Composition of prunella vulgaris extract and polypeptide zinc chelate, and use thereof in field of cosmetics

Abstract

A composition of Prunella vulgaris extract and polypeptide zinc chelate, and the use thereof in the field of cosmetics. The Prunella vulgaris extract has a saponin content of not less than 1.5 mg / mL and a flavonoid content of not less than 1.0 mg / mL. The polypeptide zinc chelate is formed by means of the chelation of a polypeptide compound [γ-Glu] n-AA with a zinc ion compound. Cosmetics containing the composition of Prunella vulgaris extract and polypeptide zinc chelate can be used for skin oil control and acne removal.

Description

Prunella vulgaris extract and polypeptide chelated zinc composition and application thereof in cosmetic field TECHNICAL FIELD

[0001] The present application relates to the field of polypeptide composition, in particular to Prunella vulgaris extract and polypeptide chelated zinc composition and application thereof in cosmetic field. BACKGROUND

[0002] Prunella vulgaris L. is a kind of perennial herb with medicinal and edible properties, and has been used as medicine for thousands of years. Prunella vulgaris L. is a kind of dry fruiting spikes of Prunella vulgaris L. belonging to Lamiaceae, which was first recorded in Shennong Bencao Jing. Prunella vulgaris L. is named after it withers after summer solstice. Prunella vulgaris L. has the effects of clearing liver and removing fire, clearing away heat and toxic material from the liver, clearing away heat from the eyes, resolving stasis and dissipating swelling. Prunella vulgaris L. has multiple pharmacological effects, such as lowering blood sugar and blood pressure, resisting bacteria and inflammation, immune suppression, removing free radicals, antioxidant, anti-tumor, inhibiting virus growth, etc. Modern research shows that Prunella vulgaris L. contains various types of chemical components, such as terpenes, phenolic acids, flavonoids, sterols, coumarins, organic acids, volatile oils and sugars. Terpenes and flavonoids are the main active substances.

[0003] The use of Prunella vulgaris extract is known in the medical field. For example, patent CN202280051935 relates to the use of a composition comprising or consisting of the aerial parts of Prunella vulgaris, aimed at preventing and / or treating disorders associated with psychological emotional stress and / or damage to the skin and hair follicles. KR101864720 teaches that Prunella vulgaris extract prevents skin atrophy by specifically inhibiting the activity of the enzyme 11 β-HSD1, which then inhibits the production of cortisol. The extract can be obtained by using water, alcohol (C1-C4) or mixtures thereof. However, this document does not mention the plant part used. In addition, the water extract of the aerial parts of Prunella vulgaris inhibits the expression of the enzyme 11 β-HSD1, which catalyzes the intracellular conversion of the inactive cortisone into the active cortisol. (K-B Roh et al., 2018, Research Article, Article ID 1762478). The extract also inhibits the cortisone-mediated reduction in collagen content, to maintain skin integrity and its homeostasis, preventing skin atrophy. Finally, although it is mentioned that the rosmarinic acid contained in the extract shows independent inhibitory activity of 11 β-HSD1, further active ingredient identification studies are needed to understand the skin diseases related to cortisol. CN106727928 discloses a water-ethanol and water-methanol concentrated extract of the stems and leaves of Prunella vulgaris, which contains caffeic acid and rosmarinic acid. The extract can be used to treat Alzheimer's disease. CN108272858 discloses an extract from the inflorescences of Prunella vulgaris containing rosmarinic acid and caffeic acid, which has antioxidant activity. Prunella vulgaris also has an oil-controlling effect, which can help regulate sebum secretion, thereby reducing the condition of oily skin and further assisting in acne removal.

