ABSTRACT
The efficacy of Uvaria chamae plant species in herbal remedies may have come as a result of trial and error. This could be as a result of poor information on the phytochemistry, antioxidant and toxicity of this plant parts. The present study compares the in vitro and in vivo antioxidant potentials and toxicities of methanol extracts of Uvaria chamae leaves and roots. Results of in vitro antioxidant potentials revealed that the methanol extract of Uvaria chamae leaves contains vitamin A (4871±79.21 I.U) and vitamin C (1.72±0.02%) while the root extract contains vitamin A (673.28±0.00I.U) and vitamin C (1.66±0.01%). Both extracts had equal contents of vitamin E (8.83±0.04 mg/100g). The leaf extract scavenged 1,1- diphenyl-2-picrylhydrazyl radical (DPPH) in a concentration dependent manner with the correlation coefficient (R2) of 0.839 and effective concentration (EC50) of 31.19 µg/ml, while the root extract scavenged DPPH with R2, 0.778 and EC50 , 14.00 µg/ml. These results were compared to the EC50 of ascorbic acid standard (25.29 µg/ml). The leaf and root extracts scavenged superoxide radical in a concentration dependent manner with EC50 of 5.93 µg/ml and 719.45 µg/ml, respectively, compared to the EC50 of ascorbic standard (30.27 µg/ml). Both the leaf and root extracts scavenged hydroxyl radical in a concentration dependent manner with EC50 of 107.89 µg/ml and 912.01 µg/ml, respectively, compared to the EC50 of vitamin E standard (106.66µg/ml). The result of the study revealed that the 1000 µg/ml root extract scavenged nitric oxide radical more than the leaf extract and vitamin E standard at the same concentration. At 500 µg/ml, the leaf extract was more effective at scavenging nitric oxide radical compared to the root extract and vitamin E standard. The leaf extract showed significantly higher (p<0.05) anti radical power (ARP) of superoxide (0.17) compared to the root extract (0.0014). However, the root extract showed significantly higher (p<0.05) ARP of DPPH (0.071) compared to the leaf extract (0.032). For the in vivo study, adult albino rats were divided into two sets (leaf and root extracts) of four groups each. Each group contained 8 rats. Comparative in vivo effects of the leaf and root extracts were determined by investigating the following parameters: catalase activity, liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alanine phosphatase (ALP), serum urea, serum creatinine, serum electrolytes (Na+, K+, and Cl-) and some haematological parameters – haemoglobin (Hb), packed cell volume (PCV) and white blood cell (WBC) count. Because the median lethal dose (LD50) investigation revealed one death at the dose of 5000 mg/kg b.w for the root extract and none for the leaf extract, both extracts were orally administered at 100, 200 and 400 mg/kg b.w. In each set (i.e leaf and root extracts), group 1 received normal saline and served as the control while groups 2, 3, and 4 received 100, 200 and 400 mg/kg b.w doses of the extracts, respectively. At day 7 post treatment, ALP, Cl- ,K+, urea, creatinine , AST and WBC count were significantly higher (p< 0.05) in both sets of treatment groups compared to the control. Serum Na+, Hb and PCV were significantly lower (p< 0.05) in the treatment groups compared to the control. While the leaf extract showed significantly higher (p< 0.05) ALT activity, the root extract showed no significant difference (p>0.05). At day 14, both extracts had significantly higher (p< 0.05) catalase activity, urea, creatinine, Cl-, Na+ and WBC count. While, the leaf extract had significantly higher (p< 0.05) ALT and K+, the root extract had significantly higher (p< 0.05) AST activity when compared to the control. At day 21, the root extract showed significantly higher (p< 0.05) ALT, AST, catalase , Cl- and Hb while the groups were not significantly (p>0.05) affected by the leaf extract when compared to the control . However, at day 28, both extracts showed significantly higher (p>0.05) ALT activity. While the root extract showed significantly higher (p< 0.05) Na+ and ALP activity, the leaf extract showed none for them when compared to the control. Histological analysis showed some levels of toxicity at doses of 100, 200 and 400 mg/kg b. w at chronic stage (beyond 14 days of extracts’ administration). These results suggest that fluctuations at the initial period were as a result of the homeostatic processes in attempt for the organism to maintain normal body functioning at the end of the 28- day administrations of both extracts. Although the leaf extract was more efficacious in maintaining the normal body metabolism; the moderate toxicity exhibited by the extracts from LD50, ALT, AST and histopathological tests could compromise its efficacy in chronic phase of treatment
