Journal Pre-proof Antidiabetic effect of Cordia morelosana, chemical and pharmacological studies Diana Giles-Rivas, Samuel Estrada-Soto, A. Berenice Aguilar-Guadarrama, Julio Almanza-Pérez, Sara García-Jiménez, Blanca Colín-Lozano, Gabriel NavarreteVázquez, Rafael Villalobos-Molina PII: S0378-8741(19)33950-9 DOI: https://doi.org/10.1016/j.jep.2020.112543 Reference: JEP 112543 To appear in: Journal of Ethnopharmacology Received Date: 2 October 2019 Revised Date: 14 December 2019 Accepted Date: 1 January 2020 Please cite this article as: Giles-Rivas, D., Estrada-Soto, S., Aguilar-Guadarrama, A.B., Almanza-Pérez, J., García-Jiménez, S., Colín-Lozano, B., Navarrete-Vázquez, G., Villalobos-Molina, R., Antidiabetic effect of Cordia morelosana, chemical and pharmacological studies, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112543. 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Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V. 6 3 *** *** 2 1 0 Control Pioglitazone MR B *** Vehicle Glibenclamide 5 mg/kg *** 4 Methyl rosmarinate 50 mg/kg 50 2 0 RA Control Pioglitazone MR RA Vehicle Glibenclamide 5 mg/kg Variation of glycemia (%) *** *** * *** * *** ** -100 1.5 2.0 *** 2 1 *** ** -50 *** *** *** 4 *** -100 0 2 1 3 Time (h) 5 Control Fenofibrate MR RA 0 Control Fenofibrate MR RA *** -100 3.0 0 1 h e m i T 3 Time (h) 5 7 () Vehicle Acarbose (3 mg/kg) 150 α-Glucosidase inhibition EECm 100 (mg/kg) 80 100 ** 50 * * * *** 0 * * * *** 60 40 Toxicity 20 Vehicle EECm 100 mg/kg 0 -50 0.5 1.0 1.5 2.0 3.0 DMSO 200 EECm Time (h ) 150 mg/dL 100 Vehicle EECm 100 mg/kg 50 Vehicle EECm 100 mg/kg 80 0 GLU 100 CHOL TG 60 40 Vehicle EECm 100 mg/kg 40 EECm Cordia morelosana PPARγ 60 80 mg/dL 0 20 20 40 0 AST ALT ALP 0 30 HDL 20 LDL 0 -10 -20 0 Vehicle EECm 100 mg/kg 0.4 VLDL 0000 10 5 10 15 Vehicle EECm 100 mg/kg 20 Days Fig.11 Weight variation. % Rel ative organ wei ght 1.0 3 0 *** *** Weight variationt (g) 0.5 4 *** 0 *** -50 U/L 0 *** 0.3 0.2 0.1 0.0 Vehicle EECm 100 mg/kg Heart % Relative organ weight *** * % Relative organ wei ght 0 *** 0 % Inhi bi ti on 100 % Variation of glycemia % Var i ati o n o f g l yc e m i a 50 200 D *** *** Expression level mRNA FATP/36B4 EECm (100 mg/kg) Expression level mRNA PPARα α /36B4 Vehicle Glibenclamide (5 mg/kg) 300 6 C 5 EECm 100 mg/kg % Variation of glycemia A *** 4 Expression level mRNA GLUT4/36B4 Expressi on l evel mRNA PPARγγ /36B4 5 0.4 0.3 0.2 0.1 0.0 Kidney 4 PPARα 3 2 1 0 Liver 7 Antidiabetic effect of Cordia morelosana, chemical and pharmacological studies Diana Giles-Rivas,1,Ψ Samuel Estrada-Soto,1,* A. Berenice Aguilar-Guadarrama,2,* Julio Almanza-Pérez,3 Sara García-Jiménez,1 Blanca Colín-Lozano,1 Gabriel NavarreteVázquez1, Rafael Villalobos-Molina4 1 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México. 2 Centro de Investigaciones Químicas, IICBA, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México. 3 Laboratorio de Farmacología, Depto. Ciencias de la Salud, D.C.B.S., Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, México. 4 Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla, Edo. de México, México Ψ Taken in part from the PhD Thesis of Diana Giles-Rivas. *Corresponding authors: Tel/Fax +52 777 329 7000. enoch@uaem.mx (S. Estrada-Soto); baguilar@uaem.mx (B. Aguilar-Guadarrama). Abstract Ethnopharmacological importance Cordia morelosana Standley (Boraginaceae) is commonly used in folk medicine for the treatment of diarrhoea, kidney inflammation, diabetes, lung pain, bronchitis, asthma, hoarseness, cough and fever. Aim Current work was conducted to develop a bio-guided isolation of antidiabetic compounds from ethanolic extract of Cordia morelosana (EECm). Material and methods The phytochemical bio-guided study was conducted by successive chromatographic techniques, and isolated compounds were characterized by 1D and 2D-NMR experiments. The in vivo antihyperglycemic and antidiabetic activities of EECm (100 mg/kg), and methyl rosmarinate (MR, 50 mg/kg) were determined on normoglycemic and diabetic murine models. Additionally, the in vitro activity was conducted to determine α-glucosidase inhibitory effect, and PPARs expression on 3T3-L1 cells by RT-PCR. Acute and sub-chronic toxicological studies for EECm were conducted on rats, following the OECD guidelines (No. 420 and 407). Results EECm promotes significant α-glucosidase inhibition (55.6%) at 1 mg/kg respect to the control. Also, EECm (100 mg/kg) showed significant antihyperglycemic effect on oral glucose tolerance test (OGTT), and in non-insulin dependent type 2 diabetes (NIDD) model, had antidiabetic activity (p<0.001) compared to controls. The bio-guided isolation allowed to obtain four known compounds described as rosmarinic acid (RA), methyl rosmarinate (MR), nicotiflorine and 1-O-methyl-scyllo-inositol. On the other hand, MR showed significant antidiabetic and anthiyperglycemic activities (p<0.05), and overexpression of PPARγ, PPARα, GLUT-4 and FAPT than control. Docking studies were conducted with PPARγ and PPARα, showing interesting binding mode profile on those targets. Finally, EECm displayed a LD50>2000 mg/kg and sub-chronic toxicological study reveals no toxic signs in animals tested compared to control. Conclusion EECm showed significant antihyperglycemic and antidiabetic actions being RA and MR the main antidiabetic metabolites. Keywords: Cordia morelosana Standley; diabetes; PPARγ; PPARα; α-glucosidase; methyl rosmarinate. 