1
Supporting Information
Rec. Nat. Prod. 8:3 (2014) 294-298
A Trimeric Proanthocyanidin from the Bark of Acacia
leucophloea Willd.
Sarfaraz Ahmed*1,4, Hsiao-Ching Lin** 2, Iram Nizam1,
Nadeem Ahmad Khan3, Shoei-Sheng Lee2 and Nizam Uddin Khan1
1Department of Chemistry, Aligarh Muslim University, Aligarh-202 002, India
2School of Pharmacy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan,
Republic of China 3GVK Biosciences, 28A, IDA, Nacharam, Hyderabad-500076, India
4Department of Pharmacognosy, College of Pharmacy, King Saud University, Post box-2457,
Riyadh-11451, Saudi Arabia
2
Table of Contents Page
Experimental 3-4
S1: 1H NMR (600 MHz, methanol-d4) Spectrum of Compound 1 5
S2: 13C NMR (600 MHz, methanol-d4) Spectrum of Compound 1 6
6
S3: COSY (600 MHz, methanol-d4) Spectrum of Compound 1 7
S4: HSQC (600 MHz, methanol-d4) Spectrum of Compound 1 8
S5: HMBC (600 MHz, methanol-d4) Spectrum of Compound 1 9
S6: Selected NOESY (600 MHz, methanol-d4) spectrum 10
of Compound 1 at δ 2.42 (A), 2.94 (B), 2.37 (C), and 6.00 (D)
S7: Selected TOCSY (600 MHz, methanol-d4) spectrum 11
of Compound 1 at δ 4.36 (A), 4.70 (B), 3.70 (C), 4.51 (D), 4.65 (E), and 3.60 (F)
S8: 1H NMR (600 MHz, chloroform-d,) spectrum of Compound of 1c 12
S9: 1H NMR (600 MHz, DMSO-d6) spectrum of Compound 1d 13
Reference 14
3
Experimental
Instrumental
The optical rotations were measured on a JASCO DIP-370 polarimeter. UV spectra were measured in
MeOH on a Hitachi 150-20 Double Beam Spectrophotometer. The CD spectra were recorded on a J-720
spectropolarimeter. 1H, 13C NMR, and 2D NMR spectra were obtained in methanol-d4 (δH 3.30 and δC 49.0)
and chloroform-d3 (δH 7.24 and δC 77.0) on a Bruker Avance III 600 NMR spectrometer, equipped with a 5
mm cryoprobe using standard pulse programs. The ESI-MS data were obtained on an Esquire 2000 ion trap
mass spectrometer (Bruker Daltonik, Bremen, Germany). The HR-ESI-MS data were measured on a
micrOTOF orthogonal ESI-TOF mass spectrometer (Bruker, Daltonik, Bremen, Germany). TLC analyses
were carried out on silica gel plates (KG60-F254, Merck). Semi-preparative HPLC was performed on a
Phenomenex® RP-18 column (Prodigy ODS-3, 250 × 10 mm, 5 µm). The microplate spectrophotometer for
bioassay was SPECTRAmax® PLUS (Molecular Devices, U.S.A).
Chemicals and reagents
CH3CN (HPLC grade) and MeOH were purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ,
USA) and deionized water was prepared from a Barnstead water purification system (Dubuque, IA, USA).
Chloroform-d3 (99.8%) was purchased from Cambridge Isotope Lab. Inc. (Andover, MA, USA).
Methanol-d4 (99.8%) was purchased from Merck KGaA (Darmstadt, Germany). α-Glucosidase type IV from
Bacillus stearothermophilus, p-nitrophenyl α-D-glucopyranoside, K2HPO4 and KH2PO4 were purchased
from Sigma-Aldrich Co. (St. Louis, MO, USA).
