Protective Effect of Phenolic Compounds Isolated from the Hooks and Stems of Uncaria sinensis on Glutamate-Induced Neuronal Death

American Journal of Chinese Medicine, Wntr, 2001 by Yutaka Shimada, Hirozo Goto, Toshiaki Kogure, Naotoshi Shibahara, Iwao Sakakibara, Hiroshi Sasaki, Katsutoshi Terasawa

(Accepted for publication September 30, 2000)

Abstract: We isolated the phenolic compounds epicatechin, catechin, procyanidin B-1, procyanidin B-2, hyperin and caffeic acid from the hooks and stems of Uncaria sinensis (HSUS), and studied their protective effects against glutamate-induced neuronal death in cultured rat cerebellar granule cells. Cell viability evaluated by MTT assay was significantly increased by application of epicatechin (100-300 [micro]M), catechin (300 [micro]M), procyanidin B-1 (30-300 [micro]M) and procyanidin B-2 (100-300 [micro]M) compared with exposure to glutamate only.[sup.45][Ca.sup.2 ] influx into cells induced by glutamate was also significantly inhibited by administration of epicatechin (300 [micro]M), catechin (300 [micro]M), procyanidin B-1 (100-300 [micro]M) and procyanidin B-2 (100-300 [micro]M). These results suggest that epicatechin, catechin, procyanidin B-1 and procyanidin B-2 are the active components of HSUS that protect against glutamate-induced neuronal death in cultured cerebellar granule cells by inhibition of [Ca.sup.2 ] influx.

Glutamate is known to be one of the mediators of neuronal death from ischemic-hypoxic injury in the brain (Choi, 1988). The extracellular concentration of glutamate elevates in the brain during ischemia (Benveniste et al., 1984). The excessive release of glutamate resulting from ischemia overstimulates glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, and induces neuronal death through [Ca.sup.2 ] influx (Rothman and Olney, 1986).

The hooks and stems of Uncaria sinensis (Oliv.) Havil. (HSUS), Uncariae Uncus Cum Ramulus, the main medicinal plant of Choto-san (Diao-Teng-San in Chinese), were used for obtaining the isolates for this study. Choto-san, which is composed of 11 crude drugs, has long been administered as a decoction to relatively aged patients suffering from headache, dizziness, vertigo, tinnitus, shoulder stiffness so forth in China and Japan. We recently revealed the usefulness of Choto-san on patients with vascular dementia by a double-blind, placebo-controlled study (Terasawa et al., 1997). We also demonstrated that the water extract of HSUS has a protective effect against glutamate-induced neuronal death in cultured cerebellar granule cells through the inhibition of [Ca.sup.2 ] influx (Shimada et al., 1998). Further, we evaluated the activities of alkaloids isolated from HSUS and revealed that such oxyindole alkaloids as isorhynchophylline, isocorynoxeine and rhynchophylline, and such indole alkaloids as hirsuteine and hirsutine possessed protective effects against glutamate-induced neuronal death by the inhibition of [Ca.sup.2 ] influx (Shimada et al., 1999).

Uncaria genus contains not only alkaloids but also phenolic compounds (Aimi et al., 1982). In the present study, we evaluated the protective effects of the HSUS-isolated phenolic compounds epicatechin, catechin, procyanidin B-1, procyanidin B-2, hyperin and caffeic acid against glutamate-induced neuronal death and their inhibitory effects on glutamate-induced [Ca.sup.2 ] influx in cultured rat cerebellar granule cells.

Materials and Methods

HPLC Analysis of Phenolic Compounds

HSUS (Guangxi district, China; 2g) was chopped and extracted with methanol (20 ml) under ultrasonication for 30 min. The solution (20 [micro]l) was filtrated (0.45 [micro]m, Toyo Roshi Co., Ltd.) and then submitted to HPLC analysis. This was performed with an LC-10A HPLC system (Shimadzu Co, Ltd.) using a TSK-gel ODS-80TM column (250 x 4.6 mm) eluting with 1% [H.sub.3][PO.sub.4]-MeOH (9:1 to 1:1, linear gradient, 60 min). The flow rate was 1.0 ml/min. The eluate was monitored at 254 nm with a UV detector (Figure 1).

[Figure 1 ILLUSTRATION OMITTED]

Extraction and Isolation of Phenolic Compounds

HSUS (1 kg) was extracted with boiling water (5 L) for 2 hr three times. Each extract was combined and lyophilized to give a brown mass (114.6 g, yield 11.5%). The water extract was chromatographed on polus polymer gel (Diaion HP-20, 2 L), eluted with water (2 L, yield 5.8%), [H.sub.2]O-methanol (1:1) (2 L, yield 2.9%), and then with methanol (2 L, yield 0.2%). The [H.sub.2]O-methanol (1:1) eluate was purified by Sephadex LH-20 column chromatography, eluted with [H.sub.2]O-ethanol (1:1) to obtain epicatechin (349 mg), catechin (509 mg), procyanidin B-1 (235 mg), procyanidin B-2 (349 mg), hyperin (194 mg) and caffeic acid (33 mg) (Figure 2). Epicatechin, catechin and caffeic acid were identified by direct comparison with authentic samples (Sigma). Procyanidin B-1, procyanidin B-2 and hyperin were identified by comparison of their spectral data in the previous literatures (Markham et al., 1978; Porter et al., 1982).

[Figure 2 ILLUSTRATION OMITTED]

As these phenolic compounds are water-insoluble, they were dissolved in dimethyl-sulfoxide (DMSO) at a concentration of 100 mM.

Cell Culture

Cerebellar granule cells were cultured in poly-L-lysine (Sigma) coated 96-well culture plates at a density of 106 cells/ml from the brains of 8-day-old Wistar rats as described previously (Gallo et al., 1982; Shimada et al., 1998). The cells were used at 7 days in vitro for the experiments.