- Email: [email protected]
Tannin production in cell cultures of Sapium sebiferum
0031-9422/92 s5.00+0.00 f_J1992PergamonPressplc.
Phytochemistry, Vol. 31, No. 3, pp. 833.-836, 1992
Printedin GreatBritain.
TANNIN PRODUCTION IN CELL CULTURES OF SAPZUM SEBIFERUM SUMANA NEERA
and KANJI ISHIMARU*
Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo, Saga, 840 Japan (Received 9 September 1991) Key Word Index-Sapium sebijhm;
Euphorbiaceac; tannin; callus; cell suspension cultures.
Abstract-Callus and cell suspension cultures of Supium sebifhun were established and the production of gallic acid, /Iglucogallin, geraniin, furosin, tercatain, chebulagic acid, chlorogenic acid, 1,2,3,6-tetra-O- and 1,2,3,4,6-penta-Ogalloyl-B-D-glucoses established. Both in the callus and cell suspension cultures, the main phenolic constituent was geraniin.
INTRODUCTION
Plants of the Euphorbiaceae are rich in phenolic compounds (especially tannins) and commonly used medicinally. Recently, tannins have been shown to be psychotropic [ 1J, to improve nitrogen metabolism [2-51 and to have anticancer activity [6,7]. Sapiumsebijkmurt,used as a source of woody biomass and oil, also contains a high content of hydrolysable tannins. While some experiments for tissue cultures of this plant have been done [S], there has been no report on the production of secondary metabolites in uitro. We succeeded in the establishment of the callus and cell suspension cultures of this plant and determined their tannin production by HPLC analysis.
Table 1. Effects of growth regulators on callus formation on leaf segments of Sapium sebifmun cultured on MS solid media for eight weeks IAA (mgl-‘)
BA (mg 1-V
Callus fr. wt* (md
0.1
0
0.1
1 0.1 1 0 0
0 0 0 3 3
0.1 0 0 0.1 0
448 954 101 44 471 0
2,4-D (meI_‘)
*Average of five samples. RESULTS AND DISCUSSlON
For the induction of the callus from the leaf segments of the parent plant of S. sebiferum we used six types of Murashige-Skoog (MS) [9] solid media containing various combinations of 24-dichlorophenoxyacetic acid (2,4D), indole-3-acetic acid (IAA) and benzyladenine (BA) (Table 1). MS medium with 1 mg l- ’ 2,4-D and 0.1 mg l- ’ BA proved to be best for the induction of the callus. The callus cultured on this medium was transferred to MS solid media supplemented with 0.5 mgl-’ 2,4D-O.5 mgl-’ BA(medium A),O.l mgl-’ 2,4-D-3 mgl-r BA (medium B) or 1 mgl- ’ naphthaleneacetic acid (NAA)-0.1 mgl-’ BA (medium C). The growth rate of the calli cultured on these solid media (10 ml medium per one tube) is shown in Fig. 1. The callus started to increase at two weeks and continued to grow until the end of the culture period (eight weeks). The growth rates of the calli cultured on A and C media were approximately three times greater than that on B medium. The callus was also transferred into MS liquid medium containing 1 mg 1- 12,4-D and 0.1 mg 1- ’ BA (medium D) to establish the cell suspension culture. The growth rate of the cells cultured in 50 ml liquid medium in a flask was determined (Fig. 1). The amount of the cells cultured in
400f--
300-
/
llcI.; 0
1
2
3
4
5
6
,
6
Time (weeks)
& callus on medium A -CM callus on medium B -w- callus on medium C -J- cells in medium 0 Fig. 1. Growth of callus and cell suspenbion cultures of Sapium
sebiferum
* Author to whom correspondence should be addressed. 833
S. NEERAand K. ISHIMARU
Time (weeks)
0
73-l --o-’
1
2
3
--•-. 3 --.- 4 -.- 5 -7 Fig. 2. Tannin production in the calli of Sapium sebiferum cultured on medium A.
