The present invention relates to antimicrobial compositions, and to uses of such
compositions as medicaments, and in methods of treating microbial infections.
The invention extends to methods of preventing microbial infections on objects
and surfaces coated with the compositions.
The growth in viral infections along with the emergence of antimicrobial drug
resistance in human bacterial pathogens is an increasing problem worldwide. As
a consequence, effective treatment and control of such micro-organisms is
becoming a greater challenge. However, bacterial resistance has appeared for
every major class of antibiotic. Since the introduction of antimicrobials, the
emergence of resistance has become increasingly evident, particularly in
important pathogens such as E.coli, Salmonella spp., Campylobacter spp.,
Enterococcus spp and Staphylococcus spp.
Over the last decade, research into the antimicrobial properties of traditional plant-
based medicines has increased. These studies have screened numerous plants
for antimicrobial properties. For example, Punica granatum L(Punicaceae), which
is referred to in English as pomegranate, has been highlighted in many of these
studies as having antimicrobial activity against a range of both Gram-positive and
Gram-negative bacteria. Recently, several studies have concentrated on the
antimicrobial properties of pomegranates. For example, one group of researchers
used different extraction methods using pomegranates against a range of six
bacteria, including S.aureus, E.coli, K.pneumoniae, P.vulgaris, B.subtllis and
S.typhi, and demonstrated good activity against all isolates tested. Another group
demonstrated that pomegranate extract was able to inhibit not only the growth of
S.aureus, but also the production of enterotoxin.
Many bacteria have advanced protective mechanisms to detoxify heavy metal
ions. However, despite this, a wide range of literature exists describing the
development of metal compounds as antimicrobial agents. Many low
molecularmass metal compounds exhibit bactericidal and/or bacteriostatic

activities. In one study, the susceptibilities of Staphylococcus strains to solutions
of metal salts (in the range of 50 μmol to 80 mmol) were determined. The
frequencies of resistance for Staphylococcus strains varied widely between
different metal salts. Accordingly, despite the growing need for new antimicrobial
therapies, the mechanism of action of many metal binding antibiotics is not
understood.
The enhancement of antimicrobial activities of various plant extracts by the
addition of metal salts has been previously investigated. For example, EP
0,744,896B1 discloses antimicrobial compositions, which are based on a
combination of ferrous salts and an extract from a plant such as pomegranate
rind, Viburnum plicatum leaves or flowers, tea leaves, or maple leaves. The
addition of ferrous salts to the plant extract was found to enhance the anti-viral
and anti-fungal activities of the composition.
However, a significant problem with these iron salt-based plant extract
compositions is that they lack stability, and therefore retain their antimicrobial
activity for only very short periods, ie up to a maximum of 30 minutes.
Accordingly, these compositions are of limited use. Another problem with these
iron-based antimicrobial compositions is that they become discoloured upon
application to a subject. It will be appreciated that antimicrobial compositions that
become discoloured are far from ideal in the majority of applications, in particular
those for topical use on patients, and also in non-therapeutic uses, such as on
surfaces prone to microbial infection in hospitals. A further disadvantage with the
iron-based compositions disclosed in EP 0,744,896B1 is that they are optimally
active at low pH (ie circa pH 4.0). Compositions which are optimally active only in
acidic conditions are disadvantageous in the majority of applications, particularly
when treating patients. Furthermore, such compositions are particularly difficult to
formulate.
It is therefore an object of the present invention to obviate or mitigate one or more
of the problems of the prior art, whether identified herein or elsewhere, and to

provide improved antimicrobial compositions, which may be used in methods for
treating microbial infections.
The inventor based his research on the anti-viral and anti-fungal compositions
reported in EP 0,744,896B1 in an attempt to solve the problems inherent with
these compositions. In order to address these problems, the inventor investigated
whether or not it was possible to substitute the ferrous ions with other metal ions
to form an antimicrobial composition, which exhibited improved properties, ie did
not turn black, had antimicrobial activity for more than 30 minutes, and was active
at a more amenable pH, such as neutral pH. Therefore, a number of other metal
ions were tested in combination with an active plant extract, for example,
pomegranate rind extract (PRE) as a model for other plant extract-based
compositions.
As described in Example 2, the metal ions that were tested for their abilities to
enhance the activity of the PRE included copper (I), copper (II), zinc (II), and
manganese (II). Iron (II) salts were also tested as a control. As shown in the
results in Figure 2, as expected, the iron (II) compositions exhibited antimicrobial
activity, thereby confirming the work disclosed in EP 0,744,896B1. However, the
inventor noticed that zinc and manganese ion-based compositions exhibited no
antimicrobial activity at all, as shown in Figure 3. Furthermore, surprisingly, the
highest activity was exhibited by Cu(il) salts upon addition to PRE. Given that
zinc and manganese-based plant extract combinations are ineffective, but copper-
based compositions are active, the inventor has suggested that there is some, as
yet unknown, mechanism of action for these metal ion-based antimicrobial
compounds.
From a consideration of the Periodic Table, the inventor has noticed a pattern
emerge concerning which metal ions are active and which are inactive when
combined with plant extracts. Given that manganese- and zinc-based
compositions are inactive, and given that iron- and copper-based compositions
are active, the inventor believes that salts of the two metals that are between iron

and copper in period 4 of the transition elements, ie cobalt and nickel, may also be
used to prepare active antimicrobial compositions.
The inventor believes that he is the first to prepare antimicrobial compositions
based on a combination of a copper salt, or a nickel salt, or a cobalt salt,
combined with a suitable plant extract, for example, pomegranate plant extract or
tea leaves.
Therefore, according to a first aspect of the invention, there is provided an
antimicrobial composition comprising (i) a copper salt and/or a cobalt salt and/or a
nickel salt; and (ii) an extract of a plant selected from a group consisting of Punica
granatum, Viburnum plicatum, Camellia sinensis, and Acer spp.
The inventor has found that compositions according to the first aspect, which
comprise an extract of a plant, such as Punica granatum (ie pomegranate),
Viburnum plicatum, Camellia sinensis (ie tea), and Acer spp. combined with salts
of copper, nickel and/or cobalt exhibit surprisingly effective antimicrobial activity,
and in some cases are more active that known iron-based compositions. This
activity could not have been predicted as the mechanism of action is unknown,
and not predictable in view of the fact that manganese- and zinc-based
compositions are inactive. From his studies, and as demonstrated in the
examples, the inventor has found that copper salts appear to be the most active.
Therefore, preferably the composition according to the first aspect comprises an
effective concentration of a copper salt and an effective concentration of an
extract of a plant selected from a group consisting of Punica granatum, Viburnum
plicatum, Camellia sinensis, and Acer spp.
A significant advantage of such copper-based compositions is that they are less
likely to become discoloured, in use, than known iron-based compositions. While
the inventor does not wish to be bound by any hypothesis, he believes that known
iron-based antimicrobial compositions become discoloured because aromatic
compounds contained within the composition are polymerized in the presence of

the iron ions. However, surprisingly, and advantageously, such polymerisation
does not occur in the presence of copper ions, and so the composition according
to the first aspect does not become discoloured. This is a significant advantage of
using copper-based compositions over the known iron-based compositions
because they may be used in many more applications, which are currently not
possible with iron-based compositions, such as topically on patients.
By the term "antimicrobial composition", we mean a substance or agent, which
kills, inhibits or slows the growth of a micro-organism. Examples of micro-
organisms, which the composition according to the invention may combat include
bacteria, viruses, fungi, or protozoa, and other pathogens and parasites.
An objective of the present invention was to stabilise and prolong the activity of
the antimicrobial compositions according to the first aspect of the invention, and
also known iron-based antimicrobial compositions. Therefore, as described in
Example 1, the inventor carried out spectroscopic metal ion binding studies to
investigate the mechanism of action of the iron-based composition disclosed in EP
0,744,896B1. Interestingly, the results of the metal binding study indicate that the
activation step for enhanced antibiotic activity (ie addition of ferrous ions to the
PRE component) results in the oxidation of the metal ion from the Fe(ll) to the
Fe(lll) oxidation state. Although the inventor does not wish to be bound by any
hypothesis, he believes that the significant loss of activity of the iron-based
antimicrobial composition, which is witnessed after 30 minutes, may be directly
attributable to this oxidation process.
This surprising realization led the inventor to investigate the effects of adding a
reducing agent to the active mixture in an attempt to re-generate the Fe(ll) by
reduction of the oxidised Fe(lll) ions to rejuvenate efficacy, and activity. To test
his hypothesis, the inventor chose Vitamin C as a reducing agent (reductant) to
see if it had the effect of extending the activity life of iron-based and also copper-
based compositions.

As described in the Examples, to his surprise, adding Vitamin C did have a
significant effect of considerably extending the activity of both iron- and copper-
based plant extract ointment compositions. The results clearly demonstrate that
enhanced bactericidal activity (against P.aeruginosa) occurs when Vitamin C was
included in both copper- and iron-based compositions.
Therefore, preferably the composition according to the first aspect comprises a
reducing agent. The inventor believes that this stabilizing or activating effect of
the reducing agent on the compositions according to the invention is an important
aspect of the invention as it not only applies to copper-based plant extract
compositions, but also to known iron-based compositions. The inventor also
believes that the benefit of adding a reducing agent could be used in relation to
cobalt- and nickel-based compositions.
Therefore, according to a second aspect of the invention, there is provided an
antimicrobial composition comprising (i) a copper salt and/or an iron salt and/or a
nickel salt and/or a cobalt salt; (ii) an extract of a plant selected from a group
consisting of Punica granatum, Viburnum plicatum, Camellia sinensis, and Acer
spp.; and (iii) a reducing agent.
While the inventor does not wish to be bound by any hypothesis, he believes that
the reducing agent has the effect of maintaining the iron in the Fe(ll) active state
and the copper in the Cu (II) active state. Examples 3, 4 and 7 demonstrate how
effective the addition of a reducing agent is to the activity of these compositions.
The inventor was surprised that the duration of the activity of iron-based and
copper-based compositions, particularly when formulated as ointments, could be
increased from 30 minutes in the absence of a reducing agent to as much as 3
months in the presence of a reducing agent. This was totally unexpected, and
most advantageous in medical applications.
By the term "reducing agent", we mean any agent or compound that donates
electrons to the metal ion, ie copper or iron or cobalt or nickel, in the composition.

The skilled technician will appreciate the various types of reducing agent or
reductant that may be combined in the composition. For example, the reducing
agent may be cysteine or glutathione. However, a preferred reducing agent
comprises Vitamin C (ie ascorbate), which is shown to be surprisingly active in the
Examples.
One, five and twenty equivalents of Vitamin C have been used relative to the
metal ion which in the Examples was normally fixed at about 4.8 millimoles.
Where a reducing agent is used, the composition preferably comprises between
about 1mM and about 200mM reducing agent, more preferably between about
2mM and about 150mM reducing agent, even more preferably between about
3mM and about 120mM reducing agent, and most preferably between about 4mM
and about 100mM reducing agent.
Figure 2 demonstrates that the effect of the reducing agent increases with
increasing concentration of Vitamin C. Hence, it is preferred that excess
concentrations of reducing agent are used compared to the metal ion.
Therefore, suitable effective concentrations of the reducing agent in compositions
according to the first and second aspect of the invention are in a weight ratio of
the reducing agent to the metal ion of at least 1:1, more suitably at least 2:1, and
even more suitably at least 5:1. Preferred weight ratios of reducing agent to metal
ion are at least 10:1, more preferably at least 20:1, and most preferably at least
50:1.
The compositions of the first and second aspect exhibit surprisingly high
antimicrobial activities. Accordingly, the inventor believes that he is the first to
discover a first medical use for the compositions according to the first and second
aspects.
Hence, in a third aspect, there is provided a composition comprising (i) a copper
salt and/or a cobalt salt and/or a nickel salt; and (ii) an extract of a plant selected
from a group consisting of Punica granatum, Viburnum plicatum, Camellia
8

