Anti-ulcer agents are medications that are used to treat and prevent ulcers, which are sores that form on the lining of the stomach or upper part of the small intestine. There are several types of anti-ulcer agents, including proton pump inhibitors (PPIs), H2 receptor blockers, and mucosal protective agents. PPIs, such as omeprazole and lansoprazole, work by reducing the amount of acid produced in the stomach. H2 receptor blockers, such as ranitidine and famotidine, block the action of histamine, a substance that stimulates acid production. Mucosal protective agents, such as sucralfate, form a protective barrier on the lining of the stomach to prevent acid and enzymes from damaging the tissue (Sun et al, 2018). 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

Peptic ulcer disease (PUD), a common disorder of the digestive system, is defined as digestive tract injury that results in a mucosal break greater than 3–5 mm (Fang et al., 2019), with a visible depth reaching the submucosa (Xie, et al., 2022), Mainly occurring in the stomach and proximal duodenum (Fang, 2019).

Some of the common symptoms of peptic ulcers include; Abdominal pain: This is the most common symptom of peptic ulcers. The pain is usually felt in the upper abdomen, and it can be either sharp or dull. The pain may be relieved by eating, but it may come back again a few hours later, Indigestion: This is a feeling of discomfort or fullness in the upper abdomen, which may be accompanied by bloating, belching, or nausea, Heartburn: This is a burning sensation in the chest or throat, which may be accompanied by a sour taste in the mouth. Loss of appetite: This is a common symptom of peptic ulcers, which may be due to the pain or discomfort associated with eating, Weight loss: This may occur if the patient avoids eating due to the pain or discomfort associated with peptic ulcers, Nausea and vomiting: These symptoms may occur in some cases, especially if the ulcer is located in the stomach and Black or tarry stools: This may indicate bleeding in the stomach or small intestine, which requires immediate medical attention (Stechmiller et al., 2019).

The pathogenesis of peptic ulcer disease involves a complex imbalance between gastric offensive mucosal factors like prostaglandins (PG’s), gastric mucus, cellular renewal, blood flow, mucosal cell shedding, glycoproteins, mucin secretion, proliferation, and antioxidant enzymes like catalase (CAT), scavenger, and Helicobacter pylori (H.pylori), as well as defensive mucosal factors like (Wattanaphraya et al., 2021), The location and severity of the disease can be used to classify peptic ulcers. The development of peptic ulcers is also influenced by a number of additional factors, such as tumor necrosis factor, reactive oxygen species (ROS), histamine release, the incidence of apoptosis, and the secretion of bile acids (Gupta et al., 2021).

Peptic ulcers are a common condition worldwide, with an estimated 10% of the global population affected at some point in their lives (Keikha, Ali-Hassanzadeh, & Karbalaei, 2020), The prevalence of peptic ulcers varies depending on the region and population studied, In general, peptic ulcers are more common in developing countries, particularly in areas with low socioeconomic status, poor sanitation, and high rates of Helicobacter pylori infection (Le et al., 2022).

Some studies suggest that the prevalence of peptic ulcers in African countries like Ghana is relatively high, with one study reporting an overall prevalence of 19.5% among patients undergoing upper gastrointestinal endoscopy. However, the prevalence may vary depending on the population studied, and more research is needed to determine the true prevalence of peptic ulcers in Ghana (Sung et al., 2009), The prevalence of peptic ulcers in Nigeria is not well documented, and the available data is limited. However, peptic ulcers are a common gastrointestinal disorder worldwide and are estimated to affect about 4% of the population in developing countries like Nigeria (Zibima et al., 2020), A study conducted in 2018 in Nigeria reported a prevalence of 16.7% of peptic ulcer disease in patients attending a tertiary hospital in Northern Nigeria. Another study conducted in the same year in a different region of Nigeria reported a prevalence of 5.5% (Kayode et al., 2019).

The prevalence of peptic ulcers in Uganda is not well established due to limited research studies in the country (Milivojevic, & Milosavljevic, 2020), However, according to a study conducted in Kampala, the capital city of Uganda, the prevalence of Helicobacter pylori infection, which is a common cause of peptic ulcers, was found to be 78.6% among dyspeptic patients (patients with indigestion, stomach pain, or discomfort). The study also found that 50.6% of the dyspeptic patients had peptic ulcers (Milivojevic, & Milosavljevic, 2020).

Another study conducted in the southwestern region of Uganda among patients with upper gastrointestinal symptoms found that 54% of the patients had H. pylori infection, and 32% had peptic ulcers, these studies suggest that peptic ulcers are a common health problem in Uganda, particularly among dyspeptic patients and those with upper gastrointestinal symptoms (Tarasconi et al., 2020).

The treatment of peptic ulcers depends on the underlying cause, which can be attributed to infection with the bacterium Helicobacter pylori, long-term use of non-steroidal anti-inflammatory drugs (NSAIDs), excessive alcohol consumption or stress, the following are some common treatment options for peptic ulcers; Antibiotics: If the ulcer is caused by H. pylori, a course of antibiotics is prescribed to kill the bacteria. Usually, two or more antibiotics are used in combination with a proton pump inhibitor (PPI) and/or bismuth subsalicylate. This is known as triple therapy or quadruple therapy (Bereda, 2022)..

Proton pump inhibitors (PPIs): These drugs reduce the amount of acid produced in the stomach and help to relieve pain and promote healing of the ulcer. PPIs are commonly used in combination with antibiotics to treat H. pylori infection, H2 blockers: These drugs also reduce the amount of acid produced in the stomach and help to relieve pain and promote healing of the ulcer, Antacids: These medications neutralize stomach acid and can provide temporary relief of symptoms. They are often used in combination with other medications for more effective treatment and Lifestyle changes: Making lifestyle changes such as reducing stress, avoiding smoking and excessive alcohol consumption, and eating a healthy diet can help to reduce the risk of developing peptic ulcers and promote healing of existing ulcers (Kowada, & Asaka, 2022).

The treatment of peptic ulcer disease typically involves a combination of medication to reduce stomach acid production and antibiotics to eliminate H. pylori infection (if present) (Chan, 2021), In some cases, surgery may be necessary Lanas, &kei Chan, (2017), It is important to note that peptic ulcer disease can lead to serious complications, such as bleeding, perforation, and obstruction of the digestive system, which require urgent medical attention (Guo et al., 2021).

The acceptance and use of herbal medicine is on the increase globally. In Africa the situation is not different, over 80 % of the population particularly in the developing countries depends directly on plants for their primary healthcare requirements (Russell et al., 2020), In the East African region countries such as Burundi and Tanzania that neighbor Uganda, the population using traditional medicine is also well above 80 % particularly in the rural areas. Plants form an important part of health care especially for the rural poor in Uganda (Kaggwa et al., 2022), The Ugandan government has specifically up scaled the use of herbal medicine and is in the process of integrating it into the main health care system. The noted increased use of herbal medicine is as a result of the confirmed therapeutic evidence of the herbal remedies (Mao et al., 2019), This has been enhanced by the consequences of limited access to modern health services in most developing countries including Uganda, high cost of modern medicine compared to the indigenous herbal medicines, wide socio-cultural acceptance of traditional medicine and the belief that natural products pose no risk (Ssenku et al., 2022).

It’s important to note that while these plants have shown anti-ulcer properties in studies, they have not been specifically proved scientifically, Aloe vera has been traditionally used as a natural remedy for various digestive issues, including ulcers. Some studies have shown that the gel from the aloevera plant can help to soothe and heal damaged digestive tissue, including ulcers. Additionally, aloevera has anti-inflammatory and antioxidant properties that can help to reduce inflammation and oxidative stress in the digestive tract, which can contribute to the development of ulcers, It’s important to note that while aloevera has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects (Dinat, Orchard, & Van Vuuren, 2022),Also, consuming aloevera orally can have negative side effects, such as abdominal cramps, diarrhea and electrolyte imbalances (Teshome et al., 2019).

Because of their cost, accessibility, desire for individualized health care, and fear of the negative effects associated with synthetic drugs, natural goods have remained the drugs of choice for some people due to their safety and efficacy. Also, usage increases as new infectious diseases emerge and conventional medications fail to treat diseases like cancer (Akinwumi, & Sonibare, 2019). In affluent nations, 80% of people utilize traditional remedies made from plants, whereas more than 30% of pharmaceuticals now have some connection to plants, either directly or indirectly. Out of the 252 drugs on the World Health Organization’s (WHO) list of essential medicines, 11% are entirely plant-based, and 122 plant-derived drugs have 80% of their uses related to their original ethnopharmacological purposes. It is estimated that 25% of all drugs prescribed globally are derived from plants (Kuna, et al., 2019).

