Application of American mint extract and product of American mint extract

Abstract

The invention provides application of an American mint extract and a product of the American mint extract. Belongs to the technical field of plant extracts. Research finds that the American mint extract has a whitening effect of inhibiting tyrosinase from catalyzing to generate melanin, is applied to preparation of whitening products, also has multiple effects of resisting oxidation, resisting inflammation and the like, and has the advantages of small toxicity, strong whitening effect and no side effect compared with the existing whitening agent. The method can play a huge role in the fields of medicine, clinic and cosmetics, is low in cost, simple to plant and easy to produce, and enriches the raw material sources of whitening products.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, in particular to the application of peppermint extract and its products. Background Art

[0002] With the improvement of living standards, consumers have higher requirements for whitening products, especially in the Asian beauty and skin care market, where whitening is still the most concerned issue for consumers. Pigmentation is a common skin problem with a complex pathogenesis involving factors such as increased melanin production, abnormal distribution and metabolic imbalance. The clinical manifestations of pigmentation are diverse, such as sun spots, freckles and chloasma, which affect appearance. Although chemical drugs such as arbutin, kojic acid, hydrocortisone and its derivatives have been used clinically, due to their low activity and easy to make the skin sensitive, the development of new natural anti-melanin drugs has become urgent.

[0003] Peppermint is an annual herbaceous plant of the genus Peppermint in the family Lamiaceae. It is native to America and has been successfully introduced to my country in recent years. Peppermint is widely used to relieve symptoms such as colds, indigestion and various skin diseases. In recent years, studies have also found that peppermint has multiple effects such as antioxidant, anti-inflammatory, antibacterial and improvement of cognitive impairment. However, there is no relevant research on the whitening effect of peppermint extract.

[0004] Therefore, how to provide a natural plant extract with whitening effect is a technical problem that technicians in this field need to solve urgently. Summary of the invention

[0005] In view of this, the present invention has found through research that American peppermint extract can significantly inhibit the activity of tyrosinase, thereby blocking the tyrosinase-catalyzed melanin production pathway, effectively inhibiting the production of melanin, and having a whitening effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] Application of American peppermint extract, the application being any one of the following:

[0008] A: Application in the preparation of whitening products;

[0009] B: Application in the preparation of products for inhibiting intracellular tyrosinase activity;

[0010] C: Application in the preparation of products for inhibiting melanin production.

[0011] Preferably, the product is a pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic having the efficacy of whitening, inhibiting intracellular tyrosinase activity or inhibiting melanin production.

[0012] Preferably, the peppermint extract is a water extract or an essential oil extract.

[0013] Another object of the present invention is to provide: a pharmaceutical raw material, a pharmaceutical product, a cosmetic raw material or a cosmetic having whitening effect, comprising the above-mentioned American peppermint extract.

[0014] Preferably, other functional active ingredients and / or auxiliary materials are also included.

[0015] Another object of the present invention is to provide: a pharmaceutical raw material, a pharmaceutical product, a cosmetic raw material or a cosmetic having the efficacy of inhibiting intracellular tyrosinase activity, comprising the above-mentioned peppermint extract.

[0016] Preferably, other functional active ingredients and / or auxiliary materials are also included.

[0017] Another object of the present invention is to provide: a pharmaceutical raw material, a pharmaceutical product, a cosmetic raw material or a cosmetic having the effect of inhibiting melanin production, comprising the above-mentioned American peppermint extract.

[0018] Preferably, other functional active ingredients and / or auxiliary materials are also included.

[0019] It can be seen from the above technical solution that compared with the prior art, the present invention has the following benefits:

[0020] Through research, the present invention finds that American peppermint extract has a whitening effect of inhibiting the production of melanin catalyzed by tyrosinase, and also has multiple functions such as anti-oxidation and anti-inflammatory. Compared with existing whitening agents, it has low toxicity and strong whitening effect, and can play a huge role in the fields of medicine, clinical practice and cosmetics. It is low in cost, simple to plant and easy to produce, thus enriching the raw material source of whitening products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 The effect of water extract of American peppermint on tyrosinase activity in vitro;

[0023] Figure 2 To: Effect of peppermint essential oil on tyrosinase activity in vitro;

[0024] Figure 3 To: Effect of peppermint essential oil on tyrosinase activity in vitro;

[0025] Figure 4 To study the effects of different concentrations of water extract of American peppermint on the hatching rate and survival rate of zebrafish embryos;

[0026] Figure 5 The following are the inhibitory effects of the same concentration of American peppermint water extract on zebrafish melanin production;

[0027] Figure 6 The effect of the same concentration of American peppermint water extract on the melanin area on the surface of zebrafish;

[0028] Figure 7 To study the effects of different concentrations of American peppermint water extract on the melanin content in zebrafish;

[0029] Figure 8 To investigate the effects of different concentrations of water extracts of American peppermint on tyrosinase activity in zebrafish.

