KR101560754B1 - Extracts from sorghum and sorghum by-product including polyphenol compounds and their manufacturing method and cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to an extract containing a polyphenolic compound derived from a water-soluble and a by-product, a method for preparing the same, and a cosmetic composition containing the same as an active ingredient. More particularly, the present invention relates to a cosmetic composition comprising a tyrosinase And polyphenol compounds showing strong inhibitory activity against elastase which is related to wrinkle improvement, it can be used as a source of functional materials, and as a new new material crop, it can be used as a new crop, And a method for producing the same and a cosmetic composition containing the polyphenol compound as an active ingredient.

Description

[0001] The present invention relates to an extract containing a polyphenolic compound derived from a water-soluble and anhydride-derived polyphenolic compound, a process for producing the extract, and a cosmetic composition containing the same as an active ingredient.

The present invention relates to an extract of tyrosinase which is related to whitening of skin and an extract of polyphenol compound which shows strong inhibitory activity to elastase which is related to wrinkle improvement from aquatic extracts and which can be applied to cosmetics and functional foods, And a process for producing the extract and a cosmetic composition containing the polyphenol compound as an active ingredient.

Sorghum (Sorghum, Sorghum bicolor L. Moench is a perennial plant of the alopecia plantus and is one of the major grains following rice, barley, wheat and corn, and is a major grain food crop in many parts of the world. And in Asia and Africa as food resources and folk remedies (Ryu et al., 2006, The Korean Journal of Food and Nutrition, 19, 176182). Sorghum is rich in secondary metabolites such as tannins, phenols, anthocyanins, plant sterols, and policosanols, which are known to significantly affect human health (Awika & Rooney, 2004, Phytochemistry, 65, 1199-1221) . Recent studies have shown that antimicrobial activity of sorghum (Choi et al., 2006, Food Chemistry, 103, 130-138), anticancer effect (Kwak et al., 2004, Journal of Korean Society of Food Science Nutrition, 33, 921-929) , And cholesterol lowering effects (Ha et al., 1998, Korean Journal of Food Science and Technology, 30, 224-229) and reported that it could reduce cardiovascular disease (Cho et al., 2000, Journal of the Federation of American Societies for Experimental Biology, 14, A249). Furthermore, sorghum has DPPH radical scavenging activity and has been reported to have an antimutagenic effect. (Kwak et al., 2004, Journal of Korean Society of Food Science Nutrition, 33, 921-929). HMG-CoA reductase inhibitory activity was detected in the methanol extract of Acer. (Ha et al., 1998, Korean Journal of Food Science and Technology, 30, 224-229). However, there is almost no information on the whitening and wrinkle-reducing effect of millet.

On the other hand, exposure to external environment and natural aging do not adequately defend the damage caused by oxidative stress due to the role of antioxidant system in the human body, and skin becomes aged due to the production of reactive oxygen species. Among them, facial skin is directly exposed to ultraviolet rays, dry outside air, etc., so that aging phenomenon such as wrinkles occurs more easily than non-skin. In particular, the main mechanism involved in the generation of skin wrinkles is that the skin is continuously exposed to oxidative stress from harmful environments such as air pollution, ultraviolet ray exposure, stress or disease, and the radicals in the body are increased, collagen, elastin, hyaluronic acid and the like may be destroyed to cause settlement (wrinkles) on certain parts of the skin. In addition, these mechanisms oxidize the lipid part of the cell membrane and cause cell destruction, which can lead to diseases such as dermatitis, acne or skin cancer. In addition, radicals are involved in the melanin formation process, which can cause stains, freckles and wrinkles.

Studies to develop antioxidant and anti-aging materials have been actively conducted in an effort to prevent or improve such skin aging. The antioxidants used to prevent free oxygen and free radical formation have been previously known as ascorbic acid, a-tocopherol, carotenoid, flavonoid, bytylated bydroxy toluene (BHT), butylated hydroxy anisole (BHA) ), And are used as general materials for cosmetics. The development of natural antioxidants that are stable in all fields such as pharmaceuticals in the development of materials can be said to be a dream of industry. BHA or BHT, which is known to have the most antioxidative effects among antioxidants, has been widely used because of its effectiveness, economy and stability. However, mutagenicity and toxicity are pointed out as well as general avoidance phenomenon of synthesis. Recently, many attempts have been made to develop a natural antioxidant having no strong safety and sensory properties and having a strong activity.

