By Oguwike, RC; Iloh, SE; Oguwike, FN (2023).

Greener Journal of Epidemiology and Public Health

ISSN: 2354-2381

Vol. 11(1), pp. 53-58, 2023

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Efficacy of Ethanolic Leaf Extract of Grona triflora on Castor Oil Induced Diarrhoea in Albino Mice

Oguwike, R.C¹; Iloh, S.E¹; Oguwike, F.N²

¹Dept. of Pharmacology, Faculty of Pharmaceutical Sciences, Chukwuemeka Odumegwu Ojukwu University Igbariam Anambra State.

²Dept. of Pharmacognosy, Faculty of Pharmaceutical sciences, Chukwuemeka Odumegwu Ojukwu University Igbariam Anambra state.

ARTICLE INFO

ABSTRACT

Article No.: 072523069

Type: Research

Full Text: PDF, PHP, HTML, EPUB, MP3

The effect of ethanol leaf extract of Grona triflora on castor oil induced diarrhea in albino mice was investigated. A total of twenty-five (25) male mice were used. The animals were divided into five groups of five animals per groups. The LD50 was calculated using Lorke’s method.

Group 1 served as the negative control. They received distilled water (10 ml/kg) orally. Group 2 served as the positive control and received a standard drug loperamide (3 mg/kg) orally. Group 3, 4 and 5 was the test groups and they received ethanolic extract of Grona triflora leaves with the doses of 250, 500 and 1000 mg/kg body weight orally. To induce diarrhea, the animals orally received castor oil, doses of extract was (1ml/kg body weight). Then one-hour post administration of castor oil, doses of extract was administered orally. The effect of castor oil to induce diarrhea, the mass, number, and frequency of stool was measured and recorded per hour for five hours.

The effects of the aqueous extract of Grona triflora leaves on intraluminal fluid accumulation were evaluated by measuring the percentage of reduction of intestinal secretion and weight of intestinal contents. The intestinal transit was also evaluated by measuring the distance travelled by the charcoal meal after an hour. The results showed that there was no mortality during the acute toxicity test.

The extract produced a significant (P<0.001) decrease across the treatment group respectively with 1000 mg/kg body weight having the highest percentage inhibition as compared to control (positive and negative) in the severity of castor oil induced diarrhea, castor oil induced enteropooling in mice and castor oil induced intestinal transit in mice. Summarily the results were dose dependent in the three models tested.

It could be deduced from this study that ethanolic extract of Grona triflora was found to be effective against castor oil induced diarrhea in experimental mice at 250, 500 and 1000 mg/kg body weight which provides evidence that could justify its traditional use.

Accepted: 25/07/2023

Published: 20/09/2023

*Corresponding Author

Prof. Francis N. Oguwike

E-mail: foguwike@ gmail.com

Phone: 08037791363

Keywords: Grona triflora, albino mice, Antidiarrheal activity, castor oil, ethanolic extract, Loperamide.

INTRODUCTION

The medicinal use of plants, leaves, roots, seeds, barks and fruit in the management and treatment of disease has been an age long practice and led to important breakthrough in pharmacology (Udubuike, 2011). Most people in sub-Saharan part of Africa mostly depend on herbal medicinal cure as some plants have been discovered to have medicinal value (Olaleye et al, 2000).

Among all the diseases causing death, diarrhea is the most common source of death especially in underdeveloped and developing countries because it affects all age grades (both the children and the elderly). Most diarrhea-associated deaths are attributed to unsafe water, inadequate sanitation, and poor hygiene and inadequate health services. The gastrointestinal (G.I) system is a system responsible for the digestion and absorption of ingested foods and liquids (Beverley et al, 2017). It starts from the mouth and ends in the anus. Each organ of the digestive system has a specific function they play and a disorder to any of them leads to a disease condition. The major symptoms of common GI disorders include recurrent abdominal pain and bloating, heartburn, indigestion/dyspepsia, nausea and vomiting, diarrhea, and constipation. (Beverley Greenwood et al, 2017). An infection of the intestine is known to be the most common cause of acute diarrhea. (kwang, 2015).

