Polygonum aviculare extract as well as preparation method and application thereof

Abstract

The invention discloses a polygonum aviculare extract as well as a preparation method and application of the polygonum aviculare extract. The preparation method comprises the following steps: crushing, extracting and filtering polygonum aviculare, then enriching by utilizing a resin column chromatography mode, and finally concentrating and drying to obtain polygonum aviculare extract powder; the prepared polygonum aviculare extract has good application value in the aspect of oil control of cosmetics, polygonum aviculare plant resources can be effectively utilized, and the application field of polygonum aviculare plants is widened; according to the preparation method, a targeted aqueous two-phase extraction technology is provided, and the preparation method has the advantages that the price is low and reagents are friendly and environmentally friendly while effective components such as polygonum aviculare flavonoids are effectively enriched and impurities are removed. The invention provides a special extraction process for polygonum aviculare, and the extraction process has the advantages of being capable of being applied to the cosmetic industry and having an oil control effect.

Description

Technical Field

[0001] This invention relates to the field of extract preparation technology, and in particular to a knotweed extract, its preparation method, and its application. Background Technology

[0002] Sebaceous glands are one of the main appendages of the skin, distributed on the surface of the skin such as the face, trunk, and scalp. Their main function is to secrete sebum to moisturize the skin and hair and prevent dryness. However, overactive sebaceous glands in oily skin can lead to excessive sebum secretion, causing a series of skin problems. Excessive sebum secretion is usually induced by androgens. In addition, high-lipid, high-sugar diets and stress factors (cortisol) are also contributing factors to stimulating sebum secretion. When sebaceous glands secrete too much sebum, it is easily oxidized to form lipid peroxides, which stimulate inflammation around the hair follicles. Simultaneously, the accumulation of excessive sebum mixed with keratinocytes further leads to skin problems such as enlarged pores and clogged pores.

[0003] Currently, the main technologies for oil control focus on cleansing or using physical absorbents to absorb oil, oral sebaceous gland activity regulators, and medical aesthetic aids such as phototherapy.

[0004] While physical absorbents can provide immediate oil absorption, long-term use without timely cleaning can clog pores and lead to more skin problems.

[0005] Oral sebaceous gland activity modulators are effective, but due to their oral nature, long-term use may affect liver function, and sebaceous gland activity will return to the pre-treatment level after discontinuation of the medication.

[0006] Photorejuvenation and other medical aesthetic techniques can reduce the activity of sebaceous glands by heating the dermis, but they are expensive and require a high level of expertise from the operators, otherwise the risk of laser burns is high.

[0007] Therefore, using cosmetics to target oil production on specific areas of the skin is a relatively inexpensive and effective method of oil control.

[0008] The main oil-controlling measures in cosmetic oil-controlling products are: 1. Controlling oil production at its source: By inhibiting the activity of sebaceous gland cells, it reduces sebum secretion and strengthens the skin barrier; 2. Unclog pores: Exfoliation with salicylic acid and other peeling agents reduces existing oil buildup. However, the strong exfoliating power of these agents can damage the skin barrier, and the skin barrier is weakened after using exfoliating ingredients like salicylic acid, requiring strict sun protection; otherwise, it can lead to further skin problems such as hyperpigmentation and darkening.

[0009] Plant extracts can effectively inhibit sebum secretion by controlling oil at its source. At the same time, their gentle and safe oil-controlling ability gives them a unique position among oil-controlling ingredients in cosmetics.

[0010] Flat-bellied ox ( Polygonum aviculare *Polygonum aviculare* (L.) is a plant belonging to the genus *Polygonum* in the family Lycoraceae of the order Caryophyllales. Its strong adaptability to soil allows it to grow at altitudes ranging from 10 to 4200 meters, while also tolerating harsh winters with temperatures as low as -40°C. As a traditional Chinese medicine, *Polygonum aviculare* is mainly used in clinical practice for its diuretic, heat-clearing, insecticidal, and antipruritic properties. Its pharmacological activity primarily derives from its flavonoids, phenylpropanoids, and phenolic acids. Researchers have extracted and isolated over 90 compounds from *Polygonum aviculare*, of which over 40 are flavonoids and their glycosides; therefore, flavonoids and their glycosides are considered the main chemical components of *Polygonum aviculare*. Modern pharmacology indicates that *Polygonum aviculare* possesses various physiological activities, including antibacterial and insecticidal effects, anti-diabetic properties, anti-obesity and anti-aging effects, anti-inflammatory effects, antioxidant effects, anti-tumor effects, wound-healing promotion, and liver protection.

[0011] Currently, existing research on Polygonum multiflorum has reported its cosmetic effects such as anti-oxidation, anti-inflammation, whitening, and anti-photoaging, but there are no studies or reports on the efficacy of Polygonum multiflorum extract in oil control. Summary of the Invention

[0012] The purpose of this invention is to provide a flat battery ( Polygonum aviculare L.) Extract, Preparation Method and Application. This invention provides a dedicated extraction process for Polygonum multiflorum, and the extracted extract can be used in the cosmetics industry and has oil-controlling effects.

[0013] The technical solution of the present invention: An extract of Polygonum aviculare, comprising at least 50-62% by weight of flavonoids.

[0014] The preparation method of the above-mentioned Polygonum hydropiper extract includes the following steps: S1. Pre-treatment: The raw medicinal material of Polygonum aviculare is pulverized using a pulverizer and then passed through a 24-mesh sieve before use; S2. Preparation of Polygonum multiflorum extract: Weigh Polygonum multiflorum powder and add it to the extraction system. Stir and mix well, then place the extract in an ultrasonic extractor to obtain Polygonum multiflorum extract. S3. Preparation of Polygonum multiflorum filtrate: After filtering the Polygonum multiflorum extract using a Buchner funnel to remove the residue, separate the liquid using a funnel to obtain the upper extract; concentrate the upper extract under reduced pressure using a rotary evaporator to a paste-like state; add ethanol solution, disperse evenly, centrifuge, and pass the supernatant through an MCE membrane to obtain Polygonum multiflorum filtrate; S4. Extract purification: The filtrate of Polygonum multiflorum was subjected to resin adsorption at a certain flow rate. After adsorption, the resin column bed was washed with deionized water in the forward direction. The adsorbed product was desorbed by passing the column through ethanol in the forward direction to obtain Polygonum multiflorum eluent. S5. Extract concentration and drying: The eluent of Polygonum multiflorum is concentrated using a rotary evaporator and then dried using a vacuum drying device. After drying, the sample is ground to obtain Polygonum multiflorum extract powder.

