Chapter one

INTRODUCTION

Many impoverished countries, notably African countries like Nigeria, use traditional medicine as their primary source of health treatment. This traditional medication is typically derived from plants [Rehan Ahmad et al, 2008; Stephen bent, 2008].

Today, approximately 88 percent of the world’s population uses plant-derived medications as their first line of defence for health maintenance and disease prevention [Kintzois et al., 2006].

Currently, there are approximately 60 species of medicinal plants that have been identified in primary health care and are classified according to their pharmacological effects such as peptic ulcers, antiflatulence, laxative, antidiarrhoea, and antihepatic [Viomolos et al, 2003].

However, in the future, the development of novel medicinal molecules will be solely based on plant origin [Perulmalsamy et al 1999]. Senna tora is an example of traditional medicine having pharmacological activities such as anticholesterolemic, antispasmodic, carninitative, emolliants, ophthalmic, and purgative [Polunin and Stanton, 1984; Yeung, 1985; Manandhar, 2002].

It can be cardiotonic, antiperiodic, anthelminitic, and expectorant, as well as used to treat cogh, leprosy, ringworm, colic, flatulence, constipation, and other illnesses [Nature Serve, 2007].

Senna tora, formerly known as cassia tora, can inhibit the formation of prostaglandins and other inflammatory agonists such as cytokines, histamine, kinin, and free radicals. Manila, 1998 supported his findings by confirming its use in Korea for the treatment of constipation, oedema, and liver protection. The liver, the largest internal organ of the human body, conducts over 500 tasks, many of which are critical to survival.

The liver can regenerate or grow back cells that have been lost by short-term injury or disease; however, if the liver is repeatedly harmed over time, it may acquire irreversible alterations that permanently impair its function. Carbon tetrachloride is a hazardous chemical that disrupts the liver’s functioning.

This means that ingesting it causes liver damage. CCL4 is stable in the presence of air and light, but it is also inflammable. Despite its toxicity to the liver, it is helpful as a grain fumigant, pesticide, and other applications.

Carbon tetrachloride alters the equilibrium of bodily fluids and secretions. For example, CCL4 inhibits or alters lipid metabolism, cholesterol metabolism, and liver biotransformation processes. Thus, carbon tetrachloride is fatal.

The liver generates enzymes such as aspartate amino transaminases, alkaline phosphatase, lactate dehydrogenase, gamma glutamyl transsferase, and others that catalyse the body’s metabolic activity; however, when CCL4 attacks the liver, these enzymes are not generated.

Free radicals are extremely reactive molecules with an unequal quantity of electrons in their outermost orbits. This can combine with biological components such as unsaturated fatty acids to produce new free radicals, causing permanent biochemical injury such as membrane damage, apotosis, and cell necrosis.

Antioxidants scavenge free radicals and inhibit subsequent processes, thereby protecting macromolecules and the cellular environment from toxicity and degeneration [Hong B.O. Shao et al, 2008]. The oxygen consumption inherent in cell growth generates a variety of reactive oxygen species [ROS].

ROS include substances such as superoxide anion radicals and hydroxyl radicals [OH]. ROS can be extremely harmful, targeting the lipids of cell membranes and causing DNA mutations, which can advance the spread of many diseases [Valontao et al 2002, Gulcin et al 2003].

Reactive oxygen species are constantly created during normal physiological activities and are eliminated by antioxidant defence mechanisms [Buyukokuroglu et al., 2001]. Many studies have demonstrated that some cassia species have developed antibacterial and antioxidant properties.

1.1 Aims of the Research

The purpose of this study is to investigate the hepatoproductive effects of sena tora leaf extract on carbon tetrachloride-induced toxicity.

CHAPTER ONE

INTRODUCTION

The most prevalent type of primary liver cancer is hepatocellular carcinoma (HCC). According to Melissa (2004), it is responsible for 60% of all cancer cases worldwide.

The existence of cirrhosis is the most important factor. HCC has a distinct age and sex distribution that is probably influenced by a particular actiology component. Within the same nation, its distribution differs among ethnic groups as well (Munoz 1989).

In south-eastern Nigeria, a high prevalence of hepatitis B and C may have had a significant role in the development of liver disease, including cirrhosis and HCC.

