Compositions and methods for inhibiting advanced glycation end products

Abstract

A composition for administration to a subject is disclosed. The composition comprises at least one botanical active ingredient. The botanical active ingredient is present in the composition in an amount effective to inhibit advanced glycation end products (AGEs) in the skin of the subject. The botanical active ingredient comprises at least one extract selected from the group consisting of i) Dendrobium officinale flower extract, ii) Gendzin extract, iii) Eucommia ulmoides extract, and iv) a combination of i)-iii). The composition may be in the form of a topical composition, e.g., for topical application. The composition may also be in the form of an oral composition, e.g., for ingestion. A method of inhibiting advanced glycation end products (AGEs) in the skin of a subject is also provided. The method comprises administering an effective amount of the composition to the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and all benefits of Chinese Patent Application No. 202210437608.5, filed on April 25, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates generally to compositions and methods for inhibiting protein glycation, and more specifically to a botanical extract-containing composition for inhibiting advanced glycation end products (AGEs) in the skin of a subject. Related methods are also provided. [Background technology]

[0003] Advanced glycation end products (AGEs) are the end products of a series of chemical reactions initiated by the binding of reducing sugars to free amino groups in proteins, lipids and nucleic acids. AGEs are generally regarded as a complex and heterogeneous group of compounds. Recently, their accumulation in vivo has been implicated as a key pathogenic process in diabetic complications, including peripheral neuropathy, nephropathy, retinopathy and cataracts, as well as other health disorders, such as atherosclerosis, Alzheimer's disease, the pathogenesis of ovarian aging, infertility due to polycystic ovarian syndrome (PCOS) and normal aging. Meanwhile, some highly reactive carbonyl species (RCS) generated during the formation of AGEs are also thought to play an important role in diabetic complications.

[0004] In view of the above, there remains an opportunity to provide new and useful advanced glycation end products (AGE) inhibitors, which may provide potential preventive and therapeutic approaches to reduce the risk of diabetes or other pathogenic complications caused by AGE formation. Thus, there remains an opportunity to provide new and useful compositions and methods for inhibiting AGEs. Summary of the Invention

[0005] A composition for administration to a subject is provided. The composition includes at least one botanical active ingredient. The botanical active ingredient is generally present in the composition in an amount effective to inhibit advanced glycation end products (AGEs) in the skin of the subject. The botanical active ingredient includes at least one extract selected from the group consisting of i) Dendrobium officinale flower extract, ii) Scrophularia ningpoensis extract, iii) Eucommia ulmoides extract, and iv) a combination of i)-iii).

[0006] In various embodiments, the composition is a topical composition formulated for topical administration to a subject, hi other embodiments, the composition is an oral composition formulated for oral administration to a subject.

[0007] The composition can be used to inhibit AGEs in the skin of a subject. A method of inhibiting AGEs in the skin of a subject includes administering to the subject an effective amount of the composition. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a plot showing the mean inhibition of fluorescent advanced glycation end products (AGEs) using rutin as a control.

[0009] [Diagram 2] FIG. 1 is a plot showing the average inhibition of fluorescent AGEs using Dendrobium officinale (flower) ("DOF")-aqueous extract.

[0010] [Diagram 3] FIG. 1 is a plot showing the average inhibition rate of fluorescent AGEs using DOF-ethanol extract.

[0011] [Figure 4] FIG. 1 is a plot showing the average inhibition of fluorescent AGEs using aqueous extract of Gentian root.

[0012] [Diagram 5] FIG. 1 is a plot showing the average inhibition rate of fluorescent AGEs using Eucommia ulmoides (male flowers)-aqueous extract.

[0013] [Figure 6] 1 is a graph showing the effect of DOF-aqueous extract on human dermal fibroblasts (HDF).

[0014] [Figure 7] 13 is an image of the anti-glycation effect of DOF-aqueous extract on HDF.

[0015] [Figure 8] 1 is a flow chart of online detection of antioxidants.

[0016] [Figure 9-1] 1 is a graph showing the antioxidant efficacy of DOF extracts. [Figure 9-2] 1 is a graph showing the antioxidant efficacy of DOF extracts.

[0017] [Figure 10] 1 is a graph showing COX-2 inhibition of DOF hot water extract ("DOF-W").

[0018] [Figure 11-1] 1 is a graph showing the anti-glycation activity of DOF-W. [Figure 11-2] 1 is a graph showing the anti-glycation activity of DOF-W.

[0019] [Figure 12-1] 13 is an image showing the effect of DOF extract on collagen expression by immunofluorescence (IF) staining. [Figure 12-2] 13 is an image showing the effect of DOF extract on collagen expression by immunofluorescence (IF) staining.

[0020] [Figure 13]This is an image of SA-β-gal stained fibroblasts.

[0021] [Figure 14] FIG. 14: Total ion chromatograms of aqueous extracts of samples in both negative and positive ion modes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] A composition for administration to a subject is disclosed. The composition comprises at least one botanical active ingredient. The composition is described below, followed by related uses and methods.

[0023] As can be seen from the present disclosure, the compositions are not particularly limited except for the botanical active ingredients, particularly the extracts thereof, and related ingredients and methods. Thus, the compositions may be formulated, for example, as topical compositions (e.g., cosmetic compositions), or as oral compositions, or as dietary supplements, pharmaceuticals, or supplements, and may be utilized as the sole therapeutic agent in itself or in combination with other therapeutic agents compatible therewith.

[0024] The compositions are useful for treating, preventing and / or ameliorating various conditions, such as conditions associated with aging. Specifically, as will be seen from the following description and examples, the compositions of the present embodiments are believed to be capable of inhibiting protein glycation, and more specifically, inhibiting advanced glycation end products (AGEs) in the skin of a subject.

[0025] Thus, the compositions can be utilized to treat (i.e., slow, prevent, reverse, etc.) age-related or other conditions generally associated with AGEs, such as diabetic complications including neuropathy, nephropathy, retinopathy, and cataracts, as well as other health disorders such as atherosclerosis, Alzheimer's disease, the pathogenesis of ovarian aging, infertility due to polycystic ovarian syndrome (PCOS), and normal aging.

[0026] As introduced above, the composition comprises a botanical active ingredient. More specifically, the botanical active ingredient comprises, optionally consists essentially of, or optionally consists of at least one extract selected from the group consisting of i) Dendrobium officinale flower extract, ii) Gentian extract, iii) Eucommia ulmoides extract, and iv) a combination of i)-iii).

[0027] In certain embodiments, only one of the three extracts i)-iii) is present in the composition. In other embodiments, only two of the three extracts i)-iii) are present in the composition. In yet other embodiments, all three of the extracts i)-iii) are present in the composition. Each of the individual extracts i)-iii) may be referred to simply as a "botanical extract" or collectively as the "botanical extracts," and are described in turn below.

[0028] The term "extract" is used herein in its conventional sense to refer to a composition obtained by fluid extraction from a raw material. Thus, the term "botanical extract" refers to a composition obtained by fluid extraction (e.g., solvent extraction, gas extraction, CO extraction, etc.) from a botanical source (i.e., plant material). 2 The botanical extract suitable for use in the composition can be obtained by any extraction method known in the art, including water extraction, steam extraction, solvent extraction, etc., or a combination of such methods. Exemplary extraction techniques are described below. However, the botanical extract is generally not limited to a specific extraction method or additional / auxiliary techniques used to obtain the botanical extract, but rather can vary according to the parameters described herein. Furthermore, an extraction step is not required to prepare the botanical active ingredient and / or composition, since suitable extracts (e.g., standardized extracts) are readily available from many commercial suppliers.

[0029] Botanical extracts suitable for use in or as botanical active ingredients include those obtained by solvent extraction using, for example, polar solvents such as alcohols (e.g., methanol, ethanol, butylene glycol, etc.), ethers (e.g., diethyl ether, methyl tert butyl ether, etc.), ketones (e.g., acetone), esters (e.g., ethyl acetate), phenol, water, and non-polar solvents such as benzene, xylene, toluene, and derivatives, modifications, and combinations thereof (e.g., solvent-water blends including alcohol-water, acetone-water, etc.). Additional and alternative extraction techniques include sequential fractionation, total hydro-ethanolic extractions, lump-sum extractions, supercritical fluid extraction (e.g., CO2 extraction, etc.), and the like. 2 (e.g., by extraction with a polar solvent), as well as sequential or secondary extractions from a first extraction (e.g., non-polar solvent extraction of a botanical extract obtained from a polar solvent extraction), or other processing techniques such as filtration, purification, distillation, dehydration, evaporation, concentration, drying, etc. Specific examples of suitable extraction methods are described in U.S. Patent No. 7,897,184, which is incorporated herein by reference.

[0030] As understood in the art, various parts or portions of plants such as bark, berries, flowers, fruits, leaves, skin, resin, rhizomes, roots, seeds and / or wood can be used to obtain essential oils and extracts. Essential oils can be obtained by a number of processes such as distillation (e.g., using steam), expression, solvent extraction, absolute oil extraction, resin tapping, and / or cold pressing.

[0031] In various embodiments, the solvent used to obtain a botanical extract suitable for the present disclosure is one in which the resulting botanical extract and / or the resulting form (e.g., botanical extract powder) is suitable for ingestion. For example, the solvent is water or ethanol.

[0032] In one example, the botanical extract can be obtained using organic solvent extraction techniques. In another example, solvent sequential fractionation can be used to obtain the botanical extract. Total water-ethanol extraction techniques can also be used to obtain the botanical extract. Generally, this is called batch extraction. The botanical extract produced by this process contains a wide variety of phytochemicals present in the extracted material, including fat and water-soluble phytochemicals. After collecting the botanical extract solution, the solvent is evaporated to obtain the botanical extract.

[0033] Total ethanol extraction can also be used. This technique uses ethanol as the solvent. This extraction technique produces a botanical extract that may contain fat-soluble and / or lipophilic compounds in addition to water-soluble compounds. Total methanol extraction can also be used in a similar manner with similar results.

[0034] Another example of an extraction technique that can be used to obtain botanical extracts is supercritical fluid carbon dioxide extraction (SFE). In this extraction procedure, the material to be extracted is not exposed to any organic solvent. Rather, the extraction solvent is carbon dioxide (CO ) with or without modifiers at supercritical conditions (e.g., >31.3°C and >73.8 bar). 2 ). One skilled in the art will appreciate that temperature and pressure conditions can be varied to obtain the best yield of botanical extract. This technique produces a botanical extract of fat-soluble and / or lipophilic compounds similar to the total hexane and ethyl acetate extraction technique, which can also be used.

[0035] Each of the above extraction methods may also include and / or be utilized in combination with one or more additional processing steps known in the art. For example, the plant material may be crushed, ground, powdered, etc. There may also be one or more filtration steps, for example, to remove cellulose / fibrous materials or other solid materials. There may also be one or more purification steps, for example, to remove certain components and / or contaminants. Such purification may be performed, for example, by distillation, evaporation, centrifugation, etc. There may also be one or more concentration and / or drying steps to remove water and / or other volatile materials, for example, alcohol, light compounds, VOCs, etc. Furthermore, acids and / or bases may be added to adjust or neutralize pH. Depending on the desired form of the final / destination botanical extract, various additional steps known in the art, for example, screening, pressing, milling, grinding, mixing, dispersion, etc., may also be utilized. It is understood that repetition and / or combination of these additional processing steps in various orders is also envisioned.

[0036] Dendrobium officinale In some embodiments, the botanical active ingredient, and therefore the composition, comprises an extract of Dendrobium officinale flowers, i.e., an extract that includes, or optionally consists essentially of, material from the flowering plant species Dendrobium officinale. The extract of Dendrobium officinale flowers is not particularly limited and may include or be any flower extract or combination of flower extracts from the Dendrobium officinale plant suitable for use in the embodiments herein. More specifically, exemplary Dendrobium officinale flower extracts include those that can inhibit AGEs or induce / exhibit any other such activity described herein as part of the botanical active ingredient.

[0037] Dendrobium officinale has been reported to contain various bioactive components such as polysaccharides, bibenzyls, phenanthrenes, and flavonoids. Dendrobium officinale may simply be referred to as D. officinale, or it may be called by various other names, such as Dendrobium officinale Kimura et. Migo, "Tie Pi Shi Hu", Tiepi Shihu, i.e., Iron Skin Dendrobium.

[0038] Sometimes, Dendrobium officinale is also called "Dendrobium candidum". As further background art, in the Chinese Pharmacopoeia (ChP) of 2005, tiepishihu (Chinese: Iron Skin Dendrobium) was a type of medicinal Dendrobium called Dendrobium candidum Wall ex. Lindl., but this Latin name later became controversial and was regarded as a synonym of Dendrobium moniliforme (L.) Sw. In the ChP of 2010, tiepishihu was renamed Dendrobium officinale Kimura et Migo (alias Dendrobium officinale, i.e., D. officinale).

[0039] Specific examples of Dendrobium officinale flower extracts are known in the art. Thus, Dendrobium officinale flower extracts may be purchased from various sources or otherwise commercially obtained, prepared (e.g., using any conventional extraction technique known in the art, e.g., any of the techniques described herein), or combinations thereof. Specific examples of commercially available extracts include "Freeze Dried Dendrobium candidum Flower Extract Powder" from DAMIN INTERNATIONAL GROUP of Fujian, China, and Dendrobium officinale Kimura et.Migo flower (water) extract powder from QINGYUN SHAN Pharmaceutical of Guangdong, China.

[0040] In certain embodiments, the extract of Dendrobium officinale flowers is obtained by water extraction (or aqueous extraction) of Dendrobium officinale flowers or flower-based plant material. In further or alternative embodiments, the extract of Dendrobium officinale flowers is obtained by alcohol extraction (e.g., ethanol extraction) of Dendrobium officinale flowers or flower-based plant material. The flowers may be fresh or dried, typically dried to prevent spoilage. The dried flowers may then be formed into a powder, which can be used as the extract itself, or more typically, the powdered flowers are further processed to form an extract as described below.

[0041] As known by those skilled in the art, Dendrobium officinale is cultivated primarily for its flowers, which may be from one or more plants and may be fresh, dried, or otherwise aged.

[0042] For example, when Dendrobium officinale flowers are ground to a uniform size in a mill, a certain extract can be obtained. The resulting powder is then extracted using water or ethanol solution. The solution is then filtered and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain the extract.

[0043] In various embodiments, the botanical active ingredient comprises an extract of Dendrobium officinale flowers. In further or alternative embodiments, the composition is substantially or completely free of ingredients derived from plant material that is not Dendrobium officinale flower-based. In these embodiments, the plant material that is not Dendrobium officinale flower-based can be, for example, the root, bark, rhizome, leaves, or stem of the Dendrobium officinale plant. Without wishing to be bound by any particular theory, it is believed that the flowers of Dendrobium officinale are most useful for anti-glycation effects (as exemplified in the Examples section below), and not other parts of the Dendrobium officinale plant.

[0044] A suitable flower extract may be processed (e.g., defatted, partially defatted, ground, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain an extract of Dendrobium officinale flowers. Similarly, Dendrobium officinale flowers may be extracted in raw form or the extract of Dendrobium officinale flowers may be processed and then extracted (e.g., may be used in raw form, suspended form, dehydrated form, concentrated form, etc.).

[0045] The amount of Dendrobium officinale extract utilized in the botanical active ingredient may vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of Dendrobium officinale extract, e.g., 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises Dendrobium officinale extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit can be selected such that the botanical active ingredient comprises an extract of Dendrobium officinale in an amount of <100 mg, <250 mg, <500 mg, <750 mg, <1000 mg, <2000 mg, <5000 mg. In various embodiments, the botanical active ingredient can comprise an amount of Dendrobium officinale extract in an amount of optionally greater than 1 wt% (wt %), optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt%, based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0046] In certain embodiments, the botanical active ingredient comprises two or more Dendrobium officinale extracts, for example, two, three, four, or more Dendrobium officinale extracts, in such embodiments, each Dendrobium officinale extract is independently selected and may be the same or different from any other Dendrobium officinale extract, each utilized in the amounts described above.