1. Introduction Type 2 Diabetes mellitus (DM2) is characterized by hyperglycemia with unusual carbohydrate, fat, and protein metabolism resulting from defective pancreatic β-cells or insulin deficiency/action. This chronic hyperglycemic circumstance is associated with longterm damage, dysfunction, and organ failure, particularly the nerves, eyes, blood vessels, heart, and kidney (Balakrishnan et al., 2018). For the treatment of DM2 several oral antidiabetic drugs exist, which are classified according to their biological effect as: insulin secretagogue (sulfonylureas, meglitinides, inhibitor DPP-4, incretinomimetic), insulin sensitizer (biguanides, thiazolidinediones), antihyperglycemic (acarbose), and inhibitor of glucose recapture (glifozine), among others (Chaudhury, 2017). In latter classification, one of the effective treatment strategies is to induce postprandial antihyperglycemic action by α-glucosidase inhibition (as an efficient adjuvant in DM2 therapeutics), and/or most important is to induce the insulin sensitization through Peroxisome Proliferator-Activated Receptors (PPARs) agonism. Thus, the αglucosidases inhibitors can reduce postprandial hyperglycemia, preventing impaired glucose tolerance, improving lipid metabolism and reducing oxidative stress indirectly (Wascher et al., 2005). However, the efficacy of α-glucosidases inhibitors in prediabetes treatment is not favourable. Results from recent clinical trials have shown that acarbose, a powerful α-glucosidase inhibitor, reduced the risk of diabetes by only 25% and had limited effects on the associated complications (Delorme and Chiasson, 2005). Recent research strategies also explore targeting the nuclear receptors (NRs), such as PPARs that are representative members of this large superfamily of NRs, which consist of three isotypes: PPARα, PPARβ/δ, and PPARγ acting as sensors of the cellular metabolic states (Brunmeir and Xu, 2018). A broad variety of natural compounds has been found to bind and activate PPAR proteins, and have been investigated as attractive therapeutic targets for DM2 (Shen and Lu, 2013). The genus Cordia encompasses about 250 species; the majority are tree- or shrub-sized and native to the Americas (Matias et al., 2015), and is distributed worldwide in warmer regions (Geller et al., 2010). Cordia morelosana Standley (Boraginaceae), is a tree of 2 to 4 meters high, with black bark and groups of white flowers; known as anacahuite, encinillo or palo prieto, which is commonly used in folk medicine for the treatment of diarrhoea, kidney inflammation, diabetes, lung pain, bronchitis, asthma, hoarseness, cough and fever (Monroy and Castillo, 2007). To date, there has not been data published about chemical composition. The aim of the present work was to develop a bio-guided isolation of antidiabetic compounds from ethanolic extract of Cordia morelosana (EECm) in order to obtain potential drugs in the treatment of DM2. 2. Materials and methods 2.1 Chemicals Streptozotocin (STZ), nicotinamide (NA), glibencz<alamide, glucose, sucrose and solvents for NMR spectra acquisition were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Acarbose and other reagents were purchased from local distributors. A Variant Inova Unity 400 spectrometer equipped with 5 mm multinuclear inverse detection probe was used to obtain 1D- and 2D-NMR experiments. 2.2 Plant material and preparation of EECm Cordia morelosana Stand I. (Boraginaceae) was collected and identified by Dr. Patricia Castillo-España in March 2005 in the “Corredor Biológico Ajusco-Chichinautzin”, Morelos, México. A specimen was deposited at the Biodiversity and Conservation Research Centre Herbarium (HUMO), Autonomous University of Morelos State, Cuernavaca, Morelos with a voucher number 19011. Aerial parts of C. morelosana (4 kg) was dried, powdered and extracted for 72 h in ethanol (three times). After filtration, the solvent was eliminated under reduced pressure below 40°C until dryness to give 70.62 g of the extract. 2.3 Phytochemical analysis EECm (49.4 g) was subjected to open column chromatography (CC) using silica gel 60 (0.063-0.200) Merck®; dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and methanol (MeOH) gradient were used as mobile phase. From this process, 170 fractions were obtained and pooled in five groups (F-I to F-V) according to the thin layer chromatography analysis. Then, the most active FII (68.3±1.2 % of α-glucosidase inhibition) fraction was subjected to several purification processes until obtain Rosmarinic acid (RA) and Methyl rosmarinate (MR). On the other hand, from FIII (21.9±2.4 % of inhibition) and FIV (48.5±1.7 of inhibition) allowed the isolation of Nicotiflorin, and 1-O-methyl-scylloinositol, respectively. All of them were identified by the analysis of spectroscopic data and compared with those reported in the literature. 2.4 Animals Six-week-old CD1 mice (25-30 g) were provided by Faculty of Medicine animal facilities, from Autonomous University of State of Morelos. On the other hand, adult male Sprague Dawley rats (200-250 g) were obtained from the Biomedicine Unit, FES Iztacala, National Autonomous University of Mexico. The animals were kept at constant laboratory conditions (12 h light/dark cycle, 25±2°C, and 45–65% humidity). Experiments were carried out in accordance with Mexican Federal Regulations for Animal Experimentation and Care (SAGARPA-NOM-062-ZOO-1999), based on US National Institutes of Health Publication No. 85-23, revised 1985. Before antidiabetic and OGTT experimentation, animals were deprived of food for 13 h but allowed free access to water. 2.5 In vivo assays 2.5.1 Oral glucose and sucrose tolerance tests Fasted normoglycemic mice were divided into three groups (n=6): vehicle group was administered with saline solution (s.s. 0.9% NaCl), reference drug glibenclamide group (5 mg/kg), or acarbose group (3 mg/kg) were used as positive controls, and EECm group received 100 mg/kg of the extract. 