History of Antibiotic growth promoters (AGPs)

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Activity of an antimicrobial compound isolated from Ginkgo biloba

Introduction

Ginkgo biloba is among the most sold medicinal plants of this world with estimates of sales in the USA of more than $249 million (de Kosky et al., 2008). Most of the sales are of special extracts from the leaves and are mainly used for the improvement of peripheral and central blood circulation (DeFeudis, 1998). Important constituents present in the leaves are terpene trilactones, i.e., ginkgolides (A, B, C, J) and bilobalide, many flavonol glycosides, biflavones, proanthocyanidins, alkylphenols, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine and polyprenols (van Beek, 2002). Egb761 is the first standardised preparation patented by Schwabe (1994) for medicinal use. The content of ginkgolic acids (alkylphenol) in this preparation should not exceed 5ppm because of suspected cytotoxic and allergenic properties (Pan, 2007).
A range of bioactivities such as antiparasitic (Atzori et al., 1993; Bombardellii and Ghione, 1993; Chen et al., 2008), antiviral (de Tommasi et al., 1990), antifungal (Watanabe et al., 1990; Anke and Sterner, 1991) and immunomodulating (Bourguet-Kondracki, 1991) activities have been displayed by compounds from G. biloba such as the terpenes (ginkgolides and bilobalides). In addition to these activities, the antibacterial activities of compounds from G. biloba leaf extracts have also been investigated. Bombardellii and Ghione (1993) reported 0.01-0.1 μg/ml bilobalide to be active in vitro against pathological strains of Trichomas vaginalis, S. aureus, E. faecalis, E. coli and Lactobacillus spp. Lee and Kim (2002) found kaempferol and quercitin (flavonol glycosides) to be active against C. perfringens and/or E. coli. In contrast to these findings Mazzanti et al (2000) and Lee and Kim (2002) found no antimicrobial activity for bilobalide and ginkgolide A and B as well as for rutin. It should however, be noted that Lee and Kim (2002) used the agar diffusion method which has severe limitations due to the inability of non-polar compounds to diffuse into the aqueous matrix of the agar (Eloff, 1998). Antibacterial activity for the alkylphenols (ginkgolic acid, ginkgols and bilobols) were observed by several investigators: Itokawa et al. (1987) observed a weak antimicrobial activity of bilobol and cardanol (alkylphenols) against S. aureus and E. faecalis. In contrast, Choi et al (2009) reported strong activity of hydroxyalkenyl salicylic acids (ginkgolic acid) as low as 2 μg/ml against vancomycin-resistant Enterococcus spp. Adawadka and El-Sohly (1981) observed activity of the anacardic acids (ginkgolic acids) against Mycobacterium smegmatis. Pan (2007) reported on the activities of ginkgolic acids as low as 25 μg/ml against Gram-positive bacteria including methicillin resistant S. aureus.
Due to the fact that antibacterial activity is apparently not confined to a single compound in G. biloba extract, the objective of the current investigation was firstly to isolate and identify the major antibacterial compound from G. biloba and secondly, to determine whether activity against E. faecalis and C. perfringens in an extract or fraction of an extract of G. biloba can be attributed to that specific compound or whether synergism or other interactions also play a role in the observed activity.

Materials and methods

Plant collection

Powders of leaves of G. biloba and H. perforatum were obtained from Biomox Pharmaceutical (Pty) Ltd – South Africa.

Isolation and identification of active compound

Column Chromatography

Two kilograms of finely ground G. biloba leaves were extracted with n-hexane. The hexane was removed using a rotary evaporator. A bioautogram of this extract developed with BEA (BEA = benzene 90 ml, ethanol 10 ml and ammonium hydroxide 1 ml) and sprayed with S. aureus revealed the presence of antibacterial zones.
The main antibacterial compound present in the hexane extract was isolated by bioassay guided fractionation using silica gel 60 Column chromatography with a chloroform-methanol gradient solvent system. Active fraction/s were combined and subjected to Sephadex LH-20 column chromatography eluting with chloroform-methanol (2:1) to isolate the active compound.

