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Revista Brasileira de Farmacognosia

Print version ISSN 0102-695XOn-line version ISSN 1981-528X

Rev. bras. farmacogn. vol.26 no.3 Curitiba May/June 2016

http://dx.doi.org/10.1016/j.bjp.2016.02.003 

Original articles

Solanum paniculatum root extract reduces diarrhea in rats

Jonh A.B. Tenórioa 

Dulciana S. do Montea 

Thelma M.G. da Silvaa 

Teresinha G. da Silvab 

Clécio S. Ramosa  * 

aDepartment of Chemistry, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil

bDepartment of Antibiotics, Universidade Federal de Pernambuco, Recife, PE, Brazil

Abstract

Solanum paniculatum L., Solanaceae, locally known as "jurubeba", is widely used in Brazil for culinary purposes, and in folk medicine to treat of diverse disorder including gastric dysfunctions. In this study we investigated the antidiarrheal activity of S. paniculatum roots extract in rats at different concentrations (125, 250 and 500 mg/kg, p.o) using different experimental models such as castor oil-induced diarrhea, enteropooling and gastrointestinal motility, determined by in vivo experimental models. The major compound of root extract was characterized as chlorogenic acid based in the IR, 1D and 2D NMR analysis. All the extract doses achieved antidiarrheal potency, as indicated by reduced weight of feces in castor oil-induced diarrhea, decreased intestinal motility and significantly inhibited castor oil-induced enteropooling compared to the vehicle group. The highest dose (500 mg/kg) produced greater anti-motility effect and better reduction of enteropooling, similar to the reference drug Loperamide (5 mg/kg). Extract from S. paniculatum L. roots had antidiarrheal activity, as shown by the lower weight of the feces as well as decrease in the accumulation of intestinal fluid and slower transit, justifying the traditional use of plant for diarrhea.

Keywords Antidiarrheal activity; Chronic diarrhea; Chlorogenic acid; Gastrointestinal mobility; Jurubeba

Introduction

The family Solanaceae A.L. Jussieu comprises more than 3000 species distributed in 106 genera, and their species have a cosmopolitan distribution. The most economically important genus of the family is Solanum, with approximately 1500 species distributed all over the world (Nurit et al., 2007). Among the species of Solanum genus, Solanum paniculatum L. is to be more that highlights for its many medicinal uses, wide distribution and especially for being recognized as herbal medicine by the Brazilian Pharmacopeia, whose roots and stems are indicated in the treatment of anemia and liver and digestive disorders. The species S. paniculatum L., known popularly either as jurubeba or jurupeba, is used commonly in Brazilian folk medicine for the treatment of diverse disorders, such as liver and gastric dysfunctions and hangover as well as for culinary purposes (Mesia-Vela et al., 2002; Botion et al., 2005; Agra et al., 2007; Sabir and Rocha, 2008; Vieira et al., 2013). The plant is a component of a variety of pharmaceutical formulations including: syrups, infusions and decoctions, ethanol extracts, and elixirs (Júnior et al., 2015). Ethnopharmacological studies have revealed that leaves, roots, stems and fruits of S. paniculatum are used to treat disorders of the liver and kidney, anemia, tuberculosis, malaria and hypertension, and in anti-inflammatory, antipyretic and stimulant formulations (Joachimovits, 1954; Ribeiro et al., 1986; Agra et al., 2007). Chemical studies revealed a variety of compound groups in S. paniculatum tissues, such as steroids, terpenes, alkaloids (including jurubebina, jubebine and solanine), and saponins (including sojuripidine, isojurubidine, isopaniculidine and jurubidine) (Vieira et al., 2010; Ramos and Ramos, 2012).

Diarrhea continues to be a major health problem throughout the world, especially causing of malnutrition in children under five years old. It is also a major cause of the high morbidity and mortality, particularly among children. In developing countries, the principal causes of diarrhea is associated with the enterotoxins that are produced and secreted from bacterial organisms like Vibrio cholerae, Salmonella, Shigella and Escherichia coli (Walker et al., 2013).

Considering the pharmacological potential of S. paniculatum and the use of plants for the treatment of diarrhea, this study investigated the activity of S. paniculatum extract against diarrhea induced by various methods in rats.

