Investigación IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Ximena Jaramillo-Fierro1*, Santiago Ojeda-Riascos1 1 Universidad Técnica Particular de Loja, Departamento de Química y Ciencias Exactas, , P.O, Loja-Ecuador. *Autor para correspondencia: e-mail: xvjaramillo@utpl.edu.ec Recibido: 2018/04/14 Aprobado: 2018/06/11 DOI: RESUMEN En el proceso de identificar plantas promisorias con actividades hipoglucémicas y antioxidantes, evaluamos las actividades inhibidoras de la α-glucosidasa y la α-amilasa, el contenido fenólico soluble total (TSPC), la actividad de eliminación de radicales libres (DPPH, ABTS) y la capacidad inhibidora de la autooxidación linoleica, de doce plantas utilizadas en la medicina tradicional del Ecuador: Siparuna eggersi (Monte de oso), Croton wagneri (Moshquera), Ilex guayusa (Guayusa), Baccharis genistelloides (Tres filos), Neonelsonia acuminada (Zanahoria blanca), Oreocallis grandiflora (Cucharillo), Verbena litoralis (Verbena), Justicia colorata (Insulina), Artocarpus altilis (Fruto del pan), Adiantun poiretii (Culantrillo), Costus comosus (Caña agria) y Piper crassinervium (Guabiduca). O. grandiflora se encontró superior a las otras plantas, especialmente en términos de su actividad inhibidora sobre α-glucosidasa (IC50 = 2.8 ± 0.4 μg / mL) y α-amilasa (IC50 = 161.5 ± 1.3 μg / mL), así como para los radicales libres (DPPH, ABTS) actividad de barrido (IC50-DPPH = 9.9 ± 0.06 μg / mL; IC50-TEAC = 6.6 ± 0.55 μg / mL). Por otro lado, A. altilis tuvo la mayor capacidad inhibidora de la autooxidación linoleica (IC50-β-CLAMS = 3.1 ± 0.20 μg / mL), y O. grandiflora, nuevamente, obtuvo el valor más alto de contenido fenólico soluble total (TSPC = 185.9 ± 0.09 GAE / g extracto). Estos hallazgos sugieren que O. grandiflora podría considerarse como un agente inhibidor y antioxidante enzimático alternativo para el tratamiento de la diabetes mellitus. Palabras clave: α-amilasa, α-glucosidasa, DPPH, TEAC, β-CLAMS, FOLIN-CIOCALTEU ABSTRACT In the course of identifying promissory plants with hypoglycemic and antioxidant activities we evaluated the α-glucosidase and α-amylase inhibitory activities, the total soluble phenolic content (TSPC), the free radicals (DPPH, ABTS) scavenging activity and the linoleic autoxidation inhibitory capacity, of twelve plants used in traditional medicine of Ecuador: Siparuna eggersi (Monte de oso), Croton wagneri (Moshquera), Ilex guayusa (Guayusa), Baccharis genistelloides (Tres filos), Neonelsonia acuminate (Zanahoria blanca), Oreocallis grandiflora (Cucharillo), Verbena litoralis (Verbena), Justicia colorata (Insulina), Artocarpus altilis (Fruto del pan), Adiantun poiretii (Culantrillo), Costus comosus (Caña agria) and Piper crassinervium (Guabiduca).The study has shown that O. grandiflora was superior in comparison to the others plants, especially, in terms of its inhibitory activity on α-glucosidase (IC50 = AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18. ISSN: 1390-6267- E-ISSN: 2550-6684 I 23 Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR 2.8 ± 0.4 µg/mL) and α-amylase (IC50 = 161.5 ± 1.3 µg/mL), as well as for free radicals (DPPH, ABTS) scavenging activity (IC50-DPPH = 9.9 ± 0.06 µg/mL; IC50-TEAC = 6.6 ± 0.55 µg/mL). On the other hand, A. altilis had the major linoleic autoxidation inhibitory capacity (IC50-β-CLAMS = 3.1 ± 0.20 µg/mL), and O. grandiflora again had the highest value of total soluble phenolic content (TSPC = 185.9 ± 0.09 GAEs/g extract). These findings suggest that O. grandiflora might be considered as an alternative enzyme inhibitory and antioxidative agent for the treatment of diabetes mellitus. Keywords: α-amylase, α-glucosidase, DPPH, TEAC, β-CLAMS, FOLIN-CIOCALTEU INTRODUCTION Diabetes is recognized as a group of heterogeneous disorders with the common elements of hyperglycemia and glucose intolerance due to insulin deficiency, impaired effectiveness of insulin action, or both (HernandezGalicia et al., 2002) (Shai et al., 2010). Anti-diabetic or hypoglycemic compound or composition, generally refers to an agent that lowers blood glucose levels. In traditional medicine, diabetes mellitus is treated with diet, physical exercise and medicinal plants (Alarcón-Aguilar, Roman-Ramos, Flores-Sánchez, & Aguirre-García, 2002). More than 1200 plant species from 725 genera and 183 families have been used in ethnopharmacology or experimentally around the world in the control of diabetes mellitus (Hasenah, Houghton, & Soumyanath, 2006); and, approximately, 30% of the traditionally used antidiabetic plants has been