Whitening additive and whitening essential oil including the whitening additive

Abstract

This invention provides a whitening essence oil comprising the following components (wt): 20%-55% rosehip oil, 10%-20% jojoba oil, 10%-20% cocoyl oleate / caprylate, 0.1%-10% meadowfoam seed oil, 5%-15% squalane, 0.1%-10% olive oil, 0.1%-10% evening primrose oil, 0.01%-0.05% glycyrrhizin, 0.1%-1.0% VC-IP, 0.1%-1% tocopheryl acetate, 0.001%-0.1% rose oil, 0.01%-0.05% tetrahydrocurcumin, and 3%-8% rose enzymatic hydrolysate. This invention also provides rose enzymatic hydrolysate as a whitening additive. The whitening essence oil provided by this invention can inhibit melanin production in human skin and has a significant whitening effect.

Description

Technical Field

[0001] The invention relates to a whitening additive and also relates to a whitening essential oil comprising the whitening additive. Background Art

[0002] Melanin is mainly produced by melanocytes in the basal layer of the human epidermis, which can reduce the damage of ultraviolet rays to the skin. The content and distribution of melanin determine the color of the skin. However, the abnormal accumulation of melanin in the basal layer can cause excessive pigmentation, causing melasma, freckles, age spots, etc., affecting people's quality of life. With the continuous deepening of research on melanin biosynthesis, researchers have found that tyrosinase plays an important role in melanin biosynthesis. It is the main rate-limiting enzyme in the conversion of tyrosine and dopa to melanin. Its overexpression is the main cause of excessive pigmentation. Therefore, inhibiting the activity of tyrosinase can block the biosynthesis reaction chain of melanin, reduce the production of melanin, and achieve a whitening effect.

[0003] In view of the pathways and influencing factors of melanin production, the main approaches to skin whitening and skin care include the following aspects: (1) reducing the number of melanocytes produced by inhibiting the proliferation of skin melanocytes; (2) reducing the biosynthesis of melanin by inhibiting the catalytic activity of tyrosinase, the key enzyme in the production of skin melanin, thereby achieving the purpose of whitening; (3) preventing the damage of ultraviolet rays in the sun to the skin (tanning and sunburn) through sun protection, and slowing down skin aging; (4) removing oxygen free radicals in the skin by using antioxidants, slowing down melanin production and slowing down skin aging.

[0004] In the prior art, in order to prevent skin from getting darker, spots, freckles, etc. and to keep the skin white, people have proposed whitening cosmetics mixed with various whitening agents such as hydroquinone, kojic acid, arbutin, etc. However, if these substances are mixed in large quantities, problems may arise in terms of feel and safety. For example, hydroquinone is very irritating to the skin; kojic acid is easy to change color and is also irritating to the skin; arbutin has a single effect and poor effect, etc.

[0005] As people pay more and more attention to skin health, the development of natural whitening agents that are safe, stable, effective and cost-effective has become one of the main research directions of the current pharmaceutical and cosmetics industries and has very good development prospects. Summary of the invention

[0006] In one aspect, the present invention provides a whitening essential oil, comprising the following components (wt): 20%-55% of rosehip oil, 10%-20% of jojoba oil, 10%-20% of coconut oil caprylate / caprate, 0.1%-10% of white meadowfoam seed oil, 5%-15% of squalane, 0.1%-10% of olive oil, 0.1%-10% of evening primrose oil, 0.01%-0.05% of glabridin, 0.1%-1.0% of VC-IP, 0.1%-1% of tocopherol acetate, 0.001%-0.1% of rose oil, 0.01%-0.05% of tetrahydrocurcumin, and 3%-8% of rose hydrolysate.

[0007] In some embodiments, the whitening essence oil includes the following components (wt): 15% jojoba oil, 15% coconut oil caprylate / caprate, 8% white meadowfoam seed oil, 8% squalane, 2.3% olive oil, 3.5% evening primrose oil, 0.03% glabridin, 0.5% VC-IP, 0.6% tocopherol acetate, 0.05% rose oil, 0.04% tetrahydrocurcumin and 5% rose hydrolysate, and the balance is made up with rosehip oil.

[0008] In some embodiments, the rose hydrolysate is prepared by the following method:

[0009] 1) Rose flower powder in aqueous solution with papain;

[0010] 2) treating the enzymatic hydrolysis product obtained in step 1) with protease alcalase;

[0011] 3) treating the enzymatic hydrolysis product obtained in step 2) with bromelain; and

[0012] 4) Centrifuge the enzymatic hydrolysis product obtained in step 3) and collect the supernatant.

[0013] In some embodiments, step 1) is performed under the conditions of papain concentration of 0.6 mg / mL, temperature of 60° C., and pH of 6.5 for 1 hour.