30 min after administration of test samples, a dose of 2 g/kg of glucose or sucrose solution was administered to each animal. Blood samples were collected from caudal vein at 0 (fasting blood glucose), 0.5, 1, 1.5, 2 and 3 h after administration. The percentage variation of glycemia was determined in relation to glucose blood level at 0 h, according to the equation: % variation of glycemia= [(Gx-G0)/G0] x 100, where G0 was the initial glycemia values, and Gx was the glycemia values at 0.5, 1.0 1.5, 2.0 and 3 h, respectively, as described (Ortiz-Andrade et al., 2007). 2.5.2 Anti-diabetic effect 2.5.2.1 Experimental Non-Insulin-Dependent diabetes (NIDD) model NIDD mice model was induced in overnight fasted mice by a single injection of 100 mg/kg of streptozotocin dissolved in citrate buffer (pH 4.5), 15 min after the i.p. administration of 40 mg/kg of nicotinamide. Hyperglycemia was established by the higher plasma glucose concentration, determined by commercial glucometer (Accu-Check, Performa). Animals with blood glucose (GLU) concentrations higher than 140 mg/kg were selected for the assay. The NIDD mice were separated into three groups (each, n=6 animals), vehicle group was orally administered with s.s. 0.9% NaCl, reference drug glibenclamide group (5 mg/kg) was used as positive control, and third group test received EECm (100 mg/kg) or 50 mg/kg of MR. Blood samples were collected from caudal vein at 0 (fasting blood glucose), 1, 3, 5 and 7 h after administration. The percentage variation of glycemia was determined in relation to glucose blood level at 0 h, according to the equation mentioned above. 2.5.3 Acute and sub-chronic oral toxicity The experiment was conducted in accordance with the recommendation of the Organisation for Economic Co-operation and Development (OECD) 420 (Acute Oral Toxicity), and 407 (Repeated Dose 28-day Oral Toxicity), with some modifications. For acute toxicity, male CD1 mice were used. Before experiments, all animals were fasted 16 h. To determine acute toxicity, animals (250-300 g) were orally administered with gradual doses (5, 50, 300, and 2,000 mg/kg body weight) of the EECm, and observed for 14 days after single dose administration. All deaths and signs of toxicity were recorded during the experiment. In sub-chronic assays, two independent groups of six animals (rats, n=6) were used. Vehicle group was orally administered by gavage, using a cannula, with s.s. 0.9% NaCl and the treated group received an oral single dose of EECm (100 mg/kg) daily, for 28 days. During assay, all animals were kept under observation to identify any signs of toxicity, and individual weights were determined. At day 29 animals were anesthetized, and blood samples were collected by cardiac puncture into clean tubes without heparin, and centrifuged (CIVEQ) at 4000 g for 10 min. Serum was carefully separated and stored frozen until required for analysis. Level of cholesterol, glucose, triglycerides, very lowdensity lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined. After that, rats were sacrificed by cervical dislocation to obtain kidney, liver and hearth from all animals in vehicle and treated groups. Then, relative organs’ weight was determined at the end of assays (29 days). The relative organ weight was calculated as follows: [relative organ weight/body weight] x 100. 2.5.4 Histopathological analysis All animals were subjected to gross necropsy and careful macroscopic observed. Tissue samples were collected from liver, kidney and heart and then were immersed in 10% formaldehyde in sodium phosphate buffer (50 mM), at pH 7.4. Tissue sample were processed separately in a Histokinette 200 apparatus (Reichert Jung, Germany). The sections were paraffin-embedded, cut into 5 µm slices and stained with hematoxylin-eosin; slices were examined under light microscope. 2.6 In vitro assays 2.6.1 Inhibition of the α-glucosidase activity Glucosidase inhibition assays were performed in quadruplicate as previously reported (Ramírez et al., 2012). Corn starch substrate (12.5 mg/mL) in 0.1M sodium phosphate buffer (pH 7.0) in a final volume of 250 µL was digested by crude enzyme, derived from a homogenate of Sprague Dawley rats’ intestinal mucosa. Substrate was premixed with C. morelosana extract and Fractions (F-I to F-V) at a concentration of 1 mg/mL, and the reaction was started by addition of 50 µl of crude enzyme. Then, the mixture was incubated at 37°C for 10 minutes, and reaction was stopped by acarbose and ice incubation and released glucose was quantified by a glucose-oxidase based clinical reagent (SPINREACT), following manufacturer’s directions. 2.6.2 Relative expression of PPARγ, PPARα, GLUT-4 and FAPT To determine the effect of RA and MR on PPARγ, PPARα, GLUT-4 and FAPT expression, in vitro assay was developed as described (Hidalgo-Figueroa et al., 2013; Almanza-Perez et al., 2010). 3T3-L1 cells (ATCC, 9 x 105 cells per well) were cultured in 6-well plates in Dulbecco’s modified Eagle’s medium supplemented with 25 mM glucose, 10% fetal bovine serum (v/v), 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM nonessential amino acids, and gentamicin, in a 5% CO2 humidified atmosphere, at 37°C. After 2 days of confluence, the cells were differentiated to the adipocyte phenotype with 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 mM dexamethasone acetate, and 80 mM bovine insulin, for 48 h, followed by insulin alone for another 48 h. The adipocytes were treated 24 h with 10 µg/ml of compounds to determine the effect on PPARγ, PPARα, GLUT-4, and FAPT expression. RNA was isolated from cultured cells and 2 µg of total RNA were reverse-transcripted in a thermocycler. The enzyme was inactivated and finally samples were cooled. Each reverse-transcripted reaction was amplified with SYBR Green master mix containing 0.5 mM of customized primers for PPARγ, PPARα, FAPT and GLUT-4. PCR was conducted and the threshold cycles (Ct) were measured in separate tubes in duplicate. The ∆Ct values were calculated in every sample for each gene of interest. Relative changes in the expression’s level of one specific gene (∆∆Ct) were calculated as described (Hidalgo-Figueroa et al., 2013). 