Metabolic profile of danshen in rats by HPLC-LTQ-Orbitrap mass spectrometry

Abstract

Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), is one of the traditional Chinese medicines (TCMs) most commonly used for the treatment of cardiovascular and cerebrovascular diseases. However, little is known about the chemical and metabolic profiles of danshen in vitro or in vivo. In particular, more information is needed in relation to the 50% ethanol extracts usually used in danshen formulations such as Fufang Xueshuantong Capsules and Fufang Danshen tablets. High-performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer (HPLC-LTQ-Orbitrap) provides a sensitive and accurate method for analyzing the composition of samples. This method was used to determine the in vitro and in vivo chemical and metabolic profiles of danshen. Sixty-nine components of danshen extract and 118 components of danshen in rat plasma, urine, feces, and bile were unambiguously or tentatively identified. These results not only revealed the material composition of danshen, but also provided a comprehensive research approach for the identification of multi-constituents in TCMs.

1. Introduction

Recently, high-performance liquid chromatography-mass spectrometry (HPLC-MS), especially for high-resolution mass spectrometry (HRMS), has become a powerful tool for detecting and identifying known and unknown metabolites of drugs owing to its high mass accuracy and high sensitivity (Liu et al., 2011; Wang et al., 2011; Liang et al., 2013). MS/MS data provide abundant information for elucidating the structure of compounds. Thus, this method provides an effective and powerful tool for the identification of compounds in complex matrices, such as traditional Chinese medicines (TCMs) and bio-samples. The linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap), an electrostatic Fourier-transform mass spectrometer, combines a high trapping capacity and MSⁿ scanning function of the linear ion trap with accurate mass measurements to within 5 ppm (parts per million) and a resolving power of up to 100 000 (Cai et al., 2015; Zhang et al., 2015). Data-dependent MS/MS scanning can obtain more fragmentation information, improving the efficiency and accuracy of identification (Wang et al., 2016).

Danshen, the dried root of Chinese sage, Salvia miltiorrhiza Bunge (Lamiaceae), is one of the TCMs most commonly used in China and elsewhere, and is used either alone or in formulations. It has been widely used in the treatment of cardiovascular and cerebrovascular diseases, such as coronary artery disease (Ji et al., 2003), myocardial infarction (Sun et al., 2005), and stroke (Lam et al., 2003). It has also been used to treat other conditions, such as renal diseases (Kang et al., 2004) and diabetes (Belin et al., 2009). Many formulations containing danshen, for instance the Fufang Danshen Dripping Pill and Fufang Xueshuantong Capsule, are now frequently used in the clinical treatment of cardiovascular diseases and eye diseases (Duan et al., 2013; Yang et al., 2014). There are two principal bioactive components in danshen: water-soluble phenolic acids and liposoluble tanshinones. The phenolic acids include danshensu, rosmarinic acid, lithospermic acid, salvianolic acid A, salvianolic acid B, and other salvianolic acids. The tanshinones include tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, 15,16-dihydrotanshinone I, and other tanshinones (Zhang et al., 2005; Wu et al., 2006).

Previous in vivo studies have focused mainly on the water decoction of danshen (Zhao et al., 2015) or its effective parts and components (Li et al., 2007; Sun et al., 2007). Danshen has often been used only as a component of ethanol extracts, especially in formulations, because of its complex composition and compatibility with other herbs. Also, there has been limited research on the excretion of danshen in feces and bile (Sun et al., 2007). Therefore, comprehensive and systematic studies are needed of the chemical and metabolic profiles of danshen in vitro and in vivo. In the present study, we analyzed the chemical profile of a 50% ethanol extract of danshen, as such extracts are often used in its formulation. The metabolic profile of danshen was determined in bio-samples from rats. An HPLC-LTQ-Orbitrap method coupled with an extracted ion chromatogram (EIC) data-processing technique was applied to elucidate the chemical and metabolic profiles. A total of 69 components of danshen extract and 118 components of danshen in rat plasma, urine, feces, and bile were unambiguously or tentatively identified. The present study provides a basis for research on the quality control and pharmacology of danshen, and establishes a comprehensive and reliable method for identification of multi-components of TCMs both in vitro and in vivo.

2. Materials and methods

2.1. Materials and reagents

Danshen crude drug was provided by the Guangdong Zhongsheng Pharmaceutical Co., Ltd. (Guangzhou, China) and was authenticated by Professor Jian-mei HUANG. Voucher specimens were deposited in the School of Chinese Materia Medica, Beijing University of Chinese Medicine, China.

Eleven reference standards, including caffeic acid, protocatechuic aldehyde, protocatechuic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid, tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone, were purchased from the Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Three reference standards of tanshinol B, danshenxinkun B, and tanshinone IIB were purchased from the Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Three authentic standards, namely salvianolic acids A, B, and C, were obtained from the School of Chinese Materia Medica, Beijing University of Chinese Medicine, China.

HPLC-grade methanol and acetonitrile, and LC/MS-grade formic acid were purchased from Fisher Scientific (Fisher, Fair Lawn, NJ, USA).

2.2. Instrumentation and analytical conditions

Chromatographic analysis was performed using a Thermo Accela 600 HPLC system (Thermo Scientific, Bremen, Germany) equipped with a binary pump and an autosampler. Samples were separated on a Waters XBridge-C18 column (5 μm, 150 mm×4.6 mm) at room temperature. A gradient elution of solvent acetonitrile (A) and water containing 0.1% formic acid (B) was applied according to the following program: 0–10 min, 5%–20% A; 10–25 min, 20%–30% A; 25–35 min, 30%–70% A; 35–60 min, 70% A. The flow rate was set at 1.0 ml/min. Sample solution (10 μl) was injected into the HPLC-MS/MS system.

MS analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). The mass spectrometer was connected to the Accela HPLC system by an electrospray ionization (ESI) source and operated in both positive and negative ion modes. Compounds were detected by full scan mass analysis from m/z 100 to 1000 at a resolving power of 30 000 with data-dependent MSⁿ (n=3) analysis. The optimized source parameters in positive (and negative) mode were as follows: capillary temperature, 350 °C; sheath gas flow, 30 arbitrary unit (arb); auxiliary gas flow, 10 arb; source voltage, 4.0 kV; capillary voltage, 35 V; tube lens voltage, 110 V. The isolation width was 2 Da, and the normalized collision energy (CE) was 35%.

2.3. Preparation of drugs

2.3.1 Preparation of danshen freeze-dried powder

Danshen freeze-dried powder was prepared by refluxing the extract twice with 50% (v/v) ethanol (100 g/700 ml for 3 h the first time, and 100 g/500 ml for 2 h the second time) after soaking in 50% ethanol for 30 min. Each decoction was mixed, filtered, vacuum-evaporated, and freeze-dried. The yield of powdered extract was about 42.3% (w/w).