pharmacologically and chemically investigated (Alarcón-Aguilar et al., 2002; Andrade-Cetto & Heinrich, 2005; Rao, Sudarshan, Rajasekhar, Nagaraju, & Rao, 2002; Soumyanath, 2006) (Deutschländer, Lall, Venter, & Dewanjee, 2012). There are more than 200 pure compounds from plant sources that have been reported to show blood glucose lowering activity (Hasenah et al., 2006). An interesting finding is that plants typically have more than one active component, often associated with more than one mode of action. Certain groups, such as alkaloids, saponins, xanthones and flavonoids, and non starch polysaccharides, appear to have effects of particular significance in diabetes treatment, therefore the identification of activities and modes of action are important for drug development, and for the validation, standardization, and rational use of traditional herbal remedies (Soumyanath, 2006). The mechanisms involved in hypoglycemic activity from antidiabetic plants are numerous, including direct competitive antagonism with insulin, stimulation of insulin secretion, stimulation of glycogenesis and hepatic glycolysis, pancreatic beta cell potassium channel blockers, cAMP stimulation, among others. (Liu et al., 2017). Another therapeutic approach of medicinal plants for treating diabetes is to decrease the post-prandial hyperglycaemia. This is done by retarding the absorption of glucose through the inhibition of carbohydrate hydrolyzing enzymes, α-amylase and α-glucosidase, in the digestive tract. It is now believed that inhibition of these enzymes involved in the digestion and absorption of carbohydrates can significantly decrease the postprandial increase of blood glucose level after a mixed carbohydrate diet, and therefore can be an important and potentially natural and safe approach in the management of type 2 diabetes as well as chronic vascular complications (McCue, Kwon, & Shetty, 2004; Shim et al., 2003). Examples of such inhibitors, which are in clinical use, are acarbose, miglitol, emigitate, voglibose, (Bailey, 2003; Onal, Timur, Okutucu, & Zihnioglu, 2005). These inhibitors are widely used, as monotherapy as well as combination therapy with other antidiabetic agents (Fujisawa, Ikegami, Inoue, Kawabata, & Ogihara, 2005). A main drawback of currently used α-glucosidase and α-amylase inhibitors are side effects such as abdominal distention, flatulence, meteorism and possibly diarrhea. It has been suggested that such adverse effects might be caused by the excessive inhibition of pancreatic α-amylase resulting in the abnormal bacterial fermentation of undigested carbohydrates in the colon (Kwon, Apostolidis, & Shetty, 2007). Therefore, it becomes necessary to identify glucosidase inhibitors, from natural sources, having lesser side-effects (Conforti et al., 2005); (Bhat, Zinjarde, Bhargava, Kumar, & Joshi, 2008). 24 I AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 Diabetes is a major risk factor for premature atherosclerosis, and oxidative stress plays an important role (Conforti et al., 2005). In fact, numerous studies demonstrated that oxidative stress, mediated mainly by hyperglycemia-induced generation of free radicals, contributes to the development and progression of diabetes and related contributions, thus it became clear that ameliorating oxidative stress through treatment with antioxidants might be an effective strategy for reducing diabetic complications (Cunningham, 1998; Johansen, Harris, Rychly, & Ergul, 2006); (Kaleem et al., 2006). Several valuable reviews on the ethnobotanical use of plants of Southern Ecuador are available (Bejár, Russman, Roa, & Sharon, 2002; Bussmann & Sharon, 2006; PFN-133, 2006; Tene et al., 2007), nevertheless, studies dedicated to either antioxidants or antidiabetics have been published so far. The objective of this investigation was to ascertain the scientific basis for the use of plants in the treatment of diabetes mellitus. Therefore, this study was designed to investigate the α-glucosidase and α-amylase inhibitory capacity, free radicals (DPPH, ABTS) scavenging activity and linoleic autoxidation inhibitory capacity of twelve plants advocated in traditional Southern Ecuador medicine. MATERIALS AND METHODS Chemicals α-amylase (E.C. 3.2.1.1) type VI-B from porcine pancreas, α-glucosidase (E.C. 3.2.1.20) type I from Saccharomyces cerevisiae, p-Nitrophenyl-α-D-glucopyranoside (pNPG) as a synthetic substrate of α-glucosidase, 2,2 Diphenyl-1-picryhydrazyl (DPPH), 6 hydroxi-2,5,7,8-tetrametil-cromo-2-carboxilico (TROLOX), 3,5-Dinitrosalisylic acid (DNS), linoleic