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.

1. 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 [2-6]. Consequently, most medicinal plants belong to higher plants have been widely to treat many human diseases in traditional folk medicine [1, 7–9]. 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.

2. Materials and Methods

2.1. Chemicals and Reagents

The COX-2 inhibitor screening assay kit was purchased from Beyotime Biotechnology (Shanghai, China). 2,2-Diphenyl-1-pircryhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), and celecoxib were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Ascorbic acid (Vc) was purchased from Aladdin (Shanghai, China). Acetonitrile and formic acid (LC-MS grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). HPLC grade water was deionized using a Milli-Q ultrapure water system (Merck Millipore, Milford, MA, USA).

2.2. 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].

2.3. Extraction Procedure

The dried and powdered roots of D. dentiger (10.0 kg) were extracted with 95% EtOH (60L × 3) and subsequently 50% EtOH (60 L ×3) by maceration at room temperature for seven days. All filtrates were combined and evaporated under reduced pressure (EYELA, Tokyo, Japan) to obtain the ethanol crude extract of D. dentiger (DD, 1275 g, 12.75%).

2.4. UHPLC-Q-TOF-MS/MS

The UHPLC-Q-TOF-MS/MS was provided in our previously published article [14]. Chromatographic separation was conducted on a Luna Omega C18 (100 × 2.1mm, 1.6 μm, Phenomenex Inc., CA, USA) keeping at 40°C. 0.1% aqueous formic acid (v/v, A) and acetonitrile (B) were used as mobile phases. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 0-15 min, 25% B; 15-18 min, 25%-55% B; 18-40 min, 55%-95% B; 40-42 min, 95% B for column cleaning, and a conditioning cycle time of 3 min with the same initial conditions of 5% B. The sample inject volume was 3 μL.

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 [2-4]. 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 (Asample). The absorbance of a blank (Ablank) and control (Acontrol) composed of only the sample and COX-2 enzyme solutions was also determined, respectively. The COX − 2inhibitoryactivity(%) = [(Acontrol–Asample)/(Acontrol–Ablank)] × 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 [2-4]. 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 (Asample). The absorbance of a blank (Ablank) and negative control (Acontrol) 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: DPPHscavengingactivity = [1 − (Asample–Ablank)/Acontrol] × 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 K2S2O8 (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 (Asample) was measured. The absorbance of a blank (Ablank) and negative control (Acontrol) 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: ABTSscavengingactivity = [1 − (Asample–Ablank)/Acontrol] × 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 ± standarddeviation (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.

3. Results and Discussion

3.1. 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 [16-44]. 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 used to further confirm the chemical structure. Furthermore, the fragmentation pathways of some representative compounds were proposed in order to facilitate structural identification. Among them, compounds 7, 10, 11, 13-32, 34-39, 41, 43-46, 48, 50-59, 61-65, 67-69, and 71-77 were reported for the first time in the Araliaceae family. Moreover, this is the first report on compounds 1-3, 5, 6, 9, 12, 40, 42, 47, 49, 60, 66, and 78 from the genus Dendropanax and compounds 4, 8, 33, and 70 from Dendropanax dentiger [12, 13].

Table 1

Compounds identified from the roots of D. dentiger by UHPLC-Q-TOF-MS/MS in positive/negative ion mode.

