RESUMEN

Objetivo:

Comparar el efecto hipoglicemiante del extracto acuoso de Moringa oleifera (moringa), Smallanthus sonchifolius (yacón) y metformina en Rattus norvegicus, variedad albina, con diabetes mellitus inducida.

Materiales y métodos:

Estudio preclínico, experimental controlado y aleatorizado. La diabetes se indujo por vía intraperitoneal con una dosis de aloxano a 130 mg/kg de peso vivo (PV); se emplearon 24 Rattus norvegicus, variedad albina, machos, cepa Holfzman (seis por grupo). Se dividieron de la siguiente manera: grupo control (sin tratamiento), grupo metformina (14 mg/kg PV), grupo M. oleifera (200 mg/kg PV), y grupo S. sonchifolius (140 mg/kg PV), los tratamientos fueron administrados mediante sonda orogástrica durante 15 días. Los niveles de glicemia fueron determinados usando un glucómetro electrónico Accu-Chek® Instant (Roche).

Resultados:

Se observó reducción de la glicemia en los tratamientos: M. oleifera (p = 0,009), S. sonchifolius (p = 0,002) y metfotmina (p = 0,002), en 313 mg/dL, 281,5 mg/dL y 415 mg/dL, respectivamente. En cuanto a la comparación de la glicemia en los grupos tratados y control, se observó que a las 24 horas y cuatro días de tratamiento no hubo diferencia (p > 0,05); mientras que al octavo (p < 0,05) y décimo quinto día (p < 0,01) los grupos tratados tuvieron menor glicemia respecto al control, pero similares entre ellos.

Conclusión:

El extracto acuoso de S. sonchifolius y de M. oleifera, y la metformina presentaron similar efecto hipoglicemiante en ratas de experimentación con diabetes inducida.

Palabras clave:
Hipoglicemiante; Diabetes Mellitus; Moringa oleífera; Ratas; Aloxano

ABSTRACT

Objective:

To compare the hypoglycemic effect of the aqueous extract of Moringa oleifera (moringa), Smallanthus sonchifolius (yacon) and metformin on Rattus norvegicus, albino variety, with induced diabetes mellitus.

Materials and methods:

Preclinical, experimental, controlled and randomized study. Diabetes was induced intraperitoneally with a dose of alloxan at 130 mg/kg. A total of 24 male Rattus norvegicus, albino variety, Holfzman strain (6 per group) were used. They were divided as follows: control group (no treatment), metformin group (14 mg/kg), M. oleifera group (200 mg/kg), and S. sonchifolius group (140 mg/kg), treatments were administered via orogastric tube for 15 days. Glycemia levels were determined using an Accu-Chek® Instant electronic glycometer (Roche).

Results:

Decreased glycemia was observed in the treatment groups: M. oleifera (p = 0.009), S. sonchifolius (p = 0.002) and metformin (p = 0.002), by 313 mg/dL, 281.5 mg/dL and 415 mg/dL, respectively. When comparing glycemia in the treated and control groups, no difference was observed (P > 0.05) at 24 hours and four days of treatment; while at the eighth (P < 0.05) and fifteenth day (P < 0.01) the treated groups had lower glycemia than the control group, but it was similar among them.

Conclusion:

The aqueous extract of S. sonchifolius, M. oleifera, and metformin presented similar hypoglycemic effect in experimental rats with induced diabetes.

Keywords:
Hypoglycemic Agents; Diabetes Mellitus; Moringa oleifera; Rats; Alloxan

