Antihelminthic activity of crude saponins and tannins isolated from Tetradenia riparia leaf extracts

Marble Nasasira; Genny Dominguez Montero; Miriela Betancourt Valladares

Antihelminthic activity of crude saponins and tannins isolated from leaf extracts of Tetradenia riparia (Hochst.) Codd (nutmeg bush)

Artículo original

Antihelminthic activity of crude saponins and tannins isolated from leaf extracts of Tetradenia riparia (Hochst.) Codd (nutmeg bush)

Actividad antihelmíntica de saponinas y taninos aislados de los extractos de las hojas de Tetradenia riparia (Hochst.) Codd. (nuez moscada)

Nasasira Marble¹ORCID: https://orcid.org/0000-0003-1785-2499
Genny Dominguez Montero²* ORCID: https://orcid.org/0000-0001-5006-7762
Miriela Betancourt Valladares³ ORCID: https://orcid.org/0000-0002-5301-4057

¹Mbarara University of Science and Technology, Uganda.
²Florida Municipal Hospital Camaguey. Cuba.
³Faculty of Dentistry, Camaguey Medical University, Camaguey. Cuba.

*Autor para la correspondencia. Correo electrónico: dominguezgenny@gmail.com

ABSTRACT

Introduction: Helminth infections affect developing countries, particularly the child population. The medicinal worth of plants lies in their active principles, which have a physiological effect on the human body. Reports have been issued of the anthelmintic activity of Tetradenia riparia (Hochst.) Codd [Lamiaceae], and its phytochemical study has revealed the presence of several metabolites which could be responsible for its pharmacological effect.
Objective: Evaluate the anthelmintic activity of saponins and tannins isolated from extracts of T. riparia leaves.
Method: 14.07 % aqueous extract and 23.0 % alcoholic extract were obtained from T. riparia dry leaves. The saponins were isolated from the aqueous extract and the tannins from the alcoholic extract after confirming their presence by foam formation for saponins and the ferric chloride test for tannins. Sixty Ascaris suum worms were divided into ten groups: eight experimental and two controls (negative and positive). The experimental groups were exposed to various extract concentrations (40 mg/ml, 80 mg/ml and 120 mg/ml) prepared with Goodwin's physiological solution, which was also used as negative control. The positive control was albendazole 80 mg/ml. The motility test was used to determine anthelmintic activity. The chi-square test was applied, and p values below 0.05 were considered significant.
Results: Saponins and tannins were detected at high and moderate concentrations in aqueous and alcoholic T. riparia leaf extracts, respectively. Crude metabolites displayed anthelmintic activity depending on the dose. Saponins and tannins displayed anthelmintic activity, and no significant differences were found between the effect of the two metabolites isolated or between them and the positive control.
Conclusions: The saponins and tannins isolated from T. riparia leaves may be responsible for the anthelmintic effect of the plant. Anthelmintic activity is similar in saponins, tannins and albendazole.

Key words: anthelmintic activity, ascaricidal activity, Tetradenia riparia, saponins, tannins, motility.

