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The Journal of Nutrition Vol. 128 No. 9 September 1998,
pp. 1512-1516
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The effect of the fruiting body and mycelium of Volvariella volvacea (straw mushroom) on the concentrations of plasma lipids, liver cholesterol, fecal neutral sterol and bile acid excretions was investigated in male Golden Syrian hamsters. The hamsters were fed a purified hypercholesterolemic diet (0.1% cholesterol, 10% fat) for 4 wk to elevate plasma lipid concentrations. Twelve hamsters with elevated plasma total cholesterol were randomly assigned to each treatment group: control (5% cellulose), mushroom fruiting body (5%) and mushroom mycelium (5%). After 4 wk of mushroom diet consumption, the plasma total cholesterol, HDL cholesterol, and combined VLDL + LDL cholesterol concentrations (mmol/L) were significantly lower than control in the group fed the fruiting body-diet (40, 38 and 43%, respectively) (P < 0.05). The liver cholesterol levels were significantly lower in both the mushroom fruiting body- and the mycelium-fed groups (28 and 21% in terms of concentration; 39 and 30% in terms of total content, respectively) (P < 0.05) than that in the control group. Fecal neutral sterol excretion in the mushroom fruiting body- and mycelium-fed groups was significantly higher (81 and 74%, respectively) (P < 0.05) than that in the control group. Although no significant differences (P > 0.05) in the excretion of fecal bile acids were observed among groups fed the mushroom diets and the control diet, the mushroom fruiting body diet-fed hamsters apparently had less bacterial degradation of cholic acid as indicated by a significantly greater proportion (P < 0.05) of fecal cholic acid than in controls. They also had a significantly lower proportion of fecal deoxycholic acid (P < 0.05). This study suggests that the fruiting body of the straw mushroom lowers elevated plasma cholesterol in hypercholesterolemic hamsters, whereas the mycelium does not.
KEY WORDS: Volvariella volvacea · mushroom · hamsters · cholesterol · bile acids
On the basis of recent knowledge, it has been demonstrated that the lowering of circulating cholesterol (especially LDL cholesterol) levels can prevent, arrest and even reverse coronary atherosclerosis (Barter and Rye 1996 Straw mushroom (Volvariella volvacea) is a widely cultivated tropical mushroom in Southeast Asia. It is not only a popular ingredient in traditional Chinese cuisine but also has a Chinese folk history concerning its pharmaceutical effects (Chang and Mao 1995 Several investigators have reported that the plasma total cholesterol concentrations in hamsters respond to hypercholesterolemic diets in a manner similar to that seen in humans fed high cholesterol diets (Nistor et al. 1987 Animal model.
Male Golden Syrian hamsters weighing 100-120 g were obtained from the University Animal House and housed individually in metal cages in a room with controlled temperature (24-25°C) and humidity (40-50%) and a 12-h light:dark cycle. Animals were allowed free access to a commercial rat ration (Rodent Laboratory Chow 5001, Purina Laboratories, St. Louis, MO) and water for 1 wk to allow adaptation to the environment. All experimental protocols complied with NIH guidelines (NRC 1985).
Diet.
At the end of the first week, all hamsters were switched to a purified, hypercholesterolemic diet (control diet) for 4 wk. The control diet was based on the AIN-76A diet (AIN 1980) with the following composition (g/kg diet): cornstarch, 496.9; casein, 200; sucrose, 100; corn oil, 100; cellulose, 50; AIN-76 mineral mix, 35; AIN-76 vitamin mix 10; DL-methionine, 3.0; choline bitartrate, 2.0; cholic acid, 2.0; cholesterol, 1.0; butylated hydroxytoluene, 0.1; and menadione sodium bisulfite complex, 0.001. In the two mushroom diets, the cellulose (50 g/kg) was replaced by an equivalent amount (50 g/kg) of the dried straw mushroom fruiting body powder or mycelium powder. Both the fruiting body and mycelium of straw mushroom were cultivated in our laboratory (Cheung 1996b
Experimental design.
