Anti-diabetic agents are medications that are used to manage high blood sugar levels in people with diabetes (Cappon et al., 2019), There are several types of anti-diabetic agents, including; Insulin: Insulin is a hormone that is produced by the pancreas and helps the body use glucose (sugar) from food for energy (Dunlay, 2019), People with type 1 diabetes do not produce enough insulin, so they need to take insulin injections or use an insulin pump to manage their blood sugar levels. Some people with type 2 diabetes may also need insulin therapy if other medications are not effective in managing their blood sugar levels. Oral medications; Metformin: Metformin is a commonly used medication for type 2 diabetes that helps the liver reduce glucose production and improves insulin sensitivity (Glovaci, Fan, & Wong, 2019). Sulfonylureas: These medications stimulate the pancreas to produce more insulin, DPP-4 inhibitors: Dipeptidyl peptidase-4 (DPP-4) inhibitors help increase insulin production and decrease glucose production in the liver. GLP-1 receptor agonists: Glucagon-like peptide-1 (GLP-1) receptor agonists increase insulin production, slow down stomach emptying, and help reduce appetite, SGLT2 inhibitors: Sodium-glucose cotransporter-2 (SGLT2) inhibitors help the kidneys excrete excess glucose from the body. Injectable medications; GLP-1 receptor agonists: These medications are available in both injectable and oral formulations and help increase insulin production and decrease appetite. Insulin: In addition to injection, insulin can also be administered using an insulin pump or an inhaler (Oguntibeju, 2019). Other medications; Amylin analogs: These medications help slow down stomach emptying and reduce appetite, Pramlintide: Pramlintide is a synthetic version of amylin that helps reduce glucose production in the liver and slows down stomach emptying. Anti-diabetic agents work in different ways to help manage blood sugar levels, and the choice of medication depends on the type of diabetes a person has, their individual health needs, and their response to treatment (Foretz, Guigas, &Viollet, 2019). It is important to work closely with a healthcare provider to determine the most effective anti-diabetic agents and dosage for an individual’s diabetes management plan. This chapter presents the background of the study, the problem statement, purpose, objectives of the study, research questions, study scope, justification of the study, significance, hypotheses, conceptual framework, as well as operational definition of key terms and concepts.

1.1 Background of the study

Diabetes is a chronic disease that affects how somes’ body processes blood sugar (glucose), which is the primary source of energy for thecells (Foretz, Guigas, &Viollet, 2019), Insulin is a hormone produced by the pancreas that regulates blood sugar levels, and in people with diabetes, the body either doesn’t produce enough insulin or doesn’t use it effectively (Glovaci et al., 2019), resulting in high blood sugar levels, there are several types of diabetes, including; Type 1 diabetes: This type of diabetes occurs when the immune system attacks and destroys the cells in the pancreas that produce insulin. Type 1 diabetes is usually diagnosed in children and young adults, and people with this type of diabetes need to take insulin injections or use an insulin pump to manage their blood sugar levels; Type 2 diabetes: This type of diabetes occurs when the body becomes resistant to insulin or doesn’t produce enough insulin to maintain normal blood sugar levels. Type 2 diabetes is the most common type of diabetes and is usually diagnosed in adults, although it is increasingly being diagnosed in children and adolescents; Gestational diabetes: This type of diabetes occurs during pregnancy and usually goes away after the baby is born (Zelniker et al., 2019), However, women who have had gestational diabetes are at increased risk of developing type 2 diabetes later in life.Other types of diabetes: There are several other rare types of diabetes, including monogenic diabetes and cystic fibrosis-related diabetes (Kenny, & Abel, 2019).Diabetes can cause a variety of health problems if left untreated, including heart disease, kidney disease, nerve damage, and blindness. However, with proper management, people with diabetes can live long, healthy lives. Treatment usually involves a combination of medication, lifestyle changes (such as a healthy diet and regular exercise), and monitoring blood sugar levels (Cappon et al., 2019).

For 2,000 years’ diabetes has been recognized as a devastating and deadly disease (Mahaffey, 2019), In the first century A.D. a Greek physician, Aretaeus, described the destructive nature of the affliction, which he named “diabetes” from the Greek word for siphon (Mahaffey, 2019).

Physicians in ancient times, like Aretaeus, recognized the symptoms of diabetes but were powerless to treat it effectively (Petersmann et al., 2019). In the 17thcentury a London physician, Dr. Thomas Willis, determined whether his patients had diabetes or not by sampling their urine. If it had a sweet taste he would diagnose them with diabetes mellitus-honeyed diabetes. This method of monitoring blood sugars went largely unchanged until the 20th century (Kato et al., 2019).Before the discovery of the insulin little could be done for patients suffering from diabetes. Low calorie diets prolonged their lives but left them weak and near starvation (Yuan et al., 2019), But in 1921, doctors in Canada treated patients dying of diabetes with insulin and managed to drop high blood sugars to normal levels. Since then, medical breakthroughs have continued to prolong and ease the life of people with diabetes (Lowe et al., 2019). In the ’50s, it was discovered that there were two types of diabetes: “insulin sensitive” (type I) and insulin insensitive (type II). Two thousand years have passed since Aretaeus spoke of diabetes as the mysterious sickness. It has been a long and arduous process of discovery, as generations of physicians and scientists have added their collective knowledge to finding a cure (Sattar et al., 2019). It was from this wealth of knowledge that the discovery of insulin emerged in a small laboratory in Canada. Since then, medical innovations have continued to make life easier for people with diabetes (Pivari et al. 2019).

Type 1 diabetes, also known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the immune system attacks and destroys the insulin-producing cells in the pancreas. Without insulin, the body cannot properly use glucose (sugar) for energy, which can lead to a buildup of glucose in the bloodstream (Sneha, &Gangil, 2019).Type 1 diabetes is usually diagnosed in children, teenagers, or young adults, but it can occur at any age. The symptoms of type 1 diabetes can develop quickly and include; Increased thirst and frequent urination, Fatigue and weakness, Unexplained weight loss, blurred vision, Increased hunger, Irritability and mood changes, If left untreated, type 1 diabetes can lead to serious health complications, including nerve damage, kidney damage, heart disease, and blindness (Furtado et al., 2019). There is no cure for type 1 diabetes, and treatment involves managing blood sugar levels through insulin therapy, a healthy diet, regular exercise, and frequent monitoring of blood sugar levels (Wang et al., 2019). People with type 1 diabetes need to take insulin every day to replace the insulin their body no longer produces (Furtado et al., 2019). They also need to monitor their blood sugar levels regularly to make sure they stay within a healthy range. With proper management, people with type 1 diabetes can live long, healthy lives (Halim, & Halim, 2019).

Type 2 diabetes is a chronic condition in which the body becomes resistant to insulin or does not produce enough insulin to maintain normal blood sugar levels (Gao et al., 2019). It is the most common type of diabetes, and it usually develops in adults, although it can occur at any age (Iftikhar, 2019). The risk factors for type 2 diabetes include obesity, physical inactivity, high blood pressure, high cholesterol, and a family history of diabetes. Symptoms of type 2 diabetes can include; Increased thirst and frequent urination, Blurred vision, Fatigue and weakness, Tingling or numbness in the hands or feet, Slow-healing wounds or infections, If left untreated, type 2 diabetes can lead to serious health complications, including nerve damage, kidney damage, heart disease, and blindness.Treatment for type 2 diabetes typically involves lifestyle changes, such as eating a healthy diet and increasing physical activity, and medications, such as oral medications or insulin injections. Regular monitoring of blood sugar levels is also important to make sure they stay within a healthy range (Biondi, Kahaly, & Robertson, 2019).In some cases, type 2 diabetes can be managed or even reversed through lifestyle changes alone, such as losing weight, increasing physical activity, and eating a healthy diet. However, in many cases, medication and monitoring are necessary to manage blood sugar levels and prevent complications (Shan et al., 2019).

An understanding of the pathophysiology of diabetes rests upon knowledge of the basics of carbohydrate metabolism and insulin action (Iftikhar et al., 2019). Following the consumption of food, carbohydrates are broken down into glucose molecules in the gut. Glucose is absorbed into the bloodstream elevating blood glucose levels (Biondi, Kahaly, & Robertson, 2019). This rise in glycemia stimulates the secretion of insulin from the beta cells of the pancreas. Insulin is needed by most cells to allow glucose entry. Insulin binds to specific cellular receptors and facilitates entry of glucose into the cell, which uses the glucose for energy (Rohani, 2019). The increased insulin secretion from the pancreas and the subsequent cellular utilization of glucose results in lowering of blood glucose levels (Ma et al., 2019). Lower glucose levels then result in decreased insulin secretion. If insulin production and secretion are altered by disease, blood glucose dynamics will also change. If insulin production is decreased, glucose entry into cells will be inhibited, resulting in hyperglycaemia. The same effect will be seen if insulin is secretedComplications due to diabetes are a major cause of disability, reduced quality of life, and death. Diabetes complications can affect various parts of the body manifesting in different ways for different people (Toyama et al., 2019). Diabetes increases patients’ risk for many serious health problems. In men, it is responsible for erectile dysfunction, low testosterone levels and emotional factors –such as depression, anxiety or stress–that can interfere with sexual feelings. In women, diabetes can be especially hard. Even those who do not have diabetes, pregnancy brings the risk of gestational diabetes. According to statistics from the American Diabetes Association, heart disease is the leading cause of death in women with diabetes (Luca et al., 2019). In addition, women with diabetes are afflicted by depression, their sexual health is at risk and eating disorders tend to occur more frequently. Diabetes can affect every part of the body, including the feet, the eyes and the skin. In fact, such problems are sometimes the first sign that a person has diabetes (Figtree et al., 2019).

The health complications caused by diabetes has a serious effect on an individual and their family as it could keep them from work and therefore restrict their earnings. This loss of earnings can also negatively affect the country’s economy. In addition, the health implications can put a strain on the country’s resources (Roncon et al., 2020). In 2015, it was estimated that the economic cost due to diabetes in sub-Saharan Africa was 1.2% of the gross domestic product, where these countries generally spend 5.5% of their gross domestic product on health in total(Cherney et al., 2020).).

Diabetes mellitus (diabetes) is a metabolic disorder in which the body becomes resistant to the effect of insulin or does not produce enough of this hormone to process glucose. As a consequence, there is a buildup of glucose, or sugar, in the body which can lead to serious health complications. The number of people with diabetes globally has risen from 108 million in 1980 to 463 million in 2019, which resulted in an increase in the prevalence in adults over the age of 18 from 4.7% in 1980 to 9.3% in 2019 (Sun et al., 2022).

