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ANTIMYCOTIC ACTIVITY AND TOXICOLOGICAL EFFECTS OF STEM BARK EXTRACT OF VITELLARIA PARADOXA IN WISTAR RATS.

Byline: Ahmed, Risikat .N and Sani, . A

ABSTRACT:

Vitellaria paradoxa (Shea butter plant) found abundant in the derived savannah zones of Nigeria and reported to possess a vast number of healing properties by indigenous communities was investigated for antimycotic activity and toxicological effects in wistar strain rats randomized into 8 groups of 3 rats each. The extract from the plant bark was prepared by cold extraction and concentrated using a rotary evaporator after which it was further reconstituted by standard procedures. The experimental rats were artificially induced with tinea by Trichophyton rubrum and later treated with the aqueous extract at doses of 50, 100, 200, 300 and 400 mg/kg body weight over a period of 14 days in order to investigate the activity of the extract. The toxicological effects of the extract was carried out using standard assay kit for the determination of some marker enzyme activities for liver, kidney and serum. Histopathological examination was conducted using haematoxylin and eosin staining techniques.

The phytochemical Screening of the crude extract was done. Data obtained were expressed as means of 3 replicates +- SD and subjected to one way Analysis of Variance (ANOVA) and Duncan's multiple range test at p (less than) 0.05. The extract showed therapeutic action on infection caused by Trichophyton rubrum without any adverse effect on the organs at 100-300 mg/kg body weight doses. However there were significant differences in enzyme levels at p (less than) 0.05 with doses of 300 and 400mg/kg bd. Wt exhibiting alteration when compared with the control group. Histopathological examination revealed slight degree of damage done on the tissues investigated. Several active constituents were got by the phytochemical screening of the extract which may have contributed to its efficacy. The possible incorporation of the Aqueous extract of the stem bark of Vitellaria paradoxa in the therapeutic treatment of dermatitis caused by T. rubrum is recommended and considered safe at doses of 50 to 300mg/kg bd. Wt.

Keywords : Vitellaria paradoxa, Trichophyton rubrum, Wistar rats, Phytochemical, Toxicity.

INTRODUCTION

The contribution of herbal product to modern medicine is well known and life in most parts of Africa begins and ends with herbal medicine. About 65-80% of world's population relies on traditional medicine for their health care needs [1].

According to the United Nations Conference on Trade and Development, 33% of total modern drugs produced by industrialized countries are plant based [2]. In Nigeria, thousands of plant species are known to have medicinal values [3] and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times [4]. As a result of better cultural acceptability and fewer side effects herbal medicine still remains the mainstay of 75-80% of the whole population in the developing countries for primary health care [5].

A medicinal plant has been described as any plant in which, one or more of its parts contain substances that can be used for therapeutic purpose or which acts as precursors for the synthesis of useful drugs [3]. During the last century, the practice of herbalism became mainstream throughout the world. This has been attributed to the recognition of the value of traditional medical systems and the identification of medicinal plants from indigenous pharmacopoeias which have significant healing powers [6][7]. Medicinal plants are a source of great economic value in the African continent [8] and the ethnomedicinal resources of Africa remain largely unexploited. Medicinal plants can be described as natures' pharmacy for nearly 80% of people living in Africa [9]. Nature has bestowed on us a very rich botanical wealth and a large number of diverse plants growing in different parts of the continent.

However the medicinal values of these plants lie in their component phytochemicals which produce definite physiological actions on the human body [10,11]. Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. It is well known that plants produce these chemicals to protect themselves and recent researches have demonstrated that they can also protect humans against diseases. These chemicals possess varied actions such as antioxidants, hormonal activity, stimulatory activities ,antimicrobial effects e.t.c. ). A vast majority of herbal medicines contain a complex mixture of chemical classes of compounds such as terpenes, alkaloids, polysaccharides or other chemicals that buffer, modulate and modify the effects of any active ingredient in the herb[12].

These active antimicrobial compounds in plants represents a useful area for development of natural products that can be used as substitutes for pathogenic bacteria and fungi resistant to existing conventional antibiotics. However, the potency of herbal remedies soon became an issue of dispute due to lack of qualitative and quantitative identification of their bioactive components[3].

Despite the popular use of herbal preparations for the treatment of infectious diseases there is limited scientific data available regarding the safety aspects of some of these remedies [13]. Many traditional medicines and foods especially in the tropical regions of Africa and Asia contain renal toxic plants. One of such food/medicine is the djenkol bean, a pungent smelling edible fruit of the hardwood tree Pithecellobium labatum [14]. In recent times published literatures have indicated the possible adverse effects and drug - herb interactions on the use of herbal remedies[15].

