抗アレルギー物質の探索を目的とする新規アッセイ法の開発と応用

卵白リゾチーム（HEL）を用いて感作したマウスのアレルギーインダクションフェーズでの血流量低下を指標とするアッセイ法を確立し、これを用いてこの血流量低下のメカニズムの解明を試みた。その結果、アナフィラキシー発症時に血流量あるいは血圧低下に関与するヒスタミン等のケミカルメディエーターは血流量低下に関与しないが、血管内皮細胞や血小板、好中球などが関与し、シクロオキシゲナーゼ（COX）-1および2、プロスタグランジン（PG）I2、トロンボキサン（TX）A2、エンドセリン（ET）-1、顆粒球エラスターゼ（GE）および誘導型NO合成酵素（iNOS）由来のNOが複雑に関連して関与していることを明らかにした。さらに天然資源から新規機能物質の探索を行う目的で、顕著な活性が認められたツリフネソウおよびスイフヨウに関して新規アッセイ法を用いてアレルギーインダクションフェーズ抑制物質の探索を行った結果、ツリフネソウ花弁の35% EtOHエキスよりluteolin（3）などに有意な改善効果を確認し、活性発現メカニズムについて検討した。次にスイフヨウ花弁MeOHエキスでは、活性成分としてquercetin-3-O- β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside（12）、および新規化合物mutabiloside（11）を明らかにした。さらに本アッセイ法における末梢血流量低下の症状が、漢方における瘀血症状と類似していることに着目し、本アッセイ法が瘀血改善薬の探索に利用可能であることを明らかにし、新たな瘀血のアッセイ法として応用できることを示した。以上、本法はアレルギーのインダクションフェーズでの多数の因子による複雑な生体内反応を総合的にとらえたものであり、天然資源より新しいメカニズムによるアレルギー予防薬のシーズやリード化合物の探索に応用できる可能性を示すと考えられる。We discovered a phenomenon in which the blood flow in vein microcirculation markedly decreases in response to hen-egg white lysozyme (HEL)-sensitization without any change in blood pressure. Using this blood flow decrease as a guide, we developed an in vivo assay method to search for substances, which can prevent allergies. The blood flow decrease appears to be regulated by various factors such as nitric oxide (NO), thromboxane (TX) A2, prostacyclin (PGI2) and endothelin (ET)-1 together with cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase (cNOS). Using this in vivo assay method, allergy-preventive activity was demonstrated for the 35% EtOH extract of flowers of Impatiens textori MIQ. and the MeOH extract of the petals of Hibiscus mutabilis L. \u27versicolor\u27 MAKINO in a continuing search for allergy-preventive substances from natural source. Among the principal compounds in IT, apigenin (1), apigenin 7-glucoside (2), luteolin (3) and luteolin 7-glucoside (10) showed significant allergy-preventive effects. Among the principal compounds in HM, quercetin-3-O- β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside (12), and mutabiloside (11) showed significant allergy-preventiveeffects. “Oketsu”, or stagnant blood syndrome, is one of the important pathological concepts in therapy with Kampo formula and drugs. We showed the effectiveness of the method for screening drugs aimed at “oketsu” treatment

Beneficial effect of Sparassis crispa on stroke through activation of Akt/eNOS pathway in brain of SHRSP

Sparassis crispa (S. crispa) is a mushroom used as a natural medicine that recently became cultivatable in Japan. In this study, we investigated not only the preventive effects of S. crispa against stroke and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP) but also the mechanism involved by using studies of the cerebral cortex at a young age. Six-week-old male SHRSP were divided into 2 groups, a control group and an S. crispa group administered 1.5% S. crispa in feed, and we then observed their survival. In addition, rats of the same age were treated with 1.5% S. crispa for 4 weeks and we measured body weight, blood pressure, blood flow from the tail, NOx production, and the levels of expression of several proteins in the cerebral cortex by western blot analysis. Our results showed that the S. crispa group had a delayed incidence of stroke and death and significantly decreased blood pressure and increased blood flow after the administration. Moreover, the quantity of urinary excretion and the nitrate/nitrite concentration in cerebral tissue were higher than those of control SHRSP rats. In the cerebral cortex, phosphor-eNOS (Ser1177) and phosphor-Akt (Ser473) in S. crispa-treated SHRSP were increased compared with those of control SHRSP rats. In conclusion, S. crispa could ameliorate cerebrovascular endothelial dysfunction by promoting recovery of Akt-dependent eNOS phosphorylation and increasing NO production in the cerebral cortex. S. crispa may be useful for preventing stroke and hypertension

Atractylodin Produces Antinociceptive Effect through a Long-Lasting TRPA1 Channel Activation

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors