Citrus Limonoids: Chemistry and Functionality

An Undergraduate Seminar byMarie Astrid C. Amboy

Chem 199

What are citrus limonoids?- are a group of highly oxygenated triterpenoids

present in the order, Rutales which includes the family Rutaceae, Meliaceae and Simarobaceae

- are a group of secondary metabolites that have not been found to have any direct function in plant growth and development, unlike primary metabolites such as amino acids and nucleotides, which have recognized roles in the processes of assimilation, respiration, transport and differentiation.

- are tetranortriterpenoids with a 4,4,8-trimethyl-17-furanylsteroidal skeleton bearing several oxygenated functions

(Vahyazit and Konar, 2010)

there is no general figure for the molecular weight of limonoids because of their diverse chemical structures.

Limonin was the first isolated from navel orange juice in 1938 (Higby); and 20 years later, its structure was first determined (Arigoni et al., 1960). The chemical composition of limonin is C26H30O8 with a molecular weight of 470.

In the Citrus genus, limonoids are present as :

1. Limonoid aglycones (LA)-are further classified into limonoid

monolactones and limonoid dilactones. The former has an open D-ring such as limonoate A-ring lactone, and the later has a closed D-ring such as limonin.

- >50 isolated from the Rutaceae (36 from Citrus & related genera)

2. Limonoid glucosides (LG) -- 17 isolated

Relationship between LA and LG1. LA: bitter, insoluble in water2. LG: non-bitter, water-soluble3. LA glucosidated to LG --fruit

maturation --- Natural Debittering process --- Occurs only in fruit tissues and

seeds

Figure 1. Natural Citrus limonoids.

Source: Ruberto, G. et. al., J. Agric. Food Chem. 2002, 50, 6766-6774

Some Major Limonoids

Some Minor limonoids

Figure 2. Structures of Limonin and limonoid glucosides.

Source: Schoch et. al. ,J. Agric. Food Chem 2001, 49, 1102-1108

More than 20 limonoid glycosides (all as glucosides) havebeen isolated and characterized from the tissues of members of the genus Citrus and related genera in the plant family Rutaceae. These tasteless, water-soluble limonoid glucosides occur in citrus fruit tissues, juice, and seeds in high concentrations.

The concentration of these compounds in citrus juice ranges from ∼150 to 300 ppm, while the limonoid Glucoside content of citrus peel and flesh solids is ∼500 ppm.

Citrus seeds contain limonoid glucosides in about 1% of their dry weight. (Manners, 2007)

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 FlavedoLow: Juice vesicles

Limonin distribution within grapefruit leaves

More concentrated in areas adjacent to the conductive tissues

flushing leaves > mature leaves

Limonoids in citrus seeds: Relative composition and concentration

Four taxonomic groups of aglycones:1. Citrus group (19 limonoids)2. Fortunella group (17 limonoids)3. Papedocitrus group (1 limonoid)4. Poncirus group (4 limonoids)

Biosynthetic pathways of each group of these limonoids have been elucidated

Biosynthesis The biosynthetic pathways of these

limonoid aglycones have been established (Hasegawa and Miyake, 1996). Emerson (1948) first isolated nomilin using a radioactive tracer technique, and limonin was later demonstrated to be the predominant limonoid synthesized and accumulated in seedlings of lemon, Valencia orange, grapefruit and tangerine (Hasegawa et al., 1984).

The phloem region of stem was found to be the major site of nomilin biosynthesis. Epicotyl, hypocotyl and root tissues were also capable of biosynthesizing nomilin, but leaves, fruits and seeds did not show this capacity (Hasegawa et al., 1986a).

In the form of nomilinoate A-ring lactone, nomilin is biosynthesized via terpenoid biosynthetic pathways from acetate and mevalonate, via farnesyl pyrophosphate (Ou et al., 1988).

This precursor is translocated from the stem to other sites such as leaves, fruit tissues, peels and seeds (Hasegawa et al., 1986b), where it is further metabolized in each tissue to the other limonoids.

Four different pathways are involved in limonoid biosynthesis:

1. the limonin pathway 2. the calamin pathway3. the ichangensin pathway and; 4. the 7-acetate limonoid pathway

(Hasegawa and Miyake, 1996).

True citrus species only contain the limonin group of limonoids.

Limonin, nomilin, obacunone and deacetylnomilin are the major limonoids found in this group.

Delayed Bitterness

delayed bitterness develops in juice of citrus fruit suffering physical damage or damage from field freeze event. Physical disruption of the juice sacs in the citrus fruit initiates the biological transformation of a tasteless limonoid aglycone precursor (limonoate A-ring lactone) to a bitter limonoid aglycone (limonin).

This biochemical transformation is catalyzed by the enzyme limonin D-ring lactone hydrolase at pH 6.5 or lower

Delayed Bitterness

This natural debittering process is catalyzed by the enzyme UDPD-glucose: limonoid glucosyltransferase (limonoid glucosyltransferase). A single enzyme appears to be responsible for the glucosidation of all the limonoid aglycones to their respective glucosides.

consequently juices extracted from seedless fruits suffer more severely from limonin bitterness than those of the seedless fruits (Hasegawa, 1980)

Figure 3. Biosynthesis of limonoids in citrus with structures of the major limonoids. Source: Moriguchi, T. et. al., 2002

The correlation of citrus limonoid structural character and perceived bitterness has established the presence of a closed D-ring, a C14–C15 epoxide, a C-7 keto group, and an acetyl ester group at C-1 in a seven-membered A-ring as requirements for bitterness.

