In Vitro Evaluation of the Antidiabetic and Antiadipogenic Potential of

Chapter 12

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In Vitro Evaluation of the Antidiabetic and Antiadipogenic Potential of Amaranth Protein Hydrolysates Aída Jimena Velarde-Salcedo,1 Elvira González de Mejía,2 and Ana Paulina Barba de la Rosa*,1 1Instituto Potosino de Investigación Científica y Tecnológica A. C., Camino a la presa San José 2055, San Luis Potosí, SLP 78216, México 2Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 228 E.R. Madigan Lab, MC-051, 1201 W. Gregory Drive, Urbana, IL 61801 *E-mail: [email protected]

Obesity, which is a major recognized risk factor for type-2 diabetes, is rapidly increasing in prevalence resulting in a “diabesity” epidemic. Diabesity represents one of the major public health problems in the 21st century. Some of the strategies that have shown to be effective in reducing type-2 diabetes incidence are exercise and a healthy diet. New drugs that have as a target the inhibition of the enzyme dipeptidyl dipeptidase IV (DPPIV) have been released. However some of these drugs have secondary effects; for that reason, the food industry is exploring the aspects related to the components present in food that promote a healthy life, such as the bioactive peptides encrypted in the proteins of several foods. Amaranth is a plant native from North, Central and South America which contains antihypertensive, antioxidant and cancer preventive peptides. Also there is evidence that amaranth has some hypoglycemic action; however, the antidiabetic potential and the effect upon body weight of the seed proteins have not been well characterized. The aim of this study was to identify the ability of amaranth peptides to inhibit the DPPIV activity and the effect of these peptides upon fat accumulation in mouse adipocyte cultures.

© 2012 American Chemical Society In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction The modern food industry is exploring the aspects related to the components present in food that promote the well-being and a healthy life in the consumer (1). It has been reported that food proteins and peptides, aside from being basic macronutrients, also present a wide variety of biological activities that could have a beneficial effect in human health acting as antioxidants, antihypertensives, antithrombotics, among others (2). Biologically active peptides are small sequences of amino acids encrypted in food proteins that are activated when released by proteolytic enzymes, during food processing or during intestinal digestion (3). Milk and its derivatives are one of the richest sources of biopeptides, but biopeptides are also found in other animal and vegetables sources like meat, eggs, fish, wheat, corn, soybean, rice, mushrooms and amaranth (2–5).

Amaranth Nutraceutical Properties Amaranth is a plant native from North, Central and South America cultivated since pre-Columbian times and in the 1980’s was declared a crop with a high biotechnological potential (NAS 1984). In the last decade amaranth has emerged not only for its nutritional value, but also for its nutraceutical properties (6). The presence of several compounds in amaranth seeds like phytosterols, flavonoids, essential oils and protein extracts have been reported. This evidence explains the use of amaranth as an hypocholesterolemic, antioxidant and hypoglucemic plant (7–11). In our research group we have identified the presence of peptides with different biological properties (5) encrypted in amaranth proteins, which include peptides with an inhibitory activity upon the angiotensin-converting enzyme (ACE) that has an antihypertensive effect. These peptides have been characterized using the coronary endothelial cell line and aortic rat rings showing the vasorelaxing effect via nitric oxide production ((12). Fritz et al. (13) have also demonstrated the blood pressure lowering effect of amaranth hydrolysates in hypertensive and normotensive rats. It has also been reported that the lunasin-like peptide is present in amaranth proteins (14). Lunasin is a novel biopeptide reported for the first time in soybean that is capable of inhibiting cancer development (15). Both soybean and amaranth lunasin contains the domains that are required for the internalization of the protein and for its binding to chromatin in cancer cells. It was determined that amaranth lunasin-like peptide internalize into the cell and reaches the nucleus more rapidly than soybean lunasin, where it prevents cell malignization in the presence of a carcinogenic agent and also inhibits H3 and H4 histone acetylation. This evidence shows the anticancer properties of amaranth seeds (14).

Amaranth and Type-2 Diabetes The dipeptidyl peptidase IV (DPPIV) is responsible for the degradation of the major insulinotropic hormones, the incretins. Incretins are peptidic hormones released to the blood stream after food intake by the enteroendocrine cells of the 190 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


small intestine (Figure 1). These hormones immediately stimulate the synthesis and excretion of insulin by pancreatic β cells and therefore decrease the glucose blood levels (16–18). However, the lifetime of incretins is very short because they are rapidly inactivated by DPPIV. Hence, the use of DPPIV inhibitors increases the time of action of these hormones and potentiate their effects (19–21). There are several DPPIV inhibitors available that have shown promising results as antidiabetic agents (22–24), and the search of natural products with a similar activity is always a viable alternative for metabolic diseases and opens up a new investigation field.

Figure 1. Schematic representation of incretin secretion and action. GIP and GLP-1 are secreted after food ingestion, and they then stimulate glucose-dependent insulin secretion. Once released, incretins are inactivated by DPPIV.

