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Effects of passion fruit peel flour (Passiflora edulis f. flavicarpa O. Deg.) in cafeteria diet-induced metabolic disorders
Journal of Ethnopharmacology 250 (2020) 112482
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Effects of passion fruit peel flour (Passiflora edulis f. flavicarpa O. Deg.) in cafeteria diet-induced metabolic disorders
T
Aline De Faveria, Renata De Faverib, Milena Fronza Broeringa, Izabel Terranova Bousfielda, Marina Jagielski Gossa, Samuel Paulo Mullerc, Raquel Oliveira Pereirad, Ana Mara de Oliveira e Silvad, Isabel Daufenback Machadoc, Nara Lins Meira Quintãoa, José Roberto Santina,∗ a
Postgraduate Program in Pharmaceutical Science, Universidade Do Vale Do Itajaí, Itajaí, Santa Catarina, Brazil Biomedicine Course, Universidade Do Vale Do Itajaí, Itajaí, Santa Catarina, Brazil Postgraduate Program in Biodiversity, Universidade Regional de Blumenau, Blumenau, Santa Catarina, Brazil d Nutrition Department (DNUT), Universidade Federal de Sergipe (UFS), São Cristóvão, Sergipe, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Passiflora edulis Metabolic syndrome Cafeteria diet Diabetes Steatosis
Ethnopharmacological relevance: Passiflora edulis f. flavicarpa O. Deg. is a native Brazilian fruit known as sour or yellow passion fruit. From its peel, mainly in the northeast of Brazil, is produced a flour that is largely used as folk medicine to treat diabetes and other metabolic conditions. Aim of the study: The aim of the study was to show the effects of P. edulis peel flour (PEPF) in metabolic disorders caused by cafeteria diet in mice. Material and methods: The antioxidant activity in vitro of PEPF extract was determined by ferric reducing/antioxidant power, β-carotene/linoleic acid system and nitric oxide scavenging activity assay. C57BL/6 mice divided in 3 groups: Control group, fed on a standard diet (AIN); Cafeteria diet (CAF) group, fed on a cafeteria diet, and PEPF group, fed on a cafeteria diet containing 15% of PEPF, during 16 weeks. The glucose tolerance and insulin sensitivity were evaluated through the glucose tolerance test (GTT) and the insulin tolerance test (ITT). After the intervention period, blood, hepatic, pancreatic and adipose tissues were collected for biochemical and histological analysis. Cholesterol, triglyceride, interleukins and antioxidant enzymes were measured in the liver tissue. Results: PEPF extract presented antioxidant activity in the higher concentrations in the performed assays. The PEPF intake decreased the body weight gain, fat deposition, predominantly in the liver, improved the glucose tolerance and insulin sensitivity in metabolic changes caused by cafeteria diet. Conclusion: Together, the data herein obtained points out that P. edulis peel flour supplementation in metabolic syndrome condition induced by CAF-diet, prevents insulin and glucose resistance, hepatic steatosis and adiposity.
1. Introduction
Generally, several symptoms cluster together, among them are insulin resistance, obesity/central obesity, dyslipidaemia and hypertension (Eslami et al., 2019). This complex pathophysiological condition is usually originated from high calorie intake and low energy expenditure. However, it can be also affected by individual epigenetic characteristics, sedentary lifestyle and also the composition of gut microbiota (Reave, 2012; Saklayen, 2018). Passion fruit (Passiflora edulis f. flavicarpa O. Deg.), plant native from Brazil, is popularly known as sour or yellow passion fruit (Dhawan
Metabolic syndrome comprehends an important condition that combine at least two risk factors occurring together, such as type 2 diabetes mellitus, cardiovascular disease, obesity and hepatic disorders, that leads to approximately 1.6-fold increase in mortality worldwide (Cornier et al., 2008; Schnack and Romani, 2017). In real numbers, more than a billion people suffer from metabolic syndrome (Saklayen, 2018).
∗ Corresponding author. Postgraduate Program in Pharmaceutical Sciences, Universidade do Vale do Itajaí, Rua Uruguai, 458, Bloco F6, ECS, Sala 316, CEP 88302901, Itajaí, SC, Brazil. E-mail address: [email protected] (J.R. Santin).
https://doi.org/10.1016/j.jep.2019.112482 Received 30 July 2019; Received in revised form 11 December 2019; Accepted 12 December 2019 Available online 19 December 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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bath at 50 °C for further reading 2 h later. The results were expressed as calculated percentage of oxidation inhibition (% OI) compared to the absorbance decay of the control (considered as 100% of oxidation). The nitric oxide (NO) scavenging activity was evaluated adding 50 μL of the extract (3, 10, 30, 100, 300 or 1000 μg/mL) or Trolox (100 μg/mL) and 50 μL of SNP (20 mmol/L; in phosphate buffer; pH 7.4) in a 96-well microplate, and then incubated at 37 °C for 1 h. After that, 100 μL of Griess reagent (2% sulphanilamide, and 0.2% napthylethylene diamine dihydrochloridediluted in 5% o-phosphoric acid) were added and the absorbance was obtained at 540 nm. A standard curve for sodium nitrite (NaNO2; 5–100 μmol/L) was plotted to obtain the results expressed as μmol of nitrite/L (Basu and Hazra, 2006).
et al., 2004). Agra et al. (2007) extensively studied the most used medicinal plants in Brazil and showed that the dried and powdered peel of P. edulis is used in the folk medicine for the treatment of diabetes. It is also important to emphasize that the fruit peel contains the amount of phenolics higher than the edible portion or pulp (Tehranifar et al., 2011), such as vitexin, isovitexin, apigenin, isoorientin, cyanidin-3-Oglycoside and quercetin-3-O-glycoside. Additionally, the edulic acid, a carboxylic acid, is also founded in the peel fruit (Yapo and Koffi, 2006; Zeraik et al. 2011, 2012; Cazarin et al., 2014). Probably, this chemical composition is responsible for the promising biological effects observed by the population. Besides the chemical compounds, the peel presents high quantity of dietary fibres, mainly the soluble ones, pectin as example (Goss et al., 2018), a nutrient that presents crucial role in the maintenance of human health, reducing the risk of developing metabolic syndrome and its complication (Bernaud and Rodrigues, 2013; Slavin, 2013; Zhang et al., 2016). Recently, Goss and co-authors (2018) have demonstrated that the passion fruit peel flour added to the rat food significantly reduced the insulin resistance and adipocyte hyperplasia induced by fructose-induced metabolic syndrome. In this study we aimed to evaluate the effects of passion fruit peel flour (PEPF) in metabolic syndrome induced by cafeteria (CAF) diet, which is an appropriate experimental model to study metabolic syndrome, once it is able to induce obesity with insulin resistance, high plasma triglyceride and steatosis (Parafati et al., 2018). Data here-obtained point out that PEPF prevents the glucose intolerance, insulin resistance and mainly the hepatic steatosis induced by CAF diet, providing a beneficial employment of this product.
2.3. Animals and treatment Male 3-week old C57BL/6 mice were obtained from the Central Vivarium of the Universidade do Vale do Itajaí. The animals were kept with free access to water and food, 4 animals per cage (20–23 °C, humidity 60%, 12 h light/dark cycle) for at least a week before the experiment. All procedures were performed according to the Brazilian Society of Science of Laboratory Animals guidelines for the appropriate care and use of experimental animals. All procedures were approved by the local Ethics Committee of UNIVALI (protocol number 016/17). The mouse weight and water and food intake values were obtained every three days. Thirty mice were distributed in 3 different groups (n = 10): (1) control: water and standard diet; (2) Cafeteria (CAF) group: water and cafeteria diet; (3) Cafeteria (CAF) plus PEPF group: water and cafeteria diet plus 15% of PEPF. All treatments were conducted daily for 16 weeks. The standard diet (AIN-93M) adopted in the research followed the recommendations of the American Institute of Nutrition for a maintenance diet of adult mice (Reves et al., 1993). To compose the cafeteria diet, AIN-93 diet was associated to hyper caloric foods rich in carbohydrates and lipids, as milk-chocolate, peanuts and sweet biscuits, in ratios of 3:2:2:1. This diet was previously standardised by Estadella et al. (2004) and analysed by Oliveira et al. (2015). For the treated group (3), 15% of PEPF were added to the CAF diet. The ingredients were homogenised, and the mixture was moistened with distilled water for manual palletisation. The different experimental diets were kept in an air-circulation oven at 50 °C for 48 h to dry and store in sealedplastic containers at 4 °C. The calorie intake was calculated considering 4 calories for protein and carbohydrates, and 9 calories for lipids. The calculation was made based on the nutritional information provided by the manufacturers considering all constituents of each treatment (Control, CAF, CAF + PEPF).
