Extract containing Kochia scoparia saponin Ic for regulating skin water-oil balance and application thereof

Abstract

The present invention relates to the technical field of skin care, and in particular to an extract containing kochia saponin Ic for regulating the water-oil balance of the skin and an application thereof. The present invention provides an extract containing kochia saponin Ic for regulating the water-oil balance of the skin. The present invention also provides the use of the extract containing kochia saponin Ic as an active ingredient in a water-oil balanced skin care product. The present invention also provides the use of the extract containing kochia saponin Ic as an active ingredient in a medicine for treating sebaceous gland secretion imbalance. The kochia saponin Ic in the present invention is a naturally occurring compound. Experiments have shown that the extract containing kochia saponin Ic is used to regulate the water-oil balance of the skin, can be used in water-oil balanced skin care products and as an active ingredient in a medicine for treating sebaceous gland secretion imbalance, and has important significance for treating excessive skin oil secretion.

Description

Technical Field

[0001] The invention relates to the technical field of skin care, and in particular to an extract containing kochia scoparia saponin Ic for regulating the water-oil balance of the skin and application thereof. Background Art

[0002] Skin water-oil balance, also known as skin lipid-water balance, refers to the proper ratio of water and oil (sebum) on the skin surface. This balance is essential for maintaining healthy skin condition and skin barrier function. When the skin's water-oil balance is broken (water-oil imbalance), there are two situations: ① more water and less oil. Because the skin's oil can help the skin lock in moisture and reduce water evaporation, once the oil is lacking, it cannot lock in moisture, resulting in water loss. The skin will soon turn into dry skin with less water and less oil; ② more oil and less water, the skin's oil increases, more moisture is locked, and the ability of the natural moisturizing factor in the skin to obtain external moisture is reduced, hindering the skin's dual absorption of water from inside and outside, causing the skin pores to enlarge, and too much oil is retained in the pores. Anaerobic bacteria will rapidly multiply inside the pores to produce monounsaturated fatty acids, which promote the formation of acne and sebaceous gland follicle inflammation.

[0003] The pathway of lipid synthesis includes the production of total sebaceous gland lipids including saturated and monounsaturated fatty acids. The key transcription factor regulating lipid synthesis, sterol regulatory element-binding protein-1c SREBP1c, controls the synthesis of sebaceous gland triglycerides, squalene and the desaturation reaction of fatty acids.

[0004] Insulin-like growth factor 1 (IGF-1) is an effective inducer of testosterone (T) and dehydroepiandrosterone (DHEA) in the gonads. IGF-1 not only promotes the synthesis of androgens in the adrenal glands and gonads, but also promotes the conversion of testosterone into dihydrotestosterone (DHT) which is 10 times more active by inducing 5α-reductase (5α-R), thereby increasing the signal transduction of androgens / androgen receptors. Both androgens and IGF-1 can promote the expression of SREBP1c, which is a key transcription factor regulating lipid production. In this way, the expression of SREBP1c is inhibited by inhibiting 5α-reductase to reduce the signal transduction of androgen receptors, thereby reducing lipid synthesis.

[0005] FoxO1 (Forkhead box O1), this gene belongs to the forkhead family of transcription factors, characterized by a unique forkhead domain. When oil secretion is normal, the expression level of SREBP1c is low due to the inhibitory effect of transcription factor FoxO1 and the low activity state of mTORC1 (mammalian target of rapamycin complex 1). When oil secretion is abnormal: insulin and IGF-1 activate AKT kinase, which reduces the nuclear localization of FoxO1 through FoxO1 phosphorylation, thereby enhancing the transcriptional activity of SREBP1c; androgens can also stimulate the transcriptional activity of SREBP1c by activating AKT; FoxO1 inhibits the transcriptional activity of androgen receptors, while hormones such as insulin, IGF-1 and androgens work synergistically with mTORC1 to upregulate the transcriptional activity of SREBP1c. In this way, abnormal oil secretion reduces the transcriptional activity of SREBP1c by upregulating the transcriptional activity of androgen receptors by upregulating FoxO1, thereby reducing lipid synthesis.

