Glucosides of Limonoids - American Chemical Society

Chapter 8

Glucosides of Limonoids 1

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Shin Hasegawa, Chi H. Fong , Zareb Herman , and Masaki Miyake 1

Fruit and Vegetable Chemistry Laboratory, U.S. Department of Agriculture, 263 South Chester Avenue, Pasadena, CA 91106 Wakayama Agricultural Biological Research Institute, Tsukatsuki, Momoyama, Wakayamaken, Japan 649—61

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Limonoids are one of the two bitter principles in Citrus. Recently, limonoids have been shown to be present as glucoside derivatives in the Rutaceae. The 17-ß-D-glucopyranosides of 17 citrus limonoids have been isolated from Citrus and its hybrids. Four such compounds have been also isolated from noncitrus members of the Rutaceae. They are all nonbitter. The glucosidation of limonoids is an enzymic conversion of bitter to nonbitter compounds and is a natural limonoid debittering process taking place in Citrus. This chapter describes the discovery, occurrence, biosynthesis and possible biological significance of limonoid glucosides in Citrus. Limonoids are a group of chemically r e l a t e d t r i t e r p e n e d e r i v a t i v e s found i n the Rutaceae and Meliaceae f a m i l i e s . Among 37 limonoid aglycones reported t o occur i n Citrus and i t s hybrids, four a r e known t o be b i t t e r i n t a s t e . These a r e limonin, nomilin, i c h a n g i n and n o m i l i n i c a c i d (I) . Limonin i s the primary cause of c i t r u s j u i c e b i t t e r n e s s . This problem occurs i n a v a r i e t y of c i t r u s j u i c e s , and i t i s economically important t o the c i t r u s i n d u s t r y because b i t t e r j u i c e s have a lower market value f o r producers. B i t t e r n e s s occurs g e n e r a l l y i n j u i c e extracted from f r u i t s harvested i n e a r l y season, but i s g r e a t l y reduced l a t e r i n the season. This i s due t o a decrease i n the concentration of limonoids i n f r u i t t i s s u e s as f r u i t maturation progresses. However, how limonoids a r e metabolized i n f r u i t during l a t e stages of f r u i t growth and maturation was not understood u n t i l r e c e n t l y when Hasegawa e t a l . (2) discovered that limonoids a l s o occur i n Citrus as n o n b i t t e r glucoside d e r i v a t i v e s . The discovery of limonoid g l u c o s i d e s f i n a l l y showed that limonoid aglycones a r e converted t o t h e i r glucosides during l a t e stages of f r u i t growth and maturation. At l e a s t 17 l i m o n o i d g l u c o s i d e s from Citrus (2-8) and 4 from non-citrus members of the Rutaceae f a m i l y (9,10) have been isolated. Limonoid g l u c o s i d e s a r e a l s o found t o be one of t h e major secondary metabolites present i n Citrus (5,11) (e.g. 320

0097-6156/92/0490-0087$06.00/0 © 1992 American Chemical Society

Teranishi et al.; Flavor Precursors ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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ppm i n commercial orange j u i c e s and 0.6% of dry weight i n seeds) , and they a r e b i o s y n t h e s i z e d i n f r u i t t i s s u e s and seeds during l a t e stages of f r u i t growth and maturation (12). Limonoid UDP-D-glucose t r a n s f e r a s e c a t a l y z e s the g l u c o s i d a t i o n of limonoids i n Citrus and i t s a c t i v i t y occurs only i n matured f r u i t t i s s u e s and seeds {12-14). The g l u c o s i d a t i o n of limonoids i s considered t o be a n a t u r a l limonoid d e b i t t e r i n g process taking p l a c e i n Citrus.


Delayed

Bitterness

Most c i t r u s f r u i t s are not b i t t e r t a s t i n g i f eaten f r e s h or i f f r e s h l y squeezed j u i c e i s consumed. However, a few hours a f t e r j u i c i n g , t h e j u i c e e x t r a c t e d from a v a r i e t y of c i t r u s f r u i t s becomes b i t t e r . T h i s phenomenon i s known as delayed b i t t e r n e s s , and the mechanism i s shown i n Figure 1.

