Patent Application
20080025930
Except in operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts or ratios of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word “about.” All amounts are by weight of the final composition, unless otherwise specified.
Compositions herein described are particularly useful in methods of treating signs of aging. As used herein, “treating the signs of aging” includes preventing, reducing, forestalling, reversing or treating the signs of aging mentioned above, whether the cause be chronological or pre-mature aging.
As used herein, “skin beneficial” means that the extract comprises an amount of certain actives that are effective at inhibiting the activity of matrix metalloproteinases-1, 2 and 9, and/or effective at scavenging peroxynitrite.
The present invention is predicated on the observation that extracts of the leaves of Menyanthes have a surprising ability to protect skin cells against the damaging effects of UV radiation. Specifically, it has been surprisingly discovered that extracts of Menyanthes leaves effectively inhibit specific matrix metalloproteinases implicated in UV damage, while also scavenging reactive oxygen species (ROS). The ROSs in question are related to proMMP activation, but are also known to degrade the skin via oxidative stress, thus posing a double threat to the skin. Specifically, while not wishing to be bound by any one theory, it is believed that such plant extracts protect against UV-induced skin damage and related oxidative stresses, by inhibiting and/or reducing MMPs-1, 2 and 9 that degrade the dermal collagen, while also scavenging peroxynitrite. As such, the compositions of the present invention would provide a double benefit in that the compositions reduce MMPs as well as scavenge ROSs.
As experiments show (see Examples 3) the primary active or effective components, capable of inhibiting MMP-1, 2 and 9, are specific phenolic acids, flavonoids and coumarins extracted from the leaves of Menyanthes trifoliata. Furthermore, it is shown herein, that the specific, active phenolic acids present in the Menyanthes trifoliata leaf extracts are ferulic acid and protocatechuic acid. Specific, active flavonoids are quercetin, iso-quercetrin and rutin. Specific, active coumarins are scoparone and scopoletin.
While components of Menyanthes have been reported as having various types of biological activity, it was unexpected that Menyanthes trifoliata leaf extracts would exhibit specific MMP-1, 2 and 9 inhibition activity and that the specifically named phenolic acids, flavonoids and coumarins would be primarily responsible for such. In addition, although the specific phenolic acids, flavonoids and coumarins are herein shown to be the principle active components in achieving inhibition of MMP-1, 2 and 9, additional components, although not necessarily very effective on their own, may be present in the plant extracts that can have some contributory activity.
In the preferred embodiment, an extract of Menyanthes trifoliata L. is used. It is expected that other species of Menyanthes may also prove useful, including, cristata Roxb., hydrophylla Lour., indica, meridionalis Willd. ex Griseb, nymphoides L., ovata L. f, pumila Douglas ex Griseb., punctata Muhl. ex Griseb and trachysperma Michx and combinations thereof. In the preferred embodiment, Menyanthes trifoliate L. is used, although other subspecies may also prove useful, including, but not limited to trifoliata fo. Brevistyla Aver., trifoliata var. minor Michx. Ex Raf., trifoliata subsp. Trifoliata, trifoliate var. trifoliata and trifoliata subsp. Verna.
“Menyanthes trifoliata extract” is a generic term describing a number of different chemical compositions that may contain several different active components. Numerous extracts are commercially available, and any one of those may prove useful in the present invention. However, particularly preferred for use is a Menyanthes trifoliata L. extract available from Monteloeder in Spain. It will be understood that the term “Menyanthes extract” as used herein shall encompass not only a Menyanthes extract per se, but also a composition to which one or more of the active components such as noted herein, are added. Such added active components may be from synthetic or natural sources, either from Menyanthes or from material other than Menyanthes, in amounts equivalent to those described in the use of the Menyanthes extract.
Menyanthes extracts containing the specific active phenolic acids, flavonoids and coumarins, are most easily obtained by contacting the plant part with a suitable solvent or solvent(s), according to methods known in the art. The choice of the solvent should be made based on the properties of the active ingredient that is to be extracted. Ultimately, the extract may be isolated from the solvent. Particularly preferred solvents are alcoholic, ethyl acetate and dichloromethane. As the examples show, these solvents produce extracts of Menyanthes trifoliata that possess the specific active components needed to inhibit MMP-1, 2 and 9 and scavenge peroxynitrite. The concentration of solvent may be adjusted by a person skilled in the art and the extraction may be repeated on the same sample to increase the yield. The alcoholic, ethyl acetate or dichloromethane extracts will contain elements other than the specific active components. Nevertheless, the extracts may be used without further refinement or, alternatively, the specific active components may be isolated from the extract.
Based on total weight of a composition according to the present invention, the composition will comprise from 0.001 to 15 wt % of the active components, whether they are added in extract or isolated form. Where cost or other factors dictate, preferable concentrations range from 0.01 to 10 wt %, or most preferably from 0. 1 to 5 wt % of the active components, whether they are added in extract or isolated form. To achieve broad spectrum efficacy, it is preferable that compositions according to the present invention comprise active components from at least two of phenolic acids, flavonoids and coumarins. Most preferably, compositions according to the present invention comprise active components from all three of phenolic acids, flavonoids and coumarins. The preferred concentration of specific phenolic acids is 0.001 to 5.00 wt-%. The preferred concentration of specific flavonoids is 0.001 to 5.00 wt-%. The preferred concentration of specific coumarins is 0.001 to 5.00 wt-%.