[0015] In the aforementioned method for preparing Polygonum aviculare extract, the preparation of Polygonum aviculare extract in step S2 is detailed as follows: S2.1 Establishing the extraction system: Prepare an ethanol solution with a concentration of 40-70%, add ammonium sulfate solid with a mass fraction of 16-24%, stir and let stand to separate the layers to obtain an aqueous two-phase extraction system; S2.2 Preparation of Polygonum multiflorum extract: Weigh Polygonum multiflorum powder and add it to the aqueous two-phase extraction system. The material-to-liquid ratio is 1:20-40. Stir for 20 minutes to mix thoroughly. Place the extract in an ultrasonic extractor and set the ultrasonic frequency to 260-400w, the ultrasonic time to 40-70min, and the ultrasonic temperature to 40℃ to obtain Polygonum multiflorum extract.

[0016] In the aforementioned method for preparing Polygonum aviculare extract, the preparation of Polygonum aviculare filtrate in step S3 is detailed below: S3.1 Place the extract of Polygonum aviculare in a Buchner funnel for vacuum filtration to remove the residue, and use a funnel to separate the layers of the permeate and take the upper layer of extract. S3.2. The upper extract was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotary evaporation conditions were set as follows: 40-80 rpm rotation speed in the eggplant flask, 80 mbar vacuum pressure, and 50℃ water bath temperature. S3.3 Add 15% (v / v) ethanol solution at a ratio of 1:5 of crude drug amount, disperse evenly, and centrifuge for 20-40 min at a speed of 8000-11000×g and a temperature of 25℃. Collect the supernatant. S3.4. Pass the supernatant through a 0.22μm MCE membrane to obtain the filtrate of Cyperus rotundus.

[0017] In the aforementioned method for preparing Polygonum aviculare extract, the purification of the extract in step S4 is detailed below: S4.1 Resin Adsorption: The filtrate of Polygonum multiflorum was packed with filler at a ratio of raw drug to column volume of 1:1.5; the filtrate was loaded into the macroporous adsorption resin by forward loading, with an adsorption flow rate of 3.5-4.5 BV / h, and the content of the target product at the outlet was detected every 1 BV to determine the adsorption state. S4.2 Water washing to remove impurities: After the resin adsorption is completed, the resin column bed is washed with deionized water in the forward direction at a flow rate of 2.5-3.5 BV / h for 2-3 hours to remove residual liquid and some water-soluble impurities in the column bed; Brix is ​​used to detect the Brix detection amount at each 1 BV outlet liquid until the Brix detection amount is ≤0.1%, and the water washing is completed. S4.3 Resin Desorption: The resin was desorbed by passing 4-6 BV of 60% ethanol through the column in a forward direction at a flow rate of 1.5-2.5 BV / h. The content of the target substance in the effluent was measured every 1 BV to determine the desorption state of the resin. Brix ≤ 0.1 at the effluent was regarded as the desorption endpoint. The resulting liquids were combined and recorded as the eluent.

[0018] In the aforementioned method for preparing Polygonum aviculare extract, the extraction concentration and drying in step S5 are detailed below: S5.1 Concentration: The eluent of Polygonum aviculare was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotation speed of the flask was set to 40-60 rpm, the vacuum pressure to 80 mbar, and the water bath temperature to 50℃. S5.2 Drying: Use a vacuum drying device to dry the sample, setting the drying vacuum degree to 40-60mb, the drying temperature to 37℃, and the drying time to 10-20h, to obtain a dried sample of Polygonum multiflorum extract; grind the dried sample of Polygonum multiflorum extract to obtain Polygonum multiflorum extract powder.

[0019] The aforementioned Polygonum hydropiper extract can be used as an active ingredient in the preparation of cosmetics with oil-controlling effects. Compared with the prior art, this application obtains the extract powder of Polygonum multiflorum by crushing, extracting, filtering, enriching it by resin column chromatography, and finally concentrating and drying it. The extract of Polygonum aviculare obtained in this application has great application value in oil control in cosmetics, and can effectively utilize Polygonum aviculare plant resources and broaden the application field of Polygonum aviculare plant. The preparation method proposed in this application presents a targeted aqueous two-phase extraction technology, which effectively enriches and removes impurities such as flavonoids from Polygonum aviculare while offering advantages such as low price, reagent-friendly and environmentally friendly properties.

[0020] Therefore, this invention provides a dedicated extraction process for Polygonum hydropiper, which has the advantages of being applicable to the cosmetics industry and possessing oil-controlling effects. Attached Figure Description

