NJ025-201

Identification of Bioactive Polyphenols from Wild-Type Phellinus linteus Using LC-MS and NMR

Papawee Saiki1, Leo J. L. D. Van Griensven2, and Toshihiko Toida3*

1Cellular and Molecular Biotechnology Research Institute, National Institute of Advance Industrial Science and Technology, Tsukuba 305-8566, Japan. 

2Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands. 3Graduate School of Pharmaceutical Sciences, Chiba University, Chiba-shi, Chiba, Japan.

Corresondence to: Toshihiko Toida, toida@faculty.chiba-u.jp

Received: February 19, 2025; Revised: April 1, 2025; Accepted: April 7, 2025; Published: April 14, 2025

NATPRO J. 2025; 2: 1-7 

Published: April 14, 2025 https://doi.org/10.23177/NJ025.201 

Copyright ©  The Asian Society of Natural Products

Abstract

Phellinus linteus (L.) Quel. has been used in traditional Asian medicine for over two centuries against a variety of diseases. Many researchers have focused on the antioxidant properties of polyphenols from Phellinus linteus (P. linteus), however, this mushroom lacks identification of its polyphenols. We demonstrated the presence of nine polyphenol compounds i.e. 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-3-buten-2-one, interfungin A, inonoblin C, phelligridin D, interfungin B, inoscavin E, inoscavin C and methylinoscavin D in the ethanol extract of P. linteus fruiting bodies by LC-MS. The chromatographic separation was performed on a common reversed-phase C18 column using a water–acetonitrile with formic acid gradient program and the detection was achieved by ESI source operated in negative ion mode. Further characterization of P. linteus polyphenols was done by proton nuclear magnetic resonance spectroscopy (1H NMR) which showed strong proton resonances of 3,4-dihydroxybenzaldehyde, interfungin A, inonoblin C, phelligridin D, inoscavin E and inoscavin C.

Keywords

LC-MS, NMR, Phellinus linteus, polyphenol

Graphic Abstract

Introduction

Phellinus linteus (P. linteus) is a well-known fungal species of the genus Phellinus from the Hymenochaetaceae family which has been used as a source of traditional herbal medicine in oriental countries such as China, Korea, and Japan for many years [1-3]. P. linteus extract was reported to have anti-angiogenic, antioxidant [4-5], anti-inflammation [6-7], anti-cancer [8], anti-bacterial [9] and immunomodulatory effects [10-11]. It was reported earlier that hispolon, a phenolic compound from P. linteus has anti-inflammatory, anti-proliferative and anti-metastatic effects [1, 12]. The group of phenolic compounds seems responsible for several bioactivities such as antioxidant effects [13-14], DPPH radical-scavenging capacity [15] and tyrosinase inhibition [16]. We reported that a polyphenol extract from Phellinus igniarius, which is a closely related species, protects against acrolein toxicity in vitro and provides protection in a mouse stroke model [11]. Acrolein (CH2=CH-CHO), an unsaturated aldehyde produced from spermine, is one of the major contributors to oxidative stress. Acrolein has been found to be more toxic than reactive oxygen species (H2O2 and •OH), and it can be easily conjugated with proteins, bringing about changes in nature of the proteins [17]. Based on these findings, we have been investigating the scavenger effects of polyphenols from mushrooms using animal stroke model and Neuro-2a cell line [17].

The polyphenol extract from P. igniarius mainly contained 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-3-buten-2-one, inonoblin C, phelligridin D, inoscavin C, phelligridin C and interfungin B [11]. We observed that the polyphenol extracts from P. linteus and P. igniarius had different protective properties for acrolein toxicity in vitro and in a mouse stroke model. Because there is only limited information available on this group of important and interesting polyphenols from P. linteus, we analyzed the DMSO soluble fraction of the P. linteus ethanol extract by electron spray ionization LC-MS. Additionally, the characterization of the P. linteus polyphenols was confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR). 

Materials and methods

Materials and Chemicals

Ethanol (99.5%) and dimethyl sulfoxide (DMSO) d6 (99.9% containing 0.05 v/v% TMS) were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). The hydrophilic 0.22 µm filters were purchased from Merck Ltd. (Tokyo, Japan).  Phenol reagent solution (Acid 1.8 N) for determination of total polyphenol content was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and gallic acid standard was purchased from Sigma–Aldrich Co. (Tokyo, Japan). Analytical reagents and standards for HPLC i.e. ethanol HPLC grade, formic acid solution, acetonitrile HPLC grade, esculetin and 3,4 dihydroxybenzaldehyde were obtained from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). 

