Composition containing Hypericum perforatum flower / leaf / stem extract and preparation method thereof

Abstract

The present invention provides a composition containing a flower / leaf / stem extract of Hypericum perforatum and a preparation method thereof, wherein the composition comprises the following raw materials in parts by weight: 5-7 parts of a flower / leaf / stem extract of Hypericum perforatum, 3-4 parts of a flower / leaf / stem extract of Hypericum perforatum, 1-2 parts of a wild mango kernel fat, 3-4 parts of acacia australis fruit extract, and 0.1-0.2 parts of a sponge spicule. The present invention extracts the flower / leaf / stem of Hypericum perforatum after treatment with alcohol extraction, Rhizopus arrhizus fermentation, and Rhodotorula glutinosus fermentation related technologies, fully extracts the active substances in the flower / leaf / stem of Hypericum perforatum, and the obtained flower / leaf / stem extract of Hypericum perforatum has a synergistic effect with the flower / leaf / stem extract of Hypericum perforatum, the wild mango kernel fat, the acacia australis fruit extract, and the sponge spicule, and can moisturize the skin while providing anti-inflammatory and oil-control effects.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic preparations, and particularly relates to a composition containing Hypericum perforatum flower / leaf / stem extracts and a preparation method thereof. Background Art

[0002] As people pay more and more attention to health and beauty, the application of natural plant extracts in the field of skin care has become increasingly important. In particular, Hypericum perforatum flower / leaf / stem extracts have attracted much attention due to their unique pharmacological effects, such as anti-depression, anti-inflammatory and oil control, and other potential skin care effects. In recent years, the research on Hypericum perforatum flower / leaf / stem extracts has continued to deepen, and its application in cosmetics and skin care products has gradually increased. In the existing technology, the extraction efficiency and purity of Hypericum perforatum flower / leaf / stem extracts are often unsatisfactory, and the product formula is single, lacks specificity, and the permeability and absorbency are also unsatisfactory. Summary of the invention

[0003] The present invention provides a composition containing a Hypericum perforatum flower / leaf / stem extract and a preparation method thereof. Active substances in the Hypericum perforatum flower / leaf / stem are fully extracted by using an alcohol extraction technique and a double fermentation technique of Rhizopus arrhizus and Rhodotorula glutinosus. The obtained Hypericum perforatum flower / leaf / stem extract is combined with Hypericum perforatum oil, wild mango kernel fat, Acacia australis fruit extract and sponge spicules to form a composition. The composition has better effects in terms of oil resistance, anti-inflammatory, moisturizing and the like.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a method for extracting a Hypericum perforatum flower / leaf / stem extract, the specific steps of which are:

[0006] S1. Wash, dry and crush the raw materials;

[0007] S2. The S1 powder is extracted with ethanol, filtered and purified to obtain a filtrate A1 and a residue A2;

[0008] S3. Drying the residue A2 and then fermenting it;

[0009] S4. Filtering the fermentation product of S3 to obtain filtrate A3, and sterilizing the filtrate A3;

[0010] S5. Mix the filtrate A1 and the filtrate A3, and perform secondary fermentation, oscillation, and filtration on the mixture of the filtrate A1 and the filtrate A3 using the strain to obtain the filtrate A4;

[0011] S6. The filtrate A4 is freeze-dried to obtain a powdered Hypericum perforatum flower / leaf / stem extract A5.

[0012] Preferably, the S1 raw material is at least one of Hypericum perforatum leaves, flowers and stems.

[0013] Preferably, the S1 pulverization is to pulverize the raw material using ultrafine powder airflow pulverization technology, and the raw material is sieved through a 600-800 mesh sieve after pulverization.

[0014] Preferably, the S2 is purified by acid precipitation using hydrochloric acid with a concentration of 1 mol / L.

[0015] Preferably, the S3 drying temperature is ≥70°C, the purpose of which is to remove ethanol.

[0016] Preferably, the strain used in the S3 fermentation is Rhizopus arrhizus, the pH is 6.5-7, the pressure is normal pressure, the temperature is 30-35°C, the fermentation time is 16h, the humidity is 60% rh-80% rh, and the amount of bacteria added is ≥ 7% of the raw material mass.

[0017] Preferably, the S5 secondary fermentation strain is red yeast, the pH is 5-6, the pressure is normal pressure, the temperature is 25-30°C, the fermentation time is 8h, the humidity is 60% rh-70% rh, and the amount of bacteria added is ≥5% of the raw material mass.

[0018] Preferably, the S5 oscillation is specifically to oscillate the unfiltered fermentation product using ultrasonic oscillation, the ultrasonic frequency is 70-100 kHz, and the ultrasonic time is 20-25 min.

