HK1238146A - Compositions and methods for stimulating magp-1 to improve the appearance of skin - Google Patents

Description

Compositions and methods for stimulating MAGP-1 to improve skin appearance

The present application is a divisional application of chinese patent application No. 201410457703.7 (original application No. 201180031240.2, PCT/US2011/040157), filed on 6/13/2011 entitled "composition and method for stimulating MAGP-1 to improve skin appearance".

RELATED APPLICATIONS

This international application claims priority to U.S. provisional patent application serial No. US61/360,083, filed on 30/6/2010, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to methods of improving the aesthetic appearance of human skin and/or improving the appearance of aged and/or photodamaged skin by applying to the skin a compound that stimulates the expression of the extracellular matrix protein MAGP-1 and provides a benefit to the skin.

Background

There is an increasing need in the cosmetics industry to develop products that can be topically applied to the skin to improve the condition and appearance of the skin. Consumers are concerned with reducing or delaying the dermatological signs of natural or hormonal aging skin and skin aging due to environmental stress, such as fine lines, wrinkles, sagging skin and other conditions caused by the progressive loss of cell growth, proliferation and functionality in the epidermal and dermal layers. During the aging process, the appearance of skin, i.e., the color and appearance of skin, deteriorates slowly from aging and/or exposure to environmental stresses such as sunlight.

Thus, there is still a general need in the cosmetics industry for products that retard or counteract the effects of aging of the skin, and more specifically for products that produce such effects without unwanted side effects. In particular, there remains a need for topically applied cosmetic compositions using natural plant materials as active ingredients that have anti-aging and skin texture benefits.

Active components or ingredients derived or extracted from plants and plant seeds are commonly used for various pharmaceutical, therapeutic and cosmetic purposes. Such active ingredients may be obtained from the whole plant or from different parts of the plant, such as seeds, needles, leaves, flowers, roots, bark, cones, stems, rhizomes, callus cells, protoplasts and meristems. The active ingredient or ingredients are incorporated into the compositions in various forms. Such forms include pure or semi-pure ingredients, solid or liquid extracts or derivatives or solid natural plant materials. The plant material may be incorporated into the composition in various sub-forms, for example in whole, chopped, ground or crushed form, otherwise in an altered physical form for incorporation into the composition.

MAGP-1 (microfibrillar associated glycoprotein 1) is a small, glycine-rich acidic protein in the extracellular matrix (ECM). MAGP-1 is a 30-33kDa member of the protein family of microfibrillar-associated glycoproteins. It was found to be a key component of the microfibers and elastic fibers in the skin. MAGP-1 forms complexes with other microfibrils, such as fibrillin. Based on animal studies, in addition to providing structural support for skin elastic fiber formation, MAGP-1 is essential for vascular integrity, wound healing, and proper body fat deposition, possibly through modulation of TGF- β signaling. MAGP-1 apparently mediates Notch extracellular domain release and is also capable of binding collagen type IV and promoting elastic fiber formation.

The present invention identifies a large number of compounds and/or plant materials and extracts that were found to be active in the simulated expression of the extracellular matrix protein MAGP-1. Among the plant materials which are preferably in the form of extracts are wood sesbania, holy-chamomile, kadsura root, longboat flower, osmidium brevicornum, osmidium odoratum, Tiliacora triandra and cassiabarktree. Suitable compounds are the tetrapeptide lysine-threonine-phenylalanine-lysine (Lys-Thr-Phe-Lys) (KTFK herein), thiodipropionic acid (TDPA) and its lower alkyl mono-and diesters, substituted 2- (2-benzyl-1-benzimidazolyl) acetamide analogs having the structure:

and an N-substituted sulfonyloxybenzylamine having the structure:

it is not known in advance that some plant species with MAGP-1 activity, namely, juniper, kava, dragon boat flower and osmanthus serratus (including extracts of these plant species) can be topically applied to the skin to improve the aesthetic appearance of the skin. Similarly, it was previously unknown that substituted 2- (2-benzyl-1-benzimidazolyl) acetamide analogs disclosed above and N-substituted sulfonyloxybenzylamines disclosed above can be topically applied to the skin to improve the aesthetic appearance of skin.

May tea (Antidesma bunius) is a fruit tree species in the Euphorbiaceae family. It is naturally produced in southeast Asia, the Philippines and northern Australia. Its common philippine name and other names include bignay, bugnay or bignai and the raisin tree. This is a variable plant which can be short and shrubbery or tall and vertical, approximately 30 meters tall. It has large oval cortex evergreen leaves up to about 20 cm long and 7 cm wide. In asia, its leaves can promote sweating and are used to treat snake bites (Morton, j.1987. bignay.p.210-212: Fruits of war animals. julia f.morton, miam, FL.).

Boxthorn (opercula turpethum) is a plant in the family of pharbitidae (synonym indian pharbitis, ipomoea turpethum) and is commonly referred to as indian pharbitis, fue vao and st. It is one of many plants that have been claimed to have activity against liver diseases as cited in the literature. Vasudesan, N, v., indianmatic Plants, vol.iv, Orient Longman Ltd, Chennai,1995,172. It also has anthelmintic, expectorant, antipyretic, anti-inflammatory and purgative properties. The literature is as above.

Dragon boat flower (Ixora chinensis) is a fairly small, dense shrub, typically only 5-10 feet tall. There are very small, dwarf plant varieties. The dragon boat flowers bloom only when a few inches high. Commonly used as hedges or small garden plants. The plant grows mainly as an ornamental plant, while its flowers are considered to have some medicinal uses, including the treatment of hypertension and the treatment of rheumatism, abscesses, bruises and wounds. The plant is naturally grown in india and srilanca.

Cleriodendron denticulatum (Cleriodendron lindleyi) is a shrub, vine and treelet, typically grown to 1-12m height, with either a pair or recurrent leaves. The genus naturally grows in tropical and temperate regions of the world, with most species occurring in tropical africa and south asia, but a few in tropical america and north oceania, and a few extending from the north to temperate regions of east asia. Cleriodenurum comes from Greek and means the "fortune Tree". Dendros means a tree. By chance is meant the possibility that the medicinal value of some plants from this genus is unknown.

Sesbania (Sesbania grandiflora) (also known as agiti, synonyms Sesbania grandiflora, aeschynomenne grandiflora) or kungfeng bird tree/scarlet wisteria is a small tree in the genus Sesbania. It is a fast growing tree with a typical adult height of 3-5 m. The leaves are regular and round, and the flowers are white and large, extremely characteristic. The fruit appeared to be flat, long and thin green beans. Trees flourish under full sunlight exposure and are extremely frost sensitive. It is believed to be derived from india or southeast asia and grows primarily in hot and humid tropical regions of the world. Bark, leaves, gums and flowers are considered to be of medicinal use. Astringent bark is used to treat smallpox and other eruptive fever (James A. Duke.1983.handbook of energy crops. not published). Sesbania has previously been disclosed in US patent US7,618,662, the disclosure of which is incorporated herein by reference.

The cassave (Thunbergia laurifolia) or blue trumpet climbing plants are invasive species and are used as ornamental plants. This plant is found in the Cerrado plant in brazil. In malaysia, sap from crushed leaves of the cassaya morning glory was taken for hypermenorrhea, placed into the ear for deafness and used for mud dressing wounds and furuncles (Burkill, I.H (1966). "a Dictionary of the ecological Products of the malay peninsula. volume II (I-Z)". Ministry of Agriculture and Cooperations, KualaLumpur.). In Thailand, leaves are used as antipyretics and detoxifiers for detoxification (Kanchanapom, T. et al (2002) 'Iridodiglucosides from Thunbergia laurifolia.'. Phytochemistry 60: 769-.

