行政院國家科學委員會補助專題研究計畫成果報告
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※ 雲實與黃蝴蝶莢果之鞣質及相關連化合物之研究 ※
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計畫類別：■個別型計畫 □整合型計畫 計畫編號：NSC 90－2320－B－038－043－
執行期間：90 年 08 月 01 日至 91 年 07 月 31 日
計畫主持人：徐 鳳 麟 共同主持人：
計畫參與人員：
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：台北醫學大學藥學院
中 華 民 國 91 年 12 月 18 日
中文摘要
從雲實莢果之含水丙酮萃出物中，分離 出 (+)-gallocatechin (1), (-)-epicatechin 3-O-gallate (2), procyanidin B-1 (3), procyanidine B-3 (4), prodelphinidine B-1 (5), gallocatechin-(48)-catechin (6), 4-O- galloyl–(-)-shikimic acid (9), 5-O- galloyl- (-)-shikimic acid (10) 等化合物。 另外，從黃 蝴 蝶 之 果 實 ， 純 化 得 到 化 合 物 1, (-)-epigallocatechin 3-O-gallate (11), (-)-epicatechin (12), 1,6-di-O-galloyl--D- glucose (16), 2,6-di-O-galloyl-D-glucose (17), 1,2,3,6-tetra-O-galloyl--D-glucose (18) 。 這 些化合物之結構是藉由光譜解析以及化學之 方法而定。其中化合物 7, 8, 13, 14 及 15 為 新穎之成分。.
關鍵詞：雲實、黃蝴蝶、鞣質、豆科
Abstract
From the aqueous acetone extract of the fruit of C. decapetala: (+)-gallocatechin (1), (-)-epicatechin 3-O-gallate (2), procyanidin B-1 (3), procyanidine B-3 (4), prodelphinidine B-1 (5), gallocatechin- (48)-catechin (6), 4-O-galloyl– (-)-shikimic acid (9), 5-O-galloyl- (-)-shikimic acid (10) were isolated. From the fruit of C. pulcherrima: 1, (-)-epigallocatechin 3-O-gallate (11), (-)-epicatechin (12), 1,6-di-O-galloyl--D-glucose (16), 2,6-di-
O-galloyl-D-glucose
(17), 1,2,3,6-tetra-O- galloyl--D-glucose (18) were obtained. The structure of those compounds was determinate base on spectral analysis and chemical evidence.Compounds 7, 8, 13, 14 and 15 were novel constituents.
Keywords:
Caesalpinia decapetala, Caesalpinia pulcherrima,
Tannin, LeguminosaeINTRODUCTION
Leguminosae, a great plant family, is widely distributed in the whole world and is rich in tannin substance. There are about 60 genera and 170 species in Taiwan, 1 and have been an important source of the economic and medicinal plants. Caesalpinia decapetala was traditionally taken as antitussives, expectorants and anthelmintics.2 On the other hand,
Caesalpinia pulcherrima have been used for
botanical remedies.3 According to the literatures, the rhizome contains cassane diterpenoids; the root contains caesalpin diterpenoids with anticarcinogenic effect;4 the seeds are rich in galactomanna.5 AS a consequence, a wide spectrum of biological activities has been reported for many condensed tannins6 for example, silymarin for liver problems,7 ginkgo flavone glycosides for circulatory disorders,8 pycnogenol from pin tree for antioxidant.9 This paper deals with the isolation and structure determination of novel condensed tannins of above-mentioned two plants.RESULTS AND DISCUSSIONS
From the aqueous acetone extract of the fruit of C. decapetala: (+)-gallocatechin (1),10 (-)-epicatechin 3-O-gallate (2),10 procyanidin B-1 (3),11 procyanidine B-3 (4),11 prodelphinidine B-1 (5)11, gallocatechin- (48)-catechin (6),12 4-O-galloyl–
(-)-shikimic acid (9),13 5-O-galloyl- (-)-shikimic acid (10)13 were isolated. From C.
pulcherrima: 1, (-)-epigallocatechin 3-O-gallate
(11),10 (-)-epicatechin (12),10 1,6-di-O-galloyl--D-glucose (16),14 2,6-di-O-galloyl-D-glucose
(17),15 1,2,3,6-tetra-O- galloyl--D-glucose (18)16 were obtained. The structure of those compound were determinate base on spectral analysis and chemical evidence.Compounds 7, 8, 13, 14 and 15 were novel constituents.
