Grain legumes and the prevention of cardiovascular disease

1 Introduction

Ripe seeds of the plant family Fabaceae, known commonly as `legumes’ or `pulses’, are major foodstuffs in most countries and are an indispensable supply of proteins for the third world population, as they contain the highest amount of proteins in edible vegetables (Belitz and Grosch, 1999).

In spite of their positive features, the consumption of grain legumes has decreased during the twentieth century in most industrialised countries: for example it was 7.3 kg per person per year in 1920 in France, whereas it is now less than 1 kg (Champ, 2001). The low consumption in Europe is due to a number of reasons, such as difficult digestibility, at least for some individuals, taste and, critically important, the long cooking time, in a society in which most people can dedicate to cooking only a very short period of time. However, in the last few years, a reduction of the consumption of food items of animal origin and a moderate increase in the consumption of vegetables, grain legumes included, has resulted as a positive consequence of the crisis of the meat market, mostly due to bovine spongiform encephalopathy (BSE).

As the incidence of cardiovascular disease, obesity and type II diabetes is increasing, it has become urgent to promote a more appropriate diet for the populations of industrialised countries. Pulses may have a very important role in this direction. In fact, based on a number of studies reporting beneficial effects induced by the use of soy proteins vs. animal proteins in the prevention of cardiovascular disease, the US Food and Drug Administration (FDA) recently validated (FDA, 1999) the health claim about the role of soy protein in reducing the risk of coronary heart diseases, mainly by reducing cholesterolaemia (Anderson et al., 1995; Bakhit et al., 1994; Sirtori et al., 1998).

This post will provide data on the nutritional value of major pulses, will discuss some problems related to the presence of antinutritional factors, and will discuss in detail the experimental and clinical literature supporting the beneficial role of soybean and other legumes in the prevention of cardiovascular diseases.

2 The main components of grain legumes

The composition of major grain legumes is shown in Table below. Soybean and lupin are particularly rich in protein, poor in starch and have a rather high content of unsaturated lipids; peanut is definitely rich in lipids, whereas the most abundant constituents of all the other legumes are digestible carbohydrates. The protein content of the least protein-rich legumes, runner bean and chickpea, is anyhow higher than 22 per cent, i.e. a value very seldom found in vegetables.

2.1 Proteins

Legume proteins are an important complement to cereal proteins in vegetarian diets or diets poor in animal proteins, because they have a satisfactory content of lysine and other essential amino acids, their only defect being the relatively low content of sulphur-containing amino acids (Gueguen and Cerletti, 1994). The bioavailability of legume proteins has been studied in detail especially with reference to their use as feed for monogastric animals. Their protein efficiency ratios (PER) are lower than those of animal proteins, such as casein and lactalbumin (Bhatty et al., 2000; Cuadrado et al., 2002; Marzo et al., 2002; Urbano et al., 2003). Indeed, when animal feeding is considered, a high value for this parameter is critically important, because increasing body weight in the shortest time is the main target. However, when dealing with human adults, the philosophy may be the opposite and a lower PER may mean less difficulty in maintaining a correct caloric intake and consequently correct body weight.

Purified legume storage proteins (chickpea 11S and 7S globulins, faba bean globulins and lupin globulins) and casein have been subjected to an in vitro enzyme (pepsin + pancreatin) digestion process. Protein digests were then used in a bicameral Caco-2 cell culture system to determine amino acid transport across cell monolayers. With digests from legume proteins, absolute amounts of aspartate, glycine and arginine transported were higher than those found in digested casein. These results confirm the in vivo observations that amino acids from legume proteins are probably absorbed at rates different from those in other proteins of animal origin such as casein (Rubio and Seiquer, 2002).

Fractionation of legume proteins using procedures based on differential solubility yields three fractions: albumins, globulins and glutelins, with globulins being predominant (Brooks and Morr, 1992). The total lack of prolamines makes legumes particularly useful for preparing gluten-free foods for coeliac individuals. Globulins in seeds function mostly as storage proteins, to be mobilised during the course of germination. Globulins can be separated by ultracentrifugation or chromatography into two major components present in all legumes: legumins (approximately 11S) and vicilins (approximately 7S). In soybean, legumins derive from a protein precursor, which is split into an acidic polypeptide A (pI : 5) and a basic polypeptide B (pI p 8.2). These two peptides are linked by a disulphide bridge between Cys (92) and Cys (424) and are regarded as subunits. Six subunits are assembled to give the 11S globulin (Belitz and Grosch, 1999). Strong homology exists between the 11S globulins of different legumes, although variable regions are found mostly in the A peptide, and the A/B cleavage site is highly conserved. Generally legumins are not glycosylated, but there are exceptions, for example the 11S fraction of lupin (Gueguen and Cerletti, 1994).

Percentage composition of main grain legumes
Systematic name Common name

Crude
protein

Lipids

Digestible carbohydrates

Crude fibre

Minerals

Arachis hypogea Peanuts 27.4 50.7 9.1 7.5 2.7
Glycine max Soybeans 39.0 19.6 7.6 16.6 5.5
Lupinus albus White lupins 44.7 12.5 13.6 38.3 5.0
Lupinus angustifolius Narrow-leaf lupins 31.1 6.0 5.3 14.7 3.5
Lupinus luteus Yellow lupins 50.2 6.1 8.4 30.7 4.1
Cicer arietinum Chickpeas 22.7 5.0 54.6 10.7 3.0
Lens culinaria Lentils 28.6 1.6 57.6 11.9 3.6
Phaseolus coccineus Runner beans 23.1 2.1 55.2 4.5 3.9
Phaseolus lunatus Lima beans 25.0 1.6 57.8 15.0 3.9
Phaseolus vulgaris Common beans 24.1 1.8 54.1 19.2 4.4
Pisum sativus Peas 25.7 1.4 53.7 18.7 3.0
Vicia faba Broad beans 25.7 1.5 46.5 10.3 5.6
Vigna unguiculata Cowpeas 24.6 1.8 52.0 5.0 3.4
Vigna radiata Mungo beans 26.9 1.6 46.3 5.1 3.6

Sources: Belitz and Grosch (1999); Chango et al. (1998); Gueguen and Cerletti (1994)

The sequences of many vicilins are well known too: they derive from the post-translational cleavage of a single precursor in some polypeptides. In the case of soybean (,3-conglycinin), these peptides are three, a, a’ and 3 and vicilins consist of three polypeptides, not linked by a disulphide bridge, which can be different or identical (hetero- and homopolymeric forms). Taking into consideration other legumes, the sequences of precursor proteins are highly homologous, but the cleavage sites are variable. The vicilins are always glycosylated, but at a different extent in different legumes (Gueguen and Cerletti, 1994).