Chromatographic separation was performed on a Hitachi LaChrom Elite HPLC ultra-high-pressure liquid chromatography (U-HPLC) system from Hitachi High-Technologies Corporation (Tokyo, Japan). The chromatographic separation was performed on an Inertsil column (ODS-3 150 mm × 4.6 mm, 5 μm from GL Sciences Inc., (Tokyo, Japan). Mass spectrum analysis was performed using a Bruker AmaZon SL Ion Trap Mass Spectrometer from Bruker Japan Co., LTD. (Yokohama, Japan) LC-MS Data were collected with Bruker Compass Data Analysis 4.2 from Bruker Japan Co., LTD. (Yokohama, Japan).

Deionized water used for all experiments was prepared by Milli-Q® Integral Water Purification System of Merck Ltd. (Tokyo, Japan).

Polyphenol Extraction

Dried fruiting body powder of wild-type Phellinus linteus was kindly provided by Amazing Grace Health Products Limited Partnership (Bangkok, Thailand). Identification of the wild types had been done by Dr. Usa Klinhom (Mahasarakham University, Thailand) and representative samples of both species were stored in the collection of the Medicinal Mushroom Museum of Mahasarakham University as published before [18]. The powder was extracted with 70% ethanol (10% w/v) at 70ºC for 16 hrs. The hydroethanolic extract was collected by centrifugation at 10,000 g for 20 min, and lyophilized. The dry powder was then dissolved in DMSO, filtered with a hydrophilic 0.22 µm filter, and stored at -20ºC for characterization and further use.

Total Phenolics Determination

The concentration of total polyphenols was estimated by the Folin-Ciocalteu assay [19]. The procedure consisted of diluting 50 µl polyphenol extract into 400 µl water followed by the addition 50 µl of the phenol reagent solution as described in Materials and chemicals. The mixture was incubated at room temperature for 5 minutes. 500 µl of 7% Na2CO3 Solution was added to the mixture, and then the mixture was incubated at room temperature for 90 minutes. Absorbance was measured at 750 nm. Gallic acid was used as a reference compound within a range of 0 to 500 μg/ml.

Analysis of Polyphenol by Liquid Chromatography Mass Spectrometry (LC-MS)

Chromatographic separation was performed on a Hitachi LaChrom Elite HPLC ultra-high-pressure liquid chromatography (U-HPLC) system fitted with a Bruker AmaZon SL Ion Trap Mass Spectrometer. The chromatographic separation was performed on an Inertsil column (ODS-3 150 mm × 4.6 mm, 5 μm). The mobile phase was composed of water (A) equilibrated with formic acid (pH 3.0) and 100% acetonitrile (B) with formic acid (pH 3.0) under gradient elution conditions at 0–60 min, 0–50% B; 60-90 min, 50-100% B and 90-100 min, 100% B. The flow rate of the mobile phase was 0.4 ml/min, and the column temperature was maintained at 30ºC. Mass spectrum analysis was performed using a Bruker AmaZon SL Ion Trap Mass Spectrometer fitted with an ESI source and operated in negative ion mode. The key optimized ESI parameters were as follows: spray voltage: -3.5 KV; capillary temperature: 220ºC; Ultra scan. Data were collected with Bruker Compass Data Analysis 4.2 and analyzed by Mass++ software [20] with the MassBank mass spectral library.

Nuclear Magnetic Resonance Spectroscopy (NMR)

One-dimensional spectra of 10 mg of dry sample in dimethyl sulfoxide-d6, 99.9% containing 0.05 v/v TMS were acquired using a Jeol 600 MHz instrument. The operation condition for 1H NMR was as follows: spin, 15 Hz; relaxation delay, 5 s; temperature, 25ºC.

