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orbitrap mass spectrometer
Journal of Chromatography B, 1025 (2016) 7–15
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Biotransformation and metabolic profile of buddleoside with human intestinal microflora by ultrahigh-performance liquid chromatography coupled to hybrid linear ion trap/orbitrap mass spectrometer Jin-hua Tao a,b , Jin-ao Duan a,∗ , Shu Jiang a , Yi-yun Qian a , Da-wei Qian a a Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, PR China b School of Pharmacy, Nantong University, Nantong 226001, PR China
a r t i c l e
i n f o
Article history: Received 19 January 2016 Received in revised form 26 April 2016 Accepted 30 April 2016 Available online 3 May 2016 Keywords: Buddleoside UPLC-LTQ/Orbitrap/MS/MS Intestinal microflora Enzyme activity
a b s t r a c t Buddleoside (also known as linarin) as the major flavonoid in Chrysanthemum morifolium Ramat., has been reported to possess a wide range of pharmacological activities. The human intestinal microbiota might have an important impact on drug metabolism and ultimately on the drug oral bioavailability. However, the interaction of the buddleoside with human intestinal bacteria remains unknown. In this study, the conversion of buddleoside by different bacteria from human feces was firstly investigated. A reliable, sensitive and rapid analytical method, ultra performance liquid chromatography was established and successfully applied to investigate the metabolites and metabolic profile of buddleoside by human intestinal bacteria. Among the isolated bacteria, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the accurate mass data and the characteristic MSn product ions, the parent and six metabolites were detected and tentatively identified compared with blank samples. The metabolites were produced by four main metabolic pathways including deglycosylation, acetylation, methylation and hydroxylation. Buddleoside could be firstly converted to its aglycon acacetin (M2) by the majority of the isolated intestinal bacteria. Subsequently, M2 was further metabolize to its methylated (M3), acetylated (M4), hydroxylated (M5) and hydrogenated product (M6). However, acacetin-7-glucosid (M1) was obtained only from the minor bacterial samples like Bacillus sp. 46. To further explain the metabolism of buddleoside, the d-glucosidase and ␣-l-rhamnosidase activities of four strains were analyzed. Bacillus sp. 46 could only produce ␣-l-rhamnosidase, while the other three strains showed two kinds of enzyme activities. Furthermore, the activities of ␣-l-rhamnosidase and -d-glucosidase reached the highest level at 12–18 h and 10–12 h, respectively. The metabolic routes and metabolites of buddleoside produced by human intestinal microflora were firstly reported in this paper. The results will be very helpful for the further investigation of the pharmacokinetic research of buddleoside and to unravel how it works in vivo. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chrysanthemum indicum, a traditional herb medicine, belongs to Compositae family. The dried flowers of C. indicum Ramat. have been used as a folk medicine for thousands of years in China. They are reported to possess the ability of clearing heat and detoxification, and in clinic traditionally used for headache, influenza, hepatic and eye diseases [1–5]. Meanwhile, Chrysanthemum has
∗ Corresponding author at: Jiangsu Key laboratory for TCM formulae Research, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, PR China. E-mail address: [email protected] (J.-a. Duan). http://dx.doi.org/10.1016/j.jchromb.2016.04.055 1570-0232/© 2016 Elsevier B.V. All rights reserved.
been widely used as a popular beverage and mixed spices in Korea since ancient times [6]. Its chemical constituents include flavonoids, volatile oil, lactone and organic acids. Buddleoside as the major flavonoid in C. indicum Ramat. has been reported to possess a wide range of pharmacological activities such as anti-hypertensive, anti-oxidative, anti-inflammatory, and antimicrobial effects [7]. It is well known that most traditional Chinese medicines (TCMs) are administered orally and ingredients of these medicines inevitably lead to contact with intestinal microflora in the gastrointestinal (GI) tract and subsequently be transformed by intestinal bacteria before being absorbed into the blood. The trillions of bacteria housed in our GI tract serve an important purpose in
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host homeostasis. Intestinal bacteria have a variety of enzymes, e.g. hydrolases, oxidoreductases, lyases and transferase. Anaerobic environment in the intestine lead to the reduction reaction to proceed smoothly, and then nitro, carbonyl, azocompounds are easily reduced. Cleaving enzyme can make the ring, C C and C N cleavage. Transferase can catalyze the methylation, acetylation, sulfation reaction. Glycosides medicine metabolism mainly involve hydrolytic enzymes, such as -glucosidase, ␣-rhamnosidase, glucuronidase and ␣-galactosidase, which release aglycones of flavonoids from their glycosides and glucuronides [8–14]. High-resolution mass spectrometry (HRMS), which allows accurate mass measurements at high resolving power, has been used for metabolite identification, contaminant analyses, and doping control. HRMS demonstrates some obvious advantages including the collection of full-scan spectra providing abundant information on sample composition, retrospective data analyses, and identification of potential metabolites [15–27]. As one of the major HRMS-based techniques, UPLC coupled with hybrid mass spectrometry, which combines the linear trap quadrupole (LTQ) and Orbitrap MS/MS mass analyser, has been widely used in qualitative and quantitative analysis of drugs and drug metabolism [18–21]. In this paper, we attempted to isolate different intestinal bacteria from human feces and carry out research on their abilities and characteristics in the metabolism of buddleoside. To further clarify the metabolic profile of buddleoside, ultra performance liquid chromatography with linear ion trap-orbitrap mass spectrometer (UPLC-LTQ/Orbitrap/MS/MS) combined with offline data processing methods of the software Xcalibur 2.1 system which included Mass FrontierTM 7.0 and MetWorks1.3 SP3 (Thermo Fisher, San Jose, CA, USA) was applied. The results will provide helpful information for further in vivo metabolism and active mechanism research on the active components. 2. Experimental 2.1. Chemicals and reagents Buddleoside standard substance (purity 99.56%) was purchased from Shanghai Winherb Medicals & T Development Co. Ltd (Shanghai, China). The UPLC-grade acetonitrile was purchased from TEDIA Company Inc. (Fairfield, USA), formic acid was obtained from Merck KGaA (Darmstadt, Germany), ultra-pure water was purified by an EPED super purification system (Nanjing, China). Other reagents were of analytical grade. AnaeroPack Rectangular Jars were purchased from Mitsubishi Gas Chemical Company Inc. (Japan). General anaerobic medium (GAM) was purchased from Shanghai Kayon Biological Technology Co. Ltd. (Shanghai, China). p-Nitrophenyl -d-glucoside (PNP--d-glu) was provided by Baomanbio Bioscience & Technology Co. (Shanghai, China). p-Nitrophenyl ␣-l-rhamnoside (PNP ␣-l-rham) was provided by Solarbio Bioscience & Technology Co. (Shanghai, China) and p-nitrophenol (PNP) was provided by Jianglai Bioscience & Technology Co. (Shanghai, China). 2.2. Characterization and isolation of the human intestinal bacteria Four grams of fresh human fecal sample from a healthy female volunteer who has not taken any antibiotics in 3 months were mixed with 20 mL of sterile physiological saline and then homogenized and suspended for 3 min. After centrifugation for 10 min at 2000g, the suspension was obtained as human intestinal bacterial mixture which was serially diluted in water and aliquot was plated on GAM agar plates. The plates were incubated in anaerobic condition at 37 ◦ C for 72 h. About 100 different bacterial colonies were isolated.
