- Email: [email protected]
Effect of a polyglucosamine on the body weight of male rats: Mechanisms of action
Food Chemistry 124 (2011) 978–982
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of a polyglucosamine on the body weight of male rats: Mechanisms of action Gianpietro Bondiolotti a, Umberto Cornelli b, Rosanna S. Strabbioli c, Natale G. Frega c, Matteo Cornelli d, Silvio R. Bareggi a,* a
Department of Pharmacology, Chemotherapy and Medical Toxicology, School of Medicine, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy Loyola University Medical School, Chicago, USA c University of Ancona, Italy d Cor. Con. Srl, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 12 February 2010 Received in revised form 7 July 2010 Accepted 14 July 2010
Keywords: Polyglucosamine Chitosan Body weight Cholesterol Lipids Glucose
a b s t r a c t Polyglucosamine (PG) is a low-molecular-weight chitosan (125 kDa) mixed with vitamin C and tartaric acid in standardised proportions. The aim of this study was to determine its effect on the body weight of male rats and clarify the mechanism of action. Three groups of 12 young male rats were fed a standard diet, or a diet containing PG (1% or 2%), for nine weeks. Body weight, food and water intake, and total cholesterol and triglyceride levels were measured before and at the end of treatment; low-density (LDL) and high-density lipoproteins (HDL), the amount of faeces, and their lipid, glucose and acetate content were also measured at the end of treatment. Total body weight increased by 234 ± 44.2 g in the controls, 233 ± 32.4 g in the rats fed PG 1%, and 206 ± 32.8 in those fed PG 2%; the weight increase was significantly less only in the PG 2% group, with the greatest difference being reached after four weeks (p < 0.01). Food intake was similar in all three groups. Twenty-four hour faecal weight/body weight was significantly higher (p < 0.05) in the animals treated with PG 2% than in the controls; faecal lipid, acetate, glucose and water content were also significantly higher in the PG 2% group. There was no significant change in the plasma lipid profiles of any of the groups. Dietary PG 2% reduces body weight, increases faecal weight (and faecal lipid and water content), and makes available fats and glucose as fuel for colon bacteria, as indicated by the higher acetate content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The biology (Muzzarelli, 1997; Singla & Chawla, 2001) and toxicology of chitosans (Kim, Park, Yang, & Han, 2001) have been extensively studied over the last decade, but their use to treat excess weight or reduce cholesterol levels is controversial, as some studies have led to positive results (Bokura & Kobayashi, 2003; Giustina & Ventura, 1995; Maezaki et al., 1993; Sciutto & Colombo, 1995; Veneroni et al., 1996) and others to uncertain or negative results (Gades & Stern, 2005; Metso et al., 2003; Mhurchu et al., 2004; Pittler & Ernst, 1998). These differences may be due to the different experimental conditions of the studies: the characteristics of the chitosans involved were not specified, although their density, molecular weight (Chiang, Yao, & Chen, 2000) and origin (from crab or crayfish chitin) may all affect their activity. The presence of ascorbic acid or other acidic components in the formulation can substantially alter their ability to bind with water and lipids (Deichi, Imasato, Kanauchi, & Kobayashi, 1994; Deichi, Imasato, Kanauchi, & * Corresponding author. Tel.: +39 02 50316946; fax: +39 02 50316949. E-mail address: [email protected] (S.R. Bareggi). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.039
Shizukuishi, 1994), and aqueous solutions of different chitosans vary widely in their ability to bind with water and emulsify in the presence of water and oil. It is therefore important to determine the ability of a well-defined chitosan to affect body weight. We evaluated the activity of a chitosan whose low molecular weight and particular preparation (e.g., mixed with given proportions of L-ascorbic and tartaric acids, as previously reported (Bondiolotti, Bareggi, Frega, Strabioli, & Cornelli, 2007)) have led to it being called ‘‘polyglucosamine” (PG). The aim of the study was to determine the effects of adding two concentrations of PG (1% and 2%) to the flour used to prepare food pellets on the body weight, water and food intake, plasma lipid profile, 24-h faecal weight, and faecal lipid and water content of rats. Faecal glucose and acetate concentrations were also measured as markers of intestinal metabolism. 2. Materials and methods 2.1. Polyglucosamine Polyglucosamine is a mixture of 96% low-molecular-weight chitosan (LMWC), 3% ascorbic acid and 1% tartaric acid. The
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
average molecular weight measured by means of size exclusion chromatography (SEC) using a triple detector array (TDA) (Bertini, Bisio, Torri, Bensi, & Terbojevich, 2005) is 125 kDa. One or two per cent of PG was added to the flour used to prepare the rat food pellets (see Section 2.3). 2.2. Animals, body weight, and food and water intake All of the experimental procedures complied with the EU international guidelines for animal experiments. Three groups of 12 3-month-old male Wistar rats (Charles River) were caged individually at a temperature of 22 °C and relative humidity of 50% under controlled lighting conditions for an initial period of three weeks. Their mean weights were 304 ± 15.5 g in the control group, 310 ± 14.3 g in the 1% PG group, and 298 ± 15.9 g in the 2% PG group. Food and water were available ad libitum and, once a week for nine weeks (every Monday), both were checked and replaced between 9:00 and 10:00 a.m.; the body weight of the animals was recorded on the same day. This is a standard method used in our laboratories (Bondiolotti et al., 2007) to determine body weight, and food and water intake in studies of fibre activity. The ratio between weekly water and food intake and weekly weight gain was also measured, in order to calculate food efficiency (conversion index or CI), and the possible interference of water intake with weight gain. 2.3. Diet The control animals were fed #48 Randoin-Causeret type pellets (the standard diet used in our laboratories), which provides 3.415 kcal/kg; 1% or 2% PG was added to the powder used to prepare the diet for the experimental animals. A fixed quantity of 25 kg of pellets was prepared for each concentration used in the trial. 