Materials and methods

Plant materials, extraction and isolation

Solanum paniculatum L., Solanaceae, specimens were collected in the city of Camaragibe, Pernambuco state, northeastern Brazil, in January 2012. Botanical identification was done by Instituto Agronômico de Pernambuco (IPA) and a voucher specimen was deposited at the Dárdano de Andrade Lima Herbarium of IPA (88503). The roots were dried at 40 °C by 72 h and the dried material was milled to a fine powder in a MACSALAB mill.

Dried roots (46 g) were firstly submitted Soxhlet extraction with 200 ml of dichloromethane for 2 h and followed by extraction using 300 ml of ethanol for a period of 4 h. The solvent was filtered and concentrated in vacuo to yield 4.4 g (9.6%) of fatty-free crude extracts with presence of precipitate. Part of extract was washed twice with chloroform, filtrated and dried to yield a white solid identified as chlorogenic acid.

Instrumentation and chromatography materials

IR spectra were measured in KBr pellets with a Varian infrared spectrometer. 1D and 2D NMR spectra were recorded using Bruker spectrometer NMR (300 MHz and 500 MHz). HPLC analyses of extracts were performed in a Shimadzu LC10 instrument using a Phenomenex RP-18 column (250 mm × 4.7 mm, 5 µm), eluted in gradient mode starting with 5% aqueous formic acid/methanol (8:2) for 15 min, raising to 30% methanol in 20 min, with detection at 325 nm and flow rate of 1 ml/min. Four mg of the crude extract was suspended in 2 ml of methanol and applied to C18 cartridges. The cartridges were eluted with 4 ml of MeOH and the eluents were filtered (0.45 µm filter) prior to analysis. The standard of chlorogenic acid was acquired of the Sigma–Aldrich (Batch: SLBB6914V).

Experimental animals

Male albino rats weighing between 200 and 250 g were used for the experiments. The animals were obtained from the animal house of the Antibiotics Department of Federal University of Pernambuco, which is registered with the Brazilian College of Animal Experimentation under no. 18. All experiments were authorized by the Ethical Committee for Animal Care (protocol number: 23076.46150/2012-23 CCB-UFPE). The animals were kept in polypropylene boxes at a temperature of 22 ± 3 °C with a light-dark cycle of 12 h and received balanced feed and water ad libitum. The animals were deprived of food before each experiment.

Castor oil induced diarrhea

The method described by Awouters et al. (1978) was followed. Healthy albino rats of either sex (200–250 g) were randomly selected and divided into five groups of five animals each. They were deprived of food for 12 h prior to the test, with free access to water. Group 1 received the vehicle alone (1% Tween 80), while those in group 2 received Loperamide (5 mg/kg) as positive control. Animals in groups 3, 4 and 5 received S. paniculatum root extracts (125, 250, 500 mg/kg) respectively. Extract administration was by the oral route. The animals were housed singly in cages lined with pre-weighed filter paper. One hour after pretreatment with the extract, the animals were then given 1 ml of castor oil orally and the time between oil administration and appearance of first diarrheal drop was noted. Thereafter, they were observed for 4 h for the presence of diarrhea and total weight of feces excreted was obtained.

Gastrointestinal motility test

Male albino rats (200–250 g) were randomly divided into five groups of five rats each. They were deprived of food for 12 h before the test, but were allowed free access to water. Group 1 rats (control) were treated with the vehicle (1% Tween 80). Group 2 rats received Loperamide (5 mg/kg), while groups 3, 4 and 5 received different doses of the S. paniculatum root extracts (125, 250 and 500 mg/kg), respectively. Thirty minutes after drug administration, 1 ml of charcoal meal (5%) was administered orally to all animals and 30 min later, all the rats were sacrificed and the abdomen was opened. The small intestine was dissected out from the pylorus to the cecum and the total distance traveled by the charcoal plug along the small intestine was estimated for both the control and the treated groups. The percentage distance traveled by the charcoal meal from the pylorus to the cecum was noted.

