Parguerol and Isoparguerol Isolated from the Sea Hare, Aplysia kurodai, Induce Neurite Outgrowth in PC-12 Cells

Abstract

The extract of the sea hare, Aplysia kurodai, showed neurotrophic activity toward rat pheochromocytoma (PC-12) cells. Bioassay-guided purification afforded two active compounds, which were subsequently identified to be parguerol and isoparguerol by spectroscopic analysis. It was found that both parguerol and isoparguerol induced neurite outgrowth in PC-12 cells at concentrations of 25 and 50 μM, respectively.

Introduction

It is well known that neurons are terminally differentiated cells. Neurotrophic factors, such as nerve growth factor (NGF) [1], brain-derived neurotrophic factor (BDNF) [2], neurotrophin 3 (NT-3) [3], and glia-derived neurotrophic factor (GDNF) [4], are necessary for the functional maintenance and organization of neurons. Among various neurotrophic factors, NGF has been extensively investigated and found to show pleiotrophic effects, such as the induction of neuronal differentiation, neural cell survival, and prevention of apoptosis of neurons, in both central and peripheral nervous systems [1]. In rat pheochromocytoma (PC-12) cells, which have been used as an in vitro model system to study the mechanisms of neuronal differentiation, NGF induces their differentiation, leading to the extension of neurites and the development of the characteristics of sympathetic neurons [5]. In the course of our search for new neurotrophic substances from natural resources [6, 7], we succeeded in isolating two neurotrophic substances and identified them to be parguerol (1) [8] and isoparguerol (2) [8]. Here we report the isolation, identification, and neurotrophic activities of the two neurotrophic substances, 1 and 2, together with inactive deoxyparguerol acetate (3) [8].

Results and Discussion

Specimens of Aplysia kurodai (1.7 kg) were collected by hand from Toyama Bay in the Japan Sea and kept frozen until extraction with MeOH. After evaporation of the solvent, the resulting aqueous residue was extracted with EtOAc. The EtOAc layer showed neurotrophic activity in PC-12 cells and was fractionated by a combination of silica gel chromatography and gel permeation on Sephadex LH-20 to afford parguerol (1), isoparguerol (2), and deoxyparguerol acetate (3) in yields of 6.5 × 10⁻⁵, 4.1 × 10⁻⁵, and 3.5 × 10⁻⁵%, respectively.

The FABMS of 1 showed 1:1 doublet ion peaks at m/z 479 and 481 [M + Na]⁺, indicating the presence of a bromine atom in the molecule. The ¹H NMR spectrum of 1 measured in CDCl₃ revealed two characteristic highfield signals assignable to a trisubstituted cyclopropane ring at δ 0.08 (t, J = 5.4 Hz) and 0.88 (d, J = 10.2 and 5.4 Hz), two singlet methyls at δ 1.07 (s) and 1.14 (s), one acetate methyl at δ 2.07 (s), two pairs of hydroxymethyls at δ 3.84 (dd, J = 12.7, 9.3 Hz)/3.95 (dd, J = 12.7, 2.9 Hz) and 3.39 (d, J = 11.7 Hz)/3.55 (d, J = 11.7 Hz), three downfield methines at δ 3.18 (dt, J = 4.9 and 11.7 Hz), 4.28 (dd, J = 9.3 and 2.9 Hz), and 5.36 (d, J = 4.9 Hz), and one trisubstituted double bond at δ 5.36 (d, J = 4.9 Hz). These data were reminiscent of parguerol [8] which had been isolated from the sea hare, Aplysia dactylomela, collected in Puerto Rico, and finally analysis of 2D NMR data confirmed 1 to be parguerol.

Compound 2 showed the same ion peaks at m/z 479 and 481 (intensity, 1:1) [M + Na]⁺ as those of 1 in the FABMS. The ¹H NMR spectrum of 2 was similar to that of 1, except for the upfield shift of H-2 at δ 4.96 (d, J = 4.0 Hz) by 0.40 ppm and the absence of the cyclopropane ring and a hydroxymethyl at C-19 in 2. The comparison of the ¹H NMR data of 2 and isoparguerol [8] in CDCl₃ identified 2 as isoparguerol.