Limonoate A - r i n g l a c t o n e (Nonbitter Precursor)

Limonin (Bitter)

Figure 1. Mechanism of Delayed B i t t e r n e s s Intact c i t r u s f r u i t s do not contain b i t t e r limonin, but rather a n o n b i t t e r p r e c u r s o r of limonin, limonoate A - r i n g lactone (LARL)(15). When the f r u i t i s j u i c e d , LARL i s g r a d u a l l y converted t o limonin under a c i d i c c o n d i t i o n s . T h i s conversion i s a c c e l e r a t e d by the a c t i o n of an endogenous enzyme, limonin D-ring lactone hydrolase. The r e s u l t i n g limonin i s extremely b i t t e r i n t a s t e . Most persons can detect limonin b i t t e r n e s s a t 5 or 6 ppm i n j u i c e . J u i c e e x t r a c t e d from e a r l y to mid-season navel oranges may contain over 30 ppm limonin. However, j u i c e from f r u i t harvested l a t e i n the season i s u s u a l l y n o n b i t t e r because i t contains l e s s than 6 ppm of limonin. D i s c o v e r y of

Limonoid

Glucosides

The procedure which has been used by the c i t r u s i n d u s t r y and research l a b o r a t o r i e s t o analyze or i s o l a t e limonoids from c i t r u s t i s s u e s and j u i c e s i n v o l v e s e x t r a c t i o n with organic solvents such as methylene c h l o r i d e and e t h y l acetate. The aqueous f r a c t i o n has been ignored because limonoids are extracted i n the organic f r a c t i o n s . Recently, we examined the aqueous e x t r a c t s of c i t r u s t i s s u e s and j u i c e s , and we discovered that Citrus contains l a r g e q u a n t i t i e s of water s o l u b l e limonoid d e r i v a t i v e s . (See Figure 2.) The f i r s t compound was i s o l a t e d from an e x t r a c t of g r a p e f r u i t seeds by column chromatography and i d e n t i f i e d as limonin 17-β-D-glucopyranoside, known as limonin g l u c o s i d e



8. HASEGAWA ET AL.

Glucosides ofLimonoids

Obacunone

Isoobacunoic acid

Ichangensin

Isolimonic acid

Epiisoobacunoic acid

trans-Obacunoic acid

R ^ A c , R =H, R =CH , R =0, R5=H Nomilinic acid R|=OH, R =H, R =CH , R4=0, R5=H Deacetylnomilinic acid R!=OH, R =CH , R =CH , R =OH, R =0 Calamin R^OH, R =H, R =CH OH, R4=0, R5=H 19-Hydroxydeacetylnomilinic acid 2

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F i g u r e 2.

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Structures

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Limonoids


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(Figure 4) (2) . One limonin molecule i s l i n k e d with one D-glucose molecule a t the 17-position of the open D-ring by a β-glucosidic l i n k a g e . Using column chromatography and NMR spectroscopy, seventeen of these compounds have been i s o l a t e d and i d e n t i f i e d from Citrus and i t s hybrids (2-8) . These a r e l i s t e d i n Table 1. Table 1. Limonoid Glucosides i n Citrus


MonQcarbQxylic a c i d s

Dicarboxylic acids

17-β-D-glucopyranosides o f :

17-&-D-glucopyranosides o f :

1. 2. 3. 4. 5. 6. 7. 8.

10. 11. 12. 13. 14. 15. 16.

limonin nomilin deacetylnomilin obacunone ichangin ichangensin calamin 6-keto-7β-deacetylnomilol

9. methyl d e a c e t y l n o m i l i n a t e

nomilinic acid deacetylnomilinic a c i d obacunoic a c i d trans-obacunoic a c i d isoobacunoic a c i d epiisoobacunoic a c i d 19-hydroxydeacety1nomilinic acid

17. i s o l i m o n i c a c i d

A l l of the compounds i s o l a t e d are e i t h e r monocarboxylic o r d i c a r b o x y l i c a c i d s . A l l contain one glucose moiety i n the same p o s i t i o n as limonin 17-β-D-glucopyranoside. Since 37 limonoid aglycones have been i d e n t i f i e d , i t i s very p o s s i b l e that each aglycone has a corresponding glucoside d e r i v a t i v e . A l l limonoid glucosides i s o l a t e d t o date are n o n b i t t e r t o t a s t e . Limonoid g l u c o s i d e s are a l s o found t o be present i n nonc i t r u s members of the Rutaceae family. The 17-β-D-glucopyranosides of limonin, l i m o n i n diosphenol and 6fi-hydroxy-5e p i l i m o n i n are present i n Tetradium rutaecarpa (9) . The 17-βD-glucopyranosides of l i m o n i n and obacunone have been i s o l a t e d from Phellodendron amurense (10) .

