Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity

Journal Pre-proofs Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity Alisson S.P. Caldeira, Ulrich C. Mbiakop, Rodrigo M. Pádua, Maryna van de Venter, Motlalepula G. Matsabisa, Priscilla R.V. Campana, Steyner F. Cortes, Fernão C. Braga PII: DOI: Reference:

S0308-8146(20)32596-6 https://doi.org/10.1016/j.foodchem.2020.128734 FOCH 128734

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

5 June 2020 12 November 2020 22 November 2020

Please cite this article as: Caldeira, A.S.P., Mbiakop, U.C., Pádua, R.M., van de Venter, M., Matsabisa, M.G., Campana, P.R.V., Cortes, S.F., Braga, F.C., Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem. 2020.128734

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Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity

Alisson S. P. Caldeira

a,#,

Ulrich C. Mbiakop

b,#,

Rodrigo M. Pádua a, Maryna van de Venter c,

Motlalepula G. Matsabisa d, Priscilla R. V. Campana a, Steyner F. Cortes b, Fernão C. Braga a,* # Contributed

a

equally to this work

Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universidade Federal de Minas

Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b

Department of Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal

de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil c Department

of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box

77000, Port Elizabeth 6031, South Africa d

Department of Pharmacology, School of Clinical Medicine, Faculty of Health Sciences, University

of the Free State, Bloemfontein, 9300, South Africa

* Corresponding author Fernão Castro Braga, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil Tel.: +55 31 3409 6951 E-mail address: [email protected]

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ABSTRACT Pequi fruit peels are an underexploited source of polyphenols. The anti-diabetic potential of an extract and fractions from the peels were evaluated in a panel of assays. The extract and fractions thereof inhibited the release of cytokines involved in insulin resistance – TNF, IL-1β, and CCL2 – by lipopolysaccharide-stimulated THP-1 cells. The ethyl acetate fraction inhibited in vitro α-glucosidase (pIC50 = 4.8 ± 0.1), an enzyme involved in the metabolization of starch and disaccharides to glucose, whereas a fraction enriched in tannins (16C) induced a more potent α-glucosidase inhibition (pIC50 = 5.3 ± 0.1). In the starch tolerance test in mice, fraction 16C reduced blood glucose level (181 ± 10 mg/dL) in comparison to the vehicle-treated group (238 ± 11 mg/dL). UPLC-DAD-ESI-MS/MS analyses disclosed phenolic acids and tannins as constituents, including corilagin and geraniin. These results highlight the potential of pequi fruit peels for developing functional foods to manage type-2 diabetes.

Keywords: Pequi fruit peels; By-product; Tannins; Anti-diabetic potential; in vitro assays; in vivo assay; Functional food.

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1. Introduction Caryocar brasiliense Camb. (Caryocaraceae) is an arborous species popularly known as pequizeiro, found in the Brazilian biome cerrado, a savannah-like vegetation (Amaral, Moriel, Foglio, & Mazzola, 2014). The species is cultivated for the economic exploitation of its fruits named pequi, being Minas Gerais state responsible for 73% of the national production (Turini, 2016). The fruit pulp is edible, and the fruits are also traditionally used as stomachic and to treat influenza, whereas a decoction from C. brasiliense leaves and flowers is used as energetic, tonic, aphrodisiac, and for treating liver diseases (De Oliveira et al., 2018). The pequi peel, composed of exocarp and mesocarp, is usually discharged during fruit processing. Fruit wastes like peels are known sources of bioactive compounds and may represent a low-cost starting material to produce functional foods. Hence, there is a growing interest in evaluating the chemical composition and biological activities of pequi peel, aiming at exploiting this by-product. Within this context, the antioxidant capacities of flours prepared from pequi peels were reported to be higher than those of fruits and fruit by-products reported in the literature (Leão, Botelho, Oliveira, & Franca, 2017). This report also described a preliminary evaluation of the chemical composition of the flours, based on the quantitation of total polyphenols, non-extractable proanthocyanidins, and carotenoid contents. The processing of pequi fruit extracts has also been investigated. The pretreatment of an aqueous pequi fruit extract with bioadsorbents like chitosan and moringa seeds, followed by microfiltration, resulted in extracts with up to 50% retention of total polyphenols (Magalhães, Cardoso, & Reis, 2018). In the same direction, treating the extract with chitosan and submitting it to sequential ultrafiltration process produced a clear permeate and retained 42% of the phenolic compounds from the feed solution (Magalhães, Sá, Cardoso, & Reis, 2019). So far, only a few studies have addressed the biological properties of C. brasiliense fruit peels. Recent publications showed that polyphenolic compounds isolated from hydroethanolic extracts of pequi fruit peels presented antifungal (Breda et al., 2016), antibacterial (Ascari, Takahashi, &

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Boaventura, 2010), and antioxidant activities (Roesler, Catharino, Malta, Eberlin, & Pastore, 2008). A hydroethanolic extract from peels inhibited, in a concentration-dependent manner, the release of tumor necrosis factor (TNF) by lipopolysaccharide (LPS)-stimulated human acute monocytic leukemia (THP-1) cells, thus indicating a potential anti-inflammatory activity (Gusman, Campana, Castro, Castilho, Teixeira, & Braga, 2015). Several edible and medicinal plants with antioxidant and anti-inflammatory activities have been shown to possess potential anti-diabetic activity attested by pre-clinical and clinical assays, especially polyphenols-rich species like Vaccinium corymbosum (Stull, Cash, Johnson, Champagne, & Cefalu, 2010) and Zingiber officinale (Arablou, Aryaeian, Valizadeh, Sharifi, Hosseini, & Djalali, 2014). Diabetes mellitus (DM) is a disease or a chronic metabolic disorder with multiple etiologies, characterized by elevated blood glucose levels, change in the metabolism of lipids, carbohydrates, and proteins (World Health Organization, 1999). DM complications cause damage to eyes (retinopathy), leading to blindness, to kidneys (nephropathy), leading to renal failure, and to nerves (neuropathy), leading to impotence. Type 2 diabetes mellitus or adult-onset diabetes is the most common form of the disease, usually affecting obese adults, resulting from insulin resistance or insufficient production of insulin by the pancreas (American Diabetes Association, 2014). Obesity is one of the main factors that contribute to the development of insulin resistance and increases the risk of type 2 diabetes. Some inflammatory mediators associated with obesity may induce insulin resistance (Gregor & Hotamisligil, 2011). Obesity causes a proinflammatory state in metabolic tissues, as evidenced by increased release of proinflammatory cytokines such as interleukin-1β (IL1β) and TNF, which may directly interfere with insulin signaling in adipocytes, hepatocytes, fibroblasts and myocytes (Ballak, Stienstra, Tack, Dinarello, & Diepen, 2015). Furthermore, macrophages were reported to be able to infiltrate adipocytes to produce IL-1β, TNF, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (CCL2), which are involved in the pathogenesis of