[0047] The Dendrobium officinale extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the botanical active ingredient, such as the particular Dendrobium officinale extract utilized, and / or the particular components of the composition. When utilized, it is understood that the Dendrobium officinale extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0048] Genjin In some embodiments, the botanical active ingredient, and thus the composition, comprises a genus extract, i.e., an extract that comprises, or optionally consists essentially of, the material from the plant species genus genus.Genus extract is not particularly limited and may comprise or be any extract or combination of extracts from genus genus suitable for use in the embodiments herein.More specifically, exemplary genus extracts include those that can inhibit AGE or induce / show any other such activity described herein as part of the botanical active ingredient.

[0049] It has been reported that S. ningpoensis contains various bioactive components such as iridoids, phenolic glycosides, phenolic acids, alkaloids, flavonoids, triterpenes, and other compounds. S. ningpoensis may be simply referred to as S. ningpoensis, or by various other names, such as Scrophularia ningpoensis Hemsl., Ningpo figwort, Chinese figwort, "Xuan Shen" (or Radix Scrohulariae), "Hu Huang Lian", "Shen Nong Ben Cao Jing" (or "Shen Nong's Herbal"), Yuan Shen, "Yuan Shen", Xuan Shen, Yuan Shen, or Xuan Shen.

[0050] Specific examples of Gendzin extracts are known in the art. Thus, Gendzin extracts may be purchased from various sources or otherwise commercially obtained, prepared (e.g., using any conventional extraction technique known in the art, such as any of the techniques described herein), or combinations thereof. In certain embodiments, Gendzin extracts are obtained by water extraction (or aqueous extraction) of Gendzin plant material. In further or alternative embodiments, Gendzin extracts are obtained by alcohol extraction (e.g., ethanol extraction) of Gendzin plant material.

[0051] As known by those skilled in the art, Gentian is mainly cultivated for its root, rhizome, and / or root part.Therefore, in various embodiments, Gentian extract is Gentian root extract.Suitable extractions include those mentioned above, for example, root extraction with water and ethanol.Root part may be from one or more plants, and may be fresh, dried, or otherwise aged.

[0052] For example, when Gentian (e.g., root) is ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain the extract.

[0053] The Gendzin extract may include material from any part or combination of parts of the plant, and is not limited to root extract. For example, the Gendzin extract may include material extracted from one or more parts of the Gendzin plant, including its root, stem, bark, rhizome, leaves, buds, flowers, seeds, and / or fruit. Such extracts may also be further processed (e.g., degreased, partially degreased, crushed, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain the Gendzin extract. Similarly, the Gendzin plant may be extracted in raw form or may be processed after the Gendzin extract process (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient includes the Gendzin extract, which includes material obtained (i.e., extracted) from the root of Gendzin.

[0054] The amount of Gendzin extract utilized in the botanical active ingredient can vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of Gendzin extract, for example, 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises Gendzin extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises gensin extract in an amount of <100 mg, <250 mg, <500 mg, <750 mg, <1000 mg, <2000 mg, <5000 mg. In various embodiments, the botanical active ingredient may comprise an amount of gensin extract in an amount optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt%, based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0055] In certain embodiments, the botanical active ingredient comprises more than one Gendzin extract, for example, two, three, four, or more Gendzin extracts, In such embodiments, each Gendzin extract is independently selected and may be the same or different from any other Gendzin extract, each utilized in the amounts described above.

[0056] The Gendzin extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the botanical active ingredient, such as the particular Gendzin extract, utilized and / or the particular components of the composition. When utilized, it is understood that the Gendzin extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0057] Eucommia In some embodiments, the botanical active ingredient, and thus the composition, comprises an Eucommia extract, i.e., an extract that includes, or optionally consists essentially of, a material from the flowering plant species Eucommia.The Eucommia extract is not particularly limited and may include or be any extract or combination of extracts from the Eucommia plant suitable for use in the embodiments herein.More specifically, exemplary Eucommia extracts include those that can inhibit AGEs or induce / show any other such activity described herein as part of the botanical active ingredient.

[0058] Eucommia has been reported to contain various bioactive components such as lignans, iridoids (e.g., iridoid glucoside geniposidic acid), phenols, steroids, terpenoids, and flavonoids. Eucommia is sometimes referred to simply as E. ulmoides, or by various other names, such as Eucommia ulmoides Oliv., duzhong, or Eucommia ulmoides.

[0059] Specific examples of Eucommia extracts are known in the art. Thus, Eucommia extracts may be purchased from various sources or otherwise commercially obtained, prepared (e.g., using any conventional extraction technique known in the art, e.g., any of the techniques described herein), or combinations thereof. In certain embodiments, Eucommia extracts are obtained by water extraction (or aqueous extraction) of Eucommia plant material. In further or alternative embodiments, Eucommia extracts are obtained by alcohol extraction (e.g., ethanol extraction) of Eucommia plant material.

[0060] As known by those skilled in the art, Eucommia is primarily cultivated for its flowers, more primarily for its male (or staminate) flowers. Thus, in various embodiments, the Eucommia extract is an extract of Eucommia male flowers. Suitable extractions include those mentioned above, for example, extraction of male flowers with water and ethanol. The male flowers may be from one or more plants and may be fresh, dried, or otherwise aged.

[0061] For example, when Eucommia ulmoides (e.g., male flowers) are ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered, and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain an extract.

[0062] An Eucommia extract may include materials from any part or combination of parts of the plant, and is not limited to (male) flower extracts. For example, an Eucommia extract may include materials extracted from one or more parts of the Eucommia plant, including its roots, stems, bark, rhizomes, leaves, buds, flowers, seeds, and / or fruits. Such extracts may also be further processed (e.g., defatted, partially defatted, crushed, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain an Eucommia extract. Similarly, an Eucommia plant may be extracted in raw form, or may be processed after Eucommia extract processing (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient comprises an Eucommia extract that includes materials obtained (i.e., extracted) from the male flowers of Eucommia.

[0063] The amount of Eucommia extract utilized in the botanical active ingredient can vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of Eucommia extract, for example, 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises Eucommia extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises an Eucommia extract in an amount of <100 mg, <250 mg, <500 mg, <750 mg, <1000 mg, <2000 mg, <5000 mg. In various embodiments, the botanical active ingredient can comprise an amount of Eucommia extract in an amount optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt%, based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0064] In certain embodiments, the botanical active ingredient comprises two or more Eucommia extracts, for example, two, three, four or more Eucommia extracts, In such embodiments, each Eucommia extract is independently selected and may be the same or different from any other Eucommia extract, each utilized in the amounts described above.

[0065] The Eucommia extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the botanical active ingredient, such as the particular Eucommia extract, utilized and / or the particular components of the composition. When utilized, it is understood that the Eucommia extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0066] supplementary extract In various embodiments, the composition further comprises one or more auxiliary extracts. More specifically, in addition to the Dendrobium officinale flower extract and / or the Gentian extract and / or the Eucommia ulmoides extract (each as described above), the botanical active ingredient (and thus the composition) further comprises, optionally further consists essentially of, or optionally further consists of at least one auxiliary extract selected from the group consisting of i) Phyllanthus emblica extract, ii) Sophora japonica extract, iii) Aronia extract, iv) Punica granatum extract, and v) a combination of i)-iv).

[0067] In certain embodiments, only one of the four auxiliary extracts i)-iv) is present in the composition. In other embodiments, only two of the four auxiliary extracts i)-iv) are present in the composition. In yet other embodiments, only three of the four auxiliary extracts i)-iv) are present in the composition. In yet other embodiments, all four of the auxiliary extracts i)-iv) are present in the composition. Each of the individual auxiliary extracts i)-iv) may be referred to simply as a "botanical extract" or collectively as the "botanical extracts," and are described in turn below.

[0068] Yukan In some embodiments, the botanical active ingredient, and therefore the composition, comprises an extract of Emblica officinalis, i.e., an extract that includes, or optionally consists essentially of, material from the fruit-bearing plant species Emblica officinalis. The Emblica officinalis extract is not particularly limited and may include or be any extract or combination of extracts from the Emblica officinalis plant suitable for use in the embodiments herein.

[0069] It has been reported that it contains various bioactive components, such as antioxidants. It may be simply called P. emblica, or by various other names, such as Phyllanthus emblica L, emblica myrobalan, myrobalan, Indian gooseberry, maracuja, amla, Yu Gan Zi, yu gan zi, Fructus Phyllanthi, or Anmarel.

[0070] Specific examples of Emblica officinalis extracts are known in the art. Thus, Emblica officinalis extracts may be purchased from various sources or otherwise commercially obtained, prepared (e.g., using any conventional extraction technique known in the art, e.g., any of the techniques described herein), or combinations thereof. In certain embodiments, Emblica officinalis extracts are obtained by water extraction (or aqueous extraction) of Emblica officinalis plant material. In further or alternative embodiments, Emblica officinalis extracts are obtained by alcohol extraction (e.g., ethanol extraction) of Emblica officinalis plant material.

[0071] As known by those skilled in the art, Yukan is primarily cultivated for its fruit. Thus, in various embodiments, the Yukan extract is an extract of Yukan fruit or pinyin (embric leafflower fruit). Suitable extractions include those mentioned above, such as extracting fruit with water and ethanol. The fruit may be from one or more plants and may be fresh, dried, or otherwise ripened.

[0072] An extract of Undaria pentaphyllum may include material from any part or combination of parts of a plant, and is not limited to a fruit extract. For example, an extract of Undaria pentaphyllum may include material extracted from one or more parts of Undaria pentaphyllum plant, including its root, stem, bark, rhizome, leaf, bud, flower, seed, and / or fruit. Such extracts may also be further processed (e.g., defatted, partially defatted, crushed, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain an extract of Undaria pentaphyllum. Similarly, Undaria pentaphyllum plant may be extracted in raw form or may be processed after Undaria pentaphyllum extract processing (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient comprises an extract of Undaria pentaphyllum that includes material obtained (i.e., extracted) from the fruit of Undaria pentaphyllum.

[0073] For example, when the Yukan (e.g., fruit) is ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered, and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain the extract.

[0074] The amount of the Yukan extract utilized in the botanical active ingredient can vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of the Yukan extract, for example, 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises the Yukan extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises an amount of Emblica officinalis extract of ≦100 mg, ≦250 mg, ≦500 mg, ≦750 mg, ≦1000 mg, ≦2000 mg, ≦5000 mg. In various embodiments, the botanical active ingredient may comprise an amount of Emblica officinalis extract of optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt% based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0075] In certain embodiments, the botanical active ingredient comprises two or more Emblica officinalis extracts, for example, two, three, four, or more Emblica officinalis extracts. In such embodiments, each Emblica officinalis extract is independently selected and may be the same or different from any other Emblica officinalis extract, each utilized in the amounts described above.

[0076] The Emblica officinalis extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, if present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the particular botanical active ingredient, such as the particular Emblica officinalis extract, utilized and / or the particular components of the composition. When utilized, it is understood that the Emblica officinalis extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0077] Japanese pagoda tree / Styphnolobium japonicum In some embodiments, the botanical active ingredient, and thus the composition, comprises a Sophora japonica extract, i.e., an extract that comprises, or optionally consists essentially of, material from the flowering plant species Sophora japonica. The Sophora japonica extract is not particularly limited and may include or be any extract or combination of extracts from the Sophora japonica plant suitable for use in the embodiments herein.

[0078] It has been reported that Sophora japonica contains various bioactive components, such as the flavonoid glycosides sophoricoside, genistin, and rutin, the flavonoid aglycones genistein, quercetin, and kaempferol, genistein and genistein glycosides, including sophorabiosides, sophoricosides, genistein-7-diglucoside, genistein-7-diglucorhamnoside, and kaempferol and the kaempferol glycosides kaempferol-3-sophoroside and kaempferol-3-rhamnodiglucoside. Sophora japonica may be simply referred to as S. Japonica, or by various other names, such as Styphnolobium japonicum (L.) Schott, pagoda tree, Chinese scholar tree, sophora japonica, and huai.

[0079] Specific examples of Sophora japonica extracts are known in the art. Thus, Sophora japonica extracts may be purchased from various sources or otherwise commercially obtained, prepared (e.g., using any conventional extraction technique known in the art, e.g., any of the techniques described herein), or combinations thereof. In certain embodiments, Sophora japonica extracts are obtained by water extraction (or aqueous extraction) of Sophora japonica plant material. In further or alternative embodiments, Sophora japonica extracts are obtained by alcohol extraction (e.g., ethanol extraction) of Sophora japonica plant material.

[0080] As known by those skilled in the art, Sophora japonica is mainly cultivated for its flowers. Thus, in various embodiments, Sophora japonica extract is an extract of Sophora japonica flower. Suitable extracts include those mentioned above, for example, water and ethanol extracts of flowers. Flowers may be from one or more plants and may be fresh, dried, or otherwise aged.

[0081] The solanum japonica extract may include materials from any part or combination of parts of the plant, and is not limited to flower extracts. For example, the solanum japonica extract may include materials extracted from one or more parts of the solanum japonica plant, including its roots, stems, bark, rhizomes, leaves, buds, flowers, seeds, and / or fruits. Such extracts may also be further processed (e.g., degreased, partially degreased, crushed, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain the solanum japonica extract. Similarly, the solanum japonica plant may be extracted in raw form or may be processed after the solanum japonica extract process (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient comprises a solanum japonica extract that includes materials obtained (i.e., extracted) from the solanum japonica flower.

[0082] For example, when Sophora japonica (e.g., flowers) are ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered, and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain an extract.

[0083] The amount of Sophora japonica extract utilized in the botanical active ingredient may vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises Sophora japonica extract in an amount of 1-2000 mg, for example, 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges may also be utilized. For example, in certain embodiments, the botanical active ingredient comprises Sophora japonica extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises Sophora japonica extract in an amount of ≦100 mg, ≦250 mg, ≦500 mg, ≦750 mg, ≦1000 mg, ≦2000 mg, ≦5000 mg. In various embodiments, the botanical active ingredient may comprise an amount of Sophora japonica extract in an amount optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt%, based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0084] In certain embodiments, the botanical active ingredient comprises two or more Sophora japonica extracts, for example, two, three, four, or more Sophora japonica extracts, in such embodiments, each Sophora japonica extract is independently selected and may be the same or different from any other Sophora japonica extract, each utilized in the amounts described above.

[0085] The Sophora japonica extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the botanical active ingredient, such as the particular Sophora japonica extract, utilized and / or the particular components of the composition. When utilized, it is understood that the Sophora japonica extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0086] Aronia In some embodiments, the botanical active ingredient, and therefore the composition, comprises an Aronia extract, i.e., an extract that includes, or optionally consists essentially of, material from the fruit-bearing plant genus Aronia. The Aronia extract is not particularly limited and may include or be any extract or combination of extracts from the Aronia plant suitable for use in the embodiments herein.

[0087] Aronia has been reported to contain various bioactive components including polyphenol compounds such as anthocyanins. Aronia is a genus of deciduous shrubs of chokeberry. Chokeberry is different from choke cherry. The Aronia genus is thought to have three species: Aronia arbutifolia (or Photinia pyrifolia), also simply called red chokeberry; Aronia melanocarpa (or Photinia melanocarpa), also simply called black chokeberry; and Aronia prunifolia (or Photinia floribunda), also simply called purple chokeberry. The black species generally contains higher levels of anthocyanins than the purple or red species, while the red and purple species are generally richer in phenolic acids and proanthocyanins. Common Chinese names for Aronia include wild chokeberry, aronia berry, and black chokeberry.

