Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46

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Short communication

Structural elucidation of in vivo metabolites of isobavachalcone in rat by LC–ESI-MSn and LC–NMR Su Su a,d , Yanan Wang c , Lu Bai a , Binbin Xia a , Xiaorong Li a,b , Yu Tang a , Pingxiang Xu a,b , Ming Xue a,b,∗ a

Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China Beijing Laboratory for Biomedical Detection Technology and Instrument, Beijing 100069, China c State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China d Department of Pharmacy, Xuanwu Hospital, Capital Medical University, Beijing 100053, China b

a r t i c l e

i n f o

Article history: Received 7 May 2014 Received in revised form 27 October 2014 Accepted 6 November 2014 Available online 15 November 2014 Keywords: Structural elucidation Isobavachalcone Metabolites LC–ESI-MSn NMR

a b s t r a c t Isobavachalcone (IBC) is a prenylated chalcone and belongs to the class of flavonoids, which is an active component isolated from Psoralea corylifolia L. IBC showed a range of significant pharmacological activities, including antibacterial, anticancer, anti-reverse transcriptase and antioxidant actions. In this research, the mass spectral fragmentation pattern of IBC was investigated to predict the in vivo metabolites, and five phase I metabolites and ten phase II metabolites of IBC in rat bile were elucidated and identified after oral administration using novel LC–ESI-MSn and LC–NMR method. The molecular structures of these metabolites were proposed on the basis of the characteristics of their precursor ions, product ions, MS/MS fragment behaviors and chromatographic retention time. The phase I metabolites were mainly biotransformed via the hydroxylation, reduction, cyclization and oxidative cleavage reactions. The phase II metabolites were mainly identified as the glucuronide conjugates and sulfated conjugates. All these findings were reported for the first time and would contribute to a further understanding of the in vivo intermediate processes and metabolic mechanism of isobavachalcone and its analogs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Isobavachalcone (IBC) is classified as chalcones and an analog of flavonoid compounds that possess the C6-C3-C6 backbone. IBC was firstly isolated from Psoralea Corylifolia in 1968 and there have been rapidly increasing reports available on this compound these years [1]. IBC has been reported to possess many pharmacological activities, including antibacterial, antifungal, anti-tuberculosis, anticancer, anti-reverse transcriptase virus and antioxidant activities [2–9]. Zhang et al. found that IBC was an A␤ aggregation inhibitor, which might provide a solution in Alzheimer’s therapy [10]. Koike et al. observed that IBC was a new potent PTP1B inhibitor to gain the potential therapeutic value for diabetes and obesity [11]. A recent research suggested that IBC exhibited a high activity against the key enzyme of SARS virus replication

∗ Corresponding author at: Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China. Tel.: +86 10 8391 1520/83950163; fax: +86 10 8391 1520. E-mail address: [email protected] (M. Xue). http://dx.doi.org/10.1016/j.jpba.2014.11.010 0731-7085/© 2014 Elsevier B.V. All rights reserved.

[4]. Since IBC showed such vital pharmacological activities, there were widely increasing focuses ondevelopment of isolation and quantitationmethod of IBC from Psoralea corylifolia utilizing liquid chromatography electrospray ionizationtandem mass spectrometry (LC–ESI-MSn ) and CE-MS [12–14]. However, the mass spectral fragmentation pattern, in vivo metabolites and metabolic pathways of IBC were absent. To better understand the in vivo metabolism characterization of IBC, the mass spectral fragmentation pattern of IBC was investigated in this article, and a novel and sensitive LC–ESI-MSn method was established to study the MS structural characteristics of IBC along with its metabolites. The HPLC-SPE-NMR was also utilized for a further analysis of some metabolites. The in vivo metabolism study of IBC might provide useful information for the further studies pharmacological activity of IBC. It is the first time for the article to provide a robust and sensitive LC–ESI-MSn and HPLCNMR method to simultaneously elucidate five phase I metabolites and ten phase II metabolites of IBC in rat bile and also present the in vivo metabolic pathways of IBC. All of these findings contributed a further understanding of the intermediate processes and metabolism mechanism of these prenylated chalcone compounds.

