*11.2.2.2 Total flavonoid content*

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**)

**Figure 1.** *IR spectra of isolate of [A] Amla fruit [B] pomegranate peel extract.*

then, the content of flavonoids in pomegranate peels of different solvent extract was expressed in terms as mg QE/g. The content of flavonoid in different solvents was as 32.88 ± 0.26, 42.11 ± 0.29 and 70.8 ± 0.1732 mg QE/g for chloroform, ethanol and ethyl acetate respectively. As compare to other solvents ethyl acetate gave more yields.

### **11.3 Techniques of isolation and purification of bioactive molecule from amla fruit and pomegranate peel extract**

#### *11.3.1 Fractionation of bioactive compound by flash chromatographic technique*

The mobile phase used as ethyl acetate: methanol 100:0 to 0:100 with flow rates were kept at 4 ml/min with wavelength for amla fruit at 270 nm and pomegranate peel extract at 263.5 nm. Column was loaded with 8.0 gm slurry (3 g extract +5 g silica gel) in 25 gm of silica gel (200–400 mesh size).

1 gm of amla extract and peels of pomegranate powder extract separately mixed with 3 gm of silica gel and triturated properly in mortar and pestle. Then, properly mixed extract samples were loaded in sample holder. The separation was completed in 15 minutes only. The Five fractions were isolated by linear gradient with peak tube volume was 14 ml and run time was 15 min. Different fractions no. FA001 to FA005 from amla extract and FP001 to FP005 from peels of pomegranate extract were isolated and dried on buchi roto evaporator (R-210 water bath B-491) for dryness.

Among all five fractions of amla extract fraction number the UV spectra of fraction no FA004 phytoconstituent which gives absorbance at 270.5 nm and this absorbance confirmed with standard gallic acid solution spectra at 272 nm. (**Figure 2A**) The percentage yield of fraction FA004 was found to be 33.4 mg/ gm. The five fractions of peels of pomegranate extract fraction no FB004 gives maximum absorbance at 263.5 nm and also this absorbance confirmed by standard quercetin sample absorbance at 345 nm (**Figure 2B**) scanning with UV Spectrophotometry summarized in **Tables 1** and **2** . The percentage yield of fraction FP004 was found to be 42.6 mg/gm. Further these two isolated fractions no FA004 and FP004 characterized for IR, H1NMR, HPLC and HPTLC techniques for better results.

#### *11.3.2 Gas chromatography*

The study represented a simple gas chromatographic method for estimation of ethyl acetate contents in both amla and pomegranate extract. The GC analysis of the crude

**263**

**Figure 3.**

**Table 1.**

**Figure 2.**

**Table 2.**

*Flash chromatography of amla extract.*

*Flash chromatography of peels of pomegranate extract.*

*Gas chromatogram of [A] Amla fruit [B] pomegranate peel extract.*

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

**Peak Start (nm) Apex (nm) End (nm) Height (Abs) Area** FA001 600.0 430.5 379.0 0.039 1.257 FA002 379.0 350.5 340.5 0.144 2.708 FA003 340.5 339.5 304.0 0.154 3.471 FA004 304.0 270.5 262.0 0.426 11.114 FA005 262.0 239.0 210.0 1.971 70.223

**Peak Start (nm) Apex (nm) End (nm) Height (Abs) Area** FP001 600.0 429.5 379.0 0.064 2.745 FP002 379.0 350.0 340.5 0.266 5.440 FP003 340.5 335.0 302.5 0.272 5.920 FP004 302.0 263.5 252.0 0.369 12.189 FP005 252.0 230.5 210.0 1.893 53.258

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

*Flash chromatogram of [A] Amla fruit [B] pomegranate peel extract.*

*Colon Available Bioactive Compounds Exhibits Anticancer Effect on* In-Vitro *Model… DOI: http://dx.doi.org/10.5772/intechopen.96632*

#### **Figure 2.**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

then, the content of flavonoids in pomegranate peels of different solvent extract was expressed in terms as mg QE/g. The content of flavonoid in different solvents was as 32.88 ± 0.26, 42.11 ± 0.29 and 70.8 ± 0.1732 mg QE/g for chloroform, ethanol and ethyl acetate respectively. As compare to other solvents ethyl acetate gave more yields.

