Preparation and application of a dandruff-removing composition containing sophora flavescens polypeptide

Abstract

The present application relates to the technical field of scalp care, and specifically provides a dandruff-removing composition containing sophora flavescens polypeptide, a preparation method and application thereof.The dandruff-removing composition contains various active ingredients such as sophora flavescens polypeptide SoFla-I, salicylic acid, selenium disulfide, glutathione and nicotinamide, and is prepared through a specific process.The amino acid sequence of the sophora flavescens polypeptide SoFla-I is shown as SEQ ID No.1, and the preparation includes steps such as freeze-drying of sophora flavescens roots, ultrasonic extraction, ammonium sulfate precipitation and dialysis purification.Experiments show that the composition can effectively inhibit malassezia, and significantly improve dandruff and pruritus symptoms, and the dandruff score and pruritus score significantly decrease after 14 days of use, and the satisfaction of the subjects is high.The composition of the present application is high in safety and significant in dandruff-removing effect, and is suitable for shampoo products, scalp care agents and the like.

Description

Technical Field

[0001] This invention relates to the field of scalp care technology, and more specifically, to the preparation and application of an anti-dandruff composition containing Sophora flavescens polypeptide. Background Technology

[0002] Dandruff is a common scalp problem, primarily caused by the overgrowth of Malassezia, accompanied by symptoms such as itching and flaking, affecting quality of life and mental health. Existing anti-dandruff products mostly rely on chemically synthesized ingredients, such as zinc pyrithione and ketoconazole. While these have some effect, long-term use may lead to drug resistance, scalp dryness, or irritation. For example, selenium sulfide and salicylic acid are commonly used anti-dandruff agents, but their effectiveness when used alone is limited, and they are insufficient for improving severe dandruff. Natural extracts such as plant peptides have attracted attention due to their good biocompatibility and few side effects; however, the application of Sophora flavescens peptides in the field of dandruff treatment has not yet been systematically reported.

[0003] Sophora flavescens, a traditional Chinese medicine, is known to possess anti-inflammatory and antibacterial activities, but research on the extraction, purification, and application of its polypeptide components in scalp care is limited. While existing methods for polypeptide extraction, such as ultrasonic disruption and chromatographic purification, are well-established, the specific sequences of Sophora flavescens polypeptides and their synergistic effects with anti-dandruff ingredients have not been explored. Furthermore, the stability of active ingredients in the composition and transdermal absorption efficiency are key challenges; for example, glutathione is easily oxidized and inactivated, requiring nano-encapsulation techniques to enhance stability.

[0004] This invention innovatively isolates the Soflavin polypeptide SoFla-I and combines it with other ingredients (such as salicylic acid and selenium disulfide), utilizing nanoemulsification technology to improve bioavailability, thus solving the problems of poor longevity and insufficient compatibility of existing anti-dandruff products. Background technology analysis indicates that developing a highly efficient and gentle anti-dandruff composition based on natural polypeptides has significant application value. Summary of the Invention

[0005] The present invention first provides an anti-dandruff composition containing Sophora flavescens polypeptide, comprising Sophora flavescens polypeptide SoFla-I, and at least one active ingredient selected from salicylic acid, selenium disulfide, glutathione and nicotinamide, wherein the amino acid sequence of Sophora flavescens polypeptide SoFla-I is shown in SEQ ID No. 1.

[0006] In some embodiments, a carrier matrix is ​​also included, the carrier matrix comprising glycerol, butanediol, water and surfactant.

[0007] In some embodiments, the mass percentage of the Sophora flavescens polypeptide SoFla-I is 0.1%-5%, preferably 0.5%.

[0008] In some embodiments, it also contains salicylic acid in a mass percentage of 1%-3%, preferably 2%.

[0009] In some embodiments, it also contains selenium disulfide in a mass percentage of 0.5%-2%, preferably 1%.

[0010] In some embodiments, the mixture also comprises glutathione and nicotinamide, wherein the mass percentage of glutathione is 0.05%-0.2%, preferably 0.1%, and the mass percentage of nicotinamide is 0.1%-0.5%, preferably 0.3%.

