Metabolic profile of Cortex Fraxini in rats using UHPLC combined with Fourier transform ion cyclotron resonance mass spectrometry

Abstract

Cortex Fraxini is a widely used traditional Chinese medicine (TCM) used for the treatment of gout or hyperuricemia. In this study, a reliable and sensitive ultra-high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) method was developed for systematical screen and identification of the metabolic profile in rats after oral administration of Cortex Fraxini. The chromatographic separation was performed on a Universil XB C18 column (150 mm × 2.1 mm, 1.8 μm; Kromat, USA) and eluted by a gradient program. The identification was achieved on a Bruker high resolution spectrometer in positive ion mode. According to the result, a total of 81 constituents, including 29 prototype compounds and 52 metabolites were tentatively identified and validated by MS data and fragment ions from MS/MS spectra. The parents of all identified metabolites were assigned, and the metabolites were mainly coumarin derivatives. In conclusion, the newly established UHPLC-FT-ICR-MS method with high resolution and sensitivity was an effective approach for the identification of prototypes and metabolites of Cortex Fraxini in vivo. These results provided significant information about the metabolic profile of Cortex Fraxini in vivo and also laid a solid foundation for further pharmacokinetic and pharmacological research of this TCM.

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1. Introduction

Traditional Chinese medicine (TCM) has been widely used to fight diseases in China for thousands of years. The identification of absorbed components and its metabolites is of great importance for clarifying the therapy mechanism of TCM. Therefore, it is important to develop a reliable and sensitive approach for illustrating metabolic profile of TCM.

Cortex Fraxini (named “Qin Pi” in China) is the dry bark of Oleaceae plant Fraxinus chinensis Roxb., Fraxinus rhynchophylla Hance., Fraxinus stylosa Lingelsh. and Fraxinus szaboana Lingelsh. Cortex Fraxini is a well-known TCM to treat gout, hyperuricemia and arthritis in clinic.^1,2 Cortex Fraxini mainly contains coumarins, iridoids, phenylpropanols and phenolic compounds,^3–5 which have various bioactivities such as anti-oxidant, anti-inflammation and so on.^6–9 Coumarins are the major bioactive constituents in Cortex Fraxini and can be used to treat gout with renal dysfunction in clinic.¹⁰ Among these coumarins, aesculin is the most abundant constitute of coumarins. However, there has been no holistic research on the absorbed constituents and metabolic profile of Cortex Fraxini in vivo.

Metabolism research plays an important role in the development process of drugs, such as the assessment of action mechanism. Drugs can produce new active or toxic species via metabolism, so it is necessary for the evaluation of efficacy and safety of a drug.¹¹ Nowadays, metabolism studies are designed to suggest new active constitutes and support further toxicology researches.¹² To obtain enough metabolic information from complex biological samples, it is essential to establish a sensitive and reliable analytical method. Till now, liquid chromatography combined with mass spectrometry (LC-MS) has been widely utilized as an efficient tool to detect and identify metabolites in complex matrixes due to its high sensitivity and selectivity.^13–16 Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) with high mass accuracy and high resolution is the most powerful tool for the various analyses.^17–20 FT-ICR-MS is specialized in the determination of elemental composition and can be used to identify metabolites.^21,22

In this present study, an ultra high performance liquid chromatography equipped with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) method was established for systematical characterization of metabolic profile in plasma, urine, bile and feces from rats after oral administration of Cortex Fraxini. The metabolic profile of Cortex Fraxini in vivo was revealed for the first time. The results of this study may provide significant information for further research on Cortex Fraxini, such as the relationship between pharmacological activities and chemical constituents in Cortex Fraxini.

2. Materials and methods

2.1 Chemicals and reagents

Cortex Fraxini (lot number: 140501; source: Shanxi, China) was bought from Guoda pharmacy (Shenyang, China) and identified by professor Jingming Jia (Department of TCM, Shenyang Pharmaceutical University, Shenyang, China). A voucher specimen (no. 2015-1012) was deposited at college of TCM, Shenyang Pharmaceutical University. HPLC-grade methanol was obtained from Fisher Scientific (Fair Lawn, NJ, USA) and the distilled water was purchased from Wahaha Co., LTD. (Hangzhou, China).

2.2 Preparation of Cortex Fraxini for animal treatment

For administration, the powder of Cortex Fraxini (200 g) was extracted in boiled water (2 L) for 3 hours. The filtrate was precipitated by 2.5 fold volume of ethanol (90%, v/v). The obtained supernatant was concentrated to 200 mL by rotary evaporation under reduced pressure. Afterwards, the concentrated solution was filtered by D101 macroporous adsorption resin and the resulting solution was dried by lyophilization. The dried powder was stored in a vacuum drying oven. The content of aesculin in dried powder was determined to be 6% by HPLC method.

2.3 Animal experiments

Male Sprague Dawley rats (200–220 g) were obtained from Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). All rats were housed in SPF grade Experimental Animal House with relative humidity (50 ± 10%), temperature (22 ± 2 °C) and 12 h light/12 h dark cycle, and with food and water freely available. All of the procedures were complied with the Regulations of Experimental Animal Administration (State Committee of Science and Technology of the People's Republic of China) and approved by the Medical Ethic Committee of Shenyang Pharmaceutical University (no. SYPU-IACUC-C2015-0074). All surgery was performed under standard conditions, and all efforts were made to minimize suffering.

The rats were divided into group I and group II randomly with six rats in each group. The rats were fasted for a night with water available before administration. The prepared Cortex Fraxini powder was administrated to rats at the dose of 1.7 g kg⁻¹ body weight (about aesculin 100 mg kg⁻¹)²³ by intragastric gavage, and it was suspended in 0.5% carboxymethyl cellulose sodium salt (CMC-Na) before administration. Rats in group I were put into metabolic cages for 24 h to collect urine and feces. Blood from group I were collected at 0.5, 1, 3, 6, 12 and 24 h after dosing, and were obtained via retro-orbital bleeding into heparinized haemospasia tubes. About 0.5 mL blood was collected at each time point, and then centrifuged at 13 000 rpm for 10 min immediately. After administration, rats in group II were anaesthetized and cannulated in bile duct. Afterwards, bile samples were collected for 12 h. Blank samples of plasma, urine bile and feces were also collected from rats without dosing. All biological samples were stored at −80 °C before analysis.

2.4 Sample preparation

The obtained plasma samples were mixed together, and the urine, feces and bile samples were treated as plasma either. As for the plasma, urine and bile samples, an aliquot of 400 μL of acetonitrile was added into 200 μL mixed samples. The mixtures were vortex mixed for 1 min and then centrifuged at 13 000 rpm for 10 min. Subsequently, the organic layer was transferred and then dried under nitrogen stream at 35 °C. Residues were reconstituted in 200 μL water–methanol (80 : 20, v/v) for UHPLC-FT-ICR-MS analysis. Feces samples were dried in shade and then grind into powder. The powder of feces samples (0.5 g) was extracted by methanol (3 mL) in an ultrasonic water bath for 30 min and then centrifuged at 13 000 rpm for 10 min. All samples were filtered through a 0.22 μm membrane, and the resulting filtrate was transferred into vials of autosampler.

2.5 Instrument and analytical conditions

The chromatographic analysis was performed on an Agilent 1260 UHPLC system (USA). LC separation was achieved on a Universil XB C18 column (150 mm × 2.1 mm, 1.8 μm; Kromat, USA) at 35 °C. The mobile phase contained water (A) and methanol (B). For the separation of samples, the gradient condition of mobile phase was: 25–25% (B) in 0–5 min, 25–40% (B) in 5–13 min, 40–65% (B) in 13–23 min, 65–75% (B) in 23–28 min, 75–75% (B) in 28–30 min. The flow rate was 0.20 mL min⁻¹ and the injection volume was 5 μL.

The mass spectra instrument consisted of a Bruker Solarix 7.0 T FT-ICR-MS system (Bruker, Germany) and a Bruker Compass-Hystar workstation (Bruker, Germany). Ionization was performed with positive electrospray ionization (ESI) mode, and the main parameters were set as follows: capillary voltage, 4.5 kV; dry gas flow, 8 L min⁻¹; dry gas temperature, 200 °C; nebulizer gas pressure, 4 bar. Full-scan mass spectrum data was recorded from m/z 100 to 1000 amu, and the collision energy was ranged from 10 eV to 30 eV for MS/MS experiments.

3. Results and discussion

3.1 Metabolic profile analysis of Cortex Fraxini based on UHPLC-FT-ICR-MS

Under the analytical conditions at “Subsection 2.5”, the base peak ion (BPI) chromatograms of plasma, urine, bile and feces samples from blank and administrated rats are shown in Fig. 1. Samples of administrated were investigated to confirm the prototypes and metabolites against blank samples, because the endogenous substances from complex biological matrices could cause interference. To exclude the interferences in BPI chromatograms, extract ion chromatograms (EICs) of FT-ICR-MS were used to extract the neat mass spectra data of each metabolite respectively, which improved the sensitivity and simplified data processing. The obtained mass spectra data contained retention time, ion mode, precise molecular weight and MS/MS data.

