TW201800100A - Application of lotus leaf extract for treating hyperpigmentation syndrome which can effectively inhibit formation and accumulation of melanin in skin cells to reduce content of melanin in skin cells - Google Patents

Abstract

This invention discloses an application of lotus leaf extract which can effectively inhibit the formation and accumulation of melanin in skin cells to reduce the content of melanin in skin cells, so as to achieve the efficacy of protecting skin cells and maintaining beauty while being able to effectively treating or improving hyperpigmentation syndrome.

Description

Use of lotus leaf extract for treating pigmentation syndrome

The present invention relates to the second use of plant extracts, in particular to the use of a lotus leaf extract to treat pigmentation syndrome.

According to the fact that melanin is a protein that exists in human skin or hair, it affects skin color, hair color, and pupil color, and its main function is to protect the skin from ultraviolet rays. Melanin is produced by melanoblasts in the basal layer of the skin which are stimulated by ultraviolet rays. Specifically, when melanocytes are stimulated, they will activate the mechanism to protect skin cells, activate tyrosinase, and then generate melanin, which means that the human system can protect skin cells through the mechanism of melanin formation. When melanin is formed, in general, melanin will be removed with keratin metabolism. However, when the skin is severely damaged and the metabolic rate is reduced, excess melanin cannot be ruled out, causing spots to appear on the skin.

Although melanin is an important line of defense for protecting the skin, it is considered beautiful to maintain flawless white skin based on modern aesthetic standards. Therefore, many studies have focused on finding effective and natural or chemically synthesized agents to improve pigmentation. However, whitening-related products developed so far have high toxicity, low stability, poor skin penetration, and poor activity. Good, but can not achieve the effect of removing stains, so that it cannot be widely used in the market.

Lotus leaves are lotus leaves. Studies have shown that lotus leaves contain various alkaloids such as nuciferine, pronuciferine, and liriodenine. Flavonoids, nelumboside, quercetin, isoquercetin and other ingredients. In the past pharmacopoeia, it was recorded that the load leaf has the functions of mild heat and detoxification, hemostasis, and cooling blood, and clinical studies have pointed out that lotus leaf alkali can effectively break down body fat and can be used as an effective ingredient in weight loss products. Therefore, in recent years, research on the efficacy of lotus leaf has focused on reducing Blood lipids, metabolic syndrome, antioxidant and other fields, that is, the current research has not pointed out or revealed clear evidence that lotus leaves or their extracts have the purpose of treating pigmentation syndrome.

The main purpose of the present invention is to provide the use of a lotus leaf extract for treating pigmentation syndrome.

Another object of the present invention is to provide the use of lotus leaf extract, which can effectively inhibit the formation and accumulation of melanin in skin cells, reduce the content of melanin in skin cells, and achieve the effects of protecting skin cells and maintaining beauty.

Accordingly, in order to achieve the above-mentioned object, the present invention discloses the use of lotus leaf extract for the preparation of a composition for treating pigmentation syndrome, wherein the pigmentation syndrome refers to a disease with pigmented skin.

Among them, the lotus leaf extract is obtained by extracting lotus leaf with water.

Among them, lotus leaf extracts can inhibit the expression of proteins related to melanin production, and the proteins related to melanin production are selected from the group consisting of tyrosinase, MITF (Microphthalmia transcription factor), TRP1 (Tyrosinase related protein-1), and CREB (cAMP response element -binding protein) or PKA (Protein kinase A).

The composition is prepared in the form of a paste or a gel, and contains lotus leaf extract of at least 1% wt, and has a better effect when it contains lotus leaf extract of 1-2% wt.

The lotus leaf extract is prepared as a solution with a concentration of at least 0.3 mg / mL, and And when lotus leaf extract is prepared as a solution with a concentration of 0.3 ~ 0.5mg / mL, it has better efficacy.

