Original Research

Volume 17, March 2022, pages 56-72

Qualitative analysis of secondary metabolites of chaga mushroom (Inonotus Obliquus): phenolics, fatty acids, and terpenoids

Chaga mushroom is an edible herbal fungus that is mostly distributed in the circumboreal region of the Northern Hemisphere. The medicinal/nutraceutical use of chaga has been recorded in different ancient cultures, including Ainu and Khanty in Northeastern Asia, and various First nations such as Wet’suwet’en, Chipewyan, Cree, and Gitxsan in North America. Chaga is a Latinized Russian word known as the sterile conk trunk rot of birch in Canada. The official binomial name of chaga is Inonotus obliquus, but other names, including Phaeoporus obliquus, Polyporus obliquus, or Fuscoporia obliqua, have also been sporadically used in the earlier literature (He et al., 2001; Reid, 1976).

Attributed by its global anecdotal evidence of medicinal properties, chaga has been used as a functional beverage (tea) or folk medicine (decoction, ointment) for the treatment of stomach diseases, intestinal worms, liver/heart ailments, dermatomycoses, joint pain, and different types of cancer in the East European countries for centuries (Babitskaya et al., 2002; Koyama, 2017; Lemieszek et al., 2011; Peng and Shahidi, 2020; Saar, 1991; Shashkina et al., 2006; Shikov et al., 2014). To date, numerous studies have claimed various bioactivities, together with related biomolecular mechanism of chaga, including antioxidant, antimicrobial, anti-cancer, hypoglycemic, antilipidemic, anti-inflammation, abirritative, immunoregulatory, and cardioprotective effects (Koyama et al., 2008; Patel, 2015; Peng and Shahidi, 2020; Shashkina et al., 2006; Zhong et al., 2009). Such a broad spectrum of biological/pharmacological functions implies the complexity of bioactive substances in chaga, although related clinical data are relatively scarce. As a result, the bioactive compounds of chaga have gradually been unveiled over the past 20 years. Many chaga-based supplements have been commercialized in the current nutraceutical market. To satisfy and sustain the increased commercial demand for chaga products, the artificial culture of chaga has been practiced for decades to overcome the long growth period of wild chaga (Ka et al., 2017; Sun et al., 2011; Zheng et al., 2010).

The diversity and content of bioactive components of this parasitic fungus varies greatly according to its nutritional and environmental conditions, including physical (e.g., UV and γ-radiation), biological (e.g., host), and chemical (e.g., pH, oxygen, heavy metals, and exogenous phytochemicals) factors. For instance, ergosterol became the dominant sterols in the cultured mycelium of chaga, rather than lanosterol and inotodiol in wild chaga. Other trace sterols of wild chaga, such as episterol, 24-methylene dihydrolanosterol, and ergosterol peroxide can not be found in cultured mycelium (Zheng et al., 2007). Similar phenomenon was observed among different wild types of chaga. For example, the Canadian and Ukrainian chaga collected by Géry et al. (2018) contained around 1% and 10% of betulin and betulinic acid content of French chaga, respectively. The total phenolic content of decoction and tincture of chaga from Thailand is around 2 and 5–10 times of those harvested from Russia and Finland, even though the DPPH scavenging efficacy of chaga tincture from Finland is about 9–20 times higher than those from Thailand and Russia. Meanwhile, the content of p-hydroxybenzoic acid of Finland and Thailand chaga is around 2 and 20 times higher than that in the Russian chaga, respectively, while gallic acid can be detected in samples from Thailand but not in those from Finland and Russia (Glamočlija et al., 2015). Apparently, chaga from different production sites usually show distinguishable chemical profiles. To achieve a comparable compositional profile of the artificial culture of chaga mycelia, various physiochemical stimulus have been applied to mimic growth environments which efficiently manipulates the production of chaga secondary metabolites (Zheng et al., 2010). More information on compositional and proportional difference, and the production monitoring of secondary metabolites of chaga is found in a recent review, in which around 220 compounds including 108 terpenoids, 64 small-molecule phenolics, 10 alkaloids, 17 amino acids, and various bioactive polymers such as polysaccharides-protein complex and allomelanins were summarized (Peng and Shahidi, 2020). In combination with newly updated 21 phenolics and 10 terpenoids from 2021 to 2022, it provides the database for the current study of primary compositional characterization of wild chaga harvested in Newfoundland (Abu-Reidah et al., 2021; Chang et al., 2022; Kou et al., 2021). In this article, numerous bioactive compounds including small-molecule phenolics, terpenoids, and hydroxylated fatty acids were qualitatively identified using full-scan ToF mass spectrometry. Intriguingly, more than half of these secondary metabolites can not be found in previous reports, implying the significance of quality discrepancy of chaga materials from different production regions and its potential influences on the products’ performance and reliability. The establishment of reproducible qualification protocols of natural food supplements and related compositional databases can effectively promote the capability of companies and authorities in supervising their quality control, product standardization, and potential counterfeits. Therefore, this contribution aimed to provide a comparative analysis between small-molecule bioactive secondary metabolites from Newfoundland wild chaga and its counterparts reported elsewhere, as well as an updated database of phenolic, fatty acid, and terpenoid compounds.