Fig. 1 Base peak intensity (BPIs) chromatograms of biological samples in positive ion modes. P0, U0, U0 and F0 represents blank samples of plasma, urine, bile, and feces from rats; P1, U1, U1 and F1 represents plasma, urine, bile, and feces samples from rats after oral administration of Cortex Fraxini.

The element compositions of each constitute were deduced by Bruker workstation, and the mass error values between experimental and theoretical below 3.0 ppm were acceptable. The chemical structure were proposed by scrutinizing the retention time, deduced molecular formula, and then further confirmed by MS/MS data and published literatures.^23–26 As a result, a total of 81 constitutes were found and identified, including 29 prototype components (P1–P29) and 52 biotransformed metabolites (M1–M52). The results of prototype components are shown in Table 1 and the results of metabolites are presented in Table 2. The tables included the information of formula, retention time, MS data, calculated m/z, error, ion mode and MS/MS data.

Table 1 UHPLC-FT-ICR-MS analysis of the prototype compound in rat plasma, urine, bile and feces^a

No.	Rt (min)	Identification	Formula	Molecular weight	Ion mode	MS (m/z)	ppm	MS/MS (m/z)	Source
a P, U, B and F represented rat plasma, urine, bile and feces samples respectively.
P1	2.73	Sinapaldehyde glucoside	C17H22O9	370.1263	[M + Na]^+	393.11593	−0.83	209.07832, 166.06245, 138.03178	B
P2	3.72	Aesculin	C15H16O9	340.0794	[M + H]^+	341.08672	−0.03	179.03455, 151.04002, 133.02970	P, U, B, F
P3	4.03	Osmanthuside H	C19H28O11	432.1631	[M + Na]^+	455.15240	−0.03	301.12958, 151.06155, 139.07636	P, U, B, F
P4	4.61	Syringin	C17H24O9	372.1420	[M + H]^+	373.14954	−0.61	364.06355, 232.23347, 185.01094	P, U, B
P5	5.12	Fraxin	C16H18O10	370.0900	[M + H]^+	371.09721	0.18	209.02670, 194.02135, 181.03957	P, U, B
P6	5.21	Fraxetin diglucoside	C22H28O15	532.1428	[M + Na]^+	555.13204	−0.03	369.07688, 207.02612, 192.00275	P, U, B
P7	5.52	Scopolin	C16H18O9	354.0950	[M + Na]^+	377.08437	−0.18	324.08195, 192.03785, 15104688	U, B
P8	6.17	Aesculetin	C9H6O4	178.0266	[M + H]^+	179.03386	0.12	151.03953, 133.29895, 123.04881	P, U, B, F
P9	6.24	6,7-Dimethoxy-coumarin	C11H10O4	206.0579	[M + H]^+	207.06521	−0.11	174.96985, 143.01934, 132.96804	U, B
