Hypocholesterolemic Effects of Nutraceuticals Produced from the Red Microalga Porphyridium sp in Rats

Red microalgae contain functional sulfated polysaccharides (containing dietary fibers), polyunsaturated fatty acids, zeaxanthin, vitamins, minerals, and proteins. Studies in rat models support the therapeutic properties of algal biomass and isolated polysaccharides. Algal products incorporated into rat diets were found to significantly improve total serum cholesterol, serum triglycerides, hepatic cholesterol levels, HDL/LDL ratios and increased fecal excretion of neutral sterols and bile acids. Morphological and metabolic changes were induced by consumption of algal products. These results suggest that red microalgae can be used as potent hypocholesterolemic agents, and they support the potential use of red microalgae as novel nutraceuticals.

In the present study, we investigated the hypocholesterolaemic effects of algal biomass and of the isolated algal polysaccharide (AP) of Porphyridium sp. in hypercholesterolemic rats and sought explanations for the observed mechanisms of action of these effects.

Materials and Methods
Algae and Culture Conditions. The unicellular red alga Porphyridium sp. (UTEX 637) was obtained from the culture collection of the University of Texas, Austin, TX, USA. The algae were cultivated outdoors for 21 days in artificial seawater [27] in polyethylene sleeves [28].
Biomass Production. Algal cultures were grown for 14 days. The entire culture was centrifuged at 17,000 × g for 20 min (Cepa Z-41, Carl Parberg, Lahr/Schwarzwald, Germany) and the pellet collected. In order to remove salts, the pellet was washed (deionized water, pH 4). This procedure was repeated and the pellet obtained was then lyophilized and stored in a desiccator until the start of the experiment (up to 3 months). The dry algal cells were designated biomass.
Algal Polysaccharide (AP) Preparation. The growth medium was centrifuged and the transparent supernatant containing soluble polysaccharides was collected. This was followed by cross flow filtration (stainless steel equipment, Bio Nes Ltd; filters, A/G Company Ltd) to remove salts. The concentrated algal polysaccharide was frozen (−20 °C), lyophilized and stored in a desiccator until the start of the experiment (up to three months).
Dietary Fiber Sources and Analyses. Four sources of dietary fiber were used: (1) cellulose (Solka-folc, James River Corp., Hackensack, NJ, USA); (2) pure citrus pectin (methoxy concentration 10 g·100 g -1 ; Sigma, St. Louis, MO, USA); (3) Porphyridium sp. cells (biomass); and (4) algal polysaccharide (AP) of Porphyridium sp. (prepared as described above). Table 1 provides analyses of the dietary fiber sources. Carbohydrate analysis was performed using the phenol-sulfuric method [29]. Fat content was measured by the Kates method [30] and protein content was determined by the Lowry method [31]. Ash content was measured in oven dried samples (70 °C, 24 h). Samples were cooled in a desiccator and weighed. The dried sample was then burned at 600 °C for 24 h. The remaining ash was cooled in a desiccator and weighed. Dietary fiber analysis was carried out by the AOAC standard method [32].
Animals. Male Sprague-Dawley rats (Harlan, Jerusalem, Israel), each weighing 130-140 g, were housed in individual stainless-steel cages suspended in a controlled environment (22-24 °C and 12 h light/dark), with free access to food and water. Animals were kept according to the guidelines set forth by the Animal Care Committee of Ben-Gurion University of the Negev.
Diets. The composition and dietary fiber content of the four different cholesterol-rich diets fed to the rats is presented in Tables 1 and 2. The algal biomass contained 27% insoluble and 8.5% soluble dietary fibers on a dry weight basis while the AP comprised 37% soluble and 8% insoluble dietary fibers. Experimental Procedures. Twenty-four rats were divided into four groups such that the mean weights of each group were similar. Each group was fed one of the four cholesterol-rich diets ( Table 2) for a 15-day period. Every two to three days the animals and feces were weighed, and food intake was assessed. On each of the last four days of the experiment, feces were collected, weighed and frozen at −20 °C. The samples were then dried by lyophilisation and stored in a desiccator for later analysis. On day 14 of the experiment, food was withheld from the rats overnight (12-14 h), and the following morning animals were anesthetized with chloral hydrate 100 g·L -1 . Blood samples were taken from the heart and placed in tubes with heparin. Samples were then centrifuged for 10 min at 1,000 × g, and plasma was frozen at −20 °C until analysis. The livers were removed, weighed and stored at −20 °C for further analysis.
Determination of Plasma Lipids. Total plasma cholesterol and lipoprotein contents were measured by enzymatic colorimetric methods [33,34]. Plasma triglyceride (TG) levels were determined by the method of Fossati and Prencipe [35].
Fecal and Hepatic Neutral Sterol Analysis. Neutral sterols were determined by the method of Searcy and Bergquest [36], with the following modifications. Dried fecal or liver samples (50 mg) were saponified with 3 mL of 10% ethanolic KOH for 1 h at 65 °C and then cooled to room temperature. Neutral lipids were extracted as follows: 3 mL of water and 5 mL of petroleum ether (b.p. 40-60 °C) were added to each sample, the contents were mixed for 30 s, and the solvent was removed. The second and third extractions were carried out with 5 mL and 3 mL, respectively, of petroleum ether, and the three extracts were pooled for each sample. The samples were then dried under nitrogen, and 3 mL of acetic acid saturated with FeSO 4 and 1 ml H 2 SO 4 were added. Absorbance was read at 490 nm to determine steroid concentrations. A standard curve was prepared with increasing concentrations of cholesterol.
Fecal Bile Acid Analysis. Dried fecal matter (100 mg) was extracted overnight with 10 mL of chloroform-methanol [37]. Two milliliters of KCl, 3.7 g·L -1 , were then added, and the sample was centrifuged at 1,500 × g for 10 min. The upper layer was evaporated to dryness, and the residue was then dissolved in 1 ml of 50% methanol. Total bile acids were determined by enzymatic analysis using a modification of the method of Sheltawy and Losowsky [38], as follows: to 100 μL of sample containing 2 mmol·L −1 NAD, in phosphate buffer, pH 10.5, were added 3α-hydroxysteroid dehydrogenase (0.1 IU) in a final volume of 1.5 mL. After 30 min at room temperature, the absorbance was read at 340 nm to determine the NADH formed.
Statistical Analysis. Data were expressed as means and standard error. Group means were compared by one-way analysis of variance (ANOVA). Means were considered significantly different at P ≤ 0.05, as determined by Fisher's projected least significant difference method.

