In-vivo antidiarrheal activity: From the crude extract and solvent fractions of Rhamnus prinoides (Rhamnaceae) leaves

Objectives The inherent toxicities of the drugs urge the search for alternative drugs that are safe and effective. Therefore, the objective of the study is to evaluate the in-vivo anti-diarrheal activity of crude extract and solvent fractions of Rhamnus prinoides leaves. Methods The Leaves of Rhamnus prinoides were macerated using absolute methanol and then fractionated. For in-vivo antidiarrheal activity evaluation of the crude extract and solvent fraction, castor oil-induced diarrhea, castor oil-induced anti-enteropolling, and intestinal transit models were used. One-way analysis of variance was used to analyze the data, followed by a Tukey post-test. The standard and negative control groups were treated with loperamide and 2% tween 80 respectively. Results A significant reduction in the frequency of wet stools and watery content of diarrhea, intestinal motility, intestinal fluid accumulation, and delaying the onset of diarrhea as compared with controls were observed in mice treated with 200 mg/kg and 400 mg/kg ME. However the effect increased dose-dependently, and the 400 mg/kg ME produced a comparable effect with the standard drug in all models. Amongst the solvent fractions, n-BF significantly delayed the time of diarrheal onset and reduced the frequency of defecation, and intestinal motility at doses of 200mg/kg and 400mg/kg. Furthermore, the maximum percentage inhibition of intestinal fluid accumulation was observed in mice treated with 400 mg/kg n-BF (p<0.01; 61.05%). Conclusion The results of this study showed that crude leaves extract and solvent fractions of Rhamnus prinoides had significant anti-diarrheal activity, providing scientific support for its traditional use as a diarrhea treatment.


Introduction 50
More than 80% of the world's population, according to the World Health Organization (WHO), 51 relies on traditional medicine for their primary healthcare requirements. It is commonly stated 52 that plants are responsible for 25% of all medications given today. 53 according to this estimate, account for a large portion of natural product-based pharmaceuticals 54 [1]. Herbal products derived from medicinal plants are chosen because they require less testing, 55 are safer, more efficient, culturally acceptable, and have fewer adverse effects [2]. 56 There have been numerous reports of traditional plants being used to treat diarrheal illnesses.

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Herbal goods are believed to be more compatible with the human body since they contain 58 chemical substances that are a part of organism physiology [2]. Plant extracts can lower 59 electrolyte release, delay gastrointestinal transit, restrict gut motility, enhance water adsorption, constipation (Loperamide), and addiction [5]. As a result, it is a must to expand research on 75 culturally preserved medicinal plants to discover alternative pharmaceuticals derived from 76 natural sources. 77 Furthermore, there is a wealth of ethnomedical information on plant usage in the scientific 78 literature that has yet to be gathered in a useable manner [1]. Rhamnus prinoides (R. prinoides) 79 (Rhamnaceae) is also known as Gesho in Ethiopia. It is used in traditional medicine to treat 80 headaches, neck ulcers, and edema [6]. It is one of the numerous herbs that have traditionally 81 been used to treat diarrhea. This study will confirm the plant's traditional use and elucidate the 82 nature of the phytochemical constituents responsible for its effect and possible ways of 83 antidiarrheal action. The results from the current study can be used to aid in the development of 84 novel anti-diarrheal agents that address issues with current anti-diarrheal medications. It will also 85 guide traditional users on how to prepare and use the plant in various ways. Furthermore, the 86 findings of this study will aid the scientific community in furthering their research into the plant 87 R. prinoides by launching advanced studies into molecular mechanisms and formulation of plant 88 source drugs, as well as identifying the specific agent responsible for the anti-diarrheal effect. 89 Phytochemical screening revealed the presence of phenols, flavonoids, alkaloids, saponin, 90 glycoside, and tannins in this medicinal plant, as well as the presence of five different labdane 91 types of diterpene lactones in Ethiopian collections of R. prinoides leaves [7]. Furthermore, R. 92 prinoides have been shown to have analgesic, anti-inflammatory, antihelmintic, and antibacterial 93 action in animals [6]. In multiple experimental models, these findings suggest that R. prinoides 94 may have anti-diarrheal potential. As a result, the aim this study is to prove the reputed anti-  To eliminate dust elements, the dried leaves of R. prinoides were first washed in distilled water.