**11.3 Techniques of isolation and purification of bioactive molecule** 

*11.3.1 Fractionation of bioactive compound by flash chromatographic technique*

The mobile phase used as ethyl acetate: methanol 100:0 to 0:100 with flow rates were kept at 4 ml/min with wavelength for amla fruit at 270 nm and pomegranate peel extract at 263.5 nm. Column was loaded with 8.0 gm slurry (3 g extract +5 g

1 gm of amla extract and peels of pomegranate powder extract separately mixed with 3 gm of silica gel and triturated properly in mortar and pestle. Then, properly mixed extract samples were loaded in sample holder. The separation was completed in 15 minutes only. The Five fractions were isolated by linear gradient with peak tube volume was 14 ml and run time was 15 min. Different fractions no. FA001 to FA005 from amla extract and FP001 to FP005 from peels of pomegranate extract were isolated and dried on buchi roto evaporator (R-210 water bath B-491) for

Among all five fractions of amla extract fraction number the UV spectra of fraction no FA004 phytoconstituent which gives absorbance at 270.5 nm and this absorbance confirmed with standard gallic acid solution spectra at 272 nm. (**Figure 2A**) The percentage yield of fraction FA004 was found to be 33.4 mg/ gm. The five fractions of peels of pomegranate extract fraction no FB004 gives maximum absorbance at 263.5 nm and also this absorbance confirmed by standard quercetin sample absorbance at 345 nm (**Figure 2B**) scanning with UV Spectrophotometry summarized in **Tables 1** and **2** . The percentage yield of fraction FP004 was found to be 42.6 mg/gm. Further these two isolated fractions no FA004 and FP004 characterized for IR, H1NMR, HPLC and HPTLC techniques

The study represented a simple gas chromatographic method for estimation of ethyl acetate contents in both amla and pomegranate extract. The GC analysis of the crude

**from amla fruit and pomegranate peel extract**

*IR spectra of isolate of [A] Amla fruit [B] pomegranate peel extract.*

silica gel) in 25 gm of silica gel (200–400 mesh size).

**262**

dryness.

**Figure 1.**

for better results.

*11.3.2 Gas chromatography*

*Flash chromatogram of [A] Amla fruit [B] pomegranate peel extract.*


#### **Table 1.**

*Flash chromatography of amla extract.*


#### **Table 2.**

*Flash chromatography of peels of pomegranate extract.*

**Figure 3.** *Gas chromatogram of [A] Amla fruit [B] pomegranate peel extract.*

ethyl acetate extracts of amla gives retention time at 4.526 min (**Figure 3A**) and for pomegranate fruit at 4.528 min. (**Figure 3B**) Ethyl acetate concentration in amla fruit was found to be 1305.376 ppm and in pomegranate fruit was found to be 1538.440 ppm. Excellent results were obtained within the worldwide accepted validation reference values and particularly taking into account the low concentration levels investigated [51].

#### **11.4 Structural clarification of the bioactive molecules**

The isolated compounds (Fraction No. FA004 from amla extract and FP004 from peels of pomegranate extract by flash chromatography) was characterized by using FT-IR, <sup>1</sup> H-NMR and quantitatively estimated by using HPLC technique [52].

#### *11.4.1 FTIR spectroscopy of the isolated compound*

FT-IR spectra of isolate of amla fruit extract resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes, C=O stretch for acid and aromatic benzonoid ring (**Figure 1A**) and FT-IR spectra of isolate of Pomegranate peel extract resulted in presence of functional groups hydroxyl (-OH) stretch at 3366 cm−1, C-H stretch of alkenes at 2945 cm−1, C=O stretch for lactone and aromatic benzonoid ring 1020 cm−1 (**Figure 3**).

#### *11.4.2 NMR spectroscopy of the isolated compound*

The analysis was done at the BRUKER instrument of 400 MHz d 9.136 (1H, H-7, s), 7.08 (1H, H-2, H-6, s) and 5.011 (1H, H-3, H-4, H-5, s). <sup>1</sup> H NMR of isolate of amla fruit showed the aromatic proton, acidic proton and hydroxyl proton and presence of 7 carbons in structure (**Figure 4A**) given molecular formula as C7H6O5 [53] <sup>1</sup> H-NMR signals of isolate of Pomegranate peel extract shows signals at 12 (S 1H OH Pyran), 6.2 (S 2H Aromatic OH), 6.9 (S1H Aromatic OH), 7.1 (S1H Aromatic OH), 7–8 (S Aromatic proton) <sup>1</sup> H NMR showed the aromatic proton and hydroxyl proton and presence of 15 carbons (**Figure 4B**) in structure given molecular formula as C15H10O7.

