Rachell Ann Bala

CHAPTER I RATIONALE Diabetes mellitus describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism, resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long-term damage, dysfunction and failure of various organs (WHO, 1999). Reaching goals in diabetes studies can be a challenge or a success depending on the reliability of the research models, the mice. Among the animals used in the research, mice comprise a majority of all experimental mammals. The remarkable genetic similarity of mice to human, combined with great convenience, perhaps accounts for mice so often being the experimental model of choice in research. This model has since been well characterized as a model of diabetes mellitus ( Aasum, et al., 2005). There are different types of hypoglycaemic agents reported such as biguanides and sulphonylurea that are available alongside insulin for the treatment of diabetes mellitus (Nyunaï. et al, 2006). Thus, plants were the major source of materials which the ancient men resorted for combating various ailments (Barawidan et al, 2010). There is a growing interest in herbal remedies because of their effectiveness, minimal side effects and clinical experience and relatively low costs. Even the World Health Organization (WHO) approves the use of plant drugs for different diseases, including diabetes mellitus (Kayode et al, 2010). 1 Ageratum conyzoides contains many bioactive compounds including flavonoids, alkaloids, cumarins, essential oils, chromenes, benzofurans, terpenoids, and tannins. Tannins as one of the bioactive component of bulak manok was reported to possess antidiabetic activity (Teotia, 1997). Therefore, our present study was to determine the hypoglycemic activity of bulak manok (Ageratum conyzoides L.) leaf extract in alloxan-induced diabetic house mice. 2 Conceptual Framework CONCEPTUAL FRAMEWORK Independent Variable Dependent Variable Control variables Group A.1 (T 1): Positive Control Mouse feeds, distilled water ad libitum and 0.5 mL glibenclamide solution Group A.2 (T 1): Negative Control Mouse feeds, distilled water ad libitum Treatments Lowering of blood sugar in alloxan-induced diabetic white mice. Group B (T 2): Alloxan monohydrate (0.2 mg/kg) + 0.5 ml of bulak manok aqueous leaf extract Group C (T 3): Alloxan monohydrate (0.2 mg/kg) + 0.3 ml of bulak manok aqueous leaf extract Group C (T 4): Alloxan monohydrate (0.2 mg/kg) + 0.1 ml of bulak manok aqueous leaf extract 3 Theoretical background This study is anchored on the theory of herbal medicine. This theory proposed that the use of chemical and biological substances from the plants and herbs as herbal plants drugs utilized in preventing and curing diseases. These herbal drugs serve as effective alternatives or adjuncts to modern medicines (Manaderan, 2007). STATEMENT OF THE PROBLEM Intake of leaf extract of bulak manok has the potential of lowering of blood sugar among alloxan- induced house mice. The study wants to find out the efficacy of bulak manok as hypoglycemic agent. Specifically, the study would answer the following questions: 1. What dose of bulak manok leaf extract is most effective in lowering blood sugar? 2. What is the difference in the level of blood sugar before and after the intake of bulak manok leaf extract? 3. What is the difference in the level of blood sugar between the administrations of each treatment? OBJECTIVES OF THE STUDY The study aimed to investigate the efficacy of bulak manok leaf extract in lowering blood sugar among alloxan- induced diabetic house mice. Specifically, it aimed to: 1. determine the dose of bulak manok leaf extract that is most effective in lowering blood sugar among alloxan – induced diabetic house mice 4 2. compare the difference between the level of blood sugar before and after the intake of bulak manok leaf extract and 3. determine the difference in the level of blood sugar between the administrations of each treatment STATEMENT OF HYPOTHESIS Research hypothesis: Bulak manok aqueous leaf extract is an effective agent for lowering blood sugar among alloxan – induced diabetic house mice. Specific hypothesis: 1. The variation of dosage of bulak manok leaf extract is effective in lowering blood sugar. 2. There is no significant difference in the level of blood sugar before and after the intake of bulak manok leaf extract. 3. There is a significant difference in the level of blood sugar between the administration of each treatment. SIGNIFICANCE OF THE STUDY The following are those in one way or another can benefit the research: For the scientific community to gain additional knowledge about the additional benefits of bulak manok leaf extract aside from its antibacterial efficacy. Business industries to formulate a product out of Ageratum conyzoides leaf extract. 