[0014] In some embodiments, step 2) is performed under the conditions of alcalase concentration of 25 mg / mL, temperature of 52° C., and pH of 8.0 for 2.5 h.

[0015] In some embodiments, step 3) is carried out for 5 hours under the conditions of 2.0 mg / mL bromelain concentration, 55° C., and pH 7.0.

[0016] The step 4) comprises centrifugation at 10000 g for 15 min.

[0017] On the other hand, the present invention provides a whitening additive, which is a rose enzymatic hydrolysate, and is prepared by the following method:

[0018] 1) Treat rose flower powder with papain;

[0019] 2) treating the enzymatic hydrolysis product obtained in step 1) with protease alcalase;

[0020] 3) treating the enzymatic hydrolysis product obtained in step 2) with bromelain; and

[0021] 4) Centrifuge the enzymatic hydrolysis product obtained in step 3) and collect the supernatant.

[0022] The whitening essential oil provided by the invention can inhibit the generation of melanin in human skin and has a significant whitening effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The MI value change curve of Example 6 is shown.

[0024] Figure 2 The ITA° value variation curve of Example 6 is shown.

[0025] Figure 3 The whitening essence oil of the present invention has a spot-lightening effect. DETAILED DESCRIPTION

[0026] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Unless otherwise specified, the reagents and experimental methods used are common reagents and experimental methods in the art.

[0027] The inventors found that by treating rose powder with papain, alcalase and bromelain in aqueous solution (buffer) in sequence, the resulting hydrolysate has the ability to inhibit tyrosinase activity, and adjusting the order of protease treatment will significantly affect the inhibitory ability of the obtained hydrolysate on tyrosinase activity. The inventors speculate that one or several peptides in the hydrolysate have tyrosinase inhibitory activity, and different protease treatment orders may affect the length and / or amino acid sequence of the peptide ultimately produced, thereby affecting its inhibitory ability. The whitening essence oil containing the hydrolysate has a better whitening effect than the control sample.

[0028] The present invention is described in detail below through specific examples.

[0029] Example 1 Preparation of Rose Enzyme Hydrolysate

[0030] Wash and dry fresh roses, grind them in an ultrafine grinder, and pass through a 80-meter wooden sieve to obtain rose powder. Take 100g of rose powder, add 1L of deionized water, disperse it with ultrasound for 10 minutes, and then add protease for enzymatic treatment according to the steps in Table 1.

[0031] Table 1 Protease treatment process

[0032] step Protease Type Working concentration (mg / mL) temperature pH time 1 Papain 0.6 60℃ 6.5 1h 2 Protease alcalase 25 52℃ 8.0 2.5 3 Bromelain (from stem) 2.0 55℃ 7.0 5h

[0033] After the protease treatment, the obtained enzymatic hydrolysate was heated to 90°C and kept warm for 20 minutes to inactivate the added protease. After the enzymatic hydrolysate was cooled, it was centrifuged at 10,000 g for 15 minutes to remove impurities and particulate matter. The obtained supernatant was the rose enzymatic hydrolysate, which was packaged and stored at low temperature.

[0034] Example 2 Tyrosinase Activity Inhibition Experiment

[0035] Experimental principle: Tyrosinase catalyzes L-dopa to generate dopachrome, which has a characteristic absorption peak at 490nm, and then the tyrosinase activity is measured.

[0036] Experimental grouping settings: 1) Positive control group: adding arbutin; 2) Experimental group: adding different amounts of rose hydrolysate prepared in Example 1; 3) Blank control group: adding only complete culture medium. Three replicate wells were set for each well.

[0037] Experimental process: Human A375 melanoma cells were diluted to a concentration of 10 5 Cells / mL. Cells were seeded in a 96-well plate, 100 μL per well, and the test substance was added after 24 hours, and the culture was continued for 48 hours. PBS was washed twice, 100 μL of TritonX-100 (10%) solution was added to each well, and the cells were vortexed for 5 minutes to dissolve the cells. After a 37°C warm bath, 10 μL of 1% L-Dopa was added to each well. Incubate at 37°C for 30 minutes. The absorbance value (OD value) was measured at a wavelength of 490 nm, and the blank well was adjusted to zero, and the enzyme activity of tyrosinase relative to the blank group was calculated.

[0038] The results are shown in Table 2.

[0039] Table 2 Tyrosinase inhibition ability

[0040]

[0041]

[0042] Relative to blank control, *p<0.05; **p<0.01; ***p<0.001.

[0043] It can be seen from the results in the table that when the usage amount gradually increases, the rose hydrolysate of the present invention has a significant tyrosinase inhibition ability, and even has an inhibitory effect better than α-arbutin when the addition amount is large.

