**9. Characterization of polyherbal nanoparticles**

#### **9.1 Particle size and zeta potential**

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 (0.22 lm) ultra pure water [43–45].

#### **9.2 By FTIR spectroscopy**

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).

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

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 The supernatant 5 ml withdrawn at specified time intervals and assayed for drug release in UV spectrophotometrically gallic acid at 270 nm and quercetin at 259 nm.

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

#### **10.1 Cell culture**

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

**261**

*Colon Available Bioactive Compounds Exhibits Anticancer Effect on* In-Vitro *Model…*

for 5 min and then re-suspended in growth medium for further experiments.

containing 10% FBS, seeded in 96-well plates at a density of 2x105

in a CO2 incubator at 37 °C with 5% CO2. After reaching confluency, cells were isolated from the dish with Trypsin–EDTA. The cell suspension was centrifuged at 1000 r/min

Cell viability was studied using an MTT assay. Cells were grown in a medium

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**.**

Soxhlet extraction method carried out for extraction of amla fruit and peels of pomegranate fruit by using three solvents as chloroform, ethanol and ethyl acetate separately. Ethyl acetate solvent gives highest yield 42.51% (Amla fruit) and 42.89% (Pomegranate peels) hence used for extraction of phenolics and flavonoids [50].

Qualitative tests of Phytochemical screening of amla fruit and pomegranate peel extract gives positive test for presence of flavonoids, alkaloids, tannins and

Calibration curve of standard gallic acid showed linear equation at y = 0.014x + 0.395, R2 = 0.996 The content of phenolics in different solvents was as 25.73 ± 0.21, 42.09 ± 0.19 and 63.76 ± 0.29 mg GAE/g for chloroform, ethanol and ethyl acetate respectively. As compare to other solvent ethyl acetate gave more yields hence this is

The concentration of flavonoid standard quercetin on the calibration line was based on the calculated absorbance at y = 0.017 × +0.412, R2 = 0.990 (**Figure 1**)

cells/well, and

*DOI: http://dx.doi.org/10.5772/intechopen.96632*

**10.2 Cell viability assay**

**11. Result and discussion**

**11.1 Soxhlet extraction method**

**11.2 Phytochemical screening**

*11.2.1 Qualitative tests*

*11.2.2 Quantitative test*

*11.2.2.1 Total phenolic content*

*11.2.2.2 Total flavonoid content*

suitable solvent for extraction of phenolics.

carbohydrate.

in a CO2 incubator at 37 °C with 5% CO2. After reaching confluency, cells were isolated from the dish with Trypsin–EDTA. The cell suspension was centrifuged at 1000 r/min for 5 min and then re-suspended in growth medium for further experiments.
