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Kinetic disposition of dietary polyphenols and methylxanthines in the rat mammary tissue
Journal of Functional Foods 61 (2019) 103516
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Kinetic disposition of dietary polyphenols and methylxanthines in the rat mammary tissue María Ángeles Ávila-Gálvez, María Romo-Vaquero, Antonio González-Sarrías, Juan Carlos Espín
T ⁎
Laboratory of Food & Health, Research Group on Quality, Safety, and Bioactivity of Plant Foods, CEBAS-CSIC, 30100 Campus de Espinardo, Murcia, Spain
ARTICLE INFO
ABSTRACT
Keywords: Mammary tissue Methylxanthine Polyphenol Breast cancer Resveratrol Plant extract
We recently showed that methylxanthines and conjugated phenolic-derived metabolites reached the mammary tissue (MT) of breast cancer patients after consuming a blend of phenolic-rich extracts. The pre-surgery fasting could prevent the detection of some (including those non-conjugated) metabolites. We investigated here the pharmacokinetics in rat plasma and MT of phenolic-derived metabolites and methylxanthines using the same blend (pomegranate, olive, cocoa, orange, lemon, and grapeseed extracts plus resveratrol; containing 37 phenolics, theobromine, and caffeine). We also compared the pharmacokinetics of resveratrol when administered in the blend or individually. We show for the first time that micromolar levels of methylxanthines and conjugatedderived (but not free) metabolites from resveratrol, dihydroresveratrol, hesperetin, urolithins, and hydroxytyrosol reached the MT. Resveratrol-3-glucuronide and resveratrol-3-sulfate showed shorter Tmax (plasma and MT) and resveratrol-3-sulfate higher Cmax (MT) when resveratrol was administered individually. This study could help to conveniently design preclinical studies using physiologically relevant conditions in (breast) cell models.
1. Introduction The new global cancer data (Bray et al., 2018) reveal that breast cancer is the most commonly diagnosed cancer in women (24.2%), and the leading cause of cancer death in women (15%) (Bray et al., 2018). It is known that the non-hereditary factors are the main drivers of incidence, including long-term endogenous hormonal exposures and exogenous hormone intake, nulliparity, early age at menarche, alcohol intake and obesity (Bray et al., 2018). Regarding protective factors, long-term breastfeeding and physical activity may be beneficial (Brinton, Gaudet, & Gierach, 2018), and some studies also suggest the protection of vegetable consumption against estrogen receptor-negative (ER-) breast cancer risk (Emaus et al., 2016). The recent Continuous Update Project (CUP) only observed a partial protective association between breast cancer risk with the intake of fruits, vegetables, tea, coffee, isoflavones, and carotenoids (WCRF/AICR, 2017). However, it has been suggested that the mixture of phytochemicals found in whole
foods, rather than isolated phytochemicals, exerts effects against carcinogenesis (Kapinova et al., 2017). Overall, these observations contrast with the abundant research in animal models that describe the preventive effect of polyphenols and polyphenol-rich plant extracts against breast cancer, including pomegranate (Bishayee, Mandal, Bhattacharyya, & Bhatia, 2016), citrus flavanones (Singhal et al., 2018), resveratrol (Sinha, Sarkar, Biswas, & Bishayee, 2016), berries (Amatori et al., 2016) and ellagic acid (Wang et al., 2012). Besides, a large number of studies in cellular models persist in the testing of plant extracts or polyphenols in molecular forms and(or) concentrations, describing many mechanisms, targets and pathways, when in fact they do not reach the mammary tissue (Ávila-Gálvez, González-Sarrías, & Espín, 2018). To shed some light on this topic, we recently conducted a clinical study in breast cancer patients that consumed a cocktail of plant extracts (pomegranate, cocoa, lemon, orange, grapeseed, and olive) plus resveratrol from diagnosis to surgical resection (Ávila-Gálvez et al.,
Abbreviations: DHRSV, dihydroresveratrol; DHRSV 3-glur, DHR 3-O-glucuronide; EIC, extracted ion chromatogram; HP, hesperetin; HP 7-glur, HP 7-O-glucuronide; HP 3′-glur, HP 3′-O-glucuronide; HP 7-sulf, HP 7-O-sulfate; HPLC-ESI-IT-MS/MS, high performance liquid chromatography-electrospray ionisation-ion trap tandem mass spectrometry; Hytyr glur, hydroxytyrosol glucuronide; MeOH, methanol; MT, mammary tissue; MX, methylxanthine; PBS, phosphate buffered saline; PM, phenolic-derived metabolites; RSV, resveratrol; RSV 3-sulf, RSV 3-O-sulfate; RSV 4′-sulf, RSV 4′-O-sulfate; RSV 3-glur, RSV 3-O-glucuronide; UPLC-ESI-QTOF-MS, ultra-high performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry; Uro, urolithin; Uro-A 3-glur, Uro-A 3-O-glucuronide; Uro-A 3-sulf, Uro-A 3-O-sulfate ⁎ Corresponding author. E-mail address: [email protected] (J.C. Espín). https://doi.org/10.1016/j.jff.2019.103516 Received 30 May 2019; Received in revised form 6 August 2019; Accepted 12 August 2019 Available online 21 August 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Phenolic compounds and methylxanthines administered to each rat.a Compound
RT (min)
m/z-
MS/MS
λmax
Source
Content (µg) per capsule
Gallic acid Punicalin* Protocatechuic acid Hydroxytyrosol Theobromineb Punicalagin Catechinc
8.71 9.80 11.56 11.63 12.80 13.12/15.18 15.72
169 781 153 153 181 1083 289
124 721/601/299 109 123 138 781/721/601 271/245/205/179
270 258/380 230/260 272 272 258/380 278
8.41 ± 0.58 40.31 ± 4.64 14.5 ± 1.45 211.12 ± 11.89 265.35 ± 8.12 604.94 ± 67.86 323.35 ± 11.02
Caffeineb Ferulic acid O-Glu* Luteolin 6,8-di-C-Glu* Epicatechinc
17.72 18.00 18.32 18.57
195 355 609 289
138/110 235/217/193/175/160/134 519/489/399/369 271/245/205/179
272 295/328 270/338 280
Ellagic acid-Glu* Apigenin 6,8-di-C-Glu (Vicenin-2)* Eriodictiol-Glu-Rhamn-Glu* Diosmetin 6,8-di-C-Glu* Gallagic acid* Chrysoeriol 6,8-di-C-Glu* Luteolin-C-Glu* Eriocitrin (eriodictiol rutinoside) Ellagic acid-rhamnoside* Apigenin 8-C-xylanopyranosil-Glu* Ellagic acid Quercetin 3-O-rutinoside (Rutin) Luteolin 7-O-rutinoside*
19.30 19.64 19.93 20.51 20.95 21.05 21.96 22.58 22.59 23.39 23.62 23.94 24.06
463 593 757 623 601 623 447 595 447 563 301 609 593
301 503/473/383/353 595/449/287 605/533/503/413/383 299/271/242 533/503/413/383 429/357/327 459/329/287 301 413/341/311/293 256/184 301 285
252/362 271/332 282/330 270/346 254/380 270/346 270/338 284/334 252/364 268/332 254/360 254/350 260/348
Epicatechin gallatec Diosmetin 8-C-Glu* Naringenin 7-O-rutinoside (narirutin)*
24.57 24.88 25.02
441 461 579
397/331/289/193/169 371/341 271
280 268/344 278/328
Catechin gallatec Limocitrin-Glu-HMG-Glu* Apigenin 7-O-rutinoside*
25.72 26.16 26.16
441 813 577
397/331/289/193/169 753/693/651/549 269
278 260/272/350 270/342
Limocitrin-neohesperidoside* Hesperetin 7-O-rutinoside (Hesperidin)
26.33 26.61
653 609
345/330/301 301
259/378 284/336
Diosmetin 7-O-rutinoside (Diosmin)*
26.90
607
299/284
270/334
Isorhamnetin rutinoside* Hesperetin-Glu* Limocitrin-HMG-Glu* Limocitrol-Glu-HMG* Trans-Resveratrol Cis-Resveratrol Isosakuranetin 7-rutinoside* Hesperetin TOTAL PHENOLICS TOTAL METHYLXANTHINES
27.05 27.80 27.98 28.16 28.55 31.68 31.88 35.60
623 463 651 681 227 227 593 301
315/299 301 549/507/345 619/579/537/375 185 185/159 285 –
254/368 284/330 274/350 260/377 306 286 284/332 288/330
Pomegranate Pomegranate Cocoa Olive Cocoa Pomegranate Cocoa Grapeseed Cocoa Lemon Lemon Cocoa Grapeseed Pomegranate Lemon Lemon Lemon Pomegranate Lemon Lemon Lemon Pomegranate Lemon Lemon Lemon Orange Lemon Grapeseed Lemon Lemon Orange Grapeseed Lemon Lemon Orange Lemon Orange Lemon Lemon Orange Orange Orange Lemon Lemon Resveratrol Resveratrol Orange Orange
6.67 ± 0.01 114.55 ± 15.37 10.73 ± 1.16 451.24 ± 13.05 29 ± 2.61 39.44 ± 0.58 22.62 ± 2.61 19.72 ± 1.16 18.27 ± 1.74 65.54 ± 1.16 9.28 ± 0.87 401.07 ± 28.42 12.18 ± 1.16 17.11 ± 2.61 1,390.55 ± 140.65 2.9 ± 0.29 1.45 ± 0.58 38.28 ± 0.87 97.73 ± 2.9 24.36 ± 0.87 NC 13.63 ± 4.64 36.54 ± 1.45 13.05 ± 0.58 NC 1.74 ± 0.58 NC 1,077.35 ± 24.65 63.51 ± 2.32 NC 29.0 ± 18.27 1.45 ± 0.87 1.16 ± 0.29 4.35 ± 0.29 NC 2,207.48 ± 207.06 6.67 ± 1.74 13.63 ± 4.64 7.54 ± 0.29 7,445.75 ± 583.77 272.02 ± 8.13
*Compounds ‘tentatively identified’ based on the their m/z, MS/MS fragmentation patterns and UV spectra; RT, retention time; NC, identified but not quantified; Glu, glucoside; Rhamn, rhamnoside; HMG, 3-hydroxy-3-methyl-glutaryl-. a Adapted from Ávila-Gálvez et al. (2019). Values are shown as mean ± SD; btheobromine and caffeine were determined in m/z+ mode; ctotal content determined by the phloroglucinolysis reaction (Kennedy & Jones., 2001).