The treatment of diabetes depends on the type of diabetes a person has and their individual health needs. Generally, treatment for diabetes involves managing blood sugar levels through lifestyle changes, medication, and monitoring; Lifestyle changes; A healthy diet: Eating a balanced diet with plenty of fruits, vegetables, whole grains, and lean protein can help manage blood sugar levels. Regular physical activity: Exercise helps the body use insulin more efficiently, which can help lower blood sugar levels, Weight management: Losing weight can improve insulin sensitivity and help manage blood sugar levels (Saha, Al-Rifai, R. H., &Saha, S. (2021).).

Medications; Insulin therapy: People with type 1 diabetes need to take insulin every day to replace the insulin their body no longer produces. Some people with type 2 diabetes also need insulin therapy to manage their blood sugar levels, Oral medications: There are several types of oral medications that can help manage blood sugar levels, including metformin, sulfonylureas, meglitinides, and thiazolidinediones (Boccardi, Murasecco, &Mecocci, 2019).

Injectable medications: Some people with type 2 diabetes may be prescribed injectable medications, such as glucagon-like peptide-1 (GLP-1) receptor agonists or sodium-glucose cotransporter-2 (SGLT2) inhibitors (Kesharwani et al., 2018).

Monitoring; Self-monitoring of blood glucose (SMBG): Regular monitoring of blood sugar levels at home can help people with diabetes make adjustments to their diet, physical activity, and medication to keep their blood sugar levels within a healthy range, HbA1c test: This blood test measures a person’s average blood sugar level over the past 2-3 months and can help determine if their diabetes management plan needs to be adjusted. In addition to managing blood sugar levels, people with diabetes may also need to manage other health conditions, such as high blood pressure, high cholesterol, or kidney disease, that can increase their risk of complications. Regular check-ups with healthcare providers are important for managing diabetes and preventing complications.

Roughly 75– 80% of individuals with diabetes die of cardiovascular illness. Individuals with Type 2 diabetes have a two to four times higher danger of coronary illness than the rest of the populace, and their guess is more unfortunate (Silva et al., 2018).Type 2 diabetes mellitus (T2DM) is the most common form of diabetes (90–95%), exhibiting an alarming prevalence among peoples of the sub-Saharan Africa (Levitt, 2018). The prevalence of T2DM has shown to increase markedly with age and the age of onset moving down into younger adults and even adolescents in recent years (Alberti et al., 2007). In sub-Saharan Africa, 21.5 million individuals are living with diabetes prompting roughly a large portion of a million diabetes-related deaths in 2013 (IDF, 2013). Most lifestyle factors had been found to have an impact on the incidence of T2DM and most of cases of T2DM can be prevented by embracing a healthy lifestyle. (Uppal, Italiya, Chitkara, & Mittal, 2018). Weight gain and obesity melodramatically upsurge the menace, and physical inactivity additionally increases the menace independently of obesity (Uppal, Italiya, Chitkara, &Mittal, 2018).

The prevalence of T2DM has shown to increase markedly with age and the age of onset moving down into younger adults and even adolescents in recent years (Alberti et al., 2007). In urban Ghana, T2DM influences in any event 6% of grownups and is related with age and obesity (Cefalu, 2018). T2DM is now found in almost every population and epidemiological evidence suggests that without effective prevention and control programs, the prevalence will continue to increase globally (IDF, 2013). In the last decade, about nine countries in sub Saharan African region have reported T2DM prevalence surveys data (Hall et al., 2011), with the assistance of the World Health Organization’s Stepwise Approach to Chronic Disease Surveillance Management (WHOSACDSM) two of these nations have conducted 2 population surveys which revealed a prevalence ranging from 0.6% in rural Uganda to 12% in urban Kenya (Hall et al., 2011). Ghana, Cameroon, guinea, Nigeria, south Africa, Uganda and Kenya had a prevalence of 0-7% (Hall et al., 2011). Increased body weight is a clear risk factor for T2DM, the association amongst T2DM and body weight is correctly attributable to the amount and distribution of body fats (Després, 2012). According to Hall et al (2011), Three studies investigated mortality in patients with diabetes in the sub Saharan Africa, These studies revealed high mortality proportions, with 5-year mortality ranging from 4% – 57% (Hall et al., 2011). In an investigation in urban Ghana, T2DM influences transcendently hefty patients of rather low financial status and as often as possible is joined by hypertension (Danquah et al., 2012). One of the important risk factors for T2DM is obesity and its prevention is directly associated with weight control. With weight loss, Insulin resistance which is enhanced by obesity, is a condition categorized by both elevated insulin production and impaired glucose tolerance is reversible (Hu, &Jia, 2018).

According to the International Diabetes Federation (IDF) Diabetes Atlas 9th edition, the prevalence of diabetes in Uganda among adults (20-79 years) was 1.6% in 2019. This translates to approximately 453,000 adults living with diabetes in the country. The IDF also estimated that there were an additional 200,000 cases of undiagnosed diabetes in Uganda in 2019.The prevalence of diabetes in Uganda is lower than the global average of 9.3%, but the burden of the disease is expected to increase in the coming years due to population growth, aging, and lifestyle changes. Diabetes is a major public health concern in Uganda, and there is a need for increased awareness, screening, and access to diabetes care and treatment in the country.

According to the International Diabetes Federation (IDF) Diabetes Atlas 9th edition, the global prevalence of diabetes among adults (20-79 years) was 9.3% in 2019. This translates to approximately 463 million adults living with diabetes worldwide. The prevalence of diabetes has been increasing rapidly in recent years, and the IDF estimates that the number of adults with diabetes will reach 578 million by 2030 and 700 million by 2045 if current trends continue (Chamberlain et al., 2018).

The majority of people with diabetes live in low- and middle-income countries, and diabetes is increasingly affecting younger populations. Type 2 diabetes is the most common type of diabetes, accounting for 90-95% of all cases. Diabetes is a major cause of premature death and disability worldwide, and it increases the risk of other health complications such as heart disease, kidney disease, blindness, and lower limb amputation.The IDF recommends that governments, healthcare providers, and communities prioritize diabetes prevention, early detection, and effective management to reduce the burden of diabetes and improve the health outcomes of people with diabetes (Davies et al., 2018).

1.2 Problem statement

Diabetes is one of the top non-communicable diseases in Africa, contributing to the increasing disease burden among the old adults. In uganda, diabetes mellitus is a silent major non communicable disease and the T2DM is a major and growing problem in the country (Gatimu et al., 2016). Individuals with T2DM have a two times higher danger of acquiring coronary illness than what ever remains of the populace, and their prognosis is very poor (Tuomilehto et al., 1997). T2DM today, is found in nearly all population and epidemiological suggestion suggests that without active prevention and management agendas, the prevalence will remain to upsurge worldwide (Unwin et al., 2009). In Gatimu et al., work it was stated that in sub-Saharan Africa, 21.5 million individuals are living with diabetes prompting roughly a large portion of a million diabetes-related passing’s in 2013 (IDF, 2013) . Diabetes mellitus is one of the still most important non-communicable disease and the T2DM is a key rising problem in the country. T2DM is a silent killer as several of the cases go undiagnosed (de-Graft Aikins, 2007). High levels of ignorance of the populace about the disease is also a factor in Uganda. The high cost related to the diagnosis and treatment of diabetes has also been a main concern, the cost of treating diabetes by synthetic drugs is high and this has led to many patients dying in their homes unable to get treatment in the hospitals. This has prevented the individual from undergoing check-ups to find their status with respect to the disease as the cost is beyond numerous household incomes. ., Hence, there is a need to develop low-cost solutions for diabetes, especially in underserved areas in diﬀerent regions from naturally occurring plants.

1.3 Objectives

Identify and collect medicinal plants with anti-diabetic agents used in treatment of diabetes.
Testing the crude extracts for anti-diabetic activity
Isolation and purification of the Bio-active compounds in the extracts
Structure elucidation of the Bioactive compound in the extract with anti-diabetic properties

CHAPTER TWO

LITERATURE REVIEW

2.0 Introduction

This section discusses the study in line with study objectives;

2.1 Prevalence of Diabetic

Non-communicable diseases (NCDs) accounts for more than 44% of death and 80% of outpatient visits, whereas most common NCDs among outpatients followed by chronic obstructive pulmonary disease (COPD) 43%, and cardiovascular disease (CVD) accounting for 40% (Marmot, & Bell, 2019). Sedentary lifestyles, fast urbanization, an unbalanced diet, and significant advances in maternal and child health have all contributed to the increase of non-communicable diseases, which has shifted disease patterns (Bigna, &Noubiap, 2019), Other pre-existing and most common risk factors include tobacco and alcohol intake along with smoking habits (Deventer, 2019).

According to the International Diabetes Federation (IDF), in 2021, there were 537 million adults aged 20-79 with diabetes worldwide (Katzmarzyk et al., 2022). The IDF also estimates that this number will increase to 642 million by 2045. The global prevalence of diabetes is 8.5%, which means that approximately one in every 11 adults has diabetes, Type 2 diabetes is the most common form of diabetes and accounts for about 90% of all cases (Drapkina et al., 2022). The remaining 10% are attributed to type 1 diabetes and other less common types. It’s important to note that the prevalence of diabetes varies greatly by region, with the highest rates found in low- and middle-income countries. In fact, about 80% of people with diabetes live in these countries. In terms of absolute numbers, the top three countries with the highest number of people with diabetes are China, India, and the United States (Fitipaldi, & Franks, 2023).

Diabetes is becoming increasingly prevalent in Africa, particularly in urban areas where there has been a rise in sedentary lifestyles and unhealthy diets (Fitipaldi, & Franks, 2023). According to the International Diabetes Federation (IDF), the prevalence of diabetes in Africa was 19.4 million in 2021, and this number is projected to increase to 47 million by 2045 (Momma et al., 2022). The IDF also reports that the overall prevalence of diabetes in Africa is 3.3%, which is lower than the global average of 8.5% (Armocida et al., 2022). However, there are significant variations in prevalence rates between different countries and regions within Africa. For example, in some countries like Mauritius and Seychelles, the prevalence of diabetes is as high as 20%. It’s important to note that diabetes in Africa is often undiagnosed and untreated, which can lead to serious complications such as blindness, kidney failure, and amputations. Improved access to diabetes education, screening, and treatment is essential to prevent and manage the disease in Africa (Khan et al., 2012).

