Isolation and quantitative determination of ergosterol peroxide in various edible mushroom species

Abstract

Ergosterol peroxide, the steroidal derivative with cytotoxic activity, has been isolated for the first time from the mycelium of edible and medicinal mushroom Hericium erinaceum (lion’s mane mushroom) together with erinacine A. The new densitometric method was applied for the quantitative determination of ergosterol peroxide in n-hexane extracts of H. erinaceum, Laetiporus sulfureus (chicken mushroom), and Morchella esculenta (common morel) mycelia, as well as in Boletus edulis (king bolete), Suillus bovinus (Jersey cow mushroom), and B. badius (bay bolete) fruiting bodies. The ergosterol peroxide content reached 15.98 ± 0.78, 10.07 ± 0.75, 13.37 ± 0.56, 29.32 ± 1.43, 17.27 ± 0.84, and 12.60 ± 0.59 mg per 100 g, respectively. What is significant was that ergosterol peroxide was identified for the first time, to the best of our knowledge, in edible mushrooms mentioned above.

Introduction

Up to now, ergosterol peroxide (EP) has been isolated from various species of edible and medicinal mushrooms – e.g., Ganoderma lucidum (El-Mekkawy et al., 1998, Ziegenbein et al., 2006), Cordyceps sinensis (Bok, Lermer, Chilton, Klingeman, & Towers, 1999; Nam, Jo, Kim, Hyun, & Kim, 2001), Volvariella volvacea (Mallavadhani et al., 2006), Armillaria mellea (Kim, Park, Min, & Yu, 1999), microscopic fungi such as Gibberella fujikuroi and Aspergillus spp. (Nemec, Jernejc, & Cimerman, 1997), yeasts such as Saccharomyces cerevisiae (Nes, Xu, & Haddon, 1989), plants such as Melilotus messanensis (Macías, Simonet, & Galindo, 1997), sponge Ascidia nigra (Gunatilaka, Gopichand, Schmitz, & Djerassi, 1981), soft coral Sinularia flexibilis (Yu, Deng, van Ofwegen, Proksch, & Lin, 2006), and halotolerant microalga Dunaliella salina (Sheffer, Fried, Gottlieb, Tietz, & Avron, 1986).

It has been proven that EP poses a wide spectrum of biological activity. This compound was shown to have antitumor activity against Walker carcinosarcoma and human mammary adenocarcinoma cell lines in vitro (Jong & Donovick, 1989), as well as against human gastric tumor cell line (SNU-1), human hepatoma cell line (SNU-354), human colorectal tumor cell line (SNU-C4), and murine sarcoma-180. The cytotoxicity of EP is assumed to be relevant to its conversion to ergosterol, accompanied by the formation of highly toxic peroxide products (Nam et al., 2001). Recent studies showed that the cytotoxicity of EP on HL60 cells results from its ability to induce apoptosis (Takei, Yoshida, Ohnishi-Kameyama, & Kobori, 2005).

Ergosterol peroxide inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory ear oedema (Yasukawa et al., 1994) and proves to be selectively active against PLA₂ enzymes secreted from Crotalus adamenteus venom (Gao et al., 2007). This suggests its possible medicinal use as an antivenom and anti-inflammatory agent (Keyzers & Davies-Coleman, 2005).

EP also shows antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, Pseudomonas sp., Candida albicans, and A. niger (Lu, Zou, Meng, Hu, & Tan, 2000). On the other hand, EP was found to be inactive against S. aureus ATTC 25923, Escherichia coli ATTC 25922, P. aeruginosa ATTC 27753, and 11 strains of Candida spp. Moreover, it showed poor activity against Mycobacterium tuberculosis H37Rv (Cateni et al., 2007).

The mechanism of EP biosynthesis and its authenticity as a metabolite has been controversial since the sixties. Adam, Campbell, and McCorkindale (1967) found this compound to be an artifact that arises from oxidation or photooxidation of ergosterol induced by air. However, the study of either Nes et al. (1989) or Nemec et al. (1997) refuted it.

Ergosterol peroxide is believed to be either an intermediate in the H₂O₂-dependent enzymatic oxidation in the biosynthetic pathway of steroidal dienes or a product of detoxification of reactive oxygen species. Its content in cells depends on many factors – on the level of reactive oxygen species or individual ratio between EP formation and its conversion back into ergosterol (Batrakov et al., 2004, da Graça Sgarbi et al., 1997).

Erinacine A is a cyathane-xyloside diterpene isolated from H. erinaceum mycelium and fruiting body and is known to have potent stimulating activity on nerve growth factor (NGF) synthesis as well as activity against Methicillin-resistant S. aureus (Kawagishi et al., 1994, Kawagishi et al., 2006).

Our paper describes the isolation and identification of EP and erinacine A from H. erinaceum mycelium. A sensitive and precise method for the determination of EP has been developed. We examined the possibility of EP formation during an UV–vis radiation. A quantitative determination of EP in medicinal and edible mushrooms H. erinaceum (lion’s mane mushroom), Boletus edulis (king bolete), Morchella esculenta (common morel), Laetiporus sulfureus (chicken mushroom), Suillus bovinus (Jersey cow mushroom), and B. badius (bay bolete) was performed.