TWI772986B - Use of mosla chinensis maxim. extract for improving skin appearance - Google Patents

Abstract

A use ofMosla chinensis Maxim. extract for improving skin appearance. In particular, theMosla chinensis Maxim. extract is obtained with an aqueous solvent.

Description

Uses of Lemongrass for skin conditioning

The present invention relates to the use of a citronella violacea extract, in particular to the use of the citronella violacea extract for preparing and improving skin. Among them, the improvement of the skin includes the improvement of skin antioxidant capacity, the promotion of anti-glycation capacity, the promotion of skin firmness, the reduction of skin redness and the tightening of skin pores.

Citronella purpurea (scientific name: Mosla chinensis Maxim. ) is also known as Shixiangyuan, Xiangyu, and Chitou lily, a species under the genus Limbicaceae of the Lamiaceae family. Lemongrass violacea is an annual herb, mainly distributed in Asia. Its stems are 9-4 cm high and the corolla is purple. It can be used to treat heat stroke, fever, stomach pain and vomiting, acute gastroenteritis and indigestion.

Skin aging can be divided into endogenous and exogenous. Up to 70% are exogenous aging, and the main cause is photoaging caused by ultraviolet rays; and 30% are endogenous aging. Of the 30%, only 10% are due to natural aging of the human body, while the other 20% are saccharified and oxidized by internal or external factors, resulting in skin damage and aging.

Excessive intake of carbohydrates in daily life is the main cause of glycation in the body. After these excess carbohydrates are combined with proteins in the body, they will cause a glycation reaction under the heating of body temperature, thereby producing glycated proteins and advanced glycation end products (AGEs). AGEs will accumulate in the body, causing oxidative stress, leading to chronic inflammation in the skin, and the collagen structure in the skin will also be damaged and broken, and aging problems such as wrinkles and sagging will also occur.

Therefore, in order to solve skin aging and improve skin condition, and to expand the development and application of Lemongrass serrata, a Lemongrass serrata extract and a skin conditioning composition are proposed, which can be used to improve the skin's antioxidant capacity and resist glycation. Use of a composition for enhancing power, enhancing skin firmness, reducing redness and tightening skin pores.

In some embodiments, Citronella nigra extract can inhibit the formation of Adavanced Glycation Endproducts (AGEs). In some embodiments, lemongrass extract can enhance the anti-glycation ability of the skin.

In some embodiments, lemongrass extract can maintain skin firming effect by enhancing the anti-glycation ability of the skin. In some embodiments, lemongrass extract can slow or prevent sagging skin.

In some embodiments, the lemongrass extract can maintain skin firmness, and wherein the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, the lemongrass extract can scavenge free radicals in cells, wherein the lemongrass extract is extracted with an aqueous solvent. In some embodiments, the cells are skin cells.

In some embodiments, the Lemongrass violetgrass extract can reduce the damage of free radicals to skin cells, so as to achieve the purpose of enhancing the antioxidant capacity of the skin.

In some embodiments, Citronella nigra extract can inhibit, slow or reduce the formation of free radicals or peroxides in cells.

In some embodiments, the lemongrass extract can tighten skin pores, and wherein the lemongrass extract is extracted with an aqueous aqueous solvent.

In some embodiments, the concentration of Citronella nigra extract is 0.5 wt%.

In some embodiments, the Citronella violacea extract has a Degrees Brix value of 10.0 or more.

In some embodiments, the total flavonoid content of the lemongrass extract is above 12,000 ppm.

In some embodiments, the use of a lemongrass extract for preparing a composition for improving or/and conditioning skin condition, wherein the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, the use of a lemongrass extract in the preparation of a composition for reducing redness of the skin, wherein the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, the use of a lemongrass extract in the preparation of a composition for soothing skin redness, wherein the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, the use of a lemongrass extract for preparing a composition for tightening skin pores, wherein the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, the use of a lemongrass extract for preparing a composition for firming skin, slowing skin, or preventing skin sagging, wherein the lemongrass extract is extracted with an aqueous solvent.

Some specific implementation aspects of this case will be described below. Without departing from the spirit of this case, this case can still be practiced in many different forms, and the scope of protection should not be limited to the conditions specifically stated in the description.