Thus after a brief honeymoon where herbs have been portrayed as "wonder drugs", we are now seeing article after article on the dangers of herbs. As in most situations, the truth lies hidden under the media hype, bad or poorly understood science, exaggerated claims, and our natural resistance to new ideas. Thus herbal medicine is still poorly understood by the public, medical practitioners and the media.

Toxicity is the capacity of a substance to poison. According to a Swiss physician Paracelsus, all things are poison but the dose determines toxicity. The toxicity of a substance therefore is the detrimental manifestation of its biochemical effects in a living system. Thus the severity of a substance is the function of its interaction with the physiology of a particular organism [16]. Lack of experience, education, and lack of good information about herbs makes consumers easy victims of marketing exploitation and herbal myths. The same reasons make many physicians and other orthodox health care providers suspicious and uncomfortable, especially with the exaggerated claims, miracle cures, and unproven remedies their patients are taking. Along with this new interest is a profound ignorance, with many people equating natural with harmlessness.

Antifungal chemotherapy is in constant need of new and effective compounds due to the variable efficacy and adverse effects of drugs in current use [17]. Antifungals should work by exploiting differences between mammalian and fungal cells to kill the organism without significantly harming the host due to similarities between fungal and mammalian cells. This however has made it difficult to design drugs that target fungi without affecting human cells. The drugs of choice for the treatment of fungal infections are primarily polyene and azole classes of compounds which have been found to be nephrotoxic and less effective in invasive mycotic infection. In an attempt to overcome the above limitations there is need to source for new antifungal agents. [18][19]. In an attempt to overcome the above limitations the need to look for additional sources with better modes of action is of pivotal importance.

According to literature several works have been done using the parts of V. paradoxa to establish claims of efficacy. However there is little in terms of the safety evaluation of the plant. The aim of this work is to investigate the antifungal efficacy of Aqueous extract of the stem bark of V. paradoxa against Trichophyton rubrum as well as determine its level of toxicity and phytochemical composition.

MATERIALS AND METHODS

2.1 Collection of Plant Materials.

Stem bark of Vitellaria paradoxa were collected from the permanent site campus of the university of Ilorin, Nigeria. The plant material was identified and sample deposited at the herbarium unit of the department of Plant Biology, University of Ilorin, Nigeria. A Voucher Specimen No : UIH/04 obtained was obtained for the plant sample.

2.2 Sample Preparation And Extraction Procedure Fresh samples of the stem bark of Vitellaria paradoxa were air-dried for a period of about two weeks and pre-crushed manually using mortar and pestle. The material was further processed into powder using a mechanical grinder. Cold water extraction was done by percolation of the powdered crude sample in distilled water and left on an orbital shaker for 48 hours; after which it was filtered using Whatman number 1 filter paper and later concentrated under vacuum by rotary evaporator (Model type 349/2,Corning Limited). The residual extract was stored at refrigerator temperature of 40C in sterile plastic bottles until required for use.

The residual filtrate was later reconstituted taking into consideration the average weight of rats, duration of extract administration and the required volume of the doses. The crude extract was diluted in cold water to obtain varying concentrations of 50, 100 200 300 and 400mg/kg Body Weight. These were refrigerated until required for use.

2.3. Determination of Antifungal Efficacy of Vitellaria paradoxa Stem Bark in Albino Wistar Rats 2.3.1 Experimental Animal Male albino Wistar rats strain of 8-12 weeks weighing between 122.0-140.0 kg were obtained from the animal- holding unit of the Biochemistry department of University of Ilorin, Nigeria. They were bred and maintained in clean metabolic cage-sand, placed in well-ventilated house conditions (temperature: 28+-20C; photoperiods: 12 hours natural light and 12 hours darkness; humidity: 45-50 %). The animals were maintained on rat pellets from Bendel Feeds And Flour Mills, Edo state, Nigeria. They were supplied with tap water ad libitum. Cleaning of the cages was done daily.

2.3.2 Selection Of Fungal Isolate.

The fungal isolate used in this study was Trichophyton rubrum and was selected on its ability to cause tinea or ring- worm infection of which Vitellaria paradoxa is traditionally used to treat. The clinical isolate was obtained from the Culture Collection Unit of Olabisi Onabanjo University Teaching Hospital, Shagamu, Ogun State, Nigeria. It was routinely sub-cultured for the purpose of purity during storage on Saboraud Dextrose Agar (SDA) slopes and stored at 40C until required for use.