2.3. Incubation of buddleoside with gut bacteria and preparation of sample solutions for UPLC-LTQ/Orbitrap/MS/MS The buddleoside standard solution was prepared by dissolving accurately weighed buddleoside in methanol to the final concentration of 5.0 mg/mL and then stored in a refrigeration at 4 ◦ C before analysis. Aliquots of 0.1 mL of the approximately one hundred different bacterial colonies were inoculated into 0.9 mL of GAM broth containing 0.1 mM buddleoside, and the media were incubated under anaerobic conditions at 37 ◦ C for 72 h. After the incubation, the incubated solution was extracted with ethyl acetate three times, and then the ethyl acetate layer was dried under vacuum. The residues were dissolved in 0.3 mL methanol, centrifuged at 13,000g for 10 min, soluble fraction was analyzed by UPLC-LTQ/Orbitrap/MS/MS. Meanwhile, in order to avoid extraction artifact, we added a blank control experiment. GAM broth (1.0 mL) containing 0.1 mM buddleoside was incubated under the same conditions and the sample preparation as noted above.
2.4. UPLC-LTQ/Orbitrap/MS conditions Chromatographic separations were performed using a UPLC system consisting of a quaternary Accela 600 pump and Accela Autosampler (Thermo Fisher Scientific). An analytical Hypersil gold C18-column (50 × 2.1 mm, 1.9 m particle size; Thermo Fisher Scientific) was used for separations. The mobile phase consisted of (A) acetonitrile and (B) water containing 0.1% formic acid using a gradient elution of 10–35% A at 0–4 min, 4–6 min, held at 35%, 35–60% A at 6–13 min, 60–90% A at 13–16 min, held at 90% A for 1 min and then an immediate reduction to 10% A at 18.2 min; 10% A for equilibration of the column. The flow rate was 200 L/min, injection volume was 2 L. The UPLC system was coupled to a linear ion trap and Orbitrap hybrid mass spectrometer (LTQ/Orbitrap) equipped with a heatedelectrospray ionization probe (HESI-II; Thermo Fisher Scientific). The mass spectrometer was operated in negative mode. Parameters of the ion source were as follows: source voltage 5 kV, capillary voltage −40 V, tube lens voltage −80 V, capillary temperature 275 ◦ C, sheath and auxiliary gas flow (N2) 42 and 11 (arbitrary units). The MS spectra were acquired by full-range acquisition covering 100–1000 m/z. A data-dependant scan was performed for the fragmentation study by deploying collision induced dissociation (CID). The normalized collision energy of the CID was set at 35 eV. Acquisition and data processing were performed with Xcalibur v.2.0 software (Thermo Fisher, San Jose, CA, USA) and Qualbrowser.
2.5. 16S rRNA gene sequencing and phylogenetic analysis Genomic DNA was extracted from the isolate using a Karroten Genomic DNA Purification kit (Karroten, China). The 16S rRNA gene sequence of the strain was amplified by employing two universal primes, 16S-1F (5 -AGA GTT TGA TCC TGG CTC AG-3 ), 16S-1R (5 -AGA AAG GAG GTG ATC C-3 ). A polymerase chain reaction (PCR) program was used for amplification as follows: 94 ◦ C for 1 min, followed by 29 cycles consisting of 94 ◦ C for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 2 min, and a single final extension step consisting of 72 ◦ C for 10 min. The PCR product was purified from the agarose gel using a Karroten gel purification kit. Sequencing of the 16S rDNA fragments was performed by Majorbio (Shanghai, China). The homology search of the16S rRNA gene sequence was performed by EzBioCloud. A phylogenetic tree was constructed using the neighbor-joining method using the CLUSTAL W program and MEGA (ver 5.0) software.
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Fig. 1. UPLC chromatogram of metabolites of buddleoside by human intestinal bacteria.
Fig. 2. Possible fragmentation and metabolic pathways of buddleoside by the human intestinal bacteria: (a) Fragmentation pathway of Parent, (b) Proposed metabolic pathways of buddleoside.
2.6. Assay for glucosidase and rhamnosidase activities The strains were cultured for 24 h in 1 mL of GAM at 37 ◦ C, then the suspensions were centrifuged at 5000g for 15 min. The supernatant was diluted five times in phosphate buffer (0.2 M, pH 7.3). The suspensions were incubated with 0.25 mM PNP--d-glu and 0.25 mM PNP-␣-l-rham at 37 ◦ C, respectively. Aliquots of the reactive solutions were collected at different time and were diluted with water (1:10) in a 96-well microplate. The absorbance of each well was measured with a microplate reader at 405 nm. The absorbance of a series of different concentrations of PNP was used to calculate the enzyme activity.
3. Results and discussion 3.1. Method optimization To improve the sensitivity and obtain appropriate retention times and symmetric peak shapes for the metabolites, different
analytical mobile phases such as acetonitrile, methanol, water and 0.1% formic acid were tested. Comparing with other solvents, acetonitrile showed more powerful separation ability, shorter retention time and lower column pressure. Additionally, 0.1% formic acid added in the water phase could help show a higher response and better peak sensitivity. Therefore, the optimal solvent system consisting of acetonitrile and 0.1% formic acid aqueous solution, which remarkably enhanced the efficiency of ionization and gave satisfactory sensitivity, was ultimately chosen as mobile phase using a gradient elution. Electrospray ionization was applied to obtain good sensitivity and fragmentation. For the heated electrospray ionization probe (HESI) conditions, both negative and positive ion detection modes were tested. Due to the more obvious fragment characteristics and higher sensitivity for the investigated compounds with the HESI negative ionization mode, the negative mode was selected and applied for analysis. Furthermore, to acquire maximum sensitivity for the analytes, parameters of the ion source such as desolvation, source voltage, capillary voltage, capillary temperature and
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desolvation gas flow were further optimized. Consequently, the optimum conditions were decided as follows: source voltage 5 kV, capillary voltage −40 V, tube lens voltage −80 V, capillary temperature 275 ◦ C, sheath and auxiliary gas flow (N2) 42 and 11 (arbitrary units). The MS spectra were acquired by full-range acquisition covering 100–1000 m/z. A data-dependant scan was performed for the fragmentation study by deploying collision induced dissociation (CID). The normalized collision energy of the CID was set at 35 eV.