EXPERIMENTAL
Plant material
The fresh pods of Caesalpinia decapetala (Roth.) Alston. and Caesalpinia pulcherrima Swartz. was collected in Chang-Jhu, Ping-Tung, Taiwan (Jul. 2000), and verified by Dr.
Feng-Chi Ho (Taiwan Forestry Research Institute, Heng-Chun Branch, Ping-Tung, Taiwan).
Extraction and isolation
[I] The fresh pods of C. decapetala (6.5 kg) were extracted four times with 70 % aqueous acetone at room temp. The extract was concentrated under reduced pressure and the resulting brown precipitates were removed by filtration. The filtrated was concentrated and subjected to Sephadex LH-20 column chromatography with water containing increasing amounts of MeOH and finally with a mixture of H2O-acetone (1:1) to give fraction 1 (16 g), fraction 2 (7 g). fraction 3 (10.6 g), fraction 4 (14 g). Fraction 1 was chromatographed over Sephadex LH-20, MCI-gel CHP-20P, Cosmosil C18-OPN (H2O-MeOH) (1:0-0:1) to yield 4-O-galloyl-
(-)-shikimic acid (9) (29 mg). Repeated chromatography of fraction 2 on Sephadex LH-20, MIC-gel CHP-20P, Fuji-gel ODS-G3 (H2O-MeOH, 1:0-0:1) to obtain 5-O-galloyl- (-)-shikimic acid (10) (32 mg). Fraction 3 was subjected to Sephdex LH-20, Fuji-ge ODS-G3, MCI-gel CHP-20P (H2O-MeOH, 1:0-0:1) to yield (+)-gallocatechin (1) (851 mg), procyanidin B-1 (3) (101 mg), procyanidin B-3 (4) (95 mg), and prodelphinidin B-3 (5) (28 mg). Fraction 4 was applied to columns of Sephadex LH-20, MIC-gel CHP-20P, Cosomsil C18-OPN, Fuji-gel ODS-G3 (H2O-MeOH, 1:0-0:1) to give (-)-epicatechin 3-O-gallate (2) (38 mg), gallocatechin-(48)-catechin (6) (63 mg), prodelphinidin B-3 3-O-gallate (7) (481 mg), and prodelphinidin B-6 3-O-gallate (8) (75 mg).
[II] The fresh pods of C. pulcherrima (2.1 kg) were extracted four times with 70 % aqueous acetone at room temp. The extract was concentrated under reduced pressure, and the resulting brown precipitates were removed by filtration. The filtrate was concentrated and subjected to Sephadex LH-20 column chromatography with H2O containing increasing amounts of MeOH and finally with a mixture of H2O:acetone (1:1) to give fraction 1 (10.2 g), fraction 2 (24.1 g), fraction 3 (16.3 g), and fraction 4 (14.6 g). Fraction 1 was chromatographed over MIC-gel CHP-20P, Sephadex LH-20 (H2O-MeOH, 1:0-0:1) to yield 1,6-di-O- galloyl--D-glucose (16) (104 mg) and 2,6-di-O-galloyl-D-glucose (17) (932 mg). Purification of Fraction 2 by MCI-gel CHP-20P, Fujigel ODS-G3, Cosmosil C18-OPN (H2O-MeOH, 1:0-0:1) to give 1,2,3,4-tetra-O-galloyl--D-glucose (18) (81
mg), 3,4,5-tri-O-galloyl-(-)-shikimic acid (15) (131 mg), and (+)-gallocatechin (1) (581 mg).