2.3.2 Preparation of danshen extract

A total of 1.05 g danshen freeze-dried powder was accurately weighed and ultrasonicated with 30 ml of 50% ethanol for 30 min. The supernatants were filtered through a 0.22-μm membrane filter. The filtrates were collected and stored at 4 °C until HPLC-MS/MS analysis.

2.3.3 Preparation of danshen suspension

Danshen freeze-dried powder was accurately weighed and suspended in deionized water to obtain a final concentration of 1.5 g/ml (crude drug) for intragastric administration.

2.3.4 Preparation of standard solutions

Individual standard stock solutions of the seventeen standards were prepared by accurately weighing and then dissolving each standard in methanol, with concentrations ranging from 0.09 to 1.20 mg/ml. A working solution of each of the seventeen standards was obtained by diluting each stock solution with methanol to the desired concentration. Working solutions were stored at 4 °C before analysis.

2.4. Animals and drug administration

Twelve male Sprague-Dawley rats, weighing (250±20) g, were purchased from the Si Bei Fu Experimental Animal Science and Technology Co., Ltd. (Beijing, China). The rats were divided into two groups: a control group (n=3, one each for blank plasma, urine and feces, and bile) and a drug group (n=9, 3 for dosed plasma, 3 for dosed urine and feces, and 3 for dosed bile). The rats were housed in a controlled environment (12-h light/12-h dark cycle, at consistent temperature and humidity) for three days before the experiment. Danshen was administered orally to the drug group once a day at a dose of 1 ml/100 g body weight for three days. An equal dose of deionized water was administered by oral gavage to the rats of the control group.

Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, and all experimental protocols were reviewed and approved by the Institutional animal Experimentation Committee of Beijing University of Chinese Medicine.

2.5. Biological sample collection

Before the last administration, the rats were deprived of food for 12 h. Blood samples (0.4 ml) were collected from the orbital vein and gathered into heparinized tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h, respectively. All blood samples were then centrifuged at 3000g for 10 min to obtain plasma samples. Plasma samples from different rats and different time points in each group were then mixed in the same proportions to produce pooled plasma samples, which were stored at −80 °C until additional extraction and analysis.

The urine and feces of rats in each group were collected over a 24-h period starting immediately after the last administration. The urine and feces samples in each group were combined separately and stored at −80 °C until additional extraction and analysis.

Rats were fixed on a wooden plate and anesthetized with ethylurethanm following the last administration. An abdominal incision was made and the common bile duct was cannulated with PE10 tubing (inside diameter (ID)=0.28 mm, San Diego, CA USA) for collection of the bile samples. Bile samples from each group were collected for 24 h and combined and stored at −80 °C until additional extraction and analysis.

2.6. Biological sample pretreatment

An aliquot of 2 ml plasma for positive ion detection was suspended in 8 ml methanol. Another aliquot of 2 ml plasma for negative ion detection was suspended in 200 µl 10% (v/v) hydrochloric acid and 8 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatants were evaporated to dryness under nitrogen gas at room temperature, and the residues were dissolved in 200 µl 70% (v/v) methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Urine sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 600 µl 70% methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Bile sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 1.5 ml 70% methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Feces were dried at 37 °C and grinded into powder. Feces sample (1.5 g) was extracted with 30 ml 70% methanol in an ultrasonic bath for 30 min, followed by filtration. Filtrate (2 ml) was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 400 µl 70% methanol. After centrifugation at 12 000g for 10 min, a 10-µl aliquot of the supernatant was injected into the HPLC-MS/MS system for analysis.

2.7. Data processing

Thermo Xcalibur 2.1 workstation (Thermo Fisher Scientific, Bremen, Germany) was used for data acquisition and processing. Metworks (Thermo Scientific, Bremen, Germany) was used for data-filtering and identification of possible metabolites. The maximum mass error between the measured and calculated values was 5 ppm.

3. Results

3.1. Analysis of the chemical profile of danshen in vitro

The results from total ion chromatography (TIC) of the danshen extract and the reference standards in positive mode and negative mode are shown in Fig. 1. Based on accurate mass measurements, MS/MS fragmentations, retention time, and reference data (Liu AH et al., 2007; Liu M et al., 2007; Su et al., 2015), a total of 69 components of danshen, including 23 phenolic acids, 33 tanshinones, and 13 unknown compounds, were identified. Their accurate mass measurements, retention time, and HPLC-MS/MS data are shown in Table 1. Among them, 16 compounds were unambiguously confirmed by comparison with reference standards. Their structures are shown in Fig. 2.

Fig. 1.

Total ion chromatography of danshen extract in negative (a) and positive (b) ion modes

Table 1.

