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Phenolic constituents of California buckeye fruit
Phytochemistry, Vol. 31, No. 11, pp. 3793-3794, Printed m Great Britain.
1992 0
0031~9422p2 s5.00+0.00 1992 Pergamon Press Ltd
PHENOLIC CONSTITUENTS OF CALIFORNIA BUCKEYE FRUIT ISAO KUBO* and B&PING YING Division of Entomology and Parasitology, College of Natural Resources, University of California, Berkeley, CA 94720, U.S.A. (Received2 March 1992) Key Word Index--Aesculus cai~omica; Hippocastanaceae; California buckeye; arbutin; (-)-epicatechin;
hydro-
quinone.
Abstract-The fresh fruit of California buckeye, Aesculus californica *was separated into husk, seed coat and endosperm. The chemical analyses exhibited differences in each part. Arbutin was isolated only from the endosperm, (-)-epicatechin from the seed coat and saponins from the husk. Hydroquinone was found to be an artifact of arbutin origin.
INTRODUCTION
In a previous paper we reported the identification of hydroquinone (1) as a plant growth regulator from the fruit of California buckeye, Aescuhts calijbrnica Nutt. (Hippocastanaceae) [l]. It was the only active principle found when the isolation was guided by lettuce and rice seedling assay [2]. Hydroquinone inhibited germination at high concentrations (above 100 ppm), but it stimulated growth at low concentrations for both lettuce and rice seedlings. Interestingly, (-)-epicatechin (2) isolated from the same source tended to counteract the growth inhibitory activity of hydroquinone [ 11. In our continuing effort to understand this, we became aware that hydroquinone is not a naturally occurring substance in the fruit of A. californica. Hydroquinone was not detected in the methanol extract prepared from the newly collected fresh fruit, although the extract inhibited germination of lettuce seed. This finding prompted us to reinvestigate the chemical constituents of the fresh fruit.
RRSULTS AND DISCUSSION
The fresh fruit of A. culifornica was separated into three
parts: husk, seed coat and endosperm. These separated parts were extracted with methanol and the extracts were subjected to chemical analysis. Despite careful chemical analysis, 1 was not detected in any part of the fresh fruit. Instead, a rather large amount (0.06%, fr. wt) of arbutin (3), a hydroquinone glucoside, was isolated from the endosperm. This result indicated that hydroquinone is not a natural product. Most likely, it was an artifact of the previous isolation procedure. The detailed bioassay with the purified arbutin did not exhibit any inhibitory activity in the lettuce seedling assay up to 1000 ppm. Thus, the toxin occurs in the plant in a safe, non-toxic, bound form. For example, arbutin has been used as a commercial whitening agent [3] which is applied to human skin repeatedly and often for long durations. *Author to whom correspondence
should be addressed.
Interestingly, arbutin was isolated from only the endosperm, but not other parts. To the best of our knowledge, this is the first report of arbutin from A. californica. In addition, a non-protein amino acid, 2-amin&methyl4(Z)-hexenoic acid (4) [4] was isolated in quantity, together with phenylalanine (9, from the same endosperm extract and not from other plant parts. Interestingly, another compound (-)-epicatechin (2) was isolated from only the seed coat, but not any other part. A large amount of condensed tannin was also detected in only the seed coat. However, bince this tannin portion did not exhibit any noticeable activity in the lettuce seedling assay up to 1000 ppm, further purification was not carried out. The fruit was eaten in great quantity, after removal of the husk and seed coat, by various Californian Indian tribes [5]. However, because it is poisonous in its natural state it had to be processed. Powdered endosperm were bleached with running water for several days before drying. Most of the bitter tasting arbutin (3) and the