ABSTRACT
This study investigated the phytochemical profile and anti-malarial property of Commiphora pedunculata, a medicinal plant used in northern Nigeria for the treatment of fever. The dried stem bark of the plant was extracted by maceration in dichloromethane: methanol (1:1). The crude extract was fractionated by vacuum liquid chromatographic (VLC), eluted with Hexane-dichloromethane (9:1), dichloromethane-ethyl acetate (20:1), ethyl acetate (100%), ethyl acetate-methanol (5:1) to yield four fractions, which were analyzed for their phytochemical profile using thin layer chromatography. The percentage yield of the crude extract was obtained as 4.82%. The VLC procedure produced different fractions of extracts from the stem bark with various percentage yields; 3.7552%, 14.2448%, 8.2116% and 4.0622%. The TLC produced 3 spots using Hexane-Ethyl acetate (7:3) as mobile phase indicating 3 potential compounds and 2 spots using the Dichloromethane-Ethyl acetate (1:1) mobile phase, indicating 2 potential compounds. The four fractions from VLC were investigated for in vivo antimalarial activity in Plasmodium berghei infected mice. It showed significant anti-malarial activity against Plasmodium berghei at low dose (50mg/kg BW) which signifies its potential of being used as an antimalarial medication. It is evident that Commiphora pedunculata contained some phytochemicals in the extract that can be recovered for beneficial purposes.
TABLE OF CONTENT
Contents
DECLARATION ii
CERTIFICATION iii
DEDICATION iv
APPRECIATION v
ABSTRACT vi
TABLE OF CONTENT vii
LIST OF TABLES xi
TABLE OF FIGURES xii
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of Malaria 1
1.2 History of Malaria Medication. 1
1.3 Commiphora Pedunculata 3
1.4 Statement of Research Problem 4
1.5 Aim of Experiment 5
1.6 Objectives of Experiment 5
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Taxonomy of The Plant 6
2.1 The Family Burseraceae (Torchwoods). 6
2.2 Uses of Commiphora Pedunculata 7
2.3 Habitat 8
2.4 Varieties of The Plant 8
2.5 Biosynthetic Pathways of Plant Extracts 8
2.5.1 Mevalonic-acid pathway. 8
2.5.2 Shikimic-acid pathway 9
2.5.3 Methylerythritol-phosphate pathway (MEP) 9
2.5.4 Malonic-acid pathway 10
2.6 Chemicals Found in Plant Extracts. 10
2.7 Uses of Plant Extracts 16
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Materials 17
3.1.1 Chemicals and reagents 17
3.1.2 Equipment and apparatus 17
3.2 Plant Material and Preparation of Extracts 18
3.2.1 Plant material 18
3.2.2 Extraction of plant material using cold maceration 18
3.3 Vacuum Liquid Chromatography (VLC) Fractionation 19
3.3.1 Materials and reagents 20
3.3.2 Procedure 20
3.4 Thin Layer Chromatography (TLC) of Sample “B” and “C” 21
3.4.1 Materials 21
3.4.2 Procedure 22
3.5. Antimalarial Activity 23
3.5.1. Source of experimental animals 23
3.5.2 Source of Plasmodium species 23
3.5.3 Experimental grouping of animals 24
3.5.4 Inoculation of experimental animal 24
3.5.5 In vivo anti plasmodial activity of Commiphora pedunculata fractions 24
CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 Results 26
4.1.1 Vacuum liquid chromatography (VLC) 26
4.1.2 Percentage yield of the extracts: 26
4.1.3 Thin layer chromatography (TLC) of fraction B (Dichloromethane-ethyl acetate, (20:1) 27
4.1.4 Anti-malarial activity 29
4.2 Discussion 30
4.2.1 Vacuum liquid chromatography (VLC) and percentage yield. 30
4.2.2 Thin layer chromatography (TLC) 31
4.2.3 Anti-malarial activity 32
CHAPTER FIVE
5.0 CONCLUSION
5.1 RECOMMENDATIONS 33
References 34
LIST OF TABLES
Table 4.1 Percentage yield of various fractions of extract from Vacuum Liquid Chromatography
TABLE OF FIGURES
Figure 1: Commiphora pedunculata plant 4
Figure 2 Molecular structure of flavone backbone 11
Figure 3 Various structures of alkaloids 12
Figure 4 Limonene; a monoterpene 13
Figure 5 Cholesterol; a steroid 13
Figure 6 Tannic acid; a tannin 14
Figure 7 A saponin 15
Figure 8 Plant material soaked in solvent during cold maceration 19
Figure 9 Vacuum liquid chromatography set up 21
Figure 10 TLC profile of sample B using Hexane: ethyl acetate (7:3) as mobile phase 28
Figure 11 TLC profile of sample B using Dichloromethane: ethyl acetate (1:1) as solvent phase 29
Figure 12 : Graph of Parasitemia against Days post infection, showing the trend in parasitemia in laboratory mice groups; IC, IT3.1CQ, IT50EC, IT100EC, 1T50DC, and IT100DC 30
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of Malaria
Malaria is a disease transmitted by the microscopic Plasmodium parasite. It is transmitted to humans through the Female Anopheles mosquito (vector). When a female anopheles mosquito carrying the parasite bites a human, the parasite is transmitted into the blood stream and relocates to the liver where they mature and reproduce (Caraballo, 2014). Malaria is a deadly disease. According to data by the United Nations International Children’s Emergency Fund (UNICEF), as of 2021, there were 247 million malaria cases globally that led to 619,000 deaths with 77 percent of these deaths being children below 5 years of age (WHO, 2023).
