- Open Access
Lipid-lowering properties of protein-rich mucuna product
© The Author(s) 2016
- Received: 10 October 2015
- Accepted: 14 April 2016
- Published: 25 May 2016
The objective of this study is to evaluate the effect of protein-rich mucuna product (PRMP) on lipid parameters of hyperlipidemic rats.
Hyperlipidemia was induced in male rats for 3 weeks through high-fat diet. After induction, 30 hyperlipidemic rats were divided into five groups of six rats: control group (CG) received casein and four groups received PRMP as protein source at different proportions: 8.2, 16.4, 24.6, and 32.8 % corresponding, respectively, to 25, 50, 75, and 100 % substitution of casein in the diet for 3 weeks. Lipid and oxidative stress parameters of rats were assessed.
There were no significant differences in food intake and body weight loss among the experimental groups. The concentrations of the serum low-density lipoprotein cholesterol, total cholesterol, and triglycerides were lower in groups fed on PRMP 50, 75, and 100 % than in the CG group (p < 0.05). Histological analysis of the liver revealed that animals fed on PRMP diets presented a lower level of steatosis than the CG group. The most significant reduction of lipid parameters was obtained when PRMP was used as unique source of protein (PRMP 100 %). PRMP also influenced oxidative stress parameters as evidenced by a decrease in malondialdehyde and an increase in catalase and superoxide dismutase.
These findings demonstrated that PRMP exerts hypolipidemic effect; it has a metabolic effect on endogenous cholesterol metabolism and a protector effect on the development of hepatic steatosis. Our results also suggest that PRMP could manage metabolic diseases associated with oxidative stress.
- Protein-rich mucuna product
- Oxidative stress
Dyslipidemia is an independent and modifiable risk factor for cardiovascular diseases . Its prevalence is growing in developed countries as well as in developing countries . Dyslipidemia is characterized by elevated low-density lipoproteins (LDL), high triglycerides and low high-density lipoproteins (HDL) level. High levels of HDL are found consistently to be associated with long life in diverse population while LDL is the principal atherogenic component of cholesterol and major constituent of atherogenic plaques and cardiovascular diseases [3, 4]. The major patho-physiological mechanism involved in atherosclerosis is oxidative stress . In this respect, oxidatively modified LDL particles are thought to promote specifically the early development of atherosclerotic lesions .
Among strategies to fight against dyslipidemia and to reduce the risk of coronary disease, dietary management has shown promising results through a regulation of HDL and LDL cholesterol levels. In particular, beneficial effects on serum lipids (cholesterol-lowering effect) have been reported on our daily food seeds and seed proteins including soy bean , faba bean , amaranth , cowpea , and pea .
The cholesterol-lowering effect of seeds and seed proteins has been associated with the action of components such as phytic acid, dietary fiber, saponins, phytosterols, proteins, peptides, and the amino acid profile of the proteins . While cholesterol-lowering effect of many conventional legume seed protein isolates and, particularly, soy proteins have been widely studied, much is still to be done for under-utilized seeds of the tropical legume likeMucuna pruriens. Mucuna seeds are very good sources of proteins (23–35 %) , but they contain appreciable amount of antinutrients tannins, trypsin inhibitors, polyphenols, and phytate that limit their consumption [13, 14]. In this respect, our recent studies reported that consumption of raw mucuna flour produces deleterious effects on growth, hematological parameters, and kidneys of rats . In order to improve the nutritional properties of mucuna beans, we successfully produced a curd by the process of decortication, milling to produce flour, extraction of protein by thermal solubilization, and protein precipitation using citric acid. The product was not only poor in antinutrients but also contained 59–61 % proteins, and based on this , it was called protein-rich mucuna product (PRMP). Rats that fed PRMP exhibited blood biochemical and hematological characteristics, liver and kidney histology, and growth performance similar to casein as protein reference . Moreover, compared to animals that fed on casein as protein source, PRMP induces a decrease in blood cholesterol; a property generally associated with consumption of legume seeds .