INTRODUCTION

The World Health Organization states that the number of people with diabetes mellitus (DM) worldwide has increased from 108 million in 1980 to 422 million in 2014 and will be the seventh leading cause of death by 2030 ¹1. Organización Mundial de la Salud. Diabetes [Internet]. OMS; 2016. [citado el 25 de marzo de 2018]. Disponible en: http://www.who.int/mediacentre/factsheets/fs312/es/.
http://www.who.int/mediacentre/factsheet... 11. Inocente-Camones M, Guija-Poma E, Zarzosa-Norabuena E, Loja Herrera B, Ponce-Pardo J. Efecto hipoglicemiante de los extractos acuoso y etanólico de Psidium guajava L. (Guayaba) en ratas diabéticas inducidas por aloxano. Horiz Med. 2015;15(2):41-48.^,22. Organización Mundial de la Salud. Día Mundial de la Salud 2016: diabetes [Internet]. OMS; 2016. [citado el 25 de marzo de 2018]. Disponible en: http://www.who.int/campaigns/world-health-day/2016/event/es/.
http://www.who.int/campaigns/world-healt... . Type 2 diabetes mellitus (DM 2) is a growing problem, 371 million adults live with this type of diabetes in the world, of which 26 million (7%) reside in Latin America ³3. Asociación Latinoamericana de Diabetes. Guías ALAD sobre el diagnóstico, control y tratamiento de la Diabetes Mellitus Tipo 2 con medicina basada en evidencia. 2013. Revista de la Asociación Latinoamericana de la Salud [Internet]. ALAD; 2013 [citado el 30 de marzo de 2018]. Disponible en: https://issuu.com/alad-diabetes/docs/guias_alad_2013.
https://issuu.com/alad-diabetes/docs/gui... . Peru’s National Institute of Statistics and Informatics (INEI) reported that nationally, 3.9% of the population aged 15 years and older was diagnosed with DM in 2019 ⁴4. Instituto Nacional de Estadística e Informática del Perú. Perú: Enfermedades no transmisibles y transmisibles, 2019 [Internet]. INEI; 2020 [citado el 10 de junio de 2020]; p. 33. Disponible en: https://proyectos.inei.gob.pe/endes/2019/SALUD/ENFERMEDADES_ENDES_2019.pdf.
https://proyectos.inei.gob.pe/endes/2019... . On the Peruvian coast, a larger population with diabetes was reported (4%) and a higher incidence in urban areas ⁵5. Instituto Nacional de Estadística e Informática del Perú. En el Perú 3 de cada 100 personas de 15 y más años reportan tener diabetes [Internet]. INEI; 2016 [citado 29 de marzo de 2018]. Disponible en: https://www.inei.gob.pe/prensa/noticias/en-el-peru-3-de-cada-100-personas-de-15-y-mas-anos-reportan-tener-diabetes-8993/.
https://www.inei.gob.pe/prensa/noticias/... .

DM is a chronic degenerative disease, its most frequent chronic complications are nephropathy, neuropathy, retinopathy, ketoacidosis, and cardiovascular disease ⁶6. Omodanisi E, Aboua Y, Oguntibeju O. Assessment of the Anti-Hyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats. Molecules. 2017;22(4):439. doi: 10.3390/molecules22040439.
https://doi.org/10.3390/molecules2204043... . Treatment for DM 2 is multifactorial and personalized, and it’s based on nutrition, physical activity, and medications, with 47.8% reported adherence ⁷7. Guzmán-Gómez G, Arce A, Saavedra H, Rojas M, Solarte J, Mina M, et al. Adherencia al tratamiento farmacológico y control glucémico en pacientes adultos con diabetes mellitus tipo 2. Alad. 2018;8(1):35-43. doi: 10.24875/ALAD.18000319.
https://doi.org/10.24875/ALAD.18000319... . International guidelines recommend metformin as the first line pharmacological treatment ⁸8. Association American Diabetes. Pharmacologic approaches to glycemic treatment: standards of medical care in diabetes-2018. Diabetes Care. 2018;41(1):S73-85. doi: 10.2337/dc18-S008.
https://doi.org/10.2337/dc18-S008... .

Medicinal plants (MP) constitute a viable therapeutic alternative due to their low cost and easy availability for many populations. More than 400 MP with great phytochemical diversity are studied for their antidiabetic potential and less or no side effects, so it is important to scientifically validate the effectiveness and safety to ensure their use ⁹9. Russo D, Valentão P, Andrade PB, Fernandez EC, Milella L. Evaluation of antioxidant, antidiabetic and anticholinesterase activities of Smallanthus sonchifolius landraces and correlation with their phytochemical profiles. Int J Mol Sci. 2015;16(8):17696-718. doi: 10.3390/ijms160817696.
https://doi.org/10.3390/ijms160817696...

10. Shah M, Keach J, Panichayupakaranant P. Antidiabetic naphthoquinones and their plant resources in Thailand. Chem Pharm Bull. 2018;66(5):483-92. doi: 10.1248/cpb.c17-00529.
https://doi.org/10.1248/cpb.c17-00529...

11. Inocente-Camones M, Guija-Poma E, Zarzosa-Norabuena E, Loja Herrera B, Ponce-Pardo J. Efecto hipoglicemiante de los extractos acuoso y etanólico de Psidium guajava L. (Guayaba) en ratas diabéticas inducidas por aloxano. Horiz Med. 2015;15(2):41-48.

12. Castañeda B, Castro de la Mata R, Manrique R, Ibánez L, Fujita R, Barnett, et al. Estudio fitoquímico y farmacológico de 4 plantas con efecto hipoglicemiante. Horiz Med. 2008;8(1):6-34.^-1313. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... . Moringa oleifera (moringa) and Smallanthus sonchifolius (yacon) are easily accessible and low-cost MP in our environment.