3.2. Characterization of the human intestinal bacteria According to Bergey’s Manual of Systematic Bacteriology, colony morphology, micromorphology, and comparative 16S rRNA gene sequence analysis, 68 Escherichia, 16 Enterococcus, and 16 Bacillus strains were identified in this study. The data analysis of the samples about the bacterial bioconversion ability indicated that all of the isolated bacterial colonies had metabolic capacity on buddleoside. Since four bacteria showed more powerful conversion
Fig. 3. Representative MS/MS spectrum: (a) M0 (m/z 591), (b) M1 (m/z 445), (c) M2 (m/z 283), (d) M3 (m/z 297), (e) M4 (m/z 325), (f) M5 (m/z 299), (g) M6 (m/z 285).
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Fig. 3. (Continued)
capability than others, they were selected as the typical bacteria to analyze the metabolic profiles. Four bacteria were identified as Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus
sp. 46 by the 16S rRNA gene sequencing. They have been deposited in GenBank under accession No. KC819112, KC819111, KC417329 and KC257405, respectively.
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Fig. 3. (Continued)
3.3. Metabolic profile of buddleoside by different human intestinal bacteria
spectrum of M2. From these data analyses, M2 could be identified as aglycone acacetin in accordance with the authentic standard.
An overview of the metabolites was obtained after optimizing the UPLC and MS conditions. Using negative ion electrospray tandem mass spectrometry and the Mass FrontierTM 7.0 and MetWorks1.3 program, the parent and six metabolites of buddleoside (Table 1 and Fig. 1) were detected in the different bacterial samples compared with blank samples. The six metabolites were likely to be acacetin-7-glucoside (M1), acacetin (M2), methylated acacetin (M3), acetylated acacetin (M4), hydroxylated acacetin (M5) and hydrogenated acacetin (M6). Additionally, the proposed metabolic pathways of buddleoside by the human intestinal bacteria were inferred (Fig. 2a and b).
3.4.4. Metabolite M3 Metabolite M3 in the UPLC chromatogram having a retention time of 16.83 min and exhibited a [M−H]− ion at m/z 297.1101 (Fig. 3d). It was 14 Da higher than that of M2, indicating the chemical structure of M3 could be the methylated product of M2. According to the fragmentation pathway (Fig. 2a), M3 lost CH2 to form m/z 283.1657. Then, m/z 269.2115 and 255.1964 were obtained from m/z 283.1657 by the loss of CH3 and CO, respectively. The m/z 269.2115 was further fragmented to be m/z 241.2169 by the loss of CO. The m/z 131.0602 was also obtained by RDA cleavage from m/z 283.1657. Thus, M3 was identified as the methylated acacetin.
3.4. Identification of the buddleoside metabolites 3.4.1. Parent compound Buddleoside (M0) was identified by comparing the UPLC retention time (5.26 min), accurate MS at m/z 591.2471 [M−H]− and MS/MS spectra with the authentic standard. As illustrated in MS/MS spectrum (Fig. 3a), the precursor ion m/z 591.2471 afforded the buddleoside aglycone (acacetin) product ion at m/z 283.1657 by the loss of rhamnose and glucose moiety. The characteristic product ion of m/z 268.0352 was the loss of CH3 from m/z 283.1657. 3.4.2. Metabolite M1 Metabolite M1 was observed in the UPLC chromatogram with a retention time at 10.20 min. MS analyses showed molecular ion at m/z 445.2161 [M−H]− , which was 146 Da lower than that of M0 buddleoside, indicating that it might be generated by the loss of rhamnose (Fig. 3b). M1 was degraded by the intestinal bacterium Bacillus sp. 46. which indicated that it could produce ␣-l-rhamnosidase but did not produce -d-glucosidase. 3.4.3. Metabolite M2 Metabolite M2 was detected as deprotonated molecular ion [M−H]− at m/z 283.1657 with retention time of 5.12 min (Fig. 3c), generated from the deglycosylation of buddleoside by the loss of m/z 308.0814. The other characteristic fragments were m/z 268.0320, 255.0295 and 239.0512 corresponding to the loss of CH3 , CO, CO2 from m/z 283.1657, respectively (Fig. 2a). Its product ion m/z 131.0602 (RDA) was also appeared in the product ion mass
3.4.5. Metabolite M4 The metabolite M4 showed a UPLC profile with a retention time of 2.40 min and an MS spectrum which gave an [M−H]− ion at m/z 325.1187 (Fig. 3e) which was 42 Da (C2 H2 O) higher than that of M2. The m/z 255.0310 was obtained from m/z 283.1657 by the loss of CO. The m/z 131.0602 was also obtained by RDA cleavage from m/z 283.1657. Thus, M4 was identified as the acetylated acacetin. 3.4.6. Metabolite M5, M6 Metabolite M5, M6 with a retention time of 5.61 min and 5.79 min produced a deprotonated molecular ion at m/z 299.0641 and m/z 285.2401, respectively (Fig. 3f and g). The fragment ion of M5 was 16 Da higher than that of M2. The m/z 255.2114, 271.0605 were obtained by the loss of CO from m/z 283.1657 and 299.0641, respectively. The fragment ion of M6 was 2 Da higher than that of M2. The m/z 285.2401 was further fragmented to be m/z 241.1552 by the loss of CO2 . The m/z 151.1118 was also obtained by RDA cleavage from m/z 285.2401. Thus, M5 and M6 were identified as the hydroxylated acacetin and reduction product, respectively. 3.5. ˛-l-rhamnosidase and ˇ-d-glucosidase activities The analysis for corresponding enzyme activity was performed using PNP--d-glu and PNP-␣-l-rham as substrates. Fig. 4a and b showed the release of PNP after incubation of PNP--d-glu and PNP-␣-l-rham with the four stains. As was reflected, Bacillus sp. 46 could produce ␣-l-rhamnosidase but did not produce -d-glucosidase or -d-glucosidase activity was so low that the
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Table 1 Buddleoside and its metabolites identified in different human intestinal bacterial samples using UPLC-LTQ/Orbitrap/MS. Peak
tR (min)
Metabolite Name
Formula
Mass Difference
m/z Found
mDa
PPm
M0 M1 M2 M3 M4 M5 M6
5.26 10.20 5.12 16.83 2.40 5.61 5.79
buddleoside acacetin-7-glucoside acacetin Methylated Methylationdaidzein Acetylated Hydroxylated Hydrogenated
C28 H32 O14 C22 H22 O10 C16 H11 O5 C17 H13 O5 C18 H13 O6 C16 H11 O6 C16 H13 O5
−0.0010 −146.0310 −162.0504 14.0010 41.9530 16.0001 2.0744
591.2471 445.2161 283.1657 297.1101 325.1187 299.0641 285.2401
−1.0 1.3 −1.1 0.4 1.0 −0.9 0.8
−2.5 −2.2 −4.4 1.5 2.2 −2.0 1.0
Fig. 4. Buddleoside deglycosylated pathway by human intestinal bacteria.
