Cuban Journal of Agricultural Science, 50(2): 253-258, 2016, ISSN: 2079-3480

 

TECHNICAL NOTE

 

Application of an in vitro methodology for detecting active saponins in plant extracts

 

Aplicación de una metodología in vitro para la detección de saponinas activas en extractos vegetales

 

 

R. Rodríguez,^I Sarai Gómez,^I M. Fondevila,^II

^IInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
^IIUniversidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, España.

 

 

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ABSTRACT

In order to evaluate the saponins presence in plant extracts in methanol (50%), obtained from the tropical shrub legumes Enterolobium cyclocarpum (enterolobium), Acacia cornigera (acacia), Albizia lebbekoides (Albizia) and Leucaena leucocephala (leucaena), a methodology based on two in vitro assays was applied: foam test and blood agar test. In both a completely randomized experimental design was applied, with five extracts per treatment (legume) and three repetitions per extract. The average value of the three repetitions per extract of each legume was considered the experimental unit. The enterolobium extracts were positive to both tests, while the other three legumes were negative (P < 0.0001). With the use of this methodology, based on the combination of these two tests, the presence of active saponins in E. cyclocarpum extracts was confirmed. Moreover, it could state that in the extracts of acacia, albizia and leucaena there were not presence of these compounds or tannins able to hydrolyzing the membrane of red blood cells.

Key words: saponins, foam test, blood agar test, tropical legumes.

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RESUMEN

Para evaluar la presencia de saponinas en extractos vegetales en metanol (50 %), obtenidos a partir de las leguminosas arbustivas tropicales Enterolobium cyclocarpum (enterolobium), Acacia cornigera (acacia), Albizia lebbekoides (albizia) y Leucaena leucocephala (leucaena), se aplicó una metodología basada en dos ensayos in vitro: ensayo de la espuma y la prueba del agar sangre. En ambos se aplicó diseño experimental completamente aleatorizado, con cinco extractos por tratamiento (leguminosa) y tres repeticiones por extracto. El valor promedio de las tres repeticiones por extracto de cada leguminosa se consideró como la unidad experimental. Los extractos de enterolobium dieron positivo a ambas pruebas, mientras que los de las otras tres leguminosas mostraron resultados negativos (P < 0.0001). Con el empleo de esta metodología, basada en la combinación de estos dos ensayos, se confirmó la presencia de saponinas activas en extractos de E. cyclocarpum. Además, se pudo afirmar que en los extractos de acacia, albizia y leucaena no hubo presencia de estos compuestos o de taninos capaces de hidrolizar la membrana de los glóbulos rojos.

Palabras clave: saponinas, ensayo de espuma, ensayo agar sangre, leguminosas tropicales.

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INTRODUCTION

Saponins are terpenic and steroid derivatives which are in many of the plants that are used as food or as a natural source for obtaining additives for ruminant diets (Wina et al. 2005). Although the wide range of biological effects of these compounds is known, most can be attributed to more or less complex interactions with biological membranes which affect their principal properties, among them permeability (Francis et al. 2002). Therefore, these secondary metabolites can be used to manipulate and improve, naturally, the processes of digestion and absorption of nutrients in animals which intake them (Makkar et al. 1998, Hart et al. 2008).

To study the biological effect of saponins there is not a chemical compound that shows higher chemical affinity for and inactivates them, as happen with polyethylene glycol which is used to evaluate the biological effect of tannins (Makkar et al. 1995). Therefore, it can not study their particular effect on the integral plant and it is necessary to previously extract these metabolites before evaluating them (Rodríguez and Fondevila 2012). To confirm the effectiveness of the extraction process and the presence of active saponins a methodology which allowed characterizing the presence of these metabolites in the extract before their use in any scientific study is needed, for avoiding unnecessary loss of time and resources. To characterize the presence of these compounds in plant extracts may apply different quantitative analysis techniques or other cheaper and faster, as the combination of in vitro tests, which allow confirm the presence of these compounds, of qualitative and semi –quantitative ways . This work had as objective to evaluate the saponins presence by two in vitro tests in extracts obtained from the tropical shrub legumes Enterolobium cyclocarpum (enterolobium), Acacia cornigera  (acacia), Albizia lebbekoides (albizia) and Leucaena leucocephala (leucaena).

The four legume species were collected in November 2012. Leaves and petioles of plants from the arboretum of the Instituto de Ciencia Animal were taken. Legumes were completely established in typical red ferrallitic soil (Hernández et al. 2015), without irrigation and fertilization. It was took, approximately, 0.2 kg of fresh material of each example at a height of 1.5 meters, simulating the browsing of animals. The collected material was grouped by species, it was dried in forced air oven, with regulated temperature at 60 °C during 72 h. Then it was grounded in a hammer mill at a particle size of 0.5 mm and stored in sealed nylon bags until its experimental use in the Laboratorio de Nutrición de la Facultad de Veterinaria de la Universidad de Zaragoza, España.

The alcoholic extraction of secondary metabolites was performed according to the procedure described by Goel et al. (2008). In beakers 10 g of each legume were weighed and were added 200 mL of methanol (50%) as an extraction solvent. This process was repeated five times per legume. The plant substrate was soaked in the solvent during 48 h, at rest, in dark at room temperature. Then it was centrifuged at 3000 x g (C 4.11 Centrifuge, Jouan, Saint-Nazaire, Francia) for 15 min and the supernatant was collected. The solid residue was washed with 100 mL of the extraction solvent and recentrifuged under the same conditions. The supernatant was mixed with the obtained in the first centrifugation.