toxic non-protein amino acid, 2-amino-4methyl-4(E)hexenoic acid (4) [4] in the endosperm can be removed by this process since both are water-soluble. Furthermore, several saponins were isolated from only the husk. The unripe fruit of the buckeye was crushed and thrown into streams by various Californian Indian tribes to stupefy fish [5]. This effect can be explained by the action of saponins together with other toxic substances. Saponins are well known as fish poisons. The saponins isolated from the husk exhibited potent molluscicidal activity against an aquatic snail, Biomphalaria glabratus [6]. Since they did not exhibit any notable activity in the lettuce seedling assay, their structures will be reported in detail elsewhere. (-)-Epicatechin (2) alone did not exhibit any inhibitory activity in the lettuce seedling assay up to 1000 ppm. However, during the course of the bioassay, we observed that when (-)-epicatechin was present in the test solution, the roots and the solution surrounding the roots of lettuce. seedlings were stained yellow to orange. Further studies of this phenomenon led us to the discovery that oxidases were released from the roots of lettuce seedlings into the rhizosphere [7]. They reduce the growth inhibit-
3793
I. KUBOand B.-P. YING
3794
ory effects of some naturally occurring phenol& through oxidation. Arbutin (3), upon hydrolysis and oxidation is believed to be converted to benzoquinone and glucose (6). The considerable toxicity of benzoquinone is widely recognized. Thus, benzoquinone at a concentration of 10 ppm completely prevented germination of lettuce seed treated with such a solution. While the toxicity of benzoquinone is high, that of hydroquinone is relatively weak. When the collected root exudate was mixed with freshly prepared 1, which is almost colourless, the mixture rapidly turned orange, similar to the case of 2 as previously reported [7]. On the other hand, when dissolved in water and allow to stand 24 hr at 25”, hydroquinone is converted to an orange solution due to the air oxidation. However, 6 was not detected in these reaction mixtures, at least not as one of the major compounds. Thus, the enzymes exuded from the root of lettuce seedlings oxidized 1, but not to 6. For plants, it is not necessary to convert the relatively nontoxic 1 to the toxic 6. Only after leaching from the fruit, or more specifically, from the endosperm and into the soils, arbutin may convert to the toxic benzoquinone after hydrolysis and oxidation, and can exert its effects. In order to exhibit toxicity, benzoquinone must persist for a reasonable length of time in the soil. This seems not to be the case for benzoquinone. Nevertheless, much remains to be learned about the toxicity of compounds such as arbutin and juglone in soil [S, 91. EXPERIMENTAL
General. Mp: uncorr. ‘H and ‘jCNMR were taken in pyridine-d, at 360 MHz for ‘H and 75 MHz for ‘V. FAB-MS were obtained using glycerol matrix. Plaru material. The fresh fruit of A. californica was collected from various locations in California. Extraction and isolation. As soon as the fruit was collected, it was separated into three portions, namely husk, seed coat and endosperm. Endosperm (1.5 kg) was ground and extracted with MeOH (3 x ) at ambient temp. After evapn of the solvent under red. pres., a brown residue (73.2g) was obtained. This was dissolved in 20 ml of MeOH-H,O (1: l), and chromatographed on Sephadex LH-20 column (3 i.d. x 38 cm) using the same solvent system as the eluent. The first eluent contained mainly amino acids which were crystallized from H,O-EtOH to yield 254 mg of 2-amino4-methyl+?)-hexenoic acid (4). In addition, from the mother liquid, 25 mg of phenylalanine (5) was obtained. Later eluent was further separated by CC under the same conditions to give 191 mg of the hydroquinone glucoside, arbutin (3). A&tin (3). ‘HNMR (pyridine-d,): 64.08 (lH, ddd, H-5’), 4.25-4.40 (3H, m, H-2’, H-3’ and H-4’), 4.40 (lH, dd, H-6’a), 4.54 (lH,dd,H-6”b),5.51(1H,d,5=BHZ,H-l’~7.12(2H,d,J=BHz, H-2) and 7.40 (2H, d, J=B Hz, H-3). 