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Open AccessArticle

Investigation of the In Vivo Metabolism of Sibirioside A and Angoroside C in Rats by HPLC-ESI-IT-TOF-MSn

Division of Pharmacognosy, School of Pharmaceutical Sciences, Peking University Health Science Center, No. 38, Xueyuan Road, Beijing 100191, China
*
Author to whom correspondence should be addressed.
Molecules 2018, 23(10), 2702; https://doi.org/10.3390/molecules23102702
Received: 19 September 2018 / Revised: 16 October 2018 / Accepted: 18 October 2018 / Published: 19 October 2018

Abstract

Sibirioside A and angoroside C are two important phenylpropanoid glycosides of the traditional Chinese medicine Scrophulariae Radix. High performance liquid chromatography, coupled with an ion trap time-of-flight multistage mass spectrometry equipped with electrospray ionization source (HPLC-ESI-IT-TOF-MSn), was applied to the profile and we identified the metabolites of sibirioside A and angoroside C in vivo in rats. A total of four metabolites of sibirioside A were identified: SM1, SM2 and SM3 which were known as new compounds. A total of 25 metabolites were detected for angoroside C: AM4, AM5, AM6, AM7, AM16, AM17, AM20, AM21, AM22, AM23 and AM25 which were identified to be new compounds. The main metabolic reactions were hydrolysis, reduction, hydroxylation, methylation, sulfation, and gluconylation. The prototype of sibirioside A was widely distributed in tissues found in the heart, liver, spleen, lung, kidney, stomach and small intestine of rats, and mainly distributed in the stomach, small intestine, kidney and liver. But for angoroside C, nothing was found in the viscera except the stomach and small intestine. The metabolites of sibirioside A were mainly eliminated from feces, while it was urine for the metabolites of angoroside C. Furthermore, 19 metabolites were likely to have bioactivities based on the ‘PharmMapper’ analysis, which roughly matched the known pharmacological activities of Scrophulariae Radix (SR) and the prototypes. One of the main pharmacological activities of SR in traditional Chinese medicine is anti-diabetes, and the predicted results showed that SM1, SM2, SM3, AM2, AM4, AM5, AM6, AM9, AM10, AM11, AM12, AM13, AM15, AM18, AM19, AM24, and AM25 might be used to cure diabetes. These findings provide a reference for studying the metabolism, distribution and pharmacological actions of phenylpropanoid glycosides in vivo.
Keywords: sibirioside A; angoroside C; metabolism; HPLC-IT-TOF-MSn; Scrophulariae Radix sibirioside A; angoroside C; metabolism; HPLC-IT-TOF-MSn; Scrophulariae Radix

1. Introduction

Phenylpropanoid glycosides are the most abundant and important constituents, secondary to iridoid glycosides in Scrophulariae Radix (SR), and the representative components include acteoside, angoroside C, sibirioside A and so on [1]. Some research reported that angoroside C can take many pharmacological roles, including anti-inflammatory [2,3,4], antioxidation [5], and cardiovascular protection [6]. For sibirioside A, there was paucity of reports on its pharmacological activity, but only a few literatures about isolation and purification [7,8,9]. SR is widely used in traditional Chinese medicine, derived from the dried roots of Scrophularia ningpoensis Hemsl. [10]. SR exhibits a variety of pharmacological actions such as anti-inflammation, analgesia, immunoregulation, blood sugar regulation, anticoagulation, cardiovascular protection, and neuroprotection [11,12,13,14,15]. The pharmacopoeia stipulates that the traditional processing method for SR is “sweating-drying” [10], and previous research indicated that the content of angoroside C decreased after traditional processes while sibirioside A was the opposite [16]. It is a novel and promising way to have some insight into the mechanism of crude drug processing in metabolic terms. The rudimentary metabolic characteristics and pathways of harpagoside [17] and harpagide [18] have been resolved. To investigate the in vivo metabolic process of phenylpropanoid glycosides, we chose sibirioside A and angoroside C as research objects.
To explore the metabolism and distribution of compounds in biological samples, high performance liquid chromatography coupled with multi-stage mass spectrometry (HPLC-MSn) is known as an advantageous method, as it provides sensitive, high-resolution and accurate information on hundreds of compounds simultaneously [17]. In this study, we applied the HPLC-IT-TOF-MSn method to investigate the metabolism of sibirioside A and angoroside C and the distribution of their metabolites in rats.

2. Results

2.1. Fragmentation Behaviors of Sibirioside A and Angoroside C

Aqueous solutions of the reference substances, sibirioside A and angoroside C, were directly injected for analysis to obtain the fragmentation characteristics of the parent compounds. Several characteristic fragments of sibirioside A and angoroside C were observed (Figure S1) in the negative ion mode mass spectrum. Sibirioside A showed quasimolecular ion peaks at m/z 517.1450 ([M + HCOO]) and m/z 471.1414 ([M − H]). Its MS2 spectra fragments m/z 323.0918 ([M-cinnamic acid − H]), m/z 309.0991 ([M − fructosyl − H]), m/z 179.0523 ([glucose − H] or [fructose − H]), m/z 161.0401 ([glucose − H2O − H] or [fructose − H2O − H]). The quasimolecular ion peak of angoroside C was m/z 783.2513 ([M − H]), and the MS2 fragments were shown as m/z 607.1993, m/z 475.1826, m/z 461.1514, m/z 443.1411, m/z 329.1078, m/z 325.0911. The fragmentation processes of sibirioside A and angoroside C were shown in Figure 1 and Figure 2.
In total, four sibirioside A metabolites and 25 angoroside C metabolites were identified, and the Liquid Chromatography Mass Spectrometry (LC-MS) results are shown in Table 1.

