EFFECT OF AQUEOUS LEAF EXTRACT OF Piliostigma thonningii ON SERUM LIPID PROFILE FOLLOWING INDOMETHACIN INDUCED GASTRIC ULCER IN WISTAR ALBINO RATS

ABSTRACT

In an attempt to determine the effect of P.thonningii leaf extract on lipid profile in male Wistar albino rats following indomethacin induced mucosa onslaught. The thirty six (36) male rats that were divided into six (6) groups of 6 rats each. Group one (1) served as control and was given 0.5ml of normal saline (vehicle). Group three (3), five (5) and six (6) were given 100, 100 and 200mg/kg body weight of the extract while group two (2) was treated with 100mg/kg body weight of standard drug (cimetidine). The vehicle and extract were administered orally while the drug was administered intra-muscularly for 12days. After 12days of administration all rats were fasted for 24 hours, gastric ulceration was then induced by the administration of 40mg/kg indomethacin orally to group 2, 4, 5 and 6. 12 hours after indomethacin administration all rats were sacrificed after been anaesthetized with chloroform, blood was collected by cardiac puncture and serum collected for the determination of lipid profile using standard methods.
The result depict a significant (P<0.05) increases in serum total cholesterol when compared with the control. Likewise the standard drug (cimetidine), the extract and other treated groups show a significant (P<0.05) increase in serum HDL when compared with the control while the group induced with ulcer without treatment showed a significant (P<0.05) decrease in serum HDL when compared with the control. The extract also produced a significant (P<0.05) decrease of serum VLDL while group treated with cimetidine and indomethacin produced a significant (P<0.05) increase when compared with the control. Similar pattern was shown with serum LDL. The treatment also produced a significant (P<0.05) increase throughout the experimental group for serum triglyceride when compared with the control.  The biochemical and physiological alterations in this study following indomethacin mediated mucosa onslaught suggest that the extract treated groups are shielded against cardiovascular related disorder  but it appears that cimetidine and indomethacin treated groups might be predisposed to cardiovascular derangements such as atherosclerosis, myocardial infarction and coronary heart diseases vial a mechanism not yet fully understood.

 CHAPTER ONE

                                                            INTRODUCTON

Medicinal plants play a significant role in providing primary health care services to rural habitats and are used by about 80% of the marginal communities around the world (Ahmad et al; 2003). Each medicinal plant species has its own nutrient composition besides having pharmacologically important phytochemicals. These nutrients are essential for the physiological functions of human body (Karthika et al; 2013). Such nutrients and biochemical like carbohydrates, fats and proteins play an important role in satisfying human needs for energy and life processes (Novak et al; 2000).Phytochemical studies have attracted the attention of plant scientists due to the development of new and sophisticated techniques. These techniques play a significant role in giving the solution to systematic problems on one hand and in the search for additional resources of raw materials for pharmaceutical industry on the other hand. Plant synthesizes a wide variety of chemical compounds, which can be sorted by their chemical class, biosynthetic origin and functional groups into primary and secondary metabolites. Knowledge of the chemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information be of value in disclosing new resources of such chemical substances. One of these plants is Piliostigma thonningii (Murder et al; 2003).

Ulcer and Cardiovascular diseases and related disorders are a major cause of mortality both in men and women all over the world (S. C. Smith, 2004). They are commonly characterized by high levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the serum. Increased total cholesterol and more significantly LDL cholesterol in the serum have been implicated in the etiology of cardiovascular diseases and are seen as primary risk factors (Edijala et al; 2005). Also, high level of lipids in the blood has been associated with hypertension and lipid peroxidation (Recknagel 1983). Orthodox medicine, though, is generally preferred and acceptable, traditional medicine is still very much relied on all over the world (Leckridge et al; 2005). This is common in the developing countries where the cost of orthodox medicine is astronomical and unaffordable to a large size of the populace (K. Busia, 2005). According to World Health Organization, about 80% of folks in developing countries depend mainly on traditional medicine for their primary health care, and about 85% of such traditional medicine involves the use of plant extracts(Farnsworth et al; 1988).Some commonly consumed herbs have been reported to promote reduction in blood lipids (Adebayo et al; 2006). P. thonningii is an under-explored leguminous plant that belongs to the family, Leguminosae-Caesalpiniodae. The tree is perennial in nature, and the petals varies from white to pink in color and are produced between November and April (Jimoh et al; 2005). Silva and colleagues (Silva et al; 1997) reported that in many African countries various parts of P. thoninngii (organs: root, bark, seed, and fruit) are used for various medicinal purposes. For instance, the plant is used to treat wounds, ulcers, gastric/heart pain, gingivitis, and as an antipyretic. In Tanzania and Zimbabwe, a cough remedy is prepared from the root bark; this fraction exhibits significant anti-inflammatory/analgesic activity. Certain compounds isolated from its leaves have been reported to elicit anti-inflammatory and antibacterial activities (Akinpelu et al; 2000).Generally, plants reported to exhibit lipid lowering activity are rich in flavonoids and tannins which play significant role in the mobilization and metabolism of lipids. Preliminary phytochemical studies on Piliostigma thonningii reveals high levels of flavonoids, tannins, and alkaloids (Akindahunsi et al; 2005). The plant is also reported to contain nutritionally important vitamins (such as C, E, and beta-carotene) and minerals (such as calcium, magnesium, zinc, and potassium) all of which contribute to its high-antioxidant properties). Against high incidence of cardiovascular diseases, there is paucity of information on scientifically verified plants with antilipidaemic and anticholesterolaemic properties (Akindahunsi et al; 2005). In light of the chemical constituents of P. thonningii, this study was designed to evaluate the effect of its ethanol leaf extract on the serum lipid profile following indomethacin induced ulcer in rats.

1.1 BACK GROUND OF STUDY:

Piliostigma thonningii is being used in Nigerian traditional medicine for the treatment of various diseases including gastric ulcer. The assemblage of different parts of this plant has been found traditionally useful. As a result, people have resulted to using parts of this plant in the management or treatment of different types of diseases. Such parts are the stem, roots, seeds and leaf (Bekele-Tesemma.,2007).The stem of this plant have been used in the management of earache, toothache, diarrhoea, dysentery, intestinal problems in tropical Africa (Akinpelu and Obuotor.,2000).The back of P.thonningii could be used to manage cough; this is done by chewing the bark or through infusion. The infusion or maceration of the bark also includes the treatment of malaria and leprosy. The analgesic properties of this plant are also ascribed to the bark (Cowan, 1999). The leaf also serves as a laxative, sometimes giving to neonates as tonic and to massage mother’s abdomen. The leaves after soaking in hot water are applied topically to wound dressing (Odukoya,2002).The barks are used in phytochemistry as elements like alkaloids, antibiotics, bacteriostatic, fungi static, tannins and astringents (Kunle,2010). The pods root and bark produces dyes, stains, inks, as bi-products. The leaves are chewed to relieve thirst, the fruits and seeds can be edible. The pods and foliage are nutritious to herbivorous like cattle and elephants (Kunle, 2010). P.thonningii is also used medicinally in many topical African countries to treat wounds, ulcers, heart pain, etc. (Siva et al., 1997).

             1.2 STATEMENT OF THE PROBLEM.                                    

Some researchers over the years have published works on the antilipidemic, hypocholesterolemic and hypotriglyceridemic effect of P. thonningii leaf. Most herbal practitioners also uses the plant to manage gastric and peptic ulcer. Since there is the possibility of people having ulcer to also suffer cardiovascular disorders such as coronary heart diseases and arteriosclerosis. Hence, paucity demand that research work on the focus on the effect of ethanol leaf extract of p.thonningii on serum lipid profile following indomethacin induced gastric ulcer in rats.

          1.3   JUSTIFICATION OF THE STUDY

This research is to access the effects of aqueous leaf extract of P. thonningii          on serum lipid profile following indomethacin induced gastric ulcer in wistar           albino rats. Although the extract of P. thonningii is known for its anti­-ulcer property, the extract is administered alongside the standard drug (cimetidine) for a short period of 12days to determine if the P. thonningii extract is more effective in ulcer control and how the extract affects the lipid profile in the ulcerated rats.

            1.4  AIM OF THE STUDY.

To ascertain the effects of aqueous leaf extract of P.thonningii on serum lipid profile following indomethacin induced gastric ulcer in wistar albino rats.

1.5  OBJECTIVES OF THE STUDY.

The objectives of this study is to access the ameliorative effect of P. thonningii on serum lipid profile of rats induced with indomethacin by monitoring the serum total cholesterol, serum- LDL, serum-HDL, and serum-VLDL and serum triglyceride.

