J Biosci Vol. 43, No. 4, September 2018, pp. 585–596 Ó Indian Academy of Sciences DOI: 10.1007/s12038-018-9774-1

Evaluation of metabolic changes induced by polyphenols in the crayfish Astacus leptodactylus by metabolomics using Fourier transformed infrared spectroscopy

1 2 3 3 MARIA GRAZIA VOLPE ,SUSAN COSTANTINI ,ELENA COCCIA ,LUCIA PARRILLO 1,3 and MARINA PAOLUCCI * 1Institute of Food Sciences –National Research Council (ISA-CNR), Via Roma 64, 83100 Avellino, Italy 2National Cancer Institute ‘Fondazione G. Pascale’ - IRCCS, Via M. Semmola 52, 80131 Naples, Italy 3Department of Science and Technologies, University of Sannio, Via Port’Arsa, 11, 82100 Benevento, Italy

*Corresponding author (Email, [email protected]) MS received 20 March 2018; accepted 25 May 2018; published online 10 July 2018

In the present study, the effects of polyphenols on the chemical composition of the hepatopancreas of the Astacus leptodactylus, a highly sought farmed crayfish, have been investigated by attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. The hepatopancreas spectrum was quite complex and contained several peaks arising from the contribution of different functional groups belonging to protein, lipids and carbohydrates. The PCA statistical analysis revealed that there were significant differences between crayfish fed a diet without polyphenols and crayfish fed a diet containing polyphenols. Such differences indicated an increase in lipids and proteins in the hepatopancreas of polyphenol-fed crayfish. In conclusion, the analysis of the infrared spectral profile of the hepatopancreas of Astacus leptodactylus, allowed us to elucidate the changes in different biomolecules in response to polyphenol treatment, and confirms the suitability of ATR-FTIR spectral data to analyze diet-induced metabolic effects. These considerations, coupled with the small amount of sample and no preparation needed, make ATR-FTIR a useful tool for routine analyses where the metabolic impact of substances is investigated, especially with a large number of samples.

Keywords. Astacus leptodactylus; ATR-FTIR; crayfish; hepatopancreas; metabolic fingerprinting; metabolomics; polyphenols; vibrational spectroscopy

