臺灣紫芝活性代謝物之最適化生產與活性探討

許凱廸

doi:10.6342/NTU201702288

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台湾紫芝活性代谢物之最适化生产与活性探讨

Optimized Production and Functional Evaluation of Bioactive Metabolites from Ganoderma formosanum

许凯廸 , 博士　　导师：郑光成　　

英文 DOI： 10.6342/NTU201702288

台湾紫芝；美白活性；酪胺酸酶薄膜层析平台；美白药物筛选；胞外多醣体； β-葡聚醣； Ganoderma formosanum ； depigmenting activity ； tyrosinase-based TLC bioautography ； anti-melanogenic drug screening ； EPS ； β-glucan.

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摘要 │ 参考文献 (182) │ 国际计量

摘要〈TOP〉

台湾紫芝为台湾特有种灵芝，约30年前在桃园地区的枫香树上首次被发现。由於其新颖性，仅有少数研究证明其药理潜力，目前研究较详尽的为台湾紫芝胞外多醣体的免疫调节与抗肿瘤活性。然而，台湾紫芝胞外多醣体的液态深层醱酵之最适化生产条件尚未确立，且台湾紫芝的潜在生理活性仍然未知。
本研究的主要目的为探讨台湾紫芝新颖之活性与增进其胞外多醣体之产量，方向分为三大部份: (1) 研究台湾紫芝菌丝体萃取物之美白活性 (2) 开发一种结合酪胺酸酶与薄膜层析法的高通量美白药物筛选平台，以利监定台湾紫芝萃取物的美白活性成份 (3) 利用统计与数学模型，最适化生产台湾紫芝胞外多醣体的液态培养条件。
	台湾紫芝菌丝体酒精萃取物之乙酸乙酯份化层 (GFE-EA) 相较於其它份化层具有最好的体外抑制酪胺酸酶活性，进一步在B16-F10细胞模式中也发现，GFE-EA透过抑制酪胺酸酶活性与其蛋白表现量的机制，来降低胞泌与胞内黑色素形成。进一步在斑马鱼模式中，发现GFE-EA仅需七份之一麴酸的剂量，便能达到同样程度的抗黑色素形成效果，且具较低的副作用。以上结果显示，GFE-EA作为化妆品应用的高度潜力。
为了分离纯化GFE-EA中具有美白活性之成分，我们建立了一种酪胺酸酶薄膜层析平台，以利监定GFE-EA份化层中的活性组成。再者，此平台赋予研究人员同时进行活性分析与监定活性物质之可能。相较以酪胺酸酶活性分析比色法侦测麴酸，酪胺酸酶薄膜层析平台不仅具备较优越的药物剂量侦测极限，也兼具较低的酵素使用量。此外，Gage R&R分析的结果显示，此平台具可靠的重复性与再现性。因此，我们利用此平台，从数个GFE-EA份化层中筛选出两个具抑制酪胺酸活性的份化层 (GFE-EA F4、F5)。值得注意的是，这两个份化层在斑马鱼模式中同样显示抗黑色素形成的效果，证实酪胺酸酶薄膜层析平台在筛选抗黑色素形成之药物的应用性。
在最适化胞外多醣体的生产部份。我们利用反应曲面法探讨起始pH值、葡萄糖与酵母萃取物对於胞外多醣生产的影响，实行Box-Behnken design (BBD) 模型的三因子三阶层分析後，发现最佳培养的条件为起始pH值5.3、葡萄糖 49.2克/每公升与酵母萃取物4.9克/每公升。利用此最适化生产条件，经九天的醱酵後，胞外多醣体的总产量达到833毫克/每公升，是使用未优化培养基的1.4倍产量，同时也是目前文献报导中最高的产量纪录。特别的是，被认为是多醣体活性来源的β-葡聚醣，在此胞外多醣体中的比例为53 ± 5.5%，显示台湾紫芝胞外多醣体拥有免疫调节活性之高度潜力。
	以上研究不仅推翻一般认为菌丝体缺乏子实体才拥有特定活性的认知，同时也建立一个优越的酵素薄膜层析平台，使研究者能同时评估混合物样品的活性与监定其活性成份。此外，这是首个阐明最适化台湾紫芝多醣体生产条件的研究，提供未来大规划工业化醱酵的基础。总结来说，我们认为台湾紫芝的活性代谢物，份别具有作为美白活性化妆品与免疫调节保健食品的潜力。

并列摘要〈TOP〉

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.

参考文献 ( 182 ) 〈TOP〉

Akbar, R., & Yam, W. K. (2011). Interaction of ganoderic acid on HIV related target: molecular docking studies. Bioinformation, 7(8), 413-417.
连结：
Ali, A., Ashraf, Z., Kumar, N., Rafiq, M., Jabeen, F., Park, J. H., . . . Attri, P. (2016). Influence of plasma-activated compounds on melanogenesis and tyrosinase activity. Scientific Reports, 6. doi:ARTN 2177910.1038/srep21779
连结：
Ates, G., Doktorova, T. Y., Pauwels, M., & Rogiers, V. (2014). Retrospective analysis of the mutagenicity/genotoxicity data of the cosmetic ingredients present on the Annexes of the Cosmetic EU legislation (200012). Mutagenesis, 29(2), 115-121. doi:10.1093/mutage/get068
连结：
Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965-977. doi:10.1016/j.talanta.2008.05.019
连结：
Bishop, K. S., Kao, C. H. J., Xu, Y. Y., Glucina, M. P., Paterson, R. R. M., & Ferguson, L. R. (2015). From 2000 years of Ganoderma lucidum to recent developments in nutraceuticals. Phytochemistry, 114, 56-65. doi:10.1016/j.phytochem.2015.02.015
连结：

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使用DOI作为永久连结

引用含有DOI的文献

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。 如想对DOI的使用与概念有进一步了解，请参考 （ ） 。

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。如想对DOI的使用与概念有进一步了解，请参考（）。