Using mass spectrometry data and the biopep database (http://www.uwm.edu.pl/biochemia/index.php/en/biopep), it was found that in addition to the hypertensive and cancer-preventive peptides reported in amaranth glutelins (5), this fraction also contained peptides related to the inhibitory activity of the DPPIV. Then the activities of these peptides were tested in vitro. Glutelins were digested with trypsin, using different enzyme:substrate ratios to obtain different degrees of hydrolysis. As shown in Figure 2A, it was found that the intact glutelins had no significant effect upon DPPIV activity, but when they were digested with trypsin the DPPIV activity decreased in a dose-dependent manner. This inhibitory activity was higher when the degree of hydrolysis increased, reaching up to 80% of inhibition of the enzyme (IC50= 1.2 mg/ml). 191 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. DPPIV inhibitory activity of tryptic glutelins hydrolysates. (A) Amaranth glutelins were digested with trypsin using different enzyme:substrate ratios; (B) fragments bigger than 10 kDa were removed by ultracentrifugation and DPPIV inhibitory activity was measured.

192 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Isolation of DPPIV inhibitory peptides from amaranth gluelins. (A) Tryptic glutelins hydrolysates were separated by size exclusion chromatography. Boxes indicate the four (1-4) fractions collected. Arrow indicates the elution volume of a known DPPIV inhibitor, a tripeptide, diprotin A, as a reference. (B) The main peaks were collected, pooled and tested for their inhibitory activity upon DPPIV.



Interestingly, when the bigger fragments of the hydrolysates were removed by ultrafiltration using a 10 kDa MWCO, it was observed that all the hydrolysates had the same activity, with no significant differences among them (Figure 2B) with IC50s between 1.1 and 1.2 mg/ml. This could indicate that big peptides (larger than three amino acids) can also inhibit DPP IV. The glutelins hydrolysates obtained at 1:5 and 1:2.5 enzyme:substrate ratio were fractionated by size exclusion chromatography using a column with a separation range between 100 and 7000 Da (Figure 3A). The same separation profile was obtained with both digestions, however as the amount of enzyme increased, the amount of released peptides also increased (peak area). The peaks were collected and pooled by freeze-drying in four fractions, peaks 3 and 4 were collected in the same fraction. Fractions 1, 2 and 3 showed the inhibitory activity upon DPPIV (Figure 3B), where the fraction 1 had the lowest values and both fraction 2 and 3 presented a very similar pattern of inhibition. No activity was found in fraction 4, perhaps because this fraction contained free amino acids, based on the elution volume of the tripeptide (shown with an arrow in Figure 3A). With this information, it was hypothesized that not only small peptides are able to inhibit DPPIV, but apparently bigger fragments can also be considered as potential inhibitors. Docking modeling strategies are useful to predict the molecular mechnisms of action and further work is being carried out.

Figure 4. DPP IV inhibitory activity of amaranth seed storage proteins. Tryptic hydrolysates from amaranth seed storage proteins were prepared and tested on the DPP IV activity assay.



Tryptic digestions of other amaranth storage proteins fractions were tested. Albumins and globulins also had an inhibitory activity upon DPPIV, but 11S globulins had the lowest value (Figure 4). The gastrointestinal digestion model using different enzymes present during regular digestion was established in order to determine if amaranth consumption could also release these inhibitory peptides. The enzymes used were pepsin released in the stomach, trypsin and pancreatin, which includes lipase, nucleases and other proteases. With this method, the glutelins hydrolysates showed a pattern similar to tryptic hydrolysates (Figure 5). Flours obtained from two amaranth species, A. hypochondriacus and A. cruentus were compared as well as the flour from popped amaranth. No differences were found among species (IC50= 1.1 mg/ml for A. hypochondriacus and 1.4 mg/ml for A. cruentus) but the inhibitory activity of flour from popped amaranth decreased considerably compared with the raw flours. This indicates that the heating process is affecting the hydrolysis and availability of bioactive peptides, maybe due to the denaturalization of the proteins, hydrolysis, cross-linking, peptide fragmentation or Maillard reactions between carbohydrates and proteins (25). Commercial amaranth supplement was also tested showing that this sample had the lowest inhibitory activity of all amaranth samples. Other seeds were tested. Black bean, which barely had an inhibitory activity upon DPPIV; soybean, which had a higher inhibitory activity than black bean but not as much as amaranth; and wheat, which presented the same pattern as the raw amaranth flours (IC50= 0.8 mg/ml). It seems that the effects upon DPPIV are shared among cereals or seeds with a similar protein distribution. Further work needs to be done.

Amaranth and Obesity It has been reported that amaranth proteins have antilipemic and weight-lowering effects in rats, indicating a possible potential of amaranth against obesity (26–28). The 3T3-L1 cell line of mouse fibroblasts that can be differentiated into adipocytes using specific hormones is widely used as a model of study. The cells were cultured in presence and absence of the tryptic glutelins hydrolysates throughout and after the differentiation process and lipid accumulation using oil red O staining was measured. Cells cultured in control conditions accumulated a considerable amount of fat, whereas the treated cells produced a lower amount (Figure 6). It was observed that when the cells were treated with the hydrolysates during the whole differentiation process, the final lipids generated decreased significantly, up to a 50% using 100 µg of hydrolysate. This effect seems to be more potent in the cells that are already differentiated where a higher effect was observed. This may indicate that the effect relies more in the lipid synthesis than in the whole signaling pathways involved in differentiation. However, these are preliminary results and more refined experiments need to be done. 195 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 5. DPPIV inhibitory activity of different seeds sources. A gastrointestinal digestion model was performed on the defatted flour of several amaranth products and other common seeds. The released peptides after digestion were used in the DPPIV activity assay.

Figure 6. Anti-lipogenic effect of amaranth tryptic glutelins hydrolysates upon 3T3-L1 cell line. *p

In Vitro Evaluation of the Antidiabetic and Antiadipogenic Potential of

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