The present study was undertaken in an attempt to confirm the probable hypolipidemic effect of PRMP, with the objective to evaluate the effect of protein substitution in the diet by PRMP protein on the serum’s lipidemic profile of hyperlipidemic rats.
Production of PRMP and amino acid profile determination
Mucuna pruriensvar. pruriensseeds were harvested from an experimental farm located at the University of Ngaoundere (Cameroon). Seeds of Mucuna were used to produce PRMP using the hydrothermal solubilization and acid precipitation of proteins as recently described by Ngatchic et al. . The amino acid composition of PRMP was determined by HPLC using RP-18.220 column (PTC RP-18.220 mm, 2, 1 mm I.D, Applied Biosystem, appelera Bio Corps, Fosters City, USA) after hydrolyzing the samples with 6 N HCl at 105 °C for 24 h.
In vitro antioxidant activity
Preparation of extract
0.5 g of PRMP powder was extracted twice with 25-mL ethanol (70 %) at 25 C for 24 h. Extract was filtered with Whatman N 1 filter paper, and the filtrate was stored at −20 C for determination of antioxidant activity.
Determination of DPPH radical scavenging activity of PRMP
Ferric iron-reducing activity
The ability of PRMP extract to reduce iron(III) to iron(II) was evaluated following the method of Oyaizu . An aliquot of 1 mL of PRMP extract was dissolved in distilled water and mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of aqueous K3Fe (CN)6(1 %) solution and incubated for 30 min at 50 C. 2.5 mL of trichloroacetic acid 10 % were added, and the mixture centrifuged for 10 min at 2000g. 2.5 mL aliquot of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of aqueous FeCl3 0.1 %. The absorbance was taken at 700 nm. Ferric iron-reducing activity was determined as ascorbic acid equivalents (mg ascorbic acid/100 g of PRMP).
Diet formulation and animal experiment
Formulation of diets for induction of hyperlipidemia
Soy bean oil
Formulation of experimental diets
Protein-rich mucuna products (%)
Soy bean oil
Blood sample collection, serum lipid profile, and histopathological analysis
At the end of the 3-week experiment, rats were fasted for 14 h, the blood samples were collected, and the liver and kidney were removed. Serum lipid profile was done as previously reported by Ngatchic et al. . In this respect, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in serum were determined as described earlier . In order to observe the steatosis, the histological examination of liver was performed; in the procedure, organs were fixed in formaldehyde (10 %) and the tissues subsequently dehydrated in upgraded concentrations of ethanol (10–90 %), cleaned in xylene, impregnated, and embedded in paraffin. Sections of 5 μm were cut using a microtome, stained with hematoxylin and eosin stains. Light microscopic (microscope OPTIKA DM-20, SN 223932 Italy; camera CM-D OLYMPUS) examination of multiple tissue sections from the liver in all animal groups was performed, and image representatives of the typical histological profile were examined.
Tissue preparation and determination of in vivo antioxidant activity
Liver and kidney removed were rinsed with NaCl (0.9 %) solution. Tissues were minced and homogenized (10 %w/v) in ice-cold potassium phosphate buffer (0.1 M, pH 7.4). The homogenate was centrifuged at 3000gfor 10 min at 4 C; the resultant supernatant was used for the determination of antioxidant activity.
Measurement of lipid peroxidation
Lipid peroxidation was evaluated with the Yagi  method. This method depends on the formation of malondialdehyde (MDA) as an end product of lipid peroxidation. The amount of malondialdehyde was determined by the spectrophotometric method at 532 nm after the reaction with thiobarbituric acid. The level of malondialdehyde was determined using the molar extinction coefficient of chromophore (1.56 × 105M−1cm−1).
Catalase activity was assayed by the method of Sinha  which is based on the formation of chromic acetate from dichromate and glacial acetic acid in the presence of hydrogen peroxide. Chromic acetate produced was measured using spectrophotometer at 620 nm. One enzyme unit was defined as the amount of enzyme which catalyzed the oxidation of 1 μmol H2O2per min under assay conditions. The activity was expressed in terms of units per milligram of protein.
