Chromatographic Techniques to Purify Individual Limonoids from Seeds and Molasses of Citrus Fruits

Limonoids, chemically related triterpinoids predominantly found in citrus and neem relatives, are known to play a pivotal role in the prevention of different types of cancer and cardiovascular diseases. Since the concentrations of these compounds are low in the plant tissues, the isolation of pure compounds is the limiting factor for the individual activity studies in animal models. In this study, combinations of chromatographic techniques were used to isolate limonoid aglycones and limonoid glucosides from citrus byproducts such as seeds and molasses. The compounds were initially extracted with different polar solvents and the concentrated extracts were passed through a series of adsorbent resin (SP-70) and ion-exchange resins (WA-30, Dowex-50, Q-sepharose) to remove further impurities. The use of increasing ionic strength of NaCl from 0 to 800 mm to release the exchanged compounds from the ion exchange columns further separated the limonoids from flavonoids, which was confirmed through TLC, UV, and analytical HPLC methods. Individual compounds were further purified using flash chromatography and preparative HPLC methods and identified by using LC-MS analysis. Direct crystallization of limonin resulted in a 17% increase in the yield as compared to the previously reported methods. The results suggest that application of these purification methods are useful for the bulk purification of compounds in order to further investigate their biological activity.