TWI567192B - Method for preparing extract of Antrodia camphorata for whitening - Google Patents

Description

Method for preparing extract of Antrodia camphorata for whitening

The present invention relates to a method for preparing an extract of Antrodia camphorata for whitening, and more particularly to a method for preparing an extract of Antrodia camphorata for whitening by temperature gradient stimulation growth treatment.

Antrodia camphorate is a fungus that is parasitic on burdock. It is a large evergreen broad-leaved tree that grows only in Taiwan and is a rare species in nature. In recent years, many scholars and units have published research data, confirming that Niobium has anti-cancer, anti-oxidation, anti-itching and immune-enhancing effects, which has made many people rush, even known as "the ruby in the scorpion". Its preciousness is self-evident.

At present, the use of faded spots or whitening spots used in medical and cosmetic cosmetics is nothing more than the three main directions of inhibiting melanin production, promoting melanin metabolism or reducing melanin. Although most of the current treatments are treated simultaneously in these three directions, the most important treatment effect is to inhibit the formation of melanin, which is the source of melanin formation. Among them, tyrosinase plays a key role in the formation of melanin. Therefore, the research and development of tyrosinase activity inhibitors has become an important development direction in the beauty and medical fields.

Several tyrosinase inhibitors commonly used in cosmetics and medical applications are terephthalic acid Hydroquinone, vitamin C, arbutin, ellagic acid, tranexamic acid, kojic acid, etc., however, some whitening ingredients often have limitations in their use. For example, vitamin C is easily oxidized in the sun or in contact with air, and is not heat-resistant. When the concentration exceeds 5%, it will cause irritation and redness to the skin. Very unstable citrate is easily oxidized and discolored and causes skin irritation. If long-term use of citrate products is excessive, cytotoxicity and pathological changes will occur, and severe DNA will be affected in the nucleus. Hydroquinone is cytotoxic, and most people who use hydroquinone-containing products have erythema symptoms, or use a concentration of more than 5%, which can cause leukoplakia or pigmentation. Therefore, the search for effective whitening and non-cytotoxic tyrosinase inhibitors has become an important trend in the development of skin lightening agents.

Among them, some studies have pointed out that after fermentation of burdock and mulberry, yam and wine cellar, the cellulite mycelium extract has the effect of inhibiting tyrosinase. Therefore, how to directly extract a large amount of tyrosine-containing ingredients which have a whitening effect in Antrodia camphorata is a major issue in the cosmetics industry.

In view of the above problems of the prior art, the object of the present invention is to provide a method for preparing a whitening burdock extract by temperature gradient stimulation growth treatment to solve the tyrosinase inhibitor for whitening in the prior art. It is a problem of unstable nature or side effects on the human body.

According to one aspect of the present invention, a method for preparing an extract of Antrodia camphorata for whitening is provided, comprising the steps of: inoculating a strain of Antrodia camphorata in a medium, culturing for a preset time to obtain an entity of Antrodia camphorata; and stimulating the body of Antrodia camphorata by temperature gradient stimulation The growth treatment is performed to obtain a fermentation product of Antrodia camphorata; and the fermentation of the Antrodia camphorata is extracted with a solvent to obtain an extract of Antrodia camphorata.

Preferably, the strain of Antrodia camphorata may comprise: a strain of Antrodia camphorata; and comprising From about 1% to about 10% glucose, from about 1% to about 10% xylose, from about 0.1% to about 1% yeast extract, from about 0.1% to about 1% peptone, and about 0.2% A culture solution of about 2% coffee grounds.

Preferably, the medium may comprise from about 5% to about 15% by weight of white rice, brown rice, purple rice, wheat, oats, oatmeal, sorghum or coix seed.

Preferably, the ratio of the strain of the aqueous solution of Antrodia camphorata to the medium may be about 0.6 liters to about 1.6 liters of the strain of Antrodia camphorata and about 1 kg of the medium.

Preferably, the preset time may be from about 7 days to about 14 days.

Preferably, the temperature gradient stimulation growth treatment may be temperature gradient increasing stimulation growth, temperature gradient decreasing stimulation growth or repeated temperature gradient stimulation growth.

Preferably, wherein the temperature gradient stimulation growth treatment comprises: a first period, which may be from about 4 ° C to about 8 ° C, from about 3 days to about 7 days; and a second period, which may be from about 10 ° C to about 16 ° C. 3 days to about 7 days; during the third period, from about 18 ° C to about 24 ° C for about 3 days to about 7 days; and for the fourth period, from about 26 ° C to about 32 ° C for about 3 days to about 7 The temperature gradient incremental stimulation growth may include sequentially passing through the first period, the second period, the third period, and the fourth period; wherein the temperature gradient decreasing stimulation growth system comprises sequentially passing through the fourth period, the third period, and the second And a first period; wherein the repeated temperature gradient stimulation growth system comprises sequentially passing through the fourth period, the third period, the second period, the first period, the second period, the third period, and the fourth period.

Preferably, the solvent may comprise water, alcohol, acetic acid or sodium bicarbonate.

Preferably, the ratio of the fermentation of the Antrodia camphorata to the solvent is about 1 kg of the fermentation of the Antrodia camphorata and about 10 liters to about 20 liters of the solvent.

Preferably, the fermentation of the Antrodia camphorata fermented material may further comprise drying and grinding the fermentation of the Antrodia camphorata powder into a powder.

According to the method of the present invention for preparing an extract of Antrodia camphorata, it may have one or more of the following advantages:

(1) This method can increase the activity of tyrosinase inhibitor in the extract by extracting the body of the Antrodia camphorata by extracting the growth process by temperature gradient, thereby reducing the intracellular melanin content, and can be applied to whitening. The manufacture of skin cosmetics.

(2) The method can stimulate the growth treatment by a temperature gradient, so that the extract of Antrodia camphorata obtained by the method does not cause toxicity to cell growth, thereby solving the problem that the whitening product prepared by the prior art is irritating to the skin or causes cells. Toxic problem.

(3) This method can effectively apply Taiwan's unique Antrodia camphorata to whitening products by stimulating growth treatment with temperature gradient to achieve the purpose of making good use of national biological resources and bringing new business opportunities to the cosmetics market.

S1~S3‧‧‧ steps

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the first embodiment of the method for preparing an extract of Antrodia camphorata.

Fig. 2a is a bar graph showing the inhibition rate of the tyrosinase monophenol measured by the extracts obtained in Examples 2 to 5 of the present invention and Comparative Examples 1 to 2.

Fig. 2b is a bar graph showing the inhibition rate of the tyrosinase bisphenol measured by the extracts obtained in Examples 2 to 5 of the present invention and Comparative Examples 1 to 2.

Fig. 3a is a bar graph showing the inhibition rate of the tyrosinase monophenol measured by the extracts obtained in Examples 6 to 9 of the present invention and Comparative Examples 1 to 2.

Figure 3b is an extract obtained in Examples 6 to 9 of the present invention and a comparative example thereof. A bar graph of the inhibition rate of tyrosinase bisphenol was measured from 1 to 2.

Fig. 4a is a bar graph showing the inhibition rate of the tyrosinase monophenol measured by the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 3.

Fig. 4b is a bar graph showing the inhibition rate of the tyrosinase bisphenol measured by the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 3.

Fig. 5a is a bar graph showing the cell survival rate of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 4.

Fig. 5b is a bar graph showing the measured intracellular tyrosidase activity of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 4.

Fig. 5c is a bar graph showing the intracellular melanin content of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 4.

Fig. 6a is a bar graph showing the measured reducing power of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1, 3 and 5.

Fig. 6b is a bar graph showing the DPPH radical scavenging rate of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1, 3 and 6.

Figure 7a is a Lineweaver-Burk plot of the inhibitory effect of the extract prepared in Example 14 on the inhibition of different concentrations of dopamine substrate by tyrosinase.

Figure 7b is an XY scatter plot of slope versus concentration for the various concentrations of the extract prepared in Example 14 in the Lineweaver-Burk plot.

Fig. 7c is an XY scatter plot of Km, app value versus concentration, obtained from different concentrations of the extract prepared in Example 14 in the Lineweaver-Burk plot.

Figure 8a is an HPLC chart of flavonoids.

Figure 8b is an HPLC chart of the extract obtained in Example 14 of the present invention.

The invention will be further described in detail by the following preferred embodiments and the accompanying drawings. It should be noted that the experimental data disclosed in the following embodiments are for explaining the technical features of the present invention, and are not intended to limit the manner in which they can be implemented.

Please refer to FIG. 1 , which is a flow chart of Embodiment 1 of the method for preparing Antrodia camphorata extract of the present invention. As can be seen from the above, the method for preparing an extract of Antrodia camphorata of the present invention may comprise the steps of inoculating a strain of Antrodia camphorata in a medium for a predetermined period of time to obtain an Antrodia camphorata entity (Step S1). Wherein, the Antrodia camphor strain solution may comprise: Antrodia strain; and comprising about 1% to about 10% glucose, about 1% to about 10% xylose, about 0.1% to about 1% yeast extract, about 0.1% Up to about 1% peptone and about 0.2% to about 2% coffee slag. The medium may comprise a mixture of white, brown, purple, wheat, oat, oatmeal, sorghum or coix seed in a weight ratio of from about 5% to about 15%. The ratio of the strain of Antrodia camphorata to the culture medium may be about 0.6 liters to about 1.6 liters of the strain of Antrodia camphorata and about 1 kg of the medium. The preset time for the culture can be from about 7 days to about 14 days. The Antrodia camphorata body is subjected to growth treatment by temperature gradient stimulation to obtain a fermentation product of Antrodia camphorata (step S2). Wherein, the temperature gradient stimulation growth treatment may comprise from about 4 ° C to about 8 ° C for a first period of from about 3 days to about 7 days, from about 10 ° C to about 16 ° C for a second period of from about 3 days to about 7 days, about From 18 ° C to about 24 ° C for a third period of from about 3 days to about 7 days and from about 26 ° C to about 32 ° C for a fourth period of from about 3 days to about 7 days. The temperature gradient stimulation growth processing may include increasing the temperature gradient by the first period, the second period, the third period, and the fourth period, or including sequentially flowing through the fourth period, the third period, and the second period. And the temperature gradient in the first period is decreased to stimulate growth; or includes the fourth period, the third period, and the The repeated temperature gradient stimulates growth during the second period, the first period, the second period, the third period, and the fourth period. The fermentation of the Antrodia camphorata is extracted with a solvent to obtain an extract of Antrodia camphorata (step S3). Among them, the solvent may include water, alcohol, acetic acid or sodium hydrogencarbonate. The ratio of the fermentation of the Antrodia camphorata to the solvent may be about 1 kg of the fermentation of the Antrodia camphorata and about 10 liters to about 20 liters of the solvent. Before the extraction, the fermentation of the Antrodia camphorata may further comprise drying and grinding the fermentation of the Antrodia camphorata into powder.

In the second embodiment of the present invention, the strain of Antrodia camphorata is first inoculated into the culture solution, and is cultured at a temperature of 25 ° C to about 30 ° C at a temperature of about 100 rpm to about 200 rpm using a shaker for about 7 days to about 21 days. Astragalus membranaceus strain solution. Approximately 0.6 liters to about 1.6 liters of Antrodia camphorata strain solution is inoculated into about 1 kg of a mixture of about 5% to about 15% by weight of the whole grain, and placed in an environment of about 25 ° C to about 30 ° C for about 7 It takes about 14 days until the mycelium is covered to obtain the body of the burdock. The burdock body is stimulated by a temperature gradient to obtain a fermentation product of Antrodia camphorata. The fermentation of the Antrodia camphorata is dried and ground into a powder. Take about 1 kg of the dried powdered Antrodia camphorata fermentation product, add about 10 liters to about 20 liters of water, and use an ultrasonic oscillator to carry out extraction at room temperature for about 2 hours to about 4 hours, and then separate the residue to Obtain the extract of Antrodia camphorata.

Embodiments 3 to 5 of the present invention are similar to the method of Example 2, except that the fermentation of the Antrodia camphorata is about 50% to about 60% alcohol, about 0.5N to about 1N acetic acid, and about 0.1M, respectively. About 0.2 M sodium bicarbonate was extracted, and after extraction, Example 3 was subjected to dialysis to remove alcohol, and Example 4 and Example 5 were adjusted to pH neutral. Comparative Example 1 is a 2% citric acid prepared by a conventional technique, and Comparative Example 2 is a 7% arbutin prepared by a conventional technique.

Followed by measuring the inhibition of tyrosinase, the method: take to test the extract solution 50 in the well 200 μ L (Well) dish, 50 to 200 μ L of potassium phosphate buffer, was added to 10 after 100 μ L tyrosine (L- or dopamine) substrate solution (at a concentration of 0.1 to 1mg / mL), placed in a shaker bath uniformly mixed 10 minutes shaking, the entire sample was added 10 to 100 μ L tyrosine Enzyme (100 to 300 units/mL), the sample is mixed with an enzyme immunoassay analyzer (ELISA) for 5 to 10 seconds before measurement, and measured at a wavelength of 475 nm. The measured initial absorbance is a blank group (A ₀ After reacting at 30 to 40 ° C for 20 to 30 minutes, the measured absorbance of the sample is (A _S ); the sample is replaced with an equal volume of potassium phosphate buffer, and the reaction is measured after 20 to 30 minutes. The absorbance value is the control group (A _b ). The tyrosinase inhibition rate was calculated as: tyrosinase inhibition rate (%) = (A _b - (A _{S -} A ₀ )) / A _b × 100%.

Comparative Examples 1, 2 and 5 to 10 times diluted extracts of Examples 2, 3, 4, and 5 were measured for their tyrosinase monophenol inhibition rate and tyrosinase bisphenol inhibition rate, respectively. As shown in Figures 2a and 2b.

Please refer to FIGS. 2a and 2b, which are strip diagrams showing the inhibition rates of the tyrosinase monophenols measured by the extracts obtained in Examples 2 to 5 of the present invention and Comparative Examples 1 to 2, respectively. The extracts obtained in Examples 2 to 5 of the invention and Comparative Examples 1 to 2 were measured as bar graphs showing the inhibition rate of tyrosinase bisphenol. As can be seen from the two figures, in Example 3, the inhibition rates (37.22% and 33.78%) of monophenol or bisphenol were better than those of the other examples, so that the latter examples all used alcohol as a solvent for extracting the fermentation of Antrodia camphorata.

Embodiments 6 to 9 of the present invention are similar to the method of Example 3 except that the mixing ratio of the medium is as shown in Table 1 below. Comparative Examples 1, 2 and 5 to 10 times diluted extracts of Examples 6, 7, 8, and 9 were measured for their tyrosinase monophenol inhibition rate and tyrosinase bisphenol inhibition rate, respectively. As shown in Figures 3a and 3b.

Please refer to FIGS. 3a and 3b, which are strip diagrams showing the inhibition rates of the tyrosinase monophenols measured by the extracts obtained in Examples 6 to 9 of the present invention and Comparative Examples 1 to 2, respectively. The extracts obtained in Examples 6 to 9 of the invention and Comparative Examples 1 to 2 were measured as bar graphs showing the inhibition rate of tyrosinase bisphenol. As can be seen from the two figures, the inhibition rate (47.22% and 37.63%) of the monophenol or bisphenol in Example 6 is better than that of the other examples, so the subsequent examples are the mixing ratios used in the embodiment 6. Whole grains are used as a medium.

Embodiments 10 to 14 of the present invention are similar to the method of Embodiment 6, except that the temperature gradient stimulation growth treatment is temperature gradient increasing stimulation growth, temperature gradient decreasing stimulation growth, repeated temperature gradient stimulation growth, and repeated 3 The secondary temperature gradient stimulated growth and repeated 4 temperature gradients stimulated growth. While Comparative Example 3 is similar to the method of Example 6, the only difference is that the Antrodia camphorata entity is not subjected to temperature gradient stimulation growth treatment. The extracts obtained in Comparative Examples 1 to 3 and 5 to 10 times diluted in Examples 10, 11, 12, 13, and 14 were respectively measured for tyrosinase monophenol inhibition rate and tyrosinase bisphenol inhibition rate. The results are shown in Figures 4a and 4b.

Please refer to FIGS. 4a and 4b, which are bar graphs showing the inhibition rates of tyrosinase monophenols measured in the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 3, respectively. Further, the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 3 were measured as bar graphs showing the inhibition ratio of tyrosinase bisphenol. From the two figures, it can be seen that Examples 12, 13 and 14 of the growth of the Antrodia camphorata body by repeated temperature gradient stimulation, compared with the inhibition rate of monophenol or bisphenol, compared with the growth rate without temperature gradient stimulation, Comparative Example 3, temperature gradient Example 10 with increasing stimulation growth and Example 11 with decreasing temperature gradient stimulation were preferred. And as the number of repeated stimuli increases, the effect is more pronounced. The inhibition rates of Example 14 such as the repeated four stimulations were 91.88% and 88.76%, respectively, and even quite close to the tannins used in the prior art (Comparative Example 1) and the arbutin in the inhibition rate of monophenol (Comparative Example 2). .

Next, mouse melanoma cells B16-F10 were cultured with the extract, and the tyrosinase activity in B16-F10 cells and the melanin content in B16-F10 cells were measured. Culture methods are: 8 × 10 ⁴ cells / mL access 6- well culture plate, the extract was added 48 hours after co-culture, the cells were washed once in DPBS, was added 260 μ L of reaction 1X trypsin-EDTA, was added 1mL culture medium and then centrifuged 1000rpm 5 minutes to collect the cells were then washed twice in DPBS to remove residual trypsin-EDTA, followed by addition of 150 μ L lysis buffer (lysis buffer) 0.1M phosphate buffer (150ml of The solution (pH 6.8) contained 1% Triton X-100 (Sigma) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), oscillated uniformly, and then placed in liquid nitrogen for 3 minutes, followed by super The sonic wave was shaken for 3 minutes, the freezing was repeated for 3 minutes, and the ultrasonic wave was shaken for 3 minutes 3 to 4 times. After the cells were broken, the cells were centrifuged at 12000 rpm for 15 minutes at 4 ° C. The method for measuring tyrosinase activity in B16-F10 cells was as follows: centrifugation after 50 μ L of supernatant with 100 μ L of 0.1% of L-phenylalanine 3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalanine, Dopa ) after mixing, the reaction at 37 ℃ 1 hour measured at 475nm Absorbance value. The method for measuring the melanin content in B16-F10 cells is as follows: the supernatant after centrifugation After the liquid was removed, 1N NaOH was added, followed by heating at 80 ° C for 1 hour, and the absorbance was analyzed at a wavelength of 405 nm.

Comparative Example 4 is a mouse melanoma cell to which no extract was added. B16-F10. The extracts obtained in Comparative Examples 1 and 2, and Comparative Example 3 and Examples 10, 11, 12, 13, and 14 were diluted with B16-, respectively, based on the values measured in Comparative Example 4. The F10 cells were cultured, and the cell viability of each extract against B16-F10, the tyrosinase activity in B16-F10 cells, and the melanin content in B16-F10 cells were measured, and the results are shown in Fig. 5a to Fig. 5c.

Please refer to FIGS. 5a to 5c, which are bar graphs showing the cell survival rate of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 4, respectively, and Example 10 of the present invention. The extracts prepared in 14 and Comparative Examples 1 to 4 were measured in the bar graph of intracellular tyrosinase activity and the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1 to 4 were measured intracellularly. Bar graph of melanin content. As can be seen from Fig. 5a, whether it is Comparative Example 3 which was not stimulated by temperature gradient stimulation, or Examples 10 to 14 which were stimulated by various temperatures, the cell viability was greater than 80%, which means Antrodia camphorata extract does not cause toxicity to cell growth. From Fig. 5b and Fig. 5c, it can be seen that Comparative Example 3, which was not stimulated by temperature gradient stimulation, Example 10 which was stimulated by temperature gradient increase stimulation, and Example 11 which was stimulated by temperature gradient decrease, decreased intracellular tyrosine The effects of enzyme activity and melanin content are not obvious. Examples 12, 13 and 14 which were stimulated by repeated temperature gradients twice, 3 times and 4 times, respectively, were effective in reducing intracellular tyrosinase activity and melanin content, which is close to the tannic acid used in the prior art (Comparative Example) 1) and arbutin (Comparative Example 2). And as the number of repeated stimuli increases, the effect is more pronounced. For example, the tyrosine activity of Example 14 is only 18.67%, while the melanin content is only 31.33%.

Next, the measurement of the antioxidant power is performed, which can be expressed by the reducing power and the DPPH radical scavenging rate. The reducing power is measured by taking 10 to 100 μL of the extract solution, adding 10 to 100 μL, 200 mM phosphate buffer (sodium phosphate, pH 6.6), and 10 to 100 μL of 1% red blood salt (potassium ferricyanide, K). ₃ Fe(CN) ₆ ) Mix well, place at 50 ° C for 20 minutes, add 10 to 100 μL of 10% trichloroacetic acid (TCA), centrifuge at 9000 rpm for 3 minutes, and take the supernatant. 10 to 100 μL of 10 to 100 μL of distilled water and 10 to 100 μL of a 0.1% ferric chloride solution (FeCl ₃ ) were added, and after sufficiently mixing, the absorbance was measured at a wavelength of 700 nm. The method of measuring the clearance rate of DPPH radical is: Fresh configuration 0.2mg / mL methanol solution of DPPH 100 to 200 μ L, was added extract solution from 50 to 150 [mu] L after uniformly mixed by shaking, protected from light for 30 minutes. The absorbance at 517 nm was measured by an enzyme immunoassay (ELISA), and 2% citric acid and 3% vitamin E were used as a control. (A _b ) is the blank group absorbance; (As) is the absorbance of the extract solution. The DPPH free radical scavenging rate is calculated as: DPPH free radical scavenging rate (%) = [1-(As/Ab))] × 100%.

Comparative Example 5 is a 0.1% vitamin C prepared by a conventional technique, and Comparative Example 6 is a 3% vitamin E prepared by a conventional technique. The extracts prepared in Comparative Example 1 and Comparative Example 5 and Examples 10, 11, 12, 13, and 14 were diluted to 0.5%, and the reducing power thereof was measured; and Comparative Example 1 and Comparative Example 6 and Example 10 were compared. The extracts prepared in 11, 12, 13, and 14 were measured for DPPH radical scavenging rate, and the results are shown in Figures 6a and 6b.

Please refer to FIGS. 6a and 6b, which are bar graphs of the extracts obtained in Examples 10 to 14 of the present invention and Comparative Examples 1, 3 and 5, respectively, and the examples of the present invention. The strips prepared from 10 to 14 and Comparative Examples 1, 3 and 6 were measured for the DPPH radical scavenging rate. From the two figures, it can be seen that Examples 12, 13 and 14 of the growth of the Antrodia camphorata body by repeated temperature gradient stimulation, compared with the growth rate or the DPPH free radical scavenging rate, compared with the temperature gradient without the temperature gradient stimulation. Example 10 with increasing stimulation growth and Example 11 with decreasing temperature gradient stimulation were preferred. Therefore, stimulation of growth by repeated temperature gradients can improve the antioxidant capacity of Antrodia camphorata extract.

Next, the enzyme inhibition kinetics were measured by formulating different concentrations of dopamine substrate (0 to 0.8 g/L) and respectively carrying out different concentrations of extract inhibitor (0 to 40 g/L) in the well plate. Aminase inhibition assay, using ELISA to monitor melanin changes over time, The maximum reaction rate between different concentrations of dopamine substrate and extract inhibitor was determined by Michaelis-Menten method, and the enzyme kinetic parameters Km, Vm, Km, app and KI were analyzed by Lineweaver-Burk double reciprocal plot. Regression of different concentrations of the extract inhibitor curve, by linear regression of the intersection of the position can be concluded that the inhibitor is competitive, noncompetitive or uncompetitive.

The extracts obtained in Example 14 were separately formulated to a concentration of 0, 0.1, 1, 5, 10, 20 and 40 mg/mL, respectively, and reacted with various concentrations of dopamine substrate, and plotted according to the Lineweaver-Burk double reciprocal. . Please refer to Figures 7a to 7c, which are Lineweaver-Burk plots of inhibitors of different concentrations of the extract prepared in Example 14 on the decomposition of different concentrations of dopamine substrate by tyrosinase, respectively, and Example 14 of different concentrations. The XY scatter plot of the slope versus concentration of the extract obtained in the Lineweaver-Burk plot and the Km obtained from the extract of Example 14 in the Lineweaver-Burk plot, the XY spread of the _app value versus the concentration Figure. As can be seen from the figure, when the concentration of the extract obtained in Example 14 is increased, the slope obtained is higher and both intersect on the Y-axis, and the maximum reaction rate (Vm value) obtained is constant as the concentration of the extract is increased. However, its K _{m, app} value will increase with the increase of extract concentration, which proves that the mechanism of inhibition of tyrosinase by Antrodia camphorata extract is competitive inhibition. However, in competitive inhibition behavior, Antrodia camphorata extract (I) interacts with free enzyme (E) to produce an enzyme-inhibitor complex (EI), while blocking tyrosinase and dopamine matrix (S) The resulting enzyme-matrix complex (ES) acts to block the formation of melanin. In addition, the slope and K _{m, app} values are linear with the concentration of the extract inhibitor.

Please refer to Figures 8a and 8b, which are HPLC images of flavonoids and HPLC images of the extracts obtained in Example 14 of the present invention, respectively. As shown in the figure, the composition of the extract of Antrodia camphorata according to any one of the embodiments of the present invention is analyzed by High Performance Liquid Chromatography (HPLC), which has a distinct peak at 4 to 8 minutes in the spectrum, and The peaks of the polyphenolic bioflavonoids are similar. HPLC map As a result, it was preliminarily judged that the extract of the extract of Antrodia camphorata contains polyphenols of bioflavonoids.

In summary, the method for preparing an extract of Antrodia camphorata of the present invention, which has the tyrosinase inhibition rate, the survival rate to B16-F10 cells, and the B16-F10 cell yoghurt extract obtained by the extract of Antrodia camphorata Amino acidase activity, melanin content in B16-F10 cells, and antioxidant capacity are comparable to those of commonly used whitening products, and even better. At the same time, the Antrodia camphorata extract obtained by the method of the four times repeated temperature gradient stimulation growth has the best performance, so the repeated temperature gradient stimulation growth in this method helps to improve the inhibition of tyramine by Antrodia camphorata extract. The ability of the acid enzyme to produce extracts using this method is most suitable as a new generation of whitening cosmetics.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

S1~S3‧‧‧ steps

Claims (8)

A method for preparing an extract of Antrodia camphorata for whitening, comprising the steps of: inoculating a strain of Antrodia camphorata in a medium for a predetermined period of time to obtain a body of Antrodia camphorata; and repeating the body of the Antrodia camphorata 2 to 4 times Temperature gradient stimulating growth treatment to obtain a burdock fermented product; and extracting the burdock fermented material in a solvent to obtain an extract of Antrodia camphorata; wherein each repeated temperature gradient stimulating growth treatment comprises sequentially following a fourth period And cultivating, wherein the first period is about 4 ° C to about 8 ° C From about 3 days to about 7 days; the second period is from about 10 ° C to about 16 ° C for from about 3 days to about 7 days; the third period is from about 18 ° C to about 24 ° C for from about 3 days to about 7 Days; and the fourth period is from about 26 ° C to about 32 ° C for from about 3 days to about 7 days. The method of claim 1, wherein the A. angustifolia strain liquid system comprises: a strain of Antrodia camphorata; and a culture solution comprising about 1% to about 10% glucose, and about 1% to about 10% wood. Sugar, from about 0.1% to about 1% yeast extract, from about 0.1% to about 1% peptone, and from about 0.2% to about 2% coffee Brown slag. The method of claim 2, wherein the medium comprises from about 5% to about 15% by weight of white rice, brown rice, purple rice, wheat, oats, oatmeal, sorghum or coix seed. The method of claim 3, wherein the ratio of the A. angustifolia strain liquid to the medium is about 0.6 liters to about 1.6 liters of the A. sinensis strain solution and about 1 kg of the medium. The method of claim 1, wherein the predetermined time is from about 7 days to about 14 days. The method of claim 1, wherein the solvent comprises water, alcohol, acetic acid or sodium hydrogencarbonate. The method of claim 6, wherein the ratio of the fermentation of the Antrodia camphorata to the solvent is about 1 kg of the fermentation of the Antrodia camphorata and about 10 liters to about 20 liters of the solvent. The method of claim 7, wherein the fermented Antrodia camphora fermented product further comprises drying and grinding the astragalus fermentate into a powder.