Supplementation with a new trypsin inhibitor from peanut is associated with reduced fasting glucose, weight control, and increased plasma CCK secretion in an animal model

Abstract

Ingestion of peanuts may have a beneficial effect on weight control, possibly due to the satietogenic action of trypsin inhibitors. The aim of this study was to isolate a new trypsin inhibitor in a typical Brazilian peanut sweet (paçoca) and evaluate its effect in biochemical parameters, weight gain and food intake in male Wistar rats. The trypsin inhibitor in peanut paçoca (AHTI) was isolated. Experimental diets were prepared with AIN-93G supplemented with AHTI. Animals had their weight and food intake monitored. Animals were anesthetized, euthanized, and their bloods collected by cardiac puncture for dosage of cholecystokinin (CCK) and other biochemical parameters. Supplementation with AHTI significantly decreased fasting glucose, body weight gain, and food intake. These effects may be attributed to increased satiety, once supplemented animals showed no evidence of impaired nutritional status and also because AHTI increased CCK production. Thus, our results indicate that AHTI, besides reducing fasting glucose, can reduce weight gain via food intake reduction.

• Arachis hypogaea L.
• bioactive proteins
• obesity
• satiety

Introduction

Obesity stands out among the diseases caused by nutritional inadequacies. It is growing in many regions of the world, in developed and underdeveloped countries, reaching epidemic levels1–3. This makes the investment in resources to find natural products for the prevention and treatment of obesity a growing field.

Thus, advances in the scientific expertise that involves quality, safety, and efficacy inherent to herbal medicinal products in recent years have placed the area of bioactive natural products in an outstanding position. In the treatment of obesity, factors such as the high cost of drugs and its side effects, have increased the demand by the population for natural products using plants with slimming actions4–6.

Oilseeds intake, regardless their high fat composition, have shown beneficial effects on health7^,8. Peanut, among these seeds, is one of the most consumed in Brazil, representing an important source of protein and fat. Its great economic impact is mainly due to the various forms of consumption in the country9.

Although peanuts present high energy content, researches have shown a relationship between its consumption and a reduction in weight gain. There are several hypotheses to justify these findings. One of them is that part of the energy is not available due to components that prevent digestion8^,10^,11. Another possibility is that the satietogenic peanut effect may be attributed to its peculiar lipid, fiber, and protein composition. Also, there are reports in the literature that postulate that the presence of trypsin inhibitors in peanuts12–16 may lead to a reduction in weight gain11. In this context, it is noteworthy that the trypsin inhibitors increase the concentration of cholecystokinin (CCK), which has a satietogenic effect17–20.

As new studies are developed investigating the effects of peanuts ingestion, various foods peanut derived are increasingly being consumed in Brazil, including: peanut oil, peanut butter, and paçoca, the latter consumed as a typical Brazilian Northeastern dessert, made with sugar, flour, and condensed milk. In addition, in 2010, a research conducted by the Brazilian Institute of Public Opinion and Statistics (Ibope) demonstrated that 66% of the Brazilian population consumes peanuts21.

Sequiz et al.11 showed that consumption of peanut paçoca, a food high in trypsin inhibitor, derived from peanut, could help to promote satiety and reduced weight gain, without causing damage to the pancreas, or cause changes in biochemical parameters analyzed. However, it is not known if the presence of trypsin inhibitor was a determining factor in this weight reduction. In this sense, it is important the isolation and characterization of this trypsin inhibitor of peanut paçoca, as well as, research if this inhibitor is responsible for weight reduction and satiety.

Experimental section

Reagents

Trypsin, Azocasein 1%, α-N-benzoyl-arginine-p-nitroanilide, dimethylformamide, bovine serum albumin (BSA), dimethyl sulfoxide, acrylamide, N′N′-methylenebisacrylamide, N,N,N,N′-tetramethylethylenediamine, Coomassie Blue G-250, and R-250 were purchased from Sigma, St. Louis, MO. CCK immunoassay kit was purchased from Phoenix Pharmaceuticals, Burlingame, CA.

Peanut paçoca (peanut sweet)

The peanut paçoca was purchased in local shops in the city of Natal, RN, northeast Brazil.

Animals

Male Wistar rats, two months old, weighing between 150 and 200 g were used. Animals were acclimated individually in metabolic cages and maintained on a photoperiod of 12/12 h at a temperature of 25 ± 2 °C and humidity between 45% and 55%, with access to water and food ad libitum. The experimental protocol of this study was submitted to the Ethics Committee on Animal Use of the Federal University of Rio Grande do Norte – UFRN (protocol N° 021/2011). For the development of this protocol all ethical principles and procedures were adopted. Experiments from this study are part of a larger project20.

