Abstract
One of the problems associated with medicinal plants in Nigeria is paucity of information on the phytochemistry and toxicity of some of these plants. The study aimed at revealing a range of phytochemicals in the plant, which are physiologically potent in ameliorating several diseases. The potent secondary metabolites in the leaves of this plant Securidaca longepedunculata , were extracted and their antioxidant and toxicological potentials were evaluated using in vitro methods and albino rats as the models. The results showed that methanol extract scavenged 1, 1- diphenyl-2- picrylhydrazyl radical (DPPH.) in a concentration dependent manner with a correlation coefficient (R2) of 0.976, indicating antioxidant activity with effective concentration that inhibits 50 percent of the radicals (EC50) of 90.02±0.2 µg/ml compared to ascorbic acid standard with EC50 of 98.01±0.2 µg/ml. Superoxide radical scavenging activity was concentration dependent with an EC50 of
350.11±0.42 µg/ml compared with ascorbic acid standard with EC50 of 812.97±0.97 µg/ml. The extract, also showed hydroxyl radical scavenging activity with an EC50 of 83.74±0.02µg/ml compared to α- tocopherol standard with EC50 of 54.16±0.01 µg/ml. There was an inverse correlation between the percentage inhibition and concentration with R2 of -0.958. The methanol extract, also scavenged nitric oxide radical in a concentration dependent manner with 500 µg/ml being more effective than
500 µg/ml of ascorbic acid standard. Comparison of the anti-radical power (ARP) of DPPH. (0.011), superoxide radical (0.003) and hydroxyl radical (0.012) of the extract revealed that the ARP of the extract against hydroxyl radical was most efficacious. The antioxidant vitamin contents of the extract showed that vitamin C was significantly high (p< 0.05), (4.62±0.14 mg/100g) when compared to
vitamin A (0.902±0.05 µg/g) and vitamin E (1.474±0.01 mg/100g). The 100, 200 and 500 mg/kg bw fed to rats significantly increased (p< 0.05) catalase activity, while in weeks two and three the catalase activity decreased significantly (p< 0.05). The extract solution showed a maximum absorption at wavelength (lambdamax) of 285nm-thus indicating that subsequent investigations using the extract would be better at UV region in absorption spectra. There was no death in the mean lethal dose (LD50) investigation. The aspartate aminotransferase (AST) showed a significant decrease (p< 0.05) in week one and an increase in other weeks. The alanine aminotransferase (ALT) showed a significant decrease (p< 0.05), in weeks one, three and four while week two showed a non-significant increase (p> 0.05). The serum alkaline phosphatase (ALP) showed a significant (P 0.05) decrease in activity in all the groups at weeks one and two. At week three only group two showed a non significant increase (P 0.05); others showed a non significant decrease (P 0.05). At week four, all the groups showed an increase, but only groups two and three were significant (P
0.05) when compared to their controls group 1. Both conjugated and unconjugated bilirubin of groups two to four of weeks two to four showed generally a significant increase (p< 0.05), compared with that of the control. The serum sodium level showed a significant increase (p< 0.05), in groups two and three of week one. Weeks two to three showed a non-significant increase (p> 0.05) compared to that of the control group. Serum potassium and chloride ion concentrations showed a significant decrease (p< 0.05) in weeks one to three and significant increase (p< 0.05) in week four. The serum urea showed overall significant decrease (p< 0.05) compared to that of the control group (p< 0.05). The serum creatinine showed a significant increase (p< 0.05) in weeks one and two, a non-significant decrease (p> 0.05) in week three and a significant decrease in week four (p< 0.05). Weeks one and two rats showed significant decrease (p< 0.05) in random blood sugar (RBS), while in weeks three and four, the rats showed significant increase (p< 0.05) in RBS concentration. The packed cell volume (PCV) and haemoglobin concentration (Hb) showed significant decrease (p< 0.05) when compared with those of the control group. The white blood cell (WBC) counts of the rats showed significant increase (p< 0.05). Histological analysis showed some level of toxicity in 100, 200 and 500 mg/kg b.w. at chronic stage (beyond 14 days of administration). These results seemed to suggest a rich phytochemical constituents, suggested a moderate antioxidant activity, a relatively safe level at acute phase (within 14 days) and a visible damage (moderate toxicity) at chronic stage.
CHAPTER ONE
INTRODUCTION
1.1 Introduction
Phytomedicinal research of indigenous plant parts is presently gaining more grounds than ever as the majority of people are now patronizing herbal medicinal treatment which is considered to be more easily accessible and cheaper than orthodox medical treatment (Ajiboye et al., 2010). The usefulness of medicinal plants is directly linked to the wide range of chemical compounds synthesized in various biochemical pathways; which are classified as secondary metabolites(Ameyawand Duker-Eshun, 2009). The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body (Edoga et al., 2005; Lavanya et al., 2007). Antioxidants can delay or inhibit the oxidation of lipids, proteins, DNA or other biomolecules by inhibiting the initiation or propagation of oxidative chain reaction, (Awah et al., 2010), thereby preventing the untimely death of a cell and some ailments. The measurement of enzyme activities in the body fluid aids the diagnosis of assault on organs and tissues and also assists the determination of the toxicity of chemical compounds or drugs (Ajiboye et al., 2010).
1.2Profile ofSecuridaca longependunculata
Securidaca longepedunculata Linn belonging to the family of polygalaceae, is a spiny semi- deciduous shrub that grows to about 12m tall (Owoyele et al., 2006). It is widely distributed in West African and South African regions (Abdullahi and Lawal, 2010). The plant is known as Rhodes’ violet wild vesteria (English), uwar magunguna (Hausa) and Atumaka (Ibo). Eaten as vegetable,Securidaca longepedunculata was reported to have analgesic, anti- inflammatory and hypoglycemic potentials (Ajiboye et al., 2010), and has high efficacy in treating malaria and rheumatism(Gill, 1992). It is also used in bacterial chemotherapy (Akinniyi et al., 1996; Mohammed et al., 2012). The plant is also used in the treatment of cough, headache, constipation, wound, sore throat and gout (Ojewale, 2008). Its anti-snake venom potential has also been reported (Wannag et al., 2005). Some secondary metabolite contents such as alkaloids and flavonoids and related compounds such as methyl salicylate has been reported (Iddagoda and Thamara, 2003;Abdullahi and Lawal, (2010).
Fig.1Securidaca longepedunculata leaf and flower
1.2.1Phytochemistry
The significance of medicinal plants is directly linked to the wide range of chemical compounds synthesized in the various biochemical pathways. These compounds are classified as secondary metabolites (Ameyaw and Duker-Eshun,2009).The importance of natural molecules in medicine lies not only in their pharmaceutical or chemotherapeutic effects but also in their role as template molecules for the production of synthetic drugs. Most anti-malarial drugs currently in use are quinoline derivatives modeled on the quinine molecule. (Ameyaw and Duker-Eshun, 2009). There are three large classes of secondary metabolites in pants: Nitrogen-containing compounds, terpenoids and phenolics. Some of these phytochemicals are alkaloids, flavonoids, saponin, resins, tannins, among others.
1.2.2 Alkaloids
Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. They have pharmacological effect and biological activities such as anti-malarial, anti-microbial,
anti-hyperglycemic and anti-inflammatory effect and are used as medications, recreational drugs, or in etheogenic rituals (Tackie and Schiff, 1993). There is, also the non-basic forms such as quaternary compounds and N-oxides (Ameyaw and Duker-Eshun, 2009). Many alkaloids are poisonous to other organisms.
1.2.3 Flavonoids
Flavonoids, carotenoids among others are among the antioxidants produced by plants for their own sustenance (Ali et al., 2010). Flavonoids have hydroxyl group (OH). The effect of hydroxyl moiety of flavonoids on protein targets varies depending on position and number of the moiety on the flavonoid skeleton (Hyunchu et al.,2010).
1.2.4 Tannin
Tannins are naturally occurring plant polyphenols. They bind and precipitate proteins. They form complexes, also with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion. The tannin phenolic group is an excellent hydrogen donor that forms strong hydrogen bonds with the protein’s carboxyl group (Reed, 1995). The anti carcinogenic and anti mutagenic potentials of tannins may be related to their antioxidant property (Chung et al., 1998). 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 (Chunget al., 1998).
1.2.5 Total Phenolics
Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Kahkonen et al., 1999). 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. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics 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 (Shahidi et al., 1992). Phenolics are the main antioxidant components of food. While 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 (Roginsky and Lissi, 2004). Polyphenolic compounds are known to have antioxidant activity. This activity is believed to be mainly due
to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Ali et al., 2010).
