ANTIDIARRHOEAL EFFECT OF UNRIPE Musa paradisiacae PULP AND PEEL HOMOGENATES ON CASTOR OIL-INDUCED DIARRHOEA IN WISTAR ALBINO RATS

ABSTRACT

Musa paradisiacae commonly known as plantain is a rhizomatous perennial crop used as a source of starchy staple for millions of people in Nigeria. Different parts of the plant have been used in the treatment of various ailments and there are claims that it has antidiarrhoeal activity. This study is therefore aimed at determining the effects of unripe Musa paradisiacae pulp and peel homogenates on castor oil-induced diarrhoea in Wistar albino rats. The qualitative phytochemical constituents of Musa paradisiacae pulp and peel were found to be flavonoids, saponins, soluble carbohydrates, tannins, reducing sugars, hydrogen cyanide, steroids, alkaloids, and glycosides. The LD₅₀ results showed no toxicity up to 5000 mg/kg body weight. Rats were divided into 7 groups of 4 rats each. The groups were pre-treated as follows: group 1: normal saline (control); group 2: 3 mg/kg lomotil (standard drug); groups 3 and 4: 200 and 400 mg/kg unripe Musa paradisiacae pulp homogenates respectively; groups 5 and 6: 200 and 400mg/kg unripe Musa paradisiacae peel homogenates respectively; group 7: combination of unripe Musa paradisiacae pulp and peel homogenates (200/400 mg/kg respectively). After the treatments, diarrhoea was induced using castor oil. Relative to the control group 1, the treatment groups 2-7 inhibited castor oil-induced frequency of defecation and wetness of stool dose dependently but non-significantly (p>0.05). Both the pulp and peel homogenates produced non-significant decreases (p>0.05) in the distances travelled by the charcoal meal (marker) in castor oil-induced diarrhoea rats compared to the control group 1. Pre-treatment of the rats with unripe Musa paradisiacae pulp and peel homogenates decreased significantly (p<0.05) enteropooling indicated by decreases in the volume and weight of the gastro-intestinal contents relative to the control group 1. Treatment with the unripe Musa paradisiacae pulp and peel homogenates led to significant decreases (p<0.05) in the bicarbonate ion concentrations except in group 3 rats while the potassium ion concentrations increased significantly (p<0.05) in all the groups except in groups 3, 4 and 7 rats which showed non-significant decreases (p>0.05) compared to the control group 1. Sodium ion concentrations of the pre-treated groups increased non-significantly (p>0.05) except in groups 4 and 7 rats which decreased non-significantly (p>0.05) relative to the control group 1.Using everted rat intestines, the pulp and peel homogenates enhanced significant (p<0.05) influx of sodium ions into the everted sacs (serosal) and significant (p<0.05) efflux of potassium ions out of the sacs (mucosal) in relation to the control group 1. These findings reveal that unripe Musa paradisiacae pulp and peel exhibit antidiarrhoeal properties by inhibiting gastro-intestinal motility, enteropooling, wetness and frequency of defecation. They have also shown abilities to facilitate transport of electrolytes across the small intestinal membrane.

CHAPTER ONE

INTRODUCTION

 

The use of traditional medicines in West Africa is probably as old as the duration of human settlement in the region (Abdul-aguye, 1997). A medicinal plant provides an important source of new chemical substances with potential therapeutic effects. These have been used in traditional medicine for the treatment of several diseases and aliments (Mukerjee et al., 1998). It is already important to the global economy with demand steadily increasing not only in developing countries but also in industrialized countries (Sofowara, 1993).

Herbalism or herbal medicine is the use of plants for medicinal purposes, and the study of such use (Briskin, 2000). Herbal medicine is still the mainstay of about 75 – 80% of the world population, mainly in the developing countries, for primary health care (Kamboj, 2000). Plants have been the basis for medical treatments through much of human history, and such traditional medicine is still widely practiced today (Briskin, 2000).  This is primarily because of the general belief that herbal drugs are without any side effects besides being cheap and locally available (Gupta and Raina, 1998). Modern medicine recognizes herbalism as a form of alternative medicine as the practice of herbalism is not strictly based on evidence gathered using the scientific method (Talalay, 2001).  According to the World Health Organization (WHO), the use of herbal remedies throughout the world exceeds that of the conventional drugs by two to three times (Evans, 1994). The use of plants for healing purposes predates human history and forms the origin of much modern medicine. Modern medicine, does, however, make use of many plant-derived compounds as the basis for evidence-tested pharmaceutical drugs, and phytotherapy works to apply modern standards of effectiveness testing to herbs and medicines that are derived from natural sources (Talalay, 2001). Examples include aspirin (willow bark), digoxin (from foxglove), quinine (from cinchona bark), and morphine (from the opium poppy) (Vickers and Zollman, 1999).  Currently, a number of medicinal plants with antidiarrhoeal and antimicrobial properties are used in traditional herbal practice in many countries of the world. So it is important to identify and evaluate commonly available natural drugs that could be used against any type of diarrhoeal disease.

A number of herbs are thought to likely have adverse effects (Talay, 2001). Furthermore, “adulteration, inappropriate formulation, or lack of understanding of plant and drug interactions have led to adverse reactions that are sometimes life threatening or lethal (Elvin-Lewis, 2001)^. Proper double-blind clinical trials are needed to determine the safety and efficacy of each plant before they can be recommended for medical use (Vickers, 2007). Although many consumers believe that herbal medicines are safe because they are “natural”, herbal medicines and synthetic drugs may interact, causing toxicity to the patient. Herbal remedies can also be dangerously contaminated, and herbal medicines without established efficacy, may unknowingly be used to replace medicines that do have corroborated efficacy (Ernst, 2007). The World Health Organization (WHO), the specialized agency of the United Nations (UN) that is concerned with international public health, published quality control methods for medicinal plant materials in 1998 in order to support WHO Member States in establishing quality standards and specifications for herbal materials, within the overall context of quality assurance and control of herbal medicines (WHO, 2010).

There are different methods of herbal preparations and the exact composition of an herbal product is influenced by the method of extraction. They are:

• Tisanes or herbal teas; are the resultant liquid of extracting herbs into water (Green, 2000). The methods used are, infusions (hot water extracts of herbs), decoctions (long term boiled extracts usually of harder substances like roots and bark) and maceration (old infusion of plants with high mucilage content) (Green, 2000).
• Tinctures; alcoholic extracts of herbs generally stronger than tisanes (Green, 2000).
• Syrups; extracts of herbs made with syrups or honey (Green, 2000).

In developing countries, diarrhoea continues to be one of the leading causes of mortality and morbidity in children less than 5 years old. According to World Health Report, diarrhoea is the cause of 3.3% of all deaths. Worldwide distribution of diarrhoea accounts for more than 5-8 million deaths each year in children. The incidence of diarrhoeal disease still remains high despite the effort by many government and international organizations to reduce it. Nigeria, the fourth largest economy in Africa with an estimated per capita income of $350 has over half of its population living in poverty (WHO, 2007). This implies that very few people can afford orthodox medicine in curing diseases. Use of traditional medicines to combat the consequences of diarrhoea has been emphasized by WHO in its Diarrhoea Control Programme. It is therefore important to identify and evaluate available natural drugs as alternatives to current antidiarrhoeal drugs, which are not always free from adverse effects. Several studies have shown the beneficial effects of a number of medicinal plants used traditionally in the treatment of diarrhoeal disease, one of such being Bombax buonopozense (Akudor et al., 2011), Vitex doniana (Ukwuani et al., 2012), Anacardium occidentale (Omoboyowa et al., 2013) etc.

Musa paradisiacae belongs to the Musaceae family and is cultivated in many tropics and subtropical countries of the world. It ranks third after yams and cassava for sustainability in Nigeria (Akomolafe and Aborisade, 2007). Musa paradisiacae is a rhizomatous perennial crop used as a source of starchy staple for millions of people in Nigeria (Adeniyi et al., 2006).Unripe Musa paradisiacae, which is the green plantain contains more starch than the ripe plantain in which the starch is converted to sugars (glucose, fructose and sucrose). It has been indicated to posses antidiabetic (Eleazu et al., 2013), antioxidant (Shodehinde and Oboh, 2012), antimicrobial (Hossain et al., 2011), and antiulcerogenic properties (Ralph et al., 1984). There have also been traditional claims that unripe Musa paradisiacae can be used in diarrhoeal treatments even though it has not been scientifically proven.

 

• Musa paradisiacae

 

1.1.1 Taxonomy of Musa paradisiacae

Kingdom–     Plantae

Division –    Spermatophyta

Sub-division – Angiospermae

Phylum – Tracheophyta

Class – Liliopsida

Order – Zingiberales

Family – Musceae

Genus – Musa

Species – Paradisiacae

 (Smith, 1977).

 

1.1.2 Common names of Musa paradisiacae

Musa paradisiacae is commonly known as plantain. Among the Igbos of Nigeria, it is known as “ogede or abrika”, in Yoruba as “ogede agbagba”, in “Igala as agbo̥”, and in Hausa as “agada or afutu”.

 

1.1.3 Origin of Musa paradisiacae

Bananas and plantains belong to the genus Musa. It was Linnaeus that first gave the scientific name Musa sapientum for all sweet bananas, and the scientific name Musa paradisiacae for plantains (Simmonds, 1962). However, Linnaeus did not know that the two species he had described were in fact hybrids and not two distinct species (Zeller, 2005). Therefore, those two names could not be relevant in modern taxonomy.