5 For the readers and future researchers, this serves as their guide for the future researches. SCOPE AND LIMITATION This study limits to the use of blood sample as the basis for the blood sugar level in mice which was used to verify the hypoglycemic efficacy of the study. This study includes the atmospheric temperature, humidity, food, ventilation and handling with regards to the factors that has affected the conditions of the mice. 6 CHAPTER II REVIEW OF RELATED LITERATURE Hypoglycaemia in Mice The retina is among the most metabolically active tissues in the body, requiring a constant supply of blood glucose to sustain function. The impact of high blood glucose on the vision of mice rendered hypoglycaemic by a null mutation of the glucagon receptor gene, Gcgr. Metabolic stress from moderate hypoglycaemia led to late-onset loss of retinal function in mice, loss of visual acuity, and eventually death of retinal cells. At 10 months of age mice began to lose visual acuity and exhibit changes in retinal anatomy, including an increase in cell death that was initially more pronounced in the inner retina (Umino et al., 2006). Bulak – manok (Ageratum conyzoides) Ageratum conyzoides L., is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also has bioactivity with insecticidal and nematocidal acitivity. This tropical species appears to be a valuable agricultural resource (Lin Chua Ming, 1999) The phytochemical studies carried out on Ageratum conyzoides showed that this plant contains many bioactive compounds and this includes: flavonoids, alkaloids, coumarin, glucosides, chromenes, terpenoids and tannins (Okunade, 2002). As reported by Oliver (1980), Mukherjee et al. (2006), glucosides, flavonoids, tannins, organic sulphurs components and alkaloids constituted the active metabolic part of hypoglycemic plants. 7 In the study of Nyember Nyunai et al., (2010) in the aqueous crude extract in the leaves of Ageratum conyzoides indicated that Ageratum conyzoides posses an important hypoglycaemic potential in streptozotocin – induced diabetic rats. The result of this study suggests that this plant may contain more than one hypoglycaemic component. Glibenclamide The findings of the study of Holstein (2011) indicated that despite glimepiride being prescribed more frequently than glibenclamide (6976 vs 6789 person-years), glimepiride induced fewer episodes of hypoglycaemia (6 vs 38 episodes); one episode occurred with a combination of the two preparations. The incidence of severe hypoglycaemia was 0.86/1000 person-years for glimepiride and 5.6/1000 person-years for glibenclamide. The characteristics of the 45 patients who presented with sulphonylurea-associated hypoglycaemia were as follows: mean age 79 years (95% CI 75.2; 82.6); glycosylated haemoglobin 5.4% (95% CI 5.1; 5.7); impaired renal function in 62%. Alloxan monohydrate Alloxan is a toxic analogue, which selectively destroys insulin-producing cells in the pancreas when administered to rodents and many other animal species. This causes an insulin-independent diabetes mellitus in animals, with characteristics similar to type 1 diabetes in humans. Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter ( Barawidan et al., 2010). 8 Definition of Terms Ageratum conyzoides – scientific name of bulak manok. compounds – a substance formed when two or more elements are chemically joined. glibenclamide - also known as glyburide (USAN), is an antidiabetic drug in a class of medications known as sulfonylureas, closely related to sulfa drugs. It is used in the treatment of type 2 diabetes. As of 2011, it is one of only two oral antidiabetics in the World Health Organization Model List of Essential Medicines (the other being metformin). hypoglycaemic – Lowering the concentration of glucose in the blood. Used of a drug. monoclonal - Monoclonal antibodies are molecularly homogeneous antibodies that are produced to target only one specific antigen (bind to only one specific receptor on a foreign protein). These monoclonal antibodies have been produced artificially by fusing a normally short-lived, antibody-producing B cell to a fast-growing cell, such as a cancer cell. streptozotocin – An alkylating nitrosurea derivative used to treat lymphomas and other CA Adverse effects Renal tubular dysfunction, glycosuria,hypoglycemia 9 CHAPTER III METHODOLOGY Research Locale The entire experiment was conducted in one of the researchers’ residence at Zone 2 Canduman, Mandaue City. Experimental Organisms The experimental organisms used in the research study were 45 straight- run white mice that were purchased at Manalili Petshop, Manalili Cebu City with ages ranging from 5 to 8 weeks old with approximately 25-28g initial body weight. This was made to ensure the uniformity in ages of the animals. All mice were placed in separate cages with three mice in every cage. The cages were uniform in size and that all mice were purchased from a single breeder. Research Design The researchers used the Complete Randomized Design (CRD). This design was intended to randomly each treatment as to avoid bias in the experiment, and to have valid and reliable results. 10 Table 1. Research Design. Control Group Treatments T1 (Alloxan Positive monohydrate Control Negative solution, (Alloxan Control mouse feeds, monohydrate, Alloxan distilled water mouse feeds, monohydrate, ad libitum, distilled water (mouse feeds, and 0.5 ml and and distilled aqueous leaf Glibenclamide water) extract of solution) Ageratum conyzoides) 9 9 9 No. of mice No. of 3 Replicates 3 3 T2 (Alloxan monohydrate solution, mouse feeds, distilled water ad libitum, and 0.3 ml aqueous leaf extract of Ageratum conyzoides) 9 T3 (Alloxan monohydrate solution, mouse feeds, distilled water ad libitum, and 0.1 ml aqueous leaf extract of Ageratum conyzoides) 9 3 3 There were 4 treatments with 3 replicates and each replicate contains 3 straight- run white mice. C A1 C B3 C B1 T A2 T A1 T C3 C B2 T B2 C A2 T A3 C A3 T B3 T C1 T B1 T C2 Legend: C A: Positive control (Alloxan monohydrate solution, mouse feeds, distilled water ad libitum and Glibencamide solution) 11 CB: Negative control (Alloxan monohydrate, mouse feeds, distilled water ad libitum) TA: Treatment 1 (Alloxan monohydrate, mouse feeds, distilled water ad libitum and 0.5 mL bulak manok aqueous leaf extract) TB: Treatment 2 (Alloxan monohydrate, mouse feeds, distilled water ad libitum and 0.3 mL bulak manok aqueous leaf extract) TC: Treatment 3 (Alloxan