Analysis of Limonoid Glucosides TLC i s the best method of d e t e c t i n g limonoid g l u c o s i d e s . I t can be used t o q u a n t i f y limonin g l u c o s i d e and the t o t a l limonoids (11). However, i t i s d i f f i c u l t t o separate i n d i v i d u a l glucosides by the method used thus f a r . The solvent system used i s EtOAc-methyl e t h y l ketone-formic a c i d (88%) -H 0 (5: 3: 1: 1) (11). The method i n v o l v e s s p o t t i n g the sample onto a s i l i c a gel p l a t e along with the appropriate limonoid glucoside standard, developing the p l a t e i n a s u i t a b l e solvent system, spraying the p l a t e with E h r l i c h ' s reagent (1% p-dimethylaminobenzaldehyde i n EtOH) , and developing the p l a t e i n a chamber of HCL gas. Limonoid g l u c o s i d e s produce d i s t i n c t i v e reddish-orange spots on the p l a t e . A HPLC method f o r the q u a n t i f i c a t i o n of limonin g l u c o s i d e i n c i t r u s j u i c e s was f i r s t developed by Fong e t a l (11) . Later, Herman e t a l (16) reported a method f o r the a n a l y s i s of i n d i v i d u a l limonoid g l u c o s i d e s i n orange j u i c e . Ozaki e t a l . 2


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Glucosides of Limonoids

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(5) reported a method f o r a n a l y s i s of seven limonoid g l u c o s i d e s of c i t r u s seeds. The method uses a C18 reverse-phase a n a l y t i c a l column and a l i n e a r gradient s t a r t i n g with 15% CH3CN i n 3 mM H P0 and ending with 26% CH CN at 33 min. The flow r a t e was 1 ml per min and the glucosides are detected by UV absorption at 210 nm (5) . 3

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Extraction and Isolation of Limonoid Glucosides Since limonoid g l u c o s i d e s a r e water s o l u b l e , they can be extracted with water from c i t r u s seeds. I t i s p o s s i b l e to e x t r a c t both limonoid aglycones and glucosides from the same batch of c i t r u s seed meal (2,3) . The meals are washed with hexane or petroleum ether t o remove o i l y m a t e r i a l s . Aglycones are extracted with acetone. Glucosides are then e x t r a c t e d with MeOH. The MeOH e x t r a c t a l s o contains some aglycones which can be separated by p a r t i t i o n i n g between water and CH2CI2. The water f r a c t i o n containing g l u c o s i d e s can be used f o r i s o l a t i o n of individual glucosides. Limonoid g l u c o s i d e s can be i s o l a t e d by open column chromatography, p r e p a r a t i v e HPLC or a combination of both (28). Since crude e x t r a c t s obtained from seeds or f r u i t t i s s u e s contain f l a v o n o i d g l u c o s i d e s which co-elute with limonoid glucosides during column chromatography, a DEAE Sephacel column i s h e l p f u l to separate these two types of g l u c o s i d e before subsequent chromatographic separation. Flavonoid glucosides have no a f f i n i t y to the column, whereas limonoid g l u c o s i d e s have weak a f f i n i t y and can be e l u t e d with 0.2 M NaCl s o l u t i o n . XAD and XAD-2 have been used f o r open column chromatography, and a C18 reverse-phase column has been used f o r p r e p a r a t i v e HPLC chromatography. In both methods, e i t h e r MeOH or CH3CN has been used f o r e l u t i n g limonoid g l u c o s i d e s .

Identification of limonoid glucosides The most u s e f u l method f o r o b t a i n i n g s t r u c t u r a l information about limonoid g l u c o s i d e s i s nuclear magnetic resonance (NMR) spectroscopy (2,3). These s p e c t r a are run i n deuterated dimethyl s u l f o x i d e , i n which the glucosides are h i g h l y s o l u b l e , at 90°. At lower temperatures the s i g n a l s are s e v e r e l y broadened. The H NMR spectrum of a new limonoid glucoside, when compared with s p e c t r a of known limonoids, can provide a good i n d i c a t i o n of the p o s s i b l e s t r u c t u r e of the aglycone. The C NMR spectrum then u s u a l l y confirms the s t r u c t u r e . T y p i c a l l y the s i g n a l s of carbons near the 17-position are c o n s i d e r a b l y s h i f t e d , while those f u r t h e r away are almost i d e n t i c a l to those of the aglycone. In d i f f i c u l t cases running 2D NMR spectra may be necessary to determine the s t r u c t u r e s . In p a r t i c u l a r , the linkage of the sugar t o the 17-position i s shown by a cross peak between H-17 and glucose H-l i n the 2D NOESY spectrum. For i d e n t i f i c a t i o n of known limonoid glucosides the H NMR spectrum i s s u f f i c i e n t . The p o s i t i o n s of the four or f i v e C-methyl s i g n a l s are s i g n i f i c a n t l y d i f f e r e n t f o r each known g l u c o s i d e . X