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obesity-induced insulin resistance in obese individuals (Xu, Barnes, Yang, Tan, Yang, & Chou, 2003). Despite a large number of hypoglycemic drugs available, most of them cause undesirable adverse effects, have a high cost, and their use alone cannot effectively control blood glucose unless lifestyle changes are adopted, such as regular physical activity, body weight control, healthy diet and smoking cessation (Hung, Qian, Morris-Natschke, Hsu, & Lee, 2012; World Health Organization, 2018). Keeping in mind the biological activities previously reported for pequi fruit peels and its composition rich in polyphenols, we herewith investigated the potential anti-diabetic activity of an extract and fractions prepared from C. brasiliense fruit peels using in vitro and in vivo assays. Additionally, we carried out the identification of the potential bioactive constituents in the peels, aiming to the future development of functional food from this by-product.

2. Materials and methods 2.1. Plant material and preparation of the ethanol extract (CBFEE) Mature fruits of C. brasiliense were purchased at a market (Mercado Central Christo Raeff) in the municipality of Montes Claros, Minas Gerais State, Brazil, in May 2019. The fruits were thoroughly washed in water, and the peels (containing the exocarp and mesocarp) were manually removed. After drying the peels at 40 °C for 72 h in a ventilated oven, the material was ground in a knife mill. A portion of the powdered material (150 g) was packed into a percolator and left to macerate with 500 mL of ethanol 96 °GL at room temperature (24 ± 2 °C) for 24 h. Afterward the percolate was collected, and the same volume of solvent was replaced in the percolator. The procedure was repeated every 24 h, for 10 days. The solvent was removed under reduced pressure in a rotatory evaporator at 50 °C to afford the C. brasiliense fruit peel ethanol extract (CBFEE, 75.4 g).

2.1.1. Fractionation of CBFEE by partition between immiscible solvents

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A portion of CBFEE (2 g) was dissolved in 100 mL water/methanol (7:3) and sequentially partitioned with n-hexane (HEX), dichloromethane (DCM), and ethyl acetate (EtOAc) (3 × 50 mL each). The solvents were removed under reduced pressure in a rotatory evaporator at a maximal temperature of 50 °C to give the HEX (2.8 mg), DCM (14.5 mg), and EtOAc (278.3 mg) fractions, along with the remaining hydromethanolic (HM) fraction (1524.2 mg).

2.2. Preparation of the aqueous acetone extract (CBFAE) The aqueous acetone extract of C. brasiliense fruit peels was prepared by exhaustive percolation of 500 g of the dried fruit peels with acetone/water (8:2) solution. The solvent was removed under reduced pressure in a rotatory evaporator at 50 °C to afford the aqueous acetone extract (CBFAE, 196.31 g).

2.2.1. Fractionation of CBFAE by column chromatography over Sephadex LH-20 A portion of CBFAE (2 g) was suspended in 200 mL ethanol and then added to the head of a glass column (73 × 3.5 cm i.d.) filled with Sephadex® LH-20 (250 g, wet weight) in ethanol. After bedding down the gel material, fractionation was conducted by successive elution with mixtures of acetone and ethanol (EtOH) in different proportions (0%, 10%, 30%, 50%, and 80% acetone). A total of 474 fractions of 8 mL were collected and analyzed by thin-layer chromatography (TLC). They were pooled according to the similarity of their TLC profiles in 20 fraction groups, termed fractions 1C20C. Fraction 15C (45.4 mg), comprising fractions eluted with 10 to 50% acetone in EtOH, and fraction16C (108.7 mg), composed of fractions eluted with 80% acetone in EtOH, were selected for biological assays due to the presence of tannins, as evidenced by UPLC-PDA-ESI-MS/MS analyses.

2.3. In vitro assays 2.3.1 Cell culture

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THP-1 cell line was purchased from American Type Culture Collection (ATCC-TIB-202, Manassas, USA). THP-1 cells were cultivated in RPMI 1640 medium containing 10% heatinactivated fetal bovine serum (FBS) at 37 ºC in a humidified 5% CO2 atmosphere. Chang liver cells (ATCC CCL-13) and immortalized rat skeletal (L6) myoblasts (ATCC CRL-1458) were obtained from Highveld Biological, South Africa.

2.3.2. Evaluation of sample cytotoxicity The cytotoxicity of CBFEE, HEX, DCM, EtOAc, and HM fractions, as well as dexamethasone, employed as a positive control, were evaluated by the thiazolyl blue tetrazolium bromide (MTT) assay to determine their maximum nontoxic concentrations in THP-1 cells (Stockert, Horobin, Colombo, & Blázquez-Castro, 2018). THP-1 cells in 2% FBS-RPMI 1640 medium were seeded in 96-well plates at a density of 1 × 106 cells/well. Prior to the assays, the THP-1 cells were differentiated into macrophages using 0.005% phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Saint Louis, USA) for 72 h. The samples (10, 30, and 90 μg/mL in 2% FBS-RPMI 1640 medium with 0.2% dimethyl sulfoxide (DMSO) in the highest concentration) or dexamethasone (0.1 μM) were added to the wells and incubated for 3 h, and then stimulated with 10 μL of LPS (1 ng/μL) for 24 h. Subsequently, 28 μL of MTT (2 mg/mL) in phosphate-buffered saline (PBS) solution was added to each well followed by incubation at 37 °C for 1.5 h, and the purple formazan formed by reduction of MTT reagent was dissolved with 130 μL of DMSO. The amount of MTT formazan product was determined by measuring the absorbance at 510 nm using a microplate reader (Tecan, Männedorf, Switzerland). The percentage of cell viability was determined by comparing the absorbance readings of the control wells with those recorded for dexamethasone or extract/fractions.

