Metabolic analysis of rhubarb extractby rat intestinal bacteria using liquidchromatography–tandem mass spectrometryRui Song,a,b Lei Xu,a,b Fengguo Xu,a,b Haijuan Dong,a,b Yuan Tiana,b andZunjian Zhanga,b*

ABSTRACT: Through investigation of the metabolism of rhubarb extract by rat intestinal bacteria, a total of 14 components inrhubarb extract were found to be biotransformed. These components included aloe-emodin-O-glucosides, emodin-O-glucosides, chrysophanol-O-glucosides, physcion-O-glucosides and the corresponding aglycones. Rhein also could bebiotransformed by rat intestinal bacteria. Twelve major metabolites were detected in the incubation sample. Under ESI tandemmass conditions, the sequential fragmentation patterns of [M – H]- ions were similar to those of free anthraquinones, thusallowing the rapid identification of the metabolites formed in incubation samples. The results suggested that the proposedhydrolysis of glycoside group followed by hydrogenation in quinoid moiety and/or further acetylation was the major biotrans-formation pathway for these anthraquinone glycosides by rat intestinal bacteria. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: anthraquinone glycosides; intestinal bacteria; metabolites; LC-DAD; LC-ESI-MS/MS

IntroductionRhubarb (Rhei rhizoma) is one of the important herbal drugs usedin oriental medicine, and has purgative, choleretic, liver protect-ing, antibacterial, anti-inflammatory and anticarcinogenic effects(Duke, 2002; Jiao and Du, 2000; Xiao et al., 1984) and containsmany compounds (Jiao and Du, 2000; Kashiwada et al., 1986a, b,1989; Lin et al., 2006; Nonaka et al; 1977, 1981; Ye et al., 2007),including free anthraquinones, anthraquinone glycosides, sen-nosides, naphthalins, stilbenes, galloyglucoses, acylglucose, phe-nylbutanones, flavan-3-ols and organic acids. Among theseconstituents, anthraquinones are antileukemic, antiseptic, antitu-bercular and antitumor components.

Sennosides and anthraquinone glycosides have purgativeeffects and they are also the most important pharmacologicalconstituents of rhubarb. Stilbenes have anticonvulsant, estro-genic, antioxidant and anticancer activity. Galloylglucoses haveantidiarrhea and antioxidant activity (Chen and Yen 1991; Jiaoand Du, 2000; Li, 1973; Xiao et al., 1984).

In traditional Chinese medicine (TCM), most remedies areadministered orally in the form of crude decoctions, and activecomponents inevitably come into contact with microflora andenzymes in the alimentary tract. For rhubarb, which containsmany glycosides with different aglycones, intestinal bacteria playimportant role in the metabolism of these glycosides. Actually,many experiments focusing on the degradation or metabolism ofsennosides and free anthraquinone in rhubarb in vitro or in vivohave been studied. It is known that sennoside A and B are easilytransformed into several deglycosylated products by intestinalmicroflora (Dreessen et al., 1981; Hattori et al., 1982, 1988; Hietalaet al., 1988; Kobashi et al., 1980; Lemli and Lemmens, 1980; Lemli,1988), including sennidin monoglucoside, sennidin, rheinan-throne and rhein. At 2.3 h after oral administration of sennoside

C, nearly equimolar amounts of aloe-emodin anthrone and rhei-nanthrone were detected in the large intestine of mice (Yamau-chi et al., 1992). Rhein, rhein-8-O-glucoside, aloe-emodin andchrysophanol could be transformed by human intestinalbacteroide species RHEIN-I and RHEIN-II (de Witte and Lemli,1988; de Witte et al., 1992; Meselhy et al., 2001). However, all theabove reports of the metabolic analysis were concerned onlywith several pure compounds isolated from rhubarb. Knowledgeconcerning the degradation and metabolism of rhubarb extractcaused by intestinal bacteria is generally lacking. Since rhubarband most of the remedies containing it are administered orally inthe form of crude decoction in TCM, metabolic analysis ofrhubarb aqueous extract with the global concept is essential andhelpful in understanding the action mechanism of the drug.Therefore, an LC-MSn approach is, for the first time, used to deter-mine the components that can be biotransformed by rat intesti-nal bacteria in rhubarb extract and elucidate the structures of thecorresponding metabolites.

* Correspondence to: Zunjiang Zhang, Center for Instrumental Analysis, ChinaPharmaceutical University, No.24 Tongjia Lane, Nanjing 210009, China.E-mail: zunjianzhangcpu@hotmail.com

a Key Laboratory of Drug Quality Control and Pharmacovigilance (China Phar-maceutical University), Ministry of Education, No. 24 Tongjia Lane, Nanjing,210009, China

b Center for Instrumental Analysis, China Pharmaceutical University, No. 24Tongjia Lane, Nanjing 210009, China

Abbreviations used: TCM, traditional Chinese medicine; CID, collision-induced dissociation; EIC, extracted ion chromatogram.