CHAPTER ONE
INTRODUCTION
Over the years, man has been facing with the challenges of preventing and eliminating diseases in the body. The discovery of the efficacy of certain plant species in herbal remedies by man, might have come as a result of trial and error. This however, has created some gaps in common beliefs on the treatment of ailments among some related and unrelated human societies of the world. Phytochemical analysis on certain plant species by modern practitioners have shown some corresponding results with already existing tradomedical information while in some cases, has differed completely thereby causing doubt in herbal treatment (Nwachukwu et al., 2011). In recent times, some plants including Uvaria chamae, have been used as herbal medicines due to the presence of phytochemicals and antioxidants in them (Riby et al., 2006). These Antioxidants are vital substances which protect the body from damages caused by free radical-induced oxidative stress (Awah et al.,
2010). However, the herb can display some toxicological properties. The assay of enzyme activities in the body fluid of any model in question, aids the diagnosis of the damages on the vital organs and as well, assists in the determination of its toxicity (Ajiboye et al., 2010).
1.1 Profile of Uvaria chamae
Uvaria chamae belongs to the family of Anonacaea. It is a climbing large shrub or small tree native to the tropical rain forest of West and Central Africa where it grows as wet and coastal shrub (Okwu and Iroabuchi, 2004). It is also known as finger root or bush banana (Omajali et al., 2011). This common name refers to the fruit growing in its small branches; the fruit carpels are in finger-like clusters, the shape giving rise to the many native names translated as bush banana, implying wildness (Irving,1961). It is commonly called by the Igala people of the eastern part of Kogi State, Nigeria as Awuloko or Ayiloko by others, Kas Kaifi by the Hausas, Mmimi Ohea/Udagu by the Igbos, Oko Oja by the Yorubas, Akotompo by the Fula- Fante people of Ghana, Boelemimbo by the Fula-Pwaar people of Guinea Bissau, Liasa by the Yoruba- Ife people of Togo (Oliver, 2010). It is an evergreen plant that grows about 3.6 to 4.5m high, cultivated as well as wild. The plant is extensively branched with sweet, aromatic and alternate leaves commonly used to cure diseases and heal injuries (Omajali et al., 2011)
Uvaria chamae in Nigeria has a wide spread reputation as a medicinal plant. The root- decoction is used as a purgative and also as a lotion. Sap from the root and stem is applied to wounds and sores; the root is made into a drink and a body wash for oedematous condition. The root bark yields an oleo- resin that is taken internally for catarrhal inflammation of mucous membranes, respiratory catarrh and gonorrhea while the root extract is used in phyto medicine for the treatment of piles ,epitasis, haematuria and haemolysis (Oliver, 2010). It is a medicinal plant used in the treatment of fever and injuries (Bukill, 1989). There are other oral claims that the plant can cure abdominal pain, used as treatment for piles, wounds, sore throat
,diarrhea etc (Bukill, 1989). In Ghana, the root with Guinea grains is used in application to the fontanelle for cerebral diseases. Among the Fulai people of Senegal, the root has a reputation as the “medicine of riches” and is taken for conditions of lassitude and senescence. It is also considered to be a woman’s medicine used for amenorrhea and to prevent miscarriage and in Togo, a root-decoction is given for pains of childbirth (Okwu and Iroabuchi, 2004). It is used for the treatment of jaundice in Ivory- coast. In Sierra Leone, the root is reputed for having purgative and febrifugal properties. In Nigeria however, the root- bark is used for the treatment of bronchitis, and gonorrhea in addition to its being used internally for catarrhal inflammation of mucous membranes (Okwu and Iroabuchi, 2004).
Fig. 1 shows the Uvaria chamae plant parts.