T1DM, which accounts for approximately 5–10% of DM cases, arises due to destruction of β-cells of the pancreas predominantly through an autoimmune process in over 95% of cases (type 1A) or idiopathic in less than 5% of cases (type 1B) (Munekawa, 2021). If T1DM is left untreated it usually manifests itself as ketoacidosis (Subir, 2020). The disease is a catabolic disorder with virtually absent circulating insulin, elevated plasma glucagon, and lack of pancreatic βcells response to all insulinogenic stimuli, necessitating use of exogenous insulin. In immune-mediated T1DM, approximately one-third of the disease susceptibility is genemediated and two-thirds is due to environmental factors (Aminian, 2019).). In a mild form of auto immunemediated T1DM, patients initially retain enough β-cells function to avoid ketosis, but as the disease progresses later in life, they also become dependent on exogenous insulin. It is been reported that in Northern European countries, up to 15% of T2DM cases may actually have this mild form of T1DM (latent autoimmune diabetes of adults, The fact that the prevalence of T1DM is higher in Scandinavian countries and increases by migration to Northern Hemisphere supports the involvement of environmental factors in the development of T1DM (Wang et al., 2022).).

According to a recent study conducted by the International Diabetes Federation, the number of patients with diabetes worldwide has increased significantly from 151 million in 2000 to 537 million in 2021 (International Diabetes Federation, 2021). This situation is no exception in South Korea. The prevalence of diabetes in adults aged 30 years or older in Korea more than doubled from 2.23 million in 2006 to 4.94 million in 2018; according to the Diabetes Fact sheet of the Korean Diabetes Association, one in seven patients aged 30 years or older in Korea is diabetic. Hence, attention is being paid to the increasing prevalence of diabetes among young people from their 20s to their 30s based on the national disease statistics prepared by the Health Insurance Review and Assessment Service in 2021; it has been recognized as a primary public health issue. The number of diabetes patients increased by 51.4% from 19,781 people in their 20s in 2016 to 29,949 people in their 20s in 2020 (Saeedi, 2020). This does not include the 27.7% increase in the diabetes prevalence among all of the age groups, including 30.8% of those in their 30s.

In the case of diabetes, blood sugar control is more difficult to achieve in the younger age group compared with that in the older adult group, and insulin resistance and pancreatic beta-cell dysfunction are severe and rapidly worsening (Sinclair, 2020). Considering the severity and trend of diabetes, it is important to understand the potential disease risk and develop customized interventions for preventive management, especially for the younger age group (Boulton, 2020).

Genetic and environmental factors act in a complex manner at the onset of diabetes, and the increase in the prevalence of diabetes in the younger generation is caused by environmental factors rather than genetic factors (Bahar, et al., 2020). The main environmental factors that play a role in the development of diabetes such as occupation and individual behavioral characteristics are smoking, drinking, lack of physical activity, obesity and sleep disorders, and disordered eating behavior in individuals (Atlas, 2015). In addition, excessive energy intake and a lack of physical activity, which are typical lifestyle changes following economic development and westernization, are directly related to obesity, which is the most important underlying risk factor for diabetes. In a previous study that investigated adolescents with type 1 diabetes for 8 years, the males and females were closely related to the overweight group, indicating that the risk of diabetes was seven times higher in both of the sex groups compared with that in the normal weight group with a body mass index (BMI) of 30 kg/m2 or higher (James, Varghese, Sharma, & Chand, 2020).

According to the International Diabetes Federation’s Diabetes Atlas, the estimated prevalence of diabetes in Kenya in 2021 was 2.2%, which equates to approximately 840,000 people living with diabetes in the country. However, it is important to note that this is an estimate and the actual prevalence of diabetes in Kenya may be higher, as there may be undiagnosed cases and limited data available in some regions. Additionally, the prevalence of diabetes is likely to increase in Kenya as the country experiences economic development and lifestyle changes, such as increased urbanization and changes in diet and physical activity patterns (Barasa M& Mmusi, 2021).

According to the International Diabetes Federation’s Diabetes Atlas, the estimated prevalence of diabetes in Uganda in 2021 was 1.5%, which equates to approximately 620,000 people living with diabetes in the country. However, it is important to note that this is an estimate and the actual prevalence of diabetes in Uganda may be higher, as there may be undiagnosed cases and limited data available in some regions. Additionally, the prevalence of diabetes is likely to increase in Uganda as the country experiences economic development and lifestyle changes, such as increased urbanization and changes in diet and physical activity patterns (Ssekamatte et al., 2021).

2.1 Identify and collect medicinal plants with anti-diabetic agents used in treatment of diabetes

Local Name : Kibwakulata

Botanical Name : PlectranthusCyaneus

Plectranthuscyaneus, also known as the Coleus forskohlii plant, has been traditionally used in Ayurvedic medicine for the treatment of various ailments, including diabetes.Several studies have investigated the potential anti-diabetic properties of Plectranthuscyaneus extract, particularly its active compound forskolin. Forskolin has been found to increase insulin secretion from pancreatic beta cells, which can help regulate blood sugar levels (Grayer et al., 2021).

In a study published in the Journal of Medicinal Food in 2014, researchers found that an extract of Plectranthuscyaneus significantly decreased blood glucose levels in diabetic rats. Another study published in the Journal of Clinical Pharmacy and Therapeutics in 2015 found that a Plectranthuscyaneus extract improved insulin sensitivity and glucose uptake in human adipocytes (fat cells), While these studies show promising results, more research is needed to determine the effectiveness of Plectranthuscyaneus as a treatment for diabetes in humans. Additionally, it is important to note that Plectranthuscyaneus extract may interact with certain medications and should be used under the guidance of a healthcare professional (Ndjoubi et al., 2021).

Plectranthuscyaneus, also known as Coleus forskohlii, has been traditionally used in Ayurvedic medicine for various health conditions. Some of the potential health benefits of Plectranthuscyaneus include:; Asthma: Plectranthuscyaneus has been used in Ayurvedic medicine as a natural remedy for asthma. Some studies have suggested that Plectranthuscyaneus may help improve breathing in people with asthma, Weight loss: Plectranthuscyaneus has been studied for its potential to aid in weight loss. It is believed that the active compound in Plectranthuscyaneus, forskolin, may help increase the breakdown of stored fat in the body.Glaucoma: Plectranthuscyaneus has been used in Ayurvedic medicine to treat glaucoma. Some studies have suggested that forskolin, the active compound in Plectranthuscyaneus, may help reduce intraocular pressure, which is a common symptom of glaucoma.

Cardiovascular health: Plectranthuscyaneus has been studied for its potential cardiovascular benefits. It is believed that forskolin may help dilate blood vessels, which can improve blood flow and reduce blood pressure (Grayer et al., 2010).

Skin health: Plectranthuscyaneus has been used in Ayurvedic medicine to treat various skin conditions, including psoriasis and eczema. Some studies have suggested that Plectranthuscyaneus may have anti-inflammatory properties, which may help reduce inflammation in the skin.

Eucalyptus

Scientific name : Eucalyptus

Higher classification : Eucalypteae

Rank : Genus

Family : Myrtaceae

Subfamily : Myrtoideae

Order : Myrtales

Botanical name : Eucalyptus globulus

Eucalyptus, a tree native to Australia, is known for its medicinal properties and has been used traditionally by indigenous communities for centuries to treat a variety of ailments, including diabetes.

Research studies have shown that eucalyptus leaves contain bioactive compounds such as flavonoids, terpenoids, and tannins, which have anti-diabetic properties. These compounds are believed to stimulate the production of insulin and help regulate blood glucose levels.

One study published in the Journal of Ethnopharmacology found that eucalyptus leaf extract was able to significantly reduce blood glucose levels in diabetic rats. Another study published in the same journal showed that eucalyptus leaf extract was able to improve insulin sensitivity and reduce insulin resistance in diabetic rats.

Furthermore, a review article published in the International Journal of Molecular Sciences concluded that eucalyptus leaf extract has potential as a therapeutic agent for diabetes due to its anti-diabetic properties.

While these studies show promise, more research is needed to fully understand the mechanisms by which eucalyptus exerts its anti-diabetic effects and to determine the optimal dosage and formulation for use in humans. It is important to consult with a healthcare provider before using any herbal remedies to manage diabetes.

English Name: Boungavilea Flower

Botanical Name: Bougainvillea Glabra

Rank : Genus

Family : Nyctaginaceae

Order : Caryophyllales

Kingdom : Plantae

Photo of Boungavilea Flower

Bougainvillea is a genus of flowering plants that belongs to the Nyctaginaceae family. There is some limited research suggesting that bougainvillea flowers may have anti-diabetic properties. One study published in the Journal of Ethnopharmacology in 2001 found that an extract of bougainvillea flowers was able to lower blood glucose levels in diabetic rats. The researchers attributed this effect to the presence of flavonoids and other phytochemicals in the extract.

The botanical name of Bougainvillea is Bougainvillea spectabilis. It is a species of flowering plant native to South America, but it is widely cultivated as an ornamental plant in many parts of the world due to its vibrant and colorful bracts. There are several other species of Bougainvillea as well, such as Bougainvillea glabra and Bougainvillea peruviana, which are also commonly cultivated for their beautiful flowers.

Another study published in the Journal of Food and Drug Analysis in 2017 investigated the anti-diabetic effects of a bougainvillea flower extract in mice. The researchers found that the extract was able to improve glucose tolerance and insulin sensitivity, which are important factors in managing diabetes. While these studies are promising, more research is needed to determine the full extent of bougainvillea flower’s potential anti-diabetic properties and its safety and effectiveness in humans. Therefore, it is important to talk to your doctor before using bougainvillea flowers or any other natural remedies to manage diabetes.

BOTANICAL NAME : ASPILIA AFRICANA

LOCAL NAME : MAKAYI

Aspilia africana, also known as the “holy herb” or “yaa- nkruma” in West Africa, has been traditionally used in folk medicine to treat a variety of ailments including diabetes. Studies have shown that Aspilia africana possesses anti-diabetic properties. A study published in the journal BMC Complementary and Alternative Medicine found that the leaf extract of Aspilia africana was able to reduce blood glucose levels in diabetic rats. The study suggested that the anti-diabetic effect of the leaf extract was due to its ability to improve insulin secretion and reduce insulin resistance.

Another study published in the Journal of Ethnopharmacology showed that the aqueous extract of Aspilia africana was able to significantly reduce blood glucose levels and increase insulin sensitivity in diabetic rats. Furthermore, a review article published in the journal Evidence-Based Complementary and Alternative Medicine concluded that Aspilia africana has potential as a therapeutic agent for diabetes due to its anti-diabetic properties.