[0006] ATF4 (Activating Transcription Factor 4) is a member of the basic leucine zipper protein family and belongs to the cAMP (cyclic adenosine monophosphate) response element binding protein (CREB (cAMP Response Element Binding protein)) family. ATF4 induces intracellular lipid accumulation by activating SREBPs (Sterol-regulatory element binding proteins). Studies have shown that ATF4-deficient mice have reduced body fat content under normal diet.

[0007] Kochia scoparia (L.) Schrad is a dried mature fruit of the Chenopodiaceae plant, mainly produced in Hebei, Shanxi, Shandong and other places. Kochia scoparia is used as a traditional Chinese medicine, with the effects of clearing heat and dampness, dispelling wind and relieving itching, and is often used to treat symptoms such as painful urination, vaginal itching and leucorrhea, urticaria, eczema, and skin itching. Kochia scoparia saponin Ic (oleanolic acid-3-O-β-D-xylopyranose (1→3) β-D-Pyranoid glucose) is a compound extracted from the dried mature fruit of Kochia scoparia, which is a triterpenoid saponin. Kochia scoparia saponin Ic has the traditional Chinese medicine effects of clearing heat and dampness, dispelling wind and relieving itching. Modern research has found that it also has anti-itching, anti-inflammatory and anti-allergic effects. Summary of the invention

[0008] The first object of the present invention is to provide a new use of Kochia scoparia saponin Ic, that is, an extract containing Kochia scoparia saponin Ic is used to regulate the water-oil balance of the skin.

[0009] The second object of the present invention is to provide an extract containing Kochia scoparia saponin Ic for use as an active ingredient in water-oil balancing skin care products.

[0010] The third object of the present invention is to provide an extract containing Kochia scoparia saponin Ic as an active ingredient in a drug for treating imbalanced sebaceous gland secretion.

[0011] To achieve the above-mentioned first objective, the present invention provides an extract containing Kochia scoparia saponin Ic for regulating the water-oil balance of the skin.

[0012] To achieve the above-mentioned second objective, the present invention provides the use of an extract containing Kochia scoparia saponin Ic as an active ingredient in water-oil balancing skin care products.

[0013] Furthermore, the skin care product is a water-oil balancing toner, a water-oil balancing essence, a water-oil balancing repair cream or a water-oil balancing oil-control shampoo.

[0014] To achieve the third objective, the present invention provides the use of an extract containing Kochia scoparia saponin Ic as an active ingredient in a drug for treating imbalanced sebaceous gland secretion.

[0015] Beneficial effects of the present invention:

[0016] Kochia scoparia saponin Ic is a naturally occurring compound. Experiments have shown that extracts containing Kochia scoparia saponin Ic can be used to regulate the water and oil balance of the skin, which is of great significance in treating excessive oil secretion of the skin.

[0017] The extract containing Kochia scoparia saponin Ic can be used in water-oil balancing skin care products.

[0018] Extracts containing Kochia scoparia saponin Ic can be used as active ingredients in medicines to treat imbalanced sebaceous gland secretion. DETAILED DESCRIPTION

[0019] The invention is further described in detail below in conjunction with specific embodiments. In the following embodiments, Kochia scoparia saponin Ic is a compound extracted from the dried mature fruit of Kochia scoparia.

[0020] Water-oil imbalance modeling: The mice were fed with modeling feed, which was a feed with a high proportion of unsaturated fatty acids added to the daily feed for mice. Specifically, 1.25g of fish oil and 25g of linoleic acid were added to every 100g of feed (fish oil: linoleic acid = 1:20), thereby inducing excessive reproduction of Malassezia on the surface of the mouse skin, changing the skin condition of the mouse, and causing symptoms of water-oil imbalance on the skin.

