Abstract Despite the potential applications of pineapple fibers, the systematic characterization of their polyphenolic composition has not been fully elucidated. In this study, UHPLC-MS and OPLS-DA were used to comprehensively analyze the polyphenolic profiles in pineapple leaf (PLF), stem (PSF) and root (PRF) fibers, while evaluating their bioactive properties. The investigation revealed that PRF contained significantly higher polyphenol content (9.55 mg/g) than PLF and PRF, and 83 phenolic compounds were identified, with distinct metabolic pathway distributions among different plant tissues, and the inhibitory effects of polyphenol on E. coli and S. aureus were optimized in PLF. These findings provide critical insights for the development of value-added applications of pineapple by-products in functional materials and antimicrobial formulations. Keywords: Pineapple leaf, Pineapple stem fiber, Polyphenols, Pineapple root fiber, Antioxidant activity Graphical abstract [35]Image 1 [36]Open in a new tab Highlights * • PRF showed highest polyphenols (9.55 mg/g) among pineapple fibers. * • PSF polyphenols exhibited strongest antioxidant activity. * • PLF polyphenols had best antibacterial effects against E. coli/S. aureus. * • 83 PCs identified, revealing tissue-specific metabolic differences. 1. Introduction Pineapple (Ananas comosus) consists of pineapple leaves (PL), pineapple stem (PS), pineapple root (PR) and pineapple fruits. Pineapple fruits were used for fruit processing, while most of leaves, stems and roots of pineapples have been pulverized and recycled. Using Bali varieties as an example, one pineapple produces an average of 0.10 kg of pineapple stems, 0.15 kg of pineapple roots and 1 kg of pineapple leaves, so the average annual production of pineapple leaves, stems and roots in Guangdong, China is more than 540,000 tons, 54,000 tons and 80,000 tons respectively. However, the effective utilization rate is currently below 1 % ([37]Liu et al., 2023). Pulverized PL have been used to prepare silage for dairy cows ([38]Du et al., 2014; [39]Sarangi et al., 2022), thereby improving milk yield, biogas production rate and pool volume ratio ([40]Mamo et al., 2019). Additionally, PL has been utilized to produce butanol through fermentation ([41]Sajjanshetty et al., 2021), synthesized sugar via enzymatic hydrolysis ([42]Nashiruddin et al., 2022), and extract fiber to improve antibacterial properties of textiles and the performance of composite materials ([43]Kueh et al., 2023; [44]Natrayan et al., 2022; [45]Najeeb et al., 2022; [46]Ridzuan et al., 2019; [47]Santosh and Suresh, 2019; [48]Zeleke et al., 2022). Pineapple leaf fiber (PLF) has a porous structure and is rich in bioactive compounds such as triterpenoids, amides, and phenylpropanoids, which exhibit significant antibacterial effects ([49]Emeka et al., 2014; [50]Liao et al., 2025). Currently, polyphenols are widely used antibacterial components ([51]Fendri et al., 2022; [52]Chaudhry et al., 2022). However, their industrial applications face limitations due to several challenges, including inconsistent extraction rates and the unclear identification of specific antibacterial components in PL. Furthermore, the variations in antibacterial compounds across different parts of pineapple plant remain poorly understood. Polyphenols inhibit growth and reproduction of microorganisms by disrupting microbial cell morphology, affecting energy metabolism and membrane potential, and inhibiting the synthesis of biomacromolecules ([53]Li et al., 2024). The aqueous extract of PLs could significantly retards the growth of yeast, Staphylococcus aureus (S. aureus), Candida albicans (C. albicans) and escherichia coli (E.coi) in the logarithmic and the stable phases at concentrations of 1.65–4.95 mg/mL, with an inhibition rate 95 % ([54]Sangita & Debasish, 2013). Additionally, researchers have identified various bioactive compounds in PLs, including two hydroxycinnamic acids, three phenylpropanoid quinin acids, four phenylpropanoid monoglycerides, eight diacylglycerides, three flavonoids, and six phenylpropanoid glycosides ([55]Ma et al., 2007). Further investigation into the chemical constituents of PLs reveled good non-toxicity toward both tumor and non-tumor cell lines ([56]Aquino et al., 