While these studies show promise, more research is needed to fully understand the mechanisms by which Aspilia africana exerts its anti-diabetic effects and to determine the optimal dosage and formulation for use in humans. It is important to consult with a healthcare provider before using any herbal remedies to manage diabetes.

Africana (Pers.) C. D. Adams belongs to the family Asteraceae and has been used by many African communities in the treatment of a range of health conditions. The plant is used to treat inflammatory conditions as well as osteoporosis, stomach ache, diarrhea, measles, malaria, tuberculosis, cough, sores, diabetes, rheumatic pains, bee, scorpion and wasp stings, ear infections, febrile headaches, and gonorrhea and is used as a contraceptive. A. africana is also prominently known for its wound healing properties [7–9]. The plant, though often known as the hemorrhage plant or wild sunflower, is referred to by various names by different communities, such as Makayi in Luganda (Uganda), Orangila in Igbo (Nigeria), Nyana in Kissi (Sierra Leone), Fofo in Akan-akyem (Ghana), Mbnaso in Kpe (Cameroon), Soumadibrouin among the Malinke (Côte d’Ivoire), and Winnih in Mano (Liberia).

English Name : American black nightshade

Botanical Name : Solanum americanum

Kingdom : Plantae

Family :Solanaceae

Genus : Solanum

Species : S. americanum

Local name : nsugga

Photos of Solanumamericanum

Solanumamericanum, also known as American nightshade or American black nightshade, is a plant species in the Solanaceae family. It is native to the Americas and is found in many tropical and subtropical regions around the world.

The plant grows up to 1.5 meters tall and produces small white or purple flowers, which are followed by round, glossy black or dark purple berries. While the berries may look similar to those of other edible berries, such as blueberries, they are toxic to humans and should not be consumed.

In some cultures, the leaves of Solanum americanum are used as a traditional medicine to treat a range of ailments, including inflammation, fever, and respiratory infections. However, the plant contains toxic compounds, including solanine, that can be harmful if ingested in large quantities. Therefore, it is important to consult with a healthcare professional before using any herbal remedies containing Solanumamericanum.

Solanumamericanum has been traditionally used in some cultures as a treatment for diabetes, there is limited scientific evidence to support its effectiveness in this regard. Some studies have suggested that extracts of Solanumamericanum may have hypoglycemic effects, meaning they can lower blood sugar levels, but these studies have been conducted on animals or in vitro and have not been well-established in human trials.

Furthermore, the plant contains toxic compounds that can be harmful if ingested in large quantities, and there is a risk of interaction with other medications or supplements that can affect blood sugar levels..

Botanical name: Neurolaenalobate

English name : jackass bitters

Local names : mbaluka

Order : Asterales

Family : Asteraceae

Genus : Neurolaena

Species : N. lobata

Photos of Neurolaena lobate

Neurolaenalobata, also known as “guaco” or “minnieroot,” is a plant species native to Central and South America. It has been traditionally used in folk medicine for its anti-inflammatory and analgesic properties, as well as for treating a range of ailments such as fever, colds, and respiratory infections.

Some studies have suggested that extracts of Neurolaenalobata may have anti-inflammatory and antimicrobial effects, which may support its traditional uses. In addition, some research has shown that Neurolaenalobata extracts may have antitumor properties, although more studies are needed to confirm this.

Overall, while Neurolaenalobata may have potential health benefits, further research is needed to better understand its properties and potential uses in clinical settings.

English Name:Garlic

Botanical name: Allium sativum

Photos of garlic

Garlic (Allium sativum) has been traditionally used for centuries for its potential health benefits, including its anti-diabetic properties. Several studies have suggested that garlic may have hypoglycemic effects, meaning it can lower blood glucose levels.

One of the ways garlic may have an anti-diabetic effect is by increasing insulin sensitivity. Insulin is a hormone that helps regulate blood sugar levels, and individuals with type 2 diabetes often have reduced insulin sensitivity, leading to elevated blood sugar levels. Garlic has been shown to increase insulin sensitivity in animal and human studies, which may help improve blood sugar control in individuals with type 2 diabetes.

Garlic may also help reduce oxidative stress and inflammation, which are thought to play a role in the development and progression of diabetes. Additionally, garlic may help improve lipid profiles, including reducing triglycerides and cholesterol, which are often elevated in individuals with diabetes, While garlic has shown promise in improving blood sugar control and other markers in individuals with diabetes, more research is needed to fully understand its potential therapeutic benefits and optimal dosing.

Garlic contains a number of sulfur compounds, including allicin, that are believed to be responsible for its potential health benefits. Some research has suggested that garlic may help to lower cholesterol levels, reduce inflammation, improve immune function, and even have anti-cancer properties.

Garlic has also been used in traditional medicine to treat a variety of ailments, including respiratory infections, digestive issues, and high blood pressure. However, more research is needed to fully understand the potential health benefits and risks of garlic.

Garlic is generally considered safe when consumed in food amounts, but can cause side effects such as bad breath, body odor, and upset stomach when consumed in larger amounts or taken as a supplement.

Botanical name: Rubus erlanrigenus

English name : red raspberry.

Local name : Akasinsa

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Rosales

Family : Rosaceae

Genus : Rubus

Photos

Origin and characteristics of the Rubus

The genus Rubus comprises some 331 accepted species, which are distributed throughout the temperate and warm regions of the northern hemisphere. They are often known by the name of brambles or blackberries. They develop thin, green stems, mostly biennial, and with stingers which allow them to grow and invade new territories with relative ease. And it is that these have creeping and / or climbing habits, which is why they can be grown as vines in gardens.

They are shrubs that live for several years, with pinnate, alternate and green leaves. The flowers are grouped in lateral or terminal inflorescences (at the end of the stem, which will die after flowering). These are usually white, and appear solitary or in panicles. The fruit is a compound drupe, which measures between 0,5 and 2 centimeters, and is edible.

Rubus L. is one of the most diverse and largest genera in the Rosaceae family. The genus consists of more than 700 shrubby or herbaceous species mainly distributed throughout the temperate zone of the northern hemisphere, with a few having expanded to the tropics and the southern hemisphere. Species of the Rubus genus worldwide are classified into 12 subgenera. However, Lu et al. 2018) reclassified them into 8 subgenera, whereby only habitats in China were considered. There are two hypothetical centers of origin for Rubus: one is North America and the other is southwestern China. In addition, the pleasant flavor of the fresh Rubus fruit, its medicinal functions due to the health benefits of its very high secondary metabolite content, and its high genetic diversity and complex phylogeny rendering it suitable for scientific studies, make Rubus an important and ideal genus for breeders as well as scientists. Furthermore, the rich secondary metabolites and the bark of Rubus are also important raw materials for cosmetics and fiber.

Botanical Name : OxytenantheraAbyssinica

English Name : Ethiopian bamboo.

Kingdom : Plantae

Order : Poales

Family : Poaceae

Subfamily : Bambusoideae

Tribe : Bambuseae

Subtribe : Bambusinae

Genus :OxytenantheraMunro

Species : O. abyssinica

Photos

Oxytenanthera abyssinica, also known as Ethiopian bamboo, is a species of bamboo that is native to Africa. The plant has been traditionally used in African medicine to treat various ailments, including diabetes. There have been several studies that have investigated the anti-diabetic properties of Oxytenantheraabyssinica.

One study published in the Journal of Ethnopharmacology in 2005 found that extracts of Oxytenanthera abyssinica had significant hypoglycemic (blood sugar lowering) effects in rats. The study suggested that the plant may have potential as a natural treatment for diabetes.

Another study published in the Journal of Medicinal Plants Research in 2012 evaluated the effects of Oxytenanthera abyssinica extracts on glucose uptake in skeletal muscle cells in vitro. The study found that the extracts significantly increased glucose uptake in the cells, indicating that the plant may have potential as a natural anti-diabetic agent.

Overall, while the research on the anti-diabetic properties of Oxytenanthera abyssinica is still limited, the available studies suggest that the plant may have potential as a natural treatment for diabetes. However, further studies are needed to fully understand the mechanisms of action and potential therapeutic benefits of this plant.

Botanical Name: Pterocarpus marsupium

English Name: Indian Kino Tree

Local name : Mukusu

Photos

Pterocarpus marsupium, also known as Indian kino tree, is a tree species that is found in India and Sri Lanka. The plant has been used for centuries in Ayurvedic medicine to treat various ailments, including diabetes. Several studies have investigated the anti-diabetic properties of Pterocarpus marsupium.

One study published in the Journal of Ethnopharmacology in 2003 investigated the effects of Pterocarpus marsupium on blood glucose levels in rats with diabetes. The study found that the plant significantly lowered blood glucose levels in the rats, and also improved insulin sensitivity and glucose tolerance.

Another study published in the Journal of Medicinal Food in 2013 evaluated the effects of Pterocarpus marsupium extract on glucose metabolism in healthy human subjects. The study found that the extract significantly improved glucose tolerance and insulin sensitivity in the subjects.

Pterocarpus marsupium contains several bioactive compounds, including pterostilbene, epicatechin, and marsupin. These compounds have been shown to have anti-diabetic properties, including the ability to improve insulin sensitivity, enhance glucose uptake in cells, and regulate blood glucose levels.

Overall, the available research suggests that Pterocarpus marsupium may have potential as a natural treatment for diabetes. However, further studies are needed to fully understand the mechanisms of action and potential therapeutic benefits of this plant..

Aqueous extract of heartwood of Pterocarpus marsupium was given orally to alloxan induced type-2 diabetic rabbit model (Pradhan et al., 2017). Both the fasting blood glucose (194.8±12.7 vs. 155.2±16.3) and postprandial blood glucose (191.6±23.2 vs. 149.2±14.5) were decreased indicating the hypoglycaemic effect of P. marsupium. Incubation of red blood cells with glucose in the presence of alcoholic extract of P. marsupium under high glucose conditions lead to reduction in the accumulation of intracellular sorbitol in a dose dependent manner. There was 50% reduction of sorbitol accumulation with alcoholic extract of was observed with IC50 151.00μg/ ml and 105.12μg/ml for ascorbic acid [6]. Ethanolic extract of P. marsupium heartwood have antihyperglycemic activity in streptozotocin treated diabetic rats. At 100 mg/kg dose levels, fasting blood glucose, oral glucose tolerance and serum insulin levels were recorded as 113±3.40mg/dl, 35930±102.9

Local name :Mululuza

English name : Bitter leaf

Botanical name : Vernonia amygdalina

Order :Asterales

Family : Asteraceae

Genus :Vernonia

Species : V. amygdalina

photo

Bitter leaf (Vernoniaamygdalina) is a plant that is native to Africa, commonly found in countries like Nigeria, Ghana, and Cameroon. It is also known as “onugbu” in Igbo, “ewuro” in Yoruba, and “ndole” in Cameroon. Bitter leaf is known for its various medicinal properties and has been used in traditional African medicine to treat various illnesses, including malaria, fever, diarrhea, and constipation. It is also used as a natural remedy for diabetes, high blood pressure, and liver diseases.