RESUMEN

Introducción: Las infecciones causadas por helmintos afectan a los países en desarrollo, especialmente a los niños. El valor medicinal de las plantas radica en sus principios activos los cuales tienen efecto fisiológico en el organismo humano. Se ha reportado la actividad antihelmíntica de Tetradenia riparia (Hochst.) Codd [Lamiaceae] y su estudio fitoquímico ha revelado la presencia de varios metabolitos que pudieran ser responsables de su efecto farmacológico.
Objetivo: Evaluar la actividad antihelmíntica de las saponinas y los taninos aislados de los extractos de las hojas de T. riparia.
Método: Se obtuvieron 14,07 % de extracto acuoso y 23,0 % de extracto alcohólico de las hojas secas de T. riparia. Las saponinas se aislaron del extracto acuoso y los taninos se aislaron del extracto alcohólico después de confirmar su presencia mediante la formación de espuma para las saponinas y la prueba con cloruro férrico para los taninos. Se dividieron 60 gusanos Ascaris suum en 10 grupos; ocho experimentales y dos controles (negativo y positivo). Los grupos experimentales fueron expuestos a diferentes concentraciones de los extractos (40 mg/ml, 80 mg/ml y 120 mg/ml) preparados con solución fisiológica Goodwin que se usó también como control negativo. El control positivo fue albendazol 80 mg/mL. Se empleó la prueba de motilidad para determinar la actividad antihelmíntica. Se realizó la prueba de Chi-cuadrado y se consideraron significativos los valores menores que 0,05.
Resultados: Las saponinas y los taninos se detectaron en concentraciones altas y moderadas en los extractos acuosos y alcohólicos de las hojas de T. riparia, respectivamente. Los metabolitos crudos revelaron acción antihelmíntica en dependencia de la dosis. Las saponinas y los taninos mostraron efecto antihelmíntico y no se encontraron diferencias significativas entre el efecto de ambos metabolitos aislados ni entre ellos y el control positivo.
Conclusiones: Las saponinas y los taninos aislados de las hojas de T. riparia pueden ser los responsables del efecto antihelmíntico de la planta. El poder antihelmíntico de las saponinas, los taninos y el albendazol es similar.

Palabras clave: actividad antihelmíntica; actividad ascaricida; Tetradenia riparia; saponinas; taninos; motilitidad.

Recibido: 24/07/2016
Aprobado: 10/08/2018

INTRODUCTION

More than one billion people are infected with helminthes in developing countries causing substantial morbidity and hundreds of thousands of deaths annually. Helminths also plague livestock in developing and developed countries.^(1,2) A study conducted in 2005 among 20,185 school children in Uganda revealed that 6.3 %, 5.0 %, 43.5 % were infected with Ascaris lumbricoides, Trichuris trichiura and hookworms respectively.⁽³⁾

Despite the impact of helminthes on public health and economies, the antihelminthic pharmacopeia is not extensive.Besides, whereas chemotherapy is available for infection with most parasitic helminthes, widespread use of antihelminthic drugs has resulted in the emergence of drug resistance.² Therefore, new treatment options for helminthes infections are urgently needed. This includes ethno-pharmacological practices such as treatment with natural plant extracts which are at a low cost, access to large amounts of raw material and easy integration into traditional cultural practice. Previous studies have indicated that several plants in Uganda are used in treatment of livestock and human's helminthes although some are considered to be more potent than others.⁽⁴⁾ A study conducted in Kampala, Uganda, identified twenty-one plants through preliminary field surveys and seven were selected for in vitro anti-helminthic activity against Ascaris suum. Of the seven plants that were initially screened, five gave appreciable positive results. Tetradenia riparia (Hochst.) Codd [Lamiaceae] was the most potent among the plants screened and the isolation of the secondary metabolites that may be responsible for the antihelminthic activity of the plant was recommended, among them saponins and tannins were included.⁽⁵⁾

The species T. riparia are a widespread herbaceous shrub occurring throughout eastern and tropical Africa; it is commonly known as nutmeg bush, ginger bush or misty plume bush.⁽⁶⁾Phytochemical studies of T. riparia reported the accumulation of essential oils in the leaves which is particularly reach in terpenes, diterpenoids and alpha-pyrones;⁽⁶⁾ other studies revealed the presence of saponins, flavonoids and tannins to be the prominent secondary metabolites present.⁽⁷⁾

A number of studies conducted with nematode parasites of ruminant livestock have demonstrated direct anthelmintic effects of extracts from tannin-containing plants in in vitro assays, with in vivo verification of these results.⁽⁸⁾ Crude saponins isolated from several plants have also demonstrated a strong antihelminthic activity.⁽⁹⁾

The above background motivated this study, aimed to evaluate the antihelminthic activity of saponins and tannins isolated from T. riparia leaf extracts.