MTT Assay

Cells were plated in 96-well microtiter plates and treated as described below. Thereafter, the medium was removed and 50 μl/ml of MTT solution in DMEM and antibiotics (penicillin and streptomycin) were added. After a 1 hr incubation, the MTT solution was replaced with 100 μl of DMSO to dissolve the tetrazolium crystals. Finally, the absorption was read at a test wavelength of 540 nm and a reference wavelength of 650 nm with a Multiskan JX microplate reader (Thermo Labsystems, UK). Cell viability (%) was calculated as [optical density (OD) of the treated wells]/[OD of the control wells] x 100 [21].

Results & Discussion

Total Phenolics Determination

The polyphenol content of the DMSO soluble fraction of the hydroethanol extract was measured by the Folin-Ciocalteau method using gallic acid as a reference. Within the range of 0 to 500 µg/ml there was a highly significant correlation (P = 0.9946) between the dilution curves of the extract and gallic acid. The DMSO fraction of the hydroethanolic extract of P. linteus contained 458.73 μg GAE (gallic acid equivalents) per ml. The crude water extract from P. linteus contained 20.18 μg GAE per ml indicating that DMSO soluble hydroethanolic extract has 23 times more GAE (i.e. antioxidant effect) than the crude water extract. As Folin-Ciocalteu measures redox dependent coloration of the reagent, the value reflects reductive, i.e. antioxidative capacity of the solution.

Analysis of Polyphenols by Liquid Chromatography Mass Spectrometry (LC-MS)

The crude polyphenol extract from P. linteus was subjected to LC-MS and NMR to characterize its components as described in Materials and methods. The total ion current (TIC) chromatogram of the polyphenol extract from P. linteus showed 10 main peaks (Figure 1). The composition of the chromatograms was analyzed by MASS++ with the MassBank library. Tentatively, content of each polyphenol was calculated using TIC intensity as shown in Figure 1.  To confirm identification, the compounds were identified by comparing their retention time and mass spectral data (Figure 1). The ESI-MS of compound 1 gave a molecular ion at m/z 136.84 [M−H]− at 25.0 min (Figure 2A) whereas the ESI-MS of 3,4-dihydroxybenzaldehyde gave a molecular ion at m/z 136.90 [M−H]− at 23.9 min (Figure 3). The molecular formula of compound 1 was determined as C7H6O3 by 1H NMR spectral data of 3,4-dihydroxybenzaldehyde (Figure 4) [11]. This strongly suggested that compound 1 is 3,4-dihydroxybenzaldehyde (Table 1).

IThe ESI-MS of compound 2 gave a molecular ion at m/z 176.84 [M−H]− at 34.9 min (Figure 2B). The ESI-MS of esculetin and 4-(3,4-dihydroxyphenyl)-3-buten-2-one gave a molecular ion at m/z 176.75 at 23.9 min (Figure 5) and 177 [M−H]− respectively [22]. 4-(3,4-Dihydroxyphenyl-3-buten-2-one has one more carbon atom than esculetin [11]. It was tentatively suggested that compound 2 is 4-(3,4-dihydroxyphenyl)-3-buten-2-one. The ESI-MS of compound 3 gave a molecular ion at m/z 463.10 [M−H]−. Weesepoel suggested that compound 3 might be davallialactone or interfungin A [22]. Proton NMR resonances of compound 3 showed strong signals of interfungin A that lack signals of davallialactone as shown in Table 2 [23]. These observations strongly suggest that compound 3 is interfungin A. The ESI-MS of compound 8 gave a molecular ion at m/z 377.05 [M−H]−. Weesepoel suggested compound 8 might be inoscavin E or phellifuropyranone A [22]. In 2013, Yoon & Paik showed that P. linteus extract contains inoscavin E [24]. Furthermore, the proton NMR resonances of compound 8 showed strong signals of inoscavin E as shown in Table 2 [23]. It was strongly suggested that compound 8 is inoscavin E.

To further explore the structural features that distinguish active compounds from inactive ones, PCA on two sets of molecular properties was performed: conventional molecular descriptors and Morgan fingerprints (Figure 2 and 3). The analysis included three datasets: active and inactive compounds from PubChem, and active compounds from BindingDB. The analysis pipeline was developed by using Python, utilizing libraries such as RDKit for molecular processing, scikit-learn for PCA, and matplotlib for visualization. For each descriptor set, we created both 2D and 3D PCA plots using matplotlib. Active compounds from PubChem were represented in red, inactive compounds in blue, and active compounds from BindingDB in green to ensure clear visualization. The PCA was conducted on standardized data to account for different feature scales. 

