Phenolic Compounds and Triterpenes from the Barks of Diospyros burmanica

Abstract

Diospyros burmanica Kurz. is an evergreen deciduous tree distributed in Mandalay of Myanmar, which belongs to the family of Ebenaceae. In Myanmar, it has been used to treat diarrhea, diabetes, diabetes and also as lumbers. In this study, seven flavonoids (1 - 7), a phenolic compound (8), and five triterpenes (9 - 13) were isolated from the barks of D. burmanica and their chemical structures were elucidated. Isolates were identified to be (+)-catechin (1), (+)-catechin 3-O-$\alpha$-L-rhamnopyranoside (2), (+)-catechin 3-O-gallate (3), (-)-epicatechin (4), (-)-epicatechin 3-O-gallate (5), (+)-afzelechin 3-O-$\alpha$-L-rhamnopyranoside (6), (+)-2,3-trans-dihydrokaempferol 3-O-$\alpha$-L-rhamnopyranoside (7), methyl gallate (8), lupeol (9), methyl lup-20(29)-en-3-on-28-oate (10), $\beta$-amyrin (11), $\alpha$-amyrin (12), $3\beta$-hydroxy-D:B-friedo-olean-5-ene (13) through MS, ¹H NMR and ¹³C NMR spectroscopic evidences.

Introduction

Diospyros burmanica Kurz (Ebenaceae) is an evergreen deciduous tree distributed in the Mandalay region of Myanmar, and local traditional practitioners have used this plant as a medicinal plant to treat diabetes, diarrhea and dysentery. Over 350 species of genus Diospyros have been known worldwide and many have been used as traditional medicines in the India, Africa and China,1 and especially D. kaki has well been investigated for its phytochemicals and biological activities.2-6 As for the study of D. burmanica, only a paper has been reported revealing bisnaphtoquinones and naphthol derivatives and their leishmanicidal inhibitory activities.7 This study focused on the further phytochemical investigation of D. burmanica and led to the isolation of seven flavonoids, a phenolic compound, and five triterpenes.

 

Experimental

General experimental procedure − 1H NMR and 13C NMR spectra were obtained on a Bruker AscendTM 500 spectrometer (Bruker, Germany). Mass spectra were recorded by using an Agilent 6530 ESI-QTOF MS (Agilent Technologies, USA) and JEOL JMS-700 spectrometer (JEOL, Japan). A Gilson preparative HPLC system (Gilson, USA) was used to isolate compounds and equipped with a GX-271 liquid handler, binary pumps, and an UV/VIS-155 detector. An MPLC system composed of an IOTA S 300 pump (ECOM, Czech Republic) and a Saphire 600 UV-VIS variable wavelength detector (ECOM, Czech Republic) were used. The preparative HPCCC (Dynamic Extractions, UK) possessed two sets of two bobbins. One bobbin was equipped with an analytical coil (11 mL, 0.8 mm ID), and the other with a preparative coil (492 ml, 4 mm ID). Deionized water was prepared by Millipore Milli-Q water purification system (Millipore, USA), and organic solvents for column chromatography were purchased from Daejung-Chemical and Metals Co. Ltd. (Kyunggi-Do, Korea). Silica gel and reversed-phase silica gel for column chromatography were Kieselgel 60 (230 - 400 mesh, Merck, Germany) and YMC RP-18 resin (YMC, Japan), respectively. HPLC column was YMC-Pack ODS-A (250 × 20 mm, 5 μm, YMC, Japan) and YMC-Pack Ph (250 × 20 mm, 5 μm, YMC, Japan).

Plant material − Barks and leaves of D. burmanica were collected from AKNP of Myanmar in February 2012, and identified by Professor Young-Dong Kim (Hallym University, Chuncheon, Korea). A voucher specimen (CU-2014-2-12) was deposited at the Herbarium of College of Pharmacy, The Catholic University of Korea.