13CNMR (pyridine-d,): 662.5 (C-6’), 71.4 (C-4’), 75.1 (C-2’), 78.6 (C-3’), 78.6 (C-S’), 103.6 (C-l’), 116.8 (C-2), 118.8 (C-3), 151.9 (C-4) and 154.1 (C-l). SI-MS: m/z 277 [M-H,O-Na] and 255 [M-H,O-H]. 2-Amino-4-methyU(E)-hcxenoic acid (4). ‘H NMR (D,O): 61.52 (3H, d, J=6.3 Hz, Me-S), 1.56 (3H, s, Me-4), 2.33 (lH, dd, J = 14.0,9.7 Hz, H-3a), 2.55 (lH, dd, J= 14.0, 3.9 Hz, H-3b), 3.71 (IH, dd, J=9.7, 3.9 Hz, H-2) and 5.36 (lH, 4, J=6.3 Hz, H-5). ‘-‘CNMR (DaO): 611.7 (C-6), 13.2 (C-7), 39.7 (C-3), 51.8 (C-2), 124.0 (C-5), 134.1 (C-4) and 173.6 (C-l). This non-protein amino acid was previously isolated from the seed of A. calijornica [43. To the best of our knowledge, the
13CNMR data have not been reported. The above assigned ‘“C NMR data was based on the following. The signal at 6 173.6 (C-l) and the other two sp’ carbon signals, 6124.0 (C-5) and 134.1(C-4), could be easily assigned, since the b 124.0signal had a cross peak with the ‘H signal at 65.4 on its 13C-lH COSY spectrum. The C-2 and C-3 signals appeared at 651.8 and 39.7, respectively. The signal at 6,39.7 correlated with the signals at 6a2.33 and 2.55, and the ‘% signal at 651.8 had a cross peak with the ‘H signal at 63.71 on the 13C-lH COSY spectrum. The chemical shifts of two methyl carbon signals were quite close. The signal at 611.7 had a cross peak with a methyl proton doublet at 61.52. Another methyl carbon signal at S 13.2 had a cross peak with the methyl proton singlet at 61.56. Therefore, these two signals could be assigned as C-6 and C-7, respectively. Phenylalanine (5). AU spectral data in accord with published results. Seed coat (17.2 g) was extracted with MeOH ( x 3) at ambient temp. After removal of solvent, the extract (3.4 g) was separated by RLCC [lo] (CHCI,MeOH-H,O, 7: 13:8, ascending mode) to give 1.1 g of crude epicatechin. 200 mg of this crude sample was purified by recycle HPLC [ill (JAI LC-09, Asahipak GS-320, MeOH, 4.8 mlmin-‘, UV detector) to give 0.173 g of the (-)epicatechin fraction which was crystallized from H,O with trace amount of acetic acid to yield 93 mg of pure ( - )-epicatechin. (-)-Epicatechin (2). Identified by direct comparison with the authentic sample previously isolated from the same source [l]. Husk (34.6 g) was found to contain rather large amounts of saponins. This saponin portion did not exhibit any recognizable activity in the lettuce seedling assay up to 1000 ppm. Bioassay. The lettuce seed (Lacruca sativn L. cv Grand Rapids) was used for the seedling assay, and the assay was carried out according to the method described in ref. [2]. Collection of lettuce root exudate. The exudate was collected as previously described [7]. Acknowledgements-The
authors measuring the NMR data and assistance. B. P. Y. thanks the disciplinary Science for financial
acknowledge Mr H. Naolu for Dr K. S. Tan for technical Takasago Institute for Intersupport.
REFERENCES 1. Kubo, I., Matsumoto, A., Taniguchi, M. and Wood, W. F. (1985)Chem. Pharm Bull. 33, 3826. 2. Kamisaka, S. (1973) Plant Cell Pkysiol. 14, 747.
3. Akiu, S., Suzuki, Y., Fujinuma, Y. and Fukuda, M. (1988) Proc. Jap. Sot. Invest. Dermatoi. 12, 138. 4. Fowden, L. and Smith, A. (1968) Phytochemistry 7, 809. 5. Callegari, J. and Durand, K. (1977) in Wild Edible and Medicinal Plants of California, p. 12. Callegari and Durand,
El Cerrito. 6. Marston. A. and Hostettmann. K. (1985) Phytochemistry 24, 639. 7. Tan, K. S. and Kubo, I. (1990) Experientia 46, 478. 8. Williamson, G. B. and Weidenhamer, J. D. (1990) J. Chem. Ecol. 16, 1739.
9. Funk, D. T., Case, P. J., Rietveld, W. J. and Phares, R. E. (1979) For. Sci. 25, 452. 10. Kubo, I., Marshall, G. T. and F. J. Hanke (1988) in Countercurrent Chromatography (Mandava, N. M. and Ito. Y. eds), pp. 493-511. Dekker, New York. 11. Kubo, I. and Nakatsu, T. (1990) LCGC, 8, 933.