1. Introduction of hydroxytyrosol and tyrosol, oleuropein, and its aglycone (Priore et al. 2015). Particular attention has been devoted to Polyphenols are a large class of natural compounds derived the hydroxytyrosol, due to its high amount and activity as a from plants (Bravo 1998; Manach et al. 2004). Epidemio- potent scavenger of superoxide anion and hydroxyl radical logical, clinical and nutritional studies strongly support the even more active than synthetic antioxidants and antioxidant evidence that dietary polyphenols are effective in the pre- vitamins (Ranalli et al. 2003; Ferna´ndez-Bolan˜oset al. 2012). vention of common diseases, including cancer, neurodegen- Fourier transform infrared spectroscopy (FTIR) is a rapid, erative diseases, gastrointestinal disorders and others (Pandey simple, high-resolution analytical method that is based on the and Rizvi 2009; Zhang and Tsao 2016). Prevention of disease vibrations of chemical and functional groups in the analyzed by polyphenols is mainly due to their antioxidative properties, components. It provides biochemical profiles containing however the reversal of epigenetic changes can have strong overlapping signals from a majority of the compounds that are effects as well (Russo et al. 2015). Polyphenols are present in present in a cell or in a tissue (Baker et al. 2014). The main almost all classes of food and agro-industrial residues (Naz- advantages of FTIR are the small amount of sample needed, in zaro et al. 2012). The by-products of fruits, vegetables, oil- the order of micrograms or even nanograms, the rapidity of seeds, nuts, and cereals have been found to contain high the analysis and the lack of long and time-consuming pre- amounts of polyphenols (Kuppusamy et al. 2015). For treatment of the sample (Dettmer et al. 2007; Corte et al. 2011; example, olive mill waste water (OMWW), which results Kawase et al. 2014). The main disadvantage is given by the from the olive oil extraction, is a rich source of different complex biochemical information requiring validated and polyphenols with a wide range of biological activities robust chemometrics to be used in order to turn data into (Hamden et al. 2009). The phenolic complex is mainly made information (Karoui et al. 2010). Whilst not as specific and http://www.ias.ac.in/jbiosci 585 586 Maria Grazia Volpe et al. sensitive as some techniques, such as GC-ToF-MS, this Srl via dell’ Industria, 8 -47843 Misano Adriatico –RN, spectroscopic tool is successfully applied to the characteri- Italy) from Lake Sevan (Armenia) and declared, according to zation of complex biological systems, providing simultane- the European Community Law, in good health and disease- ously, in a single measurement, information on the main free. Upon arrival, crayfish were weighted (25.0±4.0 g) and biomolecules, such as carbohydrates, amino acids, fatty acids, stocked in 500 L tanks (1 9 1 9 0.5 m) in a semi-recircu- lipids, proteins and polysaccharides (Goodacre et al. 2004). lating system. The system was held under natural photope- Thus, for the study of several biological systems, such as riod. Temperature and pH (pHmeter GLP 21 Crison), normal and malignant cells or tissues, and whole model dissolved oxygen, total ammonia nitrogen, nitrite nitrogen, organisms (i.e. the nematode Caenorhabditis elegans), FTIR nitrate nitrogen, phosphorus, phosphate, total chlorine and has been successfully employed (Severcan et al. 2000; Ami firmness (CaCO3) were measured twice a week with a et al. 2004; Vargas-Caraveo et al. 2014). Recently, the Hanna Instruments Photometer C200. All parameters fell in attenuated total reflectance–Fourier transform infrared (ATR- the accepted range for this species (Safari et al. 2014). FTIR) spectroscopy and Fourier transform infrared Crayfish were divided into three groups of eight animals microspectroscopy (FTIRM) have been employed to inves- each. One group was fed on the basic diet (Control Diet, or tigate the biochemical status of different types of cells like CD), the second group was fed on the basic diet enriched fibroblast, carcinogenic cells, plasmatic cells, and enterocytes with 0.05 g/100 g of the diet of polyphenols extracted from under induced oxidative stress and in response to polyphenols olive mill waste water (OMWW) (Low Concentration Diet, (Vileno et al. 2010; Oleszko et al. 2015; Barraza-Garza et al. or D1), and the third group was fed on the basic diet enri- 2016). Moreover, ATR-FTIR provides useful information ched with 0.5 g/100 g of diet of OMWW polyphenols (High when studying protein structure associated to pathological Concentration Diet, or D2). The amount of feed adminis- modification, such as amyloid proteins in amyloid diseases, tered was 5% of the body weight and was adjusted every two Parkinson’s, Huntington’s and Prion’s diseases, type II dia- weeks, according to the weight of the animal. It was betes, and also some cancers (Sarroukh et al. 2013). In this administered every other day between 8:00 and 9:00 am. All sense, ATR-FTIR has been proposed as a tool to complement animals were weighed once every two weeks and at the histopathological analysis in the clinical routine for the conclusion of the experiment. The feeding experiment lasted diagnosis of cutaneous squamous cell carcinoma, being able for 24 weeks. At the end of the experiment animals were to discriminate between normal and cancerous cells on the weighted, sacrified, and the hepatopancreas removed. The basis of metabolic changes (Lima et al. 2015). Derenne et al. hepatosomatic index (HSI) was calculated as follows: % = (2013) could detect metabolic changes in in vitro cancer cells [hepatopancreas mass / final body mass] 9 100. exposed to polyphenols by ATR-FTIR. All animal experiments complied with the ARRIVE In the present study, the effectiveness of ATR-FTIR in the guidelines and were carried out in accordance with the U.K. detection of the chemical composition of the hepatopancreas Animals (Scientific Procedures) Act 1986 and associated due to polyphenols in the diet has been investigated in the guidelines, EU Directive 2010/63/EU for animal experi- crayfish Astacus leptodactylus, a freshwater decapod crus- ments, or the National Institutes of Health guide for the care tacean actively commercialized in Europe, where crayfish are and use of Laboratory animals (NIH Publications No. 8023, perceived as luxury food and therefore highly sought. Among revised 1978). OMWW was a generous gift of a local pro- crustaceans, crayfish are an appealing food resource and sev- ducer of olive oil (Industria Olearia Biagio Mataluni S. R. L. eral species are farmed around the world (FAO report 2016). Via Benevento 82016 Montesarchio BN, Italy). Polyphenols Consequently, the research is always on the look out to find were extracted and characterized as reported in Costantini new feed ingredients to tailor diets aimed at suiting the con- et al. (2018). The main phenolic compounds identified were: sumer needs. Indeed, the diet has a strong influence on the Hydroxytyrosol, Tyrosol, Caffeic acid, Verbascoside, Ferulic product nutritional value that ultimately depends on the bio- acid, Oleuropein. Basic diet composition is reported in chemical constituents like proteins, carbohydrates, lipids, table 1. amino acids and minerals. Therefore, the availability of a rapid, cost sensitive and not invasive method of analysis of the bio- chemical changes caused by the diet is of evident utility. 2.2 ATR-FTIR spectral measurements

