Alcohol extract of Gynura Root and its applications

Abstract

The present invention discloses an ethanol extract of Gynura Root and its application. The ethanol extract of Gynura Root is prepared by using an ethanol solution with a volume fraction of 10-100% as an extraction solvent. The extracts of different polar parts of the extract have a certain degree of inhibitory effect on Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes, and can be used as raw materials for anti-aging and anti-acne cosmetics. It is expected to explore the use value of Gynura Root and provide ideas and theoretical basis for the research and promotion of functional traditional Chinese medicine cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine, and particularly relates to an alcohol extract of chrysanthemum notoginseng and application thereof. Background Art

[0002] Chrysanthemum notoginseng of the genus Chrysanthemum in the Asteraceae family ( Gynura japonica (Thunb.) Juel.) is also known as Tusanqi, Sanqi grass, Sanxuecao, and Juyesanqi. It is a tall perennial herb. Jusanqi is a traditional Chinese herbal medicine used by the Chinese people and has been recorded in many documents. It was first recorded in the "Yunnan Materia Medica", which records that Jusanqi "can be used raw to break blood, roasted to replenish blood, and can be used to stop bleeding, disperse blood, arrow wounds, cane blows, and injuries from falls. Applying the affected area can cure it." Its roots or whole plants are used as medicine, which has the effects of breaking blood and dispersing blood stasis, stopping bleeding, detoxifying and reducing swelling. It is used to treat injuries from falls, hematemesis, epistaxis, blood in the stool, metrorrhagia, sores, carbuncles, and swellings. Modern pharmacological studies have shown that Jusanqi has anti-tumor, antioxidant, hemostatic, anti-malarial, anti-inflammatory, analgesic, and local anesthetic effects. However, Chrysanthemum notoginseng contains a large amount of pyrrolizidine alkaloids, and excessive use can cause serious liver toxicity. There have been studies at home and abroad on Chrysanthemum notoginseng-induced hepatic sinusoidal obstruction syndrome (HSOS), and there are also related studies reporting that the aerial parts of Chrysanthemum notoginseng can also cause liver damage, but there are currently no reports on the antibacterial, anti-acne, and anti-aging effects of Chrysanthemum notoginseng.

[0003] Therefore, it is necessary to study the efficacy of Chrysanthemum notoginseng extracts, which is conducive to expanding the use value of Chrysanthemum notoginseng and providing ideas and theoretical basis for the research and development and promotion of functional Chrysanthemum notoginseng products. Summary of the invention

[0004] In response to the problems existing in the prior art, the present invention mainly studies the antibacterial activity, anti-inflammatory, anti-acne, anti-aging and other cosmetic-related effects of different effective parts of the whole herb of Chrysanthemum Notoginseng, determines its safe and effective action sites, and provides a certain foundation for the subsequent development and application research of Chrysanthemum Notoginseng functional ingredients.

[0005] This is achieved specifically through the following technical solutions:

[0006] The dry powder of Chrysanthemum notoginseng is extracted with 10-100% ethanol solution as an extractant, the extracts are collected and combined, and the ethanol is concentrated to obtain an extract, which is the Chrysanthemum notoginseng alcohol extract.

[0007] Specifically, take the dry powder of Gynura Root, and extract it by ultrasonic extraction with 10 - 100% ethanol. After filtering the extract through a Buchner funnel, pour it into a round-bottom flask and place it on a rotary evaporator for rotary evaporation to obtain the ethanol extract paste of Gynura Root. Separate the paste with different polar solvents such as petroleum ether, dichloromethane, ethyl acetate, n-butanol and chromatographic columns such as silica gel column and macroporous resin to obtain the extracts of different effective parts. Place the obtained paste in a vacuum drying oven for drying, seal it and store it in a desiccator for later use.

[0008] Preferably, the dry powder of Gynura Root is the dry powder of the above-ground part of Gynura Root.

