Keita Minamisaka

The capitula of Chrysanthemum indicum Linné or C. morifolium Ramatuelle (Kikuka in Japanese) are included in several formulae of Kampo medicines (traditional Japanese medicines), such as Chotosan, which is used for headache and dizziness. Luteolin, the principal constituent of C. indicum, has antioxidant and anti-inflammatory activities. However, the effects of other flavonoids on this crude drug have not yet been thoroughly investigated. To evaluate and compare anti-inflammatory effects, we used primary cultured rat hepatocytes, which produce proinflammatory mediators, such as nitric oxide (NO) and proinflammatory cytokines, in response to interleukin (IL)-1β. Eight derivatives of 5,7-dihydroxyflavone were purified and identified in the ethyl acetate-soluble fraction of a C. indicum capitulum extract: luteolin (Compound 1), apigenin (2), diosmetin (3), 5,7-dihydroxy-3′,4′,5′-trimethoxyflavone (4), acacetin (5), eupatilin (6), jaceosidin (7), and 6-methoxytricin (8). Luteolin is the most abundant compound in this fraction. All compounds significantly suppressed NO production in hepatocytes, with apigenin and acacetin showing the greatest efficacy. The comparison of the IC₅₀ values of the inhibition of NO production suggests that substitutions by hydroxyl and methoxy groups at the C-3′ and C-4′ positions of 5,7-dihydroxyflavone may be at least essential for the suppression of NO production. In hepatocytes, acacetin and luteolin decreased the levels of mRNAs encoding inducible nitric oxide synthase (iNOS), proinflammatory cytokines, including tumor necrosis factor, IL-6, and type 1 IL-1 receptor, which regulates inflammatory responses. Based on the comparison of the IC₅₀ values and the content, luteolin, jaceosidin, and diosmetin may be responsible for the anti-inflammatory effects of C. indicum capitula.

Obesity, which is characterized by excessive body fat accumulation and energy imbalance, is a major risk factor for type 2 diabetes mellitus. Boesenbergia rotunda rhizomes (known as fingerroots) exhibit a variety of pharmacological activities and are used in traditional medicine. Pinostrobin was identified as a major constituent of Boesenbergia rotunda rhizome (BR) extract and suppressed nitric oxide production in interleukin 1β-treated hepatocytes. Next, we investigated the anti-obesity effects of BR extract in ob/ob mice, a type 2 diabetes mellitus model. The ob/ob mice were treated with or without 1% BR extract for 14 days and then analyzed for serum insulin and triglyceride levels, liver histology, and liver mRNA expression. The administration of BR extract significantly decreased blood glucose concentrations and increased serum insulin concentrations in ob/ob mice. In addition, this treatment reduced triglyceride levels in both the serum and liver and decreased lipid accumulation in hepatocytes. Microarray analysis revealed that hepatic mRNA affected the expression of genes involved in inflammation, lipid metabolism, lipid synthesis, and insulin signaling, leading to improvements in obesity. Because BR extract has hypoglycemic and antidiabetic effects on ob/ob mice, it might be a promising candidate for addressing obesity and related metabolic disorders.

Traditional Japanese medicines, i.e., Kampo medicines, consist of crude drugs (mostly plants) that have empirical pharmacological functions (‘Yakuno’ in Japanese), such as clearing heat. Crude drugs with cold properties, such as Phellodendron bark, have the empirical function of clearing heat as they cool the body. Because we found that anti-inflammatory compounds were present in several crude drugs for clearing heat, it is speculated that the empirical function of clearing heat may be linked to anti-inflammatory activities. When 10 typical crude drugs were selected from 22 herbal crude drugs for clearing heat, we identified anti-inflammatory compounds in five crude drugs, including Phellodendron bark. In this study, the other crude drugs were extracted and partitioned with ethyl acetate (EtOAc) and n-butanol to obtain three crude fractions. All the EtOAc-soluble fractions, except that from Forsythia fruits, inhibited interleukin (IL)-1β-induced nitric oxide (NO) production in primary-cultured rat hepatocytes. Anti-inflammatory compounds were identified from these EtOAc-soluble fractions: baicalein from Scutellaria roots, (−)-nyasol from Anemarrhena rhizomes, and loniflavone from Lonicera leaves and stems. (+)-Phillygenin was purified from Forsythia fruits by removing cytotoxic oleanolic and betulinic acids. These compounds suppressed the production of NO and cytokines in hepatocytes. Anti-inflammatory compounds were not purified from the EtOAc-soluble fraction of Rehmannia roots because of their low abundance. Collectively, these findings indicate that anti-inflammatory compounds are present in all 10 crude drugs for clearing heat, confirming that these anti-inflammatory compounds in crude drugs provide the empirical functions for clearing heat. Other empirical functions of Kampo medicine can also be explained by modern pharmacological activities.