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P. bifurcatum is a lower plant that has been used in ethnomedicine for the treatment of inflammation, cough and hypertension. The aim of the study was to evaluate the possible anti-inflammatory and hepatoprotective properties of methanol extract of P. bifurcatum. The acute toxicity test of the methanol extract showed no toxicity up to 5000 mg/kg body weight. The effect of the extract on inflammation was evaluated using fresh egg albumin-induced paw oedema in rats while its effect on liver damage was accessed using the carbon tetrachloride (CCl4)-induced hepatic damage and the following hematological, biochemical and antioxidant parameters measured, namely: packed cell volume(PCV), red blood cell(RBC), white blood cell(WBC), hemoglobin(Hb), platelet count(PLAT), bilirubin, alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), superoxide dismutase(SOD), catalase(CAT), reduced glutathione(GSH), malondialdehyde(MDA), c-reactive protein(CRP) as well as histopathologic evaluation. Administration of the methanol extract of P. bifurcatum (1000mg/kg) caused a progressive decrease in paw oedema(0.57±0.20, 0.47±0.02 and 0.25±0.02) at 1hr,2hr and 3hr, respectively, compared to the control(0.97±0.04,0.85±0.08 and 0.67±0.08) at the same period. The extract(1000mg/kg) also caused significant(p<0.05) increases in blood parameters (PCV-49.20±1.30, Hb-16.38±0.19, RBC-6.76±0.08, PLAT-146.20±0.66), GSH and CAT, no significant changes in SOD and organ to body weight ratio but significantly(p<0.05) reduced the WBC, ALT, AST, ALP, MDA and CRP compared to the untreated control. Histological sections of the liver in the two dose groups revealed severe coagulative necrosis of the hepatocytes in the centrilobular and midzonal areas of the hepatic lobules. The periportal hepatocytes were relatively normal compared to the untreated control. It is concluded from this study that methanol extract of P.bifurcatum possesses anti-inflammatory potential and can considerably ameliorate liver damage.


Title Page                                                                                                                                         i

Declaration                                                                                                                                     ii

Certification                                                                                                                                   iii

Dedication                                                                                                                                      iv

Acknowledgement                                                                                                                         v

Table of Contents                                                                                                                         vi

List of Tables                                                                                                                                xii

List of Figures                                                                                                                             xiii

Abstract                                                                                                                                       xvi

CHAPTER 1                                                                                                            


1.1 Background of the Study                                                                                                         1

1.2 Statement of the Problem                                                                                                         3

1.3 Aim                                                                                                                                           4

1.4 Objectives                                                                                                                                 4

1.5 Justification of Study                                                                                                               5




2.1 Overview of P. bifurcatum                                                                                                       6

2.2 Taxonomy                                                                                                                                 6

2.3 Phytoconstituents of P. bifurcatum                                                                                          7

2.4 Pharmacological properties of P. bifurcatum                                                                            8

2.4.1 Antioxidant activity                                                                                                             8

2.4.2 Antimicrobial activity                                                                                                          8

2.5 Anti-Inflammatory Properties of Some Medicinal Plants                                                      9

2.5.1 Dichrostachys cinerea (Fabaceae)                                                                                      9

2.5.2 Curcuma longa Linn (Zingiberacea)                                                                                   9

2.5.3 Asparagus africanus (Liliaceae)                                                                              10

2.5.4 Zingiber officinale (Zingiberaceae)                                                                                     11

2.5.5 Moringa oleifera (Moringaceae)                                                                             11

2.6 Hepatoprotective Properties of Some Medicinal Plants                                                        12

2.6.1 Aquilaria agallocha (Thymelaeaceae)                                                                                 12

2.6.2 Phyllanthus muellarianus (Euphorbiaceae)                                                                         12

2.6.3 Caesalpinia crista (Fabaceae)                                                                                             13

2.6.4 Opuntia ficus-indica (Cactaceae)                                                                                        13

2.6.5 Convolvulus arvensis (Convolvulaceae)                                                                 13

2.7 Inflammation                                                                                                                         14

2.7.1 Mediators of inflammation                                                                                                 14 Vasoactive amines and peptides                                                                                      14 Eicosanoids                                                                                                                      15 Acute-phase protein                                                                                                         16 Inflammatory cytokines                                                                                                   16

2.8 Inflammation and Diseases                                                                                                    20

2.8.1 Inflammation and cardiovascular diseases                                                                         20

2.8.2 Inflammation and insulin resistance diabetes mellitus                                                        21

2.8.3 Inflammation and cancer                                                                                                   21

2.8.4 Inflammation and osteoarthritis                                                                                         23