Castor oil-induced enteropooling

In this method, as described by Robert et al. (1976), rats were deprived of food for 12 h before the experiment. The rats were divided into six groups of five each. The vehicle (1% Tween 80) was given to the first group. The second group received Loperamide (5 mg/kg), while groups 3, 4 and 5 received graded doses of SPRE (125, 250 and 500 mg/kg). Thirty minutes later, all the rats were treated with 1 ml of castor oil. A sham group did not receive any treatment. After 30 min, each rat was sacrificed and the whole length of the intestine from the pylorus to the cecum was dissected and the contents weighed.

Statistical analysis

Results were expressed as mean ± SEM. The significance of difference between means was determined using one-way analysis of variance and the statistical significance was set at p < 0.05. All data were analyzed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA).

Results

Chemical profile

The analysis by HPLC root extract of S. paniculatum showed a major peak with retention time at 8 min (Fig. 1). The ethanolic crude extract of the S. paniculatum roots was submitted to purification steps, resulting in the isolation of major compound as a yellow amorphous powder determined as chlorogenic acid. Structural elucidation of chlorogenic acid was based on interpretation of spectral data, mainly IR, UV 1H and 13C NMR and 2D-NMR, including comparison with values described in the literature (Agbo et al., 2014).

Fig. 1 Chemical profile obtained by HPLC of crude extract of Solanum paniculatum roots. (C18 column 250 mm × 4.7 mm, 5 µm, eluted with 5% aqueous formic acid/methanol 8:2 for 15 min, raising to 30% methanol in 20 min; λ = 325 nm; flow 1 ml/min). 

Castor oil-induced diarrhea

The highest total weight of feces was obtained in the control group (11.17 ± 0.70 g), whereas the lowest weight of feces was recorded for animals that received Loperamide (3.81 ± 0.13 g) (Table 1). All the extract doses achieved antidiarrheal potency by means of reduced weight of feces compared to the control group.

Table 1 Effect of the Solanum paniculatum roots extract on castor oil-induced diarrhea in rats. 

Group Dose (mg/kg) Total weight of feces (g) Inhibition (%)
Vehicle 11.17 ± 0.70
Loperamide 5 3.81 ± 0.13a 65.9
125 6.54 ± 0.16a 41.5
Extract 250 6.26 ± 0.28a 44.0
500 5.59 ± 0.10a 50.0

ap < 0.01 with respect to the vehicle-treated group, using one-way ANOVA followed Turkey's test.

Values are expressed as mean ± SEM (n = 5).

Gastrointestinal motility test

In the control group, charcoal meal traversed most of the small intestine (73.88 ± 1.34%). In the extract test groups (125, 250 and 500 mg/kg), significant dose-dependent decreases in the transit of charcoal meal through the small intestine were observed (64.62 ± 1.87, 61.92 ± 1.80 and 55.86 ± 3.40), respectively (Table 2). The highest dose of SPRE (500 mg/kg) produced greater anti-motility effect, similar to Loperamide (5 mg/kg).

Table 2 Effect of the Solanum paniculatum roots extract on gastrointestinal motility test. 

Group Dose (mg/kg) Distance traveled by charcoal (%) Inhibition (%)
Vehicle 73.88 ± 1.34
Loperamide 5 52.45 ± 2.28b 47.5
125 64.62 ± 1.87a 35.4
Extract 250 61.92 ± 1.80a 38.0
500 55.86 ± 3.40b 44.1

ap < 0.05 with respect to the vehicle-treated group, using one-way ANOVA followed Turkey's test.

bp < 0.01 with respect to the vehicle-treated group, using one-way ANOVA followed Turkey's test.

Values are expressed as mean ± SEM (n = 5).

Castor oil-induced enteropooling

As shown in Table 3, the intestinal weight content was much higher in the control group (3.277 ± 0.18 g) compared with sham group (2.040 ± 0.03 g). Consistently, 5 mg/kg of Loperamide reduced the intestinal content significantly compared with the control group (p < 0.01). Treatments with SPRE (125, 250 and 500 mg/kg) decreased the intestinal weight contents to 2.788 ± 0.08 g (p < 0.05), 2.657 ± 0.08 g (p < 0.05) and 2.402 ± 0.14 g (p < 0.01), respectively.

Table 3 Effect of the Solanum paniculatum roots extract on castor oil-induced enteropooling. 

