Sesquiterpenoids from the roots and rhizomes of Valeriana amurensis and their effects on NGF-induced neurite outgrowth in PC12 cells

Abstract Two new sesquiterpenoids, including a kessane-type sesquiterpenoid (1) and one bisabolane derivative (2), together with fourteen known sesquiterpenoids (3–16), were isolated from the roots and rhizomes of Valeriana amurensis. The structures of new compounds were established on the basis of extensive spectroscopic analysis. All isolates were evaluated for their effects on nerve growth factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells. As a results, four compounds including 10–12 and 15 showed potent promoting effects at the concentration of 10 µM on NGF-induced neurite outgrowth in PC12 cells with the differentiation rate of 11.84%, 12.21%, 13.77% and 12.16%, respectively. Graphical Abstract


Introduction
The genus Valeriana (family Valerianaceae) contains approximately 250 species and is widely distributed in Europe, Asia and North America (Houghton 1988(Houghton , 1999. Several members like Valeriana officinalis L., Valeriana jatamansi Jones and Valeriana edulis Nutt. Ex Torr., etc. were used as traditional medicines, most are active on the central nervous system (CNS), especially as a sleep aid and a mild sedative in many countries (Houghton 1988(Houghton , 1999. Previous phytochemical studies on Valeriana plants resulted in the isolation of structurally diverse secondary metabolites, including sesquiterpenoids (Nishiya et al. 1992(Nishiya et al. , 1994(Nishiya et al. , 1995Tori et al. 1996;Wang et al. 2009;Wang et al. 2012;Wu et al. 2014;, acylated iridoids and valepotriates (Wang et al. 2009;Yang et al. 2016;Jiang et al. 2017;Dong et al. 2018;Tian et al. 2019), flavone glycosides and lignans (Tang et al. 2003;Wang et al. 2009Wang et al. , 2012. Some of which displayed extensive biological properties such as sedative, anxiolytic, antidepressant, anticonvulsant, antimicrobial, antiviral and Cav2.2 inhibiting activities. In the course of our continual search for bioactive natural products on CNS from genus Valeriana (Wang et al. 2009(Wang et al. , 2011(Wang et al. , 2013Jiang et al. 2017;Dong et al. 2018), the roots and rhizomes of Valeriana amurensis Smir. ex Kom. were investigated herein.
V. amurensis, a perennial herb, is broadly distributed in Russian Far East, northeast China and North Korea (Houghton 1988;Huang et al. 2004). The roots and rhizomes of the plant were used as a traditional Chinese medicine (TCM) to treat neurological system diseases, including insomnia, epilepsy, neurasthenia and insanity (You 1989;. To date, there are a few published research work in chemical constituents and CNS activities of this specie, and bioactive compounds like sesquiterpenoids, iridoids and lignans were isolated and characterized (Wang et al. 2012;Wu et al. 2014). To further reveal the chemical basis for the therapeutic effect of V. amurensis on CNS, we carry out phytochemical investigations. In the present study, two new sesquiterpenoids, including a kessane-type sesquiterpenoid (1) and one bisabolane derivative (2), together with fourteen known compounds (3-16) were isolated, and their potential effects on nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells were evaluated. NGF, a neurotrophic factor, serves a significant role in the proliferation, differentiation, survival and death of nerve cells. The compounds with activities on enhancing NGF-induced differentiation in PC12 cells are expected to be important for the treatment of neurodegenerative diseases (Cosgaya et al. 1996;Heese et al. 2006). Herein, the isolation, the structural elucidation of the new compounds, and the NGFmediated neurite outgrowth activity of 1-16 in PC12 cells were described.

