A comprehensive study of pomegranate flowers polyphenols and metabolites in rat biological samples by high-performance liquid chromatography quadrupole time-of-flight mass

Pomegranate flowers is an ancient medicine that has commonly been used to treat various diseases such as diabetes. However, no reports are available on the metabolic profile of pomegranate flowers in vivo.

Journal of Chromatography A 1604 (2019) 460472 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma A comprehensive study of pomegranate flowers polyphenols and metabolites in rat biological samples by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry Zainaipuguli Yisimayili a,b,c, Rahima Abdulla a, Qiang Tian c, Yangyang Wang b,c, Mingcang Chen c, Zhaolin Sun c, Zhixiong Li c, Fang Liu c, Haji Akber Aisa a,b,∗, Chenggang Huang b,c,∗ a Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China b University of Chinese Academy of Sciences, Beijing 100049, China c Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China a r t i c l e i n f o Article history: Received 21 May 2019 Revised 14 August 2019 Accepted 21 August 2019 Available online 22 August 2019 Keywords: Punica granatum L flowers Polyphenols Ellagitannin Metabolism HPLC-Q-TOF-MS2 a b s t r a c t Pomegranate flowers is an ancient medicine that has commonly been used to treat various diseases such as diabetes However, no reports are available on the metabolic profile of pomegranate flowers in vivo In the present study, with the aid of HPLC-Q-TOF-MS2 , 67 compounds were identified in pomegranate flowers extract, including 18 ellagitannins, 14 gallic acid and galloyl derivatives, five anthocyanins and 18 flavonoids Seven compounds were firstly identified In vivo, 22 absorbed compounds and 35 metabolites were identified in rat biosamples (urine, feces, plasma and tissues) after orally administered with pomegranate flowers extract This result showed that not all compounds abundant in pomegranate flowers extract could be absorbed well in plasma and tissues This finding also suggested a potential correlation between study on metabolic profile of these compounds in vivo and study on strategy of screening bioactivity of the isolates with in vitro cell systems evaluation Notably, mono-glucuronide conjugated metabolite of ellagitannin compound (corilagin) was firstly identified In addition, this is first report to identify phase II conjugate metabolites of ellagitannins in vivo after oral administration of ellagitanninsrich extracts (or foods) Thus, characterizing its multiple constitution, absorption and metabolic fate of these compounds in vivo is helpful to better analyze the active components in pomegranate flowers © 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction Pomegranate (Punica granatum L.) is widely cultivated for its widely consumed fruit in the regions of Southeast Asia, the Mediterranean area and USA Notably, pomegranate flowers is an ancient medicine that has commonly been used to treat various diseases such as chronic diarrhea and aphthous stomatitis In Unani and Ayurvedic medicine, and in some parts of China, pomegranate flowers have widely been used to treat diabetes [1–4] According to previous studies, the health benefits of pomegranate flowers have been associated with their polyphenol content, specifically their anthocyanins, flavonoids and tannins ∗ Corresponding authors E-mail addresses: haji@ms.xjb.ac.cn (H.A Aisa), cghsimm@126.com (C Huang) content [5,6] Anthocyanins, a type of the flavonoids, are the major pigments responsible for the bright color of pomegranate flowers [5,7] Tannins are one group of natural compounds and the major compounds in pomegranate flowers Besides, the wide range of bioactivities of pomegranate flowers have been associated with the polyphenols isolated from (or present in) pomegranate flowers such as phenolics, ellagitannins and flavonoids as active components based on strategy of screening bioactivity of the isolates with in vitro cell systems evaluation [1,3,5,6,8,9,11] While it is difficult to make structure-activity correlation conclusion among the phytocompounds in the extract or exploring bioactivity of the isolates with in vitro cell systems evaluation Because the ingested compounds, at least part of them, reach the circulatory system and specific tissues to exert biological effect as a result of in vivo process of absorption, distribution, metabolism and excretion [21,25,28] Thus, further studies such as bioavailability and metabolism of these compounds in vivo would be required https://doi.org/10.1016/j.chroma.2019.460472 0021-9673/© 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 before exploring their some potential activities After oral administration of pomegranate flowers extract, the phytocompounds absorbed as native form and their derived metabolites, at least a portion of them, may be the functional components responsible for the bioactivities of pomegranate flowers such as antioxidant, anti-inflammatory, α -glucosidase inhibitory and hepatoprotective activities [1,5,6,9–11] However, no reports are available on the metabolic profile of pomegranate flowers in vivo The absence of scientific evidence for its activities may restrict its further development including clinical application Thus, characterizing its multiple constitution, absorption and metabolic fate of these compounds in vivo is necessary to better analyze the bioactive components in pomegranate flowers Therefore, in the present study, using rapid and high sensitive, high-performance liquid chromatography quadrupole time-offlight mass spectrometry (HPLC-Q-TOF-MS2 ) method, we characterized the phytochemical profile of pomegranate flowers extract Furthermore, the absorbed compounds and their metabolites in rat plasma, tissues, urine and feces after oral administration of pomegranate flowers extract were analyzed comprehensively This study will provide vital information for finding possible candidates for the real bioactive compounds in pomegranate flowers and provide a solid basis for further study of biological properties of the compounds in pomegranate flowers Materials and methods 2.1 Chemicals and reagents Reference standards (corilagin, gallic acid, ethyl gallate, ellagic acid, brevifolin, brevifolincarboxylic acid, punicalagin, apigenin, apigenin-7-O-glucoside, kaempferol, luteolin, luteolin-7O-glucoside, isoquercetin, urolithin D, urolithin C, urolithin B, urolithin A) were used to absolutely identified these compounds in pomegranate flowers and rat biosamples These standards were purchased from the Chengdu MUST Bio-Technology Co Ltd (Chengdu, China) Acetonitrile methanol and formic acid were bought from Thermo Fisher Scientific Co.Ltd (Waltham, Massachusetts, USA) Milli-Q System (Millipore, Billerica, MA, USA) was used to prepare purified water for HPLC Other chemicals were analytical-grade and bought from the Sinopharm Chemical Reagent Co Ltd (Shanghai, China) conditions