[0021] The experimental animals were 12 male ICR mice, each weighing 20-25g. The mice were kept in a common experimental animal room with a temperature of 25-32℃, a humidity of 40-60%, and no special control of the light cycle. The 12 mice were randomly divided into four groups: blank control group 1, model group, positive drug group and Kochia scoparia saponin Ic group, with 3 mice in each group. Before the experiment, the four groups of mice were allowed to adapt for 3-7 days, and then the lower abdomen of each mouse and the back 1 cm above the base of the mouse tail were depilated, and the depilation area was 2cm×2cm. The mice in each group of the model group, positive drug group and Kochia scoparia saponin Ic group were fed with the above-mentioned modeling feed for the first 15 days and passively gavaged, with a gavage dose of 0.4mL each in the morning and evening, and fed with normal feed for the next 15 days, with a feeding cycle of 30 days. The blank control group 1 was fed with normal feed for 30 days.

[0022] At 9:00 a.m. on the first and second days after the mice were fed, 50 μL of 5% DNCB ethanol solution was evenly applied to the 2 cm×2 cm area of ​​the depilatory part of the lower abdomen of the mice for sensitization. Starting from the sixth day of feeding, 25 μL of 1% DNCB ethanol solution was applied to the 2 cm×2 cm area of ​​the depilatory part of the back of the mice to stimulate inflammation. The stimulation was performed once every 3 days, for a total of 6 times, and the stimulation time was all at 9:00 a.m. From the day of mouse sensitization, the mice were given drugs, with a drug administration area of ​​2 cm×2 cm. The drug administration scheme is shown in Table 1, and the drug administration time was all at 5:00 p.m.

[0023] Table 1 Dosage regimen for each group

[0024]

[0025] 1. Evaluation and observation results of mouse skin lesions

[0026] The clinical appearance score was used to evaluate the appearance of mice in different groups. The areas to be observed were: the back of the neck (the area from the top of the head to the tail); the base of the ears and the area around the mouth; the area to verify the healing; and the area with less hair at the base of the limbs. The appearance score was calculated based on the main symptoms of sebaceous follicle inflammation (skin hair loss, scaling, rashes / papules).

[0027] The scoring range for each degree of skin lesions is 0-3 points: 0 points means no obvious symptoms; 1 point means mild lesions; 2 points means moderate lesions; 3 points means severe lesions. The specific scoring criteria are shown in Table 2.

[0028] Table 2 Clinical appearance scoring criteria for animals in the sebaceous follicle inflammation experiment

[0029]

[0030] The skin lesion scores of the four groups of mice after 30 days of the experiment are shown in Table 3.

[0031] Table 3 Clinical appearance scores of the four groups of experimental animals

[0032]

[0033] As shown in Table 3, the skin of mice in the model group showed a higher skin lesion score. After being treated with different samples, the skin lesions of mice in the positive drug group and the Kochia scoparia saponin Ic group were significantly improved compared with the model group.

[0034] 2. H&E stained skin pathology sections and observation of sections under a microscope

[0035] The levels of epidermal and dermal inflammation and skin integrity of the mice were observed.

[0036] The skin of the four groups of mice was sampled separately. After each sampling, the samples were fixed with 4% paraformaldehyde. After the fixation was in good condition, the samples were trimmed, dehydrated, embedded, sliced, H&E stained, and sealed according to the pathological laboratory detection SOP (abbreviation of "Standard Operating Procedure") procedures, and finally qualified slices were examined under a microscope. The digital slices were browsed under a microscope, and the tissue slices of each group of slices were observed in detail at 20 times and 200 times, and the basic pathological changes in the slices, such as congestion, congestion, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, inflammatory changes, etc., were described in words. The observation results are described in Table 4.

