Anti-inflammatory and anticancer activities of extracts and compounds from the mushroom Inonotus obliquus

Abstract

Mushroom Inonotus obliquus (I. obliquus) has been used as functional food and traditional Chinese herbs for long time. An efficient method for bioassay-guided preparative isolation was used for identifying the anti-inflammatory and anticancer constituents in I. obliquus. The petroleum ether and ethyl acetate fractions were found to have significant inhibition effects on NO production and NF-κB luciferase activity in macrophage RAW 264.7 cells and cytotoxicity against human prostatic carcinoma cell PC3 and breast carcinoma cell MDA-MB-231. Six main constituents were isolated from these two fractions and they were identified as lanosterol (1), 3β-hydroxy-8,24-dien-21-al (2), ergosterol (3), inotodiol (4), ergosterol peroxide (5) and trametenolic acid (6). Compound ergosterol, ergosterol peroxide and trametenolic acid showed anti-inflammatory activities and ergosterol peroxide and trametenolic acid showed obviously cytotoxicity on human prostatic carcinoma cell PC3 and breast carcinoma MDA-MB-231 cell. The results obtained in this work might contribute to understanding the biological activity of mushroom I. obliquus for food and drug application.

Introduction

Mushroom Inonotus obliquus (I. obliquus) was a white-rot fungus belonging to the family of Hymenochaetaceae Donk, habiting as a parasitism on birches in the cold latitudes of Europe and Asia (Hawksworth, Kirk, Sutton, & Pegler, 1995). In Russian, I. obliquus had been used as a traditional remedy to cure various diseases such as cancer, cerebrovascular diseases, diabetes, gastrointestinal diseases since the sixteenth century (Choi et al., 2010, Sun et al., 2008). Triterpenes, polysaccharides, polyphenols and melanin were found in I. obliquus, which were responsible for the anticancer and antitumor activities (Handa et al., 2012, Song et al., 2008), anti-inflammatory ability (Van et al., 2009), antioxidant effect (Ma, Chen, Zhang, Zhang, & Fu, 2012), hypoglycemic ability (Lu, Chen, Dong, Fu, & Zhang, 2010), immunomodulatory activity (Fan, Ding, Ai, & Deng, 2012) and anti-mutagenic properties (Ham et al., 2009). However, most of the bioactive studies were mainly focused on the extracts or fractions and the effective constituents were not well illustrated.

Chronic inflammation was implicated in the pathogenesis of series of diseases including atherosclerosis, obesity, metabolic syndrome, diabetes, neurodegenerative diseases, and even several types of cancers (Moro et al., 2012). Macrophagesacted as a crucial role in the inflammatory response and it could release a variety of factors including nitric oxide (NO), prostaglandin mediators and proinflammatory cytokines (TNF-α, IL-1β, IL-6) in response to activating stimulus such as lipopolysaccharide (LPS). Many kinds of natural products were investigated on the anti-inflammatory properties using the LPS-induced macrophage model. I. obliquus was a traditional medicinal mushroom and it had been reported to have anti-inflammatory potential on the extracts. Several studies on methanol extract and ethanol extract from I. obliquus had shown to inhibit macrophage functions by decreasing the production of inflammatory mediators such as NO, prostaglandins (PGE₂) and some cytokines (Kim et al., 2007, Park et al., 2005, Van et al., 2009). However, there were no reports concerning the active compounds on the anti-inflammatory activity. Steroids and triterpenes compounds had been reported to show significantly anti-inflammatory ability in other natural resources (Chang et al., 2008, Jiang and Dusting, 2003). However, the anti-inflammatory activities of triterpenes compounds in I. obliquus were still unknown.

Anticancer experiments with n-hexane extract and water extract of I. obliquus had been conducted and the extracts were found to exhibit anticancer effects (Kahlos et al., 1987, Youn et al., 2008). As the main chemical constituents of I. obliquus, lanostane-type triterpenes compounds were found to have potential anticancer abilities. Lanostane-type triterpenoids isolated from the sclerotium of I. obliquus such as inotodiol (De & Ourisson, 1972), 3β,22dihydroxylanosta-7,6(11),24-triene, 3β-hydroxylanosta-8,24-dien-21-al (Kirsti, Lauri, & Raimo, 1987), 22R-epoxylanost-8ene-3β,24S-diol, trametenolic acid, lanosterol (Nakata et al., 2007), inonotsulides A, B, and C, inonotsuoxides A and B, inonotsutriols A, B, and C, lanosta-8,23E-diene-3β,22R,25-triol and lanosta-7:9(11), 23E-triene-3β,22R,25-triol, spiroinonotsuoxodiol, inonotsudiol A and inonotsuoxodiol A, and inonotsutriols D and E (Handa et al., 2012) were reported to show anti-tumor promoting activities. Inotodiol was found to have a significant anticancer activity on Walker 256, MCF-7 and Hela S3 tumor cells (Kahlos et al., 1987, Rzymowska, 1998). Although some studies on the anti-cancer effects of water, methanol, petroleum ether, n-hexane extracts and some lanostane-type triterpenoids from I. obliquus against Walker 256, Hela S3, S180 cells were reported, anticancer effects using bioactivity-guided screening method against the human prostatic carcinoma cell PC3 and breast carcinoma cell MDA-MB-231 had not been found in literature.

In this study, the purpose was to isolate and identify the bioactive fraction and compounds from I. obliquus by using activity-guided isolation methods. The anti-inflammatory and anti-cancer capacities of ethanol extract (EE), petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butyl alcohol fraction (n-BF), water fraction (WF) and six compounds were comparatively studied and the structure–activity relationship of the six compounds was also discussed.