[0019] In a second aspect, the present invention provides a composition comprising the following raw materials in parts by weight:

[0020] 5-8 parts of Hypericum perforatum flower / leaf / stem extract;

[0021] 3-4 parts of Hypericum perforatum oil;

[0022] 1-2 parts of wild mango kernel butter;

[0023] 3-4 parts of Acacia acacia fruit extract;

[0024] Sponge spicules 0.1-0.2 parts.

[0025] In a third aspect, the present invention provides a leave-on skin topical preparation, wherein the mass percentage of the composition in the topical preparation is ≤8%.

[0026] Preferably, the leave-on skin topical preparation further comprises a matrix, and the topical preparation matrix comprises the following raw materials in percentage by mass: 15% glycerol, 1% sodium lauryl sulfate, 2% betaine, 2% honey, and the remainder is deionized water.

[0027] In the present invention:

[0028] The flowers / leaves / stems of Hypericum perforatum are extracted using alcohol extraction and secondary fermentation technology to fully extract the active substances in the flowers / leaves / stems of Hypericum perforatum. The flower / leaves / stem extracts of Hypericum perforatum are then combined with Hypericum perforatum oil, wild mango kernel fat, Acacia australis fruit extract, and sponge spicules to provide anti-inflammatory and oil-control effects while also moisturizing the skin.

[0029] Compared with the prior art, the invention has the following beneficial effects: the alcohol extraction and secondary fermentation technology is used to extract Hypericum perforatum, firstly, the Hypericum perforatum is alcohol extracted, the natural active substances that are easy to dissolve are extracted by ethanol, and then the alcohol extraction residue is dried, and after removing the solvent, it is fermented by Rhizopus arrhizae, and the filtrate is obtained by filtration after fermentation, and the active substances previously extracted by ethanol are mixed after sterilization, and then fermented by Rhodotorula glutinosus. Due to the reason of the previous fermentation product of Rhizopus arrhizae, it is not necessary to add relevant culture medium thereto, and then after the fermentation is completed, ultrasonic oscillation is used to break the cell membrane of Rhodotorula glutinosus, and the contents flow out, and then filtered to obtain the filtrate, and finally the filtrate is freeze-dried to obtain the final product, Hypericum perforatum flower / leaf / stem extract. The extract is rich in natural active substances such as hypericin, benzanthrone, polysaccharide, ketone, carotenoids, etc. The extract is compatible with Hypericum perforatum oil, wild mango kernel fat, beautiful acacia fruit extract, and sponge spicules, and provides anti-inflammatory and oil control effects while also providing the effect of moisturizing the skin. DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0031] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers; unless otherwise specified, percentages and parts are calculated by weight.

[0032] Some raw materials and their sources are as follows:

[0033] Hypericum perforatum L. flowers / leaves / stems: purchased from Hongsheng Professional Chinese Medicinal Materials Planting Cooperative, Qiaocheng District, Bozhou City;

[0034] Hypericum Perforatum Oil: purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.

[0035] Wild mango kernel butter (Irvingia Gabonensis Kernel Butter): purchased from Ningbo Chaoming Biotechnology Co., Ltd.;

[0036] Acacia Concinna Fruit Extract: purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0037] Hydrolyzed Sponge: Sanyuan Tianyu Biological Products Co., Ltd.;

[0038] Rhizopus arrhizus: Shanghai Guyan Industrial Co., Ltd.;

[0039] Rhodotorula Mucilaginosa: Ningbo Testo Biotechnology Co., Ltd.;

[0040] Rhizopus oryzae: Ningbo Testo Biotechnology Co., Ltd.;

[0041] Saccharomyces cerevisiae: Shanghai Guyan Industrial Co., Ltd.

[0042] The specific steps of the preparation method of Hypericum perforatum flower / leaf / stem extract are as follows:

[0043] S1. Wash, dry, crush and sieve the flowers / leaves / stems of Hypericum perforatum.

[0044] S2. The S1 powder was extracted with 40% ethanol, filtered after extraction, and then acid-precipitated with 1 mol / L hydrochloric acid to obtain a filtrate A1 and a residue A2;

[0045] S3. The filter residue A2 was dried at 70 ° C to remove the ethanol solvent, and then fermented with Rhizopus arrhizus at a fermentation temperature of 30-35 ° C, a pressure of normal pressure, a pH of 6.5-7, a fermentation time of 16 h, a fermentation humidity of 60% rh-80% rh, and a bacterial dosage of ≥ 7% of the raw material mass;

[0046] S4, filtering the fermentation product of S3 to obtain filtrate A3, and sterilizing the filtrate A3;

[0047] S5, mixing filtrate A1 and filtrate A3, and performing secondary fermentation on the mixture of filtrate A1 and filtrate A3 using red yeast, with a fermentation temperature of 25-30°C, a pressure of normal pressure, a pH of 5-6, a fermentation time of 6 hours, a humidity of 60% rh-70% rh, and a bacterial dosage of ≥5% of the raw material mass. After fermentation, ultrasonically oscillating the unfiltered fermentation liquid, and then filtering to obtain filtrate A4;

[0048] S6, freeze-drying the filtrate A4 to obtain a powdered Hypericum perforatum flower / leaf / stem extract A5.

