**10.2 Cell viability assay**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

An o/w nanoemulsion of gallic acid and quercetin was prepared by using a GMO/chitosan framework as reported by with slight modifications. Briefly, isolated gallic acid (100 mg) and quercetin (100 mg) were dissolved in molten GMO (2 g), then add 12.5 ml of 0.1% poloxamer 407 sonicated at 18 W for 3 min in probe sonicator. To this emulsion, dropwise 12.5 ml of 2.4% chitosan solution was added again using probe sonicator at 16 W for 4 min [40, 41]. Finally this phase was subjected to twelve cycles of HPH at 15,000 psi to give the nanoemulsion. Then, lyophilized with 2% mannitol as a cryoprotectant for 48 hr. Central composite design [42] was applied to examine the combined effect two variables, each at 2 levels and the pos-

Average particle size and zeta potential of the CS nanoparticles were determined by Particle Size Analyzer (Zetasizer Ver System; Malvern Instruments Ltd., Malvern, UK). To analyze particle size, nanosuspension was diluted with filtered

FTIR has proven to be a valuable tool for identification of functional groups present in compound from plants. Attenuated total reflection/Fourier transform infrared spectroscopic (ATR/FTIR) spectra was collected at room temperature by coupling ATR accessory to an FTIR spectrometer (Perkin Elmer, Spectrum 100).

CS nanoparticles were tested in various simulated fluids at different pH to evaluate the release of nanoparticles at particular pH and also to determine the drug release [46]. Four milligrams of CS nanoparticles were dispersed in a freshly prepared phosphate-buffered saline (PBS; pH = 2.0, 4.5, 6.8, 7.4) as a release

medium in a dialysis membrane sac (mw cut-off 12 kDa; Sigma Aldrich) to simulate ileo-colon conditions for 24 hr [47]. The enclosed dialysis sac was immersed in a beaker containing 50 mL of the release medium. The beaker was placed in a shaking incubator at 37 °C under mild agitation (90–100 rpm) PBS; pH = 2.0 for first four hour, pH = 4.5 for next five to nine hour, pH = 6.8 for next ten to thirteen hour and finally pH = 7.4 for fourteen to twenty-four hour. The supernatant 5 ml withdrawn at specified time intervals and assayed for drug release in UV spectrophotometri-

A human colorectal adenocarcinoma cell line (HCT116) were cultured with RPMI 1640 and McCoy's 5A medium (Fisher Scientific, Waltham, MA, USA), respectively [48]. All cell culture mediums contained 10% fetal bovine serum. Cells were incubated

**8. Formulation of nanoparticles**

sible 9 combinations of CS nanoparticles.

**9.1 Particle size and zeta potential**

(0.22 lm) ultra pure water [43–45].

**9.2 By FTIR spectroscopy**

**9.3** *In vitro* **release studies**

**9. Characterization of polyherbal nanoparticles**

cally gallic acid at 270 nm and quercetin at 259 nm.

**10.** *In vitro* **anticancer activity (cytotoxicity) by MTT assay**

**260**

**10.1 Cell culture**

Cell viability was studied using an MTT assay. Cells were grown in a medium containing 10% FBS, seeded in 96-well plates at a density of 2x105 cells/well, and incubated at 37 °C in CO2 incubator with 5% CO2 for 24 h [49]. Then, polyherbal extract, CS nanoparticles and standard cisplatin were added (final concentrations of 6.25, 12.5, 25, 50, and 100 ug/ml) to the mono-layers of cells, which were subsequently incubated for at 24 and 48 h, media were aspirated and MTT solution at a concentration of 5 mg/ml in phosphate-buffered saline (PBS) buffer was added 20 ml/well. After further incubation (3 h), the media was removed and replaced with 100 ml of DMSO. Plates were washed with 1% acetic acid, air-dried, and then 10 mM Tris base pH 7.4 (150 μl) was added to the wells to solubilize the dye. The plates were shaken vigorously for 5 min and color absorbance was measured at 540 nm using an ELISA microplate reader. (ELISA reader Denver Jasco Model 7800 UV/VIS Spectrophotometer Jasco Tokyo, Japan) Untreated cells were used as positive controls with 100% viability and cells without assay reagents were used as a blank**.**