[0021] Figure 1 This is a flowchart of the extraction method of the present invention; Figure 2 This is a typical diagram of the efficacy experiment of Experiment 2 in the embodiments of the present invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0023] Example 1. A method for preparing an extract of Polygonum aviculare, as follows: Figure 1 As shown, the steps are as follows: S1. After grinding the raw medicinal material into powder using a pulverizer, pass it through a 24-mesh sieve and weigh out 200g of powder. S2. Prepare 4000 mL of 45% ethanol solution and add 24% ammonium sulfate solid to it. Stir overnight and let it stand to separate into layers to obtain a two-phase aqueous extraction system. Add 200g of Polygonum aviculare powder to the extraction system to make the material-liquid ratio 1:20; stir for 20min to mix thoroughly, place the extract in an ultrasonic extractor, set the ultrasonic frequency to 280w, the ultrasonic time to 45min, and the ultrasonic temperature to 40℃ to complete the preparation of Polygonum aviculare extract. S3. Take out the extract of Polygonum multiflorum and place it in a Buchner funnel for vacuum filtration to remove the residue. Separate the permeate into layers using a funnel and take the upper layer of extract. The upper extract was concentrated to a paste state under reduced pressure using a rotary evaporator, with the following settings: rotation speed of 50 rpm, vacuum pressure of 80 mbar, and water bath temperature of 50℃. Add 1000 mL of 15% (v / v) ethanol solution to the crude drug at a ratio of 1:5, disperse evenly, and centrifuge for 20 min at a speed of 8500×g and a temperature of 25℃. The supernatant was collected and passed through a 0.22 μm MCE membrane to collect the filtrate, which was then used for subsequent enrichment with macroporous adsorption resin. S4. Fill the column with 350 mL of packing material. Slowly load the filtrate of Cyperus rotundus into the macroporous adsorption resin using a forward column flow method. Set the adsorption flow rate to 3.5 BV / h. Detect the content of the target product at the outlet every 1 BV to determine the adsorption state. After resin adsorption is complete, the resin column bed is washed with deionized water in a forward pass at a flow rate controlled at 2.5 BV / h for 2 hours to remove residual feed and some water-soluble impurities from the column bed; at 4 BV, the Brix detection level at the effluent is ≤0.1%; After the resin was washed with water, the product was desorbed by forward column chromatography using 4 BV of 60% ethanol. The desorption flow rate was controlled at 1.5 BV / h, and Brix ≤ 0.1 at the 3 BV outlet. The collected liquid was combined and recorded as the eluent. S5. The eluent of Polygonum aviculare was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotation speed of the flask was set to 40 rpm, the vacuum pressure to 80 mbar, and the water bath temperature to 50 ℃. The sample was dried using a vacuum drying device with a vacuum degree of 40mb, a drying temperature of 37℃, and a drying time of 10h. The dried sample of Polygonum multiflorum extract was then ground to obtain Polygonum multiflorum extract powder.

[0024] The Polygonum hydropiper extract prepared by the above method contains at least 50-65% flavonoids by mass percentage.

[0025] The extract of Polygonum hydropiper has an oil-controlling effect when used in cosmetics.

[0026] Example 2. A method for preparing an extract of Polygonum aviculare, similar to that in Example 1, comprising the following steps: S1. After grinding the raw medicinal material into powder using a pulverizer, pass it through a 24-mesh sieve and weigh out 400g of powder. S2. Prepare 12000 mL of 50% ethanol solution and add 20% ammonium sulfate solid to it. Stir overnight and let it stand to separate into layers to obtain a two-phase aqueous extraction system. Add 400g of Polygonum aviculare powder to the extraction system to make the material-liquid ratio 1:30; stir for 20 minutes to mix thoroughly, place the extract in an ultrasonic extractor, set the ultrasonic frequency to 320w, the ultrasonic time to 60min, and the ultrasonic temperature to 40℃ to complete the preparation of Polygonum aviculare extract. S3. Take out the extract of Polygonum multiflorum and place it in a Buchner funnel for vacuum filtration to remove the residue. Separate the permeate into layers using a funnel and take the upper layer of extract. The upper extract was concentrated to a paste under reduced pressure using a rotary evaporator. The rotary evaporation conditions were set as follows: 60 rpm rotation speed in an eggplant-shaped flask, 80 mbar vacuum pressure, and 50°C water bath temperature. Add 2000 mL of 15% (v / v) ethanol solution to the crude drug at a ratio of 1:5, disperse evenly, and centrifuge for 30 min at a speed of 10000×g and a temperature of 25℃. The supernatant was collected and passed through a 0.22 μm MCE membrane to collect the filtrate, which was then used for subsequent enrichment with macroporous adsorption resin. S4. Fill the column with 600 mL of packing material. Slowly load the filtrate of Cyperus rotundus into the macroporous adsorption resin using the forward column flow method. Set the adsorption flow rate to 4 BV / h. Detect the content of the target product at the outlet every 1 BV to determine the adsorption state. After resin adsorption is complete, the resin column bed is washed with deionized water in a forward pass at a flow rate of 3 BV / h for 2.5 hours to remove residual feed and some water-soluble impurities from the column bed; at 4 BV, the Brix detection level at the outlet liquid is ≤0.1%; After the resin was washed with water, the product was desorbed by forward column chromatography using 5 BV of 60% ethanol. The desorption flow rate was controlled at 2 BV / h, and Brix ≤ 0.1 at the 4 BV outlet. The collected liquid was combined and recorded as the eluent. S5. The eluent of Polygonum aviculare was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotation speed of the flask was set to 50 rpm, the vacuum pressure to 80 mbar, and the water bath temperature to 50 ℃. The sample was dried using a vacuum drying device with a vacuum level of 50 mb, a drying temperature of 37°C, and a drying time of 15 h. The dried sample of Polygonum multiflorum extract was then ground to obtain Polygonum multiflorum extract powder.

