Identification of metabolites of FR429, a potential antitumor ellagitannin, transformed by rat intestinal bacteria in vitro, based on liquid chromatography–ion trap-time of flight mass spectrometry analysis

Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 162–167

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Identification of metabolites of FR429, a potential antitumor ellagitannin, transformed by rat intestinal bacteria in vitro, based on liquid chromatography–ion trap-time of flight mass spectrometry analysis Jie Fu a,b,1 , Jing-Yi Ma a,1 , Xian-Feng Zhang c,1 , Yan Wang a,∗ , Ru Feng a , Yang-Chao Chen d,∗∗ , Xiang-Shan Tan a , Yi-Ying Zhang a , Yu-Peng Sun a , Ying Zhou a , Chao Ma a , Chi-Yu He a , Zhen-Xiong Zhao a , Xiao-Wei Du b a

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, China Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin 150040, China c Department of Neurosurgery, First Hospital, Jilin University, Changchun 130021, China d School of Biomedical Sciences, Faculty of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong Special Administrative Region b

a r t i c l e

i n f o

Article history: Received 24 May 2012 Received in revised form 5 July 2012 Accepted 25 July 2012 Available online 4 August 2012 Keywords: FR429 Ellagitannins LC–IT-TOF/MS NMR Metabolites identification

a b s t r a c t FR429 is an ellagitannin with a potential antitumor activity, isolated and purified from Polygonum capitatum Buch.-Ham.ex D.Don, which is a traditional Miao-nationality herbal medicine in Guizhou and Yunnan of China. Our preliminary result of pharmacology study has indicated that the antitumor activity of FR429. However, the metabolism of FR429 has not been reported yet. In this study, LC–ion trap-time of flight mass spectrometry (LC–IT-TOF/MS) was used to characterize unpredictable metabolites of FR429 biotransformed by intestinal bacteria in vitro. Total thirteen metabolites were detected and characterized via comparisons of their accurate molecular masses and fragment ions of each MSn stage with those of the parent drug, and four of them were also elucidated by NMR. The results demonstrated that FR429 could be transformed by intestinal bacteria in vitro, mainly via hydrolysis and reduction reaction. This work provided a basis for the further study on the biotansformation of FR429 in vivo. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Polygonum capitatum Buch.-Ham.ex D.Don, a traditional Miaonationality herbal medicine distributed mainly in Guizhou and Yunnan in China, is commonly used in the treatment of rheumatic arthritis, pyelonephritis, cystitis urinary calculi and other diseases [1]. In our previous work, FR429, reported as Davidiin [2], has been isolated, purified and identified as ellagitannins component. We have developed a high performance liquid chromatography–diode array detection (HPLC–DAD) method for simultaneous determination of three active constituents (gallic acid, FR429 and quercitrin) present in P. capitatum. It was found that FR429 was the most abundant component in ethanol extract of this plant [3]. Ellagitannins have been demonstrated to exhibit various biological activities including antitumor, antioxidant, anti-inflammatory and antimicrobial [4]. The preliminary result of pharmacology study (personal

∗ Corresponding author. Tel.: +86 10 63165238; fax: +86 10 63165238. ∗∗ Corresponding author. Tel.: +852 2696 1100; fax: +852 2696 1100. E-mail addresses: [email protected] (Y. Wang), [email protected] (Y.-C. Chen). 1 These authors made equal contribution to this work. 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.07.028

communications) has indicated that FR429 could inhibit hepatoma cells in a concentration-dependent manner, while its effect on normal liver cells was significantly decreased. The underlying mechanism of its antitumor activity may be relevant to apoptosis induction via activation of caspase-3, as well as down-regulated expression of EZH2 in hepatoma cells. Although FR429 possesses favorable activity, its metabolic characteristic is still unknown. FR429 contains multiple hydroxyl groups and chemical bonds linking moieties, which are likely to influence in undergoing extensive metabolism and producing unpredicted metabolites. It is suggested that ion-trap and triple quadrupole were applied to perform MS/MS for analytical characterization of drug metabolites [5,6]. However, the identification of metabolites with multiple and extremely small quantities in the complex biological samples still remains a great challenge only by ion-trap or triple quadrupole [7]. HPLC–ion trap-time of flight mass spectrometry (LC–IT-TOF/MS) used in our study with high sensitivity could provide the information of accurate molecular weights and the fragments of MSn spectra. In addition, nuclear magnetic resonance (NMR) was applied to identify the major metabolites herein. In our previous work, FR429 was studied in vitro by incubation with rat liver microsomes. However, no metabolites were detected by LC–IT-TOF/MS. It was presumed that FR429 could not