Isolation of a new trypsin inhibitor

The peanut paçoca was powdered in cold mill (6 °C) until the formation of a fine grained flour (40 mesh). Total proteins were extracted from this flour in 50 mM Tris-HCl buffer, pH 7.5, at a ratio of 1:10 (w/v). The mixture was subjected to constant stirring for 3 h at room temperature. The material was centrifuged at 12 000× g for 30 min at 4 °C. The supernatant and the precipitate were denominated crude extract (CE) and precipitated from crude extract (PEB), respectively. The CE was fractionated by sequential precipitation with ammonium sulfate in 0–30%, 30–60%, and 60–90% saturation ranges. Each fraction was kept for 12 h at 8 °C and centrifuged at 12 000× g for 30 min at 4 °C to obtain the precipitated proteins. The precipitates were resuspended in 50 mM Tris-HCl pH 7.5, dialyzed against the same buffer and stored at −20 °C until the experiments. About 60 mg of F2 (30–60%) were applied to a Trypsin-Sepharose 4B affinity column (10 cm × 1.5 cm) in Tris-HCL buffer 50 mM, pH 7.5. Adsorbed proteins were eluted from the matrix with 5 mM HCl solution and collected in aliquots of 3 ml at a flow rate of 2 ml/min. The protein profile was evaluated by spectrophotometry at 280 nm. The retained and eluted peak from the affinity matrix was dialyzed against Tris-HCl 50 mM, pH 7.5, lyophilized and called Arachis hypogaea L. trypsin inhibitor – AHTI.

Protein quantification and evaluation of the purity of the protein inhibitor

Proteins were quantified by the Bradford22 method using BSA as a standard. The purity degree of the inhibitor in different fractions and of the purified inhibitors was determined by SDS-PAGE 12.5% as described by Laemmli23. An aliquot containing approximately 15 µg of protein was applied on the gel. After electrophoresis, proteins were silver stained24. Molecular weight markers (Fermentas Life Sciences, Thermo Fischer Scientific, Waltham, MA) were utilized as standard.

Antitrypsin activity

The trypsin inhibitor activity of CE, PEB, F1, F2, F3, and AHTI was determined using the following methodology: 20 µL aliquots of bovine trypsin solution (0.3 mg/ml 2.5 mM HCl) were pre-incubated with 560 µL Tris-HCl 0.05 M buffer, pH 7.5, 120 µL of HCl 2.5 mM, and 100 µL of CE, PEB, F1, F2, or F3 and 20 µL of AHTI for 15 min at 37 °C. After this period, the reaction was started by adding 500 µL of a specific substrate solution (1.25 mM BApNA). The reaction proceeded for another 10 min under the same conditions of incubation and was then quenched by adding 120 µL of 30% acetic acid solution25. All assays were performed in triplicate and blank tests were performed. The absorbance was measured at 410 nm (Ultrospec™ 2100 pro, GE Healthcare Life Sciences, Piscataway, NJ). The results are expressed in IU/μg soluble protein. The IU was defined as the amount of inhibitor that can decrease by 0.01 nm the absorbance value in the trypsin inhibitor assay.

AHTI stability evaluation

To assess stability in different temperatures and pH we followed the methodology described by Gomes et al.26. Aliquots of AHTI were incubated for 30 min at 40, 60, 80, and 100 °C. After the incubation, samples were cooled to 4 °C. To evaluate the stability of AHTI after treatment at different pH inhibitor aliquots were dialyzed against buffer to the desired pH for 16 h. Such buffers were: to pH 2–4, Glycine-HCl; to pH 5–8 sodium phosphate monobasic; to pH 9–12 glycine-NaOH, in which the solute concentration was 100 mM. After 1 h incubation at 37 °C in these buffers the samples were dialyzed for 4 h in 50 mM Tris-HCl, pH 7.5, to adjust the pH of the assay. The inhibitory activity for trypsin assays were done in triplicate using 10 µg (0.3 nM) of the inhibitor.

Determination of IC₅₀ and K_i against trypsin

The concentration of AHTI that inhibits 50% of trypsin activity (IC₅₀) was determined by constructing a curve, which relates the inhibition percentage of trypsin activity and AHTI concentration. For the construction of the curve, increasing concentrations of inhibitor were added in 10 µL trypsin aliquots (0, 4 μg = 0.012 nM). To determine the mechanism of inhibition and inhibition constant (K_i), a graph was plotted according to Dixon et al.27, where the abscissa axis (X) corresponds to increasing concentrations of AHTI (0; 1.84; 3.68; 7.35; 11; 14.71; 29.41 and 58.82 nM) and the axis of ordinates (Y) corresponds to the inverse value of the maximum speed. The enzymatic assay for the determination of trypsin Ki was done using two BApNA concentrations (1.25 × 10⁻³ and 6.25 × 10⁻⁴M).