KTFK is a synthetic tetrapeptide lysine-threonine-phenylalanine-lysine (Lys-Thr-Phe-Lys), which is known to be active in collagen and fibronectin synthesis. KTFK was previously disclosed in U.S. patent application publication No. US2004/0126344, the disclosure of which is incorporated herein by reference.

Amorphophallus konjac is found in Mogaska in tropical regions of Asia, Polynesia and North Australia. The root is a wind-expelling drug, tonic, stomachic and tonic. Chopra.r.n., nayar.s.l., and chopra.i.c. gloss of industrial Medicinal Plants (including supples.) county of Scientific and industrial research, New delhi.1986; medicinal Plants of Nepal depth.of Medicinal plants.Nepal 1993. It is dry and used for the treatment of hemorrhoids and dysentery. The literature is as above. Fresh roots are used as pungent stimulants and expectorants, and most of them are used in india for treating acute rheumatism, as described in the literature. Amorphophallus konjac is previously disclosed in US patent No. US7,618,862, the disclosure of which is incorporated herein by reference.

Thiodipropionic acid (TDPA) is a white crystalline solid with a mild specific odor and exhibiting some antimicrobial and antioxidant activity (WHO/Food add./24.65FAO Nutrition measures reports series No. 38a). Thiodipropionic acid and its esters have been previously disclosed in U.S. patent application publication No. US2004/0126344, the disclosure of which is incorporated herein by reference.

Sapindus rarak (Sapindus rarak) is a component of the Sapindus genus, which contains about 5-12 species of shrubs and treelets in the Sapindus genus of the litchi family, and naturally grows in the moderate temperate to tropical regions in the old and new continents. Sapindus mukurossi extract exhibits analgesic effect. Sapindus mukurossi was previously disclosed in US patent US7,514,092, the disclosure of which is incorporated herein by reference.

Tiliacor triandra Diels, also known as Yanang, of the Tiliacor family is a flowering plant species that naturally grows in the continental east Asia, and is used for cooking, particularly in the northeast of Thailand and Laos. It is a climbing plant with mostly single, smooth, oval, dark green leaves and light yellow flowers. Among traditional southeast Asia drugs, Tiliacor triandra has been used as an herbal medicine for antipyresis, alcoholism, inflammation and bacterial/fungal infection. For example, the use of Tiliacor triandra Diels against Plasmodium falciparum (causing malaria in humans) is disclosed in Phytoher. Res.,3,215-217(1989) by Pavanand et al. The Tiliacor triandra is the subject of a separate application filed concurrently with the present application.

Safe, effective and novel composition ingredients for treating, preventing, alleviating, inhibiting and/or ameliorating signs of aging skin disorders (including environmental stress) caused by progressive deterioration of epidermal and dermal skin layers can be advantageously used in therapeutic formulations and skin products. As described herein, provided herein are new and beneficial methods and compositions for treating wrinkles and the like, as well as for personal skin care products, and modes of action thereof.

Disclosure of Invention

It is an object of the present invention to provide topical compositions having a compound, particularly a natural plant material, blends thereof or ingredients derived therefrom, in an amount sufficient to improve the aesthetic appearance of human skin and to prevent, ameliorate and/or reduce dermatological signs of naturally-aged or hormonally-aged or photoaged skin, such as fine lines, wrinkles, sagging skin and other conditions resulting from progressive deterioration of cell growth, proliferation and functionality in the epidermal and dermal layers, wherein the plant material, blends thereof or ingredients derived therefrom are in a cosmetically, dermatologically acceptable vehicle, carrier or diluent.

It is another object of the present invention to provide a composition suitable for topical application to human skin having one or more MAGP-1 active agents that stimulate MAGP-1 expression comprising: (i) a plant material, especially an extract thereof, selected from the group consisting of May tea, Kadsura longipedunculata, Kadsura japonica and Clerodendrum serratum, and mixtures thereof; and (ii) a compound selected from the group consisting of 2- (2-benzyl-1-benzimidazolyl) acetamide analogs and N-substituted sulfonyloxybenzylamines and combinations/mixtures thereof.

It is another object of the present invention to provide a method of topically applying to human skin a composition having a compound, particularly a natural plant material, blends thereof, or ingredients derived therefrom, and stimulating MAGP-1 expression.

It is another object of the present disclosure to provide a topical composition comprising two or more ingredients, wherein at least one first ingredient is selected from the group consisting of Maurera parvifolia, Kadsura Heterophylla, Kadsura japonica, Ottelia brevicorna, Ottelia brevifolia, and N-substituted sulfonyloxybenzylamines, and at least one second ingredient is selected from the group consisting of Sesbania arborescens, Amorphophallus campanulata, Sapindus mukuri, Tetrapeptide lysine-threonine-phenylalanine-lysine (KTFK), and Thiodipropionic acid (TDPA) and esters thereof, in amounts effective to treat, prevent, control, ameliorate, inhibit, and/or reduce dermatological signs of naturally-aged or hormonally-aged or photo-aged skin, such as fine lines, wrinkles, sagging skin, and other conditions resulting from progressive deterioration of cell growth, proliferation, and functionality in the epidermal and dermal layers.

It is another object of the present specification to provide a method of improving the aesthetic appearance of skin comprising treating the effects of aging in the skin by topically applying to the skin a composition of the present specification.

It is a further object of the present specification to provide screening methods for identifying compounds that stimulate the expression of the MAGP-1 protein and to provide compositions of active ingredients identified using such screening methods.

Other objects, features and advantages of the present invention will be better understood upon reading the detailed description.

In accordance with the above and other objects of the present invention, it has been unexpectedly found that expression of MAGP-1 in skin improves the appearance of aged or photodamaged skin by increasing elasticity, tightening pores, improving vascular integrity and reducing dark circles in the lower eye and reducing overall lines, wrinkles and sagging.

Drawings

Figure 1 illustrates MAGP-1 protein expression in the dermis of light-protected and light-exposed young subjects (18-25 years) and elderly subjects (40-65 years).

Figure 2 illustrates MAGP-1 expression around hair follicles and pores in young (18-25 years) and elderly (40-65 years) subjects protected from light and light exposure.

Detailed Description

Unless otherwise specified, the terms used herein are intended to have their ordinary meaning.

The present specification provides novel active agents and novel methods for improving the aesthetic appearance of human skin by topically applying newly discovered compositions effective in treating signs of aging and the consequences associated with dermatological aging of the skin, such as fine lines, wrinkles, sagging skin, and other conditions resulting from aging, including natural or hormonal aging and/or photoaging, to improve the aesthetic appearance of skin. It is understood that natural aging represents structural, functional and metabolic changes in the skin that occur in parallel with aging and degenerative changes in other body organs, whereas photoaging is a separate process and is primarily concerned with damage to collagen and elastic fibers in the skin due to exposure to the environment (e.g., sunlight). The aesthetic appearance of skin can be improved by applying these compositions topically, on a regular basis, and continuously (e.g., on a daily basis) to the skin.

As shown in fig. 1 and 2 and described in more detail in the examples, it was found that MAGP-1 in skin tissue changes with age and light exposure. Thus, aged skin shows a reduced number of MAGP-1 complexes with fibrillin and other microfibrils, accompanied by a loss of skin elasticity and a number of other properties of healthy skin. Compositions of the present invention comprising one or more active agents for increasing MAGP-1 expression are provided for reducing, alleviating, ameliorating the deleterious effects of skin aging, and helping to restore a more youthful appearance to skin.

