MODULATORY EFFECTS OF THE CRUDE METHANOL LEAF EXTRACT OF CISSAMPELOS OWARIENSIS ON SOME BIOCHEMICAL PARAMETERS IN BLEOMYCIN-INDUCED LUNG FIBROSIS IN MALE WISTAR RATS

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TABLE OF CONTENTS

TITLE PAGE

CERTIFICATION……………………………………………………………………………………………………….. ii

DEDICATION……………………………………………………………………………………………………………. iii

ACKNOWLEDGEMENT……………………………………………………………………………………………. iv

TABLE OF CONTENTS………………………………………………………………………………………………. v

LIST OF FIGURES……………………………………………………………………………………………………. viii

LIST OF TABLES………………………………………………………………………………………………………… x

ABSTRACT………………………………………………………………………………………………………………… xi

CHAPTER ONE………………………………………………………………………………… 1

  1. INTRODUCTION………………………………………………………………………………………………. 1
    1. BACKGROUND OF STUDY………………………………………………………………………………. 1
      1. BLEOMYCIN INDUCING LUNG FIBROSIS……………………………………………………….. 3
    1. GENERAL OVERVIEW OF THE PLANT……………………………………………………………. 6
      1. ORIGIN OF THE LEAVES…………………………………………………………………………………. 6
    1. OBJECTIVES OF RESEARCH WORK………………………………………………………………. 10
    1. CHROMATOGRAPHY……………………………………………………………………………………… 10
      1. GAS CHROMATOGRAPHY……………………………………………………………………………… 10
      1. VACUUM LIQUID CHROMATOGRAPHY……………………………………………………….. 11
    1. LUNGS…………………………………………………………………………………………………………….. 11
      1. STRUCTURE OF THE LUNGS………………………………………………………………………….. 11
      1. FUNCTION OF THE LUNGS……………………………………………………………………………. 12
    1. LIPID PEROXIDATION……………………………………………………………………………………. 13
      1. INITIATION…………………………………………………………………………………………………….. 13
      1. PROPAGATION……………………………………………………………………………………………… 13
      1. TERMINATION………………………………………………………………………………………………… 14
      1. TESTS………………………………………………………………………………………………………………. 14

CHAPTER TWO………………………………………………………………………………. 15

2.6       HISTOPATHOLOGY………………………………………………………………………………………. 20

CHAPTER THREE…………………………………………………………………………… 21

CHAPTER FOUR…………………………………………………………………………….. 35

REFERENCES…………………………………………………………………………………………………………… 38

APPENDIX…………………………………………………………………………………………………………………. 46

LIST OF FIGURES

Figure 1: Picture of Cissampelos owariensis leaves…………………………………………………………….. 8

Figure 2: Picture of normal and fibrotic lungs…………………………………………………………………… 13

Figure 3.2.1: Bar chart showing inhibition of FeSO4-induced lipid peroxidation by CME, EAF, MEF of C. owariensis leaves………………………………………………………………………………….. 23

Figure 3.3.1: Bar chart showing plasma alkaline phosphatase activities of BLM-induced lung fibrosis in rats and the various treatment groups……………………………………………………………….. 24

Figure 3.3.2: Bar chart showing protein concentration of bleomycin induced lung fibrosis in rats and the various treatment groups of total protein………………………………………………………… 25

Figure 3.4.1: Bar chart showing superoxide dismutase activity of BLM-induced lung fibrosis in rats and treatment groups……………………………………………………………………………………………. 26

Figure 3.5.1: Bar chart showing reduced glutathione activity (GSH) of bleomycin induced lung fibrosis in rats and treatment groups…………………………………………………………………………. 27

Figure 3.6.1: Bar chart showing status of the level of Lipid peroxidation in the lung homogenate of BLM-induced lung fibrosis and treatment with quercetin, 200 and 400mg/kg CME of C. owariensis leaves………………………………………………………………………………………….. 28

Figure 3.7.1: The GC-MS representation of the crude methanol extract of Cissampelos owariensis……………………………………………………………………………………………………………………. 29

