Journal of Natural Sciences

December 2018, Vol. 6, No. 1&2, pp. 8-12

ISSN 2334-2943 (Print) 2334-2951 (Online)
Copyright © The Author(s). All Rights Reserved.
Published by American Research Institute for Policy Development
DOI: 10.15640/jns.v6n1-2a2
URL: https://doi.org/10.15640/jns.v6n1-2a2

Antihyperglycemic Activity of Aqueous Extracts of Eight Amazonian Species

Diego Vásquez1, Marx Peña, Blanca Diaz1, Dora García2, Víctor Sotero3

Abstract

The use of plant species for the prevention, control and treatment of various pathologies is currently a
practice recommended by the WHO. This research was carried out in order to expand the knowledge related
to the antihyperglycemic activity of plant species; the objective was to evaluate the aqueous extracts of eight
plant species as potential inhibitors of the enzyme α-glucosidase. The samples were collected from various
points from Loreto Region; they were processed by standard techniques to obtain freeze-dried samples and
quantify the concentrations at 100 μg/mL 500 μg/mL and 100 μg/mL.. The species Coussapoa asperifolia
showed the highest percentage of inhibition and an IC50 of 32.1 μg/mL. Likewise, it is concluded that the
species Remijia pedunculata, Bahuinia glabra, Handroanthus obscurus and Calycophyllum spruceanum reported values
that are within the range of antihyperglycemic activity at in vitro assays.

Keywords: Antihyperglycemic, type II diabetes, α-glucosidasa.

1. Introduction
Diabetes Mellitus (DM) is one of the main causes of morbidity and the population worldwide, in our country
it is cataloged within the 10 leading causes of death; this has become a heterogeneous group of chronic metabolic
disorders, characterized mainly by an abnormally high concentration of glucose in the blood, called hyperglycemia
(FID, 2013). Likewise, in the Amazon, plant species are fundamental in the way of life of the Amazonian population,
which issued to satisfy the needs of health, being used as a natural medicine (Huamantupa et al., 2011). This
traditional knowledge is a primary source of potential information for the development of research in the field of
pharmacology (Tananta, 2014). Consequently, studies in ethnobotany report that Amazonian plants act favorably in
the treatment of hyperglycemia, attenuating their services and they matter (Bhushan et al., 2010; Avellaneda, 2013;
Gomez, 2012). Therefore, it is important to conduct a bioprospecting study of Amazonian plants with a history of
antidiabetic activity and validate this knowledge with experimental studies.
In that sense, treatment and control of DM represents a high cost for state health systems; likewise, it is
generated in people suffering from morbidity, depression for the family and a high economic impact (Gnecco 1998).
In this context, several studies have emerged in favor of the treatment and improvement of the quality of life of the
patients who suffer from it, among them several plant species that offer both prophylactic and maintenance therapies
stand out (Bhushan et al., 2010). Therefore, the in vitro tests open a space of importance in research as an
experimental support for the therapeutic treatment of DM in complementary medicine, as they provide very precise
results of the effect of metabolites of plant extracts for the treatment of DM (Corrales et al., 2014);

1 Faculty of Biology. National University of the Peruvian Amazon. Ciudad Universitaria –Zungarococha- Iquitos-Perú
E-mail: diego.vasquez.fcb@gmail.com
2 Faculty of Chemical Engineering. Universidad Nacional de la Amazonia Peruana. Freyre 632-Iquitos-Perú.

