本研究针对沙棘果渣资源化利用瓶颈,优化黄酮类化合物提取工艺,探究沙棘黄酮对UVB诱导损伤HaCaT细胞的抗氧化保护作用。采用“脱脂-复合酶解-超声协同”技术:沙棘果渣经石油醚脱脂后,以2.5%果胶酶+2.5%纤维素酶破坏细胞壁,结合55%乙醇(含0.1 mol/L柠檬酸-磷酸盐缓冲液,pH 5.0,液料比(mL∶g)30∶1),在超声功率300 W、时间50 min、温度50 ℃条件下提取黄酮;构建150 mJ/cm2 UVB损伤HaCaT细胞模型,设空白组(C)、模型组(UVB)及沙棘黄酮低/中/高剂量组(HRF-L/M/H,100,200,400 μg/mL),经沙棘黄酮处理后,通过MTT、EdU检测细胞活力与增殖,采用试剂盒法分析过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)活性及活性氧(ROS)、丙二醛(MDA)水平。本研究优化工艺后的黄酮提取率为(9.92±0.44)mg/g;与C组相比,UVB组细胞活力、EdU阳性率降低,CAT、SOD、GPX活性下降,ROS、MDA升高(P<0.05);与UVB组相比,HRF-L/M/H组细胞活力、EdU阳性率升高,CAT、SOD、GPX活性升高,ROS、MDA降低(P<0.05)。本研究表明,复合酶解-超声协同法可实现沙棘果渣黄酮高效提取;沙棘黄酮通过增强抗氧化酶活力、清除ROS、抑制脂质过氧化,缓解UVB对HaCaT细胞的损伤,本研究为沙棘果渣的资源利用及天然抗氧化剂开发提供了理论支撑。
This study aimed to address the bottleneck in the resource utilization of Hippophae rhamnoides Linn. pomace, optimize the extraction process, and investigate their antioxidant protective effects on HaCaT cells damaged by UVB irradiation. A “defatting-composite enzymatic hydrolysis-ultrasound synergistic” technique was adopted: After defatting Hippophae rhamnoides Linn. pomace with petroleum ether, a mixture of 2.5% pectinase and 2.5% cellulase was used to break the cell walls. Combined with 55% ethanol(containing 0.1 mol/L citrate-phosphate buffer, pH 5.0, liquid-to-solid ratio of 30∶1 mL/g), flavonoids were extracted under ultrasonic conditions of 300 W, for 50 min, and at 50 ℃. A HaCaT cell model damaged by 150 mJ/cm2 UVB was established, including a blank group(C), a model group(UVB), and Hippophae rhamnoides Linn. flavonoid low/middle/high dose groups(HRF-L/M/H, 100, 200, 400 μg/mL, respectively). After treatment with Hippophae rhamnoides Linn. flavonoids, cell viability and proliferation were detected by MTT and EdU assays, and the activities of catalase(CAT), superoxide dismutase(SOD), and glutathione peroxidase(GPX), as well as the levels of reactive oxygen species(ROS)and malondialdehyde(MDA)were analyzed using commercial kits. The extraction yield of flavonoids after process optimization in this study is(9.92±0.44)mg/g, which is higher than those reported in previous literatures. Compared with the C group, the cell viability and EdU positive rate in the UVB group decrease, the activities of CAT, SOD, and GPX decrease, and the levels of ROS and MDA increase(P<0.05). Compared with the UVB group, the cell viability and EdU positive rate in the HRF-L/M/H groups increase, the activities of CAT, SOD, and GPX increase, and the levels of ROS and MDA decrease(P<0.05). This study demonstrates that the composite enzymatic hydrolysis-ultrasound synergistic method can achieve efficient extraction of flavonoids from Hippophae rhamnoides Linn. pomace. Hippophae rhamnoides Linn. flavonoids alleviate UVB-induced damage to HaCaT cells by enhancing the activities of antioxidant enzymes, scavenging ROS, and inhibiting lipid peroxidation. This study provides theoretical support for the resource utilization of Hippophae rhamnoides Linn. pomace and the development of natural antioxidants.