**2.1 Extraction process**

The extraction process has been completed based on the method described by Alam et al. [32]. About 500 g of sun and shade dried black pepper has been converted into powdered material by using a blender. Powdered black pepper was then transferred into an opaque bottle, and it has been filled with 1.5 L of methanol as a mother solvent for this experiment. The plant materials were kept in the bottle for 7 days with occasional shaking for better extraction (it can be called crude black pepper extract). The extracts of black pepper have been filtered through a fresh cotton plug and finally with Whatman No.1 filter papers. The filtrates found from the extraction process had been sent into the rotary evaporator (Bibby Sterlin Ltd, UK) to get a concentrated mass of sample under reduced pressure at 50 °C.

#### **2.2 Fractionation (in between liquid-liquid solvent) process**

The obtained concentrated mass of black pepper had been partitioned in between liquid solvent as described in **Figure 3**, where ultimately, a crystal seed fraction has been found.

## **2.3 Recrystallization process**

Recrystallization is a process through which we can easily eradicate our desired compound from a mixture of undesired one by forming crystal of that compound in a favorable solvent, and simply it could be designated as an efficient method of purification [33]. As we have found a crystal seed fraction from black pepper, so we have utilized this recrystallization technique mentioned in **Figure 4** to purify crystal.

The flow diagram we have mentioned above had been followed to purify our crystal seed. The crystal seed was washed with KOH to remove resin-like compound or isomer of the crystal seed, and it was then filtered through filter paper for preliminary purification. Now it has been treated with petroleum ether for recrystallization and warmed up the combination to dissolve it at 50 °C. After that, the mixture was cooled to form crystal. The crystal is filtered for final purification.

#### **Figure 3.**

*Overall fractionation process of crude extract of black pepper in between liquid-liquid solvent.*

**Figure 4.**

*Recrystallization technique for crystal seed produced from chloroform fraction.*
