Therapeutic action of white beans by changing the digestion of carbohydrates
Correspondence Address: DOI: 10.4103/2229-5119.96952
The rates of obesity and diabetes are worrying. Some adjuvants can assist in weight loss and reduction in blood glucose. In this context, the α-amylase inhibitor is presented as a promising alternative because it interferes with digestion and thus absorption of carbohydrates from the diet. The white bean is the main source of α-amylase inhibitors protein (α-AI). The α-AI1 white kidney bean extract appears as a promising aid in the treatment of diabesity. However, there are several aspects that require further studies, such as the optimal dose and safety arising from the use of this inhibitor as well as effects from the change of carbohydrate digestion in the long term. Keywords: α-amylase inhibitor; diabetes, obesity, weight loss, white kidney bean
The rates of obesity and diabetes are worrying. The World Health Organization predicts for 2015 a population of 2.3 billion people is overweight, and of these, 700 million are obese and 300 million are diabetics. Hence, the need for adjuvants that can assist in weight loss and reduction in blood glucose. The α-amylase inhibitor is presented as a promising alternative because it interferes with digestion and, thus, absorption of carbohydrates from the diet. Such inhibition results in decreased availability of calories and postprandial blood glucose. The white bean is the main source of α-amylase inhibitors protein (α-AI).[1],[2],[3],[4] The protein inhibitors of α-amylase is widely distributed in plants, mainly cereals (wheat and barley) and beans. The use of these inhibitors has been done to reduce postprandial hyperglycemia as an auxiliary tool in the search for nutraceuticals and phytopharmaceuticals to control obesity and diabetes mellitus (DM), non-insulin-dependent. This inhibition induces carbohydrate tolerance, satiety, weight loss, and prolonged gastric emptying. [5] "Diabesity": Prevalence and social and economic impact A positive energy balance resulting from a disparity between the availability and consumption of energy by the body leads to weight gain and, eventually, obesity. Currently, the global trend in weight gain poses a major threat to global health. Adiposity (overweight and obesity) is a risk factor for a variety of chronic diseases and disorders such as dyslipidemia, hypertension, type II diabetes, cardiovascular disease, osteoarthritis, and some cancers. [6] One of the main factors related to obesity as a risk for cardiovascular mortality is that of providing the increase of new cases of diabetes, and an aggravating factor is that in obese individuals, the presence of diabetes adds an increased risk of coronary heart disease. [6] DM is a condition in which the body partially loses the power to metabolize sugars supplied by the foods eaten. As a result, hyperglycemia occurs. [7] The highest incidence of diabetes is type 2, in which the body does not produce enough insulin to control blood glucose, or body cells are unable to respond normally to the hormone. The type 2 diabetes is usually in adults over 40 years, but is becoming increasingly common among younger people. As with obesity, the incidence of diabetes has reached epidemic proportions. And the two ills keep a disastrous relationship, so much so that experts have created the neologism, "diabesity." Overweight and obesity are major modifiable risk factors for type 2 diabetes. [8] A positive association between obesity and the risk of developing type 2 diabetes has been repeatedly observed in cross-sectional and prospective studies. The risk of developing DM increases continuously with increasing body mass index (BMI) and age. In the presence of a BMI above 22 kg/m 2 , diabetes increases the risk of 25% for each increase of 1.0 kg/m 2 . Furthermore, the risk for developing chronic complications is directly related to the increase in BMI. Considering the individuals affected by diabetes, 80-90% are overweight. [9] In 2005, the World Health Organization (WHO) reported that 1.6 billion adults were classified as overweight, while at least 400 million were considered obese. The WHO predicts that over the next 10 years, an increase of 100% of the overall rates of overweight and obesity, and in 2015 there would be about 2.3 billion adults who are overweight and 700 million are obese. [6] As for diabetes, according to projections from the International Diabetes Federation, based in Brussels (Belgium), in 2025 the number of patients with this disease will reach the mark of 350 million. [8] Strategies for weight loss and control of blood sugar levels usually involve a combination of dietary changes limiting caloric intake, increased physical activity, behavior therapy, pharmacotherapy, and, in extreme cases, surgery. The availability of supplements for weight