Chemical Constituents Identified from Fruit Body of Cordyceps bassiana and Their Anti-Inflammatory Activity.

Cordyceps bassiana is one of Cordyceps species with anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities. This mushroom has recently demonstrated to have an ability to reduce 2,4-dinitrofluorobenzene-induced atopic dermatitis symptoms in NC/Nga mice. In this study, we further examined phytochemical properties of this mushroom by column chromatography and HPLC analysis. By chromatographic separation and spectroscopic analysis, 8 compounds, such as 1,9-dimethylguanine (1), adenosine (2), uridine (3), nicotinamide (4), 3-methyluracil (5), 1,7-dimethylxanthine (6), nudifloric acid (7), and mannitol (8) were identified from 6 different fractions and 4 more subfractions. Through evaluation of their anti-inflammatory activities using reporter gene assay and mRNA analysis, compound 1 was found to block luciferase activity induced by NF-κB and AP-1, suppress the mRNA levels of cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-α. Therefore, our data strongly suggests that compound 1 acts as one of major principles in Cordyceps bassiana with anti-inflammatory and anti-atopic dermatitis activities.

Syk-Mediated Suppression of Inflammatory Responses by Cordyceps bassiana.

The fruit body of artificially cultivated Cordyceps bassiana has been reported to exhibit anti-inflammatory and anticancer activities. Although it has been suggested that the fruit body has neutraceutic and pharmaceutic biomaterial potential, the exact anti-inflammatory molecular mechanism has not been fully elucidated. In this study, we demonstrated the immunopharmacologic activity of Cordyceps bassiana under in vitro conditions and investigated its anti-inflammatory mechanism. Water extract (Cm-WE) of the fruit body of artificially cultivated Cordyceps bassiana without polysaccharide fractions reduced the expression of the proinflammatory genes cyclooxygenase (COX)-2, interleukin (IL)-12, and inducible nitric oxide synthase (iNOS) and promoted the expression of the anti-inflammatory gene IL-10 in lipopolysaccharide (LPS)-treated RAW264.7 cells. In addition, this fraction suppressed proliferation and interferon (IFN)-[Formula: see text] production in splenic T lymphocytes. Cm-WE blocked the activation of nuclear factor (NF)-[Formula: see text]B and activator protein (AP)-1 and their upstream inflammatory signaling cascades, including Syk, MEK, and JNK. Using kinase assays, Syk was identified as the target enzyme most strongly inhibited by Cm-WE. These results strongly suggest that Cm-WE suppresses inflammatory responses by inhibiting Syk kinase activity, with potential implications for novel neutraceutic and pharmaceutic biomaterials.

Anti-Proliferative and Pro-Apoptotic Activities of 4-Methyl-2,6-bis(1-phenylethyl)phenol in Cancer Cells.

It has been found that 4-isopropyl-2,6-bis(1-phenylethyl)phenol (KTH-13), a novel compound isolated from Cordyceps bassiana, is able to suppress tumor cell proliferation by inducing apoptosis. To mass-produce this compound, we established a total synthesis method. Using those conditions, we further synthesized various analogs with structural similarity to KTH-13. In this study, we aimed to test their anti-cancer activity by measuring anti-proliferative and pro-apoptotic activities. Of 8 compounds tested, 4-methyl-2,6-bis(1-phenylethyl)phenol (KTH-13-Me) exhibited the strongest anti-proliferative activity toward MDA-MB 231 cells. KTH-13-Me also similarly suppressed the survival of various cancer cell lines, including C6 glioma, HCT-15, and LoVo cells. Treatment of KTH-13-Me induced several apoptotic signs in C6 glioma cells, such as morphological changes, induction of apoptotic bodies, and nuclear fragmentation and chromatin condensation. Concordantly, early-apoptotic cells were also identified by staining with FITC-Annexin V/PI. Moreover, KTH-13-Me highly enhanced the activation of caspase-3 and caspase-9, and decreased the protein level of Bcl-2. In addition, the phosphorylation levels of Src and STAT3 were diminished in KTH-13-Me-treated C6 cells. Therefore, these results suggest that KTH-13-Me can be developed as a novel anti-cancer drug capable of blocking proliferation, inducing apoptosis, and blocking cell survival signaling in cancer cells.

