EVALUATION OF THE ANTIDIABETIC EFFECTS OF WATER AND METHANOLIC EXTRACTS OF AVOCADO (PERSEA AMERICANA) SEED ON ALLOXAN-INDUCED DIABETIC RATS


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EVALUATION OF THE ANTIDIABETIC EFFECTS OF WATER AND METHANOLIC EXTRACTS OF AVOCADO (PERSEA AMERICANA) SEED ON ALLOXAN-INDUCED DIABETIC RATS

ABSTRACT: The high prevalence, complications and cost of conventional drugs in the management diabetes necessitated the search for alternative treatment. As a result, this study seeks to evaluate the composition, anti-diabetic potential toxicity and tissue-protective effects of both the water and methanolic extracts of Persea americana (avocado pear) seed on alloxan-induced diabetic albino rats were investigated. This study was conceived and designed based on information on the local use of the seed in diabetes treatment. Proximate and anti-nutritional constituents of the seed were determined and 100g of the sample was extracted with 1000ml of both water and methanol using the maceration method. The extracts were evaporated to dryness using a rotary evaporator and stored at 4oC until use. The effects of different doses (200mg/kg.b.wt., 300/kg.b.wt.) of both water and methanolic extracts of P. americana seed on alloxan-induced diabetic albino rats were compared with those of a reference drug, insulin. The glucose level and weight of the rats were measured weekly for 21 days. The liver function tests and the histopathologies of the liver, and kidneys, were investigated. Results of the proximate investigation shows that the seed is rich in carbohydrate (49.03± 0.02 g/100g), lipid (17.90± 0.14 g/100g), protein (15.55± 0.36 g/100g) moisture (15.10± 0.14 g/100g) and ash (2.26±0.23 g/100g). Anti nutritional components such as total oxalate (14.98±0.03 mg/100g), tannin (6.98±0.04 mg/100g) and phytic acid (3.18±0.16 mg/1 00g). Results also showed that both the water and methanolic extracts exhibited significant anti-diabetic effects on the experimental rats. However, the methanolic extracts showed a better anti diabetic effect than the water extracts. The extracts showed no significant effects on the liver function parameters (bilirubin, conjugate bilirubin, AST, ALP and ALT) compared with the normal control but rather reversed the histopathological damage that occurred in alloxan-induced albino diabetic rats. In conclusion, the present study provides a pharmacological basis for the traditional use of P. americana seeds extracts in the management of Diabetes mellitus. It seems P. americana seed contains substantial amount of nutrients that could warrant its utilization in animal feed or food. However, further studies are required to indentify the active ingredient responsible for the anti-diabetic properties of the seed extract..

