TWI724210B - A longan flower extract with anti-aging, whitening, anti-allergic and cell repair - Google Patents

Abstract

The present invention relates to a longan flower extract with anti-aging, whitening, anti-allergic and cell repair, which is to administer an effective dose of longan flower extract to a skin cell to remove DPPH. And ABTS. + Free radical ability, reducing power, chelating iron ions and inhibiting lipid peroxides; inhibiting tyrosinase activity; eliminating nitric oxide (NO) free radicals, inhibiting lipoxygenase-1 (LOX-1) Activity; Inhibit the ability of hyaluronidase, increase skin hyaluronic acid content, promote wound repair, reduce wrinkles; inhibit the activity of matrix metalloproteinases (MMPs), reduce collagen degradation; promote cell repair ability and improve anti-allergic ability and other mechanisms.

Description

A longan flower extract with anti-aging, whitening, anti-allergic and cell repair

The present invention relates to the application of longan flower extract, especially refers to the use of longan flower (DLFE) as an additive for cosmetics, skin care products and other external products to achieve anti-aging, anti-oxidation, anti-inflammation, inhibition of melanin, anti-wrinkle, The purpose of anti-allergic and cell repair mechanisms.

Modern Orientals have an increasing demand for fair complexion, and they also attach the same importance to health. Therefore, how to propose a healthy way to lighten spots is a development focus for the non-medical products industry. Traditional plants are aimed at beauty and maintenance. Most of them are warming and invigorating medicines for invigorating qi, activating blood and adjusting physique. There are also a few single or compound prescriptions that are directly used for skin whitening, acne removal, and anti-inflammatory. However, the analysis of the mechanism of the effective ingredients in plant extracts and their formulation design and application in quasi-medicinal products are still the focus of active development in the domestic quasi-medicine industry. Therefore, if the traditional plants can be developed into low-dose, high-efficiency spot-lightening products, it will help to enhance the technology industry of medical products.

The problem found so far is that in the existing whitening products, most of the existing whitening products use vitamin C, hydroquinone, azaleaic acid and arbutin to achieve the effect of reducing spots. Although these substances do have significant effects on blemishes, some blemishes have the effect of directly destroying melanocytes, thus causing cell cytotoxicity. Therefore, the safety and concentration of melanin production and antioxidant components are worthy of attention. The present invention extracts natural plant longan flower extract which can remove DPPH. And ABTS. + Free radical ability, reducing power, chelating iron ions and inhibiting lipid peroxides; inhibiting tyrosinase activity; eliminating nitric oxide (NO) free radicals, inhibiting lipoxygenase-1 (LOX-1) active.

Most of the literature points out that longan flower has anti-oxidant and anti-inflammatory effects, but there are few related studies on the activity of inhibiting melanin biosynthesis and DNA repair, and there is no discovery of its whitening and anti-aging functions. It contains whitening ingredients such as Vitamin C, Hydroquinone, Acelaic acid and Arbutin. Although they have significant effects, some whitening agents can directly destroy melanocytes , So it will cause cell toxicity.

In view of the fact that the above-mentioned existing whitening and anti-aging product compositions still have many shortcomings in actual use, based on the tireless spirit, with his rich professional knowledge and years of practical experience to improve, and based on this research Created the present invention.

Longan flower contains a lot of polyphenols and flavonoids, which has good antioxidant capacity, and has the effects of lowering blood lipids and inhibiting lipid synthesis. However, relevant literature discussions are rare, so the present invention uses longan flower extract to perform a series of antioxidant, DNA repair, inhibition of melanin biosynthesis and anti-inflammatory activity evaluation.

The main purpose of the present invention is to provide an application of longan flower extract, which refers to the use of longan flower ( Dimocarpus longan flower , DLF) water and ethanol extracts as cosmetic additives that have the ability to scavenge free radicals and inhibit tyrosinase activity ; In this way, longan flower extract can be used as a skin external product to prevent or alleviate the skin aging of one body, to achieve anti-aging, anti-oxidation, anti-inflammatory, inhibition of melanin, anti-wrinkle, anti-allergic and cell repair and other mechanisms .

In order to achieve the above-mentioned implementation objectives, the present invention uses a longan flower extract to apply an effective dose of the longan flower extract to a skin cell, which has the effect of scavenging free radicals, inhibiting lipid peroxides, and inhibiting lipoxygenase. The activity of lipoxygenase-1 (LOX-1) can improve anti-oxidation, inhibit melanin production, anti-inflammatory and cell repair.

