Separation And Purification Of Carnosic Acid From Rosemary Leaves And Investigatation Of Biological Activities Of Carnosic Acid

Rosemary( Rosmarinus officinalis L.), as a widely distributed medicinal herb, the leaves contain a variety of bioactive matters, among which carnosic acid has shown a series of important pharmacological and biological activities, such as antioxidant, antibacterial, anti-inflammatory, antitumor, protecting the central nervous system, protecting liver and stomach, reducing blood sugar and blood fat, and has been widely employed in food, medicine and health products, cosmetic, perfume and many other aspects. In this paper, the separation and purification processes of carnosic acid from rosemary leaves were studied. Meanwhile, the antioxidant and antibacterial activities of carnosic acid were studied by radical scavenging effect, reducing ability and tablet dilution methods.The composite method of three steps was used to get carnosic acid from rosemary leaves. The first step is getting rosemary extracts from rosemary leaves by ultrasound-assisted ethanol extraction; the second step is to get crude products of carnosic acid by ultrasonic-assisted n-hexane extraction and pH adjustment; the third step is to get purified carnosic acid from crude products of carnosic acid by silica gel column chromatography. Results of single factor experiments showed that, the reasonable conditions of ultrasound-assisted n-hexane extraction of caonosic acid from rosemary extracts were liquid/solid ratio 20:1(mL/g), ultrasonic frequency 28 k Hz, ultrasonic time 40 min, ultrasonic power 140 W. The reasonable conditions of silica gel column chromatography were 200-300 mesh silica gel 40 g, the eluent ethyl acetate: petroleum ether 4:7(v/v), the flow rate 6mL/min and 0.3 g solid loaded. The purity of carnosic acid is ≥ 95 % by this method.Then, the two-step method to get carnosic acid from rosemary leaves based on the composite method of three steps was employed. The first step was getting rosemary extracts from rosemary leaves by ultrasound-assisted ethanol extraction; the second step was to get purified carnosic acid from rosemary extracts by silica gel column chromatography. Results of single factor experiments showed that, the reasonable conditions of silica gel column chromatography were 200-300 mesh silica gel 50 g, the eluent ethyl acetate: petroleum ether 4:13(v/v), the flow rate 4 mL/min and 0.5 g solid loaded. The purity of carnosic acid was ≥ 97 % by this method. Meanwhile, the substance obtained by separation and purification was verified through UV, IR, NMR, LC-MS.The antioxidant activities of carnosic acid and other common synthetic antioxidants were compared by 3 kinds of currently accepted methods of detection and evaluation the antioxidant activities in vitro such as DPPH radical scavenging effect, ABTS radical scavenging effect andreducing ability. The results showed that, the antioxidant activity of carnosic acid was significantly higher than that of synthetic antioxidants PG, BHT, BHA, VE under the same concentration, while slightly lower than THBQ. At the same time, IC50 s of scavenging DPPH and ABTS radical of carnosic acid were 2.53 μg/mL and 51.58 μg/mL.The minimum inhibitory concentration(MIC) of carnosic acid and 3 kinds of common antibacterial agents benzalkonium bromide, ampicillin sodium enzyme and erythomycin on sixed different bacteria were tested and evaluated by tablet dilution method experiments. Antibacterial tests indicated that carnosic acid had broad-spectrum antibacterial activity. Especially for Escherichia coli and Pseudomonas aeruginosa, the MICs of carnosic acid were 4 and < 2 μg/mL, better than that of bromogeramine, sodium ampicillin and erythromycin. Moreover, for Klebsiella pneumonia, carnosic acid showed the same inhibitory effect with erythromycin, better than bromogeramine and sodium ampicillin.The separation and purification of carnosic acid from rosemaryleaves and investigation of biological activities of canosic acid laid a theoretical and application foundation for the comprehensive development and utilization of rosemary resources, and expanded broaderapplication for carnosic acid.