were as follows: capillary, 40 0 V and 350 V for positive and negative ionization modes, respectively; nozzle voltage, 500 V; nebulizer, 45 psi; gas temperature and flow rate, 300 °C and L/min; sheath gas temperature and flow rate, 350 °C and 12 L/min; fragmentor, 100 V; collision energy (CE), 15 eV, 30 eV The m/z range of full mass spectra for MS1 was 10 0–170 The m/z range was set from 100 to 1200 for MS2 experiments The HPLC-MS system operation and data analysis were carried out with the Agilent Masshunter Workstation software which contain with Data Acquisition (Version B.05.01) software and Qualitative Analysis (Version B.06.00) software 2.3 Sample preparation and pretreatment Dried pomegranate flowers (300 g) were extracted with ethanol /water (7:3, v/v) three times (solid /liquid ratio was 1:15, 1:15, 1:10, respectively) for (12 h, h, h, respectively) at 60 °C The combined extract was concentrated to 1.5 g /mL under vacuum at 60 °C 2.4 Animal experiment Sixty male Sprague-Dawly (SD) rats were purchased from Shanghai SLAC Laboratory Animal Co Ltd (Shanghai, China) Before the experiment, all the rats were maintained in house with environmentally controlled at a 12 h light-dark cycle and at 22 ± °C with relative humidity (50 ± 10%) for six days Before drug administration, all rats were fasted for 10 h and they were free to access water The rats were randomly separated into eleven groups (n = /group) Groups 1–10 were orally administered with pomegranate flowers extract at a dose of 15 g/kg and used for collecting blood and tissues samples After oral administration of pomegranate flowers extract, urine and feces (from to 48 h) were collected from the rats in group 10 which were kept separately in metabolic cages The blank biological samples were collected from the rats in group 11 After oral administration of pomegranate flowers extract, systematic blood (6–8 mL) from aorta abdominal and organs (liver, heart, kidney, spleen, and lung) were collected at 15 min, 0.5 h, h, h, h, h, h, 12 h, 24 h, 48 h (10 time points) The blood biosamples were promptly centrifuged (12,0 0 rpm, 10 min) The physiological saline water (0.9%) was used to homogenize organs 2.5 Sample preparation 2.2 Instrumentations and investigation conditions The HPLC-Q-TOF-MS2 system (Agilent Technologies, Palo Alto, CA, USA) which consisted of a HPLC system (1260 Series, coupled to an Agilent Q-TOF mass spectrometer equipped with a Dual Agilent Jet Stream Electrospray Ionization (ESI) sourse (6530 Series) was used for the identification of components in pomegranate flowers extract and its metabolites in rat biosamples The chromatographic separation for pomegranate flowers extract and biological samples were accomplished on an ACE Excel Super C18 column (100 × 2.1 mm, 3.0 μm), (Advanced Chromatography Technologies Ltd Aberdeen, Scotland) The HPLC flow rate and column temperature were set at 0.35 mL/min and at 40 °C, respectively The optimized mobile phases contain solvent A and solvent B which were 0.1% formic acid in water and 0.1% formic acid in acetonitrile An optimized mobile phase gradient elution was as follows: 0–8.0 min, 3.0% B; 8.0–16.0 min, 3.0–8.0% B; 16.0–32.0 min, 8.0% B; 32.0–54.0 min, 8.0–18.0% B; 54.0–60.0 min, 18.0% B; 60.0–65.0 min, 18.0–50.0% B; 65.0–72.0 min, 50.0–80.0% B; 72.0–76.0 min, 80.0– 95.0% B; 76.0–80.0 min, 95% B; 80.1–85.0 min, 3.0% B In this study, the mass spectrometric detection for every samples was performed in both ionization modes The detection parameters for the MS Plasma (200 μL at each time point) and 600 μL acetonitrile were mixed for and centrifuged (14,0 0 rpm, 10 min) The supernatants were separately evaporated to dryness under vacuum at 40 °C After removal of the solvent of combined residue from ten time points, 200 μL of methanol-water (7:3, v/v) was used to dissolve the residue The tissues homogenate and urine biosamples were treated respectively with the same ways as the plasma Ground feces were mixed with 10 times of methanol (v/w) and extracted in ultrasonic bath for two times (for 30 min) The clear methanol layers were evaporated to dryness under vacuum at 40 °C Methanol-water (7:3, v/v) used to dissolve the residue was 200 μL After centrifuging (14,000 rpm, 10 min) each sample, 10 μL of sample was used to analysis by HPLC-Q-TOF-MS2 Results and discussion In present study, a qualitative analysis of the polyphenols in the pomegranate flowers extract, absorbed compounds and metabolites in rats orally administered with pomegranate flowers extract were carried out by using high sensitive HPLC-Q-TOF-MS2 in both ionization modes Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 Fig Base peak chromatogram (BPC) of pomegranate flowers extract: (a) positive ion mode, (b) negative ion mode 3.1 Identification of polyphenols in pomegranate flowers extract In this study, as shown in Table 1, 67 compounds were identified in pomegranate flowers extract, including 18 ellagitannins, 14 gallic acid and galloyl derivatives, five anthocyanins and 18 flavonoids Seven compounds were firstly identified in pomegranate flowers The peak characterization was performed based on their retention time (tR ), accurate molecular mass (mass error of less than ppm), major MS/MS fragment ions Furthermore, the experimental data were compared with commercially available authentic standards for absolutely identification The base peak chromatograms (positive and negative ion mode) of pomegranate flowers extract were shown in Fig Analysis of ellagitannins, gallic acid and galloyl derivatives were performed in the negative ion mode because of stronger response in the MS spectra Both positive and negative ion mode were adopted to identify anthocyanins and flavonoids 3.1.1 Ellagitannins Ellagitannins, member of the tannin family, are characterized as hydrolyzable conjugates containing one or more hexahydroxydiphenoyl (HHDP) group(s) to esterify a sugar, usually glucose [12] During their MS/MS fragmentation, it can be observed the typical losses such as galloyl moiety (152 Da), gallic acid (170 Da), HHDP (302 Da), galloyl-glucose (332 Da), HHDP glucose (482 Da) and galloyl-HHDP-glucose (634 Da) residues Besides, in the negative ESI-IT/Q-TOF-MS2 mode, the characteristic fragment ions observed at m/z 300.99 (which is produced after the spontaneous lactonization of the HHDP unit into ellagic acid) and m/z 169.01, indicate the existence of HHDP group and galloyl group in the molecule, respectively, based on the fragmentation pattern of ellagitannins previously reported in the literatures [12–18] As shown in Fig 2, peak 25 showed a protonated molecular ion [M−H]− at m/z 633.0730 (0 ppm) with a molecular formula of C27 H22 O18 In the MS2 mode, the product ion at m/z 463.0544 [M−H−170 Da]− was occurred via the loss of a gallic acid from the molecular ion The typical fragment ion at m/z 300.9995 [M−H−332 Da]– , as a base peak, was formed from a galloyl-glucose