Dietary interventions have been used to control serum TAG and cholesterol, thus contributing to prevent CHD; in particular, diets containing soyabean and other legumes seeds have been associated with a reduction in the number of risk factors associated with these illnesses( Reference Parolini, Manzini and Busnelli 1 – Reference Anderson 4 ). Soyabean proteins in particular have been studied, and these proteins have gained official recognition as potential reducers of risk factors for CVD in 1999, when the US Food and Drug Administration (FDA)( 5 ) accepted the recommendation on the intake of 25 g of soyabean protein per d for the prevention of these diseases. The FDA, based on a survey of clinical studies, indicated that daily consumption of these levels of soyabean protein may reduce total cholesterol (TC) and LDL-cholesterol( Reference Harland and Haffner 6 , Reference Jenkins, Mirrahimi and Srichaicul 7 ). However, proteins of other legumes have also shown some action on lipid metabolism and especially on cholesterol( Reference Parolini, Manzini and Busnelli 1 , Reference Frota, Mendonça and Saldiva 8 , Reference Macarulla, Medina and De Diego 9 ), but most of these proteins have not been studied in great detail. The search of specific subfractions responsible for blood lipid-decreasing effects, as well as the mechanisms involved, is still the subject of many studies( Reference Duranti, Lovati and Dani 10 – Reference Zhong, Liu and Ma 16 ). Once again, the dyslipidaemic-controlling effect of the soyabean β-conglycinin fraction, a 7S storage globulin, has been well documented both in animal models and human subjects( Reference Scarafoni, Magni and Duranti 3 , Reference Duranti, Lovati and Dani 10 – Reference Ferreira, Silva and Demonte 12 , Reference Kohno, Hirotsuka and Kito 17 ), but still no conclusive evidence on the mechanism/s involved has been put forward.
The 7S globulins of most leguminous seeds are composed of a subunit assortment yielding a molecular mass of approximately 150–220 kDa; the constituent subunits may vary in size and number within and among species( Reference Derbyshire, Wright and Boulter 18 ). Despite these differences, a considerable degree of sequence homology, when comparing partial and complete nucleotide sequences of genes and mRNA encoding for them, has been observed( Reference Fukuda, Maruyama and Salleh 19 ). Various authors have described that the subunits of the 7S legume storage globulins have similar amino acid sequences( Reference Schuler, Ladin and Pollaco 20 ), and this may have led to the assumption that they can be cleaved in similar ways by digestive enzymes. Indeed, Nielsen et al. ( Reference Nielsen, Deshpande and Hermodson 21 ) found that the cleavage sites of broad bean phaseolin by pepsin, trypsin and chymotrypsin are consistent with those of pea vicilin and soyabean β-conglycinin, all belonging to the 7S globulin family.
Previous studies have shown that β-conglycinin displays hypocholesterolaemic and hypolipidaemic action, when added to the diet as a unique source of protein or as a single daily administration( Reference Duranti, Lovati and Dani 10 , Reference Ferreira, Silva and Demonte 12 , Reference Lovati, Manzoni and Gianaza 22 , Reference Ferreira, Silva and Demonte 23 ). More recently, various authors have demonstrated that the α' subunit is the component responsible for the activity( Reference Duranti, Lovati and Dani 10 , Reference Consonni, Lovati and Manzoni 24 ). The effects of soyabean 7S globulin were comparable with those observed with the drugs rosuvastatin, simvastatin and fibrates( Reference Duranti, Lovati and Dani 10 , Reference Ferreira, Silva and Demonte 11 , Reference Ferreira, Silva and Demonte 23 ). It is generally agreed that these effects on lipid metabolism may be due to peptides arising from the gastrointestinal digestion of protein. As a matter of fact, in silico simulated cleavage analyses showed that gastrointestinal digestion may lead to the production of peptides exhibiting similar biological activities as those detected with the soyabean 7S globulin (ES Ferreira, unpublished results). However, a unique region bearing the activity has not unequivocally been identified yet.
In the search of the biological activity of never-investigated species, we undertook the present study to monitor the effects of isolated 7S globulins from cowpeas and adzuki beans (7S-C and 7S-A, respectively), compared with simvastatin, on lipid parameters of rats fed a hyperlipidaemic and cholesterolaemic diet.
