Pleurotus florida Aqueous Extracts and Powder Influence Lipid Profile and Suppress Weight Gain in Rats Fed High Cholesterol Diet

Pleurotus florida mushroom is a nutritious food with therapeutic potential. This study investigates the effects of P. florida aqueous extracts and powder on serum lipid profile and weight gain in rats fed high cholesterol (HC) diets. Twenty five male albino rats were partitioned into five groups (n = 5): BD: basal diet; HC: High Cholesterol diet; HCPFP5: HC plus 5% P. florida powder; HC-PFE5: HC plus 5% P. florida powder extract; HC-PFE7.5: HC plus 7.5% P. florida powder extract. Animals had free access to diets and water for 4 weeks. Results showed that weight gain was significantly (P < 0.05) suppressed in rats fed high cholesterol diet supplemented with P. florida powder or its extracts, compared to rats fed high cholesterol diet alone. Additionally, supplementation with P. florida increased fecal lipid excretion, while serum triglyceride, LDL, VLDL and total cholesterol decreased. HDL-C increased and LDL-C/HDL-C ratio decreased in rats fed P. florida. These results show that P. florida extracts like powders possess antihypercholesterolemic effects and prevent weight gain, thus reducing the risk for cardiovascular diseases. Mechanisms for these effects are suggested.


Introduction
Cardiovascular diseases (CVDs) are the leading cause of death, accounting for 31% of deaths worldwide [1]. In 2008, more than 17 million deaths were attributed to CVDs alone, with more of these deaths occurring in low income countries [1]. Premature deaths attributed to CVDs ranged from 4% in high income countries to 42% in low income countries [1]. Hypercholesterolemia is a major risk factor associated with CVDs, such as atherosclerosis and its related complications [1][2][3][4]. Keeping blood serum cholesterol levels at desirable low levels therefore is one of the major preventive strategies for these diseases. In this regard, the search for natural substances (functional foods, nutraceuticals), with antihypercholesterolemic effects is desirable; as they provide a safe and natural means to combat the rising incidence of hypercholesterolemia and cardiovascular diseases especially noticeable in low income countries.
Edible mushrooms have for long been appreciated for their flavor and texture. They are recognized as nutritious foods as well as an important source of biologically active compounds of medicinal value [5][6][7]. They are rich in fiber, protein and micronutrients, and low in caloric value [8,9], making them a natural food for the prevention of cardiovascular diseases as first suggested by Traditional Chinese Medicine [10,11]. To this effect, the cholesterol lowering properties of some edible mushrooms have been reported [12][13][14].
The oyster mushroom, Pleurotus species, is a highly nutritious, edible mushroom and a common species in tropical West Africa and Southern parts of Asia [15]. They are the main species of mushroom cultivated in Cameroon (P. ostreatus, P. pulmonarius, P. florida and P. sajou-cajou) [16], where they are mostly consumed fresh after cooking, or dried and used in the preparation of stews and soups as substitute for fish or meat [17]. Oyster mushrooms are interesting as they have demonstrated immunoregulatory [18], antioxidant [5][6][7]19], and antiinflammatory [19,20] properties. These beneficial effects have been attributed to their water soluble polysaccharide component (βglucans), as well as their phytochemical composition. In particular, the antihypercholesterolemic properties of water soluble and ethanol extracts of P. ostreatus [21] as well as 5% powder incorporations of P. ostreatus [22] and P. ferulae [13] have been demonstrated in animals. With respect to P. florida, the hypocholesterolemic properties of its powder has been demonstrated in rats fed cholesterol enriched diets [23]. It's hot water extracts on the other hand have been shown to possess antioxidant effects [5,19,24]. The beneficial effect of antioxidants in regulating lipid metabolism in hypercholesterolemic rats has been reported [25]. These studies suggest that the oyster mushrooms P. florida, like others of the Pleurotus family can be an important food in the management of hypercholesterolemia. Given that the powder form of P. florida, and extracts of other species, has been shown to possess hypocholesterolemic effects, this study set out to investigate the antihypercholesterolemic properties of water extracts of P. florida in comparison with the powder. With increasing emphasis on functional and convenience foods, and the increased production and consumption of P. florida mushroom and mushroom juice in Cameroon and beyond, information on the antihypercholesterolemic property of P. florida mushroom and its juice, is necessary to promote its consumption as a health food. This study therefore had as objective to determine and compare the antihypercholesterolemic effect of aqueous extracts of P. florida mushroom with the powder form, in experimental animals, through analyses of weight gain and serum lipid profile.

