EFFECT OF AQUEOUS EXTRACT OF LEMON LEAVES ON LEAD INDUCED SPLEEN OF ADULT WISTAR RAT

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EFFECT OF AQUEOUS EXTRACT OF LEMON LEAVES ON LEAD INDUCED SPLEEN OF ADULT WISTAR RAT

Abstract

This study investigates the protective effects of aqueous extract of lemon leaves (Citrus limon) on lead-induced damage in the spleen of adult Wistar rats. Lead toxicity is a major public health concern due to its detrimental effects on various organs, including the spleen, which plays a crucial role in the immune system. Previous research has highlighted the antioxidant properties of lemon leaves, suggesting their potential therapeutic benefits against oxidative stress and toxicity.

A total of 40 adult Wistar rats were randomly divided into four groups: Control (Group I), Lead-exposed (Group II), Lead + Lemon Leaf Extract (Group III), and Lemon Leaf Extract only (Group IV). Group II was exposed to lead acetate at a dose of 100 mg/kg body weight for 28 days to induce spleen toxicity. Group III received both lead acetate and aqueous extract of lemon leaves at a dose of 200 mg/kg body weight. Group IV was administered only the lemon leaf extract.

Histopathological analysis of the spleen was performed to assess the extent of tissue damage and inflammation. Biochemical assays were conducted to measure levels of oxidative stress markers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Additionally, spleen function was evaluated by examining changes in hematological parameters.

The results showed significant histopathological changes in the spleen of lead-exposed rats, including congestion, lymphoid depletion, and increased macrophage activity. These changes were markedly reduced in the group treated with lemon leaf extract, indicating a protective effect. Biochemical analysis revealed elevated MDA levels and reduced SOD and CAT activities in the lead-exposed group, while the co-administration of lemon leaf extract significantly mitigated these effects, restoring oxidative balance. Hematological analysis also indicated improvements in spleen function with lemon leaf treatment.

In conclusion, the aqueous extract of lemon leaves exhibits significant protective effects against lead-induced spleen toxicity in adult Wistar rats. This study suggests that lemon leaves could be a potential therapeutic agent for managing lead toxicity, emphasizing the need for further research to explore their mechanisms of action and potential applications in human health.