E-.mail: doegato@hotmail.com
3 Foundation for the Sustainable Development of the Lower Jungle of Peru-FUNDESAB-PERU. Samanez Ocampo 576-Iquitos-

Perú. E-mail: proyectopalmeras@gmail.com

Vasquez et al. 9

Thus providing knowledge about the biological potential of these species. Consequently, the development of
an analytical method that determines the behavior of the extract in diminishing hyperglycemia becomes urgent.
Studies of Amazonian plants with antidiabetic activity carried out to date are widely developed in in vivo models
(Lecca & Rojas, 2011). In vitro models being an important step as a pre-test to in vivo models. Due to this, the
objective of this investigation was to determine the in vitro inhibitory activity of eight extracts of Amazonian plant
species on α-glucosidase. Also, determine the percentages of inhibition of the enzyme and perform the IC50 of each
extract
2. Materials and Methods
2.1. Collection and processing of plant material.
The species were collected, georeferenced, according to tables 1 and 2, the individuals were coded and assigned a
correlative code, and they were stored in paper bags and coded for their taxonomic identification. The exsiccates were
deposited in the Herbarium Amazonenese of the National University of the Peruvian Amazon. The processing was
carried out in the production area of the Institute of Traditional Medicine of EsSalud-Iquitos, where they were
washed with a solution with sodium hypochlorite at 6%, to then dry in an oven at 40°C.
2.2. Preparation of the lyophilized aqueous extract.
Each of the eigth plant species was processed as follows: 50 mg of the sample obtained after grinding the
plant was weighed; 500 mL of distilled water was added. This mixture was heated on an electric grill with constant
stirring. The temperature was maintained between 60 and 70°C for three hours. Subsequently, it was left to cool and
filtered with Whatman paper, and then concentrated with a rotavapor at 45°C and 40 rpm. Finally, the concentrated
solution was frozen at -10°C and then transferred to the Labotec ® lyophilizer. Subsequently, with this products of
each of the species, were prepared solutions at concentrations of 1000 μg/mL, 500 μg/mL and 100 μg/mL.
Table 1. Location of vegetable species
Code Family Scientific name Common name in Part used
Peru (Spanish)
01GLC Apocynaceae Aspidosperma excelsumBenth. Remo caspi Bark
02GLC Bignoniaceae Handroanthus obscurus (Bureau & K.Schum.) Tahuari negro Bark
Mattos
03GLC Clusiaceae Garcinia macrophylla Mart. Charichuelogrande Bark
04GLC Fabaceae Bauhinia glabra Jacq. Pata de vaca Leaves
05GLC Menispermaceae Abuta grandifolia (Mart.) Sandwith Abuta Bark
06GLC Moraceae Coussapoa asperifoliaTrécul Renacocaspi Leaves
07GLC Rubiaceae Calycophyllum spruceanum (Benth.) Hook. f. ex K. Capirona Bark
Schum.
08GLC Rubiaceae Remijia pedunculata (H. Karst.) Flueck Chullachaquicaspi Bark

Table 2. Code of plant species and collection coordinates.

Códe UTM Coordinates Location
01GLC 18M 649887 9458507 Jenaro Herrera – Requena-Loreto
02GLC 18M 680171 9575359 Arboretum “El Huayo”-Iquitos-Loreto
03GLC 18M 649586 9458325 Jenaro Herrera-Requena-Loreto
04GLC 18M 691874 9583730 Institute of Traditional Medicine of EsSalud-Iquitos - Loreto
05GLC 18M 706377 9559428 Tamshiyacu-Loreto
06GLC 18M 649718 9458368 Jenaro Herrera-Requena-Loreto
07GLC 18M 703277 9556622 Tamshiyacu - Loreto
08GLC 18M 650070 9458459 Jenaro Herrera – Requena - Loreto
10 Journal of Natural Sciences, Vol. 6(1&2), December 2018

