**4. Structural clarification of the bioactive molecules**

The flash chromatographic fractions of amla fraction no FA004 and pomegranate fraction no FP004 were filtered, dried and kept at 4 ° C for characterization of FT-IR and 1 H-NMR techniques and quantitavely estimated by HPLC technique [33, 34].

**259**

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

Only fraction A16 and Fraction P4 was additionally elucidated by <sup>1</sup>

using solvent D6 + CDCL3 MIX. The analysis was done at the BRUKER instrument

HPLC PU-2080 Plus (Systronics) with UV-2075 plus intelligent detector and HPLC C18 column (250 × 4.6 mm, 5 μm) was set at 270 nm for estimation of gallic acid and 259 nm for estimation of quercetin [36, 37]. HPLC PU-2080 Plus

(Systronics) with UV-2075 plus intelligent detector and HPLC C18 column (250 × 4.6 mm, 5 μm) was set at 270 nm for estimation of gallic acid and 259 nm for estimation of quercetin. The mobile phase Acetonitrile and 2% Acetic Acid with ratio 40:60 used for elution of both compounds. Flow of mobile phase and injection loop was set at 1.0 ml/min and 20 μL respectively. Quantitative determination of gallic acid and quercetin content in fraction concentrations (FA004 by flash chromatography of amla extract) in the range 0.01 to 0.5 mg/ml and (Fraction FP004 by flash chromatography

of pomegranate extract) in the concentration range of 0.01 to 0.5 μg/ml used.

The two isolated compounds were analyzed for their solubility in different

Melting points of two isolated biomolecule compounds was done in thermonic

2, 2-diphenyl-1-picryl-hydrazyl (DPPH) is a stable organic free radical used to estimate the antioxidant activity of various compounds. The scavenging action on DPPH radical from amla fruit and isolated fraction (FA004 by flash chromatography) and peels of pomegranate extract and isolated fraction (FP004 by flash chromatography of peels of pomegranate extract) determined by following method [38, 39]. As of different concentrations was mixed with an aliquot of DPPH (1 ml, 0.004% w/v) and analyzed at 517 nm. Then the scavenging capacity was calculated using equation number (1).

( ) ( A517 of control – A517 of sample) Scavenging activity % <sup>100</sup>

A517 of control ∆ ∆ <sup>=</sup> <sup>×</sup> <sup>∆</sup> (1)

apparatus to determine its identity and purity. The observed melting point of isolated compound was compared with the standard melting point of respective

**5. Determination of solubility of isolated compound**

solvents as in DMSO, ethanol, methanol and acetone.

**7. Antioxidant activity by DPPH method**

**6. Melting point determination**

gallic acid and quercetin.

FTIR has proven to be a valuable tool for the characterization and identification of functional groups present in compound from plants extract. Infrared spectra was collected using IR (α-ATR Bruker Germany spectrometer) operated form 4000–600 cm−1 at resolution of 4 cm−1. Data analyzed using Opus software.

HNMR by

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

**4.2 NMR spectroscopy of the isolated compound**

**4.1 FTIR spectroscopy**

of 400 MHz [35].

**4.3 HPLC of isolated compounds**

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

#### **4.1 FTIR spectroscopy**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

thermostat at 45o

bance was obtained.

**from extracts**

of silica particles [29, 30].

**3.2 Gas chromatography**

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

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 absor-

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

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

measured at 415 nm with a UV-1800 spectrophotometer [28].

min) to 250 °C at 20 °C/min. (hold up to 5 min) [31, 32].

**4. Structural clarification of the bioactive molecules**

**3. Techniques of isolation and purification of bioactive molecules** 

**3.1 Fractionation of bioactive compound by flash chromatographic technique**

Flash chromatography instrument consisting of (Analytical technologies limited, Shanghai china) consisting of TBP2H02pump along with TBD2000 UV detector and automatic fraction collector was used for analysis. System equipped with Chromo station software was used for data monitoring during the analysis. The separation was carried out on OROCHEM OROFLO-4SiHPS column made up

The gas chromatography used for estimation of residual class 3 ethyl acetate solvent in both crude extracts was performed using a Gas Chromatography system 7890 B with Agilent DB 624 column with helium gas at 1 ml/min flow mode reference solution tetrahydrofuran. GC temperature was set at 50 °C (hold for couple of

The flash chromatographic fractions of amla fraction no FA004 and pomegranate fraction no FP004 were filtered, dried and kept at 4 ° C for characterization of FT-IR

H-NMR techniques and quantitavely estimated by HPLC technique [33, 34].

C for 45 min. The same procedure was repeated for the standard

**258**

and 1

FTIR has proven to be a valuable tool for the characterization and identification of functional groups present in compound from plants extract. Infrared spectra was collected using IR (α-ATR Bruker Germany spectrometer) operated form 4000–600 cm−1 at resolution of 4 cm−1. Data analyzed using Opus software.

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

Only fraction A16 and Fraction P4 was additionally elucidated by <sup>1</sup> HNMR by using solvent D6 + CDCL3 MIX. The analysis was done at the BRUKER instrument of 400 MHz [35].

### **4.3 HPLC of isolated compounds**

HPLC PU-2080 Plus (Systronics) with UV-2075 plus intelligent detector and HPLC C18 column (250 × 4.6 mm, 5 μm) was set at 270 nm for estimation of gallic acid and 259 nm for estimation of quercetin [36, 37]. HPLC PU-2080 Plus (Systronics) with UV-2075 plus intelligent detector and HPLC C18 column (250 × 4.6 mm, 5 μm) was set at 270 nm for estimation of gallic acid and 259 nm for estimation of quercetin. The mobile phase Acetonitrile and 2% Acetic Acid with ratio 40:60 used for elution of both compounds. Flow of mobile phase and injection loop was set at 1.0 ml/min and 20 μL respectively. Quantitative determination of gallic acid and quercetin content in fraction concentrations (FA004 by flash chromatography of amla extract) in the range 0.01 to 0.5 mg/ml and (Fraction FP004 by flash chromatography of pomegranate extract) in the concentration range of 0.01 to 0.5 μg/ml used.
