Abstract
Herbs with anti-diabetic activity could initiate interactions if used concurrently with orthodox drugs. Moringa oleifera is one of such herbs usually taken as an adjunct to orthodox oral hypoglycaemic agents. This study investigated the possible pharmacological interaction between the ethanol extract of Moringa oleifera leaves (MOE) and metformin co-administered to hyperglycaemic Wistar rats and the effect of MOE on normoglycaemic rats. Hyperglycaemia was induced by administration of a single dose (150 mg/kg i.p) of alloxan. Rats having fasting blood glucose (FBG) ≥ 200 mg/dl after 72 h were considered hyperglycaemic. A dose-response study for MOE was carried out with 8 groups of hyperglycaemic rats which were administered 100, 200, 400, 800, 1000 and 2000 mg/kg MOE respectively. Blood glucose level was monitored hourly and a plot of percentage glycaemic reduction at 4h versus log dose was used to estimate the median effective dose (ED50), to be 750 mg/kg. Pharmacological interaction study was carried out for 28 days with 8 groups of hyperglycaemic rats. Group 1 served as control, groups 2, 3, and 4 received 375, 750 and 1500 mg/kg MOE corresponding to ½ ED50, ED50 and 2ED50 respectively. Groups 5, 6 and 7 received 375, 750 and 1500 mg/kg MOE but co-administered with metformin (150 mg/kg) while group 8 received metformin (150 mg/kg). For normoglycaemic rats, group 1 served as control while groups 2, 3 and 4 were administered 375, 750 and 1500 mg/kg MOE respectively. Parameters such as FBS, feed/water intake, body weight changes, lipid/protein profiles, electrolytes, creatinine and urea levels were measured in both hyperglycaemic and normoglycaemic rats. Relative organ weight (ROW) and histopathological examination of the pancreas, heart, brain, kidney, liver, lungs, spleen and stomach were also carried out. Results of the hyperglycaemic study, showed that 375mg/kg MOE significantly (p<0.05) reduced FBS on day 28, 750 and 1500 mg/kg MOE significantly (p<0.01) reduced FBS on days 21 and 28. The 375 and 750 mg/kg MOE co-administration with metformin produced significant (p<0.01), (p<0.001) reduction in FBS on days 14, 21 and 28 respectively while 1500 mg/kg MOE/metformin co-administration produced a significant (p<0.001) reduction in FBS on days 7, 14, 21 and 28 compared to hyperglycaemic control. A dose- dependent significant reduction in food and water intake was observed throughout the experiment with no significant change in body weight. Each of cholesterol, triglycerides and low density lipoprotein was dose-dependently reduced significantly (p<0.001) with a corresponding significant (p<0.001) increase in high density lipoprotein in all groups. Total protein and albumin were significantly (p<0.001) increased in all the groups except in the 750mg/kg MOE that showed significantly (p<0.05) increase. Serum aspartate transaminase, alanine transaminase, alkaline phosphatase and bilirubin levels reduced more significantly (p<0.001) in the extract/metformin co-administered groups. A significant (p<0.001) increase in serum Na+, K+ and HCO3+ levels with a corresponding decrease in Cl– ions was observed at all doses when compared to the hyperglycaemic control. The MOE/metformin co-administered groups showed the most significant (p<0.001) reduction in creatinine and urea levels. Among all the organs examined, only the liver and spleen showed a dose-dependent increase in ROWs while only the pancreas showed remarkable changes in the form of beta-cell necrosis on histopathological examination. However, regeneration of beta-cells was observed in a dose-dependent fashion in all groups. The normoglycaemic study did not show any significant difference for all the parameters examined. When compared to the metformin treated group, MOE/metformin co-administration showed significant changes in all parameters monitored. In conclusion, MOE showed additive interaction with metformin. This could be important in maximizing diabetes management
CHAPTER ONE
1.0 INRODUCTION
1.1 Statement of Research Problem
Increasing number of patients and consumers are using herbs and herbal products as complementary therapy in the treatment and management of chronic ailments such as tuberculosis, diabetes, hypertension, HIV/AIDS and cancer. Herbs are also employed in the treatment of endemic diseases especially malaria, as well as other social conditions like obesity (Obodozie et al., 2004). This upsurge in the use of phytomedicines is a global phenomenon, with more than 80% of people in Africa and Asia using herbal medicines and an increasing number in the Western world (WHO, 1999). Herbs are widely acceptable; accessible, affordable and are also considered to be safe since they are „natural‟. As a result, the use of herbs and herbal products alongside conventional therapy for better therapeutic outcome continues to grow rapidly. Herbs and herbal products contain a multiple of both active and inactive principles. This increases the possibility of interaction when co-used with orthodox drugs. Herbal-drug interaction can occur either at pharmacodynamic (PD) or pharmacokinetic (PK) level (Mohammed and Mohammed, 2009). Some diabetics use an antidiabetic herb concurrently with a conventional anti-diabetic drug for a synergistic glycaemic control (Adikwu et al., 2010). This could lead to drug-herb interaction. The clinical significance of these interactions varies; it can lead to an increase/decrease of therapeutic efficacy or even increase in the toxic effects of drug therapy (Mohammed and Mohammed, 2009). In some instances, the interaction may have a beneficial effect by increasing drug efficacy or diminishing potential side effects (Tende et al., 2011). The issue of drug-herb interactions should therefore be serious considered while a patient is combining a potent antidiabetic agent and herbal remedies (Adikwu et al., 2010).