[0088] Specific examples of Aronia extracts are known in the art. Thus, Aronia extracts may be purchased from various suppliers, or otherwise obtained commercially, or prepared (e.g., using any conventional extraction technique known in the art, such as any of the techniques described herein), or combinations thereof. In certain embodiments, the Aronia extract is obtained by water extraction of Aronia plant material. In further or other embodiments, the Aronia extract is obtained by alcohol extraction (e.g., ethanol extraction) of Aronia plant material.

[0089] As is known to those skilled in the art, Aronia is mainly cultivated for its fruits. Thus, in various embodiments, the Aronia extract is an extract of Aronia fruits. Suitable extractions include those described above, e.g., extraction of the fruits with water and ethanol. The fruits may be from one or more plants and may be fresh, dried, or otherwise ripened.

[0090] The Aronia extract may include materials from any part or combination of parts of the plant, and is not limited to fruit extracts. For example, the Aronia extract may include materials extracted from one or more parts of the Aronia plant, including its roots, stems, bark, rhizomes, leaves, buds, flowers, seeds, and / or fruit. Such extracts may also be further processed (e.g., defatted, partially defatted, milled, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain the Aronia extract. Similarly, the Aronia plant may be extracted in its raw form or may be processed after the Aronia extract process (e.g., used in its raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient comprises an Aronia extract that includes materials obtained (i.e., extracted) from the Aronia fruit. In certain embodiments, the Aronia extract is simply a powder of Aronia fruit.

[0091] For example, when aronia (e.g., fruit) is ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain the extract.

[0092] The amount of aronia extract utilized in the botanical active ingredient may vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of aronia extract, e.g., 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises an amount of aronia extract of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises Aronia extract in an amount of <100 mg, <250 mg, <500 mg, <750 mg, <1000 mg, <2000 mg, <5000 mg. In various embodiments, the botanical active ingredient may comprise an amount of Aronia extract in an amount of optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt%, based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0093] In certain embodiments, the botanical active ingredient comprises more than one Aronia extract, e.g., two, three, four, or more Aronia extracts. In such embodiments, each Aronia extract is independently selected and may be the same or different from any other Aronia extract, each utilized in the amounts described above.

[0094] The aronia extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. When utilized, the carrier vehicle is selected based on the particular botanical active ingredient, such as the particular aronia extract, and / or particular components of the composition to be utilized. When utilized, it is understood that the aronia extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0095] Pomegranate In some embodiments, the botanical active ingredient, and therefore the composition, comprises a pomegranate extract, i.e., an extract that includes, and optionally consists essentially of, material from the fruit-bearing plant species pomegranate, which is not particularly limited and may include or be any extract or combination of extracts from the pomegranate plant suitable for use in the embodiments herein.

[0096] Pomegranate is reported to contain various bioactive components including various phytochemicals. Pomegranate may simply be referred to as pomegranate or P. granatum, or by various other names such as sweet pomegranate, sweet granatum, Tian Shi Liu, Tian Jiang, Fructus Punicae Granati, pomegranate peel, or Shi Liu Pi. Generally, the most abundant phytochemicals in pomegranate juice are polyphenols, which include hydrolyzable tannins called ellagitannins that are produced when ellagic acid and gallic acid combine with carbohydrates to form punicalagin, also known as pomegranate ellagitannin. The red color of the juice is derived from anthocyanins such as delphinidin, cyanidin, and pelargonidin glycosides. Generally, juice pigmentation increases during fruit maturation. The pomegranate peel contains a large amount of polyphenols, condensed tannins, catechins, and prodelphinidins. Pomegranate seed oil contains punicic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid.

[0097] Specific examples of pomegranate extracts are known in the art. Thus, pomegranate extracts may be purchased from various suppliers, or otherwise obtained commercially, prepared (e.g., using any conventional extraction technique known in the art, such as any of the techniques described herein), or combinations thereof. In certain embodiments, the pomegranate extract is obtained by water extraction of pomegranate plant material. In further or other embodiments, the pomegranate extract is obtained by alcohol extraction (e.g., ethanol extraction) of pomegranate plant material.

[0098] As known to those skilled in the art, pomegranate is mainly cultivated for its fruit. Thus, in various embodiments, the pomegranate extract is an extract of pomegranate fruit. Suitable extractions include those described above, such as extraction of the fruit with water and ethanol. The fruit may be from one or more plants and may be fresh, dried, or otherwise aged.

[0099] Pomegranate extract may include materials from any part or combination of parts of the plant, and is not limited to fruit extract. For example, pomegranate extract may include materials extracted from one or more parts of the pomegranate plant, including its root, stem, bark, rhizome, leaves, buds, flowers, seeds, and / or fruit. Such extracts may also be further processed (e.g., defatted, partially defatted, crushed, dried, precipitated, washed, filtered, sieved, extracted, distilled, concentrated, etc.) to obtain pomegranate extract. Similarly, pomegranate may be extracted in raw form or may be processed after pomegranate extract processing (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.). In certain embodiments, the botanical active ingredient comprises a pomegranate extract that includes materials obtained (i.e., extracted) from pomegranate fruit. In certain embodiments, the pomegranate extract is simply a powder of pomegranate fruit.

[0100] For example, when pomegranate (e.g., fruit) is ground to a uniform size in a mill, a specific extract can be obtained. The resulting powder is then extracted with water or ethanol solution. The solution is then filtered and the filtrate is concentrated under reduced pressure to obtain a syrup. The syrup can then be freeze-dried to dryness to obtain the extract.

[0101] The amount of pomegranate extract utilized in the botanical active ingredient may vary and is selected based on the number and type of ingredients utilized in the botanical active ingredient. In certain embodiments, the botanical active ingredient comprises 1-2000 mg of pomegranate extract, for example, 1-1000 mg, optionally 2-800 mg, optionally 20-750 mg, or optionally 50-500 mg. However, amounts outside these ranges can also be utilized. For example, in certain embodiments, the botanical active ingredient comprises pomegranate extract in an amount of at least 1 mg, optionally at least about 10 mg, optionally at least about 20 mg, optionally at least about 50 mg, optionally at least 100 mg, optionally at least 250 mg, optionally at least 500 mg, optionally at least 1000 mg, or optionally at least 1500 mg. In these or other embodiments, the upper limit may be selected such that the botanical active ingredient comprises pomegranate extract in an amount of <100 mg, <250 mg, <500 mg, <750 mg, <1000 mg, <2000 mg, <5000 mg. In various embodiments, the botanical active ingredient can comprise an amount of pomegranate extract in an amount optionally greater than 1 wt%, optionally greater than 5 wt%, optionally greater than 10 wt%, optionally greater than 25 wt%, optionally greater than 50 wt%, optionally greater than 75 wt%, optionally greater than 80 wt%, or optionally greater than 95 wt% based on the total weight of the botanical active ingredient. In such embodiments, the upper limits can be selected to be approximately ≦10 wt%, ≦20 wt%, ≦30 wt%, ≦40 wt%, ≦50 wt%, ≦60 wt%, ≦70 wt%, ≦80 wt%, ≦90 wt%, and ≦99 wt%, respectively, based on the total weight of the botanical active ingredients.

[0102] In certain embodiments, the botanical active ingredient comprises more than one pomegranate extract, for example, two, three, four or more pomegranate extracts, In such embodiments, each pomegranate extract is independently selected and may be the same or different from any other pomegranate extract, each utilized in the amounts described above.

[0103] The pomegranate extract can be utilized in any form, such as undiluted (i.e., without the presence of a solvent, carrier vehicle, diluent, etc.) or in a carrier vehicle, such as a solvent or dispersion medium. Carrier vehicles, when present, include aqueous solvents (e.g., water), organic solvents, fluids, oils, etc., or combinations thereof. If utilized, the carrier vehicle is selected based on the botanical active ingredient, such as the particular pomegranate extract, utilized and / or the particular components of the composition. If utilized, it is understood that the pomegranate extract may be combined with the carrier vehicle before, during, or after combination with the botanical active ingredient and / or any other components of the composition.

[0104] definition In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided.

[0105] "Ameliorating at least one sign of aging" and "ameliorating a sign of aging" are used interchangeably herein to refer to preventing, arresting, reversing, improving, attenuating, and / or reducing the signs of aging. Representative signs of aging include, but are not limited to, fine lines, wrinkles, crow's feet, dark circles around the eyes, age spots, linear skin atrophy, or combinations thereof.

[0106] "Improving the appearance of skin" and "improving the aesthetic appearance of skin" are used interchangeably herein to refer to the aesthetic improvement of the appearance of skin. Representative improvements include, but are not limited to, favorable characteristics and / or properties related to skin thickness, elasticity, resilience, moisturization, smoothness, tone, texture, radiance, luster, brightness, clarity, contour, firmness, tautness, suppleness, softness, sensitivity, pore size, or combinations thereof. These terms may also be used to refer to the improvement of adverse skin conditions. Exemplary adverse conditions affected by, caused by, or resulting from such adverse skin conditions include, but are not limited to, psoriasis, eczema, seborrhea, dermatitis, sunburn, estrogen disturbances, hyperpigmentation, hypopigmentation, discoloration, sallowness, freckles, atrophy of the skin, skin breakouts, fragile skin, dryness, roughness to the touch, cracking, thinning hair, thickening, fibrosis, enlarged pores, cellulite formation, bruising, acne formation, apoptosis, cell differentiation, cell dedifferentiation, tumor induction or prevention of tumor progression, viral infection, fungal infection, bacterial infection, spider veins (telangiectasia), hirsutism, rosacea, itch, calluses, warts, corns, or combinations thereof.

[0107] The term "composition" or "formulation" refers to a product that treats, improves, promotes, enhances, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition associated with a natural state, biological process, or disease or disorder. For example, the composition or formulation improves at least one sign of aging skin in a subject and / or minimizes or inhibits glycation in the skin (e.g., mature skin). The term composition and formulation includes, but is not limited to, pharmaceutical (i.e., drug), over-the-counter (OTC), cosmetic, food, food ingredient, or dietary supplement composition that includes an effective amount of the extract, at least one component thereof, or a mixture thereof. Exemplary compositions and / or formulations include creams, cosmetic lotions, packs or powders, or emulsions, lotions, foam liniments, tablets, salves, granules, or ointments. Preferred compositions are formulated for topical application / administration and oral administration / ingestion.

[0108] As used herein, the term "effective amount" or "therapeutically effective amount" of a pure compound, composition, extract, mixture of extracts, components of an extract, and / or active agent or ingredient, or combinations thereof, refers to an amount effective at a dosage and duration sufficient to obtain a desired result. For example, "effective amount" or "therapeutically effective amount" refers to an amount of a pure compound, composition, extract, botanical extract, mixture of extracts, mixture of botanical extracts, components of an extract, and / or active agent or ingredient, or combinations thereof, sufficient when administered to a subject (e.g., a mammal such as a human) to achieve a treatment such as improvement of aged skin and / or minimization or inhibition of glycation in mature skin. The amount of a composition, extract, botanical extract, mixture of extracts, mixture of botanical extracts, components of an extract, and / or active agent or ingredient of the present disclosure that constitutes an "effective amount" or "therapeutically effective treatment" varies depending on the active agent or compound, the condition being treated and its severity, the mode of administration, the duration of the treatment, or the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art in view of his or her knowledge and the present disclosure.

[0109] The term "pharmaceutical acceptable" means drugs, medicines, extracts, or inert ingredients suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, etc., and commensurate with a reasonable benefit / risk ratio.

[0110] The terms "applying" and "administering" are defined as providing a composition to a subject via routes known in the art, including but not limited to topical, intravenous, intraarterial, oral, parenteral, buccal, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In preferred embodiments, topical and / or oral routes of administration of the described compositions are preferred.

[0111] The terms "minimize," "reduce," "suppress," "reduce," and / or "inhibit" refer to a decrease or reduction in the activity and / or expression of a protein, and / or downstream effects thereof, in the presence of a botanical (plant) component or botanical extract described herein, as compared to the activity and / or expression of the protein in the absence of a botanical component or botanical extract described herein, such as a control sample. The extent of the decrease or inhibition of the activity and / or expression of a protein, and / or downstream effects thereof, will vary depending on the nature and amount of the botanical component or botanical extract present, but will be manifested, for example, as a detectable decrease in the activity and / or expression of the protein, desirably greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, or about 99% (or any degree of decrease ranging from about 5% to about 99%), as compared to the activity and / or expression of the protein in the absence of the botanical component or botanical extract. For example, a composition including a plant component or plant extract of Dendrobium officinale may minimize or reduce glycation in skin, such as mature skin.

[0112] As used herein, the term "subject" or "individual" includes a mammal to which a composition can be administered. Non-limiting examples of mammals include humans, non-human primates, rodents (including transgenic and non-transgenic mice), and the like. In some embodiments, the subject is a mammal, and in some embodiments, the subject is a human.

[0113] Composition (or Formulation) The composition may contain any amount of botanical active ingredient, which is selected based on the number and type of ingredients utilized in the composition as a whole. Generally, the botanical active ingredient is present in the composition in an amount effective to inhibit AGEs in the skin of a subject.

[0114] In certain embodiments, the composition includes the botanical active ingredient in an amount of 1-5000 mg, optionally 2-2000 mg, optionally 5-1750 mg, optionally 10-1500 mg, optionally 15-1250 mg, optionally 20-1000 mg, optionally 25-750 mg, optionally 30-500 mg, optionally 35-500 mg, optionally 40-500 mg, optionally 45-450 mg, optionally 50-450 mg, or optionally 50-400 mg. However, amounts outside of and / or overlapping these ranges can also be utilized. For example, it is understood that the above ranges regarding the amount of each botanical extract in the botanical active ingredient can be equally applied to the amount of each botanical extract in the entire composition, such as when the botanical active ingredient consists of only one of the botanical extracts.

[0115] In a specific embodiment of the composition, the formulation comprises Dendrobium officinale flower extract, Phyllanthus officinalis fruit extract, Sophora japonica flower extract, Aronia berry powder, and Pomegranate berry powder. Further specific embodiments are illustrated in Table A below. [Table 1]

[0116] In Table A above, the flavor essence and sugar substitute may be any type of conventional ingredient known in the art, such as flavoring agents. The plant (or botanical) extract may each be as described above. Examples of suitable flavoring agents are further described below.

[0117] In general, the compositions are not limited with respect to formulation, peripheral ingredients, form, number of functions, etc., other than that they comprise botanical active ingredients and botanical extracts thereof. Rather, the compositions may vary and may be formulated in any manner consistent with the present disclosure.

[0118] Typically, the compositions are formulated or otherwise adapted for administration to a mammalian subject (e.g., a human). For example, in various embodiments, the compositions are adapted to be administered topically or ingested and / or orally to a human subject.

[0119] In certain embodiments, the composition is further defined as a topical composition formulated for topical administration to a subject.In such embodiments, the composition may also be referred to as a cosmetic composition, and typically includes at least one cosmetically acceptable carrier in addition to the bioactive agent composition.In certain embodiments, the cosmetically acceptable carrier is not naturally occurring.In other words, the carrier is not a natural product in these particular embodiments.In other embodiments, the carrier is selected from conventional carriers known in the art and can be used in conventional amounts.

