Evaluation of the Effects of Galinsoga parviflora Aqueous Leaf Extract on the Biochemical Changes in

www.idosr.org

©IDOSRPublication

InternationalDigitalOrganizationforScientificResearch

IDOSRJOURNALOFSCIENCEANDTECHNOLOGY8(1):47-59,2022.

Kyomukama et al

ISSN:2579-079X

Evaluation of the Effects of Galinsoga parviflora Aqueous Leaf Extract on the

Biochemical Changes in the Cerebellum after Mercury Chloride Exposure

John Ayuba Tabakwot, Okesina Akeem Ayodeji and Usman Ibe M

DepartmentofHumanAnatomy,KampalaInternationalUniversity,Uganda.

ABSTRACT

The cerebellum forms a major part of the brain and its function is to control all muscular movements and coordination Injury or damage to the cells of the cerebellum may lead to conditions like;ataxia, Joubertsyndrome,and manyothers.These injuries may be a result of traumatic brain injuries or toxic agents such as mercury, tin, and aluminum. For example,mercury exposure has been reported to be part of our daily lives, therefore it is important to understand the pathological implication that might follow these exposures andalsowiththehelpofalocalherb; Galinsogaparviflora (GP)tofindacheapandreadily availablemanagementsolutionto mercurytoxicityinthecerebellum. Thisstudy evaluated the effect of Galinsogaparviflora aqueous leaf extract on the biochemical changes in the cerebellum after mercury chloride exposure. Twenty-five adult male mice of an average weight of 25g were randomly divided into 5 groups of 5 rats each (n=5). The animals in Groups 1-5receivedoral administration of distilled water, 2.3mg/kg body weight of HgCl2, 2.3mg/kgHgCl2followedbyGPextract(800mg/kg),GPextract(800mg/kg)followedby2.3 mg/kg HgCl2 and GP extract (800mg/kg) followed by 2.3 mg/kg HgCl2 concurrently respectively. These treatments were followed by biochemical analysis. There was a reduction inCAT(220.28±27.84)andSOD(43.25±13.28)concentration andincrease inMDA (3.03±0.09) levels in the cerebellum of the mice. Furthermore, there was significant increase in concentration of CAT(264.52±3.61) and SOD(50.0±4.08) and reduced in level of MDAinthe cerebellum(2.96±0.20).Also,there was significantincreaseinconcentration of CAT(299.25±13.49), reduction in concentration SOD(55.0±5.40) and increase in MDA (3.19±0.15) level in the cerebellum. There was a significant increase in concentration of CAT (275.4±16.04), SOD (76.75±2.36) and increase level of MDA(2.73±0.19) in the cerebellum. In conclusion, the study revealed that mercury exposure in the cerebellum of mice reduced antioxidant activities. However, the introduction of Galinsogaparviflora in the cerebellum acted as a therapeutic agent against mercury toxicity in the cerebellum of adultmalemice.

Keywords: Galinsoga parviflora, biochemical changes, cerebellum, and mercury chloride exposure.

INTRODUCTION

Mercury exposure is associated with alteration biochemical composition of different body tissues consequently manifesting as structural and functional impairment [1] Lohren etal., [2] carried out a toxicology study on both organic and inorganic mercury species in differentiated human neurons and human astrocytes; then found that both organic and inorganic mercury can easily cross the blood-brain barrier (BBB) with its consequent detection in the CNS. The

mechanism of mercury induce toxicity in the various tissues of the body are well documented [3]. The major mechanism of mercury-induced toxicity is via induction and/or aggravation of oxidative imbalance in the various tissue of the body[4].Astudyby[5]onchronicdietary mercury exposure was linked with oxidativestress,brainlesions,andaltered behavior in Atlantic salmon. Jha etal., [6] studied the prooxidative and neurotoxic effects of mercury chloride in rats and

www.idosr.org

found that mercury administration was associated with a significant decrease in the mean levels of superoxide dismutase and glutathione-s-transferase and a significant increase in lipid peroxidation, a significant decrease in the enzymatic activity of glutathione peroxidase and catalase, a significant increase in the glutathione reductase levels in comparison to control. Aragão et al., [7] studies on hippocampal dysfunction provoked by mercury chloride exposure revealed that mercury chloride can cause oxidative stress, with consequent cytotoxicity and cell death by apoptosis. A study by [3] conducted studied the effectofascorbicacidinmercury-induced changes on the histomorphology of the cerebellumofadultWistarrats;andfound a significant increase in the mean SOD andLPOvaluesaftertheadministrationof mercuricchloride.