acid, (±)-α-tocopherol, potassium chloride, sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, sodium phosphate monobasic, potassium peroxodisulfate, 2,2’-Azio-bis (3-etilbenzotiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium sodium tartrate tetrahydrate, β-carotene Type I, sodium carbonate, were purchased from Sigma Chemical Co., and other chemicals including Tween 40 emulsifier, Folin ciocalteu and maltose were obtained from Merck Co., Sodium hydroxide and starch soluble purum were purchased from Riedel-deHaën. Finally, UltraPure Tris was obtained from Invitrogen. Selection and collection of traditional antidiabetic plants The traditional antidiabetic plants in this study were collected from various locations of Loja and Zamora Chinchipe, two provinces of southern Ecuador. Table 1 shows the scientific name, family, herbarium voucher, vernacular name(s) and therapeutical applications of each plant (Tene et al., 2007). Table 1. Medicinal plants used in treatment of diabetes in southern Ecuador No. Scientific name 1 2 3 4 5 6 7 8 9 10 Oreocallis grandiflora (Lam.) R.Br. Siparuna eggersii Hieron Artocarpus altilis (Parkinson) Fosberg Adiantum poiretii Wikstr. Costus comosus (Jacq.) Roscoe Piper crasinervium Kunth. Baccharis genistelloides (Lam.) Pers. Croton wagneri Müll. Arg. Ilex guayusa Loes. Neonelsonia acuminata (Benth.) blancaJ.M.Coult & Rose ex Drude Verbena litoralis Kinth. Justicia colorata (Nees) Wassh 11 12 Family Herbarium voucher Vernacular name(s) Proteaceae Monimiaceae Moraceae Pteridaceae Costaceae Piperaceae Asteraceae Euphorbiaceae Aquifoliaceae Apiaceae PPN-pe-001 PPN-mn-001 PPN-mo-003 PPN-pt-001 PPN-cs-001 PPN-pi-002 PPN-as-013 PPN-eu-001 PPN-aq-001 PPN-ap-007 Cucharillo Monte del oso Fruto del pan Culantrillo Caña Guabiduca Tres filos Moshquera Guayusa Zanahoria Verbenaceae Acanthaceae PPN-ve-001 PPN-ac-004 Verbena Insulina AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 I 25 Investigación Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Preparation of methanol extracts Plants were dried, pulverized and extracted with methanol 10:1 (methanol:plant) for 5 h at room temperature and constant stirring. The extracts were then concentrated using a rotary evaporator after filtration and kept at -20 °C until the assay experiments. Hypoglycemic activity Inhibition assay for α-amylase activity The α-amylase activity was determined by the method of Tsujida et al. (2006) (Takahiro & Takeshi, 2006). A volume of 125 µL of α-amylase was premixed with 125 µL of extract at various concentrations (10-1000 µg/ mL) and 125 µL of starch as a substrate was added as a 0.5 % starch solution in 20 mM phosphate buffer (pH 6.9) to start the reaction. The reaction was carried out at 25 °C for 10 min and terminated by addition of 125 µL of the DNS reagent (96 mM 3,5-dinitrosalicylic acid, 12% sodium potassium tartrate in 2 M NaOH). The reaction mixture was heated for 15 min at 100 °C and then diluted with 1 mL of distilled water in an ice bath. α-amylase activity was determined by measuring absorbance at λ=540 nm. Inhibition assay for α-glucosidase activity The inhibitory activity of total extracts against α-glucosidase was measured according to Matsui et al. (2001), (Matsui, 1996). Briefly, 35 µL of α-glucosidase (0.075 unit) was premixed with 35 µL of extract at various concentrations (10-1000 µg/mL). A volume of 930 µL of 10 mM p-nitrophenyl-D-glucopyranoside (pNPG) as a substrate in 67 mM phosphate buffer (pH = 6.9) was added to the mixture to start the reaction. The reaction was incubated at 37 °C for 15 min and stopped by adding 1 mL of 0.5 M TRIS buffer? (pH = 7.4). The α-glucosidase activity was determined by measuring the p-nitrophenol release from pNPG at λ=400 nm. The enzymatic inhibitory activity (%) in each reaction was calculated from the absorbance A, B, C and D by the following equation: % inhibition = {1-[(B-D)/(A-C)]} x 100 Where: B = Sample absorbance D = Blank 1 absorbance (without enzyme) A = Blank 2 absorbance (without inhibitor) C = Blank 3 absorbance (without inhibitor and enzyme) Total Soluble Phenolic Content of the extracts and antioxidant activity Total Soluble Phenolic Content assay (TSPC) The total phenolic content was determined by an assay modified from Shetty et al. (1995) (Kwon et al., 2007). The total extract (250 µL) was transferred into a test tube and mixed with 1.5 mL of distilled water. To each sample, 125 µL of 50% (v/v) Folin-Ciocalteu reagent was added and mixed. After 5 min, 250 µL of 20% Na2CO3 was added to the reaction mixture and allowed to stand