Methods
Flour preparation from cowpea and adzuki bean seeds
Cowpea (Vigna unguiculata, L) and adzuki bean (Vigna angularis, L) seeds were obtained from Empresa de Pesquisa Agropecuária de Minas Gerais at the Federal University of Viçosa, Minas Gerais, Brazil. The seeds were selected, soaked in distilled water (4°C/12 h), manually decorticated, dried at room temperature and powdered to 60 mesh sizes. The flours were stored to 4°C and used for protein extractions.
Isolation of cowpea 7S globulin
The procedure consisted of the extraction of the cowpea flour with 1/20 (w/v) 0·1 m-NaCl in distilled water, adjusted to pH 7·5 with 2 m-NaOH. Stirring was applied for 1 h at room temperature. The suspension was then centrifuged at 10 000 g for 40 min at the same temperature. From this step, all following procedures were carried out at 4°C. The supernatant fraction was diluted 1/1 with distilled water; its pH was adjusted to 5·0 with 2 m-HCl and then kept overnight. A further centrifugation step as above allowed us to obtain a soluble fraction (albumins) and a pellet. The pellet was homogenised in water 1:20 (w (initial weight)/v) by using a Potter-Elvehjem homogeniser and stirred at 4°C for 10 min. Then, the pH was adjusted to 5·0 and the suspension centrifuged as above. The new pellet was dissolved in 0·1 m-NaCl 1:10 (w (initial weight)/v) at pH 7·0 and stirred at 4°C for 20 min. After further centrifugation as above, the pellet was discarded and the supernatant fraction was diluted 1/1 (v/v) with distilled water; the pH was adjusted to 5·0 and the solution was kept at 4°C overnight. The latter centrifugation step allowed us to recover a pellet, mainly consisting of 7S globulin, as will be shown. The pellet was dissolved in 0·2 m-NaCl at pH 7·0, and dialysed before freeze-drying.
Isolation of adzuki bean 7S globulin
The adzuki bean flour was suspended in 1:20 (w/v) distilled water; the pH was adjusted to 7·5 and the suspension was stirred at room temperature for 30 min. The homogenate was then centrifuged as above. The supernatant fraction (S1) was kept and the pellet was solubilised in 1:10 (w (initial weight)/v) 0·5 m-NaCl, adjusted to pH 7·5 and stirred at room temperature for 30 min. Then the suspension was centrifuged as above and the supernatant fraction (S2) was mixed to S1. All subsequent procedures were performed at 4°C. The two mixed supernatant fractions were diluted 1/5 (v/v) with distilled water, homogenised as above, adjusted to pH 5 and kept at 4°C overnight. After centrifugation as above, the pellet was washed with distilled water and centrifuged once again. The new pellet was solubilised in 1:10 (w (initial weight)/v) 0·25 m-NaCl at pH 5·3 and stirred at 4°C for 20 min. After centrifugation as above, the soluble fraction was diluted 1/5 (v/v) with distilled water and homogenised as above; the pH was adjusted to 5·0 and the suspension was kept at 4°C overnight. After a final centrifugation as above, the supernatant fraction was discarded and the pellet was dissolved in 0·2 m-NaCl at pH 7·0, before dialysis and freeze-drying as above.
SDS-PAGE
The homogeneity of the isolated proteins was performed on 10 % polyacrylamide gels containing 0·1 % SDS, in a Mini Protean II cell (Bio-Rad) and stained with Coomassie Brilliant Blue (R-250). Marker proteins of known molecular weight were: rabbit muscle phosphorylase B (94 kDa), bovine serum albumin (66 kDa), hen egg white albumin (45 kDa), bovine carbonic anhydrase (29 kDa), soyabean trypsin inhibitor (21·5 kDa) and hen egg white lysozyme (14·4 kDa).