loss has risen dramatically in recent years; however there is no unanimity for efficacy (Celleno et al., 2007). Many plant species have been used experimentally for ethnopharmacology to support treatment of diabetes. Some substances may have therapeutic potential, while others may produce hypoglycemia as a side effect because of their toxicity, especially hepatotoxicity. [7] Searches for drugs that act by providing a viable alternative for weight control and blood glucose lowering pharmacological approaches that help with the treatment of obesity and diabetes have been successful, especially in the case of molecules that affect the absorption or the availability of nutrients. In this context, we highlight the α-amylase inhibitor by interfering with the digestion of carbohydrates from the diet. Carbohydrates Carbohydrates are the most abundant organic molecules in nature. Certain carbohydrates like sucrose and starch are the basis of the diet in most of the world, and their oxidation is the major metabolic pathway supplying energy to the cells. Structurally, they can be classified into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Starch is the most important source of carbohydrates for human consumption, representing 80-90% of all polysaccharides in the diet, and the main responsible for the technological properties that characterize many of the products processed. [10] Starch Structurally, starch is a homopolysaccharide composed of chains of amylose and amylopectin. Amylose consists of glucose units joined by glycosidic bonds α-1, 4, resulting in a linear chain. Since the amylopectin is formed by glucose units joined in α-1, 4 and α-1, 6 linkages, forming a branched structure. The proportions in which these structures appear to differ in botanical sources, varieties of the same species, and even within the same range, according to the level of maturity of the plant. [11] Molecular forms: Amylose Amylose is an essentially linear polymer, composed of units of α-D-glucopyranoside linked in α-1, 4, with few links α-1, 6, between 0.1% and 2.2%. [Figure 1]a This molecule has a number average degree of polymerization of 500-5000 units of glucose residues. [11]
The molecular weight is about 250,000 Daltons (1500 units of glucose), but varies widely among plant species and within species, depending on the degree of maturation. Amylose molecules of cereals are generally smaller than those from other sources (eg, tubers and legumes). [12] Molecular forms: Amylopectin The amylopectin is the branched component of starch. It is formed by residues of α-D-glucopyranoside (17 to 25 units) united in α-1, 4 linkages, and strongly branched, with 4% to 6% of bonds α-1, 6 [Figure 1]b. The molecular weight of amylopectin varies between 50 and 500 × 106 Daltons. [13] The amylopectin has a degree of polymerization of 4700 to 12,800 units of glucose residues. The individual chains vary between 10 and 100 units of glucose. The amylopectin is degraded by the digestive action of α-amylase on the links α-1-4, producing α-limit dextrins (residual chains containing the branch points) and later by the action of enzymes isoamylase and pullulanase in -connections 1-6, producing maltose.[12] Chains of amylopectin are organized in different ways, suggesting a classification of chains A, B, and C [Figure 2]. Type A is a chain composed of non-glucose-lowering links joined by α-1, 4 unbranched, being attached to a type B chain by linking α-1, 6. The chains of type B are composed of glucoses linked in α-1, 4 and α-1, 6. The chain C is unique in an amylopectin molecule, consisting of bonds α-1, 4 and α-1, 6, with terminal cluster gear.[13]
Digestion and absorption In humans, between 40% and 80% of total caloric intake of carbohydrates is represented in its various forms, representing the most important energy source. According to chemical structure, carbohydrates can be classified into forms of absorption as absorbable (undigested), digestible, fermentable and non fermentable. Absorbable carbohydrates (monosaccharides, comprising a single unit, such as glucose, galactose, fructose, xylose, and ribose), by definition, need not be digested to be transported into the blood. However, since has two or more carbohydrate units (oligo-and polysaccharides), needs to be enzymatically digested to be absorbed. [14] In the human diet, the main digestible carbohydrates are disaccharides such as sucrose (sugar) and lactose, and higher polysaccharides such as starches, which are the main source of carbohydrates in most Western diets. In contrast, fermentable carbohydrates cannot be digested and the enzymes cannot easily break the glycosidic bonds. However, in the colon, these carbohydrates are easily metabolized by bacteria through the fermentation process. Likewise, if the digestible carbohydrates such as sucrose and lactose are poorly digested