[0009] Preferably, the dry powder of Gynura Root is a powder with a particle size less than 50 mesh.

[0010] Preferably, the ethanol solution is added at a material-liquid ratio of 1:8.

[0011] Preferably, the extraction is carried out under ultrasonic conditions for 2 hours.

[0012] Application of the ethanol extract of Gynura Root in the preparation of a bacteriostatic agent against Staphylococcus aureus.

[0013] Application of the ethanol extract of Gynura Root in the preparation of a bacteriostatic agent against Staphylococcus epidermidis.

[0014] Application of the ethanol extract of Gynura Root in the preparation of a bacteriostatic agent against Propionibacterium acnes.

[0015] Application of the ethanol extract of Gynura Root in the preparation of raw materials for anti-acne cosmetics.

[0016] Application of the ethanol extract of Gynura Root in the preparation of anti-aging cosmetics.

[0017] In the present invention, Gynura Root is extracted with alcohol, and different polar extraction parts of the extract can inhibit Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes. For the first time, the efficacy of Gynura Root in cosmetics is studied, and it is found that it has cosmetic effects such as anti-acne and anti-aging, providing ideas and theoretical basis for the research and promotion of functional traditional Chinese medicine cosmetics. Description of the Drawings

[0018] Figure 1 Shows the bacteriostatic effect of the petroleum ether fraction against Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes;

[0019] (Wherein, Ⅰ Staphylococcus aureus, Ⅱ Staphylococcus epidermidis, Ⅲ Propionibacterium acnes, A 1‰ DMSO (blank control group); B petroleum ether fraction). Detailed Embodiments

[0020] The following specific technical examples further describe the present invention in detail for better understanding the technical solution.

[0021] Pretreatment before extraction of Gynura Root:

[0022] The dry aerial parts of Gynura Root are pulverized into dry powder, and after pulverization, it is sieved through a 50-mesh sieve and reserved.

[0023] Preparation of Gynura Root extracts with different polarities

[0024] Take 100 g of the dry powder of Gynura Root, ultrasonically extract it with 75% ethanol at a material-liquid ratio of 1:8 (m / v) for 2 h. After filtering the extract through a Buchner funnel, pour it into a round-bottom flask and rotate and evaporate it on a rotary evaporator to obtain the ethanol extract paste of Gynura Root. Disperse the paste at a ratio of 1:10 (m / v) in pure water, and then extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at a ratio of 1:10 (m / v) respectively until the extract is nearly colorless and transparent. Combine the extracts to obtain the petroleum ether extraction part, dichloromethane extraction part, ethyl acetate extraction part, n-butanol extraction part, and water part. After concentration under reduced pressure, obtain the extracts of different polar parts. Place the obtained extracts in a vacuum drying oven for drying, seal and store them in a desiccator for later use.

[0025] Antibacterial experiment

[0026] Treatment of the test substance: Preparation of the test solution: Accurately weigh 100.0 mg of the petroleum ether phase extract paste, dichloromethane extract paste, ethyl acetate extract paste, n-butanol extract paste, water phase extract paste, and ethanol extract paste of Gynura Root respectively, place them in a 10 mL volumetric flask, dissolve them with an appropriate amount of DMSO, and then dilute them to 10.0 mg / mL with pure water (DMSO does not exceed 1‰). Filter the prepared test solution with a concentration of 10 mg / mL through a 0.22 μm sterile filter for sterilization and reserve.

[0027] Preparation of the standard solution: Accurately weigh 100.0 mg of chlorogenic acid standard product, place it in a 10 mL volumetric flask, dissolve it with an appropriate amount of DMSO, and then dilute it to 10.0 mg / mL with pure water (DMSO does not exceed 1‰). Filter the prepared standard solution with a concentration of 10 mg / mL through a 0.22 μm sterile filter for sterilization and reserve.