[0120] In other specific embodiments, the composition is further defined as an oral composition formulated for oral administration to a subject. In such embodiments, the composition may also be referred to as an ingestible composition, and typically includes at least one pharma- ceutically acceptable additive in addition to the bioactive agent composition. In certain embodiments, the pharma-ceutically acceptable additive is not naturally occurring. In other words, the pharma-ceutically acceptable additive is not a natural product in these specific embodiments. In other embodiments, the pharma-ceutically acceptable additive is selected from conventional additives known in the art and can be used in conventional amounts.

[0121] It is therefore understood that the particular additives, carriers, adjuvants, fillers, etc. present in or combined with the composition may vary. Furthermore, the physical form of the composition is not limited and is selected based on the particular components of the composition, the desired use of the composition, etc. Thus, as will be understood in light of the description herein, the composition can be formulated as a liquid, dry powder, suspension, emulsion, gel, paste, etc., and combinations thereof. In certain embodiments, the composition is formulated as a sterile, non-pyrogenic liquid solution or suspension, coated capsule, suppository, lyophilized powder, transdermal patch, soft gel, or other form known in the art. Other examples of suitable forms include solids, gels, liquids, creams, lotions, pomades, mousses, powders, foams, sprays, ointments, or other preparations in which the botanical active ingredient is in a suitable carrier vehicle, such as any of those described herein. In certain embodiments, the composition is formulated or otherwise provided as an eye cream or mask for topical application.

[0122] The composition can be prepared by various methods. For example, the active material of the composition (such as botanical extracts) and optionally one or more inactive materials (such as one or more conventional ingredients, additives, excipients) can be mixed or blended, and compressed or compounded by utilizing various techniques known in the art. The composition of the present disclosure is not limited to a specific order of steps or method of manufacture.

[0123] In various embodiments, the composition is administered topically by application to the skin of a subject.The subject is typically a human being, and can include males and females of various ages.The method / composition of the present disclosure is not limited to a specific subject.

[0124] The composition may be in various forms. Examples of suitable forms include solids, gels, and liquids. For example, the composition may be formulated for application as a gel, cream, lotion, pomade, mousse, powder, or foam for application to the skin of a subject. In another example, the composition may be formulated for spraying onto the skin of a subject. The composition may be formulated to be sprayed as either an aerosol spray or a pump spray. In yet another example, the composition may be formulated for application using a pre-moistened towelette. In another example, the composition may be formulated as a solid that is rubbed into the skin of a subject. In another example, the composition is formulated for delivery via a patch that is adhered to the skin of a subject.

[0125] In addition to the botanical active ingredient (i.e., "active substance" or "active ingredient"), the compositions may contain pharma- ceutically acceptable additives, which are inert substances (or "inactive ingredients") including, but not limited to, excipients, such as diluents and binders, granulating agents, glidants (or flow aids), fillers, lubricants, preservatives, stabilizers, coatings, disintegrants, flavors, and dyes. The active ingredients and pharma-ceutically acceptable additives can be combined or compounded as desired to form individual doses that, when applied topically, provide a desired amount of the active ingredient to a human subject.

[0126] Optionally, the composition may include one or more additional components such as additives.Suitable additives include those known in the art, including, but not limited to, moisturizers, emollients, emulsifiers, surfactants, oils, extracts, skin protectants, disinfectants, bactericides, drugs and drug substances, analgesic compounds, anti-neuralgic compounds, antioxidants, blood circulation enhancers, antidepressant compounds, anti-anxiety compounds, anti-stress compounds, sunscreens, insect repellents, preservatives, scrubbing agents, fragrances, colorants, fillers, solvents, vehicles, carriers, other types of additives known to those skilled in the art, and combinations thereof.Such additives may be utilized alone or in combination.Generally, any additive may be of any type used in personal care products and cosmetics.

[0127] Excipients can be further classified as other ingredients. Specifically, excipients used in oral solid dosage forms have been classified into groups based on their functionality, such as diluents, disintegrants, binders, compression aids, granulating agents, glidants, lubricants, release control polymers, stabilizers (such as antioxidants, chelating agents, and pH adjusters), film coating polymers, coating agents, vehicles, plasticizers, surfactants, colorants, sweeteners, and flavoring agents.

[0128] In various embodiments, the composition comprises at least one component selected from the group consisting of binders, lubricants, glidants, and combinations thereof. In certain embodiments, the composition comprises one or more compounds including, but not limited to, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, cellulose acetate phthalate, gum arabic, gums, waxes, glycerol monostearate, acrylic acid polymers and copolymers, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, ethyl methacrylate, lactose, calcium sulfate, calcium hydrogen phosphate, sugar, microcrystalline cellulose (MCC), starch, sodium starch glycolate, polyvinylpyrrolidone, polyethylene glycol, and magnesium stearate. Combinations of such components can be utilized, and such components and other components used in conventional tablets are known in the art.

[0129] As used herein, a "diluent" can be an inert substance added to increase the bulk of a composition so that tablets are of a practical size for compression. Thus, they may also be called fillers. Commonly used diluents include, but are not limited to, microcrystalline cellulose (MCC), wood cellulose, corn starch, modified corn starch, calcium (tri)phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, (powdered) sugar, dextrose, mannitol, sorbitol, and the like. Diluents / fillers can be used alone or in various mixtures and can be utilized in any amount known in the art for oral compositions.

[0130] As used herein, a "flavoring agent" is a compound intended to impart a more palatable taste to the composition. Flavoring agents vary considerably in their chemical structure, ranging from simple esters, alcohols, and aldehydes to carbohydrates and complex volatile oils. Almost any desired type of synthetic flavor is currently available and is well known in the art. In cases where the flavoring or flavoring agent can counteract the astringent, bitter, or sour taste inherent to the raw material, the composition may include acidulants (e.g., citric acid, tartaric acid, malic acid, ascorbic acid, etc.), sweeteners (e.g., sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia, thaumatin, etc.), flavorings (e.g., various fruit flavors including lemon oil, orange oil, strawberry, etc., and yogurt, mint, menthol, etc.). Flavoring agents may be used alone or in various mixtures and may be utilized in any amount known in the art of oral compositions.

[0131] As used herein, a "lubricant" is a material that serves multiple functions with respect to the composition. In certain embodiments, as in tablet manufacturing, the lubricant serves one or more functions, such as improving the flow rate of tablet granulation, preventing adhesion of tablet material to the surface of the die or punch, reducing interparticle friction, and facilitating the ejection of the tablet from the die cavity. Examples of suitable lubricants include, but are not limited to, zinc stearate, gum arabic powder, cocoa butter, carnauba wax, carmellose calcium, carmellose sodium, caropeptide, hydrous silicon dioxide, dried aluminum hydroxide gel, glycerin, magnesium silicate, light anhydrous silicic acid, light liquid paraffin, crystalline cellulose, hydrogenated oil, synthetic aluminum silicate, sesame oil, wheat flour starch, white beeswax, magnesium oxide, dimethylpolysiloxane, potassium sodium tartrate, sucrose fatty acid ester, glycerin fatty acid ester, silicone resin, aluminum hydroxide gel, stearyl alcohol, stearic acid, aluminum stearate, stearic acid, glycerin fatty ... Examples of lubricants include calcium phosphate, polyoxyl stearate, magnesium stearate, cetanol, gelatin, talc, magnesium carbonate, precipitated calcium carbonate, corn starch, lactose, hard fat, saccharose, potato starch, hydroxypropyl cellulose, fumaric acid, sodium stearyl fumarate, polyethylene glycol, polyoxyethylene polyoxypropylene glycol, polysorbate, beeswax, magnesium aluminometasilicate, methylcellulose, Japan wax, glycerol monostearate, sodium lauryl sulfate, calcium sulfate, magnesium sulfate, liquid paraffin, phosphoric acid, palmitic acid, hydrogenated vegetable oils and fats. Lubricants can be used alone or in various mixtures and can be utilized in any amount known in the art of oral compositions.

[0132] As used herein, a "binder" is an agent used to impart cohesive qualities to powdered materials. Binders, sometimes called "granulating agents," impart cohesiveness to tablet formulations, thereby ensuring that the tablet remains intact after compression and improving free flow by the formulation of granules of the desired hardness and size. Materials commonly used as binders include starches such as cornstarch and pregelatinized starch, sugars such as sucrose, glucose, dextrose, molasses, lactose, natural and synthetic gums such as gum arabic, sodium alginate, Irish moss extract, breadwah gum, gum ghatti, psyllium mucilage, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, microcrystalline cellulose, microcrystalline dextrose, amylose, larch arabinogalactan, ethylcellulose, cellulose acetate, and the like. Binders can be used alone or in various mixtures and can be utilized in any amount known in the art of oral compositions.

[0133] As used herein, a "coloring agent" is an agent that gives the composition a more pleasing appearance and also aids the manufacturer in controlling the product during its preparation and aids the user in identifying the product. Any of the approved water-soluble FD&C dyes, mixtures thereof, or their corresponding lakes can be used to color the tablets. A color lake is the combination of a water-soluble dye by adsorption to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye. Coloring agents can be used alone or in various mixtures and can be utilized in any amount known in the art of oral compositions.

[0134] Other conventional ingredients that may optionally be present in the composition include preservatives, stabilizers, anti-adherents, or silica flow regulators or glidants such as silicon dioxide. Such ingredients may be used alone or in various mixtures and may be utilized in any amounts known in the art for oral compositions.

[0135] It is understood that certain ingredients or additives may be classified under different terminologies, and just because an ingredient or additive is classified under such a term does not mean that it is limited to that function. When utilized, an additive or additives can be present in the composition in various amounts. Additional ingredients for optional use in the compositions, for example when adapted for topical or oral administration, can be found in U.S. Pat. Nos. 5,747,006, 5,980,904, 6,994,874, 7,060,304, 7,247,321, 7,348,034, 7,364,759, 7,700,110, 7,722,904, 8,202,556, 8,916,212, 9,445,975, 9,801,809, 10,307,366, 10,532,024, and 10,537,516, as well as in U.S. Pat. Nos. 2006 / 0257509, 2007 / 0224154, 2008 / 0081082, 2008 / 0124409, 2013 / 0302265, 2017 / 0252293, 2017 / 0281666, 2018 / 0200285, 2019 / 0083566, 2019 / 0160117, 2020 / 0171117, 2020 / 0383898, 2021 / 0017240, and 2021 / 0212926, the disclosures of which are incorporated herein by reference in their entireties.

[0136] Method of administration The composition can be administered or applied daily, several times a day, or in any suitable regimen as needed to obtain the desired outcome. In the method of the present disclosure, the frequency of administration (e.g., topical application) can vary depending on several factors, including the desired level of AGE inhibition. In general, the regimen includes application of the composition once or twice a day, including morning administration and / or evening administration. The amount and / or frequency of application of the composition can vary depending on several factors, including the desired level of outcome and the specific composition.

[0137] The formulations of the present invention can be administered externally, internally, or any combination thereof to provide improved skin appearance. Preferably, the formulations of the present invention are administered with an acceptable carrier. For example, the formulations of the present invention can be administered externally in the form of gels, lotions, creams, tonics, emulsions, etc., with an acceptable carrier. As a further example, the formulations of the present invention can be administered internally in the form of pills, tablets, powders, bars, beverages, etc., with an acceptable carrier. Thus, the formulations described herein are useful in a wide variety of end products, including pharmaceutical products, food, and beverage compositions. Preferably, the products are useful for providing an improved appearance to mammalian skin.

[0138] When the formulation of the present invention is orally administered in liquid form, the liquid may be water-based, milk-based, tea-based, fruit juice-based, or some combination thereof. The solid and liquid formulations for oral administration of the present invention may further include thickening agents, including xanthan gum, carboxymethylcellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose, microcrystalline cellulose, starch, dextrin, fermented whey, tofu, maltodextrin, polyols including sugar alcohols (e.g., sorbitol and mannitol), carbohydrates (e.g., lactose), propylene glycol alginate, gellan gum, guar, pectin, tragacanth gum, arabic gum, locust bean gum, arabic gum, gelatin, and mixtures of these thickening agents. These thickening agents are typically included in the formulation of the present invention at levels up to about 0.1%, depending on the particular thickening agent involved and the viscosity effect desired.

[0139] The solid and liquid (food and beverage) formulations of the present invention can, and typically do, contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and / or artificial non-caloric / low-caloric sweeteners. The amount of sweetener used in the formulations of the present invention varies, but typically depends on the type of sweetener used and the sweetness intensity desired.

[0140] In another example, the formulations of the present invention are typically administered topically in the form of a solution, gel, lotion, cream, ointment, oil-in-water emulsion, water-in-oil emulsion, stick, spray, paste, mousse, tonic, foundation, or other suitable cosmetic and topical form.

[0141] Preferably, the formulation of the present invention suitable for topical administration is mixed with an acceptable carrier.The acceptable carrier can function variously as a solvent, carrier, diluent or dispersion medium for the components of the composition, thereby allowing the components to be applied uniformly on the surface of the skin with appropriate dilution.The acceptable carrier can also facilitate the penetration of the composition into the skin.

[0142] In one example of a formulation for topical application, the acceptable carrier comprises about 70 wt% to about 99.99 wt% of the total composition. In another example, the acceptable carrier comprises about 85 wt% to about 99.99 wt% of the total composition. The acceptable carrier may also comprise about 90 wt% to about 99.99 wt% of the total composition, or about 99.95 wt% to about 99.999 wt% of the total composition. The acceptable carrier may comprise the remainder of the composition in the absence of other cosmetic adjuvants or additives.

[0143] Various components used in carrying out the present invention may be soluble or insoluble in acceptable carrier.When all components of the formulation are soluble in acceptable carrier, the vehicle functions as a solvent.However, when all or some components of the formulation are insoluble in acceptable carrier, those components are dispersed in the vehicle, for example, by suspension, emulsion, gel, cream or paste.

[0144] Therefore, it will be clear to those skilled in the art that the range of acceptable carriers that can be used is very wide. For example, the acceptable carrier may be an emulsion, a lotion, a cream, or a tonic. The acceptable carrier may include water, ethanol, butylene glycol, or various other solvents that aid in skin penetration. Some examples of suitable vehicles are described in U.S. Patent Nos. 6,184,247 and 6,579,516, the entire contents of which are incorporated herein by reference.

[0145] Preferably, the acceptable carrier used in the practice of the invention includes water and ethanol. Optionally, the acceptable carrier also contains butylene glycol. For example, the acceptable carrier may include butylene glycol at 2-5 wt% of the composition. In the practice of the invention, preferably, the acceptable carrier is mixed with the formulation of the invention comprising 2 wt% of the total composition. In other examples, the acceptable carrier is mixed with the formulation of the invention comprising 0.001 wt% to 30 wt% of the total composition, 1 wt% to 5 wt% of the total composition, 0.01 wt% to 15 wt% of the total composition, or 0.5 wt% to 1.0 wt% of the total composition.

