 and 1000 μg/ml) of plant extracts were added to 10% suspension of rat erythrocytes. The mixture was incubated for 1hr at 37^°C temperature, cells were centrifuged and the supernatant was used to measure the absorbance of the liberated haemoglobin at 540 nm in a UV-VIS spectrophotometer (Model Shimadzu, UV 1800). Two controls were prepared without extracts; negative control received sterile phosphate buffer saline, while hydrogen peroxide (50-200 µM) was taken as positive control. The average value was calculated from triplicate assays. The cell viability for each sample was calculated by dividing sample's absorbance on negative control absorbance multiplied by hundred.

2.6.3 Cytotoxicity study

Cytotoxicity studies with  aqueous extracts of five wild edible plants were evaluated out by  3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay on isolated goat liver cells utilizing the convention as portrayed by Mosmann ¹⁸ .

Fresh goat livers procured from local market and were perfused in PBS (pH 7.4) with collagenase and the liver was then minced in minute pieces and cells were separated using cell strainer having 40µl pore size (Genetix cell strainer, S. Korea) . The cells were then washed with HBSS and centrifuge at 800 g to eliminate fine debris. The cell feasibility extends somewhere in the range of 85% to 95% as controlled by the trypan blue rejection test.  The immaculateness of hepatocytes was examined by phase-contrast microscopy. The isolated cells were taken in an eppendorf tube containing 0.5 ml RPMI and 10% FBS. The different concentrations (100,200,300,500 and1000 μg/ml) of aqueous extracts (100μl) of edible plants were added to the freshly isolated hepatocytes and incubated for 2 hrs at 37^oC in a CO₂ incubator. Medium control (blank medium) and cell control (cells without extract treatment) were also taken and incubated under same experimental condition.  All the tubes were then centrifuged and supernatant was discarded. Thiazolyl Blue Tetrazolium Bromide, MTT (5 mg/ml, in PBS, pH 4.5) (Sigma, USA) were added into tube to achieve a final concentration of 0.5 mg/ml and incubated for 1 h at 37°C until intracellular purple formazan crystals are visible under microscope. After 1 h, the culture medium with MTT was carefully removed by centrifugation and 100μl DMSO was added to it and incubated for 30 min to 1 hr to dissolve formazan crystals. The UV absorbance of resulting purple solution was spectrophotometrically measured at 570 nm in UV-VIS spectrophotometer (Model Shimadzu, UV 1800)  and the level of cell feasibility was  determined the hepatotoxicity of plant extracts.

2.6.4 Genotoxicity study                     

The genotoxic potential of the water extracts of the plants were assessed by single-cell gel electrophoresis comet examine as depicted by Singh et al ¹⁹. One (1) ml blood was collected from tail vein of a healthy rat and 100µl of heparinised whole blood was incubated with 100 µl plant extracts of different concentrations (100,200,300,500 and1000 µg/ml) and incubated at 37^°C for 2h in a CO₂ incubator. The negative and positive controls were included. 100 µl cell suspensions were embedded in 100 µl of 0.5% low melting point agarose (LMPA) and then spread on a slide pre-coated with a film of 1% normal melting point agarose (NMPA). Two slides were prepared for each sample in which agarose cell suspensions were allowed to solidify at 4^°C. After solidification, slides were immersed to cold lysis buffer, (2.5 M NaCl, 100 mM EDTA, 10 mMTris buffer, 10% DMSO, Triton X-100 0.8%, pH 10) for 1 h. The slides were removed from lysis buffer and placed on a horizontal gel electrophoresis chamber, filled with alkaline electrophoresis buffer (1mM EDTA, 0.3N NaOH, pH 13.0) for 20 min for unwinding of DNA. Then, electrophoresis was performed for 30 min at 25V/300mA and electrophoresis slides were neutralized (three times) and stained with ethidium bromide solution (20mg/ml). The stained nuclei were visualized under fluorescent microscopy and photographed. Olive Tail Moment (OTM) of individual stained nuclei was calculated using comet assay software. A higher percentage tail DNA indicated a higher level of DNA damage and higher level of genotoxicity of plant extract.

Ethical clearance for performing experiments on rats to get erythrocytes, was obtained from Institutional Animal Ethics Committee (Approval No.-04/P/S/IAEC/2017), Serampore College, West Bengal, India conforming the CPCSEA guidelines. Goat liver was procured fresh from local abattoir and brought on ice within 30 minutes of death.

2.7 Statistical analysis

All the analysis was done using triplicate samples. Experimental results were subjected to univariate analysis of variance (ANOVA), followed by Tukey test (p ≤  0.05) using the statistical package for the social sciences (SPSS version 7.5).