2.9 Screening Models for Anti-Inflammatory Activity                                                              24

2.9.1 Carrageenan-induced paw edema                                                                                      24

2.9.2 Histamine/5-Hydroxytryptamine-induced paw edema                                                     25

2.9.3 Bradykinin-induced paw edema                                                                                        26

2.9.4 Dextran induced paw edema                                                                                             26

2.9.5 Lipopolysaccharide-induced paw edema                                                                          26

2.9.6 Granular pouch model                                                                                                       26

2.9.7 Complete Freund’s adjuvant-induced arthritis                                                                  27

2.10 Liver Toxicity                                                                                                                     27

2.10.1 Stages of liver toxicity                                                                                                     28

2.10.2 Screening models for hepatoprotective agents                                                                29

2.10.3 Liver function tests                                                                                                          33 Biochemical indices                                                                                                      33 Histopathological assessment                                                                                       35 Types of histological preparations                                                                                35



3.1 Materials                                                                                                                               37

3.1.1 Collection of plant materials                                                                                              37

3.1.2 Instruments/ equipment                                                                                                     37

3.1.3   Chemicals/reagents                                                                                                          38

3.1.4   Drugs                                                                                                                                   39

3.2   Methods                                                                                                                                  40

3.2.1   Extract preparation                                                                                                              40

3.2.2   Determination of percentage yield of methanol extract P. bifurcatum leaves                     40

3.2.3    Phytochemical screening                                                                                                     40 Test for alkaloids                                                                                                               40 Test for steroids                                                                                                                 40 Test for flavonoids                                                                                                             41 Test for tannins                                                                                                                  41 Test for saponins                                                                                                                41 Test for terpenoids                                                                                                             41 Test for phenolics                                                                                                               41

3.2.4    Experimental animals                                                                                                         42

3.2.5    Acute toxicity (LD50)                                                                                                          42   Anti-inflammatory test                                                                                                     43   Hepatoprotective activity                                                                                                 44

3.2.7     Assessment of liver function                                                                                             45   Determination of alkaline phosphatase (ALP)                                                                  45   Determination of alanine aminotransferase (ALT)                                                           46 Determination of aspartate aminotransferase (AST)                                                          47   Determination of direct bilirubin (DB) and total bilirubin (TB) in serum                        48

3.2.8    Hematology parameters                                                                                                     48   Determination of erythrocyte count by hemocytometry                                                  48   Determination of total leucocyte count by hemocytometry                                                         49 Determination of packed cell volume (PCV)                                                                     50

3.2.9 Antioxidant estimation                                                                                                          51   Estimation of extent of lipid peroxidation                                                                       51   Assay of superoxide dismutase activity                                                                           52   Assay for catalase activity                                                                                                53    Reduced glutathione (GSH) estimation                                                                          53    Determination of total protein.                                                                                        54

3.2.11     Organ weight to body weight ratio                                                                                 57

3.2.12      Histopathological examination                                                                                       57    Tissue preparation                                                                                                          57    Slide examination                                                                                                          58

3.3     Statistical analysis                                                                                                                 58



4.1       Results                                                                                                                               59

4.1.1    Qualitative phytochemical screening of methanol extract of

            P. bifurcatum leaves                                                                                                          59 

4.1.2   Oral Acute Toxicity Study of methanol extract of P. bifurcatum leaves                          61

4.1.3    Effect of Methanol leaf extract of P. bifurcatum on egg albumin

            induced paw edema                                                                                                          62

4.1.4    Effect of methanol leaf extract of P. bifurcatum on packed cell volume, hemoglobin             concentration, red blood cell and platelet counts of CCl4- intoxicated rats.                    64

4.1.5    Effect of methanol leaf extract of P. bifurcatum on white blood cell count of CCl4-          intoxicated rats                                                                                                             69

4.1.6    Effect of Methanol leaf extract of P. bifurcatum on serum alanine transaminase, aspartate             transaminase, alkaline phosphatase activity and bilirubin concentration of CCl4-intoxicated           rats.                                                                                                                                            71

4.1.7    Effect of Methanol leaf extract of P. bifurcatum on catalase, superoxide dismutase activity, reduced glutathione concentration and malondialdehyde levels of CCl4-intoxicated rats.                                                                                                            77

4.1.8    Effect of Methanol leaf extract of P. bifurcatum on serum c- reactive protein activity of    CCl4-intoxicated rats.                                                                                                                 82

4.1.9    Final body weight, organ weights, and organ to body ratio                                              84

4.1.10  Histopathology of the liver                                                                                               86

4.2       Discussion                                                                                                                          92



5.1       Conclusion                                                                                                                        102

5.2       Recommendations                                                                                                            102