The obtained alcoholic extract was concentrated by solvent extraction by means of roto-evaporation at 80 °C, 100 rpm and vacuum (UNIVEBA-401, J.P. Selecta S.A., Barcelona, España). When the concentration of plant extracts process ends, the content of roto-evaporation flasks was washed with distilled water and transferred to a 250 mL volumetric flask. Finally, after ensuring the proper washing flasks, there were make up the volume to the volumetric flask. The plant extracts, free of methanol, were stored at 4 °C in the volumetric flask, in dark, hermetically sealed, up to 72 h, to prevent their degradation or contamination.

The obtained plant extracts were characterized by a methodology based on the application of two in vitro assays, one qualitative and other semi-quantitative, known as foam test and blood agar test, respectively.

The foam test was carried out according to the procedure proposed by Rossi et al. (2007). This test is based on when shaking the aqueous solution of samples which contains saponins, stable foam is form as the obtained by shaking the aqueous solution of a soap. To it, 1mL of each extract was placed in a test tube and was added 9 mL of distilled water. Then the solution was filtered, an aliquot of 1 mL was taken and transferred to test tube of 5 mL, in all cases in triplicate. The test tubes were shaken single and vigorously in a tube shaker (Top mixer AT-1, SBS Instruments, S.A., Barcelona, Spain), for half a minute and then put to rest for 15 min. After that time, it was measured with millimeter ruler the height of the formed foam. It was considered that the test was negative for the saponins presence, when the foam height was lower than 5 mm, and positive when the foam reached 5 mm or more.

The blood agar test was carried out according to the procedure proposed by Lurssen (2001). This test is based on the saponins ability of hydrolyzing the membranes of the red blood cells. For the experiment, Petri dishes were taken with blood agar medium, dispensed in the bottom (TSS BIOMERIEUX REF 43001, Spain). Each dish was divided into four and in the middle of each subdivision, a blood agar layer was eliminated with the help of a test tube. Wells of 10 mm diameter were obtained in each subdivision, always taking care not remove the blood agar from the wells bottom to prevent the extract spreading through the lower part of the Petri dish. Then, 100 µL of each legume extract was added in one of the wells, trying that in all cases the liquid fill the well, but not to pass by the upper surface of the blood agar layer. Three Petri dishes for each of the five repetitions of plant extract previously obtained were used. All Petri dishes were incubated in the dark, at 37 °C, during 24 h. Subsequently it was measured with millimeter rule the diameter of the hydrolysis halo (mm) which each extract produced around the well, known as a circular clear zone around it.

In both test a completely randomized experimental design was applied, with five extracts by treatment (legume) and three repetitions per extract. The average value of the three repetitions per extract of each legume was considered the experimental unit. The InfoStat statistical package for comparison by variance analysis (ANOVA) of the results was used (Di Rienzo et al. 2010). When differences (P <0.05) were found, the treatment means were compared by the multiple ranges Duncan test (1955).

The results of the foam test to the extracts of the four evaluated legumes are shown in figure 1. Only the enterolobium extracts showed positive results to the saponins presence, as the foam height of the extracts  of acacia, albizia and leucaena was lower  than  5 mm (P <0.0001).

In figure 2 are showed the results of blood agar test. The  enterolobium extracts had an hydrolytic effect on the membranes of red blood cells which form the blood agar, while those of the rest of legumes had no such effect (P <0.0001). This test also allowed verifying that the extracts of acacia, albizia and leucaena not either contain tannins with hydrolytic capacity, it is known that some of these polyphenolic compounds may interfere in this test, when having hydrolytic capacity on the membranes of red blood cells (Scalbert 1991, Hernández et al. 2006).

It is known that E. cyclocarpum is an  species rich in saponins (Wina et al. 2005) and the use of methanol as solvent provides good results to extracts these compounds (Sowemimo et al. 2015). However, the information on the content of secondary metabolites in the other legumes is abundant regarding the tannins, but low in relation to the saponins. Ekpenyong (1990) refers the presence of saponins in leucaena and that the bitter taste of its foliage, is partly due, to the presence of these compounds. Awe et al. (2013) observed moderate contents of saponins in this species.

There are also references of the presence of these secondary metabolites in species of the Acacia and Albizia genus (Wina et al. 2005, Kokila et al. 2013), but no specific was found for the species which were evaluated in this study.

From the point of view of ruminant nutrition, it is important to confirm the presence of active saponins in plant extracts to determine the real potential of the use of sources rich in saponins as a natural tool to manipulate ruminal fermentation towards an increase of microbial protein which is produce and reaches the duodenum, and to make a more efficient use of fermentable energy of the diet (Rodriguez and Fondevila 2012). Therefore, it is essential to ensure the quality of these extracts by confirming the active presence of these secondary metabolites in them.

It is concluded that the use of this methodology, based on the combination of these two in vitro assays, allowed confirming the presence of active saponins in E. cyclocarpum extracts and saying that there were not presence in the extracts of acacia, albizia and leucaena of these compounds or tannins capable of hydrolyzing the membrane of red blood cells.

 

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Received: 13/10/2015
Accepted: 20/6/2016

 

 

R. Rodríguez, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email: rrodrí[e-mail]