Moringa oleifera belongs to the family of Moringaceae, a fast growing drought-resistant tree, native to Sub-Himalayan tracts of Northern India but also distributed world wide in the tropics and sub tropics (Fuglie, 1999). In Nigeria, though the Moringa tree is widespread throughout the states, it is usually found around farms and compounds as fence in Northern part of the country. In Nigeria, it is cultivated for its use as an alternative green vegetable source for human consumption and other medicinal uses. The pharmacological properties of Moringa oleifera have received a lot of attention in recent years due to its wide usage in the management and treatment of a number of ailments. Ethanol extract of the leaves has been shown to possess antifungal activity against a number of dermatophytes (Chaung et al., 2007) while, the methanol extract was shown to have a CNS depressant activity (Pal et al., 1996) and the aqueous extract also has demonstrated anti-fertility activity (Prakash, 1998). Moringa oleifera is used to manage diabetes mellitus and it has also been confirmed scientifically to possess hypoglycaemic properties (Jaiswal et al., 2009; Chinwe and Isitua, 2010; Tende et al., 2011). The wide consumption of Moringa oleifera for its therapeutic and nutritional benefits poses a possibility of pharmacological interaction when co-used with a conventional first-line antidiabetic agent such as metformin. Metformin is a biguanide commonly used by type II diabetic patients because of its fewer side effects and less likelihood to cause weight gain or raise cholesterol levels. A scientific study is therefore important to ascertain the beneficial or toxicity potentials of such combination
1.2 Justification for the Study
The World Health Organization (WHO) estimates that about 80% of African and Asian populations rely on traditional medicine as primary method for their health care needs. The scenario in developed countries is very similar with 70 to 80% of the population using some form of alternative and complimentary medicine (WHO, 2008). Herbal medicines are often used under a false sense of security due to the perception that „natural‟ ensures safety (Brantley et al., 2013). This misconception is as a result of the lack of regulatory quality control and standardization enforcement for herbal products. It should be understood that herbal preparations contain active phytochemicals in varying proportions which have a tendency like any other active pharmacological substance to alter the enzymatic systems, transporters and/ or the physiologic process (Vidushi, 2013). Herbal products have increasingly been incorporated into Western health care as various reports suggest a high contemporaneous prevalence of herb-drug use in developing and developed countries (Vidushi, 2013). Herb-drug interaction is the single most important clinical consequence of this practice (Fasinu et al., 2012). Despite these consequences, a standard system for interaction predictions and evaluation is non-existent as most researchers concentrate on potential therapeutic effects and mechanism of action of medicinal plants and often ignoring their potential interactions with conventional drugs. Consequently, the mechanisms underlying herb-drug interaction remain an understudied area in pharmacology (Brantley et al., 2013). The knowledge of the interactions of herbs with prescription and/or over-the-counter drugs is essential for minimizing clinical risks (Alissa, 2014). The interaction of drugs with herbal medicines is a significant safety concern, especially for drugs with narrow therapeutic indices (e.g. warfarin and digoxin), drugs for chronic ailments and drugs used in the management of life threatening conditions due to the fact that an alteration in the pharmacokinetics and/or pharmacodynamics of the drug by herbal remedies could bring about altered efficacy and/or toxicity, adverse reactions that are sometimes life threatening or lethal (Elvin-Lewis 2011). Several clinically important drugs have been reported to interact with some herbs leading to an exaggerated pharmacological activity or toxicity. Typical examples are the bleeding observed when warfarin is combined with garlic (Allium sativum), mild serotonin syndrome in patients who mix St. John‟s Wort (Hypericum perforatum) with serotonin-reuptake inhibitors; decreased bioavailability of digoxin, theophylline and cyclosporine when these drugs are combined with St John‟s Wort; induction of mania in depressed patients who mix antidepressants and Panax ginseng and increased risk of hypertension when tricyclic antidepressants are combined with yohimbine ( Fugh-Berman, 2000
