Neuroprotective effects of resveratrol derivatives from the roots of Vitis thunbergii var. sinuate against glutamate-induced neurotoxicity in primary cultured rat cortical cells

Abstract

We observed that an aqueous extract of this medicinal plant exhibited significant neuroprotection against glutamate-induced toxicity in primary cultured rat cortical cells from methanol extracts of the roots of Vitis thunbergii var. sinuate (Vitaceae). To further clarify the underlying neuroprotective mechanisms of this observed effect, we isolated and identified various active fractions and components. By using such fractionation procedures, five resveratrol derivatives − vitisinols A (1), B (2), C (3), (+)-vitisin A (4), and (+)-vitisin C (5) − were isolated from the methanol extracts from the roots of V. thunbergii var. sinuate. Among these five resveratrol derivatives, 3 exhibited most significant neuroprotective activity against glutamate-induced neurotoxicity, as indicated by a cell viability of approximately 75%−85%, at concentrations ranging from 0.1 μM to 10 μM. These findings indicate that the neuroprotective effects of V. thunbergii var. sinuate might be due to the inhibition of glutamate-induced toxicity by resveratrol derivatives present in the plant.

Keywords

Vitaceae neuroprotective resveratrol derivatives glutamate-induced toxicity

Introduction

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders affecting many elderly people worldwide. In addition to the neuropathologic hallmarks of this disease, namely neurofibrillary tangles and amyloid plaques, it is also characterized by the loss of cholinergic neurons in the basal forebrain and a major cause of morbidity and disability in the adult population.¹ Neuronal death is an important feature of both acute and chronic neurodegenerative diseases. AD is associated with the accumulation of l-glutamate (Glu) deposits in senile plaques and neurofibrillary tangle lesions in specific areas of brain. Glutamate, a major excitatory amino-acid neurotransmitter in the central nervous system (CNS), is involved in fast synaptic transmission, neuronal plasticity, outgrowth and survival, memory, learning and behaviour.² However, glutamate-mediated excitotoxicity appears to play a crucial role in neurodegenerative disorders, particularly in Parkinson’s disease, Alzheimer’s disease, epilepsy, spinal cord trauma and ischemic stroke.³ Thus, neuroprotection against glutamate-induced toxicity has been a therapeutic strategy for preventing and/or treating both acute and chronic forms of neurodegeneration.⁴ Vitis thunbergii var. sinuate (Vitaceae) is widely distributed in China, Japan, and Korea, the roots of V. thunbergii var. sinuate in Korea as is a folk medicine ‘Gga Ma Gui Meo Ru’ for treatment of common cold, neuralgia and rheumatis.⁵ Park et al. reported cafferic acid derivatives of the roots of V. thunbergii var. sinuate.⁵ Although some natural compound has been isolated from V. thunbergii var. sinuate, its biological activity has not been fully characterized. The aim of this study was to investigate the neuroprotective properties of the roots of V. thunbergii var. sinuate. The principal component of the drugs manifesting neuroprotective activity has not yet been reported so far. In the context of our natural product chemistry program dealing with the development of new potent neuroprotection agents, we have examined the isolation of major compound as leads for novel glutamate-induced toxicity inhibitors.

Material and methods

General

Thin-layer chromatography (TLC) was performed on precoated silica gel G and GP uniplates from Analtech and visualized with 254-nm UV light. Vacuum liquid chromatography was carried out on silica gel 60 (Scientific Adsorbents Incorporated [SAI], particle size 32–63 µm, pore size 60 Å]. ¹H NMR spectra was recorded on a Bruker DPX 500 at 500 MHz; respectively. The chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane, and J–values are in Hz. DL-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNOX) were purchased from Sigma Chemical Company (St. Louis, Missouri, USA).

Plant materials and isolation of compounds

The roots of V. thunbergii var. sinuate were collected to Cheon Kwan San in August 2008 at Jangheung-gun, Jeolranamdo, South Korea. A voucher specimen (JNNRR-2008-0263) was deposited at herbarium, Jeonnam Institute of Natural Resources Research, Jangheung, South Korea. The air-dried roots of V. thunbergii var. sinuate (10 kg) were extracted with methanol by refluxing for 4 h (three times × 50 L) on a sonication bath at 35°C. The extract was filtered through a Buchner funnel using Whatman No. 1 filter paper. The combined MeOH extract was evaporated under reduced pressure to yield a black residue. Vacuum liquid chromatography (1 kg, 50 × 60 cm) of the MeOH extract (500 g), using n-hexane:CH₂Cl₂ (1:0 – 0:1) and CH₂Cl₂:MeOH (1:0–0:1) step gradients, produced 42 fractions. These were pooled by TLC profile into eight fractions (VT1–VT8), in which fraction VT 5 (23 g) eluted with petroleum ether-acetone (1:0 [500 mL], 2:1 [500 mL], 1:1 [500 mL], 1:2 [500 mL], 1:5 [500 mL], and 0:1 [500 mL]). The major three spot were purified by recrystallization with MeOH to afford compounds 1 (2.4mg), 2 (6.2 mg) 4 (11.2 mg). VT 7 (28 g) eluted with CH₂Cl₂-acetone (1:0 [500 mL], 2:1 [500 mL], 1:1 [500 mL], 1:2 [500 mL], 1:5 [500 mL], and 0:1 [500 mL]). The major two spots were purified by recrystallization with MeOH to afford compounds 3 (9.4 mg), 5 (17.2 mg).