Another embodiment of the present invention discloses a composition comprising a lotus leaf extract, water, propylene glycol and gel, wherein the weight percentage of the lotus leaf extract is 1-2. Specifically, the lotus leaf extract, water, propylene glycol and gel are obtained at 1: 4: 10: 75 or 1: 6.5: 5: 37.5.

The first graph is the result of the cell viability test.

The second graph is a standard curve for synthetic melanin.

The third figure is the melanoma accumulation results of melanoma cells B16-F1 after different treatments.

The fourth graph is the result of analyzing the melanin content of melanoma cells B16-F1 after different treatments.

The fifth graph is the result of analyzing the melanin secretion of melanoma cells B16-F1 after different treatments.

The sixth graph shows the performance of tyrosinase, MITF, and TRP1 in the cells of each group.

The seventh graph is the result of analyzing the expression levels of tyrosinase, MITF, and TRP1 in the cells of each group.

The eighth graph shows the performance of CREB and PKA in each group of cells.

The ninth graph is the result of analyzing the expression levels of CREB and PKA in the cells of each group.

The tenth graph shows the performance of tyrosinase, MITF, and TRP1 in the skin tissue of each group of mice.

The eleventh figure is the result of analyzing the expression levels of tyrosinase, MITF, and TRP1 in the skin tissue of the mice in each group.

The twelfth figure shows the performance of CREB, PKA, ERK in the skin tissue of each group of mice.

The thirteenth figure is the result of analyzing the expression levels of CREB, PKA, and ERK in the skin tissue of each group of mice.

The fourteenth figure is the result of H & E staining of the skin tissue sections of each group of mice, in which A to D are the first group to the fourth group with a magnification of 40x.

The fifteenth figure is the result of Fontana-Masson staining of the skin tissue sections of each group of mice. Middle A to D are the first group to the fourth group in order, the magnification is 40x.

The sixteenth figure is the melanin content in the skin tissue of each group of mice.

In order to verify the efficacy of the present invention, the following will be further described by way of examples and drawings.

In the following example, the data of the cell experiment is analyzed by student's t-test, and the data of the animal experiment is calculated by one way ANOVA, and then analyzed by student's t-test. The results are all expressed by means ± SD. When p <0.05, Expressed as a statistically significant difference.

The animal test disclosed in the example was reviewed and approved by the Experimental Animal Care and Use Committee of Sun Yat-sen Medical University, and the consent number was 1606.

Example 1: Preparation of lotus leaf extract

After drying the lotus leaf to make powder, take 200 grams of lotus leaf powder, add secondary water to 5 liters, stir at room temperature for one hour, leave it in the cold room for 12 hours, filter to remove impurities and collect to 15 liters. The freeze-drying treatment gives a powdered lotus leaf extract with a yield of about 1.5%. The lotus leaf extract is stored at room temperature for use in subsequent examples. The lotus leaf extract is prepared as lotus leaf extracts of various concentrations. Before use in each example, a filtration and sterilization process should be performed, for example, 0.22 μm. Filter.

Example 2: Cell Culture

Melanoma cell line B16F1 (ATCC CRL-6323) purchased from the Center for Bioresource Preservation and Application of the Food Industry Development Institute.

The melanoma cell line B16F1 was cultured in a medium containing 1.5 g / L sodium bicarbonate. 90% DMEM medium, and additionally added 5mL, 200mM glutamic acid, 5mL penicillin / streptomycin, and 10% fetal bovine serum, wherein the medium and additives were purchased from GBICO-life technologies. All cells were cultured in a constant temperature environment of 5% carbon dioxide and 37 ° C. The medium was renewed every 2-3 days, and the dilution ratio of the subculture was 1:10.

