Shoji Maehara

Ability of endophytic filamentous fungi associated with Cinchona ledgeriana to produce Cinchona alkaloids

We have investigated the ability of endophytic filamentous fungi associated with Cinchona ledgeriana (Rubiaceae) to produce Cinchona alkaloids on potato dextrose agar medium and in a synthetic liquid medium. It was found that all twenty-one endophytic fungi produce Cinchona alkaloids, despite their genetic differences.

Composition of endophytic fungi living in Cinchona ledgeriana (Rubiaceae).

A total of 21 endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in West Java, Indonesia. They were classified into six genera, namely nine Phomopsis spp., six Diaporthe spp., two Schizophyllum spp., two Penicillium spp., one Fomitopsis sp., and one Arthrinium sp. by using nucleotide sequence analysis of the internal transcribed spacers (ITS1 and ITS2) including 5.8S ribosomal DNA region and phylogenetic analysis.

Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of Cinchona alkaloids

Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

Curcuma sp.‐derived dehydrocurdione induces heme oxygenase‐1 through a Michael reaction between its α, β‐unsaturated carbonyl and Keap1

To elucidate the anti‐inflammatory mechanism of Curcuma sp., we investigated whether dehydrocurdione, a sesquiterpene contained in Curcuma sp., induces heme oxygenase (HO)‐1, an antioxidative enzyme, in RAW 264.7 macrophages. Dehydrocurdione was extracted from the rhizome of Curcuma sp., and its purity was verified by high performance liquid chromatography. Treatment with 10–100 μM dehydrocurdione transiently and concentration‐dependently increased HO‐1 mRNA and protein levels. Docking simulation suggested the presence of the Michael reaction between dehydrocurdione and Kelch‐like ECH‐associated protein (Keap)1 keeping nuclear factor‐erythroid2‐related‐factor (Nrf)2, a transcription factor, in the cytoplasm. Nrf2 that was definitely free from Keap1 was detected in the nuclei after dehydrocurdione treatment. Subsequently, the HO‐1 E2 enhancer, a target of Nrf2, was activated, resulting in HO‐1 expression. Also, an investigation using 6‐shogaol and 6‐gingerol supported the concept that the α, β‐unsaturated carbonyl structure plays an important role in the interaction with Keap1. Dehydrocurdione suppressed lipopolysaccharide‐induced NO release, a marker of inflammation. Clarification of the HO‐1 synthesis increase mechanism revealed in this study will help contribute to the development of novel phytotherapeutic strategies against inflammation‐associated diseases.

Shogaol but not gingerol has a neuroprotective effect on hemorrhagic brain injury: contribution of the α, β-unsaturated carbonyl to heme oxygenase-1 expression

Abstract We investigated the effects of shogaol, which has an &agr;, &bgr;‐unsaturated carbonyl group, and gingerol, which does not, on primary‐cultured microglia to understand how the &agr;, &bgr;‐unsaturated carbonyl interacts with Kelch‐like ECH‐associated protein (Keap)1. Shogaol (1 &mgr;M) but not the same concentration of gingerol significantly increased heme oxygenase (HO)‐1 protein levels in cultured microglia without cytotoxicity. In addition, shogaol suppressed the release of the inflammation marker nitric oxide induced by 30 U/ml thrombin treatment. A docking simulation suggested that the &agr;, &bgr;‐unsaturated carbonyl of shogaol but not gingerol interacts with Keap1. Nuclear import of nuclear factor E2‐related factor 2 and increased binding of the HO‐1 E2 enhancer support the docking‐simulation prediction. The transcription inhibitor actinomycin D (0.1 &mgr;g/ml) markedly blocked the increase of HO‐1 mRNA levels by shogaol. To evaluate whether the &agr;, &bgr;‐unsaturated carbonyl can be used for intracerebral hemorrhage (ICH) therapy, we investigated the effect of shogaol on an in vivo mouse ICH model. Intracerebroventricular injection of 0.2 nmol shogaol increased striatal HO‐1 protein levels and rescued ICH‐induced neuron loss. Thus, the &agr;, &bgr;‐unsaturated carbonyl is necessary for the interaction of compounds, such as shogaol, with Keap1, and these findings may be useful for screening novel ICH therapeutic agents that increase HO‐1 expression.