The ¹H NMR spectrum of 3 was also similar to that of 1, except for the presence of two more acetoxy methyls and the downfield shift of H-7 at δ 4.40 (dt, J = 4.9, 11.7 Hz) and H₂-16 at δ 4.28 (2H, d, J = 9.8 Hz) in 3, which implied the hydroxy groups at C-7 and C-16 in 1 were acetylated in 3. In addition, the spectrum suggested that the hydroxymethyl (δ 3.55 and 3.39) at C-19 in 1 was replaced with methyl (δ 1.02) in 3. The presence of pseudomolecular ion peaks at m/z 547/549 (intensity, 1:1) [M + Na]⁺ in the FABMS supported identification of 3 as deoxyparguerol acetate. Although 3 was derived from deoxyparguerol (4) [8], this is the first isolation of 3 from natural resources.

Parguerol (1) and isoparguerol (2) showed significant neurotrophic effects on PC-12 cells (Fig. 1): More than 70% of the cells exhibited neurite outgrowth by treatments with 1 and 2 at concentrations of 25 and 50 μg/mL, respectively, for 2 days. On the other hand, deoxyparguerol acetate (3) was unable to induce neurite extension even at a concentration of 100 μg/mL, although it possesses the same skeleton as that of 1 and 2. It should be noted that both 1 and 2 induce bipolar (two-neurite-bearing) morphology, while NGF (a positive control) induces multipolar (multiple-neurite-bearing) morphology. The relationship between the morphological change and the cellular event has been extensively studied and elucidated in the case of murine neuroblastoma Neuro 2A cells [9]. However, such relationship remains obscure in the case of PC-12 cells. Further pharmacological study is needed to understand the mechanism of 1- and 2-induced morphological changes in PC-12 cells.

It is well known that the content of sea hare digestive glands is dependent on diet that this animal ingests [10–13]. A large number of cognate metabolites have been isolated from both the red alga of the genus Laurencia and the sea hare that preys on the red alga. Metabolites isolated from sea hares exhibit various biological activities, for example, cytotoxic [14, 15], algicidal [15], antifungal [15, 16], and ichthyotoxic [16] activities: It can be inferred that sea hares contain such metabolites for the sake of their self-defense. This is the first report of sea hare metabolites that show neurotrophic activity.

Conclusions

We have isolated parguerol (1), isoparguerol (2), and deoxyparguerol acetate (3) from the sea hare, Aplysia kurdai. In rat pheochromocytoma (PC-12) cells, 1 and 2 showed significant neurotrophic activities at concentrations of 25 and 50 μg/mL, respectively, while 3 did not show such effect even at a concentration of 100 μg/mL.

NGF activates neuronal function and prevents neuronal death in the brain. When peripherally administered, however, exogeneously administered NGF is thought to be unable to cross the blood-brain barrier. Therefore, low-molecular-weight NGF-like substances such as 1 or 2 are useful for treatment of various neuronal degenerative disorders.

Experimental

General

NMR spectra were recorded on a JEOL GSX500 in CDCl₃. All chemical shifts were reported with respect to CDCl₃ (δ 7.26). Mass spectra were measured on a JEOL SX-102 mass spectrometer.

Extraction and Isolation

The frozen material (1.7 kg, wet wt) was extracted with MeOH. The extract was concentrated under reduced pressure and extracted with EtOAc. The EtOAc layer (1.4 g) was subjected to silica gel chromatography with a stepwise gradient of hexane/EtOAc/MeOH. The most potent neurotrophic fraction (216.3 mg) eluted with EtOAc/MeOH (1:1) was purified by silica gel chromatography with CHCl₃/MeOH (9:1) followed by gel permeation on Sephadex LH-20 to afford parguerol (1, 1.11 mg, 6.5 × 10⁻⁵%), isoparguerol (2, 0.69 mg, 4.1 × 10⁻⁵%), and deoxyparguerol acetate (3, 0.60 mg, 3.5 × 10⁻⁵%).