The case used Excel software for statistical analysis. Data are presented as mean ± standard deviation (SD), and differences between groups were analyzed by Student's t-test. In the graph, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference.

The numerical values used herein are approximate, all experimental data are expressed within the range of plus or minus 10%, and the optimum is within the range of plus or minus 5%.

Herein, "wt%" refers to weight percent, and "vol%" refers to volume percent.

As used herein, the term "extract" refers to a product prepared by extraction. The extract may be presented as a solution in a solvent, or the extract may be presented as a concentrate or essence free or substantially free of solvent.

As used herein, "citronella raw material" generally refers to a whole plant, wherein the whole plant may comprise the original, dried or otherwise physically processed whole plant to facilitate handling, which may further comprise whole, minced, chopped Whole plants that have been diced, milled, ground or otherwise processed to affect the size and physical integrity of the raw material.

Refer to Figure 1. In some embodiments, a lemongrass extract is extracted from a lemongrass raw material by an aqueous solvent. In some embodiments, the Citronella chinensis extract ( Mosla chinensis Maxim. extract) is obtained by sequentially performing the crushing procedure S01 , the heating procedure S02 , the filtering procedure S03 , and the concentration procedure S04 on the Citronella chinensis raw material.

In some embodiments, the lemongrass raw material can be a whole plant. In some embodiments, the lemongrass raw material can be fresh or dried whole lemongrass.

In some embodiments, the pulverizing procedure S01 refers to whipping the raw material of Lemongrass violacea until it is split into pulverized shapes. For example, comminution can be performed using a juicer, conditioner or homogenizer.

In some embodiments, the heating procedure S02 refers to mixing the pulverized lemongrass raw material with a water-containing solvent and heating for a fixed period of time. In some embodiments, heating refers to raising the temperature of the lemongrass raw material and the water-containing solvent to 50°C to 100°C. In some embodiments, a fixed period of time refers to 0.5 hours to 3 hours. For example, the temperature of lemongrass raw material and the aqueous solvent was raised to 85°C for extraction for 1 hour.

In some embodiments, the weight ratio (the solvent containing water: the raw material of Lemongrass violacea) in the heating procedure S02 is 5:1 to 20:1. For example, the aqueous solvent: citronella raw material is 10:1.

For example, 90 kilograms of lemongrass raw material, 900 kilograms of water and 0.63 kilograms of citric acid are used to mix and continue to heat to 100°C, and maintain the temperature at 100°C for 0.5 hours. For example, 90 kg of lemongrass raw material, 900 kg of water and 0.7 kg of citric acid are used to mix and then continue to heat to 85°C and maintain at 85°C for 1 hour.

In some embodiments, the filtering procedure S03 refers to passing the lemongrass raw material and the solvent after the heating procedure S02 through a screen to filter out the solids in the solvent to form a filtrate. For example, the screen may be a 400 mesh screen.

In some embodiments, a cooling process is further included between the heating process S02 and the filtering process S03, wherein the cooling process refers to allowing the heated lemongrass raw material and the solvent to stand to naturally cool down to room temperature.

In some embodiments, the concentration procedure S04 means that the filtrate obtained in the filtration procedure S03 is concentrated under reduced pressure (brand/model: BUCHI-Rotavapor R-100) to obtain a primary extract. In some embodiments, the primary extract obtained in the concentration procedure S04 may be the Lemongrass violacea extract. In some embodiments of the concentration procedure S04, the concentration under reduced pressure is performed between 40°C and 70°C. For example, the Citronella violacea extract is obtained by sequentially performing the crushing procedure S01 , the heating procedure S02 , the filtration procedure S03 and the concentration procedure S04 on the Citronella violacea raw material.

In some embodiments, the Lemongrass serrata extract is used for the preparation of an anti-glycation composition, and the Lemongrass serrata extract is extracted with an aqueous solvent.

In some embodiments, the lemongrass extract is used to prepare an antioxidant composition, and the lemongrass extract is extracted with an aqueous solvent.