S. Su et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46

Our investigation has provided much novel information on the in vivo isobavachalcone metabolism, which would help to a novel drug development, as well as a better understanding of the safety and efficacy of this drug. 2. Experimental 2.1. Chemicals and reagents IBC was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, Jiangsu, China). HPLC grade methanol was purchased from Fisher Scientific Products (Fair Lawn, NJ, USA). Ethyl acetate and formic acid were purchased from Dikma Technology Inc (Lake Forest, CA, USA). Ultra-pure water was from a Milli-Q plus water purification system (Millipore, Bedford, MA, USA). All other chemicals and reagents were of analytical grade.

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an aux/sweep gas, and ultra-high purity helium was used as collision gas for the collision induced dissociation (CID) experiments in the ion trap. The optimized parameters were as follows. In positive mode, sheath flow-rate of 40 kPa, a typical source spray voltage of 4.5 kV, a capillary voltage of 26 V and a heated capillary temperature of 320 ◦ C. In negative mode: sheath flow-rate of 40 kPa, a typical source spray voltage of 3.8 kV, a capillary voltage of −17.20 V and a heated capillary temperature of 320 ◦ C. The other parameters were optimized for maximum abundances of the interested ions by an automatic tune procedure. The LC–NMR analysis was performed on a Bruker AVANCE III HD 600 MHz with an Agilent 1260, and the chemical shits were recorded in ppm (ı) down field from the internal standard tetramethylsilane (TMS) in CD3 OD, and the HPLC method was as described below. The parent drug IBC and metabolite M4 were separated by HPLC and collected according to its UV spectra and retention time for further NMR analysis.

2.2. Animal experiments The protocol for animal experiment was approved by the Committee on the Care and Use of Laboratory Animals of China (Beijing, China). Adult male Sprague-Dawley rats weighing 250 ± 10 g were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and randomly divided into two groups (n = 4). The rats were fed with normal food and supplied with water ad libitum. Rats were fasted 12 h with free access to water before oral administration. Treated group were given doses of IBC (50 mg/kg) by intragastric administration. The control group was given an equivalent solution without IBC. The rats were anesthetized and fixed on wooden plates. Heating lamps under the wooden plates were used for keeping rats’ body warm during experiment. PE-10 tube (i.d. 0.08 cm, Becton Dickinson, USA) was used to collect bile samples. Bile samples were collected for 12 h and were stored at −80 ◦ C immediately until use. 2.3. Sample preparation 6 mL ethyl acetate was added to 2 mL bile samples. The suspension was shaken on a bench top shaker for 10 min at room temperature and centrifuged at 13,000 rpm for 10 min. The upper organic layer was transferred to a new tube. The procedure was repeated twice. The lower aqueous layer was treated with 6 mL methanol for protein precipitation and shaken for 10 min. The samples were centrifuged at 13,000 rpm for 5 min and aspirated the supernatant to a new tube. Organic and aqueous samples were dried in a centrivap vacuum concentrator (Labconco, Kansas City, USA) at 40 ◦ C. The residues were reconstituted with 500 ␮L methanol for organic samples and 2 mL methanol for aqueous samples, filtered with 0.22 ␮m syringe filters for LC–ESI-MSn and LC–NMR analysis. 2.4. Apparatus and analytical conditions The gradient elution was performed using an Agilent HPLC system (Series 1100, Agilent technology, Palo Alto, CA, USA) with a Waters Symmetry C18 column (100 × 2.1 mm, i.d., 3.5 ␮m) with a flow rate at 2 mL/min. The temperature of column oven was maintained at 40 ◦ C. The mobile phase system consisted of methanol (A) and water containing 0.1% formic acid (B) using a gradient elution: 0–20 min, 60% A, 20–30 min, 80% A. The injection volume was set to 10 ␮L. The MS analysis was performed on a Finngian LCQ Deca-XP ion trap mass spectrometer equipped with an electrospray ion source (Thermo Finnigan, San Jose, CA, USA) working in both positive and negative ion mode. Nitrogen was used as a sheath gas and

