TWI474828B - Anisomeles ovate extract for preventing melanin precipitation - Google Patents

Description

Parsnip extract for preventing melanin precipitation

This case relates to a parsnip extract for preventing melanin precipitation, in particular to an ability to inhibit tyrosinase activity, inhibit microlipid granule peroxidation, scavenge free radicals, and inhibit extracellular melanogenesis. Parsnip extract; thereby achieving the goal of improving melanin precipitation.

Although only Asian women in the traditional concept have a white ugly concept, women all over the world have the need to prevent pigmentation and speckle formation. Whether it is to prevent pigmentation or to prevent speckle formation, it is a commonly used effect to improve the composition of melanin precipitation. The mechanism of action involves the inhibition of melanin production. The literature indicates that melanin formation occurs in melanocytes, and the mechanism is as follows: melanin is formed by tyrosine (Tyrosine) in vivo to form dopa (D-Dopa), and then formed by tyrosinase again. Dopaquinone, once again undergoes a series of chemical reactions to form a mixed type of melanin; in general, the effect of improving the melanin-precipitating composition is to inhibit melanin production, which can be achieved by inhibiting melanin production and reduction in melanocytes. Melanin, promotes melanin excretion on the cell surface, and selectively exhibits cytotocixity to melanocytes; current practice Whitening ingredients are often used to improve melanin precipitation.

Compared with the current common whitening ingredients, including Vitamin C (L-ascorbic acid), Hydroquinone (Hydroquinone), and Kojic acid, these whitening ingredients have significant effects, but each has its own use. limit. 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, and long-term overuse of citric acid products can cause cytotoxicity and pathological changes. 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 and stable tyrosinase inhibitors to prevent the formation of melanin in the skin has become an important trend in the development of whitening products.

In summary, while considering the cytotoxicity and efficacy of the composition of melanin precipitation, the use of traditional Chinese medicine extract as a source of composition for improving melanin precipitation can reduce the doubts in use.

The main object of the present invention is to provide an angioside extract for preventing melanin precipitation, in particular to an ability to inhibit tyrosinase activity, to inhibit microlipid peroxidation, to scavenge free radicals, and A parsnip extract that inhibits the production of extracellular melanin; thereby achieving the goal of improving melanin precipitation.

In order to achieve the above-mentioned object, the present inventors propose a herbicide extract for preventing melanin precipitation, which is obtained by extracting parsnip with at least one step of an organic solvent; When the indicator standard is vitamin C, the parsnip extract has 0.1 to 2 times the ability to inhibit tyrosinase activity and 1 to 11 times the ability to inhibit extracellular melanin production compared to the index standard.

In one embodiment of the present invention, when the treatment standard of the indicator standard and the parsnip extract is 50 μg/mL, the parsnip extract has an ability to inhibit tyrosinase activity by 0.13 to 1.56 times compared with the index standard. And 5.52 to 10.7 times inhibition of extracellular melanin production; when the standard concentration of the standard and the parsnip extract is 100 μg/mL, the parsnip extract has 0.23 to 1.64 times inhibition of tyramine compared to the indicator standard. The ability of oxidase activity, and 1.20 to 1.77 times inhibition of extracellular melanogenesis.

In one embodiment of the present invention, the organic solvent is selected from the group consisting of esters, alcohols, alkanes, and halogenated alkanes; and the extracting of the parsnip comprises at least the following steps: Step 1: using the first organic solvent For example, a 95% by volume ethanol solution may be used to extract a parsnip material to obtain a first extract; and step 2: using a second organic solvent, for example, a 1:1 ratio of n-hexane/methanol solution. Extracting the first extract to divide a first organic phase and a second organic phase, wherein the first organic phase has a second extract and the second organic phase has a first residue; and step 3: utilizing a a third organic solvent, for example, a 2:1 by volume ethyl acetate/water solution, extracting a first residue to separate a third organic phase and a first aqueous phase, wherein the third organic phase has a third extraction And the first aqueous phase has a second residue; and step 4: using a fourth organic solvent, for example, a n-butanol/water solution having a volume ratio of 1:1, extracting the second residue to divide one Fourth organic phase and one Dihydrate phase, wherein the fourth phase has a fourth organic extract and a second aqueous phase having a third residue.

In one embodiment of the present invention, the first organic phase may be, for example, a n-hexane phase, the second organic phase may be, for example, a methanol phase, the third organic phase may be, for example, an ethyl acetate phase, and the fourth organic phase may be, for example, N-butanol phase.

