Composition for improving skin health comprising gynostemma pentaphyllum extract

Abstract

The present invention relates to a food composition for improving skin health, the food composition comprising a Gynostemma pentaphyllum extract or Damulin B. The Gynostemma pentaphyllum extract or damulin B inhibited the generation of reactive oxygen species (ROS) induced by ultraviolet irradiation in keratinocytes and increased the expression of antioxidant proteins. In addition, the Gynostemma pentaphyllum extract or damulin B also exhibited a moisturizing effect by regulating the expression of genes related to collagen synthesis and degradation in the skin, as well as the expression of hyaluronidase, and thereby reducing the hyaluronidase responsible for moisture retention in the skin. In addition, the skin wrinkle-improving, skin-moisturizing, and skin roughness-improving effects and safety of the Gynostemma pentaphyllum extract were confirmed through human clinical trials. Therefore, the Gynostemma pentaphyllum extract or damulin B according to the present invention can be used as a food composition for improving skin health.

Description

A composition for improving skin health containing Gynostemma pentaphyllum extract

[0001] The present invention relates to a composition for improving skin health comprising Gynostemma pentaphyllum extract or damulin B as an active ingredient.

[0002] The skin is the largest tissue surrounding the body and plays a protective role by defending against external stimuli and bacterial invasions, as well as regulating body temperature, providing sensory functions, and eliminating waste products. Like other organs, the skin undergoes aging. Skin aging is divided into intrinsic aging, which occurs due to the decline in bodily functions and changes in physiological functions such as hormones as one ages, and extrinsic aging, which results from continuous exposure to various environmental factors, including ultraviolet (UV) radiation. Among these, photoaging caused by UV radiation is the most direct cause of extrinsic aging. It induces various phenomena resulting from skin damage, such as reduced skin moisture due to damage to the skin barrier, consequent dryness, decreased elasticity, roughness, pigmentation, and increased epidermal thickness.

[0003] As mentioned above, the generally used method to improve photoaging caused by ultraviolet rays—that is, skin barrier damage caused by UV rays—is to replenish moisture and oil through the application of topical skin agents, such as cosmetics or ointments containing sunscreens and moisturizers. However, these topical agents have a limitation in that their moisturizing effect is temporary because they do not penetrate to the dermis and act only on the epidermis.

[0004] Furthermore, the skin's moisture balance is regulated by the epidermis. The epidermis is the layer where dead cells accumulate and plays a crucial role in maintaining skin hydration. If the moisture content of the stratum corneum falls below 10%, the skin becomes dry and skin problems may occur. Additionally, the moisture content within the stratum corneum is maintained at 10–20%, which protects the skin from external factors and inhibits moisture evaporation. In particular, due to various causes such as recent environmental changes, shifts in lifestyle patterns, various types of stress, and makeup habits, moisture in the stratum corneum is decreasing, leading to dry and rough skin.

[0005] Meanwhile, as people's desire for beauty grows, interest in "inner beauty," often referred to as "edible cosmetics," is on the rise. "Inner beauty" is a compound word formed from "inner" and "beauty," signifying the cultivation of healthy skin from within the human body. The domestic inner beauty market grew tenfold in just eight years, from 50 billion won in 2011 to 500 billion won in 2019. The market for products seeking efficacy in skin hydration, elasticity, and pigmentation through the consumption of such food is expected to continue expanding. While most moisturizing cosmetics focus on the absorption of moisture and active ingredients into the stratum corneum, these topical products have limitations in that their effects are temporary as they act only on the epidermis and fail to penetrate the dermis. In contrast, inner beauty ingredients are delivered into the body via the digestive system and blood vessels after ingestion, creating an environment conducive to skin health. By acting on the skin tissues of the entire epidermis and dermis—not just the stratum corneum—they exhibit effects such as improved hydration and elasticity.

[0006] Therefore, research on inner beauty, or beauty foods, is increasing in order to overcome the limitations of topical skin products and achieve systemic improvement of skin barrier damage. Korean Patent Publication No. 10-2006-0119384 discloses an oral composition for improving skin beauty containing soybean extract powder and red ginseng concentrate powder, and Korean Patent Publication No. 10-2009-0054723 discloses an oral composition for improving skin beauty containing curcumin as an active ingredient. However, there have been no reports regarding the effects of Gynostemma pentaphyllum extract on improving skin damage caused by ultraviolet rays and on moisturizing effects.

[0007] Accordingly, the inventors conducted research to develop an inner beauty composition capable of preventing skin damage and improving damaged skin, and confirmed that Gynostemma pentaphyllum extract or damulin B can improve skin health, thereby completing the present invention.

[0008] To achieve the above objective, one aspect of the present invention provides a food composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I:

[0009] <Chemical Formula I>

[0010] .

[0011] Another aspect of the present invention provides a feed composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the chemical formula I.

[0012] Another aspect of the present invention provides a use for improving skin health comprising an extract of *Gynostemma pentaphyllum* or damulin B represented by the chemical formula I as an active ingredient.

[0013] The Gynostemma pentaphyllum extract or Damulin B of the present invention inhibited the generation of reactive oxygen species (ROS) induced by UV irradiation in keratinocytes and increased the expression of antioxidant proteins. Furthermore, by regulating the expression of genes related to collagen synthesis and degradation in the skin, as well as the expression of hyaluronic acid degrading enzymes, it reduced the degradation of hyaluronic acid responsible for moisture retention in the skin, thereby exhibiting a moisturizing effect. In addition, the skin wrinkle improvement, skin moisturizing effects, and safety of the Gynostemma pentaphyllum extract were confirmed through human clinical trials. Therefore, the Gynostemma pentaphyllum extract or Damulin B according to the present invention can be used as an active ingredient in a moisturizing food composition that prevents and improves skin damage caused by UV rays.

[0014] Figure 1 is a graph showing the results of measuring cytotoxicity in keratinocytes treated with Gynostemma pentaphyllum extract (GPDH) or damulin B through MMT analysis.

[0015] Figure 2a is a diagram and graph showing the results of confirming the mRNA expression levels of genes involved in collagen synthesis in keratinocytes according to the treatment concentration of Gynostemma pentaphyllum extract (GPHD) via PCR. Figure 2b is a diagram and graph showing the results of confirming the mRNA expression levels of genes involved in collagen synthesis in keratinocytes according to the treatment concentration of Damiedone B in keratinocytes via PCR.

[0016] Figure 3 is a diagram and graph showing the results of measuring the expression of genes involved in collagen synthesis and degradation using PCR after irradiating keratinocytes with Gynostemma pentaphyllum extract (GPHD) and / or ultraviolet rays.

[0017] Figure 4a is a figure and graph showing the results of confirming the phosphorylation of MAPK (mitogen-activated protein kinase; ERK, JNK, p38) and p53 using Western blot after keratinocytes were irradiated with Gynostemma pentaphyllum extract (GPHD) or Damulin B and UV light. Figure 4b is a figure and graph showing the results of confirming the expression of a subunit of AP-1, a downstream protein of the MAPK signaling pathway, using Western blot after keratinocytes were irradiated with Gynostemma pentaphyllum extract (GPHD) or Damulin B and UV light. Figure 4c is a figure summarizing the results of Figures 4a and 4b.

[0018] Figure 5a is a graph showing the results of measuring the amount of reactive oxygen species (ROS) produced after keratinocytes were irradiated with Gynostemma pentaphyllum extract (GPHD) or Damulin B and ultraviolet light. Figure 5b is a graph showing the percentage of cells generating reactive oxygen species among the results of Figure 5a.

[0019] Figure 6 is a diagram and graph showing the results of confirming the expression of proteins related to antioxidant activity through Western blot after irradiating keratinocytes with Gynostemma pentaphyllum extract (GPHD) or Damulin B and ultraviolet rays.

[0020] Figure 7 is a diagram showing the mechanism of action of the present invention for preventing and improving skin damage caused by ultraviolet rays using the Gynostemma pentaphyllum extract or Damulin B.

[0021] Figure 8a is a diagram and graph showing the results of confirming the mRNA expression levels of genes involved in collagen synthesis in skin fibroblasts according to the treatment concentration of Gynostemma pentaphyllum extract (GPHD) using PCR. Figure 8b is a diagram and graph showing the results of confirming the expression of genes involved in collagen synthesis in skin fibroblasts according to the treatment concentration of damulin B using PCR. Figure 8c is a diagram summarizing the results of Figures 8a and 8b.

[0022] Figure 9a is a diagram and graph showing the results of confirming the expression of proteins related to collagen degradation and antioxidant regulation using Western blot after irradiating skin fibroblasts with Gynostemma pentaphyllum extract (GPHD) or damulin B and ultraviolet rays. Figure 9b is a diagram summarizing the results of Figure 9a.

[0023] Figure 10 is a graph showing the results of confirming the expression of genes related to skin barrier function or skin moisturization using qPCR after administering Gynostemma pentaphyllum extract (GPHD) and / or irradiating UV rays to hairless mice and collecting skin tissue from the mice.

[0024] Figure 11 is a graph showing the epidermal moisture level and transepidermal water loss (TEWL) of hairless mice after administering Gynostemma pentaphyllum extract (GPHD) and / or irradiating them with ultraviolet light.

[0025] FIG. 12 is a diagram summarizing the skin moisturizing mechanism of a composition containing Gynostemma pentaphyllum extract or damulin B according to the present invention.

[0026] Figure 13 is a schematic diagram showing the participation status of subjects in a human clinical trial.

[0027] Figures 14a to 14c are graphs showing the results of measuring the depth of wrinkles around the eyes over time (before intake, after 6 weeks, and after 12 weeks) in a group that consumed Gynostemma pentaphyllum extract (test group) and a placebo group (control group). Figure 14a is a graph measuring the average depth of wrinkles around the eyes. Figure 14b is a graph measuring the average depth of the largest wrinkles around the eyes. Figure 14c is a graph measuring the maximum depth of the largest wrinkles around the eyes.

[0028] Figure 15 shows photographs of the depth of wrinkles around the eyes of the group that consumed Gynostemma pentaphyllum extract and the placebo group over time (before consumption, after 6 weeks, and after 12 weeks).

[0029] Figures 16a to 16c are graphs showing the results of measuring skin elasticity over time (before intake, after 6 weeks, and after 12 weeks) for the group that consumed Gynostemma pentaphyllum extract and the placebo group. Figure 16a is a graph measuring the skin's recovery ability relative to its resistance to physical force. Figure 16b is a graph measuring the ratio of the skin's relaxation elasticity to its suction elasticity. Figure 16c is a graph measuring the ratio of elasticity relative to the skin's overall curve.

[0030] Figure 17 is a graph showing the results of measuring the skin moisture content of the group consuming Gynostemma pentaphyllum extract and the placebo group over time (before consumption, after 6 weeks, and after 12 weeks).

[0031] Figure 18 is a graph showing the results of measuring the amount of water loss over time (before intake, after 6 weeks, and after 12 weeks) in the group that consumed Gynostemma pentaphyllum extract and the placebo group.

[0032] Figure 19 is a graph showing the results of measuring the skin keratin content of the group consuming Gynostemma pentaphyllum extract and the placebo group over time (before consumption, after 6 weeks, and after 12 weeks).

[0033] FIGS. 20a to 20c are graphs showing the results of measuring the skin roughness of the cheek area of ​​the group consuming Gynostemma pentaphyllum extract and the placebo group over time (before consumption, after 6 weeks, and after 12 weeks). FIG. 20a is a graph measuring the arithmetic mean roughness of the cheek area, FIG. 20b is a graph measuring the maximum value of the height of the protrusion-indentation of the cheek area, and FIG. 20c is a graph measuring the average value of the five highest protrusions and the five lowest indentations.