[0011] The present invention also provides a method for preparing the above-mentioned anti-dandruff composition, comprising the following steps:

[0012] (a) Extraction of SoFla-I polypeptide from Sophora flavescens root, including freeze-drying, ultrasonic extraction, ammonium sulfate precipitation and dialysis purification;

[0013] (b) The Sophora flavescens polypeptide SoFla-I was mixed with other active ingredients and treated with nanoemulsification technology to form a uniformly dispersed system.

[0014] In some embodiments, the nanoemulsification technique includes encapsulating glutathione with liposomes and subjecting it to high-pressure homogenization at a pressure of 600-800 bar.

[0015] The present invention also provides the use of the above-described composition in the preparation of scalp care products, wherein the scalp care products are selected from shampoos, scalp care agents or anti-dandruff agents.

[0016] In some embodiments, the composition is used to inhibit Malassezia, with an inhibition rate of not less than 90%.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] (1) Highly effective antibacterial: The composition showed an inhibition rate of 96.67% against Malassezia and an inhibition zone diameter of 8.5 mm, which was significantly higher than the control composition (inhibition rate of 73.33%). This is attributed to the synergistic effect of Sophora flavescens polypeptide SoFla-I with selenium disulfide and salicylic acid.

[0019] (2) Significantly improves scalp symptoms: Clinical tests showed that after 14 days of use, the dandruff score (ASFS value) and itching score decreased significantly, the subjects were highly satisfied, and the effect was better than the control combination without Sophora flavescens peptide.

[0020] (3) High safety: The compound of natural ingredients has a high satisfaction rate among test subjects, with no irritation and is suitable for sensitive scalps.

[0021] (4) Good stability: The efficiency of ingredient encapsulation is enhanced by nano-emulsification technology to ensure the long-term stability of active ingredients. Attached Figure Description

[0022] Figure 1 This is a gel chromatography image of the purification of SoFla-I, a polypeptide from Sophora flavescens. Detailed Implementation

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] Example 1: Preparation of Sophora flavescens polypeptide

[0025] Dried Sophora flavescens roots were frozen at -20℃ for 24 hours, then removed and dried in a vacuum freeze dryer for 48 hours. The vacuum freeze dryer parameters were set as follows: cold trap temperature -80℃, freezing chamber temperature -40±5℃, and vacuum degree 0-10 Pa. The mortar and pestle were pre-cooled with liquid nitrogen, and the freeze-dried Sophora flavescens was ground into powder. 5g of the powder was weighed and dispensed into pre-cooled 50 mL centrifuge tubes. 35 mL of pre-cooled PBS solution (4℃) was added, and the mixture was vortexed. The 50 mL centrifuge tubes were placed in an ultrasonic cell disruptor, with a power of 200-300W and a temperature of 35℃, and extracted for 30-40 minutes, pausing for 1 minute every 5 minutes. After ultrasonic disruption, the cells were centrifuged at 8000 rpm for 20 minutes at 4℃. The supernatant was collected, and the residue was extracted again under the same conditions. The two supernatants were combined to obtain the crude extract. The crude extract was placed in a constant temperature water bath and incubated at 65℃ for 30 minutes, then rapidly cooled to room temperature. Centrifuge at 8000 rpm for 15 min at room temperature, discard the precipitate, and collect the supernatant in a clean beaker. While stirring, slowly add solid ammonium sulfate to the supernatant until saturation reaches 50%. Seal the beaker and let it stand overnight at 4°C. Centrifuge at 10000 rpm for 20 min, discard the precipitate, and collect the supernatant. Transfer the supernatant to a 5 kDa dialysis bag and use deionized water as the dialysate. Dialyze at 4°C for 24 h, changing the dialysate every 6 h, until no sulfate ions are present in the dialysate (i.e., no white precipitate is formed when tested with barium chloride solution), thus obtaining the desalting solution.

[0026] The desalting solution was purified using gel filtration chromatography with Sephadex G-25 pre-packed column packing. The desalting solution was filtered through a 0.22 μm filter membrane before loading. The column was equilibrated until the equilibration solution reached 1–2 mm below the surface of the chromatography bed, at which point the outlet was closed. 5 mL of the filtered desalting solution was slowly injected into the top of the column using a pipette, taking care not to disturb the gel bed. After the sample had completely penetrated the gel, elution was continued with elution buffer. The absorbance at 220 nm was monitored using a UV detector, and the main peak fraction was collected and named SoFla-I. The gel chromatography purification results of the Sophora flavescens polypeptide are shown below. Figure 1As shown in the figure, the horizontal axis represents the retention time of the peak and the vertical axis represents the absorption intensity at 220 nm. It can be seen from the figure that SoFla-I is the main peak with the highest response intensity.