Figure 3.8.1: Histological picture of the lung section of a normal rat………………………………….. 30

Figure 3.8.2: Histological representation of untreated BLM-induced lung fibrosis………………… 31

Figure 3.8.3: Histopathology picture of the lung of BLM-induced lung fibrosis and treatment with10mg/kg quercetin…………………………………………………………………………………………………. 32

Figure 3.8.4: Histopathology picture of the lung of BLM-induced lung fibrosis and treatment with 200mg/kg crude methanol extract of Cissampelos owariensis………………………………………. 33

Figure 3.8.5: Histopathology picture of the lung of BLM-induced lung fibrosis and treatment with 400mg/kg crude methanol extract of Cissampelos owariensis………………………………………. 34

Figure 4: Graph of bovine serum albumin…………………………………………………………………………. 53

LIST OF TABLES

Table 1: Shows the qualitative phytochemical screening of CME, EAF, and MEF of the leaves of

Cissampelos owariensis containing alkaloids, saponins, and tannins…………………………………… 22

Table 2: Shows the statistical data of the blood chemistry and the various oxidative stress parameters……………………………………………………………………………………………………………………. 35

ABSTRACT

Antioxidants have been shown to play protective role in the treatment of bleomycin-induced lung fibrosis. The goal of the study was to determine the effect of Cissampelos owariensis (CO) leaves, an alkaloid rich antioxidant on BLM-induced lung fibrosis in rat models. The CO (UIH- 22559) leaves were extracted using methanol and fractionated using vacuum liquid chromatography (VLC) to obtain the ethyl-acetate fraction (EAF) and methanol fraction (MEF). The active component of CME was determined using gas chromatography and mass spectrometry (GC-MS). The inhibitory role of CME, EAF and MEF were assessed on FeSO4– induced lipid peroxidation (LPO) in in-vitro assay. In the in-vivo study, twenty (20) male Wistar rats (100-150g) were administered bleomycin (0.7U/100mg) intraperitoneally. They were divided into four (4) groups of five (5) animals each and given distilled water (negative control), 10mg/kg quercetin, 200 and 400mg/kg CME orally and daily for 28days. Additional five (5) rats were given distilled water (positive control). Activities of superoxide dismutase (SOD), reduced glutathione, levels of lipid peroxidation and plasma activities of total protein and alkaline phosphatase (ALP) were determined spectrophotometrically. Histological examination on lung tissues was done microscopically.

In the in-vitro studies, CME inhibited FeSO4-induced LPO levels by 34%, 65%, 52%, and 89%; EAF by 45%, 55%, 50%, and 52%; MEF by 37%, 60%, 61%, and 64%, in 50, 200, 300 and

400µg/ml respectively. The GC-MS showed that hexanoic acid, tetradecanoic acid, 9, 12, 15- Octadecatrienoic acid methyl ester, Linoleic acid ethyl ester, phytol, and bis(2-ethylhexyl) phythalate among others were the active components present in the CME of CO.

In the in-vivo studies, CME inhibited LPO in BLM-induced lung fibrosis by up to 83.200±1.1314 and 94.200±4.2427 in 200 and 400mg/kg groups when compared with the control. Levels of SOD and GSH were statistically increased by treatment with 400mg/kg CME while plasma total serum protein and ALP were decreased statistically when treated with 200, 400mg/kg CME and 10mg/kg quercetin relative to control. Data were analyzed using one way ANOVA α0.05. Histological examinations showed hemorrhagic lesions and presence of fibrosis which reduced to mild sloughing of the epithelium by treatment groups.

The results suggest that crude methanol extract of Cissampelos owariensis leaves may serve promising role in the treatment of BLM-induced lung fibrosis.

Key words: Oxidative stress, Apoptosis, quercetin, Cissampelos owariensis, VLC, GC-MS.

MODULATORY EFFECTS OF THE CRUDE METHANOL LEAF EXTRACT OF CISSAMPELOS OWARIENSIS ON SOME BIOCHEMICAL PARAMETERS IN BLEOMYCIN-INDUCED LUNG FIBROSIS IN MALE WISTAR RATS