Studies on the antioxidants and antinutriens content of leafy vegetables cultivated at Dhapa, a Municipal Solid waste landfilling site in Kolkata, West Bengal, India

: The impact of Municipal Solid Waste (MSW) landfilling on vegetation in terms of nutritional quality is still under investigation. The main objectives of this study are to assess the antinutrients and antioxidants content of the leafy vegetables grown on Dhapa landfilled ground (DLG) and also to compare these nutritional qualities with the same leafy vegetables collected from other than landfilled ground i.e. Normal ground (NG). From the analysis it was observed that both antinutrients and antioxidants content of the leafy vegetables vary significantly (P<0.01) in between places except oxalate and tannin content (P>0.05). Furthermore, Phytate content, Antioxidant contents (Total Phenol, Flavonoid & Ascorbic acid) as well as Total Antioxidant Activity (reflected by FRAP Assay & DPPH Radical Scavenging Assay) of the samples of DLG were found to be significantly higher (P<0.001) than the samples of NG.

degenerative diseases. On the other hand Antinutrients are the compounds that may hinder the bioavailability of some most essential nutrients in the living system. But the reactivity of antinutrients with the bio-molecules such as polysaccharides, proteins, and metal ions has important nutritional and physiological consequences (Schofield et al., 2001). Nutritional quality of the vegetables entirely depends on the composition of soil used for vegetation. As Municipal solid wastes (MSW) of the city of Kolkata are generally dumped at Dhapa sites it is quite obvious that it has some effects on the quality of vegetables grown over there. In this context the present study gives detail reports on the effect of solid waste on Antioxidants and Antinutrients content of the leafy vegetables cultivated in Dhapa, Kolkata, West Bengal.  Table 1. ii.For Antioxidant Assay:-Cleaned vegetables, dried with paper towel was made into a paste and appropriate amount of each paste sample was extracted with -• 6 % metaphosphoric acid for vitamin C estimation (Joseph et al., 1944) and • 80% methanol and left it overnight, then centrifuged at 10,000 rpm for 15 min and the supernatants were decanted into polypropylene tubes and filtered hrough Whatman No.1 filter paper. The clear extracts were analyzed both for determination of phenolic contents and antioxidant activity (Zhang and Hamauzu, 2004) .

ANALYSIS: (A)
Antinutrients content : • Determination of Oxalate content: Briefly, the determination was as previously described by Baker, 1952 with some modifications. The sample extract was weighed after taking in a beaker and boiled for 15 mins. This was then adjusted to previous volume with distilled water and volume was made upto 100ml with 2N HCl. The mixture was shaken well and filtered (whatman 40). To the 25 ml of filterate , 5 ml of phosphoric tungstate reagentwas added & mixed well & once or twkept overnight . Next day the mixture was centrifuged for 10 minutes at 3000 rpm and filtered (whatman 40) . 20 ml of clear solution was then taken to a 50 ml centrifuge tube and 2-3 drops of methyl red was added & neutralized with ammonia. Then 5 ml of Calcium chloride reagentwas added to it and stirred with a fine glass rod and kept tube overnight in a refrigerator at 5 -7 0 C. Next day the mixture was again centrifuged for 10 minutes at 3000 rpm and filtered (whatman 40) .The ppt was then dissolved in distilled water followed by 5 ml of 2N sulphuric acid. Then the mixture was placed in a water bath over 80 0 C for 2 minutes and titrated the oxalic acid with N/100 potassium permanganate solution to a faint pink colour which persisted for about 30 s after which the burette reading was taken. The oxalate content was evaluated from the titre value.
The overall redox reaction is: 2MnO4 + 5C2 O 4 2 +16H + 2Mn 2 + + 8H2O + 10CO2 • Determination of Phytate content: Total phytate content of the extract was determined according to the supernatant difference method of Thompson & Erdman (1982).Sample extract (10 ml) was mixed with 4 ml FeCl3 solution and heated in a boiling water bath for 45 mins. If the supernatant is no clear after 30 mins, add one or two drops of 3% Sodium sulphate in 3% TCA and continue heating.It was then Centrifuged for 10 -15 mins and carefully decant the clear supernatant. After that precipitate was washed twice by dispensing well in 20 -25ml of 3% TCA and heated in boiling water bath for 5 -10 mins and centrifuged. The process of washing with water repeated. The precipitate was then dispersed in 27ml of water and 3 ml of 1.5N NAOH with mixing and the volume was made upto approximately 30ml with water and allowed to heat in boiling water bath for 30mins. It was then filtered hot through a moderately retentive paper Whatman No.2. The precipitate was washed with 6 -70ml hot water and the filtrate was discarded. Then the precipitate from the paper was dissolved with 40ml 3.2N HNO3 into a 100ml volumetric flask and the volume was made upto the mark. 5ml aliquot was transferred to another 100ml volumetric flask and diluted to approximately 70ml. 20ml of 1.5M KSCN was added to it and diluted to the volume. Finally read at 480nm of wavelength. Standard curve was prepared by using Fe(NO)3. The (µg) iron present in the standard curve was found out and phytate P was calculated as per the equation. Phytate P present in the sample = µg of Fe x 15 / Weight of the sample (g) • Determination of Tannin content: Tannin content in the plant extract ( extracted in boiled water) was determined as described by (Schanderi, 1970) , using tannic acid as the standard. The extract solution (1 ml) was mixed with the Folin denis reagent (5ml) and super saturated solution of Na2CO3(10 ml) and volume made upto 100ml by distilled water. After 30 minutes of incubation at room temperature, the absorbance of the reaction compound at 700 nm was measured spectrophotometer (Perkin Elmer, Lambda 25, UV/VIS Spectrophotometer) The overall tannin content was expressed as mg of tannic acid equivalents / gm dry weight .