3. Results and discussion 3.1. Mass spectral fragmentation of IBC Whether phase I reactions or phase II reactions, all of the metabolic reactions in vivo occurred on the basis of the parent compound. And the fragmentation pattern of the parent compound in vitro always performance similarly with some in vivo metabolic reaction, especially the oxidation and reduction in phase I reaction. Herein, the fragmentation pattern of the parent compound in vitro would provide useful information for predicting the metabolite structures before charactering the structure of metabolites in vivo. A full-scan mass spectra of IBC showed a protonated molecule [M+H]+ at m/z 325 in positive ion mode (Fig. 1A) and a deprotonated molecule [M−H]− at m/z 323 in negative ion mode (Fig. 1B) under the optimized MS/MS conditions and the possible fragmentation pathways of IBC in positive ion mode and negative ion mode are shown in Fig. 2A and B, respectively. In positive ion mode, IBC generated two main fragment ions at m/z 269 and 205 (Fig. 1A-a). The most abundant product ion at m/z 269 was resulted from a loss of isobutylene chain [(CH3 )2 C CH2 ] with a characteristic neutral loss of 56 Da [15]. The product ion at m/z 205 was yielded by an oxidative cleavage of a single bond to generate a B ring with the attached C O group. The daughter fragment ions at m/z 269 and 205 were the products ions at m/z 149 and 167 (Fig. 1A-b). The product ion at m/z 149 could either be generated from a neutral loss of isobutylene chain [(CH3 )2 C CH2 ] of the product ion at m/z 205, or could be generated from the most abundant product ion at m/z 269 by a direct cleavage of a 4-vinylphenol group [HO C6 H5 CH CH] (120 Da). The product ion at m/z149 underwent further fragmentation, forming the ions at m/z 121 and 93, seen in Fig. 1A-c. In negative ion mode, IBC produced two main fragment ion at m/z 203 and m/z 119 by a cleavage of the single bond between C C and C O group (Fig. 1B). The product ion at m/z 203 underwent further fragmentation, forming ions products at m/z 183, 175, 159 and 157, seen in Fig. 1B-b. The most abundant product ion at m/z 159 was eliminated a carbon monoxide [C O] and OH from the product ion at m/z 203. The product ion at m/z 185 was generated from elimination a H2 O form the product ion at m/z 203, and further eliminated a CO to form the ion at m/z 157. The product ion at m/z 175 showed the fragment ions at m/z 160, 157, 147, 133, 107 and 93 (see the Fig. 1B-d). The production at m/z 157, 147 and 133 were generated by elimination of a H2 O (18 Da), ethylene [CH2 CH2 ] (28 Da) and propylene [CH3 CH CH2 ] (42 Da), respectively, from the product ion at m/z 175, and the product ion at m/z 160 was generated via a cleavage of CH3 (15 Da).

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S. Su et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46

268.95

100

203.55

100

a

80

80

60

60

a 323.44

325.03

40

324.16

40 204.60

205 .04 20

20 204.30 205.72 268.19

121.11 149.05

0 100

150

200

250 m /z

149.07

100

324.25

342.68

300

119.62 118.68

0

100

350

221.40 235.40

159.54 201.71 150

200

250 m /z

300

b

b

107.02

80

60

350

160.14

100

80

325 .15

279.49 322.65

60 157.24

268 .95

40

166 .89

20

0

40 175.09

133.15

20

147.16

93.12

9 3 .0 2

121.01

180.76

100

150

227.09 250.99

20 0

287.94

25 0

3 2 4 .9 4

300

3 6 0 .5 9

60

35 0

m /z

149.06

100

129.23

79.21

55.34

0

80

10 0

12 0

00

160.76 175.

