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DOI: 10.1055/a-2689-8131
Quantitative Analysis and Simultaneous Characterization of Triterpenoids and Phenolics in Inonotus obliquus (Chaga) Using LC-PDA-ELSD and LC-DAD-QToF
This research is supported in part by “Science Based Authentication of Botanical Ingredients” funded by the Center for Food Safety and Applied Nutrition, US Food and Drug Administration grant number 5U01FD004246, and “Discovery & Development of Natural Products for Pharmaceutical & Agricultural Applications” funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6060-6-015.
Abstract
Inonotus obliquus is widely recognized as the Chaga mushroom. Chaga contains various bioactive compounds, including polysaccharides, triterpenoids, polyphenols, and melanin. To address the characterization and quantitative analysis of triterpenoids and phenolics in Chaga, a multi-analytical approach has been developed combining LC-PDA-ELSD and LC-DAD-QToF. These methods were designed to quantify 11 compounds, comprising seven triterpenoids and four fatty acids, using LC-PDA-ELSD, and four phenolics using the LC-DAD-QToF method. Calibration curves for these compounds demonstrated excellent linearity within the tested range. The methods exhibited high precision, with intra- and inter-day relative standard deviations below 3% and recoveries ranged from 91% to 104%. The validated methods were applied to analyze eleven sclerotia samples, one mycelium sample, three grain-based samples, and eighteen dietary supplements. Results revealed that eight of the eighteen supplements (44%) contained ground mycelium, which primarily showed the presence of fatty acids but lacks detectable levels of triterpenoid and phenolic markers characteristic of Chaga. Triterpenoids and hispidin, identified as key bioactive compounds, were detected in eight (44%) of the eighteen supplements; however, these products also contained fatty acids and/or betulin. Two (11%) of the 18 supplements showed the presence of phenolic compounds only; no triterpenoids were detected. Additionally, untargeted metabolomic screening using LC-DAD-QToF tentatively identified 103 compounds from diverse chemical groups, including nine reference compounds. These findings provide valuable insights for the quality assessment of dietary or food supplements marketed as containing Chaga.
Keywords
Inonotus obliquus - Hymenochaetaceae - dietary supplements - LC-PDA-ELSD - LC-DAD-QToF - styrylpyrones - terpenoids - phenolicsPublication History
Received: 03 June 2025
Accepted after revision: 24 August 2025
Accepted Manuscript online:
25 August 2025
Article published online:
11 September 2025
© 2025. Thieme. All rights reserved.
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References
- 1 Barros L, Cruz T, Baptista P, Estevinho LM, Ferreira ICFR. Wild and commercial mushrooms as source of nutrients and nutraceuticals. Food Chem Toxicol 2008; 46: 2742-2747
- 2 Fordjour E, Manful CF, Javed R, Galagedara LW, Cuss CW, Cheema M, Thomas R. Chaga mushroom: A super-fungus with countless facets and untapped potential. Front Pharmacol 2023; 14: 1273786
- 3 Wolfe D. Chaga: King of the Medicinal Mushrooms. 1st Ed.. Berkeley, CA, USA: North Atlantic Books; 2012: 20-32
- 4 Shikov AN, Pozharitskaya ON, Makarov VG, Wagner H, Verpoorte R, Heinrich M. Medicinal plants of the Russian pharmacopoeia; Their history and applications. J Ethnopharmacol 2014; 154: 481-536
- 5 Balandaykin ME, Zmitrovich IV. Review on chaga medicinal mushroom, Inonotus obliquus (Higher Basidiomycetes): Realm of medicinal applications and approaches on estimating its resource potential. Int J Med Mushrooms 2015; 17: 95-104
- 6 Global Industry Analysts. Inc. Chaga Mushroom-Based Products – Global Strategic Business Report. 2025: 322. https://www.researchandmarkets.com/report/chaga-mushroom
- 7 Wold CW, Christopoulos PF, Arias MA, Dzovor DE, Øynebråten I, Corthay A, Inngjerdingen KT. Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity. Commun Biol 2024; 7: 222
- 8 Glamočlija J, Ćirić A, Nikolić M, Fernandes Â, Barros L, Calhelha RC, Ferreira ICFR, Soković M, van Griensven LJLD. Chemical characterization and biological activity of Chaga (Inonotus obliquus), a medicinal “mushroom”. J Ethnopharmacol 2015; 162: 323-332
- 9 Alhallaf W, Perkins LB. The anti-inflammatory properties of chaga extracts obtained by different extraction methods against LPS-induced RAW 264.7. Molecules 2022; 27: 4207
- 10 Baek J, Roh HS, Baek KH, Lee S, Lee S, Song SS, Kim KH. Bioactivity-based analysis and chemical characterization of cytotoxic constituents from Chaga mushroom (Inonotus obliquus) that induce apoptosis in human lung adenocarcinoma cells. J Ethnopharmacol 2018; 224: 63-75