METHOD

The antihelminthic activity of crude saponins and tannins isolated from T. riparia aqueous and ethanolic leaf extracts respectively, was assessed by observing motility of Ascaris suum Goeze worms known as the large roundworms of pigs.

Plant material

Fresh plant leaves were collected from Isingiro, Western Uganda, taken to the Faculty of Science, Department of Biology for identification by the botanist Dr. Eunice Olet and it was given a voucher number: NASASIRA MARBLE 001.

Preparation of T. riparia leaf extracts and metabolites isolation

Dried leaves (1100 g) were cleaned and dried under shade for two weeks. The dried leaves were pulverized into coarse particles using an electric blender to achieve a surface area for optimal drug extraction. Saponins were isolated from the aqueous leaf extract and tannins from the ethanolic leaf extract to ensure optimal extraction of both metabolites. Previous isolation, presence of both, saponins and tannins was confirmed through froth formation test and ferric chloride test respectively.⁽¹⁰⁾

The aqueous leaf extract was obtained using 937g of the leaves powder using the maceration method as Adiukwu C. Paul and collaborators in 2011.⁽¹¹⁾ A sticky solid extract was obtained at a percentage yield of 14.07 % w/w. Saponins were isolated from this extract according to the technique followed by Odadoni and Ochuko in 2002.⁽¹²⁾ A sticky solid extract was obtained at a percentage yield of 2.6 % w/w.

The ethanolic leaf extract was obtained using 20 g of the plant powder using a Soxhlet extraction method described by Norsyamimi H.A. and collaborators in 2015.⁽¹³⁾ A sticky solid extract was obtained at a percentage yield of 23.0 % w/w. Tannins were isolated from this extract using lead acetate as presented by Hostettmann Kurt and collaborators in 1998.⁽¹⁴⁾ A sticky solid extract was obtained at a percentage yield of 36.52 % w/w.

Worms

The live roundworms (A. suum) were identified and collected from a pig abattoir in Bushenyi, Western Uganda. The worms were obtained from the small intestines of freshly slaughtered pigs; after manually straining the contents to collect the worms, they were immediately placed into a thermos flask containing freshly prepared Goodwin's solution at 37 °C and then transported to Mbarara University of Science and Technology Pharmacy Laboratory for identification by the laboratory technician Mr. Nkwangu David and later the susceptibility assay experiments to be conducted.

Antihelminthic susceptibility test

The in-vitro susceptibility was determined by performing the Motility assay. A total of 60 A. suum live worms were used. Goodwin's physiologic solution was used as the solvent to prepare a series of concentrations 40 mg/ml, 80 mg/ml and 120 mg/ml from the dried aqueous and ethanolic leaf extracts. This same physiologic solution was used as a negative control. Using the same procedure, 80 mg/ml positive control of the available standard drug albendazole was also prepared.⁽¹⁵⁾ As a quality control measure, every dilution was done twice to minimize errors and the average of the pair was used as the test solution.

Six live worms (A. suum) were introduced into 10 conical flasks containing the different extract concentrations, the positive control and negative control experiments at the same time. The flasks and their contents were then immersed in a water bath and maintained at 37 ^oC throughout the experiment. The worms were monitored at a 12 hours interval for 48 hours and observed for motility, paralysis and mortality in accordance with the following criteria;⁽¹⁶⁾

Motility (alive): When complete sinusoidal movement of the entire body occurs at 37 ^oC.
Paralysis: When only one terminal of the worm shows movement.
Death: When the worms had lost their motility when shaken vigorously or when dipped in warm water at 50 ^oC followed by fading away of their body color.

Statistical analysis

Analysis of the data was carried out using STATA version 8 Software. The antihelminthic activity of both plant extracts and the standard drug albendazole were compared using a Chi-square test and a p-value less than 0.05 was considered significant.

RESULTS

Phytochemistry

The phytochemical screening of the T. riparia aqueous and ethanolic leaf extracts confirmed the presence of saponins and tannins in high and moderate concentrations respectively.