4
5
6
7
8
Time (weeks)
2
*1 --o-, 2 --•-. 3
-.... 4 -*- 5 -7
Fig. 3. Tannin production in the calli of Sapium sebijkum this liquid medium increased from the beginning of the cultured on medium B. culture and reached a maximum level at 5 weeks when it remained at a plateau. From the 80% aqueous acetone extract of the stems of the parent plant, five phenolic compounds, /?-glucogallin 4,000 (1) [lo], chlorogenic acid (2)[ll], tercatain (3) [12], chebulagic acid (4) [13] and geraniin (5) [14] were isolated. After identification of these constituents, the phenolic compounds produced in the callus cultures were determined. From the callus cultured for two months on medium A four compounds, 5, gallic acid (6) 1,2,3,4,6penta-0-galloyl-/?-D-glucose (7) [15] and furosin (8) [16] were isolated. On the other hand, from the callus cultured on medium B seven compounds 1, 3, 5-8, and 1,2,3,6tetra-o-galloyl+D-glucose (9) [ 171 were isolated. Therefore, these calli of 5. sebijhm contain almost the same type of hydrolysable tannins as the parent plant. The amounts of these phenolic compounds l-5, and 7 in several cultures of 5. sebiferum were determined by Time (weeks) HPLC. In the callus cultured on medium A (10ml medium in one tube), compound 5 was the main phenolic -1 (Fig. 2). The amount of 5 continued to increase from the -a-, 2 early stage of the culture (121.18 pg per tube at one week) --*-, 3 until the end of the culture period to reach the highest .‘..’ 4 level (2199.33 pg per tube at eight weeks). The amounts of -5 1 and 3 gradually increased to their maximum levels (1: -7 265.32 pg per tube at six weeks, 3: 160.04 pg per tube at Fig. 4. Tannin production in the calli of Sapium seb@wm seven weeks), which were almost one-tenth the level in cultured on medium C. comparison with that of 5. Compounds 2, 4 and 7 appeared only at the early stages (one to two weeks) of this culture. The callus cultured on medium B also produced high levels of 5 (520.83 pg per tube at eight weeks) (Fig. 3). But to the highest level (3092.8 pg per tube) at eight weeks the amount of 5 in this culture was almost one-quarter (Fig. 4). For the production of 5 in the callus culture of 5. that of the one cultured on medium A. The amounts of 1, sebiferum, medium C seems to be most suitable. The 3 and 7 increased slightly throughout the culture time, amounts of 1,3 and 7 also gradually increased and but their levels were not very high (under 100 pg per tube). reached over 150 pg per tube. Compounds 2 and 4 appeared in low concentration only In the cell suspension culture in medium D, tannins at the early stage in this culture. were similar to those produced in the callus cultures In the callus cultured on medium C, the main product 5 (Fig. 5). In this culture, as in the callus cultures, 5 was the increased rapidly after two weeks culture and it reached main constituent and reached over 3OOOpg per flask at
Tannin production in cell cultures of Sapium sebifkrum 4,000
*
3,000 p
2.ooo
---A/’
0 G= 1,cooi
P
Loa 3, E $
400 I 200 lC%l 1
0
/
y
/----A
JS ..--.. I
2
3
4
5
6
7
8
Time (weeks) *l -Qo-, 2 -..-. 3 --m-’ 4
--r-5 -7
Fig. 5. Tannin production in the cell suspension culture of Sapium sebiferum cultured on medium D.