or poorly absorbed, they are fermented in the large intestine. The main end products of carbohydrate fermentation are short-chain fatty acids (acetate, propionate, and butyrate) and gases (carbon dioxide, hydrogen, and methane). [15] They can be absorbed in the large intestine (providing energy), used as substrate by bacteria, released as flatus, or excreted in the form of biomass in the feces. Some carbohydrates (such as components of plant cell wall) are neither digested/absorbed nor fermented. They pass through the gastrointestinal tract mostly unchanged and are shed in the feces. [6] The physiological process of digestion of carbohydrates occurs by the action of enzymes distributed along the gastrointestinal tract. Salivary and pancreatic amylases and glucosidases of the microvilli of the duodenal enterocytes act sequentially on the carbohydrates, reducing them to monosaccharides that are absorbed (Thomas et al., 2008). Digestion of carbohydrates begins in the mouth by the action of salivary α-amylase, which hydrolyzes bonds α-1-4 starch, whose products of this process are maltose and dextrin maltriose small. The process of digestion of starch continues in the small intestine by the action of α-amylase secreted by the pancreas.[6] The small intestine is the primary site of absorption of monosaccharides. Two mechanisms are responsible for the process: active transport and facilitated diffusion (Thomas et al., 2008). The digestion process is completed by enzymes released in the duodenal microvilli (maltase, sucrase, and lactase, also known as α-glucosidases and disaccharidases) to produce glucose, absorbable monosaccharides, fructose, and galactose. A small proportion of monosaccharides can be absorbed passively; however, a carrier protein is required. [6] Monosaccharides are absorbed into the circulation, distributed, and sent to the liver, which regulates blood glucose levels during the post-feeding and fasting periods (Thomas et al., 2008). Mechanisms of hydrolysis of the glycosidic bond The cleavage of the glycosidic bond involving glycolytic enzymes may occur by two main mechanisms: SN2 related to the direct nucleophilic substitution by water molecule in C-1 anomeric carbon of glucose with inversion of stereochemistry (I), less common, usually seen in glucoamylase or bimolecular substitution mechanism with the formation of covalent intermediate, which results in retention of configuration (II), as shown in [Figure 3]. Only enzymes that act with retention of configuration have transglicosilation activity, as several α-glucosidases.[16] In mechanism I, acts as a carboxylic acid group and another as a basis, unlike the mechanism II, in which a carboxylic acid group acts as the acid and base and the other as a nucleophile. However, some evidence has suggested the generation of ion in the transition state, the SN1 mechanism, with participation of the catalytic site amino acids containing carboxyl groups with cleavage of the link between the glycoside anomeric carbon and oxygen, producing positively charged species, such as carbocation (A) and oxocarben ion (B) [Figure 4]. The transition state necessary for hydrolysis of the glycosidic bond is characterized by pseudo-axial orientation of the CO bond to be broken and distorted conformation "skew." [17]
α-Amylase In the process of degradation of starch, the enzymes involved can be classified according to the mode of action into endoamylases, exoamylases, and debranching enzymes. The endoamylases are represented by α-amylases, which hydrolyze the starch in the inner regions. The exoamylases comprise the glucoamylase and fungal and bacterial β-amylases of cereals. These release of glucose and maltose units from the ends of the polymer. Since debranching enzymes act on the glycosidic α-1, 6 which cannot be cleaved by α-and -amylases (Smith, 2008). The α-amylase is found in plants, animals, and microorganisms, and it catalyzes the hydrolysis of complex carbohydrates into simple sugars such as maltose, maltotriose, and glucose. Biochemically, it is α-1, 4-glucan-4-glucanohidrolase classified by the acronym EC 3.2.1.1. acting on glycosidic bonds α-1, 4. The hydrolysis by α-amylase occurs in several steps, starting with the internal connection of the enzyme-substrate, followed by separation of the polymer, and later another process for hydrolytic release of several molecules of maltose and oligosaccharides (Antunes, 2008). [18] Studies of X-ray diffraction of porcine pancreatic amylase provides an exploration into the mechanism of the enzyme amylase. [19] The degradation of starch by α-amylase in vitro presents two proposed mechanisms: the attack multiple and the attack on multi-string. At first, the enzyme can bind to a specific region of the substrate and release only one molecule, while the other portion of the long polymer is retained for subsequent