Without wishing to be bound by theory, the compounds and natural plant materials and extracts described herein (hereinafter referred to as MAGP-1 active agents) stimulate MAGP-1 expression in skin and improve the appearance of aged or photodamaged skin by increasing elasticity, tightening pores, improving vascular integrity, reducing under-eye dark circles, and reducing overall fine lines, wrinkles, and skin sagging. It is believed that MAGP-1 expression forms a complex with microfibrillar protein (e.g., fibrillin). Fibrillin is a glycoprotein that is essential for the formation of elastic fibers found in connective tissue. Fibrillin is secreted by fibroblasts into the extracellular matrix and incorporates insoluble microfibrils, which apparently provide a deposition platform for elastin, a protein in connective tissue that is elastic and allows many tissues within the body to recover their shape after being stretched or contracted.

The present specification provides novel compositions and methods of use thereof that have been newly discovered to effectively treat signs of skin aging and the consequences associated with dermatological aging of the skin (including natural aging or hormonal aging and/or photoaging) by affecting one or more of the following: increased elasticity, reduced fine lines, wrinkles, sagging skin, reduced under-eye darkness/abrasion and fat pockets, grooming/reduced fat pad effect and improved skin tone, providing improved shine and healthy shine, and other conditions.

Because MAGP-1 stimulation increases the formation of fibrillins, such as fibrillin, which are secreted into the extracellular matrix by fibroblasts, another embodiment of the present invention provides a composition comprising a MAGP-1 active agent of the present invention and a plant or compound, such as a material that is active in increasing fibroblast proliferation. This combination increases collagen production; provide anchoring and increased adhesion between keratinocytes; promotes cell-cell adhesion in the epidermis and dermis; and strengthens the dermoepidermal junction. Concomitantly, the aesthetic appearance of aged skin is improved herein by one or more of an increase in skin firmness, skin clarity, skin softness, or skin thickness.

The present invention also provides methods of topically applying the compositions to skin that improve skin condition and appearance by stimulating the expression of the extracellular protein MAGP-1.

The method of the present invention incorporates a MAGP-1 active agent or combination thereof comprising a compound and/or plant extract selected from the group consisting of: sesbania, May tea, Kadsura longipedunculata, Longboat flower, Clerodendron cumingii, tetrapeptide lysine-threonine-phenylalanine-lysine (KTFK), Amorphophallus rivieri, thiodipropionic acid (TDPA) and its C1-C6 mono-and di-esters, Sapindus mukurossi, Tiliacor triandra, Ivy leaf morning glory, N-substituted sulfonyloxybenzylamines having the structural formula I, namely 4- ((N-isobutylphenylsulfonylamino) methyl) phenyl methanesulfonate (CAS 853758-16-4):

and substituted 2- (2-benzyl-1-benzimidazolyl) acetamide analogs having the structural formula II, namely 2- {2- [ (3-methylphenyl) methyl ] -1H-1, 3-benzodiazol-1-yl }1- (morpholin-4-yl) ethan-1-one (CAS 1043743-29-8):

compounds I and II were purchased from AMRI Hungary, Zahony u 7, H-1031Budapest, Hungary.

It is to be understood that the term "treating" as used herein includes reducing, ameliorating, improving, alleviating and/or eliminating the dermatological effects of aging and/or environmental stress. The compositions and methods of the present invention are suitable for treating dermatological and dermatological conditions in a large number of areas of the body, including, but not limited to, the face, forehead, lips, neck, arms, hands, legs, knees, feet, chest, back, groin, buttocks, and the like. In a preferred embodiment, the composition is applied to the face.

One embodiment of the present specification relates to novel compositions comprising a novel MAGP-1 active agent or a combination thereof selected from May tea, an N-substituted sulfonyloxybenzylamine of structure I (supra), a substituted 2- (2-benzyl-1-benzimidazolyl) acetamide analog of structure II (supra), Kalopanax septemlobus, Adinandra, and Mirabilis jalapa, and further comprising a cosmetically, dermatologically, or pharmacologically acceptable vehicle; the present specification also relates to methods of improving skin by topically applying the novel topical compositions to skin in need of treatment.

In another embodiment, the compositions of the present invention comprise one or more of the novel MAGP-1 active agents listed above in combination with one or more other MAGP-1 active agents, and a cosmetically, dermatologically, or pharmacologically acceptable vehicle; also relates to methods of improving skin by topically applying the novel topical compositions to skin in need of treatment. Among the one or more other MAGP-1 active agents, mention may be made of sesbania, KTFK, Amorphophallus konjac, Thiodipropionic acid (TDPA) and C₁－C₆Alkyl mono-and diesters, Tiliacor triandra, and Guinea.

Histological studies of the skin show that when aging occurs, the skin undergoes structural, functional and metabolic changes that occur in parallel with aging and degenerative changes in other body organs. Although chronological age and/or hormonal aging play a significant role, the amount of wrinkles present is also highly dependent on the amount of exposure to environmental stress during an individual's life, for example, over 10, 20, 30 or more years in repeated exposure to sunlight from the personal opportunity for oxidative damage from overexposure to Ultraviolet (UV) sunlight. Although there is a gradual thinning of the skin in men with aging of about 1% every year, the thickness of the skin in most women remains surprisingly constant until menopause, after which, with significant and sometimes dramatic thinning with age, the need for treatments is prompted in older women that increase the collagen content of the skin and thereby improve the firmness, thickness and clarity of the skin.

Thus, from this point of view, successful recovery of young skin must address a variety of key issues, including: viability of fibroblasts and keratinocytes, cell-cell adhesion in epidermis and dermis, cell-cell feeding of epidermis, cell-cell anchoring and adhesion between keratinocytes, communication between dermis and epidermis, collagenase overproduction, collagen replacement and mechanical properties of skin. Cosmetic ingredients that address these key issues are used in the topical compositions of the present specification.

Topical compositions

The natural plant material may be in any form including, but not limited to, whole plants, dried plants, ground plants or parts thereof, including, but not limited to, seeds, needles, leaves, roots, bark, cones, stems, rhizomes, callus cells, protoplasts, flowers, and meristems or components and/or components found in or isolated from natural plant material and/or parts of plants, or any combination thereof. In one embodiment, the natural plant material is in the form of an extract derived from a selected portion of the natural plant material. Typically, the plant material is obtained from whole plants or plant leaves. It is understood that "natural plant material" also includes components, ingredients, compositional components, or extracts derived from natural plant material.

In the case of the Tiliacor triandra, the plant extract is preferably obtained from the vine of the plant. In the case of ixora and sesbania, the extract is preferably obtained from the flowers of plants. In the case of May tea, Guiye mountain morning glory and Clerodendron trichotomum, the extract is preferably obtained from leaves. In the case of a box-berry vine, the extract is preferably obtained from the aerial parts of the plant, i.e. the whole plant, excluding the roots.

For the Tiliacor trianda compositions contemplated by the present invention, the present invention contemplates embodiments that do not include hydrogen peroxide, iodine, tarrow roots, and mixtures of tarrow roots with iodine.

Compositions comprising these plants are effective when applied topically, preferably daily.

The composition has a concentration of MAGP-1 active agent of from about 0.0001 wt% to about 90 wt%, from about 0.001 wt% to about 25 wt%, from about 0.01 wt% to about 10 wt%, from about 0.05 wt% to about 1 wt%, based on the total weight of the composition. In the case of plant extracts, the above amounts refer to the "active amount" of the plant extract. The term "active amount" or "dry weight" is used synonymously and refers to the amount of the plant/extract after removal of the solvent and/or other diluents. One of ordinary skill in the art will be able to adjust the amount of extract used depending on the particular application or effect desired.

The MAGP-1 active of the present specification is preferably contained in a cosmetically or dermatologically acceptable vehicle, medium, diluent or carrier. In one embodiment, including topical application, the compositions of the present specification comprise a medium (vehicle, diluent or carrier) compatible with human skin. These compositions may be formulated as water, alcohol or water/alcohol based solutions, ointments, lotions, gels, water-in-oil, oil-in-water, triple water-oil-water emulsions with cream or gel appearance, microemulsions or aerosols. Furthermore, these compositions may be in the form of vesicular dispersions comprising ionic and/or non-ionic lipids as described above. Dosage forms suitable for such compositions are formulated according to conventional knowledge and techniques used in the art.