3 RESULTS AND DISCUSSION:

3.1 Proximate composition of wild edible plants

The edible parts of plant materials e.g A. variegata, F.virens, B. roxburghii,  C.  serratum and R. arboreum collected from different places of Meghalaya state were taken for the examination of proximate composition. The proximate arrangement of these plants has been introduced in Table 1. The consequence of examination

Table 1. Nutraceutical  profile of wild edible plants collected from Meghalaya state in India

		Plants
Parameter studied		A. variegata	F.  virens	B.  roxburghii	C.  serratum	R..  arboreum
Proximate composition	Ash (%)	5.53±0.22^e	12.38±0.22^b	12.43±0.18^a	9.43±0.18^c	8.54±0.21^d
	Moisture (%)	86.28±0.16^b	79.24±0.31^e	81.34±0.23^d	84.48±0.20^c	87.61±0.23^a
	Crude fat (%)	1.89±0.04^c	1.74±0.41^d	2.19±0.10^a	1.16±0.04^e	2.04±0.05^b
	Crude fibre (%)	13.51±0.15^a	11.11±0.03^b	9.26±0.08^c	6.30±0.26^e	8.69±0.28^d
	Protein (%)	3.43±0.03^d	13.86±0.05^a	9.43±0.04^b	13.82±0.04^a	5.43±0.05^c
	Carbohydrate (%)	13.89±0.06^a	4.23±0.04^e	10.30±0.10^d	13.61±0.04^b	12.56±0.10^c
	Energy (kcal/100gm)	86.43±0.47^e	88.29±0.58^d	98.78±1.07^b	120.09±0.15^a	89.86±0.62^c
Mineral content (mg/100gm dry plant material)	Na	2.94±0.23^c	2.86±0.24^c	4.32±0.21^b	4.57±0.25^a	2.09±0.23^d
	K	126.96±4.34^e	481.94±5.69^a	140.52±2.79^d	452.99±6.48^b	232.25±4.25^c
	Ca	157.21±3.27^d	254.09±5.30^b	318.58±7.21^a	222.22±3.98^c	148.24±5.44^e
	Cu	0.17±0.009^b	BDL	BDL	0.82±0.004^a	BDL
	Mg	116.70±0.06^d	224.88±0.52^b	271.44±0.34^a	116.49±0.59^d	195.39±0.61^c
	Zn	1.81±0.018^d	2.43±0.022^b	1.85±0.05^d	9.94±0.25^a	2.22±0.02^c
	Fe	17.99±0.35^c	6.80±0.23^e	11.96±0.21^d	22.89±0.56^b	29.64±0.31^a
	Mn	BDL	1.38±0.09^d	2.68±0.08^b	3.92±0.13^a	2.44±0.05^c
Heavy metal content (mg/100gm dry plant material)	Pb	0.024±0.005^a	0.019±0.002^b	0.014±0.007^bc	0.018±0.003^b	0.012±0.004^c
	Cr	0.25± 0.005^a	0.16± 0.014^b	0.19± 0.015^ab	0.21± 0.005^ab	0.18± 0.014^ab
	Cd	Not detected	Not detected	Not detected	Not detected	Not detected
	Hg	Not detected	Not detected	Not detected	Not detected	Not detected
Vitamin content (mg/100gm dry plant material)	C	0.22± 0.006^c	1.42± 0.033^b	0.029± 0.003^e	0.062± 0.003^d	1.97± 0.03^a
	B1	Not detected	0.26± 0.002^a	0.052± 0.001^c	0.13± 0.003^b	Not detected
	B2	0.195± 0.003^d	0.24± 0.013^bc	0.22± 0.006^c	0.261± 0.02^b	0.31± 0.02^a
	B3	0.083± 0.002^b	Not detected	Not detected	Not detected	0.14 ±0.006^a
	B5	0.12± 0.003^b	0.049± 0.001^d	0.089± 0.002^c	0.15± 0.006^a	0.13± 0.003^ab
	B6	0.51± 0.016^b	0.51± 0.003^b	0.42 ±0.016^c	0.063± 0.001^d	0.62± 0.016^a
	B9	3.11± 0.066^b	0.54± 0.016^d	1.049± 0.033^c	1.05± 0.03^c	5.52± 0.20^a
Anti-nutritional composition      (%)	Oxalate	0.33±0.02^b	0.31±0.06^c	0.14±0.04^d	0.36±0.08^a	0.31±0.08^c
	Phytate	0.31±0.08^c	0.36±0.04^b	0.52±0.07^a	0.37±0.09^b	0.31±0.03^c
	Saponin	0.12±0.02^a	0.041±0.04^b	0.032±0.008^c	0.11±0.04^a	0.12±0.04^a
	Tannin	0.27±0.04^e	0.37±0.32^d	0.56±0.55^c	1.42±0.09^b	1.96±0.12^a
	Cyanogenic glycoside	0.00173± 0.0002^b	0.00097 ±0.0006^c	0.00216± 0.0003^a	0.00097± 0.0008^c	0.00097± 0.00003^c

Each value in the table was obtained by calculating the average of three experiments and data are presented as Mean ± Standard error of the mean (SEM).Statistical analysis were carried out by Tukeys test at 95% confidence level and statistical significance were accepted at the p < 0.05 level. The superscript letter   denotes the significant differences within same parameters of individual plant.