2019). We detected 39 and 33 phenolic-derived metabolites and 8 and 6 methylxanthines, in normal and malignant mammary tissues, respectively. The concentrations roughly reached the range of nanomolar, and all phenolic metabolites were conjugated except for two dihydroxybenzoic acid derivatives (Ávila-Gálvez et al., 2019). In that study, we identified two potential variables that could affect the results obtained: i) the fasting of patients before surgery (around 10–12 h) could prevent a higher occurrence of metabolites in mammary tissues, and ii) the simultaneous administration of many phenolics (which is a plausible dietary context) could interfere with each other in their absorption and metabolism. To answer the above open questions, we evaluated here the distribution kinetics of the same phenolics in the rat mammary tissue and determined a comparative plasma and mammary tissue pharmacokinetic study of the primary occurring phenolic and methylxanthine metabolites.
2. Materials and methods 2.1. Chemicals Uro-A 3-O-glucuronide (Uro-A 3-glur), Uro-A 3-O-sulfate (Uro-A 3sulf), HP 7-O-glucuronide (HP 7-glur), HP 3′-O-glucuronide (HP 3′glur), HP 7-O-sulfate (HP 7-sulf), were obtained from Villapharma Research S.L. (Parque Tecnológico de Fuente Alamo, Murcia, Spain). Resveratrol (RSV, 3,5,4′-trihydroxy-trans-stilbene) was obtained from Laboratorios Admira S.L. (Alcantarilla, Murcia, Spain). DHRSV 3-Oglucuronide (DHRSV 3-glur), RSV 3-O-sulfate (RSV 3-sulf), RSV 4′-Osulfate (RSV 4′-sulf), RSV 3-O-glucuronide (RSV 3-glur) were obtained as described elsewhere (Azorín-Ortuño et al., 2011). Hydroxytyrosol glucuronide (Hytyr glur) was synthesized according to Lucas, Alcantara, and Morales (2009). Phosphate buffered saline (PBS) was 2
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Fig. 1. Experimental design.
Committee (Madrid, Spain; reference 201770E081). Animals were housed 3–4 to a cage in a room with controlled temperature (22 ± 2 °C), 55 ± 10% relative humidity, and a 12 h light-dark cycle. Animals were fed with a rat standard chow (Panlab, Barcelona, Spain). Diet and tap water were administered ad libitum until 6 h before the start of the experiment. The experimental design is shown in Fig. 1. One capsule was administered by gavage to each fasting animal, and a set of 4 animals was sacrificed with carbon dioxide at 0.5, 1, 2, 4, 6, 10, and 16 h after the capsule administration. A set of four animals also received one empty capsule and served as controls.
purchased from Sigma-Aldrich (St. Louis, MO, USA), and liquid chromatography-mass (LC-MS) grade solvents from J.T. Baker (Deventer, The Netherlands). Mili-Q ultra-pure water system (Millipore Corp. Bedford, MA, USA) was used in all experiments. 2.2. Plant extracts Two formulations were used in the present study: RSV alone and a blend of RSV plus orange, lemon, pomegranate, cocoa, grapeseed, and olive extracts (hereafter termed ‘blend’). Laboratorios Admira S.L provided the plant extracts. Both formulations were encapsulated in hard gelatin capsules (size 9), specially designed for their administration to rats weighing ~250 g (Torpac, Fairfield, USA). The same blend was previously assayed in breast cancer patients (Ávila-Gálvez et al., 2019), and the detailed composition can be found in Table 1. In the present study, each capsule contained a total of 29 mg. The formulation of the blend included 2.2 mg RSV plus 6.7 mg pomegranate, 6.7 mg olive, 6.7 mg cocoa, 2.2 mg orange, 2.2 mg lemon, and 2.2 mg grapeseed extracts. Each capsule of this formulation provided 7.4 mg of phenolics, 0.3 mg theobromine, and 0.007 mg caffeine. The human equivalent doses (HED) were 336 mg, 13.6 mg and 0.3 mg of phenolics, theobromine, and caffeine, respectively, in a 70 kg person (Reagan-Shaw, Nihal, & Ahmad, 2008). Each capsule of the RSV formulation contained only 2.2 mg RSV (HED of 100 mg in a 70 kg person) plus 26.8 mg microcrystalline cellulose as an excipient. Table 1 shows the detailed formulation of phenolic compounds and methylxanthines consumed by the rats in each capsule.
2.4. Sampling procedure Blood samples were collected by cardiac puncture in EDTAtreated tubes at each time-point after sacrifice, and mammary tissues were collected and stored at −80 °C until their analysis (Fig. 1). Blood was immediately centrifuged at 14,000g for 15 min at 4 °C in a Sigma 1–13 microcentrifuge (Braun Biotech. International, Melsungen, Germany). Plasma samples were extracted with acetonitrile:formic acid (98:2, v/v), centrifuged, and the supernatant reduced to dryness in a speed vacuum concentrator. The evaporated samples were re-suspended in methanol, filtered through a 0.22 µm polyvinylidene fluoride (PVDF) filter and injected in the UPLC-ESIQTOF-MS equipment. At sacrifice, inguinal and thoracic mammary glands were removed, extensively washed with cold PBS to avoid external blood contamination, and snap frozen in liquid nitrogen. Mammary tissues (300 mg) were processed as previously described (Ávila-Gálvez et al., 2019).
2.3. Animals and experimental design
2.5. UPLC-ESI-QTOF-MS analysis of plasma and mammary tissue
Sixty 11-weeks-old female Sprague Dawley rats weighing 258 ± 29 g were purchased from the Animal Experimentation Service of the University of Murcia (Murcia, Spain). The experimental protocol complied with the Arrive guidelines, followed the Directive of the European Council 2010/63/UE, and was approved by the Animal Experimentation Ethics Committee from University of Murcia (reference ES300305440012), the local government (reference A13180503) and the Spanish National Research Council’s Bioethics
Phenolic-derived metabolites and methylxanthines were identified and quantified in plasma and mammary tissue by UPLC-ESI-QTOF-MS as previously described (Ávila-Gálvez et al., 2019). More than 180 potential compounds (phenolics present in the extracts as well as derived metabolites both unconjugated and conjugated such as glucuronides, sulfates, sulfoglucuronides, etc.), were browsed in plasma and mammary tissue samples. The method was validated for linearity, 3
Journal of Functional Foods 61 (2019) 103516
± 4.2 ± 2.7 ± 2.4
1.2 0.6 4.0 1.1 ± ± ± ±
Hytyr, hydroxytyrosol; HP, hesperetin; Uro-A, urolithin A; RSV, resveratrol; DHRSV, dihydroresveratrol; glur, glucuronide; sulf, sulfate; –, not determined; Tmax, time of maximum concentration; Tlast, time of the last measurable concentration; T1/2, time required for the concentration to reach half of its original value; Cmax, maximum concentration; AUC0-t, area under the curve from the time of dosing to the final quantifiable concentration; MRT0-t, mean residence time from the time of dosing to the time of the final quantifiable concentration. Asterisks designate a significant difference when RSV was administered in the blend or individually (*P < 0.05; ***P < 0.001).
