The pharmacological activity of natural products for medicinal use can be investigated through analytical techniques such as Nuclear Magnetic Resonance NMR spectroscopy and Mass Spectrometry MS by following these steps:1. Extraction and isolation of bioactive compounds:The first step involves extracting the bioactive compounds from the natural source e.g., plants, fungi, or marine organisms using various extraction techniques such as solvent extraction, steam distillation, or cold pressing. The crude extract obtained is then subjected to purification and isolation techniques such as column chromatography, preparative HPLC, or solid-phase extraction to obtain the pure bioactive compounds.2. Structural elucidation using NMR spectroscopy:Once the bioactive compounds are isolated, their structures need to be elucidated to understand their pharmacological activity. NMR spectroscopy is a powerful technique for determining the structure of organic molecules. It provides information about the number and types of atoms, their connectivity, and the three-dimensional arrangement of the atoms in the molecule. By analyzing the NMR spectra 1H-NMR, 13C-NMR, and 2D-NMR experiments like COSY, HSQC, and HMBC , the structure of the bioactive compound can be determined.3. Molecular weight determination using Mass spectrometry:Mass spectrometry is an analytical technique used to determine the molecular weight and structural information of a compound. The bioactive compound is ionized, and the resulting ions are separated based on their mass-to-charge ratio m/z using various mass analyzers such as time-of-flight TOF , quadrupole, or ion trap. The mass spectrum obtained provides the molecular weight of the compound and can also give information about the fragmentation pattern, which helps in confirming the structure elucidated by NMR spectroscopy.4. Biological activity testing:After the structure of the bioactive compound is determined, its pharmacological activity can be investigated by testing it against various biological targets such as enzymes, receptors, or cell lines. This can be done using in vitro assays, cell-based assays, or in vivo animal models. The results obtained from these tests help in understanding the mechanism of action, potency, and selectivity of the compound.5. Structure-activity relationship SAR studies:By synthesizing and testing a series of structurally related compounds, the relationship between the structure of the bioactive compound and its pharmacological activity can be established. This information is crucial for optimizing the compound's potency, selectivity, and pharmacokinetic properties, leading to the development of more effective and safer drugs.In conclusion, NMR spectroscopy and Mass spectrometry are essential analytical techniques for investigating the pharmacological activity of natural products for medicinal use. They provide valuable information about the structure and molecular weight of the bioactive compounds, which can be further correlated with their biological activities through SAR studies and optimization of their pharmacological properties.