乾燥檸檬皮中活性成分之研究

Studies on Some Active Components in Dried Lemon Peel

江任欣(Supochana Charoensin)

指導教授 ： 徐睿良 ； 黃卓治

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/博士(2020年)

https://doi.org/10.6346/NPUST202000292

摘要

本研究應用間歇乾燥技術於檸檬皮的乾燥，封閉系統提供了一個回火期，允許水分和基材從內部擴散到外表面，引發化學性跟酵素性反應。分析發現，大量的1,2-環氧-順檸檬烯（0.1％），1,2-環氧-反式檸檬烯（0.2％），順-對薄荷2,8-二烯-1-醇（0.6％）的揮發物中發現反式，對-薄荷腦2,8-二烯-1-醇（0.4％），香芹酮（0.6％），順式香芹酚（0.1％）和反式香芹酚（0.1％）產生，其中香芹酮被應用於芳香及另類療法醫藥。我們假設檸檬烯的生物轉化是由間歇乾燥過程中的酶促途徑引起的。另外也發現，檸檬皮中含有多量聖草次苷及橙皮苷等類黃酮醣苷，而間歇乾燥所得檸檬皮中，發現有可能由類黃酮醣苷經酵素性水解所釋出的聖草酚及橙皮素。
為了探討檸檬皮中雌黃醇等黃烷酮之功能性，本研究，使用PMA和LPS處理巨噬細胞，導致過氧亞硝酸鹽生成，改進現有類黃酮的生理活性測定法，開發一種新的更接近生理條件的細胞模型。本方法使用在B環上具有不同數目鄰二羥基基團的三個構造類似的黃烷酮：檸檬中黃烷酮(雌黃醇)及柚皮素和松皮精，探討化學結構與生理活性之關聯性。使用了二氫羅丹明123定量分析，蛋白質西方墨點法、免疫螢光分析和通過黃烷酮與IKKβ催化激酶結構域之間在ATP結合位點之間的分子對接進行的計算機分析。結果發現，雌黃醇是過氧亞硝酸鹽的生成最有效的抑製劑。西方墨點法分析和共聚焦螢光圖像顯示，雌黃醇可通過抑制NF-kB易位而抑制iNOS和p47蛋白的表達。雌黃醇的最高-CDOCKER_能量值(38.6703 kcal/mol)，證實B環上的3'，4'-鄰二羥基化在與IKKβ激酶結構域在ATP結合位點的結合中扮演關鍵作用。我們提出黃烷酮B環上的鄰二羥基基團，可能直接佔據IKKβ激酶結構域的ATP結合位點，導致創新性細胞模型中，消除PMA和LPS處理巨噬細胞中過亞硝酸鹽之形成。

關鍵字

抗氧化劑活性 ； 細胞模型 ； 黃烷酮 ； 巨噬細胞 ； 過氧亞硝酸鹽 ； 檸檬

並列摘要

A modified intermittent drying technique was developed and applied to dry the lemon peels. The closed system provided a tempering period that allows moisture and substrate diffusion from the interior to the external surface. Significant amount of 1,2-epoxy-cis-limonene (0.1%), 1,2-epoxy-trans-limonene　(0.2%), cis-p-menth-2,8-dien-1-ol (0.6%), trans-p-menth-2,8-dien-1-ol (0.4%), carvone (0.6%), cis-carveol (0.1%), and trans-carveol (0.1%) were found in the volatiles of the intermittently dried lemon peel. Among them, carvone widely used in aromatherapy and alternative medicine is deserved special attention. We postulated that biotransformation of limonene has resulted from an enzymatic pathway during the intermittent drying process. All the data obtained in this experiment indicated that the generation of characteristic Chen-pi aroma compounds from limonene was not only a chemical oxidation reaction but also an enzymatic process. In addition to carvone, significant amount of eriocitrin and hesperidin were characterized. Under the intermittent drying process, aglycones eriodictyol and hesperetin derived from the glycosides through the enzymatic reaction were identified in the dried lemon peel.
In order to study the functionalities of flavanones in lemon peels, a novel cell model using macrophage treated with a combination of PMA and LPS leading to generation of peroxynitrite was developed to provide a more real physiological condition. Three flavanones (eriodictyol, naringenin, and pinocembrin) with different number of ortho‐dihydroxyl groups on B‐ring were used to provide a more comprehensive evaluation of the role of chemical structure in the new model. Dihydrorhodamine123 assay, protein immunoblotting, immunofluorescence assay, and in silico analysis by molecular docking between the flavanones and IKKβ catalytic kinase domain at the ATP binding site were employed. Results indicated that the generation of peroxynitrite was decreased at 10 µM of flavanones; eriodictyol was the most effective inhibitor. Western blot analysis and confocal fluorescence image also showed that eriodictyol could inhibit iNOS and p47 protein expressions through the inhibition of NF-κB translocation and performed the maximal inhibition compared to that of the other groups. In addition, the highest –CDOCKER_energy values of eriodictyol (38.6703 kcal/mol) confirmed that the 3′, 4′‐ortho‐dihydroxylation on the B‐ring played a crucial role in binding with IKKβ kinase domain at ATP binding site. Finally, we propose that the ortho‐dihydroxyl groups on B‐ring of flavanone may influence directly the occupation of the ATP binding site of IKKβ kinase domain leading to the abrogation of peroxynitrite formation in the innovative cell model.

並列關鍵字

antioxidant activity ； cell model ； flavanone ； macrophage ； peroxynitrite ； lemon

參考文獻

6. References

Abou-Arab, A., M. Mamoud, and F. Abu-Salem. 2017a. Functional Properties of Citrus Peel as Affected by Drying Methods. American Journal of Food Technology 12:193-200.

Abou-Arab, A., M. Mamoud, and F. Abu-Salem. 2017b. Influences of Juice Extraction and Drying Methods on the Chemical Analysis of Lemon Peels. International Journal of Nutrition and Food Engineering 11(7): 584-589.

Abraham, T. F., E. Marcel, K. Alexis, and Z. Belkacem. 2016. Experimental drying kinetics of mango slices (Mangifera indica L.) Amelie under intermittent conditions. International Journal of Thermal Technologies 6(3):203-211.

Ahmed, N., J. Singh, H. Chauhan, P. G. A. Anjum, and H. Kour. 2013. Different drying methods: their applications and recent advances. International Journal of Food Nutrition and Safety 4(1):34-42.