2.3.3 Animal Grouping And Inoculation

A total of 16 (sixteen) male albino wistar rats strain were completely randomized into 8 groups of 2 rats each and infected with Trichophyton rubrum spores except for the positive control group. The rats were artificially induced with superficial mycosis by hair root hair invasion.The inoculation of the rats was done with 1ml of 1x106 spores suspension of the test fungus by swabbing on a clean shaved area of the fur of the animals. Tinea infection was allowed to manifest over a period of about 7-14 days.

2.3.4 Extract/Drug Administration

Upon treatment, the infected rats were orally administered as follows:

Group A (positive control): was not infected, not treated, and was administered distilled water.

Group B (negative control): was infected but not treated with any form of the extract or antibiotic.

Group C: was treated with a known standard antibiotic.

GroupD-H: were administered extracts on a daily bases with doses of 50, 100, 200, 300 and 400mg/kg body weight respectively. The administration was done orally using metal oropharyngeal cannula. The rats were sacrificed 24 hours after their 14 days daily dosing which was the time for the period of observation.

2.4 Evaluation of Toxicological Effects of Aqueous

Extract of Vitellaria paradoxa Stem in Male Wistar Rats.

2.4.1 Collection And Preparation Of Serum.

The procedure described by [20] was employed in the preparation of the serum. In this procedure, rats were anaesthetized in a glass jar containing cotton wool soaked in diethyl ether. The rats were quickly removed, and their neck region cleared of fur. Their jugular veins were cut and the blood was collected in clean, dry, plain bottles. This was allowed to stand for 15 minutes at room temperature and centrifuged at 1000rpm for 15 minutes using GallenKamp centrifuge. The clear serum supernatants were carefully collected into sample bottles using a Pasteur pipette. The samples were stored frozen and used within 12 hours of preparation.

2.4.2 Collection And Preparation Of Tissue

Homogenate

The rats described in section 2.4.1 were thereafter quickly dissected, and the liver and kidney excised and transferred into ice-cold 0.25M sucrose solution. The organs were blotted with clean tissue papers and homogenized in ice-cold 0.25M sucrose solution at 1g/5cm3 (1:5 w/v) using mortar and pestle placed on ice-blocks.

The homogenate was further centrifuged at 3000rpm for 15 minutes to obtain supernatants which was kept in specimen bottle and frozen until required for the enzyme activity assays [20]. Parts of the organs were transferred into specimen bottle containing 10% formalin for histopathological examination.

2.4.3 Determination Of Enzyme Activity.

Assay kits by Randox was used to carry out the enzyme assays For the determination of aspartate aminotransferase (AST) activity, the method described by [21] was used for assaying the activity of AST. This was measured by monitoring the concentration of oxaloacetate hydrazine formed from 2,4-dinitrophenyl hydrazine.

In the determination of alanine aminotransferase (ALT) activity, the method described by Reitman and Frankel (1995) was used for assaying the activity of glutamate pyruvate transferase. Alanine aminotransferase catalyzes the formation of pyruvate and glutamate from alanine and (alpha) -oxoglutarate.

Alkaline phosphatase activity was determined using P- nitrophenyl phosphate as substrate at pH 10.1 as describe by [22].

2.4.4 Histopathological Examination.

Fixed tissues were dehydrated through ascending grades of ethanol up to absolute alcohol (70%, 90%, 95% and absolute). They were clean in xylene, impregnated and embedded in paraffin wax and sections were cut at 5um on a rotatory microtone. The sections were floated out on clean microscope slides which had previously been lightly coated with egg albumin preparation in order to avoid detachment from slide during the staining procedures. They were dried for 2 hours at 370C. The histopathological examination was carried out by following Standard procedure.

2.5 Phytochemical screening of stem bark of V. paradoxa

The method of [23] with slight modifications was employed in the phytochemical screening of the extract for the presence of the following biologically active compounds: Alkaloids, Tannins, general glycosides, Saponins, Steroids, Flavonoids, Anthraquinones and Reducing sugars. Control experiments contained only the plant extracts without reagents.

2.4.5 Statistical Analysis

Data obtained for the toxicological analysis in this study were expressed as means of three replicates +- standard deviation (SD). The differences in values among group were subjected in one way analysis of variance (ANOVA).