Motility assay of worms in crude saponins, crude tannins and albendazole solution

The number of motile, paralyzed and dead worms in the different solutions at different times is shown in Table 1 and 2. In the negative control (Goodwin's solution), the worms were stable all throughout the experiment as expected. In general, it was observed a dose dependent response of the worms to the crude saponins, crude tannins and albendazole, meaning that as the concentration of the solution was increased, the rate of paralysis and death also increased.

Table 1 shows the antihelminthic activity of crude saponins and the positive control albendazole. After 24 hours, at 40mg/ml of saponins only two worms were motile and the rest paralyzed. As the concentration increased to 80mg/ml, all the worms were paralyzed and at 120mg/ml four worms remained paralyzed and two worms had died. After 48 hours of incubation the highest antihelminthic activity was observed at 120mg/ml of crude saponins extract where six death worms were observed while five worms were found dead in the albendazole solution.

Table 2 gives the motility test results using the crude tannins extract and albendazole. The highest antihelminthic effect was found at 80mg/ml of the crude tannins extract where, after 24 hours of exposure three worms were found paralyzed and two dead. After 36 hours of incubation it was observed three worms paralyzed and one progressed to death being three the worms that have died at this time. 48 hours later, at this same concentration, from the three previously paralyzed worms, two progressed to death for a total of five dead worms; this equal number of worms was found death in the standard drug solution. At 120mg/ml of the crude tannins extract and after 48 hours of exposure, the number of death worms remained the same (five worms). There was no difference observed with albendazole.

Comparison of the antihelminthic activity between T. riparia crude saponins, crude tannins and albendazole

The comparison of the results obtained at 80 mg/ml of the study plant extracts and albendazole, are shown in the figure. This was the concentration used for the standard drug therefore the one used for comparison purposes with the plant metabolites.

The best antihelminthic activity was shown by the crude saponins which paralyzed all the worms at 24 hours while there was still one worm motile with both tannins and albendazole. At 36 hours of incubation the two phytochemicals and the standard drug had either already paralyzed or killed all the worms.

Regarding the ascaricidal activity the best results found at 24 hours were shown by tannins which had killed two worms followed by albendazole with only one. After 36 hours albendazole showed the strongest effect killing four worms while tannins were able to kill three and saponins only two. At 48 hours five worms were observed dead in both tannins and the standard drug, giving the best ascaricidal activity compared to the saponins; however, there was no significant difference between the ascaricidal activity of crude saponins and albendazole (p= 0.125), crude tanins and albendazole (p= 0.125), and crude tannins and saponins (p= 0.091), since the p-values were greater than 0.05 at a 5 % level of significance.

DISCUSSION

The screened T. riparia confirmed the presence of saponins and tannins that is in line with the study conducted by Khuzwayo Joyce Khethiwe in 2011.⁽⁷⁾ The research showed that in T. riparia leaves, the prominent secondary metabolites present were saponins (23.3 %), flavonoids (3.7- 4.9 %) and tannins (4.1 %).

The ascaricidal effect of saponins and tannins, isolated from the study plant, increased with increasing concentration of the extract and the incubation time. After a deep search in the accessed bibliography, researches evaluating the antihelminthic activity of T. riparia were not found, however some studies have reported a potent antihelminthic activity of tannins and saponins isolated from other plants.⁽¹⁷⁾

A study conducted by Niaz Aliet and collaborators in 2011⁽⁹⁾ on cytotoxic and anthelmintic potential of crude saponins isolated from Achillea wilhelmsii C. Koch and Teucrium stocksianum Boiss, showed that crude saponins of Achillea wilhelmsii were 1.96 and 2.12 times more potent than albendazole against Pheretima posthuma and Raillietin aspiralis respectively. Similarly, at concentration 40 mg/ml, crude saponins of Teucrium stocksianum (CST) had 1.89, 1.96 and 1.37 times more antihelminthic activity than albendazole againstPheretima posthuma, Raillietina spiralis and Ascaridia galli, respectively. These results differ from the ones found in our study where saponins have no significant difference in the ascaricidal activity when compared with the same concentration of albendazole.