BDL: Below detection limit

demonstrated that the moisture substance was discovered most astounding (87.61±0.23 %) in R. arboreum and most minimal in the leaves of F. virens (79.24±0.31 %). The moisture content in nourishment includes the measure of water present in the sustenance and decides the unaffected nature of the nourishment before utilization. Moisture content influences the physical, compound highlights of sustenance which relates with the freshness and soundness for the capacity of the nourishment for an extensive stretch of time ²⁰. The assurance of moisture content likewise is important to ascertain the substance of other nutrition constituents consistently. The moisture content was found in other palatable plants were extremely near the moisture content of some regular vegetables like spinach (92.1%), cabbage (91.9%), expansive beans (82.4) and so forth, developed in India ²¹ .

Estimation of ash content in sustenance is particularly significant for  dietary assessment of nourishment. Ash substance speaks to the complete mineral substance in food. In spite of the fact that minerals speak to a little extent of dry issue, frequently under 7% of the aggregate, they assume a significant job from a physicochemical, mechanical and healthful purpose of view ²². The ash content of the vegetables was discovered most noteworthy (12.43±0.18 %) in B. roxburghii and apparent amount was evaluated in different plants which shows that the vegetables were wealthy in minerals and could give a lot of mineral components in our eating routine.

Fat is a significant part of eating routine and gives basic unsaturated fats, which are not made by the body and should be gotten from nourishment. The basic unsaturated fats are linoleic and linolenic acid which are significant for controlling irritation, blood coagulating, and mental health. It additionally fills in as the capacity substance for the body's additional calories. Aside from these, the proximity of fat in the eating regimen is significant for the retention of fat solvent vitamins like vitamin A and carotene in the body ²¹ . The critical measure of fat was recognized in B. roxburghii (2.19±0.10 %) alongside great amount in different plants under scrutiny.

Vegetables are rich wellsprings of fiber which assumes a significant job in diminishing the dangers of numerous scatters, for example, overweight, obstruction, diabetes, serum cholesterol, heart ailments, breast and colon cancer, hypertension, etc²³. The World Health Organisation (WHO) has suggested an admission of 22-23 kg of fiber for each 1000 kcal of eating routine which is fundamental for assimilation and compelling expulsion of squanders ^24,25. The crude fiber substance of the wild vegetables spoke to in Table 1 was found between 6.30±0.26 % and 13.51±0.15 %, which was most reduced in C. serratum and most astounding in A. variegata and like commercial foods grown from the ground like apple (3.2 %), broad beans (8.9 %), cabbage (2.8 %), potato (1.7 %), spinach (2.5 %). Along these lines, these wild vegetables utilized could be consumed in the human eating regimen to satisfy WHO recommendation ²⁵ .

The most noteworthy measure of carbohydrate was recognized in the inflorescence of C. serratum (13.61±0.04 %) while the most minimal was found in the leaves of F. virens (4.23±0.04 %). An awesome measure of carbohydrate were likewise present in other palatable plants and all around contrasted with some regular eatable plants like apple (13.7%), wood apple (18.1 %), potato (20.9 %), ripe mango (14.9%) etc ²¹ . So these edible plants under investigation could be a decent sustenance wellspring of starch for human utilization.

Proteins are one of the most significant supplements required by the body and ought to be provided in satisfactory sums in the eating routine. The dietary proteins are separated into amino acids which are utilized by the body to coordinate the protein required by the body for different capacities like, for the best possible working of antibodies opposing disease, for the guideline of compounds and hormones, for development, and for the fix of body tissue and so on ²¹. The measure of crude protein substance was identified most elevated in the leaves of F. virens (13.86±0.05 %) and least in the flowers of A. variegata (3.43±0.03 %). Different plants under scrutiny viz. C. serratum, B. roxburghii and R. arboreum likewise contained great measure of protein 13.82±0.04 %, 9.43±0.04 % and 5.43±0.05 % separately.

3.2 Minerals content in wild edible plants

The edible parts of all plants under scrutiny contained minerals like sodium, potassium, calcium, manganese, magnesium, iron, zinc and copper in shifting focus as appeared in Table1. High convergences of sodium (Na) were available running from 2.09±0.23 mg/100g (R. arboreum) to 4.57±0.25 mg/100g (C. serratum). The most astounding measure of potassium was identified in the eatable pieces of F. virens (481.94±5.69 mg/100g), while the most minimal was found in the flowers of A. variegata (126.96±4.34 mg/100g). Na assumes a significant job in the vehicle of metabolites and K is significant for its diuretic nature. The K/Na proportion in our body is of incredible worry to avoid hypertension and the proportion ought to be more noteworthy than one since K depresses and Na enhances blood pressure ²⁶. The proportion of K/Na were significant in the edible parts of  F. virens (168.51), R. arboreum (111.12) and C. serratum (99.12) and especially contrasted and some regular organic products (Amla 45, papaya ready 11.5, tomato 11.31, Castanea sativa 56.67,                                   Punica granatum 1400.00)²⁵. Thus the utilization of these vegetables is useful for human and may almost certainly control the hypertension of our body.

