CA2386648A1

CA2386648A1 - Compositions and methods for managing bacterial skin conditions

Info

Publication number: CA2386648A1
Application number: CA002386648A
Authority: CA
Inventors: Janice Mccoll
Original assignee: Fytokem Products Inc
Current assignee: Fytokem Products Inc
Priority date: 2002-05-16
Filing date: 2002-05-16
Publication date: 2003-11-16

Abstract

The invention provides compositions and methods for the prophylactic or therapeutic treatment of a skin condition involving Propionibacterium aches comprising an extract of Epilobium angustifolium (Canadian Willowherb) or an oenothein selected from the group consisting of oenothein A, oenothein B, or tautomers, isomers, or derivatives thereof, any pharmacologically acceptable salts thereof, and/or a mixture of three tannins characterised respectively by high performance liquid chromatography.
The oenothein for use in such compositions, such as oenothein A or oenothein B, and the mixture of three tannins may be purified from natural sources, such as plant material (e.g. Epilobium angustifolium).

Description

COMPOSITIONS AND METHODS FOR MANAGING BACTERIAL SKIN
GONDITIONS

FIELD OF THE INVENTION
The invention is in the field of compositions and methods for the prevention, treatment and management of skin conditions involving bacterial contamination, and more particularly in the field of compositions and methods for the prevention, treatment and management of acne using plant extracts.
BACKGROUND OF THE INVENTION
Bacterial contamination of the skin may contribute to various skin disorders, including acne. Acne is a very common skin disorder involving the sebaceous follicles and is characterized by blackheads, whiteheads, papules, pustules, cysts, and various sized nodules and scars. The disorder affects skin areas where the sebaceous glands are most active and bacterial infection can occur in the sebaceous follicles.
A resident anaerobic organism, Propionibacterium aches, proliferates in the environment created by the mixture of the excessive sebum and follicular cells and produces chemotactic factors and inflammatory mediators that may lead to inflammation (see Manual of Dermatologic Therapeutics. 5t" edition, 1995, Little, Brown and Company.
Boston, pp.3-15).
A variety of acne treatment methods have been developed, including both systemic and topical administration of antibiotics, and topical administration of organic peroxides, particularly benzoyl peroxide. However, many products used in the treatment of acne are irritating and may cause inflammation of the skin. Many of the current products are only alcohol-soluble. Formulations that are alcohol-based may be irritating and are often very drying to the skin. Many of the acne treatments are effective only after extended periods of time and they can have a multitude of side effects. Side effects from current anti-acne treatments such as topical clindamycin and erythromycin or retinoids include local reactions such as burning, erythema (redness), peeling, dryness, and pruritus. Some therapies such as isotretinoin may have extremely serious side effects including teratogenicity.

Several plants have been shown to exhibit antibiotic and antifungal activity in in vitro assays. For example, methanolic extracts of Epilobium angustifolium aerial parts and roots have been shown to demonstrate broad-spectrum antifungal effects as well as anti-bacterial effects against several bacteria, such as Escherichia coli, Mycobacterium phlei, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis (see McCutcheon AR, 1996). Various plant tannins, including some ellagitannins have been reported to have antimicrobial properties (see Vivas N, 2000; Kakiuchi N, 1986; and Yoshida T, 2000). However, the spectrum of antibacterial activity of tannins has been found to be unpredictable. For example, Oenothein B, an ellagitannin present in Epilobium angustifolium has been reported to exhibit antibacterial activity against Helicobacter pylori while being inactive against Staphylococcus aureus and Escherichia coli (see Yoshida T, 2000).
SUMMARY OF THE INVENTION
It has been discovered that Epilobium angustifolium (Canadian Willowherb) extracts exhibit surprising antibacterial activity against Propionibacterium aches.
Accordingly, in one aspect, the invention provides methods for the treatment of a skin condition involving Propionibacterium aches, in which a therapeutic or prophylactic dose of an extract of Epilobium angustifolium (Canadian Willowherb) is administered.
In one aspect the invention provides a composition for treating a skin condition involving Propionibacterium aches, which comprises an extract of Epilobium angustifolium (Canadian Willowherb) in a therapeutically or prophylactically effective amount sufficient to kill Propionibacterium aches, or prevent or inhibit the growth thereof. In some embodiments the compositions of the invention may include formulations for topical use.
Other aspects of the invention include compositions and methods for the prophylactic or therapeutic treatment of a skin condition involving Propionibacterium aches, comprising a prophylactically or therapeutically effective amount of an oenothein extracted or prepared from Epilobium angustifolium (Canadian Willowherb) or from any other plant source. In an alternative embodiment, the oenothein for use in

