Bioactivity and Chemical Profiling of Medicinal Fungi Inonotus cuticularis and Inocutis levis (Hymenochaetaceae) using Chromatography and Mass Spectrometry

Document Type : Research Paper

Authors

1 Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P. O. Box 3353-5111, Tehran, Iran

2 Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, Iran

3 Department of Horticultural Sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran

Abstract

Polypore fungi are among the most preventable mushroom-forming fungi with known therapeutic potential, though only a few species have been securitized for their metabolites. This study examines the biological activity and bioactive compounds of Inocutis levis and Inonotus cuticularis collected in Iran. We examined the antimicrobial, antioxidant, and cytotoxic properties of n-hexane, acetone, and ethyl acetate extracts. Chemical profiles were assessed by chromatography and mass spectroscopy techniques. The acetonic extracts exhibited the highest antibacterial effect against all tested microbial strains. The IC50 values for DPPH and ABTS assays ranged from 144.98 – 469.02 μg/mL and 128.06 – 331.52 μg/mL, respectively. The MTT assays for both fungi indicated low toxicity on normal HDF cells with IC50 values ranging from 1447 to 1908 μg/mL. HPLC-DAD analysis showed a high level of gallic acid among other detected phenolic compounds. LC-ESI-MS/MS analysis displayed the presence of various sesquiterpenoids, furans, and styrylpyrone-class compounds. Inotilone, inonotin H and C, phellinulin B and M, cinnamic acid, p-coumaric acid, caffeic acid, phelligridin A and D, hispidin, and gallic acid were found in both species. Daedalin A is reported for the first time from the fungal family Hymenochaetaceae. In addition, several volatile compounds, including alkene hydrocarbons and some fatty acids, such as linoleic acid, were detected in GC-MS analyses. We suggest that I. levis and I. cuticularis have dual antibacterial and antioxidant properties and diverse metabolites, potentially opening new windows in future natural product-based medicine.