Moringa oleifera is a tree native to the southern Himalayas ¹⁴14. Chávez J, Carvallo T, González A. Identificación de compuestos agonistas a receptores involucrados en el síndrome metabólico de un extracto de hoja de Moringa oleifera. Centro de Investigación en Alimentación y Desarrollo. 2016;1(1):6. and part of the Moringaceae family ¹⁵15. Padilla F, Cruz J. Extractos de hojas de Moringa oleífera en la prevención y tratamiento de la diabetes mellitus. Revista Cubana de Medicina Natural y Tradicional. 2018;2(1).. In addition to proteins, minerals and vitamins, the leaves of M. oleifera contain phytochemicals, such as flavonoids, phenolic acids, alkaloids and carotenoids, isothiocyanates, glucosinolates and tannins, saponins, oxalates, and phytates ¹³13. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... . Possible compounds with hypoglycemic and antioxidant effect have been identified in previous phytochemical studies ⁶6. Omodanisi E, Aboua Y, Oguntibeju O. Assessment of the Anti-Hyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats. Molecules. 2017;22(4):439. doi: 10.3390/molecules22040439.
https://doi.org/10.3390/molecules2204043... , which act by different mechanisms, including inhibition of α-amylase and α-glucosidase activities, increased glucose uptake in muscle and liver, inhibition of glucose uptake from the gut, decreased gluconeogenesis in the liver, and increased insulin secretion and sensitivity ¹³13. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... . In addition, toxicity studies in experimental animals have shown that aqueous and alcoholic extracts of M. oleifera do not have adverse effects ¹³13. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... . M. oleifera protects tissues from oxidative stress ¹⁶16. Yassa H, Tohamy A. Extract of Moringa oleifera leaves ameliorates streptozotocin-induced Diabetes mellitus in adult rats. Acta Histochemica. 2014;116(5):844-54. doi: 10.1016/j.acthis.2014.02.002.
https://doi.org/10.1016/j.acthis.2014.02... , reduces free radical activity, lipid peroxidation, and prevents the development of chronic complications ⁶6. Omodanisi E, Aboua Y, Oguntibeju O. Assessment of the Anti-Hyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats. Molecules. 2017;22(4):439. doi: 10.3390/molecules22040439.
https://doi.org/10.3390/molecules2204043... .

S. sonchifolius belongs to the family Asteraceae, native to the Andean valleys of South America, cultivated at 2,000 - 3,100 m above sea level and reaches maturity between 6 and 12 months after sowing ¹⁷17. de Almeida Paula HA, Abranches MV, de Luces Fortes Ferreira CL. Yacon (Smallanthus sonchifolius): a food with multiple functions. Crit Rev Food Sci Nutr. 2015;55(1):32-40. doi: 10.1080/10408398.2011.645259.
https://doi.org/10.1080/10408398.2011.64... . The phytochemical composition of S. sonchifolius has been previously described and has revealed high concentrations of fats and oils, phenols and tannins, alkaloids, lactones, flavonoids and anthocyanidins ¹²12. Castañeda B, Castro de la Mata R, Manrique R, Ibánez L, Fujita R, Barnett, et al. Estudio fitoquímico y farmacológico de 4 plantas con efecto hipoglicemiante. Horiz Med. 2008;8(1):6-34.. Likewise, its high safety has been reported in acute toxicity tests in experimental models, as of its atomized extracts and its alkaloids ¹²12. Castañeda B, Castro de la Mata R, Manrique R, Ibánez L, Fujita R, Barnett, et al. Estudio fitoquímico y farmacológico de 4 plantas con efecto hipoglicemiante. Horiz Med. 2008;8(1):6-34.. S. sonchilfolius has hypoglycemic effects, induces insulin release, and increases its concentration in plasma in diabetic and normal rats ¹⁸18. Gordillo G, Negrón L, Zúñiga T, Flores E, Moreyra R, Fuertes C, et al. The hypoglycemic effect of aqueous extract of leaves of Smallanthus sonchifolius (yacon) in patients with Type 2 Diabetes Mellitus. Ciencia e Investigación. 2014;15(1):42-7.. This effect has been confirmed mainly by caffeic and chlorogenic acids together with 3 isomeric dicaffeoylquinic acids, which could satisfactorily contribute to an inhibition of α-glucosidase in the brush border of the cells from the small intestine. Decoction of yacon leaves has shown in vitro and in vivo to prolong glucose absorption time, delaying rapid digestion of sucrose; an isolated component of yacon, lactone sesquiterpene, significantly decreases postprandial glycemia levels in diabetic rats ¹⁹19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... .

