Article pubs.acs.org/JAFC
Chemical Composition of Blackberry Press Cake, Polyphenolic Extract, and Defatted Seeds, and Their Effects on Cecal Fermentation, Bacterial Metabolites, and Blood Lipid Profile in Rats Monika Kosmala,*,† Adam Jurgoński,*,‡ Jerzy Juśkiewicz,‡ Elzḃ ieta Karlińska,† Jakub Macierzyński,† Edward Rój,§ and Zenon Zduńczyk‡ †
Institute of Food Technology and Analysis, Lodz University of Technology, ul. Stefanowskiego 4/10, 90-924 Lodz, Poland Division of Food Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland § New Chemical Syntheses Institute, Aleja Tysiąclecia Państwa Polskiego 13a, 24-110 Puławy, Poland ‡
ABSTRACT: Blackberry fiber (BF), extracted blackberry fiber (EBF), defatted blackberry seeds (DBS), and blackberry polyphenols (BP) were added to the diets of rats. A control diet, fiber diets (BF, EBF, DBS) with 6% addition instead of cellulose, and a polyphenol diet (BP) with 1% addition were administered for 4 weeks. BF and DBS contained polyphenols (3.6%); EBF did not. DBS was free of fat. Ellagitannins were the main phenolics, mostly dimers (sanguiin H-6 with isomers) and monomers (pedunculagin, casuarictin isomer, and sanguiin H-2 isomer). Trimers (lambertianin C with isomers) and tetramers (lambertianin D) were in the minority. EBF increased the production of propionate and butyrate in the cecum and improved the blood lipid profile. Polyphenols beneficially decreased the activity of cecal β-glucuronidase, but they may have also increased cholesterol levels in blood. Bacterial metabolism of ellagitannins in the colon gave rise to nasutin A and urolithin A. KEYWORDS: blackberry, dietary fiber, ellagitannins, rat, nasutin A
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inflammatory properties.4,16 However, the in vivo effects may be mostly caused by ellagitannin metabolites, as ellagitannins themselves do not pass into the bloodstream in an intact form. It is known that ellagitannins are hydrolyzed in the gastrointestinal tract, mostly by the microbiota, to urolithins and nasutins.9,12,15,17−19 The metabolite profile depends on the individual characteristics of the host species and especially on the composition of its microbiota. In the blood, ellagitannin metabolites are found in free form18 or as glucuronide or sulfate conjugates.18,20 Urolithins remain in the body in the portal circulation for up to 4 days following consumption of foods rich in ellagitannins. The juice production process gives rise to a press cake, which is a concentrated source of dietary fiber and polyphenols.9,21 Dietary fiber is one of the most beneficial components of fruits and vegetables.22,23 Importantly, the press cake may be extracted to obtain polyphenol concentrates and dietary fiber with decreased polyphenolic content.24,25 Moreover, blackberry seeds, which are the main component of the press cake, yield oil that is a source of polyunsaturated fatty acids and antioxidants, i.e., tocopherols, polyphenols, and phytosterols.26 Supercritical CO2 extraction of that oil gives an interesting product that is high in dietary fiber and polyphenols and low in fat.27 In the context of the presented literature overview, the aim of the study described herein was to broaden knowledge about the
INTRODUCTION Blackberry (Rubus f ruticosus), along with raspberry, belongs to the genus Rubus and the family Rosaceae. Its leaves and fruits have long been used in herbalism for treating bleeding, diarrhea, and diabetes. Recent years have seen a growing interest in the health-promoting properties of blackberry fruits.1−5 In contrast to raspberry, which is a very widespread crop, blackberry is cultivated far less often, mainly due to its lower frost hardiness and more labor-intensive management. In the previous decade, the world’s annual production of blackberries amounted to only 155 000 tons,6 but it has been recently on the increase, especially in the USA and the EU, due to new cultivars, better marketing practices, and greater availability of fresh fruits.7 One reason for the growing interest in blackberries is the intense color of the fruits, attributable to a high content of beneficial polyphenol compounds. The major polyphenolics present in blackberry fruits are anthocyanins, ellagitannins, and flavan-3-ols (31−326, 51−68, and 27 mg/100 g fresh weight, respectively), and, in lower amounts, flavonols and phenolic acids (4−30 and 7−64 mg/100 g fresh weight, respectively).2 Since anthocyanins are among the most labile polyphenols, a considerable proportion of them is lost during fruit processing,8 while ellagitannins and proanthocyanidins are more stable. Blackberries are a source of ellagitannins comparable to pomegranates, strawberries, and raspberries.2,9−13 Similarly to raspberries, blackberries contain oligomeric ellagitannins, sanguiin-H6, and lambertianin C,9 in which the monomers are joined by a sanguinsorboyl group.14 Animal models and cell culture studies have shown that ellagitannins have strong antioxidant activity,4,15 as well as anti© 2017 American Chemical Society
Received: Revised: Accepted: Published: 5470
April 24, 2017 June 19, 2017 June 20, 2017 June 20, 2017 DOI: 10.1021/acs.jafc.7b01876 J. Agric. Food Chem. 2017, 65, 5470−5479
Article
Journal of Agricultural and Food Chemistry composition of blackberry press cake products, i.e., dietary fiber and polyphenolic preparations from the native press cake, a dietary fiber preparation obtained after polyphenolic extraction, a polyphenolic extract, and a preparation obtained from seeds defatted by supercritical CO2 treatment. The study was also aimed to identify local and systemic effects on cecal function, examine the formation of ellagitannins metabolites in the colon, and determine the blood lipid profile in a rat model.