2.2. Metabolites of Sibirioside A: SM1SM4

The negative quasi-molecular ion of SM1 at m/z 229.0153 ([M − H]) was detected in feces and urine samples, and speculated as C9H10O5S. Its degrees of unsaturation were five, meaning there was only one unsaturated bond except for the phenyl group in the molecule. The MS2 spectrum showed fragment at m/z 147.0459 (speculated to be [C9H8O2 − H]), and its MS3 fragment ion at m/z 103.0348 ([C9H8O2 − CO2 − H]) indicated that C9H8O2 might be cinnamic acid. Cinnamic acid was the product of SM1 with 82 Da lost, which was considered to be H2SO3. It is well-known that the sulphated compounds could easily lose 80 Da radical group (SO3) instead of 82 Da, but the neutral molecule H2SO3 might be lost if there was active-H in the phenylpropionic acid structure. Then we could conjecture SM1 as phenylpropionic acid sulphate. The cinnamic acid first reacted with glutathione and then hydrolyzed into the cysteine adduct by peptidase, and the cysteine β-liase gave a thiol immediately after, and finally oxidized to be SM1.
SM2 detected in feces samples showed quasi-molecular ion peak at m/z 259.0293 ([M − H]), indicating C10H12O6S as the predicted formula. The characteristic fragment ion peaks were shown as m/z 177.0540 ([M − H2SO3 − H]) and m/z 133.0983 ([M − H2SO3 − CO2 − H]). Its trends of neutral loss conformed with those of SM1, and all the fragment ion peaks were 30 Da (predicted to be CH2O) larger than the correspondent peaks of SM1, which suggested the substitution of methoxyl group. The methoxyl group was the best possibility to substitute at the aromatic ring owing to the absence of methyl (14 Da) or methoxyl (30 Da) loss in MS2 spectrum. Then it could be speculated that SM2 might be 4-methoxyphenylpropanoic acid sulphate in consideration of the substitutive resistance.
The different chromatographic peak of SM3 was observed in feces samples, and showed quasi-molecular ion peak at m/z 393.0755 ([M − H]). The fragment ion at m/z 311.1019 ([M − H2SO3 − H]) suggested SM3 to be a sulphate, and it could be dissociated to get m/z 163.0583 and m/z 147.0454. There was no fragment ion at m/z 103.03 rooted from m/z 147.0454, which indicated that the ion at m/z 147.0454 was not cinnamic acid. According to the elemental composition C9H8O2, this fragment was most likely to be β-oxobenzenepropanal. In accordance with the above analysis, SM3 might be β-hydroxybenzenepropanoic anhydride sulphate.
The molecular formula of SM4 detected in stomach samples was predicted to be C27H38O17 according to the quasi-molecular ion peak at m/z 633.2014 ([M − H]). Its MS2 spectrum showed fragment ion peaks at m/z 485.1416 ([M − cinnamoyl − H]) and m/z 323.0932 ([sucrose − H]). Neutral loss of 148 Da (cinnamic acid) occurred from the parent ion to obtain m/z 485.1416, then neutral loss of 162 Da (glucosyl) occurred from m/z 485.1416 to get m/z 323.0932, which was the main fragment ion of sibirioside A predicted to be [sucrose − H]. To sum up, SM4 was conjectured to be a sibirioside A glucose conjugation.
The metabolites and metabolic pathways of sibirioside A in rats are shown in Figure 3.