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Before extraction, it was ground with a grinder and roughly powdered with a mortar and pestle.

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Seven hundred grams of powdered leaves of R. prinoides were macerated using one liter of 123 absolute methanol (1:6 w/v). The mixture was then filtered through Whatman No. 1 filter paper, 124 and the residue was re-macerated with fresh solvent exhaustively. The filtrates from the three 125 batches were mixed and concentrated in a rotary evaporator at 40°C to remove methanol. After 126 drying, black sticky residue weighing 90 grams (12.86%) of R. prinoides crude extract was 127 collected.

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In a separatory funnel, 180 ml of n-butanol was used to suspend 80 grams of the crude extract, 129 and then an equal amount of petroleum ether was added and thoroughly combined. After 130 allowing the mixture to separate into a discrete layer, the petroleum ether portion was isolated by 131 eluting the bottom layer. The leftover material was then combined with an equivalent amount of 132 chloroform and separated in the same way. For the ME and solvent fractions, mice of either sex (weighing 20-30g) were randomly allocated 149 into five and eleven groups, each with six animals. Prior to the test, the mice fasted for 18 hours 150 while having unlimited access to water. The first two groups were given 10 ml/kg of 2 percent 151 tween80 as a negative control and 3 mg/kg of loperamide as a positive control, respectively, for 152 the investigation of the antidiarrheal properties of both ME and the solvent fractions. The ME 153 and two solvent fractions of R. prinoides leaves were administered to the remaining groups at 154 test doses of 100, 200, and 400 mg/kg. In all current models, diarrhea was induced using castor 155 oil. All of the doses were administered orally.

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Phytochemical screening of the crude extract and solvent fractions 157 Standard tests were used to conduct preliminary phytoconstituent screening of secondary 158 metabolites from both ME and the solvent fractions of the leaves of R. prinoides [10,11].

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Test for saponins 160 The 5 ml distilled water was added to 0.25 grams of ME and the solvent fractions of the leaves of 161 R. prinoides. The solution was then violently shaken, and stable, continuous foam was seen.

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Saponins were detected by the formation of a steady froth that lasted about half an hour.

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Test for terpenoids 164 The 2 ml chloroform was added to 0.25 grams of ME and the solvent fractions of the leaves of R. 165 prinoides. Then, to build a coating, 3ml concentrated sulfuric acid was carefully applied. The 166 presence of terpenoids was indicated by a reddish brown coloration of the interface.

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Test for tannins 168 In a test tube, 0.25 grams of ME and the solvent fractions of the leaves of R. prinoides were 169 cooked in 10 ml water and then filtered using filter paper (Whatman No. 1). To the filtrate, a few 170 drops of 0.1 percent ferric chloride were added. Brownish green or blue-black precipitate is a 171 sign of tannin content.

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Test for flavonoids 173 10 ml of ethyl acetate, 0.2 grams of ME, and the solvent fractions of R. prinoides leaves were 174 heated in a water bath for 3 minutes. The mixture was cooled after filtering. The filtrate was then 175 combined with 1 ml of a mild ammonia solution in 4 ml and agitated. When the layers were 176 allowed to separate, the ammonia layer's yellow tint confirmed the presence of flavonoids.

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Test for cardiac glycosides 178 The 2 ml glacial acetic acid containing one drop of ferric chloride solution was added to 0.25 179 grams of ME and the solvent fractions of the leaves of R. prinoides that had been diluted with 5 180 ml of water. The 1 ml of sulfuric acid was used as a base. The presence of a deoxysugar, which 181 is characteristic of cardenolides, was identified by a brown ring at the interface.

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Test for steroids 183 The 0.25 grams of ME and the fractions of the leaves of R. prinoides were mixed with 2 ml 184 sulfuric acid and two ml acetic anhydride. Some samples changed color from violet to blue or 185 green, indicating the presence of steroids. .

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Before the experiment, mice of either sex were fasted overnight and then randomly divided into 217 five and eleven groups (6 mice per group) for ME and solvent fractions respectively. Then, the 218 mice were treated with 10 ml/kg 2% tween80, 100, 200, and 400 mg/kg of ME and solvent 219 fractions, and a standard medication (loperamide hydrochloride 3 mg/kg) orally. After 1 hour of 220 treatment administration, the animals were given castor oil, 0.5 ml. The mice were cervically 221 dislocated one hour after receiving castor oil.