Example 3: Cytotoxicity test

This example refers to the designer of the institute according to Mosmann et al. Melanoma cells B16-F1 (3 × 104 Cell) were cultured in a 24-well culture plate. Add different concentrations of the lotus leaf extract solution to the culture medium: 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL, and different concentrations of the lotus leaf extract solution were used to treat cells for 24, 48, 72 hours, each of which Replace it every 24 hours and add new medium and lotus leaf extract solution. After the test is completed, the culture plate is washed with phosphate buffer solution, and then a medium containing MTT reagent (0.5 mg / mL) is added, and the mixture is reacted for 3 hours. After removing the medium, the crystals are eluted with isopropanol and measured at OD 563nm. The change in absorbance is shown in the first graph.

From the results in the first figure, it can be known that the cell survival rate of the lotus leaf extract treated by the present invention is similar to that of the blank group, showing that the lotus leaf extract system disclosed by the present invention has safety.

Example 4: Melanin content test

This example refers to the designer of the institute according to Bellei et al.

After melanoma cells B16-F1 were seeded in a 6-well culture plate (1 × 105 Cell) for 24 hours, 10 μM α-MSH and / or 0.5 mg / mL of the lotus leaf extract solution were added. After 72 hours of incubation, remove the medium, rinse with phosphate buffer, remove the cells with trypsin, centrifuge to leave the cells, and destroy the cells with phosphate buffer containing 1% Triton X-100. And cell debris were centrifuged at 12000 rpm for 10 minutes, and the supernatant was measured for absorbance to determine the protein content; another 200 μL of 1M NaOH was added to the cell debris and placed at 70 ° C for 2 hours. After the melanin was dissolved out, it was measured spectrophotometrically Measure the absorbance at a wavelength of 405nm, compare the standard curve of synthetic melanin (0 ~ 500μg / ml) (as shown in the second figure), and then measure the total amount of melanin and the total amount of protein to get the result (μg Melanin / mg of protein). The results of this example are shown in Figures 3 to 5.

Please refer to the third to fifth graphs, because α-MSH can promote the melanin synthesis reaction in B16-F1 cells. Therefore, the cells treated with α-MSH alone have the highest melanin content. After treatment with the lotus leaf extract solution disclosed in the present invention, it was found that the content of melanin in the cells was significantly reduced, showing that the lotus leaf extract system disclosed in the present invention can inhibit melanin production, so that there is no difference between the melanin content and the blank group. In addition, the cells were treated with α-MSH and the lotus leaf extract disclosed in the present invention at the same time, and the results showed that the melanin content in the cells would not increase. It can be seen that the lotus leaf extract disclosed in the present invention can indeed inhibit the production and accumulation of melanin in cells.

Example 5: Performance of melanogenic enzyme

B16-F1 cells were divided into six groups, the first group was a blank group, and the second to sixth groups were respectively treated under the following conditions: 10 μM α-MSH, 0.5 mg / mL of the lotus leaf extract solution, 10 μM α-MSH, and 0.3 mg / mL of the lotus leaf extract solution, 10 μM α-MSH and 0.4 mg / mL of the lotus leaf extract solution, 10 μM α-MSH and 0.5 mg / mL of the lotus leaf extract. After cultured for 24 hours in each group, the tyrosinase, MITF (Microphthalmia transcription factor), TRP1 (Tyrosinase related protein-1), and phosphorylated CREB (cAMP response element-binding protein) in each group were analyzed by Western blot method. ), Phosphorylated PKA (Protein kinase A) and other expressions of enzymes related to melanin production. The western blot method is a well-known technology of those in the technical field to which the present invention belongs, so it is not redundant here. The results are shown in Figures 6-9.