1.6   SCOPE OF STUDY

This study monitored the effect of aqueous leaf extract of P. thonningii on serum lipid profile following indomethacin-induced gastric ulcer in Wistar albino rats within short-term administration.

CHAPTER TWO

                LITERATURE REVIEW

2.1      INDOMETHACIN

Indomethacin was discovered in 1963(Hart et al; 1963) and it was first approved for use in the U.S. by the Food and Drug Administration in 1965. Its mechanism of action, along with several other NSAIDs that inhibit COX, was described in 1971 (Ferreira et al; 1971). Systemic (IUPAC) name is 2-{1-[(4-Chlorophenyl) carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl} acetic acid. And its trade name is Indocin, Indocid (dodick, 2004).Indomethacin is a non-steroidal anti-inflammatory analgesic used in the treatment of disorders such as rheumatoid arthritis, ankylosing spondylitis, and osteo-arthritis. Prostaglandins have a significant stimulatory effect on established labour; because indomethacin is a potent inhibitor of prostaglandin synthesis, this agent is also used in the treatment of preterm labour. Like other prostaglandin synthetase inhibitors, indomethacin acts by inhibiting the activity of the cyclo-oxygenase enzyme necessary for the synthesis of prostaglandins, prostacyclin, and thromboxane (dodick, 2004). Unlike aspirin, which causes an irreversible inhibition of this enzyme, indomethacin results in a competitive and reversible inhibition. Indomethacin reduces fever, pain and inflammation. It is a crystalline and poorly water soluble drug and the rate of oral absorption is often controlled by the dissolution rate in the gastrointestinal tract. Although, several different methods are available to produce amorphous drugs, ball milling appears to be the most practical method to amorphization (dodick, 2004). Solubility and dissolution profiles were evaluated using powders in a USP type II dissolution apparatus. Dissolution profiles of indomethacin co-ground with Neusilin® US2 initially and at 1 to 3 months of storage at 40°C/75% RH showed a slight increase in the maximum transient concentration (MTC) from the initial sample to the sample stored for 1 month. Further storage for 2 months did not change the MTC. The maximum sustained concentration (MSC) at the start was 13 times higher than the solubility of crystalline indomethacin and increased with storage time (dodick, 2004).

Amorphous solids of Indomethacin co-ground with Neusilin® US2 (1:4 and 1:5) at 75% RH was physically stable for 3 to 6 months when stored at 40°C and 75% RH . A further investigation of pore volumes and pore diameters for the initial and stored samples revealed no difference suggesting that there is no further deposition or depletion of drug from the pores of Neusilin® US2 during storage. Other drug candidates viz. ketoprofen, naproxen, and progesterone showed complete amorphization and stability on milling indicate that Neusilin® US2 is an excellent media for amorphization of a large number of BCS class II poorly water soluble crystalline drugs (Ferreira et al; 1971).

            2.1.1   CLINICAL INDICATOR FOR INDOMETHACIN

ü  Hemicranias continua

ü  Ankylosing spondylitis

ü  Arthritic gout

ü  Barther syndrome

ü  Bursitis

ü  Cryoglobulinemia

ü  Dysmenorrhea (menstrual cramps)

ü  Exerton headache (sanders et al ; 2012)

ü  Fever and pain associated with malignant diseases (especially tumor fever associated with liver involvement, lymphogranulomatosis) (sanders et al; 2012)

ü  Hypnic

ü  Juvenile arthritis migrain

ü  Patent ductusarteriosus

ü  Nephrogenic diabetesinsipidus (prostaglandin inhibits vasopressin’s action in the kidney)

ü  Osteoarthritis

ü  Paget’s disease of bone

ü  Paroxysmal hemicranias

ü  Pericarditis

ü  Primary stabbing headache

ü  Pseudo gout

ü  Psoriatic arthritis

ü  Renal colic (pain due to kidney stones)

ü  Reactive arthritis

ü  Retinopathy of prematurity

ü  Rheumatoid arthritis

ü  Tendinitis

ü  Trigeminal autonomic cephalgias

ü  Headaches induced by or otherwise a result of valsalva maneuver(Sanders et al; 2012)

Indomethacin has also been used clinically to delay premature labor, reduce amniotic fluid in polyhydramnios, and to close patent ductusarteriosus. Indomethacin is a potent drug with many serious side effects and should not be considered an analgestic for minor aches and pains or fever. The medication is better described as an anti-inflammatory, rather than an analgesic. Indomethacin can also affect warfarin and subsequently raise INR. (Sanderset al; 2012)

2.1.1.1   CONTRAINDICATIONS

ü  Concurrent peptic ulcer, or history of ulcer disease

ü  Allergy to indomethacin, aspirin, or other NSAIDs

ü  Patients with nasal polyps reacting with an angioedema to other NSAIDs

ü  Children under 2 years of age (with the exception of neonates with patent ductusarteriosus)

ü  Severe pre-existing renal and liver damage

ü  Caution: pre-existing bone marrow damage (frequent blood cell counts are indicated)

ü  Caution: bleeding tendencies of unknown origin (indomethacin inhibits platelet aggregation)

ü  Caution: parkinson’s disease, epilepsy, psychotic disorders (indomethacin may worsen these conditions)

ü  Concurrent with potassium sparing diuretics

ü  Patients who have a patent ductusarteriosus dependent heart defect (such as transposition of the great vessels)

ü  Significant hypertension (high blood pressure) (sanders et al; 2012)

2.1.2   MECHANISM OF ACTION OF INDOMETHACIN

Indomethacin is nonselective inhibitor of cyclooxygenase (COX) 1 and 2, enzymes that participate in prostaglandins are hormone-like molecules normally found in the body, where they have a wide variety of effects, some of which lead to pain, fever, and inflammation (Giles et al; 2007).

Prostaglandins also cause uterine concentration in pregnant women. Indomethacin is an effective tocolytic agents, (Giles et al; 2007) able to delay premature labor by reducing uterine contractions through inhibition of PG synthesis in the uterus and possibly through calcium channel blockade (Giles et al; 2007).

Indomethacin has two additional modes of actions with clinical importance:

ü  It inhibits motility of polymorpho nuclear leukocytes, similar to colchicine

ü  It uncouples oxidative phosphorylation in cartilaginous (and hepatic mitochondria, like salicylates.

These additional effects account as well for the analgesic and the anti-inflammatory properties (Giles et al; 2007)

Indomethacin readily crosses the placenta and can reduce fetal urine production to treat polyhydramnios. It does so by reducing renal blood flow and increasing renal vascular resistance, possibly by enhancing the effects of vasopressin on the fetal kidneys (Vaziriet al; 1985).

2.1.2.1   ADVERSE EFFECTS

Since indomethacin inhibits both COX-1 and COX-2, it inhibits the production of prostaglandins in the stomach and intestines, which maintain the mucous lining of the gastrointestinal tract. Indomethacin, therefore, like other non-selective COX inhibitors can cause peptic ulcers. These ulcers can result in serious bleeding and/or perforation requiring hospitalization of the patient (Vaziri et al; 1985).

To reduce the possibility of peptic ulcers, indomethacin should be prescribed at the lowest dosage needed to achieve a therapeutic effect, usually between 50-200mg/day. It should always be taken with food. Nearly all patients benefit from an ulcer protective drug (e.g. highly dosed antacids, ranitidine 150mg at bedtime, or omeprazole 20mg at bedtime). Other common gastrointestinal complaints, including dyspepsia, heartburn and mild diarrhea are less serious and rarely require discontinuation of indomethacin (Vaziriet al; 1985).

Many NSAIDs, but particularly indomethacin, cause lithium retention by reducing its excretion by the kidneys. Thus, indomethacin users have an elevated risk of lithium toxicity. For patients taking lithium (e.g. for treatment of depression or bipolar disorder), less toxic NSAIDs such as sulindac or aspirin, are preferred (Vaziriet al; 1985)

Indomethacin also reduces plasma renin activity and aldosterone levels, and increases sodium and potassium retention. It also enhances the effects of vasopressin. Together these may lead to:

ü  Edema (swelling due to fluid retention)

ü  Hyperkalemia (high potassium levels

ü  Hypernatremia (high sodium levels)

ü  Hypertention (Vaziriet al; 1985).

