Method for preparing a melissa extract and melissa extract, cosmetic composition

Abstract

The application discloses a method for preparing a Melissa extract and the Melissa extract and a cosmetic composition. The method comprises the following steps: a crushing step, crushing a Melissa raw material to obtain a Melissa crushing product; an extraction step, extracting the Melissa crushing product with a mixed solution of alcohol and water to obtain a crude extract; a filtration step, filtering the crude extract with a filter membrane to obtain a filtrate; a concentration step, vacuum-concentrating the filtrate to remove part of alcohol and water, and obtaining a crude extract paste; a resin purification step, loading the crude extract paste into an adsorption resin column, performing water elution and gradient resolution of an alcohol aqueous solution, and obtaining an eluate; a decolorization step, adsorbing and decolorizing the eluate with activated carbon, and filtering to obtain a decolorized liquid; and a concentration and drying step, concentrating and drying the decolorized liquid to obtain a powder-like Melissa extract. The method is used to prepare a nano-powder-like grade Melissa extract, and the coumarin content in the extract is lower than a detection limit.

Description

Methods for preparing lemon balm extract and lemon balm extract, cosmetic compositions Technical Field

[0001] This application belongs to the field of plant extraction technology, specifically relating to a method for preparing lemon balm extract and lemon balm extract and cosmetic compositions. Background Technology

[0002] Melissa officinalis, a perennial herb belonging to the genus Melissa in the family Lamiaceae, is also known as bee balm or honey balm. Melissa can be used to treat dysentery, intestinal ulcers, swelling, joint pain, and toothache. It is also called the "happy plant."

[0003] In related technologies, extracts of lemon balm obtained from petroleum ether, chloroform, ethyl acetate, and n-butanol all exhibited moderate to strong activity against luteinized sarcoma, Staphylococcus aureus, and Bacillus cereus. However, these extracts contain components harmful to the skin, and additional impurity removal further reduces the effective components, significantly diminishing the efficacy of lemon balm extracts and limiting their application.

[0004] Therefore, finding a non-irritating and safe preparation method is the focus of research. Summary of the Invention

[0005] In view of this, this application provides a method for preparing lemon balm extract and a lemon balm extract and cosmetic composition, with the aim of improving the active ingredients in lemon balm extract.

[0006] In a first aspect, embodiments of this application provide a method for preparing lemon balm extract, comprising:

[0007] The crushing step involves crushing the lemon balm raw material to obtain lemon balm crushed material;

[0008] The extraction steps include extracting the crushed lemon balm with a mixed solution of alcohol and water to obtain a crude extract;

[0009] The filtration step includes filtering the crude extract using a filter membrane to obtain the filtrate;

[0010] The concentration step includes vacuum concentration of the filtrate to remove some of the alcohol and water, resulting in a crude extract;

[0011] The resin purification step includes loading the crude extract into an adsorption resin column, followed by sequential water elution and gradient elution with an alcohol-water solution to obtain the eluent.

[0012] The decolorization step includes adsorbing and decolorizing the eluent with activated carbon and filtering to obtain a decolorized solution;

[0013] The concentration and drying steps include concentrating and drying the decolorized solution to obtain a powdered gentian extract.

[0014] According to one embodiment of this application, in the mixed solution of alcohol and water, the mass fraction of alcohol in the mixed solution is 50-80%.

[0015] According to an embodiment of one aspect of this application, the alcohol includes one or more of ethanol, ethylene glycol, glycerol, and 1,2-butanediol.

[0016] According to an embodiment of one aspect of this application, the extraction step satisfies at least one of the following process conditions:

[0017] 1) The ratio of crushed lemon balm to the mixed solution is 1:(10-30);

[0018] 2) The extraction temperature is 40–90℃;

[0019] 3) The extraction time is 2–10 hours;

[0020] 4) Extraction must be performed at least once;

[0021] 5) Extraction is performed using at least one of the following methods: stirring extraction, solvent extraction, and ultrasonic extraction.

[0022] According to one embodiment of this application, the filtration step includes filtering the crude extract using a filter membrane under negative pressure filtration or positive pressure filtration to obtain a filtrate.

[0023] According to one embodiment of this application, the pore size of the filter membrane is 0.22 μm to 10 μm.

[0024] According to one embodiment of this application, the crude extract contains ≤10% ethanol by mass.

[0025] According to an embodiment of one aspect of this application, the adsorption resin includes at least one of D101 type macroporous adsorption resin, AB-8 type macroporous adsorption resin, and HPD100 type macroporous adsorption resin.

[0026] According to one embodiment of this application, the water volume used for water washing is 3 BV to 5 BV.

[0027] According to an embodiment of one aspect of this application, gradient elution of the alcohol-water solution includes: eluting the alcohol-water solution at a rate of 1.5-2 BV / h, eluting with a 20-30% ethanol-water solution for 4-5 BV, collecting the first eluent, eluting again with a 40-50% ethanol-water solution for 2-3 BV, discarding the eluent, eluting again with an 80-90% ethanol-water solution for 4-5 BV, collecting the second eluent, and mixing the first and second eluents to obtain the eluent.

[0028] According to an embodiment of one aspect of this application, the decolorization step satisfies at least one of the following process conditions:

[0029] The amount of activated carbon added is 1% to 5% of the eluent mass;

[0030] The decolorization time is 20–60 minutes;

[0031] The decolorization temperature is 30–70℃.

[0032] According to an embodiment of one aspect of this application, the concentration and drying steps satisfy any one of the following process conditions:

[0033] 1) Drying is performed using a spray dryer;

[0034] Optionally, the inlet temperature of the spray dryer is 100–160℃, and the outlet temperature is 60–90℃;

[0035] Optionally, the feed rate of the concentrated decolorized liquid in the spray dryer is 5–40 mL / min;

[0036] Optionally, the concentrated decolorized liquor can be dried using a spray dryer under a compressed air pressure of 0.4–0.8 MPa.

[0037] 2) Use a granulator for drying. The drying temperature when using a granulator can be 105-125℃.

[0038] Secondly, embodiments of this application provide a lemon balm extract prepared by the method of the first aspect.

[0039] According to one embodiment of this application, the lemon balm extract includes flavonoids, rosmarinic acid, and caffeic acid.

[0040] According to one embodiment of this application, the total flavonoid content in the lemon balm extract is 40%–50%, the luteolin content is 5%–10%, the rosmarinic acid content is 20%–25%, and the caffeic acid content is 2%–3%.

[0041] According to one embodiment of this application, the mass content of coumarin in lemon balm extract is below the detection limit, and may further be <4 μg / ml.