[0034] Figure 21 shows photographs of the skin roughness of the group that consumed Gynostemma pentaphyllum extract and the placebo group over time (before consumption, after 6 weeks, and after 12 weeks).

[0035] Food composition

[0036] One aspect of the present invention provides a food composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I:

[0037] <Chemical Formula I>

[0038] .

[0039] As used in this specification, the term "Gynostemma pentaphyllum" refers to a perennial vine belonging to the Cucurbitaceae family. It grows wild in forests in mountains and fields; its rhizomes spread sideways, have white hairs at the nodes, and grow in an tangled manner, although it also climbs using tendrils. Tea made from dried Gynostemma pentaphyllum leaves is commonly referred to as vine tea and is known to eliminate alopecia areata, restore the functions of various organs, and maintain healthy skin. It possesses anti-stress effects, inhibits relaxed and spastic constipation, and has antidiarrheal effects, and is effective for bronchial asthma and senile chronic bronchitis. Additionally, it is known to have antitussive and expectorant effects, is effective for stress-induced ulcers, and provides prevention of hepatitis and arteriosclerosis, as well as analgesic effects.

[0040] In the present invention, the Gynostemma pentaphyllum extract may be any one extract selected from the group consisting of flowers, leaves, stems, branches, fruits, fruit peels, roots, tissue cultures derived therefrom, and combinations thereof. In this case, the Gynostemma pentaphyllum may be fresh herb or dried herb. Preferably, the Gynostemma pentaphyllum extract may be a leaf extract.

[0041] As used herein, the term "extract" may include all substances obtained by extracting components of a natural product, regardless of the extraction method, extraction solvent, extraction conditions, extracted components, or the form of the extract. Additionally, the extract may include substances that can be obtained by processing or treating the components of the natural product by other methods after extraction. In this case, the processing or treatment may include, for example, dilution, concentration, drying, purification, fractionation, filtration, fermentation, enzymatic treatment, etc., but is not limited thereto. Accordingly, the extract may include an extract solution, a diluted or concentrated extract solution, a dried product obtained by drying the extract solution, a modified or purified product thereof, or a fraction obtained by fractionating the same.

[0042] The above extraction method may utilize known natural product extraction methods such as steam distillation extraction, hot water extraction, ethanol extraction, heating extraction, cold maceration extraction, reflux extraction, reflux cooling extraction, ultrasonic extraction, and subcritical extraction. Preferably, it may be distillation extraction.

[0043] Specifically, the above-mentioned Gynostemma extract may be extracted by a method comprising the steps of: i) mixing Gynostemma and purified water; ii) heating the mixture at high pressure and high temperature; and iii) cooling the steam generated in ii) to collect a distilled extract.

[0044] More specifically, the above-mentioned Gynostemma extract can be extracted by the following method.

[0045] First, it may include a step of mixing Gynostemma pentaphyllum and purified water.

[0046] At this time, the mixing ratio of the above-mentioned Gynostemma pentaphyllum and purified water may be about 1:10 to about 1:1000 in weight ratio (w / w). Specifically, the mixing ratio may be about 1:100 to about 1:1000, about 1:20 to about 1:800, about 1:30 to about 1:400, about 1:40 to about 100, or about 1:50 to about 1:80, but is not limited thereto. In one embodiment, the mixing ratio of the above-mentioned Gynostemma pentaphyllum and purified water may be about 1:60.

[0047] Second, the method may include a step of heating the above mixture to high pressure and high temperature.

[0048] In the present invention, the Gynostemma extract may be extracted at a high temperature and simultaneously extracted under high pressure conditions.

[0049] The extraction temperature may be approximately 80°C to approximately 180°C. More specifically, it may be approximately 80°C to approximately 180°C, approximately 90°C to approximately 150°C, or approximately 100°C to approximately 130°C. At this time, the pressure may be approximately 1.1 atm to approximately 2.5 atm, approximately 1.2 atm to approximately 2.2 atm, approximately 1.3 atm to 2.0 atm, or approximately 1.4 atm to approximately 1.8 atm, but may be appropriately selected depending on other conditions. In one embodiment, the extract may be extracted at approximately 109°C under conditions of approximately 1.5 atm.

[0050] The above extract may be performed for about 10 to about 20 hours under the above high temperature and high pressure conditions. Specifically, it may be about 10 to about 20 hours, about 11 to about 18 hours, about 12 to about 16 hours, or about 13 to about 15 hours, but may be appropriately selected depending on other conditions. In one embodiment, the Gynostemma extract may be performed for about 15 hours.

[0051] Third, the method may include a step of cooling the steam generated by heating the above method to collect the distillation extract.

[0052] At this time, cooling can be performed at room temperature.

[0053] In addition, the above Gynostemma extract may further include a step of sterilization.

[0054] At this time, sterilization can be performed by heating the extract. Specifically, sterilization can be performed by heating at about 90°C to about 100°C, specifically about 95°C. At this time, the sterilization time can be about 10 minutes to about 120 minutes, or about 30 minutes to about 80 minutes, specifically about 60 minutes.

[0055] The Gynostemma extract according to the present invention may contain damulin B.

[0056] The term "Damulin B" used in the present invention is a damarane-based saponin substance known as a standard substance of *Gynostemma pentaphyllum*, along with Damulin A. Damulin A and Damulin B are known to exhibit effects that improve obesity or diabetes by activating AMPK (AMP-activated protein kinase). Damulin B can be represented by the chemical formula I.

[0057] As used herein, the term "improvement of skin health" refers to any act that improves or beneficially alters skin health through the prevention or improvement of skin damage, skin moisturization, or improvement of skin roughness, etc.

[0058] The aforementioned “improvement of skin damage” includes all actions that result in an overall improvement or beneficial alteration of the damaged skin condition. This may include, but is not limited to, skin regeneration (wound healing), improvement / alleviation of scars, improvement of skin texture, improvement of (hyper)pigmentation, improvement of skin wrinkles, improvement of skin elasticity, and alleviation of skin irritation (relief of inflammation). Additionally, it may include the improvement of damaged skin, including burns, scars, pigmentation caused by acne, etc.

[0059] In the present invention, the skin damage may be skin damage caused by ultraviolet rays. In other words, the food composition of the present invention may be used to protect the skin from ultraviolet rays or to improve skin damage caused or aggravated by ultraviolet rays.

[0060] Specifically, skin damage caused by ultraviolet rays may be any one selected from the group consisting of aging, wrinkles, burns, erythema, sunburn, pigmentation, skin cancer, decreased immune response, and combinations thereof, but is not limited thereto.

[0061] The above term "skin moisturization" refers to any action that maintains skin moisture or prevents moisture loss.

[0062] The aforementioned "improvement of skin roughness" refers to a reduction in the irregularity and degree of wrinkles on the skin surface, resulting in a smoother and healthier state.

[0063] The aforementioned "skin regeneration" refers to a series of reactions in which skin tissue is reorganized in response to damage caused to the skin by aging, trauma, disease, etc.

[0064] The above "improvement / alleviation of scar marks" refers to a series of reactions in which marks and / or scars remaining during or after the healing of damaged skin are improved or alleviated.

[0065] The above "improvement of skin texture" refers to the improvement of the overall condition of the skin, such as a series of reactions in which the gaps between skin cells widen, furrows deepen, or rough skin becomes soft due to the accumulation of dead skin cells on the skin surface.

[0066] The above "improvement of (hyper)pigmentation" refers to a series of reactions in which a darkened skin tone becomes brighter. Specifically, it may refer to the brightening of a skin tone darkened by melasma or freckles, but is not limited thereto.

[0067] The aforementioned "improvement of skin wrinkles" refers to a series of reactions that inhibit or hinder the formation of wrinkles on the skin, or alleviate wrinkles that have already formed.

[0068] The above term, "improvement of skin elasticity," refers to a series of reactions that alleviate the degree of sagging or loosening of the skin.

[0069] The term "skin irritation relief" above refers to a series of reactions that alleviate symptoms of skin irritated by external pollutants or diseases. Specifically, it may refer to, but is not limited to, the alleviation of inflammation, itching, redness, etc. caused by irritation.

[0070] As used in this specification, the term "included as an active ingredient" means that the extract or a component of the extract described in this specification is added to an extent capable of producing the effects mentioned above, and includes formulation in various forms by adding various components as auxiliary ingredients for the delivery and stabilization of the active ingredient.

[0071] In the present invention, the food may include a health functional food.

[0072] As used herein, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with Act No. 6727 of the Health Functional Foods Act. Here, "functionality" means consuming the food for the purpose of obtaining beneficial effects for health uses, such as regulating nutrients or physiological actions regarding the structure and function of the human body. In the present invention, the terms food composition, health food, health functional food, and health functional food may be used interchangeably.

[0073] Accordingly, the Gynostemma extract or Damulin B according to the present invention can be used as a raw material for a health functional food for preventing or improving skin damage.

[0074] The content of the active ingredient, Gynostemma pentaphyllum extract or Damulin B, in the above food composition can be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0075] Specifically, in a food composition containing a Gynostemma extract as an active ingredient, the Gynostemma extract may be included in an amount of about 0.001 weight% (v / v) to about 100 weight% (v / v) based on the total weight of the food composition. Specifically, about 0.001 wt% (v / v) to about 100 wt% (v / v), about 0.01 wt% (v / v) to about 100 wt% (v / v), about 0.01 wt% (v / v) to about 100 wt% (v / v), about 0.1 wt% (v / v) to about 100 wt% (v / v), about 1 wt% (v / v) to about 100 wt% (v / v), about 10 wt% (v / v) to about 100 wt% (v / v), about 20 wt% (v / v) to about 100 wt% (v / v), about 30 wt% (v / v) to about 100 wt% (v / v), about 40 wt% (v / v) to about 100 wt% (v / v), about 50 wt% (v / v) to about 100 It may be included in weight% (v / v), about 70 weight% (v / v) to about 100 weight% (v / v) or about 90 weight% (v / v) to about 100 weight% (v / v).

[0076] Specifically, in a food composition containing damulin B as an active ingredient, the damulin B may be included in an amount of about 0.001 wt% (w / v) to about 10 wt% (w / v) with respect to the total weight of the food composition. Specifically, it may be included in an amount of about 0.001 wt% (w / v) to about 10 wt% (w / v), about 0.01 wt% (w / v) to about 8 wt% (w / v), or about 0.1 wt% (w / v) to about 5 wt% (w / v).

[0077] However, the composition according to the present invention may be used in a range smaller than the above range in the case of long-term consumption for the purpose of health and hygiene or health control.

[0078] In addition, the above-mentioned health functional food composition may contain the active ingredient, the above-mentioned Gynostemma extract or Damulin B, alone, or be used by appropriately mixing it with other foods or food ingredients according to conventional methods.

[0079] The health functional food composition according to the present invention can be formulated into a conventional health functional food formulation known in the art. The health functional food may be manufactured, for example, as a powder, granule, tablet, pill, capsule, suspension, syrup, infusion, liquid, extract, gum, tea, jelly, or beverage.

[0080] As the above-mentioned food-grade acceptable carrier or additive, any carrier or additive known in the relevant technical field to be available for use in the preparation of the formulation to be manufactured may be used. For example, in the case of a health functional food in the form of a beverage, it may contain preservatives, stabilizers, emulsifiers, dispersants, flavorings, colorings, etc., which are included in food additives, as needed.