Blood samples were taken from hamsters that were deprived of food overnight before the introduction of the hypercholesterolemic control diet to establish the baseline plasma lipid concentrations. After the hamsters consumed the hypercholesterolemic control diet for 4 wk, blood samples were taken from food-deprived hamsters to determine the new plasma lipid concentrations. Based on these values, the hamsters were ranked by plasma total cholesterol and assigned by randomized block design (Steel and Torrie 1960 Plasma cholesterol analysis.
The blood samples (~2 mL) at each sampling period were drawn from the orbital sinus of food-deprived hamsters (16 h without food) after the administration of the anesthetic ketamine (0.3 mL/animal) (Timm 1979 Liver cholesterol analysis.
At the time of exsanguination, the livers were perfused in situ with saline, removed and immediately frozen in liquid nitrogen. The cholesterol in these liver samples was extracted using the chloroform-methanol (2:1, v/v) extraction procedure of Folch et al. (1957) Fecal samples.
Fecal samples collected over 5 d for each animal were pooled and frozen at Statistical analysis.
Data represent means ± SEM. Differences among treatment group means were determined by one-way ANOVA followed by Tukey's pairwise comparisons test (Ott 1988 Baseline plasma lipid concentrations at wk 0.
The plasma lipid concentrations (mmol/L) in the hamsters at wk 0 were as follows: 3.4 ± 0.2 for total cholesterol, 2.6 ± 0.1 for HDL cholesterol, 0.85 ± 0.3 for combined VLDL and LDL cholesterol and 0.75 ± 0.2 for triacylglycerol.
Elevated plasma lipid concentrations at wk 4.
The plasma lipid concentrations (mmol/L) in the hamsters fed a hypercholesterolemic (control) diet that contained 10% fat and 0.1% cholesterol for 4 wk were as follows: 11.2 ± 0.2 for total cholesterol, 5.7 ± 0.5 for HDL cholesterol, 5.5 ± 0.8 for combined VLDL and LDL cholesterol and 3.3 ± 0.8 for triacylglycerol. This diet had successfully induced an alimentary hypercholesterolemia in all of the hamsters by an elevation in their plasma lipid concentrations that resulted in a 120% to >fivefold increase in individual plasma lipid components.
Food intake and body weight.
No significant differences in food intakes (14 ± 3 g/d) or body weights (initial: 110 ± 8 g; final: 137 ± 11 g) were detected among the three diet groups during the study.
Postfeeding plasma and hepatic lipid concentrations at wk 9.
Although the plasma total cholesterol concentration in the group fed the mushroom fruiting body was 40% lower (P < 0.05) than that in the control group, there were no significant differences between the hamsters fed the mycelium diet and the control group (Table 2). The plasma HDL cholesterol concentration was also significantly lower (P < 0.05) only in the group fed the fruiting body diet (38%) when compared with the control group. The combined plasma VLDL + LDL cholesterol level (Table 2) of the group fed the fruiting body was 43% lower (P < 0.05) than that of the control group, which did not differ from hamsters fed the mycelium diet. There were no significant differences in the plasma triacylglycerol among the three groups (Table 2). Hamsters fed both the fruiting body and mycelium diets had lower (P < 0.05) liver cholesterol concentrations (28 and 21%) and contents (39 and 30%) and liver weights (12 and 10%) compared with the control group (Table 3).
Postfeeding fecal sterols at wk 9.
Fecal neutral sterol excretion was higher (P < 0.05) in the groups fed the fruiting body and mycelium diets (80.7 and 73.6%, respectively) than in the controls (Table 4). In all of the diet groups, the bacterial metabolite, coprostanol, was the major component of fecal neutral sterols (Table 5). The mushroom fruiting body and mycelium diets resulted in lower (P < 0.05) proportions of fecal coprostanol and higher (P < 0.05) proportions of fecal cholesterol. There were no significant differences in fecal total bile acid excretion among the groups (Table 4). However, there was a greater (P < 0.05) percentage of primary bile acids (sum of cholic acid and chenodeoxycholic acid) in hamsters fed the fruiting body diet, which was largely due to a significantly greater proportion (P < 0.05) of cholic acid and a significantly smaller proportion (P < 0.05) of deoxycholic acid (Table 6).
