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This is a continuation-in-part application that claims priority to, and the benefits of, co-pending U.S. patent application Ser. No. 10/321,706, filed Dec. 17, 2002.
1. Field of the Invention
The present invention relates to topical compositions having an active ingredient naturally or synthetically derived from a plant or plant material. More particularly, the present invention relates to topical compositions that improve the appearance of skin, lips, hair and/or nails, especially by lightening the skin, lips, hair, and/or nails. Most particularly, the present invention relates to biologically active extracts for improving the aesthetic appearance of, especially by lightening, the skin, lips, hair, and/or nails.
2. Description of the Related Art
Consumers constantly seek to improve the appearance of their hair, skin, lips and nails. There is a need for products that effectively lighten and reduce pigmentation in the hair, skin, lips and nails. Common applications for such products include, for example, bleaching hyperpigmented hair, skin, lips and/or nails; reducing age spots; evening or optimizing skin discoloration; improving the appearance of dark circles under the eyes; treating melasma, cholasma, freckles, after-burn scars, and post-injury hyperpigmentation; bleaching hair on the scalp, legs, face, and other areas where bleaching and color reduction are desired; and bleaching nail stains.
Skin, hair, lip and nail pigmentation is determined by the level of melanin present in the epidermis, hair fiber and nail bed. Three different types of melanin are present in the epidermis: DHI-melanin, DHICA-melanin and pheomelanin. The different types of melanin vary in color or shade. DHI-melanin is the darkest, and is blackish in color. DHICA-melanin is brownish in color. Pheomelanin is the lightest, and is reddish in color.
Melanin is synthesized in specialized organelles called melanosomes within pigment-producing cells (melanocytes). Melanocytes respond to stimuli to regulate melanin synthesis.
Many substances have been applied to the skin to lighten the skin. Such substances include hydroquinone, kojic acid, licorice and/or its derivatives, ascorbic acid and/or its derivatives, arbutin, bearberry, Glycyrrhiza glabra and its derivatives, Chlorella vulgaris extract, perilla extract, and coconut fruit extract. Perilla extract is disclosed as a whitening agent in U.S. Pat. No. 5,980,904 and Japanese Publications Nos. 07025742, 07187989, 10265322, 2001163759, and 2001181173. Coconut fruit extract is disclosed as a whitening agent in Japanese Patent No. 2896815B2. An extract of the spongy mass of coconut tissue is employed in a tanning sunscreen composition in U.S. Pat. No. 5,756,099.
Active ingredients derived from plants and plant seeds have been employed in topical compositions for a myriad of medicinal, therapeutic and cosmetic purposes. Such active ingredients can be obtained from various parts of a plant such as seeds, needles, leaves, roots, bark, cones, stems, rhizomes, callus cells, protoplasts, organs and organ systems, and meristems. Such active ingredients are incorporated in such compositions in a variety of forms. Such forms include a pure or semi-pure component, a solid or liquid extract or derivative, or a solid plant matter. Plant matter may be incorporated in a variety of subforms such as whole, minced, ground or crushed.
Extracts of Azadirachta indica, the neem tree, as well as other plants in the family Meliaceae, are known to have insecticidal activity. Azadirachtin, a major active ingredient of many of these extracts, is a liminoid of the tetranortriterpenoid type useful in commercial insecticides. Tetranortriterpenoids have been shown to be a potent insect growth regulator and feeding deterrent. Methods for producing azadirachtin concentrates from neem seed materials are known in the art. U.S. Pat. No. 5,698,423 to Holowach-Keller et al. is directed to a method for producing azadiractin by cell culture of Azadiracta indica.
Extracts of Glycyrrhiza glabra linn. are derived from the herb, which grows perennially in subtropical and warm temperate regions. Glycyrrhiza glabra linn., commonly known as licorice, has been used in food sweetening. The root extract contains glycyrrhizic acid and glycyrrhetinic acid. The glycyrrhizic acid is known to have an anti-inflammatory effect. The extract of the licorice root and glycyrrhetinic acid have been shown to have desoxycorticosterone and ACTH-like effects. It has been used as a demulcent and mild expectorant. In vitro studies have shown the antiviral properties of both glycyrrhetinic acid and glycyrrhizin (See Badam, “In Vitro Studies on the Effect of Glycyrrhizin from Glycyrrhiza glabra linn. on Some RNA and DNA Viruses,” Ind. J. Pharma., 26, 194-199 (1994)). The extracts of Glycyrrhiza glabra linn. can be extracted by a method disclosed in U.S. Pat. No. 6,248,309 to Iyer, et al.