amounts of PNP were not traced in 24 h. The other three strains all produced ␣-l-rhamnosidase and -d-glucosidase. Furthermore, the amounts of PNP released from PNP-glu and PNP-rham reached the highest level at about 10–12 h and 12–18 h, respectively. The results also showed that Escherichia sp. 4 possessed higher enzyme activities than Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46. Based on these results, the deglycosylated route of buddleoside was proposed: buddleoside was metabolized to be acacetin-7glucoside by ␣-l-rhamnosidase produced from these bacteria, and the latter was further converted to acacetin by -d-glucosidase. The human intestinal bacteria, classified as resident and transient flora, comprised a diversity of bacterial species and had excellent enzymatic systems contributing to their enormous catalytic and hydrolytic potential [22]. The factors influencing the natural products absorption in the gastrointestinal tract included deglycosylation before absorption, conjugation in the small intestine through glucuronidation, sulfation or methylation, etc., and metabolism and degradation in the colon to smaller phenolic molecules by intestinal bacteria. The forms in which they circulated in vivo might influence their polarity, their localization and bioactivities [23]. Acacetin, deglycosylated from buddleoside, as the major metabolite, was present in many plants such as Robinia pseudoacacia (black locust) and Turnera diffusa (damiana).
Previous studies have shown that acacetin possessed anticancer activity against many types of cancer cells including T-cell leukemia, lung, prostate, and breast cancer [24–26]. Recently, acacetin was also found that it could inhibit the expression of E-selectin in part through regulation of the p38 MAPK signaling pathway and activation of NF-kB and then attenuate monocyteendothelial interactions in vitro [27]. Deglycosylation was an important step in the absorption and metabolism of glycosides [28]. The absorption rate of aglycones was higher than that of their corresponding glycosides due to their higher lipophilicities and smaller molecular sizes [29]. Hence, the human intestinal bacteria play an important role in the pharmacological effects of buddleoside in vivo.
4. Conclusion In this paper, the reliable and powerful analytical method using UPLC-LTQ/Orbitrap/MS/MS was successfully established for separation and identification of the multiple metabolites of buddleoside by human intestinal bacteria. According to the retention behaviors, changes of molecular weights, and MS/MS fragment patterns, the prototype and its six metabolites were tentatively identified. These metabolites indicated that buddleoside could be converted by four major metabolic routes including deglycosylation, acetylation,
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Fig. 5. Enzyme activities: (a) Time-dependent release of PNP after incubation of PNP-ˇ-d-glu (0.25 mM) with four strains (n = 5, mean ± SD). (b) Time-dependent release of PNP after incubation of PNP-␣-l-rham (0.25 mM)with four strains (n = 5, mean ± SD).
methylation and hydroxylation. It has been reported that methylation of flavonoids might result not only in a dramatic increase in their hepatic metabolic stability but also in great improvement of their intestinal absorption, both of which could greatly increase their oral bioavailability [30]. This indicated that our findings, such as methylated, hydroxylated, deoxygenated and acetylated metabolites of buddleoside, might lead to a higher oral absorption which contributed to improve the bioactivity of buddleoside in vivo. Meanwhile, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the deglycosylated products of four strains and their enzyme activities, the different bacteria could convert buddleoside to different metabolites due to their different enzyme system. Moreover, we could conclude that buddleoside was firstly degraded to acacetin-7-glucoside by losing rhamnose, then the latter was further metabolized to acacetin by losing glucose (Fig. 5). Acknowledgements This work was financially supported by Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (No.ZDXM-1-10), Construction Project for Jiangsu Key Laboratory for High Technology of TCM Formulae Research (BM2010576). References [1] H.D. Jiang, J. Cai, J.H. Xu, X.M. Zhou, Q. Xia, Endotheliumdependent and direct relaxation induced by ethyl acetate extract from Flos Chrysanthemi in rat thoracic aorta, J. Ethnopharmacol. 101 (2005) 221–226. [2] C.K. Lii, Y.P. Lei, H.T. Yao, Y.S. Hsieh, C.W. Tsai, K.L. Liu, H.W. Chen, Chrysanthemum morifolium Ramat. reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells, J. Ethnopharmacol. 128 (2010) 213–220. [3] T. Zhang, D. Chen, Anticomplementary principles of a Chinese multi herb remedy for the treatment and prevention of SARS, J. Ethnopharmacol. 117 (2008) 351–361.
