KR20130035325A - External composition for skin containing saponin derived from the root of camellia sinensis - Google Patents

Abstract

PURPOSE: An external use skin composition containing saponin derived from the root of Camellia sinensis is provided to improve antioxidation, anti-wrinkling, skin whitening, and moisturizing and to treat acne, skin troubles, and atopic dermatitis. CONSTITUTION: An external use skin composition contains 0.1-50 wt% of saponin derived from the root of Camellia sinensis. The saponin is triterpenoid saponin. The composition is used for antiaging and skin elasticity enhancement.

Description

External composition for skin containing saponin derived from the root of Camellia sinensis}

The present invention relates to a composition containing saponin derived from tea tree root, more specifically, by containing saponin derived from tea tree root, anti-aging effect of skin, reduction of skin wrinkles, enhancement of skin elasticity, improvement of skin moisturizing effect, anti-inflammatory effect, acne And it relates to a composition that can provide an overall skin condition improvement effect, such as skin trouble improvement effect, whitening effect and atopic symptoms improvement effect.

Human skin is changed by a number of internal and external factors as it ages. That is, internally, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decreases, thereby reducing the biosynthesis of immune proteins and constituent proteins necessary for living organisms. Due to the increase in the amount of ultraviolet rays that reach the surface of the sun's rays and intensifying environmental pollution, free radicals and free radicals increase, thereby reducing the thickness of the skin, increasing wrinkles, reducing elasticity, The color of the skin becomes dull, skin problems frequently occur, and there are various changes such as blemishes, freckles, and black mushrooms.

As aging progresses, symptoms of alteration or decrease in the content and arrangement of collagen, elastin, hyaluronic acid and glycoproteins, which are constituents of the skin, appear and are subject to oxidative stress caused by free radicals and active harmful oxygen. In addition, cyclooxygenase-2 (Cox-2, cyclooxygenase), an enzyme that produces proinflammatory cytokine, which is known to cause inflammation in most cells constituting the skin due to aging or ultraviolet rays. Biosynthesis of the enzyme increases the biosynthesis of matrix metalloproteinase (MMP), an enzyme that breaks down skin tissues by these inflammatory factors, and the production of nitric oxide (NO) by iNOS (inducible nitric oxide synthase). It is known to increase. In other words, the biosynthesis of the substrate material is reduced by the reduction of cellular activity and micro-inflammation due to naturally occurring endogenous aging, and by external factors such as the increase of stress caused by various harmful environments and the increase of active coral species caused by sunlight. As a result, decomposition and degeneration are accelerated, and the skin matrix is destroyed and thinned, and various symptoms of skin aging appear. Therefore, many studies have been conducted on active ingredients that can prevent and improve these aging phenomena.

Many factors are involved in determining the skin color of humans, including the activity of melanocytes that make melanin, the distribution of blood vessels, the thickness of the skin and the presence or absence of pigments in the body, such as carotenoids and bilirubin. It is important.

Especially, the most important factor is the black pigment called melanin which is produced by the action of various enzymes such as tyrosinase in melanocytes in the human body. The formation of melanin pigment affects genetic factors, hormonal secretion, physiological factors such as stress, and environmental factors such as ultraviolet irradiation.

The melanin pigment produced in melanocytes of body skin is a phenolic polymer substance having a complex form of black pigment and protein. It protects the subcutaneous skin organs by blocking ultraviolet rays irradiated from the sun, and at the same time, free radicals And protects proteins and genes in the skin.

As such, melanin produced by stress stimulation inside and outside the skin is a stable substance that does not disappear until it is released to the outside through skin keratinization even if the stress disappears. However, if the melanin is produced more than necessary to cause hyperpigmentation, such as blemishes, freckles, and spots, cosmetically bad results.

Today, Asian women prefer white and clean skin like white jade, and this is an important standard of beauty, increasing the demand for treatment and cosmetic problems for hyperpigmentation.

In response to this demand, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione or derivatives thereof, or substances having tyrosinase inhibitory activity have been used in combination with cosmetics or pharmaceuticals, but they are insufficient. Its use has been limited due to its whitening effect, safety issues on the skin, formulation and stability problems when formulated in cosmetics, and the like.

The most important function of the outermost epidermis of the skin is the defense against various external stimuli (physical and chemical stimulating factors such as chemicals, air pollutants, dry environment, ultraviolet rays) and moisture in the body through the skin. It is a protective function to prevent excessive divergence of, and this protective function can maintain its function only when the stratum corneum composed of keratinocytes is normally formed. The outermost stratim corneum (horney layer) of the epidermis is formed from keratinocytes and consists of the differentiated keratinocytes and the surrounding lipid layer (J. Invest. Dermatol. 1983; 80: 44 ~ 44). 49). Keratinocytes are the characteristic cells that the morphology and function of the basal cells that proliferate continuously in the lower epidermis gradually go up to the surface of the skin and rise up to the surface of the skin. Exfoliated and new keratinocytes take over the function. This repetitive process of change is called 'epidermal differentiation' or 'keratinization'. Keratinocytes during keratinization form the stratum corneum by generating natural moisturizing factor (NMF) and intercellular fat (ceramide, cholesterol, fatty acids), making the stratum corneum firm and flexible, making it a skin barrier. It has a function as).

However, the stratum corneum can easily lose its function due to excessive washing, lifestyle factors such as bathing, environmental factors such as dry air and pollutants, and endogenous diseases such as atopic or aged skin. In recent years, more and more people are complaining of dry skin symptoms and all kinds of obstacles due to various factors. Therefore, many studies have been conducted to supply moisture from the outside or to prevent water loss from the body in order to maintain proper skin moisture, and various types of moisturizers having moisture retention capabilities have been developed and mainly cosmetics. It is widely used in the field.

However, as the risk factors for humans increase and the aged population rapidly increases in the living environment, the turnover rate of the stratum corneum is slowed, the lipid synthesis ability of the keratinocytes is degraded, or the epidermis is normal in the epidermis. Insufficient cell division, maturation, and differentiation, which leads to a decrease in the amount of moisturizing factors and lipids in the stratum corneum, leading to a condition in which the skin is unable to maintain normal stratum corneum function, ie, skin barrier function is degraded. Is in the trend.

Due to the abnormal division and differentiation of epidermal cells, the skin causes various skin diseases such as dry skin, atopy and psoriasis, and these diseases can alleviate the symptoms slightly with a conventional moisturizer having only water retention function. However, it is difficult to expect radical healing.

In general, the sebum in the skin including the scalp and face plays a role in maintaining the moisturizing of the skin and preventing the invasion of microorganisms, but excessive secretion of sebum promotes hair loss, worsens acne, promotes the enlargement of pores by acne, and seborrheic dermatitis. There are a number of diseases that can occur.

These secretions are caused by a number of causes, and most importantly, sebaceous gland cells are activated by the amount of dihydrotestosterone (DHT), one of the hormones involved in promoting sebum secretion in sebaceous gland activity. Is oversecreted. That is, in hair loss, testosterone (T) is converted into dihydrotestosterone, one of the male hormones, by the 5-alpha-reductase type 2 in the cell and at the same time in the cytoplasm. In combination with the receptor protein (receptor) enters the fluid causing hair loss, but in the skin or sebaceous gland testosterone is converted to dihydrotestosterone by 5-alpha-reductase type 1 to sebaceous gland cells By activating differentiation is promoted to secrete sebum in the sebaceous gland (sebum) to cause acne (J. Invest Dermatol 105: 209-214 Diane etc.).

Skin problems, such as acne and hair loss that occur outside of excessive sebum secretion, are further exacerbated by the skin's fine inflammatory response. If you look at the development of acne, excessive sebum accumulates in the hair follicles, which activates the acne bacteria and causes inflammation.

However, as a natural material safe for skin, there are not many raw materials that reduce sebum secretion through a fundamental cause and at the same time relieve inflammation.

Symptoms of atopic dermatitis are accompanied by skin redness, skin edema, blisters, ulcers and severe itching. If left untreated, the skin may remain exposed to substances that promote skin reactions, resulting in a prolonged (chronic) condition of contact dermatitis. Chronic contact dermatitis causes the skin to thicken, peel off, and dry, changing color and even hair loss.

Atopic dermatitis suffers from a significant number of people, with 0.5 to 1 percent of the population and 5 to 10 percent of children. The onset of symptoms usually occurs between two and six months of age, especially under 1 year of age, with 85% occurring within five years of age. It is commonly known as a disease when young, but 50% of patients disappear within two stones, but 25% go to adolescence, and the remaining 25% do not go away even as adults.

Accordingly, the demand for external skin composition such as human cleanser, hair shampoo, and hair conditioner that can be used safely without irritating to atopic skin is increasing. In addition, anti-pruritic or anti-inflammatory effect of improving the condition of atopic skin is increasing. Various researches on the products have been actively conducted.

Green tea uses the leaves of Camellia sinensis and is consumed as a medical and health drink. The medical effect of green tea is mainly due to the flavonoid component of green tea. Many triterpenoid saponins extracted from green tea leaves have been reported as bitter principle, and triterpenoid saponins can also be extracted from other parts of green tea, ie seeds, buds, roots and the like.

The pharmacological and biological properties of triterpene saponins from different plant species, including fungicides, antiviral, antimutagenic, spermicide or contraception (Hostettmann et al., 1995), have been studied (Cucurbitaceae). Antitumor activity of triterpene saponins from Chinese herbal medicines (Kong et al., 1993), husks, leaves or branches, stems and / or fruit-stems, roots, seeds and bark of Wongu & Guo Although a tumor growth inhibiting composition containing triterpenoid saponin from the known is known, the effect of improving the overall skin condition of the composition containing tea root-derived saponin as an active ingredient has not been reported yet.

Accordingly, the present inventors have found that the triterpenoid saponin derived from the root of the tea tree can provide anti-aging, skin wrinkle improvement, whitening, and moisturizing effects as well as improvement of acne and skin troubles, atopic symptoms, and improvement of skin complexion and slimming effect. The present invention has been completed and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for external application of the skin containing triterpenoid saponin derived from the roots of a tea tree, which can improve the overall condition of the skin.

In order to achieve the above object, the present invention provides an anti-aging skin external composition comprising tea tree root-derived saponin as an active ingredient.

In addition, the present invention provides a moisturizing skin external composition comprising tea tree root-derived saponin as an active ingredient.

The present invention also provides an external preparation composition for improving acne containing saponin derived from tea tree root as an active ingredient.