However, a recent trend showing a rise in hepatitis and liver cirrhosis cases in our environment raises the possibility that there are more environmental factors besides hepatitis B and C that could be responsible for the recent development.

Investigating the many risk factors and causes of chronic hepatitis would be required in order to prevent the spread of liver cirrhosis and HCC in our society. The cause of cirrhosis affects the likelihood of developing HCC.

For instance, hepatitis B-induced cirrhosis has a high chance of developing into HCC, whereas those with primary biliary cirrhosis, even if they have it, have a very low risk of developing HCC. With the exception of the hepatitis B virus, which is a DNA virus, all of these human hepatitis viruses are RNA viruses.

All forms of viral hepatitis cause clinically identical diseases, despite the fact that these viruses can be differentiated based on their molecular and antigenic characteristics.

These range from subclinical persistent infections to rapidly progressive liver disease with cirrhosis and even hepatocullular carcinoma (HCC), which are common to the blood-borne types (HCV and HBV), to asymptomatic and undetectable to fulminant and fatal acute infections that are common to all types.

It is impossible to identify which hepatitis virus is causing a case of hepatitis without a particular virological test. (Park, Kathleen, and others, 2004).

ABSTRACT

Using a contemporary atomic absorption spectrophotometer, this study examined the chemical characteristics of the water extract of the zobo (Hibiscus sabdariffa) beverage. In Nigeria, particularly in the northern region, zobo drink is a popular beverage. The analysis’s findings showed that the beverage is rich in essential mineral nutrients needed for human growth and for maintaining healthy metabolic processes. When compared to other leaves, such as the well-known vegetable leaf known as awa (Piper methysticum), zobo drink has a higher concentration of iron, a key component of human haemoglobin, according to the results.

CHAPTER ONE

INTRODUCTION

Epidemiological research and related meta-analyses at the close of the 20th century strongly implied that eating a diet high in plant-based foods for an extended period of time provided some protection against chronic illnesses, particularly cancer (Wallstrom et al., 2000).

These observations brought attention to the potential role of radical scavenging and radical suppressing nutrients and non-nutrients in explaining the apparent benefit of such diets (Weisburger, 1991), as uncontrolled production of free radicals was believed to be significantly implicated in the aetiology of cancer (Guyton and Kensler 1993).

As soon as it was discovered that free radicals were also linked to the development of numerous other chronic illnesses (Kehrer, 1993; Stohs, 1995), flavonoids and the foods and drinks that contain them came into focus.

It was a regrettable but unintended consequence of some studies and publications that many researchers mistakenly believed that dietary phenols, polyphenols, and tannins (PPT) only included flavonoids, flavonols, and flavones.

Long-term consumption of diets high in plant-based foods has been linked to improved health, according to more recent epidemiological studies (Hung et al., 2004 and Jansen et al., 2004).

However, it’s unclear if this is because such diets increase intake of certain beneficial nutrients (like isothiocyanates and other sulfur-containing plant constituents, mono-unsaturated fatty acids, and poly-unsaturated fatty acids, PPT

polyacetylenes, selenium, terpenes, etc.), decrease exposure to harmful substances (like oxidised cholesterol, pyrolysis mutagens, salt, saturated fat, etc.), or some combination as recommended in the “Polymeal” concept (Franco et al., 2004 and Johnson, 2004).

According to an in vitro study, PPT and isothiocyanates may have a mechanistic basis for genuine synergy. Conversely, more recent research attempting to evaluate the proposed association between flavonoid intake and flavones, or other flavonoids, has produced far less consistent findings.

According to certain research, flavonoids may have a preventive effect against some (but not all) types of cancer (Knekt et al., 1997 and Sun et al., 2002) or vascular illnesses (Hirvonen et al., 2001 and Mennen et al., 2004).

It’s interesting to note that in order to determine the beneficial effects of isothiocyanates and/or the phenolic components of the plants (such as the dietary phenols including flavonoids) in the management of cancer growth and the initiation of various types of diabetes, a study of the association between the consumption of Hibiscus sabdariffa (zobo) and other cruciferous vegetables and the risk of breast cancer in premenopausal women needs to be conducted.

