臺灣紫芝活性代謝物之最適化生產與活性探討

Optimized Production and Functional Evaluation of Bioactive Metabolites from Ganoderma formosanum

許凱廸(Kai-Di Hsu)

指導教授 ： 鄭光成

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2017年)

https://doi.org/10.6342/NTU201702288

摘要

臺灣紫芝為臺灣特有種靈芝，約30年前在桃園地區的楓香樹上首次被發現。由於其新穎性，僅有少數研究證明其藥理潛力，目前研究較詳盡的為臺灣紫芝胞外多醣體的免疫調節與抗腫瘤活性。然而，臺灣紫芝胞外多醣體的液態深層醱酵之最適化生產條件尚未確立，且臺灣紫芝的潛在生理活性仍然未知。
本研究的主要目的為探討臺灣紫芝新穎之活性與增進其胞外多醣體之產量，方向分為三大部份: (1) 研究臺灣紫芝菌絲體萃取物之美白活性 (2) 開發一種結合酪胺酸酶與薄膜層析法的高通量美白藥物篩選平台，以利鑑定臺灣紫芝萃取物的美白活性成份 (3) 利用統計與數學模型，最適化生產臺灣紫芝胞外多醣體的液態培養條件。
臺灣紫芝菌絲體酒精萃取物之乙酸乙酯份化層 (GFE-EA) 相較於其它份化層具有最好的體外抑制酪胺酸酶活性，進一步在B16-F10細胞模式中也發現，GFE-EA透過抑制酪胺酸酶活性與其蛋白表現量的機制，來降低胞泌與胞內黑色素形成。進一步在斑馬魚模式中，發現GFE-EA僅需七份之一麴酸的劑量，便能達到同樣程度的抗黑色素形成效果，且具較低的副作用。以上結果顯示，GFE-EA作為化妝品應用的高度潛力。
為了分離純化GFE-EA中具有美白活性之成分，我們建立了一種酪胺酸酶薄膜層析平台，以利鑑定GFE-EA份化層中的活性組成。再者，此平台賦予研究人員同時進行活性分析與鑑定活性物質之可能。相較以酪胺酸酶活性分析比色法偵測麴酸，酪胺酸酶薄膜層析平台不僅具備較優越的藥物劑量偵測極限，也兼具較低的酵素使用量。此外，Gage R&R分析的結果顯示，此平台具可靠的重複性與再現性。因此，我們利用此平台，從數個GFE-EA份化層中篩選出兩個具抑制酪胺酸活性的份化層 (GFE-EA F4、F5)。值得注意的是，這兩個份化層在斑馬魚模式中同樣顯示抗黑色素形成的效果，證實酪胺酸酶薄膜層析平台在篩選抗黑色素形成之藥物的應用性。
在最適化胞外多醣體的生產部份。我們利用反應曲面法探討起始pH值、葡萄糖與酵母萃取物對於胞外多醣生產的影響，實行Box-Behnken design (BBD) 模型的三因子三階層分析後，發現最佳培養的條件為起始pH值5.3、葡萄糖 49.2克/每公升與酵母萃取物4.9克/每公升。利用此最適化生產條件，經九天的醱酵後，胞外多醣體的總產量達到833毫克/每公升，是使用未優化培養基的1.4倍產量，同時也是目前文獻報導中最高的產量紀錄。特別的是，被認為是多醣體活性來源的β-葡聚醣，在此胞外多醣體中的比例為53 ± 5.5%，顯示臺灣紫芝胞外多醣體擁有免疫調節活性之高度潛力。
以上研究不僅推翻一般認為菌絲體缺乏子實體才擁有特定活性的認知，同時也建立一個優越的酵素薄膜層析平台，使研究者能同時評估混合物樣品的活性與鑑定其活性成份。此外，這是首個闡明最適化臺灣紫芝多醣體生產條件的研究，提供未來大規劃工業化醱酵的基礎。總結來說，我們認為臺灣紫芝的活性代謝物，份別具有作為美白活性化妝品與免疫調節保健食品的潛力。