In summary, the present invention provides a parsnip extract, which is a parsnip extract obtained by the above extraction step, whereby the parsnip extract is extracted, and the extract may include the use of the extract. The above steps provide the parsnip extract of each process stage, and have the ability to inhibit tyrosinase activity and inhibit melanin production, and thus can be applied to cosmetic material compositions, food additives or pharmaceutical compositions for whitening purposes. .

First: Flow chart of the steps of the parsnip extract of the present invention

As stated above, the present invention provides an angioside extract for preventing melanin precipitation, which utilizes at least one extraction step to obtain a parsnip having the ability to inhibit tyrosinase activity and inhibit melanin production. Extracts.

As used herein, "parsnip" refers to the aerial part of the Anisomeles anaphylla (Lamiaceae). The parsnip is a traditional Chinese medicine, and the traditional Chinese medicine for the parsnip The efficacy of cognition only exists in the effects of relieving pain, strengthening bones, dissipating heat and cooling, and has not found its function of whitening. The inventor of the present invention obtains the parsnip extract by at least one step of the organic solvent. The term "at least one step" as used herein refers to the preparation of the parsnip extract by using an organic solvent system of different polarity and establishing an improved melanin. Effect of formation The efficacy of previous applications for parsnips is a very rare case.

First, the parsnip extract for preventing melanin precipitation is obtained by extracting parsnip with at least one step of an organic solvent, and the organic solvent may be, for example, an ester, an alcohol, an alkane or a halogen. Ethnic group; wherein, when the indicator standard is vitamin C, the parsnip extract has 0.1 to 2 times the ability to inhibit tyrosinase activity and 1 to 11 times the ability to inhibit extracellular melanin production; Thus, the parsnip extract can be used as a cosmetic material composition, a food additive or a pharmaceutical composition.

In an embodiment of the present invention, the parsnip is subjected to at least the following steps: Step 1 (S1): extracting a parsnip material (101) by using a first organic solvent, for example, an ethanol solution, to obtain a first An extract (103); Step 2 (S2): extracting the first extract (103) by using a second organic solvent, for example, a n-hexane/methanol solution, to separate a first organic phase and a second organic a phase wherein the first organic phase can be, for example, a n-hexane phase and has a second extract (105), and the second organic phase can be, for example, a methanol phase, and having a first residue (107); S3): extracting a first residue (107) by using a third organic solvent, for example, an ethyl acetate/water solution, to fractionate a third organic phase and a first aqueous phase, wherein the third organic phase may be, for example, An ethyl acetate phase having a third extract (109) and a first aqueous phase having a second residue (111); and a fourth step (S4): utilizing a fourth organic solvent, which may be, for example, n-butyl An alcohol/water solution, extracting a second residue (111) to divide a fourth organic phase and a first a dihydrate phase, wherein the fourth organic phase can be, for example, a n-butanol phase, having a fourth extract (113), and The second aqueous phase has a third residue (115).

In one embodiment of the present invention, the aforementioned parsnip extract is prepared by using at least one step of an organic solvent, which comprises extracting with a 95% by volume ethanol solution, followed by successive use of a volume ratio of 1:1. The parsnip extract is extracted with an organic solvent such as n-hexane/95% methanol solution, a 2:1 volume ratio of ethyl acetate/water solution, and a volume ratio of 1:1 n-butanol/water solution.

In one embodiment of the present invention, the parsnip extract has "antimelanogenic activity", that is, the parsnip extract has the ability to inhibit tyrosinase activity and inhibit melanin production; When the concentration of the parsnip extract is 50 μg/mL and 100 μg/mL, the parsnip extract has the ability to inhibit tyrosinase activity by 0.13 to 1.56 times and 0.23 to 1.64 times, respectively, compared with the index standard. 5.52 to 10.7 times and 1.20 to 1.77 times inhibit the extracellular melanin production ability; thereby, the effect of improving melanin precipitation is achieved, and in addition, the parsnip extract has low cytotoxicity to melanocytes.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Preparation of Parsnip Extract

This example is a flow chart of the steps of using the parsnip extract of the first figure. First, a first extraction step is carried out by pulverizing the parsnip material (101) with a pulverizer at room temperature or slightly lower than room temperature, and then immersing the shattered parsnip material (101) in 95. % by volume of ethanol solution. Next, a filtration step of filtering the above-mentioned 95% ethanol solution containing the pulverized turf material by gauze, filter paper or other filtration method was repeated for four times to obtain a filtrate. Then, after removing the ethanol solution by a reduced pressure concentration method, a first extract (103) is obtained, wherein the first extract (103) is a crude extract of the parsnip.

