Collection of plant sample
The leaves of Cleome viscose were obtained from the local market in Uttarakhand. The plants were authenticated by Dr. Anamika, Department of Botany, Vardhman college Bijnor, The Department of Botany identified plant material. The leaves were cleaned and dried in the shade for two to four weeks. The powdered dried leaves were kept in an airtight container. In a nutshell, 5 g powder was sequentially Soxhlet extracted for 6-7 hours, with 3-4 cycles each hour with methanol solvent. After roughly every four cycles, the extraction was verified by taking some extract from the extractor syphon tube, spotting it on a TLC plate, and seeing it in an iodine chamber. The absence of a spot signifies that extraction has been completed.
Phytochemical analysis of extract:
Plants were qualitatively tested for phytoconstituents. Alkaloid , flavonoid , Phenolic compounds , tannins , cardiac glycosides , steroids (Liebermann-Burchard reaction) , saponins , and carbohydrate tests: molisch, fehling's, and benedict's tests were performed on extracts.
Total Flavonoid content:
The total flavonoid was estimated using the aluminium chloride colorimetric technique . In a nutshell, 0.5 ml of 1.2 % aluminium chloride and 0.5 ml 1M potassium acetate were added to 0.1 ml of leaf extract. Methanol was used to dilute the reaction mixture to 3 ml, and it was incubated at room temperature for 30 minutes. The absorbance was measured at 415 nm. Blank was set by adding all reagents except the extracts. Rutin standards ranging from 0.1 mg/ml to 1 mg/ml were used to create a standard curve.
Total Phenolic content:
TPC was estimated by modified protocol of Singleton et al., 1999 . Gallic acid (GA) was used as the standard, while pure water was used as the blank sample. Briefly 0.5 mL distilled water, 2.5 mL Folin-Ciocalteu reagent, and 0.05 mL extract were mixed together. For 5 minutes, the solution was allowed to react. After that, 2 mL of a 7 % Na2CO3 solution was added, the mixture was stirred, and the volumetric flask was filled to the necessary capacity with distilled water. After 90 minutes of incubation in the dark and at room temperature (23 °C), the solution absorbance was measured at 765 nm with a spectrophotometer. Gallic acid standards ranging from 0.01 mg/ml to 0.05 mg/ml were used to create a standard curve.
Antioxidant assay of crude extract (ABTS):
The ABTS test was carried out in accordance with Pellegrini et al., 1958 . In brief, 7mM ABTS in water was combined with 2.45mM potassium persulphate (1:1) and incubated in the dark for 12-16 hours. After that, the mixture is diluted to achieve an absorbance of 0.7 at 734 nm. Extract was diluted in different concentrations i.e. 0.1875, 0.3125, 0.4375 and 0.5625 mg/ml and 3 ml of reagent were mixed together and incubated in the dark for 10 minutes. The absorbance was measured at 734 nm. The control was made by measuring the absorbance of ABTS and methanol. Ascorbic acid standard curve was constructed concurrently with standards dilution ranges of 0.1875, 0.3125, 0.4375 and 0.5625 mg/ml. The ABTS+ scavenging effect (percent) was estimated using the equation below.
Ab=Ab. Of ABTS+ Methanol
Aa= ABTS+ sample/standard
CEAC (Vitamin C Equivalents Antioxidant Capacity) or ascorbic acid content of all the extract was estimated by standard curve of ABTS+ scavenging effect (%) linear curve equation.
Antioxidant assay of crude extract (DPPH):
The DPPH free radical scavenging activity of the crude extract, based on the scavenging of the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical was determined . Samples were diluted to different concentration i.e. 0.1875, 0.3125, 0.4375 and 0.5625 mg/ml. Reaction mixture were set by adding 1.0 ml of 0.1 mM of DPPH in each dilution. The mixture was incubated in the dark for 30 min at room temperature. Degree of inhibition of DPPH by monitoring the decrease in absorbance measured at 517 nm. Ascorbic acid was used as positive control. Standard curve of ascorbic acid was prepared by diluting the ascorbic acid in same concentrations as that of samples. Radical scavenging activity was expressed as inhibition percentage of free radical by the sample and was calculated using the following formula:
where Ao was the absorbance of control (blank without sample) and at was the absorbance in presence of sample. All the tests were performed in triplicate and graph was plotted with mean values.
Antioxidant assay of crude extract (FRAP assay):
The FRAP test was carried out basically as previously described . The samples were diluted to a concentration of 1 mg/ml. An aliquot (100 µl) of the correctly diluted extract was added to 3 ml of the standard reaction solution, and absorbance at 593 nm was measured at 0 time and after 6 minutes of standing at room temperature. The measurement was made three times. The standard curve in the 200-1000 M range was created using FeSO4. The FRAP values for both the standard (ascorbic acid 1 mg/ml)) and the samples were computed and represented as µM Fe [II]/gm dry wt.
Antioxidant assay of crude extract (H2O2 assay):
H2O2 scavenging activity was determined as previously described . A 0.6 ml aliquot of 40 mM H2O2 solution was combined with 0.1 ml of diluted crude extract. 2.4 ml of phosphate buffer (0.1 M, pH 7.4) was added to the mixture, which was rapidly agitated and incubated at room temperature for 10 minutes. The absorbance of the reaction mixture was then measured at 230 nm. Positive control was ascorbic acid. The scavenging activity of H2O2 was computed as follows:
Where A1 is the ascorbic acid absorbance and A2 is the sample absorbance.
Antimicrobial activity of Crude extract:
The bacteria Salmonella Typhi Chi–603 (MTCC 3224), Vibrio cholera 569B (Classical 01) (MTCC 3904) were procured from MTCC, IMTECH, chandigarh while Staphylococcus aureus (MCC 2408) was purchased from NCCS, NCMR, Pune. The antibacterial activity assay was performed using disc diffusion techniques (CLSI 2012 standard).
Plant extracts were loaded on filter paper discs of 6 mm diameter with different dilutions (200 mg/ml, 100 mg/ml, 75 mg/ml, and 50 mg/ml) and placed on plates with MHA for bacterial strains with 2x108 microorganisms per plate. Gentamycin was diluted at 2, 5, 7, and 10 µg/ml concentrations and loaded in the same manner as the sample. Bacterial incubation periods at 37°C and 32°C ranged from 16 to 24 hours. The zone of inhibitions (zones surrounding the discs) was measured to determine antimicrobial activity and minimum inhibitory concentration (MIC). Gentamycin served as the positive control, while dimethyl sulfoxide served as the negative control .
Data were presented as mean SD for at least three separate measurements in triplicate for each experimental point. The IC50 values in antioxidant experiments were calculated using the GraphPad Prism version 8.0 software.