牛樟芝菌絲體於三種固醇類squalene、cholesterol及stigmasterol處理下之成分與抗發炎研究

Analysis of the effects of steroids-treated Antrodia cinnamomea on the chemical composition and anti-inflammatory activity.

陳筠婷(Yun-Ting Chen)

指導教授 ： 張孟基

共同指導教授 ： 盧美光(Mei-Kuang Lu)

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02739

摘要

牛樟芝(Antrodia cinnamomea)為一種藥用真菌，對多種疾病皆具有療效。在牛樟芝的有效成分中三萜類佔大部分，目前人工栽培的三萜類含量仍不及野生子實體。本實驗目的即為利用添加前驅物策略，以期提高三萜類之產率。本研究於液態培養基中添加三種不同的固醇類成分：squalene、cholesterol及stigmasterol，濃度各為2、10、50及100 μM，觀察其對牛樟芝菌絲體的成分影響。實驗結果顯示，與未加入任何處理的牛樟芝對照組相比，不同的處理對菌絲體乾重影響差異不大，乾重為5.91 ± 0.02到7.07 ± 0.02 g/L。多醣體中以半乳糖和葡萄糖為主要成份，半乳糖以50 μM stigmasterol處理得最高含量每克多醣體含603.97 ± 177.33 μmol，而葡萄糖則於100 μM stigmasterol處理得最高含量每克多醣體含405.11 ± 72.81 μmol。以HPLC分析結果，甲醇萃取物中主要可分得4,7-dimethoxy-5-methyl-1,3-benzodioxole (4,7-D)、Dehydrosulphurenic acid (De-sul)、Zhankuic acid A (ZaA)、15α-acetyl-dehydrosulphurenic acid (15α)和Dehydroeburicoic acid (De-eb)此五種化合物，其中含量以三個lanostane-type三萜類De-sul、15α和De-eb為最多。squalene處理下ZaA和15α較他種處理相對含量較高，每毫克菌絲乾重中分別為2.37到2.84 μg及38.88到48.07 μg，而cholesterol 10 μM處理下有最高的三萜類產率達22.1%。在抗發炎能力上，測試前述五種牛樟芝純化合物及Antcin C (AntC)、Antcin K (AntK)及Zhankuic acid C(ZaC)共八種純化合物與三種添加物的菌絲體甲醇萃取物，其清除DPPH自由基的效果不明顯，在抑制NADPH oxidase 活性表現中，八種牛樟芝的純化合物皆顯示濃度相關的抑制效應，可抑制14.6%~33.5%的酵素活性表現。而在不同濃度前驅物處理中，僅在squalene組中較具有濃度相關的作用。以小鼠微膠細胞BV-2經脂多醣體 (lipoplysaccharide)刺激使細胞發炎後，偵測其NO生成量、iNOS及COX-2酵素表現。抑制NO生成量在ergostane-type的三萜類中皆有顯著的抑制效果，且以ZaA抑制效果最佳IC50 為11.31 μM，在lanostane-type的三萜類則效果不明顯。三種添加物處理的牛樟芝菌絲體甲醇萃取物皆可顯著抑制NO生成，又以squalene 50 μM處理的牛樟芝菌絲體甲醇萃取物其處理抑制效果最好，IC50為11.96 μg/ml，進一步測試其抑制iNOS及COX-2蛋白質表現，發現ZaA及squalene 50μM的處理可顯著抑制iNOS及COX-2的表現，且對COX-2的抑制有濃度相關性。實驗結果推論添加squalene可提高牛樟芝中ZaA的含量，且此種三萜類具有顯著的抗發炎功效。

關鍵字

牛樟芝 ； 三萜類 ； 抗發炎

並列摘要

Niuchangchih (Antrodia cinnamomea) is a medicinal fungus that has been reported to treat different kinds of diseases. Triterpenoids make up the majority of compounds isolated from A. cinnamomea. However, the amounts of triterpenoids in cultivated mycelia were relatively low as compared with wild fruiting bodies. The aim of this study is to enhance the content of triterpenoids in cultured A. cinnamomea through the precursor-feeding strategy. A. cinnamomea were fed with three kinds of steroids, including squalene, cholesterol and stigmasterol, with concentrations of 2, 10, 50 and 100 μM. The results showed that the dry weight of mycelia after different treatments showed no difference compared to the control group. The biomass was 5.91 ± 0.02 to 7.07 ± 0.02 g/L. Galactose and glucose were the major sugar-form of polysaccharides (PS). Galactose had the maximum value of 603.97 ± 177.33 μmol/g PS whole feeding with 50 μM stigmasterol and glucose had the maximum value of 405.11 ± 72.81 μmol/g PS while feeding with 100 μM stigmasterol. Through the analysis of HPLC, there were five major components in the methanolic extracts of A. cinnamomea: 4,7-dimethoxy-5-methyl-1,3-benzodioxole (4,7-D), Dehydrosulphurenic acid (De-sul), Zhankuic acid A (ZaA), 15α-acetyl-dehydrosulphurenic acid (15α) and Dehydroeburicoic acid (De-eb). Among them, the three lanostane-type triterpenoids, De-sul, 15α and De-eb, were the major component. The squalene-feeding treatment produced more ZaA and 15α than other treatments with the amounts of 2.37 to 2.84 μg and 38.88 to 48.07μg per mg dry weight respectively. The maximum yield of triterpenoids was achieved with 10 μM cholesterol treatment at a value of 22.1%. To study the anti-inflammatory potential, five pure compounds as previously described with three other pure compounds Antcin C (AntC), Antcin K (AntK) and Zhankuic acid C (ZaC) and the methanolic extracts of three steroid-feeding A. cinnamomea were evaluated. All of the above had a minor extent to their direct radical-scavenging properties. Eight pure compounds possess anti-oxidative effects due to inhibition (14.6%–33.5%) of NADPH oxidase activity in a concentration-dependent manner. While in three different steroids-treated groups, only the squalene group showed a concentration-dependent inhibition effect. We further studied their anti-inflammatory effects using lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible NO synthetase (iNOS) and cyclooxygenase-2 (COX-2) expression in microglial cells BV-2. All the ergostane-type triterpenoids inhibited LPS-induced NO production, and ZaA possessed the most potent inhibitory effect with an IC50 value of 11.31 μM. Whereas the lanostane-type triterpenoids showed relatively weak inhibitory effect. All the methanolic extracts of three treatments inhibited NO production. Among treated, those with 50 μM squalene-treated possessed the most potent inhibitory effects with an IC50 of 11.96 μM. ZaA and A. cinnamomea treated with 50 μM squalene effectively inhibited iNOS and COX-2 upregulation in microglial cells. Our results suggest that squalene feeding can increase the content of ZaA, and this triterpenoid exhibited potent anti-inflammatory activities.

並列關鍵字

Antrodia cinnamomea ； triterpenoids ； anti-inflammation

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