P10	6.71	6-Hydroxy-7,8-dimethoxy-coumarin	C11H10O5	222.0528	[M + H]^+	223.06012	0.13	208.03751, 190.02652, 162.03250	P, U, B, F
P11	7.34	Oleoside-11-methylester	C17H24O11	404.1318	[M + Na]^+	427.12142	−0.78	265.06851, 206.05722	U, B
P12	8.42	Fraxetin	C10H8O5	208.0372	[M + H]^+	209.04436	0.42	194.02248, 181.05052, 163.03901	P, U, B, F
P13	9.08	Pinoresinol diglucoside	C32H42O16	682.2472	[M + Na]^+	705.23729	−1.1	521.19882, 359.12826, 235.12854	U, B, F
P14	9.82	Umbelliferone glucoside	C15H16O8	324.0845	[M + H]^+	325.09206	−0.83	307.08361, 181.05062, 135.04462	U
P15	10.42	Sopoletin	C10H8O4	192.0423	[M + H]^+	193.04957	−0.17	178.01950, 150.03186, 133.04562	P, U, B, F
P16	11.92	Isoscopoletin	C10H8O4	192.0423	[M + H]^+	193.04952	0.12	178.02604, 133.02886, 122.03788	P, U, B, F
P17	12.98	Pinoresinol-β-D-glucopyraside	C26H32O11	520.1944	[M + H]^+	521.20232	−1.12	381.12628, 359.12647, 235.17894	P, U, B, F
P18	13.49	Isofraxidin	C11H10O5	222.0528	[M + H]^+	223.05998	0.52	208.03711, 190.02651, 162.03226	P, U, B, F
P19	16.11	8-Hydroxy-6,7-dimethoxy-coumarin	C11H10O5	222.0528	[M + H]^+	223.06010	0.36	208.03718, 190.02624, 162.03228	P, U, B, F
P20	16.62	Umbelliferone	C9H6O3	162.0317	[M + H]^+	163.03899	0.86	145.02928, 135.04429, 117.03427	P, U
P21	16.79	Calceolariolside A	C23H26O11	478.1475	[M + H]^+	479.15552	−1.53	461.02145, 317.12146, 181.04331	P, U, B, F
P22	18.38	Plantainoside B	C23H26O11	478.1475	[M + H]^+	479.15238	0.01	461.01027, 317.11240, 181.04161	P, U, B, F
P23	19.22	Calceolariolside B	C23H26O11	478.1475	[M + H]^+	479.15533	−1.13	461.02164, 317.12183, 181.04396	P, U, B, F
P24	19.97	Plantainoside A	C23H26O11	478.1475	[M + H]^+	479.15552	−0.76	461.00157, 317.12163, 181.04481	P, U, B, F
P25	20.34	Fraxidin-8-glucoside	C17H20O10	384.1056	[M + H]^+	385.11302	−0.24	208.03748, 190.03861, 163.03907	P, B
P26	20.36	Pinoresinol	C20H22O6	358.1416	[M + H]^+	359.14910	−0.51	341.13855, 235.12579, 217.25750	U, B, F
P27	22.24	Hydroxyframoside B	C32H38O14	646.2261	[M + Na]^+	669.21479	0.87	485.16585, 155.05429, 139.06012	U, B, F
P28	22.72	Framoside	C32H38O13	630.2312	[M + Na]^+	653.22121	−1.14	491.17348, 161.06314	U, B, F
P29	23.64	Ligustroside	C25H32O12	524.1839	[M + Na]^+	547.17860	0.05	385.14471, 315.07982	P, B

