Cytotoxicity of crude acetone extracts against Vero cells

Get Complete Project Material File(s) Now! »

Introduction

Antibiotics are commonly fed to animals in livestock production systems to prevent disease and metabolic disorders. Many of these mainstream antibiotic feed additives are products developed for human pharmaceutics (Rochfort et al, 2008). An enormous number of antibiotics have been described in literature but many are highly toxic for clinical use. Antibiotics are generally low to medium (100-1500) molecular weight compounds that exhibit a variety of chemical structures, physical and biological properties (Crosby, 1991). There are over 7000 compounds that have been described in terms of their source, mode of action and chemical structure (Crosby, 1991).
The feed additives used currently on the animal markets are mainly those with antimicrobial, endectocides and anticoccidial activity, drugs which are all synthetic derivatives of natural products (Rochfort et al, 2008). Additives are now available in varying forms, from direct-fed to slow release boluses which significantly improves the efficiency of the diet even in low quality feeds. The range of additives used in the animal production industry is quite broad (vitamins, trace minerals and growth promoters). They also include a range of auxiliary substance that may not be essential in nutrition but play a role in improving palatability (Wallace & Chesson, 1995). The majority of the industrial animal feed formulations are composed of plant derived materials. The major components of feeds are cereals and their by-products, plant material which are considered proteinaceous. There are lesser components like animal by-products in feeds but the vitamins, trace metals, growth promoters and synthetic amino acids are all added to generally complete the feed. Probiotics and enzymes are also now known to feature as additives (Wallace & Chesson, 1995). There is a growing concern worldwide on the prevalence of antibiotic resistance and this has led to the development and emergence of resistant bacteria and genes (van den Bogaard & Stobberingh, 2000).
Approximately 50% of all antibacterial agents used in Europe annually in human health are also used in veterinary medication for therapy and prevention of bacterial infections. They are also used as animal feed additives to promote growth and enhance feed efficacy (van den Bogaard & Stobberingh, 2000). Nearly all of the antibiotics used for veterinary purposes are given orally as feed additives to livestock by way of mixing it into the food, water or at times fed directly to the animals. The introduction of antibiotics in the veterinary practise has led to an increase in the resistance of pathogenic bacteria and an increase in faecal flora (Wallace, 2004). In recent years public concern over the routine use of antibiotics in livestock nutrition has increased due the emergence of resistant bacteria agents that represent a risk to human health (Benchaar, et al. 2008). The banning of antibiotic feed additives by the European Union (EU) in 2006 has directed the focus to finding new natural antibiotic feed additive alternatives.
Plant extracts offer a unique opportunity in this regard since they contain secondary metabolites which have antimicrobial properties and thus making them potential alternatives to conventional antibiotics (Wallace, 2004; Benchaar et al., 2008). This prompted the EU to invest in the Framework 6 REPLACE program which aims to screen plants for a range of activities including antibacterial, nematocidal and immune stimulating effects (www.rowett.ac.uk/rumen_up/RumenUp). The plant kingdom is a reservoir of a variety of chemical compounds and serves as source of new pharmaceutical products. Nearly all plants can be expected to protect themselves against disease and animal herbivores through the use of these chemical compounds stored by the plants. Plants compounds are been considered as possible natural alternatives (Steiner, 2006). There are large numbers of herbs and spices that are now considered as natural growth promoters in animal nutrition. Herbs like oregano and cinnamon have shown broad activity in vitro against pathogenic bacteria like E. coli, Salmonella spp and Clostridium perfringens (Kamel, 2000). Some of the enormous number of compounds that plants produce as natural protection against pathogen and insect attack may also be toxic to animals while others may not be.
The diversity of plants growing worldwide along with their pharmacological uses offer a possibility of finding novel chemical agents with efficacious antibiotic properties. It has been stated that natural products and their derivatives form about 50% of most drugs in use with about 25% of them been derived from plants (Farnsworth, 1984 & Harborne, 1998). Through the European Commission funded research projects Rumen (QLK5-CT-2001-00992) and REPLACE (FOOD-CT-2004-506487), a catalogue of plant samples were established through the collection of plant material from six geographical locations in three European countries and around the world. Pterocarya fraxinifolia, one of the plant species from the catalogue, fed to pigs and poultry had good activity in replacing antibiotic growth promoters. Leaves and fruit extracts from P. fraxinifolia trees growing in Denmark, Germany and Poland had widely diverging activity in animal studies. For this reason it would make sense to isolate and characterize the antimicrobial compounds so that this can be used to select tree populations with a high activity in replacing antibiotic growth promoters and establish the possible mechanism of activity for P. fraxinifolia as replacement for antibiotic growth promoters and this was the reason why P. fraxinifolia was selected for this study. In vivo trials on non-extracted plant material of Pterocarya fraxinifolia The influence of Pterocarya fraxinifolia plant material was tested on the growth performance of piglets six weeks post weaning. In brief a standard weaner diet was formulated and four experimental treatments were prepared consisting of; i) Control (standard diet), ii) control plus 0.5 % plant material, iii) control plus 1 % plant material and iv) control plus 2 % plant material.
A total of 192 piglets from 48 litters were included in the trial. There were twelve replicates per treatment group, each consisting of a pen with four piglets each. The piglets were weaned at 28 ± 2 days and moved to the pens for the initiation of the experiment. The piglets were kept separate in each holding pen for the duration of the experiment and no mixing of piglets was allowed. The piglets were fed ad libitum (Hojberg et al, 2008). The trail was conducted over a six week period commencing on the day of weaning. The feed intake of each pen was recorded daily and the individual body weight of the piglets was recorded weekly. In the first two weeks of the study the consistency of the faeces in the each pen was recorded. Faecal samples from randomly selected piglets from each pen were taken on days 5, 13 and 41 of the experiment and this was collected directly from the rectum for analysis (Hojberg et al, 2008). The weekly feed intake improved significantly from day 5 post weaning in the four treatments used in this study and contained to increase through days 15, 20 and 28. No decrease in feed intake was observed for the duration of the experiment. The daily feed intake was also good and no decrease was observed, with the highest daily feed intake occurring from days 7-14, 14-21 and 21-28. The live body weights of the piglets increased significantly from the first day post weaning, almost doubling in body weight after 28 ± 2 days. The daily weight gain of the piglets was notably significant on days 14-24 but the overall weight gain post 28 days was less but still significant. There was a significant drop in faecal score 6 days post weaning in all four treatment parameters, with significant decrease in faecal coliform counts and pH on day 14 through to day 28 of the trail (Engberg, 2008 & Hojberg et al, 2012). The inclusion of P. fraxinifolia plant material in the feed of piglets post weaning has growth promoting effects and demonstrates significant levels of antimicrobial activity of the non-extracted plant material from different regions of Europe. This was evident in the in vivo trials and in vitro assays conducted using the non-extracted plant material of P. fraxinifolia.