No.	RT (min)	Ion mode	Molecular weight	Measured mass	Error (ppm)	Molecular formula	Fragments	Identification	Reference
Phenylpropanoids
17	5.03	[M-H]−	342.09508	341.08806	0.7	C15H18O9	191.0568a, 179.0351, 173.0458, 135.0467, 93.0375	Caffeoyl hexose	16
18	5.23	[M-H]−	354.09508	353.08806	0.7	C16H18O9	353.0872, 191.0571a, 179.0356, 161.0254	Chlorogenic acid	16
19	5.59	[M-H]−	326.10017	325.09321	1	C15H18O8	176.0497, 163.0405, 145.0302, 119.0524a, 114.0389, 59.0167	Coumaric acid glucoside	17
21	5.91	[M-H]−	180.04226	179.03629	4.3	C9H8O4	135.0461a, 134.0386, 89.0430	Caffeic acid	18
22	6.89	[M-H]−	338.10017	337.0932	0.9	C16H18O8	191.0574a, 173.0460, 163.0410, 119.0529, 93.0385	3-p-COQA	19
26	7.93	[M-H]−	368.11073	367.10358	0.3	C17H20O9	193.0511, 191.0567a, 134.0389, 93.0377	Methyl 4-caffeoylquinate	19
27	8.12	[M-H]−	504.18429	503.17658	-0.9	C22H32O13	503.1761a, 341.1243, 221.0673, 161.0474, 101.0269	Tinoscorside D	20
31	9.56	[M-H]−	742.26842	741.26178	0.9	C34H46O18	417.1571a, 402.1310, 181.0524, 166.0289	Syringaresinol-4,4′-bis-O-β-D-glucopyranoside	21
36	10.77	[M-H]−	472.19446	471.18683	-0.8	C22H32O11	189.0565, 163.0773, 134.0381, 105.0375a, 89.0250, 71.0169	Eugenol rutinoside	22
38	11.00	[M-H]−	624.20542	623.19799	-0.3	C29H36O15	623.1974a, 461.1662, 179.0354, 161.0254a, 135.0459	Verbascoside	23
40	12.49	[M-H]−	580.21559	579.20843	0.2	C28H36O13	417.1550, 402.1323, 387.1019, 181.0516a, 166.0279, 151.0047	Syringaresinol-4′-O-β-D-glucopyranoside	21
42	13.18	[M-H]−	516.12678	515.11912	-0.7	C25H24O12	353.0878, 191.0566, 179.0359, 173.0466a, 161.0255, 135.0467	3,5-di-O-Caffeoylquinic acid	24
50	15.03	[M-H]−	500.13186	499.12418	-0.8	C25H24O11	353.0878, 337.0925, 191.0569, 179.0361, 173.0467a, 135.0463	4-PCO-5-CQA	19
54	16.76	[M-H]−	208.07356	207.06732	5	C11H12O4	161.0258, 135.0466, 133.0313a	Caffeic acid ethyl ester	21
55	17.52	[M-H]−	524.22576	523.21801	-0.9	C26H36O11	361.1659a, 346.1430, 317.1404, 231.0668, 161.0257	Secoisolariciresinol hexose	16
Alkaloids
2	1.51	[M + H]+	135.0545	136.06184	0.5	C5H5N5	136.0627, 119.0356a, 107.0489, 92.0260, 91.0554a, 65.0414	Adenine	18
3	1.54	[M + H]+	181.07389	182.08102	-0.8	C9H11NO3	119.0492, 91.0561a, 77.0414, 65.0423	Tyrosine	25
4	1.59	[M + H]+	267.09675	268.10379	-0.9	C10H13N5O4	136.0622a, 119.0360	Adenosine	18
5	1.65	[M + H]+	283.09167	284.09882	-0.5	C10H13N5O5	152.0569a, 135.0308, 110.0364	Guanosine	25
6	1.71	[M + H]+	131.09463	132.10211	1.5	C6H13NO2	86.0987a, 72.9415, 69.0718, 57.0693, 55.9383, 55.0230	Isoleucine	25
