Methods of Qualitative and Quantitative Analysis of Plant Constituents

  • A. N. M. AlamgirEmail author
Part of the Progress in Drug Research book series (PDR, volume 74)


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.


Analysis of plant constituents Extraction Separation Isolation Purification Characterization and identification of plant constituents Bioassay techniques PCR-based DNA technology 


  1. Akhtar MS, Athar MA, Yaqub M (1981) Effect of Momordica charantia on blood glucose levels of normal and alloxan diabetic rabbits. Planta Med 42:205–212CrossRefPubMedGoogle Scholar
  2. Amarasingham RD, Bisset NG, Millard AH, Woods MC (1964) A phytochemical survey of Malaya III. Alkaloids and saponins. Econ Bot 18:270–278CrossRefGoogle Scholar
  3. Aplin TEH, Cannon JR (1971) Distribution of alkaloids in some western Australian plants. Eco Bot 25(4):366–380CrossRefGoogle Scholar
  4. Aquino R, De Feo V, De Simone F, Pizza C, Cirino G (1991) Plant metabolites. New compounds and anti-inflammatory activity of Uncaria tomentosa. J Nat Prod 54(2):453–459CrossRefPubMedGoogle Scholar
  5. Eberhardt TL, Li X, Shupe TF, Hse CY (2007) Chinese Tallow Tree (Sapium sebiferum) utilization: characterization of extractives and cell-wall chemistry. Wood Fiber Sci 39:319–324Google Scholar
  6. Einhelling FA, Leather GR, Hobbs LL (1985) Use of Lemna minor L. as a bioassay in allelopathy. J Chem Ecol 11(1):65–72CrossRefGoogle Scholar
  7. Farnsworth NR, Soejarto DD (1991) Global importance of medicinal plants. In: Akerele O, Heywood V, Synge H (eds) The conservation of medicinal plants. Cambridge University Press, Cambridge, UK, pp 25–51CrossRefGoogle Scholar
  8. Ferrigni NR, Putman JE, Anderson B, Jacobsen LB, Nichols DE, Moore DS et al (1982) Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaceae seeds. J Nat Prod 45:679–686CrossRefPubMedGoogle Scholar
  9. Fings CS, Tatliff CR, Dunn RT (1970) Glucose determination by o-toluidine method using acetic acid, ‘Clinical Chemistry’ by Toro C, Ackerman PG, vol 115. Little Browning and Company, BostonGoogle Scholar
  10. Goldstein A (1964) Bio-statistics, an introductory text. McMillan Co., New York, USA, pp 172–178Google Scholar
  11. Hamburger MO, Cordell GA (1987) A direct bioautographic TLC assay for compounds possessing antibacterial activity. J Nat Prod 50(1):19–22CrossRefPubMedGoogle Scholar
  12. Harborne JB (1984) Phytochemical methods: a guide to modern techniques of plant analysis, 2nd edn. Chapman and Hall ltd., London, New York, p 1984CrossRefGoogle Scholar
  13. Hazra KM, Roy RN, Sen SK, Laska S (2007) Isolation of antibacterial pentahydroxy flavones from the seeds of Mimusops elengi Linn. Afr J Biotechnol 6(12):1446–1449Google Scholar
  14. Holstege DM, Seiber JN, Galey FD (1995) Rapid multiresidue screen for alkaloids in plant material and biological samples. J Agric Food Chem 43:691–699CrossRefGoogle Scholar
  15. Hostettmann K, Kizu H, Tomimori T (1982) Molluscicidal properties of various saponins. Planta Med 44:34–35CrossRefPubMedGoogle Scholar
  16. Jacobs RS, White S, Wilson L (1981) Selective compounds derived from marine organisms: effects on cell division in fertilized sea urchin eggs. Fed Proc 39:26–29Google Scholar
  17. Kavanagh F (1963) Analytical microbiology. In: Kavanagh F (ed) Academic Press, London, pp 125–141CrossRefGoogle Scholar
  18. Kawashima K, Miwa Y, Kimura M, Mizutani K, Hayashi A, Tanaka O (1985) Diuretic action of paeonol. Planta Med 3:187–189CrossRefGoogle Scholar
  19. Kawazu K (1981) Advances in natural products chemistry. In: Natori S, Itekawa N, Suzuki M, (eds) Wiley, New York, p 249Google Scholar
  20. Kazmi SU, Siddiqui R, Shekhani S (1990) Frontiers in natural products chemistry. In: Atta-ur-Rahman (ed), Shamim Printing Press, Karachi, pp 739–754Google Scholar
  21. Kumari S, Yasmin N, Hussain MR, Babuselvam M (2015) In vitro anti-inflammatory and anti-arthritic property of Rhizopora mucronata leaves. IJPSR 6:482–485Google Scholar
  22. Leelaprakash G, Dass SM (2011) In vitro anti-Inflammatory activity of methanol extract of Enicostemma Axillare. Int J Drug Dev Res 3(3):189–196Google Scholar
  23. Leven M, Vanden Berghe DA, Mertens F, Vlietinck A, Lammens E (1979) Screening of higher plants for biological activities. I. Antimicrobial activity. Planta Medica 36(4):311–321CrossRefGoogle Scholar
  24. McLaughlin JL (1991). Methods of Plant Biochemistry. In: Hostettmann K (ed) vol. 6. Academic Press, London, pp 1–32Google Scholar
  25. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med 45:31–34CrossRefPubMedGoogle Scholar
  26. Mizushima Y, Kobayashi M (1968) Interaction ofanti-inflammatory drugs with serum preoteins, especially with some biologically active proteins. J Pharma Pharmacol 20:169–173CrossRefGoogle Scholar
  27. Rahman AU, Choudhary MI, Thomson WJ (2001) Bioassay techniques for drug development. Harwood academic publishers, Australia, Canada, France, GermanyCrossRefGoogle Scholar
  28. Sakat S, Juvekar AR, Gambhire MN (2010) In vitro antioxidant and anti-inflammatory activity ofmethanol extract of Oxalis corniculata Linn. Int J Pharma Pharmacol Sci 2(1):146–155Google Scholar
  29. Schales O, Schales SS (1941) A simple and accurate method for the determination of chloride in biological fluids. J Biol Chem 140:879–884Google Scholar
  30. Shoyama Y, Tanaka H, Fukuda N (2003) Monoclonal antibodies against naturally occurring bioactive compounds. Cytotechnology 31:9–27CrossRefGoogle Scholar
  31. Snedecor GW (1965) Statistical methods, 5th edn. The Iowa State University Press, Ames, Iowa, USAGoogle Scholar
  32. Srivastava OP (1984) Techniques for the evaluation of antimicrobial properties of natural products. In: Dhawan BN, Srimal RC (eds) The use of pharmacological techniques for the evaluation of natural products. UNESCO, New Delhi, pp 72–79Google Scholar
  33. Webb LJ (1949) An Australian phytochemical survey.1. Alkaloids and cyanogenic compounds in Queensland plants. CSIRO Bull. 260, MelbourneGoogle Scholar
  34. White SJ, Jacobs RS (1981) Inhibition of cell division and of microtubule assembly by elatone, a halogenated sesquiterpene. Mol Pharmacol 20:614–620PubMedGoogle Scholar
  35. Zarroug MA, Nugud AD, Bashir AK, Mageed AA (1988) Evaluation of Sudanese plant extracts as mosquito larvicides. Int J Crude Drug Res 26:77–80CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BotanyChittagong UniversityChittagongBangladesh

Personalised recommendations