Preferably, the longan flower extract includes a water extract DLFW and an ethanol extract DLFE.

Preferably, the longan flower extract has the ability to scavenge or reduce free radicals to prevent cell aging.

Preferably, the free radical is DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical or ABTS (2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) free radical.

Preferably, the longan flower extract is used as a whitening or anti-oxidant cosmetic material composition, food additive or medical composition.

Preferably, the longan flower extract can inhibit skin inflammation caused by UV irradiation or a skin anti-inflammatory agent.

Preferably, the longan flower extract is used as an anti-inflammatory cosmetic material composition or medical composition.

Preferably, the longan flower extract has factors related to regulating p53 and NER repair system to repair DNA damage caused by UV irradiation.

Preferably, the longan flower extract has the activity of inhibiting extracellular mushroom tyrosinase, thereby reducing melanin production.

Preferably, the longan flower extract can inhibit the activity of NO and LOX-1 to inhibit inflammation.

Preferably, the longan flower extract has the ability to inhibit hyaluronidase, increase skin hyaluronic acid content, promote wound repair, and reduce wrinkles.

Preferably, the longan flower extract has inhibitory activity of matrix metalloproteinases (MMPs) and reduces collagen degradation.

Preferably, the longan flower extract has the ability to promote cell repair.

Preferably, the longan flower extract improves the anti-allergic ability.

Figure 1 is a graph of the cytotoxicity test of the present invention.

Figure 2 is a test diagram of the free radical scavenging ability of the present invention.

Figure 3 is a test chart of the whitening efficacy of the present invention.

Figure 4 is a graph of the anti-inflammatory test of the present invention.

Figure 5 is a diagram of the DNA protection test of the present invention.

Figure 6 is a comparison diagram of DNA protection of the present invention.

Figure 7 is a graph of the anti-wrinkle test of the present invention.

Figure 8 is a graph of the present invention's ability to inhibit hyaluronidase.

Figure 9 is a graph showing the wound healing ability of the cells of the present invention.

Figure 10 is a diagram of the UVB cell damage test of the present invention.

Figure 11 is a graph of the anti-allergic ability test of the present invention.

The purpose of the present invention and its structural and functional advantages will be described based on the structure shown in the following drawings and specific embodiments, so that the review committee can have a more in-depth and specific understanding of the present invention.

First of all, the inventors prepared monohydrate DLFW and monoethanolic extract DLFE from longan flowers. The water-extracted DLFW and ethanol extract DLFE of the above materials can reduce the oxidative pressure in HaCaT cells caused _{by H 2} O _{2 stimulation.} , Increase the content of glutathione (GSH) in cells, and increase the expression of antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx).

Longan flower extract (water extract DLFW, ethanol extract DLFE) can inhibit tyrosinase activity in cells and reduce melanin production. By immunofluorescence test and western ink spot test, after longan flower extract (water extract DLFW, ethanol extract DLFE) acts on the cells, it is confirmed that it can inhibit the melanin biosynthesis process through the receptor MC1R (melanocortin-lreceptor) and MITF ( microphthalmia-associated transcription factor), and affect the performance of tyrosinase, TRP-2 (tyrosinase-related proteins-2) and TRP-1 (tyrosinase-related proteins-1), thereby inhibiting melanin production.

In the anti-inflammatory ability test, longan flower extract can inhibit the content of intracellular inflammatory factors (TNF-α, IL-1β and IL-6), and has the p38, IL-6, IL-1β, and IL-1β that reduce inflammation-related pathways. TNF-α protein performance. Animal experiment SKH-1 mice were irradiated with UVB and used immunohistochemistry (IHC) and western blotting methods to confirm that longan flower extract has reduced inflammation-related pathways p38, IL-6, IL-1β and TNF- Alpha protein performance.

In the DNA repair and protection ability test, the longan flower extract has the relevant factors of regulating the nucleotideexcision repair (NER) repair system to repair the DNA damage caused by UV irradiation, and the regulation of p53 and the relevant factors of the NER repair system to repair the damage caused by UV DNA damage caused by irradiation.

The following specific examples can further prove the scope of practical application of the present invention, but it is not intended to limit the scope of the present invention in any form.

Experiment 1: Cytotoxicity test

In view of the fact that the existing whitening agents or skin melanin inhibitors can cause cytotoxicity, the inventors first performed the cytotoxicity test experiment of this compound after obtaining this longan flower extract, using 100, 250 and 500 μg/ml concentrations After the longan flower extract was applied to HaCaT cells for 24 hours, the survival rate was measured by MTT test. The results are shown in Figure 1. Longan Flower Water Extract DLFW and ethanol extract DLFE are not toxic in cells, and are safer compared to the commonly used ingredient ascorbic acid (AA) in the market.

