Phytochemical Analysis, Anti-inflammatory, and Antioxidant Activities of Dendropanax dentiger Roots

Dendropanax dentiger root is a traditional medicinal plant in China and used to treat inflammatory diseases for centuries, but its phytochemical profiling and biological functions are still unknown. Thus, a rapid, efficient, and precise method based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was applied to rapidly analyse the phytochemical profiling of D. dentiger with anti-inflammatory and antioxidant activities in vitro. As a result, a total of 78 chemical compositions, including 15 phenylpropanoids, 15 alkaloids, 14 flavonoids, 14 fatty acids, 7 phenols, 4 steroids, 4 cyclic peptides, 3 terpenoids, and 2 others, were identified or tentatively characterized in the roots of D. dentiger. Moreover, alkaloid and cyclic peptide were reported from D. dentiger for the first time. In addition, the ethanol crude extract of D. dentiger roots exhibited remarkable anti-inflammatory activity against cyclooxygenase- (COX-) 2 inhibitory and antioxidant activities in vitro. This study is the first to explore the phytochemical analysis and COX-2 inhibitory activity of D. dentiger. This study can provide important phytochemical profiles and biological functions for the application of D. dentiger roots as a new source of natural COX-2 inhibitors and antioxidants in pharmaceutical industry.


Introduction
Over the past few years, secondary metabolites from natural products play an important role in the development of new drugs [1]. Higher plants represent sources of abundant phytochemicals with a wide range of biological effects and have attracted more attention in the past decades [2][3][4][5][6]. Consequently, most medicinal plants belong to higher plants have been widely to treat many human diseases in traditional folk medicine [1,[7][8][9]. Although numerous studies on the medicinal plants used as traditional Chinese medicines (TCMs), problems of chemical compositions and biological properties remained the main barriers in the development of modern traditional medicines or new drugs.
The genus Dendropanax (Araliaceae), known as "Shushen" in Chinese, comprises about 80 known species in tropical America and eastern Asia. In China, 16 native species have been found, which were widely cultivated in parks and/or used as folk medicine [10]. D. dentiger (Harms) Merr. is native to China and widely distributed in Guangxi, Jiangxi, Yunnan, and Guangdong provinces. In TCM, the roots of D. dentiger have been used as an important folk medicine for the treatment of inflammatory diseases [11]. Due to its potential pharmaceutical industry promoting effects, D. dentiger afforded structurally diverse and biologically active compounds, such as steroids, alkaloids, flavonoids, and monoterpenes; some of them showed potential anti-inflammatory, cytotoxic, and antioxidant activities [12,13]. Although lots of chemical compositions report on D. dentiger, the full chemical profiling and COX-2 inhibitory activity of this plant have not yet been studied so far.
This study was the first time to determine the phytochemical profiling and COX-2 inhibitory activity. In addition, it was also to evaluate the antioxidant activity, including DPPH and ABTS assays in vitro. This finding may contribute to the processing and utility of D. dentiger.