[0081] In the case of health functional foods in capsule form, hard capsules can be manufactured by filling a conventional hard capsule with a mixture of Gynostemma extract or Damulin B and additives such as excipients, or the granules thereof, or coated granules, and soft capsules can be manufactured by filling a capsule base such as gelatin with a mixture of Gynostemma extract or Damulin B and additives such as excipients. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed. Health functional foods in pill form can be prepared by molding a mixture of Gynostemma extract or Damulin B with excipients, binders, disintegrants, etc., in a suitable way, and as needed, the pills may be coated with sucrose or other suitable coating agents, or coated with starch, talc, or other suitable substances. A granular health functional food can be prepared in granular form by appropriately mixing the above-mentioned Gynostemma extract or Damulin B with excipients, binders, disintegrants, etc., and may contain flavoring agents, stimulants, etc. as needed. The excipients may be, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium styrate talc may also be used in addition to simple excipients.

[0082] In the present invention, the definitions of terms regarding excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. include those described in literature known in the art that have the same or similar functions (Commentary on the Korean Pharmacopoeia, Munseongsa, Association of Korean Colleges of Pharmacy, 5th Revised Edition, pp. 33-48, 1989).

[0083] feed composition

[0084] Another aspect of the present invention provides a feed composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I:

[0085] <Chemical Formula I>

[0086] .

[0087] The above-mentioned Gynostemma extract, Damulin B, and skin health improvement are the same as described above.

[0088] As used herein, the term "feed" means any natural or artificial prescribed food, meal, etc., or the components of said meal, intended for or suitable for animals to eat, consume, and digest.

[0089] In a feed composition containing the above-mentioned Gynostemma extract as an active ingredient, the above-mentioned Gynostemma extract may be included in an amount of about 0.001 weight% (v / v) to about 100 weight% (v / v) based on the total weight of the feed composition. Specifically, about 0.001 wt% (v / v) to about 100 wt% (v / v), about 0.01 wt% (v / v) to about 100 wt% (v / v), about 0.01 wt% (v / v) to about 100 wt% (v / v), about 0.1 wt% (v / v) to about 100 wt% (v / v), about 1 wt% (v / v) to about 100 wt% (v / v), about 10 wt% (v / v) to about 100 wt% (v / v), about 20 wt% (v / v) to about 100 wt% (v / v), about 30 wt% (v / v) to about 100 wt% (v / v), about 40 wt% (v / v) to about 100 wt% (v / v), about 50 wt% (v / v) to about 100 It may be included in weight% (v / v), about 70 weight% (v / v) to about 100 weight% (v / v) or about 90 weight% (v / v) to about 100 weight% (v / v).

[0090] In a feed composition containing the above-mentioned damulin B as an active ingredient, the above-mentioned damulin B may be included in an amount of about 0.001 wt% (w / v) to about 10 wt% (w / v) with respect to the total weight of the feed composition. Specifically, it may be included in an amount of about 0.001 wt% (w / v) to about 10 wt% (w / v), about 0.01 wt% (w / v) to about 8 wt% (w / v), or about 0.1 wt% (w / v) to about 5 wt% (w / v).

[0091] In addition to Gynostemma pentaphyllum extract or Damulin B, the above feed composition may include known carriers, stabilizers, or additives permitted for feed. For example, binders, emulsifiers, preservatives, etc. added to prevent quality degradation, and amino acid preparations, vitamins, enzymes, flavorings, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. added to the feed to increase utility. In addition, feed mixing agents, etc., may be additionally included, but are not limited thereto.

[0092] The above feed composition may include various nutrients such as vitamins, amino acids, and minerals, antioxidants, and other additives as needed, and may be in a suitable form such as powder, granules, pellets, or suspension.

[0093] The feed composition of the present invention may be supplied to a monogastric animal alone or mixed with feed. The feed of the present invention is not particularly limited and may be any feed, such as powder feed, solid feed, dry feed, wet feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc.

[0094] use

[0095] Another aspect of the present invention provides a use for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I:

[0096] <Chemical Formula I>

[0097] .

[0098] The above-mentioned Gynostemma extract, Damulin B, and skin health improvement are the same as described above.

[0099] The present invention will be explained in more detail below through the following examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples only.

[0100] Preparation Example 1. Preparation of Gynostemma pentaphyllum extract

[0101] To obtain the Gynostemma pentaphyllum extract of the present invention, 20 kg of dried Gynostemma pentaphyllum leaves and 1.2 tons of purified water were mixed and heated to 109°C for 15 hours in a steam tank at 1.5 atmospheres. The steam generated by heating was cooled and condensed in a closed-loop system to obtain the final Gynostemma pentaphyllum extract. The Gynostemma pentaphyllum extract (GPHD) obtained by the above method was sterilized by heating at 95°C for 1 hour.

[0102] Preparation Example 2. Preparation of cell culture medium containing Gynostemma pentaphyllum extract

[0103] To prepare a cell culture medium, DMEM powder, a cell medium, was directly dissolved in a 100% (v / v) concentration of Gynostemma pentaphyllum extract and treated with bicarbonate to establish a pH of 7.2. Subsequently, fetal bovine serum (FBS) and penicillin-streptomycin sulfate were added to a sterile filter at concentrations of 10% and 100 μg / mL, respectively. The medium containing the Gynostemma pentaphyllum extract prepared as described above was used as the Gynostemma pentaphyllum extract medium concentrate. The Gynostemma pentaphyllum extract medium concentrate was diluted (v / v) by mixing it with DMEM medium at concentrations corresponding to each of the following experimental conditions and used as a Gynostemma pentaphyllum extract sample.

[0104] At this time, damulin B, a component of Dolwort, was purified to over 98% using HPLC and dissolved in a DMSO solution for use.

[0105] I. Evaluation of the effect of improving skin damage caused by ultraviolet rays

[0106] Experimental Method 1. Analysis of cells damaged by ultraviolet radiation

[0107] Experimental Method 1.1. Ultraviolet irradiation of keratinocytes

[0108] Keratinocyte cell lines (HaCaT, ATCC) were treated with DMEM containing 10% fetal bovine serum and 100 μg / mL penicillin-streptomycin sulfate in 5% CO₂ 2, They were cultured at 37℃. At this time, 1 x 10⁶ keratinocytes per plate 5 Cultured at a density of cells / mL.

[0109] After pre-treating the cultured cells with the prepared Gynostemma pentaphyllum extract (GPHD) or damulin B for 6 hours, respectively, the surface of the cultured cells was washed with DPBS. Subsequently, UVB was irradiated at 20 mJ / cm² using a CN-15 UV darkroom (Vilber). 2 As a condition of investigated.

[0110] Experimental Method 1.2. Analysis of Cell Viability

[0111] Cytotoxicity was assessed by confirming cell viability through MTT analysis.

[0112] Specifically, 1 x 10⁶ keratinocytes in a 24-well plate 4After inoculating cells at a concentration of cells / well, the wells were treated with either Gynostemma pentaphyllum extract or damulin B, respectively, and cultured for an additional 24 hours. At this time, Gynostemma pentaphyllum extract was treated at concentrations of 0%, 5%, 10%, 20%, 50%, or 100% (v / v), and damulin B was treated at concentrations of 0 μM, 10 μM, 20 μM, or 40 μM. Subsequently, 500 μg / mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well and reacted at 37°C for 2 hours. After culture, the culture medium treated with MTT solution was removed, and DMSO (dimethyl sulfoxide) was added to measure the absorbance (540 nm).

[0113] Experimental Method 1.3. Western Blot

[0114] Keratinocytes 1 x 10 6 Cells were seeded into a 60 mm culture dish, treated with Gynostemma pentaphyllum extract (50% (v / v)) and Damulin B (20 μM), respectively, and cultured for 6 hours. Subsequently, UVB (20 mJ / cm²) was applied. 2 The cells were treated with ) and the cell culture medium was replaced (DMEM) and cultured for an additional 1.5 hours. Cells treated as described above were obtained, and proteins were extracted by treating with ripa buffer.

[0115] After quantifying the extracted protein using the Bradford assay method, SDS-PAGE was performed, and the protein was transferred to a PVDF membrane. The target protein was labeled by sequentially treating the membrane with a primary antibody and a secondary antibody, respectively, and reacting them. At this time, the primary antibodies are β-actin (Cell Signaling Technology, 9562S), p44 / 42 MAPK (Erk1 / 2) (Cell Signaling Technology, 4695S), phospho- p44 / 42 MAPK (Erk1 / 2) (Cell Signaling Technology, 4376S), SAPK / JNK (Cell Signaling Technology, 9252S), phospho-SAPK / JNK(Cell Signaling Technology, 4668S), p38 MAPK(Cell Signaling Technology, 9212S), phosphor-p38 MAPK(Cell Signaling Technology, 9211S), p53(Cell Signaling Technology, 9282S), phosphor-p53(Cell Signaling Technology, 9284), c-Jun(Cell Signaling Technology, 9165S), Phospho-c-Jun (Cell Signaling Technology, Antibodies against 3270S), c-Fos (Cell Signaling Technology, 2250S), phospho-c-Fos (Cell Signaling Technology, 5348S), Nrf2 (Santa Cruz Biotechnology, sc-365949), and Heme Oxygenase 1 / HMOX1 (Santa Cruz Biotechnology, sc-136960) were used.For the secondary antibody, either Goat anti-rabbit IgG, multiplex antibody (Enzo Life Sciences, ADI-SAB-300-J) or Goat anti-mouse IgG F(ab')2, multiplex antibody (Enzo Life Sciences, ADI-SAB-100-J) was used. Subsequently, target protein expression was confirmed by treatment with a chemiluminescence (ECL) reagent.

[0116] Experimental Method 1.4. mRNA Expression Analysis

[0117] Cells were obtained using the same method as in Experimental Method 1.3 above, and total RNA was extracted by treating with Trizol (1 mL). At this time, the Gynostemma extract was also obtained by treating cells that were not irradiated with UV light under the same conditions. Subsequently, after quantifying the total RNA, cDNA was synthesized using a cDNA kit (Thermo Scientific) with the total RNA as a template. The concentration of the cDNA prepared by the above method was measured, and PCR was performed using this as a template and the primers listed below. The PCR was carried out for 30 cycles at 95°C for 2 minutes, 95°C for 40 seconds, 58°C for 40 seconds, and 72°C for 40 seconds, followed by a reaction at 72°C for 2 minutes.

[0118] The PCR product obtained by the above method was loaded onto a 1.5% agarose gel containing ethidium bromide and detected. Expression data were compared based on β-actin mRNA levels in each sample. The primers used are listed in Table 1 below.

[0119] GeneDirectionSequenceMMP1Forward5'-CCCTTCTACCCGGAAGTTGA-3'(Sequence No. 1)Reverse5'-CCGTGTAGCACATTCTGTCC-3'(Sequence No. 2)MMP3Forward5'-ATTCCATGGAGCCAGGCTTTC-3'(Sequence No. 3)Reverse5'-CATTTGGGTCAAACTCCAACTGTG-3'(Sequence No. 4)COL1A1Forward5'-TGACCTCAAGATGTGCCACT-3'(Sequence No. 5)Reverse5'-CGAACCAGACATGCCTCTTG-3'(Sequence No. 6)β-actinForward5'-ACGTTGCTATCCAGGCTGTG-3'(Sequence No. 7)Reverse5'-AAGGAAGGCTGGAAGAGTGC-3'(Sequence No. 8)

[0120] Experimental Method 1.5. Measurement of Reactive Oxygen Species Production

[0121] In the same manner as in Experimental Method 1.3 above, the Gynostemma extract, Damulin B, and UV rays were reacted, then stained with the Muse Mito Potential Assay Kit (Merck Millipore) and analyzed using the Muse Cell Analyzer (Cell Analyzer).