Parguerol (1): ¹H NMR (CDCl₃) δ 0.08 (1H, t, J = 5.4 Hz, H-18endo), 0.88 (1H, dd, J = 10.2, 5.4 Hz, H-18exo), 1.04 (1H, m, H-3), 1.07 (3H, s, H₃-17), 1.14 (3H, s, H₃-20), 1.14 (2H, m, H-1 and H-5), 1.49 (1H, m, H-14), 1.82 (1H, br d, J = 18.6 Hz, H-12), 1.92 (1H, t, J = 15.6 Hz, H-1), 1.92 (1H, m, H-6), 2.07 (3H, s, CH₃COO), 2.27 (1H, m, H-8), 2.41 (1H, m, H-6), 2.42 (1H, m, H-12), 3.18 (1H, dt, J = 4.9, 11.7 Hz, H-7), 3.39 (1H, d, J = 11.7 Hz, H-19), 3.55 (1H, d, J = 11.7 Hz, H-19), 3.84 (1H, dd, J = 12.7, 9.3 Hz, H-16), 3.95 (1H, dd, J = 12.7, 2.9 Hz, H-16), 4.28 (1H, dd, J = 9.3, 2.9 Hz, H-15), and 5.36 (2H, d, J = 4.9 Hz, H-2 and H-11). FABMS (positive, glycerol matrix) m/z 479/481 (intensity, 1:1) [M + Na]⁺.

Isoparguerol (2): ¹H NMR (CDCl₃) δ 1.09 (3H, s, H₃-17), 1.22 (3H, s, H₃-20), 1.44 (1H, m, H-14), 1.67 (1H, dd, J = 15.1, 4.9 Hz, H-1), 1.76 (1H, t, J = 15.1 Hz, H-14), 1.86 (1H, m, H-6), 1.86 (1H, m, H-12), 2.03 (3H, s, CH₃COO), 2.10 (1H, br d, J = 15.1 Hz, H-1), 2.28 (1H, m, H-8), 2.45 (1H, br d, J = 18.6 Hz, H-12), 2.53 (1H, t, J = 14.7 Hz, H-6), 3.28 (1H, dt, J = 4.9, 11.7 Hz, H-7), 3.85 (1H, dd, J = 12.7, 9.8 Hz, H-16), 3.92 (1H, dd, J = 12.7, 2.9 Hz, H-16), 4.29 (1H, dd, J = 9.8, 2.9 Hz, H-15), 4.96 (1H, d, J = 4.0 Hz, H-2), and 5.45 (1H, d, J = 6.3 Hz, H-11). FABMS (positive, glycerol matrix) m/z 479/481 (intensity, 1:1) [M + Na]⁺.

Deoxyparguerol acetate (3): ¹H NMR (CDCl₃) δ 0.01 (1H, m, H-18endo), 0.68 (1H, m, H-18exo), 1.02 (3H, s, H₃-19), 1.05 (3H, s, H₃-17), 1.16 (3H, s, H₃-20), 1.90 (1H, br d, J = 15.8 Hz, H-1), 2.07 (3H, CH₃COO), 2.09 (3H, CH₃COO), 2.13 (3H, CH₃COO), 2.42 (1H, br d, J = 18.6 Hz, H-12), 4.28 (2H, d, J = 9.8 Hz, H₂-16), 4.40 (1H, dt, J = 4.9, 11.7 Hz, H-7), 4.52 (1H, dd, J = 9.8, 2.9 Hz, H-15), 5.31 (1H, br d, J = 5.0 Hz, H-2), and 5.39 (1H, br d, J = 6.5 Hz, H-11). FABMS (positive, glycerol matrix) m/z 547/549 (intensity, 1:1) [M + Na]⁺.

Neurite Outgrowth Assay

Neurite outgrowth assay was carried out using rat pheochromocytoma (PC-12) cells.⁵ PC-12 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 5% horse serum, penicillin (50 units/mL), and streptomycin (50 μg/mL) in an incubator under 5% CO₂ at 37°C. PC-12 cells were seeded onto 24-well plates (1 × 10⁵ cells/mL) and cultivated for a day. The medium was replaced with that containing 1–3 or 5 ng/mL NGF (a positive control), and PC-12 cells were further cultivated for 2 days and observed under a phase-contrast microscope. The percentage of the cells with neurites was determined by counting 300 cells.