Methods of use of MAGP-1 active agents

As understood by practitioners, for example, cosmetic treatments including compositions containing natural plant materials (including the extracts, ingredients, and/or components of the present invention) can be performed according to a regimen for administering such compositions by topically applying a cosmetic composition as described herein. The topical cosmetic composition is preferably applied once or twice daily (e.g., morning and evening) for a period of at least 1 week, but may include a period of about 2, 4, 8, or 12 weeks. Consumers desire to continue to use the composition for extended periods of time. The cosmetic composition is preferably applied to the face and neck, but may be applied to any area of the skin in need of cosmetic improvement, wherein the cosmetic composition remains on the affected skin area, preferably without being removed or rinsed off from the skin. The method applies the composition/product in the form of cream, lotion, gel, pack, serum, ointment, patch, foundation emulsion, sunscreen composition, etc. to the skin. Preferably the cosmetic composition is a topical retention formulation, wherein a spray in self-applied form is also contemplated.

In a particular embodiment of the invention, topical compositions with a MAGP-1 active agent of the invention, including compositions comprising two or more of such active agents and compositions comprising one or more of the novel active agents as defined above, alone or in combination with one or more other active agents (including ingredients or extracts derived therefrom), are used to improve the condition and aesthetic appearance of aged, particularly mature or mature skin affected skin by any of the following methods: reducing dermatological signs of natural aging, photoaging, hormonal aging, and/or actinic aging; preventing and/or reducing the appearance of fine lines and/or wrinkles; reducing the eye-catching of fine lines and wrinkles on the face, cheeks, facial wrinkles on the forehead, vertical wrinkles between the eyes, horizontal wrinkles above the eyes and wrinkles around the mouth, marionette lines and particularly deep wrinkles or folds; preventing, reducing and/or reducing the appearance and/or depth of fine lines and/or wrinkles; improving the appearance of infraorbital and/or periorbital lines; the appearance of the fishtail lines is lightened; rejuvenating and/or rejuvenating skin, particularly aged skin; reducing skin fragility; preventing and/or reversing loss of glycosaminoglycans and/or collagen; reducing skin fragility; improving the estrogen imbalance effect; prevention and/or skin atrophy; preventing, reducing and/or treating hyperpigmentation; minimizing skin discoloration; improving skin color, radiance, clarity and/or tautness; preventing, reducing and/or ameliorating skin sagging; improving skin firmness, plumpness, flexibility and/or softness; improving procollagen and/or collagen production; improving skin texture and/or promoting skin texture to recover; improving skin barrier repair and/or function; improving the appearance of skin contours; restoring skin radiance and/or brightness; minimizing dermatological signs of fatigue and/or stress; resistance to environmental stress; supplementing components in the skin that have diminished due to aging and/or menopause; improving intercellular communication of the skin; increasing cell proliferation and/or reproduction; increasing skin cell metabolism decreased by aging and/or menopause; delaying cell aging; improving the moisturization of the skin; enhancing skin thickness; increasing the elasticity and/or resiliency of the skin; promoting the peeling of the epidermis; improving microcirculation; reduce and/or prevent cellulite formation; and any combination thereof.

The active ingredients of the disclosed topical compositions are also useful for treating, preventing, retarding, ameliorating, reducing or reducing the dermatological and/or dermatological conditions of aging. Such conditions, as used herein, generally include, but are not limited to, dermatological aging (natural aging, hormonal aging, and/or actinic aging), dermatitis, skin and hair fragility, hirsutism, rosacea, skin blemishes, sensitive skin, hyperpigmentation or hypopigmentation, thinning, roughness, keratosis, skin atrophy, wrinkles, fine lines, hyperplasia, fibrosis, and any combination thereof. The active ingredients of the topical composition may also be used to enhance the general health, vitality, condition and appearance of the skin.

Gene expression can be measured, for example, by measuring RNA levels in cultured cells using techniques such as Northern blot techniques and Polymerase Chain Reaction (PCR), such as "real-time" PCR and reverse transcription PCR (RT PCR) as practiced in the art (see, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; R.Higuchi et al, 1992, Biotechnology,10: 413:417; R.Higuchi et al, 1993, Biotechnology,11: 1026-. Furthermore, gene (cDNA) arrays (microarrays or nucleic acid gene chip arrays comprising membrane, glass, plastic or silicon support materials, etc.), Serial Analyses of Gene Expression (SAGE) (e.g., V.E. Velculus et al, Science,270(5235): 484-.

Topical compositions use plant material/active ingredients that are genetically affected in association with dermatological signs of aging (e.g., fine lines, wrinkles, and sagging skin) as biomarkers for compounds that can improve the condition and appearance of affected skin. If the expression level of such nucleic acid/protein biomarkers is altered or increased in the presence of natural plant material or active ingredients derived therefrom, the natural plant material can be used in the topical compositions of the present invention to improve the condition and appearance of skin. Such expression level assays include various methods of determining the level of nucleic acid in a cell that has been exposed to one or more test substances. Suitable methods include detecting and evaluating gene activation or expression of, for example, DNA, RNA, or mRNA. As non-limiting examples, Polymerase Chain Reaction (PCR) assays (e.g., RT-PCR), Northern blotting, in situ hybridization, and other assays known and practiced in the art can be used to quantify RNA in detecting cells that are resistant to a particular treatment (see, e.g., J.O' Connell,2002, RT-PCR Protocols, Humana Press, Totolwa, NJ; R.Rapley and D.L.Manning,1998, RNA Isolation and catalysis Protocols, Humana Press; R.Rapley,2000, Nucleic Acid Protocols Handbook, Humana Press; all of which are incorporated herein by reference). According to this assay, if the level of at least one nucleic acid biomarker is elevated in the presence of one or more test substances, it may indicate that said substance may ameliorate the dermatological signs of aging. These substances or natural plant materials can then be used in topical compositions, preferably applied daily to the skin, in order to treat, prevent, ameliorate and/or reduce dermatological signs of aging, especially fine lines, wrinkles and sagging skin, thereby improving the condition and aesthetic appearance of the skin.

In another embodiment, plant material as used herein also includes "synthetic" extracts, i.e., various combinations of known plant material ingredients and/or components that are combined together so as to substantially mimic the composition and/or activity of plant extracts of natural origin. Such synthetic extracts are included in the term "plant extract". The synthetic extract has two or more, three or more, or four or more active ingredients in common with the plant. Most preferably, the synthetic extract has substantially the same amount of active ingredient as the natural extract. The correspondence in the quantitative association of active ingredients between synthetic extracts and plants or natural extracts can also be described as "percentage of commonality". Preferably, the synthetic extract has a commonality of about 50% or more than 50% of the chemical composition of the plant or natural extract. In other words, synthetic extracts have about 50% or more of the active ingredients found in plants or natural extracts. More preferably, the chemical composition of the synthetic extract may have a commonality of about 70% or more with the chemical composition of the plant or natural extract. Most preferably, the synthetic extract has a commonality of about 90% or more than 90% with the chemical composition of the plant or natural extract.

For the compositions used in the present description, the plant extract or ingredient and/or the active ingredient is preferably directly derived from a plant. The components may be in pure form, semi-pure form or impure form. In one embodiment, the ingredients may be in the form of an extract obtained by extraction using an organic solvent.

Methods of extraction using organic solvents are well known in the art and may be used in accordance with the present invention. Briefly, an organic solvent extraction process involves washing and extracting plant material with an organic solvent. Non-limiting examples of the organic solvent include methanol, ethanol, isopropanol, dichloromethane, chloroform, hexane, xylene, and petroleum ether. Methods well known in the art may be used for organic solvent extraction.

