5.1 Preparation of extracts:M. paniculata leaves were collected from the Amarkantak region, District Anuppur, Madhya Pradesh (India). The leaves were washed under tap water, shade dried and finally grinded it into fine powder form. The extraction was carried out using different solvents (hexane, acetone, chloroform, methanol and water) for about 7-8 hours using Soxhlet extractor and concentrated using rotary evaporator. The extract was dried and stored in vials at 40C till future use.
5.2 Phytochemical screening
The phytochemical screening of all extracts was carried out according to standard methods as mentioned elsewhere with minor modifications .
5.3 Total phenolic content
The total phenolic content was determined for different M. paniculata extracts using spectrophotometric method . Different extracts were taken at concentration 1 mg/mL for analysis. The reaction mixture was prepared (in triplicate) by mixing 0.5 mL of individual extracts, 2.5 mL of 10% Folin-Ciocalteu’s reagent dissolved in water and 2.5 mL 7.5% NaHCO3. The samples were incubated for 45mins at 450C and the absorbance was measured at 765 nm using spectrophotometer (Shimadzu UV-Vis spectrophotometer-1800). Based on the measured absorbance, the total phenolic content in the test samples was calculated using the calibration plot (Y= 0.00023x + 0.0380, R2= 0.998) and expressed in terms of Gallic acid equivalent (mg of GA/g of extract).
5.4 Total flavonoids content:
The total flavonoid content was performed as per Dowd method with some modifications for the different extracts . Different extracts were taken at 1 mg/mL concentration for analysis and reaction mixture prepared by mixing 0.5 mL of extracts, 10% aluminium chloride (0.1mL), 1M potassium acetate (0.1mL) and distilled water (4.3 mL). Reaction mixture was incubated at room temperature for 30 mins and absorbance was measured at 510 nm .Quercetin (1mg/mL) was used for preparing the standard calibration curve. The total flavonoid content in the test samples was calculated using the calibration plot (𝑌 = 0.004𝑥 + 0.158, 𝑅2= 0.997) and expressed as mg quercetin equivalent (QE)/g of dried plant material.
5.5 Anti oxidant assays
5.5.1 ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay: ABTS is a synthetic radical widely used for both the polar and non-polar samples . Mixture of ABTS (14mM) and potassium per sulfate (4.95mM) (1:1 v/v) was prepared and allowed to stand overnight at room temperature (RT) in dark. ABTS+ was diluted with water to obtain equilibration of absorbance 0.70 (±0.02) at 734nm and suitable blank was used without test adding samples. 50mg/mL stock solutions of plant extracts were prepared and different concentrations used for assay. The following formula was used to calculate percentage of inhibition.
5.5.2 Hydrogen peroxide (H2O2) radical scavenging assay: Hydrogen peroxide radical scavenging activity was determined according to the method of Ruch et al (1989) . 40mM H2O2 solution was prepared in 50mM phosphate buffer (pH 7.4). 3.4mL of different concentrations of extracts (1000, 2000, 3000, 4000, and 5000 μg/mL) were added to 0.6mL of H2O2 solution. The absorbance was read at 230 nm after 10 minutes of incubation at RT against a blank solution containing only phosphate buffer without H2O2.
5.5.3 Ferric Reducing Antioxidant Power (FRAP) assay: The total antioxidant potency of extracts of M. paniculata was estimated using a ferric reducing antioxidant power (FRAP) assay . FRAP reagent (straw yellow color) was prepared by mixing 30mM acetate buffer, 10mM TPTZ, 20mM FeCl3 and distilled water. The standard curve of ferrous sulfate (FeSO4.7H2O) was prepared with concentrations ranging from 0.1, 0.2, 0.4, 0.4, 0.6, 0.8 and 1.0mM/L. Different concentrations of ascorbic acid and sample (1000, 2000, 3000, 4000, and 5000 μg/mL) were prepared from 1mg/mL stock solution and 3mL of FRAP reagent added to each and the tubes were incubated for 30 minutes in the dark, read absorbance at 593nm.