2.3. Metabolites of Angoroside C: AM1AM25

AM1 showed [M − H] ion peak at m/z 233.0119 ([M − H]) in the negative MS1 spectrum, which was predicted as C8H10O6S. The fragment ion peaks at m/z 153.0583 ([M − SO3 − H]) and m/z 123.0492 ([M − SO3 − CH2O − H]) were observed in negative MS2 spectrum, and m/z 153.0583 should be the phenylethanoid unit derived from angoroside C. Therefore, AM1 was speculated to be 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.
The formula of AM2 was determined to be C9H8O6S based on its quasi-molecular ion peak at m/z 242.9965 ([M − H]). The characteristic fragment ion peaks were observed as m/z 163.0415 ([M − SO3 − H]) and m/z 119.0532 ([M − SO3 − CO2 − H]). Therefore, the ion at m/z 163.0415 was thought to be the demethoxylation product of ferulic acid, which was the hydrolysate of angoroside C. And AM2 was thus determined as p-coumaric acid sulphate. It was easy to confirm that AM3 was 4-hydroxybenzenepropanoic acid sulphate, the hydrogenation product of AM2 based on the quasi-molecular ion peak at m/z 245.0109 ([M − H]) and its MS2 fragment ion peaks at m/z 165.0574 ([M − SO3 − H]) and m/z 121.0680 ([M − SO3 − CO2 − H]).
For AM4, AM5 and AM6, the exact formula C9H12O6S was calculated based on the quasi-molecular ion peak at m/z 247.03 ([M − H]). The ion at m/z 167.07 ([M − SO3 − H]) was detected in MS2 spectrum, which suggested that they were 3,4-hydroxyphenylpropanol sulphate. And there were three reaction sites in 3,4-hydroxyphenylpropanol for sulphating, so the three metabolites were considered as isomers.
The quasi-molecular ion peak of AM7 at m/z 259.0271 ([M − H]) suggested the formula as C10H12O6S. And the fragment ion peaks at m/z 179.0726 ([M − SO3 − H]), m/z 163.0385 ([M − SO3 − CH2 − H]), m/z 147.0473 ([M − SO3 − CH4O − H], namely [cinnamic acid − H]) and m/z 119.0507 ([M − SO3 − CH2 − CO2 − H]) indicated the metabolite might be methoxybenzenepropanoic acid sulphate, which was a isomer of SM2.
AM8 and AM9 were speculated to be C9H10O7S owing to the quasi-molecular ion peak at m/z 261.01 ([M − H]) in negative ion mode MS1 spectrum. The multistage fragment ion peaks at m/z 217.0175 ([M − CO2 − H]), m/z 181.0522 ([M − SO3 − H]) and m/z 137.0633 ([M − SO3 − CO2 − H]) indicated that they were 3,4-dihydroxybenzenepropanoic acid-O-sulphate.
The quasi-molecular ion peak at m/z 275.02 ([M − H]) observed in the negative MS1 spectrum indicated the formula C10H12O7S. Then, it was speculated that AM10 was 3-methoxyl-4-hydroxybenzenepropanoic acid sulphate and that AM11 was isomer of it owing to the fragment ion peaks at m/z 195.0665 ([M − SO3 − H]), m/z 151.0799 ([M − SO3 − CO2 − H]) and m/z 177.0777 ([M − SO3 − H2O − H]) in the MSn spectrum.
The [M − H] peaks of AM12, AM13 and AM14 were observed at m/z 329.09, suggesting the formula as C14H18O9. The parent ion was cleaved into two parts m/z 175.0282 ([glucuronic acid − H2O − H]) and m/z 153.0568. And the ion at m/z 153.0568 was determined to be 3,4-dihydroxyphenylethanol similar to AM1, which has three reaction sites for gluconylation. The above data suggested AM12, AM13 and AM14 to be 4-(2-hydroxyethyl)-1,2-benzenediol glucuronide.
AM15, speculated to be C15H18O9, was detected in urine samples with quasi-molecular ion peak at m/z 341.0848 ([M − H]). The MS2 fragment ion peaks at m/z 165.0520 ([M − glucuronic acid − H]) and m/z 121.0628 ([M − glucuronic acid − CO2 − H]) were just similar to AM3, and in combination with m/z 175.0244, AM15 was determined to be 4-hydroxybenzenepropanoic acid glucuronide. And the MS1 chromatographic peaks of AM16 and AM17 were extracted in urine sample at m/z 343.1017 ([M − H]). Their fragment ion peaks at m/z 175.0265 and m/z 167.0713 indicated that they were 3,4-hydroxyphenylpropanol glucuronide, the reduzates of AM15.
The [M − H] peak of AM18 at m/z 369.0794 (C16H18O10) was detected in urine samples. The MS2 fragment ion at m/z 175.0169 suggested the existence of glucuronic acid, and m/z 193.0508 (C9H10O4) was consistent with the phenylpropanoid unit of angoroside C. Therefore, AM18 might be 3-methoxyl-4-hydroxycinnamic acid glucuronide. And the quasi-molecular ion peak at m/z 371.0988 ([M − H]) and the MS2 fragment ion peaks at m/z 175.0265 and m/z 195.0658 of AM19 suggested that it could be the hydrogenation product of AM18, namely 3-methoxyl-4-hydroxybenzenepropanoic acid glucuronide.
The ion peaks at m/z 637.21 were the quasi-molecular ions of AM20 and AM21, which was the characteristic fragment ion of angoroside C. And the MS2 fragment ion peaks at m/z 461.1636, m/z 311.1151, m/z 193.0548, m/z 167.1269 and m/z 137.0759 were also in conformity with angoroside C. Then they might be the isomers of deglycosylated angoroside C. For AM22 and AM23, the quasi-molecular ion peaks at m/z 959.30 and main MS2 fragment ion peak at m/z 783.26 ([angoroside C − H]) were observed, which indicated AM22 and AM23 to be isomers of angoroside C glucuronide.
The formula of AM24 detected in feces samples was predicted to be C20H30O12 based on the quasi-molecular ion peak at m/z 461.1701. The fragment ion peaks at m/z 443.1711 and m/z 329.1180 could also be observed in the MS2 spectrum of angoroside C. So it could be speculated that AM24 was the hydrolysate of deferuloyl-angoroside C, namely 2-(3-hydroxy-4-methoxyphenyl)ethyl 6-O-arabinopyranosyl glucopyranoside.
The negative quasi-molecular ion of AM25 at m/z 247.0266 ([M − H]) was detected in feces and plasma samples, and speculated as C9H12O6S. The fragment ion peaks at m/z 167.0750 ([M − SO3 − H]), m/z 137.0627 ([M − SO3 − CH2O − H]) and m/z 121.0264 ([M − SO3 − CH2O − CH4 − H]) suggested that AM25 was 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.
The metabolites and metabolic pathways of angoroside C in rats are shown in Figure 4.

2.4. The Distribution of Sibirioside A and Angoroside C in Rats

To investigate the distribution of the prototypes of sibirioside A and angoroside C, the corresponding quasi-molecular ion peaks at m/z 471.14 and m/z 783.25 were extracted in negative MS1 spectrum. The extract ion flow chromatography (EIC) of different biological samples are shown in Figure 5 and Figure 6. The prototype of sibirioside A was widely distributed in rats; it could be detected in urine, heart, liver, spleen, lung, kidney, stomach, and small intestine samples, and the content of sibirioside A in the stomach and small intestine is the highest, followed by the kidney and liver. Angoroside C was detected in urine, feces, stomach, and small intestine samples, while there was only one metabolite observed in plasma samples without the prototype of angoroside C.