Numerous studies conducted during this time period have indicated positive effects linked to increased intake of other dietary phenol classes. For example, increased coffee consumption has been linked with reduced incidence of type II diabetes (van Dam et al., 2002 and Saremi et al., 2003).

Similarly, increased consumption of lignans (or at least greater plasma concentrations of their metabolites)has been linked with reduced incidence of estrogen-related cancers in some (Boccardo et al., 2004 and McCann et al., 2004) but not all studies (Kilkkinen et al., 2004 and Zeleniuch –Jacquotte et al., 2004),and a prospective study was equivocal.

1.1 SIGNIFICANCE OF THE STUDY

The flavonoids in the water extract of Hibiscus sabdariffa should be extracted for further experimental and clinical research in the management of certain ailments and disorders like cancer, diabetes mellitus etc.

The mechanisms of flavonoid-protein interactions have been discovered; it is my idea that such mechanisms should be carried to the in-vivo metabolic and physiological processes aiming at understanding the exact mechanisms involved in cellular aging processes and the advancement of illnesses.

1.2 OBJECTIVES OF THE STUDY

The objective of this study is to determine the physico chemical (biochemical) and water constituent of Zobo produced from dried calyces of Hibiscus sabdariffa (Linn Roselle).

Using atomic absorption spectrophotometer (AAS). The biochemical makeup of the zobo has adverse consequences on human health when taken.

CHAPTER ONE

INTRODUCTION

The Background of the Study 

In today’s world, eating a diet rich in sucrose has become more and more common, mostly because sugary processed foods and drinks are so widely accessible.

On the other hand, consuming too much sucrose has been connected to a number of detrimental health outcomes, such as obesity, metabolic syndrome, heart disease, and neurological conditions (1,2).

The disturbance of electrolyte balance in critical organs like the heart and brain is one of the fundamental processes causing these disease disorders.

Calcium, potassium, sodium, and chloride are examples of electrolytes that are essential for preserving the physiological processes of cells and tissues. For example, calcium has a role in muscular contraction, neurotransmission, and enzyme activity regulation (3).

Maintaining the electrical excitability of cells, such as neurones and cardiomyocytes, requires potassium (4). Osmotic equilibrium, cell volume regulation, and electrical potential across cell membranes are all dependent on sodium and chloride (5,6). Any change in these electrolyte levels has the potential to cause cellular malfunction and aid in the onset of a number of illnesses.

The potential therapeutic benefits of herbal plants and their capacity to correct physiological imbalances have long been acknowledged.

Two such plants, Vernonia amygdalina (also known as “Bitter leaf”) and Telferia occidentalis (also called “Ugu” in Nigeria), are frequently utilised in traditional medicine due to their nutritional and therapeutic qualities. Bioactive substances such as vitamins, minerals, antioxidants, and phytochemicals are abundant in these plants (7,8).

Their hepatoprotective, antidiabetic, antioxidant, and anti-inflammatory qualities have all been shown in studies (9, 10). However, little research has been done on how they affect electrolyte balance, especially when a high-sucrose diet is included.

Since the brain and heart are extremely vulnerable to metabolic disruptions and are vital to general health and wellbeing, it is crucial to comprehend the possible advantages of Telferia occidentalis and Vernonia amygdalina in re-establishing electrolyte homeostasis in these organs.

According to earlier research, diets high in sugar can cause oxidative stress, inflammation, insulin resistance, and cellular damage in these organs (11, 12).

Thus, investigating how herbal supplements affect the electrolyte balance in the heart and brain can reveal important information about their possible defences against damage caused by sugar.

The purpose of this study is to examine how Vernonia amygdalina and Telferia occidentalis affect the levels of electrolytes, particularly calcium, potassium, sodium, and chloride, in the heart and brain tissues of rats given a diet high in sucrose.

Knowing how these herbal supplements affect electrolyte homeostasis will help us better understand how they might be used therapeutically to avoid or lessen the negative effects of consuming too much sugar.

In rats given a high-sucrose diet, the study’s premise is that supplementing with Vernonia amygdalina and Telferia occidentalis will restore electrolyte balance in the brain and heart tissues, lowering the risk of cellular dysfunction and associated pathological diseases.

