Chemical constituents of Cochlospermum regium (Schrank) Pilg. root and its antioxidant, antidiabetic, antiglycation, and anticholinesterase effects in Wistar rats

Biomedicine & Pharmacotherapy 111 (2019) 1383–1392

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Chemical constituents of Cochlospermum regium (Schrank) Pilg. root and its antioxidant, antidiabetic, antiglycation, and anticholinesterase effects in Wistar rats

T

Thiago Félix de Miranda Pedrosoa,1, Thaise Regine Bonamigoa, Jenifer da Silvaa, Paulo Vasconcelosa, Jessica Maria Félixa, Claudia Andrea Lima Cardosob, Roosevelt Isaias Carvalho Souzaa, Ariany Carvalho dos Santosa, Carla Roberta Ferreira Volobuffa, ⁎ Anelise Samara Nazari Formagioa, Virginia Demarchi Kappel Tricheza,1, a b

Faculty of Health Sciences, Federal University of Grande Dourados, Dourados, MS, Brazil Course of Chemistry, State University of Mato Grosso do Sul, Dourados, MS, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Cochlospermum regium Diabetes Phenolic compounds AGEs Alloxan Acetylcholinesterase

The main physiological consequence of diabetes mellitus is chronic hyperglycemia. This condition is related to the formation of free radicals including advanced glycation end products (AGES) and to an increase in inflammatory processes. Cochlospermum regium (Schrank) Pilg., part of the Bixaceae family, is a cerrado plant known for its anti-inflammatory effects. The objectives of this study were to analyze the constituent compounds of C. regium roots and to evaluate the antioxidant, antiglycation, antidiabetic, and anticholinergic effects of its hydromethanolic extract through in vitro and in vivo experimental models. The presence of phenols, flavonoids, condensed tannins, and flavonols was analyzed by liquid chromatography - photodiode array (LC/PDA) analysis. Whereas antioxidant activity was investigated via DPPH, ABTS, β-carotene/linoleic acid, and malondialdehyde colorimetric assays. Inhibition of AGEs was examined via a bovine serum albumin system whose glycosylating agent was glucose. Antidiabetic potential was examined in normoglycemic Wistar rats that received glucose overload, in alloxan-induced diabetic rats, and in rats that received a hyperglycemic diet. Disaccharidase inhibition was assessed using in vitro and in vivo methods, as was acetylcholinesterase (AChE) inhibition in brain structures. The hydromethanolic extract (CRHE) possessed a high concentration of phenolic compounds and showed antioxidant effects. The LC-DAD results revealed that CRHE contained a high concentration of phenolic acids and the majority was gallic acid. Treatment with CRHE caused significant inhibition of AGE formation. The oral glucose tolerance test (OGTT) in normoglycemic animals showed a reduction in blood glucose levels after treatment with 100 mg/kg CHRE, accompanied by an increase in hepatic glycogen content. There was also a significant reduction in the fasting glucose levels of alloxan-induced diabetic animals after 7 days of treatment with daily doses of 100 mg/kg. After 14 weeks of hyperglycemic diet, the last four or which were combined with 100 mg/kg CRHE treatment, there was a decrease in blood triglyceride levels. There was also a statistically significant decrease in the enzymatic activity of maltase, lactase and sucrase. The results demonstrate that oral administration of 30 and 100 mg/kg CRHE inhibited AChE activity in different brain structures. Thus, the extract of C. regium showed promising antioxidant, antiglycation, and antidiabetic effects that may be related to its high phenolic content.

1. Introduction Diabetes mellitus (DM) is a multifactorial disease, caused by a combination of genetic, behavioral, social, and environmental factors.

Normal glucose homeostasis is achieved by the precise regulation of insulin produced and secreted by the islets of Langerhans in the pancreas combined with an appropriate response from body tissues and organs including the liver, muscles, and adipose tissue. A absolute

⁎

Corresponding author at: Federal University of Grande Dourados, Dourados Highway - Itahum, Km 12 - University City, Zip Code 364 - CEP 79804-970, Dourados, Mato Grosso do Sul, Brazil. E-mail address: [email protected] (V.D.K. Trichez). 1 These authors equally contributed to this study. https://doi.org/10.1016/j.biopha.2019.01.005 Received 27 July 2018; Received in revised form 2 January 2019; Accepted 3 January 2019 0753-3322/ © 2019 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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roots were washed to remove dirt, cut into pieces, and placed in a kiln with circulating air current set at 40 °C for 6 days. The dried pieces were then ground using a knife mill, macerated in methanol (Dinâmica Química Contemporânea, SP, Brazil):water (7:3), filtered. and rotated at 60 °C until complete solvent evaporation, after which the material was lyophilized to produce a yield of 21.9%, of the hydromethanolic extract from C. regium (CRHE).