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Table 2 UHPLC-FT-ICR-MS analysis of the metabolites in rat plasma, urine, bile and feces^a

No.	Formula	Molecular weight	Rt (min)	MS (m/z)	ppm	Ion mode	Source	MS/MS	Metabolite description	Presumed parent
a P, U, B and F represented rat plasma, urine, bile and feces samples respectively.
M1	C15H18O11	374.0849	3.52	397.07442	−0.73	[M + Na]^+	U, B	213.03245, 168.03583, 142.01927	Hydrolysis, hydroxylation	Aesculin
M2	C15H16O13S	436.0312	3.18	437.04086	−3.00	[M + H]^+	U	357.08262, 195.03421, 149.16774	Hydroxylation, sulfation	Aesculin
M3	C15H16O11	372.0693	2.72	373.06822	−2.32	[M + H]^+	P	211.01657, 183.03941, 165.29849	Hydroxylation	Aesculin
M4	C15H16O10	356.0743	4.66	357.08178	−0.43	[M + H]^+	U	195.03478, 167.04072, 149.16678	Oxidation	Aesculin
M5	C15H16O10	356.0743	5.94	357.08176	−0.44	[M + H]^+	P	195.02147, 177.01417, 167.01265	Hydroxylation	Aesculin
M6	C15H18O10	358.0900	3.59	381.07925	−0.08	[M + Na]^+	U, B, F	197.03733, 179.12461, 127.03518	Hydrolysis	Aesculin
M7	C16H18O10	370.0900	3.04	371.09755	−0.75	[M + H]^+	U, B, F	231.02660, 216.02145, 185.03948	Hydroxylation, methylation	Aesculin
M8	C16H18O10	370.0900	4.14	371.09747	−0.52	[M + H]^+	P, U, B, F	231.02678, 216.02182, 185.03957	Hydroxylation, methylation	Aesculin
M9	C16H20O10	372.1056	5.69	373.11315	−0.60	[M + H]^+	U, B, F	359.11255, 197.04023, 179.10544	Hydrolysis, methylation	Aesculin
M10	C17H21NO11	415.1115	5.61	416.11893	−0.46	[M + H]^+	P, U, F	254.06213, 236.07512, 127.04587	Hydrolysis, glycine conjugation	Aesculin
M11	C15H14O12S	418.0206	4.79	419.04671	−0.87	[M + H]^+	U	339.09046, 179.03387, 133.02884	Oxidation, sulfation	Aesculin
M12	C15H18O13S	438.0468	4.42	439.05646	−2.91	[M + H]^+	U	359.12516, 197.03753, 179.18248	Hydrolysis, sulfation	Aesculin
M13	C21H26O16	534.1221	12.32	535.12466	2.17	[M + H]^+	P, U, B	359.10428, 197.02656, 127.03268	Hydrolysis, glucuronidation	Aesculin
M14	C15H14O9	338.0638	4.07	339.07287	−2.10	[M + H]^+	U	177.09012, 137.06004, 133.02895	Dehydrogenation	Aesculin
M15	C21H22O16	530.0907	2.80	553.08029	−0.51	[M + Na]^+	U, B	355.07767, 179.03454, 123.04387	Deglycosylation, glucuronidation	Aesculin
M16	C21H24O15	516.1115	2.98	539.10048	−0.47	[M + Na]^+	P, B	341.08676, 179.03458, 133.02977	Glucuronidation	Aesculin
M17	C11H10O6	238.0477	5.84	239.05509	−0.31	[M + H]^+	U	225.03274, 209.04452, 177.19018	Hydroxylation, methylation	Fraxetin
M18	C11H12O6	240.0634	12.64	263.05255	0.21	[M + Na]^+	P, U, B	227.03998, 211.05458, 179.21458	Hydrolysis, methylation	Fraxetin
M19	C11H10O8S	302.0096	7.49	303.01707	−0.52	[M + H]^+	P, U, B	223.05987, 208.03715, 190.02658	Methylation, sulfation	Fraxetin
M20	C16H16O11	384.0693	3.29	385.07652	0.05	[M + H]^+	U, B, F	209.04438, 194.02249, 181.05074	Glucuronidation	Fraxetin
M21	C16H16O11	384.0693	4.96	385.07660	−0.16	[M + H]^+	P, U, B, F	209.04447, 193.02248, 179.03425	Glucuronidation	Fraxetin
M22	C10H10O6	226.0477	3.40	227.05509	−0.32	[M + H]^+	B	213.03241, 195.01254, 143.02648	Hydrolysis	Fraxetin
M23	C16H20O11	388.1006	10.33	411.08995	−0.40	[M + Na]^+	U, B	227.04654, 213.03217, 143.05621	Hydrolysis, glucuronidation	Fraxetin
M24	C10H8O8S	287.9940	6.95	289.00119	0.26	[M + H]^+	P, B	209.04434, 194.02244, 181.05058	Sulfation	Fraxetin
M25	C9H10O6	214.0477	7.65	237.03705	−0.37	[M + Na]^+	U, B	196.04657, 127.03584	Hydrolysis, hydroxylation	Aesculetin
