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Supplemental material, Supplementary material, for A New Monoterpenoid Glucoindole Alkaloid From Dipsacus asper by Feng Li, Yuto Nishidono, Ken Tanaka, Shiro Watanabe and Yasuhiro Tezuka in Natural Product Communications

A 68-year-old man was admitted with high fever and dyspnea. Under the diagnoses of pneumonia and heart failure, antibiotic and diuretic therapies were performed, but elevated CRP of more than 6 mg/dl persisted. An apical type of hypertrophic cardiomyopathy-like structure was observed in echocardiography. On the 18th hospital day, he died suddenly of fatal arrhythmia, and infected apical left ventricular (LV) thrombus complicated by old inferior and acute anterior myocardial infarction was found in the autopsy. In this case, the LV mural thrombus was adhered around the apical portion of the myocardium and presented inflammatory change, which might have caused the fatal arrhythmias.

We describe rare 2 cases of minimal lung cancer with marked progression to the chest wall. The radiological findings revealed a chest wall tumor. Both cases underwent lobectomy concomitant with chest wall resection. Pathological study showed that well-differentiated carcinoma was minimal inside the visceral pleural and poorly differentiated tumor cells broke down the pleura and extended into the chest wall. Both cases died of recurrent cancer within 1 year after surgery.

The constituents of Bu-Shen-Jian-Gu-Tang and their inhibitory activities on osteoclast-Iike cell formation were investigated. Bu-Shen-Jian-Gu-Tang was fractionated by a Diaion HP-20 column with EtOH-H_2O, and the 60% and 90% EtOH-H_20 fractions showed more potent inhibitory activities on osteoclast-like cell formation and bone resorption. Chemical investigation of these two fractions resulted in the isolation of 11 compounds, for which the structures were elucidated based on spectroscopic analysis and chemical reactions. The inhibitory activities of the isolated compounds on osteoclast-like cell formation were evaluated. Among the eleven compounds, icariin (1), icarisid II (2), 2"-O-rhamnosylicariside II (3), kaempferol (8), chikusetsusaponin IVa (10) and chikusetsusaponin V (11) showed potent inhibitory activities at a concentration of 2 μM without toxic effects. The preventive effect of Bu-Shen-Jian-Gu-Tang on bone loss may be due to the inhibitory activities of these compounds. 本研究では,補腎健骨湯について,Diaion HP-20カラムクロマトグラフィを用いて,H_2O,30%,60%及び90%EtOH-H_2Oに順次分画し,各フラクションの破骨細胞形成及び骨吸収の抑制活性を検討したところ,60%及び90%EtOH-H_2O溶出部に抑制活性が強いことが判った。さらに,これらのフラクションから単離した11種の化合物について,破骨細胞形成抑制活性を検討した。11種の化合物のうち,6種の化合物(icariin (1), icarisid II (2), 2"-O-rhamnosylicariside II (3), kaempferol (8), chikusetsu-saponin IVa (10)およびchikusetsusaponin V (11))が補腎健骨湯の骨吸収抑制活性成分であることが明らかとなった。

The inhibitory activities of 19 Chinese formulae on osteoclast formation and bone resorption in vitro were investigated. The inhibitory activity on osteoclast formation was evaluated using a mouse bone marrow cell culture in which osteoclast-like multinucleated cells were formed in the presence of parathyroid hormone (PTH). The inhibitory activity on bone resoption was evaluated using PTH-induced resorption of ^Ca from ^Ca-prelabeled neonatal mouse parietal bone. Among the 19 Chinese formulae, nine showed a strong inhibitory activity on osteoclast formation at a concentration of 50 μg/ml, and three of the nine also showed a strong inhibitory activity on bone resorption. Among these three formulae, Bu-Shen-Jian-Gu-Tang (補腎健骨湯) showed the most promising activity in a dose-dependent manner on the inhibition of bone resorption. A further in vivo experiment in ovariectomized (OVX) model rats using peripheral quantitative computed tomography (pQCT) revealed its preventive effect on bone loss.本研究では19種の漢方方剤によるin vitroでの破骨細胞形成及び骨吸収の抑制活性を検討した。破骨細胞形成抑制活性はマウス骨髄細胞培養系を用い,副甲状腺ホルモン(PTH)存在下での破骨細胞様多核細胞の形成により評価した。骨吸収抑制活性は^Ca標識した新生児マウスの頭頂骨での,PTH誘導による^Ca吸収を評価した。19種の漢方方剤のうち,9処方が50μg/mlの濃度で強い破骨細胞形成抑制活性を示し,この中で,3処方は骨吸収を強く抑制した。中でも,「補腎健骨湯」は用量依存的に骨吸収を抑制し,最も有望な活性を示した。さらに,卵巣摘出(OVX)ラットを用いたin vivoでの実験では, pQCT(末梢骨定量的コンピューター断層撮影法)での評価の結果,骨減少を予防する効果が認められた。