[0027] The collected purified solution was placed in a rotary evaporator and concentrated under reduced pressure at 40°C to 1 / 5 of its original volume. The concentrate was then transferred to a freeze-drying bottle and placed in a vacuum freeze dryer. The solution was freeze-dried at -40±5°C and a vacuum of 0-10 Pa for 12 hours to obtain a white, loose powder of Sophora flavescens polypeptide SoFla-I.

[0028] Example 2: Amino acid sequence identification of Soflaxseed polypeptide SoFla-I

[0029] The obtained SoFla-I was de novo sequenced by Biotech Biotechnology Co., Ltd. The sample preparation steps were as follows: 20 μg of peptide powder was added to 100 μL of 0.1% TFA, and vortexed for 10 min until completely dissolved. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was used as the sequencing sample for mass spectrometry analysis.

[0030] Samples were analyzed using nanoLC-MS / MS. The sample was manually injected into the LC wells and eluted using a gradient elution on a C18 column (20 cm, 75 μm). Mobile phase A was sterile water containing 0.1% formic acid, and mobile phase B was acetonitrile solution containing 0.1% formic acid. The gradient was linearly increased from 5% B to 30% B (0-45 min) at a flow rate of 0.3 μL / min. The separated peptides were directly introduced into the mass spectrometer ion source for ion pair acquisition. MS1 and MS / MS spectra were acquired. Proteome Discoverer software was used to remove contaminant and impurity peaks from the raw data, extracting the effective MS / MS spectra and analyzing the amino acid sequence. The final amino acid sequence of the peptide SoFla-I was obtained as SEQ ID No. 1: KLVFFAEDVGSNKGAIIGLMVGGVVIA.

[0031] Example 3: Preparation of an anti-dandruff composition containing Sophora flavescens polypeptide

[0032] This embodiment provides a scalp care composition S1, comprising the following components by mass percentage: 0.5% Sophora flavescens polypeptide SoFla-I, 2% salicylic acid, 1% selenium disulfide, 0.1% glutathione, 0.3% nicotinamide, 0.05% phosphatidylserine, 0.05% hydrolyzed oat β-glucan, 0.3% hydrolyzed wheat protein (Shanghai Zhouyuan Biotechnology Co., Ltd., catalog number HDB03), 0.05% arginine lactate, 0.005% lipoic acid, 0.02% asiaticoside (Shanghai Yuanye Biotechnology Co., Ltd., catalog number B20586), 0.1% whey protein hydrolysate (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number L344777), and the balance being a carrier matrix to 100%.

[0033] The carrier matrix consists of the following components by weight percentage: 5% glycerol, 4% butylene glycol, 2% propylene glycol, 0.5% Tween 80, 0.1% acetylated sodium hyaluronate, 0.2% lecithin, and the remainder is water.

[0034] Glutathione was dissolved in ethanol containing 0.5% Tween 80, and then added to an organic phase prepared with hydrogenated lecithin and cholesterol at a mass ratio of 3:1. The solvent was removed by rotary evaporation to form a lipid film. Buffer solution was added for hydration, followed by sonication for 90 seconds to form nanoemulsions with a particle size of 120 nm. Sophora flavescens polypeptide SoFla-I was dissolved in an aqueous phase containing 5% glycerol and HEPES buffer, and slowly stirred at 4°C to obtain an aqueous system of Sophora flavescens polypeptide. This aqueous system was slowly added dropwise to the glutathione emulsion, and stirring continued until completely mixed. The mixture was then dispersed using a probe-type ultrasonic treatment for 10 minutes (intermittent, 150 W power), followed by two high-pressure homogenization treatments (800 bar for the first round, 600 bar for the second round) to enhance encapsulation efficiency and stability, resulting in a homogeneous composite dispersion system. Add salicylic acid, selenium disulfide, nicotinamide, phosphatidylserine, hydrolyzed oat β-glucan, hydrolyzed wheat protein, arginine lactate, lipoic acid, asiaticoside, and whey protein hydrolysate in sequence. After mixing thoroughly, adjust the pH to 6, sterilize using a 0.22μm filter membrane, aliquot quantitatively, and seal for storage.