Experiment 2: Test of the ability to scavenge free radicals

This experiment explores the effect of longan flower water extract DLFW and ethanol extract DLFE in scavenging free radicals, using DPPH. And ABTS+. The scavenging ability of two free radicals was tested, and the results are shown in Figure 2. The ethanol extract DLFE reached a scavenging rate of 50% at about 45μg/ml in the DPPH free radical scavenging test, and the ethanol extract DLFE scavenged both The ability of free radicals is better than that of water extraction DLFW, and the results show that longan flower extract has a good inhibition rate for two free radicals.

Experiment 3: Whitening efficacy test

Tyrosinase is an important enzyme in the biosynthesis pathway of melanin. The water-extracted DLFW of longan flower and the ethanol extract DLFE were tested for extracellular mushroom tyrosinase activity at different concentrations of 100, 250 and 500 μg/ml. The results are shown in Figure 3. After the action of DLFW and ethanol extract of longan flower, the activity of inhibiting extracellular mushroom tyrosinase increases with the increase of concentration, and the ethanol extract of DLFE (EC ₅₀ is 410μg/ml The inhibition rate of) is better than that of water-extracted DLFW (EC ₅₀ is 624μg/ml), which proves that longan flower extract has the activity of inhibiting extracellular mushroom tyrosinase.

Experiment 4: Anti-inflammatory test

NO is one of the main mediators involved in inflammation, and LOX-1 is the most common enzyme that causes inflammation. This test evaluates the anti-inflammatory effects of longan flower water extract DLFW and ethanol extract DLFE. The results are shown in Figure 4. Longan flower water extract DLFW and ethanol extract DLFE have a good ability to scavenge NO free radicals and inhibit the activity of LOX-1, which proves that Longan flower water extract DLFW and ethanol extract DLFE can pass Inhibit the activity of NO and LOX-1 to achieve the activity of inhibiting the inflammatory response, and has an anti-inflammatory effect.

Experiment 5: DNA protection test

UV exposure or physiological metabolism will increase oxidative stress and cause DNA damage. In this test, pUC119 DNA was _{induced with H 2} O ₂ (1mM) and irradiated with UVB (20mJ/cm ² ) to cause DNA damage. To evaluate whether the water extract of longan flower DLFW and ethanol extract DLFE can protect DNA from oxidative stress and DNA damage caused by UVB radiation. Please refer to Figure 5, pUC119 DNA was originally a supercoiled form (SC-form). After H ₂ O ₂ (1mM) induction and UVB (20mJ/cm ² ) irradiation, it will cause DNA breakage and form (Linear form, L-form) and (Open form, O-form) DNA fragments. Continuing to refer to Figure 6, when different concentrations of ethanol extract DLFE (100, 250 and 500μg/ml) are added and then _{induced by H 2} O ₂ and UVB irradiated, the results show that the ethanol extract DLFE can effectively protect DNA, and the concentration of The better the protection effect is, it is proved that longan flower can protect DNA from damage caused by oxidative stress and UVB irradiation.

Experiment 6: Anti-wrinkle test

Collagen mainly exists in the connective tissue of the dermis in the skin tissue. It is the highest protein content in the human body. It has a strong stretching force and can support the overall structure of the dermis. Collagen is also the main component of the extracellular matrix to maintain the skin. It is elastic and has the ability to repair, and collagen will be lost with aging and environmental factors, causing wrinkles and osteoporosis in the skin. Among them, matrix metalloproteinases (MMPs) are collagen degrading enzymes, and MMP-2 and MMP-9 mainly decompose type II collagen. In order to adjust the important factors of photoaging, UV exposure of fibroblasts will cause a large number of matrix metalloproteinases to degrade the extracellular matrix (ECM) and degrade the collagen of the dermal tissue.

In this experiment, the ethanol extract DLFE (100, 250 and 500μg/ml) of longan flower was treated in Hs68 cells at different concentrations and cultured for 72 hours to determine the collagen content. This test evaluates whether the ethanol extract DLFE of Longan flower has the activity of inhibiting MMPs. The results are shown in Figure 7. After the action of the ethanol extract of longan flower DLFE, compared with Control, the collagen content increased with the increase in concentration. Therefore, it can be inferred that the ethanol extract of longan flower DLFE can inhibit MMP-9 and The activity of MMP-2 can effectively increase the content of collagen produced by fibroblasts.