7	1.72	[M + H]+	275.13689	276.14402	-0.5	C12H21NO6	276.1431, 258.1327, 230.1383a, 212.1256, 87.0330	Glutarylcarnitine	26
9	2.55	[M + H]+	165.07898	166.08619	-0.4	C9H11NO2	120.0811, 13.0550, 77.0412a	Phenylalanine	27
23	7.16	[M + H]+	341.16271	342.17031	1	C20H23NO4	342.1718, 297.1133, 282.0899, 265.0867a, 222.0899, 58.0696	Magnoflorine	26
24	7.31	[M + H]+	327.14706	328.15459	0.8	C19H21NO4	328.1557, 178.0862a, 163,0627, 151,0759	Stepholidine	28
37	10.85	[M + H]+	277.11028	278.11756	0	C18H15NO2	278.1183, 263.0948, 220.1129, 204.0813a	Dehydroroemerine	27
44	13.79	[M + H]+	589.2887	590.29653	0.9	C31H43NO10	590.2971a, 572.2870, 558.2670, 540.2608, 508.2340	Benzoylmesaconine	26
48	14.94	[M + H]+	603.30435	604.31125	-0.6	C32H45NO10	604.3141a, 554.2761, 242.1194	Benzoylaconine	26
49	15.00	[M + H]+	335.11576	336.1233	0.8	C20H17NO4	336.1244, 321.1012, 320.0937a, 306.0774, 292.0981, 278.0822	Berberine	26
52	15.83	[M + H]+	573.29378	574.30155	0.9	C31H43NO9	574.3045a, 542.2772, 510.2458, 105.0336	Benzoylhypaconine	26
57	17.95	[M + H]+	615.30435	616.31177	0.2	C33H45NO10	616.3162a, 556.2911, 524.2635, 338.1757, 161.0597	Hypaconitine	26
Flavonoids
25	7.41	[M-H]−	594.15847	593.15094	-0.4	C27H30O15	593.1523, 503.1208, 473.1098, 383.0778, 353.0675a, 297.0772	Apigenin-6,8-di-C-glucoside	17
29	8.66	[M-H]−	564.14791	563.14072	0.2	C26H28O14	563.1414, 545.1304, 503.1196, 473.1092, 443.0980, 383.0781, 353.0676a, 297.0786, 173.0466, 93.0369	Schaftoside	19
30	8.91	[M-H]−	448.10056	447.09339	0.2	C21H20O11	357.0627, 339.0514, 327.0523a, 299.0569, 297.0410, 133.0307	Isoorientin	19
32	10.07	[M-H]−	432.10565	431.09862	0.6	C21H20O10	431.0991, 341.0666, 311.0567, 293.0454, 283.0616a, 161.0247, 117.0365	Vitexin	19
33	10.11	[M-H]−	610.15339	609.14652	0.7	C27H30O16	609.1490, 447.1153, 301.0363, 300.0284a, 271.0259, 255.0310, 161.0249	Rutin	19
34	10.15	[M + H]+	464.09548	465.10317	0.9	C21H20O12	303.0504a, 257.0444, 201.0546, 85.0309	Myricitrin	29
35	10.34	[M-H]−	580.14282	579.13562	0.1	C26H28O15	579.1365, 285.0411a	Luteolin-7-O-xylosyl-glucoside	19
39	11.76	[M-H]−	462.11621	461.10885	-0.2	C22H22O11	461.1063, 299.0560a, 284.0334, 256.0375	5,7,2′-Trihydroxy-6-methoxyflavone	30
45	14.18	[M-H]−	492.12678	491.11956	0.1	C23H24O12	491.1189a, 459.0923, 323.0771, 315.0732, 314.0442, 179.0361, 175.0398, 160.0178, 152.0134, 153.0208, 132.0219, 108.0286	Tricin 5-glucoside/tricin 7-glucoside	17
47	14.88	[M-H]−	446.08491	445.07734	-0.7	C21H18O11	269.0451a	Baicalin	30