Research Article

Received 8 January 2010, Received 8 April 2010, Accepted 9 April 2010 Published online in Wiley Online Library: 4 June 2010

(wileyonlinelibrary.com) DOI 10.1002/bmc.1467

417

Biomed. Chromatogr. 2011; 25: 417–426 Copyright © 2010 John Wiley & Sons, Ltd.

Experimental

Chemicals and Reagents

Emodin, chrysophanol, rhein, aloe-emodin, physcion and gallic acid werepurchased from the Chinese National Institute for the Control of Pharma-ceutical and Biological Products (Beijing, China). Methanol (HPLC grade)was purchased from VWR International Company (Darmstadt, Germany).Formic acid (analytical reagent) was purchased from the First ChemicalCompany of Nanjing (Jiangsu, China). Ethyl acetate (analytical reagent)was purchased from Nanjing Chemical Reagent Co. Ltd. Water was dis-tilled twice before use.

Rhubarb (Rheum palmatum L.) was purchased from Li County, GansuProvince, China, and authenticated by Professor Ping Li (Key Laboratory ofModern Chinese Medicines, Ministry of Education, China PharmaceuticalUniversity, Nanjing, China).

Male Sprague–Dawley rats (230 � 10 g) were supplied by the AnimalMultiplication Center of Qinglong Mountain (SCXK 2007-0007). They werekept in an environmentally controlled breeding room and fed with stan-dard laboratory food and water ad libitum.

LC-DAD Analysis of the Biotransformation Products ofRhubarb Extract

The analyses were performed using an Agilent Series 1100 liquid chro-matograph (Agilent Technologies, Palo Alto, CA, USA), equipped with adiode array detector that recorded UV spectra in the range of 190–400 nm. Chromatography was carried out on an Agilent Zorbax SB-C18column (250 ¥ 4.6mm, 5 mm). The mobile phase consisted of 0.1% formicacid (A) and methanol (B), using a gradient elution of 5% B at 0–10 min,5–20% B at 10–30 min, 20–25% B at 30–40 min, 25–45% B at 40–160 min,45–60% B at 160–180 min, 60–80% B at 180–200 min, and 80% B at 200–220 min. The flow rate was 1 mL/min. The detection wavelengths were setat 225, 254 and 280 nm. Data were acquired and processed by HP Chem-Station software.

LC-MS/MS Analysis of the Biotransformation Products ofRhubarb Extract

The LC condition for the analysis of biotransformation samples of rhubarbextract by rat intestinal bacteria was the same as described above except

Figure 1. Representative HPLC chromatograms of the incubation samples at (a) 0 h and (b) 24 h.

Figure 2. Full-scan, MS2 spectra of [M - H]- (m/z 415) of P3; UV spectra of chrysophanol and P3.

418

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Biomed. Chromatogr. 2011; 25: 417–426wileyonlinelibrary.com/journal/bmc Copyright © 2010 John Wiley & Sons, Ltd.

Tab

le1.

LC/M

S2da

taof

the

com

pon

ents

that

can

be

bio

tran

sfor

med

byra

tin

test

inal

bac

teria

inrh

ubar

bex

trac

t

No.

Rete

ntio

ntim

e(m

in)

Prec

urso

r[M

-H

]-D

ata-

dep

ende

ntM

S2da

ta(%

bas

ep

eak)

Iden

tifica

tion

P169

.98

431

MS2

-15

V[4

31]:

431(

5),3

11(2

),29

3(15

),26

9(10

0),2

65(7

)A

loe-

emod

in1

or8-

O-g

luco

side

MS2

-40

V[4

31]:

269(

100)

,239

(71)

,211

(26)

P211

8.60

431

MS2

-30

V[4

31]:

311(

1),2

82(4

),26

9(10

0),2

54(2

),24

0(16

)Em

odin

3-O

-glu

cosi

deM

S2-3

0V

[431

]:43

1(3)

,280

(4),

269(

100)

,241

(2)

P314

4.88

415

MS2

-15

V[4

15]:

277(

10),

253(

100)

Chr

ysop

hano

l1-O

-glu

cosi

deM

S2-3

0V

[415

]:25

3(10

0),2

25(7

)P4

148.

6041

5M

S2-3

0V

[415

]:27

7(7)

,253

(100

),22

5(10

)C

hrys

opha

nol8

-O-g

luco

side

P515

2.03

431

MS2

-15

V[4

31]:

431(

53),

268(

100)

Alo

e-em

odin

w-O

-glu

cosi

deM

S2-5

3V

[431

]:26

8(80

),23

9(10

0),2

11(4

9),1

95(4

)P6

155.