TABLE OF CONTENTContents    PagesTitle Page    -    -    -    -    -    -    -    -    -    -    iCertification    -    -    -    -    -    -    -    -    -    -    iiDedication    -    -    -    -    -    -    -    -    -    -    iiiAcknowledgement    -    -    -    -    -    -    -    -    -    ivAbstract    -    -    -    -    -    -    -    -    -    -    vTable of Contents    -    -    -    -    -    -    -    -    -    viList of Tables -    -    -    -    -    -    -    -    -    -    xiList of Figures -    -    -    -    -    -    -    -    -    -    xiiCHAPTER ONE1.0    Introduction    -    -    -    -    -    -    -    -    -    11.1    Background    -    -    -    -    -    -    -    -    -    1    Global Burden of Diabetes    -    -    -    -    -    -    -    2    Statement of Problem    -    -    -    -    -    -    -    3    Aims and Objectives of the Study    -    -    -    -    -    -    3CHAPTER TWO    Literature Review    -    -    -    -    -    -    -    -    4    Diabetes Mellitus    -    -    -    -    -    -    -    -    4    Types of Diabetes Mellitus    -    -    -    -    -    -    -    5    Prevention / Treatment of Diabetes Mellitus -    -    -    -    -    7    Medicinal Plants    -    -    -    -    -    -    -    -    8    Evaluation of Drug Toxicity -    -    -    -    -    -    -    13    Liver Function    -    -    -    -    -    -    -    -    16    Total Bilurubin    -    -    -    -    -    -    -    -    17    Conjugated Bilurubin -    -    -    -    -    -    -    -    19    Aspartate Transaminase (AST)    -    -    -    -    -    -    19    Alanine Transaminase (ALT)    -    -    -    -    -    -    21    Alkaline Phosphatase (ALP)    -    -    -    -    -    -    22    Histology of Liver and Kidney    -    -    -    -    -    -    24CHAPTER THREE3.0    Materials and Methods    -    -    -    -    -    -    -    263.1    Experimental Design    -    -    -    -    -    -    -    26    Chemicals and Reagents    -    -    -    -    -    -    -    26    Sample Collection and Preparation    -    -    -    -    -    -    27    Proximate Analysis    -    -    -    -    -    -    -    -    27    Protein Content    -    -    -    -    -    -    -    -    27    Crude Fat Content    -    -    -    -    -    -    -    -    28    Moisture Content    -    -    -    -    -    -    -    -    29    Ash Content Measure -    -    -    -    -    -    -    -    30    Carbohydrate Content -    -    -    -    -    -    -    -    30    Anti-nutritional Components -    -    -    -    -    -    -    30    Tannin Content Determination    -    -    -    -    -    -    30    Oxalate Determination    -    -    -    -    -    -    -    313.5.3    Phytic Acid    -    -    -    -    -    -    -    -    -    31    Animal Experiment    -    -    -    -    -    -    -    -    32    Induction of Diabetes -    -    -    -    -    -    -    -    33    Administration of Extracts    -    -    -    -    -    -    -    33    Blood Sample Collection    -    -    -    -    -    -    -    33    Liver Function Tests -    -    -    -    -    -    -    -    34    Total Bilirubin Test    -    -    -    -    -    -    -    -    34    Conjugated Bilirubin Test    -    -    -    -    -    -    -    35    Alanine Aminotransferase (ALT) Test    -    -    -    -    -    353.6.4.3 Aspartate Aminotransferase (AST) Test    -    -    -    -    -    363.6.4.5 Alkaline Phosphatase (ALP) Test    -    -    -    -    -    -    37    Histological Study    -    -    -    -    -    -    -    -    38    Histology Sample Preparation    -    -    -    -    -    -    38    Statistical Analysis of Data    -    -    -    -    -    -    39CHAPTER FOUR4.0    Results    -    -    -    -    -    -    -    -    -    40    Proximate Analysis    -    -    -    -    -    -    -    -    40    Anti-Nutritional Analysis.    -    -    -    -    -    -    -    40    Animal Experiment Results    -    -    -    -    -    -    -    41    Glucose Analysis Results    -    -    -    -    -    -    -    41    Results of Body Weight Analysis    -    -    -    -    -    -    42    Liver Function Test Results    -    -    -    -    -    -    -    434.3.4    Histology Results    -    -    -    -    -    -    -    -    44CHAPTER FIVE5.0    Discussion    -    -    -    -    -    -    -    -    -    51    Proximate and Anti-nutrients -    -    -    -    -    -    -    51    Effects of Extracts on Body Weight -    -    -    -    -    -    54    Effects of Seed Extracts on Blood Glucose    -    -    -    -    -    55    Effects of Seed Extracts on Liver Function Parameters    -    -    -    60    Histological Effects of Extracts on the Kidney and Liver    -    -    -    655.6    Conclusion    -    -    -    -    -    -    -    -    -    68References    -    -    -    -    -    -    -    -    -    69Appendix    -    -    -    -    -    -    -    -    -    -    89

LIST OF TABLESTables    PagesTable 1:    Result of the proximate analysis of P. american seed (g/100g)    -    40Table 2:    Anti-nutritional constituents of P. americana seed (mg/100g) -    -    40Table 3:    Effect of Extracts on Blood glucose levels (mmol/L) of Normal and Diabetic rats    -    -    -    -    -    -    41Table 4:    Effect of Extracts on Mean Bodyweight (g) of Normal and Diabetic rats    -    -    -    -    -    -    -    -    42Table 5:    Effect of Extracts on Mean Liver Function Parameters of Normal and Diabetic rats    -    -    -    -    -    -    -    43