2023). Moreover, PCs reduce intracellular sugar and lipid content ([57]Peng et al., 2022), and slow fat accumulation ([58]Chen et al., 2014; [59]Yan et al., 2025). The comprehensive analysis of polyphenols in pineapple fibers (leaf, stem, and root) is crucial due to their potential as sustainable sources of bioactive compounds for functional materials, nutraceuticals, and antimicrobial applications. Characterizing their polyphenolic profiles is necessary to valorize agricultural byproducts, understand tissue-specific metabolic differences, and optimize extraction for industrial use. However, the composition and distribution of polyphenols in pineapple fibers have not been fully elucidated. In this study, polyphenols from different parts of pineapples were isolated and identified using Ultra high-pressure liquid chromatography-mass spectrometry (UHPLC-MS). Their contents, antioxidant activities, and antibacterial properties were investigated and compared to elucidate the antibacterial mechanism. This study provides a reference for the effective utilization of pineapple by-products. 2. Materials and methods 2.1. Extraction of pineapple fiber Pineapple leaves, stems, and roots were soaked in water at 25 °C for 72 h, then beaten with a plastic hammer to separate pineapple leaf fibers (PLF), pineapple stem fibers (PSF), and pineapple root fibers (PRF). Subsequently, they were dried in the sun until the water content dropped below 10 %. 2.2. Extraction of PCs from pineapple fibers A 1 g of the fiber was added to 30 mL of an acidified 70 % ethanol solution (containing 1 % HCl, v/v). The solution was stirred, sealed with plastic wrap, and incubated in a water bath at 50 °C for 30 min. After incubation, the solution was centrifuged at 4500 r/min for 10 min and then filtered. This process was repeated twice, and the filtrates and supernatants were combined, respectively. Finally, the content of pineapple leaf polyphenols (PLPs) was determined. 2.3. Determination of polyphenols content Development of the standard curve for gallic acid (GA): The contents of polyphenols in different samples were calculated ([60]Aquino et al., 2023). A 5 mg sample of GA was dissolved in 1.25 mL of anhydrous ethanol, and distilled water was added to bring the solution volume to 50 mL, resulting in a standard GA solution with a concentration of 100 mg/L. Then, 1, 2, 3, 4, 5, 6, and 7 mL of this standard solution were separately added to seven 10 mL volumetric flasks, and distilled water was added to bring the solution volumes to 10 mL. This process prepared standard GA solutions with concentrations of 10, 20, 30, 40, 50, and 60 mg/L. Next, 2 mL of each standard solution was mixed with 1 mL of Folin phenol reagent. The mixture was thoroughly shaken and left undisturbed for 4 min. Afterward, 5 mL of a 7.5 % Na[2]CO[3] solution was added, and distilled water was added to bring the final volume to 25 mL. The solution was thoroughly shaken again and incubated in a water bath at 45 °C for 40 min. The absorbance (A[0]) of distilled water at 765 nm was measured as a blank. The standard curve for GA concentration (c[0], mg/L) was determined as: A[0] = 0.0098 × c[0] + 0.0085 (R^2 = 0.9993). Measurement of polyphenol content in samples: A 2 mL aliquot of the polyphenol extract solution was mixed with 1 mL of Folin phenol reagent. The mixture was thoroughly shaken and left undisturbed for 4 min. Afterward, 5 mL of a 7.5 % Na[2]CO[3] solution was added, and distilled water was added to bring the final volume to 25 mL. The solution was thoroughly shaken again and incubated in a water bath at 45 °C for 40 min. Using distilled water as the blank control, the absorbance of the extract at 765 nm was measured. The polyphenol concentration (c[1], mg/L) in the sample solution was calculated based on the standard curve for GA concentration. Consequently, the extraction rate of polyphenols could be calculated using the following formula: [MATH: (y,mg/g)=c1×V/m :MATH] where V was the total volume (mL) of the extract, and m was the sample mass (g). 