insulin deficiency (type 1 DM -T1DM) and an inappropriate response to blood insulin levels (type 2 DM – T2DM) can cause an imbalance in glucose homeostasis, leading to chronic hyperglycemia [1]. Diabetes is considered to be one of the most significant public health challenges today, notwithstanding the fact that it was only recognized as an epidemic by international organizations in the late twentieth century. Originally, diabetes was found mainly in rich countries, but the advance of globalization has spread diabetes into all continents, increasing the population average blood sugar levels in countries such as China, India, Brazil, Mexico, Pakistan, and Egypt. Several studies confirm that the diabetes epidemic can only be tackled with an intervention based on primary prevention [2]. Today, DM and its complications are one of the leading causes of death in most countries [3]. T2DM is the most common type of diabetes, and its prevalence has increased in tandem with social and cultural changes [1,3,4]. Recent researches in understanding the pathogenesis of diabetic complications point the involvement of oxidative stress and inflammation. Intracellular hyperglycemia promotes production of mitochondrial reactive oxygen species (ROS), increased formation of intracellular advanced glycation end-products (AGEs) [5], activation of protein kinase C, and increased polyol pathway flux. ROS directly increase the expression of inflammatory and adhesion factors, formation of oxidized-low density lipoprotein, and insulin resistance [6]. AGEs cause damage to vascular tissue, DNA, brain mitochondria, and increase both the inflammatory response and the concentration of free radicals. Moreover, AGEs influence the formation of β-amyloid plaques and neurofibrils that are related to the pathophysiology of dementia [7]. Other reports show that cognitive impairment itself is related to chronic diabetes, and that there is a link between DM and cardiovascular risk factors associated with cognitive disorders. In addition to vascular complications, some studies suggest a connection between hyperglycemia and oxidative stress with an accumulation of intracerebral β-amyloid substance [7]. Despite all the scientific developments that have occurred in the last few decades, there is still no cure for diabetes. In this way, management of diabetes remains a major challenge in the medical field, mostly due to the inefficacy at the progression of diabetic complications and undesired side effects of current treatments [8]. Many vegetable species are used worldwide as antidiabetic remedies, this medicinal plants are attractive since not only can they be used as complementary and alternative remedies to prevent metabolic diseases, but they also serve as sources for the development of novel safe and effective medicines for DM [9,10]. Cochlospermum regium (Schrank) Pilg., part of the Bixaceae family, popularly known as “algodãozinho” or “algodãozinho-do-campo”, is a cerrado plant known for its anti-inflammatory effects [10–12]. Different forms of extraction of the roots are widely used to treat infections e inflammations process [11]. Chemical studies showed that flavonoids and phenolic compounds are major constituents [12]. A specie from the same genus of the above-mentioned plant, such as C. planchonii, have already been evaluated for their antidiabetic potential. However, as far as we are aware, there are no reports in the literature concerning the antidiabetic properties of C. regium. Therefore, the objectives of this study were to analyze the constituent compounds of C. regium roots and to evaluate the antioxidant, antiglycation, antidiabetic, and anticholinergic effects of its hydromethanolic extract through in vitro and in vivo experimental models.

2.2. Constituent composition 2.2.1. Total phenolic concentration The total phenol concentration of the CRHE was determined using Folin-Ciocalteau reagent, as has been described previously [13].The absorbance was measured at 765 nm. Total phenolic concentration was expressed as the equivalent of gallic acid in mg per gram of extract (mg AGE/g extract). Methanol was used as a white solution, and all assays were performed in triplicate. For details see supplementary file. 2.2.2. Total flavonoid concentration The flavonoid content in the extract was measured spectrophotometrically, as previously reported [14]. The result was expressed as quercetin equivalents in mg per gram of extract (mg QE/g extract). All assays were performed in triplicate. For details see supplementary file. 2.2.3. Flavonol concentration The flavonol content in the extract was measured spectrophotometrically, as previously reported [14]. Total flavonols were expressed in mg quercetin equivalents per gram of dry weight (mg QE/g extract) using a quercetin calibration curve. For details see supplementary file. 2.2.4. Condensed tannin (TC) concentration The TC concentrations were determined using a modified version of a previously reported method [15]. The concentration was expressed as catechin equivalents in mg per gram of extract (mg CE/g extract). For details see supplementary file. 2.2.5. Liquid chromatography – diode array detection LC - (DAD) The analyses were performed as previously reported method with adaptations [16]. See supplementary file. 2.3. Antioxidant activity 2.3.1. Radical scavengers 2.3.1.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH). The DPPH were determined using a modified version of a previously reported method [15]. For details see supplementary file. 2.3.1.2. 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS). Total antioxidant activity was measured using an improved method of ABTS (Sigma-Aldrich, St. Louis, MO, USA), removal [17]. For details see supplementary file. 2.3.2. Peroxidation 2.3.2.1. β-Carotene/linoleic acid. Peroxidation was measured in vitro using β-Carotene/Linoleic Acid method [18]. See supplementary file.

2. Materials and methods 2.3.2.2. Malondialdehyde (MDA). Antioxidant activity was measured in vitro using MDA [18]. See supplementary file.