Conventional cosmetic compositions using natural antioxidants are disclosed in Korean Patent Laid-Open No. 2011-0114369, which has a high total polyphenol content and has excellent antioxidative and tyrosinase activity inhibitory effects, By weight based on the total weight of the cosmetic composition.

Korean Patent No. 10-534074 also relates to a composition comprising a grain extract containing black beans, black beans, white beans, red beans, and black sesame seeds and an extract of brown rice or rhizome germinated mushroom, wherein the composition inhibits tyrosinase activity, A cosmetic composition showing an antioxidative effect on the skin and showing a remarkably improved skin-improving effect has been proposed.

However, although these methods are antioxidants using natural substances, there is a somewhat insufficient effect on the elastase inhibitory activity, and there is a disadvantage in that the structural analysis on the polyphenol compounds separated from the natural product extract is inferior.

Functional cosmetics are cosmetics with pharmacological effects added to general cosmetics. They can be broadly divided into whitening products, wrinkle-improving products, and ultraviolet protection products. The whitening products are known to have effects such as inhibition of melanin production and inhibition of tyrosinase, skin exfoliation, skin pigmentation prevention, and wrinkle improvement products are known to enhance skin elasticity, promote collagen synthesis, promote epidermal metabolism, The effect of promoting the production and the like is known.

However, the efficacy and side effects of the wrinkle remedies, which have been developed to date, have not been thoroughly verified and are relatively expensive. For this reason, functional cosmetics using natural products capable of replacing harmful chemical components are attracting attention, and in particular, there is a need for studies on natural antioxidants associated with inhibition of skin aging without side effects.

In order to solve these problems, the inventors of the present invention have made efforts to find a skin whitening agent and a skin aging inhibitor derived from millet and sorghum by-products.

Accordingly, it is an object of the present invention to provide a fraction of a water-extract, which is separated from millet, a by-product or a mixture thereof, which exhibits skin whitening and wrinkle-reducing effects, and a method for producing the same.

It is another object of the present invention to provide a functional cosmetic composition for skin whitening which comprises the fraction of the extract of extract as an active ingredient.

It is another object of the present invention to provide a functional cosmetic composition for improving skin wrinkles containing the fraction of the extract of extract as an active ingredient.

The present invention provides fractions of aquatic extract comprising at least one polyphenol compound selected from the group consisting of ruthenorhindin represented by the following general formula (3) and hesperidin compounds represented by the following general formula (4) separated from the extract.
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[Chemical Formula 4]

The present invention also provides a functional cosmetic composition for skin whitening comprising the fraction of the extract of extract as an active ingredient.

The present invention also provides a functional cosmetic composition for improving skin wrinkles containing the fraction of the extract of extract as an active ingredient.

Further, the present invention relates to a method for producing a compound derived from an extract of Suwo.

(I) drying, pulverizing the mill, the by-product or a mixture thereof to obtain a powder;

(Ii) extracting the powder with methanol to obtain a water extract;

(Iii) suspending the water extract in water and sequentially fractionating the mixture with n-hexane, chloroform and butanol to obtain a butanol fraction; And

(Iv) eluting the butanol fraction to obtain each compound represented by the above formula (3) or (4);

(3) or (4), wherein R < 2 >

According to the present invention, it is possible to isolate polyphenolic compounds exhibiting strong inhibitory activity on elastase associated with skin whitening-related tyrosinase and wrinkle-improving, and to establish mass separation conditions of major components And it can be used as a source of functional materials and can improve the usability of new plants as new crops.

Also, it can be utilized as an expensive functional cosmetic or pharmaceutical raw material by using a separated high purity polyphenolic compound.