1.1. Antihyperglycemic activity: inhibition of the enzyme α-glucosidase.

The inhibition assay of the α-glucosidase enzyme was developed according to the method proposed by
Artanti et al., (2012) and Srianta et al., (2012)) where 100 μL of the sample at various concentrations (1000, 500 and
100 μg/mL) was added to a 10 mL test tube containing 50 μL of 0.1M pNGP (p-Nitrophenyl-D-glucopyranoside),
added 600 μL of phosphate buffer pH 6.5, and incubated for 5 minutes at 37°C. The reaction was started by adding
250 μL of enzyme solution (0.24 U / mL); then, it was incubated for 15 minutes at 37°C. The reaction was stopped by
the addition of 1000 μL of the 0.2 M Na2CO3 solution and the absorbance of p-nitrophenol resulting from pNGP at
400 nm was measured with a spectrophotometer. Acarbose was used as a positive control (from 1000 μg dissolved in
1000 μL millipore water). The percentage of inhibition of the enzyme α-glucosidase was calculated by the following
equation:
% Inh = {1- (Abs with enz - White Abs without enz)/ (White Abs with enz - White Abs without enz)} * 100
Where:
% Inh : Percentage of inhibition of the compound
Abs m with enz. : Absorbance of the sample with the enzyme
Abs White without: Absorbance white without enzyme
White Abs with enz. : White absorbance with enzyme
The percentage values of α-glucosidase inhibition were used to calculate the IC50 of the extracts. The IC50
(corresponds to the amount of extract that inhibits 50% of the α-glucosidase enzyme activity) was determined using
the Probit analysis, the Results were measured ten times and expressed in μg/mL
2. Results and Discussion
Table 3 shows the percentages of inhibition of the α-glucosidase enzyme for the eight species respectively. Of
the evaluated species, Coussapoa asperifolia, has a higher percentage of inhibition, exceeding widely the other species; the
three concentrations exceed 50% with values between 53.64 ± 3.65% and 96.15 ± 2.85% inhibition, the data obtained
postulate this species as promising and relevant, according to the Kruskal- Wallis values did not present statistical
significance, and R2 shows a positive logarithmic trend between the percentage of inhibition and the concentrations
evaluated with a value of 0.7977.
Table. 3. Percentage de inhibition of the eight vegetable species
Inhibition Percent (%)
Code Specie
1000μg/mL 500μg/mL 100μg/mL
01GLC Aspidosperma excelsum Benth. 4.4 13.51 5.94
02GLC Handroanthus obscurus (Bureau &K.Schum.) Mattos 59.35 21.61 5.48
04GLC Garcinia macrophylla Mart. 7.81 1.99 0.94
04GLC Bauhinia glabra Jacq. 79.57 21.19 6.59
05GLC Abuta grandifolia (Mart.) Sandwith 10.87 6.58 2.58
06GLC Coussapoa asperifolia Trécul 90.74 97.44 69.16
07GLC Calycophyllum spruceanum(Benth.) Hook. f. ex K. 24.56 1.57 0.0
Schum.
08GLC Remijia pedunculata (H. Karst.) Flueck 58.28 40.69 17.68
Consequently, the species Bahuinia glabra, Remijia pedunculata and Handroanthus obscurus obtained values above
50% inhibition at the concentration of 1000 μg/mL with 72.74 ± 2.72, 59.95 ± 3.22 and 50.76 ± 3 , 73 respectively;
according to the Kruskal-Wallis test, the values did not show statistical significance, even though they are considered
of pharmacological importance for future trials in the evaluation of other models and mechanisms of the study of
antidiabetics of natural origin. The data of the extracts of the species Abuta grandifolia, Aspidosperma excelsum,
Calycophyllum spruceanum and Garcinia macrophylla, are not presented since they did not show relevant inhibitory activity.
The experimental evidence shows a great similarity in terms of the results obtained for the Mexican species
Oreocallis grandiflora, under conditions similar to those of the present study (Espinosa, 2013), the results of the
percentage of inhibition are shown with a maximum of 99.6 ± 0.3%, and an IC50 of 3.0 μg/mL at a concentration of
2000 μg/mL, in relation to Coussapoa asperifolia, both evaluated species show to have a great activity in theory
inhibitory against α-glucosidase.
Vasquez et al. 11