Fraction 3 repeatedly chromatographed over sephadex LH-20, Fuji-gel ODS-G3, Cosmosil C18-OPN, MIC-gel CHP-20P (H2O-MeOH, 1:0-0:1) to yield (-)-epigallocatechin 3-O-gallate (11) (1.3 g), and (-)-epicatechin (12) (108 mg). Fraction 4 was applied to columns of MCI-gel CHP-20P, Fuji-gel ODS-G3, Cosmosil C18 OPN, Sephadex LH-20 (H2O-MeOH, 1:0-0:1) to yiel prodelphinidin B-3 3,3’-di-O-gallate (13) (338 mg), and prodelphinidin C-2 3,3’-di-O-gallate (14) (420 mg).
Prodelphinidin B-3 3-O-gallate: A tan amorphous powder, []D –140.0 (c=1.0, acetone). Anal. Calcd for C37H30O18．7/2 H2O:
C, 53.82; H, 4.52. Found: C, 53.93; H, 4.38.
negative FAB-MS: 76.1 [M-H]-. 1H-NMR (acetone-d6+D2O): 2.38-3.11 (2H, m, 4’-H), 4.21-4.82 (4H in total, m, 2, 4, 2’, 3’-H), 5.84-6.04 (4H, in total, m, 3, 6, 8, 6’-H), 6.26, 6.43, 6.53, 6.59 (4H in total, each s, 12, 16, 12’, 16’-H), 6.91, 7.00, 7.04 (2H in total, each s, galloyl H). 13C-NMR (acetone-d6+D2O): 28.7 (C-4’), 67.8 68.5 (C-3’), 73.3 (C-3), 81.5, 81.9 (C-2’), 83.4 (C-2), 95.2, 95.7, 96.1, 96.7, 97.3 (C-6, 8, 6’) 100.5, 101.3, 101.6 (C-10, 10’), 105.7 (C-8’), 108.1 (galloyl C-2, 6), 105.7, 109.9,110.2 (C-12, 16, 12’, 16’), 121.5 (galloyl C-1), 129.7, 130.9 (C-11, 11’), 133.2, 134.4 (galloyl C-4), 158.6 (C-14, 14’), 145.6, 145.8 (C-13, 15, 13’, 14’, galloyl C-3, 5), 154.7, 155.0, 155.5, 156.6, 156.7, 157.7, 158.0 (C-5, 7, 9, 5’, 7’, 9’, 5”, 7”, 9”), 165.4, 165.9 (-COO-).
Enzymatic hydrolysis of 7 (prodelphinidin B-3 3-O-gallate): A solution of 7 (80 mg) in H2O (4
ml) was incubated with tannase at 37 C for 4 hr. The reaction mixture as concentrated to dryness in vacuo and the residue was treated with MeOH. The MeOH soluble portion was purified by Sephadex LH-20 with 80% aqueous MeOH to furnish gallic acid (19.2 mg) and prodelphinidin B-3 (5) (41 mg), off-whit amorphous, []D –143.5 (c=1.0, acetone).
1H-NMR (acetone-d6+D2O): 2.58 (1H, dd, J=7, 15 Hz, 4’-H), 2.94 (1H, dd, J=5, 15 Hz, 4’-H), 4.25-4.67 (3H, m, 2, 4, 2’-H), 5.78-6.14 (3H in total, m, 6, 8,6’-H), 6.19, 6.41, 6.58, 6.62 (4H in total, each s, 12, 16, 12’, 16’-H).
Thiolytic degradation17 of 7: A mixture of 7 (312 mg), benzylmercaptan (3 ml) and acetic acid (1 ml) in EtOH (5 ml) was refluxed for 4 h.