2.7 In silico assay 2.7.1 Molecular docking with PPARα and PPARγ receptors The docking analysis of RA and MR were carried out by means of the AutoDock, v4.2 program. The crystal structures of PPARγ and PPARα, were recovered from the Protein Data Bank (PDB) with the ID: 1I7I and 5HYK, respectively. The program performs several runs (100) in each docking experiment. Each run provides one predicted binding mode. All water molecules and every co-crystal ligands were removed from the crystallographic structure and all hydrogen atoms were added. For all ligands and proteins, Gasteiger charges were assigned and non-polar hydrogen atoms were merged. All torsions could rotate during docking. The auxiliary program AutoGrid generated the latttice maps. Each grid centred at the crystallographic coordinates of the cocrystallographic ligand, with dimensions of 50 x 50 x 50 Å and 60 x 60 x 60 Å for PPARα/γ, respectively (point’s separation: 0.375 Å). The search was carried out with the Lamarckian genetic Algorithm using default parameters; The number of docking runs was 100. After molecular docking, all results were clustered into groups with root mean square deviation (RMSD) from 0.5-2 Å. MOE 2018.01, and Pymol version 1.3 were used for visualization. 2.7.2 Docking Validation The docking protocols were validated by AutoDock 4.2, based on the important interactions made by the co-crystallographic ligand with the amino acids of binding site. The RMSD obtained was less than 2.0 Å., in both cases. This value specifies that the parameters for docking simulations agree in reproducing the orientation and the conformation in the X-ray crystal coordinates of enzyme and receptors. 2.8 Statistical analysis Data are represented as mean + standard error of mean (S.E.M). Statistical analysis was performed by analysis of variance (ANOVA) followed by Bonferro ni post-test or Student´s t test. A value of p<0.05 was considered significant. 3. Results 3.1 Antidiabetic effect of EECm on experimental NIDD model EECm (100 mg/kg) provokes decrease of blood glucose concentration in diabetic mice compared to control group (p<0.05); this effect was significant from the first h and increased during the next hours after treatment (p<0.001). Also, the effect was similar to induced by glibenclamide (5 mg/kg), used as positive control (Fig. 1). 3.2 Antihyperglycemic effects of EECm over OGTT on normoglycemic mice using glucose and sucrose as substrate. After an oral glucose load (fig. 2), EECm (100 mg/kg) induced significant decrease in the percentage variation of glycemia, from 0.5 to 3 h (p<0.001) than control group. The blood glucose level lowering effect of EECm was comparable to that produced by glibenclamide (5 mg/kg). On the other hand, Fig. 3 shows that EECm (100 mg/kg) provoked a significant reducing of hyperglycemic peak and area under the curve of OGTT when was used sucrose as substrate. The decrease of the percentage variation of glycemia is similar to acarbose (3 mg/kg). 3.3 In vitro α-glucosidases inhibition of EECm EECm induced a significant intestinal α-glucosidases inhibition (55.1%) at 1 mg/kg, comparable to that showed by Camellia sinensis (green tea hydro-alcoholic extract) used as positive control. 3.4 Bio-guided fractionation of EECm using in vitro α-glucosidases inhibition EECm was subjected to a chromatographic column process. From this, five primary groups of fractions were obtained (F-I to F-V) and evaluated on α-glucosidases activity (Fig. 5). F-II was the most active fraction (68.3±1.2 % of inhibition), which after several chromatographic processes allowed the isolation and identification of two phenolic acids, Rosmarinic acid RA (33 mg) and Methyl rosmarinate MR (111 mg). On the other hand, from less active F-III (21.9±2.4 % of inhibition) and F-IV (48.5±1.7 of inhibition), Kaempferol-3-O-β-rutinoside (Nicotiflorin), a flavonoid, was isolated by spontaneous precipitation (150 mg) as yellow powder, meanwhile an inositol compound that was characterized as 1-O-methyl-scyllo-inositol (4) was isolated as a white solid (81 mg) (Fig. 6). 3.5 MR antidiabetic effect MR (50 mg/kg) induced significant decrease (p<0.001) in plasma glucose levels in NIDD mice model, the hypoglycemic effect was sustained from 3 to 7 h, and was better than glibenclamide (5 mg/kg). 3.6 PPARs, FAPT and GLUT-4 expression induced by AR and MR in 3T3-L1 MR significantly increased (about 4-fold) the relative expression for PPARγ and GLUT-4 mRNA´s, similar to pioglitazone and provoked a high of PPARα and FAPT mRNA expression (about 4 and 5-fold, respectively). In contrast, no change in relative expressions for PPARs, GLUT-4 and FAPT was observed with RA. 3.7 Docking analysis of RA and MR with PPARγγ Figure 9 (A) exhibits the interactions of RA with PPARγ residues through hydrogen bonds with Ser342, Gly284, Cys285 and Lys367, showing a binding energy of -4.53 kcal/mol. It is important to note that the interaction with Ser342 has been described as characteristic of PPARγ partial agonist (Bruning et al., 2007). On the other hand, Figure 9 (B) shows MR hydrogen bond interaction with Lys263, and an interaction between ester of carbonyl group with