| 摘要: | 本研究以KT、KF以及LJ之根、莖和葉為材料，萃取物經冷凍乾燥後，探討(KT-L、KT-L-W、KT-L-C、KT-L-H、KT-S、KT-S-W、KT-S-B、KT-S-E、KT-S-H、KT-R-C)、 (KF-L、KF-L-W、KF-L-C、KF-S、KF-S-W、KF-S-C和KF-R¬-W)與(LJ-B、LJ-C和LJ-H)之細胞毒性、抗氧化活性與美白功效評估。
於細胞存活測試結果顯示，(KT-L-W、KT-S-W和KT-S-B)、(KF-S-W、KF-R-W和KF-L-W)與(LJ-B)作用於Hs68細胞株24小時，其細胞存活度> 90%。因此根據毒殺作用結果，本研究選用無毒殺作用的萃取物進行抗氧化與美白之活性測試。
於抗氧化活性試驗中，進行DPPH與ABTS自由基清除能力、總酚與總黃酮含量、還原力、亞鐵離子螯合能力、微脂粒氧化抑制測定、細胞內活性氧自由基測定和組織脂質過氧化等測試。實驗結果顯示萃取物中，清除DPPH自由基能力隨濃度增加而增加。以KT-S-W、KT-S-B、KT-L-W、KF-S-W、KF-R-W、KF-L-W和LJ-B其清除DPPH自由基的EC50濃度分別為0.9、1.4、4.8、0.9、0.9、5.6及14.5g/ml；清除ABTS自由基的EC50濃度分別為215.4、29.8、87.4、26.9、27.7及45.3 g/ml。對照組維他命C清除自由基DPPH和ABTS的EC50濃度為0.9和4.8 g/ml。以gallic acid標準品製作之總多酚含量標準曲線，換算萃取物之總多酚含量分別為46.8、97.4、53.6、108.9、98.7、78.6及74.3 mg/g。以rutin標準品製作之黃酮含量標準曲線，換算萃取物之黃酮含量分別為6.8、26.9、12.5、35.5、30.6、22.9及55.6 mg/g。還原力試驗中，以ascorbic acid標準品製作之還原力標準曲線，換算萃取物之ascorbic acid含量分別為11.1、14.3、12.5、13.2、12.2、14.9及16.6 mg/g。亞鐵離子螯合能力的EC50濃度分別為3.9、7.7、5.8、6.7、40.1、7.6及2.2 g/ml；對照組EDTA的EC50濃度為4.6 g/ml。在ROSs誘發實驗中，加入萃取物後，其ROSs誘發的百分比與加入過氧化氫誘發的BNLCL2細胞相比，下降約8.1-18.5%。以小鼠腦組織混合萃取物作用後，於萃取物100 μg/ml時，抑制脂質過氧化為59.8-68.9%。
於美白活性試驗中，進行細胞內黑色素含量測定、酪胺酸酶測試和與美白調控機制相關之特定蛋白質表現分析。結果發現，(KT-L-W、KT-S-W和KT-S-B)、(KF-S-W、KF-R-W和KF-L-W)和(LJ-B)分別會藉由調控與抑制黑色素生成相關之黑色素刺激荷爾蒙接受器(melanocortin-1 receptor, MC1R)、MITF (microphthalmia-associated transcription factor)、酪胺酸酶，TRP-1和TRP-2 (tyrosinase-related proteins 1 and 2)的蛋白質表現，進而抑制黑色素瘤細胞(B16)之黑色素生成。
綜上所述推論KT、KF與LJ具有強力抗氧化與美白活性，在天然抗氧化物與美白保養品開發上甚具潛力。
In this study, the roots, stems and leaves of KT, KF and LJ were lyophilized and extracted. The extracts of (KT-L, KT-L-W, KT-L-C, KT-L-H, KT-S, KT-S-W, KT-S-B, KT-S-E, KT-S-H and KT-R-C),(KF-L, KF-L-W, KF-L-C, KF-S, KF-S-W, KF-S-Cand KF-R¬-W), and (LJ-B, LJ-C and LJ-H) were investigated its cytotoxicity, antioxidant capacity and whitening effects.
The cell viability of the extracts of (KT-L-W, KT-S-W and KT-S-B), (KF-S-W, KF-R-W and KF-L-W), and (LJ-B) were > 90% in human skin fibrolast Hs68 cells for 24 h, using MTS assay. Therefore, none toxicity extracts were continutied for the antioxidant and whitening activities tests in this study.
The antioxidant activities of extracts were evaluated with 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 2,2'-azino-bis[3-ethyl benzthiazoline-6-sulfonic acid] (ABTS) radical scavenging activity, total phenolic content (TPC), total flavonoid content (TFC), reducing power, ferrous ion chelating power, lipid peroxidation using liposome model, cellular reactive oxygen species (ROSs) activities, and the lipid peroxidation assay in tissue by thibarbituric acid reactive substances (TBARS) assay. In brief, the free radical-shaving capacity was evaluated by measuring the scavenging activities of examined the increasing concentrations of extracts on DPPH and ABTS radicals. The EC50 concentrations of radical scavenging activities for KT-S-W, KT-S-B, KT-L-W, KF-S-W, KF-R-W, KF-L-W and LJ-B extracts were 0.9, 1.4, 4.8, 0.9, 0.9, 5.6 and 14.5 g/ml for DPPH, and 215.4, 29.8, 87.4, 26.9, 27.7 and 45.3 g/ml for ABTS, respectively. The EC50 concentrations of DPPH and ABTS radical scavenging activities were 0.9 and 4.8 g/ml for L-ascorbic acid (a positive control). The 100 g/ml concentrations of seven extracts were contained 46.8, 97.4, 53.6, 108.9, 98.7, 78.6 and 74.3 mg/gallic acid g for TPC, and 6.8, 26.9, 12.5, 35.5, 30.6, 22.9 and 55.6 mg/rutin g for TFC. Moreover, the 100 g/ml concentrations of seven extracts included 11.1, 14.3, 12.5, 13.2, 12.2, 14.9 and 16.6 mg/L-ascorbic acid g in reducing power assay. The EC50 concentrations of ferrous ion chelating activities were 3.9, 7.7, 5.8, 6.7, 40.1, 7.6, 2.2 g/ml for seven extracts, and 4.6 g/ml for EDTA (a positive control). Additionally, the inhibition percentages of ROSs activities in H2O2-treated BNLCL2 cell for seven extracts were significantly decreased about 8.1-18.5%. The activities of seven extracts to inhibit lipid peroxidation in mice brain tissue has presented inhibition rate of 59.8-68.9% by TBARS assay.
The whitening activities of extracts were investigated with cellular melanin content, tyrosinase and the tyroinase-related proteins in skin whitening mechanisms. After treatment with seven extracts in B16 cells, the antimelanogenesis mechanism were caused by operation of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) expressions using flow cytometry.
These results demonstrated that the KT, KF and LJ extracts have antioxidant and whitening activites, and may offer opportunities for natural antioxidants and whitening skin care products. |