LIST OF FIGURESFigures    PagesFig.1:    Section of liver tissue of normal rat    -    -    -    -    -    44Fig. 2:    Cortical section of kidney tissue of normal rat    -    -    -    44Fig. 3:    Section of liver tissue of untreated diabetic rat following liver damageby alloxan.    -    -    -    -    -    -    -    -    45Fig. 4:    Section of kidney tissue of untreated diabetic rat following kidneydamage by alloxan.    -    -    -    -    -    -    -    45Fig. 5:    Section of liver tissue of rat treated with 1unit of 40µ/50g b.w./day doseof insulin following liver damage by insulin. -    -    -    -    46Fig. 6:    Section of kidney tissue of rat treated with 1unit of 40µ/50g b.w./daydose of insulin following kidney damage induced by alloxan.    -    46Fig. 7:    Section of liver tissue of rat treated with 200mg/kg of water extract of avocado seed following liver damage induced by alloxan.    -    -    47Fig. 8:    A section of kidney tissue of rat treated with 200mg/kg of water extractof avocado seed following kidney damage by alloxan.    -    -    47Fig. 9:    Section of rat liver treated with 300mg/kg water extract of avocado seedfollowing liver damage by alloxan.    -    -    -    -    -    48Fig. 10:    Section of rat kidney tissue treated with 300mg/kg water extract ofavocado seed following kidney damage by alloxan. -    -    -    48Fig. 11:    Section of liver tissue of rat treated with 200mg/kg dose of methanolic extract of avocado seed following liver damage by alloxan.    -    49Fig. 12:    Section of medulla of kidney tissue of rat treated with 200mg/kg dose of        methanolic extract of avocado seed following kidney damage by alloxan.    49Fig. 13:    Section of liver tissue of rat treated with 300mg/kg dose of methanolic        extract of avocado seed extract following liver damage by alloxan. -    50Fig. 14:    Medullary section of kidney of rat treated with 300mg/kg dose of        methanolic extract of avocado seed extract following kidney damage by        alloxan.    -    -    -    -    -    -    -    50Fig. 15:    Mean Body weight curve of normal Rats    -    -    -    -    89Fig. 16:    Mean Body weight curve of diabetic untreated Rats -    -    -    89Fig. 17:    Mean Body weight of insulin treated diabetic Rats    -    -    -    90Fig. 18:    Effect of 200mg/b.wt. water extract treatment on diabetic Rats    -    90Fig. 19:    Effect of 300mg/b.wt. water extract treatment on diabetic Rats    -    91Fig. 20:    Effect of 200mg/b.wt. methanolic extract treatment on diabetic Rats    91Fig. 21:    Effect of 300mg/b.wt. methanolic extract treatment on diabetic Rats    92Fig. 22:    Effect of Treatments on Mean Bodyweight at Various Intervals.    -    92Fig. 23:    Mean blood glucose levels of all the groups before alloxan induction.    93Fig. 24:    Mean blood glucose levels for diabetic rats treated with insulin, waterand methanol avocado seed extracts at day 1 of treatment.    -    -    93Fig. 25:    Mean blood glucose levels for diabetic rats treated with insulin, waterand methanol avocado seed extracts at day 1 of treatment.    -    -    94Fig. 26:    Mean blood glucose levels for diabetic rats treated with insulin, waterand methanol avocado seed extracts at day 14 of treatment. -    -    94Fig. 27:    Mean blood glucose levels for diabetic rats treated with insulin, waterand methanol avocado seed extracts at day 21 of treatment. -    -    95Fig. 28:    Summary of the Blood Glucose level for diabetic rats treated with insulin, water and methanol avocado seed extract.    -    -    -    -    95Fig. 29:    Liver function parameters of alloxan induced diabetic rats treated with insulin, methanol and water extracts of P. Americana seed. -    -    96Fig. 30:    Mean Total Bilirubin concentration of diabetic rats treated with insulinand varied doses of water and methanol extracts of avocado seed.   -    96Fig. 31:    Mean Conjugate Bilirubin concentration of diabetic rats treated withinsulin, and varied doses of water and methanol extracts of avocado seed. 97Fig. 32:    Mean Aspartate Transaminase concentration of diabetic rats treated with insulin, and varied doses of water and methanol extracts of avocadoseed.    -    -    -    -    -    -    -    -    -    97Fig. 33:    Mean Alanine Transaminase concentration of diabetic rats treated with insulin, and varied doses of water and methanol extracts of avocadoseed.    -    -    -    -    -    -    -    -    -    98Fig. 34:    Mean Alkaline Phosphatase concentration of diabetic rats treated with insulin, and varied doses of water and methanol extracts of avocadoseed.    -    -    -    -    -    -    -    -    -    98