53	16.06	[M-H]−	286.04774	285.04067	0.7	C15H10O6	285.0414, 257.0410, 243.0281,217.0511, 201.0205, 187.0389, 175.0420, 151.0053, 133.0311a, 132.0218, 105.0355, 83.0196, 65.0104	Luteolin	17
56	17.84	[M-H]−	300.06339	299.05669	1.9	C16H12O6	284.0313a, 256.0382, 227.0343, 151.0067	Diosmetin	19
61	19.12	[M + H]+	402.13147	403.13883	0.2	C21H22O8	403.1397, 388.1151, 373.0922a, 342.1110	Nobiletin	31
66	20.54	[M-H]−	270.05282	269.04576	0.8	C15H10O5	269.0467a, 241.0515, 225.0568, 213.0563, 197.0617, 181.0660, 171.0458	Apigenin	32
Fatty acids
16	4.98	[M-H]−	176.06847	175.06192	4.1	C7H12O5	115.0411, 113.0637, 85.0689a	Hydroxy-methylglutaric acid	33
58	18.28	[M-H]−	228.13616	227.12906	0.8	C12H20O4	183.1407a, 165.1305	Dihydroxy dodecadienoic acid	34
59	18.40	[M-H]−	329.23349	329.2336	0.8	C18H34O5	329.2329, 229.1451, 211.1348, 209.1191, 171.1038a, 139.1141, 127.1144	Trihydroxy-octadecaenoic acid	35
62	19.55	[M-H]−	310.21441	309.20744	1	C18H30O4	209.1192a, 207.1408, 185.1193, 163.1139, 137.0985, 125.0991, 99.0849, 97.0682, 57.0403	Dihydroxy-octadecatrienoic acid	35
65	20.54	[M-H]−	314.24571	313.23872	0.9	C18H34O4	313.2389, 277.2178, 201.1139a, 199.0981, 171.1029, 127.1142, 125.0980	Dihydroxy-octadecaenoic acid	35
67	20.72	[M-H]−	312.23006	311.22326	1.5	C18H32O4	311.2234, 293.2129, 275.2005, 211.1353, 201.1141, 185.1188, 171.1040a, 139.1145, 127.1155	Dihydroxy-octadecadienoic acid	35
68	21.47	[M + H]+	352.26136	353.26862	-0.1	C21H36O4	261.2204, 187.1461, 145.1020, 131.0859, 107.0870, 93.0718, 81.0721a, 67.0580	Glyceryl linolenate	36
69	23.75	[M-H]−	294.2195	293.21295	2.5	C18H30O3	293.2114a, 249.2220, 197.1180, 185.1186, 125.0981, 113.0987	Hydroxy-octadecatrienoic acid	35
71	28.55	[M + H]+	354.27701	355.28463	1	C21H38O4	337.2750, 263.2377, 245.2258, 175.1478, 161.1327, 147.1159, 109.1017, 95.0862, 81.0720a	Glyceryl linoleate	36
72	29.08	[M-H]−	356.29266	355.28536	-0.1	C21H40O4	355.2758, 355.2918, 293.2837a, 295.0241, 240.9931	Monoolein	22
74	30.78	[M + H]+	330.27701	331.28412	-0.5	C19H38O4	313.2738, 109.1018, 95.0863, 85.1026, 81.0716, 71.0880, 57.0740a	Glycerin palmitate	36
75	37.15	[M-H]−	284.27153	283.2645	0.9	C18H36O2	283.2631a, 282.3573, 265.2527, 199.8494	Tearic acid or its isomer	35
77	40.40	[M-H]−	312.30283	311.29527	-0.9	C20H40O2	311.2948a, 311.1673, 293.2749, 184.0163, 183.0121	Arachidic acid/eicosanoic acid	37
78	43.36	[M-H]−	340.33413	339.32722	1.1	C22H44O2	339.1992, 184.0199, 183.0127a, 119.0516	Behenic acid	22
Phenols
8	1.93	[M-H]−	170.02152	169.0152	4.6	C7H6O5	125.0254a, 124.0202, 97.0341, 79.0239, 69.0410	Gallic acid	38