Genetic studies have then demonstrated that all edible bananas and plantains come from a common ancestor, Musa acuminata. Plantains also carry genes from another ancestor, Musa balbisiana (Lejju et al., 2005). The genome of each ancestor could be represented respectively by the letter A and B. Then, further studies showed that edible bananas are mostly triploids and their genome would be described as AAA. This means that they carry three sets of chromosomes derived from M. acuminate (Simmonds, 1962). Different hybrid combinations have been observed, such as AAB, BBB, and tetraploid groups (AAAA) were also described.

Therefore, an accurate classification for bananas seems to be a great challenge. However, one thing sure in that banana taxonomists seem to agree that there is no single scientific name that can be attributed to all edible bananas (Zeller, 2005; Solofo and Ellis, 2009). Therefore, a new type of classification was proposed by Simmonds and that would abandon the Latin name to use instead a group indication like this: genus (Musa) + genome group (e.g. AAA) + subgroup name (e.g. Cavendish subgroup “Grand Nain”). In Panama, the sweet bananas come mostly from the Cavendish subgroup. The plantain subgroup is also triploid but has the genome group AAB (Simmonds, 1962).

 

1.1.4 Description of Musa paradisiacae plant

The common Musa paradisiacae has broad, irregular oval leaves, abruptly contracted at the base into a long broad, channelled footstalk. The fully grown blade is 1.3–2.4 meters long and about two third as broad, usually smooth, with several parallel veins. It is wind pollinated and propagates primarily by seeds which are held on the long narrow spikes which rise well above the foliage (Zeller, 2005).

Musa x paradisiaca (M. acuminata x M. balbisiana) is a sterile (without seeds or viable pollen) triploid (2n=3x=33 chromosomes) that is cultivated in warm climates for its tasty yellow-skinned fruit (Nelson et al., 2006). This is a large, fast-growing, suckering, herbaceous perennial that produces huge oblong to paddle-shaped leaves that grow to as much as 8’ long with leaf sheaths overlapping to help form a trunk-like pseudo stem (false stem). The pseudo-stem can reach up to 2-9 m tall and with short underground stem (corm) with buds, from which short rhizomes grow to produce a clump of aerial shoots (suckers) close to the parent plant. The roots are adventitious, spreading 4-5 m laterally, descending to 75cm long, but mainly in the top of 15cm and form a dense mat. It develops from the underground rhizome (Gibert, 2009).

At maturity, the rhizome gives rise to flower (inflorescence) that is carried up along a smooth elongated unbranched stem piercing through the centre of the pseudo-stem, finally emerging out at the top in between the leaf cluster. Yellow flowers with purple-red bracts appear in summer on mature plants. The flower subsequently develops to plantain bunch consisting of 3 to 20 hands each with at least 5-10 fingers (fruits) (Zeller, 2005). The plant is also monocarpic, which means that a shoot can only flower once and will die after the fruit is produced. The leaf crown will be oriented downward due to gravity.

Raw green fruits are only eaten after cooking. Each fruit measures about 3 to 10 inches or more in length depending on the cultivar type. They tend to have coarse external features with prominent edges and flat surfaces. The flesh inside is starch rich with tiny edible black seeds concentrated at its core. Ripening process however enhances flavor and sweetness since the starch converts to sugar (glucose, fructose and sucrose) (Phebe et al., 2007). The genus honors Antonia Musa, Roman physician of the 1st century B.C.

No serious insect or disease problems. In some cases, insects like aphids, mealy bugs, moths, scale, thrips, fruit flies and spider mites may attack the plant. Susceptible to anthracnose, wilt and mosaic virus (Scott et al., 1970).

 

1.1.5 Distribution of Musa paradisiacae

The plant is widely distributed throughout the tropical regions of Southeast Asia and western Pacific regions.

It is native to Southeast Asia, India and Burma through the Malay Archipelago to New Guinea, America, Australia, Samona, and tropical Africa (Ahmad et al., 2006). However, the cultivation is limited to Florida, the Canary Islands, Egypt, Southern Japan, and South Brazil. The top leaders exporting countries of plantain are Ecuador, Colombia, Costa Rica, Guatemala and Honduras. Panama occupies the 6th position. The large diversity that occurred in plantain has resulted in a variety of cultivars (Scott et al., 1970).

The number of Musa paradisiacae cultivated varieties (cultivars) has been reported to vary from one country to another. Swennen (1990) observed that at least 116 plantain cultivars exist in different parts of West and Central Africa. In Nigeria alone, more than 20 cultivars have been reported, although only a few are important commercially Swennen (1990). Musa paradisiacae is a major starch crop of importance in the human tropical zone of Africa, Asia, Central and South America. It is undoubtedly one of the oldest cultivated fruits in West and Central Africa. It is consumed as an energy yielding food and desert. It has been estimated that Musa paradisiacae and other bananas provide nearly 60 million people in Africa with more than 200 calories (food energy) per day. Fruits such as Musa paradisiacae are an important contribution to the diets of many low and middle class people in many African settings (Stover and Simmonds, 1987). Bananas and plantains constitute the fourth most important global food commodity (after rice, wheat and maize) grown in more than 100 countries over a harvested area of approximately 10 million hectares, with an annual production of 88 million tonnes (Frison and Sharrock, 1999). The all year round fruiting habit of Musa paradisiacae puts the crop in a superior position in bridging the ‘hunger gap’ between crop harvests. It therefore contributes significantly to food and income security of people engaged in its production and trade, particularly in developing countries. Musa paradisiacae is an important staple crop, supplying up to 25% of the carbohydrates for approximately 70 million people in the humid zone of sub-Saharan Africa. (IITA, 1998).

 

 

 

1.1.6 Cultivation and storage of Musa paradisiacae

Musa paradisiacae is grown in 52 countries with world production of 33 million metric tonnes (FAO, 2005). It grows more than any other plant in compacted soils, is abundant beside paths, roadside and other areas with frequent soil compaction. It is also common in grassland and as a weed among crops. Musa paradisiacae originated in the humid tropics and performs best under warm (27-30ºC) and very wet (200-220mm per month) conditions. The musa cultivars can stand warmer and drier climates (Gibert, 2009). The best soils are deep, friable loam with a good drainage and aeration. High soil fertility and organic matter content are desirable. The crop tolerates PH values of 4.5-7.5. It is sensitive to typhoons which cause blow-downs. A major problem of Musa paradisiacae is that the fruits are highly perishable (Scott et al., 1971). The most important physiological function affecting product quality during storage is respiration and transpiration. To extend storage life, these functions should be reduced. This can be done by controlling temperature, humidity, ventilation, and atmospheric composition during storage (Scott and Gandanegara, 1974).

 

1.1.7 Historical uses of Musa paradisiacae

Every part of Musa paradisiacae including root system is used widely in various treatments. The fruit of unripe Musa paradisiacae is traditionally used in the treatment of diarrhoea, dysentery, intestinal lesions in ulcerative colitis, diabetes (unripe), in sprue, uraemia, nephritis, gout, hypertension, cardiac disease (Mwangi et al., 2007).

Unripe bananas and plantain fruits are astringent, and used to treat diarrhoea. The leaves are used for cough and bronchitis. The roots can arrest haemoptysis and posses strongly astringent, and antihelmintic properties. Plantain juice is used as an antidote for snakebite. Other uses are asthma, burns, diabetes, dysentery, excessive menstrual flow, fever, gangrene, gout, headache, haemorrhage, inflammation, insomnia, intestinal parasites, sores, syphilis, tuberculosis, ulcers, and warts (Coe and Anderson, 1999). In Suriname's traditional medicine, the red protecting leaves of the bud was used against heavy menstrual bleeding (menorrhagia). Other therapeutic uses were against dysentery, migraine, hypertension, asthma and jaundice.

1.1.8 Health benefits of Musa paradisiacae

• Indeed, they are very reliable sources of starch and energy ensuring food security for millions of households worldwide (Swennen, 1990).
• It contains dietary fibre. Adequate amount of Dietary-fibre in the food helps normal bowel movements, thereby reducing constipation problems.
• Musa paradisiacae is rich in vitamin C. Consumption of foods rich in vitamin-C helps the body develop resistance against infectious agents and scavenge harmful oxygen-free radicals.
• Musa paradisiacae contains enough of vitamin A. In addition to being a powerful antioxidant, vitamin A plays a vital role in the visual cycle, maintaining healthy mucus membranes, and enhancing skin complexion.
• As in bananas, they too are rich sources of B-complex vitamins, particularly high in vitamin-B6 (pyridoxine). Pyridoxine is an important B-complex vitamin that has a beneficial role in the treatment of neuritis, anaemia, and to decrease homocystine (one of the causative factors for coronary artery disease (CHD) and stroke episodes) levels in the body. In addition, the fruit contains moderate levels of folates, niacin, riboflavin and thiamine (Ogazi, 1996).
• They also provide adequate levels of minerals such as iron, magnesium, and phosphorous. Magnesium is essential for bone strengthening and has a cardiac-protective role as well.

Musa paradisiacae are also rich in potassium. Potassium is an important component of cell and body fluids that helps control heart rate and blood pressure, countering negative effects of sodium (Ogazi, 1996).

