玫瑰茄花青素纯化、鉴定及抗氧化活性评价

Purification, identification, and antioxidant activity of anthocyanins from roselle

摘要:

以玫瑰茄为原料制备花青素，利用Amberlite XAD-7大孔树脂进行纯化、采用傅里叶红外光谱及超高效液相色谱串联飞行时间质谱对玫瑰茄花青素纯化物（Purified Roselle Anthocyanins, PRA）中花色苷进行鉴定，并评价PRA在体外和HepG2细胞内的抗氧化活性。结果表明，玫瑰茄花青素纯化后花青素含量提高了6.35倍，达到175.27 mg/g。PRA中含有五种花色苷，以飞燕草素-3-桑布双糖苷相对丰度最高，占总花色苷丰度的57.35%。PRA具有良好的体外抗氧化作用。当PRA质量浓度为0.23 mg/mL时，对1，1-二苯基-2-苦基肼自由基（1,1-diphenyl-2-picryl-hydrazil，DPPH）自由基清除率达90%；PRA质量浓度为2.0 mg/mL时，对羟自由基清除率达97.85%。PRA还具有良好的HepG2细胞内抗氧化活性。200 μg/mL PRA处理使H2O2诱导的HepG2细胞内活性氧水平从117.47%（以对照组2',7'-二氯荧光素荧光强度为100%）降低至102.09%，一氧化氮含量由53.18 nmol/mL降低至45.79 nmol/mL，超氧化物歧化酶活力由18.19 U/mg prot升高至35.05 U/mg prot，过氧化氢酶活力由10.10 U/mg prot升高至19.38 U/mg prot。综上，PRA花色苷以飞燕草素-3-桑布双糖苷为主，且具有良好的体外抗氧化活性和体内抗氧化应激能力。这些结果为玫瑰茄花青素的高值化开发以及潜在健康功效提供理论依据。

Abstract:

Roselle was used as raw material for the preparation of anthocyanins. The anthocyanins were purified using Amberlite XAD-7 macroporous resin, identified by Fourier-transform infrared spectroscopy, and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Moreover, the antioxidant activity of anthocyanins in vitro and in HepG2 cells was evaluated. The results showed that the anthocyanin content in the purified product (Purified Roselle Anthocyanins, PRA) was 6.35-fold increased with a value of 175.27 mg/g. PRA contains five anthocyanins, of which delphinidin-3-sambubioside was the most abundant, accounting for 57.35% (w/w) of the total anthocyanins. PRA possessed good antioxidant activity in vitro. The scavenging rate of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical reached 90% when the PRA concentration was 0.23 mg/mL, and the scavenging rate of hydroxyl radical reached 97.85% when the PAR concentration was 2.0 mg/mL. PRA also possessed good intracellular antioxidant activity. PRA of 200 μg/mL treatment significantly reduced the H2O2-induced HepG2 intracellular reactive oxygen species level and nitric oxide content from 117.47% (the 2',7'-dichlorofluorescein fluorescence intensity of the control group as 100%) and 53.18 nmol/mL to 102.09% and 45.79 nmol/mL, respectively. The PRA treatment significantly increased the H2O2-induced HepG2 intracellular superoxide dismutase activity and catalase activity from 18.19 U/mg prot and 10.10 U/mg prot to 35.05 U/mg prot and 19.38 U/mg prot, respectively. In conclusion, the PRA anthocyanins were dominated by delphinidin-3-sambubioside and had good antioxidant activity in vitro and anti-oxidative stress capacity in vivo. These results provide a theoretical basis for the high-value development and the potential health benefits of Roselle anthocyanins.