Ethnomedicine in infections disease treatment and the relevance of cell culture in toxicity studies of medicinal plants

Get Complete Project Material File(s) Now! »

Introduction

Over the decades, there has been increasing evidence of infections caused by viruses and other pathogenic microorganisms. To date, viral infections still remain a major threat to humans and animals. As a metabolically inert particle, viruses reproduce only when they are within the host cell and as such require the metabolic pathway of living cells to replicate. This unique nature of viruses makes it difficult to design a drug that can either attack the virion or stages in the replication cycle, without affecting the host due to poor selective toxicity within host systems. Despite these drawbacks, substantial progress has been made in the development of antiviral agents for some viral infections in humans.
On the contrary, despite the outbreaks in recent years of RNA viral infections in the livestock sector, little success has been achieved towards the development of antiviral agents against these diseases and very few, if any, are available for veterinary use. Viral infections can be controlled either by prophylaxis or therapeutically. Although vaccines are available to protect against certain viral infections and advances are being made in DNA recombinant technology to produce new and safer vaccines, a comprehensive recent review indicates a possible vaccine-induced enhancement of infection in certain viral diseases (Huisman et al., 2009).
These responses produced in vaccinates may possibly act as a deterrence in the development of vaccines against certain viral infections. Of equal concern is the use of old vaccine viral strains in the formulation of currently available vaccines in the face of emerging virulent strains in the field. Vaccines have also been associated with residual virulence and toxicity, contamination with other pathogens, allergic responses, disease in immunodeficient hosts (modified vaccines), neurological complications, and harmful effects on the foetus and vaccine failure. The situation has become even more complex in the past decade with the rise in viral latency and resistance development to existing antiviral drugs used in humans (Kott et al., 1999).

Viruses

Development of resistance to antimicrobial agents is a growing problem worldwide that causes difficulties in the treatment of important nosocomial and community-acquired infections. Currently there are available antiviral drugs 3 for the management of a range of viral infections caused by human immunodeficiency virus 1 (HIV-1), herpes simplex virus (HSV- 1 and HSV-2), cytomegalovirus (CMV), influenza A virus, respiratory syncytial virus (RSV), papilloma viruses and hepatitis B and C viruses in humans.
Although considerable progress has been achieved in the past decades in this respect, the understanding of resistance development to antiviral agents is still in rudimentary stages, in large part because of the relatively recent advent of effective antivirals. To combat the development of antiviral resistance requires knowledge of the mechanism by which these pathogens elude therapeutic agents. Prior to the discovery of antiretrovirals, an extensive and systematic analysis of herpes simplex virus and varicella zoster virus resistance to acyclovir was undertaken and these findings have provided a major insight into antiviral drug resistance (Coen, 1996; Gilbert et al., 2002). With these viruses, the mutations that lead to resistance development to antiviral agents appear to be associated with reduced virulence and ability to cause infection. This phenomenon has served as a positive outcome for the majority of antivirals used in herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections.
In contrast, the development of resistance by the human immunodeficiency virus to antiretroviral therapy results from mutations in the genome of the virus coding for structural changes in the target proteins that can affect the binding or activity of currently used antiretroviral drugs (Menéndez-Arias, 2010). Other resistance development mechanisms against effective treatment for influenza virus (Hill et al., 2009), and chronic hepatitis B virus infection (Ghany and Liang, 2007) have been extensively reviewed. That notwithstanding, the processes by which viruses develop resistance to antiviral agents are increasingly being investigated and characterized for the growing number of antiviral agents.

