2.1. List of chemicals and reagents 2,2-diphenyl-1-picrylhydrazyl (DPPH), DAPI fluorescent dye and 0.4% trypan blue dye (Sigma chemical company, USA), Ethanol and Methanol (Sigma chemical company, USA), Ascorbic acid (Merck, Germany), De-ionized water, Sodium chloride (NaCl), Mayer’s reagent, Hydrochloric acid (1.5 %v/v), Wagner’s reagent, Chloroform, Concentrated Sulphuric acid, Fehling’s solution, 5% FeCl3 solution, Ninhydrin solution, Hemagglutination or PBS buffer (20 Mm Tris-HCl buffer, 1% NaCl, 10 mM CaCl2, pH 7.8), Artemia salina leach (Brine eggs), Sea salt (NaCl) (Roth Chemicals, India).
2.2. Collection of plant samples Fresh P. acidus (L.) fruits were collected from the germplasm bank of Bangladesh Agricultural Research Institute (BARI), Rajshahi, Bangladesh in October 2017 during fruiting. The plant part was kindly provided by Dr. Alim Uddin, Chief Scientific Officer of BARI. Afterward, fruit samples were authenticated by the taxonomist of the Department of Botany, University of Rajshahi, Bangladesh.
2.3. Preparation of crude methanol extract The crude methanol extract of desired samples was prepared following the method described by Olayaki et al. (2015) with slight modification. At first, collected fruit samples were sterilized with distilled water and then fruit samples (flesh) were cut into small pieces. Then seeds were isolated from flesh and both samples were dried in the drier at 30 C for 7 days. After that dried samples were grinded to make a coarse powder and preserved at room temperature. 100 g of flesh powder and 20 g of seed powder was then extracted with 100% methanol and shacked at 140 rpm for 2 h separately. Then the shacked samples were sonicated by using a sonicator and shaken overnight at 140 rpm. Again the samples were filtered by vacuum filter and then the filtrates were evaporated by using a rotary evaporator at 35 C temperature. Finally, dark brownish-green semisolid extracts in both cases were obtained and stored at 20 C for further experiments.
2.4. Determination of antioxidant activity a) DPPH free radical scavenging assay DPPH was used to evaluate the free radical scavenging activity of crude methanol extract of P. acidus (MEPA) fruit pulp and seed following DPPH assay based on the method described by Parejo et al. (2000) with a slight modification. At first, the stock solution of DPPH was prepared by dissolving 24 mg of DPPH into 100 ml methanol and stored at 20? until use. Then, 1 mg of MEPA fruit pulp and seed was dissolved into 1 ml distilled water separately to prepare the dose for treatment. Afterward, MEPA fruit pulp, seed, and Ascorbic acid (Aa) mediated stock solutions were taken in different test tubes at different concentrations ranging from 12.5 to 200 mg/ml. After that 1.5 ml of DPPH solution was added to each test tube. Then the reaction mixture of each test tube was shaken vigorously and incubated at room temperature for 30 min in a dark place to complete the reaction. After incubation, the absorbance of decolorized DPPH was measured at 517 nm using a spectrophotometer against a blank solution (Methanol). A typical standard solution containing all reagents except desired plant extract or Ascorbic acid was considered as control. The percentage (%) of inhibition activity was calculated from the following equation; % I = {(A0 – A1)/A0} 100
Where A0 is the absorbance of the control (without extract) and A1 is the absorbance of extract. Then the scavenging percentages of DPPH free radical for different concentrations were plotted against concentration and finally, the IC50 value was calculated from the equation of the graph.
b) Determination of nitric oxide (NO) scavenging assay
Nitric oxide free radical scavenging activity of methanol extract of P. acidus fruit pulp and seed was determined according to the method described by Marcocci et al. (1994) with some modifications. A volume of 2 ml of 10 mM sodium nitroprusside dissolved in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of desired fruit pulp and seed extract at various concentrations in different test tubes. The reaction mixture was then incubated at 25 C. After 150 min of incubation, the incubated mixtures were then mixed with 0.5 ml of Griess reagent (0.33% sulfanilic acid and 0.1% NED in 20% glacial acetic acid). After that, the mixture was again incubated at room temperature for 30 min and then the absorbance of each reaction mixture was measured at 550 nm through transferring into a cuvette. Ascorbic acid was used as a reference standard. The amount of nitric oxide free radical inhibition was calculated using the following equation: Percentage (%) of inhibition of NO free radical = [{A0 – A}/ A0] 100 Where A0 is the absorbance of blank control (NO radical solution without test sample) and A is the absorbance of the test sample. Finally, the percentage (%) of inhibition was plotted.
2.5. Determination of cytotoxicity through Brine Shrimp lethality bioassay Cytotoxic activity was done by Brine Shrimp lethality bioassay following the method of Islam et al. (2018a), Meyer et al. (1982) with Artemia salina nauplii. For this experiment, at first Brine Shrimps were hatched in a 1L beaker filled with NaCl solution at the concentration of 38 g/L. Then 10 test tubes were taken and filled with 25 ml NaCl solution in each test tube. After that 20 nauplii were taken in each test tube with the help of a micropipette and then crude MEPA fruit pulp and seed were added into the test tubes at different doses. Finally, the test tubes were incubated at room temperature for 24 h and the 50% lethal concentration (LC50) value was estimated by using a regression line obtained from the plotting of mortality percentages against different concentrations.
2.6. Determination of phytoconstituents Preliminary phytochemical analysis of crude MEPA fruit pulp and seed were conducted based on the following standard protocols described by Miah et al. (2020), Bhandari et al. (2017), and Senguttuvan et al. (2014) to detect the presence of phytoconstituents including alkaloids, phenols, flavonoids, steroids, saponins, tannins, glycosides, terpenoids, proteins, and carbohydrates in studied materials.
2.7. Determination of lectin protein activity Haemagglutination is a specific form of agglutination which is involved to agglutinate the red blood cells (RBCs) in the presence of lectin proteins. The presence of lectin activity of crude MEPA fruit pulp and seed was estimated following hemagglutination assay described by the method of Islam et al. (2018a). At first, 1 mg crude extract from both samples was weighted, and then working extracts were prepared through centrifugation at 8000 rpm for 15 min. Again, 2% suspension (w/v) of RBCs was prepared with hemagglutination buffer through the centrifugation of collected mouse blood. After that hemagglutination reaction was performed in 96-well microtiter U-bottomed plates described by Hasan et al. (2019) with a slight modification. For performing this reaction, first of all, 50 ml of hemagglutination buffer was taken in every well of titer-plate, and then 50 ml prepared extracts of both samples were added to the first well of titer plate and serially diluted into the successive wells with PBS buffer (pH 7.4). Finally, an aliquot of 50 ml of 2% RBCs suspension was added to each well. PBS buffer along with 2% RBCs suspension without the extract was considered as control. Then this titer plate was shaken by a micro shake at 300 rpm for 20 min and after shaking placed on a table under room temperature. After 15–20 min, the agglutination of blood erythrocytes was observed and agglutination activity was assessed based on the rough granules within the titer plate.