Under non-denaturating conditions, the 11S and 7S globulins exhibit a tendency towards reversible dissociation/association, depending to a large extent upon the pH value and ionic strength. The 11S globulins are relatively more stable than the 7S globulins. The lower stability of 7S globulins is evident also during industrial processing. For example a comparison of native globulins of soybean with some industrial soybean protein isolates by 2D electrophoresis and MALDI-TOF (matrix-assisted laser desorption/ionisation time of flight) has shown that fragments deriving from the legumins are much more recognisable than those deriving from vicilins (Gianazza et al., 2003). This is crucially important, as vicilins are the major responsible components for the hypocholesterolaemic activity of soybean proteins (Lovati et al., 1992).

2.2 Carbohydrates

Most cultivated legumes contain 39±51 per cent starch in raw seeds, with the exception of soybean and lupin. Legume starch has a higher content of amylose (30±45 per cent) versus cereals and potato (20±30 per cent). A higher amylose content means: (a) a higher temperature for completing gelatinisation of starch granules, and (b) a higher susceptibility to retrodegradation with the consequence of increasing resistance to endogenous amylases (Champ, 2001). Starch remains undigested in the small intestine, and is fermented by the colonic microflora when it reaches the large intestine. One characteristic of the fermentation of resistant starch is the formation of short-chain fatty acids, such as butanoate, known as the main nutrient of the colonocyte and potentially involved in the protection of colon from some diseases, particularly cancer (Bird et al., 2000).

Pulses are also a source of non-starch polysaccharides (NSP), for example lentils and beans contain 10.6 and 17.3 per cent NSP (on dry seed), 12.4 and 26.3 per cent being soluble NSP, respectively (Champ, 2001). They are contained in part in the hull of seeds rich in insoluble polysaccharides, mostly cellulose, and in part in cell walls more rich of soluble fibres, such as peptic polysaccharides. These fibres decrease the bioavailability of starch determining the low glycaemic index (GI) of legumes (Guillon and Champ, 2002), varying between 18 and 56, whereas food based on cereals have GI values in the range 65±95. This is beneficial in diabetes and may be beneficial also for the prevention of cardiovascular disease. For example, a decrease of the GI of diets of hyperlipidaemic patients from 82 to 69 units for a 1-month period resulted in a significant reduction in total and low-density lipoprotein (LDL) serum cholesterol and triglycerides, compared with mean lipid values for the preceding and following months (Jenkins et al., 1985).

2.3 Lipids

Peanuts, soybeans and lupin seeds are also interesting sources of lipids, a fact that for peanuts and soybean has a significant economic impact. White lupin oil seems particularly promising because it has a composition close to dietary recommendations for cardiovascular prevention (> 50 per cent oleic acid, > 17 per cent linoleic acid, > 7 per cent linolenic acid), however, to our knowledge, it is not commercially available.

3 The non-nutritional components of legumes

Unlike animal proteins, whose nutritional value is largely determined by their amino acid composition, the full nutritional potential of legume protein is

attained only after a certain amount of heat has been applied (Liener, 1994; Wang and McIntosh, 1996). In fact there are a number of components in legumes that can exert a negative impact on the nutritional quality of proteins and have to be inactivated by heat at least in part. The heat-labile factors are protease inhibitors, lectins, goitrogens, antivitamins; the heat-stable compounds saponins, tannins, phytoestrogens, flatulence factors, phytate, allergens (Champ, 2001). In addition, the industrial procedures may produce some new compounds with toxicological relevance, such as lysinoalanine (Arnoldi, 2002).

Triglyceride percentage composition of oil-rich legumes  
Fatty acid White lupin (%) Yellow lupin (%) Narrow leaf lupin (%) Soybean (%)

Peanut

(%)

16:0 7.8 4.8 11.0

10

10

18:0 1.6 2.5 3.8

5

3

18:1 (n-9) 53.0 21.0 38.2

21

41

18:2 (n-6) 17.2 47.5 37.1

53

35.5

18:3 (n-3) 9.5 7.5 5.3

8

0

20:0 & 20:1 5.5 4.5 1.2 4.0

2.5

22:0 & 22:1 (n-11) 5.8 7.9 1.9

±

3

n-3/n-6 0.55 0.16 0.14 0.15

0

Source: data from Hudson et al. (1983); Belitz and Grosch (1999).

3.1 Inhibitors of protease

Inhibitors of proteases are present in many vegetables and in all legume seeds. In soybean there are two main inhibitors, the Kunitz inhibitor (MW 21 500 da) and the Bowman-Birk inhibitor (8 000 da), but other inhibitors have been characterised in peanuts, chickpea, common bean, runner bean, lima bean, broad bean and pea. Trypsin and a-chymotrypsin are the main targets of this activity and the inhibitor content depends on the variety, degree of ripeness and storage time. The probable function of these inhibitors in the seeds is the protection against damage by higher animals, insects and micro-organisms. They have to be destroyed by heat treatments, which improve considerably the PER determined on the growth of rats. Moreover, soybean proteases inhibitors increase the size of the acinar cells of pancreas as well as their number (hyperplasia) (Liener, 1994).