M26	C16H16O10	368.0743	4.37	369.08153	0.26	[M + H]^+	P, U, B, F	193.04952, 178.01958, 150.03184	Glucuronidation, methylation	Aesculetin
M27	C16H16O10	368.0743	5.76	369.08180	−0.48	[M + H]^+	B	193.04945, 178.02605, 133.02885	Glucuronidation, methylation	Aesculetin
M28	C15H14O13S	434.0155	2.86	457.00491	−0.38	[M + Na]^+	B	355.05844, 259.12541, 151.07592	Glucuronidation, sulfation	Aesculetin
M29	C9H8O5	196.0372	1.86	219.02672	−1.50	[M + Na]^+	P, U, B	179.06145, 153.04582	Hydrolysis	Aesculetin
M30	C9H6O7S	257.9834	6.23	280.97272	−0.29	[M + Na]^+	P, U	151.03911, 133.29805, 123.04871	Sulfation	Aesculetin
M31	C10H8O5	208.0372	5.03	209.04452	−0.35	[M + H]^+	U, B, F	195.04255, 133.29811, 123.04882	Hydroxylation, sulfation	Aesculetin
M32	C10H8O5	208.0372	5.90	209.04446	−0.05	[M + H]^+	U, B	195.04270, 133.29827, 123.04885	Hydroxylation, sulfation	Aesculetin
M33	C10H8O5	208.0372	7.45	209.04440	0.25	[M + H]^+	P, B, F	195.04252, 133.29830, 123.04877	Hydroxylation, sulfation	Aesculetin
M34	C15H14O10	354.0587	3.78	355.06582	0.42	[M + H]^+	P, U, B, F	179.03384, 151.03952, 133.29899	Glucuronidation	Aesculetin
M35	C15H14O10	354.0587	4.73	355.06582	0.42	[M + H]^+	U	179.03384, 151.03952, 133.29887	Glucuronidation	Aesculetin
M36	C11H12O5	224.0685	5.64	225.07579	−0.18	[M + H]^+	U, B	211.05284, 197.03554	Hydrolysis, methylation	Aesculetin
M37	C11H12O5	224.0685	6.56	225.07576	−0.04	[M + H]^+	U, B, F	211.05271, 197.03549	Hydrolysis, methylation	Aesculetin
M38	C10H12O6	228.0634	12.83	251.05250	0.42	[M + Na]^+	U	211.04851, 215.04781, 127.03695	Hydrolysis, hydroxylation	Sopoletin
M39	C16H18O11	386.0849	5.11	387.08495	0.16	[M + H]^+	U, B	211.05426, 197.12514, 127.08415	Hydrolysis, glucuronidation	Sopoletin
M40	C17H20O11	400.1006	4.70	423.09010	−0.75	[M + Na]^+	P, B	239.04547, 225.04587, 221.04574	Hydroxylation, methylation	Fraxin
M41	C17H20O11	400.1006	5.58	423.08973	0.13	[M + Na]^+	P, U, B, F	239.04505, 225.04675, 221.04607	Hydroxylation, methylation	Fraxin
M42	C17H22O11	402.1162	2.57	425.10596	−1.24	[M + Na]^+	P, U, B, F	241.06457, 227.04587, 171.05742	Hydrolysis, methylation	Fraxin
M43	C16H20O14S	468.0574	19.98	469.06565	−2.13	[M + H]^+	B	389.10087, 307.01285, 226.04791	Hydrolysis, sulfation	Fraxin
M44	C22H26O10	546.1221	3.46	547.13058	−2.22	[M + H]^+	B	371.09717, 231.02658, 216.02134	Glucuronidation	Fraxin
M45	C10H8O7S	271.9991	8.13	273.00631	0.14	[M + H]^+	P, U, B	193.04964, 178.01962, 150.03178	Sulfation	Sopoletin
M46	C10H8O7S	271.9991	9.46	273.00624	0.14	[M + H]^+	P	193.04948, 178.02609, 133.02878	Sulfation	Isoscopoletin
M47	C32H38O17S	726.1830	18.21	727.19193	−2.32	[M + H]^+	B	647.22619, 485.16587, 155.05434	Sulfation	Hydroxyframoside B
M48	C17H18O11	398.0849	6.98	399.09219	0.28	[M + H]^+	P, U	223.06027, 208.03764, 162.03267	Glucuronidation	6-Hydroxy-7,8-dimethoxy-coumarin
M49	C17H18O11	398.0849	10.41	399.09199	0.49	[M + H]^+	P, F	223.06024, 208.03724, 162.03264	Glucuronidation	Isofraxidin
M50	C17H18O11	398.0849	10.88	399.09202	0.40	[M + H]^+	P, B, U	223.06042, 208.03787, 162.03242	Glucuronidation	8-Hydroxy-6,7-dimethoxy-coumarin
M51	C26H30O12	534.1737	17.62	535.18127	−0.49	[M + H]^+	U, B	359.14941, 341.13867, 235.12564	Glucuronidation	Pinoresinol
M52	C38H46O20	822.2582	19.56	823.26451	1.23	[M + H]^+	F	647.22651, 485.16558, 155.05474	Glucuronidation	Hydroxyframoside B

