Article pubs.acs.org/JAFC
Chemical Composition of Defatted Strawberry and Raspberry Seeds and the Effect of These Dietary Ingredients on Polyphenol Metabolites, Intestinal Function, and Selected Serum Parameters in Rats Monika Kosmala,*,† Zenon Zduńczyk,§ Jerzy Juśkiewicz,*,§ Adam Jurgoński,§ Elzḃ ieta Karlińska,† Jakub Macierzyński,† Rafał Jańczak,# and Edward Rój# †
Institute of Chemical Technology of Food, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-748 Olsztyn, Poland # New Chemical Syntheses Institute, Aleja Tysiąclecia Państwa Polskiego 13a, 24-110 Puławy, Poland §
ABSTRACT: Strawberry and raspberry seeds were chemically analyzed and added as dietary ingredients to investigate the physiological response of rats. In both cases the main component was dietary fiber and the main polyphenols were ellagitannins (ET). The strawberry ET were mainly constituted by monomers and a dimer, agrimoniin, whereas raspberry ET were mainly constituted by a dimer, sanguiin-H-6, and a trimer, lambertianin-C. The lower content and the less polymerized structure of strawberry ET resulted in a higher cecal metabolites concentration (mainly nasutin and urolithin-A) in comparison to rats fed diet containing raspberry seeds. Dietary raspberry seeds, a source of dietary fiber, despite being richer in polyphenol compounds, were better utilized in fermentation processes, resulting in enhanced production of short-chain fatty acids. As opposed to strawberry seeds, the treatment with raspberry seeds beneficially improved the atherogenic index of a diet, mainly due to reduced triacylglycerol concentration in the serum. KEYWORDS: berry defatted seeds, dietary fiber, ellagitannin, serum, rat
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INTRODUCTION Fruits of strawberries and raspberries are rich in vitamin C, anthocyanins, dietary fiber, and polyphenolic compounds.1,2 Leaves and fruits of raspberry have long been applied in phytotherapy to prevent hemorrhages, diarrhea, and diabetes.3 Among the biologically active compounds present in fruits, polyphenolic compounds constitute the main group with potential health significance.2 Although berry polyphenols are best known for their antioxidant and anti-inflammatory actions, recent research has shown that their bioactivities extend to many other pathways as well in this activation of phase II enzymes and qualitative modification of gut flora.4,5 Berry consumption beneficially influences the lipid profile by significantly reducing total cholesterol, low-density lipoprotein cholesterol, and triglycerides levels.6 Special attention is aroused by ellagitannins, which, together with anthocyanins and flavanols, are the key polyphenols of berry fruits.2,4,6,7 Berry fruits are relatively unstable and in fresh form are consumed shortly after harvest. A short fruiting period makes a significant part of the harvest intended for the production of concentrated juices. A byproduct of fruit processing is pomace, which in the fresh form constitutes 4% of the mass of processed strawberries8 and 10% of raspberry mass.9 A quantitatively significant component of dried pomace is seeds, the content of which ranges from 40% in strawberry pomaces10 to 80% in raspberry pomaces.11 Seeds of raspberries and strawberries, as byproducts of fruit processing, have so far been perceived as a potential fatty raw © XXXX American Chemical Society
material as they contain 10% oil with an interesting chemical composition.9 Strawberry and raspberry oils are rich sources of polyunsaturated fatty acids and antioxidants, namely, tocopherols, polyphenols, and phytosterols, that may improve the antioxidative status of a consumer.9,12 For this reason, these oils may be applied as dietary supplements as well as in cosmetics.9 Oil production from fruit seeds is effective upon the use of new separation methods, such as supercritical carbon dioxide extraction.13 Apart from a high effectiveness of lipids isolation, an additional advantage of this method is selectivity against lipid substances and high quality of defatted and disintegrated seeds as byproducts in this process.13 Product s such as seeds recovered from strawberry pomace may serve as a component of gluten-free bread14 or as a raw material for the production of polyphenolic extracts. Seeds of strawberry and raspberry are rich in dietary fiber10 and ellagitannins.7,8 Although anthocyanins in strawberries and raspberries are quantitatively the most important polyphenols,5 they are located mostly in the flesh1 and transferred to juice in the process of juice production, which is why they are found in low quantities in the seeds.10 Results of ample research confirm that ellagitannins, and to be more specific urolithins metabolized from them, display antiReceived: February 4, 2015 Revised: March 6, 2015 Accepted: March 9, 2015
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DOI: 10.1021/acs.jafc.5b00648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry inflammatory15,16 and anticarcinogenic properties.17 In this context, the consumption of fruit pomaces or extracts produced from them ensures exhaustive utilization of the biological potential of the harvested fruits. The main component of strawberry ellagitannins is dimeric GOG type agrimoniin built of two monomeric units of α-1-Ogalloyl-2,3:4,6-bis-HHDP-D-glucose that are linked by two residues of gallic acid with a C−O−C bond.10,18 In turn, raspberries contain sanguiin-H-6 and lambertianin-C,19 the monomeric units that are linked with a sanguiinsorboyl group.18 This difference in the structure of ellagitannins may affect the physiological effect of polyphenols of Rosaceae family fruits as well as byproducts of fruit processing and seeds used for oil production. It is a novel product so far little recognized in terms of composition and physiological properties. In the context of the above overview of the literature, the goal of this study was to enrich knowledge about the chemical composition of interesting and rich in dietary fiber ellagitannins, byproducts of fat carbon dioxide supercritical extraction of strawberry and raspberry seeds, and to identify the local and systemic physiological effects of such products on intestinal functions, blood glucose level, and selected blood markers of lipid metabolism of experimental rats.
