Neuroprotective Iridoids and Lignans from Valeriana amurensis

Valeriana amurensis (V. amurensis) is widely distributed in Northeast China. In addition to medicines, it has also been used to prepare food, wine, tobacco, cosmetics, perfume, and functional foods. Other studies have investigated the neuroprotective effects of V. amurensis extract. As the therapeutic basis, the active constituents should be further evaluated. In this paper, six new compounds (1–6) were isolated, including five iridoids (Xiecaoiridoidside A–E) and one bisepoxylignan (Xiecaolignanside A), as well as six known compounds (7–12). The neuroprotective effects of 1–12 were also investigated with amyloid β protein 1−42 (Aβ1-42)-induced injury to rat pheochromocytoma (PC12) cells. As a result, iridoids 1 and 2 and lignans 6, 8, and 9 could markedly maintain the cells’ viability by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH) release assay.


Introduction
The World Alzheimer Report 2018 showed that the number of dementia patients was about 50 million, of whom two-thirds had Alzheimer's disease (AD). It was expected that this number would increase to 152 million by 2050 [1]. In the past 30 years, some achievements have been made in the development of therapeutic drugs, including acetylcholinesterase inhibitors, glutamate receptor regulators, cerebral circulation improvers, γ-aminobutyric acids, peptides, calcium antagonists, antioxidants, anti-inflammatory medicines, statins, β-amyloid protein vaccines, neurotrophic factors, and central cholinergic receptor agonists [2]. The pathogenesis of AD is extremely complex, and the mechanisms have not been fully revealed. However, the damage or loss of brain neurons is well known in AD patients. Therefore, scholars have increasingly focused on the development of new drugs with brain-neuron-protective activity. Undoubtedly, brain-neuron-protective drugs can prevent the progress of AD to some extent [3]. In the past decades, more than 100 natural products have been considered as potential drugs for the treatment of AD [4,5].
The extracts from Valeriana plants can be used to treat nervous system diseases, such as PD, AD, and Huntington's disease, which are mostly related to brain neuron injuries or apoptosis. Therefore, some scholars have carried out studies on the neuroprotective effects of Valeriana in recent years [6]. As a Valeriana herb, Valeriana amurensis (V. amurensis) is mainly distributed in Northeast China [7,8]. At present, the roots and rhizomes from V. amurensis have been used as medicines in China. The extracted essential oil has been well developed and mainly used for preparing food, tobacco, wine, cosmetics, perfume, and so on. As a functional food, V. amurensis essential oil is responsible for regulating sleep cycles and helping to fall asleep, such as in the form of Xinjing Valerian Tablets and Valerian Root and Passiflora Compound Nutritional Capsules [6]. In some Chinese books, many healthcare functions of V. amurensis have been recorded, including sedative/hypnotic effects, The NOESY spectrum was used to determine the stereo-configuration of 1 [11]. The NOE correlations between H-7 and H-4α indicated that H-7 was α-oriented. The NOE correlations between CH3-9 and GlcH-1/H-4β indicated that the glucopyranosyl moiety and CH3-9 were β-oriented ( Figure 1). As a result, the structure of 1 was identified as (7′E)-4-p-coumaroyl-2,6β-dimethyl-hexahydrocyclopenta-7-carboxylic acid 3-O-β-D-glucopyranoside, which was referred to as Xiecaoiridoidside A ( Figure 2). Compound 2 was obtained as a white amorphous powder. The molecular formula of 2 was assigned as C25H34O12 based on the HRESIMS at m/z [M-H] − 525.1969 (calcd. 525.1972). The presence of a D-glucose fragment was determined by the hydrolysis experiment of 2. Most of the 1 H-NMR and 13 C-NMR spectral data (Tables 1 and 2) of 2 were identical to those of 1. The differences lay in the signals at δH 6.85 (1H, d, J = 12.9 Hz, H-7′) and 5.73 (1H, d, J = 12.9 Hz, H-8′), which indicated that an olefinic bond with a Z configuration was included in 2. Therefore, the C-4 of 2 was substituted with a cis-p-coumaroyl group. The DEPT, HSQC, 1 H-1 H COSY, HMBC, and NOESY spectra ( Figure 1) were used to identify the structure of 2 as (7′Z)-4-p-coumaroyl-2,6β-dimethyl-hexahydrocyclopenta-7-carboxylic acid 3-O-β-D-glucopyranoside, which was referred to as Xiecaoiridoidside B ( Figure 2). The NOESY spectrum was used to determine the stereo-configuration of 1 [11]. The NOE correlations between H-7 and H-4α indicated that H-7 was α-oriented. The NOE correlations between CH 3 -9 and GlcH-1/H-4β indicated that the glucopyranosyl moiety and CH 3 -9 were β-oriented ( Figure 1). As a result, the structure of 1 was identified as (7 E)-4-p-coumaroyl-2,6β-dimethyl-hexahydrocyclopenta-7-carboxylic acid 3-O-β-Dglucopyranoside, which was referred to as Xiecaoiridoidside A ( Figure 2 The presence of a D-glucose fragment was determined by the hydrolysis experiment of 2. Most of the 1 H-NMR and 13 C-NMR spectral data (Tables 1 and 2) of 2 were identical to those of 1. The differences lay in the signals at δ H 6.85 (1H, d, J = 12.9 Hz, H-7 ) and 5.73 (1H, d, J = 12.9 Hz, H-8 ), which indicated that an olefinic bond with a Z configuration was included in 2. Therefore, the C-4 of 2 was substituted with a cis-p-coumaroyl group. The DEPT, HSQC, 1 H-1 H COSY, HMBC, and NOESY spectra ( Figure 1) were used to identify the structure of 2 as (7 Z)-4-p-coumaroyl-2,6β-dimethyl-hexahydrocyclopenta-7-carboxylic acid 3-O-β-D-glucopyranoside, which was referred to as Xiecaoiridoidside B (Figure 2). Compound 3 was obtained as a white amorphous powder. The molecular formula of 3 was assigned as C 21 H 32 O 12 based on the HRESIMS at m/z [M + Na] + 499.1787 (calcd. 499.1791). The presence of a D-galactose fragment was determined by the hydrolysis experiment of 3. The 1 H-NMR spectrum of 3 ( Table 1) showed signals of two methyl groups at δ H 0.94 (6H, d, J = 6.7 Hz, CH 3 -4 , 5 ). The coupling constant of H-1" (J = 8.1 Hz) confirmed the galactopyranosyl moiety as a β configuration. Twenty-one carbon signals could be observed in the 13 C-NMR and DEPT spectra of 3 (Table 2) Figure 1). In addition, the H-1" correlated with C-11, H-10 correlated with C-7, C-8, and C-9, and H-1 correlated with C-1 suggested that the β-Dgalactopyranosyl moiety was linked to C-11, hydroxymethyl was linked to C-8, and the -O-isovaleryl group was linked to C-1, respectively. Compound 3 was obtained as a white amorphous powder. The molecular formula of 3 was assigned as C21H32O12 based on the HRESIMS at m/z [M + Na] + 499.1787 (calcd. 499.1791). The presence of a D-galactose fragment was determined by the hydrolysis experiment of 3. The 1 H-NMR spectrum of 3 ( Table 1) showed signals of two methyl groups at δH 0.94 (6H, d, J = 6.7 Hz, CH3-4′, 5′). The coupling constant of H-1" (J = 8.1 Hz) confirmed the galactopyranosyl moiety as a β configuration. Twenty-one carbon signals could be observed in the 13 C-NMR and DEPT spectra of 3 (Table 2) Figure 1). The HMBC spectrum was used to establish the iridoid skeleton of 3 ( Figure 1). In addition, the H-1" correlated with C-11, H-10 correlated with C-7, C-8, and C-9, and H-1 correlated with C-1′ suggested that the β-D-galactopyranosyl moiety was linked to C-11, hydroxymethyl was linked to C-8, and the -O-isovaleryl group was linked to C-1, respectively.
Compound 5 was obtained as a white amorphous powder. The molecular formula of 5 was assigned as C 16 (Table 1) showed a signal of a methoxyl group at δ H 3.88 (3H, s, 3-OCH 3 ). The coupling constant of H-1" (J = 7.1 Hz) confirmed the glucopyranosyl moiety as a β configuration. Moreover, the 1 H-NMR spectrum of 6 also showed the signals of a 1,4-disubstituted phenyl group and a 1,3,4-trisubstituted phenyl group. Twenty-five carbon signals could be observed in the 13 C-NMR and DEPT spectra of 6 ( Table 2), including six carbon signals of a β-D-glucopyranosyl moiety, twelve carbon signals of two phenyl groups, and a methoxyl group, as well as two oxygenated quaternary groups, two oxygenated methines, and two oxygenated methylenes. These data show typical characteristics of bisepoxylignans. The HSQC and 1 H-1 H COSY spectra further confirmed the coupling sequences of two phenyl groups in 6 ( Figure 1). The HMBC spectrum was used to establish the bisepoxylignan structure of 6 ( Figure 1), and the correlation between H-1" and C-4 suggested that the β-D-glucopyranosyl moiety was located at C-4.
which was referred to as Xiecaolignanside A (Figure 2).
Iridoids and lignans are the main components of V. amurensis. The above results adequately elucidate the neuroprotective effect of V. amurensis extracts on AD model mice.
To determine the protective effects of compounds 1-12 against Aβ1-42-induced neurotoxicity, PC12 cells were treated with 1.5 μM Aβ1-42 for 20 h after pretreatment with compounds 1-12 at concentrations of 5, 12.5, and 25 μM for 4 h. As shown in Figure 4A,B, the viability of cells treated with iridoids 1 and 2 was markedly increased in a dose-independent manner compared to that of the model group, and the same was true for lignans 6, 8, and 9. This result was further confirmed by the reduction in LDH release from cells treated with compounds 1, 2, 6, 8, and 9, especially at the concentration of 25 μM. Iridoids and lignans are the main components of V. amurensis. The above results adequately elucidate the neuroprotective effect of V. amurensis extracts on AD model mice.