2.5. Biological Activity Prediction of the Metabolites of Sibirioside A and Angoroside C

The bio-activities of the metabolites of sibirioside A and angoroside C were predicted through “PharmMapper Server”, which based on a massive pharmacophore database containing relatively well-developed target information over 7000 pharmacophore models recorded in TargetBank, BindingDB, DrugBank, and other drug target databases [30,31,32]. It automatically matches the pending molecules with all targets in the database and then calculates the fit degree to list the best scoring targets. The first 50 results on human targets were chosen and then the disease-related outcomes for every metabolite were picked out. These are shown in Table S1. In total, 29 targets were picked out, of which RASH, INSR, KPYR, ANGI, BTK, FA7, NDKB, G6PI and CAH2 were identified over 10 times. Their functions include anti-cancer, anti-diabetes, anti-anemia, neuroprotection, anti-leukemia, anti-coagulopathy, and osteogenic activity. The most common target is RASH, which has GTPase activity and is considered to be related to inhibition of growth and metastasis of tumors. Defects in INSR, an insulin binding receptor with tyrosine kinase activity, could cause insulin resistance, leprechaunism, noninsulin-dependent diabetes mellitus, and familial hyperinsulinemic hypoglycemia. This target was identified as a potential target of 17 metabolites of sibirioside A and angoroside C. Defects in INSR cause insulin resistance, leprechaunism, noninsulin-dependent diabetes mellitus, and familial hyperinsulinemic hypoglycemia. Thus, SM1, SM2, SM3, AM2, AM4, AM5, AM6, AM9, AM10, AM11, AM12, AM13, AM15, AM18, AM19, AM24, AM25 might be used to cure diabetes, which was one of the main pharmacological activities of SR in traditional Chinese medicine. In addition, SM3, AM2, AM5, AM8, AM9, AM10, AM11, AM14, AM15, AM18, AM19, AM24 and AM25 may be available to target chronic leukemia; AM2, AM4, AM5, AM6, AM8, AM9, AM10, AM11, AM14 and AM24 may have osteogenic actions. The predicted outcomes were in good agreement with the actual activities reported in the literatures [11,12,13,14,15].
The metabolites of sibirioside A and angoroside C have a variety of potential pharmacological actions, which require further research.

3. Discussion

A total of 4 metabolites of sibirioside A were found in biological samples, of which SM1 was in urine, SM1SM3 in feces, and SM4 in the stomach. A total of 25 metabolites of angoroside C were found and identified, of which AM1AM23 were in urine, AM24 and AM25 were in feces, and AM25 in plasma. For sibirioside A, the main metabolic reactions were hydrogenation, hydroxylation, methylation, sulfation, glycosylation, and dimerization, while the metabolic reaction of angoroside C included hydrolyzation, hydrogenation, sulfation, gluconylation, demethylation, and dehydroxylation. The hydrolyzation reactions occurred in the sugar ester bonds, while the hydrogenation reactions were mainly in double bonds or carboxyl. The sulfation reactive site was the carboxyl group for sibirioside A, but it was the hydroxyl group for angoroside C. The results showed that gluconylation was one of the prime and representative metabolic reactions for angoroside C.
Sibirioside A is an ester glycoside consisting of cinnamic acid and sucrose, and there were less metabolites of it due to the simple molecular structure. The relatively large polarity allowed it to be easily excreted from the urine without the need for complicated metabolic processes. Catalyzed by a variety of enzymes, a portion of sibirioside A was transformed into the metabolites which were mainly sulfated products, then discharged from feces through the bile. The prototype of sibirioside A was detected in all the selected samples except for feces and plasma, which indicated that the rate of sibirioside A clearance was relatively fast in the blood, and it was mainly distributed in the heart, liver, spleen, kidney, stomach, and small intestine in the form of prototype. It was conjectured that sibirioside A might work in the original form and could be preserved for a long time in the viscera in order to extend the time for drug concentration.
Angoroside C is a phenylpropanoid glycoside consisting of three monosaccharide, a phenylpropanoid unit, and a phenylethanoid unit. For angoroside C, total of 25 metabolites were identified, much more than that of sibirioside A, of which 22 were sulfate and glucuronide conjugates. The prototype of angoroside C was detected in urine, feces, stomach, and small intestine samples, but not observed in plasma. It was speculated that the clearance rate of angoroside C in plasma might be fast like sibirioside A. A total of 22 metabolites derived from urine samples had covered all types of metabolic reactions found in this study, and most of the prototype and metabolites were rapidly excreted from the urine.

4. Materials and Methods

4.1. Chemicals and Materials

Sibirioside A (>98%, Lot MUST-16121505) and angoroside C (99%, Lot MUST-16012611) were purchased from Chendu Must Bio-Technology Co., Ltd. (Chengdu, China). Acetonitrile (Fisher, Pittsburgh, PA, USA) and formic acid (ROE, Santa Monica, CA, USA) were of HPLC-grade, and ultrapure water was prepared by Milli-Q water purification system (Millipore, Billerica, MA, USA).

4.2. Animal Experiments

Nine male Sprague-Dawley rats weighing around 250 g were obtained from the Experimental Animal Center of Peking University’s Health Science Center (Beijing, China). The rats were housed in metabolic cages (type DXL-DL, Suzhou Fengshi Laboratory Animal Equipment Co. Ltd., Suzhou, China) kept in an environmentally controlled breeding room for one week for acclimation and had unrestricted access to laboratory-made chow and distilled water to reduce the biological background interferences. Nine rats were divided into three groups, namely three rats in each group (group A, blank group; group B, sibirioside A group; group C, angoroside C group). The standards of sibirioside A and angoroside C were dissolved in ultrapure water and orally administrated respectively to group B and group C at the dose of 200 mg/kg for six continuous days. The rats in group A were orally treated with the same volume of water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. Experiments were reviewed by the Biomedical Ethical Committee of Peking University (approval no. LA2015-007). During the continuous six days of administration, the feces and urine samples were dividually collected using the metabolic cages, and the heparin anticoagulant blood sample was collected by heart puncture under anesthesia 1 h after the last administration. The hearts, livers, spleens, lungs, kidneys and small intestines were quickly scissored and lavaged by normal saline, then the tissue samples were stored at −80 °C.