The hypoglycemic effect of these MP has been demonstrated in vitro¹²12. Castañeda B, Castro de la Mata R, Manrique R, Ibánez L, Fujita R, Barnett, et al. Estudio fitoquímico y farmacológico de 4 plantas con efecto hipoglicemiante. Horiz Med. 2008;8(1):6-34.^,1313. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... ^,1818. Gordillo G, Negrón L, Zúñiga T, Flores E, Moreyra R, Fuertes C, et al. The hypoglycemic effect of aqueous extract of leaves of Smallanthus sonchifolius (yacon) in patients with Type 2 Diabetes Mellitus. Ciencia e Investigación. 2014;15(1):42-7.^,1919. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... ; however, it is not known which of the two plants has the greater effect in vivo, which would allow considering them as an alternative treatment for DM, after the subsequent and corresponding clinical trials. The aim of this study was to compare the hypoglycemic effect of the aqueous extract of M. oleifera, that of S. sonchifolius, and metformin in R. norvegicus with induced diabetes mellitus.

MATERIALS AND METHODS

Study design and experimental animals

A randomized controlled experimental study was conducted with male albino rats (Rattus norvegicus, albino variety) that developed alloxan-induced diabetes mellitus. All those that did not raise their glucose levels (at least 250 mg/dL) and those that presented some pathology were excluded.

Twenty-four male rats of the Holfzman strain were selected, approximately 12-14 weeks old and weighing 180 ± 20 g. The animals were acquired from the biotherium of the Lambayeque Regional Hospital, where they had 10 days of adaptation with balanced food from the Universidad Nacional Agraria La Molina (UNALM). They received growth diet with the following nutritional value: 10% water, 13-14% proteins, 3-4% lipids, and 0.5% calcium; and according to the manufacturer’s brochure, the food was elaborated with the following ingredients: yellow corn, soybean 48% cake, cotton pulp, sunflower cake, agro-industrial by-products, alfalfa hay, calcium carbonate, dicalcium phosphate, synthetic amino acids, organic promoters, vitamins, minerals, sodium chloride, antifungal, and antioxidants.

Procedure

Harvesting and drying of plant materials

The moringa leaves were collected from the Yurimaguas farmhouse, district of Jayanca, province of Lambayeque, approximately two months after planting. Yacon leaves were collected from the village of Montegrande Bajo, district of Huarmaca, province of Huancabamba, Piura, approximately seven months after planting. The samples were deposited in the Lambayeque PRG Herbarium, of the Botany Department of the Universidad Nacional Pedro Ruiz Gallo (UNPRG) and identified by a qualified specialist.

The moringa and yacon leaves were washed and placed in a hot-air tray dryer for two hours at 56 °C, leaf stems were separated, and only the leaves were crushed using a mortar (each plant separately). The crushed leaves of each plant were stored in separate airtight containers, insulated from light and moisture.

Treatment preparation

Preparation of the aqueous extract of M. oleifera and S. sonchifolius at 100 mg/mL

At the Research Laboratory of the Lambayeque Regional Hospital (HRL), 10 g of moringa and yacon were weighed with an analytical scale; then, in a biosafety cabin, they were mixed with 100 mL of sterile distilled water, placed in 15 mL sterile tubes, and then taken to a bain-marie for 30 minutes at 90 °C. Subsequently, they were centrifuged at 3,500 RPM for ten minutes. In the biosafety cabin, the supernatant was extracted with the help of a 10 mL sterile syringe which constituted the aqueous extract. Aliquots were prepared and stored at 2-8 °C until use over the next four days.

Preparation of metformin at 10 mg/mL

At the Research Laboratory, 1 g of metformin (Sigma, USA) was weighed and diluted in 100 mL of distilled water ²⁰20. Barreto S, Báez S, Malvetti V, Cardozo M, Gill A, Matto J, et al. Moringa oleifera acute effect on dexamethasone-induced hyperglycemia in Wistar rats. An Fac Cienc Méd. 2016;48(1):41-8. doi: 10.18004/anales/2015.048(01)41-048.
https://doi.org/10.18004/anales/2015.048... , homogenized, and stored at 2-8 °C until being used, in the next four days.