Experimental model to evaluate food intake, protein digestibility and biochemical alterations in diets supplemented with the AHTI

Wistar rats (n = 05), after 3 d of adaptation for the establishment of the average daily dietary intake, were distributed individually, randomly into four groups in metabolic cages, receiving AIN-93G according to Reves et al.28 for 11 d. These rats consumed a single dose of 100 g of one of the following diets (Table 1): (1) Standard – AIN-93G diet; (2) Standard + Water – Standard AIN-93G diet supplemented by oral gavage with water; (3) Aproteic diet; (4) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI according to Ribeiro et al.20 This latest group was included to control for the influence of the gavage procedure. The consumption of diets and water were daily monitored. Urine and feces of each animal were collected, identified and kept in a refrigerator for later analysis.

Table 1. Composition of the experimental diets used in Wistar rats in the experimental models.

Component	Standard diet	Aproteic diet	AHTI (25 mg/kg)	AHTI (50 mg/kg)
Casein (g)	100	–	100	100
Celulose (g)	50	50	50	50
Sacarose (g)	100	100	100	100
Mineral mix (mg)	35	35	35	35
Vitamins mix (mg)	10	10	10	10
L-Cystine (g)	1.8	–	1.8	1.8
Tert-butyl hydroquinone  (mg)	0.008	0.008	0.008	0.008
Choline bitartrate (mg)	2.5	2.5	2.5	2.5
Soybean oil (g)	40	40	40	40
Starch (g)	397.486	397.486	397.7	397.7

Experimental diets: (1) Standard diet – AIN-93G diet; (2) Aproteic diet; (3) AHTI (25 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (4) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI. AHTI: A. hypogaea L. trypsin inhibitor.

For the determination of protein digestibility, feces were dried with air circulation at 105 °C for 24 h. Then they were cooled, weighed, and ground in a multiprocessor for nitrogen determination using the Kjeldahl method described by AOAC (1984). Apparent and true digestibility and the apparent biological value were calculated according to Amaya et al.29

At the end of the tests with diets, the rats were fasted for 12–15 h, anesthetized with 2% xylazine and 5% ketamine and euthanized. Blood was collected by cardiac puncture and stored in Falcon tubes with EDTA anticoagulant. Serum was separated by centrifugation at 3000 × g for 10 min and used for glucose (GL), triglycerides (TG), total cholesterol (COL), high-density lipoproteins (HDL), and low-density lipoprotein (LDL) determination. The method used in the assays was the enzymatic-colorimetric (CELM® kit, São Paulo, Brazil). Blood samples were also subjected to enzymatic assays for glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) (Labtest diagnostic kit transferases, Paraná, Brazil).

Experimental model to evaluate weight gain, food intake at specific times, biochemical alterations, and plasmatic CCK in diets supplemented with the AHTI

Wistar rats (n = 05), after 3 d of adaptation for the establishment of the average daily dietary intake, were distributed individually, randomly into five groups in metabolic cages, receiving AIN-93G according to Reves et al.28 for 11 d. These rats were evaluated for changes in food intake after 1, 2, and 18 h of oral administration of a single dose of 100 g of one of the following diets: (1) Standard + Water – Standard AIN-93G diet, supplemented by oral gavage with water; (2) Casein – Standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of casein; (3) Soybean inhibitor – Standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of soybean inhibitor; (4) AHTI (25 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (5) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI. The consumption of diets and water were daily monitored, as well as weight gain per animal.

Food intake was daily monitored at 1, 2, and 18 h after diets administration. On the last day of the experiment, prior to oral administration of the diet, the animals were fasted for a period of 6 h. After this, 100 g of diet was administered and, 1 h after, animals were anesthetized with 2% xylazine and 5% ketamine and euthanized. The same biochemical parameters analyzed in the first experiment were repeated. There were also held the dosages of VLDL (CELM® Kit, Sao Paulo, Brazil), gamma GT (kit range gt Labtest Diagnostic SA®, Paraná, Brazil) and albumin (Labtest Diagnostic kit SA®, Paraná, Brazil). Plasmatic CCK was also assessed. Blood collection, plasma extraction, and immunoassay followed the specifications of the kit (Phoenix Pharmaceuticals Inc., Burlingame, CA).

