Antidiarrheal activity of Solanum asterophorum in mice

Antidiarrheal activity of Solanum asterophorum in mice

Polyana Cristina Barros Silva^{I, II}; José Clementino Neto^III; Anne Dayse S. da Silva^III; Karoline de Melo e Silva^III; Tania Maria S. Silva^IV Maria de Fátima Agra,^II Fabiana de Andrade Cavalcante^{I, III}

^IPós-graduação em Nutrição, Universidade Federal de Alagoas, Brazil

^IILaboratório de Tecnologia Farmacêutica "Prof. Delby Fernandes de Medeiros", Universidade Federal da Paraíba, Brazil

^IIIInstituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Brazil

^IVDepartamento de Química, Universidade Federal Rural de Pernambuco, Brazil

^{Correspondence}

ABSTRACT

Several species of Solanum are used in folk medicine to treat diarrhea. Therefore, the aim of this study was to investigate and compare possible antidiarrheal activity of methanol extracts from roots (Sast-MeOH_R) and leaves (Sast-MeOH_L) of Solanum asterophorum Mart., Solanaceae, in mice. Sast-MeOH_R was shown to significantly and dose-relatedly inhibit the frequency of both solid (ED50 309.6±28.5 mg/kg) and liquid (ED50 152.1±32.5 mg/kg) stools. Conversely, Sast-MeOH_L significantly inhibited solid stool frequency only when dosed at 500 and 750 mg/kg (48.7±7.4 and 42.3±9.8%, respectively), but also significantly and dose-relatedly inhibited liquid stools (ED50 268.4±35.2 mg/kg). Thus, Sast-MeOH_R was twice as potent as Sast-MeOH_L in diarrhea inhibition. Neither extracts (when dosed up to 500 mg/kg) inhibited intestinal transit. However, both extracts significantly and dose-relatedly inhibited intestinal fluids, and Sast-MeOH_R (ED50 38.3±10.4 mg/kg) was again twice as potent as Sast-MeOH_L (ED50 78.6±6.4 mg/kg). Results suggest that antidiarrheal effects of Sast-MeOH_R and Sast-MeOH_L involve changes on intestinal secretion. In addition, active metabolites with antidiarrheal activity may be more concentrated in the roots of this species. However further studies are needed to elucidate the action mechanism involved in this activity.

Keywords: antidiarrheal effect in vivo study leaves roots Solanum asterophorum

Introduction

Diarrhea is one of the most common diseases in children worldwide and is characterized by an increase in the number of stools (3 or more/24 h), increased fluidity of stools and/or the presence of blood/mucus in these (Mathan, 1998). It affects people of any age, but is in childhood that this condition may cause higher mortality. It is the third most common cause of disease in children in developing countries and is responsible for about one third of all hospitalizations among children under five years (Souza, 2000). Many plants are used as antidiarrheal in folk medicine, such as Psidium guajava ("guava"), Stachytarpheta cayenensis ("Brazilian tea"), Eugenia uniflora ("Brazilian cherry"), Anacardium occidentale ("cashew"), Mangifera indica ("mango") and Egletes viscosa ("macela") (Almeida et al., 1995; Agra et al., 2008).

There are many Angiosperm families in Northeast Brazil, among which Solanaceae A. L. Jussieu stands out, with 96 genera and 3000 species (Hunziker, 2001). The genus Solanum L. is the most representative of this family, with circa 1400 species (Bohs, 2005) and 5000 described epithets (Nee, 1999), inhabiting tropical and subtropical regions of the world (Agra & Bhattacharyya, 1999). Several species of Solanum are known in folk medicine for showing antidiarrheal activity, such as Solanum marginatum (Abebe, 1986), Solanum khasianum (Jain & Puri, 1984), Solanum torvum (Dominguez & Alcorn, 1985) and Solanum xanthocarpum (Jain & Puri, 1984).