- 11 Wold CW, Kjeldsen C, Corthay A, Rise F, Christensen BE, Duus JØ, Inngjerdingen KT. Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga). Carbohydr Polym 2018; 185: 27-40
- 12 Wold CW, Gerwick WH, Wangensteen H, Inngjerdingen KT. Bioactive triterpenoids and water-soluble melanin from Inonotus obliquus (Chaga) with immunomodulatory activity. J Funct Foods 2020; 71: 104025
- 13 Liu C, Zhao C, Pan HH, Kang J, Yu XT, Wang HQ, Li BM, Xie YZ, Chen RY. Chemical constituents from Inonotus obliquus and their biological activities. J Nat Prod 2014; 77: 35-41
- 14 Nikitina SA, Habibrakhmanova VR, Sysoeva MA. Chemical composition and biological activity of triterpenes and steroids of Chaga mushroom. Biochem (Moscow) Suppl B: Biomed Chem 2016; 10: 63-69
- 15 Alzand KI, Ünal S, Boufaris MSM. Lanostane-type triterpenes and abietane-type diterpene from the sclerotia of chaga medicinal mushroom, Inonotus obliquus (Agaricomycetes), and their biological activities. Int J Med Mushrooms 2018; 20: 507-516
- 16 Han P, Fereidoon S. Qualitative analysis of secondary metabolites of Chaga mushroom (Inonotus Obliquus): phenolics, fatty acids, and terpenoids. J Food Bioact 2022; 17: 56-72
- 17 Thomas P, Elkhateeb W, Daba G. Chaga (Inonotus obliquus): A medical marvel but a conservation dilemma?. Sydowia 2020; 72: 123-130
- 18 Windsor C, Kreynes AE, Chilton JS, Chioffi WA, Krishnamurthy A, Ishii M. Comparative study of chaga (Inonotus obliquus) dietary supplements using complementary analytical techniques. Int J Mol Sci 2025; 26: 2970
- 19 Kim JH, Park SK, Cho CW, Le BV, Kim YH, Bao H, Kim KT, Kang JS. Quality control and evaluation of Inonotus obliquus using HPLC method with novel marker compounds. J Anal Sci Technol 2020; 11: 52
- 20 Huynh N, Beltrame G, Tarvainen M, Suomela JP, Yang B. Supercritical CO2 extraction of triterpenoids from chaga sterile conk of Inonotus obliquus . Molecules 2022; 27: 1880
- 21 Upska K, Klavins L, Radenkovs V, Nikolajeva V, Faven L, Isosaari E, Lauberts M, Busa L, Viksna A, Klavins M. Extraction possibilities of lipid fraction and authentication assessment of Chaga (Inonotus obliquus). Biomass Convers Biorefin 2023; 13: 14005-14021
- 22 Géry A, Dubreule C, André V, Rioult JP, Bouchart V, Heutte N, Eldin de Pécoulas P, Krivomaz T, Garon D. Chaga (Inonotus obliquus), a future potential medicinal fungus in oncology? A chemical study and a comparison of the cytotoxicity against human lung adenocarcinoma cells (A549) and human bronchial epithelial cells (BEAS-2B). Integr Cancer Ther 2018; 17: 832-843
- 23 Gornostai TG, Borovskii GG, Kashchenko NI, Olennikov DN. Phenolic compounds of Inonotus rheades (Agaricomycetes) mycelium: RP-UPLC-DAD-ESI/MS profile and effect of light wavelength on styrylpyrone content. Int J Med Mushrooms 2018; 20: 637-645
- 24 Du D, Zhu F, Chen X, Ju X, Feng Y, Qi LW, Jiang J. Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection. Phytochem Anal 2011; 22: 419-423
- 25 Chilton J. Commercial sample identification and characterization challenges in medicinal mushroom research. Int J Med Mushrooms 2016; 18: 203-204
- 26 Sułkowska-Ziaja K, Robak J, Szczepkowski A, Gunia-Krzyżak A, Popiół J, Piotrowska J, Rospond B, Szewczyk A, Kała K, Muszyńska B. Comparison of bioactive secondary metabolites and cytotoxicity of extracts from Inonotus obliquus isolates from different host species. Molecules 2023; 28: 4907
- 27 Räsänen RM, Hieta JP, Immanen J, Nieminen K, Haavikko R, Yli-Kauhaluoma J, Kauppila TJ. Chemical profiles of birch and alder bark by ambient mass spectrometry. Anal Bioanal Chem 2019; 411: 7573-7583
- 28 Lee IK, Yun BS. Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus . Bioorg Med Chem 2007; 15: 3309-3314
- 29 Lee IK, Yun BS. Styrylpyrone-class compounds from medicinal fungi Phellinus and Inonotus spp., and their medicinal importance. J Antibiot 2011; 64: 349-359
- 30 Francescato LN, Debenedetti SL, Schwanz TG, Bassani VL, Henriques AT. Identification of phenolic compounds in Equisetum giganteum by LC–ESI-MS/MS and a new approach to total flavonoid quantification. Talanta 2013; 105: 192-203
- 31 Fiasson JL. Distribution of styrylpyrones in the basidiocarps of various Hymenochaetaceae. Biochem Syst Ecol 1982; 10: 289-296
- 32 Phan CW, Wang JK, Cheah SC, Naidu M, David P, Sabaratnam V. A review on the nucleic acid constituents in mushrooms: Nucleobases, nucleosides and nucleotides. Crit Rev Biotechnol 2018; 38: 762-777
- 33 Borman P, Elder D. Q2(R1) Validation of Analytical Procedures. In: ICH Quality Guidelines. 2017: 127 – 166. In ICH Quality Guidelines: An Implementation Guide, Chapter 5. John Wiley & Sons, Inc. Hoboken, NJ, USA. Teasdale A, Elder D, Nims RW (eds.).