When the active components are added in extract form, the concentration of Menyanthes trifoliata extract in the composition depends on the concentration of the actives in the extract. Typically, the alcoholic extract, ethyl acetate extract, dichloromethane extract or combinations thereof may be used in an amount from 0.01 to 20% of the composition to provide a skin beneficial concentration of active components. Nevertheless, larger concentrations are not outside the scope of this invention.
In an alternate embodiment, the present invention includes a sunscreen. Suitable sunscreens include water soluble sunscreens (such as Eusolex 232); oil soluble sunscreens (such as octyl methoxycinnamate); inorganic sunscreens (such as titanium dioxide, zinc oxide) and organic sunscreens (such as camphor derivatives, cinnamates, salicylates, benzophenones, triazines, PABA derivatives, diphenylacrylate derivatives, and dibenzoylmethane derivatives.) The amount will vary depending on the formulation and the performance desired. The sunscreen may be used in an amount from 0.1% to 50% by weight of the composition. Preferably, the sunscreen is used in an amount from 1% to 40% and most preferably, an amount from 5% to 30%.
The composition further comprises a cosmetically acceptable vehicle that is suitable for topical application to skin, hair and/or nails. Cosmetically acceptable vehicles are well known in the art and are selected based on the end use of the application. For example, vehicles of the present invention include, but are not limited to, those suitable for application to the skin. Such vehicles are well known to those of ordinary skill in the art, and can include one or more compatible liquid or solid filler diluents or vehicles which are suitable for application to the skin. The exact amount of vehicle will depend upon the level of any other optional ingredients that one of ordinary skill in the art would classify as distinct from the vehicle (e.g., other active components). In compositions of the present invention, the vehicle may comprise from about 75 to about 99.99 wt % of the composition.
The vehicle and the compositions herein, may be formulated in a number of ways, including but not limited to emulsions. For example, suitable emulsions include oil-in-water, water-in-oil, water-in-oil-in-water, oil-in-water-in-oil, and oil-in-water-in-silicone emulsions. Preferred compositions comprise an oil-in-water emulsion.
The compositions of the present invention can be formulated into a wide variety of product types, including shampoos, creams, waxes, pastes, lotions, milks, mousses, gels, oils, tonics and sprays. Preferred compositions are formulated into lotions, creams, gels, shampoos and sprays. These product forms may be used for a number of applications, including but not limited to, hand and body lotions, cold creams, facial moisturizers, anti-acne preparations, topical analgesics, color cosmetics including foundations, eyeshadows, lipsticks and the like. Any additional components required to formulate such products vary with product type and can be routinely chosen by one skilled in the art.
The formulation may also comprise components that are chosen depending on the carrier and/or the intended use of the formulation. Additional components include, but are not limited to antioxidants, chelating agents, emulsion stabilizers, preservatives, fragrances, flavoring agents, humectants, waterproofing agents, water soluble film-formers, oil-soluble film formers, moisturizing agents, such as cholesterol, cationic polymers, anionic polymers, vitamins, propellants and the like.
The compositions may encompass one or more additional active components, to render either a cosmetic or pharmaceutical composition. Examples of useful actives include, but are not limited to, those that improve or eradicate age spots, keratoses and wrinkles; analgesics, anesthetics, anti-acne agents, antibacterials, antiyeast agents, antifungal agents, antiviral agents, antidandruff agents, antidermatitis agents, antipruritic agents, antiemetics, antihyperkeratolytic agents, anti-dry skin agents, antiperspirants, antipsoriatic agents, antiseborrheic agents, hair conditioners and hair treatment agents, antiaging agents, antiwrinkle agents, antiasthmatic agents and bronchodilators, sunscreen agents, antihistamine agents, depigmenting agents, wound-healing agents, vitamins, corticosteroids, tanning agents or hormones.
Particularly preferred embodiments of the present formulations are skin care lotions or creams used as an anti-aging product. To that end, the present formulations are combined with agents that are moisturizers, emollients or humectants. Examples of useful combinations are oils, fats, waxes, esters, fatty acid alcohols, fatty acid ethoxylates, glycols, sugars, hyaluronic acid and hyaluronates, dimethicone, cyclomethicone, and the like. Further examples can be found in the International Cosmetic Ingredient Dictionary, CTFA, Eighth Edition, 2000.
The methods taught herein, comprise administering or topically applying a skin beneficial amount of the composition of the present invention. The amount of the composition applied and the frequency of topical application to the skin may vary widely, depending upon the individual's needs and the level of regulation desired. A preferred method of cosmetically or pharmaceutically treating signs of aging in the skin, is via chronic topical application of a skin beneficial amount of the novel composition. It is well within the purview of the skilled artisan, such as a dermatologist or other health care provider, to regulate pharmaceutical dosages according to patient needs. The method of the present invention is suitable for daily use.