The biochemical integrity of the brain is vital for the normal functioning of the central nervous system (CNS) [8]. The biochemical markers of interest in the present studies are the oxidative stress biomarkerssuchCAT,SOD,andMDA.Just like every other tissue of the body, the nervous tissue generated reactive oxygen species during their normal metabolic activities, a process which has been seen to be accelerated in mercury exposure, with consequent recline toward oxidative stress associated tissue damage [3]. However, the body relies on both endogenous and exogenous substrates in the form of enzymes and minerals in counteringoxidativestresstendencies[9] The antioxidant enzyme system includes superoxide dismutase (SOD), glyoxalase, glutathione reductase, glutathione peroxidase, and catalase (CAT) [10]. Various plant parts have been reported to contain important phytochemical discretely involved in augmenting the function of most of these antioxidant enzymes [8]. Biochemically, it is evident that different neurons have different levels of vulnerability to oxidative stress [10]. For example, hippocampus, amygdala, and cerebellar granule cells

Kyomukama et al

have been reported as the most susceptible to oxidative stress in some studies. Due to the above, it is necessary to look at possible therapeutic/ameliorative intervention for the managementofthediverseassociated deviation in the different enzymes involved in the management of oxidative stress imbalance. The present studies look into the role of GP in the management of mercury-associated biochemical deviations. To understand the possible effect of GP in mercuryinduced biochemical changes, keen attention needs to be placed on the phytochemical composition of the plant [11]. Some of the phytochemical composition GP such as flavonoid, vitamin C, and other polyphenolic compounds possess great antioxidant capacity [12]. Kumar & Khanum, [4] in their review suggested that important phytochemicals in plants can help improve environmentally induced biochemicalchangesintissues,suchasin exposure to heavy metals like mercury. A review by [13] on the beneficial role of various phytochemicals on oxidative stress and age-related disease indicated thatplant-derivedphytochemicalsmaybe useful in the fight against mercury exposure. A study by [14] on phenolics and polyphenolics in foods, beverages, and spices, linked then with strong antioxidant capability, therefore supporting our use of GP in the present studies; considering GP rich content of phenolics and polyphenolics compound, compounds known to possess the antioxidant and metal chelating capability.

Aim of the Study

This study is designed to evaluate the effects of Galinsoga parviflora aqueous leafextractonthebiochemicalchangesin the cerebellum after mercury chloride exposure.

Research Question

What are the effects of Galinsoga parviflora aqueous leaf extract on the biochemical changes in the cerebellum aftermercurychlorideexposure?

METHODOLOGY

Study Location

The study was conducted in the animal house of the Kampala International University, Institute of Biomedical Research (IBR) and Histology Laboratory of Kampala International University, Western Campus (KIU-WC); which is situated in Western Uganda in Ishaka Municipality, Bushenyi District 350KM fromKampala,Uganda.

Ethical Consideration

Ethical approval (KIU-2021-14) was obtained from the Kampala International University Research Ethics Committee before conducting the research and register in Uganda National Council for Science and Technology. The experimental study was carried out at the Institute of Biomedical Research Laboratory (IBRL) Kampala International University-Western Campus (KIU-WC) and the laboratory animal experiments were conductedfromtheKampalaInternational University Animal House. The 3Rs (Reduction,Replacement,andRefinement) were considered in determining the number of mice to be used in this study as well as the handling of animals (EuropeanMedicineAgency,2016).Atotal of 25 mice were used in this study. The study will use an animal model system and not an in vitro system to determine the effect of Galinsoga parviflora on the cerebellum after mercury chloride is introduced then the expression of antioxidant biomarkers in cells is determined. The report by ‘resource equation’ was considered when chosen the number of animals. The animals were grouped into 5 groups with five animals pergroupandwithgroups,threeandfour receiving the same doses but in reverse methodandalsoasuitabledose wasused according to literature to avoid using too many animals so that to be able to achieve the reduction principle. The researcher obtains training on how to handle laboratory animals before the study and work under the supervision of hissupervisors.Allproceduresandworks on animals were conducted following a

Kyomukama et al

guide for the care and use of laboratory animals [7].Theanimals were treatedin a humane way to prevent them from any discomfort like excessive pain and stress. The animals were housed five per group and there was fed on a daily basic morning and evening and also their beddingswerechangedweeklysothatthe animals were comfortable to avoid the fightandsoallowtorespondtotreatment inaventilatedenvironment.Attheendof 42 days, all animals were sacrificed humanely by subjecting them to light ether anesthesia followed by cervical dislocation. At the end of the study, all sacrificedanimalswereincinerated.

