Chemical Constituents and an Antineuroinflammatory Lignan, Savinin from the Roots of Acanthopanax henryi

The phytochemical investigation on the roots of Acanthopanax henryi (Araliaceae) resulted in the discovery of twenty compounds whose chemical structures were elucidated by the analysis of 1D-, 2D-NMR, mass spectrometry data, other physicochemical properties, and a comparison of the spectral data with the literature. They were identified as (-)-sesamin (1), helioxanthin (2), savinin (3), taiwanin C (4), 6-methoxy-7-hydroxycoumarin (5), behenic acid (6), 3-O-caffeoyl-quinic acid (7), 5-O-caffeoyl-quinic acid (8), 1,3-di-O-caffeoyl-quinic acid (9), 1,4-di-O-caffeoyl-quinic acid (10), 1,5-di-O-caffeoyl-quinic acid (11), (+)-threo-(7R,8R)-guaiacylglycerol-β-coniferyl aldehyde ether (12), (+)-erythro-(7S,8R)-guaiacylglycerol-β-coniferyl aldehyde ether (13), ferulic acid (14), caffeic acid (15), stigmasterol (16), β-sitosterol (17), adenosine (18), syringin (19), and trans-coniferin (20). Among these isolates, compound 3 showed inhibitory activity against lipopolysaccharide- (LPS-) induced nitric oxide (NO) and prostaglandin E2 (PGE2) production with IC50 values of 2.22 ± 0.11 and 2.28 ± 0.23 μM, respectively. The effects of compound 3 were associated with the suppression of LPS-induced expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein. Furthermore, compound 3 negatively regulated the production of interleukin- (IL-) 1β and tumor-necrosis factor- (TNF-) α at the transcriptional level in LPS-stimulated BV2 microglial cells. These antineuroinflammatory effects of compound 3 were mediated by p38 mitogen-activated protein kinase (MAPK).


Introduction
Neuroinflammatory responses are mainly mediated by microglial activation, and they are implicated in the development of neurodegenerative diseases such as Alzheimer's diseases (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS) [1]. Microglial cells are the primary resident immune cells in the central nervous system (CNS) and are associated with defensive mechanisms to maintain homeostasis against brain injury [2]. In steady state, microglia exert protective responses by regulating innate and adaptive immune responses. However, activated microglia produce excess immune reactions which are detrimental to brain tissue and can produce various proinflammatory mediators such as tumor necrosis factor-(TNF-) , interleukins (ILs), nitric oxide (NO), prostaglandin E2 (PGE 2 ), and reactive oxygen species (ROS). Released and accumulated, these proinflammatory mediators facilitate the development of neurodegenerative diseases [3,4]. Therefore, it is important to suppress the secretion of proinflammatory mediators from activated microglial cells to prevent the neuroinflammation-related development of neurodegenerative diseases.
Acanthopanax spp. is one of the well-known medicinal resources in traditional oriental medicine in China, Korea, Japan, and far-east Russia. Its dried roots and stem barks are famous traditional folk medicine for treating rheumatism, arthritis, paralysis, sinew, and bone pain [5]. Acanthopanax henryi (Oliv.) Harms, a Chinese endemic plant, has been used as a traditional remedy for the treatment of paralysis, arthritis, rheumatism, lameness, edema, injury from falls, hernia, and abdominal pain [6,7]. Previous phytochemical studies 2 Evidence-Based Complementary and Alternative Medicine on A. henryi led to the isolation and identification of more than 30 secondary metabolites, including five flavonoids, six caffeoylquinic acid derivatives [6], sixteen triterpenoid saponins [8,9], one amide, one anthraquinone, one organic acid [9], three lignans, one diterpene, one phenylpropanoid, and two phytosterols [10]. In addition, it has been reported that this plant exhibits diverse pharmacological activities due to the wide variety of chemical constituents. For example, metabolites of the leaves of A. henryi have strong antioxidant and antiacetyl cholinesterase activities [6], and the 80% methanol fraction of root bark and ciwujianoside C3, which was isolated from leaves of this plant, have significant antiinflammatory effect in lipopolysaccharide-(LPS-) induced RAW264.7 macrophage cells [5,11]. Moreover, some glycosides from the leaves of A. henryi have antiadipogenic effect, decreasing lipid accumulation through the inhibition of proliferator-activated receptor gamma (PPAR ) and CCAAT/enhancer-binding protein alpha (C/EBP ) in 3T3-L1 cells [12]. However, it has not been investigated yet whether this plant has antineuroinflammatory effects.
Therefore, in this study, we purified twenty compounds from the roots of A. henryi and evaluated their antineuroinflammatory activity in vitro to continue our approach to contribute to the drug development for inflammationmediated neurodegenerative diseases.