Solanum asterophorum Mart., Solanaceae, is a shrubby species, popularly known as "jurubeba-de-fogo". In Brazil, it is found in Paraiba and Bahia, with indications of folk medicine use against liver dysfunctions (Agra & Bhattacharyya, 1999). Pharmacological studies show that methanol extracts obtained from aerial parts and roots of Solanum astherophorum are bioactive for Artemia salina but have no bioactivity for mollusks (Silva et al., 2007). When studying acute toxicity, Oliveira (2006) showed that the methanol extract obtained from leaves (Sast-MeOH_L) of this species did not produce nor induce behavioral changes and death in mice, while Oliveira et al. (2006) demonstrated that the same extract (Sast-MeOH_L) and the alkaloid isojuripidine isolated from its aerial parts (Silva et al, 2005) did not induce erythrocyte hemolysis in rats, but induced relaxation in guinea pig ileum through voltage-dependent calcium channels type 1.2 (Ca_v1.2).

Since many Solanum species show antidiarrheal activity and S. astherophorum have shown spasmolytic activity in guinea pig ileum, i.e., reduced its contractibility, we decided to investigate and compare possible antidiarrheal activity of methanol extracts from roots (Sast-MeOH_R) and leaves (Sast-MeOH_L) of this species, using castor oil-induced diarrhea models in mice.

Material and Methods

Plant material

Solanum asterophorum Mart., Solanaceae, was collected in the municipality of Areia, Paraíba, Brazil, and identified by Dr. Maria de Fátima Agra at the Laboratório de Tecnologia Farmacêutica "Prof. Delby Fernandes de Medeiros", Universidade Federal da Paraíba. The voucher specimens are deposited in the Herbarium Prof. Lauro Pires Xavier (JPB), UFPB, and duplicates are deposited in the Reference Collection of LTF/UFPB under the identification code "Agra 6002". Powdered aerial parts and roots were extracted with MeOH in a Soxhlet apparatus. The extract was concentrated under vacuum in rotaevaporator. The crude residue was subjected to experiments.

Animals

Female and male Swiss mice (Mus musculus) from the Central Animal Laboratory, Universidade Federal de Alagoas, weighing between 25 and 35 g, were used. Previous to the experiments, the animals were kept under strict control feeding with a balanced diet based on food pellet type and had free access to water, both room ventilation and temperature (21±1 ºC) were controlled and constant. All experimental procedures with Solanum astherophorum were submitted and approved by the Ethical Committee in Research of UFAL (No. 027241/2008 11).

Substances

Atropine and Cremophor^® were obtained from Sigma-Aldrich (USA). Carboxymethylcellulose and castor oil were obtained from Formula, Brazil. Loperamide was obtained from Janssen Cilag Farmacêutica Ltda, Brazil, and the activated charcoal obtained from Proquímios, Brazil.

Preparation of extracts for pharmacological tests

The crude methanol extracts were obtained from roots (Sast-MeOH_R) and leaves (Sast-MeOH_L) of Solanum astherophorum. These were solubilized in Cremophor^® (3%) and diluted in distilled water to obtain stock solutions (500 mg/mL). Upon completion of the experimental protocols the solutions were diluted with distilled water or saline, according to the requirement of each protocol.

Pharmacological behavioral screening and determination of LD50 of Sast-MeOH_R

The Sast-MeOH_R extract was administered orally (p.o.) or intraperitoneally (i.p.) to groups of five males and five females (one dose per group), which fasted for 12 h. Simultaneously, control animals received 10 mL/kg plus Cremophor^®. General signs and symptoms of toxicity, such as analgesia, contortions, aggression and diarrhea, were recorded for 3 h. The animals were also evaluated at 24, 48 and 72 h in order to observe if there was any death.

Evaluation of antidiarrheal activity of Sast-MeOH_R and Sast-MeOH_L

Castor oil-induced diarrhea in mice (Awouters et al., 1978)

Male mice were divided into groups (n=6) and gavaged with 0.9% saline solution 10 mL/kg plus Cremophor^® (negative control), loperamide 10 mg/kg (positive control) and Sast-MeOHR or Sast-MeOHL (doses ranging from 62.5 to 750 mg/kg). After 1 h, 0.01 mL of castor oil per gram was administered orally to each animal to induce diarrhea and the counting and consistency of dung pats was assessed for 3 h, classifying them according to consistency in solids or liquids. ED50 values (dose of a substance that produces 50% of its maximum effect) were obtained by nonlinear regression and expressed as a percentage of the mean±SEM.