2.1. Preparation of plant material

2.4. AGE formation in a bovine serum albumin (BSA) system with glucose

The C. regium root was collected in the Lagoa Grande settlement, Itahum district, Dourados, Mato Grosso do Sul, Brazil, 21°59′52.75″ and 55°20′21.85″, in October 2015. The specimen was registered in the herbarium of the Federal University of Grande Dourados (5406). The

AGEs were formed in an in vitro system as described by Kappel et al. 2012, with some modifications [19]. See supplementary file. 1384

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was removed and added to 1.3 mL color reagent which was analyzed after 10 min using a spectrophotometer set to 460 nm [19].

2.5. Ex vivo tests 2.5.1. Animals Male Wistar rats aged between 50 and 55 days were obtained from the central laboratory of the Federal University of Grande Dourados. The animals were maintained with free access to feed and water and with a light and dark cycle of 12 h. All animals were carefully monitored and maintained in accordance with the recommendations of the Brazilian Council of Veterinary Medicine and the Brazilian College of Animal Experimentation (Protocol No. 30/2015 CEUA/UFGD).

2.7. Hyperglycemic diet A highly palatable diet based on the study by Naderali et al.(2001) was used, consisting of 33% PRESENCE® ground meal, 33% ITALAC® branded condensed milk, 27% water, and 7% sucrose (2.78 calories/g). The diet was kept under refrigeration until the use. The normal control group received commercial grade brand PRESENCE® [24]. The experiment was performed for a total of 14 weeks and included 20 animals. At the beginning of the diet, all animals the average age of the animals was 50 days. The animals were divided into four groups of five rats each. The first two groups received the hyperglycemic diet, one of which received vehicle (hyperglycemic control group, HC) and the other of which received the CRHE (hyperglycemia treatment group, HT). The other two groups received commercial feed without any change, one of which received water by gavage (normal control group, NC), and the other of which received CRHE by gavage (normal treatment group, NT). For the first 10 weeks, the animals received only the specified feed for each group and for the last 4 weeks the animals were fed via gavage, always at the same hour. Animals from the treatment groups received CRHE 100 mg/kg and animals from the control groups received water. Oral glucose tolerance tests were performed at the beginning and at the end of treatment. At the end of the trial, a blood sample was collected and centrifuged at 4000 rpm (4 °C) for 15 min to obtain serum which was used to determine serum glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triacylglyceride (TAG), and alanine aminotransferase (ALT) levels using commercially available diagnostic kits. The rats were then killed via decapitation. The liver and pancreas were removed and macroscopically examined. Organ samples were fixed in 10% formalin, dehydrated with alcohol and xylene, embedded in paraffin wax, sectioned, mounted on glass slides, and stained with hematoxylin/eosin. Two board-certified veterinary pathologists then analyzed all samples.

2.5.2. Disaccharidase inhibition This methodology was based on an earlier model performed by Kappel et al. 2012 [19]. See supplementary files. 2.5.3. Inhibition of acetylcholinesterase (AChE) in brain structures Three groups of six male Wistar rats each were treated separately with CRHE (30 or 100 mg/kg) or 0.9% saline (control). After 60 min, the animals were anesthetized and killed via decapitation. The cortex, hypothalamus, hippocampus, and striatum were collected and homogenized separately in 10 mmol Tris−HCl buffer (pH 7.2) with 160 mmol sucrose (1:10, w/v). Test medium containing 1.04 mmol 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB) and 24 mmol potassium phosphate buffer (pH 7.2) was incubated for 2 min at 30 °C with 25 mL of the homogenate sample. The reaction was initialized with the addition of acetylthiocholine (ATC) 0.83 mmol/L. The reaction product was determined via absorbance spectrometry at 412 nm for 2 min. Activity was expressed as mmol of hydrolyzed ATC/hr*mg protein. The protein concentration of the homogenate samples was determined via the Coomassie blue method, using BSA as a standard. 2.6. In vivo Tests 2.6.1. Oral glucose tolerance test (OGTT) We divided 25 rats divided into five equal groups 5. All animals received an oral glucose from Sigma-Aldrich (St. Louis, MO, USA) overload of 4 g/kg body weight. Thirty minutes prior to glucose administration, each group received CRHE treatment. The groups were divided as follows: the negative control group received water, three group received CRHE (30 mg/kg, 100 mg/kg, or 300 mg/kg), and the positive control group received glibenclamide 100 mg/kg. Glycemic levels were checked at 15, 30, 60, and 180 min after glucose administration with an Accu-check Performa® glucometer using blood from the tail vein. After the experiment, the rats were killed, and the livers were collected to determine total glycogen content [19].

2.8. Statistical analysis The results are expressed as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using either one-way or two-way analysis of variance (ANOVA) followed by the Bonferroni post-test using INSTAT version 2.02 or Graph Pad Prism version 5.01, or the Student-Newman-Keuls test. The differences found were considered statistically significant for "p" equal to or less than 0.05. 3. Results

2.6.2. Alloxan-induced diabetes Diabetes was induced with two intraperitoneal doses of alloxan (Sigma-Aldrich, St. Louis, MO, USA) (120 mg/kg), with the first and second doses three days apart. Four days after the second dose, animals that had fasting glucose levels above 200 mg/dL were considered to be diabetic. These animals were divided into a control group and a CRHE treatment group that received a single daily dose of 100 mg/kg for seven consecutive days. Blood sugar levels were tested 1, 2, and 3 h after starting treatment, and fasting glucose levels were evaluated at the end of the treatment (7 days) [20–23].