 REFERENCES                                                                                                                         103





                                                                LIST OF TABLES

4.1       Qualitative phytochemical screening of methanol extract of P. bifurcatum leaves            60   4.2             Effect of methanol leaf extract of P. bifurcatum on egg albumin induced

             paw edema                                                                                                                         63

4.3       Final body weight, organ weights. and organ to body ratio                                               85







4.1       Effect of methanol leaf extract of P. bifurcatum on Packed cell volume of CCl4-                                  intoxicated rats.                                                                                                                  65

4.2       Effect of methanol leaf extract of P. bifurcatum on hemoglobin concentration of CCl4-     intoxicated rats.                                                                                                             66

4.3       Effect of methanol leaf extract of P. bifurcatum on red blood cell count of CCl4-intoxicated        rats.                                                                                                                   67

4.4      Effect of methanol leaf extract of P. bifurcatum on Platelet count of CCl4-intoxicated rats.                                                                                                                                       68

 4.5      Effect of methanol leaf extract of P. bifurcatum on white blood cell count of CCl4-           intoxicated rats.                                                                                                             70

 4.6      Effect of methanol leaf extract of P. bifurcatum on serum alanine transaminase activity of             CCl4-intoxicated rats.                                                                                                                  72

 4.7      Effect of methanol leaf extract of P. bifurcatum on serum aspartate transaminase activity of CCl4-intoxicated rats.                                                                                                      73

4.8      Effect of methanol leaf extract of P. bifurcatum on serum alkaline phosphatase activity of CCl4-intoxicated rats                                                                                          74

 4.9      Effect of methanol leaf extract of P. bifurcatum on serum total bilirubin concentration of             CCl4-intoxicated rats.                                                                                                                 75

4.10    Effect of methanol leaf extract of P. bifurcatum on serum direct bilirubin concentration of CCl4-intoxicated rats.                                                                                                    76

 4.11    Effect of methanol leaf extract of P. bifurcatum on superoxide dismutase activity of         CCl4-intoxicated rats.                                                                                                                 78

 4.12    Effect of methanol leaf extract of P. bifurcatum on catalase activity of CCl4-intoxicated   rats.                                                                                                                                     79

 4.13    Effect of methanol leaf extract of P. bifurcatum on Reduced glutathione concentration of            CCl4-intoxicated rats.                                                                                                                  80

 4.14    Effect of methanol leaf extract of P. bifurcatum on Malondialdehyde concentration of     CCl4-intoxicated rats.                                                                                                                  81

4.15     Effect of methanol leaf extract of P. bifurcatum on serum c-reactive protein activity of      CCl4-intoxicated rats.                                                                                                                  83

4.16     Histological sections of the liver of rats given distilled water (normal rats)                      87

4.17    Histological sections of the liver of rats given Carbon tetrachloride (2 mg/kg)                  88

4.18     Histological sections of the liver of rats given Silymarin (50 mg/kg) and Carbon     tetrachloride (2 mg/kg)                                                                                             89

4.19     Histological sections of the liver of rats given P. bifurcatum (500 mg/kg) and Carbon         tetrachloride (2 mg/kg)                                                                                                    90

 4.20    Histological sections of the liver of rats given P. bifurcatum (1000 mg/kg) and Carbon       tetrachloride (2 mg/kg)                                                                                                    91










The body’s complex biological response to damaging stimuli such as infections, damaged cells or allergies is known as Inflammation (Ferrero-Miliani et al., 2007). Immune cells, blood vessels, and chemical mediators are all part of this protective response. Inflammation aims at clearing out damaged cells and tissues that have been destroyed by the initial injury and the inflammatory process thus initiating tissue regeneration (Abul and Andrew, 2009). Chronic inflammation produces a constant influx of reactive oxygen species, which overwhelm the antioxidant defenses, disrupt DNA, and induce ageing and diseases.

Microorganisms, physical agents, chemicals, erroneous immune responses, and tissue death can cause inflammation. Anti-inflammation is achieved by reducing inflammatory reactions with herbal remedies or non-steroidal anti-inflammatory drugs (NSAIDs). Over time, scientists have discovered that plant materials can be used to create new anti-inflammatory drugs.

The Liver is the organ responsible for drug metabolism and elimination (Singh et al., 2012). Hepatotoxicity is defined as liver impairment or damage caused by an excessive amount of drugs or xenobiotics such as acetaminophen, cadmium chloride, ethanol, carbon tetrachloride (CCl4), and galactosamine (Novarro and Senior,  2006). Hepato-toxicants are substances that cause damage to the Liver. They result from overdosing on certain medications, industrial chemicals, and natural chemicals such as microcystins, medicinal herbs and dietary supplements (Papay et al., 2009).