[0146] In general, however, acceptable carriers for the present invention include, for example, the following: water, castor oil, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, corn oil, dimethyl sulfoxide, ethylene glycol, isopropanol, soybean oil, glycerin, soluble collagen, safflower seed oil, meadowfoam seed oil, mineral oil, squalene, shea butter, borage oil, or rice bran oil, polyquaternium-10, methylparaben, PEG-8, disodium lauroamphoacetate, sodium trideceth sulfate, hexyl ether ...soluble collagen, soluble collagen, soluble collagen, soluble collagen, soluble collagen, soluble collagen Glycol, sodium methyl cocoyl taurate, TEA lauryl sulfate, lauryl betaine, sodium myristoyl sarcosine, PEG-150 distearate, anhydrous citric acid, sodium citrate dihydrate, diazolidinyl urea, disodium EDTA, propylparaben, polysorbate 60, isopropyl palmitate, octyl palmitate, C12-15 alkyl benzoate, dipropylene glycol dibenzoate, PPG-15 stearyl ether benzoate, isododecane, isoeicosane, squalane, jojoba oil, dimethicone , Glyceryl Stearate, PEG-100 Stearate, Cetyl Alcohol, Butylene Glycol, Chlorphenesin, Fragrance, Polyacrylamide, C13-14 Isoparaffin, Laureth-7, Aloe Vera Powder, Aloe Vera Gel, Hydroxyethyl Acrylate, Sodium Acryloyldimethyltaurate Copolymer, Behenyl Alcohol, Tocopheryl Acetate, Isodecyl Neopentanoate, Trioctanoylglycerol, Cetearyl Alcohol, Cetearyl Glucoside, Chamomile Flower Extract, Biosaccharide Gum-1, Pentadecalacto Ingredients: dipropylene glycol, cyclomethicone, PEG / PPG-18 / 18 dimethicone, cyclopentasiloxane, disteardimonium hectorite, SD alcohol 40, phenoxyethanol, ethylparaben, trimethylsiloxysilicate, triethoxycaprylylsilane, micronized titanium dioxide, titanium dioxide, zinc oxide, iron oxides (yellow, red, black, etc.), caprylylsilane, sodium chloride, diisopropyl dimer dilinoleate, aluminum hydroxide, stearic acid, polyethylene beads, C12-15 alkyl benzoate,Acrylates / C10-30 alkyl acrylate, xanthan gum, sorbitan laurate, panthenol, petrolatum, isopropyl isostearate, dimethicone, arginine, phenoxyethanol, acryloyldimethyltaurate copolymer, isohexadecane, polysorbate 80, hydroxyethyl acrylate, sodium acryloyldimethyltaurate copolymer, octinoxate (ethylhexyl methoxycinnamate), oxybenzone, dicaprylyl ether, isodecyl neopentanoate, cetearyl alcohol, cetearyl glucoside, benzyl alcohol, HDI / trimethylol hexyllactone cropolymer. The following may be included, but are not limited to: glyceryl stearate, ...

[0147] Additionally, acceptable carriers for use in the present invention may optionally contain one or more humectants, including, but not limited to, dibutyl phthalate, soluble collagen, sorbitol, or sodium 2-pyrrolidone-5-carboxylate. Other examples of humectants that can be used in the practice of the present invention can be found in the CFTA Cosmetic Ingredient Handbook, the relevant portions of which are incorporated herein by reference.

[0148] Additionally, acceptable carriers of the present invention include butane-1,3-diol, cetyl palmitate, dimethylpolysiloxane, glyceryl monoricinoleate, glyceryl monostearate, isobutyl palmitate, isocetyl stearate, isopropyl palmitate, isopropyl stearate, butyl stearate, isopropyl laurate, hexyl laurate, decyl oleate, isopropyl myristate, lauryl lactate, octadecyl-2-ol, caprylic triglyceride, capric triglyceride, polyethylene glycol, glyceryl stearate ... The composition may optionally include one or more emollients, including, but not limited to, ricin, propane-1,2-diol, triethylene glycol, sesame oil, coconut oil, safflower oil, isoamyl laurate, nonoxynol-9, panthenol, hydrogenated vegetable oil, tocopheryl acetate, tocopheryl linoleate, allantoin, propylene glycol, peanut oil, castor oil, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, or myristyl myristate. Other examples of emollients that can be used in the practice of the present invention can be found in the CFTA Cosmetic Ingredient Handbook, the relevant portions of which are incorporated herein by reference.

[0149] Additionally, acceptable carriers for use in the present invention may optionally contain one or more penetration enhancers, including, but not limited to, pyrrolidones such as 2-pyrrolidone, alcohols such as ethanol, alkanols such as decanol, glycols such as propylene glycol, dipropylene glycol, butylene glycol, surfactants, or terpenes.

[0150] Other acceptable carriers that can be used in the practice of the present invention will be apparent to those skilled in the art and are included within the scope of the present invention.

[0151] For example, an acceptable carrier may be a topically applied lotion that may contain carbomer 981, water, glycerin, isopropyl myristate, mineral oil, shea butter, stearic acid, glycol stearate, cetyl alcohol, dimethicone, preservatives, tea, and various other components of the formulations of the present invention.

[0152] The formulations of the present invention may also contain various known and conventional cosmetic adjuvants, so long as they do not adversely affect the desired skin improving and moisturizing effects provided by the formulation. For example, the formulations of the present invention may further include one or more additives or other optional ingredients known in the art, including fillers (e.g., solid, semi-solid, liquid, etc.), carriers, diluents, thickeners, gelling agents, vitamins, retinoids, and retinol (e.g., vitamin B 3 , vitamin A, etc.), dyes, fragrances, UV absorbers and UV scattering agents, antioxidants and radical scavengers, organic hydroxy acids, exfoliants, skin conditioners, moisturizers, ceramides, pseudoceramides, phospholipids, sphingolipids, cholesterol, glucosamine, pharma- ceutically acceptable penetrating agents (e.g., n-decylmethylsulfoxide, lecithin organogel, tyrosine, lysine, etc.), preservatives, antibacterial agents, amino acids such as proline, pyrrolidone carboxylic acid, its derivatives and salts, isomerized sugars, buffers with bases such as panthenol, triethanolamine or sodium hydroxide, waxes such as beeswax, ozokerite wax, paraffin wax, plant extracts such as aloe vera, cornflower, witch hazel, elderflower, or cucumber, and combinations thereof. Other suitable additives and / or adjuvants are described in U.S. Pat. No. 6,184,247, the entire contents of which are incorporated herein by reference.

[0153] The formulation may include additional inactive ingredients, including but not limited to surfactants, co-solvents, and excipients. Surfactants, such as hydrophilic and hydrophobic surfactants, may be included in the formulation. A particular surfactant may be used based on the overall composition of the formulation and the intended delivery of the formulation. Useful surfactants include polyethoxylated (PEG) fatty acids, PEG-fatty acid diesters, mixtures of PEG-fatty acid mono- and diesters, polyethylene glycol glycerin fatty acid esters, alcohol-oil transesterification products, polyglycerolated fatty acids, propylene glycol fatty acid esters, propylene glycol ester-glycerin ester mixtures, mono- and diglycerides, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, polysaccharide esters, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof.

[0154] The formulation may also include co-solvents, such as alcohols and polyols, polyethylene glycol ethers, amides, esters, other suitable co-solvents, and mixtures thereof. The formulation may also include excipients or additives, such as sweeteners, flavorings, coloring agents, antioxidants, preservatives, chelating agents, viscosity adjusting agents, tonicity agents, odorants, opacifying agents, suspending agents, binders, and mixtures thereof.

[0155] In general, the formulation of the present invention is administered topically or orally at least every day for a sufficient period of time to obtain the desired level of improvement in skin appearance.Topical application or oral administration of the formulation of the present invention can continue for any suitable period of time.More specifically, within a few hours to a few days after the first application or intake, the user may notice that the skin's appearance has improved.It is understood that the frequency with which the formulation of the present invention should be applied or taken varies depending on the desired level of appearance improvement.In particular, the degree of cosmetic enhancement varies directly with the total amount of the composition used.

[0156] Useful dosage forms can be prepared by methods and techniques that are well known to those skilled in the art and which may involve the use of additional ingredients in the manufacture of tablet, capsule, or liquid dosage forms.

[0157] Industrial Applicability The present disclosure provides novel and useful advanced glycation end products (AGE) inhibitors, including the botanical extracts described herein. Such AGE inhibitors can provide a potential preventive and therapeutic approach to reduce the risk of diabetes or other pathogenic complications caused by AGE formation. Thus, the compositions and methods described herein are useful for inhibiting AGEs.

[0158] The compositions and product items provided by the present disclosure generally relate to skin care and nutritional beauty products that utilize such AGE inhibitors, with specific examples including eye creams and masks that utilize such AGE inhibitors.

[0159] If left unchecked, glycation can cause significant accumulation of AGEs in long-lived macromolecules such as collagen, and the amino acids lysine and arginine can undergo glycation to form crosslinks, reducing skin elasticity, increasing the appearance of wrinkles, and causing impaired healing.Thus, the compositions and methods herein are useful for inhibiting glycation or preventing skin aging, which in turn can increase skin elasticity, reduce the appearance of wrinkles, and otherwise promote skin healing.The compositions and methods of the present disclosure also provide a potential preventive and therapeutic approach to reduce the risk of diabetes or other pathogenic complications caused by AGE formation.

[0160] The following examples are illustrative of the compositions and methods of the present disclosure and are intended to be illustrative, but not limiting, of the disclosure. EXAMPLES

[0161] Initial Testing The inhibitory effect of five aqueous and ethanolic plant extracts on nonenzymatic glycation of proteins was evaluated. Specifically, two Dendrobium officinale flower extracts, one Dendrobium officinale stem extract, one Gentian root extract, and one Eucommia ulmoides male flower extract were evaluated for their ability to inhibit AGE formation. Four of the five extracts showed inhibition as shown in the table and figure below. The Dendrobium officinale stem extract showed no inhibition. The extracts were produced in the laboratory and are also commercially available.

[0162] Nonenzymatic glycation of HSA The anti-glycation ability of the samples was evaluated by measuring the amount of fluorescent AGE. 100 μL of human serum albumin (4 mg / mL HSA) was dissolved in potassium phosphate buffer (0.2 mol / L, pH 7.4, 0.06% NaN 3 The samples were saccharified with 100 μL of D-fructose (0.05 mol / L) and 100 μL of glucose (0.05 mol / L) in the presence of 0.05% ethanol and incubated at 50° C. for 24 hours. The incubated solutions were then used in the following assays. Rutin was used as a positive control.

[0163] Measurement of fluorescent AGEs The fluorescence intensity of the incubated solution was recorded using a microplate reader at λex / λem=360 / 453 nm. The inhibition rate of fluorescent AGE was calculated using the following formula:

number

[0164] The results are shown in the table below, and the average percent inhibition can also be seen by reference to the figures. [Table 2] *See also Figure 1 [Table 3] * See also Figure 2 [Table 4] * See also Figure 3 [Table 5] * See also Figure 4 [Table 6] * See also Figure 5 [Table 7]

[0165] As shown in Table 6 above, the extract of the stem of Dendrobium officinale had little effect on the anti-glycation activity. Thus, surprisingly, the flower of Dendrobium officinale was found to be more effective in providing anti-glycation compared to the stem of Dendrobium officinale. The difference in effectiveness is believed to be mainly based on the content of polysaccharides in the stem, which is extracted by water.

[0166] Evaluation of anti-glycation activity on primary human dermal fibroblast (HDF) cells Dendrobium officinale (flower) aqueous extract was stored at 4 °C before use. Primary HDFs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C under 95% air and 5% CO. 2 The samples were kept in a humidified atmosphere at 37 °C.

[0167] HDF cells were digested from T75 flasks and seeded into several 96-well plates at a density of 10,000 cells per well. After reaching 90% confluence in the wells, the cells were treated with various concentrations of active samples (n=3). After 48 hours, the cells were washed once in Dulbecco's Phosphate Buffered Saline (DPBS) and then the viability was tested by CCK-8 kit according to the manufacturer's guidance. HDFs were further incubated at 37°C for 2 hours. The final reading was taken by measuring the absorbance at 450 nm using a microplate reader.

[0168] Evaluation of anti-glycation activity HDF cells were digested from T75 flasks and seeded at 50,000 cells per well in 12-well culture plates on slides. After 48 hours, cells were treated with 0.5 mM glyoxal for 48 hours. Then, cells were treated with or without positive control or active samples for another 48 hours. At the end of the experiment, the cell slides were washed once with phosphate-buffered saline (PBS) and fixed overnight in polyformaldehyde at 4°C. For staining of chronic myeloid leukemia (CML) biomarkers, slides were blocked in 1% bovine serum albumin (BSA) for 1 hour, then primary CML antibody (1:50) was applied overnight at 4°C. The next day, secondary antibody (1:1000) was applied for 2 hours at room temperature. Slides were stained and mounted with DAPI (4',6-diamidino-2-phenylindole), and relevant fluorescent images were taken by microscope. CML expression was quantified from different randomly selected fields in each group using Image J. Data are presented as mean ± SD and analyzed by Student's t test or one-way ANOVA. * P < 0.05, ** P < 0.01, *** P<0.001. [Table 8] * See also Figure 6

[0169] As shown in Table 7 above, no cytotoxicity was observed in cells treated with various concentrations of Dendrobium officinale (flower) aqueous extract. "AVE" is the average and "SD" is the standard deviation. [Table 9] * See also Figure 7

[0170] As shown in Table 8 above, Dendrobium officinale (flower) aqueous extract provides excellent anti-glycation effects on HDF.

[0171] Evaluation of anti-glycation activity in zebrafish All zebrafish were reared in aquaculture water provided by the fish farming center at 28 °C (water quality: 200 mg of instant sea salt added to 1 L of reverse osmosis water, conductivity 450–550 μS / cm, pH 6.5–8.5, hardness 50–100 mg / L CaCO ). 3 The license number for laboratory animals is SYXK(Zhejiang)2012-0171, and the feeding management was in accordance with the requirements of the international AAALAC certification (certificate number: 001458). Wild-type AB zebrafish were naturally bred in pairs. Five-day-post-fertilization (DPF) zebrafish were used to evaluate the anti-aging efficacy of the samples.

[0172] Five DPF wild-type AB zebrafish were randomly selected and placed into 1.5 mL centrifuge tubes, with 10 zebrafish in each tube (experimental group). Dendrobium officinale flower extract was obtained in aqueous solution with a positive control aminoguanidine hydrochloride (AG) concentration of 200 μg / mL to set up a normal control group and a model control group at the same time. The volume of each tube was 150 μL. Except for the normal control group, the other experimental groups were given 0.4 M glucose solution to establish an AGE increase model in zebrafish. Three parallel experiments were set up. After treatment in a 60 °C shaker for 24 h, the supernatant was centrifuged and the data was collected using a multifunctional microplate reader to analyze the fluorescence value of AGE in each experimental group, and the results of the statistical analysis of this indicator were used to evaluate the anti-aging efficacy of the samples. [Table 10] Comparison with other models ** p<0.01, *** p<0.001

[0173] As shown in Table 9 above, the Dendrobium officinale (flower) aqueous extract exhibits a positive anti-glycation effect. It is therefore believed that such a positive effect may also be exerted on other subjects, including humans.

[0174] Further / confirmatory testing In light of the above initial studies, and without being bound or limited to any particular theory, it is believed that Dendrobium officinale flowers (DOF) can provide many healthful and beneficial effects. Therefore, this study aimed to investigate the in vitro biological efficacy of DOF aqueous extract and screen its active ingredients. A complete summary of this study and its associated supplemental materials can be found in the following article: Zhou H, Zhou L, Li B and Yue R (2023) "Anti-cyclooxygenase, anti-glycation, and anti-skin aging effect of Dendrobium officinale flowers'aqueous extract and its phytochemical validation in aging." Front. Immunol. 14:1095848. The disclosure of this article is incorporated herein by reference in its entirety.

[0175] method Antioxidant tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing capacity (FRAP) of plasma, and intracellular reactive oxygen species (ROS) level analysis in primary human epidermal keratinocytes, anti-cyclooxygenase 2 (COX-2) assay, anti-glycation assay (both fluorescent AGE formation in BSA fructose / glucose system and glycation cell assay), and anti-aging assay (quantification of type I and III collagen, as well as SA-β-gal staining assay) were performed to determine the potential biological effects of DOF extract and its major compounds. Ultra-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight-mass spectrometry (UPLC-ESI-QTOF-MS / MS) was performed to investigate the composition of DOF extract. An online antioxidant post-column bioassay test was applied to rapidly screen the major antioxidants in DOF extract.

