Phenylpropanoid Derivatives from the Tuber of Asparagus cochinchinensis with Anti-Inflammatory Activities

Three undescribed phenylpropanoid derivatives, including two new bibenzyl constituents (1–2), one new stilbene constituent (3), together with five known compounds stilbostemin F (4), dihydropinosylvin (5), 2-(4-hydroxyphenyl)ethyl benzoate (6), 1-(4-hydroxybenzoyl)ethanone (7), and 4-hydroxy-3-prenylbenzoic acid (8), were isolated from the tuber of Asparagus cochinchinensis. The structures of 1–8 were elucidated according to UV, IR, HRMS, 1D and 2D-NMR methods together with the published literature. All of the isolated compounds were assessed for anti-inflammatory activity by acting on lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells in vitro. The results showed that compounds 2 and 5 were found to inhibit the production of nitric oxide (NO) with the IC50 value of 21.7 and 35.8 µM, respectively. In addition, further studies found that compound 2 demonstrated concentration-dependent suppression of the protein expression of iNOS and exerted anti-inflammatory activity via the NF-κB signalling pathway. The present data suggest that phenylpropanoid derivatives from the tuber of A. cochinchinensis might be used as a potential source of natural anti-inflammatory agents.


Introduction
Nitric oxide (NO) is a key signaling molecule and regulates various physiological functions in many tissues of the human body [1,2]. However, an overproduction of NO is associated with many inflammatory diseases [3]. Hence, the inhibition of excessive production of NO may have a therapeutic benefit in controlling inflammation and discovering new drugs for reducing inflammation using natural bioactive compounds plays an important role in research [4].
The genus of Asparagus has been used as a vegetable and as medicines due to its soothing flavor and wealth of health benefits [5][6][7]. Asparagus cochinchinensis is an important traditional Chinese herbal plant, and the use of its tuber is employed for treating cutaneous inflammation, aging, hyperlipidemia, cardiovascular disease, bacterial infection, diabetes, constipation, and throat pain [8,9]. Phytochemical studies have demonstrated that it contains flavonoids, phenolics, steroidal glycosides, alkaloids, and polysaccharide compounds [10][11][12][13]. 2 of 9 In this research, as part of the ongoing search for new chemical and anti-inflammatory constituents from A. cochinchinensis [14], phytochemical and biological studies of the tuber of A. cochinchinensis were carried out to explore the anti-inflammatory ingredients.
In this research, as part of the ongoing search for new chemical and anti-inflammatory constituents from A. cochinchinensis [14], phytochemical and biological studies of the tuber of A. cochinchinensis were carried out to explore the anti-inflammatory ingredients.

Inhibitory Effects of Compounds 1-8 on NO Production of LPS-Activated RAW 264.7 Cells
An MTT assay was used to evaluate the cytotoxic effects of 1-8 on RAW 264.7 cells in vitro, with aminoguanidine hydrochloride (AH) used as a positive control. The results showed that none of the compounds or positive control exhibited significant cytotoxicity at a concentration of 50 µM (over 75% cell survival). Furthermore, the inhibitory effect of 1-8 on the production of NO in LPS-induced RAW 264.7 cells was measured by the Griess method [21]. The inhibitory effect of 1-8 on NO release is shown in Table 2. The results showed that two of the isolated compounds (2 and 5) displayed NO inhibitory activity (IC 50 21.7 and 35.8 µM) (positive control: Aminoguanidine hydrochloride, IC 50 18.4 ± 2.33 µM). Especially, by comparing compound 1, 2 with 3, we found when the methoxy group linked at C-4 could cause a dramatic promotion in the inhibitory activity. The NO inhibitory activity of compound 2 and 5 versus 3 suggested the bibenzyl compounds were more active than stilbene compounds and the ∆ 1"(2") double bond offers no assistance to antiinflammatory activity. The above structure-activity relationship (SAR) was preliminary and needed to be validated.