The solvent was evaporated off under reduced pressure, and the residue was subjected to Sephadex LH-20 chromatography with chlorofom-MeOH (4:1-1:1) to yield (+)-gallocatechin (81 mg), 7b (62 mg) and 7c (75 mg). 7b is an amorphous powder, []D
+74.7 (c=0.9, acetone), 1H-NMR (acetone-d6+D2O): 3.87 (2H, s, -S-CH2-), 4.62 (1H, d, J=4Hz, 4-H), 5.32 (1H, d, J=10 Hz, 2-H), 5.46 (1H, dd, J=4,10 Hz, 3-H), 5.89, 6.59 (each 1H, d, J=2 Hz, 6, 8-H), 6.59 (2H, s, 12, 16-H), 7.14 (2H, s, galloyl-H), 7.16-7.44 (5H, m, aromatic H). Anal. Calcd for C29H24O11S．
5/2 H2O: C, 57.91; H, 4.49, Found: C, 57.94; H, 4.33. Negtive FAB-MS m/z: 579 [M-H]-.
1H-NMR (acetone-d6+D2O): 3.79, 3.89 (each 1H, d, J=12 Hz, -S-CH2-), 4.20 (1H, d, J=4 Hz, 4-H), 5.10 (1H, d, J=6 Hz, 2-H), 5.76 (1H, dd, J=4, 6 Hz, 3-H), 6.04, 6.12 (each 1H, d, J=2 Hz, 6, 8-H), 6.53 (2H, s, 12, 16-H), 7.25 (2H, S, galloyl H), 7.15-7.30 (5H, m, aromatic H).
Desulfurization: 7c (45 mg) in EtOH (8 ml)
was treated at room temperature with an EtOH slurry of Raney-nikel (W-4), for 30 min. After filtration of the catalyst, the filtrate was concentrated under reduced pressure to dryness, and the residue was purified by Sephadex LH-20 with H2O-MeOH to give (+)-galloylcatechin 3-O-gallate (20) (7.5 mg).
Prodelphinidin B-6 3-O-gallate (8):
A tan amorphous powder, []D –118.4 (c=0.1, acetone), Anal. Calcd for C37H30O18．2H2O: C, 55.46; H, 4.29. Found: C, 55.17, H, 4.40.
Negative FAB-MS m/z: 764 [M-H]-. 1H-NMR (acetone-d6+D2O): 2.40 (1H, dd, J=9, 16 Hz, 4’-H), 2.98 (1H, dd, J=6, 16 Hz, 4’-H), 4.36 (1H, d, J=8 Hz, 2’-H), 4.68 (1H, d, J=10 Hz, 4-H), 4.82 (1H, d, J=7 Hz, 2-H), 5.86-6.32 (4H, m, 3, 6, 8, 8’-H), 6.46. 6.62 (each 2H, s, 12, 16, 12’, 16’-H), 6.97 (2H, s, galloyl H). 13C-NMR (acetone-d6+D2O): 30.4 (C-4’), 35.7 (C-4), 68.1 (C-3’), 74.5 (C-3), 81.8 (C-2’), 82.4 (C-2), 96.1, 96.3, 97.3, 97.7 (C-6, 8, 8’), 107.8, 106.6 (C-10, 10’), 107.1, 107.6, 108.0 (C-12, 16, 12’, 16’), 107.9 (C-6’), 110.0, 110.3, 110.5 (galloyl C-2, 6), 121.6 (galloyl C-1), 129.6, 130.8 (C-11, 11’), 133.4, 135.5 (C-14, 14’), 138.6 (galloyl C-4), 145.6 145.9, 146.0 (C-13, 15, 13’, 15’, galloyl C-3, 5), 154.5, 154.6, 155.2, 157.3, 158.1, 158.5 (C-5, 7, 9, 5’, 7’, 9’), 165.8 (-COO-).
Enzymatic hydrolysis of 8: Hydrolysis of 8 (25 mg) by tannase as describes for previous process to give gallic acid (4 mg) and 8a (12 mg). 8a: an amorphous powder, []D –94.6
(c=1.0, acetone). 1H-NMR (acetone-d6 +D2O):
2.58-3.13 (2H, m, 4’-H), 3.95 (1H, m, 3-H), 4.30-4.50 (3H, m, 2, 4, 2’-H), 5.83-6.25 (3H in total, m, 6, 8, 6’-H), 6.48, 6.57 (each 2H, 12, 16,
12’, 16’-H).
