HETEROCYCLES

Paper | Regular issue | Vol. 78, No. 8, 2009, pp. 1993-2001
Received, 16th February, 2009, Accepted, 31st March, 2009, Published online, 1st April, 2009.
DOI: 10.3987/COM-09-11683

■ Two New Hydrolyzable Tannins, Carpinerins A and B, from Galls of Carpinus tschonoskii

Takako Ono and Hideyuki Shigemori*

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Abstract

Two new hydrolyzable tannins, carpinerins A (1) and B (2), together with ten known tannins 3-12, were isolated from the galls on bud of Carpinus tschonoskii, and the structures of 1 and 2 were elucidated using spectroscopic data, primarily NMR and MS, and chemical means. Most of isolated tannins including carpinerin B (2) exhibited inhibitory effects on the growth of the root of cress (Lepidium sativum L.) seedling at 1×10-4 mol/L.

INTRODUCTION
Galls are excrescences formed by parasitizing organism including insects, bacteria, and viruses. Although there are many kinds of galls and it is so interesting phenomenon, little have been known about galls and its ingredient. Therefore, study on constituents of galls will be expected to lead new bioactive compounds. In search for new bioactive compounds from galls, plant growth inhibitory polyacetylenes have been isolated from the insect galls of Hedera rhombea Bean.1-3 On the other hand, constituents of galls of Carpinus tschonoskii bud also have not been reported. Carpinus tschonoskii belong to Betulaceae family and this gall is formed by mite (Eriophyes sp.)-parasitizing. In this research, to search for compounds which produced by gall-form, we noticed the difference of constituents between gall and normal bud. It is found that hydrolyzable tannins were produced by gall-form. In this paper, we reported the isolation and identification of hydrolyzable tannins from gall extract of Carpinus tschonoskii and their structure activity relationship on the growth of cress (Lepidium sativum L.) seedling.

RESULTS AND DISCUSSION
Galls of Carpinus tschonoskii were homogenized and extracted with MeOH. The MeOH extract was partitioned between EtOAc and H2O and the H2O-layer was further partitioned with BuOH. Each of EtOAc-layer and BuOH-layer was separated by ODS column chromatography and ODS-HPLC to give two new tannins, carpinerins A (1, 0.0034 %) and B (2, 0.0261 %), together with 10 known tannins, 1,2,6-trigalloylglucose (3, 0.0012 %),4 1,2,3,6-tetragalloylglucose (4, 0.0021 %),4 pentagalloylglucose (5, 0.0169 %),5 casuarictin (6, 0.0228 %),6 pedunculagin (7, 0.0062 %),6 tellimagrandin Ⅱ (8, 0.0094 %) ,7 casuarinin (9, 0.0098 %),6 liquidambin (10, 0.0356 %),8 hippophaenin A (11, 0.003 %),9 and rhoipteleanin H (12, 0.0183 %).10

