Identification and mechanism prediction of mulberroside A metabolites in vivo and in vitro of rats using an integrated strategy of UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology.

Mulberroside A is a polyhydroxylated stilbene active component of Morus alba L. Studies have shown that it has antitussive, antiasthmatic, tyrosinase and antioxidation activities. However, little is known about the metabolism of it in vitro and in vivo. In our study, an integrated strategy on the basis of UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology was established to comprehensively research the metabolic characteristic of mulberroside A for the first time. Plasma, urine, feces and liver tissues of rats in the blank group and drug group were collected after intragastric administration of mulberroside A at a dose of 150 mg/kg, and rat liver microsomes were cultured for in vitro metabolism experiment. The biological samples were processed by different methods and analyzed in positive and negative ion modes using UHPLC-Q-Exactive Plus Orbitrap MS. A total of 72 metabolites were finally identified based on the accurate molecular mass, retention time, MS/MS spectra and related literatures combined with the Compound Discoverer 3.1. The metabolic pathways were mainly hydrolysis, glucuronidation, hydrogenation, sulfation, hydroxylation, methylation and their composite reactions. In addition, a network pharmacology method was used to predict the mechanism of action of mulberroside A and its metabolites. In the end, 7 metabolites with high gastrointestinal absorption and drug-likeness and 167 targets were screened by Swiss ADME and Swiss Target Prediction. 1702 items of GO analysis and 158 related signaling pathways of KEGG were enriched using Metascape. This study established a novel integrated strategy based on UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology, which could systematically analyze the metabolism behavior of mulberroside A in vivo and in vitro of rats and provide basis for the further research of mulberroside A.

1 Introduction

Mulberroside A, presenting at relatively high abundance in the roots and twigs of Morus alba L. (Zhou et al., 2013), is a natural polyhydroxylated stilbene compound (Yu et al., 2021). Furthermore, it possesses various therapeutic effects through pharmacological studies, including hypoglycemic (Li et al., 2014), antidiabetic (Xu et al., 2021), anti-inflammatory (Chung et al., 2003), analgesic (Zhang and Shi, 2010), hepatic protective function (Zhang et al., 2008), nephroprotective (Wang et al., 2011), the inhibitory effect of melanogenesis (Park et al., 2011) and neuroprotective (Wang C. P. et al., 2014) effects, etc.

Drug metabolism, also known as drug biotransformation, refers to the process of chemical structure change of drugs under the action of drug enzymes (Jiang et al., 2014). Once a drug enters the body, it is usually converted into various forms through different metabolic pathways (Prasad et al., 2011). Drug metabolism research can elucidate the scientific connotation of drugs, clarify the pharmacodynamic active ingredients, guide the rational use of clinical drugs, and promote the improvement of dosage forms and quality control, which has great significance in the drug discovery process. However, as far as we know, the in vivo and in vitro metabolism study of mulberroside A has been poorly understood. A pharmacokinetic study on mulberroside A revealed that its oral absolute bioavailability was poor in rats (Qiu et al., 1996b). In Qiu F’s previous metabolic research on mulberroside A, four metabolites were isolated from the urine and bile of rats after oral administration of mulberroside A (Qiu et al., 1996a). After that, they reported the identification of 3 metabolites of mulberroside A in the gastrointestinal contents and feces of rats (Zhaxi et al., 2010). However, whether mulberroside A is metabolized prior to be absorbed into the blood has not been reported so far. Therefore, it is important to carry out a comprehensive metabolism study of mulberroside A. Then, we could better understand its metabolic mechanism and promote research for the further potential therapeutic applications.

Contemporarily, ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS), especially high-resolution mass spectrometry (HRMS), has been widely used in drug metabolism analysis (Dong et al., 2021; Yuan et al., 2021). UHPLC has a powerful separation capability, which is currently one of the most effective tools for the separation of complex components. In recent years, network pharmacology has been widely used in the field of traditional Chinese medicine with its unique advantages. The organic combination of metabolite identification and network pharmacology could effectively reveal the mechanism of drug action in the body. In our study, an integrated strategy on the basis of UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology was developed. This strategy was cost-effective to characterize chemical transformations and further gain comprehensive knowledge about mulberroside A.

2 Materials and methods

2.1 Chemicals and reagents

The reference standard, mulberroside A, was purchased from Chengdu Must Biotechnology Co., Ltd. (Sichuan, China), with purity greater than 98% determined using HPLC-UV analysis. Rat liver microsomes were purchased from Wuxi Xinrun Biotechnology Co., Ltd. (Jiangsu, China). HPLC grade methanol, formic acid, and acetonitrile were provided by Thermo Fisher Scientific (Fair Lawn, NJ, United States). Water was obtained from Watsons (Guangdong, China). Grace Pure SPE C18-Low solid-phase extraction cartridges (200 mg/3 ml, 59 μm, 70 Å) were purchased from Grace Davison Discovery Science (Deerfield, IL, United States).

2.2 Animals and drug administration

Six male SD rats (200 ± 10 g) were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Shandong, China). The animals were housed in a controlled environment with the temperature held at 24 ± 2 °C, relative humidity held at 60±5%, and kept on a 12 h light/12 h dark regime. After 7 days of acclimation, the rats were randomly divided into drug group (n=3) and control group (n=3). Before the experiment, all rats were fasted for 12 h with free access to water. Mulberroside A was dissolved in normal saline. The rats in the drug group were given with a dose of 150 mg/kg/d orally for 3 days. A same dose of normal saline was administered to the rats in the control group.

2.3 Sample collection

2.3.1 Plasma and liver tissue sample collection

0.5 ml of blood samples were taken from the suborbital venous plexus of rats at 0.5, 1, 1.5, 2, 4, 6, 12, and 24 h after the last administration. The blood samples collected at different times were mixed and placed in a centrifuge tube coated with sodium heparin. After that, they were centrifuged for 10 min at 3,500 rpm to separate plasma sample. Then, it was frozen in a refrigerator at -80°C. The centrifugation was performed on Centrifuge 5430R (Eppendorf, Germany).

24 h after the last administration, liver tissue was obtained and quenched in liquid nitrogen and then stored at -80°C. 1 g of liver tissue was added to 10 ml of normal saline to homogenize at 4°C and then centrifuged at 3,500 rpm for 10 min to obtain the supernatant.

2.3.2 Urine and feces sample collection

Two groups of rats were housed in the metabolic cage. The urine and feces were collected within 24 h after the last administration. The urine samples were frozen in the refrigerator at -80°C after centrifuged at 12,000 rpm for 15 min, and the feces samples were freeze-dried and ground into powder.

2.3.3 Liver microsomes incubation

MgCl2 and liver microsomes were dissolved in PBS (pH=7.4), formulated into a solution with a MgCl2 concentration of 3 mM and a protein concentration of 1 mg/ml. The solution was used to dissolve mulberroside A with a final drug concentration of 0.1 mg/ml. Drug group and blank group were set up. 900 µl of the above mixture was added to each well of the 6-well plate in the drug group while the blank group was given an equal volume of drug-negative solution. After preheating at 37°C for 5 min, 100 µl NADPH (final concentration of 25 mg/ml) was added to start the reaction and continue to incubate at 37°C. At 5, 10, 15, 30, 45, 60, 120, 240 min after the start of the reaction, 100 µl of the system solution was mixed with 200 µl of cold acetonitrile to terminate the reaction. Then, the supernatant was obtained after centrifuged at 20,000 rpm for 10 min.

2.4 Sample preparation

The SPE method for precipitation and concentration of protein and solid residues was applied to pretreat all the biological samples. Before loading and elution, 2 g feces was added to 10 ml pure water, sonicated for 0.5 h, and centrifuged to obtain the supernatant. Then, plasma, urine, feces, and liver tissues samples (1 ml) were added to SPE cartridges pretreated with methanol (3 ml) and deionized water (3 ml), respectively. After that, the SPE cartridges were successively washed with deionized water (3 ml) and methanol (3 ml). The methanol eluent was collected and dried under N2 at 4°C. The residue was then redissolved in 300 µl methanol and centrifuged for 15 min at 20,000 rpm (4°C).

