Article pubs.acs.org/JAFC
Identification of Echinacoside Metabolites Produced by Human Intestinal Bacteria Using Ultraperformance Liquid Chromatography− Quadrupole Time-of-Flight Mass Spectrometry Yang Li,† Guisheng Zhou,† Shihua Xing,† Pengfei Tu,§ and Xiaobo Li*,† †
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, People’s Republic of China
§
ABSTRACT: Echinacoside (ECH) is one of the representative phenylethanoid glycosides. It is widely present in plants and exhibits various bioactivities. However, the extremely low oral bioavailability of ECH in rats implies that ECH may go through multiple hydrolysis steps in the gastrointestinal tract prior to its absorption into the blood. Therefore, the gastrointestinal metabolites of ECH are more likely to be the bioactive components. This study established an approach combining ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with MSE technology and MetaboLynx software for rapid analysis of the ECH metabolic profile produced by human intestinal bacteria. As a result, 13 ECH metabolites and 5 possible metabolic pathways (including deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation) were identified. Furthermore, hydroxytyrosol (HT) and 3-hydroxyphenylpropionic acid (3-HPP) were found to be the two bioactive metabolites of ECH produced by human intestinal bacteria. KEYWORDS: echinacoside, human intestinal bacteria, UPLC-Q-TOF-MS, metabolites, metabolic pathway
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INTRODUCTION Echinacoside (ECH) is a natural phenylethanoid glycoside (PhG) that has been recognized as the major bioactive component in Cistanches Herba1,2 and Echinacea species3 with diversified pharmacological functions such as antioxidation,4,5 anti-inflammation,6 and neuroprotection effects.7,8 As a wellknown natural product, ECH is commonly used as a bioactive ingredient for phytomedicines, functional foods, or dietary supplements worldwide.9−11 The oral route, which is the preferred administration method of most herbal products, will inescapably lead to contact with the microflora from the intestinal tract before their components are absorbed. Additionally, some of the active natural ingredients from phytomedicines actually exert their biological effects through interaction with the enriched intestinal bacteria before absorption,12−14 and ECH-containing products may not be an exception in this regard. In a previous pharmacokinetic study, the observed absolute bioavailability (F, %) of ECH was 0.83%,15 which is extremely low. This finding implies that ECH may undergo multiple routes of hydrolysis in the gastrointestinal tract and produce degradation intermediates before being absorbed into the blood.16 ECH is composed of four chemical moieties including caffeic acid (CA), hydroxytyrosol (HT; 3,4-dihydroxyphenethyl alcohol, phenylethanoid aglycone), rhamnose (Rha), and glucose (Glu).1,17 These moieties are bound by ester linkage, which is easily broken under harsh conditions such as a strong acidic or alkali environment. However, so far there are very few investigations on the metabolic process and the biological activity of ECH byproducts in the human intestinal tract. The MSE technology, which can acquire the exact mass of precursor and fragment ions in a single analytical run, is a © XXXX American Chemical Society
commonly used approach in metabolite studies. The method was first introduced by Plumb and his colleagues,18 by setting the collision energy range of MSE manually with the capability of automatic shift, to obtain information on both precursors and fragments. Most of our target metabolites require a rapid, sensitive, and accurate analytical method for structural identification because only a tiny amount of these metabolites is generated. Hence, we applied MSE technology in this research to analyze the complex ECH metabolites produced by human intestinal bacteria. In this study, we analyzed the metabolites of ECH by human intestinal bacteria and investigated the metabolic process and characteristics of ECH in vitro by ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with MSE technology and MetaboLynx software. To our knowledge, this is the first publication focusing on the metabolism of ECH by human intestinal bacteria so far.
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MATERIALS AND METHODS
Chemicals and Reagents. General anaerobic medium broth (GAM broth) was purchased from Shanghai Kayon Biological Technology Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Deionized water was prepared by distilled water through a Milli-Q water purification system (Millipore, Bedford, MA, USA). All of the other reagents and chemicals were of analytical grade. Received: June 11, 2015 Revised: July 14, 2015 Accepted: July 17, 2015
A
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. Accurate Mass Measurements of Pseudo-molecular and Fragment Ions of Seven Standards compound
tR (min)
formula
measured mass (Da)
calculated mass (Da)
error (mDa)
MS/MS fragment ions (Da)
echinacoside acteoside hydroxytyrosol caffeic acid 3,4-dihydroxybenzenepropionic acid 3-hydroxyphenylpropionic 3-phenylpropionic acid
3.02 3.78 1.57 2.33 2.26 3.39 5.71
C35H45O20 C29H35O15 C8H9O3 C9H7O4 C9H9O4 C9H9O3 C9H9O2
785.2505 623.1971 153.0562 179.0344 181.0509 165.0560 149.0612
785.2504 623.1976 153.0552 179.0344 181.0501 165.0552 149.0603
0.1 −0.5 1.0 0.0 0.8 0.8 0.9
623, 477, 461, 315, 179, 161, 153, 135 461, 315, 179, 161, 135 123 135 137 121 105
Figure 1. UPLC-MS/MS spectra and proposed fragmentation pathways of echinacoside (a) and acteoside (b). monitored at 330 nm. The column temperature was set at 35 °C. The flow rate was 1.0 mL/min, and the sample injection volume was 10 μL. Metabolism Study of ECH by Human Intestinal Bacteria. Fresh human fecal samples were obtained from six healthy volunteers (three males and three females, 22−50 years of age) who had no history of gastrointestinal disorders or antibiotics usage for at least 3 months prior to the study. The fresh fecal samples were mixed and immediately homogenized with 25 times volume of GAM broth. The sediments were removed by filtration through three pieces of gauze. The suspension was incubated at 37 °C in an anaerobic incubator in which the air had been replaced by a gas mixture (H2 5%, CO2 10%, N2 85%). Three milligrams of ECH was placed in 10 mL of human fecal suspension, and the suspension was incubated at 37 °C for 48 h. The cultured mixture was removed and extracted with water-saturated n-butanol at time points of 1, 3, 6, 9, 12, 24, and 48 h. The mixture was centrifuged at 4800 rpm for 20 min, and the supernatant was then evaporated. The residue was dissolved in 0.5 mL of methanol and centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was analyzed by UPLC-Q-TOF-MS. UPLC-MS Analysis. UPLC-Q-TOF-MS analysis was performed on a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm, Waters Corp., USA) by gradient elution using acetonitrile (A) and water (B) at a flow rate of 0.4 mL/min. The gradient profile was 0−3 min (A, 5−20%), 3−6 min (A, 20−30%), 6− 9 min (A, 30−70%), and 9−10.5 min (A, 70−99%), then held for 2 min. The gradient was recycled back to 5% in 2.5 min for the next run. The injection volume was 3 μL. The temperature of the column oven was set to 35 °C.
