(+)-Catechin-5-galloyl Ester as a Novel Natural Polyphenol from the Bark of Acacia nilotica of Sudanese Origin 1

556 Planta Medica 55(1989)

(-i-)-Catechin-5-galloyl Ester as a Novel Natural Polyphenol from the Bark of Acacia nilotica of Sudanese Origin1 £ A. Khalid2", £ M. Yagi2, P. Khristova4, and HelmutDuddeclc5 Dedicated to the late Prof. H. M. Elamin as a token for our great gratitude for his encouragement and pioneering work on Sudan Acacias 2 Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, P. 0. Box 1996, Khartoum, Sudan Address for correspondence Department of Forestry, Faculty of Agriculture, University of Khartoum, P. 0. Box 32, Khartoum, Sudan Ruhr-Universitat Bochum, Fakultat fur Chemie, Postfach 102148, D-4630 Bochum 1, Federal Republic of Germany

Received: September 25, 1988

Materials and Methods Plant material

Abstract From the bark of Acacia nilotica subsp. tomentosa a novel polyphenol, (+)-catechin-5-galloyl ester (5), was isolated along with gallic acid (1), its methyl ester (2), naringenin (3), and (+)-catechin (4). The structure of 5 was determined by use of mass spectrometry as well as 'H- and '3C-NMR spectroscopy.

Introduction Acacia nilotica (L.) Wild. ex Del. is one of the most important tanniferous plant in Africa. Recent investigations in Sudan have established its potent molluscicidal activity (1, 2). However, confusion still exists about the chemical structure of the bioactive agent(s). Ayoub (3) reported that this molluscicidal activity is mainly due to (—)-epigallocatechin-7-gal-

late and (—)-epigallocatechin-5,7-digallate isolated from the fruit and bark of Acacia nilotica. Based on HPLC analysis coupled with FAB-MS, Self et al. (4) isolated and/or detected catechin- and not epicatechin-gallates. However, the sites of esterification have not been unequivocally defined (4). They have

proposed (4) that two of their isomeric flavan-3-ol-monogallates are substituted on the catechol ring (i.e. 3'-/4'-position) while the third one is thought to be either 5- or 7-substituted. Furthermore, Self et al. found another two isomeric flavan-3ol-digallates ande they proposed that their structure may involve the presence of one galloyl moiety at C-3' or C-4' and the second gallyol unit either at the 5-OH or the 7-OH position of ring A (4). The presence of a trace of(+)-catechin-3-gallate has been demonstrated too (4).

Bark of Acacia nilotica (L.) Wilid. ex Del. subsp. tomentosa and subsp. nilotica were collected from the outskirts of Khartoum. The plant material was taxonomically identified by late Prof. H. M. Elamin and herbarium specimens were deposited at the Forest Research Institute, Khartoum, Sudan.

Physico-chemical and spectroscopic methods All NMR spectra were recorded using a Bruker AM400 spectrometer operating at 400.1 MHz for 'H and 100.6 MHz for '3C. Solvents are indicated below. The electron-impact (70 eV) mass spectra were recorded on Varian CH-4 and CH-7 instruments.

Isolation of compounds The milled bark (1 kg) ofAcacia nilotica (L.) Wilid. ex Del. subsp. tomentosa was extracted with 70% MeOH. The extract was concentrated and fractionated with light petroleum (b.p. 60—800C), then EtOAc. The concentrated EtOAc extract (62 g) was subjected to column chromatography over polyamide (WoeIlm MN 5C6). Then elution with water followed by aqueous MeOH by gradually increasing the percentage of MeOH and finally pure MeOH furnished fractions containing a mixture (4 g) of compounds 1 and 2. This mixture was further fractionated by DCCC (Model DCC-A apparatus; Tokyo Rikakikai, Tokyo, Japan) using CHC13/MeOH/H20 (7: 13 : 8) as a solvent system. Fractions were monitored by TLC and identical fractions were pooled together to yield 1 (75 mg) and 2 (35 mg). Compound 4 was eluted from the polyamide column as a relatively pure substance (0.5 g). Finally, 3 (0.65 g) was

eluted. All physico-chemical and spectroscopic data of 1—4 were in close agreement with published data.

Characterisation of isolated compounds Gallic acid (1): Greyish amorphous powder. Identical in all respects (UV, IR, 'H-NMR, 13C-NMR, MS. and co-TLC) with au-

thentic gallic acid.

In an attempt to contribute to an understanding of this species as a potential source for bioactive agents, we have examined material originating from Sudan.

Gallic acid methyl ester (2): An off-white powder. UV Xmax nm:

283; 'H-NMR b (CD3OD): 3.78 (3H, OCH3), 7.04 (2H, H-2/6);

'3C-NMRÔ(acetone-d6): 121.5 (C-i), 110.1 (C-2/6), 146.5 (C-3/5), 139.7 (C-4), 168.9 (C0), 52.3 (OCH3); MS m/z (relative intensity, %): 184(41, Mi, 153(100, M — OCH3), 125 (24), 79(20), 39(18).