Inhibitory Effects of New Compound 2 on LPS-Enhanced Inflammatory Mediators
Proinflammatory molecules, such as iNOS, are involved in inflammation-associated diseases and act as inflammatory mediators or activators of inflammatory pathways. Herein, Western blot analysis was performed to detect the protein expression of the inflammation markers iNOS as proteins of the NF-κB pathway [22]. Among all the isolated compounds, compound 2 displayed the strongest inhibition on NO release, so it was selected for further study. Protein expression levels were normalized against GAPDH. As shown in Figure 5, compound 2 treatment significantly inhibited LPS-induced expression of iNOS in RAW 264.7 cells. A down-regulation of iNOS expression in the presence of 2 at 40 µM was observed. In conclusion, these results suggested that 2 exerts anti-inflammatory activity, possibly via the NF-κB signaling pathway. However, further investigations are necessary to elucidate whether these compounds can act on other inflammatory mechanisms.
Previous research has shown that A. cochinchinensis is a potential therapeutic agent for inflammatory diseases [23,24]. iNOS was an important target for the NF-κB inflammasome pathway to prevent an inflammatory response [25]. Therefore, anti-inflammatory agent 2, with some iNOS expression inhibitory activities, and the potential anti-inflammatory constituent 5 along with the benzofuranoid norlignans (asparlignan A and B) which were isolated from the aerial parts of A. cochinchinensis in the previous study [14] might form some of the effective ingredients for A. cochinchinensis to prevent inflammatory diseases. Previous research has shown that A. cochinchinensis is a potential therapeutic agent for inflammatory diseases [23,24]. iNOS was an important target for the NF-κB inflammasome pathway to prevent an inflammatory response [25]. Therefore, anti-inflammatory agent 2, with some iNOS expression inhibitory activities, and the potential anti-inflammatory constituent 5 along with the benzofuranoid norlignans (asparlignan A and B) which were isolated from the aerial parts of A. cochinchinensis in the previous study [14] might form some of the effective ingredients for A. cochinchinensis to prevent inflammatory diseases.

Plant Materials
The tuber of A. cochinchinensis was collected in Lu'an, Anhui province, P. R. China. The plant material was identified by associate Prof. Tao Xu (West Anhui University) and a voucher specimen (TMD-10) has been deposited at the School of Pharmacy, Anhui University of Chinese Medicine.

Extraction and Isolation
The air-dried tubers of A. cochinchinensis (3 kg) were extracted three times with methanol to obtain a crude extract, then the extract was suspended in water, followed by extraction with EtOAc. The partial fraction from the EtOAc extract (12 g) was observed using a silica gel column (200-300 mesh) and eluted sequentially with CHCl2-CH3OH (100:0 to

Plant Materials
The tuber of A. cochinchinensis was collected in Lu'an, Anhui province, China. The plant material was identified by associate Prof. Tao Xu (West Anhui University) and a voucher specimen (TMD-10) has been deposited at the School of Pharmacy, Anhui University of Chinese Medicine.

Cell Culture and NO Production Measurements
The experimental procedures were followed as per the literature [26]. Cell viability was evaluated using the MTT assay (5 mg/mL). The RAW264.7 cells were seeded into 96-well plates at density of 50,000 cells/well for 24 h. Then the cells were pretreated with the tested compounds for 30 min at 37 • C, and then stimulated with LPS (100 ng/mL) for 24 h. The Griess reaction was used to detect the NO level. Momentarily, the cell culture supernatant (50 µL) and Griess reagent (50 µL) were mixed with an equal volume for 10 min, and then the absorbance was monitored at 540 nm using a microplate reader. All the tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO. Aminoguanidine hydrochloride was used as the positive control group.

Western Blot Analysis
Cells (5 × 10 5 /well) were initially treated with different concentrations (5, 10, 20, 40 µM) of compound 2 and LPS (1 µg/mL) stimulation ( Figure 5). Then the total proteins were extracted and immunoblotted as previously described [27,28]. Briefly, the RAW264.7 cells were lysed with 1% RIPA (radio-immunoprecipitation assay) (Amresco, Solon, OH, USA) to achieve the cellular lysates. The total proteins of the cellular lysates were measured by the BCA protein assay kit. Total proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). Then the membranes were washed with TBST buffer, blocking with 5% non-fat milk for 2 h at 25 • C, and then incubated with primary antibodies for 12 h at 4 • C. After being washed with TBST buffer, the membranes were treated with a secondary antibody at room temperature and the protein bands were detected.

Conclusions
In summary, three undescribed phenylpropanoid derivatives along with five known compounds were co-isolated from the tuber of A. cochinchinensis. Among them, asparbiben A-C (1-3) were identified as new bibenzyl and stilbene derivatives. In addition, these isolated compounds enrich the chemical entities of naturally occurring phenylpropanoids and the structural diversity of the Asparagus family. The phenylpropanoid constituents, especially the bibenzyl derivatives, may act as potential anti-inflammatory agents and this has attracted the attention of many researchers [29,30]. In the bioassays, all the isolated compounds were screened for anti-inflammatory effects. The screened results indicated that compounds 2 and 5 exhibited a potential inhibitory effect on NO production, with an IC 50 value of 21.7 and 35.8 µM, respectively. These results demonstrate that structurally different phenylpropanoid compounds in A. cochinchinensis may contribute its anti-inflammatory function. Importantly, the potential compound 2 decreased the protein expression levels of iNOS, indicating that 2 may be mediated via the suppression of an LPS-induced NF-κB inflammasome pathway. Taken together, phenylpropanoid derivatives are believed to be the main anti-inflammatory constituents of A. cochinchinensis. The present study lays the foundation for research into the potential therapeutic value of phenylpropanoid derivatives for inflammatory diseases.