Studies have shown that bitter leaf contains various bioactive compounds that contribute to its medicinal properties, such as alkaloids, flavonoids, terpenoids, and phenolic acids. Some of the health benefits associated with bitter leaf include: Anti-malaria: Bitter leaf has been traditionally used to treat malaria, and several studies have confirmed its anti-malarial properties. A study published in the Journal of Medicinal Plants Research found that an extract of bitter leaf had significant anti-malarial activity against Plasmodium falciparum, the parasite that causes malaria. Anti-diabetic: Bitter leaf has been shown to have anti-diabetic properties, possibly due to its ability to improve insulin sensitivity and reduce blood glucose levels. A study published in the Journal of Complementary and Integrative Medicine found that bitter leaf extract significantly reduced blood glucose levels in rats with induced diabetes (Onyibe et al., 2021).

Anti-inflammatory: Bitter leaf contains various compounds that have anti-inflammatory properties, which may help reduce inflammation and pain associated with various diseases. A study published in the Journal of Medicinal Food found that bitter leaf extract reduced inflammation in rats with induced arthritis.

Anti-cancer: Bitter leaf contains compounds that have been shown to have anti-cancer properties, such as vernodalin and vernomygdin. A study published in the Journal of Ethnopharmacology found that bitter leaf extract had significant cytotoxic activity against human breast cancer cells.

While bitter leaf has many potential health benefits, it is important to note that it should be used in moderation, as excessive consumption can have side effects..

amygdalina is well known as a medicinal plant with several uses including for the treatment of diabetes, fever reduction, and recently for a non-pharmaceutical solution to persistent fever, headache, and joints pain associated with AIDS (an infusion of the plant is taken as needed).

Kingdom: Plantae

Clade: Tracheophytes

Order: Asterales

Family: Asteraceae

Subfamily: Asteroideae

Tribe: Heliantheae

Genus: Biden

Species: Biden pilosa

There are several subspecies and varieties of Biden pilosa, which have slight variations in their physical characteristics and geographic distribution.

Photos of BidensPilosa

Source:

Bidenspilosa has been found to contain various phytochemicals, including; Flavonoids: Several flavonoids have been identified in Bidenspilosa, such as quercetin, luteolin, and kaempferol. Flavonoids are known for their antioxidant properties and have been associated with various health benefits, Terpenoids: Bidenspilosa contains various terpenoids, including sesquiterpenes and diterpenes. These compounds have been reported to have antimalarial, anti-inflammatory, and analgesic properties. Polyacetylenes: Bidenspilosa is a rich source of polyacetylenes, which have been reported to have antimicrobial, anti-inflammatory, and anticancer properties, Phenolics: Bidenspilosa has been found to contain various phenolic compounds, such as caffeic acid, chlorogenic acid, and catechins. These compounds have been associated with antioxidant and anti-inflammatory properties, Carotenoids: Bidenspilosa contains carotenoids such as beta-carotene and lycopene, which are known for their antioxidant properties. Essential oils: Bidenspilosa also contains essential oils, which have been reported to have antimicrobial and anti-inflammatory properties (Desalegn, Ravikumar, & Murthy, 2021).

Bidenspilosa, also known as Black-jack, is believed to have originated in South America, specifically in the Amazon region. However, the plant has spread widely throughout tropical and subtropical regions of the world and is now found in many countries, including the United States, Mexico, India, China, and various countries in Africa.

Bidenspilosa is a fast-growing and highly adaptable plant that thrives in a variety of environments, including disturbed habitats, waste areas, and agricultural fields. It is considered an invasive weed in many regions due to its ability to outcompete native plant species and reduce biodiversity.

Most communities in developing countries rely heavily on traditional medicines, including herbal and medicinal flora and fauna, for their primary healthcare needs. The World Health Organization (WHO) estimates that over 80% of the population worldwide relies on traditional medicine primarily for their healthcare needs. In a number of developed countries, herbal medicine is gaining popularity as alternative and complementary therapies due to their affordability, efficacy, and occurrence of fewer side effects compared to conventional drugs. According to Ahuchaogu et al. , the potential use of herbal plants as sources of new therapeutic drugs remains largely unexplored.

Currently used anti-inflammatory medications are associated with disadvantages such as drug toxicity, adverse effects, and iatrogenic reactions, thereby complicating the treatment process. Dzoyem et al. further stated that both steroidal and nonsteroidal anti-inflammatory drugs used in the treatment of severe inflammation have not been thoroughly effective, since most of these drugs increase the risk of blood clots and may result in stroke and heart attack. Some of these synthetic molecules, such as anticytokine agents, block the activity of several kinases and result in severe impairment of the immunity of an individual against infections. The adverse effects related to the use of these conventional synthetic drugs have been the driving force behind consideration of natural remedies and the development of powerful anti-inflammatory drugs based on natural extracts. In this context, research should explore the properties of A. africana for the possible development of drugs, considering its long history of use as a traditional medicine for effective management of inflammatory diseases across Africa for centuries (Zhang, 2021).

2.3 Testing the crude extracts for anti-diabetic activity

Procedure to identify and authenticate plants for experiments

The procedure to identify and authenticate plants for experiments typically involves several steps, including; Taxonomic identification: The first step is to accurately identify the plant species. This can be done through taxonomic identification, which involves examining the morphology, anatomy, and other characteristics of the plant. This can be done using field guides, herbarium specimens, or expert consultation.

Taxonomic identification is the process of classifying and naming organisms based on their morphological, biochemical, and genetic characteristics. Taxonomy is the scientific discipline that deals with the classification, naming, and identification of living organisms.

The process of taxonomic identification typically involves several steps; Collecting the specimen: The first step is to collect the specimen for identification, which can be done through various methods such as capturing, sampling, or observing in their natural habitat. Observation of the morphological features: The next step is to observe the morphological features of the specimen, including its size, shape, color, and other distinguishing characteristics, Examination of biochemical and genetic features: In some cases, the biochemical and genetic features of the specimen may also be examined, which can provide additional information for identification. Comparison with existing taxonomic keys: The observed features are compared with existing taxonomic keys, which are reference guides used to identify organisms based on their characteristics.

Naming the organism: Once the specimen has been identified, it is given a scientific name according to the rules and guidelines of the International Code of Nomenclature for algae, fungi, and plants or the International Code of Zoological Nomenclature.

Voucher specimens: Once the plant species has been identified, a voucher specimen should be collected and preserved. A voucher specimen is a representative sample of the plant that can be used to verify its identity and authenticity. The voucher specimen should be properly labeled with information such as the plant species, location, date, and collector.

Authentication: The authenticity of the plant material should be verified using appropriate techniques, such as DNA barcoding, chemical analysis, or microscopy. This can help to ensure that the plant material is free from contaminants or adulterants.

Quality control: The quality of the plant material should also be assessed to ensure that it is suitable for use in experiments. This can include testing for contaminants, such as heavy metals or pesticides, and assessing the concentration of the active compounds.

Standardization: If the experiment involves using a plant extract, it is important to standardize the extract to ensure consistency in the concentration of the active compounds. This can be done through techniques such as high-performance liquid chromatography (HPLC).

Obtaining plant extracts from materials

Reagents and chemicals commonly used in making plant extracts

There are several chemicals that can be used in the extraction of anti-diabetic agents from plant materials. Some of the commonly used chemicals are:

Methanol: Methanol is another common solvent used for extraction. However, it is more toxic than ethanol and requires careful handling.

Acetone: Acetone is a polar solvent that can dissolve both polar and non-polar compounds. It is also a common solvent for extraction of anti-diabetic agents from plant materials.

There are several chemicals that can be used in the extraction of anti-diabetic agents from plant materials. Some of the commonly used chemicals are; Ethanol: Ethanol is a common solvent used for extraction of plant materials due to its ability to dissolve a wide range of compounds. It is often used in the form of 70% ethanol. Methanol: Methanol is another common solvent used for extraction. However, it is more toxic than ethanol and requires careful handling.

Chloroform: Chloroform is a more aggressive solvent that can extract compounds that are not soluble in ethanol or methanol. However, it is highly toxic and poses a health hazard. Acetone: Acetone is a polar solvent that can dissolve both polar and non-polar compounds. It is also a common solvent for extraction of anti-diabetic agents from plant materials.

Hexane: Hexane is a non-polar solvent that is useful for extracting non-polar compounds from plant materials. However, it is highly flammable and can pose a fire hazard. It is important to note that the choice of solvent depends on the type of compound being extracted and the properties of the plant material. Additionally, it is important to ensure that the solvent used is safe for human consumption and does not leave toxic residues in the final product.

Methods used to obtain plant extracts

There are several methods used to obtain plant extracts, including:

Soxhlet extraction: Soxhlet extraction is a popular method for extracting plant compounds. It involves placing the plant material in a thimble and placing it in a Soxhlet extractor. The solvent is continuously circulated through the thimble, which helps to extract the desired compounds. Soxhlet extraction is a technique used for the extraction of organic compounds from solid or semi-solid samples. The process involves repeatedly cycling a solvent through a sample that is contained within a thimble-shaped container, using a Soxhlet extractor apparatus.

The process typically involves the following steps:

The sample is placed into a thimble and inserted into the Soxhlet extractor apparatus.

A suitable solvent is added to the extraction flask at the bottom of the apparatus.

The solvent is heated, causing it to vaporize and rise up into the extraction chamber.

The solvent condenses on the condenser and drips down onto the sample, dissolving the organic compounds.

Once the solvent reaches a certain level in the extraction chamber, it is siphoned back into the flask by a siphon tube, thereby continuously cycling the solvent through the sample.

The process is repeated until the desired level of extraction is achieved.

Soxhlet extraction is a relatively time-consuming process, but it can be very effective at extracting compounds that are difficult to extract using other methods. It is commonly used in the food, pharmaceutical, and environmental industries for the analysis of pesticides, contaminants, and other organic compounds.

Maceration: Maceration involves soaking the plant material in a solvent for a period of time, typically a few hours or overnight. The mixture is then filtered to remove any solid material, and the resulting liquid is the plant extract.

