Chem. Pharm. Bull. 55(3) 492—494 (2007)

western North America, and its aerial parts have been used as a cough suppressant and a remedy for respiratory disease by America Indians. As chemical constituents of this plant, the presence of triterpenoids, such as ursolic acid and cucurbitan-type triterpenes as inhibitors of Epstein-Barr virus early antigen (EBV-EA) activation were reported. We reported the isolation and characterization of nine tannins and related polyphenols [casuarinin (2), stachyurin (3), pterocarinin A, stenophyllanin A (4), alienanin B, casuglaunin A, ( )-epicatechin, procyanidin B-4 and ellagic acid] along with two nitrile glucosides (menisdaurin and lithospermoside) from the leaves and stems of C. mexicana. Their inhibitory effects on in vitro EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and on in vivo two-stage carcinogenesis on mouse back-skin were also reported. Further chemical investigation on a polar fraction of the plant extract has resulted in the isolation of a new complex tannin dimer named cowaniin (1) and a known nitrile glucoside. This paper describes the isolation and structure elucidation of the new tannin, along with five known polyphenols from a non-polar fraction. A concentrated 70% aqueous acetone homogenate of the dried aerial parts of C. mexicana was extracted with Et2O, EtOAc and water-saturated n-BuOH to give the respective extracts and the water-soluble portion. The Et2O extract was chromatographed over polyvinyl gels to afford five known compounds, quercetin 3-O-a-L-arabinofuranoside, and pcoumaric, p-hydroxybenzoic, protocatechuic and gallic acids. The separation and purification of the n-BuOH and watersoluble portion by polystylene and/or polyvinyl gels gave cowaniin (1) and a known nitrile glucoside, purshianin. The known compounds were identified by direct comparison with authentic samples and/or by their spectral comparisons with data reported in the literature. Cowaniin (1) was obtained as an off-white amorphous powder and exhibited an [M NH4] ion peak at m/z 2162 in electrospray ionization (ESI)-MS. The molecular formula was determined to be C97H68O57 by elemental analysis and NMR spectra along with MS data. The H-NMR spectrum of 1 exhibited two 2H singlets (d 7.09, 7.06) and seven 1H singlets (d 6.83, 6.72, 6.62, 6.55, 6.53, 6.51, 6.25), which were assignable to two galloyl and four hexahydroxydiphenoyl (HHDP) groups. These acyl groups were consistent with the appearance of ten ester carbonyl carbon signals in C-NMR (see text) and chemically confirmed by the production of methyl tri-O-methylgallate (7) and dimethyl hexamethoxydiphenate (8) upon methanolysis of the methylated derivative of 1. The odd number of aromatic 1H singlets in the HNMR spectrum of 1 suggested that one of the HHDP groups may participate in C–C bond formation with an anomeric carbon of open-chain glucose residue as characterized by C-glucosidic ellagitannins such as casuarinin (2) and stachyurin (3). The presence of two open-chain glucose residues was indicated by the coupling patterns and chemical shifts of aliphatic proton signals which were assigned by H–H correlated spectroscopy (COSY) (see Experimental). Two anomeric proton signals were observed at d 4.64 (d, J 1.5 Hz) and 5.85 (d, J 2.5 Hz), the former of which was characteristic of that of complex tannin, a condensate of Cglucosidic tannin with catechin (or epicatechin) such as 4. In fact, the occurrence of a catechin unit in the molecule was implied by two methine signals [d 4.73 (d, J 4 Hz) and 3.98 (dt, J 5, 4 Hz)] and methylene signals [d 2.61 (dd, J 4, 16 Hz) and 2.55 (dd, J 5, 16 Hz)] as well as ABX-type signals in the aromatic region of the H-NMR spectrum. CNMR resonances of glucose and catechin residues of 1, which were assigned by the heteronuclear multiple quantum connectivity (HMQC) spectrum, indicated a close resemblance to those of stachyuranin A (6) and stenophyllanin B (5) rather than stenophyllanin A (4) as shown in Table 1. These NMR spectral features and MS data, taking the absence of A-ring protons of catechin into consideration, suggested the dimeric structure of cowaniin, in which the 6and 8-positions of the catechin unit bind respectively to each anomeric carbon of ellagitannin monomers with an openchain glucose core. The binding mode of the ellagitannin monomers to catechin as shown in 1 was determined by the heteronuclear multiple bond connectivity (HMBC) spectrum. Among three oxygen-bearing carbon signals (d 150.1, 153.5, 492 Vol. 55, No. 3