HPLC grade reagents acetonitrile, ethanol, and methanol were purchased from Fisher Scientific, Ltd. (Ottawa, ON, Canada). Formic acid was purchased from ACROS Organics (Morris, IL, USA). Water was purified using a Milli-Q system, Millipore (Bedford, MA, USA).

Chaga powder was obtained locally from Dr. Aubrey Anderson from Department of Fisheries and Oceans (DFO), St. John’s, NL, Canada. For extraction, 500 mg chaga material was freeze-dried and then ultrasonically extracted twice with 10 mL solvent mixture consisting of water-ethyl acetate-acetone (20:40:40, v/v/v). The supernatants of the extracts were combined, 1 mL of which was filtered into LC sample vials without any further concentration.

The composition of extract was determined using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS), using an Agilent 1260 HPLC unit (Agilent Technologies, Palo Alto, CA, USA) with a UV diode array detector (UV-DAD) and a SUPERLCOSILTM LC-18 column (4.6 × 250 mm × 5 μm with guard column; Sigma-Aldrich, Oakville, ON, Canada) by gradient elution. The mobile phase consisted of 0.1% formic acid in deionized water (A) and 0.1% formic acid and 5% acetonitrile in methanol (B). The solvent gradient was as follows: 5–5% B, 0–6 min; 5–8% B, 6–16 min; 8–20% B, 16–40 min; 20–45% B, 40–75 min; 45–60% B, 75–85 min; 60–90% B 85–110 min; 90–100% B, 110–120 min; 100–100, 120–125 min; 100–5% B, 125–130 min. The column was thermostatically controlled at 40 °C, and the flow rate was set at 1 mL/min. The UV-visible absorbance of the peaks was monitored between 190 and 600 nm. All samples and standards were dissolved in methanol, and the sample injection volume was 5 μL. LC flow was further analyzed online by an orthogonal time-of-flight mass spectrometer (6230 TOF LC/MS system; Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ionization source (ESI). The ESI source was operated in the negative ion mode, and full scan mass spectral data were acquired over a range from m/z 100 to 1,700. The MS conditions were as follows: drying gas flow rate, 10 L/min; nebulizer pressure, 60 psi; drying gas temperature, 350 °C; ESI temperature, 400 °C; and capillary voltage, 110 V. The mass spectrum results were analyzed by the Agilent MassHunter Workstation (version B.05.01).

Secondary metabolites include a large group of specific compounds required for survival and adaptation of plants, fungi, and bacteria in their environment. They can react to biotic stresses including mutualistic (e.g., pollination) or antagonistic interactions (e.g., disease-resistance and existence competition) with other organisms, or deal with abiotic stresses such as radiation and heavy metals. Macro fungi have a well-developed secondary metabolism producing a variety of low-molecular-weight secondary metabolites that render tremendous bioactivity and medicinal properties. However, fungi secondary metabolites remain mostly underexplored compared to plant-derived ones in spite of centuries’ old application; chaga mushroom is one typical case.

The actual compositional profile of chaga extract is very complicated, which may arise from various pathways of secondary metabolism in wild chaga and its host so that a gradient with over 120 min elution duration was used to sufficiently separate them by LC C-18 system before the coupled ESI-ToF-MS analysis. The mass error for molecular ions of all identified compounds was within ±10 ppm. For example, compound A1 gave a deprotonated molecular ion [M-H]⁻ at 181.0719, with the difference of −0.76 ppm from calculated [M-H]⁻ at 181.0718, therefore indicating its molecular formula as C₆H₁₄O₆, which is tentatively identified as a sugar alcohol of hexose, such as sorbitol.