2.4. Isolation and identification of PCs Isolation and identification of PCs: PCs were isolated and identified following a previously reported method ([61]Liu et al., 2023a, [62]Liu et al., 2023b, [63]Liu et al., 2023cB). Briefly, 0.5 g of the fiber sample and 5 mL of methanol were added to 10 mL of cold acetonitrile. The mixture was shaken, sonicated at 200 W for 30 min, and centrifuged at 15,402.8×g and 4 °C for 10 min. The supernatant was collected for further analysis. For UHPLC analysis, 10 μL of the sample was injected into the UHPLC column (LC-30A, Shimadzu, Japan) maintained at 40 °C. The mobile phase was eluted with the following gradient: 95 % A (0.1 % formic acid) and 5 % B (100 % acetonitrile) for 10 min, 30 % A and 70 % B for 7 min, 100 % B for 2 min, 95 % A and 5 % B for 3 min. The mass spectrometer test parameters were set according to the details provided in the supporting information annex. 2.5. In vitro antioxidant activities of polyphenols extracted from different parts of pineapple Polyphenols from different parts of pineapple PLF, PSF and PRF were extracted using an acidic ethanol solution, and the concentrations of polyphenols in the extracts were determined. The polyphenol extracts were diluted to a concentration of 0.01 mg/mL using the acidic ethanol solution. The antioxidant capacity, including the scavenging capacities of DPPH and hydroxyl radicals, was determined for the extracts ([64]Liu et al., 2023a, [65]Liu et al., 2023b, [66]Liu et al., 2023cA). 2.6. Antibacterial effect analysis of PCs in pineapple fiber in vitro Preparation of polyphenol samples: The polyphenol extracts were prepared according to the method described in Section [67]2.3. The polyphenol extract was concentrated to a final volume of 10 mL using rotary evaporation at 45 °C and then freeze-dried at −40 °C to obtain PFP powder. Next, 0.125 g each of PLF, PSF, and PRF polyphenol powders were weighed and dissolved in 1.5 mL of sterile water to prepare an 8 % (w/v) polyphenol solution. To obtain a 4 % (w/v) polyphenol solution, 700 μL of the as-prepared 8 % solution was mixed with 700 μL of sterile water and shaken thoroughly. Similarly, polyphenol solutions with concentrations of 2 %, 1 %, and 0.5 % (w/v) were prepared using the same dilution method. Preparation of flat plate: A 10 g sample of agar powder was added to 300 mL of distilled water. The mixture was sealed and sterilized at 121 °C for 30 min. While still hot, the agar solution was poured into plates in a clean bench. After solidification, the plates were sealed with adhesive tape and stored upside down. Preparation of bacterial solution: In a sterile clean bench, 600 μL of activated E. coli or S. aureus culture solution was added to a 15 mL glass test tube, followed by the addition of sterile water. This process resulted in a bacterial solution with a concentration of 1.5 × 10^8 CFU/mL. The solution was stored at 4 °C for further use. Measurement of bacteriostatic circle: A 200 μL aliquot of E. coli or S. aureus bacterial solution was spread evenly on the surface of the agar plate using a sterile coating rod. A small well was then punched in the center of the plate. Next, 200 μL of polyphenol solutions at different concentrations were added to the wells, and the petri dishes were sealed with adhesive tape. Three replicates were prepared for each concentration, and sterile water was used as the blank control. The plates were incubated at 37 °C for 15 h (placed upright), and the diameter of the inhibition zone was measured. 2.7. Statistical analysis Mass spectrometry data processing and analysis: UHPLC-MS raw data were converted to ABF format using Analysis Base File Converter software. The converted files were processed in MS-DIAL 4.70 for peak picking, noise reduction, deconvolution, and alignment, generating a three-dimensional data matrix in CSV format. Phenolic compound identification was performed by matching extracted features against three major spectral libraries: MassBank, ReSpect, and GNPS (containing 14,951 records in total). The matching criteria included: minimum peak height (1000 amplitude), mass slice width (0.1 Da), retention time tolerance (0.05 min), MS1 tolerance (0.015 Da), and MS2 tolerance (0.05 Da). Only matches with identification scores ≥80 were considered valid. All experiments were performed in triplicate. Data analysis was conducted using Microsoft Excel 2019 and SPSS 22.0 for statistics. Data visualization was performed using OriginLab 2022 (OriginPro, Northampton, MA). 