RESULTS

In vivo antifungal efficacy of aqueous extract of V. paradoxa stem bark in albino wistar rats The treated rats were observed for clinical signs daily till the completion of the study. There were no mortality or adverse external signs of intoxication following daily dosing and during the observation period of 14 days treatment with the extract. Extract administration and griseofulvin antibiotic showed improvement in the fur and skin of rats previously infected with T. rubrum and reduction in alopecia compared with their untreated counterparts (Plate 1, 2 and 3).

According to Table 1 there was a general increase in body weight of the rats upon treatment with extract as well as the standard antibiotic(griseofulvin) which were significant compared with the untreated counterparts. Also there were significant differences in organ weight ratio of extract treated rats compared with the other control groups and this was dose and time dependent (Table 2). Showing more pronounced effects at higher doses of the extract.

Specific activity of Aspartate aminotransferase (AST) enzyme in liver, kidney and serum of rats administered aqueous extract of V. paradoxa stem bark

The effect of AST enzyme as one of the indices of organ function as investigated in the serum, liver and kidney of rats treated with aqueous extract of the stem bark of V. paradoxa is presented in table 3. There were significant differences in the values of the AST enzyme of the three organs examined when compared with the normal rats as thepositive control which were not infected and not treated with the extract as well as the negative control that were infected but not treated. The concentration of the enzyme secreted in the normal rats (positive control) was 91.73 + 3.30cd in the liver and reduced to 71.90 + 7.90ab in the liver of rats administered 400mg/kg body weight of the extract. A similar observation was noted for with the kidney. An opposite trend was observed with the serum causing an increase in the values as the doses was increased.

Specific activity of Alanine aminotransferase (ALT) enzyme in liver, kidney and serum of rats administered aqueous extract of V. paradoxa stem bark.

The effect of administration of aqueous extract of V. paradoxa on alanine aminotransferase (ALT) activity of serum, liver and kidney of T. rubrum infected and treated rats as well as the controls is shown in table 4. The liver produced the highest concentration of the enzyme with a value of 33.33 + 0.58a in the positive control (normal rats) which drastically reduced to 13.5 + 1.80e in rats administered 400mg/kg bd.wt. Both the liver and kidney showed reduction in enzyme secretion as the dose concentration of extract administered was increased. However the reverse was the case for the concentration of the enzyme in the serum which rather increased across the doses.

Specific activity of Alanine phosphate (ALP) enzyme in liver, kidney and serum of rats administered aqueous extract of V. paradoxa stem bark.

The effect of the administration of the aqueous extract on the alkaline phosphatase enzyme (ALP) over a short term period of 14 days on rats infected with T. rubrum is presented in table 5. A similar trend of result as observed with AST and ALT was also seen in the ALP analysis in terms of decreased enzyme secretion as dose concentration of extract increased. The highest value of enzyme secretion was observed in the serum which varied significantly at (p (less than) 0.05) from the controls.

Histopathological studies : Haematoxylin and eosin (H and E) staining results Plate 4 - 11 shows the histopathology of cross sections of the liver of albino wistar strain rats after 14 days of extract administration. Plate 4 served as control which was the normal rat and was neither infected nor treated with extract while the second or negative control (Plate 5) was the infected but not treated group. As observed on the histology slides, the hepatocytes of the first control (Plate 4) appeared normal and normochromic in terms of stain retention showing the hepatic lobule radiating outwards from a central vein. The nucleus appeared roundish with prominent nucleoli. There were no signs of cellular degeneration of the cytoplasm. The negative control i.e the infected but not treated group showed some signs of degeneration of the hepatocytes with inflammation of the tissues.

Plate 6 shows the histological slide of a previously infected rat after 14 days treatment with itraconazole antibiotic, with slight changes in the normal architecture of the hepatocytes compared with the first control. Plates 7 to 11 show the histopathological signs of rats administered with 50mg/kg to 400mg/kg of aqueous extract of Vitellaria paradoxa stem bark. It was observed that histopathological alterations were seen in rats as the concentration of the extract increased from 50mg/kg bd. wt to 400 mg/kg bd. wt, producing distortions varying from mild mononuclear infilterations of the hepatocytes to moderate degeneration of the liver tissues. The histological section of the kidney tissues of T. rubrum infected and extract treated albino rats after 14 days of extract administration are presented in plates 12 to 19. The positive control (normal rat) shows a cross section of liver of a control rat depicting normal renal architecture consisting of the glomeruli and bowman's capsule (Plate 12).