Subsequently, the first extract (103) is subjected to a partitioning step using a second organic solvent composed of a hexane/95% methanol solution having a volume ratio of 1:1, whereby the first organic phase and the second phase are obtained by layering. The organic phase wherein the first organic phase is the n-hexane phase and the second organic phase is the methanol phase. The first organic phase contains a second extract (105), the organic solvent is removed by vacuum concentration to obtain a second extract (105); and the second organic phase contains a first residue (107).

Further, after removing the organic solvent of the second organic phase by a reduced pressure concentration method, the first residue (107) is subjected to the third step using a third organic solvent having a composition ratio of 2:1 in an ethyl acetate/water solution ( S3), to divide the third organic phase and the first aqueous phase, the third organic phase is an ethyl acetate phase, and the third organic phase has a third extract (109), and the organic solvent is removed by a vacuum concentration method to obtain The third extract (109); further, the first aqueous phase has a second residue (111).

Finally, after the moisture of the first aqueous phase is removed by a vacuum concentration method, the second residue (111) is subjected to step four (S4) using a fourth organic solvent having a n-butanol/water solution of a volume ratio of 1:1. The fourth organic phase and the second aqueous phase are obtained by layering, wherein the fourth organic phase is a n-butanol phase, and the fourth organic phase contains a fourth extract (113), and the organic solvent is removed by a vacuum concentration method to obtain The fourth extract (113); and the second aqueous phase contains a third residue (115).

It is worth mentioning that when the partitioning step is carried out by the method of the invention, the layering is relatively easy, and the foaming is not easy, so that the processing time can be shortened; in addition, the organic solvent or moisture is removed by the vacuum concentration method between the above extraction steps. The parsnip extracts obtained in different stages of the process can be used to find the active ingredients in the parsnip that have improved melanin precipitation.

Example 2: Evaluation of cytotoxicity of parsnip extract

In this example, it was first tested whether the parsnip extract of Example 1 at different stages of the process was cytotoxic to skin keratinocytes or melanocytes. In this example, the cell strains used for testing were HaCaT human skin keratinocyte cell line (ATCC number: CCL-155) and B16 mouse melanoma cell line (ATCC number: CRL-6322), of which ATCC is typical of the United States. Abbreviation for the American Type Culture Collection (ATCC). The above cell lines are cultured in a cell culture medium by a sterile technique, wherein the cell culture medium of the HaCaT human skin keratinocyte cell line and the B16 mouse melanoma cell line is Dulbecco's Modified Eagle's Medium (DMEM). ; Gibco, Grand Island, NY) plus an additional 10% by volume of fetal bovine serum (Hazelton Product, Denver, PA, USA) and 1 volume percent of antibiotics (containing penicillin and streptomycin, penicillin-streptomycin) . The above cells were seeded at 1.0×10 ⁴ cells per cell (cell density 1.0×10 ⁵ cells/mL, and the volume added per well was 100 μL), and then cultured at 37 ° C and maintained at a humidity of 5% carbon dioxide concentration. hour. After that, the parsnip extract (first to fourth extracts) (103) (105) (109) (113) or the concentration of 100 μg/mL of parsnip extract (first to fourth extracts) at a concentration of 50 μg/mL. (103) (105) (109) (113) is added to the above cells. After 72 hours of culture, 10 μL of 3-(4,5-dimethyloxazol-2)-2,5-diphenyltetrazolium bromide [3-(4,5-dimethylthiazol-2-) was added to each well of cells. Yl)-2,5-diphenyltetrazolium bromide; MTT tetrazolium; 5 mg/mL], after 4 hours at 37 ° C, the supernatant was removed, and 100 μL of dimethyl sulfoxide (DMSO; Sigma Chemical) was added. Co, St. Louis, USA) Dissolved MTT formazan (MTT formazan; product of MTT tetrazolium after reduction), after shaking for 10 minutes, using, for example, Multi-Detection Microplate Reader; Synergy TM 2. BioTek Instruments, Inc., USA) The absorbance at a wavelength of 570 nm was measured to calculate cell viability, and the results are shown in Table 1. The cell viability (%) treated with the extract without the parsnip was used as a control group (cell survival rate was 100%), and the cell survival rate of the other treatment groups was converted to a value obtained by comparison with the untreated control group. Each value is derived from at least 3 samples or more.

