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Caffeoylquinic acid-rich extract from chicory seeds improves glycemia, atherogenic index, and antioxidant status in rats
Nutrition 28 (2012) 300–306
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Basic nutritional investigation
Caffeoylquinic acid-rich extract from chicory seeds improves glycemia, atherogenic index, and antioxidant status in rats ski R.D., Ph.D. a, *, Jerzy Juskiewicz D.Sc. a, Zenon Zdun czyk D.Sc. a, Bogus1aw Kro l D.Sc. b Adam Jurgon a b
Division of Food Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland dz, Ło dz, Poland Institute of Chemical Technology of Food, Technical University of Ło
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2010 Accepted 21 June 2011
Objective: Comparison of the effects of a high-fructose diet supplemented with rutin, a phenolic compound with well-recognized bioavailability and bioactivity, and a chicory (Cichorium intybus L.) seed extract rich in caffeoylquinic acids (CQA) on gut physiology and the development of disorders related to metabolic syndrome. Methods: A 28-d experiment was conducted on 32 young male Wistar rats. In comparison with control rats fed a standard corn starch diet (group C), the experimental group (group E) was fed a diet with an increased content of cholesterol and fructose (to 1% and 66% of the diet, respectively), as well as with oxidized soybean oil. Rats from the other two experimental groups were administered the same diet as group E during the first 2 wk of feeding, whereas at the beginning of the last 2 wk, the diet was enriched with rutin (group ER) or the CQA-rich ethanol extract from chicory seeds (9.6% of CQA, group EC), so the amount of added phenolics was equal in both dietary groups (0.15%). Results: The diet administered in group E caused hyperglycemia and increased blood serum atherogenicity in rats, but did not induce other manifestations of the metabolic syndrome, i.e., dyslipidemia and oxidative stress. Additionally, it affected gut physiology through increasing mucosal sucrase activity and disturbing fermentative processes in the cecum, such as the production of short-chain fatty acids and the activity of microbial enzymes. Similarly to rutin, the dietary addition of the chicory seed extract improved glycemia, which was comparable to that determined in group C. In addition, the extract was found to decrease the atherogenic index to the level observed in group C and to increase blood antioxidant status. Both dietary supplements reduced the content of thiobarbituric acid-reactive substances in kidney and heart tissue when compared with group E. Conclusion: The potential efficacy of the CQA-rich extract from chicory seeds in improving dietinduced metabolic disturbances proved to be better than that of rutin; thus, the extract might be considered as a dietary supplement for carrying out clinical trials. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Chicory Caffeoylquinic acids Chlorogenic acid Rutin The metabolic syndrome
Introduction Metabolic syndrome (MS) is a widespread diet-related disorder, defined as a cluster of interrelated risk factors for cardiovascular disease and type 2 diabetes. According to the new standardized criteria for clinical diagnosis of MS, these factors include central obesity, raised blood pressure, low concentration of high-density lipoprotein (HDL) cholesterol, as well as elevated
* Corresponding author. Tel.: þ48-89-523-4601; fax: þ48-89-524-0124. ski). E-mail address: [email protected] (A. Jurgon 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.010
glycemia and triglyceridemia [1]. Moreover, there is convincing evidence that oxidative stress is also involved in the development and progression of MS [2]. Most of the aforementioned manifestations can be induced experimentally in rats, in a relatively short time, by a high-fructose diet. However, the range of metabolic disturbances in response to excess fructose ingestion seems to be complex and may depend on many factors, such as age of the animals, as well as amounts of fructose and other components in the diet [3–5]. Prevention and treatment of MS are crucial for public health. In recent years, much research has been focused on dietary flavonoids, the antioxidants that can potentially improve
ski et al. / Nutrition 28 (2012) 300–306 A. Jurgon
disorders clustered in MS. In this context, one of the most investigated and relatively well-recognized flavonoids is quercetin and its glycosides, in particular, rutin (quercetin 3-Oglucorhamnoside) [6]. In experiments on rats, rutin administered orally or intraperitoneally improved glycemia, lipidemia, and antioxidant status [7–9], as well as ameliorated obesity induced by a high-fat diet [10]. Interestingly, the bioavailability of rutin after oral administration is low and it is mainly metabolized by large intestinal bacteria, among others to the aglycone form and phenolic acids [11,12]. Apart from flavonoids, there are also many other plant phenols exhibiting a vast array of biological activities. These include esters of caffeic and quinic acids, whose activity against disorders of MS has not been studied extensively thus far, to our knowledge, considering especially caffeoylquinic acids (CQA) with more than one caffeic acid residue. The most known and recognized CQA is a monocaffeoyl derivative chlorogenic acid (5-CQA), which occurs in many plants, fruits, and vegetables, such as coffee beans, apples, tomatoes, etc. [13]. For instance, chlorogenic acid has been shown to exhibit antiobesity properties and improve lipid metabolism in mice [14]. Moreover, some authors have observed delayed glucose absorption, suggesting its shift to more distal parts of the intestine, after the ingestion of chlorogenic acid-rich decaffeinated coffee [15]. However, similarly to rutin, CQA are also low-absorbable in the upper part of the gastrointestinal tract and most probably reach the large intestine in quantities [13,16]. In our laboratory, CQA have been recently isolated from different parts of chicory (Cichorium intybus L.) [17], which is a biennial plant with many applications in the food industry, for instance, it is used as an additive to coffee (roasted roots) and salads (young leaves) or as a source of prebiotic fructans (roots). The aim of this study was to compare the effects of a highfructose diet supplemented with rutin, a phenolic compound with relatively well-recognized bioavailability and bioactivity, and a CQA-rich extract from chicory seeds on gut physiology and the development of disorders in rats related to MS. To enhance the metabolic disturbances, a high-fructose diet was additionally enriched with oxidized fat and cholesterol. We speculated that, like rutin, the multiple ethanol extract from chicory seeds can reduce adverse effects of such a diet. Materials and methods Dietary supplements Seeds of chicory (C. intybus L.) were provided by Cykoria Co. (Wierzchos1awice, Poland), a company specializing in the cultivation and processing of the plant. After drying at <70 C, the seeds were ground and then extracted four times with 75% ethanol (1:3 sample-to-solvent ratio). The extraction procedure was performed in a hermetic container without access to light. Afterwards, the ethanol was removed by vacuum distillation and the extract was then freezedried for 24 h beginning at 30 C, followed by an additional drying at 40 C for 2 h. Determination of the antioxidant activity of the extract was performed using 2,2-diphenyl-1-picrylhydrazyl radical according to the modified method of Brand-Wiliams et al. [18], and the results were expressed as nanomoles of Trolox equivalent per gram of sample. Dry matter, ash, protein, and fat contents in the extract were assayed according to the Association of Analytical Communities (AOAC) official methods, whereas the determination of saccharides and phenolic compounds was conducted with high-performance liquid chromatography methods. Dicaffeoylquinic acid (diCQA) standard was isolated by semipreparative high-performance liquid chromatography and confirmed by mass spectrometry. CQA were quantified as grams of chlorogenic acid (5-caffeoylquinic acid; Sigma, St. Louis, MO, USA) equivalents per 100 g fresh mass. The procedure of extraction, as well as the analysis of the antioxidant activity and chemical composition of the extract tested, with detailed description of the methods applied, was described previously [17]. Table 1 presents chemical composition and antioxidant activity of the extract. Rutin (quercetin 3-O-glucorhamnoside, trihydrate, purity 95%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
301