Echinacoside was kindly provided by Dr. Pengfei Tu’s laboratory from Peking University (Beijing, China). Acteoside was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Hydroxytyrosol (HT), caffeic acid (CA), 3,4-dihydroxybenzenepropionic acid, 3-hydroxyphenylpropionic acid (3-HPP), and 3phenylpropionic acid were purchased from Aladdin Industrial Inc. (Shanghai, China). The purity of each compound was determined to be >95% by HPLC-UV. Stability Study of ECH in Simulated Gastric Juice and Intestinal Juice. One milligram of ECH was placed in 10 mL of simulated gastric juice (0.08 M HCl containing 50 mg of pepsin, pH 1.5) and incubated at 37 °C for 0, 1, 2, or 4 h, respectively. The reactions were quenched by water-saturated n-butanol immediately. The mixture was centrifuged at 4800 rpm for 20 min, and then the supernatant was evaporated. The residue was dissolved in 0.5 mL of methanol and centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was analyzed by HPLC. One milligram of ECH was placed in 10 mL of simulated intestinal juice (0.05 M KH2PO4 containing 50 mg of pancreatin, pH 6.8) and incubated at 37 °C for 0, 2, 4, or 6 h, respectively. The sample processing was the same as that for ECH in simulated gastric juice. HPLC Analysis. The HPLC system (Agilent 1200, USA) consisted of a multisolvent delivery pump equipped with a quaternary solvent delivery system, an autosampler, and a DAD detector, which was used to analyze the stability of ECH in simulated gastric juice and intestinal juice. A ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm i.d., 5 μm, Agilent Ltd., USA) was used. The chromatographic condition was acetonitrile (A) and water (B) with gradient elution, 0−30 min (A, 10−30%) and 30−35 min (A, 30−100%). UV absorption was B
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 2. Echinacoside (M0) and Its Metabolites (M1−M13) Identified in the Samples Incubated with Human Intestinal Bacteria for 24 h Using UPLC-Q-TOF/MS no.
tR (min)
measured mass (Da)
error (mDa)
formula
M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13
3.02 1.57 2.26 2.33 2.44 2.95 3.19 3.35 3.39 3.46 3.66 3.72 3.77 5.72
785.2482 153.0567 181.0505 179.0372 801.2433 639.1900 787.2650 769.2546 165.0563 479.1549 625.2124 827.2624 623.1945 149.0623
−2.2 1.5 0.4 2.8 −2.1 −2.5 −1.1 −0.9 1.1 −0.5 −0.9 1.4 −3.1 2.0
C35H45O20 C8H9O3 C9H9O4 C9H7O4 C35H45O21 C29H35O16 C35H47O20 C35H45O19 C9H9O3 C23H27O11 C29H37O15 C37H47O21 C29H35O15 C9H9O2
MS/MS fragment ions (Da) 623, 123 137 135 783, 477, 623, 179, 121 315, 461, 623, 461, 105
477, 461, 315, 179, 161, 153, 135
179, 179, 181, 161,
161, 135 135 153 135
181, 315, 179, 315,
153, 181, 161, 179,
137 153, 137 153, 135 161, 135
identification parent HT 3,4-dihydroxyphenylpropionic acid CA hydroxylated echinacoside M0-Rha reduction of echinacoside dehydroxylated echinasoside 3-HPP M10-Rha reduction of acteoside acetylated echinacoside acteoside 3-phenylpropionic acid
Figure 2. UPLC-MS extracted ion chromatograms (XICs) of echinacoside and its metabolites: (a) M0; (b) M1; (c) M2; (d) M3; (e) M4; (f) M5; (g) M6; (h) M7; (i) M8; (j) M9; (k) M10; (l) M11; (m) M12; (n) M13. Mass spectrometry was carried out using a Waters Synapt mass spectrometer (Waters Corp., Milford, MA, USA). Ionization was performed in the negative electrospray (ESI) mode. The MS parameters were as follows: capillary voltage, 2.8 kV; cone voltage, 35 V; source temperature, 115 °C; desolvation temperature, 350 °C; gas flows of cone and desolvation, 50 and 700 L/h, respectively. For accurate mass measurement, leucine-enkephalin was used as the lock mass. The MSE experiment in two scan functions was carried out as follows: function 1 (low energy), m/z 50−1000, 0.25 s scan time, 0.02 s interscan delay, 4 eV collision energy; function 2 (high energy), m/z 50−1000, 0.25 s scan time, 0.02 s interscan delay, collision energy ramp of 55−70 eV. The data were processed using MassLynx 4.1 software (Waters Corp.). Data Analysis. Data analysis was performed using MetaboLynx software (Waters Corp.) that employs an expected list of potential biotransformation reactions such as deglycosylation, dehydroxylation,
and acetylation. This software automates the detection and identification of metabolites by comparing the sample with the control to eliminate the interfering peaks in the sample that also appear in the control. The mass defect filter was set as 25 mDa. Results were filtered on the basis of the mass fractional deviation from the parent compound and the expected metabolites of the parent compound. The mass window of 0.05 Da was set for both expected and unexpected metabolite mass chromatograms. Apex track peak integration was used for peak detection, and the response threshold of the absolute peak height was set to 20 units.
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RESULTS Stability of ECH in Simulated Gastric Juice and Intestinal Juice. ECH was found to be stable in simulated gastric juice and intestinal juice. After incubation with artificial C
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 3. Proposed fragmentation pathways: (a) M9 (m/z 479); (b) M10 (m/z 625); (c) M11 (m/z 827).
at m/z 179 that produced ions at m/z 135 and 161 by the loss of CO2 and H2O, respectively. HT (m/z 153.0562) produced a fragment at m/z 123, which was probably through the loss of a CH2O moiety. Similarly, CA (m/z 179.0344) yielded a product ion at m/z 135 by the loss of CO2. 3,4-Dihydroxybenzenepropionic acid (m/z 181.0509) produced the ion at m/z 137 by the loss of CO2. CO2 was lost from 3-HPP (m/z 165.0560) to form the ion at m/z 121. 3-Phenylpropionic acid (m/z 149.0612) produced m/z 105 by the loss of CO2 as well. Identification of ECH Metabolites by Human Intestinal Bacteria. Compared with the blank sample, the parent compound ECH (M0) and its 13 metabolites (M1−M13), including the metabolites of parent compound (M4−M7, M9− M12) and degradation products (M1−M3, M8, M13), were identified in human intestinal bacteria samples. Detailed information on these metabolites is listed in Table 2. Deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation were considered as the main metabolic pathways for the parent compound. The extracted ion chromatograms (XICs) of the parent compound (M0) and its metabolites (M1−M13) from the total UPLC-MS chromatograms are shown in Figure 2. In addition, M9, M10, and M11 were selected as the representative metabolites, and their proposed fragmentation pathways are shown in Figure 3. Parent Compound (M0). ECH (M0) was identified by comparing the UPLC retention time (3.02 min), accurate MS
juices, the content of ECH did not change significantly over time according to the peak areas during the course of incubation (RSD < 3.0%). Fragmentation Studies. Solutions of ECH, acteoside, HT, CA, 3,4-dihydroxybenzenepropionic acid, 3-HPP, and 3phenylpropionic acid prepared in methanol were used for the fragmentation pattern study to support metabolite characterizations. The above seven standard compounds were eluted at 3.02 min (ECH), 3.78 min (acteoside), 1.57 min (HT), 2.33 min (CA), 2.26 min (3,4-dihydroxybenzenepropionic acid), 3.39 min (3-HPP), and 5.71 min (3-phenylpropionic acid), respectively (Table 1). The fragmentation patterns of ECH and acteoside were described as followed. As shown in Figure 1a, ECH (m/z 785.2505) produced a fragment at m/z 623 by loss of a CA moiety, and then the sequential losses of rhamnose (Rha), glucose (Glu), and Glu moieties yielded the respective fragment ions at m/z 477, 315, and 153. The fragment at m/ z 461 was generated through the loss of Glu and CA moieties from the precursor ion. The product ion at m/z 179, which was one of the major fragments, corresponded to the CA moiety and then produced ions at m/z 135 and 161 by the further loss of CO2 and H2O, respectively. Acteoside (m/z 623.1971) displayed a similar fragmentation pattern in the spectrum of ECH (Figure 1b). It produced the fragment ion at m/z 461 by losing the CA moiety and then produced the ion at m/z 315 by the further loss of a Rha moiety. The CA moiety was also found D
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 4. Proposed metabolic pathways of echinacoside by human intestinal bacteria.