Determination of Nitrite (NO Production)
. BV2 cells were cultured in 24-well culture plates at a density of 5 × 10 4 cells/well. BV2 cells were pretreated with the isolated compounds from A. henryi and then stimulated with LPS (1 g/mL) for 24 h. After incubation, 100 L of each supernatant was collected and mixed with the same volume of the Griess reagent. The absorbance at 540 nm wavelength was measured using a microplate reader. The detailed procedures are described in our previous report [14]. 2 Assay. The level of PGE 2 present in each sample was determined using a commercially available kit from R&D Systems (Minneapolis, MN, USA). Three independent assays were performed according to the manufacturer's instructions. Briefly, BV2 cells were seeded in 24-well culture plates at a density of 5 × 10 4 cells/well. Prior to the stimulation with LPS (1 g/mL) for 24 h, cells were treated with test compounds. After incubation, supernatant was collected and applied to the PGE 2 ELISA kit for measuring the concentration of PGE 2 .

Western Blotting Analysis.
The proteins iNOS, COX-2, and MAPK-associated proteins, including p-p38, p38, p-JNK, JNK, p-ERK, and ERK, were detected by a Western blot analysis. The procedures of this experiment were based on our previous report [14]. Cells were lysed by RIPA buffer (Thermo Fisher Scientific, USA), and normalized for equal amounts of protein using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). 30 g of protein was loaded for each sample and separated on 7.5 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Then, proteins were transferred to nitrocellulose (NC) membranes (Bio-Rad Laboratories). After that, membranes were incubated with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) with 5% skim milk (BD Difco, USA) for 1 h at 4 ∘ C. Then, membranes were probed with primary antibodies and incubated at 4 ∘ C for 90 min or overnight.
After incubation, the membranes were washed with TBS-T, and then secondary antibodies were used for detecting primary antibodies. The protein bands on the NC membranes were covered by chemiluminescence with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK).

2.9.
Assays for IL-1 , IL-6, and TNF-. The culture media were collected to determine the levels of IL-1 , IL-6, and TNF-present in each sample using appropriate ELISA kits (R&D Systems, Inc.), as per the manufacturer's instructions. Briefly, BV2 cells were seeded in 24-well culture plates at a density of 5 × 10 4 cells/well. After incubation, the supernatant was collected and applied to the cytokine ELISA kits for measuring the concentrations of IL-1 , IL-6, and TNF-.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
The detailed procedures of qRT-PCR and the primer sequences, which were used in this investigation were reported in our previous investigations [14,15]. The analysis was conducted three times independently.

Caffeic Acid
Compound was identified as -sitosterol.   62.3 (C-5 ). All spectral data agreed with those previously reported [28]. Compound was identified as adenosine. (19). . All spectral data agreed with those previously reported [29,30]. Compound was identified as syringin. . All spectral data agreed with those previously reported [30]. Compound was identified as trans-coniferin.

Trans-Coniferin
The chemical structures of compoundsare shown in Figure 1.

Effects of Compounds on NO and PGE 2 Production
in LPS-Induced BV2 Microglial Cells. Among the isolated compounds from A. henryi, we selected 13 compounds (compounds -, , , , , , , , and -) based on their number of publications, and these compounds were screened for anti-inflammatory effects, including the inhibition of NO and PGE 2 production, in LPS-stimulated BV2 microglial cells. BV2 cells were pretreated with the compounds for 3 h and stimulated with LPS (1 g/mL) for 24 h. The concentration required to inhibit the production of NO by 50% (IC 50 value) was calculated based on the concentrations of NO and PGE 2 released into the culture  Figure 2: The effect of compound on LPS-induced iNOS and COX-2 protein expression in BV2 microglial cells. Cells were pretreated with/without the indicated concentrations of compound for 3 h and then stimulated with LPS (1 g/mL) for 24 h. The levels of iNOS and COX-2 were determined by Western blot analysis. The experiment was repeated three times, and similar results were obtained. media as measured by the Griess method and PGE 2 ELISA kit, respectively. Among the tested compounds, only compound showed inhibitory effects against LPS-induced NO production with an IC 50 value of 2.22 ± 0.11 M and LPSinduced PGE 2 production with an IC 50 value of 2.28 ± 0.23 M. The IC 50 values of butein, a positive control, were 4.41 ± 0.45 M in NO production and 3.26 ± 0.53 M in PGE 2 production, respectively. At tested concentrations, all test compounds did not show cytotoxic effects to BV2 cells (data not shown). Therefore, compound was further investigated to elucidate the up-stream signaling pathways of its antineuroinflammatory effect.

Effect of Compound 3 on LPS-Induced Expression of iNOS and COX-2 Protein in BV2 Microglial
Cells. Inhibition of iNOS and COX-2 protein expression could be an important strategy to prevent neuroinflammation. In the present study, it was investigated whether compound inhibits LPS-induced overexpression of iNOS and COX-2 protein in BV2 cells. BV2 cells were pretreated with compound for 3 h and stimulated with LPS (1 g/mL) for 24 h. As a result, LPS augmented the expression of iNOS and COX-2 proteins; however, pretreatment with compound reversed these responses (Figure 2).