14 0 m /z 159.12

16 0

18 0

80 80

c

60 121.03

40

131.01

40

92.93

20 8 1 .0 6

20 1 1 1 .0 7

95.02

121.79

0 70

80

90

100

110

120 m /z

131.71 148.04 149

130

140

15 0

A 19

c 203.10

60

157.20 65.03 79 .07 93 .48 1 0 7 .1 1

0

60

80

10 0

131.08 143.17 12 0

14 0 m /z

175.13 159.96 16 0

185.19 203 18 0

20 0

159.13

100

80

20 18

6 17

16

1

12

HO

OH 11

7

4

2

13

OH

60

d

5

143.21

40

3

14

10

9

8

144.10 20

15

O

0

65.10 60

79.07 80

157.22

105.15 119.22 129.26 100

120

140

159.71

16 0

m /z

C

B

Fig. 1. The MSn spectra and chemical structure of isobavachalcone. (A) The mass spectra in positive ion mode, MS2 [325], MS3 [325 → 269] and M4 [325 → 269 → 149]. (B) The mass spectra in negative ion mode, MS2 [323], MS3 [323 → 203], MS3 [323 → 203 → 159], M4 [325 → 203 → 175]. (C) The molecular structure of isobavachalcone.

3.2. Identification of the metabolites of IBC The retention times of isobavachalcone and its metabolites were within 30 min based on an excellent separation. The structures of

these metabolites were identified by comparing MS/MS fragment patterns and change of molecular mass weight with those of IBC. The extracted ion chromatograms of IBC as well as its five phase I and ten phase II metabolites in rat bile are presented in Fig. 3.

S. Su et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46

A

O

O

OH

O

O

-C4H8

HO

OH

m/z 325

HO

O C

O

OH

m/z 269

41

m/z 149 -CO

O

O

O

C

C

O

OH

HO

m/z 121

m/z 205

m/z 93

B

O OH

HO

OH

m/z 119 CO

O O

O

CO

-OH,-CO

CO

O

-H2O

m/z 203

m/z 160

CO

CO

CO

-H2O

-C2H4

CO

m/z 157

-CH3

m/z 147

m/z 133

-CO

m/z 185

-CO

O

m/z 143

m/z 93 HO

O

CO

m/z 159

m/z 323

O

-CH4

-C3H6

CO

CO

HO

-C5H8 m/z 175

O

CO

-CO

m/z 107

m/z 79

Fig. 2. The ESI-MS fragmentation pathways of isobavachalcone in positive ion mode (A) and negative ion mode (B).

The chromatographic retention time and mass spectrometric data of isobavachalcone and its phase I and phase II metabolites in rat bile are shown in Table S1. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.11.010. 3.2.1. Phase I metabolites of IBC The retention time of IBC was 22.10 min. Interestingly, a compound with protonated molecule [M+H]+ at m/z 325 (M1) eluted at 16.19 min, exhibiting a similar mass spectrum pattern with IBC. In the results published previously [16–18], the isoliquiritigenin could

be biotransformed to a cyclized product with an earlier retention time. The structure of IBC is similar with isoliquiritigenin and IBC could exhibit a similar metabolism pathway with isoliquiritigenin. Therefore, M1 was proposed as a cyclized product of IBC, which exhibited a same MS fragmentation pattern and was eluted at an earlier retention time compared with the parent drug IBC. The mass spectrum of M1 was shown in Fig. 1A-a, and the chemical structure of M1was shown in Fig. 5. The M2 eluted at 23.07 min and gave rise to a protonated molecule [M+H]+ at m/z 269, forming from a neutral loss of isobutylene chain [(CH3 )2 C CH2 ] with a characteristic neutral loss of

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Fig. 3. Extracted ion chromatograms of isobavachalcone and its metabolites.

56 Da. Its mass spectrum pattern in positive ion mode had been discussed in Section 3.1. The mass spectra of the product ion of M2 were shown in Fig. 1A-b, and the chemical structure of M2 was shown in Fig. 5. The metabolite M3 gave rise to the protonated molecule [M+H]+ at m/z 327 with a retention time of 23.13 min. M3 was two mass

units higher than that of IBC, suggesting that M3 was formed from a hydrogenation or reduction reaction. The main ion product at m/z 327 had the ions at m/z 271 and m/z 207, which were 2 units higher than that of the product ions at m/z 269 and 205, indicating that reduction reaction occurred at the shared position of the product ion at m/z 269 and 205 and the position was had no choice but at

S. Su et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46

Fig. 4. The ESI-MSn spectra of the metabolite M3–M15 of isobavachalcone.