Statistical analysis

Weight, feed efficiency, food intake, CCK, and biochemical parameters (glucose, total cholesterol, high-density lipoproteins (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), triglycerides, Aspartate transaminase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and albumin) were analyzed using one-way ANOVA followed by Tukey’s post-hoc test to find possible differences between the groups throughout the experiment. Differences were considered significant when the p value was less than 0.05. Data were analyzed using GraphPad Prism 6.07 software.

Results

Isolation and purity evaluation of the AHTI

In the present study, a new trypsin inhibitor was identified and isolated from peanut paçoca, showing 92% inhibition for the catalytic activity of trypsin and approximately 33 kDa (Figure 1). Therefore, this new trypsin inhibitor isolated from peanut paçoca was called AHTI. The AHTI had a yield of 63.4 and was purified 267 times (Table 2).

Figure 1. Trypsin–Sepharose affinity chromatography of F2 from peanut paçoca and SDS-PAGE of AHTI. (A) Proteic profile at 280 nm (black line) and anti-trypsin activity (gray line) of retained and eluted proteins in HCl 1 mM. Constant flow of 3.5 ml/min. (B) Denaturing, discontinuous polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue R250. M – Molecular marker. EB – Crude extract from peanut paçoca. F2 – Protein fraction from peanut paçoca saturated with 30–60% ammonium sulfate. AHTI: A. hypogaea L. trypsin inhibitor.

Table 2. AHTI isolation steps.

Steps	Volume (mL)	Total protein (mg)	Total inhibitory activity (UI)	Specific activity (UI/mg)	Purification (X)	Recovery (%UI)
Crude extract	150	432.00	17.9	0.04	1	100
F2	25	160.00	18.8	0.12	2.75	95.2
AHTI	12	2.64	28.2	10.68	267	63.4

F2, precipitation of the crude extract with ammonium sulfate in 30–60% saturation. AHTI, A. hypogaea L. trypsin inhibitor.

Effect of temperature and pH on AHTI inhibitory activity

Figure 2(A) shows that AHTI at temperatures of 60 °C to 80 °C kept its maximum inhibition activity over trypsin (92%). Nevertheless, the inhibitory activity of AHTI against different pH was changed when subjected to pH values greater than 4, resulting in 20% of trypsin inhibition (Figure 2B).

Figure 2. Stability of AHTI from peanut paçoca in different temperatures and pH. (A) 10 μg (0.30 nM) of AHTI was preincubated for 30 min at 37 °C and submitted to different temperatures (40 °C, 60 °C, 80 °C, 100 °C). (B) 10 μg (0.30 nM) of AHTI was incubated for 30 min at 37 °C in different pH2^,3^,6^,8^,11^,12. The inhibitory activity on trypsin was determined using BApNA as substrate. AHTI: A. hypogaea L. trypsin inhibitor.

Determination of IC50 and K_i against trypsin

According to the titration curve (Figure 3A), AHTI IC₅₀ is about 0.15 nM (5 µg) and is competitive, according to Dixon et al.27 AHTI inhibits trypsin competitively with a Ki value of 0.085 nM as shown in Figure 3(B).

Figure 3. Determination of IC₅₀ and Ki from AHTI from peanut paçoca. (A) AHTI concentration that inhibits 50% of trypsin activity (IC₅₀) was determined using BApNA as substrate and performed using the titration curve with 12.88 µM of trypsin and increasing concentrations of AHTI (0, 1.84, 3.68, 7.35, 11, 14.71, 29.41, and 5.82 nM). (B) The inhibition constant (Ki) of AHTI over trypsin was determined using Dixon et al.²⁷ method. The Ki value was obtained from the intercepts of the two lines of the BApNA concentrations used. The rate of the reaction was determined by V = DO405 nm/h/ml. AHTI: A. hypogaea L. trypsin inhibitor.

Weight gain and food intake in Wistar rats submitted to the diet supplemented with AHTI

In this study, we also evaluated the action of AHTI in weight gain and food intake. Thus, during the 11 d of the experiment, we observed that weight gain in the control group was higher when compared to the AHTI supplemented rats (Figure 4). Also, food intake was lower in the groups treated with AHTI (Table 3).