[0042] According to one embodiment of this application, lemon balm extract has at least one of the following effects: sun protection, anti-glycation, promoting collagen production, promoting dermal-epidermal junction, and soothing.

[0043] Thirdly, embodiments of this application provide a cosmetic composition comprising a lemon balm extract prepared by the method of the first aspect or a lemon balm extract prepared by the method of the second aspect.

[0044] According to one embodiment of this application, the lemon balm extract has a mass fraction of 0.05%-10% in the cosmetic composition.

[0045] Compared with the prior art, this application has at least the following beneficial effects:

[0046] The method provided in this application can prepare nano-powdered lemon balm extract, and the coumarin content in the lemon balm extract is below the detection limit. In this method, the total flavonoid components are selectively collected through resin purification, increasing their mass content to 40%–50%; the active ingredients are also selectively collected through resin purification, and the active ingredients are identified. Furthermore, luteolin, rosmarinic acid, and caffeic acid, through the combination of resin purification and related processes, selectively remove coumarin-like substances from the lemon balm extract, ensuring the efficacy and safety of the lemon balm extract.

[0047] The cosmetic composition containing lemon balm extract prepared in this application has good sun protection, anti-glycation, collagen-boosting, dermal-epidermal junction-promoting, and soothing effects, and has a promising application prospect in cosmetics. Attached Figure Description

[0048] To more clearly illustrate the technical solutions of the implementation regulations of this application, the drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 is a schematic flowchart of the method for preparing lemon balm extract provided in Example 1 of this application. Detailed Implementation

[0050] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the implementation details described in this specification are merely for illustrative purposes and are not intended to limit the scope of this application.

[0051] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0052] In the description of this application, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0053] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0054] Method for preparing lemon balm extract

[0055] In a first aspect, embodiments of this application provide a method for preparing lemon balm extract, comprising:

[0056] The crushing step involves crushing the lemon balm raw material to obtain lemon balm crushed material;

[0057] The extraction steps include extracting the crushed lemon balm with a mixed solution of alcohol and water to obtain a crude extract;

[0058] The filtration step includes filtering the crude extract using a filter membrane to obtain the filtrate;

[0059] The concentration step includes vacuum concentration of the filtrate to remove some of the alcohol and water, resulting in a crude extract;

[0060] The resin purification step includes loading the crude extract into an adsorption resin column, followed by sequential water elution and gradient elution with an alcohol-water solution to obtain the eluent.

[0061] The decolorization step includes adsorbing and decolorizing the eluent with activated carbon and filtering to obtain a decolorized solution;

[0062] The concentration and drying steps include concentrating and drying the decolorized solution to obtain a powdered gentian extract.

[0063] According to embodiments of this application, the lemon balm raw material can be dried lemon balm or wet material with a certain moisture content. The lemon balm raw material can be broadly understood to include lemon balm flower raw material. The lemon balm raw material is crushed into powder and sieved through a sieve to obtain relatively uniform lemon balm fragments. The decolorizing solution can be a clear, yellowish-brown solution containing lemon balm extract; the powdered lemon balm extract can be nano-sized, with a particle size of approximately 50nm-200nm.

[0064] Studies have found that lemon balm extract can exert its skin barrier repair effects by reducing transepidermal water loss, reducing erythema index, and increasing skin hydration. Lemon balm extract can also enhance the vitality of keratinocytes exposed to hydrogen peroxide and UVB irradiation. Citral in lemon balm extract can inhibit TNF-α in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and inhibit IL-6 and IL-1β in LPS-stimulated peritoneal macrophages of normal mice, thus exhibiting anti-inflammatory effects.

[0065] Further research has revealed that lemon balm extract has excellent sun protection, anti-glycation, collagen-boosting, dermal-epidermal junction-promoting, and soothing effects, making it a promising candidate for cosmetic applications.

[0066] According to the embodiments of this application, the method is simple, low-cost, free of toxic and harmful solvents, recyclable, and time-saving. It has low equipment requirements, is highly compatible with existing processes, and has great potential for large-scale application.

[0067] In some embodiments, the alcohol-water mixture has a mass fraction of 50-80%. The alcohol concentration and water ratio in the mixture can affect the solubility and extraction efficiency of the extract. Different concentrations of alcohol-water mixtures may have different solubilities for the active ingredients in lemon balm, thus affecting the extraction effect. Controlling the mass fraction in the mixture within the above-mentioned range allows for the full extraction of the active ingredients from lemon balm, improving the extract yield.

[0068] In some embodiments, the alcohol includes one or more of ethanol, ethylene glycol, glycerol, and 1,2-butanediol. These alcohols are non-toxic, safe, and readily miscible with water. Mixing these alcohols with water facilitates the full extraction of the active ingredients from lemon balm, thereby increasing the yield of the extract.

[0069] In some embodiments, the extraction step satisfies at least one of the following process conditions:

[0070] 1) The ratio of crushed lemon balm to the mixed solution is 1:(10-30);

[0071] 2) The extraction temperature is 40–90℃;

[0072] 3) The extraction time is 2–10 hours;

[0073] 4) Extraction must be performed at least once;

[0074] 5) Extraction is performed using at least one of the following methods: stirring extraction, solvent extraction, and ultrasonic extraction.

[0075] According to the embodiments, the solid-liquid ratio of lemon balm crushed material to the mixed solution can affect the solubility and extraction efficiency of the extract. Different solid-liquid ratios may result in different solubilities of the active ingredients in lemon balm, thus affecting the extraction effect. Controlling the solid-liquid ratio within the above-mentioned range can fully extract the active ingredients in lemon balm and improve the yield of the extract.

[0076] According to the embodiments described herein, the extraction temperature during the extraction process can affect the chemical reactions and component release rates in the extract. Different temperature conditions may lead to different extraction effects and chemical changes. Within the aforementioned temperature range, it is possible to obtain the desired components and remove unwanted components.

[0077] According to the embodiments described herein, the duration of the extraction process can affect the concentration and composition of the extract. A longer extraction time may result in the release of more active ingredients from lemon balm, but excessive time may lead to the extraction of other unwanted components. By controlling the extraction time within the aforementioned range, a lemon balm extract with a suitable composition is obtained.

[0078] In some embodiments, the filtration step includes filtering the crude extract using a filter membrane under negative pressure filtration or positive pressure filtration to obtain a filtrate.