HPLC-MS/MS data and identification of components of danshen

No.		t R (min)	Ion	Theoretical mass (m/z)	Experimental mass (m/z)	Error (ppm)	Formula [M−H]−/[M+H]+	MS/MS fragment		Identification
Phenolic acids
1		4.83	[M−H]−	197.0444	197.0441	−1.6	C9H9O5	MS2[197]: 179(100) MS3[179]: 135(100)	Danshensua
2		5.70	[M−H]−	153.0182	153.0184	0.9	C7H5O4		Protocatechuic acida
3		7.58	[M−H]−	137.0233	137.0236	1.7	C7H5O3	MS2[137]: 137(100)	Protocatechuic aldehydea
4		9.16	[M−H]−	179.0339	179.0336	−1.3	C9H7O4	MS2[179]: 135(100)	Caffeic acida
5		10.08	[M−H]−	193.0495	193.0493	−1.2	C10H9O4	MS2[193]: 178(100), 149(38), 134(81) MS3[178]: 134(100)	Ferulic acid/isoferulic acida
6		14.42	[M−H]−	735.1556	735.1552	−0.5	C36H31O17	MS2[735]: 537(100), 519(20) MS3[519]: 519(51), 357(73), 321(65), 297(100)	Hydrated salvianolic acid B
7		14.76	[M−H]−	537.1028	537.1028	0.1	C27H21O12	MS2[537]: 339(100), 295(37) MS3[339]: 321(33), 295(100)	Salvianolic acid H/I
8		15.02	[M−H]−	735.1556	735.1553	−0.4	C36H31O17	MS2[735]: 537(100), 519(49) MS3[537]: 519(51), 339(20), 321(70), 297(100)	Hydrated salvianolic acid B
9		16.74	[M−H]−	359.0761	359.0758	−1.0	C18H15O8	MS2[359]: 197(24), 179(23), 161(100) MS3[161]: 161(38), 133(100)	Rosmarinic acida
10		17.45	[M−H]−	493.1129	493.1129	0.0	C26H21O10	MS2[493]: 295(100) MS3[295]: 280(13), 277(65), 159(100)	Salvianolic acid A isomer
11		19.07	[M−H]−	717.1450	717.1434	−2.2	C36H29O16	MS2[717]: 519(100), 321(15) MS3[519]: 339(21), 321(100)	Salvianolic acid Ba
12		20.89	[M−H]−	717.1450	717.1450	0.0	C36H29O16	MS2[717]: 519(100), 321(18) MS3[519]: 339(21), 321(100)	Salvianolic acid E
13		21.32	[M−H]−	493.1129	493.1135	1.1	C26H21O10	MS2[493]: 295(100) MS3[295]: 280(14), 277(66), 159(100)	Salvianolic acid Aa
14		22.26	[M−H]−	731.1607	731.1598	−1.2	C37H31O16	MS2[731]: 533(100) MS3[533]: 353(51), 335(100)	Methyl salvianolic acid B
15		23.68	[M−H]−	565.1341	565.1341	0.1	C29H25O12	MS2[565]: 519(87), 367(87), 339(15), 321(100)	Dimethyl lithospermate
16		24.23	[M−H]−	491.0973	491.0982	1.9	C26H19O10	MS2[491]: 311(60), 293(100) MS3[293]: 276(32), 275(11), 265(100), 249(89), 247(13)	Isosalvianolic acid C
17		24.44	[M−H]−	565.1341	565.1341	0.1	C29H25O12	MS2[565]: 367(76), 339(17), 321(100)	Dimethyl lithospermate
18		25.23	[M−H]−	565.1341	565.1345	0.7	C29H25O12	MS2[565]: 519(88), 339(15), 321(100) MS3[321]: 293(31), 277(100), 249(76)	Dimethyl lithospermate
19		26.65	[M−H]−	565.1341	565.1345	0.7	C29H25O12	MS2[565]: 367(100)	Dimethyl lithospermate
20		27.27	[M−H]−	491.0973	491.0977	0.9	C26H19O10	MS2[491]: 311(22), 293(100) MS3[293]: 276(24), 275(18), 265(100), 249(21), 247(36)	Salvianolic acid Ca
21		27.65	[M−H]−	745.1763	745.1760	−0.5	C38H33O16	MS2[745]: 547(87), 519(100), 321(73)	Dimethyl salvianolic acid B
22		28.62	[M−H]−	745.1763	745.1762	−0.1	C38H33O16	MS2[745]: 547(87), 519(100), 321(74)	Dimethyl salvianolic acid B
23		28.97	[M−H]−	313.0707	313.0709	0.8	C17H13O6	MS2[313]: 269(36), 161(100) MS3[161]: 161(29), 133(100)	Salvianolic acid F
Tanshinones
24		33.73	[M−H]−	295.0965	295.0970	1.7	C18H15O4	MS2[295]: 277(16), 267(27), 265(100) MS3[265]: 265(100)	Tanshinol Ba
25		21.24	[M+H]+	313.1071	313.1056	−4.8	C18H17O5	MS2[313]: 295(100), 267(22), 265(87) MS3[295]: 277(100), 267(40), 249(98)	Tanshindiol A/B/C
26		22.57	[M+H]+	313.1071	313.1056	−4.6	C18H17O5	MS2[313]: 295(100), 267(7) MS3[295]: 267(100)	Tanshindiol A/B/C
27		26.14	[M+H]+	313.1071	313.1056	−4.7	C18H17O5	MS2[313]: 295(100), 267(9) MS3[295]: 267(100)	Tanshindiol A/B/C
28		27.01	[M+H]+	293.0808	293.0797	−3.9	C18H13O4	MS2[293]: 249(100) MS3[249]: 234(18), 221(24), 193(100), 178(39)	Hydroxyl tanshinone I
29		28.49	[M+H]+	313.1434	313.1422	−3.9	C19H21O4	MS2[313]: 269(100) MS3[269]: 254(25), 251(16), 223(22), 199(100)	Hydroxyl cryptotanshinone
30		29.28	[M+H]+	283.0965	283.0954	−3.7	C17H15O4	MS2[283]: 265(51), 255(27), 241(15), 237(100) MS3[237]: 222(11), 219(100), 209(87), 191(35), 181(25)	Dihydronortanshinone
31		30.79	[M+H]+	293.0808	293.0799	−3.3	C18H13O4	MS2[293]: 249(100) MS3[249]: 234(18), 221(23), 193(100), 178(38)	Przewaquinone B
32		30.97	[M+H]+	295.0965	295.0953	−4.1	C18H15O4	MS2[295]: 277(100), 267(44), 24(51) MS3[277]: 259(82), 249(100), 231(27)	Hydrated tanshinone I/trijuganone A
33		32.08	[M+H]+	295.0965	295.0951	−4.8	C18H15O4	MS2[295]: 277(100), 249(11) MS3[277]: 249(100), 221(17)	Hydrated tanshinone I/trijuganone A