Extraction and isolation − Extraction and isolation - Dried barks (1.3 kg) of D. burmanica were extracted with 100 % MeOH in an ultrasonic bath for (5 L × 2 h × 3 times). After evaporating solvent in vacuo, the methanolic extract (221.5 g) was suspended in water and partitioned sequentially with CH2Cl2 (8.1 g), EtOAc (68.9 g), and n-BuOH (93.9 g). The EtOAc soluble fraction was subjected to silica gel column chromatography (CC) (CHCl3: MeOH, 10:1→1:1, v/v) to yield five subfractions (E1~E5). E1 (7.5 g) was subjected to countercurrent chromatography (CCC) with solvent composition of n-hexane-EtOAC-MeOH-water (2:8:2:8, v/v) to yield another five fractions (E1-1~E1-5). Compound 2 (243.7 mg) was purified from E1-1 by silica gel column chromatography (CHCl3-MeOH-water, 12:5:1, v/v). Fraction E1-2 was subjected to silica gel CC (CHCl3-MeOH-water, 20:5:1, v/v) to give compound 1 (282.9 mg). Fraction E1-3 was chromatographed on silica gel CC (CHCl3-MeOH, 5:1, v/v) to give four subfraction (E1-3-1~E1-3-4), and E1-3-2 was purified by RP-HPLC (YMC-Pack Ph, MeOH-water, 25:75, v/v) to give compound 8 (56.5 mg) and 7 (27.3 mg). Compound 3 and 5 were obtained from E1-3-4 through silica gel CC (CHCl3-water, 5:1, v/v) and RP-HPLC (YMC-Pack ODS-A, MeOH-water, 30:70, v/v). Compound 4 (1.7 mg) and 6 (7.6 mg) were obtained from E2 through silica gel CC (CHCl3-MeOH-water, 15:5:1, v/v) and RP-HPLC (YMC-Pack Ph, MeOH-water, 33:67, v/v). The CH2Cl2 fraction was subjected to silica gel CC (CHCl3: MeOH, 50:1 → 5:1; v/v) to provide seven subfractions (M1~M7). M1 was subjected to silica gel CC (n-hexane-EtOAc, 10:1, v/v) to yield two subfractions (M1-1, M1-2). Compound 10 (12.0 mg) was obtained from M1-1 through RP-HPLC (YMC-Pack ODS-A, MeOH-Water, 95:5, v/v). Compound 11 (3.8 mg), 12 (10.2 mg) and 13 (2.2 mg) was obtained from M1-2 through RP-HPLC (ODS-A, MeOH). Compound 9 (21.5 mg) was obtained from M2 through silica gel CC (n-hexane-EtOAc, 20:1, v/v) and RP-HPLC (YMC-Pack Ph).

(+)-Catechin (1) : +21 (c 0.2, MeOH); UV (MeOH) λmax 277 nm; ESI-QTOF MS: m/z 291.0866 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 4.56 (1H, d, J = 7.5 Hz, H-2), 3.96 (1H, m, H-3), 2.85 (1H, dd, J = 16.3, 5.3 Hz, H-4eq), 2.50 (1H, d, J = 16.2, 8.0 Hz, H-4ax), 5.85 (1H, d, J = 2.2 Hz, H-6), 5.92 (1H, d, J = 2.2 Hz, H-8), 6.84 (1H, d, J = 2.0 Hz, H-2'), 6.76 (1H, d, J = 8.0 Hz, H-5'), 6.72 (1H, dd, J = 8.0, 2.0 Hz, H-6'); 13C NMR (CD3OD, 125 MHz): δ 83.0 (C-2), 69.0 (C-3), 28.7 (C-4), 157.7 (C-5), 96.4 (C-6), 158.0 (C-7), 95.6 (C-8), 157.1 (C-9), 100.9 (C-10), 132.4 (C-1'), 115.4 (C-2'), 146.4 (C-3'), 146.4 (C-4'), 116.2 (C-5'), 120.2 (C-6')

(+)-Catechin 3-O-α-L-rhamnopyranoside (2) : −18.2 (c 0.5, MeOH); UV (MeOH) λmax 279 nm; ESIQTOF MS: m/z 437.1454 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 4.62 (1H, d, J = 7.7 Hz, H-2), 3.93 (1H, m, H-3), 2.88 (1H, dd, J = 16.2, 8.3 Hz, H-4eq), 2.64 (1H, d, J = 16.1, 8.3 Hz, H-4ax), 5.85 (1H, d, J = 2.2 Hz, H-6), 5.93 (1H, d, J = 2.4 Hz, H-8), 6.84 (1H, d, J = 1.9 Hz, H-2'), 6.76 (1H, d, J = 8.0 Hz, H-5'), 6.72 (1H, dd, J = 8.2, 1.9 Hz, H-6'), 4.29 (1H, d, J = 1.2 Hz, H-1"), 1.25 (3H, d, J = 6.1 Hz, H-6"); 13C NMR (CD3OD, 125 MHz): δ 81.3 (C-2), 76.1 (C-3), 28.1 (C-4), 157.7 (C-5), 95.7 (C-6), 158.1 (C-7), 96.6 (C-8), 157.0 (C-9), 102.3 (C-10), 132.1 (C-1'), 115.2 (C-2'), 146.4 (C-3'), 146.5 (C-4'), 116.3 (C-5'), 120.0 (C-6'), 100.8 (C-1"), 72.2 (C-2"), 72.4 (C-3"), 74.1 (C-4"), 70.5 (C-5"), 18.1 (C-6")