for 60 min. The absorbance was read at 760 nm. The absorbance values were converted to total phenolics and were expressed as milligrams of gallic acid equivalents (GAEs) per gram of extract (Zengin et al., 2015). Standard curves were established using various concentrations of gallic acid (0.625-20 µg/mL). Determination of free radical scavenging activity using DPPH assay The antioxidant activity of each total extract was determined as the ability of the extract to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals (Buenger et al., 2005). A 0.1 mM DPPH radical solution in methanol was prepared. Immediately prior to measurement, this stock solution is diluted with methanol to an absorbance of 0.70 ± 0.02, determined by UV-Vis spectrophotometry at λ=517 nm. A volume of 1960 µL of this DPPH solution was mixed with 40 µL of sample or methanol (as control), and incubated for 15 min at RT. 26 I AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 The investigated antioxidant solutions were prepared in concentrations ranging from 5-500 µg/mL. A stock solution of α-tocopherol was prepared and serially diluted to concentrations ranging from 0.1-10 µg/mL. The absorbance of each sample at λ=517 nm was measured. This antioxidant activity is given as percentage (%) of DPPH scavenging, calculated as: Where Ac = Control absorbance As = Sample absorbance DPPH scavenging (%) = {(Ac – As)/(Ac)} x 100. Determination of free radical scavenging activity using TEAC assay The determination of the antioxidative activity of substances using the TEAC assay is based on their capability to reduce the stable radical 2,2’-Azio-bis (3-etilbenzotiazoline-6-sulfonic acid) diammonium salt (ABTS), in comparison with the standard TROLOX (Buenger et al., 2005). An ABTS solution (7 mm in water) is mixed in the with potassium peroxydisulfate solution (2.45 mM in water) and incubated for 12–16 h at room temperature in darkness. Immediately prior to measurement, this stock solution is diluted with water to an absorbance of 0.70 ± 0.02. A stock solution of TROLOX was prepared and serially diluted to concentrations ranging from 0.1-10 µg/mL.The investigated antioxidant solutions were prepared to concentrations ranging from (5-500 µg/mL). Water is used as solvents for both TROLOX and the samples. For the measurement, 40 µL of the samples or standards are mixed with 1960 µL of the reaction solution and the absorbance at 734 nm was measured after exactly 6 min against the solvent. This antioxidant activity was given as percentage (%) of ABTS scavenging, calculated as: ABTS scavenging (%) = {(Ac – As)/(Ac)} x 100. Where Ac = Control absorbance As = Sample absorbance Antioxidant activities by β-Carotene-linoleic acid assay The antioxidant activity of the methanol extracts was evaluated following the method of Miller (Duarte-Almeida, Santos, Genovese, & Lajolo, 2006). Briefly, 600 µL of β-carotene (0.2 mg/mL) dissolved in chloroform was pipetted into a small round bottom flask with 500 mg of Tween 40 and 50 µL of linoleic acid. After removing the chloroform using a rotary evaporator under reduced pressure and temperature, less than 45 °C, 60 ml of H2O2 were added to the flask with vigorous shaking. Aliquots (1960 µL) of the prepared emulsion were transferred to a series of tubes each containing 40 µL of extract or positive control (α-tocopherol). The investigated antioxidant solutions were prepared to concentrations ranging from (5-500 µg/mL). A stock solution of α-tocopherol was prepared and serially diluted to concentrations ranging from 0.05-5 µg/mL. A control sample was prepared exactly as before but without adding antioxidants. Each type of sample was prepared in triplicate. The test systems were placed in a water bath at 50 °C for 2 h. The absorbance of each sample was read spectrophotometerically at 470 nm, immediately after sample preparation (0 min) and at 120 min of the experiment. The antioxidant activity expressed as antioxidant protection factor (APF) of the extracts was evaluated in terms of bleaching of β-carotene using the following formula (Duarte-Almeida et al., 2006): APF = {[(Ac0–Ac120)-(As0–As120)]/(Ac0–Ac120)} x 100 Where: Ac0 = absorbance of control at t = 0 min Ac120 = absorbance of the control at t = 120 min As0 = absorbance of the sample at t = 0 min As120 = absorbance of the sample at t = 120 min AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 I 27 Investigación Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR IC50 Calculation The IC50 value from the hypoglycemic assays was defined as the concentration