[0037] Table 4 Results of skin pathological sections observed under microscope

[0038]

[0039] From the observation results in Table 4, it can be seen that the skin of the mice in the positive drug group and the scoparia saponin Ic group had only local necrosis, while the skin of the mice in the model group had large areas of necrosis, indicating that scoparia saponin Ic has a strong repairing effect on skin damage caused by sebaceous gland follicle inflammation.

[0040] (III) Test results of water and oil content levels in mouse skin

[0041] On the last day of the experiment, a skin moisture tester and a sebum meter were used to measure the skin moisture and oil content of the four groups of mice, respectively. Then, the average ratio of the water content to the oil content (water content / oil content) of the four groups of mice was calculated. The test results are shown in Table 5.

[0042] Table 5 Water-oil content ratio of mouse skin

[0043]

[0044] As shown in Table 5, the water-oil content ratio of the mice in the model group was significantly lower than that of the blank control group 1, and the mice in the model group had severe water-oil imbalance. After drug treatment, the water-oil content ratio of the skin of the two groups of mice in the positive drug group and the Kochia scoparia saponin Ic group recovered to varying degrees, and the Kochia scoparia saponin Ic group had a significant effect of recovering the water-oil content ratio, which was closest to the blank control group 1.

[0045] (IV) Cytokine detection and its results

[0046] Skin tissue samples from each group of mice were collected, and the levels of FoxO1 and ATF4 in the skin tissues were determined by immunohistochemistry. Each tissue sample was stained with Oil Red O, and then the lipids of the mice were observed.

[0047] Analysis method: Aipathwell is a digital pathology image analysis software based on artificial intelligence learning launched by Servicebio. It uses AI deep learning principles, performs algorithm training based on massive data and integrates it into automated image analysis software. The specific process is as follows:

[0048] (1) Tracking: Automatically locate and circle the area to be tested along the tissue to be tested, and can be manually positioned according to specific requirements;

[0049] (2) Color selection: Automatically make positive judgments based on HSI, which can be manually corrected according to specific circumstances;

[0050] (3) Calculation: According to the needs, the software automatically locates the cell nucleus and expands the cytoplasm range; calculates the number and area of ​​weakly, moderately and strongly positive cells; the integrated optical density (IOD); tissue area and other different parameters.

[0051] (4) Analysis: Calculate the area to be tested step by step at high magnification. After completion, automatically calculate each item based on the original basic data and algorithm formula to obtain the analysis results and generate a report.

[0052] Evaluation items:

[0053] ①Positive cell ratio = number of positive cells / total number of cells

[0054] ②Positive cell density = number of positive cells / area of ​​tissue to be tested

[0055] ③Average optical density value = cumulative optical density IOD value / positive pixel area

[0056] ④H-SCORE=∑(pi×i)=(percentage of weak intensity ×1)+(percentageofmoderate intensity ×2)+(percentage of strong intensity ×3)

[0057] ⑤IRS=SI (positive intensity) × PP (positive cell ratio). SI can be divided into 3 levels, 0 is no positive staining, 1 is light yellow weak positive, 2 is brownish yellow medium positive, 3 is brownish brown strong positive; PP can be divided into 4 levels, 0 is 0-5%, 1 is 6%-25%, 2 is 26%-50%, 3 is 51%-75%, 4 is >75%

[0058] ⑥Positive area ratio = positive area / tissue area

[0059] ⑦Positive surface density = cumulative optical density IOD value / tissue pixel area

[0060] 1. The results of the detection of FoxO1 levels in skin tissue are shown in Table 6.

[0061] Table 6 Detection results of FoxO1 levels in mouse skin tissue

[0062]

[0063] As shown in Table 6, the positive drug group and the kochia scoparia saponin Ic group expressed higher levels of FoxO1, and these two groups were significantly different from the model group and the blank control group, indicating that kochia scoparia saponin Ic can upregulate FoxO1.

[0064] 2. The results of the detection of ATF4 levels in skin tissue are shown in Table 7.

