- Email: [email protected]
Flavanol production by Fagopyrum esculentum hairy and normal root cultures
Phytochemistry, Vol. 32, No. 4. pp. 929 931, 1993 Pnnted in Great Britam.
FLAVANOL
0031-9422/93 $6.00+0.00 (cj 1993 Pergamon Press Ltd
PRODUCTION BY FAGOPYRUM ESCULENTUM NORMAL ROOT CULTURES
F.
TROTIN,
Y.
MOUMOU* and J.
HAIRY AND
VASSEUR*
Laboratoire de Pharmacognosie, Faculti de Pharmacie, B. P. 83, 59006 Lille Cedex, France; *Laboratoire de Physiologie cellulaire et Morphogen&e Veg&ales, Universite des Sciences et Technologies de Lille I, 59655 Villeneuve d’Ascq Cedex, France (Receioed 15 June 1992)
Key Word Index-Fagopyrum (-)-epicatechin-3-0-gallate; hairy root cultures.
esculentum; Polygonaceae; procyanidin B2; procyanidin
flavanols; ( +)-catechin; (- )-epicatechin; B2-3’-0-gallate; polyphenols; normal and
Abstract-Hairy roots culture of Fagopyrum esculentum were established by infection with Agrobacterium rhizogenes strain 15834. Faster growth (four-fold higher than normal roots culture) in B5 liquid medium was observed and the synthesis of five flavanols obtained : ( +)-catechin, ( -)-epicatechin, (-)-epicatechin-3-0-gallate, procyanidin B2 and procyanidin BZ3’-0-gallate also present in normal roots culture with a common prominency of epicatechin-3-0gallate synthesis and inversion in the contents rates of (+)-catechin and (-tepicatechin. The highest content of procyanidin B2-3’-0-gallate was obtained in hairy roots which can be considered as a better production source for flavanols, especially of the two galloylated derivatives.
INTRODUCTION
value. Comparatively, the maximal growth value of normal roots is 1%fold of that of the initial one (180 mg dry wt per flask). Histochemical staining with vanillin-HCl [ 1 l] indicated accumulation of flavanols in the cultured root tips. The qualitative flavanol composition was similar in both normal and hairy roots, with (+)-catechin, (-)epicatechin, (-)-epicatechin-3-0-gallate, procyanidin B2 and procyanidin BZ3’-0-gallate (Fig. 2). These compounds were also detected in hypocotyl calli [3, 43. Nevertheless, the respective contents (expressed in mg substances showed some g - ’ dry wt) for individual differences in the two types of cultures. A common factor in normal and hairy root cultures is the prominency of (-)-epicatechin-3-O-gallate (10.5 mg g- ’ dry wt in normal roots and 10.1 mg g-l dry wt in hairy roots) and the low content in procyanidin B2 (0.8 mg g- ’ dry wt in both normal and hairy roots). For other main components, there were differences between the two cultures. In normal root cultures, contents were in decreasing order: (-)epicatechin (6,4 mg g- 1 dry wt), > procyanidin B2-3’-Ogallate (4.2 mg g- ’ dry wt) >( +)-catechin (3.6 mg g-’ dry wt). For hairy root cultures, the respective profile was procyanidin B2-3’-0-gallate (8.6 mg g- ’ dry wt) > ( +)catechin (7.8 mg g- ’ dry wt) > (-)-epicatechin (2.1 mg g- ’ dry wt). Thus, hairy root cultures seem to be characterized by a preferential synthesis of galloylated derivatives of the monomeric and dimeric classes and by inversion of the content rates of catechin and epicatechin. As the growth yield was ca four-fold higher in hairy roots than in normal roots, the flavanol productions in
Selected callus cultures produced from hypocotyls of Fagopyrum esculentum produce noticeable amounts of flavanols including catechins and procyanidins [l-4], these compounds not being detected in the original plant material. Enhanced production could then be obtained under suitable culture conditions. Flavanols are constituents of condensed tannins whose medicinal traditional uses include haemostatic, astringent and vasculoprotective effects [S]. Recent studies indio ated new interests for catechins as antitumour agents [6], antiviral potencies for procyanidins [7], and for both classes, especially in their galloyl ester forms, antioxidant and radical scavenging effects [8]. Aiming to obtain enhanced flavanol contents in F. esculentum tissue cultures, we report the establishment of normal and hairy root cultures, their growth and flavanol production and a comparison with callus and plant organs characteristics. Hairy root have already been described for producing increased amounts of secondary metabolites such as tropane alkaloids in Duboisia [9] and Hyoscyamus [lo]. RESULTS AND DISCUSSION
Hairy roots derived from hypocotyls after induction by Agrobacterium rhizogenes strain 15834 and maintained over six months culture in BS liquid medium, showed more reproducible and faster growth kinetics than normal roots cultures (Fig. 1). The growth of hairy roots reached a maximum (720 mg dry wt per flask) after 21 days with an increase of ca 70-fold of that of the initial 929
F.