moiety loss from the [M−H]− ion The typical fragment ion at m/z 169.0130, which was associated with gallic acid, was also observed Peak 25 was absolutely identified as corilagin by comparing with its commercial standard The proposed fragmentation pattern of corilagin was shown in Fig Peaks 3, 6, 12 and 21 were isomeric compounds All of the peaks had a [M−H]− ion at m/z 633.0729 (−0.63 ppm) with molecular formula of C27 H22 O18 The fragment ion at m/z 463.0544 [M−H−170 Da]– (loss of a gallic acid), the typical ions at m/z 300.9998 and m/z 169.0138 were consistent with those of corilagin Thus, peaks 3, 6, 12 and 21 were tentatively identified as galloylHHDP-glucose isomers Peak 10 had a protonated molecular ion [M−H]− at m/z 481.0620 (−1.24 ppm) with molecular formula of C20 H18 O14 , and the MS2 spectrum had fragments at m/z 463.0544 [M−H−18 Da] and characteristic fragment ion at m/zm/z 300.9995 Thus, peak 10 was tentatively identified as HHDP-glucose Peaks 16, 23, 28, 34 and 35 were isomers All of the peaks had a [M−H]− ion at m/z 785.0818 (−3.18 ppm) with molecular formula of C34 H26 O22 The fragments at m/z 615.0637 [M−H−170 Da]– (loss of a gallic acid), m/z 463.0544 [M−H−170 Da-152 Da]– (loss of a gallic acid and a galloyl moiety), the typical ions at m/z 300.9998 and m/z 169.0138 were observed in the MS2 mode Thus, peaks 16, 23, 28, 34 and 35 were tentatively identified as digalloyl-HHDPglucoside isomers As an example, the MS/MS spectra and the proposed fragmentation pattern of one digalloyl-HHDP-glucoside were shown in Figs and As shown in Fig 2, peak 53 had a [M−H]− ion at m/z 937.0910 (−4.58 ppm) with molecular formula of C41 H30 O26 The product ions at m/z 767.0748 [M−H−170 Da]– (loss of a gallic acid), m/z 615.0683[M−H−170 Da−152 Da]– (loss of a gallic acid and a galloyl moiety), m/z 465.0754 [M−H−170 Da-302 Da]– (loss of a gallic acid and HHDP moiety), the typical ions at m/z 300.9998 and m/z 169.0138 were observed in the MS2 mode Therefore, peak 53 was tentatively identified trigalloyl-HHDP-glucose Peaks 14, 40 and 46 were isomeric compounds They had a [M−H]− ion at m/z 951.0719 (−2.73 ppm) with molecular for- Table Summary of the mass spectral data of polyphenols identified in pomegranate flower extract by HPLC-Q-TOF-MS/MS 2b 10 11c 12 13 14 15 16 17b 18 19 20b 21 22b , c 23 24 25b 26 27 28 29b , c 30 31 32c tR (min) 1.46 2.06 2.55 2.92 3.40 4.45 5.01 5.96 9.79 11.98 14.60 14.68 15.26 16.93 18.18 18.22 18.56 19.74 19.89 20.19 21.54 24.25 25.36 26.46 26.50 26.91 27.17 28.13 28.54 30.30 31.29 33.29 Molecular formula C13 H16 O10 C7 H6 O5 C27 H22 O18 C20 H20 O14 C13 H16 O10 C27 H22 O18 C13 H16 O10 C20 H20 O14 C21 H10 O13 C20 H18 O14 C15 H11 O7 C27 H22 O18 C20 H20 O14 C41 H28 O27 C27 H31 O16 C34 H26 O22 C48 H28 O30 C27 H31 O15 C20 H20 O14 C13 H8 O8 C27 H22 O18 C9 H10 O5 C34 H26 O22 C34 H24 O22 C27 H22 O18 C21 H21 O11 C21 H21 O10 C34 H26 O22 C12 H8 O6 C21 H10 O13 C27 H24 O18 C14 H10 O8 Calculated 331.0671 169.0142 633.0733 483.0780 331.0671 633.0733 331.0671 483.0780 469.0049 481.0624 465.1028 633.0733 483.0780 951.0745 611.1612 785.0843 1083.0593 595.1663 483.0780 291.0146 633.0733 197.0455 785.0843 783.0686 633.0733 449.1078 433.1135 785.0843 247.0248 469.0049 635.0890 305.0303 Observed 331.0668 169.0140 633.0729 483.0782 331.0668 633.0729 331.0668 483.0782 469.0028 481.0618 465.1023 633.0729 483.0782 951.0719 611.1605 785.0818 1083.0583 595.1660 483.0782 291.0153 633.0729 197.0451 785.0818 783.0685 633.0733 449.1085 433.1133 785.0818 247.0244 469.0028 635.0881 305.0300 Ion mode − [M−H] [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M]+ [M−H]− [M−H]− [M−H]− [M]+ [M−H]− [M−H]− [M]+ [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M]+ [M]+ [M−H]− [M−H]− [M−H]− [M−H]− [M−H]− Error (ppm) MS/MS fragments −0.90 −1.18 −0.63 0.41 −0.90 −0.63 −0.90 0.41 −4.47 −1.24 −1.07 −0.63 0.41 −2.73 −1.14 −3.18 −0.92 −0.50 0.41 2.40 −0.63 −2.02 −3.18 −0.12 1.55 −0.46 −3.18 −1.61 −4.47 −1.42 −0.98 169.0136, 125.0139 463.0535, 313.0560, 169.0136, 463.0535, 169.0136, 313.0560, 425.0149, 463.0510, 303.0486 463.0535, 313.0560, 907.0821, 449.1045, 615.0602, 781.0665, 433.1133, 313.0560, 247.0250, 463.0535, 169.0144, 615.0602, 300.9989 463.0493, 287.0532, 271.0615 615.0602, 219.0305, 425.0149, 465.0677, 273.0061, Identification 125.0236 300.9998, 169.0137, 125.0236 300.9998, 125.0236 169.0137, 300.9995, 300.9995 169.0138 125.0240 300.9998, 169.0137, 783.0580, 287.0522 463.0499, 621.9980, 271.0606 169.0137, 219.0298, 300.9998, 125.0242 463.0499, 275.0204, 169.0138 125.0240 481.0534, 300.9987 275.0204, 169.0138 125.0240 169.0157, 125.0242 300.9993, 169.0130 300.9997 125.0240 191.0346 275.0204, 169.0138 300.9993, 169.0130 300.9990, 169.0152 153.0162 463.0499, 191.0348 300.9995, 313.0568, 245.0082, 300.9993, 169.0130 169.0157, 125.0242 169.0124 217.0141 Galloyl-glucoside Gallic acid Galloyl-HHDP-glucoside Digalloyl- glucoside Galloyl-glucoside Galloyl-HHDP-glucoside Galloyl-glucoside Digalloyl-glucoside Valoneic acid dilactone HHDP-glucoside Delphinidin-3-O-glucoside Galloyl-HHDP-glucoside Digalloyl- glucoside HHDP-valoneoyl-glucoside Cyaniding-3,5-O-diglucoside Digalloyl-HHDP-glucose Punicalagin Pelargonidin-3,5-O-diglucoside Digalloyl-glucoside Brevifolincarboxylic acid Galloyl-HHDP-glucoside Ethyl gallate Digalloyl-HHDP-glucoside Di-HHDP- glucoside Corilagin Cyanidin 3-O-glucoside Pelargonidin 3-O-glucoside Digalloyl-HHDP-glucoside Brevifolin Valoneic acid dilactone Trigalloyl- glucoside Methyl brevifolincarboxylate (continued on next page) Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 No Table (continued) tR (min) Molecular formula Calculated Observed Ion mode Error (ppm) MS/MS fragments 33c 34 35 36 37a 38 39a , c 40 41c 42b 43 44 45 46 47a , b , c 48a , b 49 50a 51a 52a, b 53 54 55a 56 57a 58a 59a 60a 61 33.89 34.59 37.70 42.12 43.25 43.54 44.15 45.10 46.55 46.98 47.04 47.33 48.36 49.02 49.52 50.23 52.32 53.16 54.51 55.90 56.28 57.53 58.18 58.69 60.73 61.86 62.72 63.76 64.02 C33 H28 O22 C34 H26 O22 C34 H26 O22 C21 H10 O13 C27 H30 O16 C27 H24 O18 C27 H30 O15 C41 H28 O27 C15 H12 O8 C14 H6 O8 C41 H28 O27 C27 H30 O16 C34 H28 O22 C41 H28 O27 C21 H20 O12 C21 H20 O11 C34 H28 O22 C21 H20 O11 C21 H20 O12 C21 H20 O10 C41 H30 O26 C27 H20 O17 C21 H20 O11 C41 H32 O26 C21 H20 O11 C21 H20 O12 C15 H10 O7 C21 H20 O10 C43 H34 O28 775.0999 785.0843 785.0843 469.0049 611.1607 /609.1461 635.0890 595.1657 /593.1512 951.0745 319.0459 300.9990 951.0745 611.1607/609.1461 787.0999 951.0745 465.1028 /463.0882 449.1078 /447.0993 787.0999 449.1078 /447.0993 465.1028 433.1129 /431.0984 937.0953 615.0628 449.1078 /447.0993 939.1109 449.1078 /447.0993 465.1028 303.0499 433.1129 /431.0984 997.1164 775.0937 785.0818 785.0818 469.0028 611.1605 635.0881 595.1660 951.0719 319.0459 300.9998 951.0734 611.1605 787.0972 951.0719 465.1021 449.1085 787.0972 449.1085 465.1021 433.1128 937.0910 615.0621 449.1085 939.1061 449.1085 465.1021 303.0503 433.1128 997.1138 [M−H]− [M−H]− [M−H]− [M−H]− [M+H]+ /[M−H]− [M−H]− [M+H]+ /[M−H]− [M−H]− [M−H]− [M−H]− [M−H]− [M+H]+ /[M−H]− [M−H]− [M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M−H]− [M−H]− [M+H]+ /[M−H]− [M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M−H]− −7.99 −3.18 −3.18 −4.47 −1.14 −1.42 −0.50 −2.73 2.65 −1.15 −1.14 −3.43 −2.73 −1.50 1.55 −3.43 1.55 −1.50 −0.23 −4.58 −1.13 1.55 −5.11 1.55 −1.50 1.31 −0.23 −2.60 757.0854, 615.0602, 615.0602, 425.0149, 449.1045, 465.0677, 433.1133, 907.0821, 273.0036, 283.9967, 933.0629, 449.1045, 617.0745, 907.0821, 303.0484, 287.0573, 617.0745, 287.0573, 303.0499, 271.0612 767.0682, 445.0438, 287.0569, 769.0873, 287.0569, 303.0499, 153.0193 271.0612 953.1216, 169.0149 303.0484, 953.1216, 169.0149 153.0178 153.0173 153.0175 315.0496, 62 63 a 64a , b 65a , b 66a , b 67a a b c + − 65.13 65.66 C21 H20 O12 C43 H34 O28 465.1028 /463.0882 997.1164 465.1021 997.1138 [M+H] /[M−H] [M−H]− 65.70 67.07 67.25 67.31 C15 H10 O6 C15 H10 O5 C15 H10 O6 C17 H14 O7 287.0550 271.0601 287.0550 331.0812 287.0554 271.0603 287.0555 331.0813 [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− [M+H]+ /[M−H]− /285.0405 /269.0455 /285.0405 /329.0667 −1.50 −2.60 1.39 0.73 1.74 0.30 465.0680, 463.0499, 463.0499, 300.9995, 287.0522, 313.0568, 271.0606, 783.0580, 245.0085, 257.0083, 613.0448, 287.0522, 465.0659, 783.0580, 153.0152 153.0187 465.0659, 153.0187 153.0178 Identification 300.9992, 300.9990, 300.9990, 169.0157, 153.0159 169.0124 153.0179 481.0534, 217.0141 229.0139, 463.0492, 153.0159 313.0582, 481.0534, 169.0139 169.0130 169.0130 125.0242 783.0990, 633.0715, 481.0912, 300.9994, Ellagitannin Digalloyl-HHDP-glucoside Digalloyl-HHDP-glucoside Valoneic acid dilactone Luteolin-O-diglucoside Trigalloyll-glucoside Apigenin-O-diglucoside HHDP-valoneoyl-glucoside Ethyl brevifolincarboxylate Ellagic acid Galloyl-HHDP-DHHDP-hexoside Luteolin-O-diglucoside Tetragalloyl-glucoside HHDP-valoneoyl-glucoside Isoquercetin Luteolin 7-O-glucoside Tetragalloyl-glucoside Luteolin −7-O-glucoside isomer Tricetin -O-β -glucoside Apigenin-7-O-glucoside Trigalloyl-HHDP-glucose Galloyl-ellagic acid glucoside Kaempferol-O-glucoside Pentagalloyl-glucoside Kaempferol-O-glucoside Tricetin-4 -O-β -glucoside isomer Tricetin Apigenin-7-O-glucoside isomer Punictannin A 153.0152 783.0990, 633.0715, 481.0912, 300.9994, Quercetin Punictannin B 299.0549, 270.0516, 133.1010 Luteolin Apigenin Kaempferol Tricin 300.9987 201.0181 300.9994, 169.0147 300.9987 313.0582, 169.0147 615.0477, 465.0674, 300.9999, 169.0148 300.9999, 169.0132 153.0196 617.0752, 465.0647, 313.0547, 169.0130 153.0196 153.0178 Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 No Error (ppm) and fragment ions taken from the positive ion mode (in case detected in both modes) Confirmed by using reference standard Firstly identified compounds in pomegranate flower Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 Fig The MS/MS spectra of typical ellagitannins and galloyl derivatives in pomegranate flowers Fig Proposed fragmentation pattern of typical ellagitannins and galloyl derivatives in pomegranate flowers mula of C41 H28 O27 These compounds had major fragment ions at m/z 907.0821[M−H−44 Da]– (loss of a CO2 ), m/z 783.0580, m/z 481.0534, m/z 300.9987 in their MS2 mode Therefore, peaks 14, 40 and 46 were tentatively identified as HHDP-valoneoyl-glucoside isomers 3.1.2 Gallic acid and galloyl derivatives Peak had a [M−H]− ion at m/z 169.0140 (−1.18 ppm) with molecular formula of C7 H6 O5 The product ion at m/z 125.0239 [M−H−44 Da]− was generated via the elimination of CO2 unit from the molecular ion Peak 22 showed a [M−H]− ion at m/z 197.0451 (−2.02 ppm) with molecular formula of C9 H10 O5 and the MS2 spectrum had fragment ions at m/z 169.0144 [M−H−28 Da]– (loss of a C2 H4 moiety) and m/z 125.0242 [M−H−28 Da-44 Da]– (loss of a C2 H4 and a CO2 moieties) Peaks and 22 were absolutely identified as gallic acid and ethyl gallate by comparing with their reference standards, respectively Peaks 1, and displayed molecular ion [M−H]− at m/z 331.0668 (−0.90 ppm) with molecular formula of C13 H16 O10 The fragment ion at m/z 169.0136 [M−H−162 Da]– (loss of a glucose moiety), and m/z 125.0236 [M−H−162 Da-44 Da]– were correlated to gallic acid on the basis of the MS/MS spectra data Thus, peaks 1, and were tentatively identified as galloyl-glucose isomers Peaks 4, 8, 13 and 19 were isomeric compounds All of them showed a [M−H]− ion at m/z 483.0782 (0.41 ppm) with molecular formula of C20 H20 O14 The fragment ion at m/z 313.0560 [M−H−170 Da]– (loss of a gallic acid), the characteristic ions at m/z 169.0137 and ion at m/z 125.0240 were observed in the MS2 Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 mode Therefore, peaks 4, 8, 13 and 19 were tentatively identified as digalloyl-glucose isomers Peaks 31 and 38 exhibited a [M−H]− ion at m/z 635.0881 (−1.42 ppm) with molecular formula of C27 H24 O18 The fragment ions at m/z 465.0689 [M−H−170 Da]– (loss of a gallic acid), m/z 313.0575 [M−H−170 Da-152 Da]– (loss of a gallic acid and a galloyl moiety), typical fragment ion at m/z 169.0127 and fragment ion at m/z 125.0254 were observed in the MS2 mode Therefore, Peaks 31 and 38 were tentatively identified as trigalloyl-glucoside isomers As an example, the MS/MS spectra of one trigalloyl-glucoside was shown in Fig Peaks 45 and 49 had a [M−H]− ion at m/z 787.0972 (−3.43 ppm) with molecular formula of C34 H28 O22 The MS2 spectra of these two peaks exhibited fragments at m/z 617.0772 [M−H−170 Da]– (loss of a gallic acid), m/z 465.0654 [M−H−170 Da152 Da]– (loss of a gallic acid and a galloyl moiety), m/z 313.0531[M−H−170 Da-152 Da-152 Da]– (loss of a gallic acid and two galloyl moieties), the typical ion at m/z 169.0138 Therefore, peaks 45 and 49 were tentatively identified as tetragalloylglucopyranoside As an example, the MS/MS spectra and proposed fragmentation pattern of one tetragalloyl-glucopyranoside were shown in Figs and As shown in Fig 2, peak 56 exhibited deprotonated molecular ion [M−H]− at m/z 939.1061 (−5.11 ppm) with molecular formula of C41 H32 O26 The fragments at m/z 769.0873 [M−H−170 Da]– , m/z 617.0772 [M−H−170 Da-152 Da]– , m/z 465.0654 [M−H−170 Da152 Da-152 Da]– , m/z 313.0531[M−H−170 Da-152 Da-152 Da152 Da]– , the typical fragment ion at m/z 169.0138 and fragment ion at m/z 125.01 were observed in MS2 experiment Therefore, peak 56 was tentatively identified as pentagalloyl-glucoside According to the mass fragmentation patterns of galloyl derivatives and ellagitannins, it is suggested that if galloyl derivatives or ellagitannins have one or more galloyl group(s) to esterify a sugar (usually glucose), molecular ion firstly remove one molecule gallic acid (C7 H6 O5 , 170 Da), and then continuously lose one or more galloyl group(s) (C7 H4 O4 , 152 Da) in the mass fragmentation process The typical losses during their fragmentation are a gallic acid (170 Da) and a galloyl moiety (152 Da) 3.1.3 Others Peak 