(B)
Antioxidants content: • Determination of Total Phenols : Total phenol content in the plant extract ( extracted in 80% methanol, kept overnight) was determined as described by Singlaton and Rossi (1965) , using gallic acid as the standard. The extract solution in 80% methanol (1 ml, 50 mg ml-1) was mixed with the FC reagent (10%, 1 ml) and an aqueous solution of Na2CO3(7.5% , 0.8 ml) and volume made upto 10ml by distilled water. After 30 minutes of incubation at • Email: editor@ijfmr.com

IJFMR22063893
Volume 4, Issue 6, November-December 2022 5 room temperature, the absorbance of the reaction compound at 765 nm was measured spectrophotometer (Perkin Elmer, Lambda 25, UV/VIS Spectrophotometer) The overall phenol content was expressed as mg of gallic acid equivalents (GAE)/ gm dry weight.
• Estimation of Total Flavonoids: Total flavonoid content of the extract was determined according to a modified colorimetric method of Bao , Cay et al. (2005). Sample extract (1 ml, 50 mg ml-1) was mixed with 0.15ml of a 5% NaNO2 solution. After 6 minutes , 0.15ml of 10% AlCl3.H2O solution was added. After 6 minutes, 2 ml of 4% Sodium hydroxide was added and volume made upto 10ml by distilled water. The solution was mixed well and kept for 15 min. The increase in absorbance was measured at 510 nm using a UV-Visible spectrophotometer(Perkin Elmer, Lambda 25, UV/VIS Spectrophotometer) The total flavonoid content was calculated using standard quercetin calibration curve. The results were expressed as micrograms of quercetin equivalents (QE) per gram dry weight of the sample.

•
Estimation of Ascorbic Acids: Ascorbic acid content in the plant extract was determined as described by Joseph et al., (1944) using ascorbic acid as the standard (1mg/ml). The extract solution (2 ml) was mixed with equal amount of acetate buffer (ph 4.0) and dye (sodium salt of 2,6 dichlorophenol indophenol)solution in the separating funnel. The content was mixed well and 10ml xylene was added . This was then mixed well and allowed to stand for 6 seconds for separating the layers and then the water layer was removed and the colour in xylene was measured in a spectrophotometer (Perkin Elmer, Lambda 25, UV/VIS Spectrophotometer) at 500 nm. The ascorbic acid content was calculated as.. Determination of free radical scavenging using DPPH method: Antioxidant activity was determined by the 2,2,-di-phenyl-2-picryl-hydrazyl (DPPH) method of Zhang and Hamauzu (2004) with some modifications. The concentration of the methanol (80%) extracts of fresh vegetable was adjusted to 10 mg/ml (on dry basis), which was chosen as an appropriate concentration for assessing antioxidant activity after preliminary studies of the different concentrations. An aliquot of 2 ml of 0.1 mM DPPH radical in methanol was added to a test tube with 0.1 ml of vegetable extract, at 10 mg/ml volume made upto 4ml by methanol (80%). Instead of methanolic extract of vegetables, pure methanol was used as control. The reaction mixture was vortex mixed and let to stand at room temperature in the dark for 30 min before the decrease in absorbance at 517 nm was measured at spectrophotometer (Perkin Elmer, Lambda 25, UV/VIS Spectrophotometer    Table 3    • Phytate Phytate content shows significant differences between places and found to be significantly higher in all samples of DLG (p<0.001). They also vary significantly between samples (p<0.001). Significantly highest amount of phytate was found in Ceylon Spinach and lowest in Bottle Gourd Leaves irrespective of the locations (p<0.005).
• Oxalate Vegetables of DLG & NG does not differ significantly (P>0.05) in terms of Oxalate content, but vary significantly between samples (p<0.001) except Green Amaranth -Ceylon Spinach (P= 0.032). Significantly highest amount of oxalate was found in Spinach and lowest in Bottle Gourd Leaves irrespective of the locations (p<0.005).
• Tannin Tannin content shows no significant differences (P > 0.05) between places, but they vary significantly from sample to sample (P<0.001). Significantly highest amount of tannin was found in Green Amaranth and lowest in Spinach irrespective of the locations (p<0.005). So from the present study it can be stated that garbage farming not at all affect the antinutrients level of the vegetables produced over there except Phytate. Furthermore, it may be mentioned that after extensive literature survey, no documented data regarding antinutrients content of vegetables grown on DLG are available by which the present data could be compared.

TABLE 3 & 3.1 : ANTIOXIDANTS CONTENT •
Ascorbic acid Data reveals that the Ascorbic acid content of samples of DLG were found to be significantly higher as compared with the samples of NG (P<0.01) and also vary significantly between samples (P<0.001). Significantly highest amount of Ascorbic acid was found in Red Amaranth and lowest in Spinach irrespective of the locations (p<0.005).