The drug may also cause elevation of serum creatinine and more serious renal damage such as acute renal failure, chronic nephritis and nephrotic syndrome. These conditions also often begin with edema and hyperkalemia (Vaziriet al; 1985). Additionally, indomethacin quite often causes headache (10 to 20%), sometimes with vertigo and dizziness, hearing loss, tinnitus, blurred vision (with or without retinal damage). There were reports of worsening Parkinson’s diseases, epilepsy, and psychiatric disorders but these are not substantiated (Giles et al; 2007). Cases of life- threatening shock (including angioedema, sweating, severe hypotension and tachycardia as well as acute bronchospasm), severe or lethal hepatitis and severe bone marrow damage have all been reported. Skin reactions and photosensitivity are also possible side effects. Due to its strong antipyretic activity indomethacin may obscure the clinical course of serious infections. Psychosis has also been reported with prolonged use (Ferreira et al; 1971).The frequency and severity of side effects and the availability of better-tolerated alternatives make indomethacin today a drug of second choice. Its use in acute gout attacks and in dysmenorrhea is well-established because in these indications the duration of treatment is limited to a few days only, therefore serious side effects are not likely to occur (Giles et al; 2007).

2.1.3 EXAMINATIONS DURING LONG TERM TREATMENT

Patients should undergo regular physical examination to detect edema and signs of central nervous side effects. Blood pressure checks will reveal development of hypertension (Ferreira et al; 1971)Periodic serum electrolyte (sodium, potassium, chloride) measurements, complete blood cell counts and assessment of liver enzymes as well as of creatinine (renal function) should be performed. This is particularly important if Indomethacin is given together with an ACE inhibitor or with potassium-sparing diuretics, because these combinations can lead to hyperkalemia and/or serious kidney failure. No examinations are necessary if only the topical preparations (spray or gel) are applied (Giles et al; 2007).

2.1.4   ANIMAL TOXICITY AND HUMAN OVERDOSE

Indomethacin has a high acute toxicity both for animals (in rats, 12mg/kg) and for humans. Exact human data does not exist, but some fatal human cases, particularly in children and adolescents have been seen (Vaziriet al; 1985). Generally, overdose in humans causes drowsiness, dizziness, severe headache, mental confusion, paresthesia, numbness of limbs, nausea and vomiting. Severe gastrointestinal bleedings is also possible. Cerebral edema, and cardiac arrest with fatal outcome have been seen in children (Vaziriet al; 1985). The treatment is symptomatic and largely the same as with diclofenac. However, the possibility of severe GI tract symptoms should be particularly noted. The risk of overdose after exaggerated local treatment with gel or spray is very limited (Vaziriet al; 1985).

 

2.1.4.1 USUAL DOSAGE FORMS

ü  tablets or capsules 25 and 50 mg

ü  suppositories 50 and 100 mg

ü  modified-release Capsules 75 mg

ü  syrup (25 mg/5ml) (Ferreira et al; 1971)

ü  injectable concentrate 50 mg for i.m. injection

ü  spray or gel

ü  patches containing 0.5% by weight

ü  1% topical liquid (Ferreira et al; 1971)

2.2       ULCER

Most GI bleeding comes from ulcers. An ulcer is an area of the lining of the stomach or duodenum that has been destroyed by digestive juices and stomach acid. The actual size of the ulcer can be very small (1-2 cm), but even small lesions can cause tremendous discomfort and pain. It is also a discontinuity or break in a bodily membrane that impedes the organ of which that membrane is a part from continuing its normal functions. Gastric ulcer is a major health hazard in terms of both morbidity and mortality (Chaturvediet al., 2007).Untreated gastric ulcer is capable of inducing upper gastrointestinal bleeding (Tortora and Grabowski, 2003).The etiology of gastro duodenal ulcers is influenced by various aggressive and defensive factors such as acid-pepcin secretion, parietal cell, mucosal barrier, mucus secretion, blood flow, cellular regeneration and endogenous protective agents(prostaglandins and epidermic growth factors) (Repetto and Llesuy, 2002). According to Malyshenko et al;(2005) and Kim (2008), some other factors, such as inadequate dietary habits, cigarette smoking, excessive ingestion of non-steroidal anti-inflammatory agents, stress, hereditary predisposition and infection by Helicobacter pylori, may be responsible for the development of peptic ulcer. Several pharmaceutical products have been employed for the treatment of gastroduodenal ulcer and peptic diseases, resulting in decreasing mortality and morbidity rates, but they are not completely effective and produce many adverse effects (Rates, 2001).Ulcer therapy has progressed from vagotomy to anticholinergic drugs, histamine H2 receptor antagonists, antacids and to proton pump inhibitors (Wallace and Granger, 1996). A widely used drug associated with rare idiosyncratic hepatotoxicity is the histamine H2 receptor antagonist ranitidine (RAN) (Bourd et al., 2005). It is available over the counter for oral administration or by prescription for parenteral administration for treatment of gastric ulcers, hyper secretory diseases, and gastroesophageal reflux disease. Idiosyncratic RAN hepatotoxicity occurs in few people taking the drug (Fisher and Le Couteur, 2001). Most liver reactions are mild and reversible; however, extensive liver damage have occurred in individuals undergoing RAN therapy (Cherqui et al., 1989 and Ribeiroet al., 2000).In recent years, there has also been growing interest in alternative therapies and the use of natural products, especially those derived from plants (Rates,2001 and Schmeda-Hirschmann and Yesilada, 2005).Plant extracts are some of the most attractive sources of new drugs and have been shown to produce promising results for the treatment of gastric ulcer (Alkofahi and Atta, 1999 and Schmeda-Hirschmann and Yesilada,2005).

2.2.1   SYMPTOMS OF ULCER

The most common symptom of an ulcer is a gnawing or burning pain in the abdomen located between the navel and the bottom of the breastbone. The pain often occurs between meals and sometimes awakens people from sleep. Pain may last minutes to hours, and is often relieved by eating, taking antacids or acid blockers. Less common symptoms of an ulcer include nausea, vomiting and loss of appetite and weight, and bleeding (Tortora and Grabowski, 2003).

2.2.2   CAUSES OF ULCER

In the past, ulcers were incorrectly thought to be caused by stress. As was noted above, doctors now know that there are two major causes of ulcers. Most duodenal and gastric ulcer patients are infected with the bacterium Helicobacter pylori (H. pylori). (Tortora and Grabowski, 2003).

 Others who develop ulcers are regular users of pain medications called non-steroidal anti-inflammatory drugs (NSAIDs), which include common products like aspirin, ibuprofen, naproxen sodium and ketoprofen. The excessive or inappropriate use of over-the-counter NSAIDs can cause ulceration (Tortora and Grabowski, 2003).

2.2.3   COMPLICATIONS OF ULCERS

ü  Bleeding Internal bleeding in the stomach or the duodenum.

ü  Perforation When ulcers are left untreated, digestive juices and stomach acid can literally eat a hole in the intestinal lining, a serious medical problem that requires hospitalization, and often surgery.

ü  Obstruction Swelling and scarring from an ulcer may close the outlet of the stomach, preventing food to pass and causing vomiting and weight loss. (Ribeiro et al., 2000).

2.2.4   ULCER DIAGNOSIS

The two tests most commonly used to evaluate for ulcers are an X-ray known as an Upper GI Series, or UGI, and a procedure called an Endoscopy, or EGD. (Ribeiro et al., 2000).

ü  Endoscopy This test involves insertion of a small, lighted flexible tube through the mouth into the esophagus, stomach, and small intestine (duodenum) to examine for abnormalities and remove small tissue samples (biopsy). The test is usually performed using medicines to temporarily sedate you. (Ribeiro et al., 2000).

ü  Upper GI Series Alternately, there is an X-ray test where you are given a chalky material (barium) to drink while X-rays are taken to outline the anatomy of the upper digestive tract. (Ribeiro et al., 2000).

2.2.5   TREATMENT OF ULCER

Diet In contrast to past beliefs, diet has little to do with ulcer healing. Doctors now recommend that patients with ulcers only avoid foods that worsen their symptoms.

ü  Smoking Patients who smoke cigarettes should stop. Smoking has been shown to inhibit ulcer healing and is linked to ulcer recurrence.

ü  Medical Therapy Numerous medications which inhibit acid production can rapidly heal ulcers. Antibiotic therapy for H. pylori can accelerate healing and prevent recurrence. In general, ulcer patients should not take NSAIDs unless instructed to do so by their physician.

ü  Surgical Therapy When an ulcer fails to heal, or if complications of bleeding, perforation or obstruction develop, surgery may be necessary. (Tortora and Grabowski, 2003).

 

2.3       GASTRIC ULCER

Gastric ulcers are erosions of the horses’ stomach mucosa (inner lining) that occur as a result of excessive exposure to stomach acid. Equine Gastric Ulcer Syndrome (EGUS) varies greatly in its severity from mildly inflamed but still intact mucosa to multiple large erosions that can cause bleeding into the stomach (Tortora and Grabowski, 2003).