Extracts of Morinda citrifolia are derived from the Indian Mulberry plant. Morinda citrifolia has been used in compositions for reducing oxysterol buildup in the blood and normalizing cholesterol and blood pressure in mammals as set forth in U.S. Pat. No. 6,387,370 to Yegorova. A method of extracting and purifying an essential oil product of Morinda citrifolia is disclosed in U.S. Pat. No. 6,417,157 to Wadsworth et al.
Extracts of tomato glycolipid are derived from tomato fruit. Methods of extracting and synthesizing tomato glycolipids are disclosed in U.S. Pat. No. 4,745,186 to Mudd et al.
Extracts of Butea frondosa, also known as Butea monosperma, are derived from an East Indian deciduous tree. Butea frondosa has been used as an astringent and in treating diarrhea, dysentery, and pyrosis. Use of Butea frondosa for its ocular anti-inflammatory activity has recently been tested (See Mengi, “Evaluation of Ocular Anti-Inflammatory Activity of Butea frondosa,” Ind. J. Pharma. 27, 116-119 (1995)).
Extracts of Naringi crenulata, also known as Limonia crenulata, are derived from a small tree indigenous to East India.
Stenoloma chusana is a perennial herb found in southeast Asia. Extracts from this plant are known to have uses in treating colds, influenza, bronchitis, burns, cuts, and skin sores (See A Barefoot Doctor's Manual, Running Press, Philadelphia, Pa., p. 638).
Heretofore, these extracts have not been used as an active ingredient in a composition for the purpose of lightening skin, lips, hair or nails.
It would be desirable to have compositions that employ new biological extracts that provide an improved aesthetic appearance to the skin, lips, hair and/or nails, especially that provide effective levels of lightening, bleaching, hypopigmenting, whitening and/or depigmenting (hereinafter referred to individually and collectively as “lightening” or “lighten”). It would further be desirable to have compositions that are effective in lightening hair, skin, lips, and/or nails and require minimal concentrations of the biological material.
It is an object of the present invention to provide cosmetic compositions that improve the aesthetic appearance of skin, lips, hair and/or nails including remediating the effects of aging.
It is another object of the present invention to provide compositions for lightening of hair, skin, lips, and/or nails having an active plant extract, preferably an active biological plant extract.
It is yet another object of the present invention to provide such compositions for improving the appearance, especially by lightening, of skin, lips, hair and/or nails in which the compositions having an effective amount of an active plant extract.
It is still another object of the present invention to provide such compositions for improving the appearance, especially by lightening, of hair, skin, lips, and/or nails, in which such compositions are suitable for topical application to the hair, skin, lips, and/or nails.
It is still yet another object of the present invention to provide such compositions for improving the appearance of skin and lips by ameliorating inflammation of the skin and/or lips, including inflammation induced by or capable of inducement by external agents.
It is a further object of the present invention to provide compositions for lightening hair, skin, lips, and/or nails that is suitable for oral ingestion.
It is a still further object of the present invention to provide methods of lightening hair, skin, lips, and/or nails that include topically applying such compositions to hair, skin, lips and/or nails.
These and other objects and advantages of the present invention are provided by compositions for improving the appearance, especially by lightening, of hair, skin, lips, and/or nails or by reducing, preventing, or treating inflammation of the skin or lips, including inflammation occasioned by ailment, affliction or disease of the skin or lips, or inflammation induced or inducible by an external agent, in which such compositions have an effective amount of at least one of the following active extracts: Butea frondosa, Naringi crenulata, Stenoloma chusana, or any combinations thereof.
There is also provided compositions for improving the appearance, especially by lightening, of skin, lips, hair and/or nails or by reducing, preventing, or treating inflammation of the skin or lips, including inflammation occasioned by ailment, affliction or disease of the skin or lips, or induced or inducible by an external agent, in which such compositions have an effective amount of at least one of the active extracts, and an effective amount of at least one of the following additional extracts: Azadirachta indica, Glycyrrhiza glabra Linn., Morinda citrifolia, tomato glycolipid, or any combinations thereof.