[0079] According to embodiments of this application, filtering the crude extract using a filter membrane under negative pressure filtration or positive pressure filtration conditions can affect the quality and purity of the extract. The following are possible influencing factors:

[0080] In some embodiments, the pore size of the filter membrane is 0.22 μm to 10 μm. According to embodiments of this application, the pore size and selectivity of the membrane can affect the filtration efficiency. The aforementioned pore size can effectively prevent solid particles or impurities from passing through the filter membrane, thereby improving the filtration efficiency.

[0081] In some embodiments, the mass content of ethanol in the crude extract is ≤10%. According to the embodiments of this application, controlling the mass content of ethanol in the crude extract within the above range is beneficial for controlling the degree of concentration and facilitating subsequent adsorption and decolorization.

[0082] In some embodiments, the adsorption resin includes at least one of D101 type macroporous adsorption resin, AB-8 type macroporous adsorption resin, and HPD100 type macroporous adsorption resin.

[0083] D101 macroporous adsorption resin is a polystyrene-based adsorption resin. It has a large pore size and surface area, and is commonly used in traditional Chinese medicine extraction, biopharmaceuticals, and the food industry. D101 adsorption resin can selectively adsorb and separate target compounds, such as active ingredients and enzymes in extracts.

[0084] AB-8 macroporous adsorption resin: AB-8 macroporous adsorption resin is a polystyrene-based, highly polar adsorption resin. It has a large pore size and strong hydrophilicity, and can remove heavy metal ions, pigments, etc. from water.

[0085] HPD100 macroporous adsorption resin is a polystyrene-based non-ionic adsorption resin. It has a large pore size and high adsorption capacity, and is commonly used for protein purification, enzyme separation and purification, and the adsorption and separation of other macromolecular compounds.

[0086] According to embodiments of this application, the aforementioned types of adsorption resins, based on their specific physicochemical properties and structural design, can selectively adsorb and separate molecules based on different characteristics. They can adsorb some harmful substances while maximally preserving the active ingredients of lemon balm, achieving separation and purification. They can also selectively separate or retain certain components, such as removing coumarins, thereby altering the composition of the extract.

[0087] In some embodiments, the water volume used for water elution is 3 BV to 5 BV.

[0088] According to embodiments of this application, water elution refers to the process of rinsing and removing impurities or non-target compounds adsorbed on the resin using an aqueous solution. By using the aforementioned amount of water, non-specific impurities, ions, or low-affinity compounds adsorbed on the resin can be effectively removed. It can also remove impurities, residues, and redundant reagents from the resin surface, thereby purifying the resin and restoring its adsorption performance to improve the purity of the target compound.

[0089] In some embodiments, gradient elution of the alcohol-water solution includes: eluting the alcohol-water solution at a rate of 1.5-2 BV / h, eluting with a 20-30% (w / w) ethanol-water solution for 4-5 BV, collecting the first eluent, eluting again with a 40-50% (w / w) ethanol-water solution for 2-3 BV, discarding the eluent, eluting again with an 80-90% (w / w) ethanol-water solution for 4-5 BV, collecting the second eluent, and mixing the first and second eluents to obtain the eluent.

[0090] According to embodiments of this application, gradient elution of alcohol-water solutions refers to the gradual elution of target compounds adsorbed on resin using solutions of alcohol and water at different concentrations. By controlling the gradient elution of alcohol-water solutions and adjusting the ratio and concentration of alcohol and water within the aforementioned range, the affinity of the resin for the target compounds can be altered, thereby achieving selective adsorption and desorption of different compounds, further obtaining the target compounds and removing harmful compounds, such as obtaining flavonoids, rosmarinic acid, and caffeic acid, and removing coumarin. By controlling the gradient elution of alcohol-water solutions, the polarity and elution capacity of the solution can be gradually changed, thereby gradually eluting compounds adsorbed on the resin and achieving the purification of the target compounds.

[0091] In some embodiments, the decolorization step meets at least one of the following process conditions:

[0092] The amount of activated carbon added is 1% to 5% of the eluent mass;

[0093] The decolorization time is 20–60 minutes;

[0094] The decolorization temperature is 30–70℃.

[0095] According to embodiments of this application, activated carbon possesses excellent adsorption properties, effectively removing pigments, impurities, and odorous substances from solutions. By using activated carbon for decolorization and controlling the relevant process conditions, the clarity and purity of the solution can be improved, enabling effective decolorization and purification to meet specific quality standards and application requirements.

[0096] In some embodiments, the concentration and drying steps meet any one of the following process conditions:

[0097] 1) Drying is performed using a spray dryer;

[0098] Optionally, the inlet temperature of the spray dryer is 100–160℃, and the outlet temperature is 60–90℃;

[0099] Optionally, the feed rate of the concentrated decolorized liquid in the spray dryer is 5–40 mL / min;

[0100] Optionally, the concentrated decolorized liquor can be dried using a spray dryer under a compressed air pressure of 0.4–0.8 MPa.

[0101] 2) Use a granulator for drying. The drying temperature when using a granulator can be 105-125℃.

[0102] According to embodiments of this application, controlling the temperature, time, and feed rate during the concentration and drying processes can affect the morphology, particle size distribution, and physical properties of nanoscale powders. Controlling the inlet and outlet temperatures of the spray dryer can influence the morphology and particle size distribution of the nanoscale powders. Within the aforementioned temperature range, while considering the drying rate, sufficient heat energy can be provided to accelerate the evaporation of moisture and the drying rate of solid particles, avoiding excessively high temperatures that may lead to powder agglomeration, sintering, or thermal decomposition. Controlling the feed rate of the concentrated decolorizing liquid can control the drying time, ensuring complete solvent evaporation and achieving the desired degree of dryness for the powder, avoiding over-drying and powder agglomeration. During the drying process, controlling the compressed air pressure to 0.4–0.8 MPa maintains a suitable environment within the drying equipment, contributing to uniform powder drying and preventing uneven drying and powder agglomeration.

[0103] According to the embodiments of this application, optimizing process conditions can achieve higher extraction efficiency, better extract quality, and higher yield.

[0104] Secondly, embodiments of this application provide a lemon balm extract prepared by the method of the first aspect.

[0105] In some embodiments, lemon balm extract includes flavonoids, rosmarinic acid, and caffeic acid.

[0106] In some embodiments, the total flavonoid content in the lemon balm extract is 40%–50%, the luteolin content is 5%–10%, the rosmarinic acid content is 20%–25%, and the caffeic acid content is 2%–3%.

[0107] In some embodiments, the mass ratio of luteolin, rosmarinic acid, and caffeic acid is 1:(4-5):(0.4-0.6).

[0108] In some embodiments, the coumarin content in lemon balm extract is below the detection limit, and may further be <4 μg / ml.