Group Dose (mg/kg) Weight of intestinal content (g) Inhibition (%)
Sham 2.040 ± 0.03
Vehicle 3.277 ± 0.18
Loperamide 5 2.392 ± 0.05b 27.0
125 2.788 ± 0.08a 14.9
Extract 250 2.657 ± 0.08a 18.9
500 2.402 ± 0.14b 26.7

ap < 0.05 with respect to the vehicle-treated group, using one-way ANOVA followed Turkey's test.

bp < 0.01 with respect to the vehicle-treated group, using one-way ANOVA followed Turkey's test.

Values are expressed as mean ± SEM (n = 5).

Discussion

Diarrhea is one of the main causes of infant mortality in developing countries, causing about 5 to 8 million deaths a year, mainly among children under five years of age. Medicinal plants have been used traditionally, including S. paniculatum, as antidiarrheal without any scientific base (Konaté et al., 2015). Diarrhea involves increased gastrointestinal mobility and secretion and decreased absorption of water and electrolytes (Wang et al., 2015).

The gastrointestinal model using activated charcoal as marker has been used for over 60 years as a simple and effective to assess the effects of laxatives. This method indicates the maximum distance traveled by the marker in the small intestine in a given time interval after its administration (Silva et al., 2012). According to the model, the S. paniculatum extracts at the different concentrations evaluated significantly reduced the intestinal motility, with a dose-dependent pattern when compared to the control group (Table 2). The motility in the animals that received SPRE at the concentration of 500 mg/kg was about 44% slower, demonstrating that the extract has a positive effect in reducing intestinal transit.

Castor oil induces diarrhea by causing an increase in the secretion of fluids and electrolytes in the intestinal lumen through the mucosa. This in turn causes a build-up of fluid and an aqueous lumen content that passes quickly through the gut (Wang et al., 2015). The release of ricinoleic acid produces changes in the transport of water and electrolytes, diminishing the absorption of Na+ and K+, in turn reducing the activity of Na+, K+-ATPase in the small intestine and colon, resulting in a hypersecretory response and faster intestinal transit (Dash et al., 2014). Castor oil increases the production or release of prostaglandins (Saito et al., 2002), changing the permeability and causing injuries to the intestinal mucosa. It also stimulates biosynthesis of platelet aggregation factor (Izzo et al., 1998), which can result in inflammation of the mucosa.

The extract from S. paniculatum roots in the castor oil-induced diarrhea model reduced the diarrhea significantly, at all the tested doses, when compared to the control group that only received the vehicle (Table 1). The extract caused a diarrhea reduction of about 50%, besides more feces with solid consistency and reduced evacuation frequency in relation to the group control. Another result observed in this work was the reduction of the accumulation of intestinal fluid induced by castor oil. The S. paniculatum extract reduced the accumulation of intestinal fluid at all the evaluated doses (125, 250 and 500 mg/kg), with inhibition rates of 14.9, 18.9 and 26.7%, respectively, showing a dose-dependent relationship. The dose of 500 mg/kg produced the best results, with similar inhibition rate to Loperamide (Table 2).

There are few studies of antidiarrheal activity with natural compounds isolated from plants, being widely investigated the potential antidiarrheal from crude plant extracts. The flavonoid isoliquiritigenin isolated of Trilepisium madagascariense stem bark inhibited diarrheal droppings by 84.81%, result similar to antidiarrheal compounds Loperamide (72.65%) and diphenoxylate HCl (84.81%) (Teke et al., 2010). The diterpenoid 19-deoxyicetexone isolated from Salvia ballotiflora at doses of 12.5 and 25 mg/kg reduced diarrhea for 61.8 and 89.6%, respectively (Pérez-Gutiérrez et al., 2013). A previous study, the ethanolic extract from S. paniculatum aerial parts showed antidiarrheal activity at dose of 125, 250, 500, and 750 mg/kg, and the chlorogenic acid also was identified in the aerial parts of plant (Clementino-Neto et al., 2016). The reduced accumulation of intestinal fluid promoted by the S. paniculatum extract from roots may be associated with the presence of chlorogenic acid, a known anti-inflammatory compound, since the intestinal hypersecretion of fluid involves an inflammatory response (Izzo et al., 1998; Guo et al., 2015).