Intestinal transit in mice (Rao et al., 1997)

Male mice were divided into groups (n=6) and gavaged with 0.9% saline solution 10 mL/kg plus Cremophor^® (negative control), atropine 2 mg/kg (positive control) and Sast-MeOH_R or Sast-MeOH_L (doses ranging from 62.5 to 750 mg/kg). After 30 min, 0.01 mL of activated charcoal (5%) in carboxymethylcellulose (0.5%) solution was administered orally per gram to each animal, which was euthanized by cervical dislocation after 30 min. The abdominal cavity was opened and the small intestine removed. With the aid of a ruler, its total length (distance between the pylorus and the ileocecal valve) and the distance traveled by the marker were assessed for each animal. The results were expressed as a percentage of the distance traveled by the marker in relation to the total length.

Intestinal fluid accumulation induced by castor oil in mice (DiCarlo et al., 1994)

Male mice, fasted for 24 h with free access to water, were divided into groups (n=6) and gavaged with 0.9% saline solution 10 mL/kg plus Cremophor^® (negative control) and Sast-MeOH_R or Sast-MeOH_L (doses ranging from 25 to 125 mg/kg). After 30 min, 2 mL of castor oil was administered per animal. After other 30 min, the mice were euthanized, the small intestine was dissected from pylorus throughout cecum and the contents were collected into a beaker to assess the volume of fluid. ED50 values were calculated as previously.

Statistical analysis

All results were expressed as a percentage of the mean±SEM, and differences between means were statistically analyzed through t-test (non-paired) or one-way analysis of variance (ANOVA) followed by Bonferroni correction with p<0.05 values considered significant. ED50 and Emax values were assessed through nonlinear regression. All data were analyzed using GraphPad Prism version 3.03 software (GraphPad Software Inc., San Diego, CA, USA).

Results and Discussion

In this study we investigated and compared a possible antidiarrheal activity of crude methanol extract obtained from roots (Sast-MeOH_R) and leaves (Sast-MeOH_L) of Solanum asterophorum Mart., Solanaceae, in mice. For the first time, the strong antidiarrheal activity displayed by Sast-MeOH_R and Sast-MeOH_L extracts was demonstrated.

Since many species of Solanum are used in diarrhea treatment (Abebe, 1986; Jain & Puri, 1984; Dominguez & Alcorn, 1985) and Solanum astherophorum leaf extracts exhibit antispasmodic activity (Oliveira et al., 2006), we hypothesized that these extracts might contain antidiarrheal active metabolites.

The most frequent metabolites in Solanum species are from the alkaloid family, the steroidal alkaloids solasonine and solamargine being found in more than 100 of its species (Blankemeyer et al., 1998; Mesia-Vela et al., 2002), and toxic activity is attributed to these alkaloids (Jadhay et al., 1981; Blankemeyer et al., 1998). Due to the great interest by folk medicine in compounds from Solanaceae plants, many studies have been developed trying to evaluate the toxicological activity of Solanum species (Shakunda & Chester, 1976; Maruo et al., 2003; Heo & Lim, 2005).

Oliveira (2006) showed that Sast-MeOH_L extract did not produce behavioral changes nor did it induce death in mice in an acute toxicity study. Since this is a pioneer work on S. astherophorum roots, the acute toxicological potential of Sast-MeOH_R extract was assessed and a pharmacological monitoring on diarrhea experimental models was conducted.

The administration of Sast-MeOH_R extract orally at doses between 2500 and 5000 mg/kg and intraperitoneally at doses between 1000 and 2000 mg/kg in mice of both sexes also did not produce toxicity-characteristic behavioral changes such as sedation, aggression, urination, diarrhea, convulsion, loss of corneal reflex and ear, piloerection and writhing, nor induced death. Thus, it was not possible to assess LD50 for the tested extracts. The fact that both the root and leaf extracts are devoid of systemic toxicity allowed us a safe choice of the doses used in the experimental protocols performed in order to investigate the antidiarrheal effect of these extracts.