As can be seen from the results of the first table, compared to the cell survival rate of the control cells, In the same stage of the parsnip extract, HaCaT was treated with 50 μg/mL parsnip first extract (103), second extract (105), third extract (109) and fourth extract (113), respectively. Human skin keratinocyte cell line survival rate was 5.7±2.3%, 78.5±1.5%, 5.6±2.1% and 52.8±1.2%, respectively, while 100μg/mL parsnip first extract (103), second extraction The substance (105), the third extract (109) and the fourth extract (113) respectively treated the HaCaT human skin keratinocyte cell line, and the cell survival rates were 8.5±3.1%, 41.5±2.8%, 6.8±3.8%, respectively. 28.6 ± 4.6%. However, the B16 mouse melanoma cell line was treated with 50 μg/mL parsnip first extract (103), second extract (105), third extract (109) and fourth extract (113), respectively. Cell viability was 23.51±1.2%, 76.3±4.9%, 27.6±, respectively. 3.6% and 104.6±4.1%; while treating the B16 small with the first extract (103), the second extract (105), the third extract (109) and the fourth extract (113) of 100 μg/mL The cell viability of murine melanoma cell lines was 13.6 ± 2.9%, 31.1 ± 2.4%, 16.1 ± 2.8%, and 68.0 ± 2.3%, respectively. The results showed that the parsnip extract had lower cytotoxicity to the B16 mouse melanoma cell line than the HaCaT human skin keratinocyte strain. Four kinds of parsnip extracts were compared, wherein the second extract (105) and the fourth extract (113) of Parsnip were less cytotoxic to the B16 mouse melanoma cell line.

Example 3: Evaluation of the resistance of the herbicide extract to inhibit tyrosinase activity

In this example, the B16 mouse melanoma cell line of Example 2 was used to further evaluate the activity of the parsnip extract to inhibit tyrosinase. In general, B16 mouse melanoma cell lines were seeded at 1.0 × 10 ⁵ cells per well (1 mL volume per well) in a 24-well plate, and then cultured for at least 24 hours at 37 ° C and maintaining a humidity of 5% carbon dioxide. Thereafter, 1 μM α-melanocyte stimulating hormone (α-MSH; Sigma Chemical Co, St. Louis, USA) (control group) was used, or α-MSH binding concentration was 50 μg/mL or 100 μg/mL. The parsnip extract or vitamin C was applied to the B16 mouse melanoma cell line at 37 ° C for 72 hours, and then the following assay was performed.

1. Analysis of the ability of parsnip extract to inhibit tyrosinase activity

B16 mouse melanoma cell line was treated as described above and acted at 37 ° C. After 2 hours, the culture medium of each well was removed and phosphate buffered (phosphat) e buffered saline; PBS) solution rinse, add, for example, 900 μL of sodium phosphate buffer (50 mM, containing 1% Triton X-100) (pH 6.8), using freeze-thawed The cells were frozen at -80 ° C for 30 minutes, then taken out and thawed at 25 ° C for 25 minutes and thawed at 37 ° C for 5 minutes to lyse the cells. Thereafter, for example, 100 μL of L-dopa (L-DOPA) solution (10 mM) was added to each well of cells. After the above treatment and 4 hours at 37 ° C, the absorbance of the reaction mixture of each well at 475 nm was measured by a spectrophotometer, such as a multi-functional microplate reader, and compared with the control group (final concentration of DMSO was 0.1%). ) and positive control group (vitamin C) comparison.

From the results of the second table, the relative ratio of the resistance of the herbicide extract to the tyrosinase activity of the vitamin C in the B16 mouse melanoma cell line was 50 μg/ The relative ratios of mL vitamin C, parsnip first extract (103), second extract (105) and fourth extract (113) to intracellular tyrosinase activity were 35.9 ± 3.2%, 4.5, respectively. ±2.4%, 42.2±2.6%, and 56.1±1.2%; and 100 μg/mL vitamin C, parsnip first extract (103), second extract (105), and fourth extract (113) for intracellular cheese The relative ratios of the aminase activity inhibiting ability were 40.3 ± 2.2%, 9.1 ± 1.6%, 66.1 ± 1.8%, and 60.8 ± 3.1%, respectively. The results showed that the first extract (103), the second extract (105) and the fourth extract (113) of the parsnip could inhibit the tyrosinase activity in the melanocytes, and the second extract of the parsnip ( 105) The fourth extract (113) has higher inhibition ability of tyrosinase activity than vitamin C.