Conclusion

Overall the extract of S. paniculatum L. roots had antidiarrheal activity, as shown by the lower weight of the feces as well as decrease in the accumulation of intestinal fluid and slower transit, justifying the traditional use of plant for diarrhea.

Ethical disclosures

Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Confidentiality of data. The authors declare that no patient data appear in this article.

Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgments

JABT thanks CAPES for providing a scholarship. The authors are indebted to the Centro de Apoio a Pesquisa (CENAPESQ), UFRPE, for the laboratory facilities.

References

Agbo, M.O., Nnadi, C.O., Ukwueze, N.N., Okoye, F.B.C., 2014. Phenolic constituents from Platycerium bifurcatum and their antioxidatant properties. J. Nat. Prod. 7, 48-57. [ Links ]

Agra, M.F., Baracho, G.S., Nurit, K., Basílio, I.J.L.D., Coelho, V.P.M., 2007. Medicinal and poisonous diversity of the flora of Cariri Paraibano. J. Ethnopharmacol. 111, 383-395. [ Links ]

Awouters, F., Niemegeers, C.J.E., Lenaerts, F.N., Janscen, P.A., 1978. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis. J. Pharm. Pharmacol. 30, 41-45. [ Links ]

Botion, L.M., Ferreira, A.V.M., Côrtes, S.F., Lemos, V.S., Braga, F.C., 2005. Effects of the Brazilian phytopharmaceutical product Ierobina® on lipid metabolism and intestinal tonus. J. Ethnopharmacol. 102, 137-142. [ Links ]

Clementino-Neto, J., Pereira, J.C., Vasconcelos, L.H.C., de Souza, I.L.L., Silva, A.D.S., Silva, T.M.G., Ramos, N.S.M., Pessôa, H.L.F., Silva, T.M.S., da Silva, B.A., Cavalcante, F.A., 2016. Toxicological, antidiarrheal and spasmolytic activities of Solanum paniculatum. Planta Med. 82, 58-64. [ Links ]

Dash, P.R., Nasrin, M., Raihan, S.Z., Ali, M.S., 2014. Study of antidiarrhoeal activity of two medicinal plants of Bangladesh in castor-oil induced diarrhea. Int. J. Pharm. Sci. Res. 5, 3864-3868. [ Links ]

Guo, Y.J., Luo, T., Wu, F., Mei, Y.W., Peng, J., Liu, H., Li, H.R., Zhang, S.L., Dong, J.H., Fang, Y., Zhao, L., 2015. Involvement of TLR2 and TLR9 in the anti-inflammatory effects of chlorogenic acid in HSV-1-infected microglia. Life Sci. 127, 12-18. [ Links ]

Izzo, A.A., Gaginella, T.S., Mascolo, N., Capasso, F., 1998. Recent findings on the mode of action of laxatives: the role of platelet activating factor and nitric oxide. Trends Pharmacol. Sci. 19, 403-405. [ Links ]

Joachimovits, R., 1954. The uterotonic activity of several Brazilian plants including the Brazilian form of Spartium junceum. Sci. Pharm. 22, 7-18. [ Links ]

Júnior, G.M.V., da Rocha, C.Q., de Souza Rodrigues, T., Hiruma-Lima, C.A., Vilegas, W., 2015. New steroidal saponins and antiulcer activity from Solanum paniculatum L. Food Chem. 186, 160-167. [ Links ]

Konaté, K., Yomalan, K., Sytar, O., Brestic, M., 2015. Antidiarrheal and antimicrobial profiles extracts of the leaves from Trichilia emetica Vahl. (Meliaceae). Asian Pac. J. Trop. Biomed. 5, 242-248. [ Links ]

Mesia-Vela, S., Santos, M.T., Souccar, C., Lima-Landman, M.T.R., Lapa, A.J., 2002. Solanum paniculatum L. (jurubeba): potent inhibitor of gastric acid secretion in mice. Phytomedicine 9, 508-514. [ Links ]