General Experimental Procedures
The extracts of V. amurensis were separated by column chromatography (CC) of macroporous resin (AB-8 crosslinked polystyrene, Nan Kai, Tianjin, China), silica gel (200-300 mesh, Haiyang Chemical Group Co. Ltd., Qingdao, China), and ODS-A (120A, 50 mm; YMC, Kyoto, Japan), successively. Preparative HPLC: a Waters 2535 instrument in tandem with a Waters Sunfire prep C18 OBD TM 10 μm (19 × 250 mmi.d.) column for preparing compounds, and detection with UV-2998 and RI-2414 detectors. The NMR spectra were assayed on a Bruker DPX 400 instrument (Bruker SpectroSpin, Karlsruhe, Germany). A Xero Q Tof MS spectrometer (Waters, Milford, MA, USA) was used to measure the HRESIMS. A Shimadzu FTIR-8400S (Kyoto, Japan) was used to determine the IR spectra of the isolated compounds. The sugar derivatives from isolated compounds were analyzed on GC-MS (Agilent, California, CA, USA). PC12 cells were bought from the Institute of Biochemistry and Cell Biology (Shanghai, China). The PC12 cells were grown in DMEM (HyClone, NRH0020) with a 1% antibiotic mixture of penicillin-streptomycin and 5% fetal bovine serum in an atmosphere of 5% CO2 at 37 °C. A bicinchoninic acid (BCA) protein assay kit (Nianjing Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the total protein concentrations of the PC12 cells. The colorimetric LDH level

General Experimental Procedures
The extracts of V. amurensis were separated by column chromatography (CC) of macroporous resin (AB-8 crosslinked polystyrene, Nan Kai, Tianjin, China), silica gel (200-300 mesh, Haiyang Chemical Group Co. Ltd., Qingdao, China), and ODS-A (120A, 50 mm; YMC, Kyoto, Japan), successively. Preparative HPLC: a Waters 2535 instrument in tandem with a Waters Sunfire prep C18 OBD TM 10 µm (19 × 250 mmi.d.) column for preparing compounds, and detection with UV-2998 and RI-2414 detectors. The NMR spectra were assayed on a Bruker DPX 400 instrument (Bruker SpectroSpin, Karlsruhe, Germany). A Xero Q Tof MS spectrometer (Waters, Milford, MA, USA) was used to measure the HRESIMS. A Shimadzu FTIR-8400S (Kyoto, Japan) was used to determine the IR spectra of the isolated compounds. The sugar derivatives from isolated compounds were analyzed on GC-MS (Agilent, California, CA, USA). PC12 cells were bought from the Institute of Biochemistry and Cell Biology (Shanghai, China). The PC12 cells were grown in DMEM (HyClone, NRH0020) with a 1% antibiotic mixture of penicillin-streptomycin and 5% fetal bovine serum in an atmosphere of 5% CO 2 at 37 • C. A bicinchoninic acid (BCA) protein assay kit (Nianjing Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the total protein concentrations of the PC12 cells. The colorimetric LDH level was assayed with a colorimetric LDH assay kit on a microplate reader (VICTOR TM ×3, PerkinElmer, Inc., Waltham, MA, USA).