TROTIN~~ al.
mg per flask have been cotnpared
in Fig. 3, showing that transformed roots are a better source of Ravanols. The values for ( + )-catechin were ca eight-fold higher (5.4 vs 0.7 mg per flask), those for ( -)-epicatechin-3-0-gallate
0
IO
20 30 Time (days)
40
SO
Fig. 1. Growth of Fagopyrum hairy (-U-) and normal roots culture (-- W- ) on a hormone-free BS liquid medium containing 30 g I_ ’ sucrose.
four-fold higher (7.2 vs 1.9 mg per flask), those for procyanidin BZ-3’-0-gallate eight-fold higher (5.9 vs 0.7 mg per flask), whereas the production of other Aavanols remained about the same in both types of cultures. In compa~son with previous results obtained in optimized callus cultures [2-41, in vitro flavanol synthesis in both calli and hairy root cultures are important, especially for the galloylated monomeric and dimeric derivatives, with an apparently slight advantage for the last tissue. Nevertheless, with a IO-fold higher growth factor for hairy roots than calli (hairy roots: 10 mg to ca 700 mg and calli 10 mg to ca 70mg, within 21 days) hairy roots should be considered as a better production source. The results obtained in this study indicate that the hairy root cultures of F. esculentum transformed with A. rhizogenes strain 15834 are a useful tissue for the production of catechins and procyanidins, with a higher productivity than in plant roots, in aitro normal root cultures and calli, especially for galloylated monomeric and dimeric flavanols. EXPERIMENTAL Plant material. Seeds of F. ~~~~~ent~rnMoench were sterilized by immersion in a soln of Ca(OCl),( 120 g l- ‘), foliowed by prolonged rinsing in sterile H,O. Aseptic germination was obtained on Heller agar medium [12] containing 1% sucrose, at 22”, under 18:6 light+Iark
A
0 0
7
14 21 28 Ttme (days)
35
42
7
14 21 28 Time (days)
34
42
Fig. 2. Changes (mg g-’ dry wt) in the formation of (+)catechin (W), (-)-epicatechin (a), (-)-epicatechin-3-O-gallate (q KI), procyamdin B2 (F.C-i) and procyanidin BZ-3’-0-gallate (Cd)by Fagopyrum hairy roots (A) and normal root cultures (B) during the growth cycle m darkness, on a hormone-free BS liquid medium contaimng 30 g I-’ sucrose.
0
7
14 21 28 Time (days)
35
42
Fig. 3. Comparison of flavanol production (mg per flask) in hairy roots (A) and normal root cultures (B) of Fagopyrum during the growth cycle m darkness, on a hormone-free B5 hquid medium containing 30 g I-’ sucrose. [(+)-catechm (m), (-j epicatechin (@), (- )~picatechin-3-0-gallate (,a), procyanidin B2 (0) and procyanidin B2-3’-O-gallate (RI)].
