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) 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.