sinensis, and Acerspp.; or a composition comprising (i) a copper salt and/or an
iron salt and/or a cobalt salt and/or a nickel salt; (ii) an extract of a plant selected
from a group consisting of Punica granatum, Viburnum plicatum, Camellia
sinensis, and Acerspp.; and (iii) a reducing agent; for use as a medicament.
Furthermore, the inventor believes that the compositions according to the first and
second aspects will have utility for treating microbial infections.
Therefore, in a fourth aspect there is provided a composition comprising (i) a
copper salt and/or a cobalt salt and/or a nickel salt; and (ii) an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp.; or a composition comprising (i) a copper salt
and/or an iron salt and/or a cobalt salt and/or a nickel salt; (ii) an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp.; and (iii) a reducing agent; for use in treating,
ameliorating or preventing a microbial infection.
Furthermore, in a fifth aspect, there is provided use of a composition comprising
(i) a copper salt and/or a cobalt salt and/or a nickel salt; and (ii) an extract of a
plant selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acer spp.; or a composition comprising (i) a copper salt
and/or an iron salt and/or a cobalt salt and/or a nickel salt; (ii) an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acer spp.; and (iii) a reducing agent; in the manufacture of
a medicament for the treatment, amelioration or prevention of a microbial
infection.
According to a sixth aspect, there is provided a method of treating, preventing or
ameliorating a microbial infection, the method comprising administering to a
subject in need of such treatment a therapeutically effective amount of a
composition comprising (i) a copper salt and/or a cobalt salt and/or a nickel salt;
and (ii) an extract of a plant selected from a group consisting of Punica granatum,
Viburnum plicatum, Camellia sinensis, and Acer spp.; or of a composition

comprising (i) a copper salt and/or an iron salt and/or a cobalt salt and/or a nickel
salt; (ii) an extract of a plant selected from a group consisting of Punica granatum,
Viburnum plicatum, Camellia sinensis, and Acer spp., and (iii) a reducing agent.
According to a seventh aspect, there is provided use of a composition comprising
(i) a copper salt and/or a cobalt salt and/or a nickel salt; and (ii) an extract of a
plant selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp.; or a composition comprising (i) a copper salt
and/or an iron salt and/or a cobalt salt and/or a nickel salt; (ii) an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acer spp., and (iii) a reducing agent; as an antimicrobial
agent.
The inventor has found that the compositions according to the first and second
aspects comprising an effective concentration of copper salt and/or iron salt
and/or a cobalt salt and/or a nickel salt exhibit antimicrobial activity. A preferred
effective concentration of the copper, iron, nickel, or cobalt salt is in the range of
about 0.1 mM to about 200mM, more preferably between about 0.3mM and about
100mM, even more preferably between about 0.5mM and about 50mM, still more
preferably between about 1mM and about 30mM, and most preferably between
about 2mM and about 10mM.
The nature of the metal salt (ie copper salt, iron salt, cobalt salt or nickel salt) is
not believed to be critical to the antimicrobial activities of the compositions
according to the invention. However, it is preferred that the metal salt comprises a
metal (II) salt.
For example, the nature of the anion in a copper salt is not critical to the efficacy
of the antimicrobial composition. However, the results indicate that copper (II)
sulphate may be more active than copper (I) chloride. Therefore, where the
composition comprises a copper salt, preferably the copper salt is a copper (II)
salt, ie a cupric ion. Most preferably, the copper salt is copper sulphate. A
preferred effective concentration of the copper salt is in the range of about 0.1 mM

to about 200mM, more preferably between about 0.3mM to about 100mM, even
more preferably between about 0.5mM to about 50mM, still more preferably
between about 1mM to about 30mM, and most preferably between about 2mM to
about 10mM.
Preferably, the plant extract in the composition according to the first aspect
comprises an extract of Punica granatum, and preferably pomegranate rind
extract (PRE). Therefore, preferably the composition according to the first aspect
comprises copper sulphate, combined with an extract of Punica granatum, and
preferably PRE. This preferred composition is described herein as copper
sulphate/PRE, and has shown considerable advantage over known iron-based
compositions as it does not suffer the problem that it turns black in use. In an
embodiment where the composition comprises a reducing agent, such as Vitamin
C, it is referred to herein as copper sulphate/PRE/Vitamin C.
Furthermore, where the composition according to the second aspect comprises an
iron salt, the nature of the anion in the iron salt is not critical to the efficacy of the
antimicrobial composition. However, the results demonstrate that iron (II)
sulphate exhibits greater antimicrobial activity than iron (III) chloride. Accordingly,
preferably the iron salt is an iron (II) salt, ie a ferrous ion. Most preferably, the iron
salt comprises ferrous sulphate. A preferred effective concentration of the iron
salt is in the range of about 0.1 mM and about 200mM, more preferably between
about 0.3mM and about 100mM, even more preferably between about 0.5mM and
about 50mM, still more preferably between about 1mM and about 30mM, and
most preferably between about 2mM and about 10mM.
The nature of the anion in a nickel or cobalt salt is also not critical to the efficacy
of the antimicrobial composition. However, where the composition comprises a
nickel salt or a cobalt salt, preferably the nickel salt is a nickel (II) salt, and
preferably the cobalt salt is a cobalt (II) salt. Most preferably, the nickel salt is
nickel sulphate, and most preferably the cobalt salt is cobalt sulphate. Preferred
effective concentrations of the nickel or cobalt salt are in the range of about
0.1 mM and about 200mM, more preferably between about 0.3mM and about

100mM, even more preferably between about 0.5mM and about 50mM, still more
preferably between about 1mM and about 30mM, and most preferably between
about 2mM and about 10mM.
In one embodiment, the composition according to the first aspect comprises (i)
either a copper salt or a cobalt salt or a nickel salt; and (ii) an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp. However, in a preferred embodiment, the
composition of the first aspect comprises at least two metal ions, and most
preferably at least all three metal ions in combination with the plant extract.
In one embodiment, the composition according to the second aspect comprises (i)
either a copper salt or an iron salt or a cobalt salt or a nickel salt; (ii) an extract of
a plant selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp.; and (iii) a reducing agent.
However, in a preferred embodiment, the composition according to the second
aspect comprises at least two metal ions, and more preferably at least three, and
most preferably all four of the metal ions, in combination with an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp. and a reducing agent. Most preferably, copper
and iron salts are combined with the plant extract.
Accordingly, preferred compositions according to the first or second aspect
comprise a copper (II) salt and an iron (II) salt combined with an extract of a plant
selected from a group consisting of Punica granatum, Viburnum plicatum,
Camellia sinensis, and Acerspp. Most preferred compositions according to the
second aspect comprise copper sulphate and iron sulphate combined with an
extract of a plant selected from a group consisting of Punica granatum, Viburnum
plicatum, Camellia sinensis, and Acerspp. and a reducing agent. Preferably, the
composition is formulated as an ointment, which is described hereinafter.

Preferably, the plant extract in the composition according to the second aspect
comprises an extract of Punica granatum, and preferably pomegranate rind
extract (PRE). Preferably, the reducing agent in the composition according to the
second aspect is Vitamin C. Accordingly, most preferred compositions according
to the second aspect comprise copper sulphate, iron sulphate, and an extract of
Punica granatum, and preferably PRE, and Vitamin C. This preferred composition
is described herein as copper sulphate/iron sulphate/PRE/Vitamin C. This
composition exhibits surprising antimicrobial activity, and extended shelf-life when
formulated as an ointment.
Compositions according to the invention comprise an extract of a plant selected
from Punica granatum, Viburnum plicatum, Camellia sinensis, or Acer spp.
Punica granatum is referred to as pomegranate. The inventor has found that
extracts from the whole pomegranate may be effectively used to provide
antimicrobial compositions according to the invention. However, preferably
compositions according to the invention comprise an extract from the rind of
Punica granatum, ie pomegranate rind extract (PRE).
Viburnum plicatum has been shown to have an active component. Preferably,
compositions according to the invention comprise an extract from leaves or
flowers of Viburnum plicatum.
Camellia sinensis is the taxonomic name given to common tea. Any parts of the
tea plant may be used to prepare compositions according to the invention,
although the tea leaves are preferred. Preferred teas may be green tea or black
tea. As demonstrated in Example 8, the inventors have found that a combination
of green tea and black tea has effective antimicrobial properties against
Staphylococcus aureus, Pseudomonas aeruginosa and Proteus mirabilis.
Acer spp. refers to a broad genus of maple plant. Preferably, compositions
according to the invention comprise an extract from leaves or flowers of Acer spp.

Preferred Acer species include Acer pseudoplatanus (UK acer) or Canadian
maple plant.
In order to prepare suitable plant extracts for preparing compositions according to
the invention, the chosen plant is first comminuted, preferably in a solvent, which
is then preferably boiled. A preferred solvent is water. The extract may be
fractionated, preferably by centrifugation. The fractionated extracts contain an
active compound.
Example 8 established that the preferred extraction method for green and black
tea was by boiling at about 100°C for at least 2 min, more preferably at least 4
min, even more preferably for at least 6 min, and most preferably at least 10 min.
The plant extracts may be sterilized, for example by autoclaving, and then allowed
to cool and stored at -20°C. A further purification of the plant extract (eg
pomegranate extract) to a molecular weight cut-off of below about 10,000Da may
be carried out, for example, by membrane ultrafiltration before storage.
The plant extract may be used in a concentrated form. Alternatively, the extract
may be diluted as appropriate to its intended use. Typically, about 10g of dried
plant extract may be used in about 150ml of water. This may give an effective
concentration of between about 1 and 99% (w/w) plant extract, preferably
between about 2 and 80% (w/w) plant extract, and more preferably between about
5 and 50% (w/w) plant extract. Effective compositions according to the invention
comprise 1-99% (v/v) of the metal salt solution combined with 99-1% (v/v) of the
plant extract. The compositions according to the invention may be in the form of a
solid or liquid concentrate.
Hence, in an eighth aspect, there is provided a composition according to either the
first or second aspect in the form of a solid or liquid concentrate, for dilution with
water.

Due to their increased biological activity, compositions comprising copper and/or
iron and/or nickel and/or cobalt salts, a plant extract and, in the case of
compositions according to the second aspect a reducing agent, are of utility as
antimicrobial agents. Hence, the compositions according to the first and second
aspects of the invention may be used in the treatment against any microbial
infection, such as a bacterial, viral or fungal infection.
Preferably, the compositions according to the invention are antibacterial
compositions. The bacterium may be a Gram-positive or a Gram-negative
bacterium. For example, bacteria against which the compositions in accordance
with the invention are effective may include Firmicutes, which may be Bacilli or
Clostridia, for example Clostridium botulinum. Further examples of bacteria
against which the compositions are effective may include Bacillales, such as
Bacillus subtilis, as demonstrated in Example 5.
The compositions may be effective against Staphylococcus, for example,
Staphylococcus aureus. As demonstrated in Example 6, the compositions
according to the invention are particularly effective against MRSA (methicillin-
resistant S. aureus), MSSA (multiple antibiotic-resistant methicillin-resistant S.
aureus) and Panton-Valentine Leukocidin (PVL) producing cMRSA isolates (ie
community acquired MRSA, which produce Panton-Valentine leukocidin).
Additional Bacillales with which the compositions are effective include
Streptococci, for example, Streptococcus pyogenes or Streptococcus
pneumoniae.
Further examples of bacteria against which the compositions in accordance with
the invention are effective may include Pseudomonadales, such as Pseudomonas
aeruginosa (as demonstrated in the Examples). As demonstrated in Example 7,
the compositions according to the invention are effective against multi-drug
resistant Pseudomonads (such as extended spectrum 0-lactamase Pseudomonas
aeruginosa).