(+)-Catechin 3-O-gallate (3) : +8.9 (c 0.1, MeOH); UV (MeOH) λmax 277 nm; ESI-QTOF MS: m/z 443.2216[M+H]+; 1H NMR (CD3OD, 500 MHz): δ 5.06 (1H, d, J = 6.1 Hz, H-2), 5.37 (1H, m, H-3), 2.82 (1H, dd, J = 16.5, 5.1 Hz, H-4eq), 2.71 (1H, d, J = 16.5, 6.0 Hz, H-4ax), 5.94 (1H, d, J = 2.2 Hz, H-6), 5.96 (1H, d, J = 2.4 Hz, H-8), 6.84 (1H, s, H-2'), 6.72 (2H, s, H-5', 6'), 6.96 (2H, s, H-2", 6"); 13C NMR (CD3OD, 125 MHz): δ 75.9 (C-2), 71.3 (C-3), 24.5 (C-4), 156.6 (C-5), 96.6 (C-6), 157.7 (C-7), 95.8 (C-8), 158.2 (C-9), 99.8 (C-10), 131.6 (C-1'), 114.6 (C-2'), 146.3 (C-3'), 146.4 (C-4'), 116.4 (C-5'), 119.4 (C-6'), 121.5 (C-1"), 110.3 (C-2", 6"), 146.5 (C-3", 5"), 140.0 (C-4"), 167.7 (-COO-)

(−)-Epicatechin (4) : −38.5 (c 0.9, MeOH); UV (MeOH) λmax 280 nm; ESI-QTOF MS: m/z 291.1953 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 4.82 (1H, s, H-2), 4.17 (1H, m, H-3), 2.73 (1H, dd, J = 16.5, 2.8 Hz, H-4eq), 2.86 (1H, dd, J = 16.5, 4.9 Hz, H-4ax), 5.91 (1H, d, J = 2.2 Hz, H-6), 5.94 (1H, d, J = 2.2 Hz, H-8), 6.97 (1H, d, J = 1.8 Hz, H-2'), 6.75 (1H, d, J = 8.2 Hz, H-5'), 6.79 (1H, dd, J = 8.4, 1.7 Hz, H-6'); 13C NMR (CD3OD, 125 MHz): δ 80.0 (C-2), 67.7 (C-3), 29.4 (C-4), 157.9 (C-5), 96.0 (C-6), 158.2 (C-7), 96.5 (C-8), 157.5 (C-9), 100.2 (C-10), 132.5 (C-1'), 115.5 (C-2'), 146.1 (C-3'), 146.0 (C-4'), 116.0 (C-5'), 119.5 (C-6')

(−)-Epicatechin 3-O-gallate (5) : −12.4 (c 0.1, MeOH); UV (MeOH) λmax 277 nm; ESI-QTOF MS: m/z 443.2216 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 5.03 (1H, s, H-2), 5.52 (1H, m, H-3), 2.99 (1H, dd, J = 17.3, 4.7 Hz, H-4eq), 2.85 (1H, d, J = 17.3, 2.1 Hz, H-4ax), 5.95 (1H, d, J = 2.4 Hz, H-6), 5.96 (1H, d, J = 2.4 Hz, H-8), 6.93 (1H, d, J = 1.9 Hz, H-2'), 6.69 (1H, d, J = 8.3 Hz, H-5'), 6.81 (1H, dd, J = 8.2, 1.7 Hz, H-6'), 6.95 (2H, s, H-2", 6"); 13C NMR (CD3OD, 125 MHz): δ 78.8 (C-2), 70.1 (C-3), 27.0 (C-4), 157.4 (C-5), 96.7 (C-6), 158.0 (C-7), 96.0 (C-8), 158.0 (C-9), 99.6 (C-10), 131.6 (C-1'), 115.3 (C-2'), 146.1 (C-3'), 146.1 (C-4'), 116.2 (C-5'), 119.5 (C-6'), 121.6 (C-1"), 110.4 (C-2", 6"), 146.5 (C-3", 5"), 139.9 (C-4"), 167.8 (-COO)

Fig. 1.Chemical structures of compounds 1 - 13 from D. burmanica Kurz.