#### **Figure 4.**

*NMR spectra of isolated compound and structure of compounds [A] Amla fruit (Gallic acid) [B] pomegranate peel extract (quercetin).*

**265**

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

*Chromatogram of [A] standard Gallic acid [B] isolated fraction of amla extract.*

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

**Figure 5.**

**Figure 6.**

*11.4.3 HPLC analysis of isolated compounds*

method were characterized.

**11.5. Antioxidant activity**

*11.5.1 Antioxidant activity by DPPH method*

A comparison between the spectra of fruits of amla extract (Fraction no A004 by flash chromatography) peak at 3.165 min confirmed with that of standard gallic acid peak at 3.207 min respectively (**Figure 5A** and **B**). A good linearity was found from 5–15 μg/mL gallic acid, and the linear regression equation was y = 8008x-397.0 (rc = 0.999) where y is the peak height. The gallic acid from amla fruit extract was fractionated by HPLC of which 27.15 ± 0.001 μg/mg GAE equivalent by HPLC

*Chromatogram of [A] standard quercetin [B] isolated fraction of pomegranate peel extract.*

Same comparison between the spectra of peels of pomegranate (Fraction no B004 by flash chromatography) peak at 5.242 min with that of standard quercetin confirmed that the retention time of the analyte was 5.248 min respectively (**Figure 6A** and **B**). Linearity for the developed method was found over the concentration range 3–18 μg/ml with a linear regression equation was y = 16.01x + 25628 where y is the peak height correlation coefficient of 0.999.

The DPPH is a stable free radical, which has been widely accepted as a tool for estimating free radical-scavenging activities of antioxidants. The scavenging activity on 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) radical of both the fruits extract and isolated fractions was determined by following method. The extracts of different concentrations were mixed with an aliquot of DPPH (1 ml, 0.004% w/v) [54].

*Colon Available Bioactive Compounds Exhibits Anticancer Effect on* In-Vitro *Model… DOI: http://dx.doi.org/10.5772/intechopen.96632*

**Figure 5.** *Chromatogram of [A] standard Gallic acid [B] isolated fraction of amla extract.*

**Figure 6.** *Chromatogram of [A] standard quercetin [B] isolated fraction of pomegranate peel extract.*

### *11.4.3 HPLC analysis of isolated compounds*

A comparison between the spectra of fruits of amla extract (Fraction no A004 by flash chromatography) peak at 3.165 min confirmed with that of standard gallic acid peak at 3.207 min respectively (**Figure 5A** and **B**). A good linearity was found from 5–15 μg/mL gallic acid, and the linear regression equation was y = 8008x-397.0 (rc = 0.999) where y is the peak height. The gallic acid from amla fruit extract was fractionated by HPLC of which 27.15 ± 0.001 μg/mg GAE equivalent by HPLC method were characterized.

Same comparison between the spectra of peels of pomegranate (Fraction no B004 by flash chromatography) peak at 5.242 min with that of standard quercetin confirmed that the retention time of the analyte was 5.248 min respectively (**Figure 6A** and **B**). Linearity for the developed method was found over the concentration range 3–18 μg/ml with a linear regression equation was y = 16.01x + 25628 where y is the peak height correlation coefficient of 0.999.

#### **11.5. Antioxidant activity**

#### *11.5.1 Antioxidant activity by DPPH method*

The DPPH is a stable free radical, which has been widely accepted as a tool for estimating free radical-scavenging activities of antioxidants. The scavenging activity on 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) radical of both the fruits extract and isolated fractions was determined by following method. The extracts of different concentrations were mixed with an aliquot of DPPH (1 ml, 0.004% w/v) [54].

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

**11.4 Structural clarification of the bioactive molecules**

*11.4.1 FTIR spectroscopy of the isolated compound*

matic benzonoid ring 1020 cm−1 (**Figure 3**).

Aromatic OH), 7–8 (S Aromatic proton) <sup>1</sup>

molecular formula as C15H10O7.