Further examples of bacteria against which the compositions are effective may
include Gammaproteobacteria, which may be selected from a group consisting of
Enterobacteriales, Proteus, Serratai, Pasteurellales, and Vibrionales. As
demonstrated in Example 5, suitable Enterobacteriales against which the
compositions are effective include Escherichia ssp., such as E.coli. Examples of
Proteus against which the compositions are effective include Proteus mirabilis as
described in the Examples. Examples of Serratai include Serratia marcescens.
Examples of Pasteurellales include Haemophilus influenzae. Examples of
Vibrionales include Vibrio cholerae.
Further examples of bacteria against which the compositions according to the
invention are effective may include Betaproteobacteria, including Neisseriales, for
example, Neisseria gonorrhoeae. Further examples of bacteria against which the
compositions are effective may include Delta/epsilon subdivided Proteobacteria,
including Campylobacterales, for example Helicobacter pylori. Further examples
of bacteria against which the compositions are effective may include
Actinobacteria, for example Mycobacterium tuberculosis and Nocardia asteroides.
The compositions and medicament according to the invention may be used for the
treatment of a variety of bacterial infections, including: microbial keratitis;
conjunctivitis; bronchopulmonary infections, for example, pneumonia; urinary tract
infections, for example, cystitis, pyelonephritis; ear, nose, and throat infections, for
example, otitis media, sinusitis, laryngitis, diphtheria; skin infections including
cellulitis, impetigo, wound infections, botulism, gonorrhoea; septicaemia; peptic
and duodenal ulcer; gastritis; Campylobacter infections; Proteus mirabilis
infections; meningitis; osteomyelitis; and Salmonellosis.
The compositions according to the invention may be antiviral compositions.
Compositions and medicaments according to the invention may be used in the
treatment of a number of viral infections. The virus may be any virus, and
particularly an enveloped virus. Examples of viruses against which the
compositions are effective include poxviruses, iridoviruses, togaviruses, or
toroviruses. Further examples include a filovirus, arenavirus, bunyavirus, or a

rhabdovirus. Further examples include a paramyxovirus or an orthomyxovirus. It
is envisaged that virus may be a hepadnavirus, coronavirus, flavivirus, or a
retrovirus. The virus may be a herpes virus or a lentivirus. The virus may be
Human Immunodeficiency Virus (HIV), Human herpes simplex virus type 2
(HSV2), or Human herpes simplex virus type 1 (HSV1). Alternatively, viruses
which may be combated also include bacteriophages.
Compositions according to the invention may be antifungal compositions.
Compositions and medicaments according to the invention may be used in the
treatment of a number of fungal infections. For example, fungi against which the
compositions in accordance with the invention are effective may include a
filamentous fungus, such as an Ascomycete. Examples of fungi against which the
compositions in accordance with the invention are effective may be selected from
a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus;
Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora;
Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia;
richophyton; and Ustilago.
Further examples of fungi may be selected from a group of genera consisting of
Aspergillus and Candida. The fungus may be selected from a group of species
consisting of Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans;
Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis;
Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis;
Candida tropicalis; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium
solani; Leptosphaerianodorum; Magnaporthe grisea; Phytophthora capsici;
Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia
coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia
solani; Trichophytoninterdigitale; Trichophyton rubrum; and Ustilago maydis.
Further examples of fungi include yeast, such as Saccharomyces spp, eg S.
cerevisiae, or Candida spp, and C.albicans, which is know to infect humans.
It will be appreciated that the compositions according to the invention may be
used in a monotherapy (ie use of the compositions according to the invention

alone to prevent and/or treat a microbial infection or contamination).
Alternatively, the compositions according to the invention may be used as an
adjunct to, or in combination with, known antimicrobial therapies. For example,
conventional antibiotics for combating bacterial infections include amikacin,
amoxicillin, aztreonam, cefazolin, cefepime, ceftazidime, ciprofloxacin, gentamicin,
imipenem, linezolid, nafcillin, piperacillin, quinopristin-dalfoprisin, ticarcillin,
tobramycin, and vancomycin. For example, compounds used in antiviral therapy
include acyclovir, gangcylovir, ribavirin, interferon, anti-HIV medicaments including
nucleoside, nucleotide or non-nucleoside inhibitors of reverse transcriptase,
protease inhibitors and fusion inhibitors. Hence, compositions and medicaments
according to the invention may be used in combination with such antibacterial and
antiviral agents. Conventional antifungal agents include, for example, farnesol,
clotrimazole, ketoconazole, econazole, fluconazole, calcium or zinc undecylenate,
undecylenic acid, butenafine hydrochloride, ciclopirox olaimine, miconazole
nitrate, nystatin, sulconazole, and terbinafine hydrochloride.
Compositions according to the invention may have a number of different forms
depending, in particular, on the manner in which the composition is to be used.
Thus, for example, the composition may be in the form of a powder, tablet,
capsule, liquid, ointment, gel, hydrogel, aerosol, spray, micellar solution,
transdermal patch, liposome suspension or any other suitable form that may be
administered to a person or animal. It will be appreciated that the vehicle of the
composition of the invention should be one which is well tolerated by the subject
to whom it is given.
Compositions and medicaments comprising metal ions, plant extract, and
reducing agent (for the second aspect) according to the invention may be used in
a number of ways. For instance, oral administration may be required in which
case the metal ion, plant extract, and where used, a reducing agent, may be
contained within a composition that may, for example, be ingested orally in the
form of a tablet, capsule or liquid. Alternatively, the composition may be
administered systemically by injection into the blood stream. Injections may be
intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The

compositions may also be administered by inhalation (e.g. intranasally).
Alternatively, compositions according to the invention may be administered by
aerosol, for example using an atomiser, by which the composition may be
administered nasally or via the lungs.
However, preferably the compositions may be topically applied, for example in the
form of an ointment, cream or gel or aqueous solution. Topical administration is
useful when a subject to be treated has a microbial skin infection. For instance,
ointments may be applied to the skin, areas in and around the mouth or genitals to
treat specific viral infections. The composition may be applied intravaginally (for
example, if required to protect the subject from sexually transmitted diseases), or
rectally. Intravaginal administration is effective for treating sexually transmitted
diseases (including AIDS). Topical application to the skin is particularly useful for
treating viral infections of the skin or as a means of transdermal delivery to other
tissues also.
Example 3 describes the inventor's efforts to produce a preferred formulation for
the compositions and medicaments according to the invention. The inventor set
out to enhance the stability of the product formulation and conducted preliminary
toxicity tests. In order to optimise activity and enhance stability, a number of
formulations were prepared and investigated. The antimicrobial activities were
screened for a variety of formulations (ie cream, aqueous and ointment
formulation). Hence, the composition of the invention may take the form of a
cream or aqueous (water-based) solution.
The term "cream" refers to a soft cosmetic-type preparations. Creams of the oil-in-
water (OA/V) type include preparations such as foundation creams, hand creams,
shaving creams, and the like. Creams of the water-in-oil (W/O) type include cold
creams, emollient creams, and the like. Pharmaceutically, creams are solid
emulsions containing suspensions or solutions of active ingredients for external
application.

However, to his surprise, the inventor found that even though the cream and
aqueous-based formulations exhibited useful antimicrobial activities, they did not
remain as active for as long as the ointment formulation. For reasons not fully
understood by the inventor, the activity of the ointment formulation was
significantly prolonged compared to that of the cream or aqueous formulation.
Hence, it is preferred that the compositions and medicaments in accordance with
the invention are provided as an ointment formulation.
By the term "ointment formulation", we mean a viscous, semi-solid preparation
suitable for topical use on a variety of body surfaces (eg the skin). It will be
appreciated that an ointment has an oil base containing a substantially high
concentration of lipids, and therefore tends to be immiscible in water, whereas a
cream has a lower concentration of lipids, and tends to be water soluble. Hence,
ointments are more occlusive than creams, and form a protective film over the
skin. Ointments are therefore generally composed of single-phase hydrophobic
bases, for example of pharmaceutical grades of soft paraffin or microcrystalline
paraffin wax. Ointments are generally used for the application of insoluble of oil-
soluble medicaments and leave a greasy film on the skin, inhibiting loss of
moisture and encouraging hydration of the keratin layer (Physiochemical
Principles of Pharmacy by Florence & Attwood, 1992). Preferred ointments should
be of such composition that they soften, but not necessarily melt, when applied to
the body. They serve as vehicles for the topical application of the active
ingredients and may also function as protectives and emollients for the skin.
Accordingly, the ointment formulation may comprise a substantially high
concentration of lipid or fat. Suitably, the ointment formulation comprises at least
0.1% (w/w) lipid, more suitably at least 0.5% (w/w) lipid, even more suitably at
least 1% (w/w) lipid, and most suitably at least 2% (w/w) lipid. Preferably, the
ointment formulation comprises at least 5% (w/w) lipid, more suitably at least 10%
(w/w) lipid, even more suitably at least 15% (w/w) lipid, and most suitably at least
20% (w/w) lipid. These concentrations are higher than those for cream
formulations.

The composition according to the invention may be formulated with an ointment
base, which is preferably hydrous. The ointment base may comprise "wool alcohol
ointment" which will be known to the skilled technician. One example of a suitable
ointment base which may be used in the preparation of an ointment formulation
according to the invention is shown in Table 1.

An ointment formulation may be prepared as follows. The hydrous ointment base
may be prepared by mixing the wool alcohol ointment, phenoxyethanol and
magnesium sulphate. The purified water shown in the Table was used in the
Examples as a control where no plant extract, metal salt or Vitamin C was added.
However, in order to prepare an active ointment formulation according to the
invention, the plant extract (eg PRE) and metal salt is mixed with the ointment
base instead of the water, to thereby form the ointment formulation. In
embodiments where a reducing agent is used, a solution of a suitable reducing
agent (eg Vitamin C) may also be added to the ointment base to prepare the
active ointment formulation. For example, about 0.072 g of CuSO4, and about
5.0724 g of Vitamin C may be added to the 29.1 g of PRE which is then added to
the ointment base to form the active ointment formulation.
Figure 5 and Examples 3 and 7 show a bactericidal assay of an ointment of PRE
combined with various components, such as iron or copper salts, and Vitamin C.
Figure 6 shows the same compositions, and their activity after 3 weeks. As
shown in Figures 5 and 6, the activity enhancement upon addition of Vitamin C for
either Fe(ll)/PRE or Cu(ll)/Pre compositions is retained for three weeks with no
reduction in efficacy for the ointment formulation. Hence, the ointment formulation

shown in Table 1, combined with added Vitamin C exhibited greatly enhanced
stability compared to the aqueous preparation (data not shown) showing full
retention of activity after 3 weeks. Surprisingly, these findings are in contrast to
the cream or aqueous formulations, which in the former case had poor activity and
in the latter case lost activity after only 30 minutes. Therefore, the inventor has
clearly demonstrated the surprising efficacy, and retained activity over long
periods of time, of ointment-based formulations for compositions according to the
invention. While the inventor does not wish to be bound by any hypothesis, he
believes that the components of the ointment may have some protecting effect on
the reducing agent (ie Vitamin C), perhaps preventing it from being oxidized,
thereby prolonging its activity on the metal ion and plant extract.
Accordingly, most preferred medicaments according to the invention comprise an
ointment formulation comprising copper sulphate/PRE/Vitamin C; or iron
sulphate/PRE/Vitamin C; or copper sulphate/iron sulphate/PRE/Vitamin C.
The inventor then carried out toxicity studies on mammalian cell cultures as
described in Example 3. These studies showed that the highest percentage of
viable cells was observed with PRE while the lowest was encountered after
treating the cells with PRE/FeSCVCuSOij/Vitamin C combination which was
demonstrated to be non-toxic.
The compositions according to the invention may also be incorporated within a
slow or delayed release device. Such devices may, for example, be inserted on
or under the skin, and the active compounds (ie the iron or copper ion, the plant
extract and, where applicable, the reducing agent) may be released over weeks or
even months. Such devices may be particularly advantageous when long term
treatment with a composition according to the invention is required and which
would normally require frequent administration (eg at least daily injection).
It will be appreciated that the amount of composition that is required is determined
by the biological activity and bioavailability of the active components, which in turn
depends on the mode of administration, the physicochemical properties of the

composition employed and whether the composition is being used as a
monotherapy or in a combined therapy. The frequency of administration will also
be influenced by the above-mentioned factors and particularly the half-life of the
composition and active agents thereof within the subject being treated.
Optimal dosages to be administered may be determined by those skilled in the art,
and will vary with the particular composition in use, the strength of the preparation,
the mode of administration, and the advancement of the disease condition, ie the
microbial infection or contamination. Additional factors depending on the
particular subject being treated will result in a need to adjust dosages, including
subject age, weight, gender, diet, and time of administration.
Known procedures, such as those conventionally employed by the pharmaceutical
industry (eg In vivo experimentation, clinical trials, etc.), may be used to establish
specific formulations of the compositions according to the invention and precise
therapeutic regimes (such as daily doses of the compositions and the frequency of
administration).
Generally, a daily dose of between 0.01 ug/kg of body weight and 0.5 g/kg of body
weight of compositions according to the invention may be used for the prevention
and/or treatment of a microbial infection, depending upon which composition is
used. More preferably, the daily dose is between 0.01 mg/kg of body weight and
200 mg/kg of body weight, and most preferably, between approximately 1 mg/kg
and 100 mg/kg.
Daily doses may be given as a single administration (eg a single daily injection).
Alternatively, the composition used may require administration twice or more
times during a day. As an example, compositions according to the invention may
be administered as two (or more, depending upon the severity of the condition)
daily doses of between 25 mg and 7000 mg (ie assuming a body weight of 70kg).
A patient receiving treatment may take a first dose upon waking and then a
second dose in the evening (if on a two-dose regime) or at 3- or 4-hourly intervals