4.3. Preparation of Rat Biological Samples

Blood samples were centrifuged at 5000 rpm (type 3K15, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) for 15 min to obtain plasma samples. The plasma samples were precipitated with four times methanol, then centrifuged at 10,000 rpm for 15 min. The supernatant was dried under reduced pressure at room temperature. The residue was redissolved in one-twelfth of methanol and centrifuged at 15,000 rpm for 20 min. The supernatant was collected for analysis.
Urine samples were dried under reduced pressure at 50 °C (Heidolph Laborota 4001 rotatory evaporator, Heidolph Instruments GmbH & Co., Schwabach, Germany); the residue was ultrasonically extracted in 50 mL methanol for 30 min. The suspension was centrifuged at 5000 rpm for 15 min, and the supernatant was dried under reduced pressure at 40 °C. The residue was redissolved in five times of methanol, and filtered through a 0.22 µm nylon filter.
Feces samples were dried in a thermostat oven (Shanghai Yiheng Scientific Instruments Co., Ltd., Shanghai, China) at 50 °C, and then pulverized to get powders. The feces powders were ultrasonically extracted for 30 min in five times of 50% methanol, repeated three times. The extracts were merged and dried under reduced pressure at 40 °C, and the residue was redissolved in 10 times of methanol, then filtered through a 0.22 µm nylon filter.
Organ tissues were homogenized by an ultra-turrax T10 electric homogenizer (IKA Co., Ltd., Staufen, Germany) with twice as much water, and five times of methanol was added into the homogenate. The mixture underwent ultrasonic processing for 30 min, thencentrifuged at 15,000 rpm for 20 min. The supernatant was dried under reduced pressure at room temperature. The residue was redissolved in methanol and centrifuged at 15,000 rpm for 20 min. The supernatant was collected for analysis.

4.4. Instrumental and Analytical Conditions

HPLC analysis was performed on a Shimadzu HPLC system equipped with two LC-20AD pumps, a CTO-20A column oven, an SIL-20 AC autosampler, and a CBM-20A system controller. Chromatographic separation was carried out on an Agilent Zorbax SB-C18 column (5 μm, 250 × 4.6 mm) protected by an Agilent Zorbax SB-C18 column. A 10 μL aliquot was injected for each sample. The thermostatted autosampler was maintained at 15 °C and the column oven temperature was kept at 30 °C. The column was eluted with a gradient mobile phase consisting of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) with a flow rate of 1.0 mL/min. For the sibirioside A group, the gradient program was adopted in the following manner: 5–18% B at 0–20 min, 18–37% B at 20–40 min, 37–43% B at 40–43 min, 43–56% B at 43–63 min, 56–68% B at 63–65 min, 68–81% B at 65–75 min, 81–100% B at 75–95 min and 100% B at 95–140 min. For the angoroside C group, the gradient program was adopted in the following manner: 3% B at 0–8 min, 3–5% B at 8–10 min, 5% B at 10–20 min, 5–8% B at 20–23 min, 8–19% B at 23–38 min, 19% B at 38–45 min, 19–27% B at 45–50 min, 27% B at 50–63 min, 27–55% B at 63–73 min, 55% B at 73–83 min, 55–100% B at 83–93 min and 100% B at 93–140 min. High-resolution mass spectra were recorded on an IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). The ESI source was operated in negative ion mode. The mass spectrometer was programmed to carry out a full scan over m/z 100–1000 Da (MS1) and m/z 50–1000 Da (MS2 and MS3). The other parameters were set as follows: flow rate, 0.2 mL/min (split from 1.0 mL/min HPLC effluent); heat block and curved desolvation line temperature, 200 °C; nebulizing nitrogen gas flow, 1.5 L/min; interface voltage: (+), 4.5 kV; (−), −3.5 kV; detector voltage, 1.70 kV; relative collision-induced dissociation energy, 50%. All data were recorded and processed using Shimadzu software LCMS solution version 3.60 and Formula Predictor version 1.2 (Shimadzu, Kyoto, Japan).

5. Conclusions

A total of four metabolites of sibirioside A were detected and identified, three of which were new compounds. The main metabolic reactions included hydrogenation, hydroxylation, methylation, sulfation, glycosylation, and dimerization, and the different types of reactions usually occurred simultaneously. Sibirioside A was mainly excreted from urine in the form of prototype. The in vivo metabolism might be carried out in the liver, and then excreted from feces through bile. It was speculated that sibirioside A might take effects in prototype form due to its wide distribution in the heart, liver, spleen, lung, kidney, stomach, and small intestine. There were 25 metabolites identified in vivo for angoroside C, 11 of which were new compounds. The prime metabolic reactions were hydrolyzation, hydrogenation, sulfation, glucuronidation, demethylation, and dihydroxylation. A total of 22 of the metabolites were sulfated or glucuronidization products and mainly excreted from urine just like the prototype, which was concentrically distributing in the stomach and small intestine.
In total, 29 targets were identified including INSR, RASH, KPYR, ANGI and so on, which might be related to anti-diabetes, anti-cancer, anti-anemia, neuroprotection, anti-leukemia, anti-coagulopathy, and osteogenic activities. The predicted outcomes indicated that the metabolites of sibirioside A and angoroside C have a variety of potential pharmacological activities similar to the prototypes, which require further research.
Phenylpropanoid glycosides are a large class of compounds with a simple structure and extensive pharmacological activities. Their metabolic processes in vivo are simple and predominantly they might distribute and take effect in the form of prototypes.

Supplementary Materials

Supplementary materials are available online. Figure S1: The ESI-IT-TOF MSn spectra of angoroside C (A) and sibirioside A (B), Figures S2–S20: The negative ion mode EIC, Table S1: The predicted results of bioactivities for metabolites of sibirioside A and angoroside C.