Organic solvent extraction involves harvesting the material from the plant containing the desired components, such as seeds, needles, leaves, roots, bark, cones, stems, rhizomes, callus cells, protoplasts, organs and organ systems, and meristems. These plant materials are ground to small particle sizes and then placed into an extractor through a raw material inlet with a measurable charging machine. The plant material is pushed into the extractor with a pusher and slowly moved forward. Organic solvent (e.g., ethanol) may be added to the extractor through a solvent inlet at the upper portion of the waste drain. Due to the difference in gravity and equilibrium, the solvent flows to the raw material inlet, saturates the raw material and flows out from the opposite side of the solvent inlet. Since the plant material and the solvent move in opposite directions to each other, the plant material is constantly immersed in a solution containing a low concentration of the extract. As a result of the equilibration, high yields of plant components can be obtained by continuous extraction of plant material from low concentration solutions.

It is suitable to use about 1 to 8 hours as a typical extraction time for removing plant components, more preferably about 2 to 6 hours, most preferably about 3 to 5 hours. The extraction temperature is about 30-90 deg.C, about 40-70 deg.C, and about 50-60 deg.C. The collected extract is then fine filtered to remove debris and can be used directly or concentrated, for example by distillation of the solvent or by other conventional processing, and can also be made into a dry powder.

Similarly, aqueous-organic solvent extraction involves initially collecting raw materials from plants containing the desired alkaloid, such as seeds, needles, leaves, roots, bark, cones, stems, rhizomes, callus cells, protoplasts, organs and organ systems, and meristems of the plant, which are ground to small particle size. The ground plant material is immersed in an aqueous solution, either acidic or basic, depending on the solubility and stability of the desired extract under acidic or basic conditions. For extraction under acidic conditions, an acid such as hydrochloric acid or sulfuric acid is added to the water, for example, at a concentration of about 3% (w/v). For extraction under alkaline conditions, a base such as sodium hydroxide or sodium carbonate is added to the water. The extraction time and extraction temperature are typically similar to those used in the organic solvent extraction methods described above.

The extract was then collected and subjected to fine filtration to remove debris. Depending on the acidity or basicity of the collected extract, an alkaline agent (e.g., ammonia) or an acidifying agent (e.g., sulfuric acid) may be added to the extract to neutralize the solution by adjusting the pH. The aqueous extract can be used directly, concentrated or dried. Alternatively, an organic solvent may then be added to the neutralized solution to transfer the extract active ingredients from the aqueous phase to the organic phase. Non-limiting examples of such organic solvents include, but are not limited to, ethanol, isopropanol, butanol, pentanol, hexanol, and xylene. The extract containing the transferred active ingredients of the extract dissolved in an organic solvent can be used as such, as a concentrate or dried.

The extract can also be obtained by extracting the plant material with water, ethanol or a mixture thereof. The hydroalcoholic solvent system may include from about 10% by volume to about 90% by volume ethanol and from about 10% by volume to about 90% by volume water. More typically, the solvent system comprises from about 40% by volume to about 90% by volume ethanol and from about 10% by volume to about 60% by volume water.

Different plants containing different components can be mixed and extracted together. This mixed extraction method can be preferably used for extracting those plants containing components having similar solubility in the solvent (e.g., ethanol) used for extraction. The mixture of extracts may be concentrated and stored in a suitable solvent.

The extract may be combined with a physiologically acceptable solvent for use as a concentrate, wherein a portion of the solvent or other diluent is removed by evaporation or other suitable physical means, or preferably the solvent may be removed to provide a dried extract. Preferably, the solids are removed from the extract by filtration, centrifugation or other suitable means. Drying may be carried out by lyophilization, spray drying, granulation, or other suitable methods. Spray drying may be carried out by combining the extract with a bulking agent (e.g., maltodextrin) and then spray drying.

Suitable extraction methods are disclosed in PCT publication No. WO03/079816 (describing the preparation of tomato extracts with high content of lycopene), WO04/014404 (describing the preparation of Echinacea angustifolia extracts) and WO04/014958 (describing the extraction of polysaccharides from Echinacea angustifolia roots), all of which are incorporated herein by reference in their entirety.

Vehicle and composition

According to the present description, the compositions comprising MAGP-1 active ingredient may also comprise antioxidants, anti-inflammatory agents, sunscreens, cosmetic agents, including cosmetics, anti-ageing preparations, such as creams for fine lines and/or wrinkles, topical preparations, skin penetration enhancers, sprays and the like. Such compositions comprising plant ingredients and additional ingredients may also be formulated into various product forms in accordance with the present specification. Preferably, the compositions can be prepared as targeted delivery systems, particularly for topical administration, such as creams, lotions, gels, serum, transdermal patches and the like. Targeted delivery and/or penetration enhancement can also be achieved by iontophoresis.

The present specification further provides compositions comprising plant components, preferably for topical administration or for targeted delivery without inducing significant irritation. Thus, the compositions of the present invention are particularly suitable for sensitive skin. The composition is applied to the skin for a period of time sufficient to improve the aesthetic appearance of the skin. The composition is preferably applied topically 1, 2 or more times daily, preferably 1 or 2 times daily. Preferably, daily topical administration is carried out for a period of 1 week, 2 weeks, 4 weeks, or more than 4 weeks. The compositions may be formulated into liposomes or other encapsulates or delivery vehicles, which may contain other additives or substances and/or may be modified to more specifically reach or remain at the site following administration.

The present specification also includes compositions comprising cosmetically or dermatologically acceptable formulations suitable for contact with living animal tissues, including human tissues, without substantial adverse physiological effects on the user. The compositions encompassed by the present description may be formulated in any cosmetically and/or dermatologically suitable form, preferably as a lotion or cream, but also in the form of an anhydrous or aqueous base and in the form of a sprayable liquid. Other suitable cosmetic forms for use in the compositions of the present disclosure include: such as a lotion, lipstick, lip balm, lotion, pack, ointment, mousse, patch, pomade, solution, spray, wax-based stick, or wet wipe. In addition, the compositions contemplated herein may include one or more compatible cosmetically acceptable adjuvants commonly used and known to those skilled in the art, such as colorants, fragrances, emollients, moisturizers, preservatives, vitamins, chelating agents, thickeners, anesthetics, anti-allergic agents, antifungals, antimicrobials, other anti-inflammatory agents, antioxidants, antimicrobials, depigmenting agents, film formers, anthelmintics, pharmaceutically active agents, photostabilizers, opacifiers, stabilizers, surfactants, thickeners, viscosity modifiers, and the like, as well as other botanicals (e.g., aloe, chamomile, and the like) and further described below.

Cosmetically or dermatologically acceptable vehicles that may be used in the topical compositions of the present invention include, but are not limited to, one or more aqueous systems, glycerol C1-4 alcohols, fatty ethers, fatty esters, polyols, glycols, vegetable oils, mineral oils, liposomes, lamellar lipid materials, silicone oils, water, or any combination thereof.

In the present specification, the vehicle may be in the form of an aqueous phase, an oil phase, a gel, a wax-in-water emulsion, a silicone-in-water emulsion, a water-in-silicone emulsion, an oil-in-water emulsion or a water-in-oil emulsion. The aqueous phase is a mixture of one or more water-soluble or water-dispersible ingredients, which may be liquid, semi-solid, or solid at room temperature (25 ℃). The vehicle comprises or may be in the form of a suspension, dispersion or solution of an aqueous or aqueous-alcoholic vehicle, which may comprise a thickening or gelling agent. Those skilled in the art can select suitable product forms, ingredients contained therein, and methods of making the same based on the knowledge possessed by those skilled in the art.