[0027] Example 3. A method for preparing an extract of Polygonum aviculare, similar to that in Example 1, comprising the following steps: S1. After grinding the medicinal material into powder using a pulverizer, pass it through a 24-mesh sieve and weigh out 1000g of powder. S2. Prepare 40 L of 54% ethanol solution and add 16% ammonium sulfate solid to it. Stir overnight and allow to stand to separate into layers to obtain a two-phase aqueous extraction system. Add 1000g of Polygonum aviculare powder to the extraction system to make the material-liquid ratio 1:40; stir for 20 minutes to mix thoroughly, place the extract in an ultrasonic extractor, set the ultrasonic frequency to 360w, the ultrasonic time to 70min, and the ultrasonic temperature to 40℃ to complete the preparation of Polygonum aviculare extract. S3. Take out the extract of Polygonum multiflorum and place it in a Buchner funnel for vacuum filtration to remove the residue. Separate the permeate into layers using a funnel and take the upper layer of extract. The upper extract was concentrated to a paste under reduced pressure using a rotary evaporator. The rotary evaporation conditions were set as follows: 70 rpm rotation speed in a flask, 80 mbar vacuum pressure, and 50°C water bath temperature. Add 5000 mL of 15% (v / v) ethanol solution, disperse evenly, and centrifuge for 40 min at 11000 × g and 25 °C. Collect the supernatant and pass it through a 0.22 μm MCE membrane to collect the filtrate, which will be used for subsequent enrichment with macroporous adsorption resin. S4. Fill the column with 1500 mL of packing material. Slowly load the filtrate of Cyperus rotundus into the macroporous adsorption resin using a forward column flow method. Set the adsorption flow rate to 4.5 BV / h. Detect the content of the target product at the outlet every 1 BV to determine the adsorption state. After resin adsorption is complete, the resin column bed is washed with deionized water in a forward pass at a flow rate controlled at 3.5 BV / h for 3 hours to remove residual feed and some water-soluble impurities from the column bed; at 5 BV, the Brix detection level at the effluent is ≤0.1%; After the resin was washed with water, the product was desorbed by passing 6 BV of 60% ethanol through the column in a forward direction. The desorption flow rate was controlled at 2.5 BV / h, and the Brix at the 5 BV outlet was ≤0.1. The collected liquid was combined and recorded as the euphorbia humifusa eluent. S5. The eluent of Polygonum aviculare was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotation speed of the flask was set to 60 rpm, the vacuum pressure to 80 mbar, and the water bath temperature to 50 ℃. The sample was dried using a vacuum drying device with a vacuum level of 60 mb, a drying temperature of 37°C, and a drying time of 20 h. The dried sample of Polygonum multiflorum extract was then ground to obtain Polygonum multiflorum extract powder.

[0028] Efficacy verification of Polygonum aviculare extract Experiment 1: Detection of Flavonoid Content 1. Experimental Methods: (1) Experimental method: The content of plant flavonoids was detected using a plant flavonoid content detection kit. (2) Experimental steps:

[0029] After adding reagent one, mix well and let stand at room temperature for 5 minutes. After adding reagent two, mix well and let stand at room temperature for 5 minutes. After adding all the working solution according to the above procedure, mix well, incubate in a 37°C water bath for 45 minutes, set the centrifugation force to 10000g, centrifuge for 10 minutes, collect the supernatant, take 200 μL and measure the A470 absorbance value in a 96-well plate, and calculate the flavonoid content according to the following formula.

[0030] The calculation formula is: ΔA = Adetermined - Acontrol; ΔA' = Astandard - Ablank The above formula calculates the value of ΔA'. Flavonoid content (mg / g) = (ΔA + 0.0376) / 0.6093 In the formula, the result of ΔA is substituted into the formula for calculating flavonoid content, the result of ΔA' is used to plot a standard curve and generate a standard curve equation, and the ΔA obtained from the sample measurement is substituted into the standard curve equation to obtain the flavonoid content of the sample.

[0031] The standard is a 10 mg / mL rutin standard solution.

[0032] Analysis of experimental results: (1) Construction of standard curve: A standard curve was prepared based on the absorbance results measured from dilutions of the standard to 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 mg / mL: y = 0.6093x - 0.0376, R 2 = 0.9976.

[0033] (2) The test results are shown in the table below.

[0034] Note: Statistical analysis was performed using the t-test method. Significance between sample groups is indicated by *. Compared with the blank control group, * indicates statistical significance. p <0.05; The test results of this experiment show that the Polygonum multiflorum extract prepared using this example has a high flavonoid content (>50%). The main identified components of the Polygonum multiflorum extracts obtained in Examples 1, 2, and 3 are flavonoids. The results show that there is no significant difference in flavonoid content among the Polygonum multiflorum extract products obtained through the three examples (p>0.05), proving that the differences in the components of the products obtained through different examples are small, and the efficacy can be cross-referenced.

[0035] Therefore, the following experimental examples are analyzed based on the test results of the sample in Example 1.

[0036] Experiment 2: In vitro oil control efficacy test 1. Experimental Methods: (1) Cytotoxicity assay of human sebaceous gland cells (SZ95): MTT assay Experimental steps: Cells were seeded in 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well contains 100 cells / well) and incubated at 37°C in 5% CO2 for 24 hours; Drug administration: The sample group was replaced with the corresponding concentration of culture medium containing the sample, the normal control group was replaced with fresh culture medium, and the blank control group was replaced with blank culture medium. Three biological replicates were set up and incubated at 37℃ and 5% CO2 for 24h. After incubation, add MTT solution to each well, continue culturing for 4 hours, discard the culture medium, add DMSO solution, shake to mix, and then measure the absorbance value at 490 nm (OD). 490 ).

[0037] Cell viability (%) = (OD) 490 Sample group - OD 490 Blank control group) / (OD 490 Normal control group - OD 490 (Blank control group) × 100%. The test results are shown in Table 2-1.

[0038] (2) Detection of lipid accumulation inhibition ability: Experimental steps: Cell seeding: Human sebaceous gland cells (SZ95) were seeded at a rate of 1×10⁻⁶. 5 Each sample was inoculated into a 12-well plate and cultured at 37°C in a 5% CO2 incubator for 24 h. The following groups were set up: blank control group (containing 0% sample), model control group (inducer), sample group (inducer + 0.005% sample), and positive control group (inducer + 50 μg / mL palmatine). Except for the blank group, all other groups contained 0.0188 μM of the modeling agent dihydrotestosterone (DHT). Each sample was tested in triplicate. Modeling and drug administration: After the cells adhered to the wall, the blank control group and the model control group in the above groups were replaced with fresh culture medium, and the sample group was replaced with fresh culture medium containing the sample. DHT was used to induce lipid synthesis. Then, they were placed in a 37°C, 5% CO2 incubator for 24 hours. Sample collection and detection: After incubation for 24 hours, the culture medium was discarded and the cells were washed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 10 minutes at room temperature. After fixation, the cells were stained with Nile Red (100 μg / mL) and incubated at 37°C for 10 minutes. The staining solution was removed and the cells were washed three times with PBS. The staining was observed under a fluorescence microscope. Cells with strong orange-red fluorescence were considered to be lipid-rich positive cells. The lipid content in the cytoplasm of human sebaceous gland cells (SZ95) was detected by Nile Red staining.