Author Contributions

M.-Y.S. conceived and designed the experiments and reviewed the final manuscript; Y.-F.Z. performed the experiments, analyzed the data and prepared the manuscript; L.-J.L. participated in the animal experiments; F.X. performed the LC-MS analysis; G.-X.L. and S.-Q.C. reviewed the final manuscript.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 81274074).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Xu, F.Q.; Xu, X.D.; Chen, S.L. Progress in chemical constituents and bioactivities of Scrophularia ningpoensis. Mod. Chin. Med. 2013, 9, 752–759. [Google Scholar]
  2. Li, Y.M.; Zeng, H.W.; He, X.; Jiang, Y.Y.; Jiang, S.H.; Zhu, D.Y. Iridoid and phenylpropanoid glycosides of Scrophularia ningpoensis inhibit the formation of LTB4 and platelet aggregation. Acad. J. Second Mil. Med. Univ. 1999, 20, 301–303. [Google Scholar]
  3. Díaz, A.M.; Abad, M.J.; Femández, L.; Silván, A.M.; Santos, J.D.; Bemejo, P. Phenylpropanoid glycosides from Scrophularia scorodonia: In vitro anti-inflammatory activity. Life Sci. 2004, 74, 2515–2526. [Google Scholar] [CrossRef] [PubMed]
  4. Liu, H.; Zheng, Y.F.; Li, C.Y.; Zheng, Y.Y.; Wang, D.Q.; Wu, Z.; Huang, L.; Wang, Y.G.; Li, P.B.; Peng, W.; et al. Discovery of Anti-inflammatory Ingredients in Chinese Herbal Formula Kouyanqing Granule based on Relevance Analysis between Chemical Characters and Biological Effects. Sci. Rep. 2015, 5, 18080. [Google Scholar] [CrossRef] [PubMed][Green Version]
  5. Li, Y.M.; Han, Z.H.; Jiang, S.H.; Yao, S.D.; Zhu, D.Y. Fast repairing of oxidized OH radical adducts of dAMP and dGMP by phenylpropanoid glycosides from Scrophularia ningpoensis. Acta Pharmacol. Sin. 2000, 21, 1125–1128. [Google Scholar] [PubMed]
  6. Gu, W.L.; Chen, C.X.; Huang, X.Y.; Gao, J.P. The effect of angoroside C on pressure overload-induced ventricular remodeling in rats. Phytomedicine 2015, 22, 705–712. [Google Scholar] [CrossRef] [PubMed]
  7. Li, Y.M.; Jiang, S.H.; Gao, W.Y.; Zhu, D.Y. Phenylpropanoid glycosides from Scrophularia ningpoensis. Phytochemistry 2000, 54, 923–925. [Google Scholar] [CrossRef]
  8. Jing, J.; Chan, C.O.; Xu, L.; Jin, D.; Cao, X.; Mok, D.K.; Parekh, H.S.; Chen, S. Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae. J. Pharm. Biomed. Anal. 2011, 56, 830–835. [Google Scholar] [CrossRef] [PubMed]
  9. Zhang, J.; Fanny, C.F.; Liang, Y.; Nancy, Y.; Zhong, B.L.; Lai, C.W.; Xu, S.H. A new iridiod glycoside and a new cinnamoyl glycoside from Scrophularia ningpoensis Hemsl. Nat. Prod. Res. 2017, 31, 2361–2368. [Google Scholar] [CrossRef] [PubMed]
  10. Chinese Pharmacopoeia Commission. Pharmacopoeia of the Peoples Republic of China; Chemical Industry Press: Beijing, China, 2015. [Google Scholar]
  11. Zhang, L.; Zhang, L.; Zhang, Y.D. Comparative study on the pharmacognosy and anti-inflammatory activities of Scrophularia buergeriana Miq. Var. tsinglingensis Tsoong and S. ninpoensis Hemsl. Northwest Pharm. J. 2014, 29, 264–267. [Google Scholar]
  12. Bermejo, B.P.; Diaz, L.A.; Silvan, S.A.; De Santos, G.Z.; Fernandez, M.L.; Sanz, G.A.; Abad, M.J. Effects of some iridoids from plant origin on arachidonic acid metabolism in cellular systems. Planta Med. 2000, 66, 324–328. [Google Scholar]
  13. Liu, G.L.; Fu, P.Y.; Wang, Z.Y.; Xing, D.Y. Effects of water extract of four Chinese herbal drugs on the binding of insulin with human erythroeyte insulin receptor. Chin. J. Integr. Trad. West. Med. 1991, 10, 606–607. [Google Scholar]
  14. Ni, Z.; Cai, X.Z.; Huang, Y.P.; Wang, D.J.; Bian, H.M. Effect of extracts of Scrophularia ningpoensis Hemsl. on hemorrheology, coagulation and fibrinolysis in rats. J. Chin. Microcirc. 2004, 3, 152–153. [Google Scholar]
  15. Sohn, S.; Ko, E.; Jeon, S.; Lee, B.J.; Kim, S.H.; Dong, M.S.; Lee, D.U.; Kwak, J.H.; Kim, Y.S.; Shin, M.K.; et al. The genome-wide expression profile of Scrophularia ningpoensis-treated thapsigargin-stimulated U-87MG cells. Neurotoxicology 2009, 30, 368–376. [Google Scholar] [CrossRef] [PubMed]
  16. Wang, J.Z.; Liu, Z.; Ma, L.M.; Li, J.; Shang, M.Y.; Liu, G.X.; Xu, F.; Cai, S.Q. Changes of chemical constituents in Scrophulariae Radix during processing based on UPLC-Q-TOF MS. J. Chin. Mass Spectrom. Soc. 2016, 37, 1–9. [Google Scholar]
  17. Wang, J.Z.; Zhang, Y.F.; Xu, F.; Shang, M.Y.; Liu, G.X.; Cai, S.Q. Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC-IT-TOF-MSn. Biomed. Chromatogr. 2018, e4218. [Google Scholar] [CrossRef] [PubMed]