The results of this study can offer important new information on the possible dietary interventions of Vernonia amygdalina and Telferia occidentalis for those who are already or may soon develop metabolic disorders linked to a high-sucrose diet.

Additionally, by comprehending the fundamental processes by which these herbal plants influence electrolyte balance, new therapeutic strategies that target metabolic diseases and related organ dysfunctions may be developed.

In conclusion, examining how Vernonia amygdalina and Telferia occidentalis affect electrolyte levels in the brain and heart tissues of rats given a high-sucrose diet is crucial for determining the possible defence mechanisms of

Chapter one

INTRODUCTION

Background of the study

Rhodanese, also known as thiosulphate sulphur transferase, is an enzyme involved in the detoxification of cyanide in living organisms. It facilitates the transfer of sulphur from thiosulphate to cyanide, resulting in thiocyanate, a less hazardous molecule (Pau et al., 2019). Rhodanese has been discovered in numerous animal tissues, including the liver, kidney, and brain (Eze et al., 2017).

It is responsible for the metabolism of sulfur-containing amino acids as well as the detoxification of medicines and poisons in the liver (Jahan et al., 2019). Rhodanese’s capacity to detoxify cyanide and other harmful substances makes it potentially useful in biotechnology and other industries.

Enzyme partial purification is required for research into their characteristics and applications. Enzyme purification procedures include ammonium sulphate precipitation, dialysis, chromatography, and electrophoresis (Sarma et al., 2016). Ion exchange chromatography is an effective technology for purifying proteins and enzymes according to their charge (Azevedo et al., 2017).

Temperature and heat stability experiments are necessary to find the best conditions for enzyme activity and stability. Enzymes have certain temperature ranges where they are most active, and high temperatures can cause denaturation and loss of activity (Liu et al. 2018).

Heat stability studies provide information about enzyme heat stability, which is important for industrial applications (Gupta et al., 2018).

The purpose of this study was to partially purify rhodanese from a local goat’s liver and investigate its effect on temperature and heat stability. The study will provide information on the properties and potential applications of rhodanese derived from the liver of a local goat.

Partial purification of Rhodanese:

The liver was extracted from a local goat and homogenised in ice-cold phosphate buffer (pH 7.4) with 0.1 M NaCl. The homogenate was centrifuged at 12,000 × g for 30 minutes at 4°C. The supernatant was then collected. The supernatant was then precipitated with ammonium sulphate at 60% saturation.

The precipitated protein was collected by centrifugation at 12,000 × g for 30 minutes at 4°C and dissolved in phosphate buffer (pH 7.4). The protein solution was dialysed with phosphate buffer (pH 7.4) at 4°C overnight.

The dialysed protein solution was then passed through ion exchange chromatography on a DEAE-Sepharose column equilibrated with phosphate buffer (pH 7.4).

The column was washed with equilibration solution, and the bound proteins were eluted using a linear gradient of NaCl (0.1-0.5 M) in phosphate buffer (pH 7.4) at a flow rate of 1 mL/min. Fractions with rhodanese activity were combined and dialysed against phosphate buffer (pH 7.4) overnight at 4°C.

The protein concentration was evaluated using the Bradford assay (Bradford, 1976), and rhodanese activity was determined by detecting thiosulphate production at 460 nm with a spectrophotometer (Shimadzu UV-1800) (Hissin and Hilf, 1976).

The Impact of Temperature on Rhodanese Activity:

To see how temperature affected rhodanese activity, the enzyme solution was incubated at temperatures ranging from 20°C to 80°C for 10 minutes. The enzyme activity was then assessed using the methods outlined above.

The results showed that rhodanese had an ideal temperature range of 35-40°C, with maximum activity reported at 40°C. At temperatures higher than 40°C, enzyme activity declined, and at 80°C, the enzyme was entirely denatured. The decrease in activity at high temperatures is most likely due to protein denaturation, which results in the loss of enzyme activity.

Effects of Heat Stability on Rhodanese:

To test rhodanese’s heat stability, the enzyme solution was incubated at three different temperatures (60°C, 70°C, and 80°C) for five, ten, and fifteen minutes.