The most severe form is ulcers so severe that the stomach wall is perforated. Gastric ulcers can affect any age of horse, right from a foal, and can occur in any breed. It can also be seen as a break in the tissue lining the stomach. The term 'peptic ulcer' refers to those that occur in either the stomach or the first part of the small intestine that leads out of the stomach, called the duodenum. It was once commonly thought that stress, smoking and diet were the principal causes of stomach ulcers (Hirschmann and Yesilada, 2005). However, the Helicobacter pylori (H. pylori) bacterium is now known to be responsible for most duodenal ulcers and 60 per cent of stomach ulcers. The H. pylori bacterium also prompts many symptoms of dyspepsia, or indigestion. Treatment for stomach ulcers includes the use of antibiotics to kill the infection, and acid suppressing drugs.(Rates,2001 and Schmeda-Hirschmann and Yesilada, 2005).

2.3.1   THE STOMACH

The stomach is an organ of the digestive system, located in the abdomen just below the ribs and on the left. Swallowed food is squeezed down the esophagus and pushed through a sphincter (small muscle ring) into the stomach, where it is mixed with powerful gastric juices containing enzymes and hydrochloric acid Hirschmann and Yesilada, 2005). The stomach is a muscular bag, so it can churn the food and break it down mechanically as well as chemically. Once the food is the consistency of smooth paste, it is squeezed through a second sphincter into the first part of the small intestine (duodenum). The lining of the stomach – the mucosa or gastric epithelium – is layered with multiple folds. Ulcers occur in this lining. (Fisher and Le Couteur, 2001).

2.3.2   SYMPTOMS OF STOMACH ULCERS

Some stomach ulcers don’t produce any symptoms. If present, they can include:

ü  Abdominal pain just below the ribcage

ü   Indigestion

ü  Nausea

ü   Loss of appetite

ü  Vomiting

ü  Weight loss

ü  Bright or altered blood present in vomit or bowel motions

ü  Symptoms of anaemia, such as light-headedness

ü   Shock due to blood loss – a medical emergency          (Fisher and Le Couteur, 2001).

2.3.3   CAUSES OF STOMACH ULCERS

A stomach ulcer can be caused by a variety of factors, including:

ü  Helicobacter pylori – bacteria is thought to be responsible for around 60 per cent of stomach ulcers and at least 90 per cent of duodenal ulcers.

ü  Certain medications – which include aspirin or clopidogrel, taken regularly to help prevent heart attack or stroke, and drugs for arthritis. Anti-inflammatory medications (NSAIDS) are thought to cause around two fifths of stomach ulcers.

ü  Cancer – stomach cancer can present as an ulcer, particularly in older people. (Fisher and Le Couteur, 2001).

2.3.3.1                 Helicobacter pylori

The Helicobacter pylori bacterium (H. pylori) is the main cause of peptic ulcers. The discovery of this micro-organism in 1983 revolutionized many aspects of gastroenterology, including the treatment of stomach ulcers. It is thought that about one in three people over the age of 40 years is infected with this strain of bacteria in Australia.(Hart et al; 1983)  The germs live in the lining of the stomach and the chemicals they produce cause irritation and inflammation. H. pylori directly causes one third of stomach ulcers and is a contributing factor in around three fifths of cases. Other disorders caused by this infection include inflammation of the stomach (gastritis) and dyspepsia (indigestion) (Hart et al; 1983). Researchers believe the germ could also play a contributing role in the development of stomach cancers. The infection is more common among poor people. The mode of transmission is so far unknown, but is thought to include sharing food or utensils, coming into contact with infected vomit, and sharing of water (such as well water) in undeveloped populations.(Hart et al; 1983).

2.3.4   ULCER BLEEDING

This is a serious complication of ulcer disease and is particularly deadly in the elderly or those with multiple medical problems. Bleeding from stomach ulcers is more common in people treated with blood thinning agents, such as warfarin, aspirin or clopidogrel (Plavix) and those people should also consider using regular anti-ulcer medication to prevent this complication. (Hart et al; 1983).

2.3.5   PERFORATED ULCER

A severe, untreated ulcer can sometimes burn through the wall of the stomach, allowing digestive juices and food to leak into the abdominal cavity. This medical emergency is known as a perforated ulcer. Treatment generally requires immediate surgery. (Hart et al; 1983).

2.3.6   DIAGNOSIS OF STOMACH ULCER

Diagnosing a stomach ulcer is done using a range of methods, including:

ü  Endoscopy – a thin flexible tube is threaded down the oesophagus into the stomach under light anaesthesia. The endoscope is fitted with a small camera so the physician can see if there is an ulcer.

ü  Barium meal – a chalky liquid is drunk and an x-ray is performed, showing the stomach lining. These tests are less common nowadays, but may be useful where endoscopy is unavailable.

ü  Biopsy – a small tissue sample is taken during an endoscopy and tested in a laboratory. This biopsy should always be done if a gastric ulcer is found.

ü  C14 breathe test – to check for the presence of H. pylori. The bacteria convert urea into carbon dioxide. The test involves swallowing an amount of radioactive carbon (C14) and testing the air exhaled from the lungs. A non-radioactive test can be used for children and pregnant women. (Ribeiroet al., 2000).

2.3.7   TREATMENT FOR A STOMACH ULCER

Special diets are now known to have very little impact on the prevention or treatment of stomach ulcers. Treatment options can include:

ü  Medications – including antibiotics, to destroy the H. pylori colony, and drugs to help speed the healing process. Different drugs need to be used in combination; some of the side effects can include diarrhea and rashes. Resistance to some of these antibiotics is becoming more common.

ü  Subsequent breath tests – used to make sure the H. pylori infection has been treated successfully.

ü  Changes to existing medications – the doses of arthritis medications, aspirin or other anti-inflammatory drugs can be altered slightly to reduce their contributing effects on the stomach ulcer.

ü  Reducing acid – tablets are available to reduce the acid content in the gastric juices.

ü  Lifestyle modifications – this would include quitting cigarettes, since smoking reduces the natural defenses in the stomach and impairs the healing process. (Tortora and Grabowski, 2003).

2.4       LIPID PROFILE

The lipid profile is a group of tests that are often ordered together to determine risk of coronary heart disease. The tests that make up a lipid profile are tests that have been shown to be good indicators of whether someone is likely to have a heart attack or stroke caused by blockage of blood vessels (hardening of the arteries). (Song et al; 2006).

A lipid profile is a measurement of various lipids that are found in the blood. This kind of blood test is often used to assess risk of heart disease.

There are two common concerns people have about lipids in their diet: One is their high caloric value, which may lead to undesired weight gain. The other is their association with high total cholesterol levels, which are a risk factor for cardiovascular disease. Limiting the intake of fat and oil in the diet, especially saturated fats, may help keep cholesterol levels low and thus lower one’s risk of heart disease. One reason the USDA recommends that 10% or fewer of one’s calories come from saturated fats is because the amount of saturated fat in one’s diet correlates strongly with cholesterol levels. Saturated fats are generally solid at room temperature. Fat from animals (e.g. butter and lard) is almost always saturated, but some oils from plants are saturated, too (e.g. palm oil or coconut oil.) (Song et al; 2006).

A lipid profile contains information about several different kinds of lipid that normally circulate in the blood. Values are numerical, but in order to simplify explanation, ranges of numerical values are often placed into categories such as ‘low risk,’ or ‘high risk.’ For example, a total cholesterol level over 240 mg/dl is said to be ‘high risk’, but that doesn’t mean a reading of 238 is fine. With total cholesterol and LDL cholesterol the higher the number, the higher the risk. Conversely, the lower the LDL cholesterol, the lower the risk. However, a low number is not a guarantee against heart disease (Hart et al; 1983). The population with low cholesterol is at lower risk of heart disease, but heart disease is not absent in this population. All of these lipid levels need to be evaluated in the context of other risk factors. If you have several other risk factors, a cholesterol level of 200 mg/dl might be considered a problem, while if you have no other risk factors, it might not be. Some of the other risk factors for cardiovascular disease are: smoking, high blood pressure, diabetes, age of over 45 years for males, age of over 55 years for females, and a family history of early heart disease. (Song et al; 2006).

Lipids generally included in a blood lipid profile are described below. Units for these are mg/dl, or milligrams per deciliter. A deciliter is 1/10^thof a liter. The lipid profile includes total cholesterol, HDL-cholesterol (often called good cholesterol), LDL cholesterol (often called bad cholesterol), and triglycerides. Sometimes the report will include additional calculated values such as the Cholesterol/HDL ratio or a risk score based on lipid profile results, age, sex, and other risk factors. (Stocker et al; 2004).