Table 1 Chemical composition and antioxidant activity of the ethanol extract from chicory seeds g/100 g fresh mass 95.0 8.4 10.7 14.4 34.9 12.1 21.4 1.4 1.7 9.6 2.8 6.8 505.1
Dry matter Ash Protein Fat Mono- and disaccharides Glucose Fructose Sucrose Inulin Phenolic compounds MonoCQA DiCQA Antioxidant activity (nmol/g)
0.20 0.05 0.3 0.2 0.3 0.1 0.2 0.02 0.04 0.03 0.01 0.02 6.02
CQA, caffeoylquinic acids Data are expressed as mean values SD (n ¼ 3) Animals and diets The animal protocol used in this study was approved by the Local Animal Care and Use Committee (permission no. 23/2008). The experiment was conducted on 32 young male Wistar rats, distributed into four groups of eight animals each. All animals were housed individually in standard conditions with free access to water and semipurified casein diets (Table 2). Control rats (group C) received for 28 d a diet containing among others cholesterol (0.5%), soybean oil (8%), fructose (5%), and corn starch (66.5%). In comparison with group C, rats from the experimental group (group E) were fed for 28 d a diet with increased amounts of cholesterol and fructose (to 1% and 66% of the diet, respectively), added at the expense of cornstarch, as well as with oxidized soybean oil added instead of fresh. Rats from the other two experimental groups (ER and EC) were administered the same diet as in group E for the first 2 wk of feeding, whereas at the beginning of the last 2 wk the diet was enriched with rutin (group ER) or a chicory seed extract (group EC), added at the expense of cornstarch. In all experimental groups, the diets had comparable sums of mono- and disaccharides, whereas in the ER and EC groups the diets had additionally the same amount of phenolic compounds. Oxidized soybean oil was obtained by intensive aeration while heating at 70-80 C for 72 h. The degree of oxidation was monitored daily by determining the peroxide value according to the AOAC official method. The final degree of oxidation amounted to 120 miliequivalents of O2 per kg of soybean oil. Sample collection and analysis On termination of the experiment, rats were anaesthetized with sodium pentobarbital according to the recommendations for euthanasia of experimental animals. After laparotomy, blood samples were taken from caudal vein; then gut Table 2 Composition of the diets fed to rats* Ingredient (g/100 g diet)
Casein DL-methionine Soybean oil Oxidized soybean oily Cholesterol Mineral mixz Vitamin mixz Choline chloride Rutin Chicory seed extract Sucrose Fructose Corn starch
Group C
E
ER
EC
14.6 0.2 8 d 0.5 3.5 1 0.2 d d 0.5 5.0 66.50
14.6 0.2 d 8 1 3.5 1 0.2 d d 0.5 66.0 5.00
14.6 0.2 d 8 1 3.5 1 0.2 0.15 d 0.5 66.0 4.85
14.6 0.2 d 8 1 3.5 1 0.2 d 1.57 d 66.0 3.93
* Rats from the ER and EC groups have the same diet during first 2 wk of feeding as in group E, whereas the preparation of rutin or chicory seed extract was supplemented at the beginning of the last 2 wk. In all experimental groups, the diets have comparable sum of mono- and disaccharides, whereas in the ER and EC groups the diets have additionally equal amount of phenolic compounds. y Degree of oxidation ¼ 120 meq O2/kg. z Recommended for American Institute of Nutrition-93G diet.
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(small intestine, cecum, and colon) and internal organs (liver, heart, kidneys, and lungs) were removed and weighed. The small intestine was divided into four equal sections, and a second section from the stomach side was rinsed with cold physiological saline and cut open. Mucosal samples were collected by scraping with glass slides onto an iced glass plate. After the homogenization with four parts of cold physiological saline (v/w) and centrifugation for 10 min (9500 g, 4 C), the obtained supernatant was subsequently stored at 20 C. Sucrase, maltase, and lactase activities were assayed by the method of Dahlqvist [19] with some modifications, namely, glucose oxidase reagent was from Alpha Diagnostic Ltd. (Warsaw, Poland), and 0.2 mol/L of sodium phosphate buffer (pH 7.0) was used instead of maleate. Disaccharidase activities were expressed as mmol of glucose liberated from the respective disaccharide per minute per gram of protein. Protein content of the supernatant was estimated using Lowry’s method with bovine serum albumin as a standard. The pH of intestinal digesta was measured using a microelectrode and a pH/ION meter (model 301; Hanna Instruments, Vila do Conde, Portugal), while dry matter was determined at 105 C. Fresh cecal digesta was used for analysis of ammonia concentration, whereas determinations of enzyme activity and shortchain fatty acids (SCFA) concentration were performed after storage of the samples at –70 C. Ammonia was extracted and trapped in a solution of boric acid and then determined by direct titration with sulfuric acid [20]. The enzyme activity (a- and b-glucosidase, a- and b-galactosidase, b-glucuronidase) was measured by the rate of p- or o-nitrophenol release from their nitrophenylglucosides for 10 min [21] and expressed as mmol product formed per 1 h per gram of cecal digesta. The concentration of SCFA was measured using gas chromatography under the conditions described previously [21] and expressed as mmol of individual or total SCFA per gram of digesta. The concentration of glucose, triglycerides, total cholesterol, and its HDL fraction, as well as the activity of aspartate aminotransferase (ASP) and alanine aminotransferase (ALT) in the serum, were estimated with reagents from Alpha Diagnostics Ltd. (Warsaw, Poland). The atherogenic index was calculated from the following formula: log(triglycerides/HDL cholesterol). The activity of glutathione peroxidase in the heparinized blood and superoxide dismutase in erythrocyte lysate was determined using reagents from Randox Laboratories Ltd. (Crumlin, UK). Serum antioxidant capacity of hydrophilic and lipophilic substances was determined with photochemiluminescence detection method using a Photochem (Analytik Jena AG, Jena, Germany). In the photochemiluminescence assay, the generation of free radicals was partially eliminated by the reaction with antioxidants present in serum samples, and the remaining radicals were quantified by luminescence generation. Ascorbate and Trolox calibration curves were used to evaluate antioxidant capacity of hydrophilic and lipophilic substances, respectively, and the results were expressed as mmol of equivalents per milliliter of serum. The content of thiobarbituric acid-reactive substances (TBARS) in a tissue of internal organs (heart, kidneys, and liver) was estimated after storage at 20 C according to the method of Draper and Hadley [22]. Data analysis Results are expressed as mean values standard deviation (SD). Statistical analysis was performed using a one-way analysis of variance and the Duncan’s multiple range post-hoc test. Differences were considered significant at P 0.05. All calculations were performed using Microsoft Excel software.
Results Over 4 wk of experimental feeding, diet intake was comparable between all groups (Table 3); however, after the first 2 wk it tended to be different (P ¼ 0.07). All rats also had similar initial and final body weight, whereas the relative mass of kidneys and liver (g/100 g body weight) was lower in group C than in experimental groups, that is, the experimental (group E), the experimental with rutin (group ER), and the experimental with chicory seed extract (group EC, P 0.05). Differences in the indices of gut functioning are displayed in Table 4 (P 0.05). Both the relative mass of small intestine (with content) and the pH value of ileal digesta were higher in the experimental groups when compared with group C. The activity of mucosal enzymes was similar in all groups, except for sucrase, whose activity was higher in the E and ER groups than in the C and EC groups. Relative digesta mass of the cecum (g/g cecum) was comparable in all groups; however, it tended to be different (P ¼ 0.07), whereas the dry matter of digesta was decreased in group EC in comparison with the other groups. The pH value of
Table 3 Diet intake, body weight, and mass of selected organs of rats Group* C
E
Diet intake (g/14 d) First 2 wk 193 15.6 171 Last 2 wk 253 19.2 256 Body mass (g) Initial 93.0 3.78 92.8 Final 253 10.6 239 Gain 160 12.0 146 Mass of selected organsy Heart 0.304 0.025 0.313 Kidneys 0.664 0.021b 0.744 Liver 4.10 0.232b 5.78 Lungs 0.561 0.082 0.532
ER
EC
19.2 17.8
175 18.4 247 19.2
177 14.4 246 39.9
3.81 12.1 14.1
92.3 3.98 237 13.8 145 15.1
92.5 4.11 237 24.0 145 23.7
0.018 0.316 0.028 0.309 0.031a 0.786 0.027a 0.811 0.594a 5.68 0.319a 5.53 0.046 0.555 0.049 0.535
0.011 0.085a 0.569a 0.062
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y g/100 g body weight.