*11.4.2 NMR spectroscopy of the isolated compound*

using FT-IR, <sup>1</sup>

as C7H6O5 [53] <sup>1</sup>

ethyl acetate extracts of amla gives retention time at 4.526 min (**Figure 3A**) and for pomegranate fruit at 4.528 min. (**Figure 3B**) Ethyl acetate concentration in amla fruit was found to be 1305.376 ppm and in pomegranate fruit was found to be 1538.440 ppm. Excellent results were obtained within the worldwide accepted validation reference values and particularly taking into account the low concentration levels investigated [51].

The isolated compounds (Fraction No. FA004 from amla extract and FP004 from peels of pomegranate extract by flash chromatography) was characterized by

FT-IR spectra of isolate of amla fruit extract resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes, C=O stretch for acid and aromatic benzonoid ring (**Figure 1A**) and FT-IR spectra of isolate of Pomegranate peel extract resulted in presence of functional groups hydroxyl (-OH) stretch at 3366 cm−1, C-H stretch of alkenes at 2945 cm−1, C=O stretch for lactone and aro-

The analysis was done at the BRUKER instrument of 400 MHz d 9.136

of isolate of amla fruit showed the aromatic proton, acidic proton and hydroxyl proton and presence of 7 carbons in structure (**Figure 4A**) given molecular formula

at 12 (S 1H OH Pyran), 6.2 (S 2H Aromatic OH), 6.9 (S1H Aromatic OH), 7.1 (S1H

*NMR spectra of isolated compound and structure of compounds [A] Amla fruit (Gallic acid) [B] pomegranate* 

and hydroxyl proton and presence of 15 carbons (**Figure 4B**) in structure given

H-NMR signals of isolate of Pomegranate peel extract shows signals

H NMR showed the aromatic proton

(1H, H-7, s), 7.08 (1H, H-2, H-6, s) and 5.011 (1H, H-3, H-4, H-5, s). <sup>1</sup>

H-NMR and quantitatively estimated by using HPLC technique [52].

H NMR

**264**

**Figure 4.**

*peel extract (quercetin).*

The mixtures were vigorously shaken and left to stand for 30 min in the dark at room temperature. For this method the absorbance were recorded at 517 nm. The percentages of remaining DPPH in the presence of the amla and pomegranate peel extract (**Figures 7** and **8**) and its fractions at different concentrations are shown in **Table 3**.

**Figure 7.**

*DPPH radial scavenging activity (A) Amla extract (B) isolated fraction of FA004 by flash chromatography.*

#### **Figure 8.**

*DPPH radial scavenging activity (A) peels of pomegranate extract (B) isolated fraction no FP004 by flash chromatography.*


**267**

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

The isolated compound was analyzed for their solubility in different solvents. White colored powder of amla extract (FA004 Flash chromatography) which is soluble in ether, ethanol, methanol, glycerol and acetone. Yellow colored crystalline powder of pomegranate extract (FP004 Flash chromatography) practically insoluble in water and soluble in DMSO, ethanol, methanol and acetone.

Melting point of compound was done in thermonic apparatus to determine its identity and purity. The observed melting point of isolated compound of amla extract (FA004 Flash chromatography) was 255–257 ° C compared with the standard melting point (260 °C) of respective isolated gallic acid. The observed melting point of isolated compound of pomegranate extract (FP004 Flash chromatography) was 313–316 °C compared with the standard melting point (316 °C) of respective

In this study the goals for optimization were to minimizing particle size and maximum Zeta potential. Desirability ramp showing optimum conditions to formulate CS nanoparticles as chitosan 2.4%, and Poloxamer (407) 0.1% to achieve

A mean diameter of particle size of CS nanoparticles was found to be 214.2 ± 1.28 nm with +14.7 mV zeta potential [14, 55, 56]. Chitosan on the other hand has a positive charge in acidic solutions due to the presence of protonated amino groups which was appropriate adhere negatively charged intestinal mucus layer. This