 

1.1.9 Ripening Process and the Chemical Composition of Musa paradisiacae

The chemical composition of Musa paradisiacae varies with variety, maturity, degree of ripeness and where it is grown (soil type). During the ripening process, Musa paradisiacae produce the gas ethylene, which acts as a plant hormone and indirectly affects the flavour. Among other things, ethylene stimulates the formation of amylase, an enzyme that breaks down starch into sugar, influencing the taste of bananas (Swennen, 1990).  The greener, less ripe Musa paradisiacae contain higher levels of starch and, consequently, have a “starchier” taste. On the other hand, ripe ones taste sweeter due to higher sugar concentrations. Furthermore, ethylene signals the production of pectinase, an enzyme which breaks down the pectin between the cells of the banana, causing the banana to soften as it ripens. The water content in the green plant is about 61% and increases on ripening to about 68%. The increase in water is presumably due to the breakdown of carbohydrate during respiration. Green Musa paradisiacae contains starch which is in the range 21 to 26% (Jaffe et al., 1963; Marriott and Lancaster, 1983). The starch in the unripe plantain consists of mainly amylose and amylopectin in a ratio of around 1:5. Sugars comprise only about 1.3% of the total dry matter in unripe plantain, but this rises to around 17% in the ripe fruit (Ogazi, 1996). During ripening, the sugars are in the approximate ratio of glucose, 20: fructose, 15: sucrose, 65. Only traces of other sugars are found (Swennen, 1990).  The fat content of plantains is very low, less than 0.5% and so fats do not contribute to the energy content (Jaffe et al., 1963; Marriott and Lancaster, 1983).

The protein content of unripe fruit is between 0.5 and 1.6% and no significant change in the ripening fruit has been detected. The amino acid component includes alanine amino-butyric acid, glutamine, asparagine, histidine, serine, arginine, and leucine. The ascorbic acid content is high. Although the total lipid content remains essentially unchanged during ripening, the composition of fatty acids, especially within the phospholipid fractions has been observed to change, with a decrease in their saturation (Ogazi, 1996).

 Table 1: Nutritional value of Musa paradisiacae pulp per 100g (3.5oz)

NUTRIENTS	QUANTITIES	NUTRIENTS	QUANTITIES
Energy	510 kJ (120 kcal)	Folate (vitamin B9)	22 μg (6%)
Carbohydrates	31.89 g	Choline	13.5 mg (3%)
Sugars	15 g	Vitamin C	18.4 mg (22%)
Dietary fiber	2.3 g	Vitamin E	0.14 mg (1%)
Fat	0.37 g	Vitamin K	0.7 μg (1%)
Protein	1.3 g	Calcium	3 mg (0%)
Vitamin A equiv.	56 μg (7%)	Iron	0.6 mg (5%)
beta-carotene	457 μg (4%)	Magnesium	37 mg (10%)
Thiamine (vit. B1)	0.052 mg (5%)	Phosphorus	34 mg (5%)
Riboflavin (vit. B2)	0.054 mg (5%)	Potassium	499 mg (11%)
Niacin (vit. B3)	0.686 mg (5%)	Sodium	4 mg (0%)
Pantothenic acid (B5)	0.26 mg (5%)	Zinc	0.14 mg (1%)
Vitamin B6	0.299 mg (23%)

 

Source; (Ogazi, 1996).

 

1.2 Diarrhoea

1.2.1 Definition of Diarrhoea

Diarrhoea is an alteration in the normal bowel movement, characterized by increased frequency of bowel sound and movement, wet stool, and abdominal pain (Guerrant et al., 2001). Clinically it is used to describe increased liquidity of stool, usually associated with increased stool weight and frequency (Suleiman et al., 2008). Regardless of the understanding causes, treatment and prevention of diarrhoeal diseases, an estimated 4.6 million people, with 2.5 million children, die from diarrhoea every year, particularly in developing countries (Kosek et al., 2003). Diarrhoea can be a symptom of other diseases such as cholera, irritable bowel syndrome, gastroenteritis (intestinal inflammation and ulcerative colitis) (Schiller, 2007; Baldi et al., 2009), malaria (Gale et al., 2007) and diabetes mellitus (Forbes et al., 2011).

 

1.2.2 Mechanism/pathology of diarrhoea

Diarrhoea may occur as a result of the following;

1. Hyper secretory mechanism; here, there is an imbalance between the absorption and secretory mechanisms. There is impaired absorption with increased secretion of electrolytes resulting to osmotic load within the intestine.
2. Hyper motility; this is the rapid intestinal transition of materials causing little time for absorption of nutrients into the blood.
3. Hypo motility; this is decrease in the rate of intestinal movement leading to decreased absorption of nutrients.

 

1.2.3 Classifications of diarrhoea disease

There are different classifications of diarrhoea

 

1.2.3.1 Classification based on mode of infection

Diarrhoea can be either infectious or non-infectious in nature with infectious pathogenesis responsible for the major total episode worldwide.

In infectious diarrhoea, the potential causative pathogens include bacterial agents (Mathhabe et al., 2006), rarely fungi (Robert et al., 2001), viral and pathogenic pathogens (Brijesh et al., 2006).

Non-infectious diarrhoea can be caused by adverse reactions to drugs, toxins, allergy to food, poisons and acute inflammation which promote the release of secretagogues and some enteric nervous system (ENS) receptors e.g. prostaglandins, serotonin, substance p, vasoactive intestinal peptides and hormones in the gastrointestinal tract (Wynn and Fougere, 2007).

 

1.2.3.2 Classification based on duration of symptoms

1. Acute diarrhoea; mostly caused by enteric pathogenic infections, intoxicants or food allergy. This type is self limiting without pharmacological intervention and usually resolves within 2 weeks from onset.
2. Persistent diarrhoea; usually from a secondary cause such as enteric infections or malnutrition. It lasts for more than 14 days.
3. Chronic diarrhoea; mostly result from congenital defects in digestion and absorption. This usually lasts for more than 30 days (Baldi et al., 2009).

 

1.2.3.3 Classification based on pathological mechanisms

1. Watery or secretory diarrhoea; results from increased chlorine secretion, decreased sodium absorption and increased mucosal permeability.
2. Osmotic diarrhoea; also a watery form of diarrhoea caused by ingestion of non-absorbable indigestible material (Baldi et al., 2009) or absence of brush border enzymes required for digestion of dietary carbohydrate (Podewils et al., 2004).
3. Inflammatory diarrhoea; characterized by the presence of mucus, blood and leukocytes in the stool and is usually induced by an infectious process, allergic colitis or inflammatory bowel disease (Ravikumara, 2008).

 

1.2.4 Pathophysiology of diarrhoea

A healthy gastrointestinal tract can be defined as one where a balance is reached between the bacteria colonising the environment and the immune system. Any disturbance in this homeostasis will result in gastrointestinal tract disorders like diarrhoea.

General causes of diarrhoea are; intestinal inflammation, microbial infection (bacteria, viruses and parasites), altered gastrointestinal motility as a result of damage to enteric nervous system and Immune dysfunctions (Ravikumara, 2008)

 

1.2.4.1 Intestinal inflammation and diarrhoea

Inflammation is the body’s first line of defence against infection and hazardous stimuli in case of injury or infection in the gastrointestinal tract (Iwalewa et al., 2009). This results in the activation of neutrophils and macrophages. Once activated, the immune cell (macrophages) assist with the killing of pathogenic micro-organisms and the removal of harmful and cell debris (Gilroy et al., 2007). This task is achieved through the release of numerous pro-inflammatory cytokines, chemokines and chemo attractants (Conforti et al., 2008), reactive oxygen species, reactive nitrogen species, eicosanoids e.g. prostaglandin E₂, pain provoking mediators, cyclic AMP, etc. another antimicrobial property of inflammation is disruption of the epithelial lining which limit microbial survival and colonization of the GIT in inflamed intestine due to loss of replication niche.

While inflammation process is beneficial to the body as it removes the insulting cause (Pharoah et al., 2006; Lee et al., 2007) the large recruitment and activation of neutrophils and macrophages can induce changes in gut motility, neuronal functionality and hydro electrolyte movement with resultant diarrhoea (Gelberg, 2007).

Mechanisms involved in the inflammatory modulated diarrhoea may include;

1. Epithelial barrier disruption; gastrointestinal epithelium barrier provide a physical defence against hostile environment within the intestinal lumen (Blikslager et al., 2013). The intracellular tight junctions are the most essential components of the intestinal physical barrier. Tight junctions are multiple protein complexes located around the apical end of the lateral membrane of the epithelial cells. It performs dual functions as a selective/semi permeable paracellular barrier allowing movement of ions, solutes and water through the intestinal epithelium while also preventing the translocation of luminal antigens, micro-organisms and their transport into the mucosa (Groschwitz and Hogan, 2009; Guttman and Finlay, 2009). Disruption of the intestinal tight junction barriers by inflammatory cytokines, reactive oxygen species and pathogens (Guttman et al., 2006), impair intestinal tight junction function; cause an increase in intestinal permeability resulting to diarrhoea (Schenk and Mueller, 2008).
2. Reduced absorption capacity; nutrient-coupled absorption of electrolytes take place in the brush border mirovilli (Dudeja and Ramaswamy, 2006). In an inflamed or infected intestinal tract, the total absorptive surface area is decreased due to brush border shortening resulting in mal-absorption (Cotton et al., 2011). Small intestinal mal-absorption occurs due to impaired absorption of water, glucose and electrolytes creating an osmotic gradient that draws water into the small intestinal lumen resulting to small intestinal distension and rapid peristalsis, consequently diarrhoea (Schulke et al.,2004).
3. Chloride ion hyper secretion; Diarrhoeal agents can activate inappropriate chloride ion (Cl^–) secretion from the colonic crypt epithelial cells. This hyper secretion of Cl^– is the driving force for many diarrhoea aetiologies. The underlying mechanism is the increase in intracellular levels of cyclic nucleotides (cAMP and cGMP) and cytosolic calcium. This process in turn drives the secretion of fluid and electrolytes into the intestinal lumen, which may overwhelm the intestinal absorptive mechanism, thereby resulting in secretory diarrhoea with potential effect of severe dehydration (Petri Jr et al., 2008).
4. Interference with ability to digest; inflammatory response in the intestine may negatively affect the ability of the enterocyte to digest nutritional materials. This process called maldigestion occurs due to a deficiency in various brush border enzymes especially carbohydrates and lipids (Schulke et al., 2004). The high level of undigested carbohydrate and lipids are converted to short chain fatty acids by the colonic micro biota and the amount may exceed colonic capacity for their absorption. Excess short chain fatty acids induce osmotic gradient pulling water and ions into the intestinal lumen resulting in osmotic diarrhoea of colonic origin (Field, 2003).
5. Stimulation of the enteric nervous system; inflammation causes structural changes to the enteric nervous system that ranges from axonal damage to neuronal death (Stanzel et al., 2008). The changes include altered neurotransmitter synthesis, storage and release, therefore contributing to the altered intestinal motility during the onset and progression of many gastrointestinal disorders (Stanzel et al., 2008).