Keywords


  1. Grienke U., Zöll M., Peintner U., Rollinger J.M. European medicinal polypores–A modern view on traditional uses. J. Ethnopharmacol. 2014;154(3):564-583.
  2. Thu ZM, Myo KK, Aung HT, Clericuzio M, Armijos C, Vidari G. Bioactive phytochemical constituents of wild edible mushrooms from Southeast Asia. Molecules. 2020;25(8):1972.
  3. Venturella G, Ferraro V, Cirlincione F, Gargano ML. Medicinal mushrooms: bioactive compounds, use, and clinical trials. Int. J. Mol. Sci. 2021;22(2):634.
  4. Adongbede EM, Jaiswal YS, Davis SS, Randolph PD, Huo LN, Williams LL. Antioxidant and antibacterial activity of Trametes polyzona (Pers.) Justo. Food Sci. Biotechnol. 2020;29(1):27-33.
  5. Ghobad-Nejhad M, Kotiranta H. The genus Inonotus sensu lato in Iran, with keys to Inocutis and Mensularia worldwide. InAnnales Botanici Fennici. Finnish Zoological and Botanical Publishing Board. Bio One. 2008;45(6):465-476).
  6. Ravera S, Vizzini A, Puglisi M, Adamčík S, Aleffi M, Aloise G, Boccardo F, Bonini I, Caboň M, Catalano I, De Giuseppe AB. Notulae to the Italian flora of algae, bryophytes, fungi and lichens. 9. Ital. Bot. 2020;9:35.
  7. 7.Ying, C.-c., Icons of medicinal fungi from China. 1987: Science press.
  8. 8.Zhao J. The resources and application of medicinal fungi from forests. For Sci Technol, 1996;5:40-41.
  9. Guo L, Ma JY, Ma YZ, Zhang TL, Mao SL, Kong DX, Hua Y. Orthogonal test design for optimization of the extraction of polysaccharides from inonotus cuticularis and their antioxidant activities. Chem. Biodivers. 2020;17(9):e2000326.
  10. Dai YC, Yang ZL, Cui BK, Yu CJ, Zhou LW. Species diversity and utilization of medicinal mushrooms and fungi in China. Int. J. Med. Mushrooms. 2009; 11(3).
  11. Ehsanifard Z, Mir-Mohammadrezaei F, Safarzadeh A, Ghobad-Nejhad M. Aqueous extract of Inocutis levis improves insulin resistance and glucose tolerance in high sucrose-fed Wistar rats. J. HerbMed. Pharmacol. 2017;6(4):160-164.
  12. Ehsanifard Z, Mir Mohammadrezaei F, Ghobad-Nejhad M, Safarzade AR. The effect of aqueous extract of Inocutis levis on liver histopathology and hypertriglyceridemia in high sucrose-fed Wistar rats. J. Med. Plants. 2019;18(70):181-187.
  13. Wu F, Zhou LW, Yang ZL, Bau T, Li TH, Dai YC. Resource diversity of Chinese macrofungi: edible, medicinal and poisonous species. Fungal Divers. 2019;98(1):1-76.
  14. Chaharmiri Dokhaharani S., Ghobad-Nejhad M., Moghimi H., Farazmand A., Rahmani H. Investigating Antibacterial and Antioxidant Activity of Inocutis levis Extracts and Evaluating its Phenolic Compounds. BJM. 2020;9(35):1-16.
  15. 15.Cui Y., Kim D-S., Park K-C. Antioxidant effect of Inonotus obliquus. J. Ethnopharmacol. 2005;96(1-2):79-85.
  16. 16.Wayne P. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100. Clinical and Laboratory Standards Institute, 2018.
  17. Procop G.W. M60, Performance standards for antifungal susceptibility testing of yeasts https://clsi.org/standards/products/microbiology/documents/m60/ 2020. Ed2.
  18. Béni Z, Dékány M, Kovács B, Csupor-Löffler B, Zomborszki ZP, Kerekes E, Szekeres A, Urbán E, Hohmann J, Ványolós A. Bioactivity-guided isolation of antimicrobial and antioxidant metabolites from the mushroom Tapinella atrotomentosa. Molecules. 2018;23(5):1082.
  19. Bach F., Zielinski A.A., Helm C.V., Maciel G.M., Pedro A.C., Stafussa A.P., Ávila S., Haminiuk C.W. Bio compounds of edible mushrooms: in vitro antioxidant and antimicrobial activities. Lwt.2019;107:214-220.
  20. 20.Waterhouse, A.L., Determination of total phenolics. Curr. protoc. food anal. chem. 2002; 6(1):I1-1.
  21. DuBois M., Gilles K.A., Hamilton J.K., Rebers P.T., Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956;28(3):350-356.
  22. Kolundžić M., Grozdanić N.Đ., Dodevska M., Milenković M., Sisto F., Miani A., Farronato G., Kundaković T. Antibacterial and cytotoxic activities of wild mushroom Fomes fomentarius (L.) Fr., Polyporaceae. Ind. Crops Prod. 2016;79:110-115.
  23. Dokhaharani S.C., Ghobad-Nejhad M., Moghimi H., Farazmand A., Rahmani H. Biological activities of two polypore macrofungi (Basidiomycota) and characterization of their compounds using HPLC–DAD and LC–ESI–MS/MS. Folia Microbiol. 2021;66(5):775-786.
  24. Asgharpour F., Moghadamnia A.A., Alizadeh Y, Kazemi S. Chemical Composition and antibacterial activity of hexane extract of Lycoperdon Pyriforme. S. Afr. J. Bot. 2020;131:195-199.