2.4.1   CHOLESTEROL

Cholesterol is a white, waxy, fatty substance. It is made in the liver and released into the bloodstream. You can also get cholesterol from the food you eat. It is important to get your cholesterol checked and learn how to manage your cholesterol. (Stocker et al; 2004)

Cholesterol is a necessary molecule in human metabolism. It is a component of cell membranes, and is a building block of bile, estrogen and testosterone. The cholesterol necessary for normal metabolism is manufactured by the liver. Generally, a level less than 200 mg/dl is considered desirable. Between 200 mg/dl and 240 mg/dl is considered borderline high, and over 240 mg/dl is considered high. Cholesterol is present in the blood in three forms. The three defined below are all combinations of protein, cholesterol, and triglyceride. Cholesterol is a lipid and is insoluble in water. It is transported through the blood encased in a soluble protein. (Stocker et al; 2004)

Cholesterol is a major cause of coronary heart disease (CHD), and large clinical trials show that lipid-lowering therapy substantially reduces risk for CHD. The National Institutes of Health periodically issues clinical guidelines for cholesterol testing and management as part of the National Cholesterol Education Program (NCEP). The most recent update, the third report of NCEP's Adult Treatment Panel (ATPIII), recommended that a fasting lipoprotein profile should be obtained at least every five years in all adults aged 20 years and older. (Esterbauer et al; 1996). This lipid profile consists of total, HDL, and LDL cholesterol and triglycerides. NCEP recommends that for routine patient evaluation and follow-up, LDL cholesterol should be estimated from measurement of total and HDL cholesterol and triglycerides using the Friedewald formula. While the major focus of ATPIII is on lowering LDL cholesterol, HDL cholesterol and triglycerides are identified as significant risk factors. Low HDL cholesterol values increase CHD risk where- as high HDL values protect against CHD. Elevated triglycerides are also a strong independent risk factor for CHD most often observed in individuals with metabolic syndrome. The ATP III established therapeutic goals for LDL and HDL cholesterol and triglycerides depending upon individual CHD risk factors. Follow-up measurement of these lipid parameters is necessary to ensure that individuals achieve treatment goals. (Stocker et al; 2004)

2.4.1.1            TYPES OF CHOLESTEROL

2.4.1.1.1         HDL High-density lipoprotein

HDL Cholesterol, or High density lipoprotein: This is sometimes called “good cholesterol.” The higher the number, the better. A value below 40 mg/dl is considered a risk factor. A value above 60 mg/dl is considered protective against heart disease. HDL cholesterol is cholesterol that is packaged for delivery to the liver, where the cholesterol is removed from the body. HDL protects you against heart disease by carrying the extra cholesterol out of your arteries, which lowers your chance of having a heart attack or stroke. The more HDL you have, the better. Exercise can also increase your good cholesterol. (Esterbauer et al; 1996).

2.4.1.1.2         LDL Low-density lipoprotein

LDL Cholesterol, or Low density lipoprotein: This is sometimes referred to as the “bad cholesterol.” This form contains the highest amount of cholesterol. A value between 130 – 159 mg/dl is borderline high, and over 160 mg/dl is considered ‘high.” LDL leads to a build-up of bad fat in the artery walls, which can lead to chest pain, heart disease, heart attack, stroke, and kidney and circulation problems. Triglycerides are often present in people who have diabetes or alcoholism. They increase the risk of heart disease. (Esterbauer et al; 1996).

2.4.1.1.3         Triglycerides:

This is the most common type of lipid formed in animals. Fat tissue is primarily for the storage of this form of lipid. Triglyceride levels vary quite a bit over short time periods. A meal high in sugar, fat, or alcohol can raise the triglyceride level drastically, so the most repeatable measures of this lipid are taken after 12 hours of fasting. Even though sugar and alcohol are not lipids, your body will convert any form of excess calories into triglycerides for long-term storage. A value below 150 mg/dl indicates no increased risk, 150 -200 indicates a slight risk, and over 200 mg/dl is a high risk. (Esterbauer et al; 1996).       

 

2.4.1.1.4         VLDL cholesterol

Very-low density lipoprotein; this form contains the highest amount of triglyceride. Like LDL, this is considered “bad cholesterol.” A value less than 32 mg/dl is desirable. VLDL is usually not measured directly, but is estimated from the triglyceride count by dividing the triglyceride count by 5. This mathematical way to estimate VLDL is not valid when the triglyceride is above 400 mg/dl. (Esterbauer et al; 1996).

2.4.1.2            CAUSES OF HIGH CHOLESTEROL

Foods high in saturated and trans fat increase cholesterol levels. Saturated and trans fats are found mainly in:

ü  fatty meats •  full cream dairy products (e.g. milk, cream, cheese  and butter)

ü  deep-fried take-away foods

ü  baked products (e.g. biscuits and pastries)

You should limit the amount of foods you eat that contain saturated and trans fats (Stocker et al; 2004).

2.4.1.3            TIPS TO IMPROVE CHOLESTEROL

ü  Stop smoking

ü  Limit animal fats (e.g. butter, cream, cheese,  fried foods)

ü  Eat more fibre (e.g. fruit, vegetables, cereals,  baked beans)

ü  Eat more fish

ü  Drink less alcohol (grog)

ü  Maintain a healthy weight

ü  Increase physical activity – aim for 30 minutes or more of moderate intensity physical activity every day of the week

ü   Take your medicine every day as directed by your doctor – medication can help reduce your cholesterol if it’s too high. (Stocker et al; 2004).

2.4.2   USES OF LIPID PROFILE

The lipid profile is used to guide providers in deciding how a person at risk should be treated. The results of the lipid profile are considered along with other known risk factors of heart disease to develop a plan of treatment and follow-up. (Stocker et al; 2004).

2.4.3   RISK FACTORS (IN ADDITION TO HIGH LDL) FOR CORONARY HEART DISEASE

Risk factors include: Cigarette smoking, Age (you are a male 45 years or older or a female 55 years or older), Low HDL cholesterol (less than 40 mg/dL (1.04 mmol/L)), Hypertension (Blood Pressure of 140/90 or higher or taking high blood pressure medications), Family history of premature heart disease (heart disease in a first degree male relative under age 55 or a first degree female relative under age 65) (Esterbauer et al; 1996).

Note: High HDL (60 mg/dL or above) is considered a "negative risk factor" and its presence allows the removal of one risk factor from the total. (Esterbauer et al; 1996).

2.4.4   TREATMENT OF LIPID PROFILE

Treatment is based on your overall risk of coronary heart disease. A target LDL is identified. If your LDL is above the target value, you will be treated. Your target LDL value is: LDL less than 100 mg/dL (2.59 mmol/L) if you have heart disease or diabetes. LDL less than 130 mg/dL (3.37 mmol/L) if you have 2 or more risk factors. LDL less than 160 mg/dL (4.14 mmol/L) if you have 0 or 1 risk factor. The first step in treating high LDL is targeted at changes in lifestyle specifically adopting diets low in saturated fat and participating in moderate exercise (Esterbauer et al; 1996). You may be referred to a dietician for advice in making dietary changes. If low-fat diets and exercise are not adequate to lower LDL-cholesterol to the target value, drug therapy would be the next step. There are several classes of drugs that are effective in lowering LDL. You will prescribed one of these. Your LDL will be checked at regular intervals to assure that the drug is working. If the drug does not result in reaching your target LDL-cholesterol, your doctor may increase the amount of drug or possibly add a second drug. (Esterbauer et al; 1996).

                                              

2.5       Pilliostigma thonningii

Piliostigma thonningii is a leguminous plant belonging to the family caessalpiniaceae. The tree is perennial in nature. The plant grows to a height of 8m with branches. It has a large two –lobed simple leaves without thorns or spines (Burkill, 1995). Its leathery green leaves measure up to 15 x 17 cm, bi-lobed one eighth to one third the way down with a small bristle in the notch, glossy above and heavily veined and somewhat rusty-hairy below without thorns or spines. It is a multipurpose plant of vast economic importance. Leaves of P. thonningii are edible and chewed to relieve thirst. Its fruits and seeds are also edible fodder. The pods are nutritious and are relished by cattle, antelopes and elephants in Africa (Burkill, 1995).  The preparation from the bark of the tree is used in treating cough, usually taken as an infusion in Uganda; it is used to stop diarrhea, dysentery and intestinal upset. Maceration prepared from the bark of the tree is also used in the treatment of malaria, leprosy, sore throat, toothache and earache.  The leaf decoction is a laxative given to children, newborns, and also used as an embrocating tonic to massage the abdomen of newly de- livered mothers to massage their stomach after child birth .It also serves as a lotion for lumbago (Diallo et al., 2003). The leaves are soaked in hot water and applied topically as wound dressing or to excisions in the South-West Africa region (Okoli and Iroegbu, 2004). 