at m/z 785.2482, and MS/MS spectra with those of the authentic reference standard. Characteristic product ions at m/z 623, 477, 461, 315, 179, 161, 153, and 135 were also found in the MS2 fragmentation. Metabolites of Parent Compound (M4−M7, M9− M12). In this study, the direct metabolites of parent compound were identified by the distinctive fragmentations when compared with the parent compound. M12 (m/z 623.1945, C29H35O15, 3.77 min), a loss of Glu (162 Da) from ECH (M0), was identified as acteoside through comparison of the UPLC retention time, accurate MS, and MS/MS spectra with the authentic reference standard. M10 (m/z 625.2124, C29H37O15, 3.66 min) was 2 Da (2H) higher than M12, which indicated that M10 was the reduced product of M12. In the MS/MS spectrum of this metabolite, the presence of a diagnostic fragment ion at m/z 181 implied that reduction occurred on the α′,β′-double bond of the CA moiety. Additionally, other fragment ions from M10 were consistent with the product ions of M12. The previous study on caffeic acid metabolism by bacteria from the human gastrointestinal tract indicated that CA could be reduced to dihydro-CA by human intestinal bacteria,19,20 which implied this reduction route might occur in the metabolic process of phenylethanoid glycoside and caffeoyl-containing compound. M9 (m/z 479.1549, C23H27O11, 3.46 min) was 146 Da (Rha) lower than M10 in molecular weight and was considered as the deRha product of M10. M9 produced signals at m/z 315, 181, 153, and 137, which were similar to the generated ions of M10. The proposed fragmentation pathways of M9 and M10 are shown in Figure 3. M4 (m/z 801.2433, C35H45O21, 2.44 min), a hydroxylated product of ECH, was 16 Da (O) higher than ECH in molecular weight. The characteristic product ion at m/z 783 was generated by a H2O loss from the quasi-molecular ion. Moreover, caffeic acid related fragment ions were found at m/z 179, 161, and 135, which were the same as the fragment ions of ECH (M0), suggesting that the hydroxylation position was not on the CA moiety. A previous study proposed that the hydroxylation is more likely to occur on the HT moiety to form a benzenetriol structure.21
M5 (m/z 639.1900, C29H35O16, 2.95 min) was 146 Da (Rha) lower than ECH (M0) in molecular weight and was identified as the de-Rha product of M0. The characteristic product ion at m/z 477 was shaped by the elimination of CA moiety (162 Da). The CA moiety was also found at m/z 179 and produced the ion at m/z 135 through the loss of CO2. M6 (m/z 787.2650, C35H47O20, 3.19 min) was 2 Da (2H) higher than M0 in molecular weight, which indicated that M6 was the reduced product of M0. The characteristic fragments produced by M6 at m/z 623, 181, and 153, were similar to those of M10. These results suggested that M6 was generated by the reduction of ECH, which occurred on the α′,β′-double bond of the CA moiety. M7 (m/z 769.2546, C35H45O19, 3.35 min) was 16 Da (O) lower than the protonated ion of ECH, which indicated that it was a metabolite of ECH after the loss of oxygen. In the MS/ MS spectrum of M7, fragment ions associated with CA were found at m/z 179, 161, and 135. Therefore, the dehydroxylated site of M7 was not at the CA moiety. However, the exact dehydroxylated site of M7 could not be confirmed due to the limited information obtained in our study. M11 (m/z 827.2624, C37H47O21, 3.72 min) was 42 Da (C2H2O) higher than the protonated ion of ECH, suggesting a possible conjugation of an acetyl group on ECH. In addition, the characteristic MS/MS fragment ions of M11 at m/z 623, 179, 161, 153, and 135 were observed. The proposed fragmentation pathways of M11 are shown in Figure 3c. According to the previous research, the C-2′ position might be one of the acetyl conjugation sites of M11.20 Metabolites of Degradation Products (M1−M3, M8, M13). In this study, five degradation metabolites were identified using the authentic reference standard. ECH first degraded to HT (M1) and CA (M3), and CA underwent further metabolism to its major microbial metabolite, 3-HPP (M8). M1 (m/z 153.0567, C8H9O3, 1.57 min), M2 (m/z 181.0505, C9H9O4, 2.26 min), M3 (m/z 179.0372, C9H7O4, 2.33 min), M8 (m/z 165.0563, C9H9O3, 3.39 min), and M13 (m/z 149.0623, C9H9O2, 5.72 min) were identified as HT, 3,4dihydroxyphenylpropionic acid, CA, 3-HPP, and 3-phenylE
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 3. Percentage of Relative Content and the Area of Each Metabolite at Different Time Points no.
1 h (%) (area abs)
3 h (%) (area abs)
6 h (%) (area abs)
9 h (%) (area abs)
12 h (%) (area abs)
24 h (%) (area abs)
M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13
78.84 (3008.20) 0.43 (16.50)
74.19 (3172.60) 0.08 (3.40) 0.37 (15.90)
73.04 (3066.50) 0.17 (7.00) 0.28 (11.90)
65.51 (3096.80) 0.18 (8.60) 0.17 (8.20)
0.53 1.75 0.33 3.18
0.54 1.81 0.77 3.20
0.54 1.59 0.92 3.04
0.45 1.57 0.48 2.70
52.71 (2388.60) 0.16 (7.10) 0.08 (3.60) 0.03 (1.40) 0.31 (14.00) 1.20 (54.50) 0.43 (19.60) 1.85 (83.90)
2.51 (95.70) 12.42 (473.80)
0.09 (3.70) 2.42 (103.40) 16.48 (704.80) 0.06 (2.70)
2.02 (85.00) 18.29 (767.70) 0.12 (5.00)
0.15 (7.30) 1.84 (86.90) 26.82 (1267.80) 0.13 (6.00)
0.15 (6.60) 1.23 (55.80) 41.72 (1890.40) 0.13 (5.90)
36.08 (2259.00) 1.25 (76.70) 0.20 (12.20) 0.06 (3.50) 0.27 (16.30) 1.43 (87.60) 1.49 (91.20) 1.44 (88.10) 0.91 (55.80) 2.61 (160.00) 3.21 (197.00) 0.49 (29.80) 49.60 (3044.30) 0.27 (16.60)
sum
100.00 (3815.40)
100.00 (4276.50)
100.00 (4198.40)
100.00 (4727.10)
100.00 (4531.40)
100.00 (6138.10)
(20.40) (66.90) (12.60) (121.30)
(23.10) (77.20) (32.80) (136.90)
(22.60) (66.70) (38.50) (127.50)
(21.20) (74.30) (22.50) (127.50)
48 h (%) (area abs) 1.64 (18.80) 35.97 (412.90) 0.36 (4.10) 0.77 (8.80)
38.21 (438.60) 0.13 (1.50) 0.62 (7.10) 19.61 (225.10) 2.70 (31.00) 100.00 (1147.90)
through the application of the highly selective MSE technique,18 which provides a great advantage for the rapid investigation of multiple structures. MetaboLynx is an automated software22,23 that can detect trace level of metabolites from biological samples and is a powerful tool for metabolic characterization. Previous research found that the poor membrane permeability and the extensive presystemic metabolism of ECH might be the cause of its low oral bioavailability in rats.21,24,25 Therefore, it is not surprising that the presence of bacteria and acidic/alkaline conditions in gastrointestinal ducts could initiate the hydrolysis of ECH. However, these previous studies did not provide detailed information about the metabolism of ECH in gastrointestinal ducts. Thus, we assessed whether gastric juice, intestinal juice, or intestinal microorganisms of gastrointestinal system could metabolize ECH. In our study, ECH was found to be stable in simulated gastric juice and intestinal juice, whereas it could be metabolized to HT and 3-HPP by intestinal bacteria. 3-HPP has been reported as a major microbial metabolite of CA by human intestinal bacteria in vitro,26,27 which is in agreement with our findings. Additionally, reported evidence has shown that HT and 3-HPP possess bioactivities such as antioxidant,28,29 neuroprotective,30,31 and anti-inflammatory functions.32,33 Therefore, we conclude that ECH acts as a prodrug that is transformed to bioactive compounds (HT and 3-HPP) by intestinal bacteria after oral administration, and the released compounds then exert biological activities. In conclusion, a highly selective and sensitive approach using MSE and MetaboLynx combined UPLC-Q-TOF-MS was applied to the screening and identification of ECH metabolites produced by human intestinal bacteria in vitro. Among the 13 metabolites (M1−M13) identified in this study, M5, M6, and M11 have not been previously reported to our knowledge. Deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation were the five possible metabolic pathways that led to the generation of these metabolites. The observation of human intestinal bacteria produced HT and 3-HPP, which possess biological functions similar to those of ECH, could potentially explain the fact that ECH has prominent bioactivity but poor bioavailability. This study further explored the role of intestinal bacteria in the metabolism of natural products and improved the current understanding of ECH metabolism.