  18. Liu, Z.; Xu, F.; Wang, J.Z.; Liu, G.X.; Shang, M.Y.; Cai, S.Q. Identification and analysis of harpagide metabolites in rats in vivo. Chin. Pharm. 2017, 28, 1310–1315. [Google Scholar]
  19. Ounnas, F.; Privé, F.; Salen, P.; Gaci, N.; Tottey, W.; Calani, L.; Bresciani, L.; Lopez-Gutierrez, N.; Hazane-Puch, F.; Laporte, F.; et al. Whole rye consumption lmproves blood and liver n-3 fatty acid profile and gut microbiota composition in rats. PLoS ONE 2016, 11, e0148118. [Google Scholar] [CrossRef] [PubMed]
  20. Tuck, K.L.; Hayball, P.J.; Stupans, I. Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats. J. Agric. Food Chem. 2002, 50, 2404–2409. [Google Scholar] [CrossRef] [PubMed]
  21. Zaklo, D.; Costagliola, R.; Dorio, C.; Rathahao, E.; Cravedi, J.P. In vivo metabolic fate of the xeno-estrogen 4-N-nonylphenol in Wistar rats. Drug Metab. Dispos. 2003, 31, 168–178. [Google Scholar]
  22. Wusteman, F.S.; Dodgson, K.S.; Lloyd, A.G.; Rose, F.A.; Tudball, N. Thin-layer chromatography in the study of ester sulfates. J. Chromatogr. A 1964, 16, 334–339. [Google Scholar] [CrossRef]
  23. Stalmach, A.; Mullen, W.D. Metabolite profiling of hydroxycinnamate derivatives in plasma and urine after the ingestion of coffee by humans: Identification of biomarkers of coffee consumption. Drug Metab. Dispos. 2009, 37, 1749–1758. [Google Scholar] [CrossRef] [PubMed]
  24. Fumeaux, R.; Menozzi, C.; Stalmach, A.; Munari, C.; Kraehenbuehl, K. First synthesis, characterization, and evidence for the presence of hydroxycinnamic acid sulfate and glucuronide conjugates in human biological fluids as a result of coffee consumption. Org. Biomol. Chem. 2010, 8, 5199–5211. [Google Scholar] [CrossRef] [PubMed]
  25. Fujiwara, S.; Sakurai, S.; Sugimoto, I.; Awata, N. Absorption and metabolism of γ-oryzanol in rats. Chem. Pharm. Bull. 2008, 31, 645–652. [Google Scholar] [CrossRef]
  26. De Karina, I.T.; Jauregui, O.; Castellote, A.I.; Lamuelaraventós, R.M.; Covas, M.I. Rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method for qualitative and quantitative analysis of virgin olive oil phenolic metabolites in human low-density lipoproteins. J. Chromatogr. A 2006, 1116, 69–75. [Google Scholar]
  27. Khymenets, O.; Joglar, J.; Clapés, P.; Parella, T.; Covas, M. Biocatalyzed synthesis and structural characterization of monoglucuronides of hydroxytyrosol, tyrosol, homovanillic alcohol, and 3-(4′-hydroxyphenyl)propanol. Adv. Synth. Catal. 2010, 348, 2155–2162. [Google Scholar] [CrossRef]
  28. Thibaut, R.; Debrauwer, L.; Rao, D.; Cravedi, J.P. Characterization of biliary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss). Xenobiotica 1998, 28, 745–757. [Google Scholar] [CrossRef] [PubMed]
  29. Kajimoto, T.; Hidaka, M.; Shoyama, K.; Nohara, T. Iridoids from Scrophularia ningpoensis. Phytochemistry 1989, 28, 2701–2704. [Google Scholar] [CrossRef]
  30. Liu, X.F.; Ouyang, S.S.; Yu, B.; Liu, B.; Liu, Y.B.; Huang, K.; Gong, J.Y.; Zheng, S.Y.; Li, Z.H.; Li, H.L.; et al. PharmMapper server: A web server for potential drug target identification using pharmacophore mapping approach. Nucleic Acids Res. 2010, 38, W609–W614. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, X.; Pan, C.X.; Gong, J.Y.; Liu, X.F.; Li, H.L. Enhancing the enrichment of pharmacophore-based target prediction for the polypharmacological profiles of drugs. J. Chem. Inf. Model. 2016, 56, 1175–1183. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, X.; Shen, Y.H.; Wang, S.W.; Li, S.L.; Zhang, W.L.; Liu, X.F.; Lai, L.H.; Pei, J.F.; Li, H.L. PharmMapper 2017 update: A web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Res. 2017, 45, W356–W360. [Google Scholar] [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
Figure 1. The fragmentation processes of sibirioside A.
Figure 1. The fragmentation processes of sibirioside A.
Molecules 23 02702 g001
Figure 2. The fragmentation processes of angoroside C.
Figure 2. The fragmentation processes of angoroside C.
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Figure 3. The metabolites and metabolic pathways of sibirioside A in rats.
Figure 3. The metabolites and metabolic pathways of sibirioside A in rats.
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Figure 4. The metabolites and metabolic pathways of angoroside C in rats.
Figure 4. The metabolites and metabolic pathways of angoroside C in rats.