Table 1. Tentative identification of polyphenol extract from Phellinus linteus by UPLC/ESI-MS in negative ion mode

Nuclear Magnetic Resonance Spectroscopy (NMR)

All compounds were tentatively identified by masses of the corresponding [M−H]− from Weesepoel [22]. Furthermore, the assignment of proton NMR resonances in polyphenols indicated the presence of inonoblin C (compound 4 and 5), phelligridin D (compound 6) and inoscavin C (compound 9) [25-27].

Table 2. Compounds identified in polyphenol extract from Phellinus linteus by assignment of their proton resonances 

Previously, we reported that the polyphenol extract from P. igniarius could prevent acrolein toxicity in vitro and in a mice stroke model [28]. The polyphenol extract from P. igniarius mainly contained 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-3-buten-2-one, inonoblin C, phelligridin D, inoscavin C, phelligridin C and interfungin B [28]. In this study, polyphenol extract from P. linteus mainly contains 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-3-buten-2-one, interfungin A, inonoblin C, phelligridin D, interfungin B, inoscavin E, inoscavin C and methylinoscavin D. Although, the main compounds of polyphenol extract from P. linteus and P. igniarius are almost identical, there seems only one different compound i.e. phelligridin C, which is not present in the P. linteus derived extract. However, we could not find the prevention of acrolein toxicity by the EtOH extract from P. linteus (Figure 6B). Recently, phelligridin C was reported to show cytotoxic [29,30], antioxidant and antitumor effects [25]. Phelligridin C might be a key compound for prevention of acrolein toxicity.

Further, polyphenol extract from P. linteus also has different compounds such as interfungin A, inoscavin E and methylinoscavin D. Interfungin A was reported to have antioxidant and anti-diabetes effects [31-33]. Inoscavin E was shown to have antioxidant and cytotoxic effects [31,34]. Methylinoscavin D was found to be antioxidant [35]. These compounds, interfungin A, inoscavin E and methylinoscavin D, might be related to the different biological effects of P. linteus that were not found for P. igniarius.

Neuro-2a cells exhibit distinct characteristics that make them particularly useful in research for development of new drug screening of stroke. Primarily, these cells retain the ability to differentiate into neuron-like cells under specific conditions, allowing scientists to study neuronal function and development [36]. The viable cell number of Neuro-2a cells that were treated with P. igniarius EtOH crude extract and P. linteus EtOH crude extract at 2, 5, 10, 20 and 50 µg/ml for 4 hours and treated with 8 µM acrolein. Cell viability was determined by MTT assay after 24 hrs. Each value represents the mean ± SD (n=3).

A key aspect to be explored is the structure-activity relationship of these polyphenols. Bioinformatics approaches such as molecular docking and network pharmacology can help predict interactions with biological targets involved in oxidative stress-related neurodegenerative diseases such as Alzheimer's and Parkinson's by reducing reactive oxygen species. Computational modeling can also identify functional groups responsible for bioactivity, aiding in the design of more potent derivatives. In addition, comparative studies of P. linteus and P. igniarius could reveal how structural differences influence biological functions. Integration with experimental validation in in vitro and vivo models will improve our understanding of the precise molecular mechanisms driving their activity.

Conclusion

The simple LC-MS method that we described was used to identify the molecular mass of the DMSO soluble hydroethanol extract of P. linteus fruit bodies in negative mode. The extract consisted mainly of 9 compounds that were 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-3-buten-2-one, interfungin A, inonoblin C, phelligridin D, interfungin B, inoscavin E, inoscavin C and methylinoscavin D in Figure 7. The characterization of the different components was done by assigning their proton resonances that found characteristic signals of 3,4-dihydroxybenzaldehyde, interfungin A, inonoblin C, phelligridin D, inoscavin E and inoscavin C. We suggest that the difference in the above-mentioned biological effects of P. linteus and P. igniarius might be caused by the absence respectively presence of inoscavin C and inoscavin D in the extracts. The different presence of interfungin A, inoscavin E and methylinoscavin D in P. linteus compared to P. igniarius suggests potential alternative bioactivities that may be relevant to oxidative stress, neurodegenerative diseases, vascular dysfunction and metabolic disorders.