  25. Liu K., Xiao X., Wang J., Chen C.Y., Hu H. Polyphenolic composition and antioxidant, antiproliferative, and antimicrobial activities of mushroom Inonotus sanghuang. LWT - Food Sci. Technol. 2017;82:154-161.
  26. Hur J.M., Yang CH., Han SH., Lee SH., You Y.O., Park J.C., Kim K.J. Antibacterial effect of Phellinus linteus against methicillin-resistant Staphylococcus aureus. Fitoterapia, 2004;75(6):603-605.
  27. 27.Breijyeh Z., Jubeh B., Karaman R. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, 2020; 25(6):1340.
  28. 28.Negi P. Jayaprakasha G. Antioxidant and antibacterial activities of Punica granatum peel extracts. J. Food Sci. 2003;68(4):1473-1477.
  29. Tamrakar S., Nishida M., Amen Y., Tran H.B., Suhara H., Fukami K., Parajuli G.P., Shimizu K. Antibacterial activity of Nepalese wild mushrooms against Staphylococcus aureus and Propionibacterium acnes. J Wood Sci. 2017;63(4):379-387.
  30. Angelini P., Girometta C., Tirillini B., Moretti S., Covino S., Cipriani M., D’Ellena E., Angeles G., Federici E., Savino E., Cruciani G.A. A comparative study of the antimicrobial and antioxidant activities of Inonotus hispidus fruit and their mycelia extracts. Int J Food Prop. 2019;22(1):768-783.
  31. Stajić M, Vukojević J, Ćilerdžić J. Mushrooms as potential natural cytostatics. Medicinal Mushrooms: Recent Progress in Research and Development. 2019;143-68.
  32. Song Y., Hui J., Kou W., Xin R., Jia F., Wang N., Hu F., Zhang H., Liu H.Identification of Inonotus obliquus and analysis of antioxidation and antitumor activities of polysaccharides. Curr. Microbiol. 2008;57(5):454-462.
  33. 33.Rajamanickam K., J. Yang, and M.K. Sakharkar, Gallic acid potentiates the antimicrobial activity of tulathromycin against two key bovine respiratory disease (BRD) causing-pathogens. Front. Pharmacol. 2019;9:1486.
  34. Darkal A.K., Zuraik M.M., Ney Y., Nasim M.J., Jacob C. Unleashing the biological potential of Fomes fomentarius via dry and wet milling. Antioxidants. 2021;10(2):303.
  35. Sova, M., Antioxidant and antimicrobial activities of cinnamic acid derivatives. Mini Rev. Med. Chem. 2012;12(8):749-767.
  36. Lee, I.-K. and B.-S. Yun, Styrylpyrone-class compounds from medicinal fungi Phellinus and Inonotus spp., and their medicinal importance. J. Antibiot. 2011;64(5):349-359.
  37. Liu C., Zhao C., Pan H.H., Kang J., Yu X.T., Wang H.Q., Li BM, Xie YZ, Chen RY. Chemical constituents from Inonotus obliquus and their biological activities. J. Nat. Prod. 2014;77(1):35-41.
  38. Handa, N., Yamada T., Tanaka R. An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus. Phytochemistry. 2010;71(14-15):1774-1779.
  39. Morimura K., Yamazaki C., Hattori Y., Makabe H., Kamo T., Hirota M.A tyrosinase inhibitor, Daedalin A, from mycelial culture of Daedalea dickinsii. Biosci. Biotechnol. Biochem. 2007;0710040587-0710040587.
  40. Morimura K., Hiramatsu K., Yamazaki C., Hattori Y., Makabe H., Hirota M. Daedalin A, a metabolite of Daedalea dickinsii, inhibits melanin synthesis in an in vitro human skin model. Biosci. Biotechnol. Biochem. 2009;73(3):627-632.
  41. Liu Y., Kubo M., Fukuyama Y. Nerve growth factor-potentiating benzofuran derivatives from the medicinal fungus Phellinus ribis. J. Nat. Prod. 2012;75(12):2152-2157.
  42. Han J.J., Bao L., He L.W., Zhang X.Q., Yang X.L., Li S.J., Yao Y.J., Liu H.W. Phaeolschidins A–E, five hispidin derivatives with antioxidant activity from the fruiting body of Phaeolus schweinitzii collected in the Tibetan Plateau. J. Nat. Prod. 2013; 76(8):1448-1453.
  43. Lee I.K., Han M.S., Lee M.S., Kim Y.S., Yun B.S. Styrylpyrones from the medicinal fungus Phellinus baumii and their antioxidant properties. Bioorganic Med. Chem. Lett. 2010;20(18):5459-5461.
  44. Jeon Y.E., Lee Y.S., Lim S.S., Kim S.J., Jung SH., 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 BI. 2009;52:472-479.
  45. Zhang B, Zhou J, Li Q, Gan B, Peng W, Zhang X, Tan W, Jiang L, Li X. Manganese affects the growth and metabolism of Ganoderma lucidum based on LC-MS analysis. PeerJ. 2019;7:e6846.
  46. Wang Z.X., Feng X.L., Liu C., Gao J.M., Qi J. Diverse Metabolites and Pharmacological Effects from the Basidiomycetes Inonotus hispidus. Antibiotics. 2022;11(8):1097.
  47. Bergmann P., Takenberg M., Frank C., Zschätzsch M., Werner A., Berger R.G., Ersoy F. Cultivation of Inonotus hispidus in stirred tank and wave bag bioreactors to produce the natural colorant hispidin. Fermentation. 2022;8(10):541.
  48. Nakajima Y., Nishida H., Nakamura Y., Konishi T. Prevention of hydrogen peroxide-induced oxidative stress in PC12 cells by 3, 4-dihydroxybenzalacetone isolated from Chaga (Inonotus obliquus (persoon) Pilat). Free Radic. Biol. Med. 2009;47(8):1154-1161.