The characteristic groups of chitosan at (**Figure 9A**) 3285.15 cm−1 for O-H stretching 2875.66 cm−1 for C-H stretching and 1415.23 cm−1 for amide C-N stretching. The bands at 1150.54 cm−1 for asymmetric stretching of the bond C-O-C and 1062.04 and 1023.35 cm−1 for vibrations involving the C-O bonds of primary alcohols [57]. The carbon chain of poloxamer 407 (**Figure 9B**) at 2881.11 cm−1 aliphatic C-H stretching, plane O-H bend at 1365.12 cm−1 and 1242.02 cm−1, C-O stretch at 1096.99 cm−1, CH=CR2 at 840.46 cm−1. The C=O functionality of GMO (**Figure 9C**) was seen with a strong peak at 1738 cm−1. In the spectrum of gallic acid (**Figure 9D**) there is a broad band at 3194.61 cm−1 related to OH stretching and hydrogen bonds between phenolic hydroxyl groups. The COOH stretch/bend is observed at 1255.93 cm−1 Aromatic ring stretching is observed at 1454.44 cm−1 [58]. C- O stretching is at 1021.45 cm−1 In the spectrum of quercetin (**Figure 9E**) there is a broad band at 3194.61 cm−1 related to OH stretching and hydrogen bonds between phenolic hydroxyl groups. O-H stretch at 3190.38 cm−1, =C-H stretch at 2935.23 cm−1, aromatic C=C stretch at 1454.09 cm−1 and aromatic C-O stretch at

particle size 218.33 nm and zeta potential11.50 mV with desirability 1.000.

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

**11.7 Melting point determination**

**11.8 Formulation of CS nanoparticles**

**11.9 Characterization of CS nanoparticles**

*11.9.2 FTIR of CS nanoparticles*

*11.9.1 Analysis of particle size and zeta potential*

explains that outer coating of nanoparticles was CS only.

isolated quercetin.

**11.6 Determination of solubility of isolated compound**

#### **Table 3.**

*Antioxidant activity of amla and pomegranate extract by DPPH.*

*Colon Available Bioactive Compounds Exhibits Anticancer Effect on* In-Vitro *Model… DOI: http://dx.doi.org/10.5772/intechopen.96632*

### **11.6 Determination of solubility of isolated compound**

The isolated compound was analyzed for their solubility in different solvents. White colored powder of amla extract (FA004 Flash chromatography) which is soluble in ether, ethanol, methanol, glycerol and acetone. Yellow colored crystalline powder of pomegranate extract (FP004 Flash chromatography) practically insoluble in water and soluble in DMSO, ethanol, methanol and acetone.

#### **11.7 Melting point determination**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

**Table 3**.

**Figure 7.**

**Figure 8.**

*chromatography.*

The mixtures were vigorously shaken and left to stand for 30 min in the dark at room temperature. For this method the absorbance were recorded at 517 nm. The percentages of remaining DPPH in the presence of the amla and pomegranate peel extract (**Figures 7** and **8**) and its fractions at different concentrations are shown in

*DPPH radial scavenging activity (A) Amla extract (B) isolated fraction of FA004 by flash chromatography.*

*DPPH radial scavenging activity (A) peels of pomegranate extract (B) isolated fraction no FP004 by flash* 

**Sample R2 IC50** Ascorbic acid (Standard) 0.996 8.98 μg/ml Amla extract 0.884 25.74 μg/ml Isolated Fraction [FA004] 0.904 14.44 μg/ml Pomegranate peel extract 0.863 29.89 μg/ml Isolated Fraction [FP004] 0.910 11.21 μg/ml

*Antioxidant activity of amla and pomegranate extract by DPPH.*

**266**

**Table 3.**

Melting point of compound was done in thermonic apparatus to determine its identity and purity. The observed melting point of isolated compound of amla extract (FA004 Flash chromatography) was 255–257 ° C compared with the standard melting point (260 °C) of respective isolated gallic acid. The observed melting point of isolated compound of pomegranate extract (FP004 Flash chromatography) was 313–316 °C compared with the standard melting point (316 °C) of respective isolated quercetin.

#### **11.8 Formulation of CS nanoparticles**

In this study the goals for optimization were to minimizing particle size and maximum Zeta potential. Desirability ramp showing optimum conditions to formulate CS nanoparticles as chitosan 2.4%, and Poloxamer (407) 0.1% to achieve particle size 218.33 nm and zeta potential11.50 mV with desirability 1.000.

#### **11.9 Characterization of CS nanoparticles**

#### *11.9.1 Analysis of particle size and zeta potential*

A mean diameter of particle size of CS nanoparticles was found to be 214.2 ± 1.28 nm with +14.7 mV zeta potential [14, 55, 56]. Chitosan on the other hand has a positive charge in acidic solutions due to the presence of protonated amino groups which was appropriate adhere negatively charged intestinal mucus layer. This explains that outer coating of nanoparticles was CS only.

