Edible fungi have a long history of use in Traditional Chinese Medicine [¹]. Coprinus comatus is a basidiomycete fungus claimed to benefit glycaemic control in diabetes. Many physiological functions of Coprinus comatus have been reported, such as anti-cancer properties [²], immunomodulatory [³] and hypoglycemic activity [⁴]. Stadler et al. reported that agaricoglycerides, a new class of fungal secondary metabolites isolated from several basidiomycetes, showed strong activities against neurolysin, a protease involved in the regulation of dynorphin and neurotensin metabolism, and even exhibited moderate analgesic in vivo[⁵]. The agaricoglycerides are a new class of fungal secondary metabolites that constitute esters of chlorinated 4-hydroxy benzoic acid and glycerol. Although the crude extract from Coprinus comatus was no beneficial effect at lower doses in mice subjected to acetic-acid writhing test, formalin test and hot-plate test [⁶], there has been no report on the antinociceptive activity of agaricoglycerides extract from Coprinus comatus. Therefore, the objectives of the present study were to demonstrate the production, isolation, and biological properties of triglycerides of the fermented mushroom of Coprinus comatus.

The present study demonstrates, for the first time, that the anti-inflammatory and analgesic effects of TFC. Carrageenan-induced inflammation in rats is a well-characterized experimental model of acute inflammation that permits the quantification and correlation of cellular migration with changes in other inflammatory parameters [¹⁶]. Diclofenac is a nonsteroidal anti-inflammatory drug used to treat pain andinflammation associated with arthritis. It was used as a standard. The inflammatory response involves a complex intercellular signal promoting cytokine release. Activated inflammatory cells synthesize and secrete proinflammatory cytokines such as TNF-α, IL-1β, VEGF-α, and IL-17 [^17-19]. The suppression of these proinflammatory mediators has been found to reduce the severity of the inflammatory reaction [²⁰]. Some work has been done on the effects of the mushroom on cytokines and nuclear factor kappa B (NF-κB) levels [^21-25]. The present study was undertaken to determine the effect of TFC on protein levels of TNF-α, IL-1β, VEGF-α, and IL-17 during the inflammatory process induced by carrageenan.

Treatment with TFC (30 m/kg) significantly reduced the levels of TNF-α by 58.3% (P <0 .01), IL-1β by 27% (P < 0.05),VEG F-α by 46.5% (P < 0.01) and IL-17 by 89.2% (P <0 .001), respectively (Table 1). Results of this study clearly indicate the anti-inflammatory activity of the TFC in acute inflammatory conditions. It could effectively inhibit the leukocyte migration promoted by carrageenan in rat.

O₂⁻ has been associated with tissue damage and loss of function during inflammatory episodes [²⁶]. The total antioxidant status (TAOS) is an indication of O₂⁻ and other oxidant species. We measured TAOS activity as an indirect indication of the formation of O₂⁻ and other oxidant species. The TFC (10 m/kg) groups had the lower level of TAOS activity in comparison to the saline group (P < 0.01) (Figure 1). It is consistent with the previous report on the antioxidant properties of Coprinus comatus [²⁷,²⁸]. O₂⁻ is produced by polymorphonuclear leukocytes and macrophages from the enzyme activity of NADPH oxidase and xanthine oxidase at inflammatory sites. We hypothesized that TFC produce anti-inflammatory effect through decreasing the levels of TAOS activities.

The report that no decrease in oedema induced by formalin injection was observed following treatment with C. comatus also showed that C. comatus had a peripheral antinociceptive and anti-inflammatory effect [²⁹]. In the writhing test, TFC inhibited the acetic acid-induced abdominal constrictions in a dose-dependent manner (40.2%, 69.7%, and 76.9%) (Table 2). The acetic acid-induced writhing reaction in mice has been used as a screening tool for the assessment of analgesic or anti-inflammatory properties of new agents [³⁰]. The constrictions induced by acetic acid in mice result from an acute inflammatory reaction related to the increase in the peritoneal fluid levels of PGE₂ and PGF₂α [³¹]. The fact that TFC was able to inhibit constrictions showed that TFC has a peripheral antinociceptive effect.

On the contrary, the result of the hot-plate test did not show that TFC has a central antinociceptive effect. The hot-plate test is commonly used to assess narcotic analgesics or other centrally acting drugs [¹³,¹⁴]. The hot-plate test was performed for the assessment of the central antinociceptive effect of TFC in this study. Results showed that TFC inhibited the reaction time to thermal stimuli at 30, 60, and 90 min compared to controls. However, it was not significant (Table 3). The mechanism of TFC does not exert central antinociceptive effect could be explained that it has poor permeability across the blood brain barrier (BBB). BBB is an active interface between the circulation and the central nervous system (CNS) which restricts the free movement of different substances between the two compartments and plays a crucial role in the maintenance of the homeostasis of the CNS [³²].

Complete Freund’s adjuvant (CFA) -induced hyperalgesia is frequently used as an animal model to study chronic inflammatory pain. The CFA-induced inflammation is accompanied by a tactile hyperalgesia (HA), which is robust over several days [³³]. TFC reduced dose-dependently the CFA-induced tactile hyperalgesia (Figure 2). It is likely that the antihyperalgesic effect of CFA was due to a genuine anti-inflammatory effect.

As per the OECD Guidelines-423, LD₅₀ was calculated for TFC. When there is no information of TFC on toxicity, it is recommended to use the starting dose of 250 mg/kg body weight. This involves the estimation of the median lethal dose (LD₅₀), which is the dose that will kill 50% of the animal population within 72 hours post treatment with the test substance. After intraperitoneal injection, most mice in the 400 mg/kg group were quiet and inactive. Mice died approximately 7 hr after injection. The majority of mice in the 400 mg/kg dose group (5 mice) died within 72 hr following the injection. The LD₅₀ was determined to be 400 mg/kg.

In conclusion, TFC showed anti-inflammatory, antioxidant, peripheral antinociceptive and antihyperalgesic activity in various models of inflammatory pain. The data suggest that TFC may be a viable treatment option for inflammatory pain.

The authors declare that they have no competing interests.

J-YG and J-LS were involved in the design of this study and performed laboratory analyses and statistics. JR and C-CH drafted the manuscript along with the other authors. All authors read and approved the final manuscript.

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1472-6882/12/52/prepub

This project was supported by the Key New Drugs Innovation project from Ministry of Science and Technology (2009ZX09103-381, 2009ZX09502-017, 2010ZX09102-201-018), the project from Key Laboratory of Mental Health, Chinese Academy of Sciences, project of Shandong Province Higher Educational Science and Technology Program (J08LH62), NNSF grant (30800301, 31170992) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-R-254, KSCX2-EW-Q-18 and KSCX2-EW-J-8).