At the end of the feeding trial animals were euthanized by 2. Materials and methods immersion in ground ice for about 30 min. and from each crayfish the hepatopancreas was quickly removed, rinsed 2.1 Animals and experimental design with distilled water, weighted and frozen at -80°C before being lyophilized. Hepatopancreas was lyophilized and Adult male crayfish Astacus leptodactylus were employed. analyzed without any previous treatment and placed directly Crayfish were imported by ‘LPA live seafood’ (L.P.A. Pesca on the germanium piece of the infrared spectrometer with Metabolic fingerprinting of the crayfish Astacus leptodactylus 587

Table 1. Ingredients of the basic diets (values are reported as fingerprints are compared using powerful classification mean ± SEM; three samples for each diet were analyzed) algorithms to verify the identity and quality of each material. AssureID can employ one of two algorithms, COMPARE Ingredients g/100 g and SIMCA (Soft Independent Modeling Class Algorithm), Fish meal (anchovy) 4.5 to classify new samples. In this study, we used SIMCA as a Krill meal 4.5 chemometric approach which models the variation within Soybean meal 14.0 the collection of reference spectra. SIMCA develops sepa- Corn gluten 24.0 rate models (so-called disjointed class models or SIMCA Wheat flour 26.5 Corn starch 4.0 hyperboxes) based on principal component analysis (PCA) Fish oil 4.0 for each training set category. In details, all spectra were Canola oil 4.0 subjected to baseline correction and normalization prior to Soy lecithin 5.0 the statistical analysis (PCA) that was used as an unsuper- Cholesterol 0.5 vised classification technique aiming at sorting the spectra Glucosamine 1.0 Choline 1.5 into different categories. The scores plot consisting of a Vitamin C 1.0 projection of the original data onto principal component axes Vitamin premix1 2.0 is used to visualize clustering among samples (sample pat- 2 Mineral premix 1.5 terns, groupings, or outliers). Hence, PCA was performed to Carboxymethyl cellulose 2.0 evaluate the potential of the ATR-FTIR to differentiate the 1 Zoofast, Italy spectra of polyphenol fed crayfish and control crayfish 2 hepatopancreas. Three classes were defined: CD, D1 and Mineral premix contains (mg kg-1)Mg, 100; Zn, 60; Fe, 40; Cu, 5; Co, 0.1; I, 0.1; and antioxidant (BHT), 100. Vitamin premix contains diet D2. Eight spectra for each group (CD, D1, D2) were (mg kg-1) E, 30; K, 3; thiamine, 2; riboflavin, 7; pyridoxine, 3; analyzed. For cluster analysis, the spectral ranges pantothenic acid, 18; niacin, 40; folacin, 1.5; choline, 600; biotin, 0.7 (I) 3026–2988, (II) 2952–2880, (III) 2878–2828, (IV) and cyanocobalamin, 0.02 1769–1700, (V) 1474–1429, (VI) 1422–1359 cm-1 were independently analyzed. The performance of the developed constant pressure applied. 10 mg of sample were analyzed. SIMCA model was evaluated through the interclass distance The pressure of the ATR-FTIR acquisition was 70±2 psi. between the control and diets, the three-dimensional prin- The FTIR spectra were recorded in the mid-IR region cipal component analysis, scores plot, and the recognition (4000–650 cm-1) at resolutions of 4 cm-1 with 32 scans and rejection rates of the samples. using Perkin Elmer FTIR Frontier coupled with DTGS (deuterated tri-glycine sulphate) detector (Perkin-Elmer Inc., Norwalk, CT, USA). Air background spectrum was recorded 3. Results before each sample. Eight samples for each group were analyzed and each sample was analyzed in triplicate. Prior to Polyphenols extracted from OMWW were administered to data analysis, each spectrum was baseline corrected and the the freshwater crayfish A. leptodactylus at two levels of absorbance was normalized so that peak absorbance of the inclusion (0.05 and 0.5 g/100 g of diet) in a 24-week feeding most intense band is set to unity. Spectra were then elabo- trial. Hydroxytyrosol was the most abundant polyphenol, rated using Spectrum AssureID software, purchased with the followed by Tyrosol, Caffeic acid, Verbascoside, Ferulic acid instrument. and Oleuropein. At the end of the experiment, the body weight and the hepatosomatic index were recorded. Both increased in polyphenol fed groups with respect to the 2.3 Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) and any significant difference was determined at Table 2. Percentage of increase in body weight (BW) and hep- the 0.05 level by Duncan’s multiple range test. The analyses atosomatic index (HSI) of Astacus leptodactylus fed for 24 weeks were carried out with the Statistica version 7.0 statistical on control diet, OMWW-LC containing diet and OMWW-HC package (Statsoft Inc., Tulsa, OK, USA). containing diet (n = 30; control values were set at 100%) For ATR-FTIR, statistical analysis was performed by the Diets Spectrum AssureID software (trademark of PerkinElmer, Inc. Part Number 0993 4516 Release E; Publication Date Control OMWW-LC OMWW-HC July 2006; Software Version 4.x), a sample checking system which utilizes FT-IR and FT-NIR spectroscopy to generate BW 100 110 122 HSI 100 125 136 sample specific ’fingerprints’ of production materials. These 588 Maria Grazia Volpe et al. Table 3. Band assignments for the hepatopancreas tissue spectra based on current literature (Movasaghi et al. 2008; Malek et al. 2014; Silva et al. 2014; Zohdi et al. 2015)

