Phenolic Compounds from the Roots of Valeriana officinalis var . latifolia

Uma nova neolignana benzofurânica, isovalerato de 9-di-hidrodesidrodiconiferila, além de 10 compostos fenólicos conhecidos, olivil, pinoresinol, 8-hidroxipinoresinol, pinorespiol, 8-hidroxi-7epipinoresinol, ácido trans-p-hidroxifenil-propenoico, ácido cis-p-hidroxifenil-propenoico, ácido isoferúlico e isovanilina foram isolados a partir das raízes da Valeriana officinalis var. latifolia. Suas estruturas e configurações foram elucidadas com base em métodos espectroscópicos. A atividade inibitória da acetilcolinesterase (AChE) e a atividade intensificada do fator de crescimento neural (NGF) mediada pelo crescimento de neurites em células PC12 pelos compostos isovalerato de 9-di-hidrodesidrodiconiferila e olivil foram avaliadas.


Introduction
The genus Valeriana (Valerianaceae) consists of about 200 species, and there are about 30 species distributed in China. 1,24][15][16] Lignans were another important group of components of plants from this genus, which showed antioxidative, vasorelaxant 17 and partial agonistic activity at A1 adenosine receptors. 18. officinalis is mainly distributed in Europe but does not grow in P. R. China, and is the official species used in Europe and America.V. officinalis var.latifolia is similar to V. officinalis in botanical morphology characteristics, which was mainly distributed in P. R.China and Japan, and it was always used as the alternative species of V. officinalis in folk medicine in P. R. China. 2 Our further studies on the roots of V. officinalis var.latifolia lead to the isolation of a new benzofuran neolignan, dihydrodehydrodiconiferyl alcohol 9-isovalerate (1) (Figure 1), along with ten Vol. 24, No. 9, 2013 known phenolic compounds.The known compounds were identified as olivil (2), 19 pinoresinol (3), 20 8-hydroxypinoresinol (4), 21 pinorespiol (5), 22 8-hydroxy-7-epipinoresinol (6), 23 trans-p-hydroxyphenyl-propenoic acid (7), 24 cis-p-hydroxyphenyl-propenoic acid (8), 24 ferulic acid (9), 25 isoferulic acid (10)  26 and isovanillin (11) 27 by comparison their spectroscopic data with the literature values.In this paper, we reported the isolation and structure elucidation of the new compound (1), as well as the AChE inhibitory and NGF-mediated neurite outgrowth enhancing activities of compounds 1 and 2.

General procedures
Optical rotations were taken on a Horiba SEAP-300 polarimeter.IR spectra were measured with a Bio-Rad FTS-135 spectrometer with KBr pellets.UV spectra were obtained on a Hitachi UV 210A spectrophotometer.Electron impact mass spectrometry (EIMS) (70 eV) was recorded on a VG Auto Spec-3000 spectrometer, ESIMS was recorded with an API QSTAR Pulsar i spectrometer.1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AM-400 or DRX-500 or Avance-600 NMR spectrometer with the residual solvent as the internal standard.Column chromatography was performed either on silica gel (200-300 mesh, Qindao Marine Chemical Inc., Qingdao, P. R. China) or RP-18 gel (LiChroprep, 40-63 μm, Merck, Darmstadt, Germany).Sephadex LH-20 for chromatography was purchased from Amersham Biosciences.Fractions were monitored by thin layer chromatography (TLC), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Plant material
The plants of V. officinalis var.latifolia were collected at the Badong country, Hubei province, P. R.China in October 2008.The sample was identified by Professor You-Wei Wang, School of Pharmaceutical Sciences, Wuhan University, P. R. China.A voucher specimen (KIB-XC0810) was preserved at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, the Chinese Academy of Sciences, P. R. China.

Cell culture and bioassay of neurite outgrowth-promoting activity
The neurotrophic activity of the studied compounds was examined following the assay using PC12 cells reported previously. 29Nerve growth factor (NGF) was purchased from R&D Systems, F12 medium, poly-L-lysine, fetal bovine serum (FBS) and horse serum (HS) were purchased from Sigma Chemical Company, and PC12 cells were purchased from the cell bank of Kunming Institute of Zoology, Chinese Academy of Science.Briefly, PC12 cells were maintained in F12 supplemented with 12.5% HS and 2.5% FBS, saturated atmosphere of 5% CO 2 and incubated at 37 °C.Compounds were dissolved in DMSO.For the bioassay of neurite outgrowth-promoting activity, PC12 cells were seeded at a density of 2 × 10 4 cells mL −1 in 48 well plate coated with poly-L-lysine.After 24 h, the medium was changed to test medium containing various concentrations of NGF (50 ng mL −1 for positive control, 5 ng mL −1 for the negative control and compound group), 10% HS and 5% FBS, then 50 mmol L −1 test compounds were added (the final concentration of DMSO was 0.05%), the same concentration of DMSO was added into negative control.After 72 h incubation, the neurite outgrowth was assessed under a phase-contrast microscope (Olympus X51).Neurites processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite bearing cells.The ratio of the neuritebearing cells to total cells (with at least 100 cells examined
The acetylcholinesterase (AChE) inhibitory activity of 1 and 2 was assayed by the spectrophotometric method developed by Ellman et al. 28 with slightly modification, and they do not show inhibitory activity at the concentration of 50 μmol L −1 .Tacrine (0.33 μmol L −1 ) was used as the positive control, and showed 50.1% inhibition.Compound 4 showed weak AChE inhibitory activity in our previous studies. 11ompounds 1 and 2 were also evaluated for the enhancing activity on NGF-mediated neurite outgrowth in PC12 cells. 29The result indicated that the proportion of the NGF (5 ng mL −1 )-induced neurite-bearing cells was not enhanced by these compounds at 50 μmol L −1 , respectively. of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, for the measurement of all the spectra.