Materials
The mushroom (Pleurotus florida) used in this study was purchased from a mushroom production center in Akak, at the outskirts of Yaoundé, Center Region-Cameroon, and transported to the Food Biochemistry and Biophysics laboratory of the University of Ngaoundere.

Preparation of Pleurotus florida powder and aqueous extract
The mushroom samples were sorted to remove spoils after which they were washed twice and rinsed with distilled water, sliced and allowed to drain for 30 minutes. Samples were then dried in an electric dryer (Riviera & Bar QD105A, Paris, France) at 50°C for 24 hr. The dried samples were ground into powder (1 mm) using an electric grinder (Cullati Polymix, France). The powder obtained was stored under refrigeration at 4°C until needed for analysis. Given that previous studies had shown hypocholesterolemic effects of 5% P. florida mushroom powder supplementation in rat models [23], aqueous extracts were prepared using 5 g and 7.5 g mushroom powder, and the extracts incorporated into feed to correspond to 5% and 7.5% levels of powder incorporation. Extraction conditions were as previously determined in an optimization study to maximize the extraction of soluble fibers [26]. Powders samples were extracted in distilled water at 100°C for 30 min using a 1/10 solute / solvent ratio, and thereafter centrifuged for 10 min at 2500 g.

Determination of soluble fiber
Soluble fiber content in the powder and extracts was determined using a gravimetric method following the procedure described by [27]. The Mushroom powder was first extracted by mixing with distilled water 1:10 (w/v) and the mixture boiled for 1.5 hr to extract soluble fiber. After cooling, the mixture was centrifuged at 2500 g for 10 min. The supernatant was decanted and saved, and the residue extracted twice more in boiling water for 30 min. The supernatants were pooled and analyzed for soluble fiber. Extracts from 5 and 7.5 g mushroom powder were analyzed directly for their soluble fiber content without any further extraction. For each of the extracts, a 5 ml aliquot was slowly added to three volumes of 96% ethanol and stored at 4°C overnight to precipitate the soluble fiber fraction. The samples were centrifuged anew at 2500 g for 10 min and the supernatant discarded. The precipitates obtained were washed with a mixture of water and 96% ethanol (1:3) and centrifuged as previously indicated. The precipitates were then re-suspended in distilled water to dissolve completely. Proteins were removed by adding an equal volume of 10% trichloroacetic acid and allowed to stand for 2 hr. The protein precipitates were removed by centrifugation at 2500 g for 20 min. The supernatant was collected and slowly added to three volumes of 96% ethanol and stored at 4°C overnight to precipitate the soluble fiber. The solution was centrifuged at 2500 g for 10 min, and the supernatant discarded. The precipitate was then dried at 100°C for 30 min and the soluble fiber content expressed as (mg/g).

Total polyphenols
Total polyphenols were determined according to [28] with some modification. To 0.5 g of mushroom powder, 10 ml ethanol (70%) was added and the mixture stirred for 2 hr using a Prolabo 54 433 agitator, Paris, France at 220 rpm, to extract total polyphenols. After centrifuging (DL 6000 mark, rotor 15 cm, Japan) at 3000 g for 20 min, 20 μl of the supernatant was mixed with 0.2 ml of Folin-Ciocalteu reagent diluted (1/16), 0.4 ml of sodium carbonate (20%) and 1.38 ml distilled water. The mixture was vortexed and incubated in a water bath at 40°C for 20 minutes in the dark. Mushroom extracts were analysed as such without any further extraction. Gallic acid (0.2 g/l) was used as standard and absorbance was read at 725 nm.