13

X

Occurrence Limonoid g l u c o s i d e s are one of the major secondary metabolites i n Citrus. These compounds are present i n f r u i t t i s s u e s and seeds i n h i g h concentrations (5,11,16). However, they are not present i n leaves, stems, and immature f r u i t


FLAVOR PRECURSORS


92

t i s s u e s and seeds (14). Commercial c i t r u s j u i c e s contain very high concentrations of limonoid glucosides (11). Analyses of 15 orange, 8 g r a p e f r u i t and 4 lemon j u i c e s showed that orange j u i c e contained the highest amounts, averaging 320 ppm of t o t a l limonoid glucosides, ranging from 250 to 430 ppm. Since orange j u i c e contains g e n e r a l l y 2 ppm of limonoid aglycones ( I ) , t h i s i s 125 to 215 f o l d h i g h e r than the aglycones. Grapefruit j u i c e averaged 190 ppm, ranging from 140 to 230 ppm. Lemon j u i c e contained the lowest l e v e l averaging 82 ppm, ranging 7 6 t o 93 ppm. Limonin g l u c o s i d e i s the major limonoid g l u c o s i d e i n commercial j u i c e s (11). Orange j u i c e contains an average of 180 ppm or 56% of the t o t a l . G r a p e f r u i t j u i c e averaged 54 ppm, which i s 63% of the t o t a l , while lemon j u i c e averages 54 ppm which i s 66% of the t o t a l . Analyses of i n d i v i d u a l limonoid glucosides i n orange j u i c e show that limonin g l u c o s i d e i s the predominant, followed by glucosides of n o m i l i n i c a c i d , n o m i l i n , d e a c e t y l n o m i l i n i c a c i d , deacetylnomilin and obacunone i n order of decreasing concentration (16). C i t r u s seeds a l s o c o n t a i n very high concentration of limonoid g l u c o s i d e s . In f a c t , these compounds comprise n e a r l y 1% of the f r e s h weight of g r a p e f r u i t seeds (2) . Ozaki et al.(5) determined the concentrations of i n d i v i d u a l limonoid glucosides i n the seeds of eight c i t r u s species. As shown i n Table 2, the t o t a l c o n c e n t r a t i o n of limonoid glucosides i n the seeds ranges from 0.33 to 0.89% and averages 0.61% of the dry weight. This concentration i s approximately 2 0 - f o l d higher than i n j u i c e . Table 2. Limonoid Glucosides i n C i t r u s Seeds* Seeds Fukuhara Hyuganatsu Sanbokan Shimamikan Grapefruit Lemon Valencia Tangerine

DAG

NG

NAG

0..28 0..42 0..37 0..48 0..75 0,.14 0..13 1..69

3. 22 1. 10 1. 13 1. 89 2. 01 1. 53 4. 48 0. 42

0..98 0..76 0..55 1..29 0..89 1..39 0,.98 0..96

0G 1..09 0..65 0..90 2..35 0..86 1,.49 1,.06 0..45

LG 0 .51

0 .51 0 .37 1 .48 1 .44 0 .59 0 .90

DG 1..32 0..37 0..89 0..69 0..68 0..55 1..69 0..93

Total 7 .40 . 3..31 4..36 7. .08 6..67 6..54 8,.94 5,.36

* Determined by HPLC, mg/g of dry seeds. G, g l u c o s i d e ; DA, d e a c e t y l n o m i l i n i c a c i d ; N, nomilin; 0, obacunone; L, limonin, D, deacetylnomilin (5) . Unlike f r u i t t i s s u e , where limonin glucoside predominates (11, 16), the c o n c e n t r a t i o n of limonin glucoside i s r e l a t i v e l y low i n the seeds (5). Nomilin glucoside i s the major g l u c o s i d e i n the m a j o r i t y of the seeds. P a r t i c u l a r l y , i n Fukuhara and V a l e n c i a orange, n o m i l i n g l u c o s i d e makes up 42 and 51% of the t o t a l limonoid g l u c o s i d e s , r e s p e c t i v e l y . The glucosides of obacunone and d e a c e t y l n o m i l i n are the major glucosides i n Shimamikan and tangerine, r e s p e c t i v e l y . C i t r u s seeds a r e e x c e l l e n t sources of both limonoid aglycones and g l u c o s i d e s . For example, the concentration of t o t a l limonoids i n g r a p e f r u i t seeds i s approximately 3% of the dry weight (2,5,17). The concentration of limonoid g l u c o s i d e s i n the seeds i s g e n e r a l l y lower than that of limonoid


