| 摘要: | 傳統中醫藥對於養髮有豐富的典籍記載，以「培補肝腎、益氣養血、活血化瘀」為主要治療方針。且與西藥治療雄性禿藥物造成性功能下降的副作用相反。此外中醫五色理論，認為不同顏色藥材、功效會有所差異，而目前沒有文獻針對顏色與養髮活性做探討，因此本研究利用24種養髮藥材，藉由測量植物化學成分與養髮活性進行關聯性分析 ; 並以五色進行分類，探討其與養髮活性間的關係，並找出具有開發潛力的中藥，作為日後篩選開發養髮藥物的依歸。
利用無母數方法進行養髮活性與植物化學成分含量的關聯性，結果發現，5αR (5α-reductase) 抑制活性對總多酚和縮合單寧類具有正相關 (R=0.747及0.769, p < 0.01) ; 5αR的抑制活性與清除DPPH自由基的抗氧化力間也具有相關性 (R=0.751, p < 0.01)。24種養髮藥材中，以PMC具有最佳的抗氧化力與抑制5αR之活性，且富含總多酚與縮合單寧，因此本研究將針對PMC進行養髮活性機制探討與成分分離。不同溶劑萃取PMC，以50% 乙醇萃取物 (50E-PMC) 之5αR抑制活性最佳 ; 於毛囊乳突細胞的體外試驗，具有促進毛囊細胞生長、促進Wnt10b、抑制DKK-1與AR的基因表現，提高β-catenin蛋白的表現量。在模擬雄性禿小鼠的體內試驗中，50E-PMC具有促進毛髮生長的功效、使毛髮維持在生長期的作用。隨後50E-PMC以ODS管柱與不同比例MeOH水溶液分離出六個劃分部，結果顯示PMC_30Me含有最多的多酚類及縮合單寧，具有上調β-catenin表現量的效果，可以促進Wnt/β-catenin這條路徑。以Q-TOF/MS分析50E-PMC與不同劃分部並結合全球天然物社會分子網路 (Global natural product social molecular networking, GNPS) 製作出分子網絡圖，預測活性物質屬於flavonoid glycoside、biflavonoid與polyflavonoid，屬於黃酮類，此分析結果可以作為未來分離活性物質的策略。
綜上所論，PMC為一具有良好養髮活性的植物，在體外酵素試驗具有抑制雄性素酵素的活性，體外細胞實驗顯示具有促進增生與保護細胞的效果，體內動物試驗具有促進毛髮生長之功效等多項作用，相較於臨床用藥Finasteride與Dutasteride為5αR抑制劑有更多的優勢。配合GNPS數據庫比對，預測出PMC有潛力的活性物質，可作為未來PMC開發為治療雄性禿或養髮產品潛力藥物。
Traditional Chinese medicine (TCM) had a wealth of records in ancient books about nourishing hair for hundreds of years. Through literature review, it had been sorted out that "cultivating liver and kidney, replenishing qi and nourishing blood, promoting blood circulation and removing bruising" were the main treatment guidelines. Therefore, the treatment of hair loss with kidney-tonifying drugs was inversely related to the side effects of western medicine for androgenetic alopecia (AGA) caused by decreased sexual function. In addition, due to the five-color theory of Chinese medicine, it was believed that the colors of medicinal materials and their effects will be different. However, there was currently no literature to discuss the correlation between the color of medicinal materials and the hair-raising activity. Therefore, this study found 24 kinds of hair-raising medicinal materials through the exploration of the classics. Measure the correlation analysis between the chemical composition of the plant and the hair-raising activity. Carry out classification of five-color theory, explore its relationship with hair-raising activity. The outcome of the correlation could be as a basis for screening and developing hair-raising drugs in the future.
Using the parentless method to analysis the relationship between hair-raising activity and the content of phytochemical components, it was found that 5αR (5α-reductase) inhibitory activity had a positive correlation with total polyphenols and condensed tannins (R=0.747 and 0.769, p < 0.01); Inhibition of the activity of 5αR was also correlated with the antioxidant capacity of scavenging DPPH free radicals (R=0.751, p <0.01).
Among the 24 kinds of hair nourishing medicinal materials, PMC had the best antioxidant power and the activity of inhibiting 5αR which was rich in total polyphenols and condensed tannins. Therefore, this study would focus on PMC to explore the mechanism of hair nourishing activity and separate the ingredients. First, use different solvents for PMC extraction, and extract of 50% ethanol (50E-PMC) had the best inhibitory activity against 5αR. Next, in vitro study, experiment of hair follicle dermal papilla cells (HFDPC) showed that 50E-PMC promoted the growth of HFDPC, which could promote Wnt10b gene, inhibit DKK-1 and AR gene expression, and increase the expression of β-catenin protein. In vivo study, 50E-PMC had the effect of promoting hair growth and maintaining the hair in the growth phase on the AGA mice.
In order to find the active ingredients, we used ODS column chromatography to separate 50E-PMC into six fractions. The 30% methanol and 40% methanol fractions eluted by ODS gel had stronger activity and richer contents of total polyphenols and condensed tannins. Both of them had the effect of increasing the expression of β-catenin, which could promote the Wnt/β-catenin signaling pathway.
Then, extract of 50E-PMC and the ODS-fractions were measured by Q-TOF/MS, and the data was used to create a molecular networking diagram on the Global Natural Product Social Molecular Networking (GNPS) platform. After systematic comparison and prediction, most of the predicted active substances belonged to the category of flavonoids, which could be used as a strategy for further separation of active substances in the future that could accelerate the progress of research.
In summary, PMC was a plant with good hair nourishing activity. In vitro and in vivo study, it had the effect of promoting hair growth and avoiding the hair inhibitory effect caused by testosterone. Then, compare the activity results with the huge database of GNPS to predict potential active substances, PMC has the potential to be developed as a treatment of AGA or hair care products in the future. |