1 is a TLC developed photograph of the active single component prepared in Example 1 according to the present invention.
2A is a ¹ H, ¹³ C-NMR spectrum of 4 ', 5,7-Tedla hydroxyflavone (epigenin) prepared in Example 1 according to the present invention.
2B is a ¹ H, ¹³ C-NMR spectrum of 4 ', 5,7-trihydroxyflavinium (epigeninidine) prepared in Example 1 according to the present invention.
2C is a ¹ H, ¹³ C-NMR spectrum of 3 ', 4', 5,7-tetrahydroxyflavinium (ruthelinidine) prepared in Example 1 according to the present invention.
FIG. 2d is a ¹ H, ¹³ C-NMR spectrum of the hesperetin 7-rhamnoglucoside (hesperidin) prepared in Example 1 according to the present invention.
3 is a graph showing the tyrosinase inhibitory activity of the compound prepared in Example 1 according to the present invention.
FIG. 4 is a graph showing the effect of the compound of Example 1 according to the present invention on inhibiting elastase activity.

Hereinafter, the present invention will be described in more detail as an embodiment.

The present invention is characterized by a fraction of a water extract comprising at least one polyphenol compound selected from the group consisting of ruthenorinine represented by the following formula (3) and hesperidin compound represented by the following formula (4) separated from the extract.

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[Chemical Formula 4]

According to a preferred embodiment of the present invention, the water extract can be obtained by extracting Sorghum biclor (L.) Moench and a by-product with an organic solvent. The organic solvent may be at least one selected from the group consisting of methanol, alcohol, hexane, ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, diethyl ether and butanol. . Most preferably, when 70% by weight or more of methanol or methanol aqueous solution is used, excellent skin whitening and wrinkle-reducing effects can be exhibited by optimum extraction of the polyphenol compound.

According to a preferred embodiment of the present invention, the fraction is a butanol fraction obtained by fractionating a water extract with butanol, and the water extract is preferably fractionated by adding n-hexane, chloroform and butanol in this order. At this time, the butanol fraction is preferably eluted from a silica gel column.

The present invention also features a functional cosmetic composition for skin whitening comprising the fraction of the extract of extract as an active ingredient.

According to a preferred embodiment of the present invention, the fraction exhibits a strong inhibitory activity against tyrosinase involved in skin whitening, thereby inhibiting melanin synthesis.

Further, the present invention is characterized by a functional cosmetic composition for improving skin wrinkles containing the fraction of the extract of extract as an active ingredient.

According to a preferred embodiment of the present invention, the fraction exhibits a strong inhibitory activity against elastase which is involved in skin aging, so that decomposition of elastin can be suppressed.

The present invention also relates to a process for producing (i) a process for producing a powder by drying and pulverizing a mill, a by-product or a mixture thereof;

(Ii) extracting the powder with methanol to obtain a water extract;

(Iii) suspending the water extract in water and sequentially fractionating the mixture with n-hexane, chloroform and butanol to obtain a butanol fraction; And

(Iv) eluting the butanol fraction to obtain each compound represented by the above formula (3) or (4);

(3) or (4).

Hereinafter, the present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Extraction, Separation and Purification of Polyphenolic Compounds from Syringes, By-Products or Their Mixture Extracts

The harvesting and harvesting by-products collected in 2010 were pulverized to 100 mesh using a pulverizer, and 1 kg of the millet and the by-product powder obtained from the mill was put into an extraction vessel and washed with 80% aqueous methanol solution (3 L) do. The extracted sample was filtered through a filter paper, and then concentrated under reduced pressure to obtain a cyan-blue extract (325 g).

To the suspension, 1 L of purified water was added, and the mixture was suspended and fractionated in the order of n-hexane, ethyl acetate and butanol to obtain 32 g of n-hexane fraction, 85 g of ethyl acetate fraction, 94 g of butanol fraction and 78 g of water fraction . From the butanol fraction (94 g), a mixed solvent of chloroform: methanol (45: 1 to 1:10 mixture ratio) and methanol were sequentially used as a mobile phase solvent using silica gel column chromatography (600 g, 70 to 230 mesh, Merk) To obtain five fractions (Fr.B1 to Fr.B5).