The eluted compounds are mainly non-nitrogenous compounds, including phenolics, terpenoids, and fatty acid derivatives, and this study focuses on the analysis of phenolic constituents. Figure s1–s3 shows the total ion chromatograph (TIC) of chaga extract. By comparison with the updated phenolic database (Table s1), our results classified these constituents of Newfoundland chaga into two categories, ‘known’ and ‘newly detected compounds’, along with their accurate MS¹ data given in Table 1, as discussed below.

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Table 1. Chemical constituents (saccharides and phenolics) of chaga extract scanned by HPLC-ESI-TOF-MS

Table 1 shows that the phenolic composition of chaga is a complex profile covering 111 compounds, although the actual content of soluble small-molecular-weight phenolics is the tip of the iceberg due to the abundance of (insoluble) phenolic polymers, especially melanin- and lignin-polysaccharide complexes (Wang et al., 2015; Wold et al., 2018). According to our Folin-Ciocalteu and antioxidant results of chaga extracts, the efficacy and mass equivalents of degradation products of insoluble phenolics are several times higher than those of its soluble counterpart (unpublished results). The phenolics of chaga include phenolic acids, flavonoids, coumarins, quinones, and styrylpyrones. Among the 111 phenolic compounds in Table 1, 49 of them are in accordance with previous studies, while the other 62 are newly detected.

For phenolic acids, a total of 37 compounds were identified, including 16 hydroxybenzoic acid derivatives (compounds No. P1/2/3/4/7/9/19/20/21/23/24/28/48/54/57/70/71/72) and 19 hydroxycinnamic acid derivatives (No. P12/13/14/16/17/26/34/47/50/52/53/68/74/88/89/91/92/93/110). Twenty-four of those structures have been previously been reported in chaga and the rest of the compounds, P23 (C₁₅H₂₀O₉), P48 (C₂₂H₂₆O₁₁), P50 (C₁₈H₂₂O₁₀), P52 (C₂₅H₃₀O₁₄), P53 (C₂₅H₃₂O₁₃), P54 (C₂₃H₂₈O₁₃), P68 (C₂₅H₃₀O₁₃), P71(C₂₃H₂₆O₁₃), P72 (C₂₃H₂₆O₁₃), P89 (C₃₄H₄₀O₁₈), P91 (C₃₄H₄₀O₁₈), and P93 (C₃₄H₄₀O₁₈), were tentatively identified as syringic acid rhamnoside, agnuside, sinapoylquinic acid, lippioside II, oleuropein, picroside II, grandifloroside, helonioside B, mallophenol A, mallophenol A and its isomers, smilaside B, smilaside B isomer, and jaslanceoside B, respectively. Compared to the other phenolic acids or even any other phenolic compound, hydroxybenzoic acid (P7) was reported as the most dominating phenolic in chaga (Glamočlija et al., 2015). However, in this study, it was noticed that the dominating phenolics might indeed be dihydroxybenzaldehyde (P8) instead of hydroxybenzoic acid (P7), the former’s log P is slightly lower than the latter and therefore appearing subsequently (8.812 min of P7 and 10.189 min of P8) with a very different relative abundance in TIC (total ion chromatogram). This result is in line with that of Abu-Reidah et al. (2021)’s recent quantification results of Newfoundland chaga. However, the protocatechuic acid glucoside, ellagic acid, methylellagic acid, and 2,5-dihydroxyterephthalic acid detected in Abu-Reidah et al. (2021)’s study were not found in our sample. However, the material used in our study was enriched with various benzoic and cinnamic acid derivatives. The phenolic acids with relatively higher abundance were compounds P52, P54, P68, P71, P72, P110, and especially syringic acid and its derivatives (P23/24/57/70).

Eighteen coumarin, xanthone, anthrone, and quinone derivatives can be sporadically found in the sample. Except dihydroxycoumarin (P10), coumarin (P18), and inonotphenol A (P36), the derivatives including P15 (C₉H₆O₃), P31 (C₁₄H₁₀O₅), P32 (C₁₁H₈O₅), P33 (C₁₄H₁₆O₇), P39 (C₁₂H₁₂O₅), P42 (C₁₃H₁₂O₅), P43 (C₁₃H₈O₇), P45 (C₁₄H₁₀O₄), P51 (C₁₆H₁₈O₁₀), P57 (C₁₂H₁₀O₄), P58 (C₂₄H₁₈O₉), P66 (C₁₂H₁₄O₈), P78 (C₁₄H₈O₇), P85 (C₁₄H₈O₈), and P97 (C₂₂H₁₆O₈), are all newly detected and were tentatively characterized as hydroxycoumarin, dihydroxymethoxyxanthone, purpurogallin, phellodenol F, trimethoxycoumarin, pentahydroxyxanthone, acetyl dimethoxycoumarin, leucoquinizarine, fraxin, liqcoumarin, gaboroquinone A, fulvic acid, pentahydroxyanthraquinone, hexahydroxyanthraquinone, and hydramycin, respectively. Among these derivatives, the compounds with relatively higher abundance were compounds P15, P33, P39, P42, P45, P57, and P85.