Diabetes mellitus induction in Rattus norvegicus albino strain Holfzman

Experimental diabetes was induced with a single alloxan dose (Sigma, St. Louis, MI, USA) of 130 mg/kg liveweight (LW) via the intraperitoneal route. Glycemia levels were measured at 72 hours ²¹21. Rodriguez J, Soplapuco C. Efecto del Smallanthus sonchifolius sobre los niveles de glucosa e insulina plasmática en Oryctalagus cuniculis con diabetes inducida con alloxano [Tesis para optar el título de Médico Cirujano]. Lambayeque: Universidad Nacional Pedro Ruiz Gallo; 2004. and those with a level greater than 250 mg/dL were considered diabetic²²22. Justil C, Angulo P, Justil H, Arroyo J. Evaluación de la actividad hipoglicemiante del extracto acuoso de Abuta grandifolia (Mart.) en ratas con diabetes inducida por aloxano. Rev Inv Vet Perú. 2015;26(2):206-12. doi: 10.15381/rivep.v26i2.11008.
https://doi.org/10.15381/rivep.v26i2.110... .

Study group distribution

The diabetic rats were distributed in four groups of six rats each. Each rat was numbered with an indelible marker and randomly assigned with the Epidat 4.1 program. The groups were labeled as follows: control group, which did not receive treatment, and drank water at will; metformin group, which received 14 mg/kg LW, standard treatment of DM 2 ²⁰20. Barreto S, Báez S, Malvetti V, Cardozo M, Gill A, Matto J, et al. Moringa oleifera acute effect on dexamethasone-induced hyperglycemia in Wistar rats. An Fac Cienc Méd. 2016;48(1):41-8. doi: 10.18004/anales/2015.048(01)41-048.
https://doi.org/10.18004/anales/2015.048... ; M. oleifera group, which received 200 mg/Kg LW of the aqueous extract of M. oleifera¹⁶16. Yassa H, Tohamy A. Extract of Moringa oleifera leaves ameliorates streptozotocin-induced Diabetes mellitus in adult rats. Acta Histochemica. 2014;116(5):844-54. doi: 10.1016/j.acthis.2014.02.002.
https://doi.org/10.1016/j.acthis.2014.02... ; and S. sonchifolius group, which received 140 mg/Kg LW of the aqueous extract of S. sonchifolius¹⁹19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... .

The evaluation was carried out during 15 consecutive days, the duration of the experiment. Glycemia levels were measured at 7 hours. Likewise, the substances with hypoglycemic effect were administered at 8 hours, through a disposable orogastric tube (orogastric route), except for the control group which did not receive treatment.

All experimental animals were given a 14 g ration of balanced rodent food once a day after the corresponding treatment. The day before the glycemia measurement, the food was placed in the cages at 10 a.m. and 7 p.m., then removed for the 12-hour fast with water ad libitum.

Measuring glycemia in rats

The blood glucose concentration was measured with the Accu-Chek Instant Glucose Meter (Roche). During the experiment, blood glucose was monitored on the first, fourth, eighth, and fifteenth post-treatment days. To carry out this procedure, the specimens remained in fasting for approximately 12 hours, the blood sample was collected by puncture in the apex of the tails, after antisepsis of the area with 70% alcohol, discarding the first drop and receiving the next one on the test strip. The values obtained were expressed in milligrams per deciliter (mg/dL).

According to the manufacturer’s report, the Accu-Chek® Instant system (meter and strips) is factory-calibrated from diabetics’ capillary blood (comparison of methods and accuracy), venous blood (repeatability) and control solution (reproducibility). In addition, at the start and halfway through the evaluation of rats, measurements were made with the control solution administered by the manufacturer, which contained a glucose concentration of 100 mg/dL. The equipment complies with the requirements established by the ISO 15197:2013 and ISO 15197:2015 standards for in vitro diagnostic test systems, requirements of self-diagnostic systems for blood glucose monitoring in the management of DM ²³23. ISO 15197:2013 In vitro diagnostic test systems - Requirements for blood-glucose monitoring systems for self-testing in managing diabetes mellitus [Internet]. ISO; 2013 [citado 6 de junio de 2018]. Disponible en: https://www.iso.org/standard/54976.html.
https://www.iso.org/standard/54976.html... .

Statistical analysis

The data was coded and registered in Microsoft Excel 2016, and processed with IBM SPSS 24 (IBM Corp., Armonk, N.Y., USA). After the analysis of the distribution curve and the result of the Shapiro-Wilks normality test, a descriptive analysis of the glycemia in all treatments and measurements was performed, by calculating medians and interquartile ranges.

The comparative analysis of the hypoglycemic effect of the extracts of M. oleifera, S. sonchifolius, and metformin, was carried out through the non-parametric tests of Kruskall Wallis and Dunn test (multiple comparisons). Likewise, the Wilcoxon non-parametric test was used to measure the reduction of pre-treatment glycemia with respect to the fifteenth day of treatment. A value of p < 0.05 was considered significant.