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be transformed by rat liver microsomes. According to literature [8], ellagic acid and urolithins were produced from tannins by intestinal bacterial, which contained various metabolic enzymes involving different types of drug metabolism. It was inferred FR429, a kind of tannins, also could be transformed by intestinal bacteria. Most of the traditional Chinese medicines were taken by oral, and their metabolisms were inevitably associated with the intestinal bacteria. Therefore, it was significant to study the biotransformation of FR429 by intestinal flora. In this study, the metabolites of FR429 were characterized and identified by the powerful LC–IT-TOF/MS and NMR analysis. The aim of study is to search for the active or toxic substances of FR429 and to provide a basis for the further study on the biotansformation of FR429 in vivo, through the identification and characterization of metabolites of FR429, which was transformed by rat intestinal bacteria in vitro. 2. Materials and methods 2.1. Chemicals FR429 (purity >98%) was isolated and purified from an ethyl acetate extract of P. capitatum. Ellagic acid (powder, purity >97%) and gallic acid (powder, purity >98.9%) were purchased from Alfa Asear and National Institute for the control of Pharmaceutical and Biological Products (Beijing, China), respectively. Beef extract, peptone, and nutrient ager were supplied by Beijing Aoboxing Biotech Company Ltd. HPLC grade acetonitrile and methanol were obtained from J&K Scientific Ltd. Other chemicals were of analytical grade which were purchased from Sinopharm Chemical Reagent Co., Ltd. Distilled water was Wa Ha-ha purified water. 2.2. Apparatus The on line analysis experiments were carried out by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC–IT-TOF/MS) (Shimadzu, Japan) system. The metabolites were purified by Alltech HPLC system (Alltech, USA). A Varian Inova-600 MHz 1 H NMR spectrometer and 13 C NMR spectrometer (Varian, Inc., USA) were applied for the identification of metabolites. 2.3. Preparation of intestinal bacteria culture solution According to literature [9], the anaerobic medium broth for intestinal bacteria culture was prepared. Fresh feces (1 g) from SD rats were transferred into a flask containing the anaerobic medium (20 mL). After mixed thoroughly, bacteria were cultured under an anaerobic condition of a N2 atmosphere at 37 ◦ C for 30 min, and then the culture solution of intestinal bacteria was prepared. 2.4. Metabolism of FR429 in vitro and sample preparation FR429 (10 mg) was weighed and dissolved in methanol (100 ␮L). FR429 (10 ␮L) was added into the fresh intestinal bacteria solution (1 mL), while methanol (10 ␮L) was added as negative control. The cultures with FR429 were incubated at 37 ◦ C for 0, 5, 12, 24, 48 and 72 h. After terminated the reaction with 2% formic acid (200 ␮L) and methanol (2 mL), samples were vortex mixed for 30 s and then centrifuged at 10,000 × g for 15 min. The supernatant was dried under nitrogen flow at room temperature, and then the residue was dissolved in 300 ␮L of acetonitrile/0.2% formic acid (20:80) and filtered through a 0.45 ␮m microporous membrane. The sample of 15 ␮L was analyzed by LC–IT-TOF/MS.

Fig. 1. Extracted ion chromatograms (EICs) of metabolites in the incubation extract of FR429 in the intestinal bacteria in vitro.

