Eur Food Res Technol (2014) 238:409–416DOI 10.1007/s00217-013-2116-5
ORIGINAL PAPER
Health‑promoting effects in the gut and influence on lipid metabolism of Himanthalia elongata and Gigartina pistillata in hypercholesterolaemic Wistar rats
seaweeds, due to the wide diversity of compounds they contain, are promising organisms for providing novel bio-logically active ingredients with a high impact on the food and pharmaceutical industry and public health [1–4]. Sub-stantial research is required on the toxicity aspects [5–7] before these seaweeds can actually be used as science-based dietary recommendations, and this vast untapped resource can be utilised for beneficial purposes, since these bioresources are often regarded as underexploited.
Seaweeds are a common ingredient in Asian coun-tries; however, in western countries, they are used mainly as thickening and gelling agents. However, edible sea-weeds represent new sources of dietary fibre. The Span-ish edible seaweeds selected are in fact a good source of dietary fibre (DF 29–37 g/100 g dry weight, dw), minerals (35–37 g/100 g dw) and protein (14–16 g/100 g dw), and they have a very low lipid content (0.6–0.9 g/100 g dw) [8]. DF is constituted by alginates, laminarans and the sulphated polysaccharides, fucans, in the brown seaweed, Himanthalia; and mainly by sulphated carrageenans in the red seaweed, Gigartina. Their physico-chemical prop-erties reveal that these polysaccharides could contribute to water binding, faecal bulking and decreased transit time, thus representing a good source of food fibre for human consumption [8]. In fact, various in vitro and in vivo studies have shown the potential prebiotic effect of some polysaccharides in brown seaweeds [2, 9–11], and their positive influences on gut health [12, 13]. Moreo-ver, low molecular weight extracts from different species of red seaweeds have been fermented by gut microbiota [14, 15]. Other studies suggest low or no fermentability of brown algae polysaccharides by gut microbiota [16, 17], and it was recently shown that there was no clear prebi-otic effect from dietary supplementation with the red sea-weed Mastocarpus stellatus in Wistar rats [2]. In contrast,
Abstract The intake of Himanthalia elongata and Gigartina pistillata from the Spanish Atlantic coasts was evaluated in Wistar rats. Both seaweed diets showed higher (p < 0.001) faecal excretion. Colonic fermentation increased (p < 0.001) total short-chain fatty acids (SCFAs) in Himanthalia-fed rats due to the higher (p < 0.001) levels of acetic, propionic and butyric acids. The intake of Gigar-tina increased (p < 0.001) propionic acid and decreased (p < 0.001) butyric acid. The apparent absorption and true retention of calcium and magnesium enhanced (p < 0.05) with Himanthalia diet, while Gigartina produced no signif-icant effect. The serum concentration of HDL-C increased (p < 0.01), triglycerides (TGL) decreased (p < 0.001) and bile acids diminished (p < 0.001) in faeces of Himan-thalia-fed rats. The Gigartina diet produced a decrease (p < 0.001) in TGL, total cholesterol (p < 0.01) and LDL-C (p < 0.05) in serum and reduced TGL in liver (p < 0.001). Thus, both seaweeds improved the lipid profile, and Him-anthalia increased SCFA production and the absorption and retention of Ca and Mg as a result of the gut fermentation.
Seaweeds grow in abundance in coastal areas and are avail-able all year round. A number of studies have shown that
M.-J. Villanueva · M. Morcillo · M.-D. Tenorio · I. Mateos-Aparicio (*) · V. Andrés · A. Redondo-Cuenca Dpto. Nutrición y Bromatología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spaine-mail: [email protected]
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many studies involving seaweed supplementation have demonstrated potential cholesterol-lowering effects. In fact, studies in Wistar rats fed with cholesterol-rich diets containing a mixture of seaweeds [18] or the Nori brown seaweed [19] have shown reduced levels of serum lipids. Moreover, lower levels of triglycerides and total choles-terol were observed in healthy rats fed with the red sea-weed M. stellatus [2]. Paxman et al. [20] showed that treatment with 1.5 g of alginate may re-establish the cho-lesterol and glucose values to the levels of healthy sub-jects. More research is necessary to elucidate the effects of seaweeds on gut health and lipid metabolism before promoting the use of seaweeds. This study was carried out in order to determine which kind of seaweed—brown or red—is more suitable for dietary recommendations for the purpose of developing a potential prebiotic effect to support gut health and to treat hypercholesterolaemia and consequently to reduce the risk of cardiovascular disease. Thus, the aim of this study was to evaluate the health-pro-moting effects in the gut and the influence on lipid metab-olism of the intake of brown Himanthalia elongata and red Gigartina pistillata seaweeds for 4 weeks in hyper-cholesterolaemic Wistar rats.