1.5 ± 0.3 7.6 ± 3.6 7.5 ± 3.1 − 12.0 ± 1.2 − 5.5 ± 0.4 5.2 ± 1.1 4.8 ± 0.7 4.1 ± 0.7 9.6 ± 1.6 9.0 ± 1.6 6.1 ± 0.6 5.1 ± 0.7 6.1 ± 0.7 1.6 ± 0.1 8.2 ± 2.5 8.2 ± 2.2 8.8 ± 0.8 11.5 ± 0.5 12.0 ± 0.3 4.9 ± 0.7 5.1 ± 1.1 4.9 ± 1.3 3.4 ± 1.3 9.2 ± 0.9 9.5 ± 1.3 6.5 ± 0.5 5.5 ± 0.8 4.7 ± 1.5 110.3 ± 68.7 10.3 ± 6.1 14.4 ± 3.4 − 47.9 ± 14.6 − 4,631 ± 794.2 7,256 ± 1,040 237.5 ± 150.3 399.2 ± 8.6 1,654 ± 876.7 2,571 ± 1,697 4,062 ± 386.2 68.5 ± 7.5 376.9 ± 81.1 1.4 − − − − − 2.9 2.9 5.0 3.4 − − 5.6 6.8 7.3 0.8 ± 0.1 − − − − − 2.4 ± 1.3 5.1 ± 2.8 2.1 ± 0.5 3.5 ± 0.8 − − 5.2 ± 1.5 11.7 ± 9.1 4.1 ± 1.0 1.0 ± 0.7 9.5 ± 4.7 8.0 ± 5.4 − 13.0 ± 4.2 − 3.3 ± 2.2 1.7 ± 0.6* 4.0 ± 0.0 1.5 ± 0.7*** 9.0 ± 2.0 8.7 ± 2.3 3.0 ± 2.0 3.0 ± 2.0 2.5 ± 1.0 0.9 ± 0.8 9.5 ± 4.7 10.0 ± 6.0 10.0 ± 0.0 13.0 ± 4.2 16.0 ± 0.0 3.25 ± 2.2 1.0 ± 0.7*** 4.0 ± 0.0 0.6 ± 0.3*** 8.0 ± 2.3 8.7 ± 2.3 2.3 ± 1.5 3.5 ± 1.9 2.0 ± 0.0 a
Hytyr glur HP 3-glura HP 7-glura HP 7-sulfa Uro-A 3-glura Uro-A 3-sulfa RSV 3-glura RSV 3-glurb RSV 3-sulfa RSV 3-sulfb DHRSV 3-glura DHRSV 3-glurb Theobrominea Theophyllinea Caffeinea
Plasma MT Plasma
MT
± 0.5
4.0 ± 0.0 12.0 ± 4.9 14.0 ± 3.5 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 14.5 ± 3.0 13.5 ± 5.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 10.0 ± 0.0 12.0 ± 4.9
3.0 ± 1.2 10.5 ± 4.1 11.0 ± 4.1 − 16.0 ± 0.0 − 16.0 ± 0.0 16.0 ± 0.0 13.0 ± 6.0 10.0 ± 0.0 16.0 ± 0.0 14.0 ± 3.5 16.0 ± 0.0 10.0 ± 0.0 16.0 ± 0.0
457.0 ± 147.5 29.1 ± 7.3 18.3 ± 1.2 1.2 ± 0.3 149.5 ± 16.0 5.8 ± 1.3 8,939 ± 1,538 11,209 ± 4,405 758.6 ± 94.9 1,397 ± 482.1 1,980 ± 835.6 3,162 ± 1,835 1,525 ± 348.3 39.1 ± 7.5 159.0 ± 69.5
77.2 ± 38.7 2.1 ± 0.7 2.3 ± 0.3 − 6.7 ± 3.8 − 668.4 ± 126.9 1,262 ± 597.0* 61.6 ± 28.3 112.2 ± 40.9 206.3 ± 113.1 406.2 ± 331.6 677.7 ± 186.3 10.9 ± 2.3 53.0 ± 13.1
803.0 ± 171.2 153.3 ± 87.6 146.9 ± 31.5 10.6 ± 4.0 1,408 ± 156.9 49.3 ± 3.9 63,812 ± 13,364 53,393 ± 6,067 3,356 ± 962.4 4,375 ± 820.6 16,460 ± 5,265 21,568 ± 10,654 14,351 ± 787.5 240.0 ± 26.8 828.6 ± 205.7
MT Plasma MT (pmol/g h) Plasma
MT
Plasma (nM)
MT (pmol/g)
Plasma (nM h)
MRT0-t (h) AUC0-t Cmax Tlast (h) T1/2 (h) Tmax (h) Metabolites
Table 2 Plasma and mammary tissue (MT) pharmacokinetic parameters of the primary metabolites derived from phenolics and methylxanthines after consumption of the blend (RSV plus extractsa) or RSV individuallyb.
M.Á. Ávila-Gálvez, et al.
precision, accuracy, limits of detection (LOD) and quantification (LOQ) as well as for matrix effects. Quantification of metabolites was carried out by peak area integration of their extracted ion chromatogram (EIC) using calibration curves of standards. Phenolic metabolites were quantified in negative mode and methylxanthines in positive mode (Ávila-Gálvez et al., 2019). 2.6. Pharmacokinetic analysis Plasma and mammary tissue-time data of the primary metabolites were analyzed. Pharmacokinetic parameters were determined by non-compartmental analysis using the PKSolver, a complement addin software of Microsoft Excel (Zhang, Huo, Zhou, & Xie, 2010). The parameters are shown as the mean ± standard deviation (SD). The statistical comparison of parameters (Tmax, Cmax, etc.) of metabolites derived from RSV, when administered in the blend or separately, were analyzed on the original data using the t-test or the Wilcoxon signed-rank test. Plots were performed using Sigma Plot 13.0 (Systat Software, San Jose, CA, USA). Statistical significance was set at P < 0.05. 3. Results 3.1. Plasma and mammary tissue pharmacokinetics of phenolic-derived metabolites Table 2 shows the most representative phenolic-derived metabolites and their pharmacokinetic parameters in plasma and mammary tissue after consuming the blend or RSV alone. The more abundant polyphenol families existing in the blend were RSV (2.2 mg), ellagic acid derivatives (~2.1 mg), flavanones (~1.6 mg) and procyanidins (~0.9 mg) (Table 1). The highest Cmax values were observed for RSVderived metabolites (RSV 3-glur > DHRSV 3-glur > RSV 3-sulf in both plasma and MT), reaching the highest value in plasma in the case of RSV 3-glur (11.2 ± 4.4 μM), when administered individually (Table 2, Fig. 2A). As expected, MT and plasma Tmax values were remarkably longer for the microbial-derived metabolite DHRSV 3-glur (Table 2, Fig. 2E, F). The kinetic profiles for RSV 3-glur and RSV 3-sulf were different in both plasma (Fig. 2A) and MT (Fig. 2B), when RSV was administered individually (Table 2). In this case, MT and plasma Tmax values for RSV metabolites significantly decreased while Cmax and AUC values increased (Table 2, Fig. 2), but only became statistically significant for Cmax in MT in the case of RSV 3-glur (Table 2). Regarding DHRSV 3-glur, the administration of RSV separately only increased Cmax and AUC values in MT, but not statistically significant (Table 2; Fig. 2E, F). In the case of olive Hytyr, the primary derived metabolite was Hytyr glur that peaked in both plasma and MT at 0.5 h after the capsule administration (Table 2, Fig. 3A). Hytyr sulfate was also identified in MT from some animals, but it was not quantified due to the lack of an authentic standard. The kinetics of hesperetin conjugates (3-glur, 7glur, and 7-sulf), derived from flavanones (Table 2, Fig. 3B, C), and urolithin conjugates (3-glur and 3-sulf) derived from ellagic acid and ellagitannins (Table 2, Fig. 3D), were similar in both plasma and MT (Fig. 3B-D). As these metabolites are products of the gut microbiota metabolism, an expected delay was also observed in their Tmax values (Table 2, Fig. 3B-D). Finally, the procyanidin-derived metabolite 5(3′,4′-dihydroxyphenyl)-γ-valerolactone 3′-sulfate was observed only in some animals, which prevented the corresponding pharmacokinetic analysis. 3.2. Plasma and mammary tissue pharmacokinetics of methylxanthines Fig. 4 shows the kinetic profiles of theobromine, theophylline, and caffeine in plasma (Fig. 4A) and MT (Fig. 4B). These profiles were similar, although Cmax and AUC values were much higher for 4
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A 20000
Blend
16000
RSV 3-glur (MT)
2000
Blend
1600
RSV alone
MT (pmol/g tissue)
Plasma concentration (nM)
B
RSV 3-glur (plasma)
12000 8000
RSV alone
1200
4000
800 400
0
0 0
2
4
6
8
10
12
14
16
0
2
4
6
Time (hours)
D
MT (pmol/g tissue)
Plasma concentration (nM)
14
16
12
14
16
12
14
16
Blend
Blend RSV alone
1500
RSV alone
150
100
50
0
0 0
2
4
6
8
10
12
14
0
16
2
4
6
E
F
DHRSV 3-glur (plasma)
4500
8
10
Time (hours)
Time (hours)
DHRSV 3-glur (MT)
600
Blend
Blend
500
RSV alone 3000
MT (pmol/g tissue)
Plasma concentration (nM)
12
RSV 3-sulf (MT)
200
RSV 3-sulf (plasma)
3000
10
Time (hours)
C 4500
8
1500
RSV alone
400 300 200 100
0
0 0
2
4
6
8
10
12
14
16
0
Time (hours)
2
4
6
8
10
Time (hours)
Fig. 2. Plasma (A, C, E) and mammary tissue (MT) (B, D, F) absorption-time profiles of resveratrol (RSV) metabolites after administration of the blend (●) or RSV individually (○). RSV 3-glur (resveratrol 3-O-glucuronide), RSV 3-sulf (resveratrol 3-O-sulfate), and DHRSV 3-glur (dihydroresveratrol 3-O-glucuronide). Resveratrol 4′-O-sulfate was detected in the plasma of some animals (results not shown). Results are expressed as mean ± SD (nM in plasma and pmol/g tissue in mammary tissues).
theobromine (Table 2), the main ingested cocoa-derived methylxanthine occurring in the blend (Table 1). Remarkably, theobromine showed relatively high Cmax and AUC values in MT, close to those of RSV 3-glur (Table 2), although theobromine content in the blend was ~10-fold lower than that of RSV (Table 1).