-2-such compositions and methods may for example be oenothein A, a tautomer of oenothein A or an isomer of oenothein A or any of their pharmacologically acceptable salts or oenothein B, a tautomer of oenothein B or an isomer of oenothein B
(such as, for example oenothein D and F), or a derivative of oenothein B wherein a hydroxy group is for example replaced by a carboxylic acid group (such as, for example, oenothein G) (see Yoshida T., 1995) or any of their pharmacologically acceptable salts, or mixtures thereof.
In another aspect of the invention, new plant extracts found to contain an oenothein may be used for treating a skin condition involving Propionibacterium acnes. For example, Epilobium spp. other than Epilobium angustifolium such as Epilobium parviflorum may be used for making such extracts where they contain oenothein A or oenothein B or tautomers or isomers or a derivative of oenothein B
wherein a hydroxy group is for example replaced by a carboxylic acid group (such as, for example oenothein G) or any of their pharmacologically acceptable salts, or mixtures thereof. Various Onagreaceae plants such as, for example, Oneothera laciniata or Oneothera erythrasepala, Lythracea plants such as, for example, Woodfordia fructiosa or Cuphea hyssopifolia and Myrtaceae plants such as, for example, Eucalyptus albs, Eucalyptus consideniana or Eucalyptus viminalis may also be used for making extracts containing oenothein A or oenothein B or tautomers or isomers or derivatives or oenotheins with similar chemical structures or mixtures thereof.
In another aspect, the invention provides compositions and methods for the prophylactic or therapeutic treatment of a skin condition involving Propionibacterium acnes, comprising a prophylactically or therapeutically effective amount of a mixture of oenothein and three tannins extracted from Epilobium angustifolium (Canadian Willowherb) and characterized respectively by high performance liquid chromatography column (HPLC). The retention times for the three tannins are in the ranges of about 11.42-11.49, 11.55-11.60 and 11.96-11.99 minutes upon gradient elution in a 50% aqueous methanol solution on a SupelcosilT"" LC-18 column with a water/acetonitrile eluant having an initial composition of 98% water/2%
actetonitrile and a composition of 65% water/35% acetonitrile at 10 minutes, 2% water/98%

-3-acetronitrile at 20 minutes, and final composition of 98% water/2%
acetonitrile at 30-50 minutes.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a chromatogram of a tannin mixture exhibiting antibacterial activity against Propionibacterium acnes.
DETAILED DESCRIPTION OF THE INVENTION
In some aspects, the invention provides methods of treatment of skin conditions involving Propionibacterium acnes, in which a therapeutic or prophylactic dose of an extract of Epilobium angustifoliurn (Canadian Willowherb), or an oenothein, or a tannin mixture comprising the three tannins previously identified by HP~C and optionally oenothein is administered, such as by administration of a pharmacologically acceptable formulation. Such formulations of the invention may comprise an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein or a pharmacologically acceptable salt thereof, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture and a pharmacologically acceptable excipient or carrier. In some embodiments, such formulations may comprise a therapeutically or prophylactically effective amount sufficient to reduce Propionibacterium acnes or to prevent or inhibit the growth of Propionibacterium acnes. In particular embodiments, such formulations may comprise any one of the three aforementioned tannins by itself or in combination with a pharmacologically acceptable excipient or carrier and/or an oenothein. In other embodiments, such formulations may comprise two of the three aforementioned tannins by themselves or in combination with a pharmacologically acceptable excipient or carrier and/or an oenothein.
As used herein "pharmacologically acceptable carrier" or "excipient" includes any and all creams, gels, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and do not significantly adversely affect the pharmaceutical properties (e.g. toxicity and effectiveness) of the extract of Epilobium angustifolium

-4-(Canadian Willowherb), or the oenothein, or the tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture, such as are conventionally used in the casmetic and pharmaceutical arts. In one embodiment, the carrier is suitable for topical administration. In other embodiments, the carrier is suitable for internal administration. In some embodiments, oenotheins may undergo hydrolysis in acid or base, so that pharmacologically acceptable carriers or excipients may include pH buffers to maintain an acceptable pH for pharmaceutical activity (see Daniel et al., 1991, for a discussion of the effects of pH on ellagitannins).
A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of acne. A therapeutically effective amount of an extract of Epilobium angustifolium (Canadian Willowherb), or of an oenothein, or of a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture may vary according to factors such as the acne state, age, sex, and weight of the individual, and the ability of the extract of Epilobium angustifolium (Canadian Willowherb), or of the oenothein, or of the tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the extract of Epilobium angustifolium (Canadian Willowherb), or of the oenothein, or of the tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture are outweighed by the therapeutically beneficial effects.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the growth of Propionibacterium acnes. A
prophylactically