Fr. B3 fraction (5.6 g) was adsorbed onto silica gel (10 g, 70-230 mesh) and silica gel column chromatography (100 g, 230-400 mesh) was carried out. The mobile phase solvent was chloroform: methanol To obtain a pale yellow precipitate, which was purified to obtain Formula 1 (68 mg). In addition, fractions of Fr.B4 (3.9 g) were adsorbed on silica gel (8 g, 70-230 mesh) and then subjected to silica gel column chromatography (80 g, 230-400 mesh) 40: 1 to 1:10 mixing ratio). A pale yellow precipitate was obtained by using a mixed solvent. The resulting precipitate was purified to obtain Formulas 2 (45 mg) and 3 (52 mg). In addition, silica gel column chromatography (150 g, 230-400 mesh) was performed after adsorbing Fr.B5 (8.5 g) on silica gel (15 g, 70-230 mesh), and mobile phase solvent was chloroform: methanol (30: 1 to 1:20 mixture ratio) to obtain a pale yellow precipitate, which was purified to obtain Formula 4 (36 mg).

Silica gel thin layer chromatography (TLC) of the separated polyphenol compounds was performed, and the TLC pattern corresponding thereto was shown in FIG.

Example 2: Structural analysis of polyphenolic compounds separated from extracts of millet, by-products or mixtures thereof

The molecular weight and molecular formula of the compound obtained in Example 1 were determined by HPLC-MS (mass spectrometry). The structure of the compound was determined by nuclear magnetic resonance analysis (Bruker AMX 500) by ¹ H NMR , it was done by analyzing the ¹³ C-NMR, homo koji (HOMO-COSY), HMQC ( 1 H-Detected heteronuclear Multiple-Quantum Coherence), HMBC (Heteronuclear Multiple-Bond Coherence), DEPT (Distortionless Enhancement by Polarization) spectrum.

(4 ', 5,7-trihydroxyflavone; epigenin), 4', 5,7-trihydroxyflavone (4 ', 5,7- , 5,7-Trihydroxyflavylium; epigeninide), Formula 3 is 3 ', 4', 5,7-tetrahydroxyflavylium (ruthenoridine ), And chemical formula 4 was identified as hesperetin 7-rhamnoglucoside (hesperidin).

The structures of the separated polyphenol compounds are shown in Table 1 below, and the structural characteristics of the respective compounds are as follows.

compound rescue Material name One

4 ', 5,7-trihydroxyflavone: Epigenin 2

4 ', 5,7-trihydroxyflavinium: epigenidin 3

3 ', 4', 5,7-tetrahydroxyflavinium: ruthelinidine 4

Hesperetin 7-rhamnoglucoside: hesperidin

[Chemical Formula 1] 4 ', 5,7-trihydroxyflavone (epigenin)

1) Properties: Light yellow amorphous crystals

2) Molecular weight: 270.24

3) Molecular formula: C ₁₅ H ₁₀ O ₅

^{4) 1 H-NMR (500MHz} , Methanol- d 4) δ 7.87 (2H, d, J = 14.9 Hz), 6.94 (2H, d, J = 14.9 Hz), 6.62 (1H, s), 6.47 (1H, d, J = 3.5 Hz), 6.23 (1H, d, J = 3.5 Hz)

5) ¹³ C-NMR (125 MHz, methanol- d ₄ )? 184.2, 166.8, 166.6, 163.6, 159.9, 151.4, 147.5, 124.1, 120.7, 117.2, 114.6, 105.5, 104.3, 100.6, 95.4,

4 ', 5,7-trihydroxyflavinium (epigenidin)

1) Properties: Light yellow amorphous crystals

2) Molecular weight: 255.24

3) Molecular formula: C ₁₅ H ₁₁ O ₄

^{4) 1 H-NMR (500MHz} , Methanol- d 4) δ 9.09 (1H, d, J = 14.5 Hz), 8.30 (2H, d, J = 14.9 Hz), 8.05 (1H, d, J = 14.5 Hz) , 7.08 (1H, d, J = 14.9 Hz), 6.95 (1H, d, J = 1.2 Hz), 6.66 (1H, d, J = 1.2 Hz)