Vitisinols A (1); brown powder; UV (MeOH) λ _max 277 (3.58), 230 (3.88), 201 (4.19) nm; IR (KBr) λ _max 3448 (OH), 2922, 1614, 1580, 1414, 1256, 1114 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 7.52 (4H, d, J = 8.5 Hz, H-2a, 2b, 6a, 6b), 6.90 (4H, d, J = 8.5 Hz, H-3a, 3b, 5a, 5b), 6.15 (2H, d, J = 1.5 Hz, H-14a,14b), 6.12 (2H, d, J = 1.5 Hz, H-12a, 12b), 5.43 (2H, d, J = 11.0 Hz, H-7a, 7b), 4.49 (2H, d, J = 11.0 Hz, H-8a, 8b). EIMS m/z 452 [M]⁺.

Vitisinols B (2) brown-yellow powder; UV (MeOH) λ _max 282 (4.13), 228 (4.60), 204 (4.85) nm; IR (KBr) λ _max 3417 (OH), 2923, 1650, 1504, 1435, 1341, 1256, 1098 cm⁻¹; ¹H NMR (Me₂CO-d6, 500 MHz) δ 9.31 (1H, s, CHO), 7.42 (1H, dd, J = 2.0, 8.5 Hz, H-4c), 7.12 (2H, d, J = 8.5 Hz, H-2a,6a), 7.04 (2H, d, J = 8.5 Hz, H-2b, 6b), 6.95 (1H, d, J = 8.5 Hz, H-3c), 6.76 (2H, d, J = 8.5 Hz, H-3a, 5a), 6.67 (2H, d, J = 8.5 Hz, H-3b, 5b), 6.66 (1H, d, J = 2.0 Hz, H-6c), 6.21 (1H, brs, H-14a), 6.14 (1H, d, J = 2.0 Hz, H-14b), 6.10 (1H, d, J = 2.0 Hz, H-12b), 6.02 (1H, br s, H-12a), 5.84 (1H, d, J = 11.5 Hz, H-7a), 5.57 (1H, d, J = 3.5 Hz, H-8b), 5.47 (1H, d, J = 3.5 Hz, H-7b), 4.26 (1H, d, J = 11.5 Hz, H-8a). FABMS m/z 573 [M − H] ⁻.

Vitisinols C (3) yellow powder; UV (MeOH) λ _max 358 (4.29), 279 (3.84), 226 (4.26), 202 (4.66) nm; IR (KBr) λ _max 3423 (OH), 2922, 1602, 1571, 1445, 1382, 1167 cm⁻¹; ¹H NMR (Me₂-CO-d6, 500 MHz) δ 7.36 (2H, d, J = 8.5 Hz, H-2b, 6b), 6.90(2H, d, J = 8.5 Hz, H-2a, 6a), 6.88 (1H, d, J = 17.0 Hz, H-8b), 6.79 (2H, d, J = 8.5 Hz, H-3b, 5b), 6.74 (1H, d, J = 17.0 Hz, H-7b), 6.62 (2H, d, J = 8.5 Hz, H-3a, 5a), 6.16 (1H, s, H-10b), 6.12 (2H, d, J = 2.0 Hz, H-10a, 14a), 6.10 (1H, d, J = 2.0 Hz, H-12a), 3.31 (1H, m, H-7a), 3.16 (1H, m, H-8a), 3.15 (2H, m, H-13b), 3.00 (2H, d, J = 6.0 Hz, H-12b). EIMS m/z 428 [M]⁺.