seven weeks culture. Compounds 1,3,4 and 7 were also detected with high levels in comparison with those in the callus cultures. Both in the callus and cell suspension cultures, the main phenolic was 5. On media A and C the calli grew eqecially well and produced a large amount of tannin. Taking into account the volume of media (10 ml per tube for callus cultures and 50 ml per flask for cell suspension culture), the callus cultures seem to be superior to cell suspension culture for the production of tannins. EXPERIMENTAL
Phenolic compounds were identified by comparison of their physical and spectral (270 MHz ‘HNMR) data with those mentioned in the refs. MS media used throughout contained 30 gl-’ sucrose and all media were adjusted to pH 5.7 before autoclaving at 121” for 15 min. All cultures were. placed in the dark condition at 25”. Plant material. The leaf segments of Sapium sebijerum, collected in June 1990, were sterilized and placed aseptically on MS solid media (solidified with 2.5 gl-’ gelrite) with various combinations of 2,4-D, IAA and BA (Table 1). After two months culture, the callus derived from the leaf segments cultured on MS medium containing 1 mgl-’ 2,4-D and 0.1 mgl-’ BA were transferred to 3 types of MS solid media, A-C (10 ml medium per tube), and subcultured at 2-month intervals. The callus was also transferred into MS liquid medium D (50 ml medium per 100 ml flask) and the oz.11suspension culture was established. The cells were subcultured at 2-month intervals on a rotary shaker (100 rpm). Voucher specimens are deposited at the Laboratory of Genetic Engineering, Faculty of Agriculture in Saga University. Isolation 01 tannins. (a) From intact plants, fresh stems (ca 200 g) of S. sebi@wn, collected in February 1991, were macerated and extracted ( x 2) with 80% aq. Me&O at room temp. The extract, after concn to ca 100 ml, was subjected to Sephadex LH20 CC and eluted with H,O containing increasing amount of
835
MeOH to give 3 frs. Fr. 1 was chromatographed over MCI gel CHP-20P (stepwise elution with Hz0 and 30% MeOH) to afford 1 (12.3 mg) and 3 (173.9 mg). Fr. 2 was purified with Fuji gel ODS-G3 (stepwise elution with Hz0 and 25% MeOH) to give 3 (98.9 mg) and 4 (180.2 mg). Fr. 3 was chromatographed over Sephadex LH-20 with 60% EtOH to give 5 (419.9 mg). (b) From the callus cultured on medium A, fresh calli (ca 200 g), cultured on medium A for 6 weeks, were homogenized and extracted with 90% Me&O (300 ml x 3) at room temp. The extract was coned under red. pres. (to ca 20 ml) and applied to Sephadex LH-20 CC (elution with Hz0 containing increasing amount of MeOH) to give 3 frs. Fr. 1 was chromatographed over Sephadex LH-20 (with 80% EtOH) to afford 6 (22.9 mg). Fr. 2 was purified with Bondapak C, s Porasil B (stepwise elution with H,O and MeOH) to give 5 (135.3 mg). Fr. 3 was separated with CC over Sephadex LH-20 (with 50% Me,CO) to afford 7 (4.7 mg) and 8 (38.5 mg). (c) From the callus cultured on medium C, fresh calli (ca 200 g). harvested after 6 weeks culture on medium C, were homogenized and extracted as above to give 3 frs. Fr. 1 was separated with Sephadex LH-20 (with 80% EtOH) to give 6 (12.8 mg) and 1 (45.7 mg). Fr. 2 was purified with Bondapak Cl8 Porasil B (stepwise elution with Hz0 and MeOH) to afford 3 (15.0 mg), 5 (254.5 mg), 8 (125.7 mg) and 9 (4.0 mg). Fr. 3 was separated with Sephadex LH-20 (with 80% EtOH) to give 7 (6.1 mg). Growth experiment. (a) Callus culture, a piece of callus (ca 200 mg, fr. wt) was inoculated separately on media A-C [lo ml medium in one tube (2.2 cm in the diameter)] and cultured for 8 weeks. (b) Cell suspension culture, ca 2 g (fr. wt) of cells were transferred into liquid medium D (50 ml medium in 100 ml tlask) and cultured for 7 weeks on a rotary shaker (100 rpm). These cultures were harvested periodically and after lyophilization, the dry wt was measured. The data of the growth rate are shown as the mean of three determinations (Fig. 1). Quantitative determinations of compounds of l-5 and 7 in the callus and cell suspension cultures. The calli cultured on media A-C and the cells cultured in medium D were periodically harvested, lyophilized, and used for HPLC analysis. The samples (20-60 mg) were mashed and extracted with 80% Me&O (2 ml) for 15 hr at room temp. Each extraction, after filtration with a millipore filter (0.45 pm), was injected (2-4 ~1) on to HPLC. HPLC conditions were as follows: column; Nucleosil lOO-5C,s (4.6 mm id. x 250 mm), mobile phase; MeCN-50 mM NaH,PO, column temp.: 40”, flow rate: O.Bmlmin- ‘; R,: 6 (5.23 min), 1 (6.01 min), 2 (10.30min), 5 (16.36 min), 4 (16.98 minh 3 (17.66 min) and 7 (19.08 min) (Figs 2-5). The data are shown as the mean of duplicate experiments.