8. HASEGAWA ET AL.


aglycones. On average, the r a t i o of t o t a l aglycones t o t o t a l glucosides i s approximately 2 t o 1. C i t r u s f r u i t p e e l s a l s o contain high concentrations of limonoid g l u c o s i d e s . T h e i r concentrations increase as f r u i t maturation progresses. Peels obtained from navel oranges grown i n C a l i f o r n i a and harvested on 11/2, 11/30/1987, 1/4, 2/6, 3/16 and 4/10/1988 contained 66, 202, 298 ,258, 286 and 362 ppm of limonin glucoside, r e s p e c t i v e l y (12). V a l e n c i a orange p e e l s harvested on 11/4/1988, 1/31, 3/10, 4/21, 6/22 and 7/28/1989 contained 186, 396, 426, 433, 523 and 733 ppm of t o t a l limonoid glucosides, r e s p e c t i v e l y (10).


B i o s y n t h e s i s of

Limonoid

Glucosides

As soon as limonoid g l u c o s i d e s were discovered, i t was p o s t u l a t e d that they were b i o s y n t h e s i z e d from t h e i r open D-ring limonoid precursors. This hypothesis was confirmed by r a d i o a c t i v e t r a c e r experiments i n which C - l a b e l l e d n o m i l i n was converted t o n o m i l i n 17-β-D-glucopyranoside i n lemon seeds and albedo (14) and i n navel orange albedo (18). Other t i s s u e s , such as leaves, stems, and immature f r u i t t i s s u e s and seeds e x h i b i t e d no c a p a c i t y t o b i o s y n t h e s i z e limonoid g l u c o s i d e s ( 1 4 ) . The concentration of limonoate A - r i n g lactone (LARL), which i s a precursor of limonin and i s the predominant limonoid i n c i t r u s f r u i t t i s s u e s , decreases as f r u i t maturation progresses (Figure 3) (12) . This compound has been shown t o convert t o other minor limonoids such as 17-dehydrοlimonoate A - r i n g lactone (19), deoxylimonin and deoxylimonic a c i d (20) and p o s s i b l y t o limonol and limonyl acetate. However, these compounds are very minor and alone cannot e x p l a i n the decrease of LARL during maturation. The discovery of limonin g l u c o s i d e and the formation of limonin glucoside f i n a l l y e x p l a i n how LARL disappears a t l a t e stages of f r u i t growth and maturation. Figure 3 shows t h a t i n navel oranges grown i n C a l i f o r n i a the i n i t i a t i o n and subsequent increase i n limonin g l u c o s i d e b i o s y n t h e s i s i s accompanied by a simultaneous decrease i n LARL content (12). During September when the f r u i t s were s t i l l green, limonin g l u c o s i d e began t o appear i n the f l e s h p o r t i o n , and the content increased sharply t h e r e a f t e r . The sudden increase i n the limonin g l u c o s i d e content of navel oranges during September simultaneous with a sudden decrease i n LARL confirmed that limonin glucoside i s formed by the g l u c o s i d a t i o n of LARL. Since limonin g l u c o s i d e i s n o n b i t t e r , the g l u c o s i d a t i o n of LARL t o form limonin g l u c o s i d e would be a n a t u r a l d e b i t t e r i n g process. The s t i m u l a t i o n of t h i s conversion through the use of p l a n t b i o r e g u l a t o r s or through g e n e t i c engineering could reduce the content of LARL and subsequently reduce the limonoid b i t t e r n e s s i n c i t r u s j u i c e s . As shown i n F i g u r e 3, the t o t a l limonin (LARL + limonin glucoside) content i n c r e a s e d sharply from June t o August, slowed down once i n September, increased sharply again during October and November, and remained f a i r l y constant t h e r e a f t e r . This showed unexpectedly that the b i o s y n t h e s i s of limonoids continues t o occur i n the f r u i t t i s s u e s u n t i l the end of November. I t i s of i n t e r e s t t o note that the t o t a l weight of the f r u i t increased a l s o u n t i l the end of November. Hasegawa et a l . (12) a l s o analyzed t o t a l limonoid glucosides and found that r a t i o s of limonin g l u c o s i d e t o t o t a l limonoid g l u c o s i d e s at d i f f e r e n t stages of f r u i t growth and maturation were f a i r l y i 4


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constant, 0.7. These data suggest that a l l limonoid aglycones are simultaneously converted t o t h e i r g l u c o s i d e s throughout l a t e stages of f r u i t growth and maturation.