#### *11.9.2 FTIR of CS nanoparticles*

The characteristic groups of chitosan at (**Figure 9A**) 3285.15 cm−1 for O-H stretching 2875.66 cm−1 for C-H stretching and 1415.23 cm−1 for amide C-N stretching. The bands at 1150.54 cm−1 for asymmetric stretching of the bond C-O-C and 1062.04 and 1023.35 cm−1 for vibrations involving the C-O bonds of primary alcohols [57]. The carbon chain of poloxamer 407 (**Figure 9B**) at 2881.11 cm−1 aliphatic C-H stretching, plane O-H bend at 1365.12 cm−1 and 1242.02 cm−1, C-O stretch at 1096.99 cm−1, CH=CR2 at 840.46 cm−1. The C=O functionality of GMO (**Figure 9C**) was seen with a strong peak at 1738 cm−1. In the spectrum of gallic acid (**Figure 9D**) there is a broad band at 3194.61 cm−1 related to OH stretching and hydrogen bonds between phenolic hydroxyl groups. The COOH stretch/bend is observed at 1255.93 cm−1 Aromatic ring stretching is observed at 1454.44 cm−1 [58]. C- O stretching is at 1021.45 cm−1 In the spectrum of quercetin (**Figure 9E**) there is a broad band at 3194.61 cm−1 related to OH stretching and hydrogen bonds between phenolic hydroxyl groups. O-H stretch at 3190.38 cm−1, =C-H stretch at 2935.23 cm−1, aromatic C=C stretch at 1454.09 cm−1 and aromatic C-O stretch at

#### **Figure 9.**

*IR spectra of CS nanoparticles (a) chitosan (B) Poloxamer 407 (C) GMO (D) quercetin (E) Gallic acid (E) CS nanoparticles.*

1145.06 cm−1. The COOH stretch/bend is observed at 1255.93 cm−1 The spectra of the gallic acid and quercetin loaded CS nanoparticles showed that O-H stretch of gallic acid and quercetin was disappeared (**Figure 9F**). Here all data of FTIR results conclude that encapsulation of gallic acid and quercetin into CS nanoparticles with intermolecular hydrogen bonding occurred in the nanoformulation which correlated with the less crystalline compared to both pure biomolecules.

#### *11.9.3 In vitro release studies*

As a result CS nanoparticles have indicated improved drug releases 77.56% for gallic acid 79.06% for quercetin at 24 hr. respectively. So the CS nanoparticles can be considered as a potential barrier, which can release the biomolecules at colonic pH [59]. By engineering chitosan approach gallic acid and quercetin biomolecules achieved sustained and controlled release and also benefitted by its targeting property to colonic region. To describe the mechanism of gallic acid and quercetin release from the CS nanoparticles, [60] the data was plotted into a few kinetic models and best fitted information into the Korsmeyer–Peppas power law model.

#### **11.10 Methods of anticancer activity determination**

#### *11.10.1 In vitro cytotoxicity by MTT assay*

After 24 hours of incubation, cell viability was determined by the MTT assay. The nanoparticles induced cell cytotoxicity in a concentration dependent manner, as illustrated. Cytotoxicity of polyherbal extracts, CS nanoparticles and cisplatin (Standard) was dose on HCT 116 cell lines and activity is dependent up to the concentration of 6.25–100 ug/mL. The IC50 of polyherbal extract, chitosan nanoparticles and standard after 48 h treatment it was found to be 60.32 and 36.17 and 8.915 ug/ml respectively summarized in **Table 4**.

The antiproliferative potential of all samples shown as cytotoxicity of standard cisplatin (**Figure 10A**) CS nanoparticles (**Figure 10B**) polyherbal extract (**Figure 10C**) was done on HCT 116 cell lines and activity is dependent up to the concentration of 6.25–100 ug/mL. MTT assay determined the cytotoxic effect of all samples by decreasing the cell viability of HCT116 colon cancer cells with different serial dilutions. The half maximal inhibitory concentration (IC50) was evaluated to determine the effectiveness of CS nanoparticles in inhibiting

**269**

**Figure 11.**

*(D) untreated HCT116 cell lines.*

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

IC50 = 10.55 ug/ml

IC50 = 60.32 ug/ml

IC50 = 36.173 ug/ml

**Standard (A)** 1.157 0.680 0.582 0.19 0.0885 0.06

**Polyherbal extract (B)** 1.157 1.677 1.301 0.794 0.478 0.182

**CS nanoparticles (C)** 1.157 0.906 0.892 0.79 0.141 0.028

*Concentrations used for MTT assay (A) standard (cisplatin) (B) Polyherbal extract (C) CS nanoparticles.*

