COMPARATIVE STUDIES OF ANTIOXIDANT AND TOXIOLOGICAL PROPERTIES OF METHANOL EXTRACTS OF UVARIA CHAMAE LEAVES ANDROOTS

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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 (BrigeliusFlohe 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.



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COMPARATIVE STUDIES OF ANTIOXIDANT AND TOXIOLOGICAL PROPERTIES OF METHANOL EXTRACTS OF UVARIA CHAMAE LEAVES ANDROOTS

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