The Ca is a significant full scale supplement which establishes an enormous extent deep down, human blood and extracellular liquid. It is likewise especially required for the typical working of the cardiovascular muscles, blood coagulation, milk thickening and the guideline of cell porousness ²⁷. The convergence of Ca was most elevated in the leaves of B. roxburghii (318.58±7.21mg/100g) trailed by F. virens (254.09±5.30 mg/100gm),  C. serratum (222.22±3.98mg/100gm), A. variegata (157.21±3.27 mg/100gm) and R. arboreum (148.24±5.44 mg/100g). The Ca levels of some developed vegetables, and fruitsdiverge between 10-130.0 mg/100g ²⁵. The information demonstrating that the wild vegetables utilized in this investigation are wealthy in calcium and could give a decent wellspring of Ca to our regular diet.

Copper (Cu) is a fundamental follow component that can't be shaped by the human body. It exists as a significant segment of a catalyst that helps the consolidation of iron into red platelets, averting sickliness ²⁸.  An adequate measure of Cu was available in the leaves of C. serratum (0.82±0.004 mg/100gm). The Cu focuses were underneath the location level in other palatable plants.

Zn is a fundamental component in the nourishment of individual where it works as a vital piece of certain compounds, which assume a focal job in nucleic corrosive digestion. Moreover, Zn is a layer stabilizer and a trigger of the insusceptible reaction. Its insufficiency prompts development disappointment and poor improvement of gonadal capacity ²⁸. The Zn present in the wild plants under scrutiny was discovered most elevated in the inflorescence of C. serratum (9.94±0.25 mg/100g ) and great amount of Zn were distinguished in different plants as referenced in table 1 and the Zn levels of these consumable plants are like the levels revealed in some wild and verdant vegetables in India ²⁵.

The Manganese (Mn) convergences of the plants concentrated differed between 1.38±0.09 to 3.92±0.13 mg/100g. In this way the wild palatable plants containing Mn assumes a significant job in the digestion of protein, sugar, lipid and in the generation of steroid sexual hormones ²⁹.

Fe is significant in the eating regimen for the development of haemoglobin, typical working of the focal sensory system and in the digestion of sugars, proteins and fats. It is a part of muscle and blood and is fundamental to bear oxygen the body. Normal utilization of iron rich vegetables can forestall the iron-lack frailty ²⁹. High groupings of Iron (Fe) were available in the flowers of R. arboreum (29.64±0.31 mg/100g) followed by in C. serratum (22.89±0.56 mg/100g), A.variegata (17.99±0.35 mg/100gm) and in B. roxburghii (11.96±0.21 mg/100gm), which are very much contrasted with some regular verdant vegetables. These high Fe levels in some wild eatable plants examined could be explained with various soil attributes of the developing zone.

Magnesium (Mg) is particularly fundamental in a human body to keep up ordinary nerve and muscle work. The Mg convergences of the plants contemplated extended from 116.49±0.59 to 271.44±0.34 mg/100g. So the standard utilization of these magnesium rich plants, control the blood glucose levels and bolster a sound resistant framework ²⁹.

The substantial heavy metals substance of the wild vegetables under scrutiny has recorded in Table 1. Among substantial metals lead (Pb) is a potential contamination that promptly gathers in soils and dregs. Despite the fact that Pb isn't a basic component for plants it gets effectively retained and gathered in various plants parts. The utilization of vegetables containing Pb causes both intense and ceaseless harming ³⁰. It has unfavourable impact on liver, kidney, vascular and insusceptible framework. The centralization of Pb was most minimal (0.012±0.004 mg/100g) in R. arboreum and most elevated was recognized in the flowers of A. variegata (0.024±0.005 mg/100g). The Pb content in the wild vegetables in our investigation was beneath to the WHO allowable breaking point of 0.03 mg/100g. The degree of Pb revealed in this investigation is similar to those announced for Indian basil (0.009 mg/100g), unpleasant leaf (0.014 mg/100g), cabbage (0.013 mg/100g) and water leaf (0.018 mg/100g) ²⁹.