1992 0
0031~9422p2 s5.00+0.00 1992 Pergamon Press Ltd
PHENOLIC CONSTITUENTS OF CALIFORNIA BUCKEYE FRUIT ISAO KUBO* and B&PING YING Division of Entomology and Parasitology, College of Natural Resources, University of California, Berkeley, CA 94720, U.S.A. (Received2 March 1992) Key Word Index--Aesculus cai~omica; Hippocastanaceae; California buckeye; arbutin; (-)-epicatechin;
hydro-
quinone.
Abstract-The fresh fruit of California buckeye, Aesculus californica *was separated into husk, seed coat and endosperm. The chemical analyses exhibited differences in each part. Arbutin was isolated only from the endosperm, (-)-epicatechin from the seed coat and saponins from the husk. Hydroquinone was found to be an artifact of arbutin origin.
INTRODUCTION
In a previous paper we reported the identification of hydroquinone (1) as a plant growth regulator from the fruit of California buckeye, Aescuhts calijbrnica Nutt. (Hippocastanaceae) [l]. It was the only active principle found when the isolation was guided by lettuce and rice seedling assay [2]. Hydroquinone inhibited germination at high concentrations (above 100 ppm), but it stimulated growth at low concentrations for both lettuce and rice seedlings. Interestingly, (-)-epicatechin (2) isolated from the same source tended to counteract the growth inhibitory activity of hydroquinone [ 11. In our continuing effort to understand this, we became aware that hydroquinone is not a naturally occurring substance in the fruit of A. californica. Hydroquinone was not detected in the methanol extract prepared from the newly collected fresh fruit, although the extract inhibited germination of lettuce seed. This finding prompted us to reinvestigate the chemical constituents of the fresh fruit.
RRSULTS AND DISCUSSION
The fresh fruit of A. culifornica was separated into three
parts: husk, seed coat and endosperm. These separated parts were extracted with methanol and the extracts were subjected to chemical analysis. Despite careful chemical analysis, 1 was not detected in any part of the fresh fruit. Instead, a rather large amount (0.06%, fr. wt) of arbutin (3), a hydroquinone glucoside, was isolated from the endosperm. This result indicated that hydroquinone is not a natural product. Most likely, it was an artifact of the previous isolation procedure. The detailed bioassay with the purified arbutin did not exhibit any inhibitory activity in the lettuce seedling assay up to 1000 ppm. Thus, the toxin occurs in the plant in a safe, non-toxic, bound form. For example, arbutin has been used as a commercial whitening agent [3] which is applied to human skin repeatedly and often for long durations. *Author to whom correspondence
should be addressed.
Interestingly, arbutin was isolated from only the endosperm, but not other parts. To the best of our knowledge, this is the first report of arbutin from A. californica. In addition, a non-protein amino acid, 2-amin&methyl4(Z)-hexenoic acid (4) [4] was isolated in quantity, together with phenylalanine (9, from the same endosperm extract and not from other plant parts. Interestingly, another compound (-)-epicatechin (2) was isolated from only the seed coat, but not any other part. A large amount of condensed tannin was also detected in only the seed coat. However, bince this tannin portion did not exhibit any noticeable activity in the lettuce seedling assay up to 1000 ppm, further purification was not carried out. The fruit was eaten in great quantity, after removal of the husk and seed coat, by various Californian Indian tribes [5]. However, because it is poisonous in its natural state it had to be processed. Powdered endosperm were bleached with running water for several days before drying. Most of the bitter tasting arbutin (3) and the toxic non-protein amino acid, 2-amino-4methyl-4(E)hexenoic acid (4) [4] in the endosperm can be removed by this process