3.1. Constituent composition The CRHE contained a high concentration of phenolic compounds (1443.04 ± 8.34 mg of AGE/g extract) and flavonoids (708.75 ± 12.54 mg QE/g extract), followed by a lower flavonol content (306.97 ± 10.29 mg QE/g extract) and TCs (218 ± 4.73 mg of CE/g extract). 3.2. LC-DAD

2.6.3. Evaluation of hepatic glycogen After the OGTT, parts of the rat livers were collected for hepatic glycogen analysis. These samples were placed in large tubes (˜8 mL) along with 2 mL of 30% KOH. They were then boiled (100 °C) until homogenized, cooled to room temperature, supplemented with 5 mL of 95% ethanol, vortexed, and re-heated to 70 °C for 10 min. The samples were then immediately placed on ice for 15 min and centrifuged for 10 min at 3000 rpm. The supernatant was discarded, and 1 mL of distilled water was added to the samples. A 30 μL aliquot of the sample

The LC-DAD data of the CRHE showed three phenolic acids: gallic acid (Rt = 7.33 min; 114.26 mg/g), caffeic acid (Rt = 18.17 min; 40.1 mg/g) and ellagic acid (Rt = 21.87 min; 27.9 mg/g) (Fig. 1). 3.3. Antioxidants The antioxidant effect of CRHE was determined using the β-carotene/linoleic acid bleaching test (Table 1) and the results showed potential oxidation inhibition (IC50 = 85.50 μg/mL) comparable to the 1385

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Fig. 1. Liquid chromatography - photodiode array (LC/PDA) analysis of the CRHE. Table 1 Antioxidant effects of the hydromethanolic extract of Cochlospermum regium (Schrank) Pilg. root (CRHE) as determined via DPPH, ABTS, β-carotene/linoleic acid and MDA assays. Scavenging Radicals

Peroxidation

Samples

DPPH

ABTS

β-Carotene/ linoleic acid

MDA

IC50(μg/mL) CRHE Quercetin Ascorbic acid

14.68 ± 1.05 – 11.50 ± 1.18

138.71 ± 6.65 56.76 ± 3.76 –

85.5 ± 2.23 70.65 ± 1.65 –

68.88 ± 9.44 54.32 ± 6,89 –

Values are expressed as mean ± SEM; IC50 = concentration resulting in 50% inhibition, derived from the I% plot (percent inhibition) versus concentration in μg/mL. C. regium hydromethanolic extracts (CRHE); 2,2-diphenyl-1-picrylhydrazyl (DPPH); (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS); Malondialdehyde (MDA).

natural quercetin standard (70.65 μg/mL). In the spontaneous model of lipoperoxidation using rat brain homogenate incubated under controlled conditions of temperature and oxygenation, the extract exhibited antioxidant activity with an IC50 of 68.88 μg/mL, which was comparable to the standard (54.32 μg/mL). This weaker antioxidant activity contributed to the smaller generation of MDA, which is a toxic product resulting from degradation of the cell membrane by lipid peroxidation, causing cell disruptions and mutations. Further, the data showed that CRHE exhibited greater free radical sequestration (IC50 = 14.68 μg/mL) in the DPPH assay than in the assay using ABTS reagent (Table 1).

Fig. 2. Inhibitory effect of the C. regium hydromethanolic extracts (CRHE) on the formation of fluorescent advanced glycation end products (AGES) in a bovine serum albumin (BSA)/glucose system. A: 14 days of BSA / glucose; B: 28 days of BSA / glucose. Samples from each group were tested in triplicate. # P < 0.01 relative to the BSA and BSA + Glucose groups, ** P < 0.01 relative to the BSA + Glucose group, *** P < 0.001 relative to the BSA + Glucose group. ** P < 0.01 relative to the BSA and BSA + Glucose + CRHE(100 μg/ mL) groups, *** P < 0.001 relative to the BSA and BSA + Glucose + CRHE (1000 μg/mL) groups.

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Fig. 3. In vitro effect of the C. regium hydromethanolic extracts (CRHE) on the activity of disaccharidases. A: maltase, B: lactase, and C: saccharase. The control group without treatment represents 100% specific activity of the respective disaccharidase. Values are expressed as mean ± SEM, N = 3 for each group, * P < 0.05, ** P < 0.01, *** P < 0.001.