1.3 Acute Toxicity
Acute toxicity describes the adverse effects of a substance 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. LD50 figures are frequently used as a general indicator of a substance’s acute toxicity. LD50 is dependent on the gene, animal species tested, environmental factors and mode of administration (Ernest, 2004).
1.4 Reactive Oxygen Species
Reactive oxygen species (ROS), which consist of free radicals such as hydroxyl radical (OH.), superoxide radical (O2-), Nitric oxide (NO-), peroxyl (RO2-), lipid peroxyl (LOO-) radicals and non-free radical species such as hydrogen peroxide (H2O2), singlet oxygen (O2-
1), ozone (O3), lipid peroxide (LOOH), are different forms of activated oxygen (Halliwell and
Gutteridge, 1999; Yildrin and Mavi, 2000; Gulcin et al.,2004). ROS are produced by all aerobic organisms and can easily react with most biological molecules including proteins, lipids, lipoproteins and DNA, leading to an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify or repair the resulting damage (Halliwell, 2007). During times of environmental stress, ROS levels can increase dramatically. These ROS can also generate oxidative stress. Cumulatively, ROS could lead to many pathophysiological disorders such as arthritis, diabetes, inflammation, cancer and genotoxicity (Kourounakis and Galanakis, 1999; Gulcin and Oktay, 2002).
Experiments show that ROS induce chronic inflammation by the induction of COX-2 inflammatory cytokines. These chemokines and their receptors, in turn, promote invasion and metastasis of various tumour types (Gupta et al., 2012). ROS have important roles in cell signaling and homeostasis (Davasagayam et al., 2004). Reactive oxygen species such as superoxide anions, hydroxyl radical, nitric oxide radical among others inactivate enzymes and damage the important cellular components causing injury through covalent binding and lipid peroxidation. Generation of hydroxyl radicals is crucial for the irreversible damage
inflicted by oxidative stress (Halliwell& Gutteridge, 1999). This generation mainly proceeds
via Fenton reaction:
H2O2 + Fe2+ → Fe3+ + HO– + HO•, (1)
as well as in reaction between hypochlorous acid and superoxide anion: HOCl + O2– → O2 + Cl– + HO• (2)
The rate constant of the latter reaction is greater than that of the reaction of Fe2+ with H2O2.
Decomposition of peroxynitrous acid also yields HO•: HONOO → NO2 + HO• (3)
This reaction seems to be responsible for some 20-30 % of the decay of peroxynitrite. The hydroxyl radical is an extremely reactive species and reacts at a high rate (k ~ 109–1010 M–1 · s–1) with all surrounding molecules: proteins, lipids, nucleic acidsand sugars. Because the hydroxyl radical recombinationHO• +•OH → H2O2 is also very fast (k = 5 × 109 M–1 · s–1) the steady-state concentration of hydroxyl radical is practically zero (Halliwell& Gutteridge,
1999). Nitric oxide (NO.) has also been involved in a variety of biological functions, including neurotransmission, vascular homeostasis, antimicrobial, and antitumor activities.
Despite the possible beneficial effects of NO., its contribution to oxidative damage is also reported. This is due to the fact that NO.can react with superoxide to form the peroxynitrite anion, which is a potential oxidant that can decompose to produce OH.and NO. (Rajesh and Natvar,2011). In cellular oxidation reactions, superoxide radicals are normally formed and
their effects can be magnified because they produce other kinds of cell-damaging free radicals and oxidizing agents. Superoxide is biologically important as it can form singlet oxygen and hydroxyl radical. Over production of superoxide anion radical contributes to redox imbalance, associated with harmful physiological consequences (Rajesh and Natvar,
2011).