2. Methods and materials 2.1. Sample The PEPF, a product commercially available, was kindly provided by a food industry located in Santa Catarina, Brazil (Vitalin, number lot. 11590, with 8.7 mg of fiber in 15 g of flour). For the in vitro analysis, an extract of the PEPF was obtained using 30 g of PEPF in 10 mL of methanol:water (1:1 v/v), kept stirring for 30 min at 50 °C. Then the product was filtrated using a methanol soaked paper filter, under vacuum. The obtained extract was used to verify the antioxidant activity. The chromatographical profile of PEPF extract was previously published by Goss et al. (2018). 2.2. Antioxidant activity The antioxidant activity of the PEPF extract was evaluated using in vitro methods described below. The reducing potential of PEPF extract was analysed by the ferric reducing/antioxidant power (FRAP) assay previously described by Pulido et al. (2000) with few modifications. Firstly, 9 μL of the extract, at concentrations of 3, 10, 30, 100, 300 or 1000 μg/mL, or Trolox (100 μg/mL), were added to 27 μL of distilled water plus 270 μL of freshly prepared FRAP solution (acetate buffer pH 3.6 (0.3 mmol/L)), 2,4,6-tripyridyl-s-triazine (10 mmol/L) and ferric chloride (FeCl3) in a 96-well microplate. The microplate was then incubated at 37 °C for 30 min and the absorbance was obtained at 595 nm and the results were calculated based on a ferrous sulphate (FeSO4) standard curve. The antioxidant capacity by β-carotene/linoleic acid system was determined using the method described by Miller (1971) with few modifications. Initially, an emulsion was prepared using the proportions as follow: 120 μL of β-carotene/chloroform solution (20 mg/mL), 70 μL of linoleic acid, 22 drops of Tween 40 and 1 mL of chloroform. 250 μL of this emulsion were pipetted in a 96-well microplate plus 35 μL of the extract at concentrations of 3, 10, 30, 100, 300 or 1000 μg/ mL, or the positive control Trolox (100 μg/mL). The first reading was done at 470 nm wave length and the microplate was the kept in water
2.4. Glucose tolerance test (GTT) Fasted mice (6 h between 7:00 and 13:00 h prior to the experiment, as recommended for C57BL/6 mice; Wang and Liao, 2012) were submitted to blood extraction, representing time 0. Then, glucose was orally dosed (2 g/kg) and blood was sampled at different time-points, 30, 60, 90 and 120 min. The area under the glucose decay curve (AUC) was calculated for each mouse and the mean was calculated for each group (Faulhaber-Walter et al., 2011).
2.5. Insulin tolerance test (ITT) The same fasting protocol used for GTT was adopted for this protocol. The first glucose level was measured at time 0. Then, the mice received intraperitoneal insulin injection (2 U/kg) and the glucose level was evaluated at 15, 30, 60, 90 and 120 min after the insulin injection. The area under the glucose decay curve (AUC) was calculated for each mouse and the mean was calculated for each group (Yuan et al., 2013). 2
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Fig. 1. Antioxidant activity of Passiflora edulis peel flour extract by ferric reducing/antioxidant power (FRAP) assay (A), β-carotene/linoleic acid system (B) and nitric oxide (NO) scavenging activity (C). Data are expressed as means ± S.E.M. *p < 0.05, **p < 0.01 and ***p < 0.001 vs reactional system (RS).
2.6. Biochemical parameters
2.8. Determination of the of antioxidant markers in the liver
The biochemical analysis was performed with the blood samples collected at the end of the experiment. After the fasting protocol mentioned above, the animals were anaesthetised (ketamine and xilasine) and their blood samples were taken from the brachial artery. The samples were centrifuged at 4000 g, at 4 °C for 10 min to separate the serum. The total cholesterol, triglyceride, creatinine, glucose, and the activity of aspartate transaminase (AST) and alanine transaminase (ALT) concentrations were measured using corresponding commercial kits (Labtest, Lagoa Santa, MG, Brazil) by spectrophotometry and serum insulin concentration was measured by electrochemiluminescence assay.
Liver samples were homogenised in phosphate buffer (50 mM; pH 7.0) and centrifuged at 10,000×g for obtaining the supernatant. Tissue antioxidant status was evaluated in the supernatant using the ferric reducing/antioxidant power (FRAP) assay. Results were plotted against a standard curve of ferrous sulphate (500–1500 μmol/L) and were expressed as μmol of ferrous sulphate/mg of protein. The hepatic reduced glutathione (GSH) content was measured using the method of Tietze (1969), and the GSH level was calculated by using pure GSH as standard. Catalase (CAT) activity was assessed by the decrease in absorption of H2O2 at 240 nm, due to H2O2 consumption by CAT, as described previously (Beutler, 1975). Specific activity was expressed as units of CAT/mg of protein. The Superoxide dismutase (SOD) activity was assessed verifying the production of superoxide anion by xanthine oxidase in the presence of xanthine. One unit of SOD activity was expressed as the amount of SOD required to inhibit the rate of cytochrome C reduction by 50% at 25 °C (McCord and Fridovich, 1969). Glutathione peroxidase (GPx) activity was expressed as μmol of glutathione oxidized/mg of protein/min by using the extinction coefficient for NADPH (6220 M−1/cm), as previously described by Sies et al. (1979). The tissue protein content was determined using the Bradford method (Bio-Rad® protein assay reagent).
2.7. Extraction of hepatic lipid The liver cholesterol and triglyceride were extracted according Folch's method (1957). At the end of the experiment, fresh livers were extracted and homogenised using chloroform/methanol (2:1, 3.75 mL). Chloroform and distilled water were then added to the homogenate and the solution was vortexed. After centrifugation (1500 g for 10 min), the lower organic phase was transferred to a new glass tube and lyophilised. The lyophilised powder was dissolved in a chloroform:methanol (1:2) mixture and then stored at −20 °C. Cholesterol and triglyceride concentrations were determined using diagnostic commercial kits (Labtest, Lagoa Santa, MG, Brazil) by spectrophotometry. 3
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Fig. 2. Effects of PEPF supplementation on weight gain (A and B), food consumption (C) and energetic intake (D) after 16 experimental weeks. Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). **p < 0.01 vs cafeteria group and #p < 0.05 vs control group.
2.9. Liver IL-6 and TNF levels
3. Results
Liver samples were homogenised in phosphate buffer (50 mM; pH 7.0) and centrifuged at 10,000×g to obtain the supernatant, which was used to analyze the interleukin 6 (IL-6) and tumor necrosis factor (TNF) levels using ELISA kits (R&D systems), in accordance to the manufacture instructions.
3.1. In vitro antioxidant activity of PEPF extract Data presented in Fig. 1A demonstrates that PEPF extract significantly increased the reducing potential, from concentrations of 300–1000 μg/mL, when compared with the system. The Trolox (100 μg/mL) also presented significant effect when compared with the system. Fig. 1B shows the antioxidant activity of PEPF against lipid peroxidation, and the obtained data demonstrates that incubation with PEPF (1000 μg/mL) and trolox significantly inhibited the lipid peroxidation. PEPF extract also presented significant NO scavenging activity from concentrations of 30–1000 μg/mL, when compared to the system. As a control, Trolox (100 μg/mL) reduced the nitrite production (Fig. 1C).
2.10. Histological analysis Small samples of hepatic, pancreatic and adipose tissues, extracted at the end of the experiment were fixed in 10% formalin. Then, followed the dehydration protocol, they were embedded in paraffin, sliced and stained using hematoxylin-eosin (HE). Images of the histological sections were taken using Olympus CBA® optical microscope. For analysis of the pancreas, 15 images were captured for each experimental group using a 10x objective. The image analysis was determined using Image J® software, which quantified the areas of the respective functional units of each tissue. The results were expressed in micrometres/ field. For the adipose tissue, 10 images of each experimental group were captured using 10x objective, and the images were analysed using Adiposoft® Software (NIN, USA) (Galarraga et al., 2012).