Collection and Preparation of Plant Extract

Galinsoga parviflora was obtained from the local garden in Ishaka- Bushenyi District, Uganda. A small sample of the leaves was taken to Mbarara University of Science and Technology, Faculty of Science Herbarium Unit, Mbarara, for botanical identification and deposition of voucher number. Extraction of the plant was carried out in the Biochemistry Departmental laboratory, Kampala InternationalUniversity,WesternCampus, Ishaka, Uganda. The plant’s leaves were driedatroomtemperature;groundedwith a grinding machineand followed by extraction. Sixteen grams (16g) of the powdered leaves were dissolved in 300ml of distilled water and left to stand for three days, shaken frequently until complete extraction of plant materials. Then the extracts were filtered through a filter into a funnel and the extract was kept in an oven at 400 C for three days to solidified[15].

Purchase

of Chemical Mercuric chloride (May and Bakers, England) was purchased from a chemical storeinIshakatown,WesternUganda.

Animals

The animals were procured from the animal facility of Kampala International University Western Campus (KIU-WC) twenty-five (25) adult male mice were used for this study weighing 20-35g. All

www.idosr.org

mice were housed in a well-ventilated plastic cage; the animals were left to acclimatize for two weeks with free accesstofoodandwater.

Study Sample Size

‘The resource equation’ approach was used to arrive at adequate sample size. According to the ‘resource equation’ method a value “E or DF” is measured. DF = degree of freedom of analysis of variance(ANOVA).

DF can be measured by the following formula: DF=N – k n=DF/k+1

Where: N = total subjects number; k = numberofgroups;n=numberofsubjects in a group. Minimum DF range = 10 and maximum DF range = 20 (Festing& Altman,2002;Arifin&Zahiruddin,2017).

Kyomukama et al

Thesample size forthe studywillbe:N= 25malemice;K=5groups

DF = 25 – 5 = 25, n = DF/k+1 = 20/5+1 = 5(fivemicewillbeplacedpergroup)

Sampling Technique

The mice were sampled via simple random sampling procedures. A sequence of numbers was randomly used to direct how the animals will place in groups (table 1). After the randomization, the micewere placed in five groups comprising of 5 mice each labeled mercury and gallant soldier dose (MG), gallantsoldierandmercury(GM),mercury and gallant soldier dose concurrently(MGC), positive control (mercury dose) (PC) and negative control (NC)(table2).

Table

1:Randomization

assignment

Original 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5

Randomized 2 4 3 5 1 3 1 4 3 2 2 4 4 5 3 2 1 3 5 4 4 1 3 2 5

sequence Table 2: Group placement after randomization

Experimental Design

Dosage and Administration

Mercury chloride:2.3mg/kg of Mercury Chloride was administered. The stock solution was prepared daily to prevent the risk of degradation [16]. The dilution factorwas50mgin100ml.

Galinsoga parviflora: 800mg/kg of GP was administered. The stock solution was prepared daily to prevent the risk of degradation [17]. The dilution factor was 100mg in 1ml. Mercury chloride and GP was administered orally for three weeks respectively

Reconstitution of Galinsoga parviflora Extract mercury Chloride for Animal Administration

AsuitabledoseofGPwasadministeredto the animals. The right amount of the extract which was guided by the OECD’s (organization of economic corporation

and developments) guidelineswas reconstituted and the calculated doses shall be administered according to each animal's body weight daily (between 9am and 10am). The mercury chloride on the other hand was constituted for administration on the mice according to the appropriate dose which was 800mg/kgasdescribeby[16].Thedosage of GP was prepared by dissolving in an appropriate volume of distilled water not exceeding 20 ml/kg [18]. The mercury chloride was guided by the OECD’S guideline and the powder be dissolved in distilled water daily. The mixture was kept in the refrigerator at 60C pending daily administration to the animals accordingtotheirbodyweight.

Dosage and preparation of stock solution willbecalculatedusingtheformula:

www.idosr.org

Kyomukama et al

Dosage(mg)=Bodyweightanimals(g)xdose(mg) 1000g

Dosevolume=weightofrat(g)xappropriatevolume(10ml)ormaximumvolume(20ml) 1000g

Groupings

Animalswererandomlyassignedtofive(5)groupsoffive(5)animalsineachgroupi.e.N=5 pergroup

Table 3: Animals Grouping

GROUP DOSAGE

Group1

Receivedistilledwateronly

Group2 Receivemercurychlorideonly(2.3mg/kg)

Group3 Receive mercury chloride (2.3mg/kg) followed byGalinsoga parviflora(800mg/kg)

Group4 Receive Galinsoga parviflora (800mg/kg) followed by mercury chloride(2.3mg/kg)

Group5 Receive mercury chloride (2.3mg/kg) and Galinsogaparviflora (800mg/kg)concurrently

Biochemical Determination

of mice brain tissues homogenates for oxidative markers assay

Preparation

TheBraintissueswereexcisedand0.1gof the cerebellum was weighed and then homogenized in 0.4ml of 5% sucrose solution, they were centrifuge for 10minutes at 3000rpn [19]. The supernatant was obtained and stored at200C until it is used to determine the oxidative biomarkers and acetylcholine esteraseactivities.