42 had a precursor ion [M−H]− ion at m/z 300.9998 (2.65 ppm) with molecular formula of C14 H6 O8 The product ions at m/z 283.9963 and m/z 257.0092 were yielded by the loss of H2 O (18 Da) unit and CO2 (44 Da) unit from the [M−H]− ion, respectively Furthermore, the fragments at m/z 229.0133 and m/z 201.0182 were also observed Thus, peak 42 was absolutely identified as ellagic acid by comparing with its commercial standard Peak 29 exhibited a protonated molecular ion [M−H]− at m/z 247.0244 (−1.61 ppm) with molecular formula of C12 H8 O6 The fragment ions at m/z 219.0305 and m/z 191.0348 were occurred via the removal of CO unit and continuing removal of CO unit from the molecular ion, respectively Peak 20 had molecular ion [M−H]− at m/z 291.01 (2.40 ppm) with the molecular formula of C13 H8 O8 The fragment ion m/z 247.02 was generated by the loss of CO2 unit from the molecular ion The fragment ions at m/z 219.02 and m/z 191.03 were consistent with those of compound 29 Peaks 29 and 20 and were absolutely identified as brevifolin and brevifolincarboxylic acid by comparing with their commercial standards, respectively Peak 32 had a [M−H]− ion at m/z 305.0300 (−0.98 ppm) with molecular formula of C14 H10 O8 The fragment ions at m/z 273.0061, m/z 245.0082, m/z 217.0141were observed in the MS2 mode Peak 41 displayed [M−H]− ion at m/z 319.01 (−1.31 ppm) with molecular formula of C15 H12 O8 In the MS/MS spectrum, the fragment ions at m/z 273.0036, m/z 245.0152 and m/z 217.0167 were generated by the removal of C3 H6 O2 unit and continuing removal of CO (28 Da) and two CO (28 Da) unit from the molecular ion, respectively Moreover, the fragmentation patterns of peaks 32 and 41 were in agreement with fragmentation patterns reported in the literature [13] Peaks 32 and 41 were tantetively identified as methyl brevifolincarboxylate and ethyl brevifolincarboxylate 3.1.4 Anthocyanins Anthocyanins are naturally occurring plant pigments with unique chromatographic behavior Anthocyanins carry an inherent positive charge and can easily donate protons to free radicals ([M]+ ) under (+) ESI condition [19] Peak 11 had a positively charged molecular ion [M]+ at m/z 465.1023 (−1.07 ppm) with molecular formula of C15 H11 O7 , and MS/MS fragment ions at m/z 303.0486 [M−162 Da]+ , which this characteristic matched with the loss of glucose (162 Da) and suggested that the aglycone was delphinidin Thus, peak 11 was tentatively identified as delphinidin-3-O-glucoside Peak 15 had a positively charged molecular ion [M]+ at m/z 611.1605 with molecular formula of C27 H31 O16 , yielding by fragment ions at m/z 449.1045 [M−162 Da]+ and m/z 287.0522 [M−162 Da-162 Da]+ , which suggested that the aglycone was cyanidin Thus, peak 15 was tentatively identified as cyanidin-3, 5O-diglucoside Peak 18 had a positively charged molecular ion [M]+ at m/z 595.1660 (−0.50 ppm) with molecular formula of C27 H31 O15 In MS2 mode, the fragment ions at m/z 433.1132 [M−162 Da]+ and m/z 271.0600 [M−162 Da-162 Da]+ were observed, which suggested that the aglycone was pelargonidin Thus, peak 18 was tentatively identified as pelargonidin-3, 5-diglucoside Peak 26 had a positively charged molecular ion [M]+ at m/z 449.1085 with molecular formula of C21 H21 O11 , yielding by fragment ion at m/z 287.0532 [M−162 Da]+ , which suggested that the aglycone was cyanidin Thus, peak 26 was tentatively identified as cyanidin-3-O-glucoside Peak 27 had a positively charged molecular ion [M]+ at m/z 433.1133 (−0.46 ppm) with molecular formula of C21 H21 O10 , yielding by fragment ion m/z 271.0600 [M−162 Da]+ by the loss of a glucose moiety, which suggested that the aglycone was pelargonidin Thus, peak 27 was tentatively identified as pelargonidin-3glucoside 3.1.5 Flavonoids Peaks 64 displayed the molecular ion [M+H]+ /[M−H]− at m/z 287.0554 / 285.0409 with molecular formula of C15 H10 O6 , and main MS/MS fragment ion at m/z 153.0178 Peaks 65 exhibited precursor ion [M+H]+ /[M−H]− at m/z 271.0603 /269.0455 (0.73 ppm) with molecular formula of C15 H10 O5, and main MS/MS fragment ion at m/z 153.0173 Peaks 66 showed the molecular ion [M+H]+ /[M−H]− at m/z 287.0555 / 285.0407 with molecular formula of C15 H10 O6 , and main MS/MS fragment ion at m/z 153.0175 Peaks 64, 65 and 66 were absolutely identified as luteolin, apigenin and kaempferol by compariing with their authentic standards, respectively Peaks 48 showed the molecular ion [M+H]+ /[M−H]− at m/z 449.1085 / 447.0993 with molecular formula of C21 H20 O11 In the positive MS2 mode, the fragment ion at m/z 287.0573 (matching with the aglycone of luteolin) appeared after neutral loss of a glucose (162 Da) moiety from the molecular ion Peaks 52 had [M+H]+ /[M−H]− at m/z 433.1139 / 431.0984 with molecular formula of C21 H20 O10 The positive MS2 spectrum showed the main product ion at m/z 271.0612 after neutral loss of a glucose (162 Da) moiety from the molecular ion Thus, peaks 48 and 52 were absolutely identified as luteolin-7-O-glucoside and apigenin-7-Oglucoside by comparing with their authentic standards Peaks 47 was detected in both ionization modes with molecular ion [M+H]+ /[M−H]− at m/z 465.1028/463.0882 with molecular Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 Fig Extracted ion chromatogram, the MS/MS spectra and chemical structures (from left to right) of pelargonidin 3-O-glucoside (detected only in positive ion mode) and apigenin-7-O-glucoside (detected in both positive and negative ion mode) formula of C21 H20 O12 The main fragment ion at m/z 303.0484 was observed in the MS2 mode after the loss of a glucose (162 Da) moiety from the molecular ion Peaks 47 was absolutely identified as isoquercetin by comparing with its commercial standard It is worth to note that several flavonol glycosides and anthocyanin glycosides compounds have the same molecular ions and mass fragmentation patterns in ESI (+) positive ionization mode ([M]+ of anthocyanins and [M+H]+ of flavonol glycosides are the same), occurring as mono- or di-glucosides For example, when the aglycone parts of two different species are quercetin and delphnidin or pelargonidin and apigenin or kaempferol /luteolin and cyanidin As a representative example, Fig showed that pelargonidin 3-glucoside and apigenin-7-O-glucoside share the same molecular ion, same fragmentation behavior and same major fragment ions in their positive MS/MS spectrum However, these isomeric structures in the positive ion mode could not be distinguished based on the MS1 and MS2 data Thus, the similar fragmentation patterns between these two different species complicate accurate structural elucidation and remain challenging For the accurate identification, the three strategies below were taken into account First, both positive and negative ionization mode were used to determine the molecular weight The identification of the flavones were carried out based on the observation of the protonated and deprotonated molecules ([M+H]+ and [M−H]− ions), which have also been described by other authors using IT /Q-TOF [16,17,21] Anthocyanins carry an inherent positive charge and can easily donate protons to free radicals ([M]+ ) under (+) ESI condition Thus, most reported LC/MS studies of anthocyanins were also performed in the positive ion mode because of maximum sensitivity [19–24] Anthocyanins not ionize at all because of the absence of a free hydroxyl group in the negative ion mode, and deprotonated molecular ion ([M–H]– ) for anthocyanins could not be detected because of the neutralization of the charge [19] Thus, the unique molecular ion [M–2H]– in negative ion mode analysis may provide additional information for identification of anthocyanins compounds, but [M–2H]– of anthocyanins and [M–H]– of flavonol glycosides are also the same Only a limited number of LC/MS studies of anthocyanins were carried out with the molecular ion [M– 2H]– in negative ion mode, but the full scan MS1 spectrum from negative ionization mode was complex [22–24] In this study, the molecular ion [M–2H]– for anthocyanins was not detected in the extracted ion chromatogram (EIC) when extracting this specific mass from the negative full-scan MS1 dataset, which there was no mixed peak with the similar retention time when compared with the positive ion mode Thus, for the identification of anthocyanins, the positive ion mode was used for their identification and the negative ionization mode was used for verification in this study This characteristic is especially useful for distinguishing anthocyanin glycosides from flavonol glycosides with the same ‘quasi’-molecular ions (M+ = [M+H]+ and [M–2H]– = [M–H]– ) that co-exist in some plants It becomes very simple to distinguish between two different species if negative ionization mode is employed Thus, it is easy to distinguish rapidly between pelargonidin 3-glucoside and apigenin-7-Oglucoside when comparing the full scan MS spectrum in the negative ionization mode Secondly, anthocyanins compounds have a characteristic elution order in reversed phase liquid chromatography (RP-LC), which elute before the flavonol glycosides [25] The retention times of the pelargonidin 3-O-glucoside (27.11 min) and apigenin-7-O-glucoside (55.90 min) on a reverse phase C18 column differed by 28.8 min, which indicate that peak 27 (pelargonidin 3-O-glucoside) must be an anthocyanin with much higher polarity Thirdly, apigenin-7-O-glucoside were further confirmed by comparing with the fragmentation pattern and chromatographic retention time of its authentic standard Besides, according the studies previously reported, a photodiode array detector (DAD) to measure UV/Vis molecular absorbance was used to differentiate these two different species since anthocyanins have a typical λmax at ∼330 nm and between 440 and 540 nm [5,18,20,23] The absorbance maxima for flavonol glycosides were at 250 and 370 nm Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 [21,24] These strategies are significantly vital considering that MS and MS/MS date obtained under ESI conditions not allow identification of isomeric structures for distinguishing anthocyanin glycosides from flavonol glycosides 3.2 Identification and analysis of absorbed compounds and their metabolites in rat biosamples How to find new candidates as bioactive compounds from medicinal herbs with a higher probability for research and development in drug discovery remain challenging and controversial A common strategy for identifying the active constituents from medicinal herbs in the field of separation science is to screen bioactivity of the isolates with in vitro cell systems evaluation, although there has been a steadily rise in animal and human studies [3,5,6,8,9,11] However, sometimes the biological effects of the screened compounds not live up to in vivo study Because the ingested compounds, at least part of them, reach the circulatory system and specific tissues to exert biological effect as a result of in vivo process of absorption, distribution, metabolism and excretion It is known that the absorbed compounds could be further metabolized by various drug-metabolizing enzymes in vivo [21,25,28] For example, the previous studies reported the isolation of phenolics from pomegranate flowers such as punicatannins A and B, 1, 6-di-O-galloyl-β -D-glucose and 3, 4, 6-tri-O-galloyl-β -Dglucose Furthermore, they evaluated the abilities of these isolates to inhibit α -glucosidase inhibitory activities in vitro for seeking the bioactive antidiabetic compounds from pomegranate flowers [6,11] Thus, we were interested in absorption, distribution and metabolic fate of the compounds after oral administration of pomegranate flowers extract in vivo, especially those isolates In the present study, according to accurate mass and fragmentation pattern generated by the HPLC-Q-TOF-MS2 , 22 absorbed compounds and their 35 metabolites were absolutely or tentatively identified in rat biosamples after oral administration of pomegranate flowers extract The workflow of metabolite identification take three steps, firstly, the probable metabolites were postulated based on the metabolism rules of compounds Secondly, the molecular ion [M+H]+ /[M]+ or [M−H]− for probable metabolites were extracted from the full-scan MS1 dataset of dosed rat biological samples Thirdly, the peaks detected in the EIC were further analyzed by the QTOF-MS/MS dataset of dosed rat biological samples Among them, 15 absorbed compounds and metabolites were further confirmed with their authentic standards Among the metabolites, most of them were found in urine (19 absorbed compounds and 31 metabolites), feces (21 absorbed compounds and 25 metabolites) and plasma (15 absorbed compounds and 17 metabolites) samples, only a few of them were found in tissues, respectively (Table 2) Ellagitannins were abundant in pomegranate flowers extract (18 ellagitannins), but only corilagin was detected in plasma and tissues Galloyl derivates were also abundant in pomegranate flowers extract (14 galloyl derivates), but none of them was detected in plasma and tissues Our results indicate that ellagitannins and galloyl derivates were not well absorbed in plasma and tissues It is worth to note that the isolates (punicatannins A and B, 1, 6-di-O-galloyl-β -Dglucose and 3, 4, 6-tri-O-galloyl-β -D-glucose) were not found in plasma or tissues after oral administration of pomegranate flowers extract This our in vivo finding have not totally supported the in vitro findings that these isolates were bioactive antidiabetic compounds present in pomegranate flower According to previous studies, after normal consumption of ellagitannins-rich foods or extracts, ellagitannins are rarely detected in plasma due to their low bioavailability Ellagitannins are metabolized by the intestinal flora to produce ellagic acid and urolithins metabolites [27–30] Moreover, most of metabolism studies of ellagitannins were mainly focused on