-5-effective amount can be determined as described above for the therapeutically effective amount.
In particular embodiments, a preferred range for therapeutically or prophylactically effective amounts of an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture may be 0.0003% to 99% by weight. These may be characterized by the percentage of dry matter which they contain. Dry matter is the solid residue remaining after removal of the carrier or solvent by drying, such as by drying a solution or suspension in an oven, by lyophilization or evaporation under vacuum. Dry matter may be expressed in %
and may also be referred to as plant solids concentration. Thus, a 100 gram or 100 mL
solution or suspension containing 5% dry matter by weight, yields 5 grams of solids or residue after drying. Alternative methods of drying may yield slightly different values for the percent by weight of dry matter, so that all such values recited herein are necessarily approximations. Dry matter values include suspended as well as settled solids. Any one of the three tannins previously identified by HPLC may be effective by itself in an amount of 0.0003% to 99%. In some embodiments the therapeutically or prophylactically effective amount may be 0.1 % to 50%. In other embodiments the therapeutically or prophylactically effective amount may be 0.1 % to 10%. One method of cosmetic, prophylactic or therapeutic treatment is to apply an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from other plant sources containing oenothein or such a tannin mixture of any component thereof topically to the acne area. Dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the judgement of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the methods of the invention.
_g_ In other embodiments the therapeutically or prophylactically effective amount of an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) may be combined or admixed with various therapeutically or prophylactically anti-acne agents conventionally used in the cosmetic and pharmaceutical arts.
In accordance with another aspect of the invention, therapeutic compositions of the present invention, comprising an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture, may be provided in containers having labels that provide instructions for use of the formulation to: prevent or treat acne. The labels may also disclose that the compositions comprise an extract of Epilobium angustifolium (Canadian Willowherb), or an oenothein, or a tannin mixture extracted from Epilobium angustifolium (Canadian Willowherb) or from related Epilobium species or from any other plant source containing oenothein or such a tannin mixture.
A variety of methods may be used to purify oenotheins from natural sources for use in the various aspects of the present invention. For example, U.S. Patent No.
5,525,594 discloses methods of preparing oenothein B from plants. Similarly, Ducrey et a1.,1997 discloses oenothein A and oenothein B purification and characterization methods. Alternative purification methods may be used in accordance with the present invention, provided that they may be used to produce a pharmacologically acceptable preparation of purified oenothein B suitable for use in the various aspects of the present invention.
Anti-Acne Procedures and Protocols Overnight broth cultures of the P. acnes organisms were prepared. Cultures were centrifuged, rinsed with saline and diluted to give 80% Transmittance at wavelength of 530 nm and the suspension was used as the inoculum.

Test samples were inoculated with the organisms (zero time) and mixed well.
After the appropriate time, a sarnple was mixed with the broth blank and mixed well.
Serial dilutions were performed, prepared and placed in duplicate using the appropriate agar.
A control count was performed simultaneously on the cultures and plated in duplicate.
All plates were incubated at 3711.0°C for the appropriate time and resultant colonies were then enumerated.
Example 1 This example shows the effectiveness of whole and clear extracts of Epilobium angustifolium (Canadian Willowherb) in reducing Propionibacterium acnes after one hour of exposure.
Procedure for Making Canadian Willowherb Whole Various methods may be used for making an extract from Epilobium angustifolium (Canadian Willowherb). One method is as follows. In a beaker or other vessel, dried ground plant material was extracted with a variable amount of solvent, preferably an aqueous or aqueous ethanolic mixture such as aqueous methanol.
The resultant primary extract may be heated, for example for a period of at least minutes, and preferably 1 hour, then centrifuged and/or filtered to remove a substantial portion of plant material. Alcoholic solvent may be removed and the resultant aqueous extract concentrated or diluted to the required dry matter content ranging from 0.5% to 99% by weight, preferably 5% to 20% by weight, most preferably 5% by weight. This crude or primary extract is identified herein as Canadian Willowherb Whole Extract. Guar gum or other gums may be added to the extract to obtain a homogeneous suspension. Preservatives such as antimicrobials and antifungal agents or other agents such as acids or bases needed to adjust pH
may be added to the extract.
_g_ Procedure for Making Canadian Willowherb Clear Various methods may be employed for making Canadian Willowherb Clear.
One method is detailed as follows. The crude or primary Epilobium extract, Canadian Willowherb Whole Extract, may be treated with alcohol and decolorized with charcoal or other suitable decolorization agents to remove gums, chlorophyll and other colored components, and other unwanted constituents, and then filtered to obtain a clarified extract, known as Canadian Willowherb Clear Extract. The clarified extract may be diluted or concentrated to a desired dry matter content of plant material ranging from 0.5% to 99% by weight, preferably 5% to 20% by weight, most preferably 5% by weight. Preservatives such as antimicrobials and antifungal agents or other agents such as acids or bases needed to adjust pH may be added to the extract.
Results Results are indicated in tabular form. Numbers indicate colony-forming units (CFUs) per ml, and the reduction, or kill percentage.
This example shows the antibacterial effectiveness of a Canadian Willowherb Whole Extract (CW5) and Canadian Willowherb Clear (CWSC) extracts containing 5%
of dry matter by weight and tested at a use level of 1 % against P. aches evaluated by performing time kill studies at 60 minutes.
_g_ Table 1: Time kill results of a 1 % use level of CW5 (whole extract, 5% dry matter by weight) and CWSC (clear extract, 5% dry matter by weight) extracts mixed in water after one-hour exposure time Extract Use level Organisms ; Control 60 minutes count CW5 1.0% P. aches #11827 5.2 x 10' 5.8 x 10"

~% reduction = 88.8 CWSC 1.0% P. aches #11827 5.2 x 10 3.5 x 10 ~% reduction = 99.3 Example 2 This example shows the antibacterial effectiveness of utilizing a 4% use level of an unpreserved Canadian Willowherb Whole Extract (5% dry matter content by weight) against P. aches evaluated by performing further time kill studies at 2, 10 and 30 minutes. This corresponds to a total dry matter content of 0.2% by weight in the test solution.
Table 2: Time kill results of a 4% use level of CWSC (clear extract 5% dry matter by weight; unpreserved sample) extract mixed in water Test Use Organisms Control2 10 30 Substance Level* __ count minutes minutes minutes CWSC 4% P. aches 1.7 6.0 x 1.7 x 1.2 x x 10 10 10 10 (unpreserved #11827 sam 1e % reduction 64.7 90.0 92.9 =

*Concentration of CWSC mixed with water by volume.
These results show a remarkable effectiveness of Canadian Willowherb Whole Extract as an anti-P.acnes agent. The 4% use level corresponds to a total dry matter content of 0.2%, by weight, in the test solution. Most anti-acne treatments require long time exposures to be efficient and do not kill in vitro or in vivo in 2 minutes.