2.4. Blood sampling and faeces analysis Plasma lipid profiles were determined in the morning (between 8:00 and 9:00 a.m.) before and after the nine weeks’ treatment; the animals fed normally during the night before the measurements. A 1-mm cut was made in the rats’ tails and 0.2 ml of blood were collected in heparinised mini-tubes, which were immediately centrifuged at 0.8g (2500 rpm) for 2 min; the plasma was then stored at room temperature until analysis. Total, low-density (LDL) and high-density lipoprotein (HDL) cholesterol (Allain, Poon, Chan, Richmond, & Fu, 1974) and triglyceride levels (McGowan, Ariss, Strandbergh, & Zak, 1983) were measured within 4 h of sampling, using the FREE system (Diacron, Grosseto, Italy) and dedicated reagents. Faecal weight, and lipid and water content were measured at the end of the treatment period. The faeces were collected for 24 h directly from the cages in which the animals were housed, by sifting the litter with a sieve, in order to filter out all of the sawdust; they were then stored at 20 °C until analysis, when their lipid content was measured using Folch’s method (Folch, Lees, & Sloane Stanley, 1957). Acetic acid was measured in mmol/g of faeces using an HPLC method (Fernandes, Venketesh, & Wolever, 2000). Half of the faeces from each group were pooled and dried in an oven at 45 °C for 24 h, after which they were pulverised and dried again for a further 24 h; the difference in weight before and after drying was taken as a measure of water content. To determine the effect of PG on glucose elimination, part of the faeces collected as described above was homogenised in water (1:4), and glucose levels were determined using the FREE system
979
and peroxidase method (Trinder, 1969). Acetate concentration was determined in the same samples as a measure of lipid hydrolysis by bacteria. 2.5. Statistics The data were analysed using analysis of variance (ANOVA) with Tukey’s multiple comparison test, split-plot analyses with Duncan’s multiple comparison test, and Kruskal–Wallis analyses of variance on ranks with Tukey’s multiple comparison test. 3. Results All of the animals completed the study without any gross behavioural changes or adverse effects being observed. Table 1 shows the data relating to body weight gain, food and water intake, and the ratios between food intake and body weight gain (CI), and between water intake and body weight gain. The body weight of the controls and the animals in the 1% PG group remained fairly similar, whereas the average body weight in the 2% PG group was significantly lower (split-plot analysis with Duncan’s multiple comparison test, p < 0.05) from week 1 to week 9. The body weight gain during the nine weeks was 234 ± 44.2 g in the control group, 233 ± 32.4 g in the 1% PG group (ANOVA p > 0.05 vs controls), and 206 ± 32.8 g in the 2% PG group (ANOVA p = 0.013 vs controls). The pattern of body weight gain over time was similar in the controls and the 1% PG group, but there was a statistically significant decrease in weight between weeks 2 and 7 in the 2% PG group. The average difference was almost constant between weeks 7 and 9, but the variance was very high and so statistical significance was not reached. Average weekly food intake was similar, and tended to increase in all three groups. CI was similar in the control and 1% PG groups, but higher in the 2% PG group. However, the weekly CI data were not statistically significant because of their high degree of variability. Over the entire nine-week period, the average CI values in the controls and the 1% PG group were very similar (7.5 ± 0.80 and 7.7 ± 0.86), but they were significantly higher in the group treated with 2% PG (8.4 ± 0.92; p < 0.05 vs controls). The average intake of PG in the two groups was 1.2 ± 0.13 g/kg/ day in the 1% PG group and 2.6 ± 0.24 g/kg/day in the 2% PG group. As the initial body weight of the rats was lower than their final weight, the amounts of 1% and 2% PG in terms of mg/kg were between 1.6 ± 0.17 and 0.7 mg/kg/day in the 1% PG group and between 3.5 ± 0.24 and 1.6 ± 0.19 mg/kg/day in the 2% PG group. The average weekly water intake was lower in the animals fed PG. The differences between the control group and both PG groups were statistically significant (ANOVA p < 0.05, Tukey’s test), and dose dependence was clear throughout the observation period. Once the water intake was corrected by the body weight gain, there was no difference between the groups except for the data recorded in week 5. Furthermore, the average ratios of water intake corrected by the body weight gain of the entire nine-week period were 15 ± 2.2 in the controls, 13 ± 2.8 in the 1% PG group, and 14 ± 2.2 in the 2% PG group; these differences are not statistically significant. Table 2 shows that plasma total cholesterol and triglyceride levels did not change significantly between baseline (T0) and nine weeks (T9) in any of the three groups, and that there were no differences in HDL or LDL cholesterol levels at T9. Table 3 shows the faeces analysis. Average faecal weight was lower in the controls (9.1 ± 3.3 g) than in the 1% PG group
980
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
Table 1 Weekly body weight gain, food and water intake, food efficiency and ratio between water intake and body weight (mean ± SD). Treatments
Weeks 2
3
4
5
6
7
8
9
Body weight gain (g) Controls 43.8 ± 10.1 PG 1% 37.2 ± 9.2 PG 2% 29.8 ± 8.9
1
74.3 ± 13.4 69.3 ± 12.6 59.1 ± 14.7a
104.2 ± 17.3 102.1 ± 16.6 87.4 ± 18.8a
142.8 ± 19.0 131.3 ± 18.5 115.7 ± 21.0b
161.6 ± 27.8 158.2 ± 26.3 140.4 ± 23.4a
179.5 ± 31.3 177.4 ± 30.2 155.1 ± 25.4a
202.6 ± 34.9 198.2 ± 33.7 175.2 ± 26.0a
212.5 ± 58.4 217.5 ± 59.2 190.3 ± 28.6
233.5 ± 47.0 233.4 ± 46.6 206.2 ± 32.8
Food intake g/week (g) Controls 187 ± 13.5 PG 1% 197 ± 11.5 PG 2% 184 ± 21.2
175 ± 15.4 187 ± 12.9 183 ± 19.1
187 ± 19.1 191 ± 15.4 188 ± 17.3
183 ± 20.0 196 ± 15.6 194 ± 15.7
190 ± 22.8 195 ± 13.7 189 ± 15.1
177 ± 17.6 189 ± 13.0 184 ± 16.4
201 ± 19.5 207 ± 11.7 197 ± 15.3
207 ± 21.1 209 ± 14.6 198 ± 15.1
206 ± 22.0 208 ± 15.0 200 ± 16.5
6.8 ± 1.2 6.0 ± 1.0 6.8 ± 1.2
4.9 ± 0.9 7.4 ± 3.5 7.1 ± 1.4
10.0 ± 1.3 7.6 ± 1.6 7.9 ± 1.3
8.8 ± 1.1 12.3 ± 7.4 13.6 ± 3.7
9.6 ± 1.5 10.0 ± 1.9 10.1 ± 2.1
12.9 ± 2.5 11.5 ± 3.3 13.2 ± 2.8
13.7 ± 3.1 16.8 ± 8.6 15.7 ± 11.8
399 ± 85.2 352 ± 74.4 327 ± 74.8
400 ± 64.2 356 ± 86.8 300 ± 44.2
405 ± 149.6 369 ± 108.3 316 ± 59.8
412 ± 93.4 377 ± 95.5 336 ± 80.9
342 ± 101.2 301 ± 72.6 285 ± 60.8
377 ± 86.1 312 ± 53.2 287 ± 58.6
358 ± 74.8 332 ± 71.9 302 ± 50.3
12.9 ± 4.0 10.7 ± 3.9 11.2 ± 3.2
10.8 ± 3.6 12.3 ± 4.8 10.9 ± 2.1
21.3 ± 3.1c 13.7 ± 5.2 12.6 ± 2.9
20.6 ± 4.5 19.8 ± 6.4 21.0 ± 6.2
16.2 ± 4.2 14.3 ± 3.8 15.8 ± 4.1
17.9 ± 4.9 16.4 ± 4.6 19.1 ± 5.1
23.8 ± 8.2 20.7 ± 7.9 18.7 ± 8.4
Food efficiency or CI: g food/g weight gain (g/g) Controls 4.2 ± 1.4 5.6 ± 1.5 PG1 1% 5.5 ± 1.0 5.8 ± 0.4 PG 2% 6.7 ± 2.4 6.7 ± 1.4 Water intake ml/week (ml) Controls* 353 ± 67.4 PG 1% 318 ± 53.0 PG 2% 297 ± 51.4
400 ± 84.2 344 ± 75.5 332 ± 38.2
Ratio water intake/body gain (ml/g) Controls 7.1 ± 2.5 12.9 ± 3.9 PG 1% 8.5 ± 3.3 10.7 ± 4.1 PG 2% 9.9 ± 3.4 11.4 ± 4.2
ANOVA, Tukey’s test: (a) p < 0.05; (b) p < 0.01; () controls > PG1% > PG2%, p < 0.05, ANOVA, Tukey’s test; (c) controls > PG1% > PG2%, p < 0.05, ANOVA, Tukey’s test.