Diarrhea is defined by the World Health Organization as having three or more loose or liquid stools per day, or as having more stools than is normal for that person (Diarrhea disease factsheet 2017). It often lasts for a few days and can result in dehydration due to fluid loss, signs of dehydration often begins with loss of the normal stretchiness of the skin and irritable behavior. This can progress to decrease in urination, loss of skin color, a fast heart rate, and a decrease in responsiveness as it becomes more severe

Diarrhea is a symptom of infection caused by a host of bacterial, viral and parasitic organisms most of which can be spread by contaminated water. Diarrhea in most cases is caused by three major groups of micro-organisms namely; Viruses, bacteria and protozoa or parasites.

Grona triflora known as creeping tick trefoil or three- flower beggar weed is a plant in the family Fabaceae. It is a global specie native to the tropical regions and introduced to subtropical regions including the Southern United States.

It is found on a wide range of soils and most commonly in heavily grazed or closely cut areas in pastures, plantations, roadsides and lawns.

Grona triflora is found in association with many grasses including Axonopus spp, Bothriochloa pertusa, cynodon spp, Ischaemum timorense, Heteropogon contortus, Paspalum notatum.

It is wide spread and persistent under heavy grazing and so likely to markedly improve the diet of livestock on heavily grazed native pastures such as those found on communally owned and managed grazing land (Ohashi, 2018). There is no commercial seed production due to limited or no demand, low growth habit and the extended period over which flowering and seed set occurs.

Medicinal Uses

It is considered to have medicinal properties for treating rheumatism, fever, jaundice, stomach pain, skin problems, wounds and ulcers especially in India and China. The plant is said to be antipyretic, expectorant and antiseptic. A decoction is commonly used to treat diarrhea and dysentery and to quench thirst. The decoction also is used as a mouthwash, and the crushed plant, or a poultice of the leaves is applied externally on wounds, ulcers and for general skin problems. The whole plant is used medicinally for inducing sweat and promoting digestion.

Chemical Content of Grona triflora

The plant extract is well known to contain various phytochemical constituents like alkaloids, glycosides, steroids, saponins, tannins, proteins, amino acids and flavonoids.

To ascertain the antidiarrheal effect of ethanol leaf extract of Grona triflora on castor oil induced diarrheal in healthy albino mice among others is what prompted our curiosity in embarking on this research study.

MATERIALS AND METHODS

Animals:

Twenty-five (25) albino mice were randomly selected for this study. They were housed in wire mesh cages under standard conditions (Temperature 22-25⁰c, 12-hour light and 12-hour darkness cycle). They were allowed free access to water and feed throughout the period of the experiments. However, the study was conducted in accordance with the recommendation from the declaration of Helsinki, on guiding principles in care and use of animals.

Experimental Design

Animal grouping and dosing

The experimental animals were randomly divided into five groups (negative control, positive control and three test groups) comprising of five animals in each group. Negative controls received vehicle (10 ml/kg, distilled water) and positive controls received Loperamide (3 mg/kg). The test groups (group 3, 4 and 5) received different doses (250, 500 and 1000 mg/kg respectively) of the Grona triflora orally which was determined based on the acute oral toxicity test.

Determination of anti-diarrheal activity

Castor oil induced diarrhoea

The method described by Ezekwesili et al, 2010 was employed for this study. Five groups of swiss albino mice were used (n=5 each).

Diarrhoea was induced orally in each mice using castor oil (1 ml/kg.b.wt.). After one hour of castor oil treatment, three different doses of plant extract (250, 500 and 1000 mg/kg body weight orally) was administered orally to group 3, 4 and 5 respectively. Group 1 received distilled water orally (10 ml/kg) and serve as control while group 2 serve as standard received Loperamide (3 mg/kg body weight.) respectively. The animals were housed in individual metal cages lined with white nonabsorbent paper. Faecal output were assessed by collecting the faecal material for 5 hours after drug administration, dried for 2 hours then weighed. The percentage feacal output (% FOP) was calculated as follows:

% FOP =Ft x 100

Where Ft = mean faecal weight of each group, Fc = faecal weight of control group

% Inhibition of defecation = Mo-M X 100

Where, Mo: Mean defecation of control, M: Mean defecation of test sample/standard drug.