1.2 Statement of the problem

Peptic ulcers are a type of sore that forms in the lining of the stomach, duodenum (the first part of the small intestine), or esophagus (Bereda, 2022). They are commonly caused by a bacterial infection with Helicobacter pylori or prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) (Dore, & Pes, 2021). According to the World Gastroenterology Organization, peptic ulcers are a common medical condition, affecting about 10% of the world’s population (Maniewska, & Jeżewska, 2021). The global burden of peptic ulcers is significant, with an estimated 4 million new cases diagnosed annually (Georges, et al., 2020). Peptic ulcers can have a significant impact on a person’s quality of life, causing symptoms such as abdominal pain, bloating, nausea, and vomiting. In severe cases, complications such as bleeding or perforation of the stomach or duodenal wall can occur, which can be life-threatening. Peptic ulcers also have a significant economic burden, for both people living in developed and developing countries, with the cost of treatment and lost productivity from time off work (Petta et al., 2019). In the United States alone, the annual cost of peptic ulcer disease is estimated to be over $10 billion. Approximately 3000 deaths per year in the United States are due to duodenal ulcer and 3000 to gastric ulcer (Askin, & Moore, 2022).

Studies suggest that the incidence of peptic ulcers is increasing in Uganda, likely due to the increased use of NSAIDs and a high prevalence of H. pylori infection, one study conducted in 2015 among patients attending a tertiary hospital in Uganda found that 26.5% of the participants had peptic ulcers (Angol et al., 2017), Another study conducted in 2016 among patients attending a different tertiary hospital in Uganda found that 8.8% of the participants had peptic ulcers (Nekaka, et al., 2021).The prevalence of H. pylori infection is high in Uganda, with estimates ranging from 50% to 80% in different regions of the country. This high prevalence of H. pylori infection is likely a significant contributing factor to the high incidence of peptic ulcers in Uganda, Overall, while there is limited data on the prevalence of peptic ulcers in Uganda, the available studies suggest that the condition is a significant health concern in the country (Maniragaba, & Atukuuma, 2022).

The use of synthetic drugs to treat peptic ulcers can be associated with several dangers, including:

Side effects: Synthetic drugs used to treat peptic ulcers, such as proton pump inhibitors (PPIs) and H2 blockers, can cause a range of side effects, including nausea, diarrhea, constipation, headache, and dizziness (Nautiyal et al., 2023). Long-term use of these drugs can also increase the risk of bone fractures, kidney disease, and infections. Drug interactions: Synthetic drugs used to treat peptic ulcers can interact with other medications, leading to adverse effects. For example, PPIs can interact with blood thinners, anti-seizure medications, and antidepressants, among others. Drug resistance: Overuse of synthetic drugs can lead to the development of drug-resistant strains of bacteria, including H. pylori. This can make it more challenging to treat peptic ulcers in the future. Masking of underlying conditions: Synthetic drugs can mask underlying conditions that may be causing peptic ulcers, such as H. pylori infection or gastroesophageal reflux disease (GERD). This can delay diagnosis and appropriate treatment. Reduced effectiveness over time: Synthetic drugs used to treat peptic ulcers may become less effective over time, as the body adapts to their effects. This can lead to the need for higher doses or different medications to achieve the same level of symptom relief (Huang, et al., 2021). Preventive measures, such as avoiding the use of NSAIDs and reducing the risk of H. pylori infection through improved sanitation and hygiene, can help to reduce the global burden of peptic ulcers (Schneider et al., 2021).

Herbal medicine can be important in treating peptic ulcers for several reasons, including; Fewer side effects: Herbal medicines are often considered to have fewer side effects than synthetic drugs used to treat peptic ulcers (Kuna et al., 2019). This is because they are derived from natural sources and are often used in their whole form, which contains a range of beneficial compounds that can work synergistically to provide therapeutic effects. Increased bioavailability: Herbal medicines can often be more bioavailable than synthetic drugs, meaning that they are more easily absorbed and utilized by the body. This can increase their effectiveness in treating peptic ulcers. Anti-inflammatory effects: Many herbal medicines have potent anti-inflammatory effects, which can be beneficial in treating peptic ulcers. Inflammation can exacerbate peptic ulcer symptoms, and reducing inflammation can help to alleviate these symptoms and promote healing. Antibacterial effects: Some herbal medicines have antibacterial effects, which can help to eliminate H. pylori infections that can cause peptic ulcers. This can help to prevent the recurrence of peptic ulcers and reduce the need for synthetic antibiotics. Cost-effective: Herbal medicines can be a cost-effective alternative to synthetic drugs, which can be expensive and often require ongoing treatment to maintain their effectiveness. Integrative approach: Herbal medicines can be used as part of an integrative approach to treating peptic ulcers, which may include dietary and lifestyle changes, stress reduction, and other complementary therapies. This can help to address the underlying causes of peptic ulcers and promote overall health and wellbeing. In summary, herbal medicine can be an important and effective tool in treating peptic ulcers, offering a range of benefits that can help to alleviate symptoms and promote healing (Bi, & Man, 2014).

It is therefore against this Background that this study intends to isolate and identify anti-ulcer agents from the extracts of selected medicinal plants.

1.3 Purpose of the study

The purpose of the study is isolation and identification of anti-ulcer agents from the extracts of selected medicinal plants

1.4 Objectives

Identify and collect medicinal plants with anti-ulcer agents used in treatment of peptic ulcers.
Testing the crude extracts for anti-ulcer activity

Isolation and purification of the Bio-active compounds in the extracts

Structure elucidation of the Bioactive compound in the extract with anti-ulcer properties

1.5 Research Questions

What are medicinal plants with anti-ulcer agents used in treatment of peptic ulcers?
What are the crude extracts for anti-ulcer activity?

Isolation and purification of the Bio-active compounds in the extracts

Structure elucidation of the Bioactive compound in the extract with anti-ulcer properties

1.6 Concept of the study

This section includes content scope, time scope and Geographical scope

1.6.1 Time scope

The study will be carried out for the period of 3 years.

1.6.2 Content Scope

The contents of the study will include; to identify and collect medicinal plants with anti-ulcer agents used in treatment of peptic ulcers, Testing the crude extracts for anti-ulcer activity, Isolation and purification of the Bio-active compounds in the extracts and Structure elucidation of the Bioactive compound in the extract with anti-ulcer properties.

CHAPTER TWO

LITERATURE REVIEW

2.0 Literature Review

This section presents the isolation and identification of anti-ulcer agents from the extracts of selected medicinal plants in line to the study objectives;

2.1.1 Prevalence

The lifetime risk for developing a peptic ulcer is approximately 10% (Hein et al, 2017), Globally, as of 2010, approximately 250,000 people died of peptic ulcer disease down from 320,000 in 1990 (Rickard, 2022)., while by 2020 Peptic ulcer disease (PUD) affected more than four million people worldwide annually and has an estimated lifetime prevalence of 5−10% in the general population (Kang et al, 2011) In Western countries the prevalence of Helicobacter pylori infections roughly matches age (i.e., 20% at age 20, 30% at age 30, 80% at age 80) (Kamada et al., 2021), Prevalence is higher in third world countries where it is estimated at about 70% of the population, whereas developed countries show a maximum of 40% ratio, PUD deaths are extremely high in these countries; Cambodia, 20.80%, Kiribati, 20.71%, Lesotho, 18.88%, Laos, 16.83% and Central African Republic 15.30% (Slemrod, 2019).

Global burden and demographic profiles of PUD is approximately 8.09 million (95% UI 6.79 to 9.58 million) prevalent cases of PUD in 2019 (Azhari et al., 2018), which represents an increase of 25.82% from 1990 [6.43 million (95% UI 5.41 to 7.63 million) (Lanas, & Chan, 2017), Moreover, the age-standardized prevalence rate in 2019 was 99.40 per 100,000 (95% UI 83.86 to 117.55 per 100,000) population, which represented a decrease from 1990 [143.37 per 100,000 (95% UI 120.54 to 170.25 per 100,000) (Kavitt, et al., 2019).

Between 1990 and 2019, the number of incident cases of PUD increased from 2.82 million (95% UI 2.36 to 3.30 million) to more than 3.59 million (95% UI 3.03 to 4.22), representing an increase of 27.3% in the global incident cases of PUD (Ren et al., 2022), However, the global age-standardized incidence rate of PUD showed a decreasing trend, at 63.84 (95% UI 54.09 to 75.54) per 100,000 populations in 1990 and 44.26 (95% UI 37.32 to 51.87) per 100,000 populations in 2019 (Azhari et al., 2022).

Infections with H. pylori are declining overall, especially in wealthy nations. Peptic ulcer disease is spread through food, contaminated groundwater, and human saliva (through kissing or sharing eating utensils) (Wu et al., 2021), Until the final few decades of the 20th century, when epidemiological patterns started to hint to an astonishing decline in its incidence (Botija, 2021), The identification of the condition’s source, H. pylori, and the introduction of new, efficient medications and acid suppressants are credited with the decline in peptic ulcer disease rates (Gralnek et al., 2021), Over 4 million Americans currently have active peptic ulcers, and 350,000 new cases are identified yearly (Xie et al., 2022).

Peptic ulcers are a common condition worldwide, including in Germany. According to the German Society for Digestive and Metabolic Diseases (DGVS), approximately 5% to 10% of the German population will develop a peptic ulcer at some point in their lives (Loffroy et al., 2021), The prevalence of peptic ulcers in Germany has decreased significantly over the past few decades, thanks in part to improved diagnosis and treatment (Ciubotaru, & Leferman, 2021), This decrease is primarily due to a reduction in Helicobacter pylori infection rates, which is the most common cause of peptic ulcers (Botija et al., 2021), the use of proton pump inhibitors (PPIs) has also played a role in reducing the incidence of peptic ulcers. A 2014 study published in the journal Digestion reported a prevalence of 0.75% for peptic ulcers among 33,514 participants in Germany (Okoye, 2021), the study found that peptic ulcers were more common in older individuals and in those with a history of smoking, heavy alcohol consumption, or regular use of NSAIDs (Azhari et al., 2018).