Fig. 1: Uvaria chamae plant (Schimidt, 1987)
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1.2 Phytochemistry
The medicinal value of some medicinal plants has a link with the phytochemicals in them. These phytochemicals are chemical compounds that occur naturally in plants (phyto means “plant” in Greek). Some are responsible for color, smell etc. The term is generally used to refer to those chemicals that may have biological significance. There may be as many as
4,000 different types. Example of such phytochemicals include: alkaloids, flavonoids,
saponin, tannins etc (Palermo et al., 2014).
1.2.1 Alkaloids
Alkaloids are a group of naturally occurring chemical compounds (natural products) that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral and even weakly acidic properties (Manske, 1965). Some synthetic compounds of similar structure are also termed alkaloids. In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely other elements such as chlorine, bromine, and phosphorus (Manske, 1965).
Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals. They can be purified from crude extracts of these organisms by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications, as recreational drugs, or in entheogenic rituals. Examples are the local anesthetic and stimulant cocaine, the psychedelic psilocin, the stimulant caffeine, nicotine, the analgesic morphine (Raymond et al., 2010), the antibacterial berberine, the anticancer compound vincristine, the anti hypertension agent, reserpine, the cholinomimetic galantamine, the anticholinergic agent, atropine, the vasodilator vincamine, the anti arrhythmia compound quinidine, the anti asthma therapeutic ephedrine, and the anti malarial drug quinine. Although, alkaloids act on a diversity of metabolic systems in humans and other animals, they almost uniformly invoke a bitter taste (Rhoades, 1979).
The boundary between alkaloids and other nitrogen-containing natural compounds is not clear-cut. Compounds like amino acid peptides, proteins, nucleotides, nucleic acid, amines, and antibiotics are usually not called alkaloids (Raj, 2004). Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed
to amines rather than alkaloids. Some authors, however, consider alkaloids a special case of amines (Raj, 2004).
1.2.2 Flavonoids
Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow that is, their color in nature) are a class of plant secondary metabolites. They were referred as vitamin P (probably because of the effect they had on the permeability of vascular capillaries) from the mid-1930s to early 50s, but the term has since fallen out of use (Mobh, 1938) .Flavonoids have hydroxyl group (OH). The effect of the hydroxyl moiety of flavonoids on protein targets varies depending on the position and number of the moiety on the flavonoid skeleton (Mobh,
1938). A typical flavonoid is shown in Fig. 2.
Fig. 2: Quercetin, a typical flavonoid (Amorati and Valgimigli, 2012)
1.2.3 Tannin
A tannin (also known as vegetable tannin, natural organic tannins or sometimes tannoid, i.e. a type of biomolecule, as opposed to modern synthetic tannin) is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids. They form complexes also with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion. The tannin phenolic group is an excelent hydrogen donor that forms strong hydrogen bonds with the protein’s carboxyl group (Amorati and Valgimigli, 2012). The anti carcinogenic and anti mutagenic potentials of tannins may be related to their anti oxidant property (Amorati and Valgimigli, 2012). The anti-microbial properties seemed to be associated with the hydrolysis of ester linkage between gallic acid and polyols hydrolyzed after ripening of many edible fruits (Amorati and Valgimigli, 2012).
1.2.4 Total Phenolics
In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds consisting of a hydroxyl group (—OH) bonded directly to an aromatic
hydrocarbon group. The simplest of the class is phenol, which is also called carbolic acid C6H5OH. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule (Amorati, and Valgimigli, 2012). Fig. 3 shows the
structure of total phenol.
Fig. 3: Phenol (Amorati, and Valgimigli, 2012)
Phenolic compounds are synthesized industrially; they also are produced by plants and microorganisms, with variation between and within species (Hättenschwiler and Vitousek,
2000). Although, similar to alcohols, phenols have unique properties and are not classified as
alcohols (since the hydroxyl group is not bonded to a saturated carbon atom). They have higher acidities due to the aromatic ring’s tight coupling with the oxygen and a relatively loose bond between the oxygen and hydrogen. The acidity of the hydroxyl group in phenols is commonly intermediate between that of aliphatic alcohols and carboxylic acids (their pKa is usually between 10 and 12).
