Characterization of eleutheroside B metabolites derived from an extract of Acanthopanax senticosus...

Research article

Received: 24 August 2011, Revised: 29 November 2011, Accepted: 2 December 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.2688

Characterization of eleutheroside B metabolitesderived from an extract of Acanthopanaxsenticosus Harms by high-resolution liquidchromatography/quadrupole time-of-flightmass spectrometry and automated data analysisFang Lua, Qiang Suna, Yun Baib*, Shunru Baoa, Xuzhao Lia, Guangli Yana

and Shumin Liua*

ABSTRACT: We elucidated the structure and metabolite profile of eleutheroside B, a component derived from the extract ofAcanthopanax senticosus Harms, after oral administration of the extract in rats. Samples of rat plasma were collected andanalyzed by selective high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS)automated data analysismethod. A total of 11metabolites were detected: fourwere identified, and three of those four are reportedfor the first time here. The three new plasmametabolites were identified on the basis ofmass fragmentation patterns and literaturereports. The major in vivometabolic processes associated with eleutheroside B in A. senticosus include demethylation, acetylation,oxidation and glucuronidation after deglycosylation. A fairly comprehensive metabolic pathway was proposed for eleutheroside B.Our results provide a meaningful basis for drug discovery, design and clinical applications related to A. senticosus in traditionalChinese medicine. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: extract of Acanthopanax senticosus Harms; eleutheroside B; metabolites; UPLC-Q-TOF-MS; MetaboLynx XS

* Correspondence to: Yun Bai, School Basic Medical Sciences, HeilongjiangUniversity of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China.E-mail: [email protected]

Shumin Liu, Institute of Traditional Chinese Medicine, Heilongjiang Univer-sity of Chinese Medicine, He Ping Road 24, Harbin 150040, People’s Republicof China. E-mail: [email protected]

a Institute of Traditional Chinese Medicine, Heilongjiang University ofChinese Medicine, He Ping Road 24, Harbin 150040, People’s Republic ofChina

b School of Basic Medical Sciences, Heilongjiang University of ChineseMedicine, He Ping Road 24, Harbin 150040, People’s Republic of China

IntroductionAcanthopanax senticosus (Rupr. et Maxim.) Harms is distributedwidely in regions such as the Heilongjiang, Jilin Province of thePeople’s Republic of China, the northern regions of Japan andKorea. The root and rhizome of A. senticosus are commonly usedin traditional Chinese medicine (Chinese Pharmacopoeia Commit-tee, 2010). In the past decade, many components, such as glyco-sides, polysaccharides, flavonoids, amino acids and coumarins(Dong and Li, 2011), have been detected in A. senticosus, which isassociated with many beneficial effects, such as reduction infatigue (Huang et al., 2011; Zhang et al., 2011), oxygen free radi-cals (Liang et al., 2010; Wang et al., 2010), tumor formation (Shanet al., 2004) and blood lipids (Cha et al., 2004), and enhanced anti-coagulant (Yang et al., 2009), hypoglycemic (Cha et al., 2004; Parket al., 2006; Watanabe et al., 2010) and immunoregulatory effects(Xie et al., 1989; Han et al., 2003). A. senticosus has been used clini-cally to treat several disease states, including neurasthenia (Jiaet al., 2010), cancer (Huang et al., 2005), diabetes mellitus(Watanabe et al., 2010), Parkinson’s disease (Fujikawa et al.,2005) and ischemic heart disease (Da et al., 1994).

In vivo studies on eleutheroside B are important for drug dis-covery, design and clinical application. Pharmacokinetic studieshave been conducted on plasma- and tissue-derived eleuthero-side B, after its intravenous administration in rats (Feng et al.,2006), but the metabolite profile of eleutheroside B has not yetbeen studied (Fig. 1). A. senticosus is commonly used in clinicsof traditional Chinese medicine. Oral administration is the main

Biomed. Chromatogr. 2012 Copyright © 2012 John

route of drug administration in traditional Chinese medicine.Eleutheroside B is one of the major bioactive components of A.senticosus; thus, studies need to be performed to determinethe metabolic profile of orally administered eleutheroside B.Ultra-performance liquid chromatography coupled with quad-

rupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) has apronounced capability for higher peak capacity, greater selectiv-ity and increased sensitivity. It provides a vast amount of infor-mation rapidly and efficiently, and thus, it has been widely usedin recent metabolite analysis studies on pharmaceutical drugsand natural products. MetaboLynx is a software program thatautomatically detects and compares mass spectral chromato-grams from control and metabolized samples for identificationand reporting purposes. A number of complicated chemical

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Figure 1. Structure of eleutheroside B (M0).