[0049] A method for preparing a composition comprises the following steps:

[0050] The raw materials are weighed according to the proportion, mixed, sealed, and stored away from light to obtain the composition. The specific proportion by weight is shown in Table 1.

[0051] A method for preparing a leave-on skin topical preparation comprises the following steps:

[0052] T1: Weigh glycerol, sodium lauryl sulfate, betaine and honey according to the ratio, mix well and add into deionized water.

[0053] T2: Add the composition to T1, mix well, sterilize, seal and store to obtain the leave-on skin topical preparation. The specific mass percentage ratio is shown in Table 2.

[0054] Table 1

[0055]

[0056]

[0057] Table 2

[0058]

[0059] Note: Make up the balance with deionized water.

[0060] Safety Test

[0061] With reference to the 2015 Technical Specifications for Safety of Cosmetics, the leave-on skin topical preparations of Examples 4-8 were evaluated for cosmetic irritation. The test method was a skin patch test, and 50 randomly distributed people aged 16-65 years were tested.

[0062] Test method: Put the test substance into the patch tester, the dosage is 0.020-0.025g, cover the patch tester with the test substance on the back or forearm of the subject with non-irritating cloth-based tape, and gently press it with the palm to make it evenly adhere to the skin surface for 24 hours. After removing the test patch tester, observe the skin reaction after the indentation disappears 30 minutes later. If the result is negative, observe again 24 hours and 48 hours after the patch test.

[0063] Evaluation criteria:

[0064] Grade 0: negative reaction;

[0065] Grade 1: Suspicious reaction, only slight erythema;

[0066] Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;

[0067] Grade 3: Strong positive reaction, erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;

[0068] Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent herpes, and the reaction exceeds the test area.

[0069] Test results: Skin reactions were negative in all subjects.

[0070] The above test results show that the leave-on skin topical preparation provided by the present invention is mild and non-irritating to the skin and is safe to use.

[0071] Cytotoxicity assay

[0072] Take the human immortalized epidermal cell line (HaCaT) with stable growth state and adjust the cell concentration to 1×10 5 / mL, 100μL per well. Inoculate into 96-well cell culture plates (except the outermost wells and blank control wells). Add 200μL of PBS to the outermost wells of the cell culture plate to reduce the evaporation of the culture medium. Place in a carbon dioxide incubator and culture at 37℃±1℃, CO2 concentration 5.0%±1% until the cell fusion reaches more than 90%. Add samples, the sample concentration is 10mg / L, set up blank control and sample groups, 100μL per well. Start adding samples (sample group) and PBS (blank control) at the same time interval between each two columns to ensure that the intervention time of each test substance on the cells is 5min. After 5min, remove the test substances in turn, add PBS to each well to wash the test substances, and wash twice. When using a vacuum suction pump or a discharge gun to remove the liquid in the well, be careful to avoid sucking away the surviving cells and affecting the experimental results. Add 100μL of complete culture medium and 10μL of CCK-8 solution to each well, and place in an incubator to culture for about 2h. At a wavelength of 450 nm, the absorbance (OD) value was measured and the cell viability was calculated; the results are shown in Table 3.

[0073] Formula (1)

[0074] Table 3 Cytotoxicity test

[0075]

[0076]

[0077] According to the experimental results, the cell survival rate is maintained above 95%. The combination of the present experiment has little effect on cell viability.

[0078] Detection of lipid accumulation in SZ95 cells by Nile red staining

[0079] (1) Cell inoculation: SZ95 cells with good growth status were collected, digested with trypsin and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 3*10 5 A sterile cell slide was added to each well of a 12-well plate, and 2 mL of cell suspension was evenly seeded into each well, which was then placed in a 37°C, 5% CO2 incubator for incubation.

[0080] (2) After the cells adhered to the wall, the old culture medium was discarded and the cells were washed twice with PBS. Testosterone (6ug / ml) and linoleic acid (100μM) were used to induce lipid synthesis. The model group (no sample added) and the sample group (2μL, 1mg / L sample added) were set up separately, and then placed in a 37°C, 5% CO2 incubator for further incubation for 24h.

[0081] (3) After incubation for 24 hours, discard the culture medium, wash twice with cold PBS, and fix with 4% paraformaldehyde at room temperature for 10 minutes. After fixation, stain with Nile red dye (100 μg / mL) and incubate in a 37°C incubator for 10 minutes. Aspirate the dye, then wash with PBS 3-5 times, gently remove the cell slide and place it on a slide. Observe the staining under a fluorescence microscope. Cells with strong orange-red fluorescence are lipid-rich positive cells.

