Isolation of Carotenoids from Rhinacanthus nasutus (L.) kurz by Column Chromatography and Nanoemulsion Preparation for Bioavailability Determination

• Main Authors: Ho, Nai-Hsiang, 何迺行
• Other Authors: Chen, Bing-Huei
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/94421471170913777138

碩士 === 輔仁大學 === 食品科學系碩士班 === 103 === Rhinacanthus nasutus (L.) kurz, a traditional Chinese herb, has been reported to possess several pharmacological functions including anti-cancer, anti-bacteria, anti-virus and antioxidant activities as well as reduction of blood lipid and sugar. Most recent reports on physiological studies with R. nasutus have mainly focused on naphthoquinone, however, some other functional components like carotenoids are less investigated, but may also play a vital role. The objectives of this study were to develop a preparative column chromatographic method for isolation of carotenoids from R. nasutus. In addition, the nanoemulsions of carotenoid fraction were prepared for determination of bioavailability. Results showed that about 250 mL of ethyl acetate could elute carotenoids at a flow rate of 10 mL/min from the open-column packed with 52 g of MgO-diatomaceous earth (1:3, w/w) and loaded with 3 mL hexane extract of R. nasutus. Analysis of carotenoid fraction by HPLC revealed that a total of 14 carotenoids including all-trans-neoxanthin (1.03 ± 0.01 μg/mL), all-trans-violaxanthin (8.26 ± 0.74 μg/mL), 13- or13'-cis-lutein (5.59 ± 0.88 μg/mL), 13- or13'-cis-lutein (3.35 ± 0.78 μg/mL), all-trans-lutein (50.3 ± 2.69 μg/mL), β-apo-8'-carotenal (IS), all-trans-β-cryptoxanthin (1.99 ± 0.91 μg/mL), 13- or 13'-cis-α-carotene (2.95 ± 0.32 μg/mL), 13- or 13'-cis-α-carotene (2.44 ± 0.06 μg/mL), 15- or 15'-cis-β-carotene (2.70 ± 0.15 μg/mL), 13- or 13'-cis-β-carotene (12.8 ± 0.92 μg/mL), all-trans-α-carotene (49.2 ± 2.69 μg/mL), all-trans-β-carotene (144 ± 7.78 μg/mL) and 9- or 9'-cis-β-carotene (19.6 ± 1.34 μg/mL) could be separated within 62 min by employing a YMC C30 column and a gradient mobile phase of methanol-acetonitrile-water (82:14:4, v/v/v) (A) and methylene chloride (B) with flow rate at 0.8 mL/min and detection wavelength at 450 nm. The nanoemulsions of carotenoid fraction were prepared by using CapryolTM 90, Transcutol® HP, Tween 80 as emulsifier and deionized water, with the mean particle size of 10.4 nm for oral administration and 10.7 nm for i.v. injection. Storage for 90 days at room temperature did not show significant change in particle size distribution, indicating a high stability of the carotenoid nanoemulsions. For animal experiments, both crude carotenoid fraction and carotenoid nanoemulsions were orally administered to Sprague–Dawley rats separately via tube feeding, but i.v. injection only for nanoemulsions, and the level of carotenoids in serum was assayed by HPLC method. Various pharmacokinetic parameters such as maximum concentration (Cmax), area under plasma concentration-time curve (AUC) and absolute bioavailability were determined using a WinNonlin software system. Compared to the Cmax of crude carotenoid fraction (0.0005 ± 0.0002 μg/mL), a higher Cmax of 0.0022 ± 0.0012 μg/mL for nanoemulsion group was obtained. The AUC value (1.8338 ± 1.0250 min μg/mL) for nanoemulsion group was much higher then that of the crude carotenoid fraction group (0.4609 ± 0.2926 min μg/mL). Taken together, the absolute bioavailability of nanoemulsion (0.18 ± 0.10%) was 4-fold higher than carotenoid fraction (0.04 ± 0.02%), demonstrating that nanoemulsion technology should have some impact on bioavailability enhancement.