ellagic acid, which is one of the main hydrolysates of ellagitannins, and urolithins and their derived metabiolites in plasma, urine and feces in recent years [26–28] Remarkably, in the present study, ellagitannin corilagin and nine phase II conjugate metabolites of corilagin were firstly identified in plasma and tissues after oral administration of pomegranate flowers extract As shown in Fig 5, metabolite C5 (retention time = 11.96 min) had a molecular ion [M−H]− at m/z 809.1056 (C28 H29 O26 − , 0.24 ppm), which was 176 Da higher than that of corilagin (O5), suggesting that C5 was glucuronide conjugate metabolite The fragment ion at m/z 633.0676, as a base peak, was produced by natural loss of a glucuronic acid (176 Da) from the [M−H]− ion Besides, the fragment ion at m/z 463.0532, the typical fragment ions at m/z 300.9958 and m/z 169.0138 were also consistent with those of corilagin Thus, C5 was identified as glucuronidation of corilagin This is a first report to identify monoglucuronide conjugated metabolite of ellagitannin compound in vivo Metabolites C1 and C2 had molecular ion [M−H]− at m/z 713.0298 (C27 H21 O21 S− , −0.56 ppm), which was 80 Da (SO3 ) higher than that of corilagin Thus, C1 and C2 were identified as sulfation metabolites of corilagin Metabolite C3 had a molecular ion [M−H]− at m/z 647.0894 (C28 H23 O18 − , 0.61 ppm), which was 14 Da (CH2 ) higher than that of corilagin Therefore, C3 was identified as methylation metabolite of corilagin Metabolite C4 had a molecular ion [M−H]− at m/z 661.1048 (C29 H25 O18 − , 0.30 ppm), which was 28 Da (2∗ CH2 ) higher than that of corilagin Therefore, C4 was identified as Di-methylation metabolite of corilagin Metabolites C6 and C7 had molecular ion [M−H]− at m/z 823.1208 (C34 H31 O24 − , −0.36 ppm), which was 176 Da (a glucuronic acid) and 14 Da (CH2 ) higher than that of corilagin Therefore, C6 and C7 were identified as glucuronidation and methylation metabolites of corilagin Metabolite C8 had a molecular ion [M−H]− at m/z 837.1364 (C35 H33 O24 − , −0.67 ppm), which was176 Da (a glucuronic acid) and 28 Da (2∗ CH2 ) higher than that of corilagin Therefore, C8 was identified as glucuronidation and di-methylation metabolite of corilagin The fragmentation patterns of metabolites C1–C4 and C6– C8 were similar to that those of metabolites previously reported for corilagin (Fig 5) [29] Furthermore, the binding sites of phase II conjugate metabolites (C1–C9) of corilagin were determined by combining with our previous study [29] The nine phase II conjugate metabolites of corilagin are the methylation, glucuronidation and sulfation conjugated metabolites This finding raises the possibility that phase II conjugate metabolites of ellagitannin corilagin may function as biological antioxidant, anti-inflammatory, α -glucosidase inhibitory and hepatoprotective activities after oral administration of pomegranate flowers extract Besides, 17 metabolites of ellagic acid including urolithins and their derived metabolites were identified in this study Ellagic acid and gallic acid were identified in plasma and tissues Not only ellagic acid and gallic acid are two main compounds in pomegranate flowers, as shown in Fig 6, but also two main the hydrolyzed metabolite of ellagitannins in rats after oral administration of pomegranate flowers extract This may illustrate the wide distribution of ellagic acid and gallic acid in rat biosamples Metabolites urolithin D, urolithin C, urolithin A and urolithin B were identified in plasma and some tissues and further confirmed with their standards Besides, sulfation, methylation, glucuronidation metabolites of urolithins were found in plasma but not much in different organs, some of which have been identified in previous studies [30] The fragmentation patterns of them were consistent with previous study [30] Flavonoids were also abundant in pomegranate flowers extract (18 flavonoids), and most of them was detected in plasma and tissues The free and mainly glucuronide conjugated flavonoids were identified in plasma and liver The flavonoids glucuronide metabolites showed typical loss of a 176 Da (a glu- 10 Table Summary of the mass spectral data and distribution of absorbed compounds and metabolites detected in the rat biological samples orally administrated with pomegranate flower extract tR (min) Biotransformation Formula (neutral) [M−H]− Calculated Observed MS/MS fragments Error (ppm) U F P L S K H Lg O1 G1 G2 G3 O2 O3 O4 O5a C1 C2 C3 C4 C5b C6 C7 C8 C9 O6a E1 E2 E3 E4 E5 E6 E7 E8 E9a E10 2.02 8.45 11.24 3.32 5.90 14.60 21.50 26.46 8.04 24.84 43.56 53.47 20.18 22.66 44.91 5.03 33.19 46.95 8.81 9.56 56.36 55.35 57.38 46.43 50.68 47.78 36.16 27.51 Gallic acid Methylation of gallic acid Methylation of gallic acid Di-methylation of gallic acid Digalloyl-glucoside Galloyl-HHDP-glucoside Galloyl-HHDP-glucoside Corilagin Sulfation of corilagin Sulfation of corilagin Methylation of corilagin Di-methylation of corilagin Glucuronidation of corilagin Glucuronidation and methylation of corilagin Glucuronidation and methylation of corilagin Glucuronidation and di-methylation of corilagin Di-glucuronidation of corilagin Ellagic acid Sulfation of ellagic acid Glycosylation of ellagic acid Methylation of ellagic acid Methylation and sulfation of ellagic acid Methylation and sulfation of ellagic acid Glucuronidation and methylation of ellagic acid Glucuronidation and di-methylation of ellagic acid Methylation and glycosylation of ellagic acid Urolithin D Sulfation and di-methylation of Urolithin D C7 H6 O5 C8 H8 O5 C8 H8 O5 C8 H8 O5 C20 H20 O14 C27 H22 O18 C27 H22 O18 C27 H22 O18 C27 H22 O21 S C27 H22 O21 S C28 H24 O18 C29 H26 O18 C28 H30 O26 C34 H32 O24 C34 H32 O24 C35 H34 O24 C39 H38 O30 C14 H6 O8 C14 H6 O11 S C20 H16 O13 C15 H8 O8 C15 H8 O11 S C15 H8 O11 S C21 H16 O14 C22 H18 O14 C21 H18 O13 C13 H8 O6 C15 H12 O9 S 169.0142 183.0299 183.0299 197.0455 483.0780 633.0733 633.0733 633.0733 713.0302 713.0302 647.0890 661.1046 809.1054 823.1211 823.1211 837.1367 985.1375 300.9990 380.9558 463.0518 315.0146 394.9715 394.9715 491.0467 505.0624 477.0675 259.0248 367.0129 169.0141 183.0297 183.0297 197.0452 483.0773 633.0726 633.0726 633.0733 713.0298 713.0298 647.0894 661.1048 809.1056 823.1208 823.1208 837.1364 985.1386 300.9994 380.9555 463.0522 315.0140 394.9718 394.9718 491.0463 505.0621 477.0679 259.0245 367.0129 125.0241 168.0061, 124.0165 168.0067, 124.0165 169.0126, 125.0245 313.0567, 169.0139, 125.0248 463.0514, 300.9992, 169.0144 463.0519, 300.9993, 169.0140 463.0524, 300.9991, 169.0136 633.0713, 463.0522, 300.9993, 633.0713, 463.0522, 300.9993, 463.0490, 300.9990 477.0641, 315.0149, 169.0138 633.0676,463.0532, 300.9958 647.0860, 463.0538, 300.9991 647.0860, 463.0538, 300.9991 661.1035, 477.0670, 315.0153 633.0781, 300.9973 283.9972, 257.0091, 229.0137, 300.9992, 229.0169 300.9995, 201.0220 299.9910 315.0146, 299.9905 315.0146, 299.9905 315.0146, 299.9908, 201.0213 329.0308, 314.0066, 