Plant Cell Reports
Plant Cell Reports (1992) 11:592-596
9 Springer-Verlag1992
Bioproduction of neohesperidin and naringin in callus cultures of Citrus aurantium J.A. del Rio, A. Ortufio, E R. Marin, D. Garcia Puig, and E Sabater Department of Plant Biology (Plant Physiology), University of Mureia, Espinardo, E-30071 Murcia, Spain Received February 19, 1992/Revised version received July 9, 1992 - Communicated by N. Amrhein
Abstract The a c c u m u l a t i o n o f both neohesperidin and n a r i n g i n as major flavonoids in callus c u l t u r e s o f b i t t e r orange ( C ~ r u s ~ur~n6s was d e m o n s t r a t e d u s i n g high performance liquid chromatography w i t h a diode-array d e t e c t o r . The i d e n t i t y o f b o t h compounds was confirmed by their corresponding nuclear m a g n e t i c resonance s p e c t r a . The levels of neohesperidin are higher than those of n a r i n g i n i n c a l l u s c u l t u r e , as they are in immature f r u i t , and h i g h concentrations of both are found in young tissues such as immature f r u i t s and t h e o u t e r zone o f c a l l i . Abbreu~o~s: DMSO, d i m e t h y l s u l p h o x i d e ; DW, dry weight; HPLC, high performance l i q u i d chromatography; NMR, nuclear magnetic resonance; Rt, retention time; V/UV, visible/ultraviolet.
Introduction F l a v o n o i d s c o n s t i t u t e an i m p o r t a n t group of plant s e c o n d a r y m e t a b o l i t e s which have r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i n accordance w i t h t h e i r p h y s i o l o g i c a l r o l e i n p l a n t growth (Furuya e t a l . 1962; S t e n l i n d 1963; Harborne 1967; Jacobs and Rubery 1988), their p h a r m a c o l o g i c a l e f f e c t s (B6hm 1959) and t h e i r organoleptic properties. The genus CZtr~s produces coumarins, flavanones, f l a v o n e s and flavonols which occur i n the f r e e form a n d / o r as g l y c o s i d e s (Mater and M e t z l e r 1967; H o r o w i t z and G e n t i l i 1977). Neohesperidin, the 7-~-neohesperidoside of hesperetin (3',5,7 t r i h y d r o x y - 4 - m e t h o x y f l a v a n o n e ) and n a r i n g i n , the 7-~-neohesperidoside of naringenin (4',5,7 trihydroxy flavanone) are the predominant f l a v o n o i d s i n the bitter orange (CZtr~c~ a~r~m) (Nakabayashi 1961; H o r o w i t z and G e n t i l i 1977; Kamiya e t al. 1979). These compounds have been found in f l o w e r s , l e a v e s and f r u i t s (Jourdan e t al. 1985; C a s t i l l o e t a l . 1992). E s p e c i a l l y high concentrations of naringin were found in g r a p e f r u i t and n e o h e s p e r i d i n and n a r i n g i n in b i t t e r orange ( R o u s e f f e t al. 1987). These l e v e l s were a s s o c i a t e d w i t h young d e v e l o p i n g s t a t e s i n l e a v e s (Berhow and Vandercook 1991;
Correspondence to." J. A. del Rio
Mclntosh and M a n s e l l 1983) and l e a v e s and fruits ( C a s t i l l o e t a l . 1992), r e s p e c t i v e l y . B e a r i n g i n mind t h a t n e o h e s p e r i d i n and n a r i n g i n b i o s y n t h e s i s i s o f i n t e r e s t as these f l a v o n o i d s can be c h e m i c a l l y c o n v e r t e d i n t o artificial sweeteners (Krbecheck et al. 1968), the use o f plant cell and tissue c u l t u r e s i s an i m p o r t a n t a l t e r n a t i v e to the normal p r o c e d u r e f o r the i s o l a t i o n of these compounds from young f r u i t s . Although the a b i l i t y o f C~tr~s cultures to biotransform f l a v a n o n e s (Lewinsohn e t a l . 