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Gluc

SO3

O HO

O

OH HO

OH

OH

HO

SO3

OH

OH M9

O

O M11

M15 Glucuronidation

Sulfation

Sulfation

OH OH

Gluc O

OH

OH

OH HO

HO

OH

OH

OH

HO

O

Glucuronidation

OH

OH

O

Reduction

O HO

O M1

M4

M3

M10

Cycliaztion

Hydroxylation

Gluc

OH OH O

M6

HO

OH

Gluc HO

OH

O IBC

Glucuronidation

O

Cleavage

Cleavage

O

M7 O

HO

OH

Gluc O

O

OH

HO

O

C

OH

O M2

M5

Sulfation

O M8

SO3 SO3

O HO

OH

O M12

HO

O

OH

OH

SO3 O

O

OH

O M14

M13 Fig. 5. Proposed metabolism pathways of isobavachalcone in rat.

the C O bonds between A and B ring. The mass spectra of M3 are shown in Fig. 4-M3 and the chemical structure of M3 was shown in Fig. 5. The metabolite M4, which eluted at 14.5 min, gave rise to the protonated molecule [M+H]+ at m/z 341, and was 16 Da greater than that of IBC. M4 was tentatively elucidated as the hydroxylated metabolite of IBC, by introducing a hydroxyl ( OH) group to

the parent drug. The fragment ions of the product ion at m/z 341 were the ion series at m/z 323, 269, 221, 203 and 147 (shown in Fig. 4-M4 and Fig. 5). The product ion at m/z 323 further generate the product ion at m/z 203, and the daughter product ions of m/z 203 were at m/z 147, 161, 175 and 185, which were described as featured product ions of the product ion at m/z 203 of IBC standard.

S. Su et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 38–46 Table 1 1 H NMR data of isobavachalcone and its metabolite M4 (in ppm, at 600 MHz in CD3 OD). No.

IBC

M4

1 3 4 6 7 8 14 15 16 17 19 20

6.79 (d, J = 8.30 Hz) 6.79 (d, J = 8.30 Hz) 7.56 (d, J = 8.30 Hz) 7.56 (d, J = 8.30 Hz) 7.73 (d, J = 15.48 Hz) 7.57 (d, J = 15.48 Hz) 6.38 (d, J = 8.44 Hz) 7.78 (d, J = 8.44 Hz) 3.29 (brs) 5.17 (t, J = 6.75 Hz) 1.60 (s) 1.72 (s)

7.10 (dd, J = 3.45, 7.09 Hz) 7.32 (brs) 7.24 (d, J = 7.09 Hz) 7.78 (d, J = 14.47 Hz) 7.62 (d, J = 14.47 Hz) 6.42 (d, J = 8.68 Hz) 7.82 (d, J = 8.68 Hz) 3.34 (brs) 5.22 (t, J = 6.75 Hz) 1.65 (s) 1.77 (s)