Total proteins
Proteins (N × 6.25) were determined using the microkjeldahl method of [29]. Samples were first mineralized using Kjeldahl method and the nitrogen content of the mineralisate was evaluated after a reaction with ammonia (NH 3 ) and acetyl acetone / formaldehyde. The resulting yellow complex (3,5-diacetyl-1,4-dihydrolutidin) had a maximum absorption at 412 nm.

Soluble sugar contents
Soluble sugar contents of the flours and extracts were determined according to the method of Fischer and Stein [30]. Mushroom flours were previously extracted with distilled water (1:5 w/v) for 1h at 100°C and centrifuged at 2500 g for 30 min before quantification.

Determination of antihypercholesterolemic effect of Pleurotus florida powder and extracts in rat models
Feeding experiments: Twenty five adult male albino rats (2-3 months old) weighing 204-366 g were purchased from the University of Yaoundé I animal house and transported to the National School of Agro-Industrial Sciences (ENSAI), of the University of Ngaoundere. Animals were housed individually in semi-metabolic cages in the animal house at ENSAI, on a 12 / 12 cycle of light and darkness. The average temperature and relative humidity of the animal house were 25 ± 2°C and 60-70% respectively. The animals were distributed into five groups of five animals each and acclimatized for one week on a basal diet (Corn starch 50%; Rice powder 11.25%; Vegetable oil 1%; Egg white 10%; Dried fish 8%; Cellulose 19%; Mineral mix 0.125%; Vitamin mix 0.125%; Table salt 0.5%) formulated according to Alam et al. [13]. Thereafter the rats were fed the treatment diets, and consisted of a negative control group that received the basal diet (BD); a positive control group fed a cholesterol enriched diet (HC) and 3 experimental groups that were fed the cholesterol enriched diet supplemented with 5% mushroom powder (HC-PFP); extract from 5 g (5%) mushroom powder (HC-PFE5) and extract from 7.5 g (7.5%) mushroom powder (HC-PFE7.5) respectively. The 5% mushroom powder was used as a standard to compare the effects of substituting mushroom powder with extracts from an equivalent amount of powder, given that previous studies had demonstrated the hypocholesterolemic effects of 5% mushroom powder supplementation. The different formulations are presented in Table 1.
The animals were allowed free access to food and water for 4 weeks. Food intake and body weight were measured daily and weekly respectively. Fecal matter was collected from the animals during the last four days of the experiment for estimation of fecal lipid excreted. At the end of the experimental period rats were fasted overnight, anesthetized using chloroform and sacrificed. Blood samples were collected from the carotid artery in dry tubes; and various organs (liver, kidney and heart) were rapidly excised and weighed. Determination of food intake and body mass gain: Daily food intake was evaluated as the difference between the quantity of food given to animals and that left unconsumed after a 24 hr period. Rats were weighed at the start of the experiment and then weekly. Body weight gain of rats was calculated as the difference between the initial and final weights and results expressed as a percentage (%) of the initial weight.
Determination of fecal lipid content: The fecal matter collected from the animals was dried and ground into powder. Total lipid was extracted with hexane using a Soxhlet apparatus and quantified [31].
Determination of serum lipids: Total serum cholesterol (TC) was determined using the Olympus OSR6516 kit (Olympus Diagnostica Gmbh, Ireland) fixed onto an OLYMPUS AU2700 analyzer. Low density lipoprotein (LDL) and high density lipoprotein (HDL) was determined using BioMerieux 61534 (BioMerieux France) directly connected to an automated analyzer (OLYMPUS 2700 analyzer), and total triglyceride (TG) were determined using automated method. Very low density lipoprotein cholesterol VLDL-C was obtained by difference:

Statistical analysis
Results were subjected to Analysis of variance (ANOVA) to determine variations between treatment groups and mean separation done using the Least Significant Difference test at the 5% level. The statistical software, Statgraphic Plus 5.0 (Manugistics, Rockville, Maryland, USA) was used for analysis.