Diets and experimental protocol
All experiments were conducted in accordance with the Ethical Principles in Animal Research in the Guide for the Care and Use of Laboratory Animals( 25 ) and were approved by the Ethics Committee for Animal Research of the School of Pharmaceutical Sciences, São Paulo State University (UNESP) protocol 25/2009. A total of forty-five male Wistar rats aged 2 weeks (40–50 g body weight) were obtained from the Central Laboratory for Animals of the São Paulo State University (UNESP) at Botucatu (Brazil). The rats were fed a pelletised commercial (Purina®) diet for 2 weeks after arrival until reaching 150–160 g body weight. Afterwards, the animals were housed individually in stainless-steel cages in a room with a 12 h light–12 h dark cycle and a temperature of 23 ± 2°C. The rats were then divided into five groups (nine rats per group). A standard (STD) group was fed with a normal control diet, following the recommendations of the American Institute of Nutrition (AIN-93G) for growth( Reference Reeves, Nielsen and Fahey 26 ). Four hypercholesterolaemic (HC; high cholesterol and TAG) groups, namely HC-control, 7S-C, 7S-A and simvastatin, were fed with an AIN-93G diet, modified by adding 20 g/100 g coconut oil, 1 g/100 g cholesterol and 0·5 g/100 g cholic acid( Reference Duranti, Lovati and Dani 10 , Reference Nath, Wiener and Harper 27 ) as described in Table 1. To three groups of HC animals, an oral dose of 300 mg/kg per d of the isolated 7S globulins and 50 mg/kg per d simvastatin were administered orally daily at 14·00 hours. The globulins and the drug were dissolved in saline buffer and the vehicle alone was given to one HC group. All animals were given food and water ad libitum during the experimental course (i.e. 28 d). The mentioned globulin doses were only slightly greater than those previously adopted with soyabean β-conglycinin( Reference Duranti, Lovati and Dani 10 ). Food consumption, weight gain, faecal excretion and feeding efficiency were measured each day of the trial. The feeding efficiency coefficient was calculated from the ratio of weight gain/daily intake × 100.
Table 1. Diet composition and treatments
HC diet, hypercholesterolaemic (high cholesterol and TAG) diet; HC diet + 7S-C, HC diet plus oral daily doses of cowpea 7S globulin (300 mg/kg per d); HC diet + 7S-A, HC diet plus oral daily doses of adzuki 7S globulin (300 mg/kg per d); HC diet + SVT, HC diet plus oral daily doses of simvastatin (50 mg/kg per d); AIN, American Institute of Nutrition.
* The standard diet was based on the recommendation of the AIN (AIN-93G) and the HC diet was bases on Nath's diet( Reference Nath, Wiener and Harper 27 ) by adding 200 g/kg coconut oil, 10 g/kg cholesterol and 5 g/kg cholic acid.
† Sigma-Aldrich®, Co.
‡ PragSoluções®, Co.
Blood and organ collection
On the last day, the animals were deprived of food for 12 h and euthanised by guillotine. Blood was then collected in tubes containing gel separator SST II (Vacutainer BD D®) and centrifuged at 1900 g for 15 min. The serum was separated, stored at –24°C and used for biochemical analysis. Epididymal adipose tissue, liver and heart were removed, washed immediately in cold saline buffer, dried and weighed, frozen and stored at –40°C for a period of less than 1 month for subsequent comparative analysis.
Biochemical analyses of serum
Serum TC was measured by the liquid cholesterol CHOD-PAP (cholesterol oxidase-phenol + aminophenazone) method described by Stockbridge et al. ( Reference Stockbridge, Hardy and Glueck 28 ). Serum HDL-cholesterol was measured by the HDL-cholesterol precipitation method described by Assmann( Reference Assmann 29 ). TAG were measured by the liquid TAG GPO-PAP (glycerol-3-phosphate oxidase-phenol + aminophenazone) method as described by Annoni et al. ( Reference Annoni, Botasso and Ciaci 30 ). Serum glucose concentration was determined using a liquid glucose GOD-PAP (glucose oxidase-phenol + aminophenazone) method as described by Trinder( Reference Trinder 31 ). All these colorimetric assays were carried out with commercially available kits (Laborlab® Col). The non-HDL-cholesterol fraction (LDL-cholesterol + VLDL-cholesterol) was determined by difference between TC and HDL-cholesterol, and the atherogenic indexes (TC – HDL-cholesterol/HDL-cholesterol) were calculated as proposed by Liu et al. ( Reference Liu, Wang and Chen 32 ). Hepato-somatic and visceral fat indexes were calculated by the following relationships, respectively: (liver weight/body weight) × 100, and (fat visceral weight/body weight) × 100, as described by Chen et al. ( Reference Chen, Chiou and Suetsuna 33 ). Lipoprotein lipase activity was determined as described by Roe & Byler( Reference Roe and Byler 34 ) and glutamic pyruvic transaminase was determined as described by Tonks( Reference Tonks 35 ) using a commercially available kit (Bioclin® Co.). Serum insulin levels were analysed using a commercially available ultrasensitive rat insulin ELISA kit (DGR Instruments® GmbH), as previously described previously by Korner et al. ( Reference Korner, Savontaus and Chua 36 ). This assay had 100 % cross-reactivity to rat insulin.