(+)-Vitisin A (4) amorphous powder, UV (MeOH) λ _max 283 (4.40), 320 (4.22); IR (Nujol) λ _max 3160 (OH), 1600, 1510, 1450 (aromatic); ¹H NMR (CD₃OD) δ 7.07 (2H, d, J = 8.5 Hz, H-2a,6a), 6.77 (2H, d, J = 8.5 Hz, H-3a, Sa), 5.31 (1H, d, J = 6.3 Hz, H-7a), 4.26 (1H, d, J = 6.3 Hz, H-Sa), 6.10 (2H, d, J = 2.2 Hz, H-10a,14a), 6.19 (1H, t, J = 2.2 Hz, H-12a), 5.92 (1H, d, J = 2.0 Hz, H-2b), 6.60 (1H, d, J = 8.8 Hz, H-Sb), 6.73 (1H, dd, J = 8.8, 2.0 Hz, H-6b), 6.32 (1H, d, J = 16.3 Hz, H-7b), 6.27 (1H, d, J = 16.3 Hz, H-Sb), 6.19 (1H, d, J = 2.2 Hz, H-12b), 6.44 (1H, d, J = 2.2 Hz, H-14b), 7.07 (2H, d, J = 8.8 Hz, H-2c,6c), 6.71 (2H, d, J = 8.8 Hz, H-3c,5c), 5.81 (1H, d, J = 11.5 Hz, H-7c), 4.12 (1H, d, J = 11.5 Hz, H-8c), 5.96 (1H, d, J = 2.2 Hz, H-12c), 6.13 (1H, d, J = 2.2 Hz, H-14c), 7.00 (2H, d, J = 8.8 Hz, H-2d,6d), 6.62 (2H, d, J = 8.8 Hz, H-3d, Sd), 5.25 (1H, d, J =3.2 Hz, H-7d), 5.42 (1H, d, J = 3.2 Hz, H-Sd), 6.07 (1H, d, J = 2.0 Hz, H-12d), 6.05 (1H, d, J = 2.0 Hz, H-14d). FAB MS mlz:907 [MH]⁺.

(+)-Vitisin C (5); Methylation of vitisin C, amorphous powder, IR (Nujol) λ _max 3300 br, 1605 cm^−l; ¹H NMR (CD₃OD) δ 7.04 (2H, d, J = 8.8 Hz, H-2a,6a), 6.82 (2H, d, J = 8.8 Hz, H-3a,5a), 5.31 (lH, d, J = 4.4 Hz, H-7a), 3.55 (lH, d, J = 4.4 Hz, H-8a), 5.98 (2H, d, J = 2.2 Hz, H-l0a, 14a), 6.24 (lH, t, J = 2.2 Hz, H-12a), 7.05 (2H, d, J = 8.8 Hz, H-2b,6b), 6.68 (2H, d, J = 8.8 Hz, H-3b,5b), 5.21 (lH, d, J = 9.5 Hz, H-7b), 4.26 (lH, d, J = 9.5 Hz, H-8b). 6.46 (lH, d, J = 2.2 Hz, H-12b), 6.26 (lH, d, J = 2.2 Hz, H-14b), 6.61 (lH, d, J = 2.2 Hz, H-2c), 6.69 (lH, J = 8.1 Hz, H-5), 6.98 (lH, dd, J = 8.1, 2.20 Hz, H-6), 6.50 (lH, d, J = 16.1 Hz, H-7c), 6.74 (lH, d, J = l6.1 Hz, H-8), 6.45 (lH, d, J = 2.2 Hz, H-12c), 6.66 (lH, d, J = 2.2 Hz, H-14c), 7.25 (2H, d, J = 8.8 Hz, H-2dd), 6.86 (2H, d, J = 8.8 Hz, H-3d,5d). 5.51 (lH, d, J = 5.9 Hz, H-7d), 4.48 (lH, d. J = 5.9 Hz, Hl), 6.32 (2H, d, J = 2.2 Hz, H-10d,14d), 6.33 (lH, t, J = 2.2 Hz, H-12d), 3.77 (3H, s, MeOH), 3.62 (6H, s, MeO-1 la,13a), 3.71 (3H, s, MeO-4b), 3.74 (3H, s, MeO-13b), 3.85 (3H, s, MeO-13c), 3.78 (3H, s, MeO-4d), 3.68 (6H, s, MeO-11d,13d). EIMS m/z 644 [M]⁺.

Cortical cell culture and cell viability assessment

Primary cultures of mixed cortical cells containing both neurons and glia were prepared from 17−19-day-old fetal rats (Sprague-Dawley) as described previously.¹ Cultures were allowed to mature for at least 2 weeks before being used for experiments. Test fraction and compounds were dissolved in DMSO (final concentration in culture, 0.1%). Cortical cell cultures were washed with DMEM and incubated with test compounds for 1 h. The cultures were then exposed to 100 μM glutamate and maintained for 24 h. After the incubation, the cultures were assessed for the extent of neuronal damage by measuring the efflux of lactic dehydrogenase (LDH) which reflects the integrity of cellular membrane.

Statistical analysis

The results are expressed as means ± standard errors (SE). The data were statistically analyzed by one-way analysis of variance (ANOVA). Differences with p < 0.05 were considered significant.