931
Flavanols from Fagopyrum esculentum
period (fluorescent tubes, 50 pmol, m-2sec- ‘). Aseptic plantlets were grown for 20 days. Normal roots were excised and cultured in hormone-free B5 solid medium [13] with 3% sucrose. Hairy roots were induced by coculture of either limb or hypocotyl fragments with A. rhizogenes strain 15834. Roots appeared at the inoculation site after 2 weeks. They were then transferred onto hormone-free B5 solid medium containing carbenicillin (0.3 g 1- I, Sigma) to eliminate bacteria. After two subcultures, axenic hairy roots (25 lines obtained) were excised and individually subcultured on hormone-free B5 liquid medium containing 30 g I- 1 sucrose without antibiotic. To prove transformation, opines (agropine and mannopine) were extracted and detected by paper electrophoresis [14]. All tested lines were transformed and presented the same flavanol content. The hypocotylderived hairy roots showing the fastest growth were used for further expts. Both normal and hairy roots (ca 100 mg fr. wt) were maintained in 120 ml flasks in hormone-free B5 liquid medium with 30 g l- 1 sucrose (70 ml per flask), in the dark at 22” on a rotatory shaker at 100 rpm. Three flasks were used for each expt. Calli were grown under the optimised conditions previously described [2]. Sample preparation and analysis. Frozen plant organs (field-grown roots from 3-month-old mature plants, hypocotyls), normal or hairy roots or calli (5 g fr. wt) were extracted with MeOH (3 x 50 ml). The extract was titered and 20 ml dist. H,O added. MeOH was evapd under red. prcs. and the aq. residue partitioned with EtOAc (4 x 20 ml). The EtOAc layer was evapd to dryness and dissolved in MeOH (50 ml). HPLC analysis was performed as previously described [4] on Lichrosorb RP18 (Merck), using standards :( + )-catechin, ( - )-epicatechin (Extrasynthese), and (-)-epicatechin-3-0-gallate, procyanidin B2, procyanidin BZ3’-0-gallate already isolated from F. esculentum callus cultures and characterized by us c3,41.
REFERENCES
1. Moumou, Y., Trotin, F., Pinkas, M., Dubois, J. and Vasseur J. (1987) Ann. Pharm. Fr. 45, 255. 2. Moumou, Y., Trotin, F., Dubois, J., Vasseur J. and El-Boustani, E. (1992) J. Nat. Prod. 55, 33. 3. Moumou, Y., Vasseur J., Trotin, F. and Dubois J. (1992) Phytochemistry 31, 1239. 4. Moumou Y., Trotin F., Vasseur J., Vermeersch G., Guyon, R., Dubois, J. and Pinkas, M. (1993) Planta Med. in press. 5. Bruneton, J. (1987) EZ&nents de Phytochimie et de Pharmacognosie. Technique et Documentation, Paris. 6. Yoshizawa, S., Horiuchi, T., Fujiki, H., Yoshida, T., Okuda, T. and Sugimura, T. (1987) Phytother. Res. 1, 44. 7. Kakiuchi, N., Kusumoto, I. T., Hattori, M., Namba, T., Hatano, T. and Okuda, T. (1991) Phytother. Res., 5, 270. 8. Hatano, T., Edamatsu, R., Hiramatsu,
M., Mori, A., Fujita, Y., Yasuhara, T., Yoshida, T. and Okuda, T. (1989) Chem. Pharm. Bull. 37, 2016. 9. Deno, H., Yamagata, H., Emoto, T., Yoshioka, T., Yamada, Y. and Fujita, Y. (1987) J. Plant Pkysiol. 131, 315. 10. Sauerwein, M. and Shimomura, K. (1991) Phytochemistry 10, 3277. 11. Chalker-Scott, L., and Krhamer, R. L. (1989) in Chemistry and Signijcance
of Condensed
Tannins
(Hemingway, R. W., and Karchesy, J. J., eds), p. 345. Plenum Press, London. 12. Heller, R. (1953) Ann. Sci. Nat. Bot. Viol. Vkg. 14, 1. 13. Gamborg, U. L., Miller, R. A. and Ujima, K. (1968) Exp. Cell. Res. 50, 151. 14. Petit, A., David, C., Dahl, G. A.: Ellis, J. G., Guyon, P., Casse-Delbart, F. and Tempe, J. (1983) Mol. Gen. Genet. 190,204.