3.2 Lectins

Although protease inhibitors are generally considered to be the main antinutritional factors in legumes, there is clear evidence that other components are responsible for growth inhibition. They are lectins, a class of proteins widespread in vegetables, which have the unique property of binding carbohydrate-containing substances. In particular they have the ability to agglutinate the red blood cells from various animal species, because of the interaction of multiple binding sites on the lectin molecules with specific glycoconjugate receptors on the surface of cell membranes. Most lectins are glycoproteins. When their molecular weight exceeds 30 kda, they consist of several subunits (Belitz and Grosch, 1999). Lectins bind to the epithelial cells on the intestinal wall, causing deleterious nutritional effects by interfering with nutrient absorption. The lectins of soybean and common bean are particularly toxic, whereas other legumes, such as lupin, have very small amounts of these factors (Muzquiz et al., 1998).

3.3 a-Galactosides

All legumes contain a certain amount of a-galactosides of the raffinose family, composed of a sucrose molecule linked to 1±3 molecules of galactose (raffinose, stachiose, verbascose). The a-galactosidase, necessary to hydrolyse the a1±6 linkage, is not available in the small intestine. As a consequence, these compounds reach the large intestine, where they are fermented to produce gases, giving the well-known flatulence experienced by many people when eating legumes. In this respect a-galactosides are rated as antinutrients; however, they are also prebiotic because they stimulate the growth of lactic bacteria, especially bifidobacteria, in the colon (Champ, 2001).

3.4 Other constituents

Legumes contain other minor constituents, which contribute to defend the seeds from insects or fungi, such as saponins or isoflavonoids, rather common in many legumes, or quinazolidine alkaloids, specific of lupin. They are a family of about 100 bitter compounds containing a rather uncommon bicyclic structure. Wild species may contain more than 600 mg/kg of alkaloids, but modern domesticated varieties are called `sweet’ because, by careful breeding, the alkaloid content has been reduced to less than 130 mg/kg, well below the current maximum concentration permitted in Australia of 200 mg/kg. The Australia New Zealand Food Authority has proposed a provisional tolerable daily intake of 0.035 mg/kg/ day for these substances (Australia New Zealand Food Authority, 2001). Lupins are an interesting source of protein concentrates and isolates: the acidic work-up during their separation from the flour reduces their presence to a minimum.

Isoflavonoids are congeners of genistein that have a protective role in seeds and plants and are extensively biosynthesised in response to an abiotic or biotic stress (phytoalexins). Although they have been rated as useful components of legumes for the prevention of some diseases, such as breast cancer, osteoporosis and menopausal hot flushes, in the last few years a number of papers have raised the issue of potentially serious toxicological problems (Fort et al., 1990; Kulling et al., 1999; Kumi-Diaka et al., 1999; Newbold et al., 2000; Sirtori, 2001). These data have suggested several legislative interventions to reduce their consumption (Working Group UK, 2002). However, it should be emphasised that high amounts of isoflavone are typical of soybean, while other legumes have a much lower content .

4 The use of soybean protein in the prevention of hypercholesterolaemia

Legume proteins, particularly soy proteins, reduce plasma cholesterol both in animals, when it is elevated by dietary means (high cholesterol intake, semi-synthetic diets, etc.) (Kim et al., 1980; Terpstra et al., 1982), and in patients with hypercholesterolaemia of monogenic or polygenic origin (Sirtori et al., 1998).

The soybean diet, as of now, is certainly the most effective dietary tool for treating hypercholesterolaemia and provides a unique opportunity for the management of very young patients and also for exploring new mechanisms in plasma cholesterol regulation. The validity of this therapeutic approach was recently supported by the US FDA approving the health claims about the role of soy protein in reducing the risk of coronary heart disease (FDA, 1999).

Isoflavone content of grain legumes

Legume                           Daidzein ttg/g                 Genistein ttg/g

Arachis hypogea                      0.50                              0.82

Glycine max                          105-560                       268-841

Lupinus albus                         ND                                trace

Lupinus luteus                        ND                                trace

L. angustifolius                       ND                                trace

Cicer arietinum                      0.11-1.92                     0.69-2.14

Lens culinaria                         0.03                              0.07

Phaseolus lunatus                  0.12-0.89                     0.10-0.19

Phaseolus vulgaris                 0.07-0.40                     0.07-5.20

Pisum sativum                       0.04-0.08                     ND-0.23

Vicia faba                              0.16-0.32                        trace

Vigna radiata                         0.30-0.36                     0.16-0.60

Vigna unguiculata                  0.21-0.30                     0.11-0.56

ND = not detectable.

Source: Mazur et al. (1998); Katagiri et al. (2000).

The earliest studies by Sirtori and co-workers (Sirtori et al., 1977) clearly established that patients with elevated cholesterolaemia (total cholesterol above 7.8 mmol/L) show the most favourable response to the substitution of animal proteins with isoflavone-free soybean proteins. The initial study, a crossover trial under metabolic ward conditions, showed a 20-22 per cent reduction of total cholesterol, with no change of triglycerides (TG) and a 22-25 per cent fall of low-density lipoprotein-cholesterol (LDL-C) (Sirtori et al., 1977). In a small group of patients the effect of the addition of cholesterol to the soybean protein concentrate was also investigated. They received 500 mg of cholesterol daily, either in the first 3 weeks or in the last 3 weeks of administration of the diet, this addition did not appear to influence the hypocholesterolaemic response.

The results of these metabolic ward studies were confirmed, later on, in a large investigation on 127 outpatients treated for 8 weeks with a similar soy protein regimen within a low lipid diet (Descovich et al., 1980). A mean reduction of cholesterolaemia of 23.1 per cent in the 67 participating males and of 25.3 per cent in the 60 females was detected. Again, no significant changes in plasma TG, high-density lipoprotein-cholesterol (HDL-C) or body weight were recorded. In this study, it was possible to monitor the trend for the successive return of cholesterolaemia to baseline. This occurred in most patients 6-8 weeks upon switching to a low-lipid diet with animal proteins, and was definitely accelerated in patients with familial hypercholesterolaemia (FH).