Plant Materials
The roots and rhizomes of V. amurensis were collected from the Great Xing'an Mountains area (Heilongjiang province, China) and identified by Zhenyue Wang, who is a pharmacognosy professor at Heilongjiang University of Chinese Medicine. The voucher

Monosaccharide Analysis of 1-6
Acid hydrolysis of compounds 1-6 was conducted in accordance with the method reported in reference [20], with some differences. Briefly, the monosaccharides were obtained from hydrolyzing compounds 1-6 (2.5 mg of each) with 2.0 mL of H 2 SO 4 (2 mol/L). The monosaccharides were further treated with trimethylchlorosilane to obtain the sugar derivatives of 1-6. The sugar derivatives were analyzed by GC-MS, and the monosaccharide of compounds 1, 2, and 4-6 was determined to be D-glucose (t R = 11.45 min). The monosaccharide of compound 3 was determined to be D-galactose (t R = 8.35 min).

Determination of the Cells' Viability
The culture and treatment of PC12 cells were similar to the method reported previously in [21]. Briefly, the PC12 cells were cultured in 6-well plates (6 × 10 5 cells/well) for 24 h; after that, different concentrations (5, 12.5, and 25 µM) of compounds 1-12 were added to incubate for 24 h, while different concentrations (0.1, 0.5, 1.5, 4.5, 15, and 30 µM) of Aβ  were added to incubate for 20 h. Then, the effects of compounds 1-12 on normal cells and a suitable concentration of Aβ 1-42 for inducing PC12 cells were confirmed by MTT and intracellular LDH release assay. The cell viability ratios of each group to the normal cell group (control) were calculated and recorded.
The culture and treatment of cells were prepared as described above. Then, compounds 1-12 at concentrations of 5, 12.5, and 25 µM were added to incubate for 4 h, while the control and model groups were added with equal volumes of medium. As an effective drug in a previous study, vitamin E (VE) was used as a positive control [22]. Neurotoxicity was induced in all groups of cells by 1.5 µM for 20 h, except for the control group. Then, 20 µL MTT solutions (5 mg/mL) were added to each well, and the cells were incubated at 37 • C for another 4 h. We next aspirated off the supernatants and then dissolved formazan crystals with DMSO. The microplate reader was used to measure the optical density of each well at 490 nm. Cell viability was also detected by measuring the LDH released [21]. A 50 µL culture supernatant was collected from each well for detecting the LDH activity (U/L) with a colorimetric LDH assay kit, according to the manufacturer's instructions. Colorimetric absorbance was measured on a plate reader at 570 nm. Assays of MTT and LDH were all repeated in three independent experiments, with six wells for each, and the results were expressed as a percentage of the control group, whose optical density was set at 100%.

Statistical Analysis
All data are presented as the mean ± SD. One-way analysis of variance (ANOVA) was used to perform statistical comparisons, and differences with p values < 0.05 according to the t-test were considered to be significant.

Conclusions
In conclusion, six new compounds (Xiecaoiridoidside A-E (1-5) and xiecaolignanside A (6)) and six known compounds (7)(8)(9)(10)(11)(12) were isolated from the roots and rhizomes of V. amurensis. The chemical structures of Xiecaoiridoidside A-E and xiecaolignanside A were identified by the analyses of their 1D and 2D NMR, HRESIMS, and other spectra. In addition, the neuroprotective effects of all isolated compounds were also investigated with an Aβ 1-42 -induced PC12 cell injury model, and iridoids 1 and 2, as well as lignans 6, 8, and 9, could markedly maintain the cells' viability. Therefore, some iridoids and lignans are likely responsible for the neuroprotective effects of V. amurensis.