Loss of a positive hydrogen ion (H+) from the hydroxyl group of a phenol forms a corresponding negative phenolate ion or phenoxide ion, and the corresponding salts which are called phenolates or phenoxides. As they are present in food consumed in human diets and in plants used in traditional medicine of several cultures, their role in human health and disease is a subject of research (Mishra and Tiwari, 2011).Some phenols are germicidal and are used in formulating disinfectants. Others possess estrogenic or endocrine disrupting activities. Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Mishra and Tiwari, 2011). Antioxidant activity of plant extracts is not limited to phenolics. Activity may also come from the presence of other antioxidant secondary metabolites, such as volatile oils, carotenoids and vitamins A,C and E.
are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby, improve the quality and nutritional value of food. In plants, oils are basically monophenolics such as tocopherols, water-soluble polyphenols are more typical in water-soluble products like fruits, vegetables, tea, coffee, wine, among others (Mishra and Tiwari, 2011). Polyphenolic compounds are known to have antioxidant activity. This activity is due to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Mishra and Tiwari, 2011).
1.3 Acute toxicity
Acute toxicity describes the adverse effects of a substances that result either from a single exposure or from multiple exposures in a short space of time (less than 24 hours ). Most acute toxicity data come from animal testing or in vitro testing methods (Walum, 1998). The median lethal dose (LD50) is the dose required to kill half the members of a tested population after a specified test duration (Lorke, 1983). Investigation of the acute toxicity is the first step in the toxicological investigations of an unknown substance. The index of the acute toxicity is the LD50 (Lorke, 1983). Scientific investigation of previously unknown and known plants is necessary not only because of the need to discover new drugs but to assess the toxicity faced by the users. Besides, it is important that traditionally claimed therapeutic properties of plants be confirmed and its toxicity limit determined (Prohp amd Onoagbe,
2012).
1.4 Reactive oxygen species (ROS)
These are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g. UV or heat exposure, ROS levels can increase dramatically (Devasagayam et al., 2004). This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. Reactive oxygen species are also generated by exogenous sources such as ionizing radiation. Normally, cells defend themselves against ROS damage with enzymes such as a superoxide dismutases, catalases, lactoperoxidases, glutathione peroxidases and peroxiredoxins. Small molecule antioxidants such as ascorbic acid (vitamin C), tocopherol (vitamin E) and glutathione also play important
roles as cellular antioxidants. In a similar manner, polyphenol antioxidants assist in preventing ROS damage by scavenging free radicals. In contrast, the antioxidant ability of the extracellular space is less, the most important plasma antioxidant in humans is uric acid. Effects of ROS on cell metabolism are well documented in a variety of species. These include not only roles in apoptosis (programmed cell death) but also positive effects such as the induction of host defence genes and mobilisation of ion transport systems (Rada and Leto,
2008). This implicates them in control of cellular function. In particular, platelets involved in wound repair and blood homeostasis release ROS to recruit additional platelets to sites of injury. These also provide a link to the adaptive immune system via the recruitment of leukocytes. Reactive oxygen species are implicated in cellular activity to a variety of inflammatory responses including cardiovascular disease. They may also be involved in hearing impairment via cochlea damage induced by elevated sound levels, in ototoxicity of drugs such as cisplatin, and in congenital deafness in both animals and humans. In general, harmful effects of reactive oxygen species on the cell are most often :
damage of DNA
oxidations of polyunsaturated fatty acids in lipids (lipid peroxidation)
oxidations of amino acids in proteins
oxidatively inactivate specific enzymes by oxidation of co-factors
1.4.1 Pathogen response
When a plant recognizes an attacking pathogen, one of the first induced reactions is to rapidly produce superoxide (O2−) or hydrogen peroxide (H2O2) to strengthen the cell wall. This prevents the spread of the pathogen to other parts of the plant, essentially forming a net around the pathogen to restrict movement and reproduction. In the mammalian host, ROS is
induced as an antimicrobial defense. To highlight the importance of this defense, individuals with chronic granulomatous disease who have deficiencies in generating ROS, are highly susceptible to infection by a broad range of microbes including Salmonella enterica, Staphylococcus aureus, Serratia marcescens, and Aspergillus species (Patel et al., 1999).
1.4.2 Oxidative damage
In aerobic organisms the energy needed to fuel biological functions is produced in the mitochondria via the electron transport chain. In addition to energy, reactive oxygen species (ROS) with the potential to cause cellular damage are produced. Reactive oxygen species can
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damage DNA, RNA, and proteins, which, in theory, contributes to the physiology of ageing. (Patel et al,1999).
