Brief Introduction to Citrus Limonoids

1. Brief Introduction to Citrus Limonoids Jiaxing Li TAMU-College Station TAMUK-Citrus Center

2. Bitterness problem in citrus industry • Cause 1. The flavonoids e.g. naringin-- species related to pumello • Cause 2. The limonoids e.g. limonin

3. Biochemistry of Limonoids in Citrus • A group of highly oxygenated tetracyclic triterpenoids -- Citrus & related genera • Tow forms in citrus: 1. Limonoid aglycones (LA) -- >50 isolated from the Rutaceae (36 from Citrus&related genera) 2. Limonoid glucosides (LG) -- 17 isolated

4. Biochemistry Cont. • Relation between LA and LG 1. LA: bitter, insoluble in water 2. LG: non-bitter, water-soluble 3. LA glucosidated to LG--fruit maturation --- Natural Debittering process --- Occurs only in fruit tissues and seeds

5. Delayed bitterness • PH<6.5 • Limonoid D-ring lactone hydrolase • Closed D-ring is the key structure

6. An example of natural debittering and delayed bitterness • Navel oranges and Valencia oranges in California • Early to mid-season Navel juice has delayed bitterness, Valencia dose not • Reason: Short maturation period of the Navel (2 months); 6 months for the Valencia allow: LA LG

7. Structures of some of the major Limonoids in citrus

8. Structures Cont.

9. Biosynthesis of Citrus Limonoids • Four groups of LA 1. Limonin group 2. Calamin group 3. Ichangensin group 4. 7α-acetate limonoid • Biosynthetic pathways of each group of these limonoids have been elucidated

10. Example of Biosynthetic pathways: limonin group

11. Analytical Methods of Citrus Limonoids • Thin-layer chromatography (TLC) -- for limonoid detection • Nuclear Magnetic Resonance (NMR) -- determination of limonoid structure • HPLC -- detection & quantification • Radioimmunoassay --detection&quantif. • HPLC-MS -- detection& quantification

12. Distribution of limonin in Citrus fruit and vegetative tissues -- by Radioimmunoassay • Limonin levels in fruit and vegetative tissue (White Marsh) High: Seeds Pith Lamella Albedo Flavedo Low: Juice vesicles

13. Limonin distribution within grapefruit leaves • More concentrated in areas adjacent to the conductive tissues • flushing leaves > mature leaves

14. Biological activities of citrus limonoids • Inducers of GST, a detoxifying enzyme in mice and rats: -- to inhibit formation of chemically-induced neoplasia in forestomach, small intestine, colon, lung, skin and oral of lab animals; -- to inhibit the proliferation of breast cancer cells in culture

15. Biological activities contd. • Antifeedant activity • insect repellents • growth disrupters • reproduction inhibitors

16. Limonoids and the Taxonomic Studies of Citrus • Theoretical bases 1. Plants have incorporated many secondary metabolites into specialized physiological functions such as reproduction and intracelluar signaling 2. secondary metabolites are often functionally unique at the species level. 3. Different compounds are responsible for identical functions in different species.

17. Different Limonoid groups and the Chemotaxonomy of the true-citrus tree species and hybrids

18. Future research • Nutritional research: determination of the roles phytochemicals play in the long term health of humans. -- Food additives for cancer prevention

19. The End • Thank You • References Mark A. Berhow, Shin Hasegwa, and Gary D. Manners.Citrus Limonoids -- Functional Chemicals in Agriculture and Food/ 2000, American Chemical Society.