In addition, the present invention provides a skin external preparation composition for improving color and skin tone, which contains saponin derived from tea tree root as an active ingredient.

The present invention also provides a skin external preparation composition for reducing pores, containing saponin derived from the root of the tea tree as an active ingredient.

The present invention also provides a topical skin composition for improving atopic skin containing saponin derived from the root of the tea tree.

The present invention also provides a whitening skin external composition containing saponin derived from the root of the tea tree.

The composition of the present invention can provide anti-aging, skin wrinkle improvement effect, skin whitening effect, skin moisturizing and atopic skin improvement effect, acne and skin trouble improvement effect and skin color improvement effect by containing saponin derived from tea tree root.

The external preparation composition for skin according to the present invention contains triterpenoid saponin derived from the root of Camellia sinensis as an active ingredient.

The saponins used in the present invention are ten kinds of triterpenoid saponins derived from the root of tea tree, and in the present invention, these are referred to as 'tea root derived saponins R1 to R10', respectively. These tea tree root-derived saponins R1 to R10 have the structures of the following Chemical Formulas 1 to 10.

The composition of the present invention preferably contains the saponin in an amount of 0.1 to 50% by weight based on the total weight of the composition. This is because when the content of the active ingredient is less than 0.1% by weight, the efficacy and effect by the above ingredients are weak, and when the amount of the active ingredient exceeds 50% by weight, there is a problem of skin safety or formulation.

The composition of the present invention can be used as an anti-aging skin external composition, which is excellent in improving skin elasticity and improving wrinkles.

The composition of the present invention may be used as a moisturizing skin external preparation composition, which may enhance skin barrier function and induce differentiation of skin keratinocytes. Therefore, it can be usefully used as an external composition for skin to prevent or improve skin dryness, atopic dermatitis, contact dermatitis, or psoriasis caused by incomplete epidermal differentiation.

The composition of the present invention can be used as an external preparation composition for improving acne, which is excellent in antibacterial effect, in particular, antibacterial effect against acne causing bacteria, and also provides an anti-inflammatory effect.

The composition of the present invention can be used as an external skin composition for improving color and skin tone, and when applied to the skin, it smoothly supplies nutrients to the skin by promoting capillaries and promotes blood circulation and inhibits skin aging to improve color and skin tone. The effect is excellent.

The composition of the present invention can be used as a skin external preparation composition for pore reduction, sebum control and skin trouble improvement, which suppresses excessive secretion of sebum when applied to the skin, promotes free radical removal and collagen synthesis to shrink pores. In addition, the effect of suppressing skin problems is excellent by reducing the expression of inflammatory factors. In addition, it is possible to defend against the generation of skin irritation due to its excellent antioxidant power.

The composition of the present invention can be used as a composition for improving atopic dermatitis, which inhibits the activity of protease-activated receptor-2 (PAR-2), which causes itching, to provide an excellent anti-pruritic effect. In addition to providing anti-inflammatory effects, the secretion of Interleukin-8 (IL-8) can be reduced. Therefore, the saponin of the present invention can be suitably used as an active ingredient of the topical skin composition for stabilizing sensitive, irritant or atopic skin and scalp, and to improve or alleviate itching, heat and inflammation.

The composition of the present invention can be used as a composition for whitening, which can provide an excellent whitening effect by inhibiting tyrosinase activity and inhibiting melanin production.

The composition according to the invention may be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and non- It may be provided in the form of an ionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

In addition, the composition according to the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening agents, gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, Such as fillers, emulsifiers, emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics May contain adjuvants commonly used in the field of cosmetics or dermatology. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

In addition, the composition of the present invention may contain a skin absorption promoting substance to increase the skin improving effect.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to test examples and formulation examples. However, these test examples and formulation examples are provided for illustrative purposes only for the sake of understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

Reference Example 1 Isolation of Saponin

The roots of the tea tree were collected in November 2005 at Hannam-ri, Namwon-eup, Seogwipo-si, Jeju-do. The tea tree was chosen as a 20 year old Yabukita variety.

10 kg of fresh tea tree roots were cut into small pieces and soaked in 8 L of 80% aqueous MeOH and left at room temperature for 2 days, and this extraction method was repeated twice. The extracts were combined and concentrated in vacuo to give 540 g of a gummy brown residue. 100 g of the residue was separated by chromographie using eluting MeOH in H2O (4 L each) from 0 to 100% in a C18 silica gel flash column with eluent and eleven fractions (A to K). Got. Of these, active fractions G (4 g) and H (10 g) were combined and chromatographed again on silica gel to obtain eight fractions (GH1 to GH8).

The enzyme-inhibited saponin fraction GH5 (1.02 g) in the fractions was purified by C18 HPLC (eluent: 50 to 60% aqueous MeCN) to give 9 saponins 1 (108 mg, t _R). 19.9 min), 2 (92 mg, t _R 20.9 min), 3 (37 mg, t _R 23.3 min), 4 (60 mg, t _R 32.7 min), 5 (161 mg, t _R 35.7 min), 6 (113 mg, t _R 37.8 min), 7 (48 mg, t _R 39.4 min), 8 (33 mg, t _R 41.9 min), and 9 (56 mg, t _R 47.9 min) were obtained. In addition, analytical samples 1 to 3 were obtained using eluent 40 to 49% aqueous MeCN, and samples 4 to 8 using eluent 70 to 80% aqueous MeOH at C18 HPLC (49 mg, 32 mg, 12 mg, 16 mg, 71 mg, 20 mg, 12 mg, and 9 mg). Fraction 9 was further purified by HPLC using eluent 55 to 60% aqueous MeCN to give assay sample 9 (26 mg, t _R 43.3 min) and 10 (8 mg, t _R 62.7 min).

Ten isolated triterpenoid saponins are referred to as 'tea root-derived saponins R1 to R10', respectively, and specific analysis results are as follows.

Saponin R1 from tea tree root: white solid; Melting point 228-229 ° C .; [a] ²⁰ _D : -28.83 ( c 0.43, MeOH); IR (neat): ν _max = 3406, 1738, 1714, 1447, 1249, 1039 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.69 (3H, s, H-24), 0.87 (3H, t, J = 7.2 Hz, 2MB-H-4), 0.89 (3H, s, H-29), 1.00 (5H, 3H, s, H-25 and 2H, m, H-6), 1.01 (3H, s, H-26), 1.02 (1H, m, H _a -1), 1.03 (3H, d, J = 7.2 Hz, 2MB-H-5), 1.05 (3H, s, H-30), 1.07 (1H, m, H _a -7), 1.26 (1H, t, J = 10.4 Hz , H-5), 1.35 (1H, dd, J = 13.2, 5.2 Hz, H _a -19), 1.38 (1H, m, 2MB-H _a -3), 1.48 (1H, m, H _b -7) , 1.50 (3H, s, H-27), 1.56 (1H, m, 2MB-H _b -3), 1.64 (2H, m, H _b -1, H-9), 1.74 (1H, m, H _a -2), 1.84 (1H, m, H _b -2), 1.91 (3H, s, Ac), 1.96 (3H, s, Ac), 1.98 (2H, m, H-11), 2.23 (1H, m , 2MB-H-2), 2.25 (3H, s, Ac), 2.47 (1H, t, J = 14.2 Hz, H _b -19), 2.77 (1H, dd, J = 14.0, 4.0 Hz, H-18 ), 3.10 (1H, d, J = 11.2 Hz, H _a -28), 3.26 (1H, d, J = 11.2 Hz, H _a -23), 3.36 (1H, d, J = 11.2 Hz, H _b- 28), 3.42 (1H, t J = 8.0, Hz, GlcA-H-2), 3.54 (2H, m, GlcA-H-3 & Ara-H-3), 3.57 (1H, m, GlcA-H- 4), 3.58 (1H, m, Ara-H _a -5), 3.60 (1H, d, J = 11.2 Hz, H _b -23), 3.62 (1H, m, Ara-H-2), 3.63 (1H , m, H-3), 3.80 (1H, m, Ara- H-4), 3.82 (1H, m, GlcA-H-5), 3.90 (1H, m, Ara-H _b -5), 4.51 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara -H-1), 5.16 (1H, d, J = 4.8 Hz, H-15), 5.29 (1H, d, J = 4.8 Hz, H-16), 5.30 (1H, d, J = 10.0 Hz, H -21), 5.52 (1H, d, J = 10.0 Hz, H-22), 5.59 (1H, brs, H-12); HR-TOF-ESI-MS: m / z = 1041.5258 [M + H] ⁺ (calcd. For C ₅₂ H ₈₀ O ₂₁ + H: 1041.5270).

Saponin R2 derived from the tea tree root: white solid; Melting point 229-230 ° C .; [a] ²⁰ _D : -37.61 ( c 0.545, MeOH); IR (neat): ν _max = 3314 ,, 1727, 1444, 1248, 1038 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.69 (3H, s, H-24), 0.91 (3H, s, H-29), 1.00 (3H, s, H-25), 1.01 ( 3H, s, H-26), 1.08 (3H, s, H-30), 1.26 (1H, m, H _a -19), 1.50 (3H, s, H-27), 1.71 (3H, s, Ang -H-5), 1.90 (3H, s, Ac), 1.91 (3H, d, J = 7.6 Hz, Ang-H-4), 1.93 (3H, s, Ac), 2.24 (3H, s, Ac) , 2.50 (1H, t, J = 14.0, H _b -19), 2.78 (1H, dd, J = 14.4, 4.0 Hz, H-18), 3.14 (1H, d, J = 10.8 Hz, H _a -28 ), 3.26 (1H, d, J = 10.8 Hz, H _a -23), 3.39 (1H, d, J = 10.0 Hz, H _b -28), 3.60 (1H, d, J = 10.8 Hz, H _b- 23), 3.63 (1H, m, H-3), 4.50 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara-H-1), 5.18 (1H, d, J = 4.4 Hz, H -15), 5.28 (1H, d, J = 4.0 Hz, H-16), 5.38 (1H, d, J = 10.8 Hz, H-21), 5.60 (1H, brs, H-12), 5.61 (1H , d, J = 10.8 Hz, H-22), 6.08 (1H, q, J = 7.6 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 1039.5142 [M + H] ⁺ (calcd. For C ₅₂ H ₇₈ O ₂₁ + H: 1039.5114).

