TWI484035B - For the preparation of a product with antioxidant activity and tyrosinase inhibitory activity - Google Patents

Description

Method for preparing a product having antioxidative activity and tyrosinase inhibitory activity from xanthin

The invention relates to a self-yellowing (Polygonatum) Sibiricum Red) A method of preparing a product having antioxidant activity and tyrosinase inhibitory activity. The invention also discloses a product produced by the process and a composition comprising the product.

In the aerobic respiration process of organisms, free radicals and reactive oxygen species are generated. When the free radicals accumulate abnormally, they will cause a chain reaction of free radicals, causing cells (including cell membranes, nuclei) and other molecules in the organism (for example). Proteins, sugars, and fats are destroyed, which seriously affects various normal physiological functions in living organisms. Many studies have reported the close relationship between free radicals and aging, cancer, and various chronic diseases.

In recent years, because the earth's environment has been polluted, the ozone layer has been severely damaged, causing a large amount of ultraviolet rays to directly reach the surface of the earth, causing melanocytes in the skin to be overactive to produce melanin, and severe cases are more likely to become cancerous and cause skin cancer. In melanocytes, the activity of tyrosinase is the main key to the production of melanin. The activity of tyrosinase is increased by the stimulation of UV and pituitary gland, and then through a series of enzyme reactions. Conversion of L-tyrosine to melanin, if melanin is produced If the rate is too fast or cannot be quickly metabolized, it will deposit in the skin, causing darkening and dark spots.

In order to find more effective and safely applied ingredients for antioxidant activity and tyrosinase inhibitory activity in foods and cosmetics, non-toxic edible plants are used as raw materials and novel ingredients containing such biological activities are obtained therefrom. It has gradually attracted the attention of researchers in related industries such as food, medicine and cosmetics.

Huang Jing, a plant of the genus Liliaceae, is a chicken head, also known as chicken head yellow essence, yellow chicken dish, and has different names in different parts of the mainland, for example, pen dish, claw ginseng, Tiger ginger or chicken claws. Since ancient times, Huang Jing has been regarded as a kind of traditional Chinese medicine with anti-aging, anti-aging and longevity. The Japanese and Chinese herbal medicines record, Huang Jing single service, nine steamed and nine exposed, the food is stationed in the valley. The Compendium of Materia Medica also records that Huang Jing can make up the imaginary, stop the cold and heat, and fill the essence. Plus Huang Jing is sweet and can eat and feed the hunger, also known as the rescue of poor grass.

In recent years, traditional Chinese herbal medicines have been extensively studied. Many research reports have confirmed that many polyphenols contained in Chinese herbal medicines have anti-oxidation and antibacterial functions. In order to meet the industrial utilization value, the active ingredients in Chinese herbal medicines must be further processed. In order to enhance the action intensity of its active ingredients, in the process of searching for novel ingredients having antioxidant activity and tyrosinase inhibitory activity, the inventors unexpectedly discovered that the methanol extract of the yellow essence in the herb is taken with rice bran. The bacterial mutants are fermented together, and the obtained fermentation product has high antioxidant activity and excellent tyrosinase inhibitory activity.

Accordingly, in a first aspect, an object of the present invention is to provide a process for preparing a product having antioxidative activity and tyrosinase inhibitory activity from xanthan.

Thus, the present invention is a method for preparing a product having antioxidative activity and tyrosinase inhibitory activity from xanthan, comprising the steps of: extracting an appropriate amount of xanthin with an alcohol to obtain a mixture; The yellow essence and the alcohol in the mixture are removed, thereby obtaining an alcohol extract of a yellow essence; and a microorganism selected from the genus Aspergillus is cultured in a medium containing the alcohol extract of the yellow essence In order to obtain a fermented yellow essence product.

In a second aspect, the present invention provides a product having antioxidant activity and tyrosinase inhibitory activity produced by the method as described above.

In a third aspect, the present invention provides a composition having antioxidant activity and tyrosinase inhibitory activity, which comprises a product obtained by the method as described above.

The effect of the invention is that the fermentation product obtained by culturing the alcohol extract of the Chinese herbal medicine Huang Jing together with the microorganism of the genus Fusarium can produce a more simple antioxidant activity of the alcohol extract of the yellow essence.

What is to be understood is that if any of the previous publications are here It is quoted that the previous publication does not constitute an acknowledgement; in Taiwan or any other country, the former publication forms part of the common general knowledge in the art.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

In order to make the active ingredients of traditional Chinese herbal medicines more effectively applied to cosmetics, health food and other industries to enhance their practical value, in addition to the easy-to-large-scale way to obtain antioxidant activity and tyrosinase inhibitory activity in Chinese herbal medicines. In addition to the product, it is preferred to obtain a product having a better activity performance by the treatment method employed. The inventor chose Huangjing, which has anti-aging, anti-aging and longevity, and tried to extract the yellow essence water with methanol, and then took the obtained alcohol extract of Polygonatum with the rice cultivar variant (Aspergillus oryzae var.brunneus). IFO 5239 was fermented together and tested for the fermentation product. It was found that when the alcohol extract of the extract of the extract of the yellow essence was subjected to fermentation treatment of the specific microorganism, the antioxidant activity of the fermentation product of the alcohol extract of the extract of the yellow essence can be improved, and The fermentation product still retains a good inhibitory effect on tyrosinase. Accordingly, the present invention provides a method for producing a product having antioxidative activity and tyrosinase inhibitory activity from xanthan, which comprises the steps of: extracting an appropriate amount of xanthin with an alcohol to obtain a mixture. According to the invention, the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, and combinations thereof. Preferably, the alcohol is methanol or ethanol. In this example, the yellow essence was soaked with 80% (v/v) methanol to extract the mixture. Preferably, to promote extraction efficiency, the yellow essence is chopped and immersed in the alcohol for a sufficient period of time to permit extraction. Preferably, the minced xanthin is soaked in the alcohol for 5 to 7 days. In one embodiment of the invention, the minced yellow essence is soaked in the alcohol for 5 days.

Next, the yellow essence and the alcohol in the obtained mixture are removed, thereby obtaining an alcohol extract of xanthine. In this example, a vacuum vacuum concentrator was used to remove the alcohol to obtain an alcohol extract of Rhizoma Polygonum in the form of a thick liquid.

A microorganism selected from the genus Trichophyton is cultured in a medium containing the alcohol extract of the scutellaria to obtain a fermented yellow essence product. Preferably, the microorganism is selected from the group consisting of Aspergillus oryzae, Aspergillus flavofurcatis, and Monascus purpureus. In one embodiment of the invention, the microorganism is Aspergillus oryzae var. brunneus IFO 5239. According to the present invention, the alcohol extract of the Polygonatum is added to the medium at a concentration of 500 to 1000 mg/mL. In a preferred embodiment of the invention, the alcohol extract of Polygonatum is added to the medium at a concentration of 1000 mg/mL.

In addition, the present invention further produces the product of the fermented huangjing into a high-purity product having antioxidant activity, and is firstly treated with n-hexane. After degreasing the product of the fermented yellow essence, it was adjusted to an aqueous layer of pH 3 by adding hydrochloric acid, and the aqueous layer was extracted with ethyl acetate (EtOAc), and the ethyl acetate in the extract was removed. This high purity product having antioxidant activity can be obtained. In this embodiment, the product of the fermented yellow essence is adjusted to an aqueous layer of pH 3 by adding hydrochloric acid having a concentration of 6 N, and after extracting three times with ethyl acetate, the extracts are combined, and ethyl acetate is removed by vacuum concentration. A product having high purity of antioxidant activity is obtained.

The present invention also provides a product having antioxidant activity and tyrosinase inhibitory activity which is produced by the method as described above.

In view of the wide application of antioxidant activity and tyrosinase inhibitory activity in the food industry and the cosmetics industry, the present invention also provides a composition having antioxidant activity and tyrosinase inhibitory activity, which is contained by The product produced by the method.

In the following, the following specific examples will be further described, but it should be understood that the examples are for illustrative purposes only and are not to be construed as limiting.

Experimental Materials:

A. Polygonatum sibiricum Red used in the following specific examples was purchased from Zefang Biotechnology Co., Ltd.

B. The Czapek-Dox medium used in the following specific examples has the formulation shown in the following table.

C. Aspergillus oryzae var.brunneus IFO 5239 used in the following specific examples is purchased from the Japanese independent administration. NITE Biological Resource Center (NBRC) of the National Institute of Technology and Evaluation (NITE).

D. Aspergillus oryzae var. oryzae CCRC31646 used in the following specific examples is purchased from the Bioresource Conservation and Research Center of the Food Industry Development Research Institute.

<Specific Example 1 Removal of DPPH Free Radical Activity Test (Antioxidant Activity Test)>

1. Preparation of Huangjing methanol extract : 500 g of Huang Jing was weighed at room temperature and pulverized with a mill, followed by 0.5 liter of methanol (80%) for 5 days. Thereafter, the filtrate was filtered, and the collected filtrate was again subjected to vacuum decompression rotary concentrator (EYELA, N-1000SW) to remove methanol, thereby obtaining a yellow essence methanol extract in the form of a thick liquid.

2. Preparation of IFO 5239 fermentation product of extract of Phellodendron chinense : 0.5 cm ² of the rice blast fungus IFO 5239 (containing about 5,000-10,000 spores) was inoculated into 100 mL of CzC medium, and Shake culture was carried out in a constant temperature shaking incubator (30 ° C, 104 rpm). After 24 hours of incubation, 1000 mg of the extract of Polygonactate methanol was added and mixed thoroughly, followed by fermentation in a constant temperature shaking incubator (30 ° C, 104 rpm). After 5 days, the fermentation broth was collected and concentrated under reduced pressure using a vacuum decompression rotary concentrator (EYELA, N-1000SW) to obtain a fermentation product of a yellow liquid methanol extract in the form of a thick liquid (hereinafter referred to as IFO). 5239 Huangjing fermentation product).

3. Preparation of CCR 31646 fermentation product of the extract of the yellow essence methanol : the above microorganism was replaced by CCRC31646, and the same fermentation treatment was carried out on the methanol extract of the yellow essence to obtain the fermentation product of the methanol extract of the yellow essence in the form of a thick liquid. (hereinafter referred to as CCRC 31646 Huangjing fermentation product)

In order to understand whether the antioxidant activity of the methanol extract of Polygonatum sinensis was improved after the biotransformation of the rice bran mutant IFO5239, the above-mentioned fermentation products of the yellow essence methanol, the fermentation product of IFO 5239 and the CCRC31646 huangjing fermentation product were tested. Samples were taken and subjected to the following DDPH free radical activity test. Further, a test sample of a positive control group was used as an antioxidant component α-Tocopherol which is commonly used in commercially available cosmetics.

DDPH (1,1-diphenyl-2-picryhydrazyl) is a stable free radical containing an odd number of electrons. Its methanol solution has a strong absorbance near 517 nm. When DDPH is reduced by an antioxidant or combined with another free radical, the absorbance is Will disappear, use this property to determine the ability of the sample to be tested for DPPH free radical scavenging. The lower the absorbance value, the stronger the ability of the test sample to remove DPPH free radicals.

The DPPH free radical capacity is determined by reference to Shimada et Test method described in al. (1992). That is, 0.2 mL of DPPH methanol solution (1 mM) was taken, and 0.8 mL of the sample to be tested was added. After the mixture was uniformly mixed, the reaction was allowed to stand at room temperature for 30 minutes in the dark, and the absorbance at a wavelength of 517 nm was measured with a spectrometer (model: 2100 pro, Amersham, manufactured by Hong Kong). When the DPPH radicals are scavenged more, the more the absorbance value is decreased, the percentage of the absorbance of the control sample is reduced, and the ability of each sample to be scavenged to scavenge DPPH radicals can be obtained. BHA (Butylated hydroxyanisole) was used as a standard in the experiment, and the sample was replaced with methanol as a blank control group.

The DPPH free radical scavenging ability of each sample to be tested and the positive control group was calculated by substituting the measured absorbance value (OD ₅₁₇ ) of the wavelength ₅₁₇ nm into the following formula: P (%) = [1-(A) /B)]×100

Among them, P is the ability to scavenge DPPH free radicals

A is the absorbance of the sample to be tested or the positive control group at a wavelength of 517 nm.

B is the absorbance of the blank control group at a wavelength of 517 nm.

Among them, the IFO 5239 huangjing fermentation product was firstly formulated in methanol at different concentrations of 1 mg/mL, 5 mg/mL and 10 mg/mL, and then 0.8 mL of different concentrations of IFO 5239 huangjing fermentation product and 0.2 mL were separately taken. The DPPH methanol solution was mixed to determine the ability of the IFO 5239 huangjing fermentation product to remove DPPH free radicals at different concentrations. The results showed that the higher the concentration of the IFO 5239 huangjing fermentation product, the more the free radical elimination activity Preferably, the free radical elimination activity of the IFO 5239 huangjing fermentation product has a dose dependency, and the test results show that the IFO 5239 huangjing fermentation product is removed at a concentration of 10 mg/mL. With a base activity of up to 87.4%, it has excellent antioxidant activity that meets the application requirements. In addition, in order to further compare the difference in the ability of IFO 5239 Huangjing fermentation product to eliminate free radicals at the same concentration with other kinds of test samples, other test samples were also formulated to a concentration of 10 mg/mL, and the DPPH free radical activity was tested separately.

The results are shown in Table-1 below, which shows that the extract of Phellodendron chinense L. before fermentation treatment has 77.9% scavenging activity of DPPH free radicals, and the removal of fermented product of Polygonatum sinensis after fermentation by two microorganisms: CCRC 31646 and IFO 5239 The activity of the free radicals is stronger than the activity of scavenging free radicals of the original extract of the yellow essence methanol, indicating that the present invention can enhance the oxidation activity of the extract of the extract of the yellow essence by fermentation treatment. Among them, the scavenging free radical activity of the fermentation product of the yellow essence obtained by fermentation of Aspergillus oryzae var.brunneus IFO 5239 is better than the antioxidant component (α-Tocopherol) commonly used in commercial cosmetics. It has a significantly enhanced antioxidant capacity and is extremely valuable for development and application.

<Specific Example 2 Oxidase tyrosinase inhibitory activity test>

In order to understand whether the components containing tyrosinase inhibitory activity in the methanol extract of Rhizoma Polygonati will be affected by the biotransformation of the rice bran mutant IFO 5239 To the effect, the extract of the yellow essence methanol obtained in the specific example 1 and the fermentation product of the IFO 5239 huangjing were used as the samples to be tested and subjected to the following tyrosinase inhibitory activity measurement. In addition, a whitening component tannic acid commonly used in commercially available cosmetics was used as a test sample of a positive control group.

The tyrosinase inhibitory activity assay was carried out in accordance with the method described in Chen Yu-Chi et al. (2005), Biosci. Biotechnol. Biochem., 69(5):999-1006. Briefly, take 0.1 mL of the sample to be tested (in 1% concentration in methanol), 2.4 mL of 0.1 M phosphate buffer solution, and 0.4 mL of 1.5 mM tyrosine solution (Sigma-Aldrich) and mix well. Next, 0.1 mL of tyrosinase solution (2000 U/mL in 0.05 M phosphate buffer solution) was added and the reaction was allowed to proceed at room temperature for 2 minutes. Thereafter, the absorbance was measured at a time interval of 15 seconds at a wavelength of 470 nm for 10 minutes (i.e., the total number of measurements was 40 times).

In addition, methanol was used instead of the sample to be tested for use as a control group, and methanol and 0.05 M phosphate buffer solution were used to replace the sample to be tested and the tyrosinase solution, respectively, for use as a blank control group, using a 0.05 M phosphate buffer solution. The tyrosinase solution was replaced with a tyrosinase solution and the same experiment was performed.

The absorbance (OD ₄₇₀ ) of the wavelength of 470 nm measured at different time points of each group was plotted against time and the slope was determined, and the tyrosinase inhibitory activity of the sample to be tested was obtained by The slope is calculated by substituting the following formula: A(%)=[(BC)-(DE)]/(BC)×100

Where: A = tyrosinase inhibitory activity

B = slope of the control group

C = slope of the blank control group

D = slope of the sample to be tested

E = slope of the buckle group

The results are shown in Table 2 below. It shows that the methanol extract of Rhizoma Coptidis has not decreased in the whitening ability after being fermented by the rice bran mutant IFO 5239, but it is slightly increased. Therefore, the fermentation of the extract of the yellow essence methanol by IFO 5239 is carried out. The treatment, in addition to improving the antioxidant capacity, does not diminish the tyrosinase inhibitory activity of the original extract of the yellow essence methanol, but still retains good whitening ability, and the tyrosinase inhibitory activity of the fermentation product of IFO 5239 The tannic acid is very close and has more than 70% inhibition activity, indicating that the IFO 5239 Huangjing fermentation product can also perform a good whitening effect in application.

Based on the above experimental results, the inventors believe that the fermentation product obtained by fermenting the methanol extract of Polygonatum by the rice bran mutant IFO 5239 can exhibit better antioxidant activity and still retain good cheese. Amino acidase inhibitory activity, therefore, has practical value for the development of raw materials for antioxidant products and raw materials for whitening products in the food industry and cosmetics field.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Claims (6)

A method for preparing a product having antioxidative activity and tyrosinase inhibitory activity from xanthan, comprising the steps of: extracting an appropriate amount of xanthin with an alcohol to obtain a mixture; and obtaining the mixture The yellow essence and the alcohol are removed, thereby obtaining an alcohol extract of a yellow essence; and the culture medium of Aspergillus oryzae var. brunneus IFO 5239 is cultured in an alcohol extract containing the yellow essence In order to obtain a product of fermented yellow essence, the alcohol extract of the yellow essence is added to the medium at a concentration of 500 to 1000 mg/mL. The method of claim 1, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, and combinations thereof. The method of claim 2, wherein the alcohol is methanol or ethanol. The method of claim 3, wherein the alcohol is 80% (v/v) methanol. The method of claim 4, wherein the yellow essence is soaked in the alcohol for at least 5 days to obtain the mixture. The method of any one of claims 1 to 5, wherein the product of the fermented yellow essence is further prepared into a high-purity product having antioxidant activity, and is first treated with n-hexane. After the product of the fermented yellow essence is degreased, it is adjusted with an acid to an aqueous layer of pH 3, and the aqueous layer is extracted with ethyl acetate, and then the ethyl acetate in the extract is removed to obtain the high. A product of purity having antioxidant activity.