thereafter. Alternatively, a slow release device may be used to provide optimal
doses to a patient without the need to administer repeated doses.
Compositions according to the invention generally comprise a pharmaceutically
acceptable vehicle.
The invention also provides, in a ninth aspect, a process for making the
composition according to the first aspect, the process comprising combining a
therapeutically effective amount of a copper salt and/or a cobalt salt and/or a
nickel salt, with an extract of a plant selected from a group consisting of Punica
granatum, Viburnum plicatum, Camellia sinensis, and Acerspp.; and a
pharmaceutically acceptable vehicle.
The invention also provides, in a tenth aspect, a process for making the
composition according to the second aspect, the process comprising combining a
therapeutically effective amount of a copper salt and/or an iron salt and/or a cobalt
salt and/or a nickel salt, with an extract of a plant selected from a group consisting
of Punica granatum, Viburnum plicatum, Camellia sinensis, and Acerspp., and a
reducing agent; and a pharmaceutically acceptable vehicle.
A "therapeutically effective amount" is any amount which, when administered to a
subject, provides prevention and/or treatment of a specific medical condition.
A "subject" may be a vertebrate, mammal, domestic animal or human being.
A "pharmaceutically acceptable vehicle" as referred to herein is any combination
of known compounds known to those skilled in the art to be useful in formulating
pharmaceutical compositions.
The amount of the composition used may be from about 0.01 mg to about 800
mg. Preferably, the amount of the composition is from about 0.01 mg to about
500 mg, more preferably about 0.01 mg to about 250 mg, even more preferably

from about 0.1 mg to about 60 mg, and most preferably from about 0.1 mg to
about 40 mg.
The vehicle may include one or more substances which act as flavouring agents,
lubricants, solubilisers, suspending agents, fillers, glidants, compression aids,
binders or tablet-disintegrating agents. The vehicle can also be an encapsulating
material. In powders, the vehicle may be a finely divided solid that is in admixture
with the finely divided active metal salt, plant extract and for the composition of the
second aspect, a reducing agent. In tablets, the metal salt, plant extract, and
reducing agent may be mixed with a vehicle having the necessary compression
properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain up to 99% of the active metal ion,
plant extract and reducing agent. Suitable solid vehicles include, for example
calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,
gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins.
Compositions according to the invention may have the form of solutions,
suspensions, emulsions, syrups, elixirs and pressurized compositions. The
active agents may be dissolved or suspended in a pharmaceutically acceptable
liquid vehicle such as water, an organic solvent, a mixture of both, or
pharmaceutically acceptable oils or fats. The liquid vehicle may contain other
suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,
preservatives, sweeteners, flavouring agents, suspending agents, thickening
agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable
examples of liquid vehicle for oral and parenteral administration include water
(containing additives as above, eg cellulose derivatives, alcohols (including
monohydric alcohols and polyhydric alcohols, eg glycols) and their derivatives,
and oils (eg fractionated coconut oil and arachis oil). For parenteral
administration, the vehicle may be an oily ester such as ethyl oleate and isopropyl
myristate. Sterile liquid vehicles are useful in sterile liquid-form compositions for
parenteral administration. The liquid vehicle for pressurized compositions can be
a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions or suspensions
can be utilized by, for example, intramuscular, intrathecal, epidural,
intraperitoneal, intravenous and particularly subcutaneous injection. The metal
ion combined with plant extract and reducing agent (for the composition of the
second aspect) may be prepared as a sterile solid composition that may be
dissolved or suspended at the time of administration using sterile water, saline, or
other appropriate sterile injectable medium.
Compositions according to the invention can be administered orally in the form of
a sterile solution or suspension containing other solutes or suspending agents (for
example, enough saline or glucose to make the solution isotonic), bile salts,
acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and
its anhydrides copolymerized with ethylene oxide) and the like. Compositions
according to the invention can also be administered orally either in liquid or solid
composition form. Compositions suitable for oral administration include solid
forms, such as pills, capsules, granules, tablets, and powders, and liquid forms,
such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral
administration include sterile solutions, emulsions, and suspensions.
The compositions according to the invention may be used to treat any mammal,
for example, human, livestock, pets, and may be used in other veterinary
applications.
The inventor has realized that the compositions according to the invention may be
used as a medicament, but may also be put to a number of other antimicrobial
uses (whether in a clinical context or otherwise). For instance, in addition to
administering the compositions according to the invention to a patient or subject,
they may be used for the application to, or coating of, surfaces and objects to
prevent, ameliorate or treat microbial infections or contamination.
Therefore, according to an eleventh aspect, there is provided a method of
preventing and/or treating a microbial infection or contamination, the method

comprising applying to an object or a surface with an amount of a composition that
is effective for killing or preventing growth of micro-organisms, wherein the
composition comprises (i) a copper salt and/or a nickel salt and/or a cobalt salt;
and (ii) an extract of a plant selected from a group consisting of Punica granatum,
Viburnum plicatum, Camellia sinensis, and Acerspp.; or the composition
comprises (i) a copper salt and/or an iron salt and/or a cobalt salt and/or a nickel
salt; (ii) an extract of a plant selected from a group consisting of Punica granatum,
Viburnum plicatum, Camellia sinensis, and Acerspp.; and (iii) a reducing agent.
In a twelfth aspect, there is provided an object coated with a composition
according to the first or second aspect.
It will be appreciated that the compositions may be particularly useful for
application to, or coating of, surfaces or objects that are required to be aseptic.
As discussed above, the compositions according to the invention have the
advantage that they are antiviral and/or antibacterial and/or antifungal.
Accordingly, the compositions disclosed herein have a broad antimicrobial effect.
Furthermore, as discussed in more detail below, the compositions may adhere to
surfaces and are thereby effective for longer periods of time.
The compositions according to the invention may be used for application to, or
coating of, any object or device which is used in a biological or medical situation,
such as a medical device, and for which it may be important to prevent a microbial
infection or contamination that may lead to any infection in a patient. Examples of
medical devices to which compositions according to the invention may be applied
include lenses, contact lenses, catheters, stents, wound healing dressings,
contraceptives, surgical implants and replacement joints.
The compositions are particularly useful for coating biomaterials and objects and
devices made therefrom. Microbial contamination/infection of biomaterials can be
particularly problematic because the microbe may use such material as a
substrate for growth. Biomaterials (eg collagens and other biological polymers)

may be used to surface artificial joints. Alternatively, certain implants may
substantially comprise such biomaterials.
The compositions may be used to coat surfaces in environments that are required
to be aseptic. For instance the compositions may be used in medical
environments. The compositions may be used to keep hospital wards clean.
They may be used to clean surfaces of medical equipment (eg operating tables)
in hospitals, such as operating theatres as well as operating theatre walls and
floors. The inventor believes the compositions will be useful to improve sterility in
general and also to address the spread of MRSA in particular (the inventor
believes that MRSA may be killed by the compositions of the invention).
Therefore, the method according to the eleventh aspect may comprise applying
the composition to a surface that is selected from: hospital ward surfaces,
operating theatre surfaces, kitchen surfaces and sanitary surfaces. It will be
appreciated that the above list of objects and surfaces to which compositions
according to the invention may be applied is not exhaustive. Hence, the
compositions may be administered to any surface, which is prone to a bacterial
contamination, for example kitchen and bathroom surfaces and products, such as
a toilet seat, or the toilet itself.
The compositions may be formulated into solutions for cleaning objects and
surfaces, or for spraying thereon, or in which the object or surface may be
immersed. For instance, they may be a routine constituent of physiological
solutions (for example as a constituent of physiological saline). Preferably,
coating of the object or surface may be carried out by preparing an aqueous
solution at an appropriate pH and temperature for the composition according to
the invention to retain its antimicrobial activity. The object or surface is exposed
to the composition for sufficient time to allow immobilisation or absorption of a
suitable quantity of the composition to the surface thereof or to kill the micro-
organism.

Furthermore, the compositions according to the invention may be used to
minimise, prevent or treat microbial infections or contamination, by use as, or in
conjunction with, a preservative. Hence, the compositions may be used as a
preservative in foodstuffs. In addition, the compositions may be used to minimise
or prevent microbial growth in cultures, for example in tissue culture work, either
to supplement or to replace antibiotics.
All of the features described herein (including any accompanying claims, abstract
and drawings), and/or all of the steps of any method or process so disclosed, may
be combined with any of the above aspects in any combination, except
combinations where at least some of such features and/or steps are mutually
exclusive.
For a better understanding of the invention, and to show how embodiments of the
same may be carried into effect, reference will now be made, by way of example,
to the accompanying diagrammatic drawings, in which:-
Figure 1 is a JOB plot of Fe(lll)-PRE ratio against absorbance at 563nm. The
JOB plot shows that the isolated PRE active component binds to ferric ions in the
ratio of 1:2 (Fe: PRE);
Figure 2 is a bar chart demonstrating bactericidal efficacy of the PRE-FE(II)
mixture on addition of the reducing agent Vitamin C (CFU refers to Colony
Forming Units, all CFU/ml values are in log™);
Figure 3 is a bar chart demonstrating the effects of various metal ions with
additions of PRE (all CFU/ml values are in log10);
Figure 4 is a bar chart showing bactericidal activities for mixtures at 24 and 48
hour: in equates to bactericidal mixture added directly, out refers to mixture
prepared and stored for 24 or 48 hours prior to addition (all CFU/ml values are in
logio);
Figure 5 is a bar chart showing a bactericidal assay of pomegranate ointment 1
week after formulation (all CFU/ml values are in logio);
Figure 6 is a bar chart showing a bactericidal assay of pomegranate ointment 3
weeks after formulation (all CFU/ml values are in logio);

Figure 7 is a bar chart showing preliminary toxicity screening using Trypan blue
staining;
Figure 8 is a bar chart showing infectious agent survival after 30 minutes
exposure to fresh ointment preparations of test agents shown;
Figure 9 is a bar chart showing infectious agent survival after 30 minutes
exposure to ointment preparations of test agents shown after storage at 5°C for 3
months;
Figure 10 is a bar chart showing the antimicrobial activities of PRE alone and in
combination with metal ions after a 30 minute incubation against Ps. aeruginosa,
P. mirabilis and E. coli, using Lambda buffer as a control;
Figure 11 is a bar chart showing the antimicrobial activities of PRE alone and in
combination with metal ions after a 30 minute incubation against S. aureus and B.
subtilis, using Lambda buffer as a control;
Figure 12 is a bar chart showing the antimicrobial activities of PRE/metal ion
combinations with the addition of Vitamin C after a 30 minute incubation against
all isolates tested, using Lambda buffer as a control (all CFU/ml values are in
logio);
Figure 13 shows Box Whisker statistical analysis of the viable count data achieved
in relation to the antimicrobial activities of PRE alone and in combination with
Cu(ll) ions after a 2 hour minute incubation against 10 clinical isolates of MRSA
using Lambda buffer as a control. (Box represents 25% and 75% quartiles, bar
represents median and error bars represent range. Mean cfu ml"1 value shown by
A) (all CFU/ml values are in log™);
Figure 14 shows Box Whisker statistical analysis of the viable count data achieved
in relation to the antimicrobial activities of PRE alone and in combination with
Cu(ll) ions after a 2 hour minute incubation against 10 clinical isolates of MSSA
using Lambda buffer as a control. (Box represents 25% and 75% quartiles, bar
represents median and error bars represent range. Mean cfu ml"1 value shown by
•) (all CFU/ml values are in logio);
Figure 15 shows Box Whisker statistical analysis of the viable count data achieved
in relation to the antimicrobial activities of PRE alone and in combination with
Cu(ll) ions after a 2 hour minute incubation against 10 clinical isolates of PVL
positive cMRSA using Lambda buffer as a control. (Box represents 25% and 75%

quartiles, bar represents median and error bars represent range. Mean cfu ml"1
value shown by ■) (all CFU/ml values are in logio);
Figure 16 shows the antimicrobial activities of PRE alone and in combination with
Fe(ll) or Cu(ll) ions and Vitamin C after a 30 minute incubation against 9 clinical
isolates of ES|3L Pseudomonas aeruginosa using Lambda buffer as a control.
(Box represents 25% and 75% quartiles, bar represents median and error bars
represent range. Mean cfu ml"1 value shown by *) (all CFU/ml values are in logio);
Figure 17 shows the antimicrobial activities of the ointment formulation of PRE in
combination with Fe(ll) or Cu(ll) ions and Vitamin C after a 30 minute incubation
against 9 clinical isolates of ESpL Pseudomonas aeruginosa using Lambda buffer
as a control. (Box represents 25% and 75% quartiles, bar represents median and
error bars represent range. Mean cfu ml"1 value shown by *) (all CFU/ml values
are in logio);
Figure 18 shows activities of black and green tea extracts alone or in combination
with metal salts additives against Staph, aureus using Lambda buffer as a
control. Legend: Black tea with iron (BTI), Black tea with copper (BTC), Green tea
with iron (GTI), Green tea with copper (GTC). Error bars are SEM for each
sample tested (all CFU/ml values are in logio);
Figure 19 shows activities of black and green tea extracts alone or in combination
with metal salts additives against Prot. mirabilis using Lambda buffer as a control
(abbreviations as in Figure 18). Error bars are SEM for each sample tested (all
CFU/ml values are in logio); and
Figure 20 shows activities of black and green tea extracts alone or in combination
with metal salts additives against Ps. aeruginosa using Lambda buffer as a
control (abbreviations as in Figure 18). Error bars are SEM for each sample
tested. All CFU/ml values are in log10.
Examples
The inventor based his initial experiments on the antiviral, anti-bacteriophage, and
antifungal compositions disclosed in EP 0,744,896B1. These antimicrobial
compositions all include a combination of ferrous salts and an extract from a plant
selected from pomegranate rind, Viburnum plicatum leaves or flowers, tea leaves,
or maple leaves.