2.5. LC–IT-TOF/MS conditions FR429 and its metabolites were separated by a column of Alltima C18 (150 mm × 4.6 mm, 5 ␮m). The mobile phase composed of eluent A (0.2% formic acid in water, v/v) and B (acetonitrile) in the gradient of 10% B at 0–10 min, 10–30% B at 10–30 min, 30–55% B at 30–40 min, 55–70% B at 40–45 min with post time of 10 min at a flow rate of 0.8 mL/min. The detection wavelength range and the column temperature were set at 220–360 nm and 25 ◦ C, respectively. For IT-TOF analysis, an ESI resource with negative mode was used. Nitrogen was used as the nebulizing gas, and Helium was used for MSn analyte fragmentations. Other parameters were as follows: CDL temperature, 200 ◦ C; heat block temperature, 200 ◦ C; detector voltage, 1.70 kV; nebulizing gas, 1.5 L/min; drying gas pressure, 110 kPa. The energy of CID was set at 50%. Mass spectra were acquired in the range of m/z 100–1000 for MS1 . The MSn data were collected in an automatic mode. 2.6. Preparation conditions of the metabolites of FR429 Separations were accomplished on an Alltima C18 column (250 mm × 10 mm, 10 ␮m) at a flow rate of 2 mL/min with the detection wavelength of 280 nm. The mobile phase consisted of 0.2% formic acid (A) (v/v) and methanol (B) in the gradient of 10% B at 0–10 min, 10–30% B at 10–20 min, 30–45% B at 20–45 min, 45–70% B at 45–50 min, 70–90% B at 50–55 min, 90% B at 55–60 min. The injection volume was 100 ␮L. The samples used to be detected by 1 H NMR and 13 C NMR (600 MHz) were re-dissolved in CD3 OD. 3. Results and discussion 3.1. Detection of metabolites of FR429 formed in vitro Compared with the control group cultured at 0 h, thirteen metabolites were detected by LC–IT-TOF/MS. Their extracted ion chromatograms (EICs) are shown in Fig. 1. 3.2. Identification of metabolites 3.2.1. Fragmentation of FR429 It was important to conduct a detailed fragmentation pathway study of FR429 in LC–IT-TOF/MS firstly, because the mass fragmentation patterns of metabolites were always similar to those of the parent drug. The data of molecular ion and fragment ions of FR429 are listed in Table 1. The [M−2H]2− ion at m/z 468.0393(2) (C41 H30 O26 ) was observed as the molecular ion. The fragment at m/z 446.0485(2) was formed from a CO2 (44 Da) loss from

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Table 1 LC/MSn data obtained FR429 and the metabolites from intestinal flora incubation in vitro. Peak no.

tR (min)

Fragment ion (MS1 )

Fragment ion (MS2 )

Fragment ion (MS3 )

Formula

Diff (ppm)

FR429

24.5

468.0393 (2)

275.0142, 294.0353 185.0238 465.0696 370.5473 (2), 275.0215 257.0106, 229.0133

C41 H30 O26

6.41

1 2

4.1 26.4

339.0299 300.9917

C7 H6 O5 C14 H6 O8

2.96 3.32

3 4-1

7.1 17.8

633.0697 785.0709

4.26 4.20

21.2

785.0934

257.0077, 229.0135, 185.0286 571.0692, 465.0696 257.0077 169.0200

C27 H22 O18 C34 H26 O22

4-2

C34 H26 O22

1.53

5-1

15.0

635.0838

331.0741

C27 H24 O18

0.63

5-2

20.7

635.0960

370.9982 (2) 300.9982 617.0792 446.0485 (2) 275.0216 169.0177 257.0145 229.0197 185.0246 300.9991 741.0953 275.0215 483.0719 300.9990 483.0705 313.0449 483.0808 465.0655 483.0779 313.0621 331.0654 183.0297 168.0060 257.0106 229.0133 241.0104 231.0303 215.0345 187.0436 215.0265 199.0423 171.0403