 

1.2.4.2 Oxidative damage in diarrhoea

Excessive generation of reactive oxygen species and reactive nitrogen species by the intestinal immunological system as a result of intestinal infection, irritation, inflammation and depleted endogenous antioxidant defence cause oxidative stress (Granot and Kohen, 2004). This condition has been implicated as one of the causes of diarrhoea (Granot and Kohen, 2004)).

The Pathophysiology of oxidative stress is complex and results from the normal immune response in conditions of disease and is initiated by activated mitochondrial of the leukocyte. The free radicals produced are unstable and highly reactive, function to destroy invading organisms (Dwyer et al., 2010).

However, since their effects are usually non-specific and aimed at the lipid membrane, the chain reaction initiated by the immune system will destroy the body’s macro molecules unless scavenged (terminated). At normal physiological conditions, balance is maintained between amounts of free radicals generated and endogenous antioxidant defence system that scavenge the radicals preventing their harmful effects. However, a shift in radical generation leads to oxidative stress which causes tissue injury and subsequently diseases.

Proposed mechanisms through which these products induce diarrhoea are;

• Lipid peroxidation; they are primary mechanisms for intestinal cellular malfunction and can destroy the capacity of membranes to maintain ionic gradients resulting in an aberration in ion transport, particularly affecting potassium efflux and sodium/calcium influx (Dudeja and Ramaswamy, 2006). The production of arachidonic acid metabolites in the lipid peroxidation process can also contribute to intestinal dysfunction including diarrhoea.
• Some of the reactive species such as HOCl and NH₂Cl can also act as secretagogues on their own or can evoke the release of acetylcholine or other neurotransmitters thus stimulating the ENS to cause increased contractility or motility of intestinal tract (Gaginella et al., 1992). The reactive species also induce gene expression by stimulating signal transductions such as Ca²⁺ signalling and protein phosphorylation.
• Increased production of inflammatory mediators; The onset of lipid peroxidation process leads to changes in the physiological integrity of the cell membrane. The body responds to the process by the release of pro-inflammatory eicosanoids such as (prostaglandins, prostacyclins, and leukotrienes) and pro- inflammatory cytokines (Nardi et al., 2007), tumor necrosis factor (TNF-a) and platelet activating factor (PAF) (Kunkel et al., 1997; Conforti et al., 2008).

 

1.2.4.3 Enteric Nervous System (ENS) in diarrhoea

The enteric neural network is responsible for the control of propulsive transport and segmental peristalsis in the GIT, as well as secretion and absorption across the intestinal lumen (Wood et al., 2004; Bohn and Raehal, 2006). While the ENS functions independently of the CNS, it is modulated by the parasympathetic and sympathetic autonomic nervous system (Farthing, 2003). As a unit, the ENS is a complicated physiological system with auto regulation being mediated by a number of neurotransmitters such as acetylcholine, serotonin, histamine and endorphin (Farthing, 2002). Diarrhoea can result from the alteration of these systems;

• Smooth muscle contractility; many agonists and antagonists elicit contractility in GIT smooth muscle through activation of various receptors located within the muscle (Holzer, 2004). In some cases, the activation of the smooth muscle receptors by neurotransmitters and inflammatory mediators including ROS causes relaxation (spasmolytic) while in other cases, the process leads to increase in spontaneous or induced contraction (spasmogenic). Ionic channel (Ca²⁺ and Cl^–) are also known to play important roles in smooth muscle contraction (Giorgio et al., 2007). Anion and fluid secretion into the intestinal lumen are stimulated through the activation of the receptors on enteric secretomotor pathways and epithelial cells, consequently causing secretory diarrhoea (Giorgio et al., 2007).
• Motility; intestinal motility dysfunctions include situations in which movement of materials along the GIT is repetitive and rapid (diarrhoea) and too slow (pseudo-obstruction, slow transit constipation) (Talley, 2006; Giorgio et al., 2007) are controlled by activities of the neurotransmitters in the ENS. Pathogenic bacteria overgrowth is common as a result of intestinal hypo motility or low transit time which may lead to mucosal inflammation, increased accumulation and absorption of toxins which are known pathophysiology of diarrhoea. The mechanisms may include impaired digestion as in the deconjugation of bile salts with subsequent fat mal absorption, leading to fatty acid diarrhoea or osmotic effects of malabsorption of sugars resulting in osmotic diarrhoea. Diarrhoea also results from an increase in the gut motility (hyper motility) including an accelerated transit of food intake. The net fluid absorption from the food intake is reduced due to less adequate contact time with the GIT epithelial lining for the absorption of fluids before excretion.

 

1.2.4.4 Cystic fibrosis trans-membrane conductance regulator (CFTR) regulation

CFTR is a cyclic adenylate monophosphate (cAMP) – activated Cl^– channel expressed in epithelial cells in the intestine and other fluid transporting tissues (Thiagarajah et al., 2002). Diarrhoea pathogens and their toxins can induce secretory diarrhoea by simultaneous stimulation of the active secretion of Cl^– and inhibition of Na⁺ absorption across the apical membrane of enterocyte with resulting massive fluid and electrolyte loss into GIT (Schuier et al., 2005). The cellular signalling mechanisms include an increase in cellular cAMP and cyclic guanylate monophosphate (cGMP), which may result in activation of the CFTR Cl^– channel. Pharmacological blocking of CFTR with drugs such as glibenclamide and CFTRinh- 172 inhibits salt and water loss in diarrhoea (Schuier et al., 2005).

 

1.2.5 Specific Agents of Diarrhoea

• Bacteria; bacterial causes of diarrhoea include

1. Eschericia coli ( coli); E. coli is a gram negative rod shaped bacteria that shares a symbiotic relationship with animal host as part of normal digestive intestinal flora. Under certain defined conditions, these organism or pathogenic strains of these organisms are known to induce diarrhoea (Clarke, 2001; Le Bouguenec, 2005). There are 6 main types of pathogenic E. coli associated with diarrhoea namely; enterotoxigenic Esechericia coli (ETEC), enteroinvasive Eschericia coli (EIEC), enteropathogenic Eschericia coli (EPEC), enterohaemorrhagic Eschericia coli (EHEC), enteroaggregative Eschericia coli (EAEC) and diffusively adherent Eschericia coli (DAEC) (Clarke, 2001). The basic pathophysiology of this E. coli involves their inherent ability to adhere to epithelial cells and colonize the host tissues (Le Bouguenec, 2005). Interactions from some of the strains of E. coli are self limiting and can resolve without pharmacological interventions.
2. Staphylococcus aureus; it is a gram positive coccus present in normal intestinal or skin flora of human and homoeothermic animal. Under defined conditions, the pathogenic strains produce heat stable staphylococcal enterotoxins (SETs) and toxic shock syndrome toxins (TSST-1) (De Oliveira., et al., 2010) both of which are known to induce diarrhoea.
3. Campylobacter jejuni; it is an invasive gram negative, spiral shaped rod bacteria present in the GIT of mammals, birds, and primates. (Lengsfeld et al., 2007). The clinical signs of Camphylobacter jejuni infections include pyrexia, abdominal pains, watery diarrhoea and dysentery (Podewils et al., 2004). The characteristic mechanisms of campylobacter infection involves invasion and translocation of the epithelium with a concomitant induction of inflammation (Hu et al., 2008).

Others include; Shigella species, Vibrio cholera, Bacillus cereus, Yersinia species, Listeria monocytogenes, Clostridium species, Salmonella typhimurium, Enterococuss faecalis, etc.

• Fungal induced diarrhoea; Candida albicans is a yeast fungus and exist as a member of the normal flora in the GIT and mucocutaneous membrane (Forbes and Gros, 2001). However, following the use of antibiotic therapy that result in the sterilization of the GIT flora, Candida albicans can over grow to take the place of removed organisms with the end result of diarrhoeal symptoms (Henry-Stanley et al., 2007). Other predisposing factors include the altered intestinal permeability and diminished host immunity response. Clinical signs associated with enteric candidiasis are abdominal pain, cramping, rectal irritation and absence of nausea, vomiting, bloody and mucus stool and pyrexia (Levine et al., 1993).
• Viral induced diarrhoea; the viruses involved include; rotavirus, norovirus, hepatitis A virus and human immunodeficiency virus (HIV). Rotavirus and norovirus cause diarrhoea by the production of enterotoxins which induce Na⁺ – glucose dependent malabsorption and destruction of enterocytes. They also induce intestinal tight junction (TJ) dysfunction with resultant diarrhoea through a ‘leak flux’ mechanism in which water is secreted into the lumen of the intestine (Dickman et al., 2000).