3. Results and discussion 3.1. Comparative analysis of content of polyphenols in pineapple fibers Polyphenols, as key chemical derivatives of secondary metabolites derived from various plants, and exhibit chemical compositions that are highly dependent on their plant sources ([68]Wang et al., 2025; [69]Huang et al., 2025). As illustrated in [70]Fig. 1, the contents of polyphenols in the three fibers followed the sequence of PRF > PSF > PLF. Specifically, PRF exhibited the highest polyphenol content at 9.55 ± 0.21 mg/g, while PSF and PLF showed comparable levels. Fig. 1. [71]Fig. 1 [72]Open in a new tab Polyphenol contents in PRF, PSF, and PLF. Different letters represent the significant differences at the level of 0.05. PRF, PSF, and PLF represent pineapple root fiber, pineapple stem fiber and pineapple leaf fiber, respectively. 3.2. Isolation and identification of PCs extracted from pineapple fibers The mass spectra of polyphenols in PRF, PSF and PLF are summarized in [73]Table S1. As indicated, A 83 PCs were isolated by UHPLC-MS from PRF, PSF and PLF, and 66 and 17 of them were detected under negative and positive ion modes, respectively. With the ion abundances of the 83 polyphenols as the Y-axis and the species as the X-axis, a line chart ([74]Fig. 2(a)) and a stacked histogram ([75]Fig. 2(b)) were developed. As demonstrated, polyphenols with top 10 contents were compound 46, 70, 72, 63, 11, 38, 8, 32, 54 and 19. Polyphenols with top 10 overall contents were compound 70, 72, 46, 11, 38, 32, 63, 54, 60 and 8. Compound 46, 32 and 72 had significantly high contents in PSF, compound 63 and 19 had significantly high contents in PRF, and compound 11, 8 and 38 had significantly high contents in PLF. This could be attributed to different functions of pineapple leaves, stems and roots. Pineapple leaves were mainly responsible for nutrient synthesis and metabolism, pineapple stems were responsible for nutrient transfer, and pineapple roots were responsible for transportation of water and inorganic salts, and their key metabolic pathways might be different, and the expression levels of the same metabolic pathway could be different in different parts ([76]Fu et al., 2025). Eleven phenolic acids were identified in Bali pineapple pulp samples, including isoferulic acid (834.49 μg/g), trans-cinnamic acid (219.81 μg/g), sinapic acid (77.54 μg/g), p-coumaric acid (61.01 μg/g), caffeic acid (38.35 μg/g), 3,5-dihydroxybenzoic acid (2.63 μg/g), tannic acid (0.55 μg/g), and salicylic acid (0.11 μg/g). These findings suggested significant variations in phenolic acid contents among different pineapple tissues (Liu et al., 2023C). Fig. 2a. [77]Fig. 2a [78]Open in a new tab represent the line graph of ion abundances of ion abundances of different PCs in PLF, PSF and PRF. Fig. 2b. [79]Fig. 2b [80]Open in a new tab presents the stack histogram of different PCs in PLF, PSF and PRF. PRF, PSF, and PLF represent pineapple root fiber, pineapple stem fiber and pineapple leaf fiber, respectively. The pathway enrichment analysis of the 83 PCs is presented in [81]Fig. 3. The analysis revealed that 14 PCs were involved in known metabolic pathways of in PLF, PSF and PRF, primarily associated with 10 KEGG pathways: phenylpropanoid biosynthesis, biosynthesis of secondary metabolites (Part 2), stilbenoid-diarylheptanoid and gingerol biosynthesis, flavone and flavonol biosynthesis, flavonoid biosynthesis, tyrosine metabolism, tryptophan metabolism, ubiquinone and other terpenoid-quinone biosynthesis, isoquinone alkaloid biosynthesis, and secondary metabolite biosynthesis. Among these, phenylpropanoid biosynthesis and secondary metabolite biosynthesis (Part 2) emerged as the most significant metabolic pathways. Fig. 3. [82]Fig. 3 [83]Open in a new tab Main KEGG enrichment pathway of polyphenols from three pineapple fibers. The main polyphenols with different metabolic pathways in phenylpropanoid biosynthesis included caffeic acid (63), p-coumaric acid (54), 4-vinylphenol (60) and coniferyl alcohol (20). P-coumaric acid (54) and coniferyl alcohol (20) were the main polyphenols with different metabolic pathways in biosynthesis of varied secondary metabolites-part 2. p-coumaric acid (54) inhibited the growth of S. aureus, B. subtilis, S. pneumoniae, E. coli and C. gloeosporioides by increasing the permeability of bacterial cell membranes and interfering DNA transcription ([84]Liu et al., 2023; [85]Liu et al., 2023). Caffeic acid (63) inhibited the growth of Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Salmonella enteritidis and Staphylococcus aureus by destroying the integrity of cell membrane ([86]Lukáč et al., 2024; [87]Chen et al., 2021). 