2.3.3 Glucose utilization in Chang liver cells

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The assay was performed as described by Van de Venter et al. (2008). Briefly, Chang liver cells were seeded into 96-well culture plates and left to adhere and grow in a humidified incubator with 5% CO2 at 37 °C for 3 days. Without changing the medium, a 10 µL aliquot of the plant extract was added to each well on the third day to a final extract concentration of 25 µg/mL. The cells were incubated in the presence of the extract for a further 48 h. The medium was removed and replaced with an incubation buffer containing 8 mM glucose and 50 µg/mL of the same extract. Then, the cells were incubated for 3 h before aliquots were taken for the determination of glucose concentration. The amount of glucose left in the medium was determined using a glucose oxidase assay. The difference between the glucose concentration before and after the 3 h incubation was considered as the amount of glucose captured by the cells. The positive control used in this assay was 100 µM metformin. In order to compensate for possible differences in cell number due to the chronic exposure to extracts, the MTT assay was performed on representative wells, and glucose utilization was corrected for differences in cell numbers. All results are, therefore, comparable with respect to cell numbers.

2.3.4. Glucose utilization in L6 myoblasts Glucose utilization in differentiated L6 myocytes was measured as previously described (Van de Venter et al., 2008). L6 cells were seeded at a density of 3000 cells/well into 96-well plates and allowed to reach 90% confluence. Cells were then cultured for an additional five days in Dulbecco's modified Eagle's medium (DMEM) containing 2% FBS. Forty-eight hours prior to glucose utilization assay, plant extracts were added at 25 µg/mL. Spent culture medium was removed and replaced with fresh RPMI 1640 medium, diluted to 8 mM glucose with PBS, containing 0.1% bovine serum albumin (BSA) and the extract at 50 µg/mL for 90 min. Positive control wells received 1 µM insulin. Ten μL medium was then removed from each well and used to assay the remaining glucose using a glucose oxidase based assay kit. Toxicity assays were conducted simultaneously by adding MTT (0.5 mg/mL in RPMI 1640) to the wells and incubating for 1 h. The absorbance was measured at 560 nm after

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solubilizing the formazan crystals with DMSO. MTT results were used to normalize the glucose utilization results, and differences observed are, therefore, not due to differences in cell numbers induced by chronic exposure to extract.

2.3.5. Cytokine analysis For cytokine analysis, THP-1 cells in 2% FBS-RPMI 1640 medium were seeded in 96-well plates at a density of 1 × 106 cells/well. THP-1 cells were first pretreated with CBFEE extract and fractions (10, 30, and 90 μg/mL in 2% FBS-RPMI 1640 medium with 0.2% DMSO in the highest concentration) for 3 h followed by the addition of 10 μL of LPS solution (1 ng/μL) for 24 h. After LPS stimulation, the 96-well plates were centrifuged at 399 × g for 5 min, the cell culture supernatants were collected and stored at -80 ºC until further analysis. The concentration of TNF, IL-1β, and CCL2 in the supernatant was measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions. The optical density was measured at 490 nm on a microplate reader (Tecan).

2.3.6. In vitro α-glucosidase inhibition assay The inhibitory activity of α-glucosidase was carried out spectrophotometrically in 96-wells microplates as previously described (Pereira, Arruda, Silva, Silva, Lemos, & Cortes, 2012), with slight modifications. A reaction mixture containing 20 μL of enzyme dissolved in buffer solution (0.16 U/mL), 20 μL of test samples (10-1000 μg/mL), and 20 μL of reduced glutathione (1 mM) was preincubated at 37 °C for 15 min. The reaction was started by the addition of 20 μL of p-nitrophenylα-D-glucopyranoside (pNPG, 0.85 mM) as a substrate. After 25 min incubation at 37 °C, 20 μL of sodium carbonate solution (0.2 M) was added to stop the reaction. Absorbance readings were recorded at 405 nm on a microplate reader (Tecan). Samples were dissolved in PBS containing 1% DMSO and other reagents in 0.1 mM phosphate buffer pH 6.8. Acarbose (0.01-100 μg/mL) was used

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as a reference drug. The inhibitory effect was expressed as a percentage of inhibition compared to the absorbance recorded for the control.

2.3.7. In vitro α-amylase inhibition assay The inhibitory activity on α-amylase was assayed following a previously reported method (Muhammad et al., 2017), with modifications. Reaction mixtures containing 20 μL of phosphate buffer pH 6.9 (20 mM), 40 μL of the enzyme (0.5 mg/mL) and 40 μL of test samples (60-6000 μg/mL) were preincubated in polypropylene tubes, at 37 ºC for 10 min. The reaction started after adding 40 μL of starch solution (1%) and incubation at 37 °C for 20 min. The reaction was stopped after the addition of 100 μL of color reagent 3,5-dinitrosalicylic acid (DNSA, 96 mM). The tubes were then incubated in a boiling water bath for 10 min and were cooled at room temperature. The reaction mixture was diluted with 900 μL distilled water and homogenized. Subsequently, 200 μL of the mixture was transferred from the tubes to each well of a 96-well microplate. Absorbance readings were recorded at 540 nm on a microplate reader (Tecan). Samples were dissolved in PBS containing 1% DMSO and other reagents in 20 mM phosphate buffer pH 6.9. Acarbose (0.01-100 μg/mL) was used as a reference drug. The inhibitory effect was expressed as a percentage of inhibition compared to the absorbance recorded for the control.