References 

1. Aanniz T.; Zeouk I.; Elouafy Y.; Touhtouh J.; Hassani R.; Hammani K.; Benali T.; El-Shazly M, Khalid A, Abdalla AN, Aboulaghras S, Goh KW, Ming LC, Razi P, Bakrim S, Bouyahya A. Initial report on the multiple biological and pharmacological properties of hispolon: Exploring stochastic mechanisms. Biomed. Pharmacother. 2024. 177:117072. DOI: 10.1016/j.biopha.2024.117072.

2. Chen W, Tan H, Liu Q, Zheng X, Zhang H, Liu Y, Xu L. A Review: The Bioactivities and Pharmacological Applications of Phellinus linteus. Molecules, 2019, ;24, 1888. DOI: 10.3390/molecules24101888.

3. Kou, F.; Mei, Y.; Wang, W.; Wei, X.; Xiao, H.; Wu, X. Phellinus linteus polysaccharides: A review on their preparation, structure-activity relationships, and drug delivery systems. Int. J. Biol. Macromol. 2024, 258(Pt 1), 128702. DOI: 10.1016/j.ijbiomac.2023.128702.

4. Song, Y. S.; Kim, S. H.; Sa, J. H.; Jin, C.; Lim, C. J.; Park, E. H. Anti-angiogenic, antioxidant and xanthine oxidase inhibition activities of the mushroom Phellinus linteus.  J. Ethnopharmacol. 2003, 88, 113-116. DOI: 10.1016/s0378-8741(03)00178-8.

5. Kozarski, M.; Klaus, A.; Niksic, M.; Jakovljevic, D.; Helsper, J. P.; Van Griensven, L. J. Antioxidative and immunomodulating activities of polysaccharide extracts of the medicinal mushrooms Agaricus bisporus, Agaricus brasiliensis, Ganoderma lucidum and Phellinus linteus. Food Chem. 2011, 129, 1667-75. DOI: 10.1016/j.foodchem.2011.06.029.

6. Xie, Z.L.; Wang, Y.; Huang, J.Q.; Qian, N.; Shen, G.Z.; Chen, L.H. Anti-inflammatory activity of polysaccharides from Phellinus linteus by regulating the NF-κB translocation in LPS-stimulated RAW264.7 macrophages. Int. J. Biol. Macromol. 2019, 129, 61-7. DOI: 10.1016/j.ijbiomac.2019.02.023.

7. Kim, S. H.; Song, Y. S.; Kim, S. K.; Kim, B. C.; Lim, C. J.; Park, E. H. Anti-inflammatory and related pharmacological activities of the n-BuOH subfraction of mushroom Phellinus linteus. J. Ethnopharmacol. 2004, 93, 141-6. DOI: 10.1016/j.jep.2004.03.048.

8. Hsieh, M. J.; Chien, S. Y.; Chou, Y. E.; Chen, C. J.; Chen, J.; Chen, M. K. Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway. Phytomedicine, 2014, 21, 1746-1752. DOI: 10.1016/j.phymed.2014.07.013.

9. Hur, J. M.; Yang, C. H.; Han, S. H.; Lee, S. H.; You, Y. O.; Park, J. C.; Kim, K. J. Antibacterial effect of Phellinus linteus against methicillin-resistant Staphylococcus aureus. Fitoterapia, 2004, 75, 603-5. DOI: 10.1016/j.fitote.2004.06.005.

10. Lin, C.J.; Lien, H.M.; Lin, H.J.; Huang, C.L.; Kao, M.C.; Chen, Y.A.; Wang, C.K.; Chang, H.Y.; Chang, Y.K.; Wu, H.S. Modulation of T cell response by Phellinus linteus. J. Biosci. Bioeng. 2016, 121, 84-8. DOI: 10.1016/j.jbiosc.2015.05.008.

11. Suabyakyong, P.; Nishimura, K.; Toida, T.; Van Griensven, L. J. Structural characterization and immunomodulatory effects of polysaccharides from Phellinus linteus and Phellinus igniarius on the IL-6/IL-10 cytokine balance of the mouse macrophage cell line RAW 264.7. Food Funct. 2015, 6, 2834-44. DOI: 10.1039/c5fo00491h.