Diarrhea results from rapid movement of fecal matter through the large intestine (Guyton & Hall, 2006). To restore this process, many patients require antidiarrheal therapy in order to increase flow resistance and mucosal absorption and decrease secretion (Akindele & Adeyemi, 2006). To verify the antidiarrheal effect of Sast-MeOH_R and Sast-MeOH_L, castor oil was used since it is widely employed for screening drugs with this property. One of the advantages of this model is the high reproducibility of the evacuation of stool formed in less than one hour after the administration of laxatives (Borrelli et al., 2006). The results of oral administration of loperamide (10 mg/kg) and Sast-MeOH_R (250, 500 and 750 mg/kg) showed that there was significant dose-related inhibition of defecation frequency (97.9±2.1, 34.3±5.6, 47.8±9.7 and 70.1±6.3, respectively) when compared to the negative control (0.9% saline solution+Cremophor^®) (Figure 1A). Regarding liquid stools, both loperamide (10 mg/kg) and Sast-MeOH_R (125, 250, 500, and 750 mg/kg) showed significant, dose-related inhibition percentages of 43.6±4.4, 58.0±8.5, 80.6±12.6 and 100, respectively (Figure 1B). On the other hand, Sast-MeOH_L extract (Figure 2A) showed significant inhibitory effect on defecation frequency only when dosed at 500 and 750 mg/kg (48.7±7.4 and 42.3±9.8%, respectively). Regarding liquid stools, Sast-MeOH_L extract showed inhibitory effect when dosed at 250, 500 and 750 mg/kg, with significant dose-related inhibition frequencies of 32.9±10.1, 80.6±11.1 and 88.4±5.2%, respectively (Figure 2B). Regarding defecation frequency, only Sast-MeOH_R produced dose-related inhibition, with 309.6±28.5 mg/kg ED50. However, for liquid stools, Sast-MeOH_R extract (ED50 152.1±32.5 mg/kg) was more effective than Sast-MeOH_L (ED50 268.4±35.2 mg/kg) in inhibiting diarrhea. Therefore, both Sast-MeOH_R and Sast-MeOH_L produced significant inhibitory effect on diarrhea in mice in this induction model, as observed by reducing both solid and liquid stool frequencies, suggesting that both extracts have active metabolites with antidiarrheal activity. Conversely, the root extract showed stronger antidiarrheal effect than that from the leaves, suggesting that the active antidiarrheal metabolites are more frequent in the former than in the latter.

Clinically, diarrhea is a consequence of intestinal function disorders which lead to excessive intestinal secretion, impaired intestinal absorption and/or rapid transit (Menezes et al., 1994). Therefore, the antidiarrheal effect of the extracts was studied with experimental castor oil-induced diarrhea models and castor oil-induced intestinal transit and intestinal fluid accumulation in mice. Drugs that inhibit intestinal transit in pathophysiological states may be effective in relieving diarrhea (Borrelli et al., 2006). Activated charcoal, employed in the gastrointestinal transit model as a marker, has been used for over 60 years as a simple tool for evaluating the effect of laxatives (Gaginella et al., 1994). This method is an indicator of the maximum distance traveled by the marker (activated charcoal) through its administration and the assessment of its path in the small intestine over a period of time (Gurgel, 2000). According to this model, the results of oral administration of atropine (2 mg/kg) show that there was significant inhibition of the distance traveled by charcoal in the intestine of mice of circa 32.7±3.6%, when compared to the negative control (0.9% saline+Cremophor^®). Still, neither Sast-MeOH_R nor Sast-MeOH_L extracts reduced the distance travelled by the marker when dosed at 125, 250 or 500 mg/kg (data not shown).

Though it is common belief that the small intestine plays only an absorptive role, it also secretes water and electrolytes. This enhanced secretory capacity is clearly shown through the administration of large amounts of castor oil (Gurgel, 2000). Thus, we assessed the effects of Sast-MeOH_R and Sast-MeOH_L extracts in mice using a castor oil-induced intestinal fluid accumulation model and evaluated the fluid amount in comparison to the control group. Loperamide (69.2±1.5%), Sast-MeOH_R and Sast-MeOH_L extracts inhibited significantly and dose-relatedly the intestinal fluid content induced by castor oil dosed at 25, 50, 75, 100 and 125 mg/kg in comparison to negative control (0.9% saline+Cremophor^®). When compared to the negative control, inhibition values for the Sast-MeOH_R extract were 21.6±7.5, 30.4±4.9, 42.2±2.4, 60.8±3.0 and 71.1±2.4%, respectively, and 37.2±6.4, 52.9±4.0, 51.0±4.5 and 59.3±2.5% for Sast-MeOHL, respectively (Figure 3). Sast-MeOH_R extract (ED50 38.3±10.4 mg/kg) was about twice as effective as Sast-MeOH_L (ED50 78.6±6.4 mg/kg) in inhibiting intestinal fluid content, reinforcing the idea that the secondary metabolites with antidiarrheal activity are more frequent in the roots than in the leaves.