2.4. In vivo carbohydrate tolerance test 2.4.1. Experimental animals All experimental protocols were performed in accordance with guidelines for the humane use of laboratory animals and were previously approved by the ethics committee (CETEA) of the Universidade Federal de Minas Gerais (UFMG) (protocol 77/2019). Male Swiss mice (8-12-weekold) were used. All animals were obtained from CEBIO (Centro de Bioterismo, Institute of Biological Sciences, UFMG). Mice were maintained at five per cage at constant temperature (23 °C), with 12 h

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dark/light cycle. The animals had free access to standard chow and filtered water ad libitum. All experiments were carried out using at least five animals per group.

2.4.2. Oral starch tolerance test Swiss mice were deprived of food for 6 h before the test but had free access to filtered water throughout the experiment. Blood samples were collected from the tail tip before sample administration (time 0). CBFEE, fractions, and starch were administered by oral gavage. For administration, CBFEE and fractions (100 mg/kg) were dissolved in filtered water at a concentration of 1% (w/v) at a maximal volume of 360 µL. After 20 min, starch (2 g/kg) was administrated, and blood samples were collected at 15, 30, 60, and 120 min after starch overload. Acarbose (10 mg/kg) dissolved in filtered water was used as a reference drug.

2.4.3. Drugs Unless otherwise mentioned, all reagents were purchased from Sigma-Aldrich. Blood glucose concentration was determined with an Accu-Chek® Active glucose meter (Roche Diagnostics, Jaguaré, Brazil).

2.5. Statistical analysis GraphPad Prism Software Version 7.0 (GraphPad Software, San Diego, CA) was used for analysis, and a p-value < 0.05 was considered statistically significant. For in vitro cell assays, means were compared by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test. For the enzymatic inhibition assays, means were compared by two-way ANOVA followed by Dunnett's multiple comparisons test. The results are expressed as a percentage of inhibition ± standard error of the mean (% ± SEM) for n = 12 replicates of 2 separate sets of experiments in the cytokine assays, and n = 12 replicates of 4 separate sets of experiments in the enzymatic assays. pIC50 (log of

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the sample concentration that inhibits the activity of the enzyme by 50%) values were calculated using non-linear regression curves, and the results are expressed as mean ± SEM. Significant differences between pIC50 mean values were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. In the glucose uptake assay, results are expressed as % ± SEM for n = 6 replicates of 2 separate sets of experiments. For in vivo experiments, data from each group were expressed as mean ± SEM from at least 5 animals. Data were compared by two-way ANOVA, followed by Dunnett's multiple comparisons test.

2.6. Chemical characterization by UPLC-PDA-ESI-MS/MS The chemical composition of CBFEE, EtOAc fraction, and 16C fraction was investigated by ultraperformance liquid chromatography (UPLC) coupled with a photodiode array detector (PDA) and a mass spectrometer detector (MS). Chromatographic separation was carried out using a Waters Acquity UPLC system (Waters, Milford, USA) that consisted of a binary pump, in-line degasser, autosampler, and PDA detector (200-500 nm; Waters). The system was interfaced with a TQ-XS triple quadrupole mass spectrometer (Waters Micromass, Manchester, UK) with an electrospray ionization (ESI) source. The analyses were performed on an Acquity UPLC BEH C18 column (2.1 × 50 mm i.d., 1.7 μm; Waters) using a gradient elution of water (A) and acetonitrile (B), both containing 0.1% v/v formic acid (5-95% B in 10 min; 95-5% B in 1 min, followed by 2 min of isocratic elution to re-equilibrate the column), at a flow rate of 0.3 mL/min, and temperature of 40 °C. The injected sample volume was 5 μL for all samples prepared as methanolic solutions at a concentration of 5 mg/mL. The ESI source was operated in the negative ionization mode, and the following inlet conditions were applied: capillary voltage 3.54 kV, cone gas voltage 33 V; source temperature 120 °C; cone gas flow 90 L/h; desolvation gas flow 500 L/h at 350 °C. The mass range was set at m/z 100-1500. Data acquisition was achieved with the MassLynx 4.1 software (Waters).

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Ellagic acid (≥ 95% purity) and gallic acid (≥ 98.5% purity) used as reference standard for chemical analysis were purchased from Sigma-Aldrich® (Saint Louis, USA).

3. Results and discussion 3.1 Biological assays CBFEE, tested at 25 μg/mL, increased glucose uptake in Chang liver cells (282.2 ± 3.4%) significantly when compared to the untreated control (100.0 ± 5.4%). The increase was higher than that induced by metformin (158.9 ± 4.0%), employed as a positive control (S1 Fig. 1A). When tested at the same concentration in L6 myoblast cells, CBFEE promoted a significant increase in glucose uptake (201.2 ± 7.9%) in comparison to the untreated control (100.0 ± 10.0%), whereas the positive control insulin (1 μM) produced a response of 167.6 ± 14.0% (S1 Fig. 1B). CBFEE showed no toxicity in both cell lines at the tested concentration (S1 Fig. 1CD). These preliminary results highlight the potential anti-diabetic effect of the pequi fruit peel extract since it stimulates hepatic glucose consumption, observed in Chang liver cells, and exerts insulin-mimetic activity in the L6 muscle cell line. Therefore, we further tested CBFEE in a platform of assays to explore its potential anti-diabetic effect. We initially evaluated the effect of CBFEE and fractions thereof on the release of proinflammatory cytokines related to diabetes. All samples, except the n-hexane and dichloromethane fractions, were shown to be nontoxic to THP-1 cells at all tested concentrations (10, 30, and 90 μg/mL), with cell viability > 90% (S1 Fig. 2). Hence, their effect on the release of TNF, IL-1β, and CCL2 by LPSstimulated THP-1 cells was assayed, excepting the above-mentioned two fractions, and the results are depicted in Figure 1. The extract, its ethyl acetate, and hydromethanolic fractions reduced TNF release in a concentration-dependent manner. The EtOAc fraction inhibited TNF release by 60 ± 2% at 90 μg/mL, whereas dexamethasone (0.1 μM), employed as a positive control, promoted 75 ± 1% inhibition of