Reactive oxygen species are produced as a normal product of cellular metabolism. In particular, one major contributor to oxidative damage is hydrogen peroxide (H2O2), which is converted from superoxide that leaks from the mitochondria. Catalase and superoxide dismutase ameliorate the damaging effects of hydrogen peroxide and superoxide, respectively, by converting these compounds into oxygen and hydrogen peroxide (which is later converted to water), resulting in the production of benign molecules. However, this conversion is not 100% efficient, and residual peroxides persist in the cell. While ROS are produced as products of normal cellular functioning, excessive amounts can cause deleterious effects (Patel et al., 1999). Memory capabilities decline with age, evident in human degenerative diseases such as Alzheimer’s disease, which is accompanied by an accumulation of oxidative damage. Current studies demonstrate that the accumulation of ROS can decrease an organism’s fitness because oxidative damage is a contributor to senescence. In particular, the accumulation of oxidative damage may lead to cognitive dysfunction, as demonstrated in a study in which old rats were given mitochondrial metabolites and then given cognitive tests. Results showed that the rats performed better after receiving the metabolites, suggesting that the metabolites reduced oxidative damage and improved mitochondrial function (Liu et al., 2002).Accumulating oxidative damage can then affect the efficiency of mitochondria and further increase the rate of ROS production (Stadtman, 1992). The accumulation of oxidative damage and its implications for aging depends on the particular tissue type where the damage is occurring. Additional experimental results suggest that oxidative damage is responsible for age-related decline in brain functioning. Older gerbils were found to have higher levels of oxidized protein in comparison to younger gerbils. Treatment of old and young mice with a spin trapping compound caused a decrease in the level of oxidized proteins in older gerbils but did not have an effect on younger gerbils. In addition, older gerbils performed cognitive tasks better during treatment but ceased functional capacity when treatment was discontinued, causing oxidized protein levels to increase. This led researchers to conclude that oxidation of cellular proteins is potentially important for brain function (Carney et al., 1991).
1.4.3 Classification of ROS
1.4.3.1 Exogenous ROS
Exogenous ROS can be produced from external sources such as pollutants: tobacco, smoke, drugs, xenobiotics, or radiation. Ionizing radiation can generate damaging intermediates through the interaction with water, a process termed radiolysis (Lien et al., 2008). Since water comprises 55–60% of the human body, the probability of radiolysis is quite high under the presence of ionizing radiation. In the process, water loses an electron and becomes highly reactive. Then through a three-step chain reaction, water is sequentially converted to hydroxyl radical (-OH), hydrogen peroxide (H2O2), superoxide radical (O2-) and ultimately oxygen (O2). The hydroxyl radical is extremely reactive that immediately removes electrons from any molecule in its path, turning that molecule into a free radical and so propagating a chain reaction. But hydrogen peroxide is actually more damaging to DNA than hydroxyl radical since the lower reactivity of hydrogen peroxide provides enough time for the molecule to travel into the nucleus of the cell, subsequently wreaking havoc on macromolecules such as DNA (Lien et al., 2008).
1.4.3.2 Endogenous ROS
Reactive oxygen species are produced intracellularly through multiple mechanisms and depending on the cell and tissue types, the major sources being the “professional” producers of ROS NADPH oxidase (NOX) complexes (7 distinct isoforms) in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum (Muller, 2000). Mitochondria convert energy for the cell into a usable form, adenosine triphosphate (ATP). The process in which ATP is produced, called oxidative phosphorylation, involves the transport of protons (hydrogen ions) across the inner mitochondrial membrane by means of the electron transport chain. In the electron transport chain, electrons are passed through a series of proteins via oxidation-reduction reactions, with each acceptor protein along the chain having a greater reduction potential than the previous. The last destination for an electron along this chain is an oxygen molecule. In normal conditions, the oxygen is reduced to produce water; however, in about 0.1–2% of electrons passing through the chain (this number derives from studies in isolated mitochondria, though, the exact rate in living organisms is yet to be fully agreed upon), oxygen is instead prematurely and incompletely reduced to give the superoxide radical (·O2-), most well documented for Complex I and Complex III ( Li et al., 2013). Superoxide is not particularly reactive by itself, but can inactivate specific enzymes or initiate lipid peroxidation in its protonated form, hydroperoxyl HO2·. The pKa of hydroperoxyl is 4.8. Thus, at physiological pH, the majority will exist as superoxide anion.