[0004] Polypeptide metal ion chelate refers to the carboxyl, phosphate group, amide group and amino acid on the small molecule polypeptide prepared by proteolysis, which forms a bioactive substance by coordination, and has the advantages of high bioavailability, high safety and high biological activity. Polypeptides and metal ions are widely used in the field of cosmetics. The "Catalogue of Cosmetic Ingredients in Use (2021 Edition)" contains 79 kinds of peptide raw materials, and there are also many kinds of metal salts, which are involved in many functions such as moisturizing, whitening, sunscreen, soothing, etc. However, the raw materials of polypeptide chelated metal ions are few, and most of them are amino acid chelated metal ions, among which the most famous is blue copper peptide, which is a copper bonded amino acid (Copper Peptide Complex™) GHK complex, where GHK is a glycyl-L-histidyl-L-lysine peptide, which plays a role in skin repair, anti-aging, wrinkle removal and hair growth. SUMMARY

[0005] The inventors of the present application accidentally found that when the prepared polypeptide metal chelate is used in combination with the Prunella vulgaris extract, the oil control and anti-inflammatory effects are better than those of the two used alone, and the Prunella vulgaris extract and the polypeptide metal chelate can be used to prepare cosmetics with better oil control and acne removal effects.

[0006] The first aspect of the present application discloses a Prunella vulgaris extract and polypeptide chelated zinc composition, the Prunella vulgaris extract contains not less than 1.5 mg / mL of saponins and not less than 1.0 mg / mL of flavonoids;

[0007] The polypeptide chelated zinc is formed by chelation of a polypeptide compound [γ-Glu] n-AA and a zinc ion compound, wherein n≥1, and AA is selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0008] The preparation method of the Prunella vulgaris extract is as follows:

[0009] The Prunella vulgaris spikes are added into an aqueous solution of 1,3-butanediol, stirred and soaked at 70°C for a certain time, filtered through a 100-mesh sieve, and the Prunella vulgaris extract is obtained.

[0010] The preparation method of the polypeptide chelated zinc is as follows:

[0011] The polypeptide compound [γ-Glu] n-AA is synthesized from amino acids by fermentation method, n, The polypeptide chelated zinc solution is prepared by slowly adding a zinc sulfate solution into the polypeptide solution until the pH is 6.0-8.0, and adjusting the pH with HCl or NaOH.

[0012] Preferably, AA in the polypeptide compound is glutamine, and the zinc ion compound is selected from zinc sulfate.

[0013] The second aspect of the present application discloses the application of the Prunella vulgaris extract and polypeptide chelated zinc composition in the preparation of cosmetics. The cosmetics are used for oil control and / or acne removal of the skin.

[0014] The present application has the following advantages:

[0015] The present application finds that compared with the single use of the prepared polypeptide metal chelate or the Prunella vulgaris extract, the higher dosage of the polypeptide metal chelate or the Prunella vulgaris extract, and the single use of the polypeptide metal chelate or the Prunella vulgaris extract mixed with other oil control materials, the combination of the two can exhibit better oil control and anti-inflammatory effects, and can be used to prepare cosmetics with oil control and acne removal effects in combination with other raw materials. The two have synergistic effects and unexpected technical effects. Attached Figure Description

[0016] Figure 1 shows the effect of the sample in Example 3 on cellular lipid synthesis;

[0017] Figure 2 shows the effect of the combination of PVE and PPZ on cellular lipid synthesis in Example 3;

[0018] Figure 3 shows the effect of different concentrations of PPZ and PVZ combinations on the inflammatory factor NO in Example 4;

[0019] Figure 4 shows the effect of different concentrations of PPZ and PVZ combinations on the inflammatory factor IL-6 in Example 4;

[0020] Figure 5 shows the transdermal water loss rate of the lower part under different treatments in Example 5;

[0021] Figure 6 shows the sebum secretion on the lower face under different treatments in Example 5;

[0022] Figure 7 shows the percentage of porphyrin area on the lower surface of different treatments in Example 5;

[0023] Figure 8 shows facial acne before and 28 days after using the composition sample. (Volunteer information: Top: Male, 25 years old; Bottom: Female, 21 years old). Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels.