3.4. Inhibition of AGE formation in a BSA system with glucose

3.7. Area under the curve (AUC)

The Fig. 2 shows the differences in fluorescence intensity of the BSA, BSA + glucose, and BSA + glucose + extract groups at different concentrations. There was a statistical difference between the BSA and BSA + glucose groups, both in the 14 and 28 day trials, which indicates that AGES formation was significantly increased in the presence of glucose (Fig. 2A, B). Fig. 2A demonstrates the ability of CRHE to inhibit the production of fluorescent AGEs most clearly at a concentration of 1000 μg/mL. Moreover, Fig. 2B shows that CRHE inhibited the production of AGEs at a concentration of 100 and 1000 μg/mL when allowed to incubate for 28 days.

The Fig. 4 shows the AUC calculated from the graph of the means of each glucose tolerance test versus time, whose results are described in Section 3.6. This figure demonstrates that the AUC from the 100 mg/kg dose graph was closest to that of the glibenclamide-dose graph. The 100 mg/kg dose also produced a significantly different AUC than the 30 mg/kg dose or the control. The group treated with the reference drug glibenclamide produced a significantly different AUC than that treated with control or 30, 100, and 300 mg/kg CRHE (Figs. 5 and 6).

3.5. Effect of CRHE on disaccharidase activity

There was a significant increase in hepatic glycogen storage in the groups treated with 100 mg/kg CRHE or glibenclamide relative to the control. Subjects treated with 300 mg/kg CRHE showed significantly lesser hepatic glycogen levels than those treated with 30 mg/kg CRHE. Treatment with glibenclamide caused significantly greater hepatic glycogen storage than either the control or 300 mg/kg CRHE.

3.8. Hepatic glycogen results

The Fig. 3A shows that there was a statistically significant decrease in the enzymatic activity of maltase after treatment with 1000 μg/mL CRHE compared to the control and to treatment with 100 μg/mL CRHE. Fig. 3B shows a decrease in lactase activity after treatment with either 100 μg/mL or 1000 μg/mL CRHE. Fig. 3C shows a decrease in the enzymatic activity of saccharase after treatment with 1000 μg/mL CRHE.

3.9. Diabetic animals

3.6. Oral glucose tolerance test (OGTT)

Rats with alloxan-induced diabetes had fasting serum levels of glucose above 200 mg/dL. After treatment for 7 days with CRHE, rats treated with CRHE demonstrated a reduction in serum fasting glucose levels (Table 3).

Serum glucose levels, which were evaluated before and after the administration of oral glucose (4 g/kg), increased significantly. The reference oral hypoglycemic agent was glibenclamide (100 mg/kg), a second-generation sulfonylurea, which produced a typical decrease in serum glucose at 15, 30, and 60 min post-administration relative to the negative control (Table 2). CRHE (100 mg/kg) showed a better antihyperglycemic profile at than glibenclamide at 30 and 60 min postglucose administration. The results also show that 30 and 300 mg/kg CRHE were not effective at decreasing serum glucose levels during the test. However, rats treated with 100 mg/kg CRHE had a statistically significant decrease in serum glucose levels relative to those treated with 30 or 300 mg/kg 60 min post-administration.

3.10. Hyperglycemic diet 3.10.1. OGTT in rats given a hyperglycemic diet Table 4 showed that there was an increase with statistical significance in the group of the control hyperglycemia diet, in fasting glycemia of the animals, and also in the times of 30 and 60 min. There was a decrease in glycemia in the HT diet group when compared to the HC group. 1387

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Table 2 Acute effect of C. regium hydromethanolic extracts (CRHE) on serum glucose levels (mg/dL) in the oral glucose tolerance test. Groups treated with C. regium Time (min)

Control

30 mg/kg

100 mg/kg

300 mg/kg

Glibenclamide

0 15 30 60 180

69.4 ± 0.4 169.8 ± 11.8 187.0 ± 12.1 187.4 ± 10.8 117.0 ± 8.3

80.4 ± 3.0 167.6 ± 3.9 184.6 ± 4.7 188.0 ± 3.5 131.4 ± 3.5

77.2 ± 3.1 152.4 ± 16.5 155.8 ± 12.7a**. b* 140.8 ± 7.0a. b*** 94.8 ± 2.5b**

77.8 ± 3.7 184.2 ± 15.3c* 176.4 ± 2.1 187.4 ± 13.1c*** 100.4 ± 4.8b*

86.4 ± 3.9 127.0 ± 4.9c* 139.2 ± 4.3 141.2 ± 7.3c*** 88.4 ± 8.6b*

Values are expressed as mean ± SEM; N = 5. Statistically significant differences: a, relative to control; b, relative to 30 mg/kg and 100 mg/kg; c, relative to 100 mg/ kg * P < 0.05, ** P < 0.01, *** P < 0.001.

observed. The hepatic markers AST and ALT did not show significant changes. No significant changes were observed by necropsy or histopathological analysis in all pancreas and liver samples analyzed (data not shown). 3.11. Inhibition of AChE The results demonstrate that oral administration of 30 and 100 mg/ kg CRHE inhibited acetylcholine esterase (AChE) activity in different brain structures (Fig. 8). Fig. 8A shows that the CRHE-treated group (100 mg/kg) inhibited AChE significantly in the cerebral cortex (69%), compared to the control group (p < 0.001). In addition, significant inhibition was also observed in the hippocampus of rats treated with 30 mg/kg (11.79%; p < 0.01) and 100 mg/kg (53.76%; p < 0.001) of the CRHE (Fig. 8B), and in the hypothalamus of rats treated with 30 and 100 mg/kg CRHE, inhibiting 27.78% and 42.20% of AChE activity, respectively (p < 0.001) (Fig. 7C). In the striatum, treatment with CRHE did not affect AchE inhibition (Fig. 7D).