Materials and methods
Seaweed samples
The brown seaweed (Phaeophyta) H. elongata (L.) S.F. Gray (sea spaghetti; Fucales, Himanthaliaceae) and the red seaweed (Rhodophyta) G. pistillata (S.G. Gmelin)
Stackhouse (Gigartinales, Gigartinaceae) from the Spanish Atlantic coasts were obtained from a local supplier (Porto-Muiños, A Coruña, Spain). In the industry, the seaweeds were washed with running water under the tap to remove epiphytes and sand, air-dried at 50 °C and milled to a par-ticle size of <1.0 mm. The milled samples were stored in sealed plastic bags at 4 °C until analysis.
Feeding trials
Two independent feeding trials were performed to study the effects of two different seaweeds (H. elongata and G. pistillata). For this purpose, two seaweed enriched diets and two control diets were prepared. The control diets consisted on a commercial fibre-free AIN-93M diet (Panlab, Barcelona, Spain) enriched with cholesterol, colic acid and fat. Two different control diets were used because the macronutrients were adjusted to be similar to the composition of the added seaweed in each assay. The diets of the treated groups were prepared with the same amounts of cholesterol, colic acid and fat as the control ones. They were also supplemented with 21 % of Himanthalia or 23 % of Gigartina depending on the tested seaweed in each case. Thus, the supplemented diets were enriched with 8 % of dietary fibre, and the control ones were prepared with 8 % of cellulose as the only source of fibre. The formulation of the diets is shown in Table 1.
Wistar Hannover rats weighing approximately 200 g were used in each feeding trial (Himanthalia assay and Gigartina assay). After 6 days of acclimation to the hous-ing conditions, a total of 48 rats were distributed randomly
Table 1 Composition of the experimental diets for the two independent feeding trials (g/kg dry weight)
a Seaweed: H. elongata or G. pistillata in proportion 21 or 23 %, respectively (equivalent to 8 % of dietary fibre)
Himanthalia assay Gigartina assay
Control diet Supplementeda diet Control diet Supplementeda diet
Casein 110.6 110.6 107.8 107.8
Dextrose 122.5 122.5 119.4 119.4
Sucrose 79 79 77 77
Soybean oil 31.6 31.6 30.8 30.8
t-Butylhydroquinone 0.007 0.007 0.007 0.007
Mineral mix 27.6 27.6 27.0 26.9
Vitamin mix 7.9 7.9 7.7 7.7
l-Cysteine 1.4 1.4 1.4 1.4
Choline bitartrate 1.9 2.0 1.9 1.9
Cholesterol 10 10 10 10
Sodium cholate 2 2 2 2
Fat (lard + corn oil) 60 60 60 60
Cellulose 80 – 80 –
Starch 328.4 198.4 320.1 170.1
Seaweeds – 210 – 230
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in two groups of 12 rats (control and treated) for each assay (Himanthalia assay and Gigartina assay). The rats were housed in cages in a room with controlled light (12 h, 08:00–20:00), temperature (22 ± 1 °C) and unrestricted access to food and water. The animals were maintained in accordance with the guidelines from the registered labora-tory (No. 28079-15ABC-M, Madrid, Spain) concerning the care and use of animals [21].