of α-amylase or α-glucosidase inhibitors to inhibit 50% of activity under the assay conditions (Matsui, 1996). The IC50 value from the antioxidants assays, was defined as the concentration of antioxidant that causes 50% loss of the DPPH or ABTS activity (color) or inhibit 50% of linoleic autoxidation under the assay conditions (Molyneux, 2004). Statistical analysis All experiments were performed in triplicate. Results are expressed as mean ± S.D. (Table 2) and were compared using a one-way analysis of variance (ANOVA). Comparisons between groups were made according to the Duncan and Fisher’s test. p-values less than 0.05 (p<0.05) were considered as statistically significant. The 50% inhibitory concentration (IC50) was calculated from the XLSTAT dose–response curve (statistical program) obtained by plotting the percentage of inhibition against the concentrations. RESULTS AND DISCUSSION Recently, some medicinal plants have been reported to be useful in diabetes worldwide and have been used empirically in antidiabetic and antihyperlipidemic remedies. Antihyperglycemic activity of the plants is mainly due to their ability to restore the function of pancreatic tissues by causing an increase in insulin output or inhibit the intestinal absorption of glucose or to the facilitation of metabolites in insulin dependent processes. More than 400 plant species having hypoglycemic activity have been available in literature, however, searching for new antidiabetic drugs from natural plants is still attractive because they contain substances, which demonstrate alternative and safe effects on diabetes mellitus. Most of plants contain glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc., that are frequently implicated as having antidiabetic effect (Malviya, Jain & Malviya, 2010). Pancreatic and intestinal glucosidases are the key enzymes of dietary carbohydrate digestion and inhibitors of theses enzymes may be effective in retarding glucose absorption to suppress postprandial hyperglycemia (Bhat et al., 2008). On the other hand, it is well known that a compound having antioxidant properties can prevent oxidative stress, which plays an important role in the prevention of diabetes complications (Akhter et al., 2013). The in vitro hypoglycemic and antioxidant activities of Siparuna eggersi, Croton wagneri, Ilex guayusa, Baccharis genistelloides, Neonelsonia acuminate, Oreocallis grandiflora, Verbena litoralis, Justicia colorata, Artocarpus altilis, Adiantun poiretii, Costus comosus and Piper crassinervium were investigated, and the results expressed as inhibition percentage for each concentration (µg/mL) of extract are shown in Table 2. As can be seen in the α-amylase assay, only S. eggersi, C. wagneri and O. grandiflora had activity, being the latter the most active. In comparison, all the plant extracts had activity in the α-glucosidase assay; however, O. grandiflora had very high activity and for this reason, lower concentrations (1-100 μg/ml) were used for this plant. Table 2 also shows a dose-dependent response in all antioxidant assays. 28 I AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 Table 2. Results of hypoglycemic and antioxidant activities of medicinal plants expressed as inhibition percentage ± S.D. Results from inhibition assay for α-amylase activity µg/mL Acarbose µg/mL A.p. C.c. A.a P.c. I.g. J.c. S.e. C.w. O.g. B.g. V.l. N.a. 10 10±0.8 10 NA NA NA NA NA NA 21±0.5 50 50±2.4 50 NA NA NA NA NA NA 26±0.3 27±0.3 1±1.0 NA NA NA 30±0.5 18±0.3 NA NA 100 69±2.2 100 NA NA NA NA NA NA NA 29±0.9 31±0.9 53±0.3 NA NA 500 84±2.1 500 NA NA NA NA NA NA NA 30±0.7 32±0.4 75±0.4 NA NA NA 1000 93±2.1 1000 NA NA NA NA NA NA 49±1.0 36±0.4 83±0.5 NA NA NA Results from inhibition assay for α-glucosidase activity µg/mL Acarbose µg/mL A.p. C.c. A.a P.c. I.g. J.c. S.e. C.w. O.g.* B.g. V.l. N.a. 10 3±0.9 10 1±0.9 6±1.3 7±1.0 11±0.3 6±0.6 NA 17±0.9 8±0.4 8±0.8 3±0.0 NA 13±0.9 50 7±2.0 50 55±0.7 49±0.7 60±0.7 21±0.3 8±1.2 NA 66±0.7 13±0.3 77±0.3 19±0.2 NA 15±1.3 100 13±2.0 100 91±0.9 71±1.5 84±0.5 39±0.1 15±0.7 NA 95±0.4 27±0.4 97±0.4 36±1.0 0±0.2 21±0.7 500 33±2.3 500 95±0.9 91±0.5 96±0.5 93±0.9 90±0.7 37±0.9 96±0.0 87±0.9 98±1.0 77±1.2 81±0.9 73±0.2 1000 52±2.7 1000 99±6.6 96±1.1 99±1.2 98±0.8 100±0.0 78±0.6 100±0.0 93±1.1 99±0.5 98±0.0 99±0.8 91±1.0 *Concentration: 1-5-10-50-100 µg/mL Results of free radical scavenging activity using DPPH assay µg/mL α-Tocopherol µg/mL A.p. C.c. A.a P.c. I.g. J.c. S.e. C.w. O.g. B.g. V.l. N.a 3.125 27±1.7 10 15±1.3 11±0.4 16±0.8 6±0.8 31±0.4 20±0.7 29±0.5 8±0.7 47±1.1 10±0.7 