The present invention also provides methods for improving the appearance, especially by lightening, of hair, skin, lips, and/or nails or by reducing, preventing, or treating inflammation of the skin or lips, including inflammation occasioned by ailment, affliction or disease of the skin or lips, or induced or inducible by an external agent, comprising topically applying any one of the compositions of the present invention. The present invention also provides methods for lightening hair, skin, lips, and/or nails by orally ingesting any one of the compositions of the present invention.
The present invention provides compositions for improving the appearance of hair, skin, lips, and/or nails, especially by lightening hair, skin, lips, and/or nails. The compositions are preferably topical compositions for application to the hair, skin, lips, and/or nails. However, the present compositions can be for oral ingestion. In all applications, the compositions result in a lightening of the hair, skin, lips and/or nails. The compositions of the present invention also provide an anti-inflammatory benefit when applied to skin or lips, so that the appearance of skin is improved.
The compositions have one or more of the following extracts, as an active biological plant extract or ingredient(s) or in an active amount: Butea frondosa, Naringi crenulata, Stenoloma chusana, or any combinations thereof. It has been unexpectedly found that these active extracts improve the aesthetic appearance of hair, skin, lips and/or nails and, in particular, reduce pigmentation and lighten hair, skin, lips, and/or nails. More particularly, these active extracts have been unexpectedly shown to decrease melanin production and decrease hypermelanocytic states.
It has now been also found that the addition of at least one of these active extracts, namely Butea frondosa, Naringi crenulata, Stenoloma chusana, or any combinations thereof, to at least one of the following other or additional extracts, namely Azadirachta indica, Glycyrrhiza glabra linn., Morinda citrifolia, tomato glycolipid, or any combinations thereof, can reduce melanin pigmentation of hair, skin, lips and/or nails.
Lightening of hair, skin, lips and/or nails, as used in the present invention, means one or more of the following benefits is achieved. These benefits include bleaching hyperpigmented hair, skin, lips, and/or nails; reducing age spots; evening or optimizing skin discoloration; improving the appearance of dark circles under the eyes; treating melasma, cholasma, freckles, after-burn scars, and post-injury hyperpigmentation; bleaching hair on the scalp, legs, face, and other areas where bleaching and color reduction are desired; and bleaching nail stains.
Most generally, the present invention also provides topical compositions containing an effective amount of one or more of the active ingredients of the present invention for application to the skin to improve the aesthetic appearance of skin. These benefits are manifest in any of the following: reduction in pore size; improvement in skin tone, radiance, clarity and/or tautness; promotion of anti-oxidant activity; improvement in skin firmness, plumpness, suppleness, and/or softness; improvement in procollagen and/or collagen production; improvement in skin texture and/or promotion of retexturization; improvement in skin barrier repair and/or function; improvement in appearance of skin contours; restoration of skin luster and/or brightness; replenishment of essential nutrients and/or constituents in the skin decreased by aging and/or menopause; improvement in communication among skin cells; increase in cell proliferation and/or multiplication; increase in skin cell metabolism decreased by aging and/or menopause; improvement in skin moisturization; promotion and/or acceleration of cell turnover; enhancement of skin thickness; reducing skin sensitivity; increase in skin elasticity and/or resiliency; and enhancement of exfoliation, with or without the use of alpha or beta hydroxy acids, keto acids or other exfoliants.
Collaterally, the present invention also provides for topical compositions for application to the skin that provide an anti-aging benefit. The compositions have an effective amount of one or more active ingredients of the present invention, which, when applied to human skin, prevent, treat and/or ameliorate the various signs of aging at the area or portion of skin to which they are applied. In particular, the present invention provides compositions and methods for treating skin to prevent, inhibit, reduce and/or ameliorate the signs of dermatological aging due to, for example, chronological aging, hormonal aging, and/or photoaging. Such signs of aging include, but are not limited to skin fragility; loss of collagen and/or elastin; estrogen imbalance in skin; skin atrophy; appearance and/or depth of lines and/or wrinkles, including fine lines; skin discoloration, including dark eye circles; skin sagging; skin fatigue and/or stress, e.g., skin breakout due to environmental stress, such as pollution and/or temperature changes; skin dryness; skin flakiness; cellular aging; loss of skin tone, elasticity and/or luster; loss of skin firmness; poor skin texture; loss of skin elasticity and/or resiliency; and thin skin.