10	2.80	[M-H]−	316.07943	315.07253	1.2	C13H16O9	153.0201, 152.0127, 108.0246a	3-Carboxy-4-hydroxy-phenoxy glucoside	19
11	2.94	[M-H]−	330.09508	329.08808	0.8	C14H18O9	167.0359, 152.0128a, 123.0474, 108.0251	Vanillic acid hexose	16
12	3.47	[M + H]+	198.05282	199.05998	-0.6	C9H10O5	181.0440, 140.0465, 125.0234a, 97.0297, 77.0406	Syringic acid	25
13	3.50	[M-H]−	360.10565	359.0987	0.9	C15H20O10	197.0465, 182.0234, 167.0000, 138.0344a, 123.0112, 95.0176	Methoxypolygoacetophenoside	33
14	3.82	[M-H]−	256.0583	255.05159	2.2	C11H12O7	225.0399, 196.0402,181.0514, 163.0405, 148.0190a, 135.0469, 120.0222, 109.0389, 95.0205	Piscidic acid	39
28	8.55	[M + H]+	182.05791	183.06503	-0.8	C9H10O4	140.0463, 125.0236, 95.0504, 77.0414a, 65.0421	Syringaldehyde	25
Steroids
63	19.56	[M + HCOO]−	738.41904	783.41695	1	C39H62O13	783.4257, 737.4121a	25(27)-ene-Timosaponin AIII	35
64	19.91	[M + HCOO]−	1050.52469	1095.52283	0.9	C50H82O23	1049.5222a, 917.4771, 887.4673, 593.3684	F-Gitonin	35
73	30.23	[M-H]−	340.24023	339.23303	0.2	C23H32O2	339.2323, 163.1143a	Dimethisterone	35
76	38.29	[M + H]+	412.37052	413.37809	0.7	C29H48O	413.3786, 395.3701, 255.2108, 213.1639, 173.1328, 159.1170a, 145.1017, 109.0658	α-Spinasterol	40
Cyclic peptides
41	12.63	[M + H]+	678.50438	679.51224	0.8	C36H66N6O6	679.5139a, 661.5044, 566.4337, 548.4185, 486.2149, 435.3332, 322.2482, 209.1660, 114.0932	Cyclo hexaleucyl (or isoleucyl)	35
43	13.71	[M + H]+	791.58845	792.59618	0.6	C42H77N7O7	792.5976a, 774.5873, 679.5137, 661.5069, 566.4308, 548.4199, 453.3648, 435.3336, 340.2605, 322.2510, 227.1767, 209.1654	Cyclo hetaleucyl (or isoleucyl)	35
46	14.58	[M + H]+	904.67251	905.68109	1.4	C48H88N8O8	905.6811a, 887.6715, 774.5847, 548.4196, 435.3353	Cyclo octaleucyl (or isoleucyl)	35
51	15.33	[M + H]+	1017.75658	1018.76517	1.3	C54H99N9O9	1018.7684a, 1000.7590, 887.6637	Cyclo nonaleucyl (or isoleucyl)	35
Terpenoids
15	4.70	[M-H]−	376.13695	375.12984	0.5	C16H24O10	375.1307, 213.0777a, 169.0879, 151.0776, 113.0265	Mussaenosidic acid	18
20	5.79	[M-H]−	390.11621	389.10865	-0.7	C16H22O11	389.1098, 345.1181, 209.0459, 183.0691, 165.0554, 139.0781, 121.0672, 95.0526, 69.0405a	Oleoside	41
60	18.59	[M-H]−	822.40379	821.39586	-0.8	C42H62O16	821.3970a, 351.0564	Glycyrrhizin	42
Others
1	0.90	[M-H]−	342.11621	341.10951	1.7	C12H22O11	179.0573, 161.0463, 119.0376, 113.0272, 89.0287, 71.0195, 59.0207a	Sucrose	43
70	24.37	[M + H]+	278.15181	279.1589	-0.7	C16H22O4	149.0233a, 121.0287	Dibutyl phthalate	44