cecal digesta was lower in group EC when compared to groups C and E. The activity of microbial enzymes in the cecal digesta (aand b-glucosidases, as well as a- and b-galactosidases) was observed to decrease in the experimental groups when compared with group C. The total content of short-chain fatty acids (SCFA) in the cecal digesta was the lowest in the E and ER groups, higher in group EC, and highest in group C. In comparison with group C, the acetate content was lower in all experimental groups, whereas the propionate content was lower only in the E and ER group. In the colon, the relative digesta mass (g/g colon) was demonstrated to increase in the E and EC groups when compared with group C. Differences in the serum indices of rats are provided in Table 5 (P 0.05). In group E, glucose concentration increased when compared with group C, whereas in the ER and EC groups it was comparable with that reported for both groups E and C. The lipid profile was similar in all groups; however, the concentration of triglycerides tended to be different (P ¼ 0.09). In comparison with group C, the atherogenic index was higher in the E and ER groups and similar in group EC. Superoxide dismutase activity in erythrocytes was similar in the C and E groups and higher in group EC (P 0.05), whereas in group ER it was comparable with that assayed in the other groups (Table 6). Whole-blood glutathione peroxidase activity only tended to be different between all groups (P ¼ 0.08). Serum antioxidant capacity of hydrophilic substances was higher in group EC when compared with the other groups (P 0.05). The content of TBARS in a heart tissue was lower in the ER and EC groups than in the C and E groups (P 0.05). When compared with group E, the content of TBARS in a kidney tissue was lower in the ER and EC groups (P 0.05); simultaneously, the content was similar between the ER and EC groups, as well as between the E and C groups. Discussion The ethanol extract from seeds of chicory (C. intybus L.) used as dietary supplement in our study was a rich source of phenolic compounds (9.6%), that is, mono- and especially diCQA (2.8% and 6.8%, respectively, Table 1). It is worthy of notice that chlorogenic
ski et al. / Nutrition 28 (2012) 300–306 A. Jurgon
303
Table 4 Indices of gut functioning of rats Group* C Small intestine Mass with contenty pH of ileal digesta Activity of mucosal enzymesz Maltase Sucrase Lactase Cecum Tissue massy Digesta massx Dry matter of digesta (%) Ammonia in digesta (mg/g) pH of digesta Activity of microbial enzymes{ a–Glucosidase b–Glucosidase a–Galactosidase b–Galactosidase SCFAk (mmol/g digesta) Acetate Propionate Butyrate Total content Colon Tissue massy Digesta mass** pH of digesta
E
ER
EC
2.95 0.229b 5.83 0.318b
3.51 0.231a 6.65 0.290a
3.81 0.728a 6.45 0.133a
3.85 0.445a 6.41 0.316a
27.2 2.58 4.49 0.424b 1.16 0.285
29.0 2.43 6.09 0.594a 1.22 0.228
30.7 4.55 5.70 0.786a 1.07 0.212
29.7 3.33 4.96 0.456b 1.14 0.335
0.318 2.710 17.4 0.414 7.04
0.041 0.515 0.66a 0.067 0.104a
0.290 3.576 17.6 0.365 7.05
0.041 1.166 0.71a 0.088 0.175a
0.352 3.694 17.4 0.346 6.98
0.101 0.751 0.24a 0.029 0.143a,b
0.418 3.138 16.5 0.380 6.83
0.157 0.525 0.36b 0.047 0.212b
21.9 4.41 6.92 52.5
2.53a 0.900a 3.123a 17.66a
10.6 1.88 3.62 38.2
2.96b 0.795b 1.533b 12.37b
9.2 2.72 2.86 30.5
13.10b 1.396b 0.983b 9.58b
10.4 2.09 2.77 31.6
5.83b 0.388b 1.345b 8.61b
59.5 19.5 8.82 95.3
6.80a 3.07a 3.006 10.02a
41.4 14.2 7.77 69.3
6.98b 2.39b 1.888 10.31c
41.6 14.5 7.38 69.3
3.09b 1.69b 1.667 3.87c
47.1 17.9 8.85 79.9
6.12b 1.70a 2.928 5.42b
0.495 0.049 0.392 0.156b 6.88 0.115
0.514 0.043 0.798 0.364a 6.88 0.173
0.572 0.099 0.715 0.423a,b 6.77 0.155
0.533 0.069 0.931 0.309a 6.70 0.190
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; EC, experimental with chicory seed extract; ER, experimental with rutin. y g/100 g body weight. z mmol/min/g protein. x g/g cecal tissue. { mmol/h/g digesta. k Short-chain fatty acids. ** g/g colonic tissue.
acid and diCQA (3,5-diCQA and 3,4-diCQA) have recently been isolated from the leaves of endive species (C. endivia L.) [23]. Furthermore, diCQA have so far been isolated from many plant materials, such as fennel, seeds of arabica coffee, mugwort flowering tops, sweet potato leaves [24], or artichoke, known for an abundant source of cynarin (1,3-diCQA), as well as its isomer (1,5-diCQA) [25]. Interestingly, from among natural preparations, artichoke extracts were found to have the highest antioxidant activity in vitro, and cynarin was found to be the main contributor to the activity [26]. In the present study, the antioxidant activity of the chicory seed extract amounted to 505.1 nmol/g
and was more than twofold higher when compared with chicory leaf extract, obtained in the same way [18]. It must be emphasized, however, that the seed extract contained additionally considerable amounts of sugars (34.9%), predominantly fructose (21.4%), which can also increase the antioxidant activity in vitro [27]. In our study, after 4 wk of feeding the hypertrophy of liver and kidneys was observed in rats from group E, most probably due to the increased metabolic load of these organs, as a consequence of high fructose ingestion. Fructose raises serum uric acid concentration, increases its urinary excretion, as well as stimulates
Table 5 Basic biochemical indices of rat serum Group* C Glucose (mmol/L) Aminotransferases (U/L) Aspartate (ASP) Alanine (ALT) Lipid profile (mmol/L) Total cholesterol HDL-cholesterol Triglycerides Atherogenic indexy
E
ER
EC
9.90 1.79b
13.2 3.64a
12.2 2.17a,b
10.8 1.01a,b
92.5 15.33 26.3 4.59
90.6 18.42 28.5 6.26
93.5 14.67 30.1 8.01
90.5 18.83 28.0 3.02
2.80 1.48 2.79 0.263
0.366 0.252 0.936 0.092c
2.91 1.52 3.67 0.382
0.361 0.259 0.708 0.074a
2.83 1.46 3.33 0.356
0.435 0.188 0.550 0.098a,b
3.04 1.57 2.95 0.268
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y log(triglycerides/HDL-cholesterol).