Phytochemical Analysis

The procedure for examination of the elements present in Grona triflora is as described by Harbourne (1973) and Trease and Evans method (1996). The extract was tested for alkaloids, flavonoids, saponins, tannins, reducing sugars, glycosides, resins, calcium, carbohydrates, fat and oil steroids, and acidic compounds.

Toxicity Study

The lethal dose (LD₅₀) of the extract in albino mice was determined using Lorke’s method (1983). The procedure of determining the lethal dose is by increasing the concentration of the extract given to the mice (per body weight) in each group consisting of eight (8) mice per group for five (5) days. The concentration used are 400 mg/kg, 500 mg/kg, 1000 mg/kg, 2000 mg/kg, 3000 mg/kg, 4000 mg/kg, 5000 mg/kg and 6000 mg/kg. the mortality rate was determined and a graph plotted to determine the lethal dose (LD₅₀).

Tests: to determine the effect of G. triflora on castor oil induced diarrhea in mice,

a) Castor oil induced enteropooling: The method described by Robert et al, 1976 was used to determine the effect of extract on intraluminal fluid accumulation.

b) Gastrointestinal Motility Test (Charcoal meal): The gastrointestinal motility test to ascertain the effect of Grona triflora on normal gastrointestinal transit was conducted using the procedure of Ezekwesili et al, 2010.

Statistical Analysis

All the data obtained from this study was analyzed by one-way analysis of variance (ANOVA). The values were represented as mean ± SEM. Comparison of mean values of different groups treated with extract and positive controls will be estimated by Tukey’s multiple comparison test. P < 0.05 was considered significant.

RESULTS

Table 1: Phytochemical study of Grona triflora.

Constituents of Grona triflora

Saponins, Flavenoids,

Glycosides.

Tannins, Alkaloids, Terpenoids, Steroids.

Carbohydrate

Quinones, Fats and oil, Resins, Calcium,

Phosphorus.

Degree of concentration

+++

Key: - (Not present), + (Present in small concentration), ++ (Present in moderately high concentration), +++ (Present in high concentration).

Table 2: Antidiarrheal effect of ethanol leaf extract of Grona triflora in castor oil induced Diarrheal model in mice.

Treatment/ Dose	Onset of diarrhea (Min)	No of wet feces	Total number of feces	Average weight of wet feces (gm)	Average weight of total feces (gm)	% Inhibition of diarrhea	% Weight of wet feces	% Weight of total feces
Distilled water 10 ml/kg	59.68 ± 0.79	11.13± 0.22	12.45± 1.56	0.50±0.19	0.57±0.88	---	---	---
Loperamide 3 mg/kg	173.15± 0.71	1.09 ± 2.01	5.46 ± 0.87	0.13±0.72	0.19±0.29	90.21 ***	26.00 ***	33.33 ***
Grona triflora 250 mg/kg	99.69 ± 0.76	6.41 ± 0.73	9.84 ± 0.26	0.31± 1.65	0.37±0.08	42.41*	62.00*	64.91*
Grona triflora 500 mg/kg	157.11 ±0.56	3.18 ± 0.54	5.86 ± 0.09	0.19±0.12	0.25±0.67	71.43***	38.00**	43.86**
Grona triflora 1000 mg/kg	197.34± 0.87	0.69 ± 0.99	1.09 ± 0.33	0.08±0.44	0.10±0.89	93.80***	16.00***	17.54***

Values are mean ± SEM (n = 5); * p<0.05, ** p<0.01, *** p<0.001 versus control, analysis was performed using one-way ANOVA followed by Tuckey post hoc test.