READ The influence of the African worldview on matters of illness and trauma

Table of contents :

Declaration
Acknowledgements
List of abbreviations
Abstract
Table of contents
List of figures
List of tables
Chapter Introduction
- 1.2 Hypothesis
- 1.4 Aims/objectives
  - 1.4.1 Aim
1.4.2 Objectives
Chapter Literature review
- - - 2.1 Introduction
      - 2.1.1 Pig production systems
      - 2.1.2 Weaning of pigs
      - 2.1.3 Managing gut health
      - 2.1.4 Pig diseases
      - 2.1.5 Feed and feed additives
      - 2.1.6 Antibiotics in animal feeds
      - 2.1.7 Types of antibiotics commonly used in animal production
      - 2.1.8 Antibiotic residues
      - 2.1.9 Antibiotic resistance in bacteria
      - 2.1.10 Antibiotic mode of action
      - 2.1.11 Natural growth promoters- alternatives to antibiotics
      - 2.1.12 Major groups of antimicrobial compounds from plants
      - 2.1.13 Pterocarya fraxinifolia (Lam.) Spach
      - 2.1.14 Bacterial species used in this study

Chapter
- 3.1 Introduction
- 3.2 Materials and methods
  - 3.2.1. Plant collection
  - 3.2.2 Extraction of plant material
  - 3.2.3 Solvent-solvent fractionation of extracts
  - 3.2.4 Analysis of plant extract for preliminary screening
- 3.3 Results
  - 3.3.1 Extraction
  - 3.3.2 TLC analysis of the plant extracts
  - 3.3.3 Total phenol content
  - 3.3.4 Number of antioxidant compounds (DPPH qualitative assay)
  - 3.3.5 The Trolox Equivalent Antioxidant Concentration (TEAC quantitative assay) and phenolic content
  - 3.3.6 DPPH Free Radical Scavenging Effect
- 3.4 Discussion and conclusion
Chapter
- 4.1 Introduction
  - 4.1.1 15-lipoxygenase inhibition
  - 4.1.2 Cytotoxicity
- 4.2 Materials and methods
  - 4.2.1 Anti-inflammatory activity
  - 4.2.2 Cytotoxicity of crude acetone extracts against Vero cells
- 4.3 Results
  - 4.3.1 Cytotoxicity
  - 4.3.2 15-lipoxygenase inhibitory assay
- 4.4 Discussion and conclusion
Chapter
- 5.1 Introduction
- 5.1.1 p-iodonitrotetrazolium violet (INT) reaction
- 5.2 Materials and methods
  - 5.2.1 Plant collection and extraction
  - 5.2.2 Antimicrobial assay
  - 5.2.3 Total activity
- 5.3 Results
  - 5.3.1 Minimum inhibitory concentration of crude extracts and fractions
  - 5.3.2 Total activity
- 5.4 Discussion and conclusion
Chapter
- 6.1 Introduction
- 6.2 Materials and methods
  - 6.2.1 Plant collection and preparation
  - 6.2.2 Bioautography
  - 6.2.3 Bacterial test species
  - 6.2.4 Fungal test species
- 6.3 Results
  - 6.3.1 Bacterial and fungal bioautography
- 6.4 Discussion and conclusion
Chapter
- 7.1 Introduction
- 7.2 Materials and Methods
  - 7.2.1 Plant collection
  - 7.2.2 Solvent-solvent fractionation
  - 7.2.3 Column chromatography; isolation of bioactive compounds
  - 7.2.4 Antimicrobial activity and cytotoxicity of isolated compound(s)
- 7.3 Results
- 7.3.1 Structure Elucidation of Compounds
- 7.4 Discussion and Conclusion
Chapter
- 8.1 General discussion and conclusion
  - 8.1.1 To evaluate in vitro antimicrobial activity of P. fraxinifolia tree extracts growing in different
  - regions of Europe against selected bacteria and fungi
  - 8.1.2 To determine the antioxidant and anti-inflammatory activity as possible alternative
  - mechanisms of action and also the cytotoxicity of P. fraxinifolia extracts
  - 8.1.3 To isolate the bioactive compound from the plant and determine the structure and
  - antimicrobial activity of the isolated compounds
  - 8.1.4 To determine the correlation between in vivo activity and in vitro activity of extracts from
  - different tree populations against E.coli K88, C. perfringens and C. jejuni
  - 8.1.5 To discuss the possible mode of actions and variation of activity between extracts of tree
  - populations from different areas
- 8.2 Conclusion
- References
- Appendix A