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-1β 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.
KeywordsPhellodendri Cortex Coptidis Rhizoma Limonoid Anti-inflammatory effect Nitric oxide Hepatocyte
High-performance liquid chromatography
Nuclear magnetic resonance
Half-maximal inhibitory concentration
Inducible nitric oxide synthase
Polymerase chain reaction
Electrophoretic mobility shift assay
Phellodendron amurense bark
Coptis chinensis rhizomes
We thank Ms. Yuka Yamamoto, Ms. Megumi Hashima, and Mr. Kento Uemura for technical assistance; Dr. Takehito Miura for valuable advice; and Ms. Noriko Kanazawa for secretarial assistance; and Dr. Yuji Hasegawa (Central Equipment Room, Daiichi University of Pharmacy) for the MS measurements.
MN and YI designed the experiments and wrote the manuscript. AF designed and performed the experiments as a graduate student. KW, TO, and TO performed the experiments and analyzed the data.
Compliance with ethical standards
Conflict of interest
No potential conflict of interest.
- 1.The Committee on the Japanese Pharmacopoeia (2016) The Japanese pharmacopoeia, 17th edn. The Minister of Health, Labour and Welfare, JapanGoogle Scholar
- 4.Anetai M, Hayashi T, Yamagishi T (1987) Determination of berberine alkaloids in Phellodendri Cortex. Rep Hokkaido Inst Public Health 37:18–21Google Scholar
- 5.Anetai M (1994) Chemical evaluation of Coptis rhizome—determination of berberine alkaloids by HPLC and spectrophotometry. Rep Hokkaido Inst Public Health 44:1–6Google Scholar
- 15.Kitade H, Sakitani K, Inoue K, Masu Y, Kawada N, Hiramatsu Y, Kamiyama Y, Okumura T, Ito S (1996) Interleukin-1β markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells. Hepatology 23:797–802PubMedGoogle Scholar
- 16.Ohno N, Yoshigai E, Okuyama T, Yamamoto Y, Okumura T, Ikeya Y, Nishizawa M (2012) Chlorogenic acid from the Japanese herbal medicine Kinginka (Flos Lonicerae japonicae) suppresses the expression of inducible nitric oxide synthase in rat hepatocytes. HOAJ Biol 1:2. doi: 10.7243/2050-0874-1-2 CrossRefGoogle Scholar
- 19.Miki H, Tokuhara K, Oishi M, Nakatake R, Tanaka Y, Kaibori M, Nishizawa M, Okumura T, Kon M (2016) Japanese kampo saireito has a liver-protective effect through the inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes. J Parenter Enter Nutr 40:1033–1041CrossRefGoogle Scholar
- 28.Tanemoto R, Okuyama T, Matsuo H, Okumura T, Ikeya Y, Nishizawa M (2015) The constituents of licorice (Glycyrrhiza uralensis) differentially suppress nitric oxide production in interleukin-1β-treated hepatocytes. Biochem Biophys Rep 2:153–159. doi: 10.1016/j.bbrep.2015.06.004 PubMedPubMedCentralGoogle Scholar
- 31.Kawamura T, Yokoe M, Hisata Y, Okuda K, Noro Y, Yamaguchi S, Tanaka T, Nishibe S, Wada K (2002) Diversity of the quality of Phellodendron barks from different habitats. (2). Content variation of limonoids. Nat Med 56:198–199Google Scholar