34		32.26	[M+H]+	313.1423	313.1423	−3.5	C19H21O4	MS2[313]: 295(100), 267(73) MS3[295]: 277(100), 267(55), 249(52)	Hydroxyl cryptotanshinone
35		32.44	[M+H]+	301.1434	301.1424	−3.6	C18H21O4	MS2[301]: 283(100) MS3[283]: 265(100), 255(26)	Salvianonol
36		32.61	[M+H]+	311.1278	311.1264	−4.6	C19H19O4	MS2[311]: 293(100), 283(22), 267(80), 225(12) MS3[293]: 278(16), 275(100), 265(19), 251(80)	Tanshinone IIBa
37		32.77	[M+H]+	311.1278	311.1263	−4.6	C19H19O4	MS2[311]: 293(14), 275(11), 267(100) MS3[267]: 252(100), 239(11), 225(63), 185(47)	Hydroxyl tanshinone IIA
38		33.46	[M+H]+	341.1384	341.1371	−3.8	C20H21O5	MS2[341]: 281(100), 263(43) MS3[281]: 263(100), 235(19)	Methyl dihydrotanshinonate
39		33.69	[M+H]+	327.1214	327.1248	−3.9	C19H19O5	MS2[327]: 309(100) MS3[309]: 265(100)	Hydroxyl tanshinone IIB
40		34.56	[M+H]+	281.1536	281.1524	−4.1	C19H21O2	MS2[281]: 266(38), 263(50), 253(28), 239(100) MS3[239]: 224(22), 221(100), 193(54)	Dehydromiltirone
41		34.64	[M+H]+	293.1172	293.1159	−4.4	C19H17O3	MS2[293]: 275(100), 265(11), 247(39) MS3[275]: 260(13), 247(100)	Dehydrotanshinone IIA
42		35.19	[M+H]+	279.1016	279.1004	−4.3	C18H15O3	MS2[279]: 261(100), 233(5) MS3[261]: 233(100)	Dihydrotanshinone Ia
43		35.71	[M+H]+	315.1591	315.1576	−4.7	C19H23O4	MS2[315]: 297(100) MS3[297]: 279(100), 251(57)	Neocryptotanshinone
44		35.84	[M+H]+	281.1172	281.1160	−4.2	C18H17O3	MS2[281]: 263(100), 253(7), 235(71) MS3[263]: 248(16), 245(10), 235(100)	Danshenxinkun Ba
45		35.98	[M+H]+	339.1227	339.1219	−2.3	C20H19O5	MS2[339]: 279(100) MS3[279]: 261(100)	Methyl tanshinonate
46		36.24	[M+H]+	295.1329	295.1319	−3.3	C19H19O3	MS2[295]: 277(100), 267(15), 249(47) MS3[277]: 249(100)	Dehydrocryptotanshinone
47		37.34	[M+H]+	297.1485	297.1474	−3.7	C19H21O3	MS2[297]: 279(100), 251(81) MS3[279]: 251(100)	Cryptotanshinonea
48		37.62	[M+H]+	277.0859	277.0850	−3.5	C18H13O3	MS2[277]: 249(100), 231(13)	Tanshinone Ia
49		38.84	[M+H]+	279.1016	279.1003	−4.7	C18H15O3	MS2[279]: 261(100), 233(6) MS3[261]: 233(100), 205(3)	Dihydrotanshinone I
50		38.98	[M+H]+	293.1172	293.1166	−2.2	C19H17O3	MS2[293]: 275(100), 265(11), 247(38) MS3[275]: 260(13), 247(100)	Dehydrotanshinone IIA
51		40.05	[M+H]+	281.1536	281.1524	−4.1	C19H21O2	MS2[281]: 266(17), 263(43), 253(82), 221(100) MS3[221]: 206(17), 193(100)	Dehydromiltirone
52		40.28	[M+H]+	293.1172	293.1159	−4.5	C19H17O3	MS2[293]: 275(100), 265(11), 247(37) MS3[275]: 260(13), 247(100)	Dehydrotanshinone IIA
53		41.17	[M+H]+	295.1329	295.1317	−4.0	C19H19O3	MS2[295]: 277(100), 249(14) MS3[277]: 249(100)	Tanshinone IIAa
54		41.54	[M+H]+	281.1536	281.1526	−3.7	C19H21O2	MS2[281]: 266(18), 263(43), 253(93), 221(100) MS3[221]: 206(14), 193(100)	Dehydromiltirone
55		42.34	[M+H]+	283.1693	283.1682	−3.6	C19H23O2	MS2[283]: 265(100), 241(47), 223(63) MS3[265]: 237(64), 223(100)	Miltirone
56		45.75	[M+H]+	557.1959	557.1942	−3.1	C36H29O6	MS2[557]: 539(28), 529(100), 511(48) MS3[529]: 511(100), 501(81), 483(46)	Neoprzewaquinone A
Others
Un1		9.62	[M−H]−	509.2229	509.2230	0.2	C22H37O13	MS2[509]: 463(100) MS3[463]: 331(100), 161(27)	Unknown
Un2		10.91	[M−H]−	571.1082	571.1080	−0.5	C27H23O14	MS2[571]: 527(21), 483(100), 439(73)	Unknown
Un3		11.73	[M−H]−	627.4044	627.4059	2.4	C41H55O5	MS2[627]: 610(14), 581(70), 564(100)	Unknown
Un4		12.96	[M−H]−	723.5042	723.5013	−3.9	C41H71O10	MS2[723]: 678(100) MS3[678]: 659(100), 451(25), 338(25)	Unknown
Un5		14.07	[M−H]−	836.5856	836.5854	−0.2	C44H84O14	MS2[836]: 791(100) MS3[791]: 773(100), 565(28)	Unknown
Un6		16.21	[M−H]−	717.1450	717.1452	0.3	C36H29O16	MS2[717]: 519(100), 321(16) MS3[519]: 339(22), 321(100)	Unknown
Un7		28.14	[M−H]−	341.1020	341.1023	0.9	C19H17O6	MS2[341]: 297(100), 253(14) MS3[297]: 253(100)	Unknown
Un8		28.33	[M−H]−	671.1395	671.1402	1.0	C35H27O14	MS2[671]: 473(100) MS3[473]: 429(100), 321(100)	Unknown
Un9		24.40	[M+H]+	327.1227	327.1212	−4.6	C19H19O5	MS2[327]: 283(100), 265(8) MS3[283]: 265(29), 254(100)	Unknown
Un10		25.27	[M+H]+	369.0969	369.0955	−3.7	C20H17O7	MS2[369]: 323(100), 295(77) MS3[323]: 295(100)	Unknown
Un11		34.38	[M+H]+	297.1485	297.1471	−4.7	C19H21O3	MS2[297]: 253(100) MS3[253]: 238(33), 211(100)	Unknown
Un12		42.22	[M+H]+	283.1693	283.1677	−5.4	C19H23O2	MS2[283]: 265(100), 241(47), 223(63) MS3[265]: 237(20), 223(100)	Unknown
Un13		50.83	[M+H]+	587.2064	587.2059	−0.9	C37H31O7	MS2[587]: 569(100), 541(24) MS3[569]: 551(100), 541(71)	Unknown

