Orirubenones A, B and C, novel hyaluronan-degradation inhibitors from the mushroom Tricholoma orirubens Hirokazu Kawagishi, a, * Yumiko Tonomura, a Hiroyuki Yoshida, b Shingo Sakai b and Shintaro Inoue b a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan b Basic Research Laboratory, Kanebo Ltd, 3-28, 5-Chome, Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan Received 5 August 2003; revised 10 October 2003; accepted 10 October 2003 Available online 24 June 2004 Abstract—Three new compounds, orirubenone A (1), B (2) and C (3) were isolated from the mushroom Tricholoma orirubens. Their structures were determined by spectral analyses. These compounds inhibited hyaluronan-degradation by human skin fibroblasts. q 2004 Elsevier Ltd. All rights reserved. 1. Introduction Hyaluronan (HA), a nonsulfated glycosaminoglycan composed of repeating disaccharide units of N-acetyl- glucosamine and glucuronic acid, plays an important role in inflammation, cell locomotion, and wound healing. 1 The skin has more than 50% of total body HA, and the half-life of HA is less than one day, suggesting that the velocity of HA production and depolymerization (degradation) in the skin is very rapid. 2 Moreover, HA has been reported to have many physiological activities corresponded to its different molecular size. Oligosaccharides of HA are known to stimulate angiogenesis, 3,4 and low-molecular-mass HA (less than 5£10 5 Da) has been shown to activate NF-k B expression 5 and induce chemokine gene expression 6 and nitric-oxide synthase 7 in mouse macrophages, suggesting that the regulation of HA depolymerization is important during the process of skin inflammation. The mechanism of depolymerization in skin fibroblasts remains unclear. Some reports showed that HA bound to plasma membrane of fibroblasts, 8,9 while other reports clearly indicated an absence of lysosomal hyaluronidase activity in normal human skin fibroblasts. 10 – 12 Recently, five hyaluronidase genes and one pseudogene have been cloned. 13 Three (HYAL1, HYAL2 and HYAL3) of them are expressed in human skin fibroblasts. 14 HYAL1 is widely expressed in various tissues. However, its activity outside of lysosomes seems very little because of its sharp pH optimum around. 4,7,14 HYAL2 is localized in the lysosomes or cell membranes, and can degrade high-molecular-mass HA to intermediately sized products of 20 kDa under an acidic pH optimum. 15,16 However, no hyaluronidase activity was demonstrated in the lysate of either fibroblasts or HeLa cells transfected with human HYAL2. 15 The activity of HYAL3, which was found in chondrocytes, has not been characterized. 17 On the other hand, Nakamura et al. found that an extracellular-depoly- merization enzyme, which did not produce oligo-HA as end-products like the lysosomal hyaluronidase, was at work in normal skin fibroblasts. 11 So far, there is no direct evidence that lysosomal hyaluronidase plays a role in depolymerization of HA in skin fibroblasts. Therefore, inhibitors toward HA-depolymerization by skin fibroblasts can be useful tools for the study of the HA-depolymeriza- tion. In the present study, we screened for inhibitory activity of extracts of various mushrooms against the extracellular HA depolymerization of [ 3 H]-HA in human skin fibroblast culture. Among the mushroom extracts tested, the extract from the mushroom Tricholoma orirubens showed the most potent activity, therefore, we tried to isolate the active principles from the mushroom. We wish to report here the isolation, the structure determination and the inhibitory activity against HA-degradation of three novel phenols named orirubenone A (1), B (2), and C (3). 2. Result and discussion The extract of T. orirubens was divided into a CHCl 3 fraction, an EtOAc soluble fraction and a water soluble fraction. Since only the EtOAc soluble fraction showed significant activity, this fraction was repeatedly chromato- graphed on the basis of the result of the bioassay. As a consequence, three new compounds were purified and named orirubenone A (1), B (2), and C (3). 0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2003.10.129 Tetrahedron 60 (2004) 7049–7052 * Corresponding author. Tel./fax: þ81-542384885; e-mail address: achkawa@agr.shizuoka.ac.jp Keywords: Mushroom; Tricholoma orirubens; Hyaluronan-degradation inhibitor.