As an example only, the inventor of the present invention focused his research on
compositions using pomegranate rind extract (PRE) as the active ingredient. A
significant problem with iron salt-based PRE compositions is that they lack
stability, and therefore retain their antimicrobial activity for up to a maximum of
only 30 minutes. Another problem with these iron-based antimicrobial
compositions is that they turn black because aromatics contained within the
composition are polymerized in the presence of the iron ions. Accordingly, the
focus of the present invention was to develop a stable antimicrobial formulation of
the unstable anti-viral and anti-fungal mixture reported in EP 0,744,896B1.
Having managed to achieve this goal, the inventor extended his research to
investigate and further develop other active antimicrobial compositions. The
research is described in the following examples.
Example 1
The inventor's initial objectives were to set up an in vitro model for screening,
isolating and characterizing the active compound(s) in Pomegranate Rind Extract
(PRE) in the antimicrobial composition disclosed in EP 0,744,896B1. It was also
an aim to investigate the currently unknown mechanism of action of these
available compositions in order to assist in the development of new formulations
with longer term stabilities.
Materials and Methods
Materials and methods used for isolating the active compound PRE, and for
preparing the iron-based antimicrobial compositions are disclosed in EP
0,744,896B1, which is incorporated herein by reference.
Preparation of plant extracts
Pomegranate rind, Viburnum plicatum leaves or flowers, maple leaves and
commercial tea leaves were blended in distilled water (25% w/v), and boiled for
about 10 min. After centrifugation (20,000 x g, 4°C, 30 min), supernatants were
autoclaved (121°C, 15 min), cooled and stored at -20°C. A further purification of

the pomegranate extract to a molecular weight cut-off of 10,000Da was achieved
by membrane ultrafiltration and the filtrate stored as above.
Bacteria, viruses, and fungi
The control strain was Pseudomonas aeruginosa for standard experimentation, ie
determining optimal preparations. After optimization, the inventor demonstrated
activities for the ointment against 10 multidrug-resistant Pseudomonas
aeruginosa.
Growth conditions: on nutrient agar provided by Oxoid Ltd for 24hrs at 37°C.
The inventor established a functional in vitro bactericidal assay to screen a variety
of formulations (cream, aqueous and ointment). This assay involved adding 0.5 g
of ointment to 10 ml of water and vortexing prior to a standard suspension test
using 50 microlitres of bacterial cell suspension to a turbidity of 0.5 McFarland
solution (bacterial cell suspension equal to 1.5 x 108) plus 100 microlitres of the
ointment solution. After incubation in the dark at room temperature for 30 mins,
serial dilutions (from 10"1 to 10"5) were carried out on nutrient agar.
Metal binding studies were performed on the isolated component to inform on
stability issues for the final formulation and for elucidation of the mechanism of
action. A JOB plot was conducted by tracking the maximum wavelength over the
mole ration of 0 to 1 for Fe ions and PRE active component.
Results
Referring to Figure 1, there is shown a JOB plot, which captures the results of the
spectroscopic metal ion binding studies. The Figure demonstrates:-
1. Ferric ions (ie. iron(lll) compounds) bind to the active component of
PRE giving a characteristic peak at 563 nm attributable to a
characteristic Fe(lll)-Phenolate complex;
2. Ferrous ions (ie. iron(ll) compounds) bind to the active component also
giving a characteristic peak at 563 nm indicative of oxidation of the
metal ion to the Fe(lll) state; and

3. Figure 1 shows that the isolated PRE active component binds to ferric
ions in the ratio of 1:2 (Fe:PRE).
Interestingly, the metal binding study results indicate that the activation step for
enhanced antibiotic activity (ie addition of ferrous ions to the PRE component)
results in the oxidation of the metal ion from the Fe(ll) to the Fe(lll) oxidation state.
Although the inventor does not wish to be bound by any hypothesis, he believes
that the significant loss of activity of the iron-based antimicrobial compositions,
which is witnessed after 30 minutes, may be directly attributable to this oxidation
process.
This surprising realization led the inventor to investigate the effects of adding a
reducing agent to the active mixture in an attempt to re-generate the Fe(ll) by
reduction of the oxidised Fe(lll) ions to rejuvenate efficacy, and activity. To this
end, the inventor's studies focussed on elucidation of the mechanism of action
with a view to stabilising the combined extract (Ferrous salts and PRE). To test
his hypothesis, the inventor chose Vitamin C as a reducing agent or reductant to
see if it had the effect of extending the activity life of iron-based compositions.
Preliminary results demonstrated enhanced bactericidal activity (on Pseudomonas
aeruginosa) occurs on addition of an extra component Vitamin C (a reducing
agent added to maintain the iron in the Fe(ll) active state). Surprisingly, this tri-
component formulation (PRE + Fe(ll) + Vitamin C) exhibited exemplary
bactericidal activities under the conditions used , as shown in Figure 2.
Referring to Figure 2, there is shown the bactericidal efficacy of the PRE-Fe(ll)
mixture on addition of the reducing agent Vitamin C. As can be seen in the
Figure, the value of Colony Forming Units (CFU)/ml is significantly reduced when
the PRE/Fe(ll) mixture is added immediately upon preparation. However, after 30
minutes, this preparation has lost activity, which is completely restored upon
addition of Vitamin C (140μl).

Example 2
Based on the surprising findings of Example 1, the inventor then set out to
investigate the mechanism of action of the iron-based/PRE compositions at the
molecular level with a view to enhancing the product formulation. The enhanced
activity upon addition of ferrous ions is problematic as the mixture retains activity
for short periods (<30 mins), and principally at low pH values, which is difficult to
formulate.
In order to overcome these shortcomings, the inventor investigated whether or not
it was possible to substitute the ferrous ions completely with other metal ions, and
a number of other metal ions were therefore tested. In addition, based on the
positive results seen in Example 1 with addition of a reducing agent (such as
Vitamin C), the inventor wanted to see if it was possible to prolong the activity of
antimicrobial compositions using other active metal ions by addition of a reducing
agent. Finally, the inventor set out to optimize concentrations of the various
components in the various active antimicrobial compositions.
Materials and Methods
It had already been demonstrated that iron-based compositions combined with
PRE exhibited antiviral and antifungal activities. Hence, a large range of other
metal ions were tested for their abilities to enhance the activity of the PRE,
including:- Cu(ll), Fe(ll), Cu(l), Zn(ll) and Mn(ll).
Results
The test solutions of the ions Fe(lll), Cu(ll), Fe(ll), Cu(l), Zn(ll) and Mn(ll)
revealed that the highest activities were exhibited for Fe(ll) and Cu(ll) species
upon addition to PRE as shown in Figures 2 and 3. In contrast, as shown in
Figure 3, surprisingly, addition of solutions of Zn(ll) and Mn(ll) exhibited little or no
activity (ie no significant difference from controls).
Figure 2 demonstrates that the extent of bacterial growth decreased in proportion
to the dose of reducing agent, Vitamin C, for each of the following compositions:
PRE/FeSO4. While the inventor does not wish to be bound by any hypothesis, he

believes that adding Vitamin C to PRE/FeCI3 transformed the iron from the ferric
state (ie Fe III) to the ferrous state (ie Fe II), the latter being more active, and this
resulted in a much lower level of bacterial growth compared to the ferric state.
In terms of mechanisms of action, the preference for using metal ions in the
reduced state led to the incorporation of studies using reductants to stabilise this
oxidation state, and prolong and enhance activity. Thus, the reductant Vitamin C
was added in three different doses after pre-incubating the PRE/metal ions
mixtures for 30 minutes, and the results are shown in Figure 2.
Figure 3 demonstrates that the extent of bacterial growth decreased in proportion
for each of the following compositions: CuSO4, ZnSO4, MnSO4, PRE/CuSO4,
PRE/ZnSO4, PRE/MnSO4) ('30 mins in' refers to addition immediately upon
preparation, '30 mins out' refers to a premix and 30 minutes lapse before
addition).
The longer term activities of the PRE:metal salt(s) mixtures were investigated after
a period of 24 and 48 hours, and the results are shown in Figure 4. Figure 4
shows bactericidal activities for mixtures at 24 and 48 hour, in which "in" equates
to bactericidal mixture added directly, and "out' refers to mixtures prepared and
stored for 24 or 48 hours prior to addition. The results shown are for one system
only as an example.
Figure 3 shows that the activity of the PRE/FeSO4 system is pronounced at the
zero time point. However, even with addition of CuCI to this system, after storage
for 48 hrs, considerable bacterial growth was observed as shown in Figure 4.
Surprisingly, full bactericidal activity is restored upon addition of Vitamin C at all
concentrations.
Example 3
The activity of the unstable formulation previously disclosed in EP 0,744,896B1
was tested after storage for several months. The inventor found that this known
formulation exhibited no bactericidal activity after only a short period of time.

Therefore, following on from the promising results of Example 2, the inventor then
set out to enhance the stability of the product formulation and conduct preliminary
toxicity tests. In order to optimise activity and enhance stability, a number of
formulations were prepared and investigated. The antimicrobial activities were
screened for a variety of formulations (ie cream, aqueous and ointment). The
potential for enhancing efficacy by altering the relative concentrations of active
constituents was also explored. Preliminary toxicity tests were conducted on
mammalian cell cultures in vitro.
Materials and Methods
The key objective was to develop a formulation that retained activity to produce an
OTC preparation for commercial uses.
For the ointment formulation, a hydrous ointment base was used, the components
of which are shown in Table 1. In order to prepare a control ointment formulation
having no actives, the magnesium sulphate (0.3g) and phenoxyethanol (0.6g)
were first dissolved in the purified water (29.1g) and warmed to 60°C. The wool
alcohol ointment was then melted on a separate water bath. The two
temperatures were kept the same, and the water solution containing the
magnesium sulphate and phenoxyethanol was added in small aliquots to the
ointment solution, stirring constantly until a smooth mix was formed, whilst
maintaining the temperature at 60°C. When all the water was added, the mixture
(60g) was stirred gently until the ointment formulation was at room temperature.
50 grams was packed in an ointment jar, which was stored in a cool place but not
allowed to freeze.
In order to prepare an ointment formulation having active ingredients, the ointment
base was prepared as above, except instead of using 29.1g of pure water, 29.1g
of a plant extract solution (eg PRE or tea etc) was used. Solutions of metal salts
and Vitamin C were added to the ointment base as required, to prepare the active
ointment formulation.