6043

1M

S2-3

0V

[431

]:43

1(10

0),2

69(1

8)Em

odin

1or

8-O

-glu

cosi

deM

S2-3

0V

[431

]:43

1(2)

,311

(2),

269(

100)

,225

(6)

P717

2.91

445

MS2

-30

V[4

45]:

292(

11),

283(

100)

,268

(6),

251(

12),

240(

31)

Phys

cion

-O-g

luco

side

P817

7.15

431

MS2

-30

V[4

31]:

282(

19),

269(

100)

,241

(2),

225(

3)Em

odin

1or

8-O

-glu

cosi

deP9

177.

6544

5M

S2-1

5V

[445

]:44

5(3)

,386

(2),

337(

2),2

97(6

),28

3(10

0)Ph

ysci

on-O

-glu

cosi

deM

S2-3

0V

[445

]:29

7(8)

,283

(100

),25

1(6)

,240

(64)

P10

178.

4026

9M

S2-1

5V

[269

]:26

9(10

0),2

40(7

)A

loe-

emod

inM

S2-3

0V

[269

]:26

9(25

),26

8(50

),25

2(5)

,240

(82)

,239

(100

),22

3(23

),21

1(41

),18

3(59

),16

7(15

)P1

118

9.94

283

MS2

-15

V[2

83]:

283(

15),

239(

100)

,211

(5),

183(

5)Rh

ein

MS2

-30

V[2

83]:

239(

27),

211(

42),

183(

100)

P12

200.

7526

9M

S2-3

0V

[269

]:26

9(76

),24

1(50

),22

5(10

0),2

10(2

7),1

97(2

5)Em

odin

P13

203.

9525

3M

S2-2

5V

[253

]:25

3(10

0),2

25(5

7)C

hrys

opha

nol

MS2

-35

V[2

53]:

253(

18),

225(

100)

,210

(11)

,196

(3),

182(

10)

P14

210.

2428

3M

S2-2

0V

[283

]:28

3(10

0),2

40(1

6)Ph

ysci

on

419

Metabolic analysis of rhubarb extract by rat intestinal bacteria

Biomed. Chromatogr. 2011; 25: 417–426 wileyonlinelibrary.com/journal/bmcCopyright © 2010 John Wiley & Sons, Ltd.

that the effluent was directed to source at the rate of 0.3 mL/min via aT-union splitting. LC-MS/MS analysis was performed with a Finnigan TSQQuantum Discovery MAX system (Thermo Electron, San Jose, CA, USA)connected to a Finnigan Surveyor HPLC system (Thermo Electron, SanJose, CA, USA) via an ESI interface. Data were acquired and processed byXcalibur 2.0 controlling software. The spray voltage was set at –4000 V.The capillary voltage was fixed at -15 V, and the temperature was main-tained at 350°C. The tube lens offset voltage was set at –30 V. The LC fluidwas nebulized using N2, which was also the sheath gas and the auxiliarygas set at 40 and 10 arbitrary units, respectively. The MS/MS spectra wereproduced by collision-induced dissociation (CID) of the selected precur-sor ions with argon present in the mass analyzer, the collision energy forCID was set at 15 V initially for all of the preferred ions and then modifiedaccording to the fragments with collision gas at a pressure of 1.5 mTorr.

The biotransformation analysis of rhubarb extract in rat intestinal bac-teria by LC-MS/MS was achieved by two steps:(1) Full mass range scan analysis of the control and sample of biotrans-

formation was carried out from m/z 120 to 1000. The m/z values of thecomponents in rhubarb extract and possible metabolites wereobtained according to the differences of these chromatograms. Fur-thermore, the retention times of these compounds were alsoobtained.

(2) The mass scan time segment was divided into several parts accordingto acquired retention time information of potential targets obtainedfrom step (1). Each time segment included several scan events thatwere set to acquire a targeted product scan of the pre-setting m/zvalues of the assumed parent components and related transforma-tion products.

Preparation of Rhubarb Extract

Fifteen grams of rhubarb were immersed in 150 mL distilled water for 1 hand extracted for 40 min under reflux, the aqueous extract was filteredand the residue was extracted with 150 mL distilled water for 20 minunder reflux. The two water extracts were combined and condensed to100 mL and stored at 4°C until use.

Incubation Experiments

Preparation of intestinal bacteria culture solution. Ten-gramfresh feces from rats were mixed thoroughly with 40 mL physiologicalsaline; after filtration, a 5 mL aliquot of filtrate was transferred into abaffled flask containing 250 mL incubation buffer (Yang, 2003). Aftermixing thoroughly, cultures were incubated in an anaerobic sack (MGCCompany, Japan) under a N2 atmosphere at 37°C for 12 h, the mixture wascentrifuged at 4000 rpm for 15 min, the residue was suspended in 1mLphysiological saline, and then the culture solution of intestinal bacteriawas prepared.