80 H

6/2

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10/5

11/211 /30 1ΙΑ

2/6 3/16

4/10

Dates of Sampling

Figure 3. Changes i n the limonoate A - r i n g lactone (LARL) and limonin 17-β-D-glucopyranoside (LG) contents of navel oranges during f r u i t growth and maturation. LG values are expressed as LARL by m u l t i p l y i n g by 0.7 23 (12). The limonoids present i n seeds are most l i k e l y b i o s y n t h e s i z e d independently from the b i o s y n t h e s i s o c c u r r i n g i n f r u i t t i s s u e s . The composition of limonoid g l u c o s i d e s i n the seed i s q u i t e d i f f e r e n t from that present i n the f r u i t t i s s u e . For example, limonin g l u c o s i d e i s the predominant limonoid g l u c o s i d e i n the f r u i t t i s s u e (11,16), but i n the seed i t s concentration i s very low (5). Instead, n o m i l i n g l u c o s i d e i s the major g l u c o s i d e i n the seed. Seeds possess high concentrations of both limonoid aglycones and g l u c o s i d e s . The r a t i o of t o t a l aglycones to t o t a l glucosides i n the seed averages 2 to 1. In the j u i c e t h i s r a t i o i s about I to 150. In a d d i t i o n to the above d i f f e r e n c e s between seeds and f r u i t t i s s u e s , there are at l e a s t two more major biochemical d i f f e r e n c e s between them. F i r s t , the d i l a c t o n e s are the predominant aglycones i n the seed, whereas the monolactones are predominant i n the f r u i t t i s s u e . Secondly, during l a t e stages of f r u i t growth and maturation, the aglycone content i n the f r u i t t i s s u e decreases due t o i t s conversion to g l u c o s i d e s (12). However, t h i s decrease does not occur i n the seeds (10) since the predominant d i l a c t o n e form cannot be converted t o g l u c o s i d e s . These data s t r o n g l y suggest that limonoid and t h e i r g l u c o s i d e b i o s y n t h e s i s i n seeds and f r u i t t i s s u e s occurs independent l y . The enzyme r e s p o n s i b l e f o r the g l u c o s i d a t i o n of limonoids has been i d e n t i f i e d as limonoid UDP-D-glucose t r a n s f e r a s e (Figure 4) (13). The a c t i v i t y has been demonstrated i n c e l l f r e e e x t r a c t s of c i t r u s albedo and seed t i s s u e s . When Re l a b e l l e d nomilinoate A - r i n g lactone was incubated with c e l l f r e e e x t r a c t s of orange albedo, i t was converted to l a b e l l e d


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n o m i l i n 17-β-D-glucopyranoside. The crude enzyme preparations r e q u i r e d UDP-D-glucose as substrate(10). T h i s enzyme has y e t t o be i s o l a t e d .

Limonoate A - r i n g lactone

F i g u r e 4.

Limonin 17-β-D-glucopyranoside (Nonbitter)

Limonoid UDP-D-glucose

Transferase

Biodégradation of Limonoid Glucosides There i s no evidence that limonoid glucosides are converted back t o aglycones a f t e r they are formed i n the f r u i t (18). Radioactive t r a c e r work shows that C - l a b e l l e d n o m i l i n g l u c o s i d e was not metabolized i n albedo of navel oranges and lemons. However, during germination, the limonoid glucosides i n seeds appear t o be metabolized. A species of bacterium i s o l a t e d from s o i l by enrichment on limonin g l u c o s i d e as a s o l e carbon source possesses βglucosidase which e x h i b i t s a c t i v i t y on limonoid g l u c o s i d e s (2,8,10). The enzyme attacks a l l of the limonoid g l u c o s i d e s i s o l a t e d from the Rutaceae. No commercially a v a i l a b l e glucosidase i s a c t i v e on limonoid g l u c o s i d e s .