[0122] Experimental Method 1.6. Statistical Processing

[0123] The experimental results were expressed as mean ± standard deviation (Mean ± SEM), and when testing the difference in means between the control group and the experimental group, one-way ANOVA was used, and a difference was determined to be statistically significant when the P-value was less than 0.05.

[0124] Experimental Result 1. Confirmation of changes in cells damaged by ultraviolet rays

[0125] Experimental Results 1.1. Confirmation of cytotoxicity by treatment with Gynostemma pentaphyllum extract or Damulin B in UV-treated keratinocytes

[0126] As shown in Figure 1, when keratinocytes were treated with Gynostemma pentaphyllum extract or Damulin B at different concentrations, it was confirmed that Gynostemma pentaphyllum extract had no cytotoxicity up to 50% and Damulin B up to 20 μM.

[0127] Experimental Results 1.2. Confirmation of changes in collagenase expression in keratinocytes treated with Gynostemma pentaphyllum extract or Damulin B

[0128] As shown in Figures 2a and 2b, it was confirmed that the gene expression of collagen-degrading enzymes MMP-1 (metalloproteinase-1) and MMP-2 was reduced in the group treated with Gynostemma pentaphyllum extract (50% (v / v)) and the group treated with Damulin B (20 μM).

[0129] Experimental Results 1.3. Confirmation of changes in collagen-related gene expression induced by treatment with Gynostemma pentaphyllum extract in UV-irradiated keratinocytes

[0130] As shown in Figure 3, it was confirmed that when keratinocytes were irradiated with ultraviolet light, the expression of COL1A1 (alpha-1 type I collagen), a gene related to collagen synthesis, decreased. At this time, it was confirmed that it increased when treated with Gynostemma pentaphyllum extract. In addition, it was confirmed that although the expression of collagen-degrading enzymes MMP-1 and MMP-3 increased due to ultraviolet light, it decreased again upon treatment with Gynostemma pentaphyllum extract.

[0131] Experimental Results 1.4. Confirmation of Changes in MAPK Signaling Mechanisms by Treatment with Gynostemma pentaphyllum Extract or Damulin B in UV-Irradiated Keratinocytes

[0132] As shown in Figure 4a, phosphorylation of MAPKs (mitogen-activated protein kinases) such as ERK, JNK, and p38, as well as p53, was induced by UV irradiation. On the other hand, it was confirmed that the phosphorylation was reduced in cells treated with UV irradiation and Gynostemma pentaphyllum extract.

[0133] In addition, it was confirmed that the expression of the AP-1 subunit, a downstream protein of the MAPK signaling pathway, increased due to UV irradiation but decreased again due to treatment with Gynostemma pentaphyllum extract or damulin B (Fig. 4b).

[0134] Through the above results, it was confirmed that Gynostemma pentaphyllum extract or damulin B can inhibit the expression of collagen synthesis-related genes (COL1A1) and inhibit the increase in the expression of collagenases (MMP1 or MMP3) by increasing the expression of the subunit of transcription factor AP-1 (activator protein 1), which is involved in inhibiting collagen synthesis by MAPK phosphorylated by UV irradiation (Fig. 4c).

[0135] Experimental Result 1.5. Confirmation of Antioxidant Effects of Gynostemma pentaphyllum Extract or Damulin B in UV-Irradiated Keratinocytes

[0136] As shown in Figures 5a and 5b, it was confirmed that the production of reactive oxygen species increased due to UV irradiation, but decreased due to treatment with Gynostemma pentaphyllum extract or Damulin B.

[0137] In addition, as shown in Figure 6, the expression of the antioxidant enzyme HO-1 (heme oxygenase 1) and the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), which regulates its expression, was reduced in keratinocytes treated with ultraviolet light. On the other hand, it was confirmed that the expression of HO-1 and Nrf2 proteins was restored in keratinocytes treated with ultraviolet light, and with Gynostemma pentaphyllum extract or Damulin B.

[0138] Based on the above results, it is believed that the Gynostemma pentaphyllum extract or Damulin B can inhibit skin wrinkles caused by UV radiation by reducing reactive oxygen species increased by UV irradiation, thereby reducing the activity of MAPK induced by reactive oxygen species and the expression of its downstream transcription factor AP-1, thereby reducing the expression of collagenases MMP1 and MMP3, and increasing the expression of COL1A1, a gene related to collagen synthesis (Fig. 7).

[0139] II. Evaluation of Skin Dryness Improvement Effect

[0140] Experimental Method 2. Analysis of skin moisture status damaged by ultraviolet rays

[0141] Experimental Method 2.1. Ultraviolet Irradiation of Skin Fibroblasts

[0142] The experiment was conducted by culturing skin fibroblast cell lines (Hs68, ATCC). The experimental method was performed in the same manner as experimental method 1.1 described above.

[0143] Experimental Method 2.2. Western Blot

[0144] Skin fibroblasts 1 x 10 6 Cells were seeded into a 60 mm culture dish, treated with Gynostemma pentaphyllum extract (50% (v / v)) and Damulin B (20 μM), respectively, and cultured for 6 hours. Subsequently, UVB (20 mJ / cm²) was applied. 2 ) was investigated, and after replacing the cell culture medium (DMEM), the cells were cultured for an additional 1.5 hours. Cells treated as described above were obtained, proteins were extracted by treating with Ripa buffer, and Western blot was performed in the same manner as in Experimental Method 1.3 above.

[0145] At this time, the primary antibody used was an antibody against β-actin (Cell Signaling Technology, 9562S), TIMP1 (Cell Signaling Technology, 8946S), TIMP2 (Cell Signaling Technology, 5738S), SMAD2 / 3 (Cell Signaling Technology, 8685S), phosphor-SMAD2 / 3 (Cell Signaling Technology, 8828S), or GPX1 (Cell Signaling Technology, 3206S). The secondary antibody used was a Goat anti-rabbit IgG multiplex antibody (Enzo Life Sciences, ADI-SAB-300-J) or a Goat anti-mouse IgG F(ab')2 multiplex antibody (Enzo Life Sciences, ADI-SAB-100-J).

[0146] Experimental Method 2.3. mRNA Expression Analysis

[0147] Cells were obtained using the same method as in Experimental Method 2.2 above, and total RNA was extracted by treating with trizol (1 mL). Additionally, skin tissue from experimental mice was collected, and RNA was extracted by homogenizing the tissue using the Easy Blue® kit (Intron Biotechnology). Subsequently, cDNA was synthesized using a cDNA kit (Thermo Scientific) with the RNA as a template. Using the cDNA as a template, the expression of the gene of interest was analyzed by real-time PCR using SYBR (SYBR Premix Ex Taq, Takara).

[0148] The primers used at this time are listed in Tables 2 and 3 below.

[0149] GeneDirectionSequenceHyaluronidase 1Forward5'-GCAGAACTGGGAGAGCTACA-3'(Sequence No. 9)Reverse5'-GGTAGACAGACGGGAAGAGG-3'(Sequence No. 10)COL1A1Forward5'-TGACCTCAAGATGTGCCACT-3'(Sequence No. 5)Reverse5'-CGAACCAGACATGCCTCTTG-3'(Sequence No. 6)COL1A2Forward5'-CATCCCAGCCAAGAACTGGT-3'(Sequence No. 11)Reverse5'-ATATCAAGGAAGGGCAGGCG-3'(Sequence No. 12)TIMP1Forward5'-CTCGTCATCAGGGCCAAGTT-3'(Sequence No. 13)Reverse5'-GTAGGTCTTGGTGAAGCCCC-3'(Sequence No. 14)TIMP2Forward5'-TAGTGATCAGGGCCAAAGCG-3'(Sequence No. 15)Reverse5'-CAGGCTCTTCTTCTGGGTGG-3'(Sequence No. 16)β-actinForward5'-ACGTTGCTATCCAGGCTGTG-3'(Sequence No. 7)Reverse5'-AAGGAAGGCTGGAAGAGTGC-3'(Sequence No. 8)

[0150] GeneDirectionSequenceInvolucrinForward5'-GGGTCAGTCACTTAAGCAAG-3'(Sequence No. 17)Reverse5'-CTACTTCTCCTGCTGTGTCC-3'(Sequence No. 18)FilaggrinForward5'-CTCAGGAGGAAGAGGACAGT-3'(Sequence No. 19)Reverse5'-CAAGGTGCTTTGCTGTAAAT-3'(Sequence No. 20)AQP3Forward5'-TCTGGACACTTGGATATGGTCAA-3'(Sequence No. 21)Reverse5'-CAACAATGGCCAGCACACA-3'(Sequence No. 22)HYAL-1Forward5'-TACACAGCATGCTCAGAAAG-3'(Sequence No. 23)Reverse5'-AGTGTCTCCATTCCAAACAG-3'(Sequence No. 24)HYAL-2Forward5'-AATCTGTGGAACGCTACATC-3'(Sequence No. 25)Reverse5'-GCCTGCTTCATTACTCTGTC-3'(Sequence No. 26)

[0151] Experimental Method 2.4. UV Irradiation and Administration of Gynostemma Extract to Mice

[0152] 5-week-old male HR-1 hairless mice (SLC Inc.) reared under constant temperature and humidity conditions (temperature: 22 ± 1℃, humidity: 40%–60%, light-dark cycle: 12 hours) were acclimatized for one week. The mice were randomly divided into four groups: a control group (no UV irradiation), a group irradiated only with UV, a group administered only Gynostemma pentaphyllum extract (~0.9 mL / day, orally as a liquid), or a group administered both UV irradiation and Gynostemma pentaphyllum extract (~0.9 mL / day, orally as a liquid). All groups except the control group were exposed to UVB three times a week for seven weeks, and each test substance was administered. The UV energy was 60 mJ / cm². 2 (1~2 weeks), 120 mJ / cm² 2 (3~4 weeks), 180 mJ / cm² 2 (5~6 weeks) and 240 mJ / cm² 2The investigation was conducted by gradually increasing the amount (7 weeks).

[0153] Experimental Method 2.5. Physiological Analysis of Skin Surface

[0154] After administering UV irradiation and Gynostemma pentaphyllum extract in the same manner as in Experimental Method 2.4 above, epidermal water content and transepidermal water loss (TEWL) were measured on the dorsal skin of experimental mice using a GPSkin Barrier® (GPOWER Inc.) on the last day of UV exposure. At this time, epidermal water content and TEWL values ​​were expressed in arbitrary units (AU) and g / m², respectively. 2 It was displayed as / h.

[0155] Experimental Method 2.6. Statistical Processing

[0156] It was performed in the same manner as the above-described experimental method 1.6.

[0157] Experimental Result 2. Confirmation of moisturizing effect on UV-damaged skin in skin fibroblasts

[0158] Experimental Results 2.1. Confirmation of Changes in Collagen-Related Gene Expression Caused by Treatment with Gynostemma pentaphyllum Extract or Damulin B

[0159] As shown in Figures 8a and 8b, the expression of COL1A1 (alpha-1 type I collagen) increased in both the group treated with Gynostemma pentaphyllum extract (50%) and the group treated with Damulin B (20 μM). In addition, the collagenase MMP1 (metalloproteinase-1) decreased. Furthermore, the expression of the collagenase (MMP) inhibitors TIMP-1 (TIMP metallopeptidase inhibitor 1) and TIMP-2 (TIMP metallopeptidase inhibitor 2) increased, and the expression of the hyaluronidase 1 decreased.

[0160] Based on the above results, it is believed that Gynostemma pentaphyllum extract or damulin B can increase collagen synthesis and inhibit degradation, while simultaneously inhibiting hyaluronic acid degradation (Fig. 8c).