Structural elucidation of isolated compounds
Compound 1 was obtained as a colorless oil with the molecular formula C 20 H 34 O 3 as assigned by an m/z 345.2398 [M þ Na] þ (calcd for C 20 H 34 O 3 Na, 345.2400) from the positive HRESIMS experiment, with four degrees of unsaturation. The IR (KBr, v) absorption band at 1728 cm À1 suggesting the presence of ester C ¼ O group. The 1 H NMR spectroscopic data (Table S1) displayed six methyls [at d H 1.28, 1.27, 1.19 (each 3H, each s), 0.94 (6H, d, J ¼ 6.5 Hz) and 0.82 (3H, d, J ¼ 7.1 Hz)], and an oxygenated methine [at d H 5.00 (1H, t, J ¼ 4.1 Hz). The 13 C NMR in combination with DEPT data (Table S1) exhibited twenty carbon signals, including six methyls, five methylenes, and six methines (an oxygenated), as well as three quaternary (two O-bearing and one ester C ¼ O groups) carbons. A carefully comparison of the NMR data of 1 with those of a-kessyl acetate indicated that they possessed a similar structure (Houghton 1988;Nishiya et al. 1992;Tori et al. 1996;Houghton 1999;Wang et al. 2011), the only difference was the acetoxy residue at C-2 in a-kessyl acetate was replaced by an isovaleroxy group in 1 ( Figure S21) (Houghton 1988;Nishiya et al. 1992). The substituent patterns of 1 were further confirmed by the positive-ion HRESIMS data in combination with the HMBC correlations from H-2 (d H 5.00) to C-4 (d C 30.3), C-5 (d C 36.4) and C-10 (d C 73.2); from H-6 (d H 2.02, 1.36) to C-1 (d C 53.6), C-4 (d C 30.3) and C-11 (d C 74.4); from H-8 (d H 2.18, 1.51) to C-6 (d C 32.7), C-10 (d C 73.2) and C-11 (d C 74.4); from H-12 (d H 1.28, s) to C-7 (d C 35.8) and C-13 (d C 31.1); from H-13 (d H 1.27, s) to C-7 (d C 35.8) and C-12 (d C 28.1); from H-14 (d H 1.19) to C-1 (d C 53.6), C-9 (d C 35.4) and C-10 (d C 73.2); from H-15 (d H 0.82) to C-3 (d C 42.0) and C-5 (d C 36.4); from H-4', H-5' (d H 0.94) to C-2' (d C 44.0); and from H-3' (d H 2.10) to C-1' (d C 173.2) ( Figure S2 and Supplementary data). The position of the isovaleroxy group at C-2 in 1 was assigned based on the fact that the NMR spectral data of 1 (Table S1) were almost the same as that of a-kessyl acetate (Nishiya et al. 1992). The relative configuration of 1 was established by a combination of the ROESY experiment ( Figure S22) and detailed comparison with the spectroscopic data of those reported ones (Houghton 1988;Nishiya et al. 1992;Tori et al. 1996;Houghton 1999;Wang et al. 2011). According to carefully comparison of spectroscopic data, the configurations of 1 were consistent with those known ones (Houghton 1988;Nishiya et al. 1992;Tori et al. 1996;Houghton 1999;Wang et al. 2011). Therefore, the structure of 1 was elucidated and named as a-kessyl isovalerate.

Functional characterization of isolated compounds
All isolates were evaluated for their potential neuroprotective activities on NGF-mediated neurite outgrowth in PC12 cells (Bai et al. 2015;Greene and Tischler 1976), with 50 ng/ml (positive control) NGF and 5 ng/ml (negative control), respectively. The results are summarized in Table S2. The differentiation rate of neurite-bearing cells were 19.94 and 4.19 following incubation with 50 ng/ml NGF (positive control) and 5 ng/ml NGF (negative control) after 72 h, respectively, among the tested, compounds 10, 11, 12 and 15 showed potent promoting effects on NGF-induced neurite outgrowth in PC12 cells with the differentiation rate of 11.84%, 12.21%, 13.77% and 12.16%, respectively ( Figure S25), at the concentration of 10 lM.

Conclusions
In conclusion, the EtOAc fraction from the roots and rhizomes of V. amurensis has led to the identification of sixteen sesquiterpenoids, including one new kessane-type sesquiterpenoid (1) and a new bisabolane derivative (2). Their structures were elucidated by extensive spectroscopic analysis. All isolates were evaluated for their effects on NGF-mediated neurite outgrowth in pheochromocytoma (PC12) cells and four compounds (10-12 and 15) showed potent promoting effects, they are germacrane, maaliane, patchoulane and eudesmane type sesquiterpenoids, respectively. Taken together, the results suggested that the sesquiterpenoids, especially the structures similar to the above active compounds in this plant possess the potent promoting effects on NGF-induced neurite outgrowth in PC12 cells, which may provide further valuable insight into the pharmacophores of V. amurensis for the treatment of neurodegenerative diseases.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by grants from National Natural Science Foundation of China (31860092)