There was no significant difference between the ascaricidal activity of crude tannins and that of albendazole which match with the results obtained by Williams Andrew and collaborators in 2014⁽¹⁷⁾ in a study that assessed the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum. The study explored the structural damage caused by exposure to condensed tannins. The cuticle of worms exposed to the highest concentration of condensed tannins revealed clear damage with marked irregularity of the cuticular surface, which was the same as the smooth surface observed in control worms.

No study aimed to carry out a comparison between the antihelminthic activity of tannins and saponins isolated from any plant was found after extensive literature searching. The present study showed no significant difference between the antihelminthic effect of tannins and saponins isolated from T. riparia leaves.

On the other hand, some studies have been conducted to compare plant extracts with albendazole and have shown similar or stronger antihelminthic activity than this conventional drug. Stronger effect has been reported for plants containing more than one metabolite with antihelminthic activity.^(15,18)In this study T. riparia leaves showed similar effect of saponins and tannins compared with albendazole when they are used independently; further studies should be conducted to assess the antihelminthic effect of the combination of both phytochemicals.

Taking into account our findings it can be concluded that saponins and tannins present in T. riparia leaf extracts may be responsible for the antihelminthic activity of this plant. Similar antihelminthic activity was demonstrated with saponins, tannins and albendazole.

REFERENCES

1. Bethony J, Brooker S, Albonico M, Geiger SM, Loukas A, Diement D, et al. Soil-transmitted helminth infections, ascariasis, trichuriasis, and hookworm. Lancet 2006;67(9521):1521-32.

2. Rinaldi G, Loukas A, Brindley PJ, Irelan JT, Smout MJ. Viability of developmental stages of Schistosoma mansoni quantified with xCELLigence worm real-time motility assay (xWORM). International Journal of Parasitology: Drugs and Resistance [internet]. 2015 Dec [cited 2014 Oct 10];5(3): [aprox. 24 p.]. Available from: http://www.sciencedirect.com/science/article/pii/S2211320715300063

3. Kabatereine NB, Tukahebwa EM, Kazibwe F, Twa-Twa JM, Barenzi JF, Zaramba S, et al. Soil-Transmitted Helminthiasis in Uganda: Epidemiology and Cost of Control. Trop Med Int Health 2005;10(11/Nov):1187-9

4. Nalule AS, Mbaria JM, Olila D, Kimenju JW. Ethnopharmacological practices in management of livestock helminths by pastoral communities in the drylands of Uganda. Livestock Research for Rural Development [internet]. 2011 [cited 2014 Dec 4];23(2):[aprox. 24 p.]. Available from: http://www.lrrd.org/lrrd23/2/nalu23036.htm .

5. Wasswa P, Olila D. The in-vitro ascaricidal activity of selected indigenous medicinal plants used in ethno veterinary practices in Uganda. Afr. J. Tradit. CAM [internet]. 2006 [cited 2014 Oct 10];3(2): [aprox. 22 p.]. Available from: http://www.bioline.org.br/pdf?tc06019

6. Gairola S, Naidoo Y, Bhatt A, Nichola A. An investigation of the foliar trichomes of Tetradenia riparia (Hochst.) Codd [Lamiaceae]: An important medicinal plant of Southern Africa [internet]. Flora 2009 Dec [cited 2014 Oct 24];204(4): [aprox. 15 p.]. Available from: https://www.researchgate.net/publication/223190914_An_investigation_of_the_foliar_trichomes_of_Tetradenia_riparia_Hochst_Codd_Lamiaceae_An_important_medicinal_plant_of_Southern_Africa

7. Khuzwayo JK. Essential oil composition and some biological activities of Tetradenia riparia [dissertation]. KwaZulu-Natal, South Africa: University of Zululand; 2011. Available from: http://uzspace.uzulu.ac.za/bitstream/handle/10530/1094/ESSENTIAL%20OIL%20COMPOSITION%20AND%20SOME%20BIOLOGICAL.pdf?sequence=1

8. Hoste H, Martinez Ortiz De Montellano C, Manolaraki F, Brunet S, Ojeda Robertos N. Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections. Veterinary Parasitology. 2012;186:18-27.

