| Perilla frutescens(L.) Britt originating in China, is an annual herbaceous plant of Perilla from Lamiaceae family, and now widely distributes in Yunnan, Shanxi, Jiangsu,Sichuan and Anhui Provinces of China. Perilla frutescens is often considered as both medicinal and edible plant. Its leaves contain abundant flavonoids, however, the researches of Perilla frutescens were mainly concentrated on its essential oil components. The reports about the flavonoids in the leaves of Perilla frutescens are very rare. Therefore, the present studies aimed to study on chemical compositions and antioxidant properties of the flavonoids in the leaves of Perilla frutescens, and to provide a theoretical foundation for the further development and utilization of resources of this plant.This study consists of four parts:(1) The flavonoids in the Perilla frutescens leaves were extracted by microwave-assisted technique. The extraction conditions were optimized as follows:Ethanol concentration, ratio of liquid to material, extraction temperature, extraction time and initial microwave power were 63%, 41:1(mL/g), 72 ℃, 7 min and 500 W,respectively. The yield of flavonoids was 7.81% under these optimal conditions.(2) The flavonoids in Perilla frutescens leaves were purified by AB-8 macroporous resin and preparative chromatography. The optimal purification conditions of macroporous resin were as follows: The sample solution passed through the resin column at a flow rate of 2 BV/h and pH 2. Subsequently, the column was eluted by 70% alcohol at a flow rate of3 BV/h. The purity of flavonoids has been increased from 21.24% to 53.10%. The flavonoids in Perilla frutescens leaves were further separated and pufified by Grace RevelerisTMholographic rapid purification chromatographic system. The column was packed with 12 g RevelerisTM RP C18 Cartridge. The mobile phase was 1% acetic acid aqueous solution(A) and methanol(B). The flow rate, the injection volume and the detection wavelength were 10 m L/min, 15 mL and 280 nm(UV1) and 320 nm(UV2),respectively. The gradient elution conditions were 0~3 min: 20~45%B, 3~12 min:45~75%B, and 12~20 min: 75~75%B. The experimental results showed that the preparative chromatography could preliminary separate the flavonoids in Perilla frutescens leaves into two components.(3) Qualitative and quantitative analysis of chemical compositions of Perilla frutescens leaves were carried out using HPLC-Q-TOF and HPLC-MS/MS. Thirteen compounds were identified in Perilla frutescens leaves, including citric acid(47.94 μg/mg),tanshinol(28.28 μg/mg), protocatechuic acid(6.82 μg/mg), protocatechuic aldehyde(1.57μg/mg), caffeic acid(11.81 μg/mg), scutellarin(50.08 μg/mg), rosmarinic acid(74.94μg/mg), apigenin-6,8-two-C-glucoside(41.08 μg/mg), scutellarein-7-O-diglucuronside(15.88 μg/mg), luteolin-7-O-diglucuronside(22.45 μg/mg), rosmarinic acid dimers(20.73μg/mg), apigenin-7-O-diglucuronside(13.48 μg/mg) and scutellarein-7-O-glucoside(20.74μg/mg).(4) The antioxidant properties of every component of flavonoids in Perilla frutescens leaves were evaluated by both chemical methods and cell model. Compared with ascorbic acid(Vc), the flavonoids in Perilla frutescens leaves had weaker capacity in total antioxidant ability, superoxide anions and DPPH radicals scavenging activities, while they had stronger capacity in scavenging hydroxyl radicals and inhibition ability in lipoprotein and lipid of yolk. The results showed that every component and total flavonoids in Perilla frutescens leaves were not cytotoxic to RAW264.7 cells within the concentration range of1~500 μg/mL, and the component 2 had higher antioxidant activities than the others. |
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