Anderson et al. (1995) have analysed a total of 38 studies, both in participants with elevated plasma cholesterol and in normolipidaemic volunteers, all treated for a variable length of time with a diet with partial or total substitution of animal proteins with soy proteins. The reviewed studies ranged from the evaluation of fibre-like properties of soybean proteins, to hormonal studies, to the more recent clinical reports linking LDL receptor activation to the remarkable cholesterol-lowering properties of this diet. The conclusions of this meta-analysis, confirm that serum and LDL-C concentrations are modified according to baseline cholesterolaemia, from a minimum of —3.3 per cent in subjects with cholesterol in the normal range, way up to —19.6 per cent (LDL-C —24 per cent) in people with clear-cut hypercholesterolaemia. Normolipidaemic individuals do not respond to the cholesterol-lowering effect of soy protein (Anderson et al., 1995).

An interesting area for the use of the soybean protein diet has been the treatment of pediatric hypercholesterolaemia and of hypercholesterolaemia secondary to kidney disease. In an Italian multicentre study on 18 pre-puberal children a reduction of cholesterolaemia around 25 per cent or more was constantly achieved (Gaddi et al., 1987). These findings were later confirmed by Widhalm et al. (1993), who evaluated a similar regimen in 23 children with familial or polygenic hypercholesterolaemia. The LDL-C reduction was 22 per cent when the soy protein diet preceded the standard lipid-lowering diet, and 25 per cent, when it was given as the second treatment.

4.1 Mechanism of action

Two studies have addressed, both in a direct and an indirect way, the potential of the soy protein diet to increase LDL receptor expression in humans. In the first study, with a similar protocol as in previously described investigations in rodents (Sirtori et al., 1984), hypercholesterolaemic patients were treated in a crossover protocol, with either animal proteins or soy protein concentrates (this time with the addition of cholesterol, in order to normalise dietary fat intake) (Lovati et al., 1987). Besides plasma lipid changes, LDL degradation by mononuclear cells after each diet was monitored. During the animal protein intake, there were hardly any changes in LDL-C levels or receptor activity, but with the soy protein diet, in the presence of an elevated cholesterol intake, treated patients showed a consistently raised degradation of LDL by mononuclear cells, about 8-fold higher than the reference diet. Therefore this study confirms that some factor/s in soy proteins may exert an up-regulation of LDL receptors.

It seems, therefore, reasonable to suggest that the mechanism of action of the soy protein diet in humans is not at the intestinal or endocrine level, but rather that it involves a stimulatory effect on LDL receptors, chronically depressed in hypercholesterolaemia. This is one further reason why normolipidemic individuals respond to a limited extent to the regimen; in the presence of LDL receptor down-regulation, e.g. by cholesterol loading in normolipidemics, the hypocholesterolaemic activity becomes apparent.

Based mainly on studies in monkeys (Anthony et al., 1996), the 1995 meta-analysis (Anderson et al., 1995), suggested that up to 60 per cent or more of the dietary effect on cholesterolaemia might be linked to the presence of isoflavones, i.e. genistein and daidzein, and/or their conjugates (aglycones and glucosides) (Wang and Murphy, 1994). However, the same author has very recently changed his opinion (Anderson, 2003)

The majority of the reported clinical studies in the same meta-analysis were carried out with soy concentrates or isolates, i.e. with dietary formulations containing minimal amounts of isoflavones, i.e. less than 40 µg/g (Sirtori et al., 1995, 1997). Many studies have compared soy protein isolates or concentrates with a normal isoflavone content, soy protein isolates or concentrates depleted in isoflavones and other proteins (mainly casein or lactalbumin) added with isoflavones, giving controversial results (Adlercreutz et al., 1987; Crouse et al., 1999; Greaves et al., 1999). A general problem of these studies is that none reports a detailed investigation of the consequences induced on the structure of the proteins by the processing used to eliminate the isoflavones (mostly extraction with boiling ethanol). A different approach has been used by Fukui et al. (2002) in order to investigate whether isoflavones are responsible for the hypocholesterolaemic effect of soy protein. These authors prepared an isoflavone-free soy protein isolate (IF-SPI) by column chromatography. The study was conducted in rats in comparison with casein and a standard soy protein isolate (SPI). Plasma total cholesterol concentrations of rats fed SPI and IF-SPI were comparable and significantly lower than those of rats fed casein. Thus, the cholesterol-lowering effect of SPI in rats can be attributed entirely to their protein content.

Final confirmation of the unacceptability of the isoflavone hypothesis has come from the repetition of the primate experiments in more appropriate conditions. Greaves et al. (1999) showed that addition to a casein diet of a semipurified ethanol extract of soy, rich in isoflavones, failed to improve cholesterolaemia in ovariectomised cynomolgus monkeys vs. intact soy proteins. The same authors more recently confirmed that a soy protein diet reduces cholesterolaemia in ovariectomised adult female cynomolgous monkeys, also by partially inhibiting cholesterol absorption, whereas a semipurified soy extract, rich in isoflavones, added to a casein diet does not exert any lipid lowering effect (Greaves et al., 2000). The recent lack of confirmation of the hypothesis of positive vascular effect of an isoflavone-rich diet in postmenopausal women (Simons et al., 2000), provided definitive evidence against any role of isoflavones in the beneficial effects of soy proteins, possibly suggesting that these products, of very dubious benefit and associated with potential risk, should not be freely available (Ginsburg and Prevelich, 2000).

Other studies have been devoted to evaluating the possible responsibility of proteins per se in the reduction of cholesterolaemia. This hypothesis seems reasonable in the face of evidence (Potter et al., 1996), indicating that the presence of additional components, besides protein in the diet, might not affect in a significant way the plasma cholesterol reduction achieved with the diet.