Ethical aspects

Research was conducted in accordance with the following principles: reducing the number of experimental animals, replacing experimental animals with other methods, and refining techniques to lessen suffering. The protocol was reviewed and approved by the Committee on Ethics in Research for Animal Use (CEIPUA), of the HRL. Also, the principles and aspects related to the care and use of laboratory animals detailed in Directive 2010/63/EU of the European Parliament and Council, regarding the protection of animals used for scientific purposes, and the National Law for the Protection of Animals in Captivity ²⁴24. Unión Europea. Directiva 2010/63/UE del Parlamento Europeo y del Consejo, de 22 de septiembre de 2010, relativa a la protección de los animales utilizados para fines científicos [Internet]. UE; 2010. Disponible en: http://www.madrid.org/rlma_web/html/web/FichaNormativa.icm?ID=1942.
http://www.madrid.org/rlma_web/html/web/... were respected. At the end of the experiment the rats were euthanized with sodium pentobarbital at a dose of 100 mg/kg LW.

RESULTS

The data had a non-normal distribution. Table 1 shows medians and interquartile ranges of glycemia of rats with induced diabetes mellitus, distributed according to experimental groups (control and treatments with M. oleifera, S. sonchifolius, and metformin).

A glycemia reduction of 415 mg/dL, 313 mg/dL and 281.5 mg/dL was observed in treatments with metformin, M. oleifera and S. sonchifolius, respectively (Table 2).

Figure 1 shows the trend in the experimental groups regarding glycemia levels, where it can be seen that as time passes, glycemia in the treated groups decreases when compared to the control group, which did not receive treatment.

When comparing glycemia levels in the treated and control groups, it was observed that at 24 hours and at four days of treatment there was no significant difference (p > 0.05); while at the eighth and fifteenth day, the treatment groups had lower glycemia with respect to the control one, this difference was statistically significant (Table 3 and 4).

DISCUSSION

The aqueous extracts of S. sonchifolius and M. oleifera were found to have significant hypoglycemic effect, like metformin. In this regard, previous studies have described that the aqueous extracts of yacon and moringa leaves are frequently used in humans because of multiple health benefits, among which the reduction of postprandial glucose ¹⁸18. Gordillo G, Negrón L, Zúñiga T, Flores E, Moreyra R, Fuertes C, et al. The hypoglycemic effect of aqueous extract of leaves of Smallanthus sonchifolius (yacon) in patients with Type 2 Diabetes Mellitus. Ciencia e Investigación. 2014;15(1):42-7.

19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... ^-2020. Barreto S, Báez S, Malvetti V, Cardozo M, Gill A, Matto J, et al. Moringa oleifera acute effect on dexamethasone-induced hyperglycemia in Wistar rats. An Fac Cienc Méd. 2016;48(1):41-8. doi: 10.18004/anales/2015.048(01)41-048.
https://doi.org/10.18004/anales/2015.048... stands out.

Likewise, another study found that the decoction of yacon leaves contains phenolic compounds from caffeic, chlorogenic and dicaffeoylquinic acids, such as ferulic acid, p-coumaric acid, protocatechuic acid and quercetin, all of which contribute in the hypoglycemic effect inhibiting α-glucosidase, promoting glucose regulation ¹⁹19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... ^,2525. Dos Santos KC, Bueno BG, Pereira LF, Francisqueti FV, Braz MG, Bincoleto LF, et al. Yacon (Smallanthus sonchifolius) leaf extract attenuates hyperglycemia and kkeletal muscle oxidative stress and inflammation in diabetic rats. Evid Based Complement Alternat Med. 2017;2017(9):6418048. doi: 10.1155/2017/6418048.
https://doi.org/10.1155/2017/6418048... . Its hypoglycemic action is also attributed to the ability of binding to insulin receptors and to enhance the activity of the tyrosine kinase enzyme, whose purpose is to decrease glucose levels, as well as to protect against complications produced by diabetes ¹⁸18. Gordillo G, Negrón L, Zúñiga T, Flores E, Moreyra R, Fuertes C, et al. The hypoglycemic effect of aqueous extract of leaves of Smallanthus sonchifolius (yacon) in patients with Type 2 Diabetes Mellitus. Ciencia e Investigación. 2014;15(1):42-7..