12. Huang, G. J.; Deng, J. S.; Chiu, C. S.; Liao, J. C.; Hsieh, W. T.; Sheu, M. J.; Wu, C. H. Hispolon protects against acute liver damage in the rat by inhibiting lipid peroxidation, proinflammatory Cytokine, and Oxidative stress and downregulating the expressions of iNOS, COX-2, and MMP-9. Evid. Based Complement Alternat. Med. 2012, 480714. DOI: 10.1155/2012/480714.

13. Jung, J. Y.; Lee, I. K.; Seok, S. J.; Lee, H. J.; Kim, Y. H.; Yun, B. S. Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteus. J. Appl. Microbiol. 2008, 104, 1824-1832. DOI: 10.1111/j.1365-2672.2008.03737.x.

14. Kim, M. Y.; Seguin, P.; Ahn, J. K.; Kim, J. J.; Chun, S. C.; Kim, E. H.; Seo, S. H.; Kang, E. Y.; Kim, S. L.; Park, Y. J.; Ro, H. M.;  Chung, I. M. Phenolic compound concentration and antioxidant activities of edible and medicinal mushrooms from Korea. J. Agric. Food Chem. 2008, 56, 7265-7270. DOI: 10.1021/jf8008553.

15. Jeon, Y. E.; Lee, Y. S.; Lim, S. S.; Kim, S. J.; Jung, S. H.; Bae, Y. S.; Yi, J. S.; Kang, I. J. Evaluation of the antioxidant activity of the fruiting body of Phellinus linteus using the on-line HPLC-DPPH method. J. Korean Soc. Appl. Biol. Chem, 2009, 52, 472-9. DOI:10.3839/jksabc.2009.081.

16. Kang, H. S.; Choi, J. H.; Cho, W. K.; Park, J. C.; Choi, J. S. A sphingolipid and tyrosinase inhibitors from the fruiting body of Phellinus linteus. Arch. Pharm. Res. 2004, 27, 742-50. DOI: 10.1007/BF02980143.

17. Kashiwagi, K.; Igarashi, K. Molecular Characteristics of Toxicity of Acrolein Produced from Spermine. Biomolecules, 2023, 13, 298. DOI: 10.3390/biom13020298.

18. Sittiwet, C.; Puangpronpitag, D. Antibacterial activity of Phellinus gilvus aqueous extract. Int. J. Pharmacol.  2008, 4, 500-2. DOI: 10.3923/ijp.2008.500.502.

19. Ainsworth, E. A.; Gillespie, K. M. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat. Protoc. 2007, 2, 875-7. DOI: 10.1038/nprot.2007.102.

20. Tanaka, S.; Fujita, Y.; Parry, H. E.; Yoshizawa, A. C.; Morimoto, K.; Murase, M.; Yamada, Y.; Yao, J.; Utsunomiya, S.; Kajihara, S.; Fukuda, M.; Ikawa, M.; Tabata, T.; Takahashi, K.; Aoshima, K.; Nihei, Y.; Nishioka, T.; Oda, Y.;  Tanaka, K. Mass++: A visualization and analysis tool for mass spectrometry. J. Proteome Res. 2014, 13, 3846-53. DOI: 10.1021/pr500155z.

21. Tanaka, Y.; Marumo, T.; Omura, T.; Yoshida, S. Quantitative assessments of cerebral vascular damage with a silicon rubber casting method in photochemically-induced thrombotic stroke rat models. Life Sci. 2007, 81, 1381-8. DOI: 10.1016/j.lfs.2007.09.011.

22. Weesepoel, Y.;(2009). Extraction and characterization of triterpenoids from Ganoderma lucidum and polyphenols from Phellinus linteus. Thesis for Master of Science in Food Chemistry Wageningen University and Research Centre, The Netherlands.

23. Lee, I. K.; Kim, Y. S.; Seok, S. J.; Yun, B. S. Inoscavin E, a free radical scavenger from the fruiting bodies of Inonotus xeranticus. J. Antibiot. 2007, 60, 745-7. DOI: 10.1038/ja.2007.97.

24. Yoon, H. R.; Han, A. R.; Paik, Y. S. Prolyl Endopeptidase Inhibitory Activity of Two Styrylpyranones from Phellinus linteus. J. Appl. Biol. Chem. 2023, 56, 183-5. DOI:10.3839/JABC.2013.029.