In agreement with the results of animal and human studies, a number of experiments have been performed in a hepatoma cell line (HepG2), an in vitro model of human liver cells, highly sensitive to factors regulating LDL receptor expression and cholesterol biosynthesis/breakdown, by tracking the uptake and degradation of labelled LDL, in order to identify the soy protein component/s potentially responsible for the cholesterol-lowering effect. It has been concluded that the 7S globulin directly up-regulates the LDL receptor. This effect is exerted both on HepG2 cells and also, albeit to a lesser extent, on human skin fibroblasts (HSF), and is paralleled by an enhanced LDL degradation (Lovati et al., 1992).

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Another study by the same group has examined the effect of 7S soy globulin subunits vs. the whole 7S on the up-regulation of lipoprotein uptake and degradation in Hep G2 cells. This experiment clearly indicated that incubation of cells with purified a + a’ subunits from 7S markedly increases uptake and degradation of 125I-LDL, whereas the 0-chains are ineffective (Lovati et al., 1998). These experiments may also open the way to the development of soybean varieties with different ratios among the major globulins, possibly resulting in cultivars with improved cholesterol-lowering potential. Interestingly, a soy cultivar mutant, Keburi, devoid of the a’ subunit, had no activity of this sort, thus possibly suggesting development of soy cultivars rich in the a’ subunit (Manzoni et al., 1998). This indirect result has been recently confirmed by a direct methodology. In an experiment with HepG2 cells, the up-regulation of LDL receptors by the a’ subunit was significantly greater than that found in control cells. In addition, this study revealed a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, both involved in cell protection against oxidative and other stresses (Manzoni et al., 2003).

In order to assess the final identity of the putative peptide/s responsible for the biochemical effect, experiments have been performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin, or to peptides coming from the in vitro digestion of CroksoyR70, a commercial isoflavone-poor soy concentrate, routinely used by Sirtori and co­workers in the dietary treatment of hypercholesterolaemic patients. Increased I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10-4 mol/L, MW 2271 Da) corresponding to the 127±150 positions of the a’ subunit of 7S globulins (Lovati et al., 2000). Cells exposed to CroksoyR70 enzyme digestion products showed a more marked up-regulation of LDL receptors than controls (Lovati et al., 2000). These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Evaluation of the LDL receptor stimulatory activity of the major soy isoflavone, genistein, up to concentrations of 1 mg/mL, failed to demonstrate any evident change.

In view of these results, it would be very useful to evaluate in detail whether the ethanol extraction of isoflavones from the soy protein isolate also removes some biologically active peptides. From a practical point of view, it is important to underline that these results have stimulated the industrial interest for patents for soy protein isolates and concentrates with a very high content of ~i­conglycinin (Bringe, 2001).

5 The hypocholesterolaemic activity of other legumes

In view of the high homology of legume vicilins, it is reasonable to foresee that other legumes may exert a biological activity similar to soy proteins. Experimental data in this field are rather scarce, especially considering the vastness of the literature dealing with soybean. In part this may be due to the prejudice of the need of isoflavones that are generally rather scarce in the other legumes. However, especially in the last few years some investigations have been published both on experimental animals and in humans. Most studies have been performed on growing rats fed a normal diet or on adults fed a hypercholesterolaemic one (Nath et al., 1959).

The effects ofLupinis angustifolius has been studied by Rahman et al. (1996). In rats pair-fed for 10 days on cholesterol-free diets containing lactalbumin, raw lupin seed meal or five different semi-purified lupin fractions, a significant lowering effect on total plasma cholesterol was observed in growing rats fed the seed meal fractions compared with the value obtained from the lactalbumin control. In particular a fraction, containing -y-conglutin lowered total plasma cholesterol by 34 per cent compared with the lactalbumin-fed group. Liver lipid and cholesterol were also found to be decreased in rats fed L. angustifolius seed meal and its fractions.

Yellow and white lupin meals have been studied by Chango et al (1998) in rats fed cholesterol-rich diets. Differences among the total blood serum cholesterol levels of rat groups fed these diets for 28 days were not significant. Compared to casein and yellow lupin diets, the white lupin diet decreased plasma triglyceride levels and the insulin/glucagon ratio, as well as non­esterified liver cholesterol and plasma LDL triglycerides levels. The yellow lupin diet increased plasma glucose and insulin, as well as liver total cholesterol compared to the casein and white lupin diets.

Characteristics of studies on rats fed pulses
Entry Literature Type of pulse Preparation Amount Duration Control Cholesterol
        (%) (days) ingredient content
1 Mokady & Liener, 1982 Glycine max Meal 10 28 Casein 2%
2a Lovati et al., 1992 Glycine max 7S globulins (a) 14 Casein 1%
2b Lovati et al., 1992 Glycine max 11S globulins (a) 14 Casein 1%
3 Fukui et al., 2002 Glycine max Protein isolate (c) 20 14 Casein 0.5%
4 Sirtori et al., 2004 Lupinus albus Protein isolate (b) 21 Casein 1%
5a Chango et al., 1998 Lupinus albus Meal 45 28 Casein 1%
5b Chango et al., 1998 Lupinus luteus Meal 40 28 Casein 1%
6a Rahman et al., 1996 Lupinus angustifolius Meal 36 10 Lactalbumin 0%
6b Rahman et al., 1996 L. angustifolius Protein isolate 15 10 Lactalbumin 0%
6c Rahman et al., 1996 L. angustifolius Conglutin-y 12 10 Lactalbumin 0%
7 Zulet et al., 1999 Cicer aretinum Meal 70 16 Casein 1%
8a Lasekan et al., 1995 Pisum sativum Protein isolate 24 28 Casein 1%
8b Lasekan et al., 1995 P. sativum Protein isolate 24 28 Casein 0%
9 Alonso et al., 2001 P. sativum Meal 57 15 Casein 0%
10a Macarulla et al., 2001 Vicia faba Meal 68 14 Casein 1%
109b Macarulla et al., 2001 V. faba Protein isolate 23 14 Casein 1%
11a Dabai et al., 1996 Phaseolus vulgaris Meal 33 56 Casein 1%
11b Dabai et al., 1996 Pisum sativum Meal 33 56 Casein 1%
11c Dabai et al., 1996 Lens culinaria Meal 33 56 Casein 1%
11d Dabai et al., 1996 Phaseolus lunatus Meal 33 56 Casein 1%

(a)  30mg/kg by gavage.