3.2. Effect of PEPF in weight gain and food consumption Fig. 2A shows that CAF diet induced significant increase in body weight gain in the 10th week of treatment and achieving the stabilization until the end of the experiment (16 weeks). The PEPF supplementation (15%) increased the body weight gain. However, after the 5th week, the weight gain was lower than the CAF-diet group. In addition, it is important to emphasize that animals from CAF and CAF + PEPF groups presented significant reduction in the food consumption (Fig. 2C), although the energetic intake did not modify (Fig. 2D).
2.11. Statistical analysis Results are expressed as mean ± standard error mean (SEM) of 10 mice per group. Statistical comparisons were performed using one-way analyses of variance (one way-ANOVA) followed by Tukey's test. P values less than 0.05 (P < 0.05) were considered significant. 4
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Fig. 3. Effects of PEPF supplementation in adipose tissue parameters. Abdominal adipose tissue weight (A) and relative weight (B). Epididimal adipose tissue weight (C) and relative weight (D). Adiposity index (E), illustrative images (F) and calculated area of adipocyte cells (G). Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05 and **p < 0.01 vs cafeteria group and #p < 0.05 vs control group.
correlated to glucose or insulin metabolism (Fig. 4), and consequently any alteration was visualised in the pancreas histology (Fig. 4G).
3.3. Adipose tissue parameters Data presented in Fig. 3A–D showed that CAF-fed animals presented absolute and relative increase in the abdominal and epididimal adipose tissue, which promoted significant intensification of the adiposity index (Fig. 3E), when compared to control group. In addition, data from histological analysis of the adipose tissue (Fig. 3F) showed that the adipocyte's area (Fig. 3G) increased in the CAF-fed animals. Animals that were supplemented with PEPF presented profile similar to the control group, even being exposed to CAF diet (Fig. 3A–G).
3.5. Liver parameters The increase in liver lipids from CAF diet can be responsible for inducing non-alcoholic fatty liver disease (NAFLD) and increase in cholesterol and triglyceride in the serum. In fact, the results presented in Fig. 5 showed that CAF diet induced hepatic steatosis, which can be visualised in the histopathological analysis (Fig. 5A), where a lot of areas with lipid deposition can be evidenced. The steatosis was also confirmed by the increase in the hepatic cholesterol and triglyceride (Fig. 5B and C). Additionally, the CAF diet promoted increase in cholesterol and triglycerides in the serum (Table 1). Besides, CAF diet promoted significant elevation in the liver IL-6 levels (Fig. 5E), without interfering with AST and ALT concentration in the serum (Table 1). Nevertheless, animals that received PEPF presented normal levels for all evaluated parameters, demonstrating that PEPF prevented the biochemical effects of CAF diet. In addition, it was accessed the SOD, CAT, GPx, GSH and FRAP in the liver. The obtained data demonstrates that cafeteria diet was not able to significantly modify none of the evaluated parameters (data not show).
3.4. Effect of PEPF on insulin and glucose tolerance and fasting blood glucose, and pancreas parameters The obtained data showed that CAF diet was capable of developing glucose intolerance and insulin resistance, which was marked by increase in the fasting glucose (Fig. 4A) and in the tolerance glucose test (Fig. 4B and C). Additionally, animals that received CAF diet did not respond to exogenous insulin administration, as shown in Fig. 4D and E, characterising insulin resistance. Corroborating this data, the animals from CAF group presented increase in insulin level (Fig. 4F) and Langerhans islets hyperplasia (Fig. 4G), which was confirmed by the measurement of Langerhans islets area (Fig. H). On the other hand, animals that received PEPF did not present any metabolic change 5
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Fig. 4. Effects of PEPF supplementation in glucose metabolism after 16 experimental weeks. Fasting glucose (A), oral glucose tolerance test (B and C), insulin resistence test (D and E), plasma insulin (F), illustrative images (G) and calculated area of Langherhans islets (H). Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05, **p < 0.01 and ***p < 0.001 vs cafeteria group and #p < 0.05, ## < 0.01 vs control group.
4. Discussion
agents or suppressors of singlet oxygen, which impairs the initial steps and propagation of oxidative stress (Cazarin et al., 2014). In the present manuscript, we present more data related to the in vitro antioxidant effect of PEPF extract. The obtained results showed that PEPF extract acts as a NO scavenger, which is important for the reduction of the superoxide anion and peroxynitrite production (Gow et al., 1996; Sorokin, 2016). Additionally, the PEPF extract showed considerable reducing power, as demonstrated by the FRAP method. This data is important because during the oxidative stress Fe3+ can react with O2−, resulting in the formation of Fe2+. This species can take place in Fenton's reaction and generates hydroxyl radicals, highly reactive (Shahidi and Zhong, 2015). Finally, the PEPF extract was able to inhibit the lipid peroxidation of β-carotene. Together, these results confer to the PEPF extract considerable antioxidant activity, an important attribute for substances with the aim of preventing and also treating diseases, such as metabolic syndrome. Clinicians and researchers agree that fat diet consumption over long periods can cause metabolic impairment, such as obesity, glucose and insulin disturbance, hepatic steatosis, and so on (Castell-Auví et al., 2012). Recent literatures have reported that CAF diet promotes enhance of body weight gain in animals and, consequently, obesity (Sampey et al., 2011; Gomez-Smith et al., 2016; Dalby et al., 2017; Soares et al., 2017). Definitely, our data demonstrates that CAF diet promotes weight gain, with decreasing fed intake, but without interference of energetic intake. In contrast, the group that had PEPF incorporated to their diet presented lower percentage of weight gain.
The main factor that is contributing for the global increase of obesity is focused in diet habits, caused by the increased snacking on energy-dense foods consumption (Nasreddine et al., 2018). Our group previously demonstrated the effects of PEFF on low-fructose diet in young rats, mimetizing a diet rich in carbohydrate (Goss et al., 2018). Here, we intended to use a CAF-diet, which simulates a specific human diet habit in rodents, once it is composed by lipids and carbohydrates, and improving the metabolic syndrome knowledge, currently in the spotlight (Janebro et al., 2008). In this context, here we investigated the effect of PEPF consumption in the metabolic changes evokes by CAF diet in mice, once the literature has proposed that natural products are promising tool for dealing with metabolic disorders. (Zeraik et al., 2011, 2012; Cazarin et al., 2014). Different parts of P. edulis are extensively used by the population as sedative, anxiolytic, antispasmodic, and antioxidant. The dried and powdered peel of P. edulis is especially used to empirically treat diabetes (Agra et al., 2007). In fact, the literature attributes the PEPF effects to its dietary fiber content, as well as, to the antioxidant ability of its bioactive constituents (Zeraik et al. 2011, 2012; Cazarin et al., 2014; Janebro et al., 2008). Our group has previously demonstrated the phytochemical profile of PEPF extract, showing the presence of caffeic acid and flavonoids, as well as the antioxidant effect in the DPPH assay (Goss et al., 2018). The antioxidant activity of phenolic acids and flavonoids can be accredited to their action as hydrogen donors, reducing 6
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Fig. 5. Characterization of liver tissue from animals submitted PEPF treatment. Illustrative images of liver (A), levels of cholesterol (B), triglycerides (C), TNF (D) and IL-6 (E) in the liver. Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05 vs cafeteria group and #p < 0.05 vs control group.
the epididymal and abdominal fat deposition, leading to adipocyte hypertrophy. This data corroborates with the literature, which shows that CAF diet causes adipocyte hypertrophy, an important factor associated to dyslipidemia, insulin resistance and inflammation (Sampey et al., 2011).