From the results of the sixth to the ninth graphs, it can be seen that the cells treated with α-MSH will indeed increase the amount of enzyme expression related to the melanin production pathway, indicating that α-MSH will make the cells synthesize melanin; In the cells treated with the lotus leaf extract disclosed in the present invention, the expression of enzymes in the melanin production pathway is significantly reduced, and the expression levels of tyrosinase, MITF, phosphorylated CREB and phosphorylated PKA are lower than those of the first group. It can be seen that the lotus leaf extract disclosed in the present invention can reduce the production and accumulation of melanin by the enzyme performance of the melanin production path. In addition, comparing the results of the fourth to sixth groups with the results of the second group, it is shown that under the condition of processing α-MSH, if the lotus leaf extract disclosed in the present invention is additionally given, it can effectively inhibit the melanin production path. Enzyme performance has the effect of inhibiting the production and accumulation of melanin, and as the dosage of lotus leaf extract disclosed in the present invention is higher, the effect of inhibiting the expression of enzymes related to the melanin production path is better.

Example 6: Animal test

Several female guinea pigs N: HARTLEY weighing about 300-350 grams were purchased from the National Laboratory Animal Center. They were raised at the Animal Center of Zhongshan Medical University. The environmental conditions were equipped with automatic air conditioning and automatic light control (12 hours day and 12 hours night). The average temperature is 22 ± 2 ℃ and the relative humidity is 50-55%. The feed is Guinea pig diet 5025 from LABDIET.

The lotus leaf extract powder of Example 1 and propylene glycol, gel, and water were prepared into lotus leaf gels, which contained 1% or 2% by weight lotus leaf extracts respectively, wherein the gel was water and 2% polymer gel. Composer. For example, taking 2 g of propylene glycol and 15 g of gel, adding 0.2 g of lotus leaf extract powder and 2.8 g of water to prepare a lotus leaf gel containing 1% wt lotus leaf extract, and adding 0.4 g of lotus leaf extract powder and 2.6 g of water At this time, a lotus leaf gel containing 2% wt lotus leaf extract can be completed.

After each of the guinea pigs grew to 500 grams in weight, experiments were performed with reference to the UV-induced skin-induced pigment deposition method of Li-Hua Penga et al. The guinea pigs were integrated into 4 groups, of which the first group was a blank group; the second group was a UV group; the third group was a low-dose experimental group, which was irradiated with UV and smeared lotus leaf gel containing 1% wt lotus leaf extract The fourth group is a high-dose experimental group, which is irradiated with UV and smeared lotus leaf gel containing 2% wt lotus leaf extract. The second to fourth groups of irradiation sites were divided into three areas after back hair removal. Each area (1.5 cm x 1.5 cm) was irradiated with a UVB dose of 500 mj / cm2 for 15 minutes each time (Spectroline Select ^TM Series, Philips TL / 12 lamp emitting 280-305nm), continuous irradiation for two weeks, three times a week, and the third and fourth groups were coated with lotus leaf gel with different concentrations every day after the irradiation. Two weeks later, the skin was sacrificed for subsequent examples.

Example 7: Protein expression of skin tissue

Skin protein extracts were extracted from the skin tissues taken from the guinea pigs of each group of Example 6. The western blot method was used to analyze the skin tissues of guinea pigs in each group such as tyrosinase, MITF, TRP1, phosphorylated CREB, phosphorylated PKA, etc. The results of the enzymes related to melanin production are shown in Figures 10-13.

From the results of the tenth to thirteenth graphs, it can be seen that the irradiation of UV light will indeed increase the expression of enzymes related to melanin production in skin tissues. As shown in the second group, it is shown that skin tissues are stimulated by UV to promote the production of melanin. Compared with the second group, the expression levels of the enzymes related to melanin production in the skin tissue of the third group and the fourth group of guinea pigs were significantly reduced, and the inhibitory effect of the higher concentration was better, showing the disclosure of the present invention. The lotus leaf extract system can inhibit the expression of enzymes related to melanin production in the skin tissue, and can effectively inhibit the formation of melanin and reduce the accumulation of melanin to achieve the effect of treating pigmentation.

Example 8: Skin tissue staining results

The skin tissues taken from each group of guinea pigs in Example 6 were embedded in paraffin, and then sectioned, and then stained by the H & E and Fontana-Masson methods. The tissue sections and staining are common knowledge in the technical field to which the present invention belongs. Therefore, I will not repeat them here. The results of each group of tissue staining sections are shown in Figures 14 and 15.