2. Analysis of the ability of parsnip extract to inhibit the synthesis of dopaquinone by tyrosinase

B16 mouse melanoma cell line was treated as described above and treated at 37 ° C for 72 hours, the culture medium of each well was removed, and washed with PBS solution, for example, 20 μL of sodium phosphate buffer solution (50 mM, containing 0.5%) was added. Triton X-100) (pH 6.9), frozen-thawed, frozen at -80 ° C for 30 minutes, then taken out and thawed at 25 ° C for 25 minutes and thawed at 37 ° C for 5 minutes to dissolve the cells. Next, 190 μL of the pre-warmed and freshly prepared substrate solution was added to each well of the cells, wherein the substrate solution may include 6.3 mM 3-methyl-2-benzothiazolinone hydrazone; MBTH) and 1.1 mM dopa solution (formulated in 48 mM sodium phosphate buffer solution, pH 7.1), and added 4 volume percent (% v/v) of N,N' -dimethylformamide ( N, N'- dimethylformamide). After the above treatment and 4 hours at 37 ° C, the absorbance of the reaction mixture of each well at 508 nm was measured by a spectrophotometer, such as the above-mentioned multifunctional microplate reader, and compared with the control group (the final concentration of DMSO was 0.1%) and a positive control (vitamin C) comparison. Due to the action of intracellular tyrosinase and L-dopa solution to form dopaquinone, followed by cyclization to form DOPAchrome, while DOPAchrome and MBTH react to produce a pink product, thus having a strong absorbance at 508 nm, which is determined by this method. Parsnip extract inhibits the ability of tyrosinase to synthesize dopaquinone.

From the results of the third table, it is known that the parsnip extract and vitamin C inhibit B1. The relative ratio of the ability of the mouse melanoma cell line tyrosinase to synthesize dopaquinone, 50 μg/mL vitamin C, the first extract of parsnip (103), the second extract (105) and the fourth extract ( 113) The relative ratios of inhibition ability of intracellular tyrosinase synthesis of dopaquinone were 76.2 ± 3.6%, 82.9 ± 3.2%, and 56.1 ± 3.1, respectively. % and 62.1 ± 2.7%; while 100 μg/mL vitamin C, parsnip first extract (103), second extract (105) and fourth extract (113) inhibit B16 mouse melanoma cell line tyrosine The relative ratios of the enzymes' ability to synthesize dopaquinone were 88.3 ± 3.2%, 85.5 ± 6.4%, 69.1 ± 6.4%, and 64.8 ± 3.2%, respectively. The results showed that the first extract (103), the second extract (105) and the fourth extract (113) of the parsnip could inhibit the synthesis of dopaquinone by tyrosinase; therefore, the above results confirmed that the windproof of the first example The grass extract extract can effectively inhibit the dopa oxidase activity in melanocytes, and the ability of the first extract of parsnip (103) to inhibit the synthesis of dopaquinone by tyrosinase is similar to that of vitamin C.

3. Analysis of the ability of parsnip extract to inhibit extracellular melanogenesis

This assessment measures the amount of extracellular melanin. Briefly, B16 mouse melanoma cell lines were seeded at 3.0 x 10 ⁵ cells per well in a 6-well plate (3 mL volume per well) and then incubated for at least 24 hours at 37 ° C maintaining a humidity of 5% carbon dioxide. Thereafter, dimethyl hydrazine (DMSO; Sigma Chemical Co, St. Louis, USA) (control), or α-MSH combined with a concentration of 50 μg/mL or 100 μg/mL of the first extract of parsnip extract (103) Or vitamin C treatment, B16 mouse melanoma cell line was treated at 37 ° C for 72 hours, wherein the cell culture solution DMEM did not contain phenol red. After the B16 mouse melanoma cell line was treated for 72 hours at 37 ° C, 100 μL of cell culture solution per well was added to a 96-well plate, and each well cell was measured by a spectrophotometer (for example, a multi-functional microplate reader). The absorbance of the culture solution at 405 nm was compared with the control group (final concentration of DMSO of 0.1%) and the positive control group (vitamin C).