In some embodiments, antioxidant refers to slowing or preventing oxidation. The oxidation system is a chemical reaction in which electrons are transferred from substances to oxidants, and free radicals can be generated in the process, which in turn initiates a chain reaction. When a chain reaction occurs in a cell, the cell is susceptible to destruction or apoptosis. In some embodiments, Citronella nigra extract can remove free radicals, terminate chain reactions and inhibit other oxidative reactions. In some embodiments, free radicals are generated due to light, chemicals, or the natural aging process of an organism. In some embodiments, the aforementioned chemicals comprise end products of glycation.

As mentioned earlier, free radicals such as Superoxide Anion and Hydrogen Peroxide can cause damage to skin cells. Among them, Citronella violacea extract can reduce the removal of free radicals, for example, it can promote the expression of antioxidant-related genes, increase the protein (superoxide dismutase) transcribed by it, and then increase the decomposition of superoxide into excessive amount of hydrogen oxide.

In some embodiments, more than 0.125mg/ml of Lemongrass violacea extract can achieve antioxidant effect by scavenging free radicals in cells.

In some embodiments, the Lemongrass japonica extract with a Brix value above 10.0 can achieve antioxidant effect by scavenging free radicals in cells.

In some embodiments, lemongrass extract at a concentration of 0.5 wt% or more can be used to reduce skin redness. In some embodiments, the skin redness is due to skin inflammation. In some embodiments, lemongrass extract at a concentration of 0.5 wt% or more can be used to soothe reddened skin.

In some embodiments, Lemongrass serrata extract with a Degrees Brix value above 10.0 may be used to soothe and/or reduce redness of the skin.

Hyaluronic acid (HA) exists in the connective tissue and dermis of the human body. It is a transparent jelly-like substance responsible for binding and retaining water molecules in the skin. The hyaluronic acid in the skin accounts for more than 50% of the human body's hyaluronic acid, and it is the main component of the extracellular matrix, which can maintain the elasticity of the skin itself. With the increase of age, the hyaluronic acid in the human body will gradually lose, and the hyaluronic acid content in the skin will remain less than 60% at the age of 30. This results in voids in the skin, which gradually lose elasticity and age.

In some embodiments, Lemongrass serrata extract at a concentration of 0.5 wt% or more can be used to improve skin laxity.

In some embodiments, lemongrass extract at a concentration of 0.5 wt% or more can be used to enhance skin firmness.

In some embodiments, Lemongrass violacea extract at a concentration of 0.5 wt% or more can be used to tighten skin pores.

In some embodiments, the aforementioned compositions may be pharmaceuticals. In other words, the medicinal product contains an effective content of Lemongrass violacea extract. In other words, the medicinal product contains an effective content of Lemongrass violacea extract.

In some embodiments, the aforementioned pharmaceutical products can be manufactured into a dosage form suitable for parenteral or oral administration using techniques well known to those skilled in the art. These administration forms include, but are not limited to: tablet, troche, lozenge, pill, capsule, dispersible powder or fine granules ( granule), solution, suspension, emulsion, syrup, elixir, slurry and the like.

In some embodiments, the aforementioned pharmaceutical products can be manufactured into a dosage form suitable for parenterally or topically administration using techniques well known to those skilled in the art, including , but not limited to: injection, sterile powder, external preparation and the like. In some embodiments, the medicinal product may be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, Intradermal injection and intralesional injection.

In some embodiments, the medicinal product may further comprise a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing techniques. For example, a pharmaceutically acceptable carrier may contain one or more of the following agents: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent Disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

In some embodiments, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS) , An aqueous solution containing alcohol.

In some embodiments, the aforementioned compositions may be edible compositions. In some embodiments, the edible composition can be made into a food product or can be a food additive, meaning that it is added to the food product by conventional methods during the preparation of the food product, or can be added to the food product added during the production process. Here, the food product may be a product formulated with an edible material for ingestion by humans or animals.

In some embodiments, food products may be, but are not limited to, beverages, fermented foods, bakery products, health foods, and dietary supplements.

In some embodiments, the aforementioned composition may be a skin care composition. In some embodiments, the skin care composition can be, but is not limited to, a lotion, gel, serum, lotion, cream, face or bath product, sunscreen product, whitening product, balm, ointment, mask.