Male Golden Syrian hamsters have been used widely as a model to study atherosclerosis and cholesterol metabolism in response to dietary fat manipulations because they possess many features of cholesterol metabolism similar to those of humans (Jackson et al. 1989
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, LaRosa 1994, Rosenfeld 1989, Stalmer et al. 1986). The search for natural substances capable of lowering blood cholesterol is ongoing in the field of nutrition. Edible mushrooms (fungi) are an ideal food for the dietetic prevention of atherosclerosis due to their high content of fiber, proteins, microelements and their low fat content (Crisan and Sands 1978, Kurasawa et al. 1982). In fact, the inclusion of edible mushrooms in a natural hypocholesterolemic and antisclerotic diet has been used in Oriental medicine (Sun et al. 1984). The hypcholesterolemic effect of a few mushrooms has already been studied using rats (Bobek et al. 1991a, Cheung 1996a, Kaneda and Tokuda 1966).
). Our laboratory has demonstrated that both the fruiting body (common edible form) and the mycelium (vegetative hyphae for cultivation) of straw mushroom exerted certain cholesterol-lowering effects when fed to hypercholesterolemic rats (Cheung and Chan 1995, Cheung and Tsui 1995).
, Sicart et al. 1984 and 1986, Singhal et al. 1983). The use of hamsters in the investigation of the hypocholesterolemic effect of mushroom is rare (Bobek et al. 1991b). Thus, the present study was conducted in hamsters fed diets including the fruiting body and mycelium of the straw mushroom to determine its hypocholesterolemic effect. The influence of the mushroom diets on the plasma and hepatic cholesterol concentrations and fecal neutral sterol and bile acid excretions was examined.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
), harvested, freeze-dried and ground to powder of <1 mm in diameter using a cyclotec mill (Tecator, Hoganas, Sweden). The control and the two experimental mushroom diets were analyzed for total dietary fiber (TDF) (Prosky et al. 1988), 
-glucans (McCleary and Glennie-Holmes 1985), crude protein and fat (AOAC 1990). The results of the proximate analysis of the diets are presented in Table 1.
View this table:
Table 1.
Proximate and dietary fiber analysis of the control and mushroom diets1
) to one of the three experimental diet groups (n = 12 per group): control, mushroom fruiting body and mushroom mycelium. Hamsters were allowed free access to food and water for the duration of the study. Diet consumption and body weight were measured weekly, correcting for food spillage. After 4 wk of consuming the experimental diets (at wk 9), blood samples were collected from food-deprived hamsters before they were exsanguinated under anesthesia (sodium pentobarbital, 50 mg/kg). Fecal samples for neutral sterol and bile acid analyses were collected from each hamster over a 5-d period during the last week of feeding the experimental diets.
). Blood samples were collected in tubes containing EDTA (1 g/L blood) and plasma prepared by centrifugation at 2000 × g for 30 min. Plasma total cholesterol, HDL cholesterol and triacylglycerol were analyzed in duplicate using commercially available enzymatic kits (Sigma Diagnostic Procedure nos. 352, 352-4, and 336, respectively, St Louis, MO). The combined VLDL and LDL cholesterol concentrations were calculated as the difference between the plasma total cholesterol and HDL cholesterol.
. The concentration of the liver total cholesterol was determined colorimetrically using the chemical procedure of Seary and Bergquist (1960).