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cytokine release. TNF is a proinflammatory cytokine that is secreted by visceral adipose tissue, a common characteristic of metabolic syndrome (Musialik, 2012). Elevated TNF levels are associated with insulin resistance and type 2 diabetes (Srikanthan, Feyh, Visweshwar, Shapiro, & Sodhi, 2016). The inhibitory effect of TNF release by LPS-stimulated THP-1 cells elicited by extracts from leaves and barks of C. brasiliense has been previously described by us (Gusman, Campana, Castro, Castilho, Teixeira, & Braga, 2015). The results here reported for the peel extract and fractions thereof indicate the potential anti-inflammatory activity of this pequi by-product, which may be beneficial in the management of type 2 diabetes. IL-1β release by LPS-stimulated THP-1 cells was also inhibited in a concentration-dependent manner by the samples, reaching 75 ± 4% and 72 ± 5% inhibition, respectively, for CBFEE and EtOAc fraction at the highest tested concentration (Fig. 1B). IL-1β is a major regulator of innate immune responses, playing a key role in various inflammatory processes. Experimental evidence reveals that IL-1β cytokine is important in the pathology of type 2 diabetes, mediating inflammation induced by obesity and directly aggravating insulin resistance (Ballak, Stienstra, Tack, Dinarello, & Diepen, 2015). In a prospective study of 27,500 individuals, it was found that elevated plasma levels of IL-1β and IL-6 triplicated the risk of developing type 2 diabetes (Spranger, et al., 2003). The inhibitory effect of the test samples on the release of CCL2 is depicted in Fig. 1C. CBFEE reduced CCL2 release by THP-1 cells moderately, even at the highest assayed concentration (40 ± 8% inhibition). On the other hand, the ethyl acetate and hydromethanolic fractions significantly reduced CCL2 release in a concentration-dependent manner, the inhibition reaching 85 ± 3% and 86 ± 2%, respectively, for EtOAc and HM fractions. CCL2 is a cytokine responsible for the recruitment of monocytes to inflammatory sites that plays a critical role in the development of atherosclerosis and its complications. Circulating CCL2 has been found to be significantly increased in patients with insulin resistance and type 2 diabetes (Panne, 2012).

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As far as we currently know, there is no previous report on the inhibitory effect of an extract and fractions from pequi fruit peels on the release of TNF, IL-1β, and CCL2. Different pharmacological approaches are employed to manage type 2 diabetes, including the inhibition of enzymes in the gastrointestinal tract involved in the metabolization of starch and glucose. The α-glucosidase enzyme plays a key role in the final step of carbohydrate digestion, and its inhibition represents a valid therapeutic approach currently used to manage type 2 diabetes (Alongi & Anese, 2018). The effect of CBFEE and EtOAc fraction on α-glucosidase was evaluated in vitro (Fig. 2A and B). Both extract and fraction elicited a concentration-dependent enzymatic inhibition (Fig. 2A), with similar potencies (Fig. 2B). As discussed in the next section 3.2, the ethanol extract of pequi fruit peels is composed of phenolic acids and tannins. Keeping in mind that the potential anti-diabetic activity of the peels could be related to those constituents, we prepared an aqueous acetone extract from the peels, aiming to favor their extraction. Furthermore, we fractionated this extract by column chromatography over Sephadex LH20 to obtain fractions enriched in tannins, namely 15C and 16C, which also had their inhibitory effect on α-glucosidase tested in vitro (Fig. 2A and B). Both fractions inhibited α-glucosidase activity in a concentration-dependent manner (Fig. 2A). Interestingly, the 16C fraction elicited a more potent inhibitory effect than the extract or 15C fraction, with the highest pIC50 value (5.3 ± 0.1), as illustrated in Fig. 2B. These results demonstrate the relevance of the fraction 16C as a source of α-glucosidase inhibitors. The control drug acarbose presented a pIC50 value (7.5 ± 0.1) higher than the one of 16C fraction (Fig. 2B), in a range of potency compatible with a previous report (Zhang et al., 2020). Inhibition of α-amylase reduces the digestion of amylose and amylopectin (long-chain carbohydrates of starch), reducing glucose absorption and, consequently, lowering blood glucose levels (Nasab, Homaei, & Karami, 2020). Figure 2C shows the percentage of α-amylase inhibition assayed in vitro as a function of the sample concentrations (0.003-1 mg/mL). CBFEE, EtOAc, and 16C fraction induced approximately 30% inhibition of α-amylase at 1 mg/mL, whereas 15C fraction

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promoted a lower inhibition (<10%). In its turn, the reference drug acarbose had an inhibitory effect (Figure 2C) compatible with a previous report in the literature (Zhang et al., 2020). In the sequence, we evaluated the effect of the extract and the tannin-rich fractions on the in vivo starch tolerance test (STT). The obtained results demonstrated that neither CBFEE nor EtOAc were able to decrease the plasma glucose concentration in mice after starch administration, in comparison to the control group (Fig. 3A). However, the treatment of mice with 16C fraction or acarbose reduced serum glucose level significantly at 30 and 60 min after starch ingestion, as compared to the control group (Fig. 3B). One hour after starch administration, the serum glucose peaked in all groups, after which it began to fall. Two hours after starch administration, the serum glucose concentration returned to the initial levels. The measurement of glycemic levels 60 min after starch administration indicated that the groups pretreated with 16C fraction and acarbose exhibited blood glucose levels of 181 ± 10 mg/dL and 156 ± 11 mg/dL, respectively, compared to the mice in the control group (238 ± 11 mg/dL). On the other hand, fraction 15C induced no significant reduction in the plasmatic glucose levels of STT mice (Fig. 3B). To the best of our knowledge, this is the first report on the potential anti-diabetic effect of pequi fruit peels, here demonstrated by in vitro and in vivo results. Interestingly, the in vitro inhibitory effect of 16C fraction on the digestive enzyme α-glucosidase showed a good correlation with the in vivo results from the starch tolerance test. Furthermore, our in vivo and in vitro data clearly indicate an increase in the biological potency of fraction 16C compared to CBFEE, pointing out that the compounds characterized in this fraction (see section 3.2) may contribute to the biological effect. Therefore, the in vitro and in vivo data here reported for an extract and fractions from pequi fruit peels are strong evidence to support the development of functional foods based on this by-product. The relevance of our findings can be highlighted by comparison with data reported in the literature for extracts prepared with other fruit by-products. Hence, an extract of jackfruit peel, a by-product from Artocapus heterophyllus, was found to elicit more potent in vitro inhibition of α-glucosidase