Glu343. Similarly, MR showed π-cation interaction between aromatic ring [3,4-(dihydroxyphenyl) acryloyl portion], and at the same time a hydrogen bond with Cys285. Also, a dual hydrogen bond interaction was predicted with Ser342, showing a binding energy of -4.73 kcal/mol. 3D binding model for RA and MR (Fig. 10) shows crucial interactions for PPARγ activity. Figure 11A shows interactions between RA with PPARα by hydrogen bonds with residues Ser280, Tyr314 and His440, showing a binding free energy of -5.39 kcal/mol. These amino acids are part of the network of hydrogen-bonding residues involved in the activation of the receptor. Also, MR (Fig. 11B) displays a binding energy of -4.9 kcal/mol, presenting hydrogen bonds with Ser280, Tyr314 and His440. Additionally, an interaction with Phe273 was also observed in the co-crystalized ligand, as well as the interaction with Thr279, which could improve the contact of this ligand with PPARα binding site, and thus manifesting biological activity. 3D binding model for RA and MR (Fig. 12) shows crucial interactions for PPARα activity. 3.8 Blood biochemical parameters, relative organ weight, body weight and histopathological analysis after sub-chronic toxicity study. Serum concentrations of glucose, lipid profile and transaminases were not modified compared with vehicle group (Fig. 13). Moreover, body weight in treated and untreated animals showed not changed after treatment period (Fig. 14), and the relative organ weights (organ to body weight ratio) of treated animals did not indicate changes, compared with vehicle (Fig. 15). Finally, the detailed histopathological observation and analysis of organs showed that renal cortex and medulla were without apparent alterations; however, glomerular mesangial congestion is evident in vehicle (16a) and treated (16b) group. Figures 16c and 16d represent longitudinal cardiac muscle fibbers, without alterations or damage in both vehicle and treated groups. Meanwhile, Figs. 16e and 16f, show liver with conserved cytoarchitecture. However, microscopic observation allowed to detect perivascular inflammatory infiltrate, chronic perivascular inflammation, and both with lymphocyte predominance. 4. discussion The aim of current study was to determine the potential antidiabetic effect of C. morelosana, a medicinal plant, which is commonly used in Mexican folk medicine for the treatment of diarrhoea, kidney inflammation, lung pain, bronchitis, asthma, hoarseness, cough, fever and diabetes (Monroy and Castillo, 2007). Also, to isolate and describe some of the secondary metabolites responsible of the antidiabetic action, and to establish the potential mode of action of the isolated compound MR. It is important to mention that there are not any antidiabetic pharmacological and chemical previous studies concerning to C. morelosana. As described and due that C. morelosana is used in the folk medicine for the treatment of diabetes, we decided, firstly, to determine the potential antidiabetic activity of ethanolic extract of the plant on NIDD model. As expected, EECm (100 mg/kg) induced significant antidiabetic action on diabetic mice, comparable to positive control (glibenclamide). Activity showed by the extract could be related with three most common modes of antidiabetic action, one related with postprandial activity after α-glucosidases inhibition and/or glucose co-transporters (GLUT-2 or SGLT1) inhibition related with an antihyperglycemic action, a second one linked with insulin secretion, and the third one associated with insulin sensitization (Ortiz-Andrade et al., 2007; Ortiz-Andrade et al., 2008; Chávez-Silva et al., 2018). Thus, in order to corroborate which one of them participates, we evaluated the extract in OGTT in normoglycemic mice to establish potential antihyperglycemic action. Therefore, EECm (100 mg/kg) shows significant antihyperglycemic effect after an oral load of glucose. In this framework, some actions to control postprandial hyperglycemia are related with pancreatic and/or extra pancreatic mechanisms, such as increment of insulin sensitivity in peripheral tissues, suppression of hepatic glucose output, insulin secretion, or due to glucose uptake regulation from intestinal lumen by glucose co-transporters inhibition (Ortiz-Andrade et al., 2008). With the intention to keep exploring the mechanism of action related with the antihyperglycemic postprandial effect, we performed the OGTT after a single load of sucrose (2 g/kg). It was observed that the EECm induced a marked decrease in the hyperglycemic peak, and the effect was sustained throughout the 3 h of experimentation. The decrease of the percentage variation of glycemia is similar to acarbose (3 mg/kg), an αglucosidases inhibitor used as antihyperglycemic agent in combination with oral antidiabetic drugs (metformin or glibenclamide), for the successful therapeutic strategy to control DM2 (Brunton et al., 2011). In this context, one of the therapeutic ways used for therapy of DM2 comprises the inhibition of enzymes, which can hydrolyse the polysaccharide molecules and convert them into monosaccharide units. This kind of enzymes are α-glucosidases (Taslimi and Gulçin, 2017). The glucosidases are located in the brush border of the small intestine and are required for the breakdown of carbohydrates before monosaccharide absorption. The α-glucosidases inhibitors delay the absorption of ingested carbohydrates, reducing the postprandial glycemia and insulin peaks (Fontana Pereira et al., 2011). Thus, control of postprandial glucose levels is critical in treatment of diabetic patients as well as individuals with impaired glucose tolerance (Huang et al., 2012). Therefore, we focused