Table 3: Effects of ethanol leaf extract of Grona triflora on Castor oil induced enteropooling in mice

Treatment/Dose	Volume of intestinal contents (ml)	% Inhibition	Weight of intestinal contents (gm)	% Inhibition
Distilled water 10 ml/kg	0.78 ± 1.22	____	1.18 ± 0.97	____
Loperamide 3 mg/kg	0.18 ± 1.30	76.92***	0.28 ± 1.41	76.27***
Grona triflora 250 mg/kg	0.49 ± 0.19	37.18*	0.75 ± 0.87	36.44*
Grona triflora 500 mg/kg	0.28 ± 0.79	64.10**	0.42 ± 1.33	64.41**
Grona triflora 1000 mg/kg	0.17 ± 0.65	78.21***	0.25 ± 0.25	78.81***

Values are mean ± SEM (n = 5); * p<0.05, ** p<0.01, *** p<0.001 versus control, analysis was performed using one-way ANOVA followed by Tuckey post hoc test.

Table 4: Effect of ethanol leaf extract of Grona triflora in castor oil induced intestinal transit in mice.

Treatment/Dose	Length of small intestine (cm)	Distance moved by the Charcoal meal (cm)	Peristaltic index (%)	% Inhibition
Distilled water 10 ml/kg	56.76 ± 0.39	50.12 ± 0.35	88.77	------
Loperamide 3 mg/kg	56.46 ± 1.59	10.65 ± 1.44	18.86***	78.75***
Grona triflora 250 mg/kg	56.43 ± 1.43	35.23 ±0.98	62.43*	29.71*
Grona triflora 500 mg/kg	57.77 ± 0.27	18.02 ± 0.59	31.19**	64.05**
Grona triflora 1000 mg/kg	58.29 ± 0.38	9.88 ± 1.33	16.95***	80.28***

The extract significantly (p<0.01) inhibited the intestinal transit of charcoal meal at all tested doses.

DISCUSSIONS

Efficacy of ethanolic extract of Grona triflora o castor oil induced diarrhea in albino mice has been studied. In this present study Grona triflora leaf exhibited a typical plant constituent (Table 1) during the phytochemical analysis. Phytochemical screening of the crude extract of Grona triflora displayed the presence of different chemical constituents in the plant such as glycosides, saponins and flavenoids which are moderately high in concentrations (+++); others are tannins, alkaloids, terpenoids and steroids which are also present in high concentrations (++) and carbohydrate which occurred in small concentration (+). Quinones, resins, calcium, phosphorus, fats and oil were not present at all. The alkaloids are known to be protein precipitants and they play role in the precipitation of protein (Taofeeq et al, 2005).

The result of lethal dose studies showed that the LD₅₀ in mice using the ethanol extract of Grona triflora was 5,000mg/kg. This then shows that the doses (250 mg/kg, 500 mg/kg and 1000 mg/kg) used throughout this study were safe for the test animals.

Diarrhoea was observed in all induced experimental animals after one hour of castor oil administration (Table 2). Significant (P<0.001) inhibition of diarrhea effects was produced by the ingestion of different doses of ethanol leave extract of Grona triflora across the treatment group.

The extract of Grona triflora also significantly (P<0.001) inhibited the intestinal transit of charcoal meal at all tested doses (Table 4). The data showed that the percentage reduction of gastrointestinal transit of charcoal was 29.71% (P<0.05), 64.05% (P<0.01) and 80.28% (P<0.001) at doses of 250mg/kg, 500mg/kg and 1000mg/kg respectively.

The findings in this research study have indeed supported the claim by ethno-medical doctors that the leaf extract of Grona triflora could be used to treat wounds, ulcers, skin problems, treat diarrhea and dysentery. This could be due to the presence of phytochemical contents of alkaloids, tannins and saponins in the plant.

Plant extracts containing tannins, flavonoids, alkaloids, saponins and steroids have been reported to possess anti-diarrheal activity (Shemsu et al, 2013) (Balaji et al, 2012).

These secondary metabolites are reported to have antidiarrheal and antimicrobial activities. The mechanism of action of these chemical components for their antidiarrheal effect is also reported; saponins inhibit histamine release in vitro; terpenoids inhibit release of autacoids and prostaglandins; phenols make intestinal mucosa more resistant, reduce secretion and the intestinal transit and have astringent action; and flavonoids inhibit release of autacoids and prostaglandins. (Tiwari et al, 2011, Assefa et al, 2016).