4. Discussion We have recently described the occurrence of a wide range of metabolites derived from phenolics and methylxanthines, in normal and malignant mammary tissues, from breast cancer patients after consumption of a blend containing RSV and phenolic-rich plant extracts 5
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Fig. 3. Plasma (●,▲) and mammary tissue (MT) (□) absorption-time profiles of hydroxytyrosol (A), hesperetin (B, C) and urolithin A (D) metabolites after administration of the blend. Hytyr glur (hydroxytyrosol glucuronide), HP 3′-glur (hesperetin 3′-O-glucuronide), HP 7-glur (hesperetin 7-O-glucuronide), HP 7-sulf (hesperetin 7-O-sulfate), Uro-A glur (urolithin A glucuronide), and Uro-A sulf (urolithin A sulfate). Results are expressed as mean ± SD (nM in plasma and pmol/g tissue in mammary tissues).
(Ávila-Gálvez et al., 2019). However, after that trial, we left two open questions to be answered: (i) did the fasting of the patients before the surgery prevent the appearance of a higher concentration of metabolites in the mammary tissues? And, (ii) could metabolites reach higher levels in the tissue if they are administered individually rather than in a mixture? Besides, the most representative metabolites detected in the mammary tissues were all conjugated (Ávila-Gálvez et al., 2019). Therefore, the present kinetic study was also conceived to know whether free (unconjugated) phenolic-derived metabolites could reach the mammary tissues at shorter times after the consumption of the blend. We assayed here the same mixture as that used in the human study. However, although the proportion of phenolics and methylxanthines was equivalent, the total amount of phenolics and methylxanthines was slightly lower in the rats, i.e., 474 mg phenolics and 19 mg methylxanthines in the human trial, and 336 mg phenolics and 13.9 mg methylxanthines, in the rat study (expressed as HED). In agreement with previous reports that described some differences in the metabolism of RSV depending on the species, the predominant metabolite detected in the plasma, muscle and adipose tissues is RSV 3glur (Andrés-Lacueva et al., 2012) in contrast to humans, where RSV 3sulf is more abundant in plasma and breast tissue (Table 2) (ÁvilaGálvez et al., 2019; Boocock et al., 2007). Besides, procyanidin-derived metabolites were relatively abundant in the mammary tissues of breast cancer patients (Ávila-Gálvez et al., 2019), especially dihydroxybenzoic acid derivatives and 3′,4′-dihydroxyphenyl)-γ-valerolactone 3′-sulfate, while these metabolites were hardly detected in the present rat study, which suggested differences in the metabolism of procyanidins by rats
vs humans. In the case of other metabolites produced by the action of the gut microbiota (hesperetin, urolithins, and DHRSV), the metabolites peaked in plasma and MT from 10 h to 16 h (Table 2). These delayed Tmax agree mainly with the relatively high DHRSV 3-glur concentration detected in fasting breast cancer patients (Ávila-Gálvez et al., 2019). Therefore, taking into account the MRT values or the metabolites (Table 2), the present rat study confirms that fasting of patients prevented the occurrence of some phenolic-derived metabolites such as Hytyr glur. Besides, this was the reason for not being able to detect high concentrations of others, like RSV conjugates that reached micromolar levels (Table 2) instead of nanomolar range detected in MT from breast cancer patients (Ávila-Gálvez et al., 2019). The present study shows that the kinetics of RSV-derived metabolites was different when RSV was administered in the blend or individually (Fig. 2; Table 2). The absorption was faster when RSV was delivered separately, as indicated by the significant difference in the Tmax values of the corresponding derived metabolites (Table 2). However, the rest of the pharmacokinetic parameters were not affected except for RSV 3-sulf that peaked at higher concentration in MT when administered individually (Table 2). This behaviour could depend on the specific phenolic since the absorption and excretion of monomeric flavan-3-ols have been reported to be similar when ingested individually or in a blend (Borges et al., 2010). Overall, it seems that the co-administration of a mixture of phenolics could compete for ABC transporters and delay their absorption (Planas, Alfaras, Colom, & Juan, 2012). Methylxanthines have been reported to be detected in the mammary 6
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A
Plasma
2500
Theobromine Theophylline
example, taking into account the blood distribution in healthy breast tissue (Mankoff et al., 2002), our results show a plausible physiological mixture of approximately 10 μM in MT, mainly composed of RSV 3glur, RSV 3-sulf, and DHRSV 3-glur, and varying the exact proportions depending on the animal species. We acknowledge that the assay of conjugates may discourage researchers since conjugation hampers many direct antiproliferative and proapoptotic related effects (ÁvilaGálvez, Espín, et al., 2018). However, cell cultures (from systemic tissues) should not contain free phenolics, including RSV, to describe mechanisms of action or to describe a direct effect on the cells (ÁvilaGálvez, González-Sarrías, et al., 2018).
250
200
1500
150
1000
100
5. Conclusions
50
500
0
Theophylline, caffeine (nM)
Theobromine (nM)
Caffeine 2000
The present study was conducted in rats because it is not possible, or it would be extremely challenging to perform a kinetic disposition of phenolics and derived metabolites in breast tissue in humans. However, a call of caution is needed when comparing humans and murine models. Bearing this in mind, we have answered here the open questions related to the disposition of phenolics and methylxanthines in the mammary tissue. Relatively high concentrations of conjugated, but not free, phenolic-derived metabolites and methylxanthines can reach the mammary tissue. Our results suggest a comparatively much higher bioavailability of methylxanthines than phenolics, with a particular incidence in the mammary tissue. Besides, our results also help to conveniently design preclinical studies in (breast) cell models, hoping to counter the persisting large number of studies that claim for biological effects and specific mechanisms, targets and pathways, after exposing systemic cells to plant extracts and(or) phenolics at concentrations or molecular forms irrelevant in a physiological context.
0 0
2
4
6
8
10
12
14
16
Time (hours)
B
Mammary tissue 100
Theobromine Theophylline
80
Caffeine
600
60 400 40 200
20
Theophylline, caffeine (pmol/g tissue)
Theobromine (pmol/g tissue)
800
Ethics statements This study has been firmly committed to the concept of ‘the 3Rs’ in animal welfare (replacement, reduction, and refinement). The experimental protocol was approved by the Animal Experimentation Ethics Committee from University of Murcia (reference ES300305440012), the local government (reference A13180503) and the Spanish National Research Council’s Bioethics Committee (Madrid, Spain; reference 201770E081).
0
0 0
2
4
6
8
10
12
14
16
Time (hours) Fig. 4. Plasma (A) and mammary tissue (B) absorption-time profiles of methylxanthines after administration of the blend. (●) Theobromine, (■) theophylline, (○) caffeine. Results are expressed as mean ± SD (nM in plasma and pmol/g tissue in mammary tissues).