Example 3 This example shows the antibacterial effectiveness of different concentrations of Epilobium angustifolium (Canadian Willowherb) clear extracts against P.
aches evaluated by performing further time kill studies at 30, 60 and 120 minutes.

Table 3: Time kill result of Canadian Willowherb Clear Extract (CWSC) extract tested in water at use levels of 1-5%
Test Use Organisms Control 30 60 minutes120 Substance Level* count minutes minutes CWSC 1 % P.acnes #118271.2 x 1.1 x 1.1 x 10 1.1 x reduction = 8.3 8.3 8.3 CWSC 2% P. acnes 1.2 x 9.2 x 6.8 x 10 4.5 x #11827 23.3 43.3 62.5 reduction =

CWSC 2.5% P.acnes #'118271.7 x 5.9 x 3.1 x 10 5.0 x reduction = 65.3 81.8 70.1 CWSC 3% P"acnes #118271.5 x 5.1 x 0 0 10~ 10 reduction = 99.7 100 100 CWSC 4% P.acnes #11827 6.2 x 1.5 x 10 5.5 x ( 1.5 x 10 10 10 reduction = 99.6 99.0 99.9 ~

CWSC 5% P.acnes #11827 8 x 1~ 2 x 10 1.6 x ~ 2 10~~ 10 reduction = 93.3 99.8 99.9 j 'Concentration of CWSC mixed with water by volume.
Epilobium angustifolium (Canadian Willowherb) extracts may for example be diluted for use at use levels of approximately 1 %, 2%, 2.5%, 3%, 4%, and 5%
(by volume) corresponding respectively to an approximate total dry matter content, by weight, of 0.05%, 0.10%, 0.125'%, 0.15%, 0.20% and 0.25%. CWSC clear aqueous extracts are also effective against Propionibacterium acnes, with surprisingly strong activity for extracts having at least 0.15% dry matter or solids by weight in the clear aqueous extract. Similarly, preparations having at least an equivalent concentration of oenothein B may be advantageous, such as concentrations of oenothein B of at least about 0.00075% (or in alternative embodiments, of at least about 0.00050%, 0.00055%, 0.00060%, 0.00065%, 0.00080%, 0.00085%, 0.00090%, 0.00095%, 0.001 %, or any range of values between such concentrations). In some embodiments, it may for example be desirable to use such extracts having an activity that yields a reduction of P. acnes in such tests of at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9%. In some embodiments extracts having a P. acnes reduction activity of 100% may be used.
Example 4 Concentrated extracts cantaining higher amounts of oenothein B were also tested. These extracts contained approximately 50% oenothein B (technical grade) and 90-98% oenothein B (pure grade), by weight.
Procedure for PrJ~aringi Technical Grade Oenothein B~50+%~urity) A variety of methods may be employed to prepare technical grade of oenothein B, comprising at least 50% oenathein B. Column chromatography may be employed as well as the following method. Canadian Willowherb Clear aqueous extract, containing 0.5 to 99% dry matter by weight, preferably 5 to 20% dry matter, by weight, can be concentrated by evaporation under pressure or evaporated by other method to contain about 20% dry matter, by weight. Alternatively, Canadian Willowherb Whole aqueous extract (crude or primary extract) can be concentrated by evaporation under pressure, or evaporated by other method, to contain about 20% dry matter, by weight.
Addition of base, such as 4.0 M sodium hydroxide solution, with stirring to ensure thorough mixing, will produce a k>right yellow to dark precipitate. The resultant solution can be filtered to dryness under vacuum through various filters such as a #4 Whatman filter (Whatman International L.td.). The retained yellow to brown solid is then suspended in a minimal amount of water and treated with 2.0 N hydrochloric acid, drop wise, until the solution is acidic (about pH 4). Solution can be left to dry at room temperature, or by alternate method, and then ground to obtain the dried technical grade oenothein B, comprising at least 50% oenothein B, by weight.
Alternatively, the technical grade oenothein B may be prepared by subjecting the crude (Canadian Willowherb Whole Extract) or clear (Canadian Willowherb Clear Extract) to column chromatography as outlined in the procedure for preparing oenothein B pure grade (90-98% pure oenothein B).
The purity of the technical grade oenothein B may be determined by high performance liquid chromatography using the following method. The dried ground material (20mg) was dissolved in 1 ml 50% aqueous methanol and injected onto a SupelcosiITM LC-18 (Supelco) column (the injection volume may be in the range of 1.5 to 5 microlitres). Elution was carried out by a solvent gradient at a flow rate of 1 mL/min under the following conditions (volume percentages):
~ 0-10 min 98%water with 0.1 % v/v trifluoroacetic acid (TFA);
~ 10-20 min 65% water with 0.1 % v/v TFA and 35% acetonitrile with 0.1 % v/v TFA;
~ 20-30 min 2% water with 0.1 % v/v TFA and 98% acetonitrile with 0.1 % v/v TFA;
~ 30-50 min 98% water with 0.1 % v/v TFA and 2% acetonitrile with 0.1 % v/v TFA.
Detection was carried out at wavelengths ranging from 210 nm to 400 nm. At 270 nm oenothein B has a retention time of about 11.01-11.15 minutes under these conditions.
Procedure for Preparing Pure Grade Oenothein B j90-98% purity) In a beaker or other vessel, dried ground plant material was extracted with aqueous methanol or any other suitable solvent, under heat for at least 20 minutes.
The solution was filtered and they residue was squeezed to near dry while the mixture was still warm. The methanolic extract solution was evaporated under vacuum.
Alternately, the extract could be dried down by lyophilization after removal of solvent by evaporation, or concentrated by other means. The concentrated extract solution, about 40 grams, was subjected to column chromatography using varying concentrations of aqueous-acetone, in a Diaion (HP-20) column (Mitsubishi Chemical Corporation). This provided a water eluate, a 30% acetone eluate, and a 60%
acetone eluate. The eluates were dried down. The water eluate, about 10 grams, and the 30%
acetone eluate, about 16 grams, both contain oenothein B. The combined water and 30% acetone eluate was subjected again to a Diaion column, eluting with water, and then 10% aqueous acetone. The resultant eluate was dried down, yielding about grams of material. A 2 gram sample subjected to a MCI GEL CHP20P 37-75 micron column (Mitsubishi Chemical Corporation) was eluted with 20% aqueous methanol to yield fractions A to G. Fractions A to D, combined, gave about 1.16 gram oenothein B, at a purity of about 88%. The 1.16 gram was further subjected to column chromatography in a LiChroprep RP-18 25-40 micrometer (Merck) utilizing water: methanol (9:1) as eluent, and yielding about 0.74 grams of oenothein B, of a purity about 92-94%. Further subjections to column chromatography in another MCI
GEL CHP20P 37-75 micron column eluting with 20% aqueous methanol yields fractions ranging in purity from 92-98%.
The purity of the pure grade oenothein B may be determined by high performance liquid chromatography using the following method. The dried ground material (20mg) was dissolved in 1 mL 50% aqueous methanol and injected onto a SupelcosiITM LC-18 (Supelco) Column (the injection volume may be in the range of 1.5 to 5 microlitres). Elution was carried out by a solvent gradient at a flow rate of 1 mL/min under the following conditions (volume percentages):
~ 0-10 min 98%water with 0.1 % v/v trifluoroacetic acid (TFA);
~ 10-20 min 65% water with 0.1 % v/v TFA and 35% acetonitrile with 0.1 % v/v TFA;
~ 20-30 min 2% water with 0.1 % v/v TFA and 98% acetonitrile with 0.1 % v/v TFA;
~ 30-50 min 98% water with 0.1 % v/v TFA and 2% acetonitrile with 0.1 % v/v TFA.
Detection was carried out at wavelengths ranging from 210 nm to 400 nm. At 270 nm oenothein B has a retention time of about 11.01-11.15 minutes under these conditions.