From the results of the fourth table, it is known that the parsnip extract and vitamin C inhibit B1. The relative ratio of the ability of extracellular melanin production in mouse melanoma cells, the relative ratio of 50 μg/mL vitamin C and the first extract of parsnip (103) to inhibit the extracellular melanin production of B16 mouse melanoma was 11.7±4.0, respectively. % and 84.5 ± 2.1%; and the relative ratios of 100 μg/mL of vitamin C and the first extract of parsnip (103) to inhibit the extracellular melanin production of B16 mouse melanoma were 77.2 ± 3.7% and 91.8 ± 3.4%, respectively. The results showed that the first extract of Parsnip (103) inhibited extracellular melanin production, and the first extract (103) inhibited the production of extracellular melanin more than vitamin C.

Example 4: Evaluation of the activity of the herbicide extract to scavenge free radicals

During the normal aerobic metabolism of organisms, many reactive oxygen species (ROS) are formed naturally. The active oxygen species include superoxide anion radical (O ₂ ‧ ^- ) and hydrogen peroxide (H). ₂ O ₂ ), hydroxyl radical (OH‧), s ingle oxygen (O ₂ ), etc., attack skin tissue and cause skin tissue aging.

1. Determination of DPPH‧ free radical scavenging ability by parsnip extract

This example utilizes the parsnip extract of the different process stages of the first embodiment. It is estimated that it can scavenge the scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH‧) radicals. DPPH‧ is a stable free radical with a maximum absorbance at 517 nm. The free radical inhibition rate at this concentration can be converted by measuring the change in absorbance at 517 nm by adding a sample ethanol solution. The color changes from blue-violet to pale yellow, and the absorbance decreases. Therefore, the ability of the antioxidant to scavenge free radicals can be evaluated by the change in absorbance at 517 nm. The lower the absorbance, the better the antioxidant capacity of the sample. .

This example uses vitamin C as a standard, and the parsnip obtained in the first embodiment is An extract (103), a second extract (105), a third extract (109) and a fourth extract (113) are respectively formulated into ethanol solutions of different concentrations (50, 100 μg/mL). Take 10 μL of the sample ethanol solution, add 900 μL of 100 μM DPPH ‧ ethanol solution and 90 μL of DMSO freshly, avoid the light and mix well, and then measure the absorbance of the mixture at 517 nm with a spectrophotometer and calculate the extraction according to the following formula. The percentage of DPPH‧ free radical scavenging effects (%), the results obtained are shown in Figure 2, and each value is derived from at least 3 samples:

As can be seen from the results of Table 5, 50 μg / mL of vitamin C, parsnip The percentage (%) of DPPH‧ free radical scavenging effect of one extract (103), second extract (105), third extract (109) and fourth extract (113) was 95.8±0.4%, respectively. 47.2 ± 1.3%, 2.97 ± 1.7%, 73.2 ± 1.0% and 50.9 ± 0.6%; and 100 μg / mL of vitamin C, parsnip first extract (103), second extract (105), third extract ( 109) The percentage (%) of DPPH‧ free radical scavenging effects with the fourth extract (113) were 98.7 ± 0.2%, 72.4 ± 6.1%, 4.17 ± 2.2%, 85.4 ± 2.9%, and 68.8 ± 2.4%, respectively. The results showed that the parsnip extract had the ability to scavenge DPPH‧ free radicals. And at high dose (100μg/mL), the first extract (103) and the third extract (109) of Parsnips remove DPPH‧ free radicals. High ability.

2. Parsnip extract to ABTS‧ ⁺ Cationic radical scavenging activity

2,2'-Azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) Under the catalysis of oxidase (peroxidase), ABTS‧ ⁺ radicals are produced, and a stable blue-green color is produced. Therefore, by adding antioxidants to participate in the reaction, ABTS‧ ⁺ can be reduced to ABTS to inhibit blue-green formation and pass the measurement. The change in absorbance at 734 nm allows evaluation of the antioxidant capacity of the antioxidant, and the lower the absorbance, the better the antioxidant capacity of the sample. This method is based on water-soluble vitamin E (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid;trolox) is a positive standard, and a standard curve is prepared as a control and its total antioxidant capacity is evaluated, so it is also called trolox equivalent antioxidant capacity (trolox Equivalent Antioxidant Capacity; TEAC).