Chronic diarrhoea is one of the complications of HIV infection due to multiple enteric opportunistic microbes (DuPont and Marshall, 1995). While HIV is important in secondary enteric diseases as a result of immune suppression (CD4+ T-lymphocytes destruction), the virus can result in diarrhoea directly by altering the mucosa structural arrangement (HIV-enteropathy) (Epple et al., 2009). The diarrhoea resulting from HIV appears to be caused by the release of cytokines from the infected immune cells (Schmitz et al., 2002).

Other agents of diarrhoea include;

1. Protozoa e.g. Gardia intestinalis, Entamoeba histolytica, Cryptosporodium parvum and Cyclospora cayetanensis (Linscott, 2011).
2. Parasites e.g. Trichinella spirallus (Cui et al., 2011).

• Immune disorder; compromised immune system and hyperactive immune system (Gertsch et al., 2010).

1. Antibiotic therapy which could be antibiotic toxicity, alteration of digestive functions, overgrowth of pathogenic micro-organisms, etc.
2. Diabetic complications.
3. Food allergy.

 

1.2.6 Treatment of diarrhoea

Diarrhoea is managed in a variety of ways. These include the use of drugs such as antibiotics like loperamide, diphenoxylate, atropine, and host of others. Nutrition and supplements have also been used (Ehrlich, 2010). Certain astringent herbs such as Rubus fruticosus, Rubus idaeus, Matricaria recuitta, and Althea officialis have been used for the treatment of diarrhoea (Ehrlich, 2010). Evaluating the risk of diarrhoeal diseases requires knowledge of the complex interactions between biological, socio-economic, behavioural, and environmental factors over the time (Pathela et al., 2006). Many risk factors have been analyzed, most of them have been done retrospectively, and only few of them have been able to associate the risk factors with subsequent incidence of diarrhoea. In view of this problem, the World Health Organization has initiated the Diarrhoea Disease Control Program, which includes studies of traditional medical practices together with the evaluation of health education and prevention approaches (Mukherjee et al., 1995). In spite of the importance of diarrhoea as a problem of public health, it is counted by relatively reduced number of drugs for its treatment.

Diarrhoeal disease was often treated with antimicrobial drugs, but this treatment is generally ineffective, due to the presence of drug resistance (Cid et al., 1996). Medicinal plants play fundamental role in traditional medicine. According to Cowan (1999), about 25-50% of current pharmaceutical are derived from plants. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found invitro to have antimicrobial properties. Thus, screening of essential compounds for developing new antimicrobial drugs is important (Ahmed and Beg, 2001).

1.2.7 Potential mechanisms in the control of diarrhoea

There are several mechanisms that can be involved in diarrhoea control and management. They include;

1. Oxidative damage and antioxidants; several endogenous strategies are available in the human body to combat oxidative damage. These provide ways for normal oxidative metabolism to occur in the body without damaging the cells and also allow for normal ROS/RNS – mediated cellular response such as phagocytosis and intracellular signalling (Valko et al., 2007). Therefore the possibility exist that returning the animal to a more neutral oxidative balance may promote repair of damaged membranes (Nose, 2000). As a result, antioxidants and radical scavengers may be beneficial in the attenuation of diarrhoea. Some best known antioxidants include selenium, vitamin E, vitamin C and pro anthocyanidins in red wine and resverastrol in commercial grape seed extract.
2. Inflammation and anti- inflammatory agents; as a result of the negative impact of the inflammatory cascade on the functionality of the GIT, modulation of these processes through the use of drugs may be of benefit. Possible mechanisms include attenuation of inflammatory processes through the use of anti inflammatory, antioxidative and radical scavenging mechanisms. Potential target include drugs with COX and LOX enzyme inhibitory activity. Such drugs are Non steroidal anti inflammatory drugs (NSAIDs) like indomethacin, aspirin, ibuprofen and coxibs ().
3. Enteric Nervous System (ENS); The ENS is an important target for pharmacological intervention in diarrhoea through the use of agonists and antagonists that target these ENS endogenous receptors (Gutierrez et al., 2007)
4. Plants; Due to the widespread occurrence of diarrhoea as a disease together with its prevalence coinciding with human development, plants and fungi have featured widely in the management of the disease. Their use become so common in human and veterinary medicines that a number of compounds considered to be allopathic are of natural origin. They include;
5. Antioxidants; the natural vitamins and red pigments present in plants.
6. Anti- inflammatory; salicylic acid from willow bark.

• ENS modifiers; atropine from Atropa belladoma, tincture of opium from Papaver somniferum (Gutierrez et al., 2007).

1. Antibiotics; all antibiotics are natural products of fungal origin.

These plants alleviate diarrhoea through several mechanisms. They are;

1. Anti- infectious mechanisms of plant secondary metabolites which include:
2. Anti-microbial; Many plant metabolites exhibit some level of toxicity towards micro organisms. They accomplish this through microbial enzymes inhibition, deprivation of essential growth substances and cell membrane disruption (Cowan, 1999).
3. Anti-adhesion; Adhesion of some enteric pathogens to the mucosa epithelium of the host cell is the most important step in intestinal infections that may lead to the development of diseases (Ofek and Sharon, 1990). Application of anti adhesives chemotherapy can be effective only against micro organisms that depend on the surface contact with the host cells as prerequisite for survival, multiplication and virulence (Lengsfeld et al., 2007).
4. Antitoxin; since enteric pathogens may induce diarrhoea through the production of toxins (endotoxin or cytotoxin) the neutralization with plant antidiarrhoeal compounds may be beneficial in the management of diarrhoea.
5. Immunomodulatory; plants with immune stimulating activities may help in attenuating many infectious diseases.
6. Antioxidatve mechanisms and they are;
7. Free radical scavenging; many plants have strong anti-radical activities which can antagonize the deleterious action of free radicals. The mechanisms may be electron transfer or hydrogen donating to stabilize the free radical (Mello et al., 2005)
8. Complexation of catalytic metallic ion; metallic ions e.g. Ferrous ion (Fe²⁺), cuprous ion (Cu²⁺), magnesium ion (Mg²⁺) and zinc ion (Zn ²⁺) can also generate free radicals (Kane, 1996). Many plant molecules modulate oxidation activity by complexing with the free transition metal thereby inactivating their capacity to catalyse oxidative processes.
9. Inhibition of nitric oxide (NO); NO generated by inducible NOS (iNOS) can act synergistically with other inflammatory mediators in the development of diarrhoea. The inhibition or down regulation of iNOS expression may be beneficial in the inflammatory response.
10. Immune system optimization; over expression of the immune system may cause damage to the surrounding tissues and consequently result in diarrhoea. Many medicinal plants and phytochemicals protect against oxidative stress due to immune modulatory activity (Wang et al., 2002).
11. Lipid peroxidation inhibition; scavenging of free radicals is one of the major antioxidation mechanisms to inhibit the chain reaction of lipid peroxidation and reduction of the deleterious effect of the cytotoxic products.
12. Anti inflammatory mechanisms which are;
13. Cyclooxygenase (COX) inhibition; compounds with COX enzyme inhibitory potentials could be used as anti-inflammatory agents. Some plant secondary metabolites have potential to inhibit the formation of pro-inflammatory signalling molecules such as prostaglandins (Polya, 2003).
14. Lipoxygenase (LOX) inhibition; lipoxygenase metabolites are critical mediators of inflammation and thus important in the pathogenesis of abdominal stress and diarrhoea associated with intestinal inflammation. Plant phytochemicals with LOX inhibitory potential are candidate for anti-inflammation and the resulting diarrhoea.
15. Modulation of cytokines activity; A number of diarrhoea pathogenesis cause severe intestinal inflammation with hypersecretion of pro-inflammatory cytokines (MacNaughton, 2000). Inhibition of the pro-inflammatory cytokine mediators can remove their negative activities associated with gastrointestinal disorders including diarrhoea.
16. Anti diarrhoeal mechanism of plant phytochemicals;
17. Anti spasmodic; spasmolytic agents are used in the treatment of hypermotility of the GIT while prokinetic agents are used in the treatment of hypo motility (Gilani, et al., 2005). Many phytochemicals demonstrated various ranges of spasmolytic or antispasmodic activities against spontaneous or agonist induced contraction on isolated parts of the GIT.
18. Antisecretory; microbial enterotoxins cause diarrhoea by disturbing the balance between intestinal absorption and secretion in favour of the later. Therefore, inhibition of the intestinal secretion is one therapeutic model for treating diarrhoea (Velazquez et al., 2009)

Oral rehydration therapy (ORT): Oral rehydration therapy is the use of modest amounts of sugar and salt added to water in order to prevent and/or treat dehydration (Guerrant et al., 2003). This dehydration is most commonly caused by diarrhoea. For moderate to severe dehydration, there are medically prepared and medically recommended packets which can be added to water. Such packets vary in composition, but usually contain a mixture of glucose, sodium, potassium, and citrate. These are often used in conjunction with, or instead of, intravenous fluid replacement. For homemade solutions, authorities said it should be 1 liter (34 oz) water, 6 teaspoons sugar and 1 teaspoon salt (Chatterjee, 1957). As part of oral rehydration therapy, WHO recommends supplemental zinc (10 to 20 mg daily) for 14 days, which will help to reduce the severity and duration of this diarrhoeal episode, as well as making future diarrhoea less likely in the following two to three months. WHO also recommends continuing to feed the person affected by diarrhoea, as this will help speed recovery of normal intestinal function and the child will gain some nutrients from the food, making malnutrition less likely (Bhattacharya et al., 1990).