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[24] S.T. Chien, S.S. Lin, C.K. Wang, Y.B. Lee, K.S. Chen, Y. Fong, Y.W. Shih, Acacetin inhibits the invasion and migration of human non-small cell lung cancer A549 cells by suppressing the p38 alpha MAPK signaling pathway, Mol. Cell. Biochem. 350 (2011) 135–148. [25] K. Watanabe, S. Kanno, A. Tomizawa, S. Yomogida, M. Ishikawa, Acacetin induces apoptosis in human T cell leukemia Jurkat cells via activation of a caspase cascade, Oncol. Rep. 27 (2012) 204–209. [26] T.A. Bhat, D. Nambiar, D. Tailor, A. Pal, R. Agarwal, R.P. Singh, Acacetin inhibits in vitro and in vivo angiogenesis and downregulates stat signaling and VEGF expression, Cancer Prev. Res. 6 (2013) 1128–1139. [27] N. Tanigawa, M. Hagiwara, H. Tada, T. Komatsu, S. Sugiura, K. Kobayashi, Y. Kato, N. Ishida, K. Nishida, M. Ninomiya, M. Koketsu, K. Matsushita, Acacetin inhibits expression of E-selectin on endothelial cells through regulation of the MAP kinase signaling pathway and activation of NF-kB, Immunopharmacol. Immunotoxicol. 35 (2013) 471–477. [28] Z. Chen, S. Zheng, L. Li, H. Jiang, Metabolism of flavonoids in human: a comprehensive review, Curr. Drug Metab. 15 (2014) 48–61. [29] W. Wiczkowski, J. Romaszko, A. Bucinski, D. Szawara-Nowak, J. Honke, H. Zielinski, M.K. Piskula, Quercetin from shallots (Alliumcepa L. var aggregatum) is more bioavailable than its glucosides, J. Nutr. 138 (2008) 885–888. [30] X. Wen, T. Walle, Methylated flavonoids have greatly improved intestinal absorption and metabolic stability, Drug Metab. Dispos. 34 (2006) 1786–1792.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Biotransformation and metabolic profile of buddleoside with human intestinal microflora by ultrahigh-performance liquid chromatography coupled to hybrid linear ion trap/orbitrap mass spectrometer Jin-hua Tao a,b , Jin-ao Duan a,∗ , Shu Jiang a , Yi-yun Qian a , Da-wei Qian a a Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, PR China b School of Pharmacy, Nantong University, Nantong 226001, PR China
a r t i c l e
i n f o
Article history: Received 19 January 2016 Received in revised form 26 April 2016 Accepted 30 April 2016 Available online 3 May 2016 Keywords: Buddleoside UPLC-LTQ/Orbitrap/MS/MS Intestinal microflora Enzyme activity
a b s t r a c t Buddleoside (also known as linarin) as the major flavonoid in Chrysanthemum morifolium Ramat., has been reported to possess a wide range of pharmacological activities. The human intestinal microbiota might have an important impact on drug metabolism and ultimately on the drug oral bioavailability. However, the interaction of the buddleoside with human intestinal bacteria remains unknown. In this study, the conversion of buddleoside by different bacteria from human feces was firstly investigated. A reliable, sensitive and rapid analytical method, ultra performance liquid chromatography was established and successfully applied to investigate the metabolites and metabolic profile of buddleoside by human intestinal bacteria. Among the isolated bacteria, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the accurate mass data and the characteristic MSn product ions, the parent and six metabolites were detected and tentatively identified compared with blank samples. The metabolites were produced by four main metabolic pathways including deglycosylation, acetylation, methylation and hydroxylation. Buddleoside could be firstly converted to its aglycon acacetin (M2) by the majority of the isolated intestinal bacteria. Subsequently, M2 was further metabolize to its methylated (M3), acetylated (M4), hydroxylated (M5) and hydrogenated product (M6). However, acacetin-7-glucosid (M1) was obtained only from the minor bacterial samples like Bacillus sp. 46. To further explain the metabolism of buddleoside, the d-glucosidase and ␣-l-rhamnosidase activities of four strains were analyzed. Bacillus sp. 46 could only produce ␣-l-rhamnosidase, while the other three strains showed two kinds of enzyme activities. Furthermore, the activities of ␣-l-rhamnosidase and -d-glucosidase reached the highest level at 12–18 h and 10–12 h, respectively. The metabolic routes and metabolites of buddleoside produced by human intestinal microflora were firstly reported in this paper. The results will be very helpful for the further investigation of the pharmacokinetic research of buddleoside and to unravel how it works in vivo. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chrysanthemum indicum, a traditional herb medicine, belongs to Compositae family. The dried flowers of C. indicum Ramat. have been used as a folk medicine for thousands of years in China. They are reported to possess the ability of clearing heat and detoxification, and in clinic traditionally used for headache, influenza, hepatic and eye diseases [1–5]. Meanwhile, Chrysanthemum has
∗ Corresponding author at: Jiangsu Key laboratory for TCM formulae Research, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, PR China. E-mail address: [email protected] (J.-a. Duan). http://dx.doi.org/10.1016/j.jchromb.2016.04.055 1570-0232/© 2016 Elsevier B.V. All rights reserved.
been widely used as a popular beverage and mixed spices in Korea since ancient times [6]. Its chemical constituents include flavonoids, volatile oil, lactone and organic acids. Buddleoside as the major flavonoid in C. indicum Ramat. has been reported to possess a wide range of pharmacological activities such as anti-hypertensive, anti-oxidative, anti-inflammatory, and antimicrobial effects [7]. It is well known that most traditional Chinese medicines (TCMs) are administered orally and ingredients of these medicines inevitably lead to contact with intestinal microflora in the gastrointestinal (GI) tract and subsequently be transformed by intestinal bacteria before being absorbed into the blood. The trillions of bacteria housed in our GI tract serve an important purpose in
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host homeostasis. Intestinal bacteria have a variety of enzymes, e.g. hydrolases, oxidoreductases, lyases and transferase. Anaerobic environment in the intestine lead to the reduction reaction to proceed smoothly, and then nitro, carbonyl, azocompounds are easily reduced. Cleaving enzyme can make the ring, C C and C N cleavage. Transferase can catalyze the methylation, acetylation, sulfation reaction. Glycosides medicine metabolism mainly involve hydrolytic enzymes, such as -glucosidase, ␣-rhamnosidase, glucuronidase and ␣-galactosidase, which release aglycones of flavonoids from their glycosides and glucuronides [8–14]. High-resolution mass spectrometry (HRMS), which allows accurate mass measurements at high resolving power, has been used for metabolite identification, contaminant analyses, and doping control. HRMS demonstrates some obvious advantages including the collection of full-scan spectra providing abundant information on sample composition, retrospective data analyses, and identification of potential metabolites [15–27]. As one of the major HRMS-based techniques, UPLC coupled with hybrid mass spectrometry, which combines the linear trap quadrupole (LTQ) and Orbitrap MS/MS mass analyser, has been widely used in qualitative and quantitative analysis of drugs and drug metabolism [18–21]. In this paper, we attempted to isolate different intestinal bacteria from human feces and carry out research on their abilities and characteristics in the metabolism of buddleoside. To further clarify the metabolic profile of buddleoside, ultra performance liquid chromatography with linear ion trap-orbitrap mass spectrometer (UPLC-LTQ/Orbitrap/MS/MS) combined with offline data processing methods of the software Xcalibur 2.1 system which included Mass FrontierTM 7.0 and MetWorks1.3 SP3 (Thermo Fisher, San Jose, CA, USA) was applied. The results will provide helpful information for further in vivo metabolism and active mechanism research on the active components. 2. Experimental 2.1. Chemicals and reagents Buddleoside standard substance (purity 99.56%) was purchased from Shanghai Winherb Medicals & T Development Co. Ltd (Shanghai, China). The UPLC-grade acetonitrile was purchased from TEDIA Company Inc. (Fairfield, USA), formic acid was obtained from Merck KGaA (Darmstadt, Germany), ultra-pure water was purified by an EPED super purification system (Nanjing, China). Other reagents were of analytical grade. AnaeroPack Rectangular Jars were purchased from Mitsubishi Gas Chemical Company Inc. (Japan). General anaerobic medium (GAM) was purchased from Shanghai Kayon Biological Technology Co. Ltd. (Shanghai, China). p-Nitrophenyl -d-glucoside (PNP--d-glu) was provided by Baomanbio Bioscience & Technology Co. (Shanghai, China). p-Nitrophenyl ␣-l-rhamnoside (PNP ␣-l-rham) was provided by Solarbio Bioscience & Technology Co. (Shanghai, China) and p-nitrophenol (PNP) was provided by Jianglai Bioscience & Technology Co. (Shanghai, China). 2.2. Characterization and isolation of the human intestinal bacteria Four grams of fresh human fecal sample from a healthy female volunteer who has not taken any antibiotics in 3 months were mixed with 20 mL of sterile physiological saline and then homogenized and suspended for 3 min. After centrifugation for 10 min at 2000g, the suspension was obtained as human intestinal bacterial mixture which was serially diluted in water and aliquot was plated on GAM agar plates. The plates were incubated in anaerobic condition at 37 ◦ C for 72 h. About 100 different bacterial colonies were isolated.