This example uses the water-soluble vitamin E (trolox) as a standard, and the parsnip extract obtained in the first embodiment is separately prepared into sample solutions of different concentrations (50, 100 μg/mL), and then taken at different concentration points. 1.5 L of the sample solution, added 3.5μL ddH ₂ O (deionized water) and 7mM ABTS‧ ⁺ 145μL of the solution, after mixed uniformly dark, allowed to stand at room temperature for 20 minutes. Thereafter, the absorbance of trolox at 734 nm was measured by a spectrophotometer, and a trolox standard curve was prepared to convert the TEAC (mg trolox/g) value of each sample by a standard curve, and calculate the ABTS‧ ^{+ of} each extract according to the following formula: Percentage of free radical scavenging effects, each value is derived from at least 3 sample numbers:

From the results of the sixth table, 50 μg / mL of water-soluble vitamin E (t rolox), parsnip first extract (103), second extract (105), third extract (109) and fourth extraction Percentage (%) of ABTS‧ ⁺ radical scavenging effect of substance (113), 99.3±0.8%, 25.4±2.4%, 6.91±1.7%, 37.1±1.2%, and 73.5±0.6%, respectively; and 100 μg/mL vitamin C, Percentage (%) of ABTS‧ ⁺ radical scavenging effect of the first extract (103), the second extract (105), the third extract (109) and the fourth extract (113) of the parsnip, respectively It is 99.7 ± 0.5%, 66.7 ± 3.1%, 10.1 ± 1.0%, 49.1 ± 2.1%, and 91.2 ± 1.4%. Trolox Equivalent Antioxidant Capacity (TEAC) of water-soluble vitamin E (trolox), parsnip first extract (103), parsnip third extract (109) and fourth extract (113) They are 1000, 15832, 18863 and 6209 mg trolox/g. The results showed that the concentration of the parsnip extract obtained in Example 1 was positively correlated with the percentage (%) of the ABTS‧ ⁺ radical scavenging effect. The anti-oxidation capacity value (TEAC) of the trolox equivalent of the fourth extract (113) of the parsnip is lower than the antioxidant capacity (TEAC) of the trolox equivalent of the water-soluble vitamin E (trolox), indicating the fourth extract of the parsnip ( 113) The water-soluble vitamin E has a higher antioxidant capacity.

3. Evaluation of the inhibition ability of parsnip extract on superoxide formation

This embodiment utilizes the characteristics of a cell membrane similar to that of a liposome lipid bilayer structure. In vitro lipid peroxidation experiments were tested. Malondialdehyde (MDA), a product of lipid peroxidation, forms red thiobarbituric acid (TBA) with thiobarbituric acid (TBA) at high temperatures (TBA-reactive substances; TBARS) The MDA content can be detected by measuring the absorbance at 532 nm to determine the degree of lipid peroxidation. The lower the absorbance value, the better the ability of the sample to inhibit lipid peroxidation.

The main lipid component of the above-mentioned vesicles may be, for example, lecithin choline (EPC), which is indirectly subjected to ultrasonic oscillation in a water bath environment by using EPC with a volume ratio of 1:10 and deionized water (ie, water-repellent). Ultrasonic vibration) forms a liposome having an average particle diameter of, for example, 225 nm to 250 nm. In this example, 3.2 μL of liposome solution (including 10 mM lecithin choline), 11 μL of ferrous sulfate (FeSO ₄ ; 10 mM) solution, 11 μL of vitamin C (10 mM) solution, and 151.5 μL of 50 mM were taken. Tris-HCl buffer solution (pH 7.4) and 2.5 μL of each extract of various concentrations in Example 1 were reacted at 37 ° C for 1 hour. Next, 80 μL of TCA (10%) reagent (containing 1% TBA solution) and 10.6 μL of EDTA (0.1 M) were added and heated to 100 ° C for 20 minutes. After cooling and centrifugation at 12000 rpm for 10 minutes, after precipitation, 100 μl of the supernatant was measured for its absorbance at 532 nm to obtain the MDA content.