The literature on the epidemiology of PUD shows that PUD remains a relatively common condition worldwide, with annual incidence ranging from 0.10% to 0.19% for physician-diagnosed PUD and from 0.03% to 0.17% for PUD diagnosed during hospitalization. The 1-year prevalence of physician diagnosed PUD was 0.12–1.5%, and the 1-year prevalence of PUD diagnosed during hospitalizations was 0.10–0.19% (Salari et al., 2022).

Peptic ulcer disease (PUD) is a common condition worldwide. According to the World Gastroenterology Organization, the global prevalence of peptic ulcer disease is estimated to be around 10% to 15% of the population (Kamada et al., 2021), However, the prevalence of peptic ulcer disease varies by region and country, with higher rates reported in developing countries (Ren et al., 2022). The risk factors for peptic ulcer disease include infection with Helicobacter pylori (H. pylori), long-term use of non-steroidal anti-inflammatory drugs (NSAIDs), smoking, alcohol consumption, and stress, the symptoms of peptic ulcer disease include abdominal pain, bloating, nausea, vomiting, and loss of appetite (Azhari et al., 2022).

The overall prevalence of PUD observed in this study was 4.1%; 19.5% of all PUD cases identified were asymptomatic (Bruce, Onyemailu, & Orji, 2021), Comparing this prevalence with the lower rates obtained from other studies of physician-diagnosed PUD in primary care suggests that a proportion of individuals with PUD remain undiagnosed (Dadfar, & Edna, 2020), In individuals with asymptomatic PUD, severe complications, such as gastrointestinal hemorrhage, may be the first signs of the disease (Malfertheiner & Schulz, 2020), Hemorrhage is associated with mortality approaching 10% and high recurrence, this is particularly relevant for elderly patients who are less likely to have symptoms, possibly because of the analgesic effects of ASA and NSAIDs (Salari et al., 2022).

Peptic ulcers are open sores that develop on the inside lining of the stomach (gastric ulcers) or the upper part of the small intestine (duodenal ulcers) (Kumar et al., 2020).). They can be caused by a number of factors, including infection with the bacterium Helicobacter pylori, prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs), and smoking (Ren et al., 2022).

Overall, while the available data on peptic ulcer prevalence in Nigeria is limited, it suggests that peptic ulcers are a significant health problem in the country, and further research is needed to determine their true prevalence and to develop effective prevention and treatment strategies, In Ibadan, Southern Nigeria, DU incidence was 2.3% relative to GU 9.3%, while Northern Nigeria had higher DU incidence 6.8% but lower GU incidence 2.7%. On the contrary, two separate tertiary health centres in Kenya, Nakuru and Nairobi, had similar DU incidence but different GU incidence (Nairobi: GU 8.5%, DU 9.8%; Nakuru: GU 1.9% DU 9.5% (Akinwumi, & Sonibare, 2019).

A decrease in hospital admission from PUD was caused by the availability of efficient medical treatment to eradicate H. pylori as well as trends in the use of over-the-counter H2 antagonists, proton pump inhibitors, and improved cleanliness (Teshome et al., 2019), The blood group O, smoking, and non-steroidal anti-inflammatory medicines are additional risk factors for PUD (Wu et al., 2020), Epigastric pain associated with meals, gastroesophageal reflux, dyspepsia, and melena may be clinical signs of peptic ulcer. Perforation, gastrointestinal obstruction, and malignant transformation are other consequences in addition to gastrointestinal bleeding (Sisay Zewdu, & Jemere Aragaw, 2020).

Peptic ulcer disease (PUD) affects four million people worldwide annually, and has an estimated lifetime prevalence of 5−10% in the general population (Malfertheiner, & Schulz, 2020), Although the global prevalence of PUD has dramatically decreased in the past decades the incidence of its complications has remained constant (Dadfar, & Edna, 2020).

Worldwide, there are significant heterogeneities in coping approaches of healthcare systems with PUD in terms of prevention, diagnosis, treatment, and follow-up (Byun et al., 2020), Prevention is positively correlated with the development of infrastructures and the effectiveness of healthcare systems, the choice of diagnostic test and treatment approaches mainly relies on accessibility and cost, (Sonnenberg, & Genta, 2020), Therefore, quantifying and benchmarking health systems’ performance is crucial yet challenging to provide a clearer picture of the potential global inequities in the quality of care.

2.1.2 Peptic ulcers disease

PUD accounts for an estimated lifetime prevalence of 5–10% and an annual incidence of 0.1–0.3% in the general population in Western countries. Due to nonspecific symptoms, PUD assessment and treatment requires clinical caution due to severe complications such as bleeding, perforation, penetration into adjacent organs and gastrointestinal obstruction, all of which could require acute endoscopic or surgical treatment (Bereda, 2022).

Peptic ulcers are discontinuities of the gastric or duodenal mucosa with penetration to the muscularis mucosae and exposure of the submucosa. Primitive ulcers are caused by alterations of the gastric function (i.e., increased HCl production and pepsin function); they are mainly single lesions and are usually found at the small gastric curve and at the antrum. Secondary ulcers, on the contrary, are caused by extragastric pathogenic events, that is, stress or drugs. They can be multiple and can have a spread localization within the stomach. More than 20% of patients have a family history of duodenal ulcers. In up to one third of patients with duodenal ulcers, basal acid output (BAO) and maximal acid output are increased. A study by Schubert and Peura attested that individuals are at especially high risk those with a basal acid production (BAP) greater than 15 mEq/h. In addition to the increased gastric and duodenal acidity observed in some patients with duodenal ulcers, accelerated gastric emptying is also often present. A common cause of peptic ulcers in the pediatric age is Helicobacter pylori (HP) infection, though the use of nonsteroidal anti-inflammatory agents (NSAIDs), like aspirin and ibuprofen, represents a significant cause of ulcers as well. Other treatments comprehending steroids and antineoplastic and immunosuppressive drugs can be causative of peptic ulcers. A reduction of the protective effect of prostaglandins on gastric mucosa is considered to be the main pathogenic mechanism. Other stressful events (i.e., shock, sepsis, burnings, major trauma, intracranial hypertension, surgical procedures, and chronic diseases) can provoke acute gastric ulcers, also in the pediatric age [8]. Lesions generally appear 3–6 days after the event and the main related symptoms are bleeding and

Peptic ulcers are open sores that develop on the lining of the stomach, small intestine, or esophagus, and can cause a range of symptoms. As a result of some medications such non-steroidal anti-inflammatory drugs (NSAIDS), gastric acids, and pepsin, peptic ulcers are described as a break in the continuity of the mucosa of the stomach or duodenum that eventually results in lesions in the intestinal mucosa (Verma et al., 2010). In essence, the word “peptic” comes from the Greek word “peptikos,” which has a meaning connected to digestion. According to several findings, patients in the older age groups are more likely to develop stomach ulcers. Duodenal ulcer risk is higher in younger people ( Pahwa et al., 2011).

Similar to several digestive disorders, the prevalence of PUD initially increased and then subsequently decreased, PUD epidemiological data spanning 150 years and found that the incidence of and mortality due to PUD increased markedly during the nineteenth century and then decreased steadily due to improvements in environmental hygiene and medical therapeutic strategies. During the first 50 years of the twentieth century in the United States, PUD affected approximately 10% of the adult population (Kowada, & Asaka, 2022).

Gastric colonization with Helicobacter pylori (HP) causes peptic ulcer (PU) in about 10% – 15.0%, gastric adenocarcinoma in about 1% – 30%, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma in less than 1% of cases. Helicobacter pylori-linked virulence elements, the host’s genetic and immunological parameters, and environmental parameters execute critical roles in the development of HP-induced gastrointestinal diseases (Romstad et al., 2022), The cytotoxin-associated gene A (cagA) is known as the strongest virulence agent of HP, and CagA⁺ bacteria lead to more serious consequences such as PU and malignancy. Diverse kinds of immune cells, including neutrophils, eosinophils, dendritic cells (DCs), natural killer cells, and T and B lymphocytes accumulate into the gastric mucosa during HP infection (El-Dakroury et al., 2022).

Several studies which were conducted in the past 20–30 years indicated a sharp decreasing tendency in the PUD prevalence, PUD-related hospital admissions and PUD-associated mortality due to new anti-PUD therapies application, such as Helicobacter pylori (H. pylori) eradication and proton-pump inhibitors (PPIs) using. However, the widespread use of nonsteroidal anti-inflammatory drugs (NSAIDs), histamine receptor antagonists, and selective serotonin reuptake inhibitors, as well as increased physiological stress, have been reported as risk factors and have changed the landscape of PUD in recent years. The details of the epidemiological changes caused by these relatively new risk factors are still controversial (Wattanaphraya et al., 2021).