In addition, plasma samples were treated with methanol (3 ml) and acetonitrile (3 ml), respectively. Then, they were centrifuged at 3,500 rpm. The obtained supernatant was dried under N2 at room temperature. The residue was then redissolved in 300 µl methanol and centrifuged for 15 min at 20,000 rpm (4°C).

All the biological samples from the same group were merged into a collective sample for the further instrumental analysis.

2.5 Instruments and analytical conditions

2.5.1 UHPLC parameters

The separation was performed on Vanquish UHPLC (Thermo Scientific, Germany) with a ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). The flow rate was 0.3 ml/min and the injection volume was 5 µl. The column temperature was maintained at 35°C. The mobile phase consists of acetonitrile(A) and 0.1% formic acid-water (B). The gradient elution condition was set as: 0–5.0 min, 95% B; 5.0–10.0 min, 95%–70% B; 10.0–15.0 min, 70%–50% B; 15.0–32.0 min, 50%–10% B; 32.0–32.1 min, 10%–95% B; 32.1–35.0 min, 95% B.

2.5.2 HRMS parameters

The MS analysis was performed on Q-Exactive Plus Orbitrap MS (Thermo Scientific, Germany) with a heated electrospray ionization (HESI) source. A high-resolution mass spectrum was acquired at full scan in a mass range of m/z 80–m/z 1,200 at a resolution of 70,000 with AGC target at 3e6. The dd-MS2 data were obtained at a resolution of 17,500 with AGC target at 1e6. Mass spectrometric detection was performed in positive and negative ion modes. The ion source parameters were set as follows: sheath gas and auxiliary gas of nitrogen (purity ≥99.99%) with the flow rate of 45 arb and 10 arb; capillary temperature of 320 °C; spray voltage of 3,800/3,500 V (+/-); probe heater temperature of 320°C. The stepped normalized collision energy (NCE) was set at 15, 30 and 45. S-Lens RF Level was 50.00.

2.6 Data processing and statistical analyses

Thermo Scientific Xcalibur 3.1 workstation and Compound Discoverer 3.1 software were used to process and analyze the collected mass spectrometry data. The chemical formulas attributed to the selected peaks were calculated using a formula predictor by setting the parameters as follows: C [5-40], H [5-60], O [2-30], S [0-2], N [0-3] and the ring double bond (RDB) equivalent value [3-20]. The HRMS data obtained from UHPLC-Q-Exactive Plus Orbitrap MS was imported into the Compound Discoverer, and the structure information of mulberroside A was added to Compound Discoverer at the same time. Then, Compound Discoverer gave the information of possible metabolites according to the structural characteristics of mulberroside A, HRMS information and the corresponding databases. In addition, the metabolites were selected by Swiss ADME (http://www.swissadme.ch/) and the targets were predicted using Swiss Target Prediction (http://www.swisstargetprediction.ch/). All genes were submitted to Metascape (Zhou et al., 2019) (http://metascape.org) for GO and KEGG analysis and Cytoscape 3.8.2 was used for the construction of metabolite-target network and metabolite-target-pathway network. The visual analysis of GO and KEGG enrichment results was performed using Bioinformatics online platform (http://www.bioinformatics.com.cn/).

3 Results and discussion

3.1 The establishment of an analytical strategy

In order to understand the metabolism and mechanism of mulberroside A comprehensively, an efficient and integrated strategy was established based on the UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology, as shown in Figure 1. (1) The rats were given the mulberroside A solution by intragastric administration and biological samples were collected. Then the treated biological samples and standard mulberroside A samples were injected into UHPLC-Q-Exactive Plus Orbitrap MS to obtain HRMS data. (2) The information such as accurate molecular mass, retention time and MS/MS spectra of compounds was obtained by Thermo Scientific Xcalibur 3.1 and then the compounds were identified based on the information and related literatures. After that, the metabolic pathway of mulberroside A could be predicted on the basis of metabolites and metabolic reactions. (3) Swiss ADME and Swiss Target Prediction were applied to screen the metabolites with high gastrointestinal absorption and drug-likeness and predict the targets of these selected metabolites, which were used to map the metabolite-target network by Cytoscape 3.8.2. Then, Metascape was used for GO and KEGG analysis. In the end, the metabolite-target-pathway network was also constructed by Cytoscape 3.8.2.

FIGURE 1

Summary diagram of the established analytical strategy.

3.2 Analysis of the mass spectrometry fragmentation behaviors of mulberroside A

We summarized the mass spectrometry fragmentation behaviors of mulberroside A according to related literatures reported and the MS2 information of the reference substances (Sun, 2004; Pan et al., 2015; Xu, 2015; Guo et al., 2018; Zhang, 2018; Liang et al., 2021). As shown in Figure 2 and Figure 3, the [M+H]+ and [M-H]- was generated in positive and negative ion modes at m/z 569.1846 and m/z 567.1695, respectively, in the ESI-MS spectra of mulberroside A. Characteristic fragments were detected in positive ion mode: m/z 407 [M+H-glucose]+ ([M+H-Glu]+), m/z 245 [M+H-2Glu]+, m/z 227 [M+H-2Glu-H2O]+, m/z 199 [M+H-2Glu-H2O-CO]+, m/z 135 [M+H-2Glu-C6H6O2]+. Representative ions were detected in negative ion mode as follows: m/z 405 [M-H-Glu]-, m/z 243 [M-H-2Glu]-, m/z 225 [M-H-2Glu-H2O]-, m/z 215 [M-H-2Glu-CO]-, m/z 199 [M-H-2Glu-CO2]-, m/z 181 [M-H-2Glu-CO2-H2O]-, m/z 175 [M-H-2Glu-C3O2]−, m/z 157 [M-H-2Glu-C3O2-H2O]−. The mass fragmentation behavior of mulberroside A in positive and negative ion modes are shown in Figure 4 and Figure 5 respectively.

FIGURE 2

The MS (A) and MS2 (B) spectra of Mulberroside A in positive ion mode.

FIGURE 3

The MS (A) and MS2 (B) spectra of Mulberroside A in negative ion mode.

FIGURE 4

The mass fragmentation behavior of Mulberroside A in positive ion mode.

FIGURE 5

The mass fragmentation behavior of Mulberroside A in negative ion mode.

3.3 Detection and structural elucidation of mulberroside A metabolites

A total of 76 metabolites were finally identified based on the accurate molecular mass, retention time, MS/MS spectra and related literature reports in the plasma, urine, feces, liver tissues and liver microsomes. The chromatographic and MS data of these detected metabolites were summarized in Table 1.

TABLE 1

Summary of mulberroside A metabolites in vivo and in vitro of rats.