0.373 0.260 0.679 0.066b,c
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304 Table 6 Antioxidant status of rats Group* C Activity of blood enzymes (U/mL) Glutathione peroxidase Superoxide dismutase Serum antioxidant capacity (mmol/mL) Hydrophilic substances Lypophilic substances TBARSy in tissue (nmol/g) Heart Kidney Liver
E
ER
EC
26.2 2.87 274 31.2b
24.5 2.97 278 19.5b
28.3 4.03 298 18.5a,b
27.9 2.21 313 39.4a
0.053 0.010b 0.193 0.017
0.043 0.012b 0.171 0.040
0.053 0.017b 0.200 0.058
0.094 0.033a 0.177 0.043
61.7 17.49a 173 54.5a,b 40.9 2.98
72.4 19.01a 188 27.7a 43.7 5.58
39.8 9.00b 138 36.9b,c 45.5 7.03
43.6 6.91b 119 19.9c 44.4 10.96
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y Thiobarbituric acid-reactive substances.
inflammatory processes in the kidneys [28]. Moreover, this monosaccharide is almost exclusively metabolized in the liver, contrary to glucose, which is the basic energetic substrate for many different types of tissues [3]. However, the activity of aminotransferases in group E, as well as in the other experimental groups of our study, was comparable with the control, indicating that liver functions were sustained (ASP and ALT, Table 5). The potential role of fructose in the development of gastrointestinal tract disorders has already been described in literature, mainly due to its less effective absorption in the small intestine when compared with other mono- and disaccharides [29]. Results of our experiment, such as increased small intestinal mass and pH value of ileal digesta (group E), indicate that the excess fructose intake hinders its absorption. In the small intestine of rats from group E, the increased activity of sucrase was noted as well. It is in accordance with findings of other authors, who observed that fructose was capable of directly increasing the activity and gene expression of sucrase when compared with glucose [30]. Interestingly, the diet supplemented with chicory seed extract (group EC), but not with rutin (group ER), decreased sucrase activity to the level observed in group C (Table 4). It has been shown that CQA have maltase and sucrase inhibitory activities, and their intensity depends on the number of caffeoyl residuesdthe more residues, the stronger the inhibition [31]. Nevertheless, the observed inhibition of sucrase activity might in part be the effect of other phytochemicals present in chicory seeds [32]. Worthy of emphasizing are also differences in the composition of diets administered to experimental groups in our study. In comparison with group ER, the diet used in group EC contained less cornstarch and sucrose (4.85% and 0.5% versus 3.93% and 0.03%, respectively), that is, the substrates for sucrase which exists on the brush border membrane as a sucrase-isomaltase complex. In the cecum, the extract lowered dry matter and the pH value of the cecal digesta when compared with the C and E groups, indicating more intensive fermentation of indigestible components, such as CQA and inulin, which was also present in the extract. Indeed, the total content of SCFA in the cecal digesta as the main fermentation products of the large intestinal microflora was increased in group EC when compared with the E and ER groups, however, not to the level observed in the control. In addition, cecal propionate content was normalized under the influence of the chicory seed extract. It should be mentioned that propionate may be absorbed from the large intestine and is the inhibitor of hepatic lipogenesis [33]. Furthermore, Zhu et al. demonstrated antimicrobial activities of phenolic compounds isolated from artichoke leaves, with chlorogenic acid and diCQA being relatively one of the most
potent [34]. Results achieved in our study for the activity of microbial enzymes (glucosidases and galactosidases) suggest that the addition of the CQA-rich extract from chicory seeds to the diet has no unfavorable effect on large intestinal microflora. Administration of a high-fructose diet can disturb lipid and glucose metabolism in rats [3]. It may affect blood indices within a few weeks, especially causing hypertriglyceridemia, hyperinsulinemia, as well as glucose intolerance [4,5]. In our study, the diet containing 66% of fructose in a free form increased serum glucose concentration, but did not affect significantly serum lipid profile (group E versus group C). A lack of increase in triglyceride concentration was due to the other modifications of the diet, most likely the exchange of soybean oil for the oxidized one. It was generally assumed that the more the fat is oxidized, the less of it is absorbed from the small intestine [35]; thus, in our experiment, such a rule might influence a lack of the anticipated increase in serum triglyceride concentration in group E. On the other hand, the atherogenicity of serum in group E was considerably higher than in the control. In humans, the atherogenic index of plasma predicts MS, type 2 diabetes, and coronary heart disease [36]. In our experiment, the dietary addition of the CQA-rich extract, contrary to rutin, lowered the atherogenic index to the control level. The influence of diCQA, namely cynarin, over lipid profile has been the subject of experimental and clinical studies published in the 1970s, and the results obtained were contradictory [37]. In a more recent study, authors isolated 3,4-diCQA from a Ligularia fischeri variety and identified this compound as an inhibitor of lipid synthesis in rat liver [38]. Also, rutin has been implicated to improve lipid metabolism [7, 9]. Nevertheless, this was not the case in our study. It is noteworthy that the amount of phenolic compounds added after 2 wk of feeding to the diets of the ER and EC groups was equal (0.15 g/100 g diet). Furthermore, rutin and the chicory seed extract investigated in the present study decreased serum glucose concentration to the level observed in group C. In literature, positive effects of rutin on glucose metabolism were observed after both oral and intraperitoneal administration to rats with streptozotocin-induced diabetes, more likely due to antioxidant protection of pancreatic islet cells and stimulation of glucose utilization in extrahepatic tissues [7,8]. On the other hand, the glycemia-lowering mechanism of the chicory seed extract seems to be, at least in part, the consequence of local activities in the gastrointestinal tract. Indeed, it has been reported that chlorogenic acid might exhibit an antagonistic effect on the intestinal glucose transporter [15]. Furthermore, Matsui et al. showed strong antihyperglycemic effects of Brazilian propolis extract, pointing at CQA as the key compounds that inhibit maltase and sucrase activities [31]. In addition, the inhibition of a-glucosidase
ski et al. / Nutrition 28 (2012) 300–306 A. Jurgon
activity was attributed to some triterpenoids present in the seeds of chicory [32]. Results of our study partially support these findings, for sucrase activity was normalized in group EC. In the present investigation, the extract added to the diet of group EC increased considerably the serum antioxidant capacity of hydrophilic substances and superoxide dismutase activity in erythrocytes, thus distinguishing it from rutin (versus groups C and E). The increased antioxidant capacity suggests that CQA or their metabolites have to pass into the bloodstream. Indeed, in a human study several CQA metabolites in plasma were detected after the ingestion of artichoke leaf extracts rich in mono- and diCQA, i.e., derivatives of caffeic, ferulic, isoferulic, dihydrocaffeic, and dihydroferulic acids [16]. Interestingly, plasma concentration of dihydrocaffeic and dihdroferulic acids, the main metabolites of CQA, was the highest 6-7 h after administration, suggesting predominant absorption from the colon. Despite that rutin had no impact on serum antioxidant capacity, TBARS contents in a heart and kidney tissue, as the marker of oxidative stress, were reduced in our study, likewise, under the influence of the chicory seed extract (groups ER and EC versus group E). It is in agreement with another study, where rutin administered orally to streptozotocininduced diabetic rats (100 mg/kg body weight/d) improved the antioxidant status of selected tissues, e.g., reduced TBARS contents in kidneys, liver, and brain [8]. In vivo antioxidant properties of CQA have not been studied extensively thus far; however, di- and triCQA from aerial parts of Artemisia princeps inhibited the formation of advanced glycation end products [39]. It has been suggested that the accumulation of glycated proteins in vivo catalyzes free radical generation [40]. Moreover, glycated proteins are implicated in the development and progression of MS complications, i.e., diabetes and cardiovascular diseases [39,41]. In the context of MS manifestations, it is also of interest that diand triCQA have been shown to significantly reduce blood pressure in spontaneously hypertensive rats after oral administration (10 mg/kg body weight) [42].
Conclusion The dietary supplementation with the CQA-rich extract from chicory seeds and with rutin can improve glycemia; yet contrary to rutin, the addition of the extract decreases serum atherogenicity and increases blood antioxidant status. Therefore, the potential efficacy of the extract from chicory seeds in improving diet-induced metabolic disturbances is better than rutin, and the extract might be considered a dietary supplement for carrying out clinical trials. Furthermore, rats on a diet containing high amounts of fructose, oxidized soybean oil, and cholesterol, due to the lack of additional weight gain, dyslipidemia, and oxidative stress, is not a good experimental model mimicking MS.
