**2.9 Boiling point**

The crystal was so sensitive to increased level of heat, and it had been totally destructed during checking boiling point. So, the crystal material does not have any fixed boiling point.

#### **2.10 Phytochemical contents**

Alkaloid test for crystal (found from chloroform fraction of crude black pepper extract) had shown strong positive result according to **Table 2**, which clarifies that the crystal may have alkaloid compounds [36–38].

After comparing the physicochemical properties of crystal compound with standard one [39], it could be predicted that the crystal may be piperine.

#### **3. In-vitro free radical scavenging assay with results interpretation**

#### **3.1 DPPH radical scavenging activity of different fractions of plant materials**

Free radical scavenging ability of different fractions of sample had been evaluated by DPPH radical scavenging assay according to Blois and Desmarchelier et al. [40, 41]. The hydrogen atom donating capability of the plant extractives was estimated by identifying the color change of samples with 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH exhibits made violet/purple color in samples having antioxidant property, and it was then faded into yellowish color. 2.4 ml of DPPH solution was mixed with 1.6 ml of sample solution at different concentration. The reaction mixture has been vortexed and left it for 30 min in a dark place at room temperature. The absorbance was taken for individual fractions at different concentration via spectrophotometer at 517 nm. Butylated hydroxy toluene had been used as standard. The percentage of DPPH radical scavenging activity was calculated by the following equation:

%DPPH radical scavenging activity ¼ f g ð Þ A0 � A1 *=*A0 � 100

where A0 represents the absorbance of the control, and A1 represents the absorbance of the sample materials. Then % of inhibition has been plotted against


**Table 2.**

*Phytochemical contents explored in crystal (found from chloroform fraction of crude black pepper extract).*

concentration, and the IC50 was calculated graphically. The same experiment was repeated for three times with every fraction at each concentration.

#### *3.1.1 Data interpretation of DPPH radical scavenging assay*

According to **Table 3**, it could be easily assumed that the crystal compound may have a great antioxidant power as the data for that compound had shown a nearly similar value of standard catechin. The IC50 value was so small, which was an indication for strong free radical scavenger.
