Role of the Rosa canina L. leaf extract as an antidiarrheal drug in rodents.

OBJECTIVES: The objective of the study was to investigate the effect of the leaf extract of Rosa canina L. against experimental diarrhea induced by castor oil in rodents.
MATERIALS AND METHODS: The methanol extract of Rosa canina L. (30 and 60 mg/kg body weight) was administered orally to two groups of mice (five animals per group) in order to evaluate the activity of the extract against the castor oil-induced diarrhea model in mice. Two other groups received normal saline and diphenoxylate (5 mg/kg) as positive control. The effect of the extract on intestinal transit and castor oil-induced intestinal fluid accumulation (enteropooling) was assessed. The effects of the extract on the isolated rabbit jejunum and on the isolated rat ileum were studied.
RESULTS: The preliminary phytochemical screening of the leaf extract of Rosa Canina L. revealed the presence of alkaloids, flavonoids, glycosides, saponins, and volatile oil. Intraperitoneal LD50 of the extract was found to be 455.19 ± 23 mg/kg in mice. The antidiarrheal effect of the methanolic extract exhibited a concentration-dependent inhibition of the spontaneous pendular movement of the isolated rabbit jejunum and inhibited acetylcholine-induced contraction of the rat ileum. A dose-dependent decrease in gastrointestinal transit was observed with extracts (30 and 60 mg/kg), which also protected mice against castor oil-induced diarrhea and castor oil-induced fluid accumulation, respectively.
CONCLUSIONS: The presence of some of the phytochemicals in the leaf extract may be responsible for the observed effects, and also the basis for its use in traditional medicine as an antidiarrheal drug.

Introduction

Diarrheal diseases are one of the leading causes of morbidity and mortality in developing countries and are responsible for the death of millions of people each year.[1] Despite immense technological advancement in modern medicine, many people in the developing countries still rely on the healing practices and medicinal plants for their daily health care needs.[2] Therefore, the World Health Organization encouraged studies for the treatment and prevention of diarrheal diseases depending on traditional medical practices.[3] Rosa canina L. belongs to the family Rosaceae.[4] It is a deciduous shrub normally ranging in height from 1 to 5 m, though sometimes it can scramble higher into the crowns of taller trees. A search in the NIPRALERT database did not yield any information on the pharmacological effect of the methanolic leaf extract of the plant on castor oil-induced diarrhea, small intestinal propulsion, castor oil-induced fluid accumulation, and its effect on the gastrointestinal smooth muscle.

Materials and Methods

Animals

Adult male Wistar rats weighing 150–180 g, albino mice weighing 18–22 g, and rabbits weighing 2.3–2.5 kg were used for the studies. The animals were purchased from the animal house of Sudhakarrao Naik Institute of Pharmacy, Pusad, India. The animals were housed in polypropylene cages at room temperature 28 ± 2°C and under a 12/12 h light/dark cycle. All animals were fed with a standard pellet diet and water ad libitum. Animals were treated in line with the guide and care for laboratory animals. The approval of the institutional animal ethics committee was obtained for the study.

Plant Material

Leaves of R. canina were collected from the botanical garden of S. N. Institute of Pharmacy, Pusad, India.

Preparation of Plant Extract

The leaves of R. canina were washed with clean water, air-dried, pulverized using a pestle and mortar and sieved with a 0.3-mm sieve. One hundred grams of the coarsely powdered leaves was extracted with 500 ml of methanol using soxhlet apparatus for 12 h. Methanol was evaporated using a rotary evaporator at less than 40°C. Distilled water was used to reconstitute the solid extract to obtain the desired concentration for the studies.