1.5.1 Antioxidants
Antioxidants are substances that, when present in low concentrations, compared to that of an oxidizable substrate, significantly delay or prevent oxidation of that substance (Halliwell and Gutteridge, 1989).Two classes of antioxidants are known: (1). The low molecular weight compounds such as vitamins E and C, beta-carotene, glutathione, uric acid, bilirubin, among others and (2).The proteins: albumin, transferrin, caeruloplasmin, ferritin, superoxide dismutase, glutathione peroxidase, catalase, among others (Psotova et al., 2001). Typical phenolics that possess antioxidant activity have been characterized as phenolic acid and
flavonoids (Kahkonen et al., 1999). The increasing awareness of consumers to issues regarding food additive safety, results in an enhanced effort in finding alternative additives and preservatives from natural and probably safer sources. The main disadvantage with the synthetic antioxidants is their side effects when taken in vivo.In this regard, food manufacturers have been encouraged to use natural antioxidants instead of synthetic compounds to maintain the nutritional values of their products. For example, commercial antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) can be replaced by plant extracts, particularly polyphenols (Balasundram et al., 2006). Antioxidants are added to prevent or delay its (food) oxidation, normally initiated by free radicals formed during the food’s exposure to environmental factors such as air, light and temperature (Hras et al., 2000). Generation of free radicals or reactive oxygen species (ROS) during metabolism and other activities beyond the antioxidant capacity of a biological system, gives rise to oxidative stress. Oxidative stress (OS) plays a role in heart diseases, neurodegenerative diseases, cancer and in aging process (Astley, 2003). Antioxidants oppose these effects, thus lowering the risk of diseases (Atouiet al., 2005).
1.5.2 Antioxidant Vitamins
Vitamins are compounds that are essential to the health of humans and vertebrates but cannot be synthesized by these animals and must therefore be obtained in the diet (Nelson and Cox,
2005). Vitamin C, E and Beta-carotene are among dietary antioxidants, (Sies, 1992). Vitamin C is considered the most important water soluble antioxidant in extracellular fluids. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Sies,
1992). Vitamin C is capable of regenerating vitamin E. Vitamin E, a major lipid-soluble antioxidant is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Low intakes of dietary antioxidants are linked to an increased frequency of heart disease (Hennekens and Gaziano, 1993). Antioxidants have been shown to prevent low density lipoprotein (LDL) oxidation in vitro and retard the progression of atherosclerosis in animal models. Several human studies found supplemental vitamin E levels in LDL, increased the resistance of LDL oxidation and decreased the rate of LDL oxidation; LDL oxidation is speculated in atherosclerosis (Jialal and Fuller, 1993). Beta-carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. It may work synergistically with vitamin E, (Jacob, 1995). By extension, a diet that is excessively low in fat may negatively affect beta- carotene and vitamin E absorption, as well as other fat-soluble nutrients.
1.5.3 Catalase
Catalase is an enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al., 2004)
catalase
2H2O2 2H2O + O2
It is very important in protecting the cell from oxidative damage caused by reactive oxygen species (ROS), (Goodsell, 2004). Catalase is a tetramer of four polypeptide chains, each over
500 amino acids long (Boon et al., 2007). It contains four porphyrin heme (iron) groups that allow the enzyme to react with hydrogen peroxide, (Maehly and Chance, 1954). The reactivity of the iron centre may be improved by the presence of the phenolate ligand of Tyr
357 in the fifth iron ligand which facilitates the reduction of hydrogen peroxide, (Boon et al.,
2007).
1.6 1,1-Diphenyl-2-picrylhydrazyl radical(DPPH.) Assay
Fig. 2 Structures of 1,1-diphenyl-2-picrylhydrazyl radical and 1,1-diphenyl-2-picrylhydrazine
Source: Sagar and Singh, 2011
This 1,1- Diphenyl-2-PicrylHydrazyl radical (DPPH.), (Fig. 2) is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules do not dimerise like most other free radicals. The odd electron of
nitrogen atom in DPPH.is reduced by receiving a hydrogen atom from antioxidants to the
corresponding hydrazine, (Sagar and Singh, 2011). The delocalization also gives rise to the
deep violet colour, with an absorption in ethanol solution at 515- 520nm. On mixing DPPH.solution with a substance that can donate a hydrogen atom, it gives rise to the reduced form with the loss of violet colour. It thus undergoes a barthochromic shift (which is a shift from a colour of shorter wavelength to a colour of longer wavelength) (Akpanisi, 2004).
1.7.1 Liver Function Tests
The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum can help diagnose the injury of body tissues or organs especially the heart and the liver (Huang et al., 2006). AST and ALT are enzymes found mainly in the liver, but also found in red blood cells, heart cells, muscle tissue and other organs, such as pancreas and kidney. The normal concentrations in the blood are from 5 to 40 U/L (unit/litre) for AST and from 5 to 35
U/L for ALT. When body tissues or an organ such as the liver or heart is diseased or damaged, additional AST and ALT are released into the blood stream, causing the levels of the enzyme to rise. Therefore, the amount of AST and ALT in the blood is directly related to the extent of the tissue damage (Sampson et al.,1980). Alkaline Phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. In mammals, this enzyme is found mainly in the liver and bones. Marked increase in serum ALP levels, a disease known as hyperalkalinephosphatasemia, has been associated with malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis (Kim, 2006).