Peak wavenumber of hepatopancreas (cm-1) Vibrational mode Components 3279 Amide A: mainly N–H stretching of proteins Proteins 3011 CH stretching vibration of unsaturated fatty acids Unsaturated lipids 2924 CH2 asymmetric stretch. Stretching modes of the chain CH2 and Saturated lipids and side chains of proteins =CH groups cholesterol, phospholipids/asymmetric CH2 2854 CH2 symmetric stretch. Stretching modes of the chain CH2 and Saturated lipids and side chains of proteins =CH groups 1745 C=O stretching of esters and aldehydes Lipids, phospholipids, triglycerides, cholesterol esters 1638 C=O stretch ? NH bend (amide I) Proteins beta sheet structure 1541 N–H bend ? C–N stretch (amide II) Proteins (amide II) 1456 Scissoring vibrations of the long-chain CH2 groups. Symmetric Lipids, proteins, nucleic acids and asymmetric bending modes of methyl groups in skeletal proteins 1401 CH3 deformation of aliphatic side groups of residues Proteins, Fatty acids, amino acid symmetric - COO symmetric stretch. Stretching C-O, deformation C–H and methyl (CH3) bending proteins, nucleic acids N–H and lipids Symmetric CH3 bending modes of the methyl groups. Symmetric stretching vibration of COO2 Symmetric stretch of methyl groups - 1236 PO2 asymmetric stretch Overlapping of the protein amide III DNA stretching PO2 asymmetric nucleic acid/ and the nucleic acid phosphate vibration amide III 1149 C–O stretch C–O stretch Phosphodiester stretching bands carbohydrates, glycogen, glycogen mucin (symmetric and asymmetric) 1077 C–C stretch Glycogen 1023 COH deformation Glycogen, carbohydrates 931 C–N–C stretch of ribose-phosphate skeletal vibrations Nucleic acids 847 Left-handed helix DNA (Z form) Z type DNA 765 Out of plane bending vibrations Unknown 703 Out of plane bending vibrations Unknown 663 Out of plane bending vibrations Unknown control group (table 2). The hepatopancreas was analyzed by assigned around 1450 cm-1 (band peak 1456 cm-1) and are ATR-FTIR. indicative of the presence of lipids, proteins, and nucleic acids. Indeed, the bands attributed to the methylene group are mainly due to the unsaturated chains in lipids, whereas 3.1 Interpretation of the ATR–FTIR spectra the bands due to the vibrations of the methyl groups can be found in proteins, and nucleic acids. The range 1700–1500 Figure 1 shows an example of the ATR–FTIR spectra of the cm-1 provides information on peptide bonding in proteins hepatopancreas of crayfish in the region of 4000–650 cm-1. and its secondary structure. In particular, bands peaking at The spectrum is quite complex and contains several bands 1638 and 1541 cm-1 are attributed to amide I and II arising from the contribution of different functional groups vibrations, respectively. The presence of proteins is also belonging to protein, lipids, and carbohydrates. Since there attributed to the peak at 3279 cm-1 indicating N–H are not previous studies on crayfish hepatopancreas, the stretching. The band at 1401 cm-1 can be assigned to the assignments of the bands were based on studies on biolog- bending mode of the side chains of amino acid, lipids, and ical samples (Movasaghi et al. 2008; Malek et al. 2014; nucleic acids. However, it is likely that the major contribu- Silva et al. 2014; Zohdi et al. 2015). The assignments of the tion derives from proteins due to the preponderance of the major bands are shown in table 3. The peak assignments in peaks attributed to amide I and II vibrations. The peak at -1 -1 - the range 3050–2800 (band peaks at 2924 and 2854 cm ) 1236 cm reflects PO2 asymmetric stretch and can be and around 1700 cm-1 (band peak 1745 cm-1) are assigned to DNA. In general, the spectral range of attributable to lipid stretching vibrations, while symmetric 1150–1000 cm-1 is typical for carbohydrates. Within this and asymmetric bending vibrations of methyl groups and spectral range, the peaks at 1077 and 1023 cm-1 are -1 scissoring vibrations of the long-chain CH2 groups can be assigned to glycogen. The peaks at 931 and 847 cm are Metabolic fingerprinting of the crayfish Astacus leptodactylus 589