Phytochemical screening

A preliminary phytochemical screening for the presence of alkaloids, flavonoids, tannins, glycosides, resins, phenols, volatile oils, and saponins was carried out using standard test procedures.[5]

Acute Toxicity Studies

The acute toxicity studies were carried out based on the method described by Lork.[6] A sterilized extract was administered intraperitoneally to mice, and the dose that killed 50% of the animal population was estimated as the LD50.[7]

Effect of the Extract on Intestinal Propulsion

The effect of R. canina on intestinal propulsion in rats was tested using the charcoal meal method.[8] The rats were fasted for 24 h but allowed free access to water. They were randomized and placed in three plastic cages with five animals per cage corresponding to three groups of animals. Group 1 was administered distilled water orally by orogastric cannula. Groups 2 and 3 were pretreated with the 30 and 60 mg/kg extract of R. canina, respectively. After 30 min, each rat was administered 1 ml of 5% activated charcoal suspended in 10% aqueous tragacanth orally. The rats were sacrificed 30 min later by the inhalation of chloroform and the abdomen was exposed after dissection; the distance traveled by the charcoal plug from the pylorus to the cecum was then measured.[9]

Effect of the Extract on Castor Oil-induced Diarrhea

The method of Awouter et al.[1011] was adopted in assaying for the effect of the R. canina extract on castor oil-induced diarrhea. Mice were weighed and grouped into four groups (n = 5). Group 1 received distilled water, groups 2 and 3 were administered 30 and 60 mg/kg extract respectively orally, while group 4 received diphenoxylate (5 mg/kg) orally. Each animal was then given 0.5 ml of castor oil orally after 30 min of treatment and placed in transparent cages to observe for the consistency of fecal matter and the frequency of defecation for 4 h. Feces were collected with an absorbent sheet of paper placed beneath the transparent cages.[9] The wet feces were read at the end of the experiment by lifting up the upper part of the cage containing the sheet of paper and animals.

Effect of the Extract on Castor Oil-induced Intestinal Fluid Accumulation

This was determined as described and modified by Robert et al.[12] and Dicarlo et al.[13] Briefly, the rats were fasted for 24 h but allowed free access to water. The rats were randomized and placed in three cages of four rats per cage. Group 1 was administered distilled water, while groups 2 and 3 were pretreated with 30 and 60 mg/kg of the extract, respectively. After 30 min, each rat was administered 2 ml castor oil. The rats were anesthetized 30 min later by the inhalation of chloroform. The small intestine from the pylorus to the cecum was dissected out and its content expelled into a measuring cylinder to measure the volume of the fluid.[7]

The Effect of the Extract on the Isolated Rabbit Jejunum

Each of the adult rabbits was starved of feed for about 18 h and sacrificed by a blow on the head. Each was exsanguinated and the abdomen was cut open. About 2-cm piece free of mesentery of the ileum was mounted in a 20 ml organ bath containing aerated Tyrode solution at 37°C. A tension of 0.5 g was applied, connected to Ugo Basil Unirecorder microdynamometer, sensitivity of 1.0 and the speed of 20 mm/min. The effect of 10-min incubation of the extract on the tissue responses to acetylcholine was tested.[1415]

Effect of the Extract on the Isolated Rat Ileum

Rats were starved of feed for 18 h but allowed access to water. The rats were exsanguinated and the stomach was dissected to expose the abdomen. About 2 cm of the rat ileum was removed, dissected free of the adhering mesentery, and mounted in a 20-ml organ bath containing aerated Tyrode solution. The effect of acetylcholine and histamine at a different concentration of the R. canina leaf extract was tested after 10 min of incubation at 30°C.

Statistical Analysis

Results are presented as mean ± SEM and simple percentages. Student's t-test was used to determine the significant difference between two groups (P < 0.05).

Results

The extract represents 31.12% plant material. The result of the phytochemical screening revealed the presence of alkaloids, flavonoids, glycosides, saponins, and volatile oil. The LD50 of the extract was estimated to be 455.19 mg/kg (i.p.) in mice. The effect of the methanolic extract of the leaves of R. canina on intestinal propulsion [Table 1] revealed that 30 mg/kg of the extract decreased the intestinal propulsion by 24.96%, while 60 mg/kg of the extract caused a decrease of 32.83% in intestinal propulsion. The effect of the extract on castor oil-induced diarrhea in mice [Table 2] showed a dose-dependent decrease in the number of fecal matter passed by the animals. For 30 and 60 mg/kg extracts, a significant (P < 0.05) reduction in diarrhea was observed representing 36.11% and 41.66% inhibition, respectively. Diphenoxylate inhibited the castor oil-induced diarrhea by 66.66%. Studies on intestinal fluid accumulation [Table 3] revealed that both 30 and 60 mg/kg of the extract significantly inhibited fluid accumulation at the levels of 27.16% and 40.74%, respectively. The methanolic extract of the leaves of R. canina exhibited no effect on the isolated rabbit jejunum and rat ileum at the lower doses used (0.1–0.8 mg/ml). However, at higher doses (1.6–12.8 mg/ml), a dose-dependent relaxation of the tissue was observed – a 3.2 mg/ml dose of the extract showed 49% relaxation. The contraction caused by histamine (0.8 mg/ml) and acetylcholine (0.4 mg/ml) on the tissues was reversibly inhibited by 3.2 mg/ml of the extract [Figures 1 and 2, respectively].