[0176] Results and Discussion The aqueous extract of Dendrobium officinale flowers was found to have potential antioxidant capacity, anti-cyclooxygenase 2 (COX-2) effect, anti-glycation efficacy, and anti-aging effect. A total of 34 compounds were identified using UPLC-ESI-QTOF-MS / MS. Online ABTS radical analysis showed that 1-O-caffeoyl-β-D-glucoside, vicenin-2, luteolin-6-C-β-D-xyloside-8-C-β-D-glucoside, quercetin-3-O-sophoroside, rutin, isoquercitrin, and quercetin 3-O-(6''-O-malonyl)-β-D-glucoside were the major potential antioxidants. In addition, all of the selected 16 compounds showed significant ABTS radical scavenging capacity and effective AGE inhibitory activity. However, only certain compounds such as rutin and isoquercitrin showed selective and significant antioxidant potential as indicated by DPPH and FRAP, as well as strong COX-2 inhibitory potential, while the remaining compounds showed relatively weak or no effect, indicating that certain ingredients contributed to various functionalities. These findings demonstrated that DOF and its active compounds targeted related enzymes, highlighting their potential application in anti-aging.

[0177] 1. Background technology Dendrobium officinale flowers are popular in China for their anti-aging properties and beneficial effects on the syndrome of yin and yang deficiency. However, scientific data to confirm the pharmacological effects listed above are limited in the literature. Therefore, the aim of this study was to perform a more systematic experiment on the aqueous extract of Dendrobium officinale, including its biological effects on skin aging, such as antioxidant capacity (by (DPPH), (ABTS), and (FRAP)), anti-inflammatory effect (by COX-2), anti-glycation efficacy (by inhibition of nonenzymatic glycation and inhibition of CML expression in fibroblasts), and anti-aging evaluation (by SA-β-gal staining test and collagen expression).

[0178] The phytochemical composition of Dendrobium officinale was also analyzed using UPLC-ESI-QTOF-MS / MS. Antioxidants in aqueous extracts of Dendrobium officinale flowers were identified using an online ultra-performance liquid chromatography-photodiode array detection-mass spectrometry-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (UPLC-PDA-MS-ABTS+) method, and the biological potential of specific isolated constituents was investigated (by DPPH, ABTS, FRAP, anti-COX-2, and AGE inhibitory activities).

[0179] Previous studies have shown that Dendrobium officinale flowers can alleviate brain aging and improve spatial learning ability in old rats. However, little has been reported about the anti-glycation effect of the aqueous extract of Dendrobium officinale flowers in the literature so far. The online antioxidant study of the aqueous extract of Dendrobium officinale flowers and the various biological activities of its identified compounds described in this study have not been reported before.

[0180] 2. Materials and Methods 2.1 Plant materials, solvents, and chemicals Dendrobium officinale flowers were purchased from Amway (Shanghai) Technology and were authenticated as D. officinale Kimura et Migo flower by Amway Botanical Research Center (China). Analytical grade methanol was purchased from Honeywell (Charlotte, USA). Acetic acid and liquid LS-MS-HPLC grade acetonitrile were purchased from Merck (Darmstadt, Germany). Bovine serum albumin (BSA) lyophilized powder, ABTS (98%), DPPH· (98%), 6-hydroxyl-2578-tetra-methylchroman-2-carboxylic acid (Trolox), tyrosinase, ascorbic acid, NS-398 (COX-2 inhibitor, CAS: 123653-11-2), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Darmstadt, Germany). High glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin stock solution (PS, 10,000 U / mL), phosphate-buffered saline (PBS), and pancreatin solution were purchased from Gibco (Carlsbad, CA, USA). Reference standards vicenin-2, luteolin 6-C-β-D-xyloside-8-C-β-D-glucoside, vicenin-1, luteolin-6-C-β-D-glucoside-8-C-β-D-xyloside, quercetin-3-O-sophoroside, scaftoside, luteolin-6-C-β-D-glucopyranoside, vicenin-3, apigenin-6-C-β-D-glucopyranoside, and vicenin-4, apigenin-6-C-β-D-glucopyranoside were purchased from Gibco (Carlsbad, CA, USA). 6-C-α-L-arabinoside-8-C-β-D-xyloside, quercetin 3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragalin, and isorhamnetin-3-O-glucoside were purchased from Nature Standard Biotech Co. (Shanghai, China). Aminoguanidine hydrochloride (AG) and glucose were purchased from Aladdin (Shanghai, China), and methylglyoxal (MGO) was purchased from Adamas (Delaware, USA).Cell count kit-8 (CCK-8), FRAP kit, and SA-β-gal staining kit were purchased from Beyotime Biotechnology Co. (Shanghai, China). Alexa Fluor® 488 donkey anti-mouse IgG was purchased from Thermo Fisher (Waltham, Massachusetts, USA). Carboxymethyllysine antibody, Alexa Fluor® 488 donkey anti-mouse IgG, Alexa Fluor® 488 donkey anti-rabbit IgG, Alexa Fluor® 568 goat anti-rabbit IgG, collagen type I primary antibody, collagen type III primary antibody, and mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Cambridge, UK). Collagen type I and III enzyme-linked immunosorbent assay (ELISA) kits were purchased from BIO-SWAMP Co. (Wuhan, China). Human dermal fibroblasts (HDFs) were purchased from Archgene Biotechnology Co. (Shanghai, China). Zebrafish were purchased from Hunter Biotechnology Co. (Hangzhou, China). All other chemicals were purchased from Titan Co. (Shanghai, China). Water was purified using a Milli-Q purification system (Barnstead, USA).

[0181] 2.2 Extraction procedure Aqueous extracts of Dendrobium officinale flowers were prepared using a slightly modified method. Briefly, ground flower powder was refluxed twice with distilled water at 100 °C for 1 h at a solid-to-solvent ratio of 1:12 (w / v). The extract was separated by centrifugation (RWB3220CY-2, Eppendorf, Germany) at 19,000 × g for 15 min at 4 °C. The supernatant was evaporated using a large rotary evaporator (Hei-VAP Expert, Heidorf, Schwabach, Germany) until a small volume remained and then freeze-dried using a freeze-dryer. The DOF hot water extract (DOF-W) was then stored at -18 °C until further analysis.

[0182] 2.3 Antioxidant capacity (DPPH·, ABTS·+, and FRAP assays) For in vitro evaluation of DPPH· scavenging, ABTS·+ decolorization capacity, and total antioxidant capacity of FRAP, the rapid colorimetric method was slightly modified. Stock solutions of derivatization reagents were diluted as follows before measurement. DPPH was diluted with absolute ethanol until the absorbance was 0.8 ± 0.05 at λ = 517 nm. ABTS working solution was prepared using phosphate buffer (0.2 M, pH 7.4) to show an absorbance of 0.8 ± 0.05 at λ = 729 nm based on previous tests. FRAP solution was prepared according to the instructions of the FRAP kit. Absorbance was measured using an automated microplate reader (Molecular Group Ltd., USA), and all analyses were performed using 96-well plates. All measurements were performed in triplicate, and Trolox was used as a positive control.

[0183] For DPPH trapping, 100 μL of sample solution was mixed with 100 μL of fresh DPPH ethanol solution and the absorbance of the mixture was measured at 517 nm after 10 min. Ethanol was used as a negative control.

[0184] For the ABTS decolorization ability, 100 μL of ABTS working solution was mixed with 200 μL of sample solution, and the absorbance was recorded at 729 nm after 10 min. Phosphate buffer (0.2 M, pH 7.4) was used as a negative control.

[0185] Both DPPH· and ABTS·+ radical scavenging activities of the test samples were calculated using the following equations: Inhibition %=(A control-A sample) / A 対照 ×100 where A control and A sample represent the absorbance of the control and test sample, respectively. IC 50 was calculated graphically from the dose-inhibition curves.

[0186] For total antioxidant capacity, 180 μL of FRAP solution was mixed with 5 μL of sample solution, and the absorbance was measured at 539 nm after 5 min, with PBS as the negative control. 4Solutions (0.15 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1.2 mM, and 1.5 mM) were used for the calibration curve. FRAP values ​​were calculated using the 4 The corresponding concentration of the solution (mM FeSO 4 Using the regression equation from the standard curve, the equivalent concentration 1 (EC 1 ) was calculated. EC 1 is 1.0 mmol / L FeSO 4 It is defined as the concentration of a test sample that has an absorbance equal to the absorbance of the solution, as determined by its calibration curve.

[0187] 2.4 Antioxidant activity assay in cells 2.4.1 Cell culture and UVB treatment Primary human epidermal keratinocytes (NHEKs) were purchased from Lifeline® Cell Technology (Frederick, MD, USA) and cultured in DermaLife K Keratinocyte Calcium-Free Medium (Cat. No.-0029). NHEKs were cultured at 37°C in 5% CO 2 NHEKs were incubated in a humidified incubator at 10 °C for 24 h. NHEKs were irradiated with 10 mJ / cm 2 UVB light by using a UVB lamp (Spectroline Model EB-160C, New York, NY, USA). 2 The cells were exposed to UVB radiation with a spectral peak at 312 nm at a dose of 100 μg / mL. After UVB irradiation, the cells were washed with warm PBS and then incubated for 24 h with fresh medium containing and without various concentrations of DOF-W (10 μg / mL and 40 μg / mL).

[0188] 2.4.2 Cell viability assay and measurement of ROS production Cell viability was measured using CCK-8. After 24 h of incubation, the optical density (OD) was recorded at 450 nm using a microplate reader according to the kit's instructions. Relative levels of ROS were detected using CellROX® reagent (Life Technologies, Waltham, MA, USA) based on the method reported in a previous study. Briefly, NHEKs were inoculated into 96-well microplates at 2 × 10 4 Cells / well were seeded for 48 hours, followed by UVB treatment and incubation for the times specified above. Cells were then incubated with 5 μM CellROX® for 1 hour and washed 3 times with PBS. ROS production was measured by fluorescence intensity with excitation and emission wavelengths set at 485 nm and 520 nm, respectively.

[0189] 2.5 Anti-COX-2 assay Anti-inflammatory activity was evaluated by measuring COX-2 enzyme inhibition using the COX-2 Inhibitor Screening Kit (Beyotime, no. S0168) based on the method described. The COX inhibitor celecoxib was used as a positive control. The results of anti-COX-2 activity were calculated as IC, a measure of 50% inhibition of enzyme activity by each sample. 50 Values ​​are expressed as μg / mL or μM.

[0190] 2.6 Anti-glycation ability 2.6.1 Formation of fluorescent AGEs in the BSA-fructose / glucose system The formation of total fluorescent AGEs in glycated samples was assessed by measuring the fluorescence intensity at excitation / emission wavelengths of 350 / 450 nm as previously described. In vitro glycated proteins were prepared by incubation of BSA in the presence of D-glucose (GLC) and D-fructose (FRC). DOF extracts and specific standards were dissolved in DMSO:water (1:4). BSA (4 mg / mL, 100 μL) was premixed with 50 μL of GLC (0.5 M) and 50 μL of FRC (0.5 M) in 20 mM sodium phosphate buffer (pH 7.4), then 100 μL of sample was added to a 96-well plate and incubated at 37.5° C. for 7 days. All solutions were prepared under sterile conditions and filtered using a 0.22 μm syringe before incubation. Assays were performed in triplicate and AG was used as a positive control. The percentage inhibition of the formation of fluorescent AGEs was calculated using the following formula: Inhibition (%)=1-[(IF sample-IF sample control) / (IF control-IF blank control)]×100% where IF sample is the fluorescence intensity in the presence of sample and BSA, IF sample control is the fluorescence intensity in the presence of sample without BSA, IF control is the fluorescence intensity without sample, and IF blank control is the fluorescence intensity without sample and BSA. The antiglycation activity results are expressed as IC 50 Values ​​are expressed as μg / mL or μM.

[0191] 2.6.2 Cellular assay of glycation Based on previously reported methods, human primary dermal fibroblasts (HDFs) were induced by MGO to define a cellular model of high CML expression.

[0192] HDFs were cultured in high glucose DMEM supplemented with 10% FBS containing 100 μg / mL PS at 37 °C in a humidified 5% CO 2The cells were cultured in an incubator. The cell viability was evaluated using a CCK-8 kit. HDFs were cultured in 12-well plates to approximately 70% confluence and then treated with 0.5 mmol / L MGO for 48 h to induce glycation and the formation of CML. Various concentrations of samples were then added and co-cultured with MGO for another 48 h. The cells were then washed once with PBS and fixed in polyformaldehyde overnight at 4°C. A Triton® X-100 solution (0.1%) was applied to the fixed cells for 5 min at room temperature to improve the permeability of the cell membrane. After washing twice with PBS, the cells were blocked in 1% BSA for 1 h and then incubated with the primary CML antibody (1:50) overnight at 4°C. The secondary antibody donkey anti-mouse Alexa Fluor® 488 (1:1,000) was then added for 2 h at room temperature. Antifluorescence quenching and DAPI-containing mounting fluid were used for counterstaining, followed by visualization and fluorescent image acquisition using a microscope (Leica, Germany).The expression of CML was quantified from various randomly selected images in each group using ImageJ software.

[0193] 2.7 Anti-aging assay for HDFs 2.7.1 Cell culture HDFs were cultured in DMEM containing 10% (v / v) FBS and 1% PS at 37°C in a humidified 5% CO 2 The cells were cultured in an incubator. After reaching confluence, the cells were subcultured using pancreatin solution. All experiments were performed between the 3rd and 8th passages.

[0194] 2.7.2 Quantification of collagen types I and III using immunofluorescence The synthesis of collagen types I and III was detected using IF and ELISA. HDFs were digested and seeded in 24-well culture plates on slides at a density of 50,000 cells / well in complete medium. After reaching 80% confluence, cells were starved with medium in the absence of serum for 16 hours. Cells were then treated with positive control or active samples (prepared in serum-free medium) for another 48 hours. For analysis and visualization of collagen types I and III, both the supernatant and the remaining cell slides were collected and analyzed using ELISA kits and IF staining methods, respectively.

[0195] The collected supernatant was centrifuged at 10,000 rpm for 10 min and used for collagen I and III ELISA according to the manufacturer's protocol. The remaining cell slides were washed once with PBS and fixed overnight at 4°C in polyformaldehyde. The slides were then treated with 0.5% Triton® X100 for 5 min. After washing, the cells were blocked with 1% BSA at room temperature for 1 h. The cells were then incubated with collagen I and III primary antibodies (1:100) at room temperature for 2 h. After washing, the cells were stained with secondary antibodies (1:1,000) for 1 h (Alexa Fluor® 568 goat anti-rabbit IgG and Alexa Fluor® 488 donkey anti-rabbit IgG were used to detect the fluorescence of collagen I and III, respectively). Finally, the nuclei were counterstained with DAPI and the relevant fluorescent images were acquired using a microscope. The expression of collagen was quantified from various randomly selected images in each group using ImageJ software.

[0196] 2.7.3 SA-β-gal staining assay D-galactose (D-gal)-induced senescent human dermal fibroblasts were established based on a previously reported method. HDFs were divided into a control group, a senescent model group, and a sample (senescent model with DOF extract) group. Except for the control group, HDFs were cultured in 6-well plates to approximately 70% confluence and then treated with 20 mg / mL of D-gal for 72 h to induce cellular senescence. Subsequently, various concentrations of DOF extract were added to the sample group and co-cultured with D-gal for another 72 h. Then, the medium was aspirated from the cells and the wells were washed once with PBS. SA-β-gal staining was performed using an SA-β-gal staining kit (Beyotime Co.). Fixative was added and incubated at room temperature for 10 min. The cells were then washed twice with PBS and incubated in 1 mL of SA-β-gal staining solution (freshly prepared according to the kit protocol). The plates were incubated at 4 °C for 1 h at 4 °C for 2 h at 4 °C for 1 h at 4 °C for 2 h at 4 °C. 2 The cells were kept overnight in the dark at 7 °C in a 3-humidified incubator without PBS. The next day, the staining solution was removed and the cells were kept in a final solution of PBS. The staining of SA-β-gal was observed and the relevant images were acquired using a microscope. The average value of the percentage of blue-stained cells of five randomly selected microscopic images in each group was determined using ImageJ software.

