Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 591313, 6 pages http://dx.doi.org/10.1155/2013/591313 Research Article Aqueous Extract of Annona macroprophyllata: A Potential -Glucosidase Inhibitor F. Brindis,1 M. E. González-Trujano,2 M. González-Andrade,3 E. Aguirre-Hernández,4 and R. Villalobos-Molina1 1 Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autonoma´ de Mexico,´ Avenida de los Barrios 1, Los Reyes Iztacala, 54090 Tlalnepantla, MEX, Mexico 2 Laboratorio de Neurofarmacolog´ıa de Productos Naturales de la Direccion´ de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatr´ıa Ramon´ de la Fuente Muniz,˜ Calzada Mexico-Xochimilco´ 101, Colonia San Lorenzo Huipulco, 14370 Mexico´ City, DF, Mexico 3 Instituto Nacional de Medicina Genomica´ (INMEGEN), Periferico´ sur 4809, Arenal Tepepan, Tlalpan, Secretar´ıa de Salud, 14610 Mexico´ City, DF, Mexico 4 Departamento de Ecolog´ıa y Recursos Naturales Facultad de Ciencias, Universidad Nacional Autonoma´ de Mexico,´ Ciudad Universitaria Coyoacan,´ 04510 Mexico´ City, DF, Mexico Correspondence should be addressed to F. Brindis; [email protected] Received 29 April 2013; Accepted 25 July 2013 Academic Editor: Ruth C. R. Meex Copyright © 2013 F. Brindis et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Annona genus contains plants used in folk medicine for the treatment of diabetes. In the present study, an aqueous extract prepared from Annona macroprophyllata (Annonaceae, also known as A. diversifolia)leaveswasevaluatedonboththeactivityofyeast -glucosidase (an in vitro assay) and sucrose tolerance in Wistar rats. The results have shown that the aqueous extract from A. macroprophyllata inhibits the yeast -glucosidase with an IC50 = 1.18 mg/mL, in a competitive manner with a = 0.97 mg/mL, a similar value to that of acarbose ( = 0.79 mg/mL). The inhibitory activity of A. macroprophyllata was reinforced by its antihyperglycemic effect, at doses of 100, 300, and 500 mg/kg in rats. Chromatographic analysis identified the flavonoids rutin and isoquercitrin in the most polar fractions of A. macroprophyllata crude extract, suggesting that these flavonoids are part of the active constituents in the plant. Our results support the use of A. macroprophyllata in Mexican folk medicine to control postprandial glycemia in people with diabetes mellitus, involving active constituents of flavonoid nature. 1. Introduction One important factor that arises in postprandial hyper- glycemia is the fast uptake of glucose in the intestine, in Diabetes mellitus (DM) is a chronic disease associated with which -glucosidases hydrolyze starch and oligosaccharides abnormal and constant high blood glucose (hyperglycemia) [5]. The -glucosidases, exoglycosidases, found in the luminal that results from defects in insulin secretion, action, or surface of enterocytes contain maltase/glucoamylase and both. In type 2 diabetes mellitus (DM2), postprandial hyper- sucrose/isomaltase activities [6]. It is believed that inhibition glycemia is important in the development of the disease; it of these enzymes can effectively control the postprandial is associated with micro- and macrovascular complications, elevation of blood glucose. Therefore, an important strategy and it has been proposed as an independent risk factor for managing postprandial hyperglycemia is to inhibit - for cardiovascular disease [1, 2]. The postprandial phase is glucosidase activity [7]. The -glucosidase inhibitors, such as characterizedbyarapidandincreasedglycemia.Thesepost- acarbose,havebeenusedintheclinictocontrolbloodglucose prandial “hyperglycemic spikes” may be relevant to the increase, especially postprandial, in DM2 [8]; these chemical pathophysiological conditions of late diabetes [3, 4]. drugs have strong inhibitory activity against -glucosidases 2 BioMed Research International but have the disadvantage of undesirable side effects such kaempferol, hesperidin, naringenin, naringin, rutin, iso- as abdominal distention, flatulence, meteorism, and diar- quercitrin, and quercetin. rhea [9]. Previous research indicates that side effects could be caused by excessive inhibition of pancreatic -amylase, 2.3. -Glucosidase Assay In Vitro. -Glucosidase inhibition ∘ resulting in the abnormal bacterial fermentation of undi- wasmeasuredatpH7.0and30Cusingp-nitrophenyl- gested carbohydrates in the colon. Therefore, more effective -D-glucopyranoside (pNPG) as substrate and 0.75 IU/mL inhibitors for the enzymes should have strong inhibitory of yeast -glucosidase in 0.1 M sodium phosphate buffer. effect against -glucosidase and mild inhibitory effect against Acarbose and aqueous extract were dissolved in