| 摘要: | 肺纖維化為長新冠的後遺症之一，其發生率高於 44.9 %，但目前無有效的治療方法，因此利用具有清肺熱瀉肺火功效的桑白皮探討其抗肺纖維化之有效成分。首先自零售、批發、醫療院所、中藥廠，4 種供應管道購得 36 件桑白皮，其中 20 件帶有栓皮層，不符合臺灣中藥典性狀規範。依照香港中藥材標準萃取及分析方法，以 HPLC 分別定量 mulberroside A、oxyresveratrol、kuwanon G、morusin 指標成分。其中，mulberroside A 在有無帶栓皮層的桑白皮萃取物中含量皆大於0.1 %，且 kuwanon G 在有帶栓皮層的含量比不帶栓皮層高 9 倍。並將 36 件萃取物進行抗氧化、抗發炎及抗肺纖維化活性分析。實驗結果利用 Ward.D 進行分群分析，依活性高低及成分含量多寡可分為 2 群，具有清除 DPPH 自由基、抑制 LPS 誘導 RAW 264.7 巨噬細胞產生 NO 及抑制 TGF-β1誘導 MRC-5 肺纖維母細胞產生 PAI-1 能力，且其化學組成含量較高，為帶栓皮層之桑白皮。將桑白皮的栓皮層與纖維層分開進行成分與活性分析，結果顯示: oxyresveratrol、kuwanon G、kuwanon H 及 morusin 富含於栓皮層萃取物。且其比纖維層萃取物更具抑制 RAW 264.7 細胞產生 NO 作用 (IC50 : 76.13 μg/mL)，但對 NIH/3T3 胚胎纖維母細胞及 THP-1 單核球細胞的細胞毒性亦較強。然而，在本研究中的 20 件帶有栓皮層之桑白皮中，除 R22 在 MRC-5 細胞具有細胞毒性外，其他件桑白皮並無毒性。繼而利用RT-qPCR 測定肺纖維化基因 (vimentin、fibronectin、collagen type I) 的表現量，追蹤分離帶栓皮層之桑白皮活性成分，並進行劃分部之指紋圖譜與活性相關性分析。結果顯示: 在濃度 10 μg/mL 下，各個活性成分對 3 種肺纖維化之基因均具有不同的抑制表現: mulberroside A 及 oxyresveratrol 對 vimentin 基因具抑制表現，且具高度正相關性；kuwanon H、kuwanon G、morusin 對 3 種肺纖維化基因皆具有抑制表現，其中又以 kuwanon H 為最具抗肺纖維化之作用。根據上述結果，建議用 mulberroside A 作為桑白皮活性指標成分，若要分辨是否帶有栓皮層，則可用 kuwanon G 作為鑑定指標成分。另外，mulberroside A、oxyresveratrol、kuwanon G、kuwanon H、morusin 對不同肺纖維化基因表現作用有不同強度的抑制作用，故未來可以利用桑白皮的栓皮層萃取物開發新的抗肺纖維化之植物新藥，而非單一天然物。
Fibrosis of the lungs is one of the sequelae of COVID-19, with an incidence rate exceeding 44.9%. Currently, there is no effective treatment for this condition. Therefore, the study explored the potential anti-fibrotic components of Mori Cortex, known for its ability to clear heat from the lungs. Thirty-six samples of Mori Cortex were acquired from four different sources of supply, including retail, wholesale, medical institutions, and traditional Chinese medicine manufacturers. Among them, 20 pieces had cork that did not meet the specifications of the Taiwan Herbal Pharmacopeia. Using HPLC and following the Hong Kong Chinese Materia Medica Standards for extraction and analysis, the content of mulberroside A, oxyresveratrol, kuwanon G, and morusin were quantified as the indicative components. Mulberroside A was found to be present in amounts greater than 0.1 % in both cork and non-cork Mori Cortex extracts, while kuwanon G exhibited a nine-fold higher content in cork samples compared to non-cork ones. All 36 extracts were subjected to analyses for antioxidant, anti-inflammatory, and anti-fibrotic activities. Through Ward.D clustering analysis, the extracts were divided into two groups based on their activity levels and component quantities. The group containing higher chemical composition levels was identified as the cork Mori Cortex samples, which exhibited abilities to scavenge DPPH free radicals, inhibit NO production in LPS-induced RAW 264.7 macrophages, and suppress PAI-1 production in TGF-β1-induced MRC-5 lung fibroblasts. Further analysis component and bioactivity of the seperated cork and fibrous layers of Mori Cortex for analysis revealed that oxyresveratrol, kuwanon G, kuwanon H, and morusin were abundant in the cork extracts. These components showed stronger inhibitory effects on NO production in RAW 264.7 cells (IC50: 76.13 μg/mL) but also exhibited higher cytotoxicity against NIH/3T3 embryonic fibroblasts and THP-1 monocytes. In this study, out of the 20 cork Mori Cortex samples, only R22 showed cytotoxicity against MRC-5 cells, while the others did not exhibit toxicity. Subsequently, the expression levels of fibrotic genes (vimentin, fibronectin, collagen type I) were measured using RT-qPCR to track the active components in the cork of Mori Cortex. The results revealed that at a concentration of 10 μg/mL, each active component exhibited different inhibitory effects on the three fibrotic genes. Mulberroside A and oxyresveratrol showed inhibition of vimentin gene expression with a highly positive correlation, while kuwanon H, kuwanon G, and morusin showed inhibition of all three fibrotic genes, with kuwanon H exhibiting the most potent anti-fibrotic effect. Based on the above findings, it is suggested to use mulberroside A as the indicator component for the activity of Mori Cortex. To identify whether Mori Cortex contains cork layers, kuwanon G can be used as a marker. Additionally, mulberroside A, oxyresveratrol, kuwanon G, kuwanon H, and morusin varied degrees of inhibition on the expression of different fibrotic genes, indicating the potential for developing new anti-fibrotic plant-based drugs from the cork layer extracts of Mori Cortex, rather than relying on a single natural compound. |