1.2.8    Physiological basis of diarrhoea

Fluid from the body is normally pumped into the intestinal lumen during digestion. This fluid is typically isosmotic with blood because it contains a high concentration of sodium (approx. 142 mEq/L). A healthy individual will secrete 20-30 grams of sodium per day via intestinal secretions. Nearly all of this is reabsorbed by the intestine, helping to maintain constant sodium levels in the body (homeostasis) (Guyton and Hall, 2006).

Because there is so much sodium secreted by the intestine, without intervention, heavy continuous diarrhoea can become a potentially life-threatening condition within hours. This is because liquid secreted into the intestinal lumen during diarrhoea passes through the gut so quickly that little sodium is reabsorbed, leading to dangerously low sodium levels in the body (severe hyponatremia) (Guyton and Hall, 2006). This is the motivation for sodium and water replenishment via ORT.

Sodium absorption via the intestine occurs in two stages. The first is at the outermost cells (intestinal epithelial cells) at the surface of the intestinal lumen. Sodium passes into these outermost cells by co-transport via the Sodium-Glucose Transport (SGLT) 1protein (Guyton and Hall, 2006). From there, sodium is pumped out of the cells (basal side) and into the extracellular space by active transport via the sodium potassium pump (Canadian Paediatric Society, Nutrition Committee, 2006; Guyton and Hall, 2006).

The Na⁺/K⁺ ATPase pump on the basolateral membrane of the proximal tubule cell uses ATP to move 3 sodium ions outward into the blood, while bringing in 2 potassium ions. This creates a downhill sodium gradient inside the proximal tubule cell in comparison to both the blood and the tubule (Crane et al., 1961).

The SGLT proteins use the energy from this downhill sodium gradient created by the ATPase pump to transport glucose across the apical membrane against an uphill glucose gradient. Therefore, these co-transporters are an example of secondary active transport. (The glucose transporters (GLUT) uniporters then transport the glucose across the basolateral membrane, into the peritubular capillaries.) Both SGLT1 and SGLT2 are known as symporters, since both sodium and glucose are transported in the same direction across the membrane (Crane et al., 1961)

The co-transport of glucose into the epithelial cells via the SGLT1 protein requires sodium. Two sodium ions and one molecule of glucose/galactose are transported together across the cell membrane through the SGLT1 protein. Without sodium present, intestinal glucose or galactose will not be absorbed. This is why Oral Rehydration Salts (ORSs) include both sodium and glucose. For each cycle of the transport, hundreds of water molecules move into the epithelial cell, slowly rehydrating the affected individual (Guyton and Hall, 2006)

1.3 Phytochemicals

            Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. They are non-essential nutrients, that is, they are not required by the human body for sustaining life. These phytochemicals work differently. There possible actions are; antioxidant, hormonal action, stimulation of enzymes, interference with DNA replication, anti microbial, physical action like anti adhesion, etc. These phytochemicals include; flavonoids, saponins, alkaloids, glycosides, tannins, reducing sugars, etc.

            Phytochemicals with antidiarrhoeal potentials include;

 

• Terpenoids

They are the most structurally diverse groups of natural products formed by fusion of isoprene monomers in plants. This class of plant secondary metabolites are grouped according to the number of isoprene units or number of carbon in their skeletal structure (Zwenger and Basu, 2008). The group include mono terpenes which contain 2 units of isoprene with C10 and are present as essential or volatile components of herbs, spices and flowers. Sesquiterpenes are derivatives of three (3) isoprene units containing fifteen (15) carbon atoms in their structure and are present in essential oil. This group of compounds act as phytoalexins, antimicrobial and antifeedant in plants. Diterpenes contain 20 carbon atoms derived from four units of isoprene and posess pharmacological properties e.g. anticancer and treatment of glaucoma. Triterpenes contain 30 carbon atoms. Tetraterpenes such as carotenoids contain 40 carbon atoms made of 8 isoprene units ().

Several terpenoids have been identified to have good activity in antidiarrhoeal mechanisms. Such activities include inhibitory effect on bacteria, suppression of intestinal fluid accumulation (Chen et al., 2007). They were also reported to increase phagocyte index, stimulate macrophage, increased humoral and cell mediated immune response (Khajuria et al., 2007). Some terpenoids also inhibit nitric oxide and prostaglandins invitro (Reyes et al, 2006). They have been reported to exhibit anti- allergic activities through inflammatory response modulation.

1.3.2 Alkaloids

Alkaloids refer to a group of heterocyclic nitrogenous compounds. Most alkaloids are derived from amino acids and are classified based on their structures as pyridine, tropane or pyrrolizidine alkaloids. They have been proven to exhibit remarkable physiological and pharmacological activities. (Samy and Gopalakrishnakone, 2008). Though they have many pharmacological mechanisms such as microbiocidal effects on diarrhoegenic pathogens, the main antidiarrhoeal effect is probably that of delayed intestinal transition of bowel materials (Cowan, 1999). Tubocurarine is an important muscle relaxant used in medicine. Some alkaloids like boldine have good antioxidant properties indicating the effectiveness of the compound in preventing various oxidative stress related illnesses like inflammatory cascade, immune dysfunctions, etc. however, at high concentrations, it causes cellular damage and potentiates lipid peroxidation (pro-oxidant) (Nissanka et al., 2001; Mandal et al., 2010).

 

Fig. 4: Tubocurarine (Mandal et al., 2010).

 

1.3.3 Phenolics

Phenolic compounds are characterized as aromatic metabolites that have one or more acidic hydroxyl groups attached to the phenyl ring. The consumption of diet rich in phenolic compounds has been hypothesized to be important in health promotion and disease prevention in humans and animals (Ramful et al., 2010). This group of compounds exhibit numerous biological activities directly or indirectly on intestinal epithelium which contribute to alleviation of diarrhoea symptoms.

Some phenolic compounds exhibit anti microbial activities. They have anti secretory effect on vibrio cholera toxin induced intestinal fluid accumulation. Some have been proven to inhibit histamine induced contractile response on rat ileum and acetylcholine induced contractile response on rat duodenum (Desire et al., 2010). They have also been reported to have antispasmolytic activities (Ragone et al., 2007). Some also posess superoxide radical scavenging activity and lipid peroxidation inhibition at certain concentrations (Meghashiri et al., 2010).

Some phenolics include flavonoids which are the most important plant pigment for flower colouration, producing yellow or blue pigmentation in petals designed to attract pollinator animals. In higher plants, flavonoids are involved in U.V filtration, symbiotic nitrogen fixation and floral pigmentation. They may also serve as chemical messengers, physiological regulators and cell cycle inhibitors (Yamamoto and Gaynor, 2001; Cazarolli et al., 2008). They also posess anti allergic, anti inflammatory, antioxidant ,anticancer and anti diarrhoeal effects (in vitro). In vivo, it protects the gastro intestinal mucosa against ROS generated by acute and chronic stress.

 

 

Fig. 5: Leucoanthocyanidin (Cazarolli et al., 2008)

 

1.3.4 Glycosides

A glycoside is a molecule in which a sugar is bound to another functional group through a glycosidic bond. They play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. These can be activated by enzyme hydrolysis which causes the sugar part to be broken off, making the chemical available for use (Cazarolli et al., 2008) Classifications of glycosides include alcoholic glycosides, anthraquinone glycoside, coumarin, chromone , cyanogenic, flavonoids and phenolics

Fig. 6: Cyanogenic glycoside (amygdalin) (Cazarolli et al., 2008)

 

1.3.5 Tannins

Chang et al. (2000) found that tannins have been reported to exert many physiological effects such as to accelerate blood clotting, reduce blood pressure, decrease the serum lipid level, produce liver necrosis and modulate immune responses.

 The nervous system

Source: (Furness, 2008)

Fig. 7: Organization of the nervous system

The nervous system is made up of the central nervous system and the peripheral nervous system. The peripheral nervous system is divided into the afferent and the efferent neurons. The efferent division comprises of the somatic and autonomic nervous systems.

The autonomic nervous system (ANS or visceral nervous system or involuntary nervous system) is the part of the peripheral nervous system that acts as a control system that functions largely below the level of consciousness to control visceral functions (Furness, 2008) including heart rate, digestion, respiratory rate, salivation, perspiration, pupillary dilation, micturition (urination), sexual arousal, breathing and swallowing. Most autonomous functions are involuntary but they can often work in conjunction with the somatic nervous system which provides voluntary control.

The ANS is divided into three main sub-systems: the parasympathetic nervous system (PSNS), sympathetic nervous system (SNS) and the enteric nervous system (ENS) (Furness,2008). Depending on the circumstances, these sub-systems may operate independently of each other or interact co-operatively.

 Table 2: Functions of the autonomic nervous system

Source:(Furness, 2008)

Fig. 8: Diagrammatic representation of the parasympathetic (PSNS) and sympathetic nervous systems (SNS) (Belvisi et al., 1992).

ENS consists of a mesh-like system of neurons that governs the function of the gastrointestinal system. SNS is often considered the “fight or flight” system, while the PSNS is often considered the “rest and digest” or “feed and breed” system. In many cases, PSNS and SNS have “opposite” actions where one system activates a physiological response and the other inhibits it. An older simplification of the sympathetic and parasympathetic nervous systems as “excitory” and “inhibitory” was overturned due to the many exceptions found. A more modern characterization is that the sympathetic nervous system is a “quick response mobilizing system” and the parasympathetic is a “more slowly activated dampening system”, but even this has exceptions, such as in sexual arousal and orgasm, wherein both play a role (Belvisi et al., 1992).