A total of 26 herbal medicines were examined for their inhibitory effects on cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6). A methanol extract of each herbal medicine was prepared and then preincubated with human liver microsomes in the presence of an NADPH-generating system. Residual microsomal CYP3A4 and CYP2D6 activity was then determined by measuring the N-demethylation of erythromycin and the O-demethylation of dextromethorphan, respectively. Of the 26 herbal medicines tested, 16 were found to decrease the residual CYP3A4 activity in a preincubation time-dependent manner. The extract of Evodia Fruit caused the most dramatic decrease in residual CYP3A4 activity (i.e.22.3% residual activity after 30 min preincubation). A substantial decrease in residual CYP3A4 activity was also observed from extracts of Sappan Wood, Incised Notopterygium Rhizome, Schisandra Fruit, Great Burdock Achene, Angelica Dahurica Root and Rhubarb (residual activity of 40.6, 41.2, 53.4, 47.1, 53.4 and 59.2% after 30 min preincubation, respectively). These results are comparable to those using troleandomycin, a known irreversible inhibitor of CYP3A4, which gave a residual activity of 49.4% under identical conditions.26種の生薬のシトクロムP450 3A4(CYP3A4)及び2D6(CYP2D6)に対する阻害作用を調べた。生薬エキス粉末から調製されたメタノール可溶性画分をNADPH生成系存在下, ヒト肝ミクロソームとプレインキュベーションした後のCYP3A4の残存活性(erythromycin N-demethylation活性)とCYP2D6の残存活性(dextromethorphan O-demethylation活性)を測定した。その結果, 16種の生薬がCYP3A4活性をプレインキュベーション時間依存的に低下させた。中でも, 呉茱萸によるCYP3A4活性の低下作用が最も顕著であった(30分間プレインキュベーションした後の活性の残存率は22.3%)。次いで, 蘇木, 羌活, 五味子, 牛蒡子, 白止, 大黄が顕著な低下を示した(30分間プレインキュベーションした後の活性の残存率は, それぞれ40.6, 41.2, 53.4, 47.1, 53.4, 59.2%)。これら7種生薬によるCYP3A4活性低下作用は, CYP3A4に対する不可逆的阻害剤であるtroleandomycinによる残存率(49.4%)に匹敵した。CYP2D6活性に対してプレインキュベーション時間依存的な活性の低下を示した生薬は, 5種であった。最もCYP2D6活性の低下作用が大きかった生薬は, 羌活であり, 30分間プレインキュベーションした後の活性の残存率は61.9%であった。以上の結果から, 呉茱萸, 蘇木, 羌活, 五味子, 牛蒡子, 白止, 大黄等の複数の生薬エキス中に, CYP3A4に対するmetabolism-dependent inhibitorが含まれていることが示唆された。, relation: isVersionOf: http://ci.nii.ac.jp/naid/10014223212/, 21st Century COE Program Toyama Medical and Pharmaceutical University

The inhibitory effects of 78 herbal extracts on cytochrome P450 3A4 (CYP3A4) and P450 2D6 (CYP2D6) activity were investigated using human liver microsomes. The incubation mixture contained a methanol soluble fraction prepared from the powder of each herbal water extract (equivalent to 1.65 mg of extract powder per mL). Thirty-one herbal extracts inhibited over 50% of human liver microsomal erythromycin N-demethylation, a marker reaction of CYP3A4 activity. Among the 31 herbal extracts, 8 of them (Angelica Dahurica Root, Cassia Bark, Clove, Incised Notopterygium Rhizome, Moutan Bark, Rhubarb, Sappan Wood, Schisandra Fruit) inhibited N-demethylation by over 90%. Among the herbal extracts examined, the strongest inhibition of CYP3A4 was noted with Sappan Wood, which had an IC_ value of 43 mg/mL. Rhubarb, Schisandra Fruit, Incised Notopterygium Rhizome, and Angelica Dahurica Root had IC_ values of 77, 127, 144, and 185 μg/mL, respectively. Further, 28 of the herbal extracts inhibited over 50% of human liver microsomal dextromethorphan O-demethylation, which is a marker of CYP2D6 activity. Among the 28 herbal extracts, 13 (Cassia Bark, Clove, Coptis Rhizome, Ephedra Herb, Gambir Plant, Incised Notopterygium Rhizome, Magnolia Bark, Moutan Bark, Phellodendron Bark, Rhubarb, Sappan Wood, Sinomenium Stem, Zanthoxylum Fruit) inhibited O-demethylation by over 90%. The strongest inhibition of CYP2D6 was noted with Phellodendron Bark, which had an IC_ value of 4 μg/mL. Coptis Rhizome, Sinomenium Stem, Sappan Wood, and Rhubarb showed IC_ values of 14, 40, 52, and 64 μg/mL, respectively. These results indicate that many herbal extracts have an inhibitory effect on CYP3A4 and CYP2D6. 78種の生薬エキスについて,ヒト薬物代謝酵素シトクロムP450 3A4(CYP3A4)およびP450 2D6(CYP2D6)に対する阻害作用を調べた。生薬エキス粉末からメタノール可溶性画分を調製し,添加量1.65mg/mLで反応を行った。CYP3A4の指標として用いたヒト肝ミクロソーム中のエリスロマイシンN-脱メチル化活性に対して50%以上の阻害を示した生薬は31種であり,その中の8種(白芷,桂皮,丁子,羌活,牡丹皮,大黄,蘇木,五味子)が阻害率90%以上を示した。CYP3A4活性に対する阻害の強さは,蘇木,大黄,五味子,羌活,白芷の順であり,IC_値は,それぞれ43,77,127,144および185μg/mLであった。一方,CYP2D6の指標として用いたヒト肝ミクロソーム中のデキストロメトルファンO-脱メチル化活性に対して50%以上の阻害を示した生薬は28種であり,その中の13種(桂皮,丁子,黄連,麻黄,釣藤鈎,羌活,厚朴,牡丹皮,黄柏,大黄,蘇木,防巳,山椒)が阻害率90%以上を示した。CYP2D6活性に対する阻害の強さは,黄柏,黄連,防巳,蘇木,大黄の順であり,IC_値は各々4,14,40,52および64μg/mLであった。これらの結果から,CYP3A4あるいはCYP2D6に対する阻害活性を示す生薬が複数存在することが明らかとなった。, 21st Century COE Program, Toyama Medical and Pharmaceutical University