Fig. 4. Area under the curve values were calculated using GraphPad Prism and were based on serum glucose levels after an oral glucose tolerance test in Wistar rats tests (raw data is presented in Table 2). Statistically significant differences: a: relative to the control, b: relative to 30 mg/kg, c: relative to 100 mg/kg, d: relative to 300 mg/kg, Glibencl, glibenclamide, * P < 0.05, ** P < 0.01, *** P < 0.001.

4. Discussion DM is a chronic condition that occurs due to dysfunction in the production and/or use of insulin. A lack of insulin or cellular response to insulin reduces glucose uptake into cells, which leads to excess glucose in the blood [3,5]. Over time, high glucose levels along with excess production of free radicals such as AGEs cause tissue damage [3,5]. In contrast, phenolic compounds are natural antioxidants that may protect against damage and chronic diseases linked to the excessive production of free radicals [25]. In the present study, we investigated the ability of CRHE to improve biochemical parameters, inhibit disaccharidase and AChE activity, and inhibit AGE formation, using in vitro and in vivo models. Our data demonstrated that glucose overload in the TOTG, alloxan induction, and the hyperglycemic diet models increased blood glucose levels and that treatment with CRHE ameliorated the increase. Data from the literature suggest that other members of the Cochlospermum genus such as C. tinctorium and C. vitifolium are also rich in phenolic compounds, and may reduce glycemia, act as vasorelaxants, hepatoprotectors and may ameliorate metabolic syndrome [26,27]. Other studies have shown a reduction in blood glucose, triglyceride and cholesterol levels in alloxan-induced diabetic rats treated with an aqueous extract of C. planchonii roots [28]. Phytochemical analysis describe that the root of C. regium contains tannins and other phenolic compounds, along with mucilages, saponins, steroids, triterpenes, and flavonoids. Among the constituents identified on C. regium are specific phenolic and flavonoid compounds, include kaempferol, naringenin, aromadendrine, and dihydrokaempferol-3-Oβ-glucopyranoside, gallic acid and ellagic acid all of which have been shown to possess antioxidant activity [12,29]. Our results demonstrated that CRHE has a high content of total phenolic compounds and of flavonoids, which could justify its antioxidant activity. Moreover, the

Fig. 5. Result of the quantification of hepatic glycogen after TOTG with or without pre-treatment with glibenclamide (positive control) or the CRHE. Statistically significant differences, a: relative to the control, b: relative to pretreatment with 30 mg/kg CRHE, d: relative to pre-treatment with 300 mg/kg CRHE, * P < 0.05, ** P < 0.01, *** P < 0.001.

3.10.2. Biochemical parameters Table 5 shows the results of the biochemical tests performed at the end of the hyperglycemic diet. Animals that received only the hyperglycemia diet (HC) showed a significant increase in triglycerides when compared to animals that received only the standard commercial diet (NC). Treatment with CRHE significantly decreased triglyceride levels in animals that received the hyperglycemic diet. Regarding the other biochemical parameters analyzed, no statistical differences were

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Fig. 6. Effect of treatment with the CRHE on acetylcholinesterase (AChE) activity in the A: cerebral cortex, B: hippocampus, C: hypothalamus, and D: striatum. Values are expressed as mean ± SEM, N = 6 observations per group. * P < 0.05, *** P < 0.001 relative to the control.

the same phenolic compounds gallic, caffeic and ellagic acids, which demonstrated good antioxidant activity and an intestinal glycosidase inhibitor [30]. Previous studies have also demonstrated the antidiabetic, antioxidant and anti-inflammatory potential of ellagic acid [31]. Research has revealed additional evidence that oxidative stress plays an important role in hyperglycemia-induced tissue injury and in the early development of T2DM. These reports show that the formation of AGEs, a free radical that modifies protein groups and lipids, is a contributing factor [5]. CRHE was shown to inhibit DPPH free radical activity at rates better than ascorbic acid. Indeed, CRHE antioxidant activity, when analyzed through the inhibition β-carotene/linoleic acid and MDA oxidation, was equivalent to the quercetin standard. These results suggest that phenolic compounds are likely to be the primary

Table 3 Effect of C. regium hydromethanolic extracts (CRHE) on serum glucose levels (mg/dL) in alloxan-induced diabetic animals. Values are expressed as mean ± SEM; N = 6. *** P < 0.001 relative to the control group. Time