Structure elucidation

The isolated compound was analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy using the facilities available at the Medical University of South Africa (MEDUNSA).

Plant extracts

Different methods of extraction of G. biloba leaves were tested previously (chapter 2). Two extraction methods were selected as they resulted in the best MIC and total activity results against the test pathogens (see microdilution assay). The two extraction methods and extractants used are described briefly:

Extraction of plant material

 Direct extraction

Dried material was extracted using 100% concentrations of acetone, hexane, dichloromethane (DCM) or ethyl acetate (EA). A ratio of 1:10 dried material: extractant was used in all cases. Mixtures were shaken for 10 min in a Labotec 20.2 shaking machine at high speed. The extracts were centrifuged at 1 322 x g for 10 min before decanting into labelled containers. The process was repeated three times on the same material and extractant and the extracts were combined. Extracts were dried at room temperature under a continuous stream of air.

Solvent-solvent extraction

Solvent-solvent extraction was carried out in accordance with the method describe by Lee and Kim (2002). Dried material was extracted twice with 60% aqueous acetone at a ratio of 1:10 dried material vs. extractant at room temperature and filtered. The extract was concentrated by using rotary evaporation at 45 ◦C after which the extract was sequentially partitioned into hexane, EA, butanol and H2O portions. Each step was carried out three times to ensure adequate extraction. All solvents were saturated with distilled H2O before use to ensure adequate separation. The solvent portions were concentrated by rotary evaporation at 45◦C and further dried at room temperature under a continuous stream of air.

Microdilution assay

In the previous chapter, extracts or fractions of extracts of G. biloba were found to be active against E. faecalis and C. perfringens but not E. coli, Pseudomonas aeruginosa and Salmonella enterica. Typhimurium with low activity against S. aureus. The MIC and total activity (TA) data for C. perfringens and E. faecalis are presented again in this chapter. The TA is the volume to which 1g of dried plant material (or dried extract in the case of fractions of the extract) can be diluted and still retain activity (Eloff, 2000, 2004). The TA and MIC of the active compound were determined as described in Chapter 2. Zinc-bacitracin was used as a positive control to confirm the sensitivity of the system.

Determination of the concentration of the isolated compound by use of a bioautography method

A bioautography procedure was done according to Begue and Kline (1972) in order to determine the concentration of the active compound in the different extracts/ fractions. Briefly, duplicate TLC plates (10X20cm) were loaded with 50 μg (10 μl of 5 μg/ml) of each of the extracts or fractions. On the other side of the plate, the active compound was loaded with 10 μl each of a series of 20, 15, 10, 5, and 2.5 μg pure compound. The plates were developed in a DCM:Methanol (19:1) mobile system. Chromatograms were dried for 24 h at room temperature to remove the remaining solvent. S. aureus were used as the indicator organism to determine the concentration of the active compound in the different extracts/fractions because it has shown to develop clear zones of inhibition with very few complications frequently experienced with bioautography. Cultures of S. aureus were grown on Müeller-Hinton (MH) agar and incubated at 37◦C overnight. The broth culture was prepared by transferring 2-3 bacterial colonies with a sterile swab from agar into two 250 ml Erlenmeyer flasks each containing 100 ml MH broth. Broth cultures were incubated for 24 h at 37◦C. Developed TLC plates were inoculated with a fine spray of the bacterial suspension containing approximately 108 cells/ml of actively growing bacteria in a Biosafety Class II cabinet (Labotec, SA). The plates were sprayed until they were just wet and incubated overnight in a chamber at 100% relative humidity in the dark. The plates were subsequently sprayed with a 2 mg/ml solution of INT and incubated for 2-3 h in the same chamber. White areas indicate where reduction of INT to the colored formazan did not take place due to the presence of compound/s that inhibited the growth of the test bacteria. Bioautograms were sealed in clear plastic envelopes and scanned for a permanent record.