C27 H24 O18

1.10

C27 H24 O18

8.82

169.0137

C20 H20 O14 C8 H8 O5

0.62 2.73

229.0150 201.0079 213.0196 203.0298 187.0354 143.0517 187.0315 169.0326 143.0454

C13 H8 O7

2.55

C13 H8 O6

1.93

C13 H8 O5

1.65

9.6

635.0838

6 7

4.9 13.4

8

22.8

483.0691 367.0740 183.0300 275.0213

9

28.0

259.0259

10

29.8

243.0215

5-3

the molecular ion. The fragment pattern of m/z 370.9982(2) was formed from a CO2 (44 Da) and a galloyl group (152 Da) loss from molecular ion, and this fragment also could be produced from the fragment pattern of m/z 446.0485(2). The fragments at m/z 275.0216 and m/z 300.9982 were associated with a hexahydroxydiphenoyl (HHDP) group of FR429. The molecular ion fragmented on MS2 to produce ion at m/z 617.0792 (M-44 Da–276 Da, loss a CO2 and correlation structure of HHDP). 3.2.2. Identification and characterization of the metabolites M1, eluted at 4.1 min, had a [2M−H]− at 2 m/z 339.0299, and the MS2 spectrum had fragment at m/z 169.0177 (M-170 Da, loss of a gallic acid). M2, eluted at 26.4 min, had a [M−H]− at m/z 300.9917 that fragmented on MS2 to produce ions at m/z 229.0197 (M-44 Da–28 Da, loss of a CO2 and a CO) and m/z 257.0145 (M-44 Da, loss of a CO2 ). Compared with the reference substance, M1 and M2 were identified as gallic acid and ellagic acid, respectively. M3, M4-1 and M4-2 were identified based on the mass spectra data and NMR data. M3, eluted at 7.1 min, showed the quasimolecular ion [M−H]− at m/z 633.0697. It was suggested that the mass of the quasi-molecular ion [M−H]− in negative-ion mode was 304 Da (two galloyl groups) less than that of the parent drug. The MS2 spectrum had fragment at m/z 300.9991, associated with HHDP group. Therefore M3 kept the HHDP group, and formed by

185.0238

313.0621

losing two galloyl groups from FR429. The structure of M3 was confirmed by 1 H NMR and LC–IT-TOF/MS. Compared with the 1 H NMR data of FR429, the chemical shifts of 2-H and 4-H of glucose were quite different from that of FR429. Therefore M3 was identified as Helioscopinin B. In the same way, M4-1 and M4-2 were identified. The retention time of M4-1 and M4-2 were 17.8 min and 21.2 min, respectively. The [M−H]– ions of both compounds were observed at m/z 785.0933. It was suggested that the mass of the quasi-molecular ion [M−H]− in negative-ion mode was 152 Da (a galloyl group) less than that of the parent drug. It showed that these compounds were formed from a galloyl group loss from FR429 by hydrolysis reaction. However, M4-1 and M4-2 lost different fragments in MS2 mode, which indicated that they were isomers. The loss of a CO2 from the molecular ion of M4-1 formed a fragment at m/z 741.0953, which further lost 276 Da and produced a fragment at 465.0696 on MS3 . The lost fragment of 276 Da was associated with HHDP group. As for M4-2, the fragment at m/z 483.0719 was produced by lossing 302 Da (a HHDP group) from the molecular ion of M4-2. This suggested that both of them kept the HHDP group. According to the 1 H NMR data of FR429 and the isomers, it could be conjectured that at which position of FR429 the galloyl group was lost to obtain metabolite M4-1 and M4-2. Shown from the results, the chemical shift of 4-H of glucose of M4-1 moved to lower field compared with that of FR429, while the chemical shift of 2-H of

Table 2 1 H NMR data for glucose of FR429, M4-1, M4-2 and M3 in CD3 OD. Position

FR429

M4-1

M4-2

M3

1-H 2-H 3-H 4-H 5-H 6-H

6.06 (1H, d, J = 3Hz) 5.45 (1H, dd, J1 = 6.6 Hz, J2 = 2.4Hz) 5.75 (1H, t, J = 6.6Hz) 5.09 (1H, dd, J1 = 6.6 Hz, J2 = 2.4Hz) 4.53 (1H, m) 4.36 (1H, dd, J1 = 5.4 Hz, J2 = 12Hz)