The superoxide dismutase (SOD) activity was determined by the spectrophotometric method based on the inhibition of adrenaline oxidation to adrenochrome . Briefly, 0.2 mL of sample was diluted in 3 mL of carbonate buffer, pH 0.2, and placed into quartz spectrophotometer cuvette. The reaction was started by adding 0.3 mL of adrenalin 0.3 mM solution in 10 mM HCl. Adrenaline oxidation leads to the formation of the colored product, adrenochrome, which is detected by the spectrophotometer. In the basic pH, the adrenalin was spontaneously oxidized, with the kinetics recorded by measuring the increase of absorbance at 480 nm over time. The kinetics of adrenalin oxidation in the presence of the sample was compared with the oxidation rate of adrenalin alone. A unit of SOD is defined as the amount of enzyme that inhibits the rate of adrenaline oxidation by 50 %. The result was expressed in microunit per milligram of protein. Protein was determined using the method of Lowry et al. .
Statistical and data analyses
The statistical analyses were performed using the Statgraphics software, version 5.0. The values were presented as means with their standard deviation (±SD). One-way analysis of variance (ANOVA) was performed to test the significance of differences (p < 0.05) between groups. When the difference was significant, a Duncan multiple comparison range test was used as a post hoc test. Correlation was performed between variables to evaluate their relationship (significant correlation at p < 0.001), while a principal component analysis was used for visualization of difference between treatments.
Amino acid profile of PRMP
Amino acid composition of protein-rich mucuna product (PRMP) (mg/g protein)
FAO/WHO pattern (2007)
Phenylalanine + tyrosine
Methionine + cystine
In vitro antioxidant activity
Results of this study reveal that antioxidant activity of PRMP is 6.15 ± 0.03 mg trolox equivalent/100 g of dried matter for DPPH activity and 3.68 ± 0.06 mg ascorbic acid equivalent/100 g of dried matter for ferric iron-reducing activity, showing that the antioxidant potential of PRMP is not negligible. This must be considered since it already has a capacity to scavenge free radical and reducing metal.
Effect of high-fat diet on lipid profile, body and liver weight, and antioxidant enzymes
Food intake, weight gain, and serum lipid levels of rats fed on the standard and high-fat diet for 3 weeks
Food intake (g/day)
18.9 ± 5.04a
18.9 ± 1.09a
Weight gain (g)
22.2 ± 0.57a
35.6 ± 0.65b
Total cholesterol (mg/dL)
63.2 ± 2.76a
182.1 ± 4.13b
HDL cholesterol (mg/dL)
26.6 ± 1.80a
17.0 ± 0.76b
LDL cholesterol (mg/dL)
29.6 ± 1.47a
124.5 ± 3.53b
95.2 ± 1.37a
176.7 ± 3.87b
Effect of high-fat diet on stress parameters of rats
1.78 ± 0.02a