Control

C. regium 100 mg/kg

0 1 2 3 7

382.2 ± 25.9 356.0 ± 24.2 388.2 ± 26.1 353.2 ± 21.3 387 ± 56.9

380.7 326.2 317.0 304.7 155.3

h h h h days

± ± ± ± ±

31.7 48.8 51.0 54.0 37.9***

major compounds found in the C. regium extract were the phenols gallic, caffeic, and ellagic acids. Similarly, other studies that evaluated the antioxidant capacity and content of CRHE constituents by HPLC found

Table 4 Oral Glucose Tolerance Test (OGTT) at the beginning and at the end of treatment with a hyperglycemic diet. Start

End

Time

HC

HT

NC

NT

HC

0 15 30 60 120 180

75 ± 1.8 129 ± 4.8 131 ± 5.0 135 ± 5.3 131 ± 5.9 115 ± 4.6

74 ± 2.0 155 ± 13.1 159 ± 14.5 150 ± 9.7 113 ± 8.2 102 ± 3.9

70 ± 1.9 131 ± 9.6 148 ± 14.8 139 ± 16.2 115 ± 12.9 104 ± 14.8

72 ± 5.7 145 ± 12.1 151 ± 9.7 150 ± 9.0 122 ± 7.2 108 ± 3.7

123 153 165 165 145 134

HT ± ± ± ± ± ±

5.0a*** 18.0 8.0a* 2.9a* 7.2 1.7

111 112 144 165 150 136

NC ± ± ± ± ± ±

9.5 4.6 27.0b** 27.8 10.4 8.0

107 124 168 153 136 112

NT ± ± ± ± ± ±

6.8 6.5 22.5 10.0 8.3 6.0

101 120 137 147 136 119

± ± ± ± ± ±

8.4 6.9 11.3 17.2 16.2 10.1

Values are expressed as mean ± SEM; N = 5. HC: hyperglycemic diet control; HT: hyperglycemic diet with C. regium hydromethanolic extracts (CRHE) treatment; NC: normal diet control; NT: normal diet with CRHE treatment. * P < 0.05, ** P < 0.01, *** P < 0.001, a relative to the HC group at the beginning of treatment. b relative to the group HC at the end of treatment. 1389

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The therapeutic efficacy of gallic acid extracted from Cyamopsis tetragonoloba was tested in diabetic rats induced with streptozotocin and a high-fat diet. Treatment with gallic acid led to an improvement in insulin-dependent glucose transport in adipose tissue through the translocation and activation of transporter protein 4 (GLUT4) in the PI3K/p-Akt signaling chain [43]. This result suggests that the gallic acid found in CRHE may cause glycemia reduction in both TOTG and diabetic animals, as we observed in our study with CRHE treatment. Accordingly, stimulation of postprandial glycogen production, especially in diabetic and glucose-resistant patients, has become a therapeutic target, and it appears that discovery of a substance that acts in this mechanism will be of great value [44]. Glycogen is a highly branched glucose polymer, found predominantly in the brain, muscle, heart, and liver, where it functions as a source to release or store glucose, as appropriate. Jiang et al. demonstrated that hepatic glycogen in db/db mice (an animal model of type 2 diabetes) showed similar amounts of glycogen particles in their healthy livers. The particles of diabetic mice are much more fragile than those of healthy mice, possibly due to the underlying hyperglycemia [45]. This could explain in part the result that CRHE had a significant effect on the storage of hepatic glycogen in normoglycemic animals that received glucose. Moreover, increased hepatic storage may justify the decrease in blood glucose levels in glycemic curves of the OGTT model. The results of the biochemical tests for rats given a hyperglycemic diet showed that the HT, NC and NT groups all had significantly lower serum triglyceride levels than the HC group. The hepatic markers AST and ALT, which in many studies are used as evidence of toxicity, remained unchanged, indicating that CRHE has a good safety profile. Moreover, there were no histopathological alterations at the pancreatic or hepatic levels after treatment with CRHE, supporting the suggestion that CRHE is nontoxic. However, a limitation that we observe in this hyperglycemic diet model is that the animal not developed T2DM as we expects, but a recent review [46] concludes that numerous studies have aimed to develop an animal model that replicates the natural history and metabolic characteristics of T2DM, but the ideal animal model has not yet been identified. Nankar and Dobel demonstrated that combined treatment with ellagic acid and pioglitazone led to an improvement in hyperglycemia and dyslipidemia without any change in AST or TGP levels, and to improved GLUT 4 levels [47]. Karimi-Khouzani et al. demonstrated a significant decrease in the levels of AST and ALT after treatment with ellagic acid for liver problems caused by the chronic use of fluoxetine [48]. Toledo et al. [49] analyzed serum levels of male rats treated with C. regium extracts for the following markers: cholesterol, triglycerides, total lipids, urea, albumin, total protein, creatinine, AST, ALT, alkaline phosphatase, and glucose, and found no statistically significant changes before and after treatment. In another study using a culture of insulinresistant cells, the hypoglycemic power of gallic acid and its influence on hepatic carbohydrate metabolism in rats receiving a fructose-rich diet was evaluated. The hypothesis is that gallic acid reduces hyperglycemia by improving hepatic insulin resistance, suppressing hepatic inflammation, adjusting abnormal hepatic carbohydrate metabolism, decreasing hepatic gluconeogenesis, and increasing hepatic glycogenesis and glycolysis pathways [50]. Although some studies have reported the difficulties in inducing diabetes through drugs such as alloxan, it is still widely used as an inducer of diabetes in animals due to its low cost relative to streptozotocin [51]. Alloxan has the ability to destroy pancreatic β-cells through excess free radical production. Therefore, the prophylactic administration of antioxidant substances could prevent or reverse the diabetogenic effect. Zanoello et al. [52] discussed the protective ability of Syzygium cumini to protect pancreatic β cells from the diabetogenic action of alloxan, possibly by the presence of antioxidant substances such as eugenol, gallic acid and tannins. In our study, diabetic animals after treatment with C. regium for 7 days demonstrated a reduction in fasting glycemia relative to the animals in the control group.