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Flow rate was 1.25 mL/min. Gradient was as follows: 0−5 min, 4% B; 5−12.5 min, 4−15% B; 12.5−42.5 min, 15−40% B; 42.5−51.8 min, 40−50% B; 51.8−53.4 min, 50−55% B; and 53.4−55 min, 4% B. The volume of injection was 20 μL. Detection conditions were as follows: 280 nm (agrimoniin, sanguiin-H-6, and lambertianin-C); 360 nm (ellagic acid, quercetin and kaempferol glycosides, quercetin, and kaempferol); 520 nm (anthocyanins). Standards used were ellagic acid, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, quercetin, kaempferol, pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside, kaempferol3-O-β-D-(6″-E-p-coumaroyl)-glucopyranoside (Extrasynthese, Genay, France), agrimoniin, sanguiin-H-6, and lambertianin-C (see below). Extraction and Purification of Agrimoniin, Sanguiin-H-6, and Lambertianin-C. The extraction of agrimoniin was carried out from strawberry pomace and that of sanguiin-H-6 and lambertianin-C from raspberry pomace before the separation of seeds, with 70% acetone. One hundred grams of pomace was treated with the solvent at a 1:14 weight ratio and left to macerate for 24 h. The solution was filtered through a filter paper, and acetone was evaporated by vacuum. The solution was loaded onto an Amberlite XAD-1600N column (8 cm × 2 cm), which was rinsed with 25 mL of water and eluted with 50 mL of 60% ethanol. Purified 10 mL fractions were collected. Isolation of ETs was carried by the use of a Knauer chromatograph, composed of two pumps (Knauer K-501), a Phenomenex Luna 10u C18 100a (250 × 21, 20 mm; 10 μm) (Torrance, CA, USA) column, a UV−vis detector, and a Foxy R1 Teledyne ISCO (Lincoln, NE, USA) fraction collector.7 The flow rate was 15 mL/min. Eluent A was 0.1% formic acid in water, and eluent B was 80% methanol. Gradient was as follows: 0−3 min, 20% B; 3−20 min, 20−35% B; 20−45 min, 35−70% B; 45−50 min, 70% B; 50−55 min, 70−20% B; and 55−60 min, 20% B. The volume of injection was 500 μL. Detection parameter was 280 nm. Agrimoniin, sanguiin-H-6, and lambertianin-C peaks were collected from 10 separations, then combined; methanol was removed by distillation, and the preparations were freeze-dried. The purity of agrimoniin at 280 nm was 96%, that of sanguiin-H-6, 92.5%, and that of lambertianin-C, 92.1%. The molecular masses of agrimoniin, sanguiin-H-6, and lambertianin-C were confirmed by ESI-MS/MS detector. Water solution of the substance was directly injected into the MS detector (lCQ DECa, Thermo-Finnigan). The negative ion mode was used. The source parameters were as follows: ion spray voltage, 3.00 kV; capillary temperature, 325 °C; sheath gas and auxiliary gas, 30 and 10 units/ min, respectively. To generate MS/MS data, the precursor ions were fragmented by helium gas collision in the ion trap by optimizing the collision energy to obtain an intensity of the precursor ion close to 10% of the relative scale of the spectrum.18 The main pseudomolecular ion [M − H] of agrimoniin H-6 was observed at 1869 (m/z), and the analysis of decomposition showed the presence of 1567, 1265, 935, 633, and 301 (m/z) fragment ions. The main pseudomolecular ion [M − H] of sanguiin-H-6 was observed at 1869 (m/z), and the analysis of decomposition showed the presence of 1235, 935, 933, 633, and 301 (m/z) fragment ions. The main pseudomolecular ion [M − H] of lambertianin-C was observed at 1401 [M − 2H]2 (m/z), and the analysis of decomposition showed the presence of 1869, 1567, 1265, 1103, 933, 631, and 301 (m/z) fragment ions. Proanthocyanidins and Free Catechins. Proanthocyanidin degradation in an acidic environment with an overdose of phloroglucinol was performed as in ref 21. To 20 mg of a sample was added 800 μL of a methanol solution containing phloroglucinol (75 g/L) and ascorbic acid (15 g/L). Phloroglucinolysis was started by adding 400 μL of 0.2 mol/L hydrochloric acid in methanol and was carried out for 30 min at the temperature of 50 °C and then stopped by adding 600 μL of a 40 mmol/L sodium acetate solution in an ice bath. The samples were centrifuged for 5 min at 500 rad/s and diluted with a 40 mmol/L sodium acetate solution. A Knauer Smartline chromatograph with a fluorescence detector (FD) RF-10AXL (Shimadzu, Tokyo, Japan) and a Phenomenex Gemini 5u C18 110a (250 × 4.60 mm; 5 μm) column was used. The gradient was as follows: 0−10 min, 4−7% B; 10−27 min, 7−30% B; 27−29 min, 30−70% B; 29−34 min, 70% B; 34−35 min, 70−4% B; and 35−40 min, 4% B. Phase A consisted of a 2.5%
MATERIALS AND METHODS