Incubation experiments and extraction. Complete incubationsystems contained 50 mL culture solution of intestinal bacteria, 0.5 mLrhubarb extract and 10 mL incubation buffer. The mixture was incubatedwith anaerobic culture at 37°C. At designed times (0, 1, 2, 4, 6, 8, 12 and24 h), the reaction was terminated by extraction of the mixture ofunchanged compounds in rhubarb extract and metabolites with three-fold volumes of ethyl acetate. The ethyl acetate extract was evaporated todryness in a rotary evaporator and dissolved in 400 mL methanol.

Figure 3. Representative extracted ion current chromatograms of metabolites M2–M12 formed inincubation.

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Results and Discussion

Metabolism Profile of Rhubarb Extract in Rat IntestinalBacteria by HPLC Analysis

Firstly, the change in rhubarb extract due to incubation withintestinal bacteria was determined by HPLC. After incubation,some of peaks in rhubarb extract were decreased or even com-pletely disappeared, as shown in Fig. 1(b). The rhubarb extractwas incubated without intestinal bacteria for 24 h to measure thestabilities of rhubarb extract and blank sample, which meansrhubarb extract-free, was also tested under the same incubationconditions. These results showed that the rhubarb extracts werenot altered during the incubation, and the peaks derived fromblank sample were not overlapped with peaks emerged whenrhubarb extract was incubated with intestinal bacteria (see Sup-porting Information, Fig. S-1).

Identification of the Components that can beBiotransformed by Rat Intestinal Bacteria in RhubarbExtract and the Corresponding Metabolites

In the preliminary experiments, a data-dependent scan wasadopted as the scan mode in tandem mass spectrometry. Thechallenge of studying the in vitro metabolites of rhubarb extractlies in that most of them exist in a relatively low concentration inthe incubation accompanied by large amounts of unchangedcompounds. Some metabolites were at an extremely trace leveland could only be discerned in an enlarged HPLC-UV profile orextracted ion chromatogram (EIC). Hence, the successful initia-tion of the data-dependent triggered tandem MS scan for thedeprotonated molecule of interest was very important for char-acterization of the metabolites in trace amounts. For someextremely low-level metabolites, despite the input of the m/zvalues of deprotonated molecules into the parent mass list forthe preferential data dependent MS2 analysis, the data-dependent scan was triggered by the co-eluting unchangedcompounds instead. In order to solve this problem and obtainstructure information of interest as mush as possible, we finallyadopted the strategy described above. By employing such a scanmode, altogether 12 metabolites in the incubation of rhubarbextract with rat intestinal bacteria were characterized based ontheir mass spectra. In addition, the similarity between the UVspectrum of interest and that of free anthraquinone (includingemodin, rhein, aloe-emodin, chrysophanol and physcion) werealso considered in structural elucidation if the UV spectrum ofinterest was available.

Identification of the components that can be biotransformedby rat intestinal bacteria in rhubarb extract. Compared withthe controls, 14 components in rhubarb extract were decreasedor even completely disappeared after incubation for 24 h. Thissuggests that they have been biotransformed to new com-pounds by rat intestinal bacteria. In addition to P3–P14, shown inFig. 1(a), P1 and P2 were found in the enlarged EIC (see Support-ing Information, Fig. S-2). The major product ions of the biotrans-formed compounds along with their HPLC retention times aresummarized in Table 1.

P3 was selected as an example for the stepwise elucidation ofthe molecular structure. The structural elucidations of the otherparent components were carried out similarly.

Component P3 had an HPLC retention time of 148.5 min(Fig. 1). It exhibited deprotonated molecule [M – H]- at m/z 415(Fig. 2a). Its MS/MS fragmentation was dominated by the elimi-nation of glucosyl residue to give [Aglycone – H]- ([A – H]-) at m/z253 as the base peak (Fig. 2b). The ion at m/z 225 was the furtherfragment of m/z 253 with the loss of carbon monoxide. In addi-tion, its UV spectrum was similar to that of chrysophanol (Fig. 2cand d). According to a previous study (Ye et al., 2007), P3 wasidentified as chrysophanol 1-O-glucoside.

The identification of the other parent components and elucida-tion of their structures were performed mainly based on theirMS/MS fragmentations. In addition, the similarity between UVspectrum of interest and that of free anthraquinone (includingemodin, rhein, aloe-emodin, chrysophanol and physcion; seeSupporting Information, Fig. S-3) was also considered in the struc-tural elucidation if the UV spectrum of interest was available. Theresults of identification of these components are listed in Table 1.

Identification of metabolites in incubation samples. Com-pared with the controls, 12 metabolites were detected in

Figure 4. Enlarged extracted ion chromatograms for m/z 331 of (a)rhubarb extract and (b) the incubation samples at 0 h; enlarged extractedion chromatograms for m/z 169 of (c) rhubarb extract; (d) the incubationsample at 0 h; (e) the incubation sample at 8 h; (f ) the incubation sampleat 12 h; and (g) the incubation sample at 24 h.

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Tab

le2.