Pro-Apoptotic Activity of 4-Isopropyl-2-(1-Phenylethyl) Aniline Isolated from Cordyceps bassiana.

Cordyceps species including Cordyceps bassiana are a notable anti-cancer dietary supplement. Previously, we identified several compounds with anti-cancer activity from the butanol fraction (Cb-BF) of Cordyceps bassiana. To expand the structural value of Cb-BF-derived anti-cancer drugs, we employed various chemical moieties to produce a novel Cb-BF-derived chemical derivative, KTH-13-amine-monophenyl [4-isopropyl-2-(1-phenylethyl) aniline (KTH-13-AMP)], which we tested for anti-cancer activity. KTH-13-AMP suppressed the proliferation of MDA-MB-231, HeLa, and C6 glioma cells. KTH-13-AMP also dose-dependently induced morphological changes in C6 glioma cells and time-dependently increased the level of early apoptotic cells stained with annexin V-FITC. Furthermore, the levels of the active full-length forms of caspase-3 and caspase-9 were increased. In contrast, the levels of total forms of caspases-3, caspase-8, caspase-9, and Bcl-2 were decreased in KTH-13-AMP treated-cells. We also confirmed that the phosphorylation of STAT3, Src, and PI3K/p85, which is linked to cell survival, was diminished by treatment with KTH-13-AMP. Therefore, these results strongly suggest that this compound can be used to guide the development of an anti-cancer drug or serve as a lead compound in forming another strong anti-proliferative agent.

Cultural Characteristics and Fruiting Body Production in Cordyceps bassiana.

Single ascospore isolates of Cordyceps bassiana were observed for their colony pigmentation on Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates and were inoculated in a brown rice medium for production of fruiting bodies. Colony pigmentation did not show any relationship with perithecial stromata formation. The isolates were also grown on opposite sides of SDAY agar plates and were observed for vegetative compatibility. Neither vegetative compatibility nor perithecial stromata could be found to be related to each other. It was concluded that fertile fruiting body production was independent of colony pigmentation and vegetative compatibility. Synnemata formation was found to be more common than perithecial stromata formation. This might be due to its highly conidiogenous anamorphic stage, i.e., Beauveria bassiana.

Successful Development of Cordyceps bassiana Stromata from Beauveria bassiana.

A specimen of Beauveria bassiana was collected from Yang-yang of Gangwon province, Korea in October 2006. Conidial isolates were prepared from the specimen by the dilution method and inoculated in brown rice medium for fruiting body production. After nearly two months incubation for perithecial stromata developed from single isolates as well as from their combinations. They were determined as Cordyceps bassiana by observing the stromatal characters and their conidial structures. This is the first report of the development of C. bassiana from B. bassiana cultures.

Stable Formation of Fruiting Body in Cordyceps bassiana.

In order to breed a Cordyceps bassiana isolate that stably forms fruiting body in artificial cultivation, isolates derived from subculturing and single spores were tested through mating. From C. bassiana EFCC 783, three subcultured isolates EFCC 2830, EFCC 2831 and EFCC 2832 were obtained and fourteen single conidial isolates were obtained from these three subcultured isolates. Two different morphological types were found in the fourteen single conidial isolates. One type was able to form synnemata and another type was not able to form synnemata. Since switch of morphological type was not observed despite their continuous subculturing, cross was performed between the two types and the formation of fruiting body was examined. Ascospores were obtained from a selected fruiting body formed by hybrid of the cross. Self-cross and combinational cross of the ascospore-derived isolates generated hybrids that stably produce high quality fruiting body in artificial media.

Cordyceps bassiana and Production of Stromata in vitro Showing Beauveria Anamorph in Korea.

A Cordyceps species was found with a Beauveria anamorph state on larval insect cadavers on Obong mountains in Gangwon Provinces, Republic of Korea. Cultures from discharged ascospores formed an anamorph identifiable as Beauveria bassiana. This teleomorph-anamorph connection was also confirmed by the in vitro production of fertile ascomata from conidial cultures with morphology like that of field-collected specimen. This is the first report of in vitro production of a teleomorph for any Beauveria species. The Cordyceps species has been conspecified as Cordyceps bassiana, a species described from China with B. bassiana anamorph.