Chromium is a basic follow component that upgrades insulin capacity and impacts sugar, protein and fat digestion yet unending presentation to Cr may harm liver and kidney. The grouping of Cr was least (0.16± 0.014 mg/100g) in the leaves of F. virens and most noteworthy was identified in A. variegata (0.25± 0.005mg/100g). In present examination the Cr content in the vegetables were discovered lower when contrasted with the WHO allowable breaking point of 0.29 mg/100g ²⁹.

3.3 Evaluations of water soluble vitamin by HPLC

The HPLC strategy was effectively performed for the estimation of water soluble vitamin e.g ascorbic acid (C), thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6) and folic acid (B9). A typical HPLC chromatogram of the all standard vitamin mixture recorded at 210 nm is presented in Figure 1. As shown in the chromatogram, all investigated compounds had responses at 245 nm, where they were successfully separated.

The constituents under investigation were also identified by the recorded absorption spectra, which were comparable both for plant extracts and standard substances. The amount of all vitamins of all plant materials has been communicated as mg/100gm dry plant material and information displayed in table 1.

Vitamin C is the most significant nutrient in fruits and vegetables. It is outstanding for its cell reinforcement properties and it helps the body in repressing from viral contamination, bacterial diseases and harmfulness. It is required for the anticipation of scurvy and support of sound skin, gums and veins and the lack of this nutrient causes wounding, dying, dry skin and sadness ¹¹.  The exploratory outcome demonstrated that, the measure of

Figure 1. HPLC Chromatogram  of  mixture of Standard vitamin

(C) Ascorbic acid; (B1) Thiamine; (B3) Niacin; (B6) Pyridoxine; (B5) Pantothenic acid ; (B9) Folic acid ; (B2) Riboflavin

vitamin C in five wild consumable assessed in all plants under scrutiny in the range from 0.029±0.003 to 1.97± 0.03 mg/100gm. Due to having cell reinforcement properties, vitamin C rich plant may be useful to diminish the danger of atherosclerosis and a few types of malignancy ¹¹.

Thiamine (B), is a fundamental supplement required by the body for keeping up cell work and thusly a wide cluster of organ capacities. It is crucial for vitality creation, starch digestion and nerve cell work. The inadequacy of this nutrient prompts discount degeneration of the body, especially the apprehensive and circulatory frameworks, hypertension and cardiovascular maladies¹¹ .

The thiamine content in these wild eatable plants ran from 0.052± 0.001 to 0.26±0.002 mg/100gm. The most noteworthy measure of B1 was acquired in the leaves of F. virens pursued by C. serratum and B. roxburghii and these sums are particularly like the thiamine content in some basic vegetables and fruits like apple (0.016 mg/100gm), beans (0.132mg/100gm), cauliflower (0.073 mg/100gm), spinach (0.076mg/100gm) and so on ²¹ .

Riboflavin (B2) is a crucial nutrient required for legitimate vitality digestion and a wide assortment of cell forms. It is the partner to thiamine utilized in the reinforcing of nourishment items ¹¹.

A critical variety of riboflavin substance was seen among the tried wild palatable plants. The most astounding measure of B2 was distinguished in the flowers of R. arboreum (0.31± 0.02 mg/100gm) and the least sum was detected in A. variegata (0.195± 0.003 mg/100gm). The B2 content in these plants are practically identical with some basic foods grown from the ground like almonds (1.10 mg/100g), spinach (0.24 mg/100g), beet greens (0.41 mg/100g), green beans (0.12±2 mg/100g, potato (0.023 ±1 mg/100g) and so forth ¹¹.

VitaminB3, is a significant nutrient required for preparing fat in the body, bringing down cholesterol levels, and managing glucose levels. It is significant in DNA fix, Ca digestion, intracellular breath, and biosynthesis of unsaturated fat and steroids ¹¹  and in our examination it was not recognized in wild edibles except in the flowers of A. variegata (0.083± 0.002 mg/100gm) and R. arboreum (0.14 ±0.006 mg/100gm).

Vitamin B5, or Pantothenic acid, is a basic nutrient required by the body for cell forms and ideal upkeep of fat. The insufficiency of vitamin B5 prompts fractiousness, exhaustion, lack of concern, deadness, paresthesia, and muscle spasms in individual ¹¹.  Pantothenic acid was distinguished most noteworthy in the leaves of C. serratum (0.15± 0.006 mg/100gm) and most minimal was assessed in F. virens (0.049± 0.001 mg/100gm). Different plants under scrutiny were likewise found to contain a generally excellent measure of B5.

Pyridoxine (B6) is another water solvent vitamin essential for the correct upkeep of red platelet digestion, the sensory system, the insusceptible framework, and numerous other real capacities. It additionally assumes a job in homocysteine manufactured and degradative responses ¹¹. This nutrient is found in most nourishment items and furthermore, because of its soundness, is frequently utilized for strengthening sustenance items ¹¹. It was evaluated in all the wild consumable organic products under our examination.