F. Lu et al.

components are found in A. senticosus. Therefore, UPLC/Q-TOFMS was coupled with MarkerLynx XS and used to analyzeeleutheroside B metabolites. In this paper, a total of 11 metabo-lites were detected in the rat – four metabolites were identifiedand three are reported here for the first time.

Materials and methods

Chemicals and reagents

HPLC-grade acetonitrile and methanol were purchased fromFisher (USA). Water was purchased from Watsons (China). Phos-phoric acid (analytical grade) was purchased from Beijing ReagentCompany (Beijing, China). Eleutheroside B was purchased fromthe National Institutes for Food and Drug Control (China).

Extract preparation

Extract of A. senticosus Harms (EAS) was prepared from a crudeAcanthopanax formulation; 1 g of the crude drug was added to10mL of 80% (v/v) ethanol; the mixture was separated andpurified using macroporous adsorbents. The eleutheroside Bcontent in the EAS was 7.63% (w/w) (Shao et al., 2011).

Experimental animals

Eighteen male Wistar rats that weighed 250–270 g each werepurchased from Vital River Laboratory Animal Technology Co.Ltd (Beijing, China). Animals were bred in an environmentallycontrolled breeding room (temperature 24� 2�C; relative humid-ity, 60� 5%; 12 h dark–light cycle) for 1week before the exper-iment. The rats were fed standard laboratory food and givenwater ad libitum; all rats were fasted for 12 h before the experi-ments, but had free access to water. The ethical approval forthe experiment was given by the Legislation on the Protectionof Animals Used for Experiment Purposes (Directive 86/609/EEC).

Preparation of plasma samples

Eighteen rats were divided into six groups, and each group wastreated with EAS at 60, 90, 120, 240 or 360min or left untreated asa control group. The sample was prepared for in vivo analysis by

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dissolving freeze-dried EAS powder with distilled 5% carboxymethylcellulose (CMC) aqueous to use as a stock solution. The stock so-lution was orally administered to male Wistar rats (325mg/kg),and the same volume of 5% CMC aqueous was administered tothe control group according to the dosage. The animals wereanesthetized 60, 90, 120, 240 or 360min after drug administra-tion by intraperitoneal injection with 1% pentobarbital sodium(0.15mL/100 g). Blood was collected from the hepatic portal veinand centrifuged at 10,000 rpm for 10min at 4�C. Plasma sampleswere prepared using a solid-phase extraction column.

Waters Oasis HLB C18 cartridges (3mL, 60mg) were conditionedwith methanol (2mL) and equilibrated with double-deionized wa-ter (2mL). The prepared sample (1.5mL plasma acidified with1.5mL of 4% phosphoric acid vortexed for 30 s) was loaded intothe cartridge and eluted with the solvent. Slight pressure was ap-plied to the cartridge when a spiked sample passed, thus allowingcomplete passage of the materials while maintaining a loadingspeed of 2mL/min. The cartridges were washed in 2mL of 5%methanol followed by 3mL methanol. The eluted portion of themethanol was evaporated to dryness under nitrogen gas at 40�C.The residue was dissolved in 0.2mL mobile phase and centrifugedat 13 000 rpm for 15min at 4�C. Finally, 5 mL of the resultingsolution was injected into the UPLC for analysis.

Instrumentation and conditions

UPLC conditions. Chromatography was performed using anAcquity UPLC system (Waters Corp., Milford, MA, USA) with a condi-tioned autosampler at 4�C. The separation was performed on anAcquity UPLC HSS T3 column (100� 2.1mm i.d., 1.8mm; WatersCorp., Milford, MA, USA). The column temperature was maintainedat 40�C. Analysis was conducted on the gradient elution componentusing (A) acetonitrile (containing 0.1% formic acid) and (B) water(containing 0.1% formic acid) as the mobile phase. The gradientcondition of the mobile phase was: 0–2min linear from 10 to 14%A; 2–10min, linear from 14 to 22% A; 10–12min, linear from 22 to30% A; 12–20min, linear from 30 to 60% A; and 20–24min, linearfrom 60 to 100% A. The flow-rate of UPLC/MS was 0.3mL/min,and the injection volume was 5 mL.