Flavonoid derivatives are a diverse group of phenolics that wee also detected in the current chaga sample. A total of 30 flavonoids were detected while their ion abundances were generally in trace amounts compared with the rest of phenolic groups. Zheng et al. (2008) compared the phenolic contents of wild chaga and its mycelia cultures. Mycelia cultures of chaga mainly consisted of flavonoids [e.g., naringin, ECG (epicatechin gallate), kaempferol], and lesser amounts of styrylpyrones and melanins. However, for the wild chaga, flavonoids were determined in trace amounts, while styrylpyrones (e.g., phelligridin A/D and inoscavin A/B) and melanins were dominant. In our sample, we noticed a similar phenomenon. As aforementioned, among 85 previously reported phenolic compounds, 37 were not detected in the current sample, especially various flavonoid derivatives such as EGCG (epigallocatechin gallate), ECG, EGC (epigallocatechin), naringin, apigenin, eriocitrin, rhoifolin, isorhamnetin-3-O-rutinoside, and narirutin. However, several other non-flavonoid phenolics, including resveratrol, phellxinye A, inonoblin A, methylinoscavin A/B, and phelligridin C/F were also absent. For the 31 detected flavonoid derivatives, there were 18 flavones(ols) (compounds P22/30/37/41/44/46/60/61/62/63/73/75/86/87/90/94/96), 5 flavanones(ols) (compounds P29/80/81/83/84), 3 flavans(ols) (compounds P67/69/83) as well as aurones (compound P55), chalcones (P59), isoflavones (P85), and lignoflavonoids (P77). Twenty compounds in the list were newly detected. Compound P69 (C₂₀H₂₂O₁₀) showed the highest relative abundance, and then were compounds P38 (C₁₈H₁₆O₆), and P55 (C₁₉H₁₈O₅).

Styrylpyrones, also known as hispidin derivatives, are a rare group in plant-derived phenolics. They have a skeleton core of C6-C2-C5 and can be found in macrofungi from genus Inonotus and Phellinus or primitive angiosperm from families Piperaceae, Lauraceae, Annonaceae, Ranuculaceae and Zingiberaceae (Lee and Yun, 2011). Detailed demonstration of the structural diversity, biosynthetic pathways, bioactivities, and corresponding mechanisms of styrylpyrones are found elsewhere (Lee and Yun, 2011).

A total of 16 styrylpyrones were found in the current sample, 10 of them including hispidin (compound P27), methylinoscavin C (P40), davallialactone (P65), inonoblin B (P98), phelligridin E (P101), phelligridin D (P102), inoscavin C (P103), phelligridin G (P104), inonoblin C (P106), and phelligridin C (P107) are known styrylpyrones from previous compositional studies of chaga. The newly detected styrylpyrones P25, P64, P79, P99, and P100, were tentatively identified as phelligridin J, inoscavin A, phelligridin A, phelliribsin A, inoscavin D, and methylinoscavin D, respectively. The compounds with relatively higher abundance were P102, P107, and P79. The other 6 previously known styrylpyrones including inonoblin A, phelligridin F, phelligridin H, methylinoscavin A/B, inoscavin B, and methyl davallialactone, could not be found in the current sample.

Other phenolics, including the derivatives of simple phenols (compounds P5/6), phenolic aldehydes (P8/11), neolignans (P49), stilbenes (P76), diarylheptanoids (P108), and tocopherols (P109/111), were found in the samples we examined. As mentioned earlier, compound P8 was dominant in its relative abundance compared to any other chaga phenolics in TIC, implying the antimicrobial and anti-cancer effects of phenolic extracts of chaga. Furthermore, the high content of α- and β-tocopherols previously reported in Newfoundland chaga could not be found in the current sample while the desmethyl tocotrienol and β/γ-tocotrienol were present at the end of the current elution time. Thus, either a longer elution time or a more hydrophilic mobile phase is recommended in future studies.