This is because in both assays the inhibition of the enzyme exceeds the IC50widely; this indicates that the
natural compounds of both species have a particular chemical composition. The genera Bahuinia and Remijia are a
group of plants classified as of great importance in the traditional medicine of the Amazonian peoples (Huamantupa
et al., 2011; IIAP, 2010; Rodriguez & Tuesta (2010), they are attributed antidiuretic properties, action on the
absorption of cholesterol and antidiabetic activity. This information is corroborated by ethnobotanical studies in
which plants of these genera are referred to as groups widely used in the traditional treatment of type II DM in Brazil
(Santos et al., 2012)..
Consequently, there is evidence of similar pharmacological properties between species within the same genus,
this product of the concentration of active substances of plants; likewise, these will depend on external conditions and
factors such as climate, age of the plant, time of collection, type of soil, etc. (Velasquez & Posada, 2013). The results
obtained in the present investigation corroborate the ethnobotanical information through the assays of the
antihyperglycemic activity of the species Bahuinia glabra and Remijia pedunculata, obtaining percentages of inhibition
above 50%.
Table 4 shows the calculation of the IC50 of the extracts of the evaluated plant species, showing the values
needed to inhibit 50% of the enzyme α-glucosidase under in vitro conditions. Values below 1000 μg / mL evidence of
high antihyperglycemic activity, in this sense the species Coussapoa asperifolia possesses a high inhibitory activity of the
enzyme α-glucosidase (32.1 μg/mL); Likewise, the species Remijia pedunculata (532.10 μg/mL), Bauhinia glabra (613.20
μg/mL), Handroanthus obscurus (810.50 μg/mL) and Calycophyllum spruceanum(989.14 μg/mL) show values that are in the
range acceptable antihyperglycemic activity. The species Aspidosperma excelsum, Abuta grandifolia and Garcinia macrophylla,
presented undetermined values of a low enzymatic inhibition, therefore the IC50 could not be calculated.
Table 4. Determination of the IC 50 of eight lyophilized aqueous extracts of Amazonian plant species
Concentration
Code IC50 µg/mL
100µg/mL 500µg/mL 1000µg/mL
01GLC 1.76 ± 3.94 9.06±2.62 2.59± 2.87 ND
02GLC -2.51± 3.09 14.26± 2.24 50.76±3.73 810.50
03GLC 0.7± 2.71 0.55± 1.79 7.7±1.54 ND
04GLC 3.49± 4.43 17.65± 8.21 72.74±2.72 613.20
05GLC 2.25±2.62 4.88±2.81 9.58±1.97 ND
06GLC 53.64± 3.65 68.15± 2.36 96.15±2.85 32.1
07GLC 0.15± 1.92 0.81± 2.09 26.5±3.43 989.14
08GLC 7.30± 3.45 41.97± 3.00 59.95±3.22 532.10
The results of inhibition of the species evaluated in the present investigation present wide ranges in the values
of IC50, this variation is caused by the probability that the chemical compounds of the leaves and barks of the species
studied work in synergism to exert the inhibitory effect, the conditions of the reaction medium and the concentration
of the inhibitor. However, these results give us an idea of the potentially useful species for the control of
hyperglycemia since they reduce the absorption of glucose; this due to the presence of chemical compounds that act
as competitive inhibitors of α-glucosidase in agreement with studies previous (Bhushan 2010; Artante et al., 2012;
Elya, 2012)
3. Conclusions
According to the results obtained in this study it can be concluded that the species Remijia pedunculata, Bahuinia
glabra, Handroanthus obscurus and Calycophyllum spruceanum reported values that are within the range of antihyperglycemic
activity in a model in vitro.