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Figure 1. Typical FTIR absorption spectrum of hepatopancreas of the male crayfish Astacus leptodactylus. The measured absorbance spectrum reported was baseline corrected. The wavelength of the absorbance peaks is indicated. likely to indicate the presence of nucleic acids. Finally, the by the SIMCA models. This figure helps to visualize the peaks at 765, 703, 663 cm-1 are not assigned to any class separation among control crayfish and polyphenol-fed biomolecule. crayfish. Diet 2 group gave results similar to the Diet 1 In figure 2A representative ATR-FTIR spectra of the group and therefore in Figure 4 only the results relative to hepatopancreas of control (CD) and polyphenol-fed groups D1 are shown. The boundary ellipse (hyperboxes) around (D1 and D2) are reported. In figure 2B and C the spectrum each cluster represents the 99% confidence interval, and the obtained subtracting the spectrum of the control crayfish points in each cluster represent the spectrum of each sample. (CD) from the spectrum of D1 and D2 crayfish respectively, In detail, data analysis performed in smaller ranges of the are reported. Only positive peaks were present in the dif- spectrum, corresponding to peaks reported in table 1, ference spectrum with the exception of a peak around 1000 revealed that there were some differences among groups. In cm-1 that was negative. The subtraction of two D1 and D2 particular, cluster differences were obtained in the ranges of spectra (figure 2D) evidenced no relevant differences 3026–2988, 2952–2880, 2878–2828, 1769–1700, between D1 and D2 crayfish. 1474–1429, 1422–1359 cm-1. In order to better evaluate the differences in the spectra of crayfish, the second derivative was carried out (figure 3). Results indicate that polyphenol-fed crayfish suffered few 4. Discussion modifications in their spectral profiles, in reference to the control group. In particular, three peaks at 1149, 1023 and In this study, we employed ATR- FTIR to gain insight into 764 cm-1 in the control group shifted to 1145, 1027 and 770 the spectral profile of the biomolecules of the hepatopan- cm-1 respectively, in polyphenol-fed groups. creas of crayfish Astacus leptodactylus fed for 24 weeks with OMWW polyphenols enriched diets. A mixture of polyphenols extracted from OMWW characterized by the 3.2 Principal component analysis predominance of hydroxytyrosol was employed. Hydroxy- tyrosol (3,4-dihydroxyphenethyl alcohol) is a phenolic The biochemical profiles from biological samples are compound or polyphenol, present in the fruit and leaf of the extremely complex and, consequently, ATR-FTIR data must olive (Olea europaea L.). Polyphenols are characterized by be analyzed by means of multivariate analysis. To develop the presence of large multiples of phenol structural units the discrimination models between control crayfish and with hydroxyl functional groups bound to the phenolic crayfish fed with diets added with polyphenols from ring(s) (Quideau et al. 2011). Growth performances signifi- OMWW, ATR–FTIR spectra were subjected to SIMCA cantly improved in crayfish fed polyphenol-enriched diets analysis. Figure 4 shows the 3D-PCA scores plot generated (Parrillo et al. 2017), and a stimulating effect on the general 590 Maria Grazia Volpe et al. metabolism of the crayfish Astacus leptodactylus was seen Figure 2. (A) Overlapped FTIR spectra of the hepatopancreas ofc (Costantini et al. 2018). In the crayfish, as in all crustaceans, Astacus leptodactylus of the three experimental groups. Black line the hepatopancreas is analogous to the vertebrate liver and = control diet (CD); blue line = low polyphenol concentration diet should be considered as a metabolic factory, which is the (D1); red line = high polyphenol concentration diet (D2). (B) Sub- center of lipid and carbohydrate metabolism (Mente 2003). traction spectrum (green) between D1 and CD (light colors in the background). (C) Subtraction spectrum (green) between D2 and CD Moreover, in the absence of adipose tissue in crustaceans, (light colors in the background). (D) Subtraction spectrum (green) the hepatopancreas is the main site of lipid storage between D1 and D2 (light colors in the background). (O’Connor and Gilbert 1968; Garcı´a et al. 2002). The functions of the hepatopancreas are considered to include the storage and depletion of organic substances to support var- ious significant life activities, such as ovarian maturation, vitellogenin synthesis and molting (Wen et al. 2001; Ying polyphenol-enriched diets, lipids were synthesized and et al. 2006). Thus, the hepatosomatic index (HSI), that is the stored in the hepatopancreas of Astacus leptodactylus with ratio between the hepatopancreas and the body weight, is little mobilization (Parrillo et al. 2017). Moreover, the out- widely used to evaluate differences in crayfish condition and come of the present study is in agreement with the results of nutritional status (Jussila and Mannonen 1997). In this study, the 1H-NMR metabolomic profiling of the crayfish Astacus the group fed on the polyphenol-enriched diet, showed an leptodactylus hepatopancreas subjected to polyphenol-en- HSI significantly higher compared to control crayfish (Par- riched diets where the consumption of polyphenols extracted rillo et al. 2017), suggesting better digestion and adsorption from OMWW induced higher levels of fatty acids, choles- of food, which in turn contribute to improving hepatic terol, phospholipids, and triglycerides in the hepatopancreas storage in the digestive gland. (Costantini et al. 2018). Polyphenols may interact with FTIR technique is an analytical tool used to detect the several molecular targets influencing different signaling changes in the functional groups, bonding types, and pathways and gene expression (Manach et al. 2004). Indeed, molecular conformations of biomolecules such as nucleic literature data point at a wide array of physiological func- acids, carbohydrates, proteins, and lipids, thus it holds a tions and metabolic pathways affected by polyphenols. They considerable potential as a metabolic fingerprinting tool for mainly act as antioxidants, but also show many other effects, the rapid detection of molecular pattern (Baker et al. 2014). such as the prevention of cardiovascular diseases, cancers, Since every biological molecule absorbs mid-infrared light, and neurodegenerative diseases (Scalbert et al. 2005; Zern FTIR can detect the spectral characteristics of minimum and Fernandez 2005; Sorice et al. 2016). They are also amount of samples in a short time at low cost. If used in beneficial for gut health through their effect on microbiota combination with the appropriate multivariate analysis and, last but not least, they show metabolic effects (Martin strategies, FTIR can provide useful information about the et al. 2009; Cardona et al. 2013; Sorice et al. 2016). metabolomic alterations caused by internal or external Metabolic effects of polyphenols are studied in mammals changes. The aim of this study was to demonstrate that the and humans in particular, with respect to their ability to metabolomics alterations caused by polyphenols in the diet lower cholesterol and glucose levels in blood and therefore can be detected by FTIR analysis short time, using limited to improve hyperlipidemia and diabetes (Bahadoran et al. amount of sample that did not require long, tedious and 2013; Bruckbauer and Zemel 2014). complex pre-treatements. Spectra of the hepatopancreas of the control and The hepatopancreas spectrum obtained was quite complex polyphenol-fed crayfish exhibited imperceptible differences. and contained several bands arising from the contribution of The second derivative allowed us to observe absorption different functional groups belonging to protein, lipids, and bands with fine structures that normally are masked in the carbohydrates. In order to interpret these spectral differences raw data, but that can be important markers of the metabolic and to understand the biochemical variations in the status of the hepatopancreas and can be used for comparative polyphenol-treated groups with respect to the control group, analysis between treatments. Three peaks at 1149, 1023 and difference spectra were computed by subtracting the control 764 cm-1 in the control group shifted to 1145, 1027 and 770 hepatopancreas spectra from polyphenol treated hepatopan- cm-1 respectively in the polyphenol-fed crayfish. The first creas spectra. The subtraction spectra showed a change in two peaks indicate the presence of glycogen, while for the the intensity of the lipid and protein regions in response to 770 cm-1 peak there is no assignment. Such peak is inclu- the polyphenol treatments, suggesting an increase in both ded in the fingerprint region, relative to the individual bonds macromolecules in the polyphenol-treated groups of cray- of the molecule’s skeleton, strongly coupled to each other. fish. Indeed, the areas of the absorption bands in FTIR We hypothesize that the polar groups of polyphenols spectrum are directly related to the concentration of the administered with the diet, interacting with the polar groups molecules (Venkataramana et al. 2010). Previous studies of carbohydrate molecules of the hepatopancreas, caused a carried out by us, highlight that, after six months of shift in the wavelength frequencies. Metabolic fingerprinting of the crayfish Astacus leptodactylus 591

