**2. Materials and methods**

#### **2.1 Materials**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

anti-proliferative, anti-angiogenic [10–15].

medium [16, 17].

polymeric CS nanoparticles [22].

structure in addition quercetin has anti-tumor properties, anti-inflammatory**,**

There have been particular efforts to evaluate the therapeutic role of these active constituents present in plants rather than using whole extracts. The fundamental method of reasoning behind these systems contributes greatly to enhance the targeting delivery and bioavailability of phenolic and flavonoid remarkable formulation development can be made by preparation of nanotechnology products. The eco-friendly synthesis of gallic acid and quercetin loaded chitosan nanoparticles (CS nanoparticles) through green route from plant extracts have renowned a wide range of application in the field of modern science, due to increased drug efficacy and less toxicity in the nanosized mediated drug delivery model. At the same time, the use of gallic acid and quercetin in pharmaceutical formulations is limited due to its poor water solubility, poor bioavailability and instability in physiological

Lack of site specificity is one of the major reasons for the drug in reaching the target site in therapeutic concentrations in colorectal cancer [18]. Chitosan (CS), as the only naturally occurring positive charge polysaccharide, has remarkable properties including high bioavailability, super biodegradability, high biocompatibility, non-toxicity etc., On the other hand it causes sustained release of the drug from the particle in the tumor environment [19]. The main rationale behind using these types of polymers is their ability to prevent drug degradation in the gastric environment in the stomach and their ability to release the drug after entering the distal ileum [20]. Poloxamer 407 is a hydrophilic nontoxic copolymer used for its stabilizing properties and incorporation of hydrophobic drugs capability to increase the solubility of biomolecules [21]. Here combined biomolecules synergistic activity of nanotechnology approach has been developed to improve the bioavailability as to entrap these natural biomolecules into biodegradable

The system of glyceryl monooleate (GMO)/chitosan is a surface-modified nanoparticulate system consisting of GMO as a lipid portion and chitosan as a coating polymer to target colonic area with poloxamer 407 as a stabilizer. Therefore, the purpose of this study was to formulate CS nanoparticles of where quercetin isolated from peels of pomegranate fruit and gallic acid isolated from amla fruit as a model hydrophobic biomolecules followed by lyophilization using probe sonicator and High Pressure Homogenization (HPH) method. CS nanoparticles prepared and optimized using Quality by design approach by using central composite factorial design. Optimized formulation further characterized for different parameters as particle size, zeta potential, FT-IR and. Release kinetic studies performed using

In this study, we systematically analyzed the *In vitro* anti-cancer potential of the gallic acid and quercetin loaded chitosan nanoparticles synergy approach for combined active biomolecules and compared to their activity to combined extracts on HCT 116 human colon cancer cell lines and the mechanism of action of CS nanoparticles in regulating the growth of CRC cells. The human HCT116 cell line by MTT (3-(4,5-dimethyl-2-tiazolyl)-2,5-diphenyl-2-tetrazolium bromide) assay was exposed to cytotoxicity of polyherbal extracts, chitosan nanoparticles and cisplatin (Standard) and activity is dependent up to the concentration of 6.25- 100ug/mL for 24 h followed by MTT cellular assays [23]. To determine the potential anti-cancer effect of, we synthesized CS nanoparticles using gallic acid and quercetin biomolecules, which is a phenolic and flavonoid predominantly found in amla fruit and peels of pomegranate fruit. HCT116 cells exposed to gallic acid and quercetin for 24 h exhibited significant loss of cell viability and proliferation in a

method for conventional nanoparticle release behavior assessment.

**256**

dose-dependent manner.

Poloxamer 407 from BASF, Chitosan 90% dda obtained from CIFT Cochin, GMO from Mohini organics, standard gallic acid and quercetin purchased from Loba Chemie., 10% fetal bovine serum (Invitrogen Life Technologies USA). RPMI 1640 and McCoy's 5A medium (Fisher Scientific, Waltham, USA). All the solvents and chemicals used were procured from Himedia Laboratories, Research Lab. Mumbai.

#### **2.2 Plant material**

The sample of different parts of plant of amla and pomegranate was collected from Kolhapur district and was authenticated by Dr. Madhukar Bachulkar, Principal, Arts and Science College Peth Vadagaon Kolhapur. The voucher herbarium (PSP-1 and 2) has been deposited in the department of Pharmacognosy Bharati Vidyapeeth College of Pharmacy, Kolhapur. Amla fruit and peels of pomegranate fruit were collected in season (Feb-March), were dried under shade for 10–15 days in air.

#### **2.3 Soxhlet extraction method**

In order to extract Flavonoid and phenolics from plants with a high degree of accuracy, various solvents of differing polarities were tried as chloroform, ethanol and ethyl acetate. The dried powder of amla fruit and peels of pomegranate powder extracted with 800 ml in various solvents for 6 hours separately. All extracts were filtered and evaporated to dryness under reduced pressure at 60 °C by a rotary evaporator and to determine percentage yield for all three different solvents [24].

#### **2.4 Phytochemical screening**

#### *2.4.1 Qualitative test*

Phytochemical analysis was carried out to detect the presence of primary and secondary metabolites were used to identify the biomolecules present in the plant extract [25]. The phytochemical tests carried out for amla and peels of pomegranate extract include alkaloids, glycosides, saponins, tannins, triterpenoids, steroid, flavonoids and carbohydrate [26].

#### *2.4.2 Quantitative tests*

#### *2.4.2.1 Determination of total phenolic content for amla extract*

Phenolic compounds are important plant constituents with redox properties responsible for antioxidant activity. Folin–Ciocalteu's method (FC) is a colorimetric method based on transfer of electrons between reagents and polyphenols. Different solvent extract chloroform, ethanol and ethyl acetate of amla fruit used for determination of phenolic content. The reaction mixture was prepared by mixing 1 ml of methanolic solution of all extracts, 2.5 ml of 10% Folin–Ciocalteu's reagent dissolved in water and 2.5 ml 7.5% NaHCO3. Blank was concomitantly prepared, containing 0.5 ml methanol, 2.5 ml 10% Folin–Ciocalteu's reagent dissolved in water and 2.5 ml of 7.5% of NaHCO3. The samples were thereafter incubated in a thermostat at 45o C for 45 min. The same procedure was repeated for the standard solution of gallic acid (Standard) in methanol (10to100μg/ml) and for blank then calibration line was construed and absorbance measured at λmax 765 nm [27]. The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained.

### *2.4.2.2 Determination of total flavonoid content for pomegranate peel extract*

Flavonoids are group of polyphenolic compound used for different activities and their potency depends on the number and position of free hydroxy groups. As a basis quantitative determination, flavonoid contents in pomegranate peel extract were determined using aluminum chloride colorimetric method with sufficient modification. In this process, flavonoid content was determined using quercetin standard (5 to 320 μg/mL) to make the calibration curve. Different solvent extracts chloroform, ethanol and ethyl acetate of pomegranate peel used for determination of flavonoid content. All procedure followed for preparation of different extracts of sample solution, blank and for standard and their corresponding absorbances were measured at 415 nm with a UV-1800 spectrophotometer [28].