After its discovery in the late 1800s, studies took place to find a cure to the disease due to its high mortality rate, especially during World War II. Natural products played a big role in these discoveries. In the early 20th century, the first cure for malaria was discovered from the bark of the Cinchona tree. This was the first effective treatment for malaria. Later, Chloroquine from the Cinchona tree (Cinchona officinalis) and Artemisinin discovered from the sweet wormwood plant were used as cures. Artemisinin is a common component in modern antimalarial drugs.
1.2 History of Malaria Medication.
Quinine comes from the bark of a tree native to South America. According to legend it was first brought to Europe by a Countess who had been treated with it in Peru in the 1600s. It was introduced in 1632. The bark was named cinchona in 1742 by Linnaeus. In 1820, French chemists isolated quinine from the cinchona bark and quinine became a treatment of reference for intermittent fever throughout the world. Quinine remains an important and effective treatment for malaria today, despite sporadic observations of quinine resistance.
Research by German scientists to discover a substitute for quinine led to the synthesis in 1934 of Resochin (chloroquine) and Sontochin (3-methyl-chloroquine). These compounds belonged to a new class of antimalarials, the four-amino quinolines. The German research went no further and the formula for Resochin was passed to a US company. American researchers made slight adjustments to the captured drug to enhance its efficacy. The new formulation was called chloroquine. Only after comparing chloroquine to the older and supposedly toxic Resochin, did they realize that the two chemical compounds were identical.
Following the war, chloroquine and DDT emerged as the two principal weapons in WHO’s global eradication malaria campaign. Subsequently, chloroquine resistant P. falciparum probably arose in four separate locations starting with the Thai-Cambodian border around 1957; in Venezuela and parts of Colombia around 1960; in Papua New Guinea in the mid-1970s and in Africa starting in 1978 in Kenya and Tanzania and spreading by 1983 to Sudan, Uganda, Zambia and Malawi.
Since then, various other antimalarial drugs were discovered including; Sulfadoxin, Mefloquine, Artemisinin. Sulfadoxin emerged from the anti-malarial pipe line during World War II. It was gotten from a Pyrimidine derivative, Proguanil. This led to further study of its chemical class and the development of Pyrimethamine. Mefloquine’s efficacy in preventing falciparum malaria when taken regularly was shown in 1974 and its potential as a successful treatment agent was shown soon after. Resistance to Mefloquine began to appear in Asia in 1985, around the time the drug became generally available. Artemisinin was isolated by Chinese scientists in 1972 from Artemisia annua (sweet wormwood), better known to Chinese herbalists for more than 2000 years as Qinghao. Artemisinin has been a very potent and effective antimalarial drug, especially when used in combination with other malaria medicines. Combining an Artemisinin drug with a partner drug that has a longer half-life was found to improve the efficacy of the Artemisinin. It also reduced treatment duration with the Artemisinin and appeared to reduce the likelihood of development of resistance to the partner drug (History of antimalarial drugs, 2023)
1.3 Commiphora Pedunculata
Commiphora pedunculata is a natural product of anti-malarial importance. It is from the family Burseraceae. It is an angiosperm. C. pedunculata is a savannah shrub of about 4 – 6m height but may sometimes grow to between 12 and 15m. The bark of most C. pedunculata is papery and peels off into papery flakes, revealing a green bark underneath. The leaves are mostly compound. The fruit of C. pedunculata greatly enhances the identification of the specie. When ripe, the fruit splits into halves revealing a brightly colored pseudo-aril. This fleshy appendage completely or partially encompasses the seed as part of an attachment around part of the seed. The shape of the pseudo-aril differs from species to species. The flowers may be uni or bisexual, with the unisexual flowers only being semi developed with non-functional stamens (Steyn, 2003).
Commiphora pedunculata stem bark has been reported to treat infected wound, the root is usually chewed for treating cough and it is also used for treating jaundice, nausea and yellow fever, the decoction of the leaves and stem bark is used for the treatment of dysentery and diarrhea. In Nigeria, the common names of C. pedunculata are Luban (Arabic) and Daashin jeji (Hausa).
Figure 1: Commiphora pedunculata plant
1.4 Statement of Research Problem
Since their discovery, antimalarial drugs such as chloroquine have been administered as a cure to sick patients. However, over recent years, it has been discovered that malaria parasites have begun gaining resistance to these drugs. This poses a threat to humanity. According to WHO malaria killed 619,000 people in 2021, making it important to discover effective antimalarial drugs (WHO, 2023).
Drug resistance has been observed in two out of four malaria parasites (Plasmodium falciparum and P. vivax). Plasmodium falciparum has developed resistance to anti-malarial drugs such as Chloroquine, Quinine, Sulfadoxin and Mefloquine. This has resulted in an urgent need to find new malaria drugs from new sources.
1.5 Aim of Experiment
The aim of the experiment is to identify the compounds present in Commiphora pedunculata and to determine the anti-plasmodial properties of Commiphora pedunculata in a mouse model of malaria.
1.6 Objectives of Experiment
1. Extraction of chemical components of Commiphora pedunculata through solvent extraction.
2. Chromatographic analysis to determine chemical composition.
3. Investigation of antimalarial activity of the extract in P. berghei infected mice.
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