propionic acid, respectively, after analysis of the UPLC retention time, accurate MS, and MS/MS spectra of the authentic reference standards. Analysis of the Metabolic Pathways of ECH by Human Intestinal Bacteria. According to the results of our experiment, the proposed metabolic pathways of ECH by human intestinal bacteria are displayed in Figure 4. After incubation with human intestinal bacteria, ECH was metabolized to M4, M5, M6, M7, M11, and M12. Additionally, M12 was metabolized to M10 and M9 sequentially. After the degradation metabolites M1 and M3 emerged, M3 was further metabolized to M2, M8, and M13. Analysis of Relative Content of Metabolites by Human Intestinal Bacteria. The relative contents of parent compound ECH (M0) and its metabolites (M1−M13) by human intestinal bacteria were determined at 1, 3, 6, 9, 12, 24, and 48 h, respectively, by the percentage of each metabolite peak area accounted for in the total peak area of the determined metabolites (Table 3). According to our experimental results, ECH (M0) was metabolized to 50% in 24 h and was completely metabolized after 48 h. The major metabolites in the first 12 h were M4, M5, M6, M7, and M11, which decreased significantly from 24 h until the complete disappearance. Metabolites M9, M10, and M12 generated the maximum level of their relative contents at 24 h, which then declined significantly afterward. The results above indicated that metabolites M4−M7 and M9−M12 were the intermediates of ECH metabolism by human intestinal bacteria, which were also susceptible to further metabolism. In contrast, the relative contents of metabolites M1, M2, M3, M8, and M13, especially M1 and M8, were increased remarkably at 48 h. Therefore, these metabolites might be assumed to be the bioactive constituents of ECH that were formed before being absorbed into the blood.
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DISCUSSION In this study, we established an approach to analyze the metabolites of ECH produced by human intestinal bacteria and studied the relevant metabolic pathways. The combination of UPLC-Q-TOF-MS, MSE technology, and MetaboLynx software was used to determine the metabolic profile and to detect the metabolites of ECH. Both precursor and fragment ions could be simultaneously obtained from a single analytical run F
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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ability of rats and humans. Expert Opin. Drug Metab. Toxicol. 2007, 3, 469−489. (17) Morikawa, T.; Ninomiya, K.; Imamura, M.; Akaki, J.; Fujikura, S.; Pan, Y.; Yuan, D.; Yoshikawa, M.; Jia, X.; Li, Z. Acylated phenylethanoid glycosides, echinacoside and acteoside from Cistanche tubulosa, improve glucose tolerance in mice. J. Nat. Med. 2014, 68, 561−566. (18) Plumb, R. S.; Johnson, K. A.; Rainville, P.; Smith, B. W.; Wilson, I. D.; Castro-Perez, J. M.; Nicholson, J. K. UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom. 2006, 20, 1989− 1994. (19) Peppercorn, M. A.; Goldman, P. Caffeic acid metabolism by bacteria of the human gastrointestinal tract. J. Bacteriol. 1971, 108, 996−1000. (20) Deng, R.; Xu, Y.; Feng, F.; Liu, W. Identification of poliumoside metabolites in rat feces by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014, 969, 285−296. (21) Wang, Y.; Hao, H.; Wang, G.; Tu, P.; Jiang, Y.; Liang, Y.; Dai, L.; Yang, H.; Lai, L.; Zheng, C. An approach to identifying sequential metabolites of a typical phenylethanoid glycoside, echinacoside, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis. Talanta 2009, 80, 572−580. (22) Yan, G.; Sun, H.; Sun, W.; Zhao, L.; Meng, X.; Wang, X. Rapid and global detection and characterization of aconitum alkaloids in Yin Chen Si Ni Tang, a traditional Chinese medical formula, by ultra performance liquid chromatography-high resolution mass spectrometry and automated data analysis. J. Pharm. Biomed. Anal. 2010, 53, 421−431. (23) Yang, S.; Shi, W.; Hu, D.; Zhang, S.; Zhang, H.; Wang, Z.; Cheng, L.; Sun, F.; Shen, J.; Cao, X. In vitro and in vivo metabolite profiling of valnemulin using ultraperformance liquid chromatographyquadrupole/time-of-flight hybrid mass spectrometry. J. Agric. Food Chem. 2014, 62, 9201−9210. (24) Gao, Y.; Zong, C.; Liu, F.; Fang, L.; Cai, R.; Shi, Y.; Chen, X.; Qi, Y. Evaluation of the intestinal transport of a phenylethanoid glycoside-rich extract from Cistanche deserticola across the Caco-2 cell monolayer model. PLoS One 2015, 10, e0116490. (25) Matthias, A.; Blanchfield, J. T.; Penman, K. G.; Toth, I.; Lang, C. S.; Voss, J. J.; Lehmann, R. P. Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model. J. Clin. Pharm. Ther. 2004, 29, 7−13. (26) Gonthier, M. P.; Remesy, C.; Scalbert, A.; Cheynier, V.; Souquet, J. M.; Poutanen, K.; Aura, A. M. Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro. Biomed. Pharmacother. 2006, 60, 536−540. (27) Parkar, S. G.; Trower, T. M.; Stevenson, D. E. Fecal microbial metabolism of polyphenols and its effects on human gut microbiota. Anaerobe 2013, 23, 12−19. (28) Gordon, M. H.; Paiva-Martins, F.; Almeida, M. Antioxidant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols. J. Agric. Food Chem. 2001, 49, 2480−2485. (29) Gómez-Ruiz, J. Á .; Leake, D. S.; Ames, J. M. In vitro antioxidant activity of coffee compounds and their metabolites. J. Agric. Food Chem. 2007, 55, 6962−6969. (30) Schaffer, S.; Podstawa, M.; Visioli, F.; Bogani, P.; Müller, W. E.; Eckert, G. P. Hydroxytyrosol-rich olive mill wastewater extract protects brain cells in vitro and ex vivo. J. Agric. Food Chem. 2007, 55, 5043− 5049. (31) Wang, D.; Ho, L.; Faith, J.; Ono, K.; Janle, E. M.; Lachcik, P. J.; Cooper, B. R.; Jannasch, A. H.; D’Arcy, B. R.; Williams, B. A. Role of intestinal microbiota in the generation of polyphenol-derived phenolic acid mediated attenuation of Alzheimer’s disease β-amyloid oligomerization. Mol. Nutr. Food Res. 2015, 59, 1025−1040. (32) Richard, N.; Arnold, S.; Hoeller, U.; Kilpert, C.; Wertz, K.; Schwager, J. Hydroxytyrosol is the major anti-inflammatory compound in aqueous olive extracts and impairs cytokine and chemokine production in macrophages. Planta Med. 2011, 77, 1890−1897.