62±0.4 18±0.7 6.25 46±3.1 50 61±0.8 42±0.5 56±0.3 19±0.7 88±1.0 71±1.1 82±1.1 26±0.4 90±0.9 45±0.5 89±0.8 64±1.0 12.5 77±2.1 100 92±1.0 69±1.1 87±0.3 33±0.3 92±0.9 90±0.5 90±0.5 48±0.7 91±0.7 78±1.4 95±1.3 86±0.5 25 92±1.2 500 93±1.3 91±1.0 97±0.6 75±0.8 94±0.6 99±0.2 93±0.6 83±0.5 96±0.8 97±0.9 95±0.6 94±0.5 50 95±1.1 1000 95±0.8 94±0.4 97±0.6 88±1.2 97±1.2 99±0.5 94±0.8 93±1.2 96±0.6 98±1.4 96±0.8 96±0.5 Results of free radical scavenging activity using TEAC assay µg/ mL TROLOX µg/ mL A.p. C.c. A.a P.c. I.g. J.c. S.e. C.w. O.g. B.g. V.l. N.a 0.1 5±1.0 5 17±1.3 15±1.0 17±0.6 5±1.2 20±0.7 13±0.4 25±1.1 13±1.1 40±1.2 14±0.6 20±1.0 11±0.7 0.5 15±1.0 10 27±0.3 25±0.8 29±0.4 8±1.4 36±1.1 24±0.8 43±1.1 22±0.8 66±0.4 21±1.1 38±0.8 20±0.9 1 26±0.9 50 75±0.9 64±0.4 72±0.6 28±1.6 97±0.5 77±1.0 94±1.0 55±0.8 100±0.6 60±0.7 99±0.8 70±0.7 5 98±0.9 100 90±1.2 94±0.9 95±0.2 49±1.3 99±0.8 95±0.9 99±0.6 69±0.4 100±0.6 94±1.1 100±0.2 96±1.2 10 100±0.1 500 99±0.8 100±0.7 96±0.4 96±1.1 99±0.4 98±1.0 100±0.2 97±0.4 100±0.6 100±1.0 100±0.4 100±0.2 Results of antioxidant activities by β-Carotene-linoleic acid assay µg/ mL α-Tocopherol µg/ mL A.p. C.c. A.a P.c. I.g. J.c. S.e. C.w. O.g. B.g. V.l. N.a 0.05 28±2.1 1 10±1.0 21±0.6 36±0.4 6±0.3 26±0.6 19±0.4 14±1.1 18±3.4 NA 2±0.1 22±1.1 13±0.7 0.25 67±2.1 5 15±1.3 36±0.3 53±0.9 8±0.7 37±1.2 34±1.1 27±0.6 23±3.6 NA 3±1.1 34±1.0 14±1.2 0.5 82±1.4 10 21±0.6 51±0.9 68±0.5 12±0.2 46±1.1 43±0.7 40±0.7 36±5.2 NA 11±0.8 45±0.9 18±1.1 2.5 90±1.0 50 45±1.2 78±1.2 84±0.7 42±0.2 64±0.5 59±1.1 78±0.5 74±6.2 1±1.9 20±0.7 77±0.4 55±0.6 5 92±0.5 100 57±0.6 84±1.1 92±0.6 59±0.6 74±0.8 65±0.2 88±1.0 91±2.3 39±1.6 43±0.5 82±0.6 68±1.3 Siparuna eggersi (S.e.), Croton wagneri (C.w.), Ilex guayusa (I.g.), Baccharis genistelloides (B.g.), Neonelsonia acuminate (N.a.), Oreocallis grandiflora (O.g.), Verbena litoralis (V.l.), Justicia colorata (J.c.), Artocarpus altilis (A.a.), Adiantun poiretii (A.p.), Costus comosus (C.c.) and Piper crassinervium (P.c). AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 I 29 Investigación Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR α-amylase/α-glucosidase inhibitory activity The α-glucosidase and α-amylase inhibitor effectiveness of extracts of the different plant species were compared on the basis of their resulting IC50 values (Table 3). O. grandiflora inhibited the activity of both α-glucosidase and α-amylase with an IC50 of 2.8 ± 0.40 µg/mL and 161.5 ± 1.30 µg/mL, respectively. S. eggersii and C. wagneri, also inhibited the activity of α-amylase, but their inhibitor effectiveness was lower (IC50 > 1000 ug/mL) compared to O. grandiflora. The other extracts only inhibited the activity of α-glucosidase. S.eggersii (IC50 = 28.3 ± 0.60 µg/mL) was the next best after O. grandiflora to inhibit the activity of α-glucosidase (Table 3). Table 3. IC50 values (µg/mL) of α-glucosidase (AGH) and α-amylase (AAH) inhibition assays PLANT Oreocallis grandiflora AGH (IC50) 2.8 ± 0.40 AAH (IC50) 161.5 ± 1.30 Siparuna eggersii 28.3 ± 0.60 >1000 Artocarpus altilis 40.9 ± 0.38 NA Adiantum poiretii 46.3 ± 0.92 NA Costus comosus 57.9 ± 0.71 NA Piper crasinervium 108.5 ± 1.00 NA Baccharis genistelloides 154.6 ± 1.28 NA Croton wagneri 162.4 ± 1.34 >1000 Ilex guayusa 176.5 ± 1.50 NA Neonelsonia acuminata 198.7 ± 1.59 NA Verbena litoralis 337.9 ± 1.75 NA Justicia colorata 622.1 ± 2.52 NA POSITIVE CONTROL* 964.6 ± 2.80 56.8 ± 2.50 NA: Non active. *Acarbose for AGH and AAH The extracts of I. guayusa (IC50 = 176.5 ± 1.50 µg/mL) and V. litoralis (IC50 = 337.9 ± 1.75 µg/mL), had less inhibitory activity on α-glucosidase than O. grandiflora, however these plants could also be of interest in the treatment of diabetes mellitus due to their high antioxidant activity (Table 3). Total Soluble Phenolic Content (TSPC) of the extracts and antioxidant activity To better understand the mechanism(s) of action of the extracts against α-amylase and α-glucosidase, the total soluble phenolic content and antioxidant activity were measured for all of the extracts. Table 4 shows the total soluble phenolic contents (TSPC) of the extracts determined as gallic acid equivalents per gram of extract (GAEs/g extract), and the IC50 values obtained in the antioxidant assays, DPPH, TEAC and β-CLAMS, expressed in µg/mL. 30 I AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 Table 4. IC50 values (µg/mL) of antioxidant activity by DPPH, TEAC and β-CLAMS assays and (GAEs/g extract) of TSPC assay PLANT IC50-DPPH IC50-TEAC IC50-βCLAMS TSPC 9.9 ± 0.06 6.6 ± 0.55 108.5 ± 1.00 185.9 ± 0.09 Siparuna eggersii 17.1 ± 1.19 10.5 ± 0.89 13.0 ± 1.02 143.3 ± 0.06 Artocarpus altilis 11.7 ± 0.96 19.1 ± 1.56 3.1 ± 0.20 73.5 ± 0.08 Adiantum poiretii 32.2 ± 1.51 19.9 ± 1.63 69.8 ± 0.46 73.3 ± 0.09 Costus comosus 60.3 ± 0.47 22.5 ± 1.86 8.9 ± 0.06 52.3 ± 0.08 Piper crasinervium 167.8 ± 1.36 87.3 ± 0.72 73.9 ± 0.54 37.4 ± 0.04 Bacharis genistelloides 14.8 ± 1.22 25.4 ± 0.21 176.6 ± 1.15 44.3 ± 0.04 Croton wagneri 113.1 ± 0.91 37.8 ± 0.30 13.7 ± 1.07 82.6 ± 0.04 Ilex guayusa 14.2 ± 0.99 11.8 ± 1.01 13.0 ± 0.85 116.8 ± 0.05 Neonelsonia acuminata 31.6 ± 0.24 23.0 ± 1.93 43.1 ± 0.31 81.6 ± 0.05 Verbena litoralis 3.6 ± 0.12 10.9 ± 0.95 10.6 ± 0.07 159.8 ± 0.08 Justicia colorata 26.0 ± 0.21 20.4 ± 1.69 21.8 ± 0.14 64.9 ± 0.07 POSITIVE CONTROL* 6.3 ± 0.53 1.3 ± 0.10 0.1 ± 0.01 135.8 ± 0.00 Oreocallis grandiflora *α-Tocopherol for DPPH, TEAC and β-CLAMS and TROLOX for TSPC Taking into account the complex nature of phytochemicals, the antioxidant activities of plant extract cannot be evaluated using a single method. Thus, commonly accepted assays were employed to evaluate the antioxidant effects of the methanol extracts and free radical scavenging was determined by DPPH and TEAC assays (Table 2). The effect of antioxidants on DPPH and ABTS radicals is due to their hydrogen donating ability. Though the DPPH and ABTS radical scavenging abilities of the extracts were less than that of positive controls (α-tocopherol and TROLOX, respectively). The study showed that the extracts have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. Proton radical scavenging is an important attribute of antioxidants (Adedapo, Jimoh, Koduru, Afolayan, & Masika, 2008). Although Wang et al. (1998) found that some compounds which have ABTS scavenging activity did not show DPPH scavenging activity (Adedapo et al., 2008), in this study, the extracts showed comparable scavenging activities against DPPH and ABTS radicals. This demonstrates the capability of the extracts to scavenge different free radicals in different systems, suggesting that they may be useful therapeutic agents for treating radical-related pathological damage (Adedapo et al., 2008). The antioxidant potential of the extracts also was evaluated using model systems based on β-carotene coupled with autoxidized linoleic acid. Regarding antioxidant activity, all studied extracts, except O. grandiflora (IC50= 9.9 ± 0.06 µg/mL ; IC50-TEAC = 6.6 ± 0.55 µg/mL ; IC50-β-CLAMS = 108.5.6 ± 1.00 µg/mL), B. genistelloides DPPH (IC50-DPPH = 14.8 ± 1.22 µg/mL ; IC50-TEAC = 25.4 ± 0.21 µg/mL ; IC50-β-CLAMS = 176.6 ± 1.15 µg/mL) and A. poiretii (IC50-DPPH = 32.2 ± 1.51 µg/mL ; IC50-TEAC = 19.9 ± 1.63 µg/mL ; IC50-β-CLAMS = 69.8 ± 0.40 µg/mL), showed to have high correlation between scavenging activities and β-CLAMS assays. However, O. grandiflora, B. genistelloides and A. poiretii had best scavenging activity than autoxidized linoleic acid inhibition capacity, therefore these plant acts better as primary antioxidants than as secondary. The other plants have both primary and secondary (preventive) antioxidant activity being A. altilis (IC50-DPPH = 11.7 ± 0.96 µg/mL ; IC50= 19.1 ± 1.56 µg/mL ; IC50-β-CLAMS = 3.1 ± 0.20 µg/mL), S. eggersii (IC50-DPPH = 17.1 ± 1.19 µg/mL ; IC50-TEAC TEAC = 10.5 ± 0.89 µg/mL ; IC50-β-CLAMS = 13.0 ± 1.02 µg/mL), I. guayusa (IC50-DPPH = 14.2 ± 0.99 µg/mL ; IC50-TEAC = 11.8 ± 1.01 µg/mL ; IC50-β-CLAMS = 13.0 ± 0.85 µg/mL), V. litorialis (IC50-DPPH = 3.6 ± 0.12 µg/mL ; IC50-TEAC = 10.9 ± 0.95 µg/mL ; IC50-β-CLAMS = 10.6 ± 0.07 µg/mL) and J. colorata (IC50-DPPH = 26.0 ± 0.21 µg/mL ; IC50-TEAC = 20.4 ± 1.69 µg/mL ; IC50-β-CLAMS = 21.8 ± 0.14 µg/mL) the most active (p<0.05). The free radical scavenging method is quantitatively more reliable than β-CLAMS; however, the latter assay provides an alternative mechanism AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 I 31 Investigación Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR by measuring the capability of a compound to resist peroxidation and free radical chain reaction (Joaquim Mauricio Duarte-Almeida, Negri, Salatino, Carvalho, & Lajolo, 2007). Regarding to the Total Soluble Phenolic Content (TSPC) of the extracts, the highest value of TSPC (GAEs/g extract) corresponds to O. grandiflora (185.9 ± 0.09), followed by V. litoralis (159.8 ± 0.08), S. eggersii (143.3 ± 0.06) and I. guayusa (116.8 ± 0.05), therefore these plant species are interesting since phenolic phytochemicals have positive effect on health because they counteract the effects of reactive oxygen species (ROS) generated during cellular metabolism (Huan-xia, Hai-sheng, & Shu-fang, 2014; Miguel et al., 2014). The results of the hypoglycemic and antioxidant activities obtained here, support several traditional therapeutic uses reported for the species studied (Bejár, et al., 2002; Bussmann & Sharon, 2006; PFN-133, 2006; Tene et al., 2007). Comparison of hypoglycemic and antioxidant activities and total soluble phenolic content of extracts The data suggests that high phenolic content does not always confer a high anti-amylase or anti-glucosidase activity of an extract (Figure 1). Figure 1. Comparison between α-amylase/α-glucosidase inhibitory capacity and Total Soluble Phenolic Content. It has been reported that the antioxidant activity of plant materials is well correlated with the content of their phenolic compounds (Huan-xia, et al., 2014; Miguel et al., 2014). Therefore, it is important to consider the effect of the total phenolic content on the antioxidant activity of the extracts assayed. The antioxidant activity of the extracts was monitored using the DPPH and ABTS radical inhibition assays and β-carotene-linoleate model system. In recent studies with traditional plants, polymeric polyphenols were reported as contributing to strong glucosidases inhibition (Onal et al., 2005), but in this study the antioxidant activity of the extracts assayed was not proportional to both α-amylase or α-glucosidase inhibitory activity. There is, however, a significant correlation between Total Soluble Phenolic Content and free radicals (DPPH, ABTS) scavenging activity (Figure 2), except in Croton wagneri case; and linoleic autoxidation inhibitory capacity (Figure 3), except in few cases: Verbena litoralis, Siparuna eggersii, Ilex guayusa, Croton wagneri, Artocarpus altilis, Justicia colorata, Costus comosus. 32 I AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 Investigación Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR Figure 2. Comparison between free radicals (DPPH, ABTS) scavenging activity and Total Soluble Phenolic Content. Figure 3. Comparison between linoleic autoxidation inhibitory capacity and Total Soluble Phenolic Content. AXIOMA - Revista Científica de Investigación, Docencia y Proyección Social. Enero-junio 2018. Número 18, pp 23-36. ISSN: 1390-6267- E-ISSN: 2550-6684 I 33 Ximena Jaramillo-Fierro, Santiago Ojeda-Riascos IN VITRO HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITIES OF SOME MEDICINAL PLANTS USED IN TREATMENT OF DIABETES IN SOUTHERN ECUADOR CONCLUSION In vitro hypoglycemic and antioxidant activities of extracts from twelve medicinal plants used in treatment of diabetes in southern Ecuador were investigated based on biochemical techniques. In the α-amylase assay, only S. eggersi, C. wagneri and O. grandiflora had activity, being the last the most active. All the plant extracts had activity in the α-glucosidase assay; however, O. grandiflora had very high activity. S. eggersii was the next best after O. grandiflora to inhibit the activity of α-glucosidase. Regarding antioxidant activity, almost all the extracts showed to have high correlation between scavenging activities and β-CLAMS assays; however, O. grandiflora, B. genistelloides and A. poiretii had best scavenging activity than autoxidized linoleic acid inhibition capacity, therefore these plants act better as primary antioxidants than as secondary ones. The other plants have both primary and secondary (preventive) antioxidant activity being A. altilis, S. eggersii, I. guayusa, V. litorialis and J. colorata the most active (p<0.05). The results of the hypoglycemic and antioxidant activities obtained here based on biochemical techniques, support several traditional therapeutic uses reported for the species studied. This results, suggest that all species studied, especially O. grandiflora, are excellent candidates for future research on determining the mechanisms of their hypoglycemic or antioxidant activity, as well as for the isolation and identification of active hypoglycemic and antioxidant substances. In addition, further comprehensive pharmacological investigations, including experimental chronic studies, should be carried out to assess the possible toxicological effects of these plants. ACKNOWLEDGEMENTS Financial support for this study was granted by the “Universidad Técnica Particular de Loja” (PROY_ QUI_1237). We thank the Ministerio del Ambiente Ecuador for providing the “Autorización de Investigación Científica N-006-2014-IC-FLO-DPL-MA”. CONFLICTS OF INTEREST The authors declare no conflict of interest. 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