In general, ANS functions can be divided into sensory (afferent) and motor (efferent) subsystems. Within both, there are inhibitory and excitatory synapses between neurons. Relatively recently, a third subsystem of neurons that have been named ‘non-adrenergic and non-cholinergic' neurons (because they use nitric oxide as a neurotransmitter) have been described and found to be integral in autonomic function, in particular in the gut and the lungs (Belvisi et al., 1992).

1.5       Neurotransmitters and pharmacology

At the effector organs, sympathetic ganglionic neurons release noradrenaline (norepinephrine), along with other cotransmitters such as ATP, to act on adrenergic receptors, with the exception of the sweat glands and the adrenal medulla (Costanzo, 2007):

• Acetylcholine is the preganglionic neurotransmitter for both divisions of the ANS, as well as the postganglionic neurotransmitter of parasympathetic neurons. Nerves that release acetylcholine are said to be cholinergic. In the parasympathetic system, ganglionic neurons use acetylcholine as a neurotransmitter to stimulate muscarinic receptors (Rang et al., 2003).
• At the adrenal medulla, there is no postsynaptic neuron. Instead the presynaptic neuron releases acetylcholine to act on nicotinic receptors. Stimulation of the adrenal medulla releases adrenaline (epinephrine) into the bloodstream, which acts on adrenoceptors, producing a widespread increase in sympathetic activity (Rang et al., 2003). 

1.6       Some biologically important electrolytes; sodium, potassium and bicarbonates

Sodium and potassium are electrolytes, that is the electricity conducting minerals or salts in the body that help maintain fluid balance and regulate muscle and nerve function. Many processes in the body, especially in the brain, nervous system, and muscles, require electrical signals for communication. The movement of sodium and potassium in and out of the cell is critical in generation of these electrical signals.  The normal potassium level ranges from 3.6 to 4.8mEq/L while the normal blood sodium level ranges from 135 to 145mEq/L (Donowitz et al., 1995).

 

Bicarbonate;

Fig. 9: Hydroxidodioxidocarbonate⁽¹⁻⁾ (Donowitz et al., 1995).

 

Bicarbonate (HCO₃⁻) is alkaline, and a vital component of the pH buffering system of the human body (maintaining acid-base homeostasis). 70-75% of CO₂ in the body is converted into carbonic acid (H₂CO₃), which can quickly turn into bicarbonate. With carbonic acid as the central intermediate species, bicarbonate – in conjunction with water, hydrogen ions, and carbon dioxide – forms this buffering system, which is maintained at the volatile equilibrium required to provide prompt resistance to drastic pH changes in both the acidic and basic directions. This is especially important for protecting tissues of the central nervous system, where pH changes too far outside of the normal range in either direction could prove disastrous (Donowitz et al., 1995).

Bicarbonate also acts to regulate pH in the small intestine. It is released from the pancreas in response to the hormone secretin to neutralize the acidic chyme entering the duodenum from the stomach. Disturbances in the sodium and potassium levels can lead to conditions known as hypernatremia, hyponatremia, hyperkalemia and hypokalemia (Perez et al., 1987).

 

1.6.1    Hyponatremia

1.6.1.1 Signs and Symptoms of Hyponatremia

Muscle twitching and weakness due to osmotic swelling of cells; lethargy, confusion, seizures, and coma due to altered neurotransmission ; hypertension and tachycardia due to decreased extracellular circulating volume; nausea, vomiting, and abdominal cramps due to oedema affecting receptors in the brain or vomiting centre of the brain stem and oliguria or anuria due to renal dysfunction (Perez et al., 1987).

 

1.6.1.2 Clinical diagnosis of hyponatremia

Serum sodium <135 mEq/l, decreased urine specific gravity, decreased serum osmolality, urine sodium > 100 mEq/24 hours and increased red blood cell count.

Some causes of hyponatremia include; vomiting and diarrhoea since the body loose electrolytes along with body fluids (Perez et al., 1987).

 

1.6.1.3 Causes of hypernatremia

There are numerous causes of hypernatremia; these may include kidney disease, too little water intake, and loss of water due to diarrhoea. However, under normal physiological conditions, consumption of foods or food products with high sodium content may contribute to hypernatremia (Perez et al., 1987).

 

1.6.2    Hypernatremia

1.6.2.1 Signs and symptoms of hypernatremia

Agitation, restlessness, fever, and decreased level of consciousness due to altered cellular metabolism; hypertension, tachycardia, pitting oedema, and excessive weight gain due to water shift from intracellular to extracellular fluid; thirst, increased viscosity of saliva, rough tongue due to fluid shift  and dyspnoea, respiratory arrest and death from dramatic increase in osmotic pressure (Harris, 1983).

 

1.6.2.2       Clinical diagnosis of hypernatremia

Serum sodium > 145 mEq/l, urine sodium <40 mEq/24 hours and high serum osmolality

 

1.6.3    Hypokalemia

This condition is defined as serum concentration of potassium < 3.5mEq/L. 98% of potassium is found within the cells. Intracellular concentrations range between 150-160mEq/L. the ratio of intracellular to extracellular potassium concentration is important in determining cellular resting membrane potential and influences the function of excitable tissues such as nerves and muscles (Harris, 1983). Maintenance of this concentration gradient across the membranes is achieved by the enzyme Na⁺/K⁺-ATPase that pumps 2 potassium ions into the cell in exchange for 3 Na⁺ ions pumped out. Serum potassium concentration relates both to the internal balance between the intracellular and extracellular fluids and the external balance determining the total body potassium (Cieza et al., 1995). This is achieved by the kidney mainly under the control of aldosterone secreted by the adrenal glands. Hypokalemia is often found in adults treated with thiazides or combination diuretics (Oster et al., 1980). Risk of developing hypokalemia is increased by concomitant illness particularly heart failure, alcoholism and nephritic syndrome.  The management is usually by potassium replacement either through diet or drugs.

 

1.6.3.1 Signs and symptoms of hypokalemia

Dizziness, hypotension, arrhythmias, electrocardiogram (ECG) changes, and cardiac arrest due to changes in membrane excitability; nausea, vomiting, anorexia, diarrhoea, decreased peristalsis, and abdominal distension due to decreased bowel motility; muscle weakness, fatigue, and leg cramps due to decreased neuromuscular excitability (Donowitz et al., 1995).

 

1.6.3.2 Clinical diagnosis of hypokalemia

Serum potassium < 3.5 mEq/l, coexisting low serum calcium and magnesium levels not responsive to treatment for hypokalemia usually suggest hypomagnesaemia, metabolic alkalosis, ECG changes include flattened Waves, elevated U waves, Depressed ST segment (Cieza et al., 1995)

Some possible causes of hypokalemia include; vomiting, diarrhoea, eating disorders, certain diuretics and antibiotics, laxative abuse, etc. the most common congenital cause is Gitelman’s syndrome associated with impaired renal tubular ion transport due to mutation in the Na⁺/Cl^– co-transporter gene (Cieza et al., 1995). Barter’s syndrome is closely related but presents in infancy with failure to thrive and is due to a mutation in the Cl^– channel gene.

 

 

1.6.4    Hyperkalemia

1.6.4.1 Signs and symptoms of hyperkalemia

Tachycardia changing to bradycardia, ECG changes, and cardiac arrest due to hypo polarization and alterations in repolarization ; (Cieza et al., 1995)Nausea, diarrhoea, and abdominal cramps due to decreased gastric motility and muscle weakness and flaccid paralysis due to inactivation of membrane sodium channels.

 1.6.4.2 Clinical diagnosis of hyperkalemia

Serum potassium > 5mEq/l, metabolic acidosis, ECG changes include tented and elevated T waves, widened QRS complex, prolonged PR interval, flattened or absent P waves, depressed ST segment (Agarwal et al., 1994).

1.7 Normal Physiology of Gut Fluid and Electrolyte Transport

During the course of each day, secretion as well as absorption of fluid and electrolytes occurs along the gastrointestinal tract. Normally 7 to 8 litres of fluid is secreted each day, far exceeding dietary consumption, and almost all of these secretions, as well as any ingested fluid, are absorbed by the end of the colon (Charney and Donowitz, 2005). The gastrointestinal tract is divided into sequential segments, each with a distinct group of ion transporters and channels that interact with one another to determine the electrolyte content and volume of the fluid in the gut lumen. With the exception of the stomach and the exocrine pancreas, secretion of fluid and electrolytes occurs primarily in a subset of cells in the epithelial crypts with unique ion transport properties. Throughout the gut, fluid and electrolyte transport (both absorption and secretion) is driven primarily by Na⁺/K⁺-ATPase transport activity across the basolateral membrane of epithelial cells. Several key apical membrane electrolyte transporters participate, including Cl⁻/HCO₃⁻ and Na⁺/H⁺ ion exchangers and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, all of which are present in many segments of the gut, and H⁺/K⁺-ATPases, which are confined to the stomach and colon. Disruption of function or abnormal stimulation of these ion transporters underlies a variety of gastrointestinal disorders that are associated with acid-base and electrolyte disorders (Cieza et al., 1995)

Fig. 10: Na⁺/K⁺-ATPase transport activity (Charney and Donowitz, 2005).