Analysis of hepatic and faecal lipids
Liver and faecal lipids were extracted with chloroform–methanol (2:1, v/v) according to the method previously described by Folch et al. ( Reference Folch, Lees and Sloane Stanley 37 ). TC and TAG were extracted by the method of Haug & Hostmark( Reference Haug and Hostmark 38 ). TC and TAG concentrations were measured as described earlier for serum analysis.
Statistical analysis
All data are presented as mean values with their standard errors for nine values. The statistical analyses were performed using the SigmaStat® 3.5 program (Dundas Software). Significant differences among the groups were determined by one-way ANOVA and Bonferroni t test multiple-range comparisons v. the HC group. A difference of P < 0·05 was considered statistically significant.
Results
Isolation of cowpea and adzuki bean 7S globulins for animal trials
In the present study, two novel isolation procedures of cowpea and adzuki bean main protein fractions, i.e. the 7S globulins, were used to generate sufficient amounts of proteins for animal trials (for details, see the Methods section). The results of SDS-PAGE conducted on the 7S globulins after separation from other protein components in cowpea and adzuki bean flours are shown in Fig. 1. The apparent molecular weights of the major polypeptides were about 55–56 kDa, and were consistent with the size of most polypeptide chains of the vicilin-like family( Reference Fukuda, Maruyama and Salleh 19 , Reference Nielsen, Deshpande and Hermodson 21 ). With the two procedures approximately 8 and 3 g of 7S globulins from 100 g of cowpea and adzuki bean flours were obtained, respectively. The described procedures did not make use of any chromatographic step, which would have prevented large-scale protein preparations suitable for use in the present study. These preparations were judged sufficiently homogeneous and adequate for use in the in vivo experiments.
Fig. 1. SDS-PAGE profile under reducing conditions of cowpea (a) and adzuki (b) 7S globulins. M, marker proteins; lanes 1, total protein extracts; lanes 2, isolated 7S proteins by the methods described. Loaded samples contained about 10 µg of the proteins.
Food intake, body weight and food efficiency
Effects of the daily administration of 7S-C, 7S-A and simvastatin on food consumption, weight gain, feeding efficiency ratio and faecal excretion in the animals of all groups, fed the diets for 28 d, are shown in Table 2. 7S-A and simvastatin produced a decrease of about 12 % on the final body weight of the animals, with a reduction on the average body-weight gain of 23 % with respect to the HC diet-fed animal group. 7S-C was effective too, though to a lesser extent. This fact could be attributed to the food intake reduction between groups in the period considered (Table 2). The HC group showed an increase of 72·8 and 72·2 % in the relative liver weight of rats and in the hepatosomatic index, respectively, relative to the STD diet-fed group (P < 0·001) (Table 2); on the other hand, only the 7S-A group presented a decrease of 18·6 % in the liver weight in relation to the HC group. No significant difference of the other parameters was found with respect to the HC group (Table 2).
Table 2. Body parameters measured in rats fed a hypercholesterolaemic (HC; high cholesterol and TAG) diet without and with oral daily doses of 7S globulins or simvastatin (SVT) for 4 weeks†
(Mean values with their standard errors; nine rats per group)
7S-C, cowpea 7S globulin; 7S-A, adzuki bean 7S globulin.
Mean value was significantly different from that of the HC diet group: * P < 0·05, ** P < 0·01, *** P < 0·001 (one-way ANOVA and Bonferroni t test multiple-range comparisons).
† For details of diets, see Table 1.
Serum parameters
In the rats fed the HC diet, serum lipid levels, both TC and TAG, were significantly higher than in those given the STD diet (P < 0·001) by the end of the 28 d treatment (Fig. 2). Conversely, the animals that were given the two globulins showed a reduction of 32·5 and 33 %, respectively (P < 0·001) in serum TC, while in those that received simvastatin the level was reduced by 20·3 % (P < 0·001). The levels of serum TAG were not significantly different in relation to the HC diet, except for the 7S-A group that showed a decrease of 17·8 % (P < 0·05).
Fig. 2. Serum total cholesterol (a) and TAG (b) levels of rats fed standard (STD) and hypercholesterolaemic (HC; high cholesterol and TAG) diets without (STD and HC) and with oral daily doses of cowpea 7S globulin (7S-C), adzuki 7S globulin (7S-A) or simvastatin (SVT) for 4 weeks. For details of diets, see Table 1. Values are means, with standard errors represented by vertical bars (nine rats per group). Mean value was significantly different from that of the HC diet group: * P < 0·05, *** P < 0·001 (one-way ANOVA and Bonferroni t test multiple-range comparisons).