LC/M

S2da

taof

met

abol

ites

ofrh

ubar

bex

trac

taf

ter

incu

bat

ion

with

rat

inte

stin

alb

acte

ria

No.

Rete

ntio

ntim

e(m

in)

Prec

urso

r[M

-H

]-D

ata-

dep

ende

ntM

S2da

ta(%

bas

ep

eak)

Iden

tifica

tion

M1

7.78

169

MS2

-15

V[1

69]:

169(

52),

125(

100)

,97(

5),8

1(5)

Gal

licac

id9.

35M

S2-3

0V

[169

]:12

5(10

0),9

7(12

),79

(35)

,67(

12)

M2

113.

7732

9M

S2-1

5V

[329

]:32

9(10

0),2

70(4

3)3-

Ace

toxy

–1,8

-dih

ydro

xy-6

-hyd

roxy

met

hyl-1

0-ox

anth

rano

lM

S2-3

0V

[329

]:27

0(34

),26

9(10

0),2

52(2

),23

9(7)

,226

(3),

208(

1),

197(

2)M

314

2.17

343

MS2

-15

V[3

43]:

343(

10),

284(

100)

,240

(5)

3-A

ceto

xy-6

-car

box

y-1

,8-d

ihyd

roxy

-10-

oxan

thra

nol

M4

153.

7432

9M

S2-1

5V

[329

]:32

9(10

0),2

70(6

0)1,

3,8-

Trih

ydro

xy-6

-(ac

etox

y)m

ethy

l-10-

oxan

thra

nol

MS2

-30

V[3

29]:

269(

100)

,225

(5)

M5

163.

8532

7M

S2-1

5V

[327

]:32

7(21

),27

1(10

0)3-

Ace

toxy

-1,8

-dih

ydro

xy-6

-hyd

roxy

met

hyla

nthr

aqui

none

MS2

-30

V[3

27]:

291(

9),2

71(3

8),2

69(1

00),

256(

4),2

41(1

0),2

40(2

2),

239(

5),2

25(3

),21

1(10

)M

616

5.45

313

MS2

-15

V[3

13]:

313(

100)

,295

(2),

254(

3),2

39(6

9)3-

Ace

toxy

-1,8

-dih

ydro

xy-6

-met

hyl-1

0-ox

anth

anol

MS2

-30

V[3

13]:

254(

12),

253(

100)

,239

(14)

,225

(3)

M7

182.

1134

1M

S2-1

5V

[341

]:34

1(88

),29

7(10

0),2

85(7

6),2

51(3

9),2

40(3

9),

225(

96)

2-A

ceto

xy-6

-car

box

y-1

,8-d

ihyd

roxy

anth

raqu

inon

e

MS2

-30

V[3

41]:

285(

8),2

51(1

00),

240(

74),

225(

42),

209(

15)

M8

187.

0532

7M

S2-1

5V

[327

]:32

7(10

0),2

71(6

7)2-

Form

yl-1

,8-d

ihyd

roxy

-3-h

ydro

xym

ethy

l-6-m

etho

xyan

thra

quin

one

MS2

-30

V[3

27]:

271(

38),

270(

56),

269(

100)

,256

(11)

,243

(10)

,22

5(3)

M9

187.

6534

1M

S2-1

5V

[341

]:34

1(73

),31

1(10

0),2

93(1

2)1,

8-D

ihyd

roxy

-2-(

acet

oxy)

met

hyl-3

-met

hoxy

anth

raqu

inon

eM

S2-3

0V

[341

]:32

3(6)

,311

(12)

,308

(19)

,293

(100

),29

1(46

),27

8(24

),26

9(5)

M10

197.

7835

3M

S2-3

0V

[353

]:29

5(5)

,294

(100

),29

3(62

),27

9(8)

,267

(2),

265(

3)3-

Ace

toxy

-1,8

-dih

ydro

xy-6

-(ac

etyl

)met

hyla

nthr

aqui

none

M11

200.

3532

5M

S2-1

5V

[325

]:32

5(10

0),2

83(1

4)1,

8-D

ihyd

roxy

-2-(

acet

oxy)

met

hyl-6

-met

hyla

nthr

aqui

none

MS2

-30

V[3

25]:

325(

16),

307(

13),

283(

100)

,281

(14)

,254

(59)

,23

9(19

)M

1220

1.51

327

MS2

-30

V[3

27]:

327(

68),

309(

73),

297(

10),

283(

28),

281(

84),

255(

11),

254(

100)

,253

(74)

,1,

8-D

ihyd

roxy

-2-(

1-hy

drox

yeth

oxy)

met

hyl-6

-met

hyla

nthr

aqui

none

238(

10),

226(

7),2

09(6

)

422

R. Song et al.

Biomed. Chromatogr. 2011; 25: 417–426wileyonlinelibrary.com/journal/bmc Copyright © 2010 John Wiley & Sons, Ltd.

incubation samples (Figs 3 and 4). The major product ions ofthese metabolites along with their HPLC retention times are sum-marized in Table 2.