[0028] Preparation of the blank control: Take an appropriate amount of DMSO and place it in a 10 mL volumetric flask, dilute it to 10.0 mg / mL with pure water (DMSO does not exceed 1‰). Filter the prepared blank control solution through a 0.22 μm sterile filter for sterilization and reserve.

[0029] Preparation of the bacterial suspension

[0030] Staphylococcus aureus bacterial suspension: Streak-culture Staphylococcus aureus on the slant of Staphylococcus aureus medium, incubate it in a constant temperature incubator at 37°C for 24 hours, then use an inoculation loop to pick up an appropriate amount of activated Staphylococcus aureus cells and put them into an EP tube containing 1.5 mL of sterile physiological saline. Shake well (when viewed with the naked eye, it has a frosted glass appearance, similar to a 0.5 McFarland turbidity tube), and set aside for use.

[0031] Staphylococcus epidermidis bacterial suspension: Streak-culture Staphylococcus epidermidis on the slant of Staphylococcus epidermidis medium, incubate it in a constant temperature incubator at 37°C for 24 hours, then use an inoculation loop to pick up an appropriate amount of activated Staphylococcus epidermidis cells and put them into an EP tube containing 1.5 mL of sterile physiological saline. Shake well (when viewed with the naked eye, it has a frosted glass appearance, similar to a 0.5 McFarland turbidity tube), and set aside for use.

[0032] Propionibacterium acnes bacterial suspension: In a sterile environment in a laminar flow hood, pipette 1 mL of sterile water to dissolve the freeze-dried powder of Propionibacterium acnes. Wait until it is completely dissolved, then evenly spread it on the solid medium. Then place it in an anaerobic box and incubate it in a constant temperature incubator at 37°C for 3 days. Use an inoculation loop to scrape a little of the activated strain on the already cultured medium and inoculate it onto a new solid medium using the streak plate method. Place it in an anaerobic box and incubate it in a constant temperature incubator at 37°C for 48 hours to obtain the test strain of Propionibacterium acnes. In a sterile environment in a laminar flow hood, use an inoculation loop to scrape a small amount of bacteria and add it to a sterilized test tube containing an appropriate amount of sterile physiological saline and mix well. Adjust the bacterial suspension concentration to 1*10 8 CFU / mL to obtain the bacterial suspension.

[0033] Inhibition zone experiment

[0034] The Oxford cup method was used to measure the diameter of the inhibition zone of the test solution against Staphylococcus aureus and Staphylococcus epidermidis.

[0035] Staphylococcus aureus: Add 1 mL of Staphylococcus aureus bacterial suspension to a sterilized medium (100 mL) at 40 - 50°C and shake well. Pour about 20 mL of the solid medium mixed with the bacterial suspension into a sterilized petri dish. After the medium cools and solidifies, place 4 sterile Oxford cups on the medium, ensuring an appropriate distance between the Oxford cups. Add 200 μL of the blank control and the test sample solution or the standard solution into the cups respectively. Place it in a constant temperature incubator at 37°C in an anaerobic environment and incubate for 24 hours. After 24 hours, take it out, measure the diameter of the inhibition zone with a vernier caliper, conduct 3 parallel tests, and record the data.

[0036] Staphylococcus epidermidis: Add 1 mL of Staphylococcus epidermidis suspension to a sterilized culture medium (counting 100 mL) at 40 - 50 °C and shake well. Pour about 20 mL of the solid culture medium mixed with the suspension into a sterilized Petri dish. After the culture medium cools and solidifies, place 4 sterile Oxford cups on the culture medium at appropriate intervals. Add 200 μL of blank control and test sample solution or standard solution into the cups respectively. Place it in an anaerobic environment in a 37 °C constant temperature incubator for 24 h. Take it out after 24 h, measure the diameter of the inhibition zone with a vernier caliper, conduct 3 parallel tests, and record the data.