Table 3 shows that the HC diet caused an increase of 5·86 times in the serum non-HDL-cholesterol of the animals, when compared with the STD diet (P < 0·001). Moreover, both 7S globulins reduced serum non-HDL-cholesterol by 46 % (P < 0·001), while only a 30·7 % reduction was observed with simvastatin (P < 0·001) relative to the HC diet. The HDL-cholesterol levels in the serum of the animals were affected by the HC diet that showed a decrease of 27 %, compared with the STD diet-fed group, while the 7S globulin-treated animals presented increases of 157 and 153 % for cowpeas and adzuki beans (P < 0·001), respectively, compared with the HC group, with values above the STD diet-fed group. By comparison, the animals from the simvastatin group increased these values only 18·5 % and below the values from the animals of the STD diet-fed group (Table 3). The atherogenic index, a marker of heart disease predisposition, increased 7·69 times by the effect of the hyperlipidic diet. Both proteins displayed a reducing effect on this parameter, with values 70·6 and 67 % lower than the HC group, respectively, and more efficiently than simvastatin treatment, as previously observed with soyabean proteins( Reference Ferreira, Silva and Demonte 23 , Reference Fassini, Noda and Ferreira 39 ).
Table 3. Serum, liver and faecal parameters and serum enzyme activities of rats fed a hypercholesterolaemic (HC; high cholesterol and TAG) diet without and with oral daily doses of 7S globulins or simvastatin (SVT) for 4 weeks†
(Mean values with their standard errors; nine rats per group)
7S-C, cowpea 7S globulin; 7S-A, adzuki bean 7S globulin.
Mean value was significantly different from that of the HC diet group: * P < 0·05, ** P < 0·01, *** P < 0·001 (one-way ANOVA and Bonferroni t test multiple-range comparisons).
† For details of diets, see Table 1.
‡ Atherogenic index = log10 (TAG/HDL-cholesterol).
Serum glucose levels were not significantly affected in the trial, in spite of a decrease in the insulin levels of the groups fed the HC diet relative to the STD diet (P < 0·001) (Table 3). Table 3 shows also that lipoprotein lipase activities were significantly higher in all groups that received the HC diet; however, this effect was greater in the group treated with 7S-C. The groups that received the drug simvastatin or adzuki bean protein had a lower effect and were similar to each other. The activating effect of the globulin and simvastatin on the enzyme activity reached values close to twice that observed in the animals of the HC group.
Liver parameters
The HC diet increased the levels of cholesterol and TAG in the liver relative to the STD diet (Table 3), while only the oral daily dose of 7S-C significantly reduced both parameters by 14 and 17 %, respectively (P < 0·05).
Faecal excretion
Faecal excretions of fat and cholesterol were considerably higher in the groups consuming the HC diet (Table 3). Approximately 2·50 to 2·95 times more cholesterol was eliminated by the HC diet group compared with the STD diet-fed group. TAG excretion was 11·2 and 17·6 % greater with respect to the groups receiving the proteins of cowpea and adzuki beans, respectively; while the group that ingested the drug simvastatin was 10·4 % greater (P < 0·05). In the case of cholesterol, faecal excretion had a very large increase in the hypercholesterolaemic groups compared with the STD diet-fed group; anyway, the groups receiving the drug simvastatin or the proteins still showed greater excretion than the HC group (Table 3).