AUTHOR INFORMATION
Corresponding Author
*(X.L.) Phone: +86-21-3420-4806. Fax: +86-21-3420-4804. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Lei Feng for all of her help with UPLC-Q-TOF-MS. REFERENCES
(1) Jimenez, C.; Riguera, R. Phenylethanoid glycosides in plants: structure and biological activity. Nat. Prod. Rep. 1994, 11, 591−606. (2) Fu, G.; Pang, H.; Wong, Y. H. Naturally occurring phenylethanoid glycosides: potential leads for new therapeutics. Curr. Med. Chem. 2008, 15, 2592−2613. (3) Perry, N. B.; Burgess, E. J.; Glennie, V. L. Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species. J. Agric. Food Chem. 2001, 49, 1702−1706. (4) Cervellati, R.; Renzulli, C.; Guerra, M. C.; Speroni, E. Evaluation of antioxidant activity of some natural polyphenolic compounds using the Briggs-Rauscher reaction method. J. Agric. Food Chem. 2002, 50, 7504−7509. (5) Hu, C.; Kitts, D. D. Studies on the antioxidant activity of Echinacea root extract. J. Agric. Food Chem. 2000, 48, 1466−1472. (6) Speroni, E.; Govoni, P.; Guizzardi, S.; Renzulli, C.; Guerra, M. C. Anti-inflammatory and cicatrizing activity of Echinacea pallida Nutt. root extract. J. Ethnopharmacol. 2002, 79, 265−272. (7) Zhao, Q.; Gao, J.; Li, W.; Cai, D. Neurotrophic and neurorescue effects of Echinacoside in the subacute MPTP mouse model of Parkinson’s disease. Brain Res. 2010, 1346, 224−236. (8) Geng, X.; Tian, X.; Tu, P.; Pu, X. Neuroprotective effects of echinacoside in the mouse MPTP model of Parkinson’s disease. Eur. J. Pharmacol. 2007, 564, 66−74. (9) Thomsen, M. O.; Fretté, X. C.; Christensen, K. B.; Christensen, L. P.; Grevsen, K. Seasonal variations in the concentrations of lipophilic compounds and phenolic acids in the roots of Echinacea purpurea and Echinacea pallida. J. Agric. Food Chem. 2012, 60, 12131− 12141. (10) Luo, W.; Ang, C. Y. W.; Gehring, T. A.; Heinze, T. M.; Lin, L. J.; Mattia, A. Determination of phenolic compounds in dietary supplements and tea blends containing Echinacea by liquid chromatography with coulometric electrochemical detection. J. AOAC Int. 2003, 86, 202−208. (11) Xiong, W. T.; Gu, L.; Wang, C.; Sun, H. X.; Liu, X. Antihyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice. J. Ethnopharmacol. 2013, 150, 935−945. (12) Kim, D. H.; Jung, E. A.; Sohng, I. S.; Han, J. A.; Kim, T. H.; Han, M. J. Intestinal bacterial metabolism of flavonoids and its relation to some biological activities. Arch. Pharmacal Res. 1998, 21, 17−23. (13) Akao, T.; Hayashi, T.; Kobashi, K.; Kanaoka, M.; Kato, H.; Kobayashi, M.; Takeda, S.; Oyama, T. Intestinal bacterial hydrolysis is indispensable to absorption of 18β-glycyrrhetic acid after oral administration of glycyrrhizin in rats. J. Pharm. Pharmacol. 1994, 46, 135−137. (14) Xing, S.; Peng, Y.; Wang, M.; Chen, D.; Li, X. In vitro human fecal microbial metabolism of forsythoside A and biological activities of its metabolites. Fitoterapia 2014, 99, 159−165. (15) Jia, C.; Shi, H.; Wu, X.; Li, Y.; Chen, J.; Tu, P. Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2006, 844, 308−313. (16) Hurst, S.; Loi, C. M.; Brodfuehrer, J.; El-Kattan, A. Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailG
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Identification of Echinacoside Metabolites Produced by Human Intestinal Bacteria Using Ultraperformance Liquid Chromatography− Quadrupole Time-of-Flight Mass Spectrometry Yang Li,† Guisheng Zhou,† Shihua Xing,† Pengfei Tu,§ and Xiaobo Li*,† †
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, People’s Republic of China
§
ABSTRACT: Echinacoside (ECH) is one of the representative phenylethanoid glycosides. It is widely present in plants and exhibits various bioactivities. However, the extremely low oral bioavailability of ECH in rats implies that ECH may go through multiple hydrolysis steps in the gastrointestinal tract prior to its absorption into the blood. Therefore, the gastrointestinal metabolites of ECH are more likely to be the bioactive components. This study established an approach combining ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with MSE technology and MetaboLynx software for rapid analysis of the ECH metabolic profile produced by human intestinal bacteria. As a result, 13 ECH metabolites and 5 possible metabolic pathways (including deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation) were identified. Furthermore, hydroxytyrosol (HT) and 3-hydroxyphenylpropionic acid (3-HPP) were found to be the two bioactive metabolites of ECH produced by human intestinal bacteria. KEYWORDS: echinacoside, human intestinal bacteria, UPLC-Q-TOF-MS, metabolites, metabolic pathway
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INTRODUCTION Echinacoside (ECH) is a natural phenylethanoid glycoside (PhG) that has been recognized as the major bioactive component in Cistanches Herba1,2 and Echinacea species3 with diversified pharmacological functions such as antioxidation,4,5 anti-inflammation,6 and neuroprotection effects.7,8 As a wellknown natural product, ECH is commonly used as a bioactive ingredient for phytomedicines, functional foods, or dietary supplements worldwide.9−11 The oral route, which is the preferred administration method of most herbal products, will inescapably lead to contact with the microflora from the intestinal tract before their components are absorbed. Additionally, some of the active natural ingredients from phytomedicines actually exert their biological effects through interaction with the enriched intestinal bacteria before absorption,12−14 and ECH-containing products may not be an exception in this regard. In a previous pharmacokinetic study, the observed absolute bioavailability (F, %) of ECH was 0.83%,15 which is extremely low. This finding implies that ECH may undergo multiple routes of hydrolysis in the gastrointestinal tract and produce degradation intermediates before being absorbed into the blood.16 ECH is composed of four chemical moieties including caffeic acid (CA), hydroxytyrosol (HT; 3,4-dihydroxyphenethyl alcohol, phenylethanoid aglycone), rhamnose (Rha), and glucose (Glu).1,17 These moieties are bound by ester linkage, which is easily broken under harsh conditions such as a strong acidic or alkali environment. However, so far there are very few investigations on the metabolic process and the biological activity of ECH byproducts in the human intestinal tract. The MSE technology, which can acquire the exact mass of precursor and fragment ions in a single analytical run, is a © XXXX American Chemical Society
commonly used approach in metabolite studies. The method was first introduced by Plumb and his colleagues,18 by setting the collision energy range of MSE manually with the capability of automatic shift, to obtain information on both precursors and fragments. Most of our target metabolites require a rapid, sensitive, and accurate analytical method for structural identification because only a tiny amount of these metabolites is generated. Hence, we applied MSE technology in this research to analyze the complex ECH metabolites produced by human intestinal bacteria. In this study, we analyzed the metabolites of ECH by human intestinal bacteria and investigated the metabolic process and characteristics of ECH in vitro by ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with MSE technology and MetaboLynx software. To our knowledge, this is the first publication focusing on the metabolism of ECH by human intestinal bacteria so far.