Fig. 2.

Chemical structures of confirmed compounds in danshen extract

The numbering of compounds is consistent with that in Table 1

3.2. Analysis of the metabolic profile of danshen in vivo

For the identification of original components in bio-samples, the extract ion chromatograms (EICs) combined with the accurate mass measurements, MS/MS fragmentations, and retention time were compared with those of blank samples. For the identification of possible metabolites in bio-samples, firstly, all of the possible metabolic pathways of one component were input in Metworks; secondly, all of the possible metabolites proposed by the software were summarized in an Excel table; thirdly, the EICs, mass measurements, and MS/MS fragmentations of each metabolite were compared with those of blank samples.

As a result, 118 components were unambiguously or tentatively identified, including 38 original components and 80 transformative components (Table 2). Among these components, 7 phenolic acids and 28 tanshinones were identified in rat plasma; 17 phenolic acids and 46 tanshinones were tentatively identified in rat urine; 25 phenolic acids and 37 tanshinones were identified in rat feces; and 1 phenolic acid and 17 tanshinones were identified in rat bile.

Table 2.

Metabolites identified in bio-samples from rats after oral administration of danshen extract

No.	t R (min)	Ion	Theoretical mass (m/z)	Experimental mass (m/z)	Formula [M−H]−/[M+H]+	Error (ppm)	MS/MS fragment	Identification	Plasma	Urine	Feces	Bile
Phenolic acids
1	2.11	[M−H]−	277.0013	277.0020	C9H9O8S	2.8	MS2[277]: 259(57), 215(35), 197(100)	Sulfate danshensu	×	×	√	×
2	4.26	[M−H]−	277.0013	277.0010	C9H9O8S	−0.8	MS2[277]: 215(40), 197(100) MS3[196]: 179(100)	Sulfate danshensu	×	√	×	×
3	4.77	[M−H]−	197.0444	197.0446	C9H9O5	0.6	MS2[197]: 179(100) MS3[179]: 135(100)	Danshensua,b	√	√	√	×
4	7.35	[M−H]−	211.0601	211.0603	C10H11O5	0.9	MS2[211]: 193(100), 165(23) MS3[193]: 149(29), 134(100)	Methyl danshensu	√	√	√	√
5	7.47	[M−H]−	179.0339	179.0344	C9H7O4	3.1	MS2[179]: 135(100) MS3[135]: 135(100)	Demethyl ferulic acid	×	√	×	×
6	7.52	[M−H]−	137.0233	137.0236	C7H5O3	1.7	MS2[137]: 137(100)	Protocatechuic aldehydea,b	×	×	√	×
7	7.62	[M−H]−	181.0495	181.0502	C9H9O4	3.7	MS2[181]: 163(100) MS3[162]: 119(100)	Dihydro caffeic acid	×	√	√	×
8	8.29	[M−H]−	179.0339	179.0341	C9H7O4	1.2	MS2[179]: 135(100) MS3[135]: 135(100)	Acetylated protocatechuic aldehyde	×	√	×	×
9	8.59	[M−H]−	165.0546	165.0550	C9H9O3	2.4	MS2[165]: 121(100) MS3[121]: 121(100)	Decarbonyl ferulic acid	×	√	×	×
10	9.38	[M−H]−	361.0918	361.0910	C18H17O8	−2.2	MS2[361]: 317(39), 273(41), 239(100), 221(68) MS3[239]: 195(100), 151(19)	Dihydro rosmarinic acid	×	×	√	×
11	9.86	[M−H]−	193.0495	193.0502	C10H9O4	3.5		Ferulic acid/isoferulic acida,b	×	×	√	×
12	12.03	[M−H]−	343.0812	343.0806	C18H15O7	−1.8	MS2[343]: 299(100) MS3[299]: 255(100)	Hydroxyl and methyl salvianolic acid F	×	×	√	×
13	13.62	[M−H]−	535.1082	535.1072	C24H23O14	−1.9	MS2[535]: 359(100) MS3[359]: 197(23), 179(20), 161(100)	Rosmarinic acid glucuronide conjugate	×	√	×	×
14	13.99	[M−H]−	539.1184	539.1169	C27H23O12	−2.7	MS2[539]: 399(100), 297(67) MS3[297]: 219(100), 201(70)	Dihydro salvianolic acid H/I	×	√	√	×
15	14.67	[M−H]−	537.1028	537.1025	C27H21O12	−0.4	MS2[537]: 339(100), 295(38)	Salvianolic acid H/Ib	×	×	√	×
16	15.03	[M−H]−	735.1556	735.1539	C36H31O17	−2.3		Hydrated salvianolic acid Bb	×	×	√	×
17	15.17	[M−H]−	343.0812	343.0806	C18H15O7	−1.7	MS2[343]: 255(100) MS3[255]: 237(91), 148(100)	Hydroxyl and methyl salvianolic acid F	×	×	√	×
18	16.55	[M−H]−	555.1133	555.1125	C27H23O13	−1.5	MS2[555]: 375(100), 357(39) MS3[375]: 331(100), 269(60)	Hydrated salvianolic acid H/I	×	×	√	×
19	16.71	[M−H]−	359.0761	359.0761	C18H15O8	−0.2	MS2[359]: 271(100) MS3[271]: 149(100), 135(46), 121(74)	Rosmarinic acida,b	√	×	×	×
20	17.01	[M−H]−	523.1235	523.1225	C27H23O11	−1.9	MS2[523]: 505(53), 281(100) MS3[281]: 263(26), 174(100)	Demethyl and hydroxyl salvianolic acid A	×	×	√	×
21	17.14	[M−H]−	315.0863	315.0863	C17H15O6	0.0	MS2[315]: 297(18), 285(100), 267(12)	Dihydro salvianolic acid F	×	√	×	×
22	17.46	[M−H]−	493.1129	493.1130	C26H21O10	0.1	MS2[493]: 295(100) MS3[295]: 277(56), 159(100)	Iso salvianolic acid Ab	√	√	√	×
23	19.02	[M−H]−	717.1450	717.1439	C36H29O16	−1.5	MS2[717]: 519(100), 321(17) MS3[519]: 339(22), 321(100)	Salvianolic acid Ba,b	×	√	√	×
24	20.40	[M−H]−	763.1869	763.1854	C38H35O17	−1.9	MS2[763]: 565(100), 520(57), 321(15)	Hydrated dimethyl salvianolic acid B	×	×	√	×
25	20.80	[M−H]−	717.1450	717.1441	C36H29O16	−1.3	MS2[717]: 519(100), 321(19) MS3[519]: 339(23), 321(100)	Salvianolic acid Eb	×	√	√	×
26	21.26	[M−H]−	493.1129	493.1129	C26H21O10	−0.1	MS2[493]: 295(100) MS3[295]: 277(59), 159(100)	Salvianolic acid Aa,b	√	√	√	×
27	21.70	[M−H]−	551.1184	551.1177	C28H23O12	−1.3	MS2[551]: 519(43), 371(100), 353(51), 339(73)	Methyl salvianolic acid H/I	×	×	√	×
28	22.20	[M−H]−	731.1607	731.1597	C37H31O16	−1.4	MS2[731]: 533(100) MS3[533]: 353(49), 335(100)	Methyl salvianolic acid Bb	√	√	√	×
29	24.15	[M−H]−	491.0973	491.0971	C26H19O10	−0.3	MS2[491]: 293(100) MS3[293]: 276(33), 264(100), 249(91)	Iso salvianolic acid Cb	×	×	√	×
30	24.35	[M−H]−	565.1341	565.1334	C29H25O12	−1.1	MS2[565]: 519(84), 367(78), 321(100)	Dimethyl lithospermateb	×	×	√	×
31	25.16	[M−H]−	565.1341	565.1331	C29H25O12	−1.7	MS2[565]: 519(87), 367(79), 321(100)	Dimethyl lithospermateb	√	√	√	×
32	26.60	[M−H]−	565.1341	565.1331	C29H25O12	−1.8		Dimethyl lithospermateb	×	√	×	×
33	27.21	[M−H]−	491.0973	491.0972	C26H19O10	−0.1	MS2[491]: 293(100)	Salvianolic acid Ca,b	×	×	√	×