[0161] Experimental Results 2.2. Confirmation of Changes in Collagen Synthesis-Related Gene Expression by Treatment with Gynostemma pentaphyllum Extract or Damulin B

[0162] As shown in Figure 9a, in the group treated with UV light alone, the expression of collagenase inhibitors TIMP-1 and TIMP-2 proteins was reduced in skin fibroblasts. On the other hand, in the group treated with UV light and Gynostemma pentaphyllum extract or UV light and Damulin B, the reduced protein expression of TIMP-1 and TIMP-2 increased.

[0163] Furthermore, the expression of SMAD (suppressor of mothers against decapentaplegic) protein, a transcription factor involved in regulating collagen gene synthesis, decreased in the group treated with UV light alone. Conversely, it was confirmed that SMAD protein expression increased in the groups treated with UV light and Gynostemma pentaphyllum extract, or with UV light and Damulin B.

[0164] In addition, it was confirmed that the GPX-1 (glutathione peroxidase 1) protein, which has an antioxidant effect, was reduced by UV treatment alone, but its expression increased by treatment with Gynostemma pentaphyllum extract or damulin B.

[0165] Through the above results, it was confirmed that Gynostemma pentaphyllum extract or damulin B can inhibit the excessive production of reactive oxygen species induced by ultraviolet rays in the dermis, thereby preventing the decrease in the expression of genes or proteins involved in collagen synthesis and the increase in the expression of genes or proteins involved in collagen degradation caused by excessive reactive oxygen species (Fig. 9b).

[0166] Experimental Result 3. Confirmation of improved moisturization effect on UV-damaged skin in hairless mice irradiated with UV rays

[0167] Experimental Results 3.1. Confirmation of changes in gene expression related to skin barrier function and skin hydration by administration of Gynostemma pentaphyllum extract

[0168] As shown in Figure 10, the gene expression levels of involucrin, filaggrin, and AQP3 (aquaporin 3) decreased in mice irradiated with UV light alone. On the other hand, it was confirmed that the expression of the above genes was restored in the group irradiated with UV light and administered Gynostemma pentaphyllum extract. In addition, the gene expression of hyaluronic acid degradase 1 and hyaluronic acid degradase 2 increased in the group irradiated with UV light alone, but decreased in the group irradiated with UV light and administered Gynostemma pentaphyllum extract.

[0169] Experimental Result 3.2. Confirmation of Skin Moisturizing Enhancement Effect by Administration of Gynostemma pentaphyllum Extract

[0170] As shown in Figure 11, the epidermal water content of mice irradiated with UV light alone decreased, but it was confirmed that the epidermal water content recovered in the group administered with UV light and Gynostemma pentaphyllum extract. In addition, while the transepidermal water loss of mice irradiated with UV light alone increased, it was confirmed that it decreased again in the group administered with UV light and Gynostemma pentaphyllum extract.

[0171] Based on the above results, it is believed that Gynostemma pentaphyllum extract or Damulin B can prevent photoaging caused by ultraviolet rays. In addition, it is believed that Gynostemma pentaphyllum extract or Damulin B can also exhibit a moisturizing effect on the skin by increasing the expression of collagen synthesis enzymes and collagenase inhibitors in the skin and decreasing the expression of hyaluronic acid degrading enzymes responsible for moisture retention in the skin (Fig. 12).

[0172] III. Evaluation of Skin Health Improvement Effects at the Human Level

[0173] Experimental Method 3. Analysis of skin damaged by ultraviolet radiation at the human body level

[0174] Experimental Methods 3.1. Selection of Research Participants

[0175] A human clinical trial was conducted on 100 subjects, men and women aged 30 to 60 years with a skin moisture content of 48 AU (corneometer value) or less and eye wrinkles of grade 3 or higher (Fig. 13).

[0176] At this time, subjects corresponding to the following criteria were excluded from the study:

[0177] 1) Persons with irritation or allergic reactions to raw materials similar to pharmaceuticals or products for human clinical trials; 2) Persons with sensitive or hypersensitive skin at the test site; 3) Persons who participated in a human clinical trial for functional cosmetics or the same study within 2 months of the screening date; 4) Persons who lacked the will to refrain from prohibited drugs or excessive UV exposure during the participation period in the human clinical trial; 5) Persons who continuously used (or took) retinoid-based drugs within 3 months of the screening date; 6) Persons who received dermatological procedures on the test area within 3 months of the screening date; 7) Persons who continuously used cosmetic medical devices (laser devices, high-frequency thermal energy devices, etc.) on the test area for skin improvement purposes within 3 months of the screening date; 8) Persons who took pharmaceuticals or herbal medicines that may affect the test results within 3 months of the screening date (hormone preparations, oral contraceptives, diuretics, acne treatments, etc.); 9) Persons who continuously consumed health functional foods or general foods that may affect the test results within 3 months of the screening date (health functional foods or general foods containing the test ingredient or similar ingredients, protein-containing foods, collagen-containing products, hyaluronic acid, biotin, Brewer's yeast, skin probiotics, hydrangea leaf hot water extract powder, aloe gel, konjac potato extract, products containing pomegranate, Vitamin C, ceramide, gamma-linolenic acid, sheep placenta, N-acetylglucosamine, noni, chlorella, spirulina, etc.), 10) Those who planned to become pregnant, are pregnant, or are breastfeeding within 6 months of the screening date, 11) Those with uncontrolled hypertension or diabetes (excluding those taking antihypertensive drugs with a NO synthesis mechanism), 12) Those with clinically significant diseases requiring treatment (chronic wasting diseases, cardiovascular, endocrine, digestive, urological diseases, neuropsychiatric diseases, malignant tumors) (provided that participation is possible if there has been no recurrence for more than 5 years after surgery), 13) Smokers or those who have quit smoking within the last 6 months, 14) Those showing the following results in diagnostic medical tests (those with AST, ALT > 3 times the upper limit of the reference range;15) Persons with eGFR <60 mL / min / 1.73m2 and those whom the Principal Investigator has determined to be unsuitable for participation in the study due to diagnostic laboratory test results or other reasons.;

[0178] The above human clinical trial was designed as a 12-week, randomized, double-blind, parallel-group, placebo-controlled trial. In this case, the test group is the group that consumed Gynostemma pentaphyllum extract, and the control group is the placebo group.

[0179] The above study subjects were instructed to orally consume 340 mL / day of the test substance once a day in the morning or evening for 12 weeks. The test substance is as shown in Table 4.

[0180] Item Gynostemma pentaphyllum Leaf Extract (Test Product) Placebo (Control Substance) Content (mL) Mixing Ratio (%) Content (mL) Mixing Ratio (%) Gynostemma pentaphyllum Leaf Extract 340 10000 Water Purified 00 340 100 Gynostemma pentaphyllum Leaf Extract Total 340 10000

[0181] This study evaluated the improvement in maintaining skin health against UV-induced skin damage and skin hydration following the intake of the test product. The results were compared with those of a placebo by measuring eye wrinkles, skin elasticity, skin moisture content, transepidermal water loss, skin keratinization, and skin roughness on human skin. The human clinical trial was commissioned to the KBI (Korea Biomedical Institute) and conducted using a randomized, double-blind, placebo-controlled design.

[0182] Experimental Method 3.2. Confirmation of Skin Damage in Study Subjects

[0183] Experimental Method 3.2.1. Visual Evaluation of Eye Wrinkles

[0184] At the time of screening, one researcher conducted a visual evaluation of the left and right eye wrinkles to determine eligibility for the selection criteria. The visual evaluation was performed by fixing the subject's test area under identical conditions regarding posture, illumination, and distance, and then evaluating it according to the visual evaluation criteria for eye wrinkles on a 9-point scale (0.5 increments) (Table 5, Grade 0: no wrinkles ~ Grade 9: many wrinkles).

[0185] No wrinkles-----------------------------------------------------Many wrinkles0123456789

[0186] Experimental Method 3.2.2. Device Evaluation of Eye Wrinkles

[0187] Eye wrinkles in selected test areas before product intake, 6 weeks after intake, and 12 weeks after intake PRIMOS CR After imaging using SF (Canfield), the wrinkle parameter values ​​were analyzed using an analysis program (Table 6).

[0188] Parameter Definition Av.Dep.Average depth of wrinkles(Average wrinkle depth, μm) Mean big.Dep.Mean depth biggest wrinkle(Average depth of the largest wrinkle, μm) Max.Dep.Max depth biggest wrinkle(Maximum depth of the largest wrinkle, μm)

[0189] Eye wrinkle measurements were conducted on selected subjects free from lesions, such as protrusions or depressions in the test area, that would interfere with the analysis. After wearing a hairband to keep hair tidy, the chin and forehead were secured to a jig to minimize facial movement, and measurements were taken by adjusting the facial angle according to the test area to ensure wrinkles were clearly visible. At the time point after product consumption, the same area was photographed under the same conditions, based on the image measured at the time before consumption.

[0190] The analysis was performed using the designated analysis program for the device. For crow's feet, the analysis area was defined from the beginning to the end of the wrinkle; the same area at each time point was matched to ensure a more precise and accurate alignment, and then the same area at each time point was cropped for analysis. In this context, a decrease in the values ​​for average depth of wrinkles, mean depth of biggest wrinkles, and max depth of biggest wrinkles indicates improvement in crow's feet.

[0191] Experimental Method 3.2.3. Measurement of Skin Elasticity in Study Subjects

[0192] The elasticity of selected cheek areas was measured and analyzed using a Cutometer® MPA580(C+K) at points before product intake, 6 weeks after intake, and 12 weeks after intake (Table 7).

[0193] Parameter Definitions R2 (Ua / Uf) Gross elasticity (Recovery ability relative to resistance to physical force, %) R5 (Ur / Ue) Elastic ratio after the first traction (Ratio of relaxation elasticity to suction elasticity, %) R7 (Ur / Uf) Biologic elasticity (Ratio of elasticity relative to the entire curve, %)

[0194] Skin elasticity was measured by fixing the subject in a lying position on a bed facing forward, selecting a flat area in the center of the cheek free of lesions such as protrusions or depressions that might interfere with the measurement, attaching double-sided sticking rings to the probe of the Cutometer® MPA580(C+K) to secure it so it would not detach from the measurement site, and then applying equal vertical force to take the measurement. In this case, a higher measurement value indicates higher skin elasticity.

[0195] Experimental Method 3.3. Confirmation of Skin Moisture in Study Subjects

[0196] Experimental Method 3.3.1. Measurement of Skin Moisture Content in Study Subjects

[0197] At the time points before product intake, 6 weeks after intake, and 12 weeks after intake, the moisture content of the selected test site was measured three times each using a Corneometer® CM 825(C+K), and the average value was analyzed.

[0198] Skin moisture content was measured by selecting the flattest central part of the cheek free from lesions that interfere with measurement, such as protrusions or depressions, completely removing any remaining moisture in the sensor area, and then applying equal vertical force to the skin. At this time, a higher measurement value indicates a higher skin moisture content.

[0199] Experimental Method 3.3.2. Measurement of Transepidermal Water Loss in Study Participants

[0200] Transepidermal water loss (TEWL) in selected cheek areas was measured using Tewameter® HEX(C+K) at points before product intake, 6 weeks after intake, and 12 weeks after intake, and the values ​​in the stabilization range were analyzed. In this case, transepidermal water loss (TEWL) refers to the amount of water evaporating through the skin.

[0201] Transepidermal water loss (Transepidermal Water Loss) was measured under identical temperature and humidity conditions by placing the Tewameter® HEX vertically in contact with the center of the selected test site of the study subjects at every time point. Since the Tewameter® HEX is sensitive to even minute changes, measurements were taken in a space unaffected by external environmental factors such as wind, with minimal movement. In this context, a lower measurement value indicates an improvement in Transepidermal Water Loss.