The hypoglycemic effect of the aqueous yacon extract observed in our study was similar to the one reported by Dos Santos et al., near-normal decreases in glucose levels in rats treated with hydroethanolic extracts were obtained ²⁵25. Dos Santos KC, Bueno BG, Pereira LF, Francisqueti FV, Braz MG, Bincoleto LF, et al. Yacon (Smallanthus sonchifolius) leaf extract attenuates hyperglycemia and kkeletal muscle oxidative stress and inflammation in diabetic rats. Evid Based Complement Alternat Med. 2017;2017(9):6418048. doi: 10.1155/2017/6418048.
https://doi.org/10.1155/2017/6418048... . Likewise, Mejía et al. studied the hypoglycemic effect by using whole yacon root, obtaining a lower decrease in glucose levels (27.6 mg/dL in 34 days) ²⁶26. Mejía A, Zuloeta D, Palacios F. Efecto hipoglucemiante del consumo de yacón (Smallantus sonchifolius) en ratones diabéticos tipo 2 inducidos con aloxano. Revista Científica de Ciencias de la Salud. 2016;9(1):72-8. doi: 10.17162/rccs.v9i1.549.
https://doi.org/10.17162/rccs.v9i1.549... , compared to our study where we obtained a decrease of 281.5 mg/dL in 15 days.

Similarly, the group treated with M. oleifera significantly decreased glycemia levels compared to the control, this result could have been due to its high content of polyphenols and flavonoid compounds ⁶6. Omodanisi E, Aboua Y, Oguntibeju O. Assessment of the Anti-Hyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats. Molecules. 2017;22(4):439. doi: 10.3390/molecules22040439.
https://doi.org/10.3390/molecules2204043... ^,1313. Ahmad J, Khan I, Blundell R. Moringa oleifera and glycemic control: A review of current evidence and possible mechanisms. Phytotherapy research. 2019;33(11):2841-8. doi: 10.1002/ptr.6473.
https://doi.org/10.1002/ptr.6473... , glucosinolates and isothiocyanates ²⁷27. Fahey JW, Wade KL, Stephenson KK, Shi Y, Liu H, Panjwani AA, et al. A Strategy to deliver precise oral doses of the glucosinolates or isothiocyanates from Moringa oleifera leaves for use in clinical studies. Nutrients. 2019;11(7):1547. doi: 10.3390/nu11071547.
https://doi.org/10.3390/nu11071547... , terpenoids, quercetin and kaempferol²⁸28. Abd El Latif A, El Bialy BES, Mahboub HD, Abd Eldaim MA. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of ß cells and reduction of pyruvate carboxylase expression. Biochem Cell Biol. 2014;92(5):413-9. doi: 10.1139/bcb-2014-0081.
https://doi.org/10.1139/bcb-2014-0081... found in previous phytochemical studies. The compounds act as insulin secretagogues and contribute to attenuate diabetic complications ¹⁶16. Yassa H, Tohamy A. Extract of Moringa oleifera leaves ameliorates streptozotocin-induced Diabetes mellitus in adult rats. Acta Histochemica. 2014;116(5):844-54. doi: 10.1016/j.acthis.2014.02.002.
https://doi.org/10.1016/j.acthis.2014.02... ^,2828. Abd El Latif A, El Bialy BES, Mahboub HD, Abd Eldaim MA. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of ß cells and reduction of pyruvate carboxylase expression. Biochem Cell Biol. 2014;92(5):413-9. doi: 10.1139/bcb-2014-0081.
https://doi.org/10.1139/bcb-2014-0081... since, they improve the regeneration and viability of destroyed cells; another of their mechanisms is to reduce gluconeogenesis and glycogenolysis in the liver, which is attributed to the chlorogenic acid contained in M. oleifera leaves ²⁸28. Abd El Latif A, El Bialy BES, Mahboub HD, Abd Eldaim MA. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of ß cells and reduction of pyruvate carboxylase expression. Biochem Cell Biol. 2014;92(5):413-9. doi: 10.1139/bcb-2014-0081.
https://doi.org/10.1139/bcb-2014-0081... .

Our results are congruent with those obtained by previous studies where hyperglycemic experimental rats are treated with M. oleifera extract, resulting in a significant decrease in glycemia ⁶6. Omodanisi E, Aboua Y, Oguntibeju O. Assessment of the Anti-Hyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats. Molecules. 2017;22(4):439. doi: 10.3390/molecules22040439.
https://doi.org/10.3390/molecules2204043... ^,2020. Barreto S, Báez S, Malvetti V, Cardozo M, Gill A, Matto J, et al. Moringa oleifera acute effect on dexamethasone-induced hyperglycemia in Wistar rats. An Fac Cienc Méd. 2016;48(1):41-8. doi: 10.18004/anales/2015.048(01)41-048.
https://doi.org/10.18004/anales/2015.048... ^,2929. Idakwoji P, Barnabas A, Elah S. Co-administration of ethanolic leaf extract of Moringa oleifera and metformin reverses polyphagia, polydipsia and stabilizes body weight in alloxan-induced diabetic rats. Biokemistri. 2015;27(3):129-38..