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MATERIALS AND METHODS
Chemicals and Reagents. General anaerobic medium broth (GAM broth) was purchased from Shanghai Kayon Biological Technology Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Deionized water was prepared by distilled water through a Milli-Q water purification system (Millipore, Bedford, MA, USA). All of the other reagents and chemicals were of analytical grade. Received: June 11, 2015 Revised: July 14, 2015 Accepted: July 17, 2015
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Journal of Agricultural and Food Chemistry Table 1. Accurate Mass Measurements of Pseudo-molecular and Fragment Ions of Seven Standards compound
tR (min)
formula
measured mass (Da)
calculated mass (Da)
error (mDa)
MS/MS fragment ions (Da)
echinacoside acteoside hydroxytyrosol caffeic acid 3,4-dihydroxybenzenepropionic acid 3-hydroxyphenylpropionic 3-phenylpropionic acid
3.02 3.78 1.57 2.33 2.26 3.39 5.71
C35H45O20 C29H35O15 C8H9O3 C9H7O4 C9H9O4 C9H9O3 C9H9O2
785.2505 623.1971 153.0562 179.0344 181.0509 165.0560 149.0612
785.2504 623.1976 153.0552 179.0344 181.0501 165.0552 149.0603
0.1 −0.5 1.0 0.0 0.8 0.8 0.9
623, 477, 461, 315, 179, 161, 153, 135 461, 315, 179, 161, 135 123 135 137 121 105
Figure 1. UPLC-MS/MS spectra and proposed fragmentation pathways of echinacoside (a) and acteoside (b). monitored at 330 nm. The column temperature was set at 35 °C. The flow rate was 1.0 mL/min, and the sample injection volume was 10 μL. Metabolism Study of ECH by Human Intestinal Bacteria. Fresh human fecal samples were obtained from six healthy volunteers (three males and three females, 22−50 years of age) who had no history of gastrointestinal disorders or antibiotics usage for at least 3 months prior to the study. The fresh fecal samples were mixed and immediately homogenized with 25 times volume of GAM broth. The sediments were removed by filtration through three pieces of gauze. The suspension was incubated at 37 °C in an anaerobic incubator in which the air had been replaced by a gas mixture (H2 5%, CO2 10%, N2 85%). Three milligrams of ECH was placed in 10 mL of human fecal suspension, and the suspension was incubated at 37 °C for 48 h. The cultured mixture was removed and extracted with water-saturated n-butanol at time points of 1, 3, 6, 9, 12, 24, and 48 h. The mixture was centrifuged at 4800 rpm for 20 min, and the supernatant was then evaporated. The residue was dissolved in 0.5 mL of methanol and centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was analyzed by UPLC-Q-TOF-MS. UPLC-MS Analysis. UPLC-Q-TOF-MS analysis was performed on a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm, Waters Corp., USA) by gradient elution using acetonitrile (A) and water (B) at a flow rate of 0.4 mL/min. The gradient profile was 0−3 min (A, 5−20%), 3−6 min (A, 20−30%), 6− 9 min (A, 30−70%), and 9−10.5 min (A, 70−99%), then held for 2 min. The gradient was recycled back to 5% in 2.5 min for the next run. The injection volume was 3 μL. The temperature of the column oven was set to 35 °C.
Echinacoside was kindly provided by Dr. Pengfei Tu’s laboratory from Peking University (Beijing, China). Acteoside was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Hydroxytyrosol (HT), caffeic acid (CA), 3,4-dihydroxybenzenepropionic acid, 3-hydroxyphenylpropionic acid (3-HPP), and 3phenylpropionic acid were purchased from Aladdin Industrial Inc. (Shanghai, China). The purity of each compound was determined to be >95% by HPLC-UV. Stability Study of ECH in Simulated Gastric Juice and Intestinal Juice. One milligram of ECH was placed in 10 mL of simulated gastric juice (0.08 M HCl containing 50 mg of pepsin, pH 1.5) and incubated at 37 °C for 0, 1, 2, or 4 h, respectively. The reactions were quenched by water-saturated n-butanol immediately. The mixture was centrifuged at 4800 rpm for 20 min, and then the supernatant was evaporated. The residue was dissolved in 0.5 mL of methanol and centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was analyzed by HPLC. One milligram of ECH was placed in 10 mL of simulated intestinal juice (0.05 M KH2PO4 containing 50 mg of pancreatin, pH 6.8) and incubated at 37 °C for 0, 2, 4, or 6 h, respectively. The sample processing was the same as that for ECH in simulated gastric juice. HPLC Analysis. The HPLC system (Agilent 1200, USA) consisted of a multisolvent delivery pump equipped with a quaternary solvent delivery system, an autosampler, and a DAD detector, which was used to analyze the stability of ECH in simulated gastric juice and intestinal juice. A ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm i.d., 5 μm, Agilent Ltd., USA) was used. The chromatographic condition was acetonitrile (A) and water (B) with gradient elution, 0−30 min (A, 10−30%) and 30−35 min (A, 30−100%). UV absorption was B
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 2. Echinacoside (M0) and Its Metabolites (M1−M13) Identified in the Samples Incubated with Human Intestinal Bacteria for 24 h Using UPLC-Q-TOF/MS no.
tR (min)
measured mass (Da)
error (mDa)
formula
M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13
3.02 1.57 2.26 2.33 2.44 2.95 3.19 3.35 3.39 3.46 3.66 3.72 3.77 5.72
785.2482 153.0567 181.0505 179.0372 801.2433 639.1900 787.2650 769.2546 165.0563 479.1549 625.2124 827.2624 623.1945 149.0623
−2.2 1.5 0.4 2.8 −2.1 −2.5 −1.1 −0.9 1.1 −0.5 −0.9 1.4 −3.1 2.0
C35H45O20 C8H9O3 C9H9O4 C9H7O4 C35H45O21 C29H35O16 C35H47O20 C35H45O19 C9H9O3 C23H27O11 C29H37O15 C37H47O21 C29H35O15 C9H9O2
MS/MS fragment ions (Da) 623, 123 137 135 783, 477, 623, 179, 121 315, 461, 623, 461, 105
477, 461, 315, 179, 161, 153, 135
179, 179, 181, 161,
161, 135 135 153 135
181, 315, 179, 315,
153, 181, 161, 179,
137 153, 137 153, 135 161, 135
identification parent HT 3,4-dihydroxyphenylpropionic acid CA hydroxylated echinacoside M0-Rha reduction of echinacoside dehydroxylated echinasoside 3-HPP M10-Rha reduction of acteoside acetylated echinacoside acteoside 3-phenylpropionic acid
Figure 2. UPLC-MS extracted ion chromatograms (XICs) of echinacoside and its metabolites: (a) M0; (b) M1; (c) M2; (d) M3; (e) M4; (f) M5; (g) M6; (h) M7; (i) M8; (j) M9; (k) M10; (l) M11; (m) M12; (n) M13. Mass spectrometry was carried out using a Waters Synapt mass spectrometer (Waters Corp., Milford, MA, USA). Ionization was performed in the negative electrospray (ESI) mode. The MS parameters were as follows: capillary voltage, 2.8 kV; cone voltage, 35 V; source temperature, 115 °C; desolvation temperature, 350 °C; gas flows of cone and desolvation, 50 and 700 L/h, respectively. For accurate mass measurement, leucine-enkephalin was used as the lock mass. The MSE experiment in two scan functions was carried out as follows: function 1 (low energy), m/z 50−1000, 0.25 s scan time, 0.02 s interscan delay, 4 eV collision energy; function 2 (high energy), m/z 50−1000, 0.25 s scan time, 0.02 s interscan delay, collision energy ramp of 55−70 eV. The data were processed using MassLynx 4.1 software (Waters Corp.). Data Analysis. Data analysis was performed using MetaboLynx software (Waters Corp.) that employs an expected list of potential biotransformation reactions such as deglycosylation, dehydroxylation,
and acetylation. This software automates the detection and identification of metabolites by comparing the sample with the control to eliminate the interfering peaks in the sample that also appear in the control. The mass defect filter was set as 25 mDa. Results were filtered on the basis of the mass fractional deviation from the parent compound and the expected metabolites of the parent compound. The mass window of 0.05 Da was set for both expected and unexpected metabolite mass chromatograms. Apex track peak integration was used for peak detection, and the response threshold of the absolute peak height was set to 20 units.