Peak	t R/min	Ion mode	Formula [M-H]−	Theoretical Mass (m/z)	Experimental Mass (m/z)	RDB	Error (ppm)	MS/MS fragment ions	Identification/Reactions	PS	PM	PA	U	F	L	LM	Max. Peak area
M0	8.18	N	C26H31O14	567.1708	567.1707	11.5	−0.197	243.06(100.00), 567.17(27.20), 405.12(22.51), 225.05(10.57), 199.08(7.11), 175.07(5.26)	Mulberroside A	√	√	√				√	1.53E+06 (PM)
M1*	5.55	N	C12H13O8	285.0605	285.0607	6.5	0.618	109.03(100.00), 113.02(61.96), 285.06(38.68), 175.02(18.76)	β-D-Glucopyranosiduronic Acid 3-Hydroxyphenyl	√	√	√	√				2.53E+08 (U)
M2	7.72	N	C26H31O15	583.1652	583.1658	11.5	0.143	259.06(100.00), 583.17(45.98), 421.11(32.65), 215.07(2.51)	hydroxylation							√	2.08E+06 (LM)
M3	7.98	N	C26H29O15	581.1501	581.1499	12.5	-0.338	581.15(100.00), 243.06(52.49), 225.05(6.60)	hydrolysis	√	√						2.25E+06 (PS)
									glucuronidation
M4	8.18	N	C26H33O18S	665.1382	665.1384	10.5	0.239	96.96(100.00), 243.07(5.60), 567.17(4.23), 665.15(3.58), 405.12(0.52)	hydrogenation				√				3.58E+07 (U)
									hydroxylation
									sulfation
M5	8.19	N	C27H33O16	613.1763	613.1750	11.5	-2.155	243.07(100.00), 567.17(43.17), 225.05(7.89), 405.12(6.46), 435.56(0.28), 448.96(0.28)	dihydroxylation	√	√	√					5.81E+05 (PS)
									methylation
M6	8.20	N	C26H31O15	583.1652	583.1658	11.5	-1.314	259.06(100.00), 421.11(55.47), 583.17(39.04), 241.05(18.86)	hydroxylation							√	8.12E+06 (LM)
M7	8.20	N	C26H31O16	599.1607	599.1602	11.5	-0.736	137.02(100.00), 599.16(57.05), 125.02(50.33), 437.11(45.05), 275.06(25.62)	dihydroxylation							√	3.98E+06 (LM)
M8	8.20	N	C20H21O11	437.1078	437.1079	10.5	0.042	137.02(100.00), 437.11(72.57), 257.04(56.49), 275.05(30.75)	hydrolysis							√	3.16E+06 (LM)
									dihydroxylation
M9	8.24	N	C26H33O16	601.1763	601.1769	10.5	1.046	137.02(100.00), 243.07(42.31), 601.18(33.69), 567.17(20.17), 439.12(13.78)	hydrogenation							√	2.73E+06 (LM)
									dihydroxylation
M10	8.29	N	C21H21O10	433.1129	433.1125	11.5	-1.000	313.07(100.00), 433.11(84.30), 343.08(36.15), 257.08(17.32), 243.07(10.86)	hydrolysis					√			1.39E+07 (F)
									methylation
									glucuronidation
M11	8.43	N	C26H29O16	597.1450	597.1450	12.5	0.015	597.14(100.00), 421.11(99.27), 245.08(21.33), 175.02(22.04), 135.04(9.32), 243.06(7.70), 157.01(3.09)	hydrolysis				√				5.97E+07 (U)
									hydrogenation
									bis-glucuronidation
M12	8.52	N	C20H17O10	417.0816	417.0803	12.5	−3.268	417.17(100.00), 197.13(7.56), 241.12(7.42)	hydrolysis					√			3.16E+04 (F)
									dehydrogenation
									glucuronidation
M13	8.57	N	C26H31O15	583.1658	583.1650	11.5	−1.212	583.17(100.00), 245.01(27.22), 421.11(1.19)	hydrolysis				√				8.16E+06 (U)
									hydrogenation
									glucuronidation
M14	8.62	N	C32H39O21	759.1978	759.1960	13.5	−2.364	421.11(100.00), 245.08(9.61), 175.02(7.66), 135.04(3.69), 109.03(3.24), 583.47(0.27)	hydrolysis				√				8.17E+06 (U)
									hydrogenation
									bis-glucuronidation
M15	8.76	N	C26H27O16	595.1294	595.1291	13.5	−0.455	419.10(100.00), 595.13(76.29), 243.07(75.87), 175.02(18.03)	hydrolysis	√		√	√				1.02E+08 (U)
									bis-glucuronidation
M16	8.77	N	C26H29O15	581.1501	581.1495	12.5	−0.958	581.15(100.00), 243.07(73.83), 405.12(3.42), 225.05(4.57)	Hydrolysis	√							2.86E+06 (PS)
									Glucuronidation
M17	8.80	N	C20H19O13S	499.0541	499.0544	11.5	0.706	499.05(100.00), 243.07(85.68), 323.02(55.07), 225.05(11.54), 419.09(10.13)	Hydrolysis		√	√					1.29E+06 (PM)
									Glucuronidation
									sulfation
M18	8.83	N	C20H21O13S	501.0697	501.0696	10.5	−0.315	501.07(68.38), 421.11(28.50), 325.04(100.00), 245.08(27.22)	hydrolysis				√				4.92E+06 (U)
									hydrogenation
									glucuronidation
									sulfation
M19	8.84	N	C26H29O16	597.1450	597.1448	12.5	−0.286	597.14(93.54), 421.11(77.15), 245.08(20.01), 175.02(20.91), 135.04(9.39), 243.07(7.15), 157.01(2.96)	hydrolysis				√				1.26E+08 (U)
									hydrogenation
									bis-glucuronidation
M20	8.94	N	C26H29O16	597.1450	597.1450	12.5	0.015	597.14(100.00), 421.11(58.07), 245.08(14.71), 175.02(13.24), 135.04(6.56), 157.01(2.30), 243.07(2.11)	hydrolysis				√				9.58E+07 (U)
									hydrogenation
									bis-glucuronidation
M21	8.96	N	C14H15O8	311.0761	311.0767	7.5	1.659	113.02(100.00), 175.02(22.38), 135.04(8.38), 311.08(8.25)	isomer of 6-(4-Ethenyl-2-hydroxyphenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid				√				1.10E+07 (U)
M22	9.19	N	C20H21O10	421.1129	421.1131	10.5	0.491	421.11(100.00), 175.02(19.29), 245.08(8.78), 135.04(8.06)	hydrolysis	√			√				2.52E+09 (U)
									hydrogenation
									glucuronidation
M23	9.25	N	C26H27O16	595.1294	595.1292	13.5	−0.355	419.10(100.00), 243.06(44.01), 595.13(36.57), 225.05(8.28), 175.02(7.61)	hydrolysis			√					3.85E+06 (PA)
									bis-glucuronidation
M24	9.28	N	C20H19O13S	499.0541	499.0541	11.5	−0.075	243.07(100.00), 323.02(87.60), 499.05(81.02), 419.10(17.67), 225.05(10.32)	hydrolysis	√	√	√					2.94E+05 (PM)
									glucuronidation
									sulfation
M25	9.29	N	C26H29O15	581.1501	581.1500	12.5	−0.132	581.15(100.00), 243.07(75.56), 405.12(3.07), 225.05(7.90)	hydrolysis	√	√	√					7.62E+06 (PS)
									glucuronidation
M26	9.31	N	C26H27O16	595.1294	595.1295	13.5	0.166	419.10(100.00), 243.07(53.02), 595.13(36.30), 175.02(9.17), 225.05(6.28)	hydrolysis	√	√	√	√				3.97E+08 (U)
									bis-glucuronidation
M27	9.35	N	C26H31O14	567.1708	567.1707	11.5	−0.321	243.07(100.00), 405.12(47.37), 225.05(10.26), 199.07(5.95), 175.07(4.86), 567.17(3.04)	isomer of Mulberroside A	√	√						2.20E+06 (PS)
M28	9.35	N	C26H33O18S	665.1382	665.1390	10.5	1.156	96.96(100.00), 243.07(8.96), 405.12(5.44), 665.15(4.77), 567.17(1.44)	hydroxylation						√		3.60E+06 (L)
									hydrogenation
									Sulfation
M29	9.35	N	C27H33O16	613.1763	613.1757	11.5	−0.964	243.07(100.00), 405.12(44.79), 567.17(14.36), 225.05(6.33), 199.08(4.35)	dihydroxylation	√	√	√					1.64E+06 (PS)
									methylation
M30	9.37	N	C20H21O13S	501.0697	501.0700	10.5	0.603	501.07(61.80), 421.11(22.27), 325.04(100.00), 245.08(24.44)	hydrolysis				√				7.60E+07 (U)
									hydrogenation
									glucuronidation
									Sulfation
M31	9.46	N	C20H21O12S	485.0748	485.0748	10.5	-0.048	485.07(87.69), 405.12(2.81), 323.02(7.96), 243.07(100.00), 225.05(11.22), 175.07(6.22), 199.07(5.45), 181.06(4.47)	hydrolysis	√			√				1.29E+06 (U)
									Sulfation
M32	9.54	N	C20H21O13S	501.0697	501.0700	10.5	0.424	501.07(58.15), 421.11(23.10), 325.04(100.00), 245.08(25.85)	hydrolysis				√				6.59E+07 (U)
									hydrogenation
									glucuronidation
									sulfation
M33	9.61	N	C20H19O13S	499.0541	499.0545	11.5	0.826	243.06(100.00), 323.02(84.23), 499.05(71.06), 419.10(30.24), 225.05(5.80)	hydrolysis		√	√					2.70E+05 (PM)
									glucuronidation
									sulfation
M34	9.68	N	C20H19O10	419.0973	419.0973	11.5	-0.031	419.10(100.00), 243.06(51.26), 175.02(17.18), 225.05(11.88), 199.07(9.79)	hydrolysis	√					√		5.51E+07 (PS)
									glucuronidation
