Rapid identification of chemical profile in Gandou decoction by UPLC-Q-TOF-MS^E coupled with novel informatics UNIFI platform

Abstract

Gandou decoction (GDD), a well-known traditional Chinese medicine (TCM) formula, has been widely used for decades to treat Wilson's disease (WD) in China due to its remarkable clinical effects. However, the chemical constituents of GDD still remain unclear because of their complexity. In this work, a reliable and sensitive strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS^E) and UNIFI informatics platform was applied to investigate the chemical components in GDD. In total, 96 compounds including anthraquinones, alkaloids, protostane triterpenoids, flavonoids, triterpenoid saponins, tannins, curcuminoids, etc, were identified or tentatively characterized from GDD by comparing their retention time, accurate mass within 5 ppm error and MS^E fragmentation patterns. Among them, eleven compounds were confirmed unambiguously with reference standards. Representative compounds in different chemical structure types were analyzed in fragmentation patterns and characteristic ions. Moreover, to better understand the chemical contribution of individual herbs to the whole decoction, the corresponding each herb in GDD was also detected. This study developed a rapid method for characterizing the chemical constituents in GDD, which could not only be used for chemical standardization and quality control, but also be helpful for further research of GDD in vivo.

1. Introduction

Traditional Chinese medicines (TCM) have been extensively used for the prevention and treatment of complex and chronic diseases in China [1,2]. TCM formulae, combination of medicinal plants or animal materials, collectively exert therapeutic actions by complex interactions among multiple components from different herbal medicines. Based on TCM theories, these constituents in formulae could play a multi-target, synergistic and harmless therapeutic role [3]. As the components in TCM are rather complicated, it is difficult to separate and identify multiple chemical constituents. Therefore, developing a rapid and reliable method for elucidating the composition of TCM is necessary.

Wilson's disease (WD), also known as hepatolenticular degeneration, is an autosomal recessive genetic disorder of copper metabolism caused by ATP7B gene mutation [4,5]. Excessive copper accumulation in patients suffering from WD leads to liver disease, neurological disorder, K-F rings, and osteoporosis [6]. Currently, there are several chelating agents such as d-penicillamine, dimercaptosuccinic acid, trientine, and tetrathiomolybdate for medical therapy [7]. Although Western conventional medications are highly effective, prevalent, and low-priced, a number of side effects have been observed with chelation therapy [8]. Gandou decoction (GDD), a classical TCM formula, has been used in clinics to treat WD for decades in China [9,10]. It is composed of six crude drugs, i.e., Rheum palmatum L. (Da-Huang), Coptis chinensis Franch. (Huang-Lian), Curcuma longa L. (Jiang-Huang), Lysimachia christinae Hance (Jin-Qian-Cao), Alisma orientale (Sam.) Juzep. (Ze-Xie) and Panax notoginseng (Burk.) F. H. Chen (San-Qi). The clinical studies have been proven that GDD can promote urinary copper excretion, ameliorate liver function and improve the patient's clinical symptoms [7,11]. Furthermore, GDD appears to be safe, effective, and well tolerated and has fewer adverse effects than Western conventional medications [12]. In our previous studies, we investigated the therapeutic effect and serum metabolic profiling of GDD in copper-laden rats. It was found that GDD could reduce the hepatic copper accumulation, and improve liver pathological characteristics by restoring the impaired lipid metabolism, amino metabolism and glucose metabolism [13]. However, due to multi-component systems of TCM, the chemical constituents of GDD still remain unclear. Therefore, a systematic chemical profiling research of GDD is in an urgent need.

In recent years, UPLC-Q-TOF-MS^E (where E represents collision energy) has provided a powerful approach for the efficient separation and structural characterization of TCM with the advantage of its high resolution, sensitivity and accuracy [14]. Q-TOF-MS^E capable of simultaneously acquiring accurate mass precursor ion in MS full scan and fragment ions in MS^E high-energy scan increased the credibility of analysis results [15,16]. Additionally, UNIFI software from Waters Corporation is a versatile and automated data processing platform. The software incorporates scientific library into a streamlined workflow to integrate data acquisition, library searching, MS fragment matching and report generation, which alleviates the workload from massive MS data and realizes rapid analysis of chemical components [17]. This high throughput strategy was innovatively used for screening and identification of chemical components in herbal medicines [18,19] and TCM formulae [20]. In the present study, an integrative strategy based on UPLC-Q-TOF-MS^E coupled with UNIFI informatics platform has been applied to reveal the chemical profile of GDD. The aim of this study is to develop an analytical method for elucidating the material basis of GDD and provide valuable information for the quality control and in vivo analysis.

2. Material and methods

2.1. Materials and reagents

Rheum palmatum L., Coptis chinensis Franch., Curcuma longa L., Lysimachia christinae Hance, Alisma orientale (Sam.) Juzep. and Panax notoginseng (Burk.) F. H. Chen were purchased from Beijing Tongrentang Co., Ltd. (Hefei, China) and authenticated by Doctor Rongchun Han (College of Pharmacy, Anhui University of Chinese Medicine, Hefei, China). All voucher specimens were deposited at the authors' laboratory. The reference standards, including berberine hydrochloride, physcion, emodin, alisol B 23-acetate, quercetin and notoginsenoside R₁, were obtained from the National Institutes for Food and Drug Control (Beijing, China). Chrysophanol, rhein, aloe-emodin and kaempferide were obtained from Beina Chuanglian Biotechnology Research Institute (Beijing, China). Curcumin was isolated in our laboratory with a purity of more than 98% by HPLC, and its structure and molecular weigh have been identified by using several spectral analyses and MS, respectively. Acetonitrile and methanol (LC-MS grade) were purchased from TEDIA (Fairfield, USA). Formic acid was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Ultrapure water was purified using a Milli-Q water purification system (Millipore, Billerica, MA, USA).

2.2. Standards and sample preparation

GDD consisted of six ingredients, including Rheum palmatum L. (20.0 g), Coptis chinensis Franch (20.0 g), Curcuma longa L. (20.0 g), Lysimachia christinae Hance (24.0 g), Alisma orientale (Sam.) Juzep. (24.0 g) and Panax notoginseng (Burk.) F. H. Chen (3.0 g). They were mixed together and immersed in 0.8 L distilled water (1:8, w/v) for 0.5 h. Afterwards, they were decocted twice by extracting and refluxing for 1 h each time. Finally, the two extractions were combined and concentrated to 1.0 g crude drug per milliliter, and then the solution was freeze-dried and stored in a vacuum desiccator before use. The accurately weighed 1.0 g freeze-dried powder was dispersed in 30 mL methanol and ultrasonicated in a water bath for 30 min to prepare solutions. The individual preparation of six herbs was carried out according to the same procedures as that of GDD. An aliquot of 5 μL filtrate was injected into the UPLC-Q-TOF-MS^E system for analysis after filtered through 0.22 μm filter membrane.