[0037] Propionibacterium acnes: The Oxford cup inhibition zone method was used to evaluate the antibacterial effect of the effective part of Gynura Root-Tuber on Propionibacterium acnes. Pour about 20 mL of sterilized solid culture medium of Propionibacterium acnes into a sterile Petri dish. When the culture medium cools to 40 °C, aspirate 200 μL of the bacterial suspension and add it to the culture medium, mix well. Wait for the culture medium to stand and solidify, and place sterile Oxford cups at equal intervals on the culture medium. Add 200 μL of the sample into each Oxford cup, and repeat each sample 3 times. Use the sample solvent as the blank control and culture it in a 37 °C constant temperature incubator for 48 h. Measure the diameter of the inhibition zone of each sample with a vernier caliper and take the average value.

[0038] Anti-acne effect: Using Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes as indicator bacteria, the Oxford cup method was used to conduct antibacterial and anti-acne effect experiments on different extraction parts of Gynura Root-Tuber. The results showed that the inhibition zones of the effective part of Gynura Root-Tuber against Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes were respectively greater than 15.6 ± 2.0 mm, 13.54 ± 0.49 mm, and 9.84 ± 0.61 mm, as Figure 1 。

[0039] Study on the Anti-aging Effect of Gynura Root-Tuber Extract

[0040] DPPH determination of free radical scavenging rate: Dissolve the extract with absolute ethanol and add it to 20 mL to obtain a sample solution. Then transfer 1 mL from the sample solution and make up the volume to 50 mL to obtain a test solution. On a 96-well microplate, add 100 μL of the test solution at different concentrations and 100 μL of VC solution (8 mg / mL) at different concentrations to the drug administration group. Then add 100 μL of 0.06 mmol / L DPPH ethanol solution, mix well, react in the dark at room temperature for 30 min, and measure the absorbance at a wavelength of 517 nm. Repeat three times in parallel, calculate the scavenging rate, and finally obtain an IC50 less than 120 mg / mL.

[0041] ABTS determination of free radical scavenging rate: Mix 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution evenly in a 1:1 ratio, and let it stand overnight in the dark at room temperature to prepare the ABTS stock solution. On the 96-well microplate, add 100 μL of the test solution at different concentrations and 100 μL of VC solution at different concentrations (8 mg / mL) to the drug administration group. Then add 100 μL of the ABTS stock solution, mix well, and react in the dark at room temperature for 10 min. Measure the absorbance at a wavelength of 734 nm. There are 3 replicate wells in each group. Calculate the scavenging rate, and finally obtain an IC50 less than 14 mg / mL.

Claims

1. Use of the alcohol extract of Gynura Root, characterized in that, The use refers to the application of the alcohol extract of Gynura Root in the preparation of antibacterial agents against Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, raw materials for anti-acne cosmetics or anti-aging cosmetics. The alcohol extract of Gynura Root is prepared by the following method: The dried powder of Gynura Root is extracted with an ethanol solution of 10-100% as the extractant, the extraction solutions are collected and combined, the ethanol is concentrated and removed to obtain an extract, the extract is dispersed in pure water at a ratio of 1:10 m / v, and then extracted with petroleum ether at a ratio of 1:10 m / v respectively to obtain a petroleum ether extract. The petroleum ether extract is concentrated under reduced pressure and then dried in a vacuum drying oven to obtain the alcohol extract of Gynura Root; the dried powder of Gynura Root is the dried powder of the above-ground part of Gynura Root; the Latin name of Gynura Root is: Gynura japonica (Thunb.) Juel. .

2. Use of the Gynura Root Extract according to claim 1, characterized in that The dry powder of Gynura Root is a powder with a particle size less than 50 mesh.

3. Use of the Gynura Root Extract according to claim 1, characterized in that The ethanol solution is added at a material-liquid ratio of 1:8 m / v.

4. Use of the Gynura Root Alcohol Extract according to claim 1, characterized in that The extraction is carried out under ultrasonic conditions for 2 hours.

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