Bacteria

Infectious diseases remain a leading cause of worldwide morbidity and mortality, whether in the general healthy population or in patients who are immunocompromised and are at risk of infection with invasive opportunistic pathogens. Even though antimicrobial drugs have played a major role in keeping these pathogens in check, the development of resistance to antibiotics currently remains one of the biggest challenges facing global health care systems. Available reports indicate that around 90–95% of Staphylococcus aureus strains worldwide are resistant to penicillin (Casal et al., 2005) and in most of the Asian countries 70–80% of the same strains are methicillin resistant (Chambers, 2001).
The development of resistance to antibiotics came to light from organisms that were exposed to the first commercially available antibiotics. The antimicrobial drug resistance of staphylococci to penicillin is one such example (Barber, 1947). Resistance develops either passively or actively as a result of attainment of new genetic material by the microbe or pre-existing innate mechanism (Summers, 2006; Wright, 2007). These resistance developments lead to 4 treatment failure which frequently has fatal consequences. It is worthy of note to recognize that resistance also affects the treatment of individuals with non-resistant organisms in areas with high rates of resistance thereby increasing overall treatment costs (Howard et al., 2003). The major mechanisms of antibiotic resistance include prevention of interaction of the drug with the target site, efflux of the antibiotic from the cell, and direct destruction or modification of the compound (Walsh, 2003; Levy and Marshall, 2004; Wright, 2005). Compounding the problem is the continued selective pressure by different drugs, resulting in bacteria acquiring additional kinds of resistance mechanisms that have given rise to multidrug resistance (MDR). Some of these resistance development mechanisms to antibacterial agents in Gram-positive and Gram-negative bacteria as well as molecular mechanisms of multidrug resistance have been extensively documented (Wright, 2005; Tenover, 2006; Rice, 2006; Alekshun and Levy, 2007; Matthew and Bliziotis, 2007).

Fungi

Despite the increase in the prevalence of resistance to antibacterial and antifungal agents, not much attention has been devoted to the study of antibiotic resistance. Studies of resistance development specifically to antifungal agents have lagged even further behind. Resistance development to antifungal agents is a broad concept that describes failure of a fungal infection to respond to antifungal therapy (Sheehan et al., 1999). Traditionally, this resistance development is classified as either primary (intrinsic), where the organism is resistant prior to exposure to the antifungal, or secondary (acquired), due to a stable transient genotypic modification following exposure to an antifungal agent. A third type of antifungal resistance can be described as “clinical resistance”, which arises from progression or relapse of an infection caused by a susceptible isolate in in vitro testing to an antifungal agent recommended for the treatment of the given infection. Such resistance development is common amongst immunocompromised patients or in patients where prosthetic materials have been used (Sheehan et al., 1999).
In some cases, suboptimum drug concentrations in the blood might contribute to the development of clinical resistance. The exposure of fungal pathogens to antifungal agents stimulates different responses in the metabolism of the organism. As a survival instinct, the fungal pathogen will strive to overcome the growth inhibitory effect of the antifungal agent by development of various mechanisms to counteract the inhibitory effect of the antifungal agent. These mechanisms will permit the growth of the pathogen at higher drug concentrations than is the case for normal susceptible pathogens, while in others where higher drug concentration results in growth inhibition, the fungal pathogen can alter the therapeutic potency of a given antifungal agent, which will determine if the agent will produce a static or a cidal effect (Sanglard, 2003). This property exhibited by fungal pathogens is termed antifungal drug tolerance. Prior to the validation by the United States National Committee for Clinical Laboratory Standards (NCCLS) now known as the Clinical and Laboratory Standard Institute (CLSI) in 1997, there was no widely accepted method for in vitro susceptibility testing of fungal pathogens. The method describes the determination of minimum inhibitory concentrations (MICs) of widely used antifungals against one species, which helps to evaluate with greater confidence whether in vitro susceptibilities are correlated with clinical response to therapy. Interpretive breakpoints of resistance with this standard method currently exist only for fluconazole, itraconazole, and flucytosine.

Cell cultures and toxicity studies

The development of a novel drug requires the evaluation of three major areas in drug design i.e. the efficacy, bioavailability and safety of the drug. About 30% of failures in the development of drugs have been associated with toxicity and safety issues (Kola and Landis, 2004). Among these, toxic effects imposed on the liver by these substances are one of the major issues encountered. Moreover, off-target or idiosyncratic toxicity, resulting in the post market withdrawal of drugs, is of increasing concern. This is to a certain extent due to the unavailability of adequate in vitro screening design that can effectively correlate with animal studies and its application to humans. However, Ekwall and co-workers have shown that a test battery of in vitro methods can predict human toxicity and that in vitro IC50 values correlate with in vivo LD50 data (Ekwall et al., 1998; Clemedson et al., 2000).
The rationale behind using cytotoxicity assays to predict in vivo toxicity stems from the concept of ‘basal cell cytotoxicity’. It was suggested that for most chemicals, toxicity is an end result of non-specific change in cellular functions. In light of this, assessing the cytotoxic potential of compounds may possibly give an indication of their toxic potential in vivo. Cytotoxicity has been defined as the adverse effects resulting from interference of agents with structures and processes essential for cell survival, proliferation and function (Ekwall, 1983). One major factor that warrants consideration in a drug discovery programme is the toxic potential of new chemical entities (NCEs). At this stage, the rationale behind the screening for toxicity would not be directed towards predicting the extent and nature of all possible toxic effects in vivo but at the assessment of the risk of failure in in vivo studies