Figure 5. Delayed bitterness and natural debittering of limonoids in citrus.

Source: Manners, Gary D., J. Agric. Food Chem. 2007, 55, 8285–8294

Fig 6. Biosynthetic pathways of limonoids in citrus.

Analytical methods of Citrus Limonoids

Thin-layer chromatography (TLC) -for limonoid glucosides; method utilizing

Ehrlich’s reagent as a specific detection reagent

Nuclear Magnetic Resonance (NMR) - determination of limonoid structure High Pressure Liquid Chromatography (HPLC)

- reversed phase; utilizes ultraviolet detection at 215nm, & quantification

Radioimmunoassay - detection&quantification Enzyme-linked immunoassay

Analytical methods of Citrus Limonoids High Pressure Liquid Chromatography – Mass

Spectrometry (HPLC-MS) - detection& quantification

Electrospray Ionization Liquid Chromatography- Mass Spetrometry (ESI-LC-MS)-for the analysis of limonoid glucoside mixtures from (wet and dry samples) citrus sources

X-ray crystallography – elucidation Capillary Electrophoresis – quantification

Analytical methods of Citrus Limonoids

Atmospheric pressure chemical ionization (APCI)

Supercritical fluid extraction Fractional Crystallization –

purification C-18 or styrene divinyl benzene

solid-phase extraction (SPE)

Figure 6. Mass spectral profile of a limonoid glucoside mixture: 2. limonin glucoside; 3. nomilin glucoside; 4. deacetylnomilin glucoside; 5. nomilinic acid glucoside; 6. deacetylnomilinic acid glucoside; 7. obacunone glucoside.

Source: Schoch et. al., J. Agric. Food Chem 2001, 49, 1102-1108

Figure 7. (a) RTICC of a sample containing limonoid glucosides; (b-h) SIM chromatograms of carminic acid and individual limonoid glucosides.

Source: Schoch et. al., J. Agric. Food Chem 2001, 49, 1102-1108

Figure 5. Individual calibrations curves for limonoid glucoside quantitation.

Source: Schoch et. al., J. Agric. Food Chem 2001, 49, 1102-1108

Source: Schoch et al., J. Agric. Food Chem 2001, 49, 1102-1108

Bioactivity insect antifeedant activity antibacterial, antifungal, antiviral, antimutagenic and

cytotoxic activity Shown to reduce the risk of the following cancers: oral

activity, larynx, esophagus, stomach, pancreas, lungs, colon and rectum (Bayazit and Kozar, 2010)

Potential chemopreventive agents (Jacob et. al., 2000)- display significant inhibitory activity against cancerous tumors - induce glutathione-S-transferase activity in animals- in vitro testing of these limonoids with human breast cancer cell lines has also shown high levels of inhibitory activity

antioxidants Hypocholesterolemic Activity Antineoplastic therapeutic

Limonoids and the Taxonomic Studies of Citrus

Theoretical bases1. 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.

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

Citrus Fruits available in the Philippines

Citrus aurantium L.- orange Citrus vulgaria Risso.- sour orange Citrus nobilis Lour.- mandarin Citrus nobilis var. papillaris Blanco.- tizon Citrus decumana L.- pomelo Citrus mitis Blanco - calamondin Citrus webberii – alsem Citrus webberii var. Montana –cabugao Citrus longispina – talamisan Citrus niacroph ylla. –alemow Citrus southwickii – limao

Citrus histrix DC. – cabuyao Citrus histrix var. boholensis – canci Citrus histrix var. torosa Blanco – colobot Citrus micrantha. – biasing Citrus micrantha var. microcarpa – samuyao Citrus medico. L. – citron Citrus medica var. odorata. –tihi-tihi Citrus medico, var. nanus. Citrus

pseudolimonum.- colo-colo Citrus limetta Risso.- lime Citrus limetta var. aromatic,. Citrus excelsa. –

umon real, Citrus excelsa var. davaoensis (Wetser, 1915)

Common varieties include perante, Valencia, Clementine, Satsuma, pongkan, Washington navel, pomelo, calamansi, dalanghita, dayap and many others.

ReferencesSchoch, Thomas K. ,* Manners, Gary D., and

Hasegawa,Shin, 2001,J. Agric. Food Chem. 49, 1102-1108

Berhow, Mark A., Hasegwa, Shin, and Manners, Gary D. Citrus Limonoids -- Functional Chemicals in Agriculture and Food/ 2000, American Chemical Society

Ruberto, G. et. al., J. Agric. Food Chem. 2002, 50, 6766-6774

Fong, Chi F. et. al., J. Agric. Food Chem. IW3, 41, 112-1 15

Manners, Gary D. J. Agric. Food Chem. 2007, 55, 8285–8

Hasegawa, Shin. J. Agric. Food Chem. 1980, 28, 922-925

Miller, Edward G. et. al., J. Agric. Food Chem. 2004, 52, 4908-4912