Studies On Modern Extraction And Separation Technologies And Properties Of Secondary Metabolites In Perilla Frutescens L.

Perilla (Perilla frutescens (L.) Britt. Var. arguta (Benth.) Hand.-Mazz.) is a plant traditionally used both for medicine and for food in China, and its leaves and seeds contain various kinds of secondary metabolites with high bioactivity. To fully utilize all the parts of perilla, the present work conducted a comprehensive study on the extraction, isolation, preparation and properties of three kinds of second metabolites, i.e. anthocyanin, flavonoid glycoside and perilla ketone. The fat-soluble crude extract was first extracted from perilla leaves, and then perilla ketone was isolated from the residues by means of high speed counter current chromatography (HSCCC). Subsequently, the residue was continually utilized for the extraction of anthocyanin. Lastly, the residue from the previous step was employed as material for the extraction of flavonoid glycoside. The main contents of this work are as following:(1) A rapid and high efficient method was firstly established using HSCCC to isolate the perilla ketone. The separation was performed with a two-phase solvent system composed of n-hexane-ethanol-water (6: 5: 1, V/V/V) by eluting the mobile phase at a flow rate of 1.2mL/min and a revolution speed of 800r/min. Under this condition, 28.0mg of perilla ketone with the purity of 98.8% was obtained from 1.0g of fat-soluble crude extract of perilla leaves in 6h.(2) The optimum technological conditions used for the extraction of anthocyanins by ultrasonic assisted extraction (UAE) were as follows: Acetic acid (6%) was used as the extraction solvent. The ratio of material to liquid was 1:10. It was extracted by ultrasonic wave at the power of 225W for two times under room temperature (25℃), and the extraction time was 20min for each times. Under this condition, the extracting rate of anthocyanins was 5.95% and the content of anthocyanins in crude extract was 29.44%.(3) The optimum technological conditions used for the purification of anthocyanins by XAD-7 resin were as follows: The pH of loading solution was adjusted to 3.0 with the concentration larger than 6.5mg/mL. The salt concentration was no more than 10%. The column was loaded at the flow rate of 10 BV/h for 5h. After that, the column was rinsed using 0.1% HCl until the elute did not contain contamination such as sugar. Subsequently, the column was eluted by 30% acentonitrile (pH3.0) at the flow rate of 2BV/h for 4h. Under this condition, the extracting rate of anthocyanins was 4.44% and the content of anthocyanins in the refined product was 78.18%. Under the room temperature condition, 9.8mg malonylshisonin and 4.2mg shisonin were recovered from 200mg refined product through semi-preparative C₁₈ column. The flow rate used was 2.5mL/min, and the wavelength for detection was 530nm. The mobile phase was water-acentonitrile-formic acid (80:15:5, V/V/V).(4) When the HSCCC separation was performed with a two-phase solvent system composed of n-butanol-tert-butyl methyl ether-acentonitrile-water (2:2:1:5+0.1% TFA, V/V/V/V) by eluting the mobile phase at a flow rate of 3.0mL/min and a revolution speed of 800r/min, 10.1mg malonylshisonin and 8.6mg shisonin were obtained from 1.0g of crude anthocyanins extract. The purities of malonylshisonin and shisonin were 96.7% and 97.5%, respectively.(5)The influence of temperature, pH, light, oxygen, Vc and several metal ions on the stability of malonylshisonin was investigated. The results showed that malonylshisonin was stable under lower temperature condition (<60℃), while it had lower stability under higher temperature condition (>80℃), under which its stability was higher than that of shisonin. The indoor natural light has little effect on its heat stability, whereas the irradiation of UV light leaded to a quick degradation. It was stable under acidic condition and suitable for the processing of acidic food with the pH<4.0. It was showed that oxygen had certain effect on the promoting its degradation. The oxidation of Vc will severely reduce its stability, which could be prevented by the supplement of a certain amount of thiourea. Among the metal ions investigated when treated at the concentration of 50mg/kg, Cu²⁺ has some degree of hyperchromic effect; Na⁺ has little effect; Mg²⁺,Zn²⁺,Ca²⁺,Fe³⁺,Al³⁺ reduced the retention rate of malonylshisonin while Fe³⁺ leaded to precipitation formation.(6) The optimum technological conditions used for the extraction of flavonoid glycosides by UAE were as follows: Ethanol (70%) was used as solvent, and the ratio of material to liquid was l:20(g/mL). It was extracted by ultrasonic wave at the power of 150W for two times under 60℃, every time lasted for 40min. Under this condition, the extracting rate of flavonoid glycosides was 2.93% and the content of flavonoid glycosides in crude extract was 15.46%.(7) The optimum technological conditions used for the purification of flavonoid glycosides by AB-8 resin were as follows: The loading solution was prepared at the concentration of 20mg/mL with the addition of resin at the ratio of 100mg/g. After loading, the column stood for 30min, and then was rinsed by water until colorless. Subsequently, the column was eluted by 3BV 70% ethanol at the flow rate of 2BV/h. Under this condition, the extracting rate of flavonoid glycosides was 2.15% and the content of flavonoid glycosides in the refined product was 58.74%. Under the room temperature condition, 5.6mg luteolin-7-diglucoside was recovered from 100mg refined product through semi-preparative C₁₈ column. The flow rate used was 2.5mL/min, and the wavelength for detection was 330nm. The mobile phase was water-acentonitrile-formic acid (86:9:5, V/V/V). 9.3mg apigenin-7-diglucoside was recovered from 100mg refined product when the mobile phase composed of water-acentonitrile-formic acid (83:12:5, V/V/V) was employed.(8) A two-phase solvent system composed of chloroform-methanol-water (8:10:5, V/V/V) was adopted. The flow rate of the mobile phase was 2.5mL/min and the revolution speed was 800r/min. Under this condition, 7.5mg luteolin-7-diglucoside and 12.4mg apigenin-7-diglucoside were obtained from crude flavonoid glycosides extract. The purities of luteolin-7-diglucoside and apigenin-7-diglucoside reached 96.4% and 99.1%, respectively.(9) The extracts obtained from the leaves and the peel of the seeds through different solvent system demonstrated antioxidant activity in perilla seed oil in some degree. The extracts of the seed peel through ethanol showed higher antioxidant activity, which could give an optimal result when supplemented with the ration of 0.09%; It will markedly increase the antioxidant activity when the extracts were supplemented combined with citric acid or glycine. The results demonstrated flavonoid glycosides purified from perilla leaves as an effective antioxidant component, of which the antioxidant activity of luteolin-7-diglucoside was better than that of apigenin-7-diglucoside.