MS conditions. Mass spectrometry was carried out on aMicromass Q-TOF-microTM (Waters MS Technologies, ManchesterUK) orthogonal acceleration TOF mass spectrometer equippedwith an electrospray ion source–tandem mass spectrometer(ESI-MS/MS) in a sequential configuration that operated inpositive ion or negative ion mode using a MassLynx data analysissystem. The desolvation gas rate was set to 600 L/h in the positiveion mode and 500 L/h in the negative ion mode. The cone gasrate was set to 100 L/h. Desolvation and source temperatureswere set at 110 and 300�C, respectively. The capillary voltagewas set to 3000V in the positive ion mode and 2800V in the neg-ative ion mode. The sample and extraction cone voltages were setto 35 and 3.0 V, respectively, with 12.0 V collision energy andabout 2.8� 10�3mbar argon collision gas pressure. Scan timewas set to 0.48 s with an inter-scan delay of 0.1 s for use through-out the experiment. All analyses were performed using thelockspray, which ensured accuracy and reproducibility. Leucineenkephalin was used as the lock mass (m/z 556.2771 for the posi-tive ion mode and m/z 554.2615 for the negative ion mode) ata concentration of 0.5ng/mL and a flow-rate of 60 mL/min via alockspray interface. Data were collected in the centroid mode fromm/z 100 to m/z 900 with a lockspray frequency of 5 s. Data was

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Characterization of Eleutheroside B metabolites by UPLC-MS2

averaged from 10 scans. The mass spectrometric data were col-lected in the full-scan mode.

Data analysis

Post-acquisition analyses were performed using a MetaboLynxXS (version 4.1) program (Waters Corp., Milford, MA, USA), whichrelies on an expected metabolites list of potential biotransforma-tion reactions (Table 1). Data files were processed using theMetaboLynx XS software package that was used as a platformto search expected and unexpected metabolites (compounds)with accurate mass and fragment ion information. The data fileswere named ‘analyte’ and ‘control’, respectively, to extract theions that were absent from the control from the analyte datasets. Eleutheroside B was used as the parent compound andplaced into the mass columns. Meanwhile, the compound was

(a)

(b)

Figure 2. ESI� total ion current chromatograms of rat plasma sample in negapanax senticosus Harms (EAS); and (b) blank sample.

Table 1. List of expected metabolites for which narrowwindow extracted ion chromatograms are generated in phase Iand phase II of eleutheroside B

Biotransformation reactions Formulachange

Mass change(mDa)

Formula Parent 0Reduction +H2 2.0157Hydroxylation + desaturation +O�H2 13.9793Sulfate conjugation + SO3 79.9568Hydroxymethylene loss � CH2O �30.01062�Hydroxymethylene loss � CH2O� 2 �60.0211Deglucose � C6H10O5 �162.1424Glucuronide conjugation + C6H8O6 176.0321Methylation + CH2 14.0157Demethylation � CH2 �14.0157Glucose +C6H10O5 162.1424Acetylation + C2H2O 42.0106Glycine conjugation + C2H3NO 57.0215Cysteine conjugation + C3 H5NOS 103.0092Glutathione conjugation + C10H15 N3 O5S 289.0732


used as a filtering template with mass defect ranging from�25 to +25mD to support the MDF function. In the window ofthe expected metabolites list, the parent compound alone wasadded, and the mass window was set to 0.01mD. The unex-pected metabolite chromatograms were created over the fullacquisition mass range in the mass window of 1 amu. False posi-tives were specified by adding control mass retention times,peak detection was accomplished using the ApexTrack algo-rithm, and the threshold of the peak area was set to 0.5 unit.The retention time (Rt) filter was used to extract the chromato-grams from 0.5 to 24.0min. The elemental compositions for un-expected metabolite peaks were generated on the basis of theempirical formula of the parent compound and did not allowfor the change in the number of N, S, and P atoms.

Results

Separation and identification of metabolites

In this study, rat plasma samples were obtained after oral admin-istration of EAS and compared with a blank sample using UPLC/Q-TOF MS. The UPLC–ESI MS total ion current chromatograms innegative ion mode are shown in Fig. 2; direct-viewing analysiswas not possible.

Classes of metabolites identified by using MetaboLynx XS

Metabolite data from the UPLC-TOF MS system identified us-ing Metabolynx XS. The MetaboLynx XS program was usedto analyze blank and plasma sample data. Samples wereobtained from rats following oral administration of EAS. TheMetaboLynx output browser was able to show the metabolitesof eleutheroside B by using the modified MetaboLynx screeningroutine (Fig. 3), and it targeted metabolites derived from thesearch for eleutheroside B on the basis of MetaboLynx principles(Table 2).