REFERENCES 1. Ueki, S., Nonaka, G., Nishioka, I. and Fujiwara, M. (1985) J. Med. Pharm. Sot. WAKAN- YAKU 2,502. 2. Nagasawa. T., Shibutani, S. and Oura, H. (1978) Yakugaku
Zasshi 98, 1642. 3. Nagasawa, T., Shibutani, S. and Oura, H. (1980) Y&u&u Zasshi 100,434.
4. Nagasawa, T., Oura, H., Shoyama, Y. and Nishioka, I. (1980) Chem. Pharm. Bull. 28, 1736. 5. Shibutani, S., Nagasawa, T., Oura, H., Nonaka, G. and Nishioka, I. (1983) Chem. Pharm. Bull. 31, 2378. 6. Nishizawa, M., Yamagishi, T., Dutschman, G. E., Parker, W. B., Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-C. and Lee, K.-H. (1989) J. Nat. Prod. 52, 762. 7. Nonaka, G., Nishioka, I., Nishizawa, M., Yamagishi, T.,
836
S. NEERAand K. ISHIMARLJ
Kashiwada, Y., Dutschman, G. E., Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-C. and Lee, K.-H. (1990) J. Nat. Prod. 53, 587. 8. Mridula, M. K., Gupta, P. K. and Mascarenhas, A. F. (1983) Plant Cell Tiss. Org. Cult. 2, 133. 9. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15,473. 10. Schmidt, 0. T. and Schmadel, H. (1961) Liebigs Ann. Chem. 649, 149. 11. Court, W. A. (1977) J. Chromatog. 130, 287. 12. Tanaka, T., Nonaka, G. and Nishioka, I. (1986) Chem. Pharm. Bull. 34, 1039.
13. Haddock, E. A., Gupta, R. K. and Haslam, E. (1982)J. Chem Sot., Perkin Trans I 2535. 14. Okuda, T., Yoshida, T. and Hatano, T. (1982) J. Chem. Sot., Perkin Trans I 9. 15. Nishizawa, M.. Yamagishi, T., Nonaka, G. and Nishioka, I. (1982) J. Chem. SC., Perkin Trans I 2963. 16. Saijo, R., Nonaka, G. and Nishioka, I. (1989) Chem. Pharm. Bull. 37, 2063. 17. Nishizawa, M., Yamagishi, T., Nonaka, G. and Nishioka, I. (1983) J. Chem. SOL. Perkin‘ Trans I 961.
Phytochemistry, Vol. 31, No. 3, pp. 833.-836, 1992
Printedin GreatBritain.
TANNIN PRODUCTION IN CELL CULTURES OF SAPZUM SEBIFERUM SUMANA NEERA
and KANJI ISHIMARU*
Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo, Saga, 840 Japan (Received 9 September 1991) Key Word Index-Sapium sebijhm;
Euphorbiaceac; tannin; callus; cell suspension cultures.
Abstract-Callus and cell suspension cultures of Supium sebifhun were established and the production of gallic acid, /Iglucogallin, geraniin, furosin, tercatain, chebulagic acid, chlorogenic acid, 1,2,3,6-tetra-O- and 1,2,3,4,6-penta-Ogalloyl-B-D-glucoses established. Both in the callus and cell suspension cultures, the main phenolic constituent was geraniin.
INTRODUCTION
Plants of the Euphorbiaceae are rich in phenolic compounds (especially tannins) and commonly used medicinally. Recently, tannins have been shown to be psychotropic [ 1J, to improve nitrogen metabolism [2-51 and to have anticancer activity [6,7]. Sapiumsebijkmurt,used as a source of woody biomass and oil, also contains a high content of hydrolysable tannins. While some experiments for tissue cultures of this plant have been done [S], there has been no report on the production of secondary metabolites in uitro. We succeeded in the establishment of the callus and cell suspension cultures of this plant and determined their tannin production by HPLC analysis.