The food intake, body weight and faeces of the rats were recorded weekly. Faeces collected at the beginning and at the end of the experiment were freeze-dried and stored until analysis. The animals were fed for 4 weeks and were anaesthetised with diethyl ether. The whole organs (spleen, kidneys, heart, and liver) were rapidly removed and weighed, and the liver was freeze-dried (Telstar mod. Cry-odos, Terrasa, Spain). The gastrointestinal tract was also removed and measured, and caecum weight was recorded. After clotting the blood at room temperature, it was centri-fuged at 1,500 g for 15 min and the serum was collected for its immediate analysis.
Sampling and processing of caecal fermentation
The content of each caecum was aseptically collected for the determination of pH and short-chain fatty acids (SCFA). A caecal content aliquot was diluted 1:3 in Milli-Q deionised water (Millipore Iberica, Madrid, Spain) imme-diately to measure the pH using a microelectrode Crison micro pH 2001 (Barcelona, Spain). The remaining sample was stored at −20 °C until SCFA analysis. After thawing, samples were centrifuged at 7,000g for 15 min at 4 °C, and the supernatants utilised for gas–liquid chromatography, using 4-methyl valeric acid as the internal standard. The prepared samples were injected into a gas–liquid chroma-tograph (Perkin Elmer Autosystem, MA, USA) equipped with a flame ionisation detector and a column TRB-FFAP (30 m, 0.53 mm, 1 μm) from Teknokroma (Barcelona, Spain). The carrier gas was nitrogen with a flow rate of 15 ml/min. The temperature of the injector was 170 °C, for the detector was 220 °C and the column temperature was isothermal at 120 °C [2, 3].
Mineral content in feed and biological samples
Feed and faeces samples were incinerated at a tempera-ture that increased linearly to 550 °C for 1 h and then at 550 °C for 20 h in a microwave muffle furnace (Milestone MLS-1200 Pyro, Shelton, CT, USA). The resulting ashes were dissolved in 2 ml of 12 M HCl:14.5 M HNO3 (1:1, v:v) and then diluted to 10 ml with distilled water. Cal-cium and magnesium concentrations in feed and faeces samples were measured using a Perkin Elmer Analyst 200 atomic absorption spectrophotometer (MA, USA),
previously diluted to 0.1 % (w/v) with lanthanum oxide solution [3].
Determination of biochemical parameters in serum, liver and faeces
Glucose, uric acid, albumin, cholesterol and triglycerides from the plasma were analysed in the Autoanalyzer Cobas Integra 400 plus (Roche, Basel, Switzerland). High-density lipoprotein (HDL) was measured after precipitation of the very low-density (VLDL) and low-density (LDL) lipopro-teins with phosphotungstate and magnesium using an enzy-matic method (Spinreact, Girona, Spain). LDL cholesterol was calculated as the difference between total and HDL cholesterol [22].
Total fat was extracted with petroleum ether in a Sox-tec System HT extractor (1,043 Extraction Unit) from the freeze-dried livers and faeces. The residues were dissolved with chloroform/methanol (5:2, v:v), and an aliquot was mixed with 1 % Triton X-100 in chloroform and evaporated to dryness under nitrogen. Total cholesterol and triglycer-ides were determined using specific enzymatic colorimet-ric methods (Spinreact, Girona, Spain). Cholesterol esters were hydrolysed with cholesterol esterase. Both hydrolysed and free cholesterol were oxidised to hydrogen peroxide, which reacts with phenol and 4-aminophenazone, and the resulting colour was measured at 505 nm in a spectropho-tometer Pharmacia LKB-Ultrospec Plus (Uppsala, Swe-den). Free cholesterol was analysed in the same way but without hydrolysis. Triglycerides were determined by a colorimetric reaction with 4-aminoantipyrine, p-chlorophe-nol, catalysed by peroxidase, and the absorbance was meas-ured at 505 nm. Bile acids and total nitrogen were meas-ured in faeces. Bile acids were extracted by shaking with 96 % ethanol for 24 h. After centrifugation at 9,000 g dur-ing 15 min (Hettich Zentrifugen Universal 320, Tuttligen, Germany), an aliquot from the supernatant was analysed by the enzymatic colorimetric method (Materlab, Madrid, Spain), converting the bile acids into the corresponding ketones by 3-α-hydroxysteroid dehydrogenase in presence of NAD. The NADH formed reacts with nitroblue tetrazo-lium, producing a formazan through the catalytic action of diaphorase, and the absorbance was measured at 540 nm. The nitrogen content was analysed based on the Kjedahl method using a Büchi Digestor Unit 425 and Büchi Distil-lation Unit B-316 (Flawil, Switzerland) [22].