[0039] The calculation formula is as follows: Inhibition rate (%) = (Model control group - Sample group) / Model control group × 100%; The calculation results are shown in Table 2-2, where the sample material is the sample prepared in Example 1.

[0040] Based on the cytotoxicity results, 0.006% was subsequently selected as the sample addition concentration for the lipid accumulation inhibition experiment.

[0041] Simultaneously, obtain comparative images of fluorescence staining at any three locations under a fluorescence microscope from the model group, positive control group, and sample group of Example 1, such as... Figure 1 As shown.

[0042] Analysis of experimental results: (1) Analysis of cytotoxicity results of human sebaceous gland cells (SZ95):

[0043] Note: Statistical analysis was performed using the t-test method. The significance of the sample group compared with the blank control group is indicated by *, p value < 0.05 is indicated by *, and p value < 0.01 is indicated by **.

[0044] The results of the cytotoxicity experiment showed that there was no cytotoxicity when the detection concentration was less than 0.00625%, so 0.006% was selected as the experimental concentration.

[0045] (2) Analysis of lipid accumulation inhibition results:

[0046] Note: Statistical analysis was performed using the t-test method. Significance between the sample group and the blank control group is indicated by * and #. Compared with the blank control group, ###p<0.001; compared with the model control group, ***p<0.001. The amount of sebum secreted by human sebaceous gland cells can be efficiently assessed using Nile Red staining to evaluate the lipid metabolism status of sebaceous gland cells. Analysis of the cumulative fluorescence intensity of lipids can determine the lipid metabolism level in human sebaceous gland cells after adding the sample from Example 1. Compared with the model control group, the positive control group showed a 31% inhibition rate of sebum secretion, proving the successful establishment of the model in this experiment. The sample obtained in Example 1 showed a significant reduction in the fluorescence intensity of lipid accumulation, with an inhibition rate of 21%, indicating that even at an application rate of 0.006%, the sample from Example 1 has a significant inhibitory effect on sebum secretion.

[0047] The typical experimental results of this sample are shown in the figure below. Figure 2 As shown, the fluorescence intensity of the sample in Example 1 was significantly weaker than that of the model group, indicating that the sample in Example 1 has a significant effect on inhibiting sebum secretion.

[0048] Experiment 3: 5α-Reductase Inhibition Test 1. Test method: (1) The experimental groups are shown in Table 3-1 below:

[0049] (2) Experimental steps: Add 1 mL of PBS solution to the blank control group, 1 mL of sample working solution to the sample group, and 1 mL of finasteride working solution to the positive control group. Add 1 mL of 5% ethanol to the enzyme tube. Add 1 mL of enzyme solution, 1 mL of NADPH solution, and 1 mL of testosterone solution to a test tube, set up 3 parallel tubes, gently shake, and use a pipette to add 200 μL to a 96-well ELISA plate. Place the plate in an ELISA reader and measure the absorbance at 340 nm. This is the first measurement value, A0.

[0050] After incubating at 37°C for 30 minutes, the sample was placed in an ELISA reader for detection. The absorbance was measured at 340 nm, which is the second measurement value, A30.

[0051] The calculation formula is as follows: 5α-reductase inhibition rate (%) = (1 - (A) 样品0 -A 样品30 ) / (A 酶0 -A 酶30 ))× 100% In the formula: A 样品0 The absorbance value of the first (0 min) sample group was measured. A 样品30 The absorbance values ​​of the sample group were measured for the second time (30 min). A 酶0 The average absorbance of the enzyme tubes measured for the first time (0 min); A 酶30The average absorbance of the enzyme tube measured in the second (30 min) enzyme test.

[0052] Analysis of experimental results: The test results are shown in Table 3-2 below.

[0053] Note: Statistical analysis was performed using the t-test method. The significance of the sample group compared with the blank control group is indicated by *. Compared with the blank control group, ***p<0.001; As a key regulatory enzyme for sebum secretion, 5α-reductase catalyzes the conversion of testosterone into the more potent dihydrotestosterone (DHT). DHT binds to the androgen receptor (AR) of sebaceous gland cells, and the activity of 5α-reductase can directly stimulate sebaceous gland proliferation and sebum secretion.

[0054] The data in Table 3-2 show that, compared with the blank control, the positive control finasteride achieved an inhibition rate of 40.3%.

[0055] The sample in Example 1 showed significant inhibitory activity against 5α-reductase at concentrations of 0.01% and 0.005%, with inhibition rates of 18.10% and 7.55%, respectively, which was dose-dependent.

[0056] This demonstrates that within this concentration range, the sample from Example 1 can effectively inhibit the activity of 5α-reductase, thereby achieving the effect of oil control.

[0057] Experiment 4: Inhibitory Effect of Lipid Synthesis and Secretion Regulatory Factors The expression levels of lipid metabolism-related genes (PPARγ, SREBP-1C, IGF-1R, PLIN2) in human sebaceous gland cells were detected using RT-QPCR. 1. Test method: (1) Experimental groups:

[0058] (2) Experimental steps: Human sebaceous gland cells were seeded in 6-well plates (3 × 10⁻⁶). 5 (each well) was incubated at 37 °C with 5% CO2 for 24 h.

[0059] After incubation, the culture medium was discarded, and the samples were washed 1-2 times with D-Hanks. Fresh culture medium was then added as shown in Table 4-1. No samples were given to the blank control group, the model control group (dihydrotestosterone), the sample group (Example 1 + dihydrotestosterone), and the positive control group (clavriston + dihydrotestosterone, AKT inhibitor + dihydrotestosterone). The samples were incubated at 37 °C and 5% CO2 for 24 h.