Results Table 4: Kill Test Results for Oenothein B Technical Grade (a solid which contains approximately 50% oenothein B by weight), dissolved in water for testing Final EquivalentOrganisms Control 30 60 120 ConcentrationUse Level count minutes minutes minutes of Oenotheinof CWSC*

B by Weight 0.0018% 3.6% P. acnes #118271.5 x 10 9.2 x 4.7 x 0 10" 10"

reduction = 93.9 96.9 100 0.007% 14% P. ac:nes #118278.9 x 10 9.2 x 8.7 x 7.8 x i % reduction 99.0 99.0 91.2 ~ =

0.014% 28% P. acnes #118278.9 x 10 6.1 x 7.2 x 4.8 x reduction = 99.3 99.2 94.6 0.028% 56% P. acnes #118279.75 x 8.0 x 7.7 x 8.2 x reduction = 99.2 67.2 99.9 * This is the approximate concentration of CWSC mixed with water to obtain an oenothein B concentration equivalent to the Final Concentration tested as prepared from oenothein B Technical Grade.

Table 5: Kill Test Results for Oenothein B Pure Grade (a solid which contains approximately 90% to 98% oenothein B by weight), dissolved in water for testing ConcentrationEquivalentOrganisms Control30 60 120 of OenotheinUse Level count minutes minutes minutes B, by Weightof CWSC*

0.0003% 1.2% P. acnes #11827 1.7 6.6 x 5.5 x 7.5 x x 10 10 10 reduction = 106 61.2 67.6 55.9 0.0006% 2.4% P. acnes #11827 1.5 7.6 x 4.3 x 0 x 10 10 io reduction = 106 94.9 97.1 100 0.0035% 14% P. acnes #11827 8.9 9.0 x 8.3 x 7.0 x x 10 10 10 io reduction = 106 99.0 99.1 92.1 0.007% 28% P. acnes #11827 8.9 6.8 x 6.9 x 7.3 x x 10 10 10 reduction = 106 99.2 99.2 91.8 0.014% 56% P. acnes #11827 9.75 1.5 x 7.7 x 8.2 x x 10' 10 10 io reduction = 106 98.5 I 92.1 99.1 * This is the approximate concentration of CWSC mixed with water to obtain an oenothein B concentration equivalent to the Final Concentration tested as prepared from oenothein B Pure Grade.
These results show the surprising effectiveness of oenotheins, particularly oenothein B, as an anti- P. acnes agent in the treatment of conditions involving P.
acnes such as acne and other skin conditions. Accordingly, in some embodiments it may be advantageous to use purified oenothein B to prepare medicaments for the treatment of skin conditions involving P. acnes. In some embodiments such purified oenothein B preparations may contain 0.1 % to 10% oenothein B by weight. In other embodiments such purified oenothein B preparations may contain 50% or more oenothein B by weight (such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%
oenothein B).