2.1.3 Medicinal plants with anti-ulcer properties

All around the world, traditional green vegetables are an excellent and affordable source of nourishment for a balanced diet. These vegetables also function as traditional medicines for ailments like toothache (Amaranthus viridis L.), acute abdominal pain (Celosia argentia L.), painful urination (Portulacaoleracea L.), headache (Smithia sensitiva Ait. ), diarrhea (C. mimosoides L.), rheumatism and cough (Marsileaminuta Linn), and helminthes infestation (Spinaciaoleracea Linn.) (Kuna et al., 2019).

Traditional vegetables in Uganda are plant species that are either native to the country or were introduced there in the past, are now being cultivated, and whose leaves are added as a sauce to the basic dishes. All of the country’s geographical regions support a variety of species (Crawford, 2019), However, the extent of their production and consumption varies according to the local behaviors, beliefs, and staple foods of the populace as well as soil and climate types. While some of these traditional vegetables are grown and harvested as wild or semi wild vegetation, others have been domesticated (Mintah et al., 2019).

The vegetables contain vitamins (A, B, and C) and proteins and minerals such as iron, calcium, phosphorus, iodine, and fluorine in varying amounts but adequate for normal growth and health. According to the FAO Food Balance Sheet for Uganda, traditional food plants supply about 90% energy, 76% protein and 63% fat, and most of vitamins A and C, iron, and dietary fiber (Shedoeva,, Leavesley, Upton, & Fan, 2019), These food values are vital necessities for normal growth and defense against protein/calorie malnutrition in humans. Traditional vegetables ensure a well-balanced diet in rural areas. In some cases, parts of traditional vegetable species serve as staple foods such as the mature fruits of C. maxima and the tubers of C. benghalensis, Ipomoea spp., M. esculenta, and S. edule (Zougagh et al., 2019).

Licorice (Glycyrrhiza glabra) is a plant that has been used in traditional medicine for centuries. Its root is the part that is commonly used for medicinal purposes. In traditional medicine, licorice is used for a variety of digestive issues, including ulcers. Studies have shown that licorice has anti-ulcer properties due to its ability to increase the production of mucus in the digestive tract, which can help to protect the lining of the stomach and intestines from damage. Additionally, licorice has anti-inflammatory and antioxidant properties that can help to reduce inflammation and oxidative stress in the digestive tract, which can contribute to the development of ulcers (Tabuti, 2018).

It’s important to note that while licorice has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Consuming large amounts of licorice can also have negative side effects, such as high blood pressure, water retention and potassium depletion.

Licorice (Glycyrrhiza glabra) root has been traditionally used for its anti-ulcer properties. Some studies have shown that compounds in licorice root can help to soothe and heal damaged digestive tissue, including ulcers (Musoke, 2019), Additionally, licorice has anti-inflammatory and antioxidant properties that can help to reduce inflammation and oxidative stress in the digestive tract, which can contribute to the development of ulcers (Nakaziba et al., 2021).

The main active component in licorice root responsible for its anti-ulcer properties is glycyrrhizin. Glycyrrhizin has been shown to increase the production of mucus in the digestive tract, which can help to protect the lining of the stomach and intestines from damage and reduce the risk of ulcers (Kuna et al., 2019).

It’s important to note that while licorice has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Additionally, consuming large amounts of licorice or licorice supplements can have negative side effects, such as high blood pressure, water retention and potassium depletion (Wang et al., 2019).

Ginger (Zingiber officinale) is a well-known spice that has been used in traditional medicine for centuries. Its root is the part that is commonly used for medicinal purposes. In traditional medicine, ginger is used for a variety of digestive issues, including ulcers (Shahrajabian ,& Cheng, 2019), Studies have shown that ginger has anti-ulcer properties due to its ability to reduce inflammation in the digestive tract and protect the lining of the stomach from damage. Additionally, ginger has antioxidant properties that can help to reduce oxidative stress in the digestive tract, which can contribute to the development of ulcers (Sistani et al., 2019).

It’s important to note that while ginger has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Additionally, consuming large amounts of ginger or ginger supplements can have negative side effects, such as gastrointestinal distress, heartburn, and increased bleeding risk (Balogun, deyeOluwa, & Ashafa, 2019).

Ginger (Zingiber officinale) is a well-known spice that has been traditionally used for its anti-ulcer properties. Some studies have shown that ginger can help to soothe and heal damaged digestive tissue, including ulcers. Additionally, ginger has anti-inflammatory properties that can help to reduce inflammation in the digestive tract and protect the lining of the stomach from damage, ginger also has antioxidant properties that can help to reduce oxidative stress in the digestive tract, which can contribute to the development of ulcers (Balogun, AdeyeOluwa, & Ashafa, 2019).

The anti-ulcer effects of ginger are believed to be due to the presence of compounds such as gingerols and shogaols, which have been shown to have anti-inflammatory and antioxidant properties. It’s important to note that while ginger has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Additionally, consuming large amounts of ginger or ginger supplements can have negative side effects, such as gastrointestinal distress, heartburn, and increased bleeding risk (Imo, & Za’aku, 2019).

Marshmallow (Althaea officinalis) is a plant that has been used in traditional medicine for centuries. Its root and leaves are the parts that are commonly used for medicinal purposes. In traditional medicine, marshmallow is used for a variety of digestive issues, including ulcers. Studies have shown that marshmallow has anti-ulcer properties due to its ability to soothe and protect the lining of the digestive tract, including the stomach and intestines (Aafreen et al., 2019), Marshmallow contains mucilage, a type of soluble fiber that can form a protective gel-like layer over the digestive tract, helping to reduce irritation and prevent further damage (Alsherbiny et al., 2019).

It’s important to note that while marshmallow has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Additionally, consuming large amounts of marshmallow or marshmallow supplements can have negative side effects, such as gastrointestinal distress (Jacob, 2020), As always, it’s best to consult with a healthcare professional before using marshmallow for any health purposes. Chamomile (Matricaria chamomilla) is a plant that has been used in traditional medicine for centuries, particularly for its calming and soothing properties. The flowers of the chamomile plant are the part that is commonly used for medicinal purposes. In traditional medicine, chamomile is used for a variety of digestive issues, including ulcers (Shin et al., 2020).).

Studies have shown that chamomile has anti-ulcer properties due to its ability to reduce inflammation in the digestive tract and protect the lining of the stomach and intestines. Chamomile contains compounds, such as apigenin and chamazulene, that have been shown to have anti-inflammatory and antioxidant properties (Phumthum,& Balslev, 2020).

It’s important to note that while chamomile has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects. Additionally, consuming large amounts of chamomile or chamomile supplements can have negative side effects, such as allergic reactions in some individuals. As always, it’s best to consult with a healthcare professional before using chamomile for any health purposes.

Slippery elm (Ulmus rubra) is a tree native to North America that has been used in traditional medicine for centuries. Its inner bark is the part that is commonly used for medicinal purposes. In traditional medicine, slippery elm is used for a variety of digestive issues, including ulcers. Studies have shown that slippery elm has anti-ulcer properties due to its ability to soothe and protect the lining of the digestive tract, including the stomach and intestines. Slippery elm contains mucilage, a type of soluble fiber that can form a protective gel-like layer over the digestive tract, helping to reduce irritation and prevent further damage (Phumthum, M., & Balslev, 2020).

Additionally, slippery elm has been shown to have anti-inflammatory and antioxidant properties, which can help to reduce inflammation in the digestive tract and protect against oxidative stress, which can contribute to the development of ulcers. It’s important to note that while slippery elm has shown promising results in treating ulcers in laboratory and animal studies, more research is needed to determine its efficacy in human subjects (Shahrajabian et al., 2020).).

Callistemon citrinus (Curtis) Skeels, also known as Lemon Bottlebrush, is a plant species that belongs to the family Myrtaceae. While there are some studies on the potential health benefits of Callistemon citrinus, there is currently limited scientific evidence to support its use for the treatment or prevention of ulcers (Daharia et al., 2022), That being said, some studies have suggested that Callistemon citrinus may have anti-inflammatory and antioxidant properties, which could potentially be beneficial for reducing inflammation and oxidative stress in the digestive tract. These effects may be helpful in preventing the development of ulcers or promoting their healing (Tanveer et al., 2020).

The acceptance and use of herbal medicine is on the increase globally, In Africa the situation is not different, over 80 % of the population particularly in the developing countries depends directly on plants for their primary healthcare requirements (Bhargava et al., 2020), In the East African region countries such as Burundi and Tanzania that neighbor Uganda, the population using traditional medicine is also well above 80 % particularly in the rural areas. Plants form an important part of health care especially for the rural poor in Uganda. The Ugandan government has specifically up scaled the use of herbal medicine and is in the process of integrating it into the main health care system (Daharia et al., 2022), The noted increased use of herbal medicine is as a result of the confirmed therapeutic evidence of the herbal remedies. This has been enhanced by the consequences of limited access to modern health services in most developing countries including Uganda, high cost of modern medicine compared to the indigenous herbal medicines, wide socio-cultural acceptance of traditional medicine and the belief that natural products pose no risk (Mohammed et al., 2020).