[0030] Fig. 9 Figure 3: Changes in MI values ​​over time after using the cream. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] The experimental materials and instruments used in the embodiments of the present invention are as follows:

[0033]

[0034] Example 1

[0035] Preparation of American peppermint aqueous extract:

[0036] (1) Pretreatment: Take whole American mint flowers, wash to remove impurities and dust, and dry in the sun;

[0037] (2) Separation and extraction: add whole American mint flowers to distilled water at a solid-liquid ratio of 1-8-1:10, soak at room temperature for 12-18 hours; boil at 80-85°C for 1-1.5 hours, and separate the solid and liquid; add 500 ml of distilled water to the solid separation and continue boiling for 1-1.5 hours, separate the solid and liquid, combine the liquids obtained from the two separations, pass through a 200 nm filter paper, centrifuge at 3500-4000 r / min for 10-15 minutes, and take the supernatant for later use;

[0038] (3) Purification: add 1% by mass of clay to the supernatant of step (2), heat with stirring in a water bath at 40-45° C. for 40 min, and then filter through a filter cake made of No. 300 and No. 10 silica gel in a mass ratio of 3:1;

[0039] (4) Concentration and drying: The filtrate is taken out and concentrated by rotary evaporation at 30 r / min and 75-80° C. for 40-60 min, and then spray-dried at high temperature to obtain peppermint water extract powder.

[0040] Preparation of peppermint essential oil:

[0041] (1) Pretreatment: Take fresh whole American mint flowers, wash and remove impurities and dust;

[0042] (2) Extraction: Place the whole flower of American peppermint in a round-bottom flask at a solid-liquid ratio of 1:10, and add 2 to 3 zeolites. After cold soaking for 1 hour, use a volatile oil extractor to extract the essential oil. Heat and reflux for extraction for 1 hour, separate the water layer from the essential oil, add anhydrous sodium sulfate to the essential oil for dehydration for 2 hours, and obtain American peppermint essential oil.

[0043] Preparation of Peppermint Essential Oil:

[0044] (1) Pretreatment: Take fresh peppermint, wash it to remove impurities and dust, and break it;

[0045] (2) Extraction: Place peppermint in a round-bottom flask at a solid-liquid ratio of 1:10, and add 2 to 3 zeolites. After cold soaking for 1 hour, extract the essential oil using a volatile oil extractor. Heat and reflux for extraction for 1 hour, separate the water layer from the essential oil, add anhydrous sodium sulfate to the essential oil and dehydrate it for 2 hours to obtain peppermint essential oil.

[0046] Preparation of levodopa (1 mg / mL): Weigh 10 mg by precise gram weight, dissolve in triple-distilled water and make up to 10 mL.

[0047] Preparation of tyrosinase solution (100U / ml): Add 2.5ml of triple-distilled water to a 25KU tyrosinase drug bottle to prepare a 10,000U / ml tyrosinase stock solution, then take 10ul of the stock solution and add it to 990ul of triple-distilled water to prepare a 100U / ml tyrosinase solution.

[0048] Preparation of phosphate buffer solution (0.2 mol / L, pH=6.8): Take 98.6 ml of 0.3 mol / L disodium hydrogen phosphate solution + 1.4 ml of 0.3 mol / L sodium dihydrogen phosphate solution.

[0049] Example 2

[0050] In vitro tyrosinase activity assay

[0051] Using 0.2 M phosphate buffer (PBS) as solvent, mushroom tyrosinase was configured to 100 U / mL, and 4 groups of experiments were set up as follows:

[0052] Blank group A (140 μL PBS + 100 μL L-DOPA);

[0053] Blank control group B (90 μL PBS + 50 μL mushroom tyrosinase + 100 μL L-DOPA);

[0054] Sample background control group C (20 μL sample + 120 μL PBS + 100 μL L-DOPA);

[0055] Sample group D (20uL sample + 70μL PBS + 50μL mushroom tyrosinase + 100μL L-DOPA);

[0056] Group A added 140μL PBS and 100μL L-DOPA; Group B added 90μL PBS and 100μL L-DOPA, incubated in a 37℃ incubator for 10min, and then added 50uL mushroom tyrosinase; Group C added 20μL sample, 120μL PBS and 100μL L-DOPA; Group D added 20uL sample, 70μL PBS, 100μL L-DOPA, incubated in a 37℃ incubator for 10min, and then added 50uL mushroom tyrosinase. After incubation for another 20min, the absorbance was measured at 475nm, and the experiment was repeated 3 times.