Statistical analyses
The statistical study consisted of a one-way analysis of variance (ANOVA) performed using the Statgraphics Cen-turion XVI (Warrenton, VA, USA). Values are expressed as mean ± standard deviation.
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Results
Food intake, body weight and faecal excretion
These diets had no effect on food intake, final weight, growth rate and feeding efficiency (Table 2). There were no significant differences (p < 0.05) in heart, spleen, kid-neys and liver weights between the treated and control rats. A visual inspection of the organs showed no macroscopic alterations in either group of rats.
Both experimental diets increased faecal excretion (p < 0.001), as daily faecal weight was always higher (p < 0.001) for the seaweed-fed groups than for the con-trols throughout the experiment (Fig. 1). Stool production
increased by 34 % for the Himanthalia-fed group and 74 % for Gigartina-fed rats. This was attributed mainly to higher (p < 0.001) faecal moisture in the seaweed-fed groups (Himanthalia 69.9 ± 5.1 g/100 g; Gigartina 79.1 ± 2.3 g/100 g) than in the controls (Himanthalia 44.8 ± 2.4 g/100 g; Gigartina 45.4 ± 1.7 g/100 g).
In vivo caecal fermentation of seaweed-supplemented diets
A possible trophic effect (p < 0.05) was observed in the large intestine of rats fed with Himanthalia and Gigartina. Moreover, the small intestine (p < 0.05) and the whole gas-trointestinal tract (p < 0.001) were longer in Gigartina-fed rats than in the control group (Table 3). Caecum weight increased (p < 0.001) in both groups of seaweed-fed rats (Table 3) and was 37 % greater for Himanthalia and 59 % for Gigartina.
Total SCFA of caecal contents (Table 3) were higher (p < 0.001) in Himanthalia-fed rats than in the control; however, the Gigartina diet did not produce any sig-nificant increase in total SCFA. The levels of acetate, propionate and butyrate were higher (p < 0.001) in the Himanthalia-fed group; however, in Gigartina-fed rats, only propionate increased (p < 0.001), while butyrate decreased (p < 0.001) compared to the control. There was no change in pH in the Himanthalia group, whereas a sig-nificant increase (p < 0.001) was observed in Gigartina-fed rats.
Mineral balance
The apparent absorption (AA) and true retention (TR) of Ca was higher (p < 0.01 and p < 0.001, respectively) for rats consuming the Himanthalia-supplemented diet. The mineral balance (MB) did not show any significant differ-ences between both groups. In the balance of Mg, the AA and TR were greater (p < 0.05), as was the MB (p < 0.001), in Himanthalia-fed rats (Table 4). In the case of Gigartina, no difference was observed between the control and treated groups in any of the parameters calculated for both ele-ments (Table 4).