The composition may comprise an aqueous phase, which may comprise water or a mixture of water and at least one hydrophilic organic solvent comprising an alcohol, in particular a linear or branched lower monoalcohol with 2 to 5 carbon atoms, such as ethanol or propanol; polyols, such as propylene glycol, sorbitol, glycerol, diglycerol, panthenol or polyethylene glycol and mixtures thereof. Such an aqueous phase may comprise from about 0.5 to about 99.99 wt% of the composition.

When the composition of the present description is in the form of an emulsion, it may optionally also comprise a surfactant, preferably in an amount of 0.1 to 30% and in particular about 1 to about 20% by weight of the composition.

The composition may also comprise a thickening polymer, such as an amphiphilic polyurethane, a polyacrylic acid homopolymer or copolymer, a polyester, and/or a hydrocarbon-based resin. The polymer may be dissolved or dispersed in a cosmetically acceptable vehicle and optionally combined with a plasticizer.

The compositions of the present disclosure may also comprise an oil phase comprising an oil soluble or dispersible component that is liquid at room temperature (25 ℃) and/or an oily or waxy substance that is solid at room temperature, such as waxes, semisolids, gums, and mixtures thereof. The oil phase may also comprise an organic solvent.

Suitable oily substances which are liquid at room temperature, commonly referred to as oils, include: hydrocarbon-based oils of animal origin, such as perhydrosqualene; hydrocarbon-based vegetable oils, such as liquid triglycerides of fatty acids of 4 to 10 carbon atoms, for example heptanoic acid or caprylic acid triglycerides, or oils, such as sunflower oil, corn oil, soybean oil, grapeseed oil, castor oil, avocado oil, caprylic/capric acid triglycerides, jojoba oil; linear or branched hydrocarbons of mineral or synthetic origin, such as liquid paraffin and its derivatives, vaseline; synthetic esters and ethers, in particular esters of fatty alcohols, such as isopropyl myristate, 2-ethylhexyl palmitate, 2-octyldodecyl stearate, isostearyl isostearate; hydroxylated esters, such as isostearyl lactate, octyl hydroxystearate, octyl dodecyl hydroxystearate, heptanoates, octanoates and decanoates of fatty alcohols; polyol esters such as propylene glycol dicaprylate, neopentyl glycol diheptanoate, diethylene glycol diisononanoate, and pentaerythritol esters; fatty alcohols having 12 to 26 carbon atoms, such as octyldodecanol, 2-butyloctanol, 2-hexyldecanol, 2-undecylpentadecanol, oleyl alcohol; a partially hydrocarbon-based fluoro-and/or fluorosilicone oil; silicone oils, such as volatile or non-volatile linear or cyclic Polymethylsiloxanes (PDMS) that are liquid or semi-solid at room temperature, e.g., cyclic polydimethylsiloxanes and polydimethylsiloxanes, optionally containing phenyl groups, e.g., phenyl trimethicones, siloxanes, and mixtures thereof. These oils are generally present in an amount of about 0 wt.% to about 90 wt.%, preferably about 1 wt.% to about 80 wt.%, by weight of the oil phase.

The oil phase of the compositions of the present disclosure may also comprise one or more cosmetically acceptable organic solvents. These solvents are present in an amount of about 0 wt% to about 60 wt%, preferably about 1 wt% to about 30 wt%, based on the total weight of the composition, and may be selected from lipophilic organic solvents, amphiphilic organic solvents, and mixtures thereof. Suitable solvents that may be used in the compositions of the present invention include: acetates such as methyl acetate, ethyl acetate, butyl acetate, amyl acetate or 2-methoxyethyl acetate; isopropyl acetate; hydrocarbons, such as toluene, xylene, p-xylene, hexane or heptane; ethers containing at least 3 carbon atoms and mixtures thereof.

The composition of the present specification may further comprise any component commonly used in the cosmetic field. These components include preservatives, aqueous phase thickeners (polysaccharide biopolymers, synthetic polymers) and fatty phase thickeners, fragrances, hydrophilic and lipophilic active agents and mixtures thereof. The amounts of these various components are those conventionally used in the cosmetic arts to achieve their intended purpose and generally range from about 0.01% to about 20% by weight of the total weight of the composition. The nature of these components and the amounts thereof must be compatible with the process for producing the compositions of the present description.

The compositions of the present invention may also comprise an additional particulate phase, which is generally present in an amount of from about 0% to about 30% by weight, preferably from about 0.05% to about 20% by weight, based on the total weight of the composition, and may contain pigments and/or pearlescent agents and/or fillers used in cosmetic compositions. Suitable inorganic pigments include titanium oxide and iron oxide; suitable fillers include talc, mica, polyethylene powder, boron nitride, copolymer microspheres, and silicone resin microbeads (tosearl from Toshiba).

The oil phase of the compositions of the present disclosure may comprise one or more waxes, gums, or mixtures thereof. The waxes include hydrocarbon-based waxes, fluoro waxes and/or silicone waxes and may be of vegetable, mineral, animal and/or synthetic origin. In particular, the wax has a melting point above 25 ℃, preferably above 45 ℃. The compositions of the present disclosure may comprise from 0 to about 20 weight percent of a wax, based on the total weight of the composition. Gums are typically high molecular weight PDMS or cellulose gums or polysaccharides, and semi-solid materials are typically hydrocarbon-based compounds such as, but not limited to, lanolin and its derivatives, or may be PDMS. The compositions of the present specification may comprise from 0 to about 20 wt%, typically from about 0.1 wt% to about 10 wt% of gum, based on the total weight of the composition.

In another preferred embodiment, the topical composition of the present invention may further comprise at least one of the following ingredients: skin penetration enhancers, surface smoothing agents, skin plumpers, optical diffusers, sunscreens, exfoliation promoters, and antioxidants. Detailed descriptions of these and other suitable cosmetic ingredients can be found in the following publications: "International Cosmetic Ingredient Dictionary and Handbook," 10 th edition (2004), published by Cosmetic, Toiletry, and Fragrance Association (CTFA), pp.2177-2299, which is incorporated herein by reference in its entirety.

Among the sunscreens which may be used in the compositions of the present invention are avobenzone, cinnamic acid derivatives (e.g., octylmethoxy cinnamate), octyl salicylate, oxybenzone, titanium dioxide, zinc oxide, or any mixture thereof. The opacifier is present in an amount of about 1 wt% to about 30 wt% of the total weight of the composition. The addition of a sunscreen may prevent/reduce photodegradation of the composition while packaged, and may also be used to protect the skin from ultraviolet radiation.

In addition to the above, antioxidants function to scavenge free radicals from the skin in order to protect the skin from environmental aggressors. Examples of antioxidants that may be used in the compositions of the present invention include, but are not limited to, Alpha Hydroxy Acids (AHAs); benzoyl peroxide; a beta hydroxy acid; keto acids such as pyruvic acid, 2-oxopropanoic acid, 2-oxobutanoic acid and 2-oxopentanoic acid; oxoacids disclosed in U.S. Pat. Nos. 5,847,003 and 5,834,513 (the disclosures of which are incorporated herein by reference); salicylic acid; urea; or any mixture thereof. Preferred antioxidants are 3,6, 9-trioxaundecanedioic acid, glycolic acid, lactic acid or any mixture thereof; compounds with phenolic hydroxyl functionality, such as ascorbic acid and its derivatives/esters; beta-carotene; a catechin; curcumin; tetrahydrocurcumin; tocopherol and derivatives thereof; or any mixture thereof. The compositions of the present disclosure may have an antioxidant, preferably from about 0.001 wt% to about 10 wt% and more preferably from about 0.001 wt% to about 5 wt% of the total weight of the composition.