Table 2 Plasma lipid levels (mg/dl) in the three treatment groups at the beginning (T0) and end (T9) of the study. Treatments
Controls 1% PG 2% PG
HDL
LDL
T0
Total cholesterol T9
T0
Triglycerides T9
T9
T9
91.2 ± 17.1 90.1 ± 20.0 86.3 ± 11.9
95.4 ± 12.9 96.7 ± 16.2 96.6 ± 9.9
136 ± 48.8 135 ± 29.4 144 ± 22.7
153 ± 34.9 156 ± 50.2 168 ± 49.1
42.9 ± 5.8 43.3 ± 7.3 42.5 ± 4.4
19.6 ± 10.4 19.9 ± 7.4 18.5 ± 11.8
Mean values ± SD.
Table 3 Faecal weight, faecal lipids, total lipids related to body weight, and faecal water in the three treatment groups. Treatments
Faecal weight (g/day)
Faecal weight/body weight ratio
Total lipids in faeces (g)
% faecal lipids/ body weight
Acetatemmol/ g/24 h
% water in faecesa
Glucose mg/24 h
Controls 1% PG 2% PG
9.1 ± 3.02 10.2 ± 2.69 10.3 ± 2.32
0.017 ± 0.004 0.019 ± 0.004 0.020 ± 0.004*
0.54 ± 0.20 0.65 ± 0.19 0.61 ± 0.17
0.099 ± 0.029 0.120 ± 0.032 0.121 ± 0.031*
0.5 ± 0.18 1.7 ± 0.41 5.2 ± 1.20§
9.8 ± 0.5 11.0 ± 1.7 11.1 ± 5.2
10.9 ± 4.0 15.9 ± 5.7 20.0 ± 5.4§
Mean values ± SD. * p < 0.05 vs controls; Kruskal–Wallis ANOVA on ranks, Tukey’s test; apooled faecal data. § p < 0.01 vs controls; ANOVA, Tukey’s test.
(10.2 ± 2.7 g) and 2% PG group (10.3 ± 2.3 g). Despite the small difference between the 1% and 2% PG groups, only the values in the 2% PG group were significantly higher after correcting for body weight (faecal weight/body weight ratio, p < 0.05). Total faecal lipid levels were also higher in both PG groups, but statistically significant after correction only in the 2% PG group. There were no differences in faecal water content, measured as the difference between the weight of the pooled faeces before and after drying. Acetate concentrations due to lipid metabolism by intestinal flora were significantly higher in the 2% PG group than in the other two groups. Furthermore, the animals treated with 2% PG also had significantly higher faecal glucose concentrations but, because of very high variance in all three groups, there was no difference between the controls and 1% PG group, or between the 1% PG and 2% PG groups.
4. Discussion PG is effective in reducing the body weight of rats when added to their diet at a concentration of 2%. Similar results have been obtained by other authors (Chiang et al., 2000; Kanauchi, Deichi, Imasato, & Shizukuishi, 1994) using higher chitosan doses. It has been found that low-molecular-weight (46 and 130 kDa) chitosans are effective in reducing obesity in mice fed high-fat diets (Sumiyoshi & Kimura, 2006), but a chitosan of >650 kDa had no effect on obesity in humans (Guercioline, Radu-Radulescu, Boldrin, Dalla, & Moore, 2001). We found that the lower concentration of PG (1%) had no effect on body weight, although faecal weight, water and lipid content were similar to those observed in the group receiving the higher (2%) concentration. Both groups showed cumulative fat malab-
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
sorption: about 6 g of fat over the entire study period, which is equivalent to 54 kcal and corresponds to approximately 7.2 g of adipose tissue. The fact that this was observed in both groups suggests that the loss of body weight was not due to an increase in faecally excreted water and lipids. The increased lipid excretion (0.44 g/day) in the animals treated with 2% PG does not account for the 28 g difference in body weight over 63 days, nor does the higher faecal water content. This indicates that the excretion of fats and water with faeces may not be an index of the effect of chitosans on body weight, and that other mechanisms may be involved. One possible explanation is ‘‘bacterial” energy wasting. The increased fat excretion in the group treated with 2% PG was matched by increased acetate and glucose excretion. This indicates that intestinal hydrolysis of the fats and glucose bioavailable in the colon was much higher in the 2% PG group than in the other groups, and is consistent with a larger amount of lipids and glucose being carried to the colon and made available as fuel for bacteria. PG produces an emulsion in the stomach that tends to be relatively stable once it has formed at acidic pH levels, even when pH increases in the duodenum and ileum. PG, water and lipids are completely emulsified in vitro when their ratio is 1/10/10, but the emulsification is minimal when the ratio is 0.5/10/10, which means that an appropriate concentration of PG is needed to bind water and lipids efficiently. Some authors (Rodriguez & Albertengo, 2005) have shown that chitosan dissolves at acidic pH in the stomach and emulsifies sunflower oil to the point of forming a flocculus at higher pH levels in the duodenum; the amount of oil entrapped by 1 g of chitosan was about 4 g in floccular form. It is likely that, in the case of oxidised oil (as after cooking, for example), the affinity of chitosan will be much higher, due to the increased polarity of oxidised lipids (unpublished data). The entrapped oil cannot be absorbed through the intestinal wall. The increased amounts of acetate excreted in faeces (which is higher with 2% than with 1% PG) indicates that the amount of lipids carried to the colon and used by bacteria in vivo increases with emulsion. In other words, the reduction in body weight may be mainly due to bacteria burning off some of the energy made available by the ‘‘outer” part of the PG/water/lipid emulsion, which is much more abundant with 2% PG than with 1% PG. Both glucosamine and free fatty acid (FFA) can generate insulin resistance (Wang, Liu, Barzilai, & Rossetti, 1998). The hydrolysis of polyglucosamine to glucosamine by colonic bacteria (Yun, Amakata, Matsuo, Matsuda, & Kawamukai, 2005), or the greater bioavailability of the FFA released by the emulsion, may therefore generate insulin resistance, which is not only a characteristic of muscle and fat, but can also be generated in enterocytes (Federico, Naples, & Taylor, 2006). This may be the cause of the increased faecal glucose excretion in the group fed with 2% PG. On the whole, food efficiency (the ratio between the total amount of food and the increase in body weight) in the group treated with 2% PG was about 88% lower than that observed in the control animals, thus indicating that more food is necessary for the same increase in body weight. The lower water consumption of the PG-treated animals may have been due to the greater availability of water in the distal part of the intestine, which therefore decreases the need for drinking water. The fact that the average value was higher with 2% PG than with 1% PG confirms the fact that water intake is not the cause of body weight reduction.