In its broadest aspects, the present invention is not limited by any particular characterization of the physiological and/or chemical effects of the active extract or agents. However, the active extract used in the present compositions and methods are believed to reduce melanin in hyperpigmented areas by decreasing dark pigment formation in melanocytes and shifting the melanin synthesis path towards light melanin formation by decreasing melanocyte pH, and/or direct chelation of metals involved in melanin synthesis or staining, for example, copper.
In another aspect of the present invention, the topically applied compositions of the present invention inhibit, mitigate, ameliorate, and prevent skin and lip irritation or inflammation occasioned by ailment, illness or disease, or induced by external factors such as environmental agents, chemical agents, medicinal agents and cosmetic agents. Skin irritation thus may result from exposure to wind, heat or cold, air pollutants, and cigarette smoke. Cosmetic and pharmaceutical products may have ingredients or combinations of ingredients that produce visible skin irritation as a side effect. Susceptibility to skin irritation may vary from individual to individual, and frequently limits the use of certain products or the use of concentrations of active ingredients that might produce more advantageous results at higher levels but for the production of skin irritation as a side-effect. Skin irritation symptoms or conditions include, but are not limited to, erythema, psoriasis, edema, hyper-pigmentation, hypo-pigmentation, acne, warts, wheeling, blotchiness, uneven skin tone, scaling, flaking, itching or pruritus, tightness, burning, prickling, stinging, tingling, numbing, wind irritation, temperature irritation, smoke irritation, chemical irritation, or any combinations thereof. Accordingly, the method of the present invention provides that an effective amount from about 0.001 wt % to about 20 wt % by weight of the composition, is effective in the treatment of one or more of the following: reducing or preventing loss of collagen; improving skin firmness/plumpness; improving skin texture; decreasing/preventing lines and wrinkles; improving skin tone; enhancing skin thickness; decreasing pore size; reducing skin discoloration; reducing acne; reducing psoriasis; reducing skin sensitivity; and reducing warts.
Cosmetic, dermatological and pharmaceutical products commonly have an active agent or agents that produce skin irritation. Examples of active agents having skin irritation as a side effect include, but are not limited to, hydroxylated acids and their derivatives, α-hydroxy acids (i.e., lactic, glycolic, citric, malic, tartaric, mandelic, gluconic, methyl lactic, phenyl lactic, atrolactic, glyceric, benzilic, z-hydroxyheptanoic, z-hydroxyoctanoic and any combinations thereof), β-hydroxy acids (i.e., salicylic, 5-n-octanoylsalicylic and other derivatives of salicylic), retinoids (retinoic acid and its derivatives; retinol and its esters); anthralins (i.e., dioxyanthranol), anthranoids, peroxides (i.e., benzoyl peroxide), minoxidil, lithium salts, antimetabolites, vitamin D and its derivatives, hair dyes or colorants (i.e., para-phenylenediamine and its derivatives; aminophenols), alcoholic perfuming solutions (i.e., perfumes; toilet waters; aftershaves; deodorants), antiperspirant agents (i.e., some aluminum salts), depilatory or hair permanent active agents (i.e., thiols), depigmentating agents (i.e., hydroquinone), and some insecticide active agents. If topical products had anti-irritant protection, it would be possible to increase the amount of the normal irritant active agent (i.e., AHA or BHA) in the product without producing unpleasant skin irritation or irritation side effects. The use of the present compositions makes it possible to improve the efficacy of cosmetic, dermatological or pharmaceutical products by increasing the concentration or amount of cosmetic, dermatological, or pharmaceutical active agent as compared to the amount or concentration of such agent normally used.
In a preferred embodiment of the present invention, the composition, which improves the appearance of and, in particular, lightens hair, skin, lips, and/or nails, has an effective amount of at least one of the following active extracts: Butea frondosa, Naringi crenulata, Stenoloma chusana, or any combinations thereof. An effective amount means that the one or more active extracts are present at about 0.001 percentage by weight (wt %) to about 20 wt % based on the total weight of the composition. The one or more active extracts are present preferably at about 0.05 wt % to about 10 wt %, and more preferably at about 0.5 wt % to about 5 wt %, based on the total weight of the composition. Most preferably, the one or more active extracts are present in an amount about 1 wt % or less based on the total weight of the composition.