The most elevated B6 was seen in R. arboreum (0.62±0.016 mg/100gm) though the base was recognized in C. serratum (0.063± 0.001 mg/100gm). The measure of B6 acquired in these wild edible plants were tantamount with some basic vegetable and fruits like banana (0.37 mg/100g), avocados (0.29 mg/100g), spinach (0.24 mg/100g), broccoli (0.134 mg/100g), cauliflower (0.115mg/100g), cucumber (0.2 mg/100g) and so forth ²⁵. So the customary admission of these plants would supply adequate B6 important to keep up solid body capacities.

Vitamin B9 (folic acid) is a water-solvent B nutrient with numerous rich normal sources. It is required for various body capacities including DNA combination and fix, cell division, and cell development. The lack of folate can prompt iron deficiency in grown-ups, and more slow improvement in youngsters ¹¹. It assumes a significant job as a cancer prevention agent in vivo, both by counteracting the unfavorable impact of responsive oxygen species (ROS), just as by hindering lipid peroxidation ¹¹.

The degree of B9 in five wild eatable plants extended from 0.54± 0.016 to 5.52± 0.20 mg/100gm. The critical measure of B9 was found in R. arboreum and the flowers of A. variegata contained second most astounding measure of B9 (3.11± 0.066 mg/100gm).

3.4 Antinutritional composition

The after-effects of the antinutrient piece of the palatable plants under scrutiny were introduced in Table 1.

Oxalate is an enemy of supplement and found in nature in certain plants as dissolvable and insoluble salts and as oxalic corrosive. It ties with supplements in the gastrointestinal tract, rendering them disconnected to the       body ³¹. The utilization of palatable plants with higher measures of oxalic acid may prompt the wholesome lacks. In present examination, among five plants, oxalate was most astounding in C. serratum (0.36±0.08 %) and least in B. roxburghii (0.14±0.04%). The oxalate levels in the examined plant are particularly practically identical with some regular foods grown from the ground like spinach (0.658%), almond (0.407%), amla (0.296%), amaranth (0.772%) ²¹. Oxalate is known to hinder renal calcium assimilation, particularly at centralizations of around 45 g/100 g ³². Nonetheless, the oxalate levels in the examined plants are far not exactly the worth that is thought to be destructive.

Phytic acid (myoinositol, 1, 2, 3, 4, 5, 6 hexakis-dihydrogen phosphate) and phytates are essentially found in vegetables and furthermore present in vegetables in low focus. It ties with minerals, for example, iron, zinc, calcium and magnesium and structure insoluble complex. It likewise structure edifices with proteins and      starch ³³. Phytic acid substance in wild edible plants went from 0.31±0.03% in A. variegata to 0.52±0.07% in    B. roxburghii and the levels are practically like rice (0.151%), bean (0.248) ²¹ and the qualities in this examination likewise fall inside the scope of 0.37–0.90 mg/g of fruits (guava, mango, orange, and pineapple) ³⁴ . The phytate levels got in our investigation were lower than 10-60 mg/g that has been accounted for to defeat the minerals bioavailability issues ³⁵.

Saponins are a class of synthetic mixes found in different plant species. It has a cleanser like frothing property when they are added to fluid and interfere with epithelial capacity and make other stomach related problems. It is additionally in charge of harming red platelets, hindering chemicals and meddling with thyroid capacity ³⁶. The amount of saponin in the plants contrast in extent with the species. The most astounding measure of saponin was seen both in the flowers of A. variegata and R. arboretum(0.12 %) while the leaves of B. roxburghii had the most minimal focus (0.032±0.008 %).

Tannin is a significant antinutritional figure exists the greater part of the vegetables. It is portrayed because of their harsh polyphenolic aggravates that predicament or structure accelerate with proteins and different other natural mixes, for example, alkaloids and amino acids. These tannins typically present in sustenance items which repress the enzymatic movement of amylase, lipase, trypsin and chymotrypsin. In this manner, decline the nature of protein and meddle with iron absorption ³³. The most noteworthy convergence of tannin was distinguished in the flowers of R. arboreum (1.96±0.12 %) and most minimal sum was seen in the flowers of    A. variegata (0.27±0.04 %) and these low sums may not bring on any hurtful impact on individuals.

Cyanogenic glycosides are auxiliary metabolites that are found in different plant tissues and produce HCN upon hydrolysis. They are broadly disseminated in the plant kingdom. The capacity of cyanogenic glycosides to discharge HCN is expected to their enzymatic hydrolysis which may cause cyanide harming. Accordingly, expulsion of cyanogenic glycosides is important to improve the dietary benefit and security of cyanogen containing nourishments³⁷. This examination uncovered that the cyanide substance of the explored plants go between 0.00097±0.00003 to 0.00216±0.0003 % and the centralization of cyanogenic glycosides in these plants were low and safe to devour as sustenance.