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-0.1 4000 0053 0003 0052 0002 0051 0001 650 cm-1 592 Maria Grazia Volpe et al. In order to confirm the suitability of FTIR spectral data to Figure 4. Three-dimensional principal component analysis scoresc analyze the effect of polyphenol-enriched diet, we performed plot of the hepatopancreas of Astacus leptodactylus crayfish of the a PCA analysis using the SIMCA algorithm. The use of control group and Diet 1 group derived from SIMCA. Data analysis statistical analysis is required to highlight whether the subtle was performed in smaller ranges of spectrum reported in the alterations observed are or not statistically significant. rectangles above each plot. Cluster separation was obtained in those wavelength ranges corresponding to saturated and unsaturated lipids, triglyc- erides, fatty acids, phospholipids, amino acids, and proteins, also determines if it does not belong to any class. The indicating a good separation between groups. According to classification performance (shown in the diagnostic reports the SIMCA models, the larger interclass distance among of the Fig. 4 of this study) of the optimized SIMCA model is groups, the better separation. It is generally accepted that a based on the percentage of recognition and rejection. 100% distance value higher than 3 is indicative that the samples are correct classification rate means that all samples were cor- well separated and hence different (He et al. 2007). The rectly classified, while 100% of rejection is obtained when characteristic of SIMCA is that not only determines whether all samples belong to different classes. Thus, a rejection rate a sample belongs to any of the predefined categories, but less than 100% indicates that a percentage of samples cannot