Fig. 11: Glucose transport in intestinal epithelial cells (Nelson and Cox, 2008)

1.8 Electrolyte transport in the Jejunum and Ileum

This segment of the bowel both absorbs and secretes fluid, but absorption normally predominates, reducing the total gut fluid content to approximately 1 L/d by the time it enters the colon. Absorption is driven by sodium and chloride uptake (driving water uptake) and occurs via two linked transporters, the Na⁺/H⁺ exchanger and the Cl⁻/HCO₃⁻ exchanger (Turnberg et al., 1970). These two transporters take up Na⁺ and Cl⁻ from the gut lumen and secrete H⁺ and HCO₃⁻ into it. The latter two ions combine, forming H₂CO₃, which dehydrates to form CO₂ in the intestinal lumen. The Cl⁻/HCO₃⁻ exchanger in the small intestine is the “downregulated in adenoma” (DRA) gene product, also named CLD (for chloride diarrhoea) (Alrefai et al., 2007). By the end of the ileum, Cl⁻/HCO₃⁻ exchange predominates, resulting in an alkaline solution. Chloride secretion occurs in specialized cells in the intestinal crypts via a series of apical Cl⁻ ion channels, one of which is the CFTR channel, recycling Cl⁻ into the lumen. In contrast to the colon, the small intestinal secretory cells do not have an apical K⁺ channel. Potassium movement across the membrane is accounted for by passive diffusion and solvent drag, with absorption predominating (Turnberg, 1971). These absorptive and secretory processes leave the fluid that enters the colon slightly hypotonic with a [HCO₃⁻] of approximately 30 mmol/L and with a [K⁺] of 5 to 10 mmol/L.

1.9 Castor oil

It is a vegetable oil obtained from the castor bean (castor seed) (Adeyemi and Akindele, 2006). It is obtained by pressing the seeds of the castor plant, Ricinus communis (Euphorbiaceae). Sometimes called castor bean oil, this plant is not a member of the bean family. Castor oil is a colourless to very pale yellow liquid with mild or no odour or taste. Its boiling point is 313 °C (595 °F) and its density is 961 kg/m³  (Wood, 2001). It is a triglyceride in which approximately 90 percent of fatty acid chains are ricinoleate. Oleate and linoleates are the other significant components (Adeyemi and Akindele, 2006).

Castor oil is famous as a source of ricinoleic acid, a monounsaturated, 18-carbon fatty acid. Among fatty acids, ricinoleic acid is unusual in that it has a hydroxyl functional group on the 12th carbon (Boel and Aust, 2009). This functional group causes ricinoleic acid (and castor oil) to be more polar than most fats. The chemical reactivity of the alcohol group also allows chemical derivatization that is not possible with most other seed oils. Because of its ricinoleic acid content, castor oil is a valuable chemical in feedstocks (Adeyemi and Akindele, 2006).

 

Fig. 12: Ricinoleic acid. ((9Z, 12R)-12-Hydroxyoctadec-9-enoic acid) (Adeyemi and Akindele, 2006).

1.9.1 Ricin

The castor seed contains ricin, a toxic protein. Heating during the oil extraction process denatures and inactivates the protein (Busso and Castro-Prado, 2004).

 1.9.2 Uses of castor oil

In the industry, its derivatives have applications in the manufacturing of soaps, lubricants, hydraulic and brake fluids, paints, dyes, coatings, inks, cold resistant plastics, waxes and polishes, nylon, pharmaceuticals and perfumes (Busso and Castro-Prado, 2004).

In the food industry, castor oil is used in food additives, flavourings, candy (Wilson et al., 1998) as a mould inhibitor, and in packaging. Polyoxyethylated castor oil (e.g., Kolliphor EL) is also used in the food industries (Busso and Castro-Prado, 2004).

In medicine, the United States Food and Drug Administration (FDA) has categorized castor oil as “generally recognized as safe and effective” (GRASE) for over-the-counter use as a laxative with its major site of action the small intestine where it is digested into Ricinoleic acid. It decreases fluid absorption^.   It also induces labour in pregnant females. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors. In Ayurvedic medicine it is used to enhance memory (Swami, 2004; Dua et al., 2009).  

Therapeutically, Castor oil, or a castor oil derivative such as Kolliphor EL (polyethoxylated castor oil, a nonionic surfactant), is added to many modern drugs, including: Miconazole, an antifungal agent; (Marmion et al., 1976; Fromtling, 1988). Paclitaxel, a mitotic inhibitor used in cancer chemotherapy; (Micah et al., 2006).Sandimmune (cyclosporine injection, USP), an immunosuppressant drug widely used in connection with organ transplant to reduce the activity of the patient's immune system; Nelfinavir mesylate, an HIV protease inhibitor; (Zhang et al., 2001). Saperconazole, a triazole antifungal agent (contains Emulphor EL-719P, a castor oil derivative); Tacrolimus, an immunosuppressive drug (contains HCO-60, polyoxyl 60 hydrogenated castor oil); Xenaderm ointment, a topical treatment for skin ulcers, is a combination of Peru balsam, castor oil, and trypsin (Zhang et al., 2001) ^. 

In traditional medicine, its uses include skin disorders, burns, sunburns, cuts, and abrasions. It has been used to draw out styes in the eye by pouring a small amount into the eye and allowing it to circulate around the inside of the eyelid. The oil is also used as a rub or pack for various ailments, including abdominal complaints, headaches, muscle pains, inflammatory conditions, skin eruptions, lesions, and sinusitis (Dua et al., 2009).

1.10 Antidiarrhoeal drugs

1.10.1 Nitric oxide synthase inhibitors

Nitric oxide (NO) is known to have a protective effect on the gastrointestinal tract (Mascolo et al., 1993). NO has been reported to play an important role in castor oil-induced diarrhoea. Pre-treatment with NO synthase inhibitors from l-arginine prevent castor oil-induced diarrhoea and decrease the intestinal fluid accumulation and Na⁺ secretion induced by castor oil. Thus, NO is one of the mediators of the intestinal secretion and diarrhoea induced by castor oil (Bart and Walter, 1995; Jing et al., 2009)

1.10.2 Nufenoxole, a new orally active agent possesses potent antidiarrhoeal properties in both animals and man, with activity comparable to that of diphenoxylate and loperamide (Piercey and Ruwart, 1979). Nufenoxole, however, has a longer biological half-life and shows a wider separation of gastrointestinal and central nervous system effects. Animal and human studies have revealed no evidence of dependence liability. Nufenoxole binds to opioid receptors in the brain and myenteric plexus of the intestine (Mackerer et al., 1977). Endogenous and exogenous opioids enhance water and electrolyte absorption and inhibit fluid secretion stimulated by a variety of secretagogues in mammalian intestine (Mackerer et al., 1977) Although opioids are thought to exert their antidiarrhoeal effects by inhibiting intestinal motility, such changes in transmucosal epithelial fluid transport may be at least equally as important in mediating their action (Moriarty et al., 1985).

 

1.10.3 Loperamide

Loperamide, a piperidine derivative, is an opioid drug used against diarrhoea resulting from gastroenteritis or inflammatory bowel disease. It was developed by Janssen in 1971(Hanauer, 2008). In most countries it is available generically and under brand names such as Lopex, Imodium, Dimor, Fortasec, Lopedium, Loperamide is an opioid-receptor agonist and acts on the μ-opioid receptors in the myenteric plexus of the large intestine; by itself it does not affect the central nervous system (Mackerer et al., 1977). It works similarly to morphine, by decreasing the activity of the myenteric plexus, which in turn decreases the tone of the longitudinal and circular smooth muscles of the intestinal wall. This increases the amount of time substances stay in the intestine, allowing for more water to be absorbed out of the faecal matter (Butler, 2008). Loperamide also decreases colonic mass movements and suppresses the gastro colic reflex (Stokbroekx et al., 2008).

1.10.4  Diphenoxylate (R-1132)

Diphenoxylate (R-1132) is an opioid agonist (Moriarty et al., 1985) used for the treatment of diarrhoea that acts by slowing intestinal contractions and peristalsis allowing the body to consolidate intestinal contents and prolong transit time, thus allowing the intestines to draw moisture out of them at a normal or higher rate and therefore stop the formation of loose and liquid stools (Stokbroekx et al., 1973). It is the main active ingredient in the anti-peristaltic medication Lomotil, which also contains atropine.

 

 

 

 

Fig. 15: Ethyl 1-(3-cyano-3, 3-diphenylpropyl)-4-phenylpiperidine-4-carboxylate (Stokbroekx et al., 1973)

 

1.11 Aim of the Study

There are speculations that unripe Musa paradisiacae pulp and peel could be used to alleviate diarrhoea. This work is therefore aimed at determining the effects of unripe Musa paradisiacae pulp and peel on castor oil-induced diarrhoea in Wistar albino rats.

 

1.12 The specific objectives of the study

They include determination of the:

• Median lethal dose (LD₅₀) of the aqueous extracts of unripe Paradisiacae pulp and peel.
• Quantitative and qualitative phytochemical compositions of the aqueous extracts of unripe Musa paradisiacae pulp and peel.
• Proximate composition of the aqueous extracts of unripe Musa paradisiacae pulp and peel.
• Effects of aqueous extracts of unripe Musa paradisiacae pulp and peel on:

1. Castor oil- induced diarrhoea
2. Castor oil-induced gastro-intestinal motility

• Effects of the aqueous extracts of unripe Musa paradisiacae pulp and peel on sodium, potassium and bicarbonate ion concentrations.
• Effects of the aqueous extracts of unripe Musa paradisiacae pulp and peel on the transport of ions across the intestinal membrane.

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