Methods of Qualitative and Quantitative Analysis of Plant Constituents

Abstract

Plant kingdom harbors an inexhaustible source of active drug ingredients. Phytochemical techniques plays a significant role in searching raw materials and resources for pharmaceutical industry. Drug discovery is a lengthy procedure and it involves a number of successive processes including (i) extraction, (ii) separation, (iii) isolation of the constituents of interest, (iv) purification, (v) characterization, and (vi) identification of the isolated compounds and also their quantitative estimation. Both dried and fresh plant materials may be used for extraction following different procedures with (water, ether, acetone, methanol, ethanol, chloroform, etc.) or without (expression, sublimation, and distillation) the use of solvents. Extraction with water may be without (infusion) or with boiling (decoction) and extraction with organic solvents involves maceration, percolation, soxhlet extraction, etc. Phytochemical screening of crude extract can be performed with the appropriate tests for different active ingredients, e.g., alkaloids (Dragendorff, Mayer, Hager, and Wagner’s spot test), tannins (Ferric chloride test), Anthraquinone (Borntrager’s test), flavonoids (Shinoda test-HCL test and Lead acetate test), glycosides (Fehling’s test and Glacial acetic acid test), Cardiac glycosides (Kellar–Kiliani test), terpenoids and steroids (H₂SO₄ test), saponins (Foam test), fixed oil (Spot test), Amino acids and proteins (Ninhydrin test and copper sulfate test) and terpenes (Liberman–Burchard), steroid (Liebermann–Burchard test), phenol (phenol test), and tannins (Braemer’s test), etc. High-Throughput Screening (HTS) is a recent approach to accelerated drug discovery (e.g., screening a few thousand compounds per day or per week) and consists of several steps such as target identification, reagent preparation, compound management, assay development and high-throughput library screening including combinatorial chemistry, genomics, protein, and peptide libraries. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods, e.g., LC-MS/MS. The extracted chemical constituents are separated by various separation techniques such as fractional distillation, fractional liberation, fractional crystallization, chromatography, HPLC, etc. Isolation is a crucial step in the analysis of medicinal plants and the basic operation included steps, such as prewashing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Phytochemical characterization primarily may be initiated with the help of qualitative tests for the screening of phytochemical compounds. Characterization and identification of the separated and isolated constituents are the final steps in the photochemical analysis of plants. A pure compound is characterized and identified by determining its various physical and chemical properties like Rf value, melting point, optical values, nature and type of crystals, types and number of elements and functional groups present in the molecule, etc., by the use of different chemical tests and reactions, chromatographic techniques, crystallographic and spectroscopic methods, etc. The pure compounds are further used for the determination of structure and biological activity. In addition, various non-chromatographic techniques (immunoassay—MAbs, phytochemical screening assay, and FTIR) can also be used to facilitate the identification of the bioactive compounds. Bioassay (brine shrimp toxicity assay, crown gall tumor inhibition assay, potato disc antitumor assay-PDA, animal toxicity assay, antiviral, antimicrobial and antifungal assays, antimitotic assay, etc.) is a life-based activity-directed isolation process and its goal is to isolate bioactive compounds with certain definite degree of LD/LC/IC₅₀ value as a proof of cytotoxicity. Polymerase chain reaction (PCR)-based DNA technology of molecular biology now appears to be the basic analytical procedure in molecular pharmacognosy.