70°C until analysis. These samples were freeze-dried, ground and then analyzed for neutral sterols and bile acids according to the procedure described by Vahouny et al. (1987). In brief, 0.5-g portions of the fecal samples were acid-digested and the total lipids were extracted using toluene. The extracted lipids were then subjected to enzymatic hydrolysis of the bile acid conjugates with cholylglycine hydrolase (Sigma Chemical). Neutral sterols were extracted with petroleum ether and analyzed as trimethylsilyl ethers by gas-liquid chromatography (GLC) (Hewlett-Packard 6890, Avondale, PA) using 3% OV-17 on 100/120 Supecoport (Supelco, Bellefonte, PA). Bile acids were extracted from the residue with diethyl ether followed by ethyl acetate. The combined extracts were dried and methylated with concentrated HCl and dimethoxypropane. The esterified bile acids were analyzed by GLC using a column containing 3% SP-2100 on 100/120 mesh Supecoport (Supelco). Neutral sterols and bile acids were identified by comparison of relative retention times with standards and quantified using internal standards (5
-cholestane for cholesterol and 23-nordeoxycholic acids for bile acids) corrected for differences in the flame ionization detector responses.
) using the SYSTAT system (SYSTAT, Evanston, IL). An -level of 0.05 was set to determine significance.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Plasma lipid concentrations in hamsters after consumption of the control or mushroom (50 g/kg) diets for 4 wk1,2
View this table:
Table 3.
Liver weight and cholesterol level in hamsters after consumption of the control or mushroom
(50 g/kg) diets for 4 wk1,2
View this table:
Table 4.
Fecal weights, total neutral sterols and total bile acid levels in hamsters after consumption of the control or mushroom
(50 g/kg) diets for 4 wk1,2
View this table:
Table 5.
Composition of fecal neutral sterols of hamsters after consumption of the control or mushroom
(50 g/kg) diets for 4 wk1,2
View this table:
Table 6.
Composition of fecal bile acids of hamsters after consumption of the control or mushroom (50 g/kg)
diets for 4 wk1,2
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Nistor et al. 1987, Spady and Dietschy 1983). In addition, like humans, hamsters have gall bladders and secrete apoprotein B-100 and B-48 separately by the liver and intestine, respectively (Liu et al. 1991).
, Kahlon et al. 1996).
). The involvement of dietary fiber in moderating cholesterol-lowering responses has been well documented. Despite the fact that the control diet had a higher TDF content, which contained mainly cellulose, there was no hypocholesterolemic response in hamsters fed the control diet as expected from an insoluble dietary fiber such as cellulose (Kritchevsky 1988). Moreover, although the TDF content of the fruiting body in V. volvacea. was only half that of the mycelium (Cheung 1996b), only the mushroom fruiting body lowered plasma cholesterol, as discussed above. Previous studies in rats had suggested that mushroom -glucans are effective cholesterol-lowering polysaccharides (Cheung 1996a and 1996c). It has been hypothesized that upon ingestion, soluble dietary fibers such as -glucans increase small intestinal viscosity, resulting in reduced bile acid and cholesterol or triglyceride absorption and/or reabsorption, thus lowering plasma cholesterol (Chen and Anderson 1986). In this study, the soluble dietary fiber might have been fermented in the forestomach of the hamsters (the extent of which had not been measured), diminishing its role in the cholesterol-lowering response. Nevertheless, it had been reported that isolated mushroom -glucans from Pleurotus ostreatus lowered the serum cholesterol concentration in hamsters (Bobek et al. 1991b). Moreover, mushroom phytochemicals such as eritadenine, found in the fruiting body of shiitake mushroom (Lentinus edodes), have been reported to be hypocholesterolemic agents (Sugiyama et al. 1995). It is therefore possible that the straw mushroom may also contain unknown hypolipemic substance(s).
). Moreover, the soluble dietary fiber contained in the mushroom diets consumed by the hamsters could be fermented in the pregastric pouch of the animal, producing volatile fatty acids (Gallaher et al. 1993). It has been hypothesized that the rate-limiting enzyme in the hepatic cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-CoA reductase, could be inhibited by these volatile fatty acids (Chen et al. 1984), leading to a reduction in the liver cholesterol synthesis that further lowers the hepatic cholesterol levels. Furthermore, chitin, which is present in the straw mushroom, has been found to reduce liver cholesterol in rats (Zacour et al. 1992).