Table 2 Effect of diets supplemented with seaweeds on body and feeding efficiency
Fig. 1 Faecal excretion (g of fresh weigh faeces per day) during 4 weeks of assays (a Himanthalia assay, b Gigartina assay). CD con-trol diet, HD Himanthalia diet, GD Gigartina diet. Values are pre-sented as mean ± standard deviation. ANOVA: Points marked with asterisks differ significantly (*p < 0.05, **p < 0.01, ***p < 0.001)
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Biochemical parameters in serum, liver and faeces
Himanthalia-treated rats presented a reduction (p < 0.001) in triglycerides (28 %) and greater (p < 0.01) contents of HDL-C (20 %) in the sera. The Gigartina-supplemented diet produced a significant decrease of 31 % in triglycer-ides (TGL) (p < 0.001), 18 % in TC (p < 0.01) and 16 % in
LDL-C (p < 0.05) in comparison with the control (Table 5). The results on the liver (Table 5) revealed that the addition of seaweeds to the diet did not affect the concentrations of total lipids. Free cholesterol was significantly reduced (p < 0.05) in the rats fed with both seaweeds. A major (p < 0.001) reduction in TGL (33 %) was found in Gigar-tina-fed rats than in Himanthalia-treated group (12 %)
Table 3 Effect of diets supplemented with seaweeds on intestinal parameters and SCFAs
Gastrointestinal (GI) values are expressed as mean ± standard deviation
ANOVA: values marked with asterisks differ significantly (* p < 0.05, ** p < 0.01, *** p < 0.001)
Traces of isovalerate and isobutyrate were detected in Gigartina-fed rats (<1 % of the total SCFAs value)
(p < 0.05). In the case of faeces (Table 5), the total content of protein and bile acids increased significantly (p < 0.001) in Himanthalia-fed rats (31 and 46 %, respectively); how-ever, there were not significant differences in Gigartina-fed group.
Discussion
The experimental results indicate that seaweed supple-mentation did not affect feeding efficiency or growth rate. Gomez-Ordoñez et al. [2], Amano et al. [18] and Bocanegra et al. [19] found—as in this study—that weight gain was similar between seaweed-fed rats and controls. The bulking effect can be attributed to the significant amount of water retained in faeces due to the high water-holding capacity of these seaweeds as has been described by Gomez-Ordoñez et al. [8], Jiménez-Escrig et al. [3], Gomez-Ordoñez et al. [2] and Gudiel-Urbano and Goñi [23] found similar bulk-ing results in the seaweed-fed rats. The significant trophic effect in the large intestine and the high caecum weight in Himanthalia-fed rats may be due to seaweed fermentable carbohydrates, as they are associated with an increase in mucosal cell proliferation due to increased bacterial metab-olism or a direct stimulatory effect of SCFA [24]. However, the elongated whole gastrointestinal tract in Gigartina-fed rats cannot be explained by possible fermentation, as
there was no significant increase in SCFA production. This suggests that the enlargement could be a simple adaptive response to the tendency of residual material to accumu-late within caecum as proposed before by Wyatt et al. [25]. In fact, the trophic effect found was more intense in the Gigartina assay and could be due to the greater presence of non-fermentable carbohydrates that were collected in the caecum.
There are several factors in the gut environment such as local pH conditions, available dietary substrate, oxygen and hydrogen and gut transit time that can influence the compo-sition of colonic microbiota [26]. The significant produc-tion of SCFA (Table 3) and the notable amounts of nitrogen excreted in the faeces (Table 5) attributed to the increase in faecal bacterial nitrogen [27] could be due to a proliferation of colonic microbiota in Himanthalia-fed rats. SCFA may be responsible for a reduction in intraluminal pH directly or by increasing bacteria that may lead to that decrease [28]. However, there was no change in the pH of the Him-anthalia group despite the increase in SCFA. This could be due to the characteristics of the gut environment described above and to the existence of a microclimate at the epithe-lial surface, which stabilises a neutral pH via bicarbonate secretion [29]. In the case of the Gigartina assay, SCFA were not significant between the control and treated rats; however, the pH was slightly higher in the treated group. Gomez-Ordoñez et al. [2] similarly reported an increase
Table 5 Effect of diets supplemented with seaweeds on serum, liver and faecal parameters
Values are expressed as mean ± standard deviation
TGL triglycerides, TC total cholesterol, FC free cholesterol, EC esterified cholesterol
ANOVA: values marked with asterisks differ significantly (* p < 0.05, ** p < 0.01, *** p < 0.001)
