Compounds from the Roots and Rhizomes of Valeriana amurensis Protect against Neurotoxicity in PC12 Cells

Three new germacrane-type sesquiterpenoids, heishuixiecaoline A–C (compounds 1–3), were isolated along with ten known compounds 4–13 from fraction of Valeriana amurensis roots and rhizomes effective against Alzheimer’s disease (AD). The structures of 1–3 were elucidated on the basis of their spectroscopic data. We also investigated the protective effect of compounds 1–13 on the neurotoxicity of PC12 cells induced by amyloid-beta (Aβ25–25), respectively. As a result, germacrane-type sesquiterpenoids 1–4 and lignans 5–7 were seen to afford protection against Aβ-induced toxicity in PC 12 cells. This study will contribute to revealing the chemical basis for the therapeutic effect of V. amurensis against AD.


Introduction
The genus Valeriana has been taxonomically placed in the family Valerianaceae, comprising ca. 30 species in China [1]. Valerian (Valerianacae) is a perennial herb and some species in this genus have been used widely as a mild sedative and sleep aid for centuries in Europe and North America [2][3][4]. Previous studies on genus Valeriana plants indicated anxiolytic, antidepressant, antispasmodic, sedative, antitumor, and anti-HIV activities [5][6][7][8][9]. As one species of Valeriana, Valeriana amurensis is abundantly distributed in northeast China, especially in the Great Xing'an Mountains area. However, there were no investigations about its pharmacology activity and chemical constituents except for the sedative and anti-hyperspasmia effects of V. amurensis volatile oil [10] until we reported its potential therapeutic effect in Alzheimer's disease (AD) for the first time [11,12]. We have also screened and determined the AD-effective fraction (50% EtOH fraction from AB-8 macroporous resin column of 95% EtOH extract) from the previous studies [11,12], based on which a bioassay-guided isolation and phytochemical study of V. amurensis was performed and three new and ten known compounds were obtained from the effective fraction. The structures of known compounds 4-13 were determined by detailed 1D-and 2D-NMR analyses, ESI-MS and comparison of their spectral data with literature values. In this paper, the isolation and structural elucidation of the new germacrane-type sesquiterpenoids 1-3 is described. We also investigated the neuroprotective effects of compounds 1-15 in a PC12 neuronal cell line. The PC12 cell line, derived from rat pheochromocytoma, displays phenotypic characteristics of sympathetic neurons. The cells were grown in the presence of various toxins mimicking the conditions taking place in neurodegenerative diseases, including amyloid-beta (Aβ, the peptide composing the amyloid plaques in brains of AD patients).
Compound 2 was isolated as white amorphous powder, and its molecular formula was determined to be C 15 Tables 1 and 2) of 2 were a little similar to those of 1, The differences found were the absence of the acetate group signals at δ C 172.1 (C-16) and 21.3 (C-17) in compound 2, other differences such as the NMR data in C-7, C-8, and C-9 suggested hydroxy group is linked to C-8 in compound 2. The structure of 2 was further confirmed by the correlations of HMBC and 1 H-1 H COSY spectra as shown in Figure 1.
The relative configurations at C-6, C-7, and C-8 in 2 were deduced by a ROESY experiment ( Figure 2). The syn configuration of H-7 and H-6 were determined as described in compound 1, and the β-orientation of H-7 and H-6 were assigned by the correlations of H-7/CH 3 -13 and H-6/CH 3 -13. The β-orientation of H-8 was established by the correlations of H-8/CH 3 -13, and the Z-and E-configuration of Δ 4,5 and Δ 1,10 were determined same to 1 by the correlations H-5/H-3a, H-2(a, b)/CH 3 -15, and H-5/H-1. Therefore, compound 2 was established as 10-methyl-6,7-dimethylmethylene-8α-hydroxy-4-aldehyde-(4Z,10E))-dicyclodecadiene (Figure 3), and this compound was named heishuixiecaoline B. Compound 3 could be assigned a similar structure to 2 by comparison of their 1 H-and 13 C-NMR spectra (Tables 1 and 2). The major differences observed were the absence of the trisubstituted C=C double bond between C-1 and C-10 present in 3 and the appearance of an exocyclic double bond between C-10 (δ C 149.0) and C-15 (δ C 113.4) and the oxygenated methine signal at δ C 68.5 (C-1) in 3. These were confirmed by the HMBC correlations from H-15 (δ H 5.07, 5.12) to C-1, C-9 (δ C 37.5), and C-10. The other correlations in the HMBC and 1 H-1 H COSY spectra as shown in Figure 1 confirmed the connectivities in compound 3.

Plant Material
The roots and rhizomes of V. amurensis were collected from the Great Xing'an Mountains area in 2010. The original plant was identified by Xiaowei Du of Heilongjiang University of Chinese Medicine. A voucher specimen (No. 20100806) was deposited at the Herbarium of Heilongjiang University of Chinese Medicine, China.

Determination of Cell Viability
Cell viability was measured by quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described previously [20]. Briefly, the cells were cultured at a density 5 × 10 4 cells per well in growth medium for 24 h in 96-well plates, and then preincubated without or with various concentrations (5, 12, 25 μM) of compounds 1-13, which was followed 24 h later by exposure to 20 μM aggregated Aβ 25-35 (Sigma, St. Louis, MO, USA) prepared as described previously [21]. Vitamin E was used as a reference compound [22]. 25 μL/well of MTT solution (5 mg/mL) was added and cells were incubated at 37 °C for 4h. Supernatants were then aspirated off and formazan crystals were dissolved with DMSO. The optical density of each well was determined at 490 nm using a microplate reader. Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%. For comparing the results of MTT between the study groups, the t-test was used.