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According to the results, some prototype compounds were found in all biological sources (urine, plasma, bile and feces samples), including aesculin (P2), osmanthuside H (P3), eesculetin (P8), 6-hydroxy-7,8-dimethoxy-coumarin (P10), fraxetin (P12), sopoletin (P15), isoscopoletin (P16), pinoresinol-β-D-glucopyraside (P17), isofraxidin (P18), 8-hydroxy-6,7-dimethoxy-coumarin (P19), calceolariolside A (P21), plantainoside B (P22), calceolariolside B (P23) and plantainoside A (P24). While sinapaldehyde glucoside (P1) and umbelliferone glucoside (P14) were found only in bile and urine samples respectively. Other prototype compounds could be found in different biological sources. As for the metabolites, M8, M21, M26, M34, M41 and M42 were found in urine, plasma, bile and feces samples. Notably, M2, M4, M11, M12, M14, M17, M35 and M38 were only found in urine samples.

3.2 Mass spectral fragmentation of constitutes from Cortex Fraxini

Coumarins and its derivatives got higher responses in the mass spectra under positive ion mode. Because coumarins and their related metabolites were the major components in biological samples from rats administrated with Cortex Fraxini, the positive ion mode was selected and coumarin was used as an example to illustrate the mass spectral fragmentation. For instance, fraxin was characterized by the protonated molecule ([M + H]⁺) at m/z 371.09721 (C₁₆H₁₉O₁₀⁺). The ions at m/z 209.02670 and m/z 194.02135 obtained from MS/MS spectrum suggesting the loss of hexose (162 Da) and sequentially methyl (15 Da) from the precursor ion [M + H]⁺. The ions at m/z 181.03957 indicating the loss of CO (28 Da) from the ions at m/z 209.02670. The MS/MS spectrum of fraxin and deduced fragmentation pathways are presented as Fig. 2. Phase II metabolites were the conjugations with SO₃ (80 Da) or C₆H₈O₆ (176 Da) generating sulfate or glucuronide conjugates, and the mass losses of 80 Da or 176 Da could be observed in the mass spectra of conjugates metabolites.

Fig. 2 MS2 spectrum of fraxin in positive ion mode and its proposed fragmentation pathways.

3.3 Prototype components of Cortex Fraxini in rats

The absorbed prototype components of Cortex Fraxini were complex mixtures owing pharmaceutical effects. According to the results, five types of compound structure were identified in biological samples, including coumarins, iridoid glycosides, phenylethanoid glycosides, lignans and phenylpropanoids. The chemical structures of prototype components (P1–P29) are shown as Fig. S1.† The resulting EICs of each prototype compound from plasma samples are displayed in Fig. 3. While the EICs of prototype compounds from urine, bile and feces samples are displayed in Fig. S2–S4,† respectively.

Fig. 3 Extracted ion chromatograms (EICs) for prototype compounds of Cortex Fraxini in plasma samples.

Coumarins are the major compounds in Cortex Fraxini, and 15 coumarin prototypes were identified in this study, including aesculin, fraxin, fraxetin diglucoside, scopolin, aesculetin, 6,7-dimethoxy-coumarin, 6-hydroxy-7,8-dimethoxy-coumarin, fraxetin, umbelliferone glucoside, sopoletin, isoscopoletin, isofraxidin, 8-hydroxy-6,7-dimethoxy-coumarin, umbelliferone and fraxidin-8-glucoside. Other types of prototype are listed as follows: 4 iridoid glycosides including oleoside-11-methylester, hydroxyframoside B, framoside and ligustroside; 5 phenylethanoid glycosides containing osmanthuside H, calceolariolside A, calceolariolside B, plantainoside A and plantainoside B; 3 lignans including pinoresinol diglucoside, pinoresinol-β-D-glucopyraside and pinoresinol; 2 phenylpropanoids containing sinapaldehyde glucoside and syringin.

Among these prototypes, 20 prototype components (P2–P6, P8, P10, P12, P15–P25, P29) were observed in plasma samples; 26 components (P2–P24, P26–P28) were detected in urine samples; 27 components (P1–P13, P15–P19, P21–P29) were identified in bile samples; 18 components (P2–P4, P10, P12, P13, P15, P16, P18, P19, P21–P24, P26–P28) were acquired in feces samples.

3.4 Metabolites of Cortex Fraxini in rats

The prototype components absorbed into plasma could be further metabolized by diverse metabolic enzymes. Except for the 29 prototype components, another 52 constitutes were detected and tentatively proposed to be metabolites of Cortex Fraxini in this study. Among the 52 metabolites, 20 phase I metabolites and 32 phase II metabolites were tentatively identified, and it implied that Cortex Fraxini underwent multiple metabolic reactions in rats after intragastric gavage. The phase I metabolic pathways consisted of hydrolysis, hydroxylation, deglycosylation, methylation, oxidation and dehydrogenation. Meanwhile, phase II metabolic pathways included glucuronidation, sulfation, and glycine conjugation. The chemical structures of metabolites (M1–M52) are presented as Fig. 4. The resulting EICs of each potential metabolites from plasma samples are displayed in Fig. 5. While the EICs of possible metabolites from urine, bile and feces samples are shown in Fig. S5–S7,† respectively.

Fig. 4 The chemical structures of potential metabolites detected by UHPLC-FT-ICR-MS.

Fig. 5 Extracted ion chromatograms (EICs) for metabolites of Cortex Fraxini in plasma samples.