Carpinerin A (1) was isolated as a colorless powder, [α]D -6.0° (MeOH), and determined the molecular formula as C47H34O32 by the HRESIMS [m/z 1133.0898 (M+Na)+, Δ -3.3 mmu]. The 1H NMR spectrum showed a two-proton aromatic singlet signal (δH 7.14) due to a galloyl group, three aromatic singlet proton signals (δH 6.96, 6.66, and 6.53), and eight methine and three methylene proton signals. The 1H-1H COSY connectivities of Glc-H-1 through Glc-H-6 indicated the presence of a sugar. Its 1H-1H coupling patterns (J1-2 = 8.5 Hz, J2-3 = 10.1 Hz, J3-4 = 10.1 Hz, J4-5 = 10.1 Hz, J5-6 = 6.7 Hz) were similar to those of casuarictin (6) and it suggested that a sugar of 1 was glucose (Glc). The 13C NMR spectrum indicated the presence of a galloyl group (δC 111.3, 121.0, 141.6, 147.5, 166.6) and a hexahydroxydiphenoyl (HHDP) group (δC 109.4, 109.7, 117.3, 117.4, 126.9, 138.2, 138.4, 145.4, 145.7, 146.6, 146.7, 168.6, 170.3).6 The 1H-1H COSY correlations showed the connectivities of H-4” (δH 4.50), H-5” (δH 4.34), and H-6” (δH 4.19 and 3.80). And the HMBC correlations of this methylene proton (δH 4.19) and methine proton (δH 4.50) to a hemiacetal carbon (δC 109.7, EC-C-3”) revealed the presence of a furan ring and the methine proton (δH 4.50) to a ester carbon (δH 174.2) revealed the presence of γ-lactone. Moreover, HMBC correlations of methine proton (δH 5.81, EC-H-1) and methylene proton (δH 2.21, EC-H-3) to quaternary carbon (δC 82.8, EC-C-2”) showed the γ-lactone connected with spiro carbon (EC-C-2”). And HMBC correlations were observed that methine proton (δH 5.81, EC-H-1) to quaternary carbon (δC 49.8, EC-C-2) and two benzene carbons (δC 118.8, EC-C-1’ and 149.2, EC-C-6’) and methylene proton (δH 2.21, EC-H-3) to EC-C-2 and a carbonyl carbon (δC 200.5, EC-C-4). For all of these results and past literature data11 showed the presence of a elaecarpusinoyl (EC) group. The 13C NMR chemical shifts of EC group gave close agreement with literature data of elaeocarpusin11-13 and helioscopin A14. The HMBC correlations of Glc-H-2 (δH 4.29) to EC-C-7’ (δC 168.0) and Glc-H-3 (δH 5.82) to EC-C-7 (δC 172.0) revealed that the EC group was connected to Glc-H-2 and Glc-H-3. In a similar way, the HMBC correlations of Glc-H-1 (δH 6.93) to galloyl-C-7 (δC 166.6) and Glc-H-2 and Glc-H-4 (δH 4.29 and 5.21) to HHDP-C-7 and HHDP-C-7’ (δC 170.3 and 168.6) showed the linkage of Glc-1 to galloyl group and Glc-2 and Glc-4 to HHDP group (Figure 2). The 1H NMR date of EC group were different from literature data of elaeocarpusin and helioscopin A, in spite of close agreement of 13C NMR date. In case of 1, the 1H-1H coupling constant (J = 5.0 Hz) between EC-4” and EC-5” was observed, while those of elaeocarpusin and helioscopin A were not observed.11-14 Furthermore, it was known that heating elaeocarpusin and helioscopin A in water liberated ascorbic acid.11-14 Compound 1 was heated in water at 90℃ for 24 h and the reaction mixture was subjected to HPLC analysis. The retention time (tR 5.8 min) of the reaction mixture was identical with that of erythorbic acid. This result indicated cis-form between EC-4” and EC-5” of 1. On the other hand, heating 1 in 0.1 % AcOH at 80℃ for 30 min gave 6 by HPLC analysis of the reaction mixture, supporting that 6 was the precursor of 1. This result indicated the atropisomers of HHDP and EC group were elucidated to be both of S-configurations, since both of two HHDP groups in 6 had S-configurations.6 The NOESY correlation between EC-H-6” (δH 5.46) and HHDP’-H-3 (δH 6.53) revealed that the spiro stereochemistry of EC-2” was implied S-configuration. From these results, the structure of 1 was proposed as being represented formula 1. It presumed the plausible biosynthesis of 1 is as indicated below, HHDP group which was linked to Glc-2, 3 of 6 gave dehydrohexahydroxydiphenoyl (DHHDP) group by oxidation and it condensed with erythorbic acid (Figure 2).
Carpinerin B (2) was isolated as a pale yellow powder, [α]D -50.0° (MeOH), and determined the molecular formula as C42H30O27 by the HRESIMS [m/z 991.0872 (M+Na)+, Δ +4.5 mmu]. The 1H NMR spectrum showed a two-proton aromatic singlet signal (δH 7.11) due to a galloyl group, three aromatic singlet proton signals (δH 6.89, 6.54, and 6.32), five methines and a methylene proton signals due to glucose, a benzyl methine (δH 5.33), and a carbomethoxy (δH 3.69) proton signals. The 13C NMR spectrum of 2 was highly similar to those of rhoipteleanin H (12), indicating the presence of a galloyl group, an HHDP group, and a Phenyl (Phe)-Cycropentanone (Cp) group.10 The HMBC correlations of Cp-H-1 (δH 5.33) to Cp-C-2, 3, 5, 6 and Phe-C-1, 2, 6 and the correlations of Glc-H-1 (δH 4.53) to Cp-C-3, 4, 5 were also evidence supporting the existence of Phe-Cp group. HMBC correlations of Glc-H-5 (δH 5.40) to Galloyl-C-7 (δC 166.3), Glc-H-2 (δH 5.18) to Cp-C-7 (δC 162.8), Glc-H-3 (δH 5.45) to Phe-C-7 (δC 168.9), and Glc-H-4, 6 (δH 5.73 and 4.98) to HHDP-C-7, 7’ (δC 168.4) indicated linkage of Glc-C-5 to galloyl group, Glc-C-2, 3 to Phe-Cp group, and Glc-C-4, 6 to HHDP group. In the 1H NMR spectrum, remarkable difference were chemical shift of Glc-H-1 and 1H-1H coupling constant between Glc-H-1 and Glc-H-2 of 2 (δH 4.53, J = 1.0 Hz) and 12 (δH 5.36, J = 6.3 Hz). Moreover, coupling patterns of 2 were similar to those of stachyurin,6 while 12 were similar to those of 6. It was indicated that 2 was a stereoisomer of 12 and the stereochemistry of Glc-1 is only difference between 2 and 12. The atropisomers of HHDP and Phe-Cp groups were elucidated to be S-configurations by the NOESY correlations of Glc-H-6 (δH 4.98) to HHDP-H-3 (δH 6.54) and Glc-H-6 (δH 3.94) to Phe-H-3 (δH 6.32). It observed equally both 2 and 12. From these results, the structure of 2 was proposed as being represented formula 2. It presumed 2 was formed by oxidation of a progalloyl ring attached to Glc-C-1 of stachyurin followed by benzylic acid-type rearrangement and methylation.10