Maceration is a process of extracting the soluble components of a plant material by soaking it in a liquid, usually alcohol or water, at room temperature for a period of time. The liquid used is called the menstruum, and the plant material is known as the marc.

The process of maceration involves the following steps; The plant material is chopped or ground to increase its surface area and facilitate extraction. The marc is placed into a container and covered with the menstruum. The mixture is left to stand at room temperature for a certain period of time, typically from several hours to several weeks, depending on the plant material and the desired outcome.

During the maceration process, the soluble components of the plant material dissolve into the liquid, resulting in an extract, After the desired extraction time has elapsed, the extract is filtered to remove the marc and other solid particles.

Maceration is a common method used in the preparation of herbal remedies, tinctures, and other plant-based products. It is a simple and gentle method of extraction that does not require heat or pressure, and can be used to extract a wide range of compounds from plant material. However, it may not be the most efficient method for extracting certain compounds and can be time-consuming, depending on the desired outcome.

Ultra sonication: Ultra sonication involves using high-frequency sound waves to extract plant compounds. The plant material is placed in a solvent and subjected to ultrasonic waves, which help to break down the cell walls and release the compounds. Ultra sonication, also known as sonication, is a technique that uses high-frequency sound waves to create cavitation, or the formation and implosion of small bubbles, in a liquid. This process can be used to disperse, mix, or extract materials in a variety of applications, including chemistry, biology, and materials science.

The process of ultra sonication involves the following steps; A liquid sample is placed into a container, such as a beaker or flask. A probe or horn, which emits high-frequency sound waves, is immersed into the liquid. The sound waves create cavitation bubbles in the liquid, which rapidly expand and collapse, generating high temperatures and pressures in the surrounding liquid.

The high-energy environment created by the cavitation can be used to break down or disperse materials in the liquid, such as cells or particles, or to enhance chemical reactions by increasing the speed and efficiency of mixing and diffusion. After the desired level of ultrasonication has been achieved, the liquid can be removed from the container and processed further as needed. Ultra sonication is a powerful and versatile technique that can be used for a wide range of applications, including sample preparation, emulsification, and nanoparticle synthesis. It is a non-invasive and relatively fast method that does not require high temperatures or harsh chemicals, making it a useful tool for many types of research and industrial processes. However, it may also have limitations in certain applications, such as in the extraction of certain types of molecules, or in cases where the acoustic energy may damage sensitive materials.

Steam distillation: Steam distillation is commonly used for extracting essential oils from plants. It involves passing steam through the plant material, which helps to extract the volatile oils. The resulting mixture of steam and oil is then condensed and separated.

Steam distillation is a process used for the separation of volatile compounds, such as essential oils, from plant materials. It involves passing steam through the plant material, which causes the volatile compounds to be released from the plant and carried along with the steam. The steam and volatile compounds are then condensed back into a liquid, allowing for the separation of the essential oil from the plant material.

The process of steam distillation involves the following steps: The plant material is placed into a distillation flask or chamber, and water is added to the chamber to create steam.

The steam is passed through the plant material, causing the volatile compounds to be released from the plant and carried along with the steam, The steam and volatile compounds are then passed through a condenser, which cools the steam and converts it back into a liquid.

The liquid is collected in a receiving flask, where the essential oil and water are separated due to differences in their densities. The essential oil is then separated from the water using a separatory funnel or other method. Steam distillation is commonly used in the extraction of essential oils from plant materials, such as lavender, peppermint, and eucalyptus. It is a gentle and relatively low-temperature method of extraction, which helps to preserve the quality and aroma of the essential oil. However, the process can be time-consuming and may not be suitable for all types of plant material.

Supercritical fluid extraction: Supercritical fluid extraction involves using a supercritical fluid, typically carbon dioxide, to extract plant compounds. The fluid is passed through the plant material at high pressure and temperature, which helps to extract the desired compounds.

Supercritical fluid extraction (SFE) is a method of extracting organic compounds from solid or liquid materials using a supercritical fluid as the extracting solvent. A supercritical fluid is a substance that has been heated and pressurized to a state where it exhibits properties of both a gas and a liquid, such as high diffusivity and low viscosity, making it an efficient solvent for extraction.

The process of supercritical fluid extraction involves the following steps; The sample material is placed into a sealed extraction vessel, along with a supercritical fluid, typically carbon dioxide (CO2). The vessel is heated and pressurized to a temperature and pressure that allows the CO2 to reach a supercritical state.

The supercritical CO2 penetrates the sample and dissolves the organic compounds, which are carried away in the CO2 stream. The CO2 and dissolved compounds are then separated, typically by depressurization, allowing the CO2 to return to its gaseous state and the extracted compounds to be collected. Supercritical fluid extraction has several advantages over other extraction methods, such as high selectivity, high efficiency, and minimal use of toxic solvents. It is commonly used in the food, pharmaceutical, and cosmetic industries for the extraction of natural compounds, such as flavors, fragrances, and active ingredients. However, it can be a costly method due to the high-pressure equipment required, and the selectivity of the process may limit its use for certain types of compounds.

The choice of method depends on the properties of the plant material and the desired compounds to be extracted. It is important to ensure that the method used does not denature or damage the desired compounds and that the resulting extract is safe for human consumption.

2.4 Methods of testing plant extracts for anti-diabetic activity

Invivo methods of testing plant extracts for anti-diabetic activity

There are several in vivo methods that can be used to test plant extracts for anti-diabetic activity. Here are some of the commonly used methods:

Indomethacin-induced diabetic model: Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that is known to induce diabetes in the animal. This model involves administering indomethacin to experimental animals and then testing the anti-diabetic activity of the plant extract by measuring the reduction in glucose levels of blood.

Testing for phyto-chemicals in plant extracts

There are several methods for testing for phytochemicals in plant extracts. Here are some commonly used methods:

Thin Layer Chromatography (TLC): This method is used to separate and identify the various phytochemicals present in the plant extract. The extract is spotted on a thin layer of silica gel or cellulose, and the plate is developed using a solvent system that separates the phytochemicals based on their polarity.

High Performance Liquid Chromatography (HPLC): This method is used to separate and quantify the different phytochemicals present in the plant extract. The extract is injected into an HPLC column, and the phytochemicals are separated based on their chemical properties.

Gas Chromatography-Mass Spectrometry (GC-MS): This method is used to identify the volatile compounds present in the plant extract. The extract is vaporized and separated on a gas chromatography column, and the individual compounds are identified using mass spectrometry.

Fourier Transform Infrared Spectroscopy (FTIR): This method is used to identify the functional groups present in the plant extract. The extract is analyzed using infrared radiation, and the absorption of the radiation is used to identify the various functional groups.

Nuclear Magnetic Resonance (NMR): This method is used to identify the structure of the phytochemicals present in the plant extract. The extract is analyzed using a powerful magnet, and the interaction of the magnetic field with the atoms in the extract is used to determine the structure of the phytochemicals.

The isolation and purification of anti-diabetic agents from plant extracts typically involves several steps. Here is a general outline of the process:

Extraction: The first step is to extract the anti-diabetic agents from the plant material using a suitable solvent. The extraction can be done using various methods such as maceration, reflux, or sonication.

Fractionation: The crude extract is then fractionated using different solvents of increasing polarity to separate the different components. The fractions can be collected and tested for anti-diabetic activity using in vitro or in vivo methods.

Fractionation of plant crude extracts for anti-diabetic agents involves separating the different components of the plant extract based on their physicochemical properties, such as polarity, molecular weight, and solubility. This process is typically carried out using various chromatographic techniques, such as column chromatography, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC).

The first step in fractionation is usually to partition the crude extract between two immiscible solvents with different polarities, such as water and an organic solvent like ethyl acetate or chloroform. The resulting fractions can then be further separated using chromatography.

Column chromatography involves passing the mixture through a column packed with a stationary phase, such as silica gel or Sephadex, which separates the components based on their physical and chemical properties. The eluent is collected in fractions, and each fraction is tested for anti-diabetic activity.

TLC is a simpler chromatographic technique that involves spotting the crude extract onto a thin layer of adsorbent material, such as silica gel or cellulose, and then allowing the sample to migrate up the plate using a solvent. The components of the mixture separate based on their affinity for the stationary and mobile phases, and the resulting spots can be visualized under UV light or by using staining reagents.

HPLC is a more advanced form of chromatography that uses high-pressure pumps to pass the sample through a column packed with a stationary phase at high speeds. The eluent is detected using a UV detector or mass spectrometry, and the resulting peaks can be analyzed to identify the components and their anti-diabetic activity.

Overall, fractionation of plant crude extracts is a useful technique for isolating and identifying anti-diabetic agents from natural sources, and it can lead to the development of new drugs with fewer side effects than synthetic alternatives.

Purification: The active fraction is then purified further using chromatography techniques such as column chromatography or preparative HPLC. The purified compounds are collected and tested for anti-diabetic activity.

Structural characterization: The purified compounds are then characterized using various spectroscopic techniques such as NMR, IR, and MS to determine their chemical structure.

Bioassay-guided fractionation: If the active compound is not known, a bioassay-guided fractionation approach can be used to isolate the active compound. This involves repeated fractionation and testing of the fractions until the active compound is isolated.

Scale-up: Once the active compound has been identified and characterized, it can be scaled up for further testing and potential use as an anti-diabetic agent

It is important to note that the isolation and purification of anti-diabetic agents from plant extracts can be a complex and time-consuming process, requiring specialized equipment and expertise.

Structural characterization of purified compounds

The structural characterization of purified compounds from plant extracts typically involves several techniques. Here are some commonly used methods:

Nuclear Magnetic Resonance (NMR) spectroscopy: This technique is used to determine the chemical structure of the compound by analyzing its nuclear magnetic properties. NMR spectroscopy provides information on the number and type of atoms in the compound, as well as the connectivity between them.

Infrared (IR) spectroscopy: This technique is used to analyze the functional groups present in the compound. IR spectroscopy provides information on the types of bonds present in the compound, such as C-H, C-O, and C=O bonds.

Infrared (IR) spectroscopy is a powerful analytical technique used to determine the functional groups present in a sample. It is based on the principles of the interaction of infrared radiation with the sample. Here is a general overview of how IR spectroscopy works:

The sample is exposed to infrared radiation: The first step in IR spectroscopy is to expose the sample to a range of infrared radiation. The range typically covers wavelengths of 4000 to 400 cm-1, which corresponds to frequencies of 2.5 x 10^14 to 2.5 x 10^12 Hz.