Saponin R3 from the root of the tea tree: white solid; Melting point 233-234 ° C .; [a] ²⁰ _D : -28.84 ( c 0.208, MeOH); IR (neat): ν _max = 3415, 1726, 1368, 1249, 1023 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.70 (3H, s, H-24), 0.91 (3H, s, H-29), 0.95 (3H, s, H-26), 1.01 ( 3H, s, H-25), 1.08 (3H, s, H-30), 1.35 (3H, s, H-27), 1.50 (1H, m, H _a -15), 1.74 (3H, s, Ang -H-4), 1.80 (1H , m, H b -15), 1.90 (3H, s, Ac), 1.93 (3H, d, J = 7.6 Hz, Ang-H-5), 2.20 (3H, s , Ac), 2.66 (1H, dd, J = 14.4, 4.8 Hz, H-18), 3.10 (1H, d, J = 10.8 Hz, H _a -28), 3.27 (1H, d, J = 10.8 Hz, H _a -23), 3.29 (1H, d, J = 10.8 Hz, H _b -28), 3.60 (1H, d, J = 10.8 Hz, H _b -23), 3.64 (1H, m, H-3) , 4.51 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara-H-1), 5.10 (1H, brs, H-16), 5.46 (1H, brs, H-12), 5.50 (1H , d, J = 10.4 Hz, H-21), 5.58 (1H, d, J = 10.4 Hz, H-22), 6.08 (1H, q, J = 7.6 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 981.5094 [M + H] ⁺ (calcd. For C ₅₀ H ₇₆ O ₁₉ + H: 981.5059).

Saponin R4 derived from the tea tree root: white solid; Melting point 227-228 ° C .; [a] ²⁰ _D : -12.56 ( c 0.565, MeOH); IR (neat): ν _max = 3421, 1718, 1370, 1257, 1041 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.71 (3H, s, H-24), 0.88 (3H, s, H-29), 0.86 (3H, t, J = 8.4 Hz, 2MB- H-4), 0.98 (3H, d, J = 6.8 Hz, 2MB-H-5), 1.00 (3H, s, H-26), 1.02 (3H, s, H-25), 1.07 (3H, s , H-30), 1.38 (3H, s, H-27), 1.40 (1H, m, 2MB-H _a -3), 1.58 (1H, m, 2MB-H _b -3), 1.82 (3H, s , Ang-H-5), 1.96 (3H, d, J = 7.2 Hz, Ang-H-4), 2.22 (1H, m, 2MB-H-2), 2.32 (3H, s, Ac), 2.66 ( 1H, dd, J = 14.0, 4.0 Hz, H-18), 3.12 (1H, d, J = 10.8 Hz, H _a -28), 3.27 (1H, overlapped with solvent, H _a -23), 3.37 (1H , d, J = 10.8 Hz, H _b -28), 3.60 (1H, d, J = 10.4 Hz, H _b -23), 3.64 (1H, m, H-3), 3.99 (1H, d, J = 4.0 Hz, H-15), 4.51 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara-H-1), 5.29 (1H, d, J = 4.0 Hz, H-16), 5.41 ( 1H, d, J = 10.4 Hz, H-21), 5.52 (1H, brs, H-12), 5.54 (1H, d, J = 10.4 Hz, H-22), 6.15 (1H, q, J = 6.8 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 1039.5471 [M + H] ⁺ (calcd. For C ₅₃ H ₈₂ O ₂₀ + H: 1039.5478).

Saponin R5 derived from the tea tree root: white solid; Melting point 231-232 ° C; [α] ²⁰ _D : + 4.35 ( c 0.46, MeOH); IR (neat): ν _max = 3395, 1711, 1442, 1256, 1039 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.85 (3H, s, H-29), 0.88 (3H, t, J = 7.2 Hz, 2MB-H-4), 1.01 (3H, s, H-25), 1.02 (3H, d, J = 6.0 Hz, 2MB-H-5), 1.04 (3H, s, H-26), 1.07 (3H, s, H-30), 1.13 (3H, s , H-24), 1.42 (1H, m, 2MB-H _a -3), 1.44 (3H, s, H-27), 1.60 (1H, m, 2MB-H _b -3), 1.85 (3H, s , Ang-H-5), 1.98 (3H, d, J = 6.8 Hz, Ang-H-4), 2.32 (1H, m, 2MB-H-2), 2.62 (1H, dd, J = 14.0, 4.0 Hz, H-18), 3.00 (1H, d, J = 10.4 Hz, H _a -28), 3.27 (1H, d, J = 10.8 Hz, H _b -28), 3.71 (1H, d, J = 4.0 Hz, H-15), 3.79 (1H, d, J = 4.8 Hz, H-16), 3.89 (1H, m, H-3), 4.32 (1H, d, J = 7.6 Hz, GlcA-H-1 ), 4.50 (1H, d, J = 6.4 Hz, Ara-H-1), 5.47 (1H, brs, H-12), 5.55 (1H, d, J = 10.4 Hz, H-22), 5.86 (1H , d, J = 10.4 Hz, H-21), 6.14 (1H, q, J = 7.2 Hz, Ang-H-3), 9.41 (1H, s, H-23); HR-TOF-ESI-MS: m / z = 995.5282 [M + H] ⁺ (calcd. For C ₅₁ H ₇₈ O ₁₉ + H: 995.5216).

Saponin R6 derived from the root of the tea tree: white solid; Melting point 229-230 ° C .; [a] ²⁰ _D : -9.56 ( c 0.23, MeOH); IR (neat): ν _max = 3424, 1728, 1538, 1456, 1229, 1070 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.86 (3H, t, J = 6.4 Hz, 2MB-H-4), 0.92 (3H, s, H-29), 0.99 (3H, d, J = 7.2 Hz, 2MB-H-5), 1.02 (3H, s, H-25), 1.04 (3H, s, H-26), 1.07 (3H, s, H-30), 1.10 (3H, s , H-24), 1.36 (H, dd, J = 13.6, 4.0 Hz, 2MB-H _a -3), 1.57 (1H, dd, J = 13.6, 7.2 Hz, 2MB-H _b -3), 1.39 ( 3H, s, H-27), 1.84 (3H, s, Ang-H-5), 1.97 (3H, d, J = 7.2 Hz, Ang-H-4), 2.24 (1H, m, 2MB-H- 2), 2.32 (3H, s, Ac), 2.64 (1H, dd, J = 14.4, 4.0 Hz, H-18), 3.12 (1H, d, J = 10.8 Hz, H _a -28), 3.34 (1H , d, J = 10.8 Hz, H _b -28), 3.90 (1H, dd, J = 8.8 & 2.8 Hz, H-3), 3.97 (1H, d, J = 4.8 Hz, H-15), 4.33 ( 1H, d, J = 7.6 Hz, GlcA-H-1), 4.49 (1H, d, J = 6.8 Hz, Ara-H-1), 5.28 (1H, d, J = 4.4 Hz, H-16), 5.41 (1H, d, J = 10.4 Hz, H-21), 5.54 (1H, d, J = 10.4 Hz, H-22), 5.55 (1H, brs, H-12), 6.16 (1H, q, J = 7.2 Hz, Ang-H-3), 9.40 (1H, s, H-23); HR-TOF-ESI-MS: m / z = 1037.5406 [M + H] ⁺ (calcd. For C ₅₃ H ₈₀ O ₂₀ + H: 1037.5321).

Saponin R7 derived from the tea tree root: white solid; Melting point 223-224 ° C; [α] ²⁰ _D : -13.01 ( c 0.315, MeOH); IR (neat): ν _max = 3407, 1714, 1370, 1249, 1039 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.86 (3H, t, J = 7.4 Hz, 2MB-H-4), 0.89 (3H, s, H-29), 0.98 (3H, s, H-25), 0.99 (3H, d, J = 7.2 Hz, 2MB-H-5), 1.00 (3H, s, H-26), 1.07 (3H, s, H-30), 1.16 (3H, s , H-24), 1.37 (3H, s, H-27), 1.38 (1H, m, 2MB-H _a -3), 1.56 (1H, m, 2MB-H _b -3), 1.82 (3H, s , Ang-H-5), 1.97 (3H, d, J = 7.2 Hz, Ang-H-4), 2.26 (1H, m, 2MB-H-2), 2.31 (3H, s, Ac), 2.66 ( 1H, dd, J = 14.8, 4.0 Hz, H-18), 3.12 (1H, d, J = 10.8 Hz, H _a -28), 3.34 (1H, d, J = 10.8 Hz, H _b -28), 3.68 (3H, s, 23-COOC H ₃ ), 3.96 (1H, d, J = 4.8 Hz, H-15), 4.03 (1H, dd, J = 11.6, 4.4 Hz, H-3), 4.30 (1H , d, J = 8.0 Hz, GlcA-H-1), 4.50 (1H, d, J = 6.8 Hz, Ara-H-1), 5.28 (1H, d, J = 4.4 Hz, H-16), 5.41 (1H, d, J = 10.4 Hz, H-21), 5.54 (1H, brs, H-12), 5.54 (1H, d, J = 10.8 Hz, H-22), 6.16 (1H, q, J = 7.0 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 1067.5492 [M + H] ⁺ (calcd for C ₅₄ H ₈₂ O ₂₁ + H: 1067.5427).

Saponin R8 derived from the tea tree root: white solid; Melting point 231-232 ° C; [a] ²⁰ _D : -8.62 ( c 0.105, MeOH); IR (neat): ν _max = 3411, 1722, 1441, 1242, 1073 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.84 (3H, s, H-29), 0.85 (3H, s, H-24), 0.88 (3H, t, J = 7.2 Hz, 2MB- H-4), 0.98 (3H, s, H-25), 1.01 (3H, s, H-26), 1.02 (3H, d, J = 7.2 Hz, 2MB-H-5), 1.05 (3H, s , H-23), 1.07 (3H, s, H-30), 1.41 (3H, s, H-27), 1.42 (1H, m, 2MB-H _a -3), 1.60 (H, m, 2MB- H _b -3), 1.84 (3H, s, Ang-H-5), 1.98 (3H, d, J = 7.2 Hz, Ang-H-5), 2.30 (1H, m, 2MB-H-2), 2.62 (1H, m, H-18), 2.99 (1H, d, J = 10.0 Hz, H _a -28), 3.18 (1H, dd, J = 9.2, 5.2 Hz, H-3), 3.30 (1H, overlapped with solvent, H _b -28), 3.73 (1H, d, J = 4.0 Hz, H-15), 3.80 (1H, d, J = 4.0 Hz, H-16), 4.41 (1H, d, J = 7.2 Hz, GlcA-H-1), 4.53 (1H, d, J = 6.8 Hz, Ara-H-1), 5.47 (1H, brs, H-12), 5.55 (1H, d, J = 10.0 Hz, H-22), 5.86 (1H, d, J = 10.0 Hz, H-21), 6.16 (1H, q, J = 7.2 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 981.5435 [M + H] ⁺ (calcd. For C ₅₁ H ₈₀ O ₁₈ + H: 981.5423).