11 reference standards were dissolved in methanol. Before qualitative analysis, they were mixed together to make reasonable concentration and filtered through 0.22 μm filter membrane.

2.3. Chromatography and mass spectrometry conditions

Chromatographic analysis was performed using a Waters Acquity™ UPLC system (Waters Corporation, Milford, USA). Chromatographic separation was carried out at 30 °C, using an Agilent Eclipse Plus C18 RRHD column (2.1 mm × 100 mm, 1.8 μm) with mobile phases A (0.1% formic acid in water) and B (acetonitrile). The flow rate was set at 0.3 mL/min. The gradient profile was as follows: 0–1 min, 10%–10% B; 1–4 min, 10%–20% B; 4–10 min, 20%–30% B; 10–15 min, 30%–40% B; 15–18 min, 40%–50% B; 18–23 min, 50%–75% B; 23–25 min, 75%–85% B; 25–27 min, 85%–100% B.

Mass spectrometric detection was carried out on Waters Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, USA) equipped with an ESI source. The full scan data were acquired from 50 to 1200 Da, using a capillary voltage of 3.0 kV for positive ion mode and −2.5 kV for negative ion mode, sampling cone voltage of 40 V for positive ion mode and 50 V for negative ion mode, extraction cone voltage of 4.0 V, source temperature of 120 °C (ESI⁺) or 110 °C (ESI⁻), cone gas flow of 50 L/h, desolvation gas (N₂) flow of 600 L/h and desolvation gas temperature of 350 °C. The collision voltage was set as 6.0 eV for low-energy scan and 20–80 eV for high-energy scan. Data were centroided and mass was corrected during acquisition using an external reference (Lock-Spray™) consisting of a 200 pg/mL solution of leucineenk ephalin infused at a flow rate of 10 μL/min via a lockspray interface, generating a realtime reference ion of [M+H]⁺ (m/z 556.2771) in positive ion mode and [M−H]⁻ (m/z 554.2615) in negative ion mode to ensure accurate MS analysis. All data collected in centroid mode were obtained and used to calculate the accurate mass and composition of relative target ions with MassLynx™ V4.1 software (Waters).

2.4. Establishment of a chemical compounds library of GDD

The systematic information on chemical compounds isolated from the six individual herbs in GDD was collected and sorted out by retrieving databases such as China Journals of Full-text database (CNKI), Medline, PubMed, Web of Science and ChemSpider. A self-building library of chemical compounds was established by UNIFI software, including compound name, molecular formula, chemical structure, and accurate molecular mass. Among them, the information of 356 compounds is listed in Table S1.

2.5. Data analysis by UNIFI platform

All MS data analysis was processed on the platform of UNIFI software. Minimum peak area of 200 was set for 2D peak detection. The peaks intensity of high energy over 80 counts and the peak intensity of low energy over 200 counts were the selected parameters in 3D peak detection. A margin of error up to 5 ppm for identified compounds was allowed and the matching compounds would be generated predicted fragments from structure. We selected positive adducts including H⁺, Na⁺ and negative adducts containing HCOO⁻ and H⁻. They were allowed cross adduct combinations.

3. Results and discussion

3.1. Identification and characterization of chemical compounds

The high resolution MS data of GDD were quickly acquired by UPLC-Q-TOF-MS^E method. The base peak intensity (BPI) chromatograms of GDD in positive and negative ion modes are depicted in Fig. 1. The UNIFI screening platform was utilized to process and analyze the MS data, and then automatically matched the fragment information. After further manual verification, a total of 96 compounds were identified or tentatively characterized in GDD, including 21 anthraquinones, 14 alkaloids, 17 protostane triterpenoids, 10 flavonoids, 8 triterpenoid saponins, 10 tannins, 4 curcuminoids and 12 others. The detailed MS information of these components is summarized in Table 1. Meanwhile, the chemical structures were confirmed based on accurate mass, MS^E data and related literatures. The structures of main chemical constituents in GDD are shown in Fig. 2.

Fig. 1.

The base peak intensity (BPI) chromatograms of GDD from UPLC-Q-TOF-MS^E analysis. (A) Negative scan; (B) Positive scan.

Table 1.

Identification of chemical constituents of GDD by UPLC-Q-TOF-MS^E.