Metabolite data from the UPLC/Q-TOF MS system identifiedby using MetaboLynx XS. Owing to the metabolite name inMetaboLynx, the output browser report results may be pseudo-morph. Our study modified the metabolite name using MS2.

tive ion mode: (a) rat plasma sample after oral administration of Acantho-

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(A)

(B)

(C)

(D)

Figure 3. MetaboLynx output browser showing metabolites of eleutheroside B using the modified MetaboLynx screening routine. (A) Expected meta-bolites list. (B) The chromatogram of expected metabolites in the analyte. (C) The chromatogram of expected metabolite in the control sample. (D) Themass spectrum of expected metabolite in the analyte. (Note: The mass spectrum of expected metabolite in the control is the blank.)

F. Lu et al.

M1 identified using MetaboLynx XS. At the negative ionmode, M1 (Rt = 2.351min) had an [M1�H]� at m/z 371 and MS2

yielded major ions at m/z 195 [M1�GluA]�, m/z 175 [GluA]� andm/z 113 [GluA–CO2–H2O]

�. In accordance with the data characteris-tics collected from MS/MS, reports have indicated the presence offragments ions m/z 175 and m/z 113 with the loss of H2O and CO2

at the negative ion mode (Ling et al., 2002; Jie et al., 2004), whichis strong evidence for identifying glucuronide metabolites. Fur-thermore, another report (Pei and Guo, 2006) showed glycosidehydrolyzation by b-glucosidase after the glycoside entered epithe-lial cells of the bowels and underwent systematic recirculation inconjugates of glycosides or aglycones. Glucuronide conjugation is


a major metabolic reaction in humans and animals (Liang,2007). M1was identified as [M0+ (�Glu+GluA�CH3)] (Fig. 4a).However, the locations of methylation and glucuronidation onM1 remain undetermined. M1 was detected at 60, 90, 120, 240and 360min.

M2 identified using MetaboLynx XS in MS2. In the negativeion mode, M2 (Rt = 2.610min) showed an [M2�H]� at m/z 385and MS2 yielded a major ion at m/z 209 [M2�GluA] �, m/z175 [GluA] � and m/z 113 [GluA – CO2 – H2O]

�. In accordancewith the data on the MS/MS characteristics and literature


Table 2. Targeted metabolite search for eleutherosideBusing MetaboLynx principles

Mass Metabolitename

m/z found mDa time(min)

182.0579 2*�CH2, � GLU 181.0509 0.8 2.29196.0736 � CH2, � GLU 195.0877 21.9 0.83224.0685 +O�H2, � GLU 223.0609 0.2 6.48326.1366 +CH2, 2*�CH2O 325.1307 1.9 15.82342.1315 � CH2O 341.1239 0.2 4.97344.1107 2*�CH2 343.1041 1.2 8.78344.1471 +H2, � CH2O 343.1376 �1.7 8.14358.0900 +O�H2, 2*�CH2 357.0832 1.0 1.59372.1056 +O�H2, � CH2 371.0979 0.1 2.35386.1213 +COCH3, 2*�CH2 385.1128 0.7 2.61400.1369 +COCH3, � CH2 399.1284 0.7 2.68

msms

m/z100 150 200 250 300 350

%

0

1002.30e3371

113 175116 145 167 303197 211

244 259 281 353327

msms

m/z100 150 200 250 300 350 400

%

0

100944385

209113

175116

157139 194223 341319297237 259 367

msms

m/z100 150 200 250 300 350 400

%

0

1001.22e3399

223

113 153138 175 191 330239 312265283

355 398381

msms

m/z150 160 170 180 190 200 210 220 230

%

0

100

lsm-xy-msms-12v 18 (2.351) QT (2); Cm (1:34) 2: TOF MSMS 371.10ES-



lsm-xy-msms-12v 11 (6.448) QT (2); Cm (1:29) 7: TOF MSMS 223.06ES-942223

208

164

149157

179

167 176193

183207

198 212 222

a

b

c

d

Figure 4. (A) TOF MS spectra of metabolites with MS2:M1 (m/z 371).(B) Time-of-flight mass spectrometry (TOF MS) spectra of metabolites withMS2:M2 (m/z 385). (C) TOF MS spectra of metabolites with MS2: M3(m/z 399). (D) TOF MS spectra of metabolites with MS2: M4 (m/z 233).



reports, M2 was identified as [M0+ (�Glu +GluA)] (Fig. 4b).However, the position of glucuronidation on M2 remainsundetermined. M2 was detected at 60, 90, 120 and 240 min.