The most important conclusions of this study are the inedited demonstrations that Solanum astherophorum Mart. displays antidiarrheal activity, which surely brings great contribution to the pharmacology of this species, as well as showing that the root extract has more effective antidiarrheal activity than the leaf extract. Since steroidal alkaloid isojuripidine was isolated from the aerial parts of S. asterophorum, part of this activity can be attributed to it. Further studies are needed, including verifications of the amount and type of root alkaloids.

Acknowledgment

The authors thank the financial support by PIBIC/UFAL and FAPEAL.

Received 3 Sep 2010

Accepted 20 May 2011

• Abebe W 1986. A survey of prescriptions used in traditional medicine in Gondar region, northwestern Ethiopia: general pharmaceutical practice. J Ethnopharmacol 18: 147-165.
• Agra MF, Bhattacharyya J 1999. Ethnomedicinal and phytochemical investigation on the Solanum species in the Northeast of Brazil. In: Nee M, Symon DE, Lester RN, Jessop JP. (Eds). Solanaceae IV, Royal Botanic Gardens, Kew, p. 341-343.
• Agra MF, Silva KN, Basílio IJLD, Freitas PF, Barbosa-Filho JM 2008. Survey of medicinal plants used in the region Northeast of Brazil. Rev Bras Farmacogn 18: 472-508.
• Akindele AJ, Adeyemi OO 2006. Evaluation of the antidiarrhoeal activity of Byrsocarpus coccineus. J Ethnopharmacol 108: 20-25.
• Almeida CE, Karnikowski MGO, Foleto R, Baldisserotto B 1995. Analysis of antidiarrhoeic effect of plants used in popular medicine. Rev Saude Publ 29: 428-33.
• Awouters F, Neimegeers CJE, Lenaert FM, Janssen 1978. Delay of castor oil diarrhoea in rats; A new way to evaluate inhibitors of prostaglandin's biosynthesis. J Pharm Pharmacol 30: 41-45.
• Blankemeyer JT, Mcwilliams ML, Rayburn JR, Weissenberg M, Friedman M 1998. Developmental toxicology of solamargine and solasonine glycolalkaloids in frog embryos. Food Chem Toxicol 36: 383-389.
• Bohs L 2005. Major clades in Solanum based on ndhF sequences. In Keating RC, Hollowell VC, Croat TB (eds.) Monographs in Systematic Botany from the Missouri Botanical Garden. A festschrift for William G. D'Arcy: the legacy of a taxonomist. St. Louis, Missouri Botanical Garden Press 104: 27-49.
• Borrelli F, Capasso F, Capasso R, Ascione V, Aviello G, Longo L, Izzo AA 2006. Effect of Boswellia serrata on intestinal motility in rodents: inhibition of diarrhoea without constipation. Brit J Pharmacol 148: 553-560.
• Dicarlo GD, Mascolo N, Izzo AA, Capasso F 1994. Effect of quereetine on the gastrintestinal tract in rats and mice. Phytother Res 8: 42-45.
• Dominguez XA, Alcorn JB 1985. Screening of medicinal plants used by huastec mayans of northeastern Mexico. J Ethnopharmacol 13: 139-156.
• Gaginella TS, Mascolo N, Izzo AA, Autore G, Capasso F 1994. Nitric oxide as a mediator of bisacodyl and phenolphthalein laxative action: induction of nitric oxide synthase. J Pharmacol Exp Ther 270: 1239-1245.
• Gurgel LA 2000. Avaliação experimental da atividade antidiarréica do látex do Croton urucurana Baill. Fortaleza, 146 p. Dissertação de mestrado. Departamento de Fisiologia e Farmacologia. Centro de Ciências da Saúde. Universidade Federal do Ceará