In Nigeria, the plant is locally called Kalgo in Hausa, Abafe in Yoruba, Nyihar in Tiv, Baffin in Nupe, and Obepa in Yala land Cross River Nigeria (Dasofunjo et al., 2013),and Tawin in Gwarri and commonly referred to as Monkey bread or Camel’s tool. P.thonningii grows in open woodland and savannah regions that are moist and wooded grassland in low and medium altitude. It is found growing abundantly as a wild weed in many parts of Nigeria such as Abeokuta, Bauchi, Ilorin, Plateau and Zaria (Jimoh and Oladiji, 2005).  Different parts of p.thonningii have been discovered to have medicinal value. Its roots and twigs have been used traditionally to treat fever, snake bites, wounds, respiratory ailments and skin infections (Jimoh and Oladiji, 2005)

2.5.1      Botanical classification of Piliostigma thonningii

ü  Kingdom – Plantae

ü  Division - Tracheophyta

ü  Class - Magnoliopsida

ü  Subclass - Rosidae

ü  Order - Fabales

ü  Family - Fabaceae

ü  Sub family - Caesalpinioideae

ü  Genus - Piliostigma

ü  Specie –Thonningii.(Burkill H.M 1995)

2.5.2. Biology of pilliostigma thonningii

A dioecious tree with male and female flowers on different trees. The off- white to pink fragrant flowers appear from November to March in many flowered hanging sprays. The female flowers are superseded from May to September by the large dark red-brown flattened oblong pods. The pods sometimes break up into one-seeded pieces after falling from the tree. The tree becomes nearly leafless in the dry season. The generic epithet Piliostigma, means cap-like stigma. The specific epithet commemorates Peter Thonning, the Danish plant collector who collected the type in that portion of Danish Guinea that is now part of Ghana. Piliostigma was distinguished from Bauhinia by its unisexual flowers and indehiscent pods. (Burkill H.M 1995)

2.5.3 Biophysical limits of pilliostigma thonningii

ü  Altitude:  0-1 850 m

ü  Mean annual temperature:  20 deg. C

ü  Mean annual rainfall:  700-1 400 mm

Soil type: Heavy clayey soils or medium loamy soils are preferred by this plant. It tolerates acid soils and prefers deep fluvisols or ferrasols soils. (Burkill H.M 1995)

2.5.4 Products of pilliostigma thonningii

Food:  The leaves are edible and chewed to relieve thirst. The fruit and seeds are also edible. (Akinpeluet al; 2000).

Fodder:  The pods are nutritious and relished by cattle and antelopes. This is a preferred browse species of the African elephant (Loxodontaafricana), the fruits are also taken in considerable quantities. However the feeding habits of the African elephant are destructive and do affect local plant populations. (Akinpeluet al; 2000).

Fuel:  Provides fuel wood in considerable amounts, the advantage being its shrubby habit and multi-stemmed nature.

Fibre:  The inner bark is used to make rope. (Burkill H.M 1995)

Timber:  The sapwood is straight grained and light brown, heartwood is pinkish to dark brown and contributes less bulk. Household utensils and farm implements are made from this wood. (Akinpelu et al; 2000).

Gum or resin:  A gum tapped from the bark is used in caulking.

Tannin or dyestuff:  Three dyes can be obtained from the plant, the bark produces a red-brown dye, and the pods produce a black and blue dye. The roasted seeds and root can also be used in dye production. The bark has a tannin content of 18%, though unquantified the roots have a considerably high tannin content. (Akinpelu et al; 2000).

Medicine:P. thonningii is used medicinally in many African countries to treat wounds, ulcers, gastric/heart pain, gingivitis and as an antipyretic. In Tanzania and Zimbabwe, a cough remedy is prepared from the root bark. Polyphenolic fractions of the root bark, exhibit potent antitussive activity. In experiments with mice, this fraction exhibited a significant anti-inflammatory/analgesic activity against phenyl Quinone-induced writhings. The new compounds Piliostigmin, a 2-phenoxychromone, and C-methylflavonols were isolated from leaves of P. thonningii. Extracts were screened for activity against Herpes simplex virus type 1 (HSV-1) and African swine fever virus (ASFV). The extracts had virucidal activity against HSV-1. Further studies showed that the tested extract inhibited HSV-1 infection, and had activity. In another setting P. thonningii showed blood plasma coagulating activity. (Akinpelu et al; 2000).

2.5.5     Services of pilliostigma thonningii

Erosion control:  This deep rooting species can be employed in soil protection initiatives. (Akinpeluet al; 2000).

Shade or shelter:  Provides good shade in homesteads when in full foliage.

Reclamation:  Fixes nitrogen. (Akinpelu et al; 2000).

Soil improver:  Produces considerable amounts of litter. Use of the leaf litter as mulch enhances soil fertility however P. thonningii leaves decompose slowly. (Akinpelu et al; 2000).

Ornamental:  Its showy white flowers can be aesthetically enhancing.

Boundary or barrier or support:  P. thonningii live stakes are used in supporting vines and other weaker plants in farms. Poles or posts are obtained from the plant. (Akinpelu et al; 2000).

Intercropping:  A good tree that can be grown with Annona, Grewia and Combretum spp. Competes very little with maize if left in fields and pollarded to reduce shade. (Adewuyi et al; 2009).

Other services:  The pods are used as a soap substitute. The ashes can also be used in soap making. (Adewuyi et al; 2009).

2.5.6 Management of pilliostigma thonningii

Young individuals are susceptible to annual fires. The fire resistance strategy of P. thonningii is by quick regrowth of aboveground structures.

Management practices recommended for this species include lopping, pollarding, trimming and coppicing. (Adewuyi et al; 2009).

2.5.7 Germplasm management of pilliostigma thonningii

Seed collection should be done immediately the pods turn brown to prevent insect attack. Seed drying is recommended. The different seed pretreatments include washing, soaking for 24-48 hours, hot water treatment and different degrees of removal of the seed coat of Piliostigma sp. which gives more than 80% germination. There are 7 300 seeds per kg. The seeds are difficult to extract because of the tough/ woody pod covering. (Adewuyi et al; 2009).

2.5.8 Pests and diseases of pilliostigm athonningii

The feeding habits of the African elephant, bark stripping and voracious plant biomass intake, are destructive and affect local plant population regeneration. The bruchidCaryedonserratus develops on P. thonningii seeds. (Burkill H.M 1995).

CHAPTER THREE

                                    MATERIALS AND METHODS

3.1       PLANT MATERIAL

Fresh leaves of P. thonningii were collected from Igoli Road, Cross River University of Technology, Cross River State, Nigeria. The leaves were taken to Federal College of Forestry (FCOFJ) Jos in Plateau State, Department of Herbarium for identification and authentication with the Voucher number #25 has been deposited for future reference at the department’s (FCOF J) Herbarium.

3.2       PREPARATION OF PLANT MATERIAL

Fresh leaves of P.thonnigii were air-dried at room temperature for twenty (20) days, macerated and pulverized into powdery form using the blender and then sieved.

3.3       AQUEOUS EXTRACTION

Three hundred (300)g of powdered P.thonningii, leaves were dissolved 1200mls of distilled water for 24 hours in a refrigerator. Thereafter, it was filtered with muslin cloth and filtered using whatman filter No1.The filtrate was evaporated to dryness and the percentage yield was calculated reconstituted into dosage and administered into rats.

3.4       EXPERIMENTAL ANIMAL

Thirty-Six (36) Wistar albino rats (120-200)g were obtained from the Animal Holding Unit of the Department of Medical Biochemistry, Cross River University of Technology Cross River State Nigeria. The animals were allowed to undergo acclimatization period for seven days before the commencement of the research.

Each rats were housed in a wooden cage. The animal room was ventilated and kept at room temperature and relative humidity 29 _±2⁰ C and 70 relative humidity with 12 hours natural light dark cycle and were allowed free access to standard feed and water ad libitum, Good hygiene was maintained by constant cleaning and removal of faeces and spilled feeds from cages daily.

3.5       ANTI-ULCER ACTIVITY

The experiment was carried out on 36 male rats that were divided into six (6) groups of 6 rats each. Group one (1) served as control and was given 0.5ml of normal saline (vehicle). Group three (3), five (5) and six (6) were given 100, 100 and 200mg/kg body weight of the extract while group two (2) was treated with 100mg/kg body weight. The vehicle and extract were administered orally while the drug was administered intra-muscularly for 12days. After 12days of administration all rats were fasted for 24 hours, gastric ulceration was then induced by the administration of 40mg/kg indomethacin orally to group 2, 4, 5 and 6. 12 hours after indomethacin administration all rats were sacrificed after been anaesthetized with chloroform, blood was collected by cardiac puncture.