Since it had been reported hydrolyzable tannins had potent plant-growth inhibitory activity,15 the effect of isolated tannins 2-12 and tannin derivatives 13 and 14 8, 16 were examined on root growth of cress seedlings. All compounds except 7 and 13 exhibited inhibitory effect on root at 1×10-4 mol/L (Figure 3). Especially, 6 and 8 which possessed a galloyl group at Glc-1 showed the strongest activity (80% inhibition) at 1×10-4 mol/L. It suggested that a galloyl group was important to enhance the activity and especially connecting to Glc-1 was more effective. Moreover, the comparing between 5 (45 % inhibition) with galloyl groups at Glc-4, 6 and 8 (80% inhibition) with HHDP group at Glc-4, 6 showed the HHDP group at Glc-4, 6 was important for the activity.

EXPERIMENTAL
General procedure
Optical rotations were measured with a JASCO DIP-370 polarimeter. IR spectra were recorded on a JASCO FT/IR-300 spectrometer and UV spectra on a HITACHI U-2000A spectrometer. 1H and 13C NMR spectra were measured and recorded in Acetone-d6 or MeOD on Bruker Avance 500 or 600 spectrometers. Chemical shift values(δ) are recorded in parts per million (ppm) relative to NMR solvent Acetone-d6 (δH 2.05, δC 29.8) or MeOD (δH 3.35, δC 49.8). ESIMS were recorded on Waters-Platform LC and JEOL JMS-T100LC mass spectrometers.

Plant material
Galls of C. tschonoskii induced by infection of Eriophyes sp. were collected at University of Tsukuba, Japan. A voucher specimen has been deposited at the Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan.