Komal et al, 2013 supported the claim that tannins, flavonoids, and alkaloids are the main chemical constituents that are responsible for the antidiarrheal activity of the plants and this may be due to anti-secretory effects.

It can be deduced from this study that Grona triflora plant possesses the constituents that can stop diarrhea in mice.

REFERENCES

“Diarrhoeal Disease Factsheet” (2017), (https://www.who.int/en/news room/fact sheets/detail/diarrhoeal-disease). World Health Organization 2May. Retrieved 29 October 2020.

Assefa Belay Asrie, Mohammed Brhan Abdelwuhab, Zewdneh Shewamene, Desalegn Asmelashe Gelayee, Getnet Mequanint Adinew & Eshetie Melese Birru (2016). Antidiarr-heal activity of methanolic extract of the root bark of Cordia africana, Journal of Experimental Pharmacology, 8:53‑59, DOI: 10.2147/JEP.S116155.

Balaji G, Chalamaiah M, Ramesh B, and Amarnath Y.R (2012): Antidiarrhoeal activity of ethanol and aqueous extracts of Carum copticum seeds in experimental rats. Asian Pac J Trop Biomed. 2(2):1151–1155.

Beverley Greenwood-Van Meerveld, Anthony C. Johnson, David Grundy. (2017). Gastrointestinal physiology and function. Handbook of Experimental Pharmacology 2017; 239:1-16. Doi: 10.1007/164_2016_118.

Ezekwesili J.O, Nkemdilim U.U and Okeke C.U. (2010): Mechanism of antidiarrhoeal effect of ethanolic extract of Psidium guajava leaves. Biokemistri 22(2):87.

Harourne J.B.C, (1973). Phytochemical method. Channan and Hall, London p279.

Komal Kumar S, Rana A. (2013): Herbal approaches for diarrhea: a review. Int Res J Pharm 4:31–38.

Kwang Jae Lee. (2015). Pharmacologic agents for Chronic diarrhea. Doi: 10.5217/ir.2015.13.4.306.

Lorke, D. (1983). A new approach to practical acute toxicity. Archives of Toxicology. 53:275-289.

Ohashi H, and Ohashi K (2018): Grona, a Genus separated from Desmodium (Leguminosae Tribe Desmodieae). Journal of Japanese Botany 93:104-120. Jjbotany.com/pdf/ JJB 93-2 104-120.

Olaleye S.B, Farombi F.O, Adewoye E.O, Owoyele B.V, Onaswo S.A and Elegba R.A, (2000). Analgesic and anti-inflammatory effects of Kola viron (A. garcinia) kola seed extracts. Afri-journal. Biomed. Res 3:171-174.

Robert A, Nezamis JE, Lancaster C, Hanchar AJ, Klepper MS. (1976): Enteropooling assay: a test for diarrhea produced by prostaglandins. Prostaglandins. 11(5):809–828.

Shemsu U, Alemu T, Nigatu K. (2013): Antidiarrhoeal and antimicrobial activity of Calpurnia aurea leaf extract. BMC Complement Altern Med. 2013; 13-21.

Taofeeq O, Oyeniyi M. A, Tesleem A.O, (2005): Mechanism of action of Jatropha gossypifolia stem latex as a haemostatic agent. European Journal of General Medicine. 7(2): 315-320.

Tiwari, P., Kumar, B., Kaur, M., Kaur, G., & Kaur, H. (2011). Phytochemical screening and extraction: a review. Internationale pharmaceutica sciencia, 1(1), 98-106.

Trease, G.E, and Evans, W.C. (1996). Textbook of Pharmacognosy. 14th.edition W.B Saunders London p11.

Udubuike Daberechukwu (2011). The effect of aqueous extract of Garcinia kola (Guttiferales) in pregnant rats. Dept. of Physiology Chukwuemka Odumegwu Ojukwu University. Faculty of Basic Medical Sciences Uli Anambra state.