The renal tubules are also well distinguished. The negative control which was the infected but not treated group (plate 13) shows cross section of the liver of an infected rat presenting with congested convoluted tubules which were filled with pink eosinophilic deposits with round to spindled hyperchromatic cells. The result observed in the antibiotic treated group (plate 14) also demonstrated congested tubules filled with pinkish deposits. Plates 15 to 19 present cross section of kidney of rats administered varying concentrations of the extract doses. Generally it was observed that the degree of variation from the normal rat (Positive control) was dose dependent. Hence the higher the dose administered, the more the degree of alteration on the normal renal architecture.

Phytochemical analysis of stem bark of V. paradoxa

The assessment of the phytochemicals present in the stem bark of V. paradoxa revealed the presence of the following classes of compounds : Alkaloids, General glycosides, Saponins, Steroids, Flavonoids, Reducing Sugars, Anthraquinones.

DISCUSSION

The in vivo determination of efficacy of the aqueous extract of V. paradoxa stem bark in tinea induced albino wistar rats with T. rubrum revealed that the extract produced significant healing effect upon administration in the treatment of infected rats. The extent of extract activity on the treated rats was dose and time dependent and daily observation of the treated rats showed that administration did not result in any external adverse clinical signs but rather showed improvement. It was observed that following administration of rats with the extract doses there was improvement in the skin of rats which had previously presented with lesions and red like colouration as well as alopecia. Treatment with the extract doses caused rats to regain their fur and also disappearance of skin lesions (Plate 1).

The result of the weights of the rats showed that generally there was percentage weight gain of the treated rats over the short term period of 14 days treatment, with the highest weight gain observed at the 14th day period of observation (Table 1 ). However it was observed that the increase in body weight between day 0 - 7 was greater than that observed from day 7 - 14 at doses of 100mg/kg bd.wt and 200mg/kg bd.wt contrary to other doses used. The negative control (infected but not treated) group however showed reduction in body weight throughout the period of observation which may be due to lack of treatment with the extract following infection with T. rubrum. Also the positive control group (not infected not treated) showed increased weight gain throughout the observation period.

It could be suggested from the observations made on the treated and control groups that to some extent, extract administration as well as treatment with itraconazole had significant positive effect on the weight of the treated rats. Though the percentage body weight gain of the normal rats was higher than the extract treated groups since it had not undergone any form of assault, however for the treated groups the reduction and slower rate of adding weight may be as attributed to earlier infection of such groups with T. rubrum which may have resulted in some pathological conditions in the rats such as loss of apetite. This could possibly result in previous weight loss of the rats, thereby making it quite difficult for them to regain such weights as quickly. This result is in conformity with a report by [24] that weight of rats treated with two veterinary preparations (RumbiumTM and TyrelTM) increased with dose and period of administration.

Investigation of the organ weight ratio of treated rats on post moterm examination showed that the two organs (liver and kidney) studied had reduced weight compared with the positive control (Table 2). Analysis of variance (ANOVA) showed that there was significant difference at (P (less than) 0.05) in the weights of both the liver and kidney of extract/ itraconazole treated groups when compared with the control groups of rats. This result suggests that though the organ weight of the extract and itraconazole treated rats were significantly lower than the positive control, however administration of the extract doses and itraconazole caused increase in the weight of the rats which was dose and time dependent. This is evident from the weight of the negative control group which was lower than the extract treated groups over the period of examination.

This could possibly be as a result of the infection which was left untreated and could have resulted in the constriction of the liver and kidney thereby affecting their secretory abilities which are required for normal functioning of vertebrates. For instance the liver's highly specialized tissues regulate a wide variety of high-volume biochemical reactions, including the synthesis and breakdown of small and complex molecules, many of which are necessary for normal vital functions [25]. However Physical examination of the liver is not accurate in determining the extent of liver damage. It can only reveal presence of tenderness or the size of liver, hence some additional studies are required to examine it. The biochemical indices of liver and kidney and serum monitored in this study are useful markers for assessing the functional capacities of the organs [26].

