Research on bioactive molecules for the discovery of alternative medicines is growing rapidly. Successful complementary and alternative medicine programs are based on a number of strategies. Optimal utilization of bioactive molecules for any health benefit lies in understanding the complete chemistry of the plant-derived extracts. Thousands of research papers are published annually on the biological activity of natural products and crude extracts, yet many of these lack a complete understanding of their composition and purity. Most botanical extracts exhibit higher pharmacological benefits compared to pure principle marker compounds. It is possible that the presence of multiple compounds act synergistically to enhance the efficacy of marker compounds through pharmacokinetic or pharmacodynamic interactions. Therefore, screening of botanical extracts is critical for developing active supplements to prevent chronic diseases. Preparation of pharmacologically safe extracts from botanicals employing high throughput techniques that characterize all active components is critical for the successful utilization of botanicals for prevention of colon, prostate, and other cancers. Our group has developed several optimized methods for the quantification of various bioactive compounds (1-6).

‘Land to Lab’ facilities are available for the Bioactive Molecular core (BMC) unit for conducting research

Overview and Description of Ongoing Research

The isolation of bioactive compounds generally combines various separation techniques. Their use depends on the solubility, volatility, and stability of the compounds. Choices of different separation steps and analytical-scale optimization parameters are critical. A typical technique used in the BMC for the Soxhlet extraction of bioactive compounds followed by concentration of extracts under vacuum is illustrated in Figure 1.
We have separated a number of bioactive compounds using thin-layer chromatography (TLC) (7), flash chromatography (FC) (8, 9), low-pressure liquid chromatography (10-12), and high-pressure liquid chromatography (10, 12, 13). Most of the chromatographic techniques involve multi-step purification procedures (14); these are illustrated in Figure 2 and 3.

Figure 1. Extraction, concentration, and purification of bioactive compounds

Figure 2. Different analytical and preparative HPLC instruments used for the separation and quantification of bioactive compounds

References
1. Vikram, A.; Jayaprakasha, G. K.; Patil, B. S., Simultaneous determination of citrus limonoid aglycones and glucosides by high performance liquid chromatography. Analytica Chimica Acta 2007, 590, (2), 180-186.
2. Perez, J. L.; Jayaprakasha, G. K.; Yoo, K. S.; Patil, B. S., Development of a method for the quantification of d-glucaric acid in different varieties of grapefruits by high-performance liquid chromatography and mass spectra. Journal of Chromatography A 2008, 1190, (1-2), 394-397.
3. Jayaprakasha, G. K.; Patil, B. S., Rapid HPLC method for the simultaneous analysis of limonoids, flavonoids aglycones and their glucosides from citrus. In 235th ACS National Meeting, New Orleans, LA, United States, 2008.
4. Jayaprakasha, G. K.; Jena, B. S.; Sakariah, K. K., Improved liquid chromatographic method for determination of organic acids in leaves, pulp, fruits, and rinds of Garcinia. Journal of AOAC International 2003, 86, (5), 1063-1068.
5. Jayaprakasha, G. K.; Jagan Mohan Rao, L.; Sakariah, K. K., Improved HPLC method for the determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. J. Agric. Food Chem. 2002, 50, (13), 3668-3672.
6. Girennavar, B.; Jayaprakasha, G. K.; John, L. J.; Patil, B. S., Variation of bioactive furocoumarins and flavonoids in different varieties of grapefruits and pummelo. European Food Research and Technology 2008, 10.1007/s00217-007-0654-4.
7. Jayaprakasha, G. K.; Rao, L. J.; Singh, R. P.; Sakariah, K. K., Improved chromatographic method for the purification of phenolic constituents of the lichen Parmotrema tinctorum (Nyl) Hale. Journal of Chromatographic Science 1998, 36, (1), 8-10.
8. Raman, G.; Cho, M.; Brodbelt, J. S.; Patil, B. S., Isolation and purification of closely related Citrus limonoid glucosides by flash chromatography. Phytochemical Analysis 2005, 16, (3), 155-160.
9. Raman, G.; Jayaprakasha, G. K.; Brodbelt, J.; Minhee, C.; Patil, B. S., Isolation and identification of structurally similar citrus flavonoids by flash chromatography. Analytical Letters 2004, 37, 3005-3016.
10. Jayaprakasha, G. K.; Patil, B. S.; Bhat, N. Process for isolation of limonoid glucosides from citrus. US Patent # 2007/0237885 A1; WO 2007/118143A2, CODEN: USXXCO  US  2007237885  A1  20071011 2007.
11. Mandadi, K. K.; Jayaprakasha, G. K.; Bhat, N. G.; Patil, B. S., Red Mexican grapefruit: A novel source for bioactive limonoids and their antioxidant activity. Zeitschrift Fur Naturforschung C-a Journal of Biosciences 2007, 62, (3-4), 179-188.
12. Poulose, S. M.; Jayaprakasha, G. K.; Mayer, R. T.; Girennavar, B.; Patil, B. S., Purification of citrus limonoids and their differential inhibitory effects on human cytochrome P450 enzymes, . J. Science Food & Agriculture, 2007, 87, 1699-1709.
13. Girennavar, B.; Poulose, S. M.; Jayaprakasha, G. K.; Bhat, N. G.; Patil, B. S., Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes. Bioorganic & Medicinal Chemistry 2006, 14, (8), 2606-2612.
14. Jayaprakasha, G. K.; Brodbelt, J. S.; Bhat, N. G.; Patil, B. S., Methods for the separation of limonoids from citrus. In Potential Health Benefits of Citrus, 2006; Vol. 936, pp 34-51.
15. Jayaprakasha, G. K.; Mandadi, K. K.; Poulose, S. M.; Jadegoud, Y.; Nagana Gowda, G. A.; Patil, B. S., Novel triterpenoid from Citrus aurantium L. possesses chemopreventive properties against human colon cancer cells. Bioorganic & Medicinal Chemistry 2008, 16, (11), 5939-5951.