The enzyme activity was then assessed using the methods outlined above. The results revealed that rhodanese was heat stable for 10 minutes at 60°C, keeping around 90% of its original activity.

After 10 minutes at 70°C, the enzyme retained roughly half of its initial activity. At 80 degrees Celsius for 10 minutes, the enzyme was

Chapter one

INTRODUCTION

1.1 Background of the Study.

Sickle cell disease (SCD) is a series of genetic diseases affecting the beta-hemoglobin chain. Normal haemoglobin consists of three types: haemoglobin A, A2, and F. In sickle cell disease, haemoglobin S has an aberrant beta globin chain encoded by a valine substitution for glutamic acid on chromosome 11 (Bunn, 2007).

This is an autosomal recessive condition. Sickle cell disease is a specific genotype in which a person gets one copy of the HbS gene and another gene that codes for a qualitatively or quantitatively defective beta globin chain. Sickle cell anaemia (HbSS) affects patients who are homozygous for the HbS gene, although heterozygous versions may combine HbS with genes coding for other types of defective haemoglobin, such as haemoglobin C, an autosomal recessive mutation that replaces lysine with glutamic acid.

Additionally, individuals can inherit a mix of HbS and β-thalassemia. β-thalassemias are autosomal recessive disorders that cause anaemia due to decreased or absent β-globin chains. Other genotype pairs are HbSD, HbSO-Arab, and HbSE (Meremiku, 2008).

Sickle haemoglobin in these illnesses causes affected red blood cells to polymerise under conditions of low oxygen tension, resulting in the distinctive sickle shape. Normal red cells exist in the bloodstream for roughly 120 days, but sickled red cells die after 10 to 20 days.

Because they cannot be regenerated quickly enough, the blood is chronically depleted of red blood cells, a disease known as anaemia.

Inflammation, endothelial abnormalities, and thrombophilia cause sickle cell accumulation in the microcirculation, resulting in ischaemia in end organs and tissues distal to the blockage (Hayes, 2004).

Chapter one

INTRODUCTION

Background of the study

Oxidative stress is a biological condition defined by an imbalance between the creation of reactive oxygen species (ROS) and the ability of the body’s antioxidant defence systems to neutralise them.

ROS, including free radicals, can damage cellular components like lipids, proteins, and DNA, resulting in a variety of diseases such as cancer, cardiovascular problems, neurodegenerative diseases, and aging-related conditions.

To combat the negative consequences of oxidative stress, the human body relies on endogenous antioxidant processes, but it can also benefit from exogenous antioxidants taken from dietary sources or medicinal plants.

Medicinal plants have long been recognised as rich sources of natural antioxidants, and their use in traditional medicine is predicated on their ability to treat oxidative stress-related illnesses.

Hippocreatea welwitschii Oliv., a plant unique to specific locations, has received attention for its purported therapeutic characteristics, which include antioxidant activity.

The ethanolic extract of Hippocreatea welwitschii Oliv. has traditionally been utilised for a variety of therapeutic purposes, and there is growing interest in knowing its total antioxidant capacity (TAC) in order to investigate its potential applications in healthcare.

The TAC of a drug is its ability to scavenge free radicals, prevent lipid peroxidation, and protect against oxidative damage. It is a key measure in determining the antioxidant capacity of natural goods, especially plant extracts.

Several approaches have been devised to assess TAC, each focussing on a different component of antioxidant activity. The ferric reducing antioxidant power (FRAP) assay and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay are both widely utilised.

The FRAP assay involves reducing a ferric tripyridyltriazine (Fe^3+-TPTZ) complex to its ferrous form (Fe^2+) in presence of antioxidants. The colour change from blue to bright blue reflects the reduced capability of the tested substance.

The DPPH test, on the other hand, assesses antioxidants’ ability to scavenge the stable DPPH radical, which causes a colour change from purple to yellow. Both assays provide useful information about antioxidants’ electron-donating and radical scavenging activities.

In the case of Hippocreatea welwitschii Oliv., few scientific investigations have concentrated on determining the TAC. Therefore, the current study intends to investigate the TAC of the ethanolic extract of Hippocreatea welwitschii Oliv. utilising the FRAP and DPPH assays.