3.6     PREPARATION OF SERUM.      

The animals were anaesthetized in a jar containing cotton wool soaked in ether and chloroform in ratio1:1. When the animal became unconscious, they were brought out quickly of the jar, the abdominal region was opened along the linear Alba and diaphragm cut with scalpel blade to expose the organs and blood was collected into a sterile sample container by cardiac puncture. Blood was collected into a clean, dry centrifuge tube and allowed to clot for 30 min before centrifuging at 300rpm x 10min using Uniscope Laboratory Centrifuge. The serum was thereafter aspirated into clean, dry, sample bottles using Pasteur pipette and was kept or stored in sample bottles and used within 12hours of preparation. Later it was transferred into specimen bottles before being used for analysis of the lipid profile.

3.6.1 Determination of Serum Cholesterol

Serum cholesterol was determined using Randox cholesterol Kit based on the cholesterol oxidase method as described by Richmond (1973) and modified by Allain et al., 1974.

Principle

The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidise.

Cholesterol ester+ H₂O    ^cholesterol _esteraseCholoesterol+ fatty acids

Cholesterol + O₂^{cholesterol oxidase}Cholesterol-3-one+ H₂O₂

2H₂O₂ + phenol + 4-aminoantipyrine ^Peroxidasequinoneimine + 4H₂O

Procedure

1ml of the working reagent was added to 0.01 ml of the serum and 0.001ml distilled water was added to 1ml of the working reagent in another tube to serve as blank. The contents of the tubes were mixed thoroughly and then incubated for 5 minutes at 37 ^oc. The absorbances of the serum sample were read against the reagent blank at 500 nm with a spectrophotometer.

Calculation

Conc. of cholesterol in sample = Asample× conc. of standard

Astandard

3.6.2 Determination of Serum Triglycerides (TG)

Serum triglycerides were determined using Randox TAG kit according to the colorimetric method of Tietz, 1990. This method involves the measurement of triglycerides after enzymatic hydrolysis with lipases.

Principle

Triglycerides + H₂O ^Lipases Glycerol + Fatty acids

Glycerol + ATP ^GK Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O₂^GPODihydroxyacetone phosphate + H₂O₂

2H₂O₂ + 4-aminophenazone + 4-chlorophenol ^PODQuinoneimine + HCl + 4H₂O

The indicator is quinoneimine formed from H₂O₂, 4-aminophenazone and 4-chlorophenol under the catalytic influence of peroxidase.

Procedure

The RANDOX kit procedure was adopted. The reagent used contained 40 mM pipes buffer, pH 7.6, 5.5 mM 4-chlorophenol, 17.5 mM magnesium ions, 0.5 mM 4-aminophenazone, 1 mM ATP, 150U lipases, 0.4 U glycerol kinase, 1.5 U glycerol-3-phosphate oxidase and 0.5 U peroxidase. The sample cuvette contained 0.01 ml of serum, to which was added 1ml of reagent. The blank had only 1 ml of the reagent. Thorough mixing was done and the reaction mixture was incubated for 10 minutes at room temperature. The absorbances of the sample and standard were measured against the reagent blank at 500 nm with Spectrophotometer

Calculation

          Triglyceride concentration = Absorbance _sample× conc. of standard

Absorbance _standard

3.6.3 Determination of High Density Lipoprotein Cholesterol (HDL-Cholesterol)

Serum HDL-cholesterol were determined using Randox HDL- Cholesterol kit in two stage process

PRINCIPLE:

Low density lipoproteins (LDL and VLDL) and chylomicron fractions are precipitated quantitatively by the addition of phosphotungistic acid in the presence of magnesium ions. After centrifugation, the cholesterol in the HDL (high density lipoproteins) fractions, which remains in the supernatant, is determined.

PROCEDURE:

Precipitation– step 1.

200μl of the sample was mixed with 500μl of the precipitants (phosphotungistic acid and the presence of magnesium ions) and then allowed to stand for 10minutes at room temperature. It was centrifuged for 10minutes at 400rpm. The clear supernatant was separated within 2 hours and the cholesterol content was determined by the CHOD – PAP method as describe above.

3.6.4 Serum Low Density lipoprotein Cholesterol (LDL-Cholesterol)

Serum LDL was determined using standard formula as provided in Randox HDL cholesterol Kit Determination.

Serum LDL-Cholesterol = Total Cholesterol – (HDL Cholesterol+ TAG/5)

3.7 TISSUE PROCESSING FOR HISTOLOGY.

The tissues were allowed to fix in 10% formal saline for at least 48hours. The tissues were grossed and cut into smaller pieces of 3mm thick in pre-labelled tissue cassette. They were then processed using Automatic tissue processor (LEICA TP1020) where they passed through various reagents including Alcohol (of various concentrations starting from 70%, 80%, 90%, 95%, 100%,100%) for dehydration, two changes of Xylene and three changes of molten Paraffin wax set at 65 degree centigrade. The processing time was 12 hours. The tissues were embedded in wax, forming   paraffin blocks, ready for microtomy. After which the tissues were sectioned at 4microns using Rotary microtome (LEICA RT2115), and the sections were floated on hot water bath to attach the sections to prelabelled slides. These sections were dried on hot plate and ready for staining using Haematoxylin and Eosin staining technique.

Haematoxylin and Eosin Staining Technique

Procedure;

Ø  Take section to water.

Ø  Stain in HARRIS HAEMATOXYLIN for 5 minutes.

Ø  Rinse in water.

Ø  Differentiate in 1% acid alcohol briefly.

Ø  Rinse and blue under tap water for 10 minutes.

Ø  Counter stain in 1% AQUEOUS EOSIN for 3 minutes.

Ø  Rinse in water.

Ø  Dehydrate through ascending grades of alcohol (70%, 80%, 90% and Absolute).

Ø  Clear in Xylene.

Ø  Mount with DPX mountant.

            Statistical Analysis

Statistically analysed data used was presented as mean ± SD of five (5) determinations. Statement analysis was carved out using one way analysis of variance (ANOVA). Differences were statistically significant at P<0.05 (Mahajan, 1997).

       CHAPTER FOUR

   RESULTS

The result below depict the alteration of extract of p. thoningii leaf on lipid profile of male Wister albino rat following indomethacin mediated gastric mucosa onslaught the treated dose showed a significant (P<0.05) increases in serum total cholesterol when compared with the control (fig 6). Likewise the standard drug (cimetidine), the extract and other treated groups showed a significant (P<0.05) increase in serum high density lipoprotein (HDL) when compared with the control while the group induced with ulcer without treatment showed a significant (P<0.05) decrease in serum HDL when compared with the control (fig 7). The extract also produced a significant (P<0.05) decrease of serum very low density lipoprotein (VLDL) while group treated with cimetidine and indomethacin produced a significant (P<0.05) increase when compared with the control (fig 8). Similar pattern was shown with serum low density lipoprotein (LDL) (fig 9). The treatment also produced a significant (p<0.05) increase throughout the experimental group for serum triglyceride when compared with the control (fig 10). 

Table 1: Effect of P.thonningii on serum lipid profile of male Wistar albino rats following indomethacin mediated mucosa onslaught

LIPID PROFILE	CONTROL	CIMETIDNE	EXTRACT	ULCER CONTROL	ULCER + 100Mg/kg bwt EXTRACT	ULCER + 200Mg /kgbwt EXTRACT
T.CHOL (mmol/l)	3.24±0.02	4.22±0.02*,a	4.29±0.17*,a	3.72±0.24	4.06±0.29*,a	4.18±0.40*,a
HDL (mmol/l)	1.20±0.04	1.44±0.10	1.49±0.08	0.36±0.15	1.30±0.15	1.28±0.20
LDL (mmol/l)	2.34±0.09	2.76±0.04*,a,b,c	1.03±0.02*,a,b,c	2.50±0.20*	1.74±0.23*	2.06±0.45*,a,b
VLDL (mmol/l)	0.8±0.05	1.28±0.02	0.41±0.01	1.98±0.24*,a,b,c	0.66±0.24*,a,b	0.38±0.37*,a,b,c
TG (mmol/l)	1.26±0.30	1.66±0.35	1.47±0.24	2.36±0.21*,a,b,c	1.76±0.14	1.92±0.19*,a,b,c

Values are expressed as mean ± SEM, n=5.
*= significant (p<0.05) from control
a=significant (p<0.05) from ulcer control

 

Fig 6: Effect of extract of Piliostigma thonningii leaf on serum total cholesterol following indomethacin mediated mucosa onslaught.

Values are expressed as mean ± SEM,n=5.
*=p<0.05 vs control
a=p<0.05 vs ulcer control

 

Fig 7: Effect of extract of P. thonningii leaf on serum HDL following indomethacin mediated mucosa onslaught.