299.9909, 315.0146, 299.9903, 201.0202 241.0152, 231.0299 287.0575, 259.0604 −0.59 −1.09 −1.09 −1.52 −1.44 −1.10 −1.10 −0.56 0.56 0.61 0.30 0.24 −0.36 −0.36 −0.67 −0.35 1.32 −0.78 0.86 −1.90 0.75 0.75 −0.81 −0.59 0.83 −1.15 + + + + – + + + + + + + + + + + + + + + + + + + + + + + + + + – + – + + + + + + – + + – – + + + – + + + + + + + + + + – – – – + – – + + + + + + – + – – + + – – – + + + + – – – – – – + – – + – – + + + – + + – + + + + – + – – + – – – – – – + – – – – – – – – – + – – + – – – – + – – + – – – – – – + – – – – – + + + – + – – – – – – – – – + + – – – – – – + – – – – – + + – – + – – – – – – – + – – – – – – – – – – – – – – – – – – – + – + – – + + – – – – 169.0144 169.0144 201.0192 201.0234 (continued on next page) Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 NO Table (continued) E11 E12a E13 E14 E15a E16 E17a O7a O8a O9a O10 O11 O12 O13 O14a O15a A1 O16 O17a O18a L1 L2 O19a O20 O21 T1 T2 T3 O22 a b tR (min) Biotransformation 11.96 11.14 13.77 9.37 58.98 35.17 67.47 20.23 24.32 29.12 32.34 46.64 34.66 18.83 67.11 56.44 52.79 43.25 65.75 50.29 54.99 41.62 67.29 56.40 58.65 61.29 49.28 51.81 67.30 Methylation and glucuronidation of Urolithin D Urolithin C Glucuronidation of urolithin C Di-glucuronidation of urolithin C Urolithin A Sulfation and glucuronidation of Urolithin A Urolithin B Brevifolincarboxylic acid Ethyl gallate Brevifolin Methyl brevifolincarboxylate Ethyl brevifolincarboxylate Digalloyl-HHDP-glucoside Pelargonidin 3,5-diglucoside Apigenin Apigenin-7-O-glucoside Glucuronidation of apigenin Apigenin-di-glucoside Luteolin Luteolin 7-O-glucoside Glucuronidation of luteolin Di-glucuronidation of luteolin Kaempferol Kaempferol-3-O- glucoside Tricetin Glucuronidation and di-methylation of tricetin Di-lucuronidation of tricetin Di-glucuronidation and methylation of tricetin Tricin Formula (neutral) [M−H]− Calculated Observed MS/MS fragments C21 H22 O11 C13 H8 O5 C19 H16 O11 C25 H24 O17 C13 H8 O4 C19 H16 O13 S C13 H8 O3 C13 H8 O8 C9 H10 O5 C12 H8 O6 C14 H10 O8 C15 H12 O8 C34 H26 O22 C27 H31 O15 C15 H10 O5 C21 H20 O10 C21 H18 O11 C27 H31 O15 C15 H10 O6 C21 H20 O11 C21 H18 O12 C27 H26 O18 C15 H10 O6 C21 H20 O11 C15 H10 O7 C23 H22 O13 C27 H26 O19 C28 H28 O19 C17 H14 O7 449.0725 243.0299 419.0620 595.0941 227.0350 483.0239 211.0401 291.0146 197.0455 247.0248 305.0303 319.0459 785.0843 595.1663 271.0601 433.1129 447.0922 595.1663 287.0550 449.1078 463.0871 639.1192 287.0550 449.1078 303.0499 507.1133 655.1141 669.1298 331.0812 449.0731 243.0294 419.0617 595.0936 227.0348 483.0234 211.0402 291.0146 197.0450 247.0242 305.0300 319.0455 785.0838 595.1669 271.0605 443.1133 447.0926 595.1668 287.0555 449.1085 463.0877 639.1189 287.0558 449.1088 303.0505 507.1139 655.1136 669.1292 331.0818 273.0409, 215.0349, 243.0254 419.0614, 198.0320, 403.0688, 167.0502, 247.0252, 169.0144, 219.0305, 273.0061, 273.0036, 615.0602, 433.1133, 153.0173 271.0612 271.0612 433.1133, 153.0178 287.0573, 287.0573, 463.0871, 153.0175 287.0573, 153.0193 331.0825, 479.0829, 493.0984, 315.0496, 259.0606 187.0401 243.0245 182.0371 227.0359 139.0552 219.0296, 125.0242 191.0348 245.0082, 245.0085, 463.0499, 271.0606 191.0348 217.0141 217.0141 300.9990, 169.0130 271.0606 153.0187 153.0187 287.0573, 153.0187 153.0187 303.0499 303.0499 317.0567, 303.0513 299.0549, 133.1010 Error (ppm) U F P L S K H Lg 1.33 −2.05 −0.71 −0.84 −0.88 −1.03 0.28 −2.53 −2.42 −0.98 −1.25 −0.63 1.00 1.47 0.92 0.89 0.84 1.74 1.55 1.29 −0.46 2.78 2.22 1.96 1.18 −0.76 −0.89 1.81 + + – – + – – + – + + + – + + + + + + + + + + + + + + + + + + + – – – + + + + + + + + + + + + + + + + + + + + – – + – + – – + + + + – + + + – + + + + – + + + – + – + – – – + + – – + – + + + – + + + – – + + + – + + + – + – + – – – + – – – – – – – + – + + – – – + – + – – – + – – – – – – – – – + + – – – – – – + + – – – + – – – – – – – – – – – – – – – – – + – – – + – + – – – – – – – – – – – – – – – – – – – – + – – – – – – – + – – – – – – – – – – – – – – + – – – – Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 NO Confirmed by using reference standard Firstly identified metabolites in vivo 11 12 Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 Fig Extracted ion chromatogram and the MS/MS spectra of phase II conjugate metabolites of corilagin in rats after oral administration of pomegranate flowers extract Fig The possible metabolic pathways of corilagin (ellagitannins to urolithins) in rats orally administered with pomegranate flowers extract (Me: Methylation; GluA: Glucuronidation; Glu: Glycosylation; SO3 : Sulfation) Solid and dotted box represent the confirmed and possible determination of binding sites of respective metabolites curonic acid), which were traditionally considered to be monoglucuronides However, except absorbed compound pelargonidin3, 5-O-diglucoside, there was not detected any metabolites of the four anthocyanins in plasma and tissues Interestingly, brevifolin and its three derivates were all detected in plasma and liver This result indicates that brevifolin and its derivates were well absorbed in plasma Thus, characterizing its multiple constitution, absorption and metabolic fate of these compounds in vivo is helpful to better analyze the active components in pomegranate flowers Z Yisimayili, R Abdulla and Q Tian et al / Journal of Chromatography A 1604 (2019) 460472 Conclusion In summary, the present study applied HPLC-Q-TOF-MS2 to characterize the polyphenols composition of pomegranate flowers and then their appearance as native form, including their metabolites in rat urine, feces, plasma and tissues after oral administration of pomegranate flowers extract The 67 compounds identified in pomegranate flowers, but only 22 compounds detected in rat biosamples This result showed that not all compounds abundant in pomegranate flowers extract could be absorbed well in plasma and tissues This finding also suggested a potential correlation between study on metabolic profile of these compounds in vivo and study on strategy of screening bioactivity of the isolates with in vitro cell systems evaluation To the best of our knowledge, this is the first time to analyze the metabolic profile of pomegranate flowers in vivo This study was expected to provide significant information to find possible candidates for the real bioactive compounds in pomegranate flowers and provide a solid basis for the study of quality control of pomegranate flowers Declaration of Competing Interest None Acknowledgment This present study was financially supported by The Science and 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K Aaby, G.I .A Borge, Characterization, quantification, and yearly variation of the naturally occurring polyphenols in a common red variety of curly kale (Brassica oleracea L convar acephala var... was used for the identification of components in pomegranate flowers extract and its metabolites in rat biosamples The chromatographic separation for pomegranate flowers extract and biological samples