phosphate -amylase, which can be an effective therapy for managing buffer, and serial dilutions from 1.4 to 0.2 mg/mL were postprandial hyperglycemia with minimal side effects [10]. prepared. The increments in absorption at 405 nm, due to the Annonaceae is a family of plants consisting of 2300 to hydrolysis of pNPG by the -glucosidase, were determined 2500speciesincludedinmorethan130genera;infact,itis on a microplate reader DTX 880 Multimode Detector from the largest family of the order Magnoliales. Only four genera Beckman Coulter. Ten L of acarbose or extract solution (Annona, Rollinia, Uvaria, and Asimina)produceediblefruits (in triplicate) were incubated during 5 min with 20 Lof such as annona [11–13]. Annona macroprophyllata Donn. enzyme stock. After incubation, 10 Lofsubstratewere ∘ Sm. is a species belonging to the Annona genus; it is a added and further incubated for 35 min at 30 C. Finally, common tree in central Mexico´ known as “ilama” (States thereactionwasstoppedbyadding30Lof5MNa2CO3. ofColima,Guerrero,andMexico);´ whereas in the southeast The concentration required to inhibit the enzyme activity by (Tehuantepec region and Yucatan)´ it is called “papauce” or 50% (IC50) was calculated by regression analysis, using the “anona blanca” [14, 15].Thefruitsofthisplantareusedas following equation: food, but its leaves are employed as anticonvulsant [16], as well as analgesic and anti-inflammatory agents in traditional 100 V = , (1) Mexican medicine [17]. However, so far there are no scientific 1+(/IC50) reports supporting its probable antihyperglycemic proper- ties, even though other species of the genus are known to where V is the percentage of inhibition, A100 is the maximum have those properties, that is, Annona squamosa, A. muricata, inhibition, I is the inhibitor concentration, IC50 is the concen- A. glabra, and A. cherimola [18–20]; then we have used a trationrequiredtoinhibittheenzymeactivityby50%,and chemotaxonomic criterion in order that A. macroprophyllata, is the cooperative degree. besides its described medicinal properties [14–17], was inves- tigatedtosearchforitslikelyantidiabeticproperties. 2.4. Enzyme Kinetics. The mode of inhibition for - glucosidase was determined by the Lineweaver-Burk plots. All the results are expressed as the mean of at least three 2. Materials and Methods experiments ± SEM. Kinetic parameters such as , ,and wereevaluatedbyusingthenonlinearregressionmethod, 2.1. Plant Material and Extract. Annona macroprophyllata based on the following inhibition equation: Donn. Sm. specimens were collected in Tejupilco, Guerrero V inSeptember2010.Dr.E.Cedillo-Portugal,botanistfromthe V = max , (2) Universidad Autonoma´ de Chapingo (UACH), certified the (1 + /)+(1+/ ) authenticity of the plant, and a voucher specimen (AN9702) was deposited in the herbarium “Herbario de Plantas Utiles´ where V is the initial velocity in either the Presence or absence Efra´ım Hernandez´ X” at the UACH, in the State of Mexico,´ of the inhibitor and are the concentrations of substrate and Mexico,´ for future reference. Aqueous extract was obtained inhibitor, respectively; is the maximum velocity, is the by using 50 g of dried and powdered leaves, in a process of Michaelis-Menten constant, is the competitive inhibition infusion in 500 mL of boiling water. The aqueous extract was constant, and is the uncompetitive inhibition constant. separated from the residues by gravity filtration; samples were The kinetic data were analyzed using a computer program for frozen in liquid nitrogen and then lyophilized during 12 h nonlinear regressions (Origin 8.0). using a Heto FD3 Lab lyophilizer to yield 1.11 g (2.22%). 2.5. Experimental Animals. Male Wistar normoglycemic rats, weighing 200–250 g, were obtained from our animal facilities 2.2. HPLC Quantification of Flavonoids. An HPLC analysis at Facultad de Estudios Superiores Iztacala, U.N.A.M. Pro- was performed using an Agilent Technologies Chromato- cedures involving animals and their care fulfilled the Mex- graph. The separation was performed on an ODS Hypersil ican Official Norm for Animal Care and Handling (NOM- C18 column (15 mm × 4mmi.d.and5mparticlesize).The 062-ZOO-1999) and were in compliance with international samples were injected through a 20 Lloop.Thecolumn rulesoncareanduseoflaboratoryanimals.Furthermore, ∘ was thermostatically controlled at 30 C using a 1 mL/min clearance for conducting the studies was obtained from the flow rate. The mobile phase consisted of 15 : 85 acetonitrile- Ethics Committee for the Use of Animals in Pharmacological trifluoroacetic acid solution at pH 2.5. The detection was and Toxicological Testing of