Discussion
This study presents, as the main focus, comparative results on the cholesterol-lowering activities of two purified storage proteins from cowpeas and adzuki beans, respectively. Despite the limitations of the animal model and the very high-cholesterol diet used, the results are in line with similar ones on other legume seed proteins and highlight their potential in the cholesterolaemic control in humans, too. Indeed, while the use of drugs, such as statins, to positively affect cholesterol metabolism is well established and the mechanism of action is also well known( Reference Kobayashi, Chisaki and Narumi 40 – Reference Wang, Zhang and Xue 42 ), for various reasons, including the lack of positive effect on HDL-cholesterol( Reference Kobayashi, Chisaki and Narumi 40 ), the need of alternative drugs and approaches is crucial. Among these, the interest for food components capable of effectively facing the cases of dyslipidaemia, sometimes in association with established drugs, in order to reduce their side effects, is growing( Reference Redeout, Haranding and Marinangeli 41 ). The positive effects of soyabean proteins on the biochemical parameters related to altered metabolism of cholesterol and TAG are well described( Reference Harland and Haffner 6 , Reference Jenkins, Mirrahimi and Srichaicul 7 , Reference Aoyama, Kohno and Saito 13 , Reference Fukui, Kojima and Tachibana 14 , Reference Adams, Golden and Franke 43 – Reference Lovati, Manzoni and Corsini 45 ); however, only in a few cases have these proteins, or other 7S and/or 11S globulin fractions, been administered to animals in a purified form and in small doses( Reference Duranti, Lovati and Dani 10 – Reference Ferreira, Silva and Demonte 12 , Reference Kohno, Hirotsuka and Kito 17 , Reference Lovati, Manzoni and Gianaza 22 , Reference Moriyama, Kishimoto and Nagai 44 , Reference Fassini, Ferreira and Silva 46 ). Moreover, despite interest in the use of β-conglycinin-homologous proteins from other legume seeds is great, only a few studies have been conducted on the hypocholesterolaemic effect of cowpea and adzuki bean proteins( Reference Frota, Mendonça and Saldiva 8 , Reference Mahadevappa and Raina 47 – Reference Chau, Cheung and Wong 49 ). In an experiment using cowpeas as a unique protein source, the effect of the administration of crude flour and protein isolate resulted in a significant decrease in serum TC and non-HDL-cholesterol and an increase of faecal cholesterol in hamsters( Reference Frota, Mendonça and Saldiva 8 ). Contrary to this, Olivera et al. ( Reference Olivera, Canul and Pacheco 48 ) verified that a protein isolate from cowpeas, as the single protein source, did not alter the levels of TC and TAG in the serum of rats. In this case, however, animals fed a normolipidaemic diet were used. Meanwhile, Mahadevappa & Raina( Reference Mahadevappa and Raina 47 ) observed that the addition of whole cowpea flour, as a source of protein, in a hypercholesterolaemic and hyperlipidaemic diet caused a reduction of up to 55 % in serum TC in rats. Concerning the other source of protein, namely adzuki beans, Chau et al. ( Reference Chau, Cheung and Wong 49 ) observed no changes in serum TC, LDL-cholesterol and HDL-cholesterol levels in hamsters fed a hypercholesterolaemic diet by using a protein concentrate of this seed.
In order to clarify these controversial findings, in the present study only extensively purified 7S-C and 7S-A were given as a single daily oral dose to rats submitted to a hypercholesterolaemic and hyperlipidaemic diet for 28 d. It is worth remarking here that the administered doses represented only 2·75 % of the total protein ingested daily by the animals.
A number of body and serum parameters were evaluated in the present study. Among them, a significant decrease in food intake in rats receiving 7S-C and 7S-A compared with HC and STD diet-fed groups was observed. The lower daily food intake is probably responsible for the reduction in the average weight gain and final weight of the treated rats. Interestingly, some studies( Reference Nishi, Hara and Tomita 50 ) found that soyabean β-conglycinin presented a suppressing effect on food intake in rats via an increased cholecystokinin secretion. Hira et al. ( Reference Hira, Mori and Nakamori 51 ) verified that the 3 g intake of hydrolysed β-conglycinin was effective to increase satiety and reduce feelings of hunger in healthy human subjects. Also, Kohno et al. ( Reference Kohno, Hirotsuka and Kito 17 ) in a study with 126 volunteers found that β-conglycinin consumption for 4 weeks, in the form of a sweet, resulted in a significant reduction of visceral fat, thus contributing to the prevention of obesity. Additionally, β-conglycinin-enriched soyabean seeds can provide hydrolysates that limit fat accumulation in fat cells and inflammatory pathways in vitro, and therefore warrant further studies as a healthful food( Reference Martinez-Villaluenga, Dia and Berhow 52 ).
A key point in the present study is the finding that isolated 7S-C and 7S-A are capable of reducing serum cholesterol in hypercholesterolaemic rats as efficiently as simvastatin, as previously observed with rosuvastatin and β-conglycinin( Reference Ferreira, Silva and Demonte 23 ). Remarkably, these findings were obtained with a much lower dosage than that used in studies where β-conglycinin was used as the only source of protein in the diet( Reference Harland and Haffner 6 , Reference Aoyama, Kohno and Saito 13 , Reference Fukui, Kojima and Tachibana 14 , Reference Moriyama, Kishimoto and Nagai 44 ). Similarly, Duranti et al. ( Reference Duranti, Lovati and Dani 10 ) verified that low dosages of β-conglycinin and its α' subunit administered to hypercholesterolaemic rats decreased by 50 % the serum levels of cholesterol and TAG, comparably to clofibrate. Conversely, the reduction in TAG levels was greater for both β-conglycinin and 7S-A, while 7S-C and simvastatin did not significantly affect this parameter. These differences may denote subtle but relevant differences in the mechanism of action of the different globulins.