Table of content :

• Declaration
• Acknowledgement
• List of tables
• List of figures
• List of abbreviations
• Publications from the thesis
• Abstract
• Chapter 1: Introduction
  • 1.1. Introduction
  • 1.2. Resistance development to antimicrobial agents
    • 1.2.1. Viruses
    • 1.2.2. Bacteria
    • 1.2.3. Fungi
  • 1.3. Adverse effect associated with the use of antimicrobial agents
    • 1.3.1. Antiviral agents
    • 1.3.2. Antibacterial agents
    • 1.3.3. Antifungal agents
  • 1.4. Virus
    • 1.4.1. Impact of viral disease outbreak on agriculture and livestock
• Chapter 2: Literature review on the antimicrobial activity of plant extract
  • 2.1. Ethnomedicine in infections disease treatment and the relevance of cell culture in toxicity studies of medicinal plants
  • 2.2. Use of plants as antimicrobial agents
  • 2.3. Common class of antiviral compounds present in medicinal plants
  • 2.4. Cell cultures and toxicity studies
  • 2.5. Antioxidants and cell culture
  • 2.6. Hypothesis
  • 2.7. Aims of the study
  • 2.8. Objective
• Chapter 3: Materials and Methods
  • 3.1. Plant collection
  • 3.2. Plant preparation and storage
  • 3.3. Extraction of plant material
  • 3.4. Thin layer chromatography (TLC) analysis of crude extracts
  • 3.5. Determination of qualitative antioxidant activity of extracts
  • 3.6. Solvent-solvent fractionation
  • 3.7. Antibacterial activity
    • 3.7.1. Bioautography on TLC plates
    • 3.7.2. Microdilution assay for MIC determination
  • 3.8. Antifungal assay
    • 3.8.1. Bioautography on TLC plates
    • 3.8.2. Microdilution assay for MIC determination
    • 3.8.3. Determination of total activity
  • 3.9. Determination of Cytotoxicity of extracts, fractions and pure compounds (MTT)
  • 3.10. Genotoxicity testing of isolated compounds
  • 3.11. Antiviral assay
    • 3.11.1. Cell cultures and viruses
    • 3.11.2. Virucidal assay
    • 3.11.3. Attachment assay
• Chapter 4: Comparative cytotoxicity studies of extracts of selected medicinal plants
  • on different cell types
  • 4.1. Introduction
  • 4.2. Materials and methods
    • 4.2.1. Plant collection and preparation
    • 4.2.2. Determination of qualitative antioxidant activity of extracts
    • 4.2.3. Determination of Cytotoxicity of extracts
  • 4.3. Results and discussion
    • 4.3.1. Effect of extracting solvents on yield of extracts
    • 4.3.2. Cytotoxic effect of extracts on cells
      • 4.3.2.1. Microscopic determination of cytotoxic effect of extracts of all the plants on different cell type
      • 4.3.2.2. Determination of cytotoxicity by MTT assay
      • 4.3.2.2.1. Plumbago zeylanica
      • 4.3.2.2.2. Ekebergia capensis
      • 4.3.2.2.3. Annona senegalensis
      • 4.3.2.2.4. Carissa edulis
      • 4.3.2.2.5. Podocarpus henkelii
    • 4.3.2.3. Antioxidant activity
  • 4.5. Conclusion
• Chapter 5: The antibacterial activity of different extracts of selected South African
  • plant species
  • 5.1. Introduction
  • 5.2. Materials and methods
    • 5.2.1. Thin layer chromatography (TLC) analysis of crude extracts
    • 5.2.2. Bioautography on TLC plates
    • 5.2.3. Microdilution assay for MIC determination
    • 5.2.3. Determination of cytotoxic effect of the extracts on different cell types
  • 5.3. Results and discussion
    • 5.3.1. Chemical constituents of the crude extracts
    • 5.3.2. Inhibition of bacterial growth using bioautography
    • 5.3.3. Antibacterial activity of extracts in terms of MIC values
  • 5.4. Conclusion