Saponin R9 derived from the tea tree root: white solid; Melting point 232-233 ° C; [a] ²⁰ _D : -24.15 ( c 0.31, MeOH); IR (neat): ν _max = 3443, 1724, 1459, 1234, 1072 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.71 (3H, s, H-24), 0.84 (3H, t, J = 7.2 Hz, 2MB-H-4), 0.88 (3H, s, H-29), 0.95 (3H, s, H-26), 0.98 (3H, d, J = 6.8 Hz, 2MB-H-5), 1.01 (3H, s, H-25), 1.09 (3H, s , H-30), 1.32 (1H, m, 2MB-H _a -3), 1.37 (3H, s, H-27), 1.44 (1H, m, H _a -15), 1.57 (H, m, 2MB -H _b -3), 1.80 (1H, m, H _b -15), 1.83 (3H, s, Ang-H-5), 1.97 (3H, d, J = 7.2 Hz, Ang-H-4), 2.20 (1H, m, 2MB-H-2), 2.27 (3H, s, Ac), 2.66 (1H, dd, J = 14.0, 3.6 Hz, H-18), 3.08 (1H, d, J = 10.4 Hz , H _a -28), 3.29 (1H, overlapped with solvent, H _a -23), 3.32 (1H, overlapped with solvent, H _b -28), 3.60 (1H, d, J = 10.0 Hz, H _b -23 ), 3.63 (1H, m, H-3), 4.51 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara-H-1), 5.15 (1H, brs, H-16), 5.45 ( 1H, brs, H-12), 5.54 (2H, s, H-21 and H-22), 6.16 (1H, q, J = 7.6 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 1023.5556 [M + H] ⁺ (calcd. For C ₅₃ H ₈₂ O ₁₉ + H: 1023.5529).

Saponin R10 from tea tree root: white solid; Melting point 232-233 ° C; [a] ²⁰ _D : -22.06 ( c 0.29, MeOH); IR (neat): ν _max = 3433, 1720, 1458, 1229, 1072 cm ⁻¹ ; ¹ H-NMR (400 MHz, CD ₃ OD): δ = 0.70 (3H, s, H-24), 0.85 (3H, t, J = 7.6 Hz, 2MB-H-4), 0.89 (3H, s, H-29), 0.95 (3H, s, H-26), 0.98 (3H, d, J = 6.8 Hz, 2MB-H-5), 1.01 (3H, s, H-25), 1.09 (3H, s , H-30), 1.36 (3H, s, H-27), 1.37 (1H, m, 2MB-H _a -3), 1.43 (1H, m, H _a -15), 1.55 (1H, m, 2MB -H _b -3), 1.81 (1H, m, H _b -15), 1.83 (3H, s, Ang-H-5), 1.96 (3H, d, J = 7.2 Hz, Ang-H-4), 2.22 (1H, m, 2MB-H-2), 2.27 (3H, s, Ac), 2.66 (1H, dd, J = 14.0, 4.0 Hz, H-18), 3.08 (1H, d, J = 10.8 Hz , H _a -28), 3.29 (1H, overlapped with solvent, H _a -23), 3.32 (1H, overlapped with solvent, H _b -28), 3.60 (1H, d, J = 10.8 Hz, H _b -23 ), 3.62 (1H, m, H-3), 3.77 (3H, s, GlcA-6-OC H ₃ ), 4.51 (2H, d, J = 7.2 Hz, GlcA-H-1, Ara-H-1 ), 5.16 (1H, brs, H-16), 5.46 (1H, brs, H-12), 5.54 (2H, s, H-21 and H-22), 6.16 (1H, q, J = 7.0 Hz, Ang-H-3); HR-TOF-ESI-MS: m / z = 1037.5684 [M + H] ⁺ (calcd. For C ₅₄ H ₈₄ O ₁₉ + H: 1037.5685).

Test Example 1 Measurement of Elastase Activity Inhibition Efficacy

Inhibition of elastase activity of the saponin derived from the root of the tea tree of the present invention was measured in comparison with EGCG. The elastase and substrate used were commercially purchased from Sigma-Aldrich Co., Ltd. (Cat. No. E0127), and triterpenoid saponins were used saponins R1 to R10 derived from the tea tree root obtained in Reference Example 1.

Elastase activity inhibitory activity was tested by the following test method.

50 μL of the test substance and 50 μL of the 20 μg / mL elastase type III solution shown in Table 1 were mixed with a 10 mg / L Tris-HCL buffer (pH 8.0) in a 96 well plate. 250 μM of EGCG was used as a positive control, and distilled water was used as a non-treated group as a negative control. Thereafter, 100 µL of 0.4514 mg / mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE prepared with the above buffer was added and reacted at 25 ° C for 15 minutes. After completion of the reaction, the absorbance at a wavelength of 415 nm was measured. A blank test was performed in the same manner to correct.

Calculation method of the elastase activity inhibitory activity is shown in Equation 1 below, the results are shown in Table 1 below.

A: Absorption at wavelength 415 nm with no test sample and enzyme addition

B: Absorbance at a wavelength of 415 nm with no test sample and no enzyme

C: absorbance at wavelength 415 nm at the addition of test sample and enzyme

D: Absorbance at wavelength 415 nm with test sample added and no enzyme

compound Suppression degree (%) Untreated group 0 EGCG 65 Saponin R1 from tea tree root 66 Tea tree root-derived saponin R2 68 Tea tree root-derived saponin R3 70 Tea tree root-derived saponin R4 62 Tea tree root-derived saponin R5 61 Tea tree root-derived saponin R6 63 Saponin R7 derived from tea tree root 55 Tea tree root-derived saponin R8 57 Saponin R9 from tea tree root 66 Saponin R10 from tea tree root 71

As shown in Table 1, the degree of inhibition of the elastase activity of the saponin from the roots of the tea tree was almost remarkably superior to the EGCG compared to the EGCG known as the inhibitor of the elastase activity. It can be confirmed that the effect of inhibiting elastase activity is excellent.

Test Example 2 Collagenase (MMP-1) Inhibitory Activity

Inhibition of collagenase production of saponin derived from the root of the tea tree of the present invention was measured in comparison with retinoic acid.

Place human fibroblasts at 5,000 cells / well in 96-well microtiter plates containing DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal calf serum. It was incubated in an incubator (37%) of 5% CO 2 until it grew to about 80%. The saponins derived from the roots of tea were treated with 30 ㎍ / ml for 24 hours, followed by cell culture.

The degree of collagenase production of cell cultures was measured using a commercially available collagenase measuring instrument (Amersham Pharmacia, USA, Catalog #: RPN 2610). First, the cell culture solution collected in a 96-well plate uniformly coated with primary collagenase antibody was placed in an incubator for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, color-causing substances (3,3 ', 5,5'-tetramethylbenzidine, sigma) were added to induce color development at room temperature for 15 minutes, and 1M sulfuric acid was added to stop the color reaction. The degree of yellow was different according to the degree of reaction progress.

The absorbance of the yellow 96-well plate was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated by Equation 2 below, and the results are shown in Table 2 below. Indicated. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control.

compound Expression degree (%) Untreated group 100 Retinoic acid 75 Saponin R1 from tea tree root 78 Tea tree root-derived saponin R2 81 Tea tree root-derived saponin R3 73 Tea tree root-derived saponin R4 70 Tea tree root-derived saponin R5 88 Tea tree root-derived saponin R6 71 Saponin R7 derived from tea tree root 75 Tea tree root-derived saponin R8 88 Saponin R9 from tea tree root 76 Saponin R10 from tea tree root 81

As shown in Table 2, it can be seen that the collagenase expression level of saponin is similar to the collagenase expression inhibitory effect compared to retinoic acid known as a collagenase expression inhibitor.

Through these results, it can be confirmed that the saponin of the present invention has the effect of inhibiting the proteolytic enzyme (MMP-1).

[Formulation Examples 1 to 10 and Comparative Formulation Example 1]

Nutritional cream was prepared in a conventional manner according to the composition of Table 3 (unit: wt%).

Compounding ingredient Formulation Example 1 Formulation Example 2 Formulation Example 3 Formulation Example 4 Formulation Example 5 Formulation Example 6 Formulation Example 7 Formulation Example 8 Formulation Example 9 Formulation Example 10 Comparative Formulation Example 1 Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 Saponin R1 from tea tree root 0.1 - - - - - - - - Tea tree root-derived saponin R2 - 0.1 - - - - - - - - - Tea tree root-derived saponin R3 - - 0.1 - - - - - - - - Tea tree root-derived saponin R4 - - - 0.1 - - - - - - - Tea tree root-derived saponin R5 - - - - 0.1 - - - - - - Tea tree root-derived saponin R6 - - - - - 0.1 - - - - - Saponin R7 derived from tea tree root - - - - - - 0.1 - - - - Tea tree root-derived saponin R8 - - - - - - - 0.1 - - - Saponin R9 from tea tree root - - - - - - - - 0.1 - - Saponin R10 from tea tree root - - - - - - - - - 0.1 - Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Stearic acid 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 Glycerol stearate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Stearyl alcohol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Arakidil Behenyl Alcohol & Arakidil Glucoside 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Cetylaryl Alcohol & Cetearyl Glucoside 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 PEG-100 Stearate & Glycerolate & Propylene Glycol 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Caprylic / Carric Triglycerides 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 Cyclomedicon 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 Preservative, incense Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Triethanolamine 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

 Test Example 3 Confirmation of Skin Elasticity Improvement Efficacy

In order to confirm the effect of improving skin elasticity in humans, the formulations of Examples 1 to 10 and Comparative Formulation Example 1 were evaluated as follows.

After 110 healthy women in their 30s and 40s were divided into 11 groups of 10 people for each of the 11 groups of Formulation Examples 1-10 and Comparative Formulation Example 1, the nourishing cream was applied to the left side of the face once a day for 12 weeks. Skin elasticity was measured using (Cutometer SEM 575, C + K Electronic Co., Germany). The results are shown in Table 4 below. The results in Table 4 are described as ΔR8 (R8 (left) -R8 (right)) values of Cutometer SEM 575, where R8 values represent the nature of skin viscoelasticity.