FLAVANOL
0031-9422/93 $6.00+0.00 (cj 1993 Pergamon Press Ltd
PRODUCTION BY FAGOPYRUM ESCULENTUM NORMAL ROOT CULTURES
F.
TROTIN,
Y.
MOUMOU* and J.
HAIRY AND
VASSEUR*
Laboratoire de Pharmacognosie, Faculti de Pharmacie, B. P. 83, 59006 Lille Cedex, France; *Laboratoire de Physiologie cellulaire et Morphogen&e Veg&ales, Universite des Sciences et Technologies de Lille I, 59655 Villeneuve d’Ascq Cedex, France (Receioed 15 June 1992)
Key Word Index-Fagopyrum (-)-epicatechin-3-0-gallate; hairy root cultures.
esculentum; Polygonaceae; procyanidin B2; procyanidin
flavanols; ( +)-catechin; (- )-epicatechin; B2-3’-0-gallate; polyphenols; normal and
Abstract-Hairy roots culture of Fagopyrum esculentum were established by infection with Agrobacterium rhizogenes strain 15834. Faster growth (four-fold higher than normal roots culture) in B5 liquid medium was observed and the synthesis of five flavanols obtained : ( +)-catechin, ( -)-epicatechin, (-)-epicatechin-3-0-gallate, procyanidin B2 and procyanidin BZ3’-0-gallate also present in normal roots culture with a common prominency of epicatechin-3-0gallate synthesis and inversion in the contents rates of (+)-catechin and (-tepicatechin. The highest content of procyanidin B2-3’-0-gallate was obtained in hairy roots which can be considered as a better production source for flavanols, especially of the two galloylated derivatives.
INTRODUCTION
value. Comparatively, the maximal growth value of normal roots is 1%fold of that of the initial one (180 mg dry wt per flask). Histochemical staining with vanillin-HCl [ 1 l] indicated accumulation of flavanols in the cultured root tips. The qualitative flavanol composition was similar in both normal and hairy roots, with (+)-catechin, (-)epicatechin, (-)-epicatechin-3-0-gallate, procyanidin B2 and procyanidin BZ3’-0-gallate (Fig. 2). These compounds were also detected in hypocotyl calli [3, 43. Nevertheless, the respective contents (expressed in mg substances showed some g - ’ dry wt) for individual differences in the two types of cultures. A common factor in normal and hairy root cultures is the prominency of (-)-epicatechin-3-O-gallate (10.5 mg g- ’ dry wt in normal roots and 10.1 mg g-l dry wt in hairy roots) and the low content in procyanidin B2 (0.8 mg g- ’ dry wt in both normal and hairy roots). For other main components, there were differences between the two cultures. In normal root cultures, contents were in decreasing order: (-)epicatechin (6,4 mg g- 1 dry wt), > procyanidin B2-3’-Ogallate (4.2 mg g- ’ dry wt) >( +)-catechin (3.6 mg g-’ dry wt). For hairy root cultures, the respective profile was procyanidin B2-3’-0-gallate (8.6 mg g- ’ dry wt) > ( +)catechin (7.8 mg g- ’ dry wt) > (-)-epicatechin (2.1 mg g- ’ dry wt). Thus, hairy root cultures seem to be characterized by a preferential synthesis of galloylated derivatives of the monomeric and dimeric classes and by inversion of the content rates of catechin and epicatechin. As the growth yield was ca four-fold higher in hairy roots than in normal roots, the flavanol productions in
Selected callus cultures produced from hypocotyls of Fagopyrum esculentum produce noticeable amounts of flavanols including catechins and procyanidins [l-4], these compounds not being detected in the original plant material. Enhanced production could then be obtained under suitable culture conditions. Flavanols are constituents of condensed tannins whose medicinal traditional uses include haemostatic, astringent and vasculoprotective effects [S]. Recent studies indio ated new interests for catechins as antitumour agents [6], antiviral potencies for procyanidins [7], and for both classes, especially in their galloyl ester forms, antioxidant and radical scavenging effects [8]. Aiming to obtain enhanced flavanol contents in F. esculentum tissue cultures, we report the establishment of normal and hairy root cultures, their growth and flavanol production and a comparison with callus and plant organs characteristics. Hairy root have already been described for producing increased amounts of secondary metabolites such as tropane alkaloids in Duboisia [9] and Hyoscyamus [lo]. RESULTS AND DISCUSSION
Hairy roots derived from hypocotyls after induction by Agrobacterium rhizogenes strain 15834 and maintained over six months culture in BS liquid medium, showed more reproducible and faster growth kinetics than normal roots cultures (Fig. 1). The growth of hairy roots reached a maximum (720 mg dry wt per flask) after 21 days with an increase of ca 70-fold of that of the initial 929
F.