hydrolysis. In the multi-string attack, the action of the enzyme occurs with only a hydrolytic reaction, resulting in two molecules of similar size (Muralikrishma and Nirmala, 2005). Approximately 5-6% of the total protein in pancreatic secretions are α-amylase, a glycoprotein of 512 amino acids with a molecular weight of 57.6 kDa with a single oligosaccharide chain. Unlike other pancreatic zymogens, α-amylase does not have a inactive proform. [19] In mammals and insects, the α-amylase is structured in three protein domains: domain A, which provides the framework for flexible two parts, the domain B, with formation of mixed β-sheet and α-helices, and a globular unit, which is C domain, located opposite the insertion of domain B in A [Figure 5]. [20]
The substrate-binding sub-sites in the enzyme is formed at the interface of domains A and B, with the number of sub-sites and the chemical nature varying according to the origin of α-amylase. The structural integrity of the enzyme is calcium dependent and requires chloride ions to exert catalytic activity. The binding sites of these ions, which are functionally important for enzyme activity, also involve the fields A and B. [21] The substrate-binding site contains five sub-sites, with the catalytic site positioned at sub-site 3. The substrate can be bound by the glucose residues in sub-sites 1 or 2, allowing the cleavage between the first and second or second and third glucose residues. Therefore, amylase preferentially cleaves inside the molecule, specifically the links α-1, 4. Products resulting from digestion by α-amylase is a mixture of maltose, maltotriose, and branched glucose units containing 6-8 glucose units connected both with α-1, 4 and -1, 6. Later, intestinal enzymes of brush border, maltase and isomaltase, finish the digestion of starch. [19] Inhibition of human α-amylase may hamper the digestion of complex carbohydrates, thereby reducing the absorption and a consequent reduction in the availability of calories derived from carbohydrates, and promote or assist weight loss and reduction of postprandial glucose. [22] Inhibition of α-amylase α-amylase inhibitors have been identified in many plant species. In 1943, Kneen and Sandstedt described the inhibition of enzyme activity by substances derived from wheat, rye, and sorghum. Bowman (1945) later found similar activity in aqueous extracts of white beans. Since then, the α-amylase inhibitors have been isolated from several other plant species, including many varieties of common bean, Phaseolus vulgaris (white, red and black). [22] Several types of organic compounds are known for their inhibitory activity on the α-amylase. Generally, non-protein inhibitors are molecules of low molecular weight. The protein inhibitors can be classified according to the structure. There is a group found in fungi of the genus Streptomyces and six other groups from higher plants (Smith, 2008). According to Richardson, [23] the α-amylase inhibitors can be classified according to their tertiary structure into six types: protein-CM, Kunitz, thaumatin, knotina, thionin, and lectin. The CM-proteins are also known as inhibitors of cereal origin, with up to 160 amino acid residues. Often, these proteins have activity not only on α-amylase but also on trypsin, unlike other inhibitors that have specificity only for amylases.[24] Kunitz-type inhibitors are proteins with a molecular mass of 20 kDa and were studied by specifically inhibit α-amylases of the insects and subtilisin (a serine protease). Thaumatin like protein inhibitors, thionin knotina and inhibit insect α-amylases, but not those of mammals. Corn contains zeamatina inhibitor, type thaumatin. The type is represented by knotina inhibitor isolated from amaranth (Amaranthus hypochondriacus L.), also known as stick-purple. With 32 amino acid residues, is the smallest inhibitor described above. Finally, the thionin type is represented by the inhibitors of sorghum (Sorghum bicolor L.). [24] Inhibitors of type lectin (α-AI) are typically represented by the inhibitors found in beans (Phaseolus vulgaris), and three isoforms are described. The most characterized isoform, known as α-AI1, was identified as an inhibitor of α-amylase homologous to phytohemagglutinin (PHA). The second variant of α-AI, called α-AI2, is found in some types of wild beans. These two allelic variants have different specificities of inhibition. AI1 inhibits α-amylase of mammals, as well as α-amylase of C. maculatus and C. chinensis, but does not inhibit α-amylases of the Z. subfasciatus (ZSA). In contrast, the α-AI2 does not inhibit the first three amylases mentioned, but inhibits ZSA.