Recovery of Seeds and Their Defatting Using SFE. Strawberry and raspberry seeds after removal of oils by CO2 supercritical extraction constituted research material. Seeds were separated from industrial residues of juice production. Seeds (300 g) were crushed in a roller crusher in a CO2 atmosphere to the particle size of 0.4−0.6 mm and immediately placed in a supercritical extractor. The plant (NCSI) was composed of the extractor with 1 dm3 volume, two separators with separation capacity up to 500 bar (S1) and up to 100 bar (S2), circulating pump, condenser, and CO2 tank. The following conditions were used: extraction pressure, 200, 250 bar; I grade separation level pressure, 53 bar; temperature of the inlet, Tw, 40 °C; mass of raw material, 300 g; flow rate, QCO2, 6.0−7.0 kg (one cycle duration = 180−210 min). Analyses of Preparations. Basic Composition. AOAC methods20 were used: dry matter and ash, 940.26; protein, 920.152; crude fat, 930.09; TDF, 985.29; IDF, 991.42. Polyphenols. Extraction of polyphenols was carried out according to the method in ref 1 using 70% acetone. To 500 mg of the material was added 4 mL of a solvent, which was mixed, sonified for 15 min, and centrifuged at 800 rad/s, and the solution was decanted. The procedure was repeated twice. The solutions were combined, and the acetone was evaporated by the use of a vacuum rotary evaporator. Residue was dissolved in 2 mL of 70% glycerol and analyzed with HPLC. Sum of Ellagitannins (ET). The released ellagic acid (EA) was computed from the difference between the contents of total and free EA. The total ET, expressed per monomer α-1-O-galloyl-2,3:4,6-bisHHDP-D-glucose, was calculated by multiplying the content of released EA by a conversion factor of 1.55, which takes into account the molar content of EA in α-1-O-galloyl-2,3:4,6-bis-HHDP-D-glucose, a monomer unit of agrimoniin.1 Half a milliliter of a 70% glycerin solution and 0.075 mL of trifluoroacetic acid (Fluka) were added to 0.15 g of the sample. The components were mixed and kept at a temperature of 95 °C for 18 h. After hydrolysis, the samples were filled to 5 mL with methanol, filtered through syringe Teflon filters with a pore size of 0.45 μm (Millipore, Bedford, MA, USA), and analyzed with HPLC. Polyphenol HPLC Analysis. A Smartline (Knauer, Berlin, Germany) chromatograph with a PDA detector was used. A Phenomenex Gemini 5u C18 110a (250 × 4.60 mm; 5 μm) column was kept at 35 °C. Eluent A contained 0.05% phosphoric acid in water, and eluent B contained 0.05% phosphoric acid in 80% (v/v) acetonitrile in water. B
DOI: 10.1021/acs.jafc.5b00648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry water solution (v/v) of acetic acid, and phase B consisted of 80% (v/v) acetonitrile in water. The flow rate was 1 mL/min, and the temperature was 25 °C. The identification was conducted on the basis of the retention times of standards of (−)-epicatechin, (+)-catechin, (−)-epicatechin-phloroglucinol, and (+)-catechin-phloroglucinol. Quantitative analyses of released flavonols, namely, (+)-catechin and (−)-epicatechin, and phloroglucinol adducts were conducted on the basis of chromatograms recorded with the FD set at 278 nm excitation wavelength and 360 nm emission wavelength. The mean degree of polymerization was determined on the basis of the molar ratio of all flavan-3-ol units, that is, adducts of phloroglucinol and terminal units to the sum of terminal units, which included (+)-catechin and (−)-epicatechin. All of the analyses were performed in duplicate. In Vivo Experiments. Animals and Diets. Experiments were conducted on 24 Wistar strain rats following the protocol approved by the Local Ethical Commission for Experiments on Animals in Olsztyn (permission no. 71/2012). The animals were maintained under standard conditions: temperature of 21−22 °C, relative air humidity of 50−70%, intensive ventilation of rooms (15×/h), and 12 h lighting. Individual body weights and food intakes were recorded. The experiment, conducted on three experimental groups (eight males each), lasted for 4 weeks from week 4 of age. All animals were housed individually in metabolic cages with free access to water and the experimental diets. Diets contained similar levels of protein, fat, minerals, and vitamins and a similar content of fiber. All of the diets were isocaloric. Experimental diets were prepared as modified semisynthetic AING93G diet22 and differed only in the addition of defatted seeds of strawberries (strawberry) or raspberries (raspberry), introduced instead of a respective content of maize starch. The analyzed preparations were introduced into the diet at the same dose of 6%. The preparations introduced dietary fiber (4%), a small quantity of protein (