Hepatotoxicity is caused by direct toxicity of the main compound or a reactive metabolite from an immune response affecting liver cells, biliary epithelial cells, and liver vasculature (Deng et al., 2009). Liver disease is one of the world's most serious health issues. Using several of these hepatotoxins, hepatoprotective drugs have been researched extensively. However, studying promising hepatoprotective medicinal plants using carbon tetrachloride-induced liver injury has been effective. Its toxicity is primarily due to formation of reactive oxygen species (ROS). Reactive oxygen species bind to polyunsaturated fatty acids (PUFAs) forming hydroxyl or peroxyl radicals, damaging the cell membrane and altering enzyme activity (Khan et al., 2016). Centrilobular steatosis, inflammation, apoptosis, and necrosis are all effects of CCl4 exposure (Manibusan et al., 2007).

Medicinal plants are currently in high demand and, their popularity is growing. A medicinal plant is any plant that has medicinal properties. These plants include a diversity of compounds employed in drug discovery (Rasool Hassan,  2012). Different types of seeds, roots, leaves, fruit, skin, flowers and even plants may be utilized as medicinal plant components. Most medicinal plants have active compounds with direct or indirect therapeutic effects and are employed as medicinal agents.

Phytochemicals are referred to as biologically active naturally occurring compounds that are useful to humans (Hasler, 1999). They lack nutritional value, unlike vitamins and minerals. They do, however, have an impact on several bodily functions. They function with minerals and dietary fibre to protect the body from disease, slow the ageing process, and lower the risk of various ailments, including cancer, heart disease, stroke, and high blood pressure.

The "staghorn fern," Platycerium bifurcatum (Cav) C. Chr is an epiphyte that grows naturally on tree branches and trunks in tropical, subtropical, and rain forest environments (Bode and Oyedapo, 2011). Because it lacks roots and reproduces through spores rather than flowers, P. bifurcatum is considered a lower plant. It is propagated via spores and belongs to the Polypodiaceae family. It has been utilized in ethnomedicine in addition to ornamental purposes. Young leaves of P. bifurcatum have been reported to have various uses in Nigeria which includes preventing miscarriages in women two months after conception (Flora and Ubah, 2006). The plant can also be employed for treating oedema, coughs, and hypertension (Mensah et al., 2006).


One of the most critical public health issues is liver disease. Despite the use of hepatoprotective drugs, the number of liver disorders has continued to rise. Chronic hepatic injury, primarily caused by alcohol abuse, xenobiotic toxicity, chronic infections, such as those caused by the hepatitis C virus, bile duct damage, and non-alcoholic liver disease, are the most common causes of these disorders (Younissi et al., 2018). It is the main cause of death in most developing nations, particularly among people with metabolic syndrome, obesity, and type 2 diabetes (Loomba and Sanyal, 2013).

Nonsteroidal anti-inflammatory drugs (NSAIDS) affect the gastric mucosa, kidney, cardiovascular system, liver, and hematopoietic system. The adverse effects are most likely related to Cyclooxygenase inhibition, which prevents the generation of prostaglandins (Sostres et al., 2010).

Several plant components have been used for treatment of various ailments with few or no side effects worldwide, and some of these plant materials, which contain a large number of beneficial phytoconstituents, are poorly utilized and may contain numerous pharmacological properties.

1.3 AIM

This study aims to investigate the potential anti-inflammatory and hepatoprotective effects of methanol extract of P. bifurcatum leaves on carbon tetrachloride (CCl4) induced liver damage in Sprague Dawley rats.


The objectives of the study were as follows:

1.      Determine the phytochemical properties of methanol extract of P. bifurcatum.

2.      Determine the oral acute toxicity of methanol extract of P. bifurcatum

3.      Evaluate the effect of methanol extract of P. bifurcatum on hind paw edema in rats.

4.      Determine the effect of methanol extract of P. bifurcatum on biochemical, hematological and pathophysiologic parameters of rat liver damaged with carbon tetrachloride.



Medicinal plants are said to offer a variety of pharmacological properties. These plants have been utilized, but many others, notably lower plants, have remained unexplored. Researchers have continued to investigate the systemic effects of these plant preparations in the hopes of discovering new drugs or improving the potency of those that already exist.

P. bifurcatum is one of these lower plants that has been used in traditional medicine to treat inflammation, hypertension, cough, and other diseases. The purpose of this study is to confirm the ethnomedical use of the P. bifurcatum leaf using methanol as the extraction solvent. This will also serve as a criterion for recommending the plant's continued usage in ethnopharmacology.


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