[0057] The inhibition rate of tyrosinase was calculated according to the formula (inhibition rate = [(BA) - (DC)] / (BA) x 100%).

[0058] The tyrosinase activity test was performed on peppermint water extract, peppermint essential oil and peppermint essential oil as samples, and the experimental results were as follows: Figure 1 , Figure 2 and Figure 3 shown.

[0059] Result analysis: Figure 1 It was shown that the in vitro tyrosinase activity decreased with increasing concentration of American peppermint water extract; Figure 2 The in vitro tyrosinase activity shown decreased with increasing concentrations of peppermint essential oil; Figure 3 The results showed that peppermint essential oil promoted tyrosinase activity in vitro. It showed that both the water extract and essential oil of American peppermint had an inhibitory effect on tyrosinase activity, while other types of peppermint essential oils (peppermint essential oil) did not inhibit tyrosinase activity.

[0060] Example 3

[0061] (I) Toxicity experiment of zebrafish embryos treated with test substances

[0062] In the experiment, 8 groups of American peppermint water extract treatment groups (0.4mg / mL, 0.3mg / mL, 0.2mg / mL, 0.1mg / mL, 0.05mg / mL, 0.025mg / mL, 0.0125mg / mL and 0.00625mg / mL) were set up, and the blank control was embryos cultured in zebrafish embryo culture medium without the test substance. A 24-well culture plate was used as the experimental container, 10 zebrafish embryos were placed in each well, 2000μL of embryo culture medium corresponding to the test substance concentration was added to each well, 3 duplicate wells were set up, and the morphology of zebrafish embryos in each group was observed and recorded.

[0063] After 48 hours of treatment with the test substance, 9 embryos were randomly selected for each concentration to measure the tyrosinase activity and melanin production in vivo, and the rest were cultured for 96 hours to observe the toxicity of the test substance. The experimental results are as follows: Figure 4 shown.

[0064] Result analysis: Figure 4 As shown in the figure, as the drug concentration increases, the hatching rate and survival rate of zebrafish decrease significantly, especially at high concentrations (0.40000 mg / ml) and long-term (72 hours) exposure, the toxic effect is most significant. However, at a concentration of 0.05 mg / ml, there is no obvious toxic effect on zebrafish, which provides a dosage basis for subsequent experiments.

[0065] (II) Changes in melanin content in zebrafish fry

[0066] After 48 hours of treatment with the test substance, observe the melanin production of the embryos under a microscope and take photos. Use a sterile pipette to carefully suck out the melanin and place it vertically. When the zebrafish sinks to the mouth of the tube, gently squeeze the pipette to drop it into the groove of the concave glass slide, discard the embryo culture medium, add 1 to 2 drops of carboxymethyl cellulose, and adjust the zebrafish under a microscope so that its head is facing upward and its tail is at the same level as the body. Under the same magnification and light intensity, take a clear top view of the zebrafish. The results are as follows: Figure 5 As shown; Image J software was used for image analysis, the images were converted into 8-bit grayscale images, the background was eliminated (the parameter was set to 130 pixels), the threshold parameters were set, the total area of ​​zebrafish melanin spots was calculated, and the difference in the ratio of melanin area to total area in different concentration treatment groups was compared. The average value was calculated for more than 6 samples in each group. The experimental results are shown in Figure 6 shown.

[0067] Relative content of body surface pigment (%) = S sample / S blank × 100,

[0068] Where: Sblank is the percentage of melanin area in the blank group; Ssample is the percentage of melanin area in the sample group.

[0069] Result analysis: Figure 5and Figure 6 As shown, with the increase of the concentration of American peppermint extract, the melanin in the zebrafish head was significantly reduced; the melanin area on the surface of zebrafish larvae decreased with the increase of the concentration of American peppermint extract, indicating that American peppermint extract can effectively inhibit the production of melanin.

[0070] (III) Determination of melanin content in zebrafish using NaOH lysis method

[0071] 72 hours after the test substance treatment, 100 zebrafish embryos were collected, placed at -80℃ and killed, then washed three times with PBS buffer, added with 100μL of PBS buffer containing 1% Triton X-100, ultrasonically disrupted for 5 minutes, centrifuged at 10000r / m, 4℃ for 15 minutes, and the precipitate was collected;

[0072] After centrifugation, add 300 μL of 1 mol / L NaOH solution containing 10% DMSO to resuspend the pellet and place it in an 80°C water bath for 2 hours. After cooling, transfer it to a 96-well culture plate. Use zebrafish without the test substance as a blank control and measure the absorbance value A405 at 405 nm, which is the melanin content in the zebrafish. The results are as follows: Figure 7 The relative melanin content is calculated as follows:

[0073] Relative melanin content (%) = A sample / A blank × 100,

[0074] Where: A blank is the absorbance value of the blank group; A sample is the absorbance value after adding the sample

[0075] Result analysis: Figure 7 As shown, the melanin content in zebrafish decreased with the increase of the concentration of peppermint extract, indicating that the melanin content decreased in a concentration-dependent manner.