*Microscopy imaging of cellular uptakes (A) standard (B) CS nanoparticles (C) Polyherbal extract* 

**Untreated 6.25 12.5 25 50 100**

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

*MTT data analysis of HCT-116 cell lines.*

**Table 4.**

**Figure 10.**

**Name of Samples Concentrations ug/ml**

*Colon Available Bioactive Compounds Exhibits Anticancer Effect on* In-Vitro *Model… DOI: http://dx.doi.org/10.5772/intechopen.96632*


#### **Table 4.**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

*IR spectra of CS nanoparticles (a) chitosan (B) Poloxamer 407 (C) GMO (D) quercetin (E) Gallic acid (E)* 

1145.06 cm−1. The COOH stretch/bend is observed at 1255.93 cm−1 The spectra of the gallic acid and quercetin loaded CS nanoparticles showed that O-H stretch of gallic acid and quercetin was disappeared (**Figure 9F**). Here all data of FTIR results conclude that encapsulation of gallic acid and quercetin into CS nanoparticles with intermolecular hydrogen bonding occurred in the nanoformulation which corre-

As a result CS nanoparticles have indicated improved drug releases 77.56% for gallic acid 79.06% for quercetin at 24 hr. respectively. So the CS nanoparticles can be considered as a potential barrier, which can release the biomolecules at colonic pH [59]. By engineering chitosan approach gallic acid and quercetin biomolecules achieved sustained and controlled release and also benefitted by its targeting property to colonic region. To describe the mechanism of gallic acid and quercetin release from the CS nanoparticles, [60] the data was plotted into a few kinetic models and best fitted information into the Korsmeyer–Peppas power law model.

After 24 hours of incubation, cell viability was determined by the MTT assay. The nanoparticles induced cell cytotoxicity in a concentration dependent manner, as illustrated. Cytotoxicity of polyherbal extracts, CS nanoparticles and cisplatin (Standard) was dose on HCT 116 cell lines and activity is dependent up to the concentration of 6.25–100 ug/mL. The IC50 of polyherbal extract, chitosan nanoparticles and standard after 48 h treatment it was found to be 60.32 and 36.17 and 8.915

The antiproliferative potential of all samples shown as cytotoxicity of standard cisplatin (**Figure 10A**) CS nanoparticles (**Figure 10B**) polyherbal extract (**Figure 10C**) was done on HCT 116 cell lines and activity is dependent up to the concentration of 6.25–100 ug/mL. MTT assay determined the cytotoxic effect of all samples by decreasing the cell viability of HCT116 colon cancer cells with different serial dilutions. The half maximal inhibitory concentration (IC50) was evaluated to determine the effectiveness of CS nanoparticles in inhibiting

lated with the less crystalline compared to both pure biomolecules.

**11.10 Methods of anticancer activity determination**

*11.10.1 In vitro cytotoxicity by MTT assay*

ug/ml respectively summarized in **Table 4**.

**268**

**Figure 9.**

*CS nanoparticles.*

*11.9.3 In vitro release studies*

*MTT data analysis of HCT-116 cell lines.*

#### **Figure 10.**

*Concentrations used for MTT assay (A) standard (cisplatin) (B) Polyherbal extract (C) CS nanoparticles.*

#### **Figure 11.**

*Microscopy imaging of cellular uptakes (A) standard (B) CS nanoparticles (C) Polyherbal extract (D) untreated HCT116 cell lines.*

biological or biochemical functions. CS nanoparticles shows a higher cytotoxic effect on HCT116 cells with low concentrations (IC50 = 36.173 μg/ml) than polyherbal extract (IC50 = 60.32 μg/ml) that might be due to the active biomolecules capped to the nanoparticles.

HCT 116 cell lines considered to have more prominent take-up for CS nanoparticles and more stable even at low concentrations and longer interval than polyherbal extract. Microscopy imaging of cellular uptakes shows as standard cisplatin (**Figure 11A**) CS nanoparticles (**Figure 11B**) polyherbal extract (**Figure 11C**) and untreated HCT116 cell lines (**Figure 11D**) HCT 116 cell lines subjectively were deemed to have had greater uptake for CS nanoparticles and more stable even at low concentrations than polyherbal extract expected to be longer interval than polyherbal extract.