1986, 1989) or accumulate f l a v o n o i d s (Brunet and Ibrahim 1973; B a r t h e e t a l . 1987; Gavish e t a l . 1989; Lewinsohn e t al. 1989; Vandercook and T i s s e r a t 1989) has been d e s c r i b e d , the most s u i t a b l e procedures f o r t h e i r identification and q u a n t i f i c a t i o n have not always been followed. In the present paper, we use the r e s o l v i n g power o f high performance liquid c h r o m a t o g r a p h y (HPLC) t o g e t h e r w i t h nuclear m a g n e t i c resonance (NMR) as an i d e n t i f i c a t i o n procedure to d e t e r m i n e the production of n e o h e s p e r i d i n and n a r i n g i n i n c a l l u s c u l t u r e s from C ~ r ~ s ~ r ~ t ~ m ~ ; the c o r r e s p o n d i n g c e l l m o r p h o l o g i c a l changes a s s o c i a t e d w i t h the e x p r e s s i o n o f these compounds a r e a n a l y s e d . Materials
and m e t h o d s
m~zter~u~ and c u ~ u s c ~ t u r e . Fruits of C~tr~ auruntZ~ cv. Sevillano (10-12 mm d i a m e t e r ) were o b t a i n e d from 5 - y e a r - o l d t r e e s grown a t t h e University of Murcia. Fruits were s u r f a c e s t e r i l i z e d by t r e a t m e n t w i t h 2X ( w / v ) sodium h y p o c h l o r i t e f o r I 0 min and then rinsed repeatedly with sterile water. For initiation of callus culture, these fruits were b i s e c t e d e q u a t o r i a l l y and p l a c e d w i t h the cut s u r f a c e on Murashige and Tucker ( t g b 9 ) b a s a l agar medium m o d i f i e d as f o l l o w s : 6 ~M k i n e t i n , 3 ~M 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d and 10% coconut m i l k . A pH o f 5.8, s u c r o s e (30 g / l ) and agar (9 g / l ) were used i n a l l media. The c o c o n u t m i l k was b o i l e d and f i l t e r e d b e f o r e use. The c a l l u s c u l t u r e s were c u l t u r e d under a 16 hr l i g h t / 8 hr dark regime a t 24 QC and S u b c u l t u r e d a t 4-week i n t e r v a l s . I n all the analyses, calli were s u b c u l t u r e d 8 times b e f o r e use and t h e s a m p l i n g t o o k p l a c e one P~ant
593 week a f t e r
E•163
t h e 8 th s u b c u l t u r e .
~n.
Plant Cell Reports (1992) 11:592-596
9 Springer-Verlag1992
Bioproduction of neohesperidin and naringin in callus cultures of Citrus aurantium J.A. del Rio, A. Ortufio, E R. Marin, D. Garcia Puig, and E Sabater Department of Plant Biology (Plant Physiology), University of Mureia, Espinardo, E-30071 Murcia, Spain Received February 19, 1992/Revised version received July 9, 1992 - Communicated by N. Amrhein
Abstract The a c c u m u l a t i o n o f both neohesperidin and n a r i n g i n as major flavonoids in callus c u l t u r e s o f b i t t e r orange ( C ~ r u s ~ur~n6s was d e m o n s t r a t e d u s i n g high performance liquid chromatography w i t h a diode-array d e t e c t o r . The i d e n t i t y o f b o t h compounds was confirmed by their corresponding nuclear m a g n e t i c resonance s p e c t r a . The levels of neohesperidin are higher than those of n a r i n g i n i n c a l l u s c u l t u r e , as they are in immature f r u i t , and h i g h concentrations of both are found in young tissues such as immature f r u i t s and t h e o u t e r zone o f c a l l i . Abbreu~o~s: DMSO, d i m e t h y l s u l p h o x i d e ; DW, dry weight; HPLC, high performance l i q u i d chromatography; NMR, nuclear magnetic resonance; Rt, retention time; V/UV, visible/ultraviolet.