Moreover, the PEPF treated animals did not present alteration in the adipose tissue deposition, presenting lower adipose index compared to CAF diet group. Importantly, the adipose tissue histological analysis from PEPF-treated animals did not show any change in the adipocytes structure. Differently, animals from CAF group presented increase in
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Table 1 Lipid profile and liver enzymes in the different experimental groups after 16 weeks. Groups Control CAF CAF + PEPF
Cholesterol ##
57.66 ± 7,31 mg/dL 98.33 ± 3.93 mg/dL** 54.66 ± 4.37 mg/dL
Triglycerides
AST
ALT
34.00 ± 3.78 mg/dL 85.66 ± 3.05 mg/dL*** 48.00 ± 4.09 mg/dL
63.28 ± 1.19U/L 72.87 ± 4.06U/L 52.2 ± 0.96U/L
22.4 ± 0.67U/L 21.33 ± 1.05U/L 18.2 ± 0.73U/L
Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus P. edulis Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Values express the mean ± e.p.m. Statistical analysis was performed using ANOVA followed by the Tukey post-test. ** < 0.005 vs. group CAF + PEPF. *** < 0.001 vs. group CAF + PEPF. ## < 0.005 vs. group CAF.
allied to changes in diet habits to prevent metabolic diseases and improve patients quality of life.
The CAF diet for 16 weeks leads to the development of glucose and insulin resistance, confirmed by the increase in serum glucose and insulin, and also by the increase in the Langerhans islets area in the histopathological analysis. Insulin resistance is a well-described characteristic of obese mice, and is usually associated with decrease in insulin-receptor-AKT (IR–AKT) pathway activity, which is the main recognized signaling pathway in organs, such as skeletal muscle, liver, and adipose tissue (Czech, 2017). According to Vanzela et al. (2010), the insulin-content increase and its releaser from pancreatic islets are linked to Ca2+ cellular influx changes stimulated by CAF-diet. Of note, our data demonstrated that PEPF supplementation improved insulin resistance leading to significant decrease in insulin levels, and increase glucose uptake. Added to the effects in the insulin resistance, it is known that CAF diet lead to hepatic non-alcoholic damage associated to increase in lipogenesis, which cases steatosis (increase of lipid accumulation in the liver; Maeda Júnior et al., 2018). Clearly, our data shows that CAF diet promoted cholesterol and triglycerides accumulation in the liver, which can be responsible for inducing NAFLD. Besides hepatic steatosis, this disease can progress to fibrosis, cirrhosis and at the end, end-stage liver disease (Panera et al., 2018). Other important point is that CAF diet group was characterized by the presence of ballooning hepatocytes and some lobular inflammatory loci. Regarding to the inflammatory mediators, it was observed significant increase in the IL-6 levels in the liver tissue samples from CAF group, the main pro-inflammatory cytokine typically involved in the steatosis (Zeeni et al., 2015; Thiesen et al., 2018). In fact, the IL-6 pathway regulates hepatocyte proliferation and protects against cell death and oxidative stress, thus it suggests that CAF-diet promoted damage in the liver cells (Ozaki, 2019). Elevated levels of circulatory aminotransferases (AST and ALT) are common indicators for hepatocellular damage, and AST, in particular, has been widely used as marker for NAFLD (Hadizadeh et al., 2017). However, our data demonstrate that CAF-diet did not interfered with AST and ALT concentration. In fact, patients with NAFLD may present normal ALT and AST levels, even with advanced fibrosis. Additionally, Gasperin et al. (2018) also demonstrated that cafeteria diet did not modify the activity of AST and ALT in males or females’ mice treated with CAF-diet. It emphasizes the importance of histopathological-analysis association, the gold standard for diagnose (Goesslin et al., 2008; Elizondo-Montemayor et al., 2014). Together, these findings demonstrated the important effect of PEPF supplementation in the prevention of liver inflammation and steatosis development.
Author's contributions J.R. Santin, N.L.M. Quintão and A.M.O. Silva conceived and designed the study; A. De Faveri, M.F. Broering, R. De Faveri, R. Nunes, I.T. Bousfield, M.J. Goss, S.P. Muller, I.D. Machado and R.O. Pereira performed the experiments and analysed the data. J.R. Santin, N.L.M. Quintão and A. De Faveri wrote the paper. All authors read and approved the final manuscript. Acknowledgments This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 429505/2018-3), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, cod. 001) and the Fundação de Amparo à Pesquisa e inovação do Estado de Santa Catarina (FAPESC), A.D.F, M.F.B, and I.B. were Master students, and are recipient of CAPES (Cod. 001) grants during the study. References Agra, M.F., Freitas, P.F., Barbosa-Filho, J.M., 2007. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev. Bras. Farmacogn. 17, 114–140. Basu, S., Hazra, B., 2006. Evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases. Phytother Res. 20, 896–900. https://doi.org/10.1002/ptr.1971. Bernaud, F.S.R., Rodrigues, T.C., 2013. Fibra alimentar: ingestão adequada e efeitos sobre a saúde do metabolismo. Arquivos Brasileiros Endocrinol. Metabol. 57, 397–405. Beutler, E., 1975. Red Cell Metabolism: a Manual of Biochemical Methods. Grune & Stratton, London, pp. 89–90. Castell-Auví, A., Cedó, L., Pallarès, V., Blay, M., Ardévol, A., Pinent, M., 2012. The effects of a cafeteria diet on insulin production and clearance in rats. Br. J. Nutr. 108, 1155–1162. https://doi.org/10.1017/S0007114511006623. Cazarin, C.B.B., Silva, J.K.S., Colomeu, T.C., Zollner, R.L., Maróstica Junior, M.R., 2014. Capacidade antioxidante e composição química da casca de maracujá (Passiflora edulis). Ciência Rural. 44 1699-04. Cornier, M., Dabelea, D., Hernandez, T.L., Lindstrom, R.C., Steig, A.J., Stob, N.R., Van Pelt, R.E., Wang, H., Eckel, R.H., 2008. The metabolic syndrome. Endocr. Rev. 29, 777–822. https://doi.org/10.1210/er.2008-0024. Czech, M.P., 2017. Insulin action and resistance in obesity and type 2 diabetes. Nat. Med. 23, 804–814. https://doi.org/10.1038/nm.4350. Dalby, M.J., Ross, A.W., Walker, A.W., Morgan, P.J., 2017. Dietary uncoupling of gut microbiota and energy harvesting from obesity and glucose tolerance in mice. Cell Rep. 21, 1521–1533. https://doi.org/10.1016/j.celrep.2017.10.056. Dhawan, K., Dhawan, S., Sharma, A., 2004. Passiflora: a review update. J. Ethnopharmacol. 94, 1–23. https://doi.org/10.1016/j.jep.2004.02.023. Elizondo-Montemayor, L., Ugalde-Casas, P.A., Lam-Franco, L., Bustamante-Careaga, H., Serrano-González, M., Gutiérrez, N.G., Martínez, U., 2014. Association of ALT and the metabolic syndrome among Mexican children. Obes. Res. Clin. Pract. 8, 79–87. https://doi.org/10.1016/j.orcp.2012.08.191. Eslami, O., Shidfar, F., Dehnad, A., 2019. Inverse Association of Long-term nut Consumption with weight gain and risk of overweight/obesity: a systematic review. Nutr. Res. 68, 1–8. https://doi.org/10.1016/j.nutres.2019.04.001. Estadella, D., Oyama, L.M., Dâmaso, A.R., Ribeiro, E.B., Nascimento, C.M.O., 2004. Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 20, 218–224. https://doi.org/10.1016/j.nut.2003.10.008. Faulhaber-Walter, R., Jou, W., Mizel, D., Li, L., Zhang, J., Kim, S.M., Huang, Y., Chen, M., Briggs, J.P., Gavrilova, O., Schnermann, J.B., 2011. Impaired glucose tolerance in the absence of adenosine A1 receptor signaling. Diabetes 60, 2578–2587. https://doi. org/10.2337/db11-0058. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and
5. Conclusion Together, the data herein obtained point out that P. edulis peel flour supplementation in metabolic syndrome condition, prevents insulin and glucose resistance, hepatic steatosis and adiposity induced by CAF-diet. Additionally, the PEPF was able to reduce the inflammatory process in the liver. All data obtained corroborating with its ethnopharmacological use. It is important to emphasize that CAF diet fed intake by the animals mimics human unhealthy diet, and the average consumption in this study was consistent with the eating habits in many places worldwide, reinforcing the need of finding new strategies that could be 8
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Effects of passion fruit peel flour (Passiflora edulis f. flavicarpa O. Deg.) in cafeteria diet-induced metabolic disorders
T
Aline De Faveria, Renata De Faverib, Milena Fronza Broeringa, Izabel Terranova Bousfielda, Marina Jagielski Gossa, Samuel Paulo Mullerc, Raquel Oliveira Pereirad, Ana Mara de Oliveira e Silvad, Isabel Daufenback Machadoc, Nara Lins Meira Quintãoa, José Roberto Santina,∗ a
