*2.3. Extraction of Plant Materials*

#### 2.3.1. Preparation of Aqueous Ethanolic Extract

We extracted dried powdered plant material using aqueous ethanolic solvent (50%, for 6 h at 37 ◦C), then pooled the hydroalcoholic extract, and it dried under reduced pressure at 40 ◦C with a rotator evaporator. For hydroalcoholic extracts, the percentage yields of the crude extracts were 15.58% *w*/*w*.

#### 2.3.2. Standardization of Polyphenols in *P. maderaspatensis* by HPTLC

Standard solutions of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol were applied in triplicate on silica gel 60 F254 plates, using a CAMMAG Linomat-5 Automatic Sample Spotter. The plates were developed in a solvent system, toluene/ethyl acetate/formic acid/methanol (3:3:0.8:0.2 *v*/*v*) in a CAMAG glass twin trough chamber (20 × 10 cm) up to a distance of 8 cm, dried in the air, and scanned at 254 nm. The developed plates were dried in the air and scanned at 254 nm using CAMAG TLC scanner 3 and win CATS 4 software. The peak areas were recorded. Calibration curves of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol were obtained by plotting peak areas versus applied crude extracts, and the concentration of ellagic acid, gallic acid, catechin, quercetin rutin, and kaempferol was calculated, respectively.

#### 2.3.3. Estimation of Different Markers

Each sample solution (10 μL) was applied in triplicate on silica gel 60 F254 plates with CAMAG Linomat-5 Automatic Sample Spotter and the peak areas and absorption spectra were recorded. We calculated the number of bioactive compounds in *P. maderaspatensis* using the peak areas and the absorption spectra were recorded. The number of bioactive compounds in *P. maderaspatensis* was calculated using the respective standard calibration curves of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol.

#### 2.3.4. Fractionation of Potent Aqueous Ethanolic (50% *v*/*v*) Extracts of *P. maderaspatensis*

The aqueous ethanolic extract of *P. maderaspatensis* was dissolved in 10% distilled water and was successively fractionated thrice with hexane (3 × 600 mL), chloroform (3 × 600 mL), ethyl acetate (3 × 500 mL), and water-soluble fractions. The combined fractions of *P. maderaspatensis* were evaporated to dryness under reduced pressure at 40 ◦C in a rotary evaporator.

#### 2.3.5. Nitric Oxide Estimation of Various Fractions of *P. maderaspatensis*

Nitric oxide was measured as nitrite released from mouse macrophage cells, RAW 264.7. Cells were suspended in DMEM (Sigma) supplemented with 10% FCS, seeded in a

96-well culture dish at a density of 1 × 106 cells/well, and incubated for 48 h at 37 ◦C in an atmospheric of 5% CO2 and 95% humidity. After incubation, 100 μL of the medium was transferred from the surface of the cultures of each well and replenished with the same amount of fresh medium. Further incubation for 24 h was done with concentrations of 25, 50, and 100 μg mL−<sup>1</sup> of alcoholic and aqueous-alcoholic (50% *v*/*v*) extracts/metabolites (100 and 200 μg mL−1) of *P. maderaspatensis* in the presence or absence of the indicated amount of LPS (10 μg mL<sup>−</sup>1). Nitric oxide production was estimated in terms of the amount of nitrite. After incubation, 100 μL from the surface of the cultures was transferred into a new plate and the equivalent amount of Griess reagent was added (1% sulfanilamide in 5% phosphoric acid, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% phosphoric acid). This plate was incubated for 10 min at room temperature and measured by an ELISA reader at 570nm. NO concentration was determined using a standard curve plotted using a known quantity of sodium nitrite. The outcome is obtainable in μM concentration obtained from the mean OD of triplicate wells of each group.

The percentage of NO inhibition/stimulation by the extracts was calculated as follows:

% inhibition = 100 × [([NO2 <sup>−</sup>] control − [NO2 <sup>−</sup>] sample)/[NO2 −] control

2.3.6. Isolation and Purification of Potent Fractions by Vacuum Liquid Chromatography

The air-dried whole plant of *P. maderaspatensis* was coarsely powdered, defatted with extraction procedure thrice. Using a rotary evaporator, the aqueous ethanolic extracts were pooled and dried at 40 ◦C under reduced pressure. The crude extracts of *P. maderaspatensis* were successively fractionated thrice with chloroform, ethyl acetate, and water. Ethyl acetate fraction was found to have significant immunomodulatory and hepatoprotective activity compared with other fractions. Hence, this fraction was subjected to vacuum liquid chromatography to separate various components present in the fraction. An amount of 16 gm of ethyl acetate fraction was mixed with a small amount of Silica gel G (Merck) to form a dry slurry, which was then loaded onto a sintered glass funnel filled with silica gel gas stationary phase. The column was eluted stepwise under vacuum with solvents of accelerating polarity, starting from pure toluene, and ethyl acetate mixture, to pure ethyl acetate. To elaborate, after initially eluting with 5% ethyl acetate in toluene, the ethyl acetate portion was increased by 5% increments up to 50% and then in 10% increments up to 100% ethyl acetate with a concomitant decrease in the toluene levels. After elution with ethyl acetate, elution with 5% methanol in ethyl acetate, the methanol portion was increased by 5% increments up to 100% methanol. The solvents were eluted until they ran clear of the funnel. The flow rate of solvent was monitored constantly (100 mL/min) throughout the experiment. Various fractions were collected individually and monitored by TLC to match homogeneity. Similar fractions having the same Rf values were combined and crystallized.

#### 2.3.7. Statistical Analysis

Statistical analysis was performed using Dunnett's multiple comparison tests and one-way analysis of variance (ANOVA) using Graphpad prism 5.0 (Graphpad Software, Inc., San Diego, CA, USA). The level of significance was set at *p* < 0.05.

#### 2.3.8. Maintenance of Cell Lines

HepG2 cell lines were grown in 25 cm2 tissue culture flasks containing minimum essential medium (MEM) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦C in a CO2 incubator in an atmosphere of humidified 5% CO2 and 95% air. The cells were maintained by routine sub-culturing in 25 cm<sup>2</sup> tissue culture flasks.

#### 2.3.9. Method for Passaging the Cells

All the reagents were brought to room temperature before use. Media was removed from the 80–90% confluent flasks by a 10 mL serological pipette. Cells in the T-75 flask were washed with 10 mL of PBS. Two milliliters of 0.1% trypsin EDTA was added to the flask. The flask was kept at 37 ◦C in the CO2 incubator for 2–3 min and was observed under a microscope for detachment. Six milliliters of growth medium were added to the flask for inhibition of trypsin action and re-suspended properly by pipetting. The cell suspension was collected in a 15 mL falcon tube and then centrifuged at 1200 rpm for 3 min. The supernatant was discarded and the pellet was resuspended in 3 mL of complete medium. Cells were counted, and then 0.2–0.4 million cells were kept in a T-25 flask for growing. The flasks were incubated in a CO2 incubator at 37 ◦C and the cells were periodically monitored for any morphological changes and contamination. After the formation of an 80–90% confluent monolayer, the cells were further utilized.