25. Zheng, W.; Zhang, M.; Zhao, Y.; Miao, K.; Pan, S.; Cao, F.; Dai, Y. Analysis of antioxidant metabolites by solvent extraction from sclerotia of Inonotus obliquus (Chaga). Phytochem Anal. 2011, 22, 95-102. DOI: 10.1002/pca.1225.

26. Zheng, W.; Zhao, Y.; Zheng, X.; Liu, Y.; Pan, S.; Dai, Y.; Liu, F. Production of antioxidant and antitumor metabolites by submerged cultures of Inonotus obliquus cocultured with Phellinus punctatus. Appl. Microbiol. Biotechnol. 2011, 89, 157-167. DOI: 10.1007/s00253-010-2846-2.

27. Zheng, W.; Dai, Y.; Sun, J.; Zhao, Y.; Miao, K.; Pan, S.; Zhang, M.; Wei, J. Metabonomic analysis on production of antioxidant secondary metabolites by two geographically isolated strains of Inonotus obliquus in submerged cultures. Mycosystema, 2010, 29, 897-910. DOI: 10.3724/SP.J.1238.2010.00516.

28. Suabjakyong, P.; Saiki, R.; Van Griensven, L. J.; Higashi, K.; Nishimura, K.; Igarashi, K.; Toida, T. Polyphenol Extract from Phellinus igniarius Protects against Acrolein Toxicity In Vitro and Provides Protection in a Mouse Stroke Model. PLoS One, 2015, 10, e0122733. DOI: 10.1371/journal.pone.0122733.

29. Mo, S.; Wang, S.; Zhou, G.; Yang, Y.; Li, Y.; Chen, X.; Shi, J. Phelligridins CF: Cytotoxic Pyrano [4, 3-c][2] benzopyran-1, 6-dione and Furo [3, 2-c] pyran-4-one Derivatives from the Fungus Phellinus igniarius. J. Nat. Prod. 2004, 67, 823-8. DOI: 10.1021/np030505d.

30. Nagatsu, A.; Itoh, S.; Tanaka, R.; Kato, S.; Haruna, M.; Kishimoto, K.; Hirayama, H.; Goda, Y.; Mizukami, H.; Ogihara, Y. Identification of novel substituted fused aromatic compounds, meshimakobnol A and B, from natural Phellinus linteus fruit body.Tetrahed. lett. 2004, 45, 5931-3.  DOI: 10.1016/j.tetlet.2004.05.102.

31. Lee, I. K.; Yun, B. S. Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus. Bioorg. Med. Chem. 2007, 15, 3309-14. DOI: 10.1016/j.bmc.2007.03.039.

32. Lee, Y. S.; Kang, Y. H.; Jung, J. Y.; Kang, I. J.; Han, S. N.; Chung, J. S.; Shin, H. K.; Lim, S. S. Inhibitory constituents of aldose reductase in the fruiting body of Phellinus linteus. Biol. Pharm. Bull. 2008, 31, 765-8. DOI: 10.1248/bpb.31.765.

33. Lee, Y. S.; Kang, Y. H.; Jung, J. Y.; Lee, S.; Ohuchi, K.; Shin, K. H.; Kang, I. J.; Park, J. H. Y.; Shin, H. K.; Lim, S. S. Protein glycation inhibitors from the fruiting body of Phellinus linteus. Biol. Pharm. Bull. 2008, 31, 1968-72. DOI: 10.1248/bpb.31.1968.

34. Kojima, K.; Ohno, T.; Inoue, M.; Mizukami, H.; Nagatsu, A. Phellifuropyranone A: a new furopyranone compound isolated from fruit bodies of wild Phellinus linteus. Chem. Pharm. Bull. 2008, 56, 173-5. DOI: 10.1248/cpb.56.173.

35. Lee, I. K.; Jung, J. Y.; Seok, S. J.; Kim, W. G.; Yun, B. S. Free radical scavengers from the medicinal mushroom Inonotus xeranticus and their proposed biogenesis. Bioorg. Med. Chem. Lett. 2006, 16, 5621-4. DOI: 10.1016/j.bmcl.2006.08.016.

36. LePage, K. T.; Dickey, R. W.; Gerwick, W. H.; Jester, E. L.; Murray, T. F. On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies. Crit. Rev. Neurobiol. 2005, 17, 27-50. DOI: 10.1615/critrevneurobiol.v17.i1.20.