30A-4 |
Purification of limonoids from sweet orange (Citrus sinensis) by semi-preparative HPLC |
K. SUNTHANONT, M. R. Bennink, and M. A. Uebersax. Dept. Food Science and Human Nutrition, Michigan State University, G.M.Trout Bldg., room 204, Michigan State U, East Lansing, MI 48823 Limonoids are highly oxygenated triterpenoids present in citrus fruits (Rutaceae and Meliaceae). The two major classes found in citrus are neutral (non-carboxylated) and acidic (carboxylated). Neutral limonoids are insoluble and bitter, whereas acidic limonoids are soluble and tasteless. Bitter compounds are very unfavorable for sensory quality of orange juice. However, recent studies indicate that limonoids have potential health benefits (anticancer, cardioprotective, antioxidant activities, etc). Because of the interest in these phytonutrients and lack of commercially available standards, it was necessary to purify these compounds. Our objective was to develop rapid chromatographic conditions for purification of neutral and acidic limonoids with high resolution (Rs³1.1) and yield using semi-preparative HPLC. Limonoids were extracted from orange seeds using liquid-liquid extraction and anion exchange chromatography. Nonpolar (neutral) compounds were partitioned in methylenechloride. Acidic compounds (polar fraction) were obtained using Accell Plus QMA Sep-Pak Vac. Independent purification runs were developed for acidic and neutral compounds using reverse phase HPLC (semi-preparative column: C18, 10m, 250x22mm at 10ml/min) coupled with UV detection. Resolved peaks were screened by reverse phase HPLC (analytical column) coupled with photodiode array detection and identified by positive FAB-MS. Purification of neutral limonoids was obtained using a 30% to 80% acetonitrile gradient in 100 minutes (Rs³1.1). Purification of acidic limonoids was obtained using a 10 to 20% acetonitrile gradient (at pH 2.4) in 40 minutes (Rs³1.6). The results suggested that purification of limonoids by preparative scale HPLC can be accomplished using a simple acetonitrile:water mobile phase.
Session 30A, Citrus
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