Secondary Metabolites from the Leaves of Ilex Cornuta

Abstract

Ilex (Aquifoliaceae) species are found throughout China, and some are used extensively in traditional Chinese medicine. For example, I. rotunda is antipyretic and used for the treatment of the common cold, tonsillitis, and stomach and intestinal ulcers. I. pubescens is used for the treatment of coronary disease, myocardial infarction, dysentery, and erysipelas, and I. cornuta and I. latifolia are used for the treatment of headaches, toothaches, bloodshot eyes, and tinnitus [1]. Previous studies on the Ilex genus have resulted in the isolation of essential oils [2] triterpenes, triterpene glycosides [3–7], hemiterpene glycosides [8], and phenolic compounds [9, 10]. In the present study, 12 compounds were isolated from the leaves of I. cornuta. A MeOH extract of the I. cornuta leaves was concentrated to obtain a residue, which was partitioned between CH2Cl2 and H2O. The aqueous layer was extracted with n-BuOH, and then the n-BuOH layer was concentrated and analyzed by chromatography. As a result, 12 compounds, including eight flavonoids and four phenolic carboxylic acids, were isolated. The flavonoids were quercetin [11], quercetin 3-O-D-glucopyranoside [12], quercetin 3-O-D-galactopyranoside [13], quercetin 3-O-D-glucopyranosyl-(1 6)-D-glucopyranoside [14], isorhamnetin [15], isorhamnetin 3-O-Dglucopyranoside [15], isorhamnetin 3-O-D-galactopyranoside [15], and isorhamnetin 3-O-D-glucopyranosyl-(1 6)D-glucopyranoside [15]. The phenolic carboxylic acids were caffeic acid [16], p-hydroxybenzoic acid [17], protocatechuic acid [11], and vanillic acid [16]. This is the first report of the isolation of these compounds from this source. Leaves from I. cornuta Lindl. & Paxton were collected in Kaohsiung, Taiwan, in October 2006. Plant material was identified by Dr. Fu-Yuan Lu (Department of Forestry and Natural Resources, College of Agriculture, National Chiayi University, Chiayi City, Taiwan). A voucher specimen was deposited in the School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan. The air-dried leaves of I. cornuta (4.2 kg) were extracted with MeOH (5 10 L) at room temperature, and each MeOH fraction was concentrated under reduced pressure. The MeOH fractions were concentrated by evaporation and then partitioned to yield CH2Cl2 and aqueous extracts. The aqueous extracts were concentrated by evaporation and extracted with n-BuOH to give a n-BuOH layer and an aqueous layer. The n-BuOH layer (178.6 g) was subjected to silica gel chromatography (500 g, 70–230 mesh) with EtOAc–MeOH elution. Ten fractions were obtained. Part of fraction 2 (20.3 g) was subjected to silica gel chromatography with n-hexane–EtOAc (1:1, v/v). The volume fraction of EtOAc was gradually increased to yield five fractions (2-1–2-5). Fraction 2-2 (2.2 g) was further purified on a silica gel column using an n-hexane– CH2Cl2 mixture to obtain caffeic acid (7 mg), vanillic acid (3 mg), and p-hydroxybenzoic acid (5 mg). Fraction 2-4 (5.7 g) was further purified on a silica gel column with n-hexane–EtOAc elution to obtain isorhamnetin (12 mg). Part of fraction 3 (12.7 g) was subjected to silica gel chromatography with n-hexane–EtOAc (1:8, v/v). The volume fraction of EtOAc was gradually increased to yield quercetin (20 mg) and protocatechuic acid (34 mg). Part of fraction 4 (32.5 g) was subjected to silica gel chromatography with EtOAc–MeOH (20:1, v/v). The volume fraction of MeOH was gradually increased to yield isorhamnetin 3-O-D-glucopyranoside (67 mg) and isorhamnetin 3-O-D-galactopyranoside quercetin (42 mg). Part of fraction 5 (17.3 g) was subjected to silica gel chromatography with EtOAc–MeOH (20:1). The volume fraction of MeOH was gradually increased to obtain five further fractions (5-1–5-5). Fraction 5-3 (5.8 g) was further purified on a silica gel column with EtOAc–MeOH to obtain quercetin 3-O-D-glucopyranoside (78 mg) and quercetin 3-O-D-galactopyranoside (113 mg). Fraction 6-8 (22.7 g) was eluted with EtOAc–MeOH (8:1) and was repeatedly subjected to silica gel chromatography to give quercetin 3-O-D-glucopyranosyl-(1 6)-D-glucopyranoside (56 mg) and isorhamnetin 3-O-D-glucopyranosyl-(1 6)-D-glucopyranoside (24 mg).