M35	9.72	N	C20H21O13S	501.0697	501.0698	10.5	0.044	501.07(62.29), 421.11(33.75), 325.04(100.00), 245.08(26.83)	hydrolysis				√				2.74E+07 (U)
									hydrogenation
									glucuronidation
									sulfation
M36	9.80	N	C20H19O13S	499.0541	499.0544	11.5	0.526	243.07(100.00), 323.02(86.92), 499.05(39.50), 419.10(18.18), 225.05(2.46)	hydrolysis		√	√					1.01E+06 (PM)
									glucuronidation
									sulfation
M37	9.82	N	C20H21O10	421.1129	421.1129	10.5	-0.008	421.11(100.00), 175.02(20.18), 245.08(8.93), 243.07(2.49)	hydrolysis	√			√				7.85E+09 (U)
									hydrogenation
									glucuronidation
M38	9.83	N	C20H19O9	403.1024	403.1034	11.5	2.633	403.20(100.00), 245.08(8.58), 135.04(7.70), 175.02(3.61)	hydrolysis				√				5.32E+06 (U)
									dehydroxylation
									glucuronidation
M39	9.83	N	C20H19O11	435.0922	435.0913	11.5	-2.017	193.03(100.00), 175.02(5.76), 259.06(4.62), 157.01(3.55), 435.09(2.37)	Hydrolysis				√	√	√		2.14E+07 (U)
									hydroxylation
									glucuronidation
M40	9.83	N	C20H21O11	437.1078	437.1073	10.5	-1.230	437.11(100.00), 261.08(36.71), 175.02(20.83), 243.07(6.55)	Hydrolysis				√				1.93E+07 (U)
									Hydration
									glucuronidation
M41	10.00	N	C21H23O10	435.1286	435.1271	10.5	-3.363	193.03(100.00), 435.14(15.35), 175.02(3.27), 157.01(3.16), 403.18(1.07), 241.10(0.55), 417.18(0.40)	Hydrolysis					√			1.20E+05 (F)
									hydroxylation
									Methylation
M42*	10.03	N	C14H13O7S	325.0377	325.0376	8.5	-0.153	325.04(100.00), 245.08(17.31), 135.04(11.00), 243.06(6.19)	hydrolysis	√	√		√				3.70E+07 (U)
									hydrogenation
									sulfation
M43	10.07	N	C20H19O10	419.0973	419.0973	11.5	0.112	419.10(100.00), 243.06(54.06), 175.02(28.47), 199.07(13.35), 225.05(11.16)	hydrolysis	√			√				8.03E+06 (PS)
									glucuronidation
M44	10.21	N	C20H19O10	419.0973	419.0973	11.5	0.040	419.10(100.00), 243.06(78.98), 175.02(34.79), 225.05(16.89), 199.07(11.06)	hydrolysis	√			√				1.34E+07 (PS)
									glucuronidation
M45*	10.23	N	C14H13O7S	325.0377	325.0378	8.5	0.400	325.04(100.00), 245.08(19.99), 135.04(12.68), 243.06(5.92)	hydrolysis	√			√				8.45E+07 (U)
									hydrogenation
									sulfation
M46	10.28	N	C20H19O13S	499.0541	499.0540	11.5	-0.256	243.07(100.00), 323.02(71.89), 419.10(60.92), 499.05(48.22), 225.05(9.32)	hydrolysis				√				6.55E+06 (U)
									glucuronidation
									sulfation
M47	10.44	N	C20H19O9	403.1024	403.1023	11.5	-0.245	403.20(100.00), 227.07(29.95), 175.02(8.58), 113.02(45.99)	hydrolysis	√	√		√		√		2.98E+06 (U)
									dehydroxylation
									glucuronidation
M48*	10.48	N	C14H11O7S	323.0220	323.0221	9.5	0.279	243.06(100.00), 323.02(89.74), 225.05(17.97), 199.07(17.37), 175.07(16.90)	hydrolysis	√			√	√			1.44E+07 (PS)
									sulfation
M49*	10.57	N	C14H9O4	241.0495	241.0490	10.5	-2.096	241.14(100.00), 223.13(31.09), 179.11(19.87), 197.12(11.82)	Hydrolysis	√					√		6.06E+03 (PS)
									Dehydrogenation
M50*	10.64	N	C14H13O4	245.0808	245.0813	8.5	1.773	203.08(90.41), 123.04(81.08), 245.09(68.29), 227.13(6.47), 159.09(5.34)	Hydrolysis	√							1.05E+06 (PS)
									Hydrogenation
M51	10.79	N	C21H21O10	433.1129	433.1129	11.5	-0.169	243.06(100.00), 225.05(17.49), 257.08(16.68), 373.09(15.74), 433.11(13.36), 403.10(1.65)	Hydrolysis	√							2.82E+06 (PS)
									Methylation
									Glucuronidation
M52	10.84	N	C20H21O9	405.1180	405.1180	10.5	-0.145	405.12(46.47), 229.09(32.19), 175.02(32.02),157.01(4.20), 225.15(1.84)	Oxyresveratrol-4-O-β-D-glucopyranoside	√	√	√	√	√		√	1.98E+08 (U)
M53	10.90	N	C27H31O14	579.1708	579.1706	12.5	-0.418	271.06(100.00), 579.17(35.34), 227.07(2.63), 175.00(2.47)	dehydrogenation	√							7.60E+05 (PS)
									methylation
M54	10.92	N	C28H33O16	625.1763	625.1758	12.5	-0.850	271.06(100.00), 579.17(49.37), 300.03(7.16), 315.05(3.06), 330.07(2.99)	hydroxylation						√		3.41E+07 (L)
									acetylation
M55	10.96	N	C21H21O10	433.1129	433.1127	11.5	-0.585	243.06(94.52), 433.11(80.16), 257.08(60.10), 175.02(27.25), 373.09(8.98)	hydrolysis	√					√		7.89E+05 (PS)
									methylation
									glucuronidation
M56	11.14	N	C14H9O7S	321.0064	321.0066	10.5	0.655	241.05(24.05), 321.01(14.51), 124.01(13.46), 243.06(5.76)	hydrolysis	√			√				1.52E+06 (U)
									dehydrogenation
									sulfation
M57	11.25	N	C28H33O15	609.1814	609.1805	12.5	-1.505	301.07(100.00), 609.18(18.73), 286.05(10.54), 242.06(6.48), 463.12(0.26)	acetylation							√	2.38E+08 (LM)
M58	11.35	N	C20H19O9	403.1024	403.1018	11.5	-1.286	113.02(100.00), 403.10(37.33), 227.07(52.42), 175.02(47.17)	hydrolysis	√		√		√			2.25E+06 (PS)
									dehydroxylation
									glucuronidation
M59	11.38	N	C20H21O9	405.1180	405.1177	10.5	-0.811	405.12(100.00), 113.02(53.69), 229.09(45.46), 175.02(16.66)	Oxyresveratrol-3′-O-β-D-glucopyranoside	√			√	√		√	8.43E+07 (U)
M60	11.39	N	C28H35O14	595.2021	595.2017	11.5	-0.692	297.10(100.00), 175.02(8.91), 157.01(1.65), 419.09(0.51), 449.32(0.38)	dimethylation	√			√				1.59E+08 (U)
M61*	11.43	N	C16H15O4	271.0965	271.0964	9.5	-0.278	271.15(100.00), 225.11(14.23), 197.12(7.25), 167.11(32.05), 137.10(10.95)	hydrolysis					√			7.56E+05 (F)
									dimethylation
M62	11.54	N	C20H23O10	423.1286	423.1283	9.5	-0.717	423.27(100.00), 175.02(29.80), 201.02(28.97), 247.10(16.94), 263.17(3.76)	hydrolysis	√							2.00E+06 (PS)
									hydrogenation
									hydroxylation
M63	11.85	N	C20H23O9	407.1337	407.1353	9.5	4.056	407.17(91.79), 201.11(7.99), 137.10(2.85), 227.13(1.82), 245.19(0.70)	hydrolysis					√	√		1.34E+06 (L)
									hydrogenation
M64	12.06	N	C20H23O10	423.1286	423.1285	9.5	-0.291	423.27(100.00), 421.26(45.89), 175.02(9.52), 405.26(6.22)	Hydrolysis	√				√			1.82E+06 (PS)
									hydrogenation
									hydroxylation
M65	12.28	N	C21H21O10	433.1129	433.1126	11.5	-0.862	257.08(100.00), 433.11(86.14), 175.02(28.10), 242.06(1.94)	Hydrolysis	√	√	√	√	√	√		2.62E+07 (PS)
									methylation
									glucuronidation
M66	13.01	N	C20H23O10	423.1286	423.1286	9.5	-0.008	423.27(100.00), 421.26(18.72), 175.02(2.65), 405.26(0.98)	Hydrolysis	√			√				3.23E+06 (PS)
									hydrogenation
									hydroxylation
M67	13.13	N	C26H33O14	569.1865	569.1871	10.5	1.138	569.33(100.00), 407.28(17.04), 389.27(4.56), 551.32(1.21)	hydrogenation					√			7.90E+04 (F)
M68	13.19	N	C20H23O10	423.1286	423.1282	9.5	-0.953	423.14(100.00), 421.26(85.61), 247.13(14.76), 175.02(8.46), 245.12(2.36)	hydrolysishydrogenationhydroxylation				√				3.74E+04 (U)
M69	13.63	N	C14H11O8S	339.0169	339.0176	9.5	1.963	339.18(100.00), 259.11(11.96)	hydrolysis				√				2.64E+04 (U)
									hydroxylation
									sulfation
M70*	14.30	N	C14H11O4	243.0652	243.0651	9.5	-0.392	243.17(100.00), 199.18(31.85), 225.05(2.25), 201.05(1.38)	Oxyresveratrol	√	√		√			√	1.80E+06 (PS)
M71	14.70	N	C20H23O10	423.1286	423.1282	9.5	-0.882	423.27(100.00), 407.28(10.68), 421.26(5.80), 405.26(4.50)	hydrolysis				√				5.85E+04 (U)
									hydrogenation
									hydroxylation