[0044] Example 3 Melanoma cell melanin synthesis inhibition experiment

[0045] Experimental principle: The synthesis of melanin in cells is mainly regulated by tyrosinase, which is the rate-limiting enzyme of the initial reaction of the melanin synthesis pathway. It can catalyze the hydroxylation of tyrosine to DOPA and oxidize DOPA to DOPAquinone, and finally form the reaction product melanin. In the tyrosinase activity inhibition experiment, the hydroxylation of tyrosine to DOPA or the oxidation of DOPA to DOPAquinone is affected by affecting the activity of tyrosinase, thereby affecting the synthesis of the final reaction product melanin. The melanin synthesis function of B16 mouse skin melanoma cells is basically the same as that of normal human skin melanocytes, and can be used to determine the inhibitory effect of the test substance on melanin synthesis.

[0046] Experimental process: Human B16 melanoma cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 5×10 4 Cells / mL were inoculated in a 96-well plate, 100 μL per well, and different amounts of the rose hydrolysate prepared in Example 1 were added after 24 hours, and a control group with blank complete medium was set up at the same time, and 6 replicates were set up in each well. The culture was continued for 48 hours, the supernatant was discarded, and the cells were washed twice with PBS. Three replicates were taken from each group for cell counting, and 150 μL of 1 mol / L NaOH solution was added to the remaining three replicates for culture, and the cells were incubated in a water bath at 85°C for 1 hour. The absorbance value (OD value) of each well was measured at a wavelength of 490 nm, and the melanin synthesis inhibition rate (%) in the melanocytes of each well was calculated according to the following formula.

[0047] Melanin synthesis inhibition rate (%) = [1-(OD value of drug administration group / cell number) / (OD value of blank control group / cell number)] × 100%.

[0048] The results are shown in Table 3.

[0049] Table 3 Melanoma cell melanin synthesis experimental results

[0050]

[0051] Relative to blank control, *p<0.05; **p<0.01.

[0052] It can be seen from the results in the table that when the usage amount gradually increases, the rose hydrolysate of the present invention has the ability to significantly inhibit the synthesis of melanin in melanoma cells, and as the usage amount increases, the inhibitory ability gradually increases.

[0053] Example 4 Preparation of Rose Hydrolysate (Comparative Example)

[0054] The preparation process of the rose hydrolysate in this example was carried out as described in Example 1, except that the order of protease treatment was changed (the pH, temperature and time of treatment for various proteases remained unchanged), and the tyrosinase activity inhibition experiment was carried out as in Example 2 (100 μL of lysate was added to the experimental group, and 100 μL of complete culture medium was added to the blank group).

[0055] The results are listed in Table 4.

[0056] Table 4 Tyrosinase inhibition ability of rose hydrolysates obtained by different protease treatment sequences

[0057] Experiment number Protease treatment sequence Tyrosinase relative activity 1# Papain, protease alcalase, bromelain 45.8％*** 2# Papain, bromelain, protease alcalase 77.4％* 3# Bromelain, papain, protease alcalase 99.5％ 4# Bromelain, protease alcalase, papain 92.5％ 5# Proteases alcalase, papain, bromelain 97.6％ 6# Proteases alcalase, bromelain, papain 93.5％

[0058] Relative to blank control, *p<0.05; ***p<0.001.

[0059] From the results in the table, it can be seen that the rose hydrolysates obtained from Experiment No. 1# and 2# have different degrees of tyrosinase inhibition ability, and the rose hydrolysates obtained from other protease treatment sequences basically have no tyrosinase inhibition ability.

[0060] Example 5 Preparation of whitening essential oil

[0061] Whitening essential oil composition (wt): 5% of the rose hydrolysate prepared in Example 1, 15% of jojoba oil, 15% of coconut oil-caprylate / caprate, 8% of white meadowfoam seed oil, 8% of squalane, 2.3% of olive oil, 3.5% of evening primrose oil, 0.03% of glabridin, 0.5% of VC-IP, 0.6% of tocopherol acetate, 0.05% of rose oil and 0.04% of tetrahydrocurcumin, and the balance is made up with rose fruit oil. The whitening essential oil without adding rose hydrolysate was used as a control (the balance was made up with wild rose fruit oil).

[0062] Preparation method: Mix all the raw materials, stir and dissolve at 45 rpm until yellow, transparent and clear, and filter through a 600-mesh filter cloth.

[0063] Example 6 Whitening Effect Test

[0064] The test instruments used were: skin melanin and hemoglobin tester (MexameterMX18, CK Company, Germany); multifunctional skin tester and colorimetric test probe MPA9 (CK Company, Germany).