[0025] Example 1: Preparation of Prunella vulgaris extract

[0026] This invention involves placing *Prunella vulgaris* spikes in an aqueous solution of 1,3-butanediol, stirring and mixing at 70°C for a certain period, and then filtering through a 100-mesh sieve to obtain *Prunella vulgaris* extract (PVE). The flavonoid content was determined to be no less than 1.0 mg / mL using the sodium nitrite-aluminum nitrate colorimetric method. For saponin content determination, 100 μL of PVE solution was placed in a 10 mL stoppered test tube, heated until the solvent was completely dried, cooled in an ice-water bath, and 4 mL of 77% concentrated sulfuric acid and 0.4 mL of 8% vanillin were added. After thorough shaking, the tube was heated in a 70°C water bath for 15 min, then immediately placed in an ice-water bath for 2 min. The absorbance of the sample was measured at a wavelength of 540 nm. Using oleanolic acid as a saponin standard, the saponin content in the extract was determined to be no less than 1.5 mg / mL.

[0027] Example 2 Preparation of peptide-chelated zinc

[0028] The present application synthesizes polypeptide compound [γ-Glu] n -Gln (the polypeptide compound prepared is consistent with the polypeptide compound obtained in Chinese Patent Application CN202410645442.5), wherein n≥1, Gln can be replaced by any other 19 amino acids, and the content of the polypeptide compound in the solution is about 25-35%. Slowly add zinc sulfate solution to the polypeptide solution until the pH is 6.0-8.0 to obtain a polypeptide chelated zinc solution (PPZ).

[0029] Example 3 Cell oil control experiment

[0030] Cell culture After the cryopreserved SZ95 cells were recovered, they were cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 1×10 5 U / L penicillin, 100 mg / L streptomycin at 37°C in a 5% carbon dioxide cell incubator. When the cells were adherent and the confluence was more than 80%, they were digested and subcultured with 0.25% EDTA-containing trypsin, and the logarithmic growth phase cells were used for experiments.

[0031] Nile red staining method for detecting neutral lipids in SZ95 cells The PPZ was diluted with deionized water to obtain peptide chelated zinc dilutions with mass fractions of 1%, 2%, and 5%, respectively. The PPZ was diluted with Prunella vulgaris extract to obtain PPZ and Prunella vulgaris compositions with mass fractions of 1%, 2%, and 5%, respectively. The above dilutions and compositions were diluted 500 times with DMEM medium to obtain stock solutions. Linoleic acid (LA) was dissolved in DMSO and diluted with DMEM medium to obtain a 20 μg / mL LA working solution. The 20 μg / mL LA solution was mixed with the above stock solutions or medium in equal volumes to obtain working solutions of LA mixed with different samples and pure LA, respectively, which were used for the following experiments.

[0032] The logarithmic growth phase SZ95 cells were digested and counted, and the cell suspension was adjusted to 1×10 4The samples were transferred into each sample well of a black edge 96-well plate in an amount of 100 μL per well. The next day, the medium in the well plate was aspirated, and washed once with PBS. Then, 100 μL of the working solution described above was added as the experimental group, and the medium alone was added as the blank control group. After incubation for 48 h, the DMEM complete medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of 10 μg / mL Nile red was added to each well, and the cells were incubated in a dark environment at 37 °C for 15 min. The fluorescence intensity was detected by taking a photograph under a fluorescence microscope, and by using a multifunctional enzyme label meter with a fluorescence function, with an excitation wavelength of 485 nm and an absorption wavelength of 565 nm. The proportion of lipid synthesis of the SZ95 cells was calculated according to formula (1-1).

[0033] Lipid synthesis rate (%) = (FIT / FIB) x 100% (1-1)

[0034] In the formula, FIT and FIB are the average fluorescence intensities of the experimental group and the control group, respectively.

[0035] The results are shown in FIGS. 1-2, and the results of the fluorescence microscope photographing show that the Prunella vulgaris extract and the peptide chelated zinc have inhibitory activity on sebum synthesis, and the combination of the two has a synergistic effect.