monohydrate, mouse feeds, distilled water ad libitum and 0.1 mL bulak manok aqueous leaf extract) Research materials and instruments The research instruments that were utilized in the experiment served essential roles in the study. In the maceration process, a sifter was used to separate the larger components from the fine powders of bulak manok leaves. Right after, a blender machine was utilized to homogenize the powdered bulak manok leaves. Then, a weighing scale was also used to measure the amount of powdered bulak manok leaves needed to be dissolved with a solution of ethanol. Several airtight containers/vials were used for the storage of macerated bulak manok leaves. After the maceration process, a reflux extractor was then utilized for the isolation of the macerated bulak manok leaves. Afterwards, a water bath on an electric hot plate was used for the evaporation of ethanol in the solution of the macerated bulak manok leaves. In order to fully ensure the evaporation of ethanol, an alcohol lamp was utilized to provide enough flame. Beakers with calibrations ranging from 250mL to 500 mL were used as well. Moreover, a condenser was used 12 to allow the solution to cool down. A syringe without needle that can hold up to 0.5mL was used in order to ensure that the mice will induce their respective solutions. Research Procedure Planting and Gathering of bulak manok leaves The researchers handpicked mature bulak manok leaves planted mostly in Sudlon II, Cebu City, Cebu. This was done in the morning in order to avoid transpiration. The leaves were washed under running tap water, cut and then air dried at room temperature for 2 weeks. The air drying was held in the researcher’s residence in Sudlon II, Cebu City, Cebu. Maceration of plant materials In the maceration process, the leaves were air dried for two weeks and then homogenized into fine powder using a blender machine. The powdered samples were sieved and stored in separate airtight containers until needed. A 180 g of the powdered leaves of bulak manok was weighed separately while 80% solution of ethanol was prepared. After which, the bulak manok leaves were macerated into 2 000 mL of prepared ethanol solution and were percolated for 48 hours inside a separate airtight container. The containers were labelled accordingly. Isolation of bulak manok leaf through Reflux Extraction The macerated plant materials were stored in a room temperature and were extracted for 4 hours using Reflux Extractor. The extracts were concentrated using water bath at 40O C to remove the Ethanol from the extracts (Abubakar, 2009; Kungnapa, 2003). In assuring that the 13 alcohol had totally evaporated, a flaming test was conducted in a minute sample from each plant root extract with the use of flamed match stick. Collection of Blood sugar sample and testing the level of blood sugar of straight run white mice with the use of Glucometer To obtain the mice’s blood glucose level, the researchers prepared the following equipments to be used in the procedure, such as Glucose meter (glucometer), test strips, alcohol pad, scissor and gauze pad. First, the researchers cut a small part of the mice’s tail using a scissor. Then, allow the droplet of blood enlarge to the size of BB or greater. Afterwards, slide the Glucose meter test strip collection point along the edge of the blood droplet allowing the blood to be drawn unto the strip. The researchers made sure that the blood covers the entire collection point of the test strip. After the countdown of the glucose meter, the result was flashed on the screen. The meter then displayed the level in mg/l or mmol/l. Finally, the researchers recorded the data. Administration of Glibenclamide solution In the preparation for the administration of glibenclamide solution, a 2.5 gram tablet of glibenclamide was pounded into fine powder and added to 250 mL of distilled water. Nine straight run white mice under the positive control were treated intraperitoneally with the prepared glibenclamide solution with the use of a syringe. The administration of glibenclamide solution was once a day only that lasted for one week. 14 Administration of Alloxan monohydrate Two grams of alloxan monohydrate was dissolved in 100 mL of 0.9% sodium chloride to obtain 2% alloxan monohydrate solution. Forty-five straight run white mice were orally administered with freshly prepared 2% alloxan monohydrate solution in a dose of 120 mg/kg body weight. Diabetic mice were determined four days after alloxan induction. Mice with blood glucose level above 130mg/dl were considered diabetic and used in the experiment since the normal blood glucose level ranges from 60-120 mg/dl (Barawidan et al., 2010). Administration of bulak manok leaf extract The limiting dosage corresponding to 0.1 to 0.5 mL of bulak manok leaf extract was inoculated to the mice. To determine which mice must be given with the corresponding treatment, the researchers applied draw by lots method. The mice chosen were labelled with a permanent marker in their right leg with their corresponding treatments. After which, the juice extract of the bulak manok were induced orally to the mice using a syringe without a needle. One of the researchers was assigned to perform the supplementation of the leaf extract daily throughout the experiment. This was done to avoid the differences in the way of handling the mice. 15 Statistical/ Data Analysis The data obtained were expressed as mean + standard error of mean. The statistical tool that was used is One-way ANOVA which was followed by Post Hoc’s test to determine the differences between means and with p