Postgraduate Program in Pharmaceutical Science, Universidade Do Vale Do Itajaí, Itajaí, Santa Catarina, Brazil Biomedicine Course, Universidade Do Vale Do Itajaí, Itajaí, Santa Catarina, Brazil Postgraduate Program in Biodiversity, Universidade Regional de Blumenau, Blumenau, Santa Catarina, Brazil d Nutrition Department (DNUT), Universidade Federal de Sergipe (UFS), São Cristóvão, Sergipe, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Passiflora edulis Metabolic syndrome Cafeteria diet Diabetes Steatosis
Ethnopharmacological relevance: Passiflora edulis f. flavicarpa O. Deg. is a native Brazilian fruit known as sour or yellow passion fruit. From its peel, mainly in the northeast of Brazil, is produced a flour that is largely used as folk medicine to treat diabetes and other metabolic conditions. Aim of the study: The aim of the study was to show the effects of P. edulis peel flour (PEPF) in metabolic disorders caused by cafeteria diet in mice. Material and methods: The antioxidant activity in vitro of PEPF extract was determined by ferric reducing/antioxidant power, β-carotene/linoleic acid system and nitric oxide scavenging activity assay. C57BL/6 mice divided in 3 groups: Control group, fed on a standard diet (AIN); Cafeteria diet (CAF) group, fed on a cafeteria diet, and PEPF group, fed on a cafeteria diet containing 15% of PEPF, during 16 weeks. The glucose tolerance and insulin sensitivity were evaluated through the glucose tolerance test (GTT) and the insulin tolerance test (ITT). After the intervention period, blood, hepatic, pancreatic and adipose tissues were collected for biochemical and histological analysis. Cholesterol, triglyceride, interleukins and antioxidant enzymes were measured in the liver tissue. Results: PEPF extract presented antioxidant activity in the higher concentrations in the performed assays. The PEPF intake decreased the body weight gain, fat deposition, predominantly in the liver, improved the glucose tolerance and insulin sensitivity in metabolic changes caused by cafeteria diet. Conclusion: Together, the data herein obtained points out that P. edulis peel flour supplementation in metabolic syndrome condition induced by CAF-diet, prevents insulin and glucose resistance, hepatic steatosis and adiposity.
1. Introduction
Generally, several symptoms cluster together, among them are insulin resistance, obesity/central obesity, dyslipidaemia and hypertension (Eslami et al., 2019). This complex pathophysiological condition is usually originated from high calorie intake and low energy expenditure. However, it can be also affected by individual epigenetic characteristics, sedentary lifestyle and also the composition of gut microbiota (Reave, 2012; Saklayen, 2018). Passion fruit (Passiflora edulis f. flavicarpa O. Deg.), plant native from Brazil, is popularly known as sour or yellow passion fruit (Dhawan
Metabolic syndrome comprehends an important condition that combine at least two risk factors occurring together, such as type 2 diabetes mellitus, cardiovascular disease, obesity and hepatic disorders, that leads to approximately 1.6-fold increase in mortality worldwide (Cornier et al., 2008; Schnack and Romani, 2017). In real numbers, more than a billion people suffer from metabolic syndrome (Saklayen, 2018).
∗ Corresponding author. Postgraduate Program in Pharmaceutical Sciences, Universidade do Vale do Itajaí, Rua Uruguai, 458, Bloco F6, ECS, Sala 316, CEP 88302901, Itajaí, SC, Brazil. E-mail address: [email protected] (J.R. Santin).
https://doi.org/10.1016/j.jep.2019.112482 Received 30 July 2019; Received in revised form 11 December 2019; Accepted 12 December 2019 Available online 19 December 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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bath at 50 °C for further reading 2 h later. The results were expressed as calculated percentage of oxidation inhibition (% OI) compared to the absorbance decay of the control (considered as 100% of oxidation). The nitric oxide (NO) scavenging activity was evaluated adding 50 μL of the extract (3, 10, 30, 100, 300 or 1000 μg/mL) or Trolox (100 μg/mL) and 50 μL of SNP (20 mmol/L; in phosphate buffer; pH 7.4) in a 96-well microplate, and then incubated at 37 °C for 1 h. After that, 100 μL of Griess reagent (2% sulphanilamide, and 0.2% napthylethylene diamine dihydrochloridediluted in 5% o-phosphoric acid) were added and the absorbance was obtained at 540 nm. A standard curve for sodium nitrite (NaNO2; 5–100 μmol/L) was plotted to obtain the results expressed as μmol of nitrite/L (Basu and Hazra, 2006).
et al., 2004). Agra et al. (2007) extensively studied the most used medicinal plants in Brazil and showed that the dried and powdered peel of P. edulis is used in the folk medicine for the treatment of diabetes. It is also important to emphasize that the fruit peel contains the amount of phenolics higher than the edible portion or pulp (Tehranifar et al., 2011), such as vitexin, isovitexin, apigenin, isoorientin, cyanidin-3-Oglycoside and quercetin-3-O-glycoside. Additionally, the edulic acid, a carboxylic acid, is also founded in the peel fruit (Yapo and Koffi, 2006; Zeraik et al. 2011, 2012; Cazarin et al., 2014). Probably, this chemical composition is responsible for the promising biological effects observed by the population. Besides the chemical compounds, the peel presents high quantity of dietary fibres, mainly the soluble ones, pectin as example (Goss et al., 2018), a nutrient that presents crucial role in the maintenance of human health, reducing the risk of developing metabolic syndrome and its complication (Bernaud and Rodrigues, 2013; Slavin, 2013; Zhang et al., 2016). Recently, Goss and co-authors (2018) have demonstrated that the passion fruit peel flour added to the rat food significantly reduced the insulin resistance and adipocyte hyperplasia induced by fructose-induced metabolic syndrome. In this study we aimed to evaluate the effects of passion fruit peel flour (PEPF) in metabolic syndrome induced by cafeteria (CAF) diet, which is an appropriate experimental model to study metabolic syndrome, once it is able to induce obesity with insulin resistance, high plasma triglyceride and steatosis (Parafati et al., 2018). Data here-obtained point out that PEPF prevents the glucose intolerance, insulin resistance and mainly the hepatic steatosis induced by CAF diet, providing a beneficial employment of this product.
2.3. Animals and treatment Male 3-week old C57BL/6 mice were obtained from the Central Vivarium of the Universidade do Vale do Itajaí. The animals were kept with free access to water and food, 4 animals per cage (20–23 °C, humidity 60%, 12 h light/dark cycle) for at least a week before the experiment. All procedures were performed according to the Brazilian Society of Science of Laboratory Animals guidelines for the appropriate care and use of experimental animals. All procedures were approved by the local Ethics Committee of UNIVALI (protocol number 016/17). The mouse weight and water and food intake values were obtained every three days. Thirty mice were distributed in 3 different groups (n = 10): (1) control: water and standard diet; (2) Cafeteria (CAF) group: water and cafeteria diet; (3) Cafeteria (CAF) plus PEPF group: water and cafeteria diet plus 15% of PEPF. All treatments were conducted daily for 16 weeks. The standard diet (AIN-93M) adopted in the research followed the recommendations of the American Institute of Nutrition for a maintenance diet of adult mice (Reves et al., 1993). To compose the cafeteria diet, AIN-93 diet was associated to hyper caloric foods rich in carbohydrates and lipids, as milk-chocolate, peanuts and sweet biscuits, in ratios of 3:2:2:1. This diet was previously standardised by Estadella et al. (2004) and analysed by Oliveira et al. (2015). For the treated group (3), 15% of PEPF were added to the CAF diet. The ingredients were homogenised, and the mixture was moistened with distilled water for manual palletisation. The different experimental diets were kept in an air-circulation oven at 50 °C for 48 h to dry and store in sealedplastic containers at 4 °C. The calorie intake was calculated considering 4 calories for protein and carbohydrates, and 9 calories for lipids. The calculation was made based on the nutritional information provided by the manufacturers considering all constituents of each treatment (Control, CAF, CAF + PEPF).
