以液態發酵生產韋伯靈芝(Ganoderma weberianum)菌絲體及其萃取物對於黑色素生成之影響

Abstract

近幾十年來，關於天然物成分分析之研究相當盛行，這些天然物成分常應用於日常生活中，作為食品添加物或皮膚保養等香粧品之成分。本研究中，我們探討廣泛生長於台灣低海拔闊葉樹下的靈芝－韋伯靈芝（Ganoderma weberianum），利用 5L 攪拌式醱酵槽大量生產其液態醱酵生成之靈芝菌絲體，先由 95% 酒精初步萃取後，經三氯甲烷 (Chloroform) 和乙酸乙酯 (Ethyl acetate) 進行極性分割，得到三氯甲烷層萃取物 (CE) 及乙酸乙酯層萃取物 (EE)，並以 Sephadex LH-20 進行液相層析分離(liquid-liquid partition)，再利用UV照射下，平板層析 (TLC-Thin layer chromatography) 分析之結果作為依據，分別在 CE 和 EE 中皆收集出 6 個分劃，接著進一步探討其對於黑色素生成之影響。 利用老鼠 B16-F10 黑色素瘤細胞 (mouse B16-F10 melanoma cell) 作為篩選有效抑制黑色素生成之細胞平台，實驗結果篩選出四個具有抑制效果的樣品，其中，最為有效抑制黑色素生成之樣品為三氯甲烷層中的 Fraction-3 (F3)。首先，探討其抑制黑色素生成之機制，發現能有效抑制黑色素細胞內酪胺酸脢 (tyrosinase) 之活性，進一步分析酪胺酸脢的蛋白質表現量，發現亦無顯著差異；然而，經探討其他與黑色素生成之相關基因的mRNA層級之表現量，發現其對於MITF, tyrosinase, TRP-1, TRP-2 皆無造成較顯著的差異，因此我們初步推論此靈芝萃取層析物對於抑制老鼠黑色素瘤細胞產生黑色素的途徑，是藉由影響酪胺酸酶之活性所造成之結果。 最後，利用斑馬魚動物模式 (zebrafish animal model) 進行試驗，初步看見靈芝萃取物的分離層析物 F3，對於斑馬魚胚胎孵化的黑化過程造成抑制效果。由此可知，此經由液態醱酵培養之靈芝菌絲體，其分離層析物 F3 具有抑制黑色素生成之潛力。

Nature products isolated from Chinese medical herb have been used extensively in the inhibition of melanogenesis in skin whitening cosmetic products and preventing the browning of food products. ‘‘Lingzhi’’ is a common ingredient in various cosmetic lines and may result in the whitening effect of skin. This property is highly valued by many oriental countries. Skin whitening can be partially achieved via the inhibition of tyrosinase, a key enzyme in melanogenesis. Classical inhibitors of tyrosinase such as arbutin and kojic acid are commonly used in various skin whitening products. However, due to their side effects and safety concerns, the need to find alternative inhibitors of melanogenesis with lower toxicity remains an important field in skin whitening. Several studies have identified compounds isolated from fungal sources to possess inhibitory activities towards tyrosinase as well. Here, my work focuses on the discovery of active compound(s) with anti-melanogenesis potential from the submerged culture of Ganoderma weberianum and elucidating their mechanism of action. The mycelium of the submerged culture was first extracted using 95% ethanol, followed by partition using chloroform and ethyl acetate. These two crude extracts were then further separated using a Sephadex LH-20 column into six fractions. By the B16-F10 melanoma cell model test, some specific fractions had the ability to inhibit the melanogenesis. In particular, fraction three (F3) had the strongest inhibitory effect toward melanogenesis on the in vivo tyrosinase activity without affecting the quantity. For the mRNA level, it shows no apparent difference between the control and F3 treatment for the transcription of melanognesis related gene, Microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1(TRP-1), and tyrosinase-related protein-2 (TRP-2). For the zebrafish model test, it also showed the inhibition ability on the pigmentation within the growth of embryo. Thus, our study has demonstrated that the chemical compounds isolated from Ganoderma weberianum have the potential to serve as alternative ingredients in skin whitening products.