(+)-Afzelechin 3-O-α-L-rhamnopyranoside (6) : −83.4 (c 0.3, MeOH); UV (MeOH) λmax 225, 279 nm; ESI-QTOF MS: m/z 421.2333 [M+H]+; 1H-NMR (CD3OD, 500 MHz): δ 4.66 (1H, d, J = 7.9 Hz, H-2), 3.94 (1H, m, H-3), 2.65 (1H, dd, J = 16.3, 8.9 Hz, H-4eq), 2.91 (1H, d, J = 15.9, 5.7 Hz, H-4ax), 5.85 (1H, d, J = 2.2 Hz, H-6), 5.94 (1H, d, J = 2.4 Hz, H-8), 7.23 (2H, d, J = 8.5 Hz, H-2', 6'), 6.79 (2H, d, J = 8.5 Hz, H-3', 5'), 4.25 (1H, br s, H-1"), 1.25 (3H, d, J = 6.3 Hz, H-6"); 13C NMR (CD3OD, 125 MHz): δ 81.3 (C-2), 76.4 (C-3), 28.4 (C-4), 157.1 (C-5), 95.6 (C-6), 157.7 (C-7), 96.6 (C-8), 158.1 (C-9), 100.8 (C-10), 131.4 (C-1'), 129.5 (C-2', 6'), 116.2 (C-3', 5'), 158.7 (C-4'), 102.4 (C-1"), 72.1 (C-2"), 72.4 (C-3"), 74.1 (C-4"), 70.5 (C-5"), 18.1 (C-6")

(+)-2,3-trans-Dihydrokaempferol 3-O-α-L-rhamnopyranoside (7) : −15.3 (c 0.5, MeOH); UV (MeOH) λmax 290, 332 nm; ESI-QTOF MS: m/z 435.2365 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 5.14 (1H, d, J = 11.2 Hz, H-2), 4.62 (1H, d, J = 11.2 Hz, H-3), 5.92 (1H, d, J = 2.2 Hz, H-8), 5.89 (1H, d, J = 2.2 Hz, H-6), 7.36 (2H, d, J = 8.8 Hz, H-2', 6'), 6.84 (1H, d, J = 8.8 Hz, H-3', 5'), 4.00 (1H, d, J = 1.4 Hz, H-1"), 1.18 (3H, d, J = 6.2 Hz, H-6"); 13C NMR (CD3OD, 125 MHz): δ 84.0 (C-2), 78.8 (C-3), 196.2 (C-4), 165.7 (C-5), 97.6 (C-6), 164.3 (C-7), 96.4 (C-8), 168.9 (C-9), 102.4 (C-10), 128.8 (C-1'), 130.2 (C-2', 6'), 116.6 (C-3', 5'), 159.6 (C-4'), 102.6 (C-1"), 71.9 (C-2"), 72.3 (C-3"), 73.9 (C-4"), 70.7 (C-5"), 18.0 (C-6")

Methyl gallate (8) : + 19.3 (c 0.3, CHCl3); UV (MeOH) λmax 277 nm; ESI-Q-TOF MS: m/z 185.0443 [M+H]+; 1H NMR (CD3OD, 500 MHz): δ 7.04 (2H, s, H-2, 6), 3.81 (3H, s, -OMe); 13C NMR (CD3OD, 125 MHz): δ 121.6 (C-1), 110.2 (C-2, 6), 146.6 (C-3, 5), 139.9 (C-4), 169.2 (C=O), 52.4 (-OMe)

Lupeol (9) : +31.5 (c 0.5, CHCl3); ESI-Q-TOF MS: m/z 426.3862 [M+H]+; 1H NMR (CDCl3, 500 MHz): δ 3.16 (1H, dd, J = 11.5, 5.2 Hz, H-3), 2.35 (1H, m, H-19), 1.90 (1H, m, H-21), 0.77 (3H, s, H-23), 0.81 (3H, s, H-24), 0.92 (3H, s, H-25), 0.94 (3H, s, H-26), 1.01 (3H, s, H-27), 0.74 (3H, s, H-28), 4.54 (1H, m, H-29a), 4.66 (1H, d, J = 2.4 Hz, H-29b), 1.66 (3H, s, H-30); 13C NMR (CDCl3, 125 MHz): δ 39.0 (C-1), 27.6 (C-2), 79.2 (C-3), 39.1 (C-4), 55.5 (C-5), 18.6 (C-6), 34.5 (C-7), 41.1 (C-8), 50.7 (C-9), 37.4 (C-10), 21.2 (C-11), 25.4 (C-12), 38.3 (C-13), 43.1 (C-14), 27.7 (C-15), 35.8 (C-16), 43.2 (C-17), 48.6 (C-18), 48.2 (C-19), 151.2 (C-20), 30.1 (C-21), 40.2 (C-22), 28.2 (C-23), 15.6 (C-24), 16.3 (C-25), 16.2 (C-26), 14.8 (C-27), 18.2 (C-28), 109.5 (C-29), 19.5 (C-30)