Example 5 A mixture of oenothein B and three tannins isolated from extracts of Epilobium angustifolium (Canadian Willowherb) was also shown to exhibit antibacterial activity against Propionibacterium acnes.
Procedure for Making Tannin Mixture In a beaker or other vessel, 293 g of dried ground plant material was extracted with aqueous methanol or any other suitable solvent, under heat for at least minutes. The solution was filtered and the residue was squeezed to near dry while the mixture was still warm. The methanolic extract solution was evaporated under vacuum. Alternately, the extract could be dried down by lyophilization after removal of solvent by evaporation. The concentrated extract solution, about 40 grams, was subjected to column chromatography using varying concentrations of aqueous-acetone, in a Diaion (HP-20) Column (Mitsubishi Chemical Corporation). This provided a water eluate, a 30% acetone eluate, and a 60% acetone eluate. Finally, washing the column with methanol provided a methanol eluate, which contains mainly color pigments. The eluates were dried down. The water eluate yielded 10 grams of solids, the 30% acetone eluate yielded '16 grams of material and the 60% acetone eluate yielded 7 grams of material. The dried eluates (20mg) were dissolved in 1m1 50%
aqueous methanol and injected onto a SupelcosiITM LC-18 (Supelco) column (the injection volume may be in the range of 1.5 to 5 microlitres). Elution was carried out by a solvent gradient at a flow rate of 1 mUmin under the following conditions (volume percentages):
~ 0-10 min 98%water with 0.1 °/~ v/v trifluoroacetic acid (TFA);
~ 10-20 min 65% water with 0.1 % v/v TFA and 35% acetonitrile with 0.1 % v/v TFA;
~ 20-30 min 2% water with 0.1 % v/v TFA and 98% acetonitrile with 0.1 % v/v TFA;
~ 30-50 min 98% water with 0.1 % v/v TFA and 2% acetonitrile with 0.1 % v/v TFA.
Detection was carried out at wavelengths ranging from 210 nm to 400 nm.
Figure 1 is a chromatogram at 270 nm utilizing an injection volume of 1.5 microlitres.
This gradient is the same one used for the determination of Oenothein B.

The four components referred to in this four tannin mixture are the ones outlined in Table 5. As noted below, these four components may comprise about 64%
of the total sample. The identification of one of these tannins as oenothein B
has been confirmed.
The eluates contained at least four tannins; oenothein B is one of the major tannins in the eluates. The 30% acetone eluate contains oenothein B plus three other tannins, and was dried down to obtain a quantity of 16 grams (which represents the dried solids from the eluate, comprising oenothein B plus three other tannins -about 64% of the total - as well as other solid material from the extract - about 36% of the total weight).
The 60% acetone eluate contains three tannins that are more polar than oenothein B, and was dried down to obtain a quantity of 7 grams.
The tannins may be obtained in variable amounts. For example, one mixture (the dried solids from the 30% acetone eluate) was found to contain the tannins, identified by HPLC in the following composition:
Table 6: Tannin Mixture Chromatographic Characterisation annin Retention Percent Identification Time (Minutes) Composition 1 11.150 28.56 Oenothein B

2 11.493 10.86 3 11. 598 12.26 4 11.989 12.31 Total 63.99 Rcaci il+e Table 7: Kill Test Result for Tannin Mixture Isolated from Extracts of Epilobium angustifolium (Canadian Willowherb) ConcentrationEquivalent Organisms Control 60 of CW Tannin Use bevel count minutes of Mixture* CWSC**

0.0003% 0.54% P. acnes # 118271.1 x 1.1 x 10' (unpreserved) % reduction = 99.9 * Weight percent of isolated solids dissolved in water.
** This is the approximate concentration of CWSC mixed with water needed to obtain an equivalent concentration of 'tannins 1 through 4 as in the Concentration of CW
Tannin Mixture.
These results show the surprising effectiveness of a tannin mixture of the invention in the treatment of conditions involving P. acnes, such as acne and other skin conditions. Accordingly, in some embodiments it may be advantageous to use oenothein B in combination with other tannins, particularly tannins obtainable from Epilobium angustifolium (Canadian Willowherb), to prepare medicaments for the treatment of skin conditions, such as conditions involving P. acnes. In some embodiments such preparations may contain mixtures of tannins in the following amounts (in weight percent): (1) Oenothein B any value or any range within the values of from 0.1 to 1.5% (for example from a minimum value of any one of 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% to a higher maximum value of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2%, 1.3%, 1.4% or 1.5%); (2) Tannin 2 any value or any range within the values of from 0.01-1.0% (for example from a minimum value of any one of 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20 to a higher maximum value of 0.80%, 0.81 %, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91 %, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99% or 1.00%); (3) Tannin 3 any value or any range within the values of from 0.01-1.0% (for example from a minimum value of any one of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20 to a higher maximum value of 0.80%, 0.81 %, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91 %, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99% or 1.00%); (4) Tannin 4 any value or any range within the values of from 0.01-1.0% (for example from a minimum value of any one of 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11, 0.12, 0.13, 0.14, 0.15, 0.15, 0.17, 0.18, 0.19, 0.20 to a higher maximum value of 0.80%, 0.81 %, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91 %, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99% or 1.00%).
Conclusion Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art.
Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way.
Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to".
References:
The following documents are incorporated herein by reference:
McCutcheon AR. Ethnopharmacology of Western North American Plants with Special Focus on the Genus Artemisia. Ph.D. thesis, University of British Columbia, May, 1996.
Vivas N, Augustin M, Lonvaud-Funel A. Influence of Oak Wood and Grape Tannins on the Lactic Acid Bacterium OEnococcus oeni (Leuconostoc oenos, 8413).
Journal of the Science of Food and Agriculture, 2000, 80(11 ):1675-1678.
Kakiuchi N, Hattori M, Nishizawa M, Yamagishi T, Okuda T, Namba T. Studies on Dental Caries Prevention by Traditional Medicines: VIII. Inhibitory Effect of Various Tannins on Glucan Synthesis by Glucosyl Transferase from Streptococcus Mutans.
Chemical and Pharmaceutical Bulletin (Tokyo), 1986, 34(2):720-725.