PS, plasma treated by SPE; PM, plasma treated by methanol; PA, plasma treated by acetonitrile; U, urine; F, feces; L, liver tissue; LM, liver microsome; √, detected; *, components in Table 2.

TABLE 2

Target information of 7 metabolites of mulberroside A.

No.	Target number	Structure	Identification
N1 (M61)	102		dimethylated product of oxyresveratrol
N2 (M50)	78		2,4,3′,5′-Tetrahydroxybibenzyl
N3 (M70)	76		Oxyresveratrol
N4 (M49)	65		dehydrogenation product of oxyresveratrol
N5 (M42/M45)	19		hydrogenation and sulfation product of oxyresveratrol
N6 (M1)	7		β-D-Glucopyranosiduronic Acid 3-Hydroxyphenyl
N7 (M48)	3		sulfation product of oxyresveratrol

3.3.1 Identification of mulberroside A metabolites

M0 was identified as the prototype of mulberroside A with the [M-H]- ions at m/z 567.1707 (C26H31O14, -0.197 ppm) in negative ion modes. The fragment ions at m/z 243, m/z 567, m/z 405, m/z 225 were detected, which were consistent with the mass fragmentation behavior of the reference substance of mulberroside A. Therefore, M0 was characterized as the prototype of mulberroside A. M27 possessed the same theoretical [M-H]- and product ions with M0, but compared with M0, it was eluted later than M0. So, M27 was deduced as isomer of mulberroside A.

M1 and M21 generated [M-H]- ions at m/z 285.0607 (C12H13O8, 0.618 ppm) and m/z 311.0767 (C14H15O8, 1.659 ppm) respectively. The neutral loss of 176 Da (m/z 285→m/z 109 and m/z 311→m/z 135) was generated in their ESI-MS/MS spectra, indicating that their structures contain glucuronic acid (Glu A). In addition, the product ion at m/z 175 ([Glu A-H]-) was detected, which also proved the existence of Glu A. According to pubchem database (https://pubchem.ncbi.nlm.nih.gov/) and molecular formulas, M1 was identified as β-D-Glucopyranosiduronic Acid-3-Hydroxyphenyl and M21 was characterized as isomer of 6-(4-Ethenyl-2-hydroxyphenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid. They were the glucuronidation metabolites of compounds formed by the rupture of C1-α bond or C1′-β bond in mulberroside A.

M2 and M6 possessed the same theoretical [M-H]- ions at m/z 583.1652 (C26H31O15, mass error within 2 ppm) with the retention time of 7.72 and 8.20 min, respectively. They were 16 Da more massive than M0, which suggested that they might be the hydroxylation products of mulberroside A. The fragment ion at m/z 259 ([M-H-2Glu]-) was 16 Da more massive than m/z 243, providing evidence for hydroxylation reaction. The product ions at m/z 421 ([M-H-Glu]-) was formed by losing Glu. Therefore, it was speculated that M2 and M6 were hydroxylation products of mulberroside A.

M7 was eluted at 8.20 min with the [M-H]- at m/z 599.1602 (C26H31O16, -0.736 ppm). It was 32 Da (2O) more massive than M0, indicating that it might be the dihydroxylation product of mulberroside A. The fragment ions involved m/z 137, m/z 599, m/z 125, m/z 437, and m/z 275. The [M-H]- ion lost Glu to form m/z 437 ([M-H-Glu]-), and lost another Glu to form m/z 275 ([M-H-2Glu]-). We first assumed that two benzene rings were each bound to a hydroxyl group. Then it was split by Chem Draw, and we found that the α-β bond of the product ion at m/z 275 ([M-H-2Glu]-) broke to form C7H5O3 2-, which molecular weight was exactly m/z 137. From these, it was speculated that M7 was dihydroxylation product of mulberroside A and two hydroxyl groups were bonded to the benzene rings on both sides.

M5 and M29, with the molecular formula of C27H33O16, showed the same theoretical [M-H]- ions at m/z 613.1763 and they were 46 Da (CH2O2) more massive than M0. The loss of 30 Da (m/z 435→m/z 405) suggested the existence of methoxy. In addition, the product ions at m/z 243, m/z 405, m/z 567 were representative ions of mulberroside A. As a consequence, it was deemed that M5 and M29 were dihydroxylation and methylation products of mulberroside A.

M9 eluted at 8.24 min and generated the [M-H]- at m/z 601.1769 (C26H33O16, 1.046 ppm). It was 34 Da (H2O2) more massive than M0, which indicated that it was hydrogenation and hydroxylation product of mulberroside A. The fragment ions at m/z 137, m/z 243, m/z 601, m/z 567, m/z 439 ([M-H-Glu]-) were observed in its ESI-MS/MS spectrum. The α-β bond of M9 was broken and then lost Glu to form m/z 137 (C7H5O3 −), while m/z 243 and m/z 567 were characteristic ions of mulberroside A. Therefore, M9 was identified as vicinal diols formed by dihydroxylation at α-β bond of mulberroside A.

M54, with the retention time of 10.92 min, was 58 Da (C2H2O2) more massive than M0. The fragment ion at m/z 579 was fragmented by losing of CO and H2O from [M-H]- ion, while m/z 271 was identified as C12H15O7 − formed by the fracture of the bonds C-1 and α. The neutral loss of 30 Da (m/z 330→m/z 300) indicated that methoxy was contained in the structure. Hence, M54 was finally characterized as hydroxylation and acetylation product of mulberroside A.

M57 was 42 Da (C2H2O) more massive than M0 with the molecular formula of C28H33O15. It was speculated that M57 was acetylation product of mulberroside A. In the ESI-MS/MS spectrum of M57, the fragment ion at m/z 463 removed Glu to form m/z 301, then lose -CH3 to form the ion m/z 286. The product ion at m/z 297 was 30 Da more massive than m/z 267 ([M-H-2Glu]-), which proved the existence of methoxy.

M60 owned the [M-H]- ion at m/z 595.2017 (C28H35O14, -0.692 ppm) with the retention time of 11.39 min and was 28 Da (C2H4) more massive than M0. We speculated that M60 was dimethylation product of mulberroside A. In its ESI-MS/MS spectrum, the loss of 30 Da (m/z 449→m/z 419) was detected, which confirmed our prediction. In addition, the fragment ion at m/z 297 was identified as C14H18O7 − formed by the rupture of α-β bond.