[0060] After incubation, the culture medium was removed, and the cells were washed 1-2 times with D-Hanks. Total RNA was extracted from each experimental group, cDNA was synthesized, and q-PCR was used to detect the gene expression of β-actin and the target gene.

[0061] β-actin was used as an internal reference for gene expression to calculate the relative RNA expression level of the target gene.

[0062] Calculate the relative RNA expression level of the target gene, where: relative RNA expression level = 2 ΔΔC(t) , ΔΔC(t)=ΔC(t) 模型对照组 -ΔC(t) 样本组 ΔC(t) = C(t) 目的基因 -C(t) β-actin , Downward adjustment rate (%) = (P) 模型对照组 -P 样品组 )×100% / P 模型对照组 In the formula: P: Relative gene expression level of each gene 2. Experimental Results: (1) PPARγ Gene expression results analysis:

[0063] Compared with the normal control group, # p <0.05; compared with the model control group, ** p <0.01, *** p <0.001 PPARγ Highly expressed in sebaceous gland cells, its activation can directly upregulate fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1), thereby increasing the production of free fatty acids. At the same time, it promotes the self-renewal of adipocytes by targeting genes such as LPL (lipoprotein lipase).

[0064] Lower PPARγ High expression of [the substance] can effectively reduce lipid synthesis. PPARγ As shown in Table 4-2, after stimulation of the model group PPARγ Gene expression was significantly upregulated, proving the successful construction of the model. Adding 0.0005 mg / mL clavone (positive control) resulted in a 23% downregulation rate compared to the model group. Adding 0.006% of the Example 1 sample resulted in a 53% downregulation rate compared to the model group, demonstrating highly significant downregulation and proving that the Example 1 sample has a strong gene-promoting effect. PPARγIts ability to downregulate gene expression demonstrates its potential to reduce lipid production and control oil production at the gene regulation level.

[0065] (2) SREBP-1C Gene expression results analysis:

[0066] Compared with the normal control group, ## p <0.01; compared with the model control group, ** p <0.01 As the "master switch" for lipid synthesis. SREBP-1 Sterol regulatory element-binding protein 1 (SREBP-1) is a key regulator of lipid metabolism pathways, controlling the expression of lipid synthases such as FASN, ACC, and SCD1. Therefore, to reduce excessive lipid secretion, the expression of SREBP-1 can be reduced to decrease sebaceous gland lipid synthesis.

[0067] SREBP-1C As shown in Table 4-3, after stimulation of the model group SREBP-1C Gene expression was significantly upregulated, proving the successful construction of the model. Adding 0.0078 μM of the positive control AKT inhibitor resulted in a 23% downregulation rate compared to the model group. Adding 0.006% of the Example 1 sample resulted in a 22% downregulation rate compared to the model group, demonstrating highly significant downregulation and proving that the Example 1 sample has a strong promoting effect. SREBP-1C Its ability to downregulate gene expression demonstrates its potential to reduce lipid production and control oil production at the gene regulation level.

[0068] (3) IGF-1R Gene expression results analysis:

[0069] Compared with the normal control group, ## p <0.01; compared with the model control group, ** p <0.01, *** p <0.001 Insulin-like growth factor 1 receptor (IGF-1 receptor) IGF-1R ( ) is a transmembrane receptor that indirectly controls sebum secretion by sebaceous gland cells by influencing cell proliferation and differentiation. IGF-1R It also shows a high expression trend in skin diseases such as acne and psoriasis, therefore IGF-1R It is believed that its high expression can cause excessive proliferation of sebaceous gland cells, leading to excessive sebum secretion.

[0070] IGF-1RAs shown in Table 4-4, after stimulation of the model group IGF-1R Gene expression was significantly upregulated, proving the successful construction of the model. Adding 0.0005 mg / mL clavone (positive control) resulted in a 27% downregulation rate compared to the model group. Adding 0.006% of the Example 1 sample resulted in a 20% downregulation rate compared to the model group, demonstrating highly significant downregulation and proving that the Example 1 sample has a strong gene-promoting effect. IGF-1R Gene expression downregulation ability. At the gene regulation level, it shows the ability to control oil production by inhibiting the high expression of sebaceous gland cells.

[0071] (4) PLIN2 Gene expression results analysis:

[0072] Compared with the normal control group, # p <0.05; compared with the model control group, ** p <0.01, *** p <0.001 Lipid droplet-coated protein 2 ( PLIN2 It coats the surface of lipid droplets to prevent neutral enzymes from being prematurely hydrolyzed by lipases, and is used to maintain the structure of lipid droplets. It can participate in lipid metabolism and affect lipid deposition. PLIN2 In sebaceous gland cells, PLIN2 can regulate lipid secretion by influencing lipid synthesis and catabolism. Therefore, reducing PLIN2 expression can effectively downregulate lipid synthesis and promote its metabolic breakdown.

[0073] As shown in Table 4-5, after stimulation of the model group PLIN2 Gene expression was significantly upregulated, proving the successful construction of the model. After adding the positive control 0.0005 mg / mL clavone, the downregulation rate compared to the model group reached 44%. After adding 0.006% of the Example 1 sample, the gene expression level was downregulated by 65% ​​compared to the model group. The extremely significant downregulation demonstrates that the Example 1 sample has a strong promoting effect. PLIN2 Gene expression downregulation ability. At the gene regulation level, it shows an oil-controlling effect by reducing the stability of already formed lipid droplets.

[0074] Experiment 5: Efficacy in Improving Lipid Peroxidation 1. Test method: (1) Cytotoxicity of human immortalized keratinocytes (HaCaT): MTT assay Experimental steps: Cells were seeded in 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well contains 1 cell / well) and incubated at 37°C in 5% CO2 for 24 hours.

[0075] Drug administration: The sample group was replaced with the corresponding concentration of culture medium containing the sample, the normal control group was replaced with fresh culture medium, and the blank control group was replaced with blank culture medium. Three biological replicates were set up, and the culture was incubated at 37℃ and 5% CO2 for 24 h. After incubation, MTT solution was added to each well, and the culture was continued for 4 h. The culture medium was then discarded, and DMSO solution was added. After shaking and mixing, the absorbance value at 490 nm (OD) was measured. 490 ).