5. Conclusions In conclusion, the activity of 2% PG on body weight may be due to the concurrent effects of three different mechanisms:
981
a) increased faecal lipid elimination in relation to body weight; b) increased lipid consumption by bacteria; c) increased faecal glucose elimination. However, more research is required before any definite conclusions can be drawn. Acknowledgements GB carried out the animal and biochemical studies; SRB participated in the design and coordination of the study, performed the statistical analysis, and helped draft the manuscript; UC conceived the study, participated in the data analysis, and helped draft the manuscript; RSS participated in some biochemical studies; NGF performed some biochemical studies; MC participated in the design and coordination of the study. All of the authors approved the final version of the manuscript. None of the authors has any conflicts of interest. This study was supported in part by a grant from Regione Lombardia. References Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W., & Fu, P. C. (1974). Enzymatic determination of total serum cholesterol. Clinical Biochemistry, 20, 470–475. Bertini, S., Bisio, A., Torri, G., Bensi, D., & Terbojevich, M. (2005). Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array. Biomacromolecules, 6, 168–173. Bokura, H., & Kobayashi, S. (2003). Chitosan decreases total cholesterol in women: A randomized, double-blind, placebo-controlled trial. European Journal of Clinical Nutrition, 57, 721–725. Bondiolotti, G. P., Bareggi, S. R., Frega, N. G., Strabioli, S., & Cornelli, U. (2007). Activity of two different polyglucosamines, L112 and FF45, on body weight in male rats. European Journal of Pharmacology, 567, 155–158. Chiang, M. T., Yao, H. T., & Chen, H. C. (2000). Effect of dietary chitosans with different viscosity on plasma lipids and lipids peroxidation in rats fed on a diet enriched with cholesterol. Bioscience, Biotechnology, and Biochemistry, 64, 965–971. Federico, L. M., Naples, M., & Taylor, D. (2006). Khosrow A: Intestinal insulin resistance and aberrant production of apolipoprotein B48 lipoproteins in an animal model of insulin resistance and metabolic dyslipidemia. Diabetes, 55, 1316–1325. Fernandes, J., Venketesh, R., & Wolever, T. M. S. (2000). Different substrates and methane producing status affect short-chain fatty acid profiles produced by in vitro fermentation of human faeces. Journal of Nutrition, 130, 1932–1936. Folch, J., Lees, M., & Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497–509. Gades, M. D., & Stern, J. S. (2005). Chitosan supplementation and fat absorption in men and women. Journal of the American Dietetic Association, 105, 72–77. Giustina, A., & Ventura, P. (1995). Weight-reducing regimens in obese subjects: Effects of a new dietary fiber integrator. Acta Toxicologica et Therapeutica, 9, 199–214. Guercioline, R., Radu-Radulescu, L., Boldrin, M., Dalla, J., & Moore, R. (2001). Comparative evaluation of faecal fat excretion induced by orlistat and chitosan. Obesity Research, 9, 364–367. Kanauchi, O., Deichi, K., Imasato, Y., & Kobayashi, E. (1994). Increasing effect of a chitosan and ascorbic acid mixture on faecal dietary fat excretion. Bioscience, Biotechnology, and Biochemistry, 58, 1617–1620. Kanauchi, O., Deichi, K., Imasato, Y., & Shizukuishi, M. (1994). Kobayashi E: Mechanism for the inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate. Bioscience, Biotechnology, and Biochemistry, 59, 786–790. Kim, S. K., Park, P. J., Yang, H. P., & Han, S. S. (2001). Subacute toxicity of chitosan oligosaccharide in Sprague–Dawley rats. Arzneimittel Forschung/Drug Research, 51, 769–774. Maezaki, Y., Tsuji, K., Nakagawa, Y., Kaway, Y., Akimoto, M., Tsugita, T., et al. (1993). Hypocholesterolemic effect of chitosan in adult males. Bioscience, Biotechnology, and Biochemistry, 57, 1439–1444. McGowan, M. W., Ariss, J. D., Strandbergh, D. R., & Zak, B. (1983). A peroxidasecoupled method for the colorimetric determination of serum triglycerides. Clinical Biochemistry, 29, 538–542. Metso, S., Ylitalo, R., Nikkila, M., Wuolijoki, E., Ylitalo, P., & Lehtimaki, T. (2003). The effect of long term microcrystalline chitosan therapy on plasma lipids and glucose concentration in subjects with increased plasma total cholesterol: A randomized placebo-controlled double-blind crossover trial in healthy men and women. European Journal of Clinical Pharmacology, 59, 741–746. Mhurchu, C. N., Poppitt, S. D., McGill, A. T., Leahy, F. E., Bennet, D. A., Lin, R. B., et al. (2004). Rodgers A: The effect of the dietary supplement, chitosan, on body
982
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
weight: A randomized controlled trial in 250 overweight and obese adults. International Journal of Obesity and Related Metabolic Disorders, 28, 1149–1156. Muzzarelli, R. A. A. (1997). Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cellular and Molecular Life Sciences, 53, 131–140. Pittler, E., & Ernst, M. (1998). Chitosan as a treatment for body weight reduction? A meta-analysis. Perfusion, 11, 461–465. Rodriguez, M. S., & Albertengo, L. E. (2005). Interaction between chitosan and oil under stomach and duodenal digestive chemical conditions. Bioscience, Biotechnology, and Biochemistry, 69, 2062–2507. Sciutto, A. M., & Colombo, P. (1995). Lipid-lowering effect of chitosan dietary integrator and hypocaloric diet in obese subjects. Acta Toxicologica et Therapeutica, 16, 215–230. Singla, A. K., & Chawla, M. (2001). Chitosan: some pharmaceutical and biological aspects – An update. Journal of Pharmacy and Pharmacology, 53, 1047–1067.