Composition of mushroom (P. florida) powder and extracts
The chemical composition of the mushroom (P. florida) powder and extracts are presented in Table 2.
Soluble fiber, proteins, sugar and polyphenols were significantly (p < 0.05) higher in powder samples compared to extracts. The quantities in extracts increased with the amount of powder extracted. Soluble sugars were the major constituent in the extracts. This is in part explained by their higher initial concentrations in the powder. The low recovery of soluble fiber in extracts compared to powder, maybe attributed to the rigorous extraction conditions employed in the extraction of powder for determination of soluble fiber (100°C for 1.5 hr) compared to conditions for the experimental extracts (100°C for 30 min). Values are means ± standard deviations of three repetitions. Values on the same row with different superscripts are significantly different (p < 0.05).
Polyphenols also, though in high concentrations in powders were found in smaller quantities in the extracts. The poor solubility of polyphenols in water [32] could be responsible for the low quantities in the water soluble extracts, compared to the powders that were extracted with ethanol. In addition, the high extraction temperature (100°C) for the experimental extracts could cause oxidation of some polyphenols [33]. High extraction temperature (100°C) and prolonged heating times (30 min), could lead to Maillard type reactions with precipitation of some components (sugars and proteins). These may account in part for the poor extractability of these components and hence the smaller amounts in extracts.

Effect of Pleurotus florida powder and extracts on weight gain and lipid profile in rat models
In order to understand the effects of Pleurotus florida mushroom powder and extract on lipid metabolism and total weight gain in rats, the amount of lipids excreted in feces was determined and serum lipid profile analyzed. Weight of rats was measured and that of some organs such as the heart, liver, and kidney.

Food intake and body weight gain
Results indicate that there was no statistically significant difference (P > 0.05) in food intake amongst the groups throughout the experimental period (Table 3).
However, at the end of the four week experimental period, animals fed a HC diet had a significantly (p < 0.05) higher body weight (390 ± 2.1 g) compared to animals fed the BD, HC-PFP, HC-PFE 5 and HC-PFE 7.5 diets ( Table 3).
Addition of 1% cholesterol to the basal diet favored weight gain in rats with a final increase in weight of 7.4% above rats fed a basal diet. Incorporation of mushroom powder and extracts in high cholesterol diet, on their part significantly (p < 0.05) prevented body weight gain and the animals had lower final body weights than those fed HC and the basal diets. Addition of 5% P. florida powder in the HC diet prevented body weight gain by 15.5%, whereas extracts from an equivalent 5% and 7.5% P. florida powder prevented weight gain by 12.7% and 15.3% respectively compared to those fed HC diets. Compared to the basal diet, addition of 5% mushroom powder and extracts from 5% and 7.5% powders significantly (p < 0.05) prevented weight gain by 8.1%, 5.2% and 7.8% respectively. These results suggest that P. florida powder and extracts could influence lipid metabolism and consequently be beneficial in controlling weight gain. However, no statistically significant differences (p > 0.05) were observed in the effect of powder and extracts in preventing weight gain in rats.
Obesity is characterized by the presence of excess adipose tissue and an increased percentage of body fat [34]. Worthy of note is the fact that P. florida showed positive effects in suppressing weight gain with no significant difference between the powder form and the extracts. This finding is important as obesity is a chronic disorder associated with complications in the body such as diabetes, cardiovascular diseases, respiratory abnormalities and cancer [35][36][37]. We observed that P. florida powder and extracts had similar effects in preventing weight gain suggesting that the components most active in preventing weight gain are extractable by water. Powders and extracts contain soluble fiber which is said to bind lipids in the intestinal tract resulting in their elimination in feces [36], thereby preventing weight gain. The efficacy of extracts is important as it means more versatility and convenience in the use of mushrooms for controlling obesity and its related diseases.

