Sangtitanu Taniya, Sangtanoo Papassara, Srimongkol Piroonporn, Saisavoey Tanatorn, Reamtong Onrapak, Karnchanatat Aphichart
Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.
Research Unit in Bioconversion/Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.
Food Funct. 2020 Jun 24;11(6):4927-4939. doi: 10.1039/d0fo00227e.
This research examined the antioxidant abilities of peptides derived from the Hericium erinaceus mushroom produced via three microbial proteases (Alcalase®, Neutrase®, and Flavourzyme®) at varying concentrations of 10, 25, and 50 mg mL-1. The H. erinaceus mushroom hydrolysate produced from 10 mg mL-1 Alcalase® with 4 hours' digestion resulted in the highest levels of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazl (DPPH), and nitric oxide (NO) radical scavenging activities. Fractionation of this particular hydrolysate was then performed by ultrafiltration (10, 5, 3, and 0.65 kDa molecular weight cut-off membranes). The MW < 0.65 kDa fraction demonstrated the highest level of radical scavenging activity in the context of ABTS, DPPH, and NO. Therefore, the fraction MW < 0.65 kDa was performed with RP-HPLC to obtain the four principal fractions termed F1-4. The results reveal that the F4 sub-fraction demonstrated the best radical scavenging ability. Additionally, the F4 sub-fraction was able to reduce the potential for DNA damage caused by hydroxyl radicals. This was revealed by testing using the Fenton reaction and the pUC19, pKS, and pBR322 plasmids. This outcome was demonstrated through in vitro antiproliferative activity in human lung carcinoma cell lines (Chago-K1) using MTT assay. The F4 sub-fraction was also demonstrated to induce apoptosis, as indicated using the FITC Annexin V apoptosis detection kit with PI. Furthermore, it was also found that the activity of caspase-3, -8, and -9 in Chago-K1 cells was enhanced after exposure periods of 24 and 48 hours. Finally, this sub-fraction was selected for peptide sequencing via quadrupole-time-of-flight-electron spin induction-mass spectrometry-based de novo peptide sequencing.
本研究检测了通过三种微生物蛋白酶(碱性蛋白酶、中性蛋白酶和风味蛋白酶)在10、25和50 mg mL-1不同浓度下制备的猴头菇衍生肽的抗氧化能力。用10 mg mL-1碱性蛋白酶消化4小时产生的猴头菇水解产物具有最高水平的2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)、2,2-二苯基-1-苦基肼(DPPH)和一氧化氮(NO)自由基清除活性。然后通过超滤(截留分子量为10、5、3和0.65 kDa的膜)对该特定水解产物进行分级分离。分子量<0.65 kDa的级分在ABTS、DPPH和NO方面表现出最高水平的自由基清除活性。因此,对分子量<0.65 kDa的级分进行反相高效液相色谱(RP-HPLC)以获得四个主要级分,称为F1-4。结果表明,F4亚级分表现出最佳的自由基清除能力。此外,F4亚级分能够降低羟基自由基引起的DNA损伤潜力。这通过使用芬顿反应以及pUC19、pKS和pBR322质粒进行测试得以揭示。通过使用MTT法在人肺癌细胞系(Chago-K1)中的体外抗增殖活性证明了这一结果。使用FITC膜联蛋白V凋亡检测试剂盒和碘化丙啶(PI)表明,F4亚级分也被证明可诱导细胞凋亡。此外,还发现Chago-K1细胞中半胱天冬酶-3、-8和-9的活性在暴露24和48小时后增强。最后,选择该亚级分通过基于四极杆-飞行时间-电子自旋诱导-质谱的从头肽测序进行肽测序。