[0109] In some embodiments, lemon balm extract has at least one of the following effects: sun protection, anti-glycation, promoting collagen production, promoting dermal-epidermal junction, and soothing.

[0110] Thirdly, embodiments of this application provide a cosmetic composition comprising a lemon balm extract prepared by the method of the first aspect or a lemon balm extract prepared by the method of the second aspect.

[0111] According to embodiments of this application, the cosmetic composition is safe and non-irritating, and has good sun protection, anti-glycation, collagen-boosting, and dermal-epidermal junction-promoting effects. This cosmetic composition can be used to prepare toners, cleansers, lotions, creams, serums, etc.

[0112] In some embodiments, the lemon balm extract is present in the cosmetic composition at a mass fraction of 0.05%-10%.

[0113] According to the embodiments of this application, controlling the mass fraction of lemon balm extract in the cosmetic composition within the above-mentioned range is beneficial to further ensure that the cosmetic composition has good sun protection effect, anti-glycation effect, collagen-boosting effect, and dermal-epidermal junction-promoting effect.

[0114] In some embodiments, the lemon balm cream comprises the following components by weight percentage:

[0115] Components (by weight): Deionized water (balance), Glycerin 4-6%, Sodium hyaluronate 0.05-0.2%, Xanthan gum 0.15-0.3%, 1,3-Butanediol 2-4%, Caprylic / Capric Triglycerides 4-6%, Cetearyl Alcohol 2-4%, Jojoba Seed Oil 2-4%, Shea Butter Resin 1-2.8%, Cocoyl Alcohol-Caprylate / Capric Triglyceride 1-2.8%, Glyceryl Stearate and PEG-100 Stearate 2-4%, Lemon Balm Extract 0.15-0.3%, Tocopherol Acetate 0.08-0.15%, Preservatives 0.8-2%, Citric Acid 0.04-0.06%. surface.

[0116] In some embodiments, the lemon balm cream comprises the following components by weight percentage:

[0117] Components (by weight): Deionized water (balance), Glycerin 5%, Sodium hyaluronate 0.1%, Xanthan gum 0.2%, 1,3-Butanediol 3%, Caprylic / Capric Triglyceride 5%, Cetearyl Alcohol 3%, Jojoba Seed Oil 3%, Shea Butter Resin 2%, Cocoyl Alcohol-Caprylate / Capric Triglyceride 2%, Glyceryl Stearate and PEG-100 Stearate 3%, Lemon Balm Extract 0.2%, Tocopherol Acetate 0.1%, Preservatives 1%, Citric Acid 0.05%. surface.

[0118] Example

[0119] The following embodiments describe the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on weight, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0120] Example 1

[0121] This embodiment provides a method for preparing lemon balm extract, including the following steps:

[0122] Melissa balm was pulverized in a grinder and passed through a 40-mesh sieve. 100g of powder was added to 2000mL of 70% ethanol solution and extracted at 80℃ with stirring at 200r / min for 2h. This extraction was repeated twice to obtain a crude extract of mesona chinensis. The crude extract was then filtered under reduced pressure using a 5μm pore size filter. The filtrate was concentrated under vacuum at a temperature below 50℃ until no alcohol odor remained, yielding an extract paste. This paste was loaded onto a D101 macroporous resin column and eluted with water (2BV / h, 5BV elution volume). It was then eluent with 20% ethanol solution (5BV elution volume). The eluent was collected and further processed using... Elute 3 BV with 40% ethanol aqueous solution, discard this eluent, and finally elute 5 BV with 80% ethanol aqueous solution. Collect the eluent, combine the collected eluents, add 1% activated carbon, stir and decolorize at 50℃ for 30 min, filter under reduced pressure with a filter membrane pore size of 1 μm, concentrate the filtrate to 100 ml, and granulate using a spray dryer with an inlet temperature of 160℃, an outlet temperature of 90℃, a feed rate of 5 mL / min, and a compressed air pressure of 0.4 MPa to obtain approximately 9.5 g of nano-sized lemon balm extract powder.

[0123] Example 2

[0124] Compared with Example 1, this embodiment changes the resin filler type to AB-8, while keeping other conditions unchanged.

[0125] Example 3

[0126] The difference between this embodiment and Embodiment 1 is that the extraction temperature is 65℃.

[0127] Example 4

[0128] The difference between this embodiment and Embodiment 1 is that the pore size of the filter membrane is 4 μm.

[0129] Example 5

[0130] The difference between this embodiment and Embodiment 1 is that the gradient elution of the alcohol-water solution is different. It includes: elution rate of alcohol-water solution is 1.8 BV / h, elution with 25% ethanol-water solution for 4.2 BV, collection of the first eluent, elution with 45% ethanol-water solution for 2.5 BV, discarding the eluent, elution with 85% ethanol-water solution for 4.5 BV, collection of the second eluent, and mixing the first and second eluents to obtain the eluent collection.

[0131] Example 6

[0132] The difference between this embodiment and Embodiment 1 is that the gradient elution of the alcohol-water solution is different. It includes: elution rate of alcohol-water solution is 2.0 BV / h, elution with 28% ethanol-water solution for 4.2 BV, collection of the first eluent, elution with 44% ethanol-water solution for 2.4 BV, discarding the eluent, elution with 85% ethanol-water solution for 4.2 BV, collection of the second eluent, and mixing the first and second eluents to obtain the eluent collection.

[0133] Comparative Example 1

[0134] Compared with Example 1, this comparative example changed the resin elution process to water elution of 5BV, and the gradient elution of the alcohol-water solution was different. 5BV was eluted with 50% ethanol-water solution, and the 50% ethanol-water solution eluent was collected. Other conditions remained unchanged.

[0135] Comparative Example 2

[0136] Compared with Example 1, this comparative example uses a 10% aqueous ethanol solution as the extraction solvent, while keeping other conditions unchanged.

[0137] Test section

[0138] 1) Detection of total flavonoid content in lemon balm extract

[0139] The total flavonoid content of the lemon balm extracts obtained in Examples 1, 2, 1, and 2 was determined.

[0140] Using rutin as a standard, the total flavonoid content in the obtained lemon balm powder was determined by colorimetric method.

[0141] (1) Construction of standard curve: Accurately measure 0.108 mg / mL of rutin reference standard, prepare a series of concentrations according to Table 1, develop color for half an hour, and detect absorbance at a wavelength of 510 nm.