Figure 4. Weight gain (g/day) of Wistar rats submitted to different diets for 11 d. Diets (100 g): (1) Standard diet + Water – AIN-93G diet, supplemented by oral gavage with water (2) Casein – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of casein; (3) Soybean inhibitor – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of soybean inhibitor; (4) AHTI (25 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (5) AHTI (50 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg. Groups of n = 5 animals. AHTI: A. hypogaea L. trypsin inhibitor. Data expressed as mean ± standard deviation. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA with Tukey’s post-hoc test. f, test calculation.

Table 3. Mean food intake (g/day) on day 1, day 5, and day 11 of Wistar rats under standard diet, aproteic diet, and diet supplemented with 50 mg/kg of AHTI for 11 d.

	Diets
Day	Standard	Standard + Water	Aproteic	AHTI (50 mg/kg)	ANOVA
1	9.88 ± 1.25^a	7.58 ± 1.98^a	8.53 ± 1.95^a	8.69 ± 1.25^a	f = 0.870 p = 0.514
5	14.59 ± 2.09^a	13.56 ± 1.54^a	9.58 ± 3.06^a	5.13 ± 0.58^b	f = 10.99 p = 0.001
11	13.99 ± 2.54^a	12.58 ± 2.57^a	7.45 ± 2.06^b	6.15 ± 0.98^c	f = 8.188 p = 0.003

Diets (100 g): (1) Standard diet – AIN-93G diet; (2) Standard + Water – Standard AIN-93G diet, supplemented by oral gavage with water; (3) Aproteic diet; (4) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI. Groups of n = 5 animals. Data expressed as mean ± standard deviation of 11 d. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA with Tukey’s post-hoc test was done to calculate differences between groups of diets. f, test calculation. AHTI, A. hypogaea L. trypsin inhibitor.

Determination of in vivo protein digestibility

Interestingly, animals supplemented with AHTI, showed less weight gain compared to the control group and no significant statistical differences in true and apparent protein digestibility were identified between the groups receiving casein and AHTI (Table 4).

Table 4. True digestibility, apparent digestibility, and biological value in Wistar rats submitted to standard diet, aproteic diet, standard diet + Water, and 50 mg/kg of AHTI for 11 d.

Parameters	Standard diet	Standard diet + Water	AHTI (50 mg/kg)	ANOVA
True digestibility (%)	96.36^a	96.68^a	94.40^a	f = 2.227 p = 0.137
Apparent digestibility (%)	93.70^a	93.10^a	90.68^a	f = 3.857 p = 0.067
Biological value (%)	93.50^a	93.40^a	91.60^a	f = 3.247 p = 0.060

Diets (100 g): (1) Standard diet – AIN-93G diet; (2) Standard + Water – Standard AIN-93G diet, supplemented by oral gavage with water; (3) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI. Groups of n = 5 animals. Data expressed in mean percentage (%) of 11 d. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA and Tukey’s post-hoc test; f, test calculation; AHTI, A. hypogaea L. trypsin inhibitor.

Assessment of biochemical alterations

AHTI decreased fasting glucose in both experiments. No other significant biochemical alterations were observed in animals supplemented with AHTI. Importantly, animals also showed no significant variations between groups for albumin levels (Tables 5 and 6).

Table 5. Biochemical parameters of Wistar rats submitted to standard diet, aproteic diet, and with AHTI diets for 11 d.

	Diets
Parameters	Standard	Standard + Water	Aproteic diet	AHTI (50 mg/kg)	ANOVA
Fasting glucose (mg/dl)	154.80 ± 6.83^a	147.20 ± 11.92^a	85.40 ± 9.50^b	121.40 ± 20.61^c	f = 27.87 p = 0.000
Total cholesterol (mg/dl)	50.00 ± 9.08^a	54.00 ± 4.85^a	38.40 ± 4.72^b	52.20 ± 7.66^a	f = 5.280 p = 0.010
HDL (mg/dl)	23.80 ± 2.17^a	22.00 ± 2.35^a	21.40 ± 1.34^b	23.80 ± 2.17^a	f = 1.832 p = 0.182
LDL (mg/dl)	20.00 ± 7.07^a	21.40 ± 3.97^a	15.80 ± 3.42^a	17.20 ± 3.70^a	f = 1.433 p = 0.270
Triglycerides (mg/dl)	27.00 ± 5.79^a	20.80 ± 5.22^a	19.80 ± 3.70^a	25.60 ± 6.15^a	f = 2.227 p = 0.082
Glutamic oxaloacetic transaminase (U/L)	80.40 ± 18.40^a	71.80 ± 24.35^a	100.40 ± 19.65^a	79.40 ± 10.01^a	f = 2.111 p = 0.139
Glutamic pyruvic transaminase (U/L)	21.60 ± 5.41^a	26.00 ± 4.80^a,b	13.00 ± 1.22^a,c	16.80 ± 3.11^a,c	f = 4.993 p = 0.012

Diets (100 g): (1) Standard – AIN-93G diet; (2) Standard + Water – Standard AIN-93G diet, supplemented by oral gavage with water; (3) Aproteic diet; (4) AHTI (50 mg/kg) – Standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg of AHTI. Dosages were done on the last day of experiment. Groups of n = 5 animals. Data expressed as mean ± standard deviation. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA with Tukey’s post-hoc test. f, test calculation; AHTI, A. hypogaea L. trypsin inhibitor.