Results
Body Weight Gain. Despite similar food intake levels of 6-8 grams per day per rat, significantly lower weight gain (p < 0.03) was observed in AP fed rats (81.3 g) than in the rats fed the control, biomass or pectin diets (97.6 g, 89.1 g, and 96.6 g, respectively, Figure 1). Plasma Lipids and Lipoproteins. Plasma parameters are presented in Table 3. Cholesterol levels were significantly lower (22-34%; p < 0.001) in rats fed the biomass or AP diets than in control or pectinfed rats. Similarly, plasma TG and very-low-density lipoprotein (VLDL-C) levels were also lower (by 12-39%) in animals fed AP or biomass. Low-density lipoprotein (LDL) levels were significantly decreased (by 32-53%; p < 0.005) in the polysaccharide-fed rats when compared to rats fed the pectin or control diets. The ratio of high-to low-density lipoproteins (HDL/LDL) was higher (by 31-60%; p < 0.001) in the polysaccharide and biomass groups vs. the pectin and control diets. Fecal Dry Weight. Fecal dry weight was greater in the polysaccharide-fed rats (3.17 g·day −1 ; p < 0.0001) than in the pectin and control groups (0.93 and 1.07 g·day −1 , respectively). Fecal dry weight was also higher (2.46 g·day −1 ) in the biomass-fed animals ( Table 4).

Fecal Excretion of Neutral Sterols and Bile
Acid. The amount of neutral sterols in the feces was dramatically enhanced in rats fed algal polysaccharide (1.93 mg·day −1 ; p < 0.02) in comparison to the other three treatments (1.19, 0.82 and 0.59 mg·day -1 for the biomass, pectin and control groups, respectively) ( Table 4). The fecal bile acid content was lowest in the polysaccharide-fed animals (0.33 mg·day −1 ), higher in the pectin and control groups (0.59 mg·day −1 and 0.61 mg·day −1 , respectively) and significantly higher (p < 0.0001) in the biomass group (1.31 mg·day −1 ). Bile acid excretion was more than twofold higher in the biomass-fed rats than in the control animals.  Table 5). The lowest liver weight was found in the polysaccharide-fed rats (35.39 mg·g body weight -1 ). Hepatic cholesterol was significantly higher in the control rats (3.89 mg·g liver −1 ; p < 0.0001) than in the polysaccharide-, pectin-and biomass-fed rats (1.89, 1.81 and 2.14 mg·g liver −1 , respectively).