2.3. Incubation of buddleoside with gut bacteria and preparation of sample solutions for UPLC-LTQ/Orbitrap/MS/MS The buddleoside standard solution was prepared by dissolving accurately weighed buddleoside in methanol to the final concentration of 5.0 mg/mL and then stored in a refrigeration at 4 ◦ C before analysis. Aliquots of 0.1 mL of the approximately one hundred different bacterial colonies were inoculated into 0.9 mL of GAM broth containing 0.1 mM buddleoside, and the media were incubated under anaerobic conditions at 37 ◦ C for 72 h. After the incubation, the incubated solution was extracted with ethyl acetate three times, and then the ethyl acetate layer was dried under vacuum. The residues were dissolved in 0.3 mL methanol, centrifuged at 13,000g for 10 min, soluble fraction was analyzed by UPLC-LTQ/Orbitrap/MS/MS. Meanwhile, in order to avoid extraction artifact, we added a blank control experiment. GAM broth (1.0 mL) containing 0.1 mM buddleoside was incubated under the same conditions and the sample preparation as noted above.
2.4. UPLC-LTQ/Orbitrap/MS conditions Chromatographic separations were performed using a UPLC system consisting of a quaternary Accela 600 pump and Accela Autosampler (Thermo Fisher Scientific). An analytical Hypersil gold C18-column (50 × 2.1 mm, 1.9 m particle size; Thermo Fisher Scientific) was used for separations. The mobile phase consisted of (A) acetonitrile and (B) water containing 0.1% formic acid using a gradient elution of 10–35% A at 0–4 min, 4–6 min, held at 35%, 35–60% A at 6–13 min, 60–90% A at 13–16 min, held at 90% A for 1 min and then an immediate reduction to 10% A at 18.2 min; 10% A for equilibration of the column. The flow rate was 200 L/min, injection volume was 2 L. The UPLC system was coupled to a linear ion trap and Orbitrap hybrid mass spectrometer (LTQ/Orbitrap) equipped with a heatedelectrospray ionization probe (HESI-II; Thermo Fisher Scientific). The mass spectrometer was operated in negative mode. Parameters of the ion source were as follows: source voltage 5 kV, capillary voltage −40 V, tube lens voltage −80 V, capillary temperature 275 ◦ C, sheath and auxiliary gas flow (N2) 42 and 11 (arbitrary units). The MS spectra were acquired by full-range acquisition covering 100–1000 m/z. A data-dependant scan was performed for the fragmentation study by deploying collision induced dissociation (CID). The normalized collision energy of the CID was set at 35 eV. Acquisition and data processing were performed with Xcalibur v.2.0 software (Thermo Fisher, San Jose, CA, USA) and Qualbrowser.
2.5. 16S rRNA gene sequencing and phylogenetic analysis Genomic DNA was extracted from the isolate using a Karroten Genomic DNA Purification kit (Karroten, China). The 16S rRNA gene sequence of the strain was amplified by employing two universal primes, 16S-1F (5 -AGA GTT TGA TCC TGG CTC AG-3 ), 16S-1R (5 -AGA AAG GAG GTG ATC C-3 ). A polymerase chain reaction (PCR) program was used for amplification as follows: 94 ◦ C for 1 min, followed by 29 cycles consisting of 94 ◦ C for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 2 min, and a single final extension step consisting of 72 ◦ C for 10 min. The PCR product was purified from the agarose gel using a Karroten gel purification kit. Sequencing of the 16S rDNA fragments was performed by Majorbio (Shanghai, China). The homology search of the16S rRNA gene sequence was performed by EzBioCloud. A phylogenetic tree was constructed using the neighbor-joining method using the CLUSTAL W program and MEGA (ver 5.0) software.
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Fig. 1. UPLC chromatogram of metabolites of buddleoside by human intestinal bacteria.
Fig. 2. Possible fragmentation and metabolic pathways of buddleoside by the human intestinal bacteria: (a) Fragmentation pathway of Parent, (b) Proposed metabolic pathways of buddleoside.
2.6. Assay for glucosidase and rhamnosidase activities The strains were cultured for 24 h in 1 mL of GAM at 37 ◦ C, then the suspensions were centrifuged at 5000g for 15 min. The supernatant was diluted five times in phosphate buffer (0.2 M, pH 7.3). The suspensions were incubated with 0.25 mM PNP--d-glu and 0.25 mM PNP-␣-l-rham at 37 ◦ C, respectively. Aliquots of the reactive solutions were collected at different time and were diluted with water (1:10) in a 96-well microplate. The absorbance of each well was measured with a microplate reader at 405 nm. The absorbance of a series of different concentrations of PNP was used to calculate the enzyme activity.