Declaration of Competing Interest The authors have declared no conflict of interest. Acknowledgement
tissue of breast cancer patients (Ávila-Gálvez et al., 2019) despite the fast absorption and clearance previously described for these molecules (Wikoff et al., 2017). In fact, the clinical trial suggested that methylxanthines might persist in the body much longer than previously assumed (Ávila-Gálvez et al., 2019). This agrees with our present results in the rat, at least when methylxanthines are provided within a blend of plant extracts, which seems to be a plausible dietary context (Fig. 4; Table 2). Taking into account the low amount of methylxanthines provided in both the human trial and the present rat study, as well as the comparative ratio plasma Cmax/MT Cmax, and AUC values for all the metabolites (Table 2), our results suggest that methylxanthines (especially theobromine) are much more bioavailable and target the mammary tissues at comparatively higher concentrations than phenolics. Finally, as in the case of breast cancer patients, no free (unconjugated) phenolic-derived metabolites were detected in MT, even with a high dose of phenolics and measured at a short time after consumption (Table 2). Therefore, these results confirm that the choice of concentrations and molecular forms of phenolics is crucial when designing experiments in cell models (Ávila-Gálvez, González-Sarrías, et al., 2018; Ávila-Gálvez, Espín, & González-Sarrías, 2018). For
This research has been supported by the Projects 201770E081 and 201870I028 from the Spanish National Research Council (CSIC, Spain), and AGL2015-64124-R from the Ministry of Science, Innovation, and Universities (Spain). References Amatori, S., Mazzoni, L., Álvarez-Suárez, J. M., Giampieri, F., Gasparrini, M., ForbesHernandez, T. Y., ... Battino, M. (2016). Polyphenol-rich strawberry extract (PRSE) shows in vitro and in vivo biological activity against invasive breast cancer cells. Scientific Reports, 6, 30917. https://doi.org/10.1038/srep30917. Andrés-Lacueva, C., Macarulla, M. T., Rotches-Ribalta, M., Boto-Ordóñez, M., Urpi-Sarda, M., & Portillo, M. P. (2012). Distribution of resveratrol metabolites in liver, adipose tissue, and skeletal muscle in rats fed different doses of this polyphenol. Journal of Agricultural and Food Chemistry, 60(19), 4833–4840. https://doi.org/10.1021/ jf3001108. Ávila-Gálvez, M.Á., Espín, J. C., & González-Sarrías, A. (2018). Physiological relevance of the antiproliferative and estrogenic effects of dietary polyphenol aglycones versus their phase-II metabolites on breast cancer cells: A call of caution. Journal of Agricultural and Food Chemistry, 66(32), 8547–8555. https://doi.org/10.1021/acs. jafc.8b03100. Ávila-Gálvez, M.Á., García-Villalba, R., Martínez-Díaz, F., Ocaña-Castillo, B., Monedero-
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M.Á. Ávila-Gálvez, et al. Saiz, T., Torrecillas-Sánchez, A., ... Espín, J. C. (2019). Metabolic profiling of dietary polyphenols and methylxanthines in normal and malignant mammary tissues from breast cancer patients. Molecular Nutrition & Food Research, 63(9), e1801239. https:// doi.org/10.1002/mnfr.201801239. Ávila-Gálvez, M.Á., González-Sarrías, A., & Espín, J. C. (2018). In vitro research on dietary polyphenols and health: A call of caution and a guide on how to proceed. Journal of Agricultural and Food Chemistry, 66(30), 7857–7858. https://doi.org/10. 1021/acs.jafc.8b03377. Azorín-Ortuño, M., Yáñez-Gascón, M. J., Vallejo, F., Pallarés, F. J., Larrosa, M., Lucas, R., ... Espín, J. C. (2011). Metabolites and tissue distribution of resveratrol in the pig. Molecular Nutrition & Food Research, 55(8), 1154–1168. https://doi.org/10.1002/ mnfr.201100140. Bishayee, A., Mandal, A., Bhattacharyya, P., & Bhatia, D. (2016). Pomegranate exerts chemoprevention of experimentally induced mammary tumorigenesis by suppression of cell proliferation and induction of apoptosis. Nutrition and Cancer, 68(1), 120–130. https://doi.org/10.1080/01635581.2016.1115094. Boocock, D. J., Faust, G. E., Patel, K. R., Schinas, A. M., Brown, V. A., Ducharme, M. P., ... Brenner, D. E. (2007). Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer Epidemiology, Biomarkers & Prevention, 16(6), 1246–1252. https://doi.org/10.1158/ 1055-9965.EPI-07-0022. Borges, G., Mullen, W., Mullan, A., Lean, M. E., Roberts, S. A., & Crozier, A. (2010). Bioavailability of multiple components following acute ingestion of a polyphenol-rich juice drink. Molecular Nutrition & Food Research, 54(Suppl 2), S268–S277. https://doi. org/10.1002/mnfr.200900611. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6), 394–424. https://doi.org/10.3322/caac.21492. Brinton, L. A., Gaudet, M. M., & Gierach, G. L. (2018). Breast cancer. In M. J. Thun, M. S. Linet, J. R. Cerhan, C. A. Haiman, & D. Schottenfeld (Eds.). Cancer epidemiology and prevention (pp. 861–888). (4th ed.). New York: Oxford University Press. Emaus, M. J., Peeters, P. H., Bakker, M. F., Overvad, K., Tjønneland, A., Olsen, A., ... van Gils, C. H. (2016). Vegetable and fruit consumption and the risk of hormone receptordefined breast cancer in the EPIC cohort. American Journal of Clinical Nutrition, 103(1), 168–177. https://doi.org/10.3945/ajcn.114.101436. Kapinova, A., Stefanicka, P., Kubatka, P., Zubor, P., Uramova, S., Kello, M., ... Kruzliak, P. (2017). Are plant-based functional foods better choice against cancer than single phytochemicals? A critical review of current breast cancer research. Biomedicine & Pharmacotherapy, 96, 1465–1477. https://doi.org/10.1016/j.biopha.2017.11.134.
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Kinetic disposition of dietary polyphenols and methylxanthines in the rat mammary tissue María Ángeles Ávila-Gálvez, María Romo-Vaquero, Antonio González-Sarrías, Juan Carlos Espín
T ⁎
Laboratory of Food & Health, Research Group on Quality, Safety, and Bioactivity of Plant Foods, CEBAS-CSIC, 30100 Campus de Espinardo, Murcia, Spain
ARTICLE INFO
ABSTRACT
Keywords: Mammary tissue Methylxanthine Polyphenol Breast cancer Resveratrol Plant extract
We recently showed that methylxanthines and conjugated phenolic-derived metabolites reached the mammary tissue (MT) of breast cancer patients after consuming a blend of phenolic-rich extracts. The pre-surgery fasting could prevent the detection of some (including those non-conjugated) metabolites. We investigated here the pharmacokinetics in rat plasma and MT of phenolic-derived metabolites and methylxanthines using the same blend (pomegranate, olive, cocoa, orange, lemon, and grapeseed extracts plus resveratrol; containing 37 phenolics, theobromine, and caffeine). We also compared the pharmacokinetics of resveratrol when administered in the blend or individually. We show for the first time that micromolar levels of methylxanthines and conjugatedderived (but not free) metabolites from resveratrol, dihydroresveratrol, hesperetin, urolithins, and hydroxytyrosol reached the MT. Resveratrol-3-glucuronide and resveratrol-3-sulfate showed shorter Tmax (plasma and MT) and resveratrol-3-sulfate higher Cmax (MT) when resveratrol was administered individually. This study could help to conveniently design preclinical studies using physiologically relevant conditions in (breast) cell models.
1. Introduction The new global cancer data (Bray et al., 2018) reveal that breast cancer is the most commonly diagnosed cancer in women (24.2%), and the leading cause of cancer death in women (15%) (Bray et al., 2018). It is known that the non-hereditary factors are the main drivers of incidence, including long-term endogenous hormonal exposures and exogenous hormone intake, nulliparity, early age at menarche, alcohol intake and obesity (Bray et al., 2018). Regarding protective factors, long-term breastfeeding and physical activity may be beneficial (Brinton, Gaudet, & Gierach, 2018), and some studies also suggest the protection of vegetable consumption against estrogen receptor-negative (ER-) breast cancer risk (Emaus et al., 2016). The recent Continuous Update Project (CUP) only observed a partial protective association between breast cancer risk with the intake of fruits, vegetables, tea, coffee, isoflavones, and carotenoids (WCRF/AICR, 2017). However, it has been suggested that the mixture of phytochemicals found in whole
foods, rather than isolated phytochemicals, exerts effects against carcinogenesis (Kapinova et al., 2017). Overall, these observations contrast with the abundant research in animal models that describe the preventive effect of polyphenols and polyphenol-rich plant extracts against breast cancer, including pomegranate (Bishayee, Mandal, Bhattacharyya, & Bhatia, 2016), citrus flavanones (Singhal et al., 2018), resveratrol (Sinha, Sarkar, Biswas, & Bishayee, 2016), berries (Amatori et al., 2016) and ellagic acid (Wang et al., 2012). Besides, a large number of studies in cellular models persist in the testing of plant extracts or polyphenols in molecular forms and(or) concentrations, describing many mechanisms, targets and pathways, when in fact they do not reach the mammary tissue (Ávila-Gálvez, González-Sarrías, & Espín, 2018). To shed some light on this topic, we recently conducted a clinical study in breast cancer patients that consumed a cocktail of plant extracts (pomegranate, cocoa, lemon, orange, grapeseed, and olive) plus resveratrol from diagnosis to surgical resection (Ávila-Gálvez et al.,