Measurement of activities of various enzymes such as AST, ALT and ALP in tissues and body fluids is a significant and well known aid in disease diagnosis [27]. Biochemical indices of organ function, if altered, will impair the normal functioning of such organs [28]. When compared with the control, administration of the aqueous extract of V. paradoxa stem bark at doses investigated produced alterations in the level of aspartate aminotransferase at (P (less than) 0.05) in the liver, kidney and serum of treated rats (Table 3). It was observed that compared with the positive control the activity of AST enzyme in the liver and kidney was decreased as the doses of the extract increased up to 400mg/kg body weight.

Similar trend of result was observed for the values of the alanine transferase and alkaline phosphatase enzymes in the tissues investigated (Table 4 and 5). As observed from the result there was usually a sharp increase in the values of the enzymes in the liver and kidney at 50 and 100mg/kg bd.wt as well as for itraconazole which later resulted in a decrease as the extract doses was increased. This initial rise in enzyme level may be attributed to de novo synthesis of the enzymes in an attempt of the tissues to amend the assault done on them which in this case is the extract or infection. The later decrease in the enzyme concentration at specific higher doses however could be that the tissues could no longer resist the effect of the assault and as a result there was inactivation of the enzymes by the extract. However it was generally observed that the concentration of the enzymes in the serum increased across the concentration gradient as those of the liver and kidney reduced.

Usually only low levels of AST are found in the blood, but when there is tissue damage as a result of any stress or intrusion by harmful agent , additional AST is released into the blood stream. There are many enzymes found in the serum that did not originally originate from the serum. During tissue damage, some of these enzymes find their way into the serum by altered cell membrane permeability leading to leakage of the enzymes from the tissues to the serum [29]. Alanine and aspartate aminotranferase are considered to be sensitive indicators of hepatocellular damage and within limit can provide a quantitative evaluation of the degree of damage to the liver [30]. Hence their increase in the serum is quite implied. However the dose specific decrease in the activity of aspartate aminotransferase in the tissues may further imply inhibition of the enzyme as well as selective effect of the extract on the aminotransferase.

Serum enzyme measurements are therefore a valuable tool in clinical diagnosis, providing information on the effect and nature of pathological damage to any tissue.

The increase in serum alkaline phosphatase (ALP) activities may indicate tissue damage of the rats probably by altered cell membrane permeability leading to the leakage of the enzymes from the tissues to the serum since it is a plasma membrane enzyme. Hence any disruption in the plasma membrane of the organ will consequentially lead to raised levels of the enzyme (ALP) in the serum as obtained in the present study. Loss of alkaline phosphatase from the tissues to the serum may have consequential effect on the adequate transportation of required ions or molecules across the cell membrane [31,32].

Histological examination of the liver of extract treated rats unlike the positive control indicated the presence of periportal mononuclear cell infiltration, hepatocellular vacuolar degeneration, pinocytic vesicle and necrosis (Plates 4 to 11). These lesions may have occurred as a result of metabolism of the extract by the liver which serves as the primary organ of biotransformation and also agree with the theory of target organ toxicity [33].

The histopathological study of the kidney showed degenerative and necrotic changes in the tubular epithelia of the kidney with cellular infiltration. This effect agree with the theory of target organ toxicity [33] since the kidney is the organ of excretion [34]. The dilation of the renal tubules and shrunken glomeruli may have been due to increased tubular secretion; a mechanism employed to clear harmful toxins and drugs from the body. Mild and severe degeneration of the glomeruli, acute tubular necrosis observed may have been due to adverse effect of the extract administration on the glomeruli and renal tubules leading to progressive decrease in renal flow and glomerular filtration rate. It has been reported that adverse effect caused by drugs affects morphological sites like the tubules, interstitium, glomeruli and vessels. It has also been reported that glomerular damaged impairs the flow through peritubular vascular system [33].

The evidence from this study has suggested that while some parameters such as body weight ratio and organ weight ratio of rats were not altered by the extract, the same cannot be described for the activities of the enzymes as well as the histopathological studies. Therefore, the effect of the extract may be mild as well as parameter and dose selective.

The phytochemical analysis of the crude aqueous extract of V. paradoxa plant parts revealed the presence of pharmacologically important phytoconstituents in the plant part investigated. It was observed that the plant parts contained very essential phytochemical constituents (such as alkaloids, general glycosides, saponins e.t.c.). These secondary metabolites may have been responsible for the antimycotic activity reported in this study. In some earlier reports some biologically active substances have been found to be responsible for antimicrobial activities of certain medicinal plants.