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(pIC50 of 7.30) than extracts from the pulp, flake, and seeds (Zhang et al., 2017). The peel extract presented a total phenolic content higher than extracts from other jackfruit parts. Likewise, the ethyl acetate and methanolic extracts of Punica granatum fruit peels inhibited α-glucosidase strongly (IC50 of 9.19 ± 0.49 and 10.85 ± 0.75 μg/mL, respectively). The enzyme inhibition was credited to synergistic effects between ellagitannins (punicalagin and punicalin) and ellagic acid (Papoutsis, Zhang, Bowyer, Brunton, Gibney, & Lyng, 2021). These literature data corroborate the results here described for pequi fruit peel extract and its ethyl acetate fraction, since both contain phenolic compounds (see section 3.2) and inhibit α-glucosidase significantly. Results from in vivo assays also demonstrate the hypoglycemic potential of tannins found in fruit by-products. The administration of a methanolic extract of Syzygium cumini (jambolam) seeds to alloxan-induced diabetic rats significantly lowered blood glucose levels. The anti-diabetic effect was ascribed to the high concentration of phenolic compounds like phenolic acids, flavonols, flavanones, gallotannins, and ellagitannins (Nahid, Mazumder, Rahman, Islam, Rashid, & Kerr, 2017; De Paulo Farias, Neri-Numa, Araújo, & Pastore, 2020). Another evidence on the hypoglycemic effect of tannins found in fruit by-products comes from a pilot clinical study carried out with Myrciaria jaboticaba peel powder, consumed by the subjects in a test meal. The blood glucose showed a slight decrease, and insulin levels were significantly reduced 240 min after the test meal consumption, as compared to the intake of placebo. The effects were related to the presence of the ellagitannins casuarinin, casuarictin, pedunculagin, tellimagrandin I, and tellimagrandin II (Plaza et al., 2016). These data are also aligned with our results since ellagitannins were identified in the 16C fraction from pequi fruit peels (see section 3.2), a fraction that elicited a potent hypoglycemic activity in the starch tolerance test. All above-cited extracts prepared from fruit by-products and several others like the extracts from Chaenomeles sp. and Phoenix dactylifera (date palm) fruit peels (Miao, Li, Zhao, Gao, Wang, & Gao, 2018; Maqsood, Adiamo, Ahmad, & Mudgil, 2019), Cynara scolymus (artichoke) floral stems (Mejri

18

et al., 2020), and Cichorium intybus seeds (Perovic et al., 2021) contain bioactive phenolic compounds. All these extracts can be potentially employed to formulate new functional foods useful to manage type 2 diabetes, and the pequi fruit peels extract included. Recently, a flour prepared from pequi fruit peels was shown to contain high concentrations of phenolic compounds and present strong antioxidant capacity (Leão, Franca, Oliveira, Bastos, & Coimbra, 2016). Keeping in mind that CBFEE is easily obtained at high yields by extraction with ethanol and that it possesses promising hypoglycemic activity, its incorporation into the pequi fruit peels flour could be the starting point to develop an anti-diabetic functional food. Besides, since CBFEE is a low-cost product, it can be widely used as an ingredient to formulate different new anti-diabetic functional foods.

3.2 Chemical characterization We investigated the chemical composition of the pequi peel extract (CBFEE), along with its active fractions EtOAc and 16C. To accomplish that, an UPLC-PDA-ESI-MS/MS method was developed, with MS detection performed in the negative ionization mode. The developed method provided an adequate separation of the constituents and led to the identification of seven compounds in CBFEE within 13 min (Fig. 4A-B). Gallic acid (2; Rt = 1.07 min) and ellagic acid (7; Rt = 2.96 min) were unequivocally identified by comparison with the retention time, ultraviolet (UV) spectral data, and MS fragmentation pattern of authentic standards. The putative identification of 6-O-galloyl-glucose (1; Rt = 0.71 min), corilagin (3; Rt = 1.99 min), and geraniin (4; Rt = 2.35 min) was accomplished based on the obtained UV and MS/MS spectral data, and comparison with literature records for these compounds (Yang, Kortesniemi, Liu, Karonen, & Salminem, 2012; Breda et al., 2016). Additionally, two non-identified ellagitannins (5; Rt = 2.55 min and 6; Rt = 2.71 min) were detected in CBFEE (Table 1). Proposals of fragmentation for the identified compounds are available as supplementary material (S2 Fig. 1A-5A and S2 Fig. 1B-5B).