No.	tR (min)	Idenfitication	Neutral mass (Da)	Formular	Observed neutral mass (Da)	Experimental mass (m/z)	Error (ppm)	MS and MSE data (+ or −) (m/z)	Source
1	0.98	Gallic acid-O-glc	332.0743	C13H16O10	332.0743	331.0669	-0.5	331.0669[M−H]− 169.0141[M−H-Glc]−	a
2	1.05	Gallic acid	170.0215	C7H6O5	170.0214	169.0141	-0.8	169.0141[M−H]− 125.0244[M−H-CO2]−	a
3	1.39	Progallin A	198.0528	C9H10O5	198.0528	197.0455	-0.7	197.0455[M−H]− 124.0455[M−H-2CH2-COOH]−	a
4	1.50	(+)-Catechin-5-O-β-D-glc	452.1319	C21H24O11	452.1318	451.1245	-0.7	451.1245[M−H]− 289.0717[M−H-Glc]−	a
5	1.71	Procyanidin B	578.1424	C30H26O12	578.1432	577.1359	1.4	577.1359[M−H]− 425.0873[M–H-C7H4O4]− 407.0763[M–H-C7H4O4-H2O]− 289.0712[M−H-catechin]−	a
6	2.71	Chlorogenic acid	354.0951	C16H18O9	354.0962	353.0889	3.1	353.0889[M−H]− 191.0899[M-C9H6O3]−	b,d
7	2.82	(-)-Epicatechin	290.0790	C15H14O6	290.0797	289.0724	2.3	289.0724[M−H]− 245.0821[M−H-CO2]−	a
8	3.16	Isocorydine	341.1627	C20H23NO4	341.1628	342.1707	0.1	342.1707[M+H]+ 296.1132[M+H-CH3-CH3O]+	b
9	3.19	Procyanidin B-O-gallate	730.1534	C37H30O16	730.1544	729.1471	1.3	729.1471[M−H]− 577.1350[M−H-gallate]−	a
10	3.20	Magnoflorine	342.1705	C20H24NO4	342.1730	342.1701	-1.2	342.1701[M]+ 297.1123[M-(CH3)2NH]+ 282.0876[M-(CH3)2NH-CH3]+ 265.0861[M-(CH3)2NH-CH3OH]+ 237.0908[M-(CH3)2NH-CH3OH-CO]+	b
11	3.42	Isoboldine	327.1471	C19H21NO4	327.1479	328.1552	2.7	328.1552[M+H]+ 297.1123[M+H-CH3O]+	b
12	3.79	Isovitexin	432.1056	C21H20O10	432.1065	477.1047	1.8	477.1047[M+HCOO]− 431.0982[M−H]− 311.0567[M−H-CO2-C3H8O2]−	d
13	4.75	Tetradehydroscoulerine	322.1079	C19H16NO4	322.1055	322.1052	-6.6	322.1052[M]+ 307.0839[M-CH3]+ 279.0511[M-CH3-CO]+	b
14	4.79	Procyanidin B-5,3,3'-di-O -gallate	882.1643	C44H34O20	882.1685	881.1612	4.8	881.1612[M−H]− 729.1471[M−H-gallate]− 711.0735[M−H-gallate-H2O]− 559.1341[M−H-2gallate-H2O]−	a
15	4.86	Emodin-8-O-β-D-glc	432.1056	C21H20O10	432.1065	431.0992	1.9	431.0992[M−H]− 269.0461[M−H-Glc]− 241.0493[M−H-Glc-CO]− 225.0554[M−H-Glc-CO-O]−	a
16	4.88	Rutin	610.1534	C27H30O16	610.1555	609.1483	3.6	609.1483[M−H]− 463.0815[M−H-Rha]− 301.0487[M−H-Rha-Glc]−	a,d
17	5.09	Rhein-1-O-β-D-glc	446.0849	C21H18O11	446.0860	445.0787	2.4	445.0787[M−H]− 283.0256[M−H-Glc]− 239.0356[M−H-Glc-CO2]− 211.0403[M−H-Glc-CO2-CO]−	a
18	5.11	Isolindleyin or lindleyin	478.1475	C23H26O11	478.1483	477.1410	1.7	477.1410[M−H]− 313.0552[M−H-C10H12O2]− 169.0145[M−H-C16H20O6]−	d
19	5.14	(+)-Catechin-3-O-gallate	442.0900	C22H18O10	442.0910	441.0838	2.4	441.0838[M−H]− 289.0722[M−H-C7H4O4]− 165.1245[M−H-C14H12O6]−	a
20	5.20	Ferulic acid-O-β-D-glc	356.1107	C16H20O9	356.1113	355.1040	1.5	355.1040[M−H]− 193.1033[M−H-Glc]−	d
21	5.25	Isoquercitrin	464.0955	C21H20O12	464.0972	463.0899	3.7	463.0899[M−H]− 301.0038[M−H-C6H10O5]−	d
22	5.45	Isolindleyin or lindleyin	478.1475	C23H26O11	478.1482	477.1408	1.6	477.1408[M−H]− 313.0551[M−H-C10H12O2]− 169.0144[M−H-C16H20O6]−	a
23	5.61	Kaempferol-3-O-rutinoside	594.1585	C27H30O15	594.1599	593.1521	1.6	593.1521[M−H]− 447.1591[M−H-Rha]− 285.0832[M−H-Rha-Glc]−	d
24	5.65	Resveratrol-4'-O-β-D-(2''-O-galloyl)-glc	542.1424	C27H26O12	542.1431	541.1333	1.2	541.1333[M−H]− 313.0555[M−H-Res-H2O]−	a
25	5.70	Sennoside C or D	848.2164	C42H40O19	848.2177	847.2104	1.5	847.2104[M−H]− 685.1557[M−H-Glc]−	a
26	5.89	Tetrahydropalmatine	355.1784	C21H25NO4	355.1792	356.1865	2.4	356.1865[M+H]+ 192.1019[M+H-C10H12O2]+	b
27	6.24	Sennoside A or B	862.1956	C42H38O20	862.1981	861.1908	2.8	861.1908[M−H]− 699.1358[M−H-Glc]− 655.1880[M−H-Glc-CO2]− 386.1002[M−H-Glc-CO2-C15H9O5]−	a
28	6.36	Homoorientin	448.1006	C21H20O11	448.1000	447.0927	-1.2	447.0927[M−H]−	d
29	6.52	Thalifendine	322.1079	C19H16NO4	322.1069	322.1082	-0.4	322.1082[M]+ 279.0889[M-CH3-CO]+	b
30	6.94	Hesperetin-5-O-glc	464.1319	C22H24O11	464.1313	463.1240	1.5	463.1240[M−H]− 301.1131[M−H-Glc]−	d
31	7.01	Coptisine	320.0923	C19H14NO4	320.0921	320.0921	0.6	320.0921[M]+ 292.0970[M-CO]+	b
32	7.08	Rhein-8-O-β-D-(6"-O-acetyl)-glc	488.0955	C23H20O12	488.0949	487.0876	1.5	487.0876[M−H]− 283.0246[M−H-acetyl-Glc]− 239.0347[M−H-acetyl-Glc-CO2]−	a