Example 1: Preparation of the present invention - Preparation of Lemongrass Citronella Extract

℃下萃取30分鐘。於此，萃取可以是將紫花香茅原料浸泡在水中。後續，降溫程序將粉碎後的紫花香茅原料與水降溫到室溫（25℃）。此後，過濾程序將降溫後的紫花香茅原料及水通過400網目的篩網以形成過濾液。接著，於60℃下對過濾液進行減壓濃縮而得本發明之紫花香茅萃取物。In the crushing procedure, the whole plant of Citronella violacea is used as the raw material of Citronella nigra and crushed. Here, pulverization was performed using a 10 speed blender of the osterizer brand. Next, use water as the solvent in the heating procedure, mix the pulverized lemongrass raw material and water at a weight ratio of 1:15, and then mix them at 85

Extraction at °C for 30 minutes. Here, the extraction may be soaking the raw material of Lemongrass violacea in water. Subsequently, the cooling procedure cools the pulverized lemongrass raw material and water to room temperature (25°C). After that, the filtration procedure passed the cooled raw material of Lemongrass violacea and water through a 400-mesh sieve to form a filtrate. Next, the filtrate was concentrated under reduced pressure at 60° C. to obtain the Lemongrass violacea extract of the present invention.

Example 2: Quantitative experiment - Quantitative Experiment of Total Flavonoids in Citronella violacea Extract

Dilute the Lemongrass violacea extract obtained in Example 1 by 10 times its volume with water, and take 200 μL into a test tube. Add 1000 μL of water to this test tube and mix it evenly, then add 200 μL of 5 wt% sodium nitrite (purchased from Sigma, product number 31443) aqueous solution, mix well and let stand for 6 minutes.

Next, 200 μL of 10 wt% aqueous solution of aluminum nitrate (purchased from Alfa Aesar, product number 12360) was added, and the mixture was evenly mixed and allowed to react for 6 minutes, and then 2 mL of 4 wt% aqueous sodium hydroxide solution (sodium hydroxide was purchased from Macron, product No. 7708-10) and mix well. Finally, 1.4 mL of water was added to the test tube and mixed evenly to obtain a reaction solution. 200 μL of the reaction solution in the test tube was taken into a 96-well reaction plate, and detected with a spectrophotometer (Jasco, model V-730) at a wavelength of 500 nm. absorbance value.

Standards are then formulated to make calibration lines. In this case, the equivalent of rutin (purchased from ChromaDex, commodity number ASB-00018440) was used as the expression of the relative content of total flavonoids. First prepare 200 μg/mL rutin methanol solution as rutin standard solution. 0 μL, 200 μL, 400 μL, 600 μL, 800 μL, 1000 μL and 1200 μL of the rutin standard solution were added to different test tubes, and then water was added to the test tubes to make the total volume in each test tube 1200 μL. 200 μL of 5 wt% sodium nitrite aqueous solution was added to this test tube, and the mixture was evenly mixed and allowed to react for 6 minutes. Next, 200 μL of 10 wt% aqueous solution of aluminum nitrate was added, mixed evenly, and then allowed to stand for reaction for 6 minutes, and then 2 mL of 4 wt% aqueous sodium hydroxide solution was added to make it evenly mixed. Finally, 1.4 mL of water was added to this test tube and mixed evenly to obtain a reaction solution. Take 200 μL of the reaction solution in the test tube to a 96-well reaction plate, and detect the absorbance value with a spectrophotometer at a wavelength of 500 nm. The absorbance values of six different concentrations of rutin solutions measured in the same way were plotted as calibration lines.

Next, the absorbance value of the diluted lemongrass extract was converted into the total flavonoid content using a calibration curve. Here, it can be calculated that the total flavonoid content of the lemongrass extract before dilution (ie, the extract obtained in Example 1) is 12,000 ppm.

It is known that flavonoids are not the active ingredients for improving skin condition or anti-oxidation described in this case, but since flavonoids are known to have other uses such as preventing cardiovascular diseases, the Lemongrass violacea extract obtained by the extraction method in this case has a high total content. The content of flavonoids, so the Citronella violacea extract in this case can have a variety of compound uses. In addition, in some embodiments, the total flavonoid content can also be used as a criterion for judging the concentration end point.