[0142] Table 1

[0143]

[0144] (2) Determination of the test samples: The lemon balm extract powder from Examples 1, 2, Comparative Example 1, and Comparative Example 2 was prepared into a 0.5% lemon balm extract solution, and 0.5 mL was diluted to 10 mL. 0.5 mL of the test solution was taken, and 0.3 mL of 5% sodium nitrite, 0.3 mL of 10% aluminum nitrate, 6.9 mL of 30% ethanol, and 3 mL of 1 mol / L sodium hydroxide were added respectively. After color development for half an hour, the concentration was determined, and the total flavonoid content was obtained as shown in Table 2.

[0145] Table 2

[0146]

[0147]

[0148] 2) Detection of luteolin content in lemon balm extract

[0149] The luteolin content of the lemon balm extract powders obtained in Examples 1, 2, 1, and 2 was determined.

[0150] (1) HPLC chromatographic conditions: Mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (20:80); Column: C18 5μm (4.6mm*250mm); Detection wavelength: 330nm; Column temperature:

[0151] 25℃; Flow rate: 1mL / min.

[0152] (2) Reference material: The standard was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. 6 mg of rosmarinic acid (HPLC≥98%) was added to 50% ethanol to prepare a reference solution of 120 μg / mL. 0, 5, 10, 15, 20, 30 and 40 μL were injected to obtain linear curves.

[0153] (3) Test solution: For Examples 1, 2, Comparative Example 1, and 2, 0.5 g of lemon balm powder was dissolved in 100 mL of 50% ethanol solution to obtain a 0.5% lemon balm extract. The injection volume was 10 μL, and the luteolin content in the lemon balm powder was determined as shown in Table 3.

[0154] Table 3

[0155]

[0156] 3) Detection of rosmarinic acid content in lemon balm extract

[0157] The lemon balm extracts obtained in Examples 1, 2, Comparative Example 1, and Comparative Example 2 were subjected to quantitative analysis of rosmarinic acid content.

[0158] (1) HPLC chromatographic conditions: mobile phase: methanol-0.1% phosphoric acid aqueous solution (50:50); column: C18 5μm (4.6mm*250mm); detection wavelength: 350nm; column temperature: 25℃;

[0159] Flow rate: 1 mL / min.

[0160] (2) Reference material: The standard was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. 6 mg of rosmarinic acid (HPLC≥97%) was added to 50% ethanol to prepare a reference solution of 120 μg / mL. 0, 5, 10, 15, 20, 30 and 40 μL were injected to obtain linear curves.

[0161] (3) Test solution: For Examples 1, 2, Comparative Example 1, and 2, 0.5 g of lemon balm powder was dissolved in 100 mL of 50% ethanol solution to obtain a 0.5% lemon balm extract. 1 mL of the 0.5% lemon balm extract was added and diluted to 10 mL with 50% ethanol. The injection volume was 10 μL. The rosmarinic acid content in the lemon balm powder was determined as shown in Table 4.

[0162] Table 4

[0163]

[0164] 4) Detection of caffeic acid content in lemon balm extract

[0165] The lemon balm extracts obtained in Examples 1, 2, 1, and 2 were subjected to quantitative analysis of caffeic acid content.

[0166] (1) HPLC chromatographic conditions: The mobile phase was methanol-phosphate buffer (25:75, pH=2.3);

[0167] Chromatographic column: C18 5μm (4.6mm*150mm); Detection wavelength: 323nm; Column temperature:

[0168] 25℃; Flow rate: 0.8 mL / min.

[0169] (2) Reference material: The standard was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. 5 mg of caffeic acid (HPLC≥97%) was added to 50% ethanol to prepare a 50 μg / mL reference solution. 0, 5, 10, 15, 20, 30 and 40 μL were injected to obtain linear curves.

[0170] (3) Test solution: For Examples 1, 2, Comparative Example 1, and 2, 0.5 g of lemon balm powder was dissolved in 100 mL of 50% ethanol solution to obtain a 0.5% lemon balm extract. 1 mL of the 0.5% lemon balm extract was diluted to 10 mL with 50% ethanol, and the injection volume was 10 μL. The caffeic acid content in the lemon balm powder was determined as shown in Table 5.

[0171] Table 5

[0172]

[0173] 5) Detection of coumarin content in lemon balm extract

[0174] The coumarin components of the lemon balm extracts obtained in Examples 1, 2, 1, and 2 were analyzed. The coumarin content in the lemon balm extract powders from Examples 1, 2, 1, and 2 was determined according to GB / T 35798-2018, "Determination of Coumarins and Their Derivatives in Cosmetics by High Performance Liquid Chromatography". The determined coumarin content in the lemon balm extract powders is shown in Table 6.

[0175] Note: The detection limit of this method is 4 μg / ml.

[0176] Table 6

[0177]

[0178] 6) Cytotoxicity test

[0179] Immortalized keratinocytes were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-dextrose antibody (1×10⁻⁶). 5Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO2 until confluence reached 85-95%. Logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with DMEM medium containing 10% serum. Cells were counted using a cell counting chamber, and the cell suspension was adjusted to 2 × 10⁵ / ml. Cells were seeded into 96-well plates at a ratio of 200 μL per well and incubated at 37°C and 5% CO2 until confluence reached 45-60%. The old medium was removed, and 200 μL of serum-free medium containing different concentrations of lemon balm extract was added to each sample. Each sample was tested in quadruplicates. The control group was cell-free and contained 200 μL of PBS; the comparative control group contained cells and contained 200 μL of serum-free medium. The cells were incubated at 37°C and 5% CO2 for 24 h; then 20 μL of CCK8 solution was added to each well and incubated for another 3 h. The absorbance was measured at 450 nm and the cell viability of each group was calculated.

[0180] 1. Cell viability (%) = (Sample group - Blank group) / (Control group - Blank group) * 100;

[0181] 2. The final concentrations of lemon balm extract in Examples 1-2 and Comparative Examples 1-2 of the following samples were 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 250 μg / ml, respectively.

[0182] The results of the keratinocyte toxicity test of lemon balm extract are shown in Table 7:

[0183] Table 7

[0184]

[0185]

[0186] Note: In this experiment, the higher the cell survival rate, the lower the cytotoxicity of the lemon balm extract.

[0187] The experimental results showed that: the negative control group; the lemon balm extracts of Example 1 and Example 2 were not cytotoxic, and at high concentrations, the morphology of keratinocytes was normal, and the relative cell viability value increased to about 130%. Comparative Example 1 showed slight cytotoxicity at high concentrations, while Comparative Example 2 showed no cytotoxicity.