The increased faecal excretion of TC observed in the groups receiving the two globulins and simvastatin, compared with the HC group, is in line with the reduction of serum cholesterol levels. A similar conclusion has already been put forward with β-conglycinin( Reference Fukui, Kojima and Tachibana 14 ), glycinin( Reference Moriyama, Kishimoto and Nagai 44 ), black soya peptides( Reference Rho, Park and Ahn 53 ) and other legumes( Reference Dabai, Walker and Sambrook 54 ). Since a similar cholesterol-lowering effect has been monitored with both soyabean protein hydrolysate and the intact corresponding protein( Reference Nagaoka, Kiwa and Eto 55 ), bioactive peptides may affect both cholesterol metabolism and increase in faecal excretion, similarly to dietary fibres.
Increased levels of lipoprotein lipase observed in the animals that received 7S-C and 7S-A and simvastatin may be related to the reduction of TC and TAG in the serum of the animals, as also noted by Mochizuki et al. ( Reference Mochizuki, Maebuchi and Kohno 56 ).
7S-C and 7S-A promoted the reduction of the non-HDL-cholesterol fraction, 40 % greater than with β-conglycinin( Reference Ferreira, Silva and Demonte 23 ), 60 % greater than with the two statins and close to that of fenofibrate( Reference Ferreira, Silva and Demonte 11 ), in the same experimental model.
The reduction in serum insulin levels observed in the animals of the HC diet-fed group (Table 3) could have favoured the high values of the lipoproteins (non-HDL and HDL) in the serum. Various authors have observed the LDL-reducing effect of β-conglycinin, in vivo and in vitro. The possible mechanism has been inferred from the findings that the protein/peptides lead to an increase in mRNA expression of LDL receptors( Reference Jenkins, Mirrahimi and Srichaicul 7 , Reference Zhong, Liu and Ma 16 , Reference Lovati, Manzoni and Gianaza 22 , Reference Lovati, Manzoni and Corsini 45 , Reference Mochizuki, Maebuchi and Kohno 56 , Reference Lovati, Manzoni and Pizzagalli 57 ), thus causing a more efficient removal of the particles from the bloodstream, promoting the secretion of apoB-100 and consequently decreasing the secretion of VLDL-cholesterol( Reference Mochizuki, Maebuchi and Kohno 56 ).
The HDL-cholesterol fraction plays the important function of delivering cholesterol to the liver, thus increasing its catabolism. Statins, regardless of the dose, have been shown to have a small influence on the levels of the HDL-cholesterol fraction( Reference Xydakis and Ballantyne 58 ). Conversely, the combination statin/β-conglycinin showed a positive effect, resulting in a higher level of HDL-cholesterol in the serum of rats than the drug alone( Reference Ferreira, Silva and Demonte 23 ), and suggesting a synergic effect of the two molecules on cholesterol metabolism. The most surprising effect observed with 7S-C and 7S-A was with this parameter: indeed they increased HDL-cholesterol by 56 and 53 %, respectively, while simvastatin showed an expected minor effect (Table 3). Similarly, these proteins were more efficient than simvastatin in reducing the atherogenic index, as also observed with soyabean 7S and 11S isolated proteins( Reference Ferreira, Silva and Demonte 23 , Reference Fassini, Noda and Ferreira 39 ). This finding would suggest a synergic effect of these globulins with statins, as observed for β-conglycinin. Interestingly, the HDL-cholesterol values obtained with the globulins overcome even those of the group fed the normolipidaemic diet.