The EICs for m/z 331 and m/z 169 of rhubarb extract are shownin Fig. 4(a and c). The compound showing a deprotonated mol-ecule at m/z 169 was ascertained as gallic acid by comparing theretention time and fragmentation behaviors with the referencestandard, while all of the compounds having a deprotonatedmolecule at m/z 331 were identified as glucose gallates based ontheir mass spectra. Because of the low extraction efficiency ofethyl acetate to gallic acid and glucose gallates, the peaks in EICsfor m/z 331 and m/z 169 of incubation sample at 0 h were toosmall to identify (Fig. 4b and d). However, the peak in EICs for m/z169 of the incubation sample became larger as the incubationtime was prolonged (Fig. 4e–g). The growth of this compoundcould be attributed to the transformation of glucose gallates byintestinal bacteria. Finally, the compound was confirmed as gallicacid by comparing the retention time and fragmentation behav-iors with the reference standard.

M2 and M4 all gave an [M - H]- molecule at m/z 329. A CIDproduct ion spectrum of M2 displayed fragment ions at 270, 269,252, 239, 226 and 197 (Fig. 5). The m/z 270 was formed by the lossof acetoxy radical (-59 Da from the m/z value of the deproto-nated molecule). The m/z 269 was formed by the loss ofCH3COOH, which was extended to the loss of HCHO to form thefragment ion at m/z 239 (-30 Da with a proton shift). The MS/MS

spectrum of M4 displayed ions of m/z 269 and 225. The ion at m/z269 was formed by the loss of CH3COOH (-60 Da from m/z valueof the deprotonated molecule). The ion at m/z 225 was thefurther fragment of m/z 269 with the loss of CO2. The compoundeluting at 142.17 min showed a deprotonated molecule at m/z343 (Fig. 3). The MS/MS spectrum of M3 displayed ions of m/z 284and 240. The m/z 284 was formed by the loss of the acetoxyradical, which was the extended loss of CO2 to form the fragmention at m/z 240. The compound eluting at 165.45 min showed adeprotonated molecule at m/z 313. A CID product ion spectrumof M6 displayed fragment ions at 254, 253, 239, 225 and 210. Theion at m/z 254 was formed by the loss of the acetoxy radical,which was the extended loss of the methyl radical to form thefragment ion at m/z 239. The ion at m/z 253 was formed by theloss of CH3COOH, which was the extended loss of CO to form thefragment ion at m/z 225. According to our previous studies onmetabolic difference of anthraquinone derivatives (Song et al.,2009), the UV spectra of these four metabolites indicated thatquinone moiety was reduced (Fig. 5). Therefore, M2, M3, M4 andM6 were identified as 3-acetoxy-1,8-dihydroxy-6-hydroxymethyl-10-oxanthranol, 3-acetoxy-6-carboxy-1,8-dihydroxy-10-oxan-thranol, 1,3,8-trihydroxy-6-(acetoxy)methyl-10-oxanthranol and3-acetoxy-1,8-dihydroxy-6-methyl-10-oxanthanol.

M5, M8 and M12 all gave an [M – H]- molecule at m/z 327. A CIDproduct ion spectrum of M5 displayed fragment ions at 271, 269,256, 240, 225 and 211 (Fig. 6a). The m/z 271 was formed by loss of

Figure 5. UV spectra of M2, M3, M4 and M6; MS2 spectra of [M - H]- of M2, M3, M4 and M6.

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two-molecule carbon monoxide (-56 Da from m/z value of thedeprotonated molecule), which was the extended loss of H2(-2 Da with a proton shift) to form the fragment ion at m/z 269.The m/z 256 and m/z 211 were the further fragments of m/z 271with the loss of methyl radical and CH3COOH, respectively.According to the information obtained from MS/MS spectrum ofM5, it was tentatively identified as 3-acetoxy-1,8-dihydroxy-6-hydroxymethylanthraquinone. The MS/MS spectrum of M8 dis-played ions of m/z 271, 270, 269, 256, 243 and 225 (Fig. 6c). Them/z 271 was formed by the loss of two-molecule carbon monox-ide, which was extended to the loss of H2 to form the fragmention at m/z 269. The m/z 256 and m/z 243 were the further frag-ments of m/z 271 with the loss of methyl radical and CO, respec-tively. According to the information obtained from MS/MSspectrum of M8, it was tentatively identified as 2-formyl-1,8-dihydroxy-3-hydroxymethyl-6-methoxyanthraquinone. Theobvious product ions at m/z 254 formed by loss of ethenol andformyl radical, m/z 253 [M - H - CH2CHOH - CH2O]- wereobserved in the MS2 spectrum of M12 (Fig. 6g). Accordingto the information, M12 was characterized as 1,8-dihydroxy-2-(1-hydroxyethoxy)methyl-6-methylanthraquinone.

