Study On Preparation Of High Purity Perilla Leaves Rosmarinic Acid And Caffeic Acid

Perilla is a herb that is widely planted in my country with the same medicine and food.Many functional ingredients can be extracted from perilla leaves,which will help expand the development and application of natural plant extracts in the food,cosmetics,and pharmaceutical industries.This thesis studied the separation and purification of rosmarinic acid and caffeic acid from perilla leaves by HSCCC,the separation and preparation of rosmarinic acid and caffeic acid monomers by MPLC,and HPLC on perilla leaf rosemary The detection and analysis of fragrant acid and caffeic acid provide theoretical basis and technical support for the separation and purification of active ingredients from perilla leaves.The main findings are as follows:(1)High-speed countercurrent chromatography(HSCCC)is used to quickly separate caffeic acid and rosmarinic acid in perilla leaves at one time.By measuring the partition coefficients of caffeic acid and rosmarinic acid in 5 solvent systems with different volume ratios,the optimal system for separating caffeic acid and rosmarinic acid by high-speed countercurrent chromatography was screened out: petroleum ether-Ethyl acetate-methanol-0.5% acetic acid aqueous solution(3:7:3:7,V/V/V/V);in this solvent system,compare and analyze caffeic acid,caffeic acid,The separation effect of rosmarinic acid was analyzed,and the influence,changes and reasons of loading,host speed and mobile phase flow rate on retention time and stationary phase retention rate were analyzed,and the best conditions were screened out: host speed 800 r/min,flow The phase flow rate is 2.5m L/min,the sample volume is 200 mg,the temperature is 25℃,and the detection wavelength is 280 nm.Under these circumstances,the retention time is 160 min,and the stationary phase retention rate is 52%,achieving a good separation effect.A mixture of caffeic acid and rosmarinic acid was obtained from 200 mg of crude extracts of perilla leaves preliminary purified by macroporous resin,and 22 mg of a mixture of caffeic acid and rosmarinic acid was obtained.The purity of the two targets was 64.3% and 28.8%,respectively.Research results show that HSCCC can efficiently and quickly separate caffeic acid and rosmarinic acid from perilla leaves.(2)The fraction obtained by high-speed countercurrent chromatography was used to separate the raw materials,and the reverse phase C18 was used as the filler.The medium pressure preparative liquid chromatography was used to further separate and purify rosmarinic acid and caffeic acid.The purification process only took 23.7 minutes to obtain the caffeic acid.The purity of rosmarinic acid and rosmarinic acid are 98.29% and 97.01%,respectively.The optimized medium pressure preparative liquid chromatography conditions are: injection volume 4 m L,mobile phase flow rate 15 m L/min,and the mobile phase gradient elution was methanol-0.1% glacial acetic acid aqueous solution.The method is simple,rapid,and has high separation purity,and is suitable for the preparation and separation of rosmarinic acid and caffeic acid in perilla leaves.(3)Simultaneous determination of caffeic acid and rosmarinic acid in perilla leaves by HPLC.Chromatographic conditions: Zorbax Eclipse XDB-C18 column(150×4.6 mm,5 um);flow rate: 1 m L/min;injection volume: 10 μL;detection wavelength: 280 nm;column temperature: 35°C;elution solvent A is 0.1% glacial acetic acid solution,B is pure methanol;gradient elution program: 0-5 min 20%-40%(B);5-10 min 40%-55%(B);10-15 min55%-60 %(B);15-20 min 60%-65%(B).Sample processing method: accurately weigh 100 g of perilla leaf powder,dissolve it in 1500 m L of pure water,soak for 4 h,extract by steam distillation for 4 h,keep the temperature slightly boiling,centrifuge,take the supernatant,and pass the 0.22 μm organic phase membrane.The content of caffeic acid and rosmarinic acid are within the range of 0.0135625～0.217 mg/m L(r=0.9976)and 0.0135625～0.217 mg/m L(r=0.9997),respectively,showing a good linear relationship,and the correlation coefficient(r)is greater than 0.99,the detection limit is: 0.001,0.002 mg/m L;the limit of quantification is0.01,0.01 mg/m L.The standard addition recovery rate of caffeic acid is between 94.4% and96.3%;the standard addition recovery rate of rosmarinic acid is between 97.6% and 98.7%.Conclusion: The method is simple,fast,sensitive,precise,economic,environmentally friendly and clean.It provides a valid method for the isolation and determination of caffeic acid and rosmarinic acid in perilla leaves.