Despite the many studies with isolated 7S soyabean globulins and other seed proteins, a unique mechanism of action cannot be envisaged yet. As mentioned, different authors have shown a biochemical action via the inhibition of certain enzymes in the metabolism of cholesterol and TAG( Reference Torres, Torres-Villalvazo and Tovar 2 , Reference Aoyama, Kohno and Saito 13 , Reference Fukui, Kojima and Tachibana 14 , Reference Wang, Zhang and Xue 42 ). Also an action on gene expression, specially on hepatic mRNA of some key enzymes of sterol metabolism and LDL receptors( Reference Lovati, Manzoni and Gianaza 22 , Reference Consonni, Lovati and Manzoni 24 , Reference Moriyama, Kishimoto and Nagai 44 , Reference Martinez-Villaluenga, Dia and Berhow 52 , Reference Mochizuki, Maebuchi and Kohno 56 , Reference Lovati, Manzoni and Pizzagalli 57 ), as well as through modulation of transcription factors, such as sterol regulatory element-binding protein (SREBP)-1c and SREBP-2( Reference Torres, Torres-Villalvazo and Tovar 2 , Reference Martinez-Villaluenga, Dia and Berhow 52 , Reference Mochizuki, Maebuchi and Kohno 56 ), have been described. The observed hypocholesterolaemic effects of 7S-C and 7S-A matched those of β-conglycinin, both in vivo and in vitro ( Reference Duranti, Lovati and Dani 10 – Reference Ferreira, Silva and Demonte 12 , Reference Kohno, Hirotsuka and Kito 17 , Reference Lovati, Manzoni and Gianaza 22 , Reference Ferreira, Silva and Demonte 23 , Reference Moriyama, Kishimoto and Nagai 44 , Reference Martinez-Villaluenga, Dia and Berhow 52 , Reference Mochizuki, Maebuchi and Kohno 56 , Reference Lovati, Manzoni and Pizzagalli 57 ). Still, hypothesising a mechanism of action is untimely. Nevertheless, globulin administration in an oral daily dose suggests that biologically active peptides, arising from their digestion, could be responsible for the observed effects, as already mentioned by several authors( Reference Parolini, Manzini and Busnelli 1 , Reference Zhong, Liu and Ma 16 , Reference Lovati, Manzoni and Gianaza 22 , Reference Wang, Zhang and Xue 42 , Reference Nagaoka, Kiwa and Eto 55 , Reference Mochizuki, Maebuchi and Kohno 56 ). As a matter of facts, preliminary results in our laboratory would support the conclusion that these proteins affect mRNA levels of various enzymes such as fatty acid synthase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, HMG-CoA synthetase, as well as regulatory factors including SREBP-1c, SREBP-2 and LDL receptors (ES Ferreira, unpublished results). On the other hand, the globulin-induced increased faecal cholesterol and TAG excretion associated with lower cholesterol concentrations in the liver, especially for 7S-C, speaks in favour of a decreased re-absorption of intestinal lipids, as already described for soyabean bioactive peptides( Reference Takenaka, Nakamura and Yoshikawa 59 , Reference Singh, Vij and Hati 60 ), and also focuses on possibly different mechanisms of action between the two proteins, which will require further experimental work. In this respect, the interaction of extracted seed globulins with minor amounts of non-protein components, such as fibres, phytates and saponins, cannot be excluded, though the purity of 7S-C and 7S-A was 94 and 96 %, respectively (not shown).
Although the mechanisms of the cholesterol-lowering effects of legume seed storage globulins have not been made clear yet, it can be argued that, due to the large size of these molecules and their susceptibility to proteolytic enzymes, the observed effects could be attributed to peptides derived from their gastrointestinal digestion. Once absorbed and transported to the liver, these peptides could modulate the homeostasis of cholesterol, as already discussed by other authors for some proteins( Reference Singh, Vij and Hati 60 , Reference Nässl, Rubio-Aliaga and Sailer 61 ). Alternatively, other studies concluded that the reduction of serum cholesterol might be a consequence of a direct interaction between these peptides and cholesterol, or its catabolites, thus promoting its excretion( Reference Spielmann, Stangl and Eder 62 , Reference Kahlon, Smith and Shao 63 ). Further studies are needed to unequivocally identify the mechanism of action of these proteins on lipid metabolism.
In conclusion, the present study first showed that 7S-C and 7S-A in an isolated form and at low dosages are effective in reducing serum cholesterol levels in hypercholesterolaemic rats, thus confirming that this class of proteins, regardless of the species, may exert similar biological activities in animal models.
Acknowledgements
The authors acknowledge the careful proofreading of the text by Timothy John C. Roberts (MSc).
The present study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2009/11511-0, 2011/11779-3), E. S. F.'s fellowship was financed by the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; CAPES), Science without Borders Program at the Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Italy. The FAPESP and CAPES agencies had no role in the design, analysis or writing of this article.
The author's contributions were as follows: V. A. N., A. D. and M. D. designed the study; E. S. F. and A. L. S. A. performed the study; E. S. F. had responsibility for data acquisition, processing and final content of the manuscript; J. C. and C. F. Z performed data analysis; V. A. N., A. D. and M. D. analysed the data and wrote the manuscript.
The authors declare no conflict of interest.
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