Example 3 : Test tube experiment - Anti-glycation test

When the protein undergoes glycation reaction, it will generate a variety of advanced glycation end products (AGEs), which are irreducible substances, and change and affect the normal function of the protein, resulting in the hardening of collagen, the production of spots, and the relaxation of the skin. Decreased functionality. Therefore, if the glycation of collagen can be inhibited, the breakage of the collagen chain can be prevented, the loss rate of collagen can be reduced, and the aging and relaxation of the skin can be delayed. In order to confirm whether the Lemongrass violacea extract has the effect of inhibiting the glycation of collagen, the amount of glycation of collagen was tested as follows.

Sample preparation: 1. Using the Lemongrass violacea extract prepared in the first example of the preoperative procedure, phosphate buffered saline (Phosphate-Buffered Saline, PBS, NaH ₂ PO ₃ (Honeywell, #04269) with a concentration of 200 mM was used. ), Na ₂ HPO ₄ (Sigma, #V900061) and water, with a pH of 7.4) as a solvent, to prepare a solution of Lemongrass violacea extract with a concentration of 10 mg/mL. 2. Prepare a collagen solution with a concentration of 60 mg/mL using the aforementioned phosphate buffered saline at a concentration of 200 mM and collagen powder (collagen from Rousselot, model #P2000HD). In addition, sodium azide was added to the collagen solution so that the collagen solution contained 0.06 wt % sodium azide. (Sodium azide was purchased from Sigma, #S2002) 3. Using the aforementioned concentration of 200 mM phosphate buffered saline and fructose (fructose purchased from Sigma, #F0127), prepare a solution of fructose at a concentration of 1.5M.

Collagen glycation test: 1. Mix 0.2 mL of the aforementioned Lemongrass extract solution with 0.2 mL of the aforementioned collagen solution and 0.2 mL of the aforementioned fructose solution to prepare a sample solution. 2. Mix 0.2 mL of deionized water with 0.2 mL of the aforementioned collagen solution and 0.2 mL of the aforementioned fructose solution to prepare a blank solution as a control group. 3. Use a spectrofluorometer (manufacturer/model: BioTek FLx 800) to measure the fluorescence intensity of the sample solution and blank solution before the collagen glycation reaction (excitation wavelength 360 nm, emission wavelength 460 nm). 4. The sample solution and blank solution were placed at 50°C for 24 hours, and each solution was subjected to collagen glycation reaction. 5. Measure the fluorescence intensity of the reacted sample solution and blank solution with a spectrofluorometer (excitation wavelength 360 nm, emission wavelength 460 nm). 6. Calculate the relative generation rate of protein glycation end products according to the following formula: [( _{After the fluorescence intensity of the sample reacts} - _{Before the} fluorescence intensity of the sample reacts)/( _{After the fluorescence intensity of the control group reacts} - _{Before the fluorescence intensity of the control group reacts} )]× The relative production rates of protein glycation end products for the 100% sample relative to the control group are shown in Figure 2.

As shown in FIG. 2 , after being treated with the lemongrass extract of the present invention, the generation rate of glycation end products decreased, indicating that the lemongrass extract of the present invention has the effect of inhibiting the production of glycation end products of collagen. Among them, the relative production rate of protein glycation end products in the sample solution was only 33% of the control group. It is shown that the citronella violacea extract of the present invention can indeed inhibit the glycation reaction of collagen, reduce the generation of end products of glycation, thereby reducing the loss of collagen in the body, and achieve the effect of slowing skin aging and skin function loss.

Example 4: Cell Experiment - inhibition ROS generate

Here, the fluorescent probe DCFH-DA was used in conjunction with flow cytometry to measure the changes in the content of reactive oxygen species in human dermal fibroblasts CCD-966sk treated with the Lemongrass violacea extract of the present invention.

Materials and Instruments

1. Cell line: human skin fibroblasts CCD-966sk, obtained from Biological Resources Conservation and Research Center (BCRC); Cat. 60153, hereinafter referred to as CCD-966sk cells.

2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). Among them, the basal medium is Eagle's minimum essential medium (Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319) additionally added components to make it contain 1 mM sodium pyruvate (sodium pyruvate, purchased from Gibco), 1.5 g/L sodium bicarbonate (sodium bicarbonate, purchased from Sigma, Cat. S5761-500G) and 0.1 mM non-essential amino acid solution (purchased from Gibco) were prepared.