[0202] Experimental Method 3.3.3. Measurement of Skin Keratinization in Study Subjects

[0203] Skin keratin from selected cheek areas was collected using D-Squame Standard Sampling Discs (Clinical and Derm LLC) before product intake, 6 weeks after intake, and 12 weeks after intake, and the amount of keratin was analyzed using Squame Scan 850A (Clinical and Derm LLC).

[0204] For skin keratin sampling, subjects without skin abnormalities on the face were selected, and D-Squame Standard Sampling Discs were placed on the cheek area using a pressure instrument (Cuderm) under a constant pressure (225 g / cm²). 2 After attaching it with ), it was removed.

[0205] Skin exfoliation analysis was performed using D-Squame Standard Sampling Discs with Squame Scan 850A. First, calibration was performed using a Calibration Kit, and after measuring a Blank Squame Scan 850A, the subject's Squame Scan 850A was measured to analyze the amount of exfoliation (%). In this case, a lower measurement value indicates less skin exfoliation.

[0206] Experimental Method 3.3.4. Measurement of Skin Roughness in Study Subjects

[0207] PRIMOS skin roughness of selected cheek areas at time points before product intake, 6 weeks after intake, and 12 weeks after intake CR After taking images using SF (Canfield), the roughness parameter values ​​were analyzed using an analysis program.

[0208] Skin roughness measurements were conducted on selected subjects free from lesions, such as protrusions or depressions in the test area, that would interfere with the analysis. After wearing a hairband to style the hair, the chin and forehead were secured to a jig to minimize facial movement, and the facial angle was adjusted to ensure the roughness in the test area was clearly visible during measurement. After product consumption, the same area was photographed under the same conditions, based on the image taken before consumption.

[0209] The analysis was performed using the corresponding device-designated analysis program. For skin roughness, the cheek area was designated as the analysis region, and the same area at each time point was matched to ensure a more detailed and accurate match, after which the same area at each time point was analyzed. In this case, a decrease in the values ​​of the arithmetic mean roughness Ra (Arithmetic average), maximum height Rmax (Maximum peak to valley roughness height), and 10-point average roughness Rz (Average maximum height of the profile) indicates that skin roughness is improved (Table 8).

[0210] Parameter Definitions RaArithmetic average (Arithmetic mean roughness, μm) RmaxMaximum peak to valley roughness height (Maximum height, μm) RzAverage maximum height of the profile (10-point average roughness, μm)

[0211] Experimental Methods 3.4. Evaluation of Diagnostic Laboratory Medicine and Adverse Reactions in Study Participants

[0212] Experimental Method 3.4.1. Evaluation of Diagnostic Laboratory Medicine

[0213] At the 12-week mark following screening and product intake, blood and urine samples were collected after the study participants were instructed to fast for at least 12 hours. Using the blood and urine samples, hematological tests (WBC, RBC, hemoglobin (HgB), hematocrit (Hct), platelet count), blood biochemical tests (GGT, ALT / AST, albumin, total protein, total bilirubin, BUN, creatinine, glucose, e-FGR, uric acid), urinalysis (specific gravity, pH, WBC, nitrite, protein, glucose, ketone, bilirubin, occult blood, urobilinogen), and a pregnancy test (urine HGC) were performed. These tests were commissioned to a contract analysis agency (Samkwang Medical Foundation). Additionally, the pregnancy test was conducted using urine samples only for women of childbearing age, excluding study participants who had undergone sterilization or confirmed menopause.

[0214] Experimental Method 3.4.2. Evaluation of Adverse Reactions

[0215] Adverse events were evaluated by having the researcher observe the subject's test site at each evaluation point, confirm the condition of the test site through question-and-answer sessions with the subject, and record and evaluate it using diagnostic terms. In the event of an adverse event, the subject was instructed to record the symptoms, date of occurrence, and whether it was a serious adverse event on the "My Adverse Events" page of the case report form, and the principal investigator determined the relevance to the test product.

[0216] Experimental Method 3.4.3. Evaluation of Vital Signs

[0217] Vital signs, including systolic / diastolic blood pressure and pulse rate, were measured using an automated electronic blood pressure monitor (OMRON, Vietnam) at screening, before product intake, 6 weeks after intake, and 12 weeks after intake. Vital signs were measured with the subjects' arms positioned at heart level while maintaining a seated position after resting for 5 minutes without sudden changes in posture. Systolic / diastolic blood pressure was expressed in mmHg, and pulse rate in beats per minute.

[0218] Experimental Method 3.5. Statistical Processing

[0219] Experimental results were presented as mean ± standard deviation (Mean ± SD). For categorical data, frequencies and proportions were indicated, and a normality test was performed on all data prior to analysis. In cases of missing values, such as dropouts, the LOCF (last observation carried forward) method was applied, treating the value observed immediately before the missing value as the final value.

[0220] Experimental Method 3.5.1. Evaluation of Validity

[0221] The analysis was conducted by establishing the PPS (per protocol set) group, which consists of subjects who completed the test according to the study plan and complied with all procedures in the study protocol, and the Fas (full analysis set) group, which consists of all subjects registered in the study who consumed the product at least once.

[0222] Within-group comparison tests regarding changes after product consumption relative to baseline were analyzed using the paired t-test for parametric cases and the Wilcoxon signed-rank test for non-parametric cases. Homogeneity tests regarding differences in baseline distributions between groups were analyzed using the independent two-sample t-test for parametric cases and the Mann-Whitney U test for non-parametric cases. After confirming homogeneity, between-group comparison tests regarding changes after product consumption relative to baseline were analyzed using the independent two-sample t-test for parametric cases and the Mann-Whitney U test for non-parametric cases. In this case, a P-value of less than 0.05 was determined to indicate a statistically significant difference.

[0223] Experimental Method 3.5.2. Safety Assessment

[0224] For the analysis group, the Safety Set was defined as the group of all study participants who consumed the product for human clinical trials at least once after randomization using the LOCF method. Categorical variables were expressed as frequencies and proportions. Homogeneity tests regarding differences in baseline distributions between groups were performed using the efficacy evaluation statistic for continuous variables and the Chi-square test for categorical variables; if more than 20% of the cells had an expected frequency less than 5, Fisher's exact test was used.

[0225] For within-group and between-group comparison tests regarding changes after product intake relative to baseline, the validity evaluation statistical method was the same for continuous variables; for repeated measures, RM-ANOVA was used for parametric variables and a Linear mixed model for non-parametric variables. Categorical variables were analyzed using the Chi-square test, and Fisher's exact test was used when cells with expected frequencies less than 5 accounted for 20% or more of the total.

[0226] Experimental Result 4. Confirmation of improvement in skin health damaged by UV rays

[0227] Experimental Result 4.1. Confirmation of Improvement in Eye Wrinkles

[0228] Experimental Results 4.1.1. Average Wrinkle Length

[0229] As shown in Figures 14a, 15, and Table 9, the mean depth biggest wrinkle (Av.dep.) parameter showed a decreasing trend in the test group at 6 weeks after consumption compared to before product consumption, but there was no significant difference, and it decreased significantly at 12 weeks after consumption. In the control group, it increased significantly at 6 weeks and 12 weeks after product consumption.

[0230] As a result of the inter-group comparative analysis, the Av.dep. parameter for crow's feet significantly decreased in the test group compared to the control group at 6 weeks and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0231] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Av.dep.(μm) baseline 49.31±9.95 47.31±10.9 40.1 186 weeks change from baseline p-value 48.61±10.53 -0.69±4.5 50.291 2) 49.37±11.382.06±3.62<0.0010.00112 weekschange from baselinep-value47.51±9.54-1.80±4.300.005 2) 49.76±10.962.45±3.87<0.001 3) <0.001

[0232] 1) Compared between groups; p-value by Mann-Whitney U test

[0233] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0234] 3) Compared between groups; p-value by Independent two-sample t-test

[0235] Experimental Results 4.1.2. Average depth of the largest wrinkle

[0236] As shown in Figures 14b, 15 and Table 10, the mean depth of the biggest wrinkle (Mean big,dep.) parameter significantly decreased in the test group at 6 weeks and 12 weeks after consumption compared to before product consumption. In the control group, it significantly increased at 6 weeks and 12 weeks after product consumption.

[0237] As a result of the inter-group comparative analysis, the Mean big,dep. parameter for crow's feet wrinkles was significantly reduced in the test group compared to the control group at 6 weeks and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0238] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) )Test group (n=49)Control group (n=49)Mean big.dep.(μm)baseline 67.16±24.486 5.33±18.75 0.9586 weeks change from baseline p-value 2) 63.08±21.97-4.08±8.230.00168.04±21.062.71±8.100.0230.00112 weekschange from baselinep-value 2) 60.53±22.40 -6.63±11.81<0.00169.43±21.154.10±7.840.001<0.001

[0239] 1) Compared between groups; p-value by Mann-Whitney U test

[0240] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0241] Experimental Results 4.1.3. Maximum Length of the Largest Wrinkle

[0242] As shown in Figures 14c, 15, and Table 11, the Max.dep. parameter in the test group decreased significantly at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, there was a tendency to increase at 6 weeks after product intake, but no significant difference, and it increased significantly at 12 weeks after product intake.

[0243] As a result of the inter-group comparative analysis, the Max.dep. parameter for crow's feet wrinkles was significantly reduced in the test group compared to the control group at 6 and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0244] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Max.dep.(μm) baseline 20 4.61±9 2.16 215.18±8 8.25 0.4 39 6 weeks change from baseline p-value 2) 188.57±80.87-16.04±38.560.005219.88±83.204.69±39.640.4110.00412 weekschange from baselinep-value 2) 178.45±81.70 -26.16±45.11<0.001225.94±93.6010.76±31.940.023<0.001

[0245] 1)Compared between groups; p-value by Mann-Whitney U test

[0246] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0247] Based on the above results, it is believed that the intake of Gynostemma pentaphyllum extract can improve skin wrinkles caused by ultraviolet radiation (Fig. 15).

[0248] Experimental Result 4.2. Confirmation of Improvement in Skin Elasticity in Study Participants

[0249] Experimental Results 4.2.1. Confirmation of Skin Elasticity Improvement (R2)

[0250] As shown in Figure 16a and Table 12, the R2 (gross elasticity) parameter for skin elasticity significantly increased in the test group at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, it significantly decreased at 6 weeks and 12 weeks after product intake.

[0251] As a result of the inter-group comparative analysis, the R2 parameter for skin elasticity significantly increased in the test group compared to the control group at 6 weeks and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0252] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) R2 (%) baseline 6 7.84±4.92 6 8.31±6.53 0.6476 weeks change from baseline p-value 2) 68.38±4.87 0.53±0.67<0.001 2) 67.41±6.76-0.89±0.83<0.001 3)<0.00112 weekschange from baselinep-value 2) 69.39±5.091.55±0.95<0.001 2) 66.75±6.71 -1.56±0.97<0.001 3) <0.001

[0253] 1) Compared between groups; p-value by Mann-Whitney U test

[0254] 2) Compared within groups; p-value by paired t-test

[0255] 3) Compared within groups; p-value by Wilcoxon signed-rank test

[0256] Experimental Results 4.2.2. Confirmation of Skin Elasticity Improvement (R5)

[0257] As shown in Figure 16b and Table 13, the R5 (elastic ratio after the first traction) parameter for skin elasticity significantly increased in the test group at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, it significantly decreased at 6 weeks and 12 weeks after product intake.