Methyl lup-20(29)-en-3-on-28-oate (10) : +14.3 (c 0.35, CHCl3); LRFAB MS: m/z 469 [M+H]+; 1H NMR (CDCl3, 500 MHz): δ 2.98 (1H, m, H-3), 2.47 (1H, m, H-19), 0.90 (3H, s, H-23), 0.93 (3H, s, H-24), 0.95 (3H, s, H-25), 0.99 (3H, s, H-26), 1.04 (3H, s, H-27), 4.58 (1H, m, H-29a), 4.71 (1H, m, H-29b), 1.66 (3H, s, H-30), 3.65 (3H, s, 28-OCH3); 13C NMR (CDCl3, 125 MHz): δ 38.4 (C-1), 26.6 (C-2), 218.2 (C-3), 39.7 (C-4), 56.6 (C-5), 19.7 (C-6), 33.7 (C-7), 42.5 (C-8), 55.0 (C-9), 36.9 (C-10), 21.4 (C-11), 25.6 (C-12), 37.0 (C-13), 47.0 (C-14), 29.7 (C-15), 34.2 (C-16), 47.4 (C-17), 49.9 (C-18), 49.4 (C-19), 150.5 (C-20), 32.1 (C-21), 40.6 (C-22), 30.6 (C-23), 15.8 (C-24), 19.4 (C-25), 16.0 (C-26), 14.7 (C-27), 176.7 (C-28), 109.7 (C-29), 21.1 (C-30), 51.3 (28-OCH3)

β-Amyrin (11) : +89.2 (c 0.3, CHCl3); HRFAB MS: m/z 426.3855 [M+H]+; 1H NMR (CDCl3, 500 MHz): δ 3.20 (1H, dd, J = 10.2, 4.5 Hz, H-3), 5.16 (1H, t, J = 3.6 Hz, H-12), 0.98 (3H, s, H-23), 0.77 (3H, s, H-24), 0.92 (3H, s, H-25), 0.95 (3H, s, H-26), 1.11 (3H, s, H-27), 0.81 (3H, s, H-28), 0.85 (6H, s, H-29, H-30); 13C NMR (CDCl3, 125 MHz): δ 38.8 (C-1), 27.2 (C-2), 79.3 (C-3), 39.0 (C-4), 55.4 (C-5), 18.6 (C-6), 32.9 (C-7), 41.9 (C-8), 47.9 (C-9), 37.2 (C-10), 23.9 (C-11), 121.9 (C-12), 145.4 (C-13), 40.0 (C-14), 28.3 (C-15), 26.4 (C-16), 32.9 (C-17), 47.5 (C-18), 47.1 (C-19), 32.7 (C-20), 35.0 (C-21), 37.4 (C-22), 28.6 (C-23), 15.7 (C-24), 15.8 (C-25), 17.0 (C-26), 26.2 (C-27), 27.5 (C-28), 33.6 (C-29), 23.8 (C-30)

α-Amyrin (12) : +51.6 (c 0.7, CHCl3); HRFAB MS: m/z 426.3856 [M+H]+; 1H NMR (CDCl3, 500 MHz): δ 3.21 (1H, dd, J = 10.4, 5.1 Hz, H-3), 5.10 (1H, t, J = 3.6 Hz, H-12), 0.98 (3H, s, H-23), 0.76 (3H, s, H-24), 0.93 (3H, s, H-25), 0.99 (3H, s, H-26), 1.05 (3H, s, H-27), 0.78 (3H, s, H-28), 0.78 (3H, s, H-29), 0.89 (3H, s, H-30); 13C NMR (CDCl3, 125 MHz): δ 39.0 (C-1), 27.5 (C-2), 79.3 (C-3), 39.0 (C-4), 55.4 (C-5), 18.6 (C-6), 33.2 (C-7), 40.2 (C-8), 47.9 (C-9), 37.1 (C-10), 23.6 (C-11), 124.6 (C-12), 139.8 (C-13), 42.3 (C-14), 26.8 (C-15), 28.4 (C-16), 34.0 (C-17), 59.3 (C-18), 39.9 (C-19), 39.8 (C-20), 31.5 (C-21), 41.7 (C-22), 28.3 (C-23), 15.9 (C-24), 15.8 (C-25), 17.1 (C-26), 23.5 (C-27), 29.0 (C-28), 17.7 (C-29), 21.6 (C-30)