If too much damage is present in mitochondria, a cell undergoes apoptosis or programmed cell death. Bcl-2 proteins are layered on the surface of the mitochondria, detect damage, and activate a class of proteins called Bax, which punch holes in the mitochondrial membrane, causing cytochrome C to leak out. This cytochrome C binds to Apaf-1, or apoptotic protease activating factor-1, which is free-floating in the cell’s cytoplasm. Using energy from the ATP in the mitochondrion, the Apaf-1 and cytochrome C bind together to form apoptosomes. The apoptosomes bind to and activate caspase-9, another free-floating protein. The caspase-9 then cleaves the proteins of the mitochondrial membrane, causing it to break down and start a chain reaction of protein denaturation and eventually phagocytosis of the cell (Li et al., 2013).
1.5 Antioxidant
An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents (Sies, 1997). Although, oxidation reactions are crucial for life, they can also be damaging; plants and animals maintain complex systems of multiple types of antioxidants, such as antioxidant vitamins,: vitamin C, vitamin E etc as well as anti oxidant enzymes such as catalase, superoxide dismutase and various peroxidases. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Oxidative stress is damage to cell structure and cell function by overly reactive oxygen-containing molecules and chronic excessive inflammation. Oxidative stress seems to play a significant role in many human diseases, including cancers. The use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. For these reasons, oxidative stress can be considered to be both the cause and the consequence of some diseases (Lien et al., 2008).
Antioxidant vitamins are widely used in dietar y supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials with a limited number of antioxidants detected no benefit and even suggested that excess supplementation with certain putative antioxidants may be harmful (Jha et al., 1995).
1.5.1 Ascorbic acid (vitamin C)
Ascorbic acid or “vitamin C” is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin (Smirnoff, 2001). Most other animals are able to produce this compound in their bodies and do not require it in their diets (Linster and van Schftingen, 2007). Ascorbic acid is required for the conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins (Wells et al., 1990). Ascorbic acid is a redox catalyst which can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide (Padayatty et al., 2003). In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme ascorbate peroxidase, a function that is particularly important in stress resistance in plants (Shigeoka et al., 2002).Ascorbic acid is present at high levels in all parts of plants and can reach concentrations of 20 millimolar in chloroplasts (Smirnoff and Wheeler, 2000).
1.5.2 Tocopherols (vitamin E)
Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties (Herrera and Barbas, 2001). Among these, α-tocopherol has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolising this form (Brigelius–Flohe and Traber, 1999). It has been claimed that the α-tocopherol form is the most important lipid-soluble antioxidant, and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction (Herrera and Barbas, 2001). This removes the free radical intermediates and prevents the propagation reaction from continuing. This reaction produces oxidised α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol (Wang and Quinn, 1999). This is in line with the findings showing that α-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death (Seiler et al., 2008). GPX4 is the only known enzyme that efficiently reduces lipid- hydroperoxides within biological membranes.
1.5.3 Catalase
Catalase is a common antioxidant enzyme found in nearly all living organisms exposed to oxygen (such as vegetables, fruit or animals). It catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al., 2004). It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS). Likewise, catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second (Goodsell,2004).
1.6 1,1-Diphenyl-2-picrylhydrazylradical (DPPH) assay
Fig. 4: Structure of 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) (Sagar and Singh, 2011)
The compound, 1,1-diphenyl-2-picrylhydrazyl radical, as shown in Fig. 4, is the full name for the abbreviation of an organic chemical compound DPPH. It is a dark-colored crystalline powder composed of stable free-radical molecules. The compound DPPH, has two major applications, both in laboratory research: one is a monitor of chemical reactions involving radicals, most notably, it is a common antioxidant assay and another is a standard of the position and intensity of electron paramagnetic resonance signals (Om and Tej, 2009). The compound is a well-known radical and a trap (“scavenger”) for other radicals. Therefore, rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction. Because of a strong absorption band centered at about 520 nm, DPPH radical has a deep violet color in solution, and it becomes colorless or pale yellow when xxvii neutralized. This property allows visual monitoring of the reaction, and the number of initial radicals can be counted from the change in the optical absorption at 520 nm (Om and Tej, 2009).