Experimental product Skin elasticity effect Formulation Example 1 0.17 Formulation Example 2 0.3 Formulation Example 3 0.27 Formulation Example 4 0.29 Formulation Example 5 0.28 Formulation Example 6 0.17 Formulation Example 7 0.18 Formulation Example 8 0.14 Formulation Example 9 0.27 Formulation Example 10 0.15 Comparative Formulation Example 1 0.10

As shown in Table 4, Formulation Examples 1 to 10 containing the saponin derived from the root of the tea tree of the present invention was further increased skin elasticity compared to the group to which Comparative Formula 1 was applied.

Therefore, it can be confirmed that the composition containing saponin derived from the root of the tea tree of the present invention is very effective for improving skin elasticity.

Test Example 4 Confirmation of Skin Wrinkle Improvement Efficacy

Formulation examples 1 to 10 and Comparative Formulation Example 1 were used to confirm the wrinkle improvement effect in humans by the composition of the present invention.

In order to confirm the wrinkle improvement effect of the Formulation Examples 1 to 10 and Comparative Formulation Example 1 was evaluated as follows. 110 healthy women in their 40s were divided into 11 groups of 10 people for each of 16 groups of Formulation Examples 1 to 10 and Comparative Formulation Example 1, and the nourishing cream was applied to the face once a day for 12 weeks. The state of the swelling was measured by a skin measurer (visiometer, SV600, Courage + Khazaka electronic GmbH, Germany) for image analysis. The results are shown in Table 5 below. The values in Table 5 below represent the average of each parameter value after 12 weeks of application minus the parameter value before application.

8 weeks after use
Clinical results R1 R2 R3 R4 R5 Formulation Example 1 0.15 0.14 0.14 0.02 0.02 Formulation Example 2 0.11 0.10 0.11 0.01 0.01 Formulation Example 3 0.14 0.13 0.13 0.01 0.01 Formulation Example 4 0.2 0.2 0.18 0.03 0.03 Formulation Example 5 0.17 0.17 0.16 0.02 0.02 Formulation Example 6 0.29 0.24 0.28 0.02 0.02 Formulation Example 7 0.27 0.25 0.27 0.01 0.01 Formulation Example 8 0.15 0.15 0.15 0.03 0.03 Formulation Example 9 0.17 0.14 0.14 0.01 0.01 Formulation Example 10 0.20 0.19 0.19 0.03 0.03 Comparative Formulation Example 1 0.27 0.26 0.21 0.03 0.03

R1: difference between the highest and lowest of the wrinkle contour

R2: Divide the wrinkle contours by 5 spaces, and then average the R1 values

R3: The highest value among the R1 values divided by five

R4: The mean value of the baseline of the fold contour minus the top and valley values of each angle

R5: Difference between the baseline of the wrinkle contour line minus the outline of each wrinkle

As shown in Table 5, it was found that the external preparation composition of Formulation Examples 1 to 10 is very excellent in improving skin wrinkles.

Test Example 5 Effect of Color Improvement

In order to evaluate the skin blood circulation promoting effect of the cosmetic composition according to the present invention, the degree of blood circulation in the skin was measured using a LDPI (Laser Doppler Perfusion Imager). LDPI is a widely used and widely used device for measuring blood circulation in skin. It is a highly sensitive device that can measure not only blood velocity and quantity in the capillaries of the skin, but also flow in the small artery and the parenchyma.

After washing the face with soap in a thermo-hygrostat room for 30 minutes, the initial value was measured using LDPI. Initially, initial blood flow in the lower forehead of 30 women with cold hands and feet was measured by LDPI. Then, after the formulation examples 1 to 10 and Comparative Formulation Example 1 were used for the test subjects for one week, the blood flow rate measured after the initial measurement value (skin blood flow change) is shown in Table 6 below.

LDPI Results-Skin Blood Flow Before and After Cosmetic Use Test substance % Change in skin blood flow after 1 week of use Formulation Example 1 5 Formulation Example 2 20 Formulation Example 3 13 Formulation Example 4 4 Formulation Example 5 5 Formulation Example 6 12 Formulation Example 7 8 Formulation Example 8 9 Formulation Example 9 13 Formulation Example 10 14 Comparative Formulation Example 1 5

In the results of Table 6, the cosmetic composition according to the present invention significantly increased the skin blood flow than Comparative Formulation Example 1 containing no tea tree root-derived saponin, it was confirmed that the blood color is improved through the promotion of this blood circulation. . This suggests that the cosmetic composition containing the saponin derived from the root of the tea according to the present invention may ultimately contribute to effectively delivering the nutrients of the skin, inhibiting and delaying skin aging.

[Test Example 6] Skin tone improvement effect

In order to find out the skin tone improvement effect of Formulation Examples 1 to 10 and Comparative Formulation Example 1, 30 subjects each were used (once evening / daily application, for a total of 1 week), and then, Facial Stage DM-3 (Moritex, Japan The degree of skin tone improvement was evaluated using the device. The improvement rate of the skin tone was determined as the brightness and color value of the skin and the change in the brightness and color of the skin.

Test substance Skin tone improvement rate (%) Brightness (mean ± standard deviation) Color (mean ± standard deviation) Formulation Example 1 10 ± 1.34 12 ± 2.15 Formulation Example 2 8 ± 1.15 11 ± 2.95 Formulation Example 3 12 ± 2.15 9 ± 0.25 Formulation Example 4 11 ± 2.25 13 ± 2.34 Formulation Example 5 9 ± 0.25 10 ± 0.15 Formulation Example 6 13 ± 2.34 13 ± 2.34 Formulation Example 7 11 ± 1.05 12 ± 1.05 Formulation Example 8 8 ± 1.15 8 ± 0.15 Formulation Example 9 11 ± 2.25 11 ± 2.25 Formulation Example 10 9 ± 0.25 9 ± 0.25 Comparative Formulation Example 1 5 ± 2.34 5 ± 2.05

In the results of Table 7, Comparative Formulation Example 1 containing no tea tree root-derived saponins according to the present invention showed no significant skin tone improvement effect, whereas Formulation Examples 1 to 10 containing tea tree root-derived saponin as an active ingredient were used. It was confirmed that the skin tone after use was improved more than before.

[Test Example 7] Pore reduction effect

Pore reduction effect by promoting collagen biosynthesis

The saponin derived from the root of the tea according to the present invention was measured by comparing collagen biosynthesis promoting effect with TGF-β. First, fibroblasts were seeded by 10 ⁵ per hole in 24 wells and cultured until 90% growth. After incubation for 24 hours in serum-free DMEM medium and treated with 10ng / ml each of saponin and TGF-β derived from the root of the tea of the present invention dissolved in serum-free medium and CO ₂ The incubator was incubated for 24 hours. These supernatants were removed and procollagen increased or decreased using procollagen type I ELISA kit (procollagen type (I)). The results are shown in Table 8, and the synthetic ability of collagen was compared with the non-treated group as 100.

Test substance Collagen Synthesis (%) Untreated group 100 TGF-β 183.5 Saponin R1 from tea tree root 120.1 Tea tree root-derived saponin R2 135.1 Tea tree root-derived saponin R3 138 Tea tree root-derived saponin R4 157.8 Tea tree root-derived saponin R5 115 Tea tree root-derived saponin R6 125 Saponin R7 derived from tea tree root 143.1 Tea tree root-derived saponin R8 123.4 Saponin R9 from tea tree root 168.1 Saponin R10 from tea tree root 112.1

In the results of Table 8, it was confirmed that the saponin derived from the root of the tea tree according to the present invention exhibits excellent collagen synthesis ability at the same or higher level than the positive control TGF-β. Therefore, it was confirmed that the saponin derived from the root of the tea according to the present invention can reduce the enlarged pores by increasing the amount of collagen production around the pores.

Pore Reduction Effect

To evaluate the pore reduction effect of Formulation Examples 1 to 10 and Comparative Formulation Example 1 was evaluated as follows. A total of 110 male and female subjects with large pore sizes were selected and divided into 11 groups of 10, and the nutritional creams of Formulation Examples 1 to 10 and Comparative Formulation Example 1 were applied to the face for 4 weeks each day. Determination of the effect of pore reduction was made by visual assessment of experts by taking pictures before and after 4 weeks of the experiment. The results are shown in Table 9 below (rating rating: 0-not scaled down at all; 5-scaled down).

Test substance Rating Rating Formulation Example 1 3.5 Formulation Example 2 3.1 Formulation Example 3 2.5 Formulation Example 4 2.9 Formulation Example 5 2.1 Formulation Example 6 3.6 Formulation Example 7 4.1 Formulation Example 8 3.5 Formulation Example 9 2.5 Formulation Example 10 3.1 Comparative Formulation Example 1 0.4

From the results of Table 9, Comparative Formulation Example 1 does not have a pore reduction effect, but in the case of Formulation Examples 1 to 10 shows a pore reduction effect that can be visually confirmed, the tea tree root-derived saponin according to the present invention has a pore size. It was found that the reducing effect is excellent.

Test Example 8 Effect of suppressing sebum secretion

Inhibition of skin hypersecretion through inhibition of 5α-reductase activity

In order to confirm the inhibitory effect of 5α-reductase activity, the rate at which [ ¹⁴ C] testosterone is converted to [ ¹⁴ C] dihydrotestosterone (DHT) in HEK293-5αR2 cells was measured. HEK293 cells were transfected with p3 × FLAG-CMV-5αR2 and cultured in a 24 well plate at 2.5 × 10 ⁵ cells per well (Park et al., 2003, JDS. Vol. 31, pp. 191-98). The next day, a new medium with enzyme substrate and inhibitor was added. 0.05 μCi [ ¹⁴ C] testosterone (Amersham Pharmacia biotech, UK) was used as the substrate of the medium.

In order to confirm the degree of inhibition of 5α-reductase activity, each of the saponins derived from the roots of the tea tree shown in Table 10 was added thereto, and then incubated for 2 hours at 37 ° C. and 5% CO ₂ incubator. At this time, the tea root-derived saponin was not used as a negative control group, the pinasteride (finasteride) was used as a positive control group. Thereafter, the culture medium was collected, the steroid was extracted with 800 μl ethyl acetate, the organic solvent layer was separated, dried, and the remaining residue was dissolved again with 50 μl ethyl acetate, followed by silica plastic sheet kieselgel 60 F254. ) Was developed using ethyl acetate-hexane (1: 1) as a solvent.