19

Analysis of the EtOAc fraction by UPLC-PDA-ESI-MS/MS indicated an enrichment of compounds 1-7 in the fraction (Fig. 4C-D), whose identification was carried out similarly as described for the extract. In its turn, the chromatograms obtained for the 16C fraction disclosed a less complex profile and the MS/MS analyses led to the identification of corilagin (3; Rt = 1.99 min) and geraniin (4; Rt = 2.35 min) as the major peaks (Fig. 4E-F). The biological activities reported in this work can be possibly ascribed to the above-mentioned compounds, which belong to two classes of secondary metabolites: phenolic acids and their esters (gallic acid, ellagic acid, and 6-O-galloyl-glucose) and ellagitannins (corilagin and geraniin). Our supposition is based on previous literature data, discussed in the sequence. Hence, the treatment of LPS-stimulated RAW 264.7 macrophages with gallic acid reduced the levels of TNF, IL-1β, and IL-6, via expression of the toll-like receptor 4 (TLR4) and activation of factor nuclear kappa B (NF-κB), demonstrating its potential anti-inflammatory effect (Huang, Hou, Xue, & Wang, 2016). The potential anti-inflammatory effects of corilagin and ellagic acid were demonstrated in the same model by reducing the release of TNF, IL-1β, and IL-6 by LPS-stimulated RAW 264.7 macrophages (Zhao et al., 2008; Guan, Zheng, Yu, Li, Han, & Lu, 2017). Geraniin inhibited in vitro the release of TNF by THP-1 derived macrophages, previously stimulated by LPS, with IC50 of 48.2 μM (Liu et al., 2016). These data strongly support the participation of the above-mentioned phenolic derivatives and ellagitannins in the inhibitory effect of IL-1β release elicited by CBFEE and EtOAc fraction, as well as the marked reduction in TNF release induced by the fraction. Therefore, such compounds might contribute to the potential anti-inflammatory activity of the extract and its derived fraction, an effect that benefits subjects with insulin resistance and type 2 diabetes. Literature data also support the participation of the identified compounds on the inhibition of gastrointestinal enzymes involved in the metabolization of starch and glucose. Geraniin, isolated from Nephelium lappaceum bark extract, inhibited in vitro α-glucosidase (pIC50 = 6.0) and α-amylase (pIC50 = 6.0) enzymes, suggesting hypoglycemic activity (Palanisamy, Ling, Manaharan, &

20

Appleton, 2011). Thus, geraniin has the potential to block carbohydrate digestion and glucose absorption, which subsequently suppresses postprandial hyperglycemia (Cheng, Ton, & Kadir, 2016). In another study, corilagin isolated from an aqueous extract of Phyllanthus amarus showed significant inhibitory activity on α-glucosidase (pIC50 = 5.8) (Trinh, Staerk, & Jäger, 2016). Therefore, it is feasible to suppose that geraniin and corilagin contribute to the inhibitory effects on those enzymes induced by CBFEE and its EtOAc fraction, in addition to the 16C fraction. A previous in vivo study also demonstrated the potential anti-diabetic effect of corilagin. Administration of this ellagitannin (10 and 20 mg/kg day) significantly reduced the elevated fasting blood glucose levels in streptozotocin-induced diabetic rats (Nandini & Naik, 2019). In addition, an ethanolic extract of rambutan (Nephelium lappaceum) fruit peels, containing geraniin, quercetin, and epigallocatechin-3-gallate, administered orally at 125, 250, and 500 mg/kg for 11 days, significantly decreased the blood glucose levels in alloxan-induced diabetic rats (Muhtadi, Primarianti, & Sujono, 2015). These studies are aligned with the results here obtained in the starch tolerance test, where there was a significant reduction in blood glucose of mice treated with 16C fraction, pointing out the possible contribution of corilagin and geraniin to the hypoglycemic effect in vivo.

4. Conclusion Analyses of the chemical composition of an extract and fractions from pequi fruit peels led to the identification of phenolic compounds and hydrolyzable tannins. Such compounds might account for the potential anti-diabetic activity of the extract and some fractions, here demonstrated in vitro by inhibition of α-glucosidase and α-amylase enzymes, and in vivo by reduction of the plasmatic glucose in the starch tolerance test. Furthermore, the extract and fractions reduced the release of the proinflammatory cytokines IL-1β, TNF, and CCL2, directly related to the maintenance of insulin resistance. These results demonstrate that pequi fruit peels are a source of important health-promoting

21

ingredients, and this by-product is useful to develop new functional foods potentially valuable for management of type 2 diabetes.

Conflicts of interest The authors declare no conflicts of interest.

Acknowledgments The authors thank CNPq/Brazil and TWAS for financial support, along with a Ph.D. fellowship (UCM) and research fellowships (FCB and SFC).

22

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Figure captions

Figure 1. Inhibitory effect of the ethanolic extract (CBFEE), ethyl acetate (EtOAc) and hydromethanolic (HM) fractions from C. brasiliense fruit peels on the release of TNF (A), IL-1β (B) and CCL2 (C) by LPS-stimulated THP-1 cells, assayed at different concentrations (10, 30, and 90 μg/mL). Data represent mean ± SEM (n = 12 of 2 separate sets of experiments in six replicates each time). Significant differences among LPS-stimulated cells (C) and all treatment groups using one-way ANOVA followed by Tukey´s multiple comparisons test; *p < 0.05, **p < 0.01, and ***p < 0.001. D = dexamethasone (0.1 μM; positive control) and NC = non-stimulated cells.

Figure 2. Effect of the ethanolic extract and fractions of C. brasiliense fruit peels on α-glucosidase and α-amylase activities. Inhibitory effect of the ethanolic extract (CBFEE), ethyl acetate fraction (EtOAc), 15C and 16C fractions, and the positive control acarbose on α-glucosidase activity (A). pIC50 values for CBFEE, EtOAc, 15C, 16C, and acarbose for α-glucosidase (B). Evaluation of CBFEE, EtOAc, 15C, 16C, and acarbose on α-amylase activity (C). Data represent mean ± SEM (n = 12 of 4 separate sets of experiments in triplicate each time). Significant differences among CBFEE and other treatment groups were analyzed by two-way ANOVA followed by Dunnett´s multiple comparisons test (A and C); * p < 0.05, ** p < 0.01 and *** p < 0.001. Significant differences between pIC50 mean values were analyzed by one-way ANOVA followed by Tukey´s multiple comparisons test (B); * p < 0.05 versus CBFEE, ††† p < 0.001 versus 15C, and 0.001 versus all other pIC50 values.