into evaluate the effect of EECm on α-glucosidases activity to look for a possible mode of action as anti-hyperglycemic agent. To corroborate this hypothesis, EECm was examined to determine its ability to inhibit the intestinal αglucosidases activity; the result showed that EECm significantly inhibited α-glucosidase activity by 55.6% at 1 mg/ml, and the inhibition was similar to that produced by Camellia sinensis used as positive control (Ramírez et al., 2012). Consequently, postprandial glucose, together with related hyperinsulinemia and lipidaemia, has been implicated in the development of chronic metabolic diseases like DM2 and cardiovascular diseases (Blaak et al., 2012). In diabetes, the postprandial phase is characterized by a rapid and large increase in blood glucose levels, and the possibility that the postprandial “hyperglycemic spikes” may be relevant to the onset of cardiovascular complications has received great attention (Ceriello, 2005). Thus, development of antihyperglyemic agents could help in control of diabetes and related diseases. After previous results, it was decided to do the bio-guided fractionation of the extract, which was subjected to a chromatographic column process to obtain RA, MR, Nicotiflorin and 1-O-methyl-scyllo-inositol. RA has already been found in the genus Cordia (Geller et al., 2010) and is a characteristic constituent in members of the Lamiaceae (isolated for the first time from Rosmarinus officinalis L.), and Boraginaceae families where it occurs in higher amounts (Petersen and Simmonds, 2003). As described before other species of the genus Cordia like C. dichotoma, and C. sebestena have been reported for antidiabetic activity in diabetic rats. In addition, MR has been isolated from C. dichotoma leaves and RA has been isolated from C. Americana, C. dentate, C. dichotoma and C. verbenacea (Oza and Kulkarni, 2017). Many studies have extensively evaluated the pharmacological activities of RA, particularly for antidiabetic properties. RA exhibits significant inhibition of α-glucosidases which confirms the effect showed by the EECm (Kubínobá et al., 2013); also, RA showed a potent dipeptidyl peptidase IV (DPP-IV) and protein tyrosine phosphatase 1B inhibition. Additionally, RA has been identified as AMPK activator (Jayanthy et al., 2017). In other study, oral treatment with RA (100 mg/kg) for 30 days could restored the blood glucose level, regulated the circulating adipokines and improved insulin sensitivity, in diabetic rats (Jayanthy et al., 2014). Runtuwene et al., (2016) observed that RA reduced hyperglycemia and ameliorated insulin sensitivity by increasing glucose transporter type 4 (GLUT-4) expression, and Jayanthy and Subramanian (2014), reported that RA controls blood glucose by regulating SGLT1 in intestinal brushborder membrane. Moreover, MR was found to induce significant α-glucosidases inhibition, antihyperglycemic and hypoglycemic activities (Ruiz-Vargas et al., 2019; Moharram et al., 2012). Finally, regarding nicotiflorin (3) and 1-O-methyl-scyllo-inositol (4), there is no antidiabetic reports, with exception of a very modest α-glucosidases inhibition (Adhikari-Devkota et al., 2019). These antecedents may correlate with results observed in the OGTT and NIDD models, suggesting that the antidiabetic effects of EECm are linked to more than one mechanism, which includes the postprandial antihyperglycemic action and the insulin sensitization related with the presence of RA and MR mainly. However, there are few studies about the antidiabetic and antihyperglycemic activities of MR, and almost no data about its mechanism of action; for that reason, and because it was isolated from the most active fraction of the bio-guided study, it was decided to study its antidiabetic activity and its potential mechanism of action. Antidiabetic and insulin sensitization of RA and MR: in vivo, in vitro and in silico studies As mentioned, MR could be one of the main bioactive compounds related with the antidiabetic and antihyperglycemic activities showed by EECm, in addition to RA. Thus, we decided to evaluate the antidiabetic activity of MR. As expected, MR (50 mg/kg) induced significant antidiabetic effect, which based on its structure, it could act as a prodrug (because it is a methyl ester of RA) that after administration it is metabolized by ester hydrolysis (esterases) to generate the free carboxylic acid. This effect can be deduced, since in the first hour after administration, MR did not show any effect in the decrease of glycemia, which was observed at 3 h post administration. The significant antidiabetic activity showed by MR might be linked with the antihyperglycemic action promoted by αglucosidases inhibition (83% of inhibition at 0.75 mM), as described by Ruiz-Vargas et al. (2019); however, we think that the main mechanism of action of the methyl ester is insulin sensitization. Thus, to investigate it, we evaluated the effect of main constituents isolated from EECm: RA and MR on PPARs, fatty acid transport protein (FAPT), and GLUT-4 expression. It is well known that PPARs are members of the nuclear receptor family of ligand-activated transcription factors that bind to fatty acids (FA) and their metabolites. The three PPARs isotypes: PPARα, PPARγ and PPARβ/δ have different tissue distribution patterns and ligands specificities (Gross et al., 2017). PPARα is the receptor for the fibrate class of lipid-lowering drugs, and PPARγ is the receptor for the thiazolidinedione class of antidiabetic drugs. Therefore, the PPARs are FA-activated receptors that function as key regulators of glucose, lipid, and cholesterol metabolism (Xu et al., 2001). Results summarized in