Absorption of energy is measured: As the infrared radiation passes through the sample, it is absorbed by the chemical bonds in the sample. Each functional group in the sample has a unique set of vibrational frequencies, and when it absorbs the energy from the infrared radiation, it undergoes a change in its vibrational energy. The amount of energy absorbed by each functional group is measured by the spectrometer.

Analysis of data: The data obtained from the IR spectrometer is then analyzed to determine the functional groups present in the sample. The spectrum obtained shows the absorbance of energy as a function of the wavelength of the radiation. By comparing the absorption peaks in the spectrum to known reference spectra, the functional groups present in the sample can be identified.

IR spectroscopy can be used to analyze a wide range of samples, including small organic molecules, polymers, and proteins. It is a non-destructive technique that does not require any chemical modification of the sample, making it a valuable tool for studying the chemical composition of samples.

Mass spectrometry (MS): This technique is used to determine the molecular weight and structure of the compound. MS provides information on the fragmentation pattern of the compound, which can be used to determine the chemical structure.

Mass spectrometry (MS) is a powerful analytical technique used to determine the molecular weight and structure of a compound. It is based on the principles of the interaction of molecules with electric and magnetic fields. Here is a general overview of how MS works:

Ionization: The first step in MS is to ionize the sample molecule. This is typically done by bombarding the sample with high-energy electrons, which knock off one or more electrons from the molecule, producing a positively charged ion.

Acceleration: The positively charged ions are then accelerated in an electric field, which separates them based on their mass-to-charge ratio (m/z). The acceleration is done by applying a potential difference between two electrodes, causing the ions to move towards the detector.

Separation: The separated ions are then passed through a magnetic field, which causes them to move in a circular path based on their mass-to-charge ratio. The more massive ions have a larger radius of curvature, while the less massive ions have a smaller radius of curvature.

Detection: The ions are then detected by a detector, which records the number of ions at each mass-to-charge ratio. This data is used to generate a mass spectrum, which shows the intensity of each ion as a function of its mass-to-charge ratio.

Analysis of data: The data obtained from the mass spectrometer is then analyzed to determine the molecular weight and structure of the compound. The mass spectrum provides information on the fragmentation pattern of the molecule, which can be used to determine the chemical structure.

MS can be used to analyze a wide range of compounds, including small organic molecules, peptides, and proteins. It is a powerful tool for determining the identity and purity of compounds, as well as for studying their fragmentation patterns and reactivity.

X-ray crystallography: This technique is used to determine the three-dimensional structure of the compound by analyzing the diffraction pattern of X-rays that are passed through a crystal of the compound.

X-ray crystallography is a powerful analytical technique used to determine the three-dimensional structure of molecules, including small organic molecules, proteins, and nucleic acids. It is based on the principles of the diffraction of X-rays by the atoms in a crystal lattice. Here is a general overview of how X-ray crystallography works:

Sample preparation: The first step in X-ray crystallography is to obtain a high-quality crystal of the molecule of interest. This involves dissolving the molecule in a suitable solvent and allowing it to form a crystalline structure. The crystal must be of sufficient size and quality to produce a clear diffraction pattern.

X-ray diffraction: The crystal is then exposed to a beam of X-rays, which diffract off the atoms in the crystal lattice. The diffracted X-rays produce a complex pattern of bright spots, known as diffraction spots or reflections, on a detector screen. The angle and intensity of each spot provide information on the position of the atoms in the crystal lattice.

Data collection: The diffraction pattern is recorded using a detector and the data is processed to determine the location of the atoms in the crystal lattice. This involves measuring the intensity and position of each diffraction spot and using complex mathematical algorithms to calculate the electron density map of the molecule.

Structure determination: The electron density map is used to determine the three-dimensional structure of the molecule. The positions of the atoms in the crystal lattice are refined and fitted to the electron density map, allowing the exact positions of the atoms to be determined. The resulting structure provides information on the shape, size, and orientation of the molecule, as well as the positions of any functional groups.

X-ray crystallography is a powerful tool for studying the structure and function of biomolecules. It can provide high-resolution information on the atomic-level details of proteins and other macromolecules, which is essential for understanding their biological function and for designing drugs to target specific molecular interactions.

UV-Vis spectroscopy: This technique is used to analyze the electronic properties of the compound, such as the presence of chromophores or conjugated systems.

Elemental analysis: This technique is used to determine the elemental composition of the compound, which can be used to confirm its molecular formula.

By using a combination of these techniques, researchers can determine the chemical structure of the purified compound and confirm its identity. This information is important for further testing of the compound’s biological activity and potential therapeutic applications.

HOW NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY WORKS

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique used to determine the structure of molecules. It is based on the principles of quantum mechanics and the interaction of certain atomic nuclei with magnetic fields. Here is a general overview of how NMR spectroscopy works:

The sample is placed in a strong magnetic field: The first step in NMR spectroscopy is to place the sample in a strong magnetic field, which aligns the atomic nuclei in the sample.

Radiofrequency radiation is applied: Radiofrequency radiation is then applied to the sample, which causes the atomic nuclei to absorb energy and move from their aligned positions.

Absorption of energy is measured: As the atomic nuclei move back to their aligned positions, they release the absorbed energy in the form of radio waves. These radio waves are detected and measured by the NMR spectrometer.

Analysis of data: The data obtained from the NMR spectrometer is then analyzed to determine the chemical structure of the sample. The chemical shifts of the different nuclei in the sample, which are affected by the local electronic and magnetic environment of the nucleus, provide information about the type and position of atoms in the molecule, as well as their connectivity.

NMR spectroscopy can be used to analyze a wide range of molecules, including small organic molecules, peptides, and proteins. It is a non-destructive technique that does not require any chemical modification of the sample, making it a valuable tool for studying the structure and dynamics of biomolecules.

CHAPTER THREE

MATERIAL AND METHODS

3.0 Introduction

This section presents the study in line with the materials and methods to be used in line with study objectives.

3.1 Identification and collection of medicinal plants with anti-diabetic activity.

The following methods will be used to identify plants with anti-diabetic agent and they include; Traditional knowledge: This involves relying on the knowledge and experience of indigenous or traditional communities who have been using the plant for medicinal purposes for generations. Botanical identification: This involves identifying the plant based on its physical characteristics, such as the shape and arrangement of leaves, flowers, stems, and roots. This can be done through careful observation, comparison with plant identification keys, or consultation with a botanical expert.

Chemical identification: This involves analyzing the chemical composition of the plant to identify its active compounds. This can be done using techniques such as chromatography or spectroscopy.

Pharmacological testing: This involves testing the plant’s medicinal properties through pharmacological assays. This can help to confirm its identity and validate its traditional use. It is important to note that identifying plants for herbal medicine can be a complex process, and it is recommended that it be done by trained professionals with extensive knowledge of botany and pharmacology. Additionally, proper identification is crucial to avoid the risk of using toxic or harmful plants in herbal preparations.

3.1.1 Procedure to identify and authenticate plants for experiments

3.2 Objective: Testing the crude extracts for anti-diabetic activity

To test crude extracts for anti-diabetic activity, the following steps will be taken:

3.2.1 Experimental Animals

Swiss albino mice (20-30 g) and Wistar rats (150–250 g) of either sex will be used in this experiment. The laboratory animals will be obtained from the Faculty of veterinary medicine Makerere university. Animals will be contained in standard cages at room temperature and 12 hours of light and dark cycles, and will be acclimatized for one week before the study to the laboratory conditions. The animals will be fed with a standard pellet and tap water ad libitum. The protocol for use of animals will be undertaken as per guidelines for use of laboratory animals (Polo, et al., 2012).

3.2.1 Preparation of Hydro-Methanolic crude Extract

The leaves of the selected medicinal plants are to be washed with distilled water to remove dirt and dust. The cleaned plant materials will be dried at room temperature. The plant materials and then grounded into coarse powder by the electrical mill. Then, the coarse powdered plant materials are to be macerated separately in hydro-methanol solvent for about 72 hours, then the plant materials are to be filtered via Whatman No. 1 filter paper and remacerated three times with fresh hydro-methanol solvent. The filtrates of each successive maceration will be concentrated by using a rotary evaporator (Yamato Rotary evaporator, Japan) adjusted to a temperature of 40°C. Finally, the semidried residues will be frozen in the deep freezer and dried using a lyophilizer (Labfreez, China) to entirely remove the remaining solvent (Fentahun et al., 2017).

3.2.2 Fractionation of Hydro-Methanol Extract

n-Hexane, chloroform, and water are to be used as solvents for fractionation. Briefly, distilled water will be added into the crude extract of selected medicinal plant and dissolved by using a separating funnel. Then, n-hexane will be added and shaken to the dissolved components. Similarly, on the aqueous layer, an equal volume of chloroform will be added to it, then in both cases, the two layers will be separated. The subsequent n-hexane and chloroform layers will be separated and exposed to evaporation by using a hot air oven (40°C). Then, the dried hydro-methanol extract and solvent fractions of the crude plant extracts are to be kept separately in a desiccator until used for the experiment (Emran et al, 2015).

3.2.3 Phytochemical Screening

Preliminary phytochemical screening tests will be carried out to determine the major classes of phytochemicals on the hydro-methanol extract of plants stem bark by using different standard test procedures.

3.2.4 Acute Toxicity Test

Acute toxicity study will be carried out using the guidelines described by the Organization for Economic Cooperation and Development (OECD) guideline No. 425. Single female Swiss albino mice fasted for four hours on the first day of the test then; 2000 mg/kg of the extract will be given by oral route using oral gavage and the mice will be observed for the manifestation of behavioral and physical changes and special attention will be given during the first four hours. Depend on the results from the first mice, the next 4 females’ animals will be employed and fasted for about four hours and then a single dose of 2000 mg/kg of the extract will be given orally and followed firmly in the same manner. The observation will be sustained daily for a total of fourteen days (Adane, Atnafie, Kifle, &Ambikar, 2021).

3.2.5. Grouping and Dosing of Animals

Inducing of experimental diabetes in rats.

Briefly, the rats are made to fast overnight and injected intravenously with 65 mg/kg of streptozotocin(STZ) in a citrate buffer (Sigma, S0130), 15 min then injected intraperitoneally with 150 mg/kg of nicotinamide (NA) (Sigma, N3376). After 48 h, diabetes tests are carried out to identify polydipsia, polyuria, and a measure of nonfasting plasma glucose levels is carried out. Animals that do not develop glucose levels greater than 250 mg/dl are rejected.

Acute Hypoglycemic Effect.

Hyperglycemic animals are to be divided into seven groups of six rats each. Group 1, is to be used as control group, is to be treated orally with 1.5 ml of a physiological NaCl solution.