3β-Hydroxy-D:B-friedo-olean-5-ene (13) : +73.1 (c 0.5, CHCl3); HRFAB MS: m/z 426.3853 [M+H]+; 1H NMR (CDCl3, 500 MHz): δ 3.50 (1H, m, H-3), 5.66 (1H, m, H-12), 1.08 (3H, s, H-23), 1.17 (3H, s, H-24), 0.88 (3H, s, H-25), 1.13 (3H, s, H-26), 1.04 (3H, s, H-27), 1.19 (3H, s, H-28), 1.02 (3H, s, H-29), 0.98 (3H, s, H-30); 13C NMR (CDCl3, 125 MHz): δ 18.4 (C-1), 28.0 (C-2), 76.6 (C-3), 41.1 (C-4), 141.8 (C-5), 122.3 (C-6), 23.9 (C-7), 47.7 (C-8), 35.1 (C-9), 49.9 (C-10), 34.8 (C-11), 30.6 (C-12), 38.1 (C-13), 39.5 (C-14), 32.3 (C-15), 36.2 (C-16), 30.3 (C-17), 43.3 (C-18), 35.3 (C-19), 28.5 (C-20), 33.3 (C-21), 39.1 (C-22), 29.2 (C-23), 25.7 (C-24), 16.4 (C-25), 18.6 (C-26), 19.8 (C-27), 32.2 (C-28), 32.6 (C-29), 34.7 (C-30)

 

Results and Discussion

The methanolic extract of D. burmanica was partitioned successively with CH2Cl2, EtOAc, and n-BuOH. The EtOAc and CH2Cl2 soluble fraction were subjected diverse column chromatography to give six flavan 3-ol derivatives, a dihydroflavonol glycoside, a phenolic compound and five triterpenes. Spectroscopic data of isolates were compared with those of literature values to determin (+)-catechin (1),8 (+)-catechin 3-O-α-L-rhamnopyranoside (2),8 (+)-catechin 3-O-gallate (3),9 (−)-epicatechin (4),8 (−)-epicatechin 3-O-gallate (5),10 (+)-afzelechin 3-O-α-L-rhamnopyranoside (6),11 (+)-2,3-trans-dihydrokaempferol 3-O-α-L-rhamnopyranoside (7),12 methyl gallate (8),13 lupeol (9),14-15 methyl lup-20(29)-en-3-on-28-oate (10),16 β-amyrin (11),17 α-amyrin (12),18 3β-hydroxy-D:B-friedo-olean-5-ene (13).19 To the best of our knowledge, all isolates were isolated from D. burmanica for the first time.

Compound 1 was obtained as brownish amorphous powder and the molecular formula, C15H14O6, was established by the positive ion mode ESI-QTOF MS (m/z 291.0866 [M+H]+). The 1H NMR spectrum showed the signals for 1,3,4-substituted aromatic protons [δH 6.84 (1H, d, J = 2.0, H-2'), 6.76 (1H, d, J = 8.0, H-5'), 6.72 (1H, dd, J = 8.0, 2.0, H-6')], two meta coupling protons [δH 5.92 (1H, d, J = 2.2, H-8), 5.85 (1H, d, J = 2.2, H-6)], a methene group [δH 2.85 (1H, dd, J = 16.3, 5.3, H-4eq), 2.50 (1H, dd, J = 16.2, 8.0, H-4ax)] and two methine protons [δH 4.56 (1H, d, J = 7.5, H-2), 3.96 (1H, m, H-3)]. The 2,3-trans configuration was confirmed from the large J value of H-2 (J = 7.5 Hz). The 13C NMR detected 15 carbon signals including twelve aromatic carbons, one oxygenated aliphatic carbon and two aliphatic carbons. Thus, compound 1 was identified as (+)-catechin based on the spectroscopic evidences and comparison of literature values.

Compound 2 was isolated as dark brownish amorphous powder and the molecular formula was deduced to be C21H24O10 from its ESI-QTOF MS ion peak at m/z 437.1454 [M+H]+. The 1H and 13C NMR spectra showed similar patterns with those of compound 1 except for the sugar moiety. The sugar moiety was elucidated to be α-rhamnopyranoside from the anomeric proton signal at δH 4.29 (1H, d, J = 1.2, H-1") and six alipathic carbon signals at δC 100.8 (C-1"), 72.2 (C-2"), 72.4 (C-3"), 74.1 (C-4"), 70.5 (C-5"), 18.1 (C-6"). On the basis of spectroscopic data with comparison of literature values, the structure of compound 2 was determined to be (+)-catechin 3-O-α-L-rhamnopyranoside.