[0035] Composition S2 was prepared in the same manner and comprised, by weight percentage, the following components: 2% salicylic acid, 1% selenium disulfide, 0.1% glutathione, 0.3% nicotinamide, 0.05% phosphatidylserine, 0.05% hydrolyzed oat β-glucan, 0.3% hydrolyzed wheat protein, 0.05% arginine lactate, 0.005% lipoic acid, 0.02% asiaticoside, 0.1% whey protein hydrolysate, and the balance being a carrier matrix to 100%.

[0036] The control composition S3 was prepared in the same manner and comprised the following components by mass percentage: 0.1% glutathione, 0.3% nicotinamide, 0.05% phosphatidylserine, 0.05% hydrolyzed oat β-glucan, 0.3% hydrolyzed wheat protein, 0.05% arginine lactate, 0.005% lipoic acid, 0.02% asiaticoside, 0.1% whey protein hydrolysate, and the balance being a carrier matrix to 100%.

[0037] Example 4: Evaluation of the inhibitory effect of the anti-dandruff composition on Malassezia

[0038] The modified Dixon medium was melted by heating and poured into a 9cm diameter bacterial culture dish, then allowed to cool and solidify at room temperature. 0.2mL of the activated Malassezia suspension (concentration 2*10⁻⁶) was then added. 6 The mixture (CFU / mL) was evenly spread on the surface of the culture medium and allowed to air dry. Compositions S1, S2, and S3 were diluted 10-fold with sterile water to prepare test samples, named T1, T2, and T3. Sterile filter paper discs (6 mm in diameter) were immersed in the test samples until saturated, then drained. The treated filter paper discs were then placed on the inoculated culture medium, 3 discs per dish (spaced ≥ 2 cm apart), with 10 dishes per test sample. A blank control (filter paper discs not immersed in the sample, denoted as BLK) was also provided. The mixture was incubated at 35°C in the dark for 7 days until the inhibition zone was clearly visible. The diameter of the inhibition zone (including the diameter of the filter paper disc) was measured using calipers, and the average value was taken. An inhibition zone diameter of not less than 7 mm indicates that the sample has an antibacterial effect. The inhibition rate = (number of filter paper discs with antibacterial effect / total number of filter paper discs) * 100%. The experimental results are shown in Table 1. The test samples T1 and T2 showed good inhibition rates of Malassezia. The average inhibition zone diameter was analyzed by t-test. The inhibition zone diameters of T1 and T2 were significantly different from those of T3 (p < 0.05). Among them, the average inhibition zone diameter of T1 was larger, indicating that composition S1 had a better dandruff removal effect than S2.

[0039] Table 1. Malassezia inhibition experiment

[0040]

[0041] Example 5: Evaluation of the anti-dandruff effect of the composition by the subjects

[0042] Sixty healthy subjects aged 18-60 years with symptoms of dandruff and scalp itching were randomly divided into three groups of 20 each. The dandruff-reducing effects of compositions S1, S2, and S3 were tested in each group. Subjects maintained a regular lifestyle during the testing period and did not use any cosmetics, pharmaceuticals, or health products that could affect the results. After 14 days of continuous use of the test samples, the efficacy of the compositions was evaluated using expert visual assessment (Adherent Scalp Flaking Score, ASFS value), scalp itching severity assessment, and subjective evaluation. The specific application method was as follows: After wetting the hair with warm water, subjects applied an appropriate amount of the test sample evenly to the hair, massaged the scalp appropriately, left it on for 3-5 minutes, and then rinsed with water.