TROTIN~~ al.
mg per flask have been cotnpared
in Fig. 3, showing that transformed roots are a better source of Ravanols. The values for ( + )-catechin were ca eight-fold higher (5.4 vs 0.7 mg per flask), those for ( -)-epicatechin-3-0-gallate
0
IO
20 30 Time (days)
40
SO
Fig. 1. Growth of Fagopyrum hairy (-U-) and normal roots culture (-- W- ) on a hormone-free BS liquid medium containing 30 g I_ ’ sucrose.
four-fold higher (7.2 vs 1.9 mg per flask), those for procyanidin BZ-3’-0-gallate eight-fold higher (5.9 vs 0.7 mg per flask), whereas the production of other Aavanols remained about the same in both types of cultures. In compa~son with previous results obtained in optimized callus cultures [2-41, in vitro flavanol synthesis in both calli and hairy root cultures are important, especially for the galloylated monomeric and dimeric derivatives, with an apparently slight advantage for the last tissue. Nevertheless, with a IO-fold higher growth factor for hairy roots than calli (hairy roots: 10 mg to ca 700 mg and calli 10 mg to ca 70mg, within 21 days) hairy roots should be considered as a better production source. The results obtained in this study indicate that the hairy root cultures of F. esculentum transformed with A. rhizogenes strain 15834 are a useful tissue for the production of catechins and procyanidins, with a higher productivity than in plant roots, in aitro normal root cultures and calli, especially for galloylated monomeric and dimeric flavanols. EXPERIMENTAL Plant material. Seeds of F. ~~~~~ent~rnMoench were sterilized by immersion in a soln of Ca(OCl),( 120 g l- ‘), foliowed by prolonged rinsing in sterile H,O. Aseptic germination was obtained on Heller agar medium [12] containing 1% sucrose, at 22”, under 18:6 light+Iark
A
0 0
7
14 21 28 Ttme (days)
35
42
7
14 21 28 Time (days)
34
42
Fig. 2. Changes (mg g-’ dry wt) in the formation of (+)catechin (W), (-)-epicatechin (a), (-)-epicatechin-3-O-gallate (q KI), procyamdin B2 (F.C-i) and procyanidin BZ-3’-0-gallate (Cd)by Fagopyrum hairy roots (A) and normal root cultures (B) during the growth cycle m darkness, on a hormone-free BS liquid medium contaimng 30 g I-’ sucrose.
0
7
14 21 28 Time (days)
35
42
Fig. 3. Comparison of flavanol production (mg per flask) in hairy roots (A) and normal root cultures (B) of Fagopyrum during the growth cycle m darkness, on a hormone-free B5 hquid medium containing 30 g I-’ sucrose. [(+)-catechm (m), (-j epicatechin (@), (- )~picatechin-3-0-gallate (,a), procyanidin B2 (0) and procyanidin B2-3’-O-gallate (RI)].