[0065] Table 7 Results of ATF4 level detection in mouse skin tissue

[0066]

[0067] As shown in Table 7, compared with the model group, the ATF4 expression levels in the positive drug group and the kochia scoparia saponin Ic group were significantly lower than those in the model group, indicating that kochia scoparia saponin Ic can inhibit ATF4.

[0068] 3. Oil Red O staining and test results

[0069] Oil Red O staining is a commonly used lipid staining method, mainly used to show fat in tissues. The most widely used dye for lipid staining is Sudan dye, and the most commonly used ones are Sudan III, Sudan IV, Sudan Black and Oil Red O. The solubility of Sudan Oil Red O dye in lipids is greater than that in organic solvents, so during staining, the dye is transferred from the dye solution to the stained lipids, making the lipids appear red. This model is used to judge the amount of lipid production to evaluate the amount of oil production. The test results of Oil Red O staining of mouse skin tissue in each group are shown in Table 8.

[0070] Table 8 Detection results after Oil Red O staining

[0071]

[0072] As shown in Table 8, the oil production in the Kochia scoparia saponin Ic group was much lower than that in the model group, indicating that Kochia scoparia saponin Ic can reduce oil secretion.

[0073] (V) 5α-reductase inhibition rate detection in mice and its results

[0074] The inhibition rate of the sample on 5α-reductase was detected by using the double antibody sandwich method:

[0075] (1) Take out the required multi-well plate from the aluminum foil bag after equilibration at room temperature for 20 minutes. Seal the remaining multi-well strips in a ziplock bag and place them back at 4°C.

[0076] (2) Sample addition: Divide the multi-porous strips into four groups: blank control group 2, negative control group, positive control group and sample group, with 3 parallel wells in each group. Except for blank control group 2, which did not add any reagent, 25 μL of 20 u / mL 5α-reductase standard was first added to each well of the remaining groups. Then, 25 μL of sample diluent was added to the negative control group, 25 μL of 0.5 mg / mL finasteride was added to the positive control group, and 25 μL of 15 μg / ml Kochia scoparia saponin Ic was added to the sample group. Finally, the negative control group, positive control group and sample group were placed in a constant temperature reactor at 37°C and incubated for 60 min.

[0077] (3) For the negative control group, positive control group and sample group, discard the liquid first, pat dry with absorbent paper, fill each well with washing solution, let stand for 1 min, then discard the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0078] (4) Except for the blank control group 2, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of each group, and the reaction wells were sealed with sealing film. Then, the negative control group, positive control group and sample group were placed in a constant temperature reactor at 37°C and incubated for 60 min.

[0079] (5) For the negative control group, positive control group and sample group, discard the liquid first, pat dry with absorbent paper, fill each well with washing solution, let stand for 1 min, then shake off the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0080] (6) Add 100 μL of the mixture of colorimetric solutions A and B to each of the three parallel wells of the negative control group, positive control group, and sample group, and then incubate them in a light-proof environment at 37°C for 15 min.

[0081] (7) Then, add 50 μL of stop solution to each of the three parallel wells of the negative control group, positive control group, and sample group. Within 15 minutes, measure the OD value (optical density) of each well at a wavelength of 450 nm.

[0082] 5α-reductase inhibition rate (%) calculation formula:

[0083] Positive control group: 5α-reductase inhibition rate (%) = *100%

[0084] Sample group: 5α-reductase inhibition rate (%) = *100%

[0085] The results of the 5α-reductase inhibition test are shown in Table 9.

[0086] Table 95 α-reductase inhibition test results

[0087]

[0088] As shown in Table 9, the 5α-reductase inhibition rate of Kochia scoparia saponin Ic in the sample group was 48.8%±0.9%, with a P value of 0.0001, which was significantly different from that in the negative control group, indicating that Kochia scoparia saponin Ic can inhibit 5α-reductase.

[0089] In summary, Kochia scoparia saponin Ic reduces skin oil secretion, enhances skin barrier function, and regulates skin water and oil balance by upregulating FoxO1, inhibiting ATF4, and inhibiting 5α-reductase activity.