3. Results and discussion 3.1. Method optimization To improve the sensitivity and obtain appropriate retention times and symmetric peak shapes for the metabolites, different
analytical mobile phases such as acetonitrile, methanol, water and 0.1% formic acid were tested. Comparing with other solvents, acetonitrile showed more powerful separation ability, shorter retention time and lower column pressure. Additionally, 0.1% formic acid added in the water phase could help show a higher response and better peak sensitivity. Therefore, the optimal solvent system consisting of acetonitrile and 0.1% formic acid aqueous solution, which remarkably enhanced the efficiency of ionization and gave satisfactory sensitivity, was ultimately chosen as mobile phase using a gradient elution. Electrospray ionization was applied to obtain good sensitivity and fragmentation. For the heated electrospray ionization probe (HESI) conditions, both negative and positive ion detection modes were tested. Due to the more obvious fragment characteristics and higher sensitivity for the investigated compounds with the HESI negative ionization mode, the negative mode was selected and applied for analysis. Furthermore, to acquire maximum sensitivity for the analytes, parameters of the ion source such as desolvation, source voltage, capillary voltage, capillary temperature and
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desolvation gas flow were further optimized. Consequently, the optimum conditions were decided as follows: source voltage 5 kV, capillary voltage −40 V, tube lens voltage −80 V, capillary temperature 275 ◦ C, sheath and auxiliary gas flow (N2) 42 and 11 (arbitrary units). The MS spectra were acquired by full-range acquisition covering 100–1000 m/z. A data-dependant scan was performed for the fragmentation study by deploying collision induced dissociation (CID). The normalized collision energy of the CID was set at 35 eV.
3.2. Characterization of the human intestinal bacteria According to Bergey’s Manual of Systematic Bacteriology, colony morphology, micromorphology, and comparative 16S rRNA gene sequence analysis, 68 Escherichia, 16 Enterococcus, and 16 Bacillus strains were identified in this study. The data analysis of the samples about the bacterial bioconversion ability indicated that all of the isolated bacterial colonies had metabolic capacity on buddleoside. Since four bacteria showed more powerful conversion
Fig. 3. Representative MS/MS spectrum: (a) M0 (m/z 591), (b) M1 (m/z 445), (c) M2 (m/z 283), (d) M3 (m/z 297), (e) M4 (m/z 325), (f) M5 (m/z 299), (g) M6 (m/z 285).
J.-h. Tao et al. / J. Chromatogr. B 1025 (2016) 7–15
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Fig. 3. (Continued)
capability than others, they were selected as the typical bacteria to analyze the metabolic profiles. Four bacteria were identified as Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus
sp. 46 by the 16S rRNA gene sequencing. They have been deposited in GenBank under accession No. KC819112, KC819111, KC417329 and KC257405, respectively.
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Fig. 3. (Continued)
3.3. Metabolic profile of buddleoside by different human intestinal bacteria
spectrum of M2. From these data analyses, M2 could be identified as aglycone acacetin in accordance with the authentic standard.
An overview of the metabolites was obtained after optimizing the UPLC and MS conditions. Using negative ion electrospray tandem mass spectrometry and the Mass FrontierTM 7.0 and MetWorks1.3 program, the parent and six metabolites of buddleoside (Table 1 and Fig. 1) were detected in the different bacterial samples compared with blank samples. The six metabolites were likely to be acacetin-7-glucoside (M1), acacetin (M2), methylated acacetin (M3), acetylated acacetin (M4), hydroxylated acacetin (M5) and hydrogenated acacetin (M6). Additionally, the proposed metabolic pathways of buddleoside by the human intestinal bacteria were inferred (Fig. 2a and b).
3.4.4. Metabolite M3 Metabolite M3 in the UPLC chromatogram having a retention time of 16.83 min and exhibited a [M−H]− ion at m/z 297.1101 (Fig. 3d). It was 14 Da higher than that of M2, indicating the chemical structure of M3 could be the methylated product of M2. According to the fragmentation pathway (Fig. 2a), M3 lost CH2 to form m/z 283.1657. Then, m/z 269.2115 and 255.1964 were obtained from m/z 283.1657 by the loss of CH3 and CO, respectively. The m/z 269.2115 was further fragmented to be m/z 241.2169 by the loss of CO. The m/z 131.0602 was also obtained by RDA cleavage from m/z 283.1657. Thus, M3 was identified as the methylated acacetin.
3.4. Identification of the buddleoside metabolites 3.4.1. Parent compound Buddleoside (M0) was identified by comparing the UPLC retention time (5.26 min), accurate MS at m/z 591.2471 [M−H]− and MS/MS spectra with the authentic standard. As illustrated in MS/MS spectrum (Fig. 3a), the precursor ion m/z 591.2471 afforded the buddleoside aglycone (acacetin) product ion at m/z 283.1657 by the loss of rhamnose and glucose moiety. The characteristic product ion of m/z 268.0352 was the loss of CH3 from m/z 283.1657. 3.4.2. Metabolite M1 Metabolite M1 was observed in the UPLC chromatogram with a retention time at 10.20 min. MS analyses showed molecular ion at m/z 445.2161 [M−H]− , which was 146 Da lower than that of M0 buddleoside, indicating that it might be generated by the loss of rhamnose (Fig. 3b). M1 was degraded by the intestinal bacterium Bacillus sp. 46. which indicated that it could produce ␣-l-rhamnosidase but did not produce -d-glucosidase. 3.4.3. Metabolite M2 Metabolite M2 was detected as deprotonated molecular ion [M−H]− at m/z 283.1657 with retention time of 5.12 min (Fig. 3c), generated from the deglycosylation of buddleoside by the loss of m/z 308.0814. The other characteristic fragments were m/z 268.0320, 255.0295 and 239.0512 corresponding to the loss of CH3 , CO, CO2 from m/z 283.1657, respectively (Fig. 2a). Its product ion m/z 131.0602 (RDA) was also appeared in the product ion mass