Sumiyoshi, M., & Kimura, Y. (2006). Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long term in mice. Journal of Pharmacy and Pharmacology, 58, 201–207. Trinder, P. (1969). Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology, 22, 158–161. Veneroni, G., Veneroni, F., Contos, S., Tripodi, S., De Bernardi, M., & Guarino, C. (1996). Marletta M: Effect of a new chitosan dietary integrator and hypocaloric diet on hyperlipidemia on hyperlipemia and overweight in obese patients. Acta Toxicologica et Therapeutica, 17, 53–70. Wang, J., Liu, R., Barzilai, N., & Rossetti, L. (1998). A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature, 393, 684–688. Yun, C., Amakata, D., Matsuo, Y., Matsuda, H., & Kawamukai, M. (2005). New chitosan-degrading strains that produce chitosanases similar to ChoA of Mitsuaria chitosanitabia. Applied and Environmental Microbiology, 71, 5138–5144.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of a polyglucosamine on the body weight of male rats: Mechanisms of action Gianpietro Bondiolotti a, Umberto Cornelli b, Rosanna S. Strabbioli c, Natale G. Frega c, Matteo Cornelli d, Silvio R. Bareggi a,* a
Department of Pharmacology, Chemotherapy and Medical Toxicology, School of Medicine, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy Loyola University Medical School, Chicago, USA c University of Ancona, Italy d Cor. Con. Srl, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 12 February 2010 Received in revised form 7 July 2010 Accepted 14 July 2010
Keywords: Polyglucosamine Chitosan Body weight Cholesterol Lipids Glucose
a b s t r a c t Polyglucosamine (PG) is a low-molecular-weight chitosan (125 kDa) mixed with vitamin C and tartaric acid in standardised proportions. The aim of this study was to determine its effect on the body weight of male rats and clarify the mechanism of action. Three groups of 12 young male rats were fed a standard diet, or a diet containing PG (1% or 2%), for nine weeks. Body weight, food and water intake, and total cholesterol and triglyceride levels were measured before and at the end of treatment; low-density (LDL) and high-density lipoproteins (HDL), the amount of faeces, and their lipid, glucose and acetate content were also measured at the end of treatment. Total body weight increased by 234 ± 44.2 g in the controls, 233 ± 32.4 g in the rats fed PG 1%, and 206 ± 32.8 in those fed PG 2%; the weight increase was significantly less only in the PG 2% group, with the greatest difference being reached after four weeks (p < 0.01). Food intake was similar in all three groups. Twenty-four hour faecal weight/body weight was significantly higher (p < 0.05) in the animals treated with PG 2% than in the controls; faecal lipid, acetate, glucose and water content were also significantly higher in the PG 2% group. There was no significant change in the plasma lipid profiles of any of the groups. Dietary PG 2% reduces body weight, increases faecal weight (and faecal lipid and water content), and makes available fats and glucose as fuel for colon bacteria, as indicated by the higher acetate content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The biology (Muzzarelli, 1997; Singla & Chawla, 2001) and toxicology of chitosans (Kim, Park, Yang, & Han, 2001) have been extensively studied over the last decade, but their use to treat excess weight or reduce cholesterol levels is controversial, as some studies have led to positive results (Bokura & Kobayashi, 2003; Giustina & Ventura, 1995; Maezaki et al., 1993; Sciutto & Colombo, 1995; Veneroni et al., 1996) and others to uncertain or negative results (Gades & Stern, 2005; Metso et al., 2003; Mhurchu et al., 2004; Pittler & Ernst, 1998). These differences may be due to the different experimental conditions of the studies: the characteristics of the chitosans involved were not specified, although their density, molecular weight (Chiang, Yao, & Chen, 2000) and origin (from crab or crayfish chitin) may all affect their activity. The presence of ascorbic acid or other acidic components in the formulation can substantially alter their ability to bind with water and lipids (Deichi, Imasato, Kanauchi, & Kobayashi, 1994; Deichi, Imasato, Kanauchi, & * Corresponding author. Tel.: +39 02 50316946; fax: +39 02 50316949. E-mail address: [email protected] (S.R. Bareggi). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.039
Shizukuishi, 1994), and aqueous solutions of different chitosans vary widely in their ability to bind with water and emulsify in the presence of water and oil. It is therefore important to determine the ability of a well-defined chitosan to affect body weight. We evaluated the activity of a chitosan whose low molecular weight and particular preparation (e.g., mixed with given proportions of L-ascorbic and tartaric acids, as previously reported (Bondiolotti, Bareggi, Frega, Strabioli, & Cornelli, 2007)) have led to it being called ‘‘polyglucosamine” (PG). The aim of the study was to determine the effects of adding two concentrations of PG (1% and 2%) to the flour used to prepare food pellets on the body weight, water and food intake, plasma lipid profile, 24-h faecal weight, and faecal lipid and water content of rats. Faecal glucose and acetate concentrations were also measured as markers of intestinal metabolism. 2. Materials and methods 2.1. Polyglucosamine Polyglucosamine is a mixture of 96% low-molecular-weight chitosan (LMWC), 3% ascorbic acid and 1% tartaric acid. The
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
average molecular weight measured by means of size exclusion chromatography (SEC) using a triple detector array (TDA) (Bertini, Bisio, Torri, Bensi, & Terbojevich, 2005) is 125 kDa. One or two per cent of PG was added to the flour used to prepare the rat food pellets (see Section 2.3). 2.2. Animals, body weight, and food and water intake All of the experimental procedures complied with the EU international guidelines for animal experiments. Three groups of 12 3-month-old male Wistar rats (Charles River) were caged individually at a temperature of 22 °C and relative humidity of 50% under controlled lighting conditions for an initial period of three weeks. Their mean weights were 304 ± 15.5 g in the control group, 310 ± 14.3 g in the 1% PG group, and 298 ± 15.9 g in the 2% PG group. Food and water were available ad libitum and, once a week for nine weeks (every Monday), both were checked and replaced between 9:00 and 10:00 a.m.; the body weight of the animals was recorded on the same day. This is a standard method used in our laboratories (Bondiolotti et al., 2007) to determine body weight, and food and water intake in studies of fibre activity. The ratio between weekly water and food intake and weekly weight gain was also measured, in order to calculate food efficiency (conversion index or CI), and the possible interference of water intake with weight gain. 2.3. Diet The control animals were fed #48 Randoin-Causeret type pellets (the standard diet used in our laboratories), which provides 3.415 kcal/kg; 1% or 2% PG was added to the powder used to prepare the diet for the experimental animals. A fixed quantity of 25 kg of pellets was prepared for each concentration used in the trial. 