1.7 Liver function tests (LFTs or LFs)
Liver function tests (LFTs or LFs) are groups of blood tests that give information about the state of a patient’s liver (Om and Tej, 2009). Liver transaminases (AST or SGOT and ALT or SGPT) are useful biomarkers of liver injury in a patient with some degree of intact liver function (Johnston, 1999). Most liver diseases cause only mild symptoms initially, but these diseases must be detected early. Hepatic (liver) involvement in some diseases can be of crucial importance. This testing is performed on a patient’s blood sample. Some tests are associated with functionality (e.g., albumin), some with cellular integrity (e.g., transaminase), and some with conditions linked to the biliary tract (gamma-glutamyl transferase and alkaline phosphatase). Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage, and follow the response to treatment. Some or all of these measurements are also carried out (usually about twice a year for routine cases) on those individuals taking certain medications, such as anticonvulsants, to ensure the medications are not damaging the person’s liver (Johnston, 1999).
1.8 Renal Function tests
Renal function, in nephrology, is an indication of the state of the kidney and its role in renal physiology. Glomerular filtration rate (GFR) describes the flow rate of filtered fluid through the kidney. Creatinine clearance rate (CCr ) is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for approximating the GFR. Creatinine clearance exceeds GFR due to creatinine secretion, which can be blocked by cimetidine. In alternative fashion, overestimation by older serum creatinine methods resulted in an underestimation of creatinine clearance, which provided a less biased estimate of GFR (Stevens et al., 2006). Both GFR and CCr may be accurately calculated by comparative measurements of substances in the blood and urine, or estimated by formulas using just a blood test result (eGFR and eCCr). The results of these tests are important in assessing the excretory function of the kidneys. For example, grading of chronic renal insufficiency and dosage of drugs that are excreted primarily via urine are based on GFR (or creatinine clearance). It is commonly believed to be the amount of liquid filtered out of the blood that gets processed by the kidneys. In physiological terms, these quantities (volumetric blood flow and mass removal) are related only loosely (Stevens et al., 2006).
1.9 Serum Electrolytes
1.9.1 Sodium
Sodium is the dominant extracellular cation (positive ion) and cannot freely cross from the interstitial space through the cell membrane, into the cell. Its homeostasis (stability of concentration) inside the cell is vital to the normal function of any cell. Hyponatremia is low sodium concentration in the serum. Exercise can induce hyponatremia. When sodium levels in the blood become excessively low, excess water enters the brain cells and the cells swell. This can lead to headache, nausea, vomiting and seizures ( Moritz and Ayus, 2003). The main source of body sodium is sodium chloride contained in ingested foods (Terri and Sesin, 1958). Hyponatremia is found in a variety of conditions including the following: severe polyuria, metabolic acidosis, Addison’s disease, diarrhoea and renal tubular disease. Hypernatremia (increased serum sodium level) is found in the following conditions: hyperadrenalism, severe dehydration, diabetic coma after therapy with insulin, excess treatment with sodium salts (Maruna, 1958).
1.9.2 Potassium
Potassium, a metallic inorganic ion is the most abundant cation in the body. The vast majority of potassium is in the intracellular compartment with a small amount in the extracellular space. Total body potassium is approximately 55 mEq/Kg body weight. The intracellular potassium concentration is on average 150 mEq/L. The ratio of intracellular to extracellular K+ (K1: K2) is the major determinant of the resting membrane potential and plays a crucial role in the normal functioning of all cells, especially those with inherent excitability (Arruda et al, 1981). Elevated Potassium (Hyperkalemia) is often associated with renal failure, dehydration, shock or adrenal insufficiency. Decreased Potassium concentration in the plasma (Hypokalemia) are associated with malnutrition, negative nitrogen balance, gastro intestinal fluid losses and hyperactivity of adrenal cortex (Terri and Sesin, 1958).