M67 was 2 Da more massive than M0 and generated the [M-H]- at m/z 569.1871 (C26H33O14, 1.138 ppm) with the retention time of 13.13 min, which indicated that it might be hydrogenation product of mulberroside A. The product ion at m/z 407 ([M-H-Glu]-) was 2 Da more massive than that of m/z 405. Thus, M67 was ultimately characterized as hydrogenation product of mulberroside A.

Metabolites M4 and M28, with the [M-H]- ions at m/z 665.1384 (0.239 ppm) and m/z 665.1390 (1.156 ppm) respectively, was 98 Da more massive than M0, suggesting that they might be the hydrogenation, hydroxylation and sulfation metabolite of mulberroside A. The fragment ions at m/z 96.96 ([SO4H]-) had the highest abundance, which was a base peak, indicating that S may be contained. In addition, m/z 243, m/z 567, m/z 405 were characteristic fragments of mulberroside A. Therefore, it was speculated that M4 and M28 were hydrogenation, hydroxylation and sulfation products of mulberroside A.

M52, M59 and M70 generated the [M-H]- ions at m/z 405.1180 (-0.145 ppm), m/z 405.1177 (-0.811 ppm), and m/z 243.0651 (-0.392 ppm), respectively. M52 and M59 were 162 Da less massive than M0 while M70 was 324 Da less massive than M0. Moreover, the product ion at m/z 225 was included in their ESI-MS/MS spectra and m/z 199 was detected in the ESI-MS/MS spectra of M70. It was speculated that they were hydrolysates of mulberroside A. M52 and M59 were formed by losing a molecule of Glu while M70 was formed by losing two molecules of Glu. In the experiment of Zhaxi et al., they isolated three metabolites of mulberroside A from rat intestinal contents and feces. The three metabolites were identified as oxyresveratrol-2-O-β-D-glucopyranoside, oxyresveratrol-3′-O-β-D-glucopyranoside and oxyresveratrol according to ESI-MS spectrum, 1H and 13C-NMR spectroscopy (Zhaxi et al., 2010). In their study, oxyresveratrol-3′-O-β-D-glucopyranoside (31.3 min) was eluted later than oxyresveratrol-2-O-β-D-glucopyranoside (27.8 min). However, the hydrolysis products of mulberroside A that formed by losing a Glu should be oxyresveratrol-4-O-β-D-glucopyranoside and oxyresveratrol-3′-O-β-D-glucopyranoside. Hence, it was speculated that oxyresveratrol-2-O-β-D-glucopyranoside was formed by oxyresveratrol combined with a Glu. Therefore, in our experiment, M52, M59 were deduced as oxyresveratrol-4-O-β-D-glucopyranoside and oxyresveratrol-3′-O-β-D-glucopyranoside based on the retention time. Furthermore, M70 was identified as oxyresveratrol according to the literature (Xu et al., 2014).

M53 was deemed as the dehydrogenation and methylation product of mulberroside A, with the [M-H]- ion at m/z 579.1706 (C27H31O14, -0.418 ppm), which was 12 Da more massive than M0. The DPI at m/z 271 was deduced as C12H15O7 − formed by the rupture of α-β bond.

3.3.2 Identification of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside metabolites

M3, M16 and M25, with the retention time of 7.98, 8.77 and 9.29 min, possessed the same theoretical [M-H]- ion at m/z 581.1501 (C26H29O15, mass error within 1.00 ppm). They were 176 Da more massive than M52 or M59, which suggested that they may be the glucuronidation products of M52 or M59. In the ESI-MS/MS spectrum, the product ions at m/z 243 ([M-H-Glu A-Glu]-), m/z 405 ([M-H-Glu A]-), m/z 225 ([M-H-Glu A-Glu-H2O]-) were detected. Therefore, M3, M16 and M25 were identified as glucuronidation metabolites of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.

M8, detected at 8.20 min, generated the [M-H]- ion at m/z 437.1079 (C20H21O11, 0.042 ppm) and was 32 Da more massive than M52 or M59, indicating that it may be the dihydroxylation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. The fragment ions at m/z 275 ([M-H-Glu]-) and m/z 257 ([M-H-Glu-H2O]-) were detected in its ESI-MS/MS spectrum. In addition, the fragment of m/z 137 was also detected, which was presumed to be C7H6O3 − formed by α-β bond breakage.

M13 eluted at 8.57 min, was deduced as hydrogenation and glucuronidation product of M52 or M59. The successive loss of 162 and 176 Da (m/z 583→m/z 421→m/z 245) indicated that Glu and Glu A may be contained. Besides, the DPI at m/z 245 ([M-H-Glu-Glu A]-) was 2 Da more massive than m/z 243 (characteristic ion of M52 and M59). Eventually, M13 was identified as hydrogenation and glucuronidation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. In the ESI-MS/MS spectrum of M14, the successive loss of Glu A, Glu and Glu A (m/z 759→m/z 583→m/z 421→m/z 245) was detected, which indicated that two molecules of Glu A and a molecule of Glu may be contained. So, M14 was characterized as hydrogenation and bis-glucuronidation product of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.

M31, with the [M-H]- ion at m/z 485.0748 (C20H21O12S, -0.048ppm), was 80 Da more massive than M52 or M59, which implied that it might be the sulfation product. In its ESI-MS/MS spectrum, the product ions at m/z 405 ([M-H-SO3]-) and m/z 243 ([M-H-SO3-Glu]-) confirmed the metabolite identification. Hence, M31 was tentatively characterized as sulfation product of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.

M63 was 2 Da more massive than M52 or M59, indicating that they were hydrogenation metabolites of M52 or M59. The fragment ion at m/z 201 were detected in its ESI-MS/MS spectrums, which was formed by the successive loss of Glu and CO2. In addition, the product ion at m/z 227 ([M-H-Glu-H2O]-) was also detected.

M41 possessed the [M-H]- ion at m/z 435.1271 (C21H23O10, -3.363ppm), with the retention time of 10.00 min. The DPIs at m/z 417 [M-H-H2O]-, m/z 403 and m/z 193 were generated in its ESI-MS/MS spectrum. Therefore, it was speculated that M41 was hydroxylation and methylation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. The fragment of m/z 403 was presumed to be formed by the loss of H2O from m/z 421 (m/z 405+16) and m/z 193 may be C11H14O3 − formed by benzene ring opening.

M62, M64, M66, M68 and M71, with the same molecular formula of C20H23O10, had the same theoretical [M-H]- ion at m/z 423.1286. They were 18 Da more massive than M52 or M59, suggesting that they may be hydrogenation and hydroxylation products of M52 or M59. The DPIs at m/z 405, m/z 201, m/z 263 and m/z 247 were detected, which provided evidence for the metabolite identification.

3.3.3 Identification of oxyresveratrol metabolites

M17, M24, M33, M36 and M46 were 256 Da more massive than M70 with the same theoretical [M-H]- ions at m/z 499.0541 (C20H19O13S, error<1.00 ppm). In the ESI-MS/MS spectra, the successive loss of Glu A and SO3 (m/z 499 [M-H]-→m/z 419 [M-H-SO3]-→m/z 243 [M-H-SO3-Glu A]-, m/z 499 [M-H]-→m/z 323 [M-H-Glu A]-→m/z 243 [M-H-Glu A-SO3]-) was observed. Therefore, they were eventually characterized as glucuronidation and sulfation products of oxyresveratrol.

M34, M43 and M44 were tentatively identified as glucuronidation products of M70 according to the neutral loss of 176 Da (m/z 419→m/z 243). In addition, the fragment ion at m/z 175 ([Glu A-H]-) provided evidence for the existence of glucuronic acid group. M48 was 80 Da more massive than M70, with the [M-H]- ion at m/z 323.02209 (C14H11O7S, 0.279 ppm), implying that it may be sulfation product of M70. The neutral loss of 80 Da (m/z 323→m/z 243) proved the existence of SO3 in its ESI-MS/MS spectrum.