Values are expressed as mean ± SEM, n=5
*= P<0.05 vs control

Fig 8: Effect of extract of Piliostigma thonningii leaf on serum VLDL following indomethacin mediated mucosa onslaught

Values are expressed as mean ± SEM, n=5.
*=p<0.05 vs control
a=p<0.05 vs Cimetidine
b=p<0.05 vs extract
c=p<0.05 vs ulcer + 100mg extract

 

 

Fig 9: Effect of extract of Piliostigma thonningii leaf on serum LDL following indomethacin mediated mucosa onslaught.

Values are expressed as mean ± SEM, n=5.
*=p<0.05 vs control
a=p<0.05 vs ulcer +100mg extract
b=p<0.05 vs ulcer control
c=p<0.05 vs ulcer + 200mg extract

Fig 10: Effect of extract of Piliostigma thonningii leaf on serum triglyceride following indomethacin mediated mucosa onslaught. Values are expressed as mean ± SEM, n=5.
*=p<0.05 vs control
a=p<0.05 vs Cimetidine
b=p<0.05 vs extract
c=p<0.05 vs ulcer + 100mg extract

            CHAPTER FIVE

     DISCUSSION

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are among the most commonly prescribed medications worldwide. Over 300 million patients use non-steroidal anti-inflammatory drugs (NSAIDs) in the world to treat pain, arthritis, fever and other diseases. Nearly 30% of the users suffer from gastric lesions and bleeding. Mechanisms for such actions of NSAIDs seem to be complex and multifactorial, including the inhibition of prostaglandin (PG) synthesis, induction of apoptosis and necrosis of gastric mucosal cells (Becker et al, 2004) neutrophil penetration, dysfunction of micro vessels, reduced secretion of bicarbonate and mucus, and increased gastric motility (Vane 1971). However, NSAIDs have adverse effects on the gastric mucosa, resulting in various clinical presentations, ranging from nonspecific dyspepsia to ulceration, upper gastro- intestinal bleeding, and death, summarized by the term “NSAID gastropathy” (Becker et al, 2004). NSAIDs-induced gastric damage is the major side effect of this kind of drug (Becker et al, 2004). The main action of NSAIDs is to inhibit prostaglandin synthesis. There is substantial evidence supporting the view that the ulcer genic effect of this medication correlates with its ability to suppress prostaglandin synthesis (Vane et al, 1971). Endogenous prostaglandins normally regulate mucosal blood flow, epithelial cell proliferation, epithelial restitution, mucosal immunocyte function, mucus and bicarbonate secretion, and basal acid secretion (Wallace 1997). Therefore, decreases in prostaglandins, protective factors for ulcer formation, lead to gastric mucosal injury.

In this present study, the sera obtained were used for the biochemical analyses of total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol, total cholesterol and triglycerides following indomethacin mediated gastric mucosa onslaught. The assessment of serum lipid profile reveals the clinical basis for understanding the metabolism of lipids and their role in predisposing humans to atherosclerosis, coronary heart diseases and other cardiovascular related disorders (Dasofunjo et al.,2013;Dasofunjo et al.,2014).

Lipids are generally characterized by insolubility in aqueous or polar solvents but highly soluble nonpolar or organic solvents. Biochemical reactions and transportations of molecules generally occur in aqueous medium. Hence, lipids are normally combined with specific proteins to form structures called lipoproteins which possess substantial degree of hydrophilicity. Low density lipoproteins, high- density lipoproteins, and chylomicrons which are basically composed of triglycerides are integral parts of the serum lipoproteins (Wallace et al, 1997). Except for the HDL cholesterol, high level of all lipids in the blood is arguably a high risk factor in the onset of cardiovascular disorders. High serum concentrations of triglycerides and LDLs have been reported to cause atherosclerosis and coronary heart diseases (Cantor et al, 1980). Cholesterol is the principal sterol in animal tissues and occurs mainly in the cell membrane due to its amphipathic nature. It is also found in the adrenal gland, liver, brain, and nervous system (Cantor et al, 1980). The molecule is synthesized basically from acetyl CoA in the liver from where it is distributed through the blood to extra hepatic tissues where it is utilized for the synthesis of bile acids and steroid hormones as well as regulation of membrane fluidity. However, high level of cholesterol in the blood has adverse effects on human health. It is reportedly a major cause of cardiovascular derangements such as atherosclerosis, myocardial infarction and coronary heart diseases.

The significant increase in HDL and corresponding decrease in LDL in the extract treated groups supports the antilipidemic or cholesterol lowering or hypocholesterolemic effect of P.thonningii leaf as reported by Dasofunjo et al.,2013.Therefore despite of the indomethacin mucosa ulcerations, the observed significant increase in HDL and decrease in LDL in the extract treated groups suggests that P.thonningii possess some bioactive compounds ,possibly flavonoids or alkaloids that aids such pharmacological activities .There is also the possibility that the extract possess the ability to facilitate the transport of cholesterol and triglycerides from the blood into tissues. This may have probably occurred through the induction or suppression of certain enzymes critical to the metabolism of these lipids. This observation is similar to the report of (Adebayo et al., 2006). Hence, might not be predisposed to atherosclerosis and other cardiovascular diseases. On the other hand, the observed significant increase in LDL and decrease in HDL in indomethacin and cimetidine treated group suggest that the drugs might alter the transportation of lipids in the blood which might predisposes the animals to The induced hypotriglyceridaemic effect may be due to decrease in fatty acid synthesis, enhanced catabolism of LDL, activation of Lipid catabolism and tissue lipase (Hooper et al, 2004) and or inhabitation of acetyl-CoA carboxylase and production of triglycerides precursors such as acetyl – CoA and glycerol phosphate (Bruneton 1999).

More so, the significant increase in serum cholesterol in all the treated male albino rats may be attributed to an increased concentration of acetyl-CoA is a key substrate in the biosynthesis of cholesterol (Bruneton 1999) or increase in absorption from the intestine by binding with bile acid within the intestine and increasing bile acid secretion due to the drug (Hooper et al, 2004).

The observed increased in triglyceride in all the treated groups suggest that it can induce hypertriglyceridemic effect  which may be  due to increase in fatty acid synthesis , enhanced catabolism of LDL, activation of Lipid catabolism and tissue lipase and or increased of acetyl-CoA caboxylase and production of triglycerides precursors such as acetyl-CoA and glycerol phosphate (Bruneton 1999).

5.1     CONCLUSION

The biochemical and physiological alterations in this study following indomethacin mediated mucosa onslaught suggest that the extract treated groups are shielded against cardiovascular related disorder  but it appears that cimetidine and indomethacin treated groups are predisposed to cardiovascular derangements such as atherosclerosis, myocardial infarction and coronary heart diseases vial a mechanism not yet fully understood.

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APPENDIX

Table: Effect of aqueous leaf extract of piliostigma thonningii on serum lipid profile following indomethacin induced gastric ulcer in wistar albino rats, using five (5) parameters.

GROUPS	T.CHOL (mmol/l)	HDL (mmol/l)		LDL (mmol/l	VLDL (mmol/l)	T.G (mmol/l)
A1	3.2	1.1		2.3	0.4	0.7
A2	3.3	1.3		2.2	0.6	2
A3	3.2	1.2		2.7	0.4	0.9
A4	3.2	1.1		2.3	0.4	0.7
A5	3.3	1.3		2.2	0.6	2
B1	4.2	1.2		2.7	0.3	0.8
B2	4.2	1.6		2.8	0.3	2.3
B3	4.3	1.6		2.7	0.2	2.1
B4	4.2	1.2		2.9	0.3	0.8
B5	4.2	1.6		2.7	0.3	2.3
C1	4.5	1.3		3	0.4	0.9
C2	4.43	1.63		3.03	0.43	1.73
C3	3.6	1.6		3.1	0.4	2.1
C4	4.5	1.3		3	0.4	0.9
C5	4.43	1.63		3.03	0.43	1.72
D1	4.3	1		1.5	0.4	2.1
D2	3.3	1.6		2.3	1.4	2.2
D3	3.4	1.6		2.4	1.3	3.2
D4	4.3	1		1.5	0.4	2.1
D5	3.3	1.6		2.3	1.4	2.2
E1	3.4	1.4		1.3	0.6	2.1
E2	4.3	1.5		2.3	0.7	1.5
E3	4.9	0.7		1.5	0.7	1.6
E4	3.4	1.4		1.3	0.6	2.1
E5	4.3	1.5		2.3	0.7	1.5
F1	5.1	0.8		1.6	1.1	1.6
F2	3.3	1.6		3.6	1	2.4
F3	4.1	1.6		2.4	1.2	1.6
F4	5.1	0.8		1.6	1.1	1.6
F5	3.3		1.6	3.6	1	2.4