Plant
Material. The roots of D. dentiger were collected in the town of Baidu, Baise City, Guangxi, China, in October 2016. A botanical voucher specimen of this plant (No. DD20161022) was deposited at authors' laboratory and was identified by one of the authors Ronghua Liu [10].
2.5. COX-2 Inhibitory Assay. The anti-inflammatory effect of the sample against COX-2 inhibition was determined using colorimetric COX-2 inhibitor screening assay kit (no. S0168) and using celecoxib as the positive drug [2][3][4]. Briefly, 75 μL of assay buffer, 5 μL of cofactor working solution, and 5 μL of working solution were mixed with 5 μL of the sample at different concentrations and then incubated at 37°C. After 10 min, 5 μL of probe and 5 μL of substrate were added in all wells and then incubated at 37°C for 5 min, and the absorbance was determined (A sample ). The absorbance of a blank (A blank ) and control (A control ) composed of only the sample and COX-2 enzyme solutions was also determined, respectively. The COX − 2 inhibitory activity ð%Þ = ½ðA control -A sample Þ/ðA control -A blank Þ × 100%.
2.6. Antioxidant Assay 2.6.1. DPPH Radical Scavenging Activity. The DPPH radical scavenging activity of the sample was provided in our previously published articles [2][3][4]. Briefly, 150 μL of DPPH solution (dissolved 0.2 mM in methanol) was mixed with 50 μL of the sample at different concentrations. The mixture was stirred and incubated in the dark at 30°C for 30 min, and the absorbance was determined at 517 nm (A sample ). The absorbance of a blank (A blank ) and negative control (A control ) composed of only the sample and DPPH solutions was also determined, respectively. The DPPH radical scavenging activity of the sample was calculated by the following equation: DPPH scavenging activity = ½1 − ðA sample -A blank Þ/ A control × 100%. Vc was used as a positive control in this experiment.
2.6.2. ABTS Radical Scavenging Activity. The ABTS radical scavenging activity of the sample was carried out using the method reported by Sun et al. with minor modification [15]. Briefly, 1.76 mL of K 2 S 2 O 8 (140 mM) and 100 mL of ABTS solution (7 mM) were mixed and stored in the dark at 25°C for 12 h. Then, the ABTS stock solution was diluted with PBS (0.1 M, pH 7.4) until an absorbance value of 0:70 ± 0:02 was reached at 734 nm to obtain the diluted ABTS + radical solution. Subsequently, 10 μL of the sample was mixed with 195 μL the diluted ABTS + radical solution and incubated in the dark at 25°C for 106 min, and the absorbance of the sample at 734 nm (A sample ) was measured. The absorbance of a blank (A blank ) and negative control (A control ) composed of only the sample and diluted ABTS + radical solutions was also determined, respectively. The ABTS radical scavenging activity of the sample was calculated by the following equation: ABTS scavenging activity = ½1 − ðA sample -A blank Þ/A control × 100%. Vc was used as a positive control in this experiment.
2.7. Statistical Analysis. Graphpad Prism 6 was used for statistical analysis, and the data were presented as the means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey's test were used for comparison of differences in groups. Differences with p < 0:05 indicated statistical significance.

Identification of Main Constituents in D. dentiger Root
Extract. In the present study, the phytochemical compositions were identified using UHPL-Q-TOF-MS/MS based on the existing literatures and public databases, including ChemSpider, Massbank PubChem, and mzCloud, and summarized and described in Table 1  . The base peak chromatograms of D. dentiger roots extract in positive and negative ion modes were presented in Figure 1. A total of 78 compounds, including 15 phenylpropanoids, 15 alkaloids, 14 flavonoids, 14 fatty acids, 7 phenols, 4 steroids, 4 cyclic peptides, 3 terpenoids, and 2 others, were identified. The molecular formula was accurately assigned within mass error of 5 ppm. Then, the fragment ions were 2 BioMed Research International    3.1.1. Phenylpropanoids. Phenylpropanoids were widely distributed in medicinal plants and its structures containing one or more C 6 -C 3 units, which include three structure types, including simple phenylpropanoids, coumarins, and lignans [14]. A total of 15 phenylpropanoids in the roots of D. dentiger extract were identified in negative ion mode, including 12 simple phenylpropanoids and 3 lignans (Figure 2 26, 42, 50). They combine by the quinic acid and caffeic acid with esteratic linkage and have similar cleavage pathways. The typical neutral losses of caffeoyl, quinine, H 2 O, and CO 2 were the major cleavage pathway of such compounds. Taking compound 18 as an example, it gave the same MS 2 base peak at m/z 191.0571 due to the loss of caffeic acid and a relatively intense secondary ion at m/z 179.0356, while the ion at m/z 161.0254 was produced by continuous loss of H 2 O, allowing the assignment of chlorogenic acid as reported by the reference data. The possible fragmentation mechanism was depicted in Figure S1. Besides, compound 42 also has the same fragmentation pathways.
Three compounds (31, 40, 55) belonging to the lignan, which contain two or more C 6 -C 3 units. Compound 55 with a deprotonated molecule at m/z 523.21801 showed a base peak at m/z 361.1659 resulting from the loss of a hexosyl residue and was tentatively assigned as secoisolariciresinol hexose.