The compositions of the invention may also have one or more of the following active agents, ingredients or adjuvants: an anesthetic; an anti-allergic agent; an antifungal agent; antibacterial drugs; a chelating agent; a colorant; a demulcent; an emollient; an emulsifier; a fragrance; a humectant; a lubricant; a moisturizing agent; a pH adjusting agent; a pigment altering agent; a preservative; a stabilizer; a surfactant; a thickener; a viscosity modifier; a vitamin; or any mixture thereof. The amounts of these various substances are those conventionally used in the cosmetic or pharmaceutical field, and they may for example represent from about 0.01% to about 20% of the total weight of the composition.

Non-limiting examples of active agents useful in formulating the compositions of the present disclosure include, in addition to the natural botanical active ingredients described, those agents that have an effect on the treatment of wrinkles and/or fine lines, such as keratolytic agents, i.e., active agents that have desquamation, exfoliation, or scrubbing properties, or active agents that soften the stratum corneum of the skin. Other examples of anti-wrinkle or anti-fine line actives include hydroxy acids and retinoids. For example, these active agents may be formulated in an amount of about 0.0001% to about 5% by weight relative to the total weight of the composition.

Suitable hydroxy acids include: such as glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acids, mandelic acid, salicylic acid and alkyl derivatives thereof, including 5-n-octanoylsalicylic acid, 5-n-dodecanoylsalicylic acid, 5-n-decanoylsalicylic acid, 5-n-octylsalicylic acid, 5-n-heptyloxysalicylic acid, 4-n-heptyloxysalicylic acid and 2-hydroxy-3-methylbenzoic acid or alkoxy derivatives thereof, such as 2-hydroxy-3-methoxybenzoic acid.

Typical retinoids include, but are not limited to, retinoic acid (e.g., all-trans or 13-cis) and its derivatives, retinol (vitamin a) and its esters, such as retinyl palmitate, retinyl acetate and retinyl propionate and their salts.

More specifically, the topically applied composition may be a composition for protective care of the skin, preferably for the face, neck, hands, feet or other areas of the body. Non-limiting examples include day creams or lotions, night creams or cosmetics, sun creams, lotions or oils, body lotions, cosmetics (foundations), artificial tanning compositions, depilatories, and patches.

The amount of emulsifier used typically comprises from about 0.1% to about 30% by weight and preferably from about 0.5% to about 30% by weight relative to the total weight in the emulsion composition of the present specification.

In water-based formulations, the active compound may be formulated at a pH in the range of from 1 to 8, with a pH of 2 to 7 being preferred, and a pH of 3.5 to 5.5 being most preferred.

In another embodiment of the invention, compounds for stimulating MAGP-1 protein expression are screened for activity using an assay or screening method comprising culturing normal human epidermal fibroblasts in a culture plate with DMEMD and 10% serum, followed by incubation at 37 ℃ and 10% CO₂Incubate for 24 hours. After 24 hours, stock solutions of the compounds of the invention as test substances are prepared with suitable solvents (e.g. DMSO). Then in the presence of 10% CO₂Humidified 37 ℃ incubator of (a) cells were treated with test substance diluted in growth medium or corresponding vehicle control for 48 hours. After incubation, the growth medium is typically removed and TRIzol reagent is added to the culture plate. The resulting cell lysate was collected and placed in a freezer until RNA was separated and analyzed. RNA isolation can be performed using any method known to those skilled in the art. After isolation, total RNA concentration is determined by Nanodrop (Thermo Scientific, DE) or any other well known method. The isolated RNA was then subjected to Reverse Transcription (RT) and quantitative polymerase chain reaction (qPCR) to amplify the RNA, which was then determined and calculated according to well-known methods to determine the level of MAGP-1 stimulating activity of each compound.

Another embodiment of the present invention consists in preparing a composition comprising the MAGP-1 active ingredient identified according to the screening method described above.

The following examples are meant to illustrate some aspects of the invention in a non-limiting manner.

Detailed Description

Example 1 preparation of extract

Agrimonia pilosa-the extract is obtained by extracting dried, chopped, Agrimonia pilosa plant using ethanol, and then further extracting with hexane. Briefly, chopped plants of ixora will first be ground by hand to small particles, yielding about 250 grams of powder. The ground powder was then extracted with 50% ethanol. After filtration and vacuum evaporation, the total concentrated extract was diluted with water, centrifuged and filtered. The liquid was then extracted three times with hexane, the hexane upper layer was discarded, and the aqueous layer was lyophilized to give about 90 g of extract.

Osmyl jalapa poinaria extract-is obtained by extracting the leaves of the osmyl jasmine plant using an ethanol extraction protocol. The leaves of the osmyl jasmine dragon were first ground by hand to small particles, resulting in about 250 grams of powder per flask (2 flasks). The milled powder was then extracted with 80 vol% ethanol (2X2, 000ml/flask). After filtration and evaporation in vacuo, the total concentrated extract was lyophilized to give 30.77 grams of ethanol extract.

May tea-extract was obtained from 400 grams of ground plant leaves extracted in three portions with 4450ml of a 1:1 ethanol-water solution. The extract was filtered, concentrated in vacuo to remove ethanol, diluted to 1300ml with water and centrifuged. The resulting supernatant was washed with 500ml of dichloromethane, decolorized with 10g of activated carbon, and filtered. Then concentrated to give 57.7g of extract, dried to give powder.

Boxthorn (Operculina turphetum) -extracts were obtained by extracting aerial parts of Operculinaturphetum plants using an ethanol extraction protocol. The Operculina turphetum was first ground manually into small particles, yielding approximately 250 grams of powder. The ground powder is then extracted with a water-ethanol mixture (50-50). After filtration and evaporation in vacuo, the aqueous solution is washed with dichloromethane, concentrated in vacuo and dried.

Tiliacor triandra-extracts are obtained by extracting the vines of Tiliacor triandra plants using an ethanol extraction protocol. Briefly, the vines of Tiliacor triandra Diels were first ground by hand to small particles, yielding approximately 250 grams of powder per flask (2 flasks). The milled powder was then extracted with 80% ethanol (2X2, 000ml/flask). After filtration and vacuum evaporation, the total concentrated extract was lyophilized to give 50 g of ethanol extract.

Example 2 MAGP-1 expression in dermis

Materials and methods

Skin biopsies were obtained from light-exposed and light-protected areas of 15 young subjects (18-25 years) and 15 old subjects (40-65 years). Tissues were fixed in formalin and immunohistochemical studies were performed. anti-MAGP-1 and alkaline phosphatase-conjugated secondary antibodies were thus administered to show protein immobilization in the skin. Images were taken using a ZeissAxionskop2 microimaging system.

Results

Photoaging structurally degrades the MAGP-1 protein (FIG. 1). The arrows in figure 1 indicate the absence of MAGP-1 fibrils in the papillary dermis. Asterisks indicate degradation of the MAGP-1 structure in the dermis.

Example 3-pore and perifollicular MAGP-1 expression

Using materials and methods similar to those set forth in example 2, the results herein show that MAGP-1 protein levels are significantly reduced around hair follicles/pores due to photoaging (fig. 2). The arrows in fig. 2 indicate high levels of MAGP-1 fibril structure around hair follicles/pores from young subjects; and the arrows indicate the absence of MAGP-1 around hair follicles/pores in light-exposed skin from an aging subject.

Example 4 screening method

Cell processing

Normal human epidermal fibroblasts (Cascade Biologics) were cultured in 100mm plates with 10ml DMEM and 10% serum and at 37 ℃ and 10% CO₂Incubate under conditions for 24 hours. After 24 hours, stock solutions of test compounds/plant extracts are prepared with a suitable solvent (e.g. DMSO). In the presence of 10% CO₂Humidified 37 ℃ incubator of (1) test substance or corresponding medium diluted with growth mediumThe cells were treated with vehicle control for 48 hours. After incubation, growth medium was removed from each plate and 800ul TRIzol reagent (Invitrogen) was added to the plates. The resulting cell lysate was collected and placed in a-80 ℃ freezer until RNA was separated and analyzed.