‡‡‡

p<

30

Figure 3. Effect of the ethanolic extract and fractions from C. brasiliense fruit peels on oral starch tolerance test (STT). Evaluation of the ethanolic extract (CBFEE) and ethyl acetate fraction (EtOAc) on STT (A). Evaluation of 15C and 16C fractions from CBFEE on STT (B). Data represent mean ± SEM (n = 5 animals in each group). Significant differences among control and all treatment groups using two-way ANOVA followed by Dunnett's multiple comparisons test; ** p < 0.01 and *** p < 0.001 versus control. Acarbose was employed as a positive control.

Figure 4. UPLC-PDA-ESI-MS/MS profiles of the ethanolic extract (CBFEE), ethyl acetate (EtOAc), and 16C fraction from C. brasiliense fruit peels. Chromatograms registered by PDA and ESI- for CBFEE (respectively A and B), EtOAc (respectively C and D), and 16C (respectively E and F). Identified compounds: see Table 1 for identification and S2 Fig. 1A-5A (supplementary material) for chemical structures and fragmentation proposals. Chromatographic and spectrometric conditions: see the experimental section. Rodrigo M. Pádua, Priscilla R. V. Campana, Steyner F. Cortes, and Fernão C. Braga: Conceptualization. Alisson S. P. Caldeira, Ulrich C. Mbiakop, Maryna van de Venter: Investigation. Rodrigo M. Pádua, Priscilla R. V. Campana, Motlalepula G. Matsabisa, Steyner F. Cortes, and Fernão C. Braga: Validation. Alisson S. P. Caldeira and Ulrich C. Mbiakop: Data Curation. Alisson S. P. Caldeira and Ulrich C. Mbiakop: Writing- Original Draft. Rodrigo M. Pádua, Priscilla R. V. Campana, Steyner F. Cortes, and Fernão C. Braga: Writing- Reviewing and Editing. Steyner F. Cortes and Fernão C. Braga: Funding Acquisition.

31

(A) 1500

TNF (pg/mL)

1250

**

***

1000

***

***

*** ***

***

750 ***

500 ***

10 30 90

NC C D

10 30 90

0

10 30 90

250

CBFEE

EtOAc

HM

(B) 600

IL-1 (pg/mL)

500 400

*

*

**

300 200

***

***

10 30 90

NC C D

10 30 90

0

10 30 90

100

*** ***

***

CBFEE

EtOAc

HM

(C) 600

*** ***

300 200

*** *** ***

***

***

NC C D

***

10 30 90

0

***

10 30 90

100

Figure 1.

***

400

10 30 90

CCL2 (pg/mL)

500

CBFEE

EtOAc

HM

32

(A)

(B) ***

Inhibition (%)

***

100

** ***

50

25

CBFEE EtOAC

*** *

15C 16C Acarbose

0 -8

-7

-6

75

***

-5

***

50

*** ***

25

*

*** *

***

0 -4

Log[Drugs](g/mL)

Figure 2.

CBFEE EtOAc 16C 15C Acarbose

*

***

75

** **

Inhibition (%)

100

(C)

-3

-8

-7

-6

-5

-4

Log[Drugs](g/mL)

-3

33

(A)

Figure 3.

(B)

34

(A)

(B) 4 1

6

3

2

5 7

(C)

(D) 4

1 2

6 5

7

3

(E)

(F) 4 3

Figure 4.

35

able Table 1. Compounds identified by UPLC-PDA-ESI-MS/MS in the ethanolic extract (CBFEE), ethyl acetate EtOAc) fraction, and 16C fraction of Caryocar brasiliense fruit peels ESI- (Relative abundance - %) N°

Compound (molecular weight)

Retention time (min)

λmax (nm) MS2 Fragments

[M-H]- Scan

[M-C6H10O5-H]- = 168.9 (100) [M-C4H8O4-H]- = 211.0 (50) [M-C3H6O3-H]- = 241.0 (20)

1

6-O-Galloyl-glucose a (332.26)

0.66

271.5

[M-H]-

2

Gallic acid b (170.12)

1.07

271.5

[M-H]- = 169.0 (100) [2M-H]- = 339.1 (50)

[M-CO2-H]- = 125.1 (100)

3

Corilagin a (634.08)

1.99

270.0

[M-H]-

[M-C7H6O5-C6H10O5-H]- = 301.1 (100) [M-C7H6O5-C7H8O6-H]- = 275.0 (30) [M-C7H6O5-H]- = 463.2 (25)

4

Geraniin (hemiacetal forms) (952.08)

5

= 331.1 (100)

= 633.1 (100)

2.35

275.5

[M-H]- = 951.2 (100)

[M-C27H20O18-H]- = 301.2 (100) [M-H2O-H]- = 933.0 (55) [M-C19H6O17-H]- = 445.1 (15) [M-C28H18O19-H]- = 275.2 (10)

Unknown ellagitannin A (954)

2.55

265.5

[M-H]- = 952.9 (100)

301.0 (100), 275.2 (60), 393.4 (45), 313.2 (40), 318.8 (15)

6

Unknown ellagitannin B (966)

2.71

274.5

[M-H]- = 965.3 (100)

300.8 (100), 423.0 (40), 463.0 (30), 445.0 (30), 289.9 (20)

7

Ellagic acid b (302.19)

2.96

253.5 368.5

[M-H]- = 300.9 (100) [2M-H]- = 602.9 (40)

[M-H2O]- = 284.0 (100) [M-CO2-CO-H]- = 229.2 (15) [M-CO2-H2-H]- = 255.1 (5)

a Putatively b Identified

a

identified based on fragmentation pattern and comparison with literature data. based on reference compound analyzed in the same conditions.

Highlights  The antidiabetic potential of Caryocar brasiliense fruit peel extracts was assessed  Bioactive fractions were identified by cytokines and digestive enzymes inhibition  UPLC-PDA-ESI-MS/MS analyses disclosed hydrolyzable tannins in the active fractions  A corilagin and geraniin rich fraction reduced glycaemia in starch tolerance test  Fruit peels have potential for developing functional foods to manage type-2 diabetes

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