2.62 ± 0.05b
554.42 ± 3.29a
543.06 ± 2.10b
104.08 ± 1.99a
81.82 ± 2.58b
2.11 ± 3.35a
3.11 ± 1.88b
659.22 ± 1.94a
649.09 ± 1.17b
114.50 ± 4.38a
93.74 ± 4.51b
2.06 ± 0.09a
2.83 ± 0.03b
577.67 ± 3.29a
567.55 ± 1.04b
101.42 ± 2.02a
96.64 ± 1.45b
Effect of PRMP on food intake, body, and liver to body weights of hyperlipidemic rats
Food intake, body weight loss, and liver weight/body weight ratio of rats fed on standard diet and protein-rich mucuna product
Food intake (g/d)
Body weight loss (g)
Liver weight/body weight
18.88 ± 0.83a
4.77 ± 0.44a
3.66 ± 0.06a
PRMP 25 %
18.50 ± 0.93a
4.97 ± 0.47a
3.62 ± 0.01a
PRMP 50 %
18.77 ± 1.05a
5.01 ± 0.65a
3.53 ± 0.08a
PRMP 75 %
18.87 ± 1.25a
5.21 ± 1.03a
3.50 ± 0.05a
PRMP 100 %
18.64 ± 1.4a
4.85 ± 0.98a
3.28 ± 0.04b
Effect of PRMP on serum lipid levels
Effect of PRMP on antioxidant enzymes
Effect of proportion of PRMP as protein source in their diet on the oxidative stress parameters of experimental rats
Proportion of PRMP (%)
Superoxide dismutase (μUnit/mg)
548.68 ± 3.48a
654.15 ± 5.01a
572.33 ± 5.02a
91.78 ± 1.06a
106.67 ± 1.67a
99.05 ± 0.70a
2.14 ± 0.01a
2.76 ± 0.01a
2.69 ± 0.01a
550.51 ± 5.07a
654.06 ± 6.92a
572.59 ± 6.58a
91.69 ± 1.00a
107.07 ± 1.11a
99.37 ± 0.57a
2.13 ± 0.02a
2.74 ± 0.03a
2.69 ± 0.03a
551.75 ± 5.10a
654.50 ± 6.56a
572.39 ± 6.73a
91.42 ± 1.85a
107.31 ± 0.57a
99.08 ± 0.58a
2.13 ± 0.03a
2.73 ± 0.02a
2.69 ± 0.02a
548.21 ± 5.02a
655.85 ± 4.08a
572.29 ± 6.76a
92.81 ± 1.34a
107.39 ± 0.63a
100.07 ± 1.40a
2.02 ± 0.02b
2.65 ± 0.03b
2.60 ± 0.02b
565.14 ± 3.83b
670.32 ± 5.40b
592.62 ± 6.49b
96.89 ± 1.75b
112.30 ± 1.66b
110.83 ± 1.81b
1.94 ± 0.01c
2.5 ± 0.01c
2.53 ± 0.01c
Hyperlipidemia is a major contributor to the pathogenesis of cardiovascular diseases which is a leading health problem in the world. In this respect, strategies to improve the blood lipid’s profile were associated with the reduction of these diseases . The main finding of the present study was that PRMP decreased the blood concentration of total cholesterol, LDL cholesterol, triglycerides, and the fat liver accumulation. More precisely, the total cholesterol of the rat groups fed diets with PRMP at 25, 50, 75, and 100 % substitution of casein as protein source diminished for 0.01, 3.6, 11, and 30.7 %, respectively. Similarly, the LDL cholesterol level decreased for 0.5, 4, 21, and 37.8 %, respectively, as the level of PRMP increased in the diet. Similar reduction in total and LDL cholesterol has been reported for other plant protein isolates as cowpea , amaranth , and pea . Several mechanisms of action have been suggested to explain the reduction of cholesterol and triglycerides in rat fed with protein isolates: the amino acid profile, the presence of bioactive peptides, or other molecules.
