**6.1 Analytical methodologies**

It plays an eminent role in herbal natural drug discovery, and without analytical methodologies, it is hardly impossible. Spectroscopic characterization is the backbone and pillar of herbal drug discovery. The knowledge of this plays an important role in developing the new lead, which can be used for designing new molecules with short modification. The important steps are the extraction, isolation, and characterization of active ingredients from herbal plants [16]. Different techniques of extraction are well known as extraction is the most important step toward the analysis of bioactive constituents. Microwave-assisted extraction and conventional extraction should be studied specifically, which give the ideas about the yield obtained. Further, it highlights the isolation of active molecules by chromatographic techniques like TLC, column chromatography. The most important step toward analysis of bioactive compounds present in the plant extracts is characterization, which includes phytochemical screening assays that give ideas about the presence of secondary metabolites used to cure the health problems. Highly sophisticated techniques for structure identification of lead molecule bioactive fraction are high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gas chromatography–mass spectrometry (GC–MS). These techniques are the heart and key challenges in research of natural drug discovery giving rise to natural products in drug discovery.

#### **6.2 Isolation, identification, and characterization of phytochemicals**

The combination of various types of bioactive compound or phytochemicals is usually present in different plant extracts. The different bioactive compounds have different polarities. Separation, identification, and characterization of bioactive compounds are a big challenging job in the herbal drug development process.