Tanshinones
34	9.08	[M+H]+	345.0969	345.0976	C18H17O7	2.0	MS2[345]: 327(100), 281(53) MS3[327]: 309(14), 281(100)	Demethyl and trihydroxyl tanshinone IIB	×	×	√	×
35	12.06	[M+H]+	517.1704	517.1683	C26H29O11	−2.1		Methyl dihydrotanshinonate glucuronide conjugate	×	√	×	×
36	12.77	[M+H]+	343.0812	343.0814	C18H15O7	0.5	MS2[343]: 325(92), 297(100) MS3[297]: 279(100), 255(56)	Demethyl and carboxylated tanshindiol A/B/C	×	×	√	×
37	13.06	[M+H]+	329.1020	329.1023	C18H17O6	1.1	MS2[329]: 311(100), 265(58) MS3[311]: 283(25), 265(100)	Demethyl and two hydroxyl tanshinone IIB	×	×	√	×
38	13.37	[M+H]+	620.1909	620.1917	C28H34O11N3S	1.3	MS2[620]: 602(55), 584(32), 455(100)	Tanshindiol A/B/C glutathione conjugate	×	×	×	√
39	13.66	[M+H]+	343.0812	343.0814	C18H15O7	0.5	MS2[343]: 325(95), 297(100) MS3[297]: 279(100), 255(60)	Demethyl and carboxylated tanshindiol A/B/C	×	×	√	×
40	14.04	[M+H]+	471.1286	471.1286	C24H23O10	0.1	MS2[471]: 295(57), 277(23), 267(100) MS3[267]: 249(50), 237(100)	Hydroxyl and glucuronidated dihydrotanshinone I	×	√	×	×
41	14.25	[M−H]−	487.1235	487.1232	C24H23O11	−0.6	MS2[487]: 311(100) MS3[311]: 283(30), 281(100)	Hydroxyl and glucuronidated tanshinol B	×	√	×	×
42	14.64	[M+H]+	473.1442	473.1440	C24H25O10	−0.5	MS2[473]: 297(80), 279(100), 251(39) MS3[279]: 261(100), 251(100)	Hydroxyl and glucuronidated danshenxinkun B	×	√	×	×
43	16.60	[M+H]+	327.0863	327.0866	C18H15O6	0.7	MS2[327]: 309(100), 281(67), 265(12) MS3[309]: 291(13), 281(100)	Hydroxyl and dehydro tanshindiol A/B/C	×	×	√	×
44	18.89	[M+H]+	345.1333	345.1338	C19H21O6	1.5	MS2[345]: 327(100), 309(23) MS3[327]: 309(100), 281(19)	Hydrated and hydroxyl tanshinone IIB	×	√	×	×
45	19.32	[M+H]+	301.1434	301.1433	C18H21O4	−0.4	MS2[301]: 283(100)	Demethyl neocryptotanshinone	√	×	×	×
46	19.43	[M+H]+	301.1434	301.1439	C18H21O4	1.6	MS2[301]: 283(100) MS3[283]: 265(100), 255(26)	Demethyl and two hydroxyl miltirone	×	√	×	×
47	19.81	[M−H]−	313.0707	313.0708	C17H13O6	0.4	MS2[313]: 269(27), 252(100)	Demethyl and two hydroxyl tanshinol B	×	×	√	×
48	20.40	[M+H]+	355.1176	355.1157	C20H19O6	−2.0	MS2[355]: 337(100), 309(45)	Hydroxyl methyltanshinonate	×	√	×	×
49	20.47	[M+H]+	489.1391	489.1397	C24H25O11	1.1	MS2[489]: 313(100), 295(57)	Tanshindiol A/B/C glucuronide conjugate	√	×	×	×
50	21.17	[M+H]+	313.1071	313.1077	C18H17O5	2.0	MS2[313]: 295(100), 265(78)	Tanshindiol A/B/Cb	√	×	√	×
51	21.54	[M+H]+	339.1227	339.1209	C20H19O5	−1.8	MS2[339]: 321(100)	Methyl tanshinonate	×	√	×	×
52	22.00	[M+H]+	297.1121	297.1129	C18H17O4	2.4	MS2[297]: 279(100), 251(49), 237(36)	Hydrated dihydrotanshinone I	×	√	×	×
53	22.49	[M+H]+	313.1071	313.1077	C18H17O5	2.0	MS2[313]: 295(100), 267(7) MS3[295]: 267(100)	Tanshindiol A/B/Cb	√	√	√	×
54	22.98	[M+H]+	309.0758	309.0763	C18H13O5	1.8	MS2[309]: 291(18), 265(20), 235(100) MS3[235]: 207(18), 179(100)	Dihydroxyl tanshinone I	×	√	×	×
55	23.60	[M+H]+	295.0601	295.0603	C17H11O5	0.6	MS2[295]: 267(100) MS3[267]: 239(100)	Demethyl and two hydroxyl tanshinone I	×	×	√	×
56	23.87	[M+H]+	299.1278	299.1281	C18H19O4	0.5	MS2[299]: 281(42), 271(49), 253(100)	Demethyl and hydroxyl cryptotanshinone	×	√	×	×
57	23.89	[M−H]−	311.0914	311.0915	C18H15O5	0.3	MS2[311]: 283(32), 281(100) MS3[281]: 253(100)	Hydroxyl tanshinol B	×	√	×	×
58	24.85	[M+H]+	299.1278	299.1284	C18H19O4	1.1	MS2[299]: 281(19), 271(44), 253(100)	Demethyl and hydroxyl cryptotanshinone	√	√	×	×
59	25.78	[M−H]−	325.0707	325.0706	C18H13O6	−0.3	MS2[325]: 297(31), 295(100), 268(30) MS3[294]: 267(100)	Demethyl and carboxylated tanshinol B	×	√	×	×
60	26.06	[M+H]+	313.1071	313.1074	C18H17O5	1.3	MS2[313]: 295(100)	Tanshindiol A/B/Cb	√	√	√	×
61	26.21	[M+H]+	345.1333	345.1341	C19H21O6	0.5	MS2[345]: 327(100) MS3[327]: 299(100), 281(13)	Demethyl and carboxylated neocryptotanshinone	×	√	×	×
62	28.01	[M−H]−	297.1121	297.1124	C18H17O4	0.9	MS2[297]: 253(100), 239(74), 221(26) MS3[253]: 238(100)	Dihydro tanshinol B	×	√	×	×
63	28.04	[M+H]+	343.1176	343.1184	C19H19O6	0.1	MS2[343]: 325(100)	Dihydroxyl tanshinone IIB	×	√	√	×
64	28.27	[M+H]+	372.1806	372.1811	C21H26O5N	1.7	MS2[372]: 354(100), 311(55), 283(60) MS3[354]: 326(100), 311(59)	Neocryptotanshinone glycine conjugate	×	√	√	×
65	28.44	[M+H]+	313.1434	313.1437	C19H21O4	−0.5	MS2[313]: 269(100) MS3[269]: 254(25), 223(21), 199(100), 171(74)	Hydroxyl cryptotanshinoneb	√	√	√	×
66	28.70	[M−H]−	471.1286	471.1286	C24H23O10	0.0	MS2[471]: 295(100)	Tanshinol B glucuronide conjugate	×	√	×	×
67	28.73	[M+H]+	331.1540	331.1545	C19H23O5	0.7	MS2[331]: 313(100), 295(38)	Hydroxyl and methyl salvianonol	√	×	×	×
68	28.80	[M+H]+	459.1286	459.1287	C23H23O10	1.5	MS2[459]: 283(100) MS3[283]: 265(25), 237(100)	Dihydronortanshinone glucuronide conjugate	×	√	×	×
69	28.99	[M−H]−	471.1286	471.1286	C24H23O10	0.1	MS2[471]: 295(100)	Tanshinol B glucuronide conjugate	×	√	×	×
70	29.00	[M+H]+	618.2116	618.2119	C29H36O10N3S	−0.4	MS2[618]: 471(44), 309(100)	Tanshinone IIB glutathione conjugate	×	×	×	√
71	29.01	[M+H]+	327.1227	327.1231	C19H19O5	1.6	MS2[327]: 309(24), 299(22), 281(100), 263(57) MS3[281]: 263(100), 235(36)	Hydroxyl tanshinone IIB	×	√	×	×
72	29.41	[M+H]+	309.1121	309.1121	C19H17O4	−2.0	MS2[309]: 265(100) MS3[265]: 247(51), 223(100), 195(18)	Hydroxyl and dehydro tanshinone IIA	√	×	×	×
73	29.54	[M+H]+	343.1176	343.1185	C19H19O6	1.1	MS2[343]: 325(100)	Dihydroxyl tanshinone IIB	×	√	×	×
74	30.06	[M+H]+	313.1434	313.1438	C19H21O4	1.1	MS2[313]: 269(35), 251(100) MS3[251]: 223(100)	Hydroxyl cryptotanshinone	×	√	×	×
75	30.44	[M+H]+	445.1857	445.1864	C24H29O8	−1.8	MS2[445]: 269(100) MS3[269]: 254(100), 239(14)	Decarbonylated and glucuronidated cryptotanshinone	×	×	×	√