Table 1. Effects of growth regulators on callus formation on leaf segments of Sapium sebifmun cultured on MS solid media for eight weeks IAA (mgl-‘)
BA (mg 1-V
Callus fr. wt* (md
0.1
0
0.1
1 0.1 1 0 0
0 0 0 3 3
0.1 0 0 0.1 0
448 954 101 44 471 0
2,4-D (meI_‘)
*Average of five samples. RESULTS AND DISCUSSlON
For the induction of the callus from the leaf segments of the parent plant of S. sebiferum we used six types of Murashige-Skoog (MS) [9] solid media containing various combinations of 24-dichlorophenoxyacetic acid (2,4D), indole-3-acetic acid (IAA) and benzyladenine (BA) (Table 1). MS medium with 1 mg l- ’ 2,4-D and 0.1 mg l- ’ BA proved to be best for the induction of the callus. The callus cultured on this medium was transferred to MS solid media supplemented with 0.5 mgl-’ 2,4D-O.5 mgl-’ BA(medium A),O.l mgl-’ 2,4-D-3 mgl-r BA (medium B) or 1 mgl- ’ naphthaleneacetic acid (NAA)-0.1 mgl-’ BA (medium C). The growth rate of the calli cultured on these solid media (10 ml medium per one tube) is shown in Fig. 1. The callus started to increase at two weeks and continued to grow until the end of the culture period (eight weeks). The growth rates of the calli cultured on A and C media were approximately three times greater than that on B medium. The callus was also transferred into MS liquid medium containing 1 mg 1- 12,4-D and 0.1 mg 1- ’ BA (medium D) to establish the cell suspension culture. The growth rate of the cells cultured in 50 ml liquid medium in a flask was determined (Fig. 1). The amount of the cells cultured in
400f--
300-
/
llcI.; 0
1
2
3
4
5
6
,
6
Time (weeks)
& callus on medium A -CM callus on medium B -w- callus on medium C -J- cells in medium 0 Fig. 1. Growth of callus and cell suspenbion cultures of Sapium
sebiferum
* Author to whom correspondence should be addressed. 833
S. NEERAand K. ISHIMARU
Time (weeks)
0
73-l --o-’
1
2
3
--•-. 3 --.- 4 -.- 5 -7 Fig. 2. Tannin production in the calli of Sapium sebiferum cultured on medium A.
4
5
6
7
8
Time (weeks)
2
*1 --o-, 2 --•-. 3
-.... 4 -*- 5 -7
Fig. 3. Tannin production in the calli of Sapium sebijkum this liquid medium increased from the beginning of the cultured on medium B. culture and reached a maximum level at 5 weeks when it remained at a plateau. From the 80% aqueous acetone extract of the stems of the parent plant, five phenolic compounds, /?-glucogallin 4,000 (1) [lo], chlorogenic acid (2)[ll], tercatain (3) [12], chebulagic acid (4) [13] and geraniin (5) [14] were isolated. After identification of these constituents, the phenolic compounds produced in the callus cultures were determined. From the callus cultured for two months on medium A four compounds, 5, gallic acid (6) 1,2,3,4,6penta-0-galloyl-/?-D-glucose (7) [15] and furosin (8) [16] were isolated. On the other hand, from the callus cultured on medium B seven compounds 1, 3, 5-8, and 1,2,3,6tetra-o-galloyl+D-glucose (9) [ 171 were isolated. Therefore, these calli of 5. sebijhm contain almost the same type of hydrolysable tannins as the parent plant. The amounts of these phenolic compounds l-5, and 7 in several cultures of 5. sebiferum were determined by Time (weeks) HPLC. In the callus cultured on medium A (10ml medium in one tube), compound 5 was the main phenolic -1 (Fig. 2). The amount of 5 continued to increase from the -a-, 2 early stage of the culture (121.18 pg per tube at one week) --*-, 3 until the end of the culture period to reach the highest .‘..’ 4 level (2199.33 pg per tube at eight weeks). The amounts of -5 1 and 3 gradually increased to their maximum levels (1: -7 265.32 pg per tube at six weeks, 3: 160.04 pg per tube at Fig. 4. Tannin production in the calli of Sapium seb@wm seven weeks), which were almost one-tenth the level in cultured on medium C. comparison with that of 5. Compounds 2, 4 and 7 appeared only at the early stages (one to two weeks) of this culture. The callus cultured on medium B also produced high levels of 5 (520.83 pg per tube at eight weeks) (Fig. 3). But to the highest level (3092.8 pg per tube) at eight weeks the amount of 5 in this culture was almost one-quarter (Fig. 4). For the production of 5 in the callus culture of 5. that of the one cultured on medium A. The amounts of 1, sebiferum, medium C seems to be most suitable. The 3 and 7 increased slightly throughout the culture time, amounts of 1,3 and 7 also gradually increased and but their levels were not very high (under 100 pg per tube). reached over 150 pg per tube. Compounds 2 and 4 appeared in low concentration only In the cell suspension culture in medium D, tannins at the early stage in this culture. were similar to those produced in the callus cultures In the callus cultured on medium C, the main product 5 (Fig. 5). In this culture, as in the callus cultures, 5 was the increased rapidly after two weeks culture and it reached main constituent and reached over 3OOOpg per flask at
Tannin production in cell cultures of Sapium sebifkrum 4,000
*
3,000 p
2.ooo
---A/’
0 G= 1,cooi
P
Loa 3, E $
400 I 200 lC%l 1
0
/
y
/----A
JS ..--.. I
2
3
4
5
6
7
8
Time (weeks) *l -Qo-, 2 -..-. 3 --m-’ 4
--r-5 -7
Fig. 5. Tannin production in the cell suspension culture of Sapium sebiferum cultured on medium D.