33	7.15	Jatrorrhizine	338.1392	C20H20NO4	338.1384	338.1384	2.4	338.1384[M]+ 322.0997[M-CH3-H]+ 308.1309[M-2CH3]+	b
34	7.35	Epiberberine	336.1236	C20H18NO4	336.1250	336.1250	-4.2	336.1250[M]+ 320.0935[M-CH3-H]+ 292.1314[M-CH3-H-CO]+	b
35	7.47	Aloe emodin-8-O-β-D-glc	432.1056	C21H20O10	432.1068	431.0992	0.8	431.0992[M−H]− 269.0385[M−H-Glc]− 225.0562[M−H-Glc-CO2]−	a
36	7.48	Tetrahydroberberine	339.1471	C20H21NO4	339.1466	340.1544	-1.4	340.1544[M+H]+ 176.0705[M+H-C10H12O2]+	b
37g	7.72	Notoginsenoside R1	932.5345	C47H80O18	932.5372	977.5354	2.8	977.5354[M+HCOO]− 931.5297[M−H]− 799.4856[M−H-Xyl]− 637.4314[M−H-Xyl-Glc]− 475.3782[M−H-Xyl-2Glc]−	f
38	8.09	Worenine	334.1079	C20H16NO4	334.1080	334.1080	-0.3	334.1080[M]+ 320.1093[M-CH2]+ 318.1092[M-CH2-2H]+ 292.0969[M-CH2-CO]+	b
39g	8.46	Berberine	336.1236	C20H18NO4	336.1230	336.1231	1.8	336.1231[M]+ 321.0983[M-CH3]+ 320.0918[M-CH3-H]+ 306.0763[M-2CH3]+ 292.0969[M-CH3-H-CO]+ 278.0813[M-2CH3-CO]+	b
40	8.51	Palmatine	352.1549	C21H22NO4	352.1543	352.1538	-1.7	352.1538[M]+ 337.1430[M-CH3]+ 336.0983[M-CH3-H]+ 322.1068 [M-2CH3]+ 308.1275 [M-CH3-H-CO]+	b
41	8.53	Ginsenoside Rg1	800.4922	C42H72O14	800.4939	845.4921	2.0	845.4921[M+HCOO]− 799.4863[M−H]− 637.4321[M−H-Glc]− 475.3784[M−H-2Glc]−	f
42	9.23	Kaempferol	286.0477	C15H10O6	286.0479	285.0406	0.7	285.0406[M−H]− 229.0451[M−H-C2O2]− 169.0141[M−H-C3O5]−	a,d
43g	9.46	Quercetin	302.0427	C15H10O7	302.0432	301.0360	2.0	301.0360[M−H]− 151.0451[M−H-C8H6O3]− 121.0431[M−H-C8H6O3-CH2O]− 107.0162[M−H-C8H6O3-CH2O-CH2]−	d
44	9.61	Aloin	418.1264	C21H22O9	418.1267	417.1195	0.8	417.1195[M−H]− 255.0666[M−H-Glc]− 227.0718[M−H-Glc-CO]−	a
45	9.76	Aloe emodin-1-O-β-D-glc	432.1056	C21H20O10	432.1068	431.0995	2.7	431.0995[M−H]− 269.0452[M−H-Glc]− 225.0562[M−H-Glc-CO2]−	a
46	9.91	Aloe emodin-ω-O-β-D-glc	432.1056	C21H20O10	432.1068	431.0995	2.7	431.0995[M−H]− 269.0463[M−H-Glc]− 225.0560[M−H-Glc-CO2]−	a
47	10.09	Chrysophanol-1-O-β-D-glc	416.1107	C21H20O9	416.1118	415.1045	2.6	415.1045[M−H]− 253.0515[M−H-Glc]− 225.0563[M−H-Glc-CO]−	a
48	10.91	Aloe emodin-8-(6"-O-malonyl)-glc	518.1060	C24H22O13	518.1076	517.1003	3.1	517.1003[M−H]− 473.1097[M−H-CO2]− 269.0463[M−H-malonyl-Glc]−	a
49	10.95	Calebin-A	384.1209	C21H20O7	384.1211	429.1193	0.5	429.1193[M+HCOO]−	c
50	11.16	Naringenin	272.0685	C15H12O5	272.0689	271.0616	1.5	271.0616[M−H]− 151.0665[M−H-C8H8O]−	d
51	11.25	Aloe emodin-8-O-β-D-(6"-O-acetyl)-glc	474.1162	C23H22O11	474.1169	473.1096	1.5	473.1096[M−H]− 269.0451[M−H-acetyl-Glc]− 240.0410[M−H-acetyl-Glc-CHO]−	a
52	11.77	Physcion-8-O-β-D-glc	446.1213	C22H22O10	446.1219	445.1146	1.3	445.1146[M−H]− 283.0617[M−H-Glc]− 240.0433[M−H-Glc-CO-CH3]−	a
53	11.85	Chysophanol-8-O-β-D-(6"-O-acetyl)-glc	458.1213	C23H22O10	458.1221	457.1148	1.8	457.1148[M−H]− 253.0511[M−H-acetyl-Glc]− 225.0561[M−H-acetyl-Glc-CO]−	a
54	12.18	Notoginsenoside R2	770.4816	C41H70O13	770.4843	815.4825	3.3	815.4825[M+HCOO]− 769.4768[M−H]− 637.4335[M−H-Xyl]− 475.3786[M−H-Xyl-Glc]−	f
55	12.53	Ginsenoside Rb1	1108.6029	C54H92O23	1108.6105	1107.5967	6.6	1107.5967[M−H]− 945.5469[M−H-Glc]− 783.4917[M−H-2Glc]− 621.4361[M−H-3Glc]− 459.3824[M−H-4Glc]−	f
56	12.69	Cyclocurcumin	368.1260	C21H20O6	368.1258	367.1186	-0.4	367.1186[M−H]− 175.0401[M−H-C11H12O3]−	c
57	12.74	Ginsenoside Rg2	784.4973	C42H72O13	784.4999	829.4982	3.2	829.4982[M+HCOO]− 783.4922[M−H]− 637.4326[M−H-Rha]− 475.3789[M−H-Rha-Glc]−	f
58	12.94	Ginsenoside Rh1	638.4394	C36H62O9	638.4414	683.4382	2.9	683.4382[M+HCOO]− 637.4321[M−H]− 475.3784[M−H-Glc]−	f
59	13.18	6-Methyl-rhein	298.0477	C16H10O6	298.0481	297.0408	1.1	297.0408[M−H]− 283.0251[M−H-CH2]− 239.0358[M−H-CH2-CO2]−	a
60	13.21	Physcion-8-O-β-D-(6"-O-acetyl)-glc	488.1319	C24H24O11	488.1323	487.1250	0.8	487.1250[M−H]− 283.0615[M−H-acetyl-Glc]− 255.0317[M−H-acetyl-Glc-CO]− 240.0432[M−H-acetyl-Glc-CO-CH3]−	a
61	13.51	Orientalol A	254.1882	C15H26O3	254.1907	299.1889	8.3	299.1889[M+HCOO]− 253.1905[M−H]−	c
62	14.13	16-Oxo-alisol A	504.3451	C30H48O6	504.3474	505.3223	4.3	505.3223[M+H]+ 487.3327[M+H-H2O]+ 469.3285[M+H-2H2O]+ 451.3318[M+H-3H2O]+ 415.2824[M+H-C4H10O2]+ 397.2783[M+H-C4H10O2-H2O]+	e