2.4. Blood sampling and faeces analysis Plasma lipid profiles were determined in the morning (between 8:00 and 9:00 a.m.) before and after the nine weeks’ treatment; the animals fed normally during the night before the measurements. A 1-mm cut was made in the rats’ tails and 0.2 ml of blood were collected in heparinised mini-tubes, which were immediately centrifuged at 0.8g (2500 rpm) for 2 min; the plasma was then stored at room temperature until analysis. Total, low-density (LDL) and high-density lipoprotein (HDL) cholesterol (Allain, Poon, Chan, Richmond, & Fu, 1974) and triglyceride levels (McGowan, Ariss, Strandbergh, & Zak, 1983) were measured within 4 h of sampling, using the FREE system (Diacron, Grosseto, Italy) and dedicated reagents. Faecal weight, and lipid and water content were measured at the end of the treatment period. The faeces were collected for 24 h directly from the cages in which the animals were housed, by sifting the litter with a sieve, in order to filter out all of the sawdust; they were then stored at 20 °C until analysis, when their lipid content was measured using Folch’s method (Folch, Lees, & Sloane Stanley, 1957). Acetic acid was measured in mmol/g of faeces using an HPLC method (Fernandes, Venketesh, & Wolever, 2000). Half of the faeces from each group were pooled and dried in an oven at 45 °C for 24 h, after which they were pulverised and dried again for a further 24 h; the difference in weight before and after drying was taken as a measure of water content. To determine the effect of PG on glucose elimination, part of the faeces collected as described above was homogenised in water (1:4), and glucose levels were determined using the FREE system
979
and peroxidase method (Trinder, 1969). Acetate concentration was determined in the same samples as a measure of lipid hydrolysis by bacteria. 2.5. Statistics The data were analysed using analysis of variance (ANOVA) with Tukey’s multiple comparison test, split-plot analyses with Duncan’s multiple comparison test, and Kruskal–Wallis analyses of variance on ranks with Tukey’s multiple comparison test. 3. Results All of the animals completed the study without any gross behavioural changes or adverse effects being observed. Table 1 shows the data relating to body weight gain, food and water intake, and the ratios between food intake and body weight gain (CI), and between water intake and body weight gain. The body weight of the controls and the animals in the 1% PG group remained fairly similar, whereas the average body weight in the 2% PG group was significantly lower (split-plot analysis with Duncan’s multiple comparison test, p < 0.05) from week 1 to week 9. The body weight gain during the nine weeks was 234 ± 44.2 g in the control group, 233 ± 32.4 g in the 1% PG group (ANOVA p > 0.05 vs controls), and 206 ± 32.8 g in the 2% PG group (ANOVA p = 0.013 vs controls). The pattern of body weight gain over time was similar in the controls and the 1% PG group, but there was a statistically significant decrease in weight between weeks 2 and 7 in the 2% PG group. The average difference was almost constant between weeks 7 and 9, but the variance was very high and so statistical significance was not reached. Average weekly food intake was similar, and tended to increase in all three groups. CI was similar in the control and 1% PG groups, but higher in the 2% PG group. However, the weekly CI data were not statistically significant because of their high degree of variability. Over the entire nine-week period, the average CI values in the controls and the 1% PG group were very similar (7.5 ± 0.80 and 7.7 ± 0.86), but they were significantly higher in the group treated with 2% PG (8.4 ± 0.92; p < 0.05 vs controls). The average intake of PG in the two groups was 1.2 ± 0.13 g/kg/ day in the 1% PG group and 2.6 ± 0.24 g/kg/day in the 2% PG group. As the initial body weight of the rats was lower than their final weight, the amounts of 1% and 2% PG in terms of mg/kg were between 1.6 ± 0.17 and 0.7 mg/kg/day in the 1% PG group and between 3.5 ± 0.24 and 1.6 ± 0.19 mg/kg/day in the 2% PG group. The average weekly water intake was lower in the animals fed PG. The differences between the control group and both PG groups were statistically significant (ANOVA p < 0.05, Tukey’s test), and dose dependence was clear throughout the observation period. Once the water intake was corrected by the body weight gain, there was no difference between the groups except for the data recorded in week 5. Furthermore, the average ratios of water intake corrected by the body weight gain of the entire nine-week period were 15 ± 2.2 in the controls, 13 ± 2.8 in the 1% PG group, and 14 ± 2.2 in the 2% PG group; these differences are not statistically significant. Table 2 shows that plasma total cholesterol and triglyceride levels did not change significantly between baseline (T0) and nine weeks (T9) in any of the three groups, and that there were no differences in HDL or LDL cholesterol levels at T9. Table 3 shows the faeces analysis. Average faecal weight was lower in the controls (9.1 ± 3.3 g) than in the 1% PG group
980
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
Table 1 Weekly body weight gain, food and water intake, food efficiency and ratio between water intake and body weight (mean ± SD). Treatments
Weeks 2
3
4
5
6
7
8
9
Body weight gain (g) Controls 43.8 ± 10.1 PG 1% 37.2 ± 9.2 PG 2% 29.8 ± 8.9
1
74.3 ± 13.4 69.3 ± 12.6 59.1 ± 14.7a
104.2 ± 17.3 102.1 ± 16.6 87.4 ± 18.8a
142.8 ± 19.0 131.3 ± 18.5 115.7 ± 21.0b
161.6 ± 27.8 158.2 ± 26.3 140.4 ± 23.4a
179.5 ± 31.3 177.4 ± 30.2 155.1 ± 25.4a
202.6 ± 34.9 198.2 ± 33.7 175.2 ± 26.0a
212.5 ± 58.4 217.5 ± 59.2 190.3 ± 28.6
233.5 ± 47.0 233.4 ± 46.6 206.2 ± 32.8
Food intake g/week (g) Controls 187 ± 13.5 PG 1% 197 ± 11.5 PG 2% 184 ± 21.2
175 ± 15.4 187 ± 12.9 183 ± 19.1
187 ± 19.1 191 ± 15.4 188 ± 17.3
183 ± 20.0 196 ± 15.6 194 ± 15.7
190 ± 22.8 195 ± 13.7 189 ± 15.1
177 ± 17.6 189 ± 13.0 184 ± 16.4
201 ± 19.5 207 ± 11.7 197 ± 15.3
207 ± 21.1 209 ± 14.6 198 ± 15.1
206 ± 22.0 208 ± 15.0 200 ± 16.5
6.8 ± 1.2 6.0 ± 1.0 6.8 ± 1.2
4.9 ± 0.9 7.4 ± 3.5 7.1 ± 1.4
10.0 ± 1.3 7.6 ± 1.6 7.9 ± 1.3
8.8 ± 1.1 12.3 ± 7.4 13.6 ± 3.7
9.6 ± 1.5 10.0 ± 1.9 10.1 ± 2.1
12.9 ± 2.5 11.5 ± 3.3 13.2 ± 2.8
13.7 ± 3.1 16.8 ± 8.6 15.7 ± 11.8
399 ± 85.2 352 ± 74.4 327 ± 74.8
400 ± 64.2 356 ± 86.8 300 ± 44.2
405 ± 149.6 369 ± 108.3 316 ± 59.8
412 ± 93.4 377 ± 95.5 336 ± 80.9
342 ± 101.2 301 ± 72.6 285 ± 60.8
377 ± 86.1 312 ± 53.2 287 ± 58.6
358 ± 74.8 332 ± 71.9 302 ± 50.3
12.9 ± 4.0 10.7 ± 3.9 11.2 ± 3.2
10.8 ± 3.6 12.3 ± 4.8 10.9 ± 2.1
21.3 ± 3.1c 13.7 ± 5.2 12.6 ± 2.9
20.6 ± 4.5 19.8 ± 6.4 21.0 ± 6.2
16.2 ± 4.2 14.3 ± 3.8 15.8 ± 4.1
17.9 ± 4.9 16.4 ± 4.6 19.1 ± 5.1
23.8 ± 8.2 20.7 ± 7.9 18.7 ± 8.4
Food efficiency or CI: g food/g weight gain (g/g) Controls 4.2 ± 1.4 5.6 ± 1.5 PG1 1% 5.5 ± 1.0 5.8 ± 0.4 PG 2% 6.7 ± 2.4 6.7 ± 1.4 Water intake ml/week (ml) Controls* 353 ± 67.4 PG 1% 318 ± 53.0 PG 2% 297 ± 51.4
400 ± 84.2 344 ± 75.5 332 ± 38.2
Ratio water intake/body gain (ml/g) Controls 7.1 ± 2.5 12.9 ± 3.9 PG 1% 8.5 ± 3.3 10.7 ± 4.1 PG 2% 9.9 ± 3.4 11.4 ± 4.2
ANOVA, Tukey’s test: (a) p < 0.05; (b) p < 0.01; () controls > PG1% > PG2%, p < 0.05, ANOVA, Tukey’s test; (c) controls > PG1% > PG2%, p < 0.05, ANOVA, Tukey’s test.