[0036] As can be seen from the lipid synthesis rate, different concentrations of PPZ have a concentration-dependent inhibitory effect on the lipid synthesis of skin cells. When PPZ is used in combination with PVE, there is a synergistic effect, and the effect is concentration-dependent. The sample group of 0.05% PPZ + 0.1% PVE reduces the lipid synthesis rate by 70% relative to the positive group (LA).

[0037] Example 4: Cell Anti-inflammatory Experiment

[0038] The peptide chelated zinc was diluted with deionized water to obtain peptide chelated zinc diluents with a mass fraction of 1%, 2%, 5%, and 10%, respectively. The PPZ was diluted with the Prunella vulgaris extract to obtain compositions of PPZ and Prunella vulgaris with a mass fraction of 1%, 2%, 5%, and 10%, respectively. The above diluents and composition solutions were diluted 500 times with DMEM medium to obtain stock solutions. The LPS was dissolved in DMEM medium, and a 2.0 μg / mL LPS working solution was prepared. The 2.0 μg / mL LPS solution was mixed with the above stock solutions or the medium in equal volumes to obtain working solutions of LPS mixed with different samples and pure LPS, respectively.

[0039] After the cryopreserved RAW 264.7 macrophages were recovered, they were cultured in DMEM medium containing 10% fetal bovine serum, 1 x 10 5Cells were cultured in DMEM high-glucose medium containing U / L penicillin and 100 mg / L streptomycin at 37 ℃ in a cell culture incubator with 5% carbon dioxide. When the cells adhered and reached confluence of more than 80%, they were digested and passaged with trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for experiments.

[0040] Anti-inflammatory efficacy assay: Cell pellets were collected during the logarithmic growth phase, and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL were added to each well of a 24-well cell culture plate with 500 μL of cell suspension. After culturing for 24 hours, the above working solution was added to each well as the experimental group, and culture medium alone was added as the blank control group. The plates were then incubated for another 24 hours. Following the NO and IL-6 kit procedures, each sample was repeated in triplicate. OD values ​​were measured at 540 nm using a microplate reader, and NO content was calculated. The results are shown in Figure 3. OD values ​​were measured at 450 nm and 540 nm using a microplate reader, and IL-6 content was calculated. The results are shown in Figure 4.

[0041] The NO levels indicate that different concentrations of PPZ have a concentration-dependent inhibitory effect on the synthesis of the inflammatory factor NO in cells. When used in combination with PVE, there is a synergistic effect, which is concentration-dependent on PPZ. The 0.01% PPZ + 0.1% PVE sample group reduced NO synthesis in skin cells by 56%.

[0042] The levels of the inflammatory cytokine IL-6 showed that different concentrations of PPZ had a concentration-dependent inhibitory effect on the synthesis of IL-6 in cells. When used in combination with PVE, it exhibited a synergistic effect, which was also concentration-dependent on PPZ. The 0.01% PPZ + 0.1% PVE sample group reduced IL-6 synthesis in skin cells by 85%.

[0043] In summary, the Prunella vulgaris extract, polypeptide chelated zinc, and their combination developed in this study showed a significant increase in oil-controlling and anti-inflammatory effects after combination compared to before combination.

[0044] Example 5: Human Efficacy Test

[0045] Test environment temperature: (22±1)℃; test environment humidity: (50±10)%.

[0046] Subject inclusion criteria

[0047] Healthy individuals aged 18-50, regardless of gender (except pregnant or breastfeeding women);

[0048] The facial skin has excessive oil secretion and some degree of acne.

[0049] No severe systemic disease, no immunodeficiency or autoimmune disease, no skin treatment, cosmetic and other tests that may affect the results in the test site;

[0050] No active allergic disease, no highly sensitive constitution;

[0051] No use of hormone drugs and immunosuppressants in the past month;

[0052] No participation in other clinical trials in the test site in the past three months;

[0053] This study strictly follows the Declaration of Helsinki, and all subjects understand the purpose and process of the entire experiment, and sign the informed consent form.