63	14.41	Ginsenoside Rd	946.5501	C48H82O18	946.5581	991.5563	8.0	991.5563[M+HCOO]− 945.5563[M−H]− 783.4939[M−H-Glc]− 621.4398[M−H-2Glc]− 459.3851[M−H-3Glc]−	f
64g	14.52	Aloe emodin	270.0528	C15H10O5	270.0536	269.0462	3.0	269.0462[M−H]− 240.0421[M−H-CHO]− 211.0410[M−H-2CHO]−	a
65g	15.30	Rhein	284.0321	C15H8O6	284.0332	283.0256	4.0	283.0256[M−H]− 239.0356[M−H-CO2]− 211.0403[M−H-CO2-CO]− 183.0439[M−H-CO2-CO-CO]−	a
66	16.01	Notoginsenoside R3	962.5450	C48H82O19	962.5491	961.5418	4.2	961.5418[M−H]− 799.4889[M−H-Glc]− 637.4360[M−H-2Glc]− 475.3817[M−H-3Glc]−	f
67	16.70	8-Oxoberberine	351.1107	C20H17NO5	351.1108	352.1181	0.4	352.1181[M+H]+ 337.0948[M+H-CH3]+ 294.0764[M+H-2CH3-CO]+	b
68	16.72	Alisol I	454.3447	C30H46O3	454.3460	455.3526	2.6	455.3526[M+H]+ 383.2943[M+H-C4H8O]+	e
69	17.05	Alisol C	486.3345	C30H46O5	486.3368	487.3418	4.2	487.3418[M+H]+ 469.3265[M+H-H2O]+ 451.3215[M+H-2H2O]+ 415.2841[M+H-C4H8O]+ 397.2746[M+H-C4H10O2]+	e
70	17.24	Ar-turmerone	216.1514	C15H20O	216.1516	217.1588	0.7	217.1588[M+H]+ 120.0935[M+H-C6H9O]+ 92.06212[M+H-C8H13O]+	c
71g	17.34	Kaempferide	300.0634	C16H12O6	300.0628	299.0553	-2.1	299.0553[M−H]− 284.0489[M−H-CH3]−	d
72	17.46	Bisdemethoxycurcumin	308.1049	C19H16O4	308.1058	307.0984	2.9	307.0984[M−H]− 187.0406[M−H-C8H8O]−	c
73	17.93	Demethoxycurcumin	338.1154	C20H18O5	338.1161	337.1088	2.0	337.1088[M−H]− 217.0508[M−H-C8H8O]−	c
74g	18.38	Curcumin	368.1260	C21H20O6	368.1260	367.1192	-0.1	367.1192[M−H]− 217.0524[M−H-C9H10O2]−	c
75	18.94	Alisol M 23-acetate	488.3502	C30H48O5	488.3497	489.3480	-0.8	489.3480[M+H]+ 471.2696[M+H-H2O]+	e
76g	19.25	Emodin	270.0528	C15H10O5	270.0533	269.0460	1.7	269.0460[M−H]− 241.0507[M−H-CO]− 225.0559[M−H-CO-O]−	a
77	19.54	Bisabolone oxide A	236.1776	C15H24O2	236.1776	237.1850	0.5	237.1850[M+H]+	c
78	19.65	Alisol C 23-acetate	528.3451	C32H48O6	528.3453	529.3535	1.0	529.3535[M+H]+ 511.3415[M+H-H2O]+ 469.3456[M+H-HAc]+	e
79	19.71	Alisol F	488.3502	C30H48O5	488.3497	511.3391	-1.4	511.3391[M+Na]+ 471.2696[M+H-H2O]+ 453.2687[M+H-2H2O]+ 399.3129[M+H-C4H10O2]+ 381.3091[M+H-C4H10O2-H2O]+	e
80	20.04	Alisol L 23-acetate	510.3345	C32H46O5	510.3350	511.3418	1.0	511.3418[M+H]+ 451.3201[M+H-HAc]+ 397.2739[M+H-C6H10O2]+	e
81	20.59	Procyanidin	594.1373	C30H26O13	594.1382	593.1309	0.8	593.1309[M−H]−	a
82	21.39	Neoalisol A	488.3502	C30H48O5	488.3505	489.3572	2.5	489.3572[M+H]+ 471.3571[M+H-H2O]+	e
83g	21.71	Chrysophanol	254.0579	C15H10O4	254.0579	253.0515	3.3	253.0515[M−H]− 225.0545[M−H-CO]−	a
84	21.72	16,23-Oxido alisol B	470.3396	C30H46O4	470.3395	493.3287	-0.2	493.3287[M+Na]+ 453.3362[M+H-H2O]+ 339.2686[M+H-C6H10O2-H2O]+	e
85	22.56	Gingerdione	292.1675	C17H24O4	292.1679	337.1661	1.4	337.1661[M+HCOO]− 291.1629[M−H]−	c
86g	22.80	Physcion	284.0685	C16H12O5	284.0692	283.0619	2.5	283.0619[M−H]− 255.0315[M−H-CO]− 240.0359[M−H-CO-CH3]−	a
87	22.90	Alisol A 23-acetate	532.3764	C32H52O6	532.3767	555.3749	0.6	555.3749[M+Na]+ 515.3516[M+H-H2O]+ 383.2797[M+H-C6H12O3-H2O]+	e
88	23.05	11-Deoxy-alisol C	470.3396	C30H46O4	470.3398	471.3380	0.3	471.3380[M+H]+ 453.3515[M+H-H2O]+ 399.2874[M+H-C4H8O]+	e
89	23.32	Stearic acid	284.2715	C18H36O2	284.2717	283.2645	0.7	283.2645[M−H]−	d
90	23.54	Alisol A	490.3658	C30H50O5	490.3678	491.3661	3.8	491.3661[M+H]+ 473.3515[M+H-H2O]+ 455.3621[M+H-2H2O]+ 437.3415[M+H-3H2O]+ 383.2973[M+H-H2O-C4H10O2]+	e
91	23.79	Alisol B	472.3553	C30H48O4	472.3558	473.3622	-0.7	473.3622[M+H]+ 455.3515[M+H-H2O]+ 437.3521[M+H-2H2O]+ 383.2943[M+H-H2O-C4H8O]+	e
92	23.93	Alisol L	468.3240	C30H44O4	468.3238	469.3310	-0.4	469.3310[M+H]+ 451.3188[M+H-H2O]+ 397.2745[M+H-C4H8O]+	e
93	24.47	Alisol O	512.3502	C32H48O5	512.3492	513.3581	0.3	513.3581[M+H]+ 495.3475[M+H-H2O]+ 453.3413[M+H-HAc]+ 435.3628[M+H-HAc-H2O]+	e
94	24.65	Linolenic acid	278.2246	C18H30O2	278.2245	277.2164	-1.4	277.2164[M−H]−	a,d
95	25.28	Alisol A 24-acetate	532.3764	C32H52O6	532.3761	555.3748	1.5	555.3748[M+Na]+ 515.3516[M+H-H2O]+ 497.3472[M+H-2H2O]+ 383.2797[M+H-C6H12O3-H2O]+	e
96g	25.38	Alisol B 23-acetate	514.3658	C32H50O5	514.3661	537.3554	0.5	537.3554[M+Na]+ 515.3735[M+H]+ 497.3629[M+H-H2O]+ 479.3508[M+H-2H2O]+ 437.3415[M+H-H2O-HAc]+ 383.2688[M+H-C6H12O3]+	e