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Figure 5. The extract ion flow chromatography (EIC) at m/z 471.1500 of sibirioside A in different biological samples. (A) plasma; (B) urine; (C) feces; (D) heart; (E) liver; (F) spleen; (G) lung; (H) kidney; (I) stomach; (J) small intestine.
Figure 5. The extract ion flow chromatography (EIC) at m/z 471.1500 of sibirioside A in different biological samples. (A) plasma; (B) urine; (C) feces; (D) heart; (E) liver; (F) spleen; (G) lung; (H) kidney; (I) stomach; (J) small intestine.
Molecules 23 02702 g005
Figure 6. The EIC at m/z 783.2600 of angoroside C in different biological samples. (A) plasma; (B) urine; (C) feces; (D) heart; (E) liver; (F) spleen; (G) lung; (H) kidney; (I) stomach; (J) small intestine.
Figure 6. The EIC at m/z 783.2600 of angoroside C in different biological samples. (A) plasma; (B) urine; (C) feces; (D) heart; (E) liver; (F) spleen; (G) lung; (H) kidney; (I) stomach; (J) small intestine.
Molecules 23 02702 g006
Table 1. LC-MS data obtained in the negative ion mode for the identification of the metabolites of sibirioside A and angoroside C in rats.
Table 1. LC-MS data obtained in the negative ion mode for the identification of the metabolites of sibirioside A and angoroside C in rats.
No.tR/minFormulam/zDiffDBEFragmentIdentification
SM17.813C9H10O5S229.0155−9.175147.0459, 103.0348phenylpropanoic acid sulphate [19]
SM210.328C10H12O6S259.02934.255177.0540, 133.09834-methoxyphenylpropanoic acid sulphate *
SM37.277C18H18O8S393.075510.5010311.1019, 163.0583, 147.0454β-hydroxybenzenepropanoic anhydride sulphate *
SM424.513C27H38O17633.2014−3.479485.1416, 323.0932sibirioside A glucose conjugation *
AM115.602C8H10O6S233.0119−2.574153.0583, 123.04924-(2-hydroxyethyl)-1,2-benzenediol sulphate [20]
AM232.993C9H8O6S242.9965−7.826163.0415, 119.0532p-coumaric acid sulphate [21]
AM329.057C9H10O6S245.0109−6.535165.0574, 121.06804-hydroxybenzenepropanoic acid sulphate [22]
AM417.995C9H12O6S247.0265−6.884167.0745, 137.06423,4-hydroxyphenylpropanol sulphate 1 *
AM520.260C9H12O6S247.0271−4.454167.0745, 137.06423,4-hydroxyphenylpropanol sulphate 2 *
AM622.548C9H12O6S248.028204167.0745, 137.06423,4-hydroxyphenylpropanol sulphate 3 *
AM742.685C10H12O6S259.0271−4.255179.0726, 163.0385, 147.0473, 119.0507β-methoxybenzenepropanoic acid sulphate *
AM818.630C9H10O7S261.0063−4.215217.0175, 181.0522, 137.06333,4-dihydroxybenzenepropanoic acid-3-O-sulphate [23]
AM926.887C9H10O7S261.0051−8.815217.0175, 181.0522, 137.06333,4-dihydroxybenzenepropanoic acid-3-O-sulphate [24]
AM1030.600C10H12O7S275.0210−7.645195.0665, 177.0545, 151.07993-methoxyl-4-hydroxybenzenepropanoic acid sulphate [25]
AM1138.278C10H12O7S275.0214−6.185195.0665, 177.0545, 151.07993-hydroxyl-4- methoxyl benzenepropanoic acid sulphate [24]
AM1215.712C14H18O9329.09295.16175.0282, 153.05684-(2-hydroxyethyl)-1,2-benzenediol glucuronide [26]
AM1316.507C14H18O9329.0871−2.136175.0282, 153.05684-(2-hydroxyethyl)-1,2-benzenediol glucuronide [27]
AM1417.792C14H18O9329.0857−6.386175.0282, 153.05684-(2-hydroxyethyl)-1,2-benzenediol glucuronide [20]
AM1533.103C15H18O9341.0848−8.807323.0709, 175.0244, 165.052, 121.06284-hydroxybenzenepropanoic acid glucuronide [28]
AM1624.020C15H20O9343.1017−5.256325.097, 175.0265, 167.0713, 147.03013,4-hydroxyphenylpropanol glucuronide *
AM1729.170C15H20O9343.1017−5.256325.097, 175.0265, 167.0713, 147.03013,4-hydroxyphenylpropanol glucuronide *
AM1835.262C16H18O10369.0794−8.948193.0508, 175.01693-methoxyl-4-hydroxycinnamic acid glucuronide [25]
AM1932.448C16H20O10371.09881.087195.0658, 175.02653-methoxyl-4-hydroxybenzenepropanoic acid glucuronide [25]
AM2052.445C30H38O15637.2131−1.1012461.1636, 443.1533, 311.1151, 275.0889, 193.0548, 167.1269, 137.0759deglycosylated angoroside C 1 *
AM2153.108C30H38O15637.21776.1212461.1636, 443.1533, 311.1151, 275.0889, 193.0548, 167.1269, 137.0759deglycosylated angoroside C 2 *
AM2244.933C42H56O25959.3007−3.2315783.2594, 607.2227, 461.1681, 443.1608angoroside C glucuronide 1 *
AM2347.378C42H56O25959.2941−0.3115783.2594, 607.2227, 461.1681, 443.1608angoroside C glucuronide 2 *
AM2433.343C20H30O12461.17017.816461.1701, 443.1711, 329.1180, 191.05742-(3-hydroxy-4-methoxyphenyl)ethyl 6-O-arabinopyranosyl glucopyranoside [29]
AM2527.377C9H12O6S247.0266−6.484167.0750, 137.0627, 121.02644-(2-hydroxyethyl)-1,2-benzenediol sulphate *
* means new compound.
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