M3 identified using MetaboLynx XS in MS2. In the negativeion mode, M3(Rt = 2.678 min) showed [M3�H]� at m/z 399 andMS2 yielded a major ion at m/z 223 [M3�GluA]�, m/z 175[GluA]� and m/z 113 [GluA�CO2�H2O]

�. In accordance withthe data on MS/MS characteristics and literature reports, M3was identified as [M0+ (� Glu +GluA-2�CH3 + COCH3)] (Fig. 4c).However, the positions of acetylation and glucuronidation onM3 remain undetermined. M3 was detected at 60, 90, 120, 240and 360 min.

M4 identified using MetaboLynx XS in MS2. In the negativeion mode, M4(Rt =6.488 min) showed [M�H]� atm/z 223 and MS2yielded amajor ion atm/z 208 [M4�CH3]

�,m/z193 [M4� 2�CH3]�,

m/z 164 [M4�CH3�CO2]� and m/z 149 [M4� 2�CH3�CO2]

�.In accordance with the data on MS/MS characteristics, M4 wasidentified as [M0+ (� Glu+O�H2)] (Fig. 4d). M4 was detected at60, 90, 120, 240 and 360 min.

DiscussionMany complicated chemical compositions exist within Chinesemedicine. The study of its metabolism typically involves a com-plex series of MS experiments followed by analysis of the largeamounts of data produced; hence, often the process is time-consuming. UPLC/Q-TOF MS provides faster separation with in-creased resolution and sensitivity for analysis and elucidationof drug metabolism. The MetaboLynx XS (version4.1) softwarepackage requires minimal operator intervention and is capableof batch processing of samples, automated detection and iden-tification of drug metabolites.In this study, UPLC/Q-TOF MS was used in combination with

automated data analysis software MetaboLynx XS for rapid anal-ysis of the eleutheroside B metabolites. We detected 11 metab-olites; four metabolites were identified in plasma with blanksamples on the basis of mass fragmentation behaviors and liter-ature reports, and three were reported for the first time (M1, M2and M3). However, eleutheroside B was not detected in plasma.Eleutheroside B is low in oral bioavailability, as evidenced bythe in situ perfusion method in rats (Tan and Jia, 2008),which corresponds to our results. Our results indicate thatdemethylation, acetylation oxidation and glucuronidationafter deglycosylation are the major metabolic pathways ofeleutheroside B in A. senticosus in vivo. Furthermore, the met-abolic pathway of eleutheroside B in rats is shown in Fig. 5.Four metabolites derived from eleutheroside B may be bioac-tive constituents. These results provide a meaningful basis fordrug discovery, design and clinical application in traditionalChinese medicine.Further research on the structures of metabolites is needed

to separate the metabolites from plasma and determine theirstructures using NMR spectroscopy. However, the low metabo-lite concentrations in plasma make separation of these metab-olites challenging. in addition, pre-study research indicatedthat the synthesis of glucuronide was unstable (Needs andKroon, 2006).


OH

OOO

OHO

OHOH

OH

A

M0

OH

OO

OH

B

GluA OH

OO

OH

M2

C

GluA

-CH3

OH

OO

OH

M1

D

OHHO

OH

OH GluA

COCH3

M3

E

-CH3OH

OO

OH

F

OH

OO

OH

GluA

-CH3

M1

OH

OHHO

OH

G

H

OHHO

OH

OH GluA

COCH3

M3

COOH

OO

OH

I

M4

Figure 5. Potential metabolic biotransformation pathway and the metabolites of eleutheroside B. GluA, Glucuronyl unit; COCH3, acetylation. (A)Degluconation; (B) glucuronidation; (C) demethylation; (D) demethylation + acetylation; (E) demethylation; (F) glucuronidation; (G) demethylation;(H) glucuronidation + acetylation; and (I) hydroxylation + desaturation.

F. Lu et al.

AcknowledgmentsThis article was supported by Major Projects of National Scienceand Technology (2009ZX09103-329), ‘Chunhui Plan’ co-researchprojects (Z2009-1-15032) and National Natural Science Founda-tion of Youth Science Fund (30901974).

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