Experiment 7: Test of the ability to inhibit hyaluronidase

Hyaluronic acid is present in the dermis and connective tissues of human skin tissues, reducing wrinkles and promoting wound repair. Hyaluronidase is an enzyme that degrades hyaluronic acid, disintegrating the structure of hyaluronic acid and losing its original function. . Through this test, the ethanol extract of longan flower DLFE was evaluated for its ability to inhibit hyaluronidase.

The ethanol extract of longan flower DLFE (500μg/ml) and the standard epigallocatechin (EGCG) were added to hyaluronidase (3000unit/mL) for 18 hours. The results are shown in Figure 8. The position of the ethanol extract of longan flower DLFE is much darker than that of Control, showing that DLFE can effectively prevent hyaluronic acid from being decomposed by hyaluronidase. Quantitative analysis shows that the ethanol extract of longan flower DLFE inhibits hyaluronidase activity The rate is 82%. The results confirmed that the ethanol extract of longan flower DLFE has the ability to inhibit the activity of hyaluronidase.

Experiment 8: Cell wound healing ability test

The wound healing test is used to evaluate whether DLFE has the ability to promote cell repair. First, Longan flower extract DLFE (500μg/ml) was applied to HaCaT cells for 4 hours, then the culture medium was drained, ^{irradiated with UVB (50mJ/cm 2} ), and DLFE was added. After culturing for 4 hours, observe the cell movement with a microscope The change.

The results are shown in Fig. 9. The control and the cells irradiated with UVB alone did not change much, especially the cells irradiated with UVB alone, there was almost no change from 0 to 48 hours. The cells after longan flower extract DLFE had a clear tendency to move to the middle after 12 hours, and there were no gaps in 48 hours. The results once again confirmed that the longan flower extract DLFE has the ability to promote cell repair.

Experiment 9: UVB cell damage test

Comet test is a method of gel electrophoresis and staining of cell nucleus with EtBr to detect the damage degree of DNA in a single cell after UVB irradiation. After acting on HaCaT cells with DLFE (500μg/ml) for 4 hours, drain the culture medium, ^{irradiate it with UVB (50mJ/cm 2} ), and add longan flower extract DLFE. After culturing for 4 hours, observe the cell damage with a microscope degree.

The results are shown in Figure 10. Cells irradiated by UVB alone have DNA damage and obvious tailing. Among 1,000 cells, about 50% of cells have DNA damage. After DLFE treatment, under the action of a high concentration of 500μg/ml, about 8% of the cells have DNA damage. It can be judged that DLFE can protect DNA from damage caused by UVB radiation.

CPD is the light product produced by DNA after UVB irradiation. The results are shown in Figure 10. After UVB (50mJ/cm ² ) irradiation, the fluorescence of CPD increases, and after DLFE (500μg/ml) After that, the generation of UVB photoproducts was significantly reduced, and it was again confirmed that DLFE can protect DNA and reduce DNA damage caused by UVB irradiation.

Experiment 10: Anti-allergy test

Please refer to Figure 11: β-glucuronidase degranulation test induced by carbamicin A23187 or antigen: The degree of degranulation of RBL-2H3 mast cells induced by carbamicin A23187 and antigen is determined by β-glucuronic acid The enzyme release test is determined by the following modified method: inoculate RBL-2H3 mast cells (2×10 ⁴ cells/well) in 96-well microplates and RBL-2H3 mast cells (3×10 ⁴ cells/well) in 48 The well microplates were supplied for the carbamicin A23187 induction experiment and the antigen induction experiment respectively. Various concentrations of samples were added to the cells and allowed to stand for 20 hours; cortisol (10 nM) was used as a positive control group. In the antigen induction experiment, the cells were first passively hypersensitized with anti-dinitrophenol IgE (anti-DNP IgE) (5μg/mL) for at least 2 hours. Buffer (135mM sodium chloride, 5mM potassium chloride, 1.8mM calcium chloride, 1.0mM magnesium chloride, 5.6mM glucose, 20mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.4) after thorough washing, each with Calcium ionophore A23187 (carbamicin, 1 μM) or antigen dinitrobenzene-fetal bovine serum conjugate (100ng/mL) prepared in Tyrode’s buffer was stimulated for one hour. In order to measure the total amount of β-glucuronidase released, unstimulated cells were lysed with 0.5% Triton X-100 solution, or the cells were not treated to release β-glucuronidase spontaneously; The clear solution (50 μL) and an equal amount of 1 μM polyazide-acetylglucosamine (antigen) were prepared in 0.1M citrate buffer (pH 4.5) to be used as a substrate for releasing β-glucuronidase. After incubating at 37°C for 1 hour, 100 μL of stop buffer (0.1M Na ₂ /NaHCO ₃ , pH 10.0) was added to quench the reaction. The microdisc analyzer (Multiskan Ascent, Thermo Scientific) was set to 405nm wavelength, and the absorbance was measured; the inhibition percentage of β-glucuronidase release was calculated by dividing the experimental group by the percentage of the control value (untreated cells); The maximum tolerated dose of Giatosine was 0.5%, and all experiments were repeated three times.