Table 2 Plasma lipid levels (mg/dl) in the three treatment groups at the beginning (T0) and end (T9) of the study. Treatments
Controls 1% PG 2% PG
HDL
LDL
T0
Total cholesterol T9
T0
Triglycerides T9
T9
T9
91.2 ± 17.1 90.1 ± 20.0 86.3 ± 11.9
95.4 ± 12.9 96.7 ± 16.2 96.6 ± 9.9
136 ± 48.8 135 ± 29.4 144 ± 22.7
153 ± 34.9 156 ± 50.2 168 ± 49.1
42.9 ± 5.8 43.3 ± 7.3 42.5 ± 4.4
19.6 ± 10.4 19.9 ± 7.4 18.5 ± 11.8
Mean values ± SD.
Table 3 Faecal weight, faecal lipids, total lipids related to body weight, and faecal water in the three treatment groups. Treatments
Faecal weight (g/day)
Faecal weight/body weight ratio
Total lipids in faeces (g)
% faecal lipids/ body weight
Acetatemmol/ g/24 h
% water in faecesa
Glucose mg/24 h
Controls 1% PG 2% PG
9.1 ± 3.02 10.2 ± 2.69 10.3 ± 2.32
0.017 ± 0.004 0.019 ± 0.004 0.020 ± 0.004*
0.54 ± 0.20 0.65 ± 0.19 0.61 ± 0.17
0.099 ± 0.029 0.120 ± 0.032 0.121 ± 0.031*
0.5 ± 0.18 1.7 ± 0.41 5.2 ± 1.20§
9.8 ± 0.5 11.0 ± 1.7 11.1 ± 5.2
10.9 ± 4.0 15.9 ± 5.7 20.0 ± 5.4§
Mean values ± SD. * p < 0.05 vs controls; Kruskal–Wallis ANOVA on ranks, Tukey’s test; apooled faecal data. § p < 0.01 vs controls; ANOVA, Tukey’s test.
(10.2 ± 2.7 g) and 2% PG group (10.3 ± 2.3 g). Despite the small difference between the 1% and 2% PG groups, only the values in the 2% PG group were significantly higher after correcting for body weight (faecal weight/body weight ratio, p < 0.05). Total faecal lipid levels were also higher in both PG groups, but statistically significant after correction only in the 2% PG group. There were no differences in faecal water content, measured as the difference between the weight of the pooled faeces before and after drying. Acetate concentrations due to lipid metabolism by intestinal flora were significantly higher in the 2% PG group than in the other two groups. Furthermore, the animals treated with 2% PG also had significantly higher faecal glucose concentrations but, because of very high variance in all three groups, there was no difference between the controls and 1% PG group, or between the 1% PG and 2% PG groups.
4. Discussion PG is effective in reducing the body weight of rats when added to their diet at a concentration of 2%. Similar results have been obtained by other authors (Chiang et al., 2000; Kanauchi, Deichi, Imasato, & Shizukuishi, 1994) using higher chitosan doses. It has been found that low-molecular-weight (46 and 130 kDa) chitosans are effective in reducing obesity in mice fed high-fat diets (Sumiyoshi & Kimura, 2006), but a chitosan of >650 kDa had no effect on obesity in humans (Guercioline, Radu-Radulescu, Boldrin, Dalla, & Moore, 2001). We found that the lower concentration of PG (1%) had no effect on body weight, although faecal weight, water and lipid content were similar to those observed in the group receiving the higher (2%) concentration. Both groups showed cumulative fat malab-
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
sorption: about 6 g of fat over the entire study period, which is equivalent to 54 kcal and corresponds to approximately 7.2 g of adipose tissue. The fact that this was observed in both groups suggests that the loss of body weight was not due to an increase in faecally excreted water and lipids. The increased lipid excretion (0.44 g/day) in the animals treated with 2% PG does not account for the 28 g difference in body weight over 63 days, nor does the higher faecal water content. This indicates that the excretion of fats and water with faeces may not be an index of the effect of chitosans on body weight, and that other mechanisms may be involved. One possible explanation is ‘‘bacterial” energy wasting. The increased fat excretion in the group treated with 2% PG was matched by increased acetate and glucose excretion. This indicates that intestinal hydrolysis of the fats and glucose bioavailable in the colon was much higher in the 2% PG group than in the other groups, and is consistent with a larger amount of lipids and glucose being carried to the colon and made available as fuel for bacteria. PG produces an emulsion in the stomach that tends to be relatively stable once it has formed at acidic pH levels, even when pH increases in the duodenum and ileum. PG, water and lipids are completely emulsified in vitro when their ratio is 1/10/10, but the emulsification is minimal when the ratio is 0.5/10/10, which means that an appropriate concentration of PG is needed to bind water and lipids efficiently. Some authors (Rodriguez & Albertengo, 2005) have shown that chitosan dissolves at acidic pH in the stomach and emulsifies sunflower oil to the point of forming a flocculus at higher pH levels in the duodenum; the amount of oil entrapped by 1 g of chitosan was about 4 g in floccular form. It is likely that, in the case of oxidised oil (as after cooking, for example), the affinity of chitosan will be much higher, due to the increased polarity of oxidised lipids (unpublished data). The entrapped oil cannot be absorbed through the intestinal wall. The increased amounts of acetate excreted in faeces (which is higher with 2% than with 1% PG) indicates that the amount of lipids carried to the colon and used by bacteria in vivo increases with emulsion. In other words, the reduction in body weight may be mainly due to bacteria burning off some of the energy made available by the ‘‘outer” part of the PG/water/lipid emulsion, which is much more abundant with 2% PG than with 1% PG. Both glucosamine and free fatty acid (FFA) can generate insulin resistance (Wang, Liu, Barzilai, & Rossetti, 1998). The hydrolysis of polyglucosamine to glucosamine by colonic bacteria (Yun, Amakata, Matsuo, Matsuda, & Kawamukai, 2005), or the greater bioavailability of the FFA released by the emulsion, may therefore generate insulin resistance, which is not only a characteristic of muscle and fat, but can also be generated in enterocytes (Federico, Naples, & Taylor, 2006). This may be the cause of the increased faecal glucose excretion in the group fed with 2% PG. On the whole, food efficiency (the ratio between the total amount of food and the increase in body weight) in the group treated with 2% PG was about 88% lower than that observed in the control animals, thus indicating that more food is necessary for the same increase in body weight. The lower water consumption of the PG-treated animals may have been due to the greater availability of water in the distal part of the intestine, which therefore decreases the need for drinking water. The fact that the average value was higher with 2% PG than with 1% PG confirms the fact that water intake is not the cause of body weight reduction.
