Identification of Anti-Inflammatory Compounds from Peucedanum praeruptorum Roots by Using Nitric Oxide-Producing Rat Hepatocytes Stimulated by Interleukin 1ß.

The roots of Peucedanum praeruptorum Dunn and Angelica decursiva Franchet et Savatier are designated Zenko, which is a crude drug defined by the Japanese Pharmacopoeia. This crude drug is used as an antitussive and an expectorant and is included in the Kampo formula Jinsoin, which improves cough, fever, and headache. Although the anti-inflammatory effects of this crude drug have been determined, the constituents responsible for this effect remain unknown. To investigate biologically active compounds, rat hepatocytes were used, which produce proinflammatory mediator nitric oxide (NO) in response to proinflammatory cytokine interleukin 1ß (IL-1ß). A methanol extract of P. praeruptorum roots, which suppressed IL-1ß-induced NO production, was fractionated into three crude fractions (ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions) based on hydrophobicity. The EtOAc-soluble fraction markedly inhibited NO production. After this fraction was purified, three biologically active compounds were identified as praeruptorins A, B, and E, the contents of which were high. A comparison of their activities indicated that praeruptorin B exhibited the highest potency to inhibit NO production by decreasing inducible NO synthase expression and suppressed the expression of mRNAs encoding proinflammatory cytokines. Collectively, the three praeruptorins may primarily contribute to the anti-inflammatory effects of P. praeruptorum roots.

Recombinant canine basic fibroblast growth factor-induced differentiation of canine bone marrow mesenchymal stem cells into voltage- and glutamate-responsive neuron-like cells.

INTRODUCTION: Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated. METHODS: The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca2+ concentrations after stimulation with KCl and l-glutamate were examined. RESULTS: The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca2+ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group. CONCLUSIONS: A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs.

Identification of anti-inflammatory constituents in Phellodendri Cortex and Coptidis Rhizoma by monitoring the suppression of nitric oxide production.

Phellodendri Cortex (Obaku in Japanese) and Coptidis Rhizoma (Oren), both of which contain berberine, have been used to prepare the kampo formula orengedokuto to treat inflammatory diseases, including dermatitis, gastric ulcers, and gastritis. These drugs are blended differently in other formulas, such as the use of Phellodendri Cortex in shichimotsukokato to treat hypertension and Coptidis Rhizoma in hangeshashinto to treat diarrhea and stomatitis. However, the differences in their medicinal properties are not well characterized. We prepared extracts from Phellodendron amurense bark (PAB) and Coptis chinensis rhizome (CCR) and separated them into alkaloid and non-alkaloid fractions. Anti-inflammatory effects were examined by monitoring the production of nitric oxide (NO), which is a pro-inflammatory mediator. A non-alkaloid fraction of the PAB extract suppressed NO production in hepatocytes more efficiently than that of the CCR extract. When each non-alkaloid fraction of the PAB and CCR extracts was administered to mice, the fractions of both extracts decreased the levels of mRNAs encoding inducible NO synthase and molecules in the interleukin-1ß signaling pathway. Limonin and obakunone identified in the PAB non-alkaloid fraction suppressed NO production, exhibiting IC50 values of 16 and 2.6 µM, respectively, whereas berberine and coptisine displayed IC50 values of 12 and 14 µM, respectively. Limonin and obakunone reduced the expression of the iNOS gene, probably through the transcription factor nuclear factor-κB. Therefore, both limonoids and alkaloids may be responsible for the anti-inflammatory effects of the PAB extract, whereas alkaloids may be primarily responsible for those of the CCR extract. The different composition of the constituents may modulate the anti-inflammatory effects of Phellodendri Cortex and Coptidis Rhizoma.