[0188] 8) Cell proliferation test

[0189] 1. Immortalized keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (1×10⁵ U / L penicillin and 100 mg / L streptomycin). Cells were grown in an incubator at 37°C and 5% CO₂ until the cell confluence reached 85-95%.

[0190] 2. Digest logarithmically growing cells with 0.05% trypsin, and terminate the digestion reaction with DMEM medium containing 10% serum.

[0191] 3. Use a cell counting chamber to count the cells, adjust the cell suspension to 2*10^5 / ml, and seed 200μL per well into a 96-well plate. Incubate at 37℃ and 5% CO2 for a certain period of time until the cell confluence reaches 45-60%.

[0192] 4. Remove the old culture medium and add 100 μL of serum-free culture medium containing lemon balm extract for the test sample. Perform four replicates for each test solution. The blank group is cell-free and contains 200 μL of PBS; the example combination and comparative group contain cells and contain 100 μL of serum-free culture medium. Incubate at 37°C and 5% CO2 for 24 h.

[0193] 5. Then add 20 μL of CCK8 solution to each well, incubate for 3 h, measure the absorbance at 450 nm, and calculate the cell viability of each group;

[0194] 6. Cell viability (%) = (Sample group - Blank group) / (Control group - Blank group) * 100;

[0195] 7. Raw material testing: The final concentration of lemon balm extract in Examples 1-2 and Comparative Examples 1-2 was 250 μg / ml, respectively;

[0196] The test results are shown in Table 8:

[0197] Table 8

[0198] Cell viability / % Blank group 99.14±2.67 Example 1 128.21±2.15** Example 2 130.52±1.56** Comparative example 1 88.15±3.45 Comparative example 2 100.21±4.16 surface

[0199] Experimental conclusion: At a test concentration of 250 μg / ml, Examples 1 and 2 showed a promoting effect on keratinocyte proliferation.

[0200] 9) UV-damaged keratinocyte experiment

[0201] 1. Immortalized keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (1×10⁵ U / L penicillin and 100 mg / L streptomycin). Cells were grown in an incubator at 37°C and 5% CO₂ until the cell confluence reached 85-95%.

[0202] 2. Digest logarithmically growing cells with 0.05% trypsin, and terminate the digestion reaction with DMEM medium containing 10% serum.

[0203] 3. Count the cells using a cell counting chamber, adjust the cell suspension to 2*10^5 / ml, and seed 100μL per well into a 96-well plate. Incubate at 37℃ and 5% CO2 for a certain period of time until the cell confluence reaches 85-95%.

[0204] 4. Remove the old culture medium and add 100 μL of serum-free culture medium containing lemon balm extract for the test sample. Perform four replicates for each test solution. The blank group is cell-free and contains 200 μL of PBS; the control group contains cells and contains 100 μL of serum-free culture medium. Incubate at 37°C and 5% CO2 for 24 h. The final concentration of lemon balm extract in Examples 1-2 and Comparative Examples 1-2 is 250 μg / ml, respectively.

[0205] 5. Using 8J / cm 2 Cells in the control group and the drug-treated group were irradiated with UVA.

[0206] 6. Then add 20 μL of CCK8 solution to each well, incubate for 3 h, measure the absorbance at 450 nm, and calculate the cell viability of each group;

[0207] The test results are shown in Table 9:

[0208] Table 9

[0209] Cell Viability / % Blank Group 99.14±2.67 Control Group 81.46±3.14 Example 1 102.57±1.67** Example 2 100.25±3.21** Comparative Example 1 84.16±2.15 Comparative Example 2 82.56±1.89 surface

[0210] Experimental conclusion: At a test concentration of 250 μg / ml, the lemon balm extracts of Examples 1 and 2 have a protective effect against UVA-damaged keratinocytes.

[0211] 3) ELISA histamine content determination

[0212] The ELISA histamine kit is from Sangon Biotech Co., Ltd.

[0213] 1. Adding standards: Set up standard wells and sample wells, and add 50 μL of different concentrations of standard to each standard well according to the kit instructions.

[0214] 2. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. Add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, then add 10 μL of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix. In Examples 1-2 and Comparative Examples 1-2, the final concentration of lemon balm extract was 250 μg / ml.

[0215] 3. Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank wells.

[0216] 4. Incubation: After sealing the plate with sealing film, incubate at 37°C for 60 minutes.

[0217] 5. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0218] 6. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing solution, let stand for 30 seconds and then discard. Repeat this 5 times, then pat dry.

[0219] 7. Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0220] 8. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0221] 9. Measurement: Zero the instrument using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0222] Table 10

[0223] Histamine content (pg / mL): Example 1 2.05±0.06**; Example 2 2.14±0.15**; Comparative Example 1 2.78±0.21; Comparative Example 2 2.98±0.26 surface

[0224] Experimental conclusion: At a test concentration of 250 μg / ml, the lemon balm extract in Examples 1 and 2 significantly inhibited the release of histamine from mast cells, thereby alleviating allergy symptoms and reducing histamine's irritation and damage to the skin, thus helping to improve skin health.

[0225] 11) Inhibition of inflammatory factors experiment

[0226] 1. Cell seeding: RAW264.7 cells (Beina Biotechnology Cell Bank; EpiSkin, Shanghai Sianfuno Biotechnology Co., Ltd.) were seeded into 24-well plates at a rate of 1.5 × 10⁵ and incubated overnight at 37°C in a 5% CO₂ incubator;

[0227] 2. Experimental grouping: A blank control group, a negative control group, and a sample group were set up, including Examples 1-2 and Comparative Examples 1-2;

[0228] 3. Solution preparation: Prepare the sample working solution according to the test concentration setting table;

[0229] 4. Sample Inoculation: Inoculate the cells when the cell seeding rate in the 24-well plate reaches 50-60%. For the negative control group, add culture medium containing LPS to each well; for the sample groups (Examples 1-2 and Comparative Examples 1-2), add culture medium containing LPS and the corresponding concentration of the sample to each well. The final concentration of lemon balm extract in the final samples of Examples 1-2 and Comparative Examples 1-2 is 50 μg / ml; for the blank or solvent control group, add culture medium without LPS.

[0230] 5. After drug administration, place the 24-well plate in an incubator (37℃, 5% CO2) for culture; observe cell morphology under a microscope and take pictures;

[0231] 6. Detection: After 24 hours of cell incubation, 500 μl of cell supernatant was collected for the determination of the inflammatory factor NO content. Diluted CCK8 solution with DMEM was added, and the cells were incubated at 37°C. The OD value was then read at 450 nm.