76	30.63	[M+H]+	293.0808	293.0813	C18H13O4	2.4	MS2[293]: 275(22), 263(100), 249(23) MS3[263]: 235(100)	Hydroxyl tanshinone I	√	√	√	×
77	31.05	[M+H]+	295.0965	295.0966	C18H15O4	1.8	MS2[295]: 251(100) MS3[251]: 223(97), 195(95), 169(100)	Hydrated tanshinone Ib	√	√	×	×
78	31.08	[M+H]+	297.1121	297.1124	C18H17O4	0.6	MS2[297]: 279(100), 261(36) MS3[279]: 261(100)	Demethyl and hydroxyl tanshinone IIA	√	√	√	×
79	31.30	[M+H]+	285.1485	285.1489	C18H21O3	1.0	MS2[285]: 243(100), 229(6) MS3[243]: 228(71), 225(92), 1815(100)	Demethyl and hydroxyl miltirone	√	√	×	×
80	31.32	[M−H]−	311.0914	311.0912	C18H15O5	−0.7	MS2[311]: 267(100), 223(29)	Hydroxyl tanshinol B	×	√	×	×
81	31.36	[M+H]+	473.1806	473.1806	C25H29O9	2.3	MS2[473]: 297(36), 269(100) MS3[269]: 241(100), 213(92), 199(87)	Cryptotanshinone glucuronide conjugate	×	×	√	×
82	31.61	[M+H]+	339.1227	339.1210	C20H19O5	1.2	MS2[339]: 321(100), 295(21)	Methyl tanshinonate	×	√	×	×
83	32.13	[M+H]+	459.2014	459.2012	C25H31O8	2.4	MS2[459]: 283(100)	Miltirone glucuronide conjugate	×	×	×	√
84	32.36	[M+H]+	381.1333	381.1318	C22H21O6	2.2	MS2[381]: 363(60), 337(100)	Acetylated methyltanshinone	√	×	×	×
85	32.65	[M+H]+	311.1278	311.1282	C19H19O4	1.4	MS2[311]: 293(40), 275(37), 267(100) MS3[267]: 252(100)	Tanshinone IIBa,b	√	√	√	√
86	32.84	[M+H]+	287.1642	287.1645	C18H23O3	0.8	MS2[287]: 269(100) MS3[269]: 251(58), 241(100), 213(86)	Decarbonylated neocryptotanshinone	√	×	×	×
87	33.16	[M+H]+	299.1642	299.1646	C19H23O3	1.4	MS2[299]: 281(100), 255(68), 253(62)	Hydroxyl miltirone	×	√	×	×
88	33.24	[M+H]+	267.1016	267.1022	C17H15O3	0.3	MS2[267]: 249(100), 221(22)	Decarbonylated hydrated tanshinone I	√	×	×	×
89	33.31	[M+H]+	457.1493	457.1496	C24H25O9	0.5	MS2[457]: 281(100), 263(73), 261(41) MS3[281]: 263(100)	Danshenxinkun B glucuronide conjugate	×	×	×	√
90	33.59	[M+H]+	341.1384	341.1388	C20H21O5	1.2	MS2[341]: 281(100), 263(42) MS3[281]: 263(100), 235(18)	Methyl dihydrotanshinonateb	×	×	√	×
91	33.67	[M+H]+	279.1016	279.1021	C18H15O3	−0.2	MS2[279]: 261(100) MS3[261]: 233(100), 205(13)	Dihydro tanshinone I	√	×	×	√
92	33.69	[M−H]−	295.0965	295.0968	C18H15O4	1.2	MS2[295]: 277(17), 267(28), 265(100), 238(27) MS3[265]: 237(100)	Tanshinol Ba,b	√	√	√	√
93	33.73	[M+H]+	309.1121	309.1120	C19H17O4	2.5	MS2[309]: 265(100) MS3[265]: 247(55), 223(100)	Hydroxyl and dehydro tanshinone IIA	√	√	×	×
94	34.29	[M+H]+	297.1485	297.1489	C19H21O3	0.3	MS2[297]: 253(100) MS3[253]: 238(33), 225(24), 211(100), 209(16)	Hydroxyl dehydromiltirone	√	×	×	×
95	34.61	[M+H]+	293.1172	293.1174	C19H17O3	1.5	MS2[293]: 275(100), 247(39) MS3[275]: 247(100)	Dehydrotanshinone IIAb	×	×	√	×
96	34.63	[M+H]+	297.1485	297.1490	C19H21O3	1.6	MS2[297]: 279(100), 251(81) MS3[279]: 251(100), 237(67)	Dihydro tanshinone IIA	×	×	×	√
97	34.72	[M+H]+	311.1278	311.1284	C19H19O4	1.8	MS2[311]: 283(100) MS3[283]: 265(100), 237(17)	Hydroxyl tanshinone IIA	√	×	×	×
98	35.14	[M+H]+	279.1016	279.1020	C18H15O3	1.4	MS2[279]: 261(100), 233(5) MS3[261]: 233(100), 215(7), 205(14)	Dihydrotanshinone Ib	×	√	√	√
99	35.36	[M+H]+	293.0808	293.0815	C18H13O4	2.2	MS2[293]: 249(100) MS3[249]: 221(26), 193(100), 178(52)	Hydroxyl hydrotanshinone I	×	×	×	√
100	35.69	[M+H]+	315.1591	315.1594	C19H23O4	1.0	MS2[315]: 297(100) MS3[297]: 279(100), 268(13), 254(18), 251(58)	Hydrated cryptotanshinone	√	×	×	√
101	35.83	[M+H]+	281.1172	281.1178	C18H17O3	2.2	MS2[281]: 263(100), 235(72) MS3[263]: 235(100)	Danshenxinkun Ba b	×	√	√	×
102	35.96	[M+H]+	339.1227	339.1235	C20H19O5	2.5	MS2[339]: 279(100) MS3[279]: 261(100)	Methyl tanshinonateb	×	×	√	√
103	36.22	[M+H]+	295.1329	295.1331	C19H19O3	0.9	MS2[295]: 277(100), 249(40) MS3[277]: 262(53), 249(100), 235(38)	Dehydrocryptotanshinoneb	×	×	√	×
104	36.59	[M+H]+	309.1121	309.1124	C19H17O4	0.8	MS2[309]: 265(100) MS3[265]: 247(52), 223(100)	Hydroxyl and methyl dihydrotanshinone I	×	×	×	√
105	36.67	[M+H]+	301.1798	301.1802	C19H25O3	1.4	MS2[301]: 271(100) MS3[271]: 256(100)	Hydrated miltirone	×	×	√	×
106	36.96	[M+H]+	313.1434	313.1439	C19H21O4	1.5	MS2[313]: 295(100), 277(31), 271(33), 267(25) MS3[295]: 277(100), 253(27), 249(23)	Hydroxyl cryptotanshinone	√	×	×	×
107	37.31	[M+H]+	297.1485	297.1488	C19H21O3	1.0	MS2[297]: 279(100), 251(81) MS3[279]: 251(100), 237(73)	Cryptotanshinonea b	×	√	√	×
108	37.58	[M+H]+	277.0859	277.0863	C18H13O3	1.4	MS2[277]: 249(100), 231(13) MS3[249]: 234(18), 221(89), 193(100), 178(30)	Tanshinone Ia b	√	√	√	×
109	38.82	[M+H]+	279.1016	279.1017	C18H15O3	0.6	MS2[279]: 261(100) MS3[261]: 233(100)	Dihydrotanshinone Ia b	×	√	√	√
110	38.97	[M+H]+	293.1172	293.1178	C19H17O3	2.1	MS2[293]: 275(100), 247(40) MS3[275]: 247(100)	Dehydrotanshinone IIAb	√	√	√	√
111	40.01	[M+H]+	281.1536	281.1540	C19H21O2	1.4		Dehydromiltironeb	×	×	√	×
112	40.23	[M+H]+	293.1172	293.1175	C19H17O3	1.0		Dehydrotanshinone IIAb	√	√	√	×
113	40.58	[M−H]−	277.0859	277.0865	C18H13O3	2.1	MS2[277]: 249(100), 221(53)	Dehydrated tanshinol B	×	×	√	×
114	41.15	[M+H]+	295.1329	295.1331	C19H19O3	0.9	MS2[295]: 277(100), 249(14) MS3[277]: 262(29), 249(100)	Tanshinone IIAa b	√	√	√	√
115	41.27	[M−H]−	277.0859	277.0863	C18H13O3	1.4	MS2[277]: 249(100), 221(60)	Dehydrated tanshinol B	×	×	√	×
116	42.32	[M+H]+	283.1693	283.1694	C19H23O2	0.4	MS2[283]: 265(100), 241(47), 223(63) MS3[265]: 237(62), 223(100)	Miltironeb	×	√	√	×
117	42.70	[M+H]+	269.1536	269.1537	C18H21O2	0.4	MS2[269]: 254(100) MS3[254]: 239(100)	Demethyl miltirone	×	×	√	×
118	47.15	[M+H]+	299.1642	299.1646	C19H23O3	1.3	MS2[299]: 281(100), 256(52), 253(49)	Dihydro cryptotanshinone	×	×	√	×
Total									35	63	62	18