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RESULTS Stability of ECH in Simulated Gastric Juice and Intestinal Juice. ECH was found to be stable in simulated gastric juice and intestinal juice. After incubation with artificial C
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Figure 3. Proposed fragmentation pathways: (a) M9 (m/z 479); (b) M10 (m/z 625); (c) M11 (m/z 827).
at m/z 179 that produced ions at m/z 135 and 161 by the loss of CO2 and H2O, respectively. HT (m/z 153.0562) produced a fragment at m/z 123, which was probably through the loss of a CH2O moiety. Similarly, CA (m/z 179.0344) yielded a product ion at m/z 135 by the loss of CO2. 3,4-Dihydroxybenzenepropionic acid (m/z 181.0509) produced the ion at m/z 137 by the loss of CO2. CO2 was lost from 3-HPP (m/z 165.0560) to form the ion at m/z 121. 3-Phenylpropionic acid (m/z 149.0612) produced m/z 105 by the loss of CO2 as well. Identification of ECH Metabolites by Human Intestinal Bacteria. Compared with the blank sample, the parent compound ECH (M0) and its 13 metabolites (M1−M13), including the metabolites of parent compound (M4−M7, M9− M12) and degradation products (M1−M3, M8, M13), were identified in human intestinal bacteria samples. Detailed information on these metabolites is listed in Table 2. Deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation were considered as the main metabolic pathways for the parent compound. The extracted ion chromatograms (XICs) of the parent compound (M0) and its metabolites (M1−M13) from the total UPLC-MS chromatograms are shown in Figure 2. In addition, M9, M10, and M11 were selected as the representative metabolites, and their proposed fragmentation pathways are shown in Figure 3. Parent Compound (M0). ECH (M0) was identified by comparing the UPLC retention time (3.02 min), accurate MS
juices, the content of ECH did not change significantly over time according to the peak areas during the course of incubation (RSD < 3.0%). Fragmentation Studies. Solutions of ECH, acteoside, HT, CA, 3,4-dihydroxybenzenepropionic acid, 3-HPP, and 3phenylpropionic acid prepared in methanol were used for the fragmentation pattern study to support metabolite characterizations. The above seven standard compounds were eluted at 3.02 min (ECH), 3.78 min (acteoside), 1.57 min (HT), 2.33 min (CA), 2.26 min (3,4-dihydroxybenzenepropionic acid), 3.39 min (3-HPP), and 5.71 min (3-phenylpropionic acid), respectively (Table 1). The fragmentation patterns of ECH and acteoside were described as followed. As shown in Figure 1a, ECH (m/z 785.2505) produced a fragment at m/z 623 by loss of a CA moiety, and then the sequential losses of rhamnose (Rha), glucose (Glu), and Glu moieties yielded the respective fragment ions at m/z 477, 315, and 153. The fragment at m/ z 461 was generated through the loss of Glu and CA moieties from the precursor ion. The product ion at m/z 179, which was one of the major fragments, corresponded to the CA moiety and then produced ions at m/z 135 and 161 by the further loss of CO2 and H2O, respectively. Acteoside (m/z 623.1971) displayed a similar fragmentation pattern in the spectrum of ECH (Figure 1b). It produced the fragment ion at m/z 461 by losing the CA moiety and then produced the ion at m/z 315 by the further loss of a Rha moiety. The CA moiety was also found D
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Figure 4. Proposed metabolic pathways of echinacoside by human intestinal bacteria.
at m/z 785.2482, and MS/MS spectra with those of the authentic reference standard. Characteristic product ions at m/z 623, 477, 461, 315, 179, 161, 153, and 135 were also found in the MS2 fragmentation. Metabolites of Parent Compound (M4−M7, M9− M12). In this study, the direct metabolites of parent compound were identified by the distinctive fragmentations when compared with the parent compound. M12 (m/z 623.1945, C29H35O15, 3.77 min), a loss of Glu (162 Da) from ECH (M0), was identified as acteoside through comparison of the UPLC retention time, accurate MS, and MS/MS spectra with the authentic reference standard. M10 (m/z 625.2124, C29H37O15, 3.66 min) was 2 Da (2H) higher than M12, which indicated that M10 was the reduced product of M12. In the MS/MS spectrum of this metabolite, the presence of a diagnostic fragment ion at m/z 181 implied that reduction occurred on the α′,β′-double bond of the CA moiety. Additionally, other fragment ions from M10 were consistent with the product ions of M12. The previous study on caffeic acid metabolism by bacteria from the human gastrointestinal tract indicated that CA could be reduced to dihydro-CA by human intestinal bacteria,19,20 which implied this reduction route might occur in the metabolic process of phenylethanoid glycoside and caffeoyl-containing compound. M9 (m/z 479.1549, C23H27O11, 3.46 min) was 146 Da (Rha) lower than M10 in molecular weight and was considered as the deRha product of M10. M9 produced signals at m/z 315, 181, 153, and 137, which were similar to the generated ions of M10. The proposed fragmentation pathways of M9 and M10 are shown in Figure 3. M4 (m/z 801.2433, C35H45O21, 2.44 min), a hydroxylated product of ECH, was 16 Da (O) higher than ECH in molecular weight. The characteristic product ion at m/z 783 was generated by a H2O loss from the quasi-molecular ion. Moreover, caffeic acid related fragment ions were found at m/z 179, 161, and 135, which were the same as the fragment ions of ECH (M0), suggesting that the hydroxylation position was not on the CA moiety. A previous study proposed that the hydroxylation is more likely to occur on the HT moiety to form a benzenetriol structure.21
M5 (m/z 639.1900, C29H35O16, 2.95 min) was 146 Da (Rha) lower than ECH (M0) in molecular weight and was identified as the de-Rha product of M0. The characteristic product ion at m/z 477 was shaped by the elimination of CA moiety (162 Da). The CA moiety was also found at m/z 179 and produced the ion at m/z 135 through the loss of CO2. M6 (m/z 787.2650, C35H47O20, 3.19 min) was 2 Da (2H) higher than M0 in molecular weight, which indicated that M6 was the reduced product of M0. The characteristic fragments produced by M6 at m/z 623, 181, and 153, were similar to those of M10. These results suggested that M6 was generated by the reduction of ECH, which occurred on the α′,β′-double bond of the CA moiety. M7 (m/z 769.2546, C35H45O19, 3.35 min) was 16 Da (O) lower than the protonated ion of ECH, which indicated that it was a metabolite of ECH after the loss of oxygen. In the MS/ MS spectrum of M7, fragment ions associated with CA were found at m/z 179, 161, and 135. Therefore, the dehydroxylated site of M7 was not at the CA moiety. However, the exact dehydroxylated site of M7 could not be confirmed due to the limited information obtained in our study. M11 (m/z 827.2624, C37H47O21, 3.72 min) was 42 Da (C2H2O) higher than the protonated ion of ECH, suggesting a possible conjugation of an acetyl group on ECH. In addition, the characteristic MS/MS fragment ions of M11 at m/z 623, 179, 161, 153, and 135 were observed. The proposed fragmentation pathways of M11 are shown in Figure 3c. According to the previous research, the C-2′ position might be one of the acetyl conjugation sites of M11.20 Metabolites of Degradation Products (M1−M3, M8, M13). In this study, five degradation metabolites were identified using the authentic reference standard. ECH first degraded to HT (M1) and CA (M3), and CA underwent further metabolism to its major microbial metabolite, 3-HPP (M8). M1 (m/z 153.0567, C8H9O3, 1.57 min), M2 (m/z 181.0505, C9H9O4, 2.26 min), M3 (m/z 179.0372, C9H7O4, 2.33 min), M8 (m/z 165.0563, C9H9O3, 3.39 min), and M13 (m/z 149.0623, C9H9O2, 5.72 min) were identified as HT, 3,4dihydroxyphenylpropionic acid, CA, 3-HPP, and 3-phenylE
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Journal of Agricultural and Food Chemistry Table 3. Percentage of Relative Content and the Area of Each Metabolite at Different Time Points no.