Chemical S t a b i l i t y of Limonoid Glucosides Most of the limonoid g l u c o s i d e s appear s t a b l e a t pH 2 t o pH 8, but t h i s has not been w e l l studied. There i s no s i g n i f i c a n t d i f f e r e n c e i n the t o t a l and i n d i v i d u a l limonoid g l u c o s i d e s between f r e s h l y prepared and concentrated orange j u i c e s (11). One known exception i s n o m i l i n glucoside, which converts t o n o m i l i n i c a c i d g l u c o s i d e below pH 3, and t o obacunone g l u c o s i d e at pH 7.5 and above (10) .

Biological A c t i v i t i e s of Limonoids Limonoids are a l s o being i n v e s t i g a t e d f o r p o s s i b l e a n t i - c a n c e r e f f e c t s . C e r t a i n limonoids have been found t o induce g l u t a t h i o n e S-transferase, a d e t o x i f y i n g enzyme, i n the l i v e r and small i n t e s t i n a l mucosa of mice and t o i n h i b i t benzo (a) pyrene-induced n e o p l a s i a i n forestomach of mice (2122). They a l s o i n h i b i t the formation of 7 ,12 - dimethylbenz[a]anthracene(DMBA)- induced b u c c a l pouch epidermoid carcinomas i n hamsters (23). More r e c e n t l y , limonin g l u c o s i d e has a l s o been found t o i n h i b i t tumor growth i n hamsters (24). Limonin glucoside e x h i b i t e d a s i g n i f i c a n t decrease i n average tumor burden i n hamster buccal pouch induced by DMBA. However, nomilin and


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n o m i l i n i c a c i d g l u c o s i d e s had no s i g n i f i c a n t e f f e c t s on the DMBA-induced o r a l c a r c i n o g e n e s i s . The b i o l o g i c a l f u n c t i o n of limonoids i n p l a n t s i s not y e t known. However, there i s considerable evidence that limonoids act as a n t i f e e d a n t s against a v a r i e t y of i n s e c t pests (25-31). Thus, limonoids may serve as a p r o t e c t i o n against i n s e c t damage.


Conclusion S i g n i f i c a n t progress has been made i n biochemical research on c i t r u s limonoids. We know now where, when, and how limonoids and t h e i r g l u c o s i d e d e r i v a t i v e s are biosynthesized, metabolized and accumulated i n Citrus. The d i s c o v e r y and formation of limonoid glucosides f i n a l l y e x p l a i n how limonoid aglycones, such as b i t t e r limonin, a r e metabolized and disappear i n the f r u i t during l a t e stages of f r u i t growth and maturation. The s t i m u l a t i o n of t h i s n a t u r a l d e b i t t e r i n g process by b i o r e g u l a t i o n c o u l d reduce the content of the precursors of b i t t e r limonoids and subsequently reduce the limonoid bitterness i n citrus juices. Limonoids have been shown t o have a n t i c a r c i n o g e n i c a c t i v i t y i n l a b o r a t o r y animals. Limonoid glucosides appear t o have the s i m i l a r a c t i v i t y . However, these i n v e s t i g a t i o n s a r e s t i l l a t p r e l i m i n a r y stages, and f u r t h e r research i s needed t o determine whether l e v e l s of limonoid aglycones and g l u c o s i d e s present i n c i t r u s f r u i t have s i g n i f i c a n t e f f e c t s as a n t i - c a n c e r d i e t a r y sources. On t h e other hand, c i t r u s seeds contain very high concentrations of both limonoid aglycones and glucosides, and a r e e x c e l l e n t sources of such compounds. Acknowledgment We thank Dr.Raymond Bennett f o r h e l p f u l d i s c u s s i o n s i n t h e p r e p a r a t i o n of t h i s manuscript.