Introduction F l a v o n o i d s c o n s t i t u t e an i m p o r t a n t group of plant s e c o n d a r y m e t a b o l i t e s which have r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i n accordance w i t h t h e i r p h y s i o l o g i c a l r o l e i n p l a n t growth (Furuya e t a l . 1962; S t e n l i n d 1963; Harborne 1967; Jacobs and Rubery 1988), their p h a r m a c o l o g i c a l e f f e c t s (B6hm 1959) and t h e i r organoleptic properties. The genus CZtr~s produces coumarins, flavanones, f l a v o n e s and flavonols which occur i n the f r e e form a n d / o r as g l y c o s i d e s (Mater and M e t z l e r 1967; H o r o w i t z and G e n t i l i 1977). Neohesperidin, the 7-~-neohesperidoside of hesperetin (3',5,7 t r i h y d r o x y - 4 - m e t h o x y f l a v a n o n e ) and n a r i n g i n , the 7-~-neohesperidoside of naringenin (4',5,7 trihydroxy flavanone) are the predominant f l a v o n o i d s i n the bitter orange (CZtr~c~ a~r~m) (Nakabayashi 1961; H o r o w i t z and G e n t i l i 1977; Kamiya e t al. 1979). These compounds have been found in f l o w e r s , l e a v e s and f r u i t s (Jourdan e t al. 1985; C a s t i l l o e t a l . 1992). E s p e c i a l l y high concentrations of naringin were found in g r a p e f r u i t and n e o h e s p e r i d i n and n a r i n g i n in b i t t e r orange ( R o u s e f f e t al. 1987). These l e v e l s were a s s o c i a t e d w i t h young d e v e l o p i n g s t a t e s i n l e a v e s (Berhow and Vandercook 1991;
Correspondence to." J. A. del Rio
Mclntosh and M a n s e l l 1983) and l e a v e s and fruits ( C a s t i l l o e t a l . 1992), r e s p e c t i v e l y . B e a r i n g i n mind t h a t n e o h e s p e r i d i n and n a r i n g i n b i o s y n t h e s i s i s o f i n t e r e s t as these f l a v o n o i d s can be c h e m i c a l l y c o n v e r t e d i n t o artificial sweeteners (Krbecheck et al. 1968), the use o f plant cell and tissue c u l t u r e s i s an i m p o r t a n t a l t e r n a t i v e to the normal p r o c e d u r e f o r the i s o l a t i o n of these compounds from young f r u i t s . Although the a b i l i t y o f C~tr~s cultures to biotransform f l a v a n o n e s (Lewinsohn e t a l . 1986, 1989) or accumulate f l a v o n o i d s (Brunet and Ibrahim 1973; B a r t h e e t a l . 1987; Gavish e t a l . 1989; Lewinsohn e t al. 1989; Vandercook and T i s s e r a t 1989) has been d e s c r i b e d , the most s u i t a b l e procedures f o r t h e i r identification and q u a n t i f i c a t i o n have not always been followed. In the present paper, we use the r e s o l v i n g power o f high performance liquid c h r o m a t o g r a p h y (HPLC) t o g e t h e r w i t h nuclear m a g n e t i c resonance (NMR) as an i d e n t i f i c a t i o n procedure to d e t e r m i n e the production of n e o h e s p e r i d i n and n a r i n g i n i n c a l l u s c u l t u r e s from C ~ r ~ s ~ r ~ t ~ m ~ ; the c o r r e s p o n d i n g c e l l m o r p h o l o g i c a l changes a s s o c i a t e d w i t h the e x p r e s s i o n o f these compounds a r e a n a l y s e d . Materials
and m e t h o d s
m~zter~u~ and c u ~ u s c ~ t u r e . Fruits of C~tr~ auruntZ~ cv. Sevillano (10-12 mm d i a m e t e r ) were o b t a i n e d from 5 - y e a r - o l d t r e e s grown a t t h e University of Murcia. Fruits were s u r f a c e s t e r i l i z e d by t r e a t m e n t w i t h 2X ( w / v ) sodium h y p o c h l o r i t e f o r I 0 min and then rinsed repeatedly with sterile water. For initiation of callus culture, these fruits were b i s e c t e d e q u a t o r i a l l y and p l a c e d w i t h the cut s u r f a c e on Murashige and Tucker ( t g b 9 ) b a s a l agar medium m o d i f i e d as f o l l o w s : 6 ~M k i n e t i n , 3 ~M 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d and 10% coconut m i l k . A pH o f 5.8, s u c r o s e (30 g / l ) and agar (9 g / l ) were used i n a l l media. The c o c o n u t m i l k was b o i l e d and f i l t e r e d b e f o r e use. The c a l l u s c u l t u r e s were c u l t u r e d under a 16 hr l i g h t / 8 hr dark regime a t 24 QC and S u b c u l t u r e d a t 4-week i n t e r v a l s . I n all the analyses, calli were s u b c u l t u r e d 8 times b e f o r e use and t h e s a m p l i n g t o o k p l a c e one P~ant
593 week a f t e r
E•163
t h e 8 th s u b c u l t u r e .
~n.