Table 1

Effect of the methanolic leaf extract of Rosa canina leaves on the gastrointestinal transit in rats

Table 2

Effect of the Rosa canina extract on castor oil-induced diarrhea in mice

Table 3

Effect of the Rosa canina leaf extract on castor oil-induced intestinal fluid accumulation

Figure 1

Inhibitory effect of the Rosa canina L. leaf extract (mg/ml) on histamine-induced spontaneous contraction of the rabbit jejunum

Figure 2

Effect of the Rosa canina L. leaf extract (mg/ml) on acetylcholine-induced contraction of the rat ileum

Discussion

The phytochemical screening of the methanol extract of R. canina leaves revealed the presence of alkaloids, glycosides, saponins, and volatile oils. The LD50 value of the methanol extract of R. canina leaves was found to be 455.21 mg/kg (i.p.) in mice. Above this dose, the animals exhibited tonic signs, which indicated that the extract might not be relatively safe. Values greater than 1 g had been reported to be generally safe for all practical purposes in the laboratory.[10] The present study sought to assess the antidiarrheal activity of the plant. Our results showed that the extract significantly (P < 0.05) inhibited castor oil-induced diarrhea in rats. Several mechanisms had been previously proposed to explain the diarrheal effect of castor oil. These include the inhibition of the intestinal Na+/K+ -ATPase activity, thus reducing normal fluid absorption,[16] activation of adenylate cyclase or mucosal cAMP-mediated active secretion,[17] stimulation of prostaglandin formation,[18] and platelet activating factor.[19] Most recently, nitric oxide has been claimed to contribute to the diarrheal effect of castor oil.[20] However, it is well documented that castor oil produces diarrhea due to its most active component ricinoleic acid through a hypersecretory response.[2122] Therefore, it can be assumed that the antidiarrheal action of the extract was mediated by an antisecretory mechanism. This was also evident from the inhibition of castor oil-induced fluid accumulation by the extract, and inhibitions of the gastrointestinal transit by yohimbine suggest that the effect was mediated by α-2 adrenoceptor activation. The results were comparable to those of the standard drug, diphenoxylate. Furthermore, the extract significantly reduced intestinal transit as evidenced by the decrease in the distance traveled by the charcoal meal. These results also show that the extract suppressed the propulsion of the charcoal meal thereby increasing the absorption of water and electrolytes. The inhibition of acetylcholine and histamine by higher doses of the extract (1.6–12.8 mg/ml) in the rat ileum and rabbit jejunum, respectively, suggests that the extract probably contains substances that act through cholinergic and histaminergic antagonistic mechanisms. Some of the secondary metabolites present in the leaf extract have been implicated as having an antidiarrheal activity. The inhibitory activity of flavonoids on intestinal motility in a dose-related manner was earlier reported.[1323] Saponins have also been reported to inhibit the histamine release in vitro.[24] We suggest that saponins and/or flavonoids present in the leaf extract might be responsible for its antidiarrheal activity. From this study, the use of the leaves of R. canina in traditional medicine as a nonspecific antidiarrheal agent has been justified. Further studies are, however, needed to establish the safety of the extract and to possibly isolate the active principle responsible for the observed effects.

Conclusion

The results of this investigation revealed that R. canina contains pharmacologically active substances with antidiarrheal properties. These attributes may provide the rationale for the use of R. canina in diarrhea management by traditional healers. Further research is needed to fractionate the methanol extract and isolate the molecule(s) responsible for the antidiarrheal activity observed.