Yoshida T, Hatano T, Ito H. Chemistry and Function of Vegetable Polyphenols with High Molecular Weights. Biofactors, 2000, 13(1-4):121-125.
Yoshida T., Chou T., Shingu T., Okuda T., Oenotheins D, F, and G
Hydrolysable Tannin Dimers from Oenothera laciniatia. Phytochemistry, 1995, 40(2):555-561.
Daniel EM, Ratnayake S, Kinstle T, Stoner GD. The effects of pH and rat intestinal contents on the liberation of ellagic acid from purified and crude ellagitannins. , 1991, J. Natural Products 54(4):946-952.
Ducrey B., Marston A., Gohring S., Hartmann R., Hostettmann K., Inhibition of 5a-Reductase and Aromatase by the Ellagitannins Oenothein A and Oenothein B
from Epilobium Species. Planta Medica, 1997, 63:111-114.

Claims

WHAT IS CLAIMED IS:

1. A method for the prophylaxis or treatment of a skin condition involving Propionibacterium acnes comprising administering to a subject a prophylactically or therapeutically effective amount of an extract of Epilobium angustifolium (Canadian Willowherb).

2. The method according to claim 1, wherein the extract is obtained from the flowering parts.

3. The method according to claim 1, wherein the extract is obtained from the roots.

4. The method according to any one of claims 1 to 3, wherein the extract is a whole extract comprising a plant solids concentration by weight of about 0.5%
to about 99%.

5. The method according to claim 4, wherein the plant solids concentration by weight is ranging from about 5% to about 20%.

6. The method according to claim 4 or 5, wherein the extract is aqueous, alcoholic or an aqueous-alcoholic mixture.

7. The method according to any one of claims 1 to 3, wherein the extract is a clear extract comprising a plant solids concentration by weight of about 0.5% to about 99%.

8. The method according to claim 7, wherein the plant solids concentration by weight is ranging from about 5% to 20%.

9. The method according to claim 7 or 8, wherein the extract is aqueous, alcoholic or an aqueous-alcoholic mixture.

10. The method according to any one of claims 4 to 9, wherein the extract is combined or admixed with a therapeutic or prophylactic anti-acne agent.

11. The method according to any one of claims 4 to 10 wherein the extract is combined with an inactive pharmacologically acceptable excipient, carrier or diluent.

12. The method according to any one of claims 1 to 11 wherein the extract is administered internally.

13. The method according to any one of claims 1 to 11 wherein the extract is applied topically.

14. The method according to claim 13 wherein the extract is applied topically.

15. A method for the prophylaxis or treatment of a skin condition involving Propionibacterium acnes comprising administering to a subject a prophylactically or therapeutically effective amount of an oenothein selected from the group consisting of oenothein A, oenothein B, tautomers, isomers and derivatives thereof and pharmacologically acceptable salts thereof.

16. The method of claim 15, wherein the oenothein is extracted or prepared from Epilobium angustifolium (Canadian Willowherb) or a related Epilobium species or any other plant source containing an oenothein selected from the group consisting of oenothein A, oenothein B, tautomers, isomers and derivatives thereof.

17. The method according to 15 or 16, wherein the oenothein is combined or admixed with an anti-acne agent.

18. The method according to any one of claims 15 to 17, wherein the oenothein is combined or admixed with an inactive pharmacologically acceptable excipient, carrier or diluent.

19. The method according to any one of claims 15 to 18, wherein the oenothein is administered internally.

20. The method according to any one of claims 15 to 18, wherein the oenothein is applied topically.

21. The method according to claim 20, wherein the extract is applied topically as a gel, paste, cream, lotion, solution, wash, emulsion or ointment.