As stated above, in another preferred embodiment of the present invention, the composition for lightening hair, skin, lips, and/or nails has an effective amount of at least one of the active extracts, and one or more of the following other or additional extracts: Azadirachta indica, Glycyrrhiza glabra linn., Morinda citrifolia, tomato glycolipid, or any combinations thereof, to synergistically enhance the whitening activity of the composition.
The one or more of the additional extracts are present in an amount about 0.05 wt % to about 10 wt % based on the total weight of the composition. Preferably, the one or more additional extracts in an amount about 0.5 wt % to about 5 wt % based on the total weight of the composition. Most preferably, the one or more additional extracts are present in an amount equal to or less than 1 wt % based on the total weight of the composition.
When combined with one or more of the additional extracts, the active extracts are present in the present compositions in an amount about 0.001 wt % to about 10 wt %, and the one or more additional extracts in an amount about 0.05 wt % to about 10 wt %, based on the total weight of the composition. Preferably, the active extracts are present in an amount about 0.05 wt % to about 10 wt %, and the one or more additional extracts in an amount about 0.5 wt % to about 5 wt % based on the total weight of the composition. Most preferably, the one or more active extracts are present in an amount equal to or less than about 1 wt %, and the one or more additional extracts are present in an amount equal to or less than 1 wt %, based on the total weight of the composition.
The compositions of the present invention can be used with a carrier or vehicle. The vehicle can be altered to be appropriate for the specific use of the composition without altering the beneficial lightening effects achieved by the use of the one or more active extracts set forth above. The vehicles that can be used in compositions of the present invention include, but are not limited to, water; vegetable oils; esters such as octyl palmitate, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether and dimethyl isosorbide; alcohols such as ethanol and isopropanol; fatty alcohols such as cetyl alcohol, stearyl alcohol and behenyl alcohol; isoparaffins such as isooctane, isododecane and isohexadecane; silicone oils such as dimethicones and polysiloxanes; hydrocarbon oils such as mineral oil, petrolatum, isoeicosane and polyisobutene; polyols such as propylene glycol, glycerin, butylene glycol, pentylene glycol and hexylene glycol; or any combinations thereof.
Preferably, the vehicle is present in an amount about 50 wt % to about 99.8 wt % based on the total weight of the composition. More preferably, the vehicle is present in an amount about 80 wt % to about 99.5 wt % based on the total weight of the composition.
The present compositions may also include one or more of the following ingredients: anesthetic, anti-allergenic, antifungal, antimicrobial, anti-inflammatory agent, antioxidant, antiseptic, chelating agent, colorant, depigmenting agent, emollient, emulsifier, exfollient, film former, fragrance, humectant, insect repellent, lubricant, moisturizer, pharmaceutical agent, photostabilizing agent, preservative, skin protectant, skin penetration enhancer, sunscreen, stabilizer, surfactant, thickener, viscosity modifier, vitamin, or any combinations thereof. Preferably, these ingredients are present in an amount about 0.001 wt % to about 10 wt % based on the total weight of the composition.
The present compositions may also include skin whiteners. Some examples of such suitable skin whiteners include, but are not limited to, one or more of the following: ascorbyl glucoside, vitamin C, retinol and/or its derivatives, arbutin, bearberry extract, rumex crispus extract, milk proteins including hydrolyzed milk proteins, N,N,S-tris(carboxymethyl)cysteamine, oleanolic acids, perilla oils, placenta extract, saxifragia sarmentosa, perilla extract, juniperic acid, TDPA, ligusticum chiangxiong hort., asmunda japonica thunb., stellaria medica (L.) cyr., sedum sarmentosum bunge, ligusticum lucidum Ait., ilex purpurea hassk, emblica, apigenin, ascorbyl palmitol, carruba polyphenols, hesperitin, hydroquinone, inabata polyphenol, isoliquirtigenin, kaempherol-7-neohesperidose, L-mimosine, luteolin, oil-soluble licorice extract P-T(40), oxa acid, phenyl isothiocyanate, cococin, silymarin, T4CA, teterahydro curcumin, unitrienol, ursolic-oleanolic acid, UVA/URSI, or any combinations thereof.