[0054] Test

[0055] Sample usage method: wash the face, take an appropriate amount of product and use it on the whole face, test on day 0, 14 and 28. Volunteers should not use other skin care products during the test period. The subjects are 18-50 healthy people, n=30.

[0056] The transdermal water loss rate (TWEL) of the face was tested by the multi-probe skin test system Tewameter, and the results are shown in Figure 5. The use of formulations containing the composition (5% concentration) can reduce the transdermal water loss rate, and the transdermal water loss rate is reduced by 15.8% after 28 days of use.

[0057] The amount of skin oil secretion on the forehead was tested by the multi-probe skin test system Sebumeter. The results are shown in Figure 6. The use of formulations containing the composition 0.01% PPZ + 0.1% PVE can reduce the amount of skin oil secretion, and the amount of oil secretion is reduced by 14.2% after 28 days of use.

[0058] The proportion of porphyrin area on the face was measured by the facial image analysis system Visia CR. The results are shown in Figure 7. After using the composition formulation: on the 14th day, the proportion of porphyrin area decreased by 12.0%; on the 28th day, the proportion of porphyrin area decreased by 18.4%.

[0059] The facial images of volunteers before and 28 days after using the composition sample were taken by Visia, and the acne situation was recorded. The results are shown in Figure 8. As can be seen from the image, after 28 days of use, the acne situation on the face of the volunteers has been significantly improved.

[0060] The human efficacy test shows that the summer fructus spireae extract and polypeptide chelated zinc composition developed in this study can effectively improve the transdermal water loss, oil secretion, porphyrin area proportion and acne situation on the face of volunteers, and can be applied in various cosmetics, including oil control and acne-removing cosmetics.

[0061] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A combination of a Prunella vulgaris extract and a polypeptide chelated zinc, characterized in that, The saponin content of the extract is not less than 1.5 mg / mL, and the flavone content is not less than 1.0 mg / mL. The polypeptide chelated zinc is formed by chelation of a polypeptide compound [gamma-Glu] n-AA and a zinc ion compound, wherein n is greater than or equal to 1, and AA is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

2. The composition of Prunella vulgaris extract and polypeptide chelated zinc as described in claim 1, characterized in that, The preparation method of the extract of Prunella vulgaris is as follows: The Prunella vulgaris spikes are added into a 1,3-butanediol aqueous solution, stirred and soaked at 70 DEG C, filtered through a 100-mesh sieve, and the extract of Prunella vulgaris is obtained.

3. The composition of Prunella vulgaris extract and polypeptide chelated zinc as described in claim 1, characterized in that, The preparation method of the polypeptide chelated zinc is as follows: A polypeptide compound [gamma-Glu] is synthesized by fermentation method using amino acid as raw material n, AA, slowly adding zinc sulfate solution in the polypeptide solution to pH 6.0-8.0, to prepare a solution containing polypeptide chelated zinc.

4. The composition of Prunella vulgaris extract and polypeptide chelated zinc as described in claim 3, characterized in that, In the polypeptide compound, AA is glutamine.

5. The combination of the extract of Prunella vulgaris and the polypeptide chelated zinc as claimed in claim 3, wherein the extract of Prunella vulgaris is a water extract of Prunella vulgaris. The polypeptide chelated zinc solution is prepared by slowly adding a zinc sulfate solution into a polypeptide solution, and adjusting the pH to 6.0-8.0 using HCl or NaOH.

6. Use of the combination of the extract of Prunella vulgaris and the polypeptide chelated zinc in the preparation of cosmetics according to any one of claims 1-5.

7. Use according to claim 6, characterized in that, The cosmetics are used for oil control and / or acne removal of the skin. The cosmetics are used for oil control and / or acne removal of the skin.

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