[25] The α-AIs, to reach its active form, comprising two noncovalently linked glycopeptide subunits, α and β, of 7.8 and 14 kDa, respectively, are post-translationally modified. The proteolysis that leads to activation of α-AI1 was studied by mass spectrometry. Results suggest that a cleavage occurs in simple carboxy terminal portion of Asn77, presumably by a protease Asn-specific seed. Asn79 is removed apparently by the action of a carboxypeptidase. In addition, C-terminal 19 residues of the β chain of α-AI1 are removed. α-AI2 shows similar cleavages in α-AI1, but a glycosylation pattern a little different. Both inhibitors (α-α-AI1 and AI2), present as a mature structure heterotetramer of two α chains and two β chains and are highly glycosylated.[26] The third isoform, α-AIL (also known as α-AI3), isolated from P. vulgaris cv Rico 23 has a single chain protein. This protein may represent an evolutionary intermediate between PHA, arcelin, and α-amylase inhibitors. Interestingly, it specifically inhibits fungal α-amylase and, additionally, has hemagglutinating activity, showing that these two are not mutually exclusive and that the cleavage is probably not a prerequisite for inhibition of α-amylase. α-Amylase inhibitor from white kidney beans Crude extracts containing common bean α-amylase inhibitors type lectin were used in vivo as starch blockers in early 1980 for glycemic control in individuals with DM and obesity. These early attempts were unsuccessful due to the undesirable presence of PHAs and protease inhibitors in the extract. Later, the administration of α-AI purified obtained more consistent results. This class of inhibitors has been used for its insecticidal properties to protect the seeds from insect predation, and white bean flour as an aid in the treatment of obesity and diabetes again. [25] In bean, the α-AI1 accumulates in the seeds, making up 9-11% of the total protein. This percentage can provide a substantial income, although the extraction method can limit it in the seed is concentrated in the axis, with a concentration three times higher than in cotyledons. Apparently, this is because in this region there is a more efficient glycosylation. No α-AI1 was detected in other organs of the plant. (Obiri et al ., 2008). The synthesis of the white bean AI1 occurs at the same time as the protein phaseolin and PHA, and accumulates in protein storage vacuoles. The α-AI1 is a lectin of the common bean, which is synthesized in the rough endoplasmic reticulum, modified in the Golgi complex by the removal of a peptide marker, and, after an N-glycosylation, is transported to protein storage vacuoles where it is proteolytically processed. SDS-PAGE electrophoresis, performed on microsomal fractions, showed that fractions of 30000-35000 are associated with the endoplasmic reticulum, while 14 to 19 kDa are associated with the Golgi apparatus and vacuoles of storage. [27] The α-AI1 is detectable 17 days after pollination in the cotyledons and the axis of the seed. From there, there is a steady increase and the maximum concentration occurs after 28 days to maturity. There is need for research to assess the relationship between the content of α-AI1 and maturation to define the best time for obtaining a maximum yield with greater economy (Obiri et al., 2008). The α-AI1 is characterized by a tetrameric nature, which explains the observation that the inhibitor AI1 inhibits two molecules of α-amylase inhibitor molecule, classifying them as divalent in its mode of inhibitory action. Thus, it has been reported in several studies a stoichiometric ratio of 2:1 compared to 1:1 ratio of acarbose and cyclodextrins, becoming, in vitro, a more potent inhibitor than acarbose, based on the molar concentration. However, based on molecular weight, due to the lower weight of acarbose, the rate of reaction with the enzyme is much faster because there is no need for conformational change during the connection process (Obiri et al., 2008). Physiological effects of α-amylase inhibitor from white kidney beans Increasing experimental evidence has suggested that extracts or derivatives of beans, Phaseolus vulgaris (Fabaceae), may have the ability to reduce food intake, body lipid deposit, and glucose (Maccioni, 2010). The effects of the use of α-AI have also been shown to substantially reduce postprandial glucose and insulin concentrations. Furthermore, when administered in conjunction with meals, the α-AI have shown to be effective in reducing gastrointestinal absorption of carbohydrates, without causing discomfort. [22],[28] Recently, there has been a series of experiments carried out in order to characterize the anorectic effects of a standardized dried extract of newly produced P. vulgaris, determined Beanblockw®. Its acute and chronic administration is dose-dependent reduction in food intake in rats and mice. Notably, this extract was effective in reducing the intake of highly palatable food and liquids, including butter cookies and drink chocolate milk and regular food or starch enriched, suggesting that derivatives of P. vulgaris