The increased preference of herbal medicine has consequently propelled the search for pharmaceutical remedies against different ailments from plants. The medicines are collected from the wild and this has negatively impacted on the plant resource due to unsustainable exploitation rates as well as the health of many people who cannot afford orthodox medicine. This makes documentation, sustainable utilization as well as conservation essential (Bischoff-Kont, et al., 2021), The first step in conservation is to document material traditionally used to treat an ailment. Previous studies have identified and documented numerous medicinal plants for treatment of various diseases in Uganda however these have been targeting specific ailments and are not detailed in shared use (Fakhri et al., 2021), A larger number of medicinal plants and indigenous uses have not yet been documented. The rich history of African cultures and their innovative utilization of plants as a source of remedies have been passed down through generations largely by oral tradition (Patra, et al., 2021), This knowledge is gradually being lost as the custodians die before passing on information to the younger generations. Besides the gradual loss of ethnobotanical knowledge due to lack of documentation, overharvesting of medicinal materials from their natural habitat has been one of the major threats of traditional medicine (Zougagh et al., 2019).

Callistemon citrinus (Curtis) skeels; Callistemon citrinus, also known as the lemon bottlebrush or crimson bottlebrush, is a species of flowering plant in the myrtle family Myrtaceae. It is native to Australia and is widely cultivated as an ornamental plant in many countries. The species was first described by Curtis in 1775, and the authority for its name is Curtis, followed by Skeels in 1913. The name Callistemon citrinus refers to the lemon-scented foliage and the bright red, bottlebrush-like flowers that it produces (Imade, et al., 2022).

There is limited scientific research on the anti-ulcer properties of Callistemon citrinus (Curtis) Skeels. However, some studies have reported that the plant and its extracts have anti-inflammatory and antioxidant effects, which could potentially have a beneficial effect on the prevention and treatment of ulcers.

For example, a study published in the Journal of Ethnopharmacology found that an ethanol extract of Callistemon citrinus showed potent antioxidant activity and exhibited anti-inflammatory effects in vitro. Another study published in the same journal found that a methanol extract of the plant had a protective effect against experimentally induced ulcers in rats.

It is important to note that these studies were conducted in laboratory settings and further research is needed to establish the anti-ulcer properties of Callistemon citrinus in humans. It is not recommended to self-medicate with this plant or its extracts as they may have potential side effects and interactions with other medications. If you are experiencing symptoms of an ulcer, it is best to consult with a healthcare professional for proper diagnosis and treatment.

Callistemon citrinus, commonly known as the lemon bottlebrush, has been studied for its potential health benefits, including its anti-ulcer properties.

Studies have shown that the extract of Callistemon citrinus has anti-ulcer activity, which means it may help to protect the digestive tract from developing ulcers. This is thought to be due to the presence of certain compounds in the plant, such as flavonoids and tannins, which have been shown to have anti-inflammatory and antioxidant properties.

However, it’s important to note that these studies have primarily been conducted in animals, and more research is needed to determine the effectiveness and safety of using Callistemon citrinus as an anti-ulcer treatment in humans. It is also important to remember that self-treating with the plant extract without the supervision of a healthcare provider can be dangerous and could interact with any medications you may be taking.

Callistemon citrinus (Curtis) Skeels ,

Luganda name : Mwambala butonya

Botanical name: Callistemon citrinus

Here is the classification of Callistemon citrinus:

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Eudicots

Clade: Rosids

Order: Myrtales

Family: Myrtaceae

Genus: Callistemon

Species: Callistemon citrinus

Several phytochemicals have been identified in the leaf extract of Callistemon citrinus. Here are some of the major ones; Flavonoids: The leaf extract of Callistemon citrinus is rich in flavonoids, including quercetin, kaempferol, and their glycosides. Flavonoids have been reported to have antioxidant, anti-inflammatory, and anticancer properties, Terpenoids: The leaf extract of Callistemon citrinus contains various terpenoids, including triterpenes and sesquiterpenes. Terpenoids have been reported to possess antimicrobial, antifungal, and anti-inflammatory properties. Phenolic acids: The leaf extract of Callistemon citrinus contains phenolic acids such as gallic acid and caffeic acid. Phenolic acids have been reported to have antioxidant, anti-inflammatory, and anticancer properties. Alkaloids: Some alkaloids have been identified in the leaf extract of Callistemon citrinus. Alkaloids have been reported to have analgesic, anti-inflammatory, and antispasmodic properties.

Photo of Callistemon citrinus

Source: Internet

Callistemon citrinus, commonly known as Lemon Bottlebrush, is native to Australia but has been introduced and naturalized in many other parts of the world. It is a hardy plant that can grow in a wide range of conditions, from hot and dry to cool and wet climates.

In Australia, it is commonly found in the eastern coastal regions, from Queensland to Victoria, growing in a variety of habitats, including forests, woodlands, heathlands, and along watercourses. It is often grown as an ornamental plant in gardens and parks and is widely cultivated for its attractive flowers.

Outside of Australia, Callistemon citrinus is commonly grown in Mediterranean and subtropical climates, including regions of South America, South Africa, and the Mediterranean. It is also grown in parts of Asia, including India and Malaysia.

Callistemon citrinus, commonly known as Lemon Bottlebrush, is not native to Uganda but is grown as an ornamental plant in some areas of the country. It is commonly found in gardens, parks, and along roadsides in urban areas.

Since Callistemon citrinus is not native to Uganda, it may not be widely distributed in the country, and its presence may be limited to areas where it has been specifically planted or cultivated for decorative purposes.

The plant typically grows up to 6 meters tall and has dark green, lance-shaped leaves that are about 10 centimeters long. The crimson bottlebrush is named for its distinctive, bottlebrush-like flowers that are bright red and bloom in late spring and early summer. The flowers are made up of numerous stamens, giving them a feathery appearance (Laganà et al., 2020).

The plant is popular in landscaping due to its attractive foliage and showy flowers. It prefers well-drained soil and full sun exposure, and is relatively easy to care for. In addition to its ornamental value, the crimson bottlebrush is also used in traditional medicine, with its leaves and flowers having been used to treat a variety of ailments including coughs, colds, and sore throats (El-Dakroury et al., 2022).

Callistemon citrinus, commonly known as Lemon Bottlebrush, is a popular ornamental plant due to its bright red flowers and lemon-scented leaves. While it is not widely used for medicinal purposes, some traditional medicine practitioners have used it for various ailments.

Here are some of the reported medicinal uses of Callistemon citrinus:

Antiseptic: The leaves and flowers of Callistemon citrinus are believed to possess antiseptic properties, which can be useful in treating cuts, wounds, and other skin infections.

Digestive aid: In some traditional medicine systems, a tea made from the leaves and flowers of Callistemon citrinus is believed to aid in digestion and alleviate stomach problems such as indigestion and flatulence.

Respiratory ailments: The leaves of Callistemon citrinus are sometimes used in tea form to treat respiratory ailments such as coughs, colds, and bronchitis.

Anti-inflammatory: Some studies suggest that Callistemon citrinus may have anti-inflammatory properties, which could make it useful in treating conditions such as arthritis and other inflammatory conditions.

English name: leonotis leonurus

Luganda name: Ekifumufumu

Here is the classification of Leonotis leonurus:

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Eudicots

Clade: Asterids

Order: Lamiales

Family: Lamiaceae

Genus: Leonotis

Species: Leonotis leonurus

So, Leonotis leonurus belongs to the kingdom Plantae, the order Lamiales, the family Lamiaceae, and the genus Leonotis. Its specific name is leonurus.

Photo of leonotis leonurus

Source: Internet

The leaf extract of Leonotis leonurus contains several bioactive compounds, including phytochemicals such as; Alkaloids: Leonurine, a pyridine alkaloid, has been identified in the leaf extract of Leonotis leonurus. It has been reported to have various pharmacological properties, including antioxidant, anti-inflammatory, and cardiovascular effects. Flavonoids: The plant contains several flavonoids, including quercetin, rutin, and kaempferol. Flavonoids have been reported to have antioxidant, anti-inflammatory, and anticancer properties.

Terpenes: The plant contains various terpenes, including triterpenes and diterpenes. Terpenes have been reported to have various biological activities, including antioxidant, antimicrobial, and anticancer effects. Essential oils: The leaf extract of Leonotis leonurus contains essential oils that are rich in terpenes such as β-caryophyllene, α-pinene, and limonene. These compounds are responsible for the plant’s characteristic aroma and have been reported to have various pharmacological properties, including anti-inflammatory and analgesic effects.

These phytochemicals may contribute to the potential health benefits of Leonotis leonurus, although more research is needed to understand their specific effects and mechanisms of action. It is important to note that while the plant has been traditionally used for medicinal purposes, more research is needed to understand its safety, efficacy, and potential side effects. It is always recommended to consult a healthcare professional before using this plant for medicinal purposes.

Leonotis leonurus, also known as lion’s tail or wild dagga, is a plant species that is native to southern Africa. It is a member of the mint family (Lamiaceae) and is closely related to other well-known herbs such as sage, rosemary, and thyme. Traditionally, various parts of the plant have been used for medicinal and cultural purposes. The leaves, flowers, and roots contain a range of bioactive compounds, including alkaloids, flavonoids, terpenes, and essential oils.