aBase peak. RT: retention time; 3-p-COQA: 3-O-trans-coumaroylquinic acid; 4-PCO-5-CQA: 4-O-feruloyl-5-coumaroylquinic acid.

Figure 1

The base peak chromatograms of the D. dentiger root extract by UPLC-Q-TOF-MS/MS in negative and positive ion modes.

3.1.1. Phenylpropanoids

Phenylpropanoids were widely distributed in medicinal plants and its structures containing one or more C6-C3 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).

Figure 2

Chemical structures of phenylpropanoids from D. dentiger roots.

Compounds 17-19, 21, 22, 26, 27, 36, 38, 42, 50, and 54 were simple phenylpropanoids, while compounds 27 and 36 were phenylpropanol and phenylpropene, respectively. Moreover, compounds 17, 18, 21, 26, 38, 42, 50, and 54 were caffeic acid derivatives, including 4 caffeoylquinic acid derivatives (18, 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, H2O, and CO2 were the major cleavage pathway of such compounds. Taking compound 18 as an example, it gave the same MS2 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 H2O, 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 C6-C3 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.

3.1.2. Alkaloids

In this study, a total of 15 alkaloids (Figure 3) in the roots of D. dentiger extract were identified, including 4 diterpenoid alkaloids (44, 48, 52, and 57), 4 isoquinoline alkaloids (23, 24, 37, and 49), 3 purine alkaloids (2, 4, and 5), 3 amino acid (3, 6, and 9), and 1 other alkaloid (7).

Figure 3

Chemical structures of alkaloids from D. dentiger roots.

Compounds 44, 48, 52, and 57 were diterpenoid alkaloids, which were belonging to aconitum alkaloids. In tandem mass spectrum of aconitum alkaloids commonly observe the neutral losses of H2O (18 Da), MeOH (32 Da), CO2 (44 Da), and PhCOOH (122 Da). Take the case of the 52, it gave fragment ions at m/z 574.3045, 542.2772, 510.2458, and 105.0336 in the positive mode were corresponding to [M + H]+, [M + H–CH3OH]+, [M + H–2CH3OH]+, and [M + H–C24H39NO8]+, respectively. Compared with literature data, compound 52 was identified as benzoylhypaconine, and the possible fragmentation mechanism was depicted in Figure S2.

Compounds 23, 24, 37, and 49 were isoquinoline alkaloids, which were widely distributed in medicinal plants and have high medicinal value. Compound 23 gave fragment ions at m/z 342.1718, 297.1133, 282.0899, and 265.0867 in the positive mode were corresponding to [M + H]+, [M + H–C2H6N]+, [M + H–C2H6N–CH3]+, and [M + H–C2H6N–CH3OH]+, respectively, of which ring B lost a C2H6N by a-cleavage and formed a Cp-ring; then, the ring A lost a methoxy at C-6 and formed an epoxy between C-6 and C-7. The tandem mass pattern of this compound was similar with magnoflorine. Thus, it could be identified as magnoflorine. The ESI–MS spectra of compound 24 exhibited similar quasi-molecular ions peak [M + H]+ at m/z 328.1557; their MS2 generated fragments at m/z 178.0862 and m/z 151.0759 by splitting of RDA on C-ring. Hence, compound 24 was tentatively identified as stepholidine. The [M + H]+ ion of compound 37 at m/z 278.1183 had a similar mass and fragmentation pathway to the dehydroroemerine, according to the characteristic ions at m/z 263.0948 [M + H–CH3]+, m/z 220.1129 [M + H–CH2O–CO]+, and m/z 204.0813 [M + H–CH4–CH2O–CO]+. For compound 49, the positive mode MS spectrum showed the parent ion at m/z 336.1233 [M + H]+, and MS2 spectrum showed the fragment ions at m/z 321.1012 [M + H–CH3]+, 320.0937 [M + H–CH4]+, 306.0774 [M + H–2CH3]+, 292.0981 [M + H–CH4–CO]+, and 278.0822 [M + H–2CH3–CO]+. Compared with literature data, compound 49 was identified as berberine, and the possible fragmentation mechanism was depicted in Figure S3.