^aConfirmed by reference standards;

^bOriginal components in danshen extract.

"×":detected;"√":undetected

4. Discussion

To better identify the metabolites of danshen in vivo after oral administration, the original components of danshen were identified by HPLC-MS/MS in both negative and positive modes. According to the literature (Wei et al., 2007; Lv et al., 2010), the responses of phenolic acids are more sensitive to negative mode, while those of tanshinones are more sensitive to positive mode. In this study, the phenolic acids were detected in negative mode, and exhibited their parent ions as [M−H]⁻; the tanshinones were detected in positive mode, and exhibited their parent ions of [M+H]⁺ and/or [M+Na]⁺.

Although 10% hydrochloric acid was added to rat plasma to increase the recovery ratios of phenolic acids, few phenolic acids were detected. This may have been because of the low bioavailability and transformation of phenolic acids in vivo (Gao et al., 2009; Sun et al., 2013). Under our experimental conditions, very few phenolic acids and their metabolites were detected in rat bile, except methyl danshensu. Compared with plasma and bile, many more metabolites were detected and unambiguously identified in rat urine and feces. This suggests that urine and feces might be the major route for elimination of danshen after oral administration.

From the analysis of metabolites, we found that hydroxylation (36 out of 118), methylation/demethylation (35 out of 118), glucuronidation (14 out of 118), hydration/dehydration (8 out of 118), and hydrogenation/dehydrogenation (11 out of 118) might be the main metabolic pathways of danshen in vivo. The metabolic pathway for phenolic acids was mainly methylation/demethylation (11 out of 33), while tanshinones mostly showed hydroxylation (31 out of 85) and methylation/demethylation (19 out of 85). Hydrogenation, sulfation, acetylation, and glutathione conjugation were found to be the possible metabolic pathways of danshen. This research provided a comprehensive in vitro chemical profile and in vivo metabolic profile of danshen after oral administration, which could be useful in research on the quality control and pharmacology of danshen.

5. Conclusions

Using HPLC-MS/MS methods, our research provided the most comprehensive chemical and metabolic profiles of danshen. A total of 69 compounds in danshen extract and 118 metabolites were identified, including 35 in plasma, 63 in urine, 62 in feces, and 18 in bile. This analysis of chemical and metabolic components of danshen lays a foundation for further studies of the material composition of danshen, and provides a useful means for identification of multi-components of TCMs both in vitro and in vivo.