Three New Germacrane-Type Sesquiterpenes with NGF-Potentiating Activity from Valeriana officinalis var. latiofolia

Three new germacrane-type sesquiterpenoids, volvalerenal F (1), volvalerenal G (2) and volvalerenic acid D (3), along with five known compounds 4–8, were isolated from the CHCl3 soluble partition of the ethanol extract of Valeriana officinalis var. latiofolia. The structures of the new compounds were determined on the basis of spectroscopic evidence, including their 1D- and 2D-NMR spectra, as well as mass spectrometry. The eight germacrane-type sesquiterpenoids showed nerve growth factor (NGF) potentiating activity, which mediates the neurite outgrowth in PC 12D cells. This study intends to reveal the chemical basis of the use of V. officinalis var. latiofolia as a dietary supplement.


Introduction
The genus Valeriana, from the family Valerianaceae, consists of about 250 species widely distributed all over the World. Valeriana is a perennial herb native to Europe, Asia and North
V. officinalis var. latiofolia, a variant of V. officinalis Linn, is produced mainly in the Guizhou, Sichuan and Yunnan provinces in Southwest China. Particularly in Guizhou, V. officinalis var. latiofolia is widely cultivated and has become one of the local industry pillars [1,24]. This variant shares some common pharmaceutical actions and chemical constituents with V. officinalis Linn [25]. Abundant research has been done on V. officinalis var. latiofolia so far, and it is assumed that the sesquiterpenoids from the volatile oil and iridoids are the main contributors to its antidepressant and antinervousness activities [24,25]. Pairs of active compounds have been isolated and identified from Valeriana, including germacrane-type sesquiterpenoids, volvalerenals A-E, volvalerenic acids A-C, valerianin A-B and heishuixiecaoline A-C [13,[16][17][18][19]. However, neither the active component(s) responsible for the therapeutic properties of Valeriana nor the related molecular mechanisms are clearly understood, which severely hinders the wider application of Valeriana products. Therefore, in this paper the chemical constituents of V. officinalis var. latiofolia have been systematically investigated, and eight germacrane-type sesquiterpenoids (including three new compounds, volvalerenal F (1), volvalerenal G (2) and volvalerenic acid D (3)) were isolated and identified from chloroform extracts of this herb's syrup. Additionally, the NGF-potentiating activities of the obtained products were evaluated.
Volvalerenic acid D (3) was also isolated as a colorless oil. The HR-ESI-MS of 3 indicated that its molecular formula is C 17 H 24 O 2 (m/z 293.1744 [M+H] + , calcd. for 293.1747). The IR data was completely similar to that of compound 1, which suggested that 3 was also a germacrane-type sesquiterpenoid.
The NMR spectrum of compound 3 (Table 1) showed the following signals: an acetate carbonyl carbon, four olefinic carbons, five methylenes (one oxygenated), two methines and three methyls, which were quite similar to those of compound 1. In addition, it is noteworthy that the NMR data displayed an obvious carboxyl carbon signal at δ C 172.0 (C-16). The 1 H-1 H COSY spectrum showed key correlations such as H-6/H-7 and H-8/H-9, and key long-range correlations were observed in the HMBC experiments between H-3/C-14 and H-5/C-14 (Figure 3), which suggested the carboxyl group was located to C-4.  From the above data, the structure of 3 was identified as 15-acetoxy-4-carboxy-11, 11-dimethyl-bicyclogermacren-4E (5), 10Z (1)-diene, and it compound was named Volvalerenic acid D.
The five known compounds were identified as madolin A (4) and B (7) [26], vovalerenal A (6) and B (5) [18] and heishuixiecaoline B (8) [13] by comparing their NMR spectroscopic data with the literature values. The structures of compounds 1-8 are shown in Figure 5. The propensity of compounds 1-8 to enhance the activity on nerve growth factor (NGF)-mediated neurite outgrowth in PC 12D cells was assessed as described previously [25]. The neurite-bearing cells accounted for 22.74% and 100% in the control experiments incubated with 2 and 50 ng/mL NGF after 48 h, respectively. Under 2 ng/mL NGF conditions, all eight tested compounds (at 10, 30, 100 µmol) showed NGF-potentiating activities in various levels. Compound 3 (at 100 µmol) reached 50.15%, in particular ( Table 2). 3.12% ± 0.88 Results expressed as mean ± SD (n = 6) of eight independent experiments. The 100% value was obtained from 50 ng/mL NGF in the absence of compounds. A statistically significant difference (a, b and c, p ＜ 0.01) from the control (2 ng/mL NGF) in the absence of compounds was apparent.

General
Optical rotations were measured with a Perkin-Elmer 343 polarimeter (Perkin-Elmer, Waltham, MA, USA). IR spectra were recorded on the Bio-Rad FTS-65A spectrometer (Bio-Rad, Richmond, VA, USA). UV spectra were recorded using the UV-2501PC spectromter (Shimadzu, Japan). 1  . TLC was carried out using silica gel 60 (>230 mesh, Qingdao Marine Chemical Group Co.) and GF254 plates precoated with silica gel 60. Spots on TLC were visually observed under UV light and/or by spraying with anisaldehyde-H 2 SO 4 reagent followed by heating.

Plant Material
The dry roots of V. officinalis var. latifolia were collected from the Jiangkou region of Guizhou Province, China, in April 2012. The plant was identified by Prof. Bin Li (Beijing Institute of Radiation Medicine), and a voucher specimen (KYXC-20120313) is deposited in the herbarium of the Beijing Institute of Radiation Medicine, Beijing, China.

Activity Screening in Vitro
PC 12D cell line was obtained from Insitute of Biochemistry and Cell Biology, CAS. It was cultured in Dulbecco's modified Eagles Medium (DMEM, Gibco, New York, NY, USA) with 10% fetal calf serum (Gibco, New York, NY, USA), and 5% equine serum (Gibco), and then the cells were maintained at 37.0 °C in a humidified atmosphere which contained 6% CO 2 [25]. The test cell line was seeded in 24-well culture paltes (2 × 10 4 cells/well) coated with poly-L-lysine (Gibco). After 24 h, the medium was changed to test medium containing 1% fetal calf serum, 2% equine serum and varying concentrations of NGF (50 ng/mL for positive control, 2 ng/mL for test samples and significant difference control, Sigma, St. Louis, MO, USA) and test compounds 1-8 (10, 30, 100 µmol). After incubating for 48 h, the cells were fixed with 1% glutaraldehyde (Sigma) in phosphate buffer, and the cells with neurites outgrowth were counted (with at least 100 cells examined/viewing area, three viewing areas/well, six wells/sample). The ratio of the neurite-bearing cells to total cells was determined and expressed as a percentage.

Conclusions
Three new germacrane-type sesquiterpenoids, volvalerenal F (1), volvalerenal G (2) and volvalerenic acid D (3), along with five known compounds 4-8, were isolated from the CHCl 3 soluble partition of the ethanol extract of Valeriana officinalis var. latiofolia. NGF plays a key role in the functions of the central and peripheral nervous system [29] and all the sesquiterpenoids obtained displayed certain NGF-potentiating activities. From the current investigation it can be predicted that sesquiterpenoids will be promising candidates for dietary supplements and medicines, although further studies are needed to determine the pharmacological activities and the mechanism of these eight compounds in animals.