[0232] 7. Relative cell viability (%) = (OD of sample group - OD of zero-adjustment group) / (OD of solvent control group - OD of zero-adjustment group) * 100;

[0233] 8. The test results are shown in Table 11.

[0234] Table 11

[0235] Project Release Amount (μM) Blank Group 0.04 Model Group 28.59±0.25 Example 1 0.14±0.01 **** Example 2 0.13±0.03 **** Comparative Example 1 17.31±0.15** Comparative Example 2 23.15±0.21* surface

[0236] Experimental results showed that RAW264.7 cells overexpressed NO up to 28.59 μM after LPS stimulation. After treatment with lemon balm extract at a concentration of 50 ug / mL in Examples 1 and 2, the expression of NO in mouse macrophages decreased significantly. This indicates that the lemon balm extract can effectively act on LPS-stimulated RAW264.7 cells and alleviate the cellular inflammatory response.

[0237] 12) Anti-glycation effect test

[0238] Experimental Methods: 18.22g Balb / c mice (SPF grade, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A diabetic mouse model was established to study the effects of different test substances on AGEs in organs such as the heart, liver, and kidneys. The diabetic mouse model was established by intravenous injection of alloxan (80 mg / kg) into the tail vein. Fifty eligible mice were randomly divided into a model group and an experimental group (Example 1, Example 2, Comparative Example 1, Comparative Example 2), with 10 mice in each group. Ten healthy mice were used as a control group; no diabetic mouse model was established in the control group, and no gavage was administered. The model group and experimental group were administered lemon balm extract (Example 1, Example 2, Comparative Example 1, and Comparative Example 2) by gavage, with a concentration of 30 mg / kg for each group (the experimental group used physiological saline as the solvent), once daily for 14 days. Mice were fasted for 16 hours after the last administration and then sacrificed by cervical dislocation for subsequent experiments. The heart, liver, and kidney tissues of the mice were cut into small pieces, homogenized, and centrifuged. 5 mL of chloroform-methanol (2:1) was added to the precipitate for defatting, and the mixture was shaken overnight at 4°C. Then, after washing and overnight in HEPES buffer, collagenase was added, and the mixture was digested at 37°C for 24 hours. The supernatant was obtained by centrifugation, and its fluorescence intensity was measured at 370 nm / 440 nm using a fluorescence spectrophotometer. The relative fluorescence intensity of the sample was obtained based on the HEPES buffer concentration.

[0239] The results are shown in Table 12:

[0240] Table 12: Effects of different compositions on AGE formation in different mouse tissues (mean ± s, n = 10)

[0241]

[0242] Note: Compared with Comparative Example 1, **P<0.01, ***P<0.001; compared with Comparative Example 2, ##P<0.01, ###P<0.001

[0243] The experimental results are shown in the table. The lower the AGE fluorescence value, the better the anti-glycation effect. Compared with the model group, Examples 1, 2, and Comparative Examples 1 and 2 all significantly inhibited the production of AGE in various organs of diabetic mice to varying degrees in vivo. Among them, Examples 1 and 2 showed the strongest inhibitory effect on AGE in the liver, heart, and kidney, which also indicates that Examples 1 and 2 have superior anti-glycation effects.

[0244] 13) Skincare effect test

[0245] Forty subjects with healthy skin were selected and divided into five groups. They had not used skincare products with the same function in the week prior to the test.

[0246] After cleansing the subjects' faces, no skincare products were applied. After 30 minutes, the elasticity and collagen fiber levels of the subjects' facial skin were tested, and the test data were recorded. A face cream was prepared using lemon balm extracts prepared according to Examples 1, 2, Comparative Example 1, and Comparative Example 2, respectively. The components of the face cream are as follows:

[0247]

[0248]

[0249] The face creams in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 differ in that they use lemon balm extracts prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively.

[0250] The face creams from Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were applied to the faces of the subjects twice a day, morning and evening, for 90 consecutive days. The blank control group was treated with an equal amount of distilled water. Skin elasticity and collagen fiber levels on the subjects' faces were tested on days 1, 7, 14, and 28, and the test data were recorded.

[0251] The instrument used in this experiment was the CBS-1800U skin analysis system manufactured by CBS Corporation of Taiwan.

[0252] Table 13

[0253]

[0254] Table 14

[0255]

[0256] The experimental results show that after using skincare products containing the plant extracts of this invention for 30 days, the skin elasticity increased by more than 15%, and the collagen fiber level was significantly improved.

[0257] Different concentrations of deionized aqueous solutions containing lemon balm extract (Example 2) and different concentrations of lemon balm extract (Example 2) face creams were applied to the faces of subjects twice daily, morning and evening, for 90 consecutive days. A blank control group received an equal amount of distilled water. Skin elasticity and collagen fiber levels on the subjects' faces were tested and data recorded on days 1, 7, 14, and 28. The components of the face creams were as shown in Example 2; the deionized water content was adjusted when the lemon balm content varied.

[0258] The results are shown in Table 15.

[0259] Table 15

[0260]

[0261]

[0262] 14) Tests for adhesion proteins that promote the connection between the epidermis and dermis

[0263] Sixty healthy adult Wistar rats aged 3 months (SPF grade) were used to establish a 4% total body surface area (TBSA) deep second-degree burn model according to the method described in the reference [Hao Chunguang, Wang Lingfeng, Fu Xue, et al. Therapeutic effect of selective decellularized sheepskin biological dressing on deep second-degree burn wounds in rats [J / CD]. Chinese Journal of Trauma and Repair (Electronic Edition), 2012, 7(5):476-481.]. The rats were randomly divided into three groups: iodine gauze group, Example 1 gauze group, Example 2 gauze group, Comparative Example 1 gauze group, and Comparative Example 2 gauze group, with 32 rats in each group. Example 1: Gauze group was made by soaking gauze containing 0.5% of the lemon balm extract from Example 1 in a 70% ethanol solution; Example 2: Gauze group was made by soaking gauze containing 0.5% of the lemon balm extract from Example 2 in a 70% ethanol solution; Comparative Example 1: Gauze was made by soaking gauze containing 0.5% of the lemon balm extract from Comparative Example 1 in a 70% ethanol solution; Comparative Example 2: Gauze was made by soaking gauze containing 0.5% of the lemon balm extract from Comparative Example 2 in a 70% ethanol solution; the gauze was changed once a day at regular intervals.

[0264] The surrounding area was sutured and fixed with 4-0 sutures. Then, two pieces of sterile gauze soaked in 0.9% sodium chloride solution were placed on the biological dressing or inner dressing. The dressing was then wrapped and protected with wire mesh. After the operation, 5 mL of compound sodium chloride injection was injected into the rat's peritoneum. The rat was kept warm and revived. After waking up, the rat was allowed to eat and drink freely and was housed in individual cages.

