Study On The Extraction And Purification Of Carnosic Acid From Rosemary And Its Antioxidative Activity

In the present study, the ultrasound-assisted extraction of carnosic acid (CA) from the cured leaves of Rosmarinus officinalis L.was studied, and optimized the extraction conditions through the response surface method (RSM). The process for separation and purification of CA by macroporous absorption resin and silica gel column chromatography was studied systematically. At the same time, the antioxidant capacity of CA was test, and studied the influence of oxidation stability for added different concentrations of CA (0.1 mg/g,0.2 mg/g,0.3 mg/g) in deep-sea fish oil through the acceleration experiments. The results were as follows:1.The ultrasound-assisted extraction of carnosic acid(CA) from the cured leaves of Rosmarinus officinalis L. was established, and the optimum extraction parameters were as follows:Extraction solvent:72.29% EtOH, ratio of solid to liquid:1:10.05,ultrasonic time:51.27 min, ultrasonic power:200.55 W. The extraction yield of CA was 2.70%, while the content was 21.54% in the extractum.2. The optimum purification parameters were established:H1020 resin was screened out from 9 kinds of macroporous resin; it was the optimum macroporous resin to purify CA. The results showed saturated adsorption capacity of H1020 resin was 19.84mg/g (dry resin), adsorption temperature was 25℃,saturated absorption time was 3 h, suitable desorption agent was 90%(volume fraction) ethanol solution; the flow rate was 1mL/min when the concentration of CA was 4.45 mg/mL, after reaction reached adsorption equilibrium, adsorbed CA was completely eluted by 90% ethanol solution with 2.7 BV. After dynamic purification, the purity of CA was 70.46%.CA extracts after separated by H1020 resin were purified by silica gel column chromatography. Determine the silica gel particle size for an 83-165μm, silicone moisture is 6%, a gradient elution was performed with acetic ether-hexane (4:6, v/v) on the column, diameter ratio for 10, the flow rate was 4 mL/min, and charge capacity was 0.3 g crude extract: 20 g silica gel.And after this process the purity of CA was 98.6%.3.The antioxidation ability of CA was studied:The antioxidant capacity of CA weaker than TBHQ, but greater than BHA and BHT in the reducing of DPPH radical ability tests.The results also showed that the ability to scavenge ABTS radicals was very low, even the result of CA was lower than the three artificially synthesized antioxidants (BHA, BHT, and TBHQ).The reducing power of CA was greater than the artificially synthesized antioxidants (BHA, BHT, and TBHQ).In FRAP test, the acivity of the different antioxidants sequence was TBHQ>BHA>CA>BHT, also indicated that CA has a strong reducing activity.4. The effect of CA on oxidative stability of fish oil was studied:The antioxidation of CA in fish oil on different temperature was strong function based on the peroxide value, acid value, malondialdehyde, and conjugated diene value, and the oxidization of fish oil is effected by the amount (concentration) of carnosic acid. The acivity of different content of CA sequence was 0.3 mg/g CA>0.2 mg/g CA>0.1 mg/g CA. Antioxidant effect of three content of CA were greater than VE, lower or approximate TBHQ. Best antioxidant effect can be achieved when the concentration of carnosic acid is 0.2 mg/g to prevent oxidation and ensure the quality of fish oil.Gas chromatography-mass spectrometry (GC-MS) was conducted to analyze the variation of polyunsaturated fatty acids (PUFAs) amount during the storage. With the increase of concentration of added CA, the less PUFA reduced. Its effect is better than that of VE and TBHQ.The main PUFAs:Eicosapentaenoic acid (EPA,20:5n-3) and docosahexaenoic acid (DHA,22:6n-3)content gradually go down with extending the storage. Various antioxidants have inhibitory effect on oxidation of fish oil. The range of the content of PUFAs was 0.3 mg/g CA>0.2 mg/g CA>0.1 mg/g CA>TBHQ>VE>control.The changes of cis fatty acid (CFA)>trans fatty acid (TFA) and aldehyde compounds contents were investigated by FTIR at 30℃over a period of 66 days.During the storage of 66 days, the content of trans double bond increased to 745.62% in control group and increased 361.57% and 291.65% in the samples of adding VE and TBHQ, respectively. While the contents of trans double bond increased merely 202.31%,177.20% and 124.28%, respectively, in the samples of adding CA with different doses.Through the infrared spectroscopy analysis, CA can suppress oxidization of fish oil effectively;bring down the production of detrimental aldehydes;prevent CFA transforming into TFA. The fish oil of adding CA can stabilize a better oxidation in contrast to the indicators which are synthetic antioxidants VE and TBHQ.