2. Methods and materials 2.1. Sample The PEPF, a product commercially available, was kindly provided by a food industry located in Santa Catarina, Brazil (Vitalin, number lot. 11590, with 8.7 mg of fiber in 15 g of flour). For the in vitro analysis, an extract of the PEPF was obtained using 30 g of PEPF in 10 mL of methanol:water (1:1 v/v), kept stirring for 30 min at 50 °C. Then the product was filtrated using a methanol soaked paper filter, under vacuum. The obtained extract was used to verify the antioxidant activity. The chromatographical profile of PEPF extract was previously published by Goss et al. (2018). 2.2. Antioxidant activity The antioxidant activity of the PEPF extract was evaluated using in vitro methods described below. The reducing potential of PEPF extract was analysed by the ferric reducing/antioxidant power (FRAP) assay previously described by Pulido et al. (2000) with few modifications. Firstly, 9 μL of the extract, at concentrations of 3, 10, 30, 100, 300 or 1000 μg/mL, or Trolox (100 μg/mL), were added to 27 μL of distilled water plus 270 μL of freshly prepared FRAP solution (acetate buffer pH 3.6 (0.3 mmol/L)), 2,4,6-tripyridyl-s-triazine (10 mmol/L) and ferric chloride (FeCl3) in a 96-well microplate. The microplate was then incubated at 37 °C for 30 min and the absorbance was obtained at 595 nm and the results were calculated based on a ferrous sulphate (FeSO4) standard curve. The antioxidant capacity by β-carotene/linoleic acid system was determined using the method described by Miller (1971) with few modifications. Initially, an emulsion was prepared using the proportions as follow: 120 μL of β-carotene/chloroform solution (20 mg/mL), 70 μL of linoleic acid, 22 drops of Tween 40 and 1 mL of chloroform. 250 μL of this emulsion were pipetted in a 96-well microplate plus 35 μL of the extract at concentrations of 3, 10, 30, 100, 300 or 1000 μg/ mL, or the positive control Trolox (100 μg/mL). The first reading was done at 470 nm wave length and the microplate was the kept in water
2.4. Glucose tolerance test (GTT) Fasted mice (6 h between 7:00 and 13:00 h prior to the experiment, as recommended for C57BL/6 mice; Wang and Liao, 2012) were submitted to blood extraction, representing time 0. Then, glucose was orally dosed (2 g/kg) and blood was sampled at different time-points, 30, 60, 90 and 120 min. The area under the glucose decay curve (AUC) was calculated for each mouse and the mean was calculated for each group (Faulhaber-Walter et al., 2011).
2.5. Insulin tolerance test (ITT) The same fasting protocol used for GTT was adopted for this protocol. The first glucose level was measured at time 0. Then, the mice received intraperitoneal insulin injection (2 U/kg) and the glucose level was evaluated at 15, 30, 60, 90 and 120 min after the insulin injection. The area under the glucose decay curve (AUC) was calculated for each mouse and the mean was calculated for each group (Yuan et al., 2013). 2
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Fig. 1. Antioxidant activity of Passiflora edulis peel flour extract by ferric reducing/antioxidant power (FRAP) assay (A), β-carotene/linoleic acid system (B) and nitric oxide (NO) scavenging activity (C). Data are expressed as means ± S.E.M. *p < 0.05, **p < 0.01 and ***p < 0.001 vs reactional system (RS).
2.6. Biochemical parameters
2.8. Determination of the of antioxidant markers in the liver
The biochemical analysis was performed with the blood samples collected at the end of the experiment. After the fasting protocol mentioned above, the animals were anaesthetised (ketamine and xilasine) and their blood samples were taken from the brachial artery. The samples were centrifuged at 4000 g, at 4 °C for 10 min to separate the serum. The total cholesterol, triglyceride, creatinine, glucose, and the activity of aspartate transaminase (AST) and alanine transaminase (ALT) concentrations were measured using corresponding commercial kits (Labtest, Lagoa Santa, MG, Brazil) by spectrophotometry and serum insulin concentration was measured by electrochemiluminescence assay.
Liver samples were homogenised in phosphate buffer (50 mM; pH 7.0) and centrifuged at 10,000×g for obtaining the supernatant. Tissue antioxidant status was evaluated in the supernatant using the ferric reducing/antioxidant power (FRAP) assay. Results were plotted against a standard curve of ferrous sulphate (500–1500 μmol/L) and were expressed as μmol of ferrous sulphate/mg of protein. The hepatic reduced glutathione (GSH) content was measured using the method of Tietze (1969), and the GSH level was calculated by using pure GSH as standard. Catalase (CAT) activity was assessed by the decrease in absorption of H2O2 at 240 nm, due to H2O2 consumption by CAT, as described previously (Beutler, 1975). Specific activity was expressed as units of CAT/mg of protein. The Superoxide dismutase (SOD) activity was assessed verifying the production of superoxide anion by xanthine oxidase in the presence of xanthine. One unit of SOD activity was expressed as the amount of SOD required to inhibit the rate of cytochrome C reduction by 50% at 25 °C (McCord and Fridovich, 1969). Glutathione peroxidase (GPx) activity was expressed as μmol of glutathione oxidized/mg of protein/min by using the extinction coefficient for NADPH (6220 M−1/cm), as previously described by Sies et al. (1979). The tissue protein content was determined using the Bradford method (Bio-Rad® protein assay reagent).
2.7. Extraction of hepatic lipid The liver cholesterol and triglyceride were extracted according Folch's method (1957). At the end of the experiment, fresh livers were extracted and homogenised using chloroform/methanol (2:1, 3.75 mL). Chloroform and distilled water were then added to the homogenate and the solution was vortexed. After centrifugation (1500 g for 10 min), the lower organic phase was transferred to a new glass tube and lyophilised. The lyophilised powder was dissolved in a chloroform:methanol (1:2) mixture and then stored at −20 °C. Cholesterol and triglyceride concentrations were determined using diagnostic commercial kits (Labtest, Lagoa Santa, MG, Brazil) by spectrophotometry. 3
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Fig. 2. Effects of PEPF supplementation on weight gain (A and B), food consumption (C) and energetic intake (D) after 16 experimental weeks. Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). **p < 0.01 vs cafeteria group and #p < 0.05 vs control group.
2.9. Liver IL-6 and TNF levels
3. Results
Liver samples were homogenised in phosphate buffer (50 mM; pH 7.0) and centrifuged at 10,000×g to obtain the supernatant, which was used to analyze the interleukin 6 (IL-6) and tumor necrosis factor (TNF) levels using ELISA kits (R&D systems), in accordance to the manufacture instructions.
3.1. In vitro antioxidant activity of PEPF extract Data presented in Fig. 1A demonstrates that PEPF extract significantly increased the reducing potential, from concentrations of 300–1000 μg/mL, when compared with the system. The Trolox (100 μg/mL) also presented significant effect when compared with the system. Fig. 1B shows the antioxidant activity of PEPF against lipid peroxidation, and the obtained data demonstrates that incubation with PEPF (1000 μg/mL) and trolox significantly inhibited the lipid peroxidation. PEPF extract also presented significant NO scavenging activity from concentrations of 30–1000 μg/mL, when compared to the system. As a control, Trolox (100 μg/mL) reduced the nitrite production (Fig. 1C).
2.10. Histological analysis Small samples of hepatic, pancreatic and adipose tissues, extracted at the end of the experiment were fixed in 10% formalin. Then, followed the dehydration protocol, they were embedded in paraffin, sliced and stained using hematoxylin-eosin (HE). Images of the histological sections were taken using Olympus CBA® optical microscope. For analysis of the pancreas, 15 images were captured for each experimental group using a 10x objective. The image analysis was determined using Image J® software, which quantified the areas of the respective functional units of each tissue. The results were expressed in micrometres/ field. For the adipose tissue, 10 images of each experimental group were captured using 10x objective, and the images were analysed using Adiposoft® Software (NIN, USA) (Galarraga et al., 2012).
