| Astaxanthin is an important kind of carotenoid pigment with many biological functions, such as antioxidant, anti-tumor, resistance to ultraviolet radiation, vitamin A precursor, immune stimulation, improvement of eye health and so on. The physiological functions of astaxanthin from Haematococcus pluvialis have been widely accepted, while the biological effects of astaxanthin from other sources remain unclear due to the differences in chemical structures. So the application of astaxanthin from other sources is limited, which can only be used as the feed additive at present. In the present study, the method to obtain a pair of enantiomers from natural resources has been established, it will be a useful technical support to the development of the astaxanthin used as the functional foods and marine drugs. In addition, the physiological activities of the different sources and structures of astaxanthin have been comparatively evaluated in vitro, and the mechanism of structure-activity relationship of astaxanthin is preliminary discussed, which could provide theoretical basis for the further development and utilization of astaxanthin, and technical support for promotion of functional food and marine drugs.The main contents and conclusions of the present research are as follows:1、A C30 reversed-phase high performance liquid chromatographic (RP-HPLC) method is developed for the determination of astaxanthin in Haematococcus pluvialis. In the study, the conditions of saponification and enzymatic hydrolysis are comparatively researched on saponification efficiency of astaxanthin esters from H. pluvialis. The contents of isomers of astaxanthin were calculated by calibration factor, the calibration factor of 13-cis-astaxanthin to trans-astaxanthin was 1.3, and 9-cis-astaxanthin to trans-astaxanthin was 1.1. The experimental conditions show that H. pluvialis powder is abrasively extracted with methylene chloride:methanol (V:V, 1:3) and ultrasonically extracted, and saponified by 0.5 mL 0.1 mol/L NaOH methanol at 5℃ for 12 hours avoiding light. The separation and quantification of astaxanthin are achieved by a YMC-Carotenoid C30 using a mixture of methanol、 tert-Butyl methyl ether and 1% phosphoric acid as the mobile phase and detected with an ultraviolet detector at 474 nm. We confirm that the different reserve time of astaxanthin esters and astaxanthin separated by YMC-Carotenoid C30 column could judge whether astaxanthin esters have been completely saponified. The limits of quantitation are 0.5 mg/g. There is good linear relationship between the chromatographic peak areas and the concentrations in the range of 0.1～5 mg·L-1 with the correlation coefficient of over 0.99. The precision of the method is 3.5%～14.7%, and the average recoveries are between 94.8%～107.9%. The results show that the method has good repeatability and sensitivity, and could be successfully applied in the analysis of astaxanthin from H. pluvialis.2、A simple, low-cost method of separation and purification for preparing (3S,3’S)-trans-astaxanthin from H. pluvialis is established in the study. Crude extracts are separated from dry algal cells by extraction. The extracts are then saponified at 4 ℃ for 15 h and at 22 ℃ for 3 h with 0.02 mol/L NaOH with lower isomerization of trans-astaxanthin to cis isomers. The saponified pigment extracts are then mixed with distilled water and n-hexane at 1:1:1 volume ratio. Low-pressure column chromatography of silica gel (300～400 mesh) with different eluents is used to prepare high-purity (> 80%) of all-trans-astaxanthin. Supersaturated solution of astaxanthin in acetone is then crystallized for 72 h at 4 ℃, and the purity of all-trans-astaxanthin crystalline powder is confirmed to be >96.5% by HPLC. The 3S,3’S isomers are identified and characterized using the HPLC-APCI-MS,1H NMR,13C NMR, and HPLC-UV.3、In the present study, we investigate the method of separation and purification for preparing (3 R,3’R)-trans-astaxanthin from Phaffia rhodozyma. The results show that the dry rhodozyma is first separated by using glass tissue grinder and purified by silica gel chromatography, then supersaturation of astaxanthin in acetone is crystallized, and the purity of the all-trans-astaxanthin crystalline powder is verified above 95% by using HPLC. Then, by the method of HPLC-APCI-MS,1H and 13C NMR and chiralpak IC column based on cellulose tris (3,5-dichlorophenyl carbamate) polymer, it contained the 3R,3’R isomers almost exclusively.4、The antioxidant activity of astaxanthin isomers is comparative studied in vitro. All of the astaxanthin isomers significantly exhibit radical DPPH scavenging activity, reductive capability, and inhibit rat microsomes membrane lipid peroxidation systems induced by AAPH and DHA hydroperoxides formation. For astaxanthin stereoisomers, meso-astaxanthin have a much lower antioxidant activity than other stereoisomers, (3R,3’R)-trans-astaxanthin exhibit the higher radicals scavenging and reducing power, while (3S’,3’S’)-trans-astaxanthin have higher effect on the initial stage in lipid peroxidation. The cis isomer astaxanthin has a much higher antioxidant potency than the all-trans isomer,13-cis has a much highest scavenging effect and reductive capability, and the inhibitory activity of lipid peroxidation of 9-cis is the highest. The antioxidant activities of astaxanthin is related to the structures, the cis isomer has much higher antioxidant activities. And for all-trans astaxanthin, the antioxidant activities of the 3S,3’S and 3R,3’R, are higher than the meso. The above-mentioned results suggest that compared to the astaxanthin from chemical synthesis, astaxanthin from H. pluvialis and P. rhodozyma have a much higher antioxidant potency.5、The lymphocyte proliferation capacity, neutral red phagocytosis of PECs and natural killer (NK) cell cytotoxic activity of the astaxanthin isomers are comparatively assessed. The results show that at the final concentration of 20 μmol/L, all the three astaxanthin stereoisomers significantly promot lymphocyte proliferation, phagocytic capacities of PECs, and cytotoxic activities of NK cells. In addition, the (3S,3’S)-trans-astaxanthin treatment group show a much better response than the other groups, with the order 3S,3’S>3R,3’R>meso. The cis/trans isomers of astaxanthin also significantly exhibit high immune response. Furthermore, all-trans astaxanthin exhibit a higher lymphocyte proliferation and phagocytic capacities, and the cytotoxic activities of 9-cis and 13-cis are higher, but the difference of immune activity among the astaxanthin cis/trans isomers. The immune activities of astaxanthin is also related to the structure, (3S,3’S)-trans-astaxanthin has a much highest immune potency, and those of the cis/trans isomers are not significant difference. The immune response is related to the structure of stereoisomers of astaxanthin, and the (3S,3’S)-trans-astaxanthin from H. pluvialis exhibit the highest immunological activities. We hypothesize that the astaxanthin consist mainly of the(3S,3’S)-form has a much higher immune activity. |
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