KR20160045381A - A composite having anti-oxidizing and whitening activities using inonotus obliqua inoculated rice bran - Google Patents

Abstract

The present invention relates to a composition having antioxidant and whitening efficacy, and to a production method thereof. According to the present invention, an extract is obtained by producing rice bran containing the high content of nutrition, after inoculating mycelium derived from chaga mushroom into rice bran, and the extract can be used as a cosmetic ingredient.

Description

TECHNICAL FIELD The present invention relates to a composition having an antioxidant and whitening effect using an extract of rice bran mushroom mycorrhizae as an active ingredient, and a composition having anti-oxidizing and whitening activities using inonotus obliqua inoculated rice bran.

The present invention relates to a composition having an antioxidative and whitening effect, and a method for producing the same, which comprises preparing rice bran with high nutritional content by inoculation of rice bran mushroom mycelium into raw materials for cosmetics.

In general, reactive oxygen species, known as noxious oxygen, are known as antioxidant enzymes (superoxide dismutase, catalase, clutathione peroxidase, glutathione reductase) and non-enzymatic antioxidants (tocopherol, ascorbic acid, carotenoid, flavonoid , glutathione), the normal cellular function of the human body is maintained.

However, oxidative stress caused by excessive production of active oxygen or defective function of the defense system may cause skin antioxidant destruction, initiation of lipid peroxidation reaction, protein oxidation, DNA oxidation, chain breaks such as collagen and hyaluronic acid, Creation of wrinkles by cross-linking, melanin generation, etc., accelerate skin aging.

Enzymatic substances are self-produced in the body and include antioxidant enzymes such as superoxide dismutase (SOD), catalase, and peroxidase.

Non-enzymatic substances are nutrients that have to be absorbed from the outside of the body. They are in the form of low-molecular compounds and help the action of polymeric antioxidants (enzymes) in the body or remove active oxygen by themselves. Beta-carotene, vitamin A, vitamin C (ascorbic acid), vitamin E (tocopherol) and flavonoids are representative antioxidants.

The synthetic antioxidants developed so far include butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT) and nordihydro-guaiaretic acid (NDGA). Natural antioxidants include tocopherol, gossypol, sesamol, lecitthin, cephalin, ether extract components of red pepper, extracts from wheat germ, soybean, pepper, and tomato, and extracts of mainly florboloid pigments.

However, synthetic antioxidants are toxic in vivo and are likely to cause allergies and tumors. Moreover, they are weak against temperature and are easily destroyed by heating once.

On the other hand, melanin is a brown or black polymer pigment widely distributed in animal and plant systems, and determines human skin color. It controls ultraviolet light, cytokines, growth factors and hormones. Melanocyte stimulating hormone (α-MSH) plays a major role in controlling melanin production.

In summary, α-MSH activates adenylyl cyclase by binding to MC1R (melanocortin 1 receptor), a membrane receptor that is secreted in the midgut of the pituitary gland and expressed only in melanocytes, and activates PKA (protein kinase A) And activates intracellular cAMP response element binding protein (CREB), thereby increasing the expression of MITF (microphthalmia-associated transcription factor), a specific transcription factor in melanocytes.

The MITF stimulates the expression of melanin-producing tyrosinase and its related protein (tyrosinase-related protein), thereby regulating melanin production in melanocytes. Thus, α-MSH promotes tyrosinase activity through activation of adenylyl cyclase, PKA, CREB, and MITF, thereby increasing the proliferation and pigmentation of melanocytes.

Rice contains starch, peptides, glucosides, cellulose, and various minerals in large quantities. As it becomes more gentle and smooth, it is popular as a raw material for cosmetics.

Rice corn is a crumb of crustaceans, seeds, etc. obtained when the brown rice is converted into white rice. It contains a large amount of fat, vitamin B group and high quality protein and fiber. It is known that our ancestors have used it frequently, and it is known to help prevent stains on the face when packed with rice bran, but it has problems such as causing erythema on the skin. Accordingly, there is a demand for a cosmetic composition which can induce a whitening effect without irritating the skin by using cereals.

The present invention provides a composition capable of enhancing antioxidative and whitening efficacy without side effects by using a substance derived from a natural source, and it is an object of the present invention to provide a composition for preventing intracellular melanin synthesis, The present invention provides a composition capable of preventing melanin synthesis caused by? -MSH to promote skin whitening.

The present invention provides, as means for achieving the above object,

Immersing the rice bran in hot water at 20 to 30 캜 for 10 to 30 hours, adjusting the water content of the rice bran to 25 to 50%, and sterilizing at 100 to 140 캜;

  Inoculating the rice bran mushroom into rice bran after sterilization;

Culturing the rice bran inoculated with Mycobacterium mycelium at 25 to 30 ° C;

Drying the rice bran after culturing;

The dried rice bran mushroom was mixed with distilled water and ethanol at a predetermined ratio to obtain a rice bran cultivated with mushroom (hereinafter referred to as "chagami rice"), and the resulting extract was fractionated with ethyl acetate to obtain a fraction extract .

The tea obtained according to the present invention showed excellent antioxidative effect in the fractions of EtoAc of rice bran.

The fractions of chaga rice EtoAc inhibited the synthesis of melanin stimulated by α-MSH in a concentration-dependent manner and showed an excellent inhibitory effect on melanin synthesis as compared with that of normal rice bran.

The EtOAc fraction of chrysophyte showed no inhibitory effect on tyrosinase but inhibited intracellular melanin synthesis in a concentration dependent manner.

The present invention is characterized not only by preventing intracellular melanin synthesis but also by preventing melanin synthesis caused by? -MSH, thereby enhancing skin whitening effect.

FIG. 1 is a graph showing the antioxidative effect of tea extract on rice bran,
FIG. 2 is a graph showing the effect of suppressing melanin synthesis caused by? -MSH,
Figure 3 is a graph showing the concentration-dependent inhibitory effect of EtOAc fraction on melanin synthesis induced by [alpha] -MSH,
FIG. 4 is a graph showing concentration-dependent inhibitory effects of melanin synthesis induced by α-MSH on the intracellular and intracellular EtOAc fractions,
FIG. 5 is a graph showing the effect of Mushroom tyrosinase of rice bran extracts, and
FIG. 6 is a graph showing the effect of intracellular tyrosinase activity of rice bran.

Tea pot  Preparation of extract

Distilled water and ethanol were mixed at predetermined ratios to extract the carrageenan extract, and the extract was fractionated with ethyl acetate (EtOAc) to obtain an EtOAc fraction extract.

① Distilled water extraction

10 g of distilled water was added to 50 g of rice bran, heated at 80 to 100 ° C for 3 hours, filtered, and the filtrate was vacuum-dried to obtain 7.5 g of a dried product.

② 30% ethanol extraction and EtOAc fractionation of rice bran and rice bran

30 g of ethanol was added to 50 g of each of rice bran and tea bran, and the mixture was heated at 80 to 100 캜 for 3 hours, filtered and vacuum-dried to obtain 7.1 g of dried rice bran and dried bran.

③ The process of vacuum-drying the extract obtained in (2) was dissolved in 200 mL of distilled water, and the same amount of EtOAc was added thereto to separate the fractions twice. Then, 0.20 g of EtOAc extract and 0.32 g of tea extract were obtained.

The list and yield of the extracts obtained as a result of the extraction are summarized in Table 1 below. The samples in Table 1 were used in the following experimental examples.

Sample number List yield One Chaga irriga distilled water extract 15.0% 2 Rice bran 30% ethanol extract 14.3% 3 30% Ethanol Extract of Chaga River 14.2% 4 Rice bran 30% ethanol extract EtOAc fraction 2.86% 5 30% Ethanol Extract of Carrageenan EtOAc Fraction 4.53%

Experimental Example  One. Chaga  Antioxidative effect

1-1. Experimental course

The DPPH assay is one of the most common experiments to measure antioxidant efficacy. The principle of DPPH assay is that the deep purple Diphenylpicrylhydrazyl is reduced to Diphenylpicrylhydrazine by the antioxidant and turns into a pale yellow. The stronger the antioxidant power of the antioxidant, the closer to the pale yellow.

Also, when measuring the absorbance, a dark purple color is detected but a light yellow color is not detected. Therefore, the smaller the absorbance value is, the higher the antioxidant power is. The experimental procedure is as follows.

Five groups of 0.1 mM DPPH solution (Diphenylpicrylhydrazyl) were prepared. Group 1 was set as negative control and no manipulation was performed. Group 2 was set as positive control and treated with ascorbic acid 50 μM. 3 groups were treated with 25 μg / mL, 50 μg / mL, and 100 μg / mL of tea extracts, 30% ethanolic extracts of tea leaves and 4 ethyl acetate extracts, respectively. We observed how the color of the samples changed with a light absorber at a wavelength of 520 nm.

1-2. Experiment result

Fig. 1 is a graph showing the antioxidative effect of tea extracts on the rice bran. The absorbance was expressed in terms of% in terms of the following formula. (antioxidant activity = {1- (experiment / control)} x100)

Water-fraction extracts and EtoAc fraction extracts showed a concentration-dependent antioxidative effect when compared to the negative control group. Especially, EtoAc fraction extract showed similar effect to positive control at high concentration.

Experimental Example  2. Tea pot  Inhibitory Effect of Extracts on Intracellular Melanin Synthesis

2-1. Experimental course

(1) B16F10 cells were inoculated into 6 wells of two wells, so as to be in total of 4 groups, and cultured for 24 hours for stabilization.

② Group 1 was set as a negative control group, and 2 groups were pre-treated with α-MSH at 100 nM for 24 hours to induce melanin synthesis and no other treatment was performed. Group 3 was pretreated with α-MSH for 24 hours and treated with kojic acid at 10 μM and 1 mM.

③ Group 4 was set as the experimental group, and the highest concentration without cytotoxicity was treated with 100 μg / mL of chrysophyte distilled water extract, 30% ethanol extract of Chaga mica, and 50 μg / mL of EtOAc with 30% Respectively.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

2-2 Experimental results

2 is a graph showing the effect of suppressing melanin synthesis caused by? -MSH.

As a result of measurement, melanin synthesis by α-MSH treatment suppressed melanin synthesis in a concentration-dependent manner by the positive control kojic acid.

The tea extract of Eoac fraction from rice bran showed significant inhibitory effect. Especially, EtoAc fraction showed the same inhibitory effect on melanin synthesis as kojic acid 1 mM.

Experimental Example  3. Tea pot EtOAc  Concentration-dependent inhibitory effect on intracellular melanin synthesis of the fraction

3-1. Experimental course

(1) B16F10 cells were inoculated into 6 wells of two wells, so as to be in total of 4 groups, and cultured for 24 hours for stabilization.

② Group 1 was set as a negative control group, and 2 groups were pre-treated with α-MSH at 100 nM for 24 hours to induce melanin synthesis and no other treatment was performed. Group 3 was pretreated with α-MSH for 24 hours and treated with kojic acid at 10 μM and 1 mM.

③ Group 4 was treated with 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

3-2 Experimental results

FIG. 3 is a graph showing the concentration-dependent inhibitory effect of the EtOAc fraction on the synthesis of melanin induced by? -MSH.

As a result, the melanin production by α-MSH was inhibited by EtoAc fraction in a concentration-dependent manner, and 50 μg / mL of chitosan EtOAc fraction showed similar inhibitory effect on melanin synthesis as 1 mM kojic acid.

Experimental Example 4. Inhibitory Effect of EtOAc Fraction of Rice Bran and Chrysophyceae on Melanin Synthesis

4-1. Experimental course

1) B16F10 cells were inoculated into 6-well plates in a total of 5 groups, and incubated for 24 hours to stabilize them.

② Group 1 was set as a negative control group, and 2 groups were pre-treated with α-MSH at 100 nM for 24 hours to induce melanin synthesis and no other treatment was performed. Group 3 was pretreated with α-MSH for 24 hours and treated with 10 μM kojic acid.

③ Groups 4 and 5 were set as experimental groups, and treated with 50 μg / mL of rice bran-free EtOAc fraction and chrysanthemum EtOAc fraction at the same concentration.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

4-2 Experimental results

4 is a graph showing the concentration-dependent inhibitory effect of the EtOAc fraction on melanin synthesis caused by? -MSH.

As a result of the measurement, it was confirmed that melanin production by α-MSH was significantly inhibited in the extracts of the rice bran and the rice bran EtoAc, and melanin synthesis was significantly inhibited in the fraction of EtOAc in the rice bran than in the rice bran.

Experimental Example  5. Tea pot EtOAc  Fraction Mushroom tyrosinase  Active inhibitory effect

5-1. Experimental course

Mushroom tyrosinase activity was measured in order to determine if the extracts of chrysanthemum morifolium directly inhibited tyrosinase.

② Mushroom tyrosinase was prepared by dissolving 25,000 units in 6 ml of 0.1M potassium phosphate buffer (PPB, pH 6.81) and adding 2 ml of D.W to the enzyme solution. The substrate was dissolved in D.W as 0.01% L-DOPA.

③ Chrysophyte EtOAc fraction was dissolved in 0.1M PPB and used as 0, 25, 50, 100 and 200 ㎍ / mL. As a positive control, kojic acid 0, 25, 50, 100 and 200 μM were used. 160 μl of 0.1 M PPB in 96 wells, 10 μl of a sample, 20 μl of a substrate solution and 10 μl of an enzyme solution were reacted at room temperature for 2 minutes and absorbance was measured at 475 nm.

5-2 Experimental results

FIG. 5 is a graph showing the Mushroom tyrosinase effect of tea extracts in terms of% of absorbance by the following equation. Tyrosinase activity (%) = (absorbance of sample / absorbance of control group) × 100

Kojic acid inhibited mushroom tyrosinase activity in a dose - dependent manner, but did not inhibit the activity of chaga mushroom extract.

Experimental Example  6. Tea pot EtOAc  Fraction Intracellular tyrosinase  activation

6-1. Experimental course

To investigate the effect of tea α-MSH (100 nM) on tyrosinase activity, which is an enzyme controlling melanin synthesis in tea cells, we increased melanin synthesis and extracted tyrosinase from B16F10 cells And its activity was evaluated.

② B16F10 melanoma cells were treated with α-MSH (100 nM) for 24 hours, followed by α-MSH (100 nM) and treated with 25 μg / mL of chimeric EtOAc fraction.

After culturing for 48 hours with the sample, the cells were harvested with trypsin, and the culture of the pellet was completely removed with PBS. Then, 0.1 ml of 0.1% (V / V) PMSF containing 5 mM EDTA and 1% Cells were lysed on ice for 30 min with M sodium phosphate buffer (SPB pH 6.8).

④ Tyrosinase was obtained from the supernatant by centrifugation at 15,000 rpm for 30 min at 4 ° C. The amount of protein was calculated using Bradford reagent, and the same amount of protein diluted in 0.1 M SPB (pH 6.8) and 0.1% ) L-DOPA was reacted at 37 for 1 hour and absorbance was measured at 475 nm.

6-2 Experimental results

FIG. 6 is a graph showing the effect of intracellular tyrosinase activity on the intracellular intracellular Ca2 + concentration. As shown in FIG. 6, the intracellular EtOAc fraction inhibited tyrosinase activity in a concentration-dependent manner compared with the treatment with α-MSH (100 nM).

Claims (4)

A method for producing a composition having an antioxidative and whitening effect, comprising an extract of rice bran microalgae obtained by inoculating mycelium of carrot mushroom as an active ingredient,
Immersing the rice bran in hot water at 20 to 30 캜 for 10 to 30 hours, adjusting the water content of the rice bran to 25 to 50%, and sterilizing at 100 to 140 캜;
Inoculating the rice bran mushroom into rice bran after sterilization;
Culturing the rice bran inoculated with Mycobacterium mycelium at 25 to 30 ° C;
Drying the rice bran after culturing; And
And mixing the dried rice bran mushroom mycelium with distilled water and ethanol at a predetermined ratio to extract a rice bran cultivated with mushroom and extracting the obtained extract with ethyl acetate to obtain a fraction extract, A method for producing a composition having an antioxidative and whitening effect as an active ingredient.
The method according to claim 1,
A method for preparing a composition having an antioxidative and whitening effect, wherein the extract of rice bran cultivated with the mushroom mycelium is distilled water and ethanol, and the weight ratio of the rice bran is added at a ratio of 10 times the weight of the raw rice bran.
The method according to claim 1,
Wherein the ethanol has an antioxidative and whitening effect using a carbohydrate extract using 30% ethanol as an active ingredient.
The method according to claim 1,
The step of fractionating the extracted rice bran extracts of the rice bran is carried out by adding the obtained extracts to a mixture of distilled water and ethyl acetate in the same weight ratio and repeating the extraction twice to obtain a fraction having an antioxidative and whitening effect ≪ / RTI >

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