Table 5 Biochemical results after 14 weeks of a hyperglycemic diet. Groups

ALT (U/L) AST (U/L) Cholesterol (mg/ dL) HDL (mg/dL) Triglycerides (mg/dL) VLDL (mg/dL) HbA1c (%)

HC

HT

NC

NT

48.2 ± 11.5 71.64 ± 4.2 63.0 ± 2.9

28.4 ± 5.2 67.1 ± 5.4 54.8 ± 2.9

33.1 ± 2.6 83.5 ± 6.3 63.6 ± 2.6

41.9 ± 7.5 89.3 ± 4.8 64.0 ± 5.2

55.9 ± 3.7 107 ± 6.0

46.1 ± 1.7 68 ± 5.1a*

55.7 ± 2.5 63.8 ± 4.3a**

55.6 ± 4.4 70.8 ± 8.7a*

20.48 ± 1.3 3.9 ± 0.06

16.1 ± 2.5 4.0 ± 0.05

14.3 ± 3.1 3.9 ± 0.04

14.1 ± 1.7 3.9 ± 0.04

ALT: alanine transaminase; AST: aspartate transaminase; HDL: High Density Lipoprotein; VLDL: Very Low Density Lipoprotein; HbA1c: glycated hemoglobin; HC: hyperglycemia diet control; HT: hyperglycemic diet with CRHE treatment; NC: normal diet control; NT: normal diet with CRHE treatment. * P < 0.05, ** P < 0.01, relative to HC.

antioxidant components of C. regium. Further, treatment with CRHE caused a significant decrease in the fluorescence intensity, which correlated to a decrease in the formation of AGEs in a BSA/glucose system. Kappel et al. [19] reported similar results after treatment with Musa x paradisíaca prevented AGES formation. Bahmani et al. [32] have also reported on the beneficial effects of many substances found in plants such as alkaloids, flavonoids, terpenes, and phenolic compounds on DM, and on the ability of kaempferol 3-O-β-D-glucopyranoside to prevent glycation. The antiglycation activity of CRHE implicated in our results may be linked to its antioxidant potential, as reported for the inhibition of DPPH and ABTS. Further, a study by Adisakwattana et al. [33], showed a promising inhibition of AGEs and DPPH free radical scavenging activity from the use of gallic and ascorbic acids in the formation of fructose-induced glycation and protein oxidation. Hyperglycemia is the primary pathological cause and insulin resistance is the primary risk factor for the development of IBD. Therefore, the search for pharmacological treatments to reduce the risk of IBD is of great importance in the medical field [34]. Thus, studies report that polyphenols may influence carbohydrate metabolism at various levels, improving postprandial glycemic levels, fasting blood glucose levels, acute insulin secretion, and insulin sensitivity, being that, a strategy to help prevent DM is to limit the rate of glucose absorption from the intestines into the bloodstream. Drugs currently in use that inhibit glucose absorption include acarbose, an alpha-glucosidase and alpha-amylase inhibitor, miglitol and voglibose, but these have unpleasant side effects due to the fact that they are not selective [35–37]. In a study on caffeic acid derivatives extracted from Wedelia trilobata, Ren et al. [38] reported significantly stronger inhibition of alpha glycosidases than acarbose. In addition, Rolffy, Ortiz, et al, [39] found that oral administration of C. vitifolium methanolic extract resulted in a significant decrease in glycemia levels in normoglycemic and streptozotocin-induced diabetic rats, as well as a reduction in blood glucose levels after administration of glucose and sucrose [39]. This extract also inhibited in vitro enzymatic activity of alfa glycosidases in a concentration-dependent manner and lowered blood levels of glucose, cholesterol, HDL, and triglycerides. Although the mechanisms related to C. regium activity have not yet been elucidated, they may be associated with the inhibition of carbohydrate digestion and of glucose absorption in the gut, among others, since C. regium inhibited in vitro disaccharidase activity in our study [31,40]. Yin et al. [41], demonstrated the inhibitory activity of ellagic acid and cannabol contained in mongolian oak cup extract on T2DM-related alpha-glycosidase and glycation. Oboh et al. [42] studied co-treatment of gallic acid and acarbose and found promising results in reducing the side effects of acarbose. 1390

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