1.9.3 Chloride
An abnormal elevation of chloride ion concentration in the blood is hyperchloremia. It is associated with excess fluid loss such as vomiting and diarrhoea. Diabetes exacerbates it (Cambier et al, 2004). Non steroidal anti inflammatory drugs can modulate chloride concentration in the blood. Chloride is the major negative ion in the fluid outside the body’s cells. Its main function is to maintain electrical neutrality, mostly as a counter – ion to sodium (Terri and Sesin, 1958. Elevated serum chloride values may be seen in dehydration, hyperventilation, congestive heart valve and prostatic obstruction (Skeggs and Hochstrasser, 1964).
1.10 Haematology
Haematology is the study of blood, the blood-forming organs, and blood diseases (Sidell and Kristin, 2006). Haematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases that affect the production of blood and its components, such as blood cells, haemoglobin, blood proteins, and the mechanism of coagulation.
Packed Cell Volume (PCV) or Erythrocyte Volume Fraction (EVF), is the volume percentage of red blood cells in the body. It is considered to be an integral part of a person’s complete blood count results, along with haemoglobin (Hb) concentration, white blood cell (WBC) count and platelet count. Erythropoeitin is secreted by the kidney (Jelkmann, 2004). So by extension, low level of red blood cells points to a problem with the integrity of the kidney cells (Jelkmann, 2004).
Haemoglobin is the iron-containing oxygen- transport metalloprotein in the blood cells of almost the vertebrates. It carries oxygen from the lungs to body parts for the metabolism of glucose in order to generate energy (Sidell and Kristin, 2006). If haemoglobin is low, it signifies anaemia. Anaemia is a condition in which the number of red blood cells is insufficient to meet the body’s needs (WHO, 2001). Sickle cell anaemia is the most important haemoglobinopathy (Murray et al., 2006). The functions of blood are many and varied. Besides providing material nourishments, blood also provides the necessary moisture needed by the internal organs to function properly. Insufficient blood or blood deficiencies can cause many problems ranging from weakness, inability to concentrate, hot flushes, increased
susceptibility to infection, shortness of breath, fatigue, dizziness, palpitation, anxiety, depression, insomnia, nervousness, headache and diminished sex drive. Women in particular, are especially susceptible to blood deficiencies due to their monthly menstrual cycle. In addition, because the life span of the red blood cells is relatively short, the blood needs to be constantly replenished (Murray et al., 2006). In Nigeria, the local people are known for using natural herbs and herbal formulae for addressing various kinds of blood deficiencies. In south-eastern Nigeria, the roots of Uvaria chamae among others, are considered excellent natural herbal blood boosters, used especially for debilitating conditions, acute blood loss and blood deficiency diseases (Murray et al., 2006).
1.11 Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue fragments.
1.12 Aim and Objectives of the Study
1.12.1 Aim of the Study
The aim of this study is to compare the in vitro antioxidant potentials as well as toxicity of the methanol extracts of Uvaria chamae leaves and roots with a view to suggesting which of the plant part is more beneficial to be used in the treatment of the ailments such as inflammation, respiratory tract infection, Gastro intestinal tract infection in folk medicine.
1.12.2 Specific Objectives of the study
The study was designed to achieve the following specific objectives:
To determine the phytochemical constituents of Uvaria chamae leaf and root plant parts.
To determine the antioxidant potentials of methanol extracts of Uvaria chamae leaves and roots.
To determine the possible acute and chronic toxicity of methanol extracts of Uvaria chamae leaves and roots with a view to determining safe dose of the extracts.
To determine the effects of methanol extracts of Uvaria chamae leaves and roots on the activities of liver marker enzymes and levels of some kidney function profiles.
To determine the possible effects of methanol extracts of Uvariae chamae leaves and roots on the tissues of the kidney and liver of albino rats through histological technique, to confirm the biochemical results obtained for liver and kidney functions.
To determine the effects of methanol extracts of Uvaria chamae leaves and roots on some haematological parameters.
To compare the phytochemical properties, antioxidant potential and the toxicological properties of methanol extracts of Uvaria chamae leaves and roots.
This material content is developed to serve as a GUIDE for students to conduct academic research
COMPARATIVE STUDIES OF ANTIOXIDANT AND TOXIOLOGICAL PROPERTIES OF METHANOL EXTRACTS OF UVARIA CHAMAE LEAVES ANDROOTS
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