A total of 63 derivatives of aromatic acids and fatty acids were tentatively identified. Except three compounds F1, F2, and F28, which were characterized as aromatic acids (phthalic acid, benzoic acid, and di-iso-octyl phthalate), the other 60 compounds were all aliphatic acid derivatives including monocarboxylic acids and dicarboxylic acids as well as their hydroxylated derivatives. Twenty-one of them were hydroxylated fatty acids (compounds F3/7/8/9/10/13/14/17/26/35/39/40/44/45/48/49/54/56/58/60/63), 5 are dicarboxylic acid (compounds F4/5/6/11/39), and the left 37 compounds (F12/15/16/18/19/20/21/22/23/24/26/27/29/30/31/32/33/34/36/37/38/41/42/43/46/47/50/51/52/53/55/57/59/61/62) were unmodified saturated/unsaturated fatty acids. The chain length of fatty acid derivatives ranged from 12 carbons (compound F12, lauric acid) to 27 carbons (compound F63, hydroxyheptacosanoic acid) in the current sample. The longest saturated monocarboxylic acid was docosanoic acid (compound F55, C₂₂H₄₄O₂) and longest unsaturated monocarboxylic acid was hexacosenoic acid (F62, C₂₆H₅₀O₂) which were eluted at the very end of the gradients (119.728 and 121.570 min), while in the past study using GC-MS, the longest saturated monocarboxylic acid detected was melissic acid (C₃₀H₆₀O₂) (Table s2). This was also the case for the elution of tocopherols; For LC-MS used in this study, the more extended elution gradient with a more hydrophilic organic phase was required for achieving detection of such hydrophilic compounds (melissic acid, C30:2). Hydroxylation and decarboxylation significantly increased the hydrophilicity and shortened the elution time. For fatty acids and their derivatives, 21 compounds (F1/2/3/4/5/7/8/9/10/11/13/14/16/26/35/39/45/46/49/56/63) were newly found, and the rest of the 41 fatty acids and 1 aromatic acid derivatives have been reported in the past. In both the current and previous studies, hydroxylated fatty acid derivatives, the common oxygenated metabolites of fatty acids during bacterial or fungal fermentation and a vital component of some fungal cell walls, have been abundantly detected. As given in Table 2 and supplementary database (Table s2) of chaga secondary metabolites, the fatty acid derivatives were mainly reported by Shcherbakov et al. (2022) and Sun et al. (2011). Although both studies used GC-MS to identify the fatty acid composition, different characterization results were reported for the same molecular formula. For example, in Shcherbakov et al. (2022)’s study, C₁₄H₂₈O₂ (compound F15) was identified as myristic acid/tetradecanoic acid while it was reported as ethyl dodecanoate in Sun et al. (2011)’s study. Similar cases apply to C₁₆H₃₂O₂ (compounds F25/33/41/48/52), C₁₇H₃₄O₂ (F30/32), C₁₉H₃₈O₂ (F37/38), and C₂₀H₄₀O₂ (F43), as shown in Table 2. Only using tandem mass or standard comparison can help to distinguish and confirm their accurate structures. In this study, oleic acid (F27, C₁₈H₃₄O₂), hydroxydocosanoic acid (F44, C₂₂H₄₄O₃), hydroxytricosanoic acid (F48, C₂₃H₄₆O₃), hydroxytetracosanoic acid (F54, C₂₄H₄₈O₃), hydroxypentacosanoic acid (F58, C₂₅H₅₀O₃) showed predominant relative abundance, followed by stearic acid (F24, C₁₈H₃₆O₂), hydroxyhexacosanoic acid (F60, C₂₆H₅₂O₃), linoleic acid (compounds F22, C₁₈H₃₂O₂), palmitic acid and its isomers (F25/33/41/48/52, C₁₆H₃₂O₂), hydroxyarachidic acid (F35, C₂₀H₄₀O₃), hydroxyheneicosanoic acid (F39, C₂₁H₄₂O₃), hydroxytricosenoic acid (F40, C₂₃H₄₄O₃).