3. Phosphate-buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028.

4. DCFH-DA solution: dissolve dichlorodihydrofluorescein diacetate (2,7-dichloro-dihydro-fluorescein diacetate, DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl dimethyl sulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 mg/ml solution of DCFH-DA.

5. Flow cytometry, BD Accuri C6 Plus

6. Hydrogen peroxide (H ₂ O ₂ ): purchased from Sigma-Aldrich, product number 95299-1L.

7. Trypsin (Trypsin-EDTA): 10X Trypsin-EDTA (purchased from Gibco) was diluted 10 times in 1X PBS solution.

8. Citronella japonica extract: The Citronella japonica extract used in this experiment was prepared in the way of Example 1.

Experimental procedure

The experiment will be divided into an experimental group, a blank control group (a group without the addition of the Lemongrass violacea extract of the present invention, nor treated with hydrogen peroxide), and a control group (without the addition of the Lemongrass violacea extract of the present invention, but after The hydrogen peroxide treatment group) was carried out in three groups, and each group was subjected to three repeated experiments:

1. Inoculate CCD-966sk cells in a 6-well culture dish containing 2 ml of medium per well at 1×10 ⁵ cells per well.

2. Incubate the plate in 5% CO ₂ at 37°C for 24 hours.

3. Remove the medium.

4. Add 2 mL of experimental medium to each well of the plate and incubate at 37°C for 1 hour.

The experimental medium of the experimental group was 2 mL of cell culture medium supplemented with 5 μL of the Citronella violaceum extract of the present invention obtained in Example 1 (that is, the volume percentage of the Citronella nigra extract of the present invention in the cell culture medium was 0.25%).

The experimental medium of the control group was a simple 2 mL cell culture medium (that is, without the Citronella violaceum extract of the present invention).

The experimental medium of the control group was a simple 2 mL cell culture medium (that is, without the Citronella japonica extract of the present invention).

5. Add 2 μL of DCFH-DA solution at a concentration of 5 μg/mL to the cell culture medium in each well, and let DCFH-DA treat the cells for 15 minutes.

6. After DCFH-DA treatment, add H ₂ O ₂ to the experimental medium of the experimental group and the experimental medium of the control group, respectively, and react at 37° C. for 1 hour. Specifically, 35% wt hydrogen peroxide was first diluted to 100 mM (10 μL of hydrogen peroxide was added to 990 μL of double distilled water), and 20 μL of 100 mM hydrogen peroxide was added to a 2 mL cell culture dish.

7. After the reaction, rinse each well twice with 1 mL of 1X PBS solution.

8. Add 200 μL trypsin to each well and react in the dark for 5 minutes. After the reaction, add 6 mL of cell culture medium to stop the reaction.

9. Collect the cells and cell culture medium in each well into a corresponding 1.5 mL centrifuge tube, and centrifuge the centrifuge tube containing the cells and culture medium at 400 x g for 10 minutes.

10. After centrifugation, remove the supernatant and re-lyse the cell pellet with 1X PBS solution.

11. Centrifuge again at 400 x g for 10 minutes.

12. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of 1X PBS solution in the dark to obtain the cell solution to be tested.

13. Use a flow cytometer to detect the fluorescent signal of DCFH-DA in the cell fluid to be tested in each well. The excitation wavelength for fluorescence detection is 450-490 nm, and the emission wavelength is 510-550 nm. After entering cells, DCFH-DA will be hydrolyzed to DCFH (dichlorodihydrofluorescein), and then oxidized by reactive oxygen species to DCF (dichloroluciferin) that emits green fluorescence, which is treated by DCFH-DA. The fluorescence intensity of the cells can reflect the content of reactive oxygen species in the cells, and thus the ratio of the number of cells with high expression of reactive oxygen species in the cells to the original number of cells can be known. Since the experiment was performed in duplicate, the measurement results of the duplicate experiments in each group were averaged to obtain an average value, and then the average value of the control group was taken as 1, the relative ROS generation amount, and the average value of the control group and the experimental group was calculated. Converted to the relative amount of ROS generated, as shown in Figure 3.