The compositions of the present invention can be formulated in any suitable product form. Such product forms include, but are not limited to, aerosol spray, cream, dispersion, emulsion, foam, gel, liquid, lotion, mousse, ointment, patch, pomade, powder, pump spray, solid, solution, stick, and towelette.
The present compositions provide for products, especially cosmetic products that improve lightening of skin, nail, lips, and/or hair. Also, the present compositions can be formulated to deliver a consistent level of an active ingredient, or blend of ingredients, so that a desired cosmetic effect is achieved.
The following are examples of the present invention.
Melanosome Uptake Assay (Stenolama Chusana and Naringi Crenulata) was done as follows. Confluent cultures of B16 melanocytes produce moderate levels of melanosomes. However, to induce elevated melanosome production in this cell line, semi-confluent (60%) cultures of B16 cells were treated for approximately thirty-six (36) hours with normal growth medium supplemented with 10 mM ammonium chloride (final conc.). The medium was then aspirated and the hypermelanotic cells were washed (2×2 ml) with distilled water to provide a hypotonic stress to the cells. An aliquot (2 ml) of a hypotonic lysis solution (0.02% NP-40 in water) was added to each plate and the plates were incubated for approximately five (5) minutes at room temperature. Following verification of cell lysis using light microscopy, the cellular material from three (3) culture plates were pooled in a 15 ml conical tube and centrifuged (200×g) for 5 minutes to remove cellular debris. The resulting supernatant containing melanosomes was transferred to a clean 15 ml conical tube and centrifuged (850×g) for twenty (20) minutes. The resulting pellet containing the isolated melanosomes were resuspended in 1 ml of Phosphate Buffered Saline (PBS) and stored at 4° C. until used.
The treatment of keratinocytes with melanosomes was measured as follows. The normal human epidermal keratinocytes (NHEKs) (available from Clonetics, Inc.) were plated in the wells of 24-well plates at a density of 200,000 cells/well. Approximately twenty-four (24) hours later, the growth medium was replaced with 1 ml of the appropriate growth medium (i.e., DMEM/KGM-2) containing the melanosome preparation with or without additional treatment conditions. The cells were treated with different concentrations of perilla leaf extract (powder form or aqueous form). The cells were then returned to the incubator for approximately one and one-half (1.5) hours. For these studies, each well of keratinocytes was treated with the amount of melanosomes isolated from a single plate of B16 cells.
In some experiments, the 24-well plates of treated keratinocytes were centrifuged for 15 minutes at 1,000 rpm to facilitate the deposition of the melanosomes onto the surface membranes of the keratinocytes. The plates were then returned to the incubator for 1.25 hours.
For the analysis of melanosome uptake, the cells in each well of keratinocytes were rinsed (3×1 ml) with PBS, removed from the plate using trypsin/EDTA, and washed with PBS. To analyze the uptake of melanosomes by the keratinocytes, the internalized melanin was extracted from the cells according to a modified method of Bessou-Touya, S., et al. (Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection. J. Invest. Dermatol., 111:1103-1108, 1998) and quantified spectrophoto-metrically by determining the melanin-specific absorbance at 405 nm.
The melanocytes synthesize melanin and deposits onto melanosomes. Visual manifestation of skin color is due to presence of melanin/melanosomes in keratinocytes. Melanosomes are taken up by keratinocytes and the rate of uptake, retention and processing of melanosomes in the keratinocytes is a key determinant of skin color. The internalized melanin value reflects the amount of melanin/melanosome uptake and retention by the keratinocytes. Thus, the lower internalized melanin values, particularly internalized melanin values that are less than the control with melanin, indicate that melanin uptake by the keratinocytes has been inhibited.
The results are as follows. At 0.5% volume/volume, Stenolama Chusana showed a 44% decrease in melanosome uptake as compared to the positive control. At 0.5% volume/volume, Naringi Crenulata showed a 39% decrease in melanosome uptake as compared to the positive control.
For B16 assay (Naringi Crenulata), the actives were tested in monolayer cell culture of B16 mouse melanoma cells. These cells constitutively produce melanin and are a model system for monitoring the inhibition of melanin synthesis. The cells were seeded into 96-well plates at 5×103 cells/well and culture attached for twenty-four (24) hours. The media were then replaced with fresh media containing plant extracts. Each active was applied to six (6) wells of B16 cells on 96-well plates to allow statistical analysis. The cells were dosed with medium alone as the negative control, or the test article for seven (7) days. The plates were read at 540 nM to detect melanin formation. An increase in absorbance at 540 nM reflects a higher melanin content in the well.