Glc: glucose; Rha: rhamnose; Xyl: xylose; Res: resveratrol.

^aRheum palmatum L.

^bCoptis chinensis Franch.

^cCurcuma longa L.

^dLysimachia christinae Hance.

^eAlisma orientale (Sam.) Juzep.

^fPanax notoginseng (Burk.) F. H. Chen.

^gIdentified by comparison with reference standards.

Fig. 2.

Chemical structures of compounds identified in GDD.

3.2. Analysis of GDD by UPLC-Q-TOF-MS^E

3.2.1. Anthraquinones

21 anthraquinones were detected from GDD and were also the major bioactive constituents of Rheum palmatum L. In this study, 6 free anthraquinones, 13 anthraquinone glycosides and 2 anthrones were determined based on MS database-matching. Anthraquinones have a characteristic fragmentation behavior with successive or simultaneous losses of CO, OH, CH₃ and CO₂ [21,22]. Peaks 64, 65, 76, 83 and 86 were exactly identified as aloe emodin, rhein, emodin, chrysophanol and physcion by comparing retention time and fragmentation patterns with reference standards. Rhein, the main anthraquinone in GDD, was used to characterize the fragmentation pathways (Fig. 3). Rhein showed quasi-molecular ion [M−H]⁻ at m/z 283.0256 in negative ion mode, and yielded fragment ions at m/z 239.0356 and 211.0403 by losses of CO₂ and CO, respectively. And then, the ion at m/z 211.0403 could further lose one molecule of CO to generate ion at m/z 183.0439. Aloe emodin and emodin were isomers with the same [M−H]⁻ ion at m/z 269. In high energy MS^E spectra, emodin revealed [M−H-CO]⁻ ion at m/z 241.0507 and [M−H-CO-O]⁻ ion at m/z 225.0559, while aloe emodin could be differentiated by the characteristic ion [M−H-CHO]⁻ at m/z 240.0421. Physcion showed [M−H]⁻ ion at m/z 283.0619, and the obvious fragments ions at m/z 255.0315 [M−H-CO]⁻ and 240.0359 [M−H-CO-CH₃]⁻ were further obtained. Chrysophanol showed [M−H]⁻ ion at m/z 253.0515, only one product ion at m/z 225.0545 [M−H-CO]⁻.

Fig. 3.

The MS spectra and fragmentation pathway of rhein in negative ion mode.

For anthraquinone glycosides, aglycone ions were identified based on the MS fragmentation behaviors of free anthraquinones. Peak 47 exhibited [M−H]⁻ ion at m/z 415.1045, which generated an [M−H-162Da]⁻ ion at m/z 253.0515 by eliminating the glucose residue. The further loss of CO was in accordance with the characteristic ion at m/z 225.0563 of chrysophanol. Thus, peak 47 was assigned as chrysophanol-1-O-β-D-glc. Based on these fragmentation patterns, peaks 15, 17, 32, 35, 44, 45, 46, 47, 48, 51, 52, 53, and 60 were inferred as anthraquinones glycosides. In addition, anthrones are an important type of anthraquinone. Sennosides usually gave a significant ion at m/z 386 which originated from C-10−C-10′ cleavage. Peak 27 showed [M−H]⁻ ion at m/z 861.1908, which first produced ions at m/z 699.1358 and 655.1880 by sequential loss of terminal glucose residue and CO₂, followed by the cleavage of C-10 and C-10′ forming [M−H-Glc-CO₂-C₁₅H₉O₅]⁻ ion at m/z 386.1002. Its structure was identified as sennoside A or B. These two isomers were not distinguished from each other only by their MS spectra. Similarly, peak 25 displayed the same fragmentation patterns as peak 27, so it was presumed as sennoside C or D.

3.2.2. Alkaloids

A total of 14 alkaloids were identified in positive ion mode and came from Coptis chinensis Franch, including protoberberine alkaloids, apomorphine alkaloids, and tetrahydroprotoberberine alkaloids. As reported in the literature, the neutral losses like the methyl radical (CH₃∙), hydrogen radical (H∙) and CO are the main fragment patterns of protoberberine alkaloids due to the successive cleavage of substituted methoxyl or methylenedioxyl groups on the A- and D-rings [23]. Peak 39 was unequivocally identified as berberine by contrast with a reference standard. The MS spectrum and possible fragmentation pathways of berberine are depicted in Fig. 4. Taking berberine as an example, it produced fragment ions at m/z 321.0983 [M-CH₃]⁺, 320.0918 [M-CH₃-H]⁺, 306.0763 [M-2CH₃]⁺, 292.0969 [M-CH₃-H-CO]⁺ and 278.0813 [M-2CH₃-CO]⁺. Peak 40 showed [M]⁺ ion at m/z 352.1538 and yielded characteristic ions at m/z 337.1430 [M-CH₃]⁺, 336.0983 [M-CH₃-H]⁺, 322.1068 [M-2CH₃]⁺ and 308.1275 [M-CH₃-H-CO]⁺, which indicated that it was presumed as palmatine. Likewise, the other peaks 31, 33, 34, 38 and 67 could be tentatively identified as coptisine, jatrorrhizine, epiberberine, worenine and 8-oxoberberine, respectively.

Fig. 4.

The MS spectra and fragmentation pathway of berberine in positive ion mode.