Experimental results

As shown in Figure 3, from the comparison of the results of the control group and the control group, it can be seen that the relative ROS generation (high fluorescence performance) will be greatly increased (about 5.37 times) after the hydrogen peroxide treatment, and it is statistically significant, and the p value is less than 0.001); it shows that hydrogen peroxide treatment does lead to the production of reactive oxidative substances in cells, which in turn causes subsequent damage to skin fibroblasts. On the other hand, according to the results of comparing the control group and the experimental group, when the cells were treated with the Lemongrass violacea extract of the present invention, the relative generation of ROS was significantly reduced by about 96% (statistically significant, p value less than 0.001 ), even lower than the control group; it shows that the Citronella violacea extract of the present invention can effectively reduce the production or accumulation of reactive oxidative substances in cells. In other words, the lemongrass extract of the present invention can be used as an active oxidizing substance scavenger. That is, the Citronella violaceum extract of the present invention can reduce the oxidative damage caused by the active oxidative substances in the cells by reducing the content of the active oxidative substances in the cells.

Example 5: Human trials - Topical Use of Lemongrass Extract

Here, subjects were asked to use the essence containing the Citronella violacea extract of the present invention to investigate the skin-improving effect of using the Citronella violacea extract of the present invention externally.

Samples used: Serum containing Citronella japonica extract and placebo serum (in which Citronella japonica extract was replaced with equal weight of water, and the rest of the ingredients were the same).

Essence containing citronella violaceum extract ingredients: Sanxianjiao 0.2wt%, Xinxian ketone 0.6wt%, 1,3-butanediol 5wt%, triethanolamine 0.1wt%, hexanediol 0.6wt%, purple flower fragrance The grass extract is 0.5wt%, the thickener U21 is 0.1wt%, and the rest is water. Among them, the Citronella violacea extract is obtained by the preparation method of Example 1.

Number of subjects: 8 subjects over 25-60 years old with loose skin or red skin.

Experimental way After the subjects cleaned the whole face, according to different test items, use the corresponding instruments and measurement methods to record the values of the left and right half of the face before using the essence, or take photos before use. Then, the subjects were asked to apply the essence containing citronella violacea extract and the placebo essence to the right half of the face and the left half of the face of the subjects every morning and evening after washing the face, and then use the finger pulp. Lightly massage the left and right half of the face. After using the above two serums in the morning and evening for two weeks, use the corresponding instrument and measurement method to measure or take a photo, and then compare it with the value of the original left half of the face or the right half of the face (before and after use). When the test is to be performed, the temperature and humidity of the test area where the subject is located are consistent to reduce the impact of external temperature and humidity on the skin).

Test items 1. Skin redness, 2. Skin sagging, 3. Skin pore state will be tested respectively.

1. Redness

The facial skin of the same subject before and after using the serum was detected by using Mexameter® MX18 (C+K Multi Probe Adapter System, Germany) of Courage+Khazaka electronic company in Germany. The instrument determines the amount of heme in the skin by measuring the amount of reflection of light of a specific wavelength on the human skin. The higher the measurement value, the higher the red pigment content in the skin.

2. The degree of skin laxity

The skin elasticity test probe Cutometer® MPA580 (C+K Multi Probe Adapter System, Germany) purchased from Courage+Khazaka electronic company in Germany was used to test the facial skin of the same subject before and after using the serum. The test principle is based on the principle of suction and stretching. A negative pressure is generated on the surface of the tested skin to suck the skin into a test probe, and the depth of the skin being sucked into the probe is detected through the optical test system, and the skin relaxation is calculated by software analysis. degree.

3. The state of skin pores

The VISIA high-end digital skin tester sold by Canfield in the United States was used for testing, and the facial skin of the same subject before and after using the serum was tested through a high-resolution camera lens. By irradiating with standard white light, shadows are formed in the recesses of the pores, and the pores will be darker than the surrounding skin color, so you can detect the pores, and then use the software to analyze the number and area of the pores to obtain a value, and the higher the value, the more pores are displayed. more quantity and area.