[0065] The skin melanin content (MI) value is used to characterize the melanin index. The larger the MI value, the higher the skin melanin content. The L* measured by the colorimeter represents the brightness and is used to indicate the black and white degree of the skin. The larger the value, the more white the skin color tends to be. a* represents the red-green degree, and its change indicates the change in red-green degree before and after the use of whitening products. +a* represents red, and -a* represents green. b* represents the blue-yellow degree, and its change indicates the change in blue-yellow degree of the skin before and after the use of whitening products. +b* represents yellow, and -b* represents blue. The ITA° value can be calculated from the measured L*, a*, and b* values ​​to compare the changes in skin color of the population. The larger the ITA° value, the brighter the skin, and vice versa, the darker the skin.

[0066] A total of 76 subjects, females aged 25-40, were equally divided into two groups, and the whitening effects of the whitening essence oil containing rose hydrolysate prepared in Example 5 and the whitening essence oil without rose hydrolysate were tested respectively.

[0067] The test site is the face, and the whitening essence oil is required to be evenly applied. Volunteers apply it once a day in the morning and evening for a period of 4 weeks. During the test period, no other cosmetics are used on the test site. The parameter changes of the test site are detected at intervals of one week at the beginning of the test and within 4 weeks of using the whitening essence oil. The MI and ITA° results (mean) are shown in Figure 1 and Figure 2 The results showed that both the whitening essence oil containing rose hydrolysate and the whitening essence oil without rose hydrolysate could reduce the MI value and increase the ITA° value, but the whitening essence oil with rose hydrolysate was more effective, with significant differences between the two in the third and fourth weeks (p<0.05).

[0068] Example 7 Spot Lightening Effect Test

[0069] The whitening essential oil containing rose enzymatic hydrolysate prepared in Example 5 was used as a test sample.

[0070] A total of 31 healthy female subjects, aged 18-55 years old, had at least one obvious spot with an ITA° difference of more than 10° between the test site and the surrounding adjacent skin, and a diameter of no less than 3mm (cannot be freckles, pigmented moles, etc. that are difficult to improve with topical preparations in clinical practice), and used the test samples continuously for 28 days. The results are shown in Figure 3 middle.

[0071] The results showed that after using the test sample for 14 days, the proportion of the subject's pigmentation area decreased significantly compared with the initial value (p<0.001), and the decrease rate was 5.66%. After using the test sample for 28 consecutive days, the proportion of the subject's pigmentation area decreased significantly compared with the baseline value (p<0.001), and the decrease rate was 10.71%.

Claims

1. Whitening essence oil, comprising the following components (wt): 20%-55% of rosehip oil, 10%-20% of jojoba oil, 10%-20% of coconut oil caprylate / caprate, 0.1%-10% of white meadowfoam seed oil, 5%-15% of squalane, 0.1%-10% of olive oil, 0.1%-10% of evening primrose oil, 0.01%-0.05% of glabridin, 0.1%-1.0% of VC-IP, 0.1%-1% of tocopherol acetate, 0.001%-0.1% of rose oil, 0.01%-0.05% of tetrahydrocurcumin, and 3%-8% of rose hydrolysate, The rose hydrolysate is prepared by the following method: 1) treating rose flower powder with papain in aqueous solution; 2) treating the enzymatic hydrolysis product obtained in step 1) with protease alcalase; 3) treating the enzymatic hydrolysis product obtained in step 2) with bromelain; and 4) Centrifuge the enzymatic hydrolysis product obtained in step 3) and collect the supernatant.

2. The whitening essential oil as claimed in claim 1, comprising the following components (wt): 15% jojoba oil, 15% coconut oil caprylate / caprate, 8% meadowfoam seed oil, 8% squalane, 2.3% olive oil, 3.5% evening primrose oil, 0.03% glabridin, 0.5% VC-IP, 0.6% tocopherol acetate, 0.05% rose oil, 0.04% tetrahydrocurcumin and 5% rose hydrolysate, and the balance is made up with rosehip oil.

3. The whitening essential oil as claimed in claim 1, wherein step 1) is treated for 1 hour under the conditions of the papain concentration of 0.6 mg / mL, the temperature of 60°C and the pH of 6.

5.

4. The whitening essential oil as claimed in claim 1, wherein in step 2), the alcalase concentration is 25 mg / mL, the temperature is 52°C, and the pH is 8.0 for 2.5 h.

5. The whitening essential oil as claimed in claim 1, wherein step 3) is treated for 5 hours at a bromelain concentration of 2.0 mg / mL, a temperature of 55° C. and a pH of 7.

0.

6. The whitening essential oil as claimed in claim 1, wherein the step 4) comprises centrifugation at 10000 g for 15 min.

7. Whitening additive, which is rose enzymolyzate, is prepared by the following method: 1) Treat rose flower powder with papain; 2) treating the enzymatic hydrolysis product obtained in step 1) with protease alcalase; 3) treating the enzymatic hydrolysis product obtained in step 2) with bromelain; and 4) Centrifuge the enzymatic hydrolysis product obtained in step 3) and collect the supernatant.

Images