Among the mechanisms of hypolipidemic action of legume seed protein isolates, the composition in amino acids has been widely reported. In this respect, Carroll and Kurowska  and Dabai et al.  reported that plants with low methionine concentrations and elevated arginine to lysine ratio exhibited hypolipidemic properties. The results obtained in the present work apparently follow this trend. In fact, the analysis of the amino acid profile of the PRMP proteins revealed that the arginine to lysine ratio was 1.36, higher than the value 0.46 reported for casein. Some other researchers associated with the hypocholesterolemic effect of legume seed protein isolates to the presence of bioactive peptides. Cho et al.  and Rigamonti et al.  reported incomplete digestion of legume seed proteins in the digestive tract, and liberation of specific peptides which reduce the levels of cholesterol, probably by regulation of the activity of HMG-CoA reductase, a key enzyme in the synthesis of cholesterol, and the activity of LDL receptor. It has been demonstrated that α and α’ subunits of 7S soy globulin may reduce blood cholesterol  while the peptide conglutin of protein lupin has been reported to increase LDL receptor activity . The analysis of residual peptides upon digestion of PRMP in the digestive tract and its cholesterol-lowering action has not been investigated yet. Several studies revealed that proteins do not exhibit hypolipidemic activities but rather secondary metabolites (such as phytates and polyphenols) that accompany them. Convincing experimental evidence has been presented for depressing increase in hepatic lipids and in the hepatic activities of lipogenic enzymes (HMG-CoA reductase, fatty acid synthetase) by dietary phytate in animal [32–34]. In addition, polyphenols induce metabolic hypolipidemic effect mainly by their ability to reduce cholesterol acyltransferase and HMG-CoA reductase activities. In relation to these molecules, Rigamonti et al.  reported hypotriglyceridemic effect of pea protein isolates through mechanism of inactivation of enzymes involved in fatty acid synthesis such as fatty acid synthetase and stearoyl-CoA desaturase. Our recent investigations  revealed that PRMP contained residual level of phytate (2.56 %) and polyphenols (1.04 %) which could be responsible of the hypolipidemic activity of PRMP. Based on the above, the hypolipidemic action of PRMP could result from a synergistic bioactive phytochemical phytate, polyphenols, and amino acids leading to a reduction of fat accumulation in the liver and body weight.
Accumulation of fat in the liver (steatosis) is a major characteristic of dyslipidemia and metabolic syndrome; thus, the reduction of steatosis is a good indicator of the management of dyslipidemia. The difference in hepatic lipid accumulation between the rats fed is varying. PRMP regime clearly highlighted the reduction of hepatic fat with increased level of PRMP in the diet (Fig. 1). Compared to the livers of the rats fed with 100 % casein as protein source that showed widespread fat globules of broad size, steatosis size was reduced with increasing proportion of PRMP in the diet. There was a large reduction of fat deposit areas in the liver of the animals fed with PRMP 100 %, thus justifying the decrease in liver weight. Such regulation has been demonstrated in rat hepatocytes and adipocytes following ingestion of soy proteins through modulation of the secretion of several adipokines and free fatty acids (FFA) [35, 36]. A sterol regulatory element-binding protein (SREBP)-1-dependent mechanism has been reported for the regulation of fatty acid synthesis upon ingestion of protein isolates .
A number of oxygenated compounds are produced during the attack of free radicals against membrane lipoproteins, proteins, and polyunsaturated fatty acids. One of them is malondialdehyde which can be used as an indicator of oxidative stress, as its concentration in plasma increases as the result of free-radical processes. The antioxidative system enables transformation of reactive oxygen species into inactive and harmless compounds. Antioxidant enzymes: superoxide dismutase, glutathione peroxidase, and catalase provide primary defense against reactive oxygen species. Superoxide dismutase can selectively scavenge a superoxide radical by catalyzing its dismutation to hydrogen peroxide and molecular oxygen, while glutathione peroxidase and catalase serve to decompose hydrogen peroxide to the unreactive species . Since antioxidant enzymes are primary defense against reactive oxygen species, increase of enzymes activities (catalase and superoxide dismutase) and lower levels of the malondialdehyde observed on rats fed with PRMP show that consumption of PRMP could prevent oxidation of organism compounds like low-density lipoprotein and thus prevent many diseases such as cardiovascular diseases.
The results of the present study indicate that consumption of diet rich in fat results in triglyceride deposition in the liver, increase in blood total cholesterol, LDL cholesterol, and triglycerides. Increase in proportion of protein-rich mucuna product (PRMP) as protein source in diet results in a linear decrease of LDL and total cholesterol, triglycerides, and liver steatosis. However, the mechanism of cholesterol-lowering properties needs to be investigated. In addition, since oxidative stress is generally associated with metabolism disorder, improving antioxidant activities and reduced of levels of malondialdehyde conclude that PRMP could play an important role in management of metabolic diseases, like cardiovascular diseases.
The authors gratefully acknowledge the technical assistance of the laboratory staff from the Department of Food Science and Nutrition of National School of Agro-industrial Sciences of Ngaoundere University.
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