Nurit, K., Agra, M.F., Basílio, I.J.L.D., 2007. Estudo farmacobotânico comparativo entre Solanum paniculatum L. e Solanum rhytidoandrum Sendtn. (Solanaceae). Rev. Bras. Bioci. 5, 243-245. [ Links ]

Pérez-Gutiérrez, S., Zavala-Mendoza, D., Hernández-Munive, A., Mendoza-Martínez, A., Pérez-González, C., Sánchez-Mendoza, E., 2013. Antidiarrheal activity of 19-deoxyicetexone isolated from Salvia ballotiflora Benth in mice and rats. Molecules 18, 8895-8905. [ Links ]

Ramos, N.S.M., Ramos, C.S., 2012. Volatiles from Solanum paniculatum leaves in response to mechanical damage. Chem. Nat. Compd. 49, 953-954. [ Links ]

Ribeiro, R.A., De Melo, M.M.R., De Barros, F., Gomes, C., Trolin, G., 1986. Acute antihypertensive effect in conscious rats produced by some medicinal plants used in the state of Sao Paulo. J. Ethnopharmacol. 15, 261-269. [ Links ]

Robert, A., Nezamis, J.E., Lancaster, C., Hanchar, A.J., Klepper, M.S., 1976. Enteropooling assay: a test for diarrhea produced by prostaglandins. Prostaglandins 5, 809-828. [ Links ]

Sabir, S.M., Rocha, J.B.T., 2008. Antioxidant and hepatoprotective activity of aqueous extract of Solanum fastigiatum (false Jurubeba) against paracetamol-induced liver damage in mice. J. Ethnopharmacol. 120, 226-232. [ Links ]

Saito, T., Mizutani, F., Iwanaga, Y., Morikawa, K., Kato, H., 2002. Laxative and anti-diarrheal activity of polycarbophil in mice and rats. Jpn. J. Pharmacol. 89, 133-141. [ Links ]

Silva, P.C.B., Neto, J.C., Silva, A.D.S., Silva, K.M., Silva, T.M.S., Agra, M.F., Cavalcante, F.A., 2012. Antidiarrheal activity of Solanum asterophorum in mice. Rev. Bras. Farmacogn. 22, 131-136. [ Links ]

Teke, G.N., Kuiate, J-R., Kueté, V., Teponno, R.B., Tapondjou, L.A., Vilarem, G., 2010. Antidiarrheal activity of extracts and compound from Trilepisium madagascariense stem bark. Indian J. Pharmacol. 42, 157-163. [ Links ]

Vieira, P.M., Marinho, L.P.M., Ferri, S.C.S., Chen-Chen, L., 2013. Protective effects of steroidal alkaloids isolated from Solanum paniculatum L. against mitomycin cytotoxic and genotoxic actions. An. Acad. Bras. Cienc. 85, 553-560. [ Links ]

Vieira, M., Paula, J.R., Chen-Chen, L., 2010. Solanum paniculatum L. leaf and fruit extracts: assessment of modulation of cytotoxicity and genotoxicity by micronucleus test in mice. J. Med. Food 13, 1424-1430. [ Links ]

Walker, C.L., Rudan, I., Liu, L., Nair, H., Theodoratou, E., Bhutta, Z.A., O'Brien, K.L., Campbell, H., Black, R.E., 2013. Global burden of childhood pneumonia and diarrhoea. Lancet 381, 1405-1416. [ Links ]

Wang, S., Zhao, Y., Zhang, J., Huang, X., Wang, Y., Xu, X., Zheng, B., Zhou, X., Tian, H., Liu, L., Mei, Q., 2015. Antidiarrheal effect of Alpinia oxyphylla Miq. (Zingiberaceae) in experimental mice and its possible mechanism of action. J. Ethnopharmacol. 168, 182-190. [ Links ]

Received: September 14, 2015; Accepted: February 7, 2016

* Corresponding author. E-mail:clecio.ramos@ufrpe.br (C.S. Ramos).

Conflicts of interest

The authors declare no conflicts of interest.

Authors' contributions

JABT, DSM and TGS developed the animal model. JABT and TMGS were responsible for the collection of plant sample and the chemical studies. CSR and JABT developed the analytical methodology (HPLC). CSR and TGS designed the study and supervised the laboratory work. CSR and JABT wrote the manuscript.

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