1 h (%) (area abs)
3 h (%) (area abs)
6 h (%) (area abs)
9 h (%) (area abs)
12 h (%) (area abs)
24 h (%) (area abs)
M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13
78.84 (3008.20) 0.43 (16.50)
74.19 (3172.60) 0.08 (3.40) 0.37 (15.90)
73.04 (3066.50) 0.17 (7.00) 0.28 (11.90)
65.51 (3096.80) 0.18 (8.60) 0.17 (8.20)
0.53 1.75 0.33 3.18
0.54 1.81 0.77 3.20
0.54 1.59 0.92 3.04
0.45 1.57 0.48 2.70
52.71 (2388.60) 0.16 (7.10) 0.08 (3.60) 0.03 (1.40) 0.31 (14.00) 1.20 (54.50) 0.43 (19.60) 1.85 (83.90)
2.51 (95.70) 12.42 (473.80)
0.09 (3.70) 2.42 (103.40) 16.48 (704.80) 0.06 (2.70)
2.02 (85.00) 18.29 (767.70) 0.12 (5.00)
0.15 (7.30) 1.84 (86.90) 26.82 (1267.80) 0.13 (6.00)
0.15 (6.60) 1.23 (55.80) 41.72 (1890.40) 0.13 (5.90)
36.08 (2259.00) 1.25 (76.70) 0.20 (12.20) 0.06 (3.50) 0.27 (16.30) 1.43 (87.60) 1.49 (91.20) 1.44 (88.10) 0.91 (55.80) 2.61 (160.00) 3.21 (197.00) 0.49 (29.80) 49.60 (3044.30) 0.27 (16.60)
sum
100.00 (3815.40)
100.00 (4276.50)
100.00 (4198.40)
100.00 (4727.10)
100.00 (4531.40)
100.00 (6138.10)
(20.40) (66.90) (12.60) (121.30)
(23.10) (77.20) (32.80) (136.90)
(22.60) (66.70) (38.50) (127.50)
(21.20) (74.30) (22.50) (127.50)
48 h (%) (area abs) 1.64 (18.80) 35.97 (412.90) 0.36 (4.10) 0.77 (8.80)
38.21 (438.60) 0.13 (1.50) 0.62 (7.10) 19.61 (225.10) 2.70 (31.00) 100.00 (1147.90)
through the application of the highly selective MSE technique,18 which provides a great advantage for the rapid investigation of multiple structures. MetaboLynx is an automated software22,23 that can detect trace level of metabolites from biological samples and is a powerful tool for metabolic characterization. Previous research found that the poor membrane permeability and the extensive presystemic metabolism of ECH might be the cause of its low oral bioavailability in rats.21,24,25 Therefore, it is not surprising that the presence of bacteria and acidic/alkaline conditions in gastrointestinal ducts could initiate the hydrolysis of ECH. However, these previous studies did not provide detailed information about the metabolism of ECH in gastrointestinal ducts. Thus, we assessed whether gastric juice, intestinal juice, or intestinal microorganisms of gastrointestinal system could metabolize ECH. In our study, ECH was found to be stable in simulated gastric juice and intestinal juice, whereas it could be metabolized to HT and 3-HPP by intestinal bacteria. 3-HPP has been reported as a major microbial metabolite of CA by human intestinal bacteria in vitro,26,27 which is in agreement with our findings. Additionally, reported evidence has shown that HT and 3-HPP possess bioactivities such as antioxidant,28,29 neuroprotective,30,31 and anti-inflammatory functions.32,33 Therefore, we conclude that ECH acts as a prodrug that is transformed to bioactive compounds (HT and 3-HPP) by intestinal bacteria after oral administration, and the released compounds then exert biological activities. In conclusion, a highly selective and sensitive approach using MSE and MetaboLynx combined UPLC-Q-TOF-MS was applied to the screening and identification of ECH metabolites produced by human intestinal bacteria in vitro. Among the 13 metabolites (M1−M13) identified in this study, M5, M6, and M11 have not been previously reported to our knowledge. Deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation were the five possible metabolic pathways that led to the generation of these metabolites. The observation of human intestinal bacteria produced HT and 3-HPP, which possess biological functions similar to those of ECH, could potentially explain the fact that ECH has prominent bioactivity but poor bioavailability. This study further explored the role of intestinal bacteria in the metabolism of natural products and improved the current understanding of ECH metabolism.
propionic acid, respectively, after analysis of the UPLC retention time, accurate MS, and MS/MS spectra of the authentic reference standards. Analysis of the Metabolic Pathways of ECH by Human Intestinal Bacteria. According to the results of our experiment, the proposed metabolic pathways of ECH by human intestinal bacteria are displayed in Figure 4. After incubation with human intestinal bacteria, ECH was metabolized to M4, M5, M6, M7, M11, and M12. Additionally, M12 was metabolized to M10 and M9 sequentially. After the degradation metabolites M1 and M3 emerged, M3 was further metabolized to M2, M8, and M13. Analysis of Relative Content of Metabolites by Human Intestinal Bacteria. The relative contents of parent compound ECH (M0) and its metabolites (M1−M13) by human intestinal bacteria were determined at 1, 3, 6, 9, 12, 24, and 48 h, respectively, by the percentage of each metabolite peak area accounted for in the total peak area of the determined metabolites (Table 3). According to our experimental results, ECH (M0) was metabolized to 50% in 24 h and was completely metabolized after 48 h. The major metabolites in the first 12 h were M4, M5, M6, M7, and M11, which decreased significantly from 24 h until the complete disappearance. Metabolites M9, M10, and M12 generated the maximum level of their relative contents at 24 h, which then declined significantly afterward. The results above indicated that metabolites M4−M7 and M9−M12 were the intermediates of ECH metabolism by human intestinal bacteria, which were also susceptible to further metabolism. In contrast, the relative contents of metabolites M1, M2, M3, M8, and M13, especially M1 and M8, were increased remarkably at 48 h. Therefore, these metabolites might be assumed to be the bioactive constituents of ECH that were formed before being absorbed into the blood.
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DISCUSSION In this study, we established an approach to analyze the metabolites of ECH produced by human intestinal bacteria and studied the relevant metabolic pathways. The combination of UPLC-Q-TOF-MS, MSE technology, and MetaboLynx software was used to determine the metabolic profile and to detect the metabolites of ECH. Both precursor and fragment ions could be simultaneously obtained from a single analytical run F
DOI: 10.1021/acs.jafc.5b02881 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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AUTHOR INFORMATION
Corresponding Author
*(X.L.) Phone: +86-21-3420-4806. Fax: +86-21-3420-4804. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Lei Feng for all of her help with UPLC-Q-TOF-MS. REFERENCES
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