5.5.4 Reducing power assay (RPA): The total reducing power of extracts was estimated by the method of Oyaizu et al (1986) . The different concentrations of plant extracts (1mL of 1000, 2000, 3000, 4000, and 5000 μg/mL) were mixed with 5mL of 0.2M phosphate buffer (pH-6.6), and 5mL of 1% ferricyanide was added and incubated for 20 minutes at 500C. After incubation the 10% trichloro acetic acid (TCA) added and centrifuged at 3000 rpm for 10 minutes. To the supernatant equal amount of water and 1mL of 1% ferric chloride were added, absorbance read at 700nm. Higher the absorbance value of the reaction mixture indicated better reducing power
5.6 Membrane stabilizing property
The human red blood cell (HRBC) membrane stabilization method is mainly used to estimate the anti-inflammatory activity of plant extracts . The fresh blood was collected and mixed with an equal volume of sterilized Alsever’s solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% sodium chloride in water). (Administration of NSAIDS for 2 weeks before collecting the blood was avoided). The collected blood was further centrifuged at 3000 rpm for 10 min, and the pellet (packed cells) were washed thrice with isosaline (0.85%, pH 7.2) and finally 10% (v/v) suspension was made with isosaline. To the different concentrations of plant extracts, 1 mL phosphate buffer (0.15 M, pH 7.4), 2 mL hyposaline (0.36%), and 0.5mL HRBC suspension was added. Standard and control were prepared without addition of the extracts. Indomethacin at different concentrations (1000, 2000, 3000, 4000, and 5000 μg/mL) was used as the standard drug and compared with respective concentrations of plant extract. The reaction mixtures were incubated at 370C for 30 min and centrifuged at 3000 rpm for 10 min. The hemoglobin content in the supernatant was estimated at 560 nm .
The percentage hemolysis was calculated using the following equation:
The percentage of HRBC membrane stabilization was calculated using the following equation:
Crude extracts were evaluated for their antimicrobial properties against selected bacterial (Escherichia coli MTCC 1575, Staphylococcus aureus MTCC 1144, Bacillus subtilis MTCC 2413, Pseudomonas aeruginosa MTCC 1688) and fungal (Candida albicans MTCC 854, Peniccillin chrysogenum MTCC 1996, Aspergillus niger MTCC 872 and A. flavus MTCC 1883) strains. Standard drugs Amphotericin and Ciprofloxacin were used as positive controls.
5.7.1 Agar diffusion method
The stock cultures of bacteria were revived by inoculating in broth media and grown at 37ºC for 18 hrs. The agar plates were prepared, after solidification the 100 μl (10-4 cfu) 18 h old culture inoculated and evenly spread. After 20 min, the wells were filled with test compounds at different concentrations (1000, 500, 250, 100, 50, 25 µg/mL) kept for incubation at 37ºC for of 24 h. The diameter of inhibition zones measured and noted as described earlier . Minimum inhibitory concentration (MIC) was calculated using the same plate.
5.8 Column chromatography of hexane extract
The most active crude extract (hexane extract) was allowed to separate using gravitation column chromatography. The slurry was prepared by mixing 500g of absorbent (silica gel 60-120 mesh size) in n-hexane and stirred well to remove bubbles then poured in to glass column. The sample to be loaded on column was prepared by dissolving 5g of extractin 25ml of hexane and 20g of silica. In Table S3, we review the ratio of solvents and fractions obtained in the column chromatography. Each fraction was spotted on activated TLC plate along with extract spot and mobile phase [Hexane: acetone (3:1)] used. The fractions showing more than one spots were concentrated and allowed for further purification using only different solvent mixtures (Hexane and Acetone). The column chromatography was repeated as the eluent system to obtain a single spots on TLC.
5.9 Antibacterial activity
The different fractions collected from the hexane extract were further evaluated for antibacterial activity as mentioned earlier and the zones of inhibition were recorded.
5.10 GC-MSMS analysis
The GC-MSMS analysis of PC4 fraction of M.paniculata hexane extract was carried out on Agilent technologies model 7890A GC coupled with a mass detector 5975C MS system. The Analytic column was Agilent J&W nonpolar column DB-5MS ((5%-Phenyl)-methylpolysiloxane, 30 m × 0.25mm, ID 1.8 micron thickness). Flow rate of helium gas is 1.3ml/min, used to separate components. The different GC conditions were standardized as follows; injector parameters were injection volume 1 μL under split of 3:1, while injector temperature was set at 280 °C (mass analyzer). During GC extraction the program of oven temperature was 1 min at 75°C, increased to a temperature of 300 °C at a rate of 30 °C/min for 2 mins; inlet and transfer line temperature was 250 °C and 290o C respectively. Mass spectra were taken at an ionization mode with an electron impact at 70 eV. Interpretation of mass spectrum GCMSMS analysis was done by matching list of known compound’s spectrum with Agilent’s GC/MS Chemstation, NIST MS Library and NIST’s Automated Mass Spectral Deconvolution and Identification software .
6 Statistical analysis
All datawere expressed as mean ± SD of triplicates. Comparison of mean values between various extracts of M. paniculata were performed by one way-ANOVA, correlation coefficient (r) and coefficient of determination (r2) calculated using prism 8.0.1(244). The p<0.001 was considered to be statistically significant.