[0090] Specific examples of Kochia scoparia saponin Ic used as an active ingredient in water-oil balancing skin care products and drugs for treating imbalanced sebaceous gland secretion:

[0091] Product application example 1: water-oil balancing toner

[0092] (1) The formula is as follows:

[0093]

[0094] (2) Preparation method:

[0095] S1: Add deionized water to the main pot, add phase A premix 1 while stirring, continue stirring and heat to 85℃, keep warm for 20 minutes, and then cool down;

[0096] S2: When the temperature drops to 45°C, add the raw materials of phase B in sequence (from top to bottom in the table) and stir until dissolved evenly.

[0097] Product application example 2: Water-oil balance essence

[0098] (1) The formula is as follows:

[0099]

[0100] (2) Preparation method:

[0101] S1: Add deionized water to the main pot, sprinkle in U21, stir and disperse completely after wetting, add premix while heating and stirring, heat to 85℃, homogenize at low speed for 3min and stir for 10min, stir until dissolved and then cool down;

[0102] S2: When the temperature drops to 50°C, add phase B-10% NaOH aqueous solution and stir until uniformly dispersed;

[0103] S3: When the temperature drops to 45°C, add the raw materials of phase C in sequence (i.e., from top to bottom in the table) and stir slowly until dissolved evenly.

[0104] Product application example 3: Water-oil balance repair cream

[0105] (1) The formula is as follows:

[0106]

[0107] (2) Preparation method:

[0108] S1: Add phase A deionized water and premix 1 into the main pot in sequence while stirring, raise the temperature to 85°C, and stir until completely dissolved;

[0109] S2: While heating phase A, add phase B premix 2 into the oil pan, heat and stir to 80°C until completely dissolved, add EMT10 and stir to disperse evenly, then add the dispersion into the main pot, homogenize at medium speed for 5 minutes, then add 1503 Fluid, continue homogenizing for 3-5 minutes, and start cooling;

[0110] S3: When the temperature drops to 45°C, add the C phase raw materials and stir until they are evenly dispersed and then discharge the material.

[0111] Product application example 4: Water-oil balance oil-control shampoo

[0112] (1) The formula is as follows:

[0113]

[0114] (2) Preparation method:

[0115] S1: Add deionized water to the main pot and heat it to 80°C. Add other ingredients of phase A in order (from top to bottom in the table), stir at low speed until completely dissolved, and cool down.

[0116] S2: When the temperature drops to 60°C, add phase B and stir until it is completely dissolved, and continue to cool;

[0117] S3: When the temperature drops to 45°C, add the raw materials of phase C in sequence (i.e., from top to bottom in the table) and stir slowly until fully dispersed.

[0118] Product Application Example 5: Seborrheic Dermatitis Treatment Cream

[0119] (1) The formula is as follows:

[0120]

[0121] (2) Preparation method:

[0122] S1: Add deionized water and methylparaben to the main pot and heat to 85°C, stir for 10 minutes and then cool to 60°C;

[0123] S2: Add the raw materials of phase A into the oil pan, stir and heat to 80℃ until completely dissolved, cool to 60℃, pump into the main pot, and homogenize for 6 minutes;

[0124] S3: Add Kochia scoparia saponin Ic into the main pot, stir slowly until fully dispersed, quickly cool to 40°C, and then discharge.

[0125] In the above embodiments, Kochia scoparia saponin Ic may also be an extract containing Kochia scoparia saponin Ic. The extract containing Kochia scoparia saponin Ic can also play a role in regulating the water-oil balance of the skin.

[0126] The above is only an implementation method of the invention, and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the invention, which still fall within the scope of patent protection.

Claims

1. Use of Kochia scoparia saponin Ic as the sole active ingredient in the preparation of a drug for treating sebaceous folliculitis or seborrheic dermatitis.

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