The Activity of Superoxide Dismutase (SOD)

The SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. The SOD was achieved as outlined by Misra and Fridovich [20] as cited by Afolabi and colleagues [21]. The assay is based on the capacity of SOD to inhibit the autoxidation of epinephrine (adrenaline). Zero-point two milliliters of the supernatant was added to 2.5 ml of

Percentageinhibition

1 unit of SOD activity was defined as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline toadrenochromeduring1min.

Malondialdehyde (MDA)

Lipid peroxidation is the end product of MDAwhichreacts withthiobarbituricacid (TBA) to produce a pink-colored complex that has peak absorbance at 532 nm. As

0.05 M carbonate buffer (pH 10.2) to equilibrate in the spectrophotometer. The reaction was initiated by the addition of 0.3 ml of freshly prepared 0.3mM adrenaline to the mixture which was quicklymixedbyinversion.Thereference cuvette contains 2.5 ml of 0.05 M carbonate buffer (pH 10.2), 0.3 ml of the substrate (adrenaline), and 0.2 ml of water. The increase in absorbance at 480 nm due to the adrenochrome formed was monitored every 30 sec for 150 sec. One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of epinephrine toadrenochromeduring150sec.

Calculation:

Increase in Absorbance (per min.)= (A3 A0)/2.5

A0 =absorbanceafter30seconds A3 = absorbance after 150 seconds

outlined by Yousef and colleagues, the MDA level was determined [22]. 2 ml of thiobarbituric acid (0.375) and trichloroacetic acid (TCA) (15%) reagent was mixed in an aliquot of supernatant (200 μl). The volume was made up to 3ml with double distilled water and boiled on a water bath at 950C for 20 minutes and cooled under tap water. The reaction

www.idosr.org

product (TBA–MDA complex) was extracted by adding 3 ml of n-butanol to theabovesolution.Theabsorbanceofthe pink-colored extract in n-butanol was measured at 532 nm using a spectrophotometer. The amount of MDA was calculated using a molar extinction coefficient of 1.56x105 M-1 cm-1 and expressed as moles of MDA formed per gramweightoftissue.

Catalase

Catalase activity was assessed using the method of [23], with hydrogen peroxide asasubstrate.Thecatalasepresentinthe sample reacts with hydrogen peroxide

Phytochemical Screening of Galinsoga parviflora

Kyomukama et al

(H2O2) to produce water and oxygen. The unconverted H2O2 reacts with a probe to produce a product that can be measured spectrophotometer at an absorbance of 532nm[24].

Statistical Analysis

All the data of behavior and the biochemical study was statistically evaluated with SPSS 25 using ANOVA to compare the mean. Where necessary, Tukey post hoc test was used to find where the significant difference lies across the groups. All the results were expressedas mean ±SDorSEM value was significantatp≤0.05.

RESULTS

The preliminary phytochemical screening of Galinsogaparviflora (Cav) aqueous leaf

extract revealed a positive reaction for alkaloids, carbohydrates, flavonoids, polyphenols, and glycoside as presented inTable4.

www.idosr.org

Kyomukama et al

Morphometric studies of effects of Galinsoga parviflora (Cav) aqueous leaf extract on the cerebellum after mercury chloride exposure in adult male mice Group 1 of adult male mice revealed a reduction in weight in week 1 then graduallyincreasedinweightfromweek2 to week 7. Group 2 revealed a reduction

in weight between weeks 1 and 2 but an increase in week 3 and then another reduction in week 4. Group 3 indicated a reduction in weight from week 1 to week 3 but increased from week 4 to week 7. Group 4 revealed an increase in weight in week 1but reduce in week 2 then start increasing from week 3 to week 7. In group 5 revealed a reduction in weight from week 1 to week 4 (Table 5).

www.idosr.org

Kyomukama et al

Table 5: Body weight changes following effects of Galinsoga Parviflora aqueous leave extract on the cerebellum during mercuryexposureinadultmalemice

Groups WBA(g) W1(g) W2 (g) W3(g) W4(g) W5(g) W6(g) W7(g)