4- vinylphenol (60) also inhibited the growth of Bacillus paralicheniformis ([88]Girawale et al., 2023). As an important component of lignin, coniferol (20) could form natural aromatic polymers by randomly connecting coumarol and mustard alcohol via ester bonds, β-O-4 bonds and C-C bonds, and these polymers could enhance the physical properties of paper and fiberboard ([89]Gusakova et al., 2023). 3.3. Differential analysis of polyphenols in pineapple fibers [90]Fig. 4(a)–(c) displays the volcano plots of differential polyphenol expression profiles among the three pineapple fibers. The x-axis and y-axis represent Log[2](Fold change) and -Log[10](p-value), respectively. In the plots, green, red, and gray dots denote significantly decreased, significantly increased, and non-significantly changed polyphenol levels, respectively. Fig. 4. [91]Fig. 4 [92]Open in a new tab (a) volcano plot of differential expressions in PRF compared with PLF, (b) volcano plot of differential expressions in PSF compared with PLF, (c) volcano plot of differential expression of PRF compared with PSF, (d) cluster thermal graphs of PCs with VIP >1. As shown in [93]Fig. 4(a) and (b), compared with PLF, the contents of 34 polyphenols (e.g., compound 28, 25, 36, 76 and 71) in PRF decreased significantly; the contents of 35 polyphenols (e.g., compound 32, 82, 26, 56 and 12) in PRF increased significantly. Compared with PLF, the contents of 31 polyphenols (e.g., compound 39, 59, 76, 36 and 80) in PSF decreased significantly. The contents of 29 polyphenols (e.g., compound 32, 55, 64, 16 and 50) in PSF increased significantly. As shown in [94]Fig. 4(c), compared with PSF, the contents of 31 polyphenols (e.g., compound 46, 50, 25, and 20) in PRF decreased significantly, 25 polyphenols (e.g., compound 63, 19, 82, 12 and 56) in PRF increased significantly. A cluster heatmap was generated for 48 polyphenols with variable importance in projection (VIP) scores >1. As illustrated in [95]Fig. 4(d), polyphenols within the green-dotted box (e.g., compound 31, 25, and 58) exhibited higher concentrations compared to others. Those within the purple-dotted box (e.g., compound 20, 34, and 46) showed relatively high abundance in PSF. Specific compounds, including compound 70 and 54, demonstrated elevated levels in both PSF and PRF but remained low in PLF. 3.4. In vitro analysis of antioxidant activity of polyphenols in pineapple fibers The antioxidant activities in vitro of polyphenols at a concentration of 0.01 mg/mL are presented in [96]Fig. 5. The results demonstrate that the DPPH and hydroxyl radical scavenging capacities of polyphenols followed the order PLF > PSF > PRF, while the overall antioxidant capacity exhibited the pattern PSF > PLF > PRF. Statistical analysis revealed that while PLF and PSF showed negligible differences in both DPPH and hydroxyl radical scavenging capacities, PRF displayed significantly lower scavenging capacities compared to PSF and PLF. Furthermore, the overall antioxidant capacities of PLF, PRF, and PSF were significantly distinct from each other. Fig. 5. [97]Fig. 5 [98]Open in a new tab Antioxidant activities of polyphenols in PRF, PSF and PLF. PRF, PSF, and PLF represent pineapple root fiber, pineapple stem fiber and pineapple leaf fiber, respectively. These findings align with previous studies demonstrating a positive correlation between plant extract antioxidant activities and total polyphenol content ([99]Shi et al., 2024; [100]Kabre et al.). The antioxidant mechanisms of polyphenols are primarily attributed to hydrogen atom transfer and single electron transfer mediated by hydroxyl groups in phenolic compounds ([101]Zeng et al., 2024). The observed non-proportional relationship between antioxidant capacity and polyphenol content in pineapple fibers might be explained by the differential distribution and composition of polyphenols across various pineapple tissues. 