Abbreviations: DHRSV, dihydroresveratrol; DHRSV 3-glur, DHR 3-O-glucuronide; EIC, extracted ion chromatogram; HP, hesperetin; HP 7-glur, HP 7-O-glucuronide; HP 3′-glur, HP 3′-O-glucuronide; HP 7-sulf, HP 7-O-sulfate; HPLC-ESI-IT-MS/MS, high performance liquid chromatography-electrospray ionisation-ion trap tandem mass spectrometry; Hytyr glur, hydroxytyrosol glucuronide; MeOH, methanol; MT, mammary tissue; MX, methylxanthine; PBS, phosphate buffered saline; PM, phenolic-derived metabolites; RSV, resveratrol; RSV 3-sulf, RSV 3-O-sulfate; RSV 4′-sulf, RSV 4′-O-sulfate; RSV 3-glur, RSV 3-O-glucuronide; UPLC-ESI-QTOF-MS, ultra-high performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry; Uro, urolithin; Uro-A 3-glur, Uro-A 3-O-glucuronide; Uro-A 3-sulf, Uro-A 3-O-sulfate ⁎ Corresponding author. E-mail address: [email protected] (J.C. Espín). https://doi.org/10.1016/j.jff.2019.103516 Received 30 May 2019; Received in revised form 6 August 2019; Accepted 12 August 2019 Available online 21 August 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Phenolic compounds and methylxanthines administered to each rat.a Compound
RT (min)
m/z-
MS/MS
λmax
Source
Content (µg) per capsule
Gallic acid Punicalin* Protocatechuic acid Hydroxytyrosol Theobromineb Punicalagin Catechinc
8.71 9.80 11.56 11.63 12.80 13.12/15.18 15.72
169 781 153 153 181 1083 289
124 721/601/299 109 123 138 781/721/601 271/245/205/179
270 258/380 230/260 272 272 258/380 278
8.41 ± 0.58 40.31 ± 4.64 14.5 ± 1.45 211.12 ± 11.89 265.35 ± 8.12 604.94 ± 67.86 323.35 ± 11.02
Caffeineb Ferulic acid O-Glu* Luteolin 6,8-di-C-Glu* Epicatechinc
17.72 18.00 18.32 18.57
195 355 609 289
138/110 235/217/193/175/160/134 519/489/399/369 271/245/205/179
272 295/328 270/338 280
Ellagic acid-Glu* Apigenin 6,8-di-C-Glu (Vicenin-2)* Eriodictiol-Glu-Rhamn-Glu* Diosmetin 6,8-di-C-Glu* Gallagic acid* Chrysoeriol 6,8-di-C-Glu* Luteolin-C-Glu* Eriocitrin (eriodictiol rutinoside) Ellagic acid-rhamnoside* Apigenin 8-C-xylanopyranosil-Glu* Ellagic acid Quercetin 3-O-rutinoside (Rutin) Luteolin 7-O-rutinoside*
19.30 19.64 19.93 20.51 20.95 21.05 21.96 22.58 22.59 23.39 23.62 23.94 24.06
463 593 757 623 601 623 447 595 447 563 301 609 593
301 503/473/383/353 595/449/287 605/533/503/413/383 299/271/242 533/503/413/383 429/357/327 459/329/287 301 413/341/311/293 256/184 301 285
252/362 271/332 282/330 270/346 254/380 270/346 270/338 284/334 252/364 268/332 254/360 254/350 260/348
Epicatechin gallatec Diosmetin 8-C-Glu* Naringenin 7-O-rutinoside (narirutin)*
24.57 24.88 25.02
441 461 579
397/331/289/193/169 371/341 271
280 268/344 278/328
Catechin gallatec Limocitrin-Glu-HMG-Glu* Apigenin 7-O-rutinoside*
25.72 26.16 26.16
441 813 577
397/331/289/193/169 753/693/651/549 269
278 260/272/350 270/342
Limocitrin-neohesperidoside* Hesperetin 7-O-rutinoside (Hesperidin)
26.33 26.61
653 609
345/330/301 301
259/378 284/336
Diosmetin 7-O-rutinoside (Diosmin)*
26.90
607
299/284
270/334
Isorhamnetin rutinoside* Hesperetin-Glu* Limocitrin-HMG-Glu* Limocitrol-Glu-HMG* Trans-Resveratrol Cis-Resveratrol Isosakuranetin 7-rutinoside* Hesperetin TOTAL PHENOLICS TOTAL METHYLXANTHINES
27.05 27.80 27.98 28.16 28.55 31.68 31.88 35.60
623 463 651 681 227 227 593 301
315/299 301 549/507/345 619/579/537/375 185 185/159 285 –
254/368 284/330 274/350 260/377 306 286 284/332 288/330
Pomegranate Pomegranate Cocoa Olive Cocoa Pomegranate Cocoa Grapeseed Cocoa Lemon Lemon Cocoa Grapeseed Pomegranate Lemon Lemon Lemon Pomegranate Lemon Lemon Lemon Pomegranate Lemon Lemon Lemon Orange Lemon Grapeseed Lemon Lemon Orange Grapeseed Lemon Lemon Orange Lemon Orange Lemon Lemon Orange Orange Orange Lemon Lemon Resveratrol Resveratrol Orange Orange
6.67 ± 0.01 114.55 ± 15.37 10.73 ± 1.16 451.24 ± 13.05 29 ± 2.61 39.44 ± 0.58 22.62 ± 2.61 19.72 ± 1.16 18.27 ± 1.74 65.54 ± 1.16 9.28 ± 0.87 401.07 ± 28.42 12.18 ± 1.16 17.11 ± 2.61 1,390.55 ± 140.65 2.9 ± 0.29 1.45 ± 0.58 38.28 ± 0.87 97.73 ± 2.9 24.36 ± 0.87 NC 13.63 ± 4.64 36.54 ± 1.45 13.05 ± 0.58 NC 1.74 ± 0.58 NC 1,077.35 ± 24.65 63.51 ± 2.32 NC 29.0 ± 18.27 1.45 ± 0.87 1.16 ± 0.29 4.35 ± 0.29 NC 2,207.48 ± 207.06 6.67 ± 1.74 13.63 ± 4.64 7.54 ± 0.29 7,445.75 ± 583.77 272.02 ± 8.13
*Compounds ‘tentatively identified’ based on the their m/z, MS/MS fragmentation patterns and UV spectra; RT, retention time; NC, identified but not quantified; Glu, glucoside; Rhamn, rhamnoside; HMG, 3-hydroxy-3-methyl-glutaryl-. a Adapted from Ávila-Gálvez et al. (2019). Values are shown as mean ± SD; btheobromine and caffeine were determined in m/z+ mode; ctotal content determined by the phloroglucinolysis reaction (Kennedy & Jones., 2001).
2019). We detected 39 and 33 phenolic-derived metabolites and 8 and 6 methylxanthines, in normal and malignant mammary tissues, respectively. The concentrations roughly reached the range of nanomolar, and all phenolic metabolites were conjugated except for two dihydroxybenzoic acid derivatives (Ávila-Gálvez et al., 2019). In that study, we identified two potential variables that could affect the results obtained: i) the fasting of patients before surgery (around 10–12 h) could prevent a higher occurrence of metabolites in mammary tissues, and ii) the simultaneous administration of many phenolics (which is a plausible dietary context) could interfere with each other in their absorption and metabolism. To answer the above open questions, we evaluated here the distribution kinetics of the same phenolics in the rat mammary tissue and determined a comparative plasma and mammary tissue pharmacokinetic study of the primary occurring phenolic and methylxanthine metabolites.
2. Materials and methods 2.1. Chemicals Uro-A 3-O-glucuronide (Uro-A 3-glur), Uro-A 3-O-sulfate (Uro-A 3sulf), HP 7-O-glucuronide (HP 7-glur), HP 3′-O-glucuronide (HP 3′glur), HP 7-O-sulfate (HP 7-sulf), were obtained from Villapharma Research S.L. (Parque Tecnológico de Fuente Alamo, Murcia, Spain). Resveratrol (RSV, 3,5,4′-trihydroxy-trans-stilbene) was obtained from Laboratorios Admira S.L. (Alcantarilla, Murcia, Spain). DHRSV 3-Oglucuronide (DHRSV 3-glur), RSV 3-O-sulfate (RSV 3-sulf), RSV 4′-Osulfate (RSV 4′-sulf), RSV 3-O-glucuronide (RSV 3-glur) were obtained as described elsewhere (Azorín-Ortuño et al., 2011). Hydroxytyrosol glucuronide (Hytyr glur) was synthesized according to Lucas, Alcantara, and Morales (2009). Phosphate buffered saline (PBS) was 2
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Fig. 1. Experimental design.
Committee (Madrid, Spain; reference 201770E081). Animals were housed 3–4 to a cage in a room with controlled temperature (22 ± 2 °C), 55 ± 10% relative humidity, and a 12 h light-dark cycle. Animals were fed with a rat standard chow (Panlab, Barcelona, Spain). Diet and tap water were administered ad libitum until 6 h before the start of the experiment. The experimental design is shown in Fig. 1. One capsule was administered by gavage to each fasting animal, and a set of 4 animals was sacrificed with carbon dioxide at 0.5, 1, 2, 4, 6, 10, and 16 h after the capsule administration. A set of four animals also received one empty capsule and served as controls.
purchased from Sigma-Aldrich (St. Louis, MO, USA), and liquid chromatography-mass (LC-MS) grade solvents from J.T. Baker (Deventer, The Netherlands). Mili-Q ultra-pure water system (Millipore Corp. Bedford, MA, USA) was used in all experiments. 2.2. Plant extracts Two formulations were used in the present study: RSV alone and a blend of RSV plus orange, lemon, pomegranate, cocoa, grapeseed, and olive extracts (hereafter termed ‘blend’). Laboratorios Admira S.L provided the plant extracts. Both formulations were encapsulated in hard gelatin capsules (size 9), specially designed for their administration to rats weighing ~250 g (Torpac, Fairfield, USA). The same blend was previously assayed in breast cancer patients (Ávila-Gálvez et al., 2019), and the detailed composition can be found in Table 1. In the present study, each capsule contained a total of 29 mg. The formulation of the blend included 2.2 mg RSV plus 6.7 mg pomegranate, 6.7 mg olive, 6.7 mg cocoa, 2.2 mg orange, 2.2 mg lemon, and 2.2 mg grapeseed extracts. Each capsule of this formulation provided 7.4 mg of phenolics, 0.3 mg theobromine, and 0.007 mg caffeine. The human equivalent doses (HED) were 336 mg, 13.6 mg and 0.3 mg of phenolics, theobromine, and caffeine, respectively, in a 70 kg person (Reagan-Shaw, Nihal, & Ahmad, 2008). Each capsule of the RSV formulation contained only 2.2 mg RSV (HED of 100 mg in a 70 kg person) plus 26.8 mg microcrystalline cellulose as an excipient. Table 1 shows the detailed formulation of phenolic compounds and methylxanthines consumed by the rats in each capsule.