5.91 (1H, d, J = 2.4Hz) 5.19 (1H, dd, J1 = 2.4 Hz, J2 = 5.4Hz) 5.34 (1H, t, J = 5.4Hz) 3.91 (1H, dd, J1 = 5.4 Hz, J2 = 6Hz) 4.24 (1H, m) 4.09 (1H, dd, J1 = 5.4 Hz, J2 = 12Hz)

5.84 (1H, d, J = 3Hz) 3.99 (1H, d, J = 6Hz) 5.45 (1H, t, J = 6.6Hz) 4.80 (overlap with solvent peak) 4.35 (1H, m) 4.23 (1H, dd, J1 = 5.4 Hz, J2 = 12Hz)

5.78 (1H, d, J = 3Hz) 3.84 (1H, dd, J1 = 2.4 Hz, J2 = 5.4Hz) 5.17 (1H, t, J = 5.4Hz) 3.75 (1H, dd, J1 = 4.2 Hz, J2 = 5.4Hz) 4.19 (1H, m) 4.00 (1H, dd, J1 = 5.4 Hz, J2 = 11.4Hz)

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Fig. 2. Metabolic pathway of FR429 in intestinal flora incubation in vitro.

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Table 3 NMR data for compound M8 in CD3 OD. Position 1 2 3 4 5 6 7 1 2 3 4 5 6

13

C shift (ppm)

118.5 144.0 141.9 146.4 108.1 112.4 163.8 112.9 141.0 133.3 146.8 112.4 119.1

1

H shift (ppm)

7.32 (1H, S)

6.70 (1H, d, J = 9.0 Hz) 8.39 (1H, d, J = 9.0 Hz)

glucose of M4-2 was quite different from that of FR429. On the basis of the mass spectra data and the 1 H NMR data, it could be conjectured that M4-1 and M4-2 were obtained by losing a galloyl group at C-4 position and C-2 position of FR429, respectively. M4-1 and M4-2 were new compounds. M4-1 and M4-2 were identified as d-glucopyranose, cyclic 1, 6-(4, 4 , 5, 5 , 6, 6 -hexahydroxy [1,1 biphenyl]-2, 2 -dicarboxylate) 2, 3-dis (3, 4, 5-trihydroxybenzoate) and d-glucopyranose, cyclic 1, 6-(4, 4 , 5, 5 , 6, 6 -hexahydroxy [1,1 biphenyl]-2, 2 -dicarboxylate) 3, 4-dis (3, 4, 5-trihydroxybenzoate), respectively. 1 H NMR data of glucose of FR429, M3, M4-1 and M4-2 are summarized in Table 2. M5-1, M5-2 and M5-3 were isomers. All of them had a [M−H]− ion at m/z 635.0853. The molecular weights of them were 302 Da less than that of FR429, which suggested that they were obtained by losing two galloyl groups from FR429. The loss of a galloyl group from the molecular ions formed a fragment at m/z 483.0789. The fragment at m/z 465.0696 was produced from a gallic acid loss from the molecular ions. There were not any fragments associated with HHDP group. So these compounds without HHDP group were formed by losing two galloyl groups from FR429. M6, eluted at 4.9 min, had a [M−H]− ion at m/z 483.0691. It was suggested that the mass of the quasi-molecular ion [M−H]− in negative-ion mode was 152 Da (a galloyl groups) less than that of M5. The fragment at m/z 331.0654 was formed from a galloyl group loss from the molecular ion of M6. There was not any fragment associated with HHDP group, therefore M6 without HHDP group was formed from three galloyl groups loss from FR429. M7, eluted at 13.4 min, had a [2M−H]− ion at m/z 367.0740 and a [M−H]− at 183.0300. The fragment at 168.0060, associated with gallic acid, was formed from a methyl group (15 Da) loss from the molecular ion. Compared with the molecular ion of M1 (gallic acid), the structure of M7 was speculated. It was produced by gallic acid methylation. M8, eluted at 22.8 min, had a [M−H]− ion at m/z 275.0213, and the MS2 spectrum had fragments at m/z 229.0133 and m/z 257.0106. The fragments were similar with the fragments of M2 (ellagic acid). It was suggested that the mass of the quasi-molecular ion [M−H]− of M8 in negative-ion mode is 26 Da (a CO) less than that of M2 (ellagic acid). The structure of M8 was confirmed by 1 H NMR and LCMS-IT-TOF. The 1 H NMR and 13 C NMR data are listed in Table 3. According to literature [10], M8 was identified as 3, 4, 8, 9, 10-pentahydroxydibenzo-[b,d]pyran-6-one. It was produced from M2 by reduction reaction. M9, eluted at 28.0 min, had a [M−H]– ion at m/z 259.0259. It was suggested that the mass of the quasi-molecular ion [M−H]− in negative-ion mode is 16 Da (a hydroxyl group) less than that of M8. The fragments at m/z 241.0104 and m/z 231.0303 are produced from a H2 O and a CO loss from the molecular ion of M9, respectively. M10, eluted at 29.8 min, showed the molecular ion [M−H]− at m/z 243.0215. It was suggested that the mass of the quasi-molecular ion