22. A method for the prophylaxis or treatment of a skin condition involving Propionibacterium acnes comprising administering to a subject a prophylactically or therapeutically effective amount of a mixture of an oenothein and three tannins and characterised respectively by high performance liquid chromatography column retention peaks eluting at about 11.42-11.49, 11.55-11.60 and 11.96-11.99 minutes upon gradient elution in a 50% aqueous methanol solution on a Supelcosil.TM. LC-18 column with a water/acetonitrile eluant having an initial composition of 98%water/2% actetonitrile and a composition of 65%water/35% acetonitrile at 10 minutes, 2% water/98%
acetronitrile at 20 minutes, and final composition of 98%water/2%acetonitrile at 30 to 50 minutes.

23. The method according to claim 22, wherein the oenothein is oenothein B.

24. The method according to claim 23, wherein the oenothein is extracted from Epilobium angustifolium (Canadian Willowherb) or a related Epilobium species or any other plant source containing oenothein B.

25. The method according to claim 22 or 24, wherein the three tannins are extracted from Epilobium angustifolium (Canadian Willowherb) or a related Epilobium species or any other plant source containing the three tannins.

26. The method according to any one of claims 22 to 25, wherein the mixture is combined or admixed with therapeutic or prophylactic anti-acne agent.

27. The method according to any one of claims 22 to 26, wherein the mixture is combined or admixed with an inactive pharmacologically acceptable excipient, carrier or diluent.

28. A composition for the treatment of a skin condition involving Propionibacterium acnes comprising a prophylactically or therapeutically effective amount of an extract of Epibolium angustifolium (Canadian Willowherb).

29. The composition according to claim 29, wherein the prophylactically or therapeutically effective amount of an extract of Epibolium angustifolium (Canadian Willowherb) is about 0.6% to about 99% by weight.

30. The composition according to claim 28 wherein the prophylactically or therapeutically effective amount of an extract of Epibolium angustifolium (Canadian Willowherb) is about 0.6% to about 50% by weight.

31. The composition according to claim 30 wherein the prophylactically or therapeutically effective amount of an extract of Epibolium angustifolium (Canadian Willowherb) is about 0.6% to about 10% by weight.

32. The composition according to any one of claims 28 to 31, further comprising a therapeutic or prophylactic anti-acne agent.

33. The composition according to any one of claims 28 to 32 further comprising an inactive pharmacologically acceptable excipient, carrier or diluent.

34. A composition for the treatment of a skin condition involving Propionibacterium acnes comprising a prophylactically or therapeutically effective amount of an oenothein selected from the group consisting of oenothein A, oenothein B, tautomers, isomers and derivatives thereof and pharmacologically acceptable salts thereof.

35. The composition of claim 34, wherein the oenothein is extracted or prepared from Epilobium angustifolium (Canadian Willowherb) or a related Epilobium species or any other plant source containing an oenothein selected from the group consisting of oenothein A, oenothein B, tautomers, isomers and derivatives thereof.

36. The composition according to any one of claims 34 to 35, wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 99% by weight.

37. The composition according to claim 36 wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 50% by weight.

38. The composition according to claim 37 wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 10% by weight.

39. The composition according to any one of claims 34 to 38, further comprising a therapeutic or prophylactic anti-acne agent.

40. The composition according to any one of claims 34 to 39, further comprising an inactive pharmacologically acceptable excipient, carrier or diluent.

41. A composition for the prophylaxis or treatment of a skin condition involving Propionibacterium acnes comprising a prophylactically or therapeutically effective amount of a mixture of an oenothein and three tannins characterised respectively by high performance chromatography column retention peaks eluting at about 11.42-11.49, 11,55-11.60, and 11.96-11.99 minutes upon gradient elution in a 50% aqueous methanol solution on a Supelcosil.TM. LC-18 column with a water/acetonitrile eluant having an initial composition of 98%water/2% actetonitrile and a composition of 65%water/35% acetonitrile at minutes, 2% water/98% acetronitrile at 20 minutes, and final composition of 98%water/2%acetonitrile at 30 minutes.

42. The composition according to claim 41, wherein the oenothein is oenothein B.

43. The composition according to claim 42, wherein oenothein B is extracted from Epilobium angustifolium (Canadian Willowherb) or a related Epilobium species or any other plant containing oenothein B.

44. The composition according to any one of claims 41 to 43, wherein the three tannins are extracted from Epilobium angustifolium (Canadian Willowherb), or related Epilobium species or any other plant containing the three tannins.

45. The composition according to any one of claims 40 to 43, wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 99% by weight.

46. The composition according to claim 44 wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 50% by weight.

47. The composition according to claim 45 wherein the prophylactically or therapeutically effective amount of an oenothein is about 0.6% to about 10% by weight.

48. The composition according to any one of claims 41 to 47 further comprising therapeutic and prophylactic anti-acne agent.

49. The composition according to any one of claims 40 to 46 further comprising an inactive pharmacologically acceptable excipient, carrier or diluent.

50. The use of a purified preparation of an oenothein to prepare a medicament having a therapeutically or prophylactically effective amount of the oenothein for the treatment of a skin condition involving Propionibacterium acnes.

51. The use according to claim 50, wherein the purified preparation of the oenothein has an oenothein concentration of at least 50% by weight.

52. The use according to claim 50 or 51, wherein the oenothein is oenothein A.

53. The use according to claim 50 or 51, wherein the oenothein is oenothein B.

54. The use according to any one of claims 50 to 53, wherein purified preparation is a topical preparation.

55. The use of a medicament having a therapeutically or prophylactically effective amount of an oenothein to treat a skin condition involving Propionibacterium acnes.