). In this study, a significantly greater (P < 0.05) excretion of fecal neutral sterols was caused by the two mushroom diets (Table 4). Coprostanol is a metabolite of cholesterol, formed by the action of gut microflora, and its presence may indicate fermentation activity in the large intestine. The proportions of coprostanol (as % of neutral sterols) were significantly (P < 0.05) lower in the feces of the hamsters fed the two mushroom diets than in those of the control group (Table 5). These findings suggest that there was more fermentation in the large intestine of the hamsters fed the two mushroom diets. As fermentation increased, the drop in colonic pH suppressed the 7-hydroxylase activity, resulting in less conversion of primary bile acids (mainly cholic acid) to secondary bile acids (deoxycholic acid) (Vahouny et al. 1987). However, the present results indicated that only the hamsters fed the mushroom fruiting body diet had significantly (P < 0.05) greater proportions of cholic acid and smaller proportions of deoxycholic acid (Table 6). Furthermore, the significantly (P < 0.05) greater fecal cholesterol proportions (as % of neutral sterols) found in hamsters fed the two mushroom diets (Table 5) indicated that there was a greater excretion of fecal cholesterol, which may explain in part the hypocholesterolemic effect of the mushroom. It seems likely, though not proven in these experiments, that some mushroom soluble fiber (-glucans) might have escaped fermentation in the gastric pouch of the hamsters and increased the small intestinal viscosity, resulting in reduced cholesterol absorption (Chen and Anderson 1986).
). Although the experimental and control diets contained the same amount of cholic acid (2.0 g/kg), the percentage of total bile acids in the feces of hamsters fed the mushroom fruiting body diet was significantly (P < 0.05) higher than that of hamsters fed either the mycelium or the control diet (Table 6). The enhanced cholic acid proportion in the fecal total bile acids may indicate that cholic acid binding to components of the mushroom fruiting body is relatively stronger than the mycelium. The fecal proportion of the secondary bile acid deoxycholic acid in the fecal total bile acids was lower for hamsters fed the mushroom fruiting body diet than for those fed either the mycelium or the control diet, which might indicate that there were fewer primary bile acids available for absorption in the fruiting body diet and a reduction in the production of secondary bile acid due to less 7-hydroxylase activity mentioned earlier.
, Cheung and Tsui 1995), these results suggest that only the fruiting body of straw mushroom lowers elevated plasma cholesterol levels in hypercholesterolemic hamsters, whereas the mycelium does not. Differences in cholesterol-lowering capacity of the two mushroom diets in this experiment could not be related to the fecal excretion of neutral sterols or total bile acids. However, there was evidence that the feces of hamsters fed the diet containing the mushroom fruiting body contained a significantly (P < 0.05) higher proportions of primary bile acid (cholic acid) with a concomitant reduction in the proportions of secondary bile acids. These studies together with our previous results (Cheung and Chan 1995, Cheung and Tsui 1995) suggest that a combination of mechanisms is involved in the cholesterol-lowering effect of straw mushroom. The relative importance of individual putative cholesterol-lowering components of straw mushroom such as polysaccharides and proteins was not evaluated in this study and should be investigated further.
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FOOTNOTES |
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Manuscript received 8 September 1997. Initial reviews completed 3 November 1997. Revision accepted 19 May 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-glucans in barley and malts.
J. Inst. Brew.
1985;
91:285-295This article has been cited by other articles:
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  M. Fukushima, T. Ohashi, Y. Fujiwara, K. Sonoyama, and M. Nakano Cholesterol-Lowering Effects of Maitake (Grifola frondosa) Fiber, Shiitake (Lentinus edodes) Fiber, and Enokitake (Flammulina velutipes) Fiber in Rats Experimental Biology and Medicine, September 1, 2001; 226(8): 758 - 765. [Abstract] [Full Text] [PDF]
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