At 0.05% weight/volume, Naringi Crenulata showed a 61% decrease in pigmentation as compared to the positive control
For 14C-Dopa incorporation (Butea Frondosa), melanogenic activity was measured by measuring the radioactive melanin formed as 14C DOPA is converted to the acid insoluble melanin biopolymer in B16F10 melanoma cells. Cells were seeded into 24-well plates at a density of 2×104 cells per well and cultured attached for forty-eight (48) hours. The media were then replaced with fresh media containing plant extracts and 0.2 μCi of 14C DOPA. The cells were further incubated for 24 hours. After incubation, media were discarded and the cells were rinsed with PBS, lysed by adding 0.125 ml of 1N NaOH and incubated at 37° C. for 30 minutes, and then neutralized with 0.025 ml of 5N HCL. The resulting cell lysates were transferred into liquid scintillation vials and mixed with scintillation cocktail, and the radioactivity was determined by scintillation counter. A portion of the cell lysates was kept and the protein content was determined by Lowry method. The results were normalized to the amount of protein determined for each assay.
At 0.005% weight/volume, Butea frondosa showed a 22% decrease in 14C DOPA conversion as compared to the positive control.
Various natural plant extracts of the present invention were evaluated for inhibition of the TNF-alpha protein in an in vitro studies as described below.
TNF-alpha Inhibition Protocol
To test the efficacy of Stenolama chusana Ching., Naringi Crenulata, and Butea frondosa Roxb. for the inhibition of TNF-alpha production, an enzyme linked immunoassay (ELISA) commercially available from R&D Systems was employed.
This assay employed the quantitative sandwich enzyme immunoassay technique in which a monoclonal antibody specific for TNF-alpha has been pre-coated onto a microtiter plate. Culture supernatants from cells exposed to active materials were pipetted into separate wells. TNF-alpha in the supernatant was bound to the plate via the immobilized antibody. After several washes to remove unbound antibody, an enzyme-linked polyclonal antibody specific for TNF-alpha was added to each well. Following a wash to remove unbound antibody-enzyme reagent, a substrate solution was added to the wells. Color develops in proportion to the amount of TNF-alpha bound in the initial step. The results of the tests are provided in Table I.
|Results of In Vitro Tests|
|Stenolama chusana Ching.||+++|
|Butea frondosa Roxb.||+++|
+, ++, +++, ++++ Significant inhibition (degree of inhibition quantified by number of plus signs)
(0) No change
These in vitro studies show that the compositions of the present invention containing an extract of: Stenolama chusana Ching., Naringi Crenulata, and Butea frondosa Roxb. provide benefits to the skin by inhibiting TNF-alpha protein production, whereby the signs of subjective discomfort and/or irritation caused by cosmetic, pharmaceutical or dermatological products would be reduced, thus providing an aesthetic improvement in skin appearance.
Stenolama chusana ching. and Butea fondosa were evaluated in various biopsy studies in which the natural plant extract as set forth below was incorporated into a cosmetically suitable vehicle and applied to the volar forearm of a participant in the study, at a dose of 2 mg/cm2. The forearm area to which the extract preparation was applied was then covered with a semi-occlusive patch. This procedure was repeated for 3 weeks, 5 days per week. At the end of the treatment the sites were anesthetized with lidocaine and 2 mm punch biopsies were taken from the treated and one untreated control site. Biopsies were fixed in formalin, embedded in paraffin, sectioned, and stained for relevant endpoints.
For Stenolama chusana ching., 6 of 9 panelists showed increase in keratinocyte proliferation (visualized by the antibody to KI67), and 5 of 9 panelists showed increase in viable epidermal thickness.
For Butea frondosa provided, 5 of 8 panelists showed increase in keratinocyte proliferation (visualized by the antibody to KI67), and 5 of 8 panelists showed increase in viable epidermal thickness.
It should be understood that the foregoing description is only illustrative of the present invention. Various alternatives and modifications can be devised by those skilled in the art without departing from the invention. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended claims.