8-Alkylcoumarins from the Fruits of Cnidium monnieri Protect against Hydrogen Peroxide Induced Oxidative Stress Damage

Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1), 7-methoxy-8-(3-methyl-2,3-epoxy-1-oxobutyl)chromen-2-one (2), and 3′-O-methylvaginol (3), together with seven known compounds (4–10) were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.


Introduction
Cnidium monnieri (L.) Cusson, belonging to the Umbelliferae family, is native to China and is an important traditional Chinese medicinal plant. It is widely distributed in China and is also found in Korea, Mongolia, and Russia. The dried fruits of C. monnieri, known in Chinese as "Shechuangzi", have been used as traditional remedies for the skin disease, gynecopathy, and stasis of the blood [1]. Several investigations have reported that the fruits of C. monnieri exhibited various pharmacological effects including antidermatophytic effect [2], antipruritic action [3], anti-allergic effect [4], antiosteoporosis [5], antiproliferation of vascular smooth muscle cells [6], vasorelaxation [7], antifibrotic activity in hepatic cells [8], and anti-adipogenic activity in 3T3-L1 cells [9]. In addition, the chemical constituents including coumarins [10], chromones [11], and sesquiterpens [12], have been isolated from the fruits of C. monnieri. The above-mentioned beneficial effects are suggested to be due to coumarin compounds existing in the dried fruits of C. monnieri [6] and more than twenty compounds, such as osthole, edultin bergapten, isopimpineline, cnidiadin, archangelicin, imperatorin, xanthotoxin, oroselone, colnmbianadin, O-acetylcolnmbianetin and 2'-acetylangelicin were found. The 8-alkylcoumarin compound, osthole, has been proven to regulate cardiac or hepatic oxidative stress by Zhou and Zhang [13,14]. The oxidative stress is considered to be one of the most important factors for neurodegenerative disease like Alzheimer's disease [15], thus in this study we aimed to explore the potential antioxidant candidates from the fruits of C. monnieri. Herein, we report the extraction, purification, structural elucidation, and cytoprotective activity of three new 8-alkylcoumarins (1-3) (Figure 1).

Cytoprotective Activity against Oxidative Stress
The oxidative stress is considered to be one of the most important factors for neurodegenerative disease like Alzheimer's disease [15]. H 2 O 2 -treatment has been shown to cause oxidative stress to the cells in culture [23] and impaired their proliferation. The cytoprotective activity against oxidative stress induced by hydrogen peroxide on Neuro-2a cells was assayed. All the isolates were evaluated the anti-oxidative activity by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell proliferation assay and the results showed that all these three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a cells injured by hydrogen peroxide. The range of effective cytoprotective dosage for compound 1 was from 0.25 to 1 µM. Interestingly, compound 3 dramatically increased the cytoprotective effect at a lower dosage, 0.1 µM. Compound 2 showed slight cytoprotective ability at the concentration ranging from 0.1 to 1 µM (Figure 3). The remaining seven known compounds (4-10) exhibited no significant cytoprotective activity.

Plant Material
The fruits of C. monnieri were purchased from a local medicine store in Taipei, Taiwan. The material was identified by Prof. Chao-Lin Kuo, Department of Chinese Pharmaceutical Science and Chinese Medicine Resources, China Medical University.

Cell Viability Assay
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to determine the anti-oxidative effects of three new 8-alkylcoumarins on Neuro-2a cell viability according to the manufacturer's protocol. Cells (10 4 ) were cultured in 96-well plate containing DMEM (Dulbecco's modified eagle's medium) supplemented with 10% FBS for 1 day to become nearly confluent. Then, cells were cultured with different concentrations of the three new compounds (1-3) and seven known compounds (4-10). After 1 h, cells were cultured in the presence of 700 μM hydrogen peroxide (Sigma-Aldrich) for further 7 h. After that, the cells were washed with DPBS and 120 μL MTS solution. After 2 h incubation at 37 °C, the absorbance at 490 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). All tests were performed in triplicate. Results are expressed as the percentage relative to control without H 2 O 2 treatment.

Statistical Analysis
Multiple comparisons within experimental groups were made using one-way analysis of variance (ANOVA) to determine differences between experimental treatments and control with H 2 O 2 treatment. A level of p < 0.05 was set for significance for all tests, and all values are expressed as mean ± SEM.
The three new compounds 1-3 exhibited significant cytoprotective activity (Figure 3). Compounds 1 and 3 showed stronger cytoprotective activity than compound 2. The dose range of cytoprotective effect for compound 1 was from 0.25 to 1 µM, and no significant difference was observed between the different concentrations, 0.25, 0.5, and 1 µM through the ANOVA analysis. Compound 3 showed better cytoprotective effect at the low dosage (0.1 µM), which may be due to its good scavenging ability against hydrogen peroxide. However, at the higher dosage, the cytoprotective effect of compound 3 was dramatically decreased, which may be due to its intrinsic cytotoxicity. Compound 2 showed a weaker cytoprotective effect compared to that of compounds 1 and 3 ( Figure 3). It may be explained by the intrinsic cytotoxicity of compound 2 contributed by its reactive epoxide group [24,25].