9. Niaz A, Syed Wadood A, Ismail S, Ghayour A, Mehreen G, Imran K. Cytotoxic and anthelmintic potential of crude saponins isolated from Achillea wilhelmsii C. Koch and Teucrium stocksianum Boiss. BMC Complement Altern Med [internet]. 2011 Nov [cited 2014 Oct 7];11(106): [aprox. 19 p.]. Available from: http://www.biomedcentral.com/1472-6882/11/106

10. Khandelwal KR, Vrunda S. Practical Pharmacognosy Techniques and Experiments. 23th ed. New Delhi, India: Nirali Prakashan; 2013.

11. Adiukwu CP, Agaba A, Grace N. Pharmacognostic, antiplasmodic and antipyretic evaluation of the aqueous extract of Vernonia amygalina leaf. Int. J. Biol. Chem. Sci 2011 Apr [cited 2014 Nov 1];5(2): [aprox. 19 p.]. Available from: http://www.ajol.info/index.php/ijbcs/article/view/72134/61082

12. Obadoni BO, Ochuko PO. Phytochemical studies and comparative efficacy of the crude extracts of some haemostatic plants in Edo and Delta states of Nigeria. Global J. Pure Appl. Sci 2002;8(2):203-8.

13. Norsyamimi H, Masturah M, Nurina A, Kurnia HD, Syarul NB, Normah MN. Antioxidant and antibacterial assays on Polygonum minus extracts: Different extraction methods. International Journal of Chemical Engineering. [internet]. 2015 March [cited 2015 Apr 4];2015(2015): [aprox. 21 p.]. I.D. 826709. Available from: http://www.hindawi.com/journals/ijce/2015/826709/

14. Hostettmann K, Marston A, Hostettmann M. Preparative Chromatography Techniques. Application in Natural Products Isolation. 2nd Completely Revised and Enlarged ed. New York: Springer; 1998.

15. Pallavi SA, Parmesawaran S, Chauhan Y. In vitro anthelminthic activity of stem extracts of Piper betle Linn against Pheritima posthuma. Pharmacognosy Journal 2012;4(29):61-5

16. Ghosh T, Maity TK, Bose A, Dash GK. Antihelminthic activity of Bacopa monnieri. Indian J Nat Product 2005;21(2):16-9.

17. Williams AR, Ropiak HM, Fryganas C, Desrues O, Mueller-Harvey I, Thamsborg SM. Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum. Parasites & Vectors. [internet]. 2014 Nov [cited 2015 Feb 10];7(518): [aprox. 35 p.]. Available from: http://www.parasitesandvectors.com/content/7/1/518

18. Nilutpal SB, Bibhuti BK, Barnali G. Investigation of in-vitro anthelminthic activity of Garcinia lanceifolia bark in Pheretima posthuma (Indian adult earthworm). IJAPR (Pharmanest) 2014 May-Jun;5(3):2007-10.

Contribución de los autores

Marble Nasasira fue la investigadora principal, generó el tema bajo la supervisión de su tutora y participó directamente en la elaboración íntegra del artículo.

Genny Dominguez Montero fue la tutora de la investigación que generó los resultados presentados. Participó en todos los momentos de la investigación, desde el proyecto, ejecución, confección del informe final y fue principal responsable de la elaboración del artículo científico.

Miriela Betancourt Valladares elaboró junto a la tutora e investigadora principal el artículo científico, asesoró desde el punto de vista científico y metodológico la selección de los resultados relevantes, su discusión y presentación. Fue responsable, junto a la primera autora y la tutora, de la confección del artículo.

Conflicto de intereses

Los autores expresan que no tienen conflicto de intereses.