Extraction and Isolation
The EtOH extract (174.5 g) of the dry bark of A. leucophloea (1.64 Kg) was triturated with solvents in
sequence (500mL ×3) to give fractions soluble in CHCl3, EtOAc, acetone and EtOH. The EtOAc fraction (50
g out of 55 g) was subjected to silica gel column eluted by different solvent systems in increasing order of
polarity. EtOAc eluent of column to give fraction AL-1. AL-1 (1.01 g) was dissolved in 25% MeOH–H2O
(10 ml) and the soluble part (623.5 mg) was separated on a Lobar RP-18 column (LiChroprep RP-18, size B,
310 × 25 mm; 40-63 µm, Merck), delivered by a stepwise gradient of MeOH–H2O from 15:85 to 75:25, to
give six subfractions. Part of subfr. 3 (fr.AL-1-3) (31.7 mg out of 114.4 mg) was separated on a
semi-preparative HPLC column, 3.17 mg (0.1 ml MeOH) × 10, delivered by MeCN−H2O (18:82) with a
flow rate of 2.5 mL/min and detection at 300 nm. The fraction (6.9 mg) containing the major peak (tR = 52.1
min) was further purified on a Sephadex LH-20 column (75 × 1.5 cm, MeOH) to give 1 (1.8 mg). The 25%
MeOHaq. insoluble fraction (387 mg) of AL-1 was chromatographed on a Sephadex LH-20 column (72 × 2.5
cm, MeOH–H2O 7:3) to give 1 (17.9 mg).
4
O-Methylation and Acetylation of compound 1
To the solution of compound 1 (3.0 mg) in acetone (1 mL) was added dimethyl sulfate
(200 µL), K 2CO3 (50 mg) and Cs2CO3 (20 mg). The mixture was refluxed (65 oC) under N2 for 4
hours and the reaction mixture was evaporated to give a residue which was partitioned between
H2O (10 mL) and CHCl3 (10 mL × 3). The CHCl3 extract was purified on a silica gel column
(10–55% acetone–hexane) to give 1c (0.4 mg). The solution of 1c in acetic anhydride (200 µL)
and anhydrous pyridine (100 µL) was stirred for 4 h at room temperature. After quenching with
EtOH (200 µL) for 0.5 h, the reaction mixture was evaporated to dryness and the residue was
further purified by a Sephadex LH-20 column (7:3, MeOH–CHCl3) to give 1d (0.4 mg).
(–)-Fisetinidol-(4α,8)-[(–)-fisetinidol-(4α,6)]-(+)-catechin (1)
White amorphous powder; [α]27,D +83.8 (c 0.50, MeOH); UV (MeOH) λmax (log ε):
281.5 (4.29); CD (MeOH) ∆ε215 -49.32; 1H and 13C NMR, see Table 1; HMBC, see Figure 2;
(+ )ESIMS m/z (rel int %) 857.2 (100, [M+Na]+); (+) HRESIMS m/z 857.2037 [M+Na]+, calcd
for C45H38NaO16, 857.2058.
(2R,3S)-2,3-trans-3-Hydroxy-6,8-bi-[(2R,3S,4S)-2,3-trans-3,4-trans-3-hydroxy-3',4',7
-trimethoxyflavan-4-yl]-3',4',5,7-tetramethoxyflavan (1c)
White amorphous powder; CD (MeOH) ∆ε223 –CE, ∆ε235 –CE; 1H NMR (CDCl3, 600
MHz) δ 3.99, 3.94, 3.90, 3.86, 3.86, 3.85, 3.73, 3.73, 3.72, 3.59 (s, OCH3); (+) ESIMS m/z (rel
int %) 997.4 (100, [M+Na]+ ).
(2R,3S)-2,3-trans-3-acetoxy-6,8-bi-[(2R,3S,4S)-2,3-trans-3,4-trans-3-acetoxy-3',4',7-t
rimethoxyflavan-4-yl]-3',4',5,7-tetramethoxyflavan (1d)
White amorphous powder; CD (MeOH) ∆ε223 – CE, ∆ε234 – CE; 1H NMR (DMSO-d6,
600 MHz) δ 1.85, 1.82 (s, COCH3), 1.71, 1.67 (s, 3H, COCH3), 1.54, 1.44 (s, 3H, COCH3); (+)
ESIMS m/z (rel int %) 1123.4 (68, [M+Na]+ ), 1139.4 (100, [M+K] + ).