After drying the plastic sample in air, the bath system was used to measure the isotope amount. The isotopic isomer of testosterone and dihydrotestosterone remaining in the film after one week by placing the dried plastic sheet and the x-ray film together in the bath cassette. After the amount was measured, the conversion and inhibition rates were calculated according to the following Equations 3 and 4, and the results are shown in Table 10 below.

Test substance Conversion Rate (%) Inhibition rate (%) Negative control group 48.0 - Positive control group 27.6 42.5 Saponin R1 from tea tree root 30.2 37.1 Tea tree root-derived saponin R2 34.1 29.0 Tea tree root-derived saponin R3 40.1 16.5 Tea tree root-derived saponin R4 40.6 15.5 Tea tree root-derived saponin R5 45.1 6.1 Tea tree root-derived saponin R6 30.1 37.3 Saponin R7 derived from tea tree root 34.5 29 Tea tree root-derived saponin R8 44.1 8.2 Saponin R9 from tea tree root 32.1 33.2 Saponin R10 from tea tree root 33.1 31.1

In the results of Table 10, 5α-reductase, which converts testosterone into dihydrotestosterone, binds to a receptor protein in the cytoplasm, enters the nucleus, activates sebaceous gland cells, promotes differentiation, and hypersecretes sebum in sebaceous glands. By inhibiting the activity of the saponin derived from the root of the tea tree according to the present invention it was confirmed that the conversion of testosterone to dihydrotestosterone was blocked. Therefore, it was confirmed that the saponin derived from the root of the tea according to the present invention can suppress the excessive secretion of sebum by effectively inhibiting the activity of 5α-reductase.

Sebum secretion inhibitory effect

In order to determine the sebum secretion inhibitory effect of Formulation Examples 1 to 10 and Comparative Formulation Example 1 was evaluated as follows. Thirty male and female subjects who felt sebum secretion were selected and the nutritional creams of Formulation Examples 1 to 10 and Comparative Formulation Example 1 were applied daily to the designated site for 4 weeks. The results are shown in Table 2. The results are shown in Table 11 below. The results are shown in Table 11 below. The results are shown in Table 11 below. Respectively.

Test substance Sebum reduction rate (%) After 2 weeks After 4 weeks Formulation Example 1 11 12 Formulation Example 2 14 14 Formulation Example 3 19 17 Formulation Example 4 11 17 Formulation Example 5 12 11 Formulation Example 6 7 5 Formulation Example 7 6 10 Formulation Example 8 18 11 Formulation Example 9 14 11 Formulation Example 10 15 15 Comparative Formulation Example 1 5 5

In the results of Table 11, it can be seen that Formulation Examples 1 to 10 containing the tea tree root-derived saponin as an active ingredient according to the present invention can effectively inhibit sebum secreted in excess than Comparative Formulation Example 1 that does not contain it there was.

Test Example 9 Measurement of Increased Skin Moisturizing Effect

In order to measure the effect of tea root-derived saponin on the increase in skin moisturizing power, Formulation Examples 1 to 10 and Comparative Formulation Example 1 were used, and evaluated as follows.

110 adult men and women in their 40's and 50's, classified as dry skin, were divided into 11 groups of 10 people for each of the 11 groups of Formulation Examples 1-10 and Comparative Formulation Example 1, respectively, and applied nutrition cream to the face twice a day for 4 weeks. Skin moisture meter at constant temperature and humidity conditions (24 ° C, 40% relative humidity) before start of application, after 1 week, 2 weeks, 4 weeks after application, and after 2 weeks (total 6 weeks) after application is stopped. Skin moisture content was measured by (Corneometer CM825, C + K Electronic Co., Germany). The results are shown in Table 12 below. The results in Table 12 show the percentage increase in the measured value after treatment for a period of time, based on the skin moisture meter measured immediately before the test.

Test group Moisture growth rate (%) 1 week Two weeks 4 weeks 6 weeks Formulation Example 1 27 27 27 24 Formulation Example 2 22 24 35 30 Formulation Example 3 21 23 30 30 Formulation Example 4 25 22 25 22 Formulation Example 5 26 29 31 20 Formulation Example 6 30 28 31 17 Formulation Example 7 20 28 30 21 Formulation Example 8 28 28 29 25 Formulation Example 9 30 29 32 27 Formulation Example 10 28 31 35 26 Comparative Formulation Example 1 30 32 32 15

In the results of Table 12, when the Comparative Formulation Example 1 was applied, it showed a water increase rate of about 30% up to 4 weeks after the application, but after stopping the application, the skin moisture content decreased, while saponin derived from the root of the tea tree was In the case of applying the formulation examples 1 to 10 containing it can be confirmed that even after stopping the application, most of the skin moisture increase rate of more than 30%. It can be seen that the composition of the present invention containing saponin derived from the root of the tea tree is excellent in the skin moisturizing effect.

Test Example 10 Measurement of Keratinocyte Differentiation Promotion Effect

In order to examine the differentiation promoting effect of keratinocytes of the root of saponin from tea tree, the amount of CE (Cornified Envelop) generated during the differentiation of keratinocytes was measured using absorbance.

First, the keratinocytes of the primary cultured human isolated from the epidermis of the newborn were put in a culture flask and adhered to the bottom. After the test substance of Table 13 was treated at 5 ppm in the culture solution, the cells were 70 ~ of the floor area. It was incubated for 5 days until it grew about 80%. At this time, the low calcium (0.03mM) treated group and the high calcium (1.2mM) treated group were used as negative and positive controls, respectively. Then, the cultured cells were harvested and washed with PBS (Phophate buffered saline), followed by 10 mM Tris-HCl buffer (Tris-HCl, containing 2% SDS (sodium dodecyl sulfate) and 20 mM Dithiothreitol (DTT). pH 7.4) 1 ml was added to sonication, boiling and centrifugation, and the precipitate was suspended in 1 ml of PBS again to measure absorbance at 340 nm. Separately, a portion of the solution after the sonication was taken to measure the protein content and used as a reference when evaluating the degree of cell differentiation. The results are shown in Table 13 below.

Test substance The ability to differentiate in keratinocytes (%) Low Calcium (0.03 mM) Solution
(Negative control) 100 High calcium (1.2 mM) solution
(Positive control group) 210 Saponin R1 from tea tree root 110 Tea tree root-derived saponin R2 138 Tea tree root-derived saponin R3 117 Tea tree root-derived saponin R4 100 Tea tree root-derived saponin R5 168 Tea tree root-derived saponin R6 132 Saponin R7 derived from tea tree root 151 Tea tree root-derived saponin R8 147 Saponin R9 from tea tree root 136 Saponin R10 from tea tree root 121

As shown in Table 13, when treated with saponin derived from the root of the tea tree it was confirmed that the effect of promoting the differentiation of keratinocytes.

Test Example 11 Measurement of Skin Barrier Function Restoration Effect

In order to measure the effect of saponin derived from the roots of tea tree on the recovery of damaged skin barrier function due to skin damage, the following experiment was performed. Ten adult men and women's upper and lower limbs were damaged using a tape stripping method, and each of 11 groups of Formulation Examples 11 to 20 and Comparative Formulation Example 2, which were prepared using the composition shown in Table 14 below, was applied. The recovery of transdermal moisture loss (TWEL) was measured once a day using Vapometer (Delfin, Finland). Comparative Formulation Example 2 is a vehicle as the negative control group. The experimental results are shown in Table 15 below. The results in Table 15 were compared on the basis of the 100% difference before and after barrier damage.

Compounding ingredient Formulation Example 11 Formulation Example 12 Formulation Example 13 Formulation Example 14 Formulation Example 15 Formulation Example 16 Formulation Example 17 Formulation Example 18 Formulation Example 19 Formulation Example 20 Comparative Formulation Example 2 Purified water 69 69 69 69 69 69 69 69 69 69 69 Propylene glycol 30 30 30 30 30 30 30 30 30 30 30 Saponin R1 from tea tree root One - - - - - - - - - - Tea tree root-derived saponin R2 - One - - - - - - - - - Tea tree root-derived saponin R3 - - One - - - - - - - - Tea tree root-derived saponin R4 - - - One - - - - - - - Tea tree root-derived saponin R5 - - - - One - - - - - - Tea tree root-derived saponin R6 - - - - - One - - - - - Saponin R7 derived from tea tree root - - - - - - One - - - - Tea tree root-derived saponin R8 - - - - - - - One - - - Saponin R9 from tea tree root - - - - - - - - One - - Saponin R10 from tea tree root - - - - - - - - - One -

Test group TWEL change (%) Before processing 1 day 2 days 3 days 4 days 5 days 6 days Formulation Example 11 100 125.3 105.2 84.2 60.2 48.1 40.1 Formulation Example 12 100 124.7 110.2 90.1 78.1 50.1 35.1 Formulation Example 13 100 120 104.2 88.4 64.1 57.4 32.6 Formulation Example 14 100 119.6 108.2 89.1 62.1 58.1 38.4 Formulation Example 15 100 125.4 110.2 96.1 58.1 59.6 39.1 Formulation Example 16 100 132.1 111.3 95.1 58.7 40.1 35.1 Formulation Example 17 100 114.7 112.5 90.1 60.1 43.1 32.1 Formulation Example 18 100 128.1 107.1 88.4 62.4 52.1 31.0 Formulation Example 19 100 119.7 105.1 84.1 61.2 61.2 29.1 Formulation Example 20 100 120.1 107.3 94.1 65.4 54.1 38.1 Comparative Formulation Example 2 100 121.4 112.7 98.3 70.5 62.3 43.5

As can be seen in Table 15, it can be seen that when the tea root-derived saponin is treated, the transdermal moisture loss returns to normal and barrier damage is recovered.

[Formulation Examples 21-30 and Comparative Formulation Examples 3-4]

Formulation Examples 21 to 30 and Comparative Formulation Examples 3 to 4 were prepared according to the ingredients and the content (% by weight) shown in Table 16 below. Specifically, Formulation Examples 21 to 30 are formulated with saponins derived from tea tree root, Comparative Formulation Example 3 does not contain any active ingredients for improving acne skin, Comparative Formulation Example 4 is to be used as a reference for antimicrobial activity As a standard, it contains erythromycin, which is widely used as an acne treatment.