[0197] 2.8 UPLC-PDA-QTOF-ESI-MS / MS analysis The DOF extract (1 mg) was diluted with 1 mL of distilled water, sonicated for 20 min, and filtered using a 0.45 μm syringe filter before analysis.

[0198] UPLC analysis was performed using an Agilent 1290 UPLC system (California, USA) coupled with an Agilent Q-TOF 6545 LC / MS system, sample manager, PDA detector, and binary solvent manager, and controlled using MassHunter workstation software. Chromatographic separation of 2 μL of each sample was performed using an Acquity HSS T3 reversed-phase column (2.1 × 100 mm, 1.8 μm, Waters, Milford, Massachusetts, USA) at a separation temperature of 30 °C with a wavelength scan range of 190–400 nm. Gradient elution at a flow rate of 0.2 mL / min was completed with a mobile phase consisting of solvent A (0.2% acetic acid in ultrapure water) and solvent B (acetonitrile) in the following sequence: 0-5 min; 2% B, 5-8 min; 2%-10% B, 8-12 min; 10% B, 12-20 min; 10%-15% B, 20-28 min; 15%-20% B, 28-31 min; 20% B, 31-38 min; 20-80% B, and 38-40 min; 80% B. Finally, the initial conditions were reintroduced for 2 min. Before each run, the column was equilibrated for an additional 2 min. The MS was operated in both positive and negative ion modes. The optimized MS conditions were: TOF mass range; m / z 50-1,700, ion source gas; 50 psi, curtain gas; 35 psi, ion spray voltage; 5 kV, ion source temperature; 500 °C, collision energy; 10 eV. The applied MS / MS parameters were: MS / MS mass range; 50-1,250 m / z, collision energy; 40 eV, declustering potential; 100 V, and collision energy spread; 20 eV. Compounds were identified and analyzed by comparing their retention times, fragment ions, and formulas with corresponding standards and commercial databases.

[0199] 2.9 Online profiling of antioxidants using UPLC-PDA-QDa coupled with post-column derivatization with ABTS Online identification of antioxidant components of DOF extracts was performed using a UPLC system (Milford, Massachusetts, USA) consisting of a Waters photodiode array detector and a Waters post-column derivatization system (UPLC-PDA-QDa-ABTS) supplying fresh ABTS solution. Gradient elution at a flow rate of 0.8 mL / min was completed with a mobile phase consisting of solvent A (0.2% acetic acid in ultrapure water) and solvent B (acetonitrile) in the following order: 0–5 min; 2% B, 5–8 min; 2%–10% B, 8–12 min; 10% B, 12–20 min; 10%–15% B, 20–28 min; 15%–20% B, 28–31 min; 20% B, 31–38 min; 20%–80% B, and 38–40 min; 80% B. Separation of the compounds was performed using an Acquity reversed-phase column (4.6 × 250 mm, 5 μm, Waters, Milford, Massachusetts, USA) at 30° C. The detection wavelengths were set at 280 nm and 734 nm, and the sample injection volume was 10 μL.

[0200] The technical pathway and related equipment installation are shown in Figure 8. Figure 8 is a flow chart of online detection of antioxidants.

[0201] To detect the radical scavengers, the UPLC system was coupled to a Waters pump delivering freshly prepared ABTS·+ solution to a reaction coil (15 m, 0.25 mm inner diameter) at a flow rate of 0.2 mL / min at 37 °C. Negative peaks were recorded based on the decrease in absorbance at 729 nm after the reaction of individual compounds with ABTS·+ radicals. For preliminary identification of compounds, a Waters Acquity QDa mass detector in negative ionization mode was connected in series to the PDA with the following parameters: electrospray ion source, cone voltage; 15 V, atomizer; N2, and scan range; 100-800 m / z.

[0202] 2.10 Statistical analysis Data were statistically processed using GraphPad software. Test data were expressed as mean ± standard deviation (SD), and significance was evaluated by one-way analysis of variance (ANOVA) and Tukey's test. Statistical significance was evaluated by * p<0.05 and ** The values ​​are indicated as p<0.01.

[0203] 3.Results 3.1 Antioxidant efficacy of DOF extract in vitro We note that the recognition of herbs with antioxidant properties has increased over the last decades, mainly due to the discovery of ROS closely involved in chronic non-infectious diseases. The use of several antioxidant assays and various models is essential for a more comprehensive evaluation of natural products. Therefore, in this study, DPPH, ABTS, and FRAP assays as well as intracellular ROS levels induced by UVB irradiation in NHEK cells were used to measure the antioxidant capacity of the aqueous extract of Dendrobium officinale flowers (DOF-W).

[0204] In the concentration range of 15.6-1,000 μg / mL, DOF-W exhibited antioxidant activity in a dose-dependent manner, as shown in Figure 9. In particular, Figure 9 shows the antioxidant efficacy of DOF extracts, specifically, the DPPH scavenging activity (A), ABTS scavenging activity (B), FRAP value (C), and intracellular ROS level (D) of DOF-W. With respect to Figure 9, compared to the control, ** p<0.01 compared with the UVB model. ## p<0.01.

[0205] In the DPPH and ABTS assays, the IC of DOF-W 50 The IC values ​​for Trolox were 669.7 ± 20.59 μg / mL and 224.57 ± 0.65 μg / mL, respectively. 50The EC1 values ​​were 24.7 ± 1.0 μg / mL and 27.68 ± 1.04 μg / mL. In the FRAP assay, the EC1 value of DOF-W was 4,580 ± 260 μg / mL, while the EC1 value of Trolox was 55.35 ± 5.22 μg / mL. Although DOF-W showed weaker antioxidant capacity compared to the classical antioxidant standard (Trolox), it still showed potential antioxidant activity, especially at high concentrations, which was consistent with various previous studies.

[0206] Safety and non-toxicity are essential for functional foods and skin care cosmetics. DOF-W showed no significant damage to the viability of NHEK cells in the range of 1-40 μg / mL. UVB irradiation (10 mJ / cm 2 After UVB irradiation, cell survival was significantly decreased. However, DOF-W did not further exacerbate UVB-induced NHEK cell death. As shown in Figure 9-(D), UVB irradiation leads to increased ROS levels in NHEK cells. Treatment with 40 μg / mL DOF-W significantly reduced intracellular ROS levels.

[0207] 3.2 Anti-COX-2 ability of DOF extract COX-2 is an inducible enzyme that produces prostaglandins (PGs) and is involved in the generation of ROS, so it has always been regarded as the pathological enzyme primarily involved in inflammation, and COX-2 inhibitors, which can cause a sharp drop in the amount of ROS, have also been found to be highly associated with potential antioxidant effects. In this study, the anti-inflammatory effect of DOF-W was evaluated using a COX-2 inhibition assay.

[0208] The COX-2 inhibitory activity of DOF-W was compared with that of a known selective COX-2 inhibitor (celecoxib). In the concentration range of 31.3 to 2,000 μg / mL, DOF-W exhibited excellent concentration-dependent COX-2 inhibitory activity (compared to the control, ** p<0.01). DOF-W had an IC of 133.5±27.4μg / mL. 50 value, whereas celecoxib had an IC value of 0.53 ± 0.30 μg / mL. 50Although DOF-W may show lower potency due to dilution of the active components with neutral components, it can still be used as a promising COX-2 inhibitor.

[0209] 3.3 Anti-glycation activity of DOF-W Glycosylation in tissues is a slow and complicated process. Excessive production and accumulation of AGEs in the process can cause irreversible damage to the body. Various AGEs in skin collagen, including CML, can damage skin fibroblasts, leading to skin aging and mole formation. In this study, the determination of anti-glycation activity was evaluated by various methods, including routine chemical tests, cell experiments, and zebrafish assays.

[0210] 3.3.1 Inhibition of DOF-W on total fluorescent AGEs of nonenzymatic glycation Incubation of reducing sugars with BSA induced the production of fluorescent AGEs. In this study, BSA-fructose / glucose as a model reaction system was first applied to preliminarily evaluate the effect of DOF aqueous extract on the inhibition of AGEs. As shown in Figure 11-(A), DOF-W extract showed an IC of 428.4 ± 75.1 μg / mL. 50 The synthetic AGE inhibitor, AG, was able to reduce the levels of fluorescent AGEs in a concentration-dependent manner, with an IC value of 177.3 ± 11.6 μg / mL, as expected. 50 values ​​showed effective inhibition.

[0211] Referring to Figure 11, the anti-glycation activity of DOF-W is shown therein. Specifically, Figure 11 (A) shows the inhibition of fluorescent AGE by DOF-W extract, (B) shows the cytotoxicity of DOF-W extract on fibroblasts, and (C) shows the effect of DOF-W extract on CML expression in glyoxal-induced HDF. With reference to Figure 11, compared to the MGO model, ## p<0.01 compared with control **p<0.01, AG: aminoguanidine hydrochloride, MGO: methylglyoxal, CML: N-carboxymethyllysine. See also FIG. 7, which shows representative images and statistics of HDFs by fluorescence microscopy.

[0212] 3.3.2 Anti-glycation effect of DOF-W in human primary fibroblasts The primary anti-glycation assay in BSA / reducing sugar system as above showed that DOF extract is a potential anti-glycation substance. DOF extract was then further tested in an in vitro glycation assay to confirm its activity in cells. MGO is an important and potent precursor in the formation of AGEs, which reacts with proteins to form N-glycosylation products. ε -carboxymethyllysine (CML), one of the major AGEs in skin that does not have fluorescent properties and cannot be detected by conventional assays. Fibroblasts were treated with MGO to induce glycation, which was then visualized and quantified using IF. No obvious green fluorescent staining of CML was observed in the blank control group, indicating that normal cells secrete almost no CML, while a large amount of CML was expressed in MGO-induced cells after CML IF (as shown previously in Figure 7). Treatment of cells with the positive compound AG significantly inhibited glycation by 88.11%. As shown in Figure 11-(B), treatment with both 10 μg / mL and 40 μg / mL DOF extracts in HDF significantly inhibited glycation by 77.88% and 69.70%, respectively, in a non-obvious dose-dependent manner, with no cytotoxicity observed at a concentration of 40 μg / mL (see Figure 11-(C) and Figure 7).

[0213] 3.4 Effect of DOF extract on collagen synthesis in human dermal fibroblasts Dermal fibroblasts are believed to be involved in the synthesis of various dermal ECM proteins, including fibrous collagen. Type I collagen constitutes about 80% of total collagen, while type III collagen is more abundant in young skin than in aged skin, and is particularly involved in wound healing. Since skin aging is characterized by the degradation of ECM components, such as the degradation of type I and type III collagen, the present inventors investigated whether DOF can enhance the expression of type I and type III collagen in HDF.

[0214] As shown in Figures 12-(A) and 12-(B), IF assays revealed that DOF-treated cells synthesized more collagen type I than untreated control cells after 48 hours. DOF increased the degree of collagen type I staining by 111.88% and 154.59% of the control at concentrations of 10 μg / mL and 40 μg / mL, respectively. However, the expression level of collagen type III was not statistically changed or further decreased by DOF extract, as shown in Figures 12-(C) and 12-(D). The expression of collagen types I and III was significantly increased by vitamin C as a positive drug by 190.20% and 125.90% of the control, respectively.

[0215] Referring to FIG. 12, the effect of DOF extract on collagen expression by IF staining is shown. HDFs were treated with VC (200 μg / mL, positive control group) and DOF extract (10 μg / mL and 40 μg / mL), and in the control group, HDFs were incubated without any treatment. (A) is the image of collagen type I staining (red), and (B) is the quantification. (C) is the image of collagen type III staining (green), and (D) is the quantification. Extracellular content of collagen type I (E) and type III (F) in HDFs using ELISA. For FIG. 12, the bar scale is 100 μm, compared to the control group. * p<0.05, ** p<0.01.

[0216] The extracellular content of collagen type I and type III in HDFs in culture medium for 48 hours was also examined. As shown in Figures 12-(E) and 12-(F), treatment with vitamin C (VC), 10 μg / mL DOF, and 40 μg / mL DOF increased the level of secreted collagen type I by 21.94%, 42.6%, and 57.65%, respectively, while DOF had no obvious effect on collagen type III secretion, and the corresponding significantly stronger effect on collagen type I was further confirmed. Therefore, it can be concluded that DOF extract can upregulate the level of collagen type I in HDFs, but has no positive effect on collagen type III.

[0217] 3.5 SA-β-Gal staining assay with DOF-W Cellular senescence is an irreversible physiological phenomenon in which normal cells lose their proliferation capacity but still remain viable and maintain their metabolic activity. Cellular senescence can be measured using the SA-β-gal staining assay, which is widely used to identify the location of SA-β-gal positive cells (cells stained blue) by light microscopy. X-ray, H 2 O 2 Among several existing types of aging models, such as the D-gal-induced aging model, the D-gal-induced aging model resembles natural aging but is shorter in duration than the latter.

[0218] As shown in Figure 13, few blue stained cells were observed in the control group (3.0% ± 1.2%). SA-β-gal positive cells were significantly increased (7.9% ± 2.0%) after 6 consecutive days of treatment with 20 mg / mL D-gal in the model group. When aged cells were co-incubated with 10 μg / mL and 40 μg / mL DOF extract, the percentages of SA-β-gal positive cells were 4.0% ± 0.9% and 5.6% ± 1.2%, respectively, which were significantly lower than those in the model group.

[0219] Referring to FIG. 13, there is shown an image of fibroblasts stained with SA-β-gal. Statistical results of fibroblasts stained with SA-β-gal. For FIG. 13, the bar scale is 100 μm. Compared with the model group, ** p<0.01.

[0220] 3.6 Determination of phytochemicals using UPLC-Q / TOF-MS / MS Twenty-five compounds were further characterized by structural analysis using UPLC-PDA-ESI-Q / TOF-MS / MS. Total ion chromatograms of aqueous extracts of samples in both negative and positive ion modes are shown in Figure 14. Specifically, Figure 14 shows the UPLC-ESI / MS / MS total ion chromatogram of DOF-W.

[0221] The retention times (Rt), molecular formulas, and ions and fragmented ions are shown in Table 10. The Rt and fragmentation information of compounds 13, 14, 16, 17, 19, 20, 23, 24, and 25 were compared with those of standards. The main components of DOF-W were flavonoids (see Structure 1 below). [Table 11] [Table 12-1] [Table 12-2] Structure 1: Chemical structure of the main herbal marker in DOF-W [ka] [ka] [Table 13] [ka] [Table 14]

[0222] 3.7 Determination of antioxidants by online UPLC-PDA-MS-ABTS + capture For preliminary screening of antioxidant phytochemicals in DOF-W, UPLC coupled with PDA-Qda and ABTS-based assay was performed in this study. Constituents for which MS data and reference compounds were available could be identified from the positive ion peaks. The area of ​​the sorted negative peaks in the lower chromatogram at 734 nm corresponds to the ABTS radical scavenging activity of individual compounds of DOF extract. As can be judged from the magnitude of the negative peaks in the chromatograms, 1-O-caffeoyl-β-D-glucoside (compound 5), vicenin-2 (compound 10), luteolin-6-C-β-D-xyloside-8-C-β-D-glucoside (compound 11), quercetin-3-O-sophoroside (compound 16), rutin (compound 24), isoquercitrin (compound 25), and quercetin 3-O-(6''-O-malonyl)-β-D-glucoside (compound 27) were the most potent ABTS radical scavengers in the extracts investigated. Because components 10 and 11 were not well separated, it was difficult to distinguish which component was mainly responsible for the negative peaks, and further confirmation by additional experiments is required. In a previous report, 1-O-caffeoyl-β-D-glucoside, rutin, and isoquercitrin were identified as the main components with obvious free radical scavenging ability in the methanol extract of DOF by using precolumn DPPH and ABTS assays followed by HPLC-DAD analysis.