seven weeks culture. Compounds 1,3,4 and 7 were also detected with high levels in comparison with those in the callus cultures. Both in the callus and cell suspension cultures, the main phenolic was 5. On media A and C the calli grew eqecially well and produced a large amount of tannin. Taking into account the volume of media (10 ml per tube for callus cultures and 50 ml per flask for cell suspension culture), the callus cultures seem to be superior to cell suspension culture for the production of tannins. EXPERIMENTAL
Phenolic compounds were identified by comparison of their physical and spectral (270 MHz ‘HNMR) data with those mentioned in the refs. MS media used throughout contained 30 gl-’ sucrose and all media were adjusted to pH 5.7 before autoclaving at 121” for 15 min. All cultures were. placed in the dark condition at 25”. Plant material. The leaf segments of Sapium sebijerum, collected in June 1990, were sterilized and placed aseptically on MS solid media (solidified with 2.5 gl-’ gelrite) with various combinations of 2,4-D, IAA and BA (Table 1). After two months culture, the callus derived from the leaf segments cultured on MS medium containing 1 mgl-’ 2,4-D and 0.1 mgl-’ BA were transferred to 3 types of MS solid media, A-C (10 ml medium per tube), and subcultured at 2-month intervals. The callus was also transferred into MS liquid medium D (50 ml medium per 100 ml flask) and the oz.11suspension culture was established. The cells were subcultured at 2-month intervals on a rotary shaker (100 rpm). Voucher specimens are deposited at the Laboratory of Genetic Engineering, Faculty of Agriculture in Saga University. Isolation 01 tannins. (a) From intact plants, fresh stems (ca 200 g) of S. sebi@wn, collected in February 1991, were macerated and extracted ( x 2) with 80% aq. Me&O at room temp. The extract, after concn to ca 100 ml, was subjected to Sephadex LH20 CC and eluted with H,O containing increasing amount of
835
MeOH to give 3 frs. Fr. 1 was chromatographed over MCI gel CHP-20P (stepwise elution with Hz0 and 30% MeOH) to afford 1 (12.3 mg) and 3 (173.9 mg). Fr. 2 was purified with Fuji gel ODS-G3 (stepwise elution with Hz0 and 25% MeOH) to give 3 (98.9 mg) and 4 (180.2 mg). Fr. 3 was chromatographed over Sephadex LH-20 with 60% EtOH to give 5 (419.9 mg). (b) From the callus cultured on medium A, fresh calli (ca 200 g), cultured on medium A for 6 weeks, were homogenized and extracted with 90% Me&O (300 ml x 3) at room temp. The extract was coned under red. pres. (to ca 20 ml) and applied to Sephadex LH-20 CC (elution with Hz0 containing increasing amount of MeOH) to give 3 frs. Fr. 1 was chromatographed over Sephadex LH-20 (with 80% EtOH) to afford 6 (22.9 mg). Fr. 2 was purified with Bondapak C, s Porasil B (stepwise elution with H,O and MeOH) to give 5 (135.3 mg). Fr. 3 was separated with CC over Sephadex LH-20 (with 50% Me,CO) to afford 7 (4.7 mg) and 8 (38.5 mg). (c) From the callus cultured on medium C, fresh calli (ca 200 g). harvested after 6 weeks culture on medium C, were homogenized and extracted as above to give 3 frs. Fr. 1 was separated with Sephadex LH-20 (with 80% EtOH) to give 6 (12.8 mg) and 1 (45.7 mg). Fr. 2 was