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).
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)
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.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 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).
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