[0223] 3. In Vitro Pro-Health Efficacy of Selected Compounds from 8DOF Extract Considering the time-consuming process of isolating the components in the flowers, and to further identify and confirm the biological activity of the components that may contribute to the antioxidant, anti-inflammatory, and anti-glycation properties of the extract, certain available compounds were selected for more detailed studies in DPPH, ABTS, FRAP, COX-2, and BSA / reducing sugar glycation assays. The results of these isolated compounds are shown in Table 11. [Table 15] NOTE: nd was not detected, values ​​are expressed as mean ± standard deviation (n = 3), values ​​in the same column followed by different letters are significantly different at p ≤ 0.05 according to Tukey's test.

[0224] Considering the limitations of various antioxidant methods, the use of at least two or more assays of different oxidation mechanisms is highly recommended. Three in vitro assays (DPPH, ABTS, and FRAP) were performed to comprehensively analyze the antioxidant capacity of the selected compounds. The tested compounds showed various effects on their functional activity. It was observed that all compounds showed significant scavenging capacity towards ABTS radical, with IC 50 The values ​​ranged from 6.63 ± 0.41 μM (compound 16) to 96.39 ± 2.11 μM (compound 14). However, for the DPPH radical and FRAP assays, some compounds (compounds 10, 14, 17, 19, 21, 26, 28, and 31) had IC values ​​even higher than 5,000 μM. 50 The other compounds (compounds 11, 15, 16, 18, 24, 25, and 27) showed low levels of inhibition at IC values ​​of DPPH, respectively. 50 ranged from 24.70 ± 0.96 to 226.93 ± 47.26 μM, and the EC 1 The remaining compound 32 showed exceptional inhibition with IC values ​​ranging from 205.0 ± 12.2 to 1,100.3 ± 138.4 μM for DPPH. 50 was 2,366.67 ± 87.61, and the EC 1had a relatively weak inhibition of 2,757.3 ± 103.8. Based on previous reports, flavonoids appear to exhibit anti-inflammatory properties via modulation of ROS. In accordance with the present findings, the data from the anti-COX-2 assay indeed revealed a similar trend consistent with the results observed in the DPPH radical and FRAP assays. Compounds 11, 15, 16, 18, 24, 25 and 27 had the lowest IC of 7.9 ± 2.4 μM. 50 Compound 32 showed a strong COX-2 inhibition at IC 0.01, whereas compounds 10, 14, 17, 19, 21, 26, 28, and 31 showed no inhibitory effect. Similar to the antioxidant results (DPPH and FRAP) of compound 32, its inhibitory effect on COX-2 was weak, with IC 0.01 and IC 0.01. 50 was 535.3±150.0 μM.

[0225] Analysis of the effects on AGE formation showed that all compounds had IC values ​​ranging from 209.2 ± 3.1 to 374.4 ± 18.1 μM. 50 All results were consistent with those of rutin (IC), which is not only the main component of DOF-W extract but also a known AGE inhibitor. 50 , 222.03 ± 13.49 μM), and the inhibitory effects of all compounds are even superior to that of another commonly used synthetic AGE inhibitor, aminoguanidine hydrochloride (1,604.1 ± 104.5 μM).

[0226] 4. Discussion In this study, the results demonstrated that DOF-W exhibited promising antioxidant capacity (DPPH, ABTS, FRAP, intracellular ROS levels in NHEK cells), anti-COX-2 effect, anti-glycation efficacy (inhibition of nonenzymatic glycation reactions and inhibition of CML expression in fibroblasts), and anti-aging effect (SA-β-gal staining test and collagen expression in fibroblasts). Furthermore, the chemical and cellular anti-glycation activity and anti-COX-2 effect of DOF-W were reported for the first time.

[0227] Both oxidative stress and inflammation caused by unstable free radicals, which are highly harmful to cells and skin, are major contributors to the aging process. Compared with young skin, aged skin is more sensitive to environmental stimuli and requires external support such as antioxidants. Based on chemical, enzymatic, and cellular methods, DOF-W has been demonstrated to have obvious antioxidant and anti-inflammatory effects. Previous studies have shown that DOF-W can enhance antioxidant status and inhibit inflammatory responses in alcohol-impaired mice, which is consistent with the experimental results herein.

[0228] The results of online UPLC-PDA-MS-ABTS·+ scavenging indicated that seven compounds (5, 10 or 11, 16, 24, 25, and 27) were most likely to be the major contributors to the overall antioxidant capacity of DOF-W extract. However, although all selected compounds showed good ability to scavenge ABTS radical, only compounds 11, 15, 16, 18, 21, 24, 25, and 27 showed good antioxidant potency in DPPH and FRAP assays. Due to the lack of standard 5, its reliable antioxidant capacity could not be verified at this point. Combining the results of online and traditional chemical methods, compounds 11, 16, 24, 25, and 27 were predicted to be the major antioxidants in DOF-W extract. Based on previous reports, flavonoids seem to exhibit anti-inflammatory properties through the modulation of ROS. It was indeed observed that the anti-cyclooxygenase-2 assay showed a similar trend to the results of DPPH radical and FRAP assay. Only compounds 11, 15, 16, 18, 21, 24, 25, and 27 showed significant COX-2 inhibitory effects. According to this study, the antioxidant and anti-inflammatory effects of DOF-W extract are more likely to be attributed to compounds 11, 15, 16, 18, 24, 25, and 27. Luteolin C-glycosylflavones such as compounds 11, 15, and 18 showed significant antioxidant and COX-2 inhibitory effects. Meanwhile, apigenin, one of the flavonoids most widely distributed in the plant kingdom and most frequently tested by researchers, has been characterized as a very good free radical scavenger and a notable anti-inflammatory agent, while apigenin C-glycosylflavones such as compounds 10, 14, 17, 19, 21, and 26 showed very weak biological effects. Among these compounds, compounds 11 and 15 have the same glycosidic bond as compounds 14 and 19, respectively. Therefore, it appears that the type of aglycone, rather than the C- or O-glycosides, of flavonoids had a major impact on the antioxidant and COX-2 inhibitory potency. It was also reported that both luteolin and apigenin C-glycosylflavones exhibited much lower inflammatory effects in LPS-induced RAW264.7 than those observed with their corresponding aglycones and O-glycosides.The results herein can be explained by speculation that C-glycosylation of apigenin leads to reduced antioxidant and anti-inflammatory capacity. As most flavonoids in plants exist mainly as O-glycosides, C-glycoside flavonoids have received less attention compared to flavonoid O-glycosides, especially without comprehensive studies on their biological benefits. It is more purposeful to explore the pharmacokinetic properties of flavonoid C-glycosides and their biological activities.

[0229] Many studies have also shown that external stimuli such as excessive free radicals and spontaneous generation of AGEs in vivo, which are irreversible once formed in the body, are associated with damage to skin fibroblasts, destruction of collagen and elastic fibers, dull complexion with a lack of radiance, and deterioration due to aging. DOF-W extract has a low IC 50 DOF-W showed an excellent ability to inhibit AGE formation at 100 mg / kg / day value and obviously reduced CML expression in MGO-induced fibroblasts. In addition, it was observed that all selected compounds have a strong ability to inhibit AGE formation. Therefore, the anti-glycation potential of DOF-W can be justified by the synergistic action of most of the polyphenolic compounds present in the extract. So far, several studies have reported that various flavonoids, both O- and C-glycosides, can cause a significant reduction in AGE production in several in vitro and in vivo experimental models.

[0230] Human dermal fibroblasts, which are mainly present in the dermis, can form large amounts of collagen, which is an important factor in maintaining skin elasticity. Studies have reported that skin aging may be associated with the accumulation of senescent fibroblasts in human skin. Both immunofluorescence (IF) assays and extracellular ELISA assays showed that DOF-W extracts could increase the expression of type I collagen, but had no obvious effect on type III collagen. In addition, DOF-W could also significantly reduce the proportion of D-Gal-induced senescent cells. Previous studies have reported that Dendrobium officinale flowers could alleviate brain aging and improve spatial learning ability in aged rats.

[0231] 5. Conclusion Based on the current research on in vitro testing and phytochemical analysis, it is suggested that DOF-W is potent in terms of antioxidant, antiglycation, and antiaging effects and is worthy of further research and development. Therefore, it is believed that both DOF-W and its specific compounds may be promising agents for skin antiaging.

[0232] The terms "comprising" or "comprise" are used herein in their broadest sense to mean and encompass the concepts of "including", "include", "consist(ing) essentially of" and "consist(ing) of". The use of "for example", "eg", "such as" and "including" to enumerate illustrative examples is not limited to only the enumerated examples. Thus, "for example" or "such as" means "for example, but not limited to" or "such as, but not limited to", and encompasses other similar or equivalent examples. The term "about", as used herein, serves to reasonably encompass or account for small variations in numerical values ​​measured by instrumental analysis or as a result of handling of samples. Such small variations may be on the order of ±0-10%, ±0-5%, ±0-2.5% of the numerical value. Furthermore, the term "about" applies to both numerical values ​​when associated with a range of values. Additionally, the term "about" may be applied to numerical values ​​even if not expressly stated.

[0233] Generally, as used herein, a hyphen "-" or dash "-" in a range of values ​​means "to" or "through," ">" means "above" or "more than," "≧" means "at least" or "greater than," "<" means "below" or "less than," and "≦" means "up to" or "less than." Individually, each of the foregoing patent applications, patents, and / or patent application publications is expressly incorporated herein by reference in its entirety in one or more non-limiting embodiments.

[0234] It is to be understood that the appended claims are not limited to the expressions and specific compounds, compositions, or methods described in the detailed description, which may vary among the specific embodiments falling within the scope of the appended claims. With respect to all Markush groups relied upon herein to describe specific features or aspects of various embodiments, it is to be understood that different, special, and / or unexpected results may be obtained from each element of the respective Markush group independent of all other Markush elements. Each element of the Markush group may be relied upon individually and / or in combination to provide adequate support for specific embodiments within the scope of the appended claims.

[0235] Any ranges and subranges relied upon in describing various embodiments of the present invention are understood to be within the scope of the appended claims, both individually and collectively, and to be understood to describe and contemplate all ranges, including whole and / or fractional values ​​therein, even if such values ​​are not expressly set forth herein. Those skilled in the art will readily recognize that the recited ranges and subranges fully describe and enable various embodiments of the present invention, and that such ranges and subranges may be further described into their associated halves, thirds, quarters, fifths, etc. As merely an example, a range of "0.1-0.9" may be further described into a lower third, i.e., 0.1-0.3, a middle third, i.e., 0.4-0.6, and an upper third, i.e., 0.7-0.9, which are individually and collectively within the scope of the appended claims and may be relied upon individually and / or collectively to provide adequate support for particular embodiments within the scope of the appended claims. In addition, with respect to language defining or modifying a range, such as "at least," "greater than," "less than," "less than," etc., it is understood that such language includes subranges and / or upper or lower limits. As another example, the range "at least 10" essentially includes subranges of at least 10 to 35, at least 10 to 25, 25 to 35, etc., each of which may be relied upon individually and / or collectively to provide appropriate support for particular embodiments within the appended claims. Finally, individual numbers within a disclosed range may be relied upon to provide appropriate support for particular embodiments within the appended claims. For example, the range "1 to 9" includes various individual integers, such as 3, as well as individual numbers including decimal points (or fractions), such as 4.1, which may be relied upon to provide appropriate support for particular embodiments within the appended claims.

[0236] The present invention has been described herein in an illustrative manner, with it being understood that the nature of the terminology used is intended to be words of description rather than of limitation. Numerous modifications and variations of the present invention are possible in light of the above teachings. The present invention may be practiced otherwise than as specifically described within the scope of the appended claims. The subject matter of all combinations of independent claims and dependent claims, both single and multiple dependent claims, is expressly contemplated herein.

Claims

1. A composition for administration to a subject, comprising at least one botanical active ingredient, said botanical active ingredient comprising: i) Dendrobium officinale flower extract; ii) Gentian extract; iii) Eucommia extract, and iv) Combination of i) to iii) wherein the botanical active ingredient is present in the composition in an amount effective to inhibit advanced glycation end products (AGEs) in the skin of the subject.

2. 2. The composition of claim 1, wherein said botanical active ingredients comprise an extract of said Dendrobium officinale flower, and optionally, said botanical active ingredients consist of an extract of said Dendrobium officinale flower.

3. 3. The composition according to claim 1 or 2, wherein the extract of Dendrobium officinale flowers is obtained by water extraction (or aqueous extraction) of Dendrobium officinale flowers or flower-based plant material.

4. 3. The composition according to claim 1 or 2, wherein the extract of Dendrobium officinale flowers is obtained by alcoholic extraction, optionally ethanolic extraction, of Dendrobium officinale flowers or flower-based plant material.

5. 5. A composition according to any one of claims 1 to 4, which is substantially to completely free of components obtained from plant material that is not Dendrobium officinale flower-based.

6. 6. A composition according to any one of claims 1 to 5, wherein the botanical active ingredient comprises the Gendzin extract, optionally further defined as an extract of Gendzin root.

7. 7. The composition of any one of claims 1 to 6, wherein the botanical active ingredient comprises the Eucommia ulmoides extract, optionally further defined as an extract of Eucommia ulmoides male flowers.

8. The botanical active ingredient is i) Phyllanthus officinalis extract, ii) Sophora japonica extract; iii) Aronia extract; iv) pomegranate extract, and The composition of any one of claims 1 to 7, further comprising an extract selected from the group consisting of: v) a combination of i) to iv).

9. 9. The composition of claim 8, wherein the botanical active ingredient comprises the Emblica officinalis extract, optionally further defined as an extract of Emblica officinalis fruit.

10. 10. The composition of claim 8 or 9, wherein the botanical active ingredient comprises the Sophora japonica extract, optionally further defined as an extract of Sophora japonica flower.

11. 11. The composition of any one of claims 8 to 10, wherein the botanical active ingredient comprises the Aronia extract, optionally further defined as an extract of Aronia berries.

12. 12. The composition of any one of claims 8 to 11, wherein the botanical active ingredient comprises the pomegranate extract, optionally further defined as an extract of pomegranate fruit.

13. The composition of any one of claims 1 to 12, further defined as a topical composition formulated for topical administration to a subject.

14. 14. The composition of claim 13, further comprising a cosmetically acceptable carrier, optionally wherein said cosmetically acceptable carrier is not naturally occurring.

15. The composition of any one of claims 1 to 12, further defined as an oral composition formulated for oral administration to the subject.

16. 16. The composition of claim 15, further comprising a pharma- ceutically acceptable excipient, optionally wherein the pharma- ceutically acceptable excipient is not naturally occurring.

17. 17. Use of the composition of any one of claims 1 to 16 for inhibiting advanced glycation end products (AGE) in the skin of a subject.

18. 17. A method of inhibiting advanced glycation end products (AGEs) in the skin of a subject, comprising administering to the subject an effective amount of a composition, the composition being the composition of any one of claims 1 to 16.

19. 20. The method of claim 18, wherein the composition is administered topically to the subject.

20. 20. The method of claim 18, wherein the composition is administered orally to the subject.

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