It is suggested as an example that topical application range from about once per week to about 2 or 3 times daily, preferably from about 5 times a week to about 3 times daily, most preferably about once or twice per day. The following examples further illustrate the invention, but the invention is not limited thereto.
The following extraction scheme was useful in researching the properties of Menyanthes trifoliata leaf extracts. In the first step, an alcoholic solvent was applied to the dried leaves. Thereafter, the polarity of the solvents increases from the least polar, hexane to dichloromethane to ethyl acetate to the most polar, butanol. Ultimately, the components responsible for MMP-1, 2 and 9 inhibition reside in the alcoholic extract. However, additional extractions, as described below, were performed to further isolate the effective components. Some of those extracts (specifically, ethyl acetate and dichloromethane) were found to have suitable levels of the effective components. Thus, a number of solvents may be used to obtain Menyanthes trifoliata leaf extracts that inhibit MMPs-1, 2 and 9. Any of these extracts (alcoholic, ethyl acetate or dichloromethane) are suitable for compositions and methods of the present invention.
Several extracts and sub-fractions from the leaves of Menyanthes trifoliata were prepared by liquid-liquid partitioning and fractionation on a Sephadex LH20 gel filtration column (see example 1) and evaluated for specific anti-MMP activity. In vitro specific inhibition of MMP-2 and MMP-9 activity was estimated with assay kits from Biomol®. Recombinant human MMP-1 enzyme may be obtained from any commercially available source. In table 1, MMP inhibition is expressed as IC50 values, that is, the concentration of extract that results in a 50% reduction of the measured signal. Therefore, a lower value indicates a stronger MMP inhibition.
Menyanthes trifoliata extract
As seen from Table 1 above, the highest level of activity is found in the ethyl acetate and dichloromethane extracts. These two extracts are significantly more effective at MMP-1, 2, 9 inhibition. Because of its effectiveness at inhibiting all three MMPs, the ethyl acetate extract may be preferred, but dichloromethane extract may be used effectively and is within the scope of this invention. Of course, the alcoholic extract may also be used.
Sub-fractionation of the two crude extracts (ethyl acetate and dichloromethane) by separation on a Sephadex column results in extracts with even higher anti-metalloproteinase activity. The results are shown in Table 2.
Menyanthes trifoliata
In order to determine the components responsible for the inhibition activity of the ethyl acetate and dichloromethane extracts, an HPLC compositional analysis of the extracts was performed. Table 3 shows amount of a component as a percent of the subfraction analyzed, on a weight basis. As can be seen in Table 3, phenolic acids, flavonoids and coumarins are the primary active components in ethyl acetate and dichloromethane extracts of Menyanthes trifoliata. Comparing tables 2 and 3, it is concluded that fractions with no or relatively low concentrations of phenolic acids, flavonoids and coumarins (fractions 2.1, 2.2 and 3.1), exhibit no or relatively poor MMP-1, 2, 9 inhibition activity. Conversely, those fractions with at least two of phenolic acids, flavonoids and coumarins exhibit significant inhibition activity.
To further understand which agents may be contributing to the MMP-1, 2, 9 inhibition activity, standards of the different phenol acids, flavonoids and coumarins, identified in Menyanthes trifoliata extracts, were tested for their in vitro inhibition of MMPs-1, 2 and 9. In vitro specific inhibition of MMP-2 and MMP-9 activity is estimated with assay kits from Biomol®. Recombinant human MMP-1 enzyme is obtained from any commercially available source. Results are summarized in Table 4.
As seen in Table 4, ferulic acid, but not p-hydroxy-benzoic acid, shows strong in vitro MMP-1, 2 and 9 inhibition. Flavonoids (quercetin, iso-quercetrin and rutin) and coumarins (scopoletin and scoparone) all show strong anti-metalloproteinase activity.
Several extracts and sub-fractions from the leaves of Menyanthes trifoliata were prepared by liquid-liquid partitioning and fractionation on a Sephadex column (see Example 1) and evaluated for peroxynitrite scavenging. Assay was performed with the ABEL® peroxynitrite antioxidant test kit with Pholasin®. Results are summarized in Table 5.
Menyanthes trifoliata extract
Standards of different phenolic acids and flavonoids, identified in Menyanthes trifoliata extracts, were tested for their in vitro peroxynitrite scavenging activity. Assay was performed with the ABEL® peroxynitrite antioxidant test kit with Pholasin®. Results are summarized in Table 6.
Ferulic acid and protocatechuic acid but not p-hydroxy-benzoic acid are strong scavengers of peroxynitrite. Flavonoids (quercetin, iso-quercetrin and rutin) all show strong peroxynitrite scavenging activity.
The data show that the ethyl acetate extracts are more potent inhibitors of MMPs than the dichloromethane extracts, although the dichloromethane extracts are quite useful for the purpose. On the other hand, the two extracts are similar in their ability to scavenge peroxynitrite. Either extract or a combination may be used effectively to practice the present invention. Of course, the alcoholic extract may also be used.
It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the clear teachings of the disclosure. Accordingly, reference should be made to the following appended claims in determining the full scope of the invention.