[0076] (IV) Detection of tyrosinase activity in zebrafish

[0077] 72 hours after the test substance treatment, 100 zebrafish embryos were collected, placed at -80℃ and killed, then washed three times with PBS buffer, 100 μL of PBS buffer containing 1% Triton X-100 was added, and after ultrasonic disruption for 5 minutes, the embryos were centrifuged at 10000 r / m and 4℃ for 15 minutes. The supernatant was the crude extract of zebrafish tyrosinase.

[0078] The zebrafish tyrosinase activity was determined by tyrosinase activity inhibition test. The protein content in the crude enzyme solution was determined by BCA kit, and the protein concentration of each group was adjusted to the same concentration. 50 μL of crude enzyme solution was taken into a 96-well plate, 100 μL of 1 mg / mL L-DOPA was added, and the blank group of zebrafish embryos was used as a control. The experiment was carried out according to the method of Example 2. The experimental results are shown in FIG. Figure 8 , enzyme activity was calculated as follows:

[0079] Tyrosinase activity (%) = A sample / A blank × 100

[0080] Result analysis: Figure 8 As shown, the tyrosinase activity was compared with the blank group. The enzyme activity decreased with the increase of the concentration of American peppermint extract, indicating that the enzyme activity decreased in a dose-dependent manner.

[0081] Example 4

[0082] Preparation of skin antioxidant and / or skin whitening cream:

[0083] The American peppermint water extract was mixed with other ingredients to prepare a cream, and the ingredients and proportions were as follows:

[0084] Table 1 Ingredients and proportions of skin anti-oxidation and / or skin whitening creams

[0085] Element % 1 water 79.87 2 1,3 Propylene glycol 5 3 EDTA 0.03 4 glycerin 6 5 Cetearyl Alcohol 1.2 6 Dimethicone 2 7 Methyl Glucose Sesquistearate 2.5 8 Hydroxypropyl methylcellulose 0.6 9 1,2-Pentanediol 1 10 Phenoxyethanol 0.8 11 American peppermint water extract 1

[0086] Human evaluation test

[0087] The evaluation method is as follows: Use the skin tester MPA580 to connect the melanin skin detector The MX 18 probe was used to measure the melanin value. 20 subjects were selected, aged 35-40 years old. All the subjects had darker skin and dark brown spots. The subjects were in good health, had no history of skin diseases and allergies, had non-sensitive skin, and were able to use skin care products according to specifications. The subjects sprayed the cream prepared with American peppermint water extract on their faces once in the morning and evening every day, and used it continuously. The melanin values ​​of specific parts of the skin in the third and sixth weeks of using only the cream were compared. The test was repeated 5 times, and the average value calculated after removing the highest and lowest values ​​was the data for each volunteer tested that time. Before each test, the volunteers were required to clean their faces with facial cleanser, sit quietly for 30 minutes, and then measure again. The melanin index (MI) of each group of subjects was calculated. The larger the MI value, the darker the skin. The experimental results are as follows: Fig. 9 shown.

[0088] Result analysis: Fig. 9 As shown in the figure, with the increase of usage time, the MI value of the subjects gradually decreased, and the change was significant.

[0089] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0090] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of American peppermint extract, characterized in that, The application is any of the following: A: Application in the preparation of whitening products; B: Application in the preparation of products for inhibiting intracellular tyrosinase activity; C: Application in the preparation of products for inhibiting melanin production.

2. The use according to claim 1, characterized in that The product is a pharmaceutical raw material, a pharmaceutical product, a cosmetic raw material or a cosmetic having the functions of whitening, inhibiting the activity of intracellular tyrosinase or inhibiting the production of melanin.

3. The use according to claim 1, characterized in that The peppermint extract is water-extracted or essential oil-extracted.

4. A pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic with whitening effect, characterized in that: The invention comprises the peppermint extract as claimed in claim 1.

5. The pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic according to claim 4, characterized in that: Other functional active ingredients and / or excipients are also included.

6. A pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic having the effect of inhibiting intracellular tyrosinase activity, characterized in that: The invention comprises the peppermint extract as claimed in claim 1.

7. The pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic according to claim 6, characterized in that: Other functional active ingredients and / or excipients are also included.

8. A pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic having the effect of inhibiting melanin production, characterized in that: The invention comprises the peppermint extract as claimed in claim 1.

9. The pharmaceutical raw material, pharmaceutical product, cosmetic raw material or cosmetic according to claim 8, characterized in that: Other functional active ingredients and / or excipients are also included.

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