The NMR spectra of M4 exhibited that there were the signals on the ABX pattern with the chemical shits of ı7.32 (brs), 7.24 (d, J = 7.09 Hz) and 7.10 (dd, J = 3.45, 7.09 Hz) (seen in Fig. S1 and Table 1, where the NMR spectra of IBC exhibited no such signal (seen in Fig. S1 and Table 1), suggesting that there might be a pro-substituted phenolic hydroxyl in ring A of M4. Furthermore, the steric hindrance at ring B was greater than that of ring A, resulting to the fact that the hydroxylation most probably took place at ring A. In terms of the MSn and NMR spectra of M4, along with the factor from steric hindrance, M4 was identified as4-hydroxyl-isobavachalcone, which was formed via adding a hydroxyl group at the C-4 position in ring A. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.11.010. The metabolite M5 gave rise to the deprotonated molecule [M−H]− at m/z 203 in negative ion mode with a retention time of 19.60 min. The ESI-MS of M5 was shown in Fig. 1B; the ion fragment series were at m/z 203, 185, 175, 159, 157, 143, 107, 93 and 79, which was similar with the fragmentation pattern of the parent IBC in negative ion mode. M5 was yielded by a cleavage of a single bond between the C C attached at A ring and C O group attached at B ring. The chemical structure of M5 was shown in Fig. 5. 3.2.2. Phase II metabolites of IBC The phase II conjugated metabolites of IBC were detected by ESI-MS in both positive and negative ion mode. Ten phase II metabolites had been observed, including five glucuronide conjugates (M6–M10) with neutral losses of 176 Da, and five sulfated conjugates (M11–M15) with neutral losses of 80 Da. The extracted ion chromatograms of the phase II metabolites M6–M15 are shown in Fig. 3. The metabolite M6, M7and M8 gave rise to the protonated molecule [M+H]+ at m/z 501 observed as the glucuronide conjugates of IBC, indicating that these metabolites were the isomers conjugated with glucuronic acid at different positions. The mass spectrum pattern showed a product ion at m/z 325 corresponding to a neutral loss of 176 Da from a protonated molecular ion [M+H]+ at m/z 501 (seen in Fig. 4-M6–M8). The product ion at m/z 325 showed the fragment ions of m/z 269 and 205 (seen in Fig. 4-M6–M8 MS3 501 → 325), which were the two characteristic ion products of the parent IBC in the positive ion mode (Fig. 1A). The differences among M6, M7 and M8 were the position of IBC. UV spectroscopes were used to determine the substitution for these chalcone in a previous data [16–20]. The results suggested that the glucuronidation at either the 5-OH or 11-OH position could result in a UV band shift. The 11-OH group exhibited a smaller shift than that of the 5-OH, while the 13-OH position would not affect its UV absorption. The studies combined the UV spectroscopy of different substitution of isoliquiritigenin with their retention time and found that the 5-OH position, 11-OH position and 13-OH position eluted from a reverse

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column sequentially. As IBC’s structure was similar to that of isoliquiritigenin and exhibited a resemble UV spectroscopy behavior, the retention time of isobavachalcone-5-O-glucuronide (M6), isobavachalcone-11-O-glucuronide (M7) and isobavachalcone-13O-glucuronide (M8) were 15.78, 17.76 and 21.38 min, respectively. The proposed structures of M6–M8 were shown in Fig. 5. The mass spectrum pattern of M9 and M10 showed a product ion [M+H]+ at m/z 327 corresponding to a neutral loss of 176 Da from a protonated molecular ion [M+H]+ ion at m/z 503 (seen in Fig. 4M9 and M10). The product ion at m/z 327 showed the fragment ions at m/z 271 and 207, which were the two main characteristic product ions of M3 (seen in Fig. 4-M3). According to the previous discussion, M9 and M10 observed the glucuronide conjugates at 5OH and 13-OH position of M3 and eluted at 15.80 and 21.70 min, respectively. The proposed structures of M9 and M10 were shown in Fig. 5. The metabolite M11, M12, M13 and M14 gave rise to the deprotonated molecule [M−H]– at m/z 403, which were 80 Da more than that of the parent ion at m/z 323 (seen in Fig. 4-M11–M14). The product at m/z 323 had the ion series at m/z 221, 203 and 119, which were characteristic product ions of IBC and M1 in negative ion mode, indicating that M11–M14 were the sulfated conjugates of IBC or M1. The retention time of the sulfated conjugates was earlier than that of the parent drug, as a result of that, M12-14 would not be the sulfated conjugates of M1 (RT: 16.19 min), while M11 would be the glucuronide conjugates and sulfated conjugates had a similar elution behavior, and according to the previous discussion, the metabolite M12, M13 and M14 were identified as the 5-OH, 11OH and 13-OH sulfated conjugates and eluted at 17.18, 18.30 and 20.06 min, respectively. The proposed structures of M12–M14 were shown in Fig. 5. There were marked differences between M11 and the metabolite M12-14 on the product amount and physical chemical property, such as the retention time, indicating that the various structural differences between the metabolite M11 and the metabolite M12-14 existed. According to the data and analysis, the metabolite M11 could also be a sulfated conjugate of M1. M11 was tentatively elucidated as isobavachalcone-5-O-sulfate conjugate, because the sulfation was more likely to take place at 5-OH of ring A owing to less steric hindrance. The proposed structure of M11 was shown in Fig. 5. The metabolite M15 formed a deprotonated molecular ion [M−H]– at m/z 405 with a retention time of 17.13 min, which was 80 Da higher than the product ion at m/z 325 (shown in Fig. 4-M15). The product ion at m/z 325 was 2 units higher than that of IBC (m/z 323) in negative mode, indicating that M15 was the sulfated conjugate of the metabolite M3 via a reduction to IBC.