See Figure 4. The figure shows the comparison of the red pigment content of the skin before using the citronella violacea extract of the present invention and the placebo essence and after using the aforementioned two essences for two weeks. Among them, the proportion of the number of people improved by using the citronella violacea extract essence of the present invention accounted for 87.5% of the number of subjects. Among them, when the average skin red pigment content of the subjects before using the essence is 100%, the average skin red pigment content after using the citronella violacea extract of the present invention for 2 weeks is 92.0%, which is statistically higher than Significant ("**" symbol represents p value less than 0.01 compared to before use), and 7.3% lower than placebo group compared with placebo serum ("#" symbol represents p value compared to placebo serum value less than 0.05). It can be seen that the Citronella violacea extract of the present invention has the functions of soothing skin redness and reducing skin red pigment.

See Figure 5. The figure shows the comparison of the degree of skin relaxation before using the citronella violacea extract of the present invention and the placebo essence and after using the aforementioned two essences for two weeks. Among them, the proportion of the improved number of people who used the essence containing the Citronella violacea extract of the present invention accounted for 62.5% of the number of subjects. Among them, when the average skin relaxation degree of the subjects before using the essence was 100%, the average skin relaxation degree after using the citronella violacea extract of the present invention for 2 weeks was 93.8%, which was different from that of using the placebo essence. Compared with the placebo group, it was 4.9% lower. It can be seen that the Citronella violacea extract of the present invention has the functions of improving skin relaxation and enhancing skin firmness.

See Figure 6. The figure shows the comparison of the state of skin pores before using the citronella violacea extract of the present invention and the placebo essence and after using the aforementioned two essences for two weeks. Among them, the proportion of the improved number of people who use the essence containing the Citronella violacea extract of the present invention accounts for 75% of the number of subjects. Among them, when the average skin pore volume of the subjects before using the essence is 100%, the average skin pore volume after using the citronella violacea extract of the present invention for 2 weeks is 89.9%, which is different from the placebo essence. Compared with the placebo group, it was 11% lower. It can be seen that the Citronella violacea extract of the present invention has the function of shrinking and tightening skin pores.

Although the technical content of the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person who is familiar with the art, makes some changes and modifications without departing from the spirit of the present invention, should be included in the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the appended patent application.

none.

Fig. 1 is a flow chart of the preparation method of Citronella violacea extract according to an embodiment. Figure 2 is a graph showing the relative production ratio of final glycated protein in the experimental group and the control group in Example 3. Figure 3 is a graph showing the comparison of free radicals generated in the experimental group, the control group and the control group after being stimulated by hydrogen peroxide in Example 4. FIG. 4 is a comparison chart of skin redness in the second week using the serum containing the Citronella violacea extract of the present invention and using the placebo serum in Example 5. FIG. FIG. 5 is a graph comparing the degree of skin relaxation in the second week between the use of the serum containing the Citronella violacea extract of the present invention and the use of the placebo serum in Example 5. FIG. FIG. 6 is a graph showing the comparison of skin pore volume in the second week using the essence containing the Citronella violacea extract of the present invention and using the placebo essence in Example 8. FIG.

Claims (8)

Use of a lemongrass extract for preparing a composition for conditioning skin condition, wherein the lemongrass extract is obtained by extracting a lemongrass with a water-containing solvent, and wherein the conditioning skin condition is to improve skin Anti-glycation ability, soothes redness, tightens pores, or improves skin firmness. A use of a lemongrass extract for preparing a composition for improving skin anti-glycation ability, wherein the lemongrass extract is obtained by extracting a lemongrass with a water-containing solvent, and the composition is obtained by inhibiting Or slow down the content of end-products of glycation in cells to improve the anti-glycation ability of the skin. A use of a lemongrass extract for preparing a composition for soothing skin redness, wherein the lemongrass extract is obtained by extracting a lemongrass with a water-containing solvent, and the skin redness is caused by the skin produced by inflammation. The use according to any one of claims 1 to 3, wherein the total flavonoid content of the Lemongrass violacea extract is greater than 12000 ppm. The use according to any one of claims 1 to 3, wherein the brix value of the Lemongrass violacea extract is above 10.0. The use according to any one of claims 1 to 3, wherein the percentage by weight of the Lemongrass violacea extract in the composition is 0.1% to 5%. The use according to any one of claims 1 to 3, wherein the liquid-solid ratio of the aqueous extraction solvent to the Lemongrass violacea is 5:1 to 20:1. The use according to any one of claims 1 to 3, wherein the composition is a pharmaceutical composition, a health food composition, a food composition, or a skin care product composition.

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