Group 1 25.86±1.6 7 23.64±0.98 24.48±0.91 24.31±0.82 25.54±1.09 25.66±1.09 25.74±1.09 25.84±1.34

Group 2 25.12±0.8 4 24.08±0.98 24.06±0.97 24.18±1.22 24.04±0.87

Group 3 26.70±0.7 6 25.76±0.81 26.18±0.67 26.36±0.62 26.62±0.58 26.90±0.54 26.66±0.57 26.66±0.60

Group 4 26.3±1.17 26.22±1.10 25.94±1.04 26.00±1.03 26.66±1.07 26.86±0.79 26.78±0.77 26.66±0.79

Group 5 24.1±0.69 23.68±0.91 23.30±0.82 18.78±4.76 18.92±4.82

F value P value

0.900 0.483

1.629 0.206

1.929 0.145

1.757 0.117

1.946 0.142

0.710 0.511

0.463 0.640

0.242 0.789 N=5, WBA:Before administration,W: Week. The value was considered significantlydifferent atp<0.05,A: indicates group 1is p<0.05. b: indicates significance when test group is compared against Group 2, a: indicates significance when test group is comparedagainstGroup1.

www.idosr.org

Catalase Concentrations (CAT)

There was a reduction in catalase concentrationinthecerebellumofgroup3 animalswhencomparedtogroup1(Table 6). However, there was an increase in the concentration of catalase in the cerebellum of group 4 and 5 animals when compared to group 1 (Table 6). Furthermore, there was a significant increase in catalase concentration in the cerebellum of group 4 animals when compared to group 2 animals (Table 6). Lastly, there was an increase in cerebellar catalase concentration in both groups 3 and 5 animals when compared to groups 2animals(Table6).

Malondialdehyde (MDA) level

There was a significant increase in MDA cerebellar concentration in group 4 animalswhencomparedtogroup1(Table 6). Furthermore, there was an increase in

Kyomukama et al

the cerebellar concentration of MDA in group 3 and 5 animals when compared to group 1 (Table 6). Also, there was an increase in MDA cerebellar concentration in group 4 animals when compared to group 2 (Table 6). However, there was a reductionincerebellarMDAconcentration in group 3 and 5 animals when compared togroup2animals(Table6).

Superoxide dismutase concentrations (SOD)

There was an increase in cerebellar SOD concentration in group 5 animals when compared to group 1 animals (Table 6). However, there was a reduction in cerebellar SOD concentration in group 3 and 5 animals when compared to group 1 animals (Table 8). Although, there was an increase in cerebellar SOD concentration in groups 3, 4, and 5 animals when compared to group 2 (Table 6).

Table 6: Biochemistry study ofadult male mice during the administration of Galinsoga parviflora(cav) aqueous leaf extract on the cerebellum after mercury chloride exposure Group CAT(umol/l) MDA(nmol/g) SOD (%)

Group 1 273.38±11.32 2.43±0.10 60.0±1.42 Group 2 220.28±27.84 3.03±0.09 43.25±13.28 Group 3 264.52±3.61 2.96±0.20 50.0±4.08 Group 4 299.25±13.49b 3.19±0.15a 55.0±5.40 Group 5 275.4±16.04 2.73±0.19 76.75±2.36 F value P value

3.080 0.049

3.751 0.026

2.098 0.132 N=4. The value was considered significantly different at p<0.05, b; indicates significance, whenthetestgroupiscomparedagainstGroup2,a;indicatessignificancewhentestgroup iscomparedagainstGroup1

DISCUSSION

This study was centered on assessment effects of Galinsoga parviflora (cav) aqueous leaf extract on the cerebellum during mercury exposure in adult male mice, using biochemical to observe the specific effect of this chemical on the brain using the mice as a model. The specific objective of the study was biochemical assay of SOD, CAT and MDA concentrations to assess for oxidative stress The presence of phytochemicals

such as alkaloids, carbohydrates, flavonoids, polyphenol and glycosides which are found in GP might be the reason why treated groups 3, 4 and 5 have some cells which are yet to revealed evidence of pyknosis. The biochemical assay of some oxidative markers such as malondialdehyde(MDA),whichappears to be the most mutagenic product of lipid peroxidation as well as some antioxidants, such as catalase (CAT) and