Table 6. Biochemical parameters of Wistar rats 1 h after administration of standard diet, or diets supplemented with casein, soybean inhibitor, and AHTI for 11 d.

	Diets
Parameters	Standard +  Water	Casein (25 mg/kg)	Soybean inhibitor (25 mg/kg)	AHTI (25 mg/kg)	AHTI (50 mg/kg)	ANOVA
Fasting glucose (mg/dl)	215.25 ± 19.03^a	186.80 ± 12.91^a,b	170.00 ± 10.15^b	156.00 ± 16.10^b	170.40 ± 17.77^b	f = 9.572 p = 0.000
Total cholesterol (mg/dl)	65.79 ± 7.79^a	79.25 ± 6.13^a,b	92.00 ± 8.03^b	80.00 ± 10.60^b	85.00 ± 5.79^b	f = 7.573 p = 0.001
HDL (mg/dl)	23.20 ± 2.99^a	30.00 ± 3.67^a,b	37.20 ± 6.06^b	34.70 ± 5.70^b	34.40 ± 2.88^b	f = 7.727 p = 0.000
LDL (mg/dl)	23.00 ± 2.74^a	31.00 ± 3.37^a	35.60 ± 10.57^a	33.40 ± 22.20^a	39.50 ± 3.83^a	f = 1.878 p = 0.160
VLDL (mg/dl)	17.60 ± 3.11^a	17.90 ± 3.68^a	16.86 ± 5.54^a	12.10 ± 8.73^a	14.72 ± 5.53^a	f = 0.756 p = 0.567
Triglycerides (mg/dl)	88.25 ± 14.97^a	109.20 ± 32.18^a	84.40 ± 27.81^a	61.33 ± 44.60^a	72.00 ± 31.92^a	f = 1.463 p = 0.257
Glutamic oxaloacetic transaminase (U/L)	133.50 ± 4.73^a,b	115.33 ± 16.77^a,b	105.80 ± 12.34^a,b	147.70 ± 15.90^a,b,c	154.80 ± 15.07^a,b,c	f = 7.779 p = 0.001
Glutamic pyruvic transaminase (U/L)	77.00 ± 12.11^a	53.75 ± 11.73^b	70.06 ± 8.87^a	61.04 ± 2,14^a	57.20 ± 8.90^a	f = 4.303 p = 0.015
Gama GT (U/L)	6.10 ± 0.90^a	6.40 ± 1.30^a	9.56 ± 1.69^a	9.73 ± 0.22^a	8.88 ± 1.48^a	f = 3.914 p = 0.221
Albumin (g/dL)	4.40 ± 0.22^a	4.68 ± 0.30^a	5.27 ± 0.60^b	4.50 ± 0.15^a	4.48 ± 0.17^a	f = 3.310 p = 0.042

Diets (100 g): (1) Standard + Water – standard AIN-93G diet, supplemented by oral gavage with water; (2) Casein – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of casein; (3) Soybean inhibitor – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of soybean inhibitor; (4) AHTI (25 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (5) AHTI (50 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg. Groups of n = 5 animals. Biochemical parameters measured on the last day of the experiment. Data expressed as mean ± standard deviation. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA with Tukey’s post-hoc test. f, test calculation. AHTI, A. hypogaea L. trypsin inhibitor.

Assessment of food intake at specific times and CCK alterations

In this model we observed a significant increase in plasmatic CCK in rats supplemented with 25 mg/kg and 50 mg/kg of AHTI (Figure 5A). By analyzing food intake in 1st, 2nd, and 18th h after the administration of the diets we observed greater decrease in food consumption in the groups supplemented with AHTI (Figure 5B).