3.5 Toxicity studies

The after-effects of the toxicity investigations of edible plants including the suitability of cells and level of DNA harm utilizing buffer (negative control) and hydrogen peroxide (positive control) were exhibited in Table 2.

Haemolytic examines were performed on the grounds that plants indicated powerful nutraceutical properties and these may not be devoured on the off chance that they have haemolytic impact. Moreover, this information additionally may uncover some data about the system of cytotoxicity.

Table 2. Toxicity studies of wild edible plants collected from Meghalaya state in India

Name of the plant	Concentration of the extract (µg/ml)	Haemolytic toxicity RBC cell viability (%)	Hepatotoxicity Hepatocytes cell viability (%)	Genotoxicity (% tail DNA)
A.variegata	100	90.12±0.32	95.23±0.55	3.16±0.35
	200	89.28±0.44	94.06±0.38	3.92±0.49
	300	87.22±0.56	93.26±1.05	4.26±0.18
	500	86.78±0.45	92.15±1.02	4.82±0.57
	1000	84.25±0.33	90.11±0.68	5.01±0.25
F.virens	100	94.08±0.38	98.57±0.75	3.65±0.51
	200	92.33±0.56	97.11±1.08	4.06±0.30
	300	90.11±1.08	95.32±1.23	4.53±0.48
	500	87.32±1.01	93.04±0.69	4.98±0.19
	1000	85.48±0.55	92.47±0.33	5.48±0.33
B.roxburghii	100	95.32±1.01	97.34±1.18	2.95±0.18
	200	93.02±0.33	95.35±0.46	4.02±0.56
	300	90.14±1.28	93.48±0.58	4.68±0.29
	500	88.33±0.64	91.08±0.62	4.93±0.55
	1000	86.17±0.48	90.36±1.27	5.45±0.36
C. serratum	100	96.18±0.54	99.15±0.35	3.04±0.45
	200	95.22±0.34	98.56±1.05	4.14±0.55
	300	93.35±1.12	97.44±1.08	4.78±0.68
	500	90.18±0.43	96.78±1.06	5.12±0.34
	1000	87.49±0.55	95.03±1.14	5.48±0.78
R..arboreum	100	97.01±0.38	98.15±0.33	4.12±1.08
	200	95.15±0.46	97.32±0.78	5.32±1.14
	300	93.48±0.58	95.08±0.38	5.68±0.46
	500	90.11±1.06	94.68±1.03	5.96±1.11
	1000	88.26±0.55	93.09±0.88	6.08±0.55
Negative control	0	100.88±1.03	99.56±0.56	5.68±1.81
Positive control (H2O2)	50 µM	78.34±1.14	74.28±1.08	25.18±1.06
	100 µM	67.54±1.32	66.20±1.11	55.46±1.44
	200 µM	50.11±1.05	41.25±1.05	76.35±1.48

Each value in the table was obtained by calculating the average of three experiments and data are presented as Mean ± Standard error of the mean (SEM).

3.5.1 Haemolytic toxicity

In vitro haemolytic exercises on human erythrocytes of different fixations (100,200,300, 500 and1000 µg/ml) separates got from palatable pieces of wild plants under scrutiny were performed. The 51.75% haemolysis was gotten utilizing H₂O₂ (200µM) and 100.18% cell reasonability was seen with buffer (Figure 2). The haemolysis initiated by concentrates in red platelets was fixation subordinate yet all concentrates demonstrated lower haemolytic impact on human red platelet at all focuses. The feasibility of the haemolytic cell was most extreme (88.26±0.55%) at the most astounding portion of 1000µg/ml in the event of R. arboreum and the most reduced was seen in the aq. concentrate of A. variegata (84.25±0.33 %) at same fixation (Figure 2).

Figure 2. Haemolytic toxicity of Plant extracts ( 1000 µg/ml), negative control and positive control (H₂O₂ ; 200µM)

3.5.2 Cytotoxicity

Hepatocytes were segregated from fresh goat liver and the impacts of different focuses (100,200,300,500 and 1000 µg/ml) of aq. concentrates of eatable plants were seen on the reasonability of hepatocytes cell. The C. serratum demonstrated most elevated suitability (95.03±1.14%) of the hepatocytes cell at a greatest portion of 1000µg/ml and the least (90.11±0.68 %) was seen in A. variegata at same fixation (Figure 3). The rate practicality of RBC and hepatocytes cell for all plant extricates at all fixations (100-1000 µg/ml) were especially equivalent to the negative control though the rate reasonability of both RBC and hepatocytes cell utilizing H₂O₂ (Positive control) at a focus 200 µM were less than 50%.