931
Flavanols from Fagopyrum esculentum
period (fluorescent tubes, 50 pmol, m-2sec- ‘). Aseptic plantlets were grown for 20 days. Normal roots were excised and cultured in hormone-free B5 solid medium [13] with 3% sucrose. Hairy roots were induced by coculture of either limb or hypocotyl fragments with A. rhizogenes strain 15834. Roots appeared at the inoculation site after 2 weeks. They were then transferred onto hormone-free B5 solid medium containing carbenicillin (0.3 g 1- I, Sigma) to eliminate bacteria. After two subcultures, axenic hairy roots (25 lines obtained) were excised and individually subcultured on hormone-free B5 liquid medium containing 30 g I- 1 sucrose without antibiotic. To prove transformation, opines (agropine and mannopine) were extracted and detected by paper electrophoresis [14]. All tested lines were transformed and presented the same flavanol content. The hypocotylderived hairy roots showing the fastest growth were used for further expts. Both normal and hairy roots (ca 100 mg fr. wt) were maintained in 120 ml flasks in hormone-free B5 liquid medium with 30 g l- 1 sucrose (70 ml per flask), in the dark at 22” on a rotatory shaker at 100 rpm. Three flasks were used for each expt. Calli were grown under the optimised conditions previously described [2]. Sample preparation and analysis. Frozen plant organs (field-grown roots from 3-month-old mature plants, hypocotyls), normal or hairy roots or calli (5 g fr. wt) were extracted with MeOH (3 x 50 ml). The extract was titered and 20 ml dist. H,O added. MeOH was evapd under red. prcs. and the aq. residue partitioned with EtOAc (4 x 20 ml). The EtOAc layer was evapd to dryness and dissolved in MeOH (50 ml). HPLC analysis was performed as previously described [4] on Lichrosorb RP18 (Merck), using standards :( + )-catechin, ( - )-epicatechin (Extrasynthese), and (-)-epicatechin-3-0-gallate, procyanidin B2, procyanidin BZ3’-0-gallate already isolated from F. esculentum callus cultures and characterized by us c3,41.
REFERENCES
1. Moumou, Y., Trotin, F., Pinkas, M., Dubois, J. and Vasseur J. (1987) Ann. Pharm. Fr. 45, 255. 2. Moumou, Y., Trotin, F., Dubois, J., Vasseur J. and El-Boustani, E. (1992) J. Nat. Prod. 55, 33. 3. Moumou, Y., Vasseur J., Trotin, F. and Dubois J. (1992) Phytochemistry 31, 1239. 4. Moumou Y., Trotin F., Vasseur J., Vermeersch G., Guyon, R., Dubois, J. and Pinkas, M. (1993) Planta Med. in press. 5. Bruneton, J. (1987) EZ&nents de Phytochimie et de Pharmacognosie. Technique et Documentation, Paris. 6. Yoshizawa, S., Horiuchi, T., Fujiki, H., Yoshida, T., Okuda, T. and Sugimura, T. (1987) Phytother. Res. 1, 44. 7. Kakiuchi, N., Kusumoto, I. T., Hattori, M., Namba, T., Hatano, T. and Okuda, T. (1991) Phytother. Res., 5, 270. 8. Hatano, T., Edamatsu, R., Hiramatsu,
M., Mori, A., Fujita, Y., Yasuhara, T., Yoshida, T. and Okuda, T. (1989) Chem. Pharm. Bull. 37, 2016. 9. Deno, H., Yamagata, H., Emoto, T., Yoshioka, T., Yamada, Y. and Fujita, Y. (1987) J. Plant Pkysiol. 131, 315. 10. Sauerwein, M. and Shimomura, K. (1991) Phytochemistry 10, 3277. 11. Chalker-Scott, L., and Krhamer, R. L. (1989) in Chemistry and Signijcance
of Condensed
Tannins
(Hemingway, R. W., and Karchesy, J. J., eds), p. 345. Plenum Press, London. 12. Heller, R. (1953) Ann. Sci. Nat. Bot. Viol. Vkg. 14, 1. 13. Gamborg, U. L., Miller, R. A. and Ujima, K. (1968) Exp. Cell. Res. 50, 151. 14. Petit, A., David, C., Dahl, G. A.: Ellis, J. G., Guyon, P., Casse-Delbart, F. and Tempe, J. (1983) Mol. Gen. Genet. 190,204.