2.4. Sampling procedure Blood samples were collected by cardiac puncture in EDTAtreated tubes at each time-point after sacrifice, and mammary tissues were collected and stored at −80 °C until their analysis (Fig. 1). Blood was immediately centrifuged at 14,000g for 15 min at 4 °C in a Sigma 1–13 microcentrifuge (Braun Biotech. International, Melsungen, Germany). Plasma samples were extracted with acetonitrile:formic acid (98:2, v/v), centrifuged, and the supernatant reduced to dryness in a speed vacuum concentrator. The evaporated samples were re-suspended in methanol, filtered through a 0.22 µm polyvinylidene fluoride (PVDF) filter and injected in the UPLC-ESIQTOF-MS equipment. At sacrifice, inguinal and thoracic mammary glands were removed, extensively washed with cold PBS to avoid external blood contamination, and snap frozen in liquid nitrogen. Mammary tissues (300 mg) were processed as previously described (Ávila-Gálvez et al., 2019).
2.3. Animals and experimental design
2.5. UPLC-ESI-QTOF-MS analysis of plasma and mammary tissue
Sixty 11-weeks-old female Sprague Dawley rats weighing 258 ± 29 g were purchased from the Animal Experimentation Service of the University of Murcia (Murcia, Spain). The experimental protocol complied with the Arrive guidelines, followed the Directive of the European Council 2010/63/UE, and was approved by the Animal Experimentation Ethics Committee from University of Murcia (reference ES300305440012), the local government (reference A13180503) and the Spanish National Research Council’s Bioethics
Phenolic-derived metabolites and methylxanthines were identified and quantified in plasma and mammary tissue by UPLC-ESI-QTOF-MS as previously described (Ávila-Gálvez et al., 2019). More than 180 potential compounds (phenolics present in the extracts as well as derived metabolites both unconjugated and conjugated such as glucuronides, sulfates, sulfoglucuronides, etc.), were browsed in plasma and mammary tissue samples. The method was validated for linearity, 3
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± 4.2 ± 2.7 ± 2.4
1.2 0.6 4.0 1.1 ± ± ± ±
Hytyr, hydroxytyrosol; HP, hesperetin; Uro-A, urolithin A; RSV, resveratrol; DHRSV, dihydroresveratrol; glur, glucuronide; sulf, sulfate; –, not determined; Tmax, time of maximum concentration; Tlast, time of the last measurable concentration; T1/2, time required for the concentration to reach half of its original value; Cmax, maximum concentration; AUC0-t, area under the curve from the time of dosing to the final quantifiable concentration; MRT0-t, mean residence time from the time of dosing to the time of the final quantifiable concentration. Asterisks designate a significant difference when RSV was administered in the blend or individually (*P < 0.05; ***P < 0.001).
1.5 ± 0.3 7.6 ± 3.6 7.5 ± 3.1 − 12.0 ± 1.2 − 5.5 ± 0.4 5.2 ± 1.1 4.8 ± 0.7 4.1 ± 0.7 9.6 ± 1.6 9.0 ± 1.6 6.1 ± 0.6 5.1 ± 0.7 6.1 ± 0.7 1.6 ± 0.1 8.2 ± 2.5 8.2 ± 2.2 8.8 ± 0.8 11.5 ± 0.5 12.0 ± 0.3 4.9 ± 0.7 5.1 ± 1.1 4.9 ± 1.3 3.4 ± 1.3 9.2 ± 0.9 9.5 ± 1.3 6.5 ± 0.5 5.5 ± 0.8 4.7 ± 1.5 110.3 ± 68.7 10.3 ± 6.1 14.4 ± 3.4 − 47.9 ± 14.6 − 4,631 ± 794.2 7,256 ± 1,040 237.5 ± 150.3 399.2 ± 8.6 1,654 ± 876.7 2,571 ± 1,697 4,062 ± 386.2 68.5 ± 7.5 376.9 ± 81.1 1.4 − − − − − 2.9 2.9 5.0 3.4 − − 5.6 6.8 7.3 0.8 ± 0.1 − − − − − 2.4 ± 1.3 5.1 ± 2.8 2.1 ± 0.5 3.5 ± 0.8 − − 5.2 ± 1.5 11.7 ± 9.1 4.1 ± 1.0 1.0 ± 0.7 9.5 ± 4.7 8.0 ± 5.4 − 13.0 ± 4.2 − 3.3 ± 2.2 1.7 ± 0.6* 4.0 ± 0.0 1.5 ± 0.7*** 9.0 ± 2.0 8.7 ± 2.3 3.0 ± 2.0 3.0 ± 2.0 2.5 ± 1.0 0.9 ± 0.8 9.5 ± 4.7 10.0 ± 6.0 10.0 ± 0.0 13.0 ± 4.2 16.0 ± 0.0 3.25 ± 2.2 1.0 ± 0.7*** 4.0 ± 0.0 0.6 ± 0.3*** 8.0 ± 2.3 8.7 ± 2.3 2.3 ± 1.5 3.5 ± 1.9 2.0 ± 0.0 a
Hytyr glur HP 3-glura HP 7-glura HP 7-sulfa Uro-A 3-glura Uro-A 3-sulfa RSV 3-glura RSV 3-glurb RSV 3-sulfa RSV 3-sulfb DHRSV 3-glura DHRSV 3-glurb Theobrominea Theophyllinea Caffeinea
Plasma MT Plasma
MT
± 0.5
4.0 ± 0.0 12.0 ± 4.9 14.0 ± 3.5 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 14.5 ± 3.0 13.5 ± 5.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 16.0 ± 0.0 10.0 ± 0.0 12.0 ± 4.9
3.0 ± 1.2 10.5 ± 4.1 11.0 ± 4.1 − 16.0 ± 0.0 − 16.0 ± 0.0 16.0 ± 0.0 13.0 ± 6.0 10.0 ± 0.0 16.0 ± 0.0 14.0 ± 3.5 16.0 ± 0.0 10.0 ± 0.0 16.0 ± 0.0
457.0 ± 147.5 29.1 ± 7.3 18.3 ± 1.2 1.2 ± 0.3 149.5 ± 16.0 5.8 ± 1.3 8,939 ± 1,538 11,209 ± 4,405 758.6 ± 94.9 1,397 ± 482.1 1,980 ± 835.6 3,162 ± 1,835 1,525 ± 348.3 39.1 ± 7.5 159.0 ± 69.5
77.2 ± 38.7 2.1 ± 0.7 2.3 ± 0.3 − 6.7 ± 3.8 − 668.4 ± 126.9 1,262 ± 597.0* 61.6 ± 28.3 112.2 ± 40.9 206.3 ± 113.1 406.2 ± 331.6 677.7 ± 186.3 10.9 ± 2.3 53.0 ± 13.1
803.0 ± 171.2 153.3 ± 87.6 146.9 ± 31.5 10.6 ± 4.0 1,408 ± 156.9 49.3 ± 3.9 63,812 ± 13,364 53,393 ± 6,067 3,356 ± 962.4 4,375 ± 820.6 16,460 ± 5,265 21,568 ± 10,654 14,351 ± 787.5 240.0 ± 26.8 828.6 ± 205.7
MT Plasma MT (pmol/g h) Plasma
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Table 2 Plasma and mammary tissue (MT) pharmacokinetic parameters of the primary metabolites derived from phenolics and methylxanthines after consumption of the blend (RSV plus extractsa) or RSV individuallyb.
M.Á. Ávila-Gálvez, et al.
precision, accuracy, limits of detection (LOD) and quantification (LOQ) as well as for matrix effects. Quantification of metabolites was carried out by peak area integration of their extracted ion chromatogram (EIC) using calibration curves of standards. Phenolic metabolites were quantified in negative mode and methylxanthines in positive mode (Ávila-Gálvez et al., 2019). 2.6. Pharmacokinetic analysis Plasma and mammary tissue-time data of the primary metabolites were analyzed. Pharmacokinetic parameters were determined by non-compartmental analysis using the PKSolver, a complement addin software of Microsoft Excel (Zhang, Huo, Zhou, & Xie, 2010). The parameters are shown as the mean ± standard deviation (SD). The statistical comparison of parameters (Tmax, Cmax, etc.) of metabolites derived from RSV, when administered in the blend or separately, were analyzed on the original data using the t-test or the Wilcoxon signed-rank test. Plots were performed using Sigma Plot 13.0 (Systat Software, San Jose, CA, USA). Statistical significance was set at P < 0.05. 3. Results 3.1. Plasma and mammary tissue pharmacokinetics of phenolic-derived metabolites Table 2 shows the most representative phenolic-derived metabolites and their pharmacokinetic parameters in plasma and mammary tissue after consuming the blend or RSV alone. The more abundant polyphenol families existing in the blend were RSV (2.2 mg), ellagic acid derivatives (~2.1 mg), flavanones (~1.6 mg) and procyanidins (~0.9 mg) (Table 1). The highest Cmax values were observed for RSVderived metabolites (RSV 3-glur > DHRSV 3-glur > RSV 3-sulf in both plasma and MT), reaching the highest value in plasma in the case of RSV 3-glur (11.2 ± 4.4 μM), when administered individually (Table 2, Fig. 2A). As expected, MT and plasma Tmax values were remarkably longer for the microbial-derived metabolite DHRSV 3-glur (Table 2, Fig. 2E, F). The kinetic profiles for RSV 3-glur and RSV 3-sulf were different in both plasma (Fig. 2A) and MT (Fig. 2B), when RSV was administered individually (Table 2). In this case, MT and plasma Tmax values for RSV metabolites significantly decreased while Cmax and AUC values increased (Table 2, Fig. 2), but only became statistically significant for Cmax in MT in the case of RSV 3-glur (Table 2). Regarding DHRSV 3-glur, the administration of RSV separately only increased Cmax and AUC values in MT, but not statistically significant (Table 2; Fig. 2E, F). In the case of olive Hytyr, the primary derived metabolite was Hytyr glur that peaked in both plasma and MT at 0.5 h after the capsule administration (Table 2, Fig. 3A). Hytyr sulfate was also identified in MT from some animals, but it was not quantified due to the lack of an authentic standard. The kinetics of hesperetin conjugates (3-glur, 7glur, and 7-sulf), derived from flavanones (Table 2, Fig. 3B, C), and urolithin conjugates (3-glur and 3-sulf) derived from ellagic acid and ellagitannins (Table 2, Fig. 3D), were similar in both plasma and MT (Fig. 3B-D). As these metabolites are products of the gut microbiota metabolism, an expected delay was also observed in their Tmax values (Table 2, Fig. 3B-D). Finally, the procyanidin-derived metabolite 5(3′,4′-dihydroxyphenyl)-γ-valerolactone 3′-sulfate was observed only in some animals, which prevented the corresponding pharmacokinetic analysis. 3.2. Plasma and mammary tissue pharmacokinetics of methylxanthines Fig. 4 shows the kinetic profiles of theobromine, theophylline, and caffeine in plasma (Fig. 4A) and MT (Fig. 4B). These profiles were similar, although Cmax and AUC values were much higher for 4
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Fig. 2. Plasma (A, C, E) and mammary tissue (MT) (B, D, F) absorption-time profiles of resveratrol (RSV) metabolites after administration of the blend (●) or RSV individually (○). RSV 3-glur (resveratrol 3-O-glucuronide), RSV 3-sulf (resveratrol 3-O-sulfate), and DHRSV 3-glur (dihydroresveratrol 3-O-glucuronide). Resveratrol 4′-O-sulfate was detected in the plasma of some animals (results not shown). Results are expressed as mean ± SD (nM in plasma and pmol/g tissue in mammary tissues).