www.idosr.org

superoxide dismutase (SOD) which are chemical compounds that contain monohydroxy/polyhydroxyphenol; which work to slow down the lipid peroxidation [25]wasstudied.Theresultsofthisassay revealed that SOD activity was decreased in the treatment groups compared to the control (group 1) and when compared with group 2 there was an increase that had higher values for SOD activity. CAT activity when compared to group 1 revealed reduction in group 3 then increased in groups 4 and 5. When compared with group 2 it revealed a significant increase (P<0.05) MDA levels were higher in treatment groups but only significantly increased in group 4 compared to the control. The lowered concentrations of SOD and CAT and increase MDA levels shows reduced antioxidant activities in the cells of the cerebellum which is an indicator of oxidative stress [22,26,27,28,29,30]Cells have an intact oxidation process to detoxify the cellular environment from oxidants, and as such create equilibrium in oxidants andantioxidants fromaerobic metabolism [31,32,33]. The failure in the neutralization of events of oxidative status results in oxidative stress which leads to cell death by lipid peroxidation, carbohydrate oxidation, protein oxidation and nucleic acid oxidation [26,34,35]. Oxidative stress not only causes hazardous events such as lipid peroxidation and oxidative DNA damage, but also physiologic adaptation

Kyomukama et al

phenomenaandregulationofintracellular signal transduction [36,37,38,39,40]. Various active oxygen species are generated in the body during the process of utilizing oxygen and because the body is furnished with elaborate mechanisms to remove active oxygen species and free radicals, these by-products of oxygen metabolismarenotnecessarilyathreatto the body under physiological conditions [27,41,42]. However, if active oxygen species or free radicals are generated excessively or at abnormal sites, the balancebetweenformationandremovalis lost, resulting in oxidative stress. Consequently, active oxygen species and free radicals can attack molecules in biological membranes and tissues, thereby inducing various diseases. Oxidative stress can influence many biological processes such as apoptosis, viral proliferation, and inflammatory reactions [27,38,39,40,41,42] However, the introduction of GP was able to increase the concentration of CAT, SOD and reduced the concentration of MDA in the cerebellum of these animals. This implies that the presence of phytochemicalcontainsintheGPallowsit toactasanagentoftheantioxidantagent within the cells of the cerebellum. A review by [13] on the beneficial role of various phytochemicals on oxidative stress and age-related disease indicated thatplant-derivedphytochemicalsmaybe useful in the fight against mercury exposure.

CONCLUSION

In conclusion, the study revealed that the oxidativestressobservedinthetreatment groups could lead to the pyknosis and myelin damage and also might be the reason for loss to neuromuscular damage

and motor coordination. However, the introduction of Galinsoga parviflora in the cerebellum acted as a therapeutic agent against mercury toxicity in the cerebellumofadultmalemice.

REFERENCES

1. Ibegbu, A., AnimokuAbdulrazaq, A., Micheal, A., Daniel, B., Adamu Sadeeq, A., Peter, A. and Musa, S. A. (2017). Histomorphological effect of ascorbic acid on mercury chlorideinduced changes on the cerebellum of adult Wistar rats. Journal of Morphological Sciences, 31(4),219-224.

2. Lohren, H., Blagojevic, L., Fitkau, R., Ebert, F., Schildknecht, S., Leist, M.

and Schwerdtle, T. (2015). Toxicity of organicandinorganicmercuryspecies in differentiated human neurons and human astrocytes. Journal of Trace Elements in Medicine and Biology. https://doi.org/10.1016/j.jtemb.2015. 06.008

3. Ibegbu, A. O., Animoku, A. A., Ayuba, M., Brosu, D., Adamu, S. A., Akpulu, P. and Musa, S. A. (2014). The effect of

www.idosr.org Kyomukama et al

ascorbic acid on mercury-induced changes on the histomorphology of the cerebellum of adult wistar rats. African Journal of Cellular Pathology, 3(9),9-15.

4. Kumar, G. P. and Khanum, F. (2012). Neuroprotective potential of phytochemicals. Pharmacognosy reviews,6(12),81.

5. Berntssen, M. H. G., Aatland, A. and Handy, R. D. (2003). Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr. Aquatic Toxicology https://doi.org/10.1016/S0166445X(03)00104-8

6. Jha, A., Saidullah, B. and Bubber, P. (2019). A Study on Prooxidative and Neurotoxic Effects of Mercury ChlorideinRats. ECPharmacologyand Toxicology

7. Aragão, W. A. B., Teixeira, F. B., Fagundes, N. C. F., Fernandes, R. M., Fernandes, L. M. P., Da Silva, M. C. F., Amado, L. L., Sagica, F. E. S., Oliveira, E. H. C., Crespo-Lopez, M. E., Maia, C. S. F. and Lima, R. R. (2018).Hippocampal dysfunction provoked by mercury chloride exposure: Evaluation of cognitive impairment, oxidative stress, tissue injury and nature of cell death. Oxidative Medicine and Cellular Longevity. https://doi.org/10.1155/2018/787805 0.