Thiolytic degradation and desulfurization of 8:
8 (21 mg) dissolved in EtOH (5 ml) and
thyolysis with AcOH-benzylmercaptan to give7b
(3-O-galloyl gallocatechin 4- bezylthioether (3.5 mg), 7c (3-O-galloyl gallocatechin 4-bezylthioether (2.3 mg) and (+)-gallocatechin (6 mg). As usual process after desulfurization of 7c to yield 20.Prodelphinidin B-3 3,3-di-O-gallate (13):
An amorphous powder, []D –135.2 (c=0.9, acetone), Anal. Calcd for C44H32O22．2H2O: C, 56.66; H, 3.89. Found: C, 56.52, H, 4.04.
Negative FAB-MS m/z: 913 [M-H]-. 1H-NMR (acetone-d6+D2O): 2.60-2.72 (2H, m, 4’-H), 4.54 (1H, d, J=10 Hz, 4-H), 4.84 (2H, d, J=8 Hz, 2, 2’-H), 5.17-5.37 (1H, m, 3’-H), 5.81-6.28 (4H in total, m, 3, 6, 8, 6’-H), 6.50, 6.53, 6.59, 6.66 (4H in total, each s, 12, 16, 12’, 16’-H), 7.01, 7.07, 7.17 (4H in total, each s, 2 x galloyl H). 13C-NMR (acetone-d6+D2O): 23.1 (C-4’), 36.2 (C-4), 67.2 (C-3’), 76.2 (C-3), 78.7 (C-2’), 81.8 (C-2), 95.9, 96.7, 97.8 (C-6, 8, 6’), 106.8 (C-8’), 107.0, 107.8, 110.0, 130.3 (C-11, 11’), 133.1, 133.4 (C-14, 14’), 138.7, 138.9 (galloyl C-4), 145.4, 146.3, 145.9 (C-13, 15, 13’, 15’, galloyl C-3, 5), 153.9, 154.6, 155.1, 155.4, 155.9, 156.8, 157.3, 158.1, 158.6 (C-5, 7, 9, 5’, 7’, 9’), 165.0, 166.0, 167.1 (-COO-).
Enzymatic hydrolysis of 13: Hydrolysis of 13 (35 mg) with tannase as describes for previous process to obtain gallic acid (8 mg) and prodelphinidin B-3 (14 mg).
Prodelphinidin C-2 3,3-di-O-gallate (14):
An amorphous powder, []D –188.5 (c=1.0,
acetone), Anal. Calcd for C59H46O29．8/3 H2O:
C, 55.93; H, 4.08. Found: C, 55.45, H, 4.22.
Negative FAB-MS m/z: 1217 [M-H]-. 1H-NMR (acetone-d6+D2O):2.31-3.16 (2H, m, 3”-H), 3.49-3.84 (1H, m, 3’-H), 4.31 (1H, d, J=9 Hz, 2”-H), 4.54-4.94 (4H in total, m, 2, 4, 2’, 4’-H), 5.58-6.12 (6H in total, m, 3, 6, 8, 3’, 6’, 6”-H), 6.29, 6.53, 6.67 (6H in total, each s, 12, 16, 12’,16, 12”, 16”-H), 6.85, 6.93, 7.00, 7.08 (4H in total, each s, galloyl H). 13C-NMR (acetone-d6+D2O): 28.3 (C-4”), 36.1 (C-4. 4’), 68.7 (C-3”), 72.7 (C-3, 3’), 81.4, 81.7 (C-2’, 2”), 83.7 (C-2), 96.5, 97.6 (C-6, 8, 6’, 8’), 100.7 (C-10, 10’, 10”), 105.6, 106.3 (C-8’, 8”), 106.6, 107.1, 110.0, 110.3 (C-12, 16, 12’, 16’, 12”, 16”), 121.9 (galloyl C-1), 129.6, 130.0, 130.8 (C-11, 11’, 11”), 133.6, 133.9 (C-14, 14’, 14”), 138.2, 138.6 (galloyl C-4), 145.4, 145.8 (C-13, 15, 13’, 15’, 13”, 15”, galloyl C-3, 5), 155.4,155.9, 158.0 (C-5, 7, 9, 5’, 7’, 9’, 5”, 7”, 9”), 164.9 (-COO-).
