Devise a suitable approach that you would use to determine the phenolic, polysaccharide, fixed oil, terpenoid and alkaloid content in a Medicinal plant? In doing so, please take the following points into consideration: (i) Physicochemical properties of the test analytes. (ii) Method of sample preparation prior to analysis of test analytes. (iii) Factors that you would consider when deciding on an appropriate analytical method.
Answer
EudraLex Volume 4, Annex 7 deals with the manufacture of Herbal Medicinal Products, including Quality Control (1), but does not give much information on how to determine quality attributes. Ph. Eur. monograph 1433 defines herbal drugs as “mainly whole, fragmented or broken plants or parts of plants in an unprocessed state, usually in dried form but sometimes fresh” (2). The monograph details general tests common to most drugs, such as identification, contamination (foreign matter, pesticides, heavy metals, radioactive and microbial), moisture (LOD or water content), ash (total and insoluble) as well as less common tests, such as swelling index, bitterness value, Aflatoxin B1 and Ochratoxin A. For quantitative purposes, the monograph requires dried herbal drugs be appropriately assayed, but like the EudraLex, it does not provide significant information on how this is to be done. Sampling and sample preparation are defined in Ph. Eur. method 2.8.20 (3). To achieve a homogenous representation of the batch of medicinal plant, the method requires taking multiple samples based on predefined criteria. The number of samples and total sample mass is based on batch mass and number of containers used for storage. The individual samples are then mixed to produce the final bulk sample, which is “quartered” repeatedly until the test sample weight is achieved. The sample is then milled and sieved. This sample will be the basis for the testing below, unless otherwise stated.
Phenolic Content
Phenols are hydroxyl derivatives of aromatic hydrocarbons and are secondary metabolites of many medicinal plants. They have a wide variety of uses. Methods have been described for the analysis of total phenolic content using UV analysis (4). The sample is macerated for 48 hours with methanol, filtrated and dilution to an appropriate volume, followed by a reaction with Folin-Ciocalteu reagent. The sample is then read versus a tannic acid standard curve to calculate total content. This method is not specific. Alternative, specific HPLC methods are available (5). Analytes are extracted by stirring with aqueous acetone and filtered. Acetone is removed under vacuum, then the sample is extracted again using ethyl acetate, dried and finally dissolved in methanol for HPLC analysis versus a reference standard mix (due to the presence of multiple phenolic compounds in the sample in question). The same approach could be taken with any phenol, with appropriate method optimization. Due to the specificity of this method, it could be used to calculate content of multiple known phenolic compounds in the medicinal plant.
Polysaccharide Content
Polysaccharides are sugar-based polymers, medically useful mainly as excipients in pharmaceutical preparations. The author assumes this is the reason no analytical procedures for content analysis of polysaccharides are published in the Ph. Eur. The monographs reviewed only contained identification methods. Compendial methods for identification are based on precipitation with lead salts, or hydrolysis using acid to the constituent sugars, after which thin layer chromatography may also be used to confirm the identity. CreativeBiolabs (6) describe the steps in characterization of polysaccharides: “Polysaccharides are very intricate complex, so the process of analysis is also manifold. Generally, monosaccharides composition and their linkage positions and sequence should be determined first. Then, the anomeric configuration of linkages, the ring size (furanose or pyranose), the absolute configuration (D or L), and any other substituents present should be identified. Up to now, there is no single method that can complete polysaccharide alone. Usually, separation and extraction techniques, component analysis, methylation analysis, glycosidic hydrolysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are required to determine the fine structure while size exclusion chromatography (SEC) coupled with light scattering (LS) detectors to determine the molecular size distribution.” Once characterization is complete, and assuming a small number of polysaccharide analytes of interest are in the medicinal plant, it may be possible to develop a QC appropriate method for content analysis. Polysaccharides are generally water soluble, therefore an appropriate sample preparation would be an extraction into water from the milled and sieved bulk sample. Due to the large molecule size of polysaccharides, size exclusion HPLC (or UPLC) would provide appropriate resolution of analytes. UV detection may be possible if the analyte contains one or more chromophores, otherwise derivatization of the sample would be necessary. Content could be calculated as a percentage of the dry weight of the bulk using an appropriate reference standard.