In the management of diabetes mellitus, a number of herbs have been observed to interact with oral hypoglycaemic drugs. These include enhanced anti-hyperglycemia observed with co-administration of aqueous leaf extract of Vernonia amygdalina and metformin (Adikwu et al., 2010) and co-administration of the fruit juice of Mormodica charantia with glibenclamide (Lal et al., 2011). Similar observation was made when Mormodica charantia was consumed with chlorpropamide (Aslam and Stockley, 1979). Clinical studies have also shown that dietary gums such as the gum from the guar plant (Cyamopsis tetragonobulus) reduce the absorption of metformin and glibenclamide, consequently reducing hypoglycaemic effect (Izzo, 2004). Moringa oleifera is used traditionally to manage a number of conditions. Moringa is considered an effective treatment for anaemia, loss of appetite and lactation enhancer in women. It also combats gastric discomfort, stomach ulcers, diarrhoea, dysentery, colitis and can be used as a laxative, purgative and a diuretic; it is also used to fight colds, bronchial infections, fever and head pain, rheumatic discomfort, muscular cramp and bruises, skin infections, scabies, ringworm, insect bites and also stabilizes sugar in diabetes (Nadkarni, 2009). Tende et al. (2011) established the anti-hyperglycaemic activity of the leaves of this plant in diabetic rats. Due to its wide consumption, some diabetics take Moringa oleifera as an adjunct therapy to oral hypoglycaemic agents. One of such oral hypoglycaemic agent is metformin, a biguanide which is currently used as the first drug of choice in the treatment of uncomplicated type 2 diabetes mellitus. This concurrent use of Moringa oleifera and metformin could lead to a drug-herb interaction Identification of potential herb-drug interactions is of importance for effective therapy. This can be achieved by using appropriate in vitro and in vivo testing methods. Thus, this investigation of the possible pharmacological interaction between the ethanol extract of Moringa oleifera leaves and metformin is important towards maximizing therapy and minimizing toxicity in the management of diabetes. On establishing the type of interaction, there will now be scientific data to justify or discourage the co-use of Moringa oleifera extract and metformin by diabetics
1.3 Theoretical Frame Work
1.3.1Acute toxicity studies
Acute toxicity studies in animals are conducted to determine the nature and extent of the adverse effects which might follow the administration of a single dose of a drug. The information obtained from these studies is useful in choosing doses for repeat-dose studies, providing preliminary identification of target organs of toxicity, and, occasionally, revealing delayed toxicity. Acute toxicity studies may also aid in the selection of starting doses for chronic toxicity studies. The index of acute toxicity testing is the median lethal dose (LD50), which is the dose that will cause lethality in 50% of the test animals. The method described by Lorke adopted in this study postulates that acute toxicity should be tested in two phases using the smallest number of animals per group. In the initial phase, the range of doses producing the toxic effects is established and the nature and time of all adverse effects noted. In the second phase, further specific doses based on the results of the initial phase, are administered to calculate the LD50. An appropriate LD50 value is usually adequate to estimate the risk of acute intoxication. LD50 values usually guide the choice of doses used in efficacy and toxicity studies.
1.3.2 Dose-response study
The graphical representations of quantitative measurements of drug action are called dose- response curves (DRCs). Drugs exhibit a characteristic relationship between the dose and the pharmacologic response. Graphical representation of the dose and the magnitude of effect is the best way of clarifying the meaning of dose-response relationship. Knowledge of the relationships among dose, drug concentration in blood, and clinical response is important for the safety and effective use of drugs in individual patients. This information can help identify an appropriate starting dose, the best way to adjust dosage to the needs of a particular patient, and a dose beyond which increases would be likely to provide added benefit or would produce adverse effects. A dose-response study is important to establish the safety and effectiveness of a drug therapy. Often the dose-response curve is sigmosidal in appereance. Both ends of the curve are usually representative of doses of the drug which are either without effect, 0%, or least effect and the maximal effect, 100%. Subsequent doses beyond this latter level result in a plateuing of response. Sometimes, the plateau end of the curve may represent the toxic or even the lethal point. The smallest or the largest dose that produces 50% of the maximum response obtainable is known as the „effective median dose‟ (ED50). ED50 value is used to characterize the potency of an agent.