Fig. 8, show that MR significantly increased the relative expression for PPARγ and GLUT-4 mRNA´s, also induced high of PPARα and FAPT mRNA expression. We could asseverate that MR directly activated PPARγ and PPARα. These findings are relevant, since the PPARs control the expression of networks of genes involved in adipogenesis, lipid metabolism, inflammation and maintenance of metabolic homeostasis. While PPARα has a crucial role in fatty acid oxidation in key metabolic tissues such as skeletal muscle, liver and heart; PPARγ is most highly expressed in adipose tissue, where it is a master regulator of adipogenesis as well as a potent modulator of whole-body lipid metabolism and insulin sensitivity (Ahmadian et al., 2013). On the other hand, no change in relative expressions for PPARs, GLUT-4 and FAPT was observed with RA. Based on the in vitro assays, the RA and MR were selected to explain the experimental activities on PPARγ and PPARα binding mode. They were docked into the ligand binding site of the human PPARγ (PDB ID: 1I7I) and PPARα (PDB ID: 5HYK), using the program AutoDock 4.2. Docking protocols were validated by re-docking of co-crystal ligand tesaglitazar in PPARγ, and 2-methyl-2-[naphthalen-1-yl) phenoxy] propanoic acid in PPARα After re-docking, the RMSD obtained were 0.64 Å and 0.29 Å, respectively. RA and MR showed a characteristic partial agonist binding mode, which highlights the interaction between hydrogen bond with Ser342 found on β sheet from PPARγ receptor, and the interaction with Cys285. Partial agonists represent an advantage over full agonits, since these have been related to severe adverse effects such as weight gain, edema, congestive hearth failure, bone fracture (Capelli et al., 2016), cardiotoxicity, and even cancer (Colín-Lozano et al., 2018). Is important to mention that is widely described that this mode of attachment is independent of the traditional one (His323, Ser289, His449, Tyr473). Inteactions is clearly appreciated in 3D binding model between RA and MR with PPARγ receptor. In addition, RA showed a binding free energy of -5.39 kcal/mol with PPARα receptor, and the amino acids with it is interacted are part of the network of hydrogen-bonding residues involved in the activation of the receptor. On the other hand, MR displays a binding energy of -4.9 kcal/mol, presenting hydrogen bonds with Ser280, Tyr314 and His440. Additionally, an interaction with Phe273 was also observed in the cocrystalized ligand, as well as the interaction with Thr279, which could improve the contact of this ligand with PPARα binding site, and thus manifesting biological important biological activity. 3D binding model for RA and MR shows crucial interactions for PPARα activity. These results are relevant to explain, at the molecular level, the activity of MR in the in vitro assay; on the other hand, in in vivo assay MR decreased glucose levels in a significant manner, showing a greater effect compared with glibenclamide as control. Despite RA had no effect in PPARs expression in vitro, docking study showed the interactions with crucial amino acids residues for activation of receptors, which may be could indicates a potential agonism for PPAR’s activation. Toxicity In the acute oral toxicity assay of the EECm no deaths and no other signs or symptoms of toxicity were observed. LD50 of extract was estimated to be greater than 2000 mg/kg of body weight, classified as class 5, according to the Globally Harmonised System (GHS), which indicates that any test sample classified in this category is nontoxic based on OECD guideline 420, with relatively low acute toxicity, but that under certain circumstances it may pose a hazard to vulnerable populations. Furthermore, repeated oral administration of EECm (100 mg/kg/day) for 28 days did not produce characteristic signs of toxicity in the treated or vehicle groups (OECD guideline 407). There were no animal deaths in all experimental period. Moreover, serum concentrations of biochemicals parameters such as glucose and lipid profile were altered, which indicates that the subchronic treatment did not modify the energetic metabolism of the animals assayed. Likewise, liver marker enzymes of animals treated with EECm was not modified, denoting that there is no cellular damage or death, especially in liver, heart or muscle (Ávila-Villareal et al., 2016). Results are in accord with those observed for body weight in treated and untreated animals, which were not changed after treatment period, and the relative organ weights (organ to body weight ratio) of treated animals did not indicate changes. Finally, the detailed histopathological observation and analysis of organs showed that renal cortex and medulla were without apparent alterations; however, glomerular mesangial congestion is evident in vehicle (16a) and treated (16b) group. Figures 16c and 16d represent longitudinal cardiac muscle fibers, without alterations or damage in both vehicle and treated groups. Meanwhile, Figs. 16e and 16f, show liver with conserved cytoarchitecture. However, microscopic observation allowed to detect perivascular inflammatory infiltrate, chronic perivascular inflammation, and both with lymphocyte predominance. Damage observed in the liver and kidney was observed in both treated and untreated groups, which permits to discard that extract treatment is responsible for the damage found, but possible produced by vehicle used or for potential infections produced for a variety of viral, bacterial, parasitic and fungal agents. Frequently, these organisms cause no overt sign of disease; however, many of the natural pathogens of these laboratory animals may alter host physiology (Baker, 1998). Further experiments are necessary to corroborate such asseverations. 