[0043] The specific steps for expert visual assessment of ASFS values ​​are as follows: An expert evaluator assessed and recorded the results before using the test sample (Day 0) and on day 14 (Day 14). The assessment included: 0: no visible dandruff; 1: occasional fine, slight dandruff; 2: significant dandruff on the scalp and hair; 3: a small amount of large dandruff or a large amount of fine dandruff on the scalp and hair; 4: a large amount of large dandruff. The evaluation results are shown in Table 2. The results show that compared with S3, both S1 and S2 significantly reduced ASFS values ​​and alleviated dandruff symptoms after 14 days of use. S1 showed a better dandruff-reducing effect than S2, which is related to the added Sophora flavescens polypeptide component SoFla-I. Statistical data were analyzed using a t-test. ns indicates p ≥ 0.05 (no statistical difference), * indicates 0.01 ≤ p < 0.05, ** indicates 0.001 ≤ p < 0.01, and *** indicates p < 0.001.

[0044] Table 2 Expert Visual Perception Assessment (ASFS Value)

[0045]

[0046] The specific steps for assessing the degree of scalp itching are as follows: Before using the test sample (Day 0) and on Day 14 after using the test sample (Day 14), one expert evaluator will assess and record the degree of scalp itching. The assessment includes: 1 point (0 degree): virtually no itching; 2 points (mild, Grade I): slight itching, relieved by gentle patting without scratching, no secondary rash caused by scratching; 3 points (moderate, Grade II): significant itching, irresistible to scratching, with visible mild secondary rash caused by scratching; 4 points (severe, Grade III): intense itching, affecting sleep and work, with obvious secondary rash caused by scratching visible locally; 5 points (very severe, Grade IV): severe itching, affecting sleep and work, emotional distress, with severe and numerous secondary rashes. The evaluation results are shown in Table 3. The results indicate that compared to S3, both S1 and S2 significantly reduced scalp itching scores and alleviated itching and rash symptoms after 14 days of use. S1 showed better relief, while S3 showed no statistically significant difference in the degree of scalp itching before and after use. Statistical data were analyzed using a t-test. ns indicates p ≥ 0.05 (no statistical difference), * indicates 0.01 ≤ p < 0.05, ** indicates 0.001 ≤ p < 0.01, and *** indicates p < 0.001.

[0047] Table 3 Assessment of the degree of scalp itching

[0048]

[0049] Participants rated their satisfaction with the test samples 14 days later: 1 for "very dissatisfied", 2 for "dissatisfied", 3 for "neutral", 4 for "satisfied", and 5 for "very satisfied". The percentage of participants with N ≥ 4 (satisfied group) was counted. The scores from participants using S1 and S2 were statistically significantly higher than those using S3. Statistical data were analyzed using a t-test. ns indicates p ≥ 0.05 (no statistical difference), * indicates 0.01 ≤ p < 0.05, ** indicates 0.001 ≤ p < 0.01, and *** indicates p < 0.001.

[0050] Table 4 Subject Satisfaction Evaluation

[0051]

[0052] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dandruff-reducing composition containing Sophora flavescens polypeptide, characterized in that, The product contains SoFla-I, a polypeptide from Sophora flavescens, as well as salicylic acid, selenium disulfide, glutathione, nicotinamide, and a carrier matrix. The amino acid sequence of the SoFla-I polypeptide is shown in SEQ ID No.

1. The carrier matrix comprises glycerol, butanediol, water, and surfactant; The mass percentage of the Sophora flavescens polypeptide SoFla-I is 0.1%-5%; The salicylic acid has a mass percentage of 1%-3%; The selenium disulfide has a mass percentage of 0.5%-2%; The glutathione has a mass percentage of 0.05%-0.2%, and the nicotinamide has a mass percentage of 0.1%-0.5%.

2. A method for preparing the anti-dandruff composition as described in claim 1, characterized in that, Includes the following steps: (a) Extraction of SoFla-I polypeptide from Sophora flavescens root, including freeze-drying, ultrasonic extraction, ammonium sulfate precipitation and dialysis purification; (b) The Sophora flavescens polypeptide SoFla-I was mixed with other active ingredients and treated with nanoemulsification technology to form a uniformly dispersed system.

3. The method as described in claim 2, characterized in that, The nanoemulsification technology involves encapsulating glutathione with liposomes and then subjecting it to high-pressure homogenization at a pressure of 600-800 bar.

4. The use of the composition according to claim 1 in the preparation of a scalp care product, wherein the scalp care product is a scalp care agent.

5. The application as described in claim 4, characterized in that, The composition is used to inhibit Malassezia, with an inhibition rate of not less than 90%.

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