As a result of the seventh table, 50 μg / mL of water-soluble vitamin E (t Ratio (%) of the ability of rolox), parsnip first extract (103), second extract (105), third extract (109) and fourth extract (113) to inhibit peroxidation of vesicles, 32.1±0.1%, 69.4±2.4%, 61.7±1.2%, 69.4±4.5%, and 69.4±2.7%, respectively; and 100 μg/mL vitamin C, parsnip first extract (103), and second extract (105) The ratio (%) of the ability of the third extract (109) and the fourth extract (113) to inhibit peroxidation of the liposome is 36.0 ± 0.5%, 70.6 ± 3.6%, 68.8 ± 3.5%, and 65.9 ±, respectively. 1.3% and 91.7 ± 2.8%. The results showed that the parsnip extract obtained in Example 1 was significantly more resistant to peroxidation of vesicles than to water-soluble vitamin E (trolox).

It is worth mentioning that although the invention is modified by the method of extracting parsnip extract The use of the composition of good melanin precipitation is within the scope of the patent application, but the experimental results attached to the examples show that the parsnip extract has antioxidant activity in addition to the ability to inhibit tyrosinase activity and inhibit melanin production. active. It should be noted that the present invention is exemplified by specific process conditions, specific analytical methods or specific instruments, and illustrates the application of the cordyceps extract for preventing melanin precipitation of the present invention, but any of the technical fields of the present invention are generally It is to be understood by those skilled in the art that the present invention is not limited thereto, and the parsnip extract of the present invention can be carried out using other process conditions, other analytical methods or instruments without departing from the spirit and scope of the present invention.

It can be seen from the above embodiments of the present invention that the present invention is useful for preventing melanin precipitation. The parsnip extract has the advantage that the parsnip extract can be effectively obtained by at least one extraction step to provide its effect of improving melanin precipitation. Since the partitioning step is carried out by the method of the present invention, the layering is relatively easy, the foaming is not easy, and the process time is shortened, so that the obtained parsnip extract is obtained. It can be applied to cosmetic compositions and/or food additives.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

(101) ‧‧‧Parsnip material

(103)‧‧‧First extract

(105)‧‧‧Second extract

(107) ‧ ‧ first residue

(109)‧‧‧ Third extract

(111)‧‧‧Second remainder

(113)‧‧‧Fourth extract

(115) ‧ ‧ third residue

(S1)‧‧‧Step one

(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3

(S4)‧‧‧Step four

Claims (4)

A parsnip extract for inhibiting tyrosinase activity, inhibiting microlipid peroxidation, and scavenging free radicals is prepared by the following steps: Step 1: Extracting a parsnip material with a 95% by volume ethanol solution Obtaining a first extract; Step 2: extracting the first extract with a 1:1 ratio of n-hexane/95% methanol solution to fractionate a n-hexane phase and a methanol phase, wherein the methanol phase has a second extract, and the methanol phase has a first residue; Step 3: extracting the first residue with a volume ratio of 2:1 ethyl acetate/water solution to separate an ethyl acetate phase and An aqueous phase, wherein the ethyl acetate phase has a third extract, and the first aqueous phase has a second residue; and step 4: extracting the first portion with a volume ratio of 1:1 n-butanol/water solution The remainder is divided to form an n-butanol phase and a second aqueous phase, wherein the n-butanol phase has a fourth extract. A parsnip extract for inhibiting tyrosinase activity, inhibiting lipolysis, and scavenging free radicals according to the scope of claim 1, as a cosmetic material composition, food additive or pharmaceutical group Adult. The preparation method of the parsnip extract is prepared by the following steps: Step 1: extracting a parsnip material with a 95% by volume ethanol solution to obtain a first extract; Step 2: using a volume ratio of 1:1 Extracting the first extract with a n-hexane/95% methanol solution to separate a n-hexane phase and a methanol phase, wherein the methanol phase has a second extract, and the methanol phase has a first residue; Three: extracting the first residue by using a 2:1 volume ratio of ethyl acetate/water solution to separate an ethyl acetate phase and a first aqueous phase, wherein the ethyl acetate phase has a third extract, and The first aqueous phase has a second residue; and step 4: extracting the second residue with a volume ratio of 1:1 n-butanol/water solution to separate a n-butanol phase and a second aqueous phase Wherein the n-butanol phase has a fourth extract, and the fourth extract has both the ability to inhibit tyrosinase activity, the ability to inhibit peroxidation of the vesicles, and the ability to scavenge free radicals. The method for preparing a parsnip extract according to claim 3, wherein the parsnip extract is used as a cosmetic material composition, a food additive or a pharmaceutical composition.