The full-scan spectra of M7 and M9 suggested that their

molecular mass was 342 Da. The MS2 spectrum of M7 displayedions of m/z 341, 297, 285, 251, 240 and 225 (Fig. 6b). The production at m/z 297 with the highest abundance was formed by loss ofcarbon dioxide. The m/z 285 was formed by loss of two-molecularcarbon monoxides. The m/z 240 and m/z 225 were the furtherfragments of m/z 285 with the loss of COOH radical andCH3COOH. Therefore, M7 was tentatively identified as 2-acetoxy-6-carboxy-1,8-dihydroxyanthraquinone. Fragmentation of the [M- H]- ion of M9 at m/z 341 involved initial loss of H2O to generatethe [M – H – 18]- ion at m/z 323. The product ions at m/z 293 [M– H – H2O – CH2O]- and m/z 291 [M – H – H2O – CH3OH]- in the MS2

spectrum suggested that there was a methoxy in its structure.The ion at m/z 311 was formed by loss of CH2O (-30 Da from m/zvalue of the deprotonated molecule). The ion at m/z 269 [M – H –CH2O – CH2CO]- was the result of the structural rearrangement toform the negative ion from the ion at m/z 311. Therefore, M9was tentatively identified as 1,8-dihydroxy-2-(acetoxy)methyl-3-methoxyanthraquinone.

The compound eluting at 197.78 min showed a deprotonatedmolecule at m/z 353 (Fig. 6e). MS2 experiments of M10 performedon the [M – H]- ion yielded obvious [M – H – 59]- and [M – H – 60]-

ions by loss of CH3COO radical and CH3COOH, which suggested

Figure 6. MS2 spectra of [M - H]- of (a) M5, (b) M7, (c) M8, (d) M9, (e) M10, (f ) M11, (g) M12.

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that there was an acetoxy in its structure. The precursor ion at m/z353 produced a product ion at m/z 295, assigned as [M – H –CH3COCH3]-. The m/z 267 and m/z 265 were the further frag-ments of m/z 295 and m/z 293 with the loss of CO. Accordingto the information obtained from the MS2 spectrum ofM10, it was identified as 3-acetoxy-1,8-dihydroxy-6-(acetyl)methylanthraquinone.

In the MS2 spectrum of M11, the precursor ion at m/z 325produced product ions at m/z 307 [M – H – H2O]-, m/z 283 [M – H– CH2CO]- and m/z 281 [M – H – CH2CO – H2]- (Fig. 6f ). The ion atm/z 254 was the further fragment of m/z 283 by loss of formylradical. Therefore, M11 was tentatively identified as 1,8-dihydroxy-2-(acetoxy)methyl-6-methylanthraquinone.

The Biotransformation Pathways of Rhubarb Extract by RatIntestinal Bacteria

According to the above-mentioned results, anthraquinonesand glucose gallates could be biotransformed by rat intestinalbacteria. However, anthraquinones were undoubtedly the mostprevalent parent compounds, as 11 out of 12 metaboliteswere tentatively assigned as metabolites originating fromanthraquinones. The proposed schematic biotransformationpathway of anthraquinones by rat intestinal bacteria is shown inFig. 7.

ConclusionIn conclusion, an LC-ESI-MS/MS approach was applied to thecomplex metabolites transformed from components in rhubarbextract by rat intestinal bacteria. Under experimental conditions,anthraquinones and glucose gallates in rhubarb decoction werebiotransformed after incubation. Twelve major metabolites weredetected in the incubation samples. Hydrolysis of the glycosidegroup followed by hydrogenation in quinoid moiety and/orfurther acetylation was proposed as the major biotransformationpathway for these anthraquinone glycosides. The metabolicanalysis would be helpful for further understanding metabolicnature, exposure to these components of rhubarb extract in vivoand the relevant mechanism of their therapeutic application.

Supporting InformationSupporting information can be found in the online version of thisarticle.

AcknowledgementsThis project was financially supported by Specialized ResearchFund for TCM of State Administration of Traditional Chinese Medi-

Figure 7. Proposed biotransformation pathways of anthraquinones and their glycosides by rat intestinal bacterial.

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cine (no. 06-07ZP17), Chinese Natural Science Fund (no.30672587) and Technology Innovation Program for Post-graduates of Jiangsu Province (no. CX08B_194Z). We would liketo express our cordial gratitude to the editor of Biomedical Chro-matography, Dr Famei Li, and reviewers for their excellent help inimproving the quality of our original manuscript.