Compound 3 was obtained as dark purple amorphous powder. Its molecular formula was identified as C22H18O10 from the positive ion mode ESI-QTOFMS (m/z 443.2216 [M+H]+). The 1H and 13C NMR spectra was similar to compound 1 (see Experimental) except for the presence of a gallic acid moiety at δH 6.96 (2H, s, H-2”, 6”) and δC 121.5 (C-1"), 110.3 (C-2", 6"), 146.5 (C-3", 5"), 140.0 (C-4"), 167.7 (-COO). The HMBC correlation of δH 5.37 (H-3) to δC 167.7 indicated that gallic acid was linked to C-3 of (+)-cathecin. Thus, compound 3 was confirmed to be(+)-catechin 3-O-gallate

Compound 4 was isolated as brown amorphous powder and its molecular formula was determined to be C15H14O6 by the positive ion mode ESI-QTOFMS (m/z 291.1953 [M+H]+). The 1H NMR indicated flavan 3-ol moiety including 1,3,4-substitued aromatic proton signals at δH 6.97 (1H, d, J = 1.8, H-2'), 6.75 (1H, d, J = 8.2, H-5'), 6.79 (1H, dd, J=8.4, 1.7, H-6'), two meta-coupled aromatic proton signals at δH 5.91 (1H, d, J = 2.2, H-6), 5.94 (1H, d, J = 2.2, H-8) and a methene group at δH 2.73 (1H, dd, J = 16.5, 2.8, H-4eq), 2.86 (1H, dd, J = 16.5, 4.9, H-4ax) and two methine protons at δH 4.82 (1H, s, H-2), 4.17 (1H, m, H-3). The 2,3-cis configuration was confirmed from the singlet signal of H-2. Based on the spectroscopic evidences and comparison with literature values, compound 4 was determined to be (-)-epicatechin.

Compound 5 was obtained as dark purple amorphous powder showing its molecular as C22H18O10 from ESIQTOFMS (m/z 443.2216 [M+H]+). The 1H and 13C NMR spectra detected (−)-epicathecin skeleton and additionally observed the presence of a gallic acid moiety (see Experimental). The HMBC correation of δH 5.52 (H-3) to δC 167.8 revealed that gallic acid was linked to C-3 of (−)-epicatechin. Thus, compound 5 was elucidated as (−)-epicatechin 3-O-gallate.

The molecular formula of compound 6 was determined to be C21H24O9 by at m/z 421.2333 [M+H]+ ion peak of ESI-QTOFMS spectrum. The 1H and 13C NMR spectra displayed similar patterns with those of compound 2 except for the signals of 1,4-substituted aromatic proton signals [δH 7.23 (2H, d, J = 8.5, H-2', 6'), 6.79 (2H, d, J = 8.5, H-3', 5'); δC 131.4 (C-1'), 129.5 (C-2', 6'), 116.2 (C-3', 5'), 158.7 (C-4')]. From these spectroscopic data and through comparison with literature values, compound 6 was assigned to be (+)-afzelechin 3-O-α-L-rhamnopyranoside.

Compound 7 was isolated as yellowish amorphous powder and the molecular formula was determined to be C21H22O10 by ESI-QTOFMS ion peak at m/z 435.2365 [M+H]+. The 1H NMR spectrum displayed dihydrokaempferol skeleton including 1,4-substituted aromatic proton signals [δH 7.36 (2H, d, J = 8.8, H-2', 6'), 6.84 (2H, d, J = 8.8, H-3', 5'), two meta-coupled aromatic protons at δH 5.92 (1H, d, J = 2.2, H-8), 5.89 (1H, d, J = 2.2, H-6) and two aliphatic proton signals at δH 5.14 (1H, d, J = 11.2, H-2), 4.62 (1H, d, J = 11.2, H-3). The coupling constant (J = 11.2 Hz) between H-2 and H-3 indicated that 2,3-trans configuration. Furthermore, α-rhamnopyranoside was detected at δH 4.00 (1H, d, J = 1.4, H-1") and δC 102.6 (C-1"), 71.9 (C-2"), 72.3 (C-3"), 73.9 (C-4"), 70.7 (C-5"), 18.0 (C-6"). The anomeric proton signal of rhamnose (δH 4.00) was correlated to δC 78.8 indicating sugar moiety was attached to C-3 position of dihydrokaempferol moiety. Based on the spectroscopic evidences and comparison with literature values, compound 7 was determined to be (+)-2,3-trans-dihydrokaempferol 3-O-α-L-rhamnopyranoside.