Mass of organs and fecal lipid excreted
With the exception of the liver of HC fed rats that was significantly larger (p < 0.05), there was no significant (P > 0.05) difference in the kidney, heart and liver masses of rats fed the BD, HC-PFP, HC-PFE5, and HC-PFE7.5. Similar findings have been reported by [13] while investigating the antihypercholesterolemic effect of P. ferulae. The authors observed lipid droplets in liver tissues of animals fed high cholesterol diet indicating accumulation of lipids in hepatocytes. This is possible given that lipid metabolism takes place in the liver and adipose tissues and in the event of excesses; lipids accumulate in these tissues [34]. In this respect, it can be suggested that P. florida powder as well as its extracts, like P. ferulae, are capable of preventing liver steatosis.
In the present study, no significant differences (P > 0.05) were observed in total fecal lipids excreted by rats fed HC diets alone, or incorporated with mushroom powder and extracts, although the quantities of lipid excreted increased with incorporation of mushroom powder and extracts (Table 4). Values are means ± SD of five animals. Values in the same column with different superscripts are significantly different (p < 0.05). BD: Basal Diet; HC: High Cholesterol Diet; HC -PFP: HC + P. florida powder 5%; HC -PFE 5: HC + P. florida 5% extract; HC-PFE 7.5: HC + P. florida 7.5% extract.
However, fecal lipids of rats fed HC diet coupled with mushroom extracts was significantly (P < 0.05) higher than those of rats fed the BD. The enhanced fecal lipid excretion observed with the consumption of mushroom powder and extracts in the treatment groups indicates a reduction in absorption of lipids at intestinal level. Similar findings by [38], had reported increased fecal lipid excretion in hamsters fed straw mushroom diets, indicating that this effect is common with mushroom consumption. Soluble fiber is said to bind lipids in the intestinal tract resulting in their elimination in feces [36]. The presence of soluble fiber in mushroom powder and extracts, suggests this could be one of the mechanisms by which P. florida powder and extracts control weight gain in rats fed high cholesterol diets.

Analyses of serum lipid profile
Blood serum lipid profile and especially cholesterol which is the major component of atherogenic fatty plaque can be used to measure the risk for the development of cardiovascular diseases (CVDs) and other metabolic syndromes [36,39,40].

Total serum cholesterol and total triglycerides
Addition of 1% cholesterol to the basal diet induced hypercholesterolemia in HC fed animals since the total cholesterol level rose by 26% and total triglycerides by 40% with respect to the group fed the basal diet (Table 5).  These levels were significantly (P < 0.05) lower in the treatment groups with no significant differences among treatments. P. florida powder and extracts maintained total cholesterol and triglyceride levels at those found in the control BD group. Similar observations were made by [21] after feeding whole fungus (Pleurotus ostreatus), its water and ethanol extracts to rats for 6 weeks. According to [41], mushrooms contain statins, which have been shown to inhibit the activity of the liver enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) thereby suppressing hepatic biosynthesis of cholesterol. Decrease in HMG-CoA reductase activity has been reported in rats fed high cholesterol diets with 5% oyster mushroom (Pleurotus ostreatus) [42]. All these point to the cholesterol lowering effects of mushroom. In addition, mushrooms have been shown to contain soluble fiber which binds bile acids resulting in their elimination in feces. Consequently more cholesterol from the liver will have to be used in the synthesis of bile acids thus lowering cholesterol levels [34]. The high lipid excretion observed in the treatment groups (Table 4) supports this assertion. Thus binding of cholesterol and reduction of HMG CoA reductase activity suggests underlying mechanisms by which mushrooms lower cholesterol levels.

HDL cholesterol (HDL-C)
Consumption of the cholesterol rich diet decreased the amount of HDL-C in blood serum compared to animals fed the basal diet. However, supplementation of HC diet with P. florida powder and extracts maintained HDL-C at levels initially present in the control group fed the basal diet (Table 5). High levels of plasma HDL-C in blood implies that more cholesterol from peripheral tissues is being returned to the liver for catabolism and subsequent excretion resulting in its reduction in blood [36]. The increase in HDL-C may be attributed to the inhibition of Apo D activity which is responsible for transforming cholesteryl ester (CE) into VLDL [43]. High HDL-C levels are generally associated with a protective effect against atherosclerosis and cardiovascular diseases, whereas high levels of LDL constitute a risk factor. Consequently, P florida powder and extracts can be considered as having protective effects against CVDs.