4. Conclusion This study presents the fragmentation pathways of isobavachalcone using ESI-MSn in both positive and negative ion mode and investigates the in vivo metabolites and metabolic pathways of isobavachalcone using a sensitive LC–ESI-MSn and LC–NMR method. The results indicated that five phase I metabolites of isobavachalcone were mainly biotransformed via the hydroxylation, reduction, cyclization and oxidative cleavage reactions in rats. Ten phase II metabolites were mainly identified as the glucuronide conjugates and sulfated conjugate. All these findings were reported for the first time and would contribute to a further understanding of the intermediate processes and metabolic mechanism of these prenylated chalcone compounds. Our investigation has provided much novel information on the in vivo isobavachalcone metabolism, which would help to a novel drug development, as well as a better understanding of the safety and efficacy of the drug.

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Acknowledgments This work was supported by the program of National Natural Science Foundation of China (81173121), the key program of Beijing Natural Science Foundation (KZ201110025024), the project for Academic Human Resources Development in Institutions of High Learning (PHR201007111) and the program for Beijing Laboratory for Biomedical Detection Technology and Instrument (PXM2014014226-000021). References [1] V. Kuete, L.P. Sandjo, Isobavachalcone: an overview, Chin. J. Integr. Med. 18 (2012) 543–547. [2] J.P. Dzoyem, H. Hamamoto, B. Ngameni, B.T. Ngadjui, K. Sekimizu, Antimicrobial action mechanism of flavonoids from Dorstenia species, Drug Discov. Ther. 7 (2013) 66–72. [3] V. Kuete, B. Ngameni, J.G. Tangmouo, J.M. Bolla, S. Alibert-Franco, B.T. Ngadjui, J.M. Pages, Efflux pumps are involved in the defense of Gram-negative bacteria against the natural products isobavachalcone and diospyrone, Antimicrob. Agents Chemother. 54 (2010) 1749–1752. [4] D.W. Kim, K.H. Seo, M.J. Curtis-Long, K.Y. Oh, J.W. Oh, J.K. Cho, K.H. Lee, K.H. Park, Phenolic phytochemical displaying SARS-CoV papain-like protease inhibition from the seeds of Psoralea corylifolia, J. Enzyme Inhib. Med. Chem. 29 (2014) 59–63. [5] E. Szliszka, D. Jaworska, M. Ksek, Z.P. Czuba, W. Krol, Targeting death receptor TRAIL-R2 by chalcones for TRAIL-induced apoptosis in cancer cells, Int. J. Mol. Sci. 13 (2012) 15343–15359. [6] S. Zhao, C.M. Ma, C.X. Liu, W. Wei, Y. Sun, H. Yan, Y.L. Wu, Autophagy inhibition enhances isobavachalcone-induced cell death in multiple myeloma cells, Int. J. Mol. Med. 30 (2012) 939–944. [7] H. Jing, X. Zhou, X. Dong, J. Cao, H. Zhu, J. Lou, Y. Hu, Q. He, B. Yang, Abrogation of Akt signaling by Isobavachalcone contributes to its anti-proliferative effects towards human cancer cells, Cancer Lett. 294 (2010) 167–177. [8] R. Nishimura, K. Tabata, M. Arakawa, Y. Ito, Y. Kimura, T. Akihisa, H. Nagai, A. Sakuma, H. Kohno, T. Suzuki, Isobavachalcone, a chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma, Biol. Pharm. Bull. 30 (2007) 1878–1883.

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Structural elucidation of in vivo metabolites of isobavachalcone in rat by LC-ESI-MS(n) and LC-NMR.

Isobavachalcone (IBC) is a prenylated chalcone and belongs to the class of flavonoids, which is an active component isolated from Psoralea corylifolia...
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