(b)  50 mg/kg by gavage.

Studies on rats fed diets containing pulses: serum lipids, glucose, insulin and liver cholesterol
Entry

Cholesterol

Baseline                    Change (%)
value

LDL-cholesterol

Baseline   Change (%)
value

HDL-cholesterol

Baseline            Change (%)
value

Triglycerides

Baseline    Change (%)
value

1 1.65 -48.04 ND

ND

0.21 - 5.00 2.25 -16.08
2a 4.19 -34.80 ND

ND

ND

ND

0.61 +11.47
2b 4.19 -32.70 ND

ND

ND

ND

0.61 + 4.92
3 3.84 -22.70         1.65 -10.30
4 5.60 -22.69 4.90 -30.16 0.70 +29.63 0.84 -16.22
5a 2.47 -17.81 ND

ND

ND

ND

1.11 -49.13
5b 2.47 -11.34 ND

ND

ND

ND

1.11 -12.31
6a 1.96 -15.82 ND

ND

ND

ND

0.19 -10.53
6b 1.96 -29.08 ND

ND

ND

ND

0.19 + 5.26
6c 1.96 -34.18 ND

ND

ND

ND

0.19 + 5.26
7 3.64 -34.06 2.27 -43.17 0.90 -17.78 2.36 -20.34
8a 4.14 -26.88 ND

ND

1.17 -57.78 1.78 -40.13
8b 3.97 -61.43 ND

ND

1.35 -30.67 1.51 -47.37
9 2.07 -17.50 0.54 -66.67 1.45 +5.66 0.795 -24.28
10a 3.48 -36.78 2.54 -56.30 0.92 +3.26 1.20 -39.17
10b 3.48 -29.89 2.54 -36.61 0.92 -9.78 1.20 -10.83
11a 6.72 -36.31 4.87 -52.99 0.98 +41.84 0.818 -39.12
11b 6.72 -13.99 4.87 -26.90 0.98 - 8.16 0.818 -11.37
11c 6.72 - 6.70 4.87 -32.24 0.98 -2.04 0.818 -33.25
11d 6.72 -23.96 4.87 -38.81 0.98 +40.82 0.818 -2.81

A protein isolate from Lupinus albus was analysed by Sirtori et al. (2004) using a pharmacological approach. Rats fed a hypercholesterolaemic diet containing 20 per cent casein and treated daily by gavage with 50 mg/rat of a lupin protein isolate for 14 days compared with vehicle only. Lupin-treated rats had 167 mg/dl total cholesterol and 62 mg/dL triglycerides, versus 216 mg/dL total cholesterol and 74 mg/dL triglycerides of controls, whereas glucose was unaffected. The daily dose given to animals is particularly low, comparable to that of some well-known lipid-lowering drugs, such as fibrates (Staels et al., 1992). Isolated lupin protein fractions were also able to up-regulate the LDL receptors in HepG2 cells (Sirtori et al., 2004). White lupin proteins seem therefore promising hypocholesterolaemic nutraceuticals.

The hypercholesterolaemic rat model was also used to study yellow pea (Pisum sativum): the diet contained 20 per cent pea proteins or casein. Pea proteins reduced cholesterol and triglycerides by 27 per cent and 40 per cent respectively, when cholesterol was included in diets. Plasma glucose and insulin levels were slightly lower in rats fed pea proteins versus those fed casein, apo Al level were also lower in rats fed pea proteins (Lasekan et al., 1995).

Broad beans were studied for the first time in 1985 (Mengheri et al., 1985). Recent work has compared the hypocholesterolaemic efficiency of a Vicia faba­protein isolate compared with the intact legume (Macarulla et al., 2001). The protein isolate was prepared by isoelectric precipitation and spray dried. Rats fed on Vicia faba diets showed significantly lower body weights and energy intakes than rats fed casein. The whole seed diet induced a significant reduction in plasma triglycerides. Feeding dietary hypercholesterolaemic rats with diets containing faba bean seeds, or the protein isolate, induced a significant decrease of plasma (LDL+VLDL)-cholesterol (from 2.54 mmol/L of the casein + cholesterol diet to 1.11 mmol/L and 1.61 mmol/L respectively), but not of HDL-cholesterol. Liver cholesterol and triglycerides were also reduced. The faba bean-protein isolate was useful in improving the metabolic alterations induced by feeding a hypercholesterolaemic diet, compared with casein, but the effectiveness of whole seeds was higher as that of the protein isolate (Macarulla et al., 2001).

Another legume that has been studied in detail is chickpea (Zulet et al., 1999). The study was performed in rats fed a cholesterol-rich diet for 42 days. Lipid levels were markedly improved by feeding a chickpea diet for 16 days and liver glycogen deposition was also re-established. Data concerning carbohydrate utilisation indicated potential positive effects for diabetes therapy.

Dabai et al. (1996) have compared the hypocholesterolaemic effects of diets containing four different legumes: baked beans (Phaseolus vulgaris), marrowfat peas (Pisum sativum), lentils (Lens culinaria Medik) or butter beans (Phaseolus lunatus) in hypercholesterolaemic rats fed for 8 weeks. All experimental diets were effective, but diets containing baked beans and butter beans were more potent at lowering raised cholesterol levels than diets based on marrowfat peas and lentils. Differences in cholesterol-lowering capacity of the various legume diets in this experiments were not associated with larger concentrations of faecal bile acids or neutral sterols. However, there was evidence that the inclusion of legumes in the diets reduced fecal excretion of secondary bile acids.

The general impression is that most legumes have an effectiveness very similar to soybean in rats fed hypercholesterolaemic diets and that this area is worthy of more detailed investigations in order to single out the bioactive component(s) of each legume.