• Guyton AC, Hall JE 2006. Tratado de Fisiologia Médica Elsevier. Rio de Janeiro, p. 822-823.
• Heo KS, Lim KT 2005. Glicoprotein isolated from Solanum nigrum L. modulates the apoptotic-related signals in 12-o-tetradecanoylphorbol 13-acetate-stimulated MCF-7 cells. J Med Food 8: 69-77.
• Hunziker AT 2001. Genera Solanacearum: The genera of Solanaceae illustrated, arranged according to a new system. A. R. Gantner Verlag, Köenigstein, p. 401-486.
• Jain SP, Puri HS 1984. Ethnomedicinal plants of jaunsar-bawar hills, uttar pradesh, india. J Ethnopharmacol 12: 213-222.
• Jadhay SJ, Sharma RP, Salunkhe DK 1981. Naturally occurring toxic alkaloids in foods. Toxicology 9: 21-104.
• Maruo VM, Bernardi MM, Spinosa HS 2003. Toxicological evaluations of long-term consumption of Solanum lycocarpum St. Hill fruits in male and female adult rats. Phytomedicine 10: 48-52.
• Mathan VI 1998. Diarrhoeal diseases. Brit Med Bull 54: 402-419.
• Menezes MAS, Viana GSB, Vietla SNR, Corrêa RA, Feijó SGF 1994. Estudo farmacológico. In: Viana GSB, Matos FJA, Bandeira MAM, Rao VSN. Aroeira do sertão (Myracrodruon urundeuva Fr. All.). Fortaleza, UFC 187: 83-174.
• Mesia-Vela S, Santos MT, Souccar C, Lima-Landman MTR, Lapa AJ 2002. Solanum paniculatum L. (Jurubeba): Potent inhibitor of gastric acid secretion in mice. Phytomedicine 9: 508-514.
• Nee M 1999. Synopsis of Solanum in the New World. In: Nee M, Symon DE, Lester RN, Jessop JP. Solanaceae IV: Advances in Biology and Utilization. Royal Botanic Gardens, Kew, p. 285-333.
• Oliveira RCM 2006. Efeito antiinflamatório, antinociceptivo e espasmolítico de Solanum megalonyx Sendtn. e Solanum asterophorum Mart. (Solanaceae): um estudo comparativo. João Pessoa, 123 p. Tese de doutorado, Programa de Pós-graduação em Produtos Naturais e Sintéticos Bioativos. Universidade Federal da Paraíba.
• Oliveira RCM, Lima JT, Ribeiro LAA, Silva JLV, Monteiro FS, Assis TS, Agra MF, Silva TMS, Almeida FRC, Silva BA 2006. Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum. Z Naturforsch C 61: 799-805.
• Rao VSN, Santos FA, Sobreira TT, Souza MF, Melo CL, Silveira ER 1997. Investigations on the gastroprotective and antidiarrhoeal properties of ternatin, a tetramethoxyfl avone from Egletes viscose. Planta Med 63: 146-149.
• Shakunda C, Chester AS 1976. Teratological and toxicological studies of alkaloidal and phenolic compounds from Solanum tuberosum L. Toxicol Appl Pharm 36: 227-237.
• Silva TMS, Costa RA, Oliveira EJ, Barbosa-Filho JM, Agra MF, Camara CA 2005. Complete ¹H and ¹³C NMR assignments of Isojurupidine from Solanum asterophorum Mart. J Brazil Chem Soc 16: 1467-1471.
• Silva TMS, Nascimento RJB, Batista MM, Agra MF, Camara CA 2007. Brine shrimp bioassay of some species of Solanum from Northestern Brazil. Rev Bras Farmacogn 17: 35-38.
• Souza EC 2000. Perfil etimológico das diarréias agudas em crianças atendidas em um pronto socorro de hospital regional da cidade de São Paulo São Paulo. Dissertação de Mestrado. Faculdade de Medicina/USP.

Publication Dates

• Publication in this collection
  30 Sept 2011
• Date of issue
  Feb 2012

History

• Received
  03 Sept 2010
• Accepted
  20 May 2011