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Results and discussion

Isolation and antibacterial activity of ginkgolic acid from Ginkgo biloba leaf extracts

About 36 g of hexane extract was chromatographed over silica gel with a choroform-methanol gradient solvent system resulting in 19 fractions. Fraction 7 contained the most active zone against S. aureus. This fraction (4g) was subjected to Sephadex LH-20 chromatography, eluting with chloroform:methanol (2:1), resulting in 25 fractions of 5 ml each. Fractions 11-23 were combined and subjected to Sephadex LH-20, this time eluting with chloroform:methanol (95:5) giving rise to 37 fractions (5 ml). Fractions 30 – 37 (0.4 g) were subsequently chromatographed using a Silica gel column eluting with methanol:DCM (1:9) to yield 270mg of a white, amorphous compound labelled GbHK001.
Ginkgolic acid (6-alkylsalicylic acid) is an alkylphenol with 3 different classes occurring in G. biloba i.e. ginkgolic acids, ginkgols and bilobols. Other synonyms are 2-hydroxy-6-alkylbenzoic acids and anacardic acids (van Beek, 2002). “Ginkgolic acid” wil be used in this report. Ginkgolic acid has also been isolated from various parts of the cashew fruit Anacardium occidentale (Anacardiaceae) (Kubo et al., 1995) and recently also from Brazilian propolis (Silva et al., 2008). Activity of ginkgolic acid (C15:1, C15:2 and C15:3) against Gram-positive bacteria including methicillin resistant S. aureus (Muroi et al., 2003) and the dental pathogen, Streptococcus mutans (Green et al., 2008) with values of 1.56-6.25 μg/ml have been reported. Many studies showed that ginkgolic acids has much stronger activity against Gram-positive than Gram-negative bacteria (Kubo et al., 1993; Yang et al., 2004), except for Helicobacter pylori, the causative agent of acute gastritis (Kubo et al., 1999). The structure of the above mentioned compound is similar to that of the compound isolated in the current study except for the length and the number of double bonds of the alkyl side chain.
In this investigation, the isolated ginkgolic acid (C17:1) had no activity against the Gram-negative E. coli, S. typhimurium and P. aeruginosa. Activity of 100 μg/ml, 62.6 μg/ml and 1.56 μg/ml was observed for S. aureus, E. faecalis and C. perfringens respectively (Table 3.2). This is comparable to the spectrum of activity of bacitracin (no activity against E. coli and P. aeruginosa and activity of 0.3, 20 and 40μg/ml against C. perfringens, E. faecalis and S. aureus respectively (results not shown). In general, it is observed that antimicrobial activity of ginkgolic acid is inversely proportional to the length of the C6 chain and that at a particular critical length it reaches a maximum after which activity greatly diminishes to finally become inactive (Green et al., 2008). Green et al (2008) synthesized a series of ginkgolic acids possessing different lengths of the C6 side chain and found ginkgolic acid (C12:0) exhibited the most potent bactericidal activity against S. mutans, while ginkgolic acid (C15:0) did not show any activity up to 0.8 mg/ml. They noted that although this ginkgolic acid (C15:0) was ineffective against S. mutans, it nevertheless exhibited potent antibacterial activity against Propionibacterium acnes with a MIC of 0.78 μg/ml. Daoud et al. (1983) reported that the antimicrobial activity of a series of alkyldimethylbenzylammonium chlorides was a parabolic function of their lipophilicity and maximized with alkyl chain lengths between C12 and C16. The penetration of these compounds through cell membranes depends on their lipophilic properties. Substances with low lipid solubility would be unable to cross the lipophilic barriers and remain localized in the first aqueous phase they contact. Conversely, those with high lipid solubility would remain localized in the lipid regions (Franks and Lieb, 1986). Somewhere between these extremes there would be an optimum point of lipophilicity for transversing the cell barriers. According to Muroi et al (2003) this explanation can be applied in the case of the ginkgolic acids. Ginkgolic acid with chain length between C10 and C12 appears to possess the optimum balance between hydrophilicity and lipophilicity to penetrate cell membranes. In addition to the length, the volume of the lipophilic portions, which is altered by the position, number, and stereochemistry of double bounds, also affects activity (Muroi et al., 2003).
This explanation could probably also apply in the current investigation where the long alkyl chain length of ginkgolic acid (C17:1) rendered the ginkgolic acid less effective against S. aureus while activity was previously reported to be high (Muroi et al., 2003). It also indicates that the lipophilic character of the bacterial membrane can differ (Kubo et al., 1995) which explains the high activity of this long alkyl chain ginkgolic acid against E. faecalis and C. perfringens.
The alkylphenols possess contact allergenic, cytotoxic, mutagenic and slight neurotoxic properties (Koch et al., 2000; Baron-Rupert and Luepke, 2001). It should, however, be remarked that there is no solid evidence of a strong allergic reaction when taken orally. For instance, no reports have been filed on the adverse effects of Ginkgo homeopathic mother tinctures in spite of the fact that such extracts contain 2.2% ginkgolic acids (van Beek, 2002). The biological activities of the ginkgolic acid derivates have however attracted considerable attention for its molluscicidal activity against Oncomelania hupensis (Yang et al., 2008), anti-Toxoplasma gondii activity (Chen et al., 2008), antitumor (Kubo et al., 1993), antioxidant (Kubo et al., 2005) and xanthine oxidase inhibitory action (Masuoka and Kubo, 2004).