8. Salim, S. (2017). Oxidative stress and the central nervous system. Journalof Pharmacology and Experimental Therapeutics, 360(1),201-205.

9. Kohen, R., Beit-Yannai, E., Berry, E. M. and Tirosh, O. (1999). Overall low molecular weight antioxidant activity of biological fluids and tissues by cyclic voltammetry. Methods in enzymology,300,285-296.

10.Griendling, K. K., Sorescu, D., Lassegue, B. and Ushio-Fukai, M. (2000). Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arteriosclerosis,

thrombosis, and vascular biology, 20(10),2175-2183.

11.Onyido, I., Norris, A. R. and Buncel, E. (2004). Biomolecule mercury interactions: Modalities of DNA base mercury binding mechanisms. remediation strategies. Chemical reviews,104(12),5911-5930.

12.Rolim, P. M., Fidelis, G. P., Padilha, C. E.A.,Santos,E. S.,Rocha,H. A.O.and Macedo, G. R. (2018). Phenolic profile and antioxidant activity from peels and seeds of melon (Cucumis melo L. var. reticulatus) and their antiproliferative effect in cancer cells. Brazilian Journal of Medical and BiologicalResearch,51(4).

13.Forni, C., Facchiano, F., Bartoli, M., Pieretti, S.,Facchiano, A., D’Arcangelo, D. and Jadeja, R. N. (2019). Beneficial role of phytochemicals on oxidative stress and age-related diseases. BioMedresearchinternational, 2019.

14.Shahidi, F. and Ambigaipalan, P. (2015).Phenolicsandpolyphenolicsin foods, beverages and spices: Antioxidant activity and health effects–A review.Journal of functional foods,18,820-897.

15.Bazylko, A., Boruc, K., Borzym, J. and Kiss, A. K. (2015). Aqueous and ethanolic extracts of Galinsoga parviflora and Galinsogaciliata. Investigations of caffeic acid derivatives and flavonoids by HPTLC and HPLC-DAD-MS methods. Phytochemistry Letters. https://doi.org/10.1016/j.phytol.2014 .11.005.

16.Emanuelli, B., Peraldi, P., Filloux, C., Sawka-Verhelle, D., Hilton, D. and Van Obberghen, E. (2000) SOCS-3 is an insulininduced negative regulator of insulin signaling. J. Biol. Chem. 275, 15985–15991.

17.Yadav, J. P. and Suman Lata. (2008) Ground water quality of District Jhajjar with special reference to Fluoride. Proceedings 16th National Symposium on Environment16: 107111.

18.OECD. (2000). Guidance Document on Acute Oral Toxicity. Environmental

www.idosr.org

Health and Safety Monograph Series on Testing and Assessment. 2000. No.24.

19.Akeem, Okesina. (2019). Progesterone Regulates Superoxide Dismutase, Catalase, Malondialdehyde and Cholinesterase Activities in Trimethyltin-Induced Hippocampal DamageinAdultMaleWistarRats.16.

20.Misra, H. P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972 May 25;247(10):3170–3175.

21. Afolabi, F., Alabi, M., Babaniyi, B., Obagunwa, M. and Ojo, F. (2015) Nutrient loss during traditional ogi production. Journal of Chemical and PharmaceuticalResearch.7.246*249.

22.Yousef, H., Czupalla, C. J., Lee, D., Chen, M. B., Burke, A. N. and Zera, K. A. (2019). Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelialcellVCAM1. Nat.Med. 25, 988–1000.

23.Sinha,A.K.(1972)ColorimetricAssay of Catalase. Analytical Biochemistry, 47,389-394.

24.Claiborne, A. L. (2018). Catalase activity. In CRC handbook of methods for oxygen radical research (pp. 283284).CRCpress.

25.German J. B. (1999). Food processing and Lipid Oxidation. Advanced ExperimentalMedicalBiology.459:2350.

26.Spooner,R.andYilmaz,O.(2011).The Role of Reactive-Oxygen Species in Microbial. Transaction of the Royal Society of Tropical Medicine and Hygiene. 92:430-433

27.Yoshikawa, T. and Naito, Y. (2002). What is Oxidative Stress?.Japan Medical Association Journal 45(7): 271-276.