The aqueous extracts of M. oleifera and S. sonchifolius had similar hypoglycemic effect without significant difference, probably because both plants contain similar components, such as polyphenols, which are attributed with hypoglycemic properties ¹⁹19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... ^,2525. Dos Santos KC, Bueno BG, Pereira LF, Francisqueti FV, Braz MG, Bincoleto LF, et al. Yacon (Smallanthus sonchifolius) leaf extract attenuates hyperglycemia and kkeletal muscle oxidative stress and inflammation in diabetic rats. Evid Based Complement Alternat Med. 2017;2017(9):6418048. doi: 10.1155/2017/6418048.
https://doi.org/10.1155/2017/6418048... ^,2727. Fahey JW, Wade KL, Stephenson KK, Shi Y, Liu H, Panjwani AA, et al. A Strategy to deliver precise oral doses of the glucosinolates or isothiocyanates from Moringa oleifera leaves for use in clinical studies. Nutrients. 2019;11(7):1547. doi: 10.3390/nu11071547.
https://doi.org/10.3390/nu11071547... ^,2828. Abd El Latif A, El Bialy BES, Mahboub HD, Abd Eldaim MA. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of ß cells and reduction of pyruvate carboxylase expression. Biochem Cell Biol. 2014;92(5):413-9. doi: 10.1139/bcb-2014-0081.
https://doi.org/10.1139/bcb-2014-0081... , although the mechanism of action has not yet been exactly established, and which seems to be at several levels.

Reduction of pre-treatment glycemia was observed when compared to the fifteenth day of treatment in the positive control (79.5%), treatments with M. oleifera (74%) and S. sonchifolius (70.7%). Previous studies report that groups treated with M. oleifera obtained similar values to the hypoglycemic effect produced by metformin ^(20.29). But there are no similar reports about yacon, and no studies were found comparing both MP.

The small sample size was a limitation for this study; however, this is usual in this kind of designs due to ethical and logistic aspects. Despite this, we consider that the results are valid¹⁹19. Serra-Barcellona C, Habib N, Honoré S, Sánchez S, Genta S. Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of a-Glucosidase Activity. Plant Foods Hum Nutr. 2017;72(2):156-60. doi: 10.1007/s11130-017-0600-y.
https://doi.org/10.1007/s11130-017-0600-... ^,2020. Barreto S, Báez S, Malvetti V, Cardozo M, Gill A, Matto J, et al. Moringa oleifera acute effect on dexamethasone-induced hyperglycemia in Wistar rats. An Fac Cienc Méd. 2016;48(1):41-8. doi: 10.18004/anales/2015.048(01)41-048.
https://doi.org/10.18004/anales/2015.048... ^,2929. Idakwoji P, Barnabas A, Elah S. Co-administration of ethanolic leaf extract of Moringa oleifera and metformin reverses polyphagia, polydipsia and stabilizes body weight in alloxan-induced diabetic rats. Biokemistri. 2015;27(3):129-38.^,3030. Habib N, Serra-Barcellona C, Honoré S, Genta S, Sánchez S. Yacon roots (Smallanthus sonchifolius) improve oxidative stress in diabetic rats. Pharm Biol. 2015;53(8):1183-93. doi: 10.3109/13880209.2014.970285.
https://doi.org/10.3109/13880209.2014.97... . Another limitation could be the loss of an experimental unit of the moringa group halfway through the experiment. This is a preclinical study that proves hypoglycemic effects in experimental animals, but research in humans that prove these benefits and measure other risks must be carried out. Likewise, for logistical reasons, aspects of the alloxan physiopathology and the way treatments act, as well as the characterization of phytochemicals in the extracts, were not studied.

It is concluded that the aqueous extract of Smallanthus sonchifolius at 140 mg/kg LW and of Moringa oleifera at 200 mg/kg LW, and metformin at 14 mg/kg LW did not present significant differences in their hypoglycemic effect on rats with alloxan-induced diabetes, after 8 and 15 days of treatment. For this reason, it is recommended to carry out complementary safety studies, where toxicity and cytotoxicity are evaluated, in vitro and in vivo. Then, studies could be carried out to determine if the hypoglycemic effect found in this study is similar in humans. It is also recommended to study interactions and synergies between treatments, as well as the physiopathology of their effects.

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