Fixed Oil Content
Fixed oils are glycerides of fatty acids which are liquid at room temperature. They are industrially extracted by compression. The analytical methods described in the course book are for the assessment of the identity, standardization, and adulteration. Note that fixed oils decompose by distillation, therefore the dry sample preparation described above may be inappropriate. Compression of the fresh material to produce oil for analysis may be required. In this case, content would be calculate as: xxxxxxx
Analytical techniques for fixed oils include physical measurements (melting point, relative density, refractive index and solubility), qualitative impurities testing, chemical constants by titration (acid, iodine, saponification hydroxyl and peroxide values). Generic methods are available (7) for the analysis of composition of fixed oils by Gas Liquid Chromatography. Methyl esters are formed using either boron trifloride or methanol and alkali, which are then injected onto the system and compared to reference standards. Theoretically if the standard is appropriately characterized, this method would also be appropriate to provide he %purity needed for the calculation of content outlined above. Terpenoid content Terpenoids are a large group of secondary metabolites of acetyl-coenzyme metabolism, and have multiple uses therapeutically, as disinfectants, or for their flavor and scent. Compendial physicochemical methods (such as freezing point, optical rotation, solubility in alcohol etc.) may be employed to identify terpenoids, but these methods cannot determine the quality or content of terpenoids in a medicinal plant.
Terpenoids tend to occur as volatile mixture. As such, distillation may be an appropriate method of extraction for analysis, however it would be easier to extract directly from the sample into a chromatographic friendly solvent, followed by filtration. Hexane or acetone may be used. Note, as with fixed oils, the dry sample preparation described above may be inappropriate (volatile terpenoids may be lost in this process), and “fresh” bulk sampling may be required. For content analysis of simple terpenoids, Gas Chromatography with an FID detector is ideal, as a “fingerprint chromatogram” is generated. Constituents are identified by relative retention time (and may be quantitated as a percent of the main peak). The main component may be calculated versus a suitable herbal reference standard. Any contamination is easily identified as a peak not expected in the “fingerprint”. Where complex mixtures are to be analyzed, the sample may first be separated into fractions by HPLC, and each fraction analyzed separately on the GC. If accurate identification is required, Mass Spectroscopy may be combined with GC or HPLC, although this would not benefit routine QC content quantitation
Alkaloid content
Alkaloids are organic, nitrogen containing bases, and are divided into multiple classes, depending on the parent amino acid. Morphine and nicotine are examples of pharmacologically active, naturally occurring alkaloids. Alkaloids tend to be present in medicinal plants as salts at low levels, therefore extractions must be carried out carefully to avoid loss or decomposition. Three general extraction methods are available. The first two are for alkaloids which are not heat labile and involve extractions under reflux followed by partition separation with aqueous / organic matrixes, and finally drying. The main difference between these methods is at what point the alkaloid is turned to a base. Method 1 does it towards the end of the extraction, where method 2 does it initially, within the plant material. A “cold” method involves maceration in a percolator with alkaline (to generate the base form) and organic solvent, followed by concentration in vacuum, purified by partitioning and again evaporated in vacuum. The crude alkaloid content may be determined by titration or UV absorption. For a specific methodology, HPLC analysis may be used (several compendial methods now require HPCL).
References:
1) EudraLex. Brussels; European Commission – Enterprise and Industry Directorate General; 2009. The Rules Governing Medicinal Products in the European Union -Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, Annex 7 Manufacture of Herbal Medicinal Products
2) European Pharmacopoeia, Vol. 10. France: EDQM Council of Europe; 2021. Herbal Drugs (monograph 1433)
3) European Pharmacopoeia, Vol. 10. France: EDQM Council of Europe; 2021. Herbal Drugs: Herbal Drugs: Sampling and Sample Preparation (method 2.8.20)
4) Attard, E (2013). ‘A rapid microtitre plate Folin-Ciocalteu method for the assessment of polypenols’. Central European Journal of Biology, 8(1), 2013, Pages 48 -53
5) Schieber, A. Keller P. Carle, R (2001). ‘Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography’. Journal of Chromatography, 910 (2), 2 March 200, Pages 265-273
6) Creative Biolabs. (2021). ‘Polysaccharides Analysis’. Creative Biolabs, New York, USA. Exact publication date unknown, 2021. Available from: https://www.creative-biolabs.com/glycoprotein/polysaccharides-analysis.htm
7) Food Safety and Standards Authority of India (2015). ‘Manual of Methods of Analysis of Foods – Oils and Fats’. Government of India, New Delhi, India. Exact publication date unknown, 2015. Available from: https://old.fssai.gov.in/Portals/0/Pdf/Draft_Manuals/OIAND_FAT.pd