ReferencesChen JF and Yen KY. Pharmacology of Chinese Herb Drugs. National

Research Institute of Chinese Medicine: Taipei, 1991; 213.Dreessen M, Eyssen H and Lemli J. The metabolism of sennosides A and B

by the intestinal microflara: in vitro and in vivo studies on the rat andmouse. Journal of Pharmacy and Pharmacology 1981; 33: 679–681.

de Witte P and Lemli J. Metabolism of 14C-rhein and 14C-rhein anthrone inrats. Pharmacology 1988; 36(suppl. 1): 152–157.

de Witte P, Van Hoestenberghe A and Eyssen H. In vitro reduction of rheinanthraquinone to rhein anthrone by rat cecal microflora and someintestinal bacterial strains. Biopharmaceutics and Drug Disposition1992; 13: 243–253.

Duke JA. Handbook of Medicinal Herbs, 2nd edn. CRC Press: Boca Raton, FL,2002; 621.

Hattori M, Kim G, Motoike S, Kobashi K and Namba T. Metabolism ofsennosides by intestinal flora. Chemical and Pharmaceutical Bulletin1982; 30: 1338–1346.

Hattori M, Namba T, Akao T and Kobashi K. Metabolism of sennosides byhuman intestinal bacteria. Pharmacology 1988; 36(suppl. 1): 172–179.

Hietala P, Lainonen H and Marvola M. New aspects on the metabolism ofthe sennosides. Pharmacology 1988; 36(suppl. 1): 138–143

Jiao DH and Du SJ. The Study of Rhubarb. Shanghai Science and Technol-ogy Press: Shanghai, 2000; 169.

Kobashi K, Nishimura T, Kusaka M, Hattori M and Namba T. Metabolism ofsennosides by human intestinal bacteria. Planta Medica 1980; 40:225–236.

Kashiwada Y, Nonaka G and Nishioka I. Tannins and related compounds.XLVIII. Rhubarb. (7). Isolation and characterization of new dimeric andtrimeric procyanidins. Chemical and Pharmaceutical Bulletin 1986a;34: 4083–4091.

Kashiwada Y, Nonaka G and Nishioka I. Tannins and related compounds.XLV. Rhubarb. (5). Isolation and characterization of flavan-3-ol and

procyanidin glucosides. Chemical and Pharmaceutical Bulletin 1986b;34: 3208–3222.

Kashiwada Y, Nonaka G and Nishioka I. Studies on rhubarb (RheiRhizoma). XV. Simultaneous determination of phenolic constituentsby high-performance liquid chromatography. Chemical and Pharma-ceutical Bulletin 1989; 37: 999–1004.

Lemli J. Metabolism of sennosides – an overview. Pharmacology 1988;36(suppl. 1): 126–128.

Lemli J and Lemmens L. Metabolism of sennosides and rhein in the rat.Pharmacology 1980; 20(suppl. 1): 50–57.

Li SY. Modern Chinese Herb Drugs. Chengchung Books: Taipei, 1973;252.

Lin CC, Wu CI, Lin TC and Sheu SJ. Determination of 19 rhubarb constitu-ents by high-performance liquid chromatography–ultraviolet–massspectrometry. Journal of Separation Science 2006; 29: 2584–2593.

Meselhy MR, Nishimoto E and Akao T. Human intestinal bacteroides spp.RHEIN-I and RHEIN-II capable of transforming rhein to rheinanthrone,induce rhein-dependent diarrhea in rats. Journal of Traditional Medi-cines 2001; 18: 169–176.

Nonaka G, Minami M and Nishioka I. Studies on rhubarb (Rhei Rhizoma).III. Stilbene glycosides. Chemical and Pharmaceutical Bulletin 1977; 25:2300–2305.

Nonaka G, Nishioka I, Nagasawa T and Oura H. Tannins and related com-pounds. I. Rhubarb (1). Chemical and Pharmaceutical Bulletin 1981; 29:2862–2870.

Song R, Lin H, Zhang ZJ, Li Z, Xu L and Dong HJ. Profiling themetabolic differences of anthraquinone derivatives using liquidchromatography/tandem mass spectrometry with data-dependentacquisition. Rapid Communications in Mass Spectrometry 2009; 23:537–547.

Xiao P, He L and Wang L. Ethnopharmacologic study of Chinese rhubarb.Journal of Ethnopharmacology 1984; 10: 275–293.

Yamauchi K, Shinano K, Nakajima K, Yagi T and Kuwano S. Metabolicactivation of sennoside C in mice: synergistic action of anthrones.Journal of Pharmacy and Pharmacology 1992; 44: 973–976.

Yang XW. Metabolite Analysis for Chemical Constituents of TraditionalChinese Medicines. China Science and Technology Press: Bingjing,2003; 143.

Ye M, Han J, Chen HB, Zheng JH and Guo DA. Analysis of phenolic com-pounds in rhubarbs using liquid chromatography coupled with elec-trospray ionization mass spectrometry. Journal of the American Societyfor Mass Spectrometry 2007; 18: 82–91.

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R. Song et al.

Biomed. Chromatogr. 2011; 25: 417–426wileyonlinelibrary.com/journal/bmc Copyright © 2010 John Wiley & Sons, Ltd.

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