Literature Cited 1. Maier, v. P.,; Bennett, R. D.; Hasegawa, S. In Citrus Science and Technology. Ed. Nagy, S.; Shaw, P. E.; Veldhuis, M. K. Avi Publishing Co., Westport, CT, 1977, Vol 2. 355-396. 2. Hasegawa, S.; Bennett, R.D.; Herman, Z., Fong, C.H.; Ou, P. Phytochemistry 1989, 28, 1717. 3. Bennett, R.D.; Hasegawa, S.; Herman, Ζ. Phytochemistry 1989, 28, 2777. 4. Maeda, H.; Ozaki, Y.; Miyake, M.; Ifuku, Y.; Hasegawa, S. Nippon Nogeikagaku Kaishi 1990, 64, 1231. 5. Ozaki, Y.; Fong, C.H.; Herman, Z.; Maeda, M.; Miyake, M.; Ifuku, Y.; Hasegawa, S. Agric. Biol. Chem. 1991, 55, 137. 6. Ozaki, Y.; Miyake, M.; Maeda, H.; Ifuku, Y.; Bennett, R.D.; Herman, Ζ.; Fong, C.H.a; Hasegawa, S. Phytochemistry 1991, 30, 2659. 7. Bennett, R. D.; Miyake, M.; Ozaki, Y.; Hasegawa, S. Phytochemistry 1991 (in press) 8. Miyake, M.; Ozaki, Y.;, Ayano, S.; Bennett, R. D.; Herman, Ζ.; Hasegawa, S. Phytochemistry 1991 (in Press) 9. Ozaki, Y.; Miyake, M.; Maeda, H.; Ifuku, Y.; Bennett, R.D.; Hasegawa, S. Phytochemistry 1991, 30, 2365.



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10. Hasegawa, S.; Miyake, M.; Fong, C.H. Unpublished data, Fruit and Vegetable chemistry Laboratory, USDA, ARS, Pasadena CA 91106 11. Fong, C.H.; Hasegawa, S.; Herman, Z.; Ou, P. J. Food Sci. 1990, 54, 1501. 12. Hasegawa, S.; Ou, P.; Fong, C.H.; Herman, Z.; Coggins, C.W., Jr.; Atkin, D.R. J. Agric. Food Chem. 1991, 39, 262. 13. Herman, Z.; Fong, C.H.; Hasegawa, S. Coggins, C.W.,Jr.; Atkin, D.R. Abstract, Citrus Research Conference, Pasadena, CA., 1990, P. 8. 14. Fong, C.H.; Hasegawa, S.; Herman, Z.; Ou, P. J. Sci. Food Agric. 1991, 54, 393. 15. Maier, V.P.; Beverly, G.D. J. Food Sci. 1968, 33, 488. 16. Herman, Z.; Fong, C.H.; Ou, P.; Hasegawa, S. J. Agric. Food Chem. 1990, 38, 1860. 17. Hasegawa, S.; Bennett, R. D.; Verdon, C.P. J. Agric. Food Chem. 1980, 28, 922. 18. Herman, Z.; Fong, C.H.; Hasegawa, S. Phytochemistry 1991, 30, 1487. 19. Hasegawa,S.; Maier, V. P. and Bennett,R. D. Phytochemistry 1980, 13, 103. 20. Hasegawa, S.; Bennett, R. D.; Verdon,C. P. Phytochemistry 1974,19,1445. 21. Lam, L.K.T.; Hasegawa, S. Nutrition and Cancer, 1989, 12,43. 22. Lam, L.K.T.; Li, Y.; Hasegawa, S. J. Agric. Food Chem., 1989, 37, 878. 23. Miller, E.G.; Fanous, R.; Rivera-Hidalgo, F.; Binnie,; Hasegawa, S.; Lam, L.k.T. Carcinogenesis, 1989, 10, 1535. 24. Miller, E.G., Gonzales-Sanders, A.P.; Couvillon, Α. Μ., Wright, J. M.; Hasegawa, S.; Lam, L.K.T. Nutrition and Cancer 1992 (in press) 25. Klocke, J. Α.; Kubo,I. Ent. Exp. & Appl. 1982, 32,299. 26. Alford, A. R.; Bentley, M.D. J. Econ. Entomol. 1986,79,35. 27. Alford, A.R.; Cullen, J.A.; Storch, R.H.; Bentley, M.D.J.Econ.Entomol. 1987, 80, 575. 28. Arnason, J.T.; Philogene, B.J.R.; Donskov, D.; Kubo, I Ent. Exp. & Appl. 1987,43:221. 29. Bentley, M.D.; Rajab, M.S.; Alford, A.R.; Mendel, M.J.; Hassanali, A. Ent. Exp. & Appl. 1989, 49, 189. 30. Bentley, M.D.; Rajab, M.S. Mendel, M.J.; Alford, A.R. J. Agric. Food Chem. 1990, 38, 1400. 31. Liu, Y.B.; Alford, A.R.; Rajab,R.S.; Bentley, M.D. Physiol. Entomol. 1990, 15, 37. RECEIVED December 18, 1991


Glucosides of Limonoids - American Chemical Society

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