[0258] As a result of the inter-group comparative analysis, the R5 parameter for skin elasticity significantly increased in the test group compared to the control group at 6 weeks and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0259] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1))Experiment group (n=49)Control group (n=49)R5(%) baseline 66.68±10.22 71.31±13.26 0.0756 weeks change from baseline p-value 70.00±9.91 3.32±8.37 0.008 2) 65.78±11.36-5.52±7.01<0.001 3) <0.00112 weekschange from baselinep-value74.70±10.318.02±7.16<0.001 2) 62.21±9.94-9.10±7.61<0.001 3) <0.001

[0260] 1) Compared between groups; p-value by Mann-Whitney U test

[0261] 2) Compared within groups; p-value by paired t-test

[0262] 3) Compared within groups; p-value by Wilcoxon signed-rank test

[0263] Experimental Results 4.2.3. Confirmation of Skin Elasticity Improvement (R7)

[0264] As shown in Figure 16c and Table 14, the R7 (biologic elasticity) parameter for skin elasticity significantly increased in the test group at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, it significantly decreased at 6 weeks and 12 weeks after product intake.

[0265] As a result of the inter-group comparative analysis, the R7 parameter for skin elasticity significantly increased in the test group compared to the control group at 6 and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0266] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) R7 (%) baseline 45.50±7.36 48.14±8.6 50.12 86 weeks change from baseline p-value 47.79±7.25 2.29±4.6 20.00 1 2) 45.19±7.51 -2.95±4.58<0.001 3) <0.00112 weekschange from baselinep-value49.06±6.783.57±4.42<0.001 2) 42.93±7.05-5.21±4.54<0.001 3) <0.001

[0267] 1) Compared between groups; p-value by Mann-Whitney U test

[0268] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0269] 3) Compared within groups; p-value by paired t-test

[0270] Based on the above results, it is believed that the intake of Gynostemma pentaphyllum extract can improve skin elasticity reduced by ultraviolet rays.

[0271] Experimental Result 4.3. Confirmation of moisturization of UV-damaged skin in study subjects

[0272] Experimental Results 4.3.1. Confirmation of Changes in Skin Moisture Content of Study Subjects

[0273] As shown in Figure 17 and Table 15, the parameters for skin moisture content significantly increased in the test group at 6 weeks after product intake and 12 weeks after product use compared to before product intake. In the control group, there was an increasing trend at 6 weeks after product use, but no significant difference, and a decreasing trend at 12 weeks after product use, but no significant difference.

[0274] When comparing between groups, the skin moisture parameter significantly increased in the test group compared to the control group at 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0275] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Skin moisture content (AU) baseline 36.91±4.28 37.02±3.7 30.9296 weeks change from baseline p-value 38.56±4.17 1.64±0.88<0.001 2) 37.35±4.390.33±2.400.337 3) <0.00112 weekschange from baselinep-value39.44±3.982.53±0.95<0.001 2) 36.95±3.65-0.08±1.210.663 3) <0.001

[0276] 1) Compared between groups; p-value by Independent two-sample t-test

[0277] 2) Compared within groups; p-value by paired t-test

[0278] 3) Compared between groups; p-value by Mann-Whitney U test

[0279] Based on the above results, it is believed that the consumption of Gynostemma pentaphyllum extract can improve skin hydration.

[0280] Experimental Results 4.3.2. Confirmation of Changes in Transepidermal Water Loss of Study Participants

[0281] As shown in Figure 18 and Table 16, the parameters for transepidermal water loss significantly decreased in the test group at 6 weeks and 12 weeks after product intake compared to before product intake, while the control group showed an increasing trend at 6 weeks and 12 weeks after product intake, but there was no significant difference.

[0282] When comparing between groups, the transepidermal water loss parameter was significantly reduced in the test group compared to the control group at 6 and 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0283] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Transepidermal water loss (g / h / m²) 2 )baseline18.36±4.4418.88±5.580.9296 weekschange from baselinep-value16.99±3.81-1.37±1.45<0.001 2) 19.34±4.840.46±2.100.136 3) <0.00112 weekschange from baselinep-value15.76±3.26-2.60±2.880.001 2) 19.59±4.650.71±3.020.107 3) <0.001

[0284] 1) Compared between groups; p-value by Mann-Whitney U test

[0285] 2)Compared within groups; p-value by paired t-test

[0286] 3) Compared within groups; p-value by Wilcoxon signed-rank test

[0287] Based on the above results, it is believed that consuming products containing Gynostemma pentaphyllum extract or Damulin B can inhibit transepidermal water loss.

[0288] Experimental Results 4.3.3. Confirmation of Changes in Skin Keratin Volume of Study Subjects

[0289] As shown in Figure 19 and Table 17, the parameters for skin keratin volume significantly decreased in the test group at 6 and 12 weeks after product intake compared to before product intake. In the control group, there was an increasing trend at 6 weeks after product intake, but no significant difference, and a decreasing trend at 12 weeks after intake, but no significant difference.

[0290] When comparing between groups, the skin keratinization parameter was significantly reduced in the test group compared to the control group at 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0291] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Skin keratinization (%) baseline 10.61±2.97 10.82±2.26 0.69 46 weeks change from baseline p-value 2) 10.06±2.84-0.55±0.85<0.00111.02±2.700.20±0.970.159<0.00112 weekschange from baselinep-value 2) 9.83±2.99 -0.78±0.74<0.001 10.76±2.35 -0.06±0.95 0.661<0.001

[0292] 1) Compared between groups; p-value by independent two-sample t-test

[0293] 2) Compared within groups; p-value by paired t-test

[0294] Based on the above results, it is believed that the consumption of Gynostemma pentaphyllum extract can improve skin hydration.

[0295] Experimental Result 4.4. Confirmation of Skin Roughness in Study Subjects

[0296] Experimental Results 4.4.1. Verification of Skin Roughness (Ra)

[0297] As shown in Figures 20a, 21, and Table 18, the arithmetic average (Ra) parameter for skin roughness was significantly reduced in the test group at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, there was a decreasing trend at 6 weeks after product intake, but no significant difference, and it was significantly increased at 12 weeks after product intake.

[0298] As a result of the inter-group comparative analysis, the Ra parameter for skin roughness was significantly reduced in the test group compared to the control group 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0299] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Ra(μm) baseline 18.66±4.11 17.33±4.25 0.07 66 weeks change from baseline p-value 2)17.88±4.47-0.78±1.810.00416.99±4.08-0.35±1.390.0870.13912 weekschange from baselinep-value 2) 18.08±4.51 -0.58±1.96 0.044 17.98±4.13 0.65±1.75 0.013<0.001

[0300] 1) Compared between groups; p-value by Mann-Whitney U test

[0301] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0302] Experimental Results 4.4.2. Skin Roughness Verification (Rmax)

[0303] As shown in Figures 20b, 21, and Table 19, the maximum peak to valley roughness height (Rmax) parameter for skin roughness significantly decreased in the test group at 6 and 12 weeks after product intake compared to before product intake. In the control group, a decreasing trend was observed at 6 weeks after product intake, but there was no significant difference, and an increasing trend was observed at 12 weeks after intake, but there was no significant difference.

[0304] As a result of the inter-group comparative analysis, the Rmax parameter was significantly reduced in the test group compared to the control group 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0305] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1)Test group (n=49) Control group (n=49) Rmax (μm) baseline 1 3 1.75±26.5 71 2 1.95±29.1 6 0.0 46 6 weeks change from baseline p-value 1 2 6.48±28.6 3 -5.27±10.4 0 0.0 1 2) 119.67±28.24-2.28±9.670.105 3) 0.21612 weekschange from baselinep-value126.46±28.28-5.29±12.230.004 2) 124.73±26.932.78±12.410.124 3) <0.001

[0306] 1) Compared between groups; p-value by Mann-Whitney U test

[0307] 2) Compared within groups; p-value by paired t-test

[0308] 3) Compared within groups; p-value by Wilcoxon signed-rank test

[0309] Experimental Results 4.4.3. Skin Roughness Verification (Rz)

[0310] As shown in Fig. 20c, Fig. 21 and Table 20, the average maximum height of the profile (Rz) parameter for skin roughness on a 10-point scale was significantly reduced in the test group at 6 weeks and 12 weeks after product intake compared to before product intake. In the control group, it was significantly reduced at 6 weeks after product intake and significantly increased at 12 weeks after intake.

[0311] As a result of the inter-group comparative analysis, the Rz parameter was significantly reduced in the test group compared to the control group 12 weeks after product intake (significance probability *p<0.05 vs. before product intake, †p<0.05 vs. control group).

[0312] Parameter (Unit) Evaluation Point Average Value (values) Significance Probability (p-value 1) Test group (n=49) Control group (n=49) Rz(μm) baseline 98.67±19.97 92.28±20.4 50.10 26 weeks change from baseline p-value 2) 94.49±21.68-4.17±8.570.00190.30±19.36-1.98±6.840.0480.14912 weekschange from baselinep-value 2) 95.20±22.03 -3.46±9.66 0.016 95.29±19.74 3.01±9.28 0.028<0.001

[0313] 1) Compared between groups; p-value by Mann-Whitney U test

[0314] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0315] Based on the above results, it is believed that the intake of Gynostemma pentaphyllum extract can improve skin health (Fig. 21).

[0316] Experimental Result 5. Safety Test

[0317] Experimental Results 5.1. Analysis Results of Adverse Reaction Evaluation

[0318] As a result of the analysis, as shown in Table 21, no adverse reactions were observed in either the test group or the control group during the human clinical trial.

[0319] Parameters (Units) Test Group (n=50) Control Group (n=50) Significance Probability (p-value 1)) Presence / Accident of Adverse Events (AE) 0 / 50, 0.00% / 100.00% 0 / 50, 0.00% / 100.00% 1.000 Presence / Accident of Serious Adverse Events (SAE) 0 / 50, 0.00% / 100.00% 0 / 50, 0.00% / 100.00% 1.000 Presence / Accident of Dropout Due to Adverse Events 0 / 50, 0.00% / 100.00% 0 / 50, 0.00% / 100.00% 1.000

[0320] 1) p-value by Fisher's exact test

[0321] Experimental Results 5.2. Hematological Test Analysis Results

[0322] As shown in Table 22, the analysis results showed that for all parameters, both the test group and the control group were within the normal range.

[0323] Parameter [Normal Range] (Unit) Time Point Test Group (n=50) Control Group (n=50) Significance Probability (p-value 1) )RBC[3.70-4.70](10 6 / μL)Baseline 4.27±0.35 4.36±0.41 0.223 1) 12 weeksp-value4.33±0.430.232 2) 4.42±0.330.158 2) 0.229 1) WBC[4-10](10 3 / μL)Baseline 5.74±1.39 6.21±2.02 0.288 3) 12 weeksp-value5.97±1.330.309 4) 6.19±1.850.877 4) 0.677 3) Hemoglobin[11-15](g / dL)Baseline12.81±0.8912.98±0.930.341 1) 12 weeksp-value13.09±1.000.018 2) 13.26±0.970.005 2) 0.395 1)Hematocrit[33-45](%)Baseline39.43±2.6140.16±2.800.185 1) 12 weeksp-value39.95±3.050.180 2) 40.65±2.690.172 2) 0.227 1) Platelet[150-370](10 3 / μL)Baseline 267.94±76.76279.48±58.830.401 1) 12 weeksp-value282.10±72.910.006 2) 299.44±66.420.003 2) 0.217 1)

[0324] 1) Compared between groups; p-value by independent two-sample t-test

[0325] 2) Compared within groups; p-value by paired t-test

[0326] 3) Compared between groups; p-value by Mann-Whitney U test

[0327] 4) Compared within groups; p-value by Wilcoxon signed-rank test

[0328] Experimental Results 5.3. Results of Blood Biochemical Test Analysis

[0329] As shown in Table 23, the analysis results showed that for all parameters, both the test group and the control group were within the normal range.