[0076] Cell viability (%) = (OD) 490 Sample group OD 490 Blank control group) / (OD 490 Normal control group - OD 490 (Blank control group) × 100%. The test results are shown in Table 5-2.

[0077] (2) Detection of lipid peroxide content: MDA and LPO kits were used for detection. (a) Experimental Groups:

[0078] (b) Experimental procedures: Immortalized human keratinocytes were inoculated into 6-well plates (6 × 10⁻⁶). 5 (each well) was incubated at 37 °C under 5% CO2 conditions for 24 h.

[0079] After incubation, the culture medium was discarded, and the samples were washed 1-2 times with D-Hanks. Fresh culture medium was added as shown in Table 5-1. The model control group (LPS), the sample group (Example 1 + LPS), and the positive control group (N-acetyl-L-cysteine ​​+ LPS) were incubated at 37 °C and 5% CO2 for 24 h.

[0080] After incubation, the culture medium was discarded, and the cells were washed three times with D-Hanks. The cells were then collected and analyzed according to the instructions of the MDA and LPO assay kits.

[0081] Lipid antioxidant activity (%) = (S 模型对照组 -S 样品组 )×100% / S 模型对照组 In the formula: S: MDA content, LPO content 2. Results Analysis: (1) Analysis of HaCaT cytotoxicity MTT assay results: As shown in Table 5-2 below, in HaCaT cells, the sample concentration used in Example 1 was ≤0.00312 without cytotoxicity. Therefore, in subsequent experiments, 0.003% was selected as the MDA experimental concentration and 0.001% was selected as the LPO experimental concentration.

[0082]

[0083] (2) Analysis of lipid peroxidation detection results: Lipid peroxides and their degradation products are formed when lipids are oxidized in the presence of oxygen after excessive secretion of lipids. These peroxides have extremely strong oxidizing properties, which can trigger a series of inflammatory responses.

[0084] Therefore, the content of lipid peroxides directly reflects the oxidation status of lipids after secretion. LPO decomposes to produce MDA. The levels of LPO and MDA can reflect the inhibitory effect of a sample on lipid peroxidation.

[0085] (a) MDA content detection results:

[0086] Compared with the normal control group, ## p <0.01; compared with the model control group, ** p <0.01, *** p <0.001 The results showed that, according to the model control group analysis, the MDA content increased significantly after LPS stimulation, proving that the model was successfully established.

[0087] The addition of 0.03% N-acetyl-L-cysteine ​​as a positive control significantly reduced the MDA content induced by LPS, with a reduction effect of up to 46%. The addition of 0.003% of the sample from Example 1 significantly reduced the MDA content induced by LPS, with a reduction effect of up to 21%.

[0088] This demonstrates that the sample in the example can effectively reduce MDA content at an effective concentration of 0.003%. It also demonstrates the ability to improve lipid peroxidation.

[0089] (b) LPO content detection results:

[0090] Compared with the normal control group, ### p <0.001; compared with the model control group, ** p <0.01, ***p <0.001 The results showed that, according to the model control group analysis, the LPO content increased significantly after LPS stimulation, proving that the model was successfully established.

[0091] The addition of 0.03% N-acetyl-L-cysteine ​​as a positive control significantly reduced the LPO content induced by LPS, with a reduction effect of up to 41%. The addition of 0.003% of the sample from Example 1 significantly reduced the LPO content induced by LPS, with a reduction effect of up to 24%.

[0092] This demonstrates that the sample in the example can effectively reduce LPO content at an effective concentration of 0.003%. It also demonstrates the ability to improve lipid peroxidation.

[0093] Experiment 6: In vitro inflammatory mediator inhibition efficacy 1. Experimental Methods: (1) Cytotoxicity of mouse macrophages (RAW264.7): MTT assay Experimental steps: Cells were seeded in 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well contains 1 cell / well) and incubated at 37°C in 5% CO2 for 24 hours.

[0094] Drug administration: The sample group was replaced with the corresponding concentration of culture medium containing the sample, the normal control group was replaced with fresh culture medium, and the blank control group was replaced with blank culture medium. Three biological replicates were set up, and the culture was incubated at 37℃ and 5% CO2 for 24 h. After incubation, MTT solution was added to each well, and the culture was continued for 4 h. The culture medium was then discarded, and DMSO solution was added. After shaking and mixing, the absorbance value at 490 nm (OD) was measured. 490 ).

[0095] Cell viability (%) = (OD) 490 Sample group - OD 490 Blank control group) / (OD 490 Normal control group - OD 490 (Blank control group) × 100%. The test results are shown in Table 6-2.

[0096] (2) Detection of inflammatory mediators: PGE2 detection (a) Experimental Groups: The experimental groupings are shown in Table 6-1 below:

[0097] (b) Experimental procedures: Mouse mononuclear macrophages were inoculated into 6-well plates (3 × 10⁻⁶ cells / well). 5 (each well) was incubated at 37 °C with 5% CO2 for 24 h.

[0098] After incubation, the culture medium was discarded, and the samples were washed 1-2 times with D-Hanks. Fresh culture medium was added as shown in Table 6-1. The model (LPS), sample group (Example 1 + LPS), and positive control group (dexamethasone + LPS) were incubated at 37 °C and 5% CO2 for 24 h.

[0099] Collect the cell culture supernatant into a 1.5 mL sterile centrifuge tube and perform the test according to the instructions of the ELISA kit.

[0100] PEG2 inhibition rate (%) = (S 模型对照组 -S 样品组 )×100% / S 模型对照组 In the formula: S: PGE2 content 2. Experimental Results: (1) Analysis of RAW264.7 cytotoxicity results:

[0101] The RAW264.7 cytotoxicity assay results showed that the sample from Example 1 was not cytotoxic to the cells at a concentration of <0.00156%, therefore 0.001% was selected as the experimental concentration.