Assay for α-Glucosidase Activity
The inhibitory activities against α-glucosidase type IV from Bacillus stearothermophilus
were performed following the reported method [1]. Compound 1 was dissolved in 10% MeOH.
Acarbose (Bayer) was chosen as the positive control with the IC50 value against the same enzyme
of 0.049 µM.
5
2.53.03.54.04.55.05.56.06.57.0 ppm
3.2951
3.2978
3.3001
3.3029
3.3058
4.3524
4.3739
4.4239
4.4284
4.4372
4.4431
4.4470
4.4525
4.4589
4.4628
4.4750
4.4783
4.5060
4.6139
4.6305
4.6414
4.6470
4.6834
4.7004
4.7687
4.8269
6.1672
6.1712
6.1847
6.1887
6.2405
6.2452
6.2498
6.2552
6.2599
6.2639
6.2736
6.2775
6.3098
6.3139
6.5010
6.5133
6.5161
6.5369
6.5403
6.5928
6.6065
6.6257
6.6392
6.6531
6.6825
6.6964
6.7049
6.7081
6.7128
6.7521
6.7535
6.7599
6.7625
6.7739
6.7842
6.7860
6.7891
6.7982
6.8000
6.8023
6.8243
6.9465
6.9496
6.9766
6.9798
1.02
1.17
1.39
0.81
1.05
0.89
0.97
1.54
6.96
0.99
3.61
2.13
4.36
1.04
1.23
0.41
0.89
1.00
1.05
1.13
3.85
1.44
2.33
1.83
1.56
2.45
1.49
1.83
1.80
2.61
2.68
2.55
1.96
2.87
1.60
1.74
2.07
S1: 1H NMR (600 MHz, methanol-d4) Spectrum of Compound 1
2.62.83.0 ppm
2.3963
2.4106
2.4227
2.4373
2.5530
2.5676
2.5796
2.5941
2.7043
2.7136
2.7309
2.7404
2.9286
2.9380
2.9554
2.9646
4.44.54.64.7 ppm
4.3524
4.3592
4.3739
4.4239
4.4284
4.4372
4.4431
4.4470
4.4525
4.4589
4.4628
4.4750
4.4783
4.5060
4.6139
4.6305
4.6400
4.6414
4.6470
4.6655
4.6834
4.7004
6.06.16.26.36.46.56.66.76.86.97.0 ppm
6.1672
6.1712
6.1847
6.1887
6.2405
6.2452
6.2498
6.2599
6.2736
6.2775
6.3098
6.3139
6.5133
6.5161
6.5369
6.5928
6.6065
6.6257
6.6392
6.6531
6.6825
6.6964
6.7049
6.7081
6.7128
6.7521
6.7535
6.7599
6.7625
6.7739
6.7842
6.7891
6.7982
6.8000
6.8023
6.9496
6
405060708090100110120130140150160 ppm
29.4067
30.0717
42.4586
42.5536
42.8889
43.0928
43.1367
48.5752
48.7168
48.8583
49.0002
49.1426
49.2848
49.4263
69.0827
69.2409
71.3229
71.3761
71.8263
72.4052
82.1820
82.2909
82.3775
84.1955
84.3200
84.3944
84.5964
84.6498
103.7198
103.8414
103.9449
104.3656
109.2459
109.2950
109.9254
110.2055
110.8025
110.9507
111.0670
111.6461
115.7424
115.7827
115.9250
116.0459
116.1315
116.1798
116.2370
116.4328
117.4788
118.0155
119.1688
119.4559
119.5865
120.6577
120.7466
120.9749
121.0217
129.7961
129.9015
130.0561
130.2215
131.7876
132.0813
145.7856
146.1986
146.5244
153.3389
156.5679
156.9269