The graded doses of Moringa oleifera extract were selected based on the LD50 values and the logarithm of doses plotted against the pharmacological response (percentage glycaemic reduction) to each dose level. The plot of percentage glycaemic reduction versus log dose was used to estimate the ED50 which was in turn used for the interaction studies.
1.3.3 Alloxan-induced diabetes in rats
The alloxan model of experimental diabetes was first described by Dunn, et al., (1943). Alloxan was originally prepared by the oxidation of uric acid using nitric acid. It is regarded as a strong oxidizing agent that forms a hemiacetal with its reduction product; dialuric acid. Daluric acid can be further reduced to form alloxantin. Alloxan has bee noted to exert its diabetogenic action when administered parenterally, i.e., intravenously, intraperitoneally or subcutaneously. Furthermore, the dose of alloxan required for inducing diabetes depends on the animal species, route of administration and nutritional status (Federiuk et al., 2004). Alloxan is non-toxic to the human beta-cells, even at very high doses; this might be attributed to the differing glucose uptake mechanisms in humans and rodents (Eizirik et al., 1994). Alloxan induces diabetes by selective necrosis of the insulin-producing pancreatic beta-islets. This is similar to the auto-immune destruction of beta cells of the pancreas of type 2 diabetics as lack of insulin secretion results in both cases. Induction by alloxan leads to a triphasic blood glucose response.The first phase which starts a few minutes after alloxan administration is characterized by a transient hypoglycemic phase that lasts maximally for 30 minutes. This occurs as a result of stimulation of insulin secretion (Kliber et al., 1996). The underlying mechanism of this transient hyperinsulinemia may be attributed to a temporary increase in ATP availability due to inhibition of glucose phosphorylation through glucokinase inhibition. The second phase appears one hour after administration of alloxan. This stage is characterized by an increase in blood glucose concentration with a corresponding decrease in plasma insulin concentration. This marks the first hyperglycemic phase after the contact of the pancreatic beta cells with alloxan. These changes are as a result of inhibition of insulin secretion from the pancreatic beta cell due to the beta-cell toxicity of alloxan. The third phase is again a hypoglycemic phase that occurs 4-8 hours after alloxan injection. This severe transitional hypoglycemia phase usually lasts for several hours and it occurs as a result of alloxan-induced secretory granule and cell membrane rupture (Banerjee and Bhattacharya, 1945). In addition, other subcellular organelles such as the rough endoplasmic reticulum and Golgi complex are also ruptured
1.3.4 Pharmacological interaction study
Herb-drug interaction can occur either at pharmacokinetic or pharmacodynamic level. Pharmacokinetic interaction results when there is an alteration in the absorbtion, distribution, metabolism or elimination of a conventional drug by an herbal product or vice versa (Lal et al., 2011). A pharmacodynamic drug-herb interaction results when both the herb and drug are directed at a similar receptor target or physiological system.
The resulting effect of an herb-drug interaction could be synergetic, whereby the herbal medicine potentiates the action of synthetic drugs or antagonistic whereby, the herbal medicine reduces the efficacy of synthetic drugs (Izzo et al., 2002).
1.4 Aim of the Study
The aim of the study is to investigate the pharmacological interaction between the ethanol extract of Moringa oleifera leaves and metformin when co-administered to alloxan-induced hyperglycaemic Wistar rats.
1.4.1 Specific objectives
The specific objectives of the study are as follows
- To estimate the median lethal dose (LD50) of the ethanol extract of Moringa oleifera
- To estimate the dose of the ethanol extract of Moringa oleifera leaves required for 50% glycaemic reduction in fasted hyperglycaemic rats (ED50)
- Using hyperglycaemic rats, to carry out long-term (28 days) efficacy and toxicological studies on graded doses of ethanol extract of Moringa oleifera leaves (MOE), metformin and MOE/metformin co-administration. Parameters monitored were:
- Blood glucose level
- Food and water intake
- Body weight (BW) changes.
- Serum biochemistry, lipid and protein profiles
- Serum electrolyte, creatinine and urea levels
- Relative organ weight and histopathology of the liver, kidney, heart, brain, lungs, spleen, stomach and pancreas.
(iv.) Using normoglycaemic rats, to carry out long-term (28 days) efficacy and toxicological studies on graded doses of ethanol extract of Moringa oleifera and monitor same parameters as above.
1.5 Statement of Research Hypothesis
There is no pharmacological interaction when ethanol extract of Moringa oleifera leaves and metformin are administered concurrently to hyperglycaemic rats and Moringa oleifera possess hypoglycaemic activity in normoglycaemic rats.