[0330] Parameter [Normal Range] (Unit) Time of Evaluation Test Group (n=50) Control Group (n=50) Significance Probability (p-value) AST [0-32] (U / L) Baseline 19.60±4.13 20.82±5.76 0.325 1)12 weeksp-value19.95±4.530.604 2) 20.40±5.320.814 2) 0.937 1) ALT[0-33](U / L)Baseline14.74±4.7517.28±10.950.376 1) 12 weeksp-value14.94±6.030.750 2) 16.06±8.690.673 2) 0.612 1) ALP[35-104](U / L)Baseline66.22±20.2664.52±19.870.673 1) 12 weeksp-value69.66±19.450.015 4) 69.12±21.400.001 4) 0.895 1) Albumin[3.5-5.2](g / dL)Baseline4.62±0.194.60±0.200.686 3) 12 weeksp-value4.68±0.240.051 4) 4.71±0.24<0.001 4) 0.543 3) Total Protein[6.6-8.7](g / dL)Baseline7.11±0.317.13±0.340.720 3) 12 weeksp-value7.24±0.470.015 4) 7.28±0.390.001 4) 0.623 3) GGT[6-42](U / L)Baseline17.52±11.5517.80±10.890.745 1) 12 weeksp-value17.62±10.040.751 2) 16.66±8.670.774 2) 0.833 1) Total Bilirubin[0-1.2](mg / dL)Baseline0.51±0.250.47±0.230.350 1) 12 weeksp-value0.52±0.230.720 2) 0.49±0.230.402 2)0.652 1) BUN[6-20](mg / dL)Baseline12.23±3.6811.57±2.910.488 1) 12 weeksp-value11.94±2.890.513 4) 11.37±3.090.725 2) 0.370 1) Creatinine[0.5-0.9](mg / dL)Baseline0.63±0.080.63±0.100.719 3) 12 weeksp-value0.66±0.110.108 4) 0.65±0.120.039 4) 0.789 3) Glucose[79-99](mg / dL)Baseline87.10±8.6486.32±7.270.953 1) 12 weeksp-value89.16±7.420.060 2) 88.66±7.450.138 4) 0.863 1) Uric acid[2.4-5.7]Baseline4.11±0.814.07±0.860.784 3) 12 weeksp-value3.96±0.740.076 4) 4.01±0.920.520 4) 0.774 3) GFR[0-60 mL / min / 1.73]Baseline101.70±18.06105.12±22.580.461 1) 12 weeksp-value98.88±20.610.341 2) 101.56±24.350.327 2) 0.788 1)

[0331] 1) Compared between groups; p-value by Mann-Whitney U test

[0332] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0333] 3) Compared between groups; p-value by independent two-sample t-test

[0334] 4) Compared within groups; p-value by paired t-test

[0335] Experimental Results 5.4. Urine Test Analysis Results

[0336] As shown in Table 24, the analysis results showed that for all parameters, both the test group and the control group were within the normal range.

[0337] Parameter [Normal Range] (Unit) Time Point Test Group (n=50) Control Group (n=50) Significance Probability (p-value) 1 Specific gravity[1.003-1.03]Baseline1.63±4.331.02±0.010.484 1) 12 weeksp-value1.02±0.010.769 2) 1.02±0.010.710 3) 0.534 1) 1 pH[4.5-7.5]Baseline6.09±0.925.88±1.090.061 1) 12 weeksp-value5.81±0.830.1006.16±1.010.0590.085 1) 2 WBC[Negative]BaselinePositive / Negative11 / 3922.00% / 78.00%9 / 4118.00% / 82.00%0.803 4) 12 weeksPositive / Negativep-value 5) 5 / 4510.00% / 90.00%0.0349 / 4118.00% / 82.00%1.0000.388 4) 2 Nitrite[Negative]BaselinePositive / Negative1 / 492.00% / 98.00%0 / 500.00% / 100.00%1.0004) 12 weeksPositive / Negativep-value 5) 0 / 500.00% / 100.00%1.0002 / 484.00% / 96.00%1.0000.495 4) 2 Protein[Negative]BaselinePositive / Negative1 / 492.00% / 98.00%1 / 492.00% / 98.00%1.000 4) 12 weeksPositive / Negativep-value 5) 0 / 500.00% / 100.00%1.0001 / 492.00% / 98.00%1.0001.000 4) 2 Glucose[Negative]BaselinePositive / Negative0 / 500.00% / 100.00%1 / 492.00% / 98.00%1.000 4) 12 weeksPositive / Negativep-value 5) 0 / 500.00% / 100.00%1.0001 / 492.00% / 98.00%1.0001.000 4) 2 Ketone[Negative]BaselinePositive / Negative2 / 484.00% / 96.00%4 / 468.00% / 92.00%0.678 4) 12 weeksPositive / Negativep-value 5) 2 / 484.00% / 96.00%1.0000 / 500.00% / 100.00%1.0000.495 4) 2 Urobilinogen[Normal]BaselinePositive / Negative0 / 500.00% / 100.00%0 / 500.00% / 100.00%1.000 4) 12 weeksPositive / Negativep-value 5) 0 / 500.00% / 100.00%1.0000 / 500.00% / 100.00%1.0001.000 4) 2Bilirubin[Negative]BaselinePositive / Negative0 / 500.00% / 100.00%0 / 500.00% / 100.00%1.000 4) 12 weeksPositive / Negativep-value 5) 0 / 500.00% / 100.00%1.0000 / 500.00% / 100.00%1.0001.000 4) 2 Occult blood[Negative]BaselinePositive / Negative5 / 4510.00% / 90.00%7 / 4314.00% / 86.00%0.760 4) 12 weeksPositive / Negativep-value 5) 4 / 468.00% / 92.00%0.5644 / 468.00% / 92.00%0.3171.000 4)

[0338] 1) Compared between groups; p-value by Mann-Whitney U test

[0339] 2) Compared within groups; p-value by Wilcoxon signed-rank test

[0340] 3) Compared within groups; p-value by paired t-test

[0341] 4) Compared between groups; p-value by Fisher's exact test

[0342] 5) Compared within groups; p-value by McNemar's Test

[0343] 실험결과 5.5. 임신반응검사 분석 결과

[0344] Out of a total of 100 study participants, 63 were subject to the pregnancy test, consisting of 30 in the test group and 33 in the control group. As shown in Table 25, the analysis results showed no significant difference between the test group and the control group (p>0.05).

[0345] hCG(n, %) Test group (n=30) Control group (n=33) Significance probability (p-value 1) )Baseline(Positive / Negative)0 / 300.00% / 100.00%0 / 330.00% / 100.00%1.00012 weeks(Positive / Negative)0 / 300.00% / 100.00%0 / 330.00% / 100.00%

[0346] 1) p-value by Fisher's exact test

[0347] Experimental Results 5.6. Vital Signs Analysis Results

[0348] As a result of the analysis, as shown in Table 26, there were no significant differences between the test group and the control group in all parameters regarding vital signs (p>0.05).

[0349] Parameter (Unit) Evaluation Time Point Test Group (n=50) Control Group (n=50) Significance Probability (p-value 1) Systolic Blood Pressure (mmHg) Screening 1 18.32±12.5 1 17.02±11.4 9 0.6 59 Diastolic Blood Pressure (mmHg) 78.52±6.03 79.62±7.8 30.2 9 Pulse (beats) 72.58±8.7 73.92±11.3 8 0.6 0 7 Systolic Blood Pressure (mmHg) Baseline 1 16.76±11.4 1 15.62±13.8 30.8 7 6 weeks 1 13.28±12.5 9 1 16.00±12.1 00.3 6 6 12 weeks 1 16.26±11.8 6 1 16.78±13.0 2 0.5 9 p-value 0.8 77 2) 0.401 3) 0.117 4)Diastolic blood pressure (mmHg) Baseline 78.44±6.25 77.26±8.0 20.76 46 weeks 76.70±7.8 877.42±9.4 50.28 512 weeks 76.94±7.2 78.92±7.3 10.156 p-value 0.258 2) 0.315 2) Pulse (beats) Baseline 70.76±8.76 71.92±11.28 0.70 46 weeks 74.10±8.88 71.84±9.40 0.19 212 weeks 70.96±9.03 72.72±10.77 0.583 p-value 0.915 2) 0.915 2)

[0350] 1) Compared between groups; p-value by Independent two-sample t-test

[0351] 2) Compared within groups; p-value by paired t-test (12 weeks vs baseline)

[0352] 3) Linear mixed model

[0353] 4) Compared between groups; p-value by Mann-Whitney U test

[0354] Experimental Results 6. Conclusion and Discussion

[0355] Based on the above results, it is believed that the food composition containing the Gynostemma pentaphyllum extract of the present invention helps maintain skin health and moisturizes the skin against UV-induced skin damage by improving eye wrinkles, skin elasticity, skin moisture content, transepidermal water loss, skin keratin content, and skin roughness in human skin. Furthermore, based on diagnostic medical tests and analyses of skin and systemic adverse reactions in all participating subjects, it is considered that there are no issues regarding consumption in terms of safety.

Claims

A food composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I: <Chemical Formula I> . In paragraph 1, A food composition for improving skin health, wherein the improvement of skin health is achieved by at least one selected from skin moisturization, prevention or improvement of skin damage, and improvement of skin roughness. In paragraph 2, A food composition for improving skin health, wherein the above-mentioned skin damage is caused by ultraviolet rays. In paragraph 2, A food composition for improving skin health, wherein the above-mentioned skin damage is any one selected from the group consisting of aging, wrinkles, burns, erythema, sunburn, pigmentation, skin cancer, reduced immune response, and combinations thereof. In paragraph 1, A food composition for improving skin health, wherein the above-mentioned Dolwort extract is any one extract selected from the group consisting of flowers, leaves, stems, branches, fruits, fruit peels, roots, tissue cultures derived therefrom, and combinations thereof. In paragraph 5, A food composition for improving skin health, wherein the above-mentioned Gynostemma extract is a leaf extract. In paragraph 1, A food composition for improving skin health, wherein the above-mentioned Gynostemma extract is extracted by the following method: i) A step of mixing Gynostemma pentaphyllum and purified water; ii) a step of heating the above mixture to high pressure and high temperature; and iii) A step of collecting the extract by cooling the steam generated in ii) above. In Paragraph 7, A food composition for improving skin health, comprising the above-mentioned Gynostemma extract further comprising the following method: iv) A step of sterilizing the extract of iii) above. In Paragraph 7, A food composition for improving skin health, wherein the mixing ratio of the *Gynostemma pentaphyllum* and purified water in i) above is 1:10 to 1:1000 by weight. In Paragraph 7, A food composition for improving skin health, wherein the high temperature of ii) above is 80℃ to 180℃. In Paragraph 7, A food composition for improving skin health, wherein the high pressure of ii) above is 1.1 to 2.5 atmospheres. In Paragraph 7, A food composition for improving skin health, wherein the above step ii) is performed for 10 to 20 hours. In Paragraph 7, A food composition for improving skin health, wherein the above-mentioned Gynostemma extract contains damulin B. A feed composition for improving skin health comprising, as an active ingredient, an extract of *Gynostemma pentaphyllum* or damulin B represented by the following chemical formula I: <Chemical Formula I> . Use for improving skin health comprising Gynostemma pentaphyllum extract or damulin B represented by the following chemical formula I as an active ingredient: <Chemical Formula I> .

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