Another very useful model is the pig as developed by Kingman et al. (1993). Thirty-six growing boars were randomly allocated, in groups of six to six diets, eaten continuously for 42 days. The diets fed were: (1) a semipurified (SP; control group 1) diet, (2) SP + 10 g cholesterol/kg (control group 2), and (3), (4), (5) and (6) SP + cooked legumes (70:30, wt./wt.; baked beans (P. vulgaris), peas (P. sativum), lentils (L. culinaria), and butter beans (P. lunatus)] + 10 g cholesterol/kg. Fasting blood samples were taken on days 0, 14, 28 and 42 for the detection of total plasma cholesterol, VLDL-, LDL- and HDL-cholesterol, and triglycerides. Between days 7 and 11 and days 28 and 32 complete 5-day faecal collections were made for the measurement of neutral, acidic and conjugated steroids. After 42 days, total cholesterol and VLDL + LDL­cholesterol levels (Figs 20.2) were raised significantly in all groups, but to different extents. Compared with control group 2, diet-induced hypercholesterolaemia was significantly inhibited in the groups consuming baked beans, peas, and butter beans, although HDL-cholesterol levels were unchanged. Faecal steroid excretion by the legume groups was not significantly different from that of control group 2. This agrees with the results in rats (Dabai et al., 1996) and suggests that the mechanism for the hypocholesterolaemic effect does not involve increased hepatic bile acid synthesis and increased cholesterol clearance via the intestinal route, but probably rather involves a cellular mechanism, i.e. LDL-receptor up-regulation as already observed for soybean (Lovati et al., 1987) .

5.1 Clinical studies

Anderson and Major (2002) have very recently published a meta-analysis of all published clinical studies on pulses, in total 11. The reader is recommended to read this paper to have a complete overview of available data in this field. As already indicated above, the meta-analysis on soy (Anderson et al., 1995) has clearly shown that a clear-cut hypercholesterolaemia, related to a chronic depression of LDL receptors, is necessary to achieve an evident hypo­cholesterolaemic effect. Taking this into consideration, studies on normolipidaemic subjects will not be considered here. The experimental designs of these studies are quite varied, but a common feature is that whole seeds are considered, thus not helping in singling out which component(s) is (are) responsible for the observed effects. In addition several of these studies take into consideration mixed legumes or beans and oat-bran.

Study 1 (Anderson et al., 1984) was based on oat-bran and beans. After a control diet, 20 hypercholesterolaemic men (average cholesterol value 298 mg/ dL) were randomly allocated to oat-bran or bean-supplemented diets for 21 days on a metabolic ward. Control and test diets provided equivalent energy, fat and cholesterol, but test diets had twice more total and 3-fold more soluble fibre. Bean diets decreased total cholesterol concentration by 18.5 per cent and LDL­cholesterol by 23 per cent. Triglycerides were in contrast unchanged.

Study 2 (Anderson et al., 1990) was carried out on canned beans; 24 hyperlipidaemic men (average cholesterol value 295 mg/dL) ate one of three bean diets for 21 days in a metabolic ward. Diets A and B included 227 g canned beans (120 g beans with 107 g tomato sauce) daily, in a single dose for diet A and in a divided dose for diet B. Diet C included 182 g canned beans (162 g beans with 20 g tomato sauce) daily in a divided dose. Diets B and C, the most effective, lowered total and LDL-cholesterol and triglycerides by about 10 per cent.

In study 3 (Jenkins et al., 1983) seven male mildly hyperlipidaemic patients (average cholesterol value 268 mg/dL) substituted approximately 140 g dried beans daily for other sources of starch in their diet over a 4-month period. After this, mean fasting triglycerides were reduced by 25 per cent, while total and LDL-cholesterol levels were 7 per cent lower than values during the previous five clinic attendances. While taking beans, a nonsignificant fall (0.7 kg) was seen in body weight. Nevertheless no change was seen in macronutrient intake determined by 1-week diet histories recorded both before and four times during the study, although cholesterol intake decreased by 80 mg.

   Characteristics of clinical studies on hypercholesterolaemic or mild hypercholesterolaemic subjects on diets containing pulses
Entry Literature Type of pulse Preparation

Amount
(g/day)

Number of subjects Duration (days)
1 Anderson et al., 1984 Beans Cooked

120

10

21

2 Anderson et al., 1990 Beans Canned

69

24

21

3 Jenkins et al., 1983 Mixed beans Cooked/canned

140

7

120

4 Cobiac et al., 1990 Beans Canned

145

20

28

5 Mackay and Ball, 1992 Beans + oat bran Cooked

80

39

42

6 Fruhbeck et al., 1997 Field beans Raw/cooked

90

10

30

7 Oosthuizen et al., 2000 Common beans Extruded

110

22

28

Clinical studies on hypercholesterolaemic or mild hypercholesterolaemic subjects: serum lipids responses
Entry

Cholesterol

  LDL-cholesterol HDL-cholesterol

Triglycerides

 
  Control Change Control Change Control Change Control Change
  value (%) value (%) value (%) value (%)
  (mmol/L)   (mmol/L)   (mmol/L)   (mmol/L)  
1 7.73 -18.5 5.71 -23.12 0.83 -12.05 2.63 -3.04
2 7.63 -10.4 5.19 -8.4 1.08 -6.9 2.89 -10.80
3 6.95 -7.1 4.88 -4.80 0.97 +14.50 3.03 -25.00
4 6.32 -0.47 4.63 -0.43 1.26 -3.17 1.25 +0.80
5 6.27 -0.80 4.19 0.00 1.15 +10.43 1.52 -0.66
6 6.23 -6.90 4.37 -7.32 1.05 +15.23 1.78 -32.58
7 6.15 0.00 4.87 +4.52 0.89 -5.62 1.64 +6.70

In study 4 (Cobiac et al., 1990) the plasma cholesterol-lowering potential of canned baked beans was examined in a crossover comparison with canned spaghetti. The difference in total dietary non-starch polysaccharide (NSP) of 12 g daily (6.6 g difference in soluble NSP), was insufficient to alter cholesterol, HDL-cholesterol, triglyceride and glucose concentrations in 20 borderline hypercholesterolaemic men (average cholesterol 244 mg/dl). Thus, eating an average of six 440 g cans of this source of baked beans per week, large servings, does not lower plasma cholesterol when the intake of foods of animal origin is not decreased.