Enzymatic hydrolysis of 14: Hydrolysis of 14 (50 mg) with tannase as describes for previous process to give gallic acid (8 mg) and 14a (27 mg). 14a: []D –189.5 (c=0.9, acetone), Anal.
Calcd for C45H38O21．6 H2O: C, 52.84; H, 4.92.
Found: C, 52.97, H, 4.71. Negative FAB-MS m/z: 913 [M-H]-. 1H-NMR (acetone-d6+D2O):
2.46-3.16 (2H, m, 4”-H), 4.16-4.91 (5H, m, 2, 4, 2’, 4’, 2”-H), 5.84-6.27 (4H in total, m, 6, 8, 6’, 6”-H), 6.56, 6.60, 6.63, 6.69 (6H in total, each s, 12, 16, 12’, 16’, 12”, 16”-H).
Partial thiolysis of 14: A mixture of 14 (130 mg), benzylmercaptan (4 ml), acetic acid (2 ml) and EtOH (8 ml) was heated under reflux for 1 h. Work-up as described above gave partial degradation products, (+)-gallocatechin (9 mg),
(+)-gallocatechin 4-benzylthioether (7b) and
14b (38 mg). 14b: A tan amorphous powder,
[]D –84.55 (c=0.9, acetone), Anal. Calcd for C51H40O22．4 H2O: C, 55.24; H, 4.36. Found: C, 55.56, H, 4.46. Negative FAB-MS m/z: 1035 [M-H]-. 1H-NMR (acetone-d6+D2O): 3.79 (2H, S, -S-CH2-), 4.49-5.55 (5H, m, 2, 4, 2’, 3’, 4’-H), 5.81-6.11 (4H, m, 3-H, A-ring H), 6.35, 6.42, 6.59, 6.72 (4H, m, 12, 16, 12’, 16’-H), 6.92-7.14 (5H, m, aromatic H), 7.21 (4H, s, 2 x galloyl H).Desulfurization of 14b: 14b (20 mg) dissolved in 20 % AcOH-EtOH (5 ml) then sterring with Raney nikel under room temperature for 2 h.
Filtrated the Raney nikel, concentrated the filtrate, at TLC the spot of 13 was observed [Rf:
0.25, silica gel, bezene-HCOOEt-HCOOH (1:7:1)]
3,4,5-Tri-O-galloyl-(-)-shikimic acid (15):
An off-whit amorphous powder, []D –223.1
(c=1.0, acetone), Anal. Calcd for C28H22O17．2 H2O: C, 50.45; H, 3.93. Found: C, 50.26, H, 4.17. Negative FAB-MS m/z: 621 [M-H]-.
1H-NMR (acetone-d6+D2O): 2.72 (1H, dd, J=5.6, 19.0 Hz, 2-H), 3.12 (1H, dd, J=5.1, 19.0 Hz, 2-H), 5.66 (1H, dd, J=3.8, 8.1 Hz, 4-H), 5.73 (1H, ddd, J=5.1, 5.6, 8.1 Hz, 3-H), 6.04 (1H, t, J=3.8 Hz, 5-H), 6.93 (1H, d, J=3.8 Hz, 6-H), 7.06, 7.12, 7.14 (each 2H, s, galloyl H).
Enzymatic hydrolysis of 15: 15 (45 mg) hydrolysis with tannase uder room temperature, and purified by Sephadex LH-20 to give gallic acid (22 mg) and 15a (4 mg). 15a: whit amorphous powder, []D –19.1 (c=0.9, MeOH), identified by co-TLC with authentic sample on silica gel [Rf 0.45, benzene-HCOOEt-HCOOH
(1:5:1.5)] and cellulose [Rf 0.60, n-BuOH-AcOH-H2O (4:1:5)].
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