Antiviral activity of ingredients in the fruit rind of Punica granatum L.
The research described in this thesis concerns the antiviral activity found in the fruit
rind of pomegranate, Punica granatum L., as reported by Stewart et al., (1998). The
active ingredients were not identified.
Chromatographic separation of pomegranate rind extract (PRE) by HPLC provided
fractions for antiviral testing. The process involved a large number of samples, which
required an accurate, inexpensive, fast and easy method to detect active antiviral
fractions. The method developed was based on phage lytic cycle technology.
Pseudomonas aeruginosa 10116-195 and Escherichia coli 13706-B1 bacteriophage
were used to investigate the virucidal activity of different fractions originating from
chromatographic separation, in combination with ferrous sulphate. After 3 min of
bacteriophage contact with each fraction, corresponding host bacteria were added and
the mixture placed onto a 96-well microtitre plate filled with appropriate media and the
plate incubated overnight at 37 °C. The assay was recorded as antiviral where the well
showed bacterial growth (cloudy). JSD solution, a combination of PRE and ferrous
sulphate, was use as control. Activity comparisons were made with known compounds.
Three fractions collected from preparative HPLC were found with antiviral activity. The
active fractions were isolated, purified and characterised by TLC, HPLC, mass
spectrometry, pH dependency, and chemical stability. A tentative identification of
punicalagin as the active polyphenol was also made.
The phage lytic method was used to screen seventy-four extracts from twelve
Amazonian plant species; thirteen were found with antiviral activity. The successful
screening of this large number of samples was enabled because of the high accuracy and
simplicity of the method.
Electron microscopy was used to demonstrate the destructive capability of PRE active
fractions when mixed with ferrous sulphate solution. Mechanism of action studies
indicated potentiation of PRE activity with acid pH and ferrous sulphate. Antiviral
effects are proposed to arise from free radical production and possible specific
inhibition of enzymes associated with bacteriophage activity.