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Basic nutritional investigation
Caffeoylquinic acid-rich extract from chicory seeds improves glycemia, atherogenic index, and antioxidant status in rats ski R.D., Ph.D. a, *, Jerzy Juskiewicz D.Sc. a, Zenon Zdun czyk D.Sc. a, Bogus1aw Kro l D.Sc. b Adam Jurgon a b
Division of Food Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland dz, Ło dz, Poland Institute of Chemical Technology of Food, Technical University of Ło
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2010 Accepted 21 June 2011
Objective: Comparison of the effects of a high-fructose diet supplemented with rutin, a phenolic compound with well-recognized bioavailability and bioactivity, and a chicory (Cichorium intybus L.) seed extract rich in caffeoylquinic acids (CQA) on gut physiology and the development of disorders related to metabolic syndrome. Methods: A 28-d experiment was conducted on 32 young male Wistar rats. In comparison with control rats fed a standard corn starch diet (group C), the experimental group (group E) was fed a diet with an increased content of cholesterol and fructose (to 1% and 66% of the diet, respectively), as well as with oxidized soybean oil. Rats from the other two experimental groups were administered the same diet as group E during the first 2 wk of feeding, whereas at the beginning of the last 2 wk, the diet was enriched with rutin (group ER) or the CQA-rich ethanol extract from chicory seeds (9.6% of CQA, group EC), so the amount of added phenolics was equal in both dietary groups (0.15%). Results: The diet administered in group E caused hyperglycemia and increased blood serum atherogenicity in rats, but did not induce other manifestations of the metabolic syndrome, i.e., dyslipidemia and oxidative stress. Additionally, it affected gut physiology through increasing mucosal sucrase activity and disturbing fermentative processes in the cecum, such as the production of short-chain fatty acids and the activity of microbial enzymes. Similarly to rutin, the dietary addition of the chicory seed extract improved glycemia, which was comparable to that determined in group C. In addition, the extract was found to decrease the atherogenic index to the level observed in group C and to increase blood antioxidant status. Both dietary supplements reduced the content of thiobarbituric acid-reactive substances in kidney and heart tissue when compared with group E. Conclusion: The potential efficacy of the CQA-rich extract from chicory seeds in improving dietinduced metabolic disturbances proved to be better than that of rutin; thus, the extract might be considered as a dietary supplement for carrying out clinical trials. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Chicory Caffeoylquinic acids Chlorogenic acid Rutin The metabolic syndrome
Introduction Metabolic syndrome (MS) is a widespread diet-related disorder, defined as a cluster of interrelated risk factors for cardiovascular disease and type 2 diabetes. According to the new standardized criteria for clinical diagnosis of MS, these factors include central obesity, raised blood pressure, low concentration of high-density lipoprotein (HDL) cholesterol, as well as elevated
* Corresponding author. Tel.: þ48-89-523-4601; fax: þ48-89-524-0124. ski). E-mail address: [email protected] (A. Jurgon 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.010
glycemia and triglyceridemia [1]. Moreover, there is convincing evidence that oxidative stress is also involved in the development and progression of MS [2]. Most of the aforementioned manifestations can be induced experimentally in rats, in a relatively short time, by a high-fructose diet. However, the range of metabolic disturbances in response to excess fructose ingestion seems to be complex and may depend on many factors, such as age of the animals, as well as amounts of fructose and other components in the diet [3–5]. Prevention and treatment of MS are crucial for public health. In recent years, much research has been focused on dietary flavonoids, the antioxidants that can potentially improve
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disorders clustered in MS. In this context, one of the most investigated and relatively well-recognized flavonoids is quercetin and its glycosides, in particular, rutin (quercetin 3-Oglucorhamnoside) [6]. In experiments on rats, rutin administered orally or intraperitoneally improved glycemia, lipidemia, and antioxidant status [7–9], as well as ameliorated obesity induced by a high-fat diet [10]. Interestingly, the bioavailability of rutin after oral administration is low and it is mainly metabolized by large intestinal bacteria, among others to the aglycone form and phenolic acids [11,12]. Apart from flavonoids, there are also many other plant phenols exhibiting a vast array of biological activities. These include esters of caffeic and quinic acids, whose activity against disorders of MS has not been studied extensively thus far, to our knowledge, considering especially caffeoylquinic acids (CQA) with more than one caffeic acid residue. The most known and recognized CQA is a monocaffeoyl derivative chlorogenic acid (5-CQA), which occurs in many plants, fruits, and vegetables, such as coffee beans, apples, tomatoes, etc. [13]. For instance, chlorogenic acid has been shown to exhibit antiobesity properties and improve lipid metabolism in mice [14]. Moreover, some authors have observed delayed glucose absorption, suggesting its shift to more distal parts of the intestine, after the ingestion of chlorogenic acid-rich decaffeinated coffee [15]. However, similarly to rutin, CQA are also low-absorbable in the upper part of the gastrointestinal tract and most probably reach the large intestine in quantities [13,16]. In our laboratory, CQA have been recently isolated from different parts of chicory (Cichorium intybus L.) [17], which is a biennial plant with many applications in the food industry, for instance, it is used as an additive to coffee (roasted roots) and salads (young leaves) or as a source of prebiotic fructans (roots). The aim of this study was to compare the effects of a highfructose diet supplemented with rutin, a phenolic compound with relatively well-recognized bioavailability and bioactivity, and a CQA-rich extract from chicory seeds on gut physiology and the development of disorders in rats related to MS. To enhance the metabolic disturbances, a high-fructose diet was additionally enriched with oxidized fat and cholesterol. We speculated that, like rutin, the multiple ethanol extract from chicory seeds can reduce adverse effects of such a diet. Materials and methods Dietary supplements Seeds of chicory (C. intybus L.) were provided by Cykoria Co. (Wierzchos1awice, Poland), a company specializing in the cultivation and processing of the plant. After drying at <70 C, the seeds were ground and then extracted four times with 75% ethanol (1:3 sample-to-solvent ratio). The extraction procedure was performed in a hermetic container without access to light. Afterwards, the ethanol was removed by vacuum distillation and the extract was then freezedried for 24 h beginning at 30 C, followed by an additional drying at 40 C for 2 h. Determination of the antioxidant activity of the extract was performed using 2,2-diphenyl-1-picrylhydrazyl radical according to the modified method of Brand-Wiliams et al. [18], and the results were expressed as nanomoles of Trolox equivalent per gram of sample. Dry matter, ash, protein, and fat contents in the extract were assayed according to the Association of Analytical Communities (AOAC) official methods, whereas the determination of saccharides and phenolic compounds was conducted with high-performance liquid chromatography methods. Dicaffeoylquinic acid (diCQA) standard was isolated by semipreparative high-performance liquid chromatography and confirmed by mass spectrometry. CQA were quantified as grams of chlorogenic acid (5-caffeoylquinic acid; Sigma, St. Louis, MO, USA) equivalents per 100 g fresh mass. The procedure of extraction, as well as the analysis of the antioxidant activity and chemical composition of the extract tested, with detailed description of the methods applied, was described previously [17]. Table 1 presents chemical composition and antioxidant activity of the extract. Rutin (quercetin 3-O-glucorhamnoside, trihydrate, purity 95%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
301
Table 1 Chemical composition and antioxidant activity of the ethanol extract from chicory seeds g/100 g fresh mass 95.0 8.4 10.7 14.4 34.9 12.1 21.4 1.4 1.7 9.6 2.8 6.8 505.1