The aqueous formulation was simply a water-based formulation in which the
active ingredients (plant extract, metal salt, and in some cases, reducing agent)
were dissolved in pure water.
The cream formulation had the following composition:-
Aqueous cream (for 55 grams):
• Emulsifying component 16.5 g
• Phenoxyethanol 0.55 g
• Purified water, freshly boiled and cooled 37.95 g
Aqueous cream is an emollient and can be used as a base for drugs.
Phenoxyethanol is present as an antimicrobial preservative. It was dissolved in
water warmed to 60°C. The emulsifying ointment was weighed and melted on a
water bath. Both phases were kept close to 60°C, and then the aqueous phase
was added to the melted ointment. The mixture was removed from the heat and
stirred continuously until cold. 50 grams was weighed and packed in an ointment
jar. The preparation was stored in a cool place but not allowed to freeze.
Results
Figure 5 shows a bactericidal assay of an ointment of PRE combined with various
components, such as iron or copper salts, and Vitamin C. Figure 6 shows the
same compositions, but their activity after 3 weeks. As shown in Figure 8, the
activity enhancement upon addition of Vitamin C for either Fe(ll)/PRE or Cu(ll)/Pre
compositions is retained for three weeks with no reduction in efficacy for the
formulation. Hence, the ointment formulation shown in Table 1, combined with
added Vitamin C exhibited greatly enhanced stability compared to the aqueous
preparation (data not shown) showing full retention of activity after 3 weeks.
Surprisingly, these findings are in sharp contrast to the cream or aqueous
formulations, which in the former case had no activity and in the latter lost activity
after only 30 minutes. Therefore, the inventor has clearly demonstrated the
surprising efficacy, and retained activity over long periods, of ointment-based
formulations. This could not have been predicted from previous work.

Referring to Figure 7, there are shown the results of toxicity studies that were
carried out using Trypan blue staining. Human breast cancer cells MCF7 were
used to examine the effect of pomegranate preparations on mammalian tissues.
Trypan blue staining was used to detect non-viable cells (appear blue stained
under the microscope). The MCF7 cells were grown to confluence in 75 ml
culture flasks using Dulbeccos MEM (Gibco) supplemented with 10% fetal bovine
serum (FBS), 25 μg/mL gentamicin and 200 mM L-glutamin (growth medium) and
incubated in a 95% air and 5% C02 atmosphere at 37°C. Cells were cultured for
5 days prior to treatment with the test substances. Confluent cells were detached
with 2 ml of 0.15% trypsin (Sigma) for 5 minutes, 8 ml MEM was added, and the
cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded and
the cell pellet was resuspended in 10 ml MEM and counted using a
haemocytometer. A 24 well plate was used to seed the cells using a cell
concentration of 106/ml (200 ul per well).
Next day the media was removed and the cells were washed with PBS twice,
trypsin was added to detach the cells which were then treated with the test
substances shown in Figure 5, incubated for 30 minutes at 37°C then stained with
trypan blue (10 ul of 0.2% was added to 10 ul cell suspension) for 5 minutes,
spread onto a microscope slide and covered with a coverslip then examined under
the microscope. Non-viable cells appear blue in colour because they cannot
exclude the dye.
The toxicity studies shown in Figure 7 revealed that the highest percentage of
viable cells was observed with PRE while the lowest was encountered after
treating the cells with PRE/FeSO4/CuSO4/Vitamin C combination which is still not
very toxic.
Example 4
Based in the results of Example 3, the inventor focussed on enhancing the
stability of the product formulation. A wide range of combinations were tested to
optimise the efficacy of the active preparation. Final products were identified
which retained considerable activities over a five month period. In addition, the

inventor carried out further experiments to investigate:- (i) the activation of PRE
using copper (II) salts, ii) the activation and enhancement of the PRE/Cu
combinations using Vitamin C, and iii) the optimised formulation being an
ointment.
Materials and Methods
The inventor compiled a set of seventeen test preparations for each infectious
agent tested. In all, activities were assessed against ten extended spectrum Beta
Lactams (ESBL) Pseudomonas aeruginosa. For the most active preparations, a
number of formulations were prepared including creams, aqueous preparations
and ointments were prepared. These were tested immediately after preparation
and in the ensuing months to determine the optimum retention of activity over
time.
Results
The key objective of this project was to develop a formulation that retained activity
to produce an over-the-counter (OTC) preparation for commercial uses. Referring
to Figure 8, there is shown the degree of infectious agent survival after 30 minutes
exposure to fresh ointment preparations of test agents shown. Referring to Figure
9, there is shown infectious agent survival after 30 minutes exposure to ointment
preparations of test agents shown after storage at 5°C for 3 months.
The data show that the ointment reduced cell growth as measures in colony
forming units by a factor of 104 compared to the control samples.
As can be seen in Figures 8 and 9, the ointment formulation with added Vitamin C
exhibited a greatly enhanced activity/stability profile (compared to the aqueous
preparation - results not shown) showing full retention of activity after 3 weeks, as
discussed in Example 3. In the three month study, as shown in Figure 9,
considerable activities were afforded by the three most active combinations (i.e.
PRE/FeSO4/vitamin C; PRE/CuSO4/Vitamin C; and PRE FeSO4/CuSO4/Vitamin
C). It should be noted that activities without the addition of Vitamin C were less
active in the short term and longer term for any preparation and formulation.

Example 5
The aim of Example 5 was to determine the antimicrobial activities of
combinations of pomegranate rind extracts (PRE) with metals salts and Vitamin C
against Staphylococcus aureus, Bacillus subtilis, E. coll, Pseudomonas
aeruginosa and Proteus mirabills.
Materials and methods
Pomegranate rind extract (PRE) preparation
Pomegranate rind extract was prepared by blending 15 grams of PR with 45 ml_s
distilled water for 10 min. The crude extract was filtered through muslin followed
by Whatman No. 1 filtration paper and autoclaved (121 °C for 15 mins) prior to
storage at -20 °C.
Disk diffusion assay
Overnight cultures of the Gram-positive strains (S. aureus, B. subtilis) and the
Gram-negative strains (E. coli, Ps. aeruginosa and P. mirabilis) were suspended
in Ringer's solution (Oxoid, U.K.) to a turbidity equivalent to 0.5 McFarland (1.5 x
108 CFU/ml) and 100 \iL was spread onto Mueller-Hinton agar plates (OXOID
limited, U.K.). The extract (10 ^iL) was then spotted onto Whatman no 1 filter
paper (5 mm diameter). Plates were incubated at 37 °C for 24 h, after this time
the diameter of the zone of inhibition was recorded.
Antimicrobial activity of PRE with the addition of metal salts
All reagents were purchased from Sigma-Aldrich (Poole, Dorset) and distilled
water was used throughout. Overnight cultures on nutrient agar were then
suspended in Ringer's solution (Oxoid, U.K.) to a turbidity equivalent to 0.5
McFarland (1.5 x 108 CFU/ml). An aliquot of the PRE extract (330 Μl) was added
to 700 nl of the freshly prepared solutions (4.8 mM) of metal salts (FeSO4, CuSO4,
MnSO4, ZnO). The final solution was protected from light (Stewart et al. 1998. J.
Appl Microbiol 84:777-783).

The appropriate bacterial dilution was prepared and 50 μl placed in a sterile
Eppendorf micro-centrifuge tube with a 100 μl of the extract/metal salt solution.
After exposure of the bacteria for 30 minutes at room temperature, the activity of
the bactericidal agent was neutralized by adding an equal volume of 2% (v/v)
Tween-80 (Sigma Chemical Co., UK) in Lambda buffer. Serial dilutions were
prepared in Ringer's solution (10-5), 10 μl of each dilution is spotted on nutrient
agar plate and incubated for 24 hours at 37 °C. Each assay was conducted in
triplicate.
Antimicrobial activity of PRE with the addition of metal salts and Vitamin C
The assay was carried out as described above with the following addition: Vitamin
C was added to the metal ion (FeSO4, CuSO4) solution immediately prior to
mixing with the PRE. Aliquots of Vitamin C were made to give final metal
ion.Vitamin C ratios (and Vitamin C concentrations) of 1:1 (4.8mM), 1:5 (24nM),
1:20 (96mM) (metal salt:Vitamin C). 700 μl of this solution was then added to
PRE.
Fractionated PRE assay
PRE extracts were fractionated by molecular weight using Millipore ultra-filtration
devices (nominal M. Wgt. cut-off = 5,000 a.m.u.) and the resulting extracts were
tested by the disc diffusion method outlined above.
Results
Disk diffusion assay
Using the disk diffusion method antimicrobial efficacies were examined against a
panel of five microbes. Maximum activities for PRE were observed against the S.
aureus and B. subtilis. Moderate effects (zone sizes) were seen against Ps.
aeruginosa and P. mirabilis and there was little activity against E.coli. The
average zone diameters were 17 mm, 14 mm, 9 mm and 8 mm for S. aureus, B.
subtilis, Ps. aeruginosa and P. mirabilis respectively.

Antimicrobial activity of PRE with the addition of metal salts
The antimicrobial activities of PRE extracts along with the metal salt additives
were assessed using a modified version of the adopted by Stewart et al. (1998)
for observing the additive effects of metal ions. PRE alone did not exhibit
antimicrobial activity against the majority of the bacteria tested; except B. subtilis,
which may be due to the short incubation time of 30 minutes. Against the Gram-
negative isolates the metal ions showed moderate activity, the greatest results
were seen against E. coli (as shown in Figure 10) with Cu (II) ions reducing the
cell survive population by a factor of 104 compared to the buffer. The most
striking result was seen with the combination of the PRE with cupric salts where
no detectable growth was seen. Moderate antimicrobial activity was seen with Cu
(II) ions and PRE in combination against S. aureus that reduced the surviving
population by circa 103 compared to the buffer (as shown in Figure 11).
Antimicrobial activities of PRE with the addition of metal salts and Vitamin C
Further studies were conducted to enhance the activity of the PRE/metal ion
combination and to elucidate the mechanism of action. Based upon a putative
oxidative damage mechanism afforded by the redox active metal ion, the
inventors assessed the antimicrobial efficacy upon addition of the reductant
Vitamin C. Owing to the high activity exhibited for the PRE/Cu(ll) combination
against all Gram-negative isolates, the inventors studied the addition of Vitamin C
to the PRE/Fe(ll) combinations (as shown in Figure 12). For E. coli, a decrease
in growth of 102 was seen with the addition of a stoichiometric equivalent of
Vitamin C (with respect to metal ion concentration). Addition of 5 and 20
equivalents of Vitamin C resulted in a reduction in growth of 103 and no
discernable growth respectively. For S. aureus, addition of Vitamin C to the
PRE/Cu(ll) mixture had no significant effect at one equivalent but a marked effect
at 5 and 20 equivalents of Vitamin C (no detectable growth in either).
Fractionated PRE assay
In order to investigate the mode of antimicrobial action, the PRE was subjected to
fractionation on the basis of nominal molecular weights. The fraction with a
nominal MW below 5,000 was compared to the untreated PRE assessed using

the disc diffusion method. As shown in Table 2, similar activities were exhibited
by the low molecular weight fraction in comparison to the whole PRE.
Table 2: Diameter of the zones of inhibition of the low molecular weight fraction of
PRE compared to whole PRE (± SEM) against a panel of five bacteria-

Discussion
In this study, metal salts were applied to further enhance the properties of
pomegranate. Preliminary results using the disk diffusion assay, to assess
antimicrobial activity against a panel of bacteria, showed that the PRE was most
active against the Gram-positive organisms (S. aureus and B. subtilis). Disc
diffusion assessment of the low molecular weight fraction of PRE suggest the
antimicrobial component(s) of PRE are found within <5000 Da portion of the
extract. However, in the suspension assay, the PRE alone showed little or no
antimicrobial activity against any of the bacteria tested, perhaps due to the short
incubation time.
For the Gram-negative bacteria the combination of PRE:Cu(ll) gave the best
results with no detectable growth observed with all three isolates after 30 mins.
For S. aureus the addition of 5 and 20 equivalents of Vitamin C to the PRE:Cu(ll)
result in no detectable growth after 30mins. The addition of Vitamin C to
PRE:Fe(ll) also resulted in a reduction in growth for S. aureus of circa 104log10.
In conclusion, PRE in combination with Cu(ll) ions exhibit dramatic synergistic
antimicrobial effects against E. coli, Pseudomonas aeruginosa and Proteus

mirabilis and moderate activity against S. aureus. The active component(s) in the
PRE are found in the low molecular weight fraction. The addition of high
quantities of Vitamin C markedly enhanced the activities of both PRE/Fe(ll) and
PRE/Cu(ll) mixtures against at least S. aureus.
Example 6
The aim of this Example was to explore the potential role for metal ions in
enhancing the activities of PRE against clinical isolates of S aureus. Thirty
isolates were tested which include 10 MRSA (methicillin resistant S. aureus), 10
MSSA (multiple antibiotic-resistant methicillin resistant Staphylococcus aureus)
and 10 Panton-Valentine Leukocidin (PVL) producing cMRSA isolates (community
acquired MRSA, which produce Panton-Valentine leukocidin). The example
demonstrates the antimicrobial activities of pomegranate rind extracts (PRE)
against Staphylococcus aureus (MSSA), MRSA and PVL positive cMRSA. For
MRSA and MSSA strains, exposure to copper (II) ions for 2 hours had moderate
activities of between 102 to 103 log10 reduction in growth, which was enhanced by
the addition of PRE to 104 log10 reduction in growth observed in 80% of the
isolates. However, the PVL positive cMRSA strains were surprisingly more
sensitive to copper (II) ions and had moderate activities of between 103 logio
reduction in growth for 60 % of the isolates.
Materials and methods
Pomegranate rind extract preparation
Pomegranate rind extract (PRE) was prepared firstly by cutting rind into small
squares (approximately 5mm2) which were dried at 55°C for 24 hours, and stored
in an air tight container in the dark until further use. 10g of dry rind was added to
150ml distilled water and place in a shaker (at 80 rpm) at room temperature for 24
hours. The crude extract was passed thought muslin and a Whatman filter No.1
to remove the particulate matter, prior to filter sterilising by passing through a
0.2um filter (Millipore), into a sterile bottle. The extract was stored at -20°C for
future use.