M50 was 2 Da more massive than M70 and it was preliminarily deduced as the hydrogenation metabolite of oxyresveratrol. It was produced by hydrogenation reduction reaction occur at the α-β bond according to the product ion at m/z 123, which was formed by the rupture of α-β bond. The fragment ions at m/z 203 ([[M-H-CO-CH2]−]-) and m/z 159 ([M-H-C3O2-H2O]−) were generated in its ESI-MS/MS spectra. Besides, the DPI at m/z 227 ([M-H-H2O]-) was also detected. M49 was 2 Da less massive than M70, indicating that it might be the dehydrogenation product of oxyresveratrol. The DPIs at m/z 197 ([M-H-CO2]-), m/z 179 ([M-H-CO2-H2O]-) and m/z 223 ([M-H-H2O]-) provided evidence for the metabolite identification. Finally, M50 was identified as hydrogenation metabolite of oxyresveratrol and M49 was characterized as dehydrogenation product of oxyresveratrol.

M10, M51, M55 and M65, with the same theoretical [M-H]- ion at m/z 433.1129 (C21H21O10, error ≤1.00 ppm), were 190 Da more massive than M70. The DPI at m/z 257 ([M-H-Glu A]-) and the continuous neutral loss of 30 Da (m/z 433→m/z 403→m/z 373) were detected, suggesting that Glu A and -OCH3 might be contained. Thus, they were eventually deduced as methylation and glucuronidation metabolites of oxyresveratrol.

M11, M19 and M20, eluted at 8.43 min, 8.84 min and 8.94 min respectively, were deduced as hydrogenation and bis-glucuronidation products of M70. In the ESI-MS/MS spectra, the successive loss of 176 Da (m/z 597→m/z 421→m/z 245) indicated that two molecules of Glu A might be contained. Furthermore, the DPI at m/z 175 confirmed the existence of Glu A and the DPI at m/z 245 ([M-H-2Glu A]-) was 2 Da more massive than m/z 243 ([M-H]- ion of M70). Therefore, they were eventually identified as hydrogenation and glucuronidation products of oxyresveratrol.

M12, eluted at 8.52 min, was 174 Da more massive than M70. The neutral loss of 176 Da (m/z 421→m/z 245 and m/z 417→m/z 241) was detected in both ESI-MS/MS spectra, which was suggested that Glu A might be contained in its structure. What’s more, the DPI at m/z 241 of M12 was 2 Da less massive than m/z 243 ([M-H]- ion of M70). Thus, M12 was characterized as dehydrogenation and glucuronidation metabolite of oxyresveratrol. M22 and M37, with the retention time of 9.19 and 9.82 min, were 178 Da more massive than M70. The neutral loss of 176 Da (m/z 421→m/z 245 and m/z 417→m/z 241) provided evidence for the existence of Glu A. In addition, the DPI at m/z 245 was 2 Da more massive than m/z 243. Finally, they were deduced as hydrogenation and glucuronidation product of oxyresveratrol.

M15, M23 and M26 were identified as bis-glucuronidation products of M70 according to the successive loss of Glu A (m/z 595→m/z 419→m/z 243) with the retention time of 8.76 min, 9.25 min and 9.31 min, respectively. M18, M30, M32 and M35, with the molecular formula of C20H21O13S, generated the same theoretical [M-H]- ions at m/z 501.0697. The successive loss of 176 Da (m/z 421→m/z 245) and 80 Da (m/z 501→m/z 421, m/z 325→m/z 245) was detected in their ESI-MS/MS spectra, indicating that they might be the glucuronidation and sulfation products of M70. In addition, the DPI at m/z 245 was 2 Da more massive than the [M-H]- ion of M70 (m/z 243). So, they were deduced as the hydrogenation, glucuronidation and sulfation products of oxyresveratrol.

M38, M47 and M58 were deduced as the dehydroxylation and glucuronidation metabolites of oxyresveratrol, with the molecular formula of C20H19O9. The DPIs at m/z 227 [M-H-Glu A]- (m/z 403–176 Da, m/z 243–16 Da) provided the evidence for the metabolite identification. The successive loss of 176 and 16 Da (m/z 435→m/z 259→m/z 243) was observed in the ESI-MS/MS spectrum of M39, indicating that Glu A and -OH may be contained. Therefore, M39 was identified as hydroxylation and glucuronidation product of oxyresveratrol. M40 was characterized as hydration and glucuronidation metabolite of oxyresveratrol based on the successive loss of Glu A and H2O (m/z 437→m/z 261→m/z 243).

M42 and M45 were deemed to be the hydrogenation and sulfation products of oxyresveratrol while M56 was identified as dehydrogenation and sulfation metabolite of oxyresveratrol. The DPIs at m/z 245 (m/z 325–80 Da, m/z 243+2 Da) and m/z 241 (m/z 321–80 Da, m/z 243–2 Da) provided the evidence for the metabolite identification.

M61 possessed the [M-H]- ion at m/z 271.0964 (C16H15O4, -0.278 ppm), which was 28 Da (C2H4) more massive than M70. It was presumed to be the dimethylation product of oxyresveratrol. The successive loss of 30 Da (m/z 197→m/z 167→m/z 137) provided evidence for the existence of methoxy.

M69, with the [M-H]- ion at m/z 339.0176 (C14H11O8S, 1.963 ppm), was eluted at 13.63 min. The neutral loss of 80 Da (m/z 339→m/z 259) was observed in the ESI-MS/MS spectrum, indicating that SO3 might be contained in its structure. Besides, the fragment ion at m/z 259 was 16 Da more massive than m/z 243, which suggested that it may be hydroxylation product of M70. Hence, it was speculated that M69 was hydroxylation and sulfation product of oxyresveratrol.

3.4 Analysis of metabolic reaction of mulberroside A

The metabolic reactions of mulberroside A in vivo and in vitro of rats were hydrolysis, hydrogenation, hydroxylation, methylation, acetylation, sulfation, glucuronidation and so on. In this paper, the product peak areas of each type of metabolic reaction were summarized, as shown in Figure 6. The results showed that the metabolic reactions of mulberroside A in vivo were mainly hydrolysis, glucuronidation and hydrogenation. The total peak area of hydrolysis metabolites accounted for the largest proportion. Therefore, it was preliminarily speculated that after entering the body, mulberroside A was hydrolyzed into aglycone firstly, and then a series of metabolic reactions occur. While in vitro, the most abundant compounds were hydrolysate and acetylation products of mulberroside A.

FIGURE 6

Proportion of the area of each metabolic reaction peak.

3.5 Analysis of proposed metabolic characteristics of mulberroside A

In this experiment, a total of 72 metabolites including mulberroside A were detected, 40 in blood, 37 in urine, 12 in feces, 9 in liver, and 10 in liver microsomes. The prototype component was only detected in rat plasma samples, which indicated that mulberroside A could be absorbed into the blood, and then a series of metabolic reactions occurred. There was no significant difference on the type of metabolites in plasma and urine, and the metabolites in feces were mainly glucuronidation, sulfation and methylation products of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. It was speculated that mulberroside A was metabolized in 3 pathways in vivo and in vitro of rats. (1) The metabolic reactions such as sulfation, hydroxylation and methylation were occurred on the basis of mulberroside A (M2, M4, M5, M6, M7, M9, M28, M29, etc.). In this pathway, glucuronidation reaction was undetected. (2) Mulberroside A was hydrolyzed to form oxyresveratrol-4-O-β-D-glucopyranoside (M52) or oxyresveratrol-3′-O-β-D-glucopyranoside (M59), on which metabolic reactions took place, such as M3, M8, M13, M14, M16, M25, M31 and so on. (3) Mulberroside A continued to be hydrolyzed and lose another Glu to produce oxyresveratrol (M70). Then, groups such as glucuronic acid, sulfate and methyl were bound to oxyresveratrol (M10, M11, M15, M17, M18, M19, etc.). Besides, β-D-Glucopyranosiduronic Acid 3-Hydroxyphenyl (M1) and isomer of 6-(4-Ethenyl-2-hydroxyphenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid (M21) were formed by the rupture of C1′-β bond. The proposed metabolic pathways of mulberroside A are shown in Figure 7.

FIGURE 7

The proposed metabolic pathways of mulberroside A in vivo and in vitro of rats. Note: Glu: glucose; Glu A: glucuronic acid; component with blue flame: metabolite in vivo and in vitro; component with green flame: metabolite in vitro; *: components in Table 2.