Some of the reported traditional uses of Leonotis leonurus include: Sedative and anxiolytic: The plant has been used to induce calmness and relaxation and to alleviate anxiety.Analgesic: Leonotis leonurus has been used traditionally to alleviate pain and inflammation, including headaches, menstrual cramps, and rheumatism. Anti-inflammatory: The plant has been used to alleviate inflammation, particularly in the respiratory system, such as bronchitis and asthma.

Psychoactive effects: The plant has been reported to have mild psychoactive effects, such as euphoria, relaxation, and visual changes, While Leonotis leonurus has been traditionally used for medicinal purposes, more research is needed to understand its safety, efficacy, and potential side effects. It is important to consult a healthcare professional before using this plant for medicinal purposes.

Leonotis leonurus, also known as lion’s tail or wild dagga, has been traditionally used for various medicinal purposes. Some of the reported medicinal uses of Leonotis leonurus include:

Sedative and anxiolytic: The plant has been used to induce calmness and relaxation and to alleviate anxiety, Analgesic: Leonotis leonurus has been used traditionally to alleviate pain and inflammation, including headaches, menstrual cramps, and rheumatism, Anti-inflammatory: The plant has been used to alleviate inflammation, particularly in the respiratory system, such as bronchitis and asthma, Antimicrobial: The plant has been reported to have antimicrobial properties and has been used to treat various infections. Gastrointestinal disorders: Leonotis leonurus has been used to alleviate gastrointestinal disorders, including indigestion, bloating, and diarrhea, Mild psychoactive effects: The plant has been reported to have mild psychoactive effects, such as euphoria, relaxation, and visual changes, While Leonotis leonurus has been traditionally used for medicinal purposes, more research is needed to understand its safety, efficacy, and potential side effects. It is important to consult a healthcare professional before using this plant for medicinal purposes. Additionally, it is important to note that the psychoactive effects of the plant may be undesirable for some individuals, and caution should be exercised when using it for any purpose.

leonurus has many reputed traditional medicinal applications and is mainly taken orally or per rectum and as a topical application. Hottentots were particularly fond of smoking it instead of tobacco and used a decoction of the leaf as a strong purgative and as an emmenagogue. Early colonialists employed it in the treatment of leprosy. The leaf tea has a hypnotic effect, is diuretic and relieves headache. The leaf and stem decoction or inhalations have been used internally for cough, common cold, influenza, bronchitis, wound healing and asthma. The fresh stem juice is an infusion drunk for ‘blood impurity’. The infusions made from flowers and seeds, leaves or stems are widely used as tonics for tuberculosis, jaundice, muscular cramps, high blood pressure, diabetes, viral hepatitis, dysentery, and diarrhoea . Tea made from the whole plant is used for arthritis, piles, bladder and kidney disorder, obesity, cancer and rheumatism. The leaves and stems decoction are applied topically as a treatment for eczema, skin infections and itchiness. The leaves, roots and bark are widely used as an emetic for snakebites, bee and scorpion stings. The L. Leonurus smoke has marijuana-like effects. -pungent odour and is occasionally mixed with flowers and fruits. In ethnoveterinary the roots and leaves water drink is used in poultry, against cattle gall sickness and eye inflammation. Generally, the plant is a general tonic, having reputed dermatological, hypertension, antiinflammatory, pain and wound healing properties.

English name: Bothriocline longipes

Luganda Name: Etwatwa

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Asterales

Family: Asteraceae

Genus: Bothriocline

Species: longipes

Photo

Source:

Bothriocline longipes is a species of tree in the family Asteraceae that is native to South Africa. It is commonly known as the “Mountain Cat’s Paw” due to the shape of its leaves, which resemble a cat’s paw. This tree is typically found in the mountains and hillsides of the Eastern Cape and KwaZulu-Natal provinces of South Africa, where it grows in grassland and savanna habitats. It is adapted to survive in harsh, dry conditions, and can grow in soils with low nutrient content.

In addition to its distinctive leaves, Bothriocline longipes is also known for its showy yellow flowers, which bloom in late winter and early spring. The flowers are pollinated by insects, and produce seeds that are dispersed by wind.

Bothriocline longipes is an important tree in traditional medicine in South Africa, where it is used to treat a variety of ailments. It is also valued for its wood, which is used for fuel and to make furniture and implements.

The leaf extracts of Bothriocline longipes have been reported to contain several classes of phytochemicals, including; Flavonoids: Bothriocline longipes leaves have been found to contain various flavonoids such as quercetin, luteolin, and kaempferol. Flavonoids are known for their antioxidant properties and have been associated with various health benefits. Alkaloids: Bothriocline longipes leaves have been found to contain alkaloids such as nitriles and pyridines. Alkaloids have been shown to have various pharmacological activities, including antimicrobial, anti-inflammatory, and antitumor effects. Tannins: Bothriocline longipes leaves have been found to contain tannins, which have astringent properties and can bind to proteins, amino acids, and alkaloids. Tannins have been associated with various health benefits, including antioxidant and anti-inflammatory effects. Terpenoids: Bothriocline longipes leaves have been found to contain terpenoids such as triterpenes and steroids. Terpenoids have been shown to have various pharmacological activities, including antimicrobial, antitumor, and anti-inflammatory effects. Saponins: Bothriocline longipes leaves have been found to contain saponins, which are known for their foaming properties and have been associated with various health benefits, including antioxidant and anti-inflammatory effects. These phytochemicals are believed to contribute to the medicinal properties of Bothriocline longipes and have been reported to exhibit various pharmacological activities, including antimicrobial, antioxidant, and anti-inflammatory effects. However, further research is needed to fully understand the therapeutic potential of Bothriocline longipes leaf extracts.

Bothriocline longipes, also known as the Mountain Cat’s Paw, has been used in traditional medicine in South Africa for many years. It has been reported to have various medicinal properties, including: Antimicrobial properties: Bothriocline longipes has been traditionally used to treat infections and has been reported to exhibit antimicrobial activity against various bacteria and fungi, Anti-inflammatory properties: Bothriocline longipes has been traditionally used to treat inflammation and has been reported to exhibit anti-inflammatory activity, Antioxidant properties: Bothriocline longipes has been reported to contain various phytochemicals with antioxidant properties that may protect against oxidative stress and related diseases. Wound healing properties: Bothriocline longipes has been traditionally used to treat wounds and has been reported to exhibit wound healing properties. Respiratory disorders: Bothriocline longipes has been traditionally used to treat respiratory disorders such as coughs, bronchitis, and asthma. Digestive disorders: Bothriocline longipes has been traditionally used to treat digestive disorders such as diarrhea and dysentery.Pain relief: Bothriocline longipes has been traditionally used to relieve pain and has been reported to exhibit analgesic activity. However, more research is needed to fully understand the medicinal properties of Bothriocline longipes and to determine its effectiveness and safety for various medical conditions. It is always recommended to consult with a healthcare professional before using any herbal remedies for medicinal purposes.

Botanical name : Biden pilosa

Luganda Name : sere

Biden pilosa is a species of flowering plant in the family Asteraceae. It is also commonly known as “Spanish needle” or “shepherd’s needle.” The plant is native to South and Central America but has been introduced to many other parts of the world, where it is considered an invasive weed.

Here is the taxonomic classification of Biden pilosa:

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Eudicots

Clade: Asterids

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 Bidens Pilosa

Source:

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

Bidens pilosa, 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.

Bidens pilosa 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.

Local Name: Ekajjolyenju

Botanical Name: Dracaena

Classification

Kingdom: Plantae

Clade: Tracheophytes

Division: Magnoliophyta

Class: Liliopsida

Order: Asparagales

Family: Asparagaceae

subfamily: Nolinoideae

Genus: Dracaena

Photo of Dracaena

Phytochemicals of Dracaena

Dracaena is a genus of plants that includes approximately 120 species of trees and succulent shrubs. Some of the commonly known species of Dracaena include Dracaena marginata, Dracaena fragrans, and Dracaena reflexa. Dracaena plants are known for their ornamental value, but they also contain phytochemicals that have potential health benefits. Here are some of the phytochemicals that have been identified in Dracaena plants:

Flavonoids: Flavonoids are a group of phytochemicals that have antioxidant and anti-inflammatory properties. Some of the flavonoids that have been identified in Dracaena plants include quercetin, kaempferol, and rutin.

Alkaloids: Alkaloids are a group of nitrogen-containing compounds that have a variety of biological activities. Some of the alkaloids that have been identified in Dracaena plants include dracorhodin, dracocephalumine, and draconine. Saponins: Saponins are a group of phytochemicals that have anti-inflammatory and anti-cancer properties. Some of the saponins that have been identified in Dracaena plants include dracaenin and dracogenin, Tannins: Tannins are a group of polyphenolic compounds that have astringent properties. Some of the tannins that have been identified in Dracaena plants include catechins and epicatechins, Terpenoids: Terpenoids are a large group of phytochemicals that have a variety of biological activities. Some of the terpenoids that have been identified in Dracaena plants include beta-sitosterol, camphor, and limonene. These phytochemicals have been studied for their potential health benefits, including anti-inflammatory, antioxidant, and anti-cancer effects. However, more research is needed to fully understand the potential health benefits of Dracaena plants and their phytochemicals.