From the results of the fourteenth and fifteenth figures, it can be seen that the table of the second group of guinea pigs exposed to UV The cortical layer is significantly thickened, and melanin is deposited in the epidermal layer, which shows that not only melanin will be produced on the skin after UV irradiation, but also poor skin metabolism will cause melanin to accumulate in the epidermal layer and form pigmentation disease. Compared with the second group, the thickness of the skin tissues in the third and fourth groups are significantly thinner, indicating that the metabolism of the skin tissue is better, and the accumulation of melanin in the epidermal layer is significantly improved. In other words, the lotus leaf extract disclosed in the present invention can not only inhibit the production of melanin, but also effectively improve the situation that melanin is deposited in the epidermal layer. Therefore, the lotus leaf extract disclosed in the present invention should be used for improving or treating pigmentation.

Example 9: Melanin content of skin tissue

Take 3 grams of guinea pig skin tissue from each group in Example 6, RIPA buffer and protease inhibitor, grind on ice, and then centrifuge at 4 ° C / 20,000 × g / 20min to extract the supernatant. Measure the absorbance at a wavelength of 405 nm with a spectrophotometer, compare the standard curve of synthetic melanin, and analyze the melanin content in the skin tissue of each group of guinea pigs.

From the results in Figure 16, it can be seen that the melanin content in the skin tissue of the second group of guinea pigs is significantly higher than that of the first group of mice, and compared to the second group, the melanin in the third and fourth groups of guinea pig skin tissues. The content was significantly reduced, and was similar to the first group of guinea pigs. The above results again show that the lotus leaf extract disclosed in the present invention does have the effect of reducing the production and accumulation of melanin.

According to the description of the above examples, it is confirmed that the lotus leaf extract disclosed in the present invention can regulate the enzymes related to melanin production in the cell, so that tyrosinase is not activated, melanin production is inhibited, and the metabolic reaction of skin tissue is not affected. External stimulus effects to maintain a normal metabolic response, to achieve the effect of improving or treating melanin accumulation and pigmentation. According to this, the lotus leaf extract disclosed in the present invention does have the effect of treating pigmentation syndrome, and can effectively prevent or improve the occurrence of dark spots such as yellow spots, freckles, age spots and the like on the skin. In addition, the lotus leaf extract disclosed in the present invention is a non-chemically synthesized component, so it can be safely used in the human body, and has extremely high economic value for industrial use.

references

Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65 (1-2): p. 55-63.

Bellei, B., et al., P38 regulates pigmentation via proteasomal degradation of tyrosinase. J Biol Chem, 2010. 285 (10): p. 7288-99.

Claims (10)

A use of lotus leaf extract for preparing a composition for treating pigmentation syndrome. According to the use described in the first patent application scope, wherein the pigmentation syndrome refers to a disease with pigmented skin. According to the application described in the scope of the patent application, the lotus leaf extract is obtained by extracting lotus leaf with water. According to the use described in claim 4 of the scope of patent application, the protein related to melanin production is selected from the group consisting of tyrosinase, MITF, TRP1, CREB, and PKA. The use according to item 1 of the patent application scope, wherein the composition is prepared as a paste or gel. The use according to item 5 of the patent application scope, wherein the composition contains at least 1% wt of lotus leaf extract. The use according to item 6 of the patent application scope, wherein the composition contains at least 1 to 2% by weight of lotus leaf extract. According to the use described in the first patent application, the lotus leaf extract is prepared as a solution with a concentration of at least 0.3 mg / mL. According to the use described in the patent application No. 8, the lotus leaf extract is prepared as a solution with a concentration of 0.3 to 0.5 mg / mL. A composition comprising lotus leaf extract, water, propylene glycol and gel, wherein the weight percentage of the lotus leaf extract is 1 to 2.