Bacterial isolates
Clinical isolates of methicillin resistant (n=10), methicillin sensitive (n=10)
Staphylococcus aureus (MRSA and MSSA) and Panton-Valentine Leukocidin
producing cMRSA (n=10) were used in the study. The MRSA and MSSA isolates
were collected from the Royal Marsden Hospital (London, UK) and the cMRSA
isolates were collected from the Devon and Exeter Hospital (UK). The isolates
were cultured over night on nutrient agar (Oxoid), aerobically at 37°C and then
frozen in cyrovials (Pro-labs) at -80°C until required. Prior to use all isolates were
passaged twice on nutrient agar aerobically at 37°C. In all assays culture were
prepared by using overnight cultures on nutrient agar that were then suspended in
Ringer's solution (Pro-Lab, U.K.) to a turbidity equivalent to 0.5 McFarland (1.5 x
108cfuml'1).
Antimicrobial activity of PRE with the addition of metal salts
All reagents were purchased from Sigma-Aldrich (Poole, Dorset) and distilled
water was used as a chemical diluent throughout. The method used was an
adaptation of that described by Stewart et al (1998) supra. Briefly, overnight
cultures on nutrient agar were then suspended in Ringer's solution (Oxoid, U.K.)
to a turbidity equivalent to 0.5 McFarland (1.5 x 108 CFU/ml). An aliquot of the
PRE extract (330 \i\) was added to 700 |al of the freshly prepared solutions (4.8
mM) of metal salts (FeSO4, CuSO4); the final solution was protected from light
(Stewart et al. 1998).
The appropriate bacterial dilution was prepared and 50 ^L placed in a sterile
Eppendorf micro-centrifuge tube with a 100 pL of the extract/metal salt solution.
Following treatment of the bacteria for 2 hours at room temperature, the activity of
the bactericidal agent was neutralized by adding an equal volume of 2% (v/v)
Tween-80 (Sigma Chemical Co., UK) in Lambda buffer. Serial dilutions were
prepared in Ringer's solution (10"5), 10 uL of each dilution is spotted onto nutrient
agar plate and incubated aerobically for 24 hours at 37 °C. Each assay was
carried out in triplicate.

Antimicrobial activity of PRE and metal salts with the addition of different
concentrations of Vitamin C
The antimicrobial assay was carried out as previously stated with the following
modification. Before adding the metal salts solution to PRE, Vitamin C was added
to the metal salts. Varying concentrations of Vitamin C were added comprising
the following ratios; 1:1 (4.8mM), 1:5 (24nM), 1:20 (96mM) (metal salt: Vitamin C)
was added to the metal solution, 700 \xL of this solution was then added to PRE.
Minimum inhibition concentration (MIC) determination of PRE and CuSCu
Micro-dilution plates were prepared with freeze dried PRE or CuSO4 which was
added to sterile water in a concentration of 800mg/ml. The plates were prepared
as follows, 50ul of four-times strength Iso-Sensitest broth was added to the first
row of wells and 50ul of double strength Iso-Sensitest broth was added to all
remaining wells. To the first row of wells 50 pi of the PRE was added and mixed,
50 pi of broth from row A was transferred to row B and mixed, this process was
continued to row F. Finally, 50pl of broth was removed from well F and discarded.
Then the overnight cultures were suspended in Ringers solution to a turbidly of
0.5 McFarland (1.5X108 cfu/ml). 50pl of suspension were added to well A (final
concentration of PRE in well A = 200mg/ml) through to G. All samples were
carried out in Triplicate. All plates were incubated at 37°C for 24 hours. After
incubation 10ul of broth from each well was spotted onto nutrient agar and
incubated at 37°C for 24 hours. After incubation the plates were examined to
determine breakpoints by the presence or absence of growth.
Minimum inhibition concentration determination of PRE: CuSO4_combination
The assay was carried out as above with the following changes: PRE and CUSO4
were prepared as before but using four times concentration of half the determined
MIC (ie. If the MIC was 4mg/ml, half this would be 2mg/ml and therefore stock
concentration would be 8mg/ml). Addition the CuSO4 was made to the PRE
suspension instead of sterile water.

Results and Discussion
The suspension test method outlined by Stewart et al (1998) J. Appl Microbiol
84:777-783, was adapted to assess the antimicrobial activities of PRE extracts
along with the cupric salts. For the MRSA isolates, the PRE on its own, had
marginal activity against all isolates studied (as shown in Figure 13). In contrast,
the copper (II) ions had moderate activities of between 102 to 103 log reduction in
growth. However, in combination the PRE/copper(ll) mixture surprisingly
exhibited an enhanced activity of 104 log10 reduction in growth which were
observed in 80% of the isolates. Similar results were observed for the MSSA
isolates, with no real effect for the PRE alone, but addition of Cu(ll) ions affording
enhancement of antimicrobial activity for 80% of isolates by 104 log orders (as
shown in Figure 14).
For the PVL positive cMRSA isolates, the PRE on its own, had marginal activity
against all isolates studied. In contrast to the MSSA and MRSA, the PVL positive
cMRSA isolates were even more sensitive to copper (II) ions and had moderate
activities of between 103 log reduction in growth for 60 % of the isolates. Notably,
for 40% of the isolates less reduction in growth indicated less sensitivity to Cu(ll)
ions, however, addition of PRE reduced the growth in these 40% in line with the
copper-sensitive 60% (as shown in Figure 15).
Determination of the MIC of PRE and CUSO4 individually and in combination are
shown in Table 3.
Table 3 - Minimum inhibition concentration of PRE and CUSO4 alone and in
combination against ten isolates each of MRSA, MSSA and Panton-Valentine
Leukocidin producing cMRSA.


PRE had an MIC between 25-12.5mg/ml for all isolates tested. The combination
of PRE: Cu(ll) against all isolates of S. aureus resulted in values which were half
or a quarter of the MIC of PRE or CuSO4 alone. Thus, a considerable additive
effect is seen against S. aureus for the combination.
In conclusion, PRE in combination with Cu(ll) ions exhibit surprisingly synergistic
antimicrobial effects against three classes of S. aureus. For MSSA, MRSA and
PVL positive cMRSA isolates, antimicrobial activities were exhibited by the
mixture. The inventors believe that they are the first to report of the efficacy of
pomegranate against PVL positive cMRSA isolates.
Example 7
Example 7 demonstrates the antimicrobial activities of pomegranate rind extracts
(PRE) in combination with Fe(ll) and Cu(ll) salts against multi-drug resistant (eg
extended spectrum (3-lactamase) Pseudomonas aeruginosa. Marked activities
were observed for the aqueous PRE:Cu preparations which were greatly
enhanced by addition of the reductant Vitamin C. An ointment preparation of the
PRE:Fe(ll):Vitamin C system showed moderate activity which was exceeded by
the corresponding Cu(ll) preparation over a three months period.

Materials and methods
Pomegranate rind extracts (PRE) were prepared by cutting the rind into small
cubes (approximately 5mm3) which were dried at 55°C for 24 hours. Dried rind
was stored in air tight containers in the dark until further use. Stock solutions
were prepared by adding 10g of dry rind to 150ml distilled water and shaking (at
80 rpm) at room temperature for 24 hours. The crude extract was passed through
muslin and a Whatman filter No.1 to remove the particle matter, and filter sterilised
by passing through a 0.2 um filter (Millipore) into a sterile bottle. The PRE stock
solutions were stored at -20°C.
Clinical isolates of multi-drug resistant Pseudomona aeruginosa (ESpL P.
aeruginosa), were collected at the Royal Marsden Hospital (Sutton, UK). The
isolates were grown overnight on nutrient agar (Oxoid, UK) and then frozen in
cyrovials (Pro-labs, UK) for future use.
All reagents were purchased from Sigma-Aldrich (Poole, Dorset) and distilled
water was used throughout. P. aeruginosa isolates were removed from the
freezer and first passaged on nutrient agar twice before use. Overnight cultures
on nutrient agar were then suspended in Ringer's solution (Oxoid, U.K.) to a
turbidity equivalent to 0.5 McFarland (1.5 x 108 CFU/ml). An aliquot of the PRE
extract (330 \i\) was added to 700 μl of the freshly prepared solutions (4.8 mM) of
metal salts (FeSO4, CuSO4)- The final solution was protected from light (Stewart
et al. 1998).
The appropriate bacterial dilution was prepared and 50 μl placed in a sterile
Eppendorf micro-centrifuge tube with a 100 μl of the extract/metal salt solution.
After exposure of the bacteria for 30 minutes at room temperature, the activity of
the bactericidal agent was neutralized by adding an equal volume of 2% (v/v)
Tween-80 (Sigma Chemical Co., UK) in Lambda buffer (Stewart et al. 1998).
Serial dilutions were prepared in Ringer's solution (10-5), 10 μl of each dilution is
spotted on nutrient agar plate and incubated for 24 hours at 37 °C. Each assay
was conducted in triplicate.

The assay was carried out as described above with the following addition: Vitamin
C was added to the metal ion solution immediately prior to mixing with the PRE.
Aliquots of Vitamin C were made to give final metal ion: Vitamin C ratios (and
Vitamin C concentrations) of 1:1 (4.8mM), 1:5 (24nM), 1:20 (96mM) (metal salt:
Vitamin C). 700 \x\ of this solution was then added to PRE.
Two formulations were prepared and tested: a hydrous ointment and an aqueous
cream. The composition of the hydrous ointment base was prepared as followed
(for 60 grams) 30g wool alcohol ointment, 0.6g phenoxyethanol, 0.3 dried
magnesium sulphate, 29.1g purified water. The components were mixed until
they formed a smooth ointment. The composition of the aqueous cream base was
150g emulsifying ointment, 5g phenoxyethanol and 345g purified water. The
components were mixed until they formed a smooth cream. For both formulations
containing PRE, the base preparation was carried out as above. However, PRE
was used instead of water, and solutions of metal salts and Vitamin C were added
to the base formulation.
The assay was carried out as above with the following changes. 0.5g of either
ointment or cream was first added to 10 ml of sterile water and vortex until
dissolved in water. The appropriate bacterial dilution (1.5*108CFU/ml) was
prepared and 50 jal was placed in a sterile Eppendorf micro-centrifuge tube with a
100 \x\ of the formulation solution, which was assayed as described above.
Formulations were stored in the dark at 5°C and for 3 months, prior to re-testing to
determine loss in activity.
Results
Nine multi-drug resistant clinical isolates of Pseudomonas aeruginosa were
subjected to challenge by PRE alone or in combination with metal ions. Figure 16
gives the results of the suspension test method used to assess the antimicrobial
activities of PRE extracts along with cupric and ferrous salts. For control samples,
PRE alone had no significant effect and both Fe(ll) and Cu(ll) treatments resulted
in a modest ca 101 log10 reduction in growth (mean values). A minor reduction in
growth (ca 101 log10) was observed upon treatment with the PRE:Fe(ll)
combination. As expected, treatment with Cu(ll) alone resulted in a reduction in