5. Conclusion Phytochemical study of the Cordia morelosana showed four known compounds isolated: Rosmarinic acid, Methyl rosmarinate, Nicotiflorin and 1-O-methyl-scyllo-inositol, as the major constituents in the ethanolic extract (EECm), being rosmarinic acid and methyl rosmarinate the main bioactive antidiabetic agents accredited to the plant. This study provides scientific evidence of the efficacy and security in the use of Cordia morelosana, and the active compounds identified in the extract, bound to more than one therapeutic target of DM2, which include antihyperglycemic action (by α-glucosidases and possible glucose transporter inhibition), and insulin sensitization produced by potential PPARγ and GLUT-4 activation and/or over expression, also it is possible that it could be useful for the treatment of dyslipidemia due to PPARα and FATP. Conflict of Interest The authors have no conflict of interest to declare. Authors’ contributions to the paper were as follows: study design and coordination: S. Estrada-Soto, B. Aguilar Guadarrama, D. Giles-Rivas; RT-PCR from 3T3-L1 cells studies: J. Almanza-Pérez and D. Giles-Rivas; preparation of the extracts and Phytochemical studies: S. Estrada-Soto, B. Aguilar Guadarrama, D. Giles-Rivas; in vivo studies: D. Giles-Rivas, S. Estrada-Soto and R. Villalbobos-Molina; α-glucosidases inhibition determination: R. Villalobos-Molina, D. Giles-Rivas and S. Estrada-Soto; In silico studies: B. Colin-Lozano and G. NavarreteVázquez; preparation and writing of the manuscript D. Giles-Rivas, S. Estrada-Soto and R. Villalobos-Molina; Finally, all authors contributed to the writing and revised the manuscript. Acknowledgments Authors are in debt with Dr. Patricia Castillo-España for providing the plant material and to Dr. Guillermo Ramírez-Ávila for the α-glucosidase evaluation. This work was supported by SEP-CONACYT (Proyecto de Ciencia Básica A1-S-13540). D. 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Effect of EECm extract in the in vitro α-glucosidases inhibition. The results represent the mean ± S.E.M n=5, ***p<0.001 vs. DMSO. Figure 5. Effect of primary groups of fractions in the in vitro α-glucosidases inhibition. Results represent the mean ± S.E.M n=5, ***p<0.001 vs. DMSO. Figure 6. Structures of isolated compounds from EECm. Figure 7. Effect of MR on blood glucose levels in NIDD model. Each plot represents the means ± S.E.M. for n=6. ***p<0.001, **p<0.01 compared with Vehicle Figure 8. Effect of MR and RA on PPARs, GLUT-4 and FATP mRNA expression on 3T3L1 cells. Results represent the mean ± S.E.M. (n=6) compared with Control group. Figure 9. 2D binding model of RA (A) and MR (B), respectively, into the active site of PPARγ. Figure 10. 3D binding model of RA (magenta) and MR (blue) in the active site of PPARγ. Amino acids are represented as lines. Dashed lines signify polar interactions. Figure 11. 2D binding model of RA (A) and MR (B), respectively, into the active site of PPARα. Figure 12. 3D binding model of RA (magenta) and MR (blue) in the active site of PPARα. Amino acids are represented as lines. Dashed lines signify polar interactions. Figure 13. Effect of EECm (100 mg/kg/day) in biochemical parameters after sub-chronic treatment. Results represent the mean ± S.E.M n=6, ***p<0.001 vs. control group. Figure 14. Effect of EECm (100 mg/kg/day) in weight variation after sub-chronic treatment. Results represent the mean ± S.E.M n=6, ***p<0.001 vs control group. Figure 15. Relative organ weights after EECm (100 mg/kg/day) sub-chronic treatment. Results represent the mean ± S.E.M n=6, ***p<0.001 vs control group. Figure 16. Histopathological examination of organs: kidney, heart and liver. Letter a, c and e represent the vehicle group and b, d, f represent organs of treated group with EECm. (Hematoxylin & Eosin) 40X. Figure 1. Vehicle Glibenclamide 5 mg/kg EECm 100 mg/kg Variation of glycemia (%) 50 *** 0 * *** *** -50 *** *** *** -100 0 1 3 Time (h) 5 7 Figure 2. Vehicle % Variation of glycemia 300 Glibenclamide 5 mg/kg EECm 100 mg/kg 200 *** 100 *** * *** 0 * *** *** ** 1.0 1.5 Time (h ) 2.0 -100 0 0.5 3.0 Figure 3. Vehicle Acarbose 3 mg/kg EECm 100 mg/kg % Variation of glycemia 150 100 * 50 ** * * *** 0 * * * -50 0 0.5 1.0 1.5 Time (h ) 2.0 3.0 Figure 4. 80 % Inhibition *** *** 60 40 20 0 DMSO C. sinensis EECm Figure 5. 100 80 % Inhibition *** *** 60 *** *** 40 *** 20 0 DMSO C. Sinensis F-I F-II 1 F-III F-IV F-V Figure 6. OH OH O HO HO O OH O RA MR OH HO OH HO O OH Nicotiflorin 1-O-methyl-scyllo-inositol Figure 7. Vehicle Glibenclamide 5 mg/kg Methyl rosmarinate 50 mg/kg % Variation of glycemia 50 0 *** ** -50 *** *** *** *** -100 0 1 3 Time (h) 5 7 Figure 8. Expression level mRNA PPARγγ /36B4 5 4 *** 3 *** A *** 2 1 0 Control Pioglitazone MR RA Expression level mRNA GLUT4/36B4 6 *** 5 B *** 4 3 2 1 0 Control Pioglitazone MR RA 5 Expression level α /36B4 mRNA PPARα *** C 4 3 *** 2 1 0 Control Fenofibrate Expression level mRNA FATP/36B4 6 MR RA *** 5 4 D *** 3 2 1 0 Control Fenofibrate MR RA Figure 9. A B Figure 10. Figure 11. A B Figure 12. Figure 13. Vehicle EECm 100 mg/kg 200 mg/dL 150 100 50 0 GLU CHOL TG Vehicle EECm 100 mg/kg 80 mg/dL 60 40 20 0 HDL LDL VLDL Vehicle EECm 100 mg/kg 100 U/L 80 60 40 20 0 AST ALT ALP Figure 14. Vehicle Weight variationt (g) 40 EECm 100 mg/kg 30 20 10 0 -10 -20 0 5 10 Days 15 20 Figure 15. Vehicle EECm 100 mg/kg % Relative organ weight 0.40 0.35 0.30 0.25 0.20 Heart % Relative organ weight 0.40 Vehicle EECm 100 mg/kg 0.35 0.30 0.25 0.20 Kidney Vehicle EECm 100 mg/kg % Relative organ weight 4.0 3.5 3.0 2.5 2.0 Liver Figure 16. b Kidey a d e f Liver Heart c