• Chapter 6: Evaluation of different extracts of selected South African plant species for antiviral activity
  • 6.1. Introduction
  • 6.2. Materials and Methods
    • 6.2.1. Viral pathogens used in the study
    • 6.2.2. Cell cultures
    • 6.2.3. Determination of cytotoxic effect of extracts on cells
    • 6.2.4. Virucidal assay
    • 6.2.5. Attachment assay
  • 6.3. Results and discussion
  • 6.4. Conclusion
• Chapter 7: Evaluation of different extracts of selected South African plant species for antifungal activity
  • 7.1. Introduction
  • 7.2. Materials and Methods
    • 7.2.1. Thin layer chromatography (TLC) analysis of crude extracts
    • 7.2.2. Bioautography on TLC plates
    • 7.2.3. Microdilution assay for MIC determination
    • 7.2.4. Determination of total activity
    • 7.2.5. Determination of cytotoxic effect of the extracts on different cell types
  • 7.3. Results and discussion
    • 7.3.1. Inhibition of bacterial growth using bioautography
    • 7.3.2. Antifungal activity of extracts in terms of MIC values
  • 7.4. Conclusion
• Chapter 8: Plant selection and antimicrobial activity of solvent – solvent fractions of leaf material
  • 8.1. Introduction
    • 8.1.1. Description of the plant Podocarpus henkelii stapt ex Dallim. & Jacks
    • 8.1.2. Taxonomy
    • 8.1.3. Chemotaxonomy
    • 8.1.4. Medicinal uses
  • 8.2. Material and Methods
    • 8.2.1. Solvent-solvent fractionation of leaf material
    • 8.2.2. Analysis and concentration of fractions
    • 8.2.3. Bioassay – guided fractionation
  • 8.3. Results and discussion
  • 8.4. Conclussion
• Chapter 9: Isolation and determination of chemical structure of compounds from Podocarpus Henkelii Stapt Ex Dallim. & Jacks
  • 9.1. Introduction
  • 9.2. Methods
    • 9.2.1. Column chromatography
    • 9.2.2. Structural elucidation
  • 9.3. Results and discussion
  • 9.4. Conclusion
• Chapter 10: Biological activity and toxicity studies of isolated compounds from Podocarpus Henkelii Stapf Ex Dallim. & Jacks
  • 10.1. Introduction
  • 10.2. Materials and Methods
    • 10.2.1. Determination of minimum inhibitory concentration (MIC) of isolated compounds against bacterial pathogens
    • 10.2.2. Determination of minimum inhibitory concentration (MIC) of isolated compounds against fungal pathogens
    • 10.2.3. Virucidal assay
    • 10.2.4 Attachment assay
    • 10.2.5. Cytotoxicity assay using MTT
    • 10.2.6. Genotoxicity testing of isolated compounds
  • 10.3. Results and Discussion
    • 10.3.1. Antibacterial activity of compounds
    • 10.3.2. Antifungal activity of compounds
    • 10.3.3. Antiviral activity of compounds
    • 10.3.4. Toxicity studies of compounds
  • 10.4. Conclusion
• Chapter 11: General discussion and conclusion
  • 11.1. Antibacterial and antifungal activity of different extracts of selected plant species
  • 11.2. Determining the cytotoxic effect of the different extracts on different cell types
  • 11.3. Determining the antiviral activity of different extracts of selected plant species
  • 11.4. Selection of plant species for fuxther investigation
  • 11.5. Isolation and biological activity of isolated compounds
  • 11.6. The cytotoxicity and genotoxic activity of isolated compounds
  • 11.7. Evaluating the correlation between antiviral and antimicrobial activity
• References
• Appendix

GET THE COMPLETE PROJECT
Isolation and characterization of compounds from Podocarpus henkelii (Podocarpaceae) with activity against bacterial, fungal and viral pathogens