Moreover, other 7 alkaloid compounds 2, 3, 4, 5, 6, 7, and 9 were identified as adenine, tyrosine, adenosine, guanosine, isoleucine, glutarylcarnitine, and phenylalanine, respectively. To the best of our knowledge, alkaloid was reported from D. dentiger for the first time.

3.1.3. 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 (CH3, H2O, CO, and CO2), as well as the loss of a glucuronic acid (176 Da), hexose residue (162 Da), and apiose residue (132 Da) for flavonoids glycoside [14].

In this study, 5 flavonoids (39, 53, 56, 61, and 66) and 9 flavonoid glycosides (25, 29, 30, 32, 33, 34, 35, 45, and 47) in the roots of D. dentiger extract were identified based on the molecular weight and fragmentation information (Figure 4). Compounds 39, 53, 56, 61, and 66 were belonging to flavonoids, which considered as 5,7,2′-trihydroxy-6-methoxyflavone, luteolin, diosmetin, nobiletin, and apigenin, respectively. The MS2 spectrum of 66 shown in Figure S4 was a representative example, which showed a [M–H]– ion at m/z 269.0467, in accordance with the elemental composition of C15H10O5–.

Figure 4

Chemical structures of flavonoids from D. dentiger roots.

Compounds 25, 29, 30, 32, 33, 34, 35, 45, and 47 were flavonoid glycosides, which considered as apigenin-6,8-di-C-glucoside, schaftoside, isoorientin, vitexin, rutin, myricitrin, luteolin-7-O-xylosyl-glucoside, tricin 5-glucoside, and baicalin, respectively. Compound 29 was C-glycosides, which the disaccharide substitution continuously loses 60, 90, and 120 Da fragment ions. Compound 29 showed a [M–H]– ion at m/z 563.1414, C-6 substituted hexose broke up in 0,4X0, 0,3X0, and 0,2X0 to obtain 503.1196, 473.1092, and 443.0980 fragment ions, respectively, after that C-8 site pentose fractured at 0,3X1 and 0,2X1 to get 383.0781 and 353.0676, because the C-6 substituent glycosyl groups were superior to the C-8 replacement fracture. According to the characteristics of the fragment ions, compound 29 was easily confirmed as schaftoside. However, compound 33 was O-glycosides, which typically lost the entire sugar neutral molecule with significant loss of 132, 146, 162, and 192 Da fragments. Compound 33 [M−H]−609.1490 was rutin, which could be detected aglycone ion [Y0]−301.0363 and radical aglycone ion [Y0 − H]−300.0284 after losing carbohydrate continuously in the negative ion mode, and the possible fragmentation mechanism was depicted in Figure S5.

3.1.4. Fatty Acids

In our study, a total of 14 fatty acids (peaks 16, 58-59, 62, 65, 67-69, 71, 72, 74, 75, 77, and 78) were identified based on the reference mass spectra and databases.

3.1.5. Phenols

A total of 7 phenols were identified in this study. Compounds 8, 10, 11, 12, 13, 14, and 28 were considered as gallic acid, 3-carboxy-4-hydroxy-phenoxy glucoside, vanillic acid hexose, syringic acid, methoxypolygoacetophenoside, piscidic acid, and syringaldehyde, respectively.

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 CO2. Meanwhile, the ion at m/z 152.0128 was obtained by the loss of CH3 from the precursor ion at m/z 167.0359, while the ion at m/z 108.0251 was produced by continuous loss of CO2. Based on the above fragment ions, which was obtained in the MS2 spectrum, the structure of compound 11 was easily confirmed as vanillic acid hexose.