CHAPTER ONEINTRODUCTIONBACKGROUND OF THE STUDYDuring the past decade, the traditional systems have gained importance in the field of medicine. The World Health Organization estimates that 4 billion people, 80% of the world population (WHO, 2002), presently use herbal medicine for some aspect of primary health care (Orisataoki and Oguntibeju, 2010). Eighty percent (80%) of African populations use some form of traditional herbal medicine (WHO, 2002.) and the worldwide annual market for these products approaches US$ 60 billion (Willcox and Bodeker, 2004). Herbal medicine is a major component in all indigenous people's traditional medicine. Medicinal plants have continued to attract attention in global search for effective methods of using plants parts (e.g. seeds, leaves, stems, roots, barks etc) for the treatment of many diseases affecting humans (Sofowora, 2008). This is as a result of the continuous need for less expensive means of disease control.Medicinal plants are plants which can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs (Sofowora, 2008). Many important drugs used in healthcare today are directly derived from plants due to its bioactive constituents such as; alkaloids, tannins, steroids, etc. Examples include L-Dopa derived from Mucuna spp used for anti-parkinsonism, another is Caffeine, a CNS stimulant derived from Camellia sinensis and Quinine from Cinchona ledgeriana and used for Antimalarial, antipyretic.As a matter of fact, well into the 20th century, much of the pharmacopoeia of scientific medicine was derived from the herbal lore of native people. Many drugs commonly used today are of herbal origin. Undisputedly, the history of herbology is inextricably intertwined with that of modern medicine. Many drugs listed as conventional drugs were originally derived from plants.Salicyclic acid, a precursor of aspirin, was originally derived from white willow bark and the meadowsweet plant (Zand, et al., 2003).Plants are very unique as their existence is very essential for the sustenance of the rest of the food chain. Based on the observations made through successive generations, superstition as well as traditional medicinal folklore, man has found and has been using herbs, barks, fruits, leaves, seeds, roots and stems of different plants of various climatic regions for therapeutic purposes (Sofowora, 2008).GLOBAL BURDEN OF DIABETESDiabetes mellitus is a metabolic disease caused by a deficiency in the secretion or action of insulin (Nelson and Cox, 2005). This disorder is characterized by major symptoms as; polyuria (frequent and abundant urine), glycosuria (presence of glucose in urine) and hyperglycemia (glucose rate on an empty stomach higher than 1.2g/l in plasma blood and confirmed in at least two occasion) (N’guessan et al., 2009). Basically, there are two major clinical classes of diabetes.The type 1 diabetes or insulin dependent diabetes mellitus (IDDM) and type 2 diabetes or non insulin dependent diabetes mellitus (NIDDM) also called insulin resistant diabetes. According to the International Diabetes Federation (IDF) 2014 updates, out of the world seven billion population, 387million people, aged 20–79 years worldwide are diabetic, (IDF, 2014) giving a comparative prevalence of 8.3%, while 46.3% cases are undiagnosed. In every 7 seconds, a person dies of diabetes, 4.9 million deaths in 2014. Seventy seven percent (77%) of people with diabetes live in low and middle income countries. Africa has recorded cases of 2,150,274 (5.05%) diabetic patients with over 13million undiagnosed cases. In Nigeria, there are estimated 374,651 diabetic cases, with another 172,339 undiagnosed cases. These figures account for about4.64% Nigerian adults between ages 20-79 living with diabetes (IDF, 2014). In 2014, about 105,090 Nigerians died as a result of diabetes (IDF, 2014). An average diabetic Nigerian spent about 43527.16 naira (US $178.39) in 2014 due to diabetes treatment (IDF, 2014). With this alarming prevalence rate, diabetes mellitus poses a major challenge globally and accounts for a number of disabilities and deaths globally.Currently, diabetes therapy is based on the use of hypoglycemic drugs (sulfonamides, biguanides, and insulin), on hygieno-diet measures, exercise, and requires a lifelong treatment. With the level of poverty in developing nations like Nigeria, the need for a better and cheaper medication cannot be over emphasized. Traditional medicine has always provided a cheaper and time trusted alternative for the treatment and management of various diseases over time.STATEMENT OF PROBLEMThere is an urgent need to provide alternative and cheaper means for the management and treatment of diabetes.

AIMS AND OBJECTIVES OF THE STUDYThis study is aimed at the evaluation of the anti-diabetic effects of water and mMETHANOLic extracts of Persea americana (avocado) seed on alloxan induced diabetic rats.The specific objectives of the study are:1.    To determine nutritional and some anti-nutritional constituents of seed extracts of the plant Persea americana.2.    To evaluate the anti-diabetic properties of seed extracts of P. americana in diabetic rats3.    To evaluate the effect of P. americana seed extract on liver function enzymes.4.    To investigate the histopathological effects of P. americana on both the kidney and liver of diabetic rats.

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EVALUATION OF THE ANTIDIABETIC EFFECTS OF WATER AND METHANOLIC EXTRACTS OF AVOCADO (PERSEA AMERICANA) SEED ON ALLOXAN-INDUCED DIABETIC RATS


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