Among them, 23 metabolites (M3, M5, M8, M10, M13, M16, M18, M19, M21, M24, M26, M29, M30, M33, M34, M40–M42, M45, M46, M48–M50) were observed in plasma samples; 38 metabolites (M1, M2, M4–M15, M17–M21, M23, M25, M26, M29–M32, M34–M39, M41, M42, M45, M48, M50, M51) were identified in urine samples; 36 metabolites (M1, M6–M9, M13, M15, M16, M18–M29, M31–M34, M36, M37, M39–M45, M47, M49, M51) were detected in bile samples; 17 metabolites (M6–M10, M20, M21, M26, M31–M34, M37, M40, M42, M49, M52) were acquired in feces samples.

The parent of all identified metabolites were assigned as follows: aesculin, fraxetin, aesculetin, sopoletin, fraxin and others. M1–M16 were supposed to be metabolized from aesculin. M3, M4 and M5 belonged to the hydroxylated metabolites. M2 and M8 were the sulfated and methylated products of M5, respectively, and M7 was the isomer of M8. M4 and M14 belonged to the oxidation metabolites. M6 was the hydrolyzed metabolite, and M6 could be further metabolized to M1 (hydroxylation), M9 (methylation), M10 (glycine conjugation), M12 (sulfation) and M13 (glucuronidation). In addition, aesculin could conjugate with glucuronic acid to generate M16.

M17–M24 were supposed to be metabolized from fraxetin. Fraxetin could be hydroxylated and methylated to M17. M22 was the hydrolyzed metabolite of fraxetin, and M22 could be further metabolized to M18 (methylation) and M23 (glucuronidation). M20 and M21 were the glucuronide conjugated metabolites, and M24 was the sulfated product of fraxetin. M25–M37 were supposed to be metabolized from aesculetin. M29 was the ring-opening metabolite of aesculetin, and M29 could be further metabolized to M25 (hydroxylation), M36 (glucuronidation) and M37 (methylation). M30 was the sulfated product of fraxetin and it could further conjugated with glucuronic acid to produce M28. While M31, M32 and M33 were isomers and they belonged to the hydroxylated and methylated metabolites. M34 and M35 were the glucuronide conjugated metabolites, and they could be further methylated to M26 and M27, respectively.

M38, M39 and M45 were supposed to be metabolized from sopoletin. The hydrolyzed metabolite of sopoletin could be further metabolized to M38 (hydroxylation) and M39 (glucuronidation). M45 was the sulfated product of sopoletin. M40–M44 were supposed to be metabolized from fraxin. Fraxin could be hydroxylated and methylated to generate M40 and M41. The hydrolyzed metabolite of fraxin could be further metabolized to M42 (methylation) and M43 (sulfation). M44 was the glucuronide conjugated product of fraxin. M46 was supposed to be the sulfated metabolite of isoscopoletin and M47–M52 belonged to the glucuronide conjugated or sulfated products of other prototype compound.

According to the results, phase II metabolism including O-sulphate conjugation and O-glucuronide conjugation were the major metabolic pathways of Cortex Fraxini in vivo after administrated to rats. Meanwhile, the general role of phase II metabolism is to reduce toxicity or biological activity by conjugating with endogenous substances, producing conjugations with greater water solubility.

4. Conclusion

This work systematically investigated the metabolic profile of Cortex Fraxini in rats based on UHPLC-FT-ICR-MS, and the metabolic profile of Cortex Fraxini in vivo was revealed for the first time. After oral administration of Cortex Fraxini, a total of 81 constituents, including 29 prototype compounds and 52 metabolites were tentatively found and identified in plasma, urine, feces and bile samples from rats, and validated by MS data and fragment ions from MS/MS spectra. Five types of prototype structure were identified, including coumarins, iridoid glycosides, phenylethanoid glycosides, lignans and phenylpropanoids. The metabolites were mainly coumarin derivatives. The parents of all identified metabolites were assigned to aesculin, fraxetin, aesculetin, sopoletin, fraxin and others, and 20 phase I metabolites and 32 phase II metabolites were observed. It also demonstrated that the newly established UHPLC-FT-ICR-MS method with high resolution and sensitivity was an reliable approach for the identification of prototypes and metabolites of Cortex Fraxini in biological samples. The results provided helpful information for illustrating bioactive chemical constituents as well as action mechanism of Cortex Fraxini, which would be valuable for further research of this TCM.

Conflict of interest statement

The authors have declared no conflict of interest.

Abbreviations

TCM	Traditional Chinese medicine
FT-ICR	Fourier transform ion cyclotron resonance
UHPLC	Ultra-high performance liquid chromatography

Acknowledgements

This research was supported by the National Natural Science Foundation of China (NSFC: 81503035) and the Educational Department of Liaoning Province of China (L2014394).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01428c

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