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Table 2. Chemical constituents (fatty acids, aromatic acids, and related derivatives) of chaga extract scanned by HPLC-ESI-TOF-MS

A total of 108 terpenoids were tentatively identified from the current chaga sample, but only 30 of them

can be recognized based on previous reports (Table s3, 129 known terpenoids in chaga) and the rest of the 78 were found as new terpenoids. In this study, except for the last compound T108 (C₃₀H₅₀O, 122.267 min), most of the terpenoids eluted by the current gradient were terpenes substituted with multi-oxygenated functional groups, and various terpenes and their mono-oxygenated derivatives reported previously could not be detected due to limitation of current elution period. Meanwhile, apart from compounds T1 (monoterpenoid glycoside), T8/25/55 (sesquiterpenoids), T35/38/41/45 (diterpenoids), T15(tetraterpenoid), T87 (pentaterpenoid), the rest of the 98 terpenoids identified from our sample were all triterpenoids and steroids. The compound T77 (C₃₀H₄₈O₃) showed the highest relative abundance within all the terpenoids, followed by compounds T75 (C₃₀H₄₆O₃), T76 (C₃₉H₅₆O₅), T83 (C₃₁H₅₀O₃), T84 (C₃₉H₅₆O₄), T93 (C₃₉H₅₈O₅), T103 (C₃₂H₅₂O₆), and T106 (C₃₉H₅₈O₄). It is remarkable that several hydroxycinnamoyl esters of triterpenoids, including compounds P110, T76, T80, T82, T84, T93, T101, T104, and T106, were detected, which highlights new antioxidant contributors other than pure phenolics, lignin, and melanin in chaga extracts considering their high relative abundance in TIC. However, compared to phenolic and fatty acid derivatives, the tentatively deduced structures of terpenoid compounds are more equivocal based on the primary mass data because of their higher molecular weights, which usually lead to many possible isomers. Therefore, the names of newly identified terpenoids in Table 3 were mainly from previous terpenoid studies of other fungi and only provided as preliminary results. Furthermore, numerous in vivo and in vitro studies of bioactive properties of purified chaga terpenoids have previously been reported and reviewed (Peng and Shahidi, 2020).

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Table 3. Chemical constituents (terpenoids) of chaga extract scanned by HPLC-ESI-TOF-MS

This study tentatively screened and identified over two hundred and eighty phenolic, fatty acid, and terpenoids compounds through literature review and comparison with previously reported studies of chaga. This priliminary screening of non-nitrogenous small-molecule phenolics, fatty acids, and terpenoids by HPLC-ToF-MS shows that Newfoundland chaga exhibits a unique secondary metabolites’ profile demonstrating its potential for further development as a high-quality food supplement and nutraceutical. On the other hand, although compositional studies of chaga were initiated half of a century ago, many major bioactive compounds are still waiting to be identified. Due to the parasitic feature and environmental sensitivity of chaga mushroom, its secondary metabolic pathways and related bioactive stability of commercial products can be significantly skewed and jeopardized. Therefore, strategies for artificial cultivation and standardization of chaga culturing/farming and sustainable production may be the way for future production and marketing of chaga. Additional new secondary metabolites are waiting to be screened, separated, and identified in a targeted manner for Newfoundland chaga. However, further studies are also required for identification of nitrogenous compounds (e.g., peptides and alkaloids) and polymeric compounds (e.g., polysaccharides, lignin, and melanin) of chaga and to examine their myriad of physicochemical properties and bioactivities. Moreover, to a great extent, the use of chaga has usually been randomly guided by the folk experience, and the reported cases showing potential adverse health effects (on kidney) due to extremely high quantity of oxalic acid in chaga (up to 14% of dried material) have recently provoked serious safety concerns in unguided long-term administration of wild chaga and its products (Kwon et al., 2022; Lee et al., 2020; Peng and Shahidi, 2020). The standardized quality control based on fast detection technologies, the advancement of oxalate-free processing protocols, and the dosage guideline based on quantification of the bioactives present and their safe level of use and sufficient preclinical/clinical data for its chronic toxicity are most urgently needed.

Supplementary materials for this article are available at https://doi.org/10.31665/JFB.2022.17304.

Figure s1. TIC (total ion chromatograph) of chaga extract, 0-80 min.

Figure s2. TIC (total ion chromatograph) of chaga extract, 80-109 min.

Figure s3. TIC (total ion chromatograph) of chaga extract, 109-133 min.

Table s1. Known phenolic small molecules and polymers of chaga and their purification/identification.

Table s2. Polysaccharides, fatty acids, and other compounds of chaga and their purification/identification.

Table s3. Known terpenes and terpenoids of chaga and their purification/identification.

Acknowledgments

We thank the Natural Science and Engineering Research Council (NSERC) of Canada for financial support. H.P. acknowledges the scholarship support from China Scholarship Council (CSC).