Extraction and isolation
Galls of Carpinus tschonoskii (100 g) were homogenized by blender, extracted with MeOH (250 mL) and
concentrated in vacuo. The MeOH extracts (8.3 g) were partitioned between EtOAc (250 mL×3) and H2O (250 mL) and the H2O-layer was further partitioned with BuOH (250 mL×3). The EtOAc-soluble portion (1.85 g) was chromatographed on a C18 Sep-Pak cartridge (Waters, H2O/MeOH, 10:0→0:10) to give 4 fractions (fr. 1~fr. 4). Fr. 2 (274 mg) was subjected to a C18 Sep-Pak cartridge (Waters, H2O/MeOH, 10:0→0:10) to give 8 fractions. The fraction (78.4 mg) eluted with H2O/MeOH (8:2) on the C18 Sep-Pak cartridge was further separated by reversed-phase HPLC (TSK-gel ODS-80Ts, φ4.8×200 mm, flow rate 1.0 mL/min, A: 3% AcOH in H2O, B: 80% MeCN, A/B, 90:10→85:15) to give carpinerin B (2, 26.1 mg, tR 21 min) and rhoipteleanin H (12, 18.3 mg, tR 22 min). The fraction (78.7 mg) eluted with H2O/ MeOH (7:3) on the C18 Sep-Pak cartridge was further separated by reversed-phase HPLC (Cosmosil 5C18 MS-Ⅱ, φ10×250 mm , flow rate 2.5 mL/ min, A: 0.1% AcOH in H2O, B: 80% MeCN, A/B, 84:16 →77:23 → 72:28→ 65:35) to give casuarictin (6, 22.8 mg). The fraction (50.2 mg) eluted with H2O/MeOH (6:4) on the C18 Sep-Pak cartridge was further separated by reversed-phase HPLC (Cosmosil 5C18 MS-Ⅱ,　φ10×250 mm, flow rate 2.5 mL/ min, A: 0.1% AcOH in H2O, B: 80% CH3CN, A/B, 84:16→77:23→72:28→65:35→55:45) to give trigalloylglucose (3, 1.2 mg, tR 14 min), tellimagrandin Ⅱ (8, 9.4 mg, tR 19 min), carpinerin A (1, 3.4 mg, tR 20 min), and tetragalloylglucose (4, 2.1 mg, tR 21 min). The fraction (16.9 mg) eluted with H2O/MeOH (55:45) on the C18 Sep-Pak cartridge was pentagalloylglucose (5, 16.9 mg). The BuOH-soluble portion (2.21 g) was chromatographed on ODS (Cosmosil-75 C-18OPN, φ 2.2×30 cm, H2O/MeOH, 10:0→0:10) to give 17 fractions (fr. 1~fr. 17). Fr. 6 (317 mg) was subjected to a C18 Sep-Pak cartridge (H2O/MeOH, 10:0→0:10) to give 12 fractions. The fraction (27.2 mg) eluted with H2O/MeOH (7:3) on the C18 Sep-Pak cartridge was further separated by reversed-phase HPLC (Cosmosil 5C18 MS-Ⅱ, φ10×250 mm, flow rate 2.5 mL/min, A: 0.1% AcOH in H2O, B: 80% MeCN, A/B, 85:15→80:20→72:28→65:35) to give pedunculagin (7, 6.2 mg, tR 9 min) and hippophaenin A (11, 3 mg, tR 14 min). Fr. 7 (216 mg) was subjected to the C18 Sep-Pak cartridge (H2O/MeOH, 10:0→0:10) to give 9 fractions. The fraction eluted with H2O/MeOH (7:3) on the C18 Sep-Pak cartridge was further separated by reversed-phase HPLC (Cosmosil 5C18 MS-Ⅱ, φ 10×250 mm, flow rate 2.5 mL/ min, A: 0.1% AcOH in H2O, B: 80% MeCN, A/B, 85:15→75:25→70:30→65:35) to give liquidambin (10, 35.6 mg, tR 11 min) and casuarinin (9, 9.8 mg, tR 12 min).

Carpinerin A (1): A colorless powder; [α]D -6.0° (c 0.4, MeOH); IR (KBr) νmax 3427, 1773, 1718, 1617, 1509, 1509, 1449, 1341, 1222, 1042 cm-1; UV (MeOH) λmax (log ε) 220 (4.8), 277 (4.5) nm; ESIMS 1133 (M+Na)+; HRESIMS m/z 1133.0898 (M+Na)+, Δ -3.3 mmu; 1H NMR (500 MHz, MeOD): δ 7.14 (2H, s, galloyl), 6.96 (1H, s, EC-3’), 6.93 (1H, d, J = 8.8 Hz, Glc-1) , 6.66 (1H, s, HHDP-3), 6.53 (1H, s, HHDP’-3), 5.82 (1H, dd, J = 9.8, 10.7 Hz, Glc-3) , 5.81 (1H, d, J = 2.2 Hz, EC-1), 5.46 (1H, dd, J = 7.2, 13.2 Hz, Glc-6) , 5.21 (1H, t, J = 9.8, Glc-4) , 4.50 (1H, d, J = 4.8 Hz, EC-4”), 4.38 (1H, ddd, J = 2.0, 7.2, 9.8 Hz, Glc-5), 4.34 (1H, dt, J = 5.2, 7.1 Hz, EC-5”), 4.29 (1H, dd, J = 8.8, 10.7 Hz, Glc-2) , 4.19 (1H, dd, J = 8.7, 7.1 Hz, EC-6”), 3.91 (1H, dd, J = 13.2, 2.0 Hz, Glc-6) , 3.80 (1H, dd, J = 8.7, 7.1 Hz, EC-6”), 3.00 (1H, dd, J = 19.5, 2.2 Hz, EC-3), 2.21 (1H, d, J = 19.5 Hz, EC-3); 13C NMR (500 MHz, MeOD): δ 121.0 (galloyl-1), 111.3 (galloyl-2, 6), 147.5 (galloyl-3, 5), 141.6 (galloyl-4), 166.6 (galloyl-7), 117.4, 117.3 (HHDP, HHDP’-1), 118.8 (EC-1’), 126.9 (HHDP, HHDP’-2), 120.6 (EC-2’), 109.7, 109.4 (HHDP, HHDP’-3), 113.4 (EC-3’), 146.6, 146.7, 145.7, 145.4 (HHDP, HHDP’-4, 6), 149.1, 149.2 (EC-4’, 6’), 138.4, 138.2 (HHDP, HHDP’-5), 137.5 (EC-5’), 170.3, 168.6 (HHDP, HHDP’-7), 168.0 (EC-7’), 93.8 (Glc-1), 80.2 (Glc-2), 76.0 (Glc-3), 70.1 (Glc-4), 76.1 (Glc-5), 64.4 (Glc-6), 53.0 (EC-1), 49.8 (EC-2), 38.9 (EC-3), 200.5 (EC-4), 98.1 (EC-5), 109.3 (EC-6), 172.0 (EC-7), 174.2 (EC-1”), 82.8 (EC-2”), 109.7 (EC-3”), 92.8 (EC-4”), 74.7 (EC-5”), 75.3 (EC-6”).