Dry matter Ash Protein Fat Mono- and disaccharides Glucose Fructose Sucrose Inulin Phenolic compounds MonoCQA DiCQA Antioxidant activity (nmol/g)
0.20 0.05 0.3 0.2 0.3 0.1 0.2 0.02 0.04 0.03 0.01 0.02 6.02
CQA, caffeoylquinic acids Data are expressed as mean values SD (n ¼ 3) Animals and diets The animal protocol used in this study was approved by the Local Animal Care and Use Committee (permission no. 23/2008). The experiment was conducted on 32 young male Wistar rats, distributed into four groups of eight animals each. All animals were housed individually in standard conditions with free access to water and semipurified casein diets (Table 2). Control rats (group C) received for 28 d a diet containing among others cholesterol (0.5%), soybean oil (8%), fructose (5%), and corn starch (66.5%). In comparison with group C, rats from the experimental group (group E) were fed for 28 d a diet with increased amounts of cholesterol and fructose (to 1% and 66% of the diet, respectively), added at the expense of cornstarch, as well as with oxidized soybean oil added instead of fresh. Rats from the other two experimental groups (ER and EC) were administered the same diet as in group E for the first 2 wk of feeding, whereas at the beginning of the last 2 wk the diet was enriched with rutin (group ER) or a chicory seed extract (group EC), added at the expense of cornstarch. In all experimental groups, the diets had comparable sums of mono- and disaccharides, whereas in the ER and EC groups the diets had additionally the same amount of phenolic compounds. Oxidized soybean oil was obtained by intensive aeration while heating at 70-80 C for 72 h. The degree of oxidation was monitored daily by determining the peroxide value according to the AOAC official method. The final degree of oxidation amounted to 120 miliequivalents of O2 per kg of soybean oil. Sample collection and analysis On termination of the experiment, rats were anaesthetized with sodium pentobarbital according to the recommendations for euthanasia of experimental animals. After laparotomy, blood samples were taken from caudal vein; then gut Table 2 Composition of the diets fed to rats* Ingredient (g/100 g diet)
Casein DL-methionine Soybean oil Oxidized soybean oily Cholesterol Mineral mixz Vitamin mixz Choline chloride Rutin Chicory seed extract Sucrose Fructose Corn starch
Group C
E
ER
EC
14.6 0.2 8 d 0.5 3.5 1 0.2 d d 0.5 5.0 66.50
14.6 0.2 d 8 1 3.5 1 0.2 d d 0.5 66.0 5.00
14.6 0.2 d 8 1 3.5 1 0.2 0.15 d 0.5 66.0 4.85
14.6 0.2 d 8 1 3.5 1 0.2 d 1.57 d 66.0 3.93
* Rats from the ER and EC groups have the same diet during first 2 wk of feeding as in group E, whereas the preparation of rutin or chicory seed extract was supplemented at the beginning of the last 2 wk. In all experimental groups, the diets have comparable sum of mono- and disaccharides, whereas in the ER and EC groups the diets have additionally equal amount of phenolic compounds. y Degree of oxidation ¼ 120 meq O2/kg. z Recommended for American Institute of Nutrition-93G diet.
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(small intestine, cecum, and colon) and internal organs (liver, heart, kidneys, and lungs) were removed and weighed. The small intestine was divided into four equal sections, and a second section from the stomach side was rinsed with cold physiological saline and cut open. Mucosal samples were collected by scraping with glass slides onto an iced glass plate. After the homogenization with four parts of cold physiological saline (v/w) and centrifugation for 10 min (9500 g, 4 C), the obtained supernatant was subsequently stored at 20 C. Sucrase, maltase, and lactase activities were assayed by the method of Dahlqvist [19] with some modifications, namely, glucose oxidase reagent was from Alpha Diagnostic Ltd. (Warsaw, Poland), and 0.2 mol/L of sodium phosphate buffer (pH 7.0) was used instead of maleate. Disaccharidase activities were expressed as mmol of glucose liberated from the respective disaccharide per minute per gram of protein. Protein content of the supernatant was estimated using Lowry’s method with bovine serum albumin as a standard. The pH of intestinal digesta was measured using a microelectrode and a pH/ION meter (model 301; Hanna Instruments, Vila do Conde, Portugal), while dry matter was determined at 105 C. Fresh cecal digesta was used for analysis of ammonia concentration, whereas determinations of enzyme activity and shortchain fatty acids (SCFA) concentration were performed after storage of the samples at –70 C. Ammonia was extracted and trapped in a solution of boric acid and then determined by direct titration with sulfuric acid [20]. The enzyme activity (a- and b-glucosidase, a- and b-galactosidase, b-glucuronidase) was measured by the rate of p- or o-nitrophenol release from their nitrophenylglucosides for 10 min [21] and expressed as mmol product formed per 1 h per gram of cecal digesta. The concentration of SCFA was measured using gas chromatography under the conditions described previously [21] and expressed as mmol of individual or total SCFA per gram of digesta. The concentration of glucose, triglycerides, total cholesterol, and its HDL fraction, as well as the activity of aspartate aminotransferase (ASP) and alanine aminotransferase (ALT) in the serum, were estimated with reagents from Alpha Diagnostics Ltd. (Warsaw, Poland). The atherogenic index was calculated from the following formula: log(triglycerides/HDL cholesterol). The activity of glutathione peroxidase in the heparinized blood and superoxide dismutase in erythrocyte lysate was determined using reagents from Randox Laboratories Ltd. (Crumlin, UK). Serum antioxidant capacity of hydrophilic and lipophilic substances was determined with photochemiluminescence detection method using a Photochem (Analytik Jena AG, Jena, Germany). In the photochemiluminescence assay, the generation of free radicals was partially eliminated by the reaction with antioxidants present in serum samples, and the remaining radicals were quantified by luminescence generation. Ascorbate and Trolox calibration curves were used to evaluate antioxidant capacity of hydrophilic and lipophilic substances, respectively, and the results were expressed as mmol of equivalents per milliliter of serum. The content of thiobarbituric acid-reactive substances (TBARS) in a tissue of internal organs (heart, kidneys, and liver) was estimated after storage at 20 C according to the method of Draper and Hadley [22]. Data analysis Results are expressed as mean values standard deviation (SD). Statistical analysis was performed using a one-way analysis of variance and the Duncan’s multiple range post-hoc test. Differences were considered significant at P 0.05. All calculations were performed using Microsoft Excel software.
Results Over 4 wk of experimental feeding, diet intake was comparable between all groups (Table 3); however, after the first 2 wk it tended to be different (P ¼ 0.07). All rats also had similar initial and final body weight, whereas the relative mass of kidneys and liver (g/100 g body weight) was lower in group C than in experimental groups, that is, the experimental (group E), the experimental with rutin (group ER), and the experimental with chicory seed extract (group EC, P 0.05). Differences in the indices of gut functioning are displayed in Table 4 (P 0.05). Both the relative mass of small intestine (with content) and the pH value of ileal digesta were higher in the experimental groups when compared with group C. The activity of mucosal enzymes was similar in all groups, except for sucrase, whose activity was higher in the E and ER groups than in the C and EC groups. Relative digesta mass of the cecum (g/g cecum) was comparable in all groups; however, it tended to be different (P ¼ 0.07), whereas the dry matter of digesta was decreased in group EC in comparison with the other groups. The pH value of
Table 3 Diet intake, body weight, and mass of selected organs of rats Group* C
E
Diet intake (g/14 d) First 2 wk 193 15.6 171 Last 2 wk 253 19.2 256 Body mass (g) Initial 93.0 3.78 92.8 Final 253 10.6 239 Gain 160 12.0 146 Mass of selected organsy Heart 0.304 0.025 0.313 Kidneys 0.664 0.021b 0.744 Liver 4.10 0.232b 5.78 Lungs 0.561 0.082 0.532
ER
EC
19.2 17.8
175 18.4 247 19.2
177 14.4 246 39.9
3.81 12.1 14.1
92.3 3.98 237 13.8 145 15.1
92.5 4.11 237 24.0 145 23.7
0.018 0.316 0.028 0.309 0.031a 0.786 0.027a 0.811 0.594a 5.68 0.319a 5.53 0.046 0.555 0.049 0.535
0.011 0.085a 0.569a 0.062
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y g/100 g body weight.