[0082] (4) ImageJ software was used to analyze the fluorescence intensity of the collected photos.

[0083] SPSS 19.0 statistical software package was used for analysis. The results are shown in Table 4.

[0084] Table 4 Oil control ability test

[0085]

[0086]

[0087] Result analysis: From the results of Examples 4-8, it can be seen that the oil control composition provided by the embodiments of the present invention has a good oil control effect, especially Example 4 has a better effect.

[0088] It can be seen from the results of Example 5 and Comparative Examples 7-13 that the leave-on skin topical preparation provided by the embodiments of the present invention has good oil control effect, indicating that there is a certain synergistic effect when using the components defined by the present invention. The lack or replacement of any one of them cannot achieve the effect achieved by the present invention. The fermentation process defined by the present invention can maximize the efficacy of the components defined by the present invention. Replacing any one of them cannot achieve the effect achieved by the present invention.

[0089] Inflammatory factor inhibition test

[0090] TNF-α is the most common inflammatory factor in inflammatory reactions. It can induce the phenotype of vascular endothelial cells to change to an inflammatory type, leading to changes in the body's metabolism and hemodynamics, and promoting the production of inflammatory mediators. Inflammatory mediators can increase vascular permeability, causing local congestion and edema, and may stimulate nerve endings to cause itching and pain. Therefore, the inhibition of TNF-α helps soothe and repair the skin.

[0091] RAW264.7 cells grown to the logarithmic phase were cultured at 1.5×10 5 Cells were inoculated at a density of 100 μg / mL in a 24-well plate, with 800 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to near fusion, 50 μL of the sample concentration of 10 mg / L was administered, with 3 replicates per group. The positive control was 50 μL of 10 μg / mL dexamethasone, and 1 μg / mL lipopolysaccharide (LPS) was used for stimulation 1 hour later. After incubation in a cell culture incubator for 24 hours, the cell culture fluid was transferred to a centrifuge tube, centrifuged at 5000 r / min for 10 minutes, and the cell supernatant was collected and tested according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0092] Formula (2)

[0093] Table 5 Results of RAW secretion of TNF-α

[0094]

[0095]

[0096] From the analysis of the results in Table 5: It can be seen from the results of Examples 4-8 that the compositions provided by the examples of the present invention have good TNF-α inhibitory effects, especially Example 4 has a better effect.

[0097] From the results of Example 5 and Comparative Examples 7-11, it can be seen that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved by lacking or replacing any one of them.

[0098] Comparing the results of Example 5 with those of Comparative Example 12, it can be seen that the use of the fermentation strains and processes defined in the present invention can effectively inhibit the secretion of TNF-α factor.

[0099] Comparing the results of Example 5 with those of Comparative Example 13, it can be seen that the use of the composition ratio and process defined in the present invention can effectively inhibit the secretion of TNF-α factor.

[0100] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. A makeup composition, characterized in that: It is composed of the following raw materials by weight: 5-7 parts of Hypericum perforatum flower / leaf / stem extract; 3-4 parts of Hypericum perforatum oil; 1-2 parts of wild mango kernel butter; 3-4 parts of Acacia acacia fruit extract; Sponge spicules 0.1-0.2 parts; The preparation method of the Hypericum perforatum flower / leaf / stem extract is as follows: S1. Wash, dry and crush the flowers, leaves and stems of Hypericum perforatum; S2. The S1 powder was extracted with 40% ethanol, filtered after extraction, and then acid-precipitated with 1 mol / L hydrochloric acid to obtain a filtrate A1 and a residue A2; S3. Dry the filter residue A2 at 70°C to remove the solvent ethanol, and then ferment it with Rhizopus arrhizus at a fermentation temperature of 30-35°C, a pressure of normal pressure, a pH of 6.5-7, a fermentation time of 16h, and a bacterial dosage of ≥7% of the raw material mass; S4. Filtering the fermentation product of S3 to obtain filtrate A3, and sterilizing the filtrate A3; S5. The mixed filtrate A1 and filtrate A3 were subjected to secondary fermentation using red yeast of filtrate A1 and filtrate A3 at a temperature of 25-30 ° C, a pressure of normal pressure, a pH of 5-6, a fermentation time of 6 h, and a bacterial dosage of ≥ 5% by weight of the raw material. After fermentation, the unfiltered fermentation broth was subjected to ultrasonic oscillation and then filtered to obtain filtrate A4; S6. The filtrate A4 is freeze-dried to obtain a Hypericum perforatum flower / leaf / stem extract A5.

2. The composition according to claim 1, characterized in that The S1 needs to be sieved through a 600-800 mesh sieve after being crushed.

3. A leave-on skin external preparation, characterized in that: The leave-on skin external preparation comprises the composition according to any one of claims 1 to 2.

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