Derivative Result 3 (A) 2

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Derivative Result 3 (B)

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10 -6 1819 0081 0061 0041 0021 0001 008 006 561 cm-1

Figure 3. (A) Second derivative representative spectra of the hepatopancreas of Astacus leptodactylus of control (CD) and polyphenol fed crayfish (D1 and D2). Black line = control diet (CD); blue line = low concentration diet (D1); red line = high concentration diet (D2). (B) Zoom of the region between 1800 and 600 cm-1. The labels show the peaks at 1149 1023 and 764 in the control group shifted to 1145, 1027, and 770 in D1 and D2 groups. Metabolic fingerprinting of the crayfish Astacus leptodactylus 593 594 Maria Grazia Volpe et al. be attributed to different classes. In this study, the score plots 2016 Infrared spectroscopy as a tool to study the antioxidant for selected regions of the spectrum showed that the hep- activity of polyphenolic compounds in isolated rat enterocytes. atopancreas of the crayfish fed with polyphenol-enriched Oxidative Med. Cell. Longevity http://dx.doi.org/10.1155/2016/ diets were grouped together, and segregated from the control 9245150 crayfish, indicating that treatment with polyphenols caused Bravo L 1998 Polyphenols: chemistry, dietary sources, metabolism, an IR spectral profile different from the control crayfish in and nutritional significance. Nutr. Rev. 56 317–333 Bruckbauer A and Zemel MB 2014 Synergistic effects of the case of the peaks with the wavelength corresponding to polyphenols and methylxanthines with leucine on AMPK/ lipids, phospholipids, fatty acids, free amino acids and sirtuin-mediated metabolism in muscle cells and adipocytes. proteins, in agreement with a general metabolic increase due PLoS ONE 9 e89166 to polyphenol treatment already reported by NMR Cardona F, Andre´s-Lacueva C, Tulipania S, Tinahones FJ and (Costantini et al. 2018). 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Corresponding editor: BASUTHKAR JRAO