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.