Sunday, December 6, 2015

Isolation of High-Purity Limonin Glucoside from Molasses, a By-Product Obtained from Citrus Processing Waste Streams


            In the previous post, we highlighted a study from the USDA that examined the health benefits of limonin glucoside (LG) supplementation, as indicated by circulating levels of blood lipids, glucose and biomarkers of chronic inflammatory diseases, in human subjects. As can be seen from the complicated HPLC trace shown above, recovery of pure limonoid glucosides from natural sources in order to fund these types of human studies is not a trivial undertaking. Moreover, given the molecular complexity of most limonoids, chemical synthesis may not necessarily provide a viable supply chain alternative. In my rough estimation, the USDA study required the sourcing of about one hundred grams of limonin glucoside containing very low levels of its corresponding aglycone, limonin. While limonin glucoside is tasteless, limonin tastes extremely bitter and beverage concentrations of >6 mg/liter render citrus juice unacceptable to consumers. In fact, the original isolation of LG by the USDA produced material that was insufficiently pure. Taste tests of the resulting formulated beverages indicated that they were too bitter for human consumption due to limonin aglycone contamination. In this post, we will outline and discuss the optimized citrus extraction process that was eventually developed in order to support the human LG supplementation study.
            Citrus molasses, a thick, viscous liquid that is dark brown to almost black in color, is a major by-product of citrus processing. The fresh pulp obtained after pressing the fruit is mixed with lime (calcium oxide) and pressed to remove moisture. The resulting liquid is then filtered to remove the larger particles, sterilized by heating and concentrated to produce molasses. Citrus molasses is often fed to animals or fermented to produce ethanol, yielding meager profit margins for juice producers. Interestingly, HPLC and LC/MS analyses (see Figure above, left-hand panel) have shown that limonoid glucosides are present in significant amounts in molasses. A preparative scale method for recovery of LGs from citrus molasses was disclosed in 2002 by Thomas Schoch, Gary Manners and Shin Hasegawa at the USDA.
            A schematic flow diagram of the USDA process is reproduced in the Figure above. To begin, one to two liters of crude molasses is diluted with distilled water and the resulting solution is centrifuged. The supernantant thus obtained is then loaded onto a column containing Dowex 50WX4-100 resin and the outflow of the column is directly connected to a second column containing SP70 Sepabeads. Ethanol is used to elute the partially purified mixture from the second column. The Dowex column, a sulfonic acid cation-exchange resin, is intended to remove colored compounds present in molasses. The Sepabeads, which are a divinylbenzene copolymer (basically polystyrene), subsequently absorb and remove hydrophobic components. The eluent solution from the SP70 column is then adjusted to pH 6.5 with dilute aqueous sodium hydroxide in order to maintain consistent chemical reactivity and chromatographic behavior within the partially purified LG mixture.
            The LG solution is then loaded onto a Q-Sepharose column and the column is eluted with an aqueous sodium chloride gradient. The Q-Sepharose stationary phase is a quaternary ammonium resin, designed to bind negatively charged compounds. Limonin glucosides are relatively acidic (pKa~2.8) and are therefore anionic at pH 6.5. The salt gradient elutes the negatively charged limonoids in order of decreasing pKa, enabling separation of the LGs from things like flavonoids, which are phenolic and less acidic. The resulting LG-containing fractions are then subjected to a second Dowex/SP70 treatment and the solvent is finally removed by iterative co-evaporation with ethanol to furnish a golden-brown limonoid powder. The method outlined above can produce about ten grams of >60% pure limonoid glucoside feedstock per week. Further re-purification of the powder by recrystallization from warm water yields semi-pure limonin glucoside, contaminated predominantly with unacceptably high levels of the corresponding aglycone, limonin. In order to remove the bitter limonin impurity, an HPLC ‘polishing’ method utilizing food grade solvents (aqueous ethanol) was developed. Using a 75 x 300 mm C18 column, the method is capable of processing 20 grams of material per run in less than three hours. Recovery of the purified limonin glucoside following evaporation of solvent is nearly perfect (~94%) and the limonin content is consistently reduced to <0.10% by weight. Estimating conservatively, it looks like about two liters of molasses could be processed in four to six weeks to produce around 18 grams of high-purity limonin glucoside, suitable for human studies. By industrial standards, this type of productivity would be considered relatively low-throughput, given that a dose of 500 mg/day/patient is required for beverage supplementation studies. But, in the case of limonin or a related API of similar molecular complexity, does partial or total chemical synthesis offer a more scalable alternate supply chain approach to support downstream clinical programs? In a forthcoming post, we will examine the first and only reported chemical synthesis of limonin, disclosed in the summer of 2015 by researchers at Tohoku University in Japan. We will then directly compare the synthetic efficiency achieved by the world’s best organic chemists, using state-of-the-art methods, with that of recovery from citrus processing waste streams, in terms of practicality, cost and overall effort.

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