3.1.6. Steroids

Four steroids (peaks 63, 64, 73, and 76) were identified in this study. Peaks 63 and 64 generated [M + HCOO]− ions at m/z 783.41695 and 1095.52283 in negative mode were unequivocally determined to be 25(27)-ene-timosaponin AIII and F-gitonin by comparison with the reference data. Peak 73 produced [M−H]− ions at m/z 339.2323 in ESI− mode. By comparing the quasi-molecular ions and fragmentations with MassBank and reference mass spectra data, peak 73 was tentatively identified as dimethisterone. Compound 76 had [M + H]+ ion at m/z 413.37809, and its fragments were at m/z 395.3701 [M + H–H2O]+, 255.2108 [M + H–C10H20–H2O]+, 213.1639 [M + H–C10H20–H2O–C3H4]+, 173.1328 [M + H–C10H20–H2O–2C3H4]+, and 159.1170 [M + H–C10H20–H2O–2C3H4–CH2]+, and was identified as α-spinasterol.

3.1.7. Cyclic Peptides

A total of 4 cyclic peptides (peaks 41, 43, 46, and 51) were identified in this study, and the compounds 41, 43, 46, and 51 showed [M + H]+ ion at m/z 679.51224, 792.59618, 905.68109, and 1018.76517, respectively. They have similar fragmentation pathways [14]. This is the first time to report the cyclic peptide from D. dentiger.

3.1.8. Terpenoids

In current work, 3 terpenoids (peaks 15, 20, and 60) were identified in negative ion mode.

Compound 15 had [M–H]− ion at m/z 375.1307, and its fragments were at m/z 213.0777 [M–H–Glc]–, 169.0879 [M–H–Glc–CO2]–, and 151.0776 [M–H–Glc–CO2–H2O]–. Its fragmentation process was the same as the literature. Therefore, compound 15 was identified as mussaenosidic acid. Compound 20 exhibited a pseudomolecular ion at m/z 389.1098 [M–H]− and fragment ions at m/z 345.1181 [M–H–CO2]− corresponding to decarboxylation and m/z 165.0554 [M–H–CO2–C6H12O6]− corresponding to the cleavage of elenolic acid moieties. Meanwhile, fragment ion at m/z 69.0405 was corresponding to the propiolic acid. Compound 20 was tentatively identified as oleoside. Compound 60 showed [M–H]− ion at m/z 821.3970 and the fragment ions at m/z 351.0564 were corresponding to [2GluA–H2O]−, which the fragmentation pathways were similar with glycyrrhizin.

3.1.9. Others

Compounds 1 and 70 were given [M + H]+ ions at m/z 341.10951 and 279.1589 and identified as sucrose and dibutyl phthalate, respectively, by comparing with literature.

3.2. COX-2 Inhibitory Assay

COX-2 is one of the most important proinflammatory enzyme of action for anti-inflammatory 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 IC50 value of 77.2 ± 4.2μg/mL; however, there was an indicated remarkable difference (p < 0.01) in comparison with that of celecoxib with an IC50 value of 22.4 ± 1.4ng/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].

Table 2

IC50 values of D. dentiger root extract (DDR) and standards in COX-2, DPPH, and ABTS assays.

Samples	IC50 (μg/mL)
	COX-2 assay	DPPH assay	ABTS assay
DDR	77.2 ± 4.2∗∗	255.8 ± 10.3∗∗	151.9 ± 6.5∗∗
Celecoxiba	(22.4 ± 1.4) × 10−3	NT	NT
Vca	NT	6.0 ± 0.2	1.2 ± 0.1

aPositive drug. NT: not tested. Data are shown as mean ± SD (n = 3). Differences were analyzed using ANOVA by Tukey's test. ∗∗p < 0.01 compared with the positive drug.

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 IC50 values of 255.8 ± 10.3μg/mL for DPPH assay and 151.9 ± 6.5μg/mL for ABTS assay; however, there were exhibited significant differences (p < 0.01) comparable to those of the positive control Vc with IC50 values of 6.0 ± 0.2 and 1.2 ± 0.1μg/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 IC50 values of 0.038-0.741 μM, comparable to that of Vc with an IC50 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.

4. 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.