28.PCU Okechukwu, E Nzubechukwu, ME Ogbansh, N Ezeani, MO Nworie, (2015).The Effect of Ethanol Leaf Extract of Jatropha curcas on Chloroform Induced Hepatotoxicity in Albino Rats. Global J Biotech & Biochem10,11-15

Kyomukama et al

29.AC Nwaka, MC Ikechi-Agba, PCU Okechukwu, IO Igwenyi, KN Agbafor, (2015).The effects of ethanol extracts of Jatropha curcas on some hematological parameters of chloroform intoxicated ratsAmericanEurasianJournal ofScientificResearch 10(1),45-49.

30.Ugwu Okechukwu P.C. and Amasiorah V.I.(2020).The In Vivo Antioxidant Potentials of the Crude Ethanol Root Extract and Fractions of Sphenocentrum jollyanum on Oxidative Stress Indices in Streptozotocin-Induced Diabetic. IDOSR Journal of Biology, Chemistry AndPharmacy 5(1):26-35.

31.PCU Okechukwu, CE Offor, UA Ibiam, AL Ezugwu, AJ Uraku, CN Igwe (2015).The Effect of Ethanol Extract of Jatropha curcas on Renal Markers of Chloroform Intoxicated Albino Wister Rats European Journal of Biological Sciences 7(1),21-25.

32.Ssempijja Fred, Marta Vicente-Crespo, Dare Samuel Sunday and Edmund Eriya Bukenya (2022). The effect of Imperata cylindrica (L.) P. Beauv. on Brain tissue Morphology of Drosophila Melanogaster transgenic flies overexpressing the paralytic Gene. IDOSR Journal of Biochemistry, BiotechnologyandAlliedFields 7(1): 111.

33.Ssempijja Fred, Marta Vicente-Crespo, Dare Samuel Sunday1 and Edmund Eriya Bukenya (2022).The Effect of Imperata Cylindrica (L.) P. Beauv on Learning and memory performance in a single and prolonged exposure treatmentsofDrosophilaMelanogaster transgenic flies overexpressing the paralytic gene. IDOSR Journal of Biochemistry,BiotechnologyandAllied Fields 7(1):12-22

34.Ssempijja Fred, Marta Vicente-Crespo, Dare Samuel Sunday and Edmund Eriya Bukenya (2022). The Effect of Imperata cylindrica (L.) P. Beauv. on Myelin and Nissl Substance of Drosophila melanogastertransgenic Flies Overexpressing the Paralytic Gene. IDOSR Journal of Biology, ChemistryAndPharmacy 7(1)1-13.

www.idosr.org

35.Hussein Osman Ahmed, Joy MuhumuzaandMusiimeJamesNabaasa (2022). Evaluation of the factors associated with immediate adverse maternal outcomes among referred women in labor at Kampala International University Teaching Hospital. IAA Journal of Biological Sciences8(1):228-238

36.Hussein Osman Ahmed, Joy MuhumuzaandMusiimeJamesNabaasa (2022). Factors associated with Immediate Adverse Maternal Outcomes among Referred Women in Labor attending Kampala International University Teaching Hospital. IAA Journal of Applied Sciences 8(1):117125.

37.Hussein Osman Ahmed, Joy MuhumuzaandMusiimeJamesNabaasa (2022). The composite immediate adverse maternal outcomes among women in labor referred to Kampala International University Teaching Hospital IAA Journal of Scientific Research8(1):149-156.

38.Daniel Asiimwe, Herman Lule and Izimba Daniel (2022). Epidemiology of Assault Injuries among Trauma Patients Presenting at Kampala International University Teaching Hospital and Jinja Regional Referral

Kyomukama et al

Hospital. INOSR Applied Sciences 8(1):111-119.

39.E.O.Ikuomola,O.S Akinsonmisoye, R.O. Owolabi and M. B. Okon (2022). Assessment of Toxicity Potential of Secnidazole on Reproductive System of Male Wistar Rats. INOSR Applied Sciences 8(1):120-133

40.Ugwu Okechukwu, P. C., Onwe, S. C. and Okon, M. B. (2022). The effect of Methanol Extract of Rauwolfia vomitoria on Lipid Profile of Chloroform intoxicated Wistar Albino Rats. IAA Journal of Scientific Research,8(1),73-82

41.E.O.Ikuomola,O.S.Akinsonmisoye, R.O. Owolabi and M. B. Okon (2022).Evaluation of the Effect of Secnidazole on Sperm Motility, Morphology,ViabilityandTotalSperm Count of Wistar Rats. INOSR Experimental Sciences 8(1): 74-83, 2022.

42.E.O. Ikuomola , O.S Akinsonmisoye, R.O. Owolabi and M. B. Okon (2022).Evaluation of the effect of secnidazole on the histology of the testes and epididymis of male Wistar rats. INOSR Experimental Sciences 8(1):84-94,2022.