[0102] (2) Results of PGE2 content detection:

[0103] Compared with the normal control group, ### p <0.001; compared with the model control group, *** p <0.001 Inflammatory mediators can trigger inflammation following excessive lipid secretion and oxidation. The results of PGE2 inhibition rate assays showed that the LPS-induced model exhibited a significant improvement compared to the blank control group, demonstrating successful modeling.

[0104] The addition of 0.01% dexamethasone (positive control) significantly reduced PGE2 content, with an inhibition rate of 66%. The addition of 0.001% of the sample from Example 1 significantly reduced PGE2 content, with an inhibition rate of 48%.

[0105] This demonstrates that the sample in Example 1 has the effect of inhibiting the activity of inflammatory mediators.

Claims

1. An extract of Polygonum aviculare, characterized in that: It contains at least 50-62% flavonoids by weight percentage.

2. A method for preparing an extract of Polygonum aviculare, characterized in that, Includes the following steps: S1. Pre-treatment: The raw medicinal material of Polygonum aviculare is pulverized using a pulverizer and then passed through a 24-mesh sieve for later use; S2. Preparation of Polygonum multiflorum extract: Weigh Polygonum multiflorum powder and add it to the extraction system. Stir and mix well, then place the extract in an ultrasonic extractor to obtain Polygonum multiflorum extract. S3. Preparation of Polygonum multiflorum filtrate: After removing the residue by filtration using a Buchner funnel, the upper extract is obtained by separation using a funnel. The upper extract was concentrated under reduced pressure using a rotary evaporator to a paste-like consistency; Add ethanol solution, disperse evenly, centrifuge, take the supernatant and pass it through MCE membrane to obtain the filtrate of Polygonum hydropiper; S4. Extract purification: The filtrate of Polygonum multiflorum was subjected to resin adsorption at a certain flow rate. After adsorption, the resin column bed was washed with deionized water in the forward direction. The adsorbed product was desorbed by passing the column through ethanol in the forward direction to obtain Polygonum multiflorum eluent. S5. Extract concentration and drying: The eluent of Polygonum multiflorum is concentrated using a rotary evaporator and then dried using a vacuum drying device. After drying, the sample is ground to obtain Polygonum multiflorum extract powder.

3. The method for preparing a Polygonum aviculare extract according to claim 2, characterized in that, The preparation of Polygonum aviculare extract in step S2 is detailed below: S2.1 Establishing the extraction system: Prepare an ethanol solution with a concentration of 40-70%, add ammonium sulfate solid with a mass fraction of 16-24%, stir and let stand to separate the layers to obtain an aqueous two-phase extraction system; S2.2 Preparation of Polygonum multiflorum extract: Weigh Polygonum multiflorum powder and add it to the aqueous two-phase extraction system. The material-to-liquid ratio is 1:20-40. Stir for 20 minutes to mix thoroughly. Place the extract in an ultrasonic extractor and set the ultrasonic frequency to 260-400w, the ultrasonic time to 40-70min, and the ultrasonic temperature to 40℃ to obtain Polygonum multiflorum extract.

4. The method for preparing a Polygonum hydropiper extract according to claim 2, characterized in that, The preparation of the filtrate by Cyperus rotundus in step S3 is detailed below: S3.1 Place the extract of Polygonum aviculare in a Buchner funnel for vacuum filtration to remove the residue, and use a funnel to separate the layers of the permeate and take the upper layer of extract. S3.

2. The upper extract was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotary evaporation conditions were set as follows: 40-80 rpm rotation speed in the eggplant flask, 80 mbar vacuum pressure, and 50℃ water bath temperature. S3.3 Add 15% (v / v) ethanol solution at a ratio of 1:5 of crude drug amount, disperse evenly, and centrifuge for 20-40 min at a speed of 8000-11000×g and a temperature of 25℃. Collect the supernatant. S3.

4. Pass the supernatant through a 0.22μm MCE membrane to obtain the filtrate of Cyperus rotundus.

5. The method for preparing an extract of Polygonum aviculare according to claim 2, characterized in that, The extraction of the extract described in step S4 is as follows: S4.1 Resin Adsorption: The filtrate of Polygonum multiflorum was packed with filler at a ratio of raw drug to column volume of 1:1.5; the filtrate was loaded into the macroporous adsorption resin by forward loading, with an adsorption flow rate of 3.5-4.5 BV / h, and the content of the target product at the outlet was detected every 1 BV to determine the adsorption state. S4.2 Water washing to remove impurities: After the resin adsorption is completed, the resin column bed is washed with deionized water in the forward direction at a flow rate of 2.5-3.5 BV / h for 2-3 hours to remove residual liquid and some water-soluble impurities in the column bed; Brix is ​​used to detect the Brix detection amount at each 1 BV outlet liquid until the Brix detection amount is ≤0.1%, and the water washing is completed. S4.3 Resin Desorption: The resin was desorbed by passing 4-6 BV of 60% ethanol through the column in a forward direction at a flow rate of 1.5-2.5 BV / h. The content of the target substance in the effluent was measured every 1 BV to determine the desorption state of the resin. Brix ≤ 0.1 at the effluent was regarded as the desorption endpoint. The resulting liquids were combined and recorded as the eluent.

6. The method for preparing an extract of Polygonum aviculare according to claim 2, characterized in that, The extraction and drying process described in step S5 is as follows: S5.1 Concentration: The eluent of Polygonum aviculare was concentrated to a paste state under reduced pressure using a rotary evaporator. The rotation speed of the flask was set to 40-60 rpm, the vacuum pressure to 80 mbar, and the water bath temperature to 50℃. S5.2 Drying: Use a vacuum drying device to dry the sample, setting the drying vacuum degree to 40-60mb, the drying temperature to 37℃, and the drying time to 10-20h, to obtain a dried sample of Polygonum multiflorum extract; grind the dried sample of Polygonum multiflorum extract to obtain Polygonum multiflorum extract powder.

7. The use of the Polygonum hydropiper extract according to any one of claims 1-6 as an active ingredient in the preparation of cosmetics with oil-controlling effects.

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