Figure 5. (A) CCK plasmatic concentrations (pmol/L) 1 h after gavage administration of a single dose of different trypsin inhibitors for 11 d. Diets (100 g): (1) Standard diet + Water – AIN-93G diet, supplemented by oral gavage with water (2) Casein – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of casein; (3) Soybean inhibitor – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of soybean inhibitor; (4) AHTI 25 mg/kg – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (5) AHTI 50 mg/kg – standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg. (B) Reduction in food intake 1 h, 2 h and 18 h after different diet administration for 11 d: Diets (100 g): (1) Standard diet + Water – AIN-93G diet, supplemented by oral gavage with water (2) Casein – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of casein; (3) Soybean inhibitor – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of soybean inhibitor; (4) AHIT (25 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 25 mg/kg of AHTI; (5) AHIT (50 mg/kg) – standard AIN-93G diet, supplemented by oral gavage with 50 mg/kg. Groups of n = 5 animals. AHTI: A. hypogaea L. trypsin inhibitor. Results are expressed as mean ± standard deviation. Different letters indicate statistical difference between the groups with p < 0.05. One-way ANOVA with Tukey’s post-hoc test.

In the present study, the reduction of food intake was higher in the 1st and 2nd h after the administration of inhibitors by gavage, where the 25 mg/kg AHTI group reduced the food intake by 58.3% and 39.1% in the 1st and 2nd h, respectively. The 50 mg/kg AHTI group presented a reduction in food intake of 56.8% and 54.5% in the 1st and 2nd h, respectively. These results were higher than the effect of 25 mg/kg of SBTI (with a reduction in food intake of 35.7% and 53% in the 1st and 2nd h, respectively) and the other studied groups: casein with 27.5% and 22.1% reduction in the 1st and 2nd h and control, with 16.1% and 25.79% reduction in the 1st and 2nd h.

As for serum concentrations of CCK in the studied animals (Figure 5A), increased levels were detected in the test groups when compared to the control group (5.92 ± 1.15 pmol/L). The highest values were for groups submitted to diets with 25 and 50 mg/kg of AHTI, of 16.15 ± 1.13 pmol/L and 21.33 ± 2.12 pmol/L, respectively.

Discussion

In the present study, a new trypsin inhibitor was identified and isolated from peanut paçoca, a typical Brazilian sweet. There are reports of other trypsin inhibitors that have been purified, with molecular weights of varying sizes12–14, but none with the same mass presented in this study.

AHTI, similarly to other purified and characterized trypsin inhibitors30^,31, was thermo-resistant, keeping its total inhibitory activity on the enzyme trypsin activity. This explains why, surprisingly, the trypsin inhibitor in peanut paçoca, an industrialized food product, did not lose its activity by the processes by which it was submitted (denaturation). This stability is probably due to the presence of disulfide bridges between polypeptide chains of AHTI proteins32. Nevertheless, this must be tested. It is noteworthy that the stability of proteins is important for the development of new products and biotechnological applications at the industrial level.

AHTI is competitive, according to Dixon et al.27, as other trypsin inhibitors previously described3^,14. In this type of inhibition, the effect is reversed by increasing the substrate concentration. Therefore, the mechanism of inhibition is dependent on the substrate and inhibitor concentrations. It is known that many drugs routinely used have their action based on competitive inhibition of specific enzymes33. Norioka et al.13 purified and characterized peanut protease inhibitors (AI, A-II, BI, B-II B-III) with Ki values ranging between 1.4 and 4.4 × 10⁻⁷M. Thus, AHTI has a higher affinity for bovine trypsin when compared to other trypsin inhibitors previously reported in the literature.

We have previously shown11 that the consumption of peanut paçoca helped to reduce weight gain. Thus, in the present study we evaluated the action of AHTI isolated from paçoca in weight gain and food intake, finding that the supplementation with AHTI decreased both weight gain and food intake. We also analyzed the protein digestibility in rats supplemented with AHTI. The intention was to assess whether the reduction in weight gain was due to malnutrition caused by lower protein availability in the presence of AHTI or if the effect in reducing weight gain was, in fact, due to the satiety promotion by AHTI. Interestingly, AHTI did not negatively influence the use of proteins provided by the diet. Indeed, some authors suggest that there is a compensation of the inhibition of trypsin in the gut, affecting the enzyme stability by increased secretion and increased absorption of proteins in the distal gut34, which may have occurred in the experiment.

Furthermore, according to the results shown by Sgarbieri35, purified diets containing trypsin inhibitor from Rosinha G2 bean (Phaseolus vulgaris L.) administered in Wistar rats had no additional effect on protein digestibility and, as expected, the intestinal protease activity was not affected by diet trypsin inhibitor. However, according to Peace et al.36, Wistar rats fed SBTI showed a reduction in weight gain, feed efficiency, but also reduced the digestibility when compared to a control group receiving casein.