[0265] RT-qPCR detection of LN mRNA expression in wound tissue

[0266] Eight rats in each group were sacrificed at each time point (3, 7, 14, and 28 days post-injury). Tissue samples from the surgical area were excised and preserved in liquid nitrogen. Approximately 0.3g of tissue was removed from the liquid nitrogen and ground in a pre-cooled mortar with a pestle, continuously adding liquid nitrogen until it was ground into a powder (without visible particles). Total RNA was extracted from the tissue using TRIZOL reagent. Reverse transcription and amplification reactions were performed sequentially. The reverse transcription conditions were: 37℃ for 15 min; 85℃ for 15 min; 4℃. The reaction products were stored at -20℃ for later use. Amplification reactions were performed using a real-time PCR instrument. Each sample was tested in 5 replicates. For each reaction, a sample from 28 days post-injury was used as a positive control, and RNase-free water was used as a negative control instead of template. Melting curve analysis was used to determine whether the PCR products were correct amplification products or whether primer dimers were present. Primers were designed according to standard real-time PCR primer design principles using Primer 5.0.

[0267] Table 16. RT-qPCR detection of laminin LN mRNA expression in burned rats

[0268]

[0269] The experimental results showed that gauze treatment containing the lemon balm extract of this application significantly increased LN mRNA expression at 14 and 28 days compared to the iodine-coated gauze group, indicating that the lemon balm extract of this application has a good effect on promoting dermal-epidermal junction.

[0270] Gauze soaked in 70% ethanol solutions containing lemon balm extract of different concentrations (as described in Example 2) was used to treat gauze containing lemon balm extract of different concentrations and gauze containing face cream of different concentrations, as prepared above. Other aspects were the same as described above.

[0271] The results are shown in Table 17.

[0272] Table 17

[0273]

[0274]

[0275] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing lemon balm extract, characterized in that, include: The crushing step involves crushing the lemon balm raw material to obtain lemon balm crushed material; The extraction step includes extracting lemon balm fragments with a mixed solution of alcohol and water to obtain a crude extract; the filtration step includes filtering the crude extract using a filter membrane to obtain a filtrate; and the concentration step includes vacuum concentrating the filtrate to remove some of the alcohol and water to obtain a crude extract. The resin purification step includes loading the crude extract into an adsorption resin column, sequentially performing water elution and gradient elution with an alcohol-water solution to obtain an eluent. The gradient elution with the alcohol-water solution includes: eluting the alcohol-water solution at a rate of 1.5-2 BV / h, eluting with a 20% (w / w) ethanol-water solution for 5 BV, collecting the first eluent, then eluting with a 40% (w / w) ethanol-water solution for 3 BV, discarding the eluent, then eluting with an 80% (w / w) ethanol-water solution for 5 BV, collecting the second eluent, and mixing the first and second eluents to obtain the eluent. The decolorization step includes adsorbing and decolorizing the eluent with activated carbon and filtering to obtain a decolorized solution. The concentration and drying step includes concentrating and drying the decolorized solution to obtain a powdered lemon balm extract.

2. The method according to claim 1, characterized in that, In the mixed solution of alcohol and water, the mass fraction of alcohol in the mixed solution is 50-80%; and / or, the alcohol includes one or more of ethanol, ethylene glycol, glycerol, and 1,2-butanediol.

3. The method according to claim 2, characterized in that, The extraction process must meet at least one of the following conditions: 1) the ratio of lemon balm crushed material to mixed solution is 1:(10~30); 2) the extraction temperature is 40~90℃; 3) the extraction time is 2~10h; 4) the extraction is performed at least once; 5) at least one of the following extraction methods is used: stirring extraction, solvent extraction, and ultrasonic extraction.

4. The method according to claim 1, characterized in that, The filtration step includes filtering the crude extract using a filter membrane under negative pressure filtration or positive pressure filtration to obtain a filtrate; and / or, the pore size of the filter membrane is 0.22 μm to 10 μm.

5. The method according to claim 1, characterized in that, The crude extract contains ≤10% ethanol by mass.

6. The method according to claim 5, characterized in that, The adsorption resin includes at least one of D101 type macroporous adsorption resin, AB-8 type macroporous adsorption resin, and HPD100 type macroporous adsorption resin; and / or, the water volume for water elution is 3BV to 5BV.

7. The method according to claim 1, characterized in that, The decolorization step shall meet at least one of the following process conditions: the amount of activated carbon added shall be 1% to 5% of the mass of the eluent; the decolorization time shall be 20 to 60 min; and the decolorization temperature shall be 30 to 70℃.

8. The method according to claim 1, characterized in that, The concentration and drying steps meet any one of the following process conditions: 1) Drying is performed using a spray dryer; 2) Drying is performed using a granulator.

9. The method according to claim 8, characterized in that, The inlet temperature of the spray dryer is 100~160℃, and the outlet temperature is 60~90℃.

10. The method according to claim 8, characterized in that, The feed rate of the concentrated decolorized liquid in the spray dryer is 5~40mL / min.

11. The method according to claim 8, characterized in that, The concentrated decolorized liquor was dried using a spray dryer under a compressed air pressure of 0.4~0.8MPa.

12. The method according to claim 8, characterized in that, The drying temperature when using a granulator is 105~125℃.

13. A lemon balm extract, characterized in that, It is obtained by the preparation method according to any one of claims 1-12.

14. The lemon balm extract according to claim 13, characterized in that, Lemon balm extract includes flavonoids, rosmarinic acid, and caffeic acid.

15. The lemon balm extract according to claim 13, characterized in that, The total flavonoid content of lemon balm extract is 40%~50%, the luteolin content is 5%~10%, the rosmarinic acid content is 20%~25%, and the caffeic acid content is 2%~3%.

16. The lemon balm extract according to claim 13, characterized in that, The coumarin content in lemon balm extract is <4 μg / ml.

17. The lemon balm extract according to claim 13, characterized in that, Melissa extract has at least one of the following effects: sun protection, anti-glycation, promoting collagen production, promoting dermal-epidermal junction, and soothing.

18. A cosmetic composition, characterized in that, The extract includes the lemon balm extract obtained by the preparation method according to any one of claims 1-12 or the lemon balm extract according to any one of claims 13-17.

19. The cosmetic composition according to claim 18, characterized in that, The mass fraction of lemon balm extract in cosmetic compositions is 0.05%-10%.

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