Carpinerin B (2): A pale yellow powder; [α]D -50.0° (c 0.4, MeOH); IR (KBr) νmax 3422, 1718, 1700, 1617, 1508, 1459, 1194, 1066 cm-1; UV (MeOH) λmax (log ε) 216 (4.7), 266 (4.3) nm; ESIMS m/z 991; HRESIMS m/z 991.0872 (M+Na)+, Δ +4.5 mmu; 1H NMR (500 MHz, acetone-d6 + D2O): δ 7.11 (2H, s, galloyl), 6.89 (1H, s, HHDP-3), 6.54 (1H, s, HHDP’-3), 6.32 (1H, s, Phe-3), 5.73 (1H, dd, J = 2.1, 10.0 Hz, Glc-4), 5.45 (1H, d, J = 2.1 Hz, Glc-3), 5.40 (1H, dd, J = 4.3, 10.0 Hz, Glc-5) , 5.33 (1H, s, Cp-1) , 5.18 (1H, d, J = 1.0 Hz, Glc-2), 4.98 (1H, dd, J = 4.3, 13.4 Hz, Glc-6), 4.53 (1H, d, J = 1.0 Hz, Glc-1), 3.94 (1H, d, J = 13.4 Hz, Glc-6), 3.69 (3H, s, OMe); 13C NMR (500 MHz, acetone-d6 + D2O): δ 119.9 (galloyl-1), 109.9 (galloyl-2, 6), 145.7 (galloyl-3, 5), 139.3 (galloyl-4), 166.3 (galloyl-7), 115.2, 115.7 (HHDP, HHDP’-1), 110.2 (Phe-1), 126.0, 124.0 (HHDP, HHDP’-2), 123.8 (Phe-2), 108.1, 106.9 (HHDP, HHDP’-3), 106.1 (Phe-3), 144.8, 144.3, 144.5 (HHDP, HHDP’-4, 6), 145.4 (Phe-4), 136.4, 135.7 (HHDP, HHDP’-5), 134.9 (Phe-5), 146.0 (Phe-6), 168.4 (HHDP, HHDP’-7), 168.9 (Phe-7), 60.7 (Glc-1), 84.0 (Glc-2), 69.7 (Glc-3), 72.2 (Glc-4), 69.0 (Glc-5), 64.4 (Glc-6), 45.5 (Cp-1), 83.3 (Cp-2), 201.3 (Cp-3), 143.2 (Cp-4), 157.1 (Cp-5), 170.8 (Cp-6), 162.8 (Cp-7), 53.5 (OMe).