Figure 3. Cytotoxicity of Plant extracts (1000 µg/ml), negative control and positive control (H₂O₂ ; 200µM)

3.5.3 Genotoxicity

The genotoxicity investigations of plants included the incubation of rat lymphocytes in a low-melting point agarose suspension alongside plant concentrate of various focus (100-1000µg/ml), lysis of the cells in neutral or alkaline (pH>13) conditions, and the electrophoresis of the suspended lysed cells. This was trailed by prompt visual investigation of the slides with staining under Fluorescence microscope and computing fluorescence to decide the degree of DNA harm. Olive Tail Moment (OTM) of individual stained nuclei was determined utilizing comet assay software. Negative control (entire blood and RPMI-1640) and positive controls (entire blood, 50, 100 and 200 μM H₂O₂ and RPMI-1640), were incorporated. A higher rate tail DNA demonstrated a more elevated amount of DNA harm and more elevated amount of genotoxicity of plant extract. The single cell gel electrophoresis examine (comet assay) is a cheap, basic, and quick technique for estimating DNA strand breaks and because of its affectability permits investigation at the individual cell level and the utilization of little examples ³⁸.

Figure 4.Genotoxicity of Plant extracts ( 1000 µg/ml), negative control and positive control (H₂O₂ ; 200µM)

The consequence of comet measure demonstrated that the aq. concentrate of R. arboreum at a focus 1000 µg/ml had the most elevated rate (6.08±0.55%) of tail DNA while the least rate (5.01±0.25 %) was found in A. variegata at same focus. The 5.68% of tail DNA was acquired utilizing negative control and positive control (mixture of whole blood, RPMI 1640 and 200 μM H₂O₂) indicated 76.35% of tail DNA (Figure 4). The arrangement of free radicals during natural digestion causes mutagenicity and genotoxicity. Because of  oxidative pressure hydrogen peroxide displayed portion subordinate DNA harm (25.18-76.35% of tail DNA) which was recognized by comet test. The consequence of examination uncovered that the degree of DNA harm brought about by the plant remove at various fixations were especially like the negative control. Regular mixes, particularly got from dietary sources give countless cancer prevention agents. Ongoing investigations in people have demonstrated that supplementation with cell reinforcement mixes, for example, vitamin E and C, lycopene and ß-carotene can help diminish levels of free-radical harm apply a defensive impact against degenerative issue, for example, malignant growth, by a reduction in DNA damage ³⁹. Plants have wide scope of pharmacologically compelling phytochemicals. A considerable lot of them have been accounted for supportive for the treatment of a few ailments of person, yet couple of phytochemicals like saponin, tannin, cyanogenic glycosides and so on produce destructive impacts after presentation and can go about as master oxidants, include most likely in charge of the mutagenicity and genotoxicity ⁴⁰.

4 CONCLUSION

The examination demonstrated that these wild edible plants gathered from Meghalaya state in India are wealthy in protein, fat, sugar, fiber and could give basic supplements required to keeping up typical body work. The wholesome properties of these plants were additionally all around thought about and furthermore once in a while superior to the normal vegetables. These vegetables were likewise discovered an essentially valuable wellspring of different minerals. The minerals, especially Na, K, Ca, Fe, Cu, Mg and Zn, were available in apparent amounts. The dangerous overwhelming metals Cd and Hg were not distinguished in the plant materials but rather Pb and Cr were recognized inside as far as possible as recommended by WHO and the nearness of the substantial metals in the plant probably won't be unsafe for humankind. RP-HPLC results demonstrated the plants contained a few water solvent B and C vitamins in fluctuating sums. The after-effect of examination of nutrient substance in the wild consumable plants under scrutiny will fill in as a helpful way to figure dietary admission of C and B vitamins in the overall public. The antinutritional examination demonstrated that every one of these plants contained oxalate, phytate, saponin, cyanogenic glycoside and tannin. Notwithstanding, values acquired for these plants are lower than the built up lethal level. Henceforth they can be devoured with no limitation. The consequences of haemolytic poisonous quality, cytotoxicity and genotoxicity of fluid concentrates of every single eatable plant uncovered that these are non-harmful at cell and genomic level and furthermore safe to expend. So we accept that these plants could be utilized for the healthful reason for person because of their great nourishing characteristics, and sufficient security might be gotten against maladies emerging from lack of healthy sustenance. The trial discoveries additionally uncovered that these wild eatable plants were the great wellspring of supplement for innate populace. So the development of these wild eatable species should be embraced in enormous scale, which will deliver financial advantages for poor ranchers.

5 ACKNOWLEDGEMENTS

We are highly grateful to Dr. A. A. Mao, Director, Botanical Survey of India (BSI), Kolkata,   for their encouragement and facilities. We are also thankful to Dr. R. Gogoi, SHRC, Gangtok, BSI, Howrah and Mr. R Shanpru, Scientist, BSI, Eastern Regional circle, Shillong, Meghalaya for identifying the plant specimens.

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Received on 30.06.2020         Modified on 10.07.2020

Accepted on 16.07.2020 ©AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2020; 12(3):123-134.

DOI: 10.5958/0975-4385.2020.00031.X