Results and discussion

We found that methanol extract of the roots of V. thunbergii var. sinuate might inhibit glutamate-induced toxicity in primary cultured rat cortical cells. In order to clarify the neuroprotective components of V. thunbergii var. sinuate, as part of a continued study of neuroprotection effects of V. thunbergii var. sinuate, isolation was performed to seek active fractions and components. After solvent fractionation, we compared the inhibiting effects of various fractions on neuroprotective activity and the aqueous extract of V. thunbergii var. sinuate was found to inhibit the this activity in a dose-dependent manner in the assay system using glutamate-induced toxicity in primary cultured rat cortical cells. To clarify the active substances of V. thunbergii var. sinuate, we examined the effects of the major secondary metabolites from V. thunbergii var. sinuate on neuroprotective activity. Two biological activity resveratrol derivatives were isolated from VT-7 fraction by repeated vacuum liquid chromatography and recrystallization. Compound was identified as vitisinols C (3) and (+)-vitisin C (5) (Figure 1) by comparing physicochemical and spectroscopic data with literature values.⁶ Other inactive compounds were isolated from VT-5 fraction by repeated vacuum liquid chromatography and recrystallization. Inactive compounds were identified as vitisinols A (1), B (2), and (+)-vitisin A (4) (Figure 1). All spectral data for these compounds were in good agreement with those previously reported in literature.⁶ Furthermore, to investigate and compare the neuroprotective activities of these compounds isolated from the methanol extracts of V. thunbergii var. sinuate, the activities of four compounds were evaluated in glutamate-injured primary cultured rat cortical cells at concentrations ranging from 0.1 to 10 μM (Table 1). Vitisinols C (3) and (+)-vitisin C (5) displayed activity, significantly attenuating glutamate-induced toxicity at concentrations ranging from 0.1 to 10 μM and exhibiting cell viabilities of 75%−85%. Although the cellular and molecular mechanisms that underlie the neuroprotective action of several effective resveratrol subtypes compounds revealed in the present study should be studied further, these findings about lactons subtypes compound of V. thunbergii var. sinuate containing neuroprotective activities may provide some ideas for the development of agents to ameliorate the cellular dysfunction due to glutamate-induced toxicity.

Figure 1.

Isolated compounds from Vitis thunbergii var. sinuate.

Table 1.

Neuroprotective effects of resveratrol derivatives against glutamate-induced toxicity in primary cultured rat cortical cells^a

Compounds		Cell viability^b (%)
Compounds	0.1	1	10 (μM)
Control^c		100^d
Glutamate-treated^c,e		0
Vitisinols A (1)	–	11.2 ± 0.7	32.1 ± 0.3
Vitisinols B (2)	–	6.3 ± 0.2	11.8 ± 0.3
Vitisinols C (3)	17.2 ± 0.4*	42.4 ± 0.4**	79.3 ± 0.8***
(+)-Vitisin A (4)	11.2 ± 0.6*	33.2 ± 0.6**	39.4 ± 0.3***
(+)-Vitisin C (5)	19.3 ± 0.7*	38.7 ± 0.6**	81.2 ± 0.7***
APV^f	11.5 ± 1.4	26.5 ± 2.3*	42.5 ± 3.7*
MK-801^g	51.4 ± 4.6**	63.5 ± 5.8***	78.4 ± 2.0***
CNQX^h	23.5 ± 3.7*	44.8 ± 3.5*	53.2 ± 4.6***

Abbreviation: LDH: lactic dehydrogenase.

^a Rat cortical cell cultures were incubated with test compounds for 1 h. The cultures were then exposed to 100 μM glutamate for 24 h. After the incubation, the cultures were assessed for the extent of neuronal damage.

^b Cell viability was measured by the LDH assay.

^c LDH released from control and glutamate-treated cultures were 11.7 ± 1.3 and 47.9 ± 4.0 units/mL, respectively;

^d Cell viability was calculated as 100 × (LDH released from glutamate-treated-LDH released from glutamate + test compound-treated) / (LDH released from glutamate-treated-LDH released from control). The values shown are the mean ± STD of three experiments (3−4 cultures per experiment). Results differ significantly from the glutamate-treated: *p <0.05, **p < 0.01, ***p < 0.001.

^e Glutamate-treated value differed significantly from the untreated control at the level of p < 0.001.

^f APV: DL-2-amino-5-phosphonovaleric acid, a competitive NMDA (N-methyl-D-Aspartate) receptor antagonist.

^g MK-801: dizocilpine maleate, a noncompetitive NMDA receptor.

^h CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione, non-NMDA receptor antagonist.

Footnotes

This study was partly supported by 2009 research fund of RDA.

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