Strikingly, our results have shown that AHTI has a potential to reduce fasting glucose. Other biochemical parameters in both experimental models showed little difference between the groups, with most of the values within normal ranges37–39. Other studies with Wistar rats36^,40 and also with Swiss mice41 revealed no changes in lipid profile, as well as the dosages of glucose and transaminases20^,40^,42. As for AST and ALT, it is known that they have similar functions and its elevation in serum has been widely used as an indicator of hepatotoxicity or hepatocellular injury42. These data corroborate the fact that AHTI caused no cytotoxic effect at the studied doses and conditions. The animals also showed no statistically significant variations between groups for albumin levels, corroborating the fact that the animals did not show malnutrition and lost weight by reducing food consumption.

To corroborate the hypothesis that the reduction in weight gain promoted by AHTI was due to satiety promotion, a second experimental model to assess CCK production was done. In this model, we observed a significant increase in plasmatic CCK in rats supplemented with AHTI when compared to the other studied groups, which suggests that AHTI is a protein with a potential satietogenic effect.

Similar results were also observed in animals fed diets containing concentrated protease inhibitors (PPIC) (100 mg/kg)40, SBTI (100 mg/kg)43, potato protein extract (Potein) (100 mg/kg, 200 mg/kg, and 400 mg/kg)19 and Potein® (1.0 and 1.5 g/kg)17, all administered by gavage, and in humans subjected to a proteinase inhibitor extracted from potatoes (POT II)18.

In the present study, the reduction of food intake was higher in the 1st and 2nd h after the administration of inhibitors by gavage, where the 25 and 50 mg/kg AHTI group reduced the food intake in the 1st and 2nd h.

These results were similar to those reported for oral administration of Potein wherein three different concentrations were tested (100 mg/kg, 200 mg/kg and 400 mg/kg) and SBTI (100 mg/kg), with a decrease in food consumption between the 1st and the 3rd h19. Similarly, in another study in rats, Potein® also caused reduced food consumption, though the quantities used were higher (1.0 and 1.5 g/kg)17. In other studies, such as with PPIC, in 10 d of experiment, there was a cumulative decrease in food intake40. The authors concluded that this decrease in food intake was due to the presence of the trypsin inhibitor, which would provide a satietogenic effect, especially between the 1st and 3rd h after the administration of the trypsin inhibitor.

As for serum concentrations of CCK in our study, increased levels were detected in the test groups when compared to the control group. This result represents an increase of almost 250% with the administration of 50 mg/kg AHTI. Comparing the data of the administration of 25 and 50 mg/kg of AHTI, may indicate that possibly there is saturation in the satiety response to the concentration of CCK.

Some studies also show that there was increased plasma CCK when rats were fed diets with protein and trypsin inhibitor17^,18^,20^,40^,43. In these studies, the diets were supplemented with 100 mg/kg of casein and 100 mg/kg of PPIC which resulted in an increase of approximately 2–4 pmol/L of plasma CCK after 15 min40.

A study with synthetic protease inhibitors (20 mg/kg) also induced a significant increase in basal CCK from 2.59 pmol/L to 27.6 pmol/L in the 1st h and a decrease to 15.8 pmol/L in the 2nd h44.

In humans, the effect of POT II, which increases CCK release, was evaluated on food intake in 11 lean individuals. They received 1.5 g of POT II in a soup rich in protein (70 kcal). Those who consumed the soup alone showed no significant reduction in energy consumption, while those who consumed the soup with POT II presented a reduction in energy intake of 17.5%18.

Conclusions

The experimental administration of AHTI, a new trypsin inhibitor isolated from peanut paçoca, caused a reduction in food consumption, which resulted in a reduction in weight gain. AHTI also reduced fasting glucose. No other changes in the analyzed biochemical parameters were observed. The reduction in food intake was mediated by increased levels of CCK, once the inhibitor did not cause protein loss. This puts AHTI as a potential natural product for the prevention and/or treatment of obesity and obesity related hyperglycemia. However, more studies are needed to better understand the molecular basis for the increase in CCK and decrease in glucose seen in the present study. Also, further researches in humans are needed to corroborate the effect on human CCK and define the necessary AHTI doses.

Declaration of interest

We have no conflict of interest to declare. In memorian to Mauricio Pereira de Sales who encouraged the development of this work. This project was financially supported by the National Council for Scientific and Technological Development (CNPq-Brazil) – Universal 14/2013 – Process: 483710/2013-0 and was granted research grant by the National Council for the Improvement of Higher Education (CAPES-Brazil).