While Dracaena plants are mainly grown for ornamental purposes, some of their phytochemicals have been studied for their potential health benefits. Here are some of the diseases and conditions that Dracaena phytochemicals have been studied for:

Inflammation: Dracaena phytochemicals, including flavonoids and saponins, have been shown to have anti-inflammatory properties. They may help to reduce inflammation and related symptoms in conditions such as rheumatoid arthritis, inflammatory bowel disease, and asthma.

Cancer: Some Dracaena phytochemicals, such as saponins and alkaloids, have been studied for their potential anti-cancer effects. They may help to prevent the growth and spread of cancer cells, and may enhance the effectiveness of chemotherapy treatments.

Oxidative stress: Dracaena phytochemicals, such as flavonoids and tannins, have been shown to have antioxidant properties. They may help to protect cells from damage caused by oxidative stress, which is linked to many chronic diseases such as cardiovascular disease, diabetes, and Alzheimer’s disease.

Wound healing: Dracaena phytochemicals, including alkaloids and saponins, have been studied for their potential to promote wound healing. They may help to accelerate the healing process and reduce inflammation in wounds.

It’s important to note that while Dracaena phytochemicals have shown potential for treating these conditions, more research is needed to fully understand their effectiveness and safety for use in humans. It’s also important to consult with a healthcare professional before using any herbal or plant-based remedies for health purposes.

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-ulcer 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-ulcer agents from plant materials.

There are several chemicals that can be used in the extraction of anti-ulcer 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-ulcer 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. SSSSoxhlet 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.

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Ultrasonication: Ultrasonication 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. Ultrasonication, 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 ultrasonication 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. Ultrasonication 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.

methods of testing plant extracts for anti-ulcer activitiy

There are several methods for testing plant extracts for anti-ulcer activity. Some commonly used methods include:

Gastric acid secretion inhibition assay: This method involves measuring the ability of the plant extract to inhibit the secretion of gastric acid, which is one of the primary causes of ulcers. This can be done using animal models, such as rats or mice, and measuring the amount of gastric acid produced after treatment with the extract.

The gastric acid secretion inhibition assay is a laboratory test used to determine the ability of a substance to inhibit the secretion of gastric acid in the stomach. The assay is commonly used in the development of drugs for the treatment of acid-related diseases, such as gastroesophageal reflux disease (GERD) and peptic ulcer disease. The assay involves the following steps; A sample of the substance being tested is administered to a test subject, typically a laboratory animal such as a rat or mouse. The animal is then anesthetized and a surgical procedure is performed to expose the stomach.

Gastric acid secretion is stimulated using a substance such as histamine or gastrin, which causes the stomach to produce acid, the pH of the gastric fluid is measured before and after administration of the substance being tested. The ability of the substance to inhibit gastric acid secretion is determined by comparing the pH of the gastric fluid before and after administration.

The gastric acid secretion inhibition assay can be performed using various methods, including pH measurement, titration, or colorimetric assays. The results of the assay can provide valuable information about the efficacy and safety of substances being developed for the treatment of acid-related diseases.

Gastric mucosal damage prevention assay: This method involves inducing gastric mucosal damage, either chemically or physically, and measuring the ability of the plant extract to prevent or reduce the damage. This can be done using animal models, such as rats or mice, and measuring parameters such as ulcer index or histological changes in the gastric mucosa.

The gastric mucosal damage prevention assay is a laboratory test used to evaluate the ability of a substance to prevent or reduce gastric mucosal damage caused by factors such as acid or drugs, which can lead to the development of ulcers. This assay is commonly used in the development of drugs for the treatment of ulcers and other gastric disorders. The process of the gastric mucosal damage prevention assay involves the following steps; A sample of the substance being tested is administered to a test subject, typically a laboratory animal such as a rat or mouse. The animal is then anesthetized and a surgical procedure is performed to expose the stomach. Gastric mucosal damage is induced using a substance such as aspirin or ethanol, which can cause damage to the gastric mucosa.

The extent of gastric mucosal damage is evaluated using methods such as histology, microscopy, or biochemical assays, the ability of the substance to prevent or reduce gastric mucosal damage is determined by comparing the extent of damage in animals treated with the substance to those that were not treated. The gastric mucosal damage prevention assay can be performed using various methods and protocols, depending on the specific objectives of the study and the type of substance being tested. The results of the assay can provide valuable information about the potential efficacy and safety of substances being developed for the treatment of ulcers and other gastric disorders.

Anti-inflammatory assay: Inflammation is a major component of ulcer development, and plant extracts with anti-inflammatory activity can be effective in preventing or treating ulcers. This can be tested using in vitro assays, such as measuring the production of inflammatory cytokines by immune cells, or in vivo assays using animal models of inflammation.

The anti-inflammatory assay is a laboratory test used to evaluate the ability of a substance to reduce inflammation, which is a key component of the development of ulcers. This assay is commonly used in the development of drugs for the treatment of ulcers and other gastrointestinal disorders. The process of the anti-inflammatory assay involves the following steps; A sample of the substance being tested is administered to a test subject, typically a laboratory animal such as a rat or mouse.

An inflammatory response is induced using a substance such as carrageenan or dextran sulfate, which can cause inflammation in the tissue., The extent of inflammation is evaluated using methods such as histology, microscopy, or biochemical assays. The ability of the substance to reduce inflammation is determined by comparing the extent of inflammation in animals treated with the substance to those that were not treated. The anti-inflammatory assay can be performed using various methods and protocols, depending on the specific objectives of the study and the type of substance being tested. The results of the assay can provide valuable information about the potential efficacy and safety of substances being developed for the treatment of ulcers and other gastrointestinal disorders, as well as their potential to reduce inflammation in other tissues of the body.

Antioxidant activity assay: Oxidative stress is another factor that contributes to ulcer development, and plant extracts with antioxidant activity can be effective in preventing or treating ulcers. This can be tested using in vitro assays, such as measuring the ability of the extract to scavenge free radicals, or in vivo assays using animal models of oxidative stress.

pylori inhibition assay: Helicobacter pylori is a bacteria that is a major cause of ulcers, and plant extracts with antimicrobial activity can be effective in preventing or treating H. pylori-associated ulcers. This can be tested using in vitro assays, such as measuring the ability of the extract to inhibit H. pylori growth, or in vivo assays

Invivo methods of testing plant extracts for anti-ulcer activity

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

Indomethacin-induced ulcer model: Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that is known to induce ulcers in the stomach. This model involves administering indomethacin to experimental animals and then testing the anti-ulcer activity of the plant extract by measuring the reduction in the size and number of ulcers.

Ethanol-induced ulcer model: Ethanol is a potent ulcerogen that is used to induce ulcers in experimental animals. This model involves administering ethanol to the experimental animals and then testing the anti-ulcer activity of the plant extract by measuring the reduction in the size and number of ulcers.

Stress-induced ulcer model: Stress is known to cause ulcers in experimental animals. This model involves subjecting the experimental animals to various stressors (such as cold water swimming, restraint, or isolation) and then testing the anti-ulcer activity of the plant extract by measuring the reduction in the size and number of ulcers.

pylori-induced ulcer model: Helicobacter pylori (H. pylori) is a bacterium that is known to cause ulcers in humans. This model involves infecting experimental animals with H. pylori and then testing the anti-ulcer activity of the plant extract by measuring the reduction in the size and number of ulcers.

NSAID-induced ulcer model: NSAIDs such as aspirin and ibuprofen are known to cause ulcers in the stomach. This model involves administering NSAIDs to experimental animals and then testing the anti-ulcer activity of the plant extract by measuring the reduction in the size and number of ulcers.

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-ulcer 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-ulcer 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-ulcer activity using in vitro or in vivo methods.

Fractionation of plant crude extracts for anti-ulcer 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-ulcer 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-ulcer activity.

Overall, fractionation of plant crude extracts is a useful technique for isolating and identifying anti-ulcer 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-ulcer 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-ulcer agent

It is important to note that the isolation and purification of anti-ulcer 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.

2.2 Treatment of peptic ulcers

One of the medications prescribed the most frequently for gastroduodenal ulcers is proton pump inhibitors (PPIs). Omeprazole and lansoprazole are the PPI members that are prescribed the most frequently. PPIs are prescribed for both off-label and prescription-based conditions. PPIs have been approved by the FDA for the treatment of GERD, Zollinger Ellison disease, NSAID-activated gastroduodenal ulcer, hemorrhagic ulcer, and gastroduodenal ulcer with stomach inflammation. While PPIs are effective at reducing symptoms, studies have shown that they are among overused medications. This is because they are simple to obtain, even over the counter (OTC), and up to 70% are surprisingly used outside of their intended indications (Merkhan, Abdullah, & Althanoon, 2022).

Despite renal disorders being uncommon, they are serious and might include interstitial nephritis and kidney damage. Other serious but uncommon adverse effects include osteoporosis, hypomagnesemia, anemia, and renal disorders including anemia. The greatest harmful effects of PPIs are thought to be kidney-related adverse events (Jafari et al., 2022).