Furthermore, the in vivo and in vitro metabolites of mulberroside A were mostly based on its hydrolyzed products oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside and oxyresveratrol. The main metabolic reactions were hydrolysis, hydroxylation, methylation, sulfation, glucuronidation and their compound reactions. The metabolic result showed that the main forms of mulberroside A in vivo were the glucuronidation and sulfation products of its aglycone, while in vitro they were mainly the hydroxylation and hydrolysis products of its prototype. The in vitro pharmacokinetic study of mulberroside A also showed that the glucose group on mulberroside A was rapidly hydrolyzed to generate monoglycosides and its aglycone oxyresveratrol when incubated anaerobically with intestinal bacteria (Mei et al., 2012), which was consisted with our findings.

3.6 Comparison of three different sample preparation methods

In the study of drug metabolism, deproteinization of biological samples is essential on account of a large number of proteins were contained in plasma, which interferes with the identification of metabolites. At present, the main methods of protein removal are organic solvent precipitation (methanol, acetonitrile, methanol-ethanol, methanol-acetonitrile-acetone, etc.) and solid phase extraction method (Xu et al., 2011). In our experiment, the plasma samples of rats were treated by solid phase extraction, methanol precipitation and acetonitrile precipitation respectively. In the end, 36 metabolites were detected in the plasma samples treated by SPE while 17 metabolites were detected in the plasma samples treated by methanol precipitation and 15 metabolites in the plasma samples treated by acetonitrile precipitation. It was obvious that solid phase extraction method was significantly better than the other two methods, which was consistence with the study results of Liu et al. (Liu et al., 2020). It was speculated that solid phase extraction plays the role of separation, enrichment and concentration in the process of sample pretreatment. On the contrary, biological samples may be diluted by methanol precipitation and acetonitrile precipitation. In addition, the type of organic solvents and the proportion of different solvents also play important roles in the pretreatment of biological samples.

3.7 Network pharmacology analysis of mulberroside A and its metabolites

3.7.1 Construction and analysis of metabolite-target network

7 metabolites (N1–N7) with high gastrointestinal absorption and drug-likeness were selected by Swiss ADME. The information of the 7 metabolites is shown in Table 2. However, the Swiss ADME results showed that mulberroside A had no drug-likeness and no target of mulberroside A was obtained by Swiss Target Prediction. So, mulberroside A was not included. As a result, a total of 167 targets were obtained using Swiss Target Prediction. Then, the metabolite-target network with 174 nodes and 350 edges was constructed based on the information by Cytoscape 3.8.2, as shown in Figure 8. The area of the component graph in the figure is proportional to the degree value, so it can be seen intuitively that N1 has the largest effect, followed by N2 and N3.

FIGURE 8

The metabolite-target network of mulberroside A metabolites.

3.7.2 GO enrichment analysis of target proteins

The 167 targets were submitted to the Metascape for GO enrichment analysis, including molecular functions (MF), biological processes (BP) and cellular components (CC). A total of 1702 GO items were obtained finally (164 of MF, 1427 of BP and 111 of CC) (p < 0.01). The top 10 clusters with their representative enriched terms (one per cluster) were selected and they are shown in Figure 9. The serine/threonine/tyrosine kinase activity, transmembrane receptor protein tyrosine kinase activity, non-membrane spanning protein tyrosine kinase, carbonate dehydratase activity and kinase binding were involved in MF. In the aspect of BP, there were mainly protein phosphorylation, cellular response to nitrogen compound, response to inorganic substance, positive regulation of cell migration and response to xenobiotic stimulus. The receptor complex, membrane raft, postsynapse, extrinsic component of cytoplasmic side of plasma membrane and transferase complex, transferring phosphorus-containing groups were mainly included in the category of CC.

FIGURE 9

GO enrichment analysis of mulberroside A metabolites.

3.7.3 KEGG analysis of target proteins

Furthermore, the Metascape was also applied in KEGG analysis. In the end, 158 signal pathways were enriched (p < 0.01). The result suggested that the pathways of mulberroside A metabolites were mainly involved Axon guidance, Alzheimer disease, Serotonergic synapse, NF-kappa B signaling pathway, Neuroactive ligand-receptor interaction, Epithelial cell signaling in Helicobacter pylori infection, TNF signaling pathway and so on (Figure 10, Supplementary Table S1).

FIGURE 10

KEGG analysis of mulberroside A metabolites.

3.7.4 Construction and analysis of metabolite-target-pathway network

In the end, the 7 metabolites (N1–N7), 167 targets and the top 20 pathways were imported to Cytoscape 3.8.2 for metabolite-target-pathway network. As a result, the metabolite-target-pathway network, with 194 nodes and 602 edges, was constructed, as shown in Figure 11.

FIGURE 11

The metabolite-target-pathway network of mulberroside A metabolites.

Studies have shown that mulberroside A has the effect of neuroprotection (Wang C. P. et al., 2014). From the metabolite-target-pathway network, it can be seen intuitively that the metabolites N1, N2, N3 and N4 were involved in pathways of Axon guidance, Alzheimer disease and Serotonergic synapse. Axon guidance and serotonin (5-HT) play key roles in neuronal network (Negishi et al., 2005; Migliarini et al., 2013). Therefore, it was speculated that the metabolites N1, N2, N3 and N4 exert the neuroprotective effect by Axon guidance and Serotonergic synapse. Besides, oxyresveratrol (N3) has the effect of neuroprotection (Lee et al., 2019) and can be used to treat alzheimer disease (Lakshmi et al., 2021), which proved the results. From the metabolite-target-pathway network (Figure 11), it could be seen intuitively that EGFR may play an important role. EGFR is a tyrosine kinase which can regulate cellular homeostasis. Duan’s study suggested that mulberroside A could suppress the migration and invasion of renal cancer A498 cells through inhibit the expression of p-EGFR (Duan et al., 2019). So, it was indicated that mulberroside A could exert nephroprotective effect by regulating the expression of EGFR. In the pathway of Melanogenesis, tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) are key proteins in melanin production. The study of Wang et al. showed that mulberroside A can suppress the activity of tyrosinase (Wang S. et al., 2014). Thus, it can be concluded that mulberroside A could inhibit the production of melanin by inhibiting the activity of tyrosinase.

Furthermore, metabolites N1, N2, N3 and N4 were also associated with NF-kappa B signaling pathway, TNF signaling pathway and Epithelial cell signaling in Helicobacter pylori infection. RPS6KA5 was the key target of N2, which exerts anti-inflammatory effect by decreasing the expression of TNF-α and inhibiting the activation of NF-κB (Wang C. P. et al., 2014). It should be noted that NF-kappa B signaling pathway plays a crucial role in mulberroside A-induced suppression of P-Glycoprotein (Li et al., 2014) and research has shown that insulin regulates P-glycoprotein via PKC/NF-κB pathway (Liu et al., 2009). Thus, it was speculated that mulberroside A has the inflammatory and antidiabetic effects after being metabolized to N1, N2, N3 and N4.

4 Conclusion

In the present study, an integrated strategy based on the UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology was firstly established to elucidate the metabolic mechanism of mulberroside A. In order to detect as many metabolites as possible, not only biological samples (plasma, urine, feces, liver tissues) were prepared in vivo of rats, but also the liver microsomes of rats were incubated in vitro. For the treatment of plasma samples, three methods (solid phase extraction, methanol precipitation and acetonitrile precipitation) were applied. We concluded that solid phase extraction was the most suitable method for plasma sample of mulberroside A. As a result, a total of 72 metabolites including mulberroside A were detected by UHPLC-Q-Exactive Plus Orbitrap MS. The metabolites and metabolic pathways of mulberroside A in vivo and in vitro of rats were also elaborated. The main metabolite reactions of mulberroside A were hydrolysis, glucuronidation and hydrogenation. Furthermore, the results of network pharmacology study showed that the component that exerts pharmacological effects in the body is not mulberroside A but its metabolites, which mainly include oxyresveratrol (aglycon of mulberroside A), dehydrogenation, hydrogenation, and demethylated products of oxyresveratrol.

The integrated strategy established in our study could elucidate the metabolic pathways and mechanism of mulberroside A in vivo and in vitro of rats more comprehensively, which would provide more theoretical basis for the study of mulberroside A. It would be conducive to better understand the metabolic action and promote research for the further potential therapeutic application of mulberroside A.