growth of ca 102 log™. However, in contrast to the result with PRE:Fe(ll) where
marginal effect was seen, Vitamin C addition greatly enhanced the activities of the
PRE:Cu(ll) combinations. Addition of one equivalent of Vitamin C enhanced the
growth retardation of PRE:Cu(ll) from ca 105 to ca 103 logio reductions. Five
equivalents of Vitamin C afforded a 104 logio reduction in growth and for 20
equivalents of Vitamin C no detectable growth was observed.
The results of the suspension test method used to assess the antimicrobial
activities of ointment formulations are given in Figure 17. The ointment PRE:Fe(ll)
combination with added Vitamin C gave a reduction in growth of 102 log10 in
contrast to the corresponding aqueous formulation which exhibited no significant
activity (as shown in Figure 16). In the case of the Cu(ll):PRE combination with
Vitamin C, the expected 104 logio reduction in growth was observed in line with
the results for the aqueous formulation (as shown in Figure 16). After storage for
three months, it is notable that a similar pattern of activity is retained for each
combination with a slight loss of activity of ca 101 logio reduction in growth for both
combinations. The cream based formulations were found to be considerably less
active and less stable.
Discussion
As shown in Figure 16, and in line with the results obtained by Stewart et al.
(1998) supra, the PRE:Fe(ll) system had negligible effect on the bacterial growth
with a modest retardation of growth occurring for Fe(ll) treatment alone. The lack
of antimicrobial activity exhibited by the PRE:Fe(ll) combination may arise from
the instability of ferrous ions in aerated aqueous solutions. However, in contrast,
the PRE:Cu(ll) combination exhibited a ca 102 log10 reduction compared to Cu(ll)
treatment alone.
Following this result, a further investigation involved addition of the reductant
Vitamin C to explore if the mechanism was attributable to redox-cycling. The
profound effects produced upon addition of Vitamin C indicated that reducing the
metal ion may be important. It is notable that in the aqueous preparations this
enhanced effect was mainly seen for the PRE:Cu(ll) combination. A complete

retardation in growth was observed for the PRE:Cu(ll) system on addition of 20
equivalents of Vitamin C. These results suggest that the combination of PRE:Cu
and Vitamin C may be a possible antimicrobial agent for treating multi-drug
resistant (eg ESpL) Pseudomonads that are becoming an increasingly resistant
organism to currently available antibiotics.
The key requirement for the development of a stable formulation arose as the
aqueous combinations were found to have a short active shelf life. Initial stability
and activity tests were conducted on aqueous, cream and ointment formulations
with the focus moving to the ointment as it had the optimal properties. In addition
to developing a stable and active ointment formulation of the PRE:Cu(ll)Vitamin C,
the inactivity of the aqueous preparation of the PRE:Fe(ll):Vitamin C system is
reversed and stable as an ointment.
In conclusion, the combination of aqueous solutions of PRE and Cu(ll) salts show
antimicrobial activity against Pseudomonads which is further enhanced with
addition of Vitamin C. Stable and active ointment formulations have been
developed for combinations of both PRE:Cu(ll) and PRE:Fe(ll) systems with
Vitamin C. The inventors believe that they are the first to report of the activation
of a natural product by addition of a redox-active metal ion along with the
reductant Vitamin C.
Example 8
The aim of Example 8 was to establish the optimum extraction method for green
and black tea and determine the efficacy of these extracts against Staphylococcus
aureus, Pseudomonas aeruginosa and Proteus mirabilis in the presence and
absence of metal ions.
Materials and Methods
Microorganisms and Culture conditions
Isolates of Staph, aureus, Prot. mirabilis and Ps. aeruginosa were maintained on
Brain Heart Infusion slopes (Oxoid Ltd), at room temperature, until used. Prior to
use, the organisms were inoculated into 5mL aliquots of Brain-heart infusion broth

(Oxoid Ltd) and incubated aerobically overnight at 37°C. These starter cultures
were used as inocula for the screening assays.
Development of extraction method
A preliminary screen (disc diffusion method) against Staph, aureus was
established to investigate the optimum method of extraction. Tea extracts are
prepared from Sencha green Chinese tea and Yorkshire black tea obtained from a
commercial outlet. The methods employed were hot and cold water extraction: 8
g of loose leaf green or black tea were infused with 100mL of sterilized distilled
water at 100°C or ambient temperature for recorded time intervals up to one hour
in the dark. Overnight bacterial cultures were suspended in Ringer's solution to a
cell concentration of 1 x 105 CFU/mL and swabbed evenly in three directions on
Mueller-Hinton agar plates (Oxoid Limited). The plates were then spotted with 10
uL of each test extract prior to aerobic incubation at 37°C for 24 h.
Determination of optimum pH and concentration of tea extracts
The method outlined above was used to examine the effects of pH and extract
concentration on antimicrobial activity. The optimum tea pH was determined
using 8g of tea leaves per 100 mL water at 100°C for 10 min. The pH values of
untreated tea extracts were in the range of 4.5 to 5.0 pH units. These were
adjusted using aqueous solution of NaOH (1.0 molar) by careful drop wise
addition to reach the desired pH values (+/- 0.3) of 5, 6, 7, 8, 9. The concentration
effects for both green and black leaves were examined using the hot water
extraction method (infusion between 10 and 20 minutes) for 4, 6, 8 and 10 g in
100 mL
Bactericidal assay
Tea infusions were prepared as previously with boiling sterilized distilled water.
After 10 minutes, the extracts are removed from the beaker into sterilized
universal tubes. The pH of these extracts was neutralized using a 1 M NaOH
solution. The extracts are refrigerated, used within 4 days and without further
treatment.

Overnight cultures of the Gram-positive strain (Staph, aureus) and the Gram-
negative strains (Ps. aeruginosa and Prot. mirabilis) were suspended in Ringers
solution to a turbidity equivalent to 0.5 McFarland (1.5 x 108 CFU/ml). Metal salts
were added to the optimum tea extracts immediately before the assay was
conducted. The tea extract (330 nl) was added to 700 Μl) of the freshly prepared
ferrous sulphate or cupric sulphate solution (4.8 mmol metal salt; pH 6.3); the final
solution was protected from light.
The appropriate bacterial dilution was prepared and 50 μl of that was placed in a
sterile Eppendorf micro-centrifuge tube with a 100 μl of the extract/metal salt
solution. Following 30 min incubation at room temperature, the activity of the
putative bactericidal agent was neutralized by adding an equal volume of 2% (v/v)
Tween-80 (Sigma Chemical Co., UK) in Lambda buffer (Stewart etal., 1998).
Serial dilutions were prepared in Ringer's solution, 10 uL aliquots of each dilution
were spotted onto nutrient agar plates and incubated aerobically for 24 hours at
37°C. Each assay was conducted in triplicate.
Results and Discussion
Studies on the optimum extraction methodology for the black and green teas
established the best method was extraction at 100°C for 10 minutes. Preliminary
investigations ascertained the mean zones of inhibition (in mm for triplicate runs)
for green tea varied with pH, showing optimal activities at pH 7/8 with inhibition
values of 16, 18, 21, 21, 17 mm for the pH values of 5, 6, 7, 8, 9 respectively.
Similarly, black tea exhibited optimal activity at pH 5/7 with values of 25, 27, 26,
17, 16 mm at pH 5, 6, 7, 8, 9 respectively. In both cases, a negative trend in
activity is evident in basic conditions. The investigations of the effects of pH
showed the value of pH7 to be optimal against Staph, aureus.
The antimicrobial activities of black and green tea extracts along with the metal
salts additives against Staph, aureus are shown in Figure 18. The data show
poor efficacy for both types of tea extract and for the iron and manganese salts
when tested independently. Upon addition of the cupric salt a bactericidal efficacy
(equating to a reduction in CFU/mL by 104) was observed. For both black and

green tea extracts the addition of cupric ions slightly diminished the effect
compared to the cupric salt alone. On the addition of manganese to black tea, a
minimal reduction in viability was established (reduction of 100 CFU/mL) which
was doubled in the presence of green tea. In contrast, there was an
enhancement of inhibitory activity against Staph, aureus with the addition of
ferrous ions to both tea extracts, with a reduction in CFU/ml of circa 104 for each,
equivalent to the level seen on action of cupric ions alone.
Similar antimicrobial profiles were determined for tea extracts along with the metal
salts additives against Prot. mirabilis and Ps. aeruginosa as shown in Figures 19
and 20. The data shows poor efficacy for both types of tea extract when tested
independently as seen with Staph, aureus. For the ferric and cupric salts,
moderate bactericidal efficacies (equating to a reduction in CFU/mL by 103 and
102 respectively) was observed, whilst the reduction in the presence of
manganese was a factor below that seen with cupric salts. Upon addition of ferric
ions to black tea extracts a decrease in efficacy was observed on comparison to
the ferric salt solution alone with Prot. mirabilis, whereas there was no noteworthy
change in efficacy for Ps. aeruginosa. In the case of the green tea extract when
combined with the ferric salt, an equivalent efficacy to the ferric salt alone was
observed. The action of manganese salts on their own with both Gram-negative
microorganisms was found to be minimal. In the presence of both green and
black tea no response was elicited from the inclusion of manganese ions against
Pseudomonas aeruginosa. However, a slight antagonistic effect was observed
against Prot. mirabilis in the same circumstances.
These data demonstrate that significant enhancement of natural product extracts
as antimicrobial agents can be achieved by addition of redox active metal ions. It
is notable that contrasting activity profiles are seen for different microbial strains
with green tea extract combined with cupric/ferrous/manganese salts exhibiting
least variation with the Gram-positive. This example highlights the significance of
natural extracts and their enhanced efficacy against microorganisms of medical
importance in the presence of redox active metal ions at a time when alternative

strategies for the control of microorganisms causing hospital acquired infection
(HAI's) are being sought.
Summary
The inventor has demonstrated in the Examples that the activity of iron-based
PRE compositions may be augmented and prolonged upon combination with a
reducing agent, such as Vitamin C. This was surprising given that neither the
mechanism of action nor the reason for the very short activity lifespan was
understood for iron-based compositions before the inventor began his research.
Furthermore, the inventor has shown that, surprisingly, other metal ion-based (eg
copper) PRE compositions also exhibit considerable antimicrobial activity. The
mechanism of action of copper-based compositions is not fully understood.
However, it is believed that the mechanism for copper-based compositions is
different to that of iron-based compositions. A significant advantage of using
copper as opposed to iron is that the composition does not turn black. It will be
appreciated that a composition which turns black would be undesirable for topical
use.
In addition to the isolation and identification of new formulations with long term
activities, applications against hospital acquired infections have been
demonstrated opening up further markets for exploitation of the compositions in
accordance with the invention. The identification of new additives (Copper(ll) ions
and Vitamin C) along with exemplification of the optimum formulation for long term
efficacy present considerable commercial advantages over the known
compositions disclosed in EP 0,744,896B1.

WE CLAIM :
1. An antimicrobial composition comprising (i) a copper salt and/or a cobalt salt
and/or a nickel salt; and (ii) an extract of a plant selected from a group consisting
of Punica granatum, Viburnum plicatum, Camellia sinensis, and Acer spp.
2. A composition according to Claim 1, wherein the composition comprises a
reducing agent.
3. An antimicrobial composition comprising (i) a copper salt and/or an iron salt
and/or a nickel salt and/or a cobalt salt; (ii) an extract of a plant selected from a
group consisting of Punica granatum, Viburnum plicatum, Camellia sinensis, and
Acerspp.; and (iii) a reducing agent.
4. A composition according to Claim 2 or Claim 3, wherein the reducing agent
comprises cysteine, glutathione or Vitamin C.
5. A composition according to any one of Claims 2 to 4, wherein the reducing
agent comprises Vitamin C.
6. A composition according to any preceding claim, wherein the concentration of
the copper, iron, nickel, or cobalt salt is in the range of about 0.1 mM to about
200mM.

7. A composition according to any preceding claim, wherein the composition
comprises a copper (II) salt.
8. A composition according to any preceding claim, wherein the composition
comprises copper sulphate.
9. A composition according to any one of Claims 3 to 8, wherein the composition
comprises an iron (II) salt.

10. A composition according to any preceding claim, wherein the composition
comprises an extract of Punica granatum.
11. A composition according to any preceding claim, wherein the composition
comprises pomegranate rind extract.
12. A composition according to any preceding claim, wherein the composition is
provided as an ointment formulation.
13. A solid or liquid concentrate, which on dilution with water, provides a
composition according to any one of Claims 1 to 12.
Dated this 26th day of June 2009.

The invention provides antimicrobial compositions comprising an effective
concentration of a metal salt combined with a plant extract. In some embodiments the composition comprises a copper salt and/or an iron salt and/or a nickel salt and/or a cobalt salt; and an extract of a plant selected from a group consisting of Punica granatum, Viburnum plicatum, Camellia sinensis, and Acer spp. The
invention extends to uses of such compositions as medicaments, and to methods of treating microbial infections. The invention extends to methods for preventing microbial infections by coating objects and surfaces with the compositions.