Acknowledgement
To CONCYTEC- CIENCIACTIVA program, for the financial support for this study, within the Project Circle of
Studies of Medicinal Plants from the Peruvian Amazon.
12 Journal of Natural Sciences, Vol. 6(1&2), December 2018

References
Artanti, Nv., Firmansyah, T & Darmawam, A. (2012). Bioactivities Evaluation of Indonesian Mistletoes (Dendrophthoe
pentandra (L.) Leaves Extracts. Journal of Applied Pharmaceutical Science,. 2, 24 - 27.
Avellandea, I. (2013).Evaluación de la actividad inhibitoria de la a-glucosidasa in vitro por extractos vegetales. s.l.
Universidad Tecnológica de Pereira. Colombia 126 p.
Bhushan MS, Rao, CHV, Ojha SK, Vijayakumar M, Verma A.(2010). An analytical review of plants for antidiabetic
activity with their phytoconstituent & mechanism of action. International Jounal of Pharmaceutical Sciences
Research,1, 29-46.
Corrales, I., Reyes, J. & Piña, R. (2014). Plantas medicinales de interés estomatológico. Órgano Científico Estudiantil de
Ciencias Médicas de Cuba, 53, 256, 79-98.
Elya, B., et al., (2012). Screening of a-glucosidase inhibitori activity from some plants of Apocynaceae, Clusiaceae,
Euphorbiaceae and Rubiaceae. Journal of Biomedicine and Biotechnolog,. ID 2810780, 6p.
Espinosa, A. (2013). Actividad Antioxidante Y Antihiperglicemiante De La Especie Medicinal Oreocallis grandiflora
(Lam.) Br. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas. 12, 59 - 68.
Federación Internacional de la Diabetes. (2013).Atlas de la Diabetes de la FID. Editorial Brussels, 6th Edition, 160p.
Gnecco, F. (1998).La diabetes en la Práctica. Bogota, Colombia : Cromos, 3,, 69-75.
Gomez, R. (2012). Plantas medicinales en una aldea del estado de Tabasco, Mexico. s.l. Revista Fitotécnica de México,
2012. Vol. 35, 1, 43-49.
Huamantuppa, I., et al., (2011).Riqueza, uso y origen de plantas medicinales. Revista Peruana de Biología, 18, 283-291.
Instituto De Investigaciones de la Amazonía Peruana (IIAP) (2010).Base de datos de plantas medicinales. Programa
de la Biodiversidad. Iquitos-Perú, 72p.
Lecca, J. & Rojas, J. (2011). Efecto Hipoglicemiante del extracto acuoso liofilizado de Abuta rufescens A., en ratas con
diabetes mellitus tipo 2, inducidas con estreptozotocin. IMET-ESSALUD,2011.Thesis. Universidad Nacional
de la Amazonía Peruana. Iquitos-Peru, 137p.
Rodríguez, C. & Tuesta, V. (2010). Efecto del extracto acuoso liofilizado de Abuta rufescens y Notholaena nivea sobre la
hiperglicemia inducida en ratas IMET-ESSALU.:Thesis. Universidad Nacional de la Amazonía Peruana.
Iquitos, 144p..
Santos, M., Nunes, M. & Martins, R. (2012). Uso empirico de plantas medicinales para el tratamiento de diabetes. Revista Brasilera
de Plantas Medicinales,12, 327 - 334.
Srianta, I., Kusumawati, N., Nugerahani, I., Artanti, N. & Xu, G.R.(2012).In vitro a-glucosidase inhibitory activity of
Monascus-fermented durian seed extracts. International Food Research Journal,20, 533-536.
Tananta, L. (2014). Análisis del conocimiento tradicional del uso de especies vegetales en tres comunidades de la
cuenca baja del río Ucayali, Loreto-Perú. Universidad Nacional de la Amazonía Peruana, -Iquitos-Peru,
Thesis, 92p.
Velasquez, S. & Posada, V. (2013). Actividad Anti-inflamatoria in vitro de los extractos y fracciones obtenidas de la
corteza interna de Tabebuia chrysantha (JACQ.) G. Nicholson. Universidad Tecnológica de Pereira, Thesis, 93p.