LDL and VLDL Cholesterol
Serum concentrations of LDL and VLDL cholesterol in rats fed P florida powder and extracts were significantly (p < 0.05) lower than those of rats fed the HC diet alone, with no significant differences (P > 0.05) between treatment groups. VLDL-C is the major transport form in which TG produced in the liver are transferred to peripheral tissues, and during this time it is hydrolyzed with the partial removal of TG by lipoprotein lipase forming LDL-C [14,44]. Previous work [45] had shown that consumption of P. ostreatus mushroom reduces VLDL entry into circulation and accelerates fractional turnover rate of VLDL, which possibly explains the lower serum concentration of VLDL-C compared to LDL-C levels in all the treatment groups. In addition, the low serum VLDL and LDL-cholesterol levels in animals fed HC-PFP, HC-PFE5 and HC-PFE7.5 diets may be due to elevation of hepatic LDL receptor levels allowing for greater amounts of VLDL remnants and LDL to be removed from circulation. Higher hepatic LDL receptor mRNA levels had previously been reported in rats fed mushroom (Agaricus bisporus) fiber compared to those in rats fed cellulose, and hepatic LDL receptor level correlated negatively with serum VLDL and LDL cholesterol concentrations [46]. Thus mushrooms favor the removal of LDL-C and VLDL-C from circulation, thereby reducing their concentration in serum, and consequently removing the risk for atherosclerosis and other CVDs associated with them.
Polyphenols present in mushrooms could equally constitute an important factor contributing to this decrease in cholesterol, as it has previously been shown with green tea that their polyphenols stimulate LDL receptors and reduce LDL and VLDL cholesterol levels [47,48]. Studies with polyphenols of grape seeds have shown that these lower cholesterol by inhibiting pancreatic cholesterol esterase, binding bile acids, and reducing solubility of cholesterol in micelles which delay cholesterol absorption and enhance their excretion [49]. As such, polyphenols prevent dyslipidemia and cardiovascular diseases. Polyphenols of mushroom could have similar effects.

Atherosclerosis index
The ratio HDL-C / Total cholesterol and LDL-C / HDL-C (also called atherosclerosis-index) measures the risk for developing cardiovascular diseases given the relationship between serum lipids and artherogenesis [36,50]. Results of serum LDL-C / HDL-C ratio measured in this study are shown in Figure 1. In rats fed the high cholesterol diet, this ratio increased by 85% compared to the ratio for rats fed the basal diet. On the other hand, the LDL-C / HDL-C ratio was significantly lowered by 46%, 54% and 52%, in rats fed HC-PFP, HC-PFE5 and HC-PFE7.5 diets respectively compared to rats fed the HC diet. No significant difference was observed between animals fed the BD and the treatment diets. This decrease in LDL-C / HDL-C ratio in P. florida treated animals strongly supports the anti-atherogenic activity of this mushroom previously reported by [23]. The present study further reveals that the anti-atherogenic potential of the extracts is equally important in the reduction of cardiovascular disease risk. In the light of growing consumer interest in convenience and functional foods, and the increased consumption of mushroom juice, the present findings are important in promoting mushroom juice as a functional food and provide more versatility in the consumption and use of mushrooms.

Conclusion
The present study has demonstrated that the inclusion of Pleurotus florida extracts like powder in the diets of rats fed high cholesterol diets suppresses weight gain, through increased fecal lipid excretion and a reduction in total cholesterol and total triglycerides. Other mechanisms are suggested. Consumption of P. florida increased HDL-C, and lowered HDL-C / LDL-C ratio. P. florida extracts were as effective as the powders in their effects in stimulating lipid metabolism, and reducing CVD risk. Thus P. florida extracts like powder are potential convenient functional foods in the prevention and management of hypercholesterolemia, obesity and its related complications.