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Detection was at 250 nm (ellagitannins), 360 nm (ellagic acid, quercetin, and kaempferol glycosides as well as aglycons), and 520 nm (anthocyanins). The standards applied were cyanidin-3-O-glucoside, ellagic acid, kaempferol, kaempferol-3-O-glucoside, quercetin, and quercetin-3-O-glucoside (Extrasynthese, Genay, France). The ellagitannin standards (lambertianin C, pedunculagin, and sanguiin H-6) were obtained as previously described.30 At 250 nm, purity was as 92.1%, 80%, and 92.5%, respectively. High-Performance Liquid Chromatography and Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). A Dionex UltiMate 3000 UHPLC apparatus coupled with a Thermo Scientific Q Exactive quadrupole ion trap mass spectrometer was used. Ellagitannins were separated using a Phenomenex Luna 5 μm C18 column (250 × 4.6 mm). Phase A was formic acid in water (1/99, v/v), and phase B was acetonitrile and water (80/20, v/v). The applied gradient, with the flow rate of 1.0 mL/min, was as follows: 5% of mobile phase B for the first 6.5 min, followed by 6.5−12.5 min, 5−15% B; 12.5−44 min, 15− 45% B; 44−45 min, 45−75% B; 45−50 min, 75% B; 50−52 min, 75− 5% B; and finally column equilibration from 52 to 65 min at 5% B. MS analysis was performed in negative ion mode with capillary voltage of +4 kV, sheath gas pressure of 75 au, auxiliary gas of 17 au, and 200− 2000 m/z scan rage. Analysis of Proanthocyanidins and Catechins. An acidic environment with excess phloroglucinol degradation according to the method of ref 26 was created as described previously.30 To a 20 mg sample was added 0.8 mL of methanol solution with phloroglucinol (75 g/L) and ascorbic acid (15 g/L). Addition of 0.4 mL of 0.2 mol/L hydrochloric acid in methanol started the reaction, which proceeded at 50 °C for 30 min. It was stopped by addition of 0.6 mL of a 40 mmol/L sodium acetate solution in an ice bath. After centrifugation at 3600g for 5 min and dilution with a 40 mmol/L sodium acetate solution, the samples were analyzed using a Smartline chromatograph with a P2800 UV−vis detector (both from Knauer, Berlin, Germany), an RF-10AXL fluorescence detector (FD) (Shimadzu, Tokyo, Japan), and a Gemini 110A 5 μm C18 column (250 × 4.60 mm) (Phenomenex, Torrance, USA). Phase A consisted of acetic acid and water (2.5/97.5, v/v) and phase B of acetonitile and water (80/20, v/v). The applied gradient, with the flow rate 1 mL/min at 25 °C, 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. Comparison of the retention times and UV−vis spectra with those of standards(−)epicatechin, (+)-catechin, (−)-epicatechin−phloroglucinol adduct, and (+)-catechin−phloroglucinol adductwas used for indentification. The excitation wavelength was 278 nm, and the emission wavelength was 360 nm. In Vivo Experiments. Animals and Diets. Experiments were conducted on 40 Wistar strain rats and approved by the local Ethical Committee for Experiments on Animals in Olsztyn (permission no. 71/2012). The animals were maintained at a temperature of 21−22 °C with relative air humidity of 50−70% under intensive ventilation of rooms (20 times/h) and 12 h lighting. The study involved five experimental groups containing eight males each and lasted for 4 weeks, beginning at 4 weeks of age. The animals had free access to tap water and were fed experimental diets (see Table 1). The diets were modifications of a diet recommended by the American Institute of Nutrition31 for laboratory rodents and differed only in that maize starch was replaced with blackberry fiber (BF), blackberry extracted fiber (EBF), defatted blackberry seeds (DBS), or blackberry polyphenols (BP). The analyzed dietary fiber preparations (BF, EBF, and DBS) were added at the same dose (6%). The preparations contributed dietary fiber (4%) and a small amount of protein (