cecal digesta was lower in group EC when compared to groups C and E. The activity of microbial enzymes in the cecal digesta (aand b-glucosidases, as well as a- and b-galactosidases) was observed to decrease in the experimental groups when compared with group C. The total content of short-chain fatty acids (SCFA) in the cecal digesta was the lowest in the E and ER groups, higher in group EC, and highest in group C. In comparison with group C, the acetate content was lower in all experimental groups, whereas the propionate content was lower only in the E and ER group. In the colon, the relative digesta mass (g/g colon) was demonstrated to increase in the E and EC groups when compared with group C. Differences in the serum indices of rats are provided in Table 5 (P 0.05). In group E, glucose concentration increased when compared with group C, whereas in the ER and EC groups it was comparable with that reported for both groups E and C. The lipid profile was similar in all groups; however, the concentration of triglycerides tended to be different (P ¼ 0.09). In comparison with group C, the atherogenic index was higher in the E and ER groups and similar in group EC. Superoxide dismutase activity in erythrocytes was similar in the C and E groups and higher in group EC (P 0.05), whereas in group ER it was comparable with that assayed in the other groups (Table 6). Whole-blood glutathione peroxidase activity only tended to be different between all groups (P ¼ 0.08). Serum antioxidant capacity of hydrophilic substances was higher in group EC when compared with the other groups (P 0.05). The content of TBARS in a heart tissue was lower in the ER and EC groups than in the C and E groups (P 0.05). When compared with group E, the content of TBARS in a kidney tissue was lower in the ER and EC groups (P 0.05); simultaneously, the content was similar between the ER and EC groups, as well as between the E and C groups. Discussion The ethanol extract from seeds of chicory (C. intybus L.) used as dietary supplement in our study was a rich source of phenolic compounds (9.6%), that is, mono- and especially diCQA (2.8% and 6.8%, respectively, Table 1). It is worthy of notice that chlorogenic
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303
Table 4 Indices of gut functioning of rats Group* C Small intestine Mass with contenty pH of ileal digesta Activity of mucosal enzymesz Maltase Sucrase Lactase Cecum Tissue massy Digesta massx Dry matter of digesta (%) Ammonia in digesta (mg/g) pH of digesta Activity of microbial enzymes{ a–Glucosidase b–Glucosidase a–Galactosidase b–Galactosidase SCFAk (mmol/g digesta) Acetate Propionate Butyrate Total content Colon Tissue massy Digesta mass** pH of digesta
E
ER
EC
2.95 0.229b 5.83 0.318b
3.51 0.231a 6.65 0.290a
3.81 0.728a 6.45 0.133a
3.85 0.445a 6.41 0.316a
27.2 2.58 4.49 0.424b 1.16 0.285
29.0 2.43 6.09 0.594a 1.22 0.228
30.7 4.55 5.70 0.786a 1.07 0.212
29.7 3.33 4.96 0.456b 1.14 0.335
0.318 2.710 17.4 0.414 7.04
0.041 0.515 0.66a 0.067 0.104a
0.290 3.576 17.6 0.365 7.05
0.041 1.166 0.71a 0.088 0.175a
0.352 3.694 17.4 0.346 6.98
0.101 0.751 0.24a 0.029 0.143a,b
0.418 3.138 16.5 0.380 6.83
0.157 0.525 0.36b 0.047 0.212b
21.9 4.41 6.92 52.5
2.53a 0.900a 3.123a 17.66a
10.6 1.88 3.62 38.2
2.96b 0.795b 1.533b 12.37b
9.2 2.72 2.86 30.5
13.10b 1.396b 0.983b 9.58b
10.4 2.09 2.77 31.6
5.83b 0.388b 1.345b 8.61b
59.5 19.5 8.82 95.3
6.80a 3.07a 3.006 10.02a
41.4 14.2 7.77 69.3
6.98b 2.39b 1.888 10.31c
41.6 14.5 7.38 69.3
3.09b 1.69b 1.667 3.87c
47.1 17.9 8.85 79.9
6.12b 1.70a 2.928 5.42b
0.495 0.049 0.392 0.156b 6.88 0.115
0.514 0.043 0.798 0.364a 6.88 0.173
0.572 0.099 0.715 0.423a,b 6.77 0.155
0.533 0.069 0.931 0.309a 6.70 0.190
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; EC, experimental with chicory seed extract; ER, experimental with rutin. y g/100 g body weight. z mmol/min/g protein. x g/g cecal tissue. { mmol/h/g digesta. k Short-chain fatty acids. ** g/g colonic tissue.
acid and diCQA (3,5-diCQA and 3,4-diCQA) have recently been isolated from the leaves of endive species (C. endivia L.) [23]. Furthermore, diCQA have so far been isolated from many plant materials, such as fennel, seeds of arabica coffee, mugwort flowering tops, sweet potato leaves [24], or artichoke, known for an abundant source of cynarin (1,3-diCQA), as well as its isomer (1,5-diCQA) [25]. Interestingly, from among natural preparations, artichoke extracts were found to have the highest antioxidant activity in vitro, and cynarin was found to be the main contributor to the activity [26]. In the present study, the antioxidant activity of the chicory seed extract amounted to 505.1 nmol/g
and was more than twofold higher when compared with chicory leaf extract, obtained in the same way [18]. It must be emphasized, however, that the seed extract contained additionally considerable amounts of sugars (34.9%), predominantly fructose (21.4%), which can also increase the antioxidant activity in vitro [27]. In our study, after 4 wk of feeding the hypertrophy of liver and kidneys was observed in rats from group E, most probably due to the increased metabolic load of these organs, as a consequence of high fructose ingestion. Fructose raises serum uric acid concentration, increases its urinary excretion, as well as stimulates
Table 5 Basic biochemical indices of rat serum Group* C Glucose (mmol/L) Aminotransferases (U/L) Aspartate (ASP) Alanine (ALT) Lipid profile (mmol/L) Total cholesterol HDL-cholesterol Triglycerides Atherogenic indexy
E
ER
EC
9.90 1.79b
13.2 3.64a
12.2 2.17a,b
10.8 1.01a,b
92.5 15.33 26.3 4.59
90.6 18.42 28.5 6.26
93.5 14.67 30.1 8.01
90.5 18.83 28.0 3.02
2.80 1.48 2.79 0.263
0.366 0.252 0.936 0.092c
2.91 1.52 3.67 0.382
0.361 0.259 0.708 0.074a
2.83 1.46 3.33 0.356
0.435 0.188 0.550 0.098a,b
3.04 1.57 2.95 0.268
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y log(triglycerides/HDL-cholesterol).