S2: 13C NMR (600 MHz, methanol-d4) Spectrum of Compound 1
7
S3: COSY (600 MHz, methanol-d4) Spectrum of Compound 1
8
S4: HSQC (600 MHz, methanol-d4) Spectrum of Compound 1
9
S5: HMBC (600 MHz, methanol-d4) Spectrum of Compound 1
10
S6: Selected NOESY (600 MHz, methanol-d4) Spectrum of Compound 1 at δ 2.42 (A), 2.94 (B),
2.37 (C), and 6.00 (D)
11
S7: Selected TOCSY (600 MHz, methanol-d4) Spectrum of Compound 1 at δ 4.36 (A), 4.70 (B),
3.70 (C), 4.51 (D), 4.65 (E), and 3.60 (F)
12
3.03.54.04.55.05.56.06.57.0 ppm
2.6153
2.6325
2.6420
2.6593
3.1115
3.1212
3.1385
3.1478
3.5943
3.7212
3.7297
3.7308
3.8472
3.8548
3.8996
3.9414
3.9922
4.4727
4.4841
4.4909
4.5062
4.6486
4.6634
4.7180
4.7336
4.7584
4.7738
4.7892
6.3463
6.3509
6.3538
6.3569
6.3660
6.3694
6.3799
6.3859
6.3903
6.3940
6.3983
6.4633
6.4668
6.4864
6.4896
6.5271
6.5312
6.6604
6.6633
6.6692
6.6830
6.7035
6.7087
6.7175
6.7225
6.7464
6.7605
6.7674
6.7889
6.7939
6.8077
6.8504
6.8538
6.8645
6.8674
6.9268
6.9405
7.0630
7.0894
7.0923
7.1385
7.1417
7.2400
1.21
1.84
3.36
3.62
6.15
3.87
6.62
3.02
3.40
3.00
3.19
3.19
1.57
2.28
1.22
1.68
3.59
1.75
2.33
2.99
2.67
3.03
3.50
2.52
1.60
1.32
1.19
ssl-scl-20110831 10, 1H-spectrum, p.3379210 tube 44-45 , in CDCl3, AV600-CRP, 2011/08/31
O
O
MeO
OMe
OMe
OMe
OMeMeO
OH
OH
O
OMe
MeOOMe
OH
OMe
S8: 1H NMR (600 MHz, chloroform-d3) Spectrum of Compound 1c
13
2.02.53.03.54.04.55.05.56.06.57.0 ppm
1.4391
1.5406
1.6652
1.7077
1.8173
1.8531
2.4901
2.9263
2.9773
3.3209
3.4988
3.5667
3.5865
3.6857
3.6920
3.7013
3.7104
3.7169
3.7303
3.7351
3.7488
3.7547
3.7640
4.6949
4.7031
4.7112
4.7197
4.7297
4.7839
4.7957
4.9768
4.9932
5.0138
5.0300
5.0487
5.0650
5.8968
5.9072
5.9132
5.9241
5.9294
6.0341
6.0500
6.3440
6.3589
6.3709
6.3743
6.3876
6.4516
6.4735
6.4776
6.5126
6.5438
6.5696
6.5851
6.7126
6.7375
6.7459
6.7512
3.00
2.69
3.22
2.93
3.07
2.70
4.66
5.22
18.6
59.
087.
155.
778.
025.
215.
899.
246.
92
0.88
1.15
2.73
1.57
4.06
0.74
0.76
4.80
6.69
7.37
4.84
4.86
2.79
8.82
3.86
ssl-scl-20110919 70, 1H-spectrun, p.3379211 acetylation, in DMDO-d6, AV600-CRP, 2011/10/19
O
O
MeO
OMe
OMe
OMe
OMeMeO
OAc
OAc
O
OMe
MeOOMe
OAc
OMe
S9: 1H NMR (600 MHz, DMSO-d6) Spectrum of Compound 1d
14
Reference
[1] S. S. Lee, H. C. Lin and C. K. Chen (2008). Acylated flavonol monorhamnosides, α-glucosidase
inhibitors, from Machilus philippinensis, Phytochemistry. 69, 2347–2353.