The effects of consuming oat-bran or beans were examined in 40 mildly hypercholesterolaemic men and women (average cholesterol 242 mg/dL) in study 5 (Mackay and Ball, 1992). The subjects were initially established on a low-fat background diet (29 per cent of energy from fat) and then 55 g low-fibre oat bran, 55 g high-fibre oat bran or 80 g mixed cooked beans were added to their diet in random order for 6-week periods. Total and LDL-cholesterol and triglycerides were unchanged.

In study 7 (Oosthuizen et al., 2000) 22 hyperlipidaemic men (average cholesterol 237 mg/dL) were randomly assigned to one of two groups. After a run-in period of 4 weeks, during which subjects followed their normal diet with the exclusion of dried beans, group A received 110 g/day of extruded dry beans in the form of baked products for 4 weeks, while group B continued with the run-in diet. A wash-out period of 4 weeks followed, after which the experimental intervention was crossed-over. Extruded dry beans did not have significant effects on total serum cholesterol, LDL-cholesterol, apolipoprotein A or B, plasma fibrinogen and plasma viscosity concentrations. HDL-cholesterol concentrations decreased in both the dry bean and control periods.

Study 6 (Fruhebeck et al., 1997) examined instead the effects of a 30 day dietary supplementation with broad bean flour in young men (aged 18±21 years; n = 40) with borderline hypercholesterolaemia (average cholesterol 240 mg/dl). All participants (groups A±C) consumed the same basic diet. The control group (A) consumed 90 g control flour daily, whereas the two bean diet groups received either 90 g cooked field bean flour (groups B) or 90 g raw field bean flour (group C) daily. After 30 days, total cholesterol, LDL-C and VLDL-C, triglycerides, glucose, insulin values were lower than initial ones in all subjects, who consumed the diets containing broad bean flour. The legume intake also increased glucagon and HDL-cholesterol levels.

In conclusion a clear reduction in total and LDL-cholesterol is shown only in the three studies involving subjects with initial values above 6.9mmol/L, whereas studies on borderline hypercholesterolaemic subjects (total cholesterol in the range 6.1±6.9 mmol/L) failed to show any effect. This points out once again the importance of enrolling only subjects affected by a real hyperlipidaemia, when investigating the effects of a dietary intervention on the cholesterol levels.

Unfortunately, the limited number of studies and the fact that often they used mixtures of pulses or pulses plus oat-bran do not permit the comparison of the pulses and the components responsible for the observed activity cannot be identified. Anderson and Major (2002) proposed this order of importance: soluble dietary fibre, proteins, oligosaccharides, isoflavones, phospholipids, and fatty acids, phytosterols, saponins plus, possibly, other not yet recognised factors. Considering the very low level of isoflavones in pulses, the general impression is that these results above all confirm a minimal, if any role of isoflavones in the hypocholesterolaemic activity of soybean. The similarity of the structure of the vicilins of all grain legumes and the few studies on rats fed legume protein isolates (Lasekan et al., 1995; Rahman et al., 1996; Macarulla et al., 2001; Sirtori et al., 2004) are in favour of a major role of proteins in the hypocholesterolaemic effect exactly as in soybean. A further confirmation of this hypothesis comes from the lack of increased cholesterol clearance via intestinal excretion in the study on pigs (Kingman et al., 1993) that supports a cellular mechanism, i.e. an up-regulation of LDL-receptors, as observed in soybean protein isolates (Lovati et al., 1987).

Pulses are an extraordinary source of many potentially beneficial components and their consumption should be encouraged by physicians and nutritionists as a replacement for animal proteins.

6 Future trends

In the past 30 years many investigations have been devoted to the beneficial role of several food components, such as vegetable proteins, unsaturated fatty acids, plant sterols, viscous fibres, nuts, polyphenols, etc. Further research is certainly necessary to single out which are the major beneficial components of pulses and which is their mechanisms of action. The target of these studies will be to provide consumers with new foods and food ingredients for the preparation of a variety of functional foods, possibly with improved sensory characteristics.

However, the future of research in this field is certainly best represented by studies of possible synergies between different diet components. An example in this direction may be found in a very recent paper by Jenkins et al. (2003). Forty-six healthy, hypercholesterolaemic adults (25 men and 21 postmenopausal women) with a mean age of 59 years and body mass index of 27.6, were submitted to a randomised controlled trial. Participants were randomly assigned to undergo one of three interventions on an outpatient basis for 1 month: a diet very low in saturated fat, based on milled whole-wheat cereals and low-fat dairy foods (mean initial cholesterol 6.37 mmol/L, n =16; control); the same diet plus lovastatin, 20 mg/day (mean initial cholesterol 6.64 mmol/L, n =14); or a diet high in plant sterols (1.0 g/1000 kcal), soy protein (21.4 g/1000 kcal), viscous fibres (9.8 g/1000 kcal) and almonds (14 g/1000 kcal) (mean initial cholesterol 6.94 mmol/l, n =16; dietary portfolio). The control, lovastatin and dietary portfolio groups had mean decreases in LDL-cholesterol of 8.0, 30.9, and 28.6 per cent, respectively. This experiment has shown that a well-planned dietary portfolio containing vegetable proteins from soy, plant sterols, nuts and viscous fibres may have the same effectiveness of a standard lovastatin treatment in controlling hypercholesterolaemia. This result is of extraordinary importance, because it has definitively demonstrated that a vegetarian diet may be as effective as one of the best hypocholesterolaemic drugs in reducing cardiovascular risk, without any side effects. Indeed we are now facing a completely new era in the prevention of cardiovascular risk by diet.

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