0.373 0.260 0.679 0.066b,c
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304 Table 6 Antioxidant status of rats Group* C Activity of blood enzymes (U/mL) Glutathione peroxidase Superoxide dismutase Serum antioxidant capacity (mmol/mL) Hydrophilic substances Lypophilic substances TBARSy in tissue (nmol/g) Heart Kidney Liver
E
ER
EC
26.2 2.87 274 31.2b
24.5 2.97 278 19.5b
28.3 4.03 298 18.5a,b
27.9 2.21 313 39.4a
0.053 0.010b 0.193 0.017
0.043 0.012b 0.171 0.040
0.053 0.017b 0.200 0.058
0.094 0.033a 0.177 0.043
61.7 17.49a 173 54.5a,b 40.9 2.98
72.4 19.01a 188 27.7a 43.7 5.58
39.8 9.00b 138 36.9b,c 45.5 7.03
43.6 6.91b 119 19.9c 44.4 10.96
Results are expressed as mean values SD (n ¼ 8/group). Values not sharing the same superscript letters (a, b, c) within a row are different at P 0.05 * C, control; E, experimental; ER, experimental with rutin; EC, experimental with chicory seed extract. y Thiobarbituric acid-reactive substances.
inflammatory processes in the kidneys [28]. Moreover, this monosaccharide is almost exclusively metabolized in the liver, contrary to glucose, which is the basic energetic substrate for many different types of tissues [3]. However, the activity of aminotransferases in group E, as well as in the other experimental groups of our study, was comparable with the control, indicating that liver functions were sustained (ASP and ALT, Table 5). The potential role of fructose in the development of gastrointestinal tract disorders has already been described in literature, mainly due to its less effective absorption in the small intestine when compared with other mono- and disaccharides [29]. Results of our experiment, such as increased small intestinal mass and pH value of ileal digesta (group E), indicate that the excess fructose intake hinders its absorption. In the small intestine of rats from group E, the increased activity of sucrase was noted as well. It is in accordance with findings of other authors, who observed that fructose was capable of directly increasing the activity and gene expression of sucrase when compared with glucose [30]. Interestingly, the diet supplemented with chicory seed extract (group EC), but not with rutin (group ER), decreased sucrase activity to the level observed in group C (Table 4). It has been shown that CQA have maltase and sucrase inhibitory activities, and their intensity depends on the number of caffeoyl residuesdthe more residues, the stronger the inhibition [31]. Nevertheless, the observed inhibition of sucrase activity might in part be the effect of other phytochemicals present in chicory seeds [32]. Worthy of emphasizing are also differences in the composition of diets administered to experimental groups in our study. In comparison with group ER, the diet used in group EC contained less cornstarch and sucrose (4.85% and 0.5% versus 3.93% and 0.03%, respectively), that is, the substrates for sucrase which exists on the brush border membrane as a sucrase-isomaltase complex. In the cecum, the extract lowered dry matter and the pH value of the cecal digesta when compared with the C and E groups, indicating more intensive fermentation of indigestible components, such as CQA and inulin, which was also present in the extract. Indeed, the total content of SCFA in the cecal digesta as the main fermentation products of the large intestinal microflora was increased in group EC when compared with the E and ER groups, however, not to the level observed in the control. In addition, cecal propionate content was normalized under the influence of the chicory seed extract. It should be mentioned that propionate may be absorbed from the large intestine and is the inhibitor of hepatic lipogenesis [33]. Furthermore, Zhu et al. demonstrated antimicrobial activities of phenolic compounds isolated from artichoke leaves, with chlorogenic acid and diCQA being relatively one of the most
potent [34]. Results achieved in our study for the activity of microbial enzymes (glucosidases and galactosidases) suggest that the addition of the CQA-rich extract from chicory seeds to the diet has no unfavorable effect on large intestinal microflora. Administration of a high-fructose diet can disturb lipid and glucose metabolism in rats [3]. It may affect blood indices within a few weeks, especially causing hypertriglyceridemia, hyperinsulinemia, as well as glucose intolerance [4,5]. In our study, the diet containing 66% of fructose in a free form increased serum glucose concentration, but did not affect significantly serum lipid profile (group E versus group C). A lack of increase in triglyceride concentration was due to the other modifications of the diet, most likely the exchange of soybean oil for the oxidized one. It was generally assumed that the more the fat is oxidized, the less of it is absorbed from the small intestine [35]; thus, in our experiment, such a rule might influence a lack of the anticipated increase in serum triglyceride concentration in group E. On the other hand, the atherogenicity of serum in group E was considerably higher than in the control. In humans, the atherogenic index of plasma predicts MS, type 2 diabetes, and coronary heart disease [36]. In our experiment, the dietary addition of the CQA-rich extract, contrary to rutin, lowered the atherogenic index to the control level. The influence of diCQA, namely cynarin, over lipid profile has been the subject of experimental and clinical studies published in the 1970s, and the results obtained were contradictory [37]. In a more recent study, authors isolated 3,4-diCQA from a Ligularia fischeri variety and identified this compound as an inhibitor of lipid synthesis in rat liver [38]. Also, rutin has been implicated to improve lipid metabolism [7, 9]. Nevertheless, this was not the case in our study. It is noteworthy that the amount of phenolic compounds added after 2 wk of feeding to the diets of the ER and EC groups was equal (0.15 g/100 g diet). Furthermore, rutin and the chicory seed extract investigated in the present study decreased serum glucose concentration to the level observed in group C. In literature, positive effects of rutin on glucose metabolism were observed after both oral and intraperitoneal administration to rats with streptozotocin-induced diabetes, more likely due to antioxidant protection of pancreatic islet cells and stimulation of glucose utilization in extrahepatic tissues [7,8]. On the other hand, the glycemia-lowering mechanism of the chicory seed extract seems to be, at least in part, the consequence of local activities in the gastrointestinal tract. Indeed, it has been reported that chlorogenic acid might exhibit an antagonistic effect on the intestinal glucose transporter [15]. Furthermore, Matsui et al. showed strong antihyperglycemic effects of Brazilian propolis extract, pointing at CQA as the key compounds that inhibit maltase and sucrase activities [31]. In addition, the inhibition of a-glucosidase
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activity was attributed to some triterpenoids present in the seeds of chicory [32]. Results of our study partially support these findings, for sucrase activity was normalized in group EC. In the present investigation, the extract added to the diet of group EC increased considerably the serum antioxidant capacity of hydrophilic substances and superoxide dismutase activity in erythrocytes, thus distinguishing it from rutin (versus groups C and E). The increased antioxidant capacity suggests that CQA or their metabolites have to pass into the bloodstream. Indeed, in a human study several CQA metabolites in plasma were detected after the ingestion of artichoke leaf extracts rich in mono- and diCQA, i.e., derivatives of caffeic, ferulic, isoferulic, dihydrocaffeic, and dihydroferulic acids [16]. Interestingly, plasma concentration of dihydrocaffeic and dihdroferulic acids, the main metabolites of CQA, was the highest 6-7 h after administration, suggesting predominant absorption from the colon. Despite that rutin had no impact on serum antioxidant capacity, TBARS contents in a heart and kidney tissue, as the marker of oxidative stress, were reduced in our study, likewise, under the influence of the chicory seed extract (groups ER and EC versus group E). It is in agreement with another study, where rutin administered orally to streptozotocininduced diabetic rats (100 mg/kg body weight/d) improved the antioxidant status of selected tissues, e.g., reduced TBARS contents in kidneys, liver, and brain [8]. In vivo antioxidant properties of CQA have not been studied extensively thus far; however, di- and triCQA from aerial parts of Artemisia princeps inhibited the formation of advanced glycation end products [39]. It has been suggested that the accumulation of glycated proteins in vivo catalyzes free radical generation [40]. Moreover, glycated proteins are implicated in the development and progression of MS complications, i.e., diabetes and cardiovascular diseases [39,41]. In the context of MS manifestations, it is also of interest that diand triCQA have been shown to significantly reduce blood pressure in spontaneously hypertensive rats after oral administration (10 mg/kg body weight) [42].
Conclusion The dietary supplementation with the CQA-rich extract from chicory seeds and with rutin can improve glycemia; yet contrary to rutin, the addition of the extract decreases serum atherogenicity and increases blood antioxidant status. Therefore, the potential efficacy of the extract from chicory seeds in improving diet-induced metabolic disturbances is better than rutin, and the extract might be considered a dietary supplement for carrying out clinical trials. Furthermore, rats on a diet containing high amounts of fructose, oxidized soybean oil, and cholesterol, due to the lack of additional weight gain, dyslipidemia, and oxidative stress, is not a good experimental model mimicking MS.
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