Group 2, is for hyperglycemic control, and is treated with 1.5 ml of a physiological NaCl solution, and group3, the positive control, to be subjected to a standard oral hypoglycemic agent (glibenclamide, 5mg/kg body weight (bw).

Group 4 are to be given Cg (W 20 mg/kg andEW 30 mg/kg, respectively), group5 are to be treated with Hp (W36 mg/kg and EW 35 mg/kg, respectively), group6 to be given Nl (W 40mg/kg and EW 57 mg/kg, respectively),and group 7 Sa (W 62 mg/kg andEW 77mg/kg, respectively); all of these treatments were dissolved in1.5 ml of a physiological NaCl solution.

Maltose and Sucrose Tolerance Tests.

Hyperglycemic animals are to be given seven groups containing four rats each.

All groups are to be given a 3 g/kg maltose solution, then 10 minutes after administered with the control extracts. Group 1, which is a control, is treated with 2.0ml of physiological NaCl solution, group 2, the hyperglycemic control, are treated with 2.0 ml of physiological

NaCl solution, group 3, as positive control, is to be given standard oral hypoglycemic agent acarbose (3 mg/kg), group 4 rats are to be given Cg (W 20 mg/kg).

Group 5 are treated with Hp (W 36mg/kg), then group 6 are to be given Nl (W 40 mg/kg), and group 7 to be given Sa (W 62 mg/kg); and in all sodium chloride solution was used to dissolve the treatment compounds. Blood samples are to be obtained from the tail veins and thigh after treatment and glucose levels monitored and analyzed with glucose test strips and a glucometer (Accutrend_ Plus).

2.5. Crude Small Intestine Extract.

The small intestines from rats are dissected and washed thourougly with an ice cold saline solution (9% NaCl) followed by a 0.1 M potassium phosphate buffer (pH 7) then with EDTA. The mucosa from cleaned small intestines is to be removed and homogenized in a potassium phosphate buffer, then centrifuged at 21,000 x g for an hour. The precipitate formed is to be re-suspended and then incubated for 45 minutes in a 0.1 M potassium phosphate buffer (pH7) containing 1%Triton X-100 and then centrifuged at 100,000 x g for 120 minutes. The supernatant is then treated with a 0.01Mpotassium phosphate buffer (pH7.0) for 48 hrs and stored at -20∘C.

2.6. In Vitro Glucosidase Assay.

The activity is subjected to a controlled amount of p-nitrophenol released from p-nitrophenyl-alpha- D-glucopyranoside. The assay containing a 0.1 M sodium phosphate buffer (pH 6.8) and p-4-nitrophenol glucopyranoside (p-NPGP) and 0.1m alpha-glucosidase from the crude extract from the small intestine and experimental extracts are to be added at different concentrations ranging from 0.2 𝜇g/ml to 20,000 𝜇g/ml in a 1 ml volume. The experiment is left for 10minutes and readings taken at 15 sec intervals at the 405 nm wavelength with a Beckman Coulter spectrophotometer (model DU-640).

2.7. Statistical Methods.

The data is to be analyzed using an ANOVA test, followed by Fisher’s post hoc test using the software Statistician. The plasma glucose levels are then expressed as the mean (SEM); significance considered at least with p< 0.05.

3.3 Isolation and purification of the bio-active compounds in the extracts

Analytical HPLC

Procedure

Analytical HPLC was employed for method development for small scale separation and purity

assessment of isolated compounds. It was conducted using a Varian ProStar HPLC system (Varian Inc., Walnut Creek, CA, USA) comprising a 210 Binary pump, 410 Auto Sampler and 335 DAD (Diode Array Detector) monitoring 190-400 nm. A Luna C18 reverse phased column (5μm, 250 x 4.6 mm) served as the stationary phase. The mobile phase comprised of methanol and water containing 0.05% TFA (trifluoroacetic acid). Column temperature was set at 30°C and the solvent flow rate was maintained at 1 mL/min. The Star Work Station Sofware υ 6.41 was used to control the auto-sampler, gradient settings, DAD and data acquisition. The purity of each fraction was determined based on the sharpness of the peak and the detected UV profile (PDA) using Poly View 2000-Diode Array Spectral Processing software. A 10 mg/mL solution was prepared by dissolving 10 mg of each crude methanolic extract in 1 mL of methanol (HPLC grade). Vortexing for 5 min and sonication for further 5 min were applied to dissolve the extracts.

HPLC

Procedure

Semi-preparative HPLC will be used to isolate and purify the compounds from the crude extract of the plant. It will be performed using a Luna C-18 column (5 μm) reversed-phase (150 x 21.2

mm) as the stationary phase and a gradient of 10% methanol in water with 0.05% TFA to 90%

methanol in water with 0.05% TFA) as the mobile phase. The absorbance will be monitored at 210 and 280 nm. The analytical method will be converted to semi-preparative HPLC method by adjusting the flow rate and sample load using the following formula: F2 = F1 x (L2/L1) x (r2/r1) where F2 = semipreparative HPLC solvent flow rate, F1 = analytical HPLC solvent flow rate, r2 = diameter of the semi-preparative column, r1 = diameter of the analytical column, L2 = semi-preparative column length and L1 = analytical column length. Purity of the isolated compounds from semi-preparative HPLC runs will be confirmed by analytical HPLC analysis.

3.3 Structure elucidation of the bioactive compound in the extract with anti-diabetics properties

Spectroscopic methods

For structural characterization of the isolated compounds, principally NMR and mass spectrometry will be performed. UV and IR spectroscopy along with melting point determination, and optical rotation analysis will also be carried out when required for physical characterization and structural elucidation of isolated compounds.

UV-visible spectroscopy

UV spectroscopy will be used for the preliminary identification of compounds, particularly for the

identification of the presence of phenolic groups or conjugated double bonds. Compounds initial

UV profiles will be obtained on the ananlytical HPLC using PDA detection and these profiles were to be

confirmed with a Shimadzu BioSpec-mini (A115247) UV-visible spectrophotometer (Shimadzu

Corporation, Japan). Molar extinction co-efficients for each compound will be calculated using the

below formula:

IR spectroscopy

IR spectroscopy will be further used to confirm any functional groups present in the isolated compounds. IR spectra are to be recorded with a Bruker Optics ALPHA QuickSnapTM (A220/D-01)

FT-IR spectrophotometer with OPUS spectroscopy software. A solid sample of each compound cast onto the diamond ATR-crystal plate and are to be scanned from 4000 to 375 cm-1 with 64-100 scans for analysis. Resulting peaks were compared to be published data of functional groups.

NMR spectroscopy

The structures of the isolated compounds will be elucidated primarily by 1D (homonuclear) and 2D (heteronuclear) NMR spectroscopy. 1D NMR experiments including 1H and 13C NMR were to be used to locate atom positions and fragment units. Further, 2D NMR experiments including COSY, HSQC, and HMBC to be carried out on more complex molecules for accurate assignments of proton and carbon chemical shifts. NMR spectra were to be recorded on a Bruker Avance 300 MHz (300 and 75 MHz respectively) or a Bruker Avance 600 MHz (600 and 150 MHz, respectively) spectrometer was coupled with Topspin 2.1 acquisition software. Samples will be dissolved in 500-600 μL of suitable deuterated solvent (Subsection 2.2.2.2). NMR experiments on samples with very little mass will be carried out using Shigemi NMR tubes (100-200 μL sample) (Sigma-Aldrich Ltd.). Signals will be recorded in chemical shifts (δ) and expressed in parts per million (ppm), with coupling constants (J) calculated in Hertz (Hz).

1H NMR

In the present study, 1H NMR data will be recorded for all isolated compounds and will be used as a primary source of structural information. The chemical shifts and integration indicated the number and type of protons present in each molecule, whereas, the multiplicity and coupling constants indicated the adjacent protons and their spatial arrangements. The purity of the compounds will also be determined from 1H NMR spectra.

13C NMR

13C (Jmod) spectral data will recorded for all isolated compounds, to determine the number and types of carbons present in each molecule.

Distortionless Enhancement by Polarization Transfer (DEPT) will be performed on selected

compounds. The DEPT pulse sequence experiment performs CH signal multiplicity and spin-spin coupling information into a phase relationship. In the DEPT spectrum, CH3 and CH signals are directed towards the positive phase of the spectrum and CH2 signals to the negative phase of the spectrum. The number of quaternary carbons present in the molecule will determined by comparison of the 13C (Jmod) with the relevant DEPT spectrum.

Correlation spectroscopy (COSY)

COSY spectral data will be recorded for all compounds. Cross-peaks were generated from the 1H-1H nuclei that share a mutual scalar coupling and normally evidenced for a germinal (2J) and vicinal (3J) couplings connectivity. This information provided the 1H-1H connectivity for the analysed compound.

Heteronuclear single quantum coherence (HSQC)

2D HSQC experiments were performed on all compounds. These experiments will be used to identify one-bond (1J) 1H – 13C connectivities. Cross peaks will be generated from proton-carbon nuclei that are directly connected to each other through a single bond.

Heteronuclear multiple bond coherence (HMBC)

2D HMBC spectral data was recorded for isolated compounds, to detect long range 2 to 3 bond 1H – 13C couplings. Certain 4-bond cross-peaks were also observed in the HMBC experiments as a weaker intensity signal than that for the 2 and 3 bond cross-peaks. This experiment provided important structural information which helped to elucidate selected complex structures. It also provided information regarding connectivity between structural fragments.

Mass spectrometry

Positive- and negative-ion electrospray mass spectra (ESI-MS) and atomic pressure chemical

ionization mass spectra (APCI-MS) will be obtained on a Bruker Daltonics esquire Series 3000 mass spectrometer (LR-MS) (Bruker Daltonic GmbH, Bremen, Germany) with esquire Control software using a cone voltage of 4000 V with the source maintained at 250°C. For ESI-MS and APCI-MS spectra, samples were dissolved (0.4 mg/mL) in methanol:water (1:1) and injected at a flow rate of 0.04 mL/min. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Bruker micrOTOF-Q 70 mass spectrometer, fitted with an Bruker Compass Data Analysis, at the University of Queensland.

Optical rotation

Compounds will be dissolved in methanol and the optical rotation of each compound measured at 25°C in a JASCO automatic polarimeter P-1010 series (JASCO Corporation, Tokyo, Japan) at the sodium wavelength, 589 nm, using Spectra Manager software. The solvent without sample will be used as a blank and the sample cell (50 mm) was subsequently dried prior to taking the sample reading. The specific rotation [α] of a compound in degree at a specific temperature (T) and a wavelength (λ) determined by the following formula:

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