1.8.0 Kidney Function Test
Renal function is an indication of the state of the kidney and its role in renal physiology (Stevens et al., 2006). Creatinine clearance rate is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for approximating Glomerular Filtration Rate (GFR). Blood urea nitrogen and creatinine will not be raised above the normal range until 60% of total kidney function is lost. So there is the need to check creatinine clearance rate whenever kidney disease is suspected. A high creatinine level points to kidney disease. Low urea nitrogen concentrations were alluded to over hydration, starvation, and alcohol (Gifford and Leel-Khouri, 1989). Low urea can also be due to urea cycle defects. Serum creatinine is better than urea in assessing GFR.
1.8.1 Serum Electrolytes
1.8.1.1 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 confusion 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 (Trinder, 1951 Maruna, 1958).
1.8.1.2 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. While total body potassium is lower in females and in older patients, serum potassium concentration is independent of age and sex (Adrogue and Madias, 1981). Total body potassium is approximately 55mEq/Kg body weight. The intracellular potassium
concentration is on average 150mEq/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 (Aruda et al.,
1981). Elevated potassium (hyperkalemia) is often associated with renal failure, dehydration, shock or adrenal insufficiency. Decreased potassium levels in the plasma (hypokalemia) are associated with malnutrition, negative nitrogen balance, gastrointestinal fluid losses and hyperactivity of adrenal cortex (Terri and Sesin, 1958).
1.8.1.3 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., 1998). 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). It is therefore important to the control of proper hydration, osmotic pressure and acid/base equilibrium. Low serum Chloride values are found with extensive burns, excessive vomiting, intestinal obstruction, nephritis and metabolic acidosis. Elevated serum Chloride values may be seen in dehydration, hyperventilation, congestive heart valve and prostatic obstruction (Skeggs and Hochstrasser, 1964).
1.9 Haematology
Packed cell volume (PCV) or erythrocyte volume fraction (EVF), is the volume percentage (%) of red blood cells in blood. It is normally about 45% for men and 40% for women (Purves, et al., 2004). It is considered an integral part of a person’s complete blood count results, along with haemoglobin concentration, white blood cell count and platelet count. Erythropoietin is secreted by the kidney. 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 all the vertebrates. It carries oxygen to body parts for the metabolism of glucose in order to generate energy (Sidell and Kristin, 2006). If haemoglobin is low, it is called anemia. Anemia is a condition in which the number of red blood cells (and consequently their oxygen-carrying capacity) is insufficient to meet the body’s needs, (WHO, 2001). Sickle cell anemia is the most important hemoglobinopathy, (Murray et al., 2006). Residential elevation above sea level and smoking are known to increase haemoglobin concentrations, (WHO, 2001).
1.10 Histopathology
Histopathology refers to microscopic examination of tissue in order to study the manifestation of disease. Histopathological examination starts with surgery, biopsy or autopsy. Fixative stabilized the tissue and thereby prevented decay. The most common fixative is formalin (10% formaldehyde in water). The medical diagnosis is formulated as pathology report describing the histological findings and the opinion of the pathologist.
1.11.0 Aim and Objectives
1.11.1 Aim
The outstanding disadvantages of herbal medicinal treatment are that the toxicity of the concoctions taken by patients is not determined nor the active principles standardized. The main aim of this study is to investigate,in vitro, antioxidant contents of the methanol extract of Securidaca longepedunculata and also to determine the toxicity of this plant widely used in Nigerian folk medicine to cure various ailments.
1.11.2 Objectives
The study was designed to achieve the following specific objectives:
1. To determine the phytochemical constituents of Securidaca longepedunculata
2. To determine its antioxidant potential
3. To determine possible acute toxicity(LD50) concentration of Securidaca longepedunculata with a view to determining the non-toxic safe dose of the extract.
4. To determine the effect of the extract on liver marker enzymes and kidney function profile.
5. Determination of the possible effects of Securidaca longepedunculata methanol extract on the kidney and liver cells of Wister male rats through histological technique. 6. To determine the effect of the extract on blood cells.
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PHYTOCHEMICALS ANTIOXIDANTS AND TOXICOLOGICAL PROPERTIES OF METHANOL EXTRACT OF SECURIDACA LONGEPEDUNCULATA>
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