3.2. Effect of PEPF in weight gain and food consumption Fig. 2A shows that CAF diet induced significant increase in body weight gain in the 10th week of treatment and achieving the stabilization until the end of the experiment (16 weeks). The PEPF supplementation (15%) increased the body weight gain. However, after the 5th week, the weight gain was lower than the CAF-diet group. In addition, it is important to emphasize that animals from CAF and CAF + PEPF groups presented significant reduction in the food consumption (Fig. 2C), although the energetic intake did not modify (Fig. 2D).
2.11. Statistical analysis Results are expressed as mean ± standard error mean (SEM) of 10 mice per group. Statistical comparisons were performed using one-way analyses of variance (one way-ANOVA) followed by Tukey's test. P values less than 0.05 (P < 0.05) were considered significant. 4
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Fig. 3. Effects of PEPF supplementation in adipose tissue parameters. Abdominal adipose tissue weight (A) and relative weight (B). Epididimal adipose tissue weight (C) and relative weight (D). Adiposity index (E), illustrative images (F) and calculated area of adipocyte cells (G). Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05 and **p < 0.01 vs cafeteria group and #p < 0.05 vs control group.
correlated to glucose or insulin metabolism (Fig. 4), and consequently any alteration was visualised in the pancreas histology (Fig. 4G).
3.3. Adipose tissue parameters Data presented in Fig. 3A–D showed that CAF-fed animals presented absolute and relative increase in the abdominal and epididimal adipose tissue, which promoted significant intensification of the adiposity index (Fig. 3E), when compared to control group. In addition, data from histological analysis of the adipose tissue (Fig. 3F) showed that the adipocyte's area (Fig. 3G) increased in the CAF-fed animals. Animals that were supplemented with PEPF presented profile similar to the control group, even being exposed to CAF diet (Fig. 3A–G).
3.5. Liver parameters The increase in liver lipids from CAF diet can be responsible for inducing non-alcoholic fatty liver disease (NAFLD) and increase in cholesterol and triglyceride in the serum. In fact, the results presented in Fig. 5 showed that CAF diet induced hepatic steatosis, which can be visualised in the histopathological analysis (Fig. 5A), where a lot of areas with lipid deposition can be evidenced. The steatosis was also confirmed by the increase in the hepatic cholesterol and triglyceride (Fig. 5B and C). Additionally, the CAF diet promoted increase in cholesterol and triglycerides in the serum (Table 1). Besides, CAF diet promoted significant elevation in the liver IL-6 levels (Fig. 5E), without interfering with AST and ALT concentration in the serum (Table 1). Nevertheless, animals that received PEPF presented normal levels for all evaluated parameters, demonstrating that PEPF prevented the biochemical effects of CAF diet. In addition, it was accessed the SOD, CAT, GPx, GSH and FRAP in the liver. The obtained data demonstrates that cafeteria diet was not able to significantly modify none of the evaluated parameters (data not show).
3.4. Effect of PEPF on insulin and glucose tolerance and fasting blood glucose, and pancreas parameters The obtained data showed that CAF diet was capable of developing glucose intolerance and insulin resistance, which was marked by increase in the fasting glucose (Fig. 4A) and in the tolerance glucose test (Fig. 4B and C). Additionally, animals that received CAF diet did not respond to exogenous insulin administration, as shown in Fig. 4D and E, characterising insulin resistance. Corroborating this data, the animals from CAF group presented increase in insulin level (Fig. 4F) and Langerhans islets hyperplasia (Fig. 4G), which was confirmed by the measurement of Langerhans islets area (Fig. H). On the other hand, animals that received PEPF did not present any metabolic change 5
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Fig. 4. Effects of PEPF supplementation in glucose metabolism after 16 experimental weeks. Fasting glucose (A), oral glucose tolerance test (B and C), insulin resistence test (D and E), plasma insulin (F), illustrative images (G) and calculated area of Langherhans islets (H). Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05, **p < 0.01 and ***p < 0.001 vs cafeteria group and #p < 0.05, ## < 0.01 vs control group.
4. Discussion
agents or suppressors of singlet oxygen, which impairs the initial steps and propagation of oxidative stress (Cazarin et al., 2014). In the present manuscript, we present more data related to the in vitro antioxidant effect of PEPF extract. The obtained results showed that PEPF extract acts as a NO scavenger, which is important for the reduction of the superoxide anion and peroxynitrite production (Gow et al., 1996; Sorokin, 2016). Additionally, the PEPF extract showed considerable reducing power, as demonstrated by the FRAP method. This data is important because during the oxidative stress Fe3+ can react with O2−, resulting in the formation of Fe2+. This species can take place in Fenton's reaction and generates hydroxyl radicals, highly reactive (Shahidi and Zhong, 2015). Finally, the PEPF extract was able to inhibit the lipid peroxidation of β-carotene. Together, these results confer to the PEPF extract considerable antioxidant activity, an important attribute for substances with the aim of preventing and also treating diseases, such as metabolic syndrome. Clinicians and researchers agree that fat diet consumption over long periods can cause metabolic impairment, such as obesity, glucose and insulin disturbance, hepatic steatosis, and so on (Castell-Auví et al., 2012). Recent literatures have reported that CAF diet promotes enhance of body weight gain in animals and, consequently, obesity (Sampey et al., 2011; Gomez-Smith et al., 2016; Dalby et al., 2017; Soares et al., 2017). Definitely, our data demonstrates that CAF diet promotes weight gain, with decreasing fed intake, but without interference of energetic intake. In contrast, the group that had PEPF incorporated to their diet presented lower percentage of weight gain.
The main factor that is contributing for the global increase of obesity is focused in diet habits, caused by the increased snacking on energy-dense foods consumption (Nasreddine et al., 2018). Our group previously demonstrated the effects of PEFF on low-fructose diet in young rats, mimetizing a diet rich in carbohydrate (Goss et al., 2018). Here, we intended to use a CAF-diet, which simulates a specific human diet habit in rodents, once it is composed by lipids and carbohydrates, and improving the metabolic syndrome knowledge, currently in the spotlight (Janebro et al., 2008). In this context, here we investigated the effect of PEPF consumption in the metabolic changes evokes by CAF diet in mice, once the literature has proposed that natural products are promising tool for dealing with metabolic disorders. (Zeraik et al., 2011, 2012; Cazarin et al., 2014). Different parts of P. edulis are extensively used by the population as sedative, anxiolytic, antispasmodic, and antioxidant. The dried and powdered peel of P. edulis is especially used to empirically treat diabetes (Agra et al., 2007). In fact, the literature attributes the PEPF effects to its dietary fiber content, as well as, to the antioxidant ability of its bioactive constituents (Zeraik et al. 2011, 2012; Cazarin et al., 2014; Janebro et al., 2008). Our group has previously demonstrated the phytochemical profile of PEPF extract, showing the presence of caffeic acid and flavonoids, as well as the antioxidant effect in the DPPH assay (Goss et al., 2018). The antioxidant activity of phenolic acids and flavonoids can be accredited to their action as hydrogen donors, reducing 6
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Fig. 5. Characterization of liver tissue from animals submitted PEPF treatment. Illustrative images of liver (A), levels of cholesterol (B), triglycerides (C), TNF (D) and IL-6 (E) in the liver. Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus Passiflora Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Data are expressed as means ± S.E.M, (n = 10). *p < 0.05 vs cafeteria group and #p < 0.05 vs control group.
the epididymal and abdominal fat deposition, leading to adipocyte hypertrophy. This data corroborates with the literature, which shows that CAF diet causes adipocyte hypertrophy, an important factor associated to dyslipidemia, insulin resistance and inflammation (Sampey et al., 2011).