theobromine (Table 2), the main ingested cocoa-derived methylxanthine occurring in the blend (Table 1). Remarkably, theobromine showed relatively high Cmax and AUC values in MT, close to those of RSV 3-glur (Table 2), although theobromine content in the blend was ~10-fold lower than that of RSV (Table 1).
4. Discussion We have recently described the occurrence of a wide range of metabolites derived from phenolics and methylxanthines, in normal and malignant mammary tissues, from breast cancer patients after consumption of a blend containing RSV and phenolic-rich plant extracts 5
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Fig. 3. Plasma (●,▲) and mammary tissue (MT) (□) absorption-time profiles of hydroxytyrosol (A), hesperetin (B, C) and urolithin A (D) metabolites after administration of the blend. Hytyr glur (hydroxytyrosol glucuronide), HP 3′-glur (hesperetin 3′-O-glucuronide), HP 7-glur (hesperetin 7-O-glucuronide), HP 7-sulf (hesperetin 7-O-sulfate), Uro-A glur (urolithin A glucuronide), and Uro-A sulf (urolithin A sulfate). Results are expressed as mean ± SD (nM in plasma and pmol/g tissue in mammary tissues).
(Ávila-Gálvez et al., 2019). However, after that trial, we left two open questions to be answered: (i) did the fasting of the patients before the surgery prevent the appearance of a higher concentration of metabolites in the mammary tissues? And, (ii) could metabolites reach higher levels in the tissue if they are administered individually rather than in a mixture? Besides, the most representative metabolites detected in the mammary tissues were all conjugated (Ávila-Gálvez et al., 2019). Therefore, the present kinetic study was also conceived to know whether free (unconjugated) phenolic-derived metabolites could reach the mammary tissues at shorter times after the consumption of the blend. We assayed here the same mixture as that used in the human study. However, although the proportion of phenolics and methylxanthines was equivalent, the total amount of phenolics and methylxanthines was slightly lower in the rats, i.e., 474 mg phenolics and 19 mg methylxanthines in the human trial, and 336 mg phenolics and 13.9 mg methylxanthines, in the rat study (expressed as HED). In agreement with previous reports that described some differences in the metabolism of RSV depending on the species, the predominant metabolite detected in the plasma, muscle and adipose tissues is RSV 3glur (Andrés-Lacueva et al., 2012) in contrast to humans, where RSV 3sulf is more abundant in plasma and breast tissue (Table 2) (ÁvilaGálvez et al., 2019; Boocock et al., 2007). Besides, procyanidin-derived metabolites were relatively abundant in the mammary tissues of breast cancer patients (Ávila-Gálvez et al., 2019), especially dihydroxybenzoic acid derivatives and 3′,4′-dihydroxyphenyl)-γ-valerolactone 3′-sulfate, while these metabolites were hardly detected in the present rat study, which suggested differences in the metabolism of procyanidins by rats
vs humans. In the case of other metabolites produced by the action of the gut microbiota (hesperetin, urolithins, and DHRSV), the metabolites peaked in plasma and MT from 10 h to 16 h (Table 2). These delayed Tmax agree mainly with the relatively high DHRSV 3-glur concentration detected in fasting breast cancer patients (Ávila-Gálvez et al., 2019). Therefore, taking into account the MRT values or the metabolites (Table 2), the present rat study confirms that fasting of patients prevented the occurrence of some phenolic-derived metabolites such as Hytyr glur. Besides, this was the reason for not being able to detect high concentrations of others, like RSV conjugates that reached micromolar levels (Table 2) instead of nanomolar range detected in MT from breast cancer patients (Ávila-Gálvez et al., 2019). The present study shows that the kinetics of RSV-derived metabolites was different when RSV was administered in the blend or individually (Fig. 2; Table 2). The absorption was faster when RSV was delivered separately, as indicated by the significant difference in the Tmax values of the corresponding derived metabolites (Table 2). However, the rest of the pharmacokinetic parameters were not affected except for RSV 3-sulf that peaked at higher concentration in MT when administered individually (Table 2). This behaviour could depend on the specific phenolic since the absorption and excretion of monomeric flavan-3-ols have been reported to be similar when ingested individually or in a blend (Borges et al., 2010). Overall, it seems that the co-administration of a mixture of phenolics could compete for ABC transporters and delay their absorption (Planas, Alfaras, Colom, & Juan, 2012). Methylxanthines have been reported to be detected in the mammary 6
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example, taking into account the blood distribution in healthy breast tissue (Mankoff et al., 2002), our results show a plausible physiological mixture of approximately 10 μM in MT, mainly composed of RSV 3glur, RSV 3-sulf, and DHRSV 3-glur, and varying the exact proportions depending on the animal species. We acknowledge that the assay of conjugates may discourage researchers since conjugation hampers many direct antiproliferative and proapoptotic related effects (ÁvilaGálvez, Espín, et al., 2018). However, cell cultures (from systemic tissues) should not contain free phenolics, including RSV, to describe mechanisms of action or to describe a direct effect on the cells (ÁvilaGálvez, González-Sarrías, et al., 2018).
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The present study was conducted in rats because it is not possible, or it would be extremely challenging to perform a kinetic disposition of phenolics and derived metabolites in breast tissue in humans. However, a call of caution is needed when comparing humans and murine models. Bearing this in mind, we have answered here the open questions related to the disposition of phenolics and methylxanthines in the mammary tissue. Relatively high concentrations of conjugated, but not free, phenolic-derived metabolites and methylxanthines can reach the mammary tissue. Our results suggest a comparatively much higher bioavailability of methylxanthines than phenolics, with a particular incidence in the mammary tissue. Besides, our results also help to conveniently design preclinical studies in (breast) cell models, hoping to counter the persisting large number of studies that claim for biological effects and specific mechanisms, targets and pathways, after exposing systemic cells to plant extracts and(or) phenolics at concentrations or molecular forms irrelevant in a physiological context.
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Ethics statements This study has been firmly committed to the concept of ‘the 3Rs’ in animal welfare (replacement, reduction, and refinement). The experimental protocol was approved by the Animal Experimentation Ethics Committee from University of Murcia (reference ES300305440012), the local government (reference A13180503) and the Spanish National Research Council’s Bioethics Committee (Madrid, Spain; reference 201770E081).
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Declaration of Competing Interest The authors have declared no conflict of interest. Acknowledgement
tissue of breast cancer patients (Ávila-Gálvez et al., 2019) despite the fast absorption and clearance previously described for these molecules (Wikoff et al., 2017). In fact, the clinical trial suggested that methylxanthines might persist in the body much longer than previously assumed (Ávila-Gálvez et al., 2019). This agrees with our present results in the rat, at least when methylxanthines are provided within a blend of plant extracts, which seems to be a plausible dietary context (Fig. 4; Table 2). Taking into account the low amount of methylxanthines provided in both the human trial and the present rat study, as well as the comparative ratio plasma Cmax/MT Cmax, and AUC values for all the metabolites (Table 2), our results suggest that methylxanthines (especially theobromine) are much more bioavailable and target the mammary tissues at comparatively higher concentrations than phenolics. Finally, as in the case of breast cancer patients, no free (unconjugated) phenolic-derived metabolites were detected in MT, even with a high dose of phenolics and measured at a short time after consumption (Table 2). Therefore, these results confirm that the choice of concentrations and molecular forms of phenolics is crucial when designing experiments in cell models (Ávila-Gálvez, González-Sarrías, et al., 2018; Ávila-Gálvez, Espín, & González-Sarrías, 2018). For
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