Planta Medica 55(1989) 557

(+)- Catechin-5-galloyl Ester as a Novel Natural Polyphenol

Table 1 'H-NMR spectral data of compounds Hydrogenc

4

5

6b

H-2 H-3 H-4° H.4e H-6 H-8

4.59 (d,J= 8Hz) 3.97 (m) 2.50 (dd, J= 8 and 16Hz) 2.84(dd,J= 5 and 16Hz)

4.65 (d,J= 8Hz)

4.65 (d,J= 8Hz)

4.04 (m)

4.05 (m)

2.60 (dd,J= 8and 16Hz) 2.92 (dd,J= Sand 16Hz)

2.50 (dd,J= 8and 16Hz)

5.85 (d, J= 2Hz) 5.39 (d,J= 2Hz) 6.83 (d, i = 2Hz)

6.18 (d, = 2Hz)

H-2' H-5' H-6' H-2"

6.76 (d,J= 8Hz) 6.71 (dd,J= 2 and 8Hz) —

J

6.20 (d,J= 2Hz) 6.85 (d, J = 2Hz) 6.76 (d,J= 8Hz) 6.72 (dd,J= 2 and 8Hz) 7.15

4—6°.

2.83 (dd, J= 6 and 16Hz) 6.30 (d, J= 2Hz)

6.35 (d,J= 2Hz)

7.26

a In acetone-d6 / D,0 nixture. Taken from Ref. (5).

a: quasi-axial; e: quasi-equatorial. Not reported.

Naringenin (3): U\' 'max nm: 289, (+ NaOMe) 319 and 267, (+ A1C1,) 378, 311, and 267 (+ A1C13/HC1) 378, 311, and 267, (+

NaOAc) 324 and 267, (+ NaOAc/H3B03) 291; 'H-NMR ô (acetone-d6): 2.97 (1H, dd, H-3°"°, J= 3 and 17 Hz, 3.42 (1H, dd, H-3°'°, J= 13 and 17 Hz), 5.69 (1H, dd, H-2, J= 3 and 13Hz), 6.20 (1H, d, H-6, J= 2Hz), 6.21 (1H, d, H-8, J= 2Hz), 7.15/7.64 (4H, AA'XX' spin system, H-2'/3'/ 5'/6'), 12.43 (1H, s, 5-OH, replaceable with D20); 13C-NMR 6 (acetoned6): 43.3 (C-3), 79.8 (C-2), 95.8 (C-8), 96.8 (C-6), 103.1 (C-4a), 116.1 (C3'/S'), 128.9 (C-2'/6'), 130.6 (C-i'), 158.5 (C-4'), 164.2 (C-8a), 165.1 (C5), 167.2 (C-7), 197.1 (C-4); MS m/z (relative intensity %): 273(100, M° + H), 272(31, M'j, 179(17, C9H7O), 153(46, C7H,O), 120(89, C,H,O1, 107 (23, C6H701.

(+)-Catechin (4): Crystallized from H,O, m.p. 176— 178°C. OIl a0: +16.7° (acetone). UV Ama. nm 284, (+NaOMe) 298, (+AJC13) 288, (÷AIC13/HCI) 284, (+NaOAc) 286, 330 (sh), and 402, (+NaOAc/H,BO,) 288; 'H-NMR 6 (CD,OD): see Table 1; 13C-NMR 6 (acetone-d6): 28.5 (C-4), 68.2 (C-3), 82.3 (C-2), 95.2 (C-8), 96.0 (C-6), 100.4 (C-4a), 115.0 (C-2'), 115.6 (C-5'), 119.9 (C-6'), 131.8 (C-i '), 145.4 (C-3'), 145.5 (C-4'), 156.6 (C-8a), 157.0 (C-7), 157.4 (C-5); HRMS: 290.079 (C15H,406). calculated 290.079; MS m/z (relative intensity %): 290 (22, Mi, 272 (3, M — H20), 153 (18), 152 (48, C,H70), 139(100, C7H7O), 123(42, C7H7O), 69(14).

Compound 5: Positive vanillin/HC1 reaction (pink) and violet colour with FeC!,. UV Am nm: 284; 'H-NMR 6 (CD,OD/D,O): see Table 1; 13C-NMR 6 (CD,OD/D20): 28.5 (C-4), 68.4 (C-3), 82.9 (C-2),

101.9/102.0 (C-6/8), 107.0 (C-4a), 110.5 (C-2°/6"), 115.2 (C-4'), 116.1 (C-2'), 120.0 (C-6'), 131.9 (C-i'), 146.3 (C-3'/4'), 146.7 (C-3"), 151.8 (C5), 156.8/157.6 (C-7/8a), 167.1 (C=0), 170.3(7); MS m/z (relative intensity%); 442(20, Mi, 291 (70, C14H110), 110(30).

Enzymatic hydrolysis Compound 5(10 mg) was dissolved in aqueous solution and incubated with tannase enzyme at 37°C. After 2 h the solution

was evaporated to dryness, the residue dissolved in FtOH, and chromatographed over sephadex LH-20. Elation with EtOH yielded gallic acid (1) and 4.6mg of (+)-catechin (4).

maximum at 284 nm and the IR showed an ester carbonyl at 1760 cm1. The overall appearance of the 1H-NMR spectrum of S was similar to that of 4 except for the additional singlet at ö = 7.15 ppm, integrated for two protons, which is attributable to a galloyl unit (Table 1). The deshielding of the two meta-coupled protons H-6 and H-8 on ring A in comparison with the respective atoms of 4 indicated that the site of esterification is either at the 5- or the 7-position (Table 1). The significant deshielding of both quasi-axial and quasi-equatorial H-4 resonances indicated that the galloyl moiety is rather attached to the C-S-OH group. The deshielding effect of the galloyl moiety on the C-4 methylene protons was more pronounced than that in the reported isomer 6 (Table 1) (5). The proposed strucutre was further substantiated by mass spectral evidence. The most significant feature of the mass spectrum was the presence of a prominent peak ion (65%) at ,n/z 291. This corresponds with a ring-A fragment (7) which is produced via a retro-Diels-Alder (RDA) fragmentation and accounts for the attachment of a galloyl group to ring A. This mode of fragmentation which is associated with hydrogen transfer to the A-ring fragment (RDA + H), is consistent with those reported for flavan-3-ol (6). On en-

zymatic hydrolysis with tannase enzyme in aqueous solution S yielded gallic acid (1) and (+)-catechin. The absolute configura-

tion of 5 was determined by comparison of its CD spectrum with that of (+)-catechin (4). The "C-NMR spectrum 5 showed

18 signals. Those of C-3' and C-4' are coinciding (b = 146.3 ppm) and that of C-i" is apparently hidden below that of

C-6'. All carbon resonances are in excellent agreement with the anticipated ô-values in comparison with 4 and 6 and accounting for galloyl substitution at 5-OH. However, no signal was detected around ô = 140 ppm which could be attributed to C-4".

Instead, we had an extra signal at 6 = 170.3 ppm which we could not account for either. However, it is well documented that these types of molecules are very susceptible to decomposition (7).

Results and Discussion

The ethyl acetate concentrate of the bark of Acacia nilotica subsp. tomentosa was fractionated by a combination of polyamide and sephadex LH-20 chromatography to yield gallic acid (1) and its methyl ester (2) followed by naringe-

nm (3) and (+)-catechin (4). Finally, a novel compound was eluted which was characterized by spectroscopic evidence and enzymatic hydrolysis as (+)-catechin-5-O-galloyl ester (5). The

exact mass measurement of 5 established the molecular formula C,,H150,0. This compound is a pale yellow amorphous powder which reacted positively on TLC to vanillin/HCI (pink)

and FeCl, (blue). The UV spectrum exhibited a distinct

(+)-Catechin (4) and gallic acid (1) were identified by direct comparison with authentic material; gallic acid methyl ester (2), naringenin (3), and (+)-catechin (4) were identified by analysis of their spectra data.

From the 'H- and "C-NMR spectra it was not clear if the methyl group in 2 was at 4-OH or at the carboxylic function. A two-dimensional COLOC (NMR) experiment (8)

proved that 2 is indeed gallic acid methyl ester and not a 4methyl gallic acid; there was a cross-peak due to the existence of a three-bond 'H,"C coupling between the methyl protons and the carbony) carbon.

558

S.A. Khalidetal.

Planta Medica 55(1989)

O OR

References

— OH 2

OH

HO

1RH 2R

Fitoterapia 58, 51. Ayoub, S. M. (1984) Fitoterapia 55, 343.

OH 0

OH

Self, R.. Eagles, J., Galletti, G. C., Mueller-Harvey, I., Hartley, H. D., Lea, A. G. H., Magnolato, D., Richli, U., Gujer, R., Haslam, E.(1983) Biomed. Mass. Spectrom. 13, 449.

3

CH3

OH

6

R 20 R1

5 galloyl 6 H

R2

7 (m/z r 291)

H H

galloyl

Acknowledgements We thank Prof. F. Tillequin, Department of Pharmacognosy, University René Descartes, Paris, France, for the early measurement of the 'H-NMR spectra of 4 and 5.

Drewes, S. E. (1974) Chroman and Related Compounds, Progr. Mass

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