關鍵字

臺灣紫芝 ； 美白活性 ； 酪胺酸酶薄膜層析平台 ； 美白藥物篩選 ； 胞外多醣體 ； β-葡聚醣

並列摘要

Ganoderma formosanum, an endemic Ganoderma (Lingzhi) species in Taiwan, was firstly identified from Formosan sweet gum (Liquidambar formosana) in Taoyuan County, Taiwan three decades ago. Currently, only few studies had demonstrated its pharmacological potential due to its novelty, among which the immune-modulation and anti-tumor effects of extracellular polysaccharide (EPS) is the most understood. However, optimization for the production of G. formosanum EPS in submerged fermentation has not yet been explored, and potential bioactivities of G. formosanum remain unknown.
The specific aim of this study is to explore the frontiers of bioactivity possessed by G. formosanum and to ameliorate the yield of EPS. The present research is divided into three parts as follows: first, depigmenting activity of G. formosanum mycelium extract was studied rather than Lingzhi’s traditional uses. Second, a high-throughput tyrosinase-based TLC bioautography was developed to identify active ingredients responsible for anti-melanogenic activity of G. formosanum mycelium extract. Third, a collection of statistical and mathematical approaches was used to optimize the medium composition for EPS production in submerged fermentation.
To screen depigmenting ingredients from G. formosanum, ethyl acetate fraction of G. formosanum mycelium ethanolic extract (GFE-EA) was demonstrated that exhibits the highest inhibitory activity toward cell-free tyrosinase compared with other fractions. Furthermore, the level of secreted and intracellular melanin of B16-F10 cells were reduced by GFE-EA through suppression of tyrosinase activity and its protein expression. Moreover, GFE-EA exerts similar depigmenting efficacy to kojic acid with lower dosage (approximately one-seventh of dose), but shows less adverse effect to zebrafish. It would appear that GFE-EA has great potential for application in the cosmetics industry.
In order to purify and to isolate active compound(s) responsible for anti-melanogenic property of GFE-EA, we established a tyrosinase-based thin layer chromatography (TLC) to identify bioactive ingredients from fractions of GFE-EA, and this technique enable researchers to identify active compound(s) during functional assay. Compared with colorimetric tyrosinase activity assay, tyrosinase-based TLC not only showed better detection limit for kojic acid but also cost lower usage of enzyme. In addition, results of Gage R&R study indicated the repeatability and reproducibility of this platform are reliable. Accordingly, this platform was employed to screen fractions of GFE-EA exerting tyrosinase inhibitory activity, and two fractions (GFE-EA F4, F5) were obtained. Above all, it is worth noting that these fractions also showed depigmenting activity on zebrafish, verifying the applicability of tyrosinase-based TLC for anti-melanogenic drug screening.
For optimization of EPS production, response surface methodology (RSM) was employed to study the effects of initial pH, glucose and yeast extract concentration on EPS yield. The optimum medium composition was found to be at initial pH 5.3, 49.2 g/L of glucose and 4.9 g/L of yeast extract by implementing a three-factor-three-level Box-Behnken design (BBD). After a 9-day fermentation with optimal conditions, the yield of EPS reached 833 mg/L, which is 1.4 fold-higher than that from basic medium, and it is the highest yield have been reported to date. Above all, the percentage of β-glucan, a well-recognized bioactive polysaccharide, in EPS was 53 ± 5.5%, suggesting G. formosanum EPS may possess potential immunomodulation activity.
Altogether, these results not only subvert the general notion that mycelium lacks certain bioactivities, such as depigmenting activity, possessed by fruit bodies, but also provide a superior enzyme-based TLC platform to enable researchers to conduct functional assay and identify active compound(s) from tested mixture simultaneously. Furthermore, this is also the first research to optimize the culture conditions for G. formosanum EPS production, providing fundamental basis for industrial large-scale fermentation. In this study we argue that metabolites of G. formosanum may possess skin lightening and immunomodulation potential to be a cosmetic and nutraceutical, respectively.

並列關鍵字

Ganoderma formosanum ； depigmenting activity ； tyrosinase-based TLC bioautography ； anti-melanogenic drug screening ； EPS ； β-glucan.

參考文獻

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