Sample-induced β-glucuronidase direct degranulation test: In RBL-2H3 mast cells, the amount of β-glucuronidase released by the sample is determined by the modified β-glucuronidase release test ; Simply put, the RBL-2H3 mast cells (4×104cells/well) were seeded in a 48-well microplate, and the sample was added and then allowed to stand for 10 hours. Tyrode's buffer was added to 5.6mM glucose, 2mg/mL bovine serum albumin (BSA) and 2mM glutamic acid to prepare samples and cells. 50 μL of supernatant was transferred to a 96-well microplate, and the amount of β-glucuronidase released was measured according to the aforementioned method. Carximycin A23187 (1 μM) was used as the positive control group. All experiments were repeated more than three times.

In summary, the present invention proves that the water extract of longan flower DLFW and the ethanol extract DLFE have the ability to remove active oxygen and inhibit melanin production, and have the ability to protect and repair the DNA of skin epidermal cells and inhibit the production of inflammation. The potential of skin care products or health food additives for whitening, anti-oxidation and anti-inflammatory, and the expected use effects can be achieved through the embodiments disclosed above, and the present invention has not been disclosed before the application. The provisions and requirements of the Patent Law. If you file an application for a patent for invention in accordance with the law, you are kindly requested to review and grant a quasi-patent.

However, the above-mentioned illustrations and descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. Anyone familiar with the art will do other things based on the characteristic scope of the present invention. Equivalent changes or modifications should be regarded as not departing from the design scope of the present invention.

Claims (9)

A kind of longan flower ethanol extract is used to prepare skin anti-aging, skin whitening, skin anti-allergic, skin cell repair and skin anti-wrinkle composition, wherein the composition is used at an effective dose of 100 μg/ml to 500 μg/ml The longan flower ethanol extract is administered to a skin cell to scavenge free radicals, inhibit the effectiveness of lipid peroxides, inhibit the activity of lipoxygenase-1 (LOX-1), inhibit the ability of hyaluronidase, and increase skin hyaluronic acid content. Further improve anti-oxidation, inhibit melanin production, anti-inflammatory, cell repair, promote wound repair, and reduce wrinkle production; wherein 100μg/mL epigallocatechin and the composition are each with 500μg/mL longan flower ethanol extract Do not react with 3000unit/mL hyaluronidase for 18 hours, and the hyaluronidase inhibition rate of 500μg/mL longan flower ethanol extract of the composition is 82% of the hyaluronidase inhibition rate of 100μg/mL epigallocatechin; Furthermore, the skin cells were first ^{irradiated with 50 mJ/cm 2} UVB, and the composition was then treated with 500 μg/mL longan flower ethanol extract for 4 hours, and 8% of the skin cell DNA was damaged. The use described in claim 1, wherein the free radical is DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical or ABTS (2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) base. The use according to claim 1, wherein the composition is a whitening or anti-oxidant cosmetic material composition, food additive or pharmaceutical composition. The use according to claim 1, wherein the composition is an anti-inflammatory agent for inhibiting skin inflammation caused by UV irradiation. The use according to claim 1, wherein the composition is an anti-inflammatory cosmetic material composition or a pharmaceutical composition. The use according to claim 1, wherein the composition has related factors regulating p53 and NER repair system to repair DNA damage caused by UV irradiation. The use according to claim 1, wherein the composition has the activity of inhibiting extracellular mushroom tyrosinase, thereby reducing melanin production. The use according to claim 1, wherein the composition has the activity of inhibiting NO and LOX-1 to inhibit inflammation. The use according to claim 1, wherein the composition has the activity of inhibiting matrix metalloproteinases (MMPs) to reduce collagen degradation.

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