These renal illnesses may be mistaken with other problems that coexist with the use of PPIs and may actually be the root of the renal disorder, with PPIs only being able to play an additional, extreme mode of induction. The immunological point is a common factor among all of these disorders as an underlying cause of various renal ailments. Among people with the same demographic characteristics who do not take PPIs, renal issues are three times more likely to occur among PPI users. Due to the lack of specific, objectively measurable characteristics supporting the association, the conclusions of the majority of published data are difficult to interpret, the explanation of the association between PPI use and the development of renal problems is based on the precipitation of these drugs together with their metabolites in renal interstitium (Reusens et al., 2017). These later effects stimulate local immune response resulting in T cell stimulation and propagation of immune response which might end up with tissue necrosis and this will ensure discontinuing the offending drugs and start corticosteroids to avoid initiation of renal failure (Imade et al., 2022).

2.2 Isolation and purification of the Bio-active compounds in the extracts

Isolation and purification of bioactive compounds from extracts can be a complex process, but there are some general steps that can be followed. The specific techniques used will depend on the nature of the compounds and the properties of the extract. Here is a general overview of the steps involved in isolating and purifying bioactive compounds:

Extraction: The first step is to extract the bioactive compounds from the source material using an appropriate solvent. Different solvents can be used depending on the polarity and solubility of the compounds of interest.

Fractionation: Once the compounds have been extracted, the next step is to separate them into different fractions based on their physical and chemical properties. This can be done using techniques such as liquid-liquid extraction, column chromatography, or high-performance liquid chromatography (HPLC).

Identification: After fractionation, the fractions containing the bioactive compounds are identified using analytical techniques such as UV-vis spectroscopy, mass spectrometry, or nuclear magnetic resonance (NMR) spectroscopy.

Purification: The final step is to purify the bioactive compounds from the identified fractions. This can be done using techniques such as recrystallization, distillation, or preparative HPLC.

Medicinal plants are useful in the treatment of many ailments and diseases among rural dwellers, indigenous users, traditional medicine (TM) practitioners, and livestock owners in many African countries. The traditional knowledge of medicinal plants if harnessed, can give insights into the vital role that medicinal plants play in drug development.

Often, a single medicinal plant can have multiple uses, and sometimes different parts of the same plant may be used for the treatment of more than one disease condition. Other times, the same plant could be used as an ingredient in herbal preparations for a synergistic effect. This is made possible due to the range of phytochemicals that are present in medicinal plants along with their diversities of bioactivities. Neorautanenia mitis (A. Rich) Verdc. (Fabacae), Hydnora abyssinica A. Braun (Hydnoraceae), and Senna surattensis (Burm. f.) H. Irwin and Barneby (Fabaceae), were selected based on their promising preliminary screening results, they have shown various bioactivities and are traditionally used for the treatments of many disease conditions. The roots of N. mitis, are used for the treatment of bilharzia, syphilis, diarrhea, skin infection, dysmenorrhea and neuropsychiatric conditions. They are also used as an anticonvulsant, anti-malarial, fish poison, insecticide, and for killing bilharzias-carrying fresh water snails in many African countries. The crude extracts and phytochemical constituents isolated from N. mitis have shown antidiarrheal, acaricidal, insecticidal, antinocicetive, anti-inflammatory, larvicidal, mosquitocidal, cytotoxicity, and antimicrobial, activities. H. abyssinica, is referred to as one of the strangest plants in the world, with its vegetative body consisting of only flowers, fruits and roots and has no leaves. It is not very common among botanists and plant scientists because it is rarely encountered. However, it remains a popular and valuable medicinal plant among local users and TM practitioners, and is traded by traditional medicine vendors in local markets in South Africa, Mozambique and Nigeria. In some African countries including, Sudan, Kenya, South Africa, Malawi, Mozambique and Nigeria, it has been used for, the treatment of diarrhea, severe bacterial infections such as urinary tract infection, helminthiasis, internal wounds, piles, acne and dysentery, the expulsion of retained placenta and the treatment of throat and stomach aches. Extracts and constituents from H. abyssinica showed immunosuppressive, cytotoxic, antibacterial, antioxidant, molluscidal and antidiarrheal activities.

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-ulcer agents used in treatment of peptic ulcers.

The following methods will be used to identify plants with anti-ulcer 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-ulcer activity

To test crude extracts for anti-ulcer 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

3.2.5.1 Pylorus Ligation-Induced Ulcer Model

The shay et al. model will be used with slight modification. Rats will be randomly divided into five study groups, each consisting of six animals. Group I will be the negative control (NC), which will receive a vehicle only (distilled water+6% Tween 80). Group II will be served as a positive control and rats will be pretreated with ranitidine 50 mg/kg for ten days. Groups III, IV, and V will be received 100, 200, and 400 mg/kg of hydro-methanolic extracts of plant extracts (Meng, 2019).

Rats will be fasted for 24 hours before the study but has free access to water till the last 4 hours. After 1 hour of the last drug treatment, animals will be anesthetized with diethyl ether and the abdomen will be opened by a small midline incision below the xiphoid process. The pyloric portion of the stomach will be lifted out and ligated carefully to avoid traction to the pylorus or damage to the blood supply of gastric mucosa. The stomach will be replaced carefully and the abdominal wall will be closed by interrupted sutures. After four hours of pyloric ligation, rats will be sacrificed by inhalational anesthetic ether. The abdomen will be opened, the cardiac end of the stomach will be dissected out and the content will be drained into a test tube. The gastric juice will be collected and centrifuged at 1000 rpm for 10 minutes the volume of the supernatant will be noted and taken for the determination of total acidity and pH. The stomach mucosa of each animal will be washed with saline and running water, labeled, and placed on sodium phosphate-buffered 10% formalin until it will be examined for lesions by using a hand lens (10X) and scored accordingly.

3.2.6 Ethanol-Induced Ulcer Model

The ulcer will be induced by administering ethanol following the method by Hollander et al., and previous studies to determine the antiulcer effect of repeated and single-dose administration. Animals will be randomly assigned to 10 groups each consisting of six animals. Group I and VI will receive the vehicle (distilled water+4% Tween 80 for single and repeated dose, respectively) and considered as NCs, whereas group II and VII will be served as a reference standard and pretreated with misoprostol 5 μg/kg (single and repeated for 10 days, respectively). Groups III, IV, and V will be treated with a single dose of 200 mg/kg of hydro-methanol, chloroform, and aqueous fractions, respectively. Group VIII, IX, and X will be pretreated with repeated doses of 200 mg/kg of hydro-methanol extract, chloroform, and aqueous fractions of F. thonningii, respectively. Animals fasted for 24 hours before the administration of ethanol. All pretreatments of the last dose will be given orally 1 hour before the experiment. Gastric ulcer will be induced after 60 minutes of the plant extract in 200 mg/kg doses and misoprostol 5 μg/kg treatment by administering ethanol (90% w/v) at a dose of 1 ml/200 g bodyweight (0.2 ml) to each animal and after 1 hour, animals will be sacrificed with spinal dislocation; stomach will be incised along the greater curvature and ulceration will be scored similarly to pyloric ligation-induced ulcer model.

3.2.7. Indomethacin-Induced Ulcer Model

The ulcer will be induced with indomethacin at a dose of 18 mg/kg to evaluate the ulcer healing effect of the plant extract which will be compared with the NC (vehicle) and positive (misoprostol 5 μg/kg) treated groups. The treatment groups will receive 100, 200, 400 mg/kg (once daily). The first dose will be given 6 hours after induction of ulcer with indomethacin (18 mg/kg). Four days after ulcer induction, analysis will be done.

3.2.8. Parameters for the Evaluation of an Antiulcer Activity

Macroscopic Evaluation of the Stomach

The stomach will be opened along the greater curvature, rinsed with saline and running water to remove gastric contents and blood clots; then, the mucosa of each animal will be labeled and placed on sodium phosphate-buffered 10% formalin until it will be examined by a 10x magnifier lens to assess the formation of ulcers. The numbers of ulcers will be counted. The Kulkarni method will be used for scoring the ulcer as follows: normal colored stomach (0), red coloration (0.5), spot ulcer (1), hemorrhagic streak (1.5), deep Ulcers (2), and perforation (3).

The mean ulcer score (US) for each animal will be expressed as the ulcer index (UI). UI will be measured by using following formula: UI = UN + US + UP × 10−1, where UI is the ulcer index, UN is the average number of ulcers per animal, US is the average number of severity scores, and UP is the percentage of animals with ulcers.

3.2.9 Determination of pH and Gastric Volume

An aliquot of gastric juice will be taken and the pH of the solution will be measured using a pH meter based on the method of Tan . The volume of gastric juice of each animal will be measured after centrifugation with 1000 rpm for 10 minutes and analyzed since it is one parameter for the study of the antisecretory effect of the plant extract.

3.2.10. Determination of Total Acidity

An aliquot of 1 ml gastric juice diluted with 9 ml of distilled water will be taken and two drops of phenolphthalein indicator will be added. Then, it will be titrated with 0.01 N NaOH until a permanent pink color will be observed. Based on the volume of 0.01 N NaOH consumed, the total acidity will be expressed as mEq/l by the following formula.

Total Acidity=Vol.of NaOH×N×100 mEq/l/0.1.

3.2.11 Data Analysis

SPSS version 20 will be used for data entry, coding, cleaning, and analysis of results. The result will be expressed as the mean ± SEM for each parameter. Statistical differences will be evaluated using a one-way ANOVA followed by post hoc Tukey’s multiple comparison tests. Results will be considered to be statistically significant at (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-ulcer 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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