Effect of Compound 3 on LPS-Induced Up-Regulation of Pro-Inflammatory Cytokines.
To investigate whether compound inhibits the production of LPS-induced proinflammatory cytokines including IL-1 , IL-6, and TNF-, BV2 cells were pretreated with compound for 3 h and stimulated with LPS (1 g/mL) for 24 h. Pretreatment with compound attenuated the LPS-induced production of IL-1 and TNF-, but it had little effect on IL-6 production (Figures 3(a)-3(c)). Subsequently, to elucidate how compound inhibits proinflammatory cytokine production, the mRNA levels of the cytokines were examined. BV2 cells were pretreated with compound for 3 h and stimulated with LPS (1 g/mL) for 6 h. Consistent with the results obtained from the cytokine production data, pretreatment with compound reduced the LPS-induced mRNA levels of IL-1 and TNF-, but it did not affect to mRNA level of IL-6 ( Figures 3(d)-3(f)). These results suggested that compound exhibits the antineuroinflammatory effects by negatively regulating the production of IL-1 and TNF-at the transcriptional level in LPS-challenged BV2 microglial cells.

Effect of Compound 3 on LPS-Induced Activation of MAPK Pathway in BV2 Cells.
We examined whether the antineuroinflammatory effects of compound contribute to the inactivation of MAPK in the present study. BV2 cells were pretreated with compound for 3 h and stimulated with LPS (1 g/mL) for 30 min. LPS markedly increased the phosphorylation of p38, JNK, and ERK. Pretreatment with compound inhibited the phosphorylation of p38 (Figure 4(a)), but it had no effect on the phosphorylation of JNK and ERK (Figures 4(b) and 4(c)).

Discussion
The present study demonstrated that compound (savinin), one of the isolated compounds from the roots of A. henryi exerted antineuroinflammatory effects in LPS-treated microglial cells. Compound inhibited LPS-induced the release of proinflammatory mediators, including NO, PGE 2 , iNOS, COX-2, IL-1 , and TNF-. These inhibitory effects of compound were mediated by the inactivation of the p38 MAPK pathway.
NO and PGE 2 , as well as their regulatory enzymes iNOS and COX-2, are important mediators for neuroinflammation. They are involved in the pathogenesis of various neuroinflammatory pathophysiological conditions [31]. In addition, proinflammatory cytokines, including IL-1 , IL-6, and TNF-, are important factors in CNS immune responses, and mediate neurodegeneration [3]. Microglial cells are activated by various stimuli. The activation of these cells leads to the release of these proinflammatory mediators and the exacerbation of neuroinflammation and neuroglia-induced neurotoxicity [31,32]. Our investigation showed that compound , isolated from A. henryi, suppressed NO and PGE 2 production in LPS-stimulated BV2 microglial cells and also inhibited the LPS-induced expression of the iNOS and COX-2 protein (Figure 2). Additionally, the present data indicated that compound significantly suppressed the release of IL-1 and TNF-, but not IL-6, at the protein and mRNA levels ( Figure 3).
MAPKs are a family of serine/threonine protein kinases, and they consist of three major subunits: p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). MAPKs are involved in various cellular processes, including proliferation, differentiation, stress responses, and immune responses [33]. In inflammatory responses, the activation of MAPKs leads to release of inflammatory-related  factors, such as iNOS, COX-2, ILs, and TNF- [34]. Therefore, we further investigated whether the antineuroinflammatory effects of compound in LPS-stimulated BV2 microglial cells were related to the inactivation of MAPKs. Our results showed that compound significantly inhibited the phosphorylation of p38 MAPK, but it did not affect the phosphorylation of ERK and JNK MAPKs. Nuclear factor kappa B (NF-B) is one of the major transcription factors regulating inflammatory responses. When microglial cells are stimulated with ILs, TNF-, interferons, or LPS, NF-B can be activated following increased translocation of p65 and p50, which are subunits of the NF-B dimer, into the nucleus, as well as the phosphorylation and degradation of inhibitor of kappa B (I B)-in the cytosol [35]. This response upregulates the expression of proinflammatory cytokines, chemokines, and adhesion molecules in microglial cells [1]. Therefore, NF-B can be an important target for the treatment of neuroinflammation-related neurodegenerative diseases. Interestingly, pretreatment with compound did not decrease the nuclear translocation of p65 and p50 or the phosphorylation and degradation of I B-(data not shown). Considering the effect of compound on the LPS-induced activation of NF-B and MAPK pathways, it is suggested that compound exerted antineuroinflammatory effects by specifically inactivating the p38 MAPK pathway.

Conclusions
In summary, 20 secondary metabolites were isolated from the EtOAc-, butanol-, and PE-soluble fractions of the methanol extract of Acanthopanax henryi roots. Among these compounds, savinin (compound ) was the only compound which inhibited the production of NO and PGE 2 , and the expression of iNOS and COX-2 proteins in LPS-stimulated BV2 microglial cells. In addition, this compound significantly suppressed the release of IL-1 and TNF-at the protein and mRNA levels. These inhibitory effects of compound were mediated by p38 MAPK, but not ERK and JNK MAPKs. Taken together, our investigation showed that compound can be a promising candidate for the development of treatment options for neurodegenerative diseases. Further studies elucidating the detailed mechanisms underlying the anti-inflammatory effects of compound are suggested.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.