may be able to selectively reduce the appetite for consumption of these products (Maccioni, 2010). The exact mechanism of the effect of dry extract P. vulgaris chocolaty beverage intake is still unknown. Two mechanisms of action are suggested in an attempt to elucidate, in addition to this, the effects on food intake, body weight, and blood glucose. Both mechanisms are based on the presence of two lectins: PHA and α-amylase inhibitors (three isoforms isolated from beans). These lectins, along with a third type, named arcelin, have high degrees (40-95%) of amino acid sequence similarity. Specifically, inhibition of α-amylase: (a) suppresses the metabolism of starch, resulting in a decrease in blood glucose, and (b) delayed gastric emptying and satiety, producing in turn decreasing food intake, as quoted before. However, the effect on the absorption and metabolism of carbohydrates, in the case of chocolaty drinks used in the investigation, with negligible content of starch and complex sugars, makes it unlikely that inhibition of intraluminal α-amylase plays an important role. Additional mechanisms may involve lectins, which bind to the intestinal microvilli, stimulating the release of cholecystokinin (CCK) and glucagon-like peptides that modulate the central regulation of satiety, appetite, and food intake (Maccioni, 2010). Antinutrients and white beans In addition to the α-AI, the beans contain potentially toxic antinutritional substances related to deleterious effects such as reduced feed efficiency, histopathological changes, and death in laboratory animals. Common bean has some undesirable attributes, such as phytates, flatulent factors, phenolic compounds, enzyme inhibitors, lectins, and allergens, which must be eliminated for their effective use. [29] In humans, the consumption of raw or undercooked beans has been associated with severe, but fleeting, gastrointestinal discomfort. These effects have been attributed largely to PHA, which are present at high levels in raw beans. The content of PHA depends on the variety. For example, the red bean is known to contain up to five tetrameric isolectinas: L4, L3E1, L2E2, L1E3. [30] Lectins are glycoproteins that can display varying proportions of subunits, leukocyte reactive (L-PHA) and erythrocyte reactive polypeptide (E-PHA), which are reduced during the cooking of beans. The raw white beans may contain from 20.000 to 70.000 units PHA. g-1 compared with 200-400 units of PHA in cooked beans. [22] In addition to lectins, they also include protease inhibitors, such as trypsin inhibitor. Trypsin inhibitors are protein substances that interfere with the activity of enzyme systems of the digestive tract. Proteases are enzymes that hydrolyze the peptide bonds as a first step for the absorption of proteins. This inhibition is expressed in vivo in a reduction in protein digestion. [23] The most abundant protease inhibitor in beans is the Bowman-Birk (BBI), thermostable, with a molecular weight around 9000 Da, and has two active sites, inhibiting both trypsin and chymotrypsin. The strong inhibition of trypsin has been associated with hyperplasia and/or pancreatic hypertrophy, increased by excessive secretion of trypsin by the mechanism of self-regulation by feedback. However, currently the interest in this class of protease inhibitors is mainly based on findings that BBI can act as cancer preventive agent in vitro and in vivo. BBIs also show anti-inflammatory activity by inhibition of proteases involved in inflammation. [31] Perspectives from studies of α-Ai white beans Studies have shown that butyrate reduces the incidence of colon cancer, possibly by causing an apoptotic response to DNA damage caused by genotoxic carcinogenesis in rat distal colon, promoting the removal of mutant clones that evolve to malignancy. The increased microbial activity in the gastrointestinal tract resulting from the consumption of α-AI1, may increase the fermentation of starch, resulting in elevated levels of butyric acid. Moreover, the presence of α-AI1 in the gastrointestinal tract causes an action similar to resistant starch, which has a prebiotic effect, and greater concentration of undigested starch to ferment, producing butyrate.
The α-AI1 white kidney bean extract appears as a promising aid in the treatment of diabesity. There are several aspects that require further studies, such as the optimal dose and safety arising from the use of this inhibitor, as well as effects from the change of carbohydrate digestion in the long term. Moreover, it is necessary to have a deeper understanding of the mechanism of inhibition of α-amylase that can base the prospect of synthetic inhibitors. Additionally, the α-AI1 presents fresh perspectives on the prevention of colorectal cancer.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
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