RNA isolation

RNA was isolated according to the manufacturer's protocol. Briefly, the cell lysate was brought to room temperature and 0.2ml chloroform/1 ml was addedThe tube was shaken vigorously for 15 seconds, incubated at ambient temperature for 2-3 minutes, the sample was centrifuged at 12,000 × g for 15 minutes at 2-8 ℃, after centrifugation, the mixture separated into multiple phases, with only the RNA remaining in the upper aqueous phase.

The aqueous phase was then transferred to a new tube. By mixing with 0.5ml isopropanol/1 ml for initial homogenizationThe sample is incubated at 15-30 ℃ for 10 minutes, centrifuged at 2-8 ℃ at 12,000 × g for 10 minutes, the supernatant is removed, and the RNA is precipitated from the aqueous phase by adding at least 1ml 75% ethanol/1 ml for cell lysisThe reagents were washed 1 time with 75% ethanol RNA pellet samples were mixed by vortexing and centrifugation at 7,500 × g for 5 minutes at 2-8 deg.C the RNA pellet was briefly air dried, RNA was dissolved in RNase free water and total RNA concentration was determined by using a Nanodrop (Thermo Scientific, DE).

Reverse transcriptase and qPCR

Reverse Transcription (RT) reactions were performed in a total volume of 20. mu.l. A mixture of RT containing 20. mu.l 10 XTQMAN RT buffer, 4.4. mu.l 25mM MgCl2, 4.0. mu.l dNTP mix, 1.0. mu.l random hexamer, 0.4. mu.l RNase inhibitor, 0.5. mu.l MultiScriptbe reverse transcriptase (50U/ml), 2.0. mu.g RNA and RNase-free water was prepared to make up a final volume of 20. mu.l RT mix. The reaction system was incubated at 25 ℃ for 10min, 48 ℃ for 30min, and then 95 ℃ for 5min using a Stratagene Mx 3005P QPCR machine. After the RT step, the reaction was stored at-20 ℃ for qPCR analysis.

Quantitative polymerase chain reaction (qPCR) was performed in a total volume of 20 μ l. Applied Biosystems (AB) Universal PCR master mix was used. A mixture containing 10. mu.l of Taqman Universal PCR mix, 1. mu.l of primer and probe mix, 2. mu.l of RT product and 7. mu.l of deionized water was prepared. MAGP-1 and GAPDH primers and probes were purchased from applied biosystems, and the ID of MAGP-1 was Hs00250064_ ml and the accession number of human GAPDH was 4352934E.

The temperature profile of QPCR was 50 ℃ for 2min and 95 ℃ for 10min for 1 cycle; then 95 ℃ for 15sec and 60 ℃ for 1min, 40 cycles. The values of MAGP-1mRNA and the reference gene GAPDH were collected. Values were calibrated to GAPDH to determine the change in MAGP-1mRNA after treatment. The percent increase in mRNA for MAGP-1 was calculated by comparing the test results to the vehicle control.

Table 1 lists the components determined to be active under MAGP-1 stimulated expression.

TABLE 1 MAGP-1 screening data

All percentages are by weight based on the total weight of the composition, unless otherwise indicated.

All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference for all purposes. As will be apparent to those skilled in the art, many modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims (10)

1. A method of improving the aesthetic appearance of skin comprising topically applying a MAGP-1 stimulating substance to an area of skin in need thereof in an amount effective to improve the appearance of skin, wherein the activity of the MAGP-1 stimulating substance has been identified by a MAGP-1 screening assay.

2. The method of claim 1, wherein the MAGP-1 screening assay comprises the steps of: (1) treating human epidermal fibroblasts with a candidate substance; (2) isolating MAGP-1RNA from said cells; (3) amplifying the isolated RNA; and (4) determining whether the level of MAGP-1RNA thus obtained is higher than the level of MAGP-1RNA obtained from cells treated with a control under otherwise identical conditions, wherein an increase in MAGP-1RNA stimulation following treatment with the candidate substance as compared to the level of the control indicates that the substance is a MAGP-1 stimulating substance.

3. The method of claim 1, wherein stimulation of MAGP-1RNA is determined using quantitative polymerase chain reaction.

A method for the screening assay of MAGP-1 comprising the steps of: (1) treating epidermal fibroblasts with a candidate substance to be tested; (2) isolating MAGP-1RNA from said cells; (3) amplifying the isolated RNA.

5. A composition comprising a MAGP-1 stimulating substance in a cosmetically acceptable vehicle, wherein the activity of the MAGP-1 stimulating substance has been identified by a MAGP-1 screening assay.

6. A composition for improving the aesthetic appearance of human skin comprising:

(i) a MAGP-1 active agent selected from the group consisting of:

an extract of May tea (Antidesma bunis);

an extract of boxthorn (Operculin turpethum);

extract of Ixora chinensis (Ixora chinensis);

an extract of Cleriodendron denticulatum (Cleriodendron lindleyi);

a compound having the formula I:

a compound of formula II:

and combinations thereof; and

(ii) a cosmetically acceptable vehicle.

7. The composition of claim 6, further comprising a second MAGP-1 active agent selected from the group consisting of extracts of sesbania esculenta (Sesbaniagandiflora), Amorphophallus konjac (Amorphophalus Campanulatus), Sapindus mukurossi (Sapindus rarak), Tiliaca triandra, Pharbita guinii (Thumbellifera), KFTK, thiodipropionic acid and its C₁–C₄Mono-and dialkyl esters and combinations thereof.

8. A composition for improving the aesthetic appearance of human skin comprising two or more MAGP-1 stimulators in a cosmetically acceptable vehicle.

9. A method for improving the condition or aesthetic appearance of human skin comprising topically applying to skin in need thereof an effective amount of the composition of claim 6, wherein the improvement is selected from the group consisting of:

(a) treating, reducing and/or preventing fine lines or wrinkles;

(b) reduction of skin pore size;

(c) improved skin thickness, plumpness and/or tautness;

(d) improved skin flexibility and/or softness;

(e) improved skin tone, radiance and/or clarity;

(f) improvement in procollagen and/or collagen production;

(g) improved elastin maintenance and remodeling;

(h) skin texture improvement and/or promotion of re-texturing;

(i) skin barrier repair and/or functional improvement;

(j) improvement in skin contour appearance;

(k) restoring skin radiance and/or radiance;

(l) Supplementing essential nutrients and/or ingredients in the skin;

(m) improvement in the decline in skin appearance caused by menopause;

(n) improved skin moisturization;

(o) an increase in skin elasticity and/or resiliency;

(p) treating, reducing and/or preventing skin sagging; or

(r) reduction of pigmented spots; or a combination thereof.

10. A method for improving the condition or aesthetic appearance of human skin comprising topically applying to skin in need thereof an effective amount of the composition of claim 7, wherein the improvement is selected from the group consisting of:

(a) treating, reducing and/or preventing fine lines or wrinkles;

(b) reduction of skin pore size;

(c) improved skin thickness, plumpness and/or tautness;

(d) improved skin flexibility and/or softness;

(e) improved skin tone, radiance and/or clarity;

(f) improvement in procollagen and/or collagen production;

(g) improved elastin maintenance and remodeling;

(h) skin texture improvement and/or promotion of re-texturing;

(i) skin barrier repair and/or functional improvement;

(j) improvement in skin contour appearance;

(k) restoring skin radiance and/or radiance;

(l) Supplementing essential nutrients and/or ingredients in the skin;

(m) improvement in the decline in skin appearance caused by menopause;

(n) improved skin moisturization;

(o) an increase in skin elasticity and/or resiliency;

(p) treating, reducing and/or preventing skin sagging; or

(r) reduction of pigmented spots; or a combination thereof.