purified with Bondapak Cl8 Porasil B (stepwise elution with Hz0 and MeOH) to afford 3 (15.0 mg), 5 (254.5 mg), 8 (125.7 mg) and 9 (4.0 mg). Fr. 3 was separated with Sephadex LH-20 (with 80% EtOH) to give 7 (6.1 mg). Growth experiment. (a) Callus culture, a piece of callus (ca 200 mg, fr. wt) was inoculated separately on media A-C [lo ml medium in one tube (2.2 cm in the diameter)] and cultured for 8 weeks. (b) Cell suspension culture, ca 2 g (fr. wt) of cells were transferred into liquid medium D (50 ml medium in 100 ml tlask) and cultured for 7 weeks on a rotary shaker (100 rpm). These cultures were harvested periodically and after lyophilization, the dry wt was measured. The data of the growth rate are shown as the mean of three determinations (Fig. 1). Quantitative determinations of compounds of l-5 and 7 in the callus and cell suspension cultures. The calli cultured on media A-C and the cells cultured in medium D were periodically harvested, lyophilized, and used for HPLC analysis. The samples (20-60 mg) were mashed and extracted with 80% Me&O (2 ml) for 15 hr at room temp. Each extraction, after filtration with a millipore filter (0.45 pm), was injected (2-4 ~1) on to HPLC. HPLC conditions were as follows: column; Nucleosil lOO-5C,s (4.6 mm id. x 250 mm), mobile phase; MeCN-50 mM NaH,PO, column temp.: 40”, flow rate: O.Bmlmin- ‘; R,: 6 (5.23 min), 1 (6.01 min), 2 (10.30min), 5 (16.36 min), 4 (16.98 minh 3 (17.66 min) and 7 (19.08 min) (Figs 2-5). The data are shown as the mean of duplicate experiments.
REFERENCES 1. Ueki, S., Nonaka, G., Nishioka, I. and Fujiwara, M. (1985) J. Med. Pharm. Sot. WAKAN- YAKU 2,502. 2. Nagasawa. T., Shibutani, S. and Oura, H. (1978) Yakugaku
Zasshi 98, 1642. 3. Nagasawa, T., Shibutani, S. and Oura, H. (1980) Y&u&u Zasshi 100,434.
4. Nagasawa, T., Oura, H., Shoyama, Y. and Nishioka, I. (1980) Chem. Pharm. Bull. 28, 1736. 5. Shibutani, S., Nagasawa, T., Oura, H., Nonaka, G. and Nishioka, I. (1983) Chem. Pharm. Bull. 31, 2378. 6. Nishizawa, M., Yamagishi, T., Dutschman, G. E., Parker, W. B., Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-C. and Lee, K.-H. (1989) J. Nat. Prod. 52, 762. 7. Nonaka, G., Nishioka, I., Nishizawa, M., Yamagishi, T.,
836
S. NEERAand K. ISHIMARLJ
Kashiwada, Y., Dutschman, G. E., Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-C. and Lee, K.-H. (1990) J. Nat. Prod. 53, 587. 8. Mridula, M. K., Gupta, P. K. and Mascarenhas, A. F. (1983) Plant Cell Tiss. Org. Cult. 2, 133. 9. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15,473. 10. Schmidt, 0. T. and Schmadel, H. (1961) Liebigs Ann. Chem. 649, 149. 11. Court, W. A. (1977) J. Chromatog. 130, 287. 12. Tanaka, T., Nonaka, G. and Nishioka, I. (1986) Chem. Pharm. Bull. 34, 1039.
13. Haddock, E. A., Gupta, R. K. and Haslam, E. (1982)J. Chem Sot., Perkin Trans I 2535. 14. Okuda, T., Yoshida, T. and Hatano, T. (1982) J. Chem. Sot., Perkin Trans I 9. 15. Nishizawa, M.. Yamagishi, T., Nonaka, G. and Nishioka, I. (1982) J. Chem. SC., Perkin Trans I 2963. 16. Saijo, R., Nonaka, G. and Nishioka, I. (1989) Chem. Pharm. Bull. 37, 2063. 17. Nishizawa, M., Yamagishi, T., Nonaka, G. and Nishioka, I. (1983) J. Chem. SOL. Perkin‘ Trans I 961.