Compound 8 was isolated as dark purple amorphous powder and its molecular formula was established as C8H8O5 based on the positive mode of ESI-QTOF MS (m/z 185.0443 [M+H]+). The 1H NMR spectrum showed a singlet at δH 7.04 (2H, s, H-2, 6), assignable to symmetrical protons at H-2 and H-6, and a methoxy signal at δH 3.81 (3H, s, -OCH3). The 13C NMR spectrum also showed six aromatic carbon signals at δC 121.6 (C-1), 110.2 (C-2, 6), 146.6 (C-3, 5), 139.9 (C-4), a methoxy carbon signal at δc 52.4 and a carbonyl carbon signal at δc 169.2. Based on above data with the comparison to the literature values, compound 8 was identified as methyl gallate.

Compound 9 was obtained as white amorphous powder, and displayed molecular ion peaks at m/z 426.3862 [M+H]+ on the positive ion mode HRFAB-MS showing molecular formula of C30H50O. The 1H NMR spectrum exhibited seven methyl groups at δH 0.77 (3H, s, H-23), 0.81 (3H, s, H-24), 0.92 (3H, s, H-25), 0.94 (3H, s, H-26), 1.01 (3H, s, H-27), 0.74 (3H, s, H-28) and 1.66 (3H, s, H-30), two germinal-coupled vinyl protons at 4.54 (1H, m, H-29a), 4.66 (1H, d, J = 2.4 Hz, H-29b), an oxygenated methine proton signals at δH 3.16 (1H, dd, J = 11.5, 5.2 Hz, H-3). The 13C NMR detected thirty carbon signals showing characteristic two vinyl carbons (δC 151.2, C-20; 109.5, C-29), an oxygenated carbon (δC 79.2, C-3). Therefore, compound 9 was determined to be lupeol based on the spectroscopic evidences and literature values.

The molecular formula of compound 10 was determined to be C31H48O3 by LRFAB-MS. The 1H NMR spectrum exhibited six methyl groups at δH 0.90 (3H, s, H-23), 0.93 (3H, s, H-24), 0.95 (3H, s, H-25), 0.99 (3H, s, H-26), 1.04 (3H, s, H-27) and 1.66 (3H, s, H-30), a methoxy group at δH 3.65 (3H, s, 28-OCH3) and two germinal-coupled vinyl protons at 4.58 (1H, m, H-29a), 4.71 (1H, m). In addition, the 13C NMR observed thirty carbon signals including two vinyl carbons (δC 150.5, C-20; 109.7, C-29), two carbonyl carbons at δC 218.2 (C-3) and 176.7 (C-28). From the above spectroscopic data and comparing them with published values, compound 10 was elucidated to be methyl lup-20(29)-en-3-on-28-oate.

Compound 11 was isolated as white amorphous powder, and its molecular formula was determined to be C30H50O by HR-FABMS spectroscopy. The 1H NMR showed eight methyl signals at δH 0.98 (3H, s, H-23), 0.77 (3H, s, H-24), 0.92 (3H, s, H-25), 0.95 (3H, s, H-26), 1.11 (3H, s, H-27), 0.81 (3H, s, H-28), 0.85 (6H, s, H-29, H-30), an oxygenated methine signal at δH 3.20 (1H, dd, J = 10.2, 4.5 Hz, H-3) and a olefinic proton at δH 5.16 (1H, t, J = 3.6 Hz, H-12). The 13C NMR revealed thirty carbons containing two olefinic carbons at δC 121.9 (C-12), 145.4 (C-13) and an oxygenated carbon at δC 76.6 (C-3). On the basis of above spectroscopic data and comparing them with published values, compound 11 was identified to be β-amyrin.

The MS, 1H and 13C NMR data were close to compound 12 except for the two olefinic carbon signals at δC 124.6 (C-12), 139.8 (C-13) which was characteristic in ursan skeleton. Thus, compound 12 was determined to be a-amyrin by comparing spectroscopic data with those of published values.

The positive ion mode HRFAB-MS showed pseudomolecular ion peak at m/z 426.3853 [M+H]+ for compound 13 giving molecular formula of C30H50O. The 1H NMR showed eight methyl signals at δH 1.08 (3H, s, H-23), 1.17 (3H, s, H-24), 0.88 (3H, s, H-25), 1.13 (3H, s, H-26), 1.04 (3H, s, H-27), 1.19 (3H, s, H-28), 1.02 (3H, s, H-29), 0.98 (3H, s, H-30), an oxygenated methine signal at δH ): δ 3.50 (1H, m, H-3) and a vinyl proton at δH 5.66 (1H, m, H-12). In 13C NMR, thirty carbon resonances were observed including an oxygenated methine carbon at δC 76.6 (C-3) and two olefinic carbons at δC 141.8 (C-5), 122.3 (C-6). According to MS, MS, 1H and 13C NMR of compound 13 and comparing them with literature values, compound 13 was elucidated as 3β-hydroxy-D:B-friedoolean-5-ene.