Table of Contents

Chapter One: Introduction

1.1 Background to the Study

1.2 Statement of the Problem

1.3 Objectives of the Study

1.4 Research Questions

1.5 Research Hypotheses

1.6 Significance of the Study

1.7 Scope of the Study

1.8 Organization of the Study

Chapter Two: Literature Review

2.1 Introduction

2.2 Lead Toxicity

2.2.1 Sources and Exposure Routes of Lead

2.2.2 Mechanisms of Lead Toxicity

2.2.3 Health Effects of Lead Toxicity

2.3 The Spleen: Structure and Function

2.4 Antioxidants and Their Role in Detoxification

2.5 Lemon Leaves (Citrus limon)

2.5.1 Phytochemical Composition of Lemon Leaves

2.5.2 Traditional and Medicinal Uses of Lemon Leaves

2.6 Previous Studies on Plant Extracts and Lead Toxicity

2.7 Gaps in the Literature

2.8 Conceptual Framework

Chapter Three: Research Methodology

3.1 Introduction

3.2 Research Design

3.3 Experimental Animals

3.3.1 Selection and Care of Wistar Rats

3.3.2 Ethical Considerations

3.4 Preparation of Lemon Leaf Extract

3.5 Experimental Procedure

3.5.1 Grouping and Treatment of Animals

3.5.2 Induction of Lead Toxicity

3.5.3 Administration of Lemon Leaf Extract

3.6 Data Collection

3.6.1 Histopathological Analysis

3.6.2 Biochemical Assays

3.6.3 Hematological Analysis

3.7 Data Analysis

3.8 Validity and Reliability

Chapter Four: Results and Discussion

4.1 Introduction

4.2 Histopathological Findings

4.2.1 Spleen Tissue Analysis

4.2.2 Comparison Between Groups

4.3 Biochemical Findings

4.3.1 Oxidative Stress Markers (MDA, SOD, CAT)

4.3.2 Antioxidant Enzyme Activities

4.4 Hematological Findings

4.4.1 Spleen Function Indicators

4.4.2 Comparison Between Groups

4.5 Discussion of Results

4.5.1 Interpretation of Histopathological Findings

4.5.2 Interpretation of Biochemical Findings

4.5.3 Interpretation of Hematological Findings

4.6 Comparison with Existing Literature

Chapter Five: Conclusion and Recommendations

5.1 Introduction

5.2 Summary of Key Findings

5.3 Conclusion

5.4 Recommendations

5.4.1 Recommendations for Further Research

5.4.2 Recommendations for Practice

5.5 Implications of the Study

5.5.1 Theoretical Implications

5.5.2 Practical Implications

5.6 Limitations of the Study

5.7 Suggestions for Future Research

References

Chapter Three:

Methodology

3.1 Introduction

This chapter outlines the research design, materials, and methods employed to investigate the effect of aqueous extract of lemon leaves on lead-induced spleen toxicity in adult Wistar rats. It includes detailed descriptions of the experimental procedures, animal care, preparation of extracts, administration protocols, and techniques for assessing the biochemical and histological parameters.

3.2 Research Design

The study utilized an experimental design involving four groups of adult Wistar rats. Each group received different treatments to evaluate the protective and therapeutic effects of lemon leaf extract on lead-induced spleen damage.

Group I (Control Group): Received distilled water only.

Group II (Lead-Induced Group): Received lead acetate only.

Group III (Treatment Group): Received lead acetate and aqueous extract of lemon leaves.

Group IV (Extract Only Group): Received aqueous extract of lemon leaves only.

3.3 Materials and Reagents

Animals: Adult Wistar rats (150-200g).

Chemicals: Lead acetate trihydrate, distilled water, and other analytical grade reagents.

Plant Material: Fresh lemon leaves (Citrus limon).

Equipment: Analytical balance, centrifuge, spectrophotometer, microtome, and light microscope.

3.4 Preparation of Aqueous Extract of Lemon Leaves

Collection and Authentication: Fresh lemon leaves were collected and authenticated by a botanist.

Washing and Drying: Leaves were washed thoroughly with distilled water and air-dried at room temperature.

Extraction Process: The dried leaves were powdered and soaked in distilled water (1:10 w/v) for 24 hours. The mixture was filtered, and the filtrate was concentrated using a rotary evaporator at 40°C to obtain the aqueous extract.

Storage: The extract was stored in a refrigerator at 4°C until use.

3.5 Animal Care and Handling

Housing: Rats were housed in standard laboratory conditions with a 12-hour light/dark cycle, temperature of 22±2°C, and relative humidity of 50-60%.

Diet: Rats were fed with a standard pellet diet and provided water ad libitum.

Acclimatization: All animals were acclimatized for one week before the commencement of the experiment.

3.6 Experimental Procedures

Lead Acetate Administration: Lead acetate was administered orally at a dose of 50 mg/kg body weight for 28 days to induce spleen toxicity.

Aqueous Extract Administration: The aqueous extract of lemon leaves was administered orally at a dose of 200 mg/kg body weight daily for 28 days, starting concurrently with the lead acetate administration for the treatment group.

Control Treatments: The control group received an equivalent volume of distilled water, while the extract-only group received the aqueous extract without lead exposure.

3.7 Biochemical Analysis

At the end of the treatment period, the rats were euthanized, and blood samples were collected for biochemical analysis. The following parameters were measured:

Hemoglobin (Hb): Measured using an automated hematology analyzer.

White Blood Cell (WBC) Count: Determined using a hemocytometer.

Liver Enzymes: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using a spectrophotometer.

Oxidative Stress Markers: Levels of malondialdehyde (MDA) and glutathione (GSH) in spleen tissue were determined using colorimetric assays.

3.8 Histopathological Examination

Spleen tissues were harvested and fixed in 10% formalin. The tissues were processed, embedded in paraffin, and sectioned using a microtome. Sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope for histopathological changes.

3.9 Statistical Analysis

Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test to compare differences between groups. A p-value of <0.05 was considered statistically significant.

3.10 Ethical Considerations

The study was conducted in accordance with the guidelines for the care and use of laboratory animals, and ethical approval was obtained from the Institutional Animal Care and Use Committee (IACUC).

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