Moreover, the PEPF treated animals did not present alteration in the adipose tissue deposition, presenting lower adipose index compared to CAF diet group. Importantly, the adipose tissue histological analysis from PEPF-treated animals did not show any change in the adipocytes structure. Differently, animals from CAF group presented increase in
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Table 1 Lipid profile and liver enzymes in the different experimental groups after 16 weeks. Groups Control CAF CAF + PEPF
Cholesterol ##
57.66 ± 7,31 mg/dL 98.33 ± 3.93 mg/dL** 54.66 ± 4.37 mg/dL
Triglycerides
AST
ALT
34.00 ± 3.78 mg/dL 85.66 ± 3.05 mg/dL*** 48.00 ± 4.09 mg/dL
63.28 ± 1.19U/L 72.87 ± 4.06U/L 52.2 ± 0.96U/L
22.4 ± 0.67U/L 21.33 ± 1.05U/L 18.2 ± 0.73U/L
Control group fed on AIN 93M diet; CAF, Cafeteria diet group fed on AIN 93M diet associated with hypercaloric foods; CAF + PEPF, Cafeteria diet plus P. edulis Peel Flour group fed on AIN 93M diet associated with hypercaloric foods and 15% of Passiflora edulis peel flour. Values express the mean ± e.p.m. Statistical analysis was performed using ANOVA followed by the Tukey post-test. ** < 0.005 vs. group CAF + PEPF. *** < 0.001 vs. group CAF + PEPF. ## < 0.005 vs. group CAF.
allied to changes in diet habits to prevent metabolic diseases and improve patients quality of life.
The CAF diet for 16 weeks leads to the development of glucose and insulin resistance, confirmed by the increase in serum glucose and insulin, and also by the increase in the Langerhans islets area in the histopathological analysis. Insulin resistance is a well-described characteristic of obese mice, and is usually associated with decrease in insulin-receptor-AKT (IR–AKT) pathway activity, which is the main recognized signaling pathway in organs, such as skeletal muscle, liver, and adipose tissue (Czech, 2017). According to Vanzela et al. (2010), the insulin-content increase and its releaser from pancreatic islets are linked to Ca2+ cellular influx changes stimulated by CAF-diet. Of note, our data demonstrated that PEPF supplementation improved insulin resistance leading to significant decrease in insulin levels, and increase glucose uptake. Added to the effects in the insulin resistance, it is known that CAF diet lead to hepatic non-alcoholic damage associated to increase in lipogenesis, which cases steatosis (increase of lipid accumulation in the liver; Maeda Júnior et al., 2018). Clearly, our data shows that CAF diet promoted cholesterol and triglycerides accumulation in the liver, which can be responsible for inducing NAFLD. Besides hepatic steatosis, this disease can progress to fibrosis, cirrhosis and at the end, end-stage liver disease (Panera et al., 2018). Other important point is that CAF diet group was characterized by the presence of ballooning hepatocytes and some lobular inflammatory loci. Regarding to the inflammatory mediators, it was observed significant increase in the IL-6 levels in the liver tissue samples from CAF group, the main pro-inflammatory cytokine typically involved in the steatosis (Zeeni et al., 2015; Thiesen et al., 2018). In fact, the IL-6 pathway regulates hepatocyte proliferation and protects against cell death and oxidative stress, thus it suggests that CAF-diet promoted damage in the liver cells (Ozaki, 2019). Elevated levels of circulatory aminotransferases (AST and ALT) are common indicators for hepatocellular damage, and AST, in particular, has been widely used as marker for NAFLD (Hadizadeh et al., 2017). However, our data demonstrate that CAF-diet did not interfered with AST and ALT concentration. In fact, patients with NAFLD may present normal ALT and AST levels, even with advanced fibrosis. Additionally, Gasperin et al. (2018) also demonstrated that cafeteria diet did not modify the activity of AST and ALT in males or females’ mice treated with CAF-diet. It emphasizes the importance of histopathological-analysis association, the gold standard for diagnose (Goesslin et al., 2008; Elizondo-Montemayor et al., 2014). Together, these findings demonstrated the important effect of PEPF supplementation in the prevention of liver inflammation and steatosis development.
Author's contributions J.R. Santin, N.L.M. Quintão and A.M.O. Silva conceived and designed the study; A. De Faveri, M.F. Broering, R. De Faveri, R. Nunes, I.T. Bousfield, M.J. Goss, S.P. Muller, I.D. Machado and R.O. Pereira performed the experiments and analysed the data. J.R. Santin, N.L.M. Quintão and A. De Faveri wrote the paper. All authors read and approved the final manuscript. Acknowledgments This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 429505/2018-3), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, cod. 001) and the Fundação de Amparo à Pesquisa e inovação do Estado de Santa Catarina (FAPESC), A.D.F, M.F.B, and I.B. were Master students, and are recipient of CAPES (Cod. 001) grants during the study. References Agra, M.F., Freitas, P.F., Barbosa-Filho, J.M., 2007. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev. Bras. Farmacogn. 17, 114–140. Basu, S., Hazra, B., 2006. Evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases. Phytother Res. 20, 896–900. https://doi.org/10.1002/ptr.1971. Bernaud, F.S.R., Rodrigues, T.C., 2013. Fibra alimentar: ingestão adequada e efeitos sobre a saúde do metabolismo. Arquivos Brasileiros Endocrinol. Metabol. 57, 397–405. Beutler, E., 1975. Red Cell Metabolism: a Manual of Biochemical Methods. Grune & Stratton, London, pp. 89–90. Castell-Auví, A., Cedó, L., Pallarès, V., Blay, M., Ardévol, A., Pinent, M., 2012. The effects of a cafeteria diet on insulin production and clearance in rats. Br. J. Nutr. 108, 1155–1162. https://doi.org/10.1017/S0007114511006623. Cazarin, C.B.B., Silva, J.K.S., Colomeu, T.C., Zollner, R.L., Maróstica Junior, M.R., 2014. Capacidade antioxidante e composição química da casca de maracujá (Passiflora edulis). Ciência Rural. 44 1699-04. Cornier, M., Dabelea, D., Hernandez, T.L., Lindstrom, R.C., Steig, A.J., Stob, N.R., Van Pelt, R.E., Wang, H., Eckel, R.H., 2008. The metabolic syndrome. Endocr. Rev. 29, 777–822. https://doi.org/10.1210/er.2008-0024. Czech, M.P., 2017. Insulin action and resistance in obesity and type 2 diabetes. Nat. Med. 23, 804–814. https://doi.org/10.1038/nm.4350. Dalby, M.J., Ross, A.W., Walker, A.W., Morgan, P.J., 2017. Dietary uncoupling of gut microbiota and energy harvesting from obesity and glucose tolerance in mice. Cell Rep. 21, 1521–1533. https://doi.org/10.1016/j.celrep.2017.10.056. Dhawan, K., Dhawan, S., Sharma, A., 2004. Passiflora: a review update. J. Ethnopharmacol. 94, 1–23. https://doi.org/10.1016/j.jep.2004.02.023. Elizondo-Montemayor, L., Ugalde-Casas, P.A., Lam-Franco, L., Bustamante-Careaga, H., Serrano-González, M., Gutiérrez, N.G., Martínez, U., 2014. Association of ALT and the metabolic syndrome among Mexican children. Obes. Res. Clin. Pract. 8, 79–87. https://doi.org/10.1016/j.orcp.2012.08.191. Eslami, O., Shidfar, F., Dehnad, A., 2019. Inverse Association of Long-term nut Consumption with weight gain and risk of overweight/obesity: a systematic review. Nutr. Res. 68, 1–8. https://doi.org/10.1016/j.nutres.2019.04.001. Estadella, D., Oyama, L.M., Dâmaso, A.R., Ribeiro, E.B., Nascimento, C.M.O., 2004. Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 20, 218–224. https://doi.org/10.1016/j.nut.2003.10.008. Faulhaber-Walter, R., Jou, W., Mizel, D., Li, L., Zhang, J., Kim, S.M., Huang, Y., Chen, M., Briggs, J.P., Gavrilova, O., Schnermann, J.B., 2011. Impaired glucose tolerance in the absence of adenosine A1 receptor signaling. Diabetes 60, 2578–2587. https://doi. org/10.2337/db11-0058. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and
5. Conclusion Together, the data herein obtained point out that P. edulis peel flour supplementation in metabolic syndrome condition, prevents insulin and glucose resistance, hepatic steatosis and adiposity induced by CAF-diet. Additionally, the PEPF was able to reduce the inflammatory process in the liver. All data obtained corroborating with its ethnopharmacological use. It is important to emphasize that CAF diet fed intake by the animals mimics human unhealthy diet, and the average consumption in this study was consistent with the eating habits in many places worldwide, reinforcing the need of finding new strategies that could be 8
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