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The small intestine was then taken from each animal's abdomen and knotted with thread at the 223 pyloric end and the ileocaecal junction. The contents of the dissected small intestine were milked 224 into a graduated tube and their volume was determined after the dissected small intestine was 225 weighed. After milking, the intestine's weight was determined, and the difference between the 226 weight of the intestines when they were full and empty was computed. Finally, using the 227 calculations below, the percentage inhibitions of intestinal secretion (volume and weight) were 228 estimated relative to the negative control.

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Percent of inhibition = -× 100 11 230 'A' represents the average volume or weight of the intestine in the control group, while 'B' 231 represents the average volume or weight of the intestine in the test groups [12].

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The weight was recorded as (m1-m0) g and the volume of the intestinal contents was read from 233 the graduated measuring cylinder.

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Castor oil-induced gastrointestinal motility test

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Each animal received 0.5 ml of castor oil orally 1 hour after dosing the test agents. All mice were 240 given 1ml of 10% charcoal suspension orally after 1 hour of castor oil administration and 241 slaughtered after 30 minutes. The length of the small intestine was measured after it was 242 dissected, and the distance traveled by charcoal from the pylorus to the caecum was estimated.

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Each mouse's intestine was preserved in formalin to stop peristalsis, and then rinsed in distilled 244 water before the distance covered by the charcoal was measured. The peristaltic index (PI) for 245 the charcoal movement was calculated as follows: Where 'A' is the length of the entire intestine and 'B' is the distance covered by charcoal. The  The presence of alkaloids, tannins, flavonoids, and saponins were found in the ME and the 272 solvent fractions of the leaves of R. prinoides (Table 1). 273 Acute Toxicity Test

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Physical and behavioral abnormalities, as well as mortality, were not seen in the acute toxicity  and 200 mg/kg PtEF but the magnitude of diarrhea inhibition is lower than the n-BF ( Table 2). 320 Moreover, the 100 and 200 mg/kg of PtEF lack a significant reduction in all parameters even 321 compared with the negative control ( Table 2). There was no significant difference between the 322 standard and 400 mg/kg ME of R. prinoides and n-BF, but significant differences among doses 323 of ME of R. prinoides and PtEF were examined ( Table 2).

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The effect of ME and solvent fractions of R. prinoides on castor oil-induced 325 gastrointestinal motility 326 As described in were comparable with the 3 mg/kg loperamide drug (65.90%).

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The purpose of this study was to determine the in-vivo anti-diarrheal activity of the absolute 371 methanol leave crude extract (ME) and solvent fractions of Rhamnus prinoide, as well as the 372 likely underlying mechanism. In the models employed, the plant was found to have anti-diarrheal 373 activity.

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Castor oil's induced diarrheal has been studied using a variety of methods. These include

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The standard drug, loperamide hydrochloride, not only regulates the gastrointestinal tract but 382 also slows peristalsis across the small intestine. Nowadays, loperamide is commonly utilized in 18 383 several diarrheal models to study the antidiarrheal properties of diverse experimental plants. This 384 is due to its proven antisecretory and antimotility features [19].

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In this study, the ME of R. prinoides showed anti-diarrheal activity by reducing castor oil-

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induced diarrhea in all of the models employed. The presence of various phytochemicals in the 387 ME and solvent fractions of R. prinoides is the most reasonable explanation ( Table 1). 388 Antioxidant features were found in both flavonoids and phenolic compounds [20], which are to the n-BF and CF fractions that creates concentration variation (Table-1 as compared with ME, n-BF, and CF of R. prinoides might be due to the absence of 473 phytochemicals responsible for antisecretory action ( Table 1).

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One strategy to increase the production of diarrhea is to increase intestinal motility. The study 475 used charcoal meal as a marker to test the antimotility activity of the ME of R. prinoides. The can therefore be explained by the extracts' ability to inhibit intestinal movement (Table 3). In other words, the crude extracts' inhibitory effect would be higher as intestinal motility increased.

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The significance of this discovery shouldn't be disregarded, as it means that the risk of constipation, 499 which is a common side effect of most conventional medications, including loperamide, will be 500 reduced. The presence of phytochemicals that are responsible for the anti-motility action of ME of 501 R. prinoides could be linked to the presence of phytochemicals that are responsible for the anti-