5. Conclusions In conclusion, the activity of 2% PG on body weight may be due to the concurrent effects of three different mechanisms:
981
a) increased faecal lipid elimination in relation to body weight; b) increased lipid consumption by bacteria; c) increased faecal glucose elimination. However, more research is required before any definite conclusions can be drawn. Acknowledgements GB carried out the animal and biochemical studies; SRB participated in the design and coordination of the study, performed the statistical analysis, and helped draft the manuscript; UC conceived the study, participated in the data analysis, and helped draft the manuscript; RSS participated in some biochemical studies; NGF performed some biochemical studies; MC participated in the design and coordination of the study. All of the authors approved the final version of the manuscript. None of the authors has any conflicts of interest. This study was supported in part by a grant from Regione Lombardia. References Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W., & Fu, P. C. (1974). Enzymatic determination of total serum cholesterol. Clinical Biochemistry, 20, 470–475. Bertini, S., Bisio, A., Torri, G., Bensi, D., & Terbojevich, M. (2005). Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array. Biomacromolecules, 6, 168–173. Bokura, H., & Kobayashi, S. (2003). Chitosan decreases total cholesterol in women: A randomized, double-blind, placebo-controlled trial. European Journal of Clinical Nutrition, 57, 721–725. Bondiolotti, G. P., Bareggi, S. R., Frega, N. G., Strabioli, S., & Cornelli, U. (2007). Activity of two different polyglucosamines, L112 and FF45, on body weight in male rats. European Journal of Pharmacology, 567, 155–158. Chiang, M. T., Yao, H. T., & Chen, H. C. (2000). Effect of dietary chitosans with different viscosity on plasma lipids and lipids peroxidation in rats fed on a diet enriched with cholesterol. Bioscience, Biotechnology, and Biochemistry, 64, 965–971. Federico, L. M., Naples, M., & Taylor, D. (2006). Khosrow A: Intestinal insulin resistance and aberrant production of apolipoprotein B48 lipoproteins in an animal model of insulin resistance and metabolic dyslipidemia. Diabetes, 55, 1316–1325. Fernandes, J., Venketesh, R., & Wolever, T. M. S. (2000). Different substrates and methane producing status affect short-chain fatty acid profiles produced by in vitro fermentation of human faeces. Journal of Nutrition, 130, 1932–1936. Folch, J., Lees, M., & Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497–509. Gades, M. D., & Stern, J. S. (2005). Chitosan supplementation and fat absorption in men and women. Journal of the American Dietetic Association, 105, 72–77. Giustina, A., & Ventura, P. (1995). Weight-reducing regimens in obese subjects: Effects of a new dietary fiber integrator. Acta Toxicologica et Therapeutica, 9, 199–214. Guercioline, R., Radu-Radulescu, L., Boldrin, M., Dalla, J., & Moore, R. (2001). Comparative evaluation of faecal fat excretion induced by orlistat and chitosan. Obesity Research, 9, 364–367. Kanauchi, O., Deichi, K., Imasato, Y., & Kobayashi, E. (1994). Increasing effect of a chitosan and ascorbic acid mixture on faecal dietary fat excretion. Bioscience, Biotechnology, and Biochemistry, 58, 1617–1620. Kanauchi, O., Deichi, K., Imasato, Y., & Shizukuishi, M. (1994). Kobayashi E: Mechanism for the inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate. Bioscience, Biotechnology, and Biochemistry, 59, 786–790. Kim, S. K., Park, P. J., Yang, H. P., & Han, S. S. (2001). Subacute toxicity of chitosan oligosaccharide in Sprague–Dawley rats. Arzneimittel Forschung/Drug Research, 51, 769–774. Maezaki, Y., Tsuji, K., Nakagawa, Y., Kaway, Y., Akimoto, M., Tsugita, T., et al. (1993). Hypocholesterolemic effect of chitosan in adult males. Bioscience, Biotechnology, and Biochemistry, 57, 1439–1444. McGowan, M. W., Ariss, J. D., Strandbergh, D. R., & Zak, B. (1983). A peroxidasecoupled method for the colorimetric determination of serum triglycerides. Clinical Biochemistry, 29, 538–542. Metso, S., Ylitalo, R., Nikkila, M., Wuolijoki, E., Ylitalo, P., & Lehtimaki, T. (2003). The effect of long term microcrystalline chitosan therapy on plasma lipids and glucose concentration in subjects with increased plasma total cholesterol: A randomized placebo-controlled double-blind crossover trial in healthy men and women. European Journal of Clinical Pharmacology, 59, 741–746. Mhurchu, C. N., Poppitt, S. D., McGill, A. T., Leahy, F. E., Bennet, D. A., Lin, R. B., et al. (2004). Rodgers A: The effect of the dietary supplement, chitosan, on body
982
G. Bondiolotti et al. / Food Chemistry 124 (2011) 978–982
weight: A randomized controlled trial in 250 overweight and obese adults. International Journal of Obesity and Related Metabolic Disorders, 28, 1149–1156. Muzzarelli, R. A. A. (1997). Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cellular and Molecular Life Sciences, 53, 131–140. Pittler, E., & Ernst, M. (1998). Chitosan as a treatment for body weight reduction? A meta-analysis. Perfusion, 11, 461–465. Rodriguez, M. S., & Albertengo, L. E. (2005). Interaction between chitosan and oil under stomach and duodenal digestive chemical conditions. Bioscience, Biotechnology, and Biochemistry, 69, 2062–2507. Sciutto, A. M., & Colombo, P. (1995). Lipid-lowering effect of chitosan dietary integrator and hypocaloric diet in obese subjects. Acta Toxicologica et Therapeutica, 16, 215–230. Singla, A. K., & Chawla, M. (2001). Chitosan: some pharmaceutical and biological aspects – An update. Journal of Pharmacy and Pharmacology, 53, 1047–1067.
Sumiyoshi, M., & Kimura, Y. (2006). Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long term in mice. Journal of Pharmacy and Pharmacology, 58, 201–207. Trinder, P. (1969). Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology, 22, 158–161. Veneroni, G., Veneroni, F., Contos, S., Tripodi, S., De Bernardi, M., & Guarino, C. (1996). Marletta M: Effect of a new chitosan dietary integrator and hypocaloric diet on hyperlipidemia on hyperlipemia and overweight in obese patients. Acta Toxicologica et Therapeutica, 17, 53–70. Wang, J., Liu, R., Barzilai, N., & Rossetti, L. (1998). A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature, 393, 684–688. Yun, C., Amakata, D., Matsuo, Y., Matsuda, H., & Kawamukai, M. (2005). New chitosan-degrading strains that produce chitosanases similar to ChoA of Mitsuaria chitosanitabia. Applied and Environmental Microbiology, 71, 5138–5144.