The preparation method of Formulation Examples 21-30 and Comparative Formulation Examples 3-4 is as follows. The components of phase A in Table 16 were completely dissolved, and the components of phase B were completely dissolved in a separate dissolution bath, followed by mixing solubilization by adding phase B to phase A. The ingredients of phase C were added thereto according to the blending ratios described in Table 16, homogenized, mixed and filtered to prepare the present compositions.

division Formulation Example 21 Formulation Example 22 Formulation Example 23 Formulation Example 24 Formulation Example 25 Formulation Example 26 Formulation Example 27 Formulation Example 28 Formulation Example 29 Formulation Example
30 Comparative Formulation Example 3 Comparative Formulation Example 4 A Deionized Water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 EDTA-2Na 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 glycerin 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 B ethanol 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 PEG-60 hydrogenated castor oil 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Perfume 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 C Saponin R1 from tea tree root 5.0 - - - - - - - - - - - Tea tree root-derived saponin R2 - 5.0 - - - - - - - - - - Tea tree root-derived saponin R3 - - 5.0 - - - - - - - - - Tea tree root-derived saponin R4 - - - 5.0 - - - - - - - - Tea tree root-derived saponin R5 - - - - 5.0 - - - - - - - Tea tree root-derived saponin R6 - - - - - 5.0 - - - - - - Saponin R7 derived from tea tree root - - - - - - 5.0 - - - - - Tea tree root-derived saponin R8 - - - - - - - 5.0 - - - - Saponin R9 from tea tree root - - - - - - - - 5.0 - - - Saponin R10 from tea tree root - - - - - - - - - 5.0 - - Erythromycin - - - - - - - - - - - 5.0

 Test Example 12 Antibacterial Activity Test against Acne Bacteria

With each of the cosmetic composition prepared in the formulation of Formulation Examples 21-30 and Comparative Formulations 3-4, the antibacterial activity against propionibacterium acnes <ATCC 6919: Medium-BHI broth> Tested.

The antibacterial test method for acne bacteria was as follows.

(1) Test bacteria preparation

Propionibacterium acnes was used as a culture broth inoculated in BHI broth and anaerobic culture.

(2) dilution solution preparation

0.15 ml of the test bacteria was added to 15 ml of BHI broth (pH 6.8) or LB broth (pH 4.5) and mixed well as a dilution solution.

(3) sample preparation

The cosmetic composition stock solutions prepared from Formulation Examples 21 to 30 and Comparative Formulation Examples 3 to 4 were used as samples.

(4) antimicrobial activity test

1) Insert the sample to the starting concentration in the first well of 96-well cell culture tube (96 well plate) and add 200 μl of total amount into the dilution solution.

2) Mix the mixed solution in the first row well, take 100μl into the second row, mix well, and then add 100μl to the third row and perform double dilution.

3) After incubation for 24 hours and 48 hours at 32 ℃, determine the growth of bacteria to the extent of suspension and determine the minimum concentration without growth of bacteria as MIC (Minimum Inhibitory Concentration) value. If the mixed solution is opaque and it is difficult to determine the growth of bacteria, check through a microscope.

The antimicrobial activity test results for acne bacteria are shown in Table 17 below. MIC is expressed in terms of the concentration of the active ingredient contained in the formulation.

Item pH Propionibacterium Acnes Formulation Example 21 5.7 15.4 Formulation Example 22 5.7 10.4 Formulation Example 23 5.7 8.8 Formulation Example 24 5.7 3.5 Formulation Example 25 5.7 2.5 Formulation Example 26 5.7 0.7 Formulation Example 27 5.7 2.8 Formulation Example 28 5.7 10.4 Formulation Example 29 5.7 3.5 Formulation Example 30 5.7 0.5 Comparative Formulation Example 3 5.7 Highest concentration (no antimicrobial activity) Comparative Formulation Example 4 5.7 > 100 ppm

The smaller the concentration of ppm in MIC, the more effective the antimicrobial activity against acne bacteria. In Formulation Examples 21-30, the concentration of ppm was significantly lower than that of Comparative Formulation Example 4 using erythromycin, a known acne treatment. The composition containing saponin derived from the roots of the tea tree can be confirmed to have a much better antimicrobial activity against the test bacteria.

Test Example 13 Lipogenesis Inhibition Test

3T3-L1 cells, a mouse fibroblast cell line, were loaded with DMEM (Dulbecos modified eagles medium, GIBCO BRL, Life Technologes) medium containing 10% fetal bovine serum (FBS). The well culture plate was attached at 1 × 10 ⁵ cells / well. After 2 days, it was again exchanged with fresh DMEM (containing 10% FBS) medium and incubated for 2 days. The cultured cells were then induced to differentiate with DMEM (containing 10% FBS) containing 1 μg / ml insulin, 0.5 mM IBMX, and 0.25 μM dexamethasone (containing 10% FBS), and the saponins and caffeine derived from the roots of tea After 2 days of treatment 50μM was exchanged for DMEM containing insulin and incubated for 5 days. After 5 days, the cells were exchanged with normal medium (DMEM, containing 10% FBS) and cultured while observing until the cells changed to fat cells.

Sudan III staining (S4136, sigma-aldrich) was performed using the differentiated 3T3-L1 adipocytes in order to evaluate the inhibitory effect of the saponin-derived saponin in the adipocyte fat accumulation. Adipose cells were fixed at room temperature with 4% paraformaldehyde (pH 7.2) in phosphate buffer, washed with PBS (phsphate buffered saline), stained with Sudan III, and photographed for visual comparison. As a control group, only the medium without the test substance or the comparative substance was used, and 50 μM of caffeine was treated with another control group. The degree of fat accumulation inhibition was graded by dividing the degree of staining into +++, ++, +,-. The results are shown in Table 18.

sample % Inhibition Control group +++ Comparison + Saponin R1 from tea tree root + Tea tree root-derived saponin R2 + Tea tree root-derived saponin R3 ++ Tea tree root-derived saponin R4 ++ Tea tree root-derived saponin R5 +++ Tea tree root-derived saponin R6 + Saponin R7 derived from tea tree root + Tea tree root-derived saponin R8 ++ Saponin R9 from tea tree root ++ Saponin R10 from tea tree root ++

As shown in Table 18, it can be seen that the saponin derived from the root of the tea tree used in the present invention has a lipid synthesis inhibitory effect equivalent to or better than caffeine, a known lipid synthesis inhibitor. Therefore, lipid synthesis is inhibited, and sebum is reduced, so that occurrence of acne can be suppressed.

[Test Example 14] Test for acne improvement and sebum secretion and irritation

The 120 people having acne were divided into 12 groups of 10 people, and subjects corresponding to each group were allowed to use the cosmetic composition prepared from Formulation Examples 21 to 30 and Comparative Formulations 3 to 4 for one month. The acne improvement scale ranged from 1 to 5 points, with 1 being 'no', 3 being 'normal' and 5 being 'very so'. Experimental results are shown in the average score of 10 people in Table 19 below.

Acne disappearance was based on the number of days the extinction was read, acne recurrence was based on the results after one month. Sebum secretion is reduced from 1 to 5 points, 1 point is 'No', 3 points are 'Normal' and 5 points are 'Yes.' Experimental results are shown in the average score of 10 people in Table 19 below. The presence or absence of skin irritation was determined as (number of irritants) / (total number of test subjects).

Inflammatory Acne Improvement Cotton acne extinction Acne recurrence Reduce sebum secretion Irritation Formulation Example 21 3.5 4 days radish 3.8 1/10 Formulation Example 22 4.1 5 days radish 3.5 0/10 Formulation Example 23 3.9 4 days radish 3.1 5/10 Formulation Example 24 3.1 4 days radish 3.5 4/10 Formulation Example 25 2.5 7 days radish 2.5 3/10 Formulation Example 26 3.8 4 days radish 3.8 1/10 Formulation Example 27 4 6 days radish 4.1 2/10 Formulation Example 28 3.2 4 days radish 3.8 8/10 Formulation Example 29 3.5 5 days radish 3.5 7/10 Formulation Example 30 3.5 5 days radish 3.8 5/10 Comparative Formulation Example 3 2.1 13th U 2.0 0/10 Comparative Formulation Example 4 4.2 2 days radish 4.1 9/10

As shown in Table 19, Formulation Examples 21 to 30 did not recur acne compared to Comparative Formulation Example 3, it can be seen that there is an excellent effect on the overall acne improvement. On the other hand, Comparative Formulation Example 4 containing an antimicrobial activity standard, although showing an acne-improving effect, there is a risk of skin irritation for long-term use due to the strong irritation in use, the composition according to the present invention is not irritating appear.

Test Example 15 Inflammation Improvement Effect

The anti-inflammatory effect was evaluated by the inhibitory effect of the production of prostaglandins. The effect was measured on macrophages using saponin derived from the root of the tea tree. First, aspirin was added to macrophages taken from the abdominal cavity of mice to a final concentration of 500 M, thereby irreversibly inhibiting cyclooxygenase (COX) activity remaining in the cells. Then, 100 μl of the suspension was added to each well of the 96 well cell culture tube and incubated for 2 hours in an incubator at 37 ° C. with 5% CO ₂ to attach macrophages to the container surface. Subsequently, the attached macrophages were washed three times with PBS and then used for the inflammation improvement effect test. The cultured macrophages 5x104 cells / ml by adding RPMI medium containing 1% (w / v) of LPS incubated for 12 hours to induce the production of prostaglandins and 100 μl of saponin derived from the roots of the tea tree, free prostaglandin Was quantified using an enzyme immunoassay (ELISA).

At this time, the production inhibitory activity of the prostaglandins of the saponin derived from the roots of the tea set the difference between the prostaglandins produced in the LPS-treated and untreated groups to 100%, and treated the LPS and the sample together to reduce the percentage of prostaglandins reduced. Compared with the control group, and determined, the result (prostaglandin production inhibitory effect) is shown in Table 20 below.

Blank 100% Control group (Aspirin treated group) 25.0% Saponin R1 from tea tree root 30.0% Tea tree root-derived saponin R2 53.4% Tea tree root-derived saponin R3 38.1% Tea tree root-derived saponin R4 40.1% Tea tree root-derived saponin R5 38.1% Tea tree root-derived saponin R6 39.4% Saponin R7 derived from tea tree root 65.1% Tea tree root-derived saponin R8 67.1% Saponin R9 from tea tree root 89.1% Saponin R10 from tea tree root 68.1%

As shown in Table 20, the experimental results show that even when treated with saponin derived from the roots of the tea tree as a control group treated with aspirin, the production inhibitory effect of prostaglandins is very high.

It can be seen that the saponin derived from the root of the tea tree of the present invention can provide an excellent inflammation improving effect.

In addition, it can be seen that the saponin derived from the root of tea tree can prevent and improve skin trouble by inhibiting the expression of prostaglandin, a skin inflammation factor.

 Test Example 17 Whitening Effect

<Tyrosinase inhibitory effect>

Tyrosinase enzyme was extracted from the mushroom (Mushroom) was used as the Sigma (SIGMA). First, the substrate tyrosine was dissolved in distilled water to make a solution of 0.3 mg / ml, and the solution was added to the test tube by 1.0 ml. Then, 1.0 ml of potassium-phosphate buffer solution (0.1 mol concentration, pH 6.8) and 0.7 ml of distilled water were added thereto. Added.

0.2 ml of each sample solution prepared by mixing tea root-derived saponin with an appropriate concentration in ethanol solution was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. In this case, the control group was prepared by adding only 0.2 ml of solvent instead of each sample solution, and ascorbic acid was used as a positive control group. 0.1 ml of a tyrosinase solution of 2500 units / ml was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. The reaction tube containing the reaction solution was placed in iced water, quenched to stop the reaction, and the absorbance at 475 nm was measured with a photospectrometer. The results are shown in Table 21 below. Each tyrosinase inhibitory effect was calculated by the following equation.

Test substance Tyrosinase inhibition rate (%) Control group (no addition) 0 Ascorbic acid 52 Saponin R1 from tea tree root 58.1 Tea tree root-derived saponin R2 60 Tea tree root-derived saponin R3 68.1 Tea tree root-derived saponin R4 65 Tea tree root-derived saponin R5 58.1 Tea tree root-derived saponin R6 55.1 Saponin R7 derived from tea tree root 30.1 Tea tree root-derived saponin R8 32 Saponin R9 from tea tree root 15 Saponin R10 from tea tree root 53.4

In the results of Table 21, it was found that the tyrosinase inhibition rate of the cosmetic composition of Formula 1 to 10 containing saponin derived from the root of the present invention is much higher than the known tyrosinase inhibitor ascorbic acid, which is very excellent in the whitening effect.

Inhibitory Effect of Melanin Production Using B16 / F10 Melanoma Cells

Formulation Examples 1-10, Comparative Formulation Example 1 and a sample containing 0.001% by weight of kojic acid, respectively, were added as a test substance to the culture medium of B16 / F10 melanoma cells (Korea Cell Line Bank) at a constant concentration for 3 days. After the culture was removed, washed with PBS and dissolved in 1N NaOH cells and the absorbance was measured at 405 nm. The cells without test substance were used as a control group, and the degree of inhibition of melanin production of each test substance was measured by comparing with the melanin content of the control group. According to Equation 6, the melanin production inhibition rate is calculated and the results are shown in Table 22.

Test substance Melanogenesis inhibition rate (%) Control group (no addition) 0 Kojic acid 53 Saponin R1 from tea tree root 62.1 Tea tree root-derived saponin R2 63 Tea tree root-derived saponin R3 51 Tea tree root-derived saponin R4 45 Tea tree root-derived saponin R5 62 Tea tree root-derived saponin R6 65 Saponin R7 derived from tea tree root 70 Tea tree root-derived saponin R8 35 Saponin R9 from tea tree root 65 Saponin R10 from tea tree root 55

From the results in Table 22, it can be seen that the melanin production inhibition rate of the cosmetic composition of the formulation examples 1 to 10 containing tea tree root-derived saponin according to the present invention is much higher than kojic acid, a known melanin production inhibitor, and the whitening effect is very excellent. there was.

Test Example 17 Effect of Atopic Symptoms Improvement

<Itching relief assessment>

One day prior to the experiment, keratinocytes (cell name: HaCaT obtained from ATCC) were dispensed into 96 well plates at ⁴ × 10 ⁴ cells / well, followed by 24 hours in a 37 ° C., 5% CO ₂ incubator. Incubated. After 24 hours, wash 96 well plates twice with Hanks' Balanced Salt solution (HBSS) buffer, and then add reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM probenecid) to the cells. gave. After reaction at 37 ° C., 5% CO ₂ incubator for 30 minutes, and at room temperature for 30 minutes, the cells were washed twice with HBSS buffer and treated with 0.1% saponin derived from the root of the tea tree. At this time, a medium which was not treated with the test substance was used as a control.

After reacting for 10 minutes, 2U / ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) were treated and the change in the Ca ²⁺ concentration in the cells was measured for 80 seconds. Intracellular Ca ²⁺ concentration change was measured using FlexStation3 (Molecular Device, USA). Determine the difference between the minimum and maximum values obtained by measuring flex for 80 seconds after treatment with 2U / ml Trypsin or 5μM PAR-2 active peptide (SLIGKV), and then determine the value between 2U / ml trypsin or Inhibition rate (%) of intracellular influx of calcium ions compared to the difference between the minimum value and the maximum value when treated with 5 μM PAR-2 active peptide (SLIGKV) is shown in Table 23 below.

division % Inhibition of intracellular influx of calcium ions Trypsin
(2U / ml) PAR-2 active peptide
(5 [mu] M) Untreated control 10 15 Saponin R1 from tea tree root 21 25 Tea tree root-derived saponin R2 18 19 Tea tree root-derived saponin R3 20 19 Tea tree root-derived saponin R4 11 28 Tea tree root-derived saponin R5 11 15 Tea tree root-derived saponin R6 19 20 Saponin R7 derived from tea tree root 25 21 Tea tree root-derived saponin R8 24 26 Saponin R9 from tea tree root 15 25 Saponin R10 from tea tree root 20 30

As can be seen from Table 23, it was confirmed that the influx of intracellular calcium ions by trypsin or PAR-2 active peptide (SLIGKV) was reduced when the saponin derived from the root of tea tree was treated.

Therefore, the external preparation composition for skin of the present invention can provide an excellent antipruritic effect by effectively inhibiting PAR-2 activity causing itching.

Anti-inflammatory efficacy evaluation

One day prior to the experiment, normal human skin keratinocytes (NHEK, obtained from Lonza) were dispensed in 96 well plates at 5x10 ⁴ cell number / well, followed by 37 ° C, 5% CO ₂ incubator. Incubated for 24 hours at. After 24 hours, the cells were washed twice with PBS and changed to serum-free keratinocyte basement media (KBM). Each well was treated with 30 ppm of saponin derived from the root of the tea tree for 30 minutes, and then PGSA (10 µg / ml), PGSA (50 µg / ml) and PGSA (50 µg / ml) + LPS (1 µg / ml) were added. Each was treated. At this time, a medium not treated with the test substance was used as a control. Here, PGSA (peptidoglycan from S. aureus ) is a peptidoglycan (peptidoglycan) extracted from staphylococcus aureus, a major component of the Gram-positive (+) cell wall and bacterial membrane components are known to cause inflammation, In particular, in staphylococci, about 90% of patients with atopic dermatitis have been reported to cause secondary infection. Lypopolysaccaride (LPS) is a major component of the cell membrane of Gram-negative bacteria and is known to be a major cause of inflammation.

After incubation for 24 hours at 37 ℃, 5% CO ₂ incubator, the culture medium was taken and subjected to ELISA for Interleukin-8 (IL-8), the results are shown in Table 24 below. ELISA used the experimental method of the manufacturer (BD science).

division IL-8 secretion (pg / ml) Untreated Control 935.12 PGSA (10 μg / ml) 4812.60 PGSA (50 μg / ml) 5895.08 PGSA (50 μg / ml) + LPS (1 μg / ml) 6814.91 Saponin R1 from tea tree root 6125.8 Tea tree root-derived saponin R2 5955.4 Tea tree root-derived saponin R3 6754.1 Tea tree root-derived saponin R4 6814.5 Tea tree root-derived saponin R5 5988.1 Tea tree root-derived saponin R6 6125.3 Saponin R7 derived from tea tree root 6345.1 Tea tree root-derived saponin R8 6855.1 Saponin R9 from tea tree root 6233.1 Saponin R10 from tea tree root 6533.4

In Table 24, it was confirmed that the saponin derived from the tea tree root significantly reduced and inhibited the secretion of IL-8 increased by PGSA and LPS.

Therefore, the topical skin composition of the present invention can provide an excellent anti-inflammatory effect by significantly reducing the secretion of IL-8 increased by PGSA and LPS.

Claims (20)

Skin external composition comprising saponin derived from the root of the tea tree as an active ingredient. The external preparation composition for skin according to claim 1, wherein the saponin is triterpenoid saponin. According to claim 1 or claim 2, wherein the saponin is an external composition for skin, characterized in that of the formula 1 to 10.
[Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Formula 10]

The composition for external application for skin according to claim 1 or 2, wherein the saponin is contained in an amount of 0.1 to 50% by weight based on the total weight of the composition. The composition for external application for skin according to claim 1 or 2, wherein the composition is for anti-aging. The composition for applying the external of the skin according to claim 1 or 2, wherein the composition is for promoting skin elasticity. The composition for applying the external of the skin according to claim 1 or 2, wherein the composition is for improving skin wrinkles. The composition for external application for skin according to claim 1 or 2, wherein the composition is for moisturizing. The composition for external application for skin according to claim 1 or 2, wherein the composition is for enhancing skin barrier function. The composition for applying the external of the skin according to claim 1 or 2, wherein the composition is for inducing differentiation of keratinocytes of the skin. The composition for external application for skin according to claim 1 or 2, wherein the composition is for improving acne. The composition for external application of skin according to claim 1 or 2, wherein the composition is for antibacterial use. The composition for external application of skin according to claim 1, wherein the composition is for anti-inflammatory. The composition for external application for skin according to claim 1 or 2, wherein the composition is for improving atopic skin. The external preparation composition for skin according to claim 1 or 2, wherein the composition is for improving color and skin tone. The external preparation composition for skin according to claim 1 or 2, wherein the composition is for shrinking pores. The composition for external application for skin according to claim 1 or 2, wherein the composition is for controlling sebum. The external preparation composition for skin according to claim 1 or 2, wherein the composition is for improving skin troubles. The composition for applying the external of the skin according to claim 1 or 2, wherein the composition is for protecting against skin irritation. The composition for external application for skin according to claim 1 or 2, wherein the composition is for whitening.