3.5. Analysis of antibacterial effect of polyphenols in pineapple fibers in vitro The antimicrobial efficacy of polyphenols extracted in vitro from PRF, PSF, and PLF against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated. The results, presented in [102]Fig. 6(a) and (b), respectively, demonstrate the inhibitory effects, with corresponding inhibition zone diameters quantified in [103]Table 1. Fig. 6. [104]Fig. 6 [105]Open in a new tab Inhibitory effects of phenolic compounds in three pineapple fibers on E. coli (a) and S. aureus (b), and chemical formulae of the differential phenolic compounds (c). Table 1. Antibacterial diameter of polyphenols in the three pineapple fibers. Antibacterial diameter of Staphylococcus aureus/mm Antibacterial diameter of Escherichia coli/mm Polyphenol concentration PRFP PSFP PLFP PRFP PSFP PLFP 8 % 16.35 b 13.51 c 20.71 a 0 0 13.93 4 % 14.34 b 0 15.77 a 0 0 11.42 2 % 0 0 8.05 0 0 9.55 1 % 0 0 0 0 0 0 0.5 % 0 0 0 0 0 0 [106]Open in a new tab Note: Different letters in the same row represent the significant differences at the level of 0.05. PRFP, PSFP, PLFP present pineapple root fiber polyphenols, pineapple stem fiber polyphenols, pineapple leaf fiber polyphenols, respectively. As evidenced by the data in [107]Table 1 and [108]Fig. 6(a), polyphenols from PRF and PSF exhibited negligible inhibitory activity against E. coli, while those from PLF showed concentration-dependent antibacterial activity within the 2–8 % concentration range. [109]Fig. 6(b) and [110]Table 1 revealed a concentration-dependent inhibitory pattern against S. aureus, with efficacy following the order PLF > PRF > PSF. Notably, PLF polyphenols demonstrated significant inhibition zones at 8 % concentration. Effective antimicrobial activity against S. aureus was observed at concentrations exceeding 2 % for PLF, 4 % for PRF, and 8 % for PSF. As illustrated in [111]Fig. 4(d), polyphenolic compounds in PLF comprised 27 PCs with elevated peak intensities, including caffeoyl quinic acid (39), Compound 28, isoeugenol (58), Compound 1, 35, 59, and 76. The structural configurations of these compounds are presented in [112]Fig. 6(c). Notably, caffeoyl quinic acid had been demonstrated to exhibit potent inhibitory activity against S. aureus ([113]Bisso et al., 2025; [114]Bai et al., 2022). Furthermore, isoeugenol had been shown to induce significant intracellular reactive oxygen species (ROS) accumulation, resulting in accelerated oxidative damage and subsequent bacterial cell death ([115]Findik et al., 2011; [116]Siva et al., 2019). 4. Conclusion Quantitative analysis demonstrated that pineapple root fiber exhibited the highest polyphenol content (9.55 ± 0.21 mg/g), while stem fiber derived polyphenols showed superior antioxidant activity, and leaf fiber polyphenols displayed the strongest antimicrobial effects against E. coli and S. aureus. Through UHPLC-MS analysis, eighty-three polyphenols were isolated and identified from the three pineapple fibers, with 66 and 17 compounds detected in negative and positive ion modes, respectively. The distinct polyphenol profiles and bioactivities among the fibers likely stem from differential metabolic pathway regulation in pineapple leaves, stems, and roots. These findings highlight the potential of pineapple by-products as sustainable sources of bioactive compounds for industrial applications, such as developing antibacterial, preservative paper-based materials for fruits and vegetables. In the future, it is necessary to further explore the effects of fiber extraction methods, pulping and other pulping and papermaking processes on the content of phenolic compounds in pineapple fiber, and help the healthy development of pineapple industry. CRediT authorship contribution statement Zhikai Zhuang: Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Jihua Du: Data curation, Methodology, Validation. Yangyang Qian: Methodology, Resources, Formal analysis, Data curation. Yuhan Wang: Resources, Supervision, Writing – review & editing. Jiao Jing: Conceptualization, Funding acquisition, Resources, Writing – review & editing. Yijun Liu: Funding acquisition, Project administration, Supervision, Writing – review & editing. Gang Chen: Funding acquisition, Project administration, Supervision, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements