TWI645861B - Extract of ganoderma formosanum mycelium for skin lightening and preparation method thereof - Google Patents

Abstract

The invention relates to a method for preparing a mycelium extract of Mycelia sinensis with whitening effect, comprising the steps of: extracting a Mycelium of Mycelium of Taiwan and a first solvent at a first temperature to obtain a first extract; Purifying the first extract and then dissolving in a second solvent to obtain a second extract; reacting the second extract with a third solvent to obtain an organic layer extract and an aqueous layer extract; and purifying the organic layer extract The substance is prepared to have a substance which inhibits the activity of tyrosine.

Description

 Taiwan white mycelium mycelium extract with whitening effect and preparation method thereof  

The invention provides a mycelial extract with whitening effect and a preparation method thereof, in particular to a mycelium extract of Mycelium of Taiwan having good activity for inhibiting tyrosine, and a preparation method thereof.

Human skin is often exposed to oxygen or ultraviolet light and is one of the largest organs damaged by such oxidative stress. In recent years, it has been considered that various reactive oxygen species generated by ultraviolet rays cause peroxidation of sebum or lipids, protein denaturation, enzyme inhibition, and the like, which cause skin inflammation in a short period of time and cause aging or cancer in the long term. The skin aging (such as spots, freckles) and pigmentation caused by photooxidation pressure are caused by the abnormality of hormones or the stimulation of ultraviolet rays, which causes the melanin in the epidermal pigment cells to be hyperthyroidized, thereby causing the melanin to be excessive in the epidermis.

It is currently known that the melanin of mammals is composed of two components, eumelanins and pheomelanins, in different proportions. The formation of melanin requires several enzymes and molecules to participate in the biochemical reaction, namely tyrosine, tyrosinase and oxygen. Tyrosinase oxidizes tyrosine to dihydroxyphenylalanine, which is then converted to dopachrome, to form indoles, and finally to melanin. Among them, tyrosinase catalyzes the conversion of dihydroxyphenylalanine to dopachrome as a rate limiting step, and dopachrome forms melanin via an autooxidation reaction.

In short, when tyrosinase activity is increased, melanin production is increased; when tyrosinase activity is inhibited, the ability of melanocytes to produce melanin is correspondingly reduced. There are many causes of melanin precipitation. When the skin is exposed to ultraviolet light, intracellular tyrosinase activity is enhanced, and melanin synthesis is increased. Therefore, inhibition of tyrosinase activity is one of methods for reducing melanin production.

In the past, a method for preventing the formation of spots and freckles is a method of administering a drug or a substance for inhibiting melanin production. For example, a method of administering L-ascorbic acid in a large amount, a method of partially coating using an ointment containing citric acid, cysteine, or the like, a cream, an emulsion, etc., but all have doubts about effects, stability, and safety. . In view of the above, there is a need for an agent that has excellent melanin production inhibition ability and high safety.

The present invention has been made in view of the above-mentioned problems of the prior art, and an object thereof is to provide a whitening agent having excellent melanin production inhibiting ability and high safety and a method for producing the same. Further, it is to provide a whitening agent which has excellent tyrosinase activity inhibiting ability and high safety, and is easy to prepare and can be carried out under mild conditions.

In order to achieve the above object, the inventors of the present invention have found that the extract obtained from the mycelium of Taiwan mycelium has excellent tyrosinase activity inhibition ability and high safety, and can be used as a whitening agent. Further, the melanin production inhibitory action is also clear, and thus the present invention has been completed.

The invention provides a preparation method of the mycelium extract of Mycelium of Taiwan, the steps comprising: (A) extracting a Mycelium of Mycelium of Taiwan and the first solvent at a first temperature to obtain a first extract; (B) Purifying the first extract and then dissolving in a second solvent to obtain a second extract; (C) reacting the second extract with a third solvent to obtain an organic layer extract and an aqueous layer extract; D) Purifying the organic layer extract to obtain an active substance having strontium strontium inhibiting.

According to a preferred embodiment of the present invention, in the step (A), the first solvent is selected from the group consisting of a mixture of water and a lower alcohol. The first solvent is a 95% ethanol solution and the reaction time in step (A) is at least 1 hour. The first temperature is between 60 and 100 degrees Celsius. In the step (B), the second solvent is selected from the group consisting of pure water and deionized water.

According to a preferred embodiment of the present invention, in the step (C), the third solvent is selected from the group consisting of lower alkyl esters. More preferably, the third solvent is ethyl acetate.

According to a preferred embodiment of the present invention, after the step (B), the method further comprises: (B1) extracting the second extract with a lower alkane solvent. More preferably, the lower alkane solvent is n-hexane.

The invention further provides an extract of Mycelium of Mycelium of Taiwan, which is an extract obtained by inhibiting tyrosinase activity and obtained by the following steps: a Taiwan mycelium mycelium and a 95% ethanol solution at 100 degrees Celsius Performing an extraction reaction to obtain a first extract; purifying the first extract and then dissolving in deionized water to obtain a second extract; and reacting the second extract with an ethyl acetate solvent to obtain an ethyl acetate layer extract and An aqueous layer extract; and the ethyl acetate layer extract is purified to obtain an active substance having strontium strontium inhibiting. After the step (B), the method further comprises: (B1) extracting the second extract with a lower alkane solvent. More preferably, the lower alkane solvent is n-hexane.

1 is an experimental result of the survival rate of mouse melanoma cells treated with the mycelium extract of Mycelium of Taiwan genus prepared in the examples of the present invention.

2 is a result of measuring the melanin content of mouse melanoma cells treated with the mycelium extract of Mycelium of Taiwan genus prepared in the examples of the present invention.

Fig. 3 is a graph showing the results of measuring the intracellular tyrosinase activity of mouse melanoma cells treated with the mycelium extract of Mycelium of Taiwan genus prepared in the present invention.

4A is an experimental result of inhibiting melanin production of zebrafish by the mycelium extract of Mycelium of Taiwan prepared in the examples of the present invention.

4B is an experimental result of inhibiting tyrosinase activity of zebrafish by the mycelium extract of Mycelium of Taiwan prepared in the examples of the present invention.

Fig. 5A is an experimental result of the effect of the mycelium extract of Mycelium of Taiwan on the survival rate of zebrafish prepared in the examples of the present invention.

Fig. 5B is an experimental result of the effect of the mycelium extract of Mycelium of Taiwan prepared in the examples of the present invention on the heart rate of zebrafish.

The invention inhibits the melanin production of the extract by the Mycelium of Mycelium of Taiwan, and proves that the extract has anti-tyrosine activity and reduces the effect of melanin expression by using cell experiments and fish body system. The description and the following description are for the purpose of illustrating the preferred embodiments of the invention. Other embodiments of the present invention will be readily understood by those skilled in the art from this disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention.

Cultivation and acquisition of mycelium

In the present example, the mycelium culture was carried out by using Ganoderma formosanum mycelium (ATCC 76537) for fermentation. In general, there are two types of fermentation methods: solid and liquid deep fermentation. The former is a mycelium or a dried mycelium obtained by inoculating a mycelium in a solid medium (for example, compost and wood chips) containing a carbon source or a nitrogen source. In the latter case, microorganisms are cultured in a suitable environment using a liquid medium of a fixed composition to produce biomass (Biomass) and other metabolites (Metabolite). In this embodiment, the Mycelium of Mycelium of Taiwan is selected to be placed in a bioreactor for Submerged Fermentation, and then dried to obtain a dried mycelium by freeze-drying.

Example 1 - Preparation method of Taiwan mycelium mycelium extract

Step 1: The dried mycelium of Taiwan purple sage obtained by the above method is mixed with a 95% ethanol solution at a ratio of 1:25, and reacted at 60 ° C for at least one hour, and the extract is collected, and then 95% ethanol solution is added. Repeat the above steps. Step 2: The final ethanol extraction of step one is concentrated and concentrated under reduced pressure, followed by re-dissolution with deionized water. The aqueous layer was further subjected to an extraction reaction with ethyl acetate (EA) in a volume ratio of 1:1, and the extraction reaction was repeated three times, and finally, ethyl acetate was layered. Step 3: The ethyl acetate was layered and dried under a reduced pressure concentrator, and weighed to obtain the mycelium extract of Mycelium of Taiwan.

Example 2 - Preparation method of Taiwan mycelium mycelium extract

Step 1: The dried mycelium of Taiwan purple sage obtained by the above method is mixed with a 95% ethanol solution at a ratio of 1:25, and reacted at 60 ° C for at least one hour, and the extract is collected, and then 95% ethanol solution is added. Repeat the above steps. Step 2: The final ethanol extraction of step one is concentrated and concentrated under reduced pressure, followed by re-dissolution with deionized water. The extraction reaction was then carried out in a volume ratio of 1:1 using n-hexane and deionized water, and the extraction reaction was repeated three times. After collecting the n-hexane layer, only the water layer portion was taken, and the aqueous layer was continuously extracted with ethyl acetate (EA) in a volume ratio of 1:1, and the extraction reaction was repeated three times, and finally the ethyl acetate layer was collected. . Step 3: The ethyl acetate was layered and dried under a reduced pressure concentrator, and weighed to obtain the mycelium extract of Mycelium of Taiwan.

Example 3 - Preparation method of Taiwan mycelium mycelium extract

Step 1: The dried mycelium of Taiwan purple stalk obtained by the above method is mixed with a 95% ethanol solution at a ratio of 1:25, and reacted at 95 ° C for at least one hour, and the extract is collected, and then 95% ethanol solution is added. Repeat the above steps. Step 2: The final ethanol extraction of step one is concentrated and concentrated under reduced pressure, followed by re-dissolution with deionized water. The aqueous layer was further subjected to an extraction reaction with ethyl acetate (EA) in a volume ratio of 1:1, and the extraction reaction was repeated three times, and finally, ethyl acetate was layered. Step 3: The ethyl acetate was layered and dried under a reduced pressure concentrator, and weighed to obtain the mycelium extract of Mycelium of Taiwan.

Example 4 - Preparation method of Taiwan mycelium mycelium extract

Step 1: The dried mycelium of Taiwan purple stalk obtained by the above method is mixed with a 95% ethanol solution at a ratio of 1:25, and reacted at 95 ° C for at least one hour, and the extract is collected, and then 95% ethanol solution is added. Repeat the above steps. Step 2: The final ethanol extraction of step one is concentrated and concentrated under reduced pressure, followed by re-dissolving in deionized water. The extraction reaction was then carried out in a volume ratio of 1:1 using n-hexane and deionized water, and the extraction reaction was repeated three times. After collecting the n-hexane layer, only the water layer portion was taken, and the aqueous layer was continuously extracted with ethyl acetate (EA) in a volume ratio of 1:1, and the extraction reaction was repeated three times, and finally the ethyl acetate layer was collected. . Step 3: The ethyl acetate was layered and dried under a reduced pressure concentrator, and weighed to obtain the mycelium extract of Mycelium of Taiwan.

Example 5 - Cell Test

In this example, mouse melanoma cell cells (B16-F10) were used to test the safety of the mycelium extract of Mycelium of Taiwan and the effect of inhibiting tyrosinase.

5-1 Cell Culture - Mouse Melanoma Cell Line (B16-F10)

The mouse melanoma cells (B16-F10; BCRC 60031) were cultured in DMEM medium (Dulbecco's modified Eagle's medium) , and 10% fetal bovine serum (FBS, Hyelone, Logan, UT , USA), 50 μ g / mL penicillin and 50 μ g / mL streptomycin, at 37 [deg.] C and maintained rearing tank comprises culturing in 5% CO _2.

Determination of 5-2 decellularized tyrosinase activity inhibition

Cell-free measurement according to the method proposed by Likhitwitayawuid, K. & Sritularak, B. (A new dimeric stilbene with tyrosinase inhibitiory activity from Artocarpus gomezianus. J Nat Prod. 64, 1457-1459, doi: 10.1021/np0101806, 2001) The extent to which tyrosinase activity is inhibited. A 20 μL-free decellularized tyrosinase solution from Pleurotus ostreatus (480 units/mL; Sigma, St. Louis, MO, USA) was extracted with 180 μL of different concentrations of Ganoderma formosanum obtained by the preparation method of the present invention. Mix in liquid phase. The degree of inhibition of tyrosinase activity is calculated by the following formula: (%) = [1-(CD) / (AB)] × 100. Wherein A and B respectively represent the absorption values of the control group with and without tyrosinase; C and D respectively represent the absorption values of the control experimental group with and without tyrosinase.

5-3 Comparison of extraction solvents used in the extract of the present invention

Please refer to the preparation methods of the foregoing Examples 1-4 and Table 1 below.   It can be seen that in the second step, if ethyl acetate (EA) is used for extraction, the obtained extract has the best degree of inhibition of tyrosinase activity, and it can be confirmed that extraction with ethyl acetate (EA) has a remarkable effect.

5-4 Cell viability assay

Cell viability was determined with WST-1 reagent (Roche, Mannheim, Germany). The mouse melanoma cells were seeded in a 96-well plate (10,000 cells/well). After the cells were cultured for 24 hours, the old medium was removed and the extract of Taiwan purple lucidum (concentration: 50-200 ppm) obtained by the preparation method of the present invention was used. The new medium was replaced, and after the new medium was cultured for 48 hours, the medium was removed and replaced with WST-1 reaction reagent for 30 minutes. Cell viability was determined using a measurement method - Thermo Multiskan GO (Thermo Scientific, Waltham, MA, USA) at an absorbance at a wavelength of 450 nm. The experimental results are shown in Fig. 1. It can be seen that compared with the control group, the extract of Taiwan Zizhi obtained by the preparation method of the present invention does not affect the survival rate of the cells, and the safety can be proved.

5-5 melanin content determination

The mouse melanoma cells were seeded in a 24-well plate (50,000 cells/well). After the cells were cultured for 24 hours, the old medium was removed and replaced with a new medium containing the extract of Taiwan's Violet extract obtained by the preparation method of the present invention. After 48 hours of culture, in order to analyze the secreted melanin, the collected medium was determined using an absorbance at a wavelength of 450 nm and a standard curve of synthetic melanin (Sigma, St. Louis, MO, USA). The medium was removed and replaced with WST-1 reaction reagent for 30 minutes. Cell viability was determined by the absorbance at a wavelength of 450 nm using a measurement method Thermo Multiskan GO (Thermo Scientific, Waltham, MA, USA). To measure intracellular melanin content, mouse melanoma cells were washed with PBS solution and separated by trypsin-EDTA treatment. The collected cell liquid was centrifuged and dissolved in a 1 N sodium hydroxide solution, and heated at 60 ° C for one hour, and the supernatant was taken out and its absorbance at a wavelength of 405 nm was measured. The experimental results are shown in Fig. 2. It can be seen that compared with the control group, the extract of Taiwan purple lucidum obtained by the preparation method of the present invention can inhibit the melanin production amount of the cells.

5-6 intracellular tyrosinase activity assay

The method for determining tyrosinase activity of mouse melanoma cells is mainly used by Hu, ST et al. (Oxyresveratrol and trans-dihydromorin from the twigs of Cudrania tricuspidata as hypopigmenting agents against melanogenesis. J Funct Foods 13, 375-383, doi: 10.1016/ J.jff.2015.01.010) and Kim, JH, et al. (Downregulation of melanin synthesis by haginin A and its application to in vivo lightening model. J Invest Dermatol. 128, 1227--1235, doi: 10.1038/sj.jjd. The method of 5701177) was slightly improved. After the mouse melanoma cells were treated with the reaction reagent for 24 hours, each culture plate was rinsed with a phosphate buffer solution at 0 °C. The cells were uniformly mixed with an equal volume of lysis buffer (20 mM Tris-0.1% Triton X-100), and after standing on an ice bath, it was further centrifuged for 5 minutes (10000 g). The protein content in the supernatant was determined by the Bradford assay (Bio-Rad, Richmond, CA, USA). Tyrosinase activity in the cell test in the following manner: to obtain a concentration 20mM phosphate buffer solution (pH 6.8) and the concentration of 2mM L-DOPA supernatants 50 μ g protein consisting of the reaction mixture was 100 μ L, at 37 [deg.] C under static After 1 hour, the absorbance of the resulting dopachrome at 475 nm was measured. The experimental results are shown in Fig. 3. It can be seen that compared with the control group, the extract of Taiwan purple lucidum obtained by the preparation method of the invention can significantly inhibit the tyrosinase activity and the amount of melanin production in the cells by 50%.

Example 6 - Zebrafish Animal Test

The zebrafish used in the present invention is derived from the Techcomm Zebrafish Core of the University of Taiwan, and is housed in a water tank having a circulation system and a filter, and the light and dark periods are 14 and 10 hours, respectively. Zebrafish embryos cultured in medium 28 ℃ Danieau's of the [0.45mM HEPES (pH 7.6), 5.22mM NaCl, 0.063mM KCl, 0.054mM Ca (NO3) 2,0.036mM MgSO4], and adding 50 μ g / mL of penicillin and 50 μ g / mL streptomycin.

6-1 zebrafish evaluation method

The method for evaluating wild zebrafish of the present invention adopts Choi, TY et al. (Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pigm Cell Res. 20, 120-127, doi: 10.111/j.1600-0749.2007. The method of 00365.x, 2007) has been slightly improved.

6-2 fish tyrosinase activity and melanin content

The method for determining tyrosinase activity and melanin content in the present invention is based on Choi, TY et al. (Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pigm Cell Res. 20, 120-127, doi: 10.111/ The method of j.1600-0749.2007.00365.x, 2007) is slightly improved.

Approximately 70 synchronized zebrafish embryos were treated with the Taiwan Zizhi extract obtained by the preparation method of the present invention for 7 to 55 hours, and PTU was used as a positive control group. All protein collection methods were performed using PE LBTM tissue dissolving buffer (G-BIOSCIENCES, Maryland Heights, MO, USA) and protease inhibitors, followed by centrifugation for 5 minutes (10,000 x g at 4 ° C). The quantitation of the collected protein was performed using the Bradford assay and using fetal bovine serum albumin as a standard (Bio-Rad). Then take the protein solution 1mM L-DOPA (Sigma) collected by the 50 μ g at 37 [deg.] C for 1 hour. The activity of tyrosinase at 475 nm is calculated by the following formula: (%) = [1-(CD) / (AB)] × 100. Wherein A and B respectively represent the absorption values of the control group with and without tyrosinase; C and D respectively represent the absorption values of the control experimental group with and without tyrosinase. To determine the relative melanin content, the collected cell liquid was centrifuged and dissolved in a 1 N sodium hydroxide solution, and heated at 100 ° C for one hour, and the absorbance at a wavelength of 405 nm was measured. The experimental results are shown in FIG. 4A. It can be seen that compared with the control group, the extract of Taiwan Zizhi obtained by the preparation method of the present invention can inhibit the formation of 60% melanin in zebrafish. Figure 4B, which has the same inhibitory activity as tyrosinase, and achieves the same inhibition of melanin in the zebrafish mode with only 1/7 of the dose of citrate compared to the commercially available kojic acid. Effect.

6-3 Zebrafish Safety Test

As described in 6-2, about 70 synchronized zebrafish embryos were used as materials, and the extract of Taiwan Zizhi obtained by the preparation method of the present invention was treated for 7 to 55 hours, and PTU was used as a positive control group, and commercially available tannic acid was used as a control. Group, the survival rate and heart rate were observed under a microscope. The experimental results are shown in FIG. 5A. It can be seen that there is no statistically significant difference in the survival rate of the zebrafish zebrafish extract obtained from the preparation method of the present invention compared to the control group. As detailed in Figure 5B, it was found that 20 mM citric acid significantly affected the zebrafish heartbeat, and the ethyl acetate extract of the present invention had better biosafety than citric acid.

As described above, the extract of the present invention has an activity of inhibiting tyrosinase activity and melanin production, and can be used as a skin external preparation for whitening effect. In addition to the above-mentioned essential components, the skin external preparation may be appropriately blended with components used for external preparations for skin such as general cosmetics or pharmaceuticals, such as whitening agents, moisturizers, antioxidants, oily components, ultraviolet absorbers, and surfactants. , tackifiers, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, various pharmaceutical agents, chelating agents, pH adjusters, etc.

Claims (4)

A method for preparing a mycelium extract of Mycelium of Taiwan, the method comprising the steps of: (A) extracting a Mycelium of Mycelium of Taiwan and a first solvent at a first temperature to obtain a first extract; (B) purifying the first extract An extract is then dissolved in a second solvent to obtain a second extract; (C) reacting the second extract with a third solvent to obtain an organic layer extract and an aqueous layer extract; and (D) purifying the extract The organic layer extract is used to prepare an active material having strontium strontium sulphate ; wherein the first solvent is a 95% ethanol solution, and the first temperature is between 60 and 100 degrees Celsius, the second solvent The pure solvent or deionized water, the third solvent is ethyl acetate. The preparation method according to claim 1, wherein in the step (A), the reaction time is at least 1 hour. The preparation method according to the first aspect of the invention, after the step (B), further comprises: (B1) extracting the second extract with a solvent of n-hexane. A mycelium extract of Mycelium of Taiwan, which is an extract having tyrosinase activity inhibition and extracted by the following steps: (A) a Japanese mycelium mycelium and a concentration of 95% ethanol solution at 100 degrees Celsius Extracting to obtain a first extract; (B) purifying the first extract and then dissolving in deionized water to obtain a second extract; (C) reacting the second extract with an ethyl acetate solvent to obtain a first acetic acid An ester layer extract and an aqueous layer extract; and (D) purifying the ethyl acetate layer extract to obtain an active material having strontium strontium inhibiting.