Flavonoids.
The mass spectra fragmentation patterns were widely used to provide the structural characterization of flavonoids in relation to the flavonoid aglycone and flavonoids glycoside. Moreover, the identification of the flavonoid aglycone was based on fragmentations, which related to the lost small neutral molecules and radicals (CH 3 , H 2 O, CO, and CO 2 ), as well as the loss of a glucuronic acid (176 Da), hexose residue (162 Da), and apiose residue (132 Da) for flavonoids glycoside [14].
Take compound 11 as an example, its fragment ions at m/z 167.0359, 152.0128, 123.0474, and 108.0251 were identified as vanillic acid hexose. The ion at m/z 167.0359 was obtained by the loss of hexose, while the ion at m/z 123.0474 was produced by continuous loss of CO 2 . Meanwhile, the ion at m/z 152.0128 was obtained by the loss of CH 3 from the precursor ion at m/z 167.0359, while the ion at m/z 108.0251 was produced by continuous loss of CO 2 . Based on the above fragment ions, which was obtained in the MS 2 spectrum, the structure of compound 11 was easily confirmed as vanillic acid hexose.   [14]. This is the first time to report the cyclic peptide from D. dentiger. 3.2. COX-2 Inhibitory Assay. COX-2 is one of the most important proinflammatory enzyme of action for antiinflammatory drugs, and celecoxib was a COX-2 selective inhibitor in clinical practice [2]. As observed in Table 2, the ethanol crude extract of D. dentiger roots showed significant COX-2 inhibitory effect with an IC 50 value of 77:2 ± 4:2 μg/ mL; however, there was an indicated remarkable difference (p < 0:01) in comparison with that of celecoxib with an IC 50 value of 22:4 ± 1:4 ng/mL. To the best of our knowledge, this study was the first time to determine the COX-2 inhibitory activity for D. dentiger [12,13].
3.3. Antioxidant Activity. The DPPH and ABTS free radical scavenging activity assays were mostly used to evaluate the antioxidant effect of natural antioxidants [2]. Hence, the antioxidant activity of the D. dentiger roots ethanol crude extract was evaluated using ABTS and DPPH assays, and the results are shown in Table 2. The ethanol crude extract of D. dentiger roots showed the outstanding antioxidant activity, with IC 50 values of 255:8 ± 10:3 μg/mL for DPPH assay and 151:9 ± 6:5 μg/mL for ABTS assay; however, there were exhibited significant differences (p < 0:01) comparable to those of the positive control V c with IC 50 values of 6:0 ± 0:2 and 1:2 ± 0:1 μg/mL, respectively.
To date, only one paper was reported the antioxidant activity of D. dentiger, and its ethyl acetate and n-butanol fractions showed significant against DPPH free radical scavenging activity [45]. Moreover, 7 phenolic compounds were isolated from the extract of D. dentiger and showed moderate or significant against DPPH free radical scavenging activity, with IC 50 values of 0.038-0.741 μM, comparable to that of V c with an IC 50 value of 0.059 μM [45]. Therefore, this observed antioxidant activity could be due to the greater presence of secondary bioactive metabolites belonging to the flavonoids or phenolics noticed in ethanol crude extract of D. dentiger roots.

Conclusions
To summarize our findings, this study revealed that the root of D. dentiger was rich in phenylpropanoids, alkaloids, and flavonoids by UHPLC-Q-TOF-MS/MS and showed significant anti-inflammatory and antioxidant activities. This is the first study to describe the phytochemical profiling and COX-2 inhibitory activity of this plant [12,13]. This study can provide important chemical information for the application of D. dentiger as a new source of natural COX-2 inhibitors and antioxidants in heath food and pharmaceutical industry.

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

Conflicts of Interest
The authors declare that they have no conflict of interest.

Supplementary Materials
Fig. S1: tandem mass spectra and its fragmentation of chlorogenic acid in negative ion mode. Figure S2: tandem mass spectra and its fragmentation of benzoylhypaconine in positive ion mode. Figure S3: tandem mass spectra and its fragmentation of berberine in positive ion mode. Figure S4: tandem mass spectra and its fragmentation of apigenin in positive ion mode. Figure S5: tandem mass spectra and its fragmentation of rutin in positive ion mode. (Supplementary Materials)