Peak 10 gave [M]⁺ ion at m/z 342.1701 with a molecular formula C₂₀H₂₄NO₄. The predominant ions appeared at m/z 297.1123 [M-(CH₃)₂NH]⁺, 282.0876 [M-(CH₃)₂NH-CH₃]⁺, 265.0861 [M-(CH₃)₂NH-CH₃OH]⁺, and 237.0908 [M-(CH₃)₂NH-CH₃OH-CO]⁺, which is consistent with the common structure of apomorphine alkaloids. Thus, peak 10 was considered as magnoflorine. Analogously, peaks 8 and 11 were deemed as isocorydine and isoboldine. Additionally, the tetrahydroprotoberberine alkaloids have retro-Diels-Alder (RDA) reaction, resulting in the cleavage of the terminal chain, such as -CH₃. Peak 26 gave a protonated ion at m/z 356.1865. The fragment ions at m/z 192.1019 and 165.0893 were attributed to RDA cleavage at C- and B-rings. By further loss of a methyl radical, two obtained ions generated characteristic ions at m/z 177.0774 and 150.0663, respectively. Therefore, peak 26 was tentatively identified as tetrahydropalmatine.

3.2.3. Protostane triterpenoids

17 protostane triterpenoids in GDD originated from Alisma orientale (Sam.) Juzep. Protostane triterpenoids showed [M+H]⁺ ion, adduct [M+Na]⁺ ion in positive ion mode and all possess a tetracyclic carbon skeleton. During the collision-induced dissociation (CID) process, the hydrogen rearrangement at C-23-OH resulting in C-23−C-24 bond dissociation was proposed as a characteristic CID fragmentation pathway, which can be used to further distinguish certain positional isomers containing the acetyl unit at the C-23 or C-24 position [23,24]. Such compounds usually occurred successive losses of H₂O, acetic acid group (HAc, 60 Da) and other complex groups such as C₄H₈O (72 Da), C₄H₁₀O₂ (90 Da) and C₆H₁₂O₃ (132 Da). Peaks 96 was exactly identified as alisol B 23-acetate based on retention time and fragment behavior of reference standard. The high energy MS^E spectra and the proposed fragment pathway of alisol B 23-acetate are depicted in Fig. 5. Alisol B 23-acetate showed [M+H]⁺ and [M+Na]⁺ ions at m/z 515.3735 and 537.3554, which underwent several dehydrations or deacetylations to form fragment ions at m/z 497.3629 [M + H-H₂O]⁺, 479.3508 [M + H-2H₂O]⁺, and 437.3415 [M + H-H₂O-HAc]⁺, and then dissociation of the C-23−C-24 bond and loss of H₂O gave rise to [M + H-C₆H₁₂O₃]⁺ ion at m/z 383.2688. Peaks 87, 91, and 95 showed the similar fragmentation behavior to alisol B 23-acetate, and were identified as alisol A 23-actetate, alisol B, and alisol A 24-actetate, respectively.

Fig. 5.

The MS spectra and fragmentation pathway of alisol B 23-acetate in positive ion mode.

Peak 92 had a protonated ion [M+H]⁺ at m/z 469.3310 with a molecular formula of C₃₀H₄₄O₄, and formed characteristic ions at 451.3188 [M + H-H₂O]⁺ and m/z 397.2745 [M + H-C₄H₈O]⁺ through 23-OH dehydration and C-23−C-24 dissociation. Thus, it was assigned as Alisol L. Owing to similar cleavage patterns by loss of C₄H₈O, peaks 69 and 88 were deduced to be alisol C and 11-deoxy-Alisol C, respectively. Peak 62 exhibited [M+H]⁺ ion at m/z 505.3223. Three typical dehydration ions at m/z 487.3327, 469.3285 and 451.3318 were generated from the hydroxyl groups. The dissociation of the C-23−C-24 bond via hydrogen rearrangement at C-23-OH produced diagnostic ion at m/z 415.2824 [M + H-C₄H₁₀O₂]⁺, with further loss of H₂O generating an ion at m/z 397.2783 [M + H-C₄H₁₀O₂-H₂O]⁺, so peak 62 was tentatively identified as 16-oxo-alisol A. Peak 90, 14Da less than that of 16-oxo-alisol A and similar to 16-oxo-alisol A, was further confirmed as alisol A.

3.2.4. Flavonoids

Ten flavones and their glycosides have been screened and identified in GDD using the UNIFI workflow. It is well known that the main MS behavior of flavone aglycones was RDA fragmentation pathway and losses of small molecules and/or radicals like CH₃, CO and CO₂ [25]. For flavones glycosides, the cleavage at glycosidic linkages could happen in both positive and negative ion modes, and 162 Da (Glc), 146 Da (Rha) and 308 Da (rutinoside) were the characteristic neutral loss of flavonoid-O-glycosides. The fragment ions with low m/z were the same as that of their aglycones. Among them, peaks 43 and 71 were ascertained to be quercetin and kaempferide by contrast with reference standards. Here we took quercetin and kaempferide as examples to describe the fragment patterns of these components. Quercetin displayed a deprotonated ion at m/z 301.0360 with a molecular formula of C₁₅H₁₀O₇, and the ions at m/z 151.0451 [M−H-C₈H₆O₃]⁻, 121.0431 [M−H-C₈H₆O₃-CH₂O]⁻ and 107.0162 [M−H-C₈H₆O₃-CH₂O-CH₂]⁻ resulted from RDA cleavage. Kaempferide, with the parent ion [M−H]⁻ at m/z 299.0553, exhibited a diagnostic ion [M−H-CH₃]⁻ at m/z 284.0489 and RDA cleavage ion at m/z 151.0055.

Peak 42 presented [M−H]⁻ ion at m/z 285.0406, which was 14 Da less than that of kaempferide, showing the similar fragment pathways as kaempferide. It was presumed as kaempferol. Peak 23 displayed [M−H]⁻ ion at m/z 593.1521 and produced predominant fragment ions at 447.1591 [M−H-Rha]⁻, 285.0832 [M−H-Rha-Glc]⁻ due to successive losses of glycoside fragments. Meanwhile, the ion at m/z 285.0832 further generated the characteristic ions identical to those of kaempferol, so the structure of this compound was considered as kaempferol-3-O-rutinoside. Analogously, peaks 16 and 30 were identified as rutin and hesperetin-5-O-glc, respectively.

