Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1998-08-25
2001-02-13
Tate, Christopher (Department: 1651)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S405000, C424S648000
Reexamination Certificate
active
06187316
ABSTRACT:
Bacteriophage represent a diverse group of viruses that exert both positive and negative effects in microbiology. For example, in the dairy industry bacteriophages of the lactic acid bacteria represent a major source of starter culture failure with consequent poor-quality milk fermentations. A number of measures to prevent phage infection, including implementation of hygienic procedures, rotation of starter cultures, isolation of resistant mutants and formulation of media to suppress phage proliferation have been instituted with various degrees of success [1-4]. By contrast, coliphage have proven to be valuable monitors of faecal contamination in ground water and treated drinking water [5] and are effective viral models in studies of water quality and virucidal activity [6, 7]. Recent developments with genetically recombinant bacteriophage, containing either the bacterial luciferase lux genes or the ice nucleation gene ina, have established an additional value for phage in the rapid detection of bacteria in food and environmental samples [8-12]. In the above examples, the eventual destruction of bacteriophage is important either from the perspective of maintaining effective fermentation or from the viewpoint of good microbiological practice in the disposal of contaminated material. At present, the methods available for bacteriophage destruction such as heat and chemical disinfection [13, 14], have a significant impact on the viability and survival of associated bacteria. In the case of a sterilisation regime this is of little significance but the ability to combat bacteriophage in industrial environments would benefit from an environmentally benign procedure that could differentially destroy the virus without damaging metabolically-active bacterial cells. One example is in the dairy industry as mentioned above. Another concerns spraying plants which have symbiotic bacteria to control bacteriophage infection.
There are also many situations where it would be useful to be able to combat fungus without at the same time damaging metabolically-active cells. Plant extracts have been used against disease development in banana by fungi [15].
This invention provides an antiviral or antifungal composition comprising an effective concentration of a mixture of a ferrous salt and an extract of a plant selected from pomegranate rind,
Viburnum plicatum
leaves or flowers, tea leaves, and maple leaves.
An antiviral composition is one which combats, e.g. by preventing growth or preferably killing, virus such as bacteriophage. An antifungal composition is one which combats, e.g. by preventing growth and preferably by killing fungus and destroying its spores. Preferred compositions are antiviral or antifungal, but without at the same time substantially damaging metabolically active bacterial or other cells with which the virus or fungus is associated. Preferably the composition is an aqueous solution, i.e. one in which water is the sole or the main constituent solvent.
The composition comprises a ferrous salt, which may conveniently be ferrous sulphate. The nature of the anion is however not critical, provided that the salt is non-toxic to bacterial and other cells under the intended conditions of use and is water-soluble. A preferred ferrous salt concentration range is 0.1 mM to 0.1 M, particularly from 1 to 20 mM.
The composition also contains an extract of a plant. These plant extracts may conveniently be prepared by boiling the comminuted plant part with water or other solvent. The resulting extract may be fractionated. It is probable that the extract contains one or more active components. Although such active components have remained refractive to purification, there is a uniform consistency in the extracted activity from different sources of the plant parts and from parts obtained at different times of the year. The inventors have examined many different plants and have identified the following four as active:
Pomegranate rind. Whole pomegranates can be comminuted and used, but the activity resides in the rind.
Viburnum plicatum
leaves or flowers.
Tea leaves. These may be dried or green. Other parts of the tea plant
Camellia sinensis
may be used.
Maple leaves e.g. UK
Acer psueudoplatanus
or Canadian maple leaves, or more generally leaves or flowers of any part of the genus Acer.
The plant extract may be used as is, or diluted as appropriate, e.g. by a factor of up to 100. Effective compositions generally contain 10-90% by volume of the ferrous salt solution together with correspondingly 90-10% by volume of the concentrated or diluted plant extract. The composition should preferably be stored in the dark.
The invention also includes solid or liquid concentrates which on dilution with water gives compositions as described.
The invention also includes a method of controlling virus or fungus, which method comprises contacting the virus or fungus with an effective concentration of a ferrous salt and an effective concentration of an extract of a plant selected from pomegranate rind,
Viburnum plicatum
leaves or flowers, tea leaves and maple leaves. These two components can be used in sequence in either order. Preferably, however they are used mixed together as a composition as described above.
The virus or fungus to be controlled may be contacted with, e.g. immersed in, the composition, typically for a few seconds or minutes. Where the virus or fungus is on a surface, the composition may be applied to the surface, e.g. by spraying or wiping.
The surface may be for example a work surface or a vessel or utensil used in a hospital or kitchen or an industrial environment, or an external surface of a mammal e.g. human or a plant. Or a solution containing virus or fungus may be mixed with a composition as defined.
As described in the examples below, ferrous sulphate in combination with selected plant extracts effects complete destruction of a broad range of bacteriophage infecting diverse bacterial genera. In assays incorporating both bacteriophage and bacteria at 10
12
and 10
9
/ml respectively, the bacteriophage are entirely destroyed within two minutes without affecting bacterial viability as measured by colony forming ability.
When used alone, ferrous sulphate has virucidal activity against phages, and also bactericidal activity against some bacteria. This invention is based on the observation that ferrous sulphate, either alone or in combination with certain plant extracts, offers a potent broad spectrum virucidal activity. Since the activity of relatively low levels of ferrous sulphate can be further potentiated by the addition of trace amounts of hydrogen peroxide (data not shown) it is likely that the mechanism of action involves, at least in part, a free radical system. A mechanism similar to that operating in the phagolysozome and defined by the Modified Haber-Weiss Reaction (17) is proposed.
Fe
2+
+H
2
O
2
→Fe
3+
OH
−
+—OH
While resistance of bacterial cells may be effected through free radical scavenging and repair systems, it is clear that the plant extracts also have a role and that remains to be elucidated.
Materials and Methods
Bacteria and Bacteriophage Strains
One Gram-positive
Staphylococcus aureus
NCIMB 8588 and two Gram-negative
Salmonella typhimurium
LT2 and
Pseudomonas aeruginosa
NCIMB 10548 bacteria were used. The bacteriophage with specificity for the above bacteria were NCIMB 9563 for
Staph. aureus,
Felix 01 for
S. typhimurium [
16] (obtained from Amersham International plc., Amersham, HP7 9NA, UK) and NCIMB 10116 and 10884 for
Ps. aeruginosa.
(All these bacteria and bacteriophage are available to the public).
The bacterial cells were maintained on Tryptose Phosphate broth (TPB; Oxoid) supplemented with 1% agar (TPA) and stored at 4° C. with monthly subculture. When required, cells were resuscitated in 10 ml TPB (18 h, 37° C.) or an orbital shaker operating at 240 rpm. Appropriate bacterial dilutions were made in Lambda buffer (6 mM Tris, 10 mM MgSO
4
.7H
2
Denyer Stephen Paul
Jassim Sabah Abdel Amir
Stewart Gordon Sydney Anderson Birnie
Merck Patent GmbH
Tate Christopher
Wenderoth, Lind & Ponack L.L.P
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