Rhoipteleanin H (12) : A pale yellow powder; 1H NMR (500 MHz, acetone-d6 + D2O): δ 7.11 (2H, s, galloyl), 6.83 (1H, s, HHDP-3), 6.52 (1H, s, HHDP’-3), 6.35 (1H, s, Phe-3), 5.52 (1H, dd, J = 2.5, 10.4 Hz, Glc-4) , 6.05 (1H, d, J = 2.0 Hz, Glc-3), 5.30 (1H, dd, J=3.8, 10.4 Hz, Glc-5) , 5.26 (1H, d, J = 3.3 Hz, Cp-1) , 4.91 (1H, d, J = 6.3 Hz, Glc-2), 4.98 (1H, dd, J=3.8, 13.4 Hz, Glc-6), 5.36 (1H, dd, J = 3.0, 6.3 Hz, Glc-1), 3.90 (1H, d, J = 13.4 Hz, Glc-6), 3.68 (3H, s, OMe); 13C NMR (500 MHz, acetone-d6 + D2O): δ 119.9 (galloyl-1), 109.9 (galloyl-2, 6), 145.7 (galloyl-3, 5), 139.3 (galloyl-4), 166.3 (galloyl-7), 115.2, 115.7 (HHDP, HHDP’-1), 110.2 (Phe-1), 126.0, 124.0 (HHDP, HHDP’-2), 124.2 (Phe-2), 108.1, 106.7 (HHDP, HHDP’-3), 106.7 (Phe-3), 144.8, 144.3, 144.5 (HHDP, HHDP’-4, 6), 145.4 (Phe-4), 136.4, 135.7 (HHDP, HHDP’-5), 134.9 (Phe-5), 146.0 (Phe-6), 168.7 (HHDP, HHDP’-7), 168.4 (Phe-7), 62.5 (Glc-1), 79.0 (Glc-2), 66.6 (Glc-3), 73.1 (Glc-4), 69.3 (Glc-5), 64.3 (Glc-6), 44.6(Cp-1), 82.4 (Cp-2), 200.0 (Cp-3), 142.4 (Cp-4), 155.1 (Cp-5), 171.2 (Cp-6), 162.8 (Cp-7), 53.4 (OMe).

Derivatization of 2,3;4,6-di-O-(S)-hexahydroxydiphenoyl-D-glucitol (13) from pedunculagin (7)
To a solution of 7 (5.0 mg) in MeOH (1 mL) NaBH4 (5.0 mg, 20 eq.) was added and the mixture was stired at rt for 2 h. AcOH (1 mL) was added to the reaction mixture and evaporated. The residue was dissolved in water and the pH was adjusted to 2.0 with 10 % HCl, and directly subjected to a C18 Sep-Pak cartridge (H2O/ MeOH, 10:0→0:10) to give 13 (4.0 mg).

Derivatization of 5-O-galloyl-2,3;4,6-di-O-(S)-hexahydroxydiphenoyl-D-glucitol (14) from liquidambin (10)
To a solution of 10 (5.0 mg) in MeOH (1 mL) NaBH4 (5.0 mg, 25 eq.) was added and the mixture was stired at rt for 2 h. AcOH (1 mL) was added to the reaction mixture and evaporated. The residue was dissolved in water and the pH was adjusted to 2.0 with 10 % HCl, and directly subjected to a C18 Sep-Pak cartridge (H2O/MeOH, 85:15→0:10) to give 14 (4.1 mg).

Partial hydrolysis of Carpinerin A (1)
A solution of 1 (0.2 mg) in H2O was kept at 90 °C for 24 h. The reaction mixture was analyzed by normal-phase HPLC [TSK-gel Amide-80, TOSOH, φ4.8×200 mm, flow rate 0.8 mL/min, 50 mmol/L (triethanolamine) phosphate buffer (pH 2.5) / MeCN, 30:70], and identified it as erythorbic acid (tR 5.8 min).

Derivatization of Casuarictin (6) from Carpinerin A (1)
A solution of 1 in 0.1% AcOH was kept at 80 °C for 30 min. The reaction mixture was analyzed by reverse-phase HPLC (Cosmosil 5C18 MS-Ⅱ, 10×250 mm, flow rate 2.5 mL/min, A: 0.1% AcOH in H2O, B: 80% MeCN, A/B, 84:16→77:23→72:28→65:35), and identified it as 6 (tR 22 min).

Bioassays
Each sample of tannins dissolved in MeOH (50 μL) was put in a 27 mm petri dish on filter paper. After vaporized MeOH, added distilled water (500 μL) and placed ten seeds of cress (Lepidium sativum L.). They were kept at 24 °C for 40 h in the dark and measured the length of root of cress. Percentage elongation was calculated by reference to the elongation of control.

ACKNOWLEDGEMENTS
This work was partly supported by Grant-in-aids for the Ministry of Education, Science, Sports, and Culture of Japan.

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