in pH in the rats treated with the red seaweed M. stellatus. It is known that pH may be modified by constituents other than fermentable polysaccharides that would change the metabolism of the caecal bacteria, i.e. resistant protein can increase pH and improve the productivity of total SCFA and caecal fermentability in healthy rats [30]. However, the traces (<1 %) of the branched chain fatty acids (valerate, isovalerate and isobutyrate) found only in Gigartina-treated rats indicate that amino acids were hardly fermented [31], and therefore, the increased pH may be the result of the other dietary components of these seaweeds such as pig-ments, polyphenols, etc., that are strongly related to dietary fibre. The production of SCFA did not involve any acidi-fication of the luminal content for the Himanthalia-fed group; thus, the apparent absorption and true retention of Ca and Mg was stimulated by SCFA directly. Trinidad et al. [32] reported the contribution of acetic acid to the absorp-tion of Ca in the form of calcium acetate, and Kashimura et al. [33] showed that butyric acid is the most effective SCFA in Mg absorption. On the other hand, the enlarge-ment of the large intestine found in the Himanthalia-fed group may result in a more effective absorption of Ca and Mg, as the hypertrophy of the caecal wall provides an increased surface area for the assimilation of minerals [34]. These data agree with those of Jiménez-Escrig et al. [3] obtained in Wistar rats consuming the brown seaweed Saccharina latissima. The mineral balance of Gigartina-fed rats was unaffected, and there were no significant dif-ferences in the apparent absorption and true retention of Ca and Mg. The findings are consistent with those of Bocane-gra et al. [35] in the red seaweed Nori. Gigartina-treated rats showed a diminished butyric level and non-affected acetic content, suggesting that different types and amounts of dietary fibre could induce different fermentative pat-terns, and—as a consequence—a different behaviour in the mineral bioavailability. Alginates are less degraded than laminarans; however, fucoidans and carrageenans are not fermented [36] probably due to the high sulphur content and could thereby reduce the recovery of divalent cations in the colon.
The hypercholesterolaemic rats fed with Himanthalia appeared to have lower TGL and higher HDL-C levels than non-treated ones. Jiménez-Escrig et al. [3] found only lower levels of TGL in normocholesterolemic rats fed with S. latissima. Additionally, the lower level of free cholesterol (FC) in the liver and the increase in the excre-tion of faecal bile acids in the Himanthalia-treated group could indicate the removal of cholesterol as bile salts, highlighting the potential cholesterol-lowering effect of the dietary fibre previous reported [22, 37–39]. Himan-thalia diets produced an increase in SCFA that may inhibit hepatic cholesterol biosynthesis [40] However, Carr et al. [41] observed reduced liver and plasma cholesterol levels
in hamsters and rats fed with the non-fermentable fibre hydroxypropyl methylcellulose and reported that ferment-ability may not be required to lower cholesterol. In fact, the Gigartina diet diminished the levels of TGL, LDL-C and total cholesterol (TC) in serum and TGL in liver with-out producing a greater total amount of SCFA. The sea-weed diets tested appear to present positive hypocholes-terolemic effects in 4 weeks, although better results could possibly be achieved with an intensive treatment, as the trends indicate.
In conclusion, both seaweed diets appear to maintain feeding efficiency and increase faecal excretion and the softening of the faeces, thereby facilitating their evacu-ation. Himanthalia intake may increase SCFA produc-tion and the absorption and retention of Ca and Mg and improve the lipid profile due to an increase in HDL-C and a decrease in the hepatic free cholesterol through excretion of bile acids. The Gigartina diet did not appear to be fer-mented but decreased TGL, TC and LDL-C, improving the lipid serum profile. It is worth highlighting the amelioration of the lipid profile due to the intake of the seaweeds in the study from the point of view of their potential use as an ingredient, or on their own, in the development of diet food for hypercholesterolaemic individuals.
Acknowledgments This research work was supported by the Span-ish Ministerio de Ciencia e Innovación, through Project AGL2008-00998 ALI. Thanks are given to the algal supplier Porto-Muiños (Coruña, Spain).
Conflict of interest None.
Compliance with Ethics Requirements This article does not con-tain any studies with human or animal subjects.
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