since both are water-soluble. Furthermore, several saponins were isolated from only the husk. The unripe fruit of the buckeye was crushed and thrown into streams by various Californian Indian tribes to stupefy fish [5]. This effect can be explained by the action of saponins together with other toxic substances. Saponins are well known as fish poisons. The saponins isolated from the husk exhibited potent molluscicidal activity against an aquatic snail, Biomphalaria glabratus [6]. Since they did not exhibit any notable activity in the lettuce seedling assay, their structures will be reported in detail elsewhere. (-)-Epicatechin (2) alone did not exhibit any inhibitory activity in the lettuce seedling assay up to 1000 ppm. However, during the course of the bioassay, we observed that when (-)-epicatechin was present in the test solution, the roots and the solution surrounding the roots of lettuce. seedlings were stained yellow to orange. Further studies of this phenomenon led us to the discovery that oxidases were released from the roots of lettuce seedlings into the rhizosphere [7]. They reduce the growth inhibit-
3793
I. KUBOand B.-P. YING
3794
ory effects of some naturally occurring phenol& through oxidation. Arbutin (3), upon hydrolysis and oxidation is believed to be converted to benzoquinone and glucose (6). The considerable toxicity of benzoquinone is widely recognized. Thus, benzoquinone at a concentration of 10 ppm completely prevented germination of lettuce seed treated with such a solution. While the toxicity of benzoquinone is high, that of hydroquinone is relatively weak. When the collected root exudate was mixed with freshly prepared 1, which is almost colourless, the mixture rapidly turned orange, similar to the case of 2 as previously reported [7]. On the other hand, when dissolved in water and allow to stand 24 hr at 25”, hydroquinone is converted to an orange solution due to the air oxidation. However, 6 was not detected in these reaction mixtures, at least not as one of the major compounds. Thus, the enzymes exuded from the root of lettuce seedlings oxidized 1, but not to 6. For plants, it is not necessary to convert the relatively nontoxic 1 to the toxic 6. Only after leaching from the fruit, or more specifically, from the endosperm and into the soils, arbutin may convert to the toxic benzoquinone after hydrolysis and oxidation, and can exert its effects. In order to exhibit toxicity, benzoquinone must persist for a reasonable length of time in the soil. This seems not to be the case for benzoquinone. Nevertheless, much remains to be learned about the toxicity of compounds such as arbutin and juglone in soil [S, 91. EXPERIMENTAL
General. Mp: uncorr. ‘H and ‘jCNMR were taken in pyridine-d, at 360 MHz for ‘H and 75 MHz for ‘V. FAB-MS were obtained using glycerol matrix. Plaru material. The fresh fruit of A. californica was collected from various locations in California. Extraction and isolation. As soon as the fruit was collected, it was separated into three portions, namely husk, seed coat and endosperm. Endosperm (1.5 kg) was ground and extracted with MeOH (3 x ) at ambient temp. After evapn of the solvent under red. pres., a brown residue (73.2g) was obtained. This was dissolved in 20 ml of MeOH-H,O (1: l), and chromatographed on Sephadex LH-20 column (3 i.d. x 38 cm) using the same solvent system as the eluent. The first eluent contained mainly amino acids which were crystallized from H,O-EtOH to yield 254 mg of 2-amino4-methyl+?)-hexenoic acid (4). In addition, from the mother liquid, 25 mg of phenylalanine (5) was obtained. Later eluent was further separated by CC under the same conditions to give 191 mg of the hydroquinone glucoside, arbutin (3). A&tin (3). ‘HNMR (pyridine-d,): 64.08 (lH, ddd, H-5’), 4.25-4.40 (3H, m, H-2’, H-3’ and H-4’), 4.40 (lH, dd, H-6’a), 4.54 (lH,dd,H-6”b),5.51(1H,d,5=BHZ,H-l’~7.12(2H,d,J=BHz, H-2) and 7.40 (2H, d, J=B Hz, H-3). 