  49. Glamočlija J., Ćirić A., Nikolić M., Fernandes Â., Barros L, Calhelha RC, Ferreira IC, Soković M, Van Griensven LJ. Chemical characterization and biological activity of Chaga (Inonotus obliquus), a medicinal “mushroom”. J Ethnopharmacol. 2015;162:323-332.
  50. 50.Pubchem, CID 21315698. 3-(Hexyloxy)-4-hydroxybenzaldehyde. https://pubchem.ncbi.nlm.nih.gov/compound/21315698.
  51. Nyigo V., Baraza L.D., Nkunya M.H., Mdachi S.J., Joseph C.C., Waziri A. Chemical constitutents and cytotoxicity of some Tanzanian wild mushrooms. Tanz J Sci. 2005;31(2):1-3.
  52. Lee I.K., Seok S.J., Kim W.K., Yun BS.Hispidin Derivatives from the Mushroom Inonotus x eranticus and Their Antioxidant Activity J Nat Prod. 2006;69(2):299-301.
  53. Isaka M., Yangchum A., Supothina S., Boonpratuang T., Choeyklin R., Kongsaeree P., Prabpai S. Aromadendrane and cyclofarnesane sesquiterpenoids from cultures of the basidiomycete Inonotus sp. BCC 23706. Phytochemistry. 2015;118:94-101.
  54. Chao W., Deng J.S., Li P.Y., Kuo Y.H., Huang G.J. Inotilone from Inonotus linteus suppresses lung cancer metastasis in vitro and in vivo through ROS-mediated PI3K/AKT/MAPK signaling pathways. Sci. Rep. 2019;9(1):2344.
  55. 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(2):173-175.
  56. Huang S.C., Wang P.W., Kuo P.C., Hung H.Y., Pan T.L. Hepatoprotective principles and other chemical constituents from the mycelium of Phellinus linteus. Molecules. 2018;23(7):1705.
  57. Ohyoshi T., Mitsugi K., Higuma T., Ichimura F., Yoshida M, Kigoshi H. Concise total syntheses of phelligridins A, C, and D. RSC advances, 2019;9(13):7321-7323.
  58. Serck-Hanssen K., Wikström C. Novel 7-phenylheptan-3-ones from the fungus Phellinus tremulae. Phytochemistry, 1978;17(9):1678-1679.
  59. Nelson, G.J., D.P. Matthees, and D.E. Lewis, 1-Phenylheptane-1, 5-dione from Phellinus tremulus. Journal of Natural Products, 1993;56(7):1182-1183.
  60. Reis F.S., Barreira J.C., Calhelha R.C., van Griensven L.J., Ćirić A., Glamočlija J., Soković M., Ferreira I.C. Chemical characterization of the medicinal mushroom Phellinus linteus (Berkeley & Curtis) Teng and contribution of different fractions to its bioactivity. LWT - Food Sci. Technol. 2014;58(2):478-485.
  61. Olennikov D.N., Sof'ya V.A., Tat'yana A.P., Borovski GB. Fatty acid composition of fourteen wood-decaying basidiomycete species growing in permafrost conditions. Rec. Nat. Prod. 2014;8(2):184.
  62. Deng K., Zhang Y., Ren Z., Xie L., Peng W., Gan B. Simultaneous determination of five fatty acids in Phellinus sp. by high-performance liquid chromatography with photodiode-array detection. J. Med. Plant Res. 2011;5(13):2816-2821.
  63. Vazirian M., Faramarzi M.A., Ebrahimi S.E., Esfahani H.R., Samadi N., Hosseini S.A., Asghari A., Manayi A., Mousazadeh S.A., Asef M.R., Habibi E. Antimicrobial effect of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) and its main compounds. Int J Med Mushrooms. 2014;16(1).
  64. 64.Göring, H. Vitamin D in nature: a product of synthesis and/or degradation of cell membrane components. Biochemistry (Moscow), 2018;83(11):1350-1357.
  65. Rajakumar K., Greenspan S.L., Thomas S.B., Holick M.F. SOLAR ultraviolet radiation and vitamin D: a historical perspective. Am J Public Health. 2007;97(10):1746-1754.
  66. 66.Yazawa Y., Yokota M., Sugiyama K. Antitumor promoting effect of an active component of Polyporus, ergosterol and related compounds on rat urinary bladder carcinogenesis in a short-term test with concanavalin A. Biol. Pharm. Bull. 2000;23(11):1298-1302.
  67. Sun Y., Yin T., Chen X.H., Zhang G., Curtis R.B., Lu ZH., Jiang J.H. In vitro antitumor activity and structure characterization of ethanol extracts from wild and cultivated Chaga medicinal mushroom, Inonotus obliquus (Pers.: Fr.) Pilát (Aphyllophoromycetideae). Int J Med. Mushrooms. 2011;13(2).
  68. Kusumah D., Wakui M., Murakami M., Xie X., Yukihito K., Maeda I. Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus. Biosci. Biotechnol. Biochem. 2020;84(6):1285-1290.
  69. Wu S., Krings U., Zorn H., Berger R.G. Volatile compounds from the fruiting bodies of beefsteak fungus Fistulina hepatica (Schaeffer: Fr.) Fr. Food Chem. 2005;92(2):221-226.
  70. Aisala H., Sola J., Hopia A., Linderborg K.M., Sandell M.Odor-contributing volatile compounds of wild edible Nordic mushrooms analyzed with HS–SPME–GC–MS and HS–SPME–GC–O/FID. Food Chem. 2019; 15;283:566-78.