[M−H]− in negative-ion mode is 16 Da (a hydroxyl group) less than that of M9. The MS2 spectrum had fragments at m/z 215.0265 (M28 Da, loss of a CO) and 199.0423 (M-44 Da, loss of a CO2 ). According to literature [11], both compounds had been identified as urolithin D and urolithin C, which were produced from M2 (ellagic acid) by reduction reaction. In conclusion, the mass spectra data of the metabolites are shown in Table 1. Thirteen metabolites were detected and identified, including two new compounds, M4-1 and M4-2. M1, M2, M3, M4-1, M4-2, M5-1, M5-2, M5-3 and M6 were produced by hydrolysis reaction of the parent drug. M7 was formed from M1 by reduction reaction. M8, M9 and M10 were produced from M2 by reduction reaction. 3.2.3. Proposed metabolic pathway of FR429 There were no metabolites detected in cultured process before 5 h. Moreover, M1 (gallic acid), M2 (ellagic acid) and M8 were detected in cultured process during 12–72 h, and others were detected in cultured process during 12–48 h. The proposed metabolic pathway of FR429 is shown in Fig. 2. According to the structures of metabolites, FR429 mainly underwent hydrolysis by intestinal flora, and the galloyl groups were easily removed from C-2 and/or C-4 of glucose. According to Ref. [12], the ellagic acid was produced from ellagitannin via hydrolysis reaction and the formation of intestinal lactone spontaneously under the pH level of small intestine, and subsequently was metabolized into urolithins with intestinal bacteria. Therefore, M8, M9 (urolithin D) and M10 (urolinthin C) were all the secondary metabolites of M2 (ellagic acid). M1 was the basic structural unit of FR429 and other metabolites. Thus, M1 had a large content and could be detected at each time point. 4. Conclusion In the present work, a high performance liquid chromatography-ion trap-time of flight mass spectrometry (LC–IT-TOF/MS) (Shimadzu, Japan) system was used to detect and identify the metabolites of FR429. This system with high sensitivity and accuracy can be used to analyze unknown trace metabolites. Accurate molecular weights and the fragments of MSn spectra of FR429 and metabolites could be obtained. The summarized mass fragmentation patterns of metabolites were similar with the way of the parent drug. Thirteen metabolites of FR429 were identified and characterized, including nine hydrolysis products and four reduction products. It is demonstrated that FR429 could be metabolized by rat intestinal bacteria in vitro, mainly via hydrolysis and reduction. The polarity of hydrolysis products was larger than that of the parent drug, and the reduction products had smaller polarity. It provided a basis for future study on the transformation of FR429 in vivo. Acknowledgements The project was supported by National Natural Science Foundation of China (No. 81072611), National Science and Technology Special Projects (2012ZX09301-002-001, 2012ZX09301-002-006) and the Special Fund of Chinese Central Government for Basic Scientific Research Operations (No. 2012CHX08). This study was also supported by the analytical center of the Peking branch of Japanese Shimadzu Corporation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2012.07.028.

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