Saponins are naturally occurring surface-active glycosides mainly produced by plants, but also by lower marine animals and some bacteria [35,36]. They are surface active agents which interfere with or alter the permeability of the cell wall of microorganisms thereby leading to loss of cell wall viability[37,38]. This therefore, facilitates the entry of toxic materials or causes leakages of vital constituents from the cell [39,40]. Saponins have been reported as a major components of antifungal agents. They derive their name from their ability to form stable, soap-like foams in aqueous solutions and this easily observable character has attracted human interest from ancient times. However, this could make them useful antiseptic agents in bathing or cleansing of the surface of fungi infected skin.

Also there is some correlation between the use of this plant materials by herbal healers and the results obtained from this study as herbalists claim high efficacy to the use of this plant part in the treatment of skin infections. Flavonoids are phenolic in nature and act as cytoplasmic poisons. They also have been reported to inhibit the activity of enzymes [41]. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with microbial cell walls. More lipophilic flavonoids may also disrupt microbial cell membranes [42] Alkaloids are heterocyclic nitrogen compounds [43]. The mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and harmane is attributed to their ability to intercalate with DNA [44]. Anthraquinones are aromatic rings with two ketone substitutions. They are ubiquitous in nature and are characteristically highly reactive.

The switch between diphenol (or hydroquinone) and diketone (quinone) occurs easily through redox reactions [43]. In addition to providing a source of stable free radicals, quinones are known to complex irreversibly with nucleophilic amino acids in protein often leading to inactivation of the protein and loss of function. For that reason the potential range of quinone antimicrobial effects is great. This probably suggests the very impressive effect exerted by the aqueous extract of the stem bark of the plant since it was found to contain anthraquinones.

Tannins were another class of phytochemical documented in this study. Tannins are general descriptive name for a group of polymeric phenolic derivatives and are non-nitrogenous plant constituents with astringent properties on mucous membranes [38]. Tannins in this extract were believed to have acted by coagulating the cell wall proteins [39]. Generally, these active components may have been responsible for the diverse pharmacological actions of the extracts used in this study. If the active principles identified in this study are preserved and purified, they will be a great asset to drug development which will help in indigenous health care delivery. The plant part used in this study is readily available in the tropics where the fungal infection investigated is common. The result from this study indicates that the extracts from the plant part possess a package of active principles capable of inhibiting the growth of pathogenic agent of skin infection.

However in order to be able to adopt safe and useful traditional practices that would enable its incorporation into orthodox health care system it is important to say that the effect of the extract is mild and as such may not be completely safe for oral administration as practiced by traditional folklore medicine in Nigeria. The investigation and exploitation of medicinal plants should include measures for conservation and inventory in order to guard against the gradual loss of genetic diversities of these plants. Additionally with the current status of drug dilemma (drug resistance), man more than ever before needs a re- orientation on the sustainable use of his natural resources particularly in this era of economic recession to source raw materials for medicine by harnessing the abundant rich flora for an improved primary health care delivery

Table 1: Effect of aqueous extract of Vitelleria paradoxa stem bark on body weight and percentage body weight gain in rats

Doses (mgkg-1)###Body Weight (g)###Percentage body weight gain(%)

###Day0###Day 7###Day 14###Day 0-7###Day7-14###Day 0-14

+ ve Control###130.41###145.44###166.24###11.53###14.30###27.47

- ve Control###130.82###128.24###130.10###1.97###1.45###0.55

Itraconazole###131.90###140.01###171.11###6.14###22.21###29.73

50###117.45###121.10###128.01###3.11###5.70###8.99

100###120.10###130.90###138.20###8.99###5.58###15.07

200###118.10###134.20###139.12###13.63###3.67###17.80

300###124.33###132.33###141.24###6.43###6.73###13.60

400###126.15###140.10###150.44###11.06###7.38###19.25

+ ve Control = Not infected Not Treated(Normal rat), - ve Control = Infected not Treated with Extract

n = 3, x + SD. Test values carrying superscripts different from the control along each treatment group are significantly different at (p less than 0.05)

Table 2: Effect of aqueous extract of Vitelleria paradoxa stem bark on the organ-body weight ratios of albino rats

Dose (mg/kg bd.wt)###Liver###Kidney

+ ve Control###5.62 + 0.00g###1.11 + 0.02f

- ve Control###3.89 + 0.00ab###0.74 + 0.00a

Itraconazole###5.34 + 0.01f###1.06 + 0.00e

50###3.67 + 0.32a###0.76 + 0.03a

100###4.15 + 0.13bc###0.82 + 0.01b

200###4.26 + 0.00cd###0.82 + 0.00b

300###4.43 + 0.00de###0.91 + 0.01c

400###4.56 + 0.00e###0.94 + 0.00c

+ ve Control = Not infected Not Treated(Normal rat), - ve Control = Infected not Treated with Extract

n = 3, x + SD. Test values carrying superscripts different from the control along each treatment group are significantly different at (p less than 0.05).