3.4.5. Metabolite M4 The metabolite M4 showed a UPLC profile with a retention time of 2.40 min and an MS spectrum which gave an [M−H]− ion at m/z 325.1187 (Fig. 3e) which was 42 Da (C2 H2 O) higher than that of M2. The m/z 255.0310 was obtained from m/z 283.1657 by the loss of CO. The m/z 131.0602 was also obtained by RDA cleavage from m/z 283.1657. Thus, M4 was identified as the acetylated acacetin. 3.4.6. Metabolite M5, M6 Metabolite M5, M6 with a retention time of 5.61 min and 5.79 min produced a deprotonated molecular ion at m/z 299.0641 and m/z 285.2401, respectively (Fig. 3f and g). The fragment ion of M5 was 16 Da higher than that of M2. The m/z 255.2114, 271.0605 were obtained by the loss of CO from m/z 283.1657 and 299.0641, respectively. The fragment ion of M6 was 2 Da higher than that of M2. The m/z 285.2401 was further fragmented to be m/z 241.1552 by the loss of CO2 . The m/z 151.1118 was also obtained by RDA cleavage from m/z 285.2401. Thus, M5 and M6 were identified as the hydroxylated acacetin and reduction product, respectively. 3.5. ˛-l-rhamnosidase and ˇ-d-glucosidase activities The analysis for corresponding enzyme activity was performed using PNP--d-glu and PNP-␣-l-rham as substrates. Fig. 4a and b showed the release of PNP after incubation of PNP--d-glu and PNP-␣-l-rham with the four stains. As was reflected, Bacillus sp. 46 could produce ␣-l-rhamnosidase but did not produce -d-glucosidase or -d-glucosidase activity was so low that the
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Table 1 Buddleoside and its metabolites identified in different human intestinal bacterial samples using UPLC-LTQ/Orbitrap/MS. Peak
tR (min)
Metabolite Name
Formula
Mass Difference
m/z Found
mDa
PPm
M0 M1 M2 M3 M4 M5 M6
5.26 10.20 5.12 16.83 2.40 5.61 5.79
buddleoside acacetin-7-glucoside acacetin Methylated Methylationdaidzein Acetylated Hydroxylated Hydrogenated
C28 H32 O14 C22 H22 O10 C16 H11 O5 C17 H13 O5 C18 H13 O6 C16 H11 O6 C16 H13 O5
−0.0010 −146.0310 −162.0504 14.0010 41.9530 16.0001 2.0744
591.2471 445.2161 283.1657 297.1101 325.1187 299.0641 285.2401
−1.0 1.3 −1.1 0.4 1.0 −0.9 0.8
−2.5 −2.2 −4.4 1.5 2.2 −2.0 1.0
Fig. 4. Buddleoside deglycosylated pathway by human intestinal bacteria.
amounts of PNP were not traced in 24 h. The other three strains all produced ␣-l-rhamnosidase and -d-glucosidase. Furthermore, the amounts of PNP released from PNP-glu and PNP-rham reached the highest level at about 10–12 h and 12–18 h, respectively. The results also showed that Escherichia sp. 4 possessed higher enzyme activities than Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46. Based on these results, the deglycosylated route of buddleoside was proposed: buddleoside was metabolized to be acacetin-7glucoside by ␣-l-rhamnosidase produced from these bacteria, and the latter was further converted to acacetin by -d-glucosidase. The human intestinal bacteria, classified as resident and transient flora, comprised a diversity of bacterial species and had excellent enzymatic systems contributing to their enormous catalytic and hydrolytic potential [22]. The factors influencing the natural products absorption in the gastrointestinal tract included deglycosylation before absorption, conjugation in the small intestine through glucuronidation, sulfation or methylation, etc., and metabolism and degradation in the colon to smaller phenolic molecules by intestinal bacteria. The forms in which they circulated in vivo might influence their polarity, their localization and bioactivities [23]. Acacetin, deglycosylated from buddleoside, as the major metabolite, was present in many plants such as Robinia pseudoacacia (black locust) and Turnera diffusa (damiana).
Previous studies have shown that acacetin possessed anticancer activity against many types of cancer cells including T-cell leukemia, lung, prostate, and breast cancer [24–26]. Recently, acacetin was also found that it could inhibit the expression of E-selectin in part through regulation of the p38 MAPK signaling pathway and activation of NF-kB and then attenuate monocyteendothelial interactions in vitro [27]. Deglycosylation was an important step in the absorption and metabolism of glycosides [28]. The absorption rate of aglycones was higher than that of their corresponding glycosides due to their higher lipophilicities and smaller molecular sizes [29]. Hence, the human intestinal bacteria play an important role in the pharmacological effects of buddleoside in vivo.
4. Conclusion In this paper, the reliable and powerful analytical method using UPLC-LTQ/Orbitrap/MS/MS was successfully established for separation and identification of the multiple metabolites of buddleoside by human intestinal bacteria. According to the retention behaviors, changes of molecular weights, and MS/MS fragment patterns, the prototype and its six metabolites were tentatively identified. These metabolites indicated that buddleoside could be converted by four major metabolic routes including deglycosylation, acetylation,
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Fig. 5. Enzyme activities: (a) Time-dependent release of PNP after incubation of PNP-ˇ-d-glu (0.25 mM) with four strains (n = 5, mean ± SD). (b) Time-dependent release of PNP after incubation of PNP-␣-l-rham (0.25 mM)with four strains (n = 5, mean ± SD).
methylation and hydroxylation. It has been reported that methylation of flavonoids might result not only in a dramatic increase in their hepatic metabolic stability but also in great improvement of their intestinal absorption, both of which could greatly increase their oral bioavailability [30]. This indicated that our findings, such as methylated, hydroxylated, deoxygenated and acetylated metabolites of buddleoside, might lead to a higher oral absorption which contributed to improve the bioactivity of buddleoside in vivo. Meanwhile, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the deglycosylated products of four strains and their enzyme activities, the different bacteria could convert buddleoside to different metabolites due to their different enzyme system. Moreover, we could conclude that buddleoside was firstly degraded to acacetin-7-glucoside by losing rhamnose, then the latter was further metabolized to acacetin by losing glucose (Fig. 5). Acknowledgements This work was financially supported by Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (No.ZDXM-1-10), Construction Project for Jiangsu Key Laboratory for High Technology of TCM Formulae Research (BM2010576). References [1] H.D. Jiang, J. Cai, J.H. Xu, X.M. Zhou, Q. Xia, Endotheliumdependent and direct relaxation induced by ethyl acetate extract from Flos Chrysanthemi in rat thoracic aorta, J. Ethnopharmacol. 101 (2005) 221–226. [2] C.K. Lii, Y.P. Lei, H.T. Yao, Y.S. Hsieh, C.W. Tsai, K.L. Liu, H.W. Chen, Chrysanthemum morifolium Ramat. reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells, J. Ethnopharmacol. 128 (2010) 213–220. [3] T. Zhang, D. Chen, Anticomplementary principles of a Chinese multi herb remedy for the treatment and prevention of SARS, J. Ethnopharmacol. 117 (2008) 351–361.
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