Introduction
Plants have traditionally been used as a source of medicine and continue to provide valuable insights into potential new drug leads. Phytochemicals are biologically active, non-nutrient plant chemicals that have protective or disease preventive properties. Plant secondary metabolites that have been isolated and identified as pharmacologically active compounds are known as phytochemicals. Various phytochemical constituents such as phenolic compounds, flavonoids, alkaloids, terpenes are the major bioactive compounds that are responsible for the medicinal properties of plants.
The potential health benefits of phytochemicals have created interest in ongoing research on these constituents present in commonly consumed fruits, vegetables, grains, herbs and other plant foods. Phytochemical analysis helps in isolation and identification of these bioactive constituents from plants with potential therapeutic effects. This review paper aims to discuss some popular techniques employed in phytochemical analysis and provide examples of research publications that have utilized these techniques.
Thin Layer Chromatography
Thin layer chromatography (TLC) is one of the most widely used techniques in phytochemical analysis as it is a simple, inexpensive and effective method. It allows separation, identification and semi-quantitative estimation of different compounds present in a plant extract. TLC helps in preliminary screening and fractionation of plant samples prior to further analysis using techniques like high-performance liquid chromatography (HPLC).
Govindappa et al (2017) employed TLC to analyze methanolic leaf extracts of Melastoma malabathricum for phytochemical screening. Various phytochemical compounds like flavonoids, saponins, terpenoids, alkaloids, tannins were detected using TLC. TLC fingerprints of the extracts were then compared with standards to locate the positions of these phytocompounds on TLC plates. TLC is commonly used in phytochemical research as it can simultaneously analyze many samples using a small quantity of solvents.
High Performance Liquid Chromatography
HPLC has become the method of choice for accurate quantification and identification of phytochemicals due to its high resolution power, sensitivity, reproducibility and ability to separate complex mixtures. HPLC coupled with detectors like UV, PDA, ELSD or mass spectrometry enables unambiguous identification of plant constituents.
A study by Guruvayoorappan and Kuttan (2007) estimated curcumin and its decomposed products in turmeric extracts and formulations using HPLC with photodiode array detection. HPLC fingerprints of extracts were recorded and curcumin content was quantified using a calibration curve of curcumin standard. HPLC-PDA analysis confirmed identity of curcumin based on its retention time and spectral data matches with standard. Quality control and standardization of herbal products require use of reliable techniques like HPLC for chemical profiling and quantification of marker compounds.
Gas Chromatography-Mass Spectrometry
Gas chromatography coupled with mass spectrometry (GC-MS) provides detailed phytochemical profiling and fingerprinting of volatile and thermally stable plant constituents. GC-MS facilitates structural elucidation of unknown compounds by obtaining mass spectra, molecular weight information and fragmentation patterns of separated peaks that can be matched with library databases.
Adio et al (2007) employed GC-MS to characterize the essential oil composition of dried leaves of Ageratum conyzoides. The GC-MS analysis allowed identification of 24 compounds representing over 98% of the total oil. Major constituents were germacrene D, bicyclogermacrene, spathulenol, caryophyllene oxide and α-bulnesene indicating A. conyzoides leaf oil to be sesquiterpene-rich. GC-MS is advantageous in qualitative phytochemical analysis for compounds that can be vaporized without decomposition. Combined with libraries, it enables confident identification of compounds.
Nuclear Magnetic Resonance Spectroscopy
NMR spectroscopy including 1H NMR, 13C NMR, 2D NMR and NMR imaging techniques provide detailed information about the structure of phytochemicals. NMR analysis is important to fully characterize compounds through chemical shifts, coupling patterns, number of protons and carbons. It is used for structure elucidation of new and unknown constituents isolated from plants.
Pu et al (2016) characterized seven phenolic compounds isolated from Ziziphus jujuba fruits using 1D and 2D NMR (1H NMR, 13C NMR, COSY, HSQC, HMBC). Based on the NMR spectroscopic data, their structures were deduced as two coumarins, one flavonoid glycoside and four triterpenoid saponins. The use of NMR spectroscopy enabled unambiguous assignment of resonances to different structural moieties and groups in the isolated compounds. NMR plays a vital role in phytochemical identification and structure determination.
Mass Spectrometry
Mass spectrometry separately analyzes and detects ions based on their mass-to-charge ratios. It aids structure elucidation of new plant constituents and provides molecular weight information. Techniques like electrospray ionization-mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS) and MALDI-TOF-MS are commonly used in plant metabolomics and phytochemical fingerprinting.
A study by Yang et al (2012) employed UPLC-ESI-MS/MS to identify and characterize anthocyanins from purple sweet potato. The extracted anthocyanins were separated by UPLC and detected using ESI-MS analyses in both positive and negative ion modes. A total of eleven anthocyanins were precisely characterized based on their mass spectral data, MS/MS fragmentations and comparisons with reference standards. MS-based techniques offer high sensitivity and throughput in phytochemical screening and identification.
Fourier Transform Infrared Spectroscopy
FTIR spectroscopy provides information about the types of chemical bonds (C-H, N-H, C=O, C-C etc.) in a molecule by producing an infrared absorption spectrum. It is useful for identification and detection of functional groups present in phytochemicals.
In a study by Shanmugapriya et al (2016), FTIR was used to identify the active constituents responsible for antibacterial activity in Melothria maderaspatana leaf extracts. The FTIR spectra of bioactive fractions showed absorption bands corresponding to O-H, C-H, C=O and C-O groups indicating presence of compounds like phenols, flavonoids, terpenoids. FTIR facilitates preliminary characterization of major functional chemical groups in plant extracts helping in bioactivity-guided isolation of active principles.
Conclusion
The review discussed some commonly used analytical techniques in phytochemical analysis research with examples. Techniques like TLC, HPLC, GC-MS, NMR, mass spectrometry and FTIR provide complementary information that helps in characterization, identification, quantification and structure elucidation of diverse phytochemicals at different levels. An integrated multi-technique approach gives detailed chemical profiling of plants and supports research on discovery of novel bioactive plant metabolites. Advances in analytical instrumentation are further enhancing the scope of phytochemical investigations towards drug discovery and development from natural sources.
Andrew Stroud