13CNMR (pyridine-d,): 662.5 (C-6’), 71.4 (C-4’), 75.1 (C-2’), 78.6 (C-3’), 78.6 (C-S’), 103.6 (C-l’), 116.8 (C-2), 118.8 (C-3), 151.9 (C-4) and 154.1 (C-l). SI-MS: m/z 277 [M-H,O-Na] and 255 [M-H,O-H]. 2-Amino-4-methyU(E)-hcxenoic acid (4). ‘H NMR (D,O): 61.52 (3H, d, J=6.3 Hz, Me-S), 1.56 (3H, s, Me-4), 2.33 (lH, dd, J = 14.0,9.7 Hz, H-3a), 2.55 (lH, dd, J= 14.0, 3.9 Hz, H-3b), 3.71 (IH, dd, J=9.7, 3.9 Hz, H-2) and 5.36 (lH, 4, J=6.3 Hz, H-5). ‘-‘CNMR (DaO): 611.7 (C-6), 13.2 (C-7), 39.7 (C-3), 51.8 (C-2), 124.0 (C-5), 134.1 (C-4) and 173.6 (C-l). This non-protein amino acid was previously isolated from the seed of A. calijornica [43. To the best of our knowledge, the
13CNMR data have not been reported. The above assigned ‘“C NMR data was based on the following. The signal at 6 173.6 (C-l) and the other two sp’ carbon signals, 6124.0 (C-5) and 134.1(C-4), could be easily assigned, since the b 124.0signal had a cross peak with the ‘H signal at 65.4 on its 13C-lH COSY spectrum. The C-2 and C-3 signals appeared at 651.8 and 39.7, respectively. The signal at 6,39.7 correlated with the signals at 6a2.33 and 2.55, and the ‘% signal at 651.8 had a cross peak with the ‘H signal at 63.71 on the 13C-lH COSY spectrum. The chemical shifts of two methyl carbon signals were quite close. The signal at 611.7 had a cross peak with a methyl proton doublet at 61.52. Another methyl carbon signal at S 13.2 had a cross peak with the methyl proton singlet at 61.56. Therefore, these two signals could be assigned as C-6 and C-7, respectively. Phenylalanine (5). AU spectral data in accord with published results. Seed coat (17.2 g) was extracted with MeOH ( x 3) at ambient temp. After removal of solvent, the extract (3.4 g) was separated by RLCC [lo] (CHCI,MeOH-H,O, 7: 13:8, ascending mode) to give 1.1 g of crude epicatechin. 200 mg of this crude sample was purified by recycle HPLC [ill (JAI LC-09, Asahipak GS-320, MeOH, 4.8 mlmin-‘, UV detector) to give 0.173 g of the (-)epicatechin fraction which was crystallized from H,O with trace amount of acetic acid to yield 93 mg of pure ( - )-epicatechin. (-)-Epicatechin (2). Identified by direct comparison with the authentic sample previously isolated from the same source [l]. Husk (34.6 g) was found to contain rather large amounts of saponins. This saponin portion did not exhibit any recognizable activity in the lettuce seedling assay up to 1000 ppm. Bioassay. The lettuce seed (Lacruca sativn L. cv Grand Rapids) was used for the seedling assay, and the assay was carried out according to the method described in ref. [2]. Collection of lettuce root exudate. The exudate was collected as previously described [7]. Acknowledgements-The
authors measuring the NMR data and assistance. B. P. Y. thanks the disciplinary Science for financial
acknowledge Mr H. Naolu for Dr K. S. Tan for technical Takasago Institute for Intersupport.
REFERENCES 1. Kubo, I., Matsumoto, A., Taniguchi, M. and Wood, W. F. (1985)Chem. Pharm Bull. 33, 3826. 2. Kamisaka, S. (1973) Plant Cell Pkysiol. 14, 747.
3. Akiu, S., Suzuki, Y., Fujinuma, Y. and Fukuda, M. (1988) Proc. Jap. Sot. Invest. Dermatoi. 12, 138. 4. Fowden, L. and Smith, A. (1968) Phytochemistry 7, 809. 5. Callegari, J. and Durand, K. (1977) in Wild Edible and Medicinal Plants of California, p. 12. Callegari and Durand,
El Cerrito. 6. Marston. A. and Hostettmann. K. (1985) Phytochemistry 24, 639. 7. Tan, K. S. and Kubo, I. (1990) Experientia 46, 478. 8. Williamson, G. B. and Weidenhamer, J. D. (1990) J. Chem. Ecol. 16, 1739.
9. Funk, D. T., Case, P. J., Rietveld, W. J. and Phares, R. E. (1979) For. Sci. 25, 452. 10. Kubo, I., Marshall, G. T. and F. J. Hanke (1988) in Countercurrent Chromatography (Mandava, N. M. and Ito. Y. eds), pp. 493-511. Dekker, New York. 11. Kubo, I. and Nakatsu, T. (1990) LCGC, 8, 933.