Understanding this plant extract’s antioxidant capacity can help to determine its potential therapeutic applications in preventing or treating oxidative stress-related disorders.

The ethanolic extract of Hippocreatea welwitschii Oliv. was used for this investigation because ethanol is a commonly utilised solvent for extracting bioactive components from medicinal plants.

Ethanol extracts have been shown to be effective at extracting a variety of secondary metabolites, including phenolic chemicals, flavonoids, alkaloids, and terpenoids, all of which are known to have antioxidant effects.

These phytochemicals are known for scavenging free radicals, chelating metal ions, inhibiting lipid peroxidation, and regulating antioxidant enzyme activity.

Previous research on Hippocreatea welwitschii Oliv. has shown possible antioxidant effects, however more complete studies are needed to determine its TAC. The FRAP and DPPH assays can be used to assess the ethanolic extract’s electron-donating and radical scavenging properties.

This evaluation will shed light on the antioxidant capacity of Hippocreatea welwitschii Oliv. and contribute to a better understanding of its medicinal significance.

Furthermore, analysing the TAC of the ethanolic extract of Hippocreatea welwitschii Oliv. can aid in determining the individual bioactive components responsible for its antioxidant action.

Phytochemical study of the extract can reveal the presence of numerous antioxidant components, including phenolic acids, flavonoids, tannins, and alkaloids.

These chemicals have been extensively researched for their potential to scavenge free radicals, limit oxidative damage, and influence cellular signalling pathways involved in antioxidant defence.

The antioxidant activity of Hippocreatea welwitschii Oliv. extract may also be modified by its geographical origin, ambient conditions, and extraction methods. Variations in these parameters might cause changes in the composition and concentration of bioactive molecules, hence influencing the extract’s overall antioxidant activity.

As a result, it is critical to assess the TAC of the ethanolic extract of Hippocreatea welwitschii Oliv. employed in this investigation.

Understanding the TAC of Hippocreatea welwitschii Oliv. extract is not only scientifically significant, but also has potential applications in the healthcare and pharmaceutical industries.

Antioxidants are essential for preserving cellular health, preventing oxidative stress, and lowering the risk of chronic illnesses.

Incorporating natural antioxidants into therapeutic interventions or dietary supplements can be a safer alternative to synthetic antioxidants and may give extra health advantages.

Furthermore, TAC assessment can help with the standardisation and quality control of goods containing Hippocreatea welwitschii Oliv. extract. It guarantees that.

Chapter one

INTRODUCTION

Indigofera Linn. is a vast genus of flowering plants that belong to the subfamily Papilionoideae of the Fabaceae/Leguminosae family.

They can be found all throughout the world’s tropical and subtropical regions. Burkill (1995) identified 60 species, whereas Soladoye and Lewis (2003) identified 60 species in Nigeria, with more than 60% abundance in the northern region and 27 species scattered throughout the country’s south-west.

Indigofera, which means indigo dye in Greek, is well-known for its natural blue colours derived from the herb’s leaflets and branches. The most important species are Indigofera arrecta and Indigofera tinctoria.

Indigofera spp. adapt well to a variety of settings and have different morphological and agronomic characteristics, making them useful as forage and cover crops (Hassen et al., 2006).

Some of these species, such as Indigofera tinctoria and Indigofera suffruticosa, are used to produce indigo dyes, while others have medicinal properties, such as Indigofera articulate, which is used to treat toothache, Indigofera oblongifolia, Indigofera suffruticosa, and Indigofera aspalthoides

which are used as anti-inflammatories to treat insect stings, snake bites, and swellings (Shahjahan et al., 2005), and Indigofera arrecta extract, which is used to relieve ulcer pain.

Indigofera tinctoria stems are chewed to treat coughs, and leaf decoctions are used to treat chest pains, epilepsy, neurological disorders, asthma, bronchitis, fever, and stomach, liver, kidney, and spleen ailments, particularly in Cameroon (Takawira-Nyenya and Cardon, 2005).

The twine paste heals dislocations. Warm leaves can also help to heal wounds (Ibe and Nwufo 2005). Phytochemical analysis of Indigofera species reveals that they contain a high concentration of organic and fatty acids, as well as flavonoids such as carotenoids and coumarins.