Discussion
This study examined the therapeutic effects of biomass or algal polysaccharides from the red microalga Porphyridium sp. on the lipid metabolism of rats fed diets rich in cholesterol. A significant hypocholesterolemic effect was observed accompanied by a decrease in the accumulation of hepatic cholesterol (Table 5) and by lowered plasma TG and VLDL-C levels ( Table 3). These findings are in keeping with a number of recent studies that document the cholesterol-and lipid-lowering ability of algae [18,19,39,40] and that demonstrate the potential health benefits of incorporating algae or algal byproducts into the diet. Indeed, in Asia, seaweeds are traditionally consumed as sea vegetables, but in Western countries they are used mainly as gelling or thickening agents and not as part of the daily diet [11].
Soluble dietary fibers, such as pectin, are known to have hypocholesterolemic effects and are therefore considered important factors in reducing the risk of coronary heart disease [41]. In this study, algal products were as effective as, or more effective than, pectin in lowering plasma cholesterol levels and preventing the accumulation of cholesterol in the liver. Numerous mechanisms have been proposed to account for the cholesterol-lowering action of soluble dietary fibers. One possible explanation is that they increase the viscosity of the intestinal contents, thereby interfering with nutrient absorption and micelle formation, which, in turn, decreases lipid absorption from the intestine [42]. It has also been suggested that soluble fibers act by disrupting the enterohepatic circulation of bile acids, leading to increased bile acid excretion and a subsequent decrease in plasma cholesterol levels [43,44].
Liver weight and hepatic cholesterol accumulation was significantly lower in animals that consumed pectin, AP or biomass. This is of physiological importance as fatty liver is a risk factor associated with the development of chronic liver disease [45]. Natural compounds that have the ability to prevent accumulation of fats in the liver may be used therapeutically as nutraceuticals.
In this study, consumption of AP resulted in a significant increase in fecal excretion of neutral sterols, while consumption of biomass had a marked effect on fecal excretion of bile acids and a lower but also significant impact on neutral sterol excretion (Table 4). Pectin consumption had a much lower effect on these parameters. Therefore, it is likely that both the biomass and the AG affect lipid metabolism at the level of absorption from the digestive tract. Although both biomass and AP significantly increased fecal weight and possibly interfered with micelle formation, their mechanisms of action may be quite different, due to their unique physicochemical properties (viscosity, solubility, electrical charge, etc.) [22,23,[46][47][48] and to the fact that biomass contains primarily insoluble dietary fibers, whereas the majority of fibers in the AP are water soluble. The AP appeared to be more effective in enhancing the secretion of neutral sterols, while the biomass interfered with the enterohepatic circulation of bile acids. Consumption of both biomass and AP resulted in a depletion of total cholesterol pools, which was reflected in lowered hepatic and plasma cholesterol levels. These results corroborate the results of previous studies from our laboratory [3]. In earlier studies, rats fed algal biomass had elevated plasma cholecystokinin (CCK) levels. This may indicate an increased need for bile acid secretion via the gall bladder in order to enhance cholesterol absorption from the digestive tract to compensate for the hypocholesterolemic effect of the algal product. Similarly, HMG-CoA reductase, a key enzyme in endogenous cholesterol production, was found at increased levels in animals fed soluble fibers to lower cholesterol [49]. Feeding AP at levels of 5% or 10% to hypercholesterolemic rats enhanced HMG-CoA reductase in comparison to control animals (unpublished data).
It is known that fiber-rich diets can inhibit weight gain in rats [50,51]. In our study, the lowest weight gain and a high dry fecal weight (almost three times higher than that of control animals) were observed in AG-fed rats. These findings may be due to the high viscosity of the intestinal contents following food consumption. Indeed, we have previously shown that the viscosity of the cecal contents of AP-fed rats was much higher than that of control animals or pectin-fed rats [3]. Highly viscous chyme may impede nutrient absorption (including cholesterol absorption) along the length of the intestines, leading to lower weight gain [51]. Biomass feeding did not affect weight gain. Similarly, in chickens, the addition of 5% or 10% biomass to the diet did not influence weight gain [26].
In the current study, AP (rich in soluble-fiber) significantly reduced plasma TGs and VLDL-Cs. These findings are in keeping with the generally accepted perception that plasma TG levels are lowered by the consumption of soluble dietary fibers [52] and with findings that certain sources of insoluble dietary fibers can lower TG levels, even though most insoluble dietary fibers have little or no impact on lipid metabolism [53,54]. It is also possible that the high EPA content of the biomass, in addition to the fiber content, contributed to the trend of lowered TG levels.
Other components present in algae may also play a role in the hypocholesterolemic and triglyceride lowering effects observed in this study. Although little information is available, recent studies suggest that pigments such as C-phycocyanin present in algae are bioactive compounds and may affect lipid metabolism [55].
In conclusion, our work with red microalgae in animal models provides convincing evidence that supports the ability of both biomass and isolated algal polysaccharides (AP) to lower cholesterol levels and positively affect lipid metabolism. Increased fecal bile acid and cholesterol excretion appear to be important mechanisms of action responsible for the overall physiological impact of red microalgae consumption. These results add to the growing body of evidence that the consumption of algal products have health benefits, and as such, algal products can be classified as nutraceuticals.