DECLARATION 
ACKNOWLEDGEMENTS 
ABSTRACT 
TABLE OF CONTENTS 
LIST OF TABLES 
LIST OF FIGURES 
LIST OF ABBREVIATIONS 
1 GENERAL INTRODUCTION 
1.1 History of Antibiotic growth promoters (AGPs)
1.2 Mode of action
1.3 The problem with AGPs
1.4 Consequences of banning AGPs for Animal Productivity and Health
1.5 Alternatives to AGPs
1.6 Characteristics of acceptable alternatives
1.6.1 Efficacy
1.6.2 Safety and acceptability to regulatory agencies
1.6.3 Ease of use
1.6.4 Economic considerations
1.7 Plant extracts: A viable option for replacing AGPs?
1.8 Do Ginkgo biloba and Hypericum perforatum leaf extracts have potential to be developed in AGPs?
1.8.1 Ginkgo biloba
1.8.2 Hypericum perforatum
1.9 Background on development of extracts of G. biloba and H. perforatum
1.10 Expansion of the work of Ntloedibe (2005) and Chikoto (2006)
1.11 Aim of this study
1.12 Objectives
1.13 References
2 ACTIVITY OF EXTRACTS AND FRACTIONS OF EXTRACTS OF G. BILOBA AND H. PERFORATUM ALONE OR IN COMBINATION ON BACTERIAL PATHOGENS 19
2.1 Introduction
2.2 Materials and methods
2.2.1 Plant collection
2.2.2 Plant extraction procedure
2.2.2.1 Serial extraction
2.2.2.2 Direct extraction
2.2.2.3 Solvent-solvent fractionation
2.2.3 Phytochemical analysis
2.2.4 Biological assays
2.2.4.1 Microorganisms used
2.2.4.2 Microdilution assay
2.2.4.3 Bioautography
2.2.5 Synergy / antagonistic interactions between extracts of G. biloba and H. perforatum
2.3 Results and Discussion
2.3.1 Extraction and phytochemical analysis
2.3.2 Biological assays
2.3.3 Synergistic / antagonistic interaction between extracts of G. biloba and H. perforatum
2.4 Conclusions
2.5 References
3 ACTIVITY OF ANTIMICROBIAL COMPOUND ISOLATED FROM GINKGO BILOBA 
3.1 Introduction
3.2 Materials and methods
3.3 Results and discussion
3.4 Conclusions
3.4 Acknowledgements
3.5 References
4 PHARMACODYNAMIC ASPECTS OF HEXANE EXTRACT AND GINKGOLIC ACID FROM G. BILOBA USING E. FAECALIS 
4.1 Introduction
4.2 Materials and methods
4.3 Results and discussion
4.4 Conclusion
4.5 References
5 GINKGO BILOBA AND HYPERICUM PERFORATUM EXTRACTS USED AS GROWTH PROMOTERS IN BROILERS 
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussion
5.4 Conclusions
5.5 Acknowledgements
5.6 References
6 GENERAL CONCLUSIONS 
6.1 References
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