3.2.5. Triterpenoid saponins

Eight triterpenoid saponins were detected from Panax notoginseng (Burk.) F. H. Chen in negative ion mode. These compounds offered the intense deprotonated ion [M−H]⁻ and adduct ion [M + HCOO]⁻. The primary fragmentation pattern of triterpenoid saponins was the successive losses of glycosidic unit at the site of C-20, C-3 or C-6 of ginsenosides until the formation of [Aglycon−H]⁻ ions. The species and amount of glycosyl groups were observed from MS data, in which the mass differences of 162Da, 132Da and 146Da indicated the presence of glucose (Glc), xylose (Xyl), and rhamnose (Rha), respectively [26]. Peak 37 was definitely identified as notoginsenoside R₁ with a reference standard. To facilitate characterization of these ginsenosides, the MS fragmentation pattern of notoginsenoside R₁ is investigated in detail (Fig. 6). Notoginsenoside R₁ gave [M−H]⁻ ion at m/z 931.5297 and [M + HCOO]⁻ ion at m/z 977.5354, along with three major fragment ions at m/z 799.4856 [M−H-Xyl]⁻, 637.4314 [M−H-Xyl-Glc]⁻, and 475.3782 [M−H-Xyl-2Glc]⁻ observed in high energy MS^E spectra. Peak 55 showed deprotonated ion [M−H]⁻ at m/z 1107.5967 with a molecular formula of C₅₄H₉₂O₂₃. The fragment ion at m/z 459.3824 [M−H-4Glc]⁻ represented glycosidic cleavage by loss of four glucose residues. Hence, it was tentatively characterized as ginsenoside Rb_1.

Fig. 6.

The MS spectra and fragmentation pathway of notoginsenoside R₁ in negative ion mode.

Peak 41 displayed [M−H]⁻ and [M + HCOO]⁻ ions at m/z 799.4863 and 845.4921, respectively, and produced fragment ion at m/z 475.3784 by loss of two glucose residues. Thus, it was tentatively assigned to be ginsenoside Rg₁. Peak 54 showed deprotonated ion at m/z 769.4768. The fragment ions at m/z 637.4335 [M−H-Xyl]⁻ and 475.3786 [M−H-Xyl-Glc]⁻ corresponded to successive neutral losses of xylose residue and glucose residue, indicating that peak 54 was notoginsenoside R₂. Peak 57 gave [M−H]⁻ at m/z 783.4922, which further fragmented into m/z 637.4326 [M−H-Rha]⁻ and m/z 475.3789 [M−H-Rha-Glc]⁻, so their fragment ions suggested that it was ginsenoside Rg₂. According to the cleavage of glycosidic linkages discussed above, peaks 58, 63 and 66 were tentatively identified as ginsenoside Rh₁, ginsenoside Rd, and notoginsenoside R₃, respectively.

3.2.6. Others

Four curcuminoids were recognized as the major active components in Curcuma longa L. Peak 74 was unambiguously identified as curcumin by comparison with a reference standard. Curcumin was taken as an example, which gave precursor ion at m/z 367.1192 [M−H]⁻ and diagnostic ion at m/z 217.0524 [M−H-C₉H₁₀O₂]⁻ in negative ion mode. Analogously, peaks 56, 72 and 73 were identified as cyclocurcumin, bisdemethoxycurcumin and demethoxycurcumin, respectively. The fragmentation behaviors were in accordance with those previously reported in the literature [27].

In addition, there are small amounts of tannins in GDD, of which 10 components were identified as tannins or acylglucosides by comparing with the data in library. Peak 2 showed [M−H]⁻ ion at m/z 169.0141 as base peak, and fragment ion of m/z 125.0244 corresponding to the loss of CO₂ residues. So it was tentatively identified as gallic acid. Peak 5 gave [M−H]⁻ ion at m/z 577.1359 with a molecule formula of C₃₀H₂₆O₁₂, which yielded characteristic ions at m/z 425.0873 [M−H-C₇H₄O₄]⁻, 407.0763 [M−H-C₇H₄O₄-H₂O]⁻ and 289.0712 [M−H-catechin]⁻. Thus, it was tentatively deduced to be procyanidin B. Peak 9 exhibited [M−H]⁻ ion at m/z 729.1471 and fragment ion at m/z 577.1350 [M−H-gallate]⁻, suggesting that it had an additional gallate than procyanidin B. It was tentatively identified as procyanidin B-O-gallate. Based on the similar fragment pattern, peaks 1, 4 and 19 were determined as gallic acid-O-glc, (+)-catechin-5-O-β-D-glc and (+)-catechin-3-O-gallate by matching the data in library, respectively.

3.3. Contribution of individual herbs to GDD

The established method was subsequently applied to analyze individual herbal decoctions by UPLC-Q-TOF-MS^E, and the relative sources of 96 compounds were also correspondingly confirmed. In summary, 37 components were from Rheum palmatum L., 15 components were from Coptis chinensis Franch., 9 components came from Curcuma longa L., 15 components were from Lysimachia christinae Hance, 17 components were from Alisma orientale (Sam.) Juzep. and 8 triterpenoid saponins were from Panax notoginseng (Burk.) F. H. Chen. The BPI chromatograms of six individual herbs in positive and negative ion modes are shown in Fig. 7. But each individual herb undoubtedly contributed to chemical components in GDD. Therefore, different sources and multiple types of pharmacodynamic components can exert better therapeutic effect through synergism or complementation.

Fig. 7.

The base peak intensity (BPI) chromatograms of six individual herbs in positive (A) and negative (B) ion modes. DH = Rheum palmatum L., HL = Coptis chinensis Franch., JH = Curcuma longa L., JQC = Lysimachia christinae Hance, ZX = Alisma orientale (Sam.) Juzep. and SQ = Panax notoginseng (Burk.) F. H. Chen.

4. Conclusion

In this study, an integrative strategy based on UPLC-Q-TOF-MS^E coupled with UNIFI informatics platform was applied for chemical profile analysis of GDD. To the best of our knowledge, it was the first time to reveal the constituents in GDD comprehensively. By comparison with retention time, accurate mass, fragmentation behavior, a total of 96 compounds were identified or tentatively characterized from GDD, including anthraquinones, alkaloids, protostane triterpenoids, flavonoids, triterpenoid saponins, tannins, curcuminoids and other compounds. Additionally, the ESI-MS fragmentation patterns of representative compounds in different chemical structure types were investigated. Most of the high response constitutions in individual herbs were also detected in GDD. This approach provided a rapid method for high throughput screening and characterization of constituents, and would be available in other TCM formulae analysis. What is more, the results could supply valuable information for the quality control and further study of GDD in vivo.

Moreover, we found that most of the compounds have abundant phenolic hydroxyl, especially the anthraquinones, curcumin and flavonoids. The structures of these compounds tend to be easily chelated by copper ions. Therefore, it is speculated that natural small molecules in GDD that could selectively chelate copper are able to form stable complexes to promote copper excretion. These molecules with properties would serve as a promising alternative to current treatments. This work has great guiding significance in further research and application of GDD in clinical treatment.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: