954
J. Nat. Prod. 1999, 62, 954-958
Biological Evaluation of Proanthocyanidin Dimers and Related Polyphenols Tess De Bruyne,*,† Luc Pieters,† Myriam Witvrouw,‡ Erik De Clercq,‡ Dirk Vanden Berghe,† and Arnold J. Vlietinck† Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, and Rega Institute for Medical Research, Katholieke Universiteit Leuven (KUL), Minderbroedersstraat 10, B-3000 Leuven, Belgium Received October 26, 1998
A series of dimeric procyanidins (1-9) and some related polyphenols (10-15) were chosen as model compounds in a comparative investigation for various biological activities in order to obtain structureactivity relationships. Antiviral [herpes simplex virus (HSV) and human immunodeficiency virus (HIV)], antibacterial, superoxide radical-scavenging, and complement-modulating properties were assessed. In general, more pronounced activities were seen with epicatechin-containing dimers for anti-HSV, antiHIV, and radical-scavenging effects, while the presence of ortho-trihydroxyl groups in the B-ring was important in compounds exhibiting anti-HSV and radical-scavenging effects and complement classical pathway inhibition. Double interflavan linkages gave rise to interesting antiviral effects (HSV and HIV) and complement inhibition. The influence of the degree of polymerization or the type of interflavan linkage (4f6 or 4f8) differed in the different biological systems evaluated. Only minor or moderate antibacterial effects were observed for the compounds under investigation. Condensed tannins or proanthocyanidins are widely distributed in nature and are, in many cases, the active compounds of the medicinal plants from which they can be isolated.1 Reports of several in vitro assays demonstrate potentially significant interactions with biological systems, such as antiviral, antibacterial, molluscicidal, enzymeinhibiting, antioxidant, and radical-scavenging properties.2 Their tendency to interfere with biological systems is, at least in part, due to a characteristic ability to form complexes with macromolecules, combined with a polyphenolic nature.1,2 To investigate the possible influence of individual structural and configurational parameters on some biological systems, the properties of a series of proanthocyanidin dimers, together with some related polyphenols, namely procyanidin B1 or epicatechin-(4βf8)catechin (1), procyanidin B2 or epicatechin-(4βf8)-epicatechin (2), procyanidin B3 or catechin-(4Rf8)-catechin (3), procyanidin B4 or catechin-(4Rf8)-epicatechin (4), procyanidin B5 or epicatechin-(4βf6)-epicatechin (5), procyanidin B6 or catechin-(4Rf6)-catechin (6), procyanidin B8 or catechin-(4Rf6)-epicatechin (7), proanthocyanidin A1 or epicatechin-(4βf8,2βfOf7)-catechin (8), proanthocyanidin A2 or epicatechin-(4βf8, 2βfOf7)-epicatechin (9), procyanidin C1 or epicatechin-(4βf8)-epicatechin(4βf8)-epicatechin (10), (-)-epicatechin (11), (+)-catechin (12), epigallocatechin (13), (+)-taxifolin (14), and gallocatechin-(4′fOf7)-epigallocatechin) (15) (Chart 1), were evaluated in a panel of biological screening assays, comprising antibacterial, antiviral [anti-herpes simplex (HSV) and anti-human-immunodeficiency virus (HIV)], complement modulation, and radical-scavenging test systems. Results and Discussion Compounds 1-9, 14, and 15 were obtained from various plant sources or by biomimetic synthesis,3-5 and compounds 11 and 12 were obtained from a commercial source. A comparative NMR study of the procyanidin dimers will be published elsewhere in due course.3 Procyanidin C1 (10) * To whom correspondence should be addressed: Tel. fax: +32 3 820 27 09. E-mail:
[email protected]. † University of Antwerp. ‡ Katholieke Universiteit Leuven.
10.1021/np980481o CCC: $18.00
was isolated from Theobroma cacao, and epigallocatechin (13) from “dragon’s blood”, the latex of a South American Croton species.6 The screening system used to evaluate complement modulation was based on the hemolytic properties of the complement system and involved the spectrophotometric determination of the amount of hemoglobin that was released. The 50% inhibitory concentrations (IC50) for the classical and alternative pathways of the complement system are presented in Table 1. The results of inhibition on the classical pathway clearly showed that the dimers tested were more active than the monomeric flavonoids. The trimer procyanidin C1 (10) had a still more pronounced activity. The flavanoid epigallocatechin (13) was an important exception, having an IC50 value of 19.6 µM. Comparing the dimers with single interflavanoid linkage, differences were not very pronounced, although there was a tendency for 4f6-dimers to have a better inhibitory activity than their respective 4f8-counterparts. Procyanidins B6 (6) (IC50 18.5 µM) and B8 (7) (IC50 19.7 µM) proved the most active. The doubly linked proanthocyanidin A2 (9) exhibited potent inhibitory effect on classical complement (IC50 11.6 µM). The Bridelia ferruginea biflavanoid gallocatechin-(4′fOf7)-epigallocatechin (15) also showed a relatively low IC50 value (14.6 µM). The orthotrihydroxyl group is probably responsible for the potent complement inhibition, as also demonstrated for other interactions of procyanidins and related compounds in biological systems, such as radical scavenging activity. An increasing number of processes has been associated with enhanced free-radical production, including inflammation, radiation damage, anti-cancer reaction, immunity, arteriosclerosis, myocardial ischemia, and aging. The superoxide scavenging activity of the test compounds was expressed as the IC50 value for the production of superoxide anions in an enzymatic system. The IC50 values are listed in Table 1. When comparing the results of (-)-epicatechin (11), (+)-catechin (12), epigallocatechin (13), and (+)taxifolin (14), similar conclusions, as in a previous study on flavonoids, could be reached.7 Epigallocatechin showed a pronounced activity. Three B-ring hydroxyl groups; a hydroxyl at C-3; a saturated C-2, C-3 bond, and the absence
© 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 06/25/1999
Proanthocyanidin Dimers and Phenols
Journal of Natural Products, 1999, Vol. 62, No. 7 955
Chart 1
Table 1. Biological Activities of Compounds 1-15
compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 positive control substance
superoxide scavenging activity [IC50 (µM) ( SD] n.t.b
10.5 ( 0.1 41.3 ( 5.4 30.0 ( 2.0 11.4 ( 0.9 27.3 ( 1.8 32.9 ( 2.9 14.7 ( 1.0 17.8 ( 0.7 10.7 ( 0.5 18.3 ( 0.7 13.1 ( 0.1 3.4 ( 0.3 24.2 ( 1.5 2.0 ( 0.1 (4.9 ( 0.2)10-4 d
anti-HIV
complement classical pathway [IC50 (µM)]
complement alternative pathway [IC50 (µM)]
IC50 (µg/mL)
CC50 (µg/mL)
31.3 58.0 37.7 45.5 51.7 18.5 19.7 57.1 11.6 6.0 655.5 647.2 19.6 850.3 14.6 21.9e
72.0 n.a.c n.a. n.a. n.a. n.a. 83.7 105.0 112.8 85.5 n.a. n.a. 179.4 n.a. 86.0 408.3e
n.t. 17.2 27.0 >0.3 8.3 29.9 19.6 14.0 5.8 4.3 34.1 >86.8 >16.1 >25.4 n.t. 0.0002f
n.t. 135.1 117.8 0.3 53.5 141.2 126.6 144.3 137.6 16.5 136.2 86.8 16.1 25.4 n.t. 2.5813f
a SI ) selectivity index (CC /IC ). b n.t. ) not tested. c n.a. ) not active. 50 50 acid. f Zidovudine.
of any carbonyl at C-4 contributed to a good superoxide scavenging activity. The best results for dimeric proanthocyanidins were obtained for procyanidins B2 (2) and B5 (5). The procyanidins B3 (3), B4 (4), B6 (6), and B8 (7) were about three times less active. For the dimeric procyanidins with single linkage, those with two epicatechin units (B2 and B5) gave markedly better results than products containing a catechin unit. The type of linkage had no consistent significant influence on superoxide scavenging activity. Procyanidin C1 (10) produced no higher scavenger properties than its dimeric analogue procyanidin B2. The biflavanoid gallocatechin-(4′fOf7)-epigallocatechin (15) dis-
d
SIa 8 5