Table 3: Effect of administration of aqueous extract of V. paradoxa stem bark on Aspartate aminotransferase (AST) activity of selected tissues of T. rubrum infected rats

Parameter###Dose(mglkg bd.wt)

###+- ye Control - ye Control###Itraconazole###50###100###200###300###400

Liver###91.73+-3.30a###91.43+-5.34a###9767+-10.95d###90.20+--10.20ab###69.09+-0.10f###79.90+-9.10d###84.70+-2.30c###71.90+-7.90cd

Kidney###33.10+-1.00###33.67+-0.58###58.67+-0.58e###43.07+-9.46d###31.33+-3.79bc###34.50+-1.00c###38.67+-0.58cd###29.67+-0.29b

Serum###30.67+-0.29###31.50+-2.78###65.67+-0.58f###33.67+-0.58ab###35.67+-0.29c###60.33 +-0.58d###67.33+-1.15f###68.67+-0.29e

+- ye Control = Not infected Not Treated(normal rat), - ye Control = Infected not Treated with Extract

n = 3, x+--SD. Test values carrying superscripts different from the control across each parameter are significantly different at (p less than 0.05).

Table 4: Effect of administration of aqueous extract of V. paradoxa stem bark on Alanine aminotransferase (ALT) activity of selected tissues of T.rubrum infected rats

Parameter###Dose(mg/kg bd.wt)

###+ ye Control###-ye Control###Itraconazole###50###100###200###300###400###

Liver###33.33+-0.58a###22.83+_2.0c###40.67+-0.58f###58.67+-0.58b###23.17+-0.29d###17.67+-0.29d###18.8+-0.76d###13.5+-1.80e

Kidney###27.80+-0.20a###22.20+-3.60b###27.00+-1.60a###33.80+-0.20cd###30.67+-1.53c###27.20+-0.20a###25.20+-2.00c###23.50+-0.80b

Serum###10.63+-0.55a###22.50+-2.50b###20.70+-1.80bc###21.50+-3.80b###32.80+-2.20cd###43.00+-1.50d###45.07+-3.00d###45.90+-3.90f

+ ve Control = Not infected Not Treated(normal rat)

-ve Control = Infected not Treated with Extract

n = 3, x + SD. Test values carrying superscripts different from the control across each parameter are significantly different at (p less than 0.05).

Table 5: Effect of administration of aqueous extract of V. paradoxa stem bark on Alkaline phosphatase (ALP) activity of selected tissues of T. rubrum infected albino rats

Parameter###Dose (mg/kg bd.wt)

###+- ve Control###-ve Control###Itraconazole###50###100###200###300###400

Liver###469.36+-0.58e###497.33+-0.27f###409.14+-0.05a###412.46 +-1.16b###454.30+-0.10d###439.27+-0.11c###439.33+-0.11c###411.95+-0.57b

Kidney###251.69+-1.44g###256.05+-1.73h###202.76+-0.65e###148.63+-0.63d###124.47+-0.64c###125.89+-0.60c###216.81+-0.62f###113.91+-0.60a

Serum###439.66+-0.47a###512.28+-0.58b###529.29+-0.50se###439.39+-0.01a###613.13+-0.01g###519.35+-0.58d###603.49+-0.05f###645.74+-0.47hh

+- ve Control = Not infected Not Treated(normal rat)

-ve Control = Infected not Treated with Extract

n = 3, x +- SD. Test values carrying superscripts different from the control across each parameter are significantly different at (p less than 0.05).

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1 Depatment of Microbiology, University of Ilorin, P.M.B1515, Ilorin 240003, Nigeria. Correspondence: Ahmed Risikat .N, Depatment of Microbiology, University of Ilorin, Ilorin, Nigeria. Tel: +2348063109301.

E- Mail: anrisikat@unilorin.edu.ng

Sani, Al Hassan : Depatment of Microbiology, University of Ilorin, Ilorin, Nigeria. Tel: +2348063109301.

E- Mail: anrisikat@unilorin.edu.ng
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