^{5) 13 C-NMR (125MHz} , Methanol- d 4) δ 173.2, 172.8, 167.7, 160.9, 160.4, 149.9, 133.6, 133.6, 121.7, 118.8, 118.8, 114.2, 100.8, 103.6, 96.5

3 ', 4', 5,7-tetrahydroxyflavinium (ruthelinidine)

1) Properties: Yellow amorphous crystals

2) Molecular weight: 271.24

3) Molecular formula: C ₁₅ H ₁₁ O ₅

^{4) 1 H-NMR (500MHz} , Methanol- d 4) δ 9.05 (1H, d, J = 14.3 Hz), 8.00 (1H, d, J = 14.6 Hz), 7.89 (1H, d, J = 14.0 Hz) , 7.75 (1H, s), 7.04 (1H, d, J = 14.1 Hz), 6.92

^{5) 13 C-NMR (125MHz} , Methanol- d 4) δ 173.2, 172.5, 160.9, 160.4, 156.8, 149.5, 148.7, 125.7, 122.2, 118.2, 116.4, 114.0, 111.1, 103.6, 96.4

Hesperetin 7-rhamoglucoside (hesperidin)

1) Properties: Light yellow amorphous crystals

2) Molecular weight: 612.53

3) Molecular formula: C ₂₇ H ₃₂ O ₁₆

^{4) 1 H-NMR (500MHz} , DMSO- d 6) δ 6.93 (2H, m), 6.13 (2H, m), 5.50 (1H, m), 5.39 (1H, d, J = 8.0 Hz), 5.18 ( 2H, dd, J = 7.2, 7.8 Hz), 4.99 (1H, dd, J = 7.5, 11.9 Hz), 4.69 (1H, dd, J = 5.8, 9.1 Hz), 4.62 (1H, t, J = 9.1 Hz ), 4.50 (2H, m), 3.13-3.45 (7H, O-rhamnoglucoside)

^{5) 13 C-NMR (125MHz} , DMSO- d 6) δ 197.5, 165.3, 163.5, 162.9, 148.4, 146.9, 131.4, 118.4, 114.6, 112.5, 103.8, 101.1, 99.8, 96.9, 95.8, 78.1, 76.7, 76.0 , 73.5, 72.5, 71.2, 70.7, 70.1, 68.8, 66.1, 56.2, 49.0

3 ', 4', 5,7-tetrahydroxyflavinium (ruthelinidine) and 3 kinds of functional compounds of formula (3), which are the most effective for skin whitening and wrinkle improvement, And < 13 > C-NMR spectra are shown in Fig.

Example 3 Effect of Mushroom Tyrosinase Inhibitory Activity of Polyphenolic Compounds Isolated from Syringes, By-Products or Mixture Extracts thereof

Tyrosinase is an enzyme that regulates skin melanin synthesis and can regulate melanin production in skin cells when inhibited. The enzyme is a tyrosine hydrolyzing enzyme (hyroxylase) which oxidizes tyrosine as a substrate in the melanosome of skin cells to produce L-3,4-dihydroxy-phenylalanine (DOPA) DOPAchrome (DOPA oxidase), which has two activities, which are biosynthesized to melanin polymer. Experiments were conducted using mushroom-derived tyrosinase to investigate the inhibitory activity of tyrosinase inhibitory activity of the polyphenol compounds of Example 2 and the extracts of the water-soluble, by-products or mixture thereof of Example 1. Was dissolved in phosphate buffer (0.1 M, pH 6.5) at a concentration of 1.0 unit / ml in a 96-well plate and quantified as 100 μl. Next, tyrosine, a substrate, was dissolved in phosphate buffer to a concentration of 0.05 M to quantitate 100 ㎕. The 70% ethanol extract and the polyphenol compound as a pure water-soluble compound were dissolved in DMSO And the reaction was carried out at 37 ° C for 20 minutes. The amount of melanin produced was measured at 475 nm, and the inhibitory activity of thyrosinase inhibitory activity was determined by the following equation (1) 3 or Table 2, together with the water extract of the compound of formula (I), or a mixture of water extract thereof and the 50% inhibitory concentration (IC50) of the purely isolated compound of Example 2 against tyrosinase.

Compound No. rescue Tyrosinase inhibitory activity
(IC ₅₀ = mM) - Sorghum extract 155.38 ppm - By-product extract 157.13 ppm One

40.15 2

12.05 3

8.18 4

72.63

As shown in the above Table 2, the 50% inhibitory concentrations of the extracts, the by-products, or the mixture thereof of Example 1 according to the present invention were 155.38 and 157.13 ppm, respectively, and the polyphenol compounds of Example 2 were 40.15, 12.05, 8.18, and 72.63 mM, respectively. Thus, tyrosinase inhibitory activity is excellent and melanin synthesis can be inhibited.

A: Absorbance value at 475 nm of the inhibitor added

B: Absorbance value at 475 nm of no added inhibitor

Example 4 Elastase Inhibitory Activity of Polyphenolic Compounds Isolated from Extracts of Wheat, By-Products or Their Mixtures

In skin aging, especially wrinkle formation, action by active oxygen and destruction of extracellular matrix by MMPs (such as collagenase and elastase) is considered to be the main cause. Thus, the inhibitory activity of elastase, which is one of the major MMPs, was evaluated for the extract, the by-product or mixture of extracts of Example 1 and the polyphenol compound of Example 2. As a method of measuring the activity of elastase, an enzyme that degrades elastin, NSuccinyl-ala-ala-ala-ρ-nitroaniline (Sigma), an elastase substrate, was used to measure the activity of ρ-nitroaniline The change is a method of measuring the elastase activity by measuring the absorbance at a wavelength of 405 nm.

To 40 μL of a buffer solution in which 4.0 mM of N-succinyl- (Ala) 3-p-nitroanilide was dissolved in 0.2 M of Tris-Cl (pH 8.0) as an elastase substrate, 20 μL of a solution of the following test substance in ethanol, And then incubated at 37 ° C for 10 minutes. 10 μl of the elastase solution (final concentration: 0.0025 U / ml) was added thereto, followed by incubation at 37 ° C for 10 minutes. Then, the absorbance at 405 nm Were measured. In the control group, 10 μL of the solvent used as the sample solution was added instead of the water extract. A blank was prepared by adding 40 μL of 0.2 M Tris-Cl buffer instead of the buffer solution in which N-succinyl- (Ala) 3-p-nitroanilide was dissolved, and the concentration was the same as in the experimental group. The inhibition rate of the elastase inhibitory activity was calculated by the following formula (2). The inhibitory activity of the inhibitory activity of the elastase was determined by the following formula: (IC ₅₀ ) values are shown in FIG. 4 and Table 3 below.

Compound No. rescue Elastase inhibitory activity
(IC ₅₀ = mM) - Sorghum extract 144.85 ppm - By-product extract > 200 ppm One

> 200 2

21.17 3

6.17 4

> 200

As shown in Table 3, the 50% inhibitory concentration of the water extract of Example 1 according to the present invention was 144.85 ppm, and in the polyphenol compounds of Example 2, 21.17 and 6.17 mM By exhibiting a 50% inhibition concentration value, it is possible to exhibit an effect of improving wrinkles due to excellent elastase inhibitory activity.

A: absorbance value at 405 nm of the inhibitor added

B: absorbance value at 405 nm of no addition of inhibitor

Claims (12)

delete delete delete delete delete delete delete A skin whitening agent comprising an extract of a water extract comprising at least one polyphenol compound selected from the group consisting of ruthenorhindine represented by the following formula (3) and a hesperidin compound represented by the following formula (4) Functional cosmetic composition.
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[Chemical Formula 4]

9. The functional cosmetic composition for whitening skin according to claim 8, wherein the fraction exhibits inhibitory activity